Patent Application: US-81120891-A

Abstract:
novel tricyclic steriod analogs are disclosed which are 1h - benzindene dodecahydro compounds that are useful for enhancing gaba - induced chloride currents at the gaba receptor / chloride ionophore complex and can be represented by the following structural formulas : ## str1 ## wherein r 1 = h or c 1 - c 4 alkyl or fluoroalkyl ; r 2 = h or c 1 - c 4 alkyl or fluoroalkyl , in which r 1 and r 2 can be the same or different ; r 3 = h or ch 3 ; r 4 = h or ch 3 , in which r 3 and r 4 can be the same or different ; r 5 = h ; r 6 = h ; r 5 , r 6 =═ o ; r 7 = h ; r 8 = a hydrogen bond accepting group . r 7 , r 8 =═ o ; and r &# 39 ;= an ester group .

Description:
while the specification concludes with claims particularly pointing out and distinctly claiming the subject matter regarded as forming the invention , it is believed that the invention will be better understood from the following description of preferred embodiments taken in conjunction with the accompanying drawing in which : fig1 shows the modulation of gaba currents by a representative example of the tricyclic steroid analogs of the invention . shown is the effect of the analog prepared in example 13 and table 1 , below , in which r = coch 3 on currents induced by 1 μm gaba . neurons were voltage clamped at - 5omv , and the test compound administered at 1 μm . the synthesis of the preferred compounds of generic formulas i and ii is conveniently shown in the following reaction schemes 1 and 2 . the synthetic method in reaction scheme 1 comprises a series of steps to open the a - ring of the known and readily available steroid , 19 - nortestosterone , and remove the c 1 and c 2 . the lithium , liquid ammonia reaction carried out in the first step yields the 5α - configuration at the a / b ring fusion ( 9 ). each of the steps carried out in reaction scheme 1 is carried out in high yield . whenever more than one transformation is indicated on an arrow between structures , the yield reported is the overall yield for the combined transformations . each of the compounds shown in the reaction scheme 1 has been purified to homogeneity by chromoatographic methods . these compounds have been shown to have the correct elemental composition by combustion analysis and have been characterized by infrared and nmr spectroscopy . ## str6 ## reaction scheme 2 details the remaining steps needed to preoare various 5α - analogs having the different hydrogen bond acceptor groups ( i . e ., c ═ o , cn , coch 3 , and cyclopropyl - nh group ) of the final compounds . yields shown under the arrows are yields obtained for the four preferred compounds whose evaluation are set forth in table 1 , hereinafter . the cyano group introduced in the tosmic reaction ( step c ) is introduced with ca . 70 % 17β - stereochemistry . the 17α - cyano isomer was removed by chromatography . finally , in order to synthesize the 5β - analogs , two additional steps not shown in reaction scheme 1 are carried out as shown below . the effect of these two steps is to change the side chain from the 5α - configuration to the 5β - configuration . the final comopund shown in reaction scheme 1 is converted by an α - phenylselenylation / elimination reaction into the α , β - unsaturated ester ( 43 ). catalytic hydrogenation using palladium in tetrahydrofuran and hydrobromic acid is then carried out to give the 5β - reduced tricyclic analog . these conditions are analogous to the conditions known to convert 19 - nortestosterone to 5β - dihydrotestosterone with 98 % stereospecificity ( 46 ). chromatographic methods can be used to separate mixtures of the 5α and 5β - compounds . ## str7 ## additional tricyclic steroid analogs within the scope of formulas i and ii can be prepared by various of the following reaction schemes : introduction of r 1 and r 2 alkyl or fluoroalkyl groups where r 1 = r 2 or r 1 not = r 2 can be prepared as follows : ## str8 ## examples show preparation of compounds where r 3 = r 4 = h or r 3 = h and r 4 = ch 3 preparation of compounds where r 3 = ch 3 and r 4 = h or ch 3 is as follows : ## str9 ## compounds having r 5 , r 6 =═ o could be prepared by starting with materials having a ketone at the 11 - position . for example : ## str10 ## the various derivatives of the α - hydroxyketone are prepared as follows : ## str12 ## the silyl protecting group can then be removed with tetra n - butyl ammonium fluoride . alkyl esters of carboxylic acids can be prepared from nitriles by treatment with aqhcl to convert the nitrile group to the carboxylic acid group , and then esterifying the carboxylic acid with dry hcl in the appropriate alkanol . ## str13 ## the following detailed examples will further illustrate the invention although it will be understood that the invention is not limited to these specific examples or the details described therein . as indicated in structural formulas i and ii , r 4 can be h or ch 3 . the following are general structures of compounds prepared in the following specific examples in which r can be h or ch 3 . ## str15 ## to a solution of 17β - acetyloxy - 5α - estran - 3 - one ( 1 . 595 g , 5 mmol ) in dry dichloromethane ( 30 ml ) was added with stirring at ice - water bath temperature , triethyl amine ( 2 ml ) followed by t - butyldimethylsily triflate ( 2 . 64 g , 10 mmol ). the solution was stirred for 25 min , then diluted with dichloromethane ( 20 ml ) and washed with satd . aq . nahco 3 ( 30 ml ), brine ( 30 ml ) and dried over na 2 so 4 . the solvent was removed to yield a solid , which was purified by chromatography ( silica gel , dichloromethane , pretreated with 1 % triethyl amine in hexanes ) to get 1 . 9 g ( 87 %) to pure product as colorless crystals , m . p . 89 °- 90 ° c . ir ( film , nacl ): 2927 , 2858 , 1739 , 1676 , 1472 , 1371 , 1246 cm - 1 . 1 h nmr ( cdcl 3 ): δ4 . 80 ( dd , 1h , ch ═ c ), 4 . 59 ( dd , 1h , choac ), 2 . 04 ( s , 3h , coch 3 ), 0 . 91 ( s , 9h , ( ch 3 ) 3 c ), 0 . 80 ( s , 3h , ch 3 ), 0 . 11 ( s , 6h , ( ch 3 ) 2 si ). 13 c nmr ( cdcl 3 ): δ171 . 58 ( ch 3 coo ), 149 . 88 ( c 36l ), 103 . 48 ( c 2 ), 82 . 97 ch 17 ), 25 . 51 (( ch 3 ) 3 c ), 11 . 81 ( c 18 ), - 4 . 62 and - 4 . 78 (( ch 3 ) 3 csi and ( ch 3 ) 2 si ). elemental analysis : for c 26 h 44 o 3 si . calcd : c , 72 . 17 ; h , 10 . 25 ; found : c , 72 . 49 ; h , 10 . 06 . ## str16 ## a solution of the silyl ether of example 1 ( 0 . 866 g , 2 mmol ) in dichloromethane ( 10 ml0 and methanol ( 10 ml ) was treated with o 3 at - 78 ° c . until a blue color persisted . excess o 3 was discharged by o 2 stream . nabh 4 ( 1 . 0 g ) was added with stirring . the mixture was diluted with diethyl ether ( 25 ml ) and poured into cooled 10 % aq . hcl solution 250 ml ). the organic phase was washed with water ( 25 ml ), brine ( 25 ml ), and dried over na 2 so 4 . the solvent was removed on a rotary evaporator to give a viscous liquid , which was diluted with n - hexane to give a colorless solid . another portion of product was obtained by evaporting the n - hexane solution and hydrolyzing the residue with a mixture consisting of 10 % aq . k 2 co 3 ( 10 ml ), tetrahydrofuran ( 15 ml ), and methanol ( 30 ml ) for 1 . 5 h at room temperature . the crude compound was recrystallized from methanol to give 0 . 49 g ( 70 %) pure compound as colorless crystals , m . p . 141 °- 145 ° c . ir ( film , nacl ): 3328 , 2918 , 1732 , 1705 , 1444 , 1732 , 1246 cm - 1a . 1 nmr ( cdcl 3 ): δ4 . 59 ( t , j = 8 . 4 hz , 1h , choac ), 3 . 56 - 3 . 69 ( m , 2h , ch 2 oh ), 2 . 62 ( dd , j = 15 . 8 , j = 2 . 6 hz , 1h of ch 2 cooh ), 2 . 5 ( s , 3h , ococh 3 ), 0 . 80 ( s , 3h , ch 3 ). -- c nmr ( cdcl 3 ): δ178 . 54 ( cooh ), 171 . 78 ( ch 3 coo ), 82 . 83 ( c 3 ), 59 . 79 ( ch 2 oh ), 11 . 75 ( c 3a ). elemental analysis : for c 20 h 32 o 5 . calcd : c , 68 . 15 ; h , 9 . 15 ; found : c , 68 . 01 ; h , 9 . 27 . ## str17 ## to a solution of 3 . 52 g ( 10 mmol ) of the compound of example 2 in diethyl ether ( 350 ml ), diazomethane in diethyl ether was added until a yellow color persisted at 0 ° c . the solution was allowed to stir for an additional 15 min . excess diazomethane was destroyed by addition of several drops of formic acid . the mixture was washed with 10 % aq . nahco 3 ( 100 ml ), water ( 100 ml ), and brine ( 100 ml ); and dried over na 2 so 4 . the solvent was removed to give a virtually quantitive yield of product , m . p . 72 ° 73 . 5 ° c . ir ( film , nacl ): 3455 , 2921 , 2873 , 1737 , 1437 , 1373 , 1246 cm - 1 . 