Bioorganic & Medicinal Chemistry Letters 24 (2014) 1322–1326

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Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier .com/ locate/bmcl

Switching cannabinoid response from CB2 agonists to FAAHinhibitors

http://dx.doi.org/10.1016/j.bmcl.2014.01.0560960-894X/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Tel.: +33 3 20 96 49 06; fax: +33 3 20 96 43 74.E-mail address: regis.millet@univ-lille2.fr (R. Millet).

Aurélien Tourteau a, Natascha Leleu-Chavain a, Mathilde Body-Malapel b, Virginie Andrzejak a,Amélie Barczyk a, Madjid Djouina b, Benoit Rigo c, Pierre Desreumaux b, Philippe Chavatte a, Régis Millet a,⇑a Université Lille Nord de France, EA4481, Institut de Chimie Pharmaceutique Albert Lespagnol, IFR114, 3 rue du Pr. Laguesse, BP83, F-59006 Lille, Franceb Université Lille Nord de France, U995 INSERM, Digestive Inflammatory Diseases: Pathophysiology and Development of Therapeutic Targets, IFR114, Amphis J&K, Boulevard duPr. Leclercq, F-59045 Lille, Francec Université Catholique de Lille, HEI, EA4481, Laboratoire de Pharmacochimie, 13 Rue de Toul, F-59046 Lille, France

a r t i c l e i n f o a b s t r a c t

Article history:Received 7 November 2013Revised 14 January 2014Accepted 18 January 2014Available online 28 January 2014

Keywords:CB2

FAAHIsoxazolesCannabinoidIBD

A series of 3-carboxamido-5-aryl-isoxazoles designed as CB2 agonists were evaluated as FAAH inhibitors.The pharmacological results led to identify structure–activity relationships enabling to switch cannabi-noid response from CB2 agonists to FAAH inhibitors. Two compounds were selected for their FAAHand/or CB2 activity, and evaluated in a colitis model for their anti-inflammatory activity. Results showedthat compounds 10 and 11 inhibit the development of DSS-induced acute colitis in mice and then, areinteresting leads to explore new drug candidates for IBD.

� 2014 Elsevier Ltd. All rights reserved.

The endocannabinoid system includes two G-protein-coupledreceptors (CB1 and CB2), endogenous ligands, named endocannab-inoids (ECs), that bind to these receptors and several enzymesresponsible for the biosynthesis and the degradation of these ECs(ex: fatty acid amide hydrolase (FAAH), monoacylglycerol lipase(MAGL)).1–3

It has been established that the modulation of the endocannab-inoid system through the activation of cannabinoid receptors andthe inhibition of ECs degradation enzymes consists in a promisingtherapeutic strategy. Indeed, this system has shown beneficial ef-fects in several diseases like osteoporosis,4 pain5 or chronic inflam-mation disorders.6

The CB1 receptor is mainly expressed in the central nervous sys-tem, where it is one of the most abundant receptors.7 Its centrallocation is correlated with the behavioral effects of cannabinoids:memory, cognition and motor functions.8 On the other hand, theCB2 receptor is mainly located in cells of the immune system,9

and involved in the immunomodulatory effects induced by can-nabinoids.10 Because activation of CB1 receptors is responsible formost of the neurobehavioral effects of D9-tetrahydrocannabinol(THC) (hypermotility, analgesia, hypothermia, catalepsy),11 the

development of selective CB2 agonists without psychoactivitydue to CB1 appears to be crucial. Current research on selectiveCB2 agonists have shown a broad spectrum of pharmacological ef-fects, such as anti-inflammatory (JWH-133, GW-842,166X),12 anal-gesic (AM1241)13 and anticancer (JWH-133)14 activities (Fig. 1).

FAAH is a specific enzyme of long chain fatty acids derivatives15

located in both central and peripheral levels.16 It degrades ECs asanandamide (AEA), palmitoylethanolamide (PEA) or oleoylethan-olamide (OEA). Recently, FAAH inhibitors were developed to blockthe rapid catabolism of AEA and prolong its action. Among theseFAAH inhibitors, URB597 is one of the most potent known to date(Fig. 1) and is particularly active on acute, inflammatory or neuro-pathic pain,17–19 and protects against experimental colitis.20

Our group research aims to develop new treatments for inflam-matory bowel diseases (IBD), which two main forms are Crohn’sdisease and ulcerative colitis. These diseases affect more than2.2 million people in Europe and are characterized by an inflamma-tion of the gastrointestinal tract due to a hyperactivity of the im-mune system. The endocannabinoid system is implicated in guthomeostasis, gastrointestinal motility and IBD.21,22 Recently, ithas been demonstrated that cannabinoid receptors are overexpres-sed in vivo and in patients suffering from IBD.21,23 The beneficialrole of CB2 in intestinal inflammation has been highlighted in sev-eral in vivo and in vitro studies.14,24–26 Moreover, Massa et al.

