US 2013 0217764A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2013/0217764 A1 Piomelli et al. (43) Pub. Date: Aug. 22, 2013

(54) PERIPHERALLY RESTRICTED FAAH central nervous system (CNS). Despite their relative inability INHIBITORS to access brain and spinal cord, the compounds attenuate behavioral responses indicative of persistent pain in rodent (76) Inventors: Daniele Piomelli, Irvine, CA (US); models of inflammation and peripheral nerve injury, and Sup presses noxious stimulus-evoked neuronal activation in Spi Jason R. Clapper, Irvine, CA (US); nal cord regions implicated in nociceptive processing. CBi Guillermo Moreno-Sanz, Irvine, CA receptor blockade prevents these effects. Accordingly, the (US); Andrea Duranti, Urbino (IT): invention also provides methods, and pharmaceutical com Andrea Tontini, Pesaro (IT); Marco positions for treating conditions in which the inhibition of Mor, Ghedi (IT): Giorgio Tarzia, peripheral FAAH would be of benefit. The compounds of the Petriano (IT): Todd A. Ostomel, San invention are according to the formula (I): in which R is a Francisco, CA (US) polar group. In some embodiments, R is selected from the group consisting of hydroxy and the physiologically hydro (21) Appl. No.: 13/812,777 lysable esters thereof. R. and R are independently selected from the group consisting of hydrogen and Substituted or (22) PCT Filed: Jul. 22, 2011 unsubstituted hydrocarbyl; each R is independently selected from the group consisting of halogen and Substituted or (86). PCT No.: PCT/US11A5114 unsubstituted hydrocarbyland n is an integer from 0 to 4: each S371 (c)(1), Rs is independently selected from the group consisting of (2), (4) Date: May 6, 2013 halo and substituted or unsubstituted hydrocarbyl and m is an integer from 0 to 3; and R is substituted or unsubstituted cyclohexyl; and the pharmaceutically acceptable salts Related U.S. Application Data thereof. (60) Provisional application No. 61/368,500, filed on Jul. 28, 2010. (I) Publication Classification R2 O N (51) Int. Cl. YR, C07C 271/56 (2006.01) (52) U.S. Cl. 21 CPC ...... C07C27I/56 (2013.01) O -i-(R), USPC ...... 514/484; 560/115 R1 s1S (57) ABSTRACT O 21 R Peripherally restricted inhibitors of fatty acid amide hydro (Rs) lase (FAAH) are provided. The compounds can Suppress FAAH activity and increases anandamide levels outside the Patent Application Publication Aug. 22, 2013 Sheet 1 of 29 US 2013/0217764 A1

Brain 100 O Liver 2 - aO ve9 i 850 sse

O ------T t -2.0 - 1.0 O.O 1.0 2.0 URB937(log(mg-kg), S.C.)

FIG. 1 a Patent Application Publication Aug. 22, 2013 Sheet 2 of 29 US 2013/0217764 A1

2 300 ana 2

v 2 S 150 N d ? StS 1 og U, 60 120 N. cy) O g H-o O H O 5 15 30 60 120 Time (min)

FIG 1b. Patent Application Publication Aug. 22, 2013 Sheet 3 of 29 US 2013/0217764 A1

120

C N s80

O

L Š40 0 -t-t-t-t-H O 5 15 30 60 120 Time (min)

FIG 1c Patent Application Publication Aug. 22, 2013 Sheet 4 of 29 US 2013/0217764 A1

2 5 O O N Vehicle URB937 in

2 5

O1 O

FIG. 1d. Patent Application Publication Aug. 22, 2013 Sheet 5 of 29 US 2013/0217764 A1

3 O 6 O O

3

O O1 I -l- OOO --- -l-

FIG. 1e Patent Application Publication Aug. 22, 2013 Sheet 6 of 29 US 2013/0217764 A1

3 O

FIG. 1f Patent Application Publication Aug. 22, 2013 Sheet 7 of 29 US 2013/0217764 A1

123 OOOO

FIG. 2a Patent Application Publication Aug. 22, 2013 Sheet 8 of 29 US 2013/0217764 A1

100 57 O5 2 5

O O O -- 10 20 30 Behavioral SCOre

FIG. 2b Patent Application Publication Aug. 22, 2013 Sheet 9 of 29 US 2013/0217764 A1

40 OVehicle .S. URB937 3 O OO

L.--- -l-

FIG. 2C Patent Application Publication Aug. 22, 2013 Sheet 10 of 29 US 2013/0217764 A1

6 O f 4. O

2 O

O V Rim AM630

FIG. 2d Patent Application Publication Aug. 22, 2013 Sheet 11 of 29 US 2013/0217764 A1

15 -o-Vehicle q) -- URB937

8 -- URB937+Rim CO S 1 s n 9 50.5 O d E O O O

Time (min)

FIG. 26 Patent Application Publication Aug. 22, 2013 Sheet 12 of 29 US 2013/0217764 A1

40

30

20

10

V Rim

FIG. 2f Patent Application Publication Aug. 22, 2013 Sheet 13 of 29 US 2013/0217764 A1

10 Phase 1 40 Phase 2 .S. 30 O O 3 5 20

10 O- -, E. O-ul-, E.

FIG. 2g Patent Application Publication Aug. 22, 2013 Sheet 14 of 29 US 2013/0217764 A1

Patent Application Publication Aug. 22, 2013 Sheet 15 of 29 US 2013/0217764 A1

75 N Vehicle URB937 O d 50 O O . O 9) 9, 25 C O & O i O Rim V Rim V,VI Ventral

FIG. 3d Patent Application Publication Aug. 22, 2013 Sheet 16 of 29 US 2013/0217764 A1

80 o OVehicle 60 URB937 S. 9 : E (S :

40- ; : s 20 i it

O-lyiv V V RAM V V RAM Oh 4 h 24h

FIG. 4a Patent Application Publication Aug. 22, 2013 Sheet 17 of 29 US 2013/0217764 A1

1 5

1 O

FIG. 4b Patent Application Publication Aug. 22, 2013 Sheet 18 of 29 US 2013/0217764 A1

5 3 is 4 3 3 2 t i 1 s O VT V RAM V V R AM wn-as were re-rmor" Oh 24h

FIG. 4C Patent Application Publication Aug. 22, 2013 Sheet 19 of 29 US 2013/0217764 A1

O. 4 ## O. 3

O 2

0. 1 O v-VRA v. VRAM v. VRAM Oh 4 h 24h

FIG. 4d Patent Application Publication Aug. 22, 2013 Sheet 20 of 29 US 2013/0217764 A1

60 o ce

30 i s i C s O BL V IL CL URB

FIG. 5a Patent Application Publication Aug. 22, 2013 Sheet 21 of 29 US 2013/0217764 A1

10

o # a. 9 c 5. t s s s C s BL V IL CL URB

FIG. 5b. Patent Application Publication Aug. 22, 2013 Sheet 22 of 29 US 2013/0217764 A1

6

3

O BL IL CL URB

FIG. 5c Patent Application Publication Aug. 22, 2013 Sheet 23 of 29 US 2013/0217764 A1

6 O

3 O

O ly

FIG. 5d. Patent Application Publication Aug. 22, 2013 Sheet 24 of 29 US 2013/0217764 A1

10

a. 9. 5 s s s "Cs s O BL V IL CL URB

FIG. 5e Patent Application Publication Aug. 22, 2013 Sheet 25 of 29 US 2013/0217764 A1

6

3

O BL V IL CL URB

FIG. 5f Patent Application Publication Aug. 22, 2013 Sheet 26 of 29 US 2013/0217764 A1

40 500 G Vehicle s URB937 g o 2

d 9, 250 E CC n (5 C

S s \S

F.G. 6 Patent Application Publication Aug. 22, 2013 Sheet 27 of 29 US 2013/0217764 A1

8O OVehicle o URB937

C d CD t 40 (S s s C is V V R AM Ohr 24 hr

FIG. 7a Patent Application Publication Aug. 22, 2013 Sheet 28 of 29 US 2013/0217764 A1

1 O

FIG. 7b Patent Application Publication Aug. 22, 2013 Sheet 29 of 29 US 2013/0217764 A1

O Ohr 24 hr

FIG. 7c US 2013/0217764 A1 Aug. 22, 2013

PERPHERALLY RESTRICTED FAAH Nature, 414, 209-212 (2001)) and palmitoylethanolamide INHIBITORS (Calignano, A. et al., Nature, 394, 277-281 (1998)). Mutant mice lacking the gene encoding for FAAH cannot metabolize CROSS-REFERENCES TO RELATED anandamide (Cravatt, B. F. et al., Proc. Natl. Acad. Sci. U.S. APPLICATIONS A., 98, 9371-9376 (2001)) and, though fertile and generally 0001. This application claims priority benefit of U.S. Pro normal, show signs of enhanced anandamide activity at can visional Application Ser. No. 61/368,500 filed on Jul. 28, nabinoid receptors, such as reduced pain sensation (Cravatt, 2010, the contents of which are incorporated herein by refer B. F. et al., Proc. Natl. Acad. Sci. U.S.A., 98, 9371-9376 ence in its entirety for all purposes. (2001)). This suggests the possibility that drugs targeting FAAH may heighten the tonic actions of anandamide, while STATEMENT AS TO RIGHTS TO INVENTIONS possibly avoiding the multiple, often unwanted effects pro MADE UNDER FEDERALLY SPONSORED duced by A-THC and other direct-acting cannabinoid ago RESEARCH AND DEVELOPMENT nists (Hall, W., et al., Lancet, 352, 1611-1616 (1998); Chap eron, F., et al., Crit. Rev. Neurobiol., 13, 243-281 (1999)). 0002 This invention was made with Government support 0006 Pain perception can be effectively controlled by under Grant Nos. DAO 12413, DAO 12447 and AAO17538 neurotransmitters that operate within the CNS. This modula awarded by the National Institutes of Health. The U.S. Gov tion has been well characterized in the dorsal horn of the ernment has certain rights in this invention. spinal cord, where impulses carried by nociceptive (pain REFERENCE TO A “SEQUENCE LISTING. A sensing) fibers are processed before they are transmitted to TABLE, ORACOMPUTER PROGRAM LISTING the brain. In addition to these central mechanisms, intrinsic APPENDIX SUBMITTED ON A COMPACT DISK control of pain transmission can occurat terminals of afferent nerve fibers outside the CNS. One prominent example of 0003) NOT APPLICABLE peripheral regulation is provided by the endogenous opioids, which are released from activated immune cells during BACKGROUND OF THE INVENTION inflammation and inhibit pain initiation by interacting with 0004 Peripheral cannabinoid receptors exert a powerful opioid receptors localized on sensory nerve endings'. inhibitory control over pain initiation, but the endogenous 0007. It has been proposed that endocannabinoid media cannabinoid signal that normally engages this intrinsic anal tors might serve an analogous function to that of the opioids, gesic mechanism is unknown. To address this question, we because pharmacological activation of peripheral CB and developed a peripherally restricted inhibitor of fatty acid CB cannabinoid receptors inhibits pain-related behaviors’ amide hydrolase (FAAH), the enzyme responsible for the while genetic disruption of CB receptor expression in pri degradation of the endocannabinoid anandamide. The com mary nociceptive neurons exacerbates such behaviors. pound, called URB937, suppresses FAAH activity and Moreover, there is evidence that clinical conditions associ increases anandamide levels outside the central nervous sys ated with neuropathic pain or inflammation, such as complex tem (CNS). Despite its inability to access brain and spinal regional pain syndrome and arthritis, may be accompanied by cord, URB937 attenuates behavioral responses indicative of peripheral elevations in the levels of the endocannabinoid persistent pain in rodent models of inflammation and periph anandamide''. Another major endocannabinoid ligand, eral nerve injury, and Suppresses noxious stimulus-evoked 2-arachidonoylglycerol (2-AG), has also been implicated in neuronal activation in spinal cord regions implicated in noci nociceptive signaling outside the CNS''. ceptive processing. CB receptor blockade prevents these effects. The results suggest thatanandamide-mediated signal 0008 Much attention has been directed toward the role of ing at peripheral CB receptors controls the transmission of anandamide in pain. Methods of treating pain by administer pain information to the CNS. Brain-imperimeant FAAH ing anandamide and palmitoylethanolamide are disclosed in inhibitors, which strengthen this gating mechanism, offer a U.S. Patent Application Publication No.: 20020173550. new approach to pain therapy. Methods of treating pain by administering inhibitors of 0005 Anandamide, the naturally occurring amide of FAAH are disclosed in U.S. Patent Application Publication arachidonic acid with ethanolamine, meets all key criteria of Nos. 20040127518 and 20030134894. Methods of treating an endogenous cannabinoid substance (Devane, W. A. et al. pain by administering inhibitors of anandamide transport are Science, 258, 1946-1949 (1992)): it is released upon demand disclosed in U.S. Patent Application Publication No. by stimulated neurons (Di Marzo, V. et al., Nature, 372, 2003O149082. 686-691 (1994); Giuffrida, A. et al., Nat. Neurosci., 2,358 0009. Although these findings suggest that the endocan 363 (1999)); it activates cannabinoid receptors with high nabinoid system serves an important function in the periph affinity (Devane, W.A. et al. Science, 258, 1946-1949 (1992)) eral regulation of nociception, they offer no definitive insight and it is rapidly eliminated through a two-step process con on the identity of the endogenous ligand, or ligands, involved sisting of carrier-mediated transport followed by intracellular in this function. Filling this gap is essential, however, to gain hydrolysis (DiMarzo, V. et al., Nature, 372, 686-691 (1994): a molecular understanding of the intrinsic mechanisms that Beltramo, M. et al., FEBS Lett., 403, 263-267 (1997)). Anan control pain initiation and to discover new analgesic agents damide hydrolysis is catalyzed by the enzyme fatty acid devoid of central side effects. In the present study we identi amide hydrolase (FAAH), a membrane-bound serine hydro fied and characterized a brain-imperimeant inhibitor of the lase (Cravatt, B. F. et al., Nature, 384, 83-87 (1996); Patricelli, anandamide-degrading enzyme, FAAH, with the aim of mag M. P. et al., Biochemistry, 38, 9804-9812 (1999)) (WO nifying the actions of peripheral anandamide and unmasking 98/20119) (U.S. Pat. No. 6.271,015) that also cleaves other their possible role in the control of emerging pain signals'. A bioactive fatty ethanolamides, such as oleoylethanolamide particular concern in the development and therapeutic use of (cis-9-octadecenamide)) (Rodriguez de Fonseca, F. et al. FAAH inhibitors is their ability to modulate endogenous can US 2013/0217764 A1 Aug. 22, 2013