1 h ( nmr ( cdcl 3 ): δ4 . 59 ( t , j = 7 . 7 hz , 1h , choac ), 3 . 68 ( s , 3h , cooch 3 ), 3 . 68 - 3 . 61 ( m , 1h , ch 2 oh ), 2 . 59 ( dd , j = 14 . 1 hz , j = 4 . 1 hz , 1h of ch 2 cooch 3 ), 2 . 04 ( s , 3h , ococh 3 ), 0 . 80 ( s , 3h , ch 3 ). 13 c nmr ( cdcl 3 ): δ174 . 47 ( cooch 3 ), 171 . 55 ( ch 3 coo ), 82 , 75 ( c 3 ), 60 . 02 ( ch 2 oh ), 51 . 3 ( ch 3 o ), 11 . 74 ( c 3a ). elemental analysis : c 21 h 34 o 5 . calcd : c , 68 . 82 . ; h , 9 . 35 ; found : c , 68 . 78 ; h , 9 . 65 ## str18 ## to a stirring suspension of pyridinium chlorochromatic ( 0 . 65 g , 3 . 0 mmol ) and anhydrous naoac ( 0 . 25 g , 3 . 0 mmol ) in dry dichloromethane ( 50 ml ) was added a solution of the compound of example 3 ( 0 . 73 g 2 . 0 mmol ) in dry dichloromethane ( 10 ml ) at room temperature under nitrogen . after the mixture was stirred for 2 h , diethyl ether ( 40 ml ) was added . the mixture was filtered with a bushier filter which was filled with silica gel and washed with cichloromethane . the solvent was removed to get a solid which was recrystalliced from diethyl ether to give 0 . 70 g ( 96 . 7 %) pure product as colorless crystalline needles , m . p . 105 °- 170 ° c . to a string suspension of pyridinium chlorochromatic ( 0 . 65 g , 3 . 0 mmol ) and anhydrous naoac ( 0 . 25 g , 3 . 0 mmol ) in dry dichloromethane ( 50 ml ) was added a solution of the compound of example 3 ( 0 . 73 g 2 . 0 mmol ) in dry dichloromethane ( 10 ml ) at room temperature under nitrogen . after the mixture was stirred for 2 h , diethyl ether ( 40 ml ) was added . the mixture was filtered with a bushier filter which was filled with silica gel and whased wutg dichloromethane . the solvent was removed to get a solid which was recrystallized from diethyl ether to give 0 . 70 g ( 96 . 7 & amp ;) pure product as colorless crystalline needles , m . p . 105 °- 107 ° c . ir ( film , nacl ) 2923 , 2854 , 2721 , 1737 , 1734 , 1437 , 1374 , 1246 cm - 1 . 1 h nmr ( cdcl 3 ): δ9 . 82 ( t , j = 1 . 6 hz , 1h , cho ), 4 . 59 ( t , j = 8 . 0 hz , 1h , choac ), 3 . 67 ( s , 3h , cooch 3 ), 2 . 04 ( s , 3 , ococh 3 ), 0 . 80 ( s , 3h , ch 3 ). 13 c nmr ( cdcl 3 ): δ202 . 48 ( cho ), 173 . 60 ( cooch 3 ), 171 . 40 ( ch 3 coo ), 82 . 53 ( c 3 ), 11 . 69 ( c 3a ). elemental analysis : for c 21 h 32 o 5 . calcd : c , 69 . 29 ; h , 8 . 85 ; found : c , 69 . 05 , h , 8 . 78 . ## str19 ## a solution of the compound of example 4 ( 20 g , 55 mmol ) and p - tolulenesulfonic acid ( 1 . 0 g , 5 % w / w ) in isopropenyl acetate ( 500 ml ) was gently distilled for 2 . 0 h and about 25 ml of solution was collected . after refluxing for 14 h , the mixture was gently distilled for another 2 . 0 h and about 25 ml of solution gain was collected . the reaction mixture was cooled to room temperature and poured into dichloromethane ( 500 ml ), and washed with water ( 100 ml ) satd aq . nahco 3 100 ml ), and water ( 100 ml ). the organic layer was dried over na 2 so 4 , filtered , and concentrated to give a yellow oil , which was chromatographed ( silica gel , eluting with 4 : 1 - hexane : ethyl acetate ) to give 14 g ( 62 %) pure product as colorless crystals , m . p . 122 °- 123 ° c . ir ( film nacl ): 2920 , 1750 , 1738 , 1737 , 1673 , 1436 , 1372 , 1226 cm - 1 . 1 h nmr ( cdcl 3 ): δ7 . 01 ( d , j = 1 . 25 hz , 1h , acoch ═ ch ), 5 . 01 ( dd , j = 12 . 5 hz , j = 10 . 7 hz , 1h , acoch = ch ), 4 . 59 ( t , j = 7 . 8 hz , 1h , choac ), 3 . 63 ( s , 3h , cooch 3 ), 2 . 49 ( dd , j = 15 . 4 hz , j = 4 . 4 hz , 1h , of ch 2 cooch 3 ), 2 . 04 ( s , 3h ococh 3 ), 0 . 79 ( s , 3h , ch 3 ). 13 c nmr ( cdcl 3 ): δ174 . 09 ( och ═), (═ ch ), 82 . 67 ( ch 3 cooch ═), 136 . 35 ( och ═), 117 . 35 (═ ch ), 82 . 67 ( c 3 ), 11 . 80 ( c 3a ). elemental analysis : for c 23 h 34 o 6 . calcd : c , 67 . 96 ; h , 8 . 43 . found : c , 68 . 24 ; h , 8 . 53 . ## str20 ## a solution of the compound of example 5 ( 0 . 81 g , 2 . 0 mmol ) in dichloromethane ( 50 ml ) and acetic acid ( 0 . 5 ml ) was cooled at 31 78 ° c . in an acetone -- dry ice bath and treated in o 3 until a blue color persisted . the excess o 3 was removed by bubbling o 2 . methyl sulfide ( 4 drops , ca . 4 . 0 mmol ) was added and the mixture was stirred for 1 . 0 h at 31 78 ° c . and 1 . 