WO2024020520A2 - Enzyme compositions, steroid derivatives, enzyme inhibitors, and methods of making same for pharmaceutical applications - Google Patents
Enzyme compositions, steroid derivatives, enzyme inhibitors, and methods of making same for pharmaceutical applications Download PDFInfo
- Publication number
- WO2024020520A2 WO2024020520A2 PCT/US2023/070652 US2023070652W WO2024020520A2 WO 2024020520 A2 WO2024020520 A2 WO 2024020520A2 US 2023070652 W US2023070652 W US 2023070652W WO 2024020520 A2 WO2024020520 A2 WO 2024020520A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- compound
- steroid
- inhibitor
- enzyme
- cytochrome
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 18
- 150000003431 steroids Chemical class 0.000 title claims description 39
- 239000000203 mixture Substances 0.000 title claims description 9
- 102000004190 Enzymes Human genes 0.000 title description 15
- 108090000790 Enzymes Proteins 0.000 title description 15
- 239000002532 enzyme inhibitor Substances 0.000 title description 2
- 239000003112 inhibitor Substances 0.000 claims abstract description 19
- 150000001875 compounds Chemical class 0.000 claims description 56
- 101150053185 P450 gene Proteins 0.000 claims description 51
- 108010015742 Cytochrome P-450 Enzyme System Proteins 0.000 claims description 9
- 102000002004 Cytochrome P-450 Enzyme System Human genes 0.000 claims description 9
- 229940002612 prodrug Drugs 0.000 claims description 7
- 239000000651 prodrug Substances 0.000 claims description 7
- 230000000144 pharmacologic effect Effects 0.000 claims description 5
- 125000005594 diketone group Chemical group 0.000 claims description 3
- 230000002401 inhibitory effect Effects 0.000 claims description 3
- 230000001404 mediated effect Effects 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
- 239000012279 sodium borohydride Substances 0.000 claims description 3
- 229910000033 sodium borohydride Inorganic materials 0.000 claims description 3
- 150000003839 salts Chemical class 0.000 claims 6
- 239000003814 drug Substances 0.000 claims 4
- 238000004519 manufacturing process Methods 0.000 claims 4
- 239000004480 active ingredient Substances 0.000 claims 2
- 108010058254 Steroid 12-alpha-Hydroxylase Proteins 0.000 abstract description 7
- 102000012168 Cytochrome P450 8B1 Human genes 0.000 abstract description 3
- 229940124639 Selective inhibitor Drugs 0.000 abstract description 2
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 abstract 1
- 125000002345 steroid group Chemical group 0.000 abstract 1
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 22
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 19
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 18
- 239000003446 ligand Substances 0.000 description 16
- 239000000758 substrate Substances 0.000 description 15
- 150000003278 haem Chemical class 0.000 description 11
- 230000015572 biosynthetic process Effects 0.000 description 10
- 229910052742 iron Inorganic materials 0.000 description 9
- IOIZWEJGGCZDOL-RQDYSCIWSA-N 7alpha-hydroxycholest-4-en-3-one Chemical compound C([C@H]1O)C2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 IOIZWEJGGCZDOL-RQDYSCIWSA-N 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- GZOSMCIZMLWJML-VJLLXTKPSA-N abiraterone Chemical compound C([C@H]1[C@H]2[C@@H]([C@]3(CC[C@H](O)CC3=CC2)C)CC[C@@]11C)C=C1C1=CC=CN=C1 GZOSMCIZMLWJML-VJLLXTKPSA-N 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 6
- 229960000853 abiraterone Drugs 0.000 description 6
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 6
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 6
- 238000003786 synthesis reaction Methods 0.000 description 6
- BHQCQFFYRZLCQQ-UHFFFAOYSA-N (3alpha,5alpha,7alpha,12alpha)-3,7,12-trihydroxy-cholan-24-oic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 BHQCQFFYRZLCQQ-UHFFFAOYSA-N 0.000 description 5
- 239000004380 Cholic acid Substances 0.000 description 5
- BHQCQFFYRZLCQQ-OELDTZBJSA-N cholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 BHQCQFFYRZLCQQ-OELDTZBJSA-N 0.000 description 5
- 229960002471 cholic acid Drugs 0.000 description 5
- 235000019416 cholic acid Nutrition 0.000 description 5
- KXGVEGMKQFWNSR-UHFFFAOYSA-N deoxycholic acid Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 KXGVEGMKQFWNSR-UHFFFAOYSA-N 0.000 description 5
- OSVMTWJCGUFAOD-KZQROQTASA-N formestane Chemical compound O=C1CC[C@]2(C)[C@H]3CC[C@](C)(C(CC4)=O)[C@@H]4[C@@H]3CCC2=C1O OSVMTWJCGUFAOD-KZQROQTASA-N 0.000 description 5
- 230000003993 interaction Effects 0.000 description 5
- 230000004048 modification Effects 0.000 description 5
- 238000012986 modification Methods 0.000 description 5
- RUDATBOHQWOJDD-BSWAIDMHSA-N chenodeoxycholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)CC1 RUDATBOHQWOJDD-BSWAIDMHSA-N 0.000 description 4
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 4
- 229940126214 compound 3 Drugs 0.000 description 4
- 239000013078 crystal Substances 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- 230000001225 therapeutic effect Effects 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- HSINOMROUCMIEA-FGVHQWLLSA-N (2s,4r)-4-[(3r,5s,6r,7r,8s,9s,10s,13r,14s,17r)-6-ethyl-3,7-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl]-2-methylpentanoic acid Chemical compound C([C@@]12C)C[C@@H](O)C[C@H]1[C@@H](CC)[C@@H](O)[C@@H]1[C@@H]2CC[C@]2(C)[C@@H]([C@H](C)C[C@H](C)C(O)=O)CC[C@H]21 HSINOMROUCMIEA-FGVHQWLLSA-N 0.000 description 3
- RUDATBOHQWOJDD-UHFFFAOYSA-N (3beta,5beta,7alpha)-3,7-Dihydroxycholan-24-oic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)CC2 RUDATBOHQWOJDD-UHFFFAOYSA-N 0.000 description 3
- HUUPVABNAQUEJW-UHFFFAOYSA-N 1-methylpiperidin-4-one Chemical compound CN1CCC(=O)CC1 HUUPVABNAQUEJW-UHFFFAOYSA-N 0.000 description 3
- 102000018832 Cytochromes Human genes 0.000 description 3
- 108010052832 Cytochromes Proteins 0.000 description 3
- 101000941788 Homo sapiens 7-alpha-hydroxycholest-4-en-3-one 12-alpha-hydroxylase Proteins 0.000 description 3
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- 208000008589 Obesity Diseases 0.000 description 3
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 3
- 239000003613 bile acid Substances 0.000 description 3
- 229960001091 chenodeoxycholic acid Drugs 0.000 description 3
- 238000004440 column chromatography Methods 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 235000020824 obesity Nutrition 0.000 description 3
- 125000000430 tryptophan group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C2=C([H])C([H])=C([H])C([H])=C12 0.000 description 3
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- 102000009878 3-Hydroxysteroid Dehydrogenases Human genes 0.000 description 2
- 102100032645 7-alpha-hydroxycholest-4-en-3-one 12-alpha-hydroxylase Human genes 0.000 description 2
- UQPYXHJTHPHOMM-NIBOIBLTSA-N 7alpha,12alpha-dihydroxycholest-4-en-3-one Chemical compound C([C@H]1O)C2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)[C@@H](O)C2 UQPYXHJTHPHOMM-NIBOIBLTSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 101710088194 Dehydrogenase Proteins 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 102100026795 Peroxisomal acyl-coenzyme A oxidase 2 Human genes 0.000 description 2
- ORNBQBCIOKFOEO-YQUGOWONSA-N Pregnenolone Natural products O=C(C)[C@@H]1[C@@]2(C)[C@H]([C@H]3[C@@H]([C@]4(C)C(=CC3)C[C@@H](O)CC4)CC2)CC1 ORNBQBCIOKFOEO-YQUGOWONSA-N 0.000 description 2
- RJKFOVLPORLFTN-LEKSSAKUSA-N Progesterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)CC2 RJKFOVLPORLFTN-LEKSSAKUSA-N 0.000 description 2
