Current Medicinal Chemistry 2007 14 1505-1523 1505
Molecular Probes for P2X7 Receptor Studies
Hendra Gunosewoyo1 Mark J Coster1 and Michael Kassiou12
1School of Chemistry University of Sydney NSW 2006 Australia2School of Medical Radiation Sciences Brain amp Mind Research Institute University of Sydney NSW 2050 Australia
Abstract The ionotropic P2X7 receptor (P2X7R) has become the focus of intense research interest for a number of reasons i)it is a cation selective ion channel that is modulated by extracellular ATP Upon stimulation by high concentrations of ATP itgenerates a non-selective membrane pore which is permeable to hydrophilic molecules with molecular weight up to 900 Da ii)Though its physiological function is yet to be fully understood there is high P2X7R expression in microglia Importantly thisimplies a pivotal role for the P2X7R in neuro-inflammatory and -degenerative processes In addition P2X7R-stimulated releaseof traditional neurotransmitters in the brain such as glutamate and GABA further supports the involvement of P2X7R in neuro-inflammatory and -degenerative processes P2X7-knockout animals are also found to be resistant to inflammation andneuropathic pain which suggests that P2X7 antagonists could potentially serve as all-purpose analgesics Recent advances in thedevelopment of P2X7R ligands have resulted in identification of several different classes of P2X7R antagonists including ATPanalogues (oxidized ATP) dyes (Brilliant Blue G) tyrosine derivatives (KN-62 and KN-04) cyclic imides adamantane andbenzamide derivatives A KN-62 related radioligand has also recently been reported for use in receptor binding assays A moreextensive range of potent selective P2X7R ligands is required for a better understanding of the cascade of cellular processesassociated with the P2X7R This article will review P2X7R ligands discovered to date together with their biological activity andtherapeutic potential
Keywords ATP purinergic P2X7 microglia antagonist KN-62 adamantyl amides tetrazoles
1 INTRODUCTION endothelial cells [8 25-29] Expression of the P2X7R has alsobeen demonstrated in the enteric nervous system of guinea pigsmall intestine [30] kidney and urinary tract [31] uterine [32]and more recently at the neuromuscular junction of mousemotor nerve terminals [33] Interestingly it has been shown thatonly the P2X7R was present on presynaptic motor nerveterminals from birth and no expression of P2X1-6 receptors wasdetected [33]
It is now firmly established that the purine nucleosideadenosine and its nucleotides in particular adenosine 5rsquo-triphosphate (ATP) are capable of producing a wide range ofpharmacological effects that are not directly related to theiruniversal role in energy metabolism [1-3] ATP and itsmetabolites are increasingly recognized as key molecules inintercellular signalling processes as well as the regulation ofcell growth and differentiation of the nervous cardiovascularrespiratory and immune systems by stimulating purinergicreceptors [4]
The ionotropic P2X7R has become the focus of intenseresearch interest since it is an unusual non-desensitizingcation-selective ion channel directly gated by extracellularATP The P2X7R differs from the other P2X receptor subtypes(P2X1-6) by its long (240aa) cytoplasmic carboxy-terminal tail(Fig 1A) which is necessary for its bifunctionality [34] actingas either a selective ion channel or as a non-selective poredepending on the duration and intensity of the agoniststimulation (Fig 1B) [5 34]
Purinergic receptors are cell-surface proteins that bind toATP and its metabolites When cells are damaged a considerableamount of ATP is released into the extracellular environmentthereby serving as a fast-acting ligand for purinergic receptorsbefore being quickly hydrolyzed by ecto-ATPases andectonucleotidases [5 6] There are 2 main classes of purinergicreceptors P1 and P2 receptors which bind to extracellularadenosine and ATP respectively The P2 receptors can be furtherdivided into two families the G-protein coupled receptors(denoted P2Y receptors) and the ligand-gated ion channels(denoted P2X receptors) In mammalian cells seven P2Xreceptor subtypes (P2X1-7) [7 8] and eight distinct P2Yreceptors (P2Y1 P2Y2 P2Y4 P2Y6 P2Y11 P2Y12 P2Y13 andP2Y14 ) [9] have currently been identified and are currentlyunder scrutiny as potential therapeutic targets
Brief exposure of the P2X7R to agonists such asendogenous ATP 1 (Fig 2) or synthetic nucleotide BzATP 3(2rsquo(3rsquo)-O-(4-benzoylbenzoyl)adenosine 5rsquo-triphosphate Fig3) results in a transient current through opening of cationchannels characteristic of the entire P2X receptor familyHowever repeated or prolonged agonist exposure generates asustained current through the formation of a non-selective porepermeable to large inorganic and organic cations up tomolecular weights of 900 Da (Fig 1B) such as ethidiumbromide propidium iodide and quinolinium fluorescent dyessuch as YO-PRO-1 This non-selective pore formationeventually leads to cell death The P2X7R is structurally relatedto other members of the P2X family with 35-40 amino acididentity in the first 395 amino acids Seven variants of thehuman P2X7R resulting from alternative splicing have beenidentified and characterised [35] Detailed structural analyses ofthe P2X7R [36 37] and general P2X receptors [7 38 39] havebeen previously reported
The purinergic P2X7 receptor (P2X7R) is ubiquitouslydistributed in a wide variety of cell types including i) cells ofhaematopoietic origin such as mast cells macrophagesfibroblasts human monocyte cell line THP-1 erythrocytesgranulocytes erythroleukaemia cells and lymphocytes [3] ii)central [10-12] and spinal cord neurons [8 13] iii) brain glialcells including microglia astrocytes and Muumlller cells [14 15]iv) retina cells [16-19] v) bone cells including osteoblastsosteoclasts and osteocytes [20-24] and vi) epithelial and The rat human and mouse P2X7R-encoding cDNAs were
isolated and characterized by Surprenant et al in 1996 [34]Rassendren et al in 1997 [40] and Chessell et al in 1998respectively [41] Operational differences are observed amongstthese three mammalian species in terms of the i) non-desensitizing properties in electrophysiological studies ii) dye
Address correspondence to this author at the School of Chemistry University ofSydney NSW 2006 Australia Tel +61 2 9351 0894 Fax +61 2 9351 0652E-mail mkassioumedusydeduau
1506 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
Fig (1) A The molecular topology of the P2X1-6 and the P2X7 receptors The key distinguishing feature of the P2X7R to the other P2X receptors is itslong carboxy-terminus tail B The 3 different forms of the P2X7R upon activation by ATP closed channel open channel and pore formation Brief ATPactivation leads to opening of an ion channel permeable to small ions such as Na+ K+ and Ca2+ whereas prolonged ATP exposure results in the poreformation permeable to high molecular weight organic cations eg N-methyl-D-glucamine (NMDG) and YO-PRO-1 dye
uptake profile (Table 1) and iii) binding affinity to the agonistsATP and BzATP [42] Variations in the ionic composition of thebuffer [43] and pre-treatment of cells by repeated exposure toATP [44] also significantly affect the concentration-effectcurves (Table 1) In the YO-PRO-1 uptake assay BzATP is foundto be at least 100-fold and 30-fold more potent at rat and humanP2X7Rs respectively compared to the mouse P2X7R
any other brain cells [48] Activated microglia are known torelease various cytokines (such as interleukins [49-55] viacaspase-1 enzyme) tumour necrosis factor-α nitric oxide andreactive oxygen species Alternatively they may develop intophagocytes to remove debris cells and consequently arethought to be the main cytotoxic effector cells in the CNS Thereare literature reports for the expression of both P2X and P2Yreceptors in various microglia and for the ability of the P2X7Rto induce similar responses to those seen in activated microglia[15 45-47 56-60] The role of the P2X7R in mediating cytokinerelease is of particular interest in the context ofneurodegeneration It has recently been shown that
Although the exact physiological function of P2X7R is yetto be completely understood they are abundantly expressed inbrain microglia [45-47] Microglial cells which assume arestinginactive state under normal conditions will react tovirtually any pathogen and become activated often well before
N
NN
N
NH2
O
O HOH
OPO
OH
O
POPHO
O
O H
O
OH
1 ATP
N
NN
N
NH2
O
OHOH
OPO
O-
O
POP-O
O
O-
O
O-
2 ATP4-
Fig (2) Chemical structures for endogenous P2X7R ligand ATP (1) and its tetrabasic form (2)
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1507
Table 1 Potency of BzATP and ATP at Recombinant Mouse Human and Rat P2X7R (mP2X7R hP2X7R rP2X7R) and Mouse NTW8 Microglia(mNTW8)
Buffer composition (in millimolar)aNaCl buffer (NaCl 145 KCl 2 CaCl2 05 HEPES 10 D-glucose 10 pH 73) bNa-glutamate buffer (Na-glutamate 145 KCl 2 CaCl2 05 HEPES 10 D-glucose 10 pH73) cSucrose buffer (sucrose 280 HEPES 10 NMDG 5 KCl 56 D-glucose 10 CaCl2 1 pH 74)A change from NaCl to Na-glutamate buffer generally increases the potency of the agonists at the P2X7R Pre-treatment of cells by repeated ATP exposure will induce theP2X7R to adopt the pore state The cells in the pore state are generally more sensitive towards the agonists
inflammatory processes modulated by cytokines in particularthe interleukins and generation of tumour necrosis factors andnitric oxide have important roles in the many forms ofneurodegenerative disease such as multiple sclerosisParkinsonrsquos Alzheimerrsquos and Huntingtonrsquos disease [61] Thepresence of activated microglia in the brain has thus beenconsidered a reliable marker for neurodegenerative diseases TheP2X7R has also been shown to modulate β-amyloid-inducedcytokine expression (IL-1α and IL-1β) from humanmacrophages and microglia This further supports P2X7Rinvolvement in the neuropathology of Alzheimerrsquos disease inwhich the presence of β-amyloid is often surrounded byactivated microglia and astrocytes [62] Taken together theP2X7R has a pivotal role in neuro-inflammatory and-degenerative processes In addition P2X7R can stimulaterelease of traditional neurotransmitters in the brain such asglutamate and GABA The P2X7R regulation of glutamate andGABA release in rat gerbil and mouse hippocampus [63-65] andmurine astrocytes [66 67] has been reported although it is stillnot definitively confirmed [68] More recently two distinctglutamate release pathways in rat hippocampal astrocytes bypurinergic receptors have also been reported [69]
arthritis model [72] Inhibition of the P2X7R has also beenshown to improve recovery after spinal cord injury in ratmodels [73] However functional P2X7-like proteins have beenfound in one P2X7-knockout model [74 75] highlighting thelack of understanding of the P2X7-knockout animal modelsDominant-negative P2X7R subunits have been proposed as anovel genetic tool to understand the role of P2X7R in nativesystems [76] More recently it has been demonstrated thatexpression of cyclooxygenase-2 cannabinoid receptor CB2and P2X7R are all elevated in activated microglia cells ofmultiple sclerosis and amyotrophic lateral sclerosis spinal cord[77]
Further studies into understanding the physiologicalfunctions of P2X7R in vivo and the development of detailedpharmacophore models will lead to better understanding ofcellular cascades underlying the basis of neuro-inflammatoryand -degenerative processes A number of P2X7R antagonistshave already been developed towards this goal however a moreextensive range of ligands with drug-like properties includingimproved potency and selectivity is still required Althoughthere have been a number of reviews on the pharmacology of theP2X7R [78-80] there appears to be relatively limited accessibleresources on a comprehensive listing of molecular probesacting at the receptor [81-83] This review will present anoverview of the currently known agonists and antagonistsacting at the P2X7R and comment on their usefulness asmolecular probes for microglia activation
The exact role of P2X7R in vivo remains to be elucidated andliterature studies to date have compared normal and P2X7-knockout animal models In terms of pharmacologicalintervention the most promising roles of the P2X7R in vivowould most likely be in the areas of pain regulation [70]chronic inflammation [57 71] and neurodegeneration [61]Chronic inflammatory and neuropathic hypersensitivity arecompletely abolished in P2X7-knockout mice although normalnociceptive processing still remains [70] suggesting thatpotent selective P2X7 antagonists may serve as all-purposeanalgesics P2X7-knockout mice have been reported to showreduced severity of arthritis in an anti-collagen antibody
2 ENDOGENOUS LIGANDS FOR P2X7R
ATP 1 is the natural endogenous agonist of the P2X7R withEC50 values varying from 85 to 779 microM depending on thespecies examined (rat mouse or human) as determined from
1508 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
N
NN
N
NH2
O
OH(R)(H)RO
OPO
OH
O
POPHO
O
OH
O
O H
O
O
mixture of 2 and 3 e sters
3 BzATP
R =
Fig (3) Chemical structure for BzATP (3) the most potent P2X7R agonist to date
electrophysiological assays (Table 1) The low potency of ATPin activating the P2X7R is one of the main pharmacologicalfeatures of this unique receptor that distinguish it from otherpurinergic receptors Normal cytoplasmic concentrations of ATPin cells range from 5 to 10 mM which is released to theextracellular environment upon cell injury or insult therebyactivating the P2X7R before being quickly hydrolyzed by ecto-ATPases and ectonucleotidases Reduction of the extracellularMg2+andor Ca2+ ion concentration which are thought to beallosteric inhibitors [84] results in the potentiation of ATPeffects at the P2X7R [8] It has also been suggested that thetetrabasic form of ATP 2 is the active species acting at thereceptorrsquos ATP-binding site where the presence of Mg2+ andCa2+ ions chelate ATP4- rendering it inactive [85]
pharmacophore known for P2X7R activity The effects of thesesynthetic ligands on the P2X7R are generally tested in eitherelectrophysiological or dye uptake assays exploiting thereceptorrsquos non-desensitizing property and the ability to form alarge non-selective pore
31 ATP Derivatives
BzATP 3 (Fig 3) is the most potent P2X7R agonist to datewith 10- to 100-fold higher potency compared to ATP inactivating the receptor in a number of different systems [3] Thepotency of BzATP is also known to be species-dependent EC50value of BzATP at the rat P2X7R is 2ndash7 microM in theelectrophysiological assay which is approximately 10- to 30-fold higher than at the human P2X7R [40 88] BzATP also has adifferent EC50 value (007 ndash 025 microM) for activating the ratP2X7R in the YO-PRO-1 uptake assay performed in sucrosebuffer (Table 1) The increased sensitivity of the P2X7R toBzATP compared to the endogenous ligand ATP is one of themain criteria used to define the pharmacology of the P2X7Rsubtype Unfortunately BzATP is not highly specific for theP2X7R subtype other P2X receptors are also activated byBzATP [89] but with either equal or lower potency than ATP[90] Other ATP derivatives such as 2-methylthio-ATP 4 ATP-gamma-S 5 and ADP 6 are all weak agonists of the P2X7R (Fig4) [34 90] The order of potency for P2X7R agonists is BzATP3 gtgt ATP 1 gt 2-Methylthio-ATP 4 gt ATP-γ-S 5 gtgt ADP 6 [34] 2-Methylthio-ATP 4 has an EC50 value of 505 microM against theP2X7R derived from mouse NTW8 microglial cells based on theelectrophysiological studies [60]
Some diadenosine polyphosphates such as P1 P4-diadenosine tetraphosphate were reported to be capable ofactivating the P2X7R-mediated cellular permeabilization of ratmast cells [86] possibly due to their greater resistance tometabolic breakdown and similarity to ATP4- [3] Theendogenous peptide LL-37 an antimicrobial agent causingbacteria cell wall permeabilization derived from the humancathelicidin protein hCAP18 has been demonstrated to induceinterleukin-1β processing and release via the activation of theP2X7R [87]
3 SYNTHETIC LIGANDS FOR P2X7R
Several classes of P2X7R-active synthetic compounds havebeen reported in the literature Unfortunately despite thediscovery of these ligands there is still no general
N
NN
N
NH2
O
OHOH
OPO
O H
O
POPHO
O
OH
O
OH
SMe
4 2-Me thylthio-ATP
N
NN
N
NH2
O
OHOH
OPO
OH
O
POPHS
O
OH
O
OH
5 ATP-γ-S
N
NN
N
N H2
O
OHOH
OPO
OH
O
PHO
O
OH
6 ADP (adenosine 5-diphosphate)
N
NN
N
NH2
O
OO
OPO
O H
O
POPHO
O
OH
O
OH
7 oxidize d ATP
Fig (4) Chemical structures for ATP derivatives that are weak P2X7R agonists
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1509
The 2rsquo 3rsquo-dialdehyde ATP (or oxidized ATP) 7 (Fig 4) is thefirst irreversible inhibitor of the P2X7R expressed in the mousemacrophage cell line J774 [91] Subsequent studies alsoshowed that application of 100 microM oxidized ATP irreversiblyantagonized the rat P2X7R provided the cells were pre-incubated with oxidized ATP for 1-2h indicating that itcovalently modified the receptor [34] Co-application with anagonist did not result in any inhibition There is evidence thatoxidized ATP possesses low affinity for the P2Y receptors [91]however oxidized ATP at 100 microM concentration has beenshown to be very toxic to rat cerebellar granule neurons [92]and is likely to react with other enzymes such as ecto-ATPases[91 93] Interestingly oxidized ATP at 168 microM inhibitedP2X7R-mediated inflammatory pain in arthritic rats [94 95]Due to its non-selective inhibition extra caution is required inthe use of oxidized ATP to define involvement of P2X7R in thepurinergic system [42]
in a non-selective manner [3] It is a relatively potent antagonistat the P2X1-3 P2X5 receptors (IC50 01ndash5 microM) and P2Y1receptor but only weakly active or completely inactive at the ratP2X4 P2X6 P2X7 and some P2Y receptor subtypes [96]Interestingly PPADS is reported to be reasonably active at thehuman P2X4R with IC50 value of 28 microM indicating somespecies dependence [97] PPADS is highly specific for the P2receptors up to 100 microM concentration of PPADS does notinterfere with adenosine receptors muscarinic M1 M2 or M3α1- or α2-adrenoceptors histamine H1 AMPA or NMDA and 5-HT receptors [96]
PPADS has been widely studied in the P2 receptor systemsderived from various cell lines and structure-activityrelationship (SAR) studies have also been reported [98-101]Structural modifications of PPADS to date have involved theinvestigation of substitutions at the 4-aldehyde position 5-phosphate and the azophenyl ring Some of the key PPADSderivatives are represented in Fig (6) It was initially thoughtthat both the aldehyde and phosphate moieties are important forP2 antagonism It was also proposed that the slowlyequilibrating pseudoirreversible mode of inhibition of PPADSseen at the P2X1 receptor was due to Schiff base formationbetween the aldehyde and a specific lysine residue of thereceptor [96] The cyclic phosphates MRS2219 and MRS2220were found to be relatively potent antagonists at the rat P2X1receptor (IC50 6 and 10 microM respectively) with good selectivityover P2X2-4 P2Y1 P2Y2 P2Y4 P2Y6 and adenosine receptors[99] Another PPADS derivative that appears to be a promisingantagonist at the P2X1 receptor is PPNDS which exhibited anIC50 of 15 nM and no interactions detected withectonucleotidases P2Y1 receptor α1-adrenoceptors adenosinehistamine H1 and muscarinic M3 receptors [102] Later studiesreported that replacement of the sulfonyl groups on theazophenyl ring with carboxylates or phosphates furtherincreased the potency and selectivity at the P2X1 and P2X3receptors [101] Most recently PPADS analogues MRS2211 andMRS2603 have been reported to be active at the P2Y13 receptorbeing 45- and 74-fold more active respectively than PPADS[103] Only a limited number of PPADS analogues were testedagainst the recombinant human P2X7R in these studies and forthose it was found that none of them was more potent than iso-
Overall these ATP derivatives have high molecular weightsand could potentially bind to other purinergic receptors due totheir resemblance to the endogenous ligand ATP Thereforethey are unlikely to be suitable for developing an orally activedrug specifically targeting the P2X7R
N
HO
CHO
O P O-
O-
O
9 P5P
N
O P
O
O-
O-HO
CHO
N
N
SO3--O3S
43
21
6
5
8 PPADS
Fig (5) Chemical structures for PPADS and its parent molecule P5P
Fig (7) Chemical structure for DIDS and its derivatives
PPADS which differs from PPADS by the position of thesulfonates [100]
[109] receptor DIDS dihydro-DIDS 17 and SITS 19 (Fig 7) allblocked the ATP-stimulated Ca2+ uptake in rat parotid acinarcells via activation of the P2X7R (IC50 35 ndash 125 microM) [109]DNDS 18 in which the isothiocyanate moieties are replacedwith nitro groups was found to be inactive indicating theimportance of the isothiocyanate for interaction with theP2X7R On the other hand a variety of smaller aromaticisothiocyanate compounds derived from DIDS have been testedagainst the P2X1 receptor and were found to be active with IC50values between 11 and 54 microM [108] Overall DIDS and itsderivatives are inhibitors of both the P2X1 and P2X7R withlower potency for the latter
All the SAR data generated to date on PPADS and itsderivatives demonstrate that structural modifications on thekey functional groups (aldehyde phosphate and azophenyl)increases the potency and selectivity mainly at the P2X1receptor Consequently PPADS itself is used for testing at theP2X7R where it was found to be a potent antagonist only at therat and human receptors (IC50 15 and 90 nM respectively) butnot the mouse receptor (100-fold lower potency) in the YO-PRO-1 uptake assay provided cells are pre-incubated withPPADS for 15-30 min [41] When there was no pre-incubationthe IC50 of PPADS at the rat human and mouse P2X7R decreasedto approximately 50-70 microM in the electrophysiologicalrecordings [34 40] but pre-incubation with PPADS drasticallyimproved the IC50 value to 1 microM at the human P2X7R [88] Theinhibitory action of PPADS at the P2X7R is only partiallyreversible [88] suggesting a pseudo-irreversible mode ofantagonism [104] It has been suggested that PPADS P5P andoxidized ATP may all have a common site of action which islikely to be the ATP binding site [104] Overall it can beconcluded that PPADS per se is a species-dependent P2X7Rantagonist being most potent at the rat and human P2X7R butnot the mouse receptor Derivatives of PPADS reported to dateonly exhibit improved potency and selectivity at the P2X1receptor and not the P2X7R
5-(NN-Hexamethylene)amiloride (or HMA) 20 (Fig 8)belongs to the amilorides which are better known as potassium-sparing diuretics by blocking the Na+ channel as well as beinginhibitors of Na+-H+ counter-transport pathway [112] L-typecalcium channels [113] adenosine A1 receptors [114] andpossibly Na+-HCO3
- co-transport [115] HMA possesses affinityfor the P2X7R but its effects are highly species-dependent At 40microM concentration HMA antagonized P2X7R found in humanlymphocytes as determined from large cation uptake studies[116 117] However up to 100 microM concentration it wasineffective at the rat P2X7R [34] More interestingly theelectrophysiological studies revealed that co-application ofHMA with ATP produced an immediate and maximalaugmentation of inward currents induced by mouse P2X7R [60]This effect was not apparent when HMA was applied alone orwhen the buffer solution contained physiologicalconcentrations of Mg2+ and Ca2+ ions HMA has been reportedto be an irreversible inhibitor at the hP2X7R [88] Otheranalogues of HMA such as MIBA 21 and EIPA 22 also possesssimilar properties to HMA [116] Due to its relatively lowpotency and high species dependence at the P2X7R HMA andprobably other amilorides are unlikely to be good candidatesfor further exploration of the pharmacology of the P2X7R
33 Ion Channel Blockers
44rsquo-Diisothiocyanatostilbene-22rsquo-disulfonic acid (DIDS)16 is an anion channel blocker widely used to study various iontransport mechanisms [105-107] but has also beendemonstrated to be capable of inhibiting both the P2X1 receptor[108] and P2X7R-induced pore formation and other processes inseveral cell lines with relatively low potency [3 109-111] Forexample pre-incubation of BAC12F5 murine macrophage with150 ndash 200 microM DIDS inhibited the activation of phospholipaseD mediated by the P2X7R [110] Pre-incubation of rat primaryastrocytes with 200 microM of DIDS has also been shown to inhibitP2X7R-mediated responses such as the activation ofextracellular signal receptor kinases (ERK) [111] SARinvestigations have been performed on DIDS in order toimprove its potency and selectivity at the P2X1 [108] or P2X7
34 Polysulfonated Dyes
Suramin 23 is an example of a polysulfonated dye thatexhibits non-selective antagonism at both the P2X and P2Yreceptors with relatively low potency [3] Suramin blocks theP2X1-3 and P2X5 receptors (IC50 1-15 microM) but only shows weakor no inhibition at the P2X4 P2X6 and P2X7 receptors as well
N
NCl
N NH 2
O
NH
NH
NH2
20 5-(NN-Hexa methylene )- amiloride (HMA)
N
NCl
NH2
O
NH
NH
NH2
N
21 5-(N-Methyl-N- isobutyl)- amiloride (MIBA)
N
NCl
NH2
O
NH
NH
NH 2
N
22 5-(N-ethyl-N-isopropyl)- amiloride (EIPA)
Fig (8) Chemical structures for HMA and other amilorides
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1511
as some P2Y receptor subtypes [96] In addition suramin hasbeen shown to interact with many different proteins at theseconcentrations including glutamate nicotinic GABA and 5-HTreceptors as well as various proteases and ectonucleotidases [396] Nevertheless SAR studies on suramin have been performedin order to identify the important structural features responsiblefor antagonism at the P2 receptors [96 118-124] Selected keysuramin derivatives are represented in Fig (10 )
native and recombinant P2X1 receptors respectively and hasbeen shown to be much less potent at other P2X and P2Ysubtypes including adrenoceptors histamine H1 receptors andthe muscarinic M3 receptor [120] At the human P2X1 and P2X7receptors the IC50 values for NF449 were 005 nM and 40 microMrespectively [128] which indicated its usefulness todistinguish between the two receptors Subsequent SAR studiesconfirmed that NF449 was the most potent and selective knownP2X1 receptor antagonist [121 129] NF157 an analogue ofsuramin with fluorine replacing the methyl group has also beenreported to be highly potent at the P2Y11 receptor with a Ki of45 nM [124] More recently NF110 has also been shown to be apotent P2X3 receptor antagonist indicating that both thenumber and location of the sulfonate groups may be importantfor determining P2X subtype potency and selectivity [123]
O
HN
HN
NH
NH
O O
O ONH HNSO3-
SO 3-
SO3-
-O3S
SO 3-
-O3S
23 suramin
NF279 thus far represents the most active suramin relatedantagonist at the P2X7R and displays a reasonable potency of28 microM All the SAR data obtained to date on suramindemonstrate the importance of the molecular size number andposition of the sulfonate groups for P2X subtype potency andselectivity Unfortunately except for NF279 and NF449 othersuramin analogues provide very little information which can beused for the selective structure-based drug design of P2X7Rmolecules Suramin is also unlikely to be orally active due toits high molecular weight and interaction with many otherproteins Suramin is a very weak or almost inactive antagonistat the human P2X7R with an IC50 value of 92 microM [40] It istotally inactive at both the rat and mouse P2X7R with IC50values greater than 100 microM [34 60] Due to its interference withthe fluorescence of YO-PRO-1 suramin could not be used in theYO-PRO-1 dye uptake assay [104]
Fig (9) Chemical structure for suramin a weak antagonist at the P2X7R
Early SAR analysis showed that the shortened forms ofsuramin in the form of sulfonylbenzamides decreased thepotency at P2 receptors [118] NF023 (24 ) was later discoveredto be P2X-selective compared to P2Y receptors andectonucleotidases [118] Subsequent studies have examined theeffects of NF023 in various cell types and it was reported thatNF023 is a highly selective antagonist for either rat or humanP2X1 receptors heterologously expressed in Xenopus oocyteswith an IC50 of 02 microM [125] Another suramin derivativeNF279 (25 ) also showed potent selective reversible inhibitionat the rat P2X1 receptor in Xenopus oocytes with an IC50 of 19nM [126] In studying the effects of NF279 on human P2X1 andP2X7 receptors it was reported that NF279 is almost 100-foldmore potent at the human P2X1 than the P2X7 receptor withIC50 values of 005 and 28 microM respectively [127] Other keysuramin derivatives resulting from extensive SAR studies areNF449 (27 ) NF110 (28 ) and NF157 (26 ) [120 121 123 124128 129] NF449 has an IC50 value of 70 nM and 03 nM at the
Brilliant Blue G 29 (Fig 11) is another polysulfonated dyewhich is a potent non-competitive inhibitor of the rat andhuman P2X7R with IC50 values of 10 and 200 nM respectivelyin electrophysiological studies [130] Brilliant Blue G has alsobeen shown to be highly selective for P2X7R over P2X1-5receptors [130] It has been shown that at the rat P2X7R about100 to 300 nM concentration was required to inhibit the YO-PRO-1 uptake which was 10- to 30-fold higher than thatrequired for blocking P2X7R-induced currents demonstratingits differential effect at the channel and pore forms of the P2X7R
O
HN
HN
NH
NH
O O
R R
O ONH HNSO3-
SO3-
SO3-
-O3S
SO3-
-O3S
O
HN
HN
NH
OO
NH
O ONH
NH
R R
RR
n n
Suramin n=1 R=CH3
24 NF023 n=0 R=H
25 NF279 n=1 R=H
26 NF157 n=1 R=F
27 NF449 R=24-disulfonyl28 NF110 R=4-sulfonyl
Fig (10) Suramin and its key derivatives investigated in the SAR studies
1512 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
[130] Brilliant Blue G has also been tested on the mouseP2X7R where similar antagonistic potency was found comparedto rat and human receptors [42] In this study it was alsoreported that the selectivity of Brilliant Blue G for rat overhuman P2X7R was confirmed at 22degC but lost at 37degC Thedrawback for this compound is that it could also be bound toproteins and thus would be expected to have lower potency inthe dye uptake assay than electrophysiology measurements dueto the depletion by cell monolayer effect [42] Nevertheless itrepresents a promising antagonist at rat human and mouseanalogues of the P2X7R No studies have been performed onBrilliant Blue G to further improve its potency and selectivityat the P2X7R Other polysulfonated dyes such as Evans BlueTrypan Blue reactive blue 2 reactive red 2 and their derivativeshave also been tested at the purinergic systems [131-133] butnone of them exhibited activity at the P2X7R
opening of the ion channel calmidazolium produces no effecton the YO-PRO-1 uptake The large non-selective porehowever is still formed even when the ionic currents areinhibited indicating that pore formation does not necessarilyrequire opening of the ion channels Calmidazolium wassuggested to be an allosteric modulator of the P2X7R at theagonist binding site inducing conformational change at thereceptor level that results in decreased agonist binding affinity[84] It displays less potency at inhibiting the agonist-evokedcurrents at both the mouse and human P2X7R with IC50 valuesof 1 microM and 100 nM respectively [41 88] At present no SARstudies have been reported on calmidazolium
N
N+
ClCl
O
ClCl
Cl
Cl
30 calmidazolium
NH
N
O
SO3-
N+
SO3-
Cl
29 Coomassie Brilliant Blue G
Fig (12) Chemical structure for calmidazolium a P2X7R antagonistcapable of distinguishing the channel and pore forms of the receptor
36 KN-62 and its Analogues
KN-62 31 (Fig 13) is a derivative of isoquinolinesul-fonamide known as a selective and potent inhibitor of themultifunctional calciumcalmodulin-dependent protein kinases(CaMK) with an IC50 of 09 microM at CaMK II [134] Gargett andWiley in 1997 first demonstrated that both KN-62 31 and itsanalogue KN-04 32 were potent inhibitors of P2X7R at humanlymphocytes with IC50 values of 13 and 17-37 nM respectivelyin both the ion flux and ethidium bromide uptake assays [134]The mode of inhibition is allosteric modulation similar to thatof calmidazolium
Fig (11) Chemical structure for Brilliant Blue G a potent antagonist at therat P2X7R
35 Calmidazolium
Extracellular calmidazolium 30 (Fig 12) is a potentinhibitor of BzATP-induced currents via binding to rat P2X7Rwith an IC50 of 13 nM [84] The unique feature ofcalmidazolium is its ability to differentiate between the channeland pore form of the P2X7R while it potently inhibits the
KN-62 and KN-04 are species-selective for P2X7Rantagonism They potently inhibited the ATP-induced currentsas well as the ethidium bromide uptake in HEK293 cells
Fig (13) Chemical structures for isoquinolines KN-62 and KN-04 potent P2X7R antagonists at the hP2X7R with much less effect on the rat and humananalogues
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1513
33e most active in the series but still 30-fold less potent than KN-62
Fig (14) Conformationally restricted analogues of KN-62 [136]
expressing human P2X7R at the concentration of 30 ndash 100 nMdepending on whether ATP or BzATP was used and the cellularbackground of the human P2X7R [135] However even up to 3microM concentration they had no effect at the rat P2X7R [135] andKN-62 was found to be at least 10-fold more potent at thehuman P2X7R than the recombinant mouse analogue [60] Theseisoquinolinesulfonamide derivatives may interact with theamino-terminal domain of the P2X7R since the introduction ofthe first 335 amino acids of the human P2X7R delivers KN-62sensitivity to the rat P2X7R and the counter-part chimera isfound to be insensitive to KN-62 [135] The IC50 at the humanP2X7R is 10-fold lower at 22degC than at 37degC in the ethidiumbromide uptake study [42]
tyrosine) 2) R2 (the tyrosine side chain) and 3) R3 (thesubstituent attached to the N-piperazine) (Fig 15) [137-139]
The effect of substitutions at the R1 R2 and R3 groups (Fig15 ) on the P2X7R antagonistic properties was compared on thebasis of inhibition of K+ release [137 138] At the R1 positionlarge hydrophobic moieties linked to the amino positionthrough sulfonamide (34a-b) or carbamate (34c) groups werepreferable for P2X7R inhibition In the series of derivativeswhere R2 = quinolinesulfonyl and R3 = t-butoxycarbonyl (Boc)introduction of carbobenzyloxy (Cbz) and quinolinesulfonylgroups at the R1 position were found to be the most active[137] In another series of derivatives where R1 = Cbz and R3 =Boc introduction of arylsulfonyl (34d-g) and benzoyl (34h )moieties at the R2 position were preferred [137] A free aminogroup at the R3 position (34i-j) andor opening of thepiperazine ring to an ethylene diamine greatly reduced theP2X7R activity Amongst the various acyl groups tested at theR3 position Boc and benzoyl groups (34k-m) were found to bepreferable but not sulfonyl (34n-q ) The presence of a methylgroup at the amino terminus of the tyrosine group is thereforenot an absolute pre-requisite for P2X7 antagonism [137]
R3 N N
O
N
O
R4
R1
R2
In another study undertaken by Chen et al to furtherexplore the SAR of 34m [138] it was found that nitro-substitution at the meta- or para- position of the Cbz group atR1 (34r-t) was well tolerated However introduction of anacetamido- or isothiocyanato- group at the Cbz group abolishedP2X7R antagonism (34uv) The original isoquinoline-5-sulfonyl moiety at the R2 position could be substituted withmeta- or para-substituted phenylsulfonyl groups such as tosyl(34r-t) 3-nitrophenylsulfonyl (34w) or 4-nitrophenylsulfonyl(34x) without adverse effects on the P2X7R antagonism [138]Substitution of the benzoyl group at the R3 position by a Boc(34y) or 4-aminobenzoyl group (34z) retained activity
Fig (15) General structure for the KN-62 derivatives (34a-z)
Extensive SAR studies have been performed on KN-62 inorder to enhance its potency and selectivity at the P2X7R Thefirst attempt to improve the antagonistic activity of KN-62 wasachieved by tethering the N-methyl of the tyrosine backbone tothe ortho-position of the proximal phenyl ring which resultedin a series of conformationally restricted KN-62 analogues withthe formula shown in Fig (14 ) [136] The constrained form ofKN-62 (31 vs 33a) was devoid of P2X7R antagonisticproperties Replacement of the isoquinoline-5-sulfonyl moiety(33a) with quinoline-5-sulfonyl (33b ) quinoline-8-sulfonyl(33c) or naphthalene (33d ) resulted in the loss of ability toinhibit the P2X7R-mediated Ca2+ influx [136] The most activecompound arising from this series was compound (33e) with anIC50 value of 316 nM which is 30-fold weaker than the originalKN-62 Taken together these results indicate that an extendedrather than folded conformation of KN-62 conferred by theincorporation of a methylene unit between the N-methyl of thetyrosine backbone to the ortho-position of the proximal phenylring is preferred at the P2X7R [136]
A study by Baraldi et al reported the systematic SAR profileof KN-62 specifically looking at modifications of thephenylpiperazine moiety (R2 position Fig 16 ) [139] In theinvestigation of the optimum chain linker between thepiperazine and phenyl groups it was found that one methylenespacer (35a) slightly improved the activity whereas a twomethylene spacer was not favourable (35b ) [139] The presenceof a basic nitrogen in the piperazine moiety is proven crucial forP2X7R antagonism as substitution of the piperazine ring withpiperidine (35a vs 35c) resulted in a 3-fold reduction of activity[139] Substitution of the phenyl ring with other heterocyclessuch as pyridine (35d ) or pyrimidine (35e) also decreased theP2X7R antagonistic property The para- position prefers a
Subsequent investigations on KN-62 involved extensivemodifications in three positions 1) R1 (the amino-terminus of
1514 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
small electron-withdrawing group eg fluorine (35f) and theantagonistic property decreases as the size of the electron-withdrawing group increases (35g 35h) A methyl- or nitro-group at the para- position (35i 35j) was beneficial foractivity but not evidenced with the methoxy (35k ) group As inthe case for para- substitution introduction of F Cl or CH3 butnot a methoxy (35l-o) at the ortho- position was well toleratedMoreover the meta- position could also tolerate a Cl or CF3group (35p 35q) The disubstituted compound 35r showedreduced antagonistic properties (IC50 = 1122 nM) possibly dueto the steric bulkiness of om-xylyl residue while the dichloroderivative 35s (IC50 = 34 nM) retained activity A slightdecrease in potency was observed when the methyl group on thenitrogen on the α-position of the tyrosine was removed (35n vs
35t) further confirming that this is not substantial for P2X7Rantagonism The most potent compound arising from this studywas 35f with an IC50 value of 13 nM at inhibiting calciuminflux in human monocytes compared to KN-62 which has anIC50 of 51 nM [139] It is therefore confirmed that thephenylpiperazine group of KN-62 is crucial for interaction withthe active site of the receptor and can be potentially improvedwith small halogen substitutions particularly in the paraposition
An ortho-methyl substituent on the N-phenyl ring of KN-62increased the potency by 3-fold (36 IC50 15 nM) [140]Radiolabelling of compound 36 with tritium and preliminarybinding studies to the P2X7R have also been achieved [140]
Table 2 Structures and P2X7R Antagonistic Properties of Selected KN-62 Derivatives [137138]
34a isoquinoline-5-SO2mdash isoquinoline-5-SO2mdash Bocmdash H 77 plusmn 24 ~40
34b quinoline-8-SO2mdash quinoline-8-SO2mdash Bocmdash H 61 plusmn 30 ND
34c Cbzmdash quinoline-8-SO2mdash Bocmdash H 53 plusmn 23 ~200
34d Cbzmdash PhSO2mdash Bocmdash H 59 plusmn 14 ND
34e Cbzmdash p-tolyl-SO2mdash Bocmdash H 71 plusmn 30 ~300
34f Cbzmdash p-OMe-PhSO2mdash Bocmdash H 62 plusmn 15 ND
34g Cbzmdash isoquinoline-5-SO2mdash Bocmdash H 43 ND
34h Cbzmdash benzoylmdash Bocmdash H 47 plusmn 21 ND
34i isoquinoline-5-SO2mdash isoquinoline-5-SO2mdash H H 14 plusmn 12 ND
34j quinoline-8-SO2mdash quinoline-8-SO2mdash H H 5 plusmn 9 ND
34k Cbzmdash PhSO2mdash Cbzmdash H 48 plusmn 29 ND
34l Cbzmdash PhSO2mdash benzoylmdash H 78 plusmn 22 ND
34m Cbzmdash isoquinoline-5-SO2mdash benzoylmdash H 85 plusmn 10 ~200
34n Cbzmdash PhSO2mdash p-tolyl-SO2mdash H 0 ND
34o Cbzmdash PhSO2mdash CH3SO2mdash H 0 ND
34p Cbzmdash PhSO2mdash PhSO2mdash H 0 ND
34q Cbzmdash PhSO2mdash p-OMe-PhSO2mdash H 0 ND
34r Cbzmdash p-tolyl-SO2mdash benzoylmdash H 71 plusmn 21 ND
34s p-NO2-Cbzmdash p-tolyl-SO2mdash benzoylmdash H 69 plusmn 25 ND
34t m-NO2-Cbzmdash p-tolyl-SO2mdash benzoylmdash H 72 plusmn 6 ND
34u m-acetamido-Cbzmdash p-tolyl-SO2mdash benzoylmdash H 9 plusmn 13 ND
34v m-isothiocyanato-Cbzmdash p-tolyl-SO2mdash benzoylmdash H minus4 plusmn 5 ND
34w Cbzmdash m-NO2-PhSO2mdash benzoylmdash H 75 plusmn 10 ND
34x Cbzmdash p-NO2-PhSO2mdash benzoylmdash H 78 plusmn 18 ~100
34y m-NO2-Cbzmdash p-tolyl-SO2mdash Bocmdash H 94 plusmn 28 ~100
34z Cbzmdash p-tolyl-SO2mdash p-NH2-benzoylmdash H 93 plusmn 3 gt 300
aAntagonist function was measured by the percent inhibition of the K+ release triggered by 3mM ATP in paired wells in the absence of antagonist bAntagonistic IC50values derived from dose-response curves obtained on the basis of inhibition of K+ release
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1515
R2 X N
O
N
O
S
R1
S
O
O
N
O
O
Nthe inhibition of BzATP-induced ethidium bromide uptakemediated by the P2X7R expressed endogenously on the humanpre-monocytic cell line THP-1 [142] Compound 37a (Table 4)was identified as the lead structure and modifications on theimide and 4-pyridyl moieties were investigated The cyclicimide moiety is proven essential for the P2X7R activity sincesubstitutions with simple amines acyclic amides or acycliccarbamates almost completely abolished the activity [142] Thering can be five- or six-membered in the forms of imides (37a-bh) carbamates (37c-d) thiazolidine-24-diones (37eg) andhydantoins (37f) Replacement of the 4-pyridyl moiety with 4-substituted benzamides (37g-h) retained the P2X7R activityThe presence of a 3rsquo-nitro substituent on the biphenyl subunitwas reported to convey optimum P2X7R activity The mode ofinhibition of these compounds seemed to be competitivealthough there were observable changes in the maximalresponse as the compound concentration was increased [142]
Fig (16) General structure for the KN-62 derivatives (35a-35s)
Unfortunately while these KN-62 analogues are potentiallyuseful tools to investigate the basic properties of the P2X7Rthey do not represent good starting points for the discovery oforally-active drugs due to 1) high molecular weight (gt700 Da)2) high lipophilicity (clogP gt 6) and 3) presence of ametabolically labile yet crucial sulfonate group [141]
These compounds represent a new class of ligands thatpossess activity to the P2X7R They appear good candidates asorally active drugs due to their relatively low molecular weight(~400 Da) and would be highly amenable for further SARstudies However despite the succinimides and thiazolidine-24-diones representing potent analogues they were found to bemetabolically and chemically fragile On the other handalthough the carbamates were less potent they displayedimproved stability Future SAR studies could usefully involveinvestigation of the biphenyl ring and the chain linker
37 Cyclic Imides and Analogues
Cyclic imides 37 (Fig 18) were discovered through a high-throughput screening of AstraZenecarsquos compound library for
Table 3 Structures and P2X7R Antagonistic Properties of Selected KN-62 Derivatives [139]
Entry R1 R2 X IC50 (nM)a
KN-62 mdashCH3 mdashPh N 511 plusmn 11
35a mdashCH3 mdashCH2Ph N 211 plusmn 08
35b mdashCH3 mdashCH2CH2Ph N 600 plusmn 27
35c mdashCH3 mdashCH2Ph CH 653 plusmn 08
35d mdashCH3 2-pyridinyl N 170 plusmn 39
35e mdashCH3 2-pyrimidinyl N 798 plusmn 07
35f mdashCH3 p-F-Phmdash N 13 plusmn 01
35g mdashCH3 p-Cl-Phmdash N 105 plusmn 11
35h mdashCH3 p-I-Phmdash N 176 plusmn 38
35i mdashCH3 p-Me-Phmdash N 135 plusmn 02
35j mdashCH3 p-NO2-Phmdash N 58 plusmn 06
35k mdashCH3 p-OMe-Phmdash N 132 plusmn 43
35l mdashCH3 o-F-Phmdash N 185 plusmn 04
35m mdashCH3 o-Cl-Phmdash N 158 plusmn 09
35n mdashCH3 o-Me-Phmdash N 151 plusmn 14
35o mdashCH3 o-OMe-Phmdash N 841 plusmn 5
35p mdashCH3 m-Cl-Phmdash N 141 plusmn 03
35q mdashCH3 m-CF3-Phmdash N 311 plusmn 12
35r mdashCH3 om-diMe-Phmdash N 1122 plusmn 25
35s mdashCH3 mp-diCl-Phmdash N 339 plusmn 08
35t H o-Me-Phmdash N 288 plusmn 03
aAntagonistic IC50 (nM)a values derived from dose-response curves on the basis of inhibition of Ca2+ flux in human monocytes
1516 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
N N
O
N
S
N
OO
OS O
O
N
R
31 R = H (KN-62)
36 R = 2-[3H]Me
AstraZeneca reported that no replacement for the adamantaneframework was found [141] Investigation of the chain linkerrequirements between the adamantane and the phenyl ring wasperformed where it was found that C- and N- methylationcarbonyl reduction and methylene removal (38d-g) all causedloss of potency One methylene unit extension (38h ) retainedthe potency whereas reversal of the amide connectivity (38i)slightly decreased the potency AstraZeneca prepared acombinatorial library by reacting commercially availableadamantanemethylamine with a large variety of acid chloridesor via standard amide coupling with carboxylic acids Thegeneral trends indicated that only aromatic amides had activityat the P2X7R [141] On the phenyl ring 23- and 25-disubstitutions resulted in better antagonists (38j-o) than the26- (38p ) 35- (38q ) or 24-disusbstitutions (38r) The ortho-position could also tolerate substituents such as Br OMe andMe (38s-u) A closer examination on the reverse amides (38i v-y) also revealed that 23- and 25-disubsitutions were stillpreferred for P2X7R inhibition The indazole amide (38z) wasidentified as a target lead molecule with a good metabolicprofile suitable for further optimization for clinical trial studies[141]
Fig (17) Radiolabeled analogue of KN-62 [140]
O
NY
X
O
Z
NO2
Adamantyl amides represent a class of simple moleculeswith excellent activity at the P2X7R The nature of theadamantane group could potentially be further investigatedalthough the original authors stated that no replacement for theadamantane group was found A number of patents onadamantyl-containing benzamides have been generated byAstraZeneca [143-146] and Pfizer [147] indicating the potentialof this ligand class for generating a truly potent selective andorally active P2X7R inhibitor Pfizer specifically incorporatedtriazinyl group into the adamantyl benzamide framework [147]Fig (18) General structure for the cyclic imides generated by
AstraZeneca Non-adamantane benzamides including piperidinyl andpiperazinyl benzamides [148 149] triazinyl benzamides [150-153] quinolinyl benzamides [154 155] 34-difluoroanilinebenzamides [156] aromatic acid-containing benzamides [157]pyrazolylmethyl benzamides [158] and other heteroarylamides[159-163] have also been patented by various otherpharmaceutical companies
38 Adamantyl Amides
Adamantyl amides were also discovered from a high-throughput screening of AstraZenecarsquos compound library forinhibition of the P2X7R-mediated ethidium bromide uptakeinduced by BzATP [141] N-Adamantan-1-ylmethyl-2-chloro-benzamide 38a was identified as the lead structure with anexcellent pA2 value of 81 The presence of an ortho-substituentis believed to cause a twist in the orientation of the benzamideessential for potent P2X7R antagonism whereas para-substituents (38bc) have deleterious effects on the P2X7Ractivity Selected compounds of this series are represented inTable 5
39 Diarylimidazolines
Diarylimidazolines represent another class of P2X7-activecompounds They were first discovered through a high-throughput screening of Aventisrsquo compound library forinhibition of BzATP-induced YO-PRO-1 uptake mediated byP2X7R expressed in U373 cells stably expressing the human
Table 4 Structures and P2X7R Antagonistic Properties of Selected Cyclic Imides [142]
Entry X Y Z IC50 ( M) pA2a
37a CH2 CO N - 69
37b (CH2)2 CO N - 75
37c O CH2 N 64
37d O (CH2)2 N 063 -
37e S CO N - 77
37f NH CO N 02 -
37g S CO C-CONHMe 016 -
37h CH2 CO C-CONMe2 025 -
a pA2 = the negative logarithm of a molar concentration of an antagonist that necessitates doubling of agonist concentration needed to elicit the original submaximalresponse obtained in the absence of antagonist
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1517
P2X7R [164] Compound 39a was identified as an initial hitfrom the screening with an IC50 value of 80 nM
equipotent Some degree of branching in the chain linker wasgenerally tolerated (39f-h) Configurational constraintsimposed on the chain linker as exemplified by 39i weretolerated but this was not the case for the cyclopropyl analogue39j
X
RO
NH
N
HN
38a-y 38z
Subsequent investigations focused on the substitutionpattern in the aryl ring for the benzyl (n=1) and phenethyl (n=2)series [164] Electron withdrawing and small neutral groupswere generally tolerable in both series (39k-r v-x) Howeverelectron-donating groups exemplified by OMe gave much lessactive compounds (39s-u) In the phenethyl seriessubstitutions at the ortho- and meta- positions were preferableto the corresponding para- substitutions (39kl vs 39m and39no vs 39p) Interestingly in the benzyl series meta- andpara- substitutions resulted in more active compounds than thecorresponding ortho analogues (39v vs 39wx)
Fig (19) Adamantyl amides generated by AstraZeneca
Investigation of the nature of the linker between thearomatic ring and the 2-position of the imidazole ring revealedthat a 3 or 4 carbon linker (39bc) caused a slight decrease in theactivity and no carbon linker (39d ) rendered the compoundinactive [164] One carbon linker with a methyl branching unit(39e) was tolerated It was reported that there was noenantiomeric preference of the methyl branch they were
The diarylimidazolines examined represent good candidatesfor orally active P2X7R inhibitors they are small and highlyamenable for SAR studies The 4- and 5-phenyl rings representattractive sites for further investigations
Table 5 Structures and P2X7R Antagonistic Properties of Selected Adamantyl Amides [141]
Entry R X pA2
38a 2-Cl CH2NHCO 81
38b 4-Cl CH2NHCO inactive
38c 24-Cl2 CH2NHCO 64
38d 2-Cl CHMeNHCO 64
38e 2-Cl CH2NMeCO 54
38f 2-Cl CH2NHCH2 54
38g 2-Cl NHCO inactive
38h 2-Cl (CH2)2NHCO 78
38i 2-Cl CH2CONH 63
38j 23-Cl2 CH2NHCO 88
38k 25-Cl2 CH2NHCO 83
38l 2-Cl-3-NH2 CH2NHCO 82
38m 2-Cl-5-OMe CH2NHCO 88
38n 2-Cl-5-OH CH2NHCO 80
38o 2-Cl-5-NH2 CH2NHCO 84
38p 26-Cl2 CH2NHCO 74
38q 35-Cl2 CH2NHCO 54
38r 24-Cl2 CH2NHCO 64
38s 2-Br CH2NHCO 80
38t 2-OMe CH2NHCO 71
38u 2-Me CH2NHCO 79
38v 2-Me CH2CONH 68
38w 2-Cl-5-OMe CH2CONH 72
38x 2-Me-3-OMe CH2CONH 83
38y 2-Me-5-OMe CH2CONH 80
1518 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
N
HN
XY
R
N
HN
N
HN
N
HN
( )n
39i39j
39a-ce-hk-w 39d
Fig (20) General structure of the diarylimidazolines generated by Aventis
Table 6 Structures and P2X7R Antagonistic Properties of Selected Diarylimidazolines [164]
Entry n X Y R IC50 ( M)a
39a 1 H H H 008
39b 2 H H H 054
39c 3 H H H 061
39e 0 (R)-Me H H 011
39f 1 (S)-Me H H 003
39g 1 Me Me H 001
39h 1 Ph H H 032
39k 1 H H 2-F 016
39l 1 H H 3-F 004
39m 1 H H 4-F 022
39n 1 H H 2-Cl 007
39o 1 H H 3-Cl 012
39p 1 H H 4-Cl gt1
39q 1 H H 2-Me 007
39r 1 H H 4-Me 219
39s 1 H H 2-OMe 083
39t 1 H H 3-OMe gt3
39u 1 H H 4-OMe gt3
39v 0 H H 2-F 058
39w 0 H H 3-F 008
39x 0 H H 4-F 008
aAntagonistic IC50 values derived from dose-response curves on the basis of inhibition of YO-PRO-1 dye uptake
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1519
R1N
N
NN
R2
R1
NN
NN
R2
N
N
NN
Cl
Cl
Cl
Cl
40b-t 40u-x40a
1
2
3
4
5
Fig (21) General structure of tetrazoles generated by Abbott Laboratories
310 1-Benzyl-5-Phenyltetrazoles potency The limited solubility of those compounds howeverposed a challenge to incorporate substitutions that couldincrease the solubility while maintaining activity A 3-pyridylgroup (40d ) was found to overcome this problem Moreover anextra 2-methyl substituent (40e) resulted in higher activitycompared to 40d A methylene extension (40f) caused a decreasein potency whereas other heterocycles such as 40g and 40hresulted in analogues with similar potency to 40d
1-Benzyl-5-phenyltetrazoles were discovered through ahigh-throughput screening by Abbott Laboratories for threedifferent P2X7R activities Ca2+ flux at the rat or human P2X7RYO-PRO-1 uptake and interleukin-1β release at human P2X7R[165-168] The initial hit from the screening was the tetrazole40a (Table 7) with a pIC50 value of 69 in inhibiting P2X7Rderived from the recombinant human cell line
Investigation of the nature of the substituent attached to the5-position of the tetrazole core showed that the substitutionpattern at this position was not well tolerated [165] Forexample unsubstituted mono-chlorosubstituted 25- and 34-
Initial SAR studies focused on the substituent attached tothe 1-position of the tetrazole core [165] Unsubstituted orortho-substituted benzylic group (40bc) exhibited good
Table 7 Structures and P2X7R Antagonistic Properties of Selected 1-benzyl-5-tetrazoles [165]
1520 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
disubstituted compounds (40i-m) pyridine and quinoline ring(40n-p ) analogues were not tolerated However moreconservative changes such as CF3 and F for Cl group (40q-t)resulted in an increase of potency
release and amplification of astrocytic Ca2+ signaling werereported to be due to the P2X7R and not Cx43 hemichannels[170]
Decavanadate a type of polymer of vanadium oxides hasbeen demonstrated to be a competitive rapid-acting antagonistat the P2X7R [172] It is capable of inhibiting the BzATP-stimulated ethidium bromide uptake in human mouse and ratP2X7R with pA2 values of 74 57 and 71 respectivelyDecavanadate has not been fully tested for its selectivity overother P2X receptors however it is suggested that this largemolecular species acts at the extracellular ATP binding site ofthe receptor consistent with the competitive mode of inhibition[172] A monoclonal antibody specific for the human P2X7Rhas also been shown to be a potent and selective antagonist ofthe receptor with an IC50 value of 5 nM in blocking the BzATP-evoked currents [173] Interestingly tenidap at 50 ndash 200 microMconcentration increases the sensitivity of mouse macrophagesto ATP-mediated cytotoxicity [174] 17-β-estradiol and notprogesterone or 17-α-estradiol has also been reported to blockthe agonist-induced currents at the human P2X7R with an IC50value of 3 microM in a non-genomic manner [175] Polymyxin B apotent antibiotic well-known for its lipopolysaccharide-neutralizing effects has been demonstrated to confer asynergistic effect when co-applied with ATP in a similar mannerto tenidap inducing Ca2+ influx ethidium bromide uptake andlactate dehydrogenase release via action at the P2X7R [176]
Tetrazoles with reversed connectivity (40u-x) were alsoevaluated where it was found that this connectivity reversal hadlittle effect on the potency [165] These tetrazoles were reportedto be slightly more active in the human P2X7R than the ratanalogue [165]
The tetrazole derivative 40d has been shown to be selectivefor other P2 receptors including P2X3 P2X4 and P2Y2 receptors[165] Pharmacological testing of this tetrazole suggested acompetitive mode of inhibition at the P2X7R and its efficacyhas also been evaluated in a model of neuropathic pain in rats[165] Overall these tetrazoles are found to be P2X7R-active andhighly adaptable to structural modifications These propertiesmake the tetrazoles an interesting class of compound for furtherinvestigating P2X7R activity
311 Other P2X7R-Active Ligands
Chelerythrine 41 (Fig 22) is a member of the benzophen-anthridine alkaloids known to inhibit protein kinase C withgood potency and selectivity (IC50 066 microM) as well as enzymessuch as alanine aminotransferase and Na+K+-ATPase [169] At10 microM concentrations chelerythrine non-competitivelyinhibited the BzATP-induced 86Rb+ efflux by 74 in human B-lymphocytes [169] Dye uptake studies could not be performeddue to the fluorescent nature of the compound It is yet to bedetermined whether chelerythrine inhibition of P2X7R involvesprotein kinase C [169]
More recently other distinct classes of P2X7-active ligandshave been reported and patented by the Abbott Laboratoriesacylhydrazides [177] cyanoamidines and cyanoguanidines[178 179]
4 CONCLUSIONS AND FUTURE DIRECTIONS
O
O
N
O
O
Cl
41
Although many structurally diverse class of compoundshave been reported as P2X7R antagonists with reasonablepotency the selectivity issue over other P2 receptors andproteins such as ecto-ATPases and ectonucleotidases stillprevails This is particularly an issue for oxidized ATP andsuramin SAR studies performed on PPADS and suramin mainlygave improved potency and selectivity for the P2X1 receptorIon channel blockers such as amilorides and stilbenederivatives only possess relatively low potency at the P2X7RFurther complications in studying these P2X7R antagonistsalso arise from the demonstrated species differences acrosshuman rat and mouse systems Brilliant Blue G is active for allthree species however at present there have been no SAR datareported Another fundamental factor that hampers the study ofP2X7R in detail is the relatively low potency and selectivity ofthe available agonist BzATP The discovery of a more potentselective P2X7R agonist would be ideal since in many studiesfull dose-response curves could not be generated due to the low
Fig (22) Chemical structure for chelerythrine an alkaloid withantagonistic properties at the human P2X7R
Gap junction channel blockers such as flufenamic acid 42 carbenoxolone 43 and mefloquine 44 (Fig 23) were all foundto be inhibitors of the BzATP-induced Ca2+ flux (IC50 values of655 175 and 25 nM respectively) and of YO-PRO-1 uptake in1321N1 astrocytoma cells stably expressing rat P2X7R [170]This finding poses a question to the general use of gap junctionchannel blockers as indicators of the participation of connexinhemichannels at the sites of transmitter release [171] The ATP
HN CF3
O OH
42 f lufenamic acid
HO
-O
O
O
H
OH
O
O-
N
HN
CF3
CF3
H O
H
43 carbenoxolone 44 mefloquine
Fig (23) Chemical structures for gap junction channel blockers with P2X7R antagonistic properties
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1521
potency of BzATP The nature of functional assays on theP2X7R is also found to be complex as ionic compositiontemperature and cell origins all affect the results demandingextra care be taken when antagonist potencies are directlycompared from different studies
[23] Li J Liu D Ke H Z Duncan R L Turner C H J Biol Chem2005 280 42952-42959
[24] Penolazzi L Bianchini E Lambertini E Baraldi P G RomagnoliR Piva R Gambari R J Biomed Sci 2005 12 1013-1020
[25] Gibbons S J Washburn K B Talamo B R J Auton Pharmacol2001 21 181-190
[26] Dehaye J P Moran A Marino A Arch Oral Biol 1999 44 S39-S43Four classes of small molecules with high affinity at the
P2X7R have been reported cyclic imides adamantyl amidesdiarylimidazolines and 1-benzyl-5-phenyltetrazoles Theseclasses of compounds are highly amenable to structuralmodifications indicating their good prospects as leadmolecules for further improving the potency and selectivity atP2X7R ultimately leading to orally active drugs To date norational drug design based on the knowledge of binding siteswithin this receptor has been undertaken This is a direct resultof the fact that no crystal structure or suitable homology modelhave been reported for this receptor The fact that the P2X7R canadopt either a channel or pore state causes even moredifficulties in studying receptor dynamics and at present theexact mechanism of channel-to-pore transition remains to beelucidated Undoubtedly the P2X7R is involved in severalprocesses relevant to chronic inflammation neurodegenerationand neuropathic pain and therefore discovery of more potentselective P2X7R antagonists would have a great impact in thepresent therapeutic window
[27] Li Q Luo X Zeng W Muallem S J Biol Chem 2003 27847554-47561
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ACKNOWLEDGEMENTS[38] Khakh B S Burnstock G Kennedy C King B F North A
Seguela P Voigt M Humphrey P P A Nat Rev Neurosci 20012 165-174
The authors would like to thank Dr Natasha Hungerford forproofreading the manuscript
[39] Roberts J A Vial C Digby H R Agboh K C Wen HAtterbury-Thomas A Evans R J Pflugers Arch - Eur J Physiol2006 452 486-500
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Received February 01 2007 Revised April 04 2007 Accepted April 06 2007
1506 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
Fig (1) A The molecular topology of the P2X1-6 and the P2X7 receptors The key distinguishing feature of the P2X7R to the other P2X receptors is itslong carboxy-terminus tail B The 3 different forms of the P2X7R upon activation by ATP closed channel open channel and pore formation Brief ATPactivation leads to opening of an ion channel permeable to small ions such as Na+ K+ and Ca2+ whereas prolonged ATP exposure results in the poreformation permeable to high molecular weight organic cations eg N-methyl-D-glucamine (NMDG) and YO-PRO-1 dye
uptake profile (Table 1) and iii) binding affinity to the agonistsATP and BzATP [42] Variations in the ionic composition of thebuffer [43] and pre-treatment of cells by repeated exposure toATP [44] also significantly affect the concentration-effectcurves (Table 1) In the YO-PRO-1 uptake assay BzATP is foundto be at least 100-fold and 30-fold more potent at rat and humanP2X7Rs respectively compared to the mouse P2X7R
any other brain cells [48] Activated microglia are known torelease various cytokines (such as interleukins [49-55] viacaspase-1 enzyme) tumour necrosis factor-α nitric oxide andreactive oxygen species Alternatively they may develop intophagocytes to remove debris cells and consequently arethought to be the main cytotoxic effector cells in the CNS Thereare literature reports for the expression of both P2X and P2Yreceptors in various microglia and for the ability of the P2X7Rto induce similar responses to those seen in activated microglia[15 45-47 56-60] The role of the P2X7R in mediating cytokinerelease is of particular interest in the context ofneurodegeneration It has recently been shown that
Although the exact physiological function of P2X7R is yetto be completely understood they are abundantly expressed inbrain microglia [45-47] Microglial cells which assume arestinginactive state under normal conditions will react tovirtually any pathogen and become activated often well before
N
NN
N
NH2
O
O HOH
OPO
OH
O
POPHO
O
O H
O
OH
1 ATP
N
NN
N
NH2
O
OHOH
OPO
O-
O
POP-O
O
O-
O
O-
2 ATP4-
Fig (2) Chemical structures for endogenous P2X7R ligand ATP (1) and its tetrabasic form (2)
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1507
Table 1 Potency of BzATP and ATP at Recombinant Mouse Human and Rat P2X7R (mP2X7R hP2X7R rP2X7R) and Mouse NTW8 Microglia(mNTW8)
Buffer composition (in millimolar)aNaCl buffer (NaCl 145 KCl 2 CaCl2 05 HEPES 10 D-glucose 10 pH 73) bNa-glutamate buffer (Na-glutamate 145 KCl 2 CaCl2 05 HEPES 10 D-glucose 10 pH73) cSucrose buffer (sucrose 280 HEPES 10 NMDG 5 KCl 56 D-glucose 10 CaCl2 1 pH 74)A change from NaCl to Na-glutamate buffer generally increases the potency of the agonists at the P2X7R Pre-treatment of cells by repeated ATP exposure will induce theP2X7R to adopt the pore state The cells in the pore state are generally more sensitive towards the agonists
inflammatory processes modulated by cytokines in particularthe interleukins and generation of tumour necrosis factors andnitric oxide have important roles in the many forms ofneurodegenerative disease such as multiple sclerosisParkinsonrsquos Alzheimerrsquos and Huntingtonrsquos disease [61] Thepresence of activated microglia in the brain has thus beenconsidered a reliable marker for neurodegenerative diseases TheP2X7R has also been shown to modulate β-amyloid-inducedcytokine expression (IL-1α and IL-1β) from humanmacrophages and microglia This further supports P2X7Rinvolvement in the neuropathology of Alzheimerrsquos disease inwhich the presence of β-amyloid is often surrounded byactivated microglia and astrocytes [62] Taken together theP2X7R has a pivotal role in neuro-inflammatory and-degenerative processes In addition P2X7R can stimulaterelease of traditional neurotransmitters in the brain such asglutamate and GABA The P2X7R regulation of glutamate andGABA release in rat gerbil and mouse hippocampus [63-65] andmurine astrocytes [66 67] has been reported although it is stillnot definitively confirmed [68] More recently two distinctglutamate release pathways in rat hippocampal astrocytes bypurinergic receptors have also been reported [69]
arthritis model [72] Inhibition of the P2X7R has also beenshown to improve recovery after spinal cord injury in ratmodels [73] However functional P2X7-like proteins have beenfound in one P2X7-knockout model [74 75] highlighting thelack of understanding of the P2X7-knockout animal modelsDominant-negative P2X7R subunits have been proposed as anovel genetic tool to understand the role of P2X7R in nativesystems [76] More recently it has been demonstrated thatexpression of cyclooxygenase-2 cannabinoid receptor CB2and P2X7R are all elevated in activated microglia cells ofmultiple sclerosis and amyotrophic lateral sclerosis spinal cord[77]
Further studies into understanding the physiologicalfunctions of P2X7R in vivo and the development of detailedpharmacophore models will lead to better understanding ofcellular cascades underlying the basis of neuro-inflammatoryand -degenerative processes A number of P2X7R antagonistshave already been developed towards this goal however a moreextensive range of ligands with drug-like properties includingimproved potency and selectivity is still required Althoughthere have been a number of reviews on the pharmacology of theP2X7R [78-80] there appears to be relatively limited accessibleresources on a comprehensive listing of molecular probesacting at the receptor [81-83] This review will present anoverview of the currently known agonists and antagonistsacting at the P2X7R and comment on their usefulness asmolecular probes for microglia activation
The exact role of P2X7R in vivo remains to be elucidated andliterature studies to date have compared normal and P2X7-knockout animal models In terms of pharmacologicalintervention the most promising roles of the P2X7R in vivowould most likely be in the areas of pain regulation [70]chronic inflammation [57 71] and neurodegeneration [61]Chronic inflammatory and neuropathic hypersensitivity arecompletely abolished in P2X7-knockout mice although normalnociceptive processing still remains [70] suggesting thatpotent selective P2X7 antagonists may serve as all-purposeanalgesics P2X7-knockout mice have been reported to showreduced severity of arthritis in an anti-collagen antibody
2 ENDOGENOUS LIGANDS FOR P2X7R
ATP 1 is the natural endogenous agonist of the P2X7R withEC50 values varying from 85 to 779 microM depending on thespecies examined (rat mouse or human) as determined from
1508 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
N
NN
N
NH2
O
OH(R)(H)RO
OPO
OH
O
POPHO
O
OH
O
O H
O
O
mixture of 2 and 3 e sters
3 BzATP
R =
Fig (3) Chemical structure for BzATP (3) the most potent P2X7R agonist to date
electrophysiological assays (Table 1) The low potency of ATPin activating the P2X7R is one of the main pharmacologicalfeatures of this unique receptor that distinguish it from otherpurinergic receptors Normal cytoplasmic concentrations of ATPin cells range from 5 to 10 mM which is released to theextracellular environment upon cell injury or insult therebyactivating the P2X7R before being quickly hydrolyzed by ecto-ATPases and ectonucleotidases Reduction of the extracellularMg2+andor Ca2+ ion concentration which are thought to beallosteric inhibitors [84] results in the potentiation of ATPeffects at the P2X7R [8] It has also been suggested that thetetrabasic form of ATP 2 is the active species acting at thereceptorrsquos ATP-binding site where the presence of Mg2+ andCa2+ ions chelate ATP4- rendering it inactive [85]
pharmacophore known for P2X7R activity The effects of thesesynthetic ligands on the P2X7R are generally tested in eitherelectrophysiological or dye uptake assays exploiting thereceptorrsquos non-desensitizing property and the ability to form alarge non-selective pore
31 ATP Derivatives
BzATP 3 (Fig 3) is the most potent P2X7R agonist to datewith 10- to 100-fold higher potency compared to ATP inactivating the receptor in a number of different systems [3] Thepotency of BzATP is also known to be species-dependent EC50value of BzATP at the rat P2X7R is 2ndash7 microM in theelectrophysiological assay which is approximately 10- to 30-fold higher than at the human P2X7R [40 88] BzATP also has adifferent EC50 value (007 ndash 025 microM) for activating the ratP2X7R in the YO-PRO-1 uptake assay performed in sucrosebuffer (Table 1) The increased sensitivity of the P2X7R toBzATP compared to the endogenous ligand ATP is one of themain criteria used to define the pharmacology of the P2X7Rsubtype Unfortunately BzATP is not highly specific for theP2X7R subtype other P2X receptors are also activated byBzATP [89] but with either equal or lower potency than ATP[90] Other ATP derivatives such as 2-methylthio-ATP 4 ATP-gamma-S 5 and ADP 6 are all weak agonists of the P2X7R (Fig4) [34 90] The order of potency for P2X7R agonists is BzATP3 gtgt ATP 1 gt 2-Methylthio-ATP 4 gt ATP-γ-S 5 gtgt ADP 6 [34] 2-Methylthio-ATP 4 has an EC50 value of 505 microM against theP2X7R derived from mouse NTW8 microglial cells based on theelectrophysiological studies [60]
