Progress in Medicinal Chemistry – Vol. 44, Edited by F.D. King and G. Lawton r 2006 Elsevier B.V. All rights reserved. 6 Recent Progress in Cannabinoid Research JULIA ADAM, PHILLIP M. COWLEY, TAKAO KIYOI, ANGUS J. MORRISON and CHRISTOPHER J.W. MORT Organon Research, Newhouse, Lanarkshire, ML1 5SH, Scotland, UK INTRODUCTION 208 CANNABINOID RECEPTORS AND ENDOCANNABINOIDS 208 ANANDAMIDE TRANSPORT INHIBITORS 210 The Hydrophobic Chain 211 The Carboxamide/Carboxylate Group 211 The Polar Head Group 211 FATTY AMIDE ACID HYDROLASE INHIBITORS 212 Organosulfonate and Organophosphonate Analogues 213 Trifluoromethylketones 215 a-Ketoheterocycles 216 Carbamates 217 Other Structures 219 CANNABINOID RECEPTOR AGONISTS 220 Classical Cannabinoids 220 Cannabidiol Derivatives (Resorcinols) 233 Non-classical Cannabinoids 235 Endocannabinoid Derivatives 237 Indole and its Derivatives 247 CB 2 AGONISTS 259 CB 2 Selective Classical Cannabinoids 260 Indoles and Indazoles 262 Resorcinol Derivatives 266 Benzo[c]Chromen-6-one Derivatives 268 Other Heterocyclic CB 2 Agonists 269 DOI: 10.1016/S0079-6468(05)44406-9 207
Transcript
Progress in Medicinal Chemistry –Vol. 44,Edited by F.D. King and G. Lawtonr 2006 Elsevier B.V. All rights reserved.
6 Recent Progress in Cannabinoid
Research
JULIA ADAM, PHILLIP M. COWLEY, TAKAO KIYOI, ANGUSJ. MORRISON and CHRISTOPHER J.W. MORT
Organon Research, Newhouse, Lanarkshire, ML1 5SH, Scotland, UK
INTRODUCTION
208
CANNABINOID RECEPTORS AND ENDOCANNABINOIDS
208
ANANDAMIDE TRANSPORT INHIBITORS
210
The Hydrophobic Chain
211
The Carboxamide/Carboxylate Group
211
The Polar Head Group
211
FATTY AMIDE ACID HYDROLASE INHIBITORS
212
Organosulfonate and Organophosphonate Analogues
213
Trifluoromethylketones
215
a-Ketoheterocycles
216
Carbamates
217
Other Structures
219
CANNABINOID RECEPTOR AGONISTS
220
Classical Cannabinoids
220
Cannabidiol Derivatives (Resorcinols)
233
Non-classical Cannabinoids
235
Endocannabinoid Derivatives
237
Indole and its Derivatives
247
CB2 AGONISTS
259
CB2 Selective Classical Cannabinoids
260
Indoles and Indazoles
262
Resorcinol Derivatives
266
Benzo[c]Chromen-6-one Derivatives
268
Other Heterocyclic CB2 Agonists
269
DOI: 10.1016/S0079-6468(05)44406-9 207
RECENT PROGRESS IN CANNABINOID RESEARCH208
THERAPEUTIC APPLICATIONS OF CANNABINOID AGONISTS
270
CB1 RECEPTOR ANTAGONISTS
272
1,5-Diaryl-pyrazoles
273
4,5-Dihydro-1H-pyrazole Derivatives
282
Imidazole-, Thiazole-, Pyrrole- and Triazole-based CB1 Receptor Antagonists
285
Pyridine-, Phenyl-, Pyrimidine- and Pyrazole-based CB1 Receptor Antagonists
295
Azetidine-based CB1 Receptor Antagonists
301
Substituted Amide-based CB1 Receptor Antagonists
303
Hydantoin-based CB1 Receptor Analogues
304
Recent CB1 Receptor Antagonists
307
THERAPEUTIC APPLICATIONS OF CB1 RECEPTOR ANTAGONISTS
308
CB2 RECEPTOR ANTAGONISTS
310
SUMMARY AND FUTURE PROSPECTS
313
REFERENCES
313
INTRODUCTION
This article aims to update the previous review of the cannabinoids in thisseries, which was written in 1998 by Mechoulam et al. [1]. The intervening7 year period has seen a great deal of activity in the area that has bothexpanded our knowledge of cannabinoid pharmacology and delivered novelligands and drug candidates. Cannabinoid ligands and their pharmacologyhave also been the topic of a number of recent review articles [2–7].
CANNABINOID RECEPTORS AND ENDOCANNABINOIDS
The previous review article in this series described the discovery andcloning of cannabinoid CB1 and CB2 receptors and a classification of thesereceptors was provided in 2002 [1, 6]. Cannabinoid CB1 receptors areexpressed primarily within the central nervous system (CNS), where theyare widely distributed. The CB1 receptor is also present in some peripheraltissues. The CB2 receptor is found mainly in cells of the immune systemand exhibits 48% homology with the CB1 receptor. Both receptors arecoupled to Gi/o proteins. Evidence exists for the presence of additionalcannabinoid receptor subtypes, as covered by a recent review article [8].Furthermore, a recent patent application described high-affinity binding ofa number of cannabinoid ligands to GPR55, suggesting that this might beone of the additional cannabinoid receptors responsible for the pharma-cological observations [9].
J. ADAM ET AL. 209
Since the cloning of the cannabinoid receptors, their endogenous ligands,the endocannabinoids, have received a great deal of research interest.A number of recent review articles have extensively covered the end-ocannabinoid system [10–12], so the coverage in this article will be brief.The best studied of the endocannabinoids are anandamide (N-arachidonyl-
ethanolamine, AEA)(1) and 2-arachidonylglycerol (2-AG)(2). Anandamidewas first identified from porcine brain extracts by Devane and co-workers in1992 [13], while 2-AG was first reported in 1995 to have been isolated fromcanine gut [14] and rat brain [15]. More recently, noladin ether (2-arachidonyl-glyceryl ether, 2-AGE)(3) [16], virodhamine (O-arachidonyl-ethanolamine)(4)[17] and N-arachidonyl-dopamine (NADA)(5) [18] were proposed as endog-enous ligands for the cannabinoid receptors. In a subsequent publication, theauthors failed to detect noladin ether in mammalian brains and questioned therelevance of this compound as an endocannabinoid [19]. Anandamide, noladinether and NADA have functional selectivity for CB1 receptors, virodhamine isCB2 selective and 2-AG is essentially non-selective.
(CH2)3Rn-C4H9 4
(1) Anandamide R = CONH(CH2)2OH
(2) 2-Arachidonylglycerol R = CO2CH(CH2OH)2
(3) Noladin ether R = CH2OCH(CH2OH)2
(4) Virodhamine R = CO2(CH2)2NH2
(5) N-Arachidonyl-dopamine R = CONH(CH2)2-3,4-(OH)2-Ph
The biosyntheses of anandamide and 2-AG have been studied in depth[10]. These compounds appear to be synthesised on demand in response tocertain stimuli, rather than being stored in cells. Little is known regardingthe biosynthesis of noladin ether, virodhamine or NADA.The mechanism of release of endocannabinoids from cells and their subse-
quent re-uptake remain as points of discussion. Several groups have proposedthe existence of an anandamide transporter that can selectively release andremove endocannabinoids from their site of action [20–22]. Indeed, a numberof compounds have been proposed as inhibitors of the putative anandamidetransporter as will be outlined in the following section. An alternative sugges-tion is that the release and re-uptake of endocannabinoids is a simple diffusionprocess, with concentrations and hence diffusion being driven by the enzymefatty acid amide hydrolase (FAAH) [23]. This suggestion has recently beenrefuted by Fegley and co-workers [24], who investigated anandamide internal-isation and activity in wild-type and FAAH knock-out mice treated withanandamide transport (ANT) inhibitors, concluding that anandamide uptakewas independent of FAAH activity. The discussions will only be concludedonce the putative anandamide transporter is identified and cloned.
RECENT PROGRESS IN CANNABINOID RESEARCH210
FAAH was originally purified and cloned from rat liver microsomes andis able to catalyse the hydrolysis of anandamide and 2-AG, in addition toother long-chain fatty acid amides [25]. Studies into the structure and role ofthis enzyme have generated interest in the potential therapeutic applicationsof FAAH inhibitors [26–28]. FAAH knock-out mouse brains contained 15-fold higher levels of anandamide than their wild-type counterparts and theseanimals have also been shown to be more responsive to exogenously ad-ministered anandamide [29]. These animals also showed a reduced responseto painful stimuli, supporting the hypothesis that FAAH inhibition mayprovide novel analgesics. Levels of 2-AG were not elevated in the FAAHknock-out animals, apparently due to the existence of alternative metabolicfates for this compound [30].
ANANDAMIDE TRANSPORT INHIBITORS
To date the ANT inhibitors reported in the literature are all derivatives of long-chain fatty acids. These long-chain fatty acids can be split into three regions: thehydrophobic chain, the carboxamide/carboxylate group and the polar headgroup. These regions are highlighted for anandamide (1) (Figure 6.1). Com-prehensive reviews of ANT inhibitors and their potential as drugs for CNS-related disorders have been recently reported [31, 32], therefore the followingdiscussion will only highlight some of the structure–affinity relationships (SAR)established. The interested reader is directed to the review of Lopez-Rodrıguezet al. [31] for a more detailed discussion.
NH
O
n-C5H11
OH
(1) Anandamide
hydrophobic chaincarboxamide/carboxylategroup
polar head group
Fig. 6.1 Structure of anandamide.
Table 6.1 SAR OF THE HYDROPHOBIC CHAIN [33] R-CONH(CH2)2OH
Cpd. R IC50 (mM)
(1)a
(CH2)3-4
n-C4H9
15.1
(6)
(CH2)7-2
n-C6H13
10.6
(7)
(CH2)7-n-C8H17
10.5
(8)n-C8H17
(CH2)7- >100
(9) n-C17H35 >100
aAnandamide.
J. ADAM ET AL. 211
THE HYDROPHOBIC CHAIN
A variety of long-chain hydrocarbons have been found to be tolerated inthis position as well as the parent hydrocarbon. In general, a minimum ofone double bond located near or at the mid-point of the carbon chain isrequired for activity. In addition, the double bond must be cis configured(Table 6.1) [33].
THE CARBOXAMIDE/CARBOXYLATE GROUP
A wide range of groups are tolerated in this position including esters, am-ides, thioesters, ureas, acyl hydrazines and carbamates exhibiting a widerange of potencies and microsomal stabilities (Table 6.2) [24, 33–36].
THE POLAR HEAD GROUP
The head group has been extensively studied and a diverse range of groupsare tolerated including aliphatic, cyclic, aromatic and heteroaromaticgroups [33, 34, 37]. In general, the presence of a polar head group is fa-voured but not essential. The presence of aromatic/heteroaromatic moietiesis well tolerated; in addition, bulky groups can also be accommodated(Table 6.3).The furan derivative (23) has been tested in vivo in tests of motor activity
(open-field test) and antinociception (hot-plate test) as well as its capacity toenhance the hypokinetic and/or analgesic actions of subeffective doses of
Table 6.2 SAR OF THE CARBOXAMIDE/CARBOXYLATE GROUP
(CH2)3R4
n-C4H9
Cpd. R IC50
(mM)Ref. Cpd. R IC50
(mM)Ref.
(1) –CONH(CH2)2OH 15.1 [33] (14)
NH
O
NH
OH
Me 1.78 [35]
(10) –CO2(CH2)2OH 6.7 [33] (15)O
O
NH
OH
Me
7.1 [35]
(11) –CONH2 9 [33] (16)
NH
O
OOH
Me 3.29 [35]
(12)
-CON O25 [33] (17)
NH
O
NH
OH
OMe
17.7 [36]
(13) O-C(O)SCH2
9 [34] (18)
NH
O
OH 2.5 [24]
RECENT PROGRESS IN CANNABINOID RESEARCH212
anandamide [38]. It was found that (23) was mostly inactive when admin-istered alone, but was able to potentiate the action of an exogenous dose ofanandamide that produced no effect by itself.
FATTY AMIDE ACID HYDROLASE INHIBITORS
The enzyme FAAHwas first identified in the 1980s [39], cloned in the 1990s [25]and more recently, the crystal structure [40] of FAAH bound to methylarachidonyl phosphonate has been published. The FAAH enzyme has widesubstrate specificity capable of catalysing the hydrolysis of a wide variety of
Table 6.3 SAR OF THE POLAR HEAD GROUP
(CH2)3CONHR4
n-C4H9
Cpd. R IC50
(mM)Ref. Cpd. R IC50
(mM)Ref.
(1) –(CH2)2OH 15.1 [33] (22)
OH2.2 [33]
(19) –Et 48.5 [33] (23)
O
0.8 [34]
(20)
OH
Me 10.4 [33] (24) -( CH2)2O O 16.5 [37]
(21)
OH
Me 37.7 [33]
J. ADAM ET AL. 213
bioactive fatty acid amides and esters including the endogenous cannabinoidligands anandamide and 2-AG. Several other long-chain fatty acids and amidesalso act as substrates including palmitoylethanolamide, stearoylethanolamide,oleoylethanolamide and linoleoylethanolamide [41–44]. It has been postulatedthat selective FAAH inhibitors may elevate the levels of endogenouscannabinoids while avoiding the unwanted behavioural effects associated withthe CB1 agonists. A number of comprehensive reviews on the pharmacology,possible therapeutic uses and SAR of FAAH inhibitors have been publishedrecently [26, 45, 46]. Therefore, the following discussion will focus on high-lighting the types of structures that act as FAAH inhibitors.
ORGANOSULFONATE AND ORGANOPHOSPHONATE ANALOGUES
The first compound to find widespread use as an inhibitor of AEA break-down was phenylmethylsulfone (PMSF) (25). This non-selective serine pro-tease inhibitor has been found to act as an irreversible inhibitor of FAAHpreventing AEA breakdown [41, 47], and is commonly added to binding
RECENT PROGRESS IN CANNABINOID RESEARCH214
assays to improve the metabolic stability of AEA [48, 49]. PMSF has beenfound to increase the potency of AEA in inhibiting contractions in theguinea pig myenteric plexus [50], and to potentiate the effects of anandamidein vivo [51, 52]. However, the low potency (IC50 ¼ 290 nM) [53] and poorselectivity of this ligand has led to the development of more potent andselective sulfonyl fluoride inhibitors of FAAH. A number of fatty acidsulfonyl fluorides with improved potencies have been reported [53, 54], inparticular, palmitylsulfonyl fluoride (26) and arachidonylsulfonyl fluoride(27, AM374). Both these derivatives can inhibit FAAH with IC50s in the lownanomolar range (IC50 values of 7 and 0.11 nM, respectively). In addition,both these compounds have good selectivity over the CB1 receptor (IC50
values of 520 and 304 nM, respectively). Furthermore, AM374 (27) has beenfound to produce a significant inhibition of [3H]-acetylcholine release fromhippocampal brain slices [55] and to enhance the behavioural effects of lowdoses of anandamide in vivo [56].
(CH2)4R4
n-C4H9
(25) (26) (27) R = SO2F(28) R = PO(OMe)F
(29)
PhCH2SO2F n-C16H33SO2F n-C12H25PO(OMe)F
(CH2)3COCF34
n-C3H7(CH2)10PO(OMe)F2
n-C4H9
(31)(30) (32)
n-C8H17PO(OMe)F
A number of organophosphate compounds have been found to irreversiblyinhibit FAAH, the most studied being methylarachidonyl fluorophosphonate,MAFP (28) [57]. MAFP was initially designed and developed as an inacti-vator of cytosolic phospholipase A2 (cPLA2) [58, 59]. It was found to be ahighly potent and selective inhibitor of FAAH (relative to other amide hy-drolytic enzymes). However, it also bound irreversibly to the CB1 receptorand prevented the subsequent binding of CP 55,940 [60]. A detailed study onmodifications to the alkyl chain of (28) has recently been carried out [61].Both saturated (29) and unsaturated (30) analogues inhibit FAAH. In par-ticular, the fully saturated C-12 derivative (29), gave a highly potent inhibitorof FAAH (IC50 ¼ 3 nM) and the CB1 receptor (KD ¼ 2:5 nM). Shortening ofthe alkyl chain to the C-8 derivative (31),O-1887, gave a slight drop in FAAHaffinity (IC50 ¼ 15 nM) but importantly, (31) failed to interact with the CB1
receptor (KD>10,000 nM).
J. ADAM ET AL. 215
TRIFLUOROMETHYLKETONES
The first substrate analogue inhibitors of FAAH were reported in 1994.The anandamide analogues prepared represented three classes of putativetransition-state inhibitors: a-trifluoromethyl ketones, a-ketoesters anda-ketoamides [62]. In the initial screening studies, it was found that thetrifluoromethyl ketone compounds tested were effective inhibitors of AEAhydrolysis. A selected set of a-keto esters also inhibited hydrolysis, whilea-keto amides were ineffective. In particular, arachidonyl trifluoromethylketone (32), gave almost 100% inhibition of anandamide hydrolysis. Adetailed investigation of the structural requirements for FAAH inhi-bition with a-trifluoromethyl ketones has been carried out by Boger andco-workers [63].The electrophilic carbonyl is essential for activity; replacement with a
methyl ketone results in complete loss of activity (Table 6.4, (33) and (34)).Saturated alkyl chains are tolerated but must be at least seven carbons long,shortening the chain length further results in dramatic losses of potency(Table 6.4, (36)–(39)). In contrast, extending the chain from seven throughseventeen carbons has little effect on potency (Table 6.4, (36) and (38)).Incorporation of the oleamide D9,10 cis double bond is favoured with analmost 2-fold increase in potency compared to its saturated congener (cf.(33) and (36)), the trans isomer is also tolerated (Table 6.4, (35)). Replace-ment of the oleamide D9,10 cis double bond and alkyl tail with a phenylgroup (Table 6.4, (40)) gives a further enhancement in affinity.
aInhibition of [14C]-oleamide hydrolysis in rat liver plasma membranes.
RECENT PROGRESS IN CANNABINOID RESEARCH216
a-KETOHETEROCYCLES
The Boger group [64–66] has extensively studied the use of a-ketoheterocyclesas FAAH inhibitors. In their initial studies, a range of a-ketoheterocyclesbased on oleic acid was synthesised. A range of five- and six-memberedmonocyclic heterocycles and three bicyclic heterocycles (benzothiazole, ben-zimidazole and benzoxazole) was examined. Although many of the com-pounds tested were found to inhibit FAAH activity with micromolaraffinities, the best results were obtained with heterocycles that incorporateda weakly basic nitrogen a – to the heterocycle (Table 6.5) [64].Following on from this, the benzoxazole scaffold was examined in detail
and it was found that incorporation of a second weakly basic nitrogen togive the a-keto oxalopyridone (47) led to a >150-fold enhancement inbinding affinity (Table 6.5). Using this head group, modifications to theoleyl side chain were investigated and found to parallel the SAR found withthe trifluoromethyl ketones previously studied by Boger [63] (see above), thebest results being obtained with saturated chain lengths of C12–C18. Afurther enhancement in potency was seen when a phenyl ring was introducedinto an optimally long side chain. The best compound identified from thisseries was the oxalopyridone (48) with Ki values of 94 and 200 pM in humanand rat, respectively. However, when tested in vivo, this compound failed topotentiate the effects of anandamide [67].
Following on from these studies, Boger and co-workers [65, 66] discloseda series of substituted oleyl a�ketooxazoles exploring the introductionof substituents in the 4 and 5 positions. Analogous to the observations madein the benzoxazole series, incorporation of a second heterocyclic ringcontaining a weakly basic nitrogen proximal to the oxazole substantiallyincreased FAAH inhibition. Replacement of the oleyl side chain with theoptimal side chain found in the trifluoromethyl ketone and benzoxazoleseries gave a further enhancement in FAAH inhibitiory activity. In partic-ular, the pyridyl oxazole derivative (49) had a Ki of 7 nM and was found tohave good selectivity over a panel of serine hydrolases. In addition, thecompound failed to bind to the CB1 or CB2 receptor [67]. Compound (49)has been tested in vivo in mice and found to augment not only exogenouslyadministered AEA but also the activity of endogenously produced fatty acidamide in models of antinociception. These effects could be inhibited bypretreatment with a CB1 receptor antagonist. Importantly, the anti-nociceptive properties of (49) occurred in the absence of any significanteffects on motility or motor coordination that typically accompany theglobal activation of CB1 receptors by direct agonists [67].Boger and co-workers [68] have recently extended their studies to encom-
pass heterocyclic sulfoxides and sulfones. In general, these compounds wereless potent (up to 1,000 fold) than their a-keto heterocycle congeners. The bestresults were obtained with the benzoxazole sulfoxide (50) with a Ki of 2.6mM.
