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1 US B2 (12) United States Patent Cravatt et a]. (10) Patent N0.: (45) Date of Patent: US 8,772,318 B2 Jul. 8, 2014 (54) (75) (73) (21) (22) (86) (87) (65) (60) (51) (52) (58) METHODS AND COMPOSITIONS RELATED TO TARGETING MONOACYLGLYCEROL LIPASE Inventors: Benjamin F. Cravatt, La Jolla, CA (US); Jonathan Z. Long, Solana Beach, CA (US); Weiwei Li, Sunnyvale, CA (US); Daniel K. Nomura, La Jolla, CA (Us) Assignee: The Scripps Research Institute, La Jolla, CA (US) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 U.S.C. 154(b) by 283 days. Appl. No.: 12/998,642 PCT Filed: Nov. 10, 2009 PCT No.: 371 (0X1) (2), (4) Date: PCT/US2009/ Jul. 26, 2011 PCT Pub. No.: WO2010/ PCT Pub. Date: May 20, 2010 Prior Publication Data US 2011/ A1 Nov. 10,2011 Related U.S. Application Data Provisional application No. 61/199,286,?led on Nov. 14, Int. Cl. A61K 31/443 ( ) C07D 405/06 ( ) U.S. Cl. USPC /321; 546/184; 546/192; 546/197; 514/315 Field of Classi?cation Search USPC /184, 192, 197; 514/315, 321 See application?le for complete search history. (56) References Cited 7,989,623 B2* 2006/ A1 U.S. PATENT DOCUMENTS 8/2011 Zhu /389 4/2006 Nieland et al. FOREIGN PATENT DOCUMENTS W0 WO 2010/ /2010 OTHER PUBLICATIONS Blankman, et al., A Comprehensive Pro?le of Brain Enzymes that Hydrolyze the Endocannabinoid 2-Arachid0n0ylglycerol, Chemis try & Biology 14: (2007). Dinh, et al., RNA Interference Suggests a Primary Role for Monoacylglycerol Lipase in the Degradation of the Endocan nabinoid 2-Arachid0n0yl glycerol, Molecular Pharmacology 66(5): (2004). Goparaju, et al., Anandamide amidohydrolase reacting With 2-arachid0n0ylglycerol, another cannabinoid receptor ligand F EBS Letters 422: (1998). Hohmann, et al., An endocannabinoid mechanism for stress-in duced analgesia, Nature 435: (2005). Kathuria, et al., Modulation of anxiety through blockade of anandamide hydrolysis, Nature Medicine 9(1): (2003). Li, et al., A Functional Proteomic Strategy to Discover Inhibitors for Uncharacterized Hydrolases, J. Am. Chem. Soc. 129(31): (2007). Viso, et al., The Medicinal Chemistry of Agents Targeting Monoacylglycerol Lipase. Current Topics in Medicinal Chemistry 8(3): (2008). * cited by examiner Primary Examiner * Golam M M Shameem (74) Attorney, Agent, or Firm * Hugh Wang; Thomas Fitting (57) ABSTRACT This invention provides compounds that selectively inhibit monoacylglycerol lipase (MAGL). The invention also pro vides methods of using the MAGL selective inhibitors to stimulate 2-Arachidonoylglycerol (2-AG) mediated endocannabinoid signaling in vivo, and to treat conditions that are associated With or linked to endocannabinoid signal ing. The invention additionally provides methods of treating cancer or inhibiting tumor growth by targeting MAGL With MAGL speci?c inhibitors. The invention further provides methods of screening for MAGL inhibitors With improved biochemical and pharmaceutical properties. 4 Claims, 25 Drawing Sheets

2 US. Patent Jul. 8, 2014 Sheet 1 0f 25 US 8,772,318 B2 Figure 1 R2 WWL152 OH O JZL175 CH OH JZL M 1 M mm 5>>> W we; _ mthw vanw. nommmj "w, 5 75 M 37 25

