PL237128B1 - Method for producing 6β,11α-dihydroxyprogesterone - Google Patents
Method for producing 6β,11α-dihydroxyprogesterone Download PDFInfo
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- PL237128B1 PL237128B1 PL422500A PL42250017A PL237128B1 PL 237128 B1 PL237128 B1 PL 237128B1 PL 422500 A PL422500 A PL 422500A PL 42250017 A PL42250017 A PL 42250017A PL 237128 B1 PL237128 B1 PL 237128B1
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- KDHBHXVDYRGRDL-KDZLLLCRSA-N 6beta,11alpha-Dihydroxyprogesterone Chemical compound C1([C@H](O)C2)=CC(=O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H](C(=O)C)[C@@]2(C)C[C@H]1O KDHBHXVDYRGRDL-KDZLLLCRSA-N 0.000 title claims abstract description 6
- 238000004519 manufacturing process Methods 0.000 title description 8
- 238000000034 method Methods 0.000 claims abstract description 14
- 241000030456 Isaria farinosa Species 0.000 claims abstract description 6
- 239000003960 organic solvent Substances 0.000 claims abstract description 6
- 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 claims description 18
- 239000000758 substrate Substances 0.000 claims description 10
- 239000000186 progesterone Substances 0.000 claims description 9
- 229960003387 progesterone Drugs 0.000 claims description 9
- 230000009466 transformation Effects 0.000 claims description 5
- 238000004587 chromatography analysis Methods 0.000 claims description 3
- 241000233866 Fungi Species 0.000 claims description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 abstract description 6
- 230000000694 effects Effects 0.000 abstract description 5
- 238000005805 hydroxylation reaction Methods 0.000 abstract description 5
- 108090000790 Enzymes Proteins 0.000 abstract description 4
- 102000004190 Enzymes Human genes 0.000 abstract description 4
- 230000033444 hydroxylation Effects 0.000 abstract description 4
- 244000005700 microbiome Species 0.000 abstract description 3
- 238000000605 extraction Methods 0.000 abstract description 2
- 125000004432 carbon atom Chemical group C* 0.000 abstract 1
- 150000003431 steroids Chemical class 0.000 description 6
- BFZHCUBIASXHPK-ODYOLWGQSA-N 11a-Hydroxyprogesterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CCC(C(=O)C)[C@@]1(C)C[C@H]2O BFZHCUBIASXHPK-ODYOLWGQSA-N 0.000 description 5
- 150000001875 compounds Chemical class 0.000 description 5
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 description 4
- FUFLCEKSBBHCMO-UHFFFAOYSA-N 11-dehydrocorticosterone Natural products O=C1CCC2(C)C3C(=O)CC(C)(C(CC4)C(=O)CO)C4C3CCC2=C1 FUFLCEKSBBHCMO-UHFFFAOYSA-N 0.000 description 3
- BFZHCUBIASXHPK-QJSKAATBSA-N 11alpha-hydroxyprogesterone 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)C[C@H]2O BFZHCUBIASXHPK-QJSKAATBSA-N 0.000 description 3
- 241000235389 Absidia Species 0.000 description 3
- MFYSYFVPBJMHGN-ZPOLXVRWSA-N Cortisone Chemical compound O=C1CC[C@]2(C)[C@H]3C(=O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 MFYSYFVPBJMHGN-ZPOLXVRWSA-N 0.000 description 3
- MFYSYFVPBJMHGN-UHFFFAOYSA-N Cortisone Natural products O=C1CCC2(C)C3C(=O)CC(C)(C(CC4)(O)C(=O)CO)C4C3CCC2=C1 MFYSYFVPBJMHGN-UHFFFAOYSA-N 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 241000700159 Rattus Species 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 230000036983 biotransformation Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 229960004544 cortisone Drugs 0.000 description 3
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 3
- 239000011541 reaction mixture Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- PWCLWZOSAFOXFL-CXICGXRGSA-N 6beta-hydroxyprogesterone Chemical compound C1([C@H](O)C2)=CC(=O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H](C(=O)C)[C@@]2(C)CC1 PWCLWZOSAFOXFL-CXICGXRGSA-N 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 241000228431 Acremonium chrysogenum Species 0.000 description 2
- 241000751139 Beauveria bassiana Species 0.000 description 2
- 241000235525 Rhizomucor pusillus Species 0.000 description 2
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 2
- 241000223259 Trichoderma Species 0.000 description 2
- 230000002280 anti-androgenic effect Effects 0.000 description 2
- 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 2
