Bioorganic & Medicinal Chemistry Letters 24 (2014) 1280–1284

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1-Aryl-2-((6-aryl)pyrimidin-4-yl)amino)ethanols as competitive inhibitors of fatty acid amide hydrolase John M. Keith ⇑, Natalie Hawryluk, Richard L. Apodaca, Allison Chambers, Joan M. Pierce, Mark Seierstad, James A. Palmer, Michael Webb, Mark J. Karbarz, Brian P. Scott, Sandy J. Wilson, Lin Luo, Michelle L. Wennerholm, Leon Chang, Michele Rizzolio, Sandra R. Chaplan, J. Guy Breitenbucher Janssen Pharmaceutical Companies of Johnson & Johnson, 3210 Merryfield Row, San Diego, CA 92121, United States

a r t i c l e

i n f o

Article history: Received 26 December 2013 Revised 15 January 2014 Accepted 21 January 2014 Available online 31 January 2014

a b s t r a c t A series of 1-aryl-2-(((6-aryl)pyrimidin-4-yl)amino)ethanols have been found to be competitive inhibitors of fatty acid amide hydrolase (FAAH). One member of this class, JNJ-40413269, was found to have excellent pharmacokinetic properties, demonstrated robust central target engagement, and was efficacious in a rat model of neuropathic pain. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: Fatty acid amide hydrolase (FAAH) Enzymes Crystal structure Endo-cannabinoids Analgesia

The discovery and cloning of two cannabinoid receptors CB11 and CB22 in the early 1990s facilitated the development of a rationale for the analgesic effects of cannabis preparations containing D9-THC and its derivatives. The subsequent discovery of anandamide (AEA)3 (Fig. 1), an endogenous agonist of both CB14 and CB2 receptors with analgesic properties, and fatty acid amide hydrolase (FAAH),5 the enzyme which hydrolyzes AEA, suggested a means of engaging the cannabinoid system without the use of exogenous agonists such as D9-THC. Unfortunately, AEA is not drug-like due to its poor physicochemical properties and rapid metabolism (T1/2 on the order of a few minutes)6 by FAAH to give ethanolamine and arachidonic acid. Furthermore, the synthesis and metabolism of AEA is localized, thus preventing the appearance of the side-effects (the so-called cannabinoid tetrad)7 associated with global CB1 agonism. Indeed, systemic administration of AEA gives rise to side-effects reminiscent of those observed with the use of D9-THC.8 The potential benefit to inhibiting the FAAH enzyme is that it should lead to localized increases in AEA and likely avoid the aforementioned side-effects of general CB1 activation. While FAAH very efficiently degrades AEA, it also breaks down several other ethanolamides including N-palmitoylethanolamide (PEA), N-oleoylethanolamide (OEA) and others.9 PEA is known to ⇑ Corresponding author. Tel.: +1 858 784 3275. E-mail address: [email protected] (J.M. Keith). http://dx.doi.org/10.1016/j.bmcl.2014.01.064 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

Me O H

OH

HO

N H

H Me Me

O

Me

Me 9

Δ -THC

Anandamide (AEA)

O HO

O HO

N H Me N-Palmitoylethanolamide (PEA)

N H

Me

N-Oleoylethanolamide (OEA)

Figure 1. Structures of D9-THC and endogenous substrates of FAAH.

have anti-inflammatory properties and exert analgesic effects through a non-cannabinoid pathway,10 while OEA appears to be involved in regulating satiety,11 whereas the similar oleamide (OA), another substrate of FAAH, is an important contributor to sleep induction.12 Numerous groups have reported the preparation and pharmacological testing of FAAH inhibitors (Fig. 2). Boger’s group described several series of highly potent a-keto heterocycles.13 Piomelli14 disclosed carbamate based inhibitors as did Sanofi.15 Pfizer,16 Takeda17 and Janssen18 have disclosed phenyl and heteroaryl urea inhibitors of FAAH. All of these compounds are quite potent and are thought to form covalent bonds with Ser 241 within

