Patent Application: US-99634106-A

Abstract:
the present invention relates to a anti - proliferative target for designing chemotherapeutic agents , which comprises a eif4a protein having an amino acid sequence , as defined in claim 1 .

Description:
rna helicases are the largest group of enzymes in eukaryotic rna metabolism . the dexd / h - box putative rna helicases form the helicase superfamily ii , whose members are defined by seven highly conserved amino acid motifs , making specific targeting of selected members a challenging pharmacological problem . the translation initiation factor eif4a is the prototypical dead - box rna helicase that works in conjunction with eif4b , eif4h , and as a subunit of eif4f , to prepare the mrna template for ribosome binding — possibly by unwinding secondary structure proximal to the 5 ′ m 7 gpppn cap structure . in one embodiment of the invention there is provided small molecule inhibitors of eukaryotic translation initiation wherein one of such inhibitors characterized as hippurins ( compound i , ii and iii ) act by inhibiting the rna binding and helicase activities of eif4a . r 1 , r 2 and r 3 are independently a hydrogen atom , a halogen atom , an oxygen atom of a ketone group , — or 4 , — c ( o ) h , — co 2 h , — c ( o ) r 18 , — nr 5 r 6 , — sh , c 1 - c 10 alkyl , c 2 - c 8 alkenyl , c 2 - c 8 alkynyl , c 3 - c 8 cycloalkyl , c 4 - c 10 cycloalkenyl , —( ch 2 ), c 1 - c 10 alkyl , c 2 - c 8 alkenyl , c 2 - c 8 alkynyl , c 3 - c 8 cycloalkyl , c 4 - c 10 cycloalkenyl , c 6 - c 12 aralkyl , — c ( o ) h , or a suitable protecting group for a hydroxyl group , r 5 is a hydrogen atom , c 1 - c 10 m or 18 , — oc ( o ) r 18 , — c ( o ) r 18 , —( o )( ch 2 ) m co 2 h , — co 2 r 18 , — nhc ( o ) r 18 , or — c ( o ) nr 5 r 6 , r 4 is a hydrogen atom alkyl , c 2 - c 8 alkenyl , c 2 - c 8 alkynyl , c 3 - c 8 cycloalkyl , c 4 - c 10 cycloalkenyl , c 6 - c 12 aralkyl , — c ( o ) h , or a suitable protecting group for a hydroxyl group ; r 6 and r 7 are same or different and they each represent a halogen atom ; r 8 and r 9 are independently hydrogen , c 1 - c 10 alkyl , c 2 - c 8 alkenyl , c 2 - c 8 alkynyl , c 3 - c 8 cycloalkyl , c 4 - c 10 cycloalkenyl ; r 10 is a hydrogen , c 1 - c 10 alkyl , c 2 - c 8 alkenyl , c 2 - c 8 alkynyl , c 3 - c 8 cycloalkyl , c 4 - c 10 cycloalkenyl . there is also provided the use of compound of formula i , ii or iii wherein for compound i and ii and wherein for compound iii r 1 ═ h ; r 2 ═ oc ( o ) ch 3 r 3 ═ oh . in the present invention it has been found that hippuristanol inhibits eukaryotic protein synthesis as demonstrated by the reduction of 35 s - methionine incorporation ( fig4 ). hippuristanol exerts its effect by inhibiting cap - dependent translation initiation ( fig5 and fig6 ). furthermore it was shown that hippuristanol inhibits eif4ai f rna - dependent atpase activity and the rna binding activity of eif4ai f and eif4a c ( fig7 ). the data also indicate that hippuristanol inhibits eif4ai f - mediated helicase activity ( fig8 ). analogs of hippuristanol exhibit similar inhibition of translation ( fig1 ). while any of the analogs can be used to inhibit translation , it will be appreciated that some are more efficient based on the ic 50 and the degree of helicase inhibition as shown in fig1 . thus , translation can be inhibited by contacting cells such as mammalian cells with the compounds mentioned above . in another aspect of the invention compound i can be used to treat microorganisms infections in individuals . hippuristanol has been shown in the present invention to inhibit the replication of microorganisms such as viruses . for example poliovirus replication is delayed by inhibition of eif4a in vivo ( fig9 ). in yet another aspect of the invention it was found that compounds of molecular formula iv : r 5 is a hydrogen atom , c 1 - c 10 alkyl , c 2 - c 8 alkenyl , c 2 - c 8 alkynyl , c 3 - c 8 cycloalkyl , c 4 - c 10 cycloalkenyl , c 6 - c 12 aralkyl , — c ( o ) h , or a suitable protecting group for a hydroxyl group ; r 6 and r 7 are same or different and they each represent a halogen atom ; r 8 and r 9 are independently hydrogen , c 1 - c 10 alkyl , c 2 - c 8 alkenyl , c 2 - c 8 alkynyl , c 3 - c 8 cycloalkyl , c 4 - c 10 cycloalkenyl ; r 10 is a hydrogen , c 1 - c 10 alkyl , c 2 - c 8 alkenyl , c 2 - c 8 alkynyl , c 3 - c 8 cycloalkyl , c 4 - c 10 cycloalkenyl , can also inhibit protein synthesis in cells ( fig1 ) and furthermore clofoctol has been shown in the present invention to inhibit the growth of tumor cell lines in culture ( fig1 ). the data show that in the pi3k / akt / mtor signal transduction pathway , clofoctol ( structure shown in fig3 ) affects the 4e - bp branch downstream of tor ( fig2 and 14 ). in the presence of rapamycin , clofoctol appears to act as an agonist of rapamycin with respect to dephosphorylation of 4e - bps , but antagonizes rapamycin &# 39 ; s block of s6 phosphorylation ( fig1 a , compare lane 4 to 3 ). blotting with anti - eif4e antibodies serves as a loading control indicating that equal amounts of protein were loaded in all lanes . in another aspect of the invention compounds i , ii , iii and iv and derivatives can be used as adjuvants to chemotherapeutic agents . for example hippurin - 1 can act as adjuvant to cytotoxic agents such as doxorubicin in a mouse lymphoma model ( fig1 ). thus compounds of formula i , ii , iii or iv as well as analogs can be used to treat individuals affected by hyperproliferative diseases such as but not limited to cancers , autoimmune diseases , certain skin diseases such as psoriasis or any disease that can be treated by inhibiting translation in the cells comprising the diseased tissue . it will be appreciated that any of the compounds could be administered as a prodrug . that is to say , the administered compound ( the prodrug ) may undergo chemical reactions in an organism that would result in its transformation into one of the compounds described above . the compounds of the present invention are effective over a wide dosage range , however , the exact dosage , mode of administration and form of composition depends upon the subject to be treated and is determined by the physician or veterinarian responsible for treating the subject . generally , dosages from about 0 . 025 to about 200 mg preferably from about 0 . 1 to about 100 mg , per day may be used . generally , the unit dosage form comprises about 0 . 01 to 100 mg of the compound of formula i , ii iii or iv , as an active ingredient together with a pharmaceutically acceptable carrier . a carrier may take a wide variety of forms depending on the form of preparation desired for administration , e . g ., oral or parenteral . for parenteral formulations , the carrier may comprise sterile water or aqueous sodium chloride solution in combination with other ingredients that aid dispersion , such as ethanol and other pharmaceutically acceptable solvents . injectable suspensions may also be prepared , in which case appropriate liquid carriers , suspending agents and the like may be employed . in preparing pharmaceutical compositions in oral dosage form according to the present invention , any one or more of the usual pharmaceutical media may be used . thus , for liquid oral preparations such as suspensions , elixirs and solutions , suitable carriers and additives including water , glycols , oils , alcohols , flavoring agents , preservatives , coloring agents and the like may be used . for solid oral preparations such as powders , tablets , capsules , and for solid preparations such as suppositories , suitable carriers and additives including starches , sugar carriers , such as dextrose , mannitol , lactose and related carriers , diluents , granulating agents , lubricants , binders , disintegrating agents and the like may be used . if desired , tablets or capsules may be enteric - coated or sustained release by standard techniques . it will be appreciated that appropriate solvents for the compounds described above may also be used . examples of parenteral administration routes include without being limited to intravenous , intraarterial , intraperitoneal and the like . one of the advantages of hippuristanol and its derivatives resides in the fact that its target eif4a resides downstream of rapamycin and therefore it could be expected to be more specific than rapamycin , since the target of rapamycin , tor , is implicated in many cellular physiological processes . because of its specificity for eif4a inhibition , hippuristanol can distinguish between eif4a - dependent and eif4a - independent translation ( fig1 ). the present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope . restriction endonucleases and sp6 rna polymerase were purchased from new england biolabs ( beverly , mass .). 5 -[ 3 h ]- cytidine triphosphate ( 20 . 5 ci / mmol ), [ 35 s ] methionine (& gt ; 1000 ci / mmol ), γ - 32 p - atp ( 10 ci / mmol ), α - 32 p - atp ( 3000 ci / mmol ), α - 32 p - gtp ( 3000 ci / mmol ), 5 -[ 3 h ]- uridine ( 22 ci / mmol ), 6 -[ 3 h ]- thymidine ( 10 ci / mmol ) were obtained from perkin elmer life sciences ( boston , mass .). a high throughput screen performed in krebs - 2 extracts ( 33 ) was used to identify the active ethyl acetate fraction from i . hippuris . hippuristanol was stored as a 10 mm stock in 100 % dmso at − 70 ° c . control biochemical assays always contained the same final concentration of dmso as the parallel hippuristanol - containing reactions . collection of isis hippuris and extraction of hippuristanol . hippuristanol was purified from the gorgonian i . hippuris ( 23 ). a specimen of the gorgonian i . hippuris ( 2 . 5 kg , wet ) was collected off kohama island , okinawa on july 2003 . the whole specimen was kept frozen until used for extraction . the material was cut into pieces and steeped in 10 l of meoh three times . after concentration , the combined residual material was extracted with etoac , and the organic layer gave 27 . 2 g of an oil after concentration . this extract was partitioned between hexane and 50 % aqueous meoh . the aqueous meoh layer was further partitioned with ch 2 cl 2 . the ch 2 cl 2 layer was concentrated to yield 4 . 0 g of an oil . the hexane extract was separated by vfc ( vacuum flash chromatography ) on silica gel . the third fraction ( 2 . 4 g ) eluted with hexane - etoac ( 5 - 1 ) was further separated on a silica gel column to give six fractions . the fourth fraction ( 1 . 1 g ) from vfc was successively separated on a sephadex lh20 column ( ch 2 cl 2 — meoh , 1 - 1 ), a silica gel column ( hexane - ch 2 cl 2 - etoac ), an ods column ( meoh — h 2 o ), and finally reversed phase hplc ( meoh — h 2 o , 8 - 2 ) to give hippuristanol ( 1 , 23 . 0 mg ). the identity of hippuristanol was established by 1 h and 13 c nmr measurements and mass spectrometry ( 22 ). plasmid constructions and in vitro translations . to generate pks / ff / hcv / ren , the intermediate cloning plasmid pff / hcv / rl . pa 51 ( 33 ) was digested with bamh1 and bg / ii and inserted into the bamhi site of pksii . for in vitro transcriptions , this plasmid was linearized with bamhi and transcribed with t3 rna polymerase to generate ff / hcv / ren mrna . construction of plasmids pks / ff / ren and pks / ff / emc / ren have been previously detailed , and were linearized with bamhi for in vitro transcriptions with t3 rna polymerase ( 33 ). plasmid pcdna / ren / p2 / ff was linearized with xhoi and transcribed