Patent Application: US-49346104-A

Abstract:
2 - substituted - 5 ′- o - adenosine derivatives having at position 2 a radical r1 selected from the group consisting of h ; halogen ; o - hydrocarbyl ; s - hydrocarbyl ; nr3r4 ; and hydrocarbyl optionally substituted by halogen , cn , scn , no2 , or3 , sr3 or nr3r4 ; wherein r3 and r4 are each independently h or hydrocarbyl or r3 and r4 together with the nitrogen atom to which they are attached form a saturated or unsaturated heterocyclic ring optionally containing 1 - 2 further heteroatoms selected from oxygen , nitrogen and sulfur , and pharmaceutically acceptable salts or diastereoisomers thereof or a mixture of diastereoisomers , are useful for treatment of type 2 diabetes .

Description:
the present invention provides new 2 - substituted - 5 ′- o -( 1 - boranotriphosphate )- adenosine derivatives of the formula shown hereinbefore . as used herein , the term “ halo ” includes fluoro , chloro , bromo , and iodo , and is preferably chloro or bromo , most preferably chloro . the term “ hydrocarbyl ” in any of the definitions of the different radicals r 1 - r 4 includes any saturated or unsaturated including aromatic , straight , branched or cyclic including polycyclic , radical containing carbon and hydrogen such as , but not being limited to , c 1 - c 8 alkyl , c 2 - c 8 alkenyl , c 2 - c 8 alkynyl , c 3 - c 10 cycloalkyl , aryl and ar ( c 0 - c 8 ) alkyl . the term “ c 1 - c 8 alkyl ” typically means a straight or branched hydrocarbon radical having 1 - 8 carbon atoms and includes , for example , methyl , ethyl , n - propyl , isopropyl , n - butyl , sec - butyl , isobutyl , tert - butyl , n - pentyl , 2 , 2 - dimethylpropyl , n - hexyl , n - heptyl , n - octyl , and the like . preferred are c 1 - c 6 alkyl groups , most preferably methyl . the terms “ c 2 - c 8 alkenyl ” and “ c 2 - c 8 alkynyl ” typically mean straight and branched hydrocarbon radicals having 2 - 8 carbon atoms and 1 double or triple bond , respectively , and include ethenyl , 3 - buten - 1 - yl , 2 - ethenylbutyl , 3 - octen - 1 - yl , and the like , and propynyl , 2 - butyn - 1 - yl , 3 - pentyn - 1 - yl , and the like . c 2 - c 6 alkenyl radicals are preferred . the term “ c 3 - c 10 cycloalkyl ” means a cyclic or bicyclic hydrocarbyl group such as cyclopropyl , cyclobutyl , cyclopentyl , cyclohexyl , adamantyl , bicyclo [ 3 . 2 . 1 ] octyl , bicyclo [ 2 . 2 . 1 ] heptyl , and the like . the term “ aryl ” denotes a carbocyclic aromatic radical such as phenyl and naphthyl and the term “ ar ( c 1 - c 8 ) alkyl ” denotes an arylalkyl radical such as benzyl and phenetyl . when the radical r 1 is a o - hydrocarbyl or s - hydrocarbyl radical or is hydrocarbyl substituted by a o - hydrocarbyl or s - hydrocarbyl radical , the hydrocarbyl is preferably a c 1 - c 6 alkyl , most preferably methyl , or an aryl , most preferably phenyl , or an aralkyl , most preferably benzyl , radical . in a most preferred embodiment , r 1 is sch 3 . in the group nr 3 r 4 , r 3 and r 4 are each h or hydrocarbyl as defined above or form together with the n atom to which they are attached a saturated or unsaturated , preferably a 5 - or 6 - membered , heterocyclic ring , optionally containing 1 or 2 further heteroatoms selected from nitrogen , oxygen , and sulfur . such rings may be substituted , for example with one or two c 1 - c 6 alkyl groups . examples of radicals nr 3 r 4 include , without being limited to , amino , dimethylamino , diethylamino , ethylmethylamino , phenylmethylamino , pyrrolidino , piperidino , tetrahydropyridino , piperazino , morpholino , thiazolino , and the like . preferred compounds according to the invention are those wherein r 2 is h and r 1 is cl or , more preferably , 2 - methylthio . the invention encompasses the compounds themselves , a diastereoisomer thereof or a mixture of diastereoisomers as well as pharmaceutically acceptable salts thereof such as , but not limited to , compounds wherein m + is na + , k + , nh 4 + or the cation of an amine , particularly of a tertiary amines e . g . n ( r ) 3 h + , wherein r is preferably alkyl . the compounds of the invention are prepared , for example , according to the synthesis outlined in scheme a hereinafter and exemplified in the examples herein . other compounds with different substituents are obtained by the same methods starting from suitable compounds or introducing the desired groups during the synthesis by standard methods well - known in the art . the compounds of the invention are obtained as diastereoisomers which can be separated using a semipreparative reverse - phase lichro cart 250 - 10 column and isocratic elution [ 100 mm triethylammonium acetate ( teaa ), ph 7 ( a ): meoh ( b ), 84 : 16 ] with flow rate of 6 ml / min . fractions containing the same isomer ( similar retention time ) are freeze - dried . the isomer with the shorter retention time is herein designated isomer a and the other , isomer b . isomers a of the compounds , and particularly isomer a of the compound herein identified as 2 - sme - atpαb , constitute preferred embodiments of the invention . in one preferred embodiment , the present invention relates to a diastereoisomer a of a compound of the invention , this diastereoisomer a being characterized by being the isomer with the shorter retention time ( rt ) when separated from a mixture of diastereoisomers using a semipreparative reverse - phase lichro cart 250 - 10 column and isocratic elution [ 100 mm triethylammonium acetate ( teaa ), ph 7 ( a ): meoh ( b ), 84 : 16 ] with flow rate of 6 ml / min . the 2 - substituted - 5 ′- o -( 1 - boranotriphosphate )- adenosine derivatives of the invention are potent insulin secretagogues that target beta - cell p2y - receptors , enhancing insulin secretion up to 900 %, at the nm concentration range , under slightly stimulatory glucose concentration . at these concentrations , the compounds of the invention have no or only mild vascular side effects , in contrary to the most close compounds of the prior art , the p2y - r ligands , 2 - thioether - 5 ′- o -( 1 - thiotriphosphate ) adenosine derivatives described by us previously ( fischer et al ., 1999 ), which are also potent insulin secretagogues but their poor selectivity for the insulin - secreting cell , illustrated by their ability to induce vascular effects at insulin secreting concentrations , made these derivatives a priori not suitable for drug development as potential antidiabetics , since vascular events are the major pathophysiological complications of the disease . the compounds of the invention are p2y receptor ligands with potent insulin releasing action as well as with glucose - dependent amplifying effect on insulin secretion , which limit the risk of hypoglycemia and have also limited vascular side effects , and are therefore suitable for the treatment of type 2 diabetes . thus , the present invention includes within its scope pharmaceutical compositions comprising , as an active ingredient , an effective