Patent Application: US-12549908-A

Abstract:
a novel , improved method of synthesizing alkylated bile acid derivatives is provided . such derivatives include , but are not limited to the active , potent , and selective fxr receptor agonist such as 6 - ecdca and other ca , dca and cdca derivatives . the first step of the synthesis selectively oxidates cdca , cd , or dca related starting material . an efficient combined deprotonation , trapping , ethylation , deprotection and reduction system is used to produce the desired alkylated bile acid derivatives . this practical synthesis offers a simple and economical pathway suitable for a large - scale manufacturing of alkylated bile acid derivatives including , but not limited to , 6 - ecdca .

Description:
the first step of the novel synthesis route is a selective oxidation of the bile acid starting material by pyridinium chlorochromate ( pcc ), whose mechanism is depicted in fig7 as in the 7 - oxidation of cdca . the remaining hydroxyl groups in the resultant oxidated bile acid derivative are protected . one example of such alcohol protection can be accomplished by tetrahydropyranylation using conventional organic synthesis methods . the desired alkylation occurs at the alpha carbon adjacent to the carbonyl group using alkyl halide following conventional organic synthesis methods . examples of deprotonation agent include , but are not limited to lda and n - buli . the tetrahydropyranyl groups are removed using conventional organic synthesis method . one method to remove the tetrahydropyranyl groups uses pyridinium p - toluenesulfonate ( ppts ) following the method reported in miyashita et al . finally the carbonyl group is reduced alcohol . the reduction reaction may be achieved with nabh 4 using conventional organic chemistry . the yield for the inventive synthesis of 6 - ecdca is at least 9 %. more preferably , the yield of the inventive synthesis is at least 20 %, at least 30 %, at least 40 %, at least 50 %, or at least 75 %. “ large scale production ” means production of the desired alkylated bile acid derivative such as 6 - ecdca in amounts of about 0 . 25 kilograms or greater , preferably about 0 . 5 kilograms or greater , and more preferably about 1 . 0 kilogram or greater , e . g ., multi - kilograms . “ alkyl group ” means optionally substituted saturated or unsaturated c1 - c10 alkyl group . “ optionally substituted ” means no substitution or substitution selected from the group consisting of halogen , cn , hydroxyl , and amide . general procedures . organic reagents were purchased from commercial suppliers unless otherwise noted and were used without further purification . all solvents were analytical or reagent grade . all reactions were carried out in flame - dried glassware under argon or nitrogen . melting points were determined and reported automatically by an optoelectronic sensor in open capillary tubes and were uncorrected . 1 h nmr and 13 c nmr spectra were measured at 500 mhz and 125 mhz respectively , and using cdcl 3 or cd 3 od as the solvents and tetramethylsilane ( me 4 si ) as the internal standard . flash column chromatography was performed using sigma - aldrich silica gel 60 ( 200 - 400 mesh ), carried out under moderate pressure by using columns of an appropriate size packed and eluted with appropriate eluents . all reactions were monitored by tlc on precoated plates ( silica gel hlf ). tlc spots were visualized either by exposure to iodine vapors or by irradiation with uv light . organic solvents were removed in vacuum by rotary evaporator . elemental analyses were performed by desert analytics , tucson , ariz . synthesis of 5β - cholanicacid - 3α - ol - 7 - one ( compound 1 ). to a suspension solution of chenodeoxycholic acid ( cdca ) ( 1 . 