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Discovery of potent, non-carbonyl inhibitors of fatty acid amide hydrolase (FAAH)

Sumithra Gowlugari, Jeff DeFalco, Margaret T. Nguyen, Carl Kaub, Candace Chi, Matthew A. J. Duncton, Daniel E. Emerling, Michael G. Kelly, John Kincaid and Fabien Vincent*
Received 4th June 2012, Accepted 19th July 2012 DOI: 10.1039/c2md20146a Fatty acid amide hydrolase (FAAH) inhibition is a promising target for the treatment of pain, anxiety and depression. The vast majority of FAAH inhibitors contain an electrophilic moiety and are known to react covalently with the enzyme. Herein we present the discovery of potent inhibitors, such as RN-450 29, which are based upon a novel tetrahydropyridopyridine scaffold lacking an obvious electrophilic site, and which appear to inhibit FAAH in a reversible and non-covalent manner.

Introduction
The endocannabinoid system comprises two G-protein coupled receptors, CB1 and CB2, their endogenous ligands, with anandamide (AEA 1) and 2-arachidonoyl glycerol (2-AG) being the most thoroughly characterized, and the enzymes controlling the levels of these signaling lipids through hydrolysis, fatty acid amide hydrolase (FAAH) for AEA, and monoacylglycerol lipase (MAGL) for 2-AG.1 Isolated and characterized in 1996, FAAH has become the focus of intense research efforts, both within the academic community and the pharmaceutical industry. The latter interest has arisen from FAAH inhibition promising to harness at least some of the known therapeutic benets of cannabinoids and CB1/CB2 agonism (e.g. analgesia, anti-emesis, anxiolysis and anti-inammation), while displaying none of the equally well known psychotropic side-effects.2,3 FAAH possesses a highly nucleophilic serine, S241, which together with K142 and S217, forms part of an unusual catalytic triad within the active site of the enzyme. The vast majority of known FAAH inhibitors take advantage of the powerful reactivity of this catalytic serine, and contain electrophilic moieties (i.e., activated ketone, carbamate and urea) which form a covalent bond with the enzyme.4,5 For example, OL-135 2 benets from covalent and reversible hemi-ketal formation with S241, while URB-597 3 reacts in an essentially irreversible manner with the same residue.5 Early covalent inhibitors (e.g. 25) have been used to convincingly document the potential therapeutic effects of FAAH inhibition in animal models of human disease.510 More

recently, several compounds, notably PF-4457845, and SSR-411298 have entered clinical development for pain and depression.11 While covalent modication provides undisputable advantages in terms of active site occupancy, clinical candidates and drugs bearing reactive moieties are also over-represented in cases of idiosyncratic toxicity, potentially leading to failure in clinical trials or withdrawal from the marketplace.12 As robust in vivo efcacy has been observed for reversible, competitive inhibitors that benet from covalent hemiketal formation (e.g. OL-135 2), it may also be possible to develop non-covalent, reversible FAAH inhibitors.13 While some compounds have been reported to behave in a manner consistent with a reversible mechanism of inhibition, to the best of our knowledge only a limited number of these molecules, such as compound 6, have been thoroughly characterized as reversible and non-covalent inhibitors of FAAH.14 In this paper, we present the discovery of tetrahydropyridopyridine-based inhibitors of FAAH and characterize the mechanism of action for a representative probe compound, RN-450. The data indicate that tetrahydropyridopyridines such as RN-450 inhibit FAAH in a reversible and non-covalent manner (Fig. 1).15

Results and discussion

High-throughput screen and preliminary structureactivity studies An initial screen of our compound library was conducted against hFAAH using microsomal protein preparations and a uorescent assay readout.16 Amongst the different hit series, tetrahydropyridopyrimidine derivatives such as compound 7 were conspicuous, due to the lack of an obvious electrophilic moiety. Consequently, this scaffold was prioritized for exploratory structureactivity studies aimed at producing a tool compound suitable for detailed mechanistic studies. As tetrahydropyridopyrimidines are a relatively well-known substructure within
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Renovis, Inc. (a wholly-owned subsidiary of Evotec AG), Two Corporate Drive, South San Francisco, CA 94080, USA. E-mail: fabien_vincent_us@yahoo.com Electronic supplementary information (ESI) available: Assay protocols for FAAH inhibition and biochemical evaluation. Synthetic details for preparation of compounds 1530. See DOI: 10.1039/c2md20146a Present address: Pzer, Groton, CT, USA.