with t3 rna polymerase to generate ren / p2 / ren mrna ( 41 ). plasmid pgl3 / ren / crpv / ff was linearized with bamhi and transcribed with t7 rna polymerase to generate ren / crpv / ff mrna ( 57 ). plasmids pgc / l ( renamed from pgem - cat / luc ), pgc / emc / l ( renamed from pgem - cat / emc / luc ), and pgc / ptv / l ( renamed from pgem - cat / ptv / luc ) have been previously described ( 36 , 39 ). plasmid pcdna / ren / hcv / ff was constructed by digesting puc18 - t7 - r - luc - hcv ires - f - luc ( 53 ) with bamhi and noti and subcloning the insert into pcdna3 . in vitro translations were performed using krebs - 2 extracts at a final mrna and k + concentration of 5 μg / ml and 100 mm , respectively ( 33 ). firefly and renilla luciferase activities ( rlu ) were measured on a berthold lumat lb 9507 luminometer . following in vitro translations in the presence of [ 35 s ] methionine , protein products were separated on 10 % polyacrylamide / sds gels that were treated with en 3 hance , dried , and exposed to x - omat ( kodak ) film . in vitro translation assays in rabbit reticulocyte lysates and wheat germ extracts were performed according to the manufacturer &# 39 ; s instructions ( promega ). ribosome binding and mrna crosslinking . ribosome binding assays were performed by incubating 32 p - labelled cat mrna in rabbit reticulocyte lysate or wheat germ extracts in the presence of : 600 μm cycloheximide ( chx ), 1 mm gmp - pnp , and / or 50 μm hippuristanol or vehicle for 10 min ( 33 ). following centrifugation through 10 - 30 % glycerol gradients ( sw40 ; 39 , 000 rpm / 3 . 5 hrs ), fractions from each gradient were collected using a brandel tube piercer connected to an isco fraction collector . fractions of 500 μl were collected and radioactivity was determined by scintillation counting . chemical crosslinking of initiation factor preparations to 32 p cap - labeled oxidized cat mrna was performed under standard reaction conditions ( 49 ) containing 0 . 9 mm atp . for chemical crosslinking with individual factors , 1 μg of recombinant eif4ai f or 0 . 7 μg of purified eif4f was used . after crosslinking , samples were treated with rnase a and separated on 10 - 15 % sds - page gradient gels ( initiation factor preparation ) or 10 % sds - page gels ( individual factors ). the gels were dried and exposed to x - omat ( kodak ) film . recombinant eif4ai purification and assays . recombinant murine eif4ai was expressed in escherichia coli bl21 ( de3 ) codon +, and purified using ni - nta agarose and q sepharose chromatography . atpase assays were performed using 1 μm γ - 32 p - atp , 2 . 5 μm poly ( u ) and 4 . 5 μg of recombinant eif4ai f ( 30 ). quantifications were performed on a fujix bas2000 phosphoimager with a fuji imaging screen . atp crosslinking assays were performed with 1 μg of recombinant eif4ai f or 0 . 7 μg of purified eif4f and 2 . 5 μci of α - 32 p - atp ( 38 ). poly ( u ) was added to 2 . 5 μm where indicated . gels were exposed to x - ray film ( kodak ) at − 70 ° c . for 12 hrs with an intensifying screen . helicase assays were performed with the rna - 1 / 11 duplex and 0 . 4 μm recombinant eif4ai f or 25 nm ded1p ( 45 ). atp was added to a concentration of 1 mm . gels were dried and exposed to x - ray film ( kodak ) at − 80 ° c . for 12 hrs with an intensifying screen . cell transfections . 