Some diadenosine polyphosphates such as P1 P4-diadenosine tetraphosphate were reported to be capable ofactivating the P2X7R-mediated cellular permeabilization of ratmast cells [86] possibly due to their greater resistance tometabolic breakdown and similarity to ATP4- [3] Theendogenous peptide LL-37 an antimicrobial agent causingbacteria cell wall permeabilization derived from the humancathelicidin protein hCAP18 has been demonstrated to induceinterleukin-1β processing and release via the activation of theP2X7R [87]
3 SYNTHETIC LIGANDS FOR P2X7R
Several classes of P2X7R-active synthetic compounds havebeen reported in the literature Unfortunately despite thediscovery of these ligands there is still no general
N
NN
N
NH2
O
OHOH
OPO
O H
O
POPHO
O
OH
O
OH
SMe
4 2-Me thylthio-ATP
N
NN
N
NH2
O
OHOH
OPO
OH
O
POPHS
O
OH
O
OH
5 ATP-γ-S
N
NN
N
N H2
O
OHOH
OPO
OH
O
PHO
O
OH
6 ADP (adenosine 5-diphosphate)
N
NN
N
NH2
O
OO
OPO
O H
O
POPHO
O
OH
O
OH
7 oxidize d ATP
Fig (4) Chemical structures for ATP derivatives that are weak P2X7R agonists
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1509
The 2rsquo 3rsquo-dialdehyde ATP (or oxidized ATP) 7 (Fig 4) is thefirst irreversible inhibitor of the P2X7R expressed in the mousemacrophage cell line J774 [91] Subsequent studies alsoshowed that application of 100 microM oxidized ATP irreversiblyantagonized the rat P2X7R provided the cells were pre-incubated with oxidized ATP for 1-2h indicating that itcovalently modified the receptor [34] Co-application with anagonist did not result in any inhibition There is evidence thatoxidized ATP possesses low affinity for the P2Y receptors [91]however oxidized ATP at 100 microM concentration has beenshown to be very toxic to rat cerebellar granule neurons [92]and is likely to react with other enzymes such as ecto-ATPases[91 93] Interestingly oxidized ATP at 168 microM inhibitedP2X7R-mediated inflammatory pain in arthritic rats [94 95]Due to its non-selective inhibition extra caution is required inthe use of oxidized ATP to define involvement of P2X7R in thepurinergic system [42]
in a non-selective manner [3] It is a relatively potent antagonistat the P2X1-3 P2X5 receptors (IC50 01ndash5 microM) and P2Y1receptor but only weakly active or completely inactive at the ratP2X4 P2X6 P2X7 and some P2Y receptor subtypes [96]Interestingly PPADS is reported to be reasonably active at thehuman P2X4R with IC50 value of 28 microM indicating somespecies dependence [97] PPADS is highly specific for the P2receptors up to 100 microM concentration of PPADS does notinterfere with adenosine receptors muscarinic M1 M2 or M3α1- or α2-adrenoceptors histamine H1 AMPA or NMDA and 5-HT receptors [96]
PPADS has been widely studied in the P2 receptor systemsderived from various cell lines and structure-activityrelationship (SAR) studies have also been reported [98-101]Structural modifications of PPADS to date have involved theinvestigation of substitutions at the 4-aldehyde position 5-phosphate and the azophenyl ring Some of the key PPADSderivatives are represented in Fig (6) It was initially thoughtthat both the aldehyde and phosphate moieties are important forP2 antagonism It was also proposed that the slowlyequilibrating pseudoirreversible mode of inhibition of PPADSseen at the P2X1 receptor was due to Schiff base formationbetween the aldehyde and a specific lysine residue of thereceptor [96] The cyclic phosphates MRS2219 and MRS2220were found to be relatively potent antagonists at the rat P2X1receptor (IC50 6 and 10 microM respectively) with good selectivityover P2X2-4 P2Y1 P2Y2 P2Y4 P2Y6 and adenosine receptors[99] Another PPADS derivative that appears to be a promisingantagonist at the P2X1 receptor is PPNDS which exhibited anIC50 of 15 nM and no interactions detected withectonucleotidases P2Y1 receptor α1-adrenoceptors adenosinehistamine H1 and muscarinic M3 receptors [102] Later studiesreported that replacement of the sulfonyl groups on theazophenyl ring with carboxylates or phosphates furtherincreased the potency and selectivity at the P2X1 and P2X3receptors [101] Most recently PPADS analogues MRS2211 andMRS2603 have been reported to be active at the P2Y13 receptorbeing 45- and 74-fold more active respectively than PPADS[103] Only a limited number of PPADS analogues were testedagainst the recombinant human P2X7R in these studies and forthose it was found that none of them was more potent than iso-
Overall these ATP derivatives have high molecular weightsand could potentially bind to other purinergic receptors due totheir resemblance to the endogenous ligand ATP Thereforethey are unlikely to be suitable for developing an orally activedrug specifically targeting the P2X7R
N
HO
CHO
O P O-
O-
O
9 P5P
N
O P
O
O-
O-HO
CHO
N
N
SO3--O3S
43
21
6
5
8 PPADS
Fig (5) Chemical structures for PPADS and its parent molecule P5P
Fig (7) Chemical structure for DIDS and its derivatives
PPADS which differs from PPADS by the position of thesulfonates [100]
[109] receptor DIDS dihydro-DIDS 17 and SITS 19 (Fig 7) allblocked the ATP-stimulated Ca2+ uptake in rat parotid acinarcells via activation of the P2X7R (IC50 35 ndash 125 microM) [109]DNDS 18 in which the isothiocyanate moieties are replacedwith nitro groups was found to be inactive indicating theimportance of the isothiocyanate for interaction with theP2X7R On the other hand a variety of smaller aromaticisothiocyanate compounds derived from DIDS have been testedagainst the P2X1 receptor and were found to be active with IC50values between 11 and 54 microM [108] Overall DIDS and itsderivatives are inhibitors of both the P2X1 and P2X7R withlower potency for the latter
All the SAR data generated to date on PPADS and itsderivatives demonstrate that structural modifications on thekey functional groups (aldehyde phosphate and azophenyl)increases the potency and selectivity mainly at the P2X1receptor Consequently PPADS itself is used for testing at theP2X7R where it was found to be a potent antagonist only at therat and human receptors (IC50 15 and 90 nM respectively) butnot the mouse receptor (100-fold lower potency) in the YO-PRO-1 uptake assay provided cells are pre-incubated withPPADS for 15-30 min [41] When there was no pre-incubationthe IC50 of PPADS at the rat human and mouse P2X7R decreasedto approximately 50-70 microM in the electrophysiologicalrecordings [34 40] but pre-incubation with PPADS drasticallyimproved the IC50 value to 1 microM at the human P2X7R [88] Theinhibitory action of PPADS at the P2X7R is only partiallyreversible [88] suggesting a pseudo-irreversible mode ofantagonism [104] It has been suggested that PPADS P5P andoxidized ATP may all have a common site of action which islikely to be the ATP binding site [104] Overall it can beconcluded that PPADS per se is a species-dependent P2X7Rantagonist being most potent at the rat and human P2X7R butnot the mouse receptor Derivatives of PPADS reported to dateonly exhibit improved potency and selectivity at the P2X1receptor and not the P2X7R
5-(NN-Hexamethylene)amiloride (or HMA) 20 (Fig 8)belongs to the amilorides which are better known as potassium-sparing diuretics by blocking the Na+ channel as well as beinginhibitors of Na+-H+ counter-transport pathway [112] L-typecalcium channels [113] adenosine A1 receptors [114] andpossibly Na+-HCO3
- co-transport [115] HMA possesses affinityfor the P2X7R but its effects are highly species-dependent At 40microM concentration HMA antagonized P2X7R found in humanlymphocytes as determined from large cation uptake studies[116 117] However up to 100 microM concentration it wasineffective at the rat P2X7R [34] More interestingly theelectrophysiological studies revealed that co-application ofHMA with ATP produced an immediate and maximalaugmentation of inward currents induced by mouse P2X7R [60]This effect was not apparent when HMA was applied alone orwhen the buffer solution contained physiologicalconcentrations of Mg2+ and Ca2+ ions HMA has been reportedto be an irreversible inhibitor at the hP2X7R [88] Otheranalogues of HMA such as MIBA 21 and EIPA 22 also possesssimilar properties to HMA [116] Due to its relatively lowpotency and high species dependence at the P2X7R HMA andprobably other amilorides are unlikely to be good candidatesfor further exploration of the pharmacology of the P2X7R
33 Ion Channel Blockers
44rsquo-Diisothiocyanatostilbene-22rsquo-disulfonic acid (DIDS)16 is an anion channel blocker widely used to study various iontransport mechanisms [105-107] but has also beendemonstrated to be capable of inhibiting both the P2X1 receptor[108] and P2X7R-induced pore formation and other processes inseveral cell lines with relatively low potency [3 109-111] Forexample pre-incubation of BAC12F5 murine macrophage with150 ndash 200 microM DIDS inhibited the activation of phospholipaseD mediated by the P2X7R [110] Pre-incubation of rat primaryastrocytes with 200 microM of DIDS has also been shown to inhibitP2X7R-mediated responses such as the activation ofextracellular signal receptor kinases (ERK) [111] SARinvestigations have been performed on DIDS in order toimprove its potency and selectivity at the P2X1 [108] or P2X7
34 Polysulfonated Dyes
Suramin 23 is an example of a polysulfonated dye thatexhibits non-selective antagonism at both the P2X and P2Yreceptors with relatively low potency [3] Suramin blocks theP2X1-3 and P2X5 receptors (IC50 1-15 microM) but only shows weakor no inhibition at the P2X4 P2X6 and P2X7 receptors as well
N
NCl
N NH 2
O
NH
NH
NH2
20 5-(NN-Hexa methylene )- amiloride (HMA)
N
NCl
NH2
O
NH
NH
NH2
N
21 5-(N-Methyl-N- isobutyl)- amiloride (MIBA)
N
NCl
NH2
O
NH
NH
NH 2
N
22 5-(N-ethyl-N-isopropyl)- amiloride (EIPA)
Fig (8) Chemical structures for HMA and other amilorides
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1511
as some P2Y receptor subtypes [96] In addition suramin hasbeen shown to interact with many different proteins at theseconcentrations including glutamate nicotinic GABA and 5-HTreceptors as well as various proteases and ectonucleotidases [396] Nevertheless SAR studies on suramin have been performedin order to identify the important structural features responsiblefor antagonism at the P2 receptors [96 118-124] Selected keysuramin derivatives are represented in Fig (10 )
native and recombinant P2X1 receptors respectively and hasbeen shown to be much less potent at other P2X and P2Ysubtypes including adrenoceptors histamine H1 receptors andthe muscarinic M3 receptor [120] At the human P2X1 and P2X7receptors the IC50 values for NF449 were 005 nM and 40 microMrespectively [128] which indicated its usefulness todistinguish between the two receptors Subsequent SAR studiesconfirmed that NF449 was the most potent and selective knownP2X1 receptor antagonist [121 129] NF157 an analogue ofsuramin with fluorine replacing the methyl group has also beenreported to be highly potent at the P2Y11 receptor with a Ki of45 nM [124] More recently NF110 has also been shown to be apotent P2X3 receptor antagonist indicating that both thenumber and location of the sulfonate groups may be importantfor determining P2X subtype potency and selectivity [123]
O
HN
HN
NH
NH
O O
O ONH HNSO3-
SO 3-
SO3-
-O3S
SO 3-
-O3S
23 suramin
NF279 thus far represents the most active suramin relatedantagonist at the P2X7R and displays a reasonable potency of28 microM All the SAR data obtained to date on suramindemonstrate the importance of the molecular size number andposition of the sulfonate groups for P2X subtype potency andselectivity Unfortunately except for NF279 and NF449 othersuramin analogues provide very little information which can beused for the selective structure-based drug design of P2X7Rmolecules Suramin is also unlikely to be orally active due toits high molecular weight and interaction with many otherproteins Suramin is a very weak or almost inactive antagonistat the human P2X7R with an IC50 value of 92 microM [40] It istotally inactive at both the rat and mouse P2X7R with IC50values greater than 100 microM [34 60] Due to its interference withthe fluorescence of YO-PRO-1 suramin could not be used in theYO-PRO-1 dye uptake assay [104]
Fig (9) Chemical structure for suramin a weak antagonist at the P2X7R
Early SAR analysis showed that the shortened forms ofsuramin in the form of sulfonylbenzamides decreased thepotency at P2 receptors [118] NF023 (24 ) was later discoveredto be P2X-selective compared to P2Y receptors andectonucleotidases [118] Subsequent studies have examined theeffects of NF023 in various cell types and it was reported thatNF023 is a highly selective antagonist for either rat or humanP2X1 receptors heterologously expressed in Xenopus oocyteswith an IC50 of 02 microM [125] Another suramin derivativeNF279 (25 ) also showed potent selective reversible inhibitionat the rat P2X1 receptor in Xenopus oocytes with an IC50 of 19nM [126] In studying the effects of NF279 on human P2X1 andP2X7 receptors it was reported that NF279 is almost 100-foldmore potent at the human P2X1 than the P2X7 receptor withIC50 values of 005 and 28 microM respectively [127] Other keysuramin derivatives resulting from extensive SAR studies areNF449 (27 ) NF110 (28 ) and NF157 (26 ) [120 121 123 124128 129] NF449 has an IC50 value of 70 nM and 03 nM at the
Brilliant Blue G 29 (Fig 11) is another polysulfonated dyewhich is a potent non-competitive inhibitor of the rat andhuman P2X7R with IC50 values of 10 and 200 nM respectivelyin electrophysiological studies [130] Brilliant Blue G has alsobeen shown to be highly selective for P2X7R over P2X1-5receptors [130] It has been shown that at the rat P2X7R about100 to 300 nM concentration was required to inhibit the YO-PRO-1 uptake which was 10- to 30-fold higher than thatrequired for blocking P2X7R-induced currents demonstratingits differential effect at the channel and pore forms of the P2X7R
O
HN
HN
NH
NH
O O
R R
O ONH HNSO3-
SO3-
SO3-
-O3S
SO3-
-O3S
O
HN
HN
NH
OO
NH
O ONH
NH
R R
RR
n n
Suramin n=1 R=CH3
24 NF023 n=0 R=H
25 NF279 n=1 R=H
26 NF157 n=1 R=F
27 NF449 R=24-disulfonyl28 NF110 R=4-sulfonyl
Fig (10) Suramin and its key derivatives investigated in the SAR studies
1512 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
[130] Brilliant Blue G has also been tested on the mouseP2X7R where similar antagonistic potency was found comparedto rat and human receptors [42] In this study it was alsoreported that the selectivity of Brilliant Blue G for rat overhuman P2X7R was confirmed at 22degC but lost at 37degC Thedrawback for this compound is that it could also be bound toproteins and thus would be expected to have lower potency inthe dye uptake assay than electrophysiology measurements dueto the depletion by cell monolayer effect [42] Nevertheless itrepresents a promising antagonist at rat human and mouseanalogues of the P2X7R No studies have been performed onBrilliant Blue G to further improve its potency and selectivityat the P2X7R Other polysulfonated dyes such as Evans BlueTrypan Blue reactive blue 2 reactive red 2 and their derivativeshave also been tested at the purinergic systems [131-133] butnone of them exhibited activity at the P2X7R
opening of the ion channel calmidazolium produces no effecton the YO-PRO-1 uptake The large non-selective porehowever is still formed even when the ionic currents areinhibited indicating that pore formation does not necessarilyrequire opening of the ion channels Calmidazolium wassuggested to be an allosteric modulator of the P2X7R at theagonist binding site inducing conformational change at thereceptor level that results in decreased agonist binding affinity[84] It displays less potency at inhibiting the agonist-evokedcurrents at both the mouse and human P2X7R with IC50 valuesof 1 microM and 100 nM respectively [41 88] At present no SARstudies have been reported on calmidazolium
N
N+
ClCl
O
ClCl
Cl
Cl
30 calmidazolium
NH
N
O
SO3-
N+
SO3-
Cl
29 Coomassie Brilliant Blue G
Fig (12) Chemical structure for calmidazolium a P2X7R antagonistcapable of distinguishing the channel and pore forms of the receptor
36 KN-62 and its Analogues
KN-62 31 (Fig 13) is a derivative of isoquinolinesul-fonamide known as a selective and potent inhibitor of themultifunctional calciumcalmodulin-dependent protein kinases(CaMK) with an IC50 of 09 microM at CaMK II [134] Gargett andWiley in 1997 first demonstrated that both KN-62 31 and itsanalogue KN-04 32 were potent inhibitors of P2X7R at humanlymphocytes with IC50 values of 13 and 17-37 nM respectivelyin both the ion flux and ethidium bromide uptake assays [134]The mode of inhibition is allosteric modulation similar to thatof calmidazolium
Fig (11) Chemical structure for Brilliant Blue G a potent antagonist at therat P2X7R
35 Calmidazolium
Extracellular calmidazolium 30 (Fig 12) is a potentinhibitor of BzATP-induced currents via binding to rat P2X7Rwith an IC50 of 13 nM [84] The unique feature ofcalmidazolium is its ability to differentiate between the channeland pore form of the P2X7R while it potently inhibits the
KN-62 and KN-04 are species-selective for P2X7Rantagonism They potently inhibited the ATP-induced currentsas well as the ethidium bromide uptake in HEK293 cells
Fig (13) Chemical structures for isoquinolines KN-62 and KN-04 potent P2X7R antagonists at the hP2X7R with much less effect on the rat and humananalogues
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1513
33e most active in the series but still 30-fold less potent than KN-62
Fig (14) Conformationally restricted analogues of KN-62 [136]
expressing human P2X7R at the concentration of 30 ndash 100 nMdepending on whether ATP or BzATP was used and the cellularbackground of the human P2X7R [135] However even up to 3microM concentration they had no effect at the rat P2X7R [135] andKN-62 was found to be at least 10-fold more potent at thehuman P2X7R than the recombinant mouse analogue [60] Theseisoquinolinesulfonamide derivatives may interact with theamino-terminal domain of the P2X7R since the introduction ofthe first 335 amino acids of the human P2X7R delivers KN-62sensitivity to the rat P2X7R and the counter-part chimera isfound to be insensitive to KN-62 [135] The IC50 at the humanP2X7R is 10-fold lower at 22degC than at 37degC in the ethidiumbromide uptake study [42]
tyrosine) 2) R2 (the tyrosine side chain) and 3) R3 (thesubstituent attached to the N-piperazine) (Fig 15) [137-139]
The effect of substitutions at the R1 R2 and R3 groups (Fig15 ) on the P2X7R antagonistic properties was compared on thebasis of inhibition of K+ release [137 138] At the R1 positionlarge hydrophobic moieties linked to the amino positionthrough sulfonamide (34a-b) or carbamate (34c) groups werepreferable for P2X7R inhibition In the series of derivativeswhere R2 = quinolinesulfonyl and R3 = t-butoxycarbonyl (Boc)introduction of carbobenzyloxy (Cbz) and quinolinesulfonylgroups at the R1 position were found to be the most active[137] In another series of derivatives where R1 = Cbz and R3 =Boc introduction of arylsulfonyl (34d-g) and benzoyl (34h )moieties at the R2 position were preferred [137] A free aminogroup at the R3 position (34i-j) andor opening of thepiperazine ring to an ethylene diamine greatly reduced theP2X7R activity Amongst the various acyl groups tested at theR3 position Boc and benzoyl groups (34k-m) were found to bepreferable but not sulfonyl (34n-q ) The presence of a methylgroup at the amino terminus of the tyrosine group is thereforenot an absolute pre-requisite for P2X7 antagonism [137]
R3 N N
O
N
O
R4
R1
R2
In another study undertaken by Chen et al to furtherexplore the SAR of 34m [138] it was found that nitro-substitution at the meta- or para- position of the Cbz group atR1 (34r-t) was well tolerated However introduction of anacetamido- or isothiocyanato- group at the Cbz group abolishedP2X7R antagonism (34uv) The original isoquinoline-5-sulfonyl moiety at the R2 position could be substituted withmeta- or para-substituted phenylsulfonyl groups such as tosyl(34r-t) 3-nitrophenylsulfonyl (34w) or 4-nitrophenylsulfonyl(34x) without adverse effects on the P2X7R antagonism [138]Substitution of the benzoyl group at the R3 position by a Boc(34y) or 4-aminobenzoyl group (34z) retained activity
Fig (15) General structure for the KN-62 derivatives (34a-z)
Extensive SAR studies have been performed on KN-62 inorder to enhance its potency and selectivity at the P2X7R Thefirst attempt to improve the antagonistic activity of KN-62 wasachieved by tethering the N-methyl of the tyrosine backbone tothe ortho-position of the proximal phenyl ring which resultedin a series of conformationally restricted KN-62 analogues withthe formula shown in Fig (14 ) [136] The constrained form ofKN-62 (31 vs 33a) was devoid of P2X7R antagonisticproperties Replacement of the isoquinoline-5-sulfonyl moiety(33a) with quinoline-5-sulfonyl (33b ) quinoline-8-sulfonyl(33c) or naphthalene (33d ) resulted in the loss of ability toinhibit the P2X7R-mediated Ca2+ influx [136] The most activecompound arising from this series was compound (33e) with anIC50 value of 316 nM which is 30-fold weaker than the originalKN-62 Taken together these results indicate that an extendedrather than folded conformation of KN-62 conferred by theincorporation of a methylene unit between the N-methyl of thetyrosine backbone to the ortho-position of the proximal phenylring is preferred at the P2X7R [136]
A study by Baraldi et al reported the systematic SAR profileof KN-62 specifically looking at modifications of thephenylpiperazine moiety (R2 position Fig 16 ) [139] In theinvestigation of the optimum chain linker between thepiperazine and phenyl groups it was found that one methylenespacer (35a) slightly improved the activity whereas a twomethylene spacer was not favourable (35b ) [139] The presenceof a basic nitrogen in the piperazine moiety is proven crucial forP2X7R antagonism as substitution of the piperazine ring withpiperidine (35a vs 35c) resulted in a 3-fold reduction of activity[139] Substitution of the phenyl ring with other heterocyclessuch as pyridine (35d ) or pyrimidine (35e) also decreased theP2X7R antagonistic property The para- position prefers a
Subsequent investigations on KN-62 involved extensivemodifications in three positions 1) R1 (the amino-terminus of
1514 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
small electron-withdrawing group eg fluorine (35f) and theantagonistic property decreases as the size of the electron-withdrawing group increases (35g 35h) A methyl- or nitro-group at the para- position (35i 35j) was beneficial foractivity but not evidenced with the methoxy (35k ) group As inthe case for para- substitution introduction of F Cl or CH3 butnot a methoxy (35l-o) at the ortho- position was well toleratedMoreover the meta- position could also tolerate a Cl or CF3group (35p 35q) The disubstituted compound 35r showedreduced antagonistic properties (IC50 = 1122 nM) possibly dueto the steric bulkiness of om-xylyl residue while the dichloroderivative 35s (IC50 = 34 nM) retained activity A slightdecrease in potency was observed when the methyl group on thenitrogen on the α-position of the tyrosine was removed (35n vs
35t) further confirming that this is not substantial for P2X7Rantagonism The most potent compound arising from this studywas 35f with an IC50 value of 13 nM at inhibiting calciuminflux in human monocytes compared to KN-62 which has anIC50 of 51 nM [139] It is therefore confirmed that thephenylpiperazine group of KN-62 is crucial for interaction withthe active site of the receptor and can be potentially improvedwith small halogen substitutions particularly in the paraposition
An ortho-methyl substituent on the N-phenyl ring of KN-62increased the potency by 3-fold (36 IC50 15 nM) [140]Radiolabelling of compound 36 with tritium and preliminarybinding studies to the P2X7R have also been achieved [140]
Table 2 Structures and P2X7R Antagonistic Properties of Selected KN-62 Derivatives [137138]
34a isoquinoline-5-SO2mdash isoquinoline-5-SO2mdash Bocmdash H 77 plusmn 24 ~40
34b quinoline-8-SO2mdash quinoline-8-SO2mdash Bocmdash H 61 plusmn 30 ND
34c Cbzmdash quinoline-8-SO2mdash Bocmdash H 53 plusmn 23 ~200
34d Cbzmdash PhSO2mdash Bocmdash H 59 plusmn 14 ND
34e Cbzmdash p-tolyl-SO2mdash Bocmdash H 71 plusmn 30 ~300
34f Cbzmdash p-OMe-PhSO2mdash Bocmdash H 62 plusmn 15 ND
34g Cbzmdash isoquinoline-5-SO2mdash Bocmdash H 43 ND
34h Cbzmdash benzoylmdash Bocmdash H 47 plusmn 21 ND
34i isoquinoline-5-SO2mdash isoquinoline-5-SO2mdash H H 14 plusmn 12 ND
34j quinoline-8-SO2mdash quinoline-8-SO2mdash H H 5 plusmn 9 ND
34k Cbzmdash PhSO2mdash Cbzmdash H 48 plusmn 29 ND
34l Cbzmdash PhSO2mdash benzoylmdash H 78 plusmn 22 ND
34m Cbzmdash isoquinoline-5-SO2mdash benzoylmdash H 85 plusmn 10 ~200
34n Cbzmdash PhSO2mdash p-tolyl-SO2mdash H 0 ND
34o Cbzmdash PhSO2mdash CH3SO2mdash H 0 ND
34p Cbzmdash PhSO2mdash PhSO2mdash H 0 ND
34q Cbzmdash PhSO2mdash p-OMe-PhSO2mdash H 0 ND
34r Cbzmdash p-tolyl-SO2mdash benzoylmdash H 71 plusmn 21 ND
34s p-NO2-Cbzmdash p-tolyl-SO2mdash benzoylmdash H 69 plusmn 25 ND
34t m-NO2-Cbzmdash p-tolyl-SO2mdash benzoylmdash H 72 plusmn 6 ND
34u m-acetamido-Cbzmdash p-tolyl-SO2mdash benzoylmdash H 9 plusmn 13 ND
34v m-isothiocyanato-Cbzmdash p-tolyl-SO2mdash benzoylmdash H minus4 plusmn 5 ND
34w Cbzmdash m-NO2-PhSO2mdash benzoylmdash H 75 plusmn 10 ND
34x Cbzmdash p-NO2-PhSO2mdash benzoylmdash H 78 plusmn 18 ~100
34y m-NO2-Cbzmdash p-tolyl-SO2mdash Bocmdash H 94 plusmn 28 ~100
34z Cbzmdash p-tolyl-SO2mdash p-NH2-benzoylmdash H 93 plusmn 3 gt 300
aAntagonist function was measured by the percent inhibition of the K+ release triggered by 3mM ATP in paired wells in the absence of antagonist bAntagonistic IC50values derived from dose-response curves obtained on the basis of inhibition of K+ release
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1515
R2 X N
O
N
O
S
R1
S
O
O
N
O
O
Nthe inhibition of BzATP-induced ethidium bromide uptakemediated by the P2X7R expressed endogenously on the humanpre-monocytic cell line THP-1 [142] Compound 37a (Table 4)was identified as the lead structure and modifications on theimide and 4-pyridyl moieties were investigated The cyclicimide moiety is proven essential for the P2X7R activity sincesubstitutions with simple amines acyclic amides or acycliccarbamates almost completely abolished the activity [142] Thering can be five- or six-membered in the forms of imides (37a-bh) carbamates (37c-d) thiazolidine-24-diones (37eg) andhydantoins (37f) Replacement of the 4-pyridyl moiety with 4-substituted benzamides (37g-h) retained the P2X7R activityThe presence of a 3rsquo-nitro substituent on the biphenyl subunitwas reported to convey optimum P2X7R activity The mode ofinhibition of these compounds seemed to be competitivealthough there were observable changes in the maximalresponse as the compound concentration was increased [142]
Fig (16) General structure for the KN-62 derivatives (35a-35s)
Unfortunately while these KN-62 analogues are potentiallyuseful tools to investigate the basic properties of the P2X7Rthey do not represent good starting points for the discovery oforally-active drugs due to 1) high molecular weight (gt700 Da)2) high lipophilicity (clogP gt 6) and 3) presence of ametabolically labile yet crucial sulfonate group [141]
These compounds represent a new class of ligands thatpossess activity to the P2X7R They appear good candidates asorally active drugs due to their relatively low molecular weight(~400 Da) and would be highly amenable for further SARstudies However despite the succinimides and thiazolidine-24-diones representing potent analogues they were found to bemetabolically and chemically fragile On the other handalthough the carbamates were less potent they displayedimproved stability Future SAR studies could usefully involveinvestigation of the biphenyl ring and the chain linker
37 Cyclic Imides and Analogues
Cyclic imides 37 (Fig 18) were discovered through a high-throughput screening of AstraZenecarsquos compound library for
Table 3 Structures and P2X7R Antagonistic Properties of Selected KN-62 Derivatives [139]
Entry R1 R2 X IC50 (nM)a
KN-62 mdashCH3 mdashPh N 511 plusmn 11
35a mdashCH3 mdashCH2Ph N 211 plusmn 08
35b mdashCH3 mdashCH2CH2Ph N 600 plusmn 27
35c mdashCH3 mdashCH2Ph CH 653 plusmn 08
35d mdashCH3 2-pyridinyl N 170 plusmn 39
35e mdashCH3 2-pyrimidinyl N 798 plusmn 07
35f mdashCH3 p-F-Phmdash N 13 plusmn 01
35g mdashCH3 p-Cl-Phmdash N 105 plusmn 11
35h mdashCH3 p-I-Phmdash N 176 plusmn 38
35i mdashCH3 p-Me-Phmdash N 135 plusmn 02
35j mdashCH3 p-NO2-Phmdash N 58 plusmn 06
35k mdashCH3 p-OMe-Phmdash N 132 plusmn 43
35l mdashCH3 o-F-Phmdash N 185 plusmn 04
35m mdashCH3 o-Cl-Phmdash N 158 plusmn 09
35n mdashCH3 o-Me-Phmdash N 151 plusmn 14
35o mdashCH3 o-OMe-Phmdash N 841 plusmn 5
35p mdashCH3 m-Cl-Phmdash N 141 plusmn 03
35q mdashCH3 m-CF3-Phmdash N 311 plusmn 12
35r mdashCH3 om-diMe-Phmdash N 1122 plusmn 25
35s mdashCH3 mp-diCl-Phmdash N 339 plusmn 08
35t H o-Me-Phmdash N 288 plusmn 03
aAntagonistic IC50 (nM)a values derived from dose-response curves on the basis of inhibition of Ca2+ flux in human monocytes
1516 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
N N
O
N
S
N
OO
OS O
O
N
R
31 R = H (KN-62)
36 R = 2-[3H]Me
AstraZeneca reported that no replacement for the adamantaneframework was found [141] Investigation of the chain linkerrequirements between the adamantane and the phenyl ring wasperformed where it was found that C- and N- methylationcarbonyl reduction and methylene removal (38d-g) all causedloss of potency One methylene unit extension (38h ) retainedthe potency whereas reversal of the amide connectivity (38i)slightly decreased the potency AstraZeneca prepared acombinatorial library by reacting commercially availableadamantanemethylamine with a large variety of acid chloridesor via standard amide coupling with carboxylic acids Thegeneral trends indicated that only aromatic amides had activityat the P2X7R [141] On the phenyl ring 23- and 25-disubstitutions resulted in better antagonists (38j-o) than the26- (38p ) 35- (38q ) or 24-disusbstitutions (38r) The ortho-position could also tolerate substituents such as Br OMe andMe (38s-u) A closer examination on the reverse amides (38i v-y) also revealed that 23- and 25-disubsitutions were stillpreferred for P2X7R inhibition The indazole amide (38z) wasidentified as a target lead molecule with a good metabolicprofile suitable for further optimization for clinical trial studies[141]
Fig (17) Radiolabeled analogue of KN-62 [140]
O
NY
X
O
Z
NO2
Adamantyl amides represent a class of simple moleculeswith excellent activity at the P2X7R The nature of theadamantane group could potentially be further investigatedalthough the original authors stated that no replacement for theadamantane group was found A number of patents onadamantyl-containing benzamides have been generated byAstraZeneca [143-146] and Pfizer [147] indicating the potentialof this ligand class for generating a truly potent selective andorally active P2X7R inhibitor Pfizer specifically incorporatedtriazinyl group into the adamantyl benzamide framework [147]Fig (18) General structure for the cyclic imides generated by
AstraZeneca Non-adamantane benzamides including piperidinyl andpiperazinyl benzamides [148 149] triazinyl benzamides [150-153] quinolinyl benzamides [154 155] 34-difluoroanilinebenzamides [156] aromatic acid-containing benzamides [157]pyrazolylmethyl benzamides [158] and other heteroarylamides[159-163] have also been patented by various otherpharmaceutical companies
38 Adamantyl Amides
Adamantyl amides were also discovered from a high-throughput screening of AstraZenecarsquos compound library forinhibition of the P2X7R-mediated ethidium bromide uptakeinduced by BzATP [141] N-Adamantan-1-ylmethyl-2-chloro-benzamide 38a was identified as the lead structure with anexcellent pA2 value of 81 The presence of an ortho-substituentis believed to cause a twist in the orientation of the benzamideessential for potent P2X7R antagonism whereas para-substituents (38bc) have deleterious effects on the P2X7Ractivity Selected compounds of this series are represented inTable 5
39 Diarylimidazolines
Diarylimidazolines represent another class of P2X7-activecompounds They were first discovered through a high-throughput screening of Aventisrsquo compound library forinhibition of BzATP-induced YO-PRO-1 uptake mediated byP2X7R expressed in U373 cells stably expressing the human
Table 4 Structures and P2X7R Antagonistic Properties of Selected Cyclic Imides [142]
Entry X Y Z IC50 ( M) pA2a
37a CH2 CO N - 69
37b (CH2)2 CO N - 75
37c O CH2 N 64
37d O (CH2)2 N 063 -
37e S CO N - 77
37f NH CO N 02 -
37g S CO C-CONHMe 016 -
37h CH2 CO C-CONMe2 025 -
a pA2 = the negative logarithm of a molar concentration of an antagonist that necessitates doubling of agonist concentration needed to elicit the original submaximalresponse obtained in the absence of antagonist
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1517
P2X7R [164] Compound 39a was identified as an initial hitfrom the screening with an IC50 value of 80 nM
equipotent Some degree of branching in the chain linker wasgenerally tolerated (39f-h) Configurational constraintsimposed on the chain linker as exemplified by 39i weretolerated but this was not the case for the cyclopropyl analogue39j
X
RO
NH
N
HN
38a-y 38z
Subsequent investigations focused on the substitutionpattern in the aryl ring for the benzyl (n=1) and phenethyl (n=2)series [164] Electron withdrawing and small neutral groupswere generally tolerable in both series (39k-r v-x) Howeverelectron-donating groups exemplified by OMe gave much lessactive compounds (39s-u) In the phenethyl seriessubstitutions at the ortho- and meta- positions were preferableto the corresponding para- substitutions (39kl vs 39m and39no vs 39p) Interestingly in the benzyl series meta- andpara- substitutions resulted in more active compounds than thecorresponding ortho analogues (39v vs 39wx)
Fig (19) Adamantyl amides generated by AstraZeneca
Investigation of the nature of the linker between thearomatic ring and the 2-position of the imidazole ring revealedthat a 3 or 4 carbon linker (39bc) caused a slight decrease in theactivity and no carbon linker (39d ) rendered the compoundinactive [164] One carbon linker with a methyl branching unit(39e) was tolerated It was reported that there was noenantiomeric preference of the methyl branch they were
The diarylimidazolines examined represent good candidatesfor orally active P2X7R inhibitors they are small and highlyamenable for SAR studies The 4- and 5-phenyl rings representattractive sites for further investigations
Table 5 Structures and P2X7R Antagonistic Properties of Selected Adamantyl Amides [141]
Entry R X pA2
38a 2-Cl CH2NHCO 81
38b 4-Cl CH2NHCO inactive
38c 24-Cl2 CH2NHCO 64
38d 2-Cl CHMeNHCO 64
38e 2-Cl CH2NMeCO 54
38f 2-Cl CH2NHCH2 54
38g 2-Cl NHCO inactive
38h 2-Cl (CH2)2NHCO 78
38i 2-Cl CH2CONH 63
38j 23-Cl2 CH2NHCO 88
38k 25-Cl2 CH2NHCO 83
38l 2-Cl-3-NH2 CH2NHCO 82
38m 2-Cl-5-OMe CH2NHCO 88
38n 2-Cl-5-OH CH2NHCO 80
38o 2-Cl-5-NH2 CH2NHCO 84
38p 26-Cl2 CH2NHCO 74
38q 35-Cl2 CH2NHCO 54
38r 24-Cl2 CH2NHCO 64
38s 2-Br CH2NHCO 80
38t 2-OMe CH2NHCO 71
38u 2-Me CH2NHCO 79
38v 2-Me CH2CONH 68
38w 2-Cl-5-OMe CH2CONH 72
38x 2-Me-3-OMe CH2CONH 83
38y 2-Me-5-OMe CH2CONH 80
1518 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
N
HN
XY
R
N
HN
N
HN
N
HN
( )n
39i39j
39a-ce-hk-w 39d
Fig (20) General structure of the diarylimidazolines generated by Aventis
Table 6 Structures and P2X7R Antagonistic Properties of Selected Diarylimidazolines [164]
Entry n X Y R IC50 ( M)a
39a 1 H H H 008
39b 2 H H H 054
39c 3 H H H 061
39e 0 (R)-Me H H 011
39f 1 (S)-Me H H 003
39g 1 Me Me H 001
39h 1 Ph H H 032
39k 1 H H 2-F 016
39l 1 H H 3-F 004
39m 1 H H 4-F 022
39n 1 H H 2-Cl 007
39o 1 H H 3-Cl 012
39p 1 H H 4-Cl gt1
39q 1 H H 2-Me 007
39r 1 H H 4-Me 219
39s 1 H H 2-OMe 083
39t 1 H H 3-OMe gt3
39u 1 H H 4-OMe gt3
39v 0 H H 2-F 058
39w 0 H H 3-F 008
39x 0 H H 4-F 008
aAntagonistic IC50 values derived from dose-response curves on the basis of inhibition of YO-PRO-1 dye uptake
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1519
R1N
N
NN
R2
R1
NN
NN
R2
N
N
NN
Cl
Cl
Cl
Cl
40b-t 40u-x40a
1
2
3
4
5
Fig (21) General structure of tetrazoles generated by Abbott Laboratories
310 1-Benzyl-5-Phenyltetrazoles potency The limited solubility of those compounds howeverposed a challenge to incorporate substitutions that couldincrease the solubility while maintaining activity A 3-pyridylgroup (40d ) was found to overcome this problem Moreover anextra 2-methyl substituent (40e) resulted in higher activitycompared to 40d A methylene extension (40f) caused a decreasein potency whereas other heterocycles such as 40g and 40hresulted in analogues with similar potency to 40d
1-Benzyl-5-phenyltetrazoles were discovered through ahigh-throughput screening by Abbott Laboratories for threedifferent P2X7R activities Ca2+ flux at the rat or human P2X7RYO-PRO-1 uptake and interleukin-1β release at human P2X7R[165-168] The initial hit from the screening was the tetrazole40a (Table 7) with a pIC50 value of 69 in inhibiting P2X7Rderived from the recombinant human cell line
Investigation of the nature of the substituent attached to the5-position of the tetrazole core showed that the substitutionpattern at this position was not well tolerated [165] Forexample unsubstituted mono-chlorosubstituted 25- and 34-
Initial SAR studies focused on the substituent attached tothe 1-position of the tetrazole core [165] Unsubstituted orortho-substituted benzylic group (40bc) exhibited good
Table 7 Structures and P2X7R Antagonistic Properties of Selected 1-benzyl-5-tetrazoles [165]
1520 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
disubstituted compounds (40i-m) pyridine and quinoline ring(40n-p ) analogues were not tolerated However moreconservative changes such as CF3 and F for Cl group (40q-t)resulted in an increase of potency
release and amplification of astrocytic Ca2+ signaling werereported to be due to the P2X7R and not Cx43 hemichannels[170]
Decavanadate a type of polymer of vanadium oxides hasbeen demonstrated to be a competitive rapid-acting antagonistat the P2X7R [172] It is capable of inhibiting the BzATP-stimulated ethidium bromide uptake in human mouse and ratP2X7R with pA2 values of 74 57 and 71 respectivelyDecavanadate has not been fully tested for its selectivity overother P2X receptors however it is suggested that this largemolecular species acts at the extracellular ATP binding site ofthe receptor consistent with the competitive mode of inhibition[172] A monoclonal antibody specific for the human P2X7Rhas also been shown to be a potent and selective antagonist ofthe receptor with an IC50 value of 5 nM in blocking the BzATP-evoked currents [173] Interestingly tenidap at 50 ndash 200 microMconcentration increases the sensitivity of mouse macrophagesto ATP-mediated cytotoxicity [174] 17-β-estradiol and notprogesterone or 17-α-estradiol has also been reported to blockthe agonist-induced currents at the human P2X7R with an IC50value of 3 microM in a non-genomic manner [175] Polymyxin B apotent antibiotic well-known for its lipopolysaccharide-neutralizing effects has been demonstrated to confer asynergistic effect when co-applied with ATP in a similar mannerto tenidap inducing Ca2+ influx ethidium bromide uptake andlactate dehydrogenase release via action at the P2X7R [176]
Tetrazoles with reversed connectivity (40u-x) were alsoevaluated where it was found that this connectivity reversal hadlittle effect on the potency [165] These tetrazoles were reportedto be slightly more active in the human P2X7R than the ratanalogue [165]
The tetrazole derivative 40d has been shown to be selectivefor other P2 receptors including P2X3 P2X4 and P2Y2 receptors[165] Pharmacological testing of this tetrazole suggested acompetitive mode of inhibition at the P2X7R and its efficacyhas also been evaluated in a model of neuropathic pain in rats[165] Overall these tetrazoles are found to be P2X7R-active andhighly adaptable to structural modifications These propertiesmake the tetrazoles an interesting class of compound for furtherinvestigating P2X7R activity
311 Other P2X7R-Active Ligands
Chelerythrine 41 (Fig 22) is a member of the benzophen-anthridine alkaloids known to inhibit protein kinase C withgood potency and selectivity (IC50 066 microM) as well as enzymessuch as alanine aminotransferase and Na+K+-ATPase [169] At10 microM concentrations chelerythrine non-competitivelyinhibited the BzATP-induced 86Rb+ efflux by 74 in human B-lymphocytes [169] Dye uptake studies could not be performeddue to the fluorescent nature of the compound It is yet to bedetermined whether chelerythrine inhibition of P2X7R involvesprotein kinase C [169]
More recently other distinct classes of P2X7-active ligandshave been reported and patented by the Abbott Laboratoriesacylhydrazides [177] cyanoamidines and cyanoguanidines[178 179]
4 CONCLUSIONS AND FUTURE DIRECTIONS
O
O
N
O
O
Cl
41
Although many structurally diverse class of compoundshave been reported as P2X7R antagonists with reasonablepotency the selectivity issue over other P2 receptors andproteins such as ecto-ATPases and ectonucleotidases stillprevails This is particularly an issue for oxidized ATP andsuramin SAR studies performed on PPADS and suramin mainlygave improved potency and selectivity for the P2X1 receptorIon channel blockers such as amilorides and stilbenederivatives only possess relatively low potency at the P2X7RFurther complications in studying these P2X7R antagonistsalso arise from the demonstrated species differences acrosshuman rat and mouse systems Brilliant Blue G is active for allthree species however at present there have been no SAR datareported Another fundamental factor that hampers the study ofP2X7R in detail is the relatively low potency and selectivity ofthe available agonist BzATP The discovery of a more potentselective P2X7R agonist would be ideal since in many studiesfull dose-response curves could not be generated due to the low
Fig (22) Chemical structure for chelerythrine an alkaloid withantagonistic properties at the human P2X7R
Gap junction channel blockers such as flufenamic acid 42 carbenoxolone 43 and mefloquine 44 (Fig 23) were all foundto be inhibitors of the BzATP-induced Ca2+ flux (IC50 values of655 175 and 25 nM respectively) and of YO-PRO-1 uptake in1321N1 astrocytoma cells stably expressing rat P2X7R [170]This finding poses a question to the general use of gap junctionchannel blockers as indicators of the participation of connexinhemichannels at the sites of transmitter release [171] The ATP
HN CF3
O OH
42 f lufenamic acid
HO
-O
O
O
H
OH
O
O-
N
HN
CF3
CF3
H O
H
43 carbenoxolone 44 mefloquine
Fig (23) Chemical structures for gap junction channel blockers with P2X7R antagonistic properties
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1521
potency of BzATP The nature of functional assays on theP2X7R is also found to be complex as ionic compositiontemperature and cell origins all affect the results demandingextra care be taken when antagonist potencies are directlycompared from different studies
[23] Li J Liu D Ke H Z Duncan R L Turner C H J Biol Chem2005 280 42952-42959
[24] Penolazzi L Bianchini E Lambertini E Baraldi P G RomagnoliR Piva R Gambari R J Biomed Sci 2005 12 1013-1020
[25] Gibbons S J Washburn K B Talamo B R J Auton Pharmacol2001 21 181-190
[26] Dehaye J P Moran A Marino A Arch Oral Biol 1999 44 S39-S43Four classes of small molecules with high affinity at the
P2X7R have been reported cyclic imides adamantyl amidesdiarylimidazolines and 1-benzyl-5-phenyltetrazoles Theseclasses of compounds are highly amenable to structuralmodifications indicating their good prospects as leadmolecules for further improving the potency and selectivity atP2X7R ultimately leading to orally active drugs To date norational drug design based on the knowledge of binding siteswithin this receptor has been undertaken This is a direct resultof the fact that no crystal structure or suitable homology modelhave been reported for this receptor The fact that the P2X7R canadopt either a channel or pore state causes even moredifficulties in studying receptor dynamics and at present theexact mechanism of channel-to-pore transition remains to beelucidated Undoubtedly the P2X7R is involved in severalprocesses relevant to chronic inflammation neurodegenerationand neuropathic pain and therefore discovery of more potentselective P2X7R antagonists would have a great impact in thepresent therapeutic window
[27] Li Q Luo X Zeng W Muallem S J Biol Chem 2003 27847554-47561
[28] Wang Q Wang L Feng Y-H Li X Zeng R Gorodeski GI AmJ Physiol Cell Physiol 2004 287 C1349-C1358
[29] Bardini M Lee H Y Burnstock G Cell Tissue Res 2000 299 105-113
[30] Hu H Z Gao N Lin Z Gao C Liu S Ren J Xia Y Wood JD J Comp Neurol 2001 440 299-310
[31] Hillman K A Burnstock G Unwin R J Nephron Exp Nephrol2005 101 e24-e30
[32] Koshi R Coutinho-Silva R Cascabulho C M Henrique-Pons AKnight G E Loesch A Burnstock G J Reprod Immunol 200566 127-140
[33] Moores T S Hasdemir B Vega-Riveroll L Deuchars J Parson SH Brain Res 2005 1034 40-50
[34] Surprenant A Rassendren F Kawashima E North R A Buell GScience 1996 272 735-738
[35] Cheewatrakoolpong B Gilchrest H Anthes J C Greenfeder SBiochem Biophys Res Commun 2005 332 17-27
[36] Watters J J Sommer J A Fisette P L Pfeiffer Z A Aga MPrabhu U Guerra A N Denlinger L C Bertics P J Drug Dev Res2001 53 91-104
[37] Adinolfi E Pizzirani C Idzko M Panther E Norgauer J DiVirgilio F Ferrari D Purinergic Signall 2005 1 219-227
ACKNOWLEDGEMENTS[38] Khakh B S Burnstock G Kennedy C King B F North A
Seguela P Voigt M Humphrey P P A Nat Rev Neurosci 20012 165-174
The authors would like to thank Dr Natasha Hungerford forproofreading the manuscript
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[51] Ferrari D Pizzirani C Adinolfi E Lemoli R M Curti A IdzkoM Panther E Di Virgilio F J Immunol 2006 176 3877-3883
[52] Grahames C B A Michel A D Chessell I P Humphrey P P ABr J Pharmacol 1999 127 1915-1921
[12] Deuchars S A Atkinson L Brooke R E Musa H Milligan C JBatten T F C Buckley N J Parson S H Deuchars J J Neurosci2001 21 7143-7152
[53] Shigemoto-Mogami Y Koizumi S Tsuda M Ohsawa K KohsakaS Inoue K J Neurochem 2001 78 1339-1349
[13] Zhang X-F Han P Faltynek C R Jarvis M F Shieh C-C BrainRes 2005 1052 63-70
[54] Le Feuvre R Brough D Iwakura Y Takeda K Rothwell N J JBiol Chem 2002 277 3210-3218
[14] Franke H Bringmann A Pannicke T Krugel U Grosche JReichenbach A Illes P Drug Dev Res 2001 53 140-147
[55] Brough D Le Feuvre R A Wheeler R D Solovyova N HilfikerS Rothwell N J Verkhratsky A J Immunol 2003 170 3029-3036
[15] Bianco F Fumagalli M Pravettoni E DAmbrosi N Volonte CMatteoli M Abbracchio M P Verderio C Brain Res Rev 2005 48 144-156
[56] James G Butt A M Eur J Pharmacol 2002 447 247-260[57] Di Virgilio F Falzoni S Mutini C Sanz J M Chiozzi P Drug
Dev Res 1998 45 207-213[16] Brandle U Kohler K Wheeler-Schilling T H Mol Brain Res 1998
62 106-109[58] Burnstock G Drug Dev Res 2001 53 53-59
[17] Wheeler-Schilling T H Marquordt K Kohler K Guenther E JabsR Mol Brain Res 2001 92 177-180
[59] Boucsein C Zacharias R Farber K Pavlovic S Hanisch U-KKettenmann H Eur J Neurosci 2003 17 2267-2276
[18] Puthussery T Fletcher E L J Comp Neurol 2004 472 13-23[60] Chessell I P Michel A D Humphrey P P A Br J Pharmacol
1997 121 1429-1437[19] Sugiyama T Kawamura H Yamanishi S Kobayashi M Katsumura
K Puro D G Am J Physiol Cell Physiol 2005 288 C568-C576[61] Le Feuvre R Brough D Rothwell N Eur J Pharmacol 2002 447
261-269[20] Naemsch L N Du X Sims S M Dixon S J Drug Dev Res 2001
53 130-139[62] Rampe D Wang L Ringheim G E J Neuroimmunol 2004 147
56-61[21] Steinberg T H Jorgensen N R Bong J S Henriksen Z Atal N
Lin G C Bennett B D Eriksen E F Sorensen O H Civitelli RDrug Dev Res 2001 53 126-129
[63] Sperlagh B Kofalvi A Deuchars J Atkinson L Milligan C JBuckley N J Vizi E S J Neurochem 2002 81 1196-1211
[22] Jorgensen N R Henriksen Z Sorensen O H Eriksen E FCivitelli R Steinberg T H J Biol Chem 2002 277 7574-7580
[64] Kang T-C Park S-K Hwang I K An S-J MH W Mol BrainRes 2004 121 12-18
1522 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
[65] Papp L Vizi E S Sperlagh B Neuro Report 2004 15 2387-2391 [104] Michel A D Kaur R Chessell I P Humphrey P P A Br JPharmacol 2000 130 513-520[66] Duan S Anderson C M Keung E C Chen Y Chen Y
Swanson R A J Neurosci 2003 23 1320-1328 [105] Cabantchik Z I Greger R Am J Physiol Cell Physiol 1992 262C803-C827[67] Wang C-M Chang Y-Y Kuo J-S Sun S H Glia 2002 37 8-
18 [106] Greger R Methods Enzymol 1990 191 793-810[68] Sim J A Young M T Sung H-Y North A Surprenant A J
Neurosci 2004 24 6307-6314[107] Hua S Inesi G Biophys J 1997 73 2149-2155[108] Bultmann R Pause B Wittenburg H Kurz G Starke K Naunyn-
Schmiedebergs Arch Pharmacol 1996 354 481-490[69] Fellin T Pozzan T Carmignoto G J Biol Chem 2006 281 4274-4284 [109] Soltoff S P McMillian M K Talamo B R Cantley L C Biochem
Pharmacol 1993 45 1936-1940[70] Chessell I P Hatcher J P Bountra C Michel A D Hughes J PGreen P Egerton J Murfin M Richardson J Peck W LGrahames C B A Casula M A Yiangou Y Birch R Anand PBuell G N Pain 2005 114 386-396
[110] El-Moatassim C Dubyak G R J Biol Chem 1993 268 15571-15578
[111] Panenka W Jijon H Herx L M Armstrong J N Feighan D WeiT Yong V W Ransohoff R M MacVicar B A J Neurosci 200121 7135-7142
[71] Gracie J A Clin Exp Immunol 2004 136 402-404[72] Labasi J M Petrushova N Donovan C McCurdy S Lira P
Payette M M Brissette W Wicks J R Audoly L Gabel C J JImmunol 2002 168 6436-6445
[112] Grinstein S Rotin D Mason M J Biochim Biophys Acta RevBiomembr 1989 988 73-97
[73] Wang X Arcuino G Takano T Lin J Peng W G Wan P LiP Xu Q Liu Q S Goldman S A Nedergaard M Nat Med 200410 821-827
[113] Garcia M L King V F Shevell J L Slaughter R S Suarez-KurtzG Winquist R J Kaczorowski G J J Biol Chem 1990 2653763-3771
[74] Solle M Labasi J Perregaux D G Stam E Petrushova N KollerB H Griffiths R J Gabel C A J Biol Chem 2001 276 125-132
[114] Garritsen A Ijzerman A P Beukers M W Cragoe E J J SoudjinW Biochem Pharmacol 1990 40 827-834
[75] Sanchez-Nogueiro J Marin-Garcia P Miras-Portugal M T FEBS Lett2005 579 3783-3788
[115] Neylon C B Little P J Cragoe E J J Bobik A Circ Res 199067 814-825
[76] Raouf R Chakfe Y Blais D Speelman A Boue-Grabot EHenderson D Seguela P Mol Pharmacol 2004 65 646-654
[116] Wiley J S Chen R Wiley M J Jamieson G P Arch BiochemBiophys 1992 292 411-418
[77] Yiangou Y Facer P Durrenberger P Chessell I P Naylor ABountra C Banati R R Anand P BMC Neurol 2006 6 12
[117] Wiley J S Chen R Jamieson G P Arch Biochem Biophys 1993305 54-60
[78] Sperlagh B Vizi E S Wirkner K Illes P Prog Neurobiol 200678 327-346
[118] Bultmann R Wittenburg H Pause B Kurz G Nickel P Starke KNaunyn-Schmiedebergs Arch Pharmacol 1996 354 498-504
[79] Stojilkovic S S Tomic M He M-L Yan Z Koshimizu T-AZemkova H Ann NY Acad Sci 2005 1048 116-130
[119] Damer S Niebel B Czeche S Nickel P Ardanuy U SchmalzingG Rettinger J Mutschler E Lambrecht G Eur J Pharmacol 1998350 R5-R6[80] McGaraughty S Jarvis M F Drug Dev Res 2006 67 376-388
[81] Baraldi P G Di Virgilio F Romagnoli R Curr Top Med Chem2004 4 1707-1717
[120] Braun K Rettinger J Ganso M Kassack M Hildebrandt CUllmann H Nickel P Schmalzing G Lambrecht G Naunyn-Schmiedebergs Arch Pharmacol 2001 364 285-290[82] Romagnoli R Baraldi P G Di Virgilio F Expert Opin Ther Pat
2005 15 271-287 [121] Kassack M U Braun K Ganso M Ullmann H Nickel P BoingB Muller G Lambrecht G Eur J Med Chem 2004 39 345-357[83] Shieh C-C Jarvis M F Lee C-H Perner R J Expert Opin Ther
Pat 2006 16 1113-1127 [122] Horner S Menke K Hildebrandt C Kassack M U Nickel PUllmann H Mahaut-Smith M P Lambrecht G Naunyn-Schmiedebergs Arch Pharmacol 2005 372 1-13
[84] Virginio C Church D North R A Surprenant ANeuropharmacology 1997 36 1285-1294
[85] Nuttle L C Dubyak G J Biol Chem 1994 269 13988-13996 [123] Hausmann R Rettinger J Gerevich Z Meis S Kassack M U IllesP Lambrecht G Schmalzing G Mol Pharmacol 2006 69 2058-2067
[86] Tatham P E R Cusack N J Gomperts B D Eur J Pharmacol1988 147 13-21
[87] Elssner A Duncan M Gavrilin M Wewers M D J Immunol2004 172 4987-4994
[124] Ullmann H Meis S Hongwiset D Marzian C Wiese M NickelP Communi D Boeynaems J-M Wolf C Hausmann RSchmalzing G Kassack M U J Med Chem 2005 48 7040-7048[88] Chessell I P Michel A D Humphrey P P A Br J Pharmacol
1998 124 1314-1320 [125] Soto F Lambrecht G Nickel P Stuhmer W Busch A ENeuropharmacology 1999 38 141-149[89] Bianchi B R Lynch K J Touma E Niforatos W Burgard E C
Alexander K M Park H S Yu H Metzger R Kowaluk E JarvisM F van Biesen T Eur J Pharmacol 1999 376 127-138
[126] Rettinger J Schmalzing G Damer S Muller G Nickel PLambrecht G Neuropharmacology 2000 39 2044-2053
[90] North R A Surprenant A Annu Rev Pharm Toxicol 2000 40 563-580
[127] Klapperstuck M Buttner C Nickel P Schmalzing G Lambrecht GMarkwardt F Eur J Pharmacol 2000 387 245-252
[91] Murgia M Hanau S Pizzo P Rippa M Di Virgilio F J BiolChem 1993 268 8199-8203
[128] Hulsmann M Nickel P Kassack M Schmalzing G Lambrecht GMarkwardt F Eur J Pharmacol 2003 470 1-7
[92] Craighead M W Middlehurst K M L LeFeuvre R Kimber IRothwell N J Neurosci Lett 2001 311 77-80
[129] Rettinger J Braun K Hochmann H Kassack M U Ullmann HNickel P Schmalzing G Lambrecht G Neuropharmacology 200548 461-468[93] Beigi R D Kertesy S B Aquilina G Dubyak G R Br J
Pharmacol 2003 140 507-519 [130] Jiang L-H Mackenzie A B North R A Surprenant A MolPharmacol 2000 58 82-88[94] DellAntonio G Quattrini A Cin E D Fulgenzi A Ferrero M E
Arthritis Rheum 2002 46 3378-3385 [131] Bultmann R Starke K Naunyn-Schmiedebergs Arch Pharmacol1995 352 477-482[95] DellAntonio G Quattrini A Cin E D Fulgenzi A Ferrero M E
Neurosci Lett 2002 327 87-90 [132] Tuluc F Bultmann R Glanzel M Frahm A W Starke K Naunyn-Schmiedebergs Arch Pharmacol 1998 357 111-120[96] Lambrecht G Braun K Damer S Ganso M Hildebrandt C
Ullmann H Kassack M U Nickel P Curr Pharm Des 2002 82371-2399
[133] Wittenburg H Bultmann R Pause B Ganter C Kurz G Starke KNaunyn-Schmiedebergs Arch Pharmacol 1996 354 491-497
[97] Garcia-Guzman M Soto F Gomez-Hernandez J M Lund P-EStuhmer W Mol Pharmacol 1997 51 109-118
[134] Gargett C E Wiley J S Br J Pharmacol 1997 120 1483-1490[135] Humphreys B D Virginio C Surprenant A Rice J Dubyak GR
Mol Pharmacol 1998 54 22-32[98] Kim Y-C Camaioni E Ziganshin A U Ji X-D King B FWildman S S Rychkov A Yoburn J Kim H Mohanram AHarden T K Boyer J L Burnstock G Jacobson K A Drug DevRes 1998 45 52-66
[136] Baraldi P G Romagnoli R Tabrizi M A Falzoni S Di Virgilio FBioorg Med Chem Lett 2000 10 681-684
[137] Ravi R G Kertesy S B Dubyak G R Jacobson K A Drug DevRes 2001 54 75-87[99] Jacobson K A Kim Y-C Wildman S S Mohanram A Harden T
K Boyer J L King B F Burnstock G J Med Chem 1998 41 2201-2206
[138] Chen W Ravi R G Kertesy S B Dubyak G R Jacobson K ABioconjug Chem 2002 13 1100-1111
[100] Kim Y-C Brown S G Harden T K Boyer J L Dubyak GKing B F Burnstock G Jacobson K A J Med Chem 2001 44 340-349
[139] Baraldi P G Nunez M D C A M Falzoni S Di Virgilio FRomagnoli R J Med Chem 2003 46 1318-1329
[140] Romagnoli R Baraldi P G Pavani M G Tabrizi M A MoormanA R Di Virgilio F Cattabriga E Pancaldi C Gessi S Borea P ABioorg Med Chem Lett 2004 14 5709-5712
[101] Brown S G Kim Y-C Kim S-A Jacobson K A Burnstock GKing B F Drug Dev Res 2001 53 281-291
[102] Lambrecht G Rettinger J Baumert H G Czeche S Damer SGanso M Hildebrandt C Niebel B Spatz-Kumbel G SchmalzingG Mutschler E Eur J Pharmacol 2000 387 R19-R21
[141] Baxter A Bent J Bowers K Braddock M Brough S Fagura MLawson M McInally T Mortimore M Robertson M Weaver RWebborn P Bioorg Med Chem Lett 2003 13 4047-4050
[103] Kim Y-C Lee J-S Sak K Marteau F Mamedova L BoeynaemsJ-M Jacobson K A Biochem Pharmacol 2005 70 266-274
[142] Alcaraz L Baxter A Bent J Bowers K Braddock M CladingboelD Donald D Fagura M Furber M Laurent C Lawson M
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1523
Mortimore M McCormick M Roberts N Robertson M BioorgMed Chem Lett 2003 13 4043-4046
[165] Nelson D W Gregg R J Kort M E Perez-Medrano A Voight EA Wang Y Grayson G Namovic M T Donnelly-Roberts D LNiforatos W Honore P Jarvis M F Faltynek C R Carroll W AJ Med Chem 2006 49 3659-3666
[143] Caffrey M Rhonan F Pimm A WO2004074224A1 2004[144] Evans R Rhonan F Thompson T Willis P WO2006059945A1
2006 [166] Perez-Medrano A Nelson D W Carroll W A Kort M E Gregg RJ Voight E A Jarvis M F Kowaluk E A WO2006086229A12006
[145] Rhonan F Martin B Thompson T Tomkinson N Willis PWO2006025783A1 2006
[146] Cladingboel D Rhonan F Willis P WO2005014529A1 2005 [167] Carroll W A Perez-Medrano A Florjancic A S Nelson D WPeddi S Li T Bunnelle E M Hirst G Li B CWO20060052374A1 2006
[147] Duplantier A J EP1310493A1 2003[148] Baxter A Kindon N Pairaudeau G Roberts B Thom S
WO0144213A1 2001 [168] Carroll W A Perez-Medrano A Florjancic A S Nelson D WPeddi S Bunnelle E M Hirst G C Li B WO2005111003A12005
[149] Meghani P Bennion C WO0146200A1 2001[150] Li Z B WO20050288256A1 2005[151] Leonard J A Li Z J Li Z B Urban F J WO20050288288A1
2005[169] Shemon A N Sluyter R Conigrave A D Wiley J S Br J
Pharmacol 2004 142 1015-1019[152] Leonard J A Li Z J Li Z B Urban F J WO2006003513A1
2006[170] Suadicani S O Brosnan C F Scemes E J Neurosci 2006 26
1378-1385[153] Li Z B WO2006003500A1 2006 [171] Saez J C Retamal M A Basilio D Bukauskas F F Bennett M
V L Biochim Biophys Acta 2005 1711 215-224[154] Evans R Eyssade C Rhonan F Martin B Thompson T WillisP WO2004106305A1 2004 [172] Michel A D Xing M Thompson K M Jones C A Humphrey P
P A Eur J Pharmacol 2006 534 19-29[155] Rhonan F Thompson T Willis P WO2005009968A1 2005[156] Furber M Luker T J Mortimore M P Thorne P Meghani P
WO0071529A1 2000[173] Buell G Chessell I P Michel A D Collo G Salazzo M Herren
S Gretener D Grahames C Kaur R Kosco-Vilbois M HHumphrey P P A Blood 1998 92 3521-3528[157] Thompson T Willis P WO2006080884A1 2006
[158] Concepcion A Inoue T Mochizuki Y Muramatsu A Gantner FNakashima K Urbhans K Bacon K B WO2005019182A1 2005
[174] Sanz J M Chiozzi P Di Virgilio F Eur J Pharmacol 1998 35 235-244
[159] Dombroski M A Duplantier A J Subramanyam CWO2004099146A1 2004
[175] Cario-Toumaniantz C Loirand G Ferrier L Pacaud P J Physiol1998 508 659-666
[160] Kelly M G Kincaid J WO2006102588A1 2006 [176] Ferrari D Pizzirani C Adinolfi E Forchap S Sitta B Turchet LFalzoni S Minelli M Baricordi R Di Virgilio F J Immunol 2004173 4652-4660
[161] Kelly M J Kincaid J WO2006102610A2 2006[162] Schwink L Stengelin S Gossel M Bohme T Hessler G Stahl P
Gretzke D WO2004072025A2 2004 [177] Nelson D W Jarvis M F Carroll W A WO2006110516A1 2006[163] Schwink L Stengelin S Boehme T Gossel M Hessler G Stahl P
WO2005070925A1 2005[178] Carroll W A Perez-Medrano A Peddi S Florjancic A S
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Gross A Zhao Z Rampe D Wang L Wirtz-Brugger F Harris BA Macdonald D Bioorg Med Chem Lett 2005 15 435-438
[179] Carroll W A Perez-Medrano A Jarvis M F Wang Y Peddi SWO20050171195A1 2005
Received February 01 2007 Revised April 04 2007 Accepted April 06 2007
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1507
Table 1 Potency of BzATP and ATP at Recombinant Mouse Human and Rat P2X7R (mP2X7R hP2X7R rP2X7R) and Mouse NTW8 Microglia(mNTW8)
Buffer composition (in millimolar)aNaCl buffer (NaCl 145 KCl 2 CaCl2 05 HEPES 10 D-glucose 10 pH 73) bNa-glutamate buffer (Na-glutamate 145 KCl 2 CaCl2 05 HEPES 10 D-glucose 10 pH73) cSucrose buffer (sucrose 280 HEPES 10 NMDG 5 KCl 56 D-glucose 10 CaCl2 1 pH 74)A change from NaCl to Na-glutamate buffer generally increases the potency of the agonists at the P2X7R Pre-treatment of cells by repeated ATP exposure will induce theP2X7R to adopt the pore state The cells in the pore state are generally more sensitive towards the agonists
inflammatory processes modulated by cytokines in particularthe interleukins and generation of tumour necrosis factors andnitric oxide have important roles in the many forms ofneurodegenerative disease such as multiple sclerosisParkinsonrsquos Alzheimerrsquos and Huntingtonrsquos disease [61] Thepresence of activated microglia in the brain has thus beenconsidered a reliable marker for neurodegenerative diseases TheP2X7R has also been shown to modulate β-amyloid-inducedcytokine expression (IL-1α and IL-1β) from humanmacrophages and microglia This further supports P2X7Rinvolvement in the neuropathology of Alzheimerrsquos disease inwhich the presence of β-amyloid is often surrounded byactivated microglia and astrocytes [62] Taken together theP2X7R has a pivotal role in neuro-inflammatory and-degenerative processes In addition P2X7R can stimulaterelease of traditional neurotransmitters in the brain such asglutamate and GABA The P2X7R regulation of glutamate andGABA release in rat gerbil and mouse hippocampus [63-65] andmurine astrocytes [66 67] has been reported although it is stillnot definitively confirmed [68] More recently two distinctglutamate release pathways in rat hippocampal astrocytes bypurinergic receptors have also been reported [69]
arthritis model [72] Inhibition of the P2X7R has also beenshown to improve recovery after spinal cord injury in ratmodels [73] However functional P2X7-like proteins have beenfound in one P2X7-knockout model [74 75] highlighting thelack of understanding of the P2X7-knockout animal modelsDominant-negative P2X7R subunits have been proposed as anovel genetic tool to understand the role of P2X7R in nativesystems [76] More recently it has been demonstrated thatexpression of cyclooxygenase-2 cannabinoid receptor CB2and P2X7R are all elevated in activated microglia cells ofmultiple sclerosis and amyotrophic lateral sclerosis spinal cord[77]
Further studies into understanding the physiologicalfunctions of P2X7R in vivo and the development of detailedpharmacophore models will lead to better understanding ofcellular cascades underlying the basis of neuro-inflammatoryand -degenerative processes A number of P2X7R antagonistshave already been developed towards this goal however a moreextensive range of ligands with drug-like properties includingimproved potency and selectivity is still required Althoughthere have been a number of reviews on the pharmacology of theP2X7R [78-80] there appears to be relatively limited accessibleresources on a comprehensive listing of molecular probesacting at the receptor [81-83] This review will present anoverview of the currently known agonists and antagonistsacting at the P2X7R and comment on their usefulness asmolecular probes for microglia activation
The exact role of P2X7R in vivo remains to be elucidated andliterature studies to date have compared normal and P2X7-knockout animal models In terms of pharmacologicalintervention the most promising roles of the P2X7R in vivowould most likely be in the areas of pain regulation [70]chronic inflammation [57 71] and neurodegeneration [61]Chronic inflammatory and neuropathic hypersensitivity arecompletely abolished in P2X7-knockout mice although normalnociceptive processing still remains [70] suggesting thatpotent selective P2X7 antagonists may serve as all-purposeanalgesics P2X7-knockout mice have been reported to showreduced severity of arthritis in an anti-collagen antibody
2 ENDOGENOUS LIGANDS FOR P2X7R
ATP 1 is the natural endogenous agonist of the P2X7R withEC50 values varying from 85 to 779 microM depending on thespecies examined (rat mouse or human) as determined from
1508 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
N
NN
N
NH2
O
OH(R)(H)RO
OPO
OH
O
POPHO
O
OH
O
O H
O
O
mixture of 2 and 3 e sters
3 BzATP
R =
Fig (3) Chemical structure for BzATP (3) the most potent P2X7R agonist to date
electrophysiological assays (Table 1) The low potency of ATPin activating the P2X7R is one of the main pharmacologicalfeatures of this unique receptor that distinguish it from otherpurinergic receptors Normal cytoplasmic concentrations of ATPin cells range from 5 to 10 mM which is released to theextracellular environment upon cell injury or insult therebyactivating the P2X7R before being quickly hydrolyzed by ecto-ATPases and ectonucleotidases Reduction of the extracellularMg2+andor Ca2+ ion concentration which are thought to beallosteric inhibitors [84] results in the potentiation of ATPeffects at the P2X7R [8] It has also been suggested that thetetrabasic form of ATP 2 is the active species acting at thereceptorrsquos ATP-binding site where the presence of Mg2+ andCa2+ ions chelate ATP4- rendering it inactive [85]
pharmacophore known for P2X7R activity The effects of thesesynthetic ligands on the P2X7R are generally tested in eitherelectrophysiological or dye uptake assays exploiting thereceptorrsquos non-desensitizing property and the ability to form alarge non-selective pore
31 ATP Derivatives
BzATP 3 (Fig 3) is the most potent P2X7R agonist to datewith 10- to 100-fold higher potency compared to ATP inactivating the receptor in a number of different systems [3] Thepotency of BzATP is also known to be species-dependent EC50value of BzATP at the rat P2X7R is 2ndash7 microM in theelectrophysiological assay which is approximately 10- to 30-fold higher than at the human P2X7R [40 88] BzATP also has adifferent EC50 value (007 ndash 025 microM) for activating the ratP2X7R in the YO-PRO-1 uptake assay performed in sucrosebuffer (Table 1) The increased sensitivity of the P2X7R toBzATP compared to the endogenous ligand ATP is one of themain criteria used to define the pharmacology of the P2X7Rsubtype Unfortunately BzATP is not highly specific for theP2X7R subtype other P2X receptors are also activated byBzATP [89] but with either equal or lower potency than ATP[90] Other ATP derivatives such as 2-methylthio-ATP 4 ATP-gamma-S 5 and ADP 6 are all weak agonists of the P2X7R (Fig4) [34 90] The order of potency for P2X7R agonists is BzATP3 gtgt ATP 1 gt 2-Methylthio-ATP 4 gt ATP-γ-S 5 gtgt ADP 6 [34] 2-Methylthio-ATP 4 has an EC50 value of 505 microM against theP2X7R derived from mouse NTW8 microglial cells based on theelectrophysiological studies [60]
Some diadenosine polyphosphates such as P1 P4-diadenosine tetraphosphate were reported to be capable ofactivating the P2X7R-mediated cellular permeabilization of ratmast cells [86] possibly due to their greater resistance tometabolic breakdown and similarity to ATP4- [3] Theendogenous peptide LL-37 an antimicrobial agent causingbacteria cell wall permeabilization derived from the humancathelicidin protein hCAP18 has been demonstrated to induceinterleukin-1β processing and release via the activation of theP2X7R [87]
3 SYNTHETIC LIGANDS FOR P2X7R
Several classes of P2X7R-active synthetic compounds havebeen reported in the literature Unfortunately despite thediscovery of these ligands there is still no general
N
NN
N
NH2
O
OHOH
OPO
O H
O
POPHO
O
OH
O
OH
SMe
4 2-Me thylthio-ATP
N
NN
N
NH2
O
OHOH
OPO
OH
O
POPHS
O
OH
O
OH
5 ATP-γ-S
N
NN
N
N H2
O
OHOH
OPO
OH
O
PHO
O
OH
6 ADP (adenosine 5-diphosphate)
N
NN
N
NH2
O
OO
OPO
O H
O
POPHO
O
OH
O
OH
7 oxidize d ATP
Fig (4) Chemical structures for ATP derivatives that are weak P2X7R agonists
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1509
The 2rsquo 3rsquo-dialdehyde ATP (or oxidized ATP) 7 (Fig 4) is thefirst irreversible inhibitor of the P2X7R expressed in the mousemacrophage cell line J774 [91] Subsequent studies alsoshowed that application of 100 microM oxidized ATP irreversiblyantagonized the rat P2X7R provided the cells were pre-incubated with oxidized ATP for 1-2h indicating that itcovalently modified the receptor [34] Co-application with anagonist did not result in any inhibition There is evidence thatoxidized ATP possesses low affinity for the P2Y receptors [91]however oxidized ATP at 100 microM concentration has beenshown to be very toxic to rat cerebellar granule neurons [92]and is likely to react with other enzymes such as ecto-ATPases[91 93] Interestingly oxidized ATP at 168 microM inhibitedP2X7R-mediated inflammatory pain in arthritic rats [94 95]Due to its non-selective inhibition extra caution is required inthe use of oxidized ATP to define involvement of P2X7R in thepurinergic system [42]
in a non-selective manner [3] It is a relatively potent antagonistat the P2X1-3 P2X5 receptors (IC50 01ndash5 microM) and P2Y1receptor but only weakly active or completely inactive at the ratP2X4 P2X6 P2X7 and some P2Y receptor subtypes [96]Interestingly PPADS is reported to be reasonably active at thehuman P2X4R with IC50 value of 28 microM indicating somespecies dependence [97] PPADS is highly specific for the P2receptors up to 100 microM concentration of PPADS does notinterfere with adenosine receptors muscarinic M1 M2 or M3α1- or α2-adrenoceptors histamine H1 AMPA or NMDA and 5-HT receptors [96]
PPADS has been widely studied in the P2 receptor systemsderived from various cell lines and structure-activityrelationship (SAR) studies have also been reported [98-101]Structural modifications of PPADS to date have involved theinvestigation of substitutions at the 4-aldehyde position 5-phosphate and the azophenyl ring Some of the key PPADSderivatives are represented in Fig (6) It was initially thoughtthat both the aldehyde and phosphate moieties are important forP2 antagonism It was also proposed that the slowlyequilibrating pseudoirreversible mode of inhibition of PPADSseen at the P2X1 receptor was due to Schiff base formationbetween the aldehyde and a specific lysine residue of thereceptor [96] The cyclic phosphates MRS2219 and MRS2220were found to be relatively potent antagonists at the rat P2X1receptor (IC50 6 and 10 microM respectively) with good selectivityover P2X2-4 P2Y1 P2Y2 P2Y4 P2Y6 and adenosine receptors[99] Another PPADS derivative that appears to be a promisingantagonist at the P2X1 receptor is PPNDS which exhibited anIC50 of 15 nM and no interactions detected withectonucleotidases P2Y1 receptor α1-adrenoceptors adenosinehistamine H1 and muscarinic M3 receptors [102] Later studiesreported that replacement of the sulfonyl groups on theazophenyl ring with carboxylates or phosphates furtherincreased the potency and selectivity at the P2X1 and P2X3receptors [101] Most recently PPADS analogues MRS2211 andMRS2603 have been reported to be active at the P2Y13 receptorbeing 45- and 74-fold more active respectively than PPADS[103] Only a limited number of PPADS analogues were testedagainst the recombinant human P2X7R in these studies and forthose it was found that none of them was more potent than iso-