0 h at the room temperature . the mixture was diluted with dichloromethane ( 50 ml ) and the organic layer was washed with water ( 50 ml ), satd . aq . nahco 3 ( 50 ml ), water ( 2 × 50 ml ) again ; and dried over na 2 so 4 . the organic solvent was removed to give a solid , which was recrystallized from diethyl ether to give 0 . 64 g ( 92 %) of product as colorless crystals , m . p . 123 °- 124 ° c . ir ( film , nacl ): 2924 , 2870 , 2805 , 2707 , 1735 , 1721 , 1436 , 1372 , 1244 cm 1 . 1 h nmr ( cdcl 3 ): δ9 . 44 ( d , j = 5 . 4 hz , 1h , cho ), 4 . 62 ( t , j = 8 . 5 hz , 1h , choac ), 3 . 66 ( s , 3h , cooch 3 ), 2 . 04 ( s , 3h , ococh 3 ), 0 . 80 ( s , 3h , ch 3 ). 13 c nmr ( cdcl 3 ): δ205 . 44 ( cho ), 172 . 88 ( cooch 3 ), 171 . 38 ( c 3 coo ), 82 . 41 ( c 3 ), 11 . 71 ( c 3a ). elemental analysis : for c 20 h 30 o 5 . calcd : c , 68 . 55 ; h , 8 . 63 ; found : c , 68 . 71 ; h , 8 . 81 . ## str21 ## the mixture of the compound of example 6 ( 0 . 70 g , 2 . 0 mmol ) and wilkinson &# 39 ; s catalyst ( 1 . 85 g , 2 . 0 mmol ) in benzonitrile ( 30 ml ) was heated to 160 ° c . for 20 h under nitrogen . most of the benzonitrile was removed by distillation and a mixture of ethyl acetate and hexane ( 1 : 1 v / v , 50 ml ) was added to precipitate the organometallic by - product . the yellow solid was filtered and the solid was washed with cold ethyl acetate ( 2 × 20 ml ). the combined organic layer was evaporated to give a viscous liquid , which was chromatographed ( silica gel , 1 % acetonitrile in dichloromethane ) to give 0 . 59 g ( 91 %) pure product as slightly yellow crystals , m . p . 62 °- 62 ° c . ir ( film , nacl ): 2918 , 2851 , 1741 , 1738 , 1446 , 1373 , 1246 cm - 1 . 1 h nmr ( cdcl 3 ): δ4 . 61 ( t , j = 8 . 5 hz , 1h , choac ), 3 . 67 ( s , 3h , cooch 3 ), 2 . 04 ( s , 3h , ococh 3 ), 0 . 80 ( s , 3h , ch 3 ). 13 c nmr ( cdcl 3 ): δ173 . 74 ( cooch 3 ), 171 , 44 ( ch 3 coo ), 82 . 72 ( c 3 ), 11 . 85 . ( c 3a ). elemental analysis : for c 19 h 30 o 4 . calcd : c , 70 . 77 ; h , 9 . 38 ; found c , 70 . 68 ; h , 9 . 33 . ## str22 ## to a solution of the compound of example 7 ( 100 mg , 0 . 38 mmol ) in methanol ( 10 ml ) was added an aq . solution of naoh ( 0 . 19 g in 2 ml water ). after stirring at room temperature for 18 h , aq . hcl ( 6n , 20 ml ) was added and the reactio mixture was poured into water ( 100 ml ). the product precipitated and was recovered as a white solid by filtration . it was recrystallized from aq . methanol to give 67 mg ( 81 % yield ) pure proudct as fine white crystals , m . p . 202 °- 203 ° c . ir ( film , agcl ): 3401 , 2895 , 1703 , 1338 , 1233 , 1056 cm - 1 . 1 h nmr ( cdcl 3 ): δ3 . 66 ( t , j = 8 . 3 hz , 1h , choh ), a 2 . 24 ( d , j = 6 . 8 hz , 1h of ch 2 cooh ), 0 . 75 ( s , 3h , ch 3 ), elemental analysis : for c 16 h 26 o 3 . cacd : c , 72 . 14 ; h , 9 . 84 ; found : c , 71 . 91 ; h , 9 . 62 . ## str23 ## to a stirred , cooled ( ice - water bath ) solution of the compound of example 7 ( 0 . 65 g , 2 . 0 mmol ) in dry toluene ( 50 ml ) was added diisobutyl aluminium hydride ( 1 . 0 m solution in toluene , 12 ml , 12 mmol ). after 3 . 0 h , toluene - methanol ( 1 : 1 , 4 ml ) was added and followed by 10 % aw . hcl ( 10 ml ). then the mixture was washed with water 2 × 50 ml ) and brine ( 50 ml ). the organic layer was dried over na 2 so 4 , filtered and concentrated under reduced pressure to give a solid , which was recrystallized from ethyl alcohol to yield 0 . 47 g ( 93 %) of pure product as colorless crystals , m . p . 145 °- 147 ° 6l c . ir ( film , nacl ): 3279 , 2914 , 2870 , 2858 , 2841 , 1469 , 1443 , 1381 , 1348 , 1067 , 1056 cm - 1 . 1 h nmr ( cdcl 3 ): δ3 . 73 - 3 . 65 ( m , 3h , ch 2 oh and choh ), 0 . 76 ( s , 3h , ch 3 ). 13 c nmr ( cd 3 od ): δ82 . 63 ( c 3 ), 60 . 78 ( ch 2 oh ), 11 . 71 c 3a ). elemental analysis : for c 16 h 28 o 2 . cacd : c , 76 . 14 ; h , 11 . 18 ; found : c , 75 . 96 ; h , 11 . 29 ## str24 ## to a stirred solution of the compound of example 9 ( 254 mg , 1 . 0 mmol ) in glacial acetic acid ( 5 ml ) was added dropwise a 5 . 25 % solution of sodium hypochlorite ( 1 . 5 ml , 1 . 05 mmol ) at room temperature over 10 min . after the stirring was continued for another 1 . 0 h , isopropanol ( 2 . 