3 US. Patent Jul. 8, 2014 Sheet 2 0f 25 US 8,772,318 B2 Figure 2 Bram membrane hydrolysis (% control) \I 0'! CI 0 El MAGL, IC)50 = 8 nm I FAAH, ICSO = 4 pm O i 5 6 log([jzl184]/nm) 0 Recombinant enzyme hydrolysis (% control) Q U'IN 0010 N U I u MAGL, lc5 = 6 nm I FAAH, 1cso = 4 pm O L. o. A. N 0).,,5. g] 0)

5 US. Patent Jul. 8, 2014 Sheet 4 0f 25 US 8,772,318 B2 Figure 4 30 O a mm m=oe5 0:3: 333: Awamwa m a w n o 60 m6 md. F n T a e m Q. b o e 6:50 Escou 2.60 m. N. m 9 Q m w Q v ww vw Tlme (hrs)

6 US. Patent Jul. 8, 2014 Sheet 5 0f 25 US 8,772,318 B2 Figure 5 a tail-immersion b acetic a?fidjinducecj c rectal temperature 4 n so " h'ng 6 0 Vehicle E in ## v a 3 g 60 g g 2 8 1;, 40 *' E ## a g a B '2 5 q 1 ## $1,- 20 g (D *1: Vehicle JZL184 Vehicle JZL184-5 d locomotor activity 9 hyperre?exia 2 u.9 5 Q 60 w X I Vehicle s a #j# E 40 u Rimonabant (3 mglkg) E 40 5 m D..E g 0 Vehicle JZL184 g 0 Vehicle JZL184

7 US. Patent Jul. 8, 2014 Sheet 6 0f 25 US 8,772,318 B2 Figure 6 a N \ o NO; CH3 b 2-0 o 0 Br bz _ g T + {?f%$c 0* o Q 3. EtaN, 0H \._0 002a QOCOCI <0 o Q Q o) o 0:" JZL184

8 US. Patent Jul. 8, 2014 Sheet 7 0f 25 US 8,772,318 B2 Figure 7 a A 5 0 g 45 MAGL o 10 nm g 4 O nm n. ' A 50 nm u. \ nm E Tlme (min) b 4 8 FAAH 2* E 4.6 '\ o 2.5 pm 0- : 4.4 D 5 M u. A 10 pm 0* 4.2 E O 15 pm P o 0 1'0 2'0 30 4'0 Tlme (min) c km/[i] (M134) MAGL 4400 :t 300 FAAH 13 :t 3

10 US. Patent Jul. 8, 2014 Sheet US 8,772,318 B2 Figure 9 a b ,5 '5 I Control 5 12 a 150 u 4 mglkg 5:91 58 $2,100 I 8m9/k9 E % % El 16 mg/kg 5 4 g 50 I 40 mgfkg 0 C1811 C16:0 C18:1 C16:0

11 US. Patent Jul. 8, 2014 Sheet US 8,772,318 B2 Figure 10 a o (mg/kg,p.o.) O 2-AG (nmol/g tissue) a 0: O N O O O JZL184 (mg/kg, oral) AEA (pmol/g tissue) A30 I i.p. b 9 e % 1: p i) u m 20 g 50 : JZL184 (mg/kg, oral) '2 I o 1 4 1s 40 JZL184 (mg/k9, oral) JZL184 (mg/kg)

12 US. Patent Jul. 8, 2014 Sheet US 8,772,318 B2 Figure 11 a :1 q', DC Lu-i-"s < C» E 9; 0 g - N v 00 R", 8 Time(hrs) b 12 IC18:1 UC1610 MAG (nmol/g tissue) 0 FFA (nmol/g tissue) Time (hrs)

13 US. Patent Jul. 8, 2014 Sheet 12 0f 25 US 8,772,318 B2 Figure 12 JZL184 remaining (% rel. 0.5 hrs) to O Q=7hm o 4 8 1b 1b 2b 2A Time (hrs)