Ph(CH2)5
O O
NN
Ph(CH2)5
O
O
N N
Ph(CH2)5S O
N
O
(48) (49) (50)
CARBAMATES
Tarzia et al. [69, 70] have recently reported the FAAH inhibitory activity ofa series of alkylcarbamic acid aryl esters. The starting point for their studieswas the known serine hydrolase inhibitor carbamyl (51) that had no activityat FAAH. Replacement of the small methyl group of carbamyl (51) withmore lipophilic groups and, in particular, bulky lipophilic groups resulted inincreased affinity at FAAH (Table 6.6). Exploration of replacements of thenaphthyl moiety revealed that replacement with a biphenyl group resulted inimproved affinity and in particular, the 3-biphenylyl group proved effective
Table 6.6 SAR OF CARBAMYL DERIVATIVES [69, 70]R1NHCOOR2
Compound R1 R2 IC50 (nM)
(51) Me 1-Naphthyl >30,000(52) Et p-Tolyl >30,000(53) Cyclohexyl 2-Naphthyl 324(54) Cyclohexyl 4-Biphenylyl 2,297(55) Cyclohexyl 3-Biphenylyl 63
RECENT PROGRESS IN CANNABINOID RESEARCH218
(Table 6.6). The most potent compound identified in this series was thecyclohexyl 3-biphenylyl carbamate (55) with an IC50 of 63 nM.Following on from these initial studies, a systematic exploration of phenyl
ring substitution on the distal phenyl ring was carried out [70]. A range ofgroups were tolerated giving good inhibitory activity in the nanomolar range.The compound giving best potency in this series was the amido biphenylderivative (56) with an IC50 value of 4.6 nM. In addition, this compound wasselective against three other serine hydrolases, had no effect on the AEAtransporter and failed to bind to the CB1 and CB2 receptors [71]. When testedin vivo, (56) augmented the levels of endogenous AEA and produced CB1-dependent anxiolytic and antinociceptive properties. Importantly, as with thea-keto heterocycle derivative (49), the carbamate inhibitor (56) did not induceany of the common side effects associated with direct CB agonists.
O
OMeNH
O
ONH
CONH2
(51) (56)
Bristol-Myers Squibb has recently disclosed two different series of carba-mate-based FAAH inhibitors. The first of these is a series of 4,5-diaryl-imidazoles in which 30 compounds are specifically claimed, an example beingcompound (57). This compound is reported to have an IC50 value ofo10nM. In addition, (57) was also active in vivo in rodent models of chemo-induced, thermal and neuropathic pain [72]. The second series of compoundsis based on oxime carbamoyl FAAH inhibitors such as (58). Compound (58)is reported to have an IC50 value of o10nM and activity in rodent models ofinflammatory pain, thermal pain and inflammatory oedema [73].
J. ADAM ET AL. 219
A series of dioxane carbamate inhibitors has recently been disclosed bySanofi-Aventis. No biological data are provided for specific compounds,although most compounds are described as having IC50 values for FAAHinhibition ranging from 0.005–1 mM. Compound (59) is one of over 25compounds specifically claimed [74].Sanofi-Aventis has disclosed a series of piperidine- and piperazine-alkyl
carbamates as cannabinoid and/or FAAH modulators. No compounds arespecifically claimed in the patent. Compound (60) is reported to have anIC50 value of 85 mM and is active in a mouse pain model [75].
NN
PhPh
(CH2)6
Me
NH
O
O
F NH
n-BuOO
ON
F(57) (58)
PhO
O NHCO2CH2CONH2
N N
NHCO2CH2CONHMe
Me
(59) (60)
OTHER STRUCTURES
A number of arachidonic acid substrate analogues have been found to in-hibit FAAH. Hillard and co-workers [37] found during their studies into theSAR of ANT inhibitors (see above) that these ligands also inhibit FAAH.From these studies, the most potent FAAH inhibitor identified waschloromethylarachidonyl amide (61) with an IC50 value of 14 nM. This lig-and also bound to the CB1 receptor in the presence and absence of PMSFbut with different binding affinities (84 nM and 2.6 mM, respectively) sug-gesting that it may also be acting as a substrate at FAAH. In addition, thearchetypal AEA transport inhibitor AM 404 (22) was also an excellentinhibitor of FAAH (IC50 ¼ 0:5mM) more than 5-fold lower than its bindingaffinity value for AEA uptake (IC50 ¼ 3:4mM) [37].Another arachidonyl-based inhibitor of FAAH is arachidonyl serotonin
(62), which was reported to inhibit FAAH from rat basophilic leukaemiacells with an IC50 value of 5.6 mM, and with very little affinity at CB1
receptors [76].
RECENT PROGRESS IN CANNABINOID RESEARCH220
A series of palmitoylethanolamine-derived inhibitors has been describedin the literature as FAAH inhibitors [77, 78]. This study explored the effectof shortening the chain length and replacement of the ethanolamine headgroup with primary, secondary and tertiary amide alternatives. Of the com-pounds synthesised and tested, two compounds gave reasonable affinitiesfor FAAH inhibition, palmitoyl-isopropylamide (63) (IC50 ¼ 13mM) andpalmitoyl-allylamide (64) (IC50 ¼ 3:4mM). Both these compounds had littleaffinity for either CB1 or CB2 receptors.Finally, derivatives of the endogenous compound 2-octyl-g-bromoacetate
(65) have been reported as FAAH inhibitors [79]. In a limited SAR study, itwas found that replacement of the bromine with a chlorine atom had littleeffect on affinity. The replacement of the alkyl chain with oleyl-chain mimicsresulted in an increase of affinity for FAAH (approximately 5-fold). Theremoval of the halogen and replacement with either a proton or methylresulted in inactive compounds. The most potent compound identified inthis series was compound (66) with an IC50 value of 0.6 mM [79].
(CH2)3CONH(CH2)2R4
n-C4H9
NH
OH
(61) R = Cl
(62) R =
(63) (64)
n-C15H31CONHs-Bu n-C15H31CONHallyl
O
O
O
Brn-C6H13
Me O
O
O
Br(CH2)4n-C8H17
(65) (66)
CANNABINOID RECEPTOR AGONISTS
CLASSICAL CANNABINOIDS
Before the discovery of specific cannabinoid receptors, the term ‘cannabinoid’was used to describe the biologically active constituents of the Cannabis sativaplant, including D9-THC (67), cannabidiol (68) and their analogues and de-rivatives, many of which have characteristic pharmacological effects.
aGeneral procedures for mouse behavioural experiments are described in Reference [141].SA ¼ spontaneous activity; TF ¼ tail flick; RT ¼ rectal temperature.
J. ADAM ET AL. 221
D9-THC, the main psychoactive component of cannabis, is a moderatelypotent partial agonist of the CB1 and CB2 receptors, while cannabidiol haslittle affinity for either receptor (Table 6.7). The term ‘classical cannabinoids’is used to describe cannabinoid receptor modulators structurally related to(67), which have a tricyclic dibenzopyran core. While several other structuraltypes of cannabinoid receptor modulators have been discovered in recentyears, the classical cannabinoids are still by far the most extensively studiedgroup in terms of SAR and pharmacology.
O
Me
MeMe
OH
OH
Me
OH
Me
(67) ∆9-THC (68) Cannabidiol
1'
2'
3'
4'
5'3
456
6a
10a7
8
910
11
12
7
12
3
45
6
8
10
9
1'
2' 3'
4'
5'1''
2''
3''
4''
5''
O
Me
MeMe
OH
(73) ∆8-THC
1'
2'
3'
4'
5'3
456
6a
10a7
8
910
11
12
RECENT PROGRESS IN CANNABINOID RESEARCH222
The naturally occurring isomers (67) and (73) have very similar pharma-cological profiles. Owing to the greater chemical stability of (73) and itsanalogues, SAR around this template has been much more widely studiedthan those around (67). While caution must be exercised in comparing dataobtained by different laboratories and using different assay formats, thegeneral trends that emerge provide a qualitative picture of the structuralrequirements for activity. Three pharmacophoric elements around theclassical cannabinoid template have been shown to be important for CB1
receptor binding and/or activation: the hydrocarbon side chain at C3, thesubstituent at C9 and the phenolic hydroxyl group at C1 [83–85]. The effectsof alterations to the tricyclic core have also been explored. QuantitativeSAR (QSAR) pharmacophore models have been used to rationalise theobserved SAR and to predict the activity of novel compounds [86–89].
C3 Side-chain modifications
During the 1940s, more than 15 years before the structure of (67) waselucidated as the major psychoactive constituent of marijuana [90] and 40years before the identification of specific cannabinoid receptors, Adams andco-workers [91, 92] demonstrated that a 10,20-dimethylheptyl (10,20-DMH)side chain was the optimal 3-alkyl substituent for cannabinoid activity invivo in the D6a,10a-THC series. In this early research, the ataxia test in dogswas used as a standard measure of psychoactive cannabinoid activity. Theisomeric 10,10-DMH side chain was found to be less potent than the 10,20-DMH one in the D6a,10a-THC series, but the two side chains have similarbinding affinity and potency in the D8-THC series. The 10,10-DMH sidechain has the advantage of not containing any chiral centres and has there-fore found its place as a widely used replacement for the pentyl side chain of(67) and (73) in synthetic analogues.Tables 6.8–6.11 illustrate the wide range of C3 side-chain modified D8-
THC analogues that have been reported in the literature, together withassociated in vitro and in vivo data. The affinity of classical cannabinoidanalogues for the CB1 receptor has been shown to correlate with depressionof spontaneous activity and the production of antinociception, hypothermiaand catalepsy in mice, and with psychomimetic activity in humans [93].However, in some cases, there were unexplained differences between theobserved trends in binding affinity and the trends in activity in mousebehavioural models. This may point to differences in efficacy among fullagonists, partial agonists and antagonists/inverse agonists, or may reflectdifferences in in vivo metabolism or blood–brain barrier penetration or acombination of these factors.
Table 6.8 D8-THC ALKYL SIDE CHAINS
O
Me
MeMe R
OH
Mouse ED50 (mmol/kg)
Cpd. R CB1 Ki (nM) CB2 Ki (nM) SA TF RT Ref.
(69) 1,1-Dimethylethyl 14 a >100 a [96](70) 1,1-Dimethylpropyl 14 a 25 a [96](71) n-Butyl 65 9.0 10 6.3 [98](72) 1,1-Dimethylbutyl 11 a 1.9 4.2 [96](73) n-Pentyl (D8-THC) 44 2.9 4.8 4.5 [80]
Table 6.8 shows the simple alkyl side-chain modifications of (67). Thebinding affinity of these analogues increases with increasing side-chainlength, from butyl up to octyl. Methyl substituents in the 10- and 20-positionsof the side chain have a beneficial effect on CB1 receptor affinity and in vivopotency, as previously shown by Adams. Huffman et al. [94] further dem-onstrated that methyl substitution in the 30-position resulted in compoundswith comparable binding affinity to the parent compound, while methylsubstitution in the 40-position decreased binding affinity. A trend similar tothat seen for the pentyl side chain was observed for monomethyl substi-tution on the heptyl side chain (data not shown) [95]. Reduced CB1 bindingaffinity was seen for compounds with 10,10-dimethyl-substituted side chainslonger than octyl (92–95). These compounds also showed reduced, or no invivo activity [96].Compounds with conformationally restrained side chains, unsaturated
side chains and aromatic side chains have been synthesised in order to betterdefine the steric requirements of the binding site in the side-chain region.These are shown in Table 6.9. On average, the conformationally restrained
Table 6.11 OTHER FUNCTIONALISED D8-THC SIDE CHAINS
42 10 [107](153) 5-{N-[(p-aminosulfonylphenyl) methyl]carboxamido}-1,1-dimethylpentyl 60 56 a 67 [113]
180 2.3 [107](154) 5-{N-[(p-aminosulfonylphenyl)ethyl] carboxamido}-1,1-dimethylpentyl 29 a >176 >176 [113]
246 [107]
aPartial effect reported (SA/TF max o80%; RT max decrease o4 1C).
J.ADAM
ETAL.
227
RECENT PROGRESS IN CANNABINOID RESEARCH228
10,10-dimethyl cycloalkyl side-chain analogues of (99–101) were found tohave in vitro activity similar to their straight-chain analogues (83), (85) and(91), respectively. However, there was no trend of increasing CB1 receptor-binding affinity with increasing ring size to mirror the effect of increasingchain length in the open-chain series [97].Compounds with cis double bonds in the side chain were in general found
to be more potent and efficacious than their triple-bond congeners, both inin vivo and in in vitro functional assays [98, 106, 107]. QSAR models havebeen generated for the compounds with unsaturated [108] and 10,10-dimethyl[96] side chains to determine more precisely the pharmacophoric require-ments of the receptor. It is postulated that for optimum potency, the sidechain must be of a suitable length and flexibility to have the ability to loopback so that its terminus is in proximity to the phenolic ring. The widelyused, potency enhancing 10- and 20-methyl substituents would be expected toincrease the tendency of the side chain to adopt a looped back, rather thanan extended conformation.Aromatic rings are also tolerated in the side chain, as demonstrated by
Krishnamurthy et al. [109]. The 10,10-dimethylbenzyl compound (117)showed higher binding affinity than the simple benzyl compound (116).Compound (117) also showed around 13-fold selectivity for CB2 over CB1
binding.A range of different functional groups has been introduced into the 10-
position of the C3 side chain, as shown in Table 6.10. A number of differentfunctionalities were shown to be tolerated in this position, with lipophilicgroups such as methyl and dithiolane being preferred over polar groups suchas ketones and alcohols. Ketone (118) had similar CB1 receptor affinity tothe unfunctionalised n-heptyl compound (84), while alcohol (120) had loweraffinity. The phenyl ketone (119) had lower CB1 receptor affinity than thesimple benzyl-substituted compound (116), but higher CB2 affinity, withabout 12-fold selectivity for CB2 over CB1.A dithiolane group in the 10-position has been shown to be at least as
effective as the 10,10-dimethyl group in enhancing the binding affinity of theclassical cannabinoids, as can be seen by comparing compounds (123) and(124) with compounds (83) and (85). However, the constrained dithiolanecompounds (125–127) showed decreased activity compared to their 10,10-dimethyl analogues (99–101). In contrast to its 10,10-dimethyl and ketoneanalogues, (117) and (119), the phenyl dithiolane compound (128) does notexhibit any CB2 selectivity.Surprisingly, an iodo substituent in the 10-position was not well tolerated,
suggesting that this substituent had an unfavourable interaction withthe receptor or a detrimental effect on the conformation of the side chain[110].
J. ADAM ET AL. 229
A range of 10-substituted D8-THC, D9-THC and D6a,10a-THC derivatives,including many of those in Tables 6.9 and 6.10 has been disclosed in a patentapplication by Moore et al. [111]. The compounds are described as eitheragonists or antagonists of the CB1 and/or CB2 receptors. The in vivo activityof (1-cyclohexyl-1-methyl) ethyl compound (100) in a rat haemorrhagicshock model and the in vitro cytotoxic effects of the 10,10-dimethylbenzylcompound (117) against glioma cells are described.Halogen, cyano, carboxylic acid and amide functional groups have been
introduced at the terminus of the C3 side chain, as shown in Table 6.11. Thisarea of the molecule seems to be tolerant to both lipophilic and more polargroups. Halogen substitution at the terminal carbon of the side chain ledto enhancement of affinity, with the bulkier halogens, bromine and iodine,producing the largest effects [110, 112]. The less lipophilic 5-cyano-10,10-dimethylpentyl and 5-(N,N-dimethyl-carboxamido)-10,10-dimethylpentylcompounds (141) and (147) showed high affinity and in vivo potency similarto the 5-bromo-10,10-dimethylpentyl compound (137). However, thecyanohydrin compound (143) showed decreased affinity and potency. Thismay indicate less tolerance for a hydrogen bond donor in this position. Thecorresponding carboxylic acid (146) showed very low binding affinity atCB1, but retained affinity at CB2. The piperidine hydrazide compound (149)exhibited high binding affinity despite the presence of a hydrogen bonddonor. In this case, the hydrogen bond donor was separated by a longerlinker group from the tricyclic core.Chloro- and sulfonamide-substituted aromatic amides showed decreased
binding affinity and in vivo potency compared to the simple aliphatic am-ides. Side chains with an additional (CH2)1-2 linker between the amide andthe phenylsulfonamide group resulted in partial or absent in vivo effects[113]. The (CH2)-linked compound, (153), showed around 80-fold selectivityfor CB2 over CB1 binding [107].A number of compounds have been prepared that contain both a double
or triple bond and a terminal functional group in the side chain [98, 107,114]. In general, the combined modifications reinforced the SAR trends seenfor the individual modifications.
C9 Substituent modifications
A major route of metabolism for (67) and (73) is oxidation at the C9 po-sition to form hydroxymethyl and carboxyl metabolites. The hydroxymethylmetabolites are potent CB1 agonists with pharmacological profiles similar tothe parent compounds, while the carboxy metabolites have reduced activity[115]; the 9-carboxy analogue of (73) does not bind to the CB1 receptor [93].
Table 6.12 D8-THC C9 SUBSTITUENTS
O
R
MeMe
OH
n-C5H11
Mouse ED50 (mmol/kg)
Cpd. R CB1 Ki (nM) CB2 Ki (nM) SA TF RT Ref.
(73) Methyl (D8-THC) 44 2.9 4.8 4.5 [80]126 [93]
a 4.8 49 [112]23 a [116]
(155) Hydroxymethyl 55 [93]26 7.4 [117]
(156) Carboxyl >10,000 [93]b [115]
(157) Fluoromethyl a 15 21 [112]107 [93]
(158) Isopropyl 76 a [116](159) n-Butyl 90 c [116](160) 1-Hydroxyethyl 78 a [116]
a [118](161) 1-Hydroxypropyl 109 a [116](162) Phenyl 178 a [116](163) Benzyl 82 a [116](164) 2-Phenylethyl 106 a [116]
a [118]
aPartial effect reported (SA/TF max o80%; RT max decrease o4 1C).bInactive.cSee text.
RECENT PROGRESS IN CANNABINOID RESEARCH230
Some alternative substituents have also been introduced in the C9 position,as shown in Table 6.12.Of the direct methyl group replacements reported, small groups such as
hydroxymethyl and fluoromethyl are tolerated, while larger groups in thisposition result in compounds with reduced in vivo potency and efficacy. Then-butyl derivative (159) was the only C9 methyl replacement analogue thatshowed a marked effect on hypothermia (5 1C decrease at 168 mmol/kg;ED50 not calculated) [116].
The CB1 binding affinity of the hydroxymethyl and carboxyl analoguescan be increased by substituting the C3 pentyl side chain for a dimethylheptylside chain (Table 6.13). 11-Hydroxy-10,10-DMH D8-THC, HU-210 (165), is anextremely potent cannabinoid agonist that has been widely used as a phar-macological tool [119]. Its (+) enantiomer, HU-211 (dexanabinol), which isin clinical development for the treatment of cognitive disorders, does not havehigh affinity for CB1 receptors [120].The dimethylheptyl side-chain analogue of 9-carboxy-D8-THC, ajulemic
acid, CT-3, (166) is currently in clinical development for treatment of painand inflammation [121, 122]. Compound (166) does show some affinity forCB1 and CB2 receptors, but may also exert anti-inflammatory and analgesiceffects through other mechanisms. It does not appear to be psychoactive inhumans [123].
O
R
MeMe
n-C6H13
OH
Me Me
(165) HU-210 R = CH2OH
(166) CT-3 R = COOH
C1 modifications
In general, modification or deletion of the C1 phenolic hydroxyl groupresults in significantly reduced CB1 receptor affinity [93]. A number of1-deoxy and 1-alkoxy D8-THC analogues have been shown to be selectiveligands for the CB2 receptor (see below).
RECENT PROGRESS IN CANNABINOID RESEARCH232
Core modifications
There are limited examples in the literature where substituents have beenintroduced in other positions on the tricyclic core (Table 6.14). Substitu-tion at the C2 position of the aryl ring with an iodo substituent resulted inonly a slight drop in binding affinity. However, a nitro substituent in the2-position was not tolerated. Molecular modelling studies suggested thatthis may be attributed to dramatic reductions in the electron densityaround the phenolic hydroxyl group and the oxygen in the pyran ring.Substitution at the C4 position with either a bromo or a nitro groupresulted in loss of affinity at CB1, perhaps as the result of an unfavourablesteric interaction in the binding site. The 2,4-diiodo and 2,4-dinitro com-pounds were also inactive [80].Tetracyclic compounds in which the C3 side chain is conformationally
restricted by linking to either the C2 or the C4 position have been describedby Khanolkar et al. [99]. All the tetracyclic compounds had lower CB1 andCB2 affinity than the analogous non-constrained compounds (84). The best-tolerated constraint was the ‘southbound’ constraint in compound (173).
aPartial effect reported (SA/TF max o80%; RT max decrease o4 1C).
