[8-(PHENYLSULFONYL)-3,8-DIAZABICYCLO[3.2.1]OCT-3-YL](1H-1,2,3-TRIAZOL-4-YL)METHANONES
20170342082 · 2017-11-30
Inventors
- Marcus Koppitz (Berlin, DE)
- Holger Siebeneicher (Berlin, DE)
- Holger Steuber (Berlin, DE)
- Antonius Ter Laak (Berlin, DE)
- Reinhard Nubbemeyer (Berlin, DE)
- Antje Rottmann (Berlin, DE)
- Horst Irlbacher (Berlin, DE)
- Benjamin Bader (Berlin, DE)
- Michaele PETERS (Berlin, DE)
- Andrea Wagenfeld (Berlin, DE)
- Ildiko Terebesi (Berlin, DE)
Cpc classification
A61K31/4995
HUMAN NECESSITIES
A61P29/00
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
A61P17/02
HUMAN NECESSITIES
A61P15/00
HUMAN NECESSITIES
International classification
A61K45/06
HUMAN NECESSITIES
Abstract
The present invention covers [8-(phenylsulfonyl)-3,8-diazabicyclo[3.2.1]oct-3-yl](1H-1,2,3-triazol-4-yl)methanone compounds of general formula (I):
##STR00001##
in which R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 are as defined herein, methods of preparing said compounds, intermediate compounds useful for preparing said compounds, pharmaceutical compositions and combinations comprising said compounds and the use of said compounds for manufacturing pharmaceutical compositions for the treatment or prophylaxis of disorders, in particular of gynecological disorders, hyperproliferative disorders, metabolic disorders, or inflammatory disorders as a sole agent or in combination with other active ingredients.
Claims
1. A compound of general formula (I): ##STR00137## in which: R.sup.1 represents hydrogen, halogen, C.sub.1-C.sub.3-alkyl, C.sub.1-C.sub.3-haloalkyl, C.sub.1-C.sub.3-alkoxy, C.sub.1-C.sub.3-haloalkoxy, nitro or cyano; R.sup.2 represents hydrogen, halogen, C.sub.1-C.sub.3-alkyl, C.sub.1-C.sub.3-haloalkyl, C.sub.1-C.sub.3-alkoxy, C.sub.1-C.sub.3-haloalkoxy, nitro, cyano or SF.sub.5; R.sup.3 represents hydrogen, halogen, C.sub.1-C.sub.3-alkyl, C.sub.1-C.sub.3-alkoxy, C.sub.1-C.sub.3-haloalkoxy, nitro or hydroxy; R.sup.4 represents hydrogen, halogen, C.sub.1-C.sub.3-alkyl, C.sub.1-C.sub.3-haloalkyl, C.sub.1-C.sub.3-alkoxy, C.sub.1-C.sub.3-haloalkoxy, nitro, cyano or SF.sub.5; R.sup.5 represents hydrogen, halogen, C.sub.1-C.sub.3-alkyl, C.sub.1-C.sub.3-haloalkyl, C.sub.1-C.sub.3-alkoxy, C.sub.1-C.sub.3-haloalkoxy, nitro or cyano; wherein R.sup.1 and R.sup.2 or R.sup.2 and R.sup.3 are optionally linked to one another in such a way that they jointly form a methylenedioxy, ethylenedioxy, ethyleneoxy, trimethyleneoxy or a group selected from: ##STR00138## or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same.
2. The compound according to claim 1, wherein: R.sup.1 represents hydrogen, halogen, C.sub.1-C.sub.3-alkyl, C.sub.1-C.sub.3-haloalkyl, C.sub.1-C.sub.3-alkoxy, C.sub.1-C.sub.3-haloalkoxy, nitro or cyano; R.sup.2 represents hydrogen, halogen, C.sub.1-C.sub.3-alkyl, C.sub.1-C.sub.3-haloalkyl, C.sub.1-C.sub.3-alkoxy, C.sub.1-C.sub.3-haloalkoxy, nitro, cyano or SF.sub.5; R.sup.3 represents hydrogen, halogen, C.sub.1-C.sub.3-alkyl, C.sub.1-C.sub.3-haloalkyl, C.sub.1-C.sub.3-alkoxy, C.sub.1-C.sub.3-haloalkoxy, nitro or hydroxy; R.sup.4 represents hydrogen, halogen, C.sub.1-C.sub.3-alkyl, C.sub.1-C.sub.3-haloalkyl, C.sub.1-C.sub.3-alkoxy, C.sub.1-C.sub.3-haloalkoxy, nitro, cyano or SF.sub.5; R.sup.5 represents hydrogen, halogen, C.sub.1-C.sub.3-alkyl, C.sub.1-C.sub.3-haloalkyl, C.sub.1-C.sub.3-alkoxy, C.sub.1-C.sub.3-haloalkoxy, nitro or cyano; or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same.
3. The compound according to claim 1, wherein: R.sup.1 represents hydrogen, fluoro, chloro, bromo, methyl, trifluoromethyl, methoxy, trifluoromethoxy or cyano; R.sup.2 represents hydrogen, fluoro, chloro, bromo, methyl, trifluoromethyl, methoxy, trifluoromethoxy, cyano or SF.sub.5; R.sup.3 represents hydrogen; R.sup.4 represents hydrogen, fluoro, chloro, bromo, methyl, trifluoromethyl, methoxy, trifluoromethoxy, cyano or SF.sub.5; R.sup.5 represents hydrogen, fluoro, chloro, bromo, methyl, trifluoromethyl, methoxy, trifluoromethoxy or cyano; or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same.
4. The compound according to claim 1, wherein: R.sup.1 represents hydrogen, fluoro, chloro, bromo, methyl or trifluoromethyl; R.sup.2 represents hydrogen, fluoro, chloro, bromo, methyl, trifluoromethyl or SF.sub.5; R.sup.3 represents hydrogen; R.sup.4 represents hydrogen, fluoro, chloro, bromo, methyl, trifluoromethyl or SF.sub.5; R.sup.5 represents hydrogen, fluoro, chloro, bromo, methyl or trifluoromethyl; or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same.