293 cells were maintained in dmem media supplemented with 10 % fetal calf serum . the day before calcium phosphate transfection , cells were seeded at 3 × 10 6 cells / 10 cm dish . following transfection with pcdna / ren / hcv / ff , 293 cells were incubated for 10 hrs with hippuristanol or vehicle before harvesting , and were collected 48 hrs post - transfection . luciferase assays were performed with the dual luciferase assay kit according to manufacturer &# 39 ; s instructions ( promega ). probes for northern blots were produced using the readiprime kit ( amersham ). poliovirus infections . the day prior to infection , 4 × 10 5 hela cells were plated per 35 mm dish . for absorption , cells were washed with pbs , followed by the addition of the mahoney strain of poliovirus type 1 ( 2 pfu / cell ) in serum free d - mem containing vehicle or hippuristanol . cells were incubated at room temperature for 30 minutes with gentle rocking , after which the media was removed . cells were washed with pbs , fresh media containing 10 % fetal bovine serum was added , and cells were incubated at 37 ° c . for the indicated times . thirty minutes before harvesting , [ 35 s ] methionine was added ( 50 μci / ml ) to the media . mock - infected cells were incubated at 37 ° c . for 8 hrs , while poliovirus - infected cells were incubated at 37 ° c . for the times indicated above the panel . for harvesting , cells were washed with pbs and extracts prepared in plb ( 0 . 1 % sds , 0 . 5 % sodium deoxycholate , 1 % triton x - 100 , 1 mm pmsf ) ( 7 ). the crude etoac extraction obtained from the meoh concentrate from i . hippuris was partitioned between hexane and 50 % aqueous meoh . the hexane layer was concentrated to give 18 . 3 g . the aqueous meoh layer was partitioned with ch 2 cl 2 and the ch 2 cl 2 layer concentrated to yield 4 . 0 g of an oil . both hexane and ch 2 cl 2 extracts were used to separate hippuristanol congeners . the hexane extract was first separated by vfc ( vacuum flash chromatography ) on silica gel . the third fraction ( 2 . 4 g ) eluted with hexane / etoac ( 5 : 1 , v / v ) was further separated on a silica gel column to give six fractions . the fourth fraction ( 89 . 7 mg ) eluted with ch 2 cl 2 / etoac ( 1 : 1 , v / v ) gave precipitates that were found to be compound 19 ( 73 . 5 mg ). the fourth fraction ( 1 . 1 g ) eluted with hexane / etoac ( 1 : 1 , v / v ) from vfc was successively separated on a sephadex lh20 column ( ch 2 cl 2 / meoh , 1 : 1 , v / v ), a silica gel column ( hexane / ch 2 cl 2 / etoac / meoh , 1 : 1 : 0 : 0 , 5 : 1 : 0 : 0 , 1 : 1 : 0 : 0 , 1 : 5 : 0 : 0 , 0 : 1 : 0 : 0 , 0 : 5 : 1 : 0 , 0 : 1 : 1 : 0 , 0 : 1 : 5 : 0 , 0 : 0 : 1 : 0 , 0 : 0 : 10 : 1 , 0 : 1 : 1 : 0 , v / v ), a c18 column ( meoh / h 2 o , 9 : 1 , v / v ), and finally subjected to repeated reversed phase hplc ( meoh / h 2 o , 8 : 2 , v / v ) to yield hippuristanol ( 1 , 23 . 0 mg ), epihippuristanol ( 10 , 15 . 1 mg ), hippurin - 1 ( 6 , 6 . 7 mg ), and epihippurin - 1 ( 15 , 1 . 5 mg ). the ch 2 cl 2 extract was separated on a silica vfc column ( hexane // etoac , 1 : 0 , 10 : 1 , 5 : 1 , 2 : 1 , 1 : 5 , 0 : 1 , v / v ), followed by a sephadex lh20 column ( ch 2 cl 2 / meoh , 1 : 1 , v / v ). a fraction ( 400 mg ) eluted with etoac was further separated on a silica gel column (/ etoac ) twice and finally subjected to reversed phase hplc ( meoh / h 2 o , 19 - 1 , v / v ) to give 2 - desacetyl - hippurin - 1 ( 8 , 3 . 5 mg ) and 2 - desacetyl - epihippurin - 1 ( 17 , 20 . 4 mg ). additional amount of steroids 1 , 6 , 8 , 10 , 15 , and 17 were obtained from other fractions . preparation of steroids 7 and 16 . a dried indonesian specimen of the gorgonian i . hippuris . ( 3 . 2 kg ) collected off flores island on august 2001 was extracted with acetone , and its etoac soluble portion ( 45 . 3 g ) obtained . the extract was separated on a silica gel column twice followed by hplc separation ( silica , hexane / etoac , 1 : 5 , v / v ) to give 2 - desacetyl - hippurin - 1 3 - acetate ( 7 , 17 . 5 mg ). additional amounts of 7 were obtained by purification of other fractions . compound 7 ( 24 . 6 mg ) was treated with one drop of 1m hydrochloric solution in thf ( 1 ml ) at rt for 3 hr . the mixture was partitioned between etoac and water , and the organic layer was dried over na 2 so 4 and concentrated . the crude product was purified by hplc ( c18 , meoh / h 2 o , 8 : 2 , v / v ) to give 22 . 