nabinoid systems within the CNS system to cause unwanted nolamide, Stearoylethanolamide, or palmitoylethanolamide. psychotropic or mood-altering effects. Where the fatty ethanolamide is exogenously provided, the 0010. The present invention addresses these and other fatty acid ethanolamide can be administered to the subject needs by providing peripherally restricted FAAH inhibitors before, after, or contemporaneous with the administration of and method of their use in the treatment of a variety of the compound according to the invention. In some embodi conditions, including pain and/or inflammation. ments, the Subject is in need of treatment for pain, inflamma tion, or an immune disorder. In preferred embodiments, the BRIEF SUMMARY OF THE INVENTION pain can be nociceptive, inflammatory, or neuropathic pain. 0015. In a fifth aspect, the invention provides methods of 0011. In a first aspect, the invention provides compounds, screening compounds for their ability to be extruded from and pharmaceutical compositions of the compounds, having brain via the breast cancer resistance protein (B.CRP) trans the formula: port system. Scaffolds similar to that of URB937 and the compounds according to the invention can serve as Substrates for BCRP. Accordingly, in some embodiments the invention R2 provides methods of assaying an URB937 analog and/or O N compounds according to the invention based on their ability YR, to be transported by the BCRP transport system in vitro. 21 BRIEF DESCRIPTION OF THE DRAWINGS O -i-(R), (0016 FIG. 1 URB937 is a peripherally restricted FAAH R1 s1S inhibitor. (a) FAAH activity in liver (closed circles) and brain (closed squares) 1 h after administration of various doses of O 44 R URB937 (0.03-100 mg-kg', s.c.) in Swiss mice. (b) Distri (Rs) bution of URB937 in liver (closed circles), brain (closed squares) and serum (inset) after a single injection in mice (1 mg-kg', i.p.). (c) Time-course of inhibition of FAAH activ in which R is selected from the group consisting of hydroxy ity in liver (closed circles) or brain (closed squares) after and the physiologically hydrolyzable esters thereof, SH, administration of URB937 (1 mg-kg', i.p.). (d) Effects of —O-carboxamido, —OC(O)R7, -O-CO—NRR and URB937 (1 mg-kg', i.p., closed bars) or vehicle (open bars) —NRR, wherein R, is substituted or unsubstituted hydro on anandamide and palmitoylethanolamide (PEA) levels in carbyl and Rs and R are independently selected from the liver, forebrain and hypothalamus of Swiss mice. (e) Effects group consisting of hydrogen and (C-C)alkyl; RandR are of URB937 on anandamide and PEA levels in liver of wild independently selected from the group consisting of hydro type C57B1/6 mice (+/-) and FAAH-deficient mice (-/-). (f) gen and hydrocarbyl; each R is independently selected from Lack of effect of URB937 (1 mg-kg', i.p., closed bars) on the group consisting of halogen and hydrocarbyl and n is an 2-arachidonoylglycerol (2-AG) levels in Swiss mice. Results integer from 0 to 4: each Rs is independently selected from the are expressed as meanisem; n=3; *P<0.05: ***P<0.001 vs group consisting of halo and hydrocarbyl and m is an integer vehicle. from 0 to 3; and R is substituted or unsubstituted cyclohexyl: (0017 FIG. 2 URB937 inhibits behavioral responses to and the pharmaceutically acceptable salts thereof. In pre noxious chemicals in mice and rats. (a-d) Acetic acid (HAc)- ferred embodiments, the compounds are peripherally induced pain behavior in mice. (a) Pain behavior (number of restricted in their distribution in a recipient. writhing episodes) assessed 1 h after administration of 0012. In a second aspect, the invention provides pharma vehicle (V), URB937 (URB, 1 mg-kg", i.p.) or indomethacin ceutical compositions comprising a therapeutically effective (IDM, 1 mg-kg', i.p.). Also illustrated are the effects of amount of the compounds according to the invention. The vehicle and URB937 administered without acetic acid. (b) compositions can be formulated for any route of administra Statistical correlation between antinociception and inhibition tion including the oral and parenteral routes. In addition, the of liver FAAH activity elicited by URB937 (1 mg-kg', i.p.). compositions may be in a unit dose format. (c) Effects of URB937 (1 mg-kg', i.p.) on acetic acid-in 0013. In a third aspect, the invention provides a method of duced writhing in wild-type C57B1/6 mice (+/-) and FAAH treating a subject in need of a peripherally restricted FAAH deficient mice (-/-). (d) CB antagonistrimonabant (Rim, 1 inhibitor (e.g., a FAAH inhibitory compound according to the mg-kg', s.c.), but not CB2 antagonist AM630 (1 mg-kg', invention). In preferred embodiments, the Subject is a human. s.c.), prevents the antinociceptive effects of URB937. Results In some embodiments, the need is with respect to a treatment are expressed as meants.e.m.; n=5-17. *P<0.05 vs vehicle: for pain, inflammation, or an immune disorder of the Subject. **P<0.01 vs vehicle; and ***P<0.001 vs vehicle: #P-0.01 In some embodiments, the pain can be nociceptive, inflam vs URB937; #P-0.001 vs URB937. (e-g) Formalin-in matory, or neuropathic pain. Preferably, the peripherally duced pain behavior in rats. (e) URB937 (1 mg-kg', i.p.) restricted FAAH inhibitory compound is a compound of the produced time-dependent changes in composite pain score invention. relative to vehicle, rimonabant (2 mg-kg', i.p.) or a combi 0014. In a fourth aspect, the invention provides a method nation of URB937 and rimonabant (F-1.86, P=0.039). of enhancing the peripheral activity of an endogenously pro Formalin was injected at time–0. (f) URB937 (1 mg-kg', duced (i.e., an endocannabinoid Such as anandamide, i.p.) decreased the area under the curve (AUC) of pain behav N-arachidonoyl dopamine) or exogenously provided cannab ior during the entire formalin response (F-3.32. P=0.039). inoid fatty acid amide in a subject by administering a com (g) The antinociceptive effect of URB937 was limited to pound according to the invention. Preferably, the fatty acid Phase 2 of the formalin response (10-60 min; Fis 3.05. amide is anandamide, N-arachidonoyl dopamine, oleoyletha P=0.050) whereas pain behavior during Phase 1 (0-10 min) US 2013/0217764 A1 Aug. 22, 2013 was not reliably altered (F-2.22, P=0.115). Results are (FAAH), the enzyme responsible for the degradation of the expressed as meants.e.m.; n=5-7. *P<0.05, all groups vs endocannabinoid anandamide. The compound, called URB937; #P-0.05, URB937 or URB937 plus rimonabant vs URB937, suppresses FAAH activity and increases ananda vehicle. mide levels outside the central nervous system (CNS). 0018 FIG. 3 URB937 suppresses formalin-induced Fos Despite a Surprising relative inability (the compound is also protein expression in rat lumbar (L4) Spinal cord. (a-c) Rep Surprisingly susceptible to a transport system mediated extru resentative sections showing formalin-induced Fos-positive sion from brain) to access brain and spinal cord, URB937 cells in lumbar segments after injection of (a) Vehicle; (b) attenuates behavioral responses indicative of persistent pain URB937 (1 mg-kg', i.p.); or (c) URB937 plus rimonabant (2 in rodent models of inflammation and peripheral nerve injury, mg-kg', i.p.). Calibration bar, 1 mm. (d) Quantitative analy and Suppresses noxious stimulus-evoked neuronal activation sis of the effects of vehicle (open bars), URB937 (closed in spinal cord regions implicated in nociceptive processing. bars), rimonabant, and URB937 plus rimonabant on number CB receptor blockade prevents these effects. The results of Fos-positive cells in superficial dorsal horn (lamina I, II). indicate that anandamide-mediated signaling at peripheral nucleus proprius (lamina III, IV), neck region of the dorsal CB receptors controls the transmission of pain information horn (lamina V. VI), and ventral horn. Behavioral data from to the CNS. Relatively brain-imperimeant FAAH inhibitors, the same subjects are shown in FIG. 2. Results are expressed which strengthen this gating mechanism, might offer a new as meants.e.m.; n=5-7. *P<0.05, all groups vs URB937: approach to pain therapy. iP<0.05, URB937 plus rimonabant, or rimonabant alone vs 0025 Pain perception can be effectively controlled by URB937; P-0.05, vehicle, rimonabant vs URB937. neurotransmitters that operate within the CNS. This modula 0019 FIG.4URB937 attenuates pain behaviorelicited by tion has been well characterized in the dorsal horn of the peripheral inflammation in mice. Effects of URB937 (1 mg spinal cord, where impulses carried by nociceptive (pain kg', i.p.), administered alone or in combination with sensing) fibers are processed before they are transmitted to rimonabant (R, 1 mg-kg', i.p.) or AM630 (AM, 1 mg-kg', the brain. In addition to these central mechanisms, intrinsic i.p.), on (a) carrageenan-induced mechanical hyperalgesia; control of pain transmission can occurat terminals of afferent (b) thermal hyperalgesia; (c) mechanical allodynia; and (d) nerve fibers outside the CNS. One prominent example of paw edema. Mechanical and thermal hyperalgesia were mea peripheral regulation is provided by the endogenous opioids, Sured immediately before carrageenan injection (Oh) or at 4 which are released from activated immune cells during h and 24 h after injection. Mechanical allodynia was mea inflammation and inhibit pain initiation by interacting with Sured 0 hand 24 h after carrageenan. Results are expressed as opioid receptors localized on sensory nerve endings'. meants.e.m. n=6. *P<0.05 vs vehicle: **P<0.01 vs vehicle: (0026. The compound URB937 is a potent FAAH inhibitor ***P<0.001 vs vehicle: #P-0.05 vs URB937; #P-0.01 vs that does not readily enter the CNS and thus principally URB937. interruptsanandamide deactivation only in peripheral tissues. 0020 FIG. 5 URB937 suppresses pain behavior elicited Despite this restricted range of action, URB937 causes by peripheral neural injury in mice. (a-c) Effects of single marked antinociceptive effects in rodent models of acute and administration of vehicle (shaded bars) or URB937 (closed persistent pain, which are prevented by CB cannabinoid bars; 1 mg-kg', i.p.) on (a) mechanical hyperalgesia, (b) receptor blockade. These findings suggest that inhibition of thermal hyperalgesia, and (c) mechanical allodynia induced peripheral FAAH activity magnifies an endogenous analgesic by sciatic nerve ligation. (d-f) Effects of repeated URB937 injections (1 mg-kg', i.p. once-daily for 4 consecutive days) mechanism which regulates the transmission of emerging on (d) mechanical hyperalgesia, (e) thermal hyperalgesia, and nociceptive inputs to the spinal cord and the brain. The (f) mechanical allodynia. BL, baseline (measured before liga mechanism is likely to be mediated by anandamide or another tion); IL, ipsilateral (ligated) paw; CL, contralateral (non endogenous fatty acid amide cannabinoid. ligated) paw. Results are expressed as meants.e.m.; n=6. 0027. Without being wed to theory, peripheral ananda ***P<0.001 vs baseline; #P-0.05 vs vehicle: #P-0.01 vs mide signaling is thought to serve as a diffuse paracrine vehicle: ##P-0.001 vs vehicle. system that modulates the intensity of pain stimuli as they 0021 FIG. 6 Effects of URB937 (1 mg-kg', i.p.) on anan arise in damaged tissues. Two lines of evidence Support this damide and palmitoylethanolamide (PEA) levels in tissues of idea. First, signals generated by inflammation and neural injury can trigger the local release of anandamide. For Swiss mice. Open bars, vehicle; closed bars, URB937. example, membrane depolarization and activation of TRPV-1 Results are expressed as meants.e.m., n=4-6. *, P<0.05, *, channels each stimulates anandamide production in cultures P<0.01, ***, P<0.001 vs vehicle: of sensory neurons, while activation of the pro-inflamma 0022 FIG. 7 Intraplantar injection of carrageenan does tory receptor, Toll-like receptor 4, causes a similar effect in not affect mechanical (a) and thermal hyperalgesia (b) or macrophages. These signals, and probably others that mechanical allodynia (c) in contralateral (non-injected) paws remain to be identified, may contribute to the elevations in of peripheral anandamide documented in animal models of spi I0023 Swiss mice. Rimonabant (R, 1 mg-kg', i.p.) and nal nerve injury and inflammation' as well as in painful AM630 (1 mg-kg", i.p.) had no effect. Results are expressed human conditions such as complex regional pain syndrome as mean-ES.e.m., and arthritis". Second, though particularly abundant in the brain, CB receptors are broadly distributed throughout mam DETAILED DESCRIPTION OF THE INVENTION malian tissues and organs. In particular, they are expressed in 0024 Peripheral cannabinoid receptors exert a powerful large-sized primary sensory neurons and are transported to inhibitory control over pain initiation, but the endogenous peripheral nerve endings’, where they may be both nec cannabinoid signal that normally engages this intrinsic anal essary to maintain normal pain thresholds and sufficient to gesic mechanism is unknown. We developed a novel periph exert marked antinociceptive effects. CB receptors on erally restricted inhibitor of fatty acid amide hydrolase pain-sensing terminals may mediate the analgesic actions of US 2013/0217764 A1 Aug. 22, 2013