5. The compound according to claim 1, which is selected from the group consisting of: 1 [8-(phenylsulfonyl)-3,8-diazabicyclo[3.2.1]oct-3-yl](1H-1,2,3-triazol-4-yl)methanone; 2 1H-1,2,3-triazol-4-yl[8-{[2-(trifluoromethyl)phenyl]sulfonyl}-3,8-diazabicyclo[3.2.1]oct-3-yl]methanone; 3 {8-[(3,5-difluorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone; 4 {8-[(3-fluorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone; 5 {8-[(3-chlorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone; 6 {8-[(2-methylphenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-4-yl)methanone; 7 {8-[(2-chlorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-4-yl)methanone; 8 [8-{[3-(pentafluoro-λ.sup.5-sulfanyl)phenyl]sulfonyl}-3,8-diazabicyclo[3.2.1]oct-3-yl](1H-1,2,3-triazol-4-yl)methanone; 9 {8-[(3,5-dichlorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-4-yl)methanone; 10 [8-{[3,5-bis(trifluoromethyl)phenyl]sulfonyl}-3,8-diazabicyclo[3.2.1]oct-3-yl](1H-1,2,3-triazol-5-yl)methanone; 11 {8-[(5-chlorothiophen-2-yl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-4-yl)methanone; 12 {8-[(2,5-difluorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-4-yl)methanone; 13 {8-[(3-methylphenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone; 14 {8-[(4-methylphenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone; 15 {8-[(2-fluorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone; 16 {8-[(4-fluorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone; 17 3-{[3-(1H-1,2,3-triazol-5-ylcarbonyl)-3,8-diazabicyclo[3.2.1]oct-8-yl]sulfonyl}benzonitrile; 18 {8-[(3,5-dimethylphenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone; 19 {8-[(2,5-dimethylphenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone; 20 {8-[(3-methoxyphenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone; 21 {8-[(4-methoxyphenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone; 22 {8-[(4-chlorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone; 23 {8-[(3,4-difluorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone; 24 {8-[(2,6-difluorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone; 25 {8-[(2,4-difluorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone; 26 {8-[(3-chloro-2-methylphenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone; 27 {8-[(3-chloro-2-fluorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone; 28 {8-[(3-chloro-4-fluorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone; 29 1H-1,2,3-triazol-5-yl[8-{[3-(trifluoromethyl)phenyl]sulfonyl}-3,8-diazabicyclo[3.2.1]oct-3-yl]methanone; 30 {8-[(2,5-dichlorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone; 31 {8-[(3-bromophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone; 32 {8-[(2-bromophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone; 33 1H-1,2,3-triazol-5-yl[8-{[3-(trifluoromethoxy)phenyl]sulfonyl}-3,8-diazabicyclo[3.2.1]oct-3-yl]methanone; 34 [8-{[5-chloro-2-(trifluoromethyl)phenyl]sulfonyl}-3,8-diazabicyclo[3.2.1]oct-3-yl](1H-1,2,3-triazol-5-yl)methanone; 35 2-{[3-(1H-1,2,3-triazol-5-ylcarbonyl)-3,8-diazabicyclo[3.2.1]oct-8-yl]sulfonyl}benzonitrile; 36 1H-1,2,3-triazol-5-yl[8-{[4-(trifluoromethyl)phenyl]sulfonyl}-3,8-diazabicyclo[3.2.1]oct-3-yl]methanone; 37 {8-[(4-hydroxyphenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone; 38 {8-[(4-bromophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone; 39 [8-(naphthalen-1-ylsulfonyl)-3,8-diazabicyclo[3.2.1]oct-3-yl](1H-1,2,3-triazol-5-yl)methanone; 40 [8-(quinolin-8-ylsulfonyl)-3,8-diazabicyclo[3.2.1]oct-3-yl](1H-1,2,3-triazol-5-yl)methanone; 41 1H-1,2,3-triazol-5-yl[8-{[4-(trifluoromethoxy)phenyl]sulfonyl}-3,8-diazabicyclo[3.2.1]oct-3-yl]methanone; 42 1H-1,2,3-triazol-5-yl[8-{[2-(trifluoromethoxy)phenyl]sulfonyl}-3,8-diazabicyclo[3.2.1]oct-3-yl]methanone; 43 {8-[(4-nitrophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone; 44 {8-[(3-nitrophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone; 45 1H-1,2,3-triazol-5-yl{8-[(2,4,6-trimethylphenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}methanone; 46 {8-[(2-nitrophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone; 47 {8-[(2,5-dimethoxyphenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone; 48 {8-[(3,4-dichlorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone; 49 {8-[(4-ethylphenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone; 50 {8-[(2-chloro-4-fluorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone; 51 {8-[(2-chloro-6-methylphenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone; 52 {8-[(3,4-dimethoxyphenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone; 53 {(1S)-8-[(2,3-dichlorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone; 54 [8-(2,1,3-benzothiadiazol-4-ylsulfonyl)-3,8-diazabicyclo[3.2.1]oct-3-yl](1H-1,2,3-triazol-5-yl)methanone; 55 [8-(2,1,3-benzoxadiazol-4-ylsulfonyl)-3,8-diazabicyclo[3.2.1]oct-3-yl](1H-1,2,3-triazol-5-yl)methanone; 56 1H-1,2,3-triazol-5-yl{8-[(2,4,6-trichlorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}methanone; 57 {8-[(5-chloro-2-methoxyphenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone; 58 [8-(2,1,3-benzothiadiazol-5-ylsulfonyl)-3,8-diazabicyclo[3.2.1]oct-3-yl](1H-1,2,3-triazol-5-yl)methanone; 59 1H-1,2,3-triazol-5-yl{8-[(2,3,4-trifluorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}methanone; 60 2-fluoro-5-{[3-(1H-1,2,3-triazol-5-ylcarbonyl)-3,8-diazabicyclo[3.2.1]oct-8-yl]sulfonyl}benzonitrile; 61 {8-[(5-chloro-2-fluorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone; 62 1H-1,2,3-triazol-5-yl{8-[(2,4,5-trifluorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}methanone; 63 {8-[(5-chloro-2-methylphenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone; 64 {8-[(2-methoxyphenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone; 65 {8-[(5-bromo-2-methylphenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone; 66 [8-(1,3-benzodioxol-5-ylsulfonyl)-3,8-diazabicyclo[3.2.1]oct-3-yl](1H-1,2,3-triazol-5-yl)methanone; 67 {8-[(2-methoxy-4-methylphenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone; 68 2-chloro-6-{[3-(1H-1,2,3-triazol-5-ylcarbonyl)-3,8-diazabicyclo[3.2.1]oct-8-yl]sulfonyl}benzonitrile; 69 [8-(2,3-dihydro-1-benzofuran-7-ylsulfonyl)-3,8-diazabicyclo[3.2.1]oct-3-yl](1H-1,2,3-triazol-5-yl)methanone; 70 {8-[(2-chloro-5-fluorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone; 71 {8-[(2-chloro-3-fluorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone; 72 {8-[(4-fluoro-2-methoxyphenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone; 73 4-methoxy-3-{[3-(1H-1,2,3-triazol-5-ylcarbonyl)-3,8-diazabicyclo[3.2.1]oct-8-yl]sulfonyl}benzonitrile; 74 4-chloro-3-{[3-(1H-1,2,3-triazol-5-ylcarbonyl)-3,8-diazabicyclo[3.2.1]oct-8-yl]sulfonyl}benzonitrile; 75 sodium 5-({8-[(3,5-difluorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}carbonyl)-1,2,3-triazol-1-ide; and 76 sodium 5-({8-[(3-fluorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}carbonyl)-1,2,3-triazol-1-ide; or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, or a mixture of same.
6. A method of preparing a compound of general formula (I) according to claim 1, said method comprising the step of allowing an intermediate compound of general formula (IV): ##STR00139## in which R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 are as defined for the compound of general formula (I), to react with a compound of formula (IX): ##STR00140## thereby giving a compound of general formula (I): ##STR00141## in which R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 are as defined for the compound of general formula (I).
7. A method of preparing a compound of general formula (I) according to claim 1, said method comprising the step of allowing an intermediate compound of formula (VII): ##STR00142## to react with a compound of general formula (VIII): ##STR00143## in which R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 are as defined for the compound of general formula (I), thereby giving a compound of general formula (I): ##STR00144## in which R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 are as defined for the compound of general formula (I).
8. (canceled)
9. A pharmaceutical composition comprising: a compound of general formula (I) according to claim 1 and one or more pharmaceutically acceptable excipients.
10. A pharmaceutical combination comprising: (a) one or more compounds of general formula (I) according to claim 1, and (b) one or more further active ingredients.
11. A pharmaceutical combination according to claim 10, wherein said further active ingredients are selected from the group consisting of anti-androgens, CYP17A1 inhibitors, 5 alpha reductase inhibitors, GNRHa and GNRH antagonists, LHRH agonists for example Flutamide, Bicalutamide, Nilutamide, Enzaluatmide, ODM-201, abiraterone and abiraterone metabolites, finasteride dutasteride, Leuprolide, Goserelin, Triptorelin, Histrelin, and Degarelix.
12. A pharmaceutical combination according to claim 10, wherein a further active ingredient is a chemotherapeutic agents comprising an oxo-group, which can be reduced by the enzymatic activity of AKR1C3.
13. A method of using a compound of general formula (I) according to claim 1 for treatment or prophylaxis of a disease; said method comprising administering an effective amount of the compound to a patient.
14. A method of using a combination comprising (a) one or more compounds of general formula (I), and (b) one or more further active ingredients for treatment or prophylaxis of a disease; said method comprising administering effective amounts of the compounds and the further active ingredients to a patient.
15. The method according to claim 13, wherein the disease is a gynecological disorder; a hyperproliferative disorder; a metabolic disorder; an inflammatory disorder; an endometriosis-related gynecological disorder, condition or disease; a polycystic ovary syndrome-related gynecological disorder, condition or disease; atopic dermatitis; keloids; anthracycline resistant cancer; prostate cancer, or castration-resistant prostate cancer.
16. The method according to claim 14, wherein the disease is a gynecological disorder; a hyperproliferative disorder; a metabolic disorder; an inflammatory disorder; an endometriosis-related gynecological disorder, condition or disease; a polycystic ovary syndrome-related gynecological disorder, condition or disease; atopic dermatitis; keloids; anthracycline resistant cancer; prostate cancer, or castration-resistant prostate cancer.