1 mg of epimeric compound 16 ( 89 %). compound 16 . colorless crystals , 1 h nmr ( cdcl 3 ) δ 5 . 12 ( brs , 1h ), 4 . 43 ( m , 1h ), 4 . 29 ( brs , 1h ), 3 . 89 ( m , 1h ), 2 . 24 ( m , 1h ), 2 . 12 ( s , 3h ), 1 . 34 ( s , 3h ), 1 . 28 ( s , 6h ), 1 . 06 ( s , 3h ), 0 . 98 ( s , 3h ), 0 . 94 ( d , j = 6 . 5 hz , 3h ), 0 . 83 ( dd , j = 11 . 0 , 3 . 5 hz , 1h ). acetylation of hippuristanol ( 1 ) to give hippuristanol 3 - acetate ( 2 ) and hippuristanol 3 , 11 - diacetate ( 3 ). a mixture of hippuristanol ( 1 , 10 . 0 mg ), acetic anhydride ( 0 . 2 ml ), and dry pyridine ( 0 . 3 ml ) was allowed to stand at room temperature for 13 days . the mixture was concentrated to remove excess acetic anhydride and pyridine . the crude product was separated by silica gel thin layer chromatography ( tlc ) ( chcl 3 / etoac , 3 : 1 , v / v ) to give 8 . 5 mg of hippuristanol 3 - acetate ( 2 , 8 . 5 mg , 75 %) and 1 . 7 mg of hippuristanol 3 , 11 - diacetate ( 3 , 14 %). hippuristanol 3 , 11 - diacetate ( 3 ). glass , [ α ] d 22 + 45 . 7 ° ( c 0 . 44 , chcl 3 ); 1 h nmr ( cdcl 3 ) δ 5 . 32 ( brq , j = 2 . 8 hz , 1h ), 4 . 99 ( brt , j = 2 . 6 hz , 1h ), 4 . 30 ( dt , j = 7 . 0 , 7 . 6 hz , 1h ), 3 . 11 ( s , 1h ), 2 . 37 ( dd , j = 13 . 4 , 7 . 6 hz , 1h ), 2 . 28 ( dd , j = 14 . 4 , 2 . 5 hz , 1h ), 2 . 04 ( s , 3h ), 2 . 02 ( s , 3h ), 1 . 29 ( s , 3h ), 1 . 26 ( s , 3h ), 1 . 23 ( s , 3h ), 1 . 20 ( s , 3h ), 0 . 98 ( d , j = 7 . 0 hz , 3h ), 0 . 88 ( s , 3h ); ir ( kbr ) 3500 , 1720 , 1235 cm − 1 ; eims m / z 546 ( m + , 0 . 4 ), 531 ( 3 ), 488 ( 19 ), 418 ( 13 ), 358 ( 84 ), 298 ( 100 %). acetylation of epihippuristanol ( 10 ) to give epihippuristanol 3 - acetate ( 11 ) and epihippuristanol 3 , 11 - diacetate ( 12 ). epihippuristanol ( 10 , 10 . 0 mg ) was acetylated in the same manner as described above for hippuristanol ( 1 ). the crude product was separated by silica gel tlc ( chcl 3 / etoac , 3 : 1 , v / v ) to give 8 . 8 mg of epihippuristanol 3 - acetate ( 11 , 81 %) and 1 . 7 mg of epihippuristanol 3 , 11 - diacetate ( 12 , 14 %). epihippuristanol 3 , 11 - diacetate ( 12 , hippurin - 2 ). white solid , mp 253 - 256 . 5 ° c ., 1 h nmr ( cdcl 3 ) δ 5 . 30 ( m , 1h ), 5 . 00 ( brs , 1h ), 4 . 44 ( dt , j = 5 . 5 , 7 . 5 hz , 1h ), 2 . 25 ( m , 2h ), 2 . 04 ( s , 3h ), 2 . 01 ( s , 3h ), 1 . 29 ( s , 3h ), 1 . 27 ( s , 3h ), 1 . 22 ( s , 3h ), 0 . 98 ( s , 3h ), 0 . 94 ( d , j = 7 . 0 hz , 3h ), 0 . 89 ( s , 3h ); ir ( kbr ) 3520 , 1725 , 1250 cm − 1 . oxidation of hippuristanol ( 1 ) to yield hippuristanol 11 - one ( 4 ) and hippuristanol 3 , 11 - dione ( 5 ). to an ice - cooled solution of hippuristanol ( 1 , 31 . 7 mg ) in pyridine ( 0 . 5 ml ), cornforth reagent ( 0 . 7 ml ) was added dropwise . the mixture was kept stirring for 30 min . in an ice bath , then 3 hr at rt . the mixture was taken up in ether and the suspension was filtered . the filtrate was washed with dilute hydrochloric acid , dried over na 2 so 4 , and concentrated . the resulting product was separated on silica tlc ( chcl 3 / etoac , 3 : 1 , v / v ) to give 8 . 8 mg of hippuristanol 11 - one ( 4 , 28 %) and 17 . 7 mg of hippuristanol 3 , 11 - dione ( 5 , 56 %). hippuristanol 3 , 11 - dione ( 5 ). white crystals . mp 181 - 183 . 5 ° c . ; 1 h nmr ( cdcl 3 ) δ 4 . 42 ( m , 2h ), 3 . 02 ( s , 1h ), 2 . 82 ( ddd , j = 13 . 0 , 6 . 5 , 2 . 0 hz , 1h ), 2 . 63 ( d , j = 12 . 0 hz , 1h ), 2 . 45 ( dt , j = 6 . 5 , 14 . 5 hz , 1h ), 2 . 37 ( dd , j = 13 . 0 , 8 . 0 hz , 1h ), 1 . 31 ( s , 3h ), 1 . 22 ( s , 6h ), 1 . 20 ( s , 3h ), 1 . 11 ( s , 3h ), 0 . 99 ( d , j = 7 . 0 hz , 3h ); 13 c nmr ( cdcl 3 ) δ 211 . 6 , 210 . 0 , 115 . 0 , 84 . 9 , 80 . 4 , 79 . 1 , 63 . 9 , 63 . 