amount of at least one 2 - substituted - 5 ′- o -( 1 - boranotriphosphate )- adenosine derivative of the invention , or a pharmaceutically acceptable salt thereof , or a diastereoisomer or a mixture of diastereoisomers thereof , together with a pharmaceutically acceptable carrier or diluent . pharmaceutical compositions containing a compound of the present invention may be prepared by conventional techniques , e . g . as described in remington : the science and practice of pharmacy , 19th ed ., 1995 . the compositions may appear in conventional forms , for example capsules , tablets , solutions or suspensions . the route of administration may be any route which effectively transports the active compound to the appropriate or desired site of action , the oral route being preferred . if a solid carrier is used for oral administration , the preparation may be tabletted , placed in a hard gelatin capsule in powder or pellet form or it can be in the form of a lozenge . if a liquid carrier is used , the preparation may be in the form of a syrup , emulsion or soft gelatin capsule . tablets , dragees , or capsules having talc and / or a carbohydrate carrier or binder or the like are particularly suitable for oral application . preferable carriers for tablets , dragees , or capsules include lactose , corn starch , and / or potato starch . the invention further provides a method for treatment of a type 2 diabetes patient , particularly for enhancing insulin secretion in said patient , which comprises administration of an effective amount of a 2 - substituted - 5 ′- o -( 1 - boranotriphosphate )- adenosine derivative of the invention . the dosage to be administered is in the range of 0 . 5 - 25 mg , preferably 1 - 20 mg , most preferably 1 - 10 mg , per day . the invention will now be illustrated by the following non - limitative examples . all air - and moisture - sensitive reactions were carried out in flame - dried , nitrogen flushed , two - neck flasks sealed with rubber septa , and the reagents were introduced with a syringe . progress of reactions was monitored by tlc on precoated merck silica gel plates ( 60f - 254 ). column chromatography was performed with merck silica gel 60 ( 230 - 400 mesh ). compounds were characterized by nuclear magnetic resonance ( nmr ) using brucker dpx - 300 , dmx - 600 , or ac - 200 spectrometers . 1 h nmr spectra were measured in d 2 o , and the chemical shifts are reported in ppm relative to hod ( 4 . 78 ppm ) as an internal standard . nucleotides were characterized also by 31 p nmr in d 2 o , using 85 % h 3 po 4 as an external reference . all final products were characterized on an autospec - e fision vg high - resolution mass spectrometer by chemical ionization . nucleotides were desorbed from a glycerol matrix under fab ( fast atom bombardment ) conditions in low and high resolution . primary purification of the nucleotides was achieved on an lc ( isco ua - 6 ) system using a sephadex deae - a25 column , which was swelled in 1 m nahco 3 in the cold for 1 d . final purification of the nucleotides and separation of the diastereoisomer pair was achieved on a hplc ( merck - hitachi ) system using a semipreparative reverse - phase ( lichrospher 60 , rp - select - b ) column . conditions for lc and hplc separation are described below . 2 - methylthio - adenosine was synthesized from 2 - sh - adenosine as described before ( methods in enzymology , 1992 , 215 : 137 - 142 ). 