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J. M. Keith et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1280–1284 O

O

O

N H2N

O N

O

OL-1 35 covalen t/reversible

URB597 covalent/i rreversible N

Me

O

H N

O O

N

N

Me

O

N N H

N H

N

N

CF 3

O PF-04457845 covalent /irreversible

SA-4 7 covalent/i rreversible O O S S N

H N

O N H

O

S N

Me

N H

N N

N

S N JNJ-16 61010/Take da covalen t/reversible

Abbo tt comp etitive N

Me

O

N

N N

N

HN N

O

N N

A mge n competitive

Renovis competitive

OH

Figure 2. Structures of some known FAAH inhibitors and their mechanism.

the FAAH active site. OL-135 is thought to form a reversible tetrahedral hemiketal intermediate derived from the attack of Ser 241 onto the ketone.19 URB597 and the carbamates reported by Sanofi are thought to undergo transesterification with the FAAH enzyme with the concomitant loss of a phenolic or alcoholic fragment respectively.20 The ureas also form a carbamate with the FAAH enzyme, but with the loss of an aniline or heteroaryl amine rather than an alcohol. OL-135,14 URB597,21 and JNJ-166101022 have been found to be efficacious in various animal models of pain without the motor impairment associated with direct CB1 agonism. Several competitive inhibitor classes have been reported as well. Abbott disclosed piperidinyl sulfonamides,23 Amgen 2-aminopyrimidines24 and Renovis substituted 6-aza-tetrahydroisoquinolines.25 Several compounds have been profiled to some degree in the clinic. Pfizer’s PF-0445784526 was evaluated in phase 2 as an analgesic in subjects with osteoarthritis pain, but was apparently ineffective despite evidence of peripheral target engagement. Ongoing or completed studies with PF-04457845 include evaluating the potential for treating cannabinoid dependence27 and acute and chronic pain,28 fear conditioning,29 Vernalis’ V158866 did a FIH study,30 and Janssen’s JNJ-42165279 is currently in phase 1 clinical studies.31 An HTS campaign conducted late in our FAAH program produced an interesting and potent hit compound without any obviously reactive functionality (Fig. 3). As the hit was racemic, we prepared the two enantiomers and found that the bulk of the FAAH activity comes from the R-isomer. Encouraged by these data, we explored the SAR of substituted phenyl groups directly appended to the pyrimidine ring, a sampling of which is shown in Table 1. The compounds in Table 1 were generally constructed according to Scheme 1. Displacing one of the chlorine atoms of 4, N

N N H

F3 CO

Ph

N H

OH

HTS hit hFAAH IC50 = 30 ± 13 nM rFAAH IC 50 = 159 ± 73 nM

N H

Ph R-isomer hFAAH IC50 = 17 ± 5 nM OH rFAAH IC 50 = 37 ± 8 nM Ph S-isomer hFAAH IC50 = 10 μM rFAAH IC 50 = 10 μM

OH

Figure 3. HTS hit and individual enantiomers.