0 ml ) was added to quench any excess oxidant , followed by water ( 5 . 0 ml ). the mixture was extracted with ethyl acetate ( 2 × 25 ml ). the combined organic layer was washed with water ( 25 ml ), satd . aq nahco 3 ( 25 ml ), water ( 25 ml ), and brine ; ( 25 ml ) and dried over na 2 so 4 . the solvent was removed to give an oil , which was purifief by column chromatography ( silica gel , eluted with 1 : 1 ethyl acetate - hexane ) to give 161 mg ( 64 %) of pure product as colorless crystals ( from diethyl ether - hexane ), m . p . 38 °- 40 ° c . ir ( film , nacl ): 3435 , 2917 , 1739 , 1452 , 1406 , 1373 , 1258 , 1097 , 1046 , cm - 1 . 1 h nmr ( cdcl 3 ): δ3 . 71 ( t , j = 6 . 5 hz , 2h , ch 2 oh ), 0 . 87 ( s , 3h , ch 3 ). 13 c nmr ( cdcl 3 ): δ222 . 05 ( co ), 60 . 07 ( ch 2 oh ), 13 . 44 ( c 3a ). elemental analysis : for c 16 h 26 o 2 . calcd : 76 . 75 ; h , 10 . 47 ; found ; c , 76 . 52 ; h , 10 . 24 ## str25 ## to a solution of anhydrous cyclopropylamine ( 170 mg , 3 . 0 mmol ) in absolute methanol ( 10 ml ) was added a solution of the compound of example 10 ( 10 mg , 0 . 6 mmol ) in methanol ( 5 . 0 ml ) and then nabh 3 cn ( 62 mg , 1 . 0 mmol ) at room temperature under nitrogen . the ph value of the mixture was adjusted to equal about 6 with methanolic hcl solution . after the mixture was stirred for 72 h at room temperature , the solvent was evaporated under vacuum and the residue was dissolved in 6 n aq . hcl and extracted with diethyl ether ( 3 × 25 ml ). the solution was made basic with satd . naoh solution and was saturated with nacl . the free amine crystallized on the surface of the solution and was extracted with diethyl ether ( 33 × 50 ml ). the combined solutions were evaporated . the residue was dissolved in methanol ( 10 ml ) and bubbled with hcl gas until strongly acidic . most of the methanol was removed under vacuum to give a residue which was recrystallized from methanol - ethyl acetate - hexane to give 120 mg ( 61 %) of pure product as colorless crystalline needles , m . p . 220 °- 222 ° c . ir ( kbr ): 3400 , 3379 , 3051 , 2918 , 2845 , 2795 , 2732 , 1591 , 1446 , 1052 , 1036 cm - 1 . 1 h nmr ( cd 3 od ) δ3 . 52 ( t , j = 6 . 5 hz , 2h ch 2 oh ), 0 . 83 ( s , 3h , ch 3 ). 13 c nmr ( cd 3 od ): δ70 . 39 ( c 3 ), 60 . 70 ( ch 2 oh ), 12 . 28 ( c 3a ). elemental analysis : for c 19 h 34 clno . calcd : c , 69 . 59 ; h , 10 . 45 ; n , 4 . 27 ; cl , 10 . 81 . found : c , 69 . 46 ; h , 10 . 47 ; n , 4 . 24 ; cl , 11 . 00 . ## str26 ## a solution of the compound of example 10 ( 320 mg 1 . 28 mmol ) in dimethoxyethane ( 32 ml ) was treated with a 1 . 0 m solution of t - buok in dimethyoxyethane ( 12 . 8 ml , 12 . 8 mmol ) and ethanol ( 2 . 0 ml ). a solution of tosylmethyl isocyanide ( 500 mg , 2 . 56 mmol ) in dimethoxyethane ( 6 . 5 ml ) was added very slowly by means of a syringe over 20 min with stirring at room temperature . after 3 . 0 h , the mixture was quenched with water ( 50 ml ) and extracted with ethyl acetate ( 3 × 50 ml ). the organic layers were combined and washed with water ( 2 × 50 ml ) and bringe ( 50 ml ); and dried over na 2 so 4 . the solvent was removed to get an oil , which was purified by chromatography ( silica gel , eluted with 10 % acetonitrile in dichloromethane ) to give 200 mg ( 60 %) of a mixture of 17α - and 17β - nitrile isomers as a colorless oil . 13 c nmr spectra of this mixture showed two peaks δ 122 . 22 and δ 121 . 27 respectively , and their ratio was 38 : 62 . the isomers were separated by hplc ( ultrasphere - si , 5 μ , 250 mm × 10 mm column eluted with 30 % ethyl acetate in hexane at 3 . 0 ml / min ) to give 90 mg ( 27 %) of pure product ( β isomer ), which was recrystallized from diethyl ether and hexane as colorless crystals , m . p . 82 °- 83 ° c . ir ( film , nacl ): 3294 , 2917 , 2853 , 2233 , 1470 , 1384 , 1338 , 1056 , 1021 cm - 1 . 1 h nmr ( cdcl 3 ): δ 3 . 64 - 3 . 61 ( m , 2h , ch 2 oh ), 0 . 88 ( s , 3h , ch 3 ). 13 c nmr ( cdcl 3 ): δ 121 . 43 ( cn ), 60 . 30 ( ch 2 oh ), 14 . 06 ( c 3a ). elemental analysis : for c 17 h 27 no . calcd : c , 78 . 11 ; h , 10 . 51 ; n , 5 . 36 ; found : c , 78 . 38 ; h , 10 . 26 ; n , 5 . 28 . ## str27 ## to a solution of methylmagnesium iodide ( 3 . 0 m solution is diethyl ether , 1 . 7 ml , 5 . 