- 101000744001 Ruminococcus gnavus (strain ATCC 29149 / VPI C7-9) 3beta-hydroxysteroid dehydrogenase Proteins 0.000 description 2
- 102100021588 Sterol carrier protein 2 Human genes 0.000 description 2
- MUCRYNWJQNHDJH-OADIDDRXSA-N Ursonic acid Chemical compound C1CC(=O)C(C)(C)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(C(O)=O)CC[C@@H](C)[C@H](C)[C@H]5C4=CC[C@@H]3[C@]21C MUCRYNWJQNHDJH-OADIDDRXSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- SMZOGRDCAXLAAR-UHFFFAOYSA-N aluminium isopropoxide Chemical compound [Al+3].CC(C)[O-].CC(C)[O-].CC(C)[O-] SMZOGRDCAXLAAR-UHFFFAOYSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 210000000941 bile Anatomy 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 125000004432 carbon atom Chemical group C* 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 235000012000 cholesterol Nutrition 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- UACIBCPNAKBWHX-CTBOZYAPSA-N gonane Chemical group C1CCC[C@@H]2[C@H]3CC[C@@H]4CCC[C@H]4[C@@H]3CCC21 UACIBCPNAKBWHX-CTBOZYAPSA-N 0.000 description 2
- 238000005805 hydroxylation reaction Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- GHXZPUGJZVBLGC-UHFFFAOYSA-N iodoethene Chemical compound IC=C GHXZPUGJZVBLGC-UHFFFAOYSA-N 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 125000004433 nitrogen atom Chemical group N* 0.000 description 2
- 208000008338 non-alcoholic fatty liver disease Diseases 0.000 description 2
- 239000003921 oil Substances 0.000 description 2
- 239000012044 organic layer Substances 0.000 description 2
- 229960000249 pregnenolone Drugs 0.000 description 2
- ORNBQBCIOKFOEO-QGVNFLHTSA-N pregnenolone Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)CC2 ORNBQBCIOKFOEO-QGVNFLHTSA-N 0.000 description 2
- 238000002864 sequence alignment Methods 0.000 description 2
- 230000003595 spectral effect Effects 0.000 description 2
- 108010058363 sterol carrier proteins Proteins 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 2
- 238000010626 work up procedure Methods 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- 108700020831 3-Hydroxyacyl-CoA Dehydrogenase Proteins 0.000 description 1
- 108030006229 3-alpha,7-alpha,12-alpha-trihydroxy-5-beta-cholestanoyl-CoA 24-hydroxylases Proteins 0.000 description 1
- 102100021834 3-hydroxyacyl-CoA dehydrogenase Human genes 0.000 description 1
- 108030001538 3-oxo-5-beta-steroid 4-dehydrogenases Proteins 0.000 description 1
- VJGNBLOGSXHTLR-JFXJTJEYSA-N 7a,12a-dihydroxy-cholestene-3-one Chemical compound C([C@H]1O)C2CC(=O)C=C[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)[C@@H](O)C2 VJGNBLOGSXHTLR-JFXJTJEYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 101710120269 Acyl-CoA thioester hydrolase YbgC Proteins 0.000 description 1
- 102100024086 Aldo-keto reductase family 1 member D1 Human genes 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 101150065749 Churc1 gene Proteins 0.000 description 1
- 208000003311 Cytochrome P-450 Enzyme Inhibitors Diseases 0.000 description 1
- 108090000371 Esterases Proteins 0.000 description 1
- 206010019708 Hepatic steatosis Diseases 0.000 description 1
- 101001045218 Homo sapiens Peroxisomal multifunctional enzyme type 2 Proteins 0.000 description 1
- 108090000856 Lyases Proteins 0.000 description 1
- 102000004317 Lyases Human genes 0.000 description 1
- 102000008109 Mixed Function Oxygenases Human genes 0.000 description 1
- 108010074633 Mixed Function Oxygenases Proteins 0.000 description 1
- 102100022587 Peroxisomal multifunctional enzyme type 2 Human genes 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 102000004879 Racemases and epimerases Human genes 0.000 description 1
- 108090001066 Racemases and epimerases Proteins 0.000 description 1
- 102000037054 SLC-Transporter Human genes 0.000 description 1
- 108091006207 SLC-Transporter Proteins 0.000 description 1
- 108010015330 Steroid 17-alpha-Hydroxylase Proteins 0.000 description 1
- 102000001854 Steroid 17-alpha-Hydroxylase Human genes 0.000 description 1
- 238000006069 Suzuki reaction reaction Methods 0.000 description 1
- UVIQSJCZCSLXRZ-UBUQANBQSA-N abiraterone acetate Chemical compound C([C@@H]1[C@]2(C)CC[C@@H]3[C@@]4(C)CC[C@@H](CC4=CC[C@H]31)OC(=O)C)C=C2C1=CC=CN=C1 UVIQSJCZCSLXRZ-UBUQANBQSA-N 0.000 description 1
- 229960004103 abiraterone acetate Drugs 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000003560 cancer drug Substances 0.000 description 1
- GGCLNOIGPMGLDB-GYKMGIIDSA-N cholest-5-en-3-one Chemical compound C1C=C2CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 GGCLNOIGPMGLDB-GYKMGIIDSA-N 0.000 description 1
- NYOXRYYXRWJDKP-UHFFFAOYSA-N cholestenone Natural products C1CC2=CC(=O)CCC2(C)C2C1C1CCC(C(C)CCCC(C)C)C1(C)CC2 NYOXRYYXRWJDKP-UHFFFAOYSA-N 0.000 description 1
- 230000001906 cholesterol absorption Effects 0.000 description 1
- 229940125904 compound 1 Drugs 0.000 description 1
- 229940125898 compound 5 Drugs 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 239000010779 crude oil Substances 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- FMGSKLZLMKYGDP-USOAJAOKSA-N dehydroepiandrosterone Chemical class C1[C@@H](O)CC[C@]2(C)[C@H]3CC[C@](C)(C(CC4)=O)[C@@H]4[C@@H]3CC=C21 FMGSKLZLMKYGDP-USOAJAOKSA-N 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003596 drug target Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- OAYLNYINCPYISS-UHFFFAOYSA-N ethyl acetate;hexane Chemical class CCCCCC.CCOC(C)=O OAYLNYINCPYISS-UHFFFAOYSA-N 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 230000014101 glucose homeostasis Effects 0.000 description 1
- 230000033444 hydroxylation Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- GCYXWQUSHADNBF-AAEALURTSA-N preproglucagon 78-108 Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1N=CNC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 GCYXWQUSHADNBF-AAEALURTSA-N 0.000 description 1
- 239000000186 progesterone Substances 0.000 description 1
- 229960003387 progesterone Drugs 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- ABMYEXAYWZJVOV-UHFFFAOYSA-N pyridin-3-ylboronic acid Chemical compound OB(O)C1=CC=CN=C1 ABMYEXAYWZJVOV-UHFFFAOYSA-N 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 230000003637 steroidlike Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000000844 transformation Methods 0.000 description 1
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 1
- 230000004584 weight gain Effects 0.000 description 1
- 235000019786 weight gain Nutrition 0.000 description 1
- 238000002424 x-ray crystallography Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J43/00—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton
- C07J43/003—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton not condensed
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/04—Anorexiants; Antiobesity agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
Definitions
- Human Cytochrome P450 8B1 is the oxysterol 12 ⁇ -hydroxylase enzyme that converts 7 ⁇ -hydroxy-cholest-4-en-3-one to 7 a, 12 a -dihydroxycholest-4-en-3-one. This enzymatic activity ultimately results in the formation of cholic acid, the bile add with enhanced cholesterol absorption properties. Studies implicated this enzyme as a good drug target for nonalcoholic fatty liver disease and type 2 diabetes, but there are no selective inhibitors known for this enzyme and no structures to guide inhibitor development. As a sub-type of human cytochrome P450, the CYP8B1 active site may be compared with other similar P450 enzymes to identify features that may be useful in design of selective cytochrome P450 inhibitors across various sub-types.