17. A pharmaceutical combination according to claim 12, wherein the chemotherapeutic agent comprises anthracycline.
18. A compound of general formula (III), (IV) or (VII) ##STR00145## in which R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 are as defined for a compound of general formula (I) according to claim 1; wherein at least one of R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 is different from hydrogen.
19. A method of using a compound of general formula (III), (IV) or (VII) according to claim 18 ##STR00146## for preparation of the compound of general formula (I).
Description
EXPERIMENTAL SECTION
[0265] NMR peak forms are stated as they appear in the spectra, possible higher order effects have not been considered.
[0266] The .sup.1H-NMR data of selected examples are listed in the form of .sup.1H-NMR peaklists. For each signal peak the δ value in ppm is given, followed by the signal intensity, reported in round brackets. The δ value-signal intensity pairs from different peaks are separated by commas. Therefore, a peaklist is described by the general form: δ.sub.1 (intensity.sub.1), δ.sub.2 (intensity.sub.2), δ.sub.i (intensity.sub.i), δ.sub.n (intensity.sub.n).
[0267] The intensity of a sharp signal correlates with the height (in cm) of the signal in a printed NMR spectrum. When compared with other signals, this data can be correlated to the real ratios of the signal intensities. In the case of broad signals, more than one peak, or the center of the signal along with their relative intensity, compared to the most intense signal displayed in the spectrum, are shown. A .sup.1H-NMR peaklist is similar to a classical .sup.1H-NMR readout, and thus usually contains all the peaks listed in a classical NMR interpretation. Moreover, similar to classical .sup.1H-NMR printouts, peaklists can show solvent signals, signals derived from stereoisomers of target compounds (also the subject of the invention), and/or peaks of impurities. The peaks of stereoisomers, and/or peaks of impurities are typically displayed with a lower intensity compared to the peaks of the target compounds (e.g., with a purity of >90%). Such stereoisomers and/or impurities may be typical for the particular manufacturing process, and therefore their peaks may help to identify the reproduction of our manufacturing process on the basis of “by-product fingerprints”. An expert who calculates the peaks of the target compounds by known methods (MestReC, ACD simulation, or by use of empirically evaluated expectation values), can isolate the peaks of target compounds as required, optionally using additional intensity filters. Such an operation would be similar to peak-picking in classical .sup.1H-NMR interpretation. A detailed description of the reporting of NMR data in the form of peaklists can be found in the publication “Citation of NMR Peaklist Data within Patent Applications” (cf. Research Disclosure Database Number 605005, 2014, 1 Aug. 2014, or http://www.researchdisclosure.com/searching-disclosures). In the peak picking routine, as described in the Research Disclosure Database Number 605005, the parameter “MinimumHeight” can be adjusted between 1% and 4%. Depending on the chemical structure and/or depending on the concentration of the measured compound it may be reasonable to set the parameter “MinimumHeight”<1%.
[0268] Chemical names were generated using the ACD/Name software from ACD/Labs. In some cases generally accepted names of commercially available reagents were used in place of ACD/Name generated names.
[0269] The following table 1 lists the abbreviations used in this paragraph and in the Examples section as far as they are not explained within the text body. Other abbreviations have their meanings customary per se to the skilled person.
TABLE-US-00002 TABLE 1 Abbreviations Abbreviation Meaning Ac.sub.2O acetic anhydride AcOH acetic acid (ethanoic acid) aq. aqueous Boc tert-butoxycarbonyl br broad (.sup.1H-NMR signal) cat. catalytic conc. concentrated CI chemical ionisation d doublet DAD diode array detector DBU 1,8-diazabicyclo(5.4.0)undec-7-ene DCC N,N′-dicyclohexylcarbodiimide DCM dichloromethane dd double-doublet DIC N,N′-diisopropylcarbodiimide DIPEA diisopropylethylamine DMA N,N-dimethylacetamide DMF N,N-dimethylformamide DMSO dimethylsulfoxide dt double-triplet EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide ELSD Evaporative Light Scattering Detector EtOAc ethyl acetate EtOH ethanol eq. equivalent ESI electrospray (ES) ionisation h hour(s) HATU 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5- b]pyridinium 3-oxid hexafluorophosphate HBTU (o-benzotriazole-10yl)-N,N,N′,N,-tetramethyluronium hexafluorophosphate HCl hydrochloric acid HPLC high performance liquid chromatography LC-MS liquid chromatography mass spectrometry m multiplet min minute(s) MeCN acetonitrile MeOH methanol MS mass spectrometry NBS N-bromosuccinimide NCS N-chlorosuccinimide NMR nuclear magnetic resonance spectroscopy: chemical shifts (δ) are given in ppm. The chemical shifts were corrected by setting the DMSO signal to 2.50 ppm unless otherwise stated. PDA Photo Diode Array Pd/C palladium on activated charcoal PdCl.sub.2(dppf) [1,1′- bis(diphenylphosphino)ferrocene]dichloropalladium(II) Pd(dba).sub.2 bis(dibenzylideneacetone)palladium q quartet r.t. or rt or RT room temperature rac racemic Rt retention time (as measured either with HPLC or UPLC) in minutes s singlet sat. saturated SIBX stabilized 2-iodoxybenzoic acid SM starting material SQD Single-Quadrupole-Detector t triplet T3P propylphosphonic anhydride TBAF tetra-n-butylammonium fluoride TBDMS tert-butyldimethylsilyl TBTU N-[(1H-benzotriazol-1-yloxy)(dimethylamino)- methylene]-N-methylmethanaminium tetrafluoroborate td triple-doublet TEA triethylamine TFA trifluoroacetic acid THF tetrahydrofuran UPLC ultra performance liquid chromatography
[0270] Other abbreviations have their meanings customary per se to the skilled person.
[0271] The various aspects of the invention described in this application are illustrated by the following examples which are not meant to limit the invention in any way.
[0272] The example testing experiments described herein serve to illustrate the present invention and the invention is not limited to the examples given.
EXPERIMENTAL SECTION—GENERAL PART
[0273] All reagents, for which the synthesis is not described in the experimental part, are either commercially available, or are known compounds or may be formed from known compounds by known methods by a person skilled in the art.
[0274] The compounds and intermediates produced according to the methods of the invention may require purification. Purification of organic compounds is well known to the person skilled in the art and there may be several ways of purifying the same compound. In some cases, no purification may be necessary. In some cases, the compounds may be purified by crystallization. In some cases, impurities may be stirred out using a suitable solvent. In some cases, the compounds may be purified by chromatography, particularly flash column chromatography, using for example prepacked silica gel cartridges, e.g. Biotage SNAP cartidges KP-Sil® or KP-NH® in combination with a Biotage autopurifier system (SP4® or Isolera Four®) and eluents such as gradients of hexane/ethyl acetate or DCM/methanol. In some cases, the compounds may be purified by preparative HPLC using for example a Waters autopurifier equipped with a diode array detector and/or on-line electrospray ionization mass spectrometer in combination with a suitable prepacked reverse phase column and eluents such as gradients of water and acetonitrile which may contain additives such as trifluoroacetic acid, formic acid or aqueous ammonia.
[0275] In some cases, purification methods as described above can provide those compounds of the present invention which possess a sufficiently basic or acidic functionality in the form of a salt, such as, in the case of a compound of the present invention which is sufficiently basic, a trifluoroacetate or formate salt for example, or, in the case of a compound of the present invention which is sufficiently acidic, an ammonium salt for example. A salt of this type can either be transformed into its free base or free acid form, respectively, by various methods known to the person skilled in the art, or be used as salts in subsequent biological assays. It is to be understood that the specific form (e.g. salt, free base etc.) of a compound of the present invention as isolated and as described herein is not necessarily the only form in which said compound can be applied to a biological assay in order to quantify the specific biological activity.
[0276] UPLC-MS Standard Procedures
[0277] Analytical UPLC-MS was performed as described below. The masses (m/z) are reported from the positive mode electrospray ionisation unless the negative mode is indicated (ESI−). In most of the cases method 1 is used. If not, it is indicated.