6-dichloropyrimidine with (R)-1-phenylethanol-2-amine gave the mono-chloro intermediate in high yield. Subsequent Suzuki coupling with substituted aryl boronic acids or esters gave the final products. Alternatively, if the ethanolamine side chain was to be introduced last, then the molecules were generally constructed according to Scheme 2. Coupling of 3-chloro-4-trifluoromethylphenylboronic acid with 4,6-dichloropyrimidine followed by substitution of the remaining pyrimidyl chloride with the desired aryl ethanolamine yielded the final product. Many of the 1-arylethanol-2-amines had to be prepared. Some were constructed by forming a silyl cyanohydrin and then reducing the nitrile in situ to give the racemic product (Scheme 3). This approach worked best for those 1-arylethanol-2-amines bearing substituents stable to metal hydride reducing conditions. The amino alcohols bearing nitriles were prepared according to Scheme 4. The corresponding cyano bromoacetophenones were treated with BH3
THF at 0 °C for 1 h followed by aqueous ammonia to give the desired intermediate. We were surprised to find that an absence of substitution on the phenyl ring directly appended to the pyrimidine yields an inactive compound (1). Substituents in the 2-position hurt activity,32 while 3- and 4-substitution with lipophilic groups tended to improve potency. The most potent analogs possessed 3,4-disubstitution with non-polar functionality. Unfortunately, this region of the molecule was very sensitive to oxidative metabolism. Many of the most potent compounds (4, 11, 12, 24, 26, 28) exhibited low bioavailability and high clearance in the rat. Fortunately, we found that when the 3-chloro-4-trifluoromethylphenyl substituent was introduced, a metabolically robust and potent molecule was obtained (31; JNJ40413269). With a suitable substitution pattern on the aryl ring appended to the pyrimidine, we next explored the SAR of substituents on the phenyl ring of the ethanolamine side-chain (Table 2). Most changes made to this part of the molecule resulted in a modest to large decrease in activity. We obtained an X-ray crystal structure of humanized rat FAAH bound to one of our FAAH inhibitors (Fig. 4).33 Rationalizing the greater potency of the (R)-inhibitors is now possible as the alcohol

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Table 1 SAR of substituted phenyl ring appended to the pyrimidine N

N

Ar

N H

Ph OH

Compd

Ar

hFAAH IC50 (nM)

rFAAH IC50 (nM)

Compd

1⁄

Ph

>10,000

>10,000

17⁄

Ar

hFAAH IC50 (nM)

rFAAH IC50 (nM)

598 ± 132

1850 ± 650

317 ± 96

1267 ± 216

1433 ± 363

>10000

Cl Me

2

445 ± 95

18⁄

3500 ± 500

F3 C

3⁄

Et

32 ± 2

302 ± 30

19 MeO

4

5±2

100 ± 20

20

>10000

>10000

5⁄

72 ± 16

680 ± 93

21⁄

477 ± 122

2075 ± 460

23 ± 3

94 ± 12

22

49 ± 9

461 ± 97

291 ± 75

2902 ± 886

23

15 ± 3

39 ± 6

3±1

7±3

1.5 ± 0.5

3±1

15 ± 4

43 ± 5

6.3 ± 2.5

86 ± 15

3.2 ± 0.8

5.4 ± 1.6

12 ± 4

34 ± 8

55 ± 14

210 ± 51

5.3 ± 3.2

6.3 ± 3.7

6

7⁄

F3C

MeO

MeO

F3 CO F

8⁄

EtO

EtO

54 ± 10

345 ± 40

24 F3 C

F3 C O

9

18 ± 5

263 ± 34

EtO

25

Cl

10⁄

MeS

55 ± 17

633 ± 83

EtO

26

F

EtS

1.3 ± 0.2

11

12

13 ± 1

O

27

F3C

F

Ac

F3CS

8 ± 0.6

21 ± 6

28 F

13⁄

Cl

783 ± 265

3433 ± 1683

F3C

29

F

14

Cl

F

1784 ± 183

>10,000

30 F3C

NC

15

505 ± 145

750 ± 350

F3 C

31

Cl

16⁄ *

3340 ± 338

>10,000

Denotes racemate; IC50s are ± standard error.

N Cl

N

N

a Cl

Cl

N N H

Ph OH

N

b R

N N H

Ph OH

Scheme 1. Reagents and conditions: (a) 1:1 4,6-dichloropyrimidine and (R)-1-phenylethanolamine, 6 equiv NaHCO3, 1,4-dioxane, 100 °C, 17 h, 81%; (b) 2 equiv arylboronic acid or ester (Ar defined in Table 1), 3–10 mol % Pd(PPh3)4, K3PO4, DME, 85 °C, 15–18 h, yields varied.