- a steroid that contains a C12-pyridine ring was designed and synthesized as a possible inhibitor for cytochrome P450 8B1.
- This inhibitor possessed a 3 ⁇ -hydroxy-D 5 - steroid system, which contrasts with the 3-keto-D 4 -steroid backbone of the physiological substrate.
- two other variations of the originally designed inhibitor were synthesized: one with a 3-keto-D 4 steroid backbone and the other with a 3-hydroxy-D 4 -steroid backbone.
- Cytochrome P4508B1 (P450 8B1 or CYP8B1) is the oxysterol 12 ⁇ -hydroxylase enzyme responsible for converting its endogenous substrate, 7 ⁇ -hydroxycholest-4-en-3-one, to 7 ⁇ , 12 ⁇ - dihydroxycholest-4-en-3-one ( Figure 1, compounds 3 and 4, respectively).
- This activity results in the formation of cholic acid as shown in Figure 1.
- Chenodeoxycholic acid is the primary bile acid that is formed without P450 8B1 activity as shown in Figure 1.
- Mice lacking the gene that encodes for P450 8B1 resist weight gain and have improved glucose homeostasis through an increase of glucagon like peptide- 1 (GLP-1). Furthermore, knockdown led to regression in hepatic steatosis.
- the inhibition of P450 8B1 is a potential therapeutic strategy to treat obesity and cardiovascular diseases.
- Abiraterone is a steroid inhibitor for P450 17A1, the 17 ⁇ -hydroxylase enzyme that hydroxylates the 17-position of its steroid substrates, pregnenolone and progesterone.
- abiraterone bears (i) a 3 ⁇ -hydroxy D 3 -steroid backbone, reminiscent of pregnenolone, the substrate for P450 17A1, and (ii) a pyridine at the 17-position, which contains the nitrogen lone pair that can coordinate to the iron active site of P450 17A1.
- a pyridine substituent was incorporated at the 12- position of a steroid molecule through a Suzuki cross coupling between 3-pyridylboronic acid and the vinyl iodide at the 12-position of the steroid.
- the vinyl iodide was synthesized from a dehydroepiandrosterone (DHEA) derivative, which contained a 3 ⁇ -hydroxy-D 5 steroid backbone.
- DHEA dehydroepiandrosterone
- a P450 8B1 inhibitor with the 3 ⁇ -hydroxy-D 5 steroid backbone, as shown in Figure 2 was used in mice, it was likely that this compound undergoes various transformations in vivo due to the presence of catalytically accommodating steroid metabolizing enzymes (3 ⁇ - hydroxysteroid dehydrogenase and 170-hydroxysteroid dehydrogenase).
- catalytically accommodating steroid metabolizing enzymes (3 ⁇ - hydroxysteroid dehydrogenase and 170-hydroxysteroid dehydrogenase).
- abiraterone is delivered in vivo as a prodrug, abiraterone acetate, where its acetate at C3 is cleaved by esterases to its active form, abiraterone.
- abiraterone is oxidized and isomerized to the 3-keto-D 4 backbone by the action of 3 ⁇ -hydroxysteroid dehydrogenase.
- Other downstream steroid metabolizing enzymes convert the steroid backbone of the abiraterone metabolite further (e.g. 5a-reductase) and have different biological activities from its parent compound.
- the steroid AB ring of the originally designed P4508B1 inhibitor which possessed the 3 ⁇ -hydroxy-D 5 AB ring system, was converted to the 3-keto-D 4 steroid backbone, as shown in Figure 2, compunds 7 and 8, in order to mimic the physiological substrate, 7 ⁇ -hydroxycholest-4-en-3-one (Figure 1, compound 3).
- the 3-keto-D 4 steroid (compound 8) was reduced to the 3,17-dihydroxy D 4 steroid (compound 9) to determine if the different oxidation states at the 3- and 17-positions of the ligand potentially alter the interaction between the ligand and the enzyme.
- Fig. 1 is an illustration of cholic acid and chenodeoxycholic acid biosynthesis from cholesterol.
- FIG. 2 is an illustration of the synthesis of 12-pyridine containing steroid analogs as putative inhibitors of P450 8B1.
- Fig. 3A is a three-dimensional structure of a 12-pyridine steroid analog showing distance calculation between C6 and the nitrogen atom and the furthest carbon atom of the pyridine heterocycle at Cl 2.
- Fig. 3B is a three dimensional structure of a 12-pyridine steroid analog showing distance calculation between the C12 position and the C5 position of the P450 8B1 substrate 7 ⁇ - hydroxycholest-4-en-3 -one.
- Fig. 4 is a primary sequence alignment between P450 8B1 (NP 004385.2), P450 8 Al (NP 00952.1), P4507A1 (NP 000771.2), P4507B1 (NP0048U.1), and P450 39A1 (NP 057677.2)), showing the conserved tryptophan residue.
- Y174, D211, W281, and R479 are marked with an asterisk (*).
- Fig. 5 A is a crystal structure illustration of P450 8 Al with an inhibitor showing the distance between the heme iron and the tryptophan-281 in the I-helix.
- Fig. 5B is a crystal structure illustration of P450 7A1 with cholestenone showing the distance between the heme iron and the tryptophan-284 in the I-helix.
- Fig. 6A is a NMR spectra of steroid analog compound 8.
- Fig. 6B is a NMR spectra of steroid analog compound 8.
- Fig. 7 A is a NMR spectra of steroid analog compound 9.
- Fig., 7B is a NMR spectra of steroid analog compound 9.
- substantially is intended to mean a quantity, property, or value that is present to a great or significant extent and less than, more than or equal to total.
- substantially vertical may be less than, greater than, or equal to completely vertical.
- references to “embodiment” or “variant”, e.g., “one embodiment,” “an embodiment,” “example embodiment,” “various embodiments,” etc., may indicate that the embodiment(s) or variants) of the invention so described may include a particular feature, structure, or characteristic, but not every embodiment necessarily includes the particular feature, structure, or characteristic. Further, repeated use of the phrase “in one embodiment,” or “in an exemplary embodiment,” do not necessarily refer to the same embodiment or variant, although they may.
- the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those maimers, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts. Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is in no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.
- steroid nomenclature used herein is intended to be understood with reference to the gonane structure having 17 carbon atoms arranged in four rings s conventionally denoted by the letters A, B, C and D.
- the gonane parent structure or steroid nucleus is capable of being modified in practically unlimited manners by removal, replacement or addition of various moeties at various positions, including the saturation and unsaturation of bonds within the steroid nucleus.
- all structures are intended to be disclosed in either or both the cis or trans isomers of the same.
- FIG. 1 there is illustrated the biosynthesis of cholic acid from cholesterol (compound 1).
- P450 8B1 incorporates the 12 ⁇ -hydroxy group in its substrate, 7 ⁇ -hydroxy- cholest-4-en-3-one (compound 3), to yield 7 ⁇ ,12 ⁇ -dihydroxy-cholest-4-en-3-one (compound 4).
- the tryptophan in the I-helix (W281), which is conserved in P450 8A1 (19), P450 7A1, P4507B1, and P45039A1, as reflected in the sequence alignment in Figure 4, plays a role in an interdomain interaction between the I-helix, D211 in the F-G loop, Y174 in the E-helix (Y169 in P450 8A1), and R479 beyond the L-helix (R480 in P450 8A1).