[0278] Method 1:
[0279] Instrument: Waters Acquity UPLCMS SingleQuad; Column: Acquity UPLC BEH C18 1.7 μm, 50×2.1 mm; eluent A: water+0.1 vol % formic acid (99%), eluent B: acetonitrile; gradient: 0-1.6 min 1-99% B, 1.6-2.0 min 99% B; flow 0.8 ml/min; temperature: 60° C.; DAD scan: 210-400 nm.
[0280] Method 2:
[0281] Instrument: Waters Acquity UPLCMS SingleQuad; Column: Acquity UPLC BEH C18 1.7 μm, 50×2.1 mm; eluent A: water+0.2 vol % aqueous ammonia (32%), eluent B: acetonitrile; gradient: 0-1.6 min 1-99% B, 1.6-2.0 min 99% B; flow 0.8 ml/min; temperature: 60° C.; DAD scan: 210-400 nm.
[0282] Method 3:
[0283] Instrument: Waters Acquity UPLCMS SingleQuad; Column: Acquity UPLC BEH C18 1.7 μm 50×2.1 mm; eluent A: water+0.1 vol % formic acid (99%), eluent B: acetonitrile; gradient: 0-1.6 min 1-99% B, 1.6-2.0 min 99% B; flow 0.8 ml/min; temperature: 60° C.; DAD scan: 210-400 nm.
[0284] Method 4:
[0285] System: UPLC Acquity (Waters) with PDA Detector and Waters ZQ mass spectrometer; Column: Acquity BEH C18 1.7 μm 2.1×50 mm; Temperature: 60° C.; Solvent A: Water+0.1% Formic Acid; Solvent B: Acetonitrile; Gradient: 99% A to 1% A (1.6 min) to 1% A (0.4 min); Flow: 0.8 mL/min; Injektion Volume: 1.0 μl (0.1 mg-1 mg/mL Sample Concentration); Detection: PDA Scan Region 210-400 nm—plus fixed wavelength 254 nm; MS ESI (+), Scan region 170-800 m/z
[0286] Method 5:
[0287] System: Waters Aqcuity UPC2: Solvent Manager, Sample Manager, Column Manager, PDA, QDa MS; Column: Viridis BEH 2-EP 5 μm 100×4.6 mm; Solvent: A=CO2 B=Methanol+0.5% Vol. NH3 (32%); Flow: 4.0 mL/min; Gradient: 0-7 min 5-55% B; Pressure: 100 bar; Temperature: 40° C.; Detection: DAD 254 nm
[0288] Preparative Chromatography on HPLC Systems:
[0289] For the purification of some intermediates and examples preparative reversed phase or normal phase systems were used. Available systems were:
[0290] Labomatic, Pump: HD-5000, Fraction Collector: LABOCOL Vario-4000, UV-Detector: Knauer UVD 2.1S; Column: Chromatorex RP C18 10 μm 125×30 mm, eluent: A: water+0.1 vol % formic acid (99%), eluent B: acetonitrile; detection: UV 254 nm; software: SCPA PrepCon5.
[0291] Waters autopurification system: Pump 2545, Sample Manager 2767, CFO, DAD 2996, ELSD 2424, SQD; Column: XBrigde C18 5 μm 100×30 mm; eluent A: water+0.1% Vol. formic acid, eluent B: acetonitrile; flow: 50 mL/min; temperature: room temperature; detection: DAD scan range 210-400 nm; MS ESI+, ESI−, scan range 160-1000 m/z.
[0292] Waters autopurification system: Pump 2545, Sample Manager 2767, CFO, DAD 2996, ELSD 2424, SQD; Column: XBrigde C18 5 μm 100×30 mm; eluent A: water+0.2 vol % aqueous ammonia (32%), eluent B: acetonitrile; flow: 50 mL/min; temperature: room temperature; detection: DAD scan range 210-400 nm; MS ESI+, ESI−, scan range 160-1000 m/z.
[0293] Column Chromatography on Silica Gel:
[0294] For the purification of some intermediates and examples a column chromatography (“flash chromatography”) on silica gel was performed using devices (Isolera®) from the company Biotage. Cartridges prefilled with silica gel in different sizes were used, for example “SNAP Cartridge, KP_SIL” from the company Biotage or “Interchim Puriflash Silica HP 15UM flash column” from the company Interchim.
EXPERIMENTAL SECTION—INTERMEDIATES
Intermediate 1
3,8-diazabicyclo[3.2.1]oct-3-yl(1H-1,2,3-triazol-4-yl)methanone
[0295] ##STR00015##
Step 1.1: tert-butyl 3-(1H-1,2,3-triazol-4-ylcarbonyl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate
[0296] ##STR00016##
[0297] To a stirred solution of 500 mg (2.36 mmol) tert-butyl 3,8-diazabicyclo[3.2.1]octane-8-carboxylate in 12 mL NMP were added 532 mg 1H-1,2,3-triazole-4-carboxylic acid (4.71 mmol, 2 eq), 1.23 mL DIPEA (7.07 mmol, 3 eq) and 1.791 g HATU (4.71 mmol, 2 eq). After stirring overnight at RT, the solution was subjected to preparative HPLC to yield 378 mg (52%) tert-butyl 3-(1H-1,2,3-triazol-4-ylcarbonyl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate.
[0298] LC-MS (Method 3): Rt=0.91 min; MS (ESIpos): m/z=308 [M+H].sup.+
[0299] .sup.1H-NMR (500 MHz, DMSO-d6) δ[ppm]: 1.43 (9H), 1.53-1.72 (2H), 1.83 (2H), 2.92 (1H), 3.33 (1H), 4.14 (1H), 4.21 (1H), 4.30 (1H), 4.40 (1H), 8.29 (1H), 15.22 (1H).
Step 2: 3,8-diazabicyclo[3.2.1]oct-3-yl(1H-1,2,3-triazol-4-yl)methanone
[0300] ##STR00017##
[0301] To a stirred and cooled (ice-bath) solution of 272 mg (887 μmop tert-butyl-3-(1H-1,2,3-triazol-4-ylcarbonyl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate in 6 mL DCM were added 0.3 mL water and 3 mL TFA. After 3 h, the mixture was evaporated in vaccuo, triturated with toluene and reevaporated to yield 376 mg (200%) crude 3,8-diazabicyclo[3.2.1]oct-3-yl(1H-1,2,3-triazol-4-yl)methanone as TFA adduct which was used without further purification in the next step.
[0302] SFC-MS (Method 5): Rt=1.74 min; MS (ESIpos): m/z=208 [M+H].sup.+
[0303] .sup.1H-NMR (500 MHz, DMSO-d6) δ[ppm]: 1.67-2.00 (4H), 3.19 (1H), 3.61 (1H), 4.11 (2H), 4.40 (1H), 4.70 (1H), 8.40 (1H).
Intermediate 2
8-(phenylsulfonyl)-3,8-diazabicyclo[3.2.1]octane
[0304] ##STR00018##
Step 2.1: tert-butyl-8-(phenylsulfonyl)-3,8-diazabicyclo[3.2.1]octane-3-carboxylate
[0305] ##STR00019##
[0306] To a stirred solution of 100 mg (0.47 mmol) tert-butyl-3,8-diazabicyclo[3.2.1]octane-3-carboxylate in 2 ml DCE were added at RT 246 μL DIPEA (1.41 mmol, 3 eq) and 90 μL benzenesulfonyl chloride (125 mg, 0.71 mmol, 1.5 eq) and the mixture was stirred overnight at RT. The organic phase was washed three times with aqueous NaHCO3 solution (10%) and two times with water, dried and evaporated to yield 168 mg (101%) of the title compound.
[0307] LC-MS (Method 3): Rt=1.26 min; MS (ESIpos): m/z=353 [M+H].sup.+
[0308] .sup.1H-NMR (500 MHz, DMSO-d6) δ[ppm]: 1.19 (2H), 1.36 (11H), 2.87 (1H), 3.01 (1H), 3.75 (2H), 4.19 (2H), 7.60 (2H), 7.70 (1H), 7.87 (2H).