locally produced anandamide, and might also be implicated Natl. Acad. Sci. U.S.A. (2002)) brings about the cessation of in the anti-inflammatory activity of this lipid mediator the cellular effects of these autacoids. Owing to the various through their inhibitory influence on the release of excitatory and important physiological roles of fatty acid ethanola neuropeptides’. Nevertheless, it is reasonable to assume that mides, classes of Small-molecule compounds able to block other cannabinoid and cannabinoid-like receptors also con FAAH or FAAHs but not bind to other endocannabinoid tribute, directly or indirectly, to anandamide signaling in metabolizing enzymes, e.g. monoglyceride lipase (MGL), response to injury. Two likely candidates are CB receptors, (Dinh, T. P. et al., Proc. Natl. Acad. Sci. U.S.A. 99, 10819 which can be activated either by anandamide or 2-AG', and 10824 (2002)) or cannabinoid receptors, would be advanta type-aperoxisome proliferator-activated receptors, which are geous both as pharmacological tools and as prototypes for activated by PEA and other lipid-derived mediators'''. drug development projects (Piomelli, D., et al. Trends Phar These receptors and their endogenous ligands are present in macol. Sci. 21, 218-224 (2000); Bisogno, T., et al., Curr. peripheral sensory neurons and immune cells, and have been Pharm. Des. 8,533-547 (2002):Yarnell, A., Chem. Eng. News implicated in the modulation of nociception and inflamma 80(49), 32 (2002); Smith, A., Nat. Rev. Drug Discov. 2, 92 tion21,3132 (2003); Wendeler, M., et al. Angew. Chem. Int. Ed. 42,2938 0028 Mutant mice in which FAAH is selectively deleted 2941 (2003)). in non-neuronal cells, but is preserved in peripheral and cen 0032. The term “pharmaceutically acceptable carrier' tral neurons, display a striking phenotype in which normal encompasses any of the standard pharmaceutical carriers, nociceptive transmission is accompanied by reduced respon buffers and excipients, including phosphate-buffered saline siveness to proinflammatory triggers. A possible explana Solution, water, and emulsions (such as an oil/water or water/ tion for this finding, which is consistent with the present oil emulsion), and various types of wetting agents and/or results, is that the signaling activity of anandamide at periph adjuvants. Suitable pharmaceutical carriers and their formu eral nociceptors is regulated by FAAH localized to the noci lations are described in Remington’s Pharmaceutical Sci ceptors themselves, rather than to neighboring non-neural ences (Mack Publishing Co., Easton, 19th ed. 1995). Pre cells. This is consistent with the observation that peripheral ferred pharmaceutical carriers depend upon the intended aXotomy induces FAAH expression in large-sized sensory mode of administration of the active agent. Typical modes of neurons, a response that is expected to expand the colocal administration are described below. ization of FAAH with CB receptors. 0033. The term “effective amount’ means a dosage suffi 0029. Direct-acting agonists of opioid receptors exert pro cient to produce a desired result with respect to the indicated found analgesic effects in animal and human experimental disorder, condition, or mental state. The desired result may pain models’. Our results indicate that is possible to comprise a Subjective or objective improvement in the recipi achieve significant analgesia also by magnifying the activity ent of the dosage. With respect to pain, the improvement may of an anandamide-based mechanism involved in maintaining be decreased sign or symptom of pain. nociceptive homeostasis. These findings provide new insights 0034. The terms “treatment”, “therapy” and the like into the intrinsic control of pain and can be exploited thera include, but are not limited to, methods and manipulations to peutically to develop effective analgesics largely devoid of produce beneficial changes in a recipient’s health status. The central side effects. changes can be either Subjective or objective and can relate to features such as Symptoms or signs of the disease, disorder or Definitions condition being treated. For example, if the patient notes 0030. It is noted here that as used in this specification and decreased pain, then Successful treatment of pain has the appended claims, the singular forms “a,” “an and “the occurred. For example, if a decrease in the amount o Swelling include plural reference unless the context clearly dictates has occurred, then a beneficial treatment of inflammation has otherwise. occurred. Similarly, if the clinician notes objective changes, 0031 “FAAH' denotes a mammalian Fatty Acid Amide Such as improved range of motion, then treatment for a pain or Hydrolase and includes, but is not limited to, the human, rat, inflammation which had been impairing the motion has also and mouse forms of the enzyme. U.S. Pat. No. 6,271,015 been beneficial. Preventing the deterioration of a recipients discloses isolated and purified forms of FAAH. In one set of status is also included by the term. embodiments, the FAAH ICs of the subject compounds is 0035. Therapeutic benefit includes any of a number of defined according to inhibition of the rat enzyme under physi subjective or objective factors indicating a beneficial ologically relevant conditions. Fatty Amide Hydrolases response or improvement of the condition being treated as (FAAHs) (Deutsch, D. G. et al., Prostaglandins Leukot. discussed herein. Essent. Fatty Acid, 66, 201-210 (2002)) are enzymes respon 0036 “Pharmaceutically-acceptable' or “therapeutically sible for the degradation of lipid ethanolamides, (Fowler, C. acceptable' refers to a substance which does not interfere J., et al., Biochem. Pharmacol. 62,517-526 (2001); Patricelli, with the effectiveness or the biological activity of the active M. P. et al. Vitam. Horm., 62. 663-674 (2001)) e.g. ananda ingredients and which is not toxic to the hosts in the amounts mide (AEA, 1, FIG. 1), (Devane, W. A., et al., Science 258, used, and which hosts may be either humans or animals to 1946-1949 (1992)) oleoylethanolamide, (Rodriguez de Fon which it is to be administered. seca, F., et al. Nature (London) 414, 209-212 (2001); Fu, J., et 0037. “Therapeutically-effective amount” refers to the al., Nature (London) 425, 90-93 (2003)) and palmitoyletha amount of an active agent Sufficient to induce a desired bio nolamide, (Calignano, A., et al. Nature (London) 394, 277 logical or clinical result. That result may be alleviation of the 281 (1998); Lambert, D. M., et al., Curr. Med. Chem. 9, signs, symptoms, or causes of a disease, or any other desired 663-674 (2002)) a biochemical process which, along with alteration of a biological system. The term “therapeutically selective trasport into cells in the case of AEA, (DiMarzo, V., effective amount” is used herein to denote any amount of the Nature (London) 372, 686-691 (1994); Beltrama, M., et al., formulation which causes a Substantial improvement in a Science 277, 1094-1097 (1997); Piomelli, D., et al., Proc. disease, disorder or condition when administered to a Subject. US 2013/0217764 A1 Aug. 22, 2013