2 - sh - adenosine was obtained from adenosine in three steps according to the procedure previously reported ( j . med . chem . 1973 , 16 : 1381 - 1388 ; chem . pharm . bull . 1977 , 25 : 1959 - 1969 ). 2 - chloro - adenosine was prepared in four steps from guanosine ( synthesis 1982 , 670 - 672 ) through 2 , 6 - cl - 9β -( 2 ′, 3 ′, 5 ′- tri - o - acetyl )- d - ribofuranosylpurine ( can . j . chem . 1981 , 59 : 2601 - 2606 ), by treatment of the latter with nh 3 in etoh in a sealed ampule at 100 ° c . for 24 h . [ heading - 0061 ] 1 . 3 typical procedure for the preparation of 2 ′, 3 ′- o - methoxymethylidene adenosine derivatives ( compounds 2 in scheme a ) p - tsoh ( 2 mmol , 2 eq ) was added to a dry adenosine derivative ( 1 mmol , 1 eq ) ( compound 1 ) in a two - neck flask under n 2 , followed by addition of dry dmf ( 4 ml ). then , trimethylorthoformate ( 50 eq ) was added and the resulting solution was stirred at room temperature for 1 day . the mixture was cooled to 0 ° c . and dowex mwa - 1 ( weakly basic anion exchanger , 6 eq ) was added . stirring continued at room temperature for additional 3 h . the dowex resin was filtered out in vacuo ; the filtrate was concentrated under reduced pressure and coevaporated several times with meoh to remove residual dmf . the residue was dissolved in chcl 3 and extracted with saturated nahco 3 . the organic phase was dried with na 2 so 4 and evaporated to give pure protected adenosine derivative 2 . 1 . 3 . a ) 2 ′, 3 ′- o - methoxymethylidene adenosine ( compound 2 wherein r is h ) was obtained from adenosine and trimethylorthoformate in 80 % yield . 1 h nmr ( dmso - d 6 , 300 mhz ): δ 8 . 42 , 8 . 38 ( 2s , h - 8 , 1h ), 8 . 21 , 8 . 20 ( 2s , h - 2 , 1h ), 7 . 42 ( br . s , nh 2 , 2h ), 6 . 30 , 6 . 20 ( 2d , j = 3 hz and j = 2 . 8 hz , h - 1 ′, 1h ), 6 . 22 , 6 . 11 ( 2s , ch — och 3 , 1h ), 5 . 53 , 5 . 47 ( 2dd , j = 2 . 8 , 6 hz and j = 3 , 7 hz , h - 2 ′, 1h ), 5 . 26 , 5 . 20 ( 2t , j = 5 . 5 hz , oh - 5 ′, 1h ), 5 . 11 , 5 . 03 ( 2dd , j = 2 . 8 , 6 hz and j = 3 , 7 hz , h - 3 ′, 1h ), 4 . 31 , 4 . 24 ( 2dt , j = 3 , 5 hz , h - 4 ′, 1h ), 3 . 51 - 3 . 68 ( m , h - 5 ′, 2h ), 3 . 40 ( s , o — ch 3 , 3h ) ppm . 13 c nmr ( dmso - d 6 , 300 mhz ): δ 156 . 16 ( c - 6 ), 152 . 73 ( ch - 2 ), 148 . 88 , 148 . 84 ( c - 4 ), 139 . 79 , 139 . 69 ( ch - 8 ), 119 . 04 , 119 . 00 ( c - 5 ), 118 . 44 , 116 . 93 ( ch — ome ), 89 . 40 , 88 . 76 ( ch - 1 ′), 86 . 93 , 85 . 90 ( ch - 2 ′), 83 . 47 , 82 . 45 ( ch - 3 ′), 81 . 08 , 80 . 72 ( ch - 4 ′), 61 . 58 , 61 . 32 ( ch 2 - 5 ′), 51 . 87 , 50 . 40 ( och 3 ) ppm . ms ci / nh 3 m / z : 310 ( mh + ). 1 . 3 . b ) 2 - methylthio -( 2 ′, 3 ′- o - methoxymethylidene ) adenosine ( compound 2 wherein r is sch 3 ) was obtained from 2 - methylthioadenosine and trimethylorthoformate in 75 % yield . 1 h nmr ( cdcl 3 , 300 mhz ): δ 7 . 96 , 7 . 93 ( 2s , h - 8 , 1h ), 6 . 17 , 5 . 91 ( 2d , j = 3 . 6 hz , j = 3 . 9 hz , h - 1 ′, 1h ), 6 . 05 , 5 . 97 ( 2s , ch — ome , 1h ), 5 . 47 , 5 . 39 ( 2dd , j = 3 . 9 , 6 hz , h - 2 ′ and j = 3 . 6 , 7 hz , h - 2 ′, 1h ), 5 . 18 - 5 . 22 ( m , h - 3 ′, 1h ), 4 . 55 , 4 . 48 ( 2 “ q ”, j = 2 . 5 hz , j = 1 . 8 hz , h - 4 ′, 1h ), 3 . 18 - 4 . 03 ( m , h - 5 ′, 2h ), 3 . 46 , 3 . 35 ( 2s , och 3 , 3h ), 2 . 57 , 2 . 56 ( 2s , s — ch 3 , 3h ) ppm . 13 c nmr ( cdcl 3 , 300 mhz ): δ 165 . 86 ( c - 2 ), 154 . 31 , 154 . 24 ( c - 6 ), 149 . 52 ( c - 4 ), 139 . 35 ( c - 8 ), 119 . 48 , 117 . 74 ( ch — ome ), 117 . 41 , 117 . 33 ( c - 5 ), 92 . 33 , 92 . 03 ( ch - 1 ′), 87 . 41 , 86 . 10 ( ch - 2 ′), 83 . 