Step 2.2: 8-(phenylsulfonyl)-3,8-diazabicyclo[3.2.1]octane
[0309] ##STR00020##
[0310] To a stirred solution of 166 mg (0.47 mmol) tert-butyl-8-(phenylsulfonyl)-3,8-diazabicyclo[3.2.1]octane-3-carboxylate in 1 mL DCM were added at RT 0.1 mL water and 3 mL TFA. After 3 h, the mixture was evaporated in vaccuo, redissolved in tert-butanol and freeze-dried to yield 171 mg (143%) crude 8-(phenylsulfonyl)-3,8-diazabicyclo[3.2.1]octane as TFA-aduct which was used without further purification in the next step.
[0311] LC-MS (Method 1): Rt=0.54 min; MS (ESIpos): m/z=253 [M+H].sup.+
[0312] .sup.1H-NMR (500 MHz, DMSO-d6) δ[ppm]: 1.40 (2H), 1.77 (2H), 3.19-3.51 (4H), 4.37 (2H), 7.55-7.66 (2H), 7.74 (1H), 7.90 (1H), 8.25-9.50 (2H).
Intermediate 3
8-{[2-(trifluoromethyl)phenyl]sulfonyl}-3,8-diazabicyclo[3.2.1]octane
[0313] ##STR00021##
Step 3.1: tert-butyl-8-{[2-(trifluoromethyl)phenyl]sulfonyl}-3,8-diazabicyclo[3.2.1]octane-3-carboxylate
[0314] ##STR00022##
[0315] To a stirred solution of 4.246 g (20 mmol) tert-butyl-3,8-diazabicyclo[3.2.1]octane-3-carboxylate in 100 ml DCM were added 14 ml DIPEA (80 mmol, 4 eq) and 9.78 g 2-(trifluoromethyl)benzenesulfonyl chloride (40.0 mmol, 2 eq) and the mixture was stirred overnight at RT. The organic phase was removed in vaccuo and the residue was subjected to flash chromatography (ethyl acetate/hexane) to yield 8.18 g (97%) of the title compound.
[0316] LC-MS (Method 1): Rt=1.36 min; MS (ESIpos): m/z=365 [M-tBu+H].sup.+
[0317] .sup.1H-NMR (500 MHz, DMSO-d6) δ[ppm]: 1.38 (9H), 1.51-1.71 (4H), 2.83 (1H), 3.01 (1H), 3.76 (2H), 4.23 (2H), 7.91 (2H), 8.05 (1H), 8.27 (1H).
Step 3.2: 8-{[2-(trifluoromethyl)phenyl]sulfonyl}-3,8-diazabicyclo[3.2.1]octane
[0318] ##STR00023##
[0319] To a stirred solution of 8 g (19.45 mmol) tert-buty-8-{[2-(trifluoromethyl)phenyl]sulfonyl}-3,8-diazabicyclo[3.2.1]octane-3-carboxylate in 64 mL DCM were added 3.3 mL water and 64 mL TFA. After 3 h, the mixture was evaporated in vaccuo, triturated with toluene and reevaporated. The residue was redissolved in DCM, washed with aqueous NaHCO.sub.3 solution (10%) and water and the organic phase was dried and evaporated to yield 6.20 g (95%) of the title compound 8-{[2-(trifluoromethyl)phenyl]sulfonyl}-3,8-diazabicyclo[3.2.1]octane.
[0320] LC-MS (Method 1): Rt=0.69 min; MS (ESIpos): m/z=321 [M+H].sup.+
[0321] .sup.1H-NMR (500 MHz, DMSO-d6) δ[ppm]: 1.54 (2H), 1.79 (2H), 2.60 (2H), 2.71 (2H), 4.02 (2H), 7.90 (2H), 8.03 (1H), 8.27 (1H).
Intermediate 4
8-[(3,5-difluorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]octane hydrochloride (1:1)
[0322] ##STR00024##
Step 4.1: tert-butyl-8-[(3,5-difluorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]octane-3-carboxylate
[0323] ##STR00025##
[0324] To a stirred solution of 100 mg (0.47 mmol) tert-butyl-3,8-diazabicyclo[3.2.1]octane-3-carboxylate in 2 ml DCE were added at RT 246 μL DIPEA (1.41 mmol, 3 eq) and 150 mg 3,5-difluorobenzenesulfonyl chloride (0.71 mmol, 1.5 eq) and the mixture was stirred overnight at RT. The organic phase was washed three times with aqueous NaHCO.sub.3 solution (10%) and one time with water, dried and evaporated to yield 188 mg (103%) of the title compound.
[0325] LC-MS (Method 1): Rt=1.34 min; MS (ESIpos): m/z=389 [M+H].sup.+
[0326] .sup.1H-NMR (500 MHz, DMSO-d6) δ[ppm]: 1.30 (2H), 1.38 (9H), 1.45 (2H), 2.89 (1H), 3.03 (1H), 3.76 (2H), 4.29 (2H), 7.68 (3H).
Step 4.2: 8-[(3,5-difluorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]octane hydrochloride (1:1)
[0327] ##STR00026##
[0328] To a stirred solution of 183 mg (0.47 mmol) tert-butyl 8-[(3,5-difluorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]octane-3-carboxylate in 3 mL ethanol were added with ice-cooling 1.8 mL HCl 4M in dioxane (7 mmol, 15 eq) and the mixture was stirred for 2 h at RT. Another 1 mL HCl 4M in dioxane were added and after 3 h stirring at RT, the mixture was evaporated in vaccuo and freeze-dried from tert-butanol to yield 157 mg (103%) of the title compound 8-[(3,5-difluorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]octane hydrochloride (1:1).
[0329] LC-MS (Method 2): R.sub.t=0.64 min; MS (ESIpos): m/z=289 [M+H].sup.+
[0330] .sup.1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 1.54 (2H) 1.94 (2H) 3.11 (2H) 3.18 (2H), 4.45 (2H) 7.72 (3H), 8.87-9.92 (2H)
Intermediate 5
8-[(3-fluorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]octane hydrochloride (1:1)
[0331] ##STR00027##
Step 5.1: tert-butyl-8-[(3-fluorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]octane-3-carboxylate
[0332] ##STR00028##
[0333] To a stirred solution of 500 mg (2.36 mmol) tert-butyl 3,8-diazabicyclo[3.2.1]octane-3-carboxylate in DCM were added 1.6 ml DIPEA (9.4 mmol, 4 eq) and 380 μL 3-fluorobenzenesulfonyl chloride (2.82 mmol, 1.2 eq) and the mixture was stirred overnight at RT. The organic phase was removed in vaccuo and the residue was subjected to flash chromatography (ethyl acetate/hexane) to yield 844 mg (97%) of the title compound.
[0334] LC-MS (Method 2): R.sub.t=1.28 min; MS (ESIpos): m/z=315 M-t-Bu.sup.+
[0335] .sup.1H-NMR (400 MHz, CHLOROFORM-d) δ [ppm]: 7.68 (1H), 7.58 (1H), 7.51 (1H), 7.35-7.28 (1H), 4.19 (2H), 3.93 (1H), 3.77 (1H), 3.19-2.96 (2H), 1.69-1.54 (4H), 1.44 (s, 9H)
Step 5.2: 8-[(3-fluorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]octane hydrochloride (1:1)
[0336] ##STR00029##
[0337] To a stirred solution of 840 mg (2.27 mmol) 8-[(3-fluorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]octane-3-carboxylate in 10 mL ethanol were added 8.5 mL HCl 4M in dioxane (34 mmol, 15 eq) and the mixture was stirred for 72 h at RT. The mixture was evaporated in vaccuo, triturated with diethylether, filtered and dried in vaccuo to yield 735 mg (105%) of the title compound 8-[(3-fluorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]octane hydrochloride (1:1).
[0338] LC-MS (Method 2): R.sub.t=0.84 min; MS (ESIpos): m/z=271 [M+H].sup.+
[0339] .sup.1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 1.36-1.54 (2H) 1.81-1.97 (2H) 2.98-3.13 (2H) 3.13-3.25 (2H), 4.40 (2H) 7.57-7.65 (1H), 7.66-7.73 (1H) 7.73-7.83 (2H), 9.10-9.90 (1H)
[0340] The following intermediates were synthesized in analogy to procedures given for intermediates 2 to 5 using the appropriate sulfonyl chloride.