- the distance between the tryptophan residue (W281) and the iron in the active site was about 7-8 A, which is smaller than the distance between the C5-position and the nitrogen atom in the pyridine heterocycle in the ligand at 9 A. Therefore, the wild type P450 8B1 should not be able to accommodate the space in the active site for the ligand in P450 8B1.
- the crystal structures of P450 8 Al and P4507A1 are available, and this conserved tryptophan residue is 7-9 A away from the iron in the heme active site, confirming that the key tryptophan may play a role in substrate recognition and binding to the active site.
- the crude oil was purified by column chromatography (50 % ethyl acetate hexanes — > 100 % ethyl acetate) to yield the 3-keto-D 4 -pyridine product 8 (1 g) with the l-methyl-4-piperidone as a major contaminant.
- Figures 7 A and 7B are the respective NMR spectra for Compound 9.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Diabetes (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Engineering & Computer Science (AREA)
- Hematology (AREA)
- Obesity (AREA)
- Gastroenterology & Hepatology (AREA)
- Emergency Medicine (AREA)
- Endocrinology (AREA)
- Child & Adolescent Psychology (AREA)
- Cardiology (AREA)
- Heart & Thoracic Surgery (AREA)
- Steroid Compounds (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The present disclosure provides for a synthetic strategy to incorporate a C12α-hydroxy group from the methylene (-CH2-) in a steroid backbone, combining synthetic chemistry and enzymology techniques to develop a selective inhibitor for cytochrome P450 8B1, and developing a selective P450 8B1 inhibitor, which can be used as a tool to study P450 8B1 and treat health issues.
Description
Title:
[001] ENZYME COMPOSITIONS, STEROID DERIVATIVES, ENZYME INHIBITORS, AND METHODS OF MAKING SAME FOR PHARMACEUTICAL APPLICATIONS
Background of the Disclosure
[002] Human Cytochrome P450 8B1 is the oxysterol 12α-hydroxylase enzyme that converts 7α-hydroxy-cholest-4-en-3-one to 7 a, 12 a -dihydroxycholest-4-en-3-one. This enzymatic activity ultimately results in the formation of cholic acid, the bile add with enhanced cholesterol absorption properties. Studies implicated this enzyme as a good drug target for nonalcoholic fatty liver disease and type 2 diabetes, but there are no selective inhibitors known for this enzyme and no structures to guide inhibitor development. As a sub-type of human cytochrome P450, the CYP8B1 active site may be compared with other similar P450 enzymes to identify features that may be useful in design of selective cytochrome P450 inhibitors across various sub-types.
Accordingly, while the present disclosure references compounds that inhibit P4508B1 activity, it is postulated within the scope of the present disclosure that the referenced compounds may exhibit inhibition activity across other P450 sub-types.
[003] Previously, a steroid that contains a C12-pyridine ring was designed and synthesized as a possible inhibitor for cytochrome P450 8B1. This inhibitor possessed a 3 β-hydroxy-D5- steroid system, which contrasts with the 3-keto-D4-steroid backbone of the physiological substrate. In order to determine if binding affinity changes when the ligand obtains a 3-keto-D4-steroid backbone in the A ring, two other variations of the originally designed inhibitor were synthesized: one with a 3-keto-D4 steroid backbone and the other with a 3-hydroxy-D4-steroid backbone.
[004] None of the compounds elicited UV-Vis spectral changes of the Soret band when added to the enzyme, suggesting a lack of interaction between the ligand and the active site of the P450. However, out of the three compounds tested, only the steroid with the 3-keto-D4 steroid backbone successfully crystallized with cytochrome P450 8B1. The ligand was not bound directly to the heme active site of P450 8B1, which was consistent with the binding experiments monitored with UV-Vis spectroscopy. Although the 12-pyridine compounds were not directly bound to the wild type P450 8B1 enzyme as monitored by UV-Vis absorbance assays and X-ray
crystallography, these compounds served as tools to enhance our understanding of the structurefunction relationship of P450 enzymes.
[005] Cytochrome P4508B1 (P450 8B1 or CYP8B1) is the oxysterol 12α-hydroxylase enzyme responsible for converting its endogenous substrate, 7α-hydroxycholest-4-en-3-one, to 7α, 12α- dihydroxycholest-4-en-3-one (Figure 1, compounds 3 and 4, respectively). This activity results in the formation of cholic acid as shown in Figure 1. Chenodeoxycholic acid is the primary bile acid that is formed without P450 8B1 activity as shown in Figure 1. Mice lacking the gene that encodes for P450 8B1 resist weight gain and have improved glucose homeostasis through an increase of glucagon like peptide- 1 (GLP-1). Furthermore,
knockdown led to regression in hepatic steatosis. Thus, the inhibition of P450 8B1 is a potential therapeutic strategy to treat obesity and cardiovascular diseases.
[006] Towards the efforts to develop a P450 8B1 inhibitor, a pyridine-containing steroid
was synthesized as reported by Chung, E., et al., (2022) A synthesis of a rationally designed inhibitor of cytochrome P4508B1, a therapeutic target to treat obesity. Steroids 178, 108952, which was inspired based on the strategy for abiraterone, a prostate cancer drug, Barrie, S. E., et al, (1994) Pharmacology of novel steroidal inhibitors of cytochrome P45017a (17a- hydroxylase/C 17-20 lyase). J. Ster. Biochem. Mol. Biol. 50, 267-273. Abiraterone is a steroid inhibitor for P450 17A1, the 17α-hydroxylase enzyme that hydroxylates the 17-position of its steroid substrates, pregnenolone and progesterone. In order to target P450 17A1 activity, abiraterone bears (i) a 3β-hydroxy D3-steroid backbone, reminiscent of pregnenolone, the substrate for P450 17A1, and (ii) a pyridine at the 17-position, which contains the nitrogen lone pair that can coordinate to the iron active site of P450 17A1. Therefore, in order to access a rationally designed inhibitor for P450 8B1, a pyridine substituent was incorporated at the 12- position of a steroid molecule through a Suzuki cross coupling between 3-pyridylboronic acid and the vinyl iodide at the 12-position of the steroid. The vinyl iodide was synthesized from a dehydroepiandrosterone (DHEA) derivative, which contained a 3β-hydroxy-D5 steroid backbone. [007] Previously, a P450 8B1 inhibitor with the 3β-hydroxy-D5 steroid backbone, as shown in Figure 2 was used in mice, it was likely that this compound undergoes various transformations in vivo due to the presence of catalytically accommodating steroid metabolizing enzymes (3β- hydroxysteroid dehydrogenase and 170-hydroxysteroid dehydrogenase). For instance, abiraterone is delivered in vivo as a prodrug, abiraterone acetate, where its acetate at C3 is
cleaved by esterases to its active form, abiraterone. In turn, abiraterone is oxidized and isomerized to the 3-keto-D4 backbone by the action of 3β-hydroxysteroid dehydrogenase. Other downstream steroid metabolizing enzymes convert the steroid backbone of the abiraterone metabolite further (e.g. 5a-reductase) and have different biological activities from its parent compound.
[008] In the present disclosure, the steroid AB ring of the originally designed P4508B1 inhibitor, which possessed the 3β-hydroxy-D5 AB ring system, was converted to the 3-keto-D4 steroid backbone, as shown in Figure 2, compunds 7 and 8, in order to mimic the physiological substrate, 7α-hydroxycholest-4-en-3-one (Figure 1, compound 3). Furthermore, the 3-keto-D4 steroid (compound 8) was reduced to the 3,17-dihydroxy D4 steroid (compound 9) to determine if the different oxidation states at the 3- and 17-positions of the ligand potentially alter the interaction between the ligand and the enzyme.