The amount will vary with the condition being treated, the molecule through a carbonatom of the heteroalkyl group and stage of advancement of the condition, and the type and the heteroatoms of the heteroalkyl are not contiguous with concentration of formulation applied. Appropriate amounts another heteroatom. in any given instance will be readily apparent to those skilled 0045. The term “heteroalkenyl” derives its name from the in the art or capable of determination by routine experimen corresponding alkenyl group but differs in having 1, 2, or 3 tation. heteroatoms Substituting for a carbon of the alkenyl group. 0038 A "prophylactic treatment” is a treatment adminis The heteroatom nitrogen and Sulfur atoms are optionally oxi tered to a subject who does not exhibit signs of a neurological dized, and the nitrogenatom(s) are optionally quaternized. A or psychological disorder or condition or exhibits only early heteroatom can form a double bond with a carbon atom. A or slight signs of such a disorder or condition, wherein treat heteroalkenyl group is attached to the remainder of the mol ment is administered for the purpose of decreasing the risk of ecule through a carbon atom of to hydrocarbyl and the het developing a pathology or worsening of disorder or condi eroatoms of the hydrocarbyl are not contiguous with another tion. The compounds of the invention may be given as a heteroatom. prophylactic treatment to prevent undesirable or unwanted 0046. As used herein, the term “cycloalkyl refers to a anxiety or panic attacks, or to reduce the level of anxiety saturated monocyclic hydrocarbon radical comprising from should worsening occur. about 3 to about 8 carbon atoms, and more preferably 3 to 6 0039. The term “subject’ as used herein includes any ani carbonatoms. The term “cycloalkenyl refers to monocyclic, mal, including, but not limited to, mammals (e.g., rat, mouse, non-aromatic hydrocarbon radical comprising from about 5 cat, dog) including humans to which a treatment is to be to about 6 carbon atoms and having at least one double bond. given. Exemplary cycloalkyl groups and cycloalkenyl groups 0040. As used herein, the term “hydrocarbyl refers to a include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, (C-C) hydrocarbon radical that is a (C-Cs)alkyl, (C-Cs) cyclohexenyl, cyclohepta-1,3-dienyl, and the like. alkenyl, (C-C)cycloalkyl, (C-C)cycloalkenyl, (C-Cs) 0047. As used herein, the term "heterocycloalkyl refers heteroalkyl, (C-Cs)heteroalkenyl, (C-Cs)heterocycloalkyl, to a saturated or partially unsaturated monocyclic hydrocar or (C-C)heterocycloalkenyl radical. More preferably, the bon radical comprising from about 3 to about 8 carbonatoms, hydrocarbyl in each instance is a substituted or unsubstituted and more preferably 3 to 6 carbon atoms in which 1, 2 or 3 of (C. to C), (C. to C), or (C. to C.)hydrocarbyl, and more the carbonatoms are independently replaced by a heteroatom preferably still an unsubstituted (C to C.)alkyl. Still more independently selected from O, N, or S. Nitrogen and sulfur preferably the hydrocarbylineach instance is methyl or ethyl atoms are optionally oxidized, and the nitrogen atom(s) are or trifluoromethyl. The term “hydrocarbyl also includes optionally quaternized. Sulfur maybe in the thio, sulfinyl or those groups having up to 1, 2, or 3 atoms of a hydrocarbyl sulfonyl oxidation state. The term "heterocycloalkenyl group as set forth above replaced by a heteroatom with the refers to heterocycloalkyl group having at least one double proviso that the heteroatoms of the hydrocarbyl are not con bond. A heterocycloalkyl or heterocycloalkenyl group is tiguous to each other and the hydrocarbyl is not attached to attached to the remainder of the molecule through a carbon the remainder of the compound by a heteroatom of the hydro atom, respectively, of the heterocycloalkyl or heterocycloalk carbyl. enyl group; and the heteroatoms of the heterocycloalkyl or 0041 As used herein, the term “alkyl, by itself or as part heterocycloalkenyl are not contiguous with another heteroa of another Substituent, means, unless otherwise stated, a tom of the heterocycloalkyl or heterocycloalkenyl. straight or branched chain, Saturated, hydrocarbon radical, 0048. As used herein, the term "heteroatom' is meant to having the number of carbon atoms designated (i.e. (C-C) include oxygen (O), nitrogen (N), and Sulfur (S)). means one to six carbons). Examples of alkyl groups include 0049. As used herein, the term “halogen' or “halo” refers methyl, ethyl, n-propyl, isopropyl. n-butyl, t-butyl, isobutyl, to iodine (I), bromine (Br), chlorine (Cl), and/or fluorine (F). sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. 0050. The above hydrocarbyl, alkyl, alkenyl, cycloalkyl, 0042. As used herein, the term “alkoxy' represents an cycloalkenyl, heteroalkyl, heteroalkenyl, cycloheteroalkyl, alkyl moiety joined to the remainder of the molecule by the and cycloheteroalkenyl radicals can each be substituted with oxygenatom of the alkoxy. Accordingly, examples of alkoxy one, two or three substituents independently selected from would include, but not be limited to, methoxy, ethoxy, pro unsubstituted (C-C) or (C-C)alkyl, unsubstituted (C-C) poxy and the like. or (C-C)alkoxy, unsubstituted amino, unsubstituted (C- 0043. The term “alkenyl is derived from the name of the C) or (C-C) alkylamino, di-unsubstituted (C-C) or (C- corresponding alkyl group but differs in possessing one or C.)alkylamino, hydroxy, halo, unsubstituted carboxamido, more double bonds. Similarly, “alkynyl groups are named unsubstituted (C-C) or (C-C)alkylcarboxamido, oxo, and with respect to their corresponding alkyl group but differs in nitro. Non-limiting examples of alkoxy groups include meth possessing one or more triple bonds. Non-limiting examples oxy, ethoxy, t-butoxy, cyclopentyloxy, trifluoromethoxy, and of Such unsaturated alkenyl groups and alkynyl groups the like. As used herein, the term “oxo’ refers to —O. As used include vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadi herein, the term "amino” refers to NH. In some embodi enyl), 2.4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and ments, each of the hydrocarbyl groups are unsubstituted. In 3-propynyl, 3-butynyl, and the higher homologs and isomers. Some embodiments, each of the hydrocarbyl, alkyl, alkenyl, 0044 As used herein, the term "heteroalkyl derives its cycloalkyl, cycloalkenyl, heteroalkyl, heteroalkenyl, cyclo name from the corresponding alkyl group but differs in con heteroalkyl, and cycloheteroalkenyl groups are unsubsti taining one, two, or three heteroatoms independently selected tuted. from N, O, and S each substituting for a carbon of an alkyl 0051 A peripherally restricted compound is one which group. The heteroatom nitrogen and Sulfur atoms are option poorly penetrates the blood brain barrier or is extruded more ally oxidized, and the nitrogen atom(s) are optionally quater rapidly from the brain. Accordingly, a peripherally restricted nized. A heteroalkyl group is attached to the remainder of the compound according to the invention can be administered at US 2013/0217764 A1 Aug. 22, 2013 dosages which inhibit FAAH activity in the periphery to a far hydrogen or unsubstituted C to C. hydrocarbyl. In some greater extent than centrally (e.g., in brain). In some embodi further embodiments, each R and Rs member are indepen ments, the FAAH inhibitor according to the invention has a dently halogen or C to Chydrocarbyl. Preferably, the above subcutaneously, intravenously, or orally administered EDs compounds of the invention are peripherally restricted. for inhibiting peripheral FAAH activity(e.g., liver) which is 0054. In some further embodiments that are applicable to no more than 4, /s, or /10 of the EDs for inhibiting brain any of the above, m is 0 and n is 0, 1, 2, 3, or 4. In other further FAAH activity in the mouse. Preferably, the peripherally embodiments, m is 1 and n is 0, 1, 2, 3, or 4. In still other restricted FAAH inhibitor is one which reduces FAAH activ embodiments, m is 2 and n is 0,1,2,3, or 4. In yet still further ity in the periphery by at least 3, 4, 5, 7-, 8-fold, or 10-fold embodiments, m is 3, and n is 0, 1, 2, 3, or 4. In some further more than it reduced FAAH activity centrally (e.g., in the embodiments, the sum of mand n is 0, 1, 2, or 3. Instill further brain) of the test mammal. For instance, FAAH activity levels embodiments, of each of the above, each hydrocarbyl mem in the periphery can be inhibited by 80% (20% of the baseline ber is unsubstituted. or uninhibited level of FAAH activity remains) while central FAAH activity would be inhibited by 10% (90% of the base 0055 Preferably, R is hydroxy or a physiologically line or uninhibited level of FAAH activity remains) providing hydrolysable ester of the hydroxy. These esters include those for a 80%/10% or 8-fold difference in FAAH inhibition. of the formula —OC(O)R, wherein R, is substituted or 0052 A physiologically cleavable ester is one which is a unsubstituted hydrocarbyl, more preferably, substituted or substrate for carboxyesterases in vivo. Physiologically cleav unsubstituted alkyl, alkenyl, cycloalkyl, heteroalkyl, hetero able esters are typically rapidly hydrolyzed such that the cycloalkyl, heteroalkenyl, heterocycloalkenyl, and cycoalk concentration of the corresponding alcohol comes to exceed enyl and still more preferably, substituted or unsubstituted that of the ester in blood or plasma. For instance, a physi (C-C)alkyl (e.g., methyl, ethyl, propyl, trifluoromethyl) or a ologically cleavable ester is one which is rapidly hydrolyzed substituted or unsubstituted (C-C) hydrocarbyl selected to the corresponding alcohol and acid in vivo with a halftime from alkenyl, cycloalkyl, heteroalkyl, heterocycloalkyl, het of less than /2, 1, 2 or 3 hours at a therapeutically relevant eroalkenyl, heterocycloalkenyl, and cycloalkenyl. In further dosages. of these embodiments, m is 0 and n is 0, 1, 2., m is 1 and n is 0, 1, or 2; or m is 2 and n is 0,1, or 2. Compounds of the Invention 0056. In further embodiments that are applicable to any of the above, R and R are hydrogen or a Substituted or unsub 0053 The compounds of the invention are according to the stituted (C-C)hydrocarbyl selected from alkyl, alkenyl, formula: cycloalkyl, heteroalkyl, heterocycloalkyl, heteroalkenyl, het erocycloalkenyl, and cycoalkenyl. In further of these embodi ments, m is 0 and n is 0, 1, or 2; m is 1 and n is 0, 1, or 2; or m is 2 and n is 0, 1, or 2. In some further embodiments of such, R at least one or both of R and R is hydrogen. In further of O N these embodiments, m is 0 and n is 0, 1, 2; m is 1 and n is 0. YR, 1, or 2; or m is 2 and n is 0,1, or 2. Instill further embodiments, the R and/or Rhydrocarbyl member is unsubstituted. 21 0057. In further embodiments that are applicable to any of O -i-(R4), the above, R is hydroxy and at least one of R and R is R1 N1S hydrogen. In still further embodiments of such, both of R and Rs are hydrogen. In other embodiments in which R is O 44 R hydroxy, R and R are independently selected from substi (R5) in tuted or unsubstituted (C-C)alkyl (e.g., methyl, ethyl, pro pyl), and H. In further of these embodiments, m is 0 and n is 0, 1, 2; m is 1 and n is 0, 1, 2; or m is 2 and n is 0,1, 2. in which R is a polar group. In some embodiments, R is selected from the group consisting of hydroxy and the physi 0058. In yet still further embodiments that are applicable ologically hydrolysable esters thereof, —SH, —O-carboxa to any of the above, R is substituted or unsubstituted cyclo mido. —OC(O)R 7, O CO. NRR and —NRR, hexyl. Substituents for the cyclohexyl include alkyl (e.g., wherein R, is substituted or unsubstituted hydrocarbyl and Rs methyl, ethyl), halo (F, Cl, I, Brand preferably F or Cl), and and R are independently selected from the group consisting trifluoromethyl. In yet other of these embodiments, m is 0 and of hydrogen and substituted or unsubstituted hydrocarbyl, R n is 0, 1, 2; m is 1 and n is 0, 1, 2; or m is 2 and n is 0,1, 2. and R are independently selected from the group consisting 0059. In other embodiments that are applicable to any of of hydrogen and substituted or unsubstituted hydrocarbyl: the above, R is substituted or unsubstituted alkyl, alkenyl, each R is independently selected from the group consisting cycloalkyl, heteroalkyl, heterocycloalkyl, heteroalkenyl, het of halogen and substituted or unsubstituted hydrocarbyland n erocycloalkenyl, or cycloalkenyl and still more preferably, is an integer from 0 to 4: each Rs is independently selected Substituted or unsubstituted (C-C)alkyl (e.g., methyl, ethyl, from the group consisting of halo and Substituted or unsub propyl, trifluoromethyl) or a substituted or unsubstituted (C- stituted hydrocarbyl and m is an integer from 0 to 3; and R is C) hydrocarbyl selected from alkenyl, cycloalkyl, het Substituted or unsubstituted cyclohexyl, and the pharmaceu eroalkyl, heterocycloalkyl, heteroalkenyl, heterocycloalk tically acceptable salts thereof. In some embodiments each of enyl, and cycoalkenyl. In other embodiments, R is selected R. R. R. Rs, and Rare independently selected from hydro from (C-C)alkyl (e.g., methyl, ethyl, propyl), and n is 0, 1, gen and unsubstituted hydrocarbyl. In some further embodi 2, or 3. In still further Such embodiments, each R is halogen ments, each of R. R. R. Rs, and Ro are independently or haloalkyl (e.g., trifluoromethyl). In yet further such US 2013/0217764 A1 Aug. 22, 2013