Overall these ATP derivatives have high molecular weightsand could potentially bind to other purinergic receptors due totheir resemblance to the endogenous ligand ATP Thereforethey are unlikely to be suitable for developing an orally activedrug specifically targeting the P2X7R
N
HO
CHO
O P O-
O-
O
9 P5P
N
O P
O
O-
O-HO
CHO
N
N
SO3--O3S
43
21
6
5
8 PPADS
Fig (5) Chemical structures for PPADS and its parent molecule P5P
Fig (7) Chemical structure for DIDS and its derivatives
PPADS which differs from PPADS by the position of thesulfonates [100]
[109] receptor DIDS dihydro-DIDS 17 and SITS 19 (Fig 7) allblocked the ATP-stimulated Ca2+ uptake in rat parotid acinarcells via activation of the P2X7R (IC50 35 ndash 125 microM) [109]DNDS 18 in which the isothiocyanate moieties are replacedwith nitro groups was found to be inactive indicating theimportance of the isothiocyanate for interaction with theP2X7R On the other hand a variety of smaller aromaticisothiocyanate compounds derived from DIDS have been testedagainst the P2X1 receptor and were found to be active with IC50values between 11 and 54 microM [108] Overall DIDS and itsderivatives are inhibitors of both the P2X1 and P2X7R withlower potency for the latter
All the SAR data generated to date on PPADS and itsderivatives demonstrate that structural modifications on thekey functional groups (aldehyde phosphate and azophenyl)increases the potency and selectivity mainly at the P2X1receptor Consequently PPADS itself is used for testing at theP2X7R where it was found to be a potent antagonist only at therat and human receptors (IC50 15 and 90 nM respectively) butnot the mouse receptor (100-fold lower potency) in the YO-PRO-1 uptake assay provided cells are pre-incubated withPPADS for 15-30 min [41] When there was no pre-incubationthe IC50 of PPADS at the rat human and mouse P2X7R decreasedto approximately 50-70 microM in the electrophysiologicalrecordings [34 40] but pre-incubation with PPADS drasticallyimproved the IC50 value to 1 microM at the human P2X7R [88] Theinhibitory action of PPADS at the P2X7R is only partiallyreversible [88] suggesting a pseudo-irreversible mode ofantagonism [104] It has been suggested that PPADS P5P andoxidized ATP may all have a common site of action which islikely to be the ATP binding site [104] Overall it can beconcluded that PPADS per se is a species-dependent P2X7Rantagonist being most potent at the rat and human P2X7R butnot the mouse receptor Derivatives of PPADS reported to dateonly exhibit improved potency and selectivity at the P2X1receptor and not the P2X7R
5-(NN-Hexamethylene)amiloride (or HMA) 20 (Fig 8)belongs to the amilorides which are better known as potassium-sparing diuretics by blocking the Na+ channel as well as beinginhibitors of Na+-H+ counter-transport pathway [112] L-typecalcium channels [113] adenosine A1 receptors [114] andpossibly Na+-HCO3
- co-transport [115] HMA possesses affinityfor the P2X7R but its effects are highly species-dependent At 40microM concentration HMA antagonized P2X7R found in humanlymphocytes as determined from large cation uptake studies[116 117] However up to 100 microM concentration it wasineffective at the rat P2X7R [34] More interestingly theelectrophysiological studies revealed that co-application ofHMA with ATP produced an immediate and maximalaugmentation of inward currents induced by mouse P2X7R [60]This effect was not apparent when HMA was applied alone orwhen the buffer solution contained physiologicalconcentrations of Mg2+ and Ca2+ ions HMA has been reportedto be an irreversible inhibitor at the hP2X7R [88] Otheranalogues of HMA such as MIBA 21 and EIPA 22 also possesssimilar properties to HMA [116] Due to its relatively lowpotency and high species dependence at the P2X7R HMA andprobably other amilorides are unlikely to be good candidatesfor further exploration of the pharmacology of the P2X7R
33 Ion Channel Blockers
44rsquo-Diisothiocyanatostilbene-22rsquo-disulfonic acid (DIDS)16 is an anion channel blocker widely used to study various iontransport mechanisms [105-107] but has also beendemonstrated to be capable of inhibiting both the P2X1 receptor[108] and P2X7R-induced pore formation and other processes inseveral cell lines with relatively low potency [3 109-111] Forexample pre-incubation of BAC12F5 murine macrophage with150 ndash 200 microM DIDS inhibited the activation of phospholipaseD mediated by the P2X7R [110] Pre-incubation of rat primaryastrocytes with 200 microM of DIDS has also been shown to inhibitP2X7R-mediated responses such as the activation ofextracellular signal receptor kinases (ERK) [111] SARinvestigations have been performed on DIDS in order toimprove its potency and selectivity at the P2X1 [108] or P2X7
34 Polysulfonated Dyes
Suramin 23 is an example of a polysulfonated dye thatexhibits non-selective antagonism at both the P2X and P2Yreceptors with relatively low potency [3] Suramin blocks theP2X1-3 and P2X5 receptors (IC50 1-15 microM) but only shows weakor no inhibition at the P2X4 P2X6 and P2X7 receptors as well
N
NCl
N NH 2
O
NH
NH
NH2
20 5-(NN-Hexa methylene )- amiloride (HMA)
N
NCl
NH2
O
NH
NH
NH2
N
21 5-(N-Methyl-N- isobutyl)- amiloride (MIBA)
N
NCl
NH2
O
NH
NH
NH 2
N
22 5-(N-ethyl-N-isopropyl)- amiloride (EIPA)
Fig (8) Chemical structures for HMA and other amilorides
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1511
as some P2Y receptor subtypes [96] In addition suramin hasbeen shown to interact with many different proteins at theseconcentrations including glutamate nicotinic GABA and 5-HTreceptors as well as various proteases and ectonucleotidases [396] Nevertheless SAR studies on suramin have been performedin order to identify the important structural features responsiblefor antagonism at the P2 receptors [96 118-124] Selected keysuramin derivatives are represented in Fig (10 )
native and recombinant P2X1 receptors respectively and hasbeen shown to be much less potent at other P2X and P2Ysubtypes including adrenoceptors histamine H1 receptors andthe muscarinic M3 receptor [120] At the human P2X1 and P2X7receptors the IC50 values for NF449 were 005 nM and 40 microMrespectively [128] which indicated its usefulness todistinguish between the two receptors Subsequent SAR studiesconfirmed that NF449 was the most potent and selective knownP2X1 receptor antagonist [121 129] NF157 an analogue ofsuramin with fluorine replacing the methyl group has also beenreported to be highly potent at the P2Y11 receptor with a Ki of45 nM [124] More recently NF110 has also been shown to be apotent P2X3 receptor antagonist indicating that both thenumber and location of the sulfonate groups may be importantfor determining P2X subtype potency and selectivity [123]
O
HN
HN
NH
NH
O O
O ONH HNSO3-
SO 3-
SO3-
-O3S
SO 3-
-O3S
23 suramin
NF279 thus far represents the most active suramin relatedantagonist at the P2X7R and displays a reasonable potency of28 microM All the SAR data obtained to date on suramindemonstrate the importance of the molecular size number andposition of the sulfonate groups for P2X subtype potency andselectivity Unfortunately except for NF279 and NF449 othersuramin analogues provide very little information which can beused for the selective structure-based drug design of P2X7Rmolecules Suramin is also unlikely to be orally active due toits high molecular weight and interaction with many otherproteins Suramin is a very weak or almost inactive antagonistat the human P2X7R with an IC50 value of 92 microM [40] It istotally inactive at both the rat and mouse P2X7R with IC50values greater than 100 microM [34 60] Due to its interference withthe fluorescence of YO-PRO-1 suramin could not be used in theYO-PRO-1 dye uptake assay [104]
Fig (9) Chemical structure for suramin a weak antagonist at the P2X7R
Early SAR analysis showed that the shortened forms ofsuramin in the form of sulfonylbenzamides decreased thepotency at P2 receptors [118] NF023 (24 ) was later discoveredto be P2X-selective compared to P2Y receptors andectonucleotidases [118] Subsequent studies have examined theeffects of NF023 in various cell types and it was reported thatNF023 is a highly selective antagonist for either rat or humanP2X1 receptors heterologously expressed in Xenopus oocyteswith an IC50 of 02 microM [125] Another suramin derivativeNF279 (25 ) also showed potent selective reversible inhibitionat the rat P2X1 receptor in Xenopus oocytes with an IC50 of 19nM [126] In studying the effects of NF279 on human P2X1 andP2X7 receptors it was reported that NF279 is almost 100-foldmore potent at the human P2X1 than the P2X7 receptor withIC50 values of 005 and 28 microM respectively [127] Other keysuramin derivatives resulting from extensive SAR studies areNF449 (27 ) NF110 (28 ) and NF157 (26 ) [120 121 123 124128 129] NF449 has an IC50 value of 70 nM and 03 nM at the
Brilliant Blue G 29 (Fig 11) is another polysulfonated dyewhich is a potent non-competitive inhibitor of the rat andhuman P2X7R with IC50 values of 10 and 200 nM respectivelyin electrophysiological studies [130] Brilliant Blue G has alsobeen shown to be highly selective for P2X7R over P2X1-5receptors [130] It has been shown that at the rat P2X7R about100 to 300 nM concentration was required to inhibit the YO-PRO-1 uptake which was 10- to 30-fold higher than thatrequired for blocking P2X7R-induced currents demonstratingits differential effect at the channel and pore forms of the P2X7R
O
HN
HN
NH
NH
O O
R R
O ONH HNSO3-
SO3-
SO3-
-O3S
SO3-
-O3S
O
HN
HN
NH
OO
NH
O ONH
NH
R R
RR
n n
Suramin n=1 R=CH3
24 NF023 n=0 R=H
25 NF279 n=1 R=H
26 NF157 n=1 R=F
27 NF449 R=24-disulfonyl28 NF110 R=4-sulfonyl
Fig (10) Suramin and its key derivatives investigated in the SAR studies
1512 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
[130] Brilliant Blue G has also been tested on the mouseP2X7R where similar antagonistic potency was found comparedto rat and human receptors [42] In this study it was alsoreported that the selectivity of Brilliant Blue G for rat overhuman P2X7R was confirmed at 22degC but lost at 37degC Thedrawback for this compound is that it could also be bound toproteins and thus would be expected to have lower potency inthe dye uptake assay than electrophysiology measurements dueto the depletion by cell monolayer effect [42] Nevertheless itrepresents a promising antagonist at rat human and mouseanalogues of the P2X7R No studies have been performed onBrilliant Blue G to further improve its potency and selectivityat the P2X7R Other polysulfonated dyes such as Evans BlueTrypan Blue reactive blue 2 reactive red 2 and their derivativeshave also been tested at the purinergic systems [131-133] butnone of them exhibited activity at the P2X7R
opening of the ion channel calmidazolium produces no effecton the YO-PRO-1 uptake The large non-selective porehowever is still formed even when the ionic currents areinhibited indicating that pore formation does not necessarilyrequire opening of the ion channels Calmidazolium wassuggested to be an allosteric modulator of the P2X7R at theagonist binding site inducing conformational change at thereceptor level that results in decreased agonist binding affinity[84] It displays less potency at inhibiting the agonist-evokedcurrents at both the mouse and human P2X7R with IC50 valuesof 1 microM and 100 nM respectively [41 88] At present no SARstudies have been reported on calmidazolium
N
N+
ClCl
O
ClCl
Cl
Cl
30 calmidazolium
NH
N
O
SO3-
N+
SO3-
Cl
29 Coomassie Brilliant Blue G
Fig (12) Chemical structure for calmidazolium a P2X7R antagonistcapable of distinguishing the channel and pore forms of the receptor
36 KN-62 and its Analogues
KN-62 31 (Fig 13) is a derivative of isoquinolinesul-fonamide known as a selective and potent inhibitor of themultifunctional calciumcalmodulin-dependent protein kinases(CaMK) with an IC50 of 09 microM at CaMK II [134] Gargett andWiley in 1997 first demonstrated that both KN-62 31 and itsanalogue KN-04 32 were potent inhibitors of P2X7R at humanlymphocytes with IC50 values of 13 and 17-37 nM respectivelyin both the ion flux and ethidium bromide uptake assays [134]The mode of inhibition is allosteric modulation similar to thatof calmidazolium
Fig (11) Chemical structure for Brilliant Blue G a potent antagonist at therat P2X7R
35 Calmidazolium
Extracellular calmidazolium 30 (Fig 12) is a potentinhibitor of BzATP-induced currents via binding to rat P2X7Rwith an IC50 of 13 nM [84] The unique feature ofcalmidazolium is its ability to differentiate between the channeland pore form of the P2X7R while it potently inhibits the
KN-62 and KN-04 are species-selective for P2X7Rantagonism They potently inhibited the ATP-induced currentsas well as the ethidium bromide uptake in HEK293 cells
Fig (13) Chemical structures for isoquinolines KN-62 and KN-04 potent P2X7R antagonists at the hP2X7R with much less effect on the rat and humananalogues
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1513
33e most active in the series but still 30-fold less potent than KN-62
Fig (14) Conformationally restricted analogues of KN-62 [136]
expressing human P2X7R at the concentration of 30 ndash 100 nMdepending on whether ATP or BzATP was used and the cellularbackground of the human P2X7R [135] However even up to 3microM concentration they had no effect at the rat P2X7R [135] andKN-62 was found to be at least 10-fold more potent at thehuman P2X7R than the recombinant mouse analogue [60] Theseisoquinolinesulfonamide derivatives may interact with theamino-terminal domain of the P2X7R since the introduction ofthe first 335 amino acids of the human P2X7R delivers KN-62sensitivity to the rat P2X7R and the counter-part chimera isfound to be insensitive to KN-62 [135] The IC50 at the humanP2X7R is 10-fold lower at 22degC than at 37degC in the ethidiumbromide uptake study [42]
tyrosine) 2) R2 (the tyrosine side chain) and 3) R3 (thesubstituent attached to the N-piperazine) (Fig 15) [137-139]
The effect of substitutions at the R1 R2 and R3 groups (Fig15 ) on the P2X7R antagonistic properties was compared on thebasis of inhibition of K+ release [137 138] At the R1 positionlarge hydrophobic moieties linked to the amino positionthrough sulfonamide (34a-b) or carbamate (34c) groups werepreferable for P2X7R inhibition In the series of derivativeswhere R2 = quinolinesulfonyl and R3 = t-butoxycarbonyl (Boc)introduction of carbobenzyloxy (Cbz) and quinolinesulfonylgroups at the R1 position were found to be the most active[137] In another series of derivatives where R1 = Cbz and R3 =Boc introduction of arylsulfonyl (34d-g) and benzoyl (34h )moieties at the R2 position were preferred [137] A free aminogroup at the R3 position (34i-j) andor opening of thepiperazine ring to an ethylene diamine greatly reduced theP2X7R activity Amongst the various acyl groups tested at theR3 position Boc and benzoyl groups (34k-m) were found to bepreferable but not sulfonyl (34n-q ) The presence of a methylgroup at the amino terminus of the tyrosine group is thereforenot an absolute pre-requisite for P2X7 antagonism [137]
R3 N N
O
N
O
R4
R1
R2
In another study undertaken by Chen et al to furtherexplore the SAR of 34m [138] it was found that nitro-substitution at the meta- or para- position of the Cbz group atR1 (34r-t) was well tolerated However introduction of anacetamido- or isothiocyanato- group at the Cbz group abolishedP2X7R antagonism (34uv) The original isoquinoline-5-sulfonyl moiety at the R2 position could be substituted withmeta- or para-substituted phenylsulfonyl groups such as tosyl(34r-t) 3-nitrophenylsulfonyl (34w) or 4-nitrophenylsulfonyl(34x) without adverse effects on the P2X7R antagonism [138]Substitution of the benzoyl group at the R3 position by a Boc(34y) or 4-aminobenzoyl group (34z) retained activity
Fig (15) General structure for the KN-62 derivatives (34a-z)
Extensive SAR studies have been performed on KN-62 inorder to enhance its potency and selectivity at the P2X7R Thefirst attempt to improve the antagonistic activity of KN-62 wasachieved by tethering the N-methyl of the tyrosine backbone tothe ortho-position of the proximal phenyl ring which resultedin a series of conformationally restricted KN-62 analogues withthe formula shown in Fig (14 ) [136] The constrained form ofKN-62 (31 vs 33a) was devoid of P2X7R antagonisticproperties Replacement of the isoquinoline-5-sulfonyl moiety(33a) with quinoline-5-sulfonyl (33b ) quinoline-8-sulfonyl(33c) or naphthalene (33d ) resulted in the loss of ability toinhibit the P2X7R-mediated Ca2+ influx [136] The most activecompound arising from this series was compound (33e) with anIC50 value of 316 nM which is 30-fold weaker than the originalKN-62 Taken together these results indicate that an extendedrather than folded conformation of KN-62 conferred by theincorporation of a methylene unit between the N-methyl of thetyrosine backbone to the ortho-position of the proximal phenylring is preferred at the P2X7R [136]
A study by Baraldi et al reported the systematic SAR profileof KN-62 specifically looking at modifications of thephenylpiperazine moiety (R2 position Fig 16 ) [139] In theinvestigation of the optimum chain linker between thepiperazine and phenyl groups it was found that one methylenespacer (35a) slightly improved the activity whereas a twomethylene spacer was not favourable (35b ) [139] The presenceof a basic nitrogen in the piperazine moiety is proven crucial forP2X7R antagonism as substitution of the piperazine ring withpiperidine (35a vs 35c) resulted in a 3-fold reduction of activity[139] Substitution of the phenyl ring with other heterocyclessuch as pyridine (35d ) or pyrimidine (35e) also decreased theP2X7R antagonistic property The para- position prefers a
Subsequent investigations on KN-62 involved extensivemodifications in three positions 1) R1 (the amino-terminus of
1514 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
small electron-withdrawing group eg fluorine (35f) and theantagonistic property decreases as the size of the electron-withdrawing group increases (35g 35h) A methyl- or nitro-group at the para- position (35i 35j) was beneficial foractivity but not evidenced with the methoxy (35k ) group As inthe case for para- substitution introduction of F Cl or CH3 butnot a methoxy (35l-o) at the ortho- position was well toleratedMoreover the meta- position could also tolerate a Cl or CF3group (35p 35q) The disubstituted compound 35r showedreduced antagonistic properties (IC50 = 1122 nM) possibly dueto the steric bulkiness of om-xylyl residue while the dichloroderivative 35s (IC50 = 34 nM) retained activity A slightdecrease in potency was observed when the methyl group on thenitrogen on the α-position of the tyrosine was removed (35n vs
35t) further confirming that this is not substantial for P2X7Rantagonism The most potent compound arising from this studywas 35f with an IC50 value of 13 nM at inhibiting calciuminflux in human monocytes compared to KN-62 which has anIC50 of 51 nM [139] It is therefore confirmed that thephenylpiperazine group of KN-62 is crucial for interaction withthe active site of the receptor and can be potentially improvedwith small halogen substitutions particularly in the paraposition
An ortho-methyl substituent on the N-phenyl ring of KN-62increased the potency by 3-fold (36 IC50 15 nM) [140]Radiolabelling of compound 36 with tritium and preliminarybinding studies to the P2X7R have also been achieved [140]
Table 2 Structures and P2X7R Antagonistic Properties of Selected KN-62 Derivatives [137138]
34a isoquinoline-5-SO2mdash isoquinoline-5-SO2mdash Bocmdash H 77 plusmn 24 ~40
34b quinoline-8-SO2mdash quinoline-8-SO2mdash Bocmdash H 61 plusmn 30 ND
34c Cbzmdash quinoline-8-SO2mdash Bocmdash H 53 plusmn 23 ~200
34d Cbzmdash PhSO2mdash Bocmdash H 59 plusmn 14 ND
34e Cbzmdash p-tolyl-SO2mdash Bocmdash H 71 plusmn 30 ~300
34f Cbzmdash p-OMe-PhSO2mdash Bocmdash H 62 plusmn 15 ND
34g Cbzmdash isoquinoline-5-SO2mdash Bocmdash H 43 ND
34h Cbzmdash benzoylmdash Bocmdash H 47 plusmn 21 ND
34i isoquinoline-5-SO2mdash isoquinoline-5-SO2mdash H H 14 plusmn 12 ND
34j quinoline-8-SO2mdash quinoline-8-SO2mdash H H 5 plusmn 9 ND
34k Cbzmdash PhSO2mdash Cbzmdash H 48 plusmn 29 ND
34l Cbzmdash PhSO2mdash benzoylmdash H 78 plusmn 22 ND
34m Cbzmdash isoquinoline-5-SO2mdash benzoylmdash H 85 plusmn 10 ~200
34n Cbzmdash PhSO2mdash p-tolyl-SO2mdash H 0 ND
34o Cbzmdash PhSO2mdash CH3SO2mdash H 0 ND
34p Cbzmdash PhSO2mdash PhSO2mdash H 0 ND
34q Cbzmdash PhSO2mdash p-OMe-PhSO2mdash H 0 ND
34r Cbzmdash p-tolyl-SO2mdash benzoylmdash H 71 plusmn 21 ND
34s p-NO2-Cbzmdash p-tolyl-SO2mdash benzoylmdash H 69 plusmn 25 ND
34t m-NO2-Cbzmdash p-tolyl-SO2mdash benzoylmdash H 72 plusmn 6 ND
34u m-acetamido-Cbzmdash p-tolyl-SO2mdash benzoylmdash H 9 plusmn 13 ND
34v m-isothiocyanato-Cbzmdash p-tolyl-SO2mdash benzoylmdash H minus4 plusmn 5 ND
34w Cbzmdash m-NO2-PhSO2mdash benzoylmdash H 75 plusmn 10 ND
34x Cbzmdash p-NO2-PhSO2mdash benzoylmdash H 78 plusmn 18 ~100
34y m-NO2-Cbzmdash p-tolyl-SO2mdash Bocmdash H 94 plusmn 28 ~100
34z Cbzmdash p-tolyl-SO2mdash p-NH2-benzoylmdash H 93 plusmn 3 gt 300
aAntagonist function was measured by the percent inhibition of the K+ release triggered by 3mM ATP in paired wells in the absence of antagonist bAntagonistic IC50values derived from dose-response curves obtained on the basis of inhibition of K+ release
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1515
R2 X N
O
N
O
S
R1
S
O
O
N
O
O
Nthe inhibition of BzATP-induced ethidium bromide uptakemediated by the P2X7R expressed endogenously on the humanpre-monocytic cell line THP-1 [142] Compound 37a (Table 4)was identified as the lead structure and modifications on theimide and 4-pyridyl moieties were investigated The cyclicimide moiety is proven essential for the P2X7R activity sincesubstitutions with simple amines acyclic amides or acycliccarbamates almost completely abolished the activity [142] Thering can be five- or six-membered in the forms of imides (37a-bh) carbamates (37c-d) thiazolidine-24-diones (37eg) andhydantoins (37f) Replacement of the 4-pyridyl moiety with 4-substituted benzamides (37g-h) retained the P2X7R activityThe presence of a 3rsquo-nitro substituent on the biphenyl subunitwas reported to convey optimum P2X7R activity The mode ofinhibition of these compounds seemed to be competitivealthough there were observable changes in the maximalresponse as the compound concentration was increased [142]
Fig (16) General structure for the KN-62 derivatives (35a-35s)
Unfortunately while these KN-62 analogues are potentiallyuseful tools to investigate the basic properties of the P2X7Rthey do not represent good starting points for the discovery oforally-active drugs due to 1) high molecular weight (gt700 Da)2) high lipophilicity (clogP gt 6) and 3) presence of ametabolically labile yet crucial sulfonate group [141]
These compounds represent a new class of ligands thatpossess activity to the P2X7R They appear good candidates asorally active drugs due to their relatively low molecular weight(~400 Da) and would be highly amenable for further SARstudies However despite the succinimides and thiazolidine-24-diones representing potent analogues they were found to bemetabolically and chemically fragile On the other handalthough the carbamates were less potent they displayedimproved stability Future SAR studies could usefully involveinvestigation of the biphenyl ring and the chain linker
37 Cyclic Imides and Analogues
Cyclic imides 37 (Fig 18) were discovered through a high-throughput screening of AstraZenecarsquos compound library for
Table 3 Structures and P2X7R Antagonistic Properties of Selected KN-62 Derivatives [139]
Entry R1 R2 X IC50 (nM)a
KN-62 mdashCH3 mdashPh N 511 plusmn 11
35a mdashCH3 mdashCH2Ph N 211 plusmn 08
35b mdashCH3 mdashCH2CH2Ph N 600 plusmn 27
35c mdashCH3 mdashCH2Ph CH 653 plusmn 08
35d mdashCH3 2-pyridinyl N 170 plusmn 39
35e mdashCH3 2-pyrimidinyl N 798 plusmn 07
35f mdashCH3 p-F-Phmdash N 13 plusmn 01
35g mdashCH3 p-Cl-Phmdash N 105 plusmn 11
35h mdashCH3 p-I-Phmdash N 176 plusmn 38
35i mdashCH3 p-Me-Phmdash N 135 plusmn 02
35j mdashCH3 p-NO2-Phmdash N 58 plusmn 06
35k mdashCH3 p-OMe-Phmdash N 132 plusmn 43
35l mdashCH3 o-F-Phmdash N 185 plusmn 04
35m mdashCH3 o-Cl-Phmdash N 158 plusmn 09
35n mdashCH3 o-Me-Phmdash N 151 plusmn 14
35o mdashCH3 o-OMe-Phmdash N 841 plusmn 5
35p mdashCH3 m-Cl-Phmdash N 141 plusmn 03
35q mdashCH3 m-CF3-Phmdash N 311 plusmn 12
35r mdashCH3 om-diMe-Phmdash N 1122 plusmn 25
35s mdashCH3 mp-diCl-Phmdash N 339 plusmn 08
35t H o-Me-Phmdash N 288 plusmn 03
aAntagonistic IC50 (nM)a values derived from dose-response curves on the basis of inhibition of Ca2+ flux in human monocytes
1516 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
N N
O
N
S
N
OO
OS O
O
N
R
31 R = H (KN-62)
36 R = 2-[3H]Me
AstraZeneca reported that no replacement for the adamantaneframework was found [141] Investigation of the chain linkerrequirements between the adamantane and the phenyl ring wasperformed where it was found that C- and N- methylationcarbonyl reduction and methylene removal (38d-g) all causedloss of potency One methylene unit extension (38h ) retainedthe potency whereas reversal of the amide connectivity (38i)slightly decreased the potency AstraZeneca prepared acombinatorial library by reacting commercially availableadamantanemethylamine with a large variety of acid chloridesor via standard amide coupling with carboxylic acids Thegeneral trends indicated that only aromatic amides had activityat the P2X7R [141] On the phenyl ring 23- and 25-disubstitutions resulted in better antagonists (38j-o) than the26- (38p ) 35- (38q ) or 24-disusbstitutions (38r) The ortho-position could also tolerate substituents such as Br OMe andMe (38s-u) A closer examination on the reverse amides (38i v-y) also revealed that 23- and 25-disubsitutions were stillpreferred for P2X7R inhibition The indazole amide (38z) wasidentified as a target lead molecule with a good metabolicprofile suitable for further optimization for clinical trial studies[141]
Fig (17) Radiolabeled analogue of KN-62 [140]
O
NY
X
O
Z
NO2
Adamantyl amides represent a class of simple moleculeswith excellent activity at the P2X7R The nature of theadamantane group could potentially be further investigatedalthough the original authors stated that no replacement for theadamantane group was found A number of patents onadamantyl-containing benzamides have been generated byAstraZeneca [143-146] and Pfizer [147] indicating the potentialof this ligand class for generating a truly potent selective andorally active P2X7R inhibitor Pfizer specifically incorporatedtriazinyl group into the adamantyl benzamide framework [147]Fig (18) General structure for the cyclic imides generated by
AstraZeneca Non-adamantane benzamides including piperidinyl andpiperazinyl benzamides [148 149] triazinyl benzamides [150-153] quinolinyl benzamides [154 155] 34-difluoroanilinebenzamides [156] aromatic acid-containing benzamides [157]pyrazolylmethyl benzamides [158] and other heteroarylamides[159-163] have also been patented by various otherpharmaceutical companies
38 Adamantyl Amides
Adamantyl amides were also discovered from a high-throughput screening of AstraZenecarsquos compound library forinhibition of the P2X7R-mediated ethidium bromide uptakeinduced by BzATP [141] N-Adamantan-1-ylmethyl-2-chloro-benzamide 38a was identified as the lead structure with anexcellent pA2 value of 81 The presence of an ortho-substituentis believed to cause a twist in the orientation of the benzamideessential for potent P2X7R antagonism whereas para-substituents (38bc) have deleterious effects on the P2X7Ractivity Selected compounds of this series are represented inTable 5
39 Diarylimidazolines
Diarylimidazolines represent another class of P2X7-activecompounds They were first discovered through a high-throughput screening of Aventisrsquo compound library forinhibition of BzATP-induced YO-PRO-1 uptake mediated byP2X7R expressed in U373 cells stably expressing the human
Table 4 Structures and P2X7R Antagonistic Properties of Selected Cyclic Imides [142]
Entry X Y Z IC50 ( M) pA2a
37a CH2 CO N - 69
37b (CH2)2 CO N - 75
37c O CH2 N 64
37d O (CH2)2 N 063 -
37e S CO N - 77
37f NH CO N 02 -
37g S CO C-CONHMe 016 -
37h CH2 CO C-CONMe2 025 -
a pA2 = the negative logarithm of a molar concentration of an antagonist that necessitates doubling of agonist concentration needed to elicit the original submaximalresponse obtained in the absence of antagonist
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1517
P2X7R [164] Compound 39a was identified as an initial hitfrom the screening with an IC50 value of 80 nM
equipotent Some degree of branching in the chain linker wasgenerally tolerated (39f-h) Configurational constraintsimposed on the chain linker as exemplified by 39i weretolerated but this was not the case for the cyclopropyl analogue39j
X
RO
NH
N
HN
38a-y 38z
Subsequent investigations focused on the substitutionpattern in the aryl ring for the benzyl (n=1) and phenethyl (n=2)series [164] Electron withdrawing and small neutral groupswere generally tolerable in both series (39k-r v-x) Howeverelectron-donating groups exemplified by OMe gave much lessactive compounds (39s-u) In the phenethyl seriessubstitutions at the ortho- and meta- positions were preferableto the corresponding para- substitutions (39kl vs 39m and39no vs 39p) Interestingly in the benzyl series meta- andpara- substitutions resulted in more active compounds than thecorresponding ortho analogues (39v vs 39wx)
Fig (19) Adamantyl amides generated by AstraZeneca
Investigation of the nature of the linker between thearomatic ring and the 2-position of the imidazole ring revealedthat a 3 or 4 carbon linker (39bc) caused a slight decrease in theactivity and no carbon linker (39d ) rendered the compoundinactive [164] One carbon linker with a methyl branching unit(39e) was tolerated It was reported that there was noenantiomeric preference of the methyl branch they were
The diarylimidazolines examined represent good candidatesfor orally active P2X7R inhibitors they are small and highlyamenable for SAR studies The 4- and 5-phenyl rings representattractive sites for further investigations
Table 5 Structures and P2X7R Antagonistic Properties of Selected Adamantyl Amides [141]
Entry R X pA2
38a 2-Cl CH2NHCO 81
38b 4-Cl CH2NHCO inactive
38c 24-Cl2 CH2NHCO 64
38d 2-Cl CHMeNHCO 64
38e 2-Cl CH2NMeCO 54
38f 2-Cl CH2NHCH2 54
38g 2-Cl NHCO inactive
38h 2-Cl (CH2)2NHCO 78
38i 2-Cl CH2CONH 63
38j 23-Cl2 CH2NHCO 88
38k 25-Cl2 CH2NHCO 83
38l 2-Cl-3-NH2 CH2NHCO 82
38m 2-Cl-5-OMe CH2NHCO 88
38n 2-Cl-5-OH CH2NHCO 80
38o 2-Cl-5-NH2 CH2NHCO 84
38p 26-Cl2 CH2NHCO 74
38q 35-Cl2 CH2NHCO 54
38r 24-Cl2 CH2NHCO 64
38s 2-Br CH2NHCO 80
38t 2-OMe CH2NHCO 71
38u 2-Me CH2NHCO 79
38v 2-Me CH2CONH 68
38w 2-Cl-5-OMe CH2CONH 72
38x 2-Me-3-OMe CH2CONH 83
38y 2-Me-5-OMe CH2CONH 80
1518 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
N
HN
XY
R
N
HN
N
HN
N
HN
( )n
39i39j
39a-ce-hk-w 39d
Fig (20) General structure of the diarylimidazolines generated by Aventis
Table 6 Structures and P2X7R Antagonistic Properties of Selected Diarylimidazolines [164]
Entry n X Y R IC50 ( M)a
39a 1 H H H 008
39b 2 H H H 054
39c 3 H H H 061
39e 0 (R)-Me H H 011
39f 1 (S)-Me H H 003
39g 1 Me Me H 001
39h 1 Ph H H 032
39k 1 H H 2-F 016
39l 1 H H 3-F 004
39m 1 H H 4-F 022
39n 1 H H 2-Cl 007
39o 1 H H 3-Cl 012
39p 1 H H 4-Cl gt1
39q 1 H H 2-Me 007
39r 1 H H 4-Me 219
39s 1 H H 2-OMe 083
39t 1 H H 3-OMe gt3
39u 1 H H 4-OMe gt3
39v 0 H H 2-F 058
39w 0 H H 3-F 008
39x 0 H H 4-F 008
aAntagonistic IC50 values derived from dose-response curves on the basis of inhibition of YO-PRO-1 dye uptake
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1519
R1N
N
NN
R2
R1
NN
NN
R2
N
N
NN
Cl
Cl
Cl
Cl
40b-t 40u-x40a
1
2
3
4
5
Fig (21) General structure of tetrazoles generated by Abbott Laboratories
310 1-Benzyl-5-Phenyltetrazoles potency The limited solubility of those compounds howeverposed a challenge to incorporate substitutions that couldincrease the solubility while maintaining activity A 3-pyridylgroup (40d ) was found to overcome this problem Moreover anextra 2-methyl substituent (40e) resulted in higher activitycompared to 40d A methylene extension (40f) caused a decreasein potency whereas other heterocycles such as 40g and 40hresulted in analogues with similar potency to 40d
1-Benzyl-5-phenyltetrazoles were discovered through ahigh-throughput screening by Abbott Laboratories for threedifferent P2X7R activities Ca2+ flux at the rat or human P2X7RYO-PRO-1 uptake and interleukin-1β release at human P2X7R[165-168] The initial hit from the screening was the tetrazole40a (Table 7) with a pIC50 value of 69 in inhibiting P2X7Rderived from the recombinant human cell line
Investigation of the nature of the substituent attached to the5-position of the tetrazole core showed that the substitutionpattern at this position was not well tolerated [165] Forexample unsubstituted mono-chlorosubstituted 25- and 34-
Initial SAR studies focused on the substituent attached tothe 1-position of the tetrazole core [165] Unsubstituted orortho-substituted benzylic group (40bc) exhibited good
Table 7 Structures and P2X7R Antagonistic Properties of Selected 1-benzyl-5-tetrazoles [165]
1520 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
disubstituted compounds (40i-m) pyridine and quinoline ring(40n-p ) analogues were not tolerated However moreconservative changes such as CF3 and F for Cl group (40q-t)resulted in an increase of potency
release and amplification of astrocytic Ca2+ signaling werereported to be due to the P2X7R and not Cx43 hemichannels[170]
Decavanadate a type of polymer of vanadium oxides hasbeen demonstrated to be a competitive rapid-acting antagonistat the P2X7R [172] It is capable of inhibiting the BzATP-stimulated ethidium bromide uptake in human mouse and ratP2X7R with pA2 values of 74 57 and 71 respectivelyDecavanadate has not been fully tested for its selectivity overother P2X receptors however it is suggested that this largemolecular species acts at the extracellular ATP binding site ofthe receptor consistent with the competitive mode of inhibition[172] A monoclonal antibody specific for the human P2X7Rhas also been shown to be a potent and selective antagonist ofthe receptor with an IC50 value of 5 nM in blocking the BzATP-evoked currents [173] Interestingly tenidap at 50 ndash 200 microMconcentration increases the sensitivity of mouse macrophagesto ATP-mediated cytotoxicity [174] 17-β-estradiol and notprogesterone or 17-α-estradiol has also been reported to blockthe agonist-induced currents at the human P2X7R with an IC50value of 3 microM in a non-genomic manner [175] Polymyxin B apotent antibiotic well-known for its lipopolysaccharide-neutralizing effects has been demonstrated to confer asynergistic effect when co-applied with ATP in a similar mannerto tenidap inducing Ca2+ influx ethidium bromide uptake andlactate dehydrogenase release via action at the P2X7R [176]
Tetrazoles with reversed connectivity (40u-x) were alsoevaluated where it was found that this connectivity reversal hadlittle effect on the potency [165] These tetrazoles were reportedto be slightly more active in the human P2X7R than the ratanalogue [165]
The tetrazole derivative 40d has been shown to be selectivefor other P2 receptors including P2X3 P2X4 and P2Y2 receptors[165] Pharmacological testing of this tetrazole suggested acompetitive mode of inhibition at the P2X7R and its efficacyhas also been evaluated in a model of neuropathic pain in rats[165] Overall these tetrazoles are found to be P2X7R-active andhighly adaptable to structural modifications These propertiesmake the tetrazoles an interesting class of compound for furtherinvestigating P2X7R activity
311 Other P2X7R-Active Ligands
Chelerythrine 41 (Fig 22) is a member of the benzophen-anthridine alkaloids known to inhibit protein kinase C withgood potency and selectivity (IC50 066 microM) as well as enzymessuch as alanine aminotransferase and Na+K+-ATPase [169] At10 microM concentrations chelerythrine non-competitivelyinhibited the BzATP-induced 86Rb+ efflux by 74 in human B-lymphocytes [169] Dye uptake studies could not be performeddue to the fluorescent nature of the compound It is yet to bedetermined whether chelerythrine inhibition of P2X7R involvesprotein kinase C [169]
More recently other distinct classes of P2X7-active ligandshave been reported and patented by the Abbott Laboratoriesacylhydrazides [177] cyanoamidines and cyanoguanidines[178 179]
4 CONCLUSIONS AND FUTURE DIRECTIONS
O
O
N
O
O
Cl
41
Although many structurally diverse class of compoundshave been reported as P2X7R antagonists with reasonablepotency the selectivity issue over other P2 receptors andproteins such as ecto-ATPases and ectonucleotidases stillprevails This is particularly an issue for oxidized ATP andsuramin SAR studies performed on PPADS and suramin mainlygave improved potency and selectivity for the P2X1 receptorIon channel blockers such as amilorides and stilbenederivatives only possess relatively low potency at the P2X7RFurther complications in studying these P2X7R antagonistsalso arise from the demonstrated species differences acrosshuman rat and mouse systems Brilliant Blue G is active for allthree species however at present there have been no SAR datareported Another fundamental factor that hampers the study ofP2X7R in detail is the relatively low potency and selectivity ofthe available agonist BzATP The discovery of a more potentselective P2X7R agonist would be ideal since in many studiesfull dose-response curves could not be generated due to the low
Fig (22) Chemical structure for chelerythrine an alkaloid withantagonistic properties at the human P2X7R
Gap junction channel blockers such as flufenamic acid 42 carbenoxolone 43 and mefloquine 44 (Fig 23) were all foundto be inhibitors of the BzATP-induced Ca2+ flux (IC50 values of655 175 and 25 nM respectively) and of YO-PRO-1 uptake in1321N1 astrocytoma cells stably expressing rat P2X7R [170]This finding poses a question to the general use of gap junctionchannel blockers as indicators of the participation of connexinhemichannels at the sites of transmitter release [171] The ATP
HN CF3
O OH
42 f lufenamic acid
HO
-O
O
O
H
OH
O
O-
N
HN
CF3
CF3
H O
H
43 carbenoxolone 44 mefloquine
Fig (23) Chemical structures for gap junction channel blockers with P2X7R antagonistic properties
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1521
potency of BzATP The nature of functional assays on theP2X7R is also found to be complex as ionic compositiontemperature and cell origins all affect the results demandingextra care be taken when antagonist potencies are directlycompared from different studies
[23] Li J Liu D Ke H Z Duncan R L Turner C H J Biol Chem2005 280 42952-42959
[24] Penolazzi L Bianchini E Lambertini E Baraldi P G RomagnoliR Piva R Gambari R J Biomed Sci 2005 12 1013-1020
[25] Gibbons S J Washburn K B Talamo B R J Auton Pharmacol2001 21 181-190
[26] Dehaye J P Moran A Marino A Arch Oral Biol 1999 44 S39-S43Four classes of small molecules with high affinity at the
P2X7R have been reported cyclic imides adamantyl amidesdiarylimidazolines and 1-benzyl-5-phenyltetrazoles Theseclasses of compounds are highly amenable to structuralmodifications indicating their good prospects as leadmolecules for further improving the potency and selectivity atP2X7R ultimately leading to orally active drugs To date norational drug design based on the knowledge of binding siteswithin this receptor has been undertaken This is a direct resultof the fact that no crystal structure or suitable homology modelhave been reported for this receptor The fact that the P2X7R canadopt either a channel or pore state causes even moredifficulties in studying receptor dynamics and at present theexact mechanism of channel-to-pore transition remains to beelucidated Undoubtedly the P2X7R is involved in severalprocesses relevant to chronic inflammation neurodegenerationand neuropathic pain and therefore discovery of more potentselective P2X7R antagonists would have a great impact in thepresent therapeutic window
[27] Li Q Luo X Zeng W Muallem S J Biol Chem 2003 27847554-47561
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ACKNOWLEDGEMENTS[38] Khakh B S Burnstock G Kennedy C King B F North A
Seguela P Voigt M Humphrey P P A Nat Rev Neurosci 20012 165-174
The authors would like to thank Dr Natasha Hungerford forproofreading the manuscript
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[137] Ravi R G Kertesy S B Dubyak G R Jacobson K A Drug DevRes 2001 54 75-87[99] Jacobson K A Kim Y-C Wildman S S Mohanram A Harden T
K Boyer J L King B F Burnstock G J Med Chem 1998 41 2201-2206
[138] Chen W Ravi R G Kertesy S B Dubyak G R Jacobson K ABioconjug Chem 2002 13 1100-1111
[100] Kim Y-C Brown S G Harden T K Boyer J L Dubyak GKing B F Burnstock G Jacobson K A J Med Chem 2001 44 340-349
[139] Baraldi P G Nunez M D C A M Falzoni S Di Virgilio FRomagnoli R J Med Chem 2003 46 1318-1329
[140] Romagnoli R Baraldi P G Pavani M G Tabrizi M A MoormanA R Di Virgilio F Cattabriga E Pancaldi C Gessi S Borea P ABioorg Med Chem Lett 2004 14 5709-5712
[101] Brown S G Kim Y-C Kim S-A Jacobson K A Burnstock GKing B F Drug Dev Res 2001 53 281-291
[102] Lambrecht G Rettinger J Baumert H G Czeche S Damer SGanso M Hildebrandt C Niebel B Spatz-Kumbel G SchmalzingG Mutschler E Eur J Pharmacol 2000 387 R19-R21
[141] Baxter A Bent J Bowers K Braddock M Brough S Fagura MLawson M McInally T Mortimore M Robertson M Weaver RWebborn P Bioorg Med Chem Lett 2003 13 4047-4050
[103] Kim Y-C Lee J-S Sak K Marteau F Mamedova L BoeynaemsJ-M Jacobson K A Biochem Pharmacol 2005 70 266-274
[142] Alcaraz L Baxter A Bent J Bowers K Braddock M CladingboelD Donald D Fagura M Furber M Laurent C Lawson M
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1523
Mortimore M McCormick M Roberts N Robertson M BioorgMed Chem Lett 2003 13 4043-4046
[165] Nelson D W Gregg R J Kort M E Perez-Medrano A Voight EA Wang Y Grayson G Namovic M T Donnelly-Roberts D LNiforatos W Honore P Jarvis M F Faltynek C R Carroll W AJ Med Chem 2006 49 3659-3666
[143] Caffrey M Rhonan F Pimm A WO2004074224A1 2004[144] Evans R Rhonan F Thompson T Willis P WO2006059945A1
2006 [166] Perez-Medrano A Nelson D W Carroll W A Kort M E Gregg RJ Voight E A Jarvis M F Kowaluk E A WO2006086229A12006
[145] Rhonan F Martin B Thompson T Tomkinson N Willis PWO2006025783A1 2006
[146] Cladingboel D Rhonan F Willis P WO2005014529A1 2005 [167] Carroll W A Perez-Medrano A Florjancic A S Nelson D WPeddi S Li T Bunnelle E M Hirst G Li B CWO20060052374A1 2006
[147] Duplantier A J EP1310493A1 2003[148] Baxter A Kindon N Pairaudeau G Roberts B Thom S
WO0144213A1 2001 [168] Carroll W A Perez-Medrano A Florjancic A S Nelson D WPeddi S Bunnelle E M Hirst G C Li B WO2005111003A12005
[149] Meghani P Bennion C WO0146200A1 2001[150] Li Z B WO20050288256A1 2005[151] Leonard J A Li Z J Li Z B Urban F J WO20050288288A1
2005[169] Shemon A N Sluyter R Conigrave A D Wiley J S Br J
Pharmacol 2004 142 1015-1019[152] Leonard J A Li Z J Li Z B Urban F J WO2006003513A1
2006[170] Suadicani S O Brosnan C F Scemes E J Neurosci 2006 26
1378-1385[153] Li Z B WO2006003500A1 2006 [171] Saez J C Retamal M A Basilio D Bukauskas F F Bennett M
V L Biochim Biophys Acta 2005 1711 215-224[154] Evans R Eyssade C Rhonan F Martin B Thompson T WillisP WO2004106305A1 2004 [172] Michel A D Xing M Thompson K M Jones C A Humphrey P
P A Eur J Pharmacol 2006 534 19-29[155] Rhonan F Thompson T Willis P WO2005009968A1 2005[156] Furber M Luker T J Mortimore M P Thorne P Meghani P
WO0071529A1 2000[173] Buell G Chessell I P Michel A D Collo G Salazzo M Herren
S Gretener D Grahames C Kaur R Kosco-Vilbois M HHumphrey P P A Blood 1998 92 3521-3528[157] Thompson T Willis P WO2006080884A1 2006
[158] Concepcion A Inoue T Mochizuki Y Muramatsu A Gantner FNakashima K Urbhans K Bacon K B WO2005019182A1 2005
[174] Sanz J M Chiozzi P Di Virgilio F Eur J Pharmacol 1998 35 235-244
[159] Dombroski M A Duplantier A J Subramanyam CWO2004099146A1 2004
[175] Cario-Toumaniantz C Loirand G Ferrier L Pacaud P J Physiol1998 508 659-666
[160] Kelly M G Kincaid J WO2006102588A1 2006 [176] Ferrari D Pizzirani C Adinolfi E Forchap S Sitta B Turchet LFalzoni S Minelli M Baricordi R Di Virgilio F J Immunol 2004173 4652-4660
[161] Kelly M J Kincaid J WO2006102610A2 2006[162] Schwink L Stengelin S Gossel M Bohme T Hessler G Stahl P
Gretzke D WO2004072025A2 2004 [177] Nelson D W Jarvis M F Carroll W A WO2006110516A1 2006[163] Schwink L Stengelin S Boehme T Gossel M Hessler G Stahl P
WO2005070925A1 2005[178] Carroll W A Perez-Medrano A Peddi S Florjancic A S
WO20060025614A1 2006[164] Merriman G H Ma L Shum P McGarry D Volz F Sabol J S
Gross A Zhao Z Rampe D Wang L Wirtz-Brugger F Harris BA Macdonald D Bioorg Med Chem Lett 2005 15 435-438
[179] Carroll W A Perez-Medrano A Jarvis M F Wang Y Peddi SWO20050171195A1 2005
Received February 01 2007 Revised April 04 2007 Accepted April 06 2007
1508 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
N
NN
N
NH2
O
OH(R)(H)RO
OPO
OH
O
POPHO
O
OH
O
O H
O
O
mixture of 2 and 3 e sters
3 BzATP
R =
Fig (3) Chemical structure for BzATP (3) the most potent P2X7R agonist to date
electrophysiological assays (Table 1) The low potency of ATPin activating the P2X7R is one of the main pharmacologicalfeatures of this unique receptor that distinguish it from otherpurinergic receptors Normal cytoplasmic concentrations of ATPin cells range from 5 to 10 mM which is released to theextracellular environment upon cell injury or insult therebyactivating the P2X7R before being quickly hydrolyzed by ecto-ATPases and ectonucleotidases Reduction of the extracellularMg2+andor Ca2+ ion concentration which are thought to beallosteric inhibitors [84] results in the potentiation of ATPeffects at the P2X7R [8] It has also been suggested that thetetrabasic form of ATP 2 is the active species acting at thereceptorrsquos ATP-binding site where the presence of Mg2+ andCa2+ ions chelate ATP4- rendering it inactive [85]
pharmacophore known for P2X7R activity The effects of thesesynthetic ligands on the P2X7R are generally tested in eitherelectrophysiological or dye uptake assays exploiting thereceptorrsquos non-desensitizing property and the ability to form alarge non-selective pore
31 ATP Derivatives
BzATP 3 (Fig 3) is the most potent P2X7R agonist to datewith 10- to 100-fold higher potency compared to ATP inactivating the receptor in a number of different systems [3] Thepotency of BzATP is also known to be species-dependent EC50value of BzATP at the rat P2X7R is 2ndash7 microM in theelectrophysiological assay which is approximately 10- to 30-fold higher than at the human P2X7R [40 88] BzATP also has adifferent EC50 value (007 ndash 025 microM) for activating the ratP2X7R in the YO-PRO-1 uptake assay performed in sucrosebuffer (Table 1) The increased sensitivity of the P2X7R toBzATP compared to the endogenous ligand ATP is one of themain criteria used to define the pharmacology of the P2X7Rsubtype Unfortunately BzATP is not highly specific for theP2X7R subtype other P2X receptors are also activated byBzATP [89] but with either equal or lower potency than ATP[90] Other ATP derivatives such as 2-methylthio-ATP 4 ATP-gamma-S 5 and ADP 6 are all weak agonists of the P2X7R (Fig4) [34 90] The order of potency for P2X7R agonists is BzATP3 gtgt ATP 1 gt 2-Methylthio-ATP 4 gt ATP-γ-S 5 gtgt ADP 6 [34] 2-Methylthio-ATP 4 has an EC50 value of 505 microM against theP2X7R derived from mouse NTW8 microglial cells based on theelectrophysiological studies [60]
Some diadenosine polyphosphates such as P1 P4-diadenosine tetraphosphate were reported to be capable ofactivating the P2X7R-mediated cellular permeabilization of ratmast cells [86] possibly due to their greater resistance tometabolic breakdown and similarity to ATP4- [3] Theendogenous peptide LL-37 an antimicrobial agent causingbacteria cell wall permeabilization derived from the humancathelicidin protein hCAP18 has been demonstrated to induceinterleukin-1β processing and release via the activation of theP2X7R [87]
3 SYNTHETIC LIGANDS FOR P2X7R
Several classes of P2X7R-active synthetic compounds havebeen reported in the literature Unfortunately despite thediscovery of these ligands there is still no general
N
NN
N
NH2
O
OHOH
OPO
O H
O
POPHO
O
OH
O
OH
SMe
4 2-Me thylthio-ATP
N
NN
N
NH2
O
OHOH
OPO
OH
O
POPHS
O
OH
O
OH
5 ATP-γ-S
N
NN
N
N H2
O
OHOH
OPO
OH
O
PHO
O
OH
6 ADP (adenosine 5-diphosphate)
N
NN
N
NH2
O
OO
OPO
O H
O
POPHO
O
OH
O
OH
7 oxidize d ATP
Fig (4) Chemical structures for ATP derivatives that are weak P2X7R agonists
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1509
The 2rsquo 3rsquo-dialdehyde ATP (or oxidized ATP) 7 (Fig 4) is thefirst irreversible inhibitor of the P2X7R expressed in the mousemacrophage cell line J774 [91] Subsequent studies alsoshowed that application of 100 microM oxidized ATP irreversiblyantagonized the rat P2X7R provided the cells were pre-incubated with oxidized ATP for 1-2h indicating that itcovalently modified the receptor [34] Co-application with anagonist did not result in any inhibition There is evidence thatoxidized ATP possesses low affinity for the P2Y receptors [91]however oxidized ATP at 100 microM concentration has beenshown to be very toxic to rat cerebellar granule neurons [92]and is likely to react with other enzymes such as ecto-ATPases[91 93] Interestingly oxidized ATP at 168 microM inhibitedP2X7R-mediated inflammatory pain in arthritic rats [94 95]Due to its non-selective inhibition extra caution is required inthe use of oxidized ATP to define involvement of P2X7R in thepurinergic system [42]
in a non-selective manner [3] It is a relatively potent antagonistat the P2X1-3 P2X5 receptors (IC50 01ndash5 microM) and P2Y1receptor but only weakly active or completely inactive at the ratP2X4 P2X6 P2X7 and some P2Y receptor subtypes [96]Interestingly PPADS is reported to be reasonably active at thehuman P2X4R with IC50 value of 28 microM indicating somespecies dependence [97] PPADS is highly specific for the P2receptors up to 100 microM concentration of PPADS does notinterfere with adenosine receptors muscarinic M1 M2 or M3α1- or α2-adrenoceptors histamine H1 AMPA or NMDA and 5-HT receptors [96]
PPADS has been widely studied in the P2 receptor systemsderived from various cell lines and structure-activityrelationship (SAR) studies have also been reported [98-101]Structural modifications of PPADS to date have involved theinvestigation of substitutions at the 4-aldehyde position 5-phosphate and the azophenyl ring Some of the key PPADSderivatives are represented in Fig (6) It was initially thoughtthat both the aldehyde and phosphate moieties are important forP2 antagonism It was also proposed that the slowlyequilibrating pseudoirreversible mode of inhibition of PPADSseen at the P2X1 receptor was due to Schiff base formationbetween the aldehyde and a specific lysine residue of thereceptor [96] The cyclic phosphates MRS2219 and MRS2220were found to be relatively potent antagonists at the rat P2X1receptor (IC50 6 and 10 microM respectively) with good selectivityover P2X2-4 P2Y1 P2Y2 P2Y4 P2Y6 and adenosine receptors[99] Another PPADS derivative that appears to be a promisingantagonist at the P2X1 receptor is PPNDS which exhibited anIC50 of 15 nM and no interactions detected withectonucleotidases P2Y1 receptor α1-adrenoceptors adenosinehistamine H1 and muscarinic M3 receptors [102] Later studiesreported that replacement of the sulfonyl groups on theazophenyl ring with carboxylates or phosphates furtherincreased the potency and selectivity at the P2X1 and P2X3receptors [101] Most recently PPADS analogues MRS2211 andMRS2603 have been reported to be active at the P2Y13 receptorbeing 45- and 74-fold more active respectively than PPADS[103] Only a limited number of PPADS analogues were testedagainst the recombinant human P2X7R in these studies and forthose it was found that none of them was more potent than iso-
Overall these ATP derivatives have high molecular weightsand could potentially bind to other purinergic receptors due totheir resemblance to the endogenous ligand ATP Thereforethey are unlikely to be suitable for developing an orally activedrug specifically targeting the P2X7R
N
HO
CHO
O P O-
O-
O
9 P5P
N
O P
O
O-
O-HO
CHO
N
N
SO3--O3S
43
21
6
5
8 PPADS
Fig (5) Chemical structures for PPADS and its parent molecule P5P
Fig (7) Chemical structure for DIDS and its derivatives
PPADS which differs from PPADS by the position of thesulfonates [100]
[109] receptor DIDS dihydro-DIDS 17 and SITS 19 (Fig 7) allblocked the ATP-stimulated Ca2+ uptake in rat parotid acinarcells via activation of the P2X7R (IC50 35 ndash 125 microM) [109]DNDS 18 in which the isothiocyanate moieties are replacedwith nitro groups was found to be inactive indicating theimportance of the isothiocyanate for interaction with theP2X7R On the other hand a variety of smaller aromaticisothiocyanate compounds derived from DIDS have been testedagainst the P2X1 receptor and were found to be active with IC50values between 11 and 54 microM [108] Overall DIDS and itsderivatives are inhibitors of both the P2X1 and P2X7R withlower potency for the latter
All the SAR data generated to date on PPADS and itsderivatives demonstrate that structural modifications on thekey functional groups (aldehyde phosphate and azophenyl)increases the potency and selectivity mainly at the P2X1receptor Consequently PPADS itself is used for testing at theP2X7R where it was found to be a potent antagonist only at therat and human receptors (IC50 15 and 90 nM respectively) butnot the mouse receptor (100-fold lower potency) in the YO-PRO-1 uptake assay provided cells are pre-incubated withPPADS for 15-30 min [41] When there was no pre-incubationthe IC50 of PPADS at the rat human and mouse P2X7R decreasedto approximately 50-70 microM in the electrophysiologicalrecordings [34 40] but pre-incubation with PPADS drasticallyimproved the IC50 value to 1 microM at the human P2X7R [88] Theinhibitory action of PPADS at the P2X7R is only partiallyreversible [88] suggesting a pseudo-irreversible mode ofantagonism [104] It has been suggested that PPADS P5P andoxidized ATP may all have a common site of action which islikely to be the ATP binding site [104] Overall it can beconcluded that PPADS per se is a species-dependent P2X7Rantagonist being most potent at the rat and human P2X7R butnot the mouse receptor Derivatives of PPADS reported to dateonly exhibit improved potency and selectivity at the P2X1receptor and not the P2X7R
5-(NN-Hexamethylene)amiloride (or HMA) 20 (Fig 8)belongs to the amilorides which are better known as potassium-sparing diuretics by blocking the Na+ channel as well as beinginhibitors of Na+-H+ counter-transport pathway [112] L-typecalcium channels [113] adenosine A1 receptors [114] andpossibly Na+-HCO3
- co-transport [115] HMA possesses affinityfor the P2X7R but its effects are highly species-dependent At 40microM concentration HMA antagonized P2X7R found in humanlymphocytes as determined from large cation uptake studies[116 117] However up to 100 microM concentration it wasineffective at the rat P2X7R [34] More interestingly theelectrophysiological studies revealed that co-application ofHMA with ATP produced an immediate and maximalaugmentation of inward currents induced by mouse P2X7R [60]This effect was not apparent when HMA was applied alone orwhen the buffer solution contained physiologicalconcentrations of Mg2+ and Ca2+ ions HMA has been reportedto be an irreversible inhibitor at the hP2X7R [88] Otheranalogues of HMA such as MIBA 21 and EIPA 22 also possesssimilar properties to HMA [116] Due to its relatively lowpotency and high species dependence at the P2X7R HMA andprobably other amilorides are unlikely to be good candidatesfor further exploration of the pharmacology of the P2X7R
33 Ion Channel Blockers
44rsquo-Diisothiocyanatostilbene-22rsquo-disulfonic acid (DIDS)16 is an anion channel blocker widely used to study various iontransport mechanisms [105-107] but has also beendemonstrated to be capable of inhibiting both the P2X1 receptor[108] and P2X7R-induced pore formation and other processes inseveral cell lines with relatively low potency [3 109-111] Forexample pre-incubation of BAC12F5 murine macrophage with150 ndash 200 microM DIDS inhibited the activation of phospholipaseD mediated by the P2X7R [110] Pre-incubation of rat primaryastrocytes with 200 microM of DIDS has also been shown to inhibitP2X7R-mediated responses such as the activation ofextracellular signal receptor kinases (ERK) [111] SARinvestigations have been performed on DIDS in order toimprove its potency and selectivity at the P2X1 [108] or P2X7
34 Polysulfonated Dyes
Suramin 23 is an example of a polysulfonated dye thatexhibits non-selective antagonism at both the P2X and P2Yreceptors with relatively low potency [3] Suramin blocks theP2X1-3 and P2X5 receptors (IC50 1-15 microM) but only shows weakor no inhibition at the P2X4 P2X6 and P2X7 receptors as well
N
NCl
N NH 2
O
NH
NH
NH2
20 5-(NN-Hexa methylene )- amiloride (HMA)
N
NCl
NH2
O
NH
NH
NH2
N
21 5-(N-Methyl-N- isobutyl)- amiloride (MIBA)
N
NCl
NH2
O
NH
NH
NH 2
N
22 5-(N-ethyl-N-isopropyl)- amiloride (EIPA)
Fig (8) Chemical structures for HMA and other amilorides
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1511
as some P2Y receptor subtypes [96] In addition suramin hasbeen shown to interact with many different proteins at theseconcentrations including glutamate nicotinic GABA and 5-HTreceptors as well as various proteases and ectonucleotidases [396] Nevertheless SAR studies on suramin have been performedin order to identify the important structural features responsiblefor antagonism at the P2 receptors [96 118-124] Selected keysuramin derivatives are represented in Fig (10 )
native and recombinant P2X1 receptors respectively and hasbeen shown to be much less potent at other P2X and P2Ysubtypes including adrenoceptors histamine H1 receptors andthe muscarinic M3 receptor [120] At the human P2X1 and P2X7receptors the IC50 values for NF449 were 005 nM and 40 microMrespectively [128] which indicated its usefulness todistinguish between the two receptors Subsequent SAR studiesconfirmed that NF449 was the most potent and selective knownP2X1 receptor antagonist [121 129] NF157 an analogue ofsuramin with fluorine replacing the methyl group has also beenreported to be highly potent at the P2Y11 receptor with a Ki of45 nM [124] More recently NF110 has also been shown to be apotent P2X3 receptor antagonist indicating that both thenumber and location of the sulfonate groups may be importantfor determining P2X subtype potency and selectivity [123]
O
HN
HN
NH
NH
O O
O ONH HNSO3-
SO 3-
SO3-
-O3S
SO 3-
-O3S
23 suramin
NF279 thus far represents the most active suramin relatedantagonist at the P2X7R and displays a reasonable potency of28 microM All the SAR data obtained to date on suramindemonstrate the importance of the molecular size number andposition of the sulfonate groups for P2X subtype potency andselectivity Unfortunately except for NF279 and NF449 othersuramin analogues provide very little information which can beused for the selective structure-based drug design of P2X7Rmolecules Suramin is also unlikely to be orally active due toits high molecular weight and interaction with many otherproteins Suramin is a very weak or almost inactive antagonistat the human P2X7R with an IC50 value of 92 microM [40] It istotally inactive at both the rat and mouse P2X7R with IC50values greater than 100 microM [34 60] Due to its interference withthe fluorescence of YO-PRO-1 suramin could not be used in theYO-PRO-1 dye uptake assay [104]
Fig (9) Chemical structure for suramin a weak antagonist at the P2X7R
Early SAR analysis showed that the shortened forms ofsuramin in the form of sulfonylbenzamides decreased thepotency at P2 receptors [118] NF023 (24 ) was later discoveredto be P2X-selective compared to P2Y receptors andectonucleotidases [118] Subsequent studies have examined theeffects of NF023 in various cell types and it was reported thatNF023 is a highly selective antagonist for either rat or humanP2X1 receptors heterologously expressed in Xenopus oocyteswith an IC50 of 02 microM [125] Another suramin derivativeNF279 (25 ) also showed potent selective reversible inhibitionat the rat P2X1 receptor in Xenopus oocytes with an IC50 of 19nM [126] In studying the effects of NF279 on human P2X1 andP2X7 receptors it was reported that NF279 is almost 100-foldmore potent at the human P2X1 than the P2X7 receptor withIC50 values of 005 and 28 microM respectively [127] Other keysuramin derivatives resulting from extensive SAR studies areNF449 (27 ) NF110 (28 ) and NF157 (26 ) [120 121 123 124128 129] NF449 has an IC50 value of 70 nM and 03 nM at the
Brilliant Blue G 29 (Fig 11) is another polysulfonated dyewhich is a potent non-competitive inhibitor of the rat andhuman P2X7R with IC50 values of 10 and 200 nM respectivelyin electrophysiological studies [130] Brilliant Blue G has alsobeen shown to be highly selective for P2X7R over P2X1-5receptors [130] It has been shown that at the rat P2X7R about100 to 300 nM concentration was required to inhibit the YO-PRO-1 uptake which was 10- to 30-fold higher than thatrequired for blocking P2X7R-induced currents demonstratingits differential effect at the channel and pore forms of the P2X7R
O
HN
HN
NH
NH
O O
R R
O ONH HNSO3-
SO3-
SO3-
-O3S
SO3-
-O3S
O
HN
HN
NH
OO
NH
O ONH
NH
R R
RR
n n
Suramin n=1 R=CH3
24 NF023 n=0 R=H
25 NF279 n=1 R=H
26 NF157 n=1 R=F
27 NF449 R=24-disulfonyl28 NF110 R=4-sulfonyl
Fig (10) Suramin and its key derivatives investigated in the SAR studies
1512 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
[130] Brilliant Blue G has also been tested on the mouseP2X7R where similar antagonistic potency was found comparedto rat and human receptors [42] In this study it was alsoreported that the selectivity of Brilliant Blue G for rat overhuman P2X7R was confirmed at 22degC but lost at 37degC Thedrawback for this compound is that it could also be bound toproteins and thus would be expected to have lower potency inthe dye uptake assay than electrophysiology measurements dueto the depletion by cell monolayer effect [42] Nevertheless itrepresents a promising antagonist at rat human and mouseanalogues of the P2X7R No studies have been performed onBrilliant Blue G to further improve its potency and selectivityat the P2X7R Other polysulfonated dyes such as Evans BlueTrypan Blue reactive blue 2 reactive red 2 and their derivativeshave also been tested at the purinergic systems [131-133] butnone of them exhibited activity at the P2X7R
opening of the ion channel calmidazolium produces no effecton the YO-PRO-1 uptake The large non-selective porehowever is still formed even when the ionic currents areinhibited indicating that pore formation does not necessarilyrequire opening of the ion channels Calmidazolium wassuggested to be an allosteric modulator of the P2X7R at theagonist binding site inducing conformational change at thereceptor level that results in decreased agonist binding affinity[84] It displays less potency at inhibiting the agonist-evokedcurrents at both the mouse and human P2X7R with IC50 valuesof 1 microM and 100 nM respectively [41 88] At present no SARstudies have been reported on calmidazolium
N
N+
ClCl
O
ClCl
Cl
Cl
30 calmidazolium
NH
N
O
SO3-
N+
SO3-
Cl
29 Coomassie Brilliant Blue G
Fig (12) Chemical structure for calmidazolium a P2X7R antagonistcapable of distinguishing the channel and pore forms of the receptor
36 KN-62 and its Analogues
KN-62 31 (Fig 13) is a derivative of isoquinolinesul-fonamide known as a selective and potent inhibitor of themultifunctional calciumcalmodulin-dependent protein kinases(CaMK) with an IC50 of 09 microM at CaMK II [134] Gargett andWiley in 1997 first demonstrated that both KN-62 31 and itsanalogue KN-04 32 were potent inhibitors of P2X7R at humanlymphocytes with IC50 values of 13 and 17-37 nM respectivelyin both the ion flux and ethidium bromide uptake assays [134]The mode of inhibition is allosteric modulation similar to thatof calmidazolium
Fig (11) Chemical structure for Brilliant Blue G a potent antagonist at therat P2X7R
35 Calmidazolium
Extracellular calmidazolium 30 (Fig 12) is a potentinhibitor of BzATP-induced currents via binding to rat P2X7Rwith an IC50 of 13 nM [84] The unique feature ofcalmidazolium is its ability to differentiate between the channeland pore form of the P2X7R while it potently inhibits the
KN-62 and KN-04 are species-selective for P2X7Rantagonism They potently inhibited the ATP-induced currentsas well as the ethidium bromide uptake in HEK293 cells
Fig (13) Chemical structures for isoquinolines KN-62 and KN-04 potent P2X7R antagonists at the hP2X7R with much less effect on the rat and humananalogues
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1513
33e most active in the series but still 30-fold less potent than KN-62
Fig (14) Conformationally restricted analogues of KN-62 [136]
expressing human P2X7R at the concentration of 30 ndash 100 nMdepending on whether ATP or BzATP was used and the cellularbackground of the human P2X7R [135] However even up to 3microM concentration they had no effect at the rat P2X7R [135] andKN-62 was found to be at least 10-fold more potent at thehuman P2X7R than the recombinant mouse analogue [60] Theseisoquinolinesulfonamide derivatives may interact with theamino-terminal domain of the P2X7R since the introduction ofthe first 335 amino acids of the human P2X7R delivers KN-62sensitivity to the rat P2X7R and the counter-part chimera isfound to be insensitive to KN-62 [135] The IC50 at the humanP2X7R is 10-fold lower at 22degC than at 37degC in the ethidiumbromide uptake study [42]
tyrosine) 2) R2 (the tyrosine side chain) and 3) R3 (thesubstituent attached to the N-piperazine) (Fig 15) [137-139]
The effect of substitutions at the R1 R2 and R3 groups (Fig15 ) on the P2X7R antagonistic properties was compared on thebasis of inhibition of K+ release [137 138] At the R1 positionlarge hydrophobic moieties linked to the amino positionthrough sulfonamide (34a-b) or carbamate (34c) groups werepreferable for P2X7R inhibition In the series of derivativeswhere R2 = quinolinesulfonyl and R3 = t-butoxycarbonyl (Boc)introduction of carbobenzyloxy (Cbz) and quinolinesulfonylgroups at the R1 position were found to be the most active[137] In another series of derivatives where R1 = Cbz and R3 =Boc introduction of arylsulfonyl (34d-g) and benzoyl (34h )moieties at the R2 position were preferred [137] A free aminogroup at the R3 position (34i-j) andor opening of thepiperazine ring to an ethylene diamine greatly reduced theP2X7R activity Amongst the various acyl groups tested at theR3 position Boc and benzoyl groups (34k-m) were found to bepreferable but not sulfonyl (34n-q ) The presence of a methylgroup at the amino terminus of the tyrosine group is thereforenot an absolute pre-requisite for P2X7 antagonism [137]
R3 N N
O
N
O
R4
R1
R2
In another study undertaken by Chen et al to furtherexplore the SAR of 34m [138] it was found that nitro-substitution at the meta- or para- position of the Cbz group atR1 (34r-t) was well tolerated However introduction of anacetamido- or isothiocyanato- group at the Cbz group abolishedP2X7R antagonism (34uv) The original isoquinoline-5-sulfonyl moiety at the R2 position could be substituted withmeta- or para-substituted phenylsulfonyl groups such as tosyl(34r-t) 3-nitrophenylsulfonyl (34w) or 4-nitrophenylsulfonyl(34x) without adverse effects on the P2X7R antagonism [138]Substitution of the benzoyl group at the R3 position by a Boc(34y) or 4-aminobenzoyl group (34z) retained activity
Fig (15) General structure for the KN-62 derivatives (34a-z)
Extensive SAR studies have been performed on KN-62 inorder to enhance its potency and selectivity at the P2X7R Thefirst attempt to improve the antagonistic activity of KN-62 wasachieved by tethering the N-methyl of the tyrosine backbone tothe ortho-position of the proximal phenyl ring which resultedin a series of conformationally restricted KN-62 analogues withthe formula shown in Fig (14 ) [136] The constrained form ofKN-62 (31 vs 33a) was devoid of P2X7R antagonisticproperties Replacement of the isoquinoline-5-sulfonyl moiety(33a) with quinoline-5-sulfonyl (33b ) quinoline-8-sulfonyl(33c) or naphthalene (33d ) resulted in the loss of ability toinhibit the P2X7R-mediated Ca2+ influx [136] The most activecompound arising from this series was compound (33e) with anIC50 value of 316 nM which is 30-fold weaker than the originalKN-62 Taken together these results indicate that an extendedrather than folded conformation of KN-62 conferred by theincorporation of a methylene unit between the N-methyl of thetyrosine backbone to the ortho-position of the proximal phenylring is preferred at the P2X7R [136]