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Fig. 1 Anandamide and representative inhibitors of FAAH.

medicinal chemistry programmes,17 the corresponding tetrahydropyridopyridine scaffold (e.g. 15) was investigated rst. The synthesis of the tetrahydropyridopyridine core and of representative compounds with substituents at the 1-position of the tetrahydropyridopyridine ring are shown in Scheme 1. The results from our biological testing indicated that the FAAH enzyme was very sensitive to changes at this position (Table 1). For example, changing from quinolin-3-yl in compound 15 to quinolin-6-yl (compound 16) resulted in a greater than ve-fold reduction in potency. Additionally, removing the benzenoid ring in compound 15, to give rise to pyridine analogues such as 18 and 19, resulted in a dramatic decrease in FAAH inhibition. A similar dramatic decrease in potency was also observed when the secondary nitrogen in compound 15 was alkylated with a methyl group to give compound 20, thus showing the importance of an anilino NH to the activity of compound 15. We then proceeded to examine replacement of the benzyl group in lead structure 15. Thus, sulfonamides 2226, and alkyl derivatives 2730 were prepared by reaction of amine 21 with sulfonyl chlorides and alkylation respectively (Scheme 2). Some structureactivity relationships for sulfonamide and alkyl analogues are shown in Tables 2 and 3 respectively. Amide and urea derivatives of amine 21 were also prepared, but the resulting analogues displayed poor activity (not shown). As shown in Tables 13, discrepancies in potency between human and rat FAAH were apparent for numerous compounds. This phenomenon has been noted previously for a diverse range of inhibitors.18,19 For example, discrepancies amongst literature urea-based inhibitors such as PF-750 4 were demonstrated to

originate from differences in up to six amino acid residues between the two species.18 Accordingly, on the basis of its potency against both rat and human FAAH, we chose compound 29 (RN-450) as a tool compound with which to investigate the mechanism of action of inhibitors based on a tetrahydropyridopyridine scaffold. Biochemical investigations with RN-450 A variety of biochemical approaches were employed to characterize the interaction of RN-450 29 with hFAAH.20 First, its mechanism of inhibition was investigated using enzyme kinetics (Fig. 2A). The activity pattern observed in the presence of increasing concentrations of inhibitor (increased KM, stable Vmax) is characteristic of a reversible and competitive inhibitor (Ki 14.3 nM, 95% CI: 9.818.8 nM). Predictably, URB-597 3 displayed a reduced Vmax under similar conditions, an apparent non-competitive pattern also observed with irreversible enzyme inhibitors.20 Next, the effect of pre-incubation of compounds with hFAAH was evaluated (Fig. 2B). As expected, the IC50 value of carbamate URB-597 3 shifted signicantly (365 fold) when a 3 h pre-incubation with hFAAH was introduced. Such a lengthy pre-incubation, however, had no effect on the potency of RN-450 29 (1.7 fold shift), a result consistent with it being a reversible FAAH inhibitor. Dilution was the third biochemical approach employed to study the mechanism of RN-450 (Fig. 2C).20 Following an incubation of hFAAH with IC90 concentrations of select inhibitors, the reaction mixture was diluted 300 fold and the recovered enzymatic activity was then

Scheme 1 Synthesis of tetrahydropyridopyridines. Reagents and conditions: (a) EtI, CH2Cl2, rt, 86%. (b) NaCN, K2CO3, 50  C. (c) DMF.DMA, pyrrolidine, 130  C. (d) 48% aq. HBr, EtOH, reux, 40%. (e) BnBr, K2CO3, EtOH, H2O, reux, then (f) NaBH4, 0  C, 32% over 2 steps. (g) POCl3, reux, 80%, (h) R1NH2, Pd2(dba)3, NaOtBu, tBuOH, 120  C. (i) MeI, CH2Cl2.