O

N

NNH

O

NH

CF3

O

Cl

Cl

N

OI

O2NN

JWH-133 GW-842,166X

AM1241 SR144,528

NN

ONH

Cl

OHN

O

O NH2

URB597

O

ON

ONH

ALIAE809 (compound 13)

Figure 1. Structures of representative selective CB2 ligands (GW-842,166X,JWH-133, AM1241, SR144, 528, ALIAE809) and the FAAH inhibitor URB597.

A. Tourteau et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1322–1326 1323

showed that the genetic invalidation of FAAH in animals with 2,4-dinitrobenzene sulfonic acid (DNBS)-induced colitis significantlyreduced tissue inflammation.27 Storr et al. showed that URB597 in-creased survival and decreased inflammation in mice sufferingfrom trinitrobenzene sulfonic acid (TNBS)-induced colitis.20,28

According to the implication of the endocannabinoid system inIBD, we decided in our laboratory to develop selective CB2 agonistsand FAAH inhibitors to treat these diseases. Recently, we described3-carboxamido-5-aryl-isoxazoles as selective CB2 agonists.29 Thisseries of isoxazoles possesses a 2-substituted phenyl group at po-sition 5 and a bulky aliphatic group on the carboxamide function.Our best selective CB2 agonist is compound 13 (ALIAE809, Fig. 1)with a Ki of 9.0 nM on hCB2 receptors.

Then, we decided to further explore our 3-carboxamido-5-aryl-isoxazoles selective CB2 agonists for their capacity to inhibit FAAH.Indeed, because CB2 agonists and FAAH inhibitors are very potentmodulators of the endocannabinoid system, it would be very inter-esting to combine both activities in one molecule in order to obtaina synergistic activity of the compound and enhance its therapeuticproperties. Thus, 18 of our previously described isoxazoles weretested for their FAAH inhibition potential. A comparison of theCB2 affinity and FAAH inhibition capacity of these compoundswas carried out and structure–activity relationships wereestablished.

As previously described,29 the 3-carboxamido-5-aryl-isoxazoles1–18 were obtained in four steps from various ketones. Their syn-thesis consists in a Claisen condensation of the corresponding ke-tones, followed by a cyclization into ethyl isoxazole-3-carboxylates. The target compounds 1–18 were finally obtainedby saponification of the ethyl ester function followed by amidation.

The 3-carboxamido-5-aryl-isoxazoles 1–18 were evaluated fortheir ability to inhibit the hydrolysis of [3H]-AEA by a recombinanthuman FAAH preparation (expressed in Escherichia coli).17–19 Allcompounds were first screened at a concentration of 10 lM fortheir inhibitory activity toward the AEA-degradation enzyme. The

half maximal inhibitory concentrations (IC50) were determinedfor compounds exhibiting a specific inhibition superior to 50% forFAAH. In Table 1, all biological results towards CB2 and FAAH weresummarized.

As previously described,29 the best selective CB2 agonistsamong these 3-carboxamido-5-aryl-isoxazoles are compoundsbearing an O-alkyl chain at the ortho position of the phenyl groupat position 5 (R1) and a bulky aliphatic group on the carboxamidefunction at position 3 (R2) (Table 1). The optimal length for the O-alkyl chain is 5–6 carbons.

As shown in Table 1 and 7 isoxazoles possess an IC50 on FAAHless than 10 lM. Among these 7 molecules, compounds 10 and11, which bear an adamantyl group on the carboxamide functionand an O-pentyl chain at the meta and para position on the phenylgroup at position 5 respectively, showed the best FAAH inhibitoryactivities with Ki values of 63 nM and 8 nM. On the other hand,compound 9 with the O-pentyl chain at the ortho position inducedno FAAH inhibition. In contrast with the results obtained with thein vitro CB2 affinity assay, an O-pentyl chain at the meta and paraposition but not at the ortho position of the phenyl group is favor-able for FAAH inhibitory activity.

Furthermore, compounds 1 and 2, which possessed no affinityfor the CB2 receptor, present a weak FAAH inhibitory activity. Weobserved that compound 1, with a 1-adamantyl group on the car-boxamide function and a non-substituted phenyl group at position5, present a 10-fold better FAAH inhibitory activity compared tocompound 2 with a 2-adamantyl group on the carboxamide func-tion. When the 1-adamantyl group is replaced by a 1-adamantylm-ethyl group (compound 3), the FAAH inhibitory activity is lost.