TABLE-US-00003 Inter- Structure mediates IUPAC-Name LC-MS 6
EXPERIMENTAL SECTION—EXAMPLES
Example 1
[8-(phenylsulfonyl)-3,8-diazabicyclo[3.2.1]oct-3-yl](1H-1,2,3-triazol-4-yl)methanone
[0341] ##STR00061##
[0342] Procedure 1.1:
[0343] To a stirred and cooled solution of 1.87 g (57% purity, 5.11 mmol) -3,8-diazabicyclo[3.2.1]oct-3-yl(1H-1,2,3-triazol-4-yl)methanone (Intermediate 1) in 25 mL NMP were added at 0° C. 4.45 mL (5 eq, 25.5 mmol) DIPEA and 0.63 g (0.7 eq, 3.57 mmol) benzenesulfonyl chloride and the mixture was stirred for 1 h at 0° C. After stirring overnight at RT, the mixture was subjected to preparative HPLC to yield 215 mg (0.61 mmol, 12%) of the title compound 3,8-diazabicyclo[3.2.1]oct-3-yl(1H-1,2,3-triazol-4-yl)methanone.
[0344] LC-MS (Method 1): Rt=0.85 min; MS (ESIpos): m/z=348 [M+H].sup.+
[0345] .sup.1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 1.193 (3.38), 1.211 (5.65), 1.228 (4.02), 1.262 (0.76), 1.382 (1.39), 1.406 (3.26), 1.427 (1.85), 1.510 (1.94), 2.323 (0.45), 2.327 (0.62), 2.331 (0.43), 2.518 (2.26), 2.523 (1.43), 2.665 (0.45), 2.669 (0.63), 2.673 (0.45), 2.963 (3.11), 2.992 (3.35), 3.397 (2.90), 4.282 (3.57), 4.337 (7.53), 4.368 (3.35), 7.593 (6.59), 7.597 (2.73), 7.611 (16.00), 7.631 (11.11), 7.690 (3.31), 7.693 (6.32), 7.696 (3.91), 7.707 (2.79), 7.711 (8.65), 7.716 (2.33), 7.727 (1.88), 7.730 (2.99), 7.885 (11.98), 7.889 (15.82), 7.894 (4.11), 7.907 (12.63).
[0346] Procedure 1.2:
[0347] To a stirred solution of 504 mg (2 mmol) 8-(phenylsulfonyl)-3,8-diazabicyclo[3.2.1]octane (Intermediate 2) in 6 mL NMP were added at RT 452 mg (2 eq, 4 mmol) 1H-1,2,3-triazole-5-carboxylic acid, 1045 μL (3 eq, 6 mmol) DIPEA and 1.52 g (2 eq, 4 mmol) HATU and the mixture was stirred for 6 h. The mixture was taken up in ethyl acetate, washed with water, dried with sodium sulfate, evaporated and the residue was subjected to flash chromatography using ethyl acetate and hexane to yield 787 mg (1.93 mmol, 96%) of the title compound 3,8-diazabicyclo[3.2.1]oct-3-yl(1H-1,2,3-triazol-4-yl)methanone.
[0348] Procedure 1.3:
[0349] To a solution of tert-butyl-3,8-diazabicyclo[3.2.1]octane-3-carboxylate (0.3 mmol, 750 μL, 0.4 M) in DCE were added benzenesulfonyl chloride (0.45 mmol, 900 μL, 0.5M, 1.5 eq) in DCE and 0.9 mmol DIPEA (156 μL, 3 eq) and the mixture was shaken overnight at RT. 2 mL TFA/DCE 3:1 were added and the mixture was shaken at RT for 3 h. After evaporation of the solvent, 1H-1,2,3-triazole-5-carboxylic acid (0.6 mmol, 1.2 mL, 2 eq, 0.5M) in NMP, 928 μL DIPEA (3.6 mmol, 12 eq; adjustment of pH to 8) and HATU (0.6 mmol, 1.2 mL, 2 eq, 0.5 M) in NMP were added and the mixture was shaken overnight to yield after preparative HPLC 26 mg (25%) of the title compound 3,8-diazabicyclo[3.2.1]oct-3-yl(1H-1,2,3-triazol-4-yl)methanone.
Example 2
1H-1,2,3-triazol-4-yl[8-{[2-(trifluoromethyl)phenyl]sulfonyl}-3,8-diazabicyclo[3.2.1]oct-3-yl]methanone
[0350] ##STR00062##
[0351] 1.92 g (50% purity, 3.00 mmol) 8-{[2-(trifluoromethyl)phenyl]sulfonyl}-3,8-diazabicyclo[3.2.1]octane (Intermediate 3) were reacted in analogy to example 1, procedure 1.2 with 0.678 g (6 mmol, 2 eq) 1H-1,2,3-triazole-5-carboxylic acid to yield after work-up and purification 792 mg (62%) of the title compound 1H-1,2,3-triazol-4-yl[(8-{[2-(trifluoromethyl)phenyl]sulfonyl}-3,8-diazabicyclo[3.2.1]oct-3-yl]methanone.
[0352] LC-MS (Method 1): Rt=0.96 min; MS (ESIpos): m/z=416 [M+H].sup.+
[0353] .sup.1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 1.602 (2.82), 1.629 (2.52), 1.689 (12.16), 1.980 (0.53), 2.073 (1.54), 2.327 (1.17), 2.669 (1.20), 2.673 (0.87), 2.934 (3.84), 2.964 (4.10), 3.379 (5.16), 4.293 (5.08), 4.355 (9.64), 4.386 (4.07), 4.519 (0.72), 7.890 (1.47), 7.908 (5.87), 7.913 (7.23), 7.920 (16.00), 7.927 (6.81), 7.932 (7.53), 7.936 (7.64), 7.950 (2.37), 7.955 (1.54), 8.018 (0.79), 8.030 (7.57), 8.037 (6.89), 8.053 (5.61), 8.295 (7.27), 8.311 (6.51), 8.317 (6.21), 15.524 (0.53).
Example 3
{8-[(3,5-difluorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone
[0354] ##STR00063##
[0355] 3.46 g (12.00 mmol) 8-[(3,5-difluorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]octane (Intermediate 4) were reacted in analogy to example 1, procedure 1.2 with 2.714 g (12 mmol, 2 eq) 1H-1,2,3-triazole-5-carboxylic acid to yield after work-up and purification 1.93 g (42%) of the title compound {8-[(3,5-difluorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone.
[0356] LC-MS (Method 1): Rt=0.93 min; MS (ESIpos): m/z=384 [M+H].sup.+
[0357] .sup.1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 1.106 (1.26), 1.153 (0.74), 1.171 (1.48), 1.188 (0.74), 1.317 (4.22), 1.336 (6.93), 1.352 (4.93), 1.384 (1.15), 1.452 (1.67), 1.475 (4.11), 1.496 (2.26), 1.539 (1.26), 1.561 (1.63), 1.619 (1.48), 1.979 (1.26), 1.986 (2.74), 2.322 (1.07), 2.326 (1.48), 2.331 (1.04), 2.518 (6.26), 2.522 (4.19), 2.664 (1.07), 2.668 (1.52), 2.673 (1.07), 2.685 (0.59), 2.692 (1.00), 2.982 (3.59), 3.014 (3.67), 3.282 (0.52), 3.301 (1.00), 3.382 (3.74), 3.412 (3.81), 4.016 (0.63), 4.034 (0.59), 4.345 (6.04), 4.374 (8.00), 4.432 (4.74), 4.739 (1.33), 4.770 (1.26), 7.678 (12.00), 7.694 (15.93), 7.698 (16.00), 7.716 (4.00), 7.722 (3.56), 7.727 (1.59), 8.084 (3.89), 8.541 (4.07), 15.373 (1.67), 15.690 (1.00).
Example 4
{8-[(3-fluorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone
[0358] ##STR00064##
[0359] 3.24 g (12.00 mmol) 8-[(3-fluorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]octane (Intermediate 5) were reacted in analogy to example 1, procedure 1.2 with 2.714 g (12 mmol, 2 eq) 1H-1,2,3-triazole-5-carboxylic acid to yield after work-up and purification 1.15 g (26%) of the title compound {8-[(3-fluorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone.