0 g , 0 . 0025 mol ) and silica gel ( 4 g , 200 - 400 mesh , aldrich ) in anhydrous chcl 3 ( 2 ml ), ch 2 cl 2 ( 25 ml ) was added pyridinium chlorochromate ( 0 . 81 g , 0 . 038 mol ) in portions and the reaction mixture was stirred at room temperature for 15 min . the mixture was filtered and the filtrate was washed with water ( 20 ml ) and brine ( 20 ml ). the organic layer was dried over na 2 so 4 and concentrated . the crude oil was purified by flash column chromatography ( ch 2 cl 2 / meoh 95 : 5 ) to produce 1 as a solid ( 0 . 76 g , in 78 % yield ), mp : 201 . 1 ° c . ( lit . 1 mp : 203 - 204 ° c .). 1 h nmr ( cd 3 od ) δ 3 . 50 ( m , 1h ), 2 . 94 ( m , 1h ), 2 . 52 ( t , 1h ), 2 . 30 ( m , 2h ), 2 . 19 ( m , 6h ), 1 . 70 ( m , 2h ), 1 . 43 ( m , 4h ), 1 . 31 ( m , 6h ), 1 . 19 ( s , 3h ), 1 . 12 ( m , 4h ), 0 . 92 ( d , 3h ), 0 . 67 ( s , 3h ). 13 c nmr ( cd 3 od ) δ 213 . 7 , 176 . 8 , 70 . 1 , 54 . 8 , 49 . 2 , 48 . 9 , 47 . 7 , 46 . 0 , 44 . 9 , 43 . 0 , 42 . 4 , 38 . 9 , 36 . 8 , 35 . 1 , 34 . 9 , 33 . 7 , 31 . 0 , 30 . 6 , 29 . 2 , 27 . 8 , 24 . 3 , 22 . 0 , 21 . 4 , 17 . 3 , 10 . 5 . anal . calcd for c 24 h 38 o 4 : c , 73 . 81 ; h , 9 . 81 . found : c , 73 . 50 ; h , 9 . 63 . synthesizing 3α - tetrahydropyranyloxy - 7 - keto - 5β - cholan - 24 - oic acid ( compound 2 ). to a solution of 5β - cholanicacid - 3α - ol - 7 - one ( 1 ) ( 0 . 5 g , 0 . 0013 mol ) in chcl 3 / cl 2 ch 2 / ether ( 1 : 1 : 2 , 16 ml ), p - toluensulfonic acid ( 0 . 06 g , 0 . 0003 mol ), and 3 , 4 - dihydro - 2h - pyrane ( 0 . 41 g , 0 . 005 mol ) were added . the reaction mixture was stirred at room temperature for 60 min and water ( 10 ml ) was added . the reaction mixture was extracted with etoac ( 3 × 30 ml ) and washed with saturated nahco 3 and brine . after concentration to remove solvent , the crude oil was purified by flash column chromatography ( ch 2 cl 2 / ether 1 : 2 ) to produce 2 as a white solid ( 0 . 47 g . in 76 % yield ), mp : 160 . 8 ° c . ( lit . 1 mp : 157 - 159 ° c .). 1 h nmr ( cdcl 3 ) δ 4 . 73 ( d , 1h ), 3 . 86 ( m , 1h ), 3 . 59 ( m , 1h ), 3 . 46 ( m , 1h ), 2 . 82 ( m , 1h ), 1 . 17 ( s , 3h ), 0 . 92 ( d , 3h ), 0 . 63 ( s , 3h ). 13 c nmr ( cdcl 3 ) δ 212 . 3 , 179 . 8 , 96 . 4 , 62 . 8 , 62 . 1 , 19 . 8 , 18 . 1 , 11 . 4 . anal . calcd for c 29 h 46 o 5 : c , 73 . 38 ; h , 9 . 77 . found : c , 73 . 30 ; h , 9 . 76 . synthesizing 3α - hydroxy - 6α - ethyl - 7 - keto - 5β - cholan - 24 - oic acid ( compound 3 ). to a solution of 3α - tetrahydropyranyloxy - 7 - keto - 5β - cholan - 24 - oic acid ( 2 ) ( 0 . 3 g , 0 . 00063 mol ) and hmpa ( 0 . 7 g , 0 . 004 mol ) in dry thf ( 20 ml ), lda ( 1 . 8 m in tetrahydrofuran / heptane / ethylbenzene ) ( 2 . 0 ml , 0 . 0036 mol ) was added dropwise at − 78 ° c . the reaction mixture was stirred for 30 min . ethyl iodide ( 2 . 0 g , 0 . 013 mol ) was slowly added and the reaction mixture was allowed to warm overnight to room temperature . after concentration to remove solvent , water and ether was added . the reaction mixture was acidified with 10 % hcl , extracted with etoac ( 5 × 20 ml ), washed with brine , dried over na 2 so 4 , and concentrated to give yellow oil . after a short column by ch 2 cl 2 / ether 1 : 2 , the crude semi - solid was dissolved in chloroform ( 5 ml ) and ppts ( 0 . 015 g , 0 . 00006 mol ) was added . the reaction mixture was stirred at 55 ° c . for 7 h . the solvent was evaporated in vacuo , the crude semi - solid ( 0 . 08 g ) was obtained through a quick column and was passed to the next step without further purification . synthesizing 3α , 7α - dihydroxy - 6α - ethyl - 5β - cholan - 24 - oic acid ( 6 - ecdca ) ( compound 4 : desired end product ). to a solution of 3α - hydroxy - 6α - ethyl - 7 - keto - 5β - cholan - 24 - oic acid ( 3 ). ( 0 . 