[009] Only one of the three ligands bearing the 12-pyridine moiety in the steroid backbone (i.e. the 3-keto-D4 compound 8), crystallized with P4508B1. Although it was determined that the 12- pyridyl 3-keto-D4 steroid did not bind directly to the iron in the heme active site of P450 8B1, the structure of this complex was informative. The compound that crystallized with P4508B1, the 3-keto-D4 compound 8, was the compound that resembles most closely to the endogenous substrate of P450 8B1 with its 3-ketone and double bond at the 4-5 position (i.e. compound 3).
Brief Description of the Figures
[010] Fig. 1 is an illustration of cholic acid and chenodeoxycholic acid biosynthesis from cholesterol.
[Oil] Fig. 2 is an illustration of the synthesis of 12-pyridine containing steroid analogs as putative inhibitors of P450 8B1.
[012] Fig. 3A is a three-dimensional structure of a 12-pyridine steroid analog showing distance calculation between C6 and the nitrogen atom and the furthest carbon atom of the pyridine heterocycle at Cl 2.
[013] Fig. 3B is a three dimensional structure of a 12-pyridine steroid analog showing distance calculation between the C12 position and the C5 position of the P450 8B1 substrate 7α- hydroxycholest-4-en-3 -one.
[014] Fig. 4 is a primary sequence alignment between P450 8B1 (NP 004385.2), P450 8 Al (NP 00952.1), P4507A1 (NP 000771.2), P4507B1 (NP0048U.1), and P450 39A1 (NP 057677.2)), showing the conserved tryptophan residue. Y174, D211, W281, and R479 are marked with an asterisk (*).
[015] Fig. 5 A is a crystal structure illustration of P450 8 Al with an inhibitor showing the distance between the heme iron and the tryptophan-281 in the I-helix.
[016] Fig. 5B is a crystal structure illustration of P450 7A1 with cholestenone showing the distance between the heme iron and the tryptophan-284 in the I-helix.
[017] Fig. 6A is a NMR spectra of steroid analog compound 8.
[018] Fig. 6B is a NMR spectra of steroid analog compound 8.
[019] Fig. 7 A is a NMR spectra of steroid analog compound 9.
[020] Fig., 7B is a NMR spectra of steroid analog compound 9.
Detailed Description of the Disclosure
[021] The terminology used herein is for the purpose of describing example embodiments only and is not intended to be limiting. As used herein, the singular forms “a,: “an,” and “the” may be intended to include the plural forms as well, unless the context clearly indicates otherwise. The terms “comprises,” “comprising,” “including,” and “having,” are inclusive and therefore specify the presence of stated features, integers, steps, operations, cells, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, cells, components, and/or groups thereof. The method steps, processes, and operations described herein are not to be construed as necessarily requiring their performance in the order discussed or illustrated, unless specifically identified as an order of performance. It is also to be understood that additional or alternative steps may be employed.
[022] “Substantially” is intended to mean a quantity, property, or value that is present to a great or significant extent and less than, more than or equal to total. For example, “substantially vertical” may be less than, greater than, or equal to completely vertical.
[023] “About” is intended to mean a quantity, property, or value that is present at ±10%.
Throughout this disclosure, the numerical values represent approximate measures or limits to ranges to encompass minor deviations from the given values and embodiments having about the value mentioned as well as those having exactly the value mentioned. Other than in the working
examples provided at the end of the detailed description, all numerical values of parameters (e.g., of quantities or conditions) in this specification, including the appended claims, are to be understood as being modified in all instances by the term “about” whether or not “about” actually appears before the numerical value. “About” indicates that the stated numerical value allows some slight imprecision (with some approach to exactness in the value; approximately or reasonably close to the value; nearly). If the imprecision provided by “about” is not otherwise understood in the art with this ordinary meaning, then “about” as used herein indicates at least variations that may arise from ordinary methods of measuring and using such parameters. In addition, disclosure of ranges includes disclosure of all values and further divided ranges within the entire range, including endpoints given for the ranges.
[024] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the recited range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.
[025] References to “embodiment” or “variant”, e.g., “one embodiment,” “an embodiment,” “example embodiment,” “various embodiments,” etc., may indicate that the embodiment(s) or variants) of the invention so described may include a particular feature, structure, or characteristic, but not every embodiment necessarily includes the particular feature, structure, or characteristic. Further, repeated use of the phrase “in one embodiment,” or “in an exemplary embodiment,” do not necessarily refer to the same embodiment or variant, although they may. [026] As used herein the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those maimers, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts. Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is in no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from
grammatical organization or punctuation, or the number or type of aspects described in the specification.
[027] Chemical nomenclature used herein is intended to be understood with reference to commonly employed nomenclature developed by the International Union of Pure and Applied Chemistry (IUPAC).
[028] Finally, steroid nomenclature used herein is intended to be understood with reference to the gonane structure having 17 carbon atoms arranged in four rings s conventionally denoted by the letters A, B, C and D. The gonane parent structure or steroid nucleus, is capable of being modified in practically unlimited manners by removal, replacement or addition of various moeties at various positions, including the saturation and unsaturation of bonds within the steroid nucleus. As used herein, all structures are intended to be disclosed in either or both the cis or trans isomers of the same.
[029] This detailed description of exemplary embodiments makes reference to the accompanying drawings, which show exemplary embodiments by way of illustration. While these exemplary embodiments are described in sufficient detail to enable those skilled in the art to practice the disclosure, it should be understood that other embodiments may be realized and that logical changes and adaptations in design and construction may be made in accordance with this disclosure and the teachings herein without departing from the spirit and scope of the disclosure. Thus, the detailed description herein is presented for purposes of illustration only and not for purposes of limitation.
[030] Turning to Figure 1, there is illustrated the biosynthesis of cholic acid from cholesterol (compound 1). P450 8B1 incorporates the 12α-hydroxy group in its substrate, 7α-hydroxy- cholest-4-en-3-one (compound 3), to yield 7α,12α-dihydroxy-cholest-4-en-3-one (compound 4). Nine subsequent steps modify the A-ring and truncate the side chain to yield cholic acid (compound 5) (or chenodeoxycholic acid (compound 6) with no P450 8B1 activity) involve: (i) 3-oxo-5β-steroid 4-dehydrogenase, that reduces the C4-double bond), (ii) 3a-hydroxysteroid dehydrogenase to reduce the 3-ketone, (iii) P45027A1, (iv) solute carrier family member 27 member 5 (SLC27A5), (v) a-methylacyl-CoA racemase (AMCR), (vi) 3α,7α,12α-trihydroxy-5β- cholestanoyl-CoA 24-hydroxylase (ACOX2), (vii) 3-hydroxyacyl-CoA dehydrogenase (HSD17B4), (viii) sterol carrier protein 2 (SCP2), and (ix) acyl-CoA thioesterase, as described by Chevre, R., et al., (2018) Therapeutic modulation of the bile add pool bv
knockdown
protects against nonalcoholic fatty liver disease in mice. FASEB.J. 32, 3792-3802; Russell, D. W., et al., (1992) Bile Acid Biosynthesis. Biochemistry 31, 4737-4749, and/or Russell, D. W. (2003) The Enzymes, Regulation, and Genetics of Bile Acid Synthesis. Annu. Rev. Biochem. 72, 137-174.
Chemical Synthesis of Compounds
[031] A illustrated in Figure 2, the 3β-hydroxy-D5 steroid with a 12-pyridine moiety (compound 7), was treated under Oppenauer conditions to yield the 3,17-diketone (compound 8). This resulting diketone was treated with NaBH4 to yield the 3,17-dihydroxy product (compound 9). These three compounds where then experimentally tested for P450 8B1 inhibition activity.
Experiments with 12-Pyridine Substituted Steroids with P4508B1
[032] When the ligands bearing a 12-pyridine moiety were added to purified P450 8B1, no shift in the Soret band was detected. Traditionally, inhibitors that containing nitrogen heteroatoms bind to P450 enzymes cause a type n binding difference spectra when monitoring the Soret band via UV-Vis spectroscopy. In contrast, addition of substrates to P450 enzymes usually result in type I binding difference spectra. Hence, the fact that none of the 12-pyridine bearing steroids caused any spectral changes when added to P450 8B1, suggested that none of these ligands directly bind to the heme active site of the enzyme.