[0360] LC-MS (Method 1): Rt=0.89 min; MS (ESIpos): m/z=366 [M+H].sup.+
[0361] .sup.1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 1.154 (1.28), 1.172 (2.67), 1.189 (3.43), 1.205 (2.55), 1.217 (0.78), 1.248 (4.37), 1.269 (6.76), 1.284 (4.95), 1.316 (1.10), 1.419 (1.60), 1.442 (3.75), 1.463 (2.05), 1.532 (2.35), 1.552 (3.45), 1.578 (1.30), 1.987 (4.67), 2.074 (0.74), 2.323 (0.84), 2.327 (1.16), 2.331 (0.86), 2.523 (3.65), 2.665 (0.90), 2.669 (1.20), 2.673 (0.90), 2.686 (2.75), 2.972 (3.99), 3.003 (4.11), 3.376 (4.95), 3.409 (4.43), 4.016 (1.08), 4.034 (1.00), 4.342 (7.38), 4.377 (7.64), 4.524 (1.96), 4.552 (1.84), 7.563 (1.84), 7.568 (2.49), 7.573 (2.29), 7.587 (4.79), 7.591 (5.25), 7.606 (2.93), 7.609 (3.27), 7.612 (3.43), 7.615 (3.09), 7.650 (2.65), 7.666 (3.73), 7.671 (5.97), 7.685 (5.69), 7.692 (3.89), 7.705 (3.25), 7.749 (16.00), 7.752 (13.33), 7.758 (4.71), 7.769 (11.91), 8.307 (5.49).
[0362] The following examples were prepared in analogy to Example 1, Procedure 1.1 using 3,8-diazabicyclo[3.2.1]oct-3-yl(1H-1,2,3-triazol-4-yl)methanone (Intermediate 1):
TABLE-US-00004 Ex- Structure am- IUPAC-Name ple LC-MS NMR Data 5
[0363] The following examples were prepared in analogy to Example 1, Procedure 1.2 using the given intermediate:
TABLE-US-00005 Structure Example IUPAC-Name Intermediate LC-MS NMR Data 7 Int 6
[0364] The following examples were prepared in analogy to Example 1, Procedure 1.3 using tert-butyl-3,8-diazabicyclo[3.2.1]octane-3-carboxylate:
TABLE-US-00006 Structure Example IUPAC-Name LC-MS Data 38
Example 75
sodium 5-({8-[(3,5-difluorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}carbonyl)-1,2,3-triazol-1-ide
[0365] ##STR00135##
[0366] To a stirred solution of 6.16 g (16 mmol) {8-[(3,5-difluorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone in 170 mL MeOH and 70 mL THF were added a solution of sodium methanolate (16 mmol, 1 eq, 30% in MeOH) at RT. After stirring for 2 h at RT, 250 mL diethylether were added to precipitate the product. After cooling and filtration, the solid was dried in vaccuo to yield 4.88 g (13 mmol, 87%) of the title compound sodium 5-({8-[(3,5-difluorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}carbonyl)-1,2,3-triazol-1-ide.
[0367] LC-MS (Method 1): Rt=0.93 min; MS (ESIpos): m/z=384 [M-Na.sup.++H].sup.+
[0368] .sup.1H-NMR (500 MHz, DMSO-d6) δ[ppm]: 1.088 (0.43), 1.255 (3.70), 1.426 (0.96), 1.623 (0.98), 2.361 (0.88), 2.634 (0.81), 2.838 (0.93), 3.172 (0.95), 3.373 (0.48), 4.349 (7.53), 5.654 (0.91), 7.582 (16.00), 7.655 (1.88), 7.660 (1.72), 7.673 (3.97), 7.677 (4.37), 7.690 (7.92), 7.699 (8.03).
Example 76
sodium 5-({8-[(3-fluorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}carbonyl)-1,2,3-triazol-1-ide
[0369] ##STR00136##
[0370] A stirred solution of 4.99 g (13.7 mmol) {8-[(3-fluorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}(1H-1,2,3-triazol-5-yl)methanone was reacted in analogy to example 78 with sodium methanolate to yield 4.89 g (12.5 mmol, 92%) of the title compound sodium 5-({8-[(3-fluorophenyl)sulfonyl]-3,8-diazabicyclo[3.2.1]oct-3-yl}carbonyl)-1,2,3-triazol-1-ide.
[0371] LC-MS: R.sub.t=0.88 min; MS (ESIpos): m/z=365 [M-Na.sup.++H].sup.+
[0372] .sup.1H-NMR (600 MHz, METHANOL-d4) δ [ppm]: −0.005 (1.50), 0.006 (1.47), 1.177 (0.54), 1.442 (3.45), 1.450 (3.48), 1.614 (2.20), 1.628 (3.21), 1.635 (3.51), 1.648 (1.93), 3.055 (1.86), 3.077 (1.92), 3.481 (0.45), 3.493 (0.93), 3.502 (1.89), 3.523 (1.92), 4.253 (2.38), 4.363 (2.38), 4.537 (1.86), 4.558 (2.10), 4.586 (2.10), 4.607 (1.77), 7.409 (1.86), 7.410 (2.05), 7.414 (2.17), 7.415 (2.18), 7.424 (4.26), 7.428 (4.51), 7.429 (4.41), 7.439 (2.31), 7.442 (2.45), 7.443 (2.31), 7.593 (3.06), 7.602 (3.24), 7.607 (5.51), 7.616 (5.57), 7.621 (3.33), 7.630 (3.16), 7.671 (3.28), 7.675 (4.38), 7.678 (3.52), 7.685 (3.35), 7.687 (4.35), 7.692 (3.33), 7.749 (5.52), 7.751 (6.36), 7.754 (5.22), 7.762 (4.66), 7.764 (5.38), 7.765 (5.31), 7.766 (4.19), 7.802 (15.79), 7.803 (16.00), 7.892 (1.93).
EXPERIMENTAL SECTION—BIOLOGICAL ASSAYS
[0373] Examples were tested in selected biological assays one or more times. When tested more than once, data are reported as either average values or as median values, wherein [0374] the average value, also referred to as the arithmetic mean value, represents the sum of the values obtained divided by the number of times tested, and [0375] the median value represents the middle number of the group of values when ranked in ascending or descending order. If the number of values in the data set is odd, the median is the middle value. If the number of values in the data set is even, the median is the arithmetic mean of the two middle values.
[0376] Examples were synthesized one or more times. When synthesized more than once, data from biological assays represent average values or median values calculated utilizing data sets obtained from testing of one or more synthetic batch.
[0377] The in vitro activity of the compounds of the present invention can be demonstrated in the following assays:
[0378] AKR1C3-Inhibitory Activity Assay
[0379] The AKR1C3-inhibitory activity of the substances of the present invention was measured in the AKR1C3 assay described in the paragraphs below.
[0380] Essentially, the enzyme activity is measured by quantification of the generation of Coumberol from Coumberone (Halim et al. J. AM. CHEM. SOC. 2008, 130:14123-14128 and Yee et al. Proc. Natl. Acad. Sci. USA 2006, 103:13304-13309). In this test, the increase of the highly fluorescent Coumberol by NADPH—(nicotinamide adenine dinucleotide phosphate)-dependent reduction of the non-fluorescent Coumberone by AKR1C3 was determined.
[0381] The enzyme used was recombinant human AKR1C3 (Aldo-keto reductase family 1 member C3; GenBank Accession No. NM_003739). This was expressed in E. coli as GST (glutathione S transferase) fusion protein and purified by glutathione Sepharose affinity chromatography. The GST was removed by digestion with thrombin and subsequent size exclusion chromatography (Dufort, I., Rheault, P., Huang, X F., Soucy, P., and Luu-The, V., Endocrinology 140, 568-574 (1999)).