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Table 1 Tetrahydropyridopyridine analogues as FAAH inhibitorsa

Table 2 SAR for tetrahydropyridopyridine sulfonamides

Compound

R1

R2

hFAAH IC50 (nM)a

rFAAH IC50 (nM)a hFAAH IC50 (nM)a rFAAH IC50 (nM)a

15

76 5.6

446 72

Compound

R1

22 16 H 479 1486

190

939

17

>10 000

ndb

23

93 17

638

24 18 H 2124 >10 000 25 19 H >10 000 nd 26 20 CH3 >10 000 nd

45

614

45

969

21 6

249

a Values are presented means sem of independent experiments, except when only an n 1 was obtained; URB-597 displayed an average IC50 of 3 nM under the conditions used. b nd: not determined.

a Values are presented means sem of independent experiments, except when only an n 1 was obtained; URB-597 displayed an average IC50 of 3 nM under the conditions used.

measured. Enzyme treated with carbamate URB-597 3 and urea PF-750 4 did not show any measurable activity recovery, thus conrming the irreversibility of their inhibition. On the other hand, enzyme treated with oleyl triuoromethyl ketone (OlCF3), a covalent yet reversible inhibitor of hFAAH, recovered a signicant amount of activity ($35%). Finally, treatment with

RN-450 29 led to $65% enzymatic activity recovery, demonstrating the reversible nature of its inhibition of hFAAH. As mass spectrometry has been used to document the covalent adduction of PF-750 4 to rFAAH,5 we used this same approach to study RN-450 29 (Fig. 3). As expected, enzyme incubated with PF-750 4 displayed a modied 213243 peptide, with a molecular mass consistent with the carbamoylation of catalytic serine S241. On the other hand, no such modication was detected using

Scheme 2 Preparation of sulfonamide and alkyl derivatives. Reagents and conditions: (a) H2, Pd/C, EtOH, rt, 18 h, (b) R0 SO2Cl, CH2Cl2, DIPEA, (c) CsCO3, NMP, RX.

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Fig. 2 Modication of rFAAH with PF-750 4 and RN-450 29. Biochemical studies on the mechanism of action of RN-450 29: (A) Enzyme kinetic analysis of hFAAH inhibition by URB-597 3 (upper panel) and RN-450 29 (lower panel); (B) Time dependency of hFAAH inhibition by URB-597 3 and RN-450 29; (C) Dilution experiment with hFAAH for URB-597 3, PF-750 4, RN-450 29 and O1CF3 recovered activity after dilution shown.

Fig. 3 Modication of rFAAH with PF-750 4 and RN-450 29. Mass spectrometry analysis of peptide 213243 from rFAAH protein treated with (A) RN-450 29, (B) DMSO (control) and (C) PF-750 4.

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Table 3 SAR for alkylated tetrahydropyridopyridines

Compound

hFAAH IC50 (nM)a 76 5.6 28 6

rFAAH IC50 (nM)a 446 72 449 98

15

27

28

2.6 0.8

606 64

29

13 3

25 11

30

>10 000

ndb

a Values are presented means sem of independent experiments, except when only an n 1 was obtained; URB-597 displayed an average IC50 of 3 nM under the conditions used. b nd: not determined.

RN-450 29. Similar results were observed following the use of an alternative method, 2D electrophoresis, to detect covalent bond formation (data not shown).