is well positioned to make a hydrogen bond with Th488 which is not readily accessible to the corresponding (S)-configured analogs. The arene attached to the pyrimidine ring projects toward the catalytic machinery, but not so close as to interfere with binding. Owing to its excellent FAAH potency, we elected to perform further profiling of JNJ-40413269 in vivo in the rat. The compound was seen to exhibit good pharmacokinetic properties (Fig. 5) and

a good blood brain barrier (BBB) penetration profile (Fig. 6). JNJ-40413269 was cleared at a moderate rate, exhibited good oral bioavailability and generated concentrations in the brain that were similar to those seen in the plasma (brain-to-plasma ratio  1). This compound appeared to be an excellent candidate, from a pharmacokinetic perspective, for efficacy studies in pain models. Therefore JNJ-40413269 was profiled in the rat spinal nerve ligation (Chung) model of neuropathic pain (Fig. 7).

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J. M. Keith et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1280–1284

N

B(OH) 2

N

Cl

Cl

+

N a

N b

Cl

F3 C Cl

R

F 3C

H2N

Cl

OH

R N

N N H

F3C

OH

Cl

Scheme 2. Reagents and conditions: (a) Pd(OAc)2, PPh3, ACN/water (3:1), K3PO4, rt, 2 h, 35%; (b) 6 equiv NaHCO3, 1,4-dioxane, 100 °C, 15–18 h, yields varied (R defined in Table 2).

OTMS O

a

NH 2

b

CN

R

Figure 4. An aryl-pyrimidine bound in the active site of FAAH

OH

R

R

Scheme 3. Reagents and conditions: (a) TMSCN, 10 mol % ZnI2, neat, 0 °C, 2– 15 min; (b) 1.0 M LiAlH4, THF, 0–50 °C, yields varied.

OH

a

Br

NC

OH Br

b

NC

1

Cp (µM )

O

NH 2

0.1

0.01

NC

0.001 0

Scheme 4. Reagents and conditions: (a) 1.2 equiv. BH3
THF, THF, 1 h, 0 °C; (b) concentrated NH3 (aq), rt, 12 h, 34% for two steps.

4

8

12 Time (h)

16

20

24

Figure 5. Rat pharmacokinetic data for JNJ-40413269: Cl = 8 mL/min/kg, Vss = 1.7 L/ kg, T1/2 = 4.1 h, %F = 94%. (iv vehicle = 50% glycofurol/saline; p.o. vehicle = 5% pharmasolve, 20% cremaphore, 75% dextrose solution (5%)).

Table 2 SAR of aryl substituents on ethanolamine side-chain N

p.o. 10 mg/kg i.v. 1 mg/kg

10

N N H

F3 C

Ar

10

OH

Compd

Ar

32⁄

hFAAH IC50 (nM)

rFAAH IC50 (nM)

73 ± 23

29 ± 6

61 ± 7

65 ± 21

110 ± 44

34 ± 9

228 ± 79

23 ± 5

271 ± 83

21 ± 3

57 ± 5

18 ± 0.4

50 ± 19

26 ± 9

14 ± 3

7 ± 1.5

59 ± 14

19 ± 2

33 ± 3

8±2

F

33⁄ F

34

1

Plasma Brain

0.1

0.01 0

⁄

SMe

35⁄ CF3

36⁄ OCF3

37⁄

CN

38⁄ CN

F

39 F Cl

40⁄ F

F

41⁄ Cl *

Cp (µM)

Cl

Denotes racemate; IC50s are ± standard error.

1

2

3

4

5

6

7

Time (h) Figure 6. Blood brain barrier penetration data for JNJ-40413269 (3 mg/kg p.o., vehicle = 5% pharmasolve, 20% cremaphore, 75% dextrose solution (5%)). The brain to plasma ratio was 1–1.5 across a full dose range.