[0382] For the assay, 50 nl of a 100-fold concentrated solution of the test substance in DMSO were pipetted into a black low-volume 384-well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2.5 μl of a solution of AKR1C3 in assay buffer [50 mM potassium phosphate buffer pH 7, 1 mM DTT, 0.0022% (w/v) Pluronic F-127, 0.01% BSA (w/v) and protease inhibitor cocktail (Complete, EDTA-free Protease Inhibitor Cocktail from Roche)] were added and the mixture was incubated for 15 min to allow pre-binding of the substances to the enzyme prior to the enzyme reaction. The enzyme reaction was then started by addition of 2.5 μl of a solution of NADPH (20 μM.fwdarw.final concentration in 5 μl of assay volume is 10 μM) and Coumberone (0.6 μM.fwdarw.final concentration in 5 μl of assay volume is 0.3 μM) in assay buffer, and the resulting mixture was incubated at 22° C. for the reaction time of typically 90 min. The concentration of the AKR1C3 and the reaction time was adapted to the respective activity of the enzyme preparation and adjusted such that the assay was carried out in the linear range. Typical AKR1C3 concentrations were in the region of 1 nM. The reaction was stopped by addition of 2.5 μl of a stop solution consisting of 3 μM EM-1404 as inhibitor (U.S. Pat. No. 6,541,463) in 50 mM HEPES pH7.5 (3 μM EM-1404.fwdarw.final concentration in 7.5 μl of assay volume is 1 μM). The fluorescence of the Coumberole was then measured at 520 nm (excitation at 380 nm) using a suitable measuring instrument (Pherastar from BMG Labtechnologies). The intensity of the fluorescence was used as a measure of the amount of Coumberole formed and thus of the enzyme activity of AKR1C3. The data were normalized (enzyme reaction without inhibitor=0% inhibition; all other assay components, but no enzyme=100% inhibition). Usually, the test substances were tested on the same microtiter plate at 11 different concentrations in the range from 20 μM to 73 pM (20 μM, 5.7 μM, 1.6 μM, 0.47 μM, 0.13 μM, 38 nM, 10.9 nM, 3.1 nM, 0.9 nM, 0.25 nM and 73 pM, the dilution series were prepared prior to the assay on the level of the 100-fold concentrated solution by serial 1:3 dilutions with 100% DMSO) in duplicates for each concentration, and the IC.sub.50 values were calculated using a 4-parameter fit.
[0383] As described, the pharmacological substances claimed were examined for their inhibitory activity on the AKR1C3 enzyme (see table 2). For the major part of the structural range claimed, these substances show strong inhibition of AKR1C3 in vitro with IC.sub.50 values of less than 10 nM and predominantly even with IC.sub.50 values around 1 nM.
TABLE-US-00007 TABLE 2 AKR1C3-inhibitory activity: IC.sub.50 values of examples Example IC.sub.50 human AKR1C3 [nM] 1 3.7 2 1.0 3 1.4 4 1.2 5 1.2 6 1.7 7 1.4 8 1.3 9 0.9 10 7.3 11 1.1 12 1.1 13 1.4 14 1.9 15 1.5 16 2.2 17 2.9 18 1.0 19 1.1 20 1.0 21 2.0 22 1.5 23 2.2 24 1.8 25 2.6 26 1.1 27 0.9 28 1.2 29 0.9 30 0.6 31 0.5 32 0.7 33 1.8 34 1.9 35 6.0 36 3.3 37 2.8 38 1.5 39 1.3 40 3.4 41 6.5 42 1.1 43 9.1 44 1.9 45 1.4 46 0.9 47 29.8 48 1.1 49 1.2 50 0.9 51 0.8 52 2.5 53 0.8 54 1.2 55 2.7 56 1.4 57 9.7 58 1.2 59 2.2 60 28.4 61 1.5 62 2.8 63 1.7 64 1.3 65 0.5 66 0.9 67 1.0 68 1.3 69 0.6 70 0.5 71 0.8 72 1.1 73 28.0 74 5.5 75 0.7 76 1.1
[0384] Compound number 4 in table 1 of WO 2007/111921 (comparative example) was analysed in the same assay to determine the AKR1C3-inhibitory activity of this compound. The IC.sub.50 of compound number 4 in table 1 of WO 2007/111921 was 1810 nM.
[0385] Inhibition of Testosterone Formation from Androstenedione in Human Primary Adipocytes
[0386] Human primary preadipocytes from 2 donors with a Body Mass Index (BMI) of 26 and 30, respectively were differentiated into mature adipocytes (ordered by ZenBio, Cat# SA-1012-2 12 well Platte; Cat# SA-1012-3 12 well Platte). Adipocytes were incubated in Adipocyte Basal Medium (Fa. ZenBio, Cat# BM-1)+1% FCS+2.5 μg/ml Amphotericin B (Fa. Sigma, Cat# A2942) supplemented with 1 μM androstenedione and 1 μM, 10 μM compound 76 or vehicle for 48 h. Androstenedione served as a substrate for the formation into testosterone. After the incubation adipocytes were collected and testosterone and androstenedione concentrations were determined by LC/MS at the “Bioanalytical Service and research provider Pharm-Analyt”. Inhibition of the conversion of androstenedione to testosterone by compound 76 is determined as Testosterone/Androstenedione ratio [%]. That shows that the formation of testosterone from androstenedione is inhibited in human primary adipocytes by compound 76 (see
[0387] Callithrix jacchus Endometriosis Model
[0388] The in vivo efficacy of compound 76 was tested in a non-human primate endometriosis model in marmoset monkeys.
[0389] Marmoset monkeys (Callithrix jacchus) are non-menstruating species in which endometriosis was induced by injection of endometrial tissue into the peritoneal cavity (Einspanier, Lieder et al, 2006). 6-12 year old female common marmosets with established endometriosis were employed (body weight between 358 g and 520 g) and distributed into 2 groups, with a size of n=5 animals per group. Prior to the actual start of the treatment, the animals were subjected to a laparotomy and examined for the presence of endometriotic lesions on the bladder, the uterus and the ovaries to measure each lesion's area. The randomization of animals into the two treatment groups was performed with respect to the initial lesion status at the first laparotomy. The endometriosis severity based on total lesion area per animal was distributed similarly across the groups.
[0390] For both the treatment group (5 mg/kg of compound 76) and vehicle treated group, the sum of total lesion area/size [cm.sup.2] per animal, with respect to lesion area and lesion number, was determined and defined as pre-treatment status. 6 weeks later the treatment was started. The test compounds were administered orally once per day in capsules (PC Caps® capsules, Capsugel). To adapt for the intended dose, a trituration of the active compound with lactose was prepared and the exact dose was filled into the individual capsules. Each capsule was separately coated with salvia resistant coating (Eudragit EPO; Evonik). After the end of a 6 weeks treatment period, a second laparotomy was carried out and the number and size of the lesions on uterus, ovaries and bladder was determined with respect to total lesion size/area and defined as post-treatment status.
[0391] Total lesion size/area pre-treatment and post-treatment is shown in
[0392] The reduction of total lesion size after treatment is visualized as ratio of total lesion size/area post-versus pre-treatment in the two groups (vehicle group, 5 mg/kg example 76 group). A ratio of 1, corresponds to a stable lesion size. Ratios above 1 show an increase in total lesion size over the course of the experiment, while ratios below 1 show a decrease in total lesion size. The results displayed in
[0393] Interference with Anthracycline Resistance in Cancer Cells by AKR1C3 Inhibition
[0394] A549 lung cancer cells are expressing AKR1C3. A549 cells are plated 24 h prior the start of the experiment. After 24 h the medium is replaced with fresh medium, which contains 1, 10, 50, 100, 200, 500, and 1000 nM daunorubicin, doxorubicin and idarubicin, with or without 1 μM, 10 μM, 30 μM of compound 76. Cell viability is determined following 72 h of incubation at standard conditions (37° C., 5% CO.sub.2). Cell viability is measured by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromid; Sigma-Aldrich) solution in PBS is added to the cells to a final concentration of 1 mg/ml, and the cells are subsequently incubated at standard conditions for 4 h. The medium is aspirated and the cells are lysed with dimethyl sulfoxide on an automatic shaker for 15 min. Absorbance is measured at 570 nm and 690 nm using a microplate reader.
DESCRIPTION OF FIGURES
[0395]
[0396]
[0397] (A) The pre-treatment and post treatment total lesion size of individual marmosets with established endometriosis (n=5) at bladder, ovaries and uterus of the vehicle treated group (left box) and the compound (5 mg/kg of example 76) treated group (right box) is shown.
[0398] (B) The ratio of post-/pre-treatment total lesion size at bladder, ovaries and uterus in marmosets with established endometriosis treated with vehicle (left box) or 5 mg/kg of example 76 (right box) is shown. The dotted line represents status at the beginning of the study (first laparotomy).