05 g , 0 . 00012 mol ) in dry meoh ( 5 ml ), nabh 4 ( 0 . 03 g , 0 . 00084 mol ) was added in a small portion at 0 ° c . the reaction mixture was stirred at room temperature for 3 hr . h 2 o ( 10 ml ) was slowly added . the reaction mixture was partially concentrated to remove solvent and extracted with etoac ( 3 × 20 ml ). the combined organic extracts were washed with brine , dried over na 2 so 4 , and concentrated to give a solid . the crude product was purified by flash column chromatography ( ch 2 cl 2 / ether 1 : 2 ) to give the desired product 0 . 04 g in 80 % yield . 1 h nmr ( cdcl 3 ) δ 3 . 65 ( brs , 1h ), 3 . 31 ( m , 1h ), 2 . 33 ( m , 1h ), 2 . 20 ( m , 1 h ), 0 . 97 ( d , 3h ), 0 . 89 ( m , 6h ), 0 . 69 ( s , 3h ). 13 c nmr ( cdcl 3 ) δ 177 . 0 , 71 . 8 , 69 . 7 , 55 . 9 , 50 . 2 , 45 . 5 , 42 . 3 , 41 . 7 , 40 . 1 , 39 . 6 , 35 . 3 , 35 . 2 , 33 . 1 , 33 . 0 , 31 . 0 , 29 . 8 , 27 . 8 , 23 . 1 , 22 . 3 , 22 . 1 , 20 . 5 , 17 . 3 , 11 . 0 , 10 . 6 . anal . calcd for c 26 h 44 o 4 . ¼h 2 o : c , 73 . 44 ; h , 10 . 43 . found : c , 73 . 24 ; h , 10 . 66 . synthesis of 6 - modified cdca derivative having the general formula cdca - i from cdca starting material having the general formula cdca - i . to a suspension solution of cdca - ii ( 0 . 0025 mol ) and silica gel ( 4 g , 200 - 400 mesh , aldrich ) in anhydrous chcl 3 ( 2 ml ), ch 2 cl 2 ( 25 ml ) is added pyridinium chlorochromate ( 0 . 81 g , 0 . 038 mol ) in portions and the reaction mixture is stirred at room temperature for 15 min . the mixture is filtered and the filtrate is washed with water ( 20 ml ) and brine ( 20 ml ). the organic layer is dried over na 2 so 4 and concentrated to a crude product . the crude product is purified by flash column chromatography to produce a product having the general formula cdca - iii . to a solution of cdca - iii ( 0 . 0013 mol ) in chcl 3 / cl 2 ch 2 / ether ( 1 : 1 : 2 , 16 ml ), p - toluensulfonic acid ( 0 . 06 g , 0 . 0003 mol ), and 3 , 4 - dihydro - 2h - pyrane ( 0 . 41 g , 0 . 005 mol ) are added . the reaction mixture is stirred at room temperature for 60 min , and water ( 10 ml ) is added . the reaction mixture is extracted with etoac ( 3 × 30 ml ) and washed with saturated nahco 3 and brine . after concentration to remove solvent , the crude product is purified by flash column chromatography to produce a compound having the general formula cdca - iv . to a solution of cdca - iv ( 0 . 00063 mol ) and hmpa ( 0 . 7 g , 0 . 004 mol ) in dry thf ( 20 ml ), lda ( 1 . 8 m in tetrahydrofuran / heptane / ethylbenzene ) ( 2 . 0 ml , 0 . 0036 mol ) is added dropwise at − 78 ° c . the reaction mixture is stirred for 30 min . the corresponding alkyl halide r 4 — x ( 0 . 013 mol ) is slowly added and the reaction mixture is allowed to warm overnight to room temperature . after concentration to remove solvent , water and ether is added . the reaction mixture is acidified with 10 % hcl extracted with etoac ( 5 × 20 ml ), washed with brine , dried over na 2 so 4 , and concentrated to give a crude product . after a short column , the crude product is dissolved in chloroform ( 5 ml ), and ppts ( 0 . 015 g , 0 . 00006 mol ) is added . the reaction mixture is stirred at 55 ° c . for 7 hours . the solvent is evaporated in vacuo . the crude product having the general formula of cdca - v is obtained through a quick column and passed to the next step without further purification . to a solution of cdca - v ( 0 . 00012 mol ) in dry meoh ( 5 ml ), nabh 4 ( 0 . 03 g , 0 . 