0 mmol ) was added a solution of the compound of example 12 ( 260 mg , 1 . 0 mmol ) in dry tetrahydrofuran ( 10 ml ) at ice - water bath temperature under nitrogen . then the mixture was refluxed for 24 h . after the reaction was cooled down to 0 ° c ., satd . aq . nh 4 cl solution was added to destroy any excess grignard reagent . the mixture was extracted with ethyl acetate ( 3 × 25 ml ). the combined organic layer was dried over na 2 so 4 and evaporated to give an oil , which was purified by chromatography ( 10 % acetonitrile in dichloromethane ) to give 249 mg ( 90 %) of pure product as colorless crystals ( from ethyl ether and hexane ), m . p . 61 °- 62 ° c . ir ( film , nacl ): 3391 , 2916 , 1705 , 1447 , 1384 , 1056 cm - 1 . 1 h nmr ( cdcl 3 ) δ 3 . 69 ( t , j = 6 . 6 hz , 2h , ch 2 oh ), 2 . 12 ( s , 3h , coch 3 ), 0 . 62 ( s , 3h , ch 3 ). 13 c nmr ( cdcl 3 ): δ 210 . 34 ( co , 60 . 54 ( ch 2 oh ), 13 . 22 ( c 3a ). elemental analysis : for c 18 h 30 o 2 . calcd : c , 77 . 65 ; h , 10 . 86 ; found : c , 77 . 68 ; h , 10 . 83 . ## str28 ## a solution of an approximately 8 : 2 mixture of 3 , 17β - diacetoxyandrost - 3 - ene and 3 , 17β - diacetoxyandrost - 2 - ene 9 . 7 g , 26 . 9 mmol ) in dichloromethane ( 400 ml ) and acetic acid ( 30 ml ) was treated with o 3 at - 78 ° c . until a blue color persisted . excess o 3 was discharged by an o 2 stream until colorless and then the addition of methyl sulfide ( 1 drop ). the dichloromethane was removed on a rotary evaporator , and water ( 90 ml ) and acetic acid ( 200 ml ) was added to the remaining solution . after stirring overnight to hydrolyze the anhydride group generated during ozonolysis , water ( 200 ml ) and diethyl ether ( 200 ml ) were added . the diethyl ether layer was repeatedly washed with water to remove acetic acid and then dried over mgso 4 . solvent removal yielded a slightly yellow solid ( 10 . 7 g ) that dissolved in methanol ( 200 ml ) cooled to 0 ° c ., and reacted with slowly added portion of nabh 4 ( 14 . 3 g ). fifteen min after nabh 4 addition was completed , 10 % aq . hcl was added until the solution became acidic . water ( 250 ml ) was added , and methanol removal on a rotary evaporator was accompanied by the precipitation of the steroid product . after filtration and air drying the crude product ( 8 . 2 g , 87 %) was obtained as a white solid . recrystallization from methanol yielded the pure product which hand m . p . 182 °- 184 . 5 ° c . ir ( film , agcl ) 3319 , 1247 , 1214 , 1729 , 1692 cm - 1 . 1 h nmr ( cdcl 3 ) δ 4 . 58 ( t , j = 10 . 8 hz , 1h , choac ), 3 . 72 ( m , 2h , ch 2 oh ), 2 . 62 ( d , j = 12 . 9 hz , 1h of ch 2 cooh ), 2 . 04 ( s , 3h , ococh 3 ), 0 . 77 ( s , 6h , ch 3 ( c 3a ) and ch 3 ( c 6 )). elemental analysis : for c 21 h 34 o 5 . calcd : c , 68 . 92 ; h , 9 . 35 . found : c , 69 . 29 ; h , 9 . 29 . ## str29 ## to a solution of 5 . 4 g ( 17 . 8 mmol ) of the compound of example 14 in diethyl ether ( 150 ml ), diazomethane in diethyl ether was added until a yellow color persisted . the solution was allowed to stir for an additional 15 min . excess diazomethane was destroyed by addition of several drops of formic acid . the solvent was removed on a rotary evaporator and the crude product was purified by chromatography ( silica gel eluted with 40 % ethyl acetate in hexane ). the purified product ( 4 . 7 g , 83 %) was obtained as a solid which after recrystallization from a mixture of diethyl and hexane had m . p . 115 . 5 °- 117 . 5 ° c . ir ( film , agcl ) 3453 , 2934 , 1732 , 1438 , 1373 , 1246 cm - 1 . 1 h nmr ( cdcl 3 ) δ 4 . 58 ( t , 1h , j = 8 . 5 hz , choac ), 3 . 73 ( m , 1h of ch 2 oh ), 3 . 68 ( s , 3h , cooch 3 ), 3 . 63 ( m , 1h of ch 2 oh ), 2 . 56 ( dd , j = 14 . 4 hz , 2 . 4 hz , 1h of ch 2 cooch 3 ), 2 . 04 ( s , 3h , ococh 3 ), 0 . 77 ( s , 6h , ch 3 ( c 3a ) and ch 3 ( c 6 )). elemental analysis : for c 22 h 36 o 5 . calcd : c , 69 . 44 ; h , 9 . 54 . found : c , 69 . 23 ; h , 9 . 52 . ## str30 ## a solution of the compound of example 15 ( 4 . 7 g , 12 . 3 mmol ) in dichloromethane ( 50 ml ) was added rapidly to a suspension of pyridinium chloroformate ( 10 . 81 g , 50 . 