A study by Baraldi et al reported the systematic SAR profileof KN-62 specifically looking at modifications of thephenylpiperazine moiety (R2 position Fig 16 ) [139] In theinvestigation of the optimum chain linker between thepiperazine and phenyl groups it was found that one methylenespacer (35a) slightly improved the activity whereas a twomethylene spacer was not favourable (35b ) [139] The presenceof a basic nitrogen in the piperazine moiety is proven crucial forP2X7R antagonism as substitution of the piperazine ring withpiperidine (35a vs 35c) resulted in a 3-fold reduction of activity[139] Substitution of the phenyl ring with other heterocyclessuch as pyridine (35d ) or pyrimidine (35e) also decreased theP2X7R antagonistic property The para- position prefers a
Subsequent investigations on KN-62 involved extensivemodifications in three positions 1) R1 (the amino-terminus of
1514 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
small electron-withdrawing group eg fluorine (35f) and theantagonistic property decreases as the size of the electron-withdrawing group increases (35g 35h) A methyl- or nitro-group at the para- position (35i 35j) was beneficial foractivity but not evidenced with the methoxy (35k ) group As inthe case for para- substitution introduction of F Cl or CH3 butnot a methoxy (35l-o) at the ortho- position was well toleratedMoreover the meta- position could also tolerate a Cl or CF3group (35p 35q) The disubstituted compound 35r showedreduced antagonistic properties (IC50 = 1122 nM) possibly dueto the steric bulkiness of om-xylyl residue while the dichloroderivative 35s (IC50 = 34 nM) retained activity A slightdecrease in potency was observed when the methyl group on thenitrogen on the α-position of the tyrosine was removed (35n vs
35t) further confirming that this is not substantial for P2X7Rantagonism The most potent compound arising from this studywas 35f with an IC50 value of 13 nM at inhibiting calciuminflux in human monocytes compared to KN-62 which has anIC50 of 51 nM [139] It is therefore confirmed that thephenylpiperazine group of KN-62 is crucial for interaction withthe active site of the receptor and can be potentially improvedwith small halogen substitutions particularly in the paraposition
An ortho-methyl substituent on the N-phenyl ring of KN-62increased the potency by 3-fold (36 IC50 15 nM) [140]Radiolabelling of compound 36 with tritium and preliminarybinding studies to the P2X7R have also been achieved [140]
Table 2 Structures and P2X7R Antagonistic Properties of Selected KN-62 Derivatives [137138]
34a isoquinoline-5-SO2mdash isoquinoline-5-SO2mdash Bocmdash H 77 plusmn 24 ~40
34b quinoline-8-SO2mdash quinoline-8-SO2mdash Bocmdash H 61 plusmn 30 ND
34c Cbzmdash quinoline-8-SO2mdash Bocmdash H 53 plusmn 23 ~200
34d Cbzmdash PhSO2mdash Bocmdash H 59 plusmn 14 ND
34e Cbzmdash p-tolyl-SO2mdash Bocmdash H 71 plusmn 30 ~300
34f Cbzmdash p-OMe-PhSO2mdash Bocmdash H 62 plusmn 15 ND
34g Cbzmdash isoquinoline-5-SO2mdash Bocmdash H 43 ND
34h Cbzmdash benzoylmdash Bocmdash H 47 plusmn 21 ND
34i isoquinoline-5-SO2mdash isoquinoline-5-SO2mdash H H 14 plusmn 12 ND
34j quinoline-8-SO2mdash quinoline-8-SO2mdash H H 5 plusmn 9 ND
34k Cbzmdash PhSO2mdash Cbzmdash H 48 plusmn 29 ND
34l Cbzmdash PhSO2mdash benzoylmdash H 78 plusmn 22 ND
34m Cbzmdash isoquinoline-5-SO2mdash benzoylmdash H 85 plusmn 10 ~200
34n Cbzmdash PhSO2mdash p-tolyl-SO2mdash H 0 ND
34o Cbzmdash PhSO2mdash CH3SO2mdash H 0 ND
34p Cbzmdash PhSO2mdash PhSO2mdash H 0 ND
34q Cbzmdash PhSO2mdash p-OMe-PhSO2mdash H 0 ND
34r Cbzmdash p-tolyl-SO2mdash benzoylmdash H 71 plusmn 21 ND
34s p-NO2-Cbzmdash p-tolyl-SO2mdash benzoylmdash H 69 plusmn 25 ND
34t m-NO2-Cbzmdash p-tolyl-SO2mdash benzoylmdash H 72 plusmn 6 ND
34u m-acetamido-Cbzmdash p-tolyl-SO2mdash benzoylmdash H 9 plusmn 13 ND
34v m-isothiocyanato-Cbzmdash p-tolyl-SO2mdash benzoylmdash H minus4 plusmn 5 ND
34w Cbzmdash m-NO2-PhSO2mdash benzoylmdash H 75 plusmn 10 ND
34x Cbzmdash p-NO2-PhSO2mdash benzoylmdash H 78 plusmn 18 ~100
34y m-NO2-Cbzmdash p-tolyl-SO2mdash Bocmdash H 94 plusmn 28 ~100
34z Cbzmdash p-tolyl-SO2mdash p-NH2-benzoylmdash H 93 plusmn 3 gt 300
aAntagonist function was measured by the percent inhibition of the K+ release triggered by 3mM ATP in paired wells in the absence of antagonist bAntagonistic IC50values derived from dose-response curves obtained on the basis of inhibition of K+ release
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1515
R2 X N
O
N
O
S
R1
S
O
O
N
O
O
Nthe inhibition of BzATP-induced ethidium bromide uptakemediated by the P2X7R expressed endogenously on the humanpre-monocytic cell line THP-1 [142] Compound 37a (Table 4)was identified as the lead structure and modifications on theimide and 4-pyridyl moieties were investigated The cyclicimide moiety is proven essential for the P2X7R activity sincesubstitutions with simple amines acyclic amides or acycliccarbamates almost completely abolished the activity [142] Thering can be five- or six-membered in the forms of imides (37a-bh) carbamates (37c-d) thiazolidine-24-diones (37eg) andhydantoins (37f) Replacement of the 4-pyridyl moiety with 4-substituted benzamides (37g-h) retained the P2X7R activityThe presence of a 3rsquo-nitro substituent on the biphenyl subunitwas reported to convey optimum P2X7R activity The mode ofinhibition of these compounds seemed to be competitivealthough there were observable changes in the maximalresponse as the compound concentration was increased [142]
Fig (16) General structure for the KN-62 derivatives (35a-35s)
Unfortunately while these KN-62 analogues are potentiallyuseful tools to investigate the basic properties of the P2X7Rthey do not represent good starting points for the discovery oforally-active drugs due to 1) high molecular weight (gt700 Da)2) high lipophilicity (clogP gt 6) and 3) presence of ametabolically labile yet crucial sulfonate group [141]
These compounds represent a new class of ligands thatpossess activity to the P2X7R They appear good candidates asorally active drugs due to their relatively low molecular weight(~400 Da) and would be highly amenable for further SARstudies However despite the succinimides and thiazolidine-24-diones representing potent analogues they were found to bemetabolically and chemically fragile On the other handalthough the carbamates were less potent they displayedimproved stability Future SAR studies could usefully involveinvestigation of the biphenyl ring and the chain linker
37 Cyclic Imides and Analogues
Cyclic imides 37 (Fig 18) were discovered through a high-throughput screening of AstraZenecarsquos compound library for
Table 3 Structures and P2X7R Antagonistic Properties of Selected KN-62 Derivatives [139]
Entry R1 R2 X IC50 (nM)a
KN-62 mdashCH3 mdashPh N 511 plusmn 11
35a mdashCH3 mdashCH2Ph N 211 plusmn 08
35b mdashCH3 mdashCH2CH2Ph N 600 plusmn 27
35c mdashCH3 mdashCH2Ph CH 653 plusmn 08
35d mdashCH3 2-pyridinyl N 170 plusmn 39
35e mdashCH3 2-pyrimidinyl N 798 plusmn 07
35f mdashCH3 p-F-Phmdash N 13 plusmn 01
35g mdashCH3 p-Cl-Phmdash N 105 plusmn 11
35h mdashCH3 p-I-Phmdash N 176 plusmn 38
35i mdashCH3 p-Me-Phmdash N 135 plusmn 02
35j mdashCH3 p-NO2-Phmdash N 58 plusmn 06
35k mdashCH3 p-OMe-Phmdash N 132 plusmn 43
35l mdashCH3 o-F-Phmdash N 185 plusmn 04
35m mdashCH3 o-Cl-Phmdash N 158 plusmn 09
35n mdashCH3 o-Me-Phmdash N 151 plusmn 14
35o mdashCH3 o-OMe-Phmdash N 841 plusmn 5
35p mdashCH3 m-Cl-Phmdash N 141 plusmn 03
35q mdashCH3 m-CF3-Phmdash N 311 plusmn 12
35r mdashCH3 om-diMe-Phmdash N 1122 plusmn 25
35s mdashCH3 mp-diCl-Phmdash N 339 plusmn 08
35t H o-Me-Phmdash N 288 plusmn 03
aAntagonistic IC50 (nM)a values derived from dose-response curves on the basis of inhibition of Ca2+ flux in human monocytes
1516 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
N N
O
N
S
N
OO
OS O
O
N
R
31 R = H (KN-62)
36 R = 2-[3H]Me
AstraZeneca reported that no replacement for the adamantaneframework was found [141] Investigation of the chain linkerrequirements between the adamantane and the phenyl ring wasperformed where it was found that C- and N- methylationcarbonyl reduction and methylene removal (38d-g) all causedloss of potency One methylene unit extension (38h ) retainedthe potency whereas reversal of the amide connectivity (38i)slightly decreased the potency AstraZeneca prepared acombinatorial library by reacting commercially availableadamantanemethylamine with a large variety of acid chloridesor via standard amide coupling with carboxylic acids Thegeneral trends indicated that only aromatic amides had activityat the P2X7R [141] On the phenyl ring 23- and 25-disubstitutions resulted in better antagonists (38j-o) than the26- (38p ) 35- (38q ) or 24-disusbstitutions (38r) The ortho-position could also tolerate substituents such as Br OMe andMe (38s-u) A closer examination on the reverse amides (38i v-y) also revealed that 23- and 25-disubsitutions were stillpreferred for P2X7R inhibition The indazole amide (38z) wasidentified as a target lead molecule with a good metabolicprofile suitable for further optimization for clinical trial studies[141]
Fig (17) Radiolabeled analogue of KN-62 [140]
O
NY
X
O
Z
NO2
Adamantyl amides represent a class of simple moleculeswith excellent activity at the P2X7R The nature of theadamantane group could potentially be further investigatedalthough the original authors stated that no replacement for theadamantane group was found A number of patents onadamantyl-containing benzamides have been generated byAstraZeneca [143-146] and Pfizer [147] indicating the potentialof this ligand class for generating a truly potent selective andorally active P2X7R inhibitor Pfizer specifically incorporatedtriazinyl group into the adamantyl benzamide framework [147]Fig (18) General structure for the cyclic imides generated by
AstraZeneca Non-adamantane benzamides including piperidinyl andpiperazinyl benzamides [148 149] triazinyl benzamides [150-153] quinolinyl benzamides [154 155] 34-difluoroanilinebenzamides [156] aromatic acid-containing benzamides [157]pyrazolylmethyl benzamides [158] and other heteroarylamides[159-163] have also been patented by various otherpharmaceutical companies
38 Adamantyl Amides
Adamantyl amides were also discovered from a high-throughput screening of AstraZenecarsquos compound library forinhibition of the P2X7R-mediated ethidium bromide uptakeinduced by BzATP [141] N-Adamantan-1-ylmethyl-2-chloro-benzamide 38a was identified as the lead structure with anexcellent pA2 value of 81 The presence of an ortho-substituentis believed to cause a twist in the orientation of the benzamideessential for potent P2X7R antagonism whereas para-substituents (38bc) have deleterious effects on the P2X7Ractivity Selected compounds of this series are represented inTable 5
39 Diarylimidazolines
Diarylimidazolines represent another class of P2X7-activecompounds They were first discovered through a high-throughput screening of Aventisrsquo compound library forinhibition of BzATP-induced YO-PRO-1 uptake mediated byP2X7R expressed in U373 cells stably expressing the human
Table 4 Structures and P2X7R Antagonistic Properties of Selected Cyclic Imides [142]
Entry X Y Z IC50 ( M) pA2a
37a CH2 CO N - 69
37b (CH2)2 CO N - 75
37c O CH2 N 64
37d O (CH2)2 N 063 -
37e S CO N - 77
37f NH CO N 02 -
37g S CO C-CONHMe 016 -
37h CH2 CO C-CONMe2 025 -
a pA2 = the negative logarithm of a molar concentration of an antagonist that necessitates doubling of agonist concentration needed to elicit the original submaximalresponse obtained in the absence of antagonist
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1517
P2X7R [164] Compound 39a was identified as an initial hitfrom the screening with an IC50 value of 80 nM
equipotent Some degree of branching in the chain linker wasgenerally tolerated (39f-h) Configurational constraintsimposed on the chain linker as exemplified by 39i weretolerated but this was not the case for the cyclopropyl analogue39j
X
RO
NH
N
HN
38a-y 38z
Subsequent investigations focused on the substitutionpattern in the aryl ring for the benzyl (n=1) and phenethyl (n=2)series [164] Electron withdrawing and small neutral groupswere generally tolerable in both series (39k-r v-x) Howeverelectron-donating groups exemplified by OMe gave much lessactive compounds (39s-u) In the phenethyl seriessubstitutions at the ortho- and meta- positions were preferableto the corresponding para- substitutions (39kl vs 39m and39no vs 39p) Interestingly in the benzyl series meta- andpara- substitutions resulted in more active compounds than thecorresponding ortho analogues (39v vs 39wx)
Fig (19) Adamantyl amides generated by AstraZeneca
Investigation of the nature of the linker between thearomatic ring and the 2-position of the imidazole ring revealedthat a 3 or 4 carbon linker (39bc) caused a slight decrease in theactivity and no carbon linker (39d ) rendered the compoundinactive [164] One carbon linker with a methyl branching unit(39e) was tolerated It was reported that there was noenantiomeric preference of the methyl branch they were
The diarylimidazolines examined represent good candidatesfor orally active P2X7R inhibitors they are small and highlyamenable for SAR studies The 4- and 5-phenyl rings representattractive sites for further investigations
Table 5 Structures and P2X7R Antagonistic Properties of Selected Adamantyl Amides [141]
Entry R X pA2
38a 2-Cl CH2NHCO 81
38b 4-Cl CH2NHCO inactive
38c 24-Cl2 CH2NHCO 64
38d 2-Cl CHMeNHCO 64
38e 2-Cl CH2NMeCO 54
38f 2-Cl CH2NHCH2 54
38g 2-Cl NHCO inactive
38h 2-Cl (CH2)2NHCO 78
38i 2-Cl CH2CONH 63
38j 23-Cl2 CH2NHCO 88
38k 25-Cl2 CH2NHCO 83
38l 2-Cl-3-NH2 CH2NHCO 82
38m 2-Cl-5-OMe CH2NHCO 88
38n 2-Cl-5-OH CH2NHCO 80
38o 2-Cl-5-NH2 CH2NHCO 84
38p 26-Cl2 CH2NHCO 74
38q 35-Cl2 CH2NHCO 54
38r 24-Cl2 CH2NHCO 64
38s 2-Br CH2NHCO 80
38t 2-OMe CH2NHCO 71
38u 2-Me CH2NHCO 79
38v 2-Me CH2CONH 68
38w 2-Cl-5-OMe CH2CONH 72
38x 2-Me-3-OMe CH2CONH 83
38y 2-Me-5-OMe CH2CONH 80
1518 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
N
HN
XY
R
N
HN
N
HN
N
HN
( )n
39i39j
39a-ce-hk-w 39d
Fig (20) General structure of the diarylimidazolines generated by Aventis
Table 6 Structures and P2X7R Antagonistic Properties of Selected Diarylimidazolines [164]
Entry n X Y R IC50 ( M)a
39a 1 H H H 008
39b 2 H H H 054
39c 3 H H H 061
39e 0 (R)-Me H H 011
39f 1 (S)-Me H H 003
39g 1 Me Me H 001
39h 1 Ph H H 032
39k 1 H H 2-F 016
39l 1 H H 3-F 004
39m 1 H H 4-F 022
39n 1 H H 2-Cl 007
39o 1 H H 3-Cl 012
39p 1 H H 4-Cl gt1
39q 1 H H 2-Me 007
39r 1 H H 4-Me 219
39s 1 H H 2-OMe 083
39t 1 H H 3-OMe gt3
39u 1 H H 4-OMe gt3
39v 0 H H 2-F 058
39w 0 H H 3-F 008
39x 0 H H 4-F 008
aAntagonistic IC50 values derived from dose-response curves on the basis of inhibition of YO-PRO-1 dye uptake
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1519
R1N
N
NN
R2
R1
NN
NN
R2
N
N
NN
Cl
Cl
Cl
Cl
40b-t 40u-x40a
1
2
3
4
5
Fig (21) General structure of tetrazoles generated by Abbott Laboratories
310 1-Benzyl-5-Phenyltetrazoles potency The limited solubility of those compounds howeverposed a challenge to incorporate substitutions that couldincrease the solubility while maintaining activity A 3-pyridylgroup (40d ) was found to overcome this problem Moreover anextra 2-methyl substituent (40e) resulted in higher activitycompared to 40d A methylene extension (40f) caused a decreasein potency whereas other heterocycles such as 40g and 40hresulted in analogues with similar potency to 40d
1-Benzyl-5-phenyltetrazoles were discovered through ahigh-throughput screening by Abbott Laboratories for threedifferent P2X7R activities Ca2+ flux at the rat or human P2X7RYO-PRO-1 uptake and interleukin-1β release at human P2X7R[165-168] The initial hit from the screening was the tetrazole40a (Table 7) with a pIC50 value of 69 in inhibiting P2X7Rderived from the recombinant human cell line
Investigation of the nature of the substituent attached to the5-position of the tetrazole core showed that the substitutionpattern at this position was not well tolerated [165] Forexample unsubstituted mono-chlorosubstituted 25- and 34-
Initial SAR studies focused on the substituent attached tothe 1-position of the tetrazole core [165] Unsubstituted orortho-substituted benzylic group (40bc) exhibited good
Table 7 Structures and P2X7R Antagonistic Properties of Selected 1-benzyl-5-tetrazoles [165]
1520 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
disubstituted compounds (40i-m) pyridine and quinoline ring(40n-p ) analogues were not tolerated However moreconservative changes such as CF3 and F for Cl group (40q-t)resulted in an increase of potency
release and amplification of astrocytic Ca2+ signaling werereported to be due to the P2X7R and not Cx43 hemichannels[170]
Decavanadate a type of polymer of vanadium oxides hasbeen demonstrated to be a competitive rapid-acting antagonistat the P2X7R [172] It is capable of inhibiting the BzATP-stimulated ethidium bromide uptake in human mouse and ratP2X7R with pA2 values of 74 57 and 71 respectivelyDecavanadate has not been fully tested for its selectivity overother P2X receptors however it is suggested that this largemolecular species acts at the extracellular ATP binding site ofthe receptor consistent with the competitive mode of inhibition[172] A monoclonal antibody specific for the human P2X7Rhas also been shown to be a potent and selective antagonist ofthe receptor with an IC50 value of 5 nM in blocking the BzATP-evoked currents [173] Interestingly tenidap at 50 ndash 200 microMconcentration increases the sensitivity of mouse macrophagesto ATP-mediated cytotoxicity [174] 17-β-estradiol and notprogesterone or 17-α-estradiol has also been reported to blockthe agonist-induced currents at the human P2X7R with an IC50value of 3 microM in a non-genomic manner [175] Polymyxin B apotent antibiotic well-known for its lipopolysaccharide-neutralizing effects has been demonstrated to confer asynergistic effect when co-applied with ATP in a similar mannerto tenidap inducing Ca2+ influx ethidium bromide uptake andlactate dehydrogenase release via action at the P2X7R [176]
Tetrazoles with reversed connectivity (40u-x) were alsoevaluated where it was found that this connectivity reversal hadlittle effect on the potency [165] These tetrazoles were reportedto be slightly more active in the human P2X7R than the ratanalogue [165]
The tetrazole derivative 40d has been shown to be selectivefor other P2 receptors including P2X3 P2X4 and P2Y2 receptors[165] Pharmacological testing of this tetrazole suggested acompetitive mode of inhibition at the P2X7R and its efficacyhas also been evaluated in a model of neuropathic pain in rats[165] Overall these tetrazoles are found to be P2X7R-active andhighly adaptable to structural modifications These propertiesmake the tetrazoles an interesting class of compound for furtherinvestigating P2X7R activity
311 Other P2X7R-Active Ligands
Chelerythrine 41 (Fig 22) is a member of the benzophen-anthridine alkaloids known to inhibit protein kinase C withgood potency and selectivity (IC50 066 microM) as well as enzymessuch as alanine aminotransferase and Na+K+-ATPase [169] At10 microM concentrations chelerythrine non-competitivelyinhibited the BzATP-induced 86Rb+ efflux by 74 in human B-lymphocytes [169] Dye uptake studies could not be performeddue to the fluorescent nature of the compound It is yet to bedetermined whether chelerythrine inhibition of P2X7R involvesprotein kinase C [169]
More recently other distinct classes of P2X7-active ligandshave been reported and patented by the Abbott Laboratoriesacylhydrazides [177] cyanoamidines and cyanoguanidines[178 179]
4 CONCLUSIONS AND FUTURE DIRECTIONS
O
O
N
O
O
Cl
41
Although many structurally diverse class of compoundshave been reported as P2X7R antagonists with reasonablepotency the selectivity issue over other P2 receptors andproteins such as ecto-ATPases and ectonucleotidases stillprevails This is particularly an issue for oxidized ATP andsuramin SAR studies performed on PPADS and suramin mainlygave improved potency and selectivity for the P2X1 receptorIon channel blockers such as amilorides and stilbenederivatives only possess relatively low potency at the P2X7RFurther complications in studying these P2X7R antagonistsalso arise from the demonstrated species differences acrosshuman rat and mouse systems Brilliant Blue G is active for allthree species however at present there have been no SAR datareported Another fundamental factor that hampers the study ofP2X7R in detail is the relatively low potency and selectivity ofthe available agonist BzATP The discovery of a more potentselective P2X7R agonist would be ideal since in many studiesfull dose-response curves could not be generated due to the low
Fig (22) Chemical structure for chelerythrine an alkaloid withantagonistic properties at the human P2X7R
Gap junction channel blockers such as flufenamic acid 42 carbenoxolone 43 and mefloquine 44 (Fig 23) were all foundto be inhibitors of the BzATP-induced Ca2+ flux (IC50 values of655 175 and 25 nM respectively) and of YO-PRO-1 uptake in1321N1 astrocytoma cells stably expressing rat P2X7R [170]This finding poses a question to the general use of gap junctionchannel blockers as indicators of the participation of connexinhemichannels at the sites of transmitter release [171] The ATP
HN CF3
O OH
42 f lufenamic acid
HO
-O
O
O
H
OH
O
O-
N
HN
CF3
CF3
H O
H
43 carbenoxolone 44 mefloquine
Fig (23) Chemical structures for gap junction channel blockers with P2X7R antagonistic properties
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1521
potency of BzATP The nature of functional assays on theP2X7R is also found to be complex as ionic compositiontemperature and cell origins all affect the results demandingextra care be taken when antagonist potencies are directlycompared from different studies
[23] Li J Liu D Ke H Z Duncan R L Turner C H J Biol Chem2005 280 42952-42959
[24] Penolazzi L Bianchini E Lambertini E Baraldi P G RomagnoliR Piva R Gambari R J Biomed Sci 2005 12 1013-1020
[25] Gibbons S J Washburn K B Talamo B R J Auton Pharmacol2001 21 181-190
[26] Dehaye J P Moran A Marino A Arch Oral Biol 1999 44 S39-S43Four classes of small molecules with high affinity at the
P2X7R have been reported cyclic imides adamantyl amidesdiarylimidazolines and 1-benzyl-5-phenyltetrazoles Theseclasses of compounds are highly amenable to structuralmodifications indicating their good prospects as leadmolecules for further improving the potency and selectivity atP2X7R ultimately leading to orally active drugs To date norational drug design based on the knowledge of binding siteswithin this receptor has been undertaken This is a direct resultof the fact that no crystal structure or suitable homology modelhave been reported for this receptor The fact that the P2X7R canadopt either a channel or pore state causes even moredifficulties in studying receptor dynamics and at present theexact mechanism of channel-to-pore transition remains to beelucidated Undoubtedly the P2X7R is involved in severalprocesses relevant to chronic inflammation neurodegenerationand neuropathic pain and therefore discovery of more potentselective P2X7R antagonists would have a great impact in thepresent therapeutic window
[27] Li Q Luo X Zeng W Muallem S J Biol Chem 2003 27847554-47561
[28] Wang Q Wang L Feng Y-H Li X Zeng R Gorodeski GI AmJ Physiol Cell Physiol 2004 287 C1349-C1358
[29] Bardini M Lee H Y Burnstock G Cell Tissue Res 2000 299 105-113
[30] Hu H Z Gao N Lin Z Gao C Liu S Ren J Xia Y Wood JD J Comp Neurol 2001 440 299-310
[31] Hillman K A Burnstock G Unwin R J Nephron Exp Nephrol2005 101 e24-e30
[32] Koshi R Coutinho-Silva R Cascabulho C M Henrique-Pons AKnight G E Loesch A Burnstock G J Reprod Immunol 200566 127-140
[33] Moores T S Hasdemir B Vega-Riveroll L Deuchars J Parson SH Brain Res 2005 1034 40-50
[34] Surprenant A Rassendren F Kawashima E North R A Buell GScience 1996 272 735-738
[35] Cheewatrakoolpong B Gilchrest H Anthes J C Greenfeder SBiochem Biophys Res Commun 2005 332 17-27
[36] Watters J J Sommer J A Fisette P L Pfeiffer Z A Aga MPrabhu U Guerra A N Denlinger L C Bertics P J Drug Dev Res2001 53 91-104
[37] Adinolfi E Pizzirani C Idzko M Panther E Norgauer J DiVirgilio F Ferrari D Purinergic Signall 2005 1 219-227
ACKNOWLEDGEMENTS[38] Khakh B S Burnstock G Kennedy C King B F North A
Seguela P Voigt M Humphrey P P A Nat Rev Neurosci 20012 165-174
The authors would like to thank Dr Natasha Hungerford forproofreading the manuscript
[39] Roberts J A Vial C Digby H R Agboh K C Wen HAtterbury-Thomas A Evans R J Pflugers Arch - Eur J Physiol2006 452 486-500
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[146] Cladingboel D Rhonan F Willis P WO2005014529A1 2005 [167] Carroll W A Perez-Medrano A Florjancic A S Nelson D WPeddi S Li T Bunnelle E M Hirst G Li B CWO20060052374A1 2006
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Received February 01 2007 Revised April 04 2007 Accepted April 06 2007
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1509
The 2rsquo 3rsquo-dialdehyde ATP (or oxidized ATP) 7 (Fig 4) is thefirst irreversible inhibitor of the P2X7R expressed in the mousemacrophage cell line J774 [91] Subsequent studies alsoshowed that application of 100 microM oxidized ATP irreversiblyantagonized the rat P2X7R provided the cells were pre-incubated with oxidized ATP for 1-2h indicating that itcovalently modified the receptor [34] Co-application with anagonist did not result in any inhibition There is evidence thatoxidized ATP possesses low affinity for the P2Y receptors [91]however oxidized ATP at 100 microM concentration has beenshown to be very toxic to rat cerebellar granule neurons [92]and is likely to react with other enzymes such as ecto-ATPases[91 93] Interestingly oxidized ATP at 168 microM inhibitedP2X7R-mediated inflammatory pain in arthritic rats [94 95]Due to its non-selective inhibition extra caution is required inthe use of oxidized ATP to define involvement of P2X7R in thepurinergic system [42]
in a non-selective manner [3] It is a relatively potent antagonistat the P2X1-3 P2X5 receptors (IC50 01ndash5 microM) and P2Y1receptor but only weakly active or completely inactive at the ratP2X4 P2X6 P2X7 and some P2Y receptor subtypes [96]Interestingly PPADS is reported to be reasonably active at thehuman P2X4R with IC50 value of 28 microM indicating somespecies dependence [97] PPADS is highly specific for the P2receptors up to 100 microM concentration of PPADS does notinterfere with adenosine receptors muscarinic M1 M2 or M3α1- or α2-adrenoceptors histamine H1 AMPA or NMDA and 5-HT receptors [96]
PPADS has been widely studied in the P2 receptor systemsderived from various cell lines and structure-activityrelationship (SAR) studies have also been reported [98-101]Structural modifications of PPADS to date have involved theinvestigation of substitutions at the 4-aldehyde position 5-phosphate and the azophenyl ring Some of the key PPADSderivatives are represented in Fig (6) It was initially thoughtthat both the aldehyde and phosphate moieties are important forP2 antagonism It was also proposed that the slowlyequilibrating pseudoirreversible mode of inhibition of PPADSseen at the P2X1 receptor was due to Schiff base formationbetween the aldehyde and a specific lysine residue of thereceptor [96] The cyclic phosphates MRS2219 and MRS2220were found to be relatively potent antagonists at the rat P2X1receptor (IC50 6 and 10 microM respectively) with good selectivityover P2X2-4 P2Y1 P2Y2 P2Y4 P2Y6 and adenosine receptors[99] Another PPADS derivative that appears to be a promisingantagonist at the P2X1 receptor is PPNDS which exhibited anIC50 of 15 nM and no interactions detected withectonucleotidases P2Y1 receptor α1-adrenoceptors adenosinehistamine H1 and muscarinic M3 receptors [102] Later studiesreported that replacement of the sulfonyl groups on theazophenyl ring with carboxylates or phosphates furtherincreased the potency and selectivity at the P2X1 and P2X3receptors [101] Most recently PPADS analogues MRS2211 andMRS2603 have been reported to be active at the P2Y13 receptorbeing 45- and 74-fold more active respectively than PPADS[103] Only a limited number of PPADS analogues were testedagainst the recombinant human P2X7R in these studies and forthose it was found that none of them was more potent than iso-
Overall these ATP derivatives have high molecular weightsand could potentially bind to other purinergic receptors due totheir resemblance to the endogenous ligand ATP Thereforethey are unlikely to be suitable for developing an orally activedrug specifically targeting the P2X7R
N
HO
CHO
O P O-
O-
O
9 P5P
N
O P
O
O-
O-HO
CHO
N
N
SO3--O3S
43
21
6
5
8 PPADS
Fig (5) Chemical structures for PPADS and its parent molecule P5P
Fig (7) Chemical structure for DIDS and its derivatives
PPADS which differs from PPADS by the position of thesulfonates [100]
[109] receptor DIDS dihydro-DIDS 17 and SITS 19 (Fig 7) allblocked the ATP-stimulated Ca2+ uptake in rat parotid acinarcells via activation of the P2X7R (IC50 35 ndash 125 microM) [109]DNDS 18 in which the isothiocyanate moieties are replacedwith nitro groups was found to be inactive indicating theimportance of the isothiocyanate for interaction with theP2X7R On the other hand a variety of smaller aromaticisothiocyanate compounds derived from DIDS have been testedagainst the P2X1 receptor and were found to be active with IC50values between 11 and 54 microM [108] Overall DIDS and itsderivatives are inhibitors of both the P2X1 and P2X7R withlower potency for the latter
All the SAR data generated to date on PPADS and itsderivatives demonstrate that structural modifications on thekey functional groups (aldehyde phosphate and azophenyl)increases the potency and selectivity mainly at the P2X1receptor Consequently PPADS itself is used for testing at theP2X7R where it was found to be a potent antagonist only at therat and human receptors (IC50 15 and 90 nM respectively) butnot the mouse receptor (100-fold lower potency) in the YO-PRO-1 uptake assay provided cells are pre-incubated withPPADS for 15-30 min [41] When there was no pre-incubationthe IC50 of PPADS at the rat human and mouse P2X7R decreasedto approximately 50-70 microM in the electrophysiologicalrecordings [34 40] but pre-incubation with PPADS drasticallyimproved the IC50 value to 1 microM at the human P2X7R [88] Theinhibitory action of PPADS at the P2X7R is only partiallyreversible [88] suggesting a pseudo-irreversible mode ofantagonism [104] It has been suggested that PPADS P5P andoxidized ATP may all have a common site of action which islikely to be the ATP binding site [104] Overall it can beconcluded that PPADS per se is a species-dependent P2X7Rantagonist being most potent at the rat and human P2X7R butnot the mouse receptor Derivatives of PPADS reported to dateonly exhibit improved potency and selectivity at the P2X1receptor and not the P2X7R
5-(NN-Hexamethylene)amiloride (or HMA) 20 (Fig 8)belongs to the amilorides which are better known as potassium-sparing diuretics by blocking the Na+ channel as well as beinginhibitors of Na+-H+ counter-transport pathway [112] L-typecalcium channels [113] adenosine A1 receptors [114] andpossibly Na+-HCO3
- co-transport [115] HMA possesses affinityfor the P2X7R but its effects are highly species-dependent At 40microM concentration HMA antagonized P2X7R found in humanlymphocytes as determined from large cation uptake studies[116 117] However up to 100 microM concentration it wasineffective at the rat P2X7R [34] More interestingly theelectrophysiological studies revealed that co-application ofHMA with ATP produced an immediate and maximalaugmentation of inward currents induced by mouse P2X7R [60]This effect was not apparent when HMA was applied alone orwhen the buffer solution contained physiologicalconcentrations of Mg2+ and Ca2+ ions HMA has been reportedto be an irreversible inhibitor at the hP2X7R [88] Otheranalogues of HMA such as MIBA 21 and EIPA 22 also possesssimilar properties to HMA [116] Due to its relatively lowpotency and high species dependence at the P2X7R HMA andprobably other amilorides are unlikely to be good candidatesfor further exploration of the pharmacology of the P2X7R
33 Ion Channel Blockers
44rsquo-Diisothiocyanatostilbene-22rsquo-disulfonic acid (DIDS)16 is an anion channel blocker widely used to study various iontransport mechanisms [105-107] but has also beendemonstrated to be capable of inhibiting both the P2X1 receptor[108] and P2X7R-induced pore formation and other processes inseveral cell lines with relatively low potency [3 109-111] Forexample pre-incubation of BAC12F5 murine macrophage with150 ndash 200 microM DIDS inhibited the activation of phospholipaseD mediated by the P2X7R [110] Pre-incubation of rat primaryastrocytes with 200 microM of DIDS has also been shown to inhibitP2X7R-mediated responses such as the activation ofextracellular signal receptor kinases (ERK) [111] SARinvestigations have been performed on DIDS in order toimprove its potency and selectivity at the P2X1 [108] or P2X7
34 Polysulfonated Dyes
Suramin 23 is an example of a polysulfonated dye thatexhibits non-selective antagonism at both the P2X and P2Yreceptors with relatively low potency [3] Suramin blocks theP2X1-3 and P2X5 receptors (IC50 1-15 microM) but only shows weakor no inhibition at the P2X4 P2X6 and P2X7 receptors as well
N
NCl
N NH 2
O
NH
NH
NH2
20 5-(NN-Hexa methylene )- amiloride (HMA)
N
NCl
NH2
O
NH
NH
NH2
N
21 5-(N-Methyl-N- isobutyl)- amiloride (MIBA)
N
NCl
NH2
O
NH
NH
NH 2
N
22 5-(N-ethyl-N-isopropyl)- amiloride (EIPA)
Fig (8) Chemical structures for HMA and other amilorides
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1511
as some P2Y receptor subtypes [96] In addition suramin hasbeen shown to interact with many different proteins at theseconcentrations including glutamate nicotinic GABA and 5-HTreceptors as well as various proteases and ectonucleotidases [396] Nevertheless SAR studies on suramin have been performedin order to identify the important structural features responsiblefor antagonism at the P2 receptors [96 118-124] Selected keysuramin derivatives are represented in Fig (10 )
native and recombinant P2X1 receptors respectively and hasbeen shown to be much less potent at other P2X and P2Ysubtypes including adrenoceptors histamine H1 receptors andthe muscarinic M3 receptor [120] At the human P2X1 and P2X7receptors the IC50 values for NF449 were 005 nM and 40 microMrespectively [128] which indicated its usefulness todistinguish between the two receptors Subsequent SAR studiesconfirmed that NF449 was the most potent and selective knownP2X1 receptor antagonist [121 129] NF157 an analogue ofsuramin with fluorine replacing the methyl group has also beenreported to be highly potent at the P2Y11 receptor with a Ki of45 nM [124] More recently NF110 has also been shown to be apotent P2X3 receptor antagonist indicating that both thenumber and location of the sulfonate groups may be importantfor determining P2X subtype potency and selectivity [123]
O
HN
HN
NH
NH
O O
O ONH HNSO3-
SO 3-
SO3-
-O3S
SO 3-
-O3S
23 suramin
NF279 thus far represents the most active suramin relatedantagonist at the P2X7R and displays a reasonable potency of28 microM All the SAR data obtained to date on suramindemonstrate the importance of the molecular size number andposition of the sulfonate groups for P2X subtype potency andselectivity Unfortunately except for NF279 and NF449 othersuramin analogues provide very little information which can beused for the selective structure-based drug design of P2X7Rmolecules Suramin is also unlikely to be orally active due toits high molecular weight and interaction with many otherproteins Suramin is a very weak or almost inactive antagonistat the human P2X7R with an IC50 value of 92 microM [40] It istotally inactive at both the rat and mouse P2X7R with IC50values greater than 100 microM [34 60] Due to its interference withthe fluorescence of YO-PRO-1 suramin could not be used in theYO-PRO-1 dye uptake assay [104]
Fig (9) Chemical structure for suramin a weak antagonist at the P2X7R
Early SAR analysis showed that the shortened forms ofsuramin in the form of sulfonylbenzamides decreased thepotency at P2 receptors [118] NF023 (24 ) was later discoveredto be P2X-selective compared to P2Y receptors andectonucleotidases [118] Subsequent studies have examined theeffects of NF023 in various cell types and it was reported thatNF023 is a highly selective antagonist for either rat or humanP2X1 receptors heterologously expressed in Xenopus oocyteswith an IC50 of 02 microM [125] Another suramin derivativeNF279 (25 ) also showed potent selective reversible inhibitionat the rat P2X1 receptor in Xenopus oocytes with an IC50 of 19nM [126] In studying the effects of NF279 on human P2X1 andP2X7 receptors it was reported that NF279 is almost 100-foldmore potent at the human P2X1 than the P2X7 receptor withIC50 values of 005 and 28 microM respectively [127] Other keysuramin derivatives resulting from extensive SAR studies areNF449 (27 ) NF110 (28 ) and NF157 (26 ) [120 121 123 124128 129] NF449 has an IC50 value of 70 nM and 03 nM at the
Brilliant Blue G 29 (Fig 11) is another polysulfonated dyewhich is a potent non-competitive inhibitor of the rat andhuman P2X7R with IC50 values of 10 and 200 nM respectivelyin electrophysiological studies [130] Brilliant Blue G has alsobeen shown to be highly selective for P2X7R over P2X1-5receptors [130] It has been shown that at the rat P2X7R about100 to 300 nM concentration was required to inhibit the YO-PRO-1 uptake which was 10- to 30-fold higher than thatrequired for blocking P2X7R-induced currents demonstratingits differential effect at the channel and pore forms of the P2X7R
O
HN
HN
NH
NH
O O
R R
O ONH HNSO3-
SO3-
SO3-
-O3S
SO3-
-O3S
O
HN
HN
NH
OO
NH
O ONH
NH
R R
RR
n n
Suramin n=1 R=CH3
24 NF023 n=0 R=H
25 NF279 n=1 R=H
26 NF157 n=1 R=F
27 NF449 R=24-disulfonyl28 NF110 R=4-sulfonyl
Fig (10) Suramin and its key derivatives investigated in the SAR studies
1512 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
[130] Brilliant Blue G has also been tested on the mouseP2X7R where similar antagonistic potency was found comparedto rat and human receptors [42] In this study it was alsoreported that the selectivity of Brilliant Blue G for rat overhuman P2X7R was confirmed at 22degC but lost at 37degC Thedrawback for this compound is that it could also be bound toproteins and thus would be expected to have lower potency inthe dye uptake assay than electrophysiology measurements dueto the depletion by cell monolayer effect [42] Nevertheless itrepresents a promising antagonist at rat human and mouseanalogues of the P2X7R No studies have been performed onBrilliant Blue G to further improve its potency and selectivityat the P2X7R Other polysulfonated dyes such as Evans BlueTrypan Blue reactive blue 2 reactive red 2 and their derivativeshave also been tested at the purinergic systems [131-133] butnone of them exhibited activity at the P2X7R
opening of the ion channel calmidazolium produces no effecton the YO-PRO-1 uptake The large non-selective porehowever is still formed even when the ionic currents areinhibited indicating that pore formation does not necessarilyrequire opening of the ion channels Calmidazolium wassuggested to be an allosteric modulator of the P2X7R at theagonist binding site inducing conformational change at thereceptor level that results in decreased agonist binding affinity[84] It displays less potency at inhibiting the agonist-evokedcurrents at both the mouse and human P2X7R with IC50 valuesof 1 microM and 100 nM respectively [41 88] At present no SARstudies have been reported on calmidazolium
N
N+
ClCl
O
ClCl
Cl
Cl
30 calmidazolium
NH
N
O
SO3-
N+
SO3-
Cl
29 Coomassie Brilliant Blue G
Fig (12) Chemical structure for calmidazolium a P2X7R antagonistcapable of distinguishing the channel and pore forms of the receptor
36 KN-62 and its Analogues
KN-62 31 (Fig 13) is a derivative of isoquinolinesul-fonamide known as a selective and potent inhibitor of themultifunctional calciumcalmodulin-dependent protein kinases(CaMK) with an IC50 of 09 microM at CaMK II [134] Gargett andWiley in 1997 first demonstrated that both KN-62 31 and itsanalogue KN-04 32 were potent inhibitors of P2X7R at humanlymphocytes with IC50 values of 13 and 17-37 nM respectivelyin both the ion flux and ethidium bromide uptake assays [134]The mode of inhibition is allosteric modulation similar to thatof calmidazolium
Fig (11) Chemical structure for Brilliant Blue G a potent antagonist at therat P2X7R
35 Calmidazolium
Extracellular calmidazolium 30 (Fig 12) is a potentinhibitor of BzATP-induced currents via binding to rat P2X7Rwith an IC50 of 13 nM [84] The unique feature ofcalmidazolium is its ability to differentiate between the channeland pore form of the P2X7R while it potently inhibits the
KN-62 and KN-04 are species-selective for P2X7Rantagonism They potently inhibited the ATP-induced currentsas well as the ethidium bromide uptake in HEK293 cells
Fig (13) Chemical structures for isoquinolines KN-62 and KN-04 potent P2X7R antagonists at the hP2X7R with much less effect on the rat and humananalogues
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1513
33e most active in the series but still 30-fold less potent than KN-62
Fig (14) Conformationally restricted analogues of KN-62 [136]
expressing human P2X7R at the concentration of 30 ndash 100 nMdepending on whether ATP or BzATP was used and the cellularbackground of the human P2X7R [135] However even up to 3microM concentration they had no effect at the rat P2X7R [135] andKN-62 was found to be at least 10-fold more potent at thehuman P2X7R than the recombinant mouse analogue [60] Theseisoquinolinesulfonamide derivatives may interact with theamino-terminal domain of the P2X7R since the introduction ofthe first 335 amino acids of the human P2X7R delivers KN-62sensitivity to the rat P2X7R and the counter-part chimera isfound to be insensitive to KN-62 [135] The IC50 at the humanP2X7R is 10-fold lower at 22degC than at 37degC in the ethidiumbromide uptake study [42]
tyrosine) 2) R2 (the tyrosine side chain) and 3) R3 (thesubstituent attached to the N-piperazine) (Fig 15) [137-139]
The effect of substitutions at the R1 R2 and R3 groups (Fig15 ) on the P2X7R antagonistic properties was compared on thebasis of inhibition of K+ release [137 138] At the R1 positionlarge hydrophobic moieties linked to the amino positionthrough sulfonamide (34a-b) or carbamate (34c) groups werepreferable for P2X7R inhibition In the series of derivativeswhere R2 = quinolinesulfonyl and R3 = t-butoxycarbonyl (Boc)introduction of carbobenzyloxy (Cbz) and quinolinesulfonylgroups at the R1 position were found to be the most active[137] In another series of derivatives where R1 = Cbz and R3 =Boc introduction of arylsulfonyl (34d-g) and benzoyl (34h )moieties at the R2 position were preferred [137] A free aminogroup at the R3 position (34i-j) andor opening of thepiperazine ring to an ethylene diamine greatly reduced theP2X7R activity Amongst the various acyl groups tested at theR3 position Boc and benzoyl groups (34k-m) were found to bepreferable but not sulfonyl (34n-q ) The presence of a methylgroup at the amino terminus of the tyrosine group is thereforenot an absolute pre-requisite for P2X7 antagonism [137]
R3 N N
O
N
O
R4
R1
R2
In another study undertaken by Chen et al to furtherexplore the SAR of 34m [138] it was found that nitro-substitution at the meta- or para- position of the Cbz group atR1 (34r-t) was well tolerated However introduction of anacetamido- or isothiocyanato- group at the Cbz group abolishedP2X7R antagonism (34uv) The original isoquinoline-5-sulfonyl moiety at the R2 position could be substituted withmeta- or para-substituted phenylsulfonyl groups such as tosyl(34r-t) 3-nitrophenylsulfonyl (34w) or 4-nitrophenylsulfonyl(34x) without adverse effects on the P2X7R antagonism [138]Substitution of the benzoyl group at the R3 position by a Boc(34y) or 4-aminobenzoyl group (34z) retained activity
Fig (15) General structure for the KN-62 derivatives (34a-z)
Extensive SAR studies have been performed on KN-62 inorder to enhance its potency and selectivity at the P2X7R Thefirst attempt to improve the antagonistic activity of KN-62 wasachieved by tethering the N-methyl of the tyrosine backbone tothe ortho-position of the proximal phenyl ring which resultedin a series of conformationally restricted KN-62 analogues withthe formula shown in Fig (14 ) [136] The constrained form ofKN-62 (31 vs 33a) was devoid of P2X7R antagonisticproperties Replacement of the isoquinoline-5-sulfonyl moiety(33a) with quinoline-5-sulfonyl (33b ) quinoline-8-sulfonyl(33c) or naphthalene (33d ) resulted in the loss of ability toinhibit the P2X7R-mediated Ca2+ influx [136] The most activecompound arising from this series was compound (33e) with anIC50 value of 316 nM which is 30-fold weaker than the originalKN-62 Taken together these results indicate that an extendedrather than folded conformation of KN-62 conferred by theincorporation of a methylene unit between the N-methyl of thetyrosine backbone to the ortho-position of the proximal phenylring is preferred at the P2X7R [136]
A study by Baraldi et al reported the systematic SAR profileof KN-62 specifically looking at modifications of thephenylpiperazine moiety (R2 position Fig 16 ) [139] In theinvestigation of the optimum chain linker between thepiperazine and phenyl groups it was found that one methylenespacer (35a) slightly improved the activity whereas a twomethylene spacer was not favourable (35b ) [139] The presenceof a basic nitrogen in the piperazine moiety is proven crucial forP2X7R antagonism as substitution of the piperazine ring withpiperidine (35a vs 35c) resulted in a 3-fold reduction of activity[139] Substitution of the phenyl ring with other heterocyclessuch as pyridine (35d ) or pyrimidine (35e) also decreased theP2X7R antagonistic property The para- position prefers a
Subsequent investigations on KN-62 involved extensivemodifications in three positions 1) R1 (the amino-terminus of
1514 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
small electron-withdrawing group eg fluorine (35f) and theantagonistic property decreases as the size of the electron-withdrawing group increases (35g 35h) A methyl- or nitro-group at the para- position (35i 35j) was beneficial foractivity but not evidenced with the methoxy (35k ) group As inthe case for para- substitution introduction of F Cl or CH3 butnot a methoxy (35l-o) at the ortho- position was well toleratedMoreover the meta- position could also tolerate a Cl or CF3group (35p 35q) The disubstituted compound 35r showedreduced antagonistic properties (IC50 = 1122 nM) possibly dueto the steric bulkiness of om-xylyl residue while the dichloroderivative 35s (IC50 = 34 nM) retained activity A slightdecrease in potency was observed when the methyl group on thenitrogen on the α-position of the tyrosine was removed (35n vs
35t) further confirming that this is not substantial for P2X7Rantagonism The most potent compound arising from this studywas 35f with an IC50 value of 13 nM at inhibiting calciuminflux in human monocytes compared to KN-62 which has anIC50 of 51 nM [139] It is therefore confirmed that thephenylpiperazine group of KN-62 is crucial for interaction withthe active site of the receptor and can be potentially improvedwith small halogen substitutions particularly in the paraposition
An ortho-methyl substituent on the N-phenyl ring of KN-62increased the potency by 3-fold (36 IC50 15 nM) [140]Radiolabelling of compound 36 with tritium and preliminarybinding studies to the P2X7R have also been achieved [140]
Table 2 Structures and P2X7R Antagonistic Properties of Selected KN-62 Derivatives [137138]
34a isoquinoline-5-SO2mdash isoquinoline-5-SO2mdash Bocmdash H 77 plusmn 24 ~40
34b quinoline-8-SO2mdash quinoline-8-SO2mdash Bocmdash H 61 plusmn 30 ND
34c Cbzmdash quinoline-8-SO2mdash Bocmdash H 53 plusmn 23 ~200
34d Cbzmdash PhSO2mdash Bocmdash H 59 plusmn 14 ND
34e Cbzmdash p-tolyl-SO2mdash Bocmdash H 71 plusmn 30 ~300
34f Cbzmdash p-OMe-PhSO2mdash Bocmdash H 62 plusmn 15 ND
34g Cbzmdash isoquinoline-5-SO2mdash Bocmdash H 43 ND
34h Cbzmdash benzoylmdash Bocmdash H 47 plusmn 21 ND
34i isoquinoline-5-SO2mdash isoquinoline-5-SO2mdash H H 14 plusmn 12 ND
34j quinoline-8-SO2mdash quinoline-8-SO2mdash H H 5 plusmn 9 ND
34k Cbzmdash PhSO2mdash Cbzmdash H 48 plusmn 29 ND
34l Cbzmdash PhSO2mdash benzoylmdash H 78 plusmn 22 ND
34m Cbzmdash isoquinoline-5-SO2mdash benzoylmdash H 85 plusmn 10 ~200
34n Cbzmdash PhSO2mdash p-tolyl-SO2mdash H 0 ND
34o Cbzmdash PhSO2mdash CH3SO2mdash H 0 ND
34p Cbzmdash PhSO2mdash PhSO2mdash H 0 ND
34q Cbzmdash PhSO2mdash p-OMe-PhSO2mdash H 0 ND
34r Cbzmdash p-tolyl-SO2mdash benzoylmdash H 71 plusmn 21 ND
34s p-NO2-Cbzmdash p-tolyl-SO2mdash benzoylmdash H 69 plusmn 25 ND
34t m-NO2-Cbzmdash p-tolyl-SO2mdash benzoylmdash H 72 plusmn 6 ND
34u m-acetamido-Cbzmdash p-tolyl-SO2mdash benzoylmdash H 9 plusmn 13 ND
34v m-isothiocyanato-Cbzmdash p-tolyl-SO2mdash benzoylmdash H minus4 plusmn 5 ND
34w Cbzmdash m-NO2-PhSO2mdash benzoylmdash H 75 plusmn 10 ND
34x Cbzmdash p-NO2-PhSO2mdash benzoylmdash H 78 plusmn 18 ~100
34y m-NO2-Cbzmdash p-tolyl-SO2mdash Bocmdash H 94 plusmn 28 ~100
34z Cbzmdash p-tolyl-SO2mdash p-NH2-benzoylmdash H 93 plusmn 3 gt 300
aAntagonist function was measured by the percent inhibition of the K+ release triggered by 3mM ATP in paired wells in the absence of antagonist bAntagonistic IC50values derived from dose-response curves obtained on the basis of inhibition of K+ release
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1515
R2 X N
O
N
O
S
R1
S
O
O
N
O
O
Nthe inhibition of BzATP-induced ethidium bromide uptakemediated by the P2X7R expressed endogenously on the humanpre-monocytic cell line THP-1 [142] Compound 37a (Table 4)was identified as the lead structure and modifications on theimide and 4-pyridyl moieties were investigated The cyclicimide moiety is proven essential for the P2X7R activity sincesubstitutions with simple amines acyclic amides or acycliccarbamates almost completely abolished the activity [142] Thering can be five- or six-membered in the forms of imides (37a-bh) carbamates (37c-d) thiazolidine-24-diones (37eg) andhydantoins (37f) Replacement of the 4-pyridyl moiety with 4-substituted benzamides (37g-h) retained the P2X7R activityThe presence of a 3rsquo-nitro substituent on the biphenyl subunitwas reported to convey optimum P2X7R activity The mode ofinhibition of these compounds seemed to be competitivealthough there were observable changes in the maximalresponse as the compound concentration was increased [142]
Fig (16) General structure for the KN-62 derivatives (35a-35s)
Unfortunately while these KN-62 analogues are potentiallyuseful tools to investigate the basic properties of the P2X7Rthey do not represent good starting points for the discovery oforally-active drugs due to 1) high molecular weight (gt700 Da)2) high lipophilicity (clogP gt 6) and 3) presence of ametabolically labile yet crucial sulfonate group [141]
These compounds represent a new class of ligands thatpossess activity to the P2X7R They appear good candidates asorally active drugs due to their relatively low molecular weight(~400 Da) and would be highly amenable for further SARstudies However despite the succinimides and thiazolidine-24-diones representing potent analogues they were found to bemetabolically and chemically fragile On the other handalthough the carbamates were less potent they displayedimproved stability Future SAR studies could usefully involveinvestigation of the biphenyl ring and the chain linker
37 Cyclic Imides and Analogues
Cyclic imides 37 (Fig 18) were discovered through a high-throughput screening of AstraZenecarsquos compound library for
Table 3 Structures and P2X7R Antagonistic Properties of Selected KN-62 Derivatives [139]
Entry R1 R2 X IC50 (nM)a
KN-62 mdashCH3 mdashPh N 511 plusmn 11
35a mdashCH3 mdashCH2Ph N 211 plusmn 08
35b mdashCH3 mdashCH2CH2Ph N 600 plusmn 27
35c mdashCH3 mdashCH2Ph CH 653 plusmn 08
35d mdashCH3 2-pyridinyl N 170 plusmn 39
35e mdashCH3 2-pyrimidinyl N 798 plusmn 07
35f mdashCH3 p-F-Phmdash N 13 plusmn 01
35g mdashCH3 p-Cl-Phmdash N 105 plusmn 11
35h mdashCH3 p-I-Phmdash N 176 plusmn 38
35i mdashCH3 p-Me-Phmdash N 135 plusmn 02
35j mdashCH3 p-NO2-Phmdash N 58 plusmn 06
35k mdashCH3 p-OMe-Phmdash N 132 plusmn 43
35l mdashCH3 o-F-Phmdash N 185 plusmn 04
35m mdashCH3 o-Cl-Phmdash N 158 plusmn 09
35n mdashCH3 o-Me-Phmdash N 151 plusmn 14
35o mdashCH3 o-OMe-Phmdash N 841 plusmn 5
35p mdashCH3 m-Cl-Phmdash N 141 plusmn 03
35q mdashCH3 m-CF3-Phmdash N 311 plusmn 12
35r mdashCH3 om-diMe-Phmdash N 1122 plusmn 25
35s mdashCH3 mp-diCl-Phmdash N 339 plusmn 08
35t H o-Me-Phmdash N 288 plusmn 03
aAntagonistic IC50 (nM)a values derived from dose-response curves on the basis of inhibition of Ca2+ flux in human monocytes
1516 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
N N
O
N
S
N
OO
OS O
O
N
R
31 R = H (KN-62)
36 R = 2-[3H]Me
AstraZeneca reported that no replacement for the adamantaneframework was found [141] Investigation of the chain linkerrequirements between the adamantane and the phenyl ring wasperformed where it was found that C- and N- methylationcarbonyl reduction and methylene removal (38d-g) all causedloss of potency One methylene unit extension (38h ) retainedthe potency whereas reversal of the amide connectivity (38i)slightly decreased the potency AstraZeneca prepared acombinatorial library by reacting commercially availableadamantanemethylamine with a large variety of acid chloridesor via standard amide coupling with carboxylic acids Thegeneral trends indicated that only aromatic amides had activityat the P2X7R [141] On the phenyl ring 23- and 25-disubstitutions resulted in better antagonists (38j-o) than the26- (38p ) 35- (38q ) or 24-disusbstitutions (38r) The ortho-position could also tolerate substituents such as Br OMe andMe (38s-u) A closer examination on the reverse amides (38i v-y) also revealed that 23- and 25-disubsitutions were stillpreferred for P2X7R inhibition The indazole amide (38z) wasidentified as a target lead molecule with a good metabolicprofile suitable for further optimization for clinical trial studies[141]
Fig (17) Radiolabeled analogue of KN-62 [140]
O
NY
X
O
Z
NO2
Adamantyl amides represent a class of simple moleculeswith excellent activity at the P2X7R The nature of theadamantane group could potentially be further investigatedalthough the original authors stated that no replacement for theadamantane group was found A number of patents onadamantyl-containing benzamides have been generated byAstraZeneca [143-146] and Pfizer [147] indicating the potentialof this ligand class for generating a truly potent selective andorally active P2X7R inhibitor Pfizer specifically incorporatedtriazinyl group into the adamantyl benzamide framework [147]Fig (18) General structure for the cyclic imides generated by
AstraZeneca Non-adamantane benzamides including piperidinyl andpiperazinyl benzamides [148 149] triazinyl benzamides [150-153] quinolinyl benzamides [154 155] 34-difluoroanilinebenzamides [156] aromatic acid-containing benzamides [157]pyrazolylmethyl benzamides [158] and other heteroarylamides[159-163] have also been patented by various otherpharmaceutical companies
38 Adamantyl Amides
Adamantyl amides were also discovered from a high-throughput screening of AstraZenecarsquos compound library forinhibition of the P2X7R-mediated ethidium bromide uptakeinduced by BzATP [141] N-Adamantan-1-ylmethyl-2-chloro-benzamide 38a was identified as the lead structure with anexcellent pA2 value of 81 The presence of an ortho-substituentis believed to cause a twist in the orientation of the benzamideessential for potent P2X7R antagonism whereas para-substituents (38bc) have deleterious effects on the P2X7Ractivity Selected compounds of this series are represented inTable 5
39 Diarylimidazolines
Diarylimidazolines represent another class of P2X7-activecompounds They were first discovered through a high-throughput screening of Aventisrsquo compound library forinhibition of BzATP-induced YO-PRO-1 uptake mediated byP2X7R expressed in U373 cells stably expressing the human
Table 4 Structures and P2X7R Antagonistic Properties of Selected Cyclic Imides [142]
Entry X Y Z IC50 ( M) pA2a
37a CH2 CO N - 69
37b (CH2)2 CO N - 75
37c O CH2 N 64
37d O (CH2)2 N 063 -
37e S CO N - 77
37f NH CO N 02 -
37g S CO C-CONHMe 016 -
37h CH2 CO C-CONMe2 025 -
a pA2 = the negative logarithm of a molar concentration of an antagonist that necessitates doubling of agonist concentration needed to elicit the original submaximalresponse obtained in the absence of antagonist
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1517
P2X7R [164] Compound 39a was identified as an initial hitfrom the screening with an IC50 value of 80 nM
equipotent Some degree of branching in the chain linker wasgenerally tolerated (39f-h) Configurational constraintsimposed on the chain linker as exemplified by 39i weretolerated but this was not the case for the cyclopropyl analogue39j
X
RO
NH
N
HN
38a-y 38z
Subsequent investigations focused on the substitutionpattern in the aryl ring for the benzyl (n=1) and phenethyl (n=2)series [164] Electron withdrawing and small neutral groupswere generally tolerable in both series (39k-r v-x) Howeverelectron-donating groups exemplified by OMe gave much lessactive compounds (39s-u) In the phenethyl seriessubstitutions at the ortho- and meta- positions were preferableto the corresponding para- substitutions (39kl vs 39m and39no vs 39p) Interestingly in the benzyl series meta- andpara- substitutions resulted in more active compounds than thecorresponding ortho analogues (39v vs 39wx)
Fig (19) Adamantyl amides generated by AstraZeneca
Investigation of the nature of the linker between thearomatic ring and the 2-position of the imidazole ring revealedthat a 3 or 4 carbon linker (39bc) caused a slight decrease in theactivity and no carbon linker (39d ) rendered the compoundinactive [164] One carbon linker with a methyl branching unit(39e) was tolerated It was reported that there was noenantiomeric preference of the methyl branch they were
The diarylimidazolines examined represent good candidatesfor orally active P2X7R inhibitors they are small and highlyamenable for SAR studies The 4- and 5-phenyl rings representattractive sites for further investigations
Table 5 Structures and P2X7R Antagonistic Properties of Selected Adamantyl Amides [141]
Entry R X pA2
38a 2-Cl CH2NHCO 81
38b 4-Cl CH2NHCO inactive
38c 24-Cl2 CH2NHCO 64
38d 2-Cl CHMeNHCO 64
38e 2-Cl CH2NMeCO 54
38f 2-Cl CH2NHCH2 54
38g 2-Cl NHCO inactive
38h 2-Cl (CH2)2NHCO 78
38i 2-Cl CH2CONH 63
38j 23-Cl2 CH2NHCO 88
38k 25-Cl2 CH2NHCO 83
38l 2-Cl-3-NH2 CH2NHCO 82
38m 2-Cl-5-OMe CH2NHCO 88
38n 2-Cl-5-OH CH2NHCO 80
38o 2-Cl-5-NH2 CH2NHCO 84
38p 26-Cl2 CH2NHCO 74
38q 35-Cl2 CH2NHCO 54
38r 24-Cl2 CH2NHCO 64
38s 2-Br CH2NHCO 80
38t 2-OMe CH2NHCO 71
38u 2-Me CH2NHCO 79
38v 2-Me CH2CONH 68
38w 2-Cl-5-OMe CH2CONH 72
38x 2-Me-3-OMe CH2CONH 83
38y 2-Me-5-OMe CH2CONH 80
1518 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
N
HN
XY
R
N
HN
N
HN
N
HN
( )n
39i39j
39a-ce-hk-w 39d
Fig (20) General structure of the diarylimidazolines generated by Aventis
Table 6 Structures and P2X7R Antagonistic Properties of Selected Diarylimidazolines [164]
Entry n X Y R IC50 ( M)a
39a 1 H H H 008
39b 2 H H H 054
39c 3 H H H 061
39e 0 (R)-Me H H 011
39f 1 (S)-Me H H 003
39g 1 Me Me H 001
39h 1 Ph H H 032
39k 1 H H 2-F 016
39l 1 H H 3-F 004
39m 1 H H 4-F 022
39n 1 H H 2-Cl 007
39o 1 H H 3-Cl 012
39p 1 H H 4-Cl gt1
39q 1 H H 2-Me 007
39r 1 H H 4-Me 219
39s 1 H H 2-OMe 083
39t 1 H H 3-OMe gt3
39u 1 H H 4-OMe gt3
39v 0 H H 2-F 058
39w 0 H H 3-F 008
39x 0 H H 4-F 008
aAntagonistic IC50 values derived from dose-response curves on the basis of inhibition of YO-PRO-1 dye uptake
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1519
R1N
N
NN
R2
R1
NN
NN
R2
N
N
NN
Cl
Cl
Cl
Cl
40b-t 40u-x40a
1
2
3
4
5
Fig (21) General structure of tetrazoles generated by Abbott Laboratories
310 1-Benzyl-5-Phenyltetrazoles potency The limited solubility of those compounds howeverposed a challenge to incorporate substitutions that couldincrease the solubility while maintaining activity A 3-pyridylgroup (40d ) was found to overcome this problem Moreover anextra 2-methyl substituent (40e) resulted in higher activitycompared to 40d A methylene extension (40f) caused a decreasein potency whereas other heterocycles such as 40g and 40hresulted in analogues with similar potency to 40d
1-Benzyl-5-phenyltetrazoles were discovered through ahigh-throughput screening by Abbott Laboratories for threedifferent P2X7R activities Ca2+ flux at the rat or human P2X7RYO-PRO-1 uptake and interleukin-1β release at human P2X7R[165-168] The initial hit from the screening was the tetrazole40a (Table 7) with a pIC50 value of 69 in inhibiting P2X7Rderived from the recombinant human cell line
Investigation of the nature of the substituent attached to the5-position of the tetrazole core showed that the substitutionpattern at this position was not well tolerated [165] Forexample unsubstituted mono-chlorosubstituted 25- and 34-
Initial SAR studies focused on the substituent attached tothe 1-position of the tetrazole core [165] Unsubstituted orortho-substituted benzylic group (40bc) exhibited good
Table 7 Structures and P2X7R Antagonistic Properties of Selected 1-benzyl-5-tetrazoles [165]
1520 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
disubstituted compounds (40i-m) pyridine and quinoline ring(40n-p ) analogues were not tolerated However moreconservative changes such as CF3 and F for Cl group (40q-t)resulted in an increase of potency
release and amplification of astrocytic Ca2+ signaling werereported to be due to the P2X7R and not Cx43 hemichannels[170]
Decavanadate a type of polymer of vanadium oxides hasbeen demonstrated to be a competitive rapid-acting antagonistat the P2X7R [172] It is capable of inhibiting the BzATP-stimulated ethidium bromide uptake in human mouse and ratP2X7R with pA2 values of 74 57 and 71 respectivelyDecavanadate has not been fully tested for its selectivity overother P2X receptors however it is suggested that this largemolecular species acts at the extracellular ATP binding site ofthe receptor consistent with the competitive mode of inhibition[172] A monoclonal antibody specific for the human P2X7Rhas also been shown to be a potent and selective antagonist ofthe receptor with an IC50 value of 5 nM in blocking the BzATP-evoked currents [173] Interestingly tenidap at 50 ndash 200 microMconcentration increases the sensitivity of mouse macrophagesto ATP-mediated cytotoxicity [174] 17-β-estradiol and notprogesterone or 17-α-estradiol has also been reported to blockthe agonist-induced currents at the human P2X7R with an IC50value of 3 microM in a non-genomic manner [175] Polymyxin B apotent antibiotic well-known for its lipopolysaccharide-neutralizing effects has been demonstrated to confer asynergistic effect when co-applied with ATP in a similar mannerto tenidap inducing Ca2+ influx ethidium bromide uptake andlactate dehydrogenase release via action at the P2X7R [176]
Tetrazoles with reversed connectivity (40u-x) were alsoevaluated where it was found that this connectivity reversal hadlittle effect on the potency [165] These tetrazoles were reportedto be slightly more active in the human P2X7R than the ratanalogue [165]
The tetrazole derivative 40d has been shown to be selectivefor other P2 receptors including P2X3 P2X4 and P2Y2 receptors[165] Pharmacological testing of this tetrazole suggested acompetitive mode of inhibition at the P2X7R and its efficacyhas also been evaluated in a model of neuropathic pain in rats[165] Overall these tetrazoles are found to be P2X7R-active andhighly adaptable to structural modifications These propertiesmake the tetrazoles an interesting class of compound for furtherinvestigating P2X7R activity
311 Other P2X7R-Active Ligands
Chelerythrine 41 (Fig 22) is a member of the benzophen-anthridine alkaloids known to inhibit protein kinase C withgood potency and selectivity (IC50 066 microM) as well as enzymessuch as alanine aminotransferase and Na+K+-ATPase [169] At10 microM concentrations chelerythrine non-competitivelyinhibited the BzATP-induced 86Rb+ efflux by 74 in human B-lymphocytes [169] Dye uptake studies could not be performeddue to the fluorescent nature of the compound It is yet to bedetermined whether chelerythrine inhibition of P2X7R involvesprotein kinase C [169]
More recently other distinct classes of P2X7-active ligandshave been reported and patented by the Abbott Laboratoriesacylhydrazides [177] cyanoamidines and cyanoguanidines[178 179]
4 CONCLUSIONS AND FUTURE DIRECTIONS
O
O
N
O
O
Cl
41
Although many structurally diverse class of compoundshave been reported as P2X7R antagonists with reasonablepotency the selectivity issue over other P2 receptors andproteins such as ecto-ATPases and ectonucleotidases stillprevails This is particularly an issue for oxidized ATP andsuramin SAR studies performed on PPADS and suramin mainlygave improved potency and selectivity for the P2X1 receptorIon channel blockers such as amilorides and stilbenederivatives only possess relatively low potency at the P2X7RFurther complications in studying these P2X7R antagonistsalso arise from the demonstrated species differences acrosshuman rat and mouse systems Brilliant Blue G is active for allthree species however at present there have been no SAR datareported Another fundamental factor that hampers the study ofP2X7R in detail is the relatively low potency and selectivity ofthe available agonist BzATP The discovery of a more potentselective P2X7R agonist would be ideal since in many studiesfull dose-response curves could not be generated due to the low
Fig (22) Chemical structure for chelerythrine an alkaloid withantagonistic properties at the human P2X7R
Gap junction channel blockers such as flufenamic acid 42 carbenoxolone 43 and mefloquine 44 (Fig 23) were all foundto be inhibitors of the BzATP-induced Ca2+ flux (IC50 values of655 175 and 25 nM respectively) and of YO-PRO-1 uptake in1321N1 astrocytoma cells stably expressing rat P2X7R [170]This finding poses a question to the general use of gap junctionchannel blockers as indicators of the participation of connexinhemichannels at the sites of transmitter release [171] The ATP
HN CF3
O OH
42 f lufenamic acid
HO
-O
O
O
H
OH
O
O-
N
HN
CF3
CF3
H O
H
43 carbenoxolone 44 mefloquine
Fig (23) Chemical structures for gap junction channel blockers with P2X7R antagonistic properties
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1521
potency of BzATP The nature of functional assays on theP2X7R is also found to be complex as ionic compositiontemperature and cell origins all affect the results demandingextra care be taken when antagonist potencies are directlycompared from different studies
[23] Li J Liu D Ke H Z Duncan R L Turner C H J Biol Chem2005 280 42952-42959
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[25] Gibbons S J Washburn K B Talamo B R J Auton Pharmacol2001 21 181-190
[26] Dehaye J P Moran A Marino A Arch Oral Biol 1999 44 S39-S43Four classes of small molecules with high affinity at the
P2X7R have been reported cyclic imides adamantyl amidesdiarylimidazolines and 1-benzyl-5-phenyltetrazoles Theseclasses of compounds are highly amenable to structuralmodifications indicating their good prospects as leadmolecules for further improving the potency and selectivity atP2X7R ultimately leading to orally active drugs To date norational drug design based on the knowledge of binding siteswithin this receptor has been undertaken This is a direct resultof the fact that no crystal structure or suitable homology modelhave been reported for this receptor The fact that the P2X7R canadopt either a channel or pore state causes even moredifficulties in studying receptor dynamics and at present theexact mechanism of channel-to-pore transition remains to beelucidated Undoubtedly the P2X7R is involved in severalprocesses relevant to chronic inflammation neurodegenerationand neuropathic pain and therefore discovery of more potentselective P2X7R antagonists would have a great impact in thepresent therapeutic window
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ACKNOWLEDGEMENTS[38] Khakh B S Burnstock G Kennedy C King B F North A
Seguela P Voigt M Humphrey P P A Nat Rev Neurosci 20012 165-174
The authors would like to thank Dr Natasha Hungerford forproofreading the manuscript
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[161] Kelly M J Kincaid J WO2006102610A2 2006[162] Schwink L Stengelin S Gossel M Bohme T Hessler G Stahl P
Gretzke D WO2004072025A2 2004 [177] Nelson D W Jarvis M F Carroll W A WO2006110516A1 2006[163] Schwink L Stengelin S Boehme T Gossel M Hessler G Stahl P
WO2005070925A1 2005[178] Carroll W A Perez-Medrano A Peddi S Florjancic A S
WO20060025614A1 2006[164] Merriman G H Ma L Shum P McGarry D Volz F Sabol J S
Gross A Zhao Z Rampe D Wang L Wirtz-Brugger F Harris BA Macdonald D Bioorg Med Chem Lett 2005 15 435-438
[179] Carroll W A Perez-Medrano A Jarvis M F Wang Y Peddi SWO20050171195A1 2005
Received February 01 2007 Revised April 04 2007 Accepted April 06 2007
1510 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
Fig (7) Chemical structure for DIDS and its derivatives
PPADS which differs from PPADS by the position of thesulfonates [100]
[109] receptor DIDS dihydro-DIDS 17 and SITS 19 (Fig 7) allblocked the ATP-stimulated Ca2+ uptake in rat parotid acinarcells via activation of the P2X7R (IC50 35 ndash 125 microM) [109]DNDS 18 in which the isothiocyanate moieties are replacedwith nitro groups was found to be inactive indicating theimportance of the isothiocyanate for interaction with theP2X7R On the other hand a variety of smaller aromaticisothiocyanate compounds derived from DIDS have been testedagainst the P2X1 receptor and were found to be active with IC50values between 11 and 54 microM [108] Overall DIDS and itsderivatives are inhibitors of both the P2X1 and P2X7R withlower potency for the latter