J. ADAM ET AL. 233
O
Me
MeMe
OH
R2
R1 O
Me
MeMe
OH
n-C6H13
(173) R1 = H, R2 = n-C6H13
(174) R1 = n-C5H11, R2 = H(175)
CANNABIDIOL DERIVATIVES (RESORCINOLS)
Cannabidiol has low affinity for CB1 and CB2 receptors and is not psycho-active, but has nevertheless shown a number of pharmacological activities ofits own including anti-inflammatory and neuroprotective effects. Some sidechain-modified cannabidiol derivatives have also been evaluated for can-nabinoid receptor affinity and these are shown in Table 6.15.Bisogno et al. [124] have shown that, in contrast to classical cannabinoid
derivatives, the unnatural (+) enantiomers of cannabidiol derivatives havehigher affinity for CB1 and CB2 receptors than the natural (�) enantiomers,as shown in Table 6.16. Within both the (�) series and the (+) series, theSAR showed some similarity to those in the classical cannabinoid series, withextended and halogenated side chains tending to increase binding affinity. The100,100-dimethylheptyl, 7-hydroxy (+)-cannabidiol analogue (187) exhibitedparticularly high CB1 binding affinity. In the same study, cannabidiol andsome of its analogues were also found to be weak agonists of VR1 receptorsand weak inhibitors of anandamide uptake and degradation [124].Further (+)-cannabidiol analogues were prepared and evaluated by Fride
et al. [125], who showed that carboxylic acid derivatives (189) and (191) alsohave high CB1 receptor affinity [126]. In vivo activity was assessed in lo-comotion, rearing, catalepsy, analgesia (hot plate), hypothermia and inhi-bition of intestinal motility in mice as a percentage of the maximum possibleeffect at a single dose. The (+)-cannabidiol analogues were shown to inhibitdefecation and reduce arachidonic acid-induced ear swelling in mice. Fewcentral cannabinoid effects were observed [125]. The carboxylic acid andalcohol derivatives have been described in a patent application by the samegroup, as therapeutically useful modulators/regulators of the immune sys-tem and gastrointestinal tract [127].
Oxidation of the phenol ring of cannabidiol (73) or cannabinol to a qui-none ring has been shown to afford compounds with anti-tumour activity.However, these compounds do not bind to the CB1 receptor and theirmechanism of action is unclear [128].
NON-CLASSICAL CANNABINOIDS
The term ‘non-classical cannabinoids’ is applied to a group of bicycliccompounds identified by researchers at Pfizer in the 1980s [129]. Thesecompounds lack the pyran ring of the classical cannabinoids and the secondphenolic hydroxyl group of the cannabidiols, resulting in a simplified sub-structure represented by CP 47,497 (192) [130, 131]. The non-classical can-nabinoids still retain the three main pharmacophoric elements describedabove for the classical cannabinoids and the SAR in these regions parallelsthat of the classical cannabinoids [132].A fourth important pharmacophoric element was established for the non-
classical cannabinoid series in the form of a southern aliphatic hydroxylgroup. Addition of this group to (192) resulted in the high-affinity CB1 andCB2 receptor full agonist CP 55,940 (193) [129, 133], the tritiated form ofwhich was used to first demonstrate specific cannabinoid binding sites inbrain tissue [134]. Its enantiomer, CP 56,667 (194) has lower affinity for theCB1 receptor (Table 6.17).
aPartial effect reported (SA/TF max o80%; RT max decrease o4 1C).
RECENT PROGRESS IN CANNABINOID RESEARCH236
Melvin et al. [136] have shown that the positioning of the southern al-iphatic hydroxyl group is critical for optimal binding. The constrained an-alogue of (193), (-)-CP 55,244 (195) showed very high CB1 binding affinityand complete enantioselectivity. Conformational studies with diastereoiso-mers of (195) have suggested that the ability to form an intramolecularhydrogen bond between the phenolic hydroxyl group and the southern al-iphatic hydroxyl group may be important for receptor binding [138]. How-ever, molecular docking of these compounds in a homology model based onthe bovine rhodopsin X-ray crystal structure has suggested that these twohydroxyl groups could form intermolecular hydrogen bonds with differentresidues in the receptor-binding site [139].
n-C6H13
OH
OH
OHMe Me
n-C6H13
OH
OH
OHMe Me
(195) (-)-CP 55,244 (196) (+)-CP 55,244
Classical/non-classical hybrid cannabinoids, such as (197) and (198), havebeen described by Tius et al. [137, 140]. In these compounds, a southern
J. ADAM ET AL. 237
aliphatic hydroxyl group was added to the classical cannabinoid template.Addition of a C-6a hydroxyethyl substituent resulted in a loss of bindingaffinity, while the introduction of C-6b hydroxyethyl or hydroxypropylgroups resulted in increased affinity compared to the corresponding ethyl orpropyl analogues.
n-C5H11
OH
OH
OH
O
Me
n-C5H11
OH
OH
Me OOH
(197) (198)
ENDOCANNABINOID DERIVATIVES
The development of SAR for endocannabinoid-derived structures has pri-marily focused on the anandamide skeleton (1) with a large number ofpublications addressing the requirements for activity and stability of thisscaffold. More recently, some SAR has begun to emerge for the other end-ocannabinoids, in particular 2-AG (2). The following discussion will focuson highlighting some of the main features that contribute to affinity and/orstability; each endocannabinoid will be treated separately. A number ofdetailed reviews on this subject have been published [142–146].A number of protocols are available for measuring cannabinoid-binding
affinity and as such there is a variation in reported Ki values for end-ocannabinoids across labs. For this reason, wherever possible, the relativeaffinity compared to AEA (measured in that protocol) will be given in anattempt to provide a benchmark for comparison.
Anandamide derivatives
As outlined earlier, anandamide was the first among the endogenous can-nabinoid receptor agonists to be identified. It exhibits higher binding affinityfor the CB1 receptor (K i ¼ 89 nM) than for the CB2 receptor (Ki ¼ 371 nM)[81]. Anandamide has typical cannabinoid activities including decreasedspontaneous motor activity, immobility and production of hypothermia andanalgesia [147, 148]. However, this action in vivo is of shorter duration than
RECENT PROGRESS IN CANNABINOID RESEARCH238
that of some of the classical cannabinoids. The low potency and short-lasting activity in vivo was recognised to be due to AEA susceptibility toenzymatic hydrolysis by FAAH (see above). Anandamide SAR studies canbe split into three categories; modifications to the fatty acid chain, mod-ifications to the carboxamide group and modifications to the polar headgroup.
The fatty acid chain
In general for the C20 series, maximal activity is achieved with amides ofarachidonic acid (1) [81], mead acid (199) [149], and dihomo-g-linoleic acid(200) [150] (see Table 6.18). Decreasing the unsaturation (201), (202), orabolishment of the n-pentyl chain (203) [150] led to less active or inactivecompounds. Variable results were seen with longer chains. The C22:4 n-6analogue (204) is as active as AEA (1) whereas the C22:6 n-3 analogue (205)is less active than the C20:5 n-3 analogue (203) [150]. Replacement of thedouble bonds with triple bonds (206) resulted in loss of activity [150] (seeTable 6.18). Forcing the fatty acid chain into a hairpin conformation bycyclisation (207) also resulted in inactive compounds [151].In a study looking at oxygenated metabolites of AEA and their inter-
action with the cannabinoid system, a series of hydroxylated alkyl chainswas prepared using different lipoxygenases as biocatalysts [152–154]. Of theseven AEA derivatives prepared, only the 5R-hydroxy (208), 12S-hydroxy(209) and 15S-hydroxy (210) derivatives had any affinity for the CB1 re-ceptor. Interestingly, the 13S-hydroxy compound (211) that was inactive atthe CB1 receptor displayed some affinity for the CB2 receptor [152] (seeTable 6.18).To improve the metabolic stability of AEA analogues, a number of mod-
ifications to the C2 site of AEA have been explored [150, 155, 156]. Intro-duction of a methyl (212) or dimethyl group (213) in this position gave amodest increase in binding affinity and enhanced metabolic stability (cf.with and without PMSF). In contrast, use of an ethyl (214) or isopropylgroup (215) resulted in a drop in binding affinity. Introduction of a chiralmethyl group into the C-2 position had little effect on binding affinity butthe R-isomer (216) had improved metabolic stability compared to itsenantiomer (217) [157]. In addition, both (216) and (217) retained theirselectivity over the CB2 receptor (see Table 6.18).Several groups have suggested that the C16–C20 portion of AEA and the
C-3 pentyl side chain of (67) may play a similar role in the binding site of theCB receptor [158–161]. To explore this, a number of groups have substitutedthe C16–C20 pentyl side chain with a 10,10-dimethylheptyl chain, a widely
Table 6.18 AEA DERIVATIVES – THE FATTY ACID CHAIN SARRCONH(CH2)2OH
Cpd. R CB1 (nM)+PMSF
CB1 (nM)�PMSF
CB2 (nM) Relative CB1 Ki
(Ki/KiAEA)Ref.
(1)
(CH2)3-4
n-C4H9
89 5,400 371 1 [81]
(199)
(CH2)3-3
n-C7H15753 1,810 0.96 [149]
(200)
(CH2)6-3
n-C4H9
53 1.4 [150]
(201)
(CH2)9-2
n-C4H9
1,500 38 [150]
(202)(CH2)9-n-C8H17
>1,000 >26 [150]
(203)
(CH2)3-5
Me162 4 [150]
(204)
(CH2)5-4
n-C4H9
34 0.9 [150]
(205)
(CH2)2-6
Me324 8.3 [150]
(206)(CH2)3-
4n-C4H9
No activity [150]
(Continued )
J.ADAM
ETAL.
239
(207) O
n-Bu
(CH2)3-
OH
No activity [151]
(208)
3
OH
(CH2)3-n-C4H9
680 710 7.6 [152]
(209) n-C5H11
2
OH
(CH2)3-150 500 1.7 [152]
(210)
3
n-C4H9
OH
(CH2)3-600 >1,000 6.7 [152]
Table 6.18 CONTINUED
Cpd. R CB1 (nM)+PMSF
CB1 (nM)�PMSF
CB2 (nM) Relative CB1 Ki
(Ki/KiAEA)Ref.
RECENTPROGRESSIN
CANNABIN
OID
RESEARCH
240
(211) (CH2)7-
2
n-C5H11
OH
>1,000 600 >11 [152]
(212)
4 Me
n-C4H9
53 137 0.6 [155]
(213)
4 Me
Men-C4H9
47 41 0.5 [155]
(214)
4 Et
n-C4H9
461 285 5.2 [155]
(215)
4 i-Pr
n-C4H9
4,030 2,920 45 [155]
(216)
4 Me
n-C4H9
54 457 4,905 0.7 [157]
(217)
4 Me
n-C4H9
35 3,941 4,259 0.45 [157]
(218)
4
n-C6H13
Me
Me(CH2)3-
7 0.08 [158]
J.ADAM
ETAL.
241
RECENT PROGRESS IN CANNABINOID RESEARCH242
used replacement in classical cannabinoids (see above). While the replace-ment of this portion of the molecule did not result in as great an enhance-ment as with the classical cannabinoids, the dimethylheptyl derivative (218)(Table 6.18) did produce a 13-fold enhancement in CB1 binding affinitycompared with AEA [158].In summary, the receptor recognises unsaturated and partially saturated
fatty acyl chains 20–22 carbons long. In addition to the receptor-bindingstudies detailed here, a large body of work examining the conformat-ional requirements and potential folding arrangements of the fatty acidchain via a number of different computational methods have been published[162].
The carboxamide group
A limited series of amide replacements have been examined includingthioamides (219) [163], reverse amides (220) [164], heterocycles (221) [164],ethers (222) [165], carbamates (223), (224) [165], ureas (225) [165] andketones (226) [166] (see Table 6.19). In general, replacement of the amideresults in a loss of binding affinity compared to AEA. In some cases, re-placement of the amide results in increased stability with regard to hydro-lysis by FAAH [164, 165].
Table 6.19 AEA DERIVATIVES – THE CARBOXAMIDE GROUP SAR
The most extensive investigation of SAR has been carried out in this regionof AEA. The following SAR has been established. In general, a secondaryamide is optimal. The primary amide is inactive (227) and tertiary amidesare less active than AEA or inactive (228), (229) [37, 150, 163]. The hydroxylgroup is not required for activity and a wide range of replacements aretolerated such as hydrogen (230), small alkyl (231), halogen (232), (233),ether (234), phosphate (235) or aromatic (236) (see Table 6.20). In partic-ular, the chloroethyl derivative (232) was shown to have 11-fold improvedaffinity compared to AEA.The introduction of a methyl into the head group at the C10-position and
in particular the R-isomer (237) resulted in 4-fold higher affinity than AEA,while the S- isomer (238) had 2-fold lower affinity than AEA [48, 157], theR-isomer (237) was also resistant to enzymatic hydrolysis [157]. However,introduction of larger alkyl groups into the C10 position such as the i-butylanalogue (239) leads to a large drop in activity [155]. Methylation of the C20
position also leads to improved affinity compared to AEA. In particular, theR-isomer (240) has 4-fold enhanced affinity; this derivative is also stable toenzymatic hydrolysis by FAAH [48] (see Table 6.20).Extension of the head group by insertion of a methylene group is tol-
erated, the N-propanol derivative (241) having a slight increase in bindingaffinity compared to AEA. Further extension of the carbon chain, such asthe butyl derivative (242), led to a decrease in activity [150, 151].Replacement of the ethanolamine head group is also well tolerated. Sub-
stitution with a cyclopropyl (243) [37], allyl (244) or propargyl group (245)[164] all led to an increase in binding affinity compared to AEA. Replace-ment of the head group with aromatics is also allowed. The phenyl deriv-ative (246) retains affinity at the CB1 receptor [37], whereas the 2-substitutedN-methyl pyrrole (247) has a 2-fold improved affinity compared to AEA[167]. Interestingly, the 3-substituted furan derivative (23) that has micro-molar affinity for the AEA transporter (see above) does not bind to the CB1
receptor, but has good affinity for the CB2 receptor [167]. These results aresummarised in Table 6.20.Overall, these results suggest that the hydroxyl group is not vital for
binding and that both hydrophobic and hydrophilic head groups canbe accommodated. The cavity in which the head group binds is relativelysmall as only modest variations in this position lead to high-affinity lig-ands. With regard to CB2 selectivity, very few reports have looked in detailat the requirements for CB2 binding in AEA derivatives and most ligandssynthesised to date have tended to be relatively selective for the CB1
receptor.
Table 6.20 AEA DERIVATIVES – THE POLAR HEAD GROUP SAR
(CH2)3COR4
n-C4H9
Cpd. R CB1 (nM) +PMSF CB1 (nM) - PMSF CB2 (nM) Relative CB1 Ki (Ki/Ki AEA) Ref.
2-AG (2) was first isolated in 1995 from canine gut [14] and rat brains [15] andshown to be an endogenous CB ligand [14]. (2) is present in the rat brain inamounts 170–1,000 times greater than AEA [15, 168] and has been shown to bea potent full agonist at both the CB1 and CB2 receptors (CB1: K i ¼ 472 nMand CB2: K i ¼ 1; 400 nM) [14, 169]. In addition, this endocannabinoid hastypical cannabinoid-like activities including decreased spontaneous motor ac-tivity, immobility and production of hypothermia and analgesia [14]. TheFAAH enzyme has been reported to hydrolyse (2) four times faster than itshydrolysis of AEA [170]. In addition, it has been proposed that the majorenzymatic hydrolysis pathway of (2) occurs via the monoacylglycerol lipaserather than FAAH [171, 172].Compound (2) has been reported to induce a rapid transient increase of
intracellular free Ca2+ concentration in NG108-15 and HL-60 cells througha cannabinoid receptor-dependent mechanism [173, 174]. Furthermore, theCa2+ increase in HL-60 cells appeared to be CB2 mediated as the effectcould be blocked with a CB2 antagonist but not a CB1 antagonist [174]. Verylittle work has been carried out on the SAR of this endocannabinoid. Ingeneral, for the CB1 receptor, arachidonic acid was found to be the preferredfatty acid moiety, although some partially saturated structures had almostcomparable activities [173]. It appears that the presence of a double bond atthe D5-position is crucial for activity. With respect to the head group, the2-glycerol isomer is preferable over both the 1- and 3-analogues. The estermoiety can be replaced by a ketone but agonistic activity in NG108-15 cellsdrops approximately 100-fold [175]. With regard to the CB2 receptor, muchof the SAR overlaps with the results obtained for the CB1 receptor with theexception that glycerol esters of C22 fatty acids do not show appreciableactivity in the CB1 assay, but do show appreciable activity in the CB2 assay[173].
Noladin ether
Noladin ether (3) was recently isolated from porcine brain [16] and found tobind to the CB1 receptor (K i ¼ 21:2 nM), to bind weakly to the CB2 receptor(Ki>3 mM) and it causes typical cannabinoid-like effects such as sedation,hypothermia, intestinal immobility and mild antinociception in mice [16].This endocannabinoid had previously been synthesised independently byboth Mechoulam and co-workers [176] and Sugiura et al. [173]. SAR studiesof this endocannabinoid are lacking in the literature, however, a recentpublication highlighted the importance of the tetra-unsaturated C20 chain
J. ADAM ET AL. 247
for high affinity at the CB1 receptor, increased saturation or replacement ofthe double bonds (with, for example, a cyclopropyl) had a detrimental effecton binding affinity [177].
INDOLE AND ITS DERIVATIVES
Indoles
The discovery and cloning of the CB1 and CB2 receptors has opened the wayfor the pharmaceutical industry to identify cannabinoid agonists with com-pletely new templates. However, the first novel class of cannabinoid agonists(i.e. those not derived from THC) was discovered accidentally by theSterling-Winthrop Research Group while studying conformationally re-strained analogues of pravadoline (248) [178]. These pravadoline analoguesdisplayed reduced ability to behave as non-steroidal anti-inflammatoryagents that inhibit cyclooxygenase but increased ability to bind to the CB1
In this initial study they found that the 4-methoxyphenyl ring could bereplaced by a number of other aromatic rings, in particular, the 1-naphthylring was found to impart good potency. In addition, small substituents inthe indole 2-position were favoured and importantly that potency resided inonly one enantiomer of the aminoalkyl indole (AAI) (see Table 6.21). Onecompound of particular importance synthesised at this time is WIN 55,212-2(254a), a potent agonist at both the CB1 and CB2 receptors. Although thiscompound was not developed as a drug, it has been widely used as a phar-macological tool in cannabinoid research. The Sterling-Winthrop researchgroup [179] went on to carry out an extensive SAR study on unconstrainedaminoalkyl indoles with over 100 analogues synthesised and tested, and
Table 6.21 AMINOALKYL INDOLES – SAR OF THE BENZOXAZINE CORE [178]
N
O N
O
O
R
Ar
Cpd. Ar R MVD activity IC50 (nM)a CB1 IC50 (nM)
(249) 4-MeO-C6H4 H 44.5 249(250) 4-MeO-C6H4 Me 123 152(251) 4-MeO-C6H4 Et 28% @ 10 mM 27% @ 1mM(252) 2-F-C6H4 Me 76 1,426(253) 1-Naphthyl H 2 7.37(254) 1-Naphthyl Me 6 5.56(255) 5-Quinolinyl Me 21 132.5(R)-(+)-(254a) 0.43 2.77(S)-(�)-(254b) >10,000 8,002
aMouse vas deferens inhibitory activity.
RECENT PROGRESS IN CANNABINOID RESEARCH248
extended their previously reported SAR to include variations in the natureof the aminoalkyl substituent, the substituents on the naphthyl group andsubstituents on the indole nucleus. In general, similar trends were seen withregard to the key structural features required for cannabinoid activity i.e.an aromatic (bi)aroyl ring in the 3-position, a small substituent in the2-position and a cyclic aminoethyl substituent in the 1-position.Following on from this they demonstrated that the aminoethyl side chain
could also be replaced (see Table 6.22) [180], the most potent ligands bearinga naphthoyl moiety. In particular, the piperidinyl analogue (258) was foundto have a high affinity in the binding assay and potency in the mouse vasdeferens assay.In an attempt to rationalise these results with a pharmacophore model
that would fit both the classical cannabinoids and AAIs, Huffmann and co-workers [181] suggested that the ketonic oxygen of WIN 55,212-2 wasaligned with the phenolic hydroxyl of (67). In this arrangement, the naph-thyl moiety of the AAIs would overlay the cyclohexyl and pyran rings of theclassical cannabinoid structure. The indole nitrogen and substituent at-tached to it would then be placed in a corresponding position to the alkylchain on C3. To test this hypothesis, Huffman designed a number of AAIsin which the aminoethyl chain was replaced with other substituents and in
Table 6.22 AMINOALKYL INDOLES – SAR OF THE N-1 SUBSTITUENT [180]
NR'
O
R
Cpd. R R0 MVD activity IC50 (nM) CB1 IC50 (nM)
(256)
N
Me
-CH2
Me 2.4 6.5
(257)
N-CH2
Me
Me 1.2 5.4
(258)
N-CH2
Me
H 0.47 1.2
J. ADAM ET AL. 249
particular, carbon chains. Short side chains resulted in inactive compounds,whereas chains with four to six carbons produced best results [181] (seeTable 6.23). In particular, the n-pentyl substituent (263) was optimal ex-hibiting 2-fold greater affinity than WIN 55,212-2 and 4-fold greater affinitythan (67) in the in vitro binding assay. In addition, (263) also displayedpotent in vivo activity.Following on from this, and to further exemplify this pharmacophore
model, Huffman [182] described a novel hybrid structure that combined thehydroxydibenzopyran ring of THC and the indole moiety of the AAIs intoone molecule. It was found that the hybrid molecule (270) had a similaraffinity (19 nM) for the CB1 receptor in vitro as (67) (41 nM). The compoundwas also active in vivo in the mouse tetrad model of cannabimimetic activityand had comparable potency to (67) [182].An alternative binding model has been proposed by Xie et al. [183] using
2D-NMR spectroscopy and molecular modelling with (254a) and 9-nor-9b-OH-hexahydrocannabinol (HHC). In this case it is suggested that the ke-tonic group of (254a) corresponds to the phenolic hydroxy group, thenaphthyl moiety to the C3 alkyl side chain and the nitrogen of the morph-olinyl group to the C9 hydroxyl of HHC. A similar structural alignment has
a‘‘No max’’ indicates that the compound produced only slightly greater than 50% of thepresumed maximal effect.bWIN 55,212-2.cD9-THC.