JNJ-40413269 was seen to produce a robust dose-dependent decrease in mechanical allodynia in the spinal nerve ligation model of neuropathic pain. The highest level of reversal was seen 30 min after compound dosing and was about 55% of the maximal possible effect (MPE). A similar maximal degree of efficacy was seen with the 20, 40, and 100 mg/kg doses, but it is noteworthy that substantial efficacy was retained for at least 4 h with the higher doses tested. In an effort to determine the degree of target engagement in the brain, concentrations of three fatty acid amide (FAA) substrates of FAAH [AEA, PEA and OEA] were measured at several time-points after oral dosing of JNJ-40413269. JNJ-40413269 strongly raises the brain levels of all three substrates (Fig. 8) with peak concentrations occurring between 2 and 4 h post-dose. Further detailed

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J. M. Keith et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1280–1284 100 mg/kg JNJ-40413269 40 mg/kg JNJ-40413269 100

20 mg/kg JNJ-40413269 6 mg/kg JNJ-40413269 2 mg/kg JNJ-40413269

75

MPE (%)

Vehicle 50 25 0

1

Baseline

2

3

4

Time (h) Figure 7. Dose-response and time-course of the efficacy of JNJ-40413269 in the rat spinal nerve ligation model of neuropathic pain. Vehicle = 5% pharmasolve, 20% cremaphore, 75% dextrose solution (5%).

% control FAA levels

300

AEA

OEA

PEA

Cp

10 1

200

0.1

100

0.01

0

0.001 0 1 2

4

6

24

Plasma [JNJ-40413269], (μM)

400

Time (h) Figure 8. Time course of the elevations in brain fatty acid amide (FAA) levels and corresponding plasma compound concentrations following administration of JNJ40413269 (3 mg/kg p.o.; vehicle = 5% pharmasolve, 20% cremaphore, 75% dextrose solution (5%)).

pharmacological profiling of JNJ-40413269 will be reported elsewhere. In conclusion, we have discovered a class of potent phenyl ethanolaminopyrimidines that act as potent competitive inhibitors of the FAAH enzyme. JNJ-40413269 was found to possess good pharmacokinetic properties and was efficacious in the spinal nerve ligation model of neuropathic pain in the rat. References and notes 1. Matsuda, L. A.; Lolait, S. J.; Brownstein, M. J.; Young, A. C.; Bonner, T. I. Nature 1990, 346, 561. 2. Munro, S.; Thomas, K. L.; Abu-Shaar, M. Nature 1993, 365, 61. 3. Devane, W. A.; Hanus, L.; Breuer, A.; Pertwee, R. G.; Stevenson, L. A.; Griffin, G.; Gibson, D.; Mandelbaum, A.; Etinger, A.; Mechoulam, R. Science 1992, 258, 1946. 4. (a) Lichtman, A. H.; Hawkins, E. G.; Griffin, G.; Cravatt, B. F. JPET 2002, 302, 73; (b) Steffens, M.; Zentner, J.; Honegger, J.; Feuerstein, T. J. Biochem. Pharmacol. 2005, 69, 169; (c) Cravatt, B. F.; Giang, D. K.; Mayfield, S. P.; Boger, D. L. Nature (London, U.K.) 1996, 384, 83. 5. Cravatt, B. F.; Demarest, K.; Patricelli, M. P.; Bracey, M. H.; Giang, D. K.; Martin, B. R.; Lichtman, A. H. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 9371. 6. Willoughby, K. A.; Moore, S. F.; Martin, B. R.; Ellis, E. F. J. Pharmacol. Exp. Ther. 1997, 282, 243. 7. The cannabinoid tetrad: catalepsy, hypomotility, hypothermia and analgesia. Hyperphagia is another side-effect commonly observed. 8. The studies were carried out in FAAH( / ) mice: Cravatt, B. F.; Demarest, K.; Patricelli, M. P.; Bracey, M. H.; Giang, D. K.; Martin, B. R.; Lichtman, A. H. PNAS 2001, 98, 9371. 9. (a) Patricelli, M. P.; Cravatt, B. F. Biochemistry 1999, 38, 14125; (b) Boger, D. L.; Fecik, R. A.; Patterson, J. E.; Miyauchi, H.; Patricelli, M. P.; Cravatt, B. F. Bioorg. Med. Chem. Lett. 2000, 10, 2613.