All the SAR data generated to date on PPADS and itsderivatives demonstrate that structural modifications on thekey functional groups (aldehyde phosphate and azophenyl)increases the potency and selectivity mainly at the P2X1receptor Consequently PPADS itself is used for testing at theP2X7R where it was found to be a potent antagonist only at therat and human receptors (IC50 15 and 90 nM respectively) butnot the mouse receptor (100-fold lower potency) in the YO-PRO-1 uptake assay provided cells are pre-incubated withPPADS for 15-30 min [41] When there was no pre-incubationthe IC50 of PPADS at the rat human and mouse P2X7R decreasedto approximately 50-70 microM in the electrophysiologicalrecordings [34 40] but pre-incubation with PPADS drasticallyimproved the IC50 value to 1 microM at the human P2X7R [88] Theinhibitory action of PPADS at the P2X7R is only partiallyreversible [88] suggesting a pseudo-irreversible mode ofantagonism [104] It has been suggested that PPADS P5P andoxidized ATP may all have a common site of action which islikely to be the ATP binding site [104] Overall it can beconcluded that PPADS per se is a species-dependent P2X7Rantagonist being most potent at the rat and human P2X7R butnot the mouse receptor Derivatives of PPADS reported to dateonly exhibit improved potency and selectivity at the P2X1receptor and not the P2X7R
5-(NN-Hexamethylene)amiloride (or HMA) 20 (Fig 8)belongs to the amilorides which are better known as potassium-sparing diuretics by blocking the Na+ channel as well as beinginhibitors of Na+-H+ counter-transport pathway [112] L-typecalcium channels [113] adenosine A1 receptors [114] andpossibly Na+-HCO3
- co-transport [115] HMA possesses affinityfor the P2X7R but its effects are highly species-dependent At 40microM concentration HMA antagonized P2X7R found in humanlymphocytes as determined from large cation uptake studies[116 117] However up to 100 microM concentration it wasineffective at the rat P2X7R [34] More interestingly theelectrophysiological studies revealed that co-application ofHMA with ATP produced an immediate and maximalaugmentation of inward currents induced by mouse P2X7R [60]This effect was not apparent when HMA was applied alone orwhen the buffer solution contained physiologicalconcentrations of Mg2+ and Ca2+ ions HMA has been reportedto be an irreversible inhibitor at the hP2X7R [88] Otheranalogues of HMA such as MIBA 21 and EIPA 22 also possesssimilar properties to HMA [116] Due to its relatively lowpotency and high species dependence at the P2X7R HMA andprobably other amilorides are unlikely to be good candidatesfor further exploration of the pharmacology of the P2X7R
33 Ion Channel Blockers
44rsquo-Diisothiocyanatostilbene-22rsquo-disulfonic acid (DIDS)16 is an anion channel blocker widely used to study various iontransport mechanisms [105-107] but has also beendemonstrated to be capable of inhibiting both the P2X1 receptor[108] and P2X7R-induced pore formation and other processes inseveral cell lines with relatively low potency [3 109-111] Forexample pre-incubation of BAC12F5 murine macrophage with150 ndash 200 microM DIDS inhibited the activation of phospholipaseD mediated by the P2X7R [110] Pre-incubation of rat primaryastrocytes with 200 microM of DIDS has also been shown to inhibitP2X7R-mediated responses such as the activation ofextracellular signal receptor kinases (ERK) [111] SARinvestigations have been performed on DIDS in order toimprove its potency and selectivity at the P2X1 [108] or P2X7
34 Polysulfonated Dyes
Suramin 23 is an example of a polysulfonated dye thatexhibits non-selective antagonism at both the P2X and P2Yreceptors with relatively low potency [3] Suramin blocks theP2X1-3 and P2X5 receptors (IC50 1-15 microM) but only shows weakor no inhibition at the P2X4 P2X6 and P2X7 receptors as well
N
NCl
N NH 2
O
NH
NH
NH2
20 5-(NN-Hexa methylene )- amiloride (HMA)
N
NCl
NH2
O
NH
NH
NH2
N
21 5-(N-Methyl-N- isobutyl)- amiloride (MIBA)
N
NCl
NH2
O
NH
NH
NH 2
N
22 5-(N-ethyl-N-isopropyl)- amiloride (EIPA)
Fig (8) Chemical structures for HMA and other amilorides
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1511
as some P2Y receptor subtypes [96] In addition suramin hasbeen shown to interact with many different proteins at theseconcentrations including glutamate nicotinic GABA and 5-HTreceptors as well as various proteases and ectonucleotidases [396] Nevertheless SAR studies on suramin have been performedin order to identify the important structural features responsiblefor antagonism at the P2 receptors [96 118-124] Selected keysuramin derivatives are represented in Fig (10 )
native and recombinant P2X1 receptors respectively and hasbeen shown to be much less potent at other P2X and P2Ysubtypes including adrenoceptors histamine H1 receptors andthe muscarinic M3 receptor [120] At the human P2X1 and P2X7receptors the IC50 values for NF449 were 005 nM and 40 microMrespectively [128] which indicated its usefulness todistinguish between the two receptors Subsequent SAR studiesconfirmed that NF449 was the most potent and selective knownP2X1 receptor antagonist [121 129] NF157 an analogue ofsuramin with fluorine replacing the methyl group has also beenreported to be highly potent at the P2Y11 receptor with a Ki of45 nM [124] More recently NF110 has also been shown to be apotent P2X3 receptor antagonist indicating that both thenumber and location of the sulfonate groups may be importantfor determining P2X subtype potency and selectivity [123]
O
HN
HN
NH
NH
O O
O ONH HNSO3-
SO 3-
SO3-
-O3S
SO 3-
-O3S
23 suramin
NF279 thus far represents the most active suramin relatedantagonist at the P2X7R and displays a reasonable potency of28 microM All the SAR data obtained to date on suramindemonstrate the importance of the molecular size number andposition of the sulfonate groups for P2X subtype potency andselectivity Unfortunately except for NF279 and NF449 othersuramin analogues provide very little information which can beused for the selective structure-based drug design of P2X7Rmolecules Suramin is also unlikely to be orally active due toits high molecular weight and interaction with many otherproteins Suramin is a very weak or almost inactive antagonistat the human P2X7R with an IC50 value of 92 microM [40] It istotally inactive at both the rat and mouse P2X7R with IC50values greater than 100 microM [34 60] Due to its interference withthe fluorescence of YO-PRO-1 suramin could not be used in theYO-PRO-1 dye uptake assay [104]
Fig (9) Chemical structure for suramin a weak antagonist at the P2X7R
Early SAR analysis showed that the shortened forms ofsuramin in the form of sulfonylbenzamides decreased thepotency at P2 receptors [118] NF023 (24 ) was later discoveredto be P2X-selective compared to P2Y receptors andectonucleotidases [118] Subsequent studies have examined theeffects of NF023 in various cell types and it was reported thatNF023 is a highly selective antagonist for either rat or humanP2X1 receptors heterologously expressed in Xenopus oocyteswith an IC50 of 02 microM [125] Another suramin derivativeNF279 (25 ) also showed potent selective reversible inhibitionat the rat P2X1 receptor in Xenopus oocytes with an IC50 of 19nM [126] In studying the effects of NF279 on human P2X1 andP2X7 receptors it was reported that NF279 is almost 100-foldmore potent at the human P2X1 than the P2X7 receptor withIC50 values of 005 and 28 microM respectively [127] Other keysuramin derivatives resulting from extensive SAR studies areNF449 (27 ) NF110 (28 ) and NF157 (26 ) [120 121 123 124128 129] NF449 has an IC50 value of 70 nM and 03 nM at the
Brilliant Blue G 29 (Fig 11) is another polysulfonated dyewhich is a potent non-competitive inhibitor of the rat andhuman P2X7R with IC50 values of 10 and 200 nM respectivelyin electrophysiological studies [130] Brilliant Blue G has alsobeen shown to be highly selective for P2X7R over P2X1-5receptors [130] It has been shown that at the rat P2X7R about100 to 300 nM concentration was required to inhibit the YO-PRO-1 uptake which was 10- to 30-fold higher than thatrequired for blocking P2X7R-induced currents demonstratingits differential effect at the channel and pore forms of the P2X7R
O
HN
HN
NH
NH
O O
R R
O ONH HNSO3-
SO3-
SO3-
-O3S
SO3-
-O3S
O
HN
HN
NH
OO
NH
O ONH
NH
R R
RR
n n
Suramin n=1 R=CH3
24 NF023 n=0 R=H
25 NF279 n=1 R=H
26 NF157 n=1 R=F
27 NF449 R=24-disulfonyl28 NF110 R=4-sulfonyl
Fig (10) Suramin and its key derivatives investigated in the SAR studies
1512 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
[130] Brilliant Blue G has also been tested on the mouseP2X7R where similar antagonistic potency was found comparedto rat and human receptors [42] In this study it was alsoreported that the selectivity of Brilliant Blue G for rat overhuman P2X7R was confirmed at 22degC but lost at 37degC Thedrawback for this compound is that it could also be bound toproteins and thus would be expected to have lower potency inthe dye uptake assay than electrophysiology measurements dueto the depletion by cell monolayer effect [42] Nevertheless itrepresents a promising antagonist at rat human and mouseanalogues of the P2X7R No studies have been performed onBrilliant Blue G to further improve its potency and selectivityat the P2X7R Other polysulfonated dyes such as Evans BlueTrypan Blue reactive blue 2 reactive red 2 and their derivativeshave also been tested at the purinergic systems [131-133] butnone of them exhibited activity at the P2X7R
opening of the ion channel calmidazolium produces no effecton the YO-PRO-1 uptake The large non-selective porehowever is still formed even when the ionic currents areinhibited indicating that pore formation does not necessarilyrequire opening of the ion channels Calmidazolium wassuggested to be an allosteric modulator of the P2X7R at theagonist binding site inducing conformational change at thereceptor level that results in decreased agonist binding affinity[84] It displays less potency at inhibiting the agonist-evokedcurrents at both the mouse and human P2X7R with IC50 valuesof 1 microM and 100 nM respectively [41 88] At present no SARstudies have been reported on calmidazolium
N
N+
ClCl
O
ClCl
Cl
Cl
30 calmidazolium
NH
N
O
SO3-
N+
SO3-
Cl
29 Coomassie Brilliant Blue G
Fig (12) Chemical structure for calmidazolium a P2X7R antagonistcapable of distinguishing the channel and pore forms of the receptor
36 KN-62 and its Analogues
KN-62 31 (Fig 13) is a derivative of isoquinolinesul-fonamide known as a selective and potent inhibitor of themultifunctional calciumcalmodulin-dependent protein kinases(CaMK) with an IC50 of 09 microM at CaMK II [134] Gargett andWiley in 1997 first demonstrated that both KN-62 31 and itsanalogue KN-04 32 were potent inhibitors of P2X7R at humanlymphocytes with IC50 values of 13 and 17-37 nM respectivelyin both the ion flux and ethidium bromide uptake assays [134]The mode of inhibition is allosteric modulation similar to thatof calmidazolium
Fig (11) Chemical structure for Brilliant Blue G a potent antagonist at therat P2X7R
35 Calmidazolium
Extracellular calmidazolium 30 (Fig 12) is a potentinhibitor of BzATP-induced currents via binding to rat P2X7Rwith an IC50 of 13 nM [84] The unique feature ofcalmidazolium is its ability to differentiate between the channeland pore form of the P2X7R while it potently inhibits the
KN-62 and KN-04 are species-selective for P2X7Rantagonism They potently inhibited the ATP-induced currentsas well as the ethidium bromide uptake in HEK293 cells
Fig (13) Chemical structures for isoquinolines KN-62 and KN-04 potent P2X7R antagonists at the hP2X7R with much less effect on the rat and humananalogues
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1513
33e most active in the series but still 30-fold less potent than KN-62
Fig (14) Conformationally restricted analogues of KN-62 [136]
expressing human P2X7R at the concentration of 30 ndash 100 nMdepending on whether ATP or BzATP was used and the cellularbackground of the human P2X7R [135] However even up to 3microM concentration they had no effect at the rat P2X7R [135] andKN-62 was found to be at least 10-fold more potent at thehuman P2X7R than the recombinant mouse analogue [60] Theseisoquinolinesulfonamide derivatives may interact with theamino-terminal domain of the P2X7R since the introduction ofthe first 335 amino acids of the human P2X7R delivers KN-62sensitivity to the rat P2X7R and the counter-part chimera isfound to be insensitive to KN-62 [135] The IC50 at the humanP2X7R is 10-fold lower at 22degC than at 37degC in the ethidiumbromide uptake study [42]
tyrosine) 2) R2 (the tyrosine side chain) and 3) R3 (thesubstituent attached to the N-piperazine) (Fig 15) [137-139]
The effect of substitutions at the R1 R2 and R3 groups (Fig15 ) on the P2X7R antagonistic properties was compared on thebasis of inhibition of K+ release [137 138] At the R1 positionlarge hydrophobic moieties linked to the amino positionthrough sulfonamide (34a-b) or carbamate (34c) groups werepreferable for P2X7R inhibition In the series of derivativeswhere R2 = quinolinesulfonyl and R3 = t-butoxycarbonyl (Boc)introduction of carbobenzyloxy (Cbz) and quinolinesulfonylgroups at the R1 position were found to be the most active[137] In another series of derivatives where R1 = Cbz and R3 =Boc introduction of arylsulfonyl (34d-g) and benzoyl (34h )moieties at the R2 position were preferred [137] A free aminogroup at the R3 position (34i-j) andor opening of thepiperazine ring to an ethylene diamine greatly reduced theP2X7R activity Amongst the various acyl groups tested at theR3 position Boc and benzoyl groups (34k-m) were found to bepreferable but not sulfonyl (34n-q ) The presence of a methylgroup at the amino terminus of the tyrosine group is thereforenot an absolute pre-requisite for P2X7 antagonism [137]
R3 N N
O
N
O
R4
R1
R2
In another study undertaken by Chen et al to furtherexplore the SAR of 34m [138] it was found that nitro-substitution at the meta- or para- position of the Cbz group atR1 (34r-t) was well tolerated However introduction of anacetamido- or isothiocyanato- group at the Cbz group abolishedP2X7R antagonism (34uv) The original isoquinoline-5-sulfonyl moiety at the R2 position could be substituted withmeta- or para-substituted phenylsulfonyl groups such as tosyl(34r-t) 3-nitrophenylsulfonyl (34w) or 4-nitrophenylsulfonyl(34x) without adverse effects on the P2X7R antagonism [138]Substitution of the benzoyl group at the R3 position by a Boc(34y) or 4-aminobenzoyl group (34z) retained activity
Fig (15) General structure for the KN-62 derivatives (34a-z)
Extensive SAR studies have been performed on KN-62 inorder to enhance its potency and selectivity at the P2X7R Thefirst attempt to improve the antagonistic activity of KN-62 wasachieved by tethering the N-methyl of the tyrosine backbone tothe ortho-position of the proximal phenyl ring which resultedin a series of conformationally restricted KN-62 analogues withthe formula shown in Fig (14 ) [136] The constrained form ofKN-62 (31 vs 33a) was devoid of P2X7R antagonisticproperties Replacement of the isoquinoline-5-sulfonyl moiety(33a) with quinoline-5-sulfonyl (33b ) quinoline-8-sulfonyl(33c) or naphthalene (33d ) resulted in the loss of ability toinhibit the P2X7R-mediated Ca2+ influx [136] The most activecompound arising from this series was compound (33e) with anIC50 value of 316 nM which is 30-fold weaker than the originalKN-62 Taken together these results indicate that an extendedrather than folded conformation of KN-62 conferred by theincorporation of a methylene unit between the N-methyl of thetyrosine backbone to the ortho-position of the proximal phenylring is preferred at the P2X7R [136]
A study by Baraldi et al reported the systematic SAR profileof KN-62 specifically looking at modifications of thephenylpiperazine moiety (R2 position Fig 16 ) [139] In theinvestigation of the optimum chain linker between thepiperazine and phenyl groups it was found that one methylenespacer (35a) slightly improved the activity whereas a twomethylene spacer was not favourable (35b ) [139] The presenceof a basic nitrogen in the piperazine moiety is proven crucial forP2X7R antagonism as substitution of the piperazine ring withpiperidine (35a vs 35c) resulted in a 3-fold reduction of activity[139] Substitution of the phenyl ring with other heterocyclessuch as pyridine (35d ) or pyrimidine (35e) also decreased theP2X7R antagonistic property The para- position prefers a
Subsequent investigations on KN-62 involved extensivemodifications in three positions 1) R1 (the amino-terminus of
1514 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
small electron-withdrawing group eg fluorine (35f) and theantagonistic property decreases as the size of the electron-withdrawing group increases (35g 35h) A methyl- or nitro-group at the para- position (35i 35j) was beneficial foractivity but not evidenced with the methoxy (35k ) group As inthe case for para- substitution introduction of F Cl or CH3 butnot a methoxy (35l-o) at the ortho- position was well toleratedMoreover the meta- position could also tolerate a Cl or CF3group (35p 35q) The disubstituted compound 35r showedreduced antagonistic properties (IC50 = 1122 nM) possibly dueto the steric bulkiness of om-xylyl residue while the dichloroderivative 35s (IC50 = 34 nM) retained activity A slightdecrease in potency was observed when the methyl group on thenitrogen on the α-position of the tyrosine was removed (35n vs
35t) further confirming that this is not substantial for P2X7Rantagonism The most potent compound arising from this studywas 35f with an IC50 value of 13 nM at inhibiting calciuminflux in human monocytes compared to KN-62 which has anIC50 of 51 nM [139] It is therefore confirmed that thephenylpiperazine group of KN-62 is crucial for interaction withthe active site of the receptor and can be potentially improvedwith small halogen substitutions particularly in the paraposition
An ortho-methyl substituent on the N-phenyl ring of KN-62increased the potency by 3-fold (36 IC50 15 nM) [140]Radiolabelling of compound 36 with tritium and preliminarybinding studies to the P2X7R have also been achieved [140]
Table 2 Structures and P2X7R Antagonistic Properties of Selected KN-62 Derivatives [137138]
34a isoquinoline-5-SO2mdash isoquinoline-5-SO2mdash Bocmdash H 77 plusmn 24 ~40
34b quinoline-8-SO2mdash quinoline-8-SO2mdash Bocmdash H 61 plusmn 30 ND
34c Cbzmdash quinoline-8-SO2mdash Bocmdash H 53 plusmn 23 ~200
34d Cbzmdash PhSO2mdash Bocmdash H 59 plusmn 14 ND
34e Cbzmdash p-tolyl-SO2mdash Bocmdash H 71 plusmn 30 ~300
34f Cbzmdash p-OMe-PhSO2mdash Bocmdash H 62 plusmn 15 ND
34g Cbzmdash isoquinoline-5-SO2mdash Bocmdash H 43 ND
34h Cbzmdash benzoylmdash Bocmdash H 47 plusmn 21 ND
34i isoquinoline-5-SO2mdash isoquinoline-5-SO2mdash H H 14 plusmn 12 ND
34j quinoline-8-SO2mdash quinoline-8-SO2mdash H H 5 plusmn 9 ND
34k Cbzmdash PhSO2mdash Cbzmdash H 48 plusmn 29 ND
34l Cbzmdash PhSO2mdash benzoylmdash H 78 plusmn 22 ND
34m Cbzmdash isoquinoline-5-SO2mdash benzoylmdash H 85 plusmn 10 ~200
34n Cbzmdash PhSO2mdash p-tolyl-SO2mdash H 0 ND
34o Cbzmdash PhSO2mdash CH3SO2mdash H 0 ND
34p Cbzmdash PhSO2mdash PhSO2mdash H 0 ND
34q Cbzmdash PhSO2mdash p-OMe-PhSO2mdash H 0 ND
34r Cbzmdash p-tolyl-SO2mdash benzoylmdash H 71 plusmn 21 ND
34s p-NO2-Cbzmdash p-tolyl-SO2mdash benzoylmdash H 69 plusmn 25 ND
34t m-NO2-Cbzmdash p-tolyl-SO2mdash benzoylmdash H 72 plusmn 6 ND
34u m-acetamido-Cbzmdash p-tolyl-SO2mdash benzoylmdash H 9 plusmn 13 ND
34v m-isothiocyanato-Cbzmdash p-tolyl-SO2mdash benzoylmdash H minus4 plusmn 5 ND
34w Cbzmdash m-NO2-PhSO2mdash benzoylmdash H 75 plusmn 10 ND
34x Cbzmdash p-NO2-PhSO2mdash benzoylmdash H 78 plusmn 18 ~100
34y m-NO2-Cbzmdash p-tolyl-SO2mdash Bocmdash H 94 plusmn 28 ~100
34z Cbzmdash p-tolyl-SO2mdash p-NH2-benzoylmdash H 93 plusmn 3 gt 300
aAntagonist function was measured by the percent inhibition of the K+ release triggered by 3mM ATP in paired wells in the absence of antagonist bAntagonistic IC50values derived from dose-response curves obtained on the basis of inhibition of K+ release
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1515
R2 X N
O
N
O
S
R1
S
O
O
N
O
O
Nthe inhibition of BzATP-induced ethidium bromide uptakemediated by the P2X7R expressed endogenously on the humanpre-monocytic cell line THP-1 [142] Compound 37a (Table 4)was identified as the lead structure and modifications on theimide and 4-pyridyl moieties were investigated The cyclicimide moiety is proven essential for the P2X7R activity sincesubstitutions with simple amines acyclic amides or acycliccarbamates almost completely abolished the activity [142] Thering can be five- or six-membered in the forms of imides (37a-bh) carbamates (37c-d) thiazolidine-24-diones (37eg) andhydantoins (37f) Replacement of the 4-pyridyl moiety with 4-substituted benzamides (37g-h) retained the P2X7R activityThe presence of a 3rsquo-nitro substituent on the biphenyl subunitwas reported to convey optimum P2X7R activity The mode ofinhibition of these compounds seemed to be competitivealthough there were observable changes in the maximalresponse as the compound concentration was increased [142]
Fig (16) General structure for the KN-62 derivatives (35a-35s)
Unfortunately while these KN-62 analogues are potentiallyuseful tools to investigate the basic properties of the P2X7Rthey do not represent good starting points for the discovery oforally-active drugs due to 1) high molecular weight (gt700 Da)2) high lipophilicity (clogP gt 6) and 3) presence of ametabolically labile yet crucial sulfonate group [141]
These compounds represent a new class of ligands thatpossess activity to the P2X7R They appear good candidates asorally active drugs due to their relatively low molecular weight(~400 Da) and would be highly amenable for further SARstudies However despite the succinimides and thiazolidine-24-diones representing potent analogues they were found to bemetabolically and chemically fragile On the other handalthough the carbamates were less potent they displayedimproved stability Future SAR studies could usefully involveinvestigation of the biphenyl ring and the chain linker
37 Cyclic Imides and Analogues
Cyclic imides 37 (Fig 18) were discovered through a high-throughput screening of AstraZenecarsquos compound library for
Table 3 Structures and P2X7R Antagonistic Properties of Selected KN-62 Derivatives [139]
Entry R1 R2 X IC50 (nM)a
KN-62 mdashCH3 mdashPh N 511 plusmn 11
35a mdashCH3 mdashCH2Ph N 211 plusmn 08
35b mdashCH3 mdashCH2CH2Ph N 600 plusmn 27
35c mdashCH3 mdashCH2Ph CH 653 plusmn 08
35d mdashCH3 2-pyridinyl N 170 plusmn 39
35e mdashCH3 2-pyrimidinyl N 798 plusmn 07
35f mdashCH3 p-F-Phmdash N 13 plusmn 01
35g mdashCH3 p-Cl-Phmdash N 105 plusmn 11
35h mdashCH3 p-I-Phmdash N 176 plusmn 38
35i mdashCH3 p-Me-Phmdash N 135 plusmn 02
35j mdashCH3 p-NO2-Phmdash N 58 plusmn 06
35k mdashCH3 p-OMe-Phmdash N 132 plusmn 43
35l mdashCH3 o-F-Phmdash N 185 plusmn 04
35m mdashCH3 o-Cl-Phmdash N 158 plusmn 09
35n mdashCH3 o-Me-Phmdash N 151 plusmn 14
35o mdashCH3 o-OMe-Phmdash N 841 plusmn 5
35p mdashCH3 m-Cl-Phmdash N 141 plusmn 03
35q mdashCH3 m-CF3-Phmdash N 311 plusmn 12
35r mdashCH3 om-diMe-Phmdash N 1122 plusmn 25
35s mdashCH3 mp-diCl-Phmdash N 339 plusmn 08
35t H o-Me-Phmdash N 288 plusmn 03
aAntagonistic IC50 (nM)a values derived from dose-response curves on the basis of inhibition of Ca2+ flux in human monocytes
1516 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
N N
O
N
S
N
OO
OS O
O
N
R
31 R = H (KN-62)
36 R = 2-[3H]Me
AstraZeneca reported that no replacement for the adamantaneframework was found [141] Investigation of the chain linkerrequirements between the adamantane and the phenyl ring wasperformed where it was found that C- and N- methylationcarbonyl reduction and methylene removal (38d-g) all causedloss of potency One methylene unit extension (38h ) retainedthe potency whereas reversal of the amide connectivity (38i)slightly decreased the potency AstraZeneca prepared acombinatorial library by reacting commercially availableadamantanemethylamine with a large variety of acid chloridesor via standard amide coupling with carboxylic acids Thegeneral trends indicated that only aromatic amides had activityat the P2X7R [141] On the phenyl ring 23- and 25-disubstitutions resulted in better antagonists (38j-o) than the26- (38p ) 35- (38q ) or 24-disusbstitutions (38r) The ortho-position could also tolerate substituents such as Br OMe andMe (38s-u) A closer examination on the reverse amides (38i v-y) also revealed that 23- and 25-disubsitutions were stillpreferred for P2X7R inhibition The indazole amide (38z) wasidentified as a target lead molecule with a good metabolicprofile suitable for further optimization for clinical trial studies[141]
Fig (17) Radiolabeled analogue of KN-62 [140]
O
NY
X
O
Z
NO2
Adamantyl amides represent a class of simple moleculeswith excellent activity at the P2X7R The nature of theadamantane group could potentially be further investigatedalthough the original authors stated that no replacement for theadamantane group was found A number of patents onadamantyl-containing benzamides have been generated byAstraZeneca [143-146] and Pfizer [147] indicating the potentialof this ligand class for generating a truly potent selective andorally active P2X7R inhibitor Pfizer specifically incorporatedtriazinyl group into the adamantyl benzamide framework [147]Fig (18) General structure for the cyclic imides generated by
AstraZeneca Non-adamantane benzamides including piperidinyl andpiperazinyl benzamides [148 149] triazinyl benzamides [150-153] quinolinyl benzamides [154 155] 34-difluoroanilinebenzamides [156] aromatic acid-containing benzamides [157]pyrazolylmethyl benzamides [158] and other heteroarylamides[159-163] have also been patented by various otherpharmaceutical companies
38 Adamantyl Amides
Adamantyl amides were also discovered from a high-throughput screening of AstraZenecarsquos compound library forinhibition of the P2X7R-mediated ethidium bromide uptakeinduced by BzATP [141] N-Adamantan-1-ylmethyl-2-chloro-benzamide 38a was identified as the lead structure with anexcellent pA2 value of 81 The presence of an ortho-substituentis believed to cause a twist in the orientation of the benzamideessential for potent P2X7R antagonism whereas para-substituents (38bc) have deleterious effects on the P2X7Ractivity Selected compounds of this series are represented inTable 5
39 Diarylimidazolines
Diarylimidazolines represent another class of P2X7-activecompounds They were first discovered through a high-throughput screening of Aventisrsquo compound library forinhibition of BzATP-induced YO-PRO-1 uptake mediated byP2X7R expressed in U373 cells stably expressing the human
Table 4 Structures and P2X7R Antagonistic Properties of Selected Cyclic Imides [142]
Entry X Y Z IC50 ( M) pA2a
37a CH2 CO N - 69
37b (CH2)2 CO N - 75
37c O CH2 N 64
37d O (CH2)2 N 063 -
37e S CO N - 77
37f NH CO N 02 -
37g S CO C-CONHMe 016 -
37h CH2 CO C-CONMe2 025 -
a pA2 = the negative logarithm of a molar concentration of an antagonist that necessitates doubling of agonist concentration needed to elicit the original submaximalresponse obtained in the absence of antagonist
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1517
P2X7R [164] Compound 39a was identified as an initial hitfrom the screening with an IC50 value of 80 nM
equipotent Some degree of branching in the chain linker wasgenerally tolerated (39f-h) Configurational constraintsimposed on the chain linker as exemplified by 39i weretolerated but this was not the case for the cyclopropyl analogue39j
X
RO
NH
N
HN
38a-y 38z
Subsequent investigations focused on the substitutionpattern in the aryl ring for the benzyl (n=1) and phenethyl (n=2)series [164] Electron withdrawing and small neutral groupswere generally tolerable in both series (39k-r v-x) Howeverelectron-donating groups exemplified by OMe gave much lessactive compounds (39s-u) In the phenethyl seriessubstitutions at the ortho- and meta- positions were preferableto the corresponding para- substitutions (39kl vs 39m and39no vs 39p) Interestingly in the benzyl series meta- andpara- substitutions resulted in more active compounds than thecorresponding ortho analogues (39v vs 39wx)
Fig (19) Adamantyl amides generated by AstraZeneca
Investigation of the nature of the linker between thearomatic ring and the 2-position of the imidazole ring revealedthat a 3 or 4 carbon linker (39bc) caused a slight decrease in theactivity and no carbon linker (39d ) rendered the compoundinactive [164] One carbon linker with a methyl branching unit(39e) was tolerated It was reported that there was noenantiomeric preference of the methyl branch they were
The diarylimidazolines examined represent good candidatesfor orally active P2X7R inhibitors they are small and highlyamenable for SAR studies The 4- and 5-phenyl rings representattractive sites for further investigations
Table 5 Structures and P2X7R Antagonistic Properties of Selected Adamantyl Amides [141]
Entry R X pA2
38a 2-Cl CH2NHCO 81
38b 4-Cl CH2NHCO inactive
38c 24-Cl2 CH2NHCO 64
38d 2-Cl CHMeNHCO 64
38e 2-Cl CH2NMeCO 54
38f 2-Cl CH2NHCH2 54
38g 2-Cl NHCO inactive
38h 2-Cl (CH2)2NHCO 78
38i 2-Cl CH2CONH 63
38j 23-Cl2 CH2NHCO 88
38k 25-Cl2 CH2NHCO 83
38l 2-Cl-3-NH2 CH2NHCO 82
38m 2-Cl-5-OMe CH2NHCO 88
38n 2-Cl-5-OH CH2NHCO 80
38o 2-Cl-5-NH2 CH2NHCO 84
38p 26-Cl2 CH2NHCO 74
38q 35-Cl2 CH2NHCO 54
38r 24-Cl2 CH2NHCO 64
38s 2-Br CH2NHCO 80
38t 2-OMe CH2NHCO 71
38u 2-Me CH2NHCO 79
38v 2-Me CH2CONH 68
38w 2-Cl-5-OMe CH2CONH 72
38x 2-Me-3-OMe CH2CONH 83
38y 2-Me-5-OMe CH2CONH 80
1518 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
N
HN
XY
R
N
HN
N
HN
N
HN
( )n
39i39j
39a-ce-hk-w 39d
Fig (20) General structure of the diarylimidazolines generated by Aventis
Table 6 Structures and P2X7R Antagonistic Properties of Selected Diarylimidazolines [164]
Entry n X Y R IC50 ( M)a
39a 1 H H H 008
39b 2 H H H 054
39c 3 H H H 061
39e 0 (R)-Me H H 011
39f 1 (S)-Me H H 003
39g 1 Me Me H 001
39h 1 Ph H H 032
39k 1 H H 2-F 016
39l 1 H H 3-F 004
39m 1 H H 4-F 022
39n 1 H H 2-Cl 007
39o 1 H H 3-Cl 012
39p 1 H H 4-Cl gt1
39q 1 H H 2-Me 007
39r 1 H H 4-Me 219
39s 1 H H 2-OMe 083
39t 1 H H 3-OMe gt3
39u 1 H H 4-OMe gt3
39v 0 H H 2-F 058
39w 0 H H 3-F 008
39x 0 H H 4-F 008
aAntagonistic IC50 values derived from dose-response curves on the basis of inhibition of YO-PRO-1 dye uptake
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1519
R1N
N
NN
R2
R1
NN
NN
R2
N
N
NN
Cl
Cl
Cl
Cl
40b-t 40u-x40a
1
2
3
4
5
Fig (21) General structure of tetrazoles generated by Abbott Laboratories
310 1-Benzyl-5-Phenyltetrazoles potency The limited solubility of those compounds howeverposed a challenge to incorporate substitutions that couldincrease the solubility while maintaining activity A 3-pyridylgroup (40d ) was found to overcome this problem Moreover anextra 2-methyl substituent (40e) resulted in higher activitycompared to 40d A methylene extension (40f) caused a decreasein potency whereas other heterocycles such as 40g and 40hresulted in analogues with similar potency to 40d
1-Benzyl-5-phenyltetrazoles were discovered through ahigh-throughput screening by Abbott Laboratories for threedifferent P2X7R activities Ca2+ flux at the rat or human P2X7RYO-PRO-1 uptake and interleukin-1β release at human P2X7R[165-168] The initial hit from the screening was the tetrazole40a (Table 7) with a pIC50 value of 69 in inhibiting P2X7Rderived from the recombinant human cell line
Investigation of the nature of the substituent attached to the5-position of the tetrazole core showed that the substitutionpattern at this position was not well tolerated [165] Forexample unsubstituted mono-chlorosubstituted 25- and 34-
Initial SAR studies focused on the substituent attached tothe 1-position of the tetrazole core [165] Unsubstituted orortho-substituted benzylic group (40bc) exhibited good
Table 7 Structures and P2X7R Antagonistic Properties of Selected 1-benzyl-5-tetrazoles [165]
1520 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
disubstituted compounds (40i-m) pyridine and quinoline ring(40n-p ) analogues were not tolerated However moreconservative changes such as CF3 and F for Cl group (40q-t)resulted in an increase of potency
release and amplification of astrocytic Ca2+ signaling werereported to be due to the P2X7R and not Cx43 hemichannels[170]
Decavanadate a type of polymer of vanadium oxides hasbeen demonstrated to be a competitive rapid-acting antagonistat the P2X7R [172] It is capable of inhibiting the BzATP-stimulated ethidium bromide uptake in human mouse and ratP2X7R with pA2 values of 74 57 and 71 respectivelyDecavanadate has not been fully tested for its selectivity overother P2X receptors however it is suggested that this largemolecular species acts at the extracellular ATP binding site ofthe receptor consistent with the competitive mode of inhibition[172] A monoclonal antibody specific for the human P2X7Rhas also been shown to be a potent and selective antagonist ofthe receptor with an IC50 value of 5 nM in blocking the BzATP-evoked currents [173] Interestingly tenidap at 50 ndash 200 microMconcentration increases the sensitivity of mouse macrophagesto ATP-mediated cytotoxicity [174] 17-β-estradiol and notprogesterone or 17-α-estradiol has also been reported to blockthe agonist-induced currents at the human P2X7R with an IC50value of 3 microM in a non-genomic manner [175] Polymyxin B apotent antibiotic well-known for its lipopolysaccharide-neutralizing effects has been demonstrated to confer asynergistic effect when co-applied with ATP in a similar mannerto tenidap inducing Ca2+ influx ethidium bromide uptake andlactate dehydrogenase release via action at the P2X7R [176]
Tetrazoles with reversed connectivity (40u-x) were alsoevaluated where it was found that this connectivity reversal hadlittle effect on the potency [165] These tetrazoles were reportedto be slightly more active in the human P2X7R than the ratanalogue [165]
The tetrazole derivative 40d has been shown to be selectivefor other P2 receptors including P2X3 P2X4 and P2Y2 receptors[165] Pharmacological testing of this tetrazole suggested acompetitive mode of inhibition at the P2X7R and its efficacyhas also been evaluated in a model of neuropathic pain in rats[165] Overall these tetrazoles are found to be P2X7R-active andhighly adaptable to structural modifications These propertiesmake the tetrazoles an interesting class of compound for furtherinvestigating P2X7R activity
311 Other P2X7R-Active Ligands
Chelerythrine 41 (Fig 22) is a member of the benzophen-anthridine alkaloids known to inhibit protein kinase C withgood potency and selectivity (IC50 066 microM) as well as enzymessuch as alanine aminotransferase and Na+K+-ATPase [169] At10 microM concentrations chelerythrine non-competitivelyinhibited the BzATP-induced 86Rb+ efflux by 74 in human B-lymphocytes [169] Dye uptake studies could not be performeddue to the fluorescent nature of the compound It is yet to bedetermined whether chelerythrine inhibition of P2X7R involvesprotein kinase C [169]
More recently other distinct classes of P2X7-active ligandshave been reported and patented by the Abbott Laboratoriesacylhydrazides [177] cyanoamidines and cyanoguanidines[178 179]
4 CONCLUSIONS AND FUTURE DIRECTIONS
O
O
N
O
O
Cl
41
Although many structurally diverse class of compoundshave been reported as P2X7R antagonists with reasonablepotency the selectivity issue over other P2 receptors andproteins such as ecto-ATPases and ectonucleotidases stillprevails This is particularly an issue for oxidized ATP andsuramin SAR studies performed on PPADS and suramin mainlygave improved potency and selectivity for the P2X1 receptorIon channel blockers such as amilorides and stilbenederivatives only possess relatively low potency at the P2X7RFurther complications in studying these P2X7R antagonistsalso arise from the demonstrated species differences acrosshuman rat and mouse systems Brilliant Blue G is active for allthree species however at present there have been no SAR datareported Another fundamental factor that hampers the study ofP2X7R in detail is the relatively low potency and selectivity ofthe available agonist BzATP The discovery of a more potentselective P2X7R agonist would be ideal since in many studiesfull dose-response curves could not be generated due to the low
Fig (22) Chemical structure for chelerythrine an alkaloid withantagonistic properties at the human P2X7R
Gap junction channel blockers such as flufenamic acid 42 carbenoxolone 43 and mefloquine 44 (Fig 23) were all foundto be inhibitors of the BzATP-induced Ca2+ flux (IC50 values of655 175 and 25 nM respectively) and of YO-PRO-1 uptake in1321N1 astrocytoma cells stably expressing rat P2X7R [170]This finding poses a question to the general use of gap junctionchannel blockers as indicators of the participation of connexinhemichannels at the sites of transmitter release [171] The ATP
HN CF3
O OH
42 f lufenamic acid
HO
-O
O
O
H
OH
O
O-
N
HN
CF3
CF3
H O
H
43 carbenoxolone 44 mefloquine
Fig (23) Chemical structures for gap junction channel blockers with P2X7R antagonistic properties
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1521
potency of BzATP The nature of functional assays on theP2X7R is also found to be complex as ionic compositiontemperature and cell origins all affect the results demandingextra care be taken when antagonist potencies are directlycompared from different studies
[23] Li J Liu D Ke H Z Duncan R L Turner C H J Biol Chem2005 280 42952-42959
[24] Penolazzi L Bianchini E Lambertini E Baraldi P G RomagnoliR Piva R Gambari R J Biomed Sci 2005 12 1013-1020
[25] Gibbons S J Washburn K B Talamo B R J Auton Pharmacol2001 21 181-190
[26] Dehaye J P Moran A Marino A Arch Oral Biol 1999 44 S39-S43Four classes of small molecules with high affinity at the
P2X7R have been reported cyclic imides adamantyl amidesdiarylimidazolines and 1-benzyl-5-phenyltetrazoles Theseclasses of compounds are highly amenable to structuralmodifications indicating their good prospects as leadmolecules for further improving the potency and selectivity atP2X7R ultimately leading to orally active drugs To date norational drug design based on the knowledge of binding siteswithin this receptor has been undertaken This is a direct resultof the fact that no crystal structure or suitable homology modelhave been reported for this receptor The fact that the P2X7R canadopt either a channel or pore state causes even moredifficulties in studying receptor dynamics and at present theexact mechanism of channel-to-pore transition remains to beelucidated Undoubtedly the P2X7R is involved in severalprocesses relevant to chronic inflammation neurodegenerationand neuropathic pain and therefore discovery of more potentselective P2X7R antagonists would have a great impact in thepresent therapeutic window
[27] Li Q Luo X Zeng W Muallem S J Biol Chem 2003 27847554-47561
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ACKNOWLEDGEMENTS[38] Khakh B S Burnstock G Kennedy C King B F North A
Seguela P Voigt M Humphrey P P A Nat Rev Neurosci 20012 165-174
The authors would like to thank Dr Natasha Hungerford forproofreading the manuscript
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Neurosci Lett 2002 327 87-90 [132] Tuluc F Bultmann R Glanzel M Frahm A W Starke K Naunyn-Schmiedebergs Arch Pharmacol 1998 357 111-120[96] Lambrecht G Braun K Damer S Ganso M Hildebrandt C
Ullmann H Kassack M U Nickel P Curr Pharm Des 2002 82371-2399
[133] Wittenburg H Bultmann R Pause B Ganter C Kurz G Starke KNaunyn-Schmiedebergs Arch Pharmacol 1996 354 491-497
[97] Garcia-Guzman M Soto F Gomez-Hernandez J M Lund P-EStuhmer W Mol Pharmacol 1997 51 109-118
[134] Gargett C E Wiley J S Br J Pharmacol 1997 120 1483-1490[135] Humphreys B D Virginio C Surprenant A Rice J Dubyak GR
Mol Pharmacol 1998 54 22-32[98] Kim Y-C Camaioni E Ziganshin A U Ji X-D King B FWildman S S Rychkov A Yoburn J Kim H Mohanram AHarden T K Boyer J L Burnstock G Jacobson K A Drug DevRes 1998 45 52-66
[136] Baraldi P G Romagnoli R Tabrizi M A Falzoni S Di Virgilio FBioorg Med Chem Lett 2000 10 681-684
[137] Ravi R G Kertesy S B Dubyak G R Jacobson K A Drug DevRes 2001 54 75-87[99] Jacobson K A Kim Y-C Wildman S S Mohanram A Harden T
K Boyer J L King B F Burnstock G J Med Chem 1998 41 2201-2206
[138] Chen W Ravi R G Kertesy S B Dubyak G R Jacobson K ABioconjug Chem 2002 13 1100-1111
[100] Kim Y-C Brown S G Harden T K Boyer J L Dubyak GKing B F Burnstock G Jacobson K A J Med Chem 2001 44 340-349
[139] Baraldi P G Nunez M D C A M Falzoni S Di Virgilio FRomagnoli R J Med Chem 2003 46 1318-1329
[140] Romagnoli R Baraldi P G Pavani M G Tabrizi M A MoormanA R Di Virgilio F Cattabriga E Pancaldi C Gessi S Borea P ABioorg Med Chem Lett 2004 14 5709-5712
[101] Brown S G Kim Y-C Kim S-A Jacobson K A Burnstock GKing B F Drug Dev Res 2001 53 281-291
[102] Lambrecht G Rettinger J Baumert H G Czeche S Damer SGanso M Hildebrandt C Niebel B Spatz-Kumbel G SchmalzingG Mutschler E Eur J Pharmacol 2000 387 R19-R21
[141] Baxter A Bent J Bowers K Braddock M Brough S Fagura MLawson M McInally T Mortimore M Robertson M Weaver RWebborn P Bioorg Med Chem Lett 2003 13 4047-4050
[103] Kim Y-C Lee J-S Sak K Marteau F Mamedova L BoeynaemsJ-M Jacobson K A Biochem Pharmacol 2005 70 266-274
[142] Alcaraz L Baxter A Bent J Bowers K Braddock M CladingboelD Donald D Fagura M Furber M Laurent C Lawson M
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1523
Mortimore M McCormick M Roberts N Robertson M BioorgMed Chem Lett 2003 13 4043-4046
[165] Nelson D W Gregg R J Kort M E Perez-Medrano A Voight EA Wang Y Grayson G Namovic M T Donnelly-Roberts D LNiforatos W Honore P Jarvis M F Faltynek C R Carroll W AJ Med Chem 2006 49 3659-3666
[143] Caffrey M Rhonan F Pimm A WO2004074224A1 2004[144] Evans R Rhonan F Thompson T Willis P WO2006059945A1
2006 [166] Perez-Medrano A Nelson D W Carroll W A Kort M E Gregg RJ Voight E A Jarvis M F Kowaluk E A WO2006086229A12006
[145] Rhonan F Martin B Thompson T Tomkinson N Willis PWO2006025783A1 2006
[146] Cladingboel D Rhonan F Willis P WO2005014529A1 2005 [167] Carroll W A Perez-Medrano A Florjancic A S Nelson D WPeddi S Li T Bunnelle E M Hirst G Li B CWO20060052374A1 2006
[147] Duplantier A J EP1310493A1 2003[148] Baxter A Kindon N Pairaudeau G Roberts B Thom S
WO0144213A1 2001 [168] Carroll W A Perez-Medrano A Florjancic A S Nelson D WPeddi S Bunnelle E M Hirst G C Li B WO2005111003A12005
[149] Meghani P Bennion C WO0146200A1 2001[150] Li Z B WO20050288256A1 2005[151] Leonard J A Li Z J Li Z B Urban F J WO20050288288A1
2005[169] Shemon A N Sluyter R Conigrave A D Wiley J S Br J
Pharmacol 2004 142 1015-1019[152] Leonard J A Li Z J Li Z B Urban F J WO2006003513A1
2006[170] Suadicani S O Brosnan C F Scemes E J Neurosci 2006 26
1378-1385[153] Li Z B WO2006003500A1 2006 [171] Saez J C Retamal M A Basilio D Bukauskas F F Bennett M
V L Biochim Biophys Acta 2005 1711 215-224[154] Evans R Eyssade C Rhonan F Martin B Thompson T WillisP WO2004106305A1 2004 [172] Michel A D Xing M Thompson K M Jones C A Humphrey P
P A Eur J Pharmacol 2006 534 19-29[155] Rhonan F Thompson T Willis P WO2005009968A1 2005[156] Furber M Luker T J Mortimore M P Thorne P Meghani P
WO0071529A1 2000[173] Buell G Chessell I P Michel A D Collo G Salazzo M Herren
S Gretener D Grahames C Kaur R Kosco-Vilbois M HHumphrey P P A Blood 1998 92 3521-3528[157] Thompson T Willis P WO2006080884A1 2006
[158] Concepcion A Inoue T Mochizuki Y Muramatsu A Gantner FNakashima K Urbhans K Bacon K B WO2005019182A1 2005
[174] Sanz J M Chiozzi P Di Virgilio F Eur J Pharmacol 1998 35 235-244
[159] Dombroski M A Duplantier A J Subramanyam CWO2004099146A1 2004
[175] Cario-Toumaniantz C Loirand G Ferrier L Pacaud P J Physiol1998 508 659-666
[160] Kelly M G Kincaid J WO2006102588A1 2006 [176] Ferrari D Pizzirani C Adinolfi E Forchap S Sitta B Turchet LFalzoni S Minelli M Baricordi R Di Virgilio F J Immunol 2004173 4652-4660
[161] Kelly M J Kincaid J WO2006102610A2 2006[162] Schwink L Stengelin S Gossel M Bohme T Hessler G Stahl P
Gretzke D WO2004072025A2 2004 [177] Nelson D W Jarvis M F Carroll W A WO2006110516A1 2006[163] Schwink L Stengelin S Boehme T Gossel M Hessler G Stahl P
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[179] Carroll W A Perez-Medrano A Jarvis M F Wang Y Peddi SWO20050171195A1 2005
Received February 01 2007 Revised April 04 2007 Accepted April 06 2007
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1511
as some P2Y receptor subtypes [96] In addition suramin hasbeen shown to interact with many different proteins at theseconcentrations including glutamate nicotinic GABA and 5-HTreceptors as well as various proteases and ectonucleotidases [396] Nevertheless SAR studies on suramin have been performedin order to identify the important structural features responsiblefor antagonism at the P2 receptors [96 118-124] Selected keysuramin derivatives are represented in Fig (10 )
native and recombinant P2X1 receptors respectively and hasbeen shown to be much less potent at other P2X and P2Ysubtypes including adrenoceptors histamine H1 receptors andthe muscarinic M3 receptor [120] At the human P2X1 and P2X7receptors the IC50 values for NF449 were 005 nM and 40 microMrespectively [128] which indicated its usefulness todistinguish between the two receptors Subsequent SAR studiesconfirmed that NF449 was the most potent and selective knownP2X1 receptor antagonist [121 129] NF157 an analogue ofsuramin with fluorine replacing the methyl group has also beenreported to be highly potent at the P2Y11 receptor with a Ki of45 nM [124] More recently NF110 has also been shown to be apotent P2X3 receptor antagonist indicating that both thenumber and location of the sulfonate groups may be importantfor determining P2X subtype potency and selectivity [123]
O
HN
HN
NH
NH
O O
O ONH HNSO3-
SO 3-
SO3-
-O3S
SO 3-
-O3S
23 suramin
NF279 thus far represents the most active suramin relatedantagonist at the P2X7R and displays a reasonable potency of28 microM All the SAR data obtained to date on suramindemonstrate the importance of the molecular size number andposition of the sulfonate groups for P2X subtype potency andselectivity Unfortunately except for NF279 and NF449 othersuramin analogues provide very little information which can beused for the selective structure-based drug design of P2X7Rmolecules Suramin is also unlikely to be orally active due toits high molecular weight and interaction with many otherproteins Suramin is a very weak or almost inactive antagonistat the human P2X7R with an IC50 value of 92 microM [40] It istotally inactive at both the rat and mouse P2X7R with IC50values greater than 100 microM [34 60] Due to its interference withthe fluorescence of YO-PRO-1 suramin could not be used in theYO-PRO-1 dye uptake assay [104]
Fig (9) Chemical structure for suramin a weak antagonist at the P2X7R
Early SAR analysis showed that the shortened forms ofsuramin in the form of sulfonylbenzamides decreased thepotency at P2 receptors [118] NF023 (24 ) was later discoveredto be P2X-selective compared to P2Y receptors andectonucleotidases [118] Subsequent studies have examined theeffects of NF023 in various cell types and it was reported thatNF023 is a highly selective antagonist for either rat or humanP2X1 receptors heterologously expressed in Xenopus oocyteswith an IC50 of 02 microM [125] Another suramin derivativeNF279 (25 ) also showed potent selective reversible inhibitionat the rat P2X1 receptor in Xenopus oocytes with an IC50 of 19nM [126] In studying the effects of NF279 on human P2X1 andP2X7 receptors it was reported that NF279 is almost 100-foldmore potent at the human P2X1 than the P2X7 receptor withIC50 values of 005 and 28 microM respectively [127] Other keysuramin derivatives resulting from extensive SAR studies areNF449 (27 ) NF110 (28 ) and NF157 (26 ) [120 121 123 124128 129] NF449 has an IC50 value of 70 nM and 03 nM at the
Brilliant Blue G 29 (Fig 11) is another polysulfonated dyewhich is a potent non-competitive inhibitor of the rat andhuman P2X7R with IC50 values of 10 and 200 nM respectivelyin electrophysiological studies [130] Brilliant Blue G has alsobeen shown to be highly selective for P2X7R over P2X1-5receptors [130] It has been shown that at the rat P2X7R about100 to 300 nM concentration was required to inhibit the YO-PRO-1 uptake which was 10- to 30-fold higher than thatrequired for blocking P2X7R-induced currents demonstratingits differential effect at the channel and pore forms of the P2X7R
O
HN
HN
NH
NH
O O
R R
O ONH HNSO3-
SO3-
SO3-
-O3S
SO3-
-O3S
O
HN
HN
NH
OO
NH
O ONH
NH
R R
RR
n n
Suramin n=1 R=CH3
24 NF023 n=0 R=H
25 NF279 n=1 R=H
26 NF157 n=1 R=F
27 NF449 R=24-disulfonyl28 NF110 R=4-sulfonyl
Fig (10) Suramin and its key derivatives investigated in the SAR studies
1512 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
[130] Brilliant Blue G has also been tested on the mouseP2X7R where similar antagonistic potency was found comparedto rat and human receptors [42] In this study it was alsoreported that the selectivity of Brilliant Blue G for rat overhuman P2X7R was confirmed at 22degC but lost at 37degC Thedrawback for this compound is that it could also be bound toproteins and thus would be expected to have lower potency inthe dye uptake assay than electrophysiology measurements dueto the depletion by cell monolayer effect [42] Nevertheless itrepresents a promising antagonist at rat human and mouseanalogues of the P2X7R No studies have been performed onBrilliant Blue G to further improve its potency and selectivityat the P2X7R Other polysulfonated dyes such as Evans BlueTrypan Blue reactive blue 2 reactive red 2 and their derivativeshave also been tested at the purinergic systems [131-133] butnone of them exhibited activity at the P2X7R
opening of the ion channel calmidazolium produces no effecton the YO-PRO-1 uptake The large non-selective porehowever is still formed even when the ionic currents areinhibited indicating that pore formation does not necessarilyrequire opening of the ion channels Calmidazolium wassuggested to be an allosteric modulator of the P2X7R at theagonist binding site inducing conformational change at thereceptor level that results in decreased agonist binding affinity[84] It displays less potency at inhibiting the agonist-evokedcurrents at both the mouse and human P2X7R with IC50 valuesof 1 microM and 100 nM respectively [41 88] At present no SARstudies have been reported on calmidazolium
N
N+
ClCl
O
ClCl
Cl
Cl
30 calmidazolium
NH
N
O
SO3-
N+
SO3-
Cl
29 Coomassie Brilliant Blue G
Fig (12) Chemical structure for calmidazolium a P2X7R antagonistcapable of distinguishing the channel and pore forms of the receptor
36 KN-62 and its Analogues
KN-62 31 (Fig 13) is a derivative of isoquinolinesul-fonamide known as a selective and potent inhibitor of themultifunctional calciumcalmodulin-dependent protein kinases(CaMK) with an IC50 of 09 microM at CaMK II [134] Gargett andWiley in 1997 first demonstrated that both KN-62 31 and itsanalogue KN-04 32 were potent inhibitors of P2X7R at humanlymphocytes with IC50 values of 13 and 17-37 nM respectivelyin both the ion flux and ethidium bromide uptake assays [134]The mode of inhibition is allosteric modulation similar to thatof calmidazolium
Fig (11) Chemical structure for Brilliant Blue G a potent antagonist at therat P2X7R
35 Calmidazolium
Extracellular calmidazolium 30 (Fig 12) is a potentinhibitor of BzATP-induced currents via binding to rat P2X7Rwith an IC50 of 13 nM [84] The unique feature ofcalmidazolium is its ability to differentiate between the channeland pore form of the P2X7R while it potently inhibits the
KN-62 and KN-04 are species-selective for P2X7Rantagonism They potently inhibited the ATP-induced currentsas well as the ethidium bromide uptake in HEK293 cells
Fig (13) Chemical structures for isoquinolines KN-62 and KN-04 potent P2X7R antagonists at the hP2X7R with much less effect on the rat and humananalogues
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1513
33e most active in the series but still 30-fold less potent than KN-62
Fig (14) Conformationally restricted analogues of KN-62 [136]
expressing human P2X7R at the concentration of 30 ndash 100 nMdepending on whether ATP or BzATP was used and the cellularbackground of the human P2X7R [135] However even up to 3microM concentration they had no effect at the rat P2X7R [135] andKN-62 was found to be at least 10-fold more potent at thehuman P2X7R than the recombinant mouse analogue [60] Theseisoquinolinesulfonamide derivatives may interact with theamino-terminal domain of the P2X7R since the introduction ofthe first 335 amino acids of the human P2X7R delivers KN-62sensitivity to the rat P2X7R and the counter-part chimera isfound to be insensitive to KN-62 [135] The IC50 at the humanP2X7R is 10-fold lower at 22degC than at 37degC in the ethidiumbromide uptake study [42]
tyrosine) 2) R2 (the tyrosine side chain) and 3) R3 (thesubstituent attached to the N-piperazine) (Fig 15) [137-139]
The effect of substitutions at the R1 R2 and R3 groups (Fig15 ) on the P2X7R antagonistic properties was compared on thebasis of inhibition of K+ release [137 138] At the R1 positionlarge hydrophobic moieties linked to the amino positionthrough sulfonamide (34a-b) or carbamate (34c) groups werepreferable for P2X7R inhibition In the series of derivativeswhere R2 = quinolinesulfonyl and R3 = t-butoxycarbonyl (Boc)introduction of carbobenzyloxy (Cbz) and quinolinesulfonylgroups at the R1 position were found to be the most active[137] In another series of derivatives where R1 = Cbz and R3 =Boc introduction of arylsulfonyl (34d-g) and benzoyl (34h )moieties at the R2 position were preferred [137] A free aminogroup at the R3 position (34i-j) andor opening of thepiperazine ring to an ethylene diamine greatly reduced theP2X7R activity Amongst the various acyl groups tested at theR3 position Boc and benzoyl groups (34k-m) were found to bepreferable but not sulfonyl (34n-q ) The presence of a methylgroup at the amino terminus of the tyrosine group is thereforenot an absolute pre-requisite for P2X7 antagonism [137]
R3 N N
O
N
O
R4
R1
R2
In another study undertaken by Chen et al to furtherexplore the SAR of 34m [138] it was found that nitro-substitution at the meta- or para- position of the Cbz group atR1 (34r-t) was well tolerated However introduction of anacetamido- or isothiocyanato- group at the Cbz group abolishedP2X7R antagonism (34uv) The original isoquinoline-5-sulfonyl moiety at the R2 position could be substituted withmeta- or para-substituted phenylsulfonyl groups such as tosyl(34r-t) 3-nitrophenylsulfonyl (34w) or 4-nitrophenylsulfonyl(34x) without adverse effects on the P2X7R antagonism [138]Substitution of the benzoyl group at the R3 position by a Boc(34y) or 4-aminobenzoyl group (34z) retained activity
Fig (15) General structure for the KN-62 derivatives (34a-z)
Extensive SAR studies have been performed on KN-62 inorder to enhance its potency and selectivity at the P2X7R Thefirst attempt to improve the antagonistic activity of KN-62 wasachieved by tethering the N-methyl of the tyrosine backbone tothe ortho-position of the proximal phenyl ring which resultedin a series of conformationally restricted KN-62 analogues withthe formula shown in Fig (14 ) [136] The constrained form ofKN-62 (31 vs 33a) was devoid of P2X7R antagonisticproperties Replacement of the isoquinoline-5-sulfonyl moiety(33a) with quinoline-5-sulfonyl (33b ) quinoline-8-sulfonyl(33c) or naphthalene (33d ) resulted in the loss of ability toinhibit the P2X7R-mediated Ca2+ influx [136] The most activecompound arising from this series was compound (33e) with anIC50 value of 316 nM which is 30-fold weaker than the originalKN-62 Taken together these results indicate that an extendedrather than folded conformation of KN-62 conferred by theincorporation of a methylene unit between the N-methyl of thetyrosine backbone to the ortho-position of the proximal phenylring is preferred at the P2X7R [136]
A study by Baraldi et al reported the systematic SAR profileof KN-62 specifically looking at modifications of thephenylpiperazine moiety (R2 position Fig 16 ) [139] In theinvestigation of the optimum chain linker between thepiperazine and phenyl groups it was found that one methylenespacer (35a) slightly improved the activity whereas a twomethylene spacer was not favourable (35b ) [139] The presenceof a basic nitrogen in the piperazine moiety is proven crucial forP2X7R antagonism as substitution of the piperazine ring withpiperidine (35a vs 35c) resulted in a 3-fold reduction of activity[139] Substitution of the phenyl ring with other heterocyclessuch as pyridine (35d ) or pyrimidine (35e) also decreased theP2X7R antagonistic property The para- position prefers a
Subsequent investigations on KN-62 involved extensivemodifications in three positions 1) R1 (the amino-terminus of
1514 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
small electron-withdrawing group eg fluorine (35f) and theantagonistic property decreases as the size of the electron-withdrawing group increases (35g 35h) A methyl- or nitro-group at the para- position (35i 35j) was beneficial foractivity but not evidenced with the methoxy (35k ) group As inthe case for para- substitution introduction of F Cl or CH3 butnot a methoxy (35l-o) at the ortho- position was well toleratedMoreover the meta- position could also tolerate a Cl or CF3group (35p 35q) The disubstituted compound 35r showedreduced antagonistic properties (IC50 = 1122 nM) possibly dueto the steric bulkiness of om-xylyl residue while the dichloroderivative 35s (IC50 = 34 nM) retained activity A slightdecrease in potency was observed when the methyl group on thenitrogen on the α-position of the tyrosine was removed (35n vs
35t) further confirming that this is not substantial for P2X7Rantagonism The most potent compound arising from this studywas 35f with an IC50 value of 13 nM at inhibiting calciuminflux in human monocytes compared to KN-62 which has anIC50 of 51 nM [139] It is therefore confirmed that thephenylpiperazine group of KN-62 is crucial for interaction withthe active site of the receptor and can be potentially improvedwith small halogen substitutions particularly in the paraposition
An ortho-methyl substituent on the N-phenyl ring of KN-62increased the potency by 3-fold (36 IC50 15 nM) [140]Radiolabelling of compound 36 with tritium and preliminarybinding studies to the P2X7R have also been achieved [140]
Table 2 Structures and P2X7R Antagonistic Properties of Selected KN-62 Derivatives [137138]
34a isoquinoline-5-SO2mdash isoquinoline-5-SO2mdash Bocmdash H 77 plusmn 24 ~40
34b quinoline-8-SO2mdash quinoline-8-SO2mdash Bocmdash H 61 plusmn 30 ND
34c Cbzmdash quinoline-8-SO2mdash Bocmdash H 53 plusmn 23 ~200
34d Cbzmdash PhSO2mdash Bocmdash H 59 plusmn 14 ND
34e Cbzmdash p-tolyl-SO2mdash Bocmdash H 71 plusmn 30 ~300
34f Cbzmdash p-OMe-PhSO2mdash Bocmdash H 62 plusmn 15 ND
34g Cbzmdash isoquinoline-5-SO2mdash Bocmdash H 43 ND
34h Cbzmdash benzoylmdash Bocmdash H 47 plusmn 21 ND
34i isoquinoline-5-SO2mdash isoquinoline-5-SO2mdash H H 14 plusmn 12 ND
34j quinoline-8-SO2mdash quinoline-8-SO2mdash H H 5 plusmn 9 ND
34k Cbzmdash PhSO2mdash Cbzmdash H 48 plusmn 29 ND
34l Cbzmdash PhSO2mdash benzoylmdash H 78 plusmn 22 ND
34m Cbzmdash isoquinoline-5-SO2mdash benzoylmdash H 85 plusmn 10 ~200
34n Cbzmdash PhSO2mdash p-tolyl-SO2mdash H 0 ND
34o Cbzmdash PhSO2mdash CH3SO2mdash H 0 ND
34p Cbzmdash PhSO2mdash PhSO2mdash H 0 ND
34q Cbzmdash PhSO2mdash p-OMe-PhSO2mdash H 0 ND
34r Cbzmdash p-tolyl-SO2mdash benzoylmdash H 71 plusmn 21 ND
34s p-NO2-Cbzmdash p-tolyl-SO2mdash benzoylmdash H 69 plusmn 25 ND
34t m-NO2-Cbzmdash p-tolyl-SO2mdash benzoylmdash H 72 plusmn 6 ND
34u m-acetamido-Cbzmdash p-tolyl-SO2mdash benzoylmdash H 9 plusmn 13 ND
34v m-isothiocyanato-Cbzmdash p-tolyl-SO2mdash benzoylmdash H minus4 plusmn 5 ND
34w Cbzmdash m-NO2-PhSO2mdash benzoylmdash H 75 plusmn 10 ND
34x Cbzmdash p-NO2-PhSO2mdash benzoylmdash H 78 plusmn 18 ~100
34y m-NO2-Cbzmdash p-tolyl-SO2mdash Bocmdash H 94 plusmn 28 ~100
34z Cbzmdash p-tolyl-SO2mdash p-NH2-benzoylmdash H 93 plusmn 3 gt 300
aAntagonist function was measured by the percent inhibition of the K+ release triggered by 3mM ATP in paired wells in the absence of antagonist bAntagonistic IC50values derived from dose-response curves obtained on the basis of inhibition of K+ release
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1515
R2 X N
O
N
O
S
R1
S
O
O
N
O
O
Nthe inhibition of BzATP-induced ethidium bromide uptakemediated by the P2X7R expressed endogenously on the humanpre-monocytic cell line THP-1 [142] Compound 37a (Table 4)was identified as the lead structure and modifications on theimide and 4-pyridyl moieties were investigated The cyclicimide moiety is proven essential for the P2X7R activity sincesubstitutions with simple amines acyclic amides or acycliccarbamates almost completely abolished the activity [142] Thering can be five- or six-membered in the forms of imides (37a-bh) carbamates (37c-d) thiazolidine-24-diones (37eg) andhydantoins (37f) Replacement of the 4-pyridyl moiety with 4-substituted benzamides (37g-h) retained the P2X7R activityThe presence of a 3rsquo-nitro substituent on the biphenyl subunitwas reported to convey optimum P2X7R activity The mode ofinhibition of these compounds seemed to be competitivealthough there were observable changes in the maximalresponse as the compound concentration was increased [142]
Fig (16) General structure for the KN-62 derivatives (35a-35s)
Unfortunately while these KN-62 analogues are potentiallyuseful tools to investigate the basic properties of the P2X7Rthey do not represent good starting points for the discovery oforally-active drugs due to 1) high molecular weight (gt700 Da)2) high lipophilicity (clogP gt 6) and 3) presence of ametabolically labile yet crucial sulfonate group [141]
These compounds represent a new class of ligands thatpossess activity to the P2X7R They appear good candidates asorally active drugs due to their relatively low molecular weight(~400 Da) and would be highly amenable for further SARstudies However despite the succinimides and thiazolidine-24-diones representing potent analogues they were found to bemetabolically and chemically fragile On the other handalthough the carbamates were less potent they displayedimproved stability Future SAR studies could usefully involveinvestigation of the biphenyl ring and the chain linker
37 Cyclic Imides and Analogues
Cyclic imides 37 (Fig 18) were discovered through a high-throughput screening of AstraZenecarsquos compound library for
Table 3 Structures and P2X7R Antagonistic Properties of Selected KN-62 Derivatives [139]
Entry R1 R2 X IC50 (nM)a
KN-62 mdashCH3 mdashPh N 511 plusmn 11
35a mdashCH3 mdashCH2Ph N 211 plusmn 08
35b mdashCH3 mdashCH2CH2Ph N 600 plusmn 27
35c mdashCH3 mdashCH2Ph CH 653 plusmn 08
35d mdashCH3 2-pyridinyl N 170 plusmn 39
35e mdashCH3 2-pyrimidinyl N 798 plusmn 07
35f mdashCH3 p-F-Phmdash N 13 plusmn 01
35g mdashCH3 p-Cl-Phmdash N 105 plusmn 11
35h mdashCH3 p-I-Phmdash N 176 plusmn 38
35i mdashCH3 p-Me-Phmdash N 135 plusmn 02
35j mdashCH3 p-NO2-Phmdash N 58 plusmn 06
35k mdashCH3 p-OMe-Phmdash N 132 plusmn 43
35l mdashCH3 o-F-Phmdash N 185 plusmn 04
35m mdashCH3 o-Cl-Phmdash N 158 plusmn 09
35n mdashCH3 o-Me-Phmdash N 151 plusmn 14
35o mdashCH3 o-OMe-Phmdash N 841 plusmn 5
35p mdashCH3 m-Cl-Phmdash N 141 plusmn 03
35q mdashCH3 m-CF3-Phmdash N 311 plusmn 12
35r mdashCH3 om-diMe-Phmdash N 1122 plusmn 25
35s mdashCH3 mp-diCl-Phmdash N 339 plusmn 08
35t H o-Me-Phmdash N 288 plusmn 03
aAntagonistic IC50 (nM)a values derived from dose-response curves on the basis of inhibition of Ca2+ flux in human monocytes
1516 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
N N
O
N
S
N
OO
OS O
O
N
R
31 R = H (KN-62)
36 R = 2-[3H]Me
AstraZeneca reported that no replacement for the adamantaneframework was found [141] Investigation of the chain linkerrequirements between the adamantane and the phenyl ring wasperformed where it was found that C- and N- methylationcarbonyl reduction and methylene removal (38d-g) all causedloss of potency One methylene unit extension (38h ) retainedthe potency whereas reversal of the amide connectivity (38i)slightly decreased the potency AstraZeneca prepared acombinatorial library by reacting commercially availableadamantanemethylamine with a large variety of acid chloridesor via standard amide coupling with carboxylic acids Thegeneral trends indicated that only aromatic amides had activityat the P2X7R [141] On the phenyl ring 23- and 25-disubstitutions resulted in better antagonists (38j-o) than the26- (38p ) 35- (38q ) or 24-disusbstitutions (38r) The ortho-position could also tolerate substituents such as Br OMe andMe (38s-u) A closer examination on the reverse amides (38i v-y) also revealed that 23- and 25-disubsitutions were stillpreferred for P2X7R inhibition The indazole amide (38z) wasidentified as a target lead molecule with a good metabolicprofile suitable for further optimization for clinical trial studies[141]
Fig (17) Radiolabeled analogue of KN-62 [140]
O
NY
X
O
Z
NO2
Adamantyl amides represent a class of simple moleculeswith excellent activity at the P2X7R The nature of theadamantane group could potentially be further investigatedalthough the original authors stated that no replacement for theadamantane group was found A number of patents onadamantyl-containing benzamides have been generated byAstraZeneca [143-146] and Pfizer [147] indicating the potentialof this ligand class for generating a truly potent selective andorally active P2X7R inhibitor Pfizer specifically incorporatedtriazinyl group into the adamantyl benzamide framework [147]Fig (18) General structure for the cyclic imides generated by
AstraZeneca Non-adamantane benzamides including piperidinyl andpiperazinyl benzamides [148 149] triazinyl benzamides [150-153] quinolinyl benzamides [154 155] 34-difluoroanilinebenzamides [156] aromatic acid-containing benzamides [157]pyrazolylmethyl benzamides [158] and other heteroarylamides[159-163] have also been patented by various otherpharmaceutical companies
38 Adamantyl Amides
Adamantyl amides were also discovered from a high-throughput screening of AstraZenecarsquos compound library forinhibition of the P2X7R-mediated ethidium bromide uptakeinduced by BzATP [141] N-Adamantan-1-ylmethyl-2-chloro-benzamide 38a was identified as the lead structure with anexcellent pA2 value of 81 The presence of an ortho-substituentis believed to cause a twist in the orientation of the benzamideessential for potent P2X7R antagonism whereas para-substituents (38bc) have deleterious effects on the P2X7Ractivity Selected compounds of this series are represented inTable 5
39 Diarylimidazolines
Diarylimidazolines represent another class of P2X7-activecompounds They were first discovered through a high-throughput screening of Aventisrsquo compound library forinhibition of BzATP-induced YO-PRO-1 uptake mediated byP2X7R expressed in U373 cells stably expressing the human
Table 4 Structures and P2X7R Antagonistic Properties of Selected Cyclic Imides [142]
Entry X Y Z IC50 ( M) pA2a
37a CH2 CO N - 69
37b (CH2)2 CO N - 75
37c O CH2 N 64
37d O (CH2)2 N 063 -
37e S CO N - 77
37f NH CO N 02 -
37g S CO C-CONHMe 016 -
37h CH2 CO C-CONMe2 025 -
a pA2 = the negative logarithm of a molar concentration of an antagonist that necessitates doubling of agonist concentration needed to elicit the original submaximalresponse obtained in the absence of antagonist
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1517
P2X7R [164] Compound 39a was identified as an initial hitfrom the screening with an IC50 value of 80 nM
equipotent Some degree of branching in the chain linker wasgenerally tolerated (39f-h) Configurational constraintsimposed on the chain linker as exemplified by 39i weretolerated but this was not the case for the cyclopropyl analogue39j
X
RO
NH
N
HN
38a-y 38z
Subsequent investigations focused on the substitutionpattern in the aryl ring for the benzyl (n=1) and phenethyl (n=2)series [164] Electron withdrawing and small neutral groupswere generally tolerable in both series (39k-r v-x) Howeverelectron-donating groups exemplified by OMe gave much lessactive compounds (39s-u) In the phenethyl seriessubstitutions at the ortho- and meta- positions were preferableto the corresponding para- substitutions (39kl vs 39m and39no vs 39p) Interestingly in the benzyl series meta- andpara- substitutions resulted in more active compounds than thecorresponding ortho analogues (39v vs 39wx)
Fig (19) Adamantyl amides generated by AstraZeneca
Investigation of the nature of the linker between thearomatic ring and the 2-position of the imidazole ring revealedthat a 3 or 4 carbon linker (39bc) caused a slight decrease in theactivity and no carbon linker (39d ) rendered the compoundinactive [164] One carbon linker with a methyl branching unit(39e) was tolerated It was reported that there was noenantiomeric preference of the methyl branch they were
The diarylimidazolines examined represent good candidatesfor orally active P2X7R inhibitors they are small and highlyamenable for SAR studies The 4- and 5-phenyl rings representattractive sites for further investigations
Table 5 Structures and P2X7R Antagonistic Properties of Selected Adamantyl Amides [141]
Entry R X pA2
38a 2-Cl CH2NHCO 81
38b 4-Cl CH2NHCO inactive
38c 24-Cl2 CH2NHCO 64
38d 2-Cl CHMeNHCO 64
38e 2-Cl CH2NMeCO 54
38f 2-Cl CH2NHCH2 54
38g 2-Cl NHCO inactive
38h 2-Cl (CH2)2NHCO 78
38i 2-Cl CH2CONH 63
38j 23-Cl2 CH2NHCO 88
38k 25-Cl2 CH2NHCO 83
38l 2-Cl-3-NH2 CH2NHCO 82
38m 2-Cl-5-OMe CH2NHCO 88
38n 2-Cl-5-OH CH2NHCO 80
38o 2-Cl-5-NH2 CH2NHCO 84
38p 26-Cl2 CH2NHCO 74
38q 35-Cl2 CH2NHCO 54
38r 24-Cl2 CH2NHCO 64
38s 2-Br CH2NHCO 80