[033] Crystal structure analysis was conducted and suggested that tryptophan-281 (W281) in the I-helix of P450 8B1 hovers about 7-8 A above the heme active site, limiting the size of the ligand that can access the active site of the enzyme. Therefore, since the distance between W281 and the iron in the heme active site of P450 8B1 and the distance between C6 and the furthest carbon in the pyridine is 9.2 A (Figure 3A), it is possible that the C12-pyridine steroids are too large to access the active site of P450 8B1. In contrast, the distance between C12 and C5 of the P450 8B1 substrate, 7α-hydroxycholest-4-en-3-one, is 5-6 A (Figure 3B).
[034] As shown in Figure 3 A, the tryptophan in the I-helix (W281), which is conserved in P450 8A1 (19), P450 7A1, P4507B1, and P45039A1, as reflected in the sequence alignment in Figure 4, plays a role in an interdomain interaction between the I-helix, D211 in the F-G loop, Y174 in the E-helix (Y169 in P450 8A1), and R479 beyond the L-helix (R480 in P450 8A1).
[035] Furthermore, the distance between the tryptophan residue (W281) and the iron in the active site was about 7-8 A, which is smaller than the distance between the C5-position and the nitrogen atom in the pyridine heterocycle in the ligand at 9 A. Therefore, the wild type P450 8B1 should not be able to accommodate the space in the active site for the ligand in P450 8B1. The crystal structures of P450 8 Al and P4507A1 are available, and this conserved tryptophan residue is 7-9 A away from the iron in the heme active site, confirming that the key tryptophan may play a role in substrate recognition and binding to the active site.
[036] Although none of compounds 7, 8 or 9, yielded type II binding difference spectra with P450 8B1, inhibition assays suggested that the compounds have moderate inhibitory activity towards 12α-hydroxylation activity of 7α-hydroxycholest-4-en-3-one by P450 8B1.
W281F Mutation in P4508BI Has Enhanced Interaction "with I2-Pyridine Substituted Steroids
[037] To determine whether W281 of P450 8B1 plays a role in substrate (oxysterol) binding at the active site, a W281F site directed mutant was expressed and purified. When all three of the 12-pyridyl steroid compounds were added to the W281F mutant while monitoring by UV-Vis absorbance spectroscopy, a type 11 binding difference spectrum resulted. These observations confirm that the tryptophan plays a role in substrate binding above the heme active site. The W281F variant of P450 8B1 is not as active as the wild type, i.e. higher KM towards 12α- hydroxylation with the substrate, 7α-hydroxycholest-4-en-3-one). There were no alternative products identified by HPLC-UV analysis, scanning with 240 nm, when enzyme incubation extracts were analyzed. However, these observations do not rule out the possibility of other products formed by the P450 8B1 W281F variant, e.g. conjugation of the double bond could be lost if the enzyme epoxidized the double bond at C4 of compound 3.
[038] Three 12-pyridyl steroid compounds were synthesized and their interaction tested against human cytochrome P450 8B1. One of the compounds, bearing a 3-keto-D4 steroid backbone (compound 8), was successfully crystallized with the enzyme. The compound was not bound to the heme active site, but the structure of the complex with the ligand allowed for a deeper understanding of how P450 8B1 binds with its substrate. Tryptophan-281 in the I-helix of P450 8B1, which interacted with D211 in the F-G loop and hovered 8 A, above the iron in the active site, was identified as a residue that may limit the length of the ligand for entry to the active site.
Furthermore, binding studies with the W281F variant of P450 8B1 suggest that the ligand cannot reach the active site of the heme iron due to the W281 residue. Therefore, the 12-pyridyl steroid ligands are likely too long at about 9 A to be accommodated to bind in the active site of P450 8B1. Nevertheless, the 12-pyridine containing ligands are ample tools to investigate and understand the structure-function properties of human cytochrome P450 8B1 as a potential therapeutic target to treat obesity as well as other disorders mediated by P4508B1.
Synthesis of 12-(3 ’ -pyridyl) -androsta-4,1 l-dien-3, 17-dione (Compound 8)
[039] Aluminum isopropoxide (6.46 g, 31.7 mmol, 10 eq) and 1 -methyl -4-piperidone (7.78 ml, 63.3 mmol, 20 eq) were added to a 500 ml round bottom flask containing the C12-pyridine containing 3β-hydroxy-D5 steroid 7 (1.15 g, 3.16 mmol, 1 eq) in toluene (250 ml). The reaction was refluxed using a Dean-Stark apparatus where toluene was removed in portions (3 x 20 ml). The volume of the reaction was never reduced below 150 ml. The progress of the reaction was monitored by TLC and NMR. After 24 h, a second portion of 1 -methyl -4-piperidone (10 ml, 86.6 mmol, 27 eq) was added to the reaction and refluxing continued for another 24 h. The reaction solution was diluted with ethyl acetate (200 ml). The resulting solution was washed with deionized water (3 x 200 ml). The aluminum isopropoxide suspension that crashed out during the workup was filtered off with a fritted filter funnel under vacuum. The organic layer was concentrated under reduced pressure to form a crude yellow oil. The crude oil was purified by column chromatography (50 % ethyl acetate hexanes — > 100 % ethyl acetate) to yield the 3-keto-D4-pyridine product 8 (1 g) with the l-methyl-4-piperidone as a major contaminant. This mixture was repurified by column chromatography (100 % ethyl acetate) to afford the 3-keto-D4-pyridine product 8 as a white fluffy solid (180 mg, 0.497 mmol, 16%); R/ 0.39 (100 % ethyl acetate); [α]D 20 + 239.3° [5.6 x 104 in CHCh]; IR (ATR) 3459.13, 3025.93, 2927.84, 1736.33, 1663.04, 1447.04 cm"1; lH NMR (500 MHz, CDCh) 8 8.56 (s, 1H), 8.46 (s, 1H), 7.69 (d, J= 7.35 Hz, 1H), 7.25 (m, 1H), 5.83 (s, 1H), 5.55 (s, 1H), 2.61 - 2.34 (m, 6H), 2.26 - 1.90 (m, 6H), 1.88 - 1.79 (m, 1H), 1.70 (m, 1H), 1.38 - 1.29 (m, 1H), 1.27 (s, 3H), 1.22 (s, 3H); 13C NMR (500 MHz, CDCh) 5 214.60, 199.08, 168.41, 149.18, 148.06, 143.06, 136.92, 136.26, 127.88, 125.77, 122.39, 56.42, 51.26, 49.11, 39.18, 36.57, 35.32, 33.74, 33.54, 33.18, 29.47, 20.39, 19.40, 17.96. The yield of this reaction was low due to the water workup step to remove the l-methyl-4-piperidone. Because both the pyridine product (8) and the 1 -methyl-4-piperidone reagent, which was in excess, are soluble in water, some
of the desired product is lost in the aqueous layer. Furthermore, during the purification by silica gel column chromatography, the reagent (l-methyl-4-piperidone) co-elutes with the desired product. These mixtures were not isolated to move forward with pure compound in the next step. [040] Figures 6A and 6B are the respective NMR spectra for Compound 8.