Interestingly, 3 of our selective CB2 ligands (4, 17 and 18) inhibitFAAH in moderate concentrations (4: IC50 = 0.912 lM; 17:IC50 = 5.43 lM; 18: IC50 = 2.33 lM). Compound 4, with an adaman-tyl group on the carboxamide function at position 5 and an O-methyl moiety on the phenyl group at the ortho position, possesseda FAAH inhibitory activity with an IC50 value of 0.912 lM and agood CB2 affinity with a Ki value of 70.1 ± 5.4 nM. Herein, we canconsider compound 4 as a dual drug in spite of a moderate FAAHactivity.

Considering their good FAAH inhibitory activity, compounds 10and 11 have been selected for an in vivo study in a DSS-inducedcolitis model. Specific Pathogen Free male 7 weeks old C57/Bl6mice received 2.5% DSS in drinking water during 8 days. Concomi-tantly, they were administrated daily intraperitoneally with com-pounds 10 or 11 in hydroxypropyl b cyclodextrine (150 mM) atthe dosage of 10 mg/kg body weight. Positive control mice were in-jected with the known FAAH inhibitor URB597 dissolved in thesame vehicle at the same dosage. Control mice were injected withvehicle only. DSS administration leads to progressive weight loss,which is one of the main symptoms of colitis in mice, or in humansuffering from IBD. After 8 days of DSS administration, body weightvariation and colon weight/size ratio both reflecting colitis inten-sity, were measured. Compared with the body weight variationin the vehicle-treated group, mouse body weight was higher inmice treated with 11 (11: 106.3 ± 1.2% vs vehicle: 100.0 ± 1.8%,p 6 0.001, Fig. 2A). Body weight variation was also improved inURB597 and 10 treated mice, without reaching significant thresh-old. Besides, mice treated with 10 and 11 presented a colonweight/size ratio significantly lower than vehicle-treated mice(100.0 ± 7.0% for vehicle, 90.3 ± 2.2% for 10 (p = 0.02),83.2 ± 13.1% for 11 (p = 0.009), Fig. 2B), revealing a less severeintensity of colitis.

Furthermore, histological sections of colon from mice treatedwith vehicle, URB597, 10 and 11 were scored for histological dam-ages (Fig. 3A). The extent of colon inflammation was decreased inmice treated with 11 (58.8 ± 7.7%, p = 0.02) relative to mice treatedwith vehicle (100.0 ± 6.8%). Colons of mice treated with vehicle

Table 1Affinities (Ki values) towards hCB2 and hCB1, half-maximal effective concentration (EC50) and maximum efficacy (Emax) on hCB2 and half-maximum inhibitory concentration (IC50)towards FAAH of compounds 1–18 and reference compounds (WIN-55,212-2, URB597)

ON

NHO

R1

R2

Compounds R1 R2 hCB2 hCB1 [35S]-GTPcS (hCB2) FAAH

Ki (nM) Ki (nM) EC50 (nM) Emax (%) IC50 (lM)

1 Ph 1-Adam >1000 ND ND ND 0.459 ± 0.0302 Ph 2-Adam >1000 ND ND ND 4.0 ± 0.23 Ph 1-(Adam)methyl >1000 ND ND ND >104 2-CH3OPh 1-Adam 70.1 ± 5.4 >3000 2.6 ± 0.3 155 0.912 ± 0.0445 2-C2H5OPh 1-Adam >1000 ND ND ND >106 2-C3H7OPh 1-Adam 79.6 ± 5.8 >1000 2.2 ± 0.3 144 >107 2-C4H9OPh 1-Adam 369 ± 62 >3000 2.9 ± 0.3 147 >108 2-C5H11OPh 1-Adam 36.0 ± 3.4 >3000 2.9 ± 0.3 151 >109 2-C6H13OPh 1-Adam 30.5 ± 6.4 >3000 3.4 ± 0.3 157 >10

10 3-C5H11OPh 1-Adam >1000 ND ND ND 0.063 ± 0.00411 4-C5H11OPh 1-Adam >1000 ND ND ND 0.008 ± 0.00112 2-C5H11OPh 2-Adam 60.1 ± 6.2 >3000 14.8 ± 3.8 270 >1013 2-C5H11OPh 3-Noradam 9.0 ± 0.6 >1000 4.8 ± 0.3 234 >1014 2-C5H11OPh Cyclobutyl 412 ± 71 >1000 4.4 ± 0.4 184 >1015 2-C5H11OPh Cyclopentyl 60.9 ± 0.9 >1000 2.1 ± 0.3 160 >1016 2-C5H11OPh Cyclohexyl 22.8 ± 4.6 >3000 1.9 ± 0.3 161 >1017 2-C5H11OPh Cycloheptyl 60.8 ± 6.9 >3000 11.1 ± 3.4 233 5.43 ± 0.3018 2-C5H11OPh Cyclooctyl 73.5 ± 6.8 >3000 1.8 ± 0.3 167 2.33 ± 0.20WIN-55,212-2 9.1 ± 0.8 16.1 ± 6.0 2.0 ± 0.3 243URB597 0.015 ± 0.5

Vehicl

e

URB597 10 11

0

50

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****

Col

on w

eigh

t/siz

e ra

tio

Vehicl

e

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0

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Mou

se b

ody

wei

ght

A B

Figure 2. Effects of URB597, 10 and 11 daily treatment (10 mg/kg, IP) on mouse body weight change on day 8 (A) and colon weight/size ratio (B) in DSS-induced acute colitis.Values are expressed as a mean of percentage of variation compared with vehicle-treated mice ± SEM, n = 10 per group, ⁄⁄p <0.01, ⁄⁄⁄p <0.001.