RECENT PROGRESS IN CANNABINOID RESEARCH250
been proposed by Razdan and co-workers [184]. To test the hypothesis, aseries of 4-substituted indoles was prepared containing the key structuralfeatures of the AAIs except that the naphthyl substituent was transposedfrom position 3 to position 4 via an oxygen linker. A total of nine com-pounds were synthesised and tested. Alkyl chain-substituted compoundsfailed to show any affinity either in vitro or in vivo (see Table 6.24(271)–(273)). On the other hand, introduction of a naphthyl moiety eitherdirectly or via a number of different linkers (274)–(279), in particular, aketomethylene group (277), led to compounds with moderate potency invitro and in vivo [184].The validity of pharmacophore models that overlay the classical can-
nabinoids and AAIs has been brought into question by recent mutation workon the CB1 receptor. It was found that mutation of Lys-192 in the third
Table 6.24 AMINOALKYL INDOLES – SAR 4-SUBSTITUTED INDOLES [184]
transmembrane domain to an Ala resulted in a complete loss of binding forthe classical cannabinoid (165), in contrast to this; binding of (254a) was onlyslightly affected [185]. These results would suggest that the AAIs interact withthe CB1 receptor in a somewhat different fashion from the dibenzopyrancannabinoids.One alternative that has been proposed is that the main binding arrange-
ment of the AAIs to the receptor is through aromatic stacking. To explorethis proposal a number of naphthyl methanes have been prepared [186].These compounds lack the ketone functionality that is thought to play thesame role as the phenoxy group in the classical cannabinoids. Hence there isno viable possibility for hydrogen-bonding interactions between the receptorand the ligand at this position. Substitutions in both the 4-position of thenaphthyl and the 2-position of the indole were explored (see Table 6.25). Inthe case of the unsubstituted indole, binding affinity was maintained withonly a small drop in binding affinity being observed compared to theirnaphthoyl congeners (280)–(282). In contrast to this, substitution in the
Table 6.25 AMINOALKYL INDOLES – SAR OF SUBSTITUTED NAPHTHYLMETH-ANES [186]
R'
N
C5H11
R
Cpd. R R0 CB1 Ki (nM) Cpd. R R0 CB1 Ki (nM)
(280) H H 22 (284) Me Me 127(281) H Me 23 (285) Me OMe 323(282) H OMe 17 (254a)a 9.9(283) Me H 151 (67)b 41
aWIN 55,212-2.bD9-THC.
RECENT PROGRESS IN CANNABINOID RESEARCH252
2-position of the indole had a marked effect on binding, with affinity drop-ping up to 19-fold (cf. (282) and (285)).The high binding affinities obtained for the unsubstituted indole com-
pounds thus support the hypothesis that H-bonding may not be the mainbinding feature of the AAIs and potentially aromatic stacking may be thedominant interaction. To explain the sudden drop in potency for the2-methyl substituted indoles, the authors initiated a molecular modellingand receptor-docking study. From this study, they conclude that the lossin binding affinity is due to the loss of an aromatic stacking interaction[186].More recently, the utility of the indole group as a scaffold for cannabinoid
agonists has been demonstrated by a number of new patent applicationsappearing in the literature (286)–(290) [187–190]. Of particular note is com-pound (286) that is reported to have 18-fold selectivity for the CB1 receptor(CB1: K i ¼ 0:08 nM; CB2: K i ¼ 1:44 nM). In addition to the indole scaffold,a number of patent applications by AstraZeneca claim indole-like scaffoldssuch as benzimidazoles (289) [191–193] and azaindoles (290) [194]. Althoughthese compounds bind to both CB1 and CB2 receptors, the inventors claimthat they may be useful in treating diseases without the associated CNS sideeffects.
J. ADAM ET AL. 253
NN
O
NN
Ph
N
O
(CH2)5F
I
N
NH
O
OMe
O
Ph
N
O
MeO(288)(286) (287)
N N
Ph
t-BuCONH
OEt
N
Nt-Bu
O
(F3CCH2)2N
(290)(289)
Pyrrole and its derivatives
In Huffman’s [181] overlay model of the classical cannabinoids and AAIs,the benzenoid portion of the indole ring did not participate in the overlapand only the pyrrole ring was involved. In order to investigate this, a ho-mologous series of pyrroles was prepared and their pharmacology examined(see Table 6.26). In analogy to the indoles, the best results were obtainedwith the n-pentyl analogue (295), although this compound was approxi-mately 10-fold less potent than its indole analogue.Following on from this Tarzia et al. [195] designed and synthesised a
range of pyrrole compounds based on the Huffman overlay. A range ofsubstituents was explored around the pyrrole ring, the selections are detailedin Table 6.27. As seen with the AAIs, replacement of the naphthyl moietywith a monocyclic aromatic ring was detrimental to affinity. In particular,replacement with a phenyl ring resulted in complete loss of activity (299).A similar result was seen when the naphthyl moiety was replaced with analiphatic chain (301). Replacement of the ketone group with an amide istolerated, albeit with a loss in binding affinity. In addition, depending on the
(291) Me >10,000 106 No maxa 53.3(292) Et >10,000 84 No maxa 77.2(293) n-Pr >10,000 86 81.8 90.1(294) n-Bu 666 No maxa No maxa >108(295) n-Pentyl 87 3.6 1.2 78.8(296) n-Hexyl 399 8.8 9.6 62.8(297) n-Heptyl 309 11 9.7 52.1(254a)b 24 0.19 1.4 1.5(67)c 41 0.92 2.7 2.5
a‘‘No max’’ indicates that the compound produced only slightly greater than 50% of thepresumed maximal effect.bWIN 55,212-2.cD9-THC.
Table 6.27 PYRROLE DERIVATIVES – SAR OF SUBSTITUTED PYRROLES [195]
N
n-C5H11
MeMe
COR1
R2
Cpd. R1 R2 CB1 Ki (nM) CB2 EC50 (nM)
(298) 1-Naphthyl H 45 10(299) Ph H >1,000 >1,000(300) 1-Naphthyl Br 13 6.8(301) HO(CH2)3 H >3,000 >10,000(302) o-(Ac)C6H4NH H 367 >1,000(303) c-HexylNH H 415 483
RECENT PROGRESS IN CANNABINOID RESEARCH254
J. ADAM ET AL. 255
amide substitution an interesting selectivity profile was seen, for example,the substituted aniline compound (302) gave a moderate binding affinity atthe CB1 receptor (367 nM) but lost all affinity at the CB2 receptor(>1,000 nM). In contrast, the aminocyclohexyl amide (303) retained affin-ity at both receptors albeit reduced compared to the naphthyl analogues.The best results were obtained with the 4-bromo substituted compound(300) giving good affinity at both the CB1 (13 nM) and CB2 (6.8 nM) re-ceptor. The substituted pyrrole (300) was also tested for intrinsic activity atthe CB1 receptor using [35S]-GTPgS binding in rat cerebellar membranesand found to behave as a full agonist with an EC50 value of 140 nM.The pyrazole scaffold is usually associated with cannabinoid antagonists/
inverse agonists and in particular, rimonabant (382) (see below). However,recent work examining a series of (382) analogues for antagonist activitygave rise to a number of compounds that also displayed cannabimimeticeffects in vivo [196]. In particular, replacement of the N-piperazine in (382)with carbon chains that more directly correspond to the lipophilic side chainof (67) gave compounds exhibiting agonistic properties in the mouse triadmodel of cannabimimetic activity (see Table 6.28). The best results wereobtained with 2-fluoroethyl amide (306) that had a relatively low bindingaffinity (852 nM), but had a moderate potency in vivo [approximately 5-foldless active than (67)].
Indenes
In the early clinical trials of Pravadoline (248), the Sterling-Winthrop Re-search group observed CNS side effects. In an effort to circumvent theseissues, indenes were explored as an alternative core [197]. The rationalebehind this approach came from the observation that sulindac (308), anindene analogue of indomethacin (309), had anti-inflammatory propertiescomparable to (309) but without the CNS-associated side effects.
SOMe
Me
CO2H
F
NMe
CO2H
MeO
O Cl
(308) Sulindac (309) Indomethacin
Table 6.28 PYRROLE DERIVATIVES – SAR OF SUBSTITUTED PYRAZOLES [196]
A series of 11 compounds was prepared with and without a 2-Me subs-tituent. Owing to the method of preparation, the 2-Me substituted com-pounds were prepared as a mixture of stereoisomers (4:1; E:Z), whereas theunsubstituted compounds had predominantly E configuration (>95%). Itwas found that, as with the AAIs, naphthyl-substituted derivatives werepotent agonists in vitro and in vivo (see Table 6.29). Following on from thisReggio et al. [198] extended this work by preparing the stereo-defined in-denes in a bid to identify the bioactive conformation of the AAIs. Throughconformational analysis it had been established that the indene E-isomermimics the s-trans conformation of (254a) while the Z-isomer mimics thes-cis conformation (Figure 6.2). Measurement in CB1/CB2 binding assaysrevealed that the E-naphthylindene isomer had significantly higher bindingaffinity than the Z-isomer (see Table 6.29). This would then suggest that thes-trans conformer of (254a) and related AAIs is the bioactive conformer atthe cannabinoid receptors.
Table 6.29 INDENES – SAR OF SUBSTITUTED INDENES
N
O
R
R'
Cpd. R R0 CB1 IC50 (nM) CB2 Ki (nM) Ref.
(310) Ph H, >95% E 33%@1mM [197](311) 1-Naphthyl H, >95% E 1 [197](312) 1-Naphthyl Me, 4:1 E:Z 10 [197](313) 1-Naphthyl H, 100% E 2.72 2.72 [198](314) 1-Naphthyl H, 100% Z 148 132 [198](315) 1-Naphthyl Me, 100% E 2.89 2.05 [198](316) 1-Naphthyl Me, 100% Z 1945 658 [198](254a)a 5.56 [197]
2.48 0.28 [198]
aWIN 55,212-2.
N
O
N
O
O
N
O
N
O
O
N
O
N
O
s-cis s-trans Z-isomer E-isomer
Fig. 6.2 Conformational analysis of indene isomers.
J. ADAM ET AL. 257
RECENT PROGRESS IN CANNABINOID RESEARCH258
Other structures
A number of new scaffolds unrelated to the classical/non-classical cannabinoidsand AAIs have been reported in the literature to deliver cannabinoid agonists.Bayer [199–203] has claimed in a series of patents a number of aryl
sulfonyl esters as cannabinoid agonists for the treatment and prophylaxis ofneurodegenerative diseases. Following on from this a number of publica-tions detailing the in vitro and in vivo profiles on two of these compounds,BAY 38-7271 (317) and BAY 59-3074 (318), have been published.Compound (317) was found to have a high affinity for rat and human CB1
receptors (K i ¼ 0:46 and 1.09 nM, respectively), as well as for recombinanthuman CB1 and CB2 receptors (K i ¼ 1:85 and 5.96 nM, respectively) andalso exhibited full agonist activity using the [35S]-GTPgS technique with anEC50 value at human CB1 receptors of 15.8 nM (no data reported for theCB2 receptor). (317) also displayed typical cannabinoid-like behaviour inthe rat hypothermia model and drug discrimination assays which could bereversed by the selective CB1 antagonist, rimonabant (382). In addition,(317) also proved effective in a rat traumatic brain injury model reducinginfarct volume by 70% at 100 ng/kg/h infusion starting immediately aftersubdural haematoma. It also reduced infarct volume (27% at 1,000 ng/kg/h)in a rat model of focal cerebral ischaemia. Phase I studies concluded that(317) was safe and well tolerated when administered intravenously for either1 or 24 h in humans and (317) is currently being developed for the treatmentof traumatic brain injury and cerebral ischaemia [204, 205].The structurally related compound BAY 59-3074 (318) also has high
affinity at human CB1/CB2 receptors (K i ¼ 48:3 and 45.5 nM, respectively).However, and in contrast to (317), (318) exhibited partial agonist activity athCB1 using the [35S]-GTPgS technique with an EC50 of 142.7 nM [206, 207].The compound also behaved as a partial agonist at hCB2 receptors with anEC50 of 15.8 nM. BAY 59-3074 (318) also displayed typical cannabinoidactivity in vivo in rat hypothermia and drug discrimination assays that couldbe reversed by the selective CB1 antagonist rimonabant (382). At doses of0.3–3mg/kg p.o., (318) induced antihyperalgesic and antiallodynic effectsagainst thermal or mechanical stimuli in rat models of chronic neuropathyand inflammatory pain. The antiallodynic efficacy was maintained for 2weeks of daily administration, and no withdrawal symptoms were seenduring the 2 days after the last dosing regimen [206, 207].Novartis AG has filed a patent application on novel naphthalene deriv-
atives as potent cannabinoid agonists, especially at the CB1 receptor [208].One compound was specifically claimed, the naphthalene derivative (319),which exhibited CB1 binding with a Ki value of 15 nM. This compound wasalso active in an in vivo model of neuropathic pain, reversing hyperalgesia
J. ADAM ET AL. 259
caused by partial ligation of the sciatic nerve in Wistar rats after oral dosingwith an ED50 of 0.18mg/kg.In addition, Novartis filed a patent application on a series of quinazolines
as cannabinoid agonists [209]. Compound (320) is one of the two com-pounds specifically claimed and exhibited CB1 and CB2 binding with Ki
values of 34 and 11 nM, respectively. It was shown to be a full agonist at theCB1 receptor with an EC50 of 132 nM (no functional data for the CB2
receptor). Compound (320) was also active in the neuropathic pain modeldescribed above with an ED50 of 0.5mg/kg after oral dosing.AstraZeneca has filed a patent application on novel bis-aryl compounds
as CB1/CB2 agonists that lack CNS penetration and thus avoid the un-wanted side effects associated with activation of central CB1 receptors [210].Over 100 compounds are specifically claimed e.g. (321). Compounds weretested in receptor binding assays using human CB1 and CB2 receptor prep-arations. Respective Ki values were in the ranges 50–5,000 and 15–2,800 nM,although no specific data were presented.Shionogi [211] has filed a patent application on a series of thiazine de-
rivatives; no compounds are specifically claimed. The thiazine derivative(322) was active in vivo in the formalin-induced licking-and-biting model inICR mice with an ED50 of 1.5mg/kg after oral dosing.
O OSO2(CH2)3CF3
HO
OCN
F3C OSO2(CH2)3CF3 O
On-C5H11
(317) BAY 38-7271 (318) BAY 59-3074 (319)
O
HO(CH2)2NHN
N
O
OSO2NHMe
Me
Me
EtO2C
Me
N
Me OH
Ph
N
S N
S
SMe
(320) (321) (322)
CB2 AGONISTS
Receptor subtype selective CB2 agonists are seen as potential candidates forthe treatment of a variety of diseases, including pain-related indications. Thepromise of useful therapeutic effects without unwanted CNS side effectsmakes the development of CB2 selective compounds a particularly attractive
RECENT PROGRESS IN CANNABINOID RESEARCH260
proposition. Several classes of CB2 selective agonists, including classicalcannabinoids, resorcinols, indoles, indazoles and other heterocyclic deriv-atives and their SAR studies have been reported.
CB2 SELECTIVE CLASSICAL CANNABINOIDS
Of the three main pharmacophoric elements in the classical cannabinoidtemplate discussed previously, the phenolic hydroxyl group at C1 has beenshown to be the most important for CB2 selectivity. In 1996, a group atMerck Frosst [212] and the Huffman group [213] independently demon-strated that removal of this hydroxyl group in the 10,10-dimethylheptyl seriesleads to compounds with high CB2 receptor affinity and between 8- and 40-fold selectivity for CB2 over CB1 binding affinity (Table 6.30). The MerckFrosst paper further describes modification of the phenol to a methoxygroup, resulting in higher CB2 selectivity [212].
The CB2 receptor has been shown to tolerate shorter C3 side chains thanthe CB1 receptor and the Huffman group has exploited this in combinationwith C1 modifications to develop highly selective CB2 receptor ligands (Ta-bles 6.31 and 6.32) [214, 215].The 11-hydroxy analogues of the 1010-dimethyl compounds in Tables 6.31
and 6.32 were also prepared by the Huffman group. In general, theseshowed higher affinity for both CB1 and CB2 receptors than the simplemethyl analogues, but reduced CB2 selectivity (data not shown) [215].
Table 6.30 1-DEOXY AND 1-METHOXY D8-THC DERIVATIVES
Compounds where the C1 hydroxyl group is linked to the C2 position bya cyclic ether link, with a range of different groups in the C9 position, wereprepared by Reggio et al. [216]. These compounds showed only modest CB2
selectivity. Compound (341) was the most potent and selective with a bind-ing affinity (Ki) of 5.8 nM at CB2 and 26 nM at CB1.A patent application from Merck Frosst, published in 1997, discloses CB2
selective compounds with modified core structures where the pyran oxygenis replaced by nitrogen, such as (342), which has a Ki of around 64 nM atCB2 and around 25,000 nM at CB1 [217].
O
OH
MeMe n-C5H11
O
N
Me
MeMe OMe
Me(341)
(342)
INDOLES AND INDAZOLES
Indomethacin morpholinyl amide (343) was found as a weak but CB2 se-lective agonist through a topological similarity search using WIN 55,212-2(254a) as the template [218]. The binding affinity (Ki) of (343) was 435 and>20,000 nM for hCB2 and hCB1, respectively. A subsequent SAR studyrevealed that N-(2,3-dichlorobenzoyl) and N-naphthoyl indole derivativesshowed improved activity. Further optimisation of the substituents on theindole C3-position was also performed, and the more selective and potentCB2 agonists, L-768,242 (344) and L-759,787 (345) were discovered. Thesecompounds showed >100-fold CB2 selectivity with significant potency (Ki
During the study of N-alkylindole derivatives as cannabinoid ligands,JWH-015 (346) was found as a potent CB2 selective agonist, Ki (CB2)13.8 nM with modest selectivity, ðCB1Þ : ðCB2Þ ¼ 28 [81]. For the optimi-sation study of (346), more than 40 indoles were prepared and their CB1 andCB2 receptor affinities were determined [219]. In most cases, N-pentyl in-doles showed significant activity but low receptor subtype selectivity,whereas N-propyl derivatives had better selectivity for CB2. The SAR of thesubstituents on the naphthyl ring was also studied, and it is noteworthy thatintroduction of a methoxy group in the 2-position of the naphthyl ringreduced the binding affinity only for the CB1 receptor, even though thecompound bears a pentyl group on the indole ring. As a result, three CB2
selective compounds, JWH-120 (347), JWH-151 (348) and JWH-267 (349)were found; JWH-151 was a full agonist whereas JWH-120 and JWH-267exhibited partial activity in GTPgS binding assay.
The group of Bristol-Myers Squibb found a C3 amido-indole (350) as alead compound for their CB2 agonist program [220]. Compound (350)demonstrated moderate binding affinity for CB2 (Ki 250 nM), which wasimproved dramatically to a Ki of 8 nM by the introduction of methoxygroup on the C7 of the indole core. The 7-methoxy indole (351) showedgood receptor subtype selectivity, CB1 : CB2 ¼ 500, however, the methylester on the phenylalanine moiety was susceptible to microsomal hydrolysis.In an effort to discover non-ester CB2 agonists, compound (352), having a(1S)-fenchyl group instead of the phenylalanine methyl ester, was prepared.The introduction of a larger alkyl group onto the C2 position of the indolering decreased the CB2 binding affinity (H>Me>Et>n-Pr; Table 6.33).The non-substituted compound (353) showed dose-dependent inhibitoryactivity in an in vivo anti-inflammatory model [lipopolysaccharide (LPS)-induced TNF-a production in mice] after i.v. administration with an ED50
of 5mg/kg. Unfortunately, (353) was inactive after oral administration, and
Table 6.33 1-AMINOALKYLINDOLE ANALOGUES
N
NH
O
NO
R
Me
Me
Me
OMe
Cpd. R CB1 Ki (nM) CB2 Ki (nM) CB1/CB2 Ref.
(352) Me 10,000 30 333 [220](353) H 245 11 22 [221](354) Et 103 [220](355) n-Pr 6% @ 100nM [220]
RECENT PROGRESS IN CANNABINOID RESEARCH264
the poor oral efficacy was thought to be caused by metabolical instability,e.g. de-alkylation at the N1 position.