Synthesis of 12-(3 ’-pyridyl)-androsta-4,l 1 -diene 3a,17b-diol (Compound 9)
[041] In a 50 ml round bottom flask containing the 3-keto-D4-steroid (8) (39 mg, 0.107 mmol, 1 eq) in methanol and THF solution (10 ml, 1:1 v/v) was added NaBH4 (67 mg, 1.77 mmol, 16.5 eq). The reaction was stirred for 20 minutes. The reaction was quenched with the addition of water (20 ml) and extracted with ethyl acetate (3 x 20 ml). The organic layers were combined and concentrated under reduced pressure to afford a crude white solid residue. The crude solid was purified by column chromatography (100 % ethyl acetate — > 10 % MeOH in CH2CI2) to afford the C3, C17 -diol product 9 as a white solid (17.3 mg, 0.047 mmol, 44 %); mp 194.6 - 198.5 °C; R/ 0.33 (100 % ethyl acetate); [a]D 20 - 2050° [2 x 10-5 in CHCI3]; IR (ATR) 3282.61, 2918.36, 2849.79, 1588.03, 1435.00, 1259.65 cm-1; 1HNMR (500 MHz, CDCh) 88.55 (broad s, 2H), 7.71 (d, J= 10.15 Hz, 1H), 7.31 (m, 1H), 5.44 (s, 1H), 5.38 (s, 1H), 4.18 (m, 1H), 4.09 (m, 1H), 2.32 (apparent t, J= 11.81 Hz, 2H), 2.21 - 2.14 (m, 3H), 2.03 - 1.84 (m, 2H), 1.82 - 1.74 (m, 3H), 1.66 - 1.58 (m, 3H), 1.52 - 1.33 (m, 4H), 1.08 (s, 3H), 1.07 (s, 3H); 13C NMR (500 MHz, CDCh) 8 148.86, 147.71, 146.18, 145.83, 136.33, 127.66, 125.55, 123.04, 76.80, 68.02, 57.73, 49.22, 48.21, 38.12, 35.06, 34.24, 32.89, 31.33, 30.94, 29.28, 22.10, 19.87, 15.66.
[042] Figures 7 A and 7B are the respective NMR spectra for Compound 9.
[043] While preferred embodiments have been described in detail, it should be apparent that modifications and variations thereto are possible, all of which fall within the true spirit and scope of the invention. With respect to the above description then, it is to be realized that the optimum dimensional relationships for the parts of the invention, to include variations in size, materials, shape, form, function and manner of operation, assembly and use, are deemed readily apparent to one skilled in the art, and all equivalent relationships to those illustrated in the drawings and described in the specification are intended to be encompassed by the present invention.
[044] Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising" or the term "includes" or variations, thereof, or the term "having" or variations thereof will be understood to imply the
inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers. In this regard, in construing the claim scope, an embodiment where one or more features is added to any of the claims is to be regarded as within the scope of the invention given that the essential features of the invention as claimed are included in such an embodiment.
[045] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications that fall within its spirit and scope. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.
[046] Therefore, the foregoing is considered as illustrative only of the principles of the invention. Further, since numerous modifications and changes will readily occur to those skilled in the art, it is not desired to limit the invention to the exact construction and operation shown and described, and accordingly, all suitable modifications and equivalents may be resorted to, falling within the scope of the invention.
References
1. Murakami, K., Okada, Y., and Okuda, K. (1982) Purification and characterization of 7alpha-hydroxy-4-cholesten-3-one 12 alpha-mono oxygenase. J. Biol. Chem. 257, 8030-8035
2. Ishida, H., Noshiro, M., Okuda, K., and Coon, M. J. (1992) Purification and characterization of 7alpha-hydroxy-4-cholesten-3-one 12alpha-hydroxylase. J. Biol. Chem. 267, 21319-21323
3. Wang, D. Q.-H., Lammert, F., Cohen, D. E., Paigen, B., and Carey, M. C. (1999) American Journal of Physiology. Cholic acid aids absorption, biliary secretion, and phase transitions of cholesterol in murine cholelithogenesis 276, G751-G760
4. Woollett, L. A., Buckley, D. D., Yao, L., Jones, P. J. H., Granholm, N. A., Tolley, E. A., Tso, P., and Heubi, H. E. (2004) Cholic acid supplementation enhances cholesterol absorption in humans. Gastroenterology 126, 724-731
5. Reynier, M. O., Montet, J. C., Gerolami, A., Marteau, C., Crotte, C., Montet, A. M., and Mathieu, S. (1981) Comparative effects of cholic, chenodeoxycholic, and ursodeoxycholic adds on micellar solubilization and intestinal absorption of cholesterol. J. Lipid Res. 22, 467-473
6. Bertaggia, E., Jensen, K. K., Castro-Perez, J., Xu, Y., Di Paolo, G, Chan, R. B., Wang,
L., and Haeusler, R. A. (2017) Cyp8bl ablation prevents Weste diet-induced weight gain and hepatic steatosis because of impaired fat absorption. Am. J. Physiol. Endocrinol. Metab. 313, E121-E133
7. Kaur, A., Patankar, J. V., de Haan, W., Ruddle, P., Wijesekara, N., Groen, A. K., Verchere, C. B., Singaraja, R. R., and Hayden, M. R. (2014) Loss of Cyp8bl Improves Glucose Homeostasis by Increasing GLP-1. Diabetes 64, 1168-1179
8. Chevre, R., Trigueros-Motos, L., Castano, D., Chua, T., Corliano, M., Patankar, J. V., Sng, L., Sim, L., Juin, T. L., Carissimo, G, Ng, L. F. P., Yi, C. N. J., Eliathamby, C. C., Groen, A. K., Hayden, M. R., and Singaraja, R. R (2018) Therapeutic modulation of the bile acid pool by Cyp8bl knockdown protects against nonalcoholic fatty liver disease in mice FASEB J. 32, 3792-3802
9. Russell, D. W., and Setchell, K. D. R. (1992) Bile Acid Biosynthesis. Biochemistry 31, 4737-4749
10. Russell, D. W. (2003) The Enzymes, Regulation, and Genetics of Bile Acid Synthesis. Annu Rev. Biochem. 72, 137-174
11. Chung, E., Offei, S. D., Jia, U.-T. A., Estevez, J., Perez, ¥., Arman, H. D., and Yoshimoto, F. K. (2022) A synthesis of a rationally designed inhibitor of cytochrome P4508B1, a therapeutic target to treat obesity. Steroids 178, 108952
12. Barrie, S. E., Potter, G. A., Goddard, P. M., Haynes, B. P., Dowsett, M., and Jarman, M. (1994) Pharmacology of novel steroidal inhibitors of cytochrome P45017α (17α- hydroxylase/C 17-20 lyase). J. Ster. Biochem. Mol. Biol. 50, 267-273
13. Penning, T. M. (2003) Hydroxysteroid dehydrogenases and pre-receptor regulation of steroid hormone action. Human Reproduction Update 9, 193-205
14. Stappaerts, J., Geboers, S., Snoeys, J., Brouwers, J., Tack, J., Annaert, P., and Augustjins,
P. (2015) Rapid conversion of the ester prodrug abiraterone acetate results in intestinal supersaturation and enhanced absorption of abiraterone: in vitro, rat in situ and human in vivo studies Eur. J. Pharm. Biopharm. 90, 1-7
15. Li, Z., Bishop, A. C., Alyamani, M., Garcia, J. A., Dreicer, R., Bunch, D., Liu, J., Uoadhyay, S. K., Auchus, R. J., and Sharifi, N. (2015) Conversion of abiraterone to D4A drives anti-tumour activity in prostate cancer. Nature 523, 347-351
16. Li, Z., Alyamani, M., Li, J., Rogacki, K., Abazeed, M., Upadhyay, S. K., Balk, S. P., Taplin, M.-E., Auchus, R. J., and Sharifi, N. (2016) Conversion of abiraterone to D4A drives anti-tumour activity in prostate cancer. Nature 533 , 547-551
17. Dahal, U. P., Joswig-Jones, C., and Jones, J. P. (2012) Comparative Study of the Affinity and Metabolism of Type I and Type II Binding Quinoline Carboxamide Analogues by Cytochrome P4503 A4. J. Med. Chem. 55, 280-290
18. Offei, S. D., Arman, H. D., and Yoshimoto, F. K. (2019) Chemical synthesis of 7α- hydroxycholest-4-en-3-one, a biomarker for irritable bowel syndrome and bile acid malabsorption. Steroids 151, 108559
19. Li, Y.-C., Chiang, C.-W., Yeh, H.-C., Hsu, P. Y., Whitby, F. G., Wang, L.-H., and Chan, N.-L. (2007) Structures of Prostacyclin Synthase and Its Complexes with Substrate Analog and Inhibitor Reveal a Ligand-specific Heme Conformation Change. J. Biol. Chem. 283, 2917-2926
20. Tempel, W., Grabovec, I., MacKenzie, F., Dichenko, Y. V., Usanov, S. A., Gilep, A. A., Park, H.-W., and Strushkevich, N. (2014) Structural characterization of human cholesterol 7α- hydroxylase J. Lipid Res. 55, 1925-1932
Claims
2. The compound of Claim 1, wherein the compound exhibits pharmacological activity as an inhibitor of Cytochrome P450.
3. The compound of any of Claims 1 or 2, compound exhibits pharmacological activity as an inhibitor of Cytocrome P450 8B1.