38t 2-OMe CH2NHCO 71
38u 2-Me CH2NHCO 79
38v 2-Me CH2CONH 68
38w 2-Cl-5-OMe CH2CONH 72
38x 2-Me-3-OMe CH2CONH 83
38y 2-Me-5-OMe CH2CONH 80
1518 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
N
HN
XY
R
N
HN
N
HN
N
HN
( )n
39i39j
39a-ce-hk-w 39d
Fig (20) General structure of the diarylimidazolines generated by Aventis
Table 6 Structures and P2X7R Antagonistic Properties of Selected Diarylimidazolines [164]
Entry n X Y R IC50 ( M)a
39a 1 H H H 008
39b 2 H H H 054
39c 3 H H H 061
39e 0 (R)-Me H H 011
39f 1 (S)-Me H H 003
39g 1 Me Me H 001
39h 1 Ph H H 032
39k 1 H H 2-F 016
39l 1 H H 3-F 004
39m 1 H H 4-F 022
39n 1 H H 2-Cl 007
39o 1 H H 3-Cl 012
39p 1 H H 4-Cl gt1
39q 1 H H 2-Me 007
39r 1 H H 4-Me 219
39s 1 H H 2-OMe 083
39t 1 H H 3-OMe gt3
39u 1 H H 4-OMe gt3
39v 0 H H 2-F 058
39w 0 H H 3-F 008
39x 0 H H 4-F 008
aAntagonistic IC50 values derived from dose-response curves on the basis of inhibition of YO-PRO-1 dye uptake
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1519
R1N
N
NN
R2
R1
NN
NN
R2
N
N
NN
Cl
Cl
Cl
Cl
40b-t 40u-x40a
1
2
3
4
5
Fig (21) General structure of tetrazoles generated by Abbott Laboratories
310 1-Benzyl-5-Phenyltetrazoles potency The limited solubility of those compounds howeverposed a challenge to incorporate substitutions that couldincrease the solubility while maintaining activity A 3-pyridylgroup (40d ) was found to overcome this problem Moreover anextra 2-methyl substituent (40e) resulted in higher activitycompared to 40d A methylene extension (40f) caused a decreasein potency whereas other heterocycles such as 40g and 40hresulted in analogues with similar potency to 40d
1-Benzyl-5-phenyltetrazoles were discovered through ahigh-throughput screening by Abbott Laboratories for threedifferent P2X7R activities Ca2+ flux at the rat or human P2X7RYO-PRO-1 uptake and interleukin-1β release at human P2X7R[165-168] The initial hit from the screening was the tetrazole40a (Table 7) with a pIC50 value of 69 in inhibiting P2X7Rderived from the recombinant human cell line
Investigation of the nature of the substituent attached to the5-position of the tetrazole core showed that the substitutionpattern at this position was not well tolerated [165] Forexample unsubstituted mono-chlorosubstituted 25- and 34-
Initial SAR studies focused on the substituent attached tothe 1-position of the tetrazole core [165] Unsubstituted orortho-substituted benzylic group (40bc) exhibited good
Table 7 Structures and P2X7R Antagonistic Properties of Selected 1-benzyl-5-tetrazoles [165]
1520 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
disubstituted compounds (40i-m) pyridine and quinoline ring(40n-p ) analogues were not tolerated However moreconservative changes such as CF3 and F for Cl group (40q-t)resulted in an increase of potency
release and amplification of astrocytic Ca2+ signaling werereported to be due to the P2X7R and not Cx43 hemichannels[170]
Decavanadate a type of polymer of vanadium oxides hasbeen demonstrated to be a competitive rapid-acting antagonistat the P2X7R [172] It is capable of inhibiting the BzATP-stimulated ethidium bromide uptake in human mouse and ratP2X7R with pA2 values of 74 57 and 71 respectivelyDecavanadate has not been fully tested for its selectivity overother P2X receptors however it is suggested that this largemolecular species acts at the extracellular ATP binding site ofthe receptor consistent with the competitive mode of inhibition[172] A monoclonal antibody specific for the human P2X7Rhas also been shown to be a potent and selective antagonist ofthe receptor with an IC50 value of 5 nM in blocking the BzATP-evoked currents [173] Interestingly tenidap at 50 ndash 200 microMconcentration increases the sensitivity of mouse macrophagesto ATP-mediated cytotoxicity [174] 17-β-estradiol and notprogesterone or 17-α-estradiol has also been reported to blockthe agonist-induced currents at the human P2X7R with an IC50value of 3 microM in a non-genomic manner [175] Polymyxin B apotent antibiotic well-known for its lipopolysaccharide-neutralizing effects has been demonstrated to confer asynergistic effect when co-applied with ATP in a similar mannerto tenidap inducing Ca2+ influx ethidium bromide uptake andlactate dehydrogenase release via action at the P2X7R [176]
Tetrazoles with reversed connectivity (40u-x) were alsoevaluated where it was found that this connectivity reversal hadlittle effect on the potency [165] These tetrazoles were reportedto be slightly more active in the human P2X7R than the ratanalogue [165]
The tetrazole derivative 40d has been shown to be selectivefor other P2 receptors including P2X3 P2X4 and P2Y2 receptors[165] Pharmacological testing of this tetrazole suggested acompetitive mode of inhibition at the P2X7R and its efficacyhas also been evaluated in a model of neuropathic pain in rats[165] Overall these tetrazoles are found to be P2X7R-active andhighly adaptable to structural modifications These propertiesmake the tetrazoles an interesting class of compound for furtherinvestigating P2X7R activity
311 Other P2X7R-Active Ligands
Chelerythrine 41 (Fig 22) is a member of the benzophen-anthridine alkaloids known to inhibit protein kinase C withgood potency and selectivity (IC50 066 microM) as well as enzymessuch as alanine aminotransferase and Na+K+-ATPase [169] At10 microM concentrations chelerythrine non-competitivelyinhibited the BzATP-induced 86Rb+ efflux by 74 in human B-lymphocytes [169] Dye uptake studies could not be performeddue to the fluorescent nature of the compound It is yet to bedetermined whether chelerythrine inhibition of P2X7R involvesprotein kinase C [169]
More recently other distinct classes of P2X7-active ligandshave been reported and patented by the Abbott Laboratoriesacylhydrazides [177] cyanoamidines and cyanoguanidines[178 179]
4 CONCLUSIONS AND FUTURE DIRECTIONS
O
O
N
O
O
Cl
41
Although many structurally diverse class of compoundshave been reported as P2X7R antagonists with reasonablepotency the selectivity issue over other P2 receptors andproteins such as ecto-ATPases and ectonucleotidases stillprevails This is particularly an issue for oxidized ATP andsuramin SAR studies performed on PPADS and suramin mainlygave improved potency and selectivity for the P2X1 receptorIon channel blockers such as amilorides and stilbenederivatives only possess relatively low potency at the P2X7RFurther complications in studying these P2X7R antagonistsalso arise from the demonstrated species differences acrosshuman rat and mouse systems Brilliant Blue G is active for allthree species however at present there have been no SAR datareported Another fundamental factor that hampers the study ofP2X7R in detail is the relatively low potency and selectivity ofthe available agonist BzATP The discovery of a more potentselective P2X7R agonist would be ideal since in many studiesfull dose-response curves could not be generated due to the low
Fig (22) Chemical structure for chelerythrine an alkaloid withantagonistic properties at the human P2X7R
Gap junction channel blockers such as flufenamic acid 42 carbenoxolone 43 and mefloquine 44 (Fig 23) were all foundto be inhibitors of the BzATP-induced Ca2+ flux (IC50 values of655 175 and 25 nM respectively) and of YO-PRO-1 uptake in1321N1 astrocytoma cells stably expressing rat P2X7R [170]This finding poses a question to the general use of gap junctionchannel blockers as indicators of the participation of connexinhemichannels at the sites of transmitter release [171] The ATP
HN CF3
O OH
42 f lufenamic acid
HO
-O
O
O
H
OH
O
O-
N
HN
CF3
CF3
H O
H
43 carbenoxolone 44 mefloquine
Fig (23) Chemical structures for gap junction channel blockers with P2X7R antagonistic properties
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1521
potency of BzATP The nature of functional assays on theP2X7R is also found to be complex as ionic compositiontemperature and cell origins all affect the results demandingextra care be taken when antagonist potencies are directlycompared from different studies
[23] Li J Liu D Ke H Z Duncan R L Turner C H J Biol Chem2005 280 42952-42959
[24] Penolazzi L Bianchini E Lambertini E Baraldi P G RomagnoliR Piva R Gambari R J Biomed Sci 2005 12 1013-1020
[25] Gibbons S J Washburn K B Talamo B R J Auton Pharmacol2001 21 181-190
[26] Dehaye J P Moran A Marino A Arch Oral Biol 1999 44 S39-S43Four classes of small molecules with high affinity at the
P2X7R have been reported cyclic imides adamantyl amidesdiarylimidazolines and 1-benzyl-5-phenyltetrazoles Theseclasses of compounds are highly amenable to structuralmodifications indicating their good prospects as leadmolecules for further improving the potency and selectivity atP2X7R ultimately leading to orally active drugs To date norational drug design based on the knowledge of binding siteswithin this receptor has been undertaken This is a direct resultof the fact that no crystal structure or suitable homology modelhave been reported for this receptor The fact that the P2X7R canadopt either a channel or pore state causes even moredifficulties in studying receptor dynamics and at present theexact mechanism of channel-to-pore transition remains to beelucidated Undoubtedly the P2X7R is involved in severalprocesses relevant to chronic inflammation neurodegenerationand neuropathic pain and therefore discovery of more potentselective P2X7R antagonists would have a great impact in thepresent therapeutic window
[27] Li Q Luo X Zeng W Muallem S J Biol Chem 2003 27847554-47561
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ACKNOWLEDGEMENTS[38] Khakh B S Burnstock G Kennedy C King B F North A
Seguela P Voigt M Humphrey P P A Nat Rev Neurosci 20012 165-174
The authors would like to thank Dr Natasha Hungerford forproofreading the manuscript
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1998 124 1314-1320 [125] Soto F Lambrecht G Nickel P Stuhmer W Busch A ENeuropharmacology 1999 38 141-149[89] Bianchi B R Lynch K J Touma E Niforatos W Burgard E C
Alexander K M Park H S Yu H Metzger R Kowaluk E JarvisM F van Biesen T Eur J Pharmacol 1999 376 127-138
[126] Rettinger J Schmalzing G Damer S Muller G Nickel PLambrecht G Neuropharmacology 2000 39 2044-2053
[90] North R A Surprenant A Annu Rev Pharm Toxicol 2000 40 563-580
[127] Klapperstuck M Buttner C Nickel P Schmalzing G Lambrecht GMarkwardt F Eur J Pharmacol 2000 387 245-252
[91] Murgia M Hanau S Pizzo P Rippa M Di Virgilio F J BiolChem 1993 268 8199-8203
[128] Hulsmann M Nickel P Kassack M Schmalzing G Lambrecht GMarkwardt F Eur J Pharmacol 2003 470 1-7
[92] Craighead M W Middlehurst K M L LeFeuvre R Kimber IRothwell N J Neurosci Lett 2001 311 77-80
[129] Rettinger J Braun K Hochmann H Kassack M U Ullmann HNickel P Schmalzing G Lambrecht G Neuropharmacology 200548 461-468[93] Beigi R D Kertesy S B Aquilina G Dubyak G R Br J
Pharmacol 2003 140 507-519 [130] Jiang L-H Mackenzie A B North R A Surprenant A MolPharmacol 2000 58 82-88[94] DellAntonio G Quattrini A Cin E D Fulgenzi A Ferrero M E
Arthritis Rheum 2002 46 3378-3385 [131] Bultmann R Starke K Naunyn-Schmiedebergs Arch Pharmacol1995 352 477-482[95] DellAntonio G Quattrini A Cin E D Fulgenzi A Ferrero M E
Neurosci Lett 2002 327 87-90 [132] Tuluc F Bultmann R Glanzel M Frahm A W Starke K Naunyn-Schmiedebergs Arch Pharmacol 1998 357 111-120[96] Lambrecht G Braun K Damer S Ganso M Hildebrandt C
Ullmann H Kassack M U Nickel P Curr Pharm Des 2002 82371-2399
[133] Wittenburg H Bultmann R Pause B Ganter C Kurz G Starke KNaunyn-Schmiedebergs Arch Pharmacol 1996 354 491-497
[97] Garcia-Guzman M Soto F Gomez-Hernandez J M Lund P-EStuhmer W Mol Pharmacol 1997 51 109-118
[134] Gargett C E Wiley J S Br J Pharmacol 1997 120 1483-1490[135] Humphreys B D Virginio C Surprenant A Rice J Dubyak GR
Mol Pharmacol 1998 54 22-32[98] Kim Y-C Camaioni E Ziganshin A U Ji X-D King B FWildman S S Rychkov A Yoburn J Kim H Mohanram AHarden T K Boyer J L Burnstock G Jacobson K A Drug DevRes 1998 45 52-66
[136] Baraldi P G Romagnoli R Tabrizi M A Falzoni S Di Virgilio FBioorg Med Chem Lett 2000 10 681-684
[137] Ravi R G Kertesy S B Dubyak G R Jacobson K A Drug DevRes 2001 54 75-87[99] Jacobson K A Kim Y-C Wildman S S Mohanram A Harden T
K Boyer J L King B F Burnstock G J Med Chem 1998 41 2201-2206
[138] Chen W Ravi R G Kertesy S B Dubyak G R Jacobson K ABioconjug Chem 2002 13 1100-1111
[100] Kim Y-C Brown S G Harden T K Boyer J L Dubyak GKing B F Burnstock G Jacobson K A J Med Chem 2001 44 340-349
[139] Baraldi P G Nunez M D C A M Falzoni S Di Virgilio FRomagnoli R J Med Chem 2003 46 1318-1329
[140] Romagnoli R Baraldi P G Pavani M G Tabrizi M A MoormanA R Di Virgilio F Cattabriga E Pancaldi C Gessi S Borea P ABioorg Med Chem Lett 2004 14 5709-5712
[101] Brown S G Kim Y-C Kim S-A Jacobson K A Burnstock GKing B F Drug Dev Res 2001 53 281-291
[102] Lambrecht G Rettinger J Baumert H G Czeche S Damer SGanso M Hildebrandt C Niebel B Spatz-Kumbel G SchmalzingG Mutschler E Eur J Pharmacol 2000 387 R19-R21
[141] Baxter A Bent J Bowers K Braddock M Brough S Fagura MLawson M McInally T Mortimore M Robertson M Weaver RWebborn P Bioorg Med Chem Lett 2003 13 4047-4050
[103] Kim Y-C Lee J-S Sak K Marteau F Mamedova L BoeynaemsJ-M Jacobson K A Biochem Pharmacol 2005 70 266-274
[142] Alcaraz L Baxter A Bent J Bowers K Braddock M CladingboelD Donald D Fagura M Furber M Laurent C Lawson M
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1523
Mortimore M McCormick M Roberts N Robertson M BioorgMed Chem Lett 2003 13 4043-4046
[165] Nelson D W Gregg R J Kort M E Perez-Medrano A Voight EA Wang Y Grayson G Namovic M T Donnelly-Roberts D LNiforatos W Honore P Jarvis M F Faltynek C R Carroll W AJ Med Chem 2006 49 3659-3666
[143] Caffrey M Rhonan F Pimm A WO2004074224A1 2004[144] Evans R Rhonan F Thompson T Willis P WO2006059945A1
2006 [166] Perez-Medrano A Nelson D W Carroll W A Kort M E Gregg RJ Voight E A Jarvis M F Kowaluk E A WO2006086229A12006
[145] Rhonan F Martin B Thompson T Tomkinson N Willis PWO2006025783A1 2006
[146] Cladingboel D Rhonan F Willis P WO2005014529A1 2005 [167] Carroll W A Perez-Medrano A Florjancic A S Nelson D WPeddi S Li T Bunnelle E M Hirst G Li B CWO20060052374A1 2006
[147] Duplantier A J EP1310493A1 2003[148] Baxter A Kindon N Pairaudeau G Roberts B Thom S
WO0144213A1 2001 [168] Carroll W A Perez-Medrano A Florjancic A S Nelson D WPeddi S Bunnelle E M Hirst G C Li B WO2005111003A12005
[149] Meghani P Bennion C WO0146200A1 2001[150] Li Z B WO20050288256A1 2005[151] Leonard J A Li Z J Li Z B Urban F J WO20050288288A1
2005[169] Shemon A N Sluyter R Conigrave A D Wiley J S Br J
Pharmacol 2004 142 1015-1019[152] Leonard J A Li Z J Li Z B Urban F J WO2006003513A1
2006[170] Suadicani S O Brosnan C F Scemes E J Neurosci 2006 26
1378-1385[153] Li Z B WO2006003500A1 2006 [171] Saez J C Retamal M A Basilio D Bukauskas F F Bennett M
V L Biochim Biophys Acta 2005 1711 215-224[154] Evans R Eyssade C Rhonan F Martin B Thompson T WillisP WO2004106305A1 2004 [172] Michel A D Xing M Thompson K M Jones C A Humphrey P
P A Eur J Pharmacol 2006 534 19-29[155] Rhonan F Thompson T Willis P WO2005009968A1 2005[156] Furber M Luker T J Mortimore M P Thorne P Meghani P
WO0071529A1 2000[173] Buell G Chessell I P Michel A D Collo G Salazzo M Herren
S Gretener D Grahames C Kaur R Kosco-Vilbois M HHumphrey P P A Blood 1998 92 3521-3528[157] Thompson T Willis P WO2006080884A1 2006
[158] Concepcion A Inoue T Mochizuki Y Muramatsu A Gantner FNakashima K Urbhans K Bacon K B WO2005019182A1 2005
[174] Sanz J M Chiozzi P Di Virgilio F Eur J Pharmacol 1998 35 235-244
[159] Dombroski M A Duplantier A J Subramanyam CWO2004099146A1 2004
[175] Cario-Toumaniantz C Loirand G Ferrier L Pacaud P J Physiol1998 508 659-666
[160] Kelly M G Kincaid J WO2006102588A1 2006 [176] Ferrari D Pizzirani C Adinolfi E Forchap S Sitta B Turchet LFalzoni S Minelli M Baricordi R Di Virgilio F J Immunol 2004173 4652-4660
[161] Kelly M J Kincaid J WO2006102610A2 2006[162] Schwink L Stengelin S Gossel M Bohme T Hessler G Stahl P
Gretzke D WO2004072025A2 2004 [177] Nelson D W Jarvis M F Carroll W A WO2006110516A1 2006[163] Schwink L Stengelin S Boehme T Gossel M Hessler G Stahl P
WO2005070925A1 2005[178] Carroll W A Perez-Medrano A Peddi S Florjancic A S
WO20060025614A1 2006[164] Merriman G H Ma L Shum P McGarry D Volz F Sabol J S
Gross A Zhao Z Rampe D Wang L Wirtz-Brugger F Harris BA Macdonald D Bioorg Med Chem Lett 2005 15 435-438
[179] Carroll W A Perez-Medrano A Jarvis M F Wang Y Peddi SWO20050171195A1 2005
Received February 01 2007 Revised April 04 2007 Accepted April 06 2007
1512 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
[130] Brilliant Blue G has also been tested on the mouseP2X7R where similar antagonistic potency was found comparedto rat and human receptors [42] In this study it was alsoreported that the selectivity of Brilliant Blue G for rat overhuman P2X7R was confirmed at 22degC but lost at 37degC Thedrawback for this compound is that it could also be bound toproteins and thus would be expected to have lower potency inthe dye uptake assay than electrophysiology measurements dueto the depletion by cell monolayer effect [42] Nevertheless itrepresents a promising antagonist at rat human and mouseanalogues of the P2X7R No studies have been performed onBrilliant Blue G to further improve its potency and selectivityat the P2X7R Other polysulfonated dyes such as Evans BlueTrypan Blue reactive blue 2 reactive red 2 and their derivativeshave also been tested at the purinergic systems [131-133] butnone of them exhibited activity at the P2X7R
opening of the ion channel calmidazolium produces no effecton the YO-PRO-1 uptake The large non-selective porehowever is still formed even when the ionic currents areinhibited indicating that pore formation does not necessarilyrequire opening of the ion channels Calmidazolium wassuggested to be an allosteric modulator of the P2X7R at theagonist binding site inducing conformational change at thereceptor level that results in decreased agonist binding affinity[84] It displays less potency at inhibiting the agonist-evokedcurrents at both the mouse and human P2X7R with IC50 valuesof 1 microM and 100 nM respectively [41 88] At present no SARstudies have been reported on calmidazolium
N
N+
ClCl
O
ClCl
Cl
Cl
30 calmidazolium
NH
N
O
SO3-
N+
SO3-
Cl
29 Coomassie Brilliant Blue G
Fig (12) Chemical structure for calmidazolium a P2X7R antagonistcapable of distinguishing the channel and pore forms of the receptor
36 KN-62 and its Analogues
KN-62 31 (Fig 13) is a derivative of isoquinolinesul-fonamide known as a selective and potent inhibitor of themultifunctional calciumcalmodulin-dependent protein kinases(CaMK) with an IC50 of 09 microM at CaMK II [134] Gargett andWiley in 1997 first demonstrated that both KN-62 31 and itsanalogue KN-04 32 were potent inhibitors of P2X7R at humanlymphocytes with IC50 values of 13 and 17-37 nM respectivelyin both the ion flux and ethidium bromide uptake assays [134]The mode of inhibition is allosteric modulation similar to thatof calmidazolium
Fig (11) Chemical structure for Brilliant Blue G a potent antagonist at therat P2X7R
35 Calmidazolium
Extracellular calmidazolium 30 (Fig 12) is a potentinhibitor of BzATP-induced currents via binding to rat P2X7Rwith an IC50 of 13 nM [84] The unique feature ofcalmidazolium is its ability to differentiate between the channeland pore form of the P2X7R while it potently inhibits the
KN-62 and KN-04 are species-selective for P2X7Rantagonism They potently inhibited the ATP-induced currentsas well as the ethidium bromide uptake in HEK293 cells
Fig (13) Chemical structures for isoquinolines KN-62 and KN-04 potent P2X7R antagonists at the hP2X7R with much less effect on the rat and humananalogues
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1513
33e most active in the series but still 30-fold less potent than KN-62
Fig (14) Conformationally restricted analogues of KN-62 [136]
expressing human P2X7R at the concentration of 30 ndash 100 nMdepending on whether ATP or BzATP was used and the cellularbackground of the human P2X7R [135] However even up to 3microM concentration they had no effect at the rat P2X7R [135] andKN-62 was found to be at least 10-fold more potent at thehuman P2X7R than the recombinant mouse analogue [60] Theseisoquinolinesulfonamide derivatives may interact with theamino-terminal domain of the P2X7R since the introduction ofthe first 335 amino acids of the human P2X7R delivers KN-62sensitivity to the rat P2X7R and the counter-part chimera isfound to be insensitive to KN-62 [135] The IC50 at the humanP2X7R is 10-fold lower at 22degC than at 37degC in the ethidiumbromide uptake study [42]
tyrosine) 2) R2 (the tyrosine side chain) and 3) R3 (thesubstituent attached to the N-piperazine) (Fig 15) [137-139]
The effect of substitutions at the R1 R2 and R3 groups (Fig15 ) on the P2X7R antagonistic properties was compared on thebasis of inhibition of K+ release [137 138] At the R1 positionlarge hydrophobic moieties linked to the amino positionthrough sulfonamide (34a-b) or carbamate (34c) groups werepreferable for P2X7R inhibition In the series of derivativeswhere R2 = quinolinesulfonyl and R3 = t-butoxycarbonyl (Boc)introduction of carbobenzyloxy (Cbz) and quinolinesulfonylgroups at the R1 position were found to be the most active[137] In another series of derivatives where R1 = Cbz and R3 =Boc introduction of arylsulfonyl (34d-g) and benzoyl (34h )moieties at the R2 position were preferred [137] A free aminogroup at the R3 position (34i-j) andor opening of thepiperazine ring to an ethylene diamine greatly reduced theP2X7R activity Amongst the various acyl groups tested at theR3 position Boc and benzoyl groups (34k-m) were found to bepreferable but not sulfonyl (34n-q ) The presence of a methylgroup at the amino terminus of the tyrosine group is thereforenot an absolute pre-requisite for P2X7 antagonism [137]
R3 N N
O
N
O
R4
R1
R2
In another study undertaken by Chen et al to furtherexplore the SAR of 34m [138] it was found that nitro-substitution at the meta- or para- position of the Cbz group atR1 (34r-t) was well tolerated However introduction of anacetamido- or isothiocyanato- group at the Cbz group abolishedP2X7R antagonism (34uv) The original isoquinoline-5-sulfonyl moiety at the R2 position could be substituted withmeta- or para-substituted phenylsulfonyl groups such as tosyl(34r-t) 3-nitrophenylsulfonyl (34w) or 4-nitrophenylsulfonyl(34x) without adverse effects on the P2X7R antagonism [138]Substitution of the benzoyl group at the R3 position by a Boc(34y) or 4-aminobenzoyl group (34z) retained activity
Fig (15) General structure for the KN-62 derivatives (34a-z)
Extensive SAR studies have been performed on KN-62 inorder to enhance its potency and selectivity at the P2X7R Thefirst attempt to improve the antagonistic activity of KN-62 wasachieved by tethering the N-methyl of the tyrosine backbone tothe ortho-position of the proximal phenyl ring which resultedin a series of conformationally restricted KN-62 analogues withthe formula shown in Fig (14 ) [136] The constrained form ofKN-62 (31 vs 33a) was devoid of P2X7R antagonisticproperties Replacement of the isoquinoline-5-sulfonyl moiety(33a) with quinoline-5-sulfonyl (33b ) quinoline-8-sulfonyl(33c) or naphthalene (33d ) resulted in the loss of ability toinhibit the P2X7R-mediated Ca2+ influx [136] The most activecompound arising from this series was compound (33e) with anIC50 value of 316 nM which is 30-fold weaker than the originalKN-62 Taken together these results indicate that an extendedrather than folded conformation of KN-62 conferred by theincorporation of a methylene unit between the N-methyl of thetyrosine backbone to the ortho-position of the proximal phenylring is preferred at the P2X7R [136]
A study by Baraldi et al reported the systematic SAR profileof KN-62 specifically looking at modifications of thephenylpiperazine moiety (R2 position Fig 16 ) [139] In theinvestigation of the optimum chain linker between thepiperazine and phenyl groups it was found that one methylenespacer (35a) slightly improved the activity whereas a twomethylene spacer was not favourable (35b ) [139] The presenceof a basic nitrogen in the piperazine moiety is proven crucial forP2X7R antagonism as substitution of the piperazine ring withpiperidine (35a vs 35c) resulted in a 3-fold reduction of activity[139] Substitution of the phenyl ring with other heterocyclessuch as pyridine (35d ) or pyrimidine (35e) also decreased theP2X7R antagonistic property The para- position prefers a
Subsequent investigations on KN-62 involved extensivemodifications in three positions 1) R1 (the amino-terminus of
1514 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
small electron-withdrawing group eg fluorine (35f) and theantagonistic property decreases as the size of the electron-withdrawing group increases (35g 35h) A methyl- or nitro-group at the para- position (35i 35j) was beneficial foractivity but not evidenced with the methoxy (35k ) group As inthe case for para- substitution introduction of F Cl or CH3 butnot a methoxy (35l-o) at the ortho- position was well toleratedMoreover the meta- position could also tolerate a Cl or CF3group (35p 35q) The disubstituted compound 35r showedreduced antagonistic properties (IC50 = 1122 nM) possibly dueto the steric bulkiness of om-xylyl residue while the dichloroderivative 35s (IC50 = 34 nM) retained activity A slightdecrease in potency was observed when the methyl group on thenitrogen on the α-position of the tyrosine was removed (35n vs
35t) further confirming that this is not substantial for P2X7Rantagonism The most potent compound arising from this studywas 35f with an IC50 value of 13 nM at inhibiting calciuminflux in human monocytes compared to KN-62 which has anIC50 of 51 nM [139] It is therefore confirmed that thephenylpiperazine group of KN-62 is crucial for interaction withthe active site of the receptor and can be potentially improvedwith small halogen substitutions particularly in the paraposition
An ortho-methyl substituent on the N-phenyl ring of KN-62increased the potency by 3-fold (36 IC50 15 nM) [140]Radiolabelling of compound 36 with tritium and preliminarybinding studies to the P2X7R have also been achieved [140]
Table 2 Structures and P2X7R Antagonistic Properties of Selected KN-62 Derivatives [137138]
34a isoquinoline-5-SO2mdash isoquinoline-5-SO2mdash Bocmdash H 77 plusmn 24 ~40
34b quinoline-8-SO2mdash quinoline-8-SO2mdash Bocmdash H 61 plusmn 30 ND
34c Cbzmdash quinoline-8-SO2mdash Bocmdash H 53 plusmn 23 ~200
34d Cbzmdash PhSO2mdash Bocmdash H 59 plusmn 14 ND
34e Cbzmdash p-tolyl-SO2mdash Bocmdash H 71 plusmn 30 ~300
34f Cbzmdash p-OMe-PhSO2mdash Bocmdash H 62 plusmn 15 ND
34g Cbzmdash isoquinoline-5-SO2mdash Bocmdash H 43 ND
34h Cbzmdash benzoylmdash Bocmdash H 47 plusmn 21 ND
34i isoquinoline-5-SO2mdash isoquinoline-5-SO2mdash H H 14 plusmn 12 ND
34j quinoline-8-SO2mdash quinoline-8-SO2mdash H H 5 plusmn 9 ND
34k Cbzmdash PhSO2mdash Cbzmdash H 48 plusmn 29 ND
34l Cbzmdash PhSO2mdash benzoylmdash H 78 plusmn 22 ND
34m Cbzmdash isoquinoline-5-SO2mdash benzoylmdash H 85 plusmn 10 ~200
34n Cbzmdash PhSO2mdash p-tolyl-SO2mdash H 0 ND
34o Cbzmdash PhSO2mdash CH3SO2mdash H 0 ND
34p Cbzmdash PhSO2mdash PhSO2mdash H 0 ND
34q Cbzmdash PhSO2mdash p-OMe-PhSO2mdash H 0 ND
34r Cbzmdash p-tolyl-SO2mdash benzoylmdash H 71 plusmn 21 ND
34s p-NO2-Cbzmdash p-tolyl-SO2mdash benzoylmdash H 69 plusmn 25 ND
34t m-NO2-Cbzmdash p-tolyl-SO2mdash benzoylmdash H 72 plusmn 6 ND
34u m-acetamido-Cbzmdash p-tolyl-SO2mdash benzoylmdash H 9 plusmn 13 ND
34v m-isothiocyanato-Cbzmdash p-tolyl-SO2mdash benzoylmdash H minus4 plusmn 5 ND
34w Cbzmdash m-NO2-PhSO2mdash benzoylmdash H 75 plusmn 10 ND
34x Cbzmdash p-NO2-PhSO2mdash benzoylmdash H 78 plusmn 18 ~100
34y m-NO2-Cbzmdash p-tolyl-SO2mdash Bocmdash H 94 plusmn 28 ~100
34z Cbzmdash p-tolyl-SO2mdash p-NH2-benzoylmdash H 93 plusmn 3 gt 300
aAntagonist function was measured by the percent inhibition of the K+ release triggered by 3mM ATP in paired wells in the absence of antagonist bAntagonistic IC50values derived from dose-response curves obtained on the basis of inhibition of K+ release
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1515
R2 X N
O
N
O
S
R1
S
O
O
N
O
O
Nthe inhibition of BzATP-induced ethidium bromide uptakemediated by the P2X7R expressed endogenously on the humanpre-monocytic cell line THP-1 [142] Compound 37a (Table 4)was identified as the lead structure and modifications on theimide and 4-pyridyl moieties were investigated The cyclicimide moiety is proven essential for the P2X7R activity sincesubstitutions with simple amines acyclic amides or acycliccarbamates almost completely abolished the activity [142] Thering can be five- or six-membered in the forms of imides (37a-bh) carbamates (37c-d) thiazolidine-24-diones (37eg) andhydantoins (37f) Replacement of the 4-pyridyl moiety with 4-substituted benzamides (37g-h) retained the P2X7R activityThe presence of a 3rsquo-nitro substituent on the biphenyl subunitwas reported to convey optimum P2X7R activity The mode ofinhibition of these compounds seemed to be competitivealthough there were observable changes in the maximalresponse as the compound concentration was increased [142]
Fig (16) General structure for the KN-62 derivatives (35a-35s)
Unfortunately while these KN-62 analogues are potentiallyuseful tools to investigate the basic properties of the P2X7Rthey do not represent good starting points for the discovery oforally-active drugs due to 1) high molecular weight (gt700 Da)2) high lipophilicity (clogP gt 6) and 3) presence of ametabolically labile yet crucial sulfonate group [141]
These compounds represent a new class of ligands thatpossess activity to the P2X7R They appear good candidates asorally active drugs due to their relatively low molecular weight(~400 Da) and would be highly amenable for further SARstudies However despite the succinimides and thiazolidine-24-diones representing potent analogues they were found to bemetabolically and chemically fragile On the other handalthough the carbamates were less potent they displayedimproved stability Future SAR studies could usefully involveinvestigation of the biphenyl ring and the chain linker
37 Cyclic Imides and Analogues
Cyclic imides 37 (Fig 18) were discovered through a high-throughput screening of AstraZenecarsquos compound library for
Table 3 Structures and P2X7R Antagonistic Properties of Selected KN-62 Derivatives [139]
Entry R1 R2 X IC50 (nM)a
KN-62 mdashCH3 mdashPh N 511 plusmn 11
35a mdashCH3 mdashCH2Ph N 211 plusmn 08
35b mdashCH3 mdashCH2CH2Ph N 600 plusmn 27
35c mdashCH3 mdashCH2Ph CH 653 plusmn 08
35d mdashCH3 2-pyridinyl N 170 plusmn 39
35e mdashCH3 2-pyrimidinyl N 798 plusmn 07
35f mdashCH3 p-F-Phmdash N 13 plusmn 01
35g mdashCH3 p-Cl-Phmdash N 105 plusmn 11
35h mdashCH3 p-I-Phmdash N 176 plusmn 38
35i mdashCH3 p-Me-Phmdash N 135 plusmn 02
35j mdashCH3 p-NO2-Phmdash N 58 plusmn 06
35k mdashCH3 p-OMe-Phmdash N 132 plusmn 43
35l mdashCH3 o-F-Phmdash N 185 plusmn 04
35m mdashCH3 o-Cl-Phmdash N 158 plusmn 09
35n mdashCH3 o-Me-Phmdash N 151 plusmn 14
35o mdashCH3 o-OMe-Phmdash N 841 plusmn 5
35p mdashCH3 m-Cl-Phmdash N 141 plusmn 03
35q mdashCH3 m-CF3-Phmdash N 311 plusmn 12
35r mdashCH3 om-diMe-Phmdash N 1122 plusmn 25
35s mdashCH3 mp-diCl-Phmdash N 339 plusmn 08
35t H o-Me-Phmdash N 288 plusmn 03
aAntagonistic IC50 (nM)a values derived from dose-response curves on the basis of inhibition of Ca2+ flux in human monocytes
1516 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
N N
O
N
S
N
OO
OS O
O
N
R
31 R = H (KN-62)
36 R = 2-[3H]Me
AstraZeneca reported that no replacement for the adamantaneframework was found [141] Investigation of the chain linkerrequirements between the adamantane and the phenyl ring wasperformed where it was found that C- and N- methylationcarbonyl reduction and methylene removal (38d-g) all causedloss of potency One methylene unit extension (38h ) retainedthe potency whereas reversal of the amide connectivity (38i)slightly decreased the potency AstraZeneca prepared acombinatorial library by reacting commercially availableadamantanemethylamine with a large variety of acid chloridesor via standard amide coupling with carboxylic acids Thegeneral trends indicated that only aromatic amides had activityat the P2X7R [141] On the phenyl ring 23- and 25-disubstitutions resulted in better antagonists (38j-o) than the26- (38p ) 35- (38q ) or 24-disusbstitutions (38r) The ortho-position could also tolerate substituents such as Br OMe andMe (38s-u) A closer examination on the reverse amides (38i v-y) also revealed that 23- and 25-disubsitutions were stillpreferred for P2X7R inhibition The indazole amide (38z) wasidentified as a target lead molecule with a good metabolicprofile suitable for further optimization for clinical trial studies[141]
Fig (17) Radiolabeled analogue of KN-62 [140]
O
NY
X
O
Z
NO2
Adamantyl amides represent a class of simple moleculeswith excellent activity at the P2X7R The nature of theadamantane group could potentially be further investigatedalthough the original authors stated that no replacement for theadamantane group was found A number of patents onadamantyl-containing benzamides have been generated byAstraZeneca [143-146] and Pfizer [147] indicating the potentialof this ligand class for generating a truly potent selective andorally active P2X7R inhibitor Pfizer specifically incorporatedtriazinyl group into the adamantyl benzamide framework [147]Fig (18) General structure for the cyclic imides generated by
AstraZeneca Non-adamantane benzamides including piperidinyl andpiperazinyl benzamides [148 149] triazinyl benzamides [150-153] quinolinyl benzamides [154 155] 34-difluoroanilinebenzamides [156] aromatic acid-containing benzamides [157]pyrazolylmethyl benzamides [158] and other heteroarylamides[159-163] have also been patented by various otherpharmaceutical companies
38 Adamantyl Amides
Adamantyl amides were also discovered from a high-throughput screening of AstraZenecarsquos compound library forinhibition of the P2X7R-mediated ethidium bromide uptakeinduced by BzATP [141] N-Adamantan-1-ylmethyl-2-chloro-benzamide 38a was identified as the lead structure with anexcellent pA2 value of 81 The presence of an ortho-substituentis believed to cause a twist in the orientation of the benzamideessential for potent P2X7R antagonism whereas para-substituents (38bc) have deleterious effects on the P2X7Ractivity Selected compounds of this series are represented inTable 5
39 Diarylimidazolines
Diarylimidazolines represent another class of P2X7-activecompounds They were first discovered through a high-throughput screening of Aventisrsquo compound library forinhibition of BzATP-induced YO-PRO-1 uptake mediated byP2X7R expressed in U373 cells stably expressing the human
Table 4 Structures and P2X7R Antagonistic Properties of Selected Cyclic Imides [142]
Entry X Y Z IC50 ( M) pA2a
37a CH2 CO N - 69
37b (CH2)2 CO N - 75
37c O CH2 N 64
37d O (CH2)2 N 063 -
37e S CO N - 77
37f NH CO N 02 -
37g S CO C-CONHMe 016 -
37h CH2 CO C-CONMe2 025 -
a pA2 = the negative logarithm of a molar concentration of an antagonist that necessitates doubling of agonist concentration needed to elicit the original submaximalresponse obtained in the absence of antagonist
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1517
P2X7R [164] Compound 39a was identified as an initial hitfrom the screening with an IC50 value of 80 nM
equipotent Some degree of branching in the chain linker wasgenerally tolerated (39f-h) Configurational constraintsimposed on the chain linker as exemplified by 39i weretolerated but this was not the case for the cyclopropyl analogue39j
X
RO
NH
N
HN
38a-y 38z
Subsequent investigations focused on the substitutionpattern in the aryl ring for the benzyl (n=1) and phenethyl (n=2)series [164] Electron withdrawing and small neutral groupswere generally tolerable in both series (39k-r v-x) Howeverelectron-donating groups exemplified by OMe gave much lessactive compounds (39s-u) In the phenethyl seriessubstitutions at the ortho- and meta- positions were preferableto the corresponding para- substitutions (39kl vs 39m and39no vs 39p) Interestingly in the benzyl series meta- andpara- substitutions resulted in more active compounds than thecorresponding ortho analogues (39v vs 39wx)
Fig (19) Adamantyl amides generated by AstraZeneca
Investigation of the nature of the linker between thearomatic ring and the 2-position of the imidazole ring revealedthat a 3 or 4 carbon linker (39bc) caused a slight decrease in theactivity and no carbon linker (39d ) rendered the compoundinactive [164] One carbon linker with a methyl branching unit(39e) was tolerated It was reported that there was noenantiomeric preference of the methyl branch they were
The diarylimidazolines examined represent good candidatesfor orally active P2X7R inhibitors they are small and highlyamenable for SAR studies The 4- and 5-phenyl rings representattractive sites for further investigations
Table 5 Structures and P2X7R Antagonistic Properties of Selected Adamantyl Amides [141]
Entry R X pA2
38a 2-Cl CH2NHCO 81
38b 4-Cl CH2NHCO inactive
38c 24-Cl2 CH2NHCO 64
38d 2-Cl CHMeNHCO 64
38e 2-Cl CH2NMeCO 54
38f 2-Cl CH2NHCH2 54
38g 2-Cl NHCO inactive
38h 2-Cl (CH2)2NHCO 78
38i 2-Cl CH2CONH 63
38j 23-Cl2 CH2NHCO 88
38k 25-Cl2 CH2NHCO 83
38l 2-Cl-3-NH2 CH2NHCO 82
38m 2-Cl-5-OMe CH2NHCO 88
38n 2-Cl-5-OH CH2NHCO 80
38o 2-Cl-5-NH2 CH2NHCO 84
38p 26-Cl2 CH2NHCO 74
38q 35-Cl2 CH2NHCO 54
38r 24-Cl2 CH2NHCO 64
38s 2-Br CH2NHCO 80
38t 2-OMe CH2NHCO 71
38u 2-Me CH2NHCO 79
38v 2-Me CH2CONH 68
38w 2-Cl-5-OMe CH2CONH 72
38x 2-Me-3-OMe CH2CONH 83
38y 2-Me-5-OMe CH2CONH 80
1518 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
N
HN
XY
R
N
HN
N
HN
N
HN
( )n
39i39j
39a-ce-hk-w 39d
Fig (20) General structure of the diarylimidazolines generated by Aventis
Table 6 Structures and P2X7R Antagonistic Properties of Selected Diarylimidazolines [164]
Entry n X Y R IC50 ( M)a
39a 1 H H H 008
39b 2 H H H 054
39c 3 H H H 061
39e 0 (R)-Me H H 011
39f 1 (S)-Me H H 003
39g 1 Me Me H 001
39h 1 Ph H H 032
39k 1 H H 2-F 016
39l 1 H H 3-F 004
39m 1 H H 4-F 022
39n 1 H H 2-Cl 007
39o 1 H H 3-Cl 012
39p 1 H H 4-Cl gt1
39q 1 H H 2-Me 007
39r 1 H H 4-Me 219
39s 1 H H 2-OMe 083
39t 1 H H 3-OMe gt3
39u 1 H H 4-OMe gt3
39v 0 H H 2-F 058
39w 0 H H 3-F 008
39x 0 H H 4-F 008
aAntagonistic IC50 values derived from dose-response curves on the basis of inhibition of YO-PRO-1 dye uptake
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1519
R1N
N
NN
R2
R1
NN
NN
R2
N
N
NN
Cl
Cl
Cl
Cl
40b-t 40u-x40a
1
2
3
4
5
Fig (21) General structure of tetrazoles generated by Abbott Laboratories
310 1-Benzyl-5-Phenyltetrazoles potency The limited solubility of those compounds howeverposed a challenge to incorporate substitutions that couldincrease the solubility while maintaining activity A 3-pyridylgroup (40d ) was found to overcome this problem Moreover anextra 2-methyl substituent (40e) resulted in higher activitycompared to 40d A methylene extension (40f) caused a decreasein potency whereas other heterocycles such as 40g and 40hresulted in analogues with similar potency to 40d
1-Benzyl-5-phenyltetrazoles were discovered through ahigh-throughput screening by Abbott Laboratories for threedifferent P2X7R activities Ca2+ flux at the rat or human P2X7RYO-PRO-1 uptake and interleukin-1β release at human P2X7R[165-168] The initial hit from the screening was the tetrazole40a (Table 7) with a pIC50 value of 69 in inhibiting P2X7Rderived from the recombinant human cell line
Investigation of the nature of the substituent attached to the5-position of the tetrazole core showed that the substitutionpattern at this position was not well tolerated [165] Forexample unsubstituted mono-chlorosubstituted 25- and 34-
Initial SAR studies focused on the substituent attached tothe 1-position of the tetrazole core [165] Unsubstituted orortho-substituted benzylic group (40bc) exhibited good
Table 7 Structures and P2X7R Antagonistic Properties of Selected 1-benzyl-5-tetrazoles [165]
1520 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
disubstituted compounds (40i-m) pyridine and quinoline ring(40n-p ) analogues were not tolerated However moreconservative changes such as CF3 and F for Cl group (40q-t)resulted in an increase of potency
release and amplification of astrocytic Ca2+ signaling werereported to be due to the P2X7R and not Cx43 hemichannels[170]
Decavanadate a type of polymer of vanadium oxides hasbeen demonstrated to be a competitive rapid-acting antagonistat the P2X7R [172] It is capable of inhibiting the BzATP-stimulated ethidium bromide uptake in human mouse and ratP2X7R with pA2 values of 74 57 and 71 respectivelyDecavanadate has not been fully tested for its selectivity overother P2X receptors however it is suggested that this largemolecular species acts at the extracellular ATP binding site ofthe receptor consistent with the competitive mode of inhibition[172] A monoclonal antibody specific for the human P2X7Rhas also been shown to be a potent and selective antagonist ofthe receptor with an IC50 value of 5 nM in blocking the BzATP-evoked currents [173] Interestingly tenidap at 50 ndash 200 microMconcentration increases the sensitivity of mouse macrophagesto ATP-mediated cytotoxicity [174] 17-β-estradiol and notprogesterone or 17-α-estradiol has also been reported to blockthe agonist-induced currents at the human P2X7R with an IC50value of 3 microM in a non-genomic manner [175] Polymyxin B apotent antibiotic well-known for its lipopolysaccharide-neutralizing effects has been demonstrated to confer asynergistic effect when co-applied with ATP in a similar mannerto tenidap inducing Ca2+ influx ethidium bromide uptake andlactate dehydrogenase release via action at the P2X7R [176]
Tetrazoles with reversed connectivity (40u-x) were alsoevaluated where it was found that this connectivity reversal hadlittle effect on the potency [165] These tetrazoles were reportedto be slightly more active in the human P2X7R than the ratanalogue [165]
The tetrazole derivative 40d has been shown to be selectivefor other P2 receptors including P2X3 P2X4 and P2Y2 receptors[165] Pharmacological testing of this tetrazole suggested acompetitive mode of inhibition at the P2X7R and its efficacyhas also been evaluated in a model of neuropathic pain in rats[165] Overall these tetrazoles are found to be P2X7R-active andhighly adaptable to structural modifications These propertiesmake the tetrazoles an interesting class of compound for furtherinvestigating P2X7R activity
311 Other P2X7R-Active Ligands
Chelerythrine 41 (Fig 22) is a member of the benzophen-anthridine alkaloids known to inhibit protein kinase C withgood potency and selectivity (IC50 066 microM) as well as enzymessuch as alanine aminotransferase and Na+K+-ATPase [169] At10 microM concentrations chelerythrine non-competitivelyinhibited the BzATP-induced 86Rb+ efflux by 74 in human B-lymphocytes [169] Dye uptake studies could not be performeddue to the fluorescent nature of the compound It is yet to bedetermined whether chelerythrine inhibition of P2X7R involvesprotein kinase C [169]
More recently other distinct classes of P2X7-active ligandshave been reported and patented by the Abbott Laboratoriesacylhydrazides [177] cyanoamidines and cyanoguanidines[178 179]
4 CONCLUSIONS AND FUTURE DIRECTIONS
O
O
N
O
O
Cl
41
Although many structurally diverse class of compoundshave been reported as P2X7R antagonists with reasonablepotency the selectivity issue over other P2 receptors andproteins such as ecto-ATPases and ectonucleotidases stillprevails This is particularly an issue for oxidized ATP andsuramin SAR studies performed on PPADS and suramin mainlygave improved potency and selectivity for the P2X1 receptorIon channel blockers such as amilorides and stilbenederivatives only possess relatively low potency at the P2X7RFurther complications in studying these P2X7R antagonistsalso arise from the demonstrated species differences acrosshuman rat and mouse systems Brilliant Blue G is active for allthree species however at present there have been no SAR datareported Another fundamental factor that hampers the study ofP2X7R in detail is the relatively low potency and selectivity ofthe available agonist BzATP The discovery of a more potentselective P2X7R agonist would be ideal since in many studiesfull dose-response curves could not be generated due to the low
Fig (22) Chemical structure for chelerythrine an alkaloid withantagonistic properties at the human P2X7R
Gap junction channel blockers such as flufenamic acid 42 carbenoxolone 43 and mefloquine 44 (Fig 23) were all foundto be inhibitors of the BzATP-induced Ca2+ flux (IC50 values of655 175 and 25 nM respectively) and of YO-PRO-1 uptake in1321N1 astrocytoma cells stably expressing rat P2X7R [170]This finding poses a question to the general use of gap junctionchannel blockers as indicators of the participation of connexinhemichannels at the sites of transmitter release [171] The ATP
HN CF3
O OH
42 f lufenamic acid
HO
-O
O
O
H
OH
O
O-
N
HN
CF3
CF3
H O
H
43 carbenoxolone 44 mefloquine
Fig (23) Chemical structures for gap junction channel blockers with P2X7R antagonistic properties
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1521
potency of BzATP The nature of functional assays on theP2X7R is also found to be complex as ionic compositiontemperature and cell origins all affect the results demandingextra care be taken when antagonist potencies are directlycompared from different studies
[23] Li J Liu D Ke H Z Duncan R L Turner C H J Biol Chem2005 280 42952-42959
[24] Penolazzi L Bianchini E Lambertini E Baraldi P G RomagnoliR Piva R Gambari R J Biomed Sci 2005 12 1013-1020
[25] Gibbons S J Washburn K B Talamo B R J Auton Pharmacol2001 21 181-190
[26] Dehaye J P Moran A Marino A Arch Oral Biol 1999 44 S39-S43Four classes of small molecules with high affinity at the
P2X7R have been reported cyclic imides adamantyl amidesdiarylimidazolines and 1-benzyl-5-phenyltetrazoles Theseclasses of compounds are highly amenable to structuralmodifications indicating their good prospects as leadmolecules for further improving the potency and selectivity atP2X7R ultimately leading to orally active drugs To date norational drug design based on the knowledge of binding siteswithin this receptor has been undertaken This is a direct resultof the fact that no crystal structure or suitable homology modelhave been reported for this receptor The fact that the P2X7R canadopt either a channel or pore state causes even moredifficulties in studying receptor dynamics and at present theexact mechanism of channel-to-pore transition remains to beelucidated Undoubtedly the P2X7R is involved in severalprocesses relevant to chronic inflammation neurodegenerationand neuropathic pain and therefore discovery of more potentselective P2X7R antagonists would have a great impact in thepresent therapeutic window
[27] Li Q Luo X Zeng W Muallem S J Biol Chem 2003 27847554-47561
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ACKNOWLEDGEMENTS[38] Khakh B S Burnstock G Kennedy C King B F North A
Seguela P Voigt M Humphrey P P A Nat Rev Neurosci 20012 165-174
The authors would like to thank Dr Natasha Hungerford forproofreading the manuscript
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[138] Chen W Ravi R G Kertesy S B Dubyak G R Jacobson K ABioconjug Chem 2002 13 1100-1111
[100] Kim Y-C Brown S G Harden T K Boyer J L Dubyak GKing B F Burnstock G Jacobson K A J Med Chem 2001 44 340-349
[139] Baraldi P G Nunez M D C A M Falzoni S Di Virgilio FRomagnoli R J Med Chem 2003 46 1318-1329
[140] Romagnoli R Baraldi P G Pavani M G Tabrizi M A MoormanA R Di Virgilio F Cattabriga E Pancaldi C Gessi S Borea P ABioorg Med Chem Lett 2004 14 5709-5712
[101] Brown S G Kim Y-C Kim S-A Jacobson K A Burnstock GKing B F Drug Dev Res 2001 53 281-291
[102] Lambrecht G Rettinger J Baumert H G Czeche S Damer SGanso M Hildebrandt C Niebel B Spatz-Kumbel G SchmalzingG Mutschler E Eur J Pharmacol 2000 387 R19-R21
[141] Baxter A Bent J Bowers K Braddock M Brough S Fagura MLawson M McInally T Mortimore M Robertson M Weaver RWebborn P Bioorg Med Chem Lett 2003 13 4047-4050
[103] Kim Y-C Lee J-S Sak K Marteau F Mamedova L BoeynaemsJ-M Jacobson K A Biochem Pharmacol 2005 70 266-274
[142] Alcaraz L Baxter A Bent J Bowers K Braddock M CladingboelD Donald D Fagura M Furber M Laurent C Lawson M
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1523
Mortimore M McCormick M Roberts N Robertson M BioorgMed Chem Lett 2003 13 4043-4046
[165] Nelson D W Gregg R J Kort M E Perez-Medrano A Voight EA Wang Y Grayson G Namovic M T Donnelly-Roberts D LNiforatos W Honore P Jarvis M F Faltynek C R Carroll W AJ Med Chem 2006 49 3659-3666
[143] Caffrey M Rhonan F Pimm A WO2004074224A1 2004[144] Evans R Rhonan F Thompson T Willis P WO2006059945A1
2006 [166] Perez-Medrano A Nelson D W Carroll W A Kort M E Gregg RJ Voight E A Jarvis M F Kowaluk E A WO2006086229A12006
[145] Rhonan F Martin B Thompson T Tomkinson N Willis PWO2006025783A1 2006
[146] Cladingboel D Rhonan F Willis P WO2005014529A1 2005 [167] Carroll W A Perez-Medrano A Florjancic A S Nelson D WPeddi S Li T Bunnelle E M Hirst G Li B CWO20060052374A1 2006
[147] Duplantier A J EP1310493A1 2003[148] Baxter A Kindon N Pairaudeau G Roberts B Thom S
WO0144213A1 2001 [168] Carroll W A Perez-Medrano A Florjancic A S Nelson D WPeddi S Bunnelle E M Hirst G C Li B WO2005111003A12005
[149] Meghani P Bennion C WO0146200A1 2001[150] Li Z B WO20050288256A1 2005[151] Leonard J A Li Z J Li Z B Urban F J WO20050288288A1
2005[169] Shemon A N Sluyter R Conigrave A D Wiley J S Br J
Pharmacol 2004 142 1015-1019[152] Leonard J A Li Z J Li Z B Urban F J WO2006003513A1
2006[170] Suadicani S O Brosnan C F Scemes E J Neurosci 2006 26
1378-1385[153] Li Z B WO2006003500A1 2006 [171] Saez J C Retamal M A Basilio D Bukauskas F F Bennett M
V L Biochim Biophys Acta 2005 1711 215-224[154] Evans R Eyssade C Rhonan F Martin B Thompson T WillisP WO2004106305A1 2004 [172] Michel A D Xing M Thompson K M Jones C A Humphrey P
P A Eur J Pharmacol 2006 534 19-29[155] Rhonan F Thompson T Willis P WO2005009968A1 2005[156] Furber M Luker T J Mortimore M P Thorne P Meghani P
WO0071529A1 2000[173] Buell G Chessell I P Michel A D Collo G Salazzo M Herren
S Gretener D Grahames C Kaur R Kosco-Vilbois M HHumphrey P P A Blood 1998 92 3521-3528[157] Thompson T Willis P WO2006080884A1 2006
[158] Concepcion A Inoue T Mochizuki Y Muramatsu A Gantner FNakashima K Urbhans K Bacon K B WO2005019182A1 2005
[174] Sanz J M Chiozzi P Di Virgilio F Eur J Pharmacol 1998 35 235-244
[159] Dombroski M A Duplantier A J Subramanyam CWO2004099146A1 2004
[175] Cario-Toumaniantz C Loirand G Ferrier L Pacaud P J Physiol1998 508 659-666
[160] Kelly M G Kincaid J WO2006102588A1 2006 [176] Ferrari D Pizzirani C Adinolfi E Forchap S Sitta B Turchet LFalzoni S Minelli M Baricordi R Di Virgilio F J Immunol 2004173 4652-4660
[161] Kelly M J Kincaid J WO2006102610A2 2006[162] Schwink L Stengelin S Gossel M Bohme T Hessler G Stahl P
Gretzke D WO2004072025A2 2004 [177] Nelson D W Jarvis M F Carroll W A WO2006110516A1 2006[163] Schwink L Stengelin S Boehme T Gossel M Hessler G Stahl P
WO2005070925A1 2005[178] Carroll W A Perez-Medrano A Peddi S Florjancic A S
WO20060025614A1 2006[164] Merriman G H Ma L Shum P McGarry D Volz F Sabol J S
Gross A Zhao Z Rampe D Wang L Wirtz-Brugger F Harris BA Macdonald D Bioorg Med Chem Lett 2005 15 435-438
[179] Carroll W A Perez-Medrano A Jarvis M F Wang Y Peddi SWO20050171195A1 2005
Received February 01 2007 Revised April 04 2007 Accepted April 06 2007
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1513
33e most active in the series but still 30-fold less potent than KN-62
Fig (14) Conformationally restricted analogues of KN-62 [136]
expressing human P2X7R at the concentration of 30 ndash 100 nMdepending on whether ATP or BzATP was used and the cellularbackground of the human P2X7R [135] However even up to 3microM concentration they had no effect at the rat P2X7R [135] andKN-62 was found to be at least 10-fold more potent at thehuman P2X7R than the recombinant mouse analogue [60] Theseisoquinolinesulfonamide derivatives may interact with theamino-terminal domain of the P2X7R since the introduction ofthe first 335 amino acids of the human P2X7R delivers KN-62sensitivity to the rat P2X7R and the counter-part chimera isfound to be insensitive to KN-62 [135] The IC50 at the humanP2X7R is 10-fold lower at 22degC than at 37degC in the ethidiumbromide uptake study [42]
tyrosine) 2) R2 (the tyrosine side chain) and 3) R3 (thesubstituent attached to the N-piperazine) (Fig 15) [137-139]
The effect of substitutions at the R1 R2 and R3 groups (Fig15 ) on the P2X7R antagonistic properties was compared on thebasis of inhibition of K+ release [137 138] At the R1 positionlarge hydrophobic moieties linked to the amino positionthrough sulfonamide (34a-b) or carbamate (34c) groups werepreferable for P2X7R inhibition In the series of derivativeswhere R2 = quinolinesulfonyl and R3 = t-butoxycarbonyl (Boc)introduction of carbobenzyloxy (Cbz) and quinolinesulfonylgroups at the R1 position were found to be the most active[137] In another series of derivatives where R1 = Cbz and R3 =Boc introduction of arylsulfonyl (34d-g) and benzoyl (34h )moieties at the R2 position were preferred [137] A free aminogroup at the R3 position (34i-j) andor opening of thepiperazine ring to an ethylene diamine greatly reduced theP2X7R activity Amongst the various acyl groups tested at theR3 position Boc and benzoyl groups (34k-m) were found to bepreferable but not sulfonyl (34n-q ) The presence of a methylgroup at the amino terminus of the tyrosine group is thereforenot an absolute pre-requisite for P2X7 antagonism [137]
R3 N N
O
N
O
R4
R1
R2
In another study undertaken by Chen et al to furtherexplore the SAR of 34m [138] it was found that nitro-substitution at the meta- or para- position of the Cbz group atR1 (34r-t) was well tolerated However introduction of anacetamido- or isothiocyanato- group at the Cbz group abolishedP2X7R antagonism (34uv) The original isoquinoline-5-sulfonyl moiety at the R2 position could be substituted withmeta- or para-substituted phenylsulfonyl groups such as tosyl(34r-t) 3-nitrophenylsulfonyl (34w) or 4-nitrophenylsulfonyl(34x) without adverse effects on the P2X7R antagonism [138]Substitution of the benzoyl group at the R3 position by a Boc(34y) or 4-aminobenzoyl group (34z) retained activity
Fig (15) General structure for the KN-62 derivatives (34a-z)
Extensive SAR studies have been performed on KN-62 inorder to enhance its potency and selectivity at the P2X7R Thefirst attempt to improve the antagonistic activity of KN-62 wasachieved by tethering the N-methyl of the tyrosine backbone tothe ortho-position of the proximal phenyl ring which resultedin a series of conformationally restricted KN-62 analogues withthe formula shown in Fig (14 ) [136] The constrained form ofKN-62 (31 vs 33a) was devoid of P2X7R antagonisticproperties Replacement of the isoquinoline-5-sulfonyl moiety(33a) with quinoline-5-sulfonyl (33b ) quinoline-8-sulfonyl(33c) or naphthalene (33d ) resulted in the loss of ability toinhibit the P2X7R-mediated Ca2+ influx [136] The most activecompound arising from this series was compound (33e) with anIC50 value of 316 nM which is 30-fold weaker than the originalKN-62 Taken together these results indicate that an extendedrather than folded conformation of KN-62 conferred by theincorporation of a methylene unit between the N-methyl of thetyrosine backbone to the ortho-position of the proximal phenylring is preferred at the P2X7R [136]
A study by Baraldi et al reported the systematic SAR profileof KN-62 specifically looking at modifications of thephenylpiperazine moiety (R2 position Fig 16 ) [139] In theinvestigation of the optimum chain linker between thepiperazine and phenyl groups it was found that one methylenespacer (35a) slightly improved the activity whereas a twomethylene spacer was not favourable (35b ) [139] The presenceof a basic nitrogen in the piperazine moiety is proven crucial forP2X7R antagonism as substitution of the piperazine ring withpiperidine (35a vs 35c) resulted in a 3-fold reduction of activity[139] Substitution of the phenyl ring with other heterocyclessuch as pyridine (35d ) or pyrimidine (35e) also decreased theP2X7R antagonistic property The para- position prefers a
Subsequent investigations on KN-62 involved extensivemodifications in three positions 1) R1 (the amino-terminus of
1514 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
small electron-withdrawing group eg fluorine (35f) and theantagonistic property decreases as the size of the electron-withdrawing group increases (35g 35h) A methyl- or nitro-group at the para- position (35i 35j) was beneficial foractivity but not evidenced with the methoxy (35k ) group As inthe case for para- substitution introduction of F Cl or CH3 butnot a methoxy (35l-o) at the ortho- position was well toleratedMoreover the meta- position could also tolerate a Cl or CF3group (35p 35q) The disubstituted compound 35r showedreduced antagonistic properties (IC50 = 1122 nM) possibly dueto the steric bulkiness of om-xylyl residue while the dichloroderivative 35s (IC50 = 34 nM) retained activity A slightdecrease in potency was observed when the methyl group on thenitrogen on the α-position of the tyrosine was removed (35n vs
35t) further confirming that this is not substantial for P2X7Rantagonism The most potent compound arising from this studywas 35f with an IC50 value of 13 nM at inhibiting calciuminflux in human monocytes compared to KN-62 which has anIC50 of 51 nM [139] It is therefore confirmed that thephenylpiperazine group of KN-62 is crucial for interaction withthe active site of the receptor and can be potentially improvedwith small halogen substitutions particularly in the paraposition
An ortho-methyl substituent on the N-phenyl ring of KN-62increased the potency by 3-fold (36 IC50 15 nM) [140]Radiolabelling of compound 36 with tritium and preliminarybinding studies to the P2X7R have also been achieved [140]
Table 2 Structures and P2X7R Antagonistic Properties of Selected KN-62 Derivatives [137138]
34a isoquinoline-5-SO2mdash isoquinoline-5-SO2mdash Bocmdash H 77 plusmn 24 ~40
34b quinoline-8-SO2mdash quinoline-8-SO2mdash Bocmdash H 61 plusmn 30 ND
34c Cbzmdash quinoline-8-SO2mdash Bocmdash H 53 plusmn 23 ~200
34d Cbzmdash PhSO2mdash Bocmdash H 59 plusmn 14 ND
34e Cbzmdash p-tolyl-SO2mdash Bocmdash H 71 plusmn 30 ~300
34f Cbzmdash p-OMe-PhSO2mdash Bocmdash H 62 plusmn 15 ND
34g Cbzmdash isoquinoline-5-SO2mdash Bocmdash H 43 ND
34h Cbzmdash benzoylmdash Bocmdash H 47 plusmn 21 ND
34i isoquinoline-5-SO2mdash isoquinoline-5-SO2mdash H H 14 plusmn 12 ND
34j quinoline-8-SO2mdash quinoline-8-SO2mdash H H 5 plusmn 9 ND
34k Cbzmdash PhSO2mdash Cbzmdash H 48 plusmn 29 ND
34l Cbzmdash PhSO2mdash benzoylmdash H 78 plusmn 22 ND
34m Cbzmdash isoquinoline-5-SO2mdash benzoylmdash H 85 plusmn 10 ~200
34n Cbzmdash PhSO2mdash p-tolyl-SO2mdash H 0 ND
34o Cbzmdash PhSO2mdash CH3SO2mdash H 0 ND
34p Cbzmdash PhSO2mdash PhSO2mdash H 0 ND
34q Cbzmdash PhSO2mdash p-OMe-PhSO2mdash H 0 ND
34r Cbzmdash p-tolyl-SO2mdash benzoylmdash H 71 plusmn 21 ND
34s p-NO2-Cbzmdash p-tolyl-SO2mdash benzoylmdash H 69 plusmn 25 ND
34t m-NO2-Cbzmdash p-tolyl-SO2mdash benzoylmdash H 72 plusmn 6 ND
34u m-acetamido-Cbzmdash p-tolyl-SO2mdash benzoylmdash H 9 plusmn 13 ND
34v m-isothiocyanato-Cbzmdash p-tolyl-SO2mdash benzoylmdash H minus4 plusmn 5 ND
34w Cbzmdash m-NO2-PhSO2mdash benzoylmdash H 75 plusmn 10 ND
34x Cbzmdash p-NO2-PhSO2mdash benzoylmdash H 78 plusmn 18 ~100
34y m-NO2-Cbzmdash p-tolyl-SO2mdash Bocmdash H 94 plusmn 28 ~100
34z Cbzmdash p-tolyl-SO2mdash p-NH2-benzoylmdash H 93 plusmn 3 gt 300
aAntagonist function was measured by the percent inhibition of the K+ release triggered by 3mM ATP in paired wells in the absence of antagonist bAntagonistic IC50values derived from dose-response curves obtained on the basis of inhibition of K+ release
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1515
R2 X N
O
N
O
S
R1
S
O
O
N
O
O
Nthe inhibition of BzATP-induced ethidium bromide uptakemediated by the P2X7R expressed endogenously on the humanpre-monocytic cell line THP-1 [142] Compound 37a (Table 4)was identified as the lead structure and modifications on theimide and 4-pyridyl moieties were investigated The cyclicimide moiety is proven essential for the P2X7R activity sincesubstitutions with simple amines acyclic amides or acycliccarbamates almost completely abolished the activity [142] Thering can be five- or six-membered in the forms of imides (37a-bh) carbamates (37c-d) thiazolidine-24-diones (37eg) andhydantoins (37f) Replacement of the 4-pyridyl moiety with 4-substituted benzamides (37g-h) retained the P2X7R activityThe presence of a 3rsquo-nitro substituent on the biphenyl subunitwas reported to convey optimum P2X7R activity The mode ofinhibition of these compounds seemed to be competitivealthough there were observable changes in the maximalresponse as the compound concentration was increased [142]
Fig (16) General structure for the KN-62 derivatives (35a-35s)
Unfortunately while these KN-62 analogues are potentiallyuseful tools to investigate the basic properties of the P2X7Rthey do not represent good starting points for the discovery oforally-active drugs due to 1) high molecular weight (gt700 Da)2) high lipophilicity (clogP gt 6) and 3) presence of ametabolically labile yet crucial sulfonate group [141]
These compounds represent a new class of ligands thatpossess activity to the P2X7R They appear good candidates asorally active drugs due to their relatively low molecular weight(~400 Da) and would be highly amenable for further SARstudies However despite the succinimides and thiazolidine-24-diones representing potent analogues they were found to bemetabolically and chemically fragile On the other handalthough the carbamates were less potent they displayedimproved stability Future SAR studies could usefully involveinvestigation of the biphenyl ring and the chain linker
37 Cyclic Imides and Analogues
Cyclic imides 37 (Fig 18) were discovered through a high-throughput screening of AstraZenecarsquos compound library for
Table 3 Structures and P2X7R Antagonistic Properties of Selected KN-62 Derivatives [139]
Entry R1 R2 X IC50 (nM)a
KN-62 mdashCH3 mdashPh N 511 plusmn 11
35a mdashCH3 mdashCH2Ph N 211 plusmn 08
35b mdashCH3 mdashCH2CH2Ph N 600 plusmn 27
35c mdashCH3 mdashCH2Ph CH 653 plusmn 08
35d mdashCH3 2-pyridinyl N 170 plusmn 39
35e mdashCH3 2-pyrimidinyl N 798 plusmn 07
35f mdashCH3 p-F-Phmdash N 13 plusmn 01
35g mdashCH3 p-Cl-Phmdash N 105 plusmn 11
35h mdashCH3 p-I-Phmdash N 176 plusmn 38
35i mdashCH3 p-Me-Phmdash N 135 plusmn 02
35j mdashCH3 p-NO2-Phmdash N 58 plusmn 06
35k mdashCH3 p-OMe-Phmdash N 132 plusmn 43
35l mdashCH3 o-F-Phmdash N 185 plusmn 04
35m mdashCH3 o-Cl-Phmdash N 158 plusmn 09
35n mdashCH3 o-Me-Phmdash N 151 plusmn 14
35o mdashCH3 o-OMe-Phmdash N 841 plusmn 5
35p mdashCH3 m-Cl-Phmdash N 141 plusmn 03
35q mdashCH3 m-CF3-Phmdash N 311 plusmn 12
35r mdashCH3 om-diMe-Phmdash N 1122 plusmn 25
35s mdashCH3 mp-diCl-Phmdash N 339 plusmn 08
35t H o-Me-Phmdash N 288 plusmn 03
aAntagonistic IC50 (nM)a values derived from dose-response curves on the basis of inhibition of Ca2+ flux in human monocytes
1516 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
N N
O
N
S
N
OO
OS O
O
N
R
31 R = H (KN-62)
36 R = 2-[3H]Me
AstraZeneca reported that no replacement for the adamantaneframework was found [141] Investigation of the chain linkerrequirements between the adamantane and the phenyl ring wasperformed where it was found that C- and N- methylationcarbonyl reduction and methylene removal (38d-g) all causedloss of potency One methylene unit extension (38h ) retainedthe potency whereas reversal of the amide connectivity (38i)slightly decreased the potency AstraZeneca prepared acombinatorial library by reacting commercially availableadamantanemethylamine with a large variety of acid chloridesor via standard amide coupling with carboxylic acids Thegeneral trends indicated that only aromatic amides had activityat the P2X7R [141] On the phenyl ring 23- and 25-disubstitutions resulted in better antagonists (38j-o) than the26- (38p ) 35- (38q ) or 24-disusbstitutions (38r) The ortho-position could also tolerate substituents such as Br OMe andMe (38s-u) A closer examination on the reverse amides (38i v-y) also revealed that 23- and 25-disubsitutions were stillpreferred for P2X7R inhibition The indazole amide (38z) wasidentified as a target lead molecule with a good metabolicprofile suitable for further optimization for clinical trial studies[141]
Fig (17) Radiolabeled analogue of KN-62 [140]
O
NY
X
O
Z
NO2
Adamantyl amides represent a class of simple moleculeswith excellent activity at the P2X7R The nature of theadamantane group could potentially be further investigatedalthough the original authors stated that no replacement for theadamantane group was found A number of patents onadamantyl-containing benzamides have been generated byAstraZeneca [143-146] and Pfizer [147] indicating the potentialof this ligand class for generating a truly potent selective andorally active P2X7R inhibitor Pfizer specifically incorporatedtriazinyl group into the adamantyl benzamide framework [147]Fig (18) General structure for the cyclic imides generated by
AstraZeneca Non-adamantane benzamides including piperidinyl andpiperazinyl benzamides [148 149] triazinyl benzamides [150-153] quinolinyl benzamides [154 155] 34-difluoroanilinebenzamides [156] aromatic acid-containing benzamides [157]pyrazolylmethyl benzamides [158] and other heteroarylamides[159-163] have also been patented by various otherpharmaceutical companies
38 Adamantyl Amides
Adamantyl amides were also discovered from a high-throughput screening of AstraZenecarsquos compound library forinhibition of the P2X7R-mediated ethidium bromide uptakeinduced by BzATP [141] N-Adamantan-1-ylmethyl-2-chloro-benzamide 38a was identified as the lead structure with anexcellent pA2 value of 81 The presence of an ortho-substituentis believed to cause a twist in the orientation of the benzamideessential for potent P2X7R antagonism whereas para-substituents (38bc) have deleterious effects on the P2X7Ractivity Selected compounds of this series are represented inTable 5
39 Diarylimidazolines
Diarylimidazolines represent another class of P2X7-activecompounds They were first discovered through a high-throughput screening of Aventisrsquo compound library forinhibition of BzATP-induced YO-PRO-1 uptake mediated byP2X7R expressed in U373 cells stably expressing the human
Table 4 Structures and P2X7R Antagonistic Properties of Selected Cyclic Imides [142]
Entry X Y Z IC50 ( M) pA2a
37a CH2 CO N - 69
37b (CH2)2 CO N - 75
37c O CH2 N 64
37d O (CH2)2 N 063 -
37e S CO N - 77
37f NH CO N 02 -
37g S CO C-CONHMe 016 -
37h CH2 CO C-CONMe2 025 -
a pA2 = the negative logarithm of a molar concentration of an antagonist that necessitates doubling of agonist concentration needed to elicit the original submaximalresponse obtained in the absence of antagonist
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1517
P2X7R [164] Compound 39a was identified as an initial hitfrom the screening with an IC50 value of 80 nM
equipotent Some degree of branching in the chain linker wasgenerally tolerated (39f-h) Configurational constraintsimposed on the chain linker as exemplified by 39i weretolerated but this was not the case for the cyclopropyl analogue39j
X
RO
NH
N
HN
38a-y 38z
Subsequent investigations focused on the substitutionpattern in the aryl ring for the benzyl (n=1) and phenethyl (n=2)series [164] Electron withdrawing and small neutral groupswere generally tolerable in both series (39k-r v-x) Howeverelectron-donating groups exemplified by OMe gave much lessactive compounds (39s-u) In the phenethyl seriessubstitutions at the ortho- and meta- positions were preferableto the corresponding para- substitutions (39kl vs 39m and39no vs 39p) Interestingly in the benzyl series meta- andpara- substitutions resulted in more active compounds than thecorresponding ortho analogues (39v vs 39wx)
Fig (19) Adamantyl amides generated by AstraZeneca
Investigation of the nature of the linker between thearomatic ring and the 2-position of the imidazole ring revealedthat a 3 or 4 carbon linker (39bc) caused a slight decrease in theactivity and no carbon linker (39d ) rendered the compoundinactive [164] One carbon linker with a methyl branching unit(39e) was tolerated It was reported that there was noenantiomeric preference of the methyl branch they were
The diarylimidazolines examined represent good candidatesfor orally active P2X7R inhibitors they are small and highlyamenable for SAR studies The 4- and 5-phenyl rings representattractive sites for further investigations
Table 5 Structures and P2X7R Antagonistic Properties of Selected Adamantyl Amides [141]
Entry R X pA2
38a 2-Cl CH2NHCO 81
38b 4-Cl CH2NHCO inactive
38c 24-Cl2 CH2NHCO 64
38d 2-Cl CHMeNHCO 64
38e 2-Cl CH2NMeCO 54
38f 2-Cl CH2NHCH2 54
38g 2-Cl NHCO inactive
38h 2-Cl (CH2)2NHCO 78
38i 2-Cl CH2CONH 63
38j 23-Cl2 CH2NHCO 88
38k 25-Cl2 CH2NHCO 83
38l 2-Cl-3-NH2 CH2NHCO 82
38m 2-Cl-5-OMe CH2NHCO 88
38n 2-Cl-5-OH CH2NHCO 80
38o 2-Cl-5-NH2 CH2NHCO 84
38p 26-Cl2 CH2NHCO 74
38q 35-Cl2 CH2NHCO 54
38r 24-Cl2 CH2NHCO 64
38s 2-Br CH2NHCO 80
38t 2-OMe CH2NHCO 71
38u 2-Me CH2NHCO 79
38v 2-Me CH2CONH 68
38w 2-Cl-5-OMe CH2CONH 72
38x 2-Me-3-OMe CH2CONH 83
38y 2-Me-5-OMe CH2CONH 80
1518 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
N
HN
XY
R
N
HN
N
HN
N
HN
( )n
39i39j
39a-ce-hk-w 39d
Fig (20) General structure of the diarylimidazolines generated by Aventis
Table 6 Structures and P2X7R Antagonistic Properties of Selected Diarylimidazolines [164]