16 US. Patent Jul. 8, 2014 Sheet 15 0f 25 US 8,772,318 B2 Figure QO AEEEEREES :4 hydroiytic activity high-grade ngm A. 68mm» F85 A 53m A Kmomm» A to $8 A d www A mm? A W5 B MAGL activity 0 tumor fatty acids C1630 FFA C18ZO FFA C1811 FFA :9ch wumhmimi

17 US. Patent Jul. 8, 2014 Sheet 16 0f 25 US 8,772,318 B2 Figure 16 melanoma ovarian. > migration T] migration 100 h on M g ~ 8 8 "0 1; 60 Q g Q E 40., <21 'E Q E 20 I shmagl.. 0 o w v' 8 g z o 4 <12 5 z (/J (E E E u) a, w a a, ~ B invasion C cell survival G invasion H cell survival 160 E w 100 r i, _ 1001 g 120, *,3. so Q g 80 8 so 8 60 W g a > a >.E 40 w\ 20 5 x 20 0 ~- _ NM 0 ~ -~ m m H g 0? J > g g g (5:) (:2 g g g (2 hours 0 g E E OshMAGLl m g g g OSKOW w " " E3 shcontrol L1 shmaglz w w " [ shmaglz D lumorgrowth E tumor growth l tumor growth J tumor growth 1; ~12 1; 11; g g 400, g m 300 w 300 m 300 E E E E g 200 g 200 g 200 g g 100 g 100 g 100 i =1 ~ l O E O E 0 A. E O 1:. $130 E 3 3 :1 :J *" w days days days days -e parental we" vehicle e> parental sxow -e* sxovs vehicle ~e os1e1 JZL184 new SKOV3 ~6~ $1<ov31211a4 <9~ shmagl mg/kg oral ~ i~ shmagl SKOV3 40 mg/kg oral

18 US. Patent Jul. 8, 2014 Sheet 17 0f 25 US 8,772,318 B2 Figure 17 A B monoacyiglyoerois D migration MAG c1010 MAG 018:0 MAG c1811 MAG E H EV central MAGL-OE 3 51 $2 22 ~ , > : 3 " ' =1: 60 ' " E a E 015 QB mm 8 "m! "c; o o P 10 P 40 r H z 10 E 40 % 0 30 H % - a 0, 0, ' > g 5 M 20 g 2 E 5 *- g MAGL, 0 18 Y _ 0 >, 0 s" 43:" a u: * Lu <01 ' m 85:13:15, % 852% ééa? 8000i)?! 8mg?! ,! a 2% a 2'51? ] 0 g '5' MAGL C faiiy acids E invasion F tumor growth 020:4 FFA (216:0 FFA FFA 010:1 FFA * v A H a? 40 g. g * * g 12 1* E ~55 30, a: 400 w 0 a, U Q m w 200 B E 300 1?g 20 E E 6 3 w < 0 E 0 10 E 1 6 E E1000 E "6120 E 30 g E 4 91_ 200? fr.) $5 a a 000 l a 500 a 40 ~ 2 g 100 mm» In. _+ 1 _ + 1 g _ 0 ggggé _ 0 q 25% 0 a, 358% 0 258% j h Tm 1:1 8pm 5mm ~'0 5" 54, E, g DJ 1, 0)ng >wg3 >103: 1:ng days E g 3 2% W 5% J E g 5 2 g "1'" parantai u w (9 --*- S122A a % ~-*>~- MUMZC MAGL-OE

19 US. Patent Jul. 8, 2014 Sheet 18 0f 25 US 8,772,318 B2 Figure 18 A SKOV3 B wmwwo :8 36 E. 36 Eu.EEQQ W 36 ( a., i EN 36 w MUMZC 260 w=mu uesqe w OVCAR3 C BEE days "WQ" shcontroi normal chow ~ r~ shmagl, normal chow ShMAGL HFD 620:4 FFA 016:0 FFA tumor fatty acids 411, C1820 FFA 7 war. "92005 mil Oz 70%? 1 2 O C1811 FFA Till Ex # i mm ; Etcownw, Qz Tosa?,,, wohmmv/ia

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