embodiments, each R is halogen or unsubstituted (C-C) 0063. In preferred embodiments, any of the above com alkyl (e.g., methyl, ethyl, propyl). In further Such embodi pounds are peripherally restricted compounds. ments, m is 0 or 1. 0064 Compounds of the invention may contain one or 0060. In other embodiments that are applicable to any of more asymmetric centers and canthus occuras racemates and the above, Rs is substituted or unsubstituted alkyl, alkenyl, racemic mixtures, single enantiomers, diastereomeric mix cycloalkyl, heteroalkyl, heterocycloalkyl, heteroalkenyl, het tures and individual diastereomers. The present invention is erocycloalkenyl, or cycloalkenyl and still more preferably, meant to comprehend all Such isomeric forms of the inventive Substituted or unsubstituted (C-C)alkyl (e.g., methyl, ethyl, compounds. propyl, trifluoromethyl) or a substituted or unsubstituted (C- 0065 Compounds of the invention include any diastereoi C) hydrocarbyl selected from alkenyl, cycloalkyl, het Somers or pairs of any enantiomers. Diastereomers for eroalkyl, heterocycloalkyl, heteroalkenyl, heterocycloalk example, can be obtained by fractional crystallization from a enyl, and cycoalkenyl. In other embodiments of any of the suitable solvent, for example methanol or ethyl acetate or a above, Rs is selected from (C-C)alkyl (e.g., methyl, ethyl, mixture thereof. The pair of enantiomers thus obtained may propyl), and m is 1, 2, or 3. In still further such embodiments, be separated into individual Stereoisomers by conventional each Rs is halogen or haloalkyl (e.g., trifluoromethyl). In still means, for example by the use of an optically active acid as a further such embodiments, each Rs is halogen or unsubsti resolving agent. tuted (C-C)alkyl (e.g., methyl, ethyl, propyl). In further 0.066 Alternatively, any enantiomer of such a compound such embodiments, n is 0 or 1. of the invention may be obtained by stereospecific synthesis 0061. In a particularly preferred embodiment, R is using optically pure starting materials of known configura hydroxy or a physiologically hydrolyzable ester thereof in tion. which the hydrolysis releases the corresponding compound 0067. The compounds of the present invention may have wherein R is hydroxy, R is unsubstituted cyclohexyl, m is 0 unnatural ratios of atomic isotopes at one or more of their and n is 0, 1, or 2; or m is 1 and n is 0, 1, or 2, or m is 2 and in atoms. For example, the compounds may be radiolabeled is 0, 1, or 2. In some embodiments of such the ester is of the with isotopes, such as tritium or carbon-14. All isotopic varia formula —OC(O)R, wherein R, is substituted or unsubsti tions of the compounds of the present invention, whether tuted hydrocarbyl, more preferably, substituted or unsubsti radioactive or not, are within the scope of the present inven tuted alkyl, alkenyl, cycloalkyl, heteroalkyl, heterocy tion. cloalkyl, heteroalkenyl, heterocycloalkenyl, and 0068. The instant compounds may be isolated in the form cycloalkenyl and still more preferably, substituted or unsub of their pharmaceutically acceptable acid addition salts, such stituted (C-C)alkyl (e.g., methyl, ethyl, propyl, trifluorom as the salts derived from using inorganic and organic acids. ethyl) or a substituted or unsubstituted (C-C) hydrocarbyl Such acids may include hydrochloric, nitric, Sulfuric, phos selected from alkenyl, cycloalkyl, heteroalkyl, heterocy phoric, formic, acetic, trifluoroacetic, propionic, maleic, suc cloalkyl, heteroalkenyl, heterocycloalkenyl, and cycoalk cinic, malonic and the like. In addition, certain compounds enyl. In some further embodiments, R, is unsubstituted containing an acidic function can be in the form of their hydrocarbyl, unsubstituted alkyl, unsubstituted alkenyl, inorganic salt in which the counterion can be selected from unsubstituted cycloalkyl, unsubstituted heteroalkyl, unsub Sodium, potassium, lithium, calcium, magnesium and the stituted heterocycloalkyl, unsubstituted heteroalkenyl, like, as well as from organic bases. The term “pharmaceuti unsubstituted heterocycloalkenyl, or unsubstituted cycoalk cally acceptable salts' refers to salts prepared from pharma enyl: or unsubstituted (C-C)alkyl (e.g., methyl, ethyl, pro ceutically acceptable non-toxic bases or acids including inor pyl, trifluoromethyl) or unsubstituted (C-C) hydrocarbyl ganic bases or acids and organic bases or acids. selected from alkenyl, cycloalkyl, heteroalkyl, heterocy 0069. The invention also encompasses prodrugs of the cloalkyl, heteroalkenyl, heterocycloalkenyl, and cycoalk present compounds, which on administration undergo chemi enyl. cal conversion by metabolic processes before becoming 0062. In a particularly preferred embodiment, the com active pharmacological Substances. In general. Such prodrugs pound has the formula: will be derivatives of the present compounds that are readily convertible in vivo into a functional compound of the inven tion. Conventional procedures for the selection and prepara II tion of suitable prodrug derivatives are described, for example, in “Design of Prodrugs’, ed. H. Bundgaard, Elsevier, 1985. The invention also encompasses active metabolites of the present compounds. 0070. Some of the compounds described herein contain olefinic double bonds, and unless specified otherwise, are meant to include both E and Z geometric isomers. O 0071. Some of the compounds described herein may exist with different points of attachment of hydrogen, referred to as tautomers. Such an example may be a ketone and its enol form OH known as keto-enol tautomers. The individual tautomers as well as mixture thereof are encompassed by the inventive 3'-carbamoyl-6-hydroxybiphenyl-3-yl cyclohexylcarbamate Formulas. or is a physiologically hydrolyzable ester thereof in which the High throughput FAAH Inhibition Assays hydrolysis releases 3'-carbamoyl-6-hydroxybiphenyl-3-yl 0072 The assays for compounds described herein are cyclohexylcarbamate and the pharmaceutically acceptable amenable to high throughput Screening. Preferred assays thus salts thereof. detect binding of the inhibitor to FAAH or the release of a US 2013/0217764 A1 Aug. 22, 2013 reaction product (e.g., fatty acid amide or ethanolamine) pro in part on the nature and severity of the conditions being duced by the hydrolysis of a substrate such as oleoylethano treated and on the nature of the active ingredient. An exem lamide oranandamide. The substrate may be labeled to facili plary route of administration is the oral route. The composi tate detection of the released reaction products. High tions may be conveniently presented in unit dosage form and throughput assays for the presence, absence, or quantification prepared by any of the methods well-known in the art of of particular reaction products are well known to those of skill pharmacy. in the art. Thus, for example, U.S. Pat. No. 5,559.410 dis closes high throughput Screening methods for proteins, and 0077. In practical use, the compounds of the invention can U.S. Pat. No. 5,576,220 and No.5,541,061 disclose high be combined as the active ingredient in intimate admixture throughput methods of Screening for ligand/antibody bind with a pharmaceutical carrier according to conventional phar 1ng. maceutical compounding techniques. The carrier may take a 0073. In addition, high throughput screening systems are wide variety of forms depending on the form of preparation commercially available (see, e.g., Zymark Corp., Hopkinton, desired for administration, e.g., oral or parenteral (including Mass.; Air Technical Industries, Mentor, Ohio; Beckman intravenous). In preparing the compositions for oral dosage Instruments, Inc. Fullerton, Calif.; Precision Systems, Inc., form, any of the usual pharmaceutical media may be Natick, Mass., etc.). These systems typically automate entire employed, such as, for example, water, glycols, oils, alcohols, procedures including all sample and reagent pipetting, liquid flavoring agents, preservatives, coloring agents and the like in dispensing, timed incubations, and final readings of the the case of oral liquid preparations, such as, for example, microplate in detector(s) appropriate for the assay. These Suspensions, elixirs and solutions; or carriers such as configurable systems provide high throughput and rapid start starches, Sugars, microcrystalline cellulose, diluents, granu up as well as a high degree of flexibility and customization. lating agents, lubricants, binders, disintegrating agents and The manufacturers of such systems provide detailed proto the like in the case of oral Solid preparations such as, for cols the various high throughput. Thus, for example, Zymark example, powders, hard and Soft capsules and tablets, with the Corp. provides technical bulletins describing screening sys Solid oral preparations being preferred over the liquid prepa tems for detecting the modulation of gene transcription, rations. ligand binding, and the like. 0078 Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit Methods for Screening Compounds for Antinociceptive form in which case solid pharmaceutical carriers are obvi Activity. ously employed. If desired, tablets may be coated by standard aqueous or nonaqueous techniques. Such compositions and 0074 Methods for screening FAAH inhibitors for an anti preparations can contain at least 0.1 percent of active com nociceptive effect are well known to one of ordinary in the art. pound. The percentage of active compound in these compo For instance, the test compounds can be administered to the sitions may, of course, be varied and may conveniently be Subject animals in the mouse hot-plate test and the mouse between about 2 percent to about 60 percent of the weight of formalin test and the nociceptive reactions to thermal or the unit. The amount of active compound in Such therapeuti chemical tissue damage measured. See also U.S. Pat. No. cally useful compositions is such that a therapeutically effec 6,326, 156 which teaches methods of screening for antinoci tive dosage will be obtained. The active compounds can also ceptive activity. See Cravatt etal. Proc. Natl. Acad. Sci. U.S.A. be administered intranasally as, for example, liquid drops or 98:9371-9376 (2001). Spray. Pharmaceutical Compositions 007.9 The tablets, pills, capsules, and the like may also contain a binder Such as gum tragacanth, acacia, corn starch 0075. The invention also provides pharmaceutical compo or gelatin; excipients such as dicalcium phosphate; a disinte sitions of the above peripherally restricted FAAH inhibitory grating agent Such as corn starch, potato starch, alginic acid: compounds. The term “composition', as in pharmaceutical a lubricant Such as magnesium Stearate; and a Sweetening composition, is intended to encompass a product comprising agent such as Sucrose, lactose or saccharin. When a dosage the active ingredient(s), and the inertingredient(s) that make unit form is a capsule, it may contain, in addition to materials up the carrier, as well as any product which results, directly or of the above type, a liquid carrier Such as a fatty oil. indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of 0080 Various other materials may be present as coatings one or more of the ingredients, or from other types of reac or to modify the physical form of the dosage unit. For tions or interactions of one or more of the ingredients. instance, tablets may be coated with shellac, Sugar or both. A Accordingly, the pharmaceutical compositions of the present syrup or elixir may contain, in addition to the active ingredi invention encompass any composition made by admixing a ent, Sucrose as a Sweetening agent, methyl and propylpara compound of the present invention and a pharmaceutically bens as preservatives, a dye and a flavoring such as cherry or acceptable carrier. The term “pharmaceutical composition' orange flavor. To prevent breakdown during transit through indicates a composition Suitable for pharmaceutical use in a the upper portion of the GI tract, the composition may be an Subject, including an animal or human. A pharmaceutical enteric coated formulation. composition generally comprises an effective amount of an I0081. With respect to formulations with respect to any active agent and a pharmaceutically acceptable carrier. variety of routes of administration, methods and formulations 0076. The compositions include compositions suitable for for the administration of drugs are disclosed in Remington's oral, rectal, topical, parenteral (including Subcutaneous, Pharmaceutical Sciences, 17th Edition, (Gennaro et al. Eds. intramuscular, and intravenous), ocular (ophthalmic), pulmo Mack Publishing Co., 1985). Remington's Pharmaceutical nary (nasal or buccal inhalation), or nasal administration, Sciences, Gennaro AR ed. 20th edition, 2000: Williams & although the most Suitable route in any given case will depend Wilkins Pa., USA. US 2013/0217764 A1 Aug. 22, 2013

Administration 0087. Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of 0082. The compounds of the invention may also be admin the packaged nucleic acid Suspended in diluents, such as istered parenterally. Solutions or Suspensions of these active water, saline or PEG 400; (b) capsules, sachets or tablets, each compounds can be prepared in water Suitably mixed with a containing a predetermined amount of the active ingredient, Surfactant such as hydroxypropylcellulose. Dispersions can as liquids, Solids, granules or gelatin; (c) Suspensions in an also be prepared in glycerol, liquid polyethylene glycols and appropriate liquid; and (d) Suitable emulsions. Tablet forms mixtures thereof in oils. Under ordinary conditions of storage can include one or more of lactose, Sucrose, mannitol, Sorbi and use, these preparations contain a preservative to prevent tol, calcium phosphates, corn starch, potato starch, microc the growth of microorganisms. rystalline cellulose, gelatin, colloidal silicon dioxide, talc, 0083. The pharmaceutical forms suitable for injectable magnesium Stearate, Stearic acid, and other excipients, colo use include sterile aqueous solutions or dispersions and ster rants, fillers, binders, diluents, buffering agents, moistening ile powders for the extemporaneous preparation of sterile agents, preservatives, flavoring agents, dyes, disintegrating injectable solutions or dispersions. In all cases, the form must besterile and must be fluid to the extent that easy syringability agents, and pharmaceutically compatible carriers. Lozenge exists. It must be stable under the conditions of manufacture forms can comprise the active ingredient in a flavor, e.g., and storage and must be preserved against the contaminating Sucrose, as well as pastilles comprising the active ingredient action of microorganisms such as bacteria and fungi. The in an inert base. Such as gelatin and glycerin or Sucrose and carrier can be a solvent or dispersion medium containing, for acacia emulsions, gels, and the like containing, in addition to example, water, ethanol, polyol (e.g. glycerol, propylene gly the active ingredient, carriers known in the art. col and liquid polyethylene glycol), Suitable mixtures thereof, I0088. Injection solutions and suspensions can be prepared and vegetable oils. from sterile powders, granules, and tablets of the kind previ 0084. The compounds of the invention can be effective ously described. Formulations suitable for parenteral admin over a wide dosage range. For example, in the treatment of istration, such as, for example, by intraarticular (in the joints), adult humans, dosages from about 10 to about 1000 mg, about intravenous, intramuscular, intradermal, intraperitoneal, and 100 to about 500 mg or about 1 to about 100 mg may be Subcutaneous routes, include aqueous and non-aqueous, iso needed. Doses of the 0.05 to about 100 mg, and more prefer tonic sterile injection solutions, which can contain antioxi ably from about 0.1 to about 100 mg, per day may be used. A dants, buffers, bacteriostats, and solutes that render the for most preferable dosage is about 0.1 mg to about 70 mg per mulation isotonic with the blood of the intended recipient, day. In choosing a regimen for patients, it may frequently be and aqueous and non-aqueous sterile suspensions that can necessary to begin with a dosage of from about 2 to about 70 include Suspending agents, solubilizers, thickening agents, mg per day and when the condition is under control to reduce stabilizers, and preservatives. the dosage as low as from about 0.1 to about 10 mg per day. I0089. With respect to transdermal routes of administra For example, in the treatment of adult humans, dosages from tion, methods for transdermal administration of drugs are about 0.05 to about 100 mg, preferably from about 0.1 to disclosed in Remington’s Pharmaceutical Sciences, Gennaro about 100 mg, per day may be used. The exact dosage will A Red. 20th edition, 2000: Williams & Wilkins PA, USA. depend upon the mode of administration, on the therapy Dermal or skin patches are a preferred means for transdermal desired, form in which administered, the subject to be treated delivery of the compounds of the invention. Patches prefer and the body weight of the subject to be treated, and the ably provide an absorption enhancer such as DMSO to preference and experience of the physician or veterinarian in increase the absorption of the compounds. Other methods for charge. transdermal drug delivery are disclosed in U.S. Pat. No. 0085 Generally, the compounds of the present invention 5,962,012, 6,261,595, and 6,261,595. Each of which is incor can be dispensed in unit dosage form comprising preferably porated by reference in its entirety. from about 0.1 to about 100 mg of active ingredient together (0090 Preferred patches include those that control the rate with a pharmaceutically acceptable carrier per unit dosage. of drug delivery to the skin. Patches may provide a variety of Usually, dosage forms suitable for oral, nasal, pulmonary or dosing systems including a reservoir system or a monolithic transdermal administration comprise from about 0.001 mg to system, respectively. The reservoir design may, for example, about 100 mg, preferably from about 0.01 mg to about 50 mg have four layers: the adhesive layer that directly contacts the of the compounds admixed with a pharmaceutically accept skin, the control membrane, which controls the diffusion of able carrier or diluent. For storage and use, these preparations drug molecules, the reservoir of drug molecules, and a water preferably contain a preservative to prevent the growth of resistant backing. microorganisms. 0091 Such a design delivers uniform amounts of the drug I0086 Administration of an appropriate amount the candi over a specified time period, the rate of delivery has to be less date compound may be by any means known in the art Such than the saturation limit of different types of skin. as, for example, oral or rectal, parenteral, intraperitoneal, 0092. The monolithic design, for example, typically has intravenous, Subcutaneous, Subdermal, intranasal, or intra only three layers: the adhesive layer, a polymer matrix con muscular. In some embodiments, administration is transder taining the compound, and a water-proofbacking This design mal. An appropriate amount or dose of the candidate com brings a Saturating amount of drug to the skin. Thereby, pound may be determined empirically as is known in the art. delivery is controlled by the skin. As the drug amount An appropriate or therapeutic amount is an amount Sufficient decreases in the patch to below the saturating level, the deliv to provided the desired therapeutic effect (e.g., treat or alle ery rate falls. viate pain or treat or reduce inflammation). The candidate 0093 Compounds of the invention may be used in com compound can be administered as often as required to allevi bination with other compounds of the invention or with other ate the pain or reduce the inflammation, for example, hourly, drugs that may also be useful in the treatment, prevention, every six, eight, twelve, or eighteen hours, daily, or weekly Suppression of pain, inflammation, or immune disorders. In US 2013/0217764 A1 Aug. 22, 2013