15 mmole ) in dichloromethane and stirred at room temperature under nitrogen . after 3 h , the volume of dichloromethane was reduced to 40 ml on a rotary evaporator and then poured into diethyl ether ( 750 ml ). the ether was passed through a small column of florisil and additional diethyl ether was used to wash the florisil and elute the steroid . the diethyl ether was removed on a rotary evaporator and the crude aldehyde product ( 4 . 6 g ) immediately combined with isopropenyl acetate ( 60 ml ) and p - toluenesulfonic acid ( 0 . 5 g ) and reflexed for 16 h . the solution was then gently distilled for 1 h until about 10 ml of distillate was collected and then cooled to a room temperature . the solution was poured into dichloromethane and washed with water ( 50 ml ), 5 % aq . nahco 3 , and water ( 3 × 100 ml ). the organic layer was dried over mgso 4 , filtered , and removed on a rotary evaporator to give slightly yellow crystals ( 5 . 6 g ), which were purified by chromatography ( silica gel eluted with 4 % ethyl acetate in dichloromethane ) to give 3 . 7 g of product . recrystallization from a mixture of ethyl acetate and hexane gave 1 . 83 g ( 36 %) of a mixture of the e and z enol acetates . the enol acetate isomers were separated by high performance liquid chromatography ( econosil 5 micron , 250 mm × 4 . 6 mm , eluted at 2 . 0 ml / min with 10 % ethyl acetate in hexane ). ir ( film , agcl ): 2918 , 1756 , 1733 , 1666 , 1451 , 1373 , 1227 cm - 1 . 1 h nmr ( cdcl 3 ): δ 6 . 98 ( d , j = 12 . 7 hz , 1h , acoch = ch ), 5 . 15 ( d , j = 12 . 7 hz , 1h , acoch = ch ), 4 . 56 ( t , j = 8 . 5 hz , 1h , choac ), 3 . 63 ( s , 3h , cooch 3 ), 2 . 41 ( dd , j = 15 . 2 hz , j = 2 . 8 hz , 1h of ch 2 cooch 3 ), 2 . 03 ( s , 3h , ococh 3 ), 2 . 11 ( s , 3h , ococh 3 ), 0 . 87 ( s , 3h , ch 3 ( c 6 )), 0 . 76 ( s , 3h , ch 3 c 3a )). elemental analysis : for c 24 h 36 o 6 . calcd : c , 68 . 55 ; h , 8 . 63 . found : c , 68 . 75 ; h , 8 . 70 ## str31 ## ir ( film , agcl ): 2936 , 1759 , 1735 , 1668 , 1437 , 1371 , 1246 , 1217 cm - 1 . 1 h nmr ( cdcl 3 ): δ 6 . 70 ( d , j = 7 . 6 hz , 1h , acoch = ch ), 4 . 58 ( t , j = 8 . 5 hz , 1h , choac ), 4 . 39 ( d , j = 7 . 5 hz , 1h , acoch = ch ), 3 . 64 ( s , 3h , cooch 3 ), 2 . 44 ( d , j = 14 . 3 hz , 1h of ch 2 cooch 3 ) 2 . 11 ( s , 3h , ococh 3 ), 2 . 03 ( s , 3h , ococh 3 ), 1 . 06 ( s , 3h , ch 3 ( c 6 )), 0 . 79 ( s , 3h , ch 3 ( c 3a )). elemental analysis : for c 24 h 36 o 6 . calcd : c , 68 . 55 ; h , 8 . 63 . found : c , 68 . 93 ; h , 8 . 73 . ## str32 ## a solution of a mixture of the enol acetates of example 16 ( 200 mg , 0 . 48 mmol ) in dichloromethane ( 25 ml ) and acetic acid ( 0 . 5 ml ) was cooled to - 78 ° c . in an acetone -- dry ice bath and treated with o 3 until a blue color persisted . the excess o 3 was removed by bubbling the solution with o 3 . after adding methyl sulfide ( 1 drop ), dichloromethane ( 75 ml ) was added and the solution was washed with water , 5 % aq . nahco 3 , and water again , the dichloromethane was drived over ngso 4 , filtered , and removed on a rotary evaporator to yield a yellow oil ( 158 mg ). the oil was combined with wilkinson &# 39 ; s catalyst ( 3 × 500 mg added in equal portions , initially and after 72 and 120 h ) in benzonitrile ( 25 ml ) and heated to 150 °- 180 ° c . for 144 h under nitrogen . after cooling , ethyl acetate ( 50 ml ) was added and the mixture was filtered . removal of the solvents from the filtrate yielded a brown sludge ( 2 . 0 g ) which was chromatographed ( silica gel eluted with hexane / ethyl acetate mixtures ) to give 25 . 4 mg ( 17 . 5 %) pure produce as a white solid , m . p . 61 °- 63 ° c . 1 h nmr ( cdcl 3 ): δ 4 . 61 ( t , j = 7 . 82 hz , 1h , choac ), 3 . 67 ( s , 3h , cooch 3 ), 2 . 56 ( s , 3h , ococh 3 ), 0 . 78 ( s , 3h , ch 3 ), 0 . 77 ( s , 3h , ch 3 ). elemental analysis : for c 20 h 32 o 4 . calcd : c , 71 . 39 ; h , 9 . 59 ; found : c , 70 . 96 ; h , 9 . 62 . ## str33 ## to a solution of the compound of example 17 ( 674 mg , 2 . 0 mmol ) in methanol ( 50 ml ) was added an aq . solution of naoh ( 0 . 38 g in 5 ml water ). after stirring at room temperature for 18 h , aq . hcl ( 6n , 20 ml ) was added , and the reaction mixture was puored into water ( 10 ml ). the produce precipiated and was recovered as a white solid by filtration . it was recrystallized from aq . methanol to give 418 mg ( 74 %) pure product as fine white crystals , m . p . 