87 , 82 . 72 ( ch - 3 ′), 80 . 97 , 80 . 73 ( ch - 4 ′), 62 . 79 , 62 . 72 ( ch 2 - 5 ′), 52 . 95 , 51 . 72 ( o — ch 3 ), 14 . 47 , 14 . 41 ( s — ch 3 ) ppm . fab ( positive mode ) m / z : 356 . 035 ( mh + ). hr fab ( positive mode ) m / z : calcd for c 13 h 17 n 5 o 5 s ( mh + ) 356 . 1028 , found 356 . 1038 . 1 . 3 . c ) 2 - chloro -( 2 ′, 3 ′- o - methoxymethylidene ) adenosine ( compound 2 wherein r is cl ) was obtained from 2 - chloro - adenosine and trimethylorthoformate in 81 % yield . 1 h nmr ( cdcl 3 , 300 mhz ): δ 7 . 70 ( h - 8 , 1h ), 6 . 69 ( s , nh 2 , 2h ), 6 . 16 , 5 . 85 ( 2d , 3 = 3 . 6 hz , 3 = 3 . 9 hz , h - 1 ′, 1h ), 6 . 03 , 5 . 95 ( 2s , ch — ome , 1h ), 5 . 35 - 5 . 17 ( 2m , h - 2 ′ and h - 3 ′, 2h ), 4 . 53 , 4 . 49 ( 2 “ br s ”, h - 4 ′, 1h ), 4 . 02 - 3 . 8 ( m , h - 5 ′, 2h ), 3 . 47 , 3 . 32 ( 2s , och 3 , 3h ) ppm . 13 c nmr ( cdcl 3 , 300 mhz ): δ 163 . 54 ( c - 2 ), 156 . 45 ( c - 6 ), 154 . 15 ( c - 4 ), 130 . 86 ( c - 8 ), 119 . 54 , 117 . 67 ( ch — ome ), 117 . 85 ( c - 5 ), 92 . 71 , 92 . 44 ( ch - 1 ′), 87 . 55 , 85 . 94 ( ch - 2 ′), 83 . 98 , 82 . 68 ( ch - 3 ′), 80 . 95 , 80 . 73 ( ch - 4 ′), 62 . 97 , 62 . 88 ( ch 2 - 5 ′), 53 . 04 , 51 . 72 ( o — ch 3 ) ppm . ms ci / nh 3 m / z : 344 ( mh + ). hrms m / z : calcd for c 12 h 14 cln 5 o 5 343 . 0683 , found 343 . 0671 . general procedure for the preparation of derivatives of adenosine - 5 ′- o -( 1 - boranotriphosphate ) ( according to scheme a ) protected nucleoside 2 ( 0 . 5 mmol ) was dissolved in dry chcl 3 ( 7 ml ) in a flame - dried , two - neck flask under n 2 . ( ipr ) 2 net ( 0 . 11 ml , 1 . 3 eq ) was added at room temperature and the solution was stirred for 30 min . the mixture was cooled to 0 ° c . and [( ipr ) 2 n ] 2 pcl ( 148 mg , 1 . 1 eq ), dissolved in chcl 3 ( 2 ml ), was slowly added with a syringe ( step a ), to give derivative 3 . the resulting solution of derivative 3 was stirred at 0 ° c . for 2 h followed by the addition of a 1 m solution of h 2 p 2 o 7 − 2 ( + hnbu 3 ) 2 in dmf ( 0 . 75 ml , 1 . 5 eq ) ( step b ), to produce compound 4 . this solution was kept at room temperature for additional 4 h and then cooled to 0 ° c . a 2 m solution of bh 3 — sme 2 complex in thf ( 2 . 52 ml , 10 eq ) was added ( step c ). after 15 min of stirring at room temperature , deionized water ( 8 ml ) was added and the resulting mixture was stirred for 1 h ( step d ) and then freeze - dried . compound 6 , obtained as a semisolid , was dissolved in water and extracted with chcl 3 . the aqueous phase was freeze - dried and the resulting residue was applied on an activated sephadex deae - a25 column ( 0 - 0 . 7 m nh 4 hco 3 , total volume & gt ; 2000 ml ). the relevant fractions were collected and freeze - dried ; excess nh 4 hco 3 was removed by repeated freeze - drying with deionized water to yield compound 6 as the tris ammonium salt . the methoxymethylidene protecting group was removed by acidic hydrolysis ( 10 % hcl solution was added till ph 2 . 