00084 mol ) is added in a small portion at 0 ° c . the reaction mixture is stirred at room temperature for 3 hours as water ( 10 ml ) is slowly added . the reaction mixture is partially concentrated to remove solvent and extracted with etoac ( 3 × 20 ml ). the combined organic extracts are washed with brine , dried over na 2 so 4 , and concentrated to give a crude product . the crude product is purified by flash column chromatography to give the desired alkylated product having the general formula of cdca - i . synthesis of 6 - modified ca derivative having the general formula ca - i from ca starting material having the general formula ca - ii . to a suspension solution of ca - ii ( 0 . 0025 mol ) and silica gel ( 4 g , 200 - 400 mesh , aldrich ) in anhydrous chcl 3 ( 2 ml ), ch 2 cl 2 ( 25 ml ) is added pyridinium chlorochromate ( 0 . 81 g , 0 . 038 mol ) in portions and the reaction mixture is stirred at room temperature for 15 min . the mixture is filtered and the filtrate is washed with water ( 20 ml ) and brine ( 20 ml ). the organic layer is dried over na 2 so 4 and concentrated . the crude product is purified by flash column chromatography to produce a product having the general formula of ca - iii . to a solution of ca - iii ( 0 . 0013 mol ) in chcl 3 / cl 2 ch 2 / ether ( 1 : 1 : 2 , 16 ml ), p - toluensulfonic acid ( 0 . 06 g , 0 . 0003 mol ), and 3 , 4 - dihydro - 2h - pyrane ( 0 . 41 g , 0 . 005 mol ) are added . the reaction mixture is stirred at room temperature for 60 min and water ( 10 ml ) is added . the reaction mixture is extracted with etoac ( 3 × 30 ml ) and washed with saturated nahco 3 and brine . after concentration to remove solvent , the crude product is purified by flash column chromatography to produce compound having the general formula of ca - iv . to a solution of ca - iv ( 0 . 00063 mol ) and hmpa ( 0 . 7 g , 0 . 004 mol ) in dry thf ( 20 ml ), lda ( 1 . 8 m in tetrahydrofuran / heptane / ethylbenzene ) ( 2 . 0 ml , 0 . 0036 mol ) is added dropwise at − 78 ° c . the reaction mixture is stirred for 30 min . the corresponding alkyl halide r 4 — x ( 0 . 013 mol ) is slowly added and the reaction mixture is allowed to warm overnight to room temperature . after concentration to remove solvent , water and ether is added . the reaction mixture is acidified with 10 % hcl , extracted with etoac ( 5 × 20 ml ), washed with brine , dried over na 2 so 4 , and concentrated to give a crude product . after a short column , the crude product is dissolved in chloroform ( 5 ml ) and ppts ( 0 . 015 g , 0 . 00006 mol ) is added . the reaction mixture is stirred at 55 ° c . for 7 h . the solvent is evaporated in vacuo . the crude product having the general formula ca - v is obtained through a quick column and passed to the next step without further purification . to a solution of ca - v ( 0 . 00012 mol ) in dry meoh ( 5 ml ), nabh 4 ( 0 . 03 g , 0 . 00084 mol ) is added in a small portion at 0 ° c . the reaction mixture is stirred at room temperature for 3 hr . h 2 o ( 10 ml ) is slowly added . the reaction mixture is partially concentrated to remove solvent and extracted with etoac ( 3 × 20 ml ). the combined organic extracts are washed with brine , dried over na 2 so 4 , and concentrated to give a crude product . the crude product is purified by flash column chromatography to give a desired 6 - alkylated ca derivative having the general formula of ca - i . synthesis of 11 - modified dca derivative having the general formula of dca - i from dca starting material having the general formula of dca - i . to a suspension solution of a compound having the general formula of dca - ii ( 0 . 0025 mol ) and silica gel ( 4 g , 200 - 400 mesh , aldrich ) in anhydrous chcl 3 ( 2 ml ) and ch 2 cl 2 ( 25 ml ) is added pyridinium chlorochromate ( 0 . 