10. (a) Lambert, D. M.; Vandevoorde, S.; Jonsson, K. O.; Fowler, C. J. Curr. Med. Chem. 2002, 9, 663; (b) Lo Verme, J.; Fu, J.; Astarita, G.; La Rana, G.; Russo, R.; Calignano, A.; Piomelli, D. Mol. Pharmacol. 2005, 67, 15. 11. Thabuis, C.; Destaillats, F.; Tissot-Favre, D.; Martin, J.-C. Lip. Technol. 2007, 19, 225. 12. Boger, D. L.; Henriksen, S. J.; Cravatt, B. F. Curr. Pharm. Des. 1998, 4, 303. 13. (a) Boger, D. L.; Miyauchi, H.; Du, W.; Hardouin, C.; Fecik, R. A.; Cheng, H.; Hwang, I.; Hedrick, M. P.; Leung, D.; Acevedo, O.; Guimaraes, C. R.; Jorgensen, W. L.; Cravatt, B. F. J. Med. Chem. 2005, 48, 1849; (b) Romero, F. A.; Du, W.; Hwang, I.; Rayl, T. J.; Kimball, F. S.; Leung, D.; Hoover, H. S.; Apodaca, R. L.; Breitenbucher, J. G.; Cravatt, B. F.; Boger, D. L. J. Med. Chem. 2007, 50, 1058. 14. Fegley, D.; Gaetani, S.; Duranti, A.; Tontini, A.; Mor, M.; Tarzia, G.; Piomelli, D. J. Pharmacol. Exp. Ther. 2005, 313, 352. 15. Abouabdellah, A.; Burnier, P.; Hoornaert, C.; Jeunesse, J.; Puech, F. PCT Int. Appl. 2004 WO 2004099176. 16. (a) Ahn, K.; Johnson, D. S.; Fitzgerald, L. R.; Liimatta, M.; Arendse, A.; Stevenson, T.; Lund, E. T.; Nugent, R. A.; Nomanbhoy, T. K.; Alexander, J. P.; Cravatt, B. F. Biochemistry 2007, 46, 13019; (b) Johnson, D. S.; Ahn, K.; Kesten, S.; Lazerwith, S. E.; Song, Y.; Morris, M.; Fay, L.; Gregory, T.; Stiff, C.; Dunbar, J. B.; Liimatta, M.; Beidler, D.; Smith, S.; Nomanbhoy, T. K.; Cravatt, B. F. Bioorg. Med. Chem. Lett. 2009, 19, 2865; (c) Johnson, D. S.; Stiff, C.; Lazerwith, S. E.; Kesten, S. R.; Fay, L. K.; Morris, M.; Beidler, D.; Liimatta, M. B.; Smith, S. E.; Dudley, D. T.; Sadagopan, N.; Bhattachar, S. N.; Kesten, S. J.; Nomanbhoy, T. K.; Cravatt, B. F.; Ahn, K. ACS Med. Chem. Lett. 2011, 2, 91. 17. Matsumoto, T.; Kori, M.; Miyazaki, J.; Kiyota, Y. PCT Int. Appl. 2006, WO 2006054652. 18. (a) Keith, J. M.; Apodaca, R.; Xiao, W.; Seierstad, M.; Pattabiraman, K.; Wu, J.; Webb, M.; Karbarz, M. J.; Brown, S.; Wilson, S.; Scott, B.; Tham, C.-S.; Luo, L.; Palmer, J.; Wennerholm, M.; Chaplan, S.; Breitenbucher, J. G. Bioorg. Med. Chem. Lett. 2008, 18, 4838; (b) Tichenor, M. S.; Keith, J. M.; Jones, W. M.; Pierce, J. M.; Merit, J.; Hawryluk, N.; Seierstad, M.; Palmer, J. A.; Webb, M.; Karbarz, M. J.; Wilson, S. J.; Michelle., L.; Wennerholm, M. L.; Woestenborghs, F.; Beerens, D.; Luo, L.; Brown, S. M.; De Boeck, M.; Sandra, R.; Chaplan, S. R.; Breitenbucher, J. G. Bioorg. Med. Chem. Lett. 2012, 22, 7357; (c) Keith, J. M.; Apodaca, R.; Tichenor, M.; Xiao, W.; Jones, W.; Pierce, J.; Seierstad, M.; Palmer, J.; Webb, M.; Karbarz, M.; Scott, B.; Wilson, S.; Luo, L.; Wennerholm, M.; Chang, L.; Brown, S.; Rizzolio, M.; Rynberg, R.; Chaplan, S.; Breitenbucher, J. G. ACS Med. Chem. Lett. 2012, 3, 823. 