3 was obtained ). after 3 h at room temperature , the ph was rapidly raised to 9 by the addition of nh 4 oh solution ( ph 11 ) and the solution was kept at room temperature for 40 min ( step e ). the desired adenosine - 5 ′- o -( 1 - boranotriphosphate ) derivative , herein designated “ atpαb derivative ” ( compound 7 ), was obtained after freeze - drying of the solution . final purification and separation of diastereoisomers of 7 was achieved on a semipreparative hplc column . the triethylammonium counterions were exchanged for na + by passing the pure diastereoisomer through sephadex - cm c - 25 column . the title compound , herein identified as atpαb [ vk 39 ], was obtained according to the procedure in example 1 starting from tetrabenzoyladenosine 8 , in 19 % yield . the title compound , herein identified as 2 - sme - atpαb [ vk 38 ], was obtained according to the procedure in example 1 starting from 2 - thiomethyl -( 2 ′, 3 ′- o - methoxymethylidene ) adenosine , in 38 % yield . the title compound , herein identified as 2 - cl - atpαb [ vk 44 ], was obtained according to the procedure in example 1 starting from 2 - chloro -( 2 ′, 3 ′- o - methoxymethylidene ) adenosine , in 43 % yield . the separation of diastereoisomers was accomplished using a semipreparative reverse - phase lichro cart 250 - 10 column and isocratic elution [ 100 mm triethylammonium acetate ( teaa ), ph 7 ( a ): meoh ( b ), 84 : 16 ] with flow rate of 6 ml / min . fractions containing the same isomer ( similar retention time ) were freeze - dried . the excess buffer was removed by repeated freeze - drying with deionized water . the isomer with the shorter retention time ( rt ) is herein designated isomer a and the other , isomer b . atpαb , isomer a [ vk 39a ] ( rt 10 . 4 min ), ph 6 . 5 : 1 h nmr ( d 2 o , 200 mhz ): δ 8 . 62 ( s , h - 8 , 1h ), 8 . 25 ( s , h - 2 , 1h ), 6 . 16 ( d , j = 7 hz , h - 1 ′, 1h ), 4 . 79 ( m , h - 2 ′, 1h ), 4 . 65 ( m , h - 3 ′, 1h ), 4 . 42 ( m , h - 4 ′, 1h ), 4 . 25 ( m , h - 5 ′, 2h ), 0 . 36 ( m , bh 3 , 3h ) ppm . 31 p nmr ( d 2 o , 200 mhz ): δ 83 . 88 ( m , p α — bh 3 ), − 9 . 42 ( d , p γ ), − 22 . 23 ( t , p β ) ppm . fab ( negative mode ) m / z : 526 . 162 ( m 4 − + 2h + + na + ). isomer b [ vk 39b ]( rt 12 . 4 min ), ph 6 . 5 : 1 h nmr ( d 2 o , 200 mhz ): δ 8 . 58 ( s , h - 8 , 1h ), 8 . 25 ( s , h - 2 , 1h ), 6 . 15 ( d , j = 7 hz , h - 1 ′, 1h ), 4 . 77 ( m , h - 2 ′, 1h ), 4 . 56 ( m , h - 3 ′, 1h ), 4 . 41 ( m , h - 4 ′, 1h ), 4 . 23 ( m , h - 5 ′, 2h ), 0 . 36 ( m , bh 3 , 3h ) ppm . 31 p nmr ( d 2 o , 200 mhz ): δ 84 . 5 ( m , p α — bh 3 ), − 9 . 34 ( d , p γ ), − 22 . 2 ( t , p β ) ppm . fab ( negative mode ) m / z : 504 . 094 . 2 - sme - atpαb , isomer a [ vk 38a ]( rt 13 . 4 min ) na + form , ph 7 . 5 ): 1 h nmr ( d 2 o , 300 mhz ): δ 8 . 46 ( s , h - 8 , 1h ), 6 . 14 ( d , j = 5 . 3 hz , h - 1 ′, 1h ), 4 . 69 ( dd , j = 3 . 8 , 4 . 9 hz , h - 3 ′, 1h ), 4 . 38 ( m , h - 4 ′, 1h ), 4 . 35 , 4 . 14 ( am , h - 5 ′, 2h ), 2 . 59 ( s , ch 3 — s , 3h ), 0 . 47 ( m , bh 3 , 3h ) ppm . 31 p nmr ( d 2 o , 200 mhz ): δ 82 . 7 ( m , p α — bh 3 ), − 6 . 5 ( d , p γ ), − 21 . 5 ( t , p β ) ppm . fab ( negative mode ) m / z : 550 . 172 . isomer b [ vk 38b ]( rt 15 . 6 min ) ( na + form , ph 7 . 5 ): 1 h nmr ( d 2 o , 300 mhz ): δ 8 . 42 ( s , h - 8 , 1h ), 6 . 13 ( d , j = 5 . 6 hz , 1h ), 4 . 86 ( dd , j = 5 , 5 . 6 hz , h - 2 ′, 1h ), 4 . 61 ( dd , j = 3 . 6 , 5 hz , h - 3 ′, 1h ), 4 . 39 ( q , j = 3 . 6 , 6 hz , h - 4 ′, 1h ), 4 . 29 ( ddd , j = 2 . 9 , 7 . 4 , 11 . 8 hz , h - 5 ′, 1h ), 4 . 19 ( ddd , j = 2 . 9 , 5 . 5 , 11 . 8 hz , h - 5 ′, 1h ), 2 . 59 ( s , ch 3 — s , 3h ), 0 . 46 ( m , bh 3 , 3h ) ppm . 31 p nmr ( d 2 o , 200 mhz ) δ 83 . 9 ( m , p α — bh 3 ), − 6 . 8 ( d , p γ ), − 21 . 6 ( t , p β ) ppm . fab ( negative mode ) m / z : 550 . 202 . 2 - chloro - atpαb , isomer a [ vk 44a ]( rt 10 . 2 min ) ( na + form , ph 7 . 5 ): 1 h nmr ( d 2 o , 300 mhz ): δ 8 . 59 ( s , h - 8 , 1h ), 6 . 07 ( d , j = 5 hz , h - 1 ′, 1h ), 4 . 