N
NH
O
N
O
Ph
CO2Me
Me
R
(350) R = H, Ki(CB2) = 250 nM
(351) R = OMe, Ki(CB2) = 8nM, CB1/CB2 = 500
It was hypothesised that the increased activity of indoles bearing smallergroups at the C2 position described above would be derived from the con-formational preference of conformer A over conformer B as a result of steric
N
NH
O
N
O
R
Me
Me
Me
OMe
N
ONH
N
O
R
OMe
MeMe
Me
Amide conformer A Amide conformer B
Fig. 6.3 Structures of amide conformers A and B.
J. ADAM ET AL. 265
effects (Figure 6.3) [221]. Based on this hypothesis, indazoles and indo-lopyridones, such as (356) and (357) were designed. For (356), it was pre-dicted that conformer A would be preferred over conformer B, as the resultof an intramolecular stereoelectronic interaction. Actually, (356) was morepotent than all of the indole derivatives mentioned above. The conforma-tionally constrained indolopyridone (357) had the most potent activity in theseries [K iðCB2Þ ¼ 1:0 nM]. In the in vivo model, (357) exhibited inhibitionactivity against LPS-induced TNF-a release both after i.v. and oral admin-istration. CB2-selective indazole derivatives, such as (358), were also re-ported from the research group of the University of Connecticut [222]. TheKi values of (358) for CB2 and CB1 were 0.15 and 6.84 nM, respectively.Another type of C3 amido-indole, exemplified by (359), was disclosed bySanofi Synthelabo [223].
NN
NO
N
O
Me
Me
Me
OMe
H
N
NO
N
O
Me
Me
Me
OMe
(356) Ki(CB2) = 2 nM (357) Ki(CB2) = 1 nM
RECENT PROGRESS IN CANNABINOID RESEARCH266
NN
NH
O
N
ON
(CH2)3NHSO2MeCl
NHO
Me
MeMe
Me
(358) (359)
N
O
I
NMe
NO2
N
SO
O
F
S
OO
NHSO2Me
Me
(360) AM1241 (361)
AM1241 (360) exhibited high affinity and selectivity for CB2 [K iðCB1Þ ¼
280 nM, Ki ðCB2Þ ¼ 3:4 nM]. (360) Dose dependently inhibited experimentalneuropathic pain in a spinal nerve ligation-induced tactile and thermal hyper-sensitivity model [224]. Other indole derivatives bearing sulfonamide moietieson the side chain, such as compound (361), were disclosed [225]. Though 67derivatives including pyridyl and other heteroaromatics instead of the indolecore were listed, no specific biological data were shown.
RESORCINOL DERIVATIVES
As previously discussed, phenol derivatives bearing a six-membered carbo-cyclic ring next to the hydroxy group of the phenol represent a major classof cannabinoid ligands. These compounds correspond to pyran ring-openedanalogues of (67). Several efforts have been made to find CB2 selectiveagonists among the series.
J. ADAM ET AL. 267
MeO
CH2OH
OMe
n-C6H13
Me Me
Me
Me
HU-308 (362)
One of the conspicuous resorcinols is HU-308 (362), which is a CB2-specific agonist; this compound does not bind to CB1 (Ki>10 mM), but has asignificant affinity for CB2 (K i ¼ 22:7 nM) [226]. HU-308 elicited analgesicactivity in a formalin-induced peripheral pain model and an anti-inflam-matory effect on arachidonic acid-induced ear inflammation, though itshowed no activity in a tetrad of behavioural tests, which are linked to CNSactivity (Table 6.34).In order to study the SAR of the resorcinol derivatives, about 40 com-
pounds were synthesised and tested in biological assays [227]. The 2-cyclohexylresorcinol derivative, O-1422 (363), showed binding affinity forboth CB1 and CB2 receptors, with a moderate CB2 selectivity [K iðCB1Þ ¼
11 nM and K iðCB2Þ ¼ 1:5 nM]. Reducing the ring size of the carbon ring tocyclopentyl (364) decreased CB1 affinity by 9-fold and CB2 affinity by5-fold, though increasing the ring size to cycloheptyl (365) did not affect thebinding affinity. Introduction of a methyl group at the 2- or 3-position of
the cyclohexyl ring did not dramatically alter the affinity. However, movingthe methyl group to the 4-position (368) decreased affinity for both can-nabinoid receptors. Conversion of the two phenolic hydroxy groups of (367)to methoxy groups (369) reduced both CB1 and CB2 binding activity, CB1
affinity was decreased more than 700-fold. Further efforts to find more CB2
selective agonists in the dimethoxyphenyl series resulted in discovery of O-1966A (370), which has good affinity and selectivity for CB2 [K iðCB1Þ ¼
5; 055 nM and K iðCB2Þ ¼ 23 nM, respectively] (Table 6.35).
OH
O
OH
(CH2)4
Me Me
MeMe
AM1703 (371)
Another resorcinol derivative, AM 1703 (371), which has a carbon–car-bon triple bond at the end of the alkyl chain, was reported to be a highlypotent and selective CB2 agonist (K iðCB2Þ ¼ 0:59 pM, with 500-fold selec-tivity against CB1) [228].
BENZO[C]CHROMEN-6-ONE DERIVATIVES
Benzo[c]chromen-6-one derivatives, such as AM 1710 (372), were found tobe CB2 selective agonists [229]. Although these compounds are structurallysimilar to the THC derivatives, the dimethyl group on 6-position of the
J. ADAM ET AL. 269
THCs is converted to a carbonyl group and the C-ring is aromatised. Speciesdifferences in CB2 binding activities of these compounds were observed:(372) and AM 1714 (373) showed 10- to 15-fold higher affinities at rat CB2
than human CB2 [230].
OOn-C6H13
Me Me
OH
OR
(372) AM1710 R = Me Ki = 28nM (humanCB2) Ki = 2.0nM (ratCB2)
(373) AM1714 R = H Ki = 29nM (humanCB2) Ki = 1.9nM (ratCB2)
OTHER HETEROCYCLIC CB2 AGONISTS
Benzimidazole-5-carboxamide-based agonists have been reported [231], forexample, compound (374) showed good activity and about 100-fold selec-tivity for CB2 [K iðCB1Þ ¼ 84:8 nM, K iðCB2Þ ¼ 0:8 nM]. Thirty-five relatedcompounds were also disclosed along with (374). Tetrahydroquinolone(375a) also exhibited CB2 agonist activity [232] and the CB2 binding affinitywas 17-fold enhanced by the conversion of the six-membered carbon ring tothe corresponding eight-membered ring (375b). Tetrahydroisoquinoline(376) and its derivatives were reported as CB2 agonists [233]. To estimate theCB2 agonist activity of (376), the inhibition activity on forskolin-stimulatedcyclic AMP production was tested and the IC50 was 0.4 nM.
NH
c-Hep
O
MeMe
N
N
c-Hex
OEt
O
(CF3CH2)2NN
NH(CH2)2Ph
X
O
n-Bu
O
(376) IC50(CB2) = 0.4nM
(374)
Ki(CB1) = 908nM Ki(CB2) = 1.6nM
(375a) X = CH2
(375b) X = (CH2)3
Ki(CB1) = 2851nM Ki(CB2) = 28nM
RECENT PROGRESS IN CANNABINOID RESEARCH270
Several types of CB2 agonists with monocyclic core structures have beenreported, these compounds being structurally distinct from other series ofCB2 agonists. Tetrazine derivative (377) exhibited 90-fold selectivity for CB2
and 2-imino-1,3-thiazolidine (378) showed more than 500-fold selectivity[234, 235]. A pyridone analogue (379) of a bicyclic CB1 agonist, CP 47,497(192) showed CB2 selective activity [K iðCB1Þ ¼ 973 nM, K iðCB2Þ ¼ 56 nM)][236], though the origin of the selectivity was unclear. The pyridine deriv-atives such as (380) were disclosed by Glaxo; (380) was more than 100 timesselective for CB2 over CB1 receptors [237].
N
S
N
OSEt
i-Pr
N
N N
N
CO2Et
CO2Et
Ph
Ph
N
O
OH
n-Pr
n-C5H11
Ki(CB1) = >5000nM
Ki(CB2) = 9nM
(378)
Ki(CB1) = 1672nM
Ki(CB2) = 19nM
(377) (379)
N
NH
O
NNH
Cl
CF3
(380)
THERAPEUTIC APPLICATIONS OF CANNABINOID AGONISTS
Standardised preparations of cannabinoid agonists are available for ther-apeutic use in some countries [238]. Dronabinol (MarinolTM), an oral prep-aration of D9-THC (67), is used clinically as an appetite stimulant in AIDSpatients and an antiemetic in cancer chemotherapy. A synthetic analogue of(67), nabilone (CesametTM), (381), is also used to suppress nausea andvomiting in cancer chemotherapy.There have been a number of studies to evaluate the therapeutic effect of
cannabinoids against spastic disorders, including multiple sclerosis and spinalcord injury. For example, a randomised placebo-controlled trial in more than
J. ADAM ET AL. 271
600 multiple sclerosis patients with oral cannabis extract or (67) was performedand an objective improvement in mobility was observed in the study [239].
O
O
OH
n-C6H13
Me MeMe
MeO
CH2OH
OH
n-C6H13
Me MeMe
Me
(165) HU-210 (381) Nabilone
Though cannabinoids exhibit a wide range of antinociceptive activities in avariety of animal models, e.g. neuropathic pain [240], hyperalgesia [241] andinflammatory pain [242], there are a limited number of reports in human trialson the use of cannabinoids against pain disorders. One of the early clinicaltrials was a preliminary double-blind study of oral (67) in patients experi-encing cancer pain, which was published in 1975 [243]. While pain relief wasdemonstrated to be significantly superior to placebo after administration of15 and 20mg of (67), some CNS side effects including substantial sedationand mental clouding were observed. A more recent clinical study revealed thatthe maximally tolerated dose of cannabis extract in 40 cancer patients was0.15mg/kg [238]. There have been very few clinical trials of cannabinoids forthe treatment of neuropathic pain and it was reported that an overall benefitof oral dronabinol was not obtained on refractory neuropathic pain becauseof the CNS side effects [244]. One of the potential uses of cannabinoids is toreduce the dosage of morphine administered to treat chronic pain disorders.Holdcroft et al. [245] reported that cannabis extract decreased the require-ment of morphine in a familial Mediterranean fever patient. The CNS sideeffects of cannabinoid agonists are thought to be caused by CB1 receptoractivation in brain. Recently, it was reported that CB2 selective agonistsshowed antinociceptive effects in various pre-clinical pain models includingmodels of hyperalgesia and neuropathic pain without any CNS cannabinoideffects [246]. To date, the mechanism of inhibition of pain responses by CB2
agonists has not been resolved completely, however, an indirect action oninflammatory processes and stimulation of the release of endogenous opioidshave been proposed [247]. An alternative approach is topical administrationof a cannabinoid agonist. Analgesic and anti-hyperalgesic effects of a top-ically applied CB1/CB2 agonist HU-210 (165) to human skin were reported[248] and no psychotomimetic side effects were observed.
RECENT PROGRESS IN CANNABINOID RESEARCH272
It has been revealed that cannabinoids exhibit neuroprotectant activitiesin both in vitro and in vivo models [249]. The neuroprotective effects aremainly based on regulation of transmitter release, modulation of calciumhomeostasis, anti-oxidant properties and modulation of immune responses.A number of neurological disorders, including brain trauma, cerebral is-chaemia, Parkinson’s disease and Alzheimer’s disease represent possibletherapeutic areas for cannabinoids with neuroprotective properties. Can-nabinoids are also suggested to have potential against glaucoma due to theirneuroprotective nature and lowering of intraocular pressure [250].Other therapeutic uses of cannabinoid agonists have been reported. The
potential of cannabinoids as a treatment for asthma is supported experi-mentally. A CB1 agonist, (R)-methanandamide (21), inhibited nerve growthfactor (NGF)-induced airway hyperresponsiveness in vivo [251]. The anti-pruritic effect of cannabinoids has been reported, the action being mediatedby both CB1 and CB2 pathways [252]. Treatment with cannabis extractimproved urinary tract symptoms of multiple sclerosis patients significantlyin an open-label pilot study [253].CB2 selective agonists are thought to be promising agents against a va-
riety of disorders related to the immune system, because of their immuno-modulatory properties and lack of psychotropic effects. Myocardialischaemia-reperfusion injury [254], atherosclerosis [255], glioma [256], leu-kaemia/lymphoma [257] and osteoporosis [258] are suggested as target dis-eases for CB2 agonists. Cough reflex was also inhibited by a CB2 agonistJWH-133 (329) in a guinea pig model of cough [259].
NH
OH
MeO
(CH2)34
n-C4H9
O
Me
n-Pr
Me MeMe
Me
(21) R-Methanandamide
(329) JWH-133
CB1 RECEPTOR ANTAGONISTS
In recent years pre-clinical testing and, in particular, clinical data onSR141716A (Rimonabant, Acomplia) (382) from Sanofi-Aventis (previously
J. ADAM ET AL. 273
Sanofi-Synthelabo) has stimulated a great deal of interest in the discovery ofcannabinoid CB1 receptor antagonists. This section aims to cover the struc-tures and therapeutic applications of CB1 antagonists published to date.Reviews of CB1 receptor antagonists and their therapeutic applications haverecently appeared in the literature [260–262].
1,5-DIARYL-PYRAZOLES
The most important class of CB1 receptor antagonists identified to date arethe 1,5-diaryl-pyrazoles. This class includes rimonabant (382), first describedin patent applications from Sanofi just over a decade ago [263, 264]. Rimo-nabant has proved invaluable in the elucidation of cannabinoid receptorpharmacology, as described in the section on therapeutic applications below.Several groups have published on structural analogues of (382), one of the
earliest being the disclosure of CP 272871 (383) from Pfizer, which displayslower affinity for the CB1 receptor than (382), in addition to reduced se-lectivity over the CB2 receptor subtype. Both (382) and (383) have beenshown to act as inverse agonists rather than neutral antagonists in vitro[265]. A recently published patent application from Sanofi-Aventis claims aseries of 4-cyanopyrazole analogues of (382), with 42 specific examples [266].Makriyannis and co-workers [267] have published on SAR within the 1,5-
diaryl-pyrazole class of compounds. These studies indicated a requirementfor a para-substituted phenyl ring at the 5-position of the pyrazole, iodo-phenyl proving optimal in terms of potency. Retention of the 3-carboxamidemoiety was important, as was retention of the 2,4-dichlorophenyl substitu-ent at the 1-position. The most potent compound in the series, AM 251(384), showed slightly higher affinity for the CB1 receptor than rimonabantcoupled with excellent selectivity over the CB2 receptor. Furthermore, thepresence of the iodophenyl moiety allows for use of this compound as asingle photon emission computed tomography (SPECT) ligand for imagingstudies [268].SR147778 (385) is a close structural analogue of (382), first described in a
patent application from Sanofi-Synthelabo [269]. In this application, (385) isclaimed to have an improved duration of action over (382) following oraldosing in mice. This was demonstrated through receptor occupancy studiesand the ability of the compounds to reverse cannabinoid agonist-inducedhypothermia. Compound (385) also displayed slightly higher affinity forCB1 receptors than (382) (Ki values of 5.4 and 34 nM, respectively) whileretaining excellent selectivity over CB2 receptors. A subsequent publicationfrom Sanofi-Synthelabo further described the in vitro and in vivo profilesof (385) [270]. The compound reversed WIN 55,212-2-mediated effects
RECENT PROGRESS IN CANNABINOID RESEARCH274
(hypothermia, analgesia and gastrointestinal transit) in mice following bothi.p. and p.o. administration. (385) also reduced ethanol or sucrose con-sumption in mice and rats and food intake in fasted and non-deprived rats.The compound is currently in Phase II clinical trials.
NN
Me
Cl
Cl
R1
NH
O N
NN
NC
Cl
NHPh
O
MeO
NN
Et
Cl
Cl
Br
NH
O N
R
(382) SR141716A (Rimonabant) R1 = Cl
(384) AM 251 R1 = I
(383) CP-272871
(385) R = H, SR147778
(386) R = OH
A close analogue of (385), the 4-hydroxypiperidine analogue (386) wasrecently described in a patent application from Sanofi-Synthelabo [271] andperhaps formed as a metabolite of the parent compound.Martin and co-workers [196] have also published on the discovery and
SAR of pyrazole cannabinoids as described in the CB1 agonist section. Theanalogues were tested for CB1 receptor binding affinity and in a battery of in
J. ADAM ET AL. 275
vivo tests, including hypomobility, antinociception and hypothermia. Var-iations of the pyrazole substitution pattern in positions 1, 4 and 5 led tocompounds with varying affinity that consistently behaved as antagonists invivo. Introduction of ether moieties into the 3-position of the pyrazole coreled to the identification of both partial agonists and antagonists. For ex-ample, compound O-1043 (387) retained a Ki of 53 nM and demonstratedantagonism in vivo. An analogue, in which the amide was replaced with aketone (O-1271) (388), retained a Ki of 82 nM and again demonstrated an-tagonism in vivo.Makriyannis and Liu claimed a series of pyrazole analogues in a patent
application published in 2003 [272]. Of the 29 compounds specifically ex-emplified in the patent application, compound (389) was demonstrated toreduce lever presses when administered to rats that were trained to expectdelivery of a food pellet as the outcome. It was proposed that the reductionin lever pressing was the result of decreased appetite brought about by CB1
receptor antagonism.
NN
Me
Cl
Cl
Cl
R
NN
R1
Cl
Cl
R2
NH
O N
(387) O-1043 R = OCH2-2,4-diF-phenyl(388) O-1271 R = CO(CH2)4Me
Horti and co-workers [273] published on their studies to produce ana-logues of rimonabant with reduced lipophilicity. The aim of the work was toproduce ligands for positron emission tomography (PET) studies in hu-mans. Highly lipophilic ligands were noted to be a problem due to resultinghigh levels of non-specific binding. An additional limiting factor in the workwas the need to be able to introduce an appropriate atom for a PET la-belling study (18F, 76Br or 11C). NIDA-41020 (390) retained high CB1 re-ceptor affinity (K i ¼ 4:1 nM, (382) K i ¼ 1:8 nM in the same assay format)and a reduced lipophilicity in comparison with (382) (e logDoct values of4.78 and 5.36, respectively).
RECENT PROGRESS IN CANNABINOID RESEARCH276
Several studies have been reported on the application of conformationalrestraint to the 1,5-diaryl-pyrazole series in an attempt to provide com-pounds with modified properties. In one approach, a Sanofi-Synthelabopatent application claimed a series of conformationally restrained com-pounds, exemplified by compound (391). Compounds of the invention werestated to be CB1 receptor antagonists with Ki values below 5� 10�7M andselectivity over CB2 receptors of at least 10-fold [274].Lange and co-workers [275] published on conformationally restrained
analogues of rimonabant (382), again constraining the pyrazole 4-positionand the 5-aryl substituent into a ring system. The highest affinity compoundwithin the series was compound (392), which displayed a pKi value of 7.2((382) pKi 7.6 in the same assay). This compound (392) was found to beslightly more potent than (382) in a cell-based functional assay for CB1
antagonism (pA2 values of 8.8 and 8.6, respectively). In a subsequent pub-lication, Ruiu et al. [276] synthesised and characterised NESS-0327 (393).This compound had also been prepared and tested in the publication fromLange et al. (393) was reported to show higher affinity for the CB1 receptorthan (382) (Ki values of 350 fM and 1.8 nM, respectively), in addition tohigher affinity for the CB2 receptor (Ki values of 21 and 514 nM, respec-tively). Selectivity for the CB1 receptor over the CB2 receptor was calculatedat more than 60,000-fold for (393). Potent antagonism was demonstrated ina variety of in vitro and in vivo paradigms for (393).
NN
Cl
Cl
Cl
NH
O NS
NN
Cl
Cl
NH
O N
R
X
N
NH
O NMe
N
Cl
Cl
(391) (392) X = (CH2)2, R = NO2
(393) NESS-0327 X = CH2, R = Cl
(394)
Thomas and co-workers [277] reported that irradiation of (382) with a450W high-pressure mercury lamp brought about photocyclisation to aconstrained analogue (394). The structure of the product was elucidatedthrough NMR and X-ray diffraction analysis. The compound retained highaffinity for the CB1 receptor (K i ¼ 48 nM) and good selectivity over the CB2
receptor (K i ¼ 3; 340 nM).
J. ADAM ET AL. 277
A series of 21 compounds, differing from (382) in the amide region, hasbeen synthesised by Franciso et al. [278] and their biological propertiesstudied.The biological activity of the compounds was assessed in two binding as-
says using [3H]-CP 55,940 (see Table 6.36) and [3H]-rimonabant (data notshown), and their functional activity determined using a GTPgS assay. Upon
Table 6.36 MODIFIED AMIDE SUBSTITUENT ANALOGUES OF RIMONABANT(382) – IN VITRO DATA
NN
Cl
Cl
Cl
Me CONHR
Cpd R CB1 Ki (nM) CB2 Ki (nM) [ 35S] GTP–gS EC50 (nM)
aValue greater than the highest point on the displacement curve.