Conclusions
In summary, a novel class of FAAH inhibitors based on a tetrahydropyridopyridine scaffold lacking reactive electrophilic moieties was identied. Compound 29 (RN-450) displayed low nM potency against hFAAH, and after extensive biochemical characterization was found to be a reversible, non covalent, inhibitor of the FAAH enzyme. Compounds such as 29, by virtue of their structural novelty and unique mechanism of inhibition, represent attractive tools with which to study FAAH and the endocannabinoid pathway.

Notes and references

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7 S. Kathuria, S. Gaetani, D. Fegley, F. Valino, A. Duranti, A. Tontini, M. Mor, G. Tarzia, G. La Rana, A. Calignano, A. Giustino, M. Tattoli, M. Palmery, V. Cuomo and D. Piomelli, Nat. Med., 2003, 9, 76. 8 A. Abouabdellah, P. Burnier, C. Hoornaert, J. Jeunesse and F. Puech, US20060089344, 2006; Chem. Abstr., 2006, 142, 198102. 9 (a) T. Matsumoto, M. Kori, J. Miyazaki and Y. Kiyota, WO 2006054652, 2006; Chem. Abstr., 2006, 145, 8189; (b) J. M. Keith, R. Apodaca, W. Xiao, M. Seierstad, K. Pattabiraman, J. Wu, M. Webb, M. J. Karbarz, S. Brown, S. Wilson, B. Scott, C.-S. Tham, L. Luo, J. Palmer, M. Wennerholm, S. Chaplan and J. G. Breitenbucher, Bioorg. Med. Chem. Lett., 2008, 18, 4838. 10 Other recent non-depicted FAAH inhibitors: (a) G. C. Muccioli, N. Fazio, G. K. E. Scriba, W. Poppitz, F. Cannata, J. H. Poupaert, J. Wouters and D. M. Lambert, J. Med. Chem., 2006, 49, 417; (b) J. Adams, M. L. Behnke, A. C. Castro, C. A. Evans, L. Grenier, M. J. Grogan, T. Liu, D. A. Snyder and T. T. Tibbits, WO2008063300, 2008; Chem. Abstr., 2008, 149, 10119; (c) A. Minkkila, S. M. Saario, H. Kasnanen, J. Leppanen, A. Poso and T. Nevalainen, J. Med. Chem., 2008, 51, 7057; (d) F. Vincent, M. T. Nguyen, D. E. Emerling, M. G. Kelly and M. A. J. Duncton, Bioorg. Med. Chem. Lett., 2009, 19, 6793. 11 (a) www.clinicaltrials.gov, identier NCT00822744 (SSR-411298); (b) Pzer pipeline (PF-4457845), 27th January 2010, http://media.pzer.com/ les/research/pipeline/2010_0127/pipeline_2010_0127.pdf. 12 (a) D. C. Swinney, Nat. Rev. Drug Discovery, 2004, 3, 801; (b) D. S. Johnson, E. Weerapana and B. F. Cravatt, Future Med. Chem., 2010, 2, 949; (c) D. Zhang, A. Saraf, T. Kolasa, P. Bhatia, G. Z. Zheng, M. Patel, G. S. Lannoye, P. Richardson, A. Stewart, J. C. Rogers, J. D. Brioni and C. S. Surowy, Neuropharmacology, 2007, 52, 1095. 13 (a) A. H. Lichtman, A. Donmienne, C. C. Shelton, A. Saghatelian, C. Hardouin, D. L. Boger and B. F. Cravatt, J. Pharmacol. Exp. Ther., 2004, 311, 441; (b) L. Chang, L. Luo, J. A. Palmer, S. Sutton, S. J. Wilson, A. Barbier, J. G. Breitenbucher, S. R. Chaplan and M. Webb, Br. J. Pharmacol., 2006, 148, 102; (c) J. A. Palmer, E. S. Higuera, L. Chang and S. R. Chaplan, Neuroscience, 2008, 154, 1554; (d) A. Timmons, M. Seierstad, R. Apodaca, M. Epperson, D. Pippel, S. Brown, L. Chang, B. Scott, M. Webb, S. R. Chaplan and J. G. Breitenbucher, Bioorg. Med. Chem. Lett., 2008, 18, 2109. 