one embodiment, the second drug is not a FAAH inhibitor and mediators are released which strengthen nociception. As a is directed toward the same disorder as the FAAH inhibitor. result primary hyperalgesia (increased sensitivity to pain) Such other drugs may be administered, by a route and in an occurring in the area of injury and a secondary hyperalgesia amount commonly used therefor, contemporaneously or occurring in the tissue Surrounding the injury ensue. The sequentially with a compound of the invention. When a com hyperalgesia Subsides with the inflammation as the tissue is pound of the invention is used contemporaneously with one healed. In some further embodiments, the inflammation is or more other drugs, a pharmaceutical composition in unit associated with pulmonary edema, kidney Stones, minor inju dosage form containing such other drugs and the compound is ries, wound healing, skin wound healing, vaginitis, candidi preferred. When used in combination with one or more other asis, lumbar spondylanhrosis, lumbar spondylarthrosis, vas active ingredients, the compound of the present invention and cular diseases, migraine headaches, sinus headaches, tension the other active ingredients may be used in lower doses than headaches, dental pain, periarteritis nodosa, thyroiditis, when each is used singly. Accordingly, the pharmaceutical aplastic anemia, Hodgkin’s disease, Sclerodoma, rheumatic compositions of the present invention include those that con fever, type I diabetes, type II diabetes, myasthenia gravis, tain one or more other active ingredients, in addition to the multiple Sclerosis, sarcoidosis, nephrotic syndrome, Behcet’s compounds disclosed above. syndrome, polymyositis, gingivitis, hypersensitivity, Swell 0094. In the pharmaceutical compositions of the present ing occurring after injury, or myocardialischemia, or osteoar invention for oral, Sublingual, Subcutaneous, intramuscular, thritis. intravenous, transdermal, local or rectal administration, the active principle, by itself or in association with another active Control of Inflammation principle, can be administered to animals and humans in unit forms of administration mixed with conventional pharmaceu 0098. In some embodiments, the compounds of Formula I tical carriers. The appropriate unit forms of administration and II may be administered to alleviate inflammation in a include oral forms such as tablets, gelatin capsules, powders, Subject. The treatment may be prophylactic or therapeutic. granules and solutions or Suspensions to be taken orally, The treatment may be administered to a human subject. The Sublingual and buccal forms of administration, aerosols, compounds and compositions of the invention may be admin implants, Subcutaneous, intramuscular, intravenous, intrana istered solely for the purposes of reducing the severity or sal or intraocular forms of administration and rectal forms of frequency or extent of the inflammation. The treatment may administration. be administered in a combination therapy with another pain 0095. In other embodiments, the pharmaceutical compo reliever or anti-inflammatory agent. sitions of the present invention, the active principle or active principles are generally formulated in dosage units. The dos Control of Immune Disorders age unit contains from 0.5 to 1000 mg, advantageously from 0099. The following examples are provided for illustrative 1 to 500 mg and preferably from 2 to 200 mg of FAAH purposes, and are not intended to limit the scope of the inven inhibitor per dosage unit for daily administration. tion as claimed herein. Any variations in the exemplified articles and/or methods which occur to the skilled artisan are Methods of Treatment intended to fall within the scope of the present invention. Control of Pain EXAMPLES 0096. In some embodiments, the compounds of Formula I and II may be administered to alleviate or treat pain in a Materials and Methods Subject. The treatment may be prophylactic or therapeutic. 0100 Drug distribution coefficients. We determined Log The treatment may be administered to a human subject. The D, a values at room temperature (25tl°C.) partitioning compounds and compositions of the invention may be admin the solutes between n-octanol and an aqueous solution buff istered solely for the purposes of reducing the severity or ered at pH 7.4. frequency or extent of pain. The treatment may be adminis tered in a combination therapy with another pain reliever or 0101 Enzyme assays. We conducted standard FAAH and anti-inflammatory agent. In some embodiments, the pain can monoacylglycerol lipase assays as described', using as be a neuropathic pain selected from the group consisting of substrates H1-anandamide (a gift of the National Institute post trigeminal neuralgia, neuropathic low back pain, periph on Drug Abuse) and 2-oleoyl-sn-glycerol (Nu-Check Prep, eral or polyneuropathic pain, complex regional pain Syn Elysian, Minn.), respectively. drome (causalgia and reflex sympathetic dystrophy), diabetic 0102 Drug transport assays. Assays were performed at neuropathy, toxic neuropathy, and chronic neuropathy caused Cerep Inc. (Redmond, Wash.), following protocols outlined by chemotherapeutic agents. In other embodiments, the pain in the company's web-site (http://www.cerep.fr). is renal and liver colic pain or fibromyalgia. In some neuro 0103 Tissue analyses. We performed tissue extractions pathic pain embodiments, the primary lesion or dysfunction and liquid chromatography/mass spectrometry analyses of of the nervous system is caused by a mechanical injury to a endocannabinoids as described. Similar procedures were nerve of the subject. In a further embodiment, the mechanical utilized for the tissue extraction and LC/MS quantification of injury is due to compression of a nerve, transection of nerve, URB937, as detailed further below. causalgia, Spinal cord injury, post Surgical pain, phantom 0104 Fos expression. We measured Fos protein levels by limb pain, or scar formation in the Subject. quantitative immunocytochemistry on slices of lumbar (L4/ 0097. In other embodiments, the pain is a pain caused by L5) spinal cord from male Sprague-Dawley rats. inflammation or injury of a tissue. Inflammatory pain devel 0105 Surgery. We performed sciatic nerve ligations in ops in response to tissue damage occurring from the noxious male Swiss mice, as previously described for rats' with stimuli. In response to the tissue injury, cytokines and other minor modifications. US 2013/0217764 A1 Aug. 22, 2013

0106 Behavioral tests. We measured nocifensive and the University of Georgia, Athens, and were in compli responses elicited by i.p. injection of acetic acid in male Swiss ance with the European Community Council Directive 86 and C57B1/6 (wild-type or FAAH-deficient) mice', intra (609) EEC and the experimental protocol was carried out in plantar injection of carrageenan in male Swiss mice', intra compliance with Italian regulations (DL 116/92). plantar injection of formalin in male Sprague-Dawley rats', 0111 Tissue extractions Mice were sacrificed with isoflu and sciatic nerve ligation in male Swiss mice. rane and tissues were collected and immediately frozen in 0107 Synthesis of FAAH inhibitors URB937 was synthe liquid nitrogen. Frozen tissues were weighed and homog sized largely following a published procedure. The com enized in methanol (1 mL) containing Ha-anandamide, pound was obtained in five steps starting from 3-bromo-4- H.-PEA, Hs-2-AG, and N-cyclohexylbiphenyl-3-ylac hydroxybenzaldehyde, which was benzylated (BZC1, DMF, etamide as internal standards. Analytes were extracted with CsCO, rt, 3 h), then oxidized and hydrolyzed (m-CPBA, chloroform (2 vol) and washed with water (1 vol). Organic CHCl, 40°C., 72 h; NaOMe, EtOH, rt, 1 h) to 4-benzyloxy phases were collected and dried under nitrogen. The non 3-bromophenol; the latter was elaborated by Suzuki coupling fractionated organic extract was used for quantification of 3-carbamoylphenylboronic acid, toluene, Pd(PPh), URB937. For other analyses the organic extract was fraction NaCO/HO, reflux,2h), carbamation (c-CHCNO, Et N. ated by open-bed silica gel column chromatography, as toluene/CHCN 1:1, reflux, 18 h) and hydrogenative depro described. Briefly, the extract was dissolved in chloroform tection to the desired compound. N-cyclohexyl-O-biphenyl and loaded onto Small glass columns packed with Silica Gel 3-yl carbamates 1c-e were synthesized by reaction of the G (60-A 230-400 Mesh ASTM; Whatman, Clifton, N.J.). opportune 3'-carbamoyl-4-substituted phenol with cyclo Anandamide, PEA and 2-AG were eluted with chloroform/ hexyl isocyanate, while 1 f was obtained by a Pd/C catalyzed methanol (9:1, vol/vol). hydrogenation of the corresponding nitrocarbamate precur 0112 Serum extractions Trunk blood was collected from sor, which results from the suitable phenol derivative. All decapitated mice, allowed to clot and placed on ice. The biphenols were synthesized by a Suzuki cross-coupling reac clotted blood was centrifuged at 18,000xg for 10 min at 4°C. tion between 3-carbamoylphenylboronic acid and the corre and the serum was transferred to glass vials and diluted with sponding 3-bromo-4-substituted phenols (in the case of the distilled water to 1 mL. Proteins were precipitated with ice precursors of 1c,d) or 3-chloro-4-fluorophenol (for those of cold acetone (1 mL) containing N-cyclohexyl biphenyl-3- 1e). Detailed synthetic procedures for all the compounds will ylacetamide as an internal standard, and the precipitate was be reported elsewhere. removed by centrifugation at 3000xg for 10 min at 4°C. The 01.08 Synthesis of cyclohexylcarbamic acid 3'-carbam samples were dried under nitrogen to remove acetone, and oyl-6-hydroxybiphenyl-3-yl ester (URB937). To a stirred extracted with chloroform/methanol as described above. Suspension of cyclohexylcarbamic acid 3'-carbamoyl-6-ben 0113 Liquid chromatography/mass spectrometry (LC/ Zyloxybiphenyl-3-yl ester (222 mg: 0.5 mmol) in EtOAc (2.5 MS) Tissue levels of anandamide, PEA, 2-AG and URB937 mL) and EtOH (2.5 mL), 10% Pd/C (22 mg) was added. The were determined using an 1100-LC system coupled to a mixture was hydrogenated at 4 atm at 50° C. for 4 h, cooled, 1946A-MS detector (Agilent Technologies, Inc., Palo Alto, filtered on Celite and concentrated. Purification of the residue Calif.) equipped with an electrospray ionization interface. by column chromatography (cyclohexane/EtOAc 1:9) and URB937 and N-cyclohexyl biphenyl-3-ylacetamide recrystallization gave URB937 as a white solid. Yield: 92% (m/z=294) were eluted on an XDB Eclipse C8 column (50x (0.163 g). Mp: 128-130° C. (CHC1/n-hexane). MS (ESI) 4.6-mm inner diameter, 1.8 um, Zorbax) using a linear gra m/z. 355.2 (M+H). H NMR (200 MHz, CDC1) 8: =1.13-2. dient of 60% to 100% of A in B Over 3 minata flow rate of 1.0 02 (m. 10H), 3.55 (m, 1H), 5.13 (brd, 1H), 5.85 (brs, 1H), mL-min'. Mobile phase A consisted of methanol containing 6.59 (brs, 1H), 6.74-6.95 (m,3H), 7.07 (s, 1H), 7.34-7.41 (m, 0.25% acetic acid, 5 mMammonium acetate; mobile phase B 1H), 7.56 (m, 1H), 7.68-7.75 (m, 2H) ppm. IR (Nujol) n: consisted of water containing 0.25% acetic acid, 5 mM 3333, 1701, 1655 cm. ammonium acetate. Anandamide, 2-AG and PEA were eluted I0109) Other chemicals Hi-Anandamide was purchased with a gradient of methanol in water (from 85% to 90% from American Radiolabeled Chemicals, Inc. (St. Louis, methanol in 2.5 min) at a flow rate of 01.0 mL-min'. Col Mo.). 2-Hs-AG and AM630 were from Cayman Chemical umn temperature was kept at 40°C. MS detection was in the (Ann Arbor, Mich.). Anandamide, Ha-anandamide and positive ionization mode, capillary Voltage was set at 3 kV. PEA were synthesized in the laboratory'. Rimonabant and and fragmentor voltage was varied from 120 to 140 V. Nitro N-cyclohexyl biphenyl-3-ylacetamide were kind gifts of the gen was used as drying gas at a flow rate of 13 L-min' and a National Institute on Drug Abuse and Kadmus Pharmaceuti temperature of 350° C. Nebulizer pressure was set at 60 psi. cals Inc., respectively. Na" adducts (IM+Na") of analytes and internal standards 0110 Animals We used male Swiss Webster mice were monitored in the selective ion-monitoring mode. Limit (Charles River, 20-30g), male C57B 1/6 (Jackson Laboratory, of quantification was 0.4 pmol. 20-25 g), male FAAH-deficient mice (25-35 g) back crossed 0114 Drug transport assays Transport assays were con more than 10 times on a C57B1/6 background, male Wistar ducted at Cerep Inc. (Redmond, Wash.). Cellular permeabil rats (Charles River, 250-300 g) and male Sprague-Dawley ity was determined in the apical (A) to basal (B) and the B to (SD) rats (Harlan Laboratories, 275-350 g). Mice and Wistar A direction in the presence of 10 uM compound in Hanks rats were group-housed in standard cages at room tempera buffered salt solution containing 1% dimethylsulfoxide ture on a 12:12 h light:dark cycle with unlimited access to (DMSO). Efflux ratios were calculated as the ratio of B-A to water and standard chow pellets. Wistar rats were typically A-B permeabilities. used for the FAAH studies. All experiments met the National 0115 Fos immunohistochemistry Two hours after forma Institutes of Health guidelines for the care and use of labora lin injection into the dorsal paw, rats received a lethal dose of tory animals, were approved by the Institutional Animal Care Nembutal (50 mg-mL). They were perfused through the and Use Committee of the University of California, Irvine, heart with 100 mL of 1% heparinized phosphate buffered US 2013/0217764 A1 Aug. 22, 2013