RECENT PROGRESS IN CANNABINOID RESEARCH278
increasing the length of the alkyl chain, the CB1 binding affinity increases withi-butyl and n-pentyl analogues (399) (400) showing the highest affinity. In-creasing the chain length beyond this (e.g. n-hexyl (401)) resulted in a decreasein affinity compared with (399) and (400). In the hydroxyalkylamide series(404)–(409) and the alkylhydrazide series, this trend was also evident. Theintroduction of a hydroxyl group led to a marked decrease in affinity (cf.(395) and (405), (396) and (406)). There was little difference in binding affinityobserved between the two hydroxymethylethyl enantiomers (408) and (409)with the incorporation of the hydroxyl group again proving to have a del-eterious effect on CB1 binding. The morpholinyl analogue of rimonabant(403) again had comparatively low affinity (K i ¼ 22:9 nM), confirming thatthe presence of a polar oxygen was not particularly well tolerated. Within theseries, the most potent side chain was found to be cyclohexyl (402). Theaffinity of (402) was only slightly improved compared with (382), implyingthat the piperidine nitrogen is not likely to be involved in any crucial elec-trostatic or hydrogen-bond formation with the receptor. Throughout the se-ries, the CB2 receptor affinity was modest with CB1/CB2 selectivity rangingfrom 5-fold (404) to 378-fold (401), and all the compounds were found to beinverse agonists. Five compounds with reasonably high CB1 receptor affinities(399)–(403) were investigated for their ability to antagonise the CB1 agonist-mediated inhibition of electrically evoked contraction of mouse vas deferenstissue. All the compounds tested were found to produce antagonism. In linewith the earlier obtained binding data, (402) was found to be the most potentin the vas deferens experiment. Compounds (400), (402) and (403) also dis-played a noticeable inverse effect, enhancing the amplitude of the twitch priorto addition of the cannabinoid agonist WIN 55,212-2, an effect not shown by(399) and (401).A QSAR study was performed to understand the pharmacophoric re-
quirements of the aminopiperidine region. A number of conformations ofeach molecule were generated using a quenched molecular dynamics ap-proach and the resultant conformers overlaid by superimposing theirpyrazole rings using a root mean square minimisation procedure. A com-parative molecular field analysis (CoMFA) approach was then used to de-rive a QSAR and allow visualisation of the regions where changes in stericor electrostatic properties resulted in changes in measured biological prop-erty (CB1, CB2 and GTPgS binding). From this, it was observed that a sidechain no longer than 3 A in length could be tolerated in the aminopiperidineregion. Furthermore, increased affinity and potency were predicted forcompounds having a substituent bearing a positive charge density in thisregion.CoMFA has also been used by Shim and co-workers [279] to help un-
derstand the interaction between rimonabant (382) and the CB1 receptor.
J. ADAM ET AL. 279
Conformational analysis of (382) was first performed using the AM1 cal-culation method. Four distinct low-energy conformers (Tg, Ts, Cg, Cs) wereidentified corresponding to either trans (T) or cis (C) geometry around theamide bond and either skew (s) or gauche (g) depending on their torsionangle around the {(CQO)–N–N–C} bond in the aminopiperidine region. Inthe unprotonated form of (382), the Tg conformer was found to be the mostenergetically favourable followed by the Cg, Ts and Cs forms, which was inagreement with the AM1 calculated values. In the protonated state, the moststable conformation was Ts, followed by Tg, which was more stable than Cs
and Cg. Both protomeric states were examined in the subsequent QSARmodel since the extensive conjugation of the molecule made it uncertain asto which form would predominate under physiological conditions. ADISCO-derived pharmacophore model was developed using a previouslypublished superposition model [280] of cannabinoid agonists CP 55,244(196) and WIN 55,212-2 (254a). Construction of a 3D-QSAR CoMFAmodel for (382) and a series of 31 closely related structural analogues wasperformed using binding affinities determined from displacement of [3H]-CP55,940 in rat brain membranes. The Tg, Cg and Ts conformers only wereconsidered for the neutral species, while the Ts, Tg and Cs conformers in theprotonated species were used in the model. The values obtained for the r2
and cross-validated r2 were indicative of statistical robustness and predict-ivity in all cases except for the protonated Tg conformer where the r2cv valuefell to 0.36, below the accepted threshold for internal predictive ability. Thedata obtained from the studies suggested a crucial role for the N1 aromaticring in forming a hydrophobic interaction with the CB1 receptor. It was alsosuggested that the second aryl ring may prevent the change in receptorconformation necessary for agonist activity. In addition, it was postulatedthat the C3 amide substituent might interact with the Lys-192 residue, be-lieved to be participating in a specific ligand-binding interaction, therebyprohibiting agonist activity and also inducing or stabilising the receptorconformation required for inverse agonism.This latter theme was subsequently explored by Hurst et al. [281]. Receptor
models of the R and R* forms of CB1 were constructed using the 2.8 A crystalstructure of bovine rhodopsin. Rimonabant (382) and a conformationallyconstrained analogue (VCHSR), in which the (N-piperidinyl)-3-carboxamidemoiety of rimonabant was replaced by 3-[(E)-2-cyclohexylethenyl], wereevaluated in these models, and in vitro, in both the wild-type CB1 receptor andin a mutant in which the Lys residue at position 192 had been mutated to Ala.The two ligands were docked into the aromatic transmembrane helix (TMH)3-4-5-6 regions of the active and inactive TMH bundle, and the energy mi-minised using the AMBER force field. In the inactive R complex, (382)was observed to form a hydrogen bond with Lys-192 (TM3.28) which is part
RECENT PROGRESS IN CANNABINOID RESEARCH280
of a salt bridge with Asp-366 (TM6.58). In addition, aromatic stackinginteractions were formed with Trp-279 (TM5.43) with the dichlorophenyl ringinteracting with Phe-200 (TM3.36) and the monochlorophenyl ring interact-ing with Tyr-275 (TM5.39). In the active state, the relative positions of keyTMH3 and TMH6 residues Lys-192 and Asp-366 were observed to havechanged, breaking their salt bridges and preventing the interaction betweenLys-192 and the C3-substituent of (382). In the VCHSR/CB1 R complex, themodelled interactions were very similar to those of (382), but lacked thehydrogen-bonding interaction between Lys-192 and the C3 substituent.Analysis of the VCHSR/CB1 R* complex suggested that VCHSR shouldhave approximately equal affinity for both the R and R* states, conferring theproperty of neutral antagonism. Consistent with this hypothesis, it was foundthat VCHSR behaved as a neutral antagonist at the wild-type CB1 receptor,as witnessed by the attenuation of the inhibition of the calcium current by(254a) in superior cervical ganglion neurons. It was therefore proposed thatthe interaction between the Lys-192 residue and the C3 substituent of (382)was the principal determinant in the high affinity for the inactive R state andhence the experimentally observed inverse agonism.Molecular modelling and automated docking studies by Salo et al. [282]
also confirmed that Lys-192 played a key role in the binding mode ofHU210 (165), CP 55,940 (193), rimonabant (382), WIN 55,212-2 (254a) and2-AG. Further mutagenesis and molecular modelling work [283] comparedthe interaction of (382), (254a) and anandamide (1) with CB1 wild type andF3.25A, F3.36A, W5.43 and W6.48 mutants. The binding of (382) and(254a) was found to be affected by the F3.36A, W5.43 and W6.48 muta-tions, implying their presence in the binding site of these two ligands. Incontrast, only the F3.25A mutation was found to influence the binding of(1), suggesting the existence of a distinct binding site. It was therefore sug-gested that an aromatic micro domain in the TMH3-4-5-6 region comprisingF3.36A, W4.64, Y5.39, W5.43 and W6.48 was responsible for binding both(382) and (254a), but that the endocannabinoid-binding pocket may onlypartially overlap with the former lipophilic domain.Patent applications from Pfizer disclosed 1,5-diaryl-pyrazoles bearing
bioisosteric replacements for the 3-carboxamide moiety. One applicationshowed that the amide could be replaced by a-aminoketones as exemplifiedby compound (416) [284]. The corresponding alcohols and their ethers werealso described, including compounds that allowed the amine substituent andether to form a ring system, such as a morpholine unit. This application alsoallowed for the replacement of the 1,5-diaryl-pyrazole by a 1,2-diaryl-imidazole bearing a 3-carbonyl substituent, as exemplified by compound(417). A further patent application from Pfizer claims compounds in whichimidazoles replace the 3-carboxamide moiety in the 1,5-diaryl-pyrazole
J. ADAM ET AL. 281
series [285]. Compound (418), one of the compounds specifically claimed inthe patent, showed a CB1 receptor affinity of 371 nM.
NN
Me
ClCl
NN
Ph
Me
N N
ClCl
O N
Cl
NN
ClCl
O N
(418)(417)(416)
In addition, Pfizer has identified a number of related, fused bicyclicpyrazole analogues [286–292]. These compounds, of which structures(419–425) are specified examples, are claimed to be of use in the treatmentof a number of diseases including alcoholism, psychosis, tobacco abuse,Parkinson’s disease and obesity.
N
N
NN
OCl
Cl
PhCl
(419)
N
N
NN
NCl
Cl
Cl
N
Me
(420)
NN
Cl
FF
F
N
O
i-Pr NN
Cl
Cl
N i-Pr
O
(421) (422)
RECENT PROGRESS IN CANNABINOID RESEARCH282
N
NN
N
NCl
F
H2NCO NHi-Pr
(425)
X
N
NN
NCl
Cl
N
Me
(423) X = CH(424) X = N
4,5-DIHYDRO-1H-PYRAZOLE DERIVATIVES
Solvay Pharmaceuticals have claimed 4,5-dihydro-1H-pyrazole derivativesin a series of patent applications [293–296], compounds (426–429) representexamples of the structures involved.
NN
Ph
Cl N
SMe
SNEt2
O O
NN
Ph
Cl N
NH
N
SO O
Cl(426) R = Cl (SLV-319)(427) R = CF3 (SLV-326)
(428)
(429)
N
N
Ph
Cl N
NHMe
S
O O
R
Solvay subsequently published in vitro, in vivo, computational and X-raydiffraction data relating to this series of 3,4-diarylpyrazolines [297]. In this
J. ADAM ET AL. 283
publication, a number of compounds were evaluated, in the first instance invitro at the human CB1 and CB2 receptors, expressed into Chinese hamsterovary (CHO) cells by radioligand binding. In addition, the CB1 functionalactivity was measured via an arachidonic acid release functional assay. Re-sults for a number of the compounds are shown in Table 6.37.For compounds (431–434), a significant improvement in binding affinity
was observed when the 4-Me group in (430) was substituted with either Cl(431), 2,4,6-trimethyl (433) or F (434). Replacement of 4-Me with 4-OMe(432) failed to improve the affinity or functional activity. In the amidine-substituted series (435)–(447), dimethyl compounds (435) and (436) both
Table 6.37 PYRAZOLINE DERIVATIVES AND RIMONABANT (382) – IN VITRODATA [297]
N N
Cl
N
N
R2R3
S OO
R1
Cpd. R1 R2 R3 CB1 Ki (nM) CB1 pA2 CB2 Ki (nM)
(382) 25 8.6 1,580(430) 4-Me H H 197 8.4 >1,000(431) 4-Cl H H 16.1 9.5 >1,000(432) 4-OMe H H 196 8.3 >1,000(433) 2,4,6-Me3 H H 24.2 9.4 >1,000(434) 4-F H H 52.6 9.0 >1,000(435) 4-Cl Me Me 280 8.5 >1,000(436) 4-F Me Me >1,000 o7.5 >1,000(437) 2-Cl H Me 75.4 8.3 >1,000(438) 3-Cl H Me 13.9 8.6 >1,000(439) 4-CF3 H Me 221 9.3 >1,000(440) 4-Cl H Me 25.2 8.7 >1,000(441) H H Me 170 7.5 >1,000(442) 4-F H Me 338 8.5(443) 4-Me H Me 119 8.6 >1,000(444) 3-CF3 H Me 36.5 9.1(445) 2,4,6-Me3 H Me 54.2 9.4(446) 4-OMe H Me 22.9 8.0(447) 3,4-Benzo H Me 21.8 8.5
RECENT PROGRESS IN CANNABINOID RESEARCH284
show reduced affinity compared with their unsubstituted counterparts (431)and (434), respectively. The affinity of the amidine-NH2 analogues wasbroadly similar to that of the amidine-NHMe compounds. 3-Chloro (438)and 4-chloro (440) were approximately equipotent, however, there was someloss of activity witnessed when the substituent was moved to the 2-position(437). In contrast to (432), the 4-methoxy analogue (446) in the amidine-NHMe series was found to have excellent CB1 affinity (22.9 nM) while tri-fluoromethyl substitution at the 3-position (444) also yielded a more potentCB1 ligand than the 4-CF3 congener (439). A bulky 2-naphthyl-substitutedanalogue also proved to have excellent affinity. The amidine functionalitywas shown to be crucial for good CB1 affinity, since replacement by anamide drastically reduced affinity, whereas the substitution of the amidine-NH2 group for SMe, was more tolerated. Racemic (439) and (440) wereresolved and each of the enantiomers tested for their CB1 affinity. In eachcase, the levorotatory (4S-) eutomers were found to be more potent thantheir dextrorotatory equivalents; 4S-(439) (427) showed an affinity of35.9 nM and 4S-(440) (426) had an affinity of 7.8 nM. Throughout the se-ries, good CB1/CB2 selectivity was evident.Table 6.38 describes some of the key in vivo data for the significant
pyrazoline compounds in a CB1 agonist-induced hypotension rat model andhypothermia mouse model, compared with the Sanofi-Aventis CB1 antag-onist, rimonabant (382). From the initial disappointing discovery that pro-genitors (430) and (431) were inactive when administered orally in bothassays, it was found that (435), the amidine-NMe2 analogue of (431) didexhibit in vivo effects. Subsequently, (437)–(439) were also found to have invivo efficacy in both assays when administered orally. In common with thein vitro data, the 4S-analogues of (439) and (440) were found to be active invivo, while the 4R-enantiomers failed to display CB1 activity.
Table 6.38 PYRAZOLINE DERIVATIVES AND RIMONABANT(382) – IN VIVO DATA
aAntagonism of CB agonist (CP 55,940)-induced hypotension in rat, expressed as ED50 (mg/kg,p.o.).bAntagonism of CB agonist (WIN 55,212-2)-induced hypothermia in mouse, expressed as leasteffective dose (LED) (mg/kg, p.o.).
J. ADAM ET AL. 285
An X-ray diffraction study of (426) revealed the existence of a hydrogenbond between the N-atom at the 2-position of the dihydropyrazole ring andthe hydrogen atom of the amidine. A series of Confort-generated conform-ers of (426) were generated and minimised (MOPAC, PM3) and then com-pared with the favoured trans-gauche Tg conformer [279] of rimonabant(382). The conformer of (426) which overlapped best with the Tg conformerof (382) was then docked into a reconstructed model of the CB1 receptor[281]. It was found that hydrogen bond was formed between one of theoxygen atoms of SO2 and the Asp-366:Lys-192 salt bridge. An additionalH-bond was also observed between Ser-383 and the remaining SO2 oxygenatom. A stacking interaction could be envisaged between the 4-Cl-phenylring of (426) and the Phe-170 residue. Two hydrophobic pockets, defined byTrp-279, Phe-200, Trp-356 and Tyr-275, Trp-255, Phe-278 bind the two4-Cl-phenyl rings.Investigation of the differences in crystal packing between (431) and (426)
from comparison of their respective X-ray structures, revealed that (431)was more tightly packed than (442), reflected in their respective meltingpoints of 235 and 170 1C. It was postulated that the absence of in vivoactivity for (431) may be explained by the resultant reduction in watersolubility and dissolution rate compared with (426). The comparatively highcalculated polar surface area of (431) (122.5 A2) compared with (426)(89.3 A2) was also proposed as a factor influencing the marked difference inbioavailability between the two related compounds. Compound (426) (SLV-319) is currently being developed with Bristol-Myers Squibb for the poten-tial treatment of obesity and other metabolic disorders. Phase I trials forobesity were started in April 2004. Earlier Phase I clinical trials for thetreatment of schizophrenia and psychosis, which commenced in April 2002,appear to have been abandoned.
IMIDAZOLE-, THIAZOLE-, PYRROLE- AND TRIAZOLE-BASEDCB1 RECEPTOR ANTAGONISTS
4,5-Diarylimidazole derivatives, exemplified by (448) have been claimed byMerck as either antagonists or inverse agonists of the CB1 receptor [298]. In thisseries of compounds, the central imidazole motif can be considered a bioisost-ere of the pyrazole core of rimonabant. A similar approach was utilised bySolvay Pharmaceuticals [299] in a patent application published shortly afterthat of Merck. The isomeric 1,2-diaryl-4-carboxamide nucleus (449) can againbe considered as a structural isostere, serving to orient the two substituted arylgroups and carboxamide group in geometry analogous to that of (382). Analternative methylimidazole core, 1,2-diaryl-5-methylimidazole (450), was also
RECENT PROGRESS IN CANNABINOID RESEARCH286
utilised by Merck [300]. More recently, Pfizer has disclosed a series of novelimidazole derivatives useful in the treatment of obesity, alcoholism and de-pression [301]. The specified compound (451) was stated to have CB1 bindingaffinity in the range of 1–10nM.
N
N
Me
NH
O
Cl
Cl
Cl N
NNH
O
Cl
Cl
Cl
N
(448) (449)
N
N
O
NH
Cl
Cl
Cl
Me
N
NN
i-PrNH
O
ClCl
(450) (451)
Novel 6-aminopurine compounds, for example, (452) have been claimed byPfizer as particularly useful in the treatment of obesity and alcohol abuse [302].In conjunction with an opioid receptor antagonist, the related compounds suchas (453) are claimed as a combination therapy for treating alcohol, cocaine ortobacco addiction or dependence, reducing alcohol withdrawal symptoms andaiding in the cessation or lessening of alcohol abuse, substance abuse or be-havioural dependency, including gambling [303]. Compound (453) has alsobeen claimed to have utility in combination with a nicotinic acetylcholine ago-nist in the treatment of obesity or compulsive overeating [304] and compulsivegambling, nicotine and drug dependence [305].
J. ADAM ET AL. 287
N
N N
N
N Cl
Cl
N
Cl
N
N N
N
N
CONH2EtNH
Cl
Cl
S
N
O
NH
X
Cl
Cl
Cl
(452) (453)(454) X = CH(455) X = N
Thiazole-based cannabinoid CB1 modulators were claimed in two patentapplications by AstraZeneca [306, 307] where the strategy of bioisostericpyrazole-replacement is evidenced by the specified compounds (454) and(455), respectively. No specific biological data were presented but the spec-ified compounds were stated to have IC50 values o200 nM. A centralthiazole motif was also utilised by Solvay Pharmaceuticals [308]. In vivoantagonism was evaluated using CP 55,940-induced hypotension in rats,however no specific results were given. Compound (455) was shown to havebroadly similar affinity at both CB1 and CB2 receptor subtypes with pKi
values of 7.8 and 8.1, respectively.A thiazole core has also been utilised by Solvay Pharmaceuticals [309] in
the search for a novel bioisosteric replacement of the rimonabant (382)pyrazole core. The affinity of several compounds for the human CB1 re-ceptor was determined in transfected CHO cells using tritium-labelled CP55940. Antagonism was determined in the same cell line by WIN 55212-2-induced release of arachidonic acid. The pKi of (456) was found to be 6.9,while the pA2 value was measured as 8.7. A series of six 1,2,4-triazole an-alogues has been prepared by Jagerovic et al. [310], via their correspondingN-acylbenzamides (Table 6.39).