Entry n X Y R IC50 ( M)a
39a 1 H H H 008
39b 2 H H H 054
39c 3 H H H 061
39e 0 (R)-Me H H 011
39f 1 (S)-Me H H 003
39g 1 Me Me H 001
39h 1 Ph H H 032
39k 1 H H 2-F 016
39l 1 H H 3-F 004
39m 1 H H 4-F 022
39n 1 H H 2-Cl 007
39o 1 H H 3-Cl 012
39p 1 H H 4-Cl gt1
39q 1 H H 2-Me 007
39r 1 H H 4-Me 219
39s 1 H H 2-OMe 083
39t 1 H H 3-OMe gt3
39u 1 H H 4-OMe gt3
39v 0 H H 2-F 058
39w 0 H H 3-F 008
39x 0 H H 4-F 008
aAntagonistic IC50 values derived from dose-response curves on the basis of inhibition of YO-PRO-1 dye uptake
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1519
R1N
N
NN
R2
R1
NN
NN
R2
N
N
NN
Cl
Cl
Cl
Cl
40b-t 40u-x40a
1
2
3
4
5
Fig (21) General structure of tetrazoles generated by Abbott Laboratories
310 1-Benzyl-5-Phenyltetrazoles potency The limited solubility of those compounds howeverposed a challenge to incorporate substitutions that couldincrease the solubility while maintaining activity A 3-pyridylgroup (40d ) was found to overcome this problem Moreover anextra 2-methyl substituent (40e) resulted in higher activitycompared to 40d A methylene extension (40f) caused a decreasein potency whereas other heterocycles such as 40g and 40hresulted in analogues with similar potency to 40d
1-Benzyl-5-phenyltetrazoles were discovered through ahigh-throughput screening by Abbott Laboratories for threedifferent P2X7R activities Ca2+ flux at the rat or human P2X7RYO-PRO-1 uptake and interleukin-1β release at human P2X7R[165-168] The initial hit from the screening was the tetrazole40a (Table 7) with a pIC50 value of 69 in inhibiting P2X7Rderived from the recombinant human cell line
Investigation of the nature of the substituent attached to the5-position of the tetrazole core showed that the substitutionpattern at this position was not well tolerated [165] Forexample unsubstituted mono-chlorosubstituted 25- and 34-
Initial SAR studies focused on the substituent attached tothe 1-position of the tetrazole core [165] Unsubstituted orortho-substituted benzylic group (40bc) exhibited good
Table 7 Structures and P2X7R Antagonistic Properties of Selected 1-benzyl-5-tetrazoles [165]
1520 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
disubstituted compounds (40i-m) pyridine and quinoline ring(40n-p ) analogues were not tolerated However moreconservative changes such as CF3 and F for Cl group (40q-t)resulted in an increase of potency
release and amplification of astrocytic Ca2+ signaling werereported to be due to the P2X7R and not Cx43 hemichannels[170]
Decavanadate a type of polymer of vanadium oxides hasbeen demonstrated to be a competitive rapid-acting antagonistat the P2X7R [172] It is capable of inhibiting the BzATP-stimulated ethidium bromide uptake in human mouse and ratP2X7R with pA2 values of 74 57 and 71 respectivelyDecavanadate has not been fully tested for its selectivity overother P2X receptors however it is suggested that this largemolecular species acts at the extracellular ATP binding site ofthe receptor consistent with the competitive mode of inhibition[172] A monoclonal antibody specific for the human P2X7Rhas also been shown to be a potent and selective antagonist ofthe receptor with an IC50 value of 5 nM in blocking the BzATP-evoked currents [173] Interestingly tenidap at 50 ndash 200 microMconcentration increases the sensitivity of mouse macrophagesto ATP-mediated cytotoxicity [174] 17-β-estradiol and notprogesterone or 17-α-estradiol has also been reported to blockthe agonist-induced currents at the human P2X7R with an IC50value of 3 microM in a non-genomic manner [175] Polymyxin B apotent antibiotic well-known for its lipopolysaccharide-neutralizing effects has been demonstrated to confer asynergistic effect when co-applied with ATP in a similar mannerto tenidap inducing Ca2+ influx ethidium bromide uptake andlactate dehydrogenase release via action at the P2X7R [176]
Tetrazoles with reversed connectivity (40u-x) were alsoevaluated where it was found that this connectivity reversal hadlittle effect on the potency [165] These tetrazoles were reportedto be slightly more active in the human P2X7R than the ratanalogue [165]
The tetrazole derivative 40d has been shown to be selectivefor other P2 receptors including P2X3 P2X4 and P2Y2 receptors[165] Pharmacological testing of this tetrazole suggested acompetitive mode of inhibition at the P2X7R and its efficacyhas also been evaluated in a model of neuropathic pain in rats[165] Overall these tetrazoles are found to be P2X7R-active andhighly adaptable to structural modifications These propertiesmake the tetrazoles an interesting class of compound for furtherinvestigating P2X7R activity
311 Other P2X7R-Active Ligands
Chelerythrine 41 (Fig 22) is a member of the benzophen-anthridine alkaloids known to inhibit protein kinase C withgood potency and selectivity (IC50 066 microM) as well as enzymessuch as alanine aminotransferase and Na+K+-ATPase [169] At10 microM concentrations chelerythrine non-competitivelyinhibited the BzATP-induced 86Rb+ efflux by 74 in human B-lymphocytes [169] Dye uptake studies could not be performeddue to the fluorescent nature of the compound It is yet to bedetermined whether chelerythrine inhibition of P2X7R involvesprotein kinase C [169]
More recently other distinct classes of P2X7-active ligandshave been reported and patented by the Abbott Laboratoriesacylhydrazides [177] cyanoamidines and cyanoguanidines[178 179]
4 CONCLUSIONS AND FUTURE DIRECTIONS
O
O
N
O
O
Cl
41
Although many structurally diverse class of compoundshave been reported as P2X7R antagonists with reasonablepotency the selectivity issue over other P2 receptors andproteins such as ecto-ATPases and ectonucleotidases stillprevails This is particularly an issue for oxidized ATP andsuramin SAR studies performed on PPADS and suramin mainlygave improved potency and selectivity for the P2X1 receptorIon channel blockers such as amilorides and stilbenederivatives only possess relatively low potency at the P2X7RFurther complications in studying these P2X7R antagonistsalso arise from the demonstrated species differences acrosshuman rat and mouse systems Brilliant Blue G is active for allthree species however at present there have been no SAR datareported Another fundamental factor that hampers the study ofP2X7R in detail is the relatively low potency and selectivity ofthe available agonist BzATP The discovery of a more potentselective P2X7R agonist would be ideal since in many studiesfull dose-response curves could not be generated due to the low
Fig (22) Chemical structure for chelerythrine an alkaloid withantagonistic properties at the human P2X7R
Gap junction channel blockers such as flufenamic acid 42 carbenoxolone 43 and mefloquine 44 (Fig 23) were all foundto be inhibitors of the BzATP-induced Ca2+ flux (IC50 values of655 175 and 25 nM respectively) and of YO-PRO-1 uptake in1321N1 astrocytoma cells stably expressing rat P2X7R [170]This finding poses a question to the general use of gap junctionchannel blockers as indicators of the participation of connexinhemichannels at the sites of transmitter release [171] The ATP
HN CF3
O OH
42 f lufenamic acid
HO
-O
O
O
H
OH
O
O-
N
HN
CF3
CF3
H O
H
43 carbenoxolone 44 mefloquine
Fig (23) Chemical structures for gap junction channel blockers with P2X7R antagonistic properties
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1521
potency of BzATP The nature of functional assays on theP2X7R is also found to be complex as ionic compositiontemperature and cell origins all affect the results demandingextra care be taken when antagonist potencies are directlycompared from different studies
[23] Li J Liu D Ke H Z Duncan R L Turner C H J Biol Chem2005 280 42952-42959
[24] Penolazzi L Bianchini E Lambertini E Baraldi P G RomagnoliR Piva R Gambari R J Biomed Sci 2005 12 1013-1020
[25] Gibbons S J Washburn K B Talamo B R J Auton Pharmacol2001 21 181-190
[26] Dehaye J P Moran A Marino A Arch Oral Biol 1999 44 S39-S43Four classes of small molecules with high affinity at the
P2X7R have been reported cyclic imides adamantyl amidesdiarylimidazolines and 1-benzyl-5-phenyltetrazoles Theseclasses of compounds are highly amenable to structuralmodifications indicating their good prospects as leadmolecules for further improving the potency and selectivity atP2X7R ultimately leading to orally active drugs To date norational drug design based on the knowledge of binding siteswithin this receptor has been undertaken This is a direct resultof the fact that no crystal structure or suitable homology modelhave been reported for this receptor The fact that the P2X7R canadopt either a channel or pore state causes even moredifficulties in studying receptor dynamics and at present theexact mechanism of channel-to-pore transition remains to beelucidated Undoubtedly the P2X7R is involved in severalprocesses relevant to chronic inflammation neurodegenerationand neuropathic pain and therefore discovery of more potentselective P2X7R antagonists would have a great impact in thepresent therapeutic window
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ACKNOWLEDGEMENTS[38] Khakh B S Burnstock G Kennedy C King B F North A
Seguela P Voigt M Humphrey P P A Nat Rev Neurosci 20012 165-174
The authors would like to thank Dr Natasha Hungerford forproofreading the manuscript
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Received February 01 2007 Revised April 04 2007 Accepted April 06 2007
1514 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
small electron-withdrawing group eg fluorine (35f) and theantagonistic property decreases as the size of the electron-withdrawing group increases (35g 35h) A methyl- or nitro-group at the para- position (35i 35j) was beneficial foractivity but not evidenced with the methoxy (35k ) group As inthe case for para- substitution introduction of F Cl or CH3 butnot a methoxy (35l-o) at the ortho- position was well toleratedMoreover the meta- position could also tolerate a Cl or CF3group (35p 35q) The disubstituted compound 35r showedreduced antagonistic properties (IC50 = 1122 nM) possibly dueto the steric bulkiness of om-xylyl residue while the dichloroderivative 35s (IC50 = 34 nM) retained activity A slightdecrease in potency was observed when the methyl group on thenitrogen on the α-position of the tyrosine was removed (35n vs
35t) further confirming that this is not substantial for P2X7Rantagonism The most potent compound arising from this studywas 35f with an IC50 value of 13 nM at inhibiting calciuminflux in human monocytes compared to KN-62 which has anIC50 of 51 nM [139] It is therefore confirmed that thephenylpiperazine group of KN-62 is crucial for interaction withthe active site of the receptor and can be potentially improvedwith small halogen substitutions particularly in the paraposition
An ortho-methyl substituent on the N-phenyl ring of KN-62increased the potency by 3-fold (36 IC50 15 nM) [140]Radiolabelling of compound 36 with tritium and preliminarybinding studies to the P2X7R have also been achieved [140]
Table 2 Structures and P2X7R Antagonistic Properties of Selected KN-62 Derivatives [137138]
34a isoquinoline-5-SO2mdash isoquinoline-5-SO2mdash Bocmdash H 77 plusmn 24 ~40
34b quinoline-8-SO2mdash quinoline-8-SO2mdash Bocmdash H 61 plusmn 30 ND
34c Cbzmdash quinoline-8-SO2mdash Bocmdash H 53 plusmn 23 ~200
34d Cbzmdash PhSO2mdash Bocmdash H 59 plusmn 14 ND
34e Cbzmdash p-tolyl-SO2mdash Bocmdash H 71 plusmn 30 ~300
34f Cbzmdash p-OMe-PhSO2mdash Bocmdash H 62 plusmn 15 ND
34g Cbzmdash isoquinoline-5-SO2mdash Bocmdash H 43 ND
34h Cbzmdash benzoylmdash Bocmdash H 47 plusmn 21 ND
34i isoquinoline-5-SO2mdash isoquinoline-5-SO2mdash H H 14 plusmn 12 ND
34j quinoline-8-SO2mdash quinoline-8-SO2mdash H H 5 plusmn 9 ND
34k Cbzmdash PhSO2mdash Cbzmdash H 48 plusmn 29 ND
34l Cbzmdash PhSO2mdash benzoylmdash H 78 plusmn 22 ND
34m Cbzmdash isoquinoline-5-SO2mdash benzoylmdash H 85 plusmn 10 ~200
34n Cbzmdash PhSO2mdash p-tolyl-SO2mdash H 0 ND
34o Cbzmdash PhSO2mdash CH3SO2mdash H 0 ND
34p Cbzmdash PhSO2mdash PhSO2mdash H 0 ND
34q Cbzmdash PhSO2mdash p-OMe-PhSO2mdash H 0 ND
34r Cbzmdash p-tolyl-SO2mdash benzoylmdash H 71 plusmn 21 ND
34s p-NO2-Cbzmdash p-tolyl-SO2mdash benzoylmdash H 69 plusmn 25 ND
34t m-NO2-Cbzmdash p-tolyl-SO2mdash benzoylmdash H 72 plusmn 6 ND
34u m-acetamido-Cbzmdash p-tolyl-SO2mdash benzoylmdash H 9 plusmn 13 ND
34v m-isothiocyanato-Cbzmdash p-tolyl-SO2mdash benzoylmdash H minus4 plusmn 5 ND
34w Cbzmdash m-NO2-PhSO2mdash benzoylmdash H 75 plusmn 10 ND
34x Cbzmdash p-NO2-PhSO2mdash benzoylmdash H 78 plusmn 18 ~100
34y m-NO2-Cbzmdash p-tolyl-SO2mdash Bocmdash H 94 plusmn 28 ~100
34z Cbzmdash p-tolyl-SO2mdash p-NH2-benzoylmdash H 93 plusmn 3 gt 300
aAntagonist function was measured by the percent inhibition of the K+ release triggered by 3mM ATP in paired wells in the absence of antagonist bAntagonistic IC50values derived from dose-response curves obtained on the basis of inhibition of K+ release
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1515
R2 X N
O
N
O
S
R1
S
O
O
N
O
O
Nthe inhibition of BzATP-induced ethidium bromide uptakemediated by the P2X7R expressed endogenously on the humanpre-monocytic cell line THP-1 [142] Compound 37a (Table 4)was identified as the lead structure and modifications on theimide and 4-pyridyl moieties were investigated The cyclicimide moiety is proven essential for the P2X7R activity sincesubstitutions with simple amines acyclic amides or acycliccarbamates almost completely abolished the activity [142] Thering can be five- or six-membered in the forms of imides (37a-bh) carbamates (37c-d) thiazolidine-24-diones (37eg) andhydantoins (37f) Replacement of the 4-pyridyl moiety with 4-substituted benzamides (37g-h) retained the P2X7R activityThe presence of a 3rsquo-nitro substituent on the biphenyl subunitwas reported to convey optimum P2X7R activity The mode ofinhibition of these compounds seemed to be competitivealthough there were observable changes in the maximalresponse as the compound concentration was increased [142]
Fig (16) General structure for the KN-62 derivatives (35a-35s)
Unfortunately while these KN-62 analogues are potentiallyuseful tools to investigate the basic properties of the P2X7Rthey do not represent good starting points for the discovery oforally-active drugs due to 1) high molecular weight (gt700 Da)2) high lipophilicity (clogP gt 6) and 3) presence of ametabolically labile yet crucial sulfonate group [141]
These compounds represent a new class of ligands thatpossess activity to the P2X7R They appear good candidates asorally active drugs due to their relatively low molecular weight(~400 Da) and would be highly amenable for further SARstudies However despite the succinimides and thiazolidine-24-diones representing potent analogues they were found to bemetabolically and chemically fragile On the other handalthough the carbamates were less potent they displayedimproved stability Future SAR studies could usefully involveinvestigation of the biphenyl ring and the chain linker
37 Cyclic Imides and Analogues
Cyclic imides 37 (Fig 18) were discovered through a high-throughput screening of AstraZenecarsquos compound library for
Table 3 Structures and P2X7R Antagonistic Properties of Selected KN-62 Derivatives [139]
Entry R1 R2 X IC50 (nM)a
KN-62 mdashCH3 mdashPh N 511 plusmn 11
35a mdashCH3 mdashCH2Ph N 211 plusmn 08
35b mdashCH3 mdashCH2CH2Ph N 600 plusmn 27
35c mdashCH3 mdashCH2Ph CH 653 plusmn 08
35d mdashCH3 2-pyridinyl N 170 plusmn 39
35e mdashCH3 2-pyrimidinyl N 798 plusmn 07
35f mdashCH3 p-F-Phmdash N 13 plusmn 01
35g mdashCH3 p-Cl-Phmdash N 105 plusmn 11
35h mdashCH3 p-I-Phmdash N 176 plusmn 38
35i mdashCH3 p-Me-Phmdash N 135 plusmn 02
35j mdashCH3 p-NO2-Phmdash N 58 plusmn 06
35k mdashCH3 p-OMe-Phmdash N 132 plusmn 43
35l mdashCH3 o-F-Phmdash N 185 plusmn 04
35m mdashCH3 o-Cl-Phmdash N 158 plusmn 09
35n mdashCH3 o-Me-Phmdash N 151 plusmn 14
35o mdashCH3 o-OMe-Phmdash N 841 plusmn 5
35p mdashCH3 m-Cl-Phmdash N 141 plusmn 03
35q mdashCH3 m-CF3-Phmdash N 311 plusmn 12
35r mdashCH3 om-diMe-Phmdash N 1122 plusmn 25
35s mdashCH3 mp-diCl-Phmdash N 339 plusmn 08
35t H o-Me-Phmdash N 288 plusmn 03
aAntagonistic IC50 (nM)a values derived from dose-response curves on the basis of inhibition of Ca2+ flux in human monocytes
1516 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
N N
O
N
S
N
OO
OS O
O
N
R
31 R = H (KN-62)
36 R = 2-[3H]Me
AstraZeneca reported that no replacement for the adamantaneframework was found [141] Investigation of the chain linkerrequirements between the adamantane and the phenyl ring wasperformed where it was found that C- and N- methylationcarbonyl reduction and methylene removal (38d-g) all causedloss of potency One methylene unit extension (38h ) retainedthe potency whereas reversal of the amide connectivity (38i)slightly decreased the potency AstraZeneca prepared acombinatorial library by reacting commercially availableadamantanemethylamine with a large variety of acid chloridesor via standard amide coupling with carboxylic acids Thegeneral trends indicated that only aromatic amides had activityat the P2X7R [141] On the phenyl ring 23- and 25-disubstitutions resulted in better antagonists (38j-o) than the26- (38p ) 35- (38q ) or 24-disusbstitutions (38r) The ortho-position could also tolerate substituents such as Br OMe andMe (38s-u) A closer examination on the reverse amides (38i v-y) also revealed that 23- and 25-disubsitutions were stillpreferred for P2X7R inhibition The indazole amide (38z) wasidentified as a target lead molecule with a good metabolicprofile suitable for further optimization for clinical trial studies[141]
Fig (17) Radiolabeled analogue of KN-62 [140]
O
NY
X
O
Z
NO2
Adamantyl amides represent a class of simple moleculeswith excellent activity at the P2X7R The nature of theadamantane group could potentially be further investigatedalthough the original authors stated that no replacement for theadamantane group was found A number of patents onadamantyl-containing benzamides have been generated byAstraZeneca [143-146] and Pfizer [147] indicating the potentialof this ligand class for generating a truly potent selective andorally active P2X7R inhibitor Pfizer specifically incorporatedtriazinyl group into the adamantyl benzamide framework [147]Fig (18) General structure for the cyclic imides generated by
AstraZeneca Non-adamantane benzamides including piperidinyl andpiperazinyl benzamides [148 149] triazinyl benzamides [150-153] quinolinyl benzamides [154 155] 34-difluoroanilinebenzamides [156] aromatic acid-containing benzamides [157]pyrazolylmethyl benzamides [158] and other heteroarylamides[159-163] have also been patented by various otherpharmaceutical companies
38 Adamantyl Amides
Adamantyl amides were also discovered from a high-throughput screening of AstraZenecarsquos compound library forinhibition of the P2X7R-mediated ethidium bromide uptakeinduced by BzATP [141] N-Adamantan-1-ylmethyl-2-chloro-benzamide 38a was identified as the lead structure with anexcellent pA2 value of 81 The presence of an ortho-substituentis believed to cause a twist in the orientation of the benzamideessential for potent P2X7R antagonism whereas para-substituents (38bc) have deleterious effects on the P2X7Ractivity Selected compounds of this series are represented inTable 5
39 Diarylimidazolines
Diarylimidazolines represent another class of P2X7-activecompounds They were first discovered through a high-throughput screening of Aventisrsquo compound library forinhibition of BzATP-induced YO-PRO-1 uptake mediated byP2X7R expressed in U373 cells stably expressing the human
Table 4 Structures and P2X7R Antagonistic Properties of Selected Cyclic Imides [142]
Entry X Y Z IC50 ( M) pA2a
37a CH2 CO N - 69
37b (CH2)2 CO N - 75
37c O CH2 N 64
37d O (CH2)2 N 063 -
37e S CO N - 77
37f NH CO N 02 -
37g S CO C-CONHMe 016 -
37h CH2 CO C-CONMe2 025 -
a pA2 = the negative logarithm of a molar concentration of an antagonist that necessitates doubling of agonist concentration needed to elicit the original submaximalresponse obtained in the absence of antagonist
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1517
P2X7R [164] Compound 39a was identified as an initial hitfrom the screening with an IC50 value of 80 nM
equipotent Some degree of branching in the chain linker wasgenerally tolerated (39f-h) Configurational constraintsimposed on the chain linker as exemplified by 39i weretolerated but this was not the case for the cyclopropyl analogue39j
X
RO
NH
N
HN
38a-y 38z
Subsequent investigations focused on the substitutionpattern in the aryl ring for the benzyl (n=1) and phenethyl (n=2)series [164] Electron withdrawing and small neutral groupswere generally tolerable in both series (39k-r v-x) Howeverelectron-donating groups exemplified by OMe gave much lessactive compounds (39s-u) In the phenethyl seriessubstitutions at the ortho- and meta- positions were preferableto the corresponding para- substitutions (39kl vs 39m and39no vs 39p) Interestingly in the benzyl series meta- andpara- substitutions resulted in more active compounds than thecorresponding ortho analogues (39v vs 39wx)
Fig (19) Adamantyl amides generated by AstraZeneca
Investigation of the nature of the linker between thearomatic ring and the 2-position of the imidazole ring revealedthat a 3 or 4 carbon linker (39bc) caused a slight decrease in theactivity and no carbon linker (39d ) rendered the compoundinactive [164] One carbon linker with a methyl branching unit(39e) was tolerated It was reported that there was noenantiomeric preference of the methyl branch they were
The diarylimidazolines examined represent good candidatesfor orally active P2X7R inhibitors they are small and highlyamenable for SAR studies The 4- and 5-phenyl rings representattractive sites for further investigations
Table 5 Structures and P2X7R Antagonistic Properties of Selected Adamantyl Amides [141]
Entry R X pA2
38a 2-Cl CH2NHCO 81
38b 4-Cl CH2NHCO inactive
38c 24-Cl2 CH2NHCO 64
38d 2-Cl CHMeNHCO 64
38e 2-Cl CH2NMeCO 54
38f 2-Cl CH2NHCH2 54
38g 2-Cl NHCO inactive
38h 2-Cl (CH2)2NHCO 78
38i 2-Cl CH2CONH 63
38j 23-Cl2 CH2NHCO 88
38k 25-Cl2 CH2NHCO 83
38l 2-Cl-3-NH2 CH2NHCO 82
38m 2-Cl-5-OMe CH2NHCO 88
38n 2-Cl-5-OH CH2NHCO 80
38o 2-Cl-5-NH2 CH2NHCO 84
38p 26-Cl2 CH2NHCO 74
38q 35-Cl2 CH2NHCO 54
38r 24-Cl2 CH2NHCO 64
38s 2-Br CH2NHCO 80
38t 2-OMe CH2NHCO 71
38u 2-Me CH2NHCO 79
38v 2-Me CH2CONH 68
38w 2-Cl-5-OMe CH2CONH 72
38x 2-Me-3-OMe CH2CONH 83
38y 2-Me-5-OMe CH2CONH 80
1518 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
N
HN
XY
R
N
HN
N
HN
N
HN
( )n
39i39j
39a-ce-hk-w 39d
Fig (20) General structure of the diarylimidazolines generated by Aventis
Table 6 Structures and P2X7R Antagonistic Properties of Selected Diarylimidazolines [164]
Entry n X Y R IC50 ( M)a
39a 1 H H H 008
39b 2 H H H 054
39c 3 H H H 061
39e 0 (R)-Me H H 011
39f 1 (S)-Me H H 003
39g 1 Me Me H 001
39h 1 Ph H H 032
39k 1 H H 2-F 016
39l 1 H H 3-F 004
39m 1 H H 4-F 022
39n 1 H H 2-Cl 007
39o 1 H H 3-Cl 012
39p 1 H H 4-Cl gt1
39q 1 H H 2-Me 007
39r 1 H H 4-Me 219
39s 1 H H 2-OMe 083
39t 1 H H 3-OMe gt3
39u 1 H H 4-OMe gt3
39v 0 H H 2-F 058
39w 0 H H 3-F 008
39x 0 H H 4-F 008
aAntagonistic IC50 values derived from dose-response curves on the basis of inhibition of YO-PRO-1 dye uptake
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1519
R1N
N
NN
R2
R1
NN
NN
R2
N
N
NN
Cl
Cl
Cl
Cl
40b-t 40u-x40a
1
2
3
4
5
Fig (21) General structure of tetrazoles generated by Abbott Laboratories
310 1-Benzyl-5-Phenyltetrazoles potency The limited solubility of those compounds howeverposed a challenge to incorporate substitutions that couldincrease the solubility while maintaining activity A 3-pyridylgroup (40d ) was found to overcome this problem Moreover anextra 2-methyl substituent (40e) resulted in higher activitycompared to 40d A methylene extension (40f) caused a decreasein potency whereas other heterocycles such as 40g and 40hresulted in analogues with similar potency to 40d
1-Benzyl-5-phenyltetrazoles were discovered through ahigh-throughput screening by Abbott Laboratories for threedifferent P2X7R activities Ca2+ flux at the rat or human P2X7RYO-PRO-1 uptake and interleukin-1β release at human P2X7R[165-168] The initial hit from the screening was the tetrazole40a (Table 7) with a pIC50 value of 69 in inhibiting P2X7Rderived from the recombinant human cell line
Investigation of the nature of the substituent attached to the5-position of the tetrazole core showed that the substitutionpattern at this position was not well tolerated [165] Forexample unsubstituted mono-chlorosubstituted 25- and 34-
Initial SAR studies focused on the substituent attached tothe 1-position of the tetrazole core [165] Unsubstituted orortho-substituted benzylic group (40bc) exhibited good
Table 7 Structures and P2X7R Antagonistic Properties of Selected 1-benzyl-5-tetrazoles [165]
1520 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
disubstituted compounds (40i-m) pyridine and quinoline ring(40n-p ) analogues were not tolerated However moreconservative changes such as CF3 and F for Cl group (40q-t)resulted in an increase of potency
release and amplification of astrocytic Ca2+ signaling werereported to be due to the P2X7R and not Cx43 hemichannels[170]
Decavanadate a type of polymer of vanadium oxides hasbeen demonstrated to be a competitive rapid-acting antagonistat the P2X7R [172] It is capable of inhibiting the BzATP-stimulated ethidium bromide uptake in human mouse and ratP2X7R with pA2 values of 74 57 and 71 respectivelyDecavanadate has not been fully tested for its selectivity overother P2X receptors however it is suggested that this largemolecular species acts at the extracellular ATP binding site ofthe receptor consistent with the competitive mode of inhibition[172] A monoclonal antibody specific for the human P2X7Rhas also been shown to be a potent and selective antagonist ofthe receptor with an IC50 value of 5 nM in blocking the BzATP-evoked currents [173] Interestingly tenidap at 50 ndash 200 microMconcentration increases the sensitivity of mouse macrophagesto ATP-mediated cytotoxicity [174] 17-β-estradiol and notprogesterone or 17-α-estradiol has also been reported to blockthe agonist-induced currents at the human P2X7R with an IC50value of 3 microM in a non-genomic manner [175] Polymyxin B apotent antibiotic well-known for its lipopolysaccharide-neutralizing effects has been demonstrated to confer asynergistic effect when co-applied with ATP in a similar mannerto tenidap inducing Ca2+ influx ethidium bromide uptake andlactate dehydrogenase release via action at the P2X7R [176]
Tetrazoles with reversed connectivity (40u-x) were alsoevaluated where it was found that this connectivity reversal hadlittle effect on the potency [165] These tetrazoles were reportedto be slightly more active in the human P2X7R than the ratanalogue [165]
The tetrazole derivative 40d has been shown to be selectivefor other P2 receptors including P2X3 P2X4 and P2Y2 receptors[165] Pharmacological testing of this tetrazole suggested acompetitive mode of inhibition at the P2X7R and its efficacyhas also been evaluated in a model of neuropathic pain in rats[165] Overall these tetrazoles are found to be P2X7R-active andhighly adaptable to structural modifications These propertiesmake the tetrazoles an interesting class of compound for furtherinvestigating P2X7R activity
311 Other P2X7R-Active Ligands
Chelerythrine 41 (Fig 22) is a member of the benzophen-anthridine alkaloids known to inhibit protein kinase C withgood potency and selectivity (IC50 066 microM) as well as enzymessuch as alanine aminotransferase and Na+K+-ATPase [169] At10 microM concentrations chelerythrine non-competitivelyinhibited the BzATP-induced 86Rb+ efflux by 74 in human B-lymphocytes [169] Dye uptake studies could not be performeddue to the fluorescent nature of the compound It is yet to bedetermined whether chelerythrine inhibition of P2X7R involvesprotein kinase C [169]
More recently other distinct classes of P2X7-active ligandshave been reported and patented by the Abbott Laboratoriesacylhydrazides [177] cyanoamidines and cyanoguanidines[178 179]
4 CONCLUSIONS AND FUTURE DIRECTIONS
O
O
N
O
O
Cl
41
Although many structurally diverse class of compoundshave been reported as P2X7R antagonists with reasonablepotency the selectivity issue over other P2 receptors andproteins such as ecto-ATPases and ectonucleotidases stillprevails This is particularly an issue for oxidized ATP andsuramin SAR studies performed on PPADS and suramin mainlygave improved potency and selectivity for the P2X1 receptorIon channel blockers such as amilorides and stilbenederivatives only possess relatively low potency at the P2X7RFurther complications in studying these P2X7R antagonistsalso arise from the demonstrated species differences acrosshuman rat and mouse systems Brilliant Blue G is active for allthree species however at present there have been no SAR datareported Another fundamental factor that hampers the study ofP2X7R in detail is the relatively low potency and selectivity ofthe available agonist BzATP The discovery of a more potentselective P2X7R agonist would be ideal since in many studiesfull dose-response curves could not be generated due to the low
Fig (22) Chemical structure for chelerythrine an alkaloid withantagonistic properties at the human P2X7R
Gap junction channel blockers such as flufenamic acid 42 carbenoxolone 43 and mefloquine 44 (Fig 23) were all foundto be inhibitors of the BzATP-induced Ca2+ flux (IC50 values of655 175 and 25 nM respectively) and of YO-PRO-1 uptake in1321N1 astrocytoma cells stably expressing rat P2X7R [170]This finding poses a question to the general use of gap junctionchannel blockers as indicators of the participation of connexinhemichannels at the sites of transmitter release [171] The ATP
HN CF3
O OH
42 f lufenamic acid
HO
-O
O
O
H
OH
O
O-
N
HN
CF3
CF3
H O
H
43 carbenoxolone 44 mefloquine
Fig (23) Chemical structures for gap junction channel blockers with P2X7R antagonistic properties
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1521
potency of BzATP The nature of functional assays on theP2X7R is also found to be complex as ionic compositiontemperature and cell origins all affect the results demandingextra care be taken when antagonist potencies are directlycompared from different studies
[23] Li J Liu D Ke H Z Duncan R L Turner C H J Biol Chem2005 280 42952-42959
[24] Penolazzi L Bianchini E Lambertini E Baraldi P G RomagnoliR Piva R Gambari R J Biomed Sci 2005 12 1013-1020
[25] Gibbons S J Washburn K B Talamo B R J Auton Pharmacol2001 21 181-190
[26] Dehaye J P Moran A Marino A Arch Oral Biol 1999 44 S39-S43Four classes of small molecules with high affinity at the
P2X7R have been reported cyclic imides adamantyl amidesdiarylimidazolines and 1-benzyl-5-phenyltetrazoles Theseclasses of compounds are highly amenable to structuralmodifications indicating their good prospects as leadmolecules for further improving the potency and selectivity atP2X7R ultimately leading to orally active drugs To date norational drug design based on the knowledge of binding siteswithin this receptor has been undertaken This is a direct resultof the fact that no crystal structure or suitable homology modelhave been reported for this receptor The fact that the P2X7R canadopt either a channel or pore state causes even moredifficulties in studying receptor dynamics and at present theexact mechanism of channel-to-pore transition remains to beelucidated Undoubtedly the P2X7R is involved in severalprocesses relevant to chronic inflammation neurodegenerationand neuropathic pain and therefore discovery of more potentselective P2X7R antagonists would have a great impact in thepresent therapeutic window
[27] Li Q Luo X Zeng W Muallem S J Biol Chem 2003 27847554-47561
[28] Wang Q Wang L Feng Y-H Li X Zeng R Gorodeski GI AmJ Physiol Cell Physiol 2004 287 C1349-C1358
[29] Bardini M Lee H Y Burnstock G Cell Tissue Res 2000 299 105-113
[30] Hu H Z Gao N Lin Z Gao C Liu S Ren J Xia Y Wood JD J Comp Neurol 2001 440 299-310
[31] Hillman K A Burnstock G Unwin R J Nephron Exp Nephrol2005 101 e24-e30
[32] Koshi R Coutinho-Silva R Cascabulho C M Henrique-Pons AKnight G E Loesch A Burnstock G J Reprod Immunol 200566 127-140
[33] Moores T S Hasdemir B Vega-Riveroll L Deuchars J Parson SH Brain Res 2005 1034 40-50
[34] Surprenant A Rassendren F Kawashima E North R A Buell GScience 1996 272 735-738
[35] Cheewatrakoolpong B Gilchrest H Anthes J C Greenfeder SBiochem Biophys Res Commun 2005 332 17-27
[36] Watters J J Sommer J A Fisette P L Pfeiffer Z A Aga MPrabhu U Guerra A N Denlinger L C Bertics P J Drug Dev Res2001 53 91-104
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ACKNOWLEDGEMENTS[38] Khakh B S Burnstock G Kennedy C King B F North A
Seguela P Voigt M Humphrey P P A Nat Rev Neurosci 20012 165-174
The authors would like to thank Dr Natasha Hungerford forproofreading the manuscript
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[137] Ravi R G Kertesy S B Dubyak G R Jacobson K A Drug DevRes 2001 54 75-87[99] Jacobson K A Kim Y-C Wildman S S Mohanram A Harden T
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[140] Romagnoli R Baraldi P G Pavani M G Tabrizi M A MoormanA R Di Virgilio F Cattabriga E Pancaldi C Gessi S Borea P ABioorg Med Chem Lett 2004 14 5709-5712
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Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1523
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[147] Duplantier A J EP1310493A1 2003[148] Baxter A Kindon N Pairaudeau G Roberts B Thom S
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Received February 01 2007 Revised April 04 2007 Accepted April 06 2007
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1515
R2 X N
O
N
O
S
R1
S
O
O
N
O
O
Nthe inhibition of BzATP-induced ethidium bromide uptakemediated by the P2X7R expressed endogenously on the humanpre-monocytic cell line THP-1 [142] Compound 37a (Table 4)was identified as the lead structure and modifications on theimide and 4-pyridyl moieties were investigated The cyclicimide moiety is proven essential for the P2X7R activity sincesubstitutions with simple amines acyclic amides or acycliccarbamates almost completely abolished the activity [142] Thering can be five- or six-membered in the forms of imides (37a-bh) carbamates (37c-d) thiazolidine-24-diones (37eg) andhydantoins (37f) Replacement of the 4-pyridyl moiety with 4-substituted benzamides (37g-h) retained the P2X7R activityThe presence of a 3rsquo-nitro substituent on the biphenyl subunitwas reported to convey optimum P2X7R activity The mode ofinhibition of these compounds seemed to be competitivealthough there were observable changes in the maximalresponse as the compound concentration was increased [142]
Fig (16) General structure for the KN-62 derivatives (35a-35s)
Unfortunately while these KN-62 analogues are potentiallyuseful tools to investigate the basic properties of the P2X7Rthey do not represent good starting points for the discovery oforally-active drugs due to 1) high molecular weight (gt700 Da)2) high lipophilicity (clogP gt 6) and 3) presence of ametabolically labile yet crucial sulfonate group [141]
These compounds represent a new class of ligands thatpossess activity to the P2X7R They appear good candidates asorally active drugs due to their relatively low molecular weight(~400 Da) and would be highly amenable for further SARstudies However despite the succinimides and thiazolidine-24-diones representing potent analogues they were found to bemetabolically and chemically fragile On the other handalthough the carbamates were less potent they displayedimproved stability Future SAR studies could usefully involveinvestigation of the biphenyl ring and the chain linker
37 Cyclic Imides and Analogues
Cyclic imides 37 (Fig 18) were discovered through a high-throughput screening of AstraZenecarsquos compound library for
Table 3 Structures and P2X7R Antagonistic Properties of Selected KN-62 Derivatives [139]
Entry R1 R2 X IC50 (nM)a
KN-62 mdashCH3 mdashPh N 511 plusmn 11
35a mdashCH3 mdashCH2Ph N 211 plusmn 08
35b mdashCH3 mdashCH2CH2Ph N 600 plusmn 27
35c mdashCH3 mdashCH2Ph CH 653 plusmn 08
35d mdashCH3 2-pyridinyl N 170 plusmn 39
35e mdashCH3 2-pyrimidinyl N 798 plusmn 07
35f mdashCH3 p-F-Phmdash N 13 plusmn 01
35g mdashCH3 p-Cl-Phmdash N 105 plusmn 11
35h mdashCH3 p-I-Phmdash N 176 plusmn 38
35i mdashCH3 p-Me-Phmdash N 135 plusmn 02
35j mdashCH3 p-NO2-Phmdash N 58 plusmn 06
35k mdashCH3 p-OMe-Phmdash N 132 plusmn 43
35l mdashCH3 o-F-Phmdash N 185 plusmn 04
35m mdashCH3 o-Cl-Phmdash N 158 plusmn 09
35n mdashCH3 o-Me-Phmdash N 151 plusmn 14
35o mdashCH3 o-OMe-Phmdash N 841 plusmn 5
35p mdashCH3 m-Cl-Phmdash N 141 plusmn 03
35q mdashCH3 m-CF3-Phmdash N 311 plusmn 12
35r mdashCH3 om-diMe-Phmdash N 1122 plusmn 25
35s mdashCH3 mp-diCl-Phmdash N 339 plusmn 08
35t H o-Me-Phmdash N 288 plusmn 03
aAntagonistic IC50 (nM)a values derived from dose-response curves on the basis of inhibition of Ca2+ flux in human monocytes
1516 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
N N
O
N
S
N
OO
OS O
O
N
R
31 R = H (KN-62)
36 R = 2-[3H]Me
AstraZeneca reported that no replacement for the adamantaneframework was found [141] Investigation of the chain linkerrequirements between the adamantane and the phenyl ring wasperformed where it was found that C- and N- methylationcarbonyl reduction and methylene removal (38d-g) all causedloss of potency One methylene unit extension (38h ) retainedthe potency whereas reversal of the amide connectivity (38i)slightly decreased the potency AstraZeneca prepared acombinatorial library by reacting commercially availableadamantanemethylamine with a large variety of acid chloridesor via standard amide coupling with carboxylic acids Thegeneral trends indicated that only aromatic amides had activityat the P2X7R [141] On the phenyl ring 23- and 25-disubstitutions resulted in better antagonists (38j-o) than the26- (38p ) 35- (38q ) or 24-disusbstitutions (38r) The ortho-position could also tolerate substituents such as Br OMe andMe (38s-u) A closer examination on the reverse amides (38i v-y) also revealed that 23- and 25-disubsitutions were stillpreferred for P2X7R inhibition The indazole amide (38z) wasidentified as a target lead molecule with a good metabolicprofile suitable for further optimization for clinical trial studies[141]
Fig (17) Radiolabeled analogue of KN-62 [140]
O
NY
X
O
Z
NO2
Adamantyl amides represent a class of simple moleculeswith excellent activity at the P2X7R The nature of theadamantane group could potentially be further investigatedalthough the original authors stated that no replacement for theadamantane group was found A number of patents onadamantyl-containing benzamides have been generated byAstraZeneca [143-146] and Pfizer [147] indicating the potentialof this ligand class for generating a truly potent selective andorally active P2X7R inhibitor Pfizer specifically incorporatedtriazinyl group into the adamantyl benzamide framework [147]Fig (18) General structure for the cyclic imides generated by
AstraZeneca Non-adamantane benzamides including piperidinyl andpiperazinyl benzamides [148 149] triazinyl benzamides [150-153] quinolinyl benzamides [154 155] 34-difluoroanilinebenzamides [156] aromatic acid-containing benzamides [157]pyrazolylmethyl benzamides [158] and other heteroarylamides[159-163] have also been patented by various otherpharmaceutical companies
38 Adamantyl Amides
Adamantyl amides were also discovered from a high-throughput screening of AstraZenecarsquos compound library forinhibition of the P2X7R-mediated ethidium bromide uptakeinduced by BzATP [141] N-Adamantan-1-ylmethyl-2-chloro-benzamide 38a was identified as the lead structure with anexcellent pA2 value of 81 The presence of an ortho-substituentis believed to cause a twist in the orientation of the benzamideessential for potent P2X7R antagonism whereas para-substituents (38bc) have deleterious effects on the P2X7Ractivity Selected compounds of this series are represented inTable 5
39 Diarylimidazolines
Diarylimidazolines represent another class of P2X7-activecompounds They were first discovered through a high-throughput screening of Aventisrsquo compound library forinhibition of BzATP-induced YO-PRO-1 uptake mediated byP2X7R expressed in U373 cells stably expressing the human
Table 4 Structures and P2X7R Antagonistic Properties of Selected Cyclic Imides [142]
Entry X Y Z IC50 ( M) pA2a
37a CH2 CO N - 69
37b (CH2)2 CO N - 75
37c O CH2 N 64
37d O (CH2)2 N 063 -
37e S CO N - 77
37f NH CO N 02 -
37g S CO C-CONHMe 016 -
37h CH2 CO C-CONMe2 025 -
a pA2 = the negative logarithm of a molar concentration of an antagonist that necessitates doubling of agonist concentration needed to elicit the original submaximalresponse obtained in the absence of antagonist
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1517
P2X7R [164] Compound 39a was identified as an initial hitfrom the screening with an IC50 value of 80 nM
equipotent Some degree of branching in the chain linker wasgenerally tolerated (39f-h) Configurational constraintsimposed on the chain linker as exemplified by 39i weretolerated but this was not the case for the cyclopropyl analogue39j
X
RO
NH
N
HN
38a-y 38z
Subsequent investigations focused on the substitutionpattern in the aryl ring for the benzyl (n=1) and phenethyl (n=2)series [164] Electron withdrawing and small neutral groupswere generally tolerable in both series (39k-r v-x) Howeverelectron-donating groups exemplified by OMe gave much lessactive compounds (39s-u) In the phenethyl seriessubstitutions at the ortho- and meta- positions were preferableto the corresponding para- substitutions (39kl vs 39m and39no vs 39p) Interestingly in the benzyl series meta- andpara- substitutions resulted in more active compounds than thecorresponding ortho analogues (39v vs 39wx)
Fig (19) Adamantyl amides generated by AstraZeneca
Investigation of the nature of the linker between thearomatic ring and the 2-position of the imidazole ring revealedthat a 3 or 4 carbon linker (39bc) caused a slight decrease in theactivity and no carbon linker (39d ) rendered the compoundinactive [164] One carbon linker with a methyl branching unit(39e) was tolerated It was reported that there was noenantiomeric preference of the methyl branch they were
The diarylimidazolines examined represent good candidatesfor orally active P2X7R inhibitors they are small and highlyamenable for SAR studies The 4- and 5-phenyl rings representattractive sites for further investigations
Table 5 Structures and P2X7R Antagonistic Properties of Selected Adamantyl Amides [141]
Entry R X pA2
38a 2-Cl CH2NHCO 81
38b 4-Cl CH2NHCO inactive
38c 24-Cl2 CH2NHCO 64
38d 2-Cl CHMeNHCO 64
38e 2-Cl CH2NMeCO 54
38f 2-Cl CH2NHCH2 54
38g 2-Cl NHCO inactive
38h 2-Cl (CH2)2NHCO 78
38i 2-Cl CH2CONH 63
38j 23-Cl2 CH2NHCO 88
38k 25-Cl2 CH2NHCO 83
38l 2-Cl-3-NH2 CH2NHCO 82
38m 2-Cl-5-OMe CH2NHCO 88
38n 2-Cl-5-OH CH2NHCO 80
38o 2-Cl-5-NH2 CH2NHCO 84
38p 26-Cl2 CH2NHCO 74
38q 35-Cl2 CH2NHCO 54
38r 24-Cl2 CH2NHCO 64
38s 2-Br CH2NHCO 80
38t 2-OMe CH2NHCO 71
38u 2-Me CH2NHCO 79
38v 2-Me CH2CONH 68
38w 2-Cl-5-OMe CH2CONH 72
38x 2-Me-3-OMe CH2CONH 83
38y 2-Me-5-OMe CH2CONH 80
1518 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
N
HN
XY
R
N
HN
N
HN
N
HN
( )n
39i39j
39a-ce-hk-w 39d
Fig (20) General structure of the diarylimidazolines generated by Aventis
Table 6 Structures and P2X7R Antagonistic Properties of Selected Diarylimidazolines [164]
Entry n X Y R IC50 ( M)a
39a 1 H H H 008
39b 2 H H H 054
39c 3 H H H 061
39e 0 (R)-Me H H 011
39f 1 (S)-Me H H 003
39g 1 Me Me H 001
39h 1 Ph H H 032
39k 1 H H 2-F 016
39l 1 H H 3-F 004
39m 1 H H 4-F 022
39n 1 H H 2-Cl 007
39o 1 H H 3-Cl 012
39p 1 H H 4-Cl gt1
39q 1 H H 2-Me 007
39r 1 H H 4-Me 219
39s 1 H H 2-OMe 083
39t 1 H H 3-OMe gt3
39u 1 H H 4-OMe gt3
39v 0 H H 2-F 058
39w 0 H H 3-F 008
39x 0 H H 4-F 008
aAntagonistic IC50 values derived from dose-response curves on the basis of inhibition of YO-PRO-1 dye uptake
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1519
R1N
N
NN
R2
R1
NN
NN
R2
N
N
NN
Cl
Cl
Cl
Cl
40b-t 40u-x40a
1
2
3
4
5
Fig (21) General structure of tetrazoles generated by Abbott Laboratories
310 1-Benzyl-5-Phenyltetrazoles potency The limited solubility of those compounds howeverposed a challenge to incorporate substitutions that couldincrease the solubility while maintaining activity A 3-pyridylgroup (40d ) was found to overcome this problem Moreover anextra 2-methyl substituent (40e) resulted in higher activitycompared to 40d A methylene extension (40f) caused a decreasein potency whereas other heterocycles such as 40g and 40hresulted in analogues with similar potency to 40d
1-Benzyl-5-phenyltetrazoles were discovered through ahigh-throughput screening by Abbott Laboratories for threedifferent P2X7R activities Ca2+ flux at the rat or human P2X7RYO-PRO-1 uptake and interleukin-1β release at human P2X7R[165-168] The initial hit from the screening was the tetrazole40a (Table 7) with a pIC50 value of 69 in inhibiting P2X7Rderived from the recombinant human cell line
Investigation of the nature of the substituent attached to the5-position of the tetrazole core showed that the substitutionpattern at this position was not well tolerated [165] Forexample unsubstituted mono-chlorosubstituted 25- and 34-
Initial SAR studies focused on the substituent attached tothe 1-position of the tetrazole core [165] Unsubstituted orortho-substituted benzylic group (40bc) exhibited good
Table 7 Structures and P2X7R Antagonistic Properties of Selected 1-benzyl-5-tetrazoles [165]
1520 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
disubstituted compounds (40i-m) pyridine and quinoline ring(40n-p ) analogues were not tolerated However moreconservative changes such as CF3 and F for Cl group (40q-t)resulted in an increase of potency
release and amplification of astrocytic Ca2+ signaling werereported to be due to the P2X7R and not Cx43 hemichannels[170]
Decavanadate a type of polymer of vanadium oxides hasbeen demonstrated to be a competitive rapid-acting antagonistat the P2X7R [172] It is capable of inhibiting the BzATP-stimulated ethidium bromide uptake in human mouse and ratP2X7R with pA2 values of 74 57 and 71 respectivelyDecavanadate has not been fully tested for its selectivity overother P2X receptors however it is suggested that this largemolecular species acts at the extracellular ATP binding site ofthe receptor consistent with the competitive mode of inhibition[172] A monoclonal antibody specific for the human P2X7Rhas also been shown to be a potent and selective antagonist ofthe receptor with an IC50 value of 5 nM in blocking the BzATP-evoked currents [173] Interestingly tenidap at 50 ndash 200 microMconcentration increases the sensitivity of mouse macrophagesto ATP-mediated cytotoxicity [174] 17-β-estradiol and notprogesterone or 17-α-estradiol has also been reported to blockthe agonist-induced currents at the human P2X7R with an IC50value of 3 microM in a non-genomic manner [175] Polymyxin B apotent antibiotic well-known for its lipopolysaccharide-neutralizing effects has been demonstrated to confer asynergistic effect when co-applied with ATP in a similar mannerto tenidap inducing Ca2+ influx ethidium bromide uptake andlactate dehydrogenase release via action at the P2X7R [176]
Tetrazoles with reversed connectivity (40u-x) were alsoevaluated where it was found that this connectivity reversal hadlittle effect on the potency [165] These tetrazoles were reportedto be slightly more active in the human P2X7R than the ratanalogue [165]
The tetrazole derivative 40d has been shown to be selectivefor other P2 receptors including P2X3 P2X4 and P2Y2 receptors[165] Pharmacological testing of this tetrazole suggested acompetitive mode of inhibition at the P2X7R and its efficacyhas also been evaluated in a model of neuropathic pain in rats[165] Overall these tetrazoles are found to be P2X7R-active andhighly adaptable to structural modifications These propertiesmake the tetrazoles an interesting class of compound for furtherinvestigating P2X7R activity
311 Other P2X7R-Active Ligands
Chelerythrine 41 (Fig 22) is a member of the benzophen-anthridine alkaloids known to inhibit protein kinase C withgood potency and selectivity (IC50 066 microM) as well as enzymessuch as alanine aminotransferase and Na+K+-ATPase [169] At10 microM concentrations chelerythrine non-competitivelyinhibited the BzATP-induced 86Rb+ efflux by 74 in human B-lymphocytes [169] Dye uptake studies could not be performeddue to the fluorescent nature of the compound It is yet to bedetermined whether chelerythrine inhibition of P2X7R involvesprotein kinase C [169]
More recently other distinct classes of P2X7-active ligandshave been reported and patented by the Abbott Laboratoriesacylhydrazides [177] cyanoamidines and cyanoguanidines[178 179]
4 CONCLUSIONS AND FUTURE DIRECTIONS
O
O
N
O
O
Cl
41
Although many structurally diverse class of compoundshave been reported as P2X7R antagonists with reasonablepotency the selectivity issue over other P2 receptors andproteins such as ecto-ATPases and ectonucleotidases stillprevails This is particularly an issue for oxidized ATP andsuramin SAR studies performed on PPADS and suramin mainlygave improved potency and selectivity for the P2X1 receptorIon channel blockers such as amilorides and stilbenederivatives only possess relatively low potency at the P2X7RFurther complications in studying these P2X7R antagonistsalso arise from the demonstrated species differences acrosshuman rat and mouse systems Brilliant Blue G is active for allthree species however at present there have been no SAR datareported Another fundamental factor that hampers the study ofP2X7R in detail is the relatively low potency and selectivity ofthe available agonist BzATP The discovery of a more potentselective P2X7R agonist would be ideal since in many studiesfull dose-response curves could not be generated due to the low
Fig (22) Chemical structure for chelerythrine an alkaloid withantagonistic properties at the human P2X7R
Gap junction channel blockers such as flufenamic acid 42 carbenoxolone 43 and mefloquine 44 (Fig 23) were all foundto be inhibitors of the BzATP-induced Ca2+ flux (IC50 values of655 175 and 25 nM respectively) and of YO-PRO-1 uptake in1321N1 astrocytoma cells stably expressing rat P2X7R [170]This finding poses a question to the general use of gap junctionchannel blockers as indicators of the participation of connexinhemichannels at the sites of transmitter release [171] The ATP
HN CF3
O OH
42 f lufenamic acid
HO
-O
O
O
H
OH
O
O-
N
HN
CF3
CF3
H O
H
43 carbenoxolone 44 mefloquine
Fig (23) Chemical structures for gap junction channel blockers with P2X7R antagonistic properties
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1521
potency of BzATP The nature of functional assays on theP2X7R is also found to be complex as ionic compositiontemperature and cell origins all affect the results demandingextra care be taken when antagonist potencies are directlycompared from different studies
[23] Li J Liu D Ke H Z Duncan R L Turner C H J Biol Chem2005 280 42952-42959
[24] Penolazzi L Bianchini E Lambertini E Baraldi P G RomagnoliR Piva R Gambari R J Biomed Sci 2005 12 1013-1020
[25] Gibbons S J Washburn K B Talamo B R J Auton Pharmacol2001 21 181-190
[26] Dehaye J P Moran A Marino A Arch Oral Biol 1999 44 S39-S43Four classes of small molecules with high affinity at the
P2X7R have been reported cyclic imides adamantyl amidesdiarylimidazolines and 1-benzyl-5-phenyltetrazoles Theseclasses of compounds are highly amenable to structuralmodifications indicating their good prospects as leadmolecules for further improving the potency and selectivity atP2X7R ultimately leading to orally active drugs To date norational drug design based on the knowledge of binding siteswithin this receptor has been undertaken This is a direct resultof the fact that no crystal structure or suitable homology modelhave been reported for this receptor The fact that the P2X7R canadopt either a channel or pore state causes even moredifficulties in studying receptor dynamics and at present theexact mechanism of channel-to-pore transition remains to beelucidated Undoubtedly the P2X7R is involved in severalprocesses relevant to chronic inflammation neurodegenerationand neuropathic pain and therefore discovery of more potentselective P2X7R antagonists would have a great impact in thepresent therapeutic window
[27] Li Q Luo X Zeng W Muallem S J Biol Chem 2003 27847554-47561
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ACKNOWLEDGEMENTS[38] Khakh B S Burnstock G Kennedy C King B F North A
Seguela P Voigt M Humphrey P P A Nat Rev Neurosci 20012 165-174
The authors would like to thank Dr Natasha Hungerford forproofreading the manuscript
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S Gretener D Grahames C Kaur R Kosco-Vilbois M HHumphrey P P A Blood 1998 92 3521-3528[157] Thompson T Willis P WO2006080884A1 2006
[158] Concepcion A Inoue T Mochizuki Y Muramatsu A Gantner FNakashima K Urbhans K Bacon K B WO2005019182A1 2005
[174] Sanz J M Chiozzi P Di Virgilio F Eur J Pharmacol 1998 35 235-244
[159] Dombroski M A Duplantier A J Subramanyam CWO2004099146A1 2004
[175] Cario-Toumaniantz C Loirand G Ferrier L Pacaud P J Physiol1998 508 659-666
[160] Kelly M G Kincaid J WO2006102588A1 2006 [176] Ferrari D Pizzirani C Adinolfi E Forchap S Sitta B Turchet LFalzoni S Minelli M Baricordi R Di Virgilio F J Immunol 2004173 4652-4660
[161] Kelly M J Kincaid J WO2006102610A2 2006[162] Schwink L Stengelin S Gossel M Bohme T Hessler G Stahl P
Gretzke D WO2004072025A2 2004 [177] Nelson D W Jarvis M F Carroll W A WO2006110516A1 2006[163] Schwink L Stengelin S Boehme T Gossel M Hessler G Stahl P
WO2005070925A1 2005[178] Carroll W A Perez-Medrano A Peddi S Florjancic A S
WO20060025614A1 2006[164] Merriman G H Ma L Shum P McGarry D Volz F Sabol J S
Gross A Zhao Z Rampe D Wang L Wirtz-Brugger F Harris BA Macdonald D Bioorg Med Chem Lett 2005 15 435-438
[179] Carroll W A Perez-Medrano A Jarvis M F Wang Y Peddi SWO20050171195A1 2005
Received February 01 2007 Revised April 04 2007 Accepted April 06 2007
1516 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
N N
O
N
S
N
OO
OS O
O
N
R
31 R = H (KN-62)
36 R = 2-[3H]Me
AstraZeneca reported that no replacement for the adamantaneframework was found [141] Investigation of the chain linkerrequirements between the adamantane and the phenyl ring wasperformed where it was found that C- and N- methylationcarbonyl reduction and methylene removal (38d-g) all causedloss of potency One methylene unit extension (38h ) retainedthe potency whereas reversal of the amide connectivity (38i)slightly decreased the potency AstraZeneca prepared acombinatorial library by reacting commercially availableadamantanemethylamine with a large variety of acid chloridesor via standard amide coupling with carboxylic acids Thegeneral trends indicated that only aromatic amides had activityat the P2X7R [141] On the phenyl ring 23- and 25-disubstitutions resulted in better antagonists (38j-o) than the26- (38p ) 35- (38q ) or 24-disusbstitutions (38r) The ortho-position could also tolerate substituents such as Br OMe andMe (38s-u) A closer examination on the reverse amides (38i v-y) also revealed that 23- and 25-disubsitutions were stillpreferred for P2X7R inhibition The indazole amide (38z) wasidentified as a target lead molecule with a good metabolicprofile suitable for further optimization for clinical trial studies[141]
Fig (17) Radiolabeled analogue of KN-62 [140]
O
NY
X
O
Z
NO2
Adamantyl amides represent a class of simple moleculeswith excellent activity at the P2X7R The nature of theadamantane group could potentially be further investigatedalthough the original authors stated that no replacement for theadamantane group was found A number of patents onadamantyl-containing benzamides have been generated byAstraZeneca [143-146] and Pfizer [147] indicating the potentialof this ligand class for generating a truly potent selective andorally active P2X7R inhibitor Pfizer specifically incorporatedtriazinyl group into the adamantyl benzamide framework [147]Fig (18) General structure for the cyclic imides generated by
AstraZeneca Non-adamantane benzamides including piperidinyl andpiperazinyl benzamides [148 149] triazinyl benzamides [150-153] quinolinyl benzamides [154 155] 34-difluoroanilinebenzamides [156] aromatic acid-containing benzamides [157]pyrazolylmethyl benzamides [158] and other heteroarylamides[159-163] have also been patented by various otherpharmaceutical companies
38 Adamantyl Amides
Adamantyl amides were also discovered from a high-throughput screening of AstraZenecarsquos compound library forinhibition of the P2X7R-mediated ethidium bromide uptakeinduced by BzATP [141] N-Adamantan-1-ylmethyl-2-chloro-benzamide 38a was identified as the lead structure with anexcellent pA2 value of 81 The presence of an ortho-substituentis believed to cause a twist in the orientation of the benzamideessential for potent P2X7R antagonism whereas para-substituents (38bc) have deleterious effects on the P2X7Ractivity Selected compounds of this series are represented inTable 5
39 Diarylimidazolines
Diarylimidazolines represent another class of P2X7-activecompounds They were first discovered through a high-throughput screening of Aventisrsquo compound library forinhibition of BzATP-induced YO-PRO-1 uptake mediated byP2X7R expressed in U373 cells stably expressing the human
Table 4 Structures and P2X7R Antagonistic Properties of Selected Cyclic Imides [142]
Entry X Y Z IC50 ( M) pA2a
37a CH2 CO N - 69
37b (CH2)2 CO N - 75
37c O CH2 N 64
37d O (CH2)2 N 063 -
37e S CO N - 77
37f NH CO N 02 -
37g S CO C-CONHMe 016 -
37h CH2 CO C-CONMe2 025 -
a pA2 = the negative logarithm of a molar concentration of an antagonist that necessitates doubling of agonist concentration needed to elicit the original submaximalresponse obtained in the absence of antagonist
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1517
P2X7R [164] Compound 39a was identified as an initial hitfrom the screening with an IC50 value of 80 nM
equipotent Some degree of branching in the chain linker wasgenerally tolerated (39f-h) Configurational constraintsimposed on the chain linker as exemplified by 39i weretolerated but this was not the case for the cyclopropyl analogue39j
X
RO
NH
N
HN
38a-y 38z
Subsequent investigations focused on the substitutionpattern in the aryl ring for the benzyl (n=1) and phenethyl (n=2)series [164] Electron withdrawing and small neutral groupswere generally tolerable in both series (39k-r v-x) Howeverelectron-donating groups exemplified by OMe gave much lessactive compounds (39s-u) In the phenethyl seriessubstitutions at the ortho- and meta- positions were preferableto the corresponding para- substitutions (39kl vs 39m and39no vs 39p) Interestingly in the benzyl series meta- andpara- substitutions resulted in more active compounds than thecorresponding ortho analogues (39v vs 39wx)
Fig (19) Adamantyl amides generated by AstraZeneca
Investigation of the nature of the linker between thearomatic ring and the 2-position of the imidazole ring revealedthat a 3 or 4 carbon linker (39bc) caused a slight decrease in theactivity and no carbon linker (39d ) rendered the compoundinactive [164] One carbon linker with a methyl branching unit(39e) was tolerated It was reported that there was noenantiomeric preference of the methyl branch they were
The diarylimidazolines examined represent good candidatesfor orally active P2X7R inhibitors they are small and highlyamenable for SAR studies The 4- and 5-phenyl rings representattractive sites for further investigations
Table 5 Structures and P2X7R Antagonistic Properties of Selected Adamantyl Amides [141]
Entry R X pA2
38a 2-Cl CH2NHCO 81
38b 4-Cl CH2NHCO inactive
38c 24-Cl2 CH2NHCO 64
38d 2-Cl CHMeNHCO 64
38e 2-Cl CH2NMeCO 54
38f 2-Cl CH2NHCH2 54
38g 2-Cl NHCO inactive
38h 2-Cl (CH2)2NHCO 78
38i 2-Cl CH2CONH 63
38j 23-Cl2 CH2NHCO 88
38k 25-Cl2 CH2NHCO 83
38l 2-Cl-3-NH2 CH2NHCO 82
38m 2-Cl-5-OMe CH2NHCO 88
38n 2-Cl-5-OH CH2NHCO 80
38o 2-Cl-5-NH2 CH2NHCO 84
38p 26-Cl2 CH2NHCO 74
38q 35-Cl2 CH2NHCO 54
38r 24-Cl2 CH2NHCO 64
38s 2-Br CH2NHCO 80
38t 2-OMe CH2NHCO 71
38u 2-Me CH2NHCO 79
38v 2-Me CH2CONH 68
38w 2-Cl-5-OMe CH2CONH 72
38x 2-Me-3-OMe CH2CONH 83
38y 2-Me-5-OMe CH2CONH 80
1518 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
N
HN
XY
R
N
HN
N
HN
N
HN
( )n
39i39j
39a-ce-hk-w 39d
Fig (20) General structure of the diarylimidazolines generated by Aventis
Table 6 Structures and P2X7R Antagonistic Properties of Selected Diarylimidazolines [164]
Entry n X Y R IC50 ( M)a
39a 1 H H H 008
39b 2 H H H 054
39c 3 H H H 061
39e 0 (R)-Me H H 011
39f 1 (S)-Me H H 003
39g 1 Me Me H 001
39h 1 Ph H H 032
39k 1 H H 2-F 016
39l 1 H H 3-F 004
39m 1 H H 4-F 022
39n 1 H H 2-Cl 007
39o 1 H H 3-Cl 012
39p 1 H H 4-Cl gt1
39q 1 H H 2-Me 007
39r 1 H H 4-Me 219
39s 1 H H 2-OMe 083
39t 1 H H 3-OMe gt3
39u 1 H H 4-OMe gt3
39v 0 H H 2-F 058
39w 0 H H 3-F 008
39x 0 H H 4-F 008
aAntagonistic IC50 values derived from dose-response curves on the basis of inhibition of YO-PRO-1 dye uptake
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1519
R1N
N
NN
R2
R1
NN
NN
R2
N
N
NN
Cl
Cl
Cl
Cl
40b-t 40u-x40a
1
2
3
4
5
Fig (21) General structure of tetrazoles generated by Abbott Laboratories
310 1-Benzyl-5-Phenyltetrazoles potency The limited solubility of those compounds howeverposed a challenge to incorporate substitutions that couldincrease the solubility while maintaining activity A 3-pyridylgroup (40d ) was found to overcome this problem Moreover anextra 2-methyl substituent (40e) resulted in higher activitycompared to 40d A methylene extension (40f) caused a decreasein potency whereas other heterocycles such as 40g and 40hresulted in analogues with similar potency to 40d
1-Benzyl-5-phenyltetrazoles were discovered through ahigh-throughput screening by Abbott Laboratories for threedifferent P2X7R activities Ca2+ flux at the rat or human P2X7RYO-PRO-1 uptake and interleukin-1β release at human P2X7R[165-168] The initial hit from the screening was the tetrazole40a (Table 7) with a pIC50 value of 69 in inhibiting P2X7Rderived from the recombinant human cell line
Investigation of the nature of the substituent attached to the5-position of the tetrazole core showed that the substitutionpattern at this position was not well tolerated [165] Forexample unsubstituted mono-chlorosubstituted 25- and 34-
Initial SAR studies focused on the substituent attached tothe 1-position of the tetrazole core [165] Unsubstituted orortho-substituted benzylic group (40bc) exhibited good
Table 7 Structures and P2X7R Antagonistic Properties of Selected 1-benzyl-5-tetrazoles [165]
1520 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
disubstituted compounds (40i-m) pyridine and quinoline ring(40n-p ) analogues were not tolerated However moreconservative changes such as CF3 and F for Cl group (40q-t)resulted in an increase of potency
release and amplification of astrocytic Ca2+ signaling werereported to be due to the P2X7R and not Cx43 hemichannels[170]
Decavanadate a type of polymer of vanadium oxides hasbeen demonstrated to be a competitive rapid-acting antagonistat the P2X7R [172] It is capable of inhibiting the BzATP-stimulated ethidium bromide uptake in human mouse and ratP2X7R with pA2 values of 74 57 and 71 respectivelyDecavanadate has not been fully tested for its selectivity overother P2X receptors however it is suggested that this largemolecular species acts at the extracellular ATP binding site ofthe receptor consistent with the competitive mode of inhibition[172] A monoclonal antibody specific for the human P2X7Rhas also been shown to be a potent and selective antagonist ofthe receptor with an IC50 value of 5 nM in blocking the BzATP-evoked currents [173] Interestingly tenidap at 50 ndash 200 microMconcentration increases the sensitivity of mouse macrophagesto ATP-mediated cytotoxicity [174] 17-β-estradiol and notprogesterone or 17-α-estradiol has also been reported to blockthe agonist-induced currents at the human P2X7R with an IC50value of 3 microM in a non-genomic manner [175] Polymyxin B apotent antibiotic well-known for its lipopolysaccharide-neutralizing effects has been demonstrated to confer asynergistic effect when co-applied with ATP in a similar mannerto tenidap inducing Ca2+ influx ethidium bromide uptake andlactate dehydrogenase release via action at the P2X7R [176]
Tetrazoles with reversed connectivity (40u-x) were alsoevaluated where it was found that this connectivity reversal hadlittle effect on the potency [165] These tetrazoles were reportedto be slightly more active in the human P2X7R than the ratanalogue [165]
The tetrazole derivative 40d has been shown to be selectivefor other P2 receptors including P2X3 P2X4 and P2Y2 receptors[165] Pharmacological testing of this tetrazole suggested acompetitive mode of inhibition at the P2X7R and its efficacyhas also been evaluated in a model of neuropathic pain in rats[165] Overall these tetrazoles are found to be P2X7R-active andhighly adaptable to structural modifications These propertiesmake the tetrazoles an interesting class of compound for furtherinvestigating P2X7R activity
311 Other P2X7R-Active Ligands
Chelerythrine 41 (Fig 22) is a member of the benzophen-anthridine alkaloids known to inhibit protein kinase C withgood potency and selectivity (IC50 066 microM) as well as enzymessuch as alanine aminotransferase and Na+K+-ATPase [169] At10 microM concentrations chelerythrine non-competitivelyinhibited the BzATP-induced 86Rb+ efflux by 74 in human B-lymphocytes [169] Dye uptake studies could not be performeddue to the fluorescent nature of the compound It is yet to bedetermined whether chelerythrine inhibition of P2X7R involvesprotein kinase C [169]
More recently other distinct classes of P2X7-active ligandshave been reported and patented by the Abbott Laboratoriesacylhydrazides [177] cyanoamidines and cyanoguanidines[178 179]
4 CONCLUSIONS AND FUTURE DIRECTIONS
O
O
N
O
O
Cl
41
Although many structurally diverse class of compoundshave been reported as P2X7R antagonists with reasonablepotency the selectivity issue over other P2 receptors andproteins such as ecto-ATPases and ectonucleotidases stillprevails This is particularly an issue for oxidized ATP andsuramin SAR studies performed on PPADS and suramin mainlygave improved potency and selectivity for the P2X1 receptorIon channel blockers such as amilorides and stilbenederivatives only possess relatively low potency at the P2X7RFurther complications in studying these P2X7R antagonistsalso arise from the demonstrated species differences acrosshuman rat and mouse systems Brilliant Blue G is active for allthree species however at present there have been no SAR datareported Another fundamental factor that hampers the study ofP2X7R in detail is the relatively low potency and selectivity ofthe available agonist BzATP The discovery of a more potentselective P2X7R agonist would be ideal since in many studiesfull dose-response curves could not be generated due to the low
Fig (22) Chemical structure for chelerythrine an alkaloid withantagonistic properties at the human P2X7R
Gap junction channel blockers such as flufenamic acid 42 carbenoxolone 43 and mefloquine 44 (Fig 23) were all foundto be inhibitors of the BzATP-induced Ca2+ flux (IC50 values of655 175 and 25 nM respectively) and of YO-PRO-1 uptake in1321N1 astrocytoma cells stably expressing rat P2X7R [170]This finding poses a question to the general use of gap junctionchannel blockers as indicators of the participation of connexinhemichannels at the sites of transmitter release [171] The ATP
HN CF3
O OH
42 f lufenamic acid
HO
-O
O
O
H
OH
O
O-
N
HN
CF3
CF3
H O
H
43 carbenoxolone 44 mefloquine
Fig (23) Chemical structures for gap junction channel blockers with P2X7R antagonistic properties
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1521
potency of BzATP The nature of functional assays on theP2X7R is also found to be complex as ionic compositiontemperature and cell origins all affect the results demandingextra care be taken when antagonist potencies are directlycompared from different studies
[23] Li J Liu D Ke H Z Duncan R L Turner C H J Biol Chem2005 280 42952-42959
[24] Penolazzi L Bianchini E Lambertini E Baraldi P G RomagnoliR Piva R Gambari R J Biomed Sci 2005 12 1013-1020
[25] Gibbons S J Washburn K B Talamo B R J Auton Pharmacol2001 21 181-190
[26] Dehaye J P Moran A Marino A Arch Oral Biol 1999 44 S39-S43Four classes of small molecules with high affinity at the
P2X7R have been reported cyclic imides adamantyl amidesdiarylimidazolines and 1-benzyl-5-phenyltetrazoles Theseclasses of compounds are highly amenable to structuralmodifications indicating their good prospects as leadmolecules for further improving the potency and selectivity atP2X7R ultimately leading to orally active drugs To date norational drug design based on the knowledge of binding siteswithin this receptor has been undertaken This is a direct resultof the fact that no crystal structure or suitable homology modelhave been reported for this receptor The fact that the P2X7R canadopt either a channel or pore state causes even moredifficulties in studying receptor dynamics and at present theexact mechanism of channel-to-pore transition remains to beelucidated Undoubtedly the P2X7R is involved in severalprocesses relevant to chronic inflammation neurodegenerationand neuropathic pain and therefore discovery of more potentselective P2X7R antagonists would have a great impact in thepresent therapeutic window
[27] Li Q Luo X Zeng W Muallem S J Biol Chem 2003 27847554-47561
[28] Wang Q Wang L Feng Y-H Li X Zeng R Gorodeski GI AmJ Physiol Cell Physiol 2004 287 C1349-C1358
[29] Bardini M Lee H Y Burnstock G Cell Tissue Res 2000 299 105-113