saline (PBS) followed by 300 mL of ice-cold 4% paraform below the horizontal plane. A 22-gauge stainless-steel guide aldehyde. The lumbar-sacral region of the spinal cord was cannula was implanted into the right lateral cerebral Ventricle collected from each rat. Spinal cords were post fixed in 4% 7 days prior to experiments. Coordinates for implantation paraformaldehyde at 4°C. for 24h and then cryoprotected in (-0.9 mm antero/posterior and -1.5 mm medio/lateral rela 30% sucrose at 4° C. for 24-48 hours. Spinal cords were tive to bregma, and 3.8 mm below the surface of the skull) cryostat-cut into 40 um transverse sections at the level of the were determined using the Paxinos and Watson rat brain lumbar enlargement (L4/L5). Free floating alternating sec atlas". Guide cannulae were anchored to the skull with 3 tions were kept in a series of wells filled with PBS. Every stainless-steel screws and dental cement, and were kept fourth section was processed for Fos immunoreactivity to patent until injection by insertion of a dummy stylet. For ensure that the same cell would not be counted twice. Endog injections, the stylet was removed and drug or vehicle were enous peroxidases were inactivated by incubation in hydro infused in a volume of 5 uL with a 10 uL Hamilton microsy gen peroxide. Sections were blocked with goat serum to pre ringe connected to a 28 gauge stainless-steel injector, which vent non-specific binding and were incubated in the presence protruded 1 mm beyond the tip of the guide cannula, by a of Fos primary antibody (1:20,000, Abcam, Cambridge, PTFE 24G catheter (Small parts Inc, Logansport, Ind.) filled Mass., USA) diluted in PBS containing 0.4% Triton (for 1 h with PBS. A small air bubble (3 uL) was drawn at the distal at 37° C. and then 48 h at 4°C.). Tissue was incubated in the end of the PTFE.24G catheter to separate the injected solution presence of biotinylated goat anti-rabbit IgG (1:600, Vector from the PBS and for visual inspection of the injection. Injec Laboratories, Burlingame, Calif., USA) secondary antibody tions were performed over a 1-min period and the injector was for 1 h at 37° C. Subsequently, sections were incubated in a kept in place for an additional 1 min to prevent back flow Vectastain Elite ABC reagent (1:200, Vector Laboratories) for leakage. Placement of the cannulae was verified at the end of 1 h, followed by a 5 min incubation in 2% nickel intensified the experiments by injection of trypan blue (5 LL) before the diaminobenzidine. Sections were mounted onto glass slides, rats were euthanized. Only animals with proper placements air dried, dehydrated in ascending concentrations of ethanol, were included in the study. Rats were allowed to recover for cleared in Xylene, and protected by a glass coverslip affixed 7-10 days before experiments. Sciatic nerve ligation was with Permount. All conditions in the same experiment were performed in Swiss mice, using an adaptation of the method processed concurrently, to control for variance in immun of Bennett and Xie. Mice were anesthetized with xylazine ostaining across different runs. Immunostaining specificity (10 mg-kg', i.p.) and ketamine (100 mg-kg', i.p.), the left was established by primary antibody omission from the thigh was shaved and scrubbed with Betadine(R), and a small immunostaining protocol and by demonstration that pread incision (2 cm in length) was made in the middle of the left sorbtion with the peptide antiserum blocked specific stain thigh to expose the sciatic nerve. The nerve was loosely tied ing. at two distinct sites (spaced at a 2-mm interval) around the 0116. Immunoreactivity quantification Under a micro entire diameter of the nerve using silk sutures (7-0). The Scope, three L4 sections that qualitatively exhibited the great Surgical area was dusted with streptomycin powder and est number of Fos-positive cells were selected for quantifica closed with a single muscle Suture and two skin clips and tion. Selection of sections and quantification of the number of finally scrubbed with Betadine(R). In sham-operated animals, Fos-positive cells was performed by an observer blinded to the nerve was exposed but left untied. The animals were experimental conditions. Sections were captured at 5x mag placed under a heat lamp until they awakened. nification using a DMLB light microscope and a 1,300 digital 0119 Behavioral tests Acetic acid-induced writhing was camera under similar brightness/contrast settings to demon measured in Swiss mice or C57B 1/6 mice (wild-type and strate comparable background staining Laminar Subdivisions FAAH-deficient), as described" with minor modifications. were drawn on all sections using the Image J Software (U.S. Briefly, the mice were acclimated to the experimental room National Institutes of Health, Bethesda, Md., USA). Subdi for 2 h. Each animal was injected with acetic acid (150 uL. visions of the spinal gray matter were defined as the Superfi 0.6% in Saline) and placed into a glass cylinder. Abdominal cial laminae (laminae I and II), nucleus proprius (laminae III stretches (extension of body and hindlimbs) were counted for and IV), neck of the dorsal horn (laminae V and VI), and 20 min, starting 5 min after acetic acid injection. URB937, ventral horn (laminae, VII, VIII, IX, and X). Using Image J, rimonabant and AM630 were administered by s.c. injection 1 Fos expressing cells were counted in each Subdivision, h before acetic acid. Behavior was scored by an observer regardless of staining intensity, by an observer blinded to blinded to the treatment conditions. Formalin-induced noci experimental treatments. The intra-rater reliability ranged ception was assessed in Sprague-Dawley rats, as described'. from 93% in the superficial lamina to 81% across all lamina The rats were singly housed and kept in a shared holding subdivisions. room under a 12:12 hlight:dark cycle. They were given free 0117 Drug preparation for in vivo experiments Drugs access to food and water, and allowed to acclimate to the were dissolved in polyethylene glycol 400/Tween-80/saline facility for a week before testing. One h prior to formalin (1/1/18; by volume) and administered by i.p. (5-10 mL-kg) administration, the rats received i.p. injections of vehicle, or s.c. injection (10 mL-kg'). For lateral cerebral ventricle URB937 (1 mg-kg', i.p.), rimonabant (2 mg-kg i.p.) or a injections, URB937 was dissolved in 100% DMSO and combination of URB937 and rimonabant. They were accli injected in a volume of 5 uL. mated to the observation container (clear plexiglas box, 0118 Surgeries All surgeries were conducted under asep 29x29x25 cm) for 15 min before receiving an injection of tical conditions. Implantation of cannulae for intracere formalin (50 uL, 5% in saline) into the dorsal surface of the broVentricular (i.c.V.) drug administration. SD rats were anes right hind paw. Immediately after formalin injection, the rats thesized using a mixture of ketamine (70 mg-kg', i.p.) and were returned to the observation container and nocifensive xylazine (9.33 mg-kg', i.p.). They were placed in a stereo behavior was recorded for 60 min with a video camera. taxic frame and stabilized by ear bars (David Kopf Instru Recordings were analyzed by observers blinded to treatment ments, Tujunga, Calif., USA) with the incisor bar set 2.4 mm conditions. Nocifensive behavior was measured continuously US 2013/0217764 A1 Aug. 22, 2013 for 60 min'. The total time spent by the animals in three The new compounds had comparable potencies when tested different behavioural categories (0, 1, 2) was recorded in in membrane preparations of rat brain FAAH, and were 5-min bins where: (0) the rat exhibits normal posture, (1) the equally effective at blocking liver FAAH activity when injected paw is raised, or (2) the injected paw is licked, shaken administered systemically in mice (1 mg-kg', intraperito or bitten. Each 5-min bin was analyzed for time spent (1) neal, i.p.) (Table 1, 1b-1f). They markedly differed, however, lifting and (2) licking orbiting the injected paw. Nocifensive in their ability to access the CNS. In particular, the p-hydrox behavior was analyzed using the composite pain score yphenyl derivative URB937 (Table 1, 1b) suppressed FAAH weighted scores technique (CPS-WST1.2) calculated for the activity in peripheral tissues of mice and rats, yet failed to entire time of observation (0-60 min) and, separately, for the affect brain FAAH activity (Table 1, Table 2). A dose-explo first (0-10 min) and second phase (10-60 min) of the behav ration study in mice showed that the median effective dose ioral response". The area under the curve (AUC) correspond (EDs) of URB937 for FAAH inhibition in brain was 200 ing to CPS-WST1.2 was calculated using the trapezoidal rule. times higher than the EDs for FAAH inhibition in liver (FIG. Paw edema was induced in mice by injection into the right 1a). Moreover, after systemic administration, URB937 (1 hind paws of 50 uL of sterile Saline containing 1% w-carrag mg-kg', i.p.) distributed rapidly in serum and liver, but eenan. Paw Volumes were measured using a plethysmometer remained undetectable in brain tissue (FIG.1b). As seen with (Ugo Basile, Milan, Italy). Vehicle or URB937 (1 mg kg, other O-arylcarbamates, which are known to interact with i.p.) were injected immediately before carrageenan. Rimona FAAH through a covalent mechanism'', URB937 inhib bant and AM630 (1 mgkg', i.p.) were injected 30min before ited peripheral FAAH activity in vivo both rapidly and last carrageenan. Mechanical hyperalgesia was assessed by mea ingly (FIG. 1c). Suring the latency(s) to withdraw the paw from a constant I0122) Mechanism of peripheral segregation Exploratory mechanical pressure exerted onto its dorsal Surface. A 15-g structure-activity relationship analyses indicate that the calibrated glass cylindrical rod (diameter=10 mm) chamfered polarity of the p-hydroxyphenyl moiety is an essential con to a conical point (diameter 3 mm) was used to exert the tributor to the peripheral segregation of URB937. Table 1 mechanical force. The weight was suspended vertically shows that analogs in which the R substituent was weakly between two rings attached to a stand and was free to move polar or apolar—compounds 1c. 1d and le—readily entered vertically. A cutoff time of 3 min was used. Thermal hyper the brain after systemic administration in mice, whereas an algesia was assessed as described', measuring the latency to analog in which R consisted of a polar amino group, com withdraw the hind paw from a focused beam of radiant heat pound 1 f, was largely excluded. Nevertheless, the relatively (thermal intensity: infrared 3.0) applied to the plantar surface, high lipophilicity of URB937 (distribution coefficient, Log using a commercial apparatus (Ugo Basile, Varese, Italy). The D. : URB937, 3.03+0.01; URB597, 3.71+0.01; cutoff time was set at 30s. Mechanical allodynia was assessed meanisem, n=3) should permit the passive diffusion of the by applying a graded force to the plantar hind paw Surface molecule into the CNS unless this process is actively coun with a Von Frey filament, using a Dynamic Plantar Anesthe tered. As a first test of this idea, we determined the perme siometer (Ugo Basile). The cutoff force was set at 50 g. ability and efflux ratios of URB937 through polarized mono 0120 Statistical Analyses Results are expressed as the layers of human epithelial TC7 cells, which express various meants.e.m. Statistical significance was determined by Stu protein transporters involved in the extrusion of drugs from dents t test, one-way, or two-way analysis of variance the brain'7. URB937 did not equally distribute across the (ANOVA) followed by Bonferroni post hoc test when appro apical (A) and basal (B) compartments of TC7 monolayers, as priate. A separate univariate analysis of variance was per would be expected of a lipophilic molecule moving by pas formed to determine the effects of experimental treatment on sive diffusion. Rather, the compound accumulated into the A formalin-induced nocifensive behavior as measured by area compartment permeability, in nim-s' (% recovery) A-B, 38 under the curve. A repeated measures (TreatmentxTime re (83%); B-A, 371 (95%); efflux ratio, 9.8; mean of 2 indepen peated factor) analysis of variance was performed on forma dent experiments, through a mechanism that was insensitive lin-induced composite pain score. The Greenhouse-Geisser to the permeability-glycoprotein (Pgp) inhibitor Verapamil correction was applied to all repeated factors. For each lami (100 uM) B-A permeability in nim-s' (% recovery): 322 nar Subdivision at L4/L5 of the spinal cord, a univariate analy (94%). These findings suggest that URB937 is extruded sis of variance was performed to determine the effects of from the CNS by a transport system that is pharmacologically experimental treatment on the number of Fos-expressing distinct from Pgp. Consistent with this interpretation, injec cells. Fisher's LSD and Tukey post hoc tests were performed tion of a sub-maximaldose of URB937 (0.1 mg-kg') into the on behavioral and Fos immunostaining data, respectively. lateral cerebral ventricles of rats produced within 1 h an Post hoc comparisons that did not meet the equal variance almost complete inhibition of liver FAAH (residual activity: assumption were corrected by fractional adjustment of the 11.3+1.9% of control; meanisem, n=3). Conversely, sys degrees of freedom. Analyses were performed using SPSS temic administration of a 10 times higher dose of drug (1 statistical software (version 17.0: SPSS Incorporated, Chi mg-kg', i.p.) had no detectable effect on rat brain FAAH cago, Ill., USA). (Table 2). Results I0123 To investigate the identity of the transport system involved in the extrusion of URB937 from the CNS, we 0121 Discovery of a peripherally restricted FAAH inhibi administered in rats various pharmacological inhibitors of tor Current FAAH inhibitors readily cross the blood-brain blood-brainbarrier transporters along with the highest system barrier'. To produce inhibitors with restricted access to the dose of URB937 that does not achieve brain penetration (25 CNS, we added chemical groups of varying polarity to the mg-kg', i.p.)(FIG.1a). Co-administration of the compound proximal phenyl ring of the O-arylcarbamate URB597''' Ko-143, an inhibitor of breast cancer resistance protein (Table 1, 1a), in a position where small-sized hydrophilic (BCRP, ABCG1) caused a dose-dependent increase in the substituents are not expected to impair biological activity'. access of URB937 to the brain. By contrast, co-administra US 2013/0217764 A1 Aug. 22, 2013