69 ( dd , j = 3 . 6 , 4 . 5 hz , h - 3 ′, 1h ), 4 . 41 ( m , h - 4 ′, 1h ), 4 . 17 , 4 . 37 ( am , h - 5 ′, 2h ), 0 . 5 ( m , bh 3 , 3h ) ppm . 31 p nmr ( d 2 o , 200 mhz ) δ 82 . 9 ( m , p α — bh 3 ), − 6 . 01 ( d , p γ ), − 21 . 4 ( t , p β ) ppm . fab ( negative mode ) m / z : 559 . 023 ( m 4 − + h + + na + ). isomer b [ vk 44b ]( rt 12 . 6 min ) ( na + form , ph 7 . 5 ): 1 h nmr ( d 2 o , 300 mhz ): δ 8 . 54 ( s , h - 8 , 1h ), 6 . 04 ( d , j = 5 . 6 hz , h - 1 ′, 1h ), 4 . 57 ( dd , 3 = 3 . 5 , 4 . 7 hz , h - 3 ′, 1h ), 4 . 40 ( m , h - 4 ′, 1h ), 4 . 30 ( ddd , j = 2 . 6 , 7 . 5 , 11 . 5 hz , h - 5 ′, 1h ), 4 . 18 ( ddd , j = 2 . 9 , 5 , 11 . 5 hz , h - 5 ′, 1h ), 0 . 45 ( m , bh 3 , 3h ) ppm . 31 p nmr ( d 2 o , 200 mhz ): δ 84 . 0 ( m , p α — bh 3 ), − 6 . 4 ( d , p γ ), − 21 . 6 ( t , p β ) ppm . fab ( negative mode ) m / z : 559 . 765 ( m 4 − + h + + na + ). the profile , efficacy and potency of the insulin response induced by the synthetic ligands of the invention was evaluated in vitro in the model of rat isolated pancreas . the effects of the compounds on pancreatic vascular resistance were also simultaneously recorded . the effects of the compounds on insulin secretion and vascular resistance in the rat isolated and perfused pancreas were evaluated in the presence of a slightly stimulating glucose concentration ( 8 . 3 mmol / l ). experiments were performed in vitro in isolated perfused pancreas from male wistar albino rats fed ad libitum and weighing 300 - 350 g . the pancreas was completely isolated according to a technique previously described ( loubatières et al ., diabetologia , 1969 , 5 , 1 - 10 ) and perfused through its own arterial system with a krebs - ringer bicarbonate buffer containing 8 . 3 mmol / l glucose and 2 g / l bovine serum albumin . a mixture of o 2 ( 95 %) and co 2 ( 5 %) was bubbled through this medium at atmospheric pressure . the ph of the solution was 7 . 35 . the preparation was maintained at 37 . 5 ° c . each organ was perfused at a constant pressure ( 40 - 50 cm water ) selected so as to produce a flow rate of 2 . 5 ml / min at the start of the experiment ; in these conditions , any change in the flow rate reflects a change in vascular resistance ( hillaire - buys et al ., eur . j . pharmacol ., 1991 , 199 , 309 - 314 ). a 30 min adaptation period was allowed before the first sample was taken for insulin assay . a sample was taken 15 min later , at time 45 min . then , an infusion of atp analogues was performed during 30 min . the pancreatic flow rate was recorded and insulin was measured in the effluent fractions . insulin was assayed by the radioimmunological method of herbert et al . ( j . clin . endocrinol . metab ., 1965 , 25 , 1375 - 1384 ) using a purified rat insulin as standard ( linco research , st . charles , mo ., usa ) and anti - insulin serum ( icn biochemicals , miles , puteaux , france ). the assay sensitivity was 0 . 1 ng / ml . insulin output from perfused pancreas is expressed as ng / min and was determined by multiplying the hormone concentration in the effluent fraction by the flow rate . results are expressed as means ± standard error of the mean ( sem ). for the kinetics of insulin secretion and vascular flow rate , the results are expressed as changes in relation to the value at time 45 min taken as 100 %. for the determination of the concentration - response relationship , the mean insulin output rate was calculated as follows : the area under the curve for the drug infusion period divided by the number of minutes ( auc / 30 ). the results below show that both diastereoisomers are pharmacologically active but isomers a are more potent and selective than corresponding isomers b . the administration of the atpαb derivative 2 - methylthio - atpαb , isomer a , identified as vk 38a , induced an immediate and concentration - dependent insulin response in the range of 0 . 