208 °- 210 ° c . ir ( film , nacl ): 3367 , 2926 , 1710 , 1352 , 1055 , 1032 cm - 1 . 1 h nmr ( cdcl 3 ); δ 3 . 62 ( t , j = 8 . 8 hz , 1h , choh ), 0 . 76 ( d , j = 6 . 1 hz , 3h , ch 3 ( c 6 )), 0 . 71 ( s , 3h , ch 3 ( c 3a )). elemental analysis : for c 17 h 28 o 3 . calcd : c , 72 . 82 ; h , 10 . 06 ; found : c , 73 . 00 ; h , 10 . 18 . ## str34 ## under halothane anesthesia , 1 day old albino rat pups are sacrificed by rapid decapitation and the hippocampi are dissected and dissociated with papain ( 1 mg / ml in oxygenated l - 15 media for 30 min at 35 ° c .) and mechanical trituration ( 18 ). cells are plated on collagen coated - culture dishes at a density of 300 , 000 cells / ml . the growth media consists of eagles minimal essential media ( mem ) supplemented with 5 % fetal calf serum , 5 % horse serum , 17 mm glucose , 0 . 4 mm glutamine , 50 u / ml penicillin and 50 μg / ml streptomycin . after three days in culture cells are treated with 10 μm cytosine arabinoside ( ara - c ) to suppress glial growth . cells are subsequently fed with fresh media once per week . for recording purposes the growth media is replaced with a solution containing ( in mm ): 140 nacl , 5 kcl , 2 cacl 2 , 1 mgcl 2 , 10 glucose , 10 hepes , 0 . 001 tetrodotoxin , ph = 7 . 3 . neurons are studied using patch clamp recording techniques ( 12 ) with pipettes containing ( in mm ): 145 cscl , 5 bapta , 5 nacl , 0 . 5 cacl 2 , 2 mgatp , 10 hepes , ph = 7 . 3 . this cscl intracellular solution sets the cl - 1 equilibrium potential ( e cl ) at 0 mv ( i . e . symmetric transmembrane concentrations of cl - ). this allows reliable recording of cl - 1 currents without concern for shifts in transmembrane cl - 1 concentration which can alter gaba responses during longer agonist exposures ( 1 ). in some tests examining effects on gaba iv curves , e cl is manipulated by replacing cscl with csmeso 4 . this shifts e cl to - 81 mv . by comparing changes in iv curves in the two intracellular solutions greater confidence is had that responses are mediated by cl - - selective ion channels . bapta and mgatp are included in the recording pipette to prevent problems with response rundown which sometimes occurs in whole - cell recording ( 44 ). the tests are conducted at room temperature ( 22 ° c .). table 1 , below , sets forth the results obtained in the potential of gaba currents with the four illustrative preferred tricyclic steroid compounds compared to the control compound , 3α - oh - dhp . table 1__________________________________________________________________________potentiation of gaba currents by tricyclic analogs concentrations tested concentration for directcompound 0 . 1 μm ( n ) 1 . 0 μm ( n ) cl . sup .- current__________________________________________________________________________ activation ## str35 ## 110 ± 5 ( 3 ) 195 ± 16 ( 9 ) & gt ; 1 μm ## str36 ## 95 ± 3 ( 6 ) 176 ± 9 ( 8 ) 237 ± 18 ( 7 ) 297 ± 7 ( 11 ) 127 ± 5 ( 6 ) 346 ± 2 ( 6 ) 433 ± 20 ( 8 ) 489 ± 19 ( 12 ) * * * * __________________________________________________________________________ * no response up to 10 μm . values represent mean ± sem ; results are expressed as percent of control response to 1 μm gaba . various other examples will be apparent to the person skilled in the art after reading the present disclosure without departing from the spirit and scope of the invention . it is intended that all such other examples be included within the scope of the appended claims . 1 . akaike n , inomata n , tokutomi n ( 1987 ). contribution of chloride shifts to the fade of γ - aminobutyric acid - gated currents in frog dorsal root ganglion cells . j . phyisol , ( london ) 391 , 219 - 234 . 2 . baker k , yang j , covey d f , clifford d b , zorumski c f ( 1988 ). alpha substituted thiobutyrolactones potentiate gaba currents in voltage clamped chick spinal cord neurons . neurosci . lett ., 87 , 133 - 138 . 3 . barker j l , harrison n l , lange , g d , owen d g ( 1987 ). potentiation of gamma - aminobutyric - acid - activated chloride conductance by a steroid anaesthetic in cultured rat spinal neurones . j . physiol . 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