NH
H
H
H N
S Ph
n-C5H11
O N
N
N
O
NH
Cl
Cl
Cl
(456)(457)
Table 6.39 STRUCTURES OF 1,2,4-TRIAZOLE ANALOGUES OFRIMONABANT (382)
N
NN (CH2) MeR
2
R1
n
Cpd. R1 R2 n Cpd. R1 R2 n
(458) 4-Cl H 1 (461) 4-Cl 2,4-di-Cl 5(459) 4-Cl 4-Cl 1 (462) 2,4-di-Cl 2,4-di-Cl 5(460) 4-Cl 4-Cl 5 (463) H H 6
RECENT PROGRESS IN CANNABINOID RESEARCH288
The biological activity of the compounds was examined by assessing theirability to antagonise WIN 55,212-2 induced inhibition of electrically in-duced contractions in mouse vas deferens and guinea pig ileum. Compounds(459)–(463) were not seen to induce modification of the contractile responsein either the vas deferens or ileum tissues when administered alone, effec-tively ruling out the possibility that these compounds behaved as agonists.Compound (458) was found to inhibit contractile activity in the guinea pigileum, an effect that was not antagonised by (382), thus precluding can-nabinoid agonist activity, however the mechanism underlying the observedinhibition was not investigated further. Upon incubation of (458–463) in thepresence of WIN 55,212-2, analogue (461), bearing the familiar (4-chloro-phenyl)-(2,4-dichlorophenyl) substitution, induced a significant and dose-dependent decrease of the inhibition produced by the aminoalkylindoleagonist in both tissues. The binding affinity of (461) in rat cerebellularmembranes using [3H]-rimonabant and [3H]-WIN 55,212-2 was found to bemoderate (K i ¼ 855:6 and 748 nM, respectively) compared with rimonabant(382) (K i ¼ 4 nM). The effects of (461) in vivo were also assessed in thecannabinoid tetrad behavioural tests (nociception, temperature, spontane-ous motility and catalepsy). Compound (461), administered intraperitone-ally (1mg/kg) was found to behave as a cannabinoid antagonist, reversingthe effects of WIN 55,212-2 (1.5mg/kg, i.p.).In addition to triazole-based biosteres of (382), thiazoles and imidazoles
have also been investigated by Lange et al. [311]. The three series of com-pounds were assessed on their CB1 binding affinity and functional activity
J. ADAM ET AL. 289
and their selectivity over the CB2 receptor in CHO cells stably transfectedwith human CB1 and CB2 receptor.Comparison of thiazole-based regioisomers (464) and (465) has revealed a
marked preference for the phenyl substitution of the former, a trend alsoobserved in the subsequent imidazole series (data not shown), but not in thetriazole series as evident from the data presented for (466) and (467) (Table6.40). The thiazole (464) was approximately 8-fold less potent than thecorresponding imidazole (470) (Table 6.41) suggesting that the seeminglysubtle change of the central scaffold imparts a marked change in CB1 re-ceptor recognition.Ethyl substitution at the imidazole 5-position (469) was found to increase
potency over the unsubstituted analogue (468), while methyl substitution(470) had a slightly deleterious effect on binding (Table 6.41). Chloro (491),bromo (492), cyano (493) and fluoromethyl (494) substitution at this positionwere all well tolerated (Table 6.43). Introduction of a chloro-substitutedpyridine (475) in place of the more usual p-chlorophenyl group (470) resultedin a slight loss of affinity for the CB1 receptor, as did replacement of the p-chloro group of (470) with bromo (471), fluoro (472) and in particular, met-hoxy (473). Trifluoromethyl substitution (474) however, was well tolerated.The effect of replacing the carboxamide N-piperidinyl group of (470) was
also investigated. Reducing the ring of the piperidinyl group by one carbon
Table 6.40 THIAZOLE- AND TRIAZOLE-BASED BIOISOSTERES OF RIMONABANT(382) – IN VITRO DATA [311]
S
N
N
O
N
R1
Cl
R2
Cl
N N
N
N
O
N
R4
Cl
R3
Cl
Cpd. R1 R2 R3 R4 CB1 Ki (nM) CB1 pA2 CB2 Ki (nM)
(382) 25 8.6 1,580(464) Cl H 227 8.1 5,841(465) H Cl >1,000 7.2 4,668(466) Cl H 356 8.3 3,562(467) H Cl 382 7.6 5,444
Table 6.41 IMIDAZOLE-BASED BIOISOSTERES OF RIMONABANT (382) – IN VITRODATA [311]
N
N
N
O
N
X
R3
Cl
R1
R2
Cpd. R1 R2 R3 X CB1 Ki (nM) CB1 pA2 CB2 Ki (nM)
(468) Cl H Cl C 23 8.2 542(469) Cl Et Cl C 14 9.0 430(470) Cl Me Cl C 30 8.6 608(471) Br Me Cl C 60 8.5 489(472) F Me Cl C 52 7.7 765(473) OMe Me Cl C 106 8.7 326(474) CF3 Me Cl C 29 8.6 634(475) Cl Me Cl N 55 8.4 758
RECENT PROGRESS IN CANNABINOID RESEARCH290
(476) yielded a compound approximately equipotent with (470) whileincreasing the ring (477) or introducing a morpholine group (478) reducedthe binding affinity (Table 6.42). Larger bicyclic substituents were well tol-erated, as evidenced by (479), (485) and (486). Cyclopentylamine (480),cyclohexylamine (481) and cycloheptylamine (482) substitutents were alsoaccommodated in the lipophilic binding pocket. However, the presence of ahydroxyl group resulted in a marked decrease in affinity, for example, (483)and (484).The CB1 functional activity of a number of the imidazole-based com-
pounds reported exceeds that measured for rimonabant (382) (Table 6.43).While marked CB1/CB2 selectivity is witnessed throughout the series, theselectivity of direct (382) analogue (470) was found to be approximately 3-fold lower than for (382) itself (Table 6.44).The in vivo properties of compounds (464), (466) and (468)–(470) relative
to (382) were investigated in two animal models: a CB1 agonist-inducedhypotension rat model and a CB1 agonist-induced hypothermia mousemodel. Shown to be only moderately active in the in vitro screens, thiazole(464) and triazole (466) analogues failed to demonstrate convincing oralactivity. Compound (468) was active in the hypotension model but showed
Table 6.42 IMIDAZOLE-BASED BIOISOSTERES OF RIMONABANT (382) – IN VITRODATA [311]
aAntagonism of CB agonist (CP 55,940)-induced hypotension in rat, expressed as ED50 (mg/kg,p.o.).bAntagonism of CB agonist (WIN 55,212-2)-induced hypothermia in mouse, expressed as leasteffective dose (LED) (mg/kg, p.o.).
RECENT PROGRESS IN CANNABINOID RESEARCH292
no activity in the mouse hypothermia model. The 5-ethyl (469) and in par-ticular, the 5-methyl (470) analogues showed in vivo efficacy in both models,strongly suggesting that the 5-methylimidazole scaffold behaves as a truebioisostere of the pyrazole ring of (382).Reconstruction [297] of the CB1 homology model reported by Hurst et al.
[281] was carried out, and ligands (464), (466) and (470) docked into thereceptor along with rimonabant (382). A perfect 3D overlap of (382) and(470) was observed. In the case of the thiazole (464) and triazole (466), the
J. ADAM ET AL. 293
carboxamide and aryl rings were found to overlap well with (382), althoughthe central heterocycle deviated slightly from the orientation adopted by (382)and (470). The importance of the substitution patterns on the phenyl ringswas also investigated. It was been reported that (382) binds into the active siteof the CB1 protein in its most stable so-called transdichlorophenyl (TDC) con-formation, in which the dichlorophenyl ring is oriented trans with respect tothe carbonyl of the carboxamide group. It has been proposed that the saltbridge formed between residues Lys-192 and Asp-366 is stabilised by a hy-drogen-bonding interaction with the carboxamide carbonyl of (382) and iscrucial for conferring inverse agonism [281]. The 4-chlorophenyl ring wasobserved to participate in direct stacking interactions with Trp-255, Tyr-275,Phe-278, and Trp-279 forms a stacking interaction with both aromatic ringsof the ligand. In addition, the 2,4-dichloro ring is located in the proximity offurther two aromatic residues, Trp-356 and Phe-200, where a small lipophilicpocket enhances the binding interaction with the 2-chloro substituent. It wasproposed that the TDC conformation of (382) is stabilised by a weak hydro-gen-bond interaction between the carboxamide NH and the proximal nitro-gen atom in the pyrazole core. It was postulated that the TDC conformationof (472) is also stabilised by such an interaction, favouring this conformationover the alternative cisdichlorophenyl (CDC) conformer, which would potentiallygive rise to steric occlusion between the ortho-chloro substituent on the 2,4-dichlorophenyl ring and the residues Phe-278 or Val-364.A series of 4,5-diarylimidazole-2-carboxamides has been reported as hu-
man CB1 inverse agonists [312]. The initial HTS hit, (495), based on ananalogous scaffold to (382), was found to have moderate affinity for the CB1
receptor (7,000 nM).
N
NH
Ph
Ph SAc
Ph
(495)
The relatively low affinity was ascribed to the absence of substitution onthe phenyl rings and the absence of an amide group to form an interactionwith the receptor. Among the compounds synthesised during the hit opt-imisation program were (496–507) (Table 6.45).The trichloro derivative corresponding most closely to rimonabant (382),
(496) was found to have excellent CB1 affinity (IC50 ¼ 6:1 nM) (cf. (382):
Table 6.45 4,5-DIARYLIMIDAZOLE DERIVATIVES – IN VITRO DATA [312]
N
N
Cl
ClR2
R3
NHR1
OR4
Cpd. R1 R2 R3 R4 CB1 IC50 (nM)
(496) N-Piperidinyl H Cl Me 6.1(497) c-Hexyl H Cl Me 4.0(498) N-Morpholinyl H Cl Me 170(499) c-Pentyl H Cl Me 17(500) c-Heptyl H Cl Me 8.0(501) c-Hexyl H Cl Me 70(502) Ph H Cl Me 60(503) c-Hexyl H Cl Et 4.1(504) N-Piperidinyl Cl H Me 190(505) c-Hexyl Cl H Me 46(506) N-Piperidinyl Cl Cl Me 16(507) c-Hexyl Cl Cl Me 5.2
RECENT PROGRESS IN CANNABINOID RESEARCH294
K i ¼ 6 nM). The closely related cyclohexyl analogue (497) was found to beslightly more potent than both. However, as witnessed in the analogous seriesby Lange et al. [311], replacement of the N-piperidinyl group by N-morph-olinyl was detrimental to the CB1 binding affinity, suggesting that polarfunctionality was not well tolerated into the amidoalkyl region. A cycloheptylgroup at this position was relatively well tolerated (500), with a more sub-stantial decrease in affinity reported upon reduction of the cycloalkyl ring tocyclopentyl (499). The isomer of (496) in which the phenyl substitution wasswapped between the two aryl rings (504) was found to be considerably lesspotent than (496) and indeed less potent than either of the tetrachloro an-alogues (506) and (507). Replacement of the imidazole N-Me group by N-Et(503) resulted in a broadly equipotent analogue.A number of oxazole derivatives were also synthesised, of which (508) was
among the most potent (IC50 ¼ 80 nM). In general however, the bindingaffinities reported for the oxazole compounds were at best modest, andcomparable with the related imidazole NH series (data not shown).
J. ADAM ET AL. 295
N
O
Cl
Cl
Cl
NHc-Hex
O
(508)
In vivo studies on the leading compounds (496) and (497) were carriedout. Preliminary rat PK studies (1mg/kg i.v., 2mg/kg p.o.) for (496) indi-cated a good profile for i.v. administration (AUCnom ¼ 1:32 mMhkg=mg;Clp ¼ 27:5ml=min=kg; Vdss ¼ 3:7L=kg; t1=2 ¼ 2:4 h) and oral absorption(F ¼ 50%), in addition to a high brain:plasma ratio (B:P ¼ 2.99–3.40 be-tween 0.25 and 4 h). An immediate, dose-dependent and prolonged reduc-tion in food intake was subsequently observed in a body weight loss studycarried out using diet-induced obese rats. At doses of 1, 3 and 10mg/kg p.o.,the reduction was reported to be �13.4%, �30.6% and �67.5% relative tothe vehicle-treated animals. At 18 h after dosing of (496) at 10mg/kg, acumulative, statistically significant dose-dependent weight loss of 13 g wasobserved, compared with a 6 g gain for the vehicle-treated animals. A poorerresult was obtained with (497), with a net weight loss of 2 g versus a 10 gweight gain for the vehicle-treated animals. This result was, however, in linewith a poorer PK profile, resulting in lower bioavailability (F ¼ 30%) andslower brain penetration (B : P ¼ 0:8422:61 between 0.25 and 4 h).Stated to be particularly useful in the treatment of obesity, a series of 1,5-
diaryl-pyrrole-based compounds from AstraZeneca was claimed in a recentpatent application [313], including compound (509). Although no specificbiological data were presented, the CB1 receptor affinities are claimed to beless than 1 mM and preferably less than 200 nM.
PYRIDINE-, PHENYL-, PYRIMIDINE- AND PYRAZOLE-BASEDCB1 RECEPTOR ANTAGONISTS
Six-membered heterocycles have also been extensively used to mimic the centralpyrazole scaffold of rimonabant (382). Merck and Co. has utilised pyridines(510) [314] , and pyrimidines (511) [315] in this capacity. Sanofi-Aventis has alsoclaimed a series of pyridine-based analogues [316], as exemplified by (512) andadditionally the non-heteroatom containing terphenyl (513) [317].Pfizer too, has filed a patent application to protect a series of closely related
pyrimidine-2-carboxamides [318]. Although no compounds are specificallyclaimed, the examples include compound (514) in which the chloro-substituted
RECENT PROGRESS IN CANNABINOID RESEARCH296
diaryl motif is once again present. The affinity of this analogue in the CB1
GTPgS binding assay was claimed to be less than 20nM.
N MePh
Ph
O
NHPh
N
Ph
OCH2Ph
Cl
OMe
O
N
N
Cl
Cl
Cl
O
F
(509)
(511)
(510)
O
NH
N
X
Cl
ClCl
N
N
N
O
Ph
Ac
Cl
ClCl
(512) X = N(513) X = CH
(514)
In a recent communication from Merck [319], the synthesis and biologicalactivities of several 5,6-diarylpyridine derivatives as CB1 inverse agonistswere described. From the initial HTS screening on the Merck sample col-lection was identified (515) which was found to have moderate activity(IC50 ¼ 530 nM) at the human CB1 receptor. It was proposed that thepyridine ring behaved as an alternative scaffold to the pyrazole ring of (382),with the pyridine nitrogen overlapping with the N2 atom of the pyrazole. Inaddition, it was speculated that the 3-cyano group might act as a bioisostere
J. ADAM ET AL. 297
for the carboxamide of (382). Investigation of the phenyl substitution of thisseries yielded significant improvement in CB1 potency (Table 6.46).
(515)
N
O
NMe2
CN
MeO
The initial 4-Cl analogue (516) was found to exhibit only modest affinity,however derivative (517) showed CB1 inverse agonist activity comparablewith (382) while also demonstrating good selectivity over the CB2 receptor.
Table 6.46 5,6-DIARYLPYRIDINE DERIVATIVES – IN VITRO BINDING DATA [319]
(382) 6.2 (522) Cl Cl Cl 3-Cl 5.8(516) H H Cl H 2,800 (523) Cl Cl Cl 4-CF3 3.4(517) Cl Cl Cl H 11 (524) Cl H Cl 3,4-di-F 18(518) Cl Cl Cl 4-F 3.1 (525) Cl Cl H 3,4-di-F 8(519) Cl Cl Cl 3,5-di-F 1.8 (526) F Cl Cl 3,4-di-F 7.2(520) Cl Cl Cl 2,4-di-F 2.7 (527) Me Cl Cl 3,4-di-F 6.3(521) Cl Cl Cl 3,4-di-F 1.3 (528) F F F 3,4-di-F 66
RECENT PROGRESS IN CANNABINOID RESEARCH298
The 4-fluoro (518), 4-trifluoromethyl (523) and 3-chloro derivatives (522)also showed improved binding. 3,5-Disubstitution was also well tolerated(519), however the 3,4-difluoro compound (521) had the most potent inverseagonism of the series with an EC50 of 8 nM, with an average 110% maximalresponse and 400-fold selectivity over CB2. In addition to the crucial role ofthe 4-chloro substituent on the 5-phenyl ring, the 2,4-dichloro substitutionon the 6-phenyl ring, which mimics that of (382), was also found to beoptimal.Replacement of the 3-cyano functionality was also investigated with a
series of 3-amido derivatives (Table 6.47). Of these, the 3,4-difluorobenzylanalogues were the most potent. Modification, including lengthening andbranching the R2 and R3 alkyl groups have comparatively little effect onpotency (cf. (529)–(531)). Replacement of the 3,4-difluorobenzyl motif withn-butyl did however considerably reduce the CB1 binding affinity.Cyclohexylmethyl did appear to be tolerated in this position with com-pounds approximately equipotent with their benzyl counterparts (cf. (529)and (537)) suggesting that this group binds in a lipophilic pocket rather thanmaking a direct aromatic interaction.
Table 6.47 5,6-DIARYL-3-AMIDOPYRIDINE DERIVATIVES – IN VITRO BINDINGDATA [319]
N
Cl
OR1
Cl
Cl
O NHR2
Cpd. R1 R2 CB1 IC50 (nM) Cpd. R1 R2 CB1 IC50 (nM)
(529) 3,4-di-F-Bn Me 3.1 (534) n-Bu H 32(530) 3,4-di-F-Bn Et 1.9 (535) n-Bu Me 17(531) 3,4-di-F-Bn n-Pr 1.7 (536) n-Bu n-Pr 21(532) 3,4-di-F-Bn (CH2)2F 1.5 (537) CH2(c-Hexyl) Me 3.4(533) 3,4-di-F-Bn i-Pr 1.8 (538) CH2(c-Hexyl) n-Pr 5.2
Table 6.48 5,6-DIARYL 2-AMINOPYRIDINE DERIVATIVES – IN VITRO BINDINGDATA [319]
N
ClCl
Cl
NR1R2
O
Cpd. R1 R2 CB1 IC50 (nM) Cpd. R1 R2 CB1 C50 (nM)
(539) –(CH2)5– 340 (544) H c-Heptyl 35(540) H n-Hexyl 43 (545) H Piperidin-1-yl 56(541) H n-Hept-4-yl 32 (546) H Ph 210(542) H c-Pentyl 53 (547) H Bn 31(543) H c-Hexyl 95
J. ADAM ET AL. 299
Investigation of a 2-amido series was also carried out by Meurer et al.(Table 6.48). These compounds were found to be generally less potent thanthe 2-alkoxy-3-amido series with (545), the closest analogue of (382), amongthe most potent. In addition, the n-hexyl (540) and 4-heptyl (541) analogueswere found to have comparable affinity, as was the benzyl congener (547).The relatively poor binding of the 2- and 3-amido pyridine series comparedwith (382) was ascribed to either the difference in ring size between pyridineand pyrazole resulting in a slight change in relative orientation of substitu-ents, and/or the basicity of the nitrogen and finally the absence of a neigh-bouring methyl group to force the amide group of rimonabant into aposition perpendicular to the pyrazole ring.In vivo studies were also performed on those compounds deemed to have
the most favourable binding and functional assay data, (521) and (531).Pharmacokinetic studies carried out on Sprague-Dawley rats (1mg/kg i.v.,2mg/kg p.o.) using compound (521) suggested favourable i.v. PK properties(AUCnom ¼ 9:3mMhkg=mg; Clp ¼ 3:6ml=min=kg; Vdss ¼ 0:8L=kg; t1=2 ¼3:6 h), moderate oral absorption (F ¼ 27%) but slow brain penetration anda low brain:plasma ratio (B : P ¼ 0:0320:26 at 0.25–4 h). Compound (521)was subsequently investigated in a food intake and body weight loss
RECENT PROGRESS IN CANNABINOID RESEARCH300
study using diet-induced obese rats. In contrast to (382), oral administrationof (521) and 1, 3 and 10mg/kg did not lead to an immediate reduction infood intake at any of the doses, however after 18 h after dosing at 10mg/kg,a cumulative but non-significant (p >0.05) 22% reduction in food intakeand a dose-dependent weight loss (1 g compared with an 8 g gain for thevehicle-treated animals) was observed. It was postulated that the slow CNSpenetration witnessed in the earlier PK study was preventing an immediateeffect on the food intake.Pyrazines have been utilised first by AstraZeneca [320–325] and subse-
quently by Bristol-Myers Squibb [326]. Although no specific biological datawere presented in the initial two AstraZeneca patent applications, com-pounds including the specified (548) are claimed to have IC50 values lessthan 1 mM with the most preferred compounds having an IC50 value of lessthan 200 nM. Nine compounds are specifically claimed in the later As-traZeneca application [322] including (549) in which the N-piperidin-1-ylcarboxamide of (382) is replaced with the bioisosteric piperidin-1-yl-oxy-carbonyl. Subsequently, a tetrazole-containing 5,6-bis(4-methylphenyl)-pyrazine derivative (550) was reported to have an IC50 of 1.4 nM [323].A related tetrasubstituted compound, (551), which had been disclosed ge-nerically in an earlier AstraZeneca application [321], has been specificallyclaimed [324]. The affinity of this compound for the CB1 receptor was de-scribed only as less than 1 mM. Finally, from AstraZeneca, a third series oftetrasubstituted pyrazines was revealed [327], of which compound (552) wasstated to have excellent CB1 affinity (1.8 nM). The now-familiar diaryl-pyrazine carboxamides have more recently been the subject of a patentapplication from Bristol-Myers Squibb [326]. Of the several compoundsclaimed, (553) shows that a hydroxymethyl functionality is tolerated adja-cent to the carboxamide nitrogen but no specific data were presented in theapplication.
N
N
X
O
N
R1
R1
N
N
NH
O
N
R1
R1
R2
NN N
N
N
O
(548) R1 = H, X = NH
(549) R1 = Cl, X = O
(550) R1 = Me, R2 =
(551) R1 = Cl, R2 =
J. ADAM ET AL. 301
N
N
Me
Me
NH
O
Me
MeOH
N
N
Ot-Bu
O
Cl
Cl
O
N
(553)(552)
Merck has recently utilised a furo[2,3-b]pyridine core (554) as abioisosteric replacement for the pyrazole scaffold of rimonabant (382)[328]. The same basic pharmacophore, that of two halo-substituted arylgroups and a third hydrophobic motif proximal to a hydrogen-bond ac-ceptor, can be witnessed in the benzodioxole-based compounds, such as(555), disclosed by Roche [329].