0015 - 5 . 0 μmol / l , as shown in fig1 . the increase in glucose - induced insulin release was first in a peak form followed by a second phase of sustained secretion ( biphasic pattern ), except for the lowest concentration ( 1 . 5 nmol / l ) at which the drug induced a 230 % transient monophasic insulin response . the maximal effect is obtained between 0 . 5 and 5 . 0 mmol / l and reaches approximately 900 % ( auc for 30 min in % per min ), with an ec 50 between 15 and 50 nmol / l . concerning the vascular effects , no significant effect was observed till 150 nmol / l ; a slight and transient reduction in pancreatic flow rate ( increased vascular resistance ) was observed at 0 . 5 , 1 . 5 and 5 . 0 μmol / l , reaching − 6 ± 3 %, − 10 ± 3 % and − 8 ± 6 %, respectively ( fig2 ). the administration of isomer b of 2 - methylthio - atpαb , identified as vk 38b , induced an insulin response of similar pattern , although less potent than that induced by isomer a ( fig3 ). moreover , in contrast to isomer a , vk 38b induced a clear and transient − 27 ± 5 % reduction in pancreatic flow rate ( increase in vascular resistance ) from the concentration of 15 nmol / l ( fig4 ). the administration of isomer a of 2 - chloro - atpαb , identified as vk 44a , induced an insulin response which seems comparable to that of vk 38a ( fig5 ). however , it also induced a slight and sustained increase in pancreatic flow rate ( decreased vascular resistance ), reaching + 8 ± 3 %, + 16 ± 5 % and + 12 ± 5 % at 0 . 015 , 0 . 15 and 1 . 5 μmol / l , respectively ( fig6 ). the administration of the parent compound atpαb ( isomer a ), identified as vk 39a , induced a biphasic insulin response clearly less potent than that of vk 38 a ( fig7 ); it also induced a slight and sustained vascular response , increasing the pancreatic flow rate by + 7 ± 3 % and + 10 ± 3 % at 0 . 5 and 5 . 0 μmol / l , respectively ( fig8 ). glucose - dependence of the insulin response triggered by vk 38a in the isolated rat pancreas the rat isolated pancreas is perfused in vitro with a physiological medium containing different concentrations of glucose . vk 38a is added during 20 minutes at 20 nmol / l . insulin response is determined by the area under the concentration - time curve during vk 38a administration and is expressed as mean ± sem . the results are shown in table 1 . normal ( non diabetic ) wistar rats were treated with a single oral dose of vk 38a ( 0 . 2 mg / kg ) or placebo ( vehicle ) administered just before a glucose tolerance test , in a cross - over experimental design with a 7 - day wash - out period . glucose ( 2 g / kg ) was administered by intraperitoneal injection . blood samples were taken before and 10 , 20 , 30 , 60 minutes after glucose load to measure glycemia . the area under the curve of plasma glucose concentrations ( in percent of baseline values ) was calculated . results in four animals are given in table 2 ( baseline glucose concentrations were 1 . 26 ± 0 . 02 and 1 . 26 ± 0 . 04 g / l in control and treated animals , respectively ). brady , p . a . ; 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