4. A pharmacologically active composition comprising the compound of Claim 1, a prodrug thereof, or pharmaceutically actable salt thereof as an active ingredient of the composition.
5. A medicament comprising the compound of Claim 1, a prodrug thereof, or a pharmaceutically active salt thereof.
7. The compound of Claim 1, wherein the compound exhibits pharmacological activity as an inhibitor of Cytochrome P450.
8. The compound of any of Claims 1 or 2, compound exhibits pharmacological activity as an inhibitor of Cytocrome P450 8B1.
9. A pharmacologically active composition comprising the compound of Claim 1, a prodrug thereof, or pharmaceutically actable salt thereof as an active ingredient of the composition.
10. A medicament comprising the compound of Claim 1, a prodrug thereof, or a pharmaceutically active salt thereof.
11. A process for preparing the compound of Claim 1, comprising the steps of treating 3β- hydroxy-D5 steroid with a 12-pyridine moiety under Oppenauer conditions to yield a 3,17- diketone thereof.
12. The process of Claim 10, further comprising treating the 3, 17 diketone with NaBH< to yield a 3,17-dihydroxy substituted compound thereof.
13. A process for preparing the compound of Claim 6, comprising the steps of treating 3β- hydroxy-D5 steroid with a 12-pyridine moiety under Oppenauer conditions to yield a 3,17- diketone thereof.
14. The process of Claim 13, further comprising treating the 3, 17 diketone with NaBH4 to yield a 3,17-dihydroxy substituted compound thereof.
15. The use of the compound of Claim 1 in manufacturing a medicament for inhibiting Cytochrome P450-mediated disorders.
16. The use of the compound of Claim 6 in manufacturing a medicament for inhibiting Cytochrome P450-mediated disorders.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US202263390966P | 2022-07-20 | 2022-07-20 | |
US63/390,966 | 2022-07-20 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2024020520A2 true WO2024020520A2 (en) | 2024-01-25 |
WO2024020520A3 WO2024020520A3 (en) | 2024-03-28 |
Family
ID=89618526
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2023/070652 WO2024020520A2 (en) | 2022-07-20 | 2023-07-20 | Enzyme compositions, steroid derivatives, enzyme inhibitors, and methods of making same for pharmaceutical applications |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2024020520A2 (en) |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011088160A2 (en) * | 2010-01-15 | 2011-07-21 | Biomarin Pharmaceutical Inc. | Novel cyp17 inhibitors |
US20130252930A1 (en) * | 2010-12-16 | 2013-09-26 | Biomarin Pharmaceutical Inc. | Cyp11b, cyp17, and/or cyp21 inhibitors |
EP2688901B1 (en) * | 2011-03-25 | 2019-05-08 | Université Laval | INHIBITORS OF 17ß-HSD1, 17ß-HSD3 AND 17ß-HSD10 |
-
2023
- 2023-07-20 WO PCT/US2023/070652 patent/WO2024020520A2/en unknown
Also Published As
Publication number | Publication date |
---|---|
WO2024020520A3 (en) | 2024-03-28 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Vaz et al. | Bile acid analysis in human disorders of bile acid biosynthesis | |
Schroepfer Jr | Oxysterols: modulators of cholesterol metabolism and other processes | |
JP5709743B2 (en) | Process for the preparation of 17-alkynyl-7-hydroxysteroids and related compounds | |
JP2008504338A (en) | Novel 2-substituted estra-1,3,5 (10) -trien-17-ones as inhibitors of 17β-hydroxysteroid dehydrogenase type 1 | |
Bydal et al. | Inhibition of type 2 17β-hydroxysteroid dehydrogenase by estradiol derivatives bearing a lactone on the D-ring: structure–activity relationships | |
Pascal et al. | Plant sterol biosynthesis. Identification and characterization of two distinct microsomal oxidative enzymatic systems involved in sterol C4-demethylation | |
Janeczko et al. | Novel metabolites of dehydroepiandrosterone and progesterone obtained in Didymosphearia igniaria KCH 6670 culture | |
Hunter et al. | Transformation of 5-ene steroids by the fungus Aspergillus tamarii KITA: Mixed molecular fate in lactonization and hydroxylation pathways with identification of a putative 3β-hydroxy-steroid dehydrogenase/Δ5–Δ4 isomerase pathway | |
JP3816004B2 (en) | Process for producing Diels-Alder adducts of sterol 5,7-diene using unsaturated fatty acid catalysts | |
JP2643943B2 (en) | Novel 6- or 7-methyleneandrosta-1,4-diene-3,17-dione derivative and method for producing the same | |
Kim et al. | The human cytochrome P4507B1: catalytic activity studies | |
WO2024020520A2 (en) | Enzyme compositions, steroid derivatives, enzyme inhibitors, and methods of making same for pharmaceutical applications | |
Yoshimoto et al. | Epoxidation activities of human cytochromes P450c17 and P450c21 | |
Guo et al. | Chemical synthesis of 7-and 8-dehydro derivatives of pregnane-3, 17α, 20-triols, potential steroid metabolites in Smith–Lemli–Opitz syndrome | |
Hunter et al. | Predominant allylic hydroxylation at carbons 6 and 7 of 4 and 5-ene functionalized steroids by the thermophilic fungus Rhizomucor tauricus IMI23312 | |
Pascal et al. | Plant sterol biosynthesis: identification of a NADPH dependent sterone reductase involved in sterol-4 demethylation | |
Offei et al. | Chemical synthesis of 7α-hydroxycholest-4-en-3-one, a biomarker for irritable bowel syndrome and bile acid malabsorption | |
Christakoudi et al. | Steroids excreted in urine by neonates with 21-hydroxylase deficiency: characterization, using GC–MS and GC–MS/MS, of the D-ring and side chain structure of pregnanes and pregnenes | |
Lieberman et al. | 17-Hydroxylase: an evaluation of the present view of its catalytic role in steroidogenesis | |
Trzaskos et al. | Substrate-based inhibitors of lanosterol 14. alpha.-methyl demethylase: I. Assessment of inhibitor structure-activity relationship and cholesterol biosynthesis inhibition properties | |
Ogawa et al. | An efficient synthesis of 7α, 12α-dihydroxy-4-cholesten-3-one and its biological precursor 7α-hydroxy-4-cholesten-3-one: Key intermediates in bile acid biosynthesis | |
de Sain‐van der Velden et al. | Elevated cholesterol precursors other than cholestanol can also be a hallmark for CTX | |
Khomutov et al. | Laccase‐mediated oxidation of steroid alcohols in the presence of methylated β‐cyclodextrin: from inhibition to selective synthesis | |
Viger et al. | 18-Substituted progesterone derivatives as inhibitors of aldosterone biosynthesis | |
Ponce et al. | Singlet-oxygen ene reaction with 3β-substituted stigmastanes. An alternative pathway for the classical Schenck rearrangement |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 23843905 Country of ref document: EP Kind code of ref document: A2 |