1324 A. Tourteau et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1322–1326

only exhibited intense mononuclear and granulocytic infiltratewithin the mucosa and the submucosa and severe damages tothe mucosa (Fig. 3B). This inflammatory infiltrate was reduced incolons of the mice treated with URB597, 10 and 11. In the micehaving received the compound 11, the epithelial architecture waspreserved, with a drastic decrease of epithelial lesions observedcompared with the vehicle-treated group.

Myeloperoxidase activity reflecting neutrophil infiltration wasalso significantly diminished in colons of mice treated with 10compared to mice treated with vehicle (52.7 ± 6.5% vs100.0 ± 11.1% respectively, p = 0.03, Fig. 4A). Finally, colon levelsof major inflammatory mediators were quantified by real-timePCR (Fig. 4B). Treatment with compound 11 decreased significa-tively colon mRNA levels of IL17a (p = 0.007) and iNOS (p = 0.03).In conclusion, URB597 treatment decreased all the colitis parame-ters analyzed, but this amelioration never reached significant

threshold. Treatment with 10 also improved all the colitis markersanalyzed, and significantly decreased MPO activity in colon. Final-ly, compound 11 reduced significantly several hallmarks of colitisseverity, demonstrating its efficiency against DSS-induced acutecolitis in mice.

Then, 3-carboxamido-5-aryl-isoxazole is a good scaffold to de-sign selective CB2 agonists and FAAH inhibitors. According to thesubstituents on the heterocycle, these molecules showed an activ-ity either on CB2 or on FAAH. For selective CB2 agonists, 2 keystructural elements are essential: the presence of an alkoxy chainof 5 or 6 carbons at the ortho position of the phenyl group at posi-tion 5 and a bulky aliphatic group (adamantyl, noradamantyl) onthe carboxamide function at position 3. The presence and positionof the alkoxy chain is crucial to determine the biological activity ofthe molecule. When this chain is absent, no activity on CB2 is ob-served but the molecules showed an FAAH inhibition potential.

Vehicl

e

URB597 10 11

0

50

100

150

*H

isto

logi

cal S

core

A B

Figure 3. Histological scoring of colons from DSS-treated mice administrated with vehicle, URB597, 10 and 11 (A). Values are expressed as a mean of percentage of variationcompared with vehicle-treated mice ± SEM, n = 10 per group. ⁄p <0.05. Representative MGG stained sections of distal colon of vehicle, URB597, 10 and 11 treated mice (B,original magnification �5).

Tnf-alpha

Il1-beta Il1

7aiNOS

0

50

100

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200

250VehicleURB5971011

mRN

A re

lativ

e le

vels

Vehicl

e

URB597 10 11

0

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150

Col

onic

MPO

act

ivity

A B

Figure 4. Quantification in colons of mice with DSS-induced colitis treated with vehicle, URB597, 10 and 11 of: (A) myeloperoxidase activity, (B) Tnf-a, Il1-b, Il17a and iNOSmRNA expression levels. Values are expressed as a mean of percentage of variation compared with vehicle-treated mice, n = 10 per group. ⁄p <0.05, ⁄⁄p <0.01.

A. Tourteau et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1322–1326 1325

When this chain is at the ortho position, the molecules are goodselective CB2 agonists. While when this chain is at the meta or paraposition, the molecules are potent FAAH inhibitors. Herein, we alsoidentified several molecules targeting CB2 and FAAH together.

In addition, in vivo results showed that 3-carboxamido-5-aryl-isoxazole is particularly attractive for developing effective com-pounds to treat colitis. The first in vivo results (compounds 10and 11) confirm the potential of selective FAAH inhibitors in aDSS-induced colitis model.

Acknowledgments

The authors thank Ms. Perrine Six and Frederique Klupsch forthe LC–MS analyses, NMR and CUMA department of the Facultyof Pharmacy of the University of Lille 2 for the biophysics analyses,and the binding platform of ICPAL for the biological evaluations.

This work was financially supported by the ‘Conseil RégionalNord Pas De Calais’ (CPER PRIM).

Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.bmcl.2014.01.056.

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