N
O
NH2Ph
O
Cl
Cl
Cl
O
O
SN
O O
F
Cl
Cl
(554) (555)
AZETIDINE-BASED CB1 RECEPTOR ANTAGONISTS
A number of azetidine-based compounds have been disclosed in patent ap-plications from Aventis Pharma for CB1-modulated treatment of diseasessuch as obesity, Parkinson’s disease, schizophrenia, respiratory and neuro-logical diseases [330–334]. Compound (556) was specifically claimed for usein two formulation patent applications [330, 331] for a stable semi-solidcomposition and oral emulsion composition, respectively. The optional co-administration of an agent that activates norepinephrinergic and se-rotoninergic neurotransmission (for example, sibutramine) or dopaminergicneurotransmission was also claimed for the treatment of obesity. The op-tional use of a dopamine agonist (for example, levodopa) was claimed
RECENT PROGRESS IN CANNABINOID RESEARCH302
additionally for the reduction of dyskinesia in such neurological diseases asParkinson’s disease. Earlier Aventis patent applications disclose relatedazetidine derivatives such as (557) [334], (558) [333] and (559) [332]. Thestructural similarity to (382), of the three exemplified compounds shown, isless overt than the scaffold-hopping approaches discussed above, howeverthe p-chloro-substituted diaryl motif is preserved. The sulfonamide oxygenatoms may be performing the same hydrogen-bond acceptor role as thecarbonyl in rimonabant.
N
Cl Cl
NMeO2S R
N
Cl Cl
NHS
O O
S
N
ClCl
MeO2S
F
F
(557) R = 3,5-diF-phenyl(559) R = 5-Cl-2-pyridyl (558)(556)
O
ClCl
N NH
O
O
Cl
N N
O
N
Cl
BOC
(560) (561)
An azetidine motif was also present in two series of CB1 antagonist com-pounds disclosed by Vernalis Research [335, 336]. In the former, compound(560) was claimed to have an affinity of 285 nM in transfected HEK293 cellsusing tritium-labelled (382). Among the preferred indications were psycho-sis, schizophrenia, smoking cessation and eating disorders associated withexcessive food intake. Compound (561) was claimed to have an affinity of0.8 nM in the same binding assay [336].
J. ADAM ET AL. 303
In two related applications from Roche, a pyrrole/imidazole [337] or 2-methylpyrrole [338] perform the role of central scaffold. Among the com-pounds specifically claimed are (562) and (563), stated to be antagonists orinverse agonists. Interestingly, a cyclohexylmethyl group is present whereone might expect the more usual halo-substituted phenyl, and in this respect,may share a related pharmacophore to the pyrrole-based CB1 agonists de-scribed by Wiley et al. [181] in which the most potent analogues bear anextended pyrrole N-alkyl substituent.
N
O
NO
O
N
S N
O
O
N
(562) (563)
SUBSTITUTED AMIDE-BASED CB1 RECEPTOR ANTAGONISTS
A number of distinct CB1 receptor modulators have been claimed by Merckfor indications including eating disorder, obesity, psychosis and drug de-pendence. In the earliest [339], the familiar biaryl pharmacophore is pre-served, although a substituted acyclic propyl chain, rather than aheterocyclic one, serves to orient the phenyl rings and carboxamide groupin the required configuration (564). Two closely related series were alsodescribed in applications from Merck [340, 341]. Again, no specific biolog-ical data were presented, however the benzofuran (565) and benzodioxananalogues (566) typify the series. More recently, Merck has claimed a fur-ther two series of CB1 antagonists [342, 343] of which (567) and (568) werespecifically claimed in each of the series, respectively.
NH
NH
Cl
NC
Me O
Et NH
O
Me
Cl
Cl R O
O
O
(564)
(565) R =
(566) R =
RECENT PROGRESS IN CANNABINOID RESEARCH304
NH
O
Ph
Me O
Me
Me
ClCl
ONH
N
CF3
Me
MeOMe
OH
CN
Cl
(567) (568)
HYDANTOIN-BASED CB1 RECEPTOR ANALOGUES
A series of 29 3-alkyl 5-arylimidazolidinones, or hydantoins, active at theCB1 receptor has been published by Kanyonyo et al. [344] with a subsequentpublication describing the relationship between the experimentally derivedlipophilicity and proposed modes of binding for non-polar and polar hyd-antoins derivatives [345] (Table 6.49).The binding data for compounds (569)–(592) were obtained by screening
at a concentration of 10 mM in a competitive displacement experiment usingtritium-labelled (382) in CHO membranes transfected with the human CB1
receptor. Within the series, affinity was seen to improve as the alkyl chainlength, and hence lipophilicity was increased. Compounds (588) and (589)were, however, surprisingly active for their calculated lipophilicity. The lip-ophilicity of the hydantoin analogues was measured by reverse-phase HPLCand found to correlate most closely with the values calculated by the CLIP[346] rather than CLOGP method [347]. In addition, para-substitution ofthe two phenyl rings improved potency, with 5,5’-bis(4-bromophenyl) sub-stitution, compounds (588)–(592), being optimal. For the non-polar hyd-antoins, the potency at the CB1 receptor decreased Br>OMe>F>Me>H,whereas no such relationship was evident for the non-polar compounds.Although certain polar groups, for example, morpholine (588) and hydroxyl(589) were well tolerated, the more basic amine group in compounds (570)and (571) had a strongly negative influence on binding. Molecular modellingstudies comparing the hydantoins with the non-classical cannabinoid HHCutilised molecular lipophilic potential and hydogen bond potential to gen-erate a superposition model. Two distinct binding modes, for polar and non-polar hydantoins, were identified. The three most potent compounds (588),(589) and (591) were further characterised using a [35S]-GTPgS binding as-say [348]. This assay allowed the test ligands to be defined as agonists (pos-itive intrinsic activity), partial agonists (partial positive intrinsic activity),antagonist (no intrinsic activity) or inverse agonists (negative intrinsicactivity). All three hydantoin ligands were found to behave as neutral
Table 6.49 3-ALKYL-5,50-DIPHENYLIMIDAZOLIDINEDIONES – IN VITRO DATA[344,345]
N
NH
O
O
R1
R1(CH2) R
2n
Cpd. R1 R2 n c logPa % Displacement at 10 mMb
(569) H N-morpholine 2 2.16 o5(570) H N-piperidine 2 3.83 o15(571) H NMe2 2 2.83 o5(572) H Me 2 3.84 o20(573) H Me 3 4.26 25.1(574) H Me 4 4.8 35.4(575) H Me 5 5.37 35.6(576) H Me 7 6.50 61.2(577) H Ph 1 4.11 40.6(578) H i-Pr 0 2.89 o5(579) Me N-morpholine 2 3.49 23.9(580) Me Me 5 6.48 46.8(581) Me Me 6 7.04 51.3(582) OMe N-morpholine 2 2.73 21.7(583) OMe Me 5 5.81 66.6(584) F N-morpholine 2 2.81 30.3(585) F Me 5 5.81 40.6(586) F Me 6 6.42 51.4(587) F Me 7 6.94 62.5(588) Br N-morpholine 2 3.86 91.2(589) Br OH 3 3.76 88.4(590) Br Me 5 6.87 72.1(591) Br Me 6 7.45 89.2(592) Br Me 7 7.99 80.0
aLipophilicity calculated using the CLIP method.bResults expressed as the percentages of the displaced specific binding of [ 3H]-SR-141716A(mean7SEM, n ¼ 325).
J. ADAM ET AL. 305
antagonists with no intrinsic activity, but competitively inhibiting HU210-induced [35S]-GTPgS binding in rat cerebellum homogenates.A series of closely related thiohydantoins was subsequently investigated
by Muccioli et al. [349]. The key data are summarised in Table 6.50.
RECENT PROGRESS IN CANNABINOID RESEARCH306
Replacement of the oxygen in the 2-position of the hydantoin ring withsulfur was found to increase CB1 receptor potency between 2- and 4-fold. Asin the preceding series, para-substitution of the phenyl rings was found toincrease potency, with iodo-susbstitution, e.g. (602), showing greater affinitythan Br-substitution, e.g. (598), which in turn was more potent than thecorresponding chloro analogue (593) (R2 ¼ iBu). Substitution on the N3-position was also found to have a pronounced influence on CB1 bindingaffinity. Increasing the chain length to over six carbon atoms resulted in amarked loss of activity. Benzyl substitution (594) was better tolerated thanthe corresponding phenyl ethyl congener (595), while the truncated allylgroup, typified by analogues (600) and (603), was found to be optimal in thisseries. The most potent thiohydantoin analogue identified was found to beconsiderably less potent than rimonabant (382) (K i ¼ 589 and 5.4 nM, re-spectively), however, the compounds were selective over the CB2 receptor.Compounds (593), (594) and (600)–(602) were examined in the [35S]-GTPgSbinding assay and, like the related hydantoin series, found to behave asinverse agonists at the CB1 receptor.
Table 6.50 3-ALKYL-5,50-DIPHENYLTHIOXOIMIDAZOLIDIN-4-ONES – IN VITRODATA [349]
An analogue of (-)-cannabidiol, O-2654 (604) has recently been disclosed asa high-affinity CB1 antagonist ligand [350]. Compound (604) was found to besignificantly more potent (K i ¼ 114 nM) than cannabidiol (K i ¼ 4:9mM) inthe displacement of tritium-labelled CP 55,940 in mouse brain tissues. Thevalue obtained for (604), while less potent than that reported for (382)(1.98–12.3 nM), is comparable with the early Lilly antagonist LY320135(K i ¼ 141 nM) [351]. In addition, (604) was found to antagonise WIN 55,212-2 by competing with the agonist for the CB1 receptors. Unlike (382), thecannabidiol analogue is thought to behave as a neutral antagonist.
RECENT CB1 RECEPTOR ANTAGONISTS
A recent patent application from Roche [352] described a 2-amino-benzothiazole series. Roche claimed that compound (605) exhibited anIC50 value of 0.73 mM at CB1, and showed in excess of 10-fold selectivityover the CB2 receptor. The compounds were described as being of potentialuse in the treatment of a range of diseases, including CNS and psychiatricdisorders, type-2 diabetes, gastrointestinal diseases, cardiovascular disor-ders, infertility disorders, inflammation, cancer, atherosclerosis, cerebralvascular incidents and cranial trauma.A series of 12 tetrahydroquinoline compounds, functioning as antagonists
or inverse agonists of the CB1 receptor, has been disclosed [353]. The req-uisite three hydrophobic groups, in the example (606), three unsubstitutedphenyl groups are present, as is an adjacent hydrogen-bond acceptor (eitherthe 2-oxo group of the central heterocycle, or the sulfonamide oxygen). Asulfonamide moiety was also present in a patent application, which at thetime of writing is the most recent disclosure from Merck and Co. [354].Again, a branched butyl chain forms the backbone from which two arylgroups are appended (607). CB1 functional activity for the compounds wasclaimed to be lower than 1 mM while the CB2 functional activity was re-ported to be higher than 1 mM.
Our current understanding of the potential therapeutic applications of CB1
antagonists owes a great deal to the discovery of rimonabant (382). Indeed,clinical data demonstrating the efficacy of (382) in the treatment of obesityand nicotine addiction has provided a substantial driving force for the ex-panding research effort into this approach.The increase in appetite resulting from cannabis use has long been rec-
ognised and has also been observed in animals that have been administeredcannabinoid agonists [355]. The endocannabinoid system also appears toplay a key role in the regulation of appetite [356]. Endocannabinoid levels inthe limbic forebrain have been shown to increase with food deprivation,while 2-AG levels in the hypothalamus reduce during feeding [357]. Anand-amide (1) administration into the ventromedial hypothalamus has also beenshown to stimulate appetite in rats [358]. The hypothalamus uses leptin asthe primary signal to modulate food intake and energy balance and there isevidence linking leptin and endocannabinoids in the regulation of food in-take [359]. Di Marzo and co-workers associated defective leptin signallingwith elevated hypothalamic levels of endocannabinoids in obese db/db andob/ob mice and Zucker rats, cerebellar endocannabinoid levels were notaffected. Acute leptin treatment of normal rats and ob/ob mice reduced (1)and 2-AG levels in the hypothalamus.In additon to the central role of endocannabinoids in the regulation of
feeding behaviour, a peripheral role has also been described. Gomez and co-workers [360] reported that food deprivation produced a 7-fold reduction in(1) levels in the small intestine of rats, but not in the brain or stomach.Intestinal (1) levels returned to normal when feeding resumed. The authorsalso showed that peripheral, but not central administration of (382) reducedfood intake. The endocannabinoid system has also been reported to regulateperipheral lipogenesis [361].
J. ADAM ET AL. 309
The role of the endocannabinoids and exogenously administered agonistsoutlined above led to an interest in testing the effects of selective CB1 receptorantagonists on feeding behaviour [261, 362, 363]. Rimonabant (382) andSR147778 (385) have been shown to reduce food intake and weight gain inordinary rats without altering water intake [270, 364–366]. Rimonabant (382)has also been shown to cause a transient decrease in food intake accompaniedby a sustained decrease in bodyweight and adiposity in dietary-induced obesemice [367]. Leptin, insulin and glucose levels were all normalised in these micefollowing (382) treatment, together with an increase in serum adiponectinlevels [367, 368]. While (382) did not modify HDL-cholesterol and had mod-est effects on total cholesterol, it significantly reduced triglycerides and LDL-cholesterol and increased the HDL/LDL-cholesterol ratio.CB1 knock-out mice exhibit resistance to dietary-induced obesity in terms
of food intake, weight gain, body fat composition and the development ofdietary-induced insulin resistance [369]. The effects of (382) on feeding be-haviour and body weight are not observed in these animals, confirming thatthese are mediated through the CB1 receptor [359, 367]. CB1 knock-out micedo not demonstrate a decrease in food intake under free feeding conditions,but food intake is reduced following food deprivation.Clinical trials with (382) have provided the most exciting evidence for the
utility of CB1 antagonists in the treatment of obesity, results of the Rimo-nabant in Obesity (RIO)-Europe trial having been published very recently[370, 371]. In this trial, 1,507 patients with body-mass index of 30 kg/m2 orgreater, or body-mass index greater than 27 kg/m2 with treated or untreateddyslipidaemia, hypertension or both, were randomised to receive double-blind treatment with placebo, 5mg (382) or 20mg (382) once daily in ad-dition to a mild hypocaloric diet (600 kcal/day deficit). The primary efficacyendpoint was weight change from baseline after 1-year treatment in theintention-to-treat population. Weight loss at 1 year was significantly greaterin patients treated with (382) 5mg (mean �3.4 kg) and 20mg (�6.6 kg)compared to placebo (�1.8 kg). Significantly more patients treated with(382) 20mg than placebo achieved weight loss of 5% or greater (50.9%versus 19.2%) and 10% or greater (27.4% versus 7.3%). Rimonabant (382),20mg, produced significantly greater improvements than placebo in waistcircumference, HDL-cholesterol, triglycerides, insulin resistance and prev-alence of metabolic syndrome. The effects of (382) 5mg were of less sig-nificance. (382) was generally well tolerated with mild and transient sideeffects including nausea, dizziness, diarrhoea and vomiting.Rimonabant (382) has also shown promise in pre-clinical and clinical
studies as an aid to smoking cessation. (382) decreases nicotine self-admin-istration in rats and nicotine-induced dopamine release in nucleus acumbens[372], and also reversed nicotine-seeking behaviour in rats several weeks
RECENT PROGRESS IN CANNABINOID RESEARCH310
after nicotine withdrawal [373]. In a 10-week, placebo-controlled trial, (382)was shown to prolong abstinence rates from tobacco during the final 4weeks of the treatment period [370]. Furthermore, while patients in placebogroups in several studies gained weight, (382) patients lost weight or expe-rienced less weight gain than those on placebo. Weight gain following nic-otine withdrawal is a major factor in reducing abstinence rates. In additionto their utility as aids to smoking cessation, CB1 receptor antagonists haveshown potential pre-clinically in the treatment of addiction to other sub-stances including morphine, heroin and alcohol [374–377].Rimonabant (382) was also included in a clinical study to assess the safety
and efficacy of four novel compounds for the treatment of schizophreniaand psychoaffective disorder [378]. The other compounds included in thetrial were a neurokinin NK3 antagonist, a serotonin 2A/2C antagonist and aneurotensin NTS1 antagonist. Haloperidol and placebo groups were used ascontrols in the study. Sixty-nine patients received (382) (20mg once perday), which failed to demonstrate efficacy in this trial. The reasons for thelack of efficacy may be due to inadequate dosing or an indication that CB1
antagonism is not appropriate in the treatment of this condition.Pre-clinical data support the potential application of CB1 antagonists in
the treatment of various other conditions. These include memory disorders[379], sexual dysfunction [380], neuro-inflammation [381] and asthma [382].
CB2 RECEPTOR ANTAGONISTS
CB2 receptor antagonists have received much less attention than their CB1
counterparts, with only a relatively small number of compounds availableand less clarity on their potential therapeutic role. A selection of the avail-able compounds that have been shown to act as antagonists of the CB2
receptor and their suggested utilities will be covered in this section.As observed in the CB1 antagonist section, pyrazole derivatives again
form an important class of antagonists for the CB2 receptor. A structuralanalogue of rimonabant (382), SR144528 (608), is a potent CB2 antagonist/inverse agonist identified by Sanofi [383]. This compound has proved to be auseful tool in determining the function of CB2 receptors. The compound hassubnanomolar affinity for CB2 receptors with 700-fold selectivity over CB1
receptors. Following oral administration, (608) totally displaced the ex vivo[3H]-CP 55,940 binding to mouse spleen membranes without interactingwith CB1 receptors in the brain.Conformationally restricted pyrazole-derived CB2 selective antagonists
were described in a patent application from Sanofi-Synthelabo [384]. Com-pounds included in the application, exemplified by compound (609) are
J. ADAM ET AL. 311
claimed to act as antagonists at CB2 receptors (Kio5� 10�7M) with se-lectivity of at least 10-fold over CB1 receptors.Moving away from pyrazole-derived compounds, an aminoalkyl-indole,
AM630 (610), acts as a CB2 receptor antagonist/inverse agonist with a CB2 Ki
value of 31.2nM and 165-fold selectivity over the CB1 receptor subtype [385].It is interesting to note that this compound has been shown to act as a low-affinity partial agonist, antagonist or inverse agonist at CB1 receptors [6].Iwamura and Ueda [386] described compound (611) as a CB2 selective
inverse agonist in a patent application. The potential therapeutic roles ofCB2 antagonists are not clearly defined at the moment, although roles inregulation of the immune system and inflammation have been widely pro-posed. This patent application describes that activity of compound (611) ina mouse model of asthma, in which the compound suppressed immediateand late-phase asthmatic response and airway hyper-responsiveness.
NN
Cl
NH
O
NN
Cl
NH
O N
Cl
Cl
S
(608) SR144528 (609)
NI
N
O
O
MeO
NH
NH
O
O
O
O
O
O
(610) AM630 (611)
RECENT PROGRESS IN CANNABINOID RESEARCH312
A series of tricyclic pyrazoles has been synthesised and their CB1 and CB2
affinity determined in mouse cerebellum membranes and mouse spleen ho-mogenate, respectively [387]. Table 6.51 describes some of the pertinent data.Despite the close structural similarity between rimonabant (382) and the
fused pyrazole analogues described by Mussinu and co-workers [276], themajority of the latter show a moderate-to-strong preference for the CB2
receptor. In particular, (612), the closest homologue to (382) within theseries, shows very low nanomolar affinity (K i ¼ 0:34 nM) at CB2, while onlycomparatively modest CB1 affinity (2,050 nM). 6-Fluoro (613) and 6-bromo(614) analogues were also potent and selective CB2 ligands, while substi-tution of the larger 6-iodo group (615) resulted in a decrease in both affinityand selectivity. The unsubstituted analogue (618) exhibited a profile similar
to that of the 6-Cl congener (612). Movement of the 6-Cl substituent to C5slightly increased CB2 affinity, while the 7-Cl analogue (617) witnessed aslight decrease in CB2 binding. The 6-Me analogue (619) was found to be themost potent and selective ligand, eclipsing the 250-fold CB2-selective Sanofi-Aventis ligand, SR144528 (608).
SUMMARY AND FUTURE PROSPECTS
It is hoped that the scope of this review article has given the reader a goodimpression of the rate at which cannabinoid research is progressing. The lastdecade has seen a rapid growth in cannabinoid research, which looks set tocontinue. The therapeutic potential of cannabinoid agonists has long beenacknowledged through the use of cannabis for medicinal purposes. How-ever, the accompanying psychotropic effects have restricted the usefulness ofthis approach. We are now starting to observe that at least some of thebenefits of cannabinoids can be realised without CNS side effects throughstrategies such as peripheral restriction of compounds, CB2 subtype selec-tivity and subtle modulation of the endocannabinoid system. Improved drugdelivery systems are also proving beneficial in this regard, although beyondthe scope of this review.Antagonism of cannabinoid receptors is now attracting a huge amount of
attention, following on the successful rimonabant (382) clinical trials. At thetime of writing, a launch date in 2006 is anticipated for (382) and it will beintriguing to observe the future of this mechanistic approach in the treat-ment of various conditions, particularly obesity. With an ever-growingknowledge of the endocannabinoid system, and the possible identification offurther receptor subtypes, the future of cannabinoid research seems verybright indeed.
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