14 (a) D. J. Gustin, Z. Ma, X. Min, Y. Li, C. Hedberg, C. Guimaraes, A. C. Porter, M. Lindstrom, D. Lester-Zeiner, G. Xu, T. J. Carlson, S. Xiao, C. Meleza, R. Connors, Z. Wang and F. Kayser, Bioorg. Med. Chem. Lett., 2011, 21, 2492; (b) X. Min, S. T. Thibault, A. C. Porter, D. J. Gustin, T. J. Carlson, H. Xu, M. Lindstrom, G. Xu, C. Uyeda, Z. Ma, Y. Li, F. Kayser, N. P. C. Walker and Z. Wang, Proc. Natl. Acad. Sci. U. S. A., 2011, 108, 7379. See also; (c) X. Wang, K. Sarris, K. Sage, D. Zhang, S. P. Brown, T. Kolasa, C. Surowy, O. F. El Kouhen, S. W. Muchmore, J. D. Brioni and A. O. Stewart, J. Med. Chem., 2009, 52, 170. 15 (a) M. G. Kelly, J. Kincaid, S. Gowlugari and K. Caub, WO2009011904, 2009; Chem. Abstr., 2009, 150, 144453; (b) J. DeFalco, M. T. Nguyen, S. Gowlugari, K. Caub, D. Steiger, D. OMahony, M. Cox, A. Estiarte, R. Johnson, J. Kincaid, D. E. Emerling, M. G. Kelly, M. Duncton and F. Vincent, Characterization of a Novel Class of Reversible FAAH Inhibitors presented at World Pharmaceutical Congress, Philadelphia, USA, June 910th 2009. 16 M. K. Ramarao, E. A. Murphy, M. W. Shen, Y. Wang, K. N. Bushell, N. Huang, N. Pan, C. Williams and J. D. Clarck, Anal. Biochem., 2005, 343, 143. 17 For examplesfrom our own laboratories see (a) J. Kincaid, Y. Cao, K. Caub, D. Lonergan and M. G. Kelly, WO2007028022, 2007; Chem. Abstr., 2007, 146, 316929; (b) M. G. Kelly and J. Kincaid, WO2006102610, 2006; Chem. Abstr., 2007, 145, 377378; (c) M. G. Kelly, S. Janagani, G. Wu, J. Kincaid, D. Lonergan, Y. Fang and Z.-L. Wei, US2005277643, 2005; Chem. Abstr., 2005, 144, 51599; (d) M. G. Kelly, S. Janagani, G. Wu, J. Kincaid, D. Lonergan, Y. Fang and Z.-L. Wei, WO2005066171, 2005; Chem. Abstr., 2005, 143, 153393. 18 M. Mileni, D. S. Johnson, Z. Wang, D. Everdeen, M. Liimatta, B. Pabst, K. Bhattacharya, R. A. Nugent, S. Kamtekar,

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B. F. Cravatt, K. Ahn and R. C. Stevens, Proc. Natl. Acad. Sci. U. S. A., 2008, 105, 12820. 19 For a recent example of discrepancies in potency between human and rat FAAH for a series of 2-amino-5-arylbenzoxazole derivatives from our laboratories see M. A. Estiarte, R. J. Johnson, C. J. Kaub,

S. Gowlugari, D. J. R. OMahony, M. T. Nguyen, D. E. Emerling, M. G. Kelly, J. Kincaid, F. Vincent and M. A. J. Duncton, Med. Chem. Commun., 2012, 3, 611. 20 R. A. Copeland, in Enzymes A Practical Introduction to Structure, Mechanism and Data Analysis, Wiley, New York, 2nd edn, 2000.

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