- 229960000890 hydrocortisone Drugs 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 210000002374 sebum Anatomy 0.000 description 2
- UDHAKZRCGHAPAZ-NFBMXZEVSA-N (6r,8r,9s,10r,13s,14r)-6,14-dihydroxy-10,13-dimethyl-1,2,6,7,8,9,11,12,15,16-decahydrocyclopenta[a]phenanthrene-3,17-dione Chemical compound O=C1CC[C@]2(C)[C@H]3CC[C@](C)(C(CC4)=O)[C@]4(O)[C@@H]3C[C@@H](O)C2=C1 UDHAKZRCGHAPAZ-NFBMXZEVSA-N 0.000 description 1
- XEYANIGWTWXVBC-BSWVEEBUSA-N (8r,9s,10r,13s,14r)-14-hydroxy-10,13-dimethyl-1,2,6,7,8,9,11,12,15,16-decahydrocyclopenta[a]phenanthrene-3,17-dione Chemical compound O=C1CC[C@]2(C)[C@H]3CC[C@](C)(C(CC4)=O)[C@]4(O)[C@@H]3CCC2=C1 XEYANIGWTWXVBC-BSWVEEBUSA-N 0.000 description 1
- 102000006739 11-beta-Hydroxysteroid Dehydrogenase Type 2 Human genes 0.000 description 1
- 108010086356 11-beta-Hydroxysteroid Dehydrogenase Type 2 Proteins 0.000 description 1
- 102000004277 11-beta-hydroxysteroid dehydrogenases Human genes 0.000 description 1
- 108090000874 11-beta-hydroxysteroid dehydrogenases Proteins 0.000 description 1
- BFZHCUBIASXHPK-UHFFFAOYSA-N 11beta-hydroxy-progesterone Natural products C1CC2=CC(=O)CCC2(C)C2C1C1CCC(C(=O)C)C1(C)CC2O BFZHCUBIASXHPK-UHFFFAOYSA-N 0.000 description 1
- BFZHCUBIASXHPK-ATWVFEABSA-N 11beta-hydroxyprogesterone 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)C[C@@H]2O BFZHCUBIASXHPK-ATWVFEABSA-N 0.000 description 1
- 108010029908 3-oxo-5-alpha-steroid 4-dehydrogenase Proteins 0.000 description 1
- 102000001779 3-oxo-5-alpha-steroid 4-dehydrogenase Human genes 0.000 description 1
- 201000004384 Alopecia Diseases 0.000 description 1
- 238000006220 Baeyer-Villiger oxidation reaction Methods 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 241001440741 CHER virus Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- ITRJWOMZKQRYTA-RFZYENFJSA-N Cortisone acetate Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@@](C(=O)COC(=O)C)(O)[C@@]1(C)CC2=O ITRJWOMZKQRYTA-RFZYENFJSA-N 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 1
- 241000235395 Mucor Species 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 241000908011 Trichoderma aureoviride Species 0.000 description 1
- 241000227728 Trichoderma hamatum Species 0.000 description 1
- 241000378866 Trichoderma koningii Species 0.000 description 1
- 241001304120 Trichoderma pseudokoningii Species 0.000 description 1
- 230000001919 adrenal effect Effects 0.000 description 1
- 206010068168 androgenetic alopecia Diseases 0.000 description 1
- 201000002996 androgenic alopecia Diseases 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 239000000051 antiandrogen Substances 0.000 description 1
- 239000011942 biocatalyst Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- ALEXXDVDDISNDU-JZYPGELDSA-N cortisol 21-acetate Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@@](C(=O)COC(=O)C)(O)[C@@]1(C)C[C@@H]2O ALEXXDVDDISNDU-JZYPGELDSA-N 0.000 description 1
- 229960003290 cortisone acetate Drugs 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 230000001076 estrogenic effect Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000003779 hair growth Effects 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229960001067 hydrocortisone acetate Drugs 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000003002 hypertensinogenic effect Effects 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 150000002596 lactones Chemical class 0.000 description 1
- 210000001853 liver microsome Anatomy 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000013048 microbiological method Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 230000029865 regulation of blood pressure Effects 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 230000003637 steroidlike Effects 0.000 description 1
- 210000004291 uterus Anatomy 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Steroid Compounds (AREA)
Abstract
Zgłoszenie dotyczy sposobu wytwarzania 6β,11α-dihydroksyprogesteronu o wzorze 2. Sposób polega na tym, że w wyniku działania układu enzymatycznego zawartego w komórkach szczepu Isaria farinosa KCh KW1.1 o sekwencji 1, następuje hydroksylacja przy nieaktywowanych atomach węgla C-6 i C-11. Uzyskany w ten sposób produkt wydziela się z