tion of Verapamil, a permeability-glycoprotein (Pgp) inhibi I0128. To determine whether enhancement of peripheral tor, or probenecid, an organic anion transport protein inhibi anandamide activity alters the spinal processing of nocicep tor, had no such effect. The results suggest that URB937 is tive inputs, we measured formalin-induced Fos expression in extruded from the CNS by a transporter protein that is phar the same animals subjected to behavioral testing. URB937 macologically indistinguishable from BCRP. lowered the Fos response to formalin (FIG.3a,b), decreasing 0124 Enhancement of peripheral anandamide signaling the number of Fos-positive cells in the Superficial dorsal horn Administration of URB937 (1 mg-kg', i.p.) in mice (lamina I, II), nucleus proprius (lamina III, IV), neck region of increasedanandamide levels in the periphery, not inforebrain the dorsal horn (lamina V. VI), and ventral horn (FIG. 3d). or hypothalamus (FIG. 1d. FIG. 6). This effect was caused by This effect was prevented by the CB, antagonist rimonabant selective inhibition of FAAH activity because (i) it was (2 mg-kg', i.p.), which did not significantly alter Fos levels accompanied by elevations in other endogenous FAAH Sub when administered without URB937 (FIG.3c,d). The ability strates, such as palmitoylethanolamide (PEA) (FGI. 1d); and of URB937 to suppress spinal nociceptive processing, despite (ii) it was not observed in mutant mice in which expression of its lack of CNS penetration, Suggests that peripheral ananda the faah gene had been disrupted by homologous recombina mide modulates pain inputs before they enter the spinal cord. tion' (FIG. 1e). Importantly, URB937 did not affect monoa I0129. Modulation of inflammatory and neuropathic pain cylglycerollipase activity in vitro (median inhibitory concen We also asked whether peripheral inhibition of FAAH activity tration, ICs >100 uM; n=3) and did not alter tissue levels of might influence persistent pain responses caused by inflam its endocannabinoid substrate, 2-AG, in vivo (FIG. 1f). mation or nerve damage. We induced an inflammatory reac 0.125 Modulation of visceral pain Brain-penetrating tion in mice by injecting the polysaccharide carrageenan into FAAH inhibitors attenuate behavioral responses to noxious one of their hind paws. This resulted in the development of stimuli in rodents, a property that is generally attributed to mechanical and thermal hyperalgesia (heightened sensitivity their ability to augmentanandamide signaling in the brainand to noxious stimuli) as well as local edema (FIG. 4). A single spinal cord'*'. To test whether peripheral anandamide con systemic injection of URB937 (1 mg-kg', i.p.), administered tributes to these actions, we examined the effects of URB937 at the same time as carrageenan, caused a significant decrease on the nocifensive (pain-avoiding) response evoked by injec in mechanical and thermal hyperalgesia, assessed 4 hand 24 tion of acetic acid into the peritoneal cavity of mice. Subcu h following carrageenan treatment (FIG. 4a,b). The drug also taneous administration of URB937 reduced acetic acid-in Suppressed mechanical allodynia (pain from non-noxious duced writhing with an EDso of 0.1 mg-kg (FIG.2a and stimuli) measured 24 h after carrageenan (FIG. 4c). These data not shown). This effect was (i) comparable in efficacy to actions were restricted to the inflamed paws (FIG.7) and were that elicited by the potent non-steroidal analgesic indometha prevented by the CB antagonist rimonabant, but not by the cin (1 mg-kg', s.c.) (FIG.2a); (ii) correlated with the degree CB antagonist AM630 (each at 1 mg-kg', i.p.) (FIG. 4a-c). of peripheral FAAH inhibition (Pearson correlation coeffi URB937 did not affect paw edema 4 h after carrageenan cient, r=0.96, FIG. 2b); and (iii) absent in mutant FAAH injection, but reversed it 24 h after the injection through a deficient mice (FIG. 2c). The antinociceptive effects of mechanism that was sensitive both to CB and CB receptor URB937 were blocked by the CB antagonistrimonabant, but blockade (FIG. 4d). not by the CB antagonist AM630 (each at 1 mg-kg, s.c.) 0.130. In another group of mice, we produced peripheral nerve damage by loosely tying the left sciatic nerve. A (FIG. 2d). single dose of URB937 (1 mg-kg', i.p., 2 h before testing), 0126 Although anandamide is an agonist of Vanilloid administered one week after Surgery, attenuated thermal type-1 transient receptor potential (TRPV-1) channels'’. hyperalgesia and Suppressed mechanical hyperalgesia and URB937 evoked no detectable nocifensive behavior when mechanical allodynia in the operated paws (FIG.5a-c). Nota administered alone (1 mg-kg) (FIG. 2a), which suggests bly, this effect was not accompanied by changes in the reac that the tissue concentrations reached by endogenous anan tivity of non-operated paws, indicating that URB937 selec damide following peripheral FAAH inhibition are unable to tively normalized mechanical and thermal thresholds altered activate TRPV-1 channels. Moreover, stimulation of type-a by nerve injury (FIG.5a-c). Finally, we examined the impact peroxisome proliferator-activated receptors, which can be of repeated injections of URB937 (1 mg-kg", i.p., 2 h before induced by PEA''' (FIG. 1d), is unlikely to explain the testing) once daily for 4 days, starting 3 days after nerve antinociceptive effects of URB937 because such effects were ligation. The treatment elicited an antinociceptive effect that prevented by CB receptor blockade (FIG. 2d). was undistinguishable from that caused by single drug dosing 0127. Modulation of tissue injury-induced pain. In another series of experiments, we assessed the impact of URB937 (1 (FIG. 5d-f), which is suggestive that URB937 alleviates mg-kg', i.p.) in a tissue injury model of persistent pain established neuropathic pain without inducing tolerance. produced by administration of formalin into the dorsal hind REFERENCES paw of rats. Formalin injection elicited a marked nocifensive response, which was attenuated in a time-dependent manner I0131 1 Stein, C. 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Proc Natl Acad Sci lase inhibitors: synthesis, quantitative structure-activity rela USA 98 (16), 9371-9376 (2001). tionships, and molecular modeling studies. J Med Chem 47 0149 19 Starowicz, K., Nigam, S., & DiMarzo, V., Bio (21), 4998-5008 (2004). chemistry and pharmacology of endovanilloids. Pharmacol 0.167 37 King, A. R. et al., URB602 inhibits monoacylg Ther 114 (1), 13-33 (2007). lycerol lipase and selectively blocks 2-arachidonoylglycerol 0150 20 LoVerme, J., La Rana, G., Russo, R., Calignano, degradation in intact brain slices. Chem Biol 14 (12), 1357 A., & Piomelli, D., The search for the palmitoylethanolamide 1365 (2007). receptor. Life Sci 77 (14), 1685-1698 (2005). (0168 38 Astarita, G., Ahmed, F., & Piomelli, D., Identifi 0151. 21 Sagar, D. R. Kendall, D. A., & Chapman, V. cation of biosynthetic precursors for the endocannabinoid Inhibition of fatty acid amide hydrolase produces PPAR anandamide in the rat brain. 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phenyl-3-yl ester) reduces neuropathic pain after oral admin TABLE 2 istration in mice. J Pharmacol Exp Ther 322 (1), 236-242 (2007). % of FAAH inhibition after URB937 1 mg/Kg i.p. 0170 40 Calignano, A., La Rana, G., & Piomelli, D., administration Antinociceptive activity of the endogenous fatty acid amide, Mice Rat palmitylethanolamide. Eur J Pharmacol 419 (2-3), 191-198 Brain -3.0 - 8.0 O.2 6.7 (2001). Colon 84.7 0.3*** 90.8 2.O** 0171 41 Tolsen, A., Berge, O.G., Hunskaar, S. Rosland, Duodenum 843 - 14*** 89.4 0.1*** Hypothalamus 18.4 13.8 10.25.8 J. H., & Hole, K., The formalin test: an evaluation of the IIleum 86.8 2.4*** 91.5 2.8*** method. Pain 51 (1), 5-17 (1992). Jejunum 90.0 - 14*** 90.3 - 0.7*** 0172 42 Fegley, D. et al., Characterization of the fatty acid Kidney 88.3 10*** 914 - 0.5*** amide hydrolase inhibitor cyclohexyl carbamic acid 3'-car Liver 91.7 - 0.7*** 92.1 + O.3*** Lungs N.D. 93.5 + 1.8** bamoyl-biphenyl-3-yl ester (URB597): effects on ananda Spinal cord 17.7 10.6 19.2 15.7 mide and oleoylethanolamide deactivation. J Pharmacol Exp Spleen 72.5 - 0.7*** 86.1 2.1** Ther 313 (1), 352-358 (2005). FAAH activity was not detectable (N.D.) in heart, skeletal, muscle, pancreas or skin. 0173 43 Cadas, H., di Tomaso, E., & Piomelli, D., Occur ***P<0.001; rence and biosynthesis of endogenous cannabinoid precursor, **P - 0.01; N-arachidonoyl phosphatidylethanolamine, in rat brain. J n = 3 Neurosci 17 (4), 1226-1242 (1997). (0174 44 Presley, R. W., Menetrey, D., Levine, J. D., & 1. A compound having the formula: Basbaum, A. I. Systemic morphine Suppresses noxious stimulus-evoked Fos protein-like immunoreactivity in the rat spinal cord. J Neurosci 10 (1), 323-335 (1990). R2 (0175 45 Paxinos, G. & Watson, C., The rat brain in ster O N eotaxic coordinates, 6th ed. (Academic Press/Elsevier, NR, Amsterdam; Boston; 2007). 0176 46 Ruda, M.A., Ling, Q. D., Hohmann, A.G., Peng, 21 Y. B., & Tachibana, T. Altered nociceptive neuronal circuits O -i-(R). after neonatal peripheral inflammation. Science 289 (5479), R1 S1S 628-631 (2000). (0177 47 Hargreaves, K., Dubner, R., Brown, F., Flores, O C., & Joris, J.. A new and sensitive method for measuring (Rs)A4N thermal nociception in cutaneous hyperalgesia. Pain 32 (1), 77-88 (1988). wherein R is selected from the group consisting of (0178 48. Greenhouse and Geisser, 1959. Psychometrika. hydroxy and the physiologically hydrolyzable esters 24:95-112. thereof, —O-carboxamido. —SH, —OC(O)R-7, —O CO. NRR and -NRR, wherein R, is substi TABLE 1. tuted or unsubstituted hydrocarbyl and Rs and R are In vitro and in vivo characterization of O-arylcarbamate FAAH independently selected from the group consisting of inhibitors hydrogen and substituted or unsubstituted hydrocarbyl; R and R are independently selected from the group con CONH sisting of hydrogen and Substituted or unsubstituted hydrocarbyl, each R is independently selected from the group consist H ing of halogen and Substituted or unsubstituted hydro carbyl and n is an integer from 0 to 4. each Rs is independently selected from the group consist N”O ing of halo and substituted or unsubstituted hydrocarbyl R and m is an integer from 0 to 3: FAAH Inhibition FAAH Inhibition R is substituted or unsubstituted cyclohexyl; and Compound R ICso (nM) in liver (%) in brain (%) the pharmaceutically acceptable salts thereof. 1a. H 7.71.5 N.D. 96.204 2. The compound of claim 1, wherein R is —OC(O)R7, (URB597) wherein R, is substituted or unsubstituted hydrocarbyl. 1b OH 26.8 4.9 91.7 O.7 -3.0 - 8.0 3. The compound of claim 1, wherein R is O-carboxa (URB937) mido. 1c OCH 45.3 + 14.1 94.6 0.7 86.421 1d CH, 20.5 + 0.6 93.01.1 91.9 1.5 4. The compound of claim 1, wherein both of RandR are 1e F 49.75.8 90.7 - 1.2 89.7 1.3 independently selected from (C-C)alkyl and H. 1f NH, 42.5 + 4.2 92.20.6 23.22.1 5. The compound of claim 1, wherein R is unsubstituted ICso measured in membrane preparation of rat brain cyclohexyl. FAAH inhibition measured ex vivo 1 h after a single injection in mice (1 mg-kg', 6. The compound of claim 1, wherein R and Rs are each independently selected from halogen and Substituted or unsubstituted (C-C)alkyl. US 2013/0217764 A1 Aug. 22, 2013

7. The compound of claim 1, wherein R, is substituted or 20. A pharmaceutical composition for selectively inhibit unsubstituted (C-C)alkyl. ing peripheral Fatty Acid Amide Hydrolase (FAAH), said 8. The compound of claim 1, wherein R is the physiologi composition comprising a compound of claim 1 and a phar cally hydrolysable ester. maceutically acceptable excipient. 9. The compound of claim 1, wherein R is hydroxy and at 21. A pharmaceutical composition for treating a condition least one of R and R is hydrogen. selected from pain, inflammation, and an immune disorder, 10. The compound of claim 1, wherein R is hydroxy and said composition comprising a compound of claim 1 and a both of R and R are hydrogen pharmaceutically acceptable excipient 11. The compound of claim 1 in which m is 0. 22. A method of treating pain and/or inflammation by 12. The compound of claim 1 in which n is 0. administering to a mammal in need thereof, a therapeutically 13. The compound of claim 1 wherein the sum of m and n effective amount of a compound of claim 1. is 1, 2, or 3. 23. The method of claim 22, wherein the inflammation is 14. The compound of claim 1, wherein the compound has treated. the formula: 24. The method of claim 22 wherein the pain is treated. 25. A method of treating a condition selected from the group consisting of pain, inflammation, and an immune dis order by administering to a mammal in need thereofathera peutically effective amount of a compound of claim 1. 26. A method of increasing in a mammal in need thereof peripheral levels of anandamide, oleoylethanolamide (OEA), palmitylethanolamide (PEA), or stearoylethanolamide (SEA) by administering a compound of claim 1. 27. The method of claim 26, wherein the OEA, PEA, SEA O oranandamide is endogenous to the mammal. 28. The method of claim 26, wherein the OEA, PEA, SEA oranandamide was administered to the mammal before, after, OH or contemporaneous with the administration of the com pound. and the pharmaceutically acceptable salts thereof. 29. The method of claim 26, wherein the administration of 15. A pharmaceutical composition comprising a com the OEA, PEA, SEA oranandamide is contemporaneous with pound of claim 1. the administration of the compound. 16. (canceled) 30. The method of claim 26, wherein the mammal is need 17. (canceled) for treatment of a pain, an inflammation, or an immune dis 18. (canceled) order. 19. (canceled)