4 , 58 . 8 , 54 . 5 , 46 . 9 , 46 . 3 , 44 . 3 , 41 . 8 , 40 . 5 , 38 . 0 , 37 . 0 , 35 . 5 , 35 . 2 , 33 . 7 , 32 . 4 , 28 . 7 , 28 . 5 , 28 . 2 , 23 . 0 , 17 . 3 , 14 . 8 , 11 . 0 ; ir ( kbr ) 3510 , 1695 cm − 1 ; eims m / z 458 ( m + , 5 ), 316 ( 92 ), 84 ( 100 %). oxidation of epihippuristanol ( 10 ) to give epihippuristanol 11 - one ( 13 ) and epihippuristanol 3 , 11 - dione ( 14 ). epihippuristanol ( 10 , 31 . 4 mg ) was oxidized in the same fashion as described above for hippuristanol ( 1 ). the crude product was separated by tlc ( chcl 3 / etoac , 1 : 1 , v / v ) to give 12 . 6 mg of epihippuristanol 11 - one ( 13 , 40 %) and 14 . 0 mg of epihippuristanol 3 , 11 - dione ( 14 , 45 %). epihippuristanol 3 , 11 - dione ( 14 ). white crystals , mp 226 - 229 ° c . ; 1 h nmr ( cdcl 3 ) δ 4 . 48 ( brq , j = 7 . 0 hz , 1h ), 2 . 82 ( ddd , j = 13 , 6 . 5 , 2 hz , 1h ), 2 . 57 ( d , j = 12 hz , 1h ), 2 . 45 ( dt , j = 6 . 5 , 14 hz , 1h ), 1 . 30 ( s , 3h ), 1 . 28 ( s , 3h ), 1 . 22 ( s , 3h ), 1 . 08 ( s , 3h ), 0 . 98 ( s , 3h ), 0 . 94 ( d , j = 7 hz , 3h ); 13 c nmr ( cdcl 3 ) δ 211 . 4 , 209 . 9 , 118 . 6 , 84 . 5 , 81 . 9 , 79 . 0 , 64 . 1 , 63 . 2 , 58 . 2 , 55 . 8 , 47 . 0 , 45 . 9 , 44 . 3 , 41 . 0 , 39 . 6 , 38 . 0 , 37 . 1 , 35 . 8 ( 2c ), 35 . 3 , 32 . 3 , 31 . 5 , 29 . 0 , 28 . 2 , 26 . 0 , 23 . 0 , 17 . 6 , 14 . 0 , 11 . 1 ; ir ( kbr ) 3450 , 1700 cm − 1 ; eims m / z 458 ( m + , 6 ), 443 ( 16 ), 400 ( 7 ), 330 ( 32 ), 315 ( 100 %). preparation of 2 - desacetyl - hippurin - 1 2 - glutarate ( 9 ) and 2 - desacetyl - epihippurin - 1 2 - glutarate ( 18 ). a mixture of 2 - desacetyl - hippruin - 1 ( 8 , 9 . 6 mg ), glutaric anhydride ( 14 mg ), and pyridine ( 0 . 1 ml ) was allowed to stand at 70 ° c . for two days . after removal of pyridine , the crude product was separated by silica tlc ( etoac ), then by hplc ( c18 , meoh / h 2 o , 9 : 1 , v / v ) to give 2 . 9 mg of glutarate ( 9 , 24 %). compound 18 was prepared by treating compound 17 ( 23 . 6 mg ) with glutaric anhydride and pyridine in the same way as described above for 2 - desacetyl - hippruin - 1 ( 8 ). the product was separated by silica tlc ( etoac ) followed by silica hplc ( hexane / etoac , 1 : 4 , v / v ) to give 3 . 4 mg of glutarate ( 18 , 11 %). amorphous solid , 1 h nmr ( cdcl 3 ) δ 5 . 00 ( m , 1h ), 4 . 43 ( dt , j = 7 . 5 , 5 . 5 hz , 1h ), 4 . 26 ( brs , 1h ), 4 . 06 ( brs , 1h ), 2 . 42 ( m , 2h ), 2 . 25 ( m , 1h ), 2 . 14 ( dd , j = 14 . 5 , 2 . 5 hz , 1h ), 1 . 33 ( s , 3h ), 1 . 30 ( s , 3h ), 1 . 27 ( s , 3h ), 1 . 10 ( s , 3h ), 0 . 98 ( s , 3h ), 0 . 94 ( d , j = 7 . 0 hz , 3h ). steroids 1 , 6 , 8 , 10 , 15 , and 17steroids were identified by comparing their 1 h and 13 c nmr data with those previously isolated by higa et al . hippuristanol 3 - acetate ( 2 ). amorphous solid , 1 h nmr ( cdcl 3 ) δ 5 . 00 ( brs , 1h ), 4 . 29 ( 2h , m ), 3 . 19 ( s , 1h ), 2 . 37 ( dd , j = 13 . 5 , 7 . 5 hz , 1h ), 2 . 20 ( dd , j = 14 . 0 , 2 . 5 hz , 1h ), 2 . 04 ( s , 3h ), 1 . 39 ( s , 3h ), 1 . 31 ( s , 3h ), 1 . 22 ( s , 3h ), 1 . 19 ( s , 3h ), 1 . 05 ( s , 3h ), 0 . 98 ( d , j = 7 . 0 hz , 3h ); ir ( kbr ) 3510 , 1715 , 1240 cm − 1 ; eims m / z 505 ([ m + 1 ] + ), 489 ( 2 ), 283 ( 100 %). hippuristanol 11 - one ( 4 ). white crystals . mp 205 . 5 - 208 ° c ., 1 h nmr ( cdcl 3 ) δ 4 . 42 ( brdt , j = 7 . 0 , 7 . 5 hz , 1h ), 4 . 03 ( brs , 1h ), 3 . 01 ( s , 1h ), 2 . 57 ( d , j = 11 . 9 hz , 1h ), 2 . 36 ( dd , j = 13 . 7 , 8 . 0 hz , 1h ), 2 . 26 ( dt , j = 13 . 4 , 3 . 0 hz , 1h ), 2 . 19 ( d , j = 11 . 9 hz , 1h ), 2 . 09 ( m , 1h ), 2 . 03 ( d , j = 8 . 8 hz , 1h ), 1 . 31 ( s , 3h ), 1 . 21 ( s , 3h ), 1 . 19 ( s , 3h ), 1 . 08 ( s , 3h ), 1 . 01 ( s , 3h ), 0 . 98 ( d , j = 7 . 0 hz , 3h ); 13 c nmr ( cdcl 3 ) δ 210 . 6 , 115 . 0 , 84 . 8 , 80 . 5 , 79 . 1 , 66 . 3 , 64 . 5 , 63 . 4 , 59 . 