81 g , 0 . 038 mol ) in portions . the reaction mixture is stirred at room temperature for 15 min . the mixture is filtered and the filtrate is washed with water ( 20 ml ) and brine ( 20 ml ). the organic layer is dried over na 2 so 4 and concentrated . the crude product is purified by flash column chromatography to produce a compound having the general formula of dca - iii , to a solution of dca - iii ( 0 . 0013 mol ) in chcl 3 / cl 2 ch 2 / ether ( 1 : 1 : 2 , 16 ml ), p - toluensulfonic acid ( 0 . 06 g , 0 . 0003 mol ), and 3 , 4 - dihydro - 2h - pyrane ( 0 . 41 g , 0 . 005 mol ) are added . the reaction mixture is stirred at room temperature for 60 min and water ( 10 ml ) is added . the reaction mixture is extracted with etoac ( 3 × 30 ml ) and washed with saturated nahco 3 and brine . after concentration to remove solvent , the crude product is purified by flash column chromatography to produce a compound having the general formula of dca - iv . to a solution of dca - iv ( 0 . 00063 mol ) and hmpa ( 0 . 7 g , 0 . 004 mol ) in dry thf ( 20 ml ), lda ( 1 . 8 m in tetrahydrofuran / heptane / ethylbenzene ) ( 2 . 0 ml , 0 . 0036 mol ) is added dropwise at − 78 ° c . the reaction mixture is stirred for 30 min . the corresponding alkyl halide r 4 — x ( 0 . 013 mol ) is slowly added and the reaction mixture is allowed to warm overnight to room temperature . after concentration to remove solvent , water and ether is added . the reaction mixture is acidified with 10 % hcl , extracted with etoac ( 5 × 20 ml ), washed with brine , dried over na 2 so 4 , and concentrated to give a crude product . after a short column , the crude product is dissolved in chloroform ( 5 ml ) and ppts ( 0 . 015 g , 0 . 00006 mol ) is added . the reaction mixture is stirred at 55 ° c . for 7 h . the solvent is evaporated in vacuo . the obtained crude compound having the general formula of dca - v is obtained through a quick column and is passed to the next step without further purification . to a solution of dca - v ( 0 . 00012 mol ) in dry meoh ( 5 ml ), nabh 4 ( 0 . 03 g , 0 . 00084 mol ) is added in a small portion at 0 ° c . the reaction mixture is stirred at room temperature for 3 hr . h 2 o ( 10 ml ) is slowly added . the reaction mixture is partially concentrated to remove solvent and then extracted with etoac ( 3 × 20 ml ). the combined organic extracts are washed with brine , dried over na 2 so 4 , and concentrated to give a crude product . the crude product is purified by flash column chromatography to give a desired 11 - alkylated dca derivative having the general formula of dca - i . while the description above refers to particular embodiments of the present invention , it will be understood that many modifications may be made without departing from the spirit thereof . the accompanying claims are intended to cover such modifications as would fall within the true scope and spirit of the present invention . the presently disclosed embodiments are therefore to be considered in all respects as illustrative and not restrictive , the scope of the invention being indicated by the appended claims , rather than the foregoing description , and all changes that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein . additionally , all publications are incorporated by reference in their entirety . 1 . forman , b . m ., e . goode , j . chen , a . e . oro , d . j . bradley , t . perlman , d . j . noonan , l . t . burka , t . mcmorris , w . w . lamph , r . m . evans , and c . w . weinberger . 1995 . identification of a nuclear receptor that is activator by farnesol metabolites , cell 81 , 687 - 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