19. (a) Mileni, M.; Garfunkle, J.; DeMartino, J. K.; Cravatt, B. F.; Boger, D. L.; Stevens, R. C. J. Am. Chem. Soc. 2009, 131, 10497; (b) Guimaraes, C. R.; Boger, D. L.; Jorgensen, W. L. J. Am. Chem. Soc. 2005, 127, 17377. 20. Alexander, J. P.; Cravatt, B. F. Chem. Biol. 2005, 12, 1179. 21. Piomelli, D.; Tarzia, G.; Duranti, A.; Tontini, A.; Mor, M.; Compton, T. R.; Dasse, O.; Monaghan, E. P.; Parrott, J. A.; Putman, D. CNS Drug Rev. 2006, 12, 21. 22. See Ref. 16 Karbarz, M. J.; Luo, L.; Chang, L.; Tham, C.-S.; Palmer, J. A.; Wilson, S. J.; Wennerholm, M. L.; Brown, S. M.; Scott, B. P.; Apodaca, R. L.; Keith, J. M.; Wu, J.; Breitenbucher, J. G.; Chaplan, S. R.; Webb, M. Anesth. Analg. 2008, 108, 316. 23. Wang, X.; Sarris, K.; Katerina, Z.; Karen; Di, S. P.; Brown, T.; Kolasa; Surowy, E.; Carol, O. F.; Kouhen, S. W.; Muchmore, J. D.; Brioni, A. O.; Stewart J. Med. Chem. 2009, 52, 170. 24. Gustin, D. J.; Ma, Z.; Min, X.; Li, Y.; Hedberg, C.; Guimaraes, C.; Porter, A. C.; Lindstrom, M.; Lester-Zeiner, D.; Xu, G.; Carlson, T. J.; Xiao, S.; Meleza, C.; Connors, R.; Wang, Z.; Kayser, F. Bioorg. Med. Chem. Lett. 2011, 21, 2492. 25. Kelly, M. G.; Kincaid, J.; Gowlugari, S.; Kaub, C. PCT Int. Appl. 2009, WO 2009011904. 26. Huggins, J. P.; Smart, T. S.; Langman, S.; Taylor, L.; Young, T. Pain 2012, 153, 1837. 27. From Clinicaltrials.gov as of 2013 12-26: http://www.clinicaltrials.gov/ct2/ show/NCT01618656?term=FAAH&rank=1. 28. From Clinicaltrials.gov as of 2013 12-26: http://www.clinicaltrials.gov/ct2/ show/NCT00836082?term=FAAH&rank=3. 29. From Clinicaltrials.gov as of 2013 12-26: http://www.clinicaltrials.gov/ct2/ show/NCT01665573?term=FAAH&rank=5. 30. From Clinicaltrials.gov as of 2013 12-26: http://www.clinicaltrials.gov/ct2/ show/NCT01634529?term=FAAH&rank=6. 31. From Clinicaltrials.gov as of 2013 12-26: a) http://www.clinicaltrials.gov/ct2/ show/NCT01964651?term=FAAH&rank=9; b) http://www.clinicaltrials.gov/ ct2/show/NCT01650597?term=fatty+acid+amide+hydrolase&rank=5. 32. Data not shown. 33. This compound was used for crystallography studies because it is more soluble than most other analogs: N O O

N N H

Ph OH

hFAAH IC 50 = 28 ± 8 rFAAH IC 50 = 270 ± 93

.