0 , 54 . 9 , 46 . 4 , 41 . 8 , 40 . 5 , 38 . 9 , 35 . 8 , 35 . 6 , 35 . 3 , 33 . 6 , 32 . 8 , 30 . 9 , 28 . 9 , 28 . 7 , 28 . 5 , 27 . 9 , 23 . 0 , 17 . 2 , 14 . 8 , 10 . 8 ; ir ( kbr ) 3470 , 1705 cm − 1 ; eims m / z 460 ( m + , 1 ), 402 ( 8 ), 317 ( 99 ), 129 ( 100 %). glutarate 9 . amorphous solid , 1 h nmr ( cdcl 3 ) δ 5 . 00 ( ddd , j = 11 . 8 , 4 . 5 , 3 . 0 hz , 1h ), 4 . 30 ( brq , j = 6 . 5 hz , 1h ), 4 . 25 ( brs , 1h ), 4 . 05 ( brs , 1h ), 3 . 21 ( brs , 1h ), 2 . 46 ( m , 3h ), 2 . 37 ( dd , j = 7 . 6 , 13 . 4 hz , 1 h ), 2 . 17 ( brd , j = 14 . 0 hz , 1h ), 2 . 00 ( m , 1h ), 1 . 37 ( s , 3h ), 1 . 31 ( s , 3h ), 1 . 21 ( s , 3h ), 1 . 19 ( s , 3h ), 1 . 10 ( s , 3h ), 0 . 97 ( d , j = 7 hz , 3h ); 13 c nmr ( cdcl 3 ) δ 175 . 9 , 172 . 1 , 118 . 6 , 84 . 2 , 82 . 7 , 79 . 0 , 72 . 9 , 68 . 0 , 67 . 4 , 66 . 3 , 60 . 4 , 58 . 2 , 57 . 9 , 48 . 8 , 42 . 1 , 41 . 0 , 39 . 9 , 39 . 0 , 37 . 4 , 36 . 7 , 33 . 5 , 32 . 7 , 32 . 1 , 31 . 6 , 29 . 6 , 29 . 1 , 27 . 1 , 26 . 9 , 23 . 0 , 20 . 0 , 19 . 4 , 15 . 1 , 13 . 9 . epihippuristanol 3 - acetate ( 11 ). white solid , mp 193 . 5 - 195 ° c ., 1 h nmr ( cdcl 3 ) δ 5 . 01 ( brs , 1h ), 4 . 44 ( dt , j = 5 . 0 , 7 . 5 hz , 1h ), 4 . 30 ( brs , 1h ), 2 . 26 ( m , 1h ), 2 . 16 ( dd , j = 2 . 5 , 14 . 0 hz , 1h ), 2 . 04 ( s , 3h ), 1 . 34 ( s , 3h ), 1 . 30 ( s , 3h ), 1 . 28 ( s , 3h ), 1 . 04 ( s , 3h ), 0 . 99 ( s , 3h ), 0 . 94 ( d , j = 7 . 0 hz , 3h ); ir ( kbr ) 3490 , 1710 , 1245 cm − 1 ; eims m / z 504 ( m + , 2 ), 489 ( 3 ), 446 ( 16 ), 376 ( 60 ), 358 ( 100 %). epihippuristanol 3 , 11 - diacetate ( 12 , hippurin - 2 ). white solid , mp 253 - 256 . 5 ° c ., 1 h nmr ( cdcl 3 ) δ 5 . 30 ( m , 1h ), 5 . 00 ( brs , 1h ), 4 . 44 ( dt , j = 5 . 5 , 7 . 5 hz , 1h ), 2 . 25 ( m , 2h ), 2 . 04 ( s , 3h ), 2 . 01 ( s , 3h ), 1 . 29 ( s , 3h ), 1 . 27 ( s , 3h ), 1 . 22 ( s , 3h ), 0 . 98 ( s , 3h ), 0 . 94 ( d , j = 7 . 0 hz , 3h ), 0 . 89 ( s , 3h ); ir ( kbr ) 3520 , 1725 , 1250 cm − 1 . epihippuristanol 11 - one ( 13 ). white crystals , mp 254 - 257 ° c . ; 1 h nmr ( cdcl 3 ) δ 4 . 49 ( q , j = 7 . 1 hz , 1h ), 4 . 04 ( brs , 1h ), 2 . 51 ( d , j = 11 . 9 hz , 1h ), 2 . 25 ( d , j = 11 . 9 hz , 1h ), 1 . 29 ( s , 3h ), 1 . 28 ( s , 3h ), 1 . 05 ( s , 3h ), 1 . 01 ( s , 3h ), 0 . 98 ( s , 3h ), 0 . 94 ( d , j = 7 . 0 hz , 3h ); 13 c nmr ( cdcl 3 ) δ 210 . 5 , 118 . 6 , 84 . 4 , 81 . 9 , 79 . 1 , 66 . 3 , 64 . 6 , 63 . 1 , 58 . 4 , 56 . 1 , 45 . 9 , 41 . 0 , 39 . 6 , 39 . 0 , 35 . 8 , 35 . 3 , 32 . 7 , 31 . 4 , 30 . 9 , 29 . 0 , 28 . 9 , 27 . 9 , 25 . 8 , 23 . 0 , 17 . 6 , 14 . 0 , 10 . 9 ; ir ( kbr ) 3545 , 3480 , 1690 cm − 1 ; eims m / z 460 ( m + , 3 ), 445 ( 0 . 7 ), 402 ( 6 ), 332 ( 41 ), 317 ( 100 %). compound 19 . colorless crystals , mp : 240 ° c . ; [ α ] d 24 − 38 . 0 ° ( c 2 . 41 , chcl 3 ); 1 h nmr ( cdcl 3 ) δ 5 . 31 ( s , 1h ), 5 . 00 ( brs 1 h ), 4 . 53 ( q , j = 7 hz , 1h ), 4 . 24 ( brs 1h ), 2 . 69 ( dd , j = 14 . 5 , 3 . 5 hz , 1h ), 2 . 61 ( d , j = 6 . 5 hz , 1h ), 2 . 28 ( m , 1h ), 2 . 12 ( m , 1h ), 2 . 06 ( m , 1h ), 2 . 04 ( s , 3h ), 1 . 87 ( m , 1h ), 1 . 82 ( m , 2h ), 1 . 74 ( m , 1h ), 1 . 71 ( m , 2h ), 1 . 64 ( m , 1h ), 1 . 61 ( dd , j = 14 . 5 , 4 hz , 1h ), 1 . 48 ( m , 3h ), 1 . 41 ( s , 3h ), 1 . 41 ( m , 1h ), 1 . 34 ( m , 1h ), 1 . 29 ( s , 3h ), 1 . 27 ( m , 1h ), 1 . 18 ( m , 2h ), 1 . 00 ( s , 3h ), 096 ( s , 3h ), 0 . 93 ( d , j = 7 hz , 3h ), 0 . 79 ( m , 1h ); 13 c nmr ( cdcl 3 ) δ 170 . 6 s , 117 . 9 s , 101 . 3 d , 90 . 7 s , 84 . 7 s , 80 . 6 d , 70 . 0 d , 66 . 0 d , 64 . 3 d , 58 . 5 d , 56 . 1 s , 56 . 0 d , 41 . 1 d , 40 . 5 d , 39 . 2 t , 38 . 7 t , 35 . 9 s , 32 . 4 t , 33 . 7 t , 32 . 1 t , 32 . 1 t , 31 . 0 d , 29 . 0 q , 27 . 4 t , 25 . 6 t , 22 . 8 q , 21 . 4 q , 19 . 6 q , 14 . 4 q , 13 . 9 q ; positive noes : h - 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