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[31] Hillman K A Burnstock G Unwin R J Nephron Exp Nephrol2005 101 e24-e30
[32] Koshi R Coutinho-Silva R Cascabulho C M Henrique-Pons AKnight G E Loesch A Burnstock G J Reprod Immunol 200566 127-140
[33] Moores T S Hasdemir B Vega-Riveroll L Deuchars J Parson SH Brain Res 2005 1034 40-50
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ACKNOWLEDGEMENTS[38] Khakh B S Burnstock G Kennedy C King B F North A
Seguela P Voigt M Humphrey P P A Nat Rev Neurosci 20012 165-174
The authors would like to thank Dr Natasha Hungerford forproofreading the manuscript
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Pharmacol 2003 140 507-519 [130] Jiang L-H Mackenzie A B North R A Surprenant A MolPharmacol 2000 58 82-88[94] DellAntonio G Quattrini A Cin E D Fulgenzi A Ferrero M E
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Neurosci Lett 2002 327 87-90 [132] Tuluc F Bultmann R Glanzel M Frahm A W Starke K Naunyn-Schmiedebergs Arch Pharmacol 1998 357 111-120[96] Lambrecht G Braun K Damer S Ganso M Hildebrandt C
Ullmann H Kassack M U Nickel P Curr Pharm Des 2002 82371-2399
[133] Wittenburg H Bultmann R Pause B Ganter C Kurz G Starke KNaunyn-Schmiedebergs Arch Pharmacol 1996 354 491-497
[97] Garcia-Guzman M Soto F Gomez-Hernandez J M Lund P-EStuhmer W Mol Pharmacol 1997 51 109-118
[134] Gargett C E Wiley J S Br J Pharmacol 1997 120 1483-1490[135] Humphreys B D Virginio C Surprenant A Rice J Dubyak GR
Mol Pharmacol 1998 54 22-32[98] Kim Y-C Camaioni E Ziganshin A U Ji X-D King B FWildman S S Rychkov A Yoburn J Kim H Mohanram AHarden T K Boyer J L Burnstock G Jacobson K A Drug DevRes 1998 45 52-66
[136] Baraldi P G Romagnoli R Tabrizi M A Falzoni S Di Virgilio FBioorg Med Chem Lett 2000 10 681-684
[137] Ravi R G Kertesy S B Dubyak G R Jacobson K A Drug DevRes 2001 54 75-87[99] Jacobson K A Kim Y-C Wildman S S Mohanram A Harden T
K Boyer J L King B F Burnstock G J Med Chem 1998 41 2201-2206
[138] Chen W Ravi R G Kertesy S B Dubyak G R Jacobson K ABioconjug Chem 2002 13 1100-1111
[100] Kim Y-C Brown S G Harden T K Boyer J L Dubyak GKing B F Burnstock G Jacobson K A J Med Chem 2001 44 340-349
[139] Baraldi P G Nunez M D C A M Falzoni S Di Virgilio FRomagnoli R J Med Chem 2003 46 1318-1329
[140] Romagnoli R Baraldi P G Pavani M G Tabrizi M A MoormanA R Di Virgilio F Cattabriga E Pancaldi C Gessi S Borea P ABioorg Med Chem Lett 2004 14 5709-5712
[101] Brown S G Kim Y-C Kim S-A Jacobson K A Burnstock GKing B F Drug Dev Res 2001 53 281-291
[102] Lambrecht G Rettinger J Baumert H G Czeche S Damer SGanso M Hildebrandt C Niebel B Spatz-Kumbel G SchmalzingG Mutschler E Eur J Pharmacol 2000 387 R19-R21
[141] Baxter A Bent J Bowers K Braddock M Brough S Fagura MLawson M McInally T Mortimore M Robertson M Weaver RWebborn P Bioorg Med Chem Lett 2003 13 4047-4050
[103] Kim Y-C Lee J-S Sak K Marteau F Mamedova L BoeynaemsJ-M Jacobson K A Biochem Pharmacol 2005 70 266-274
[142] Alcaraz L Baxter A Bent J Bowers K Braddock M CladingboelD Donald D Fagura M Furber M Laurent C Lawson M
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1523
Mortimore M McCormick M Roberts N Robertson M BioorgMed Chem Lett 2003 13 4043-4046
[165] Nelson D W Gregg R J Kort M E Perez-Medrano A Voight EA Wang Y Grayson G Namovic M T Donnelly-Roberts D LNiforatos W Honore P Jarvis M F Faltynek C R Carroll W AJ Med Chem 2006 49 3659-3666
[143] Caffrey M Rhonan F Pimm A WO2004074224A1 2004[144] Evans R Rhonan F Thompson T Willis P WO2006059945A1
2006 [166] Perez-Medrano A Nelson D W Carroll W A Kort M E Gregg RJ Voight E A Jarvis M F Kowaluk E A WO2006086229A12006
[145] Rhonan F Martin B Thompson T Tomkinson N Willis PWO2006025783A1 2006
[146] Cladingboel D Rhonan F Willis P WO2005014529A1 2005 [167] Carroll W A Perez-Medrano A Florjancic A S Nelson D WPeddi S Li T Bunnelle E M Hirst G Li B CWO20060052374A1 2006
[147] Duplantier A J EP1310493A1 2003[148] Baxter A Kindon N Pairaudeau G Roberts B Thom S
WO0144213A1 2001 [168] Carroll W A Perez-Medrano A Florjancic A S Nelson D WPeddi S Bunnelle E M Hirst G C Li B WO2005111003A12005
[149] Meghani P Bennion C WO0146200A1 2001[150] Li Z B WO20050288256A1 2005[151] Leonard J A Li Z J Li Z B Urban F J WO20050288288A1
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1378-1385[153] Li Z B WO2006003500A1 2006 [171] Saez J C Retamal M A Basilio D Bukauskas F F Bennett M
V L Biochim Biophys Acta 2005 1711 215-224[154] Evans R Eyssade C Rhonan F Martin B Thompson T WillisP WO2004106305A1 2004 [172] Michel A D Xing M Thompson K M Jones C A Humphrey P
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[161] Kelly M J Kincaid J WO2006102610A2 2006[162] Schwink L Stengelin S Gossel M Bohme T Hessler G Stahl P
Gretzke D WO2004072025A2 2004 [177] Nelson D W Jarvis M F Carroll W A WO2006110516A1 2006[163] Schwink L Stengelin S Boehme T Gossel M Hessler G Stahl P
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Gross A Zhao Z Rampe D Wang L Wirtz-Brugger F Harris BA Macdonald D Bioorg Med Chem Lett 2005 15 435-438
[179] Carroll W A Perez-Medrano A Jarvis M F Wang Y Peddi SWO20050171195A1 2005
Received February 01 2007 Revised April 04 2007 Accepted April 06 2007
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1517
P2X7R [164] Compound 39a was identified as an initial hitfrom the screening with an IC50 value of 80 nM
equipotent Some degree of branching in the chain linker wasgenerally tolerated (39f-h) Configurational constraintsimposed on the chain linker as exemplified by 39i weretolerated but this was not the case for the cyclopropyl analogue39j
X
RO
NH
N
HN
38a-y 38z
Subsequent investigations focused on the substitutionpattern in the aryl ring for the benzyl (n=1) and phenethyl (n=2)series [164] Electron withdrawing and small neutral groupswere generally tolerable in both series (39k-r v-x) Howeverelectron-donating groups exemplified by OMe gave much lessactive compounds (39s-u) In the phenethyl seriessubstitutions at the ortho- and meta- positions were preferableto the corresponding para- substitutions (39kl vs 39m and39no vs 39p) Interestingly in the benzyl series meta- andpara- substitutions resulted in more active compounds than thecorresponding ortho analogues (39v vs 39wx)
Fig (19) Adamantyl amides generated by AstraZeneca
Investigation of the nature of the linker between thearomatic ring and the 2-position of the imidazole ring revealedthat a 3 or 4 carbon linker (39bc) caused a slight decrease in theactivity and no carbon linker (39d ) rendered the compoundinactive [164] One carbon linker with a methyl branching unit(39e) was tolerated It was reported that there was noenantiomeric preference of the methyl branch they were
The diarylimidazolines examined represent good candidatesfor orally active P2X7R inhibitors they are small and highlyamenable for SAR studies The 4- and 5-phenyl rings representattractive sites for further investigations
Table 5 Structures and P2X7R Antagonistic Properties of Selected Adamantyl Amides [141]
Entry R X pA2
38a 2-Cl CH2NHCO 81
38b 4-Cl CH2NHCO inactive
38c 24-Cl2 CH2NHCO 64
38d 2-Cl CHMeNHCO 64
38e 2-Cl CH2NMeCO 54
38f 2-Cl CH2NHCH2 54
38g 2-Cl NHCO inactive
38h 2-Cl (CH2)2NHCO 78
38i 2-Cl CH2CONH 63
38j 23-Cl2 CH2NHCO 88
38k 25-Cl2 CH2NHCO 83
38l 2-Cl-3-NH2 CH2NHCO 82
38m 2-Cl-5-OMe CH2NHCO 88
38n 2-Cl-5-OH CH2NHCO 80
38o 2-Cl-5-NH2 CH2NHCO 84
38p 26-Cl2 CH2NHCO 74
38q 35-Cl2 CH2NHCO 54
38r 24-Cl2 CH2NHCO 64
38s 2-Br CH2NHCO 80
38t 2-OMe CH2NHCO 71
38u 2-Me CH2NHCO 79
38v 2-Me CH2CONH 68
38w 2-Cl-5-OMe CH2CONH 72
38x 2-Me-3-OMe CH2CONH 83
38y 2-Me-5-OMe CH2CONH 80
1518 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
N
HN
XY
R
N
HN
N
HN
N
HN
( )n
39i39j
39a-ce-hk-w 39d
Fig (20) General structure of the diarylimidazolines generated by Aventis
Table 6 Structures and P2X7R Antagonistic Properties of Selected Diarylimidazolines [164]
Entry n X Y R IC50 ( M)a
39a 1 H H H 008
39b 2 H H H 054
39c 3 H H H 061
39e 0 (R)-Me H H 011
39f 1 (S)-Me H H 003
39g 1 Me Me H 001
39h 1 Ph H H 032
39k 1 H H 2-F 016
39l 1 H H 3-F 004
39m 1 H H 4-F 022
39n 1 H H 2-Cl 007
39o 1 H H 3-Cl 012
39p 1 H H 4-Cl gt1
39q 1 H H 2-Me 007
39r 1 H H 4-Me 219
39s 1 H H 2-OMe 083
39t 1 H H 3-OMe gt3
39u 1 H H 4-OMe gt3
39v 0 H H 2-F 058
39w 0 H H 3-F 008
39x 0 H H 4-F 008
aAntagonistic IC50 values derived from dose-response curves on the basis of inhibition of YO-PRO-1 dye uptake
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1519
R1N
N
NN
R2
R1
NN
NN
R2
N
N
NN
Cl
Cl
Cl
Cl
40b-t 40u-x40a
1
2
3
4
5
Fig (21) General structure of tetrazoles generated by Abbott Laboratories
310 1-Benzyl-5-Phenyltetrazoles potency The limited solubility of those compounds howeverposed a challenge to incorporate substitutions that couldincrease the solubility while maintaining activity A 3-pyridylgroup (40d ) was found to overcome this problem Moreover anextra 2-methyl substituent (40e) resulted in higher activitycompared to 40d A methylene extension (40f) caused a decreasein potency whereas other heterocycles such as 40g and 40hresulted in analogues with similar potency to 40d
1-Benzyl-5-phenyltetrazoles were discovered through ahigh-throughput screening by Abbott Laboratories for threedifferent P2X7R activities Ca2+ flux at the rat or human P2X7RYO-PRO-1 uptake and interleukin-1β release at human P2X7R[165-168] The initial hit from the screening was the tetrazole40a (Table 7) with a pIC50 value of 69 in inhibiting P2X7Rderived from the recombinant human cell line
Investigation of the nature of the substituent attached to the5-position of the tetrazole core showed that the substitutionpattern at this position was not well tolerated [165] Forexample unsubstituted mono-chlorosubstituted 25- and 34-
Initial SAR studies focused on the substituent attached tothe 1-position of the tetrazole core [165] Unsubstituted orortho-substituted benzylic group (40bc) exhibited good
Table 7 Structures and P2X7R Antagonistic Properties of Selected 1-benzyl-5-tetrazoles [165]
1520 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
disubstituted compounds (40i-m) pyridine and quinoline ring(40n-p ) analogues were not tolerated However moreconservative changes such as CF3 and F for Cl group (40q-t)resulted in an increase of potency
release and amplification of astrocytic Ca2+ signaling werereported to be due to the P2X7R and not Cx43 hemichannels[170]
Decavanadate a type of polymer of vanadium oxides hasbeen demonstrated to be a competitive rapid-acting antagonistat the P2X7R [172] It is capable of inhibiting the BzATP-stimulated ethidium bromide uptake in human mouse and ratP2X7R with pA2 values of 74 57 and 71 respectivelyDecavanadate has not been fully tested for its selectivity overother P2X receptors however it is suggested that this largemolecular species acts at the extracellular ATP binding site ofthe receptor consistent with the competitive mode of inhibition[172] A monoclonal antibody specific for the human P2X7Rhas also been shown to be a potent and selective antagonist ofthe receptor with an IC50 value of 5 nM in blocking the BzATP-evoked currents [173] Interestingly tenidap at 50 ndash 200 microMconcentration increases the sensitivity of mouse macrophagesto ATP-mediated cytotoxicity [174] 17-β-estradiol and notprogesterone or 17-α-estradiol has also been reported to blockthe agonist-induced currents at the human P2X7R with an IC50value of 3 microM in a non-genomic manner [175] Polymyxin B apotent antibiotic well-known for its lipopolysaccharide-neutralizing effects has been demonstrated to confer asynergistic effect when co-applied with ATP in a similar mannerto tenidap inducing Ca2+ influx ethidium bromide uptake andlactate dehydrogenase release via action at the P2X7R [176]
Tetrazoles with reversed connectivity (40u-x) were alsoevaluated where it was found that this connectivity reversal hadlittle effect on the potency [165] These tetrazoles were reportedto be slightly more active in the human P2X7R than the ratanalogue [165]
The tetrazole derivative 40d has been shown to be selectivefor other P2 receptors including P2X3 P2X4 and P2Y2 receptors[165] Pharmacological testing of this tetrazole suggested acompetitive mode of inhibition at the P2X7R and its efficacyhas also been evaluated in a model of neuropathic pain in rats[165] Overall these tetrazoles are found to be P2X7R-active andhighly adaptable to structural modifications These propertiesmake the tetrazoles an interesting class of compound for furtherinvestigating P2X7R activity
311 Other P2X7R-Active Ligands
Chelerythrine 41 (Fig 22) is a member of the benzophen-anthridine alkaloids known to inhibit protein kinase C withgood potency and selectivity (IC50 066 microM) as well as enzymessuch as alanine aminotransferase and Na+K+-ATPase [169] At10 microM concentrations chelerythrine non-competitivelyinhibited the BzATP-induced 86Rb+ efflux by 74 in human B-lymphocytes [169] Dye uptake studies could not be performeddue to the fluorescent nature of the compound It is yet to bedetermined whether chelerythrine inhibition of P2X7R involvesprotein kinase C [169]
More recently other distinct classes of P2X7-active ligandshave been reported and patented by the Abbott Laboratoriesacylhydrazides [177] cyanoamidines and cyanoguanidines[178 179]
4 CONCLUSIONS AND FUTURE DIRECTIONS
O
O
N
O
O
Cl
41
Although many structurally diverse class of compoundshave been reported as P2X7R antagonists with reasonablepotency the selectivity issue over other P2 receptors andproteins such as ecto-ATPases and ectonucleotidases stillprevails This is particularly an issue for oxidized ATP andsuramin SAR studies performed on PPADS and suramin mainlygave improved potency and selectivity for the P2X1 receptorIon channel blockers such as amilorides and stilbenederivatives only possess relatively low potency at the P2X7RFurther complications in studying these P2X7R antagonistsalso arise from the demonstrated species differences acrosshuman rat and mouse systems Brilliant Blue G is active for allthree species however at present there have been no SAR datareported Another fundamental factor that hampers the study ofP2X7R in detail is the relatively low potency and selectivity ofthe available agonist BzATP The discovery of a more potentselective P2X7R agonist would be ideal since in many studiesfull dose-response curves could not be generated due to the low
Fig (22) Chemical structure for chelerythrine an alkaloid withantagonistic properties at the human P2X7R
Gap junction channel blockers such as flufenamic acid 42 carbenoxolone 43 and mefloquine 44 (Fig 23) were all foundto be inhibitors of the BzATP-induced Ca2+ flux (IC50 values of655 175 and 25 nM respectively) and of YO-PRO-1 uptake in1321N1 astrocytoma cells stably expressing rat P2X7R [170]This finding poses a question to the general use of gap junctionchannel blockers as indicators of the participation of connexinhemichannels at the sites of transmitter release [171] The ATP
HN CF3
O OH
42 f lufenamic acid
HO
-O
O
O
H
OH
O
O-
N
HN
CF3
CF3
H O
H
43 carbenoxolone 44 mefloquine
Fig (23) Chemical structures for gap junction channel blockers with P2X7R antagonistic properties
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1521
potency of BzATP The nature of functional assays on theP2X7R is also found to be complex as ionic compositiontemperature and cell origins all affect the results demandingextra care be taken when antagonist potencies are directlycompared from different studies
[23] Li J Liu D Ke H Z Duncan R L Turner C H J Biol Chem2005 280 42952-42959
[24] Penolazzi L Bianchini E Lambertini E Baraldi P G RomagnoliR Piva R Gambari R J Biomed Sci 2005 12 1013-1020
[25] Gibbons S J Washburn K B Talamo B R J Auton Pharmacol2001 21 181-190
[26] Dehaye J P Moran A Marino A Arch Oral Biol 1999 44 S39-S43Four classes of small molecules with high affinity at the
P2X7R have been reported cyclic imides adamantyl amidesdiarylimidazolines and 1-benzyl-5-phenyltetrazoles Theseclasses of compounds are highly amenable to structuralmodifications indicating their good prospects as leadmolecules for further improving the potency and selectivity atP2X7R ultimately leading to orally active drugs To date norational drug design based on the knowledge of binding siteswithin this receptor has been undertaken This is a direct resultof the fact that no crystal structure or suitable homology modelhave been reported for this receptor The fact that the P2X7R canadopt either a channel or pore state causes even moredifficulties in studying receptor dynamics and at present theexact mechanism of channel-to-pore transition remains to beelucidated Undoubtedly the P2X7R is involved in severalprocesses relevant to chronic inflammation neurodegenerationand neuropathic pain and therefore discovery of more potentselective P2X7R antagonists would have a great impact in thepresent therapeutic window
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ACKNOWLEDGEMENTS[38] Khakh B S Burnstock G Kennedy C King B F North A
Seguela P Voigt M Humphrey P P A Nat Rev Neurosci 20012 165-174
The authors would like to thank Dr Natasha Hungerford forproofreading the manuscript
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Ullmann H Kassack M U Nickel P Curr Pharm Des 2002 82371-2399
[133] Wittenburg H Bultmann R Pause B Ganter C Kurz G Starke KNaunyn-Schmiedebergs Arch Pharmacol 1996 354 491-497
[97] Garcia-Guzman M Soto F Gomez-Hernandez J M Lund P-EStuhmer W Mol Pharmacol 1997 51 109-118
[134] Gargett C E Wiley J S Br J Pharmacol 1997 120 1483-1490[135] Humphreys B D Virginio C Surprenant A Rice J Dubyak GR
Mol Pharmacol 1998 54 22-32[98] Kim Y-C Camaioni E Ziganshin A U Ji X-D King B FWildman S S Rychkov A Yoburn J Kim H Mohanram AHarden T K Boyer J L Burnstock G Jacobson K A Drug DevRes 1998 45 52-66
[136] Baraldi P G Romagnoli R Tabrizi M A Falzoni S Di Virgilio FBioorg Med Chem Lett 2000 10 681-684
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K Boyer J L King B F Burnstock G J Med Chem 1998 41 2201-2206
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[139] Baraldi P G Nunez M D C A M Falzoni S Di Virgilio FRomagnoli R J Med Chem 2003 46 1318-1329
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[101] Brown S G Kim Y-C Kim S-A Jacobson K A Burnstock GKing B F Drug Dev Res 2001 53 281-291
[102] Lambrecht G Rettinger J Baumert H G Czeche S Damer SGanso M Hildebrandt C Niebel B Spatz-Kumbel G SchmalzingG Mutschler E Eur J Pharmacol 2000 387 R19-R21
[141] Baxter A Bent J Bowers K Braddock M Brough S Fagura MLawson M McInally T Mortimore M Robertson M Weaver RWebborn P Bioorg Med Chem Lett 2003 13 4047-4050
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[142] Alcaraz L Baxter A Bent J Bowers K Braddock M CladingboelD Donald D Fagura M Furber M Laurent C Lawson M
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1523
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[165] Nelson D W Gregg R J Kort M E Perez-Medrano A Voight EA Wang Y Grayson G Namovic M T Donnelly-Roberts D LNiforatos W Honore P Jarvis M F Faltynek C R Carroll W AJ Med Chem 2006 49 3659-3666
[143] Caffrey M Rhonan F Pimm A WO2004074224A1 2004[144] Evans R Rhonan F Thompson T Willis P WO2006059945A1
2006 [166] Perez-Medrano A Nelson D W Carroll W A Kort M E Gregg RJ Voight E A Jarvis M F Kowaluk E A WO2006086229A12006
[145] Rhonan F Martin B Thompson T Tomkinson N Willis PWO2006025783A1 2006
[146] Cladingboel D Rhonan F Willis P WO2005014529A1 2005 [167] Carroll W A Perez-Medrano A Florjancic A S Nelson D WPeddi S Li T Bunnelle E M Hirst G Li B CWO20060052374A1 2006
[147] Duplantier A J EP1310493A1 2003[148] Baxter A Kindon N Pairaudeau G Roberts B Thom S
WO0144213A1 2001 [168] Carroll W A Perez-Medrano A Florjancic A S Nelson D WPeddi S Bunnelle E M Hirst G C Li B WO2005111003A12005
[149] Meghani P Bennion C WO0146200A1 2001[150] Li Z B WO20050288256A1 2005[151] Leonard J A Li Z J Li Z B Urban F J WO20050288288A1
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V L Biochim Biophys Acta 2005 1711 215-224[154] Evans R Eyssade C Rhonan F Martin B Thompson T WillisP WO2004106305A1 2004 [172] Michel A D Xing M Thompson K M Jones C A Humphrey P
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Received February 01 2007 Revised April 04 2007 Accepted April 06 2007
1518 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
N
HN
XY
R
N
HN
N
HN
N
HN
( )n
39i39j
39a-ce-hk-w 39d
Fig (20) General structure of the diarylimidazolines generated by Aventis
Table 6 Structures and P2X7R Antagonistic Properties of Selected Diarylimidazolines [164]
Entry n X Y R IC50 ( M)a
39a 1 H H H 008
39b 2 H H H 054
39c 3 H H H 061
39e 0 (R)-Me H H 011
39f 1 (S)-Me H H 003
39g 1 Me Me H 001
39h 1 Ph H H 032
39k 1 H H 2-F 016
39l 1 H H 3-F 004
39m 1 H H 4-F 022
39n 1 H H 2-Cl 007
39o 1 H H 3-Cl 012
39p 1 H H 4-Cl gt1
39q 1 H H 2-Me 007
39r 1 H H 4-Me 219
39s 1 H H 2-OMe 083
39t 1 H H 3-OMe gt3
39u 1 H H 4-OMe gt3
39v 0 H H 2-F 058
39w 0 H H 3-F 008
39x 0 H H 4-F 008
aAntagonistic IC50 values derived from dose-response curves on the basis of inhibition of YO-PRO-1 dye uptake
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1519
R1N
N
NN
R2
R1
NN
NN
R2
N
N
NN
Cl
Cl
Cl
Cl
40b-t 40u-x40a
1
2
3
4
5
Fig (21) General structure of tetrazoles generated by Abbott Laboratories
310 1-Benzyl-5-Phenyltetrazoles potency The limited solubility of those compounds howeverposed a challenge to incorporate substitutions that couldincrease the solubility while maintaining activity A 3-pyridylgroup (40d ) was found to overcome this problem Moreover anextra 2-methyl substituent (40e) resulted in higher activitycompared to 40d A methylene extension (40f) caused a decreasein potency whereas other heterocycles such as 40g and 40hresulted in analogues with similar potency to 40d
1-Benzyl-5-phenyltetrazoles were discovered through ahigh-throughput screening by Abbott Laboratories for threedifferent P2X7R activities Ca2+ flux at the rat or human P2X7RYO-PRO-1 uptake and interleukin-1β release at human P2X7R[165-168] The initial hit from the screening was the tetrazole40a (Table 7) with a pIC50 value of 69 in inhibiting P2X7Rderived from the recombinant human cell line
Investigation of the nature of the substituent attached to the5-position of the tetrazole core showed that the substitutionpattern at this position was not well tolerated [165] Forexample unsubstituted mono-chlorosubstituted 25- and 34-
Initial SAR studies focused on the substituent attached tothe 1-position of the tetrazole core [165] Unsubstituted orortho-substituted benzylic group (40bc) exhibited good
Table 7 Structures and P2X7R Antagonistic Properties of Selected 1-benzyl-5-tetrazoles [165]
1520 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
disubstituted compounds (40i-m) pyridine and quinoline ring(40n-p ) analogues were not tolerated However moreconservative changes such as CF3 and F for Cl group (40q-t)resulted in an increase of potency
release and amplification of astrocytic Ca2+ signaling werereported to be due to the P2X7R and not Cx43 hemichannels[170]
Decavanadate a type of polymer of vanadium oxides hasbeen demonstrated to be a competitive rapid-acting antagonistat the P2X7R [172] It is capable of inhibiting the BzATP-stimulated ethidium bromide uptake in human mouse and ratP2X7R with pA2 values of 74 57 and 71 respectivelyDecavanadate has not been fully tested for its selectivity overother P2X receptors however it is suggested that this largemolecular species acts at the extracellular ATP binding site ofthe receptor consistent with the competitive mode of inhibition[172] A monoclonal antibody specific for the human P2X7Rhas also been shown to be a potent and selective antagonist ofthe receptor with an IC50 value of 5 nM in blocking the BzATP-evoked currents [173] Interestingly tenidap at 50 ndash 200 microMconcentration increases the sensitivity of mouse macrophagesto ATP-mediated cytotoxicity [174] 17-β-estradiol and notprogesterone or 17-α-estradiol has also been reported to blockthe agonist-induced currents at the human P2X7R with an IC50value of 3 microM in a non-genomic manner [175] Polymyxin B apotent antibiotic well-known for its lipopolysaccharide-neutralizing effects has been demonstrated to confer asynergistic effect when co-applied with ATP in a similar mannerto tenidap inducing Ca2+ influx ethidium bromide uptake andlactate dehydrogenase release via action at the P2X7R [176]
Tetrazoles with reversed connectivity (40u-x) were alsoevaluated where it was found that this connectivity reversal hadlittle effect on the potency [165] These tetrazoles were reportedto be slightly more active in the human P2X7R than the ratanalogue [165]
The tetrazole derivative 40d has been shown to be selectivefor other P2 receptors including P2X3 P2X4 and P2Y2 receptors[165] Pharmacological testing of this tetrazole suggested acompetitive mode of inhibition at the P2X7R and its efficacyhas also been evaluated in a model of neuropathic pain in rats[165] Overall these tetrazoles are found to be P2X7R-active andhighly adaptable to structural modifications These propertiesmake the tetrazoles an interesting class of compound for furtherinvestigating P2X7R activity
311 Other P2X7R-Active Ligands
Chelerythrine 41 (Fig 22) is a member of the benzophen-anthridine alkaloids known to inhibit protein kinase C withgood potency and selectivity (IC50 066 microM) as well as enzymessuch as alanine aminotransferase and Na+K+-ATPase [169] At10 microM concentrations chelerythrine non-competitivelyinhibited the BzATP-induced 86Rb+ efflux by 74 in human B-lymphocytes [169] Dye uptake studies could not be performeddue to the fluorescent nature of the compound It is yet to bedetermined whether chelerythrine inhibition of P2X7R involvesprotein kinase C [169]
More recently other distinct classes of P2X7-active ligandshave been reported and patented by the Abbott Laboratoriesacylhydrazides [177] cyanoamidines and cyanoguanidines[178 179]
4 CONCLUSIONS AND FUTURE DIRECTIONS
O
O
N
O
O
Cl
41
Although many structurally diverse class of compoundshave been reported as P2X7R antagonists with reasonablepotency the selectivity issue over other P2 receptors andproteins such as ecto-ATPases and ectonucleotidases stillprevails This is particularly an issue for oxidized ATP andsuramin SAR studies performed on PPADS and suramin mainlygave improved potency and selectivity for the P2X1 receptorIon channel blockers such as amilorides and stilbenederivatives only possess relatively low potency at the P2X7RFurther complications in studying these P2X7R antagonistsalso arise from the demonstrated species differences acrosshuman rat and mouse systems Brilliant Blue G is active for allthree species however at present there have been no SAR datareported Another fundamental factor that hampers the study ofP2X7R in detail is the relatively low potency and selectivity ofthe available agonist BzATP The discovery of a more potentselective P2X7R agonist would be ideal since in many studiesfull dose-response curves could not be generated due to the low
Fig (22) Chemical structure for chelerythrine an alkaloid withantagonistic properties at the human P2X7R
Gap junction channel blockers such as flufenamic acid 42 carbenoxolone 43 and mefloquine 44 (Fig 23) were all foundto be inhibitors of the BzATP-induced Ca2+ flux (IC50 values of655 175 and 25 nM respectively) and of YO-PRO-1 uptake in1321N1 astrocytoma cells stably expressing rat P2X7R [170]This finding poses a question to the general use of gap junctionchannel blockers as indicators of the participation of connexinhemichannels at the sites of transmitter release [171] The ATP
HN CF3
O OH
42 f lufenamic acid
HO
-O
O
O
H
OH
O
O-
N
HN
CF3
CF3
H O
H
43 carbenoxolone 44 mefloquine
Fig (23) Chemical structures for gap junction channel blockers with P2X7R antagonistic properties
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1521
potency of BzATP The nature of functional assays on theP2X7R is also found to be complex as ionic compositiontemperature and cell origins all affect the results demandingextra care be taken when antagonist potencies are directlycompared from different studies
[23] Li J Liu D Ke H Z Duncan R L Turner C H J Biol Chem2005 280 42952-42959
[24] Penolazzi L Bianchini E Lambertini E Baraldi P G RomagnoliR Piva R Gambari R J Biomed Sci 2005 12 1013-1020
[25] Gibbons S J Washburn K B Talamo B R J Auton Pharmacol2001 21 181-190
[26] Dehaye J P Moran A Marino A Arch Oral Biol 1999 44 S39-S43Four classes of small molecules with high affinity at the
P2X7R have been reported cyclic imides adamantyl amidesdiarylimidazolines and 1-benzyl-5-phenyltetrazoles Theseclasses of compounds are highly amenable to structuralmodifications indicating their good prospects as leadmolecules for further improving the potency and selectivity atP2X7R ultimately leading to orally active drugs To date norational drug design based on the knowledge of binding siteswithin this receptor has been undertaken This is a direct resultof the fact that no crystal structure or suitable homology modelhave been reported for this receptor The fact that the P2X7R canadopt either a channel or pore state causes even moredifficulties in studying receptor dynamics and at present theexact mechanism of channel-to-pore transition remains to beelucidated Undoubtedly the P2X7R is involved in severalprocesses relevant to chronic inflammation neurodegenerationand neuropathic pain and therefore discovery of more potentselective P2X7R antagonists would have a great impact in thepresent therapeutic window
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ACKNOWLEDGEMENTS[38] Khakh B S Burnstock G Kennedy C King B F North A
Seguela P Voigt M Humphrey P P A Nat Rev Neurosci 20012 165-174
The authors would like to thank Dr Natasha Hungerford forproofreading the manuscript
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Mol Pharmacol 1998 54 22-32[98] Kim Y-C Camaioni E Ziganshin A U Ji X-D King B FWildman S S Rychkov A Yoburn J Kim H Mohanram AHarden T K Boyer J L Burnstock G Jacobson K A Drug DevRes 1998 45 52-66
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[138] Chen W Ravi R G Kertesy S B Dubyak G R Jacobson K ABioconjug Chem 2002 13 1100-1111
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[139] Baraldi P G Nunez M D C A M Falzoni S Di Virgilio FRomagnoli R J Med Chem 2003 46 1318-1329
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[101] Brown S G Kim Y-C Kim S-A Jacobson K A Burnstock GKing B F Drug Dev Res 2001 53 281-291
[102] Lambrecht G Rettinger J Baumert H G Czeche S Damer SGanso M Hildebrandt C Niebel B Spatz-Kumbel G SchmalzingG Mutschler E Eur J Pharmacol 2000 387 R19-R21
[141] Baxter A Bent J Bowers K Braddock M Brough S Fagura MLawson M McInally T Mortimore M Robertson M Weaver RWebborn P Bioorg Med Chem Lett 2003 13 4047-4050
[103] Kim Y-C Lee J-S Sak K Marteau F Mamedova L BoeynaemsJ-M Jacobson K A Biochem Pharmacol 2005 70 266-274
[142] Alcaraz L Baxter A Bent J Bowers K Braddock M CladingboelD Donald D Fagura M Furber M Laurent C Lawson M
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1523
Mortimore M McCormick M Roberts N Robertson M BioorgMed Chem Lett 2003 13 4043-4046
[165] Nelson D W Gregg R J Kort M E Perez-Medrano A Voight EA Wang Y Grayson G Namovic M T Donnelly-Roberts D LNiforatos W Honore P Jarvis M F Faltynek C R Carroll W AJ Med Chem 2006 49 3659-3666
[143] Caffrey M Rhonan F Pimm A WO2004074224A1 2004[144] Evans R Rhonan F Thompson T Willis P WO2006059945A1
2006 [166] Perez-Medrano A Nelson D W Carroll W A Kort M E Gregg RJ Voight E A Jarvis M F Kowaluk E A WO2006086229A12006
[145] Rhonan F Martin B Thompson T Tomkinson N Willis PWO2006025783A1 2006
[146] Cladingboel D Rhonan F Willis P WO2005014529A1 2005 [167] Carroll W A Perez-Medrano A Florjancic A S Nelson D WPeddi S Li T Bunnelle E M Hirst G Li B CWO20060052374A1 2006
[147] Duplantier A J EP1310493A1 2003[148] Baxter A Kindon N Pairaudeau G Roberts B Thom S
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Received February 01 2007 Revised April 04 2007 Accepted April 06 2007
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1519
R1N
N
NN
R2
R1
NN
NN
R2
N
N
NN
Cl
Cl
Cl
Cl
40b-t 40u-x40a
1
2
3
4
5
Fig (21) General structure of tetrazoles generated by Abbott Laboratories
310 1-Benzyl-5-Phenyltetrazoles potency The limited solubility of those compounds howeverposed a challenge to incorporate substitutions that couldincrease the solubility while maintaining activity A 3-pyridylgroup (40d ) was found to overcome this problem Moreover anextra 2-methyl substituent (40e) resulted in higher activitycompared to 40d A methylene extension (40f) caused a decreasein potency whereas other heterocycles such as 40g and 40hresulted in analogues with similar potency to 40d
1-Benzyl-5-phenyltetrazoles were discovered through ahigh-throughput screening by Abbott Laboratories for threedifferent P2X7R activities Ca2+ flux at the rat or human P2X7RYO-PRO-1 uptake and interleukin-1β release at human P2X7R[165-168] The initial hit from the screening was the tetrazole40a (Table 7) with a pIC50 value of 69 in inhibiting P2X7Rderived from the recombinant human cell line
Investigation of the nature of the substituent attached to the5-position of the tetrazole core showed that the substitutionpattern at this position was not well tolerated [165] Forexample unsubstituted mono-chlorosubstituted 25- and 34-
Initial SAR studies focused on the substituent attached tothe 1-position of the tetrazole core [165] Unsubstituted orortho-substituted benzylic group (40bc) exhibited good
Table 7 Structures and P2X7R Antagonistic Properties of Selected 1-benzyl-5-tetrazoles [165]
1520 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
disubstituted compounds (40i-m) pyridine and quinoline ring(40n-p ) analogues were not tolerated However moreconservative changes such as CF3 and F for Cl group (40q-t)resulted in an increase of potency
release and amplification of astrocytic Ca2+ signaling werereported to be due to the P2X7R and not Cx43 hemichannels[170]
Decavanadate a type of polymer of vanadium oxides hasbeen demonstrated to be a competitive rapid-acting antagonistat the P2X7R [172] It is capable of inhibiting the BzATP-stimulated ethidium bromide uptake in human mouse and ratP2X7R with pA2 values of 74 57 and 71 respectivelyDecavanadate has not been fully tested for its selectivity overother P2X receptors however it is suggested that this largemolecular species acts at the extracellular ATP binding site ofthe receptor consistent with the competitive mode of inhibition[172] A monoclonal antibody specific for the human P2X7Rhas also been shown to be a potent and selective antagonist ofthe receptor with an IC50 value of 5 nM in blocking the BzATP-evoked currents [173] Interestingly tenidap at 50 ndash 200 microMconcentration increases the sensitivity of mouse macrophagesto ATP-mediated cytotoxicity [174] 17-β-estradiol and notprogesterone or 17-α-estradiol has also been reported to blockthe agonist-induced currents at the human P2X7R with an IC50value of 3 microM in a non-genomic manner [175] Polymyxin B apotent antibiotic well-known for its lipopolysaccharide-neutralizing effects has been demonstrated to confer asynergistic effect when co-applied with ATP in a similar mannerto tenidap inducing Ca2+ influx ethidium bromide uptake andlactate dehydrogenase release via action at the P2X7R [176]
Tetrazoles with reversed connectivity (40u-x) were alsoevaluated where it was found that this connectivity reversal hadlittle effect on the potency [165] These tetrazoles were reportedto be slightly more active in the human P2X7R than the ratanalogue [165]
The tetrazole derivative 40d has been shown to be selectivefor other P2 receptors including P2X3 P2X4 and P2Y2 receptors[165] Pharmacological testing of this tetrazole suggested acompetitive mode of inhibition at the P2X7R and its efficacyhas also been evaluated in a model of neuropathic pain in rats[165] Overall these tetrazoles are found to be P2X7R-active andhighly adaptable to structural modifications These propertiesmake the tetrazoles an interesting class of compound for furtherinvestigating P2X7R activity
311 Other P2X7R-Active Ligands
Chelerythrine 41 (Fig 22) is a member of the benzophen-anthridine alkaloids known to inhibit protein kinase C withgood potency and selectivity (IC50 066 microM) as well as enzymessuch as alanine aminotransferase and Na+K+-ATPase [169] At10 microM concentrations chelerythrine non-competitivelyinhibited the BzATP-induced 86Rb+ efflux by 74 in human B-lymphocytes [169] Dye uptake studies could not be performeddue to the fluorescent nature of the compound It is yet to bedetermined whether chelerythrine inhibition of P2X7R involvesprotein kinase C [169]
More recently other distinct classes of P2X7-active ligandshave been reported and patented by the Abbott Laboratoriesacylhydrazides [177] cyanoamidines and cyanoguanidines[178 179]
4 CONCLUSIONS AND FUTURE DIRECTIONS
O
O
N
O
O
Cl
41
Although many structurally diverse class of compoundshave been reported as P2X7R antagonists with reasonablepotency the selectivity issue over other P2 receptors andproteins such as ecto-ATPases and ectonucleotidases stillprevails This is particularly an issue for oxidized ATP andsuramin SAR studies performed on PPADS and suramin mainlygave improved potency and selectivity for the P2X1 receptorIon channel blockers such as amilorides and stilbenederivatives only possess relatively low potency at the P2X7RFurther complications in studying these P2X7R antagonistsalso arise from the demonstrated species differences acrosshuman rat and mouse systems Brilliant Blue G is active for allthree species however at present there have been no SAR datareported Another fundamental factor that hampers the study ofP2X7R in detail is the relatively low potency and selectivity ofthe available agonist BzATP The discovery of a more potentselective P2X7R agonist would be ideal since in many studiesfull dose-response curves could not be generated due to the low
Fig (22) Chemical structure for chelerythrine an alkaloid withantagonistic properties at the human P2X7R
Gap junction channel blockers such as flufenamic acid 42 carbenoxolone 43 and mefloquine 44 (Fig 23) were all foundto be inhibitors of the BzATP-induced Ca2+ flux (IC50 values of655 175 and 25 nM respectively) and of YO-PRO-1 uptake in1321N1 astrocytoma cells stably expressing rat P2X7R [170]This finding poses a question to the general use of gap junctionchannel blockers as indicators of the participation of connexinhemichannels at the sites of transmitter release [171] The ATP
HN CF3
O OH
42 f lufenamic acid
HO
-O
O
O
H
OH
O
O-
N
HN
CF3
CF3
H O
H
43 carbenoxolone 44 mefloquine
Fig (23) Chemical structures for gap junction channel blockers with P2X7R antagonistic properties
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1521
potency of BzATP The nature of functional assays on theP2X7R is also found to be complex as ionic compositiontemperature and cell origins all affect the results demandingextra care be taken when antagonist potencies are directlycompared from different studies
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P2X7R have been reported cyclic imides adamantyl amidesdiarylimidazolines and 1-benzyl-5-phenyltetrazoles Theseclasses of compounds are highly amenable to structuralmodifications indicating their good prospects as leadmolecules for further improving the potency and selectivity atP2X7R ultimately leading to orally active drugs To date norational drug design based on the knowledge of binding siteswithin this receptor has been undertaken This is a direct resultof the fact that no crystal structure or suitable homology modelhave been reported for this receptor The fact that the P2X7R canadopt either a channel or pore state causes even moredifficulties in studying receptor dynamics and at present theexact mechanism of channel-to-pore transition remains to beelucidated Undoubtedly the P2X7R is involved in severalprocesses relevant to chronic inflammation neurodegenerationand neuropathic pain and therefore discovery of more potentselective P2X7R antagonists would have a great impact in thepresent therapeutic window
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[36] Watters J J Sommer J A Fisette P L Pfeiffer Z A Aga MPrabhu U Guerra A N Denlinger L C Bertics P J Drug Dev Res2001 53 91-104
[37] Adinolfi E Pizzirani C Idzko M Panther E Norgauer J DiVirgilio F Ferrari D Purinergic Signall 2005 1 219-227
ACKNOWLEDGEMENTS[38] Khakh B S Burnstock G Kennedy C King B F North A
Seguela P Voigt M Humphrey P P A Nat Rev Neurosci 20012 165-174
The authors would like to thank Dr Natasha Hungerford forproofreading the manuscript
[39] Roberts J A Vial C Digby H R Agboh K C Wen HAtterbury-Thomas A Evans R J Pflugers Arch - Eur J Physiol2006 452 486-500
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Received February 01 2007 Revised April 04 2007 Accepted April 06 2007
1520 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
disubstituted compounds (40i-m) pyridine and quinoline ring(40n-p ) analogues were not tolerated However moreconservative changes such as CF3 and F for Cl group (40q-t)resulted in an increase of potency
release and amplification of astrocytic Ca2+ signaling werereported to be due to the P2X7R and not Cx43 hemichannels[170]
Decavanadate a type of polymer of vanadium oxides hasbeen demonstrated to be a competitive rapid-acting antagonistat the P2X7R [172] It is capable of inhibiting the BzATP-stimulated ethidium bromide uptake in human mouse and ratP2X7R with pA2 values of 74 57 and 71 respectivelyDecavanadate has not been fully tested for its selectivity overother P2X receptors however it is suggested that this largemolecular species acts at the extracellular ATP binding site ofthe receptor consistent with the competitive mode of inhibition[172] A monoclonal antibody specific for the human P2X7Rhas also been shown to be a potent and selective antagonist ofthe receptor with an IC50 value of 5 nM in blocking the BzATP-evoked currents [173] Interestingly tenidap at 50 ndash 200 microMconcentration increases the sensitivity of mouse macrophagesto ATP-mediated cytotoxicity [174] 17-β-estradiol and notprogesterone or 17-α-estradiol has also been reported to blockthe agonist-induced currents at the human P2X7R with an IC50value of 3 microM in a non-genomic manner [175] Polymyxin B apotent antibiotic well-known for its lipopolysaccharide-neutralizing effects has been demonstrated to confer asynergistic effect when co-applied with ATP in a similar mannerto tenidap inducing Ca2+ influx ethidium bromide uptake andlactate dehydrogenase release via action at the P2X7R [176]
Tetrazoles with reversed connectivity (40u-x) were alsoevaluated where it was found that this connectivity reversal hadlittle effect on the potency [165] These tetrazoles were reportedto be slightly more active in the human P2X7R than the ratanalogue [165]
The tetrazole derivative 40d has been shown to be selectivefor other P2 receptors including P2X3 P2X4 and P2Y2 receptors[165] Pharmacological testing of this tetrazole suggested acompetitive mode of inhibition at the P2X7R and its efficacyhas also been evaluated in a model of neuropathic pain in rats[165] Overall these tetrazoles are found to be P2X7R-active andhighly adaptable to structural modifications These propertiesmake the tetrazoles an interesting class of compound for furtherinvestigating P2X7R activity
311 Other P2X7R-Active Ligands
Chelerythrine 41 (Fig 22) is a member of the benzophen-anthridine alkaloids known to inhibit protein kinase C withgood potency and selectivity (IC50 066 microM) as well as enzymessuch as alanine aminotransferase and Na+K+-ATPase [169] At10 microM concentrations chelerythrine non-competitivelyinhibited the BzATP-induced 86Rb+ efflux by 74 in human B-lymphocytes [169] Dye uptake studies could not be performeddue to the fluorescent nature of the compound It is yet to bedetermined whether chelerythrine inhibition of P2X7R involvesprotein kinase C [169]
More recently other distinct classes of P2X7-active ligandshave been reported and patented by the Abbott Laboratoriesacylhydrazides [177] cyanoamidines and cyanoguanidines[178 179]
4 CONCLUSIONS AND FUTURE DIRECTIONS
O
O
N
O
O
Cl
41
Although many structurally diverse class of compoundshave been reported as P2X7R antagonists with reasonablepotency the selectivity issue over other P2 receptors andproteins such as ecto-ATPases and ectonucleotidases stillprevails This is particularly an issue for oxidized ATP andsuramin SAR studies performed on PPADS and suramin mainlygave improved potency and selectivity for the P2X1 receptorIon channel blockers such as amilorides and stilbenederivatives only possess relatively low potency at the P2X7RFurther complications in studying these P2X7R antagonistsalso arise from the demonstrated species differences acrosshuman rat and mouse systems Brilliant Blue G is active for allthree species however at present there have been no SAR datareported Another fundamental factor that hampers the study ofP2X7R in detail is the relatively low potency and selectivity ofthe available agonist BzATP The discovery of a more potentselective P2X7R agonist would be ideal since in many studiesfull dose-response curves could not be generated due to the low
Fig (22) Chemical structure for chelerythrine an alkaloid withantagonistic properties at the human P2X7R
Gap junction channel blockers such as flufenamic acid 42 carbenoxolone 43 and mefloquine 44 (Fig 23) were all foundto be inhibitors of the BzATP-induced Ca2+ flux (IC50 values of655 175 and 25 nM respectively) and of YO-PRO-1 uptake in1321N1 astrocytoma cells stably expressing rat P2X7R [170]This finding poses a question to the general use of gap junctionchannel blockers as indicators of the participation of connexinhemichannels at the sites of transmitter release [171] The ATP
HN CF3
O OH
42 f lufenamic acid
HO
-O
O
O
H
OH
O
O-
N
HN
CF3
CF3
H O
H
43 carbenoxolone 44 mefloquine
Fig (23) Chemical structures for gap junction channel blockers with P2X7R antagonistic properties
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1521
potency of BzATP The nature of functional assays on theP2X7R is also found to be complex as ionic compositiontemperature and cell origins all affect the results demandingextra care be taken when antagonist potencies are directlycompared from different studies
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P2X7R have been reported cyclic imides adamantyl amidesdiarylimidazolines and 1-benzyl-5-phenyltetrazoles Theseclasses of compounds are highly amenable to structuralmodifications indicating their good prospects as leadmolecules for further improving the potency and selectivity atP2X7R ultimately leading to orally active drugs To date norational drug design based on the knowledge of binding siteswithin this receptor has been undertaken This is a direct resultof the fact that no crystal structure or suitable homology modelhave been reported for this receptor The fact that the P2X7R canadopt either a channel or pore state causes even moredifficulties in studying receptor dynamics and at present theexact mechanism of channel-to-pore transition remains to beelucidated Undoubtedly the P2X7R is involved in severalprocesses relevant to chronic inflammation neurodegenerationand neuropathic pain and therefore discovery of more potentselective P2X7R antagonists would have a great impact in thepresent therapeutic window
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Seguela P Voigt M Humphrey P P A Nat Rev Neurosci 20012 165-174
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potency of BzATP The nature of functional assays on theP2X7R is also found to be complex as ionic compositiontemperature and cell origins all affect the results demandingextra care be taken when antagonist potencies are directlycompared from different studies
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P2X7R have been reported cyclic imides adamantyl amidesdiarylimidazolines and 1-benzyl-5-phenyltetrazoles Theseclasses of compounds are highly amenable to structuralmodifications indicating their good prospects as leadmolecules for further improving the potency and selectivity atP2X7R ultimately leading to orally active drugs To date norational drug design based on the knowledge of binding siteswithin this receptor has been undertaken This is a direct resultof the fact that no crystal structure or suitable homology modelhave been reported for this receptor The fact that the P2X7R canadopt either a channel or pore state causes even moredifficulties in studying receptor dynamics and at present theexact mechanism of channel-to-pore transition remains to beelucidated Undoubtedly the P2X7R is involved in severalprocesses relevant to chronic inflammation neurodegenerationand neuropathic pain and therefore discovery of more potentselective P2X7R antagonists would have a great impact in thepresent therapeutic window
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1997 121 1429-1437[19] Sugiyama T Kawamura H Yamanishi S Kobayashi M Katsumura
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Lin G C Bennett B D Eriksen E F Sorensen O H Civitelli RDrug Dev Res 2001 53 126-129
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[65] Papp L Vizi E S Sperlagh B Neuro Report 2004 15 2387-2391 [104] Michel A D Kaur R Chessell I P Humphrey P P A Br JPharmacol 2000 130 513-520[66] Duan S Anderson C M Keung E C Chen Y Chen Y
Swanson R A J Neurosci 2003 23 1320-1328 [105] Cabantchik Z I Greger R Am J Physiol Cell Physiol 1992 262C803-C827[67] Wang C-M Chang Y-Y Kuo J-S Sun S H Glia 2002 37 8-
18 [106] Greger R Methods Enzymol 1990 191 793-810[68] Sim J A Young M T Sung H-Y North A Surprenant A J
Neurosci 2004 24 6307-6314[107] Hua S Inesi G Biophys J 1997 73 2149-2155[108] Bultmann R Pause B Wittenburg H Kurz G Starke K Naunyn-
Schmiedebergs Arch Pharmacol 1996 354 481-490[69] Fellin T Pozzan T Carmignoto G J Biol Chem 2006 281 4274-4284 [109] Soltoff S P McMillian M K Talamo B R Cantley L C Biochem
Pharmacol 1993 45 1936-1940[70] Chessell I P Hatcher J P Bountra C Michel A D Hughes J PGreen P Egerton J Murfin M Richardson J Peck W LGrahames C B A Casula M A Yiangou Y Birch R Anand PBuell G N Pain 2005 114 386-396
[110] El-Moatassim C Dubyak G R J Biol Chem 1993 268 15571-15578
[111] Panenka W Jijon H Herx L M Armstrong J N Feighan D WeiT Yong V W Ransohoff R M MacVicar B A J Neurosci 200121 7135-7142
[71] Gracie J A Clin Exp Immunol 2004 136 402-404[72] Labasi J M Petrushova N Donovan C McCurdy S Lira P
Payette M M Brissette W Wicks J R Audoly L Gabel C J JImmunol 2002 168 6436-6445
[112] Grinstein S Rotin D Mason M J Biochim Biophys Acta RevBiomembr 1989 988 73-97
[73] Wang X Arcuino G Takano T Lin J Peng W G Wan P LiP Xu Q Liu Q S Goldman S A Nedergaard M Nat Med 200410 821-827
[113] Garcia M L King V F Shevell J L Slaughter R S Suarez-KurtzG Winquist R J Kaczorowski G J J Biol Chem 1990 2653763-3771
[74] Solle M Labasi J Perregaux D G Stam E Petrushova N KollerB H Griffiths R J Gabel C A J Biol Chem 2001 276 125-132
[114] Garritsen A Ijzerman A P Beukers M W Cragoe E J J SoudjinW Biochem Pharmacol 1990 40 827-834
[75] Sanchez-Nogueiro J Marin-Garcia P Miras-Portugal M T FEBS Lett2005 579 3783-3788
[115] Neylon C B Little P J Cragoe E J J Bobik A Circ Res 199067 814-825
[76] Raouf R Chakfe Y Blais D Speelman A Boue-Grabot EHenderson D Seguela P Mol Pharmacol 2004 65 646-654
[116] Wiley J S Chen R Wiley M J Jamieson G P Arch BiochemBiophys 1992 292 411-418
[77] Yiangou Y Facer P Durrenberger P Chessell I P Naylor ABountra C Banati R R Anand P BMC Neurol 2006 6 12
[117] Wiley J S Chen R Jamieson G P Arch Biochem Biophys 1993305 54-60
[78] Sperlagh B Vizi E S Wirkner K Illes P Prog Neurobiol 200678 327-346
[118] Bultmann R Wittenburg H Pause B Kurz G Nickel P Starke KNaunyn-Schmiedebergs Arch Pharmacol 1996 354 498-504
[79] Stojilkovic S S Tomic M He M-L Yan Z Koshimizu T-AZemkova H Ann NY Acad Sci 2005 1048 116-130
[119] Damer S Niebel B Czeche S Nickel P Ardanuy U SchmalzingG Rettinger J Mutschler E Lambrecht G Eur J Pharmacol 1998350 R5-R6[80] McGaraughty S Jarvis M F Drug Dev Res 2006 67 376-388
[81] Baraldi P G Di Virgilio F Romagnoli R Curr Top Med Chem2004 4 1707-1717
[120] Braun K Rettinger J Ganso M Kassack M Hildebrandt CUllmann H Nickel P Schmalzing G Lambrecht G Naunyn-Schmiedebergs Arch Pharmacol 2001 364 285-290[82] Romagnoli R Baraldi P G Di Virgilio F Expert Opin Ther Pat
2005 15 271-287 [121] Kassack M U Braun K Ganso M Ullmann H Nickel P BoingB Muller G Lambrecht G Eur J Med Chem 2004 39 345-357[83] Shieh C-C Jarvis M F Lee C-H Perner R J Expert Opin Ther
Pat 2006 16 1113-1127 [122] Horner S Menke K Hildebrandt C Kassack M U Nickel PUllmann H Mahaut-Smith M P Lambrecht G Naunyn-Schmiedebergs Arch Pharmacol 2005 372 1-13
[84] Virginio C Church D North R A Surprenant ANeuropharmacology 1997 36 1285-1294
[85] Nuttle L C Dubyak G J Biol Chem 1994 269 13988-13996 [123] Hausmann R Rettinger J Gerevich Z Meis S Kassack M U IllesP Lambrecht G Schmalzing G Mol Pharmacol 2006 69 2058-2067
[86] Tatham P E R Cusack N J Gomperts B D Eur J Pharmacol1988 147 13-21
[87] Elssner A Duncan M Gavrilin M Wewers M D J Immunol2004 172 4987-4994
[124] Ullmann H Meis S Hongwiset D Marzian C Wiese M NickelP Communi D Boeynaems J-M Wolf C Hausmann RSchmalzing G Kassack M U J Med Chem 2005 48 7040-7048[88] Chessell I P Michel A D Humphrey P P A Br J Pharmacol
1998 124 1314-1320 [125] Soto F Lambrecht G Nickel P Stuhmer W Busch A ENeuropharmacology 1999 38 141-149[89] Bianchi B R Lynch K J Touma E Niforatos W Burgard E C
Alexander K M Park H S Yu H Metzger R Kowaluk E JarvisM F van Biesen T Eur J Pharmacol 1999 376 127-138
[126] Rettinger J Schmalzing G Damer S Muller G Nickel PLambrecht G Neuropharmacology 2000 39 2044-2053
[90] North R A Surprenant A Annu Rev Pharm Toxicol 2000 40 563-580
[127] Klapperstuck M Buttner C Nickel P Schmalzing G Lambrecht GMarkwardt F Eur J Pharmacol 2000 387 245-252
[91] Murgia M Hanau S Pizzo P Rippa M Di Virgilio F J BiolChem 1993 268 8199-8203
[128] Hulsmann M Nickel P Kassack M Schmalzing G Lambrecht GMarkwardt F Eur J Pharmacol 2003 470 1-7
[92] Craighead M W Middlehurst K M L LeFeuvre R Kimber IRothwell N J Neurosci Lett 2001 311 77-80
[129] Rettinger J Braun K Hochmann H Kassack M U Ullmann HNickel P Schmalzing G Lambrecht G Neuropharmacology 200548 461-468[93] Beigi R D Kertesy S B Aquilina G Dubyak G R Br J
Pharmacol 2003 140 507-519 [130] Jiang L-H Mackenzie A B North R A Surprenant A MolPharmacol 2000 58 82-88[94] DellAntonio G Quattrini A Cin E D Fulgenzi A Ferrero M E
Arthritis Rheum 2002 46 3378-3385 [131] Bultmann R Starke K Naunyn-Schmiedebergs Arch Pharmacol1995 352 477-482[95] DellAntonio G Quattrini A Cin E D Fulgenzi A Ferrero M E
Neurosci Lett 2002 327 87-90 [132] Tuluc F Bultmann R Glanzel M Frahm A W Starke K Naunyn-Schmiedebergs Arch Pharmacol 1998 357 111-120[96] Lambrecht G Braun K Damer S Ganso M Hildebrandt C
Ullmann H Kassack M U Nickel P Curr Pharm Des 2002 82371-2399
[133] Wittenburg H Bultmann R Pause B Ganter C Kurz G Starke KNaunyn-Schmiedebergs Arch Pharmacol 1996 354 491-497
[97] Garcia-Guzman M Soto F Gomez-Hernandez J M Lund P-EStuhmer W Mol Pharmacol 1997 51 109-118
[134] Gargett C E Wiley J S Br J Pharmacol 1997 120 1483-1490[135] Humphreys B D Virginio C Surprenant A Rice J Dubyak GR
Mol Pharmacol 1998 54 22-32[98] Kim Y-C Camaioni E Ziganshin A U Ji X-D King B FWildman S S Rychkov A Yoburn J Kim H Mohanram AHarden T K Boyer J L Burnstock G Jacobson K A Drug DevRes 1998 45 52-66
[136] Baraldi P G Romagnoli R Tabrizi M A Falzoni S Di Virgilio FBioorg Med Chem Lett 2000 10 681-684
[137] Ravi R G Kertesy S B Dubyak G R Jacobson K A Drug DevRes 2001 54 75-87[99] Jacobson K A Kim Y-C Wildman S S Mohanram A Harden T
K Boyer J L King B F Burnstock G J Med Chem 1998 41 2201-2206
[138] Chen W Ravi R G Kertesy S B Dubyak G R Jacobson K ABioconjug Chem 2002 13 1100-1111
[100] Kim Y-C Brown S G Harden T K Boyer J L Dubyak GKing B F Burnstock G Jacobson K A J Med Chem 2001 44 340-349
[139] Baraldi P G Nunez M D C A M Falzoni S Di Virgilio FRomagnoli R J Med Chem 2003 46 1318-1329
[140] Romagnoli R Baraldi P G Pavani M G Tabrizi M A MoormanA R Di Virgilio F Cattabriga E Pancaldi C Gessi S Borea P ABioorg Med Chem Lett 2004 14 5709-5712
[101] Brown S G Kim Y-C Kim S-A Jacobson K A Burnstock GKing B F Drug Dev Res 2001 53 281-291
[102] Lambrecht G Rettinger J Baumert H G Czeche S Damer SGanso M Hildebrandt C Niebel B Spatz-Kumbel G SchmalzingG Mutschler E Eur J Pharmacol 2000 387 R19-R21
[141] Baxter A Bent J Bowers K Braddock M Brough S Fagura MLawson M McInally T Mortimore M Robertson M Weaver RWebborn P Bioorg Med Chem Lett 2003 13 4047-4050
[103] Kim Y-C Lee J-S Sak K Marteau F Mamedova L BoeynaemsJ-M Jacobson K A Biochem Pharmacol 2005 70 266-274
[142] Alcaraz L Baxter A Bent J Bowers K Braddock M CladingboelD Donald D Fagura M Furber M Laurent C Lawson M
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1523
Mortimore M McCormick M Roberts N Robertson M BioorgMed Chem Lett 2003 13 4043-4046
[165] Nelson D W Gregg R J Kort M E Perez-Medrano A Voight EA Wang Y Grayson G Namovic M T Donnelly-Roberts D LNiforatos W Honore P Jarvis M F Faltynek C R Carroll W AJ Med Chem 2006 49 3659-3666
[143] Caffrey M Rhonan F Pimm A WO2004074224A1 2004[144] Evans R Rhonan F Thompson T Willis P WO2006059945A1
2006 [166] Perez-Medrano A Nelson D W Carroll W A Kort M E Gregg RJ Voight E A Jarvis M F Kowaluk E A WO2006086229A12006
[145] Rhonan F Martin B Thompson T Tomkinson N Willis PWO2006025783A1 2006
[146] Cladingboel D Rhonan F Willis P WO2005014529A1 2005 [167] Carroll W A Perez-Medrano A Florjancic A S Nelson D WPeddi S Li T Bunnelle E M Hirst G Li B CWO20060052374A1 2006
[147] Duplantier A J EP1310493A1 2003[148] Baxter A Kindon N Pairaudeau G Roberts B Thom S
WO0144213A1 2001 [168] Carroll W A Perez-Medrano A Florjancic A S Nelson D WPeddi S Bunnelle E M Hirst G C Li B WO2005111003A12005
[149] Meghani P Bennion C WO0146200A1 2001[150] Li Z B WO20050288256A1 2005[151] Leonard J A Li Z J Li Z B Urban F J WO20050288288A1
2005[169] Shemon A N Sluyter R Conigrave A D Wiley J S Br J
Pharmacol 2004 142 1015-1019[152] Leonard J A Li Z J Li Z B Urban F J WO2006003513A1
2006[170] Suadicani S O Brosnan C F Scemes E J Neurosci 2006 26
1378-1385[153] Li Z B WO2006003500A1 2006 [171] Saez J C Retamal M A Basilio D Bukauskas F F Bennett M
V L Biochim Biophys Acta 2005 1711 215-224[154] Evans R Eyssade C Rhonan F Martin B Thompson T WillisP WO2004106305A1 2004 [172] Michel A D Xing M Thompson K M Jones C A Humphrey P
P A Eur J Pharmacol 2006 534 19-29[155] Rhonan F Thompson T Willis P WO2005009968A1 2005[156] Furber M Luker T J Mortimore M P Thorne P Meghani P
WO0071529A1 2000[173] Buell G Chessell I P Michel A D Collo G Salazzo M Herren
S Gretener D Grahames C Kaur R Kosco-Vilbois M HHumphrey P P A Blood 1998 92 3521-3528[157] Thompson T Willis P WO2006080884A1 2006
[158] Concepcion A Inoue T Mochizuki Y Muramatsu A Gantner FNakashima K Urbhans K Bacon K B WO2005019182A1 2005
[174] Sanz J M Chiozzi P Di Virgilio F Eur J Pharmacol 1998 35 235-244
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[175] Cario-Toumaniantz C Loirand G Ferrier L Pacaud P J Physiol1998 508 659-666
[160] Kelly M G Kincaid J WO2006102588A1 2006 [176] Ferrari D Pizzirani C Adinolfi E Forchap S Sitta B Turchet LFalzoni S Minelli M Baricordi R Di Virgilio F J Immunol 2004173 4652-4660
[161] Kelly M J Kincaid J WO2006102610A2 2006[162] Schwink L Stengelin S Gossel M Bohme T Hessler G Stahl P
Gretzke D WO2004072025A2 2004 [177] Nelson D W Jarvis M F Carroll W A WO2006110516A1 2006[163] Schwink L Stengelin S Boehme T Gossel M Hessler G Stahl P
WO2005070925A1 2005[178] Carroll W A Perez-Medrano A Peddi S Florjancic A S
WO20060025614A1 2006[164] Merriman G H Ma L Shum P McGarry D Volz F Sabol J S
Gross A Zhao Z Rampe D Wang L Wirtz-Brugger F Harris BA Macdonald D Bioorg Med Chem Lett 2005 15 435-438
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Received February 01 2007 Revised April 04 2007 Accepted April 06 2007
1522 Current Medicinal Chemistry 2007 Vol 14 No 14 Gunosewoyo et al
[65] Papp L Vizi E S Sperlagh B Neuro Report 2004 15 2387-2391 [104] Michel A D Kaur R Chessell I P Humphrey P P A Br JPharmacol 2000 130 513-520[66] Duan S Anderson C M Keung E C Chen Y Chen Y
Swanson R A J Neurosci 2003 23 1320-1328 [105] Cabantchik Z I Greger R Am J Physiol Cell Physiol 1992 262C803-C827[67] Wang C-M Chang Y-Y Kuo J-S Sun S H Glia 2002 37 8-
18 [106] Greger R Methods Enzymol 1990 191 793-810[68] Sim J A Young M T Sung H-Y North A Surprenant A J
Neurosci 2004 24 6307-6314[107] Hua S Inesi G Biophys J 1997 73 2149-2155[108] Bultmann R Pause B Wittenburg H Kurz G Starke K Naunyn-
Schmiedebergs Arch Pharmacol 1996 354 481-490[69] Fellin T Pozzan T Carmignoto G J Biol Chem 2006 281 4274-4284 [109] Soltoff S P McMillian M K Talamo B R Cantley L C Biochem
Pharmacol 1993 45 1936-1940[70] Chessell I P Hatcher J P Bountra C Michel A D Hughes J PGreen P Egerton J Murfin M Richardson J Peck W LGrahames C B A Casula M A Yiangou Y Birch R Anand PBuell G N Pain 2005 114 386-396
[110] El-Moatassim C Dubyak G R J Biol Chem 1993 268 15571-15578
[111] Panenka W Jijon H Herx L M Armstrong J N Feighan D WeiT Yong V W Ransohoff R M MacVicar B A J Neurosci 200121 7135-7142
[71] Gracie J A Clin Exp Immunol 2004 136 402-404[72] Labasi J M Petrushova N Donovan C McCurdy S Lira P
Payette M M Brissette W Wicks J R Audoly L Gabel C J JImmunol 2002 168 6436-6445
[112] Grinstein S Rotin D Mason M J Biochim Biophys Acta RevBiomembr 1989 988 73-97
[73] Wang X Arcuino G Takano T Lin J Peng W G Wan P LiP Xu Q Liu Q S Goldman S A Nedergaard M Nat Med 200410 821-827
[113] Garcia M L King V F Shevell J L Slaughter R S Suarez-KurtzG Winquist R J Kaczorowski G J J Biol Chem 1990 2653763-3771
[74] Solle M Labasi J Perregaux D G Stam E Petrushova N KollerB H Griffiths R J Gabel C A J Biol Chem 2001 276 125-132
[114] Garritsen A Ijzerman A P Beukers M W Cragoe E J J SoudjinW Biochem Pharmacol 1990 40 827-834
[75] Sanchez-Nogueiro J Marin-Garcia P Miras-Portugal M T FEBS Lett2005 579 3783-3788
[115] Neylon C B Little P J Cragoe E J J Bobik A Circ Res 199067 814-825
[76] Raouf R Chakfe Y Blais D Speelman A Boue-Grabot EHenderson D Seguela P Mol Pharmacol 2004 65 646-654
[116] Wiley J S Chen R Wiley M J Jamieson G P Arch BiochemBiophys 1992 292 411-418
[77] Yiangou Y Facer P Durrenberger P Chessell I P Naylor ABountra C Banati R R Anand P BMC Neurol 2006 6 12
[117] Wiley J S Chen R Jamieson G P Arch Biochem Biophys 1993305 54-60
[78] Sperlagh B Vizi E S Wirkner K Illes P Prog Neurobiol 200678 327-346
[118] Bultmann R Wittenburg H Pause B Kurz G Nickel P Starke KNaunyn-Schmiedebergs Arch Pharmacol 1996 354 498-504
[79] Stojilkovic S S Tomic M He M-L Yan Z Koshimizu T-AZemkova H Ann NY Acad Sci 2005 1048 116-130
[119] Damer S Niebel B Czeche S Nickel P Ardanuy U SchmalzingG Rettinger J Mutschler E Lambrecht G Eur J Pharmacol 1998350 R5-R6[80] McGaraughty S Jarvis M F Drug Dev Res 2006 67 376-388
[81] Baraldi P G Di Virgilio F Romagnoli R Curr Top Med Chem2004 4 1707-1717
[120] Braun K Rettinger J Ganso M Kassack M Hildebrandt CUllmann H Nickel P Schmalzing G Lambrecht G Naunyn-Schmiedebergs Arch Pharmacol 2001 364 285-290[82] Romagnoli R Baraldi P G Di Virgilio F Expert Opin Ther Pat
2005 15 271-287 [121] Kassack M U Braun K Ganso M Ullmann H Nickel P BoingB Muller G Lambrecht G Eur J Med Chem 2004 39 345-357[83] Shieh C-C Jarvis M F Lee C-H Perner R J Expert Opin Ther
Pat 2006 16 1113-1127 [122] Horner S Menke K Hildebrandt C Kassack M U Nickel PUllmann H Mahaut-Smith M P Lambrecht G Naunyn-Schmiedebergs Arch Pharmacol 2005 372 1-13
[84] Virginio C Church D North R A Surprenant ANeuropharmacology 1997 36 1285-1294
[85] Nuttle L C Dubyak G J Biol Chem 1994 269 13988-13996 [123] Hausmann R Rettinger J Gerevich Z Meis S Kassack M U IllesP Lambrecht G Schmalzing G Mol Pharmacol 2006 69 2058-2067
[86] Tatham P E R Cusack N J Gomperts B D Eur J Pharmacol1988 147 13-21
[87] Elssner A Duncan M Gavrilin M Wewers M D J Immunol2004 172 4987-4994
[124] Ullmann H Meis S Hongwiset D Marzian C Wiese M NickelP Communi D Boeynaems J-M Wolf C Hausmann RSchmalzing G Kassack M U J Med Chem 2005 48 7040-7048[88] Chessell I P Michel A D Humphrey P P A Br J Pharmacol
1998 124 1314-1320 [125] Soto F Lambrecht G Nickel P Stuhmer W Busch A ENeuropharmacology 1999 38 141-149[89] Bianchi B R Lynch K J Touma E Niforatos W Burgard E C
Alexander K M Park H S Yu H Metzger R Kowaluk E JarvisM F van Biesen T Eur J Pharmacol 1999 376 127-138
[126] Rettinger J Schmalzing G Damer S Muller G Nickel PLambrecht G Neuropharmacology 2000 39 2044-2053
[90] North R A Surprenant A Annu Rev Pharm Toxicol 2000 40 563-580
[127] Klapperstuck M Buttner C Nickel P Schmalzing G Lambrecht GMarkwardt F Eur J Pharmacol 2000 387 245-252
[91] Murgia M Hanau S Pizzo P Rippa M Di Virgilio F J BiolChem 1993 268 8199-8203
[128] Hulsmann M Nickel P Kassack M Schmalzing G Lambrecht GMarkwardt F Eur J Pharmacol 2003 470 1-7
[92] Craighead M W Middlehurst K M L LeFeuvre R Kimber IRothwell N J Neurosci Lett 2001 311 77-80
[129] Rettinger J Braun K Hochmann H Kassack M U Ullmann HNickel P Schmalzing G Lambrecht G Neuropharmacology 200548 461-468[93] Beigi R D Kertesy S B Aquilina G Dubyak G R Br J
Pharmacol 2003 140 507-519 [130] Jiang L-H Mackenzie A B North R A Surprenant A MolPharmacol 2000 58 82-88[94] DellAntonio G Quattrini A Cin E D Fulgenzi A Ferrero M E
Arthritis Rheum 2002 46 3378-3385 [131] Bultmann R Starke K Naunyn-Schmiedebergs Arch Pharmacol1995 352 477-482[95] DellAntonio G Quattrini A Cin E D Fulgenzi A Ferrero M E
Neurosci Lett 2002 327 87-90 [132] Tuluc F Bultmann R Glanzel M Frahm A W Starke K Naunyn-Schmiedebergs Arch Pharmacol 1998 357 111-120[96] Lambrecht G Braun K Damer S Ganso M Hildebrandt C
Ullmann H Kassack M U Nickel P Curr Pharm Des 2002 82371-2399
[133] Wittenburg H Bultmann R Pause B Ganter C Kurz G Starke KNaunyn-Schmiedebergs Arch Pharmacol 1996 354 491-497
[97] Garcia-Guzman M Soto F Gomez-Hernandez J M Lund P-EStuhmer W Mol Pharmacol 1997 51 109-118
[134] Gargett C E Wiley J S Br J Pharmacol 1997 120 1483-1490[135] Humphreys B D Virginio C Surprenant A Rice J Dubyak GR
Mol Pharmacol 1998 54 22-32[98] Kim Y-C Camaioni E Ziganshin A U Ji X-D King B FWildman S S Rychkov A Yoburn J Kim H Mohanram AHarden T K Boyer J L Burnstock G Jacobson K A Drug DevRes 1998 45 52-66
[136] Baraldi P G Romagnoli R Tabrizi M A Falzoni S Di Virgilio FBioorg Med Chem Lett 2000 10 681-684
[137] Ravi R G Kertesy S B Dubyak G R Jacobson K A Drug DevRes 2001 54 75-87[99] Jacobson K A Kim Y-C Wildman S S Mohanram A Harden T
K Boyer J L King B F Burnstock G J Med Chem 1998 41 2201-2206
[138] Chen W Ravi R G Kertesy S B Dubyak G R Jacobson K ABioconjug Chem 2002 13 1100-1111
[100] Kim Y-C Brown S G Harden T K Boyer J L Dubyak GKing B F Burnstock G Jacobson K A J Med Chem 2001 44 340-349
[139] Baraldi P G Nunez M D C A M Falzoni S Di Virgilio FRomagnoli R J Med Chem 2003 46 1318-1329
[140] Romagnoli R Baraldi P G Pavani M G Tabrizi M A MoormanA R Di Virgilio F Cattabriga E Pancaldi C Gessi S Borea P ABioorg Med Chem Lett 2004 14 5709-5712
[101] Brown S G Kim Y-C Kim S-A Jacobson K A Burnstock GKing B F Drug Dev Res 2001 53 281-291
[102] Lambrecht G Rettinger J Baumert H G Czeche S Damer SGanso M Hildebrandt C Niebel B Spatz-Kumbel G SchmalzingG Mutschler E Eur J Pharmacol 2000 387 R19-R21
[141] Baxter A Bent J Bowers K Braddock M Brough S Fagura MLawson M McInally T Mortimore M Robertson M Weaver RWebborn P Bioorg Med Chem Lett 2003 13 4047-4050
[103] Kim Y-C Lee J-S Sak K Marteau F Mamedova L BoeynaemsJ-M Jacobson K A Biochem Pharmacol 2005 70 266-274
[142] Alcaraz L Baxter A Bent J Bowers K Braddock M CladingboelD Donald D Fagura M Furber M Laurent C Lawson M
Molecular Probes for P2X7 Receptor Studies Current Medicinal Chemistry 2007 Vol 14 No 14 1523
Mortimore M McCormick M Roberts N Robertson M BioorgMed Chem Lett 2003 13 4043-4046
[165] Nelson D W Gregg R J Kort M E Perez-Medrano A Voight EA Wang Y Grayson G Namovic M T Donnelly-Roberts D LNiforatos W Honore P Jarvis M F Faltynek C R Carroll W AJ Med Chem 2006 49 3659-3666
[143] Caffrey M Rhonan F Pimm A WO2004074224A1 2004[144] Evans R Rhonan F Thompson T Willis P WO2006059945A1
2006 [166] Perez-Medrano A Nelson D W Carroll W A Kort M E Gregg RJ Voight E A Jarvis M F Kowaluk E A WO2006086229A12006
[145] Rhonan F Martin B Thompson T Tomkinson N Willis PWO2006025783A1 2006
[146] Cladingboel D Rhonan F Willis P WO2005014529A1 2005 [167] Carroll W A Perez-Medrano A Florjancic A S Nelson D WPeddi S Li T Bunnelle E M Hirst G Li B CWO20060052374A1 2006
[147] Duplantier A J EP1310493A1 2003[148] Baxter A Kindon N Pairaudeau G Roberts B Thom S
WO0144213A1 2001 [168] Carroll W A Perez-Medrano A Florjancic A S Nelson D WPeddi S Bunnelle E M Hirst G C Li B WO2005111003A12005
[149] Meghani P Bennion C WO0146200A1 2001[150] Li Z B WO20050288256A1 2005[151] Leonard J A Li Z J Li Z B Urban F J WO20050288288A1
2005[169] Shemon A N Sluyter R Conigrave A D Wiley J S Br J
Pharmacol 2004 142 1015-1019[152] Leonard J A Li Z J Li Z B Urban F J WO2006003513A1
2006[170] Suadicani S O Brosnan C F Scemes E J Neurosci 2006 26
1378-1385[153] Li Z B WO2006003500A1 2006 [171] Saez J C Retamal M A Basilio D Bukauskas F F Bennett M
V L Biochim Biophys Acta 2005 1711 215-224[154] Evans R Eyssade C Rhonan F Martin B Thompson T WillisP WO2004106305A1 2004 [172] Michel A D Xing M Thompson K M Jones C A Humphrey P
P A Eur J Pharmacol 2006 534 19-29[155] Rhonan F Thompson T Willis P WO2005009968A1 2005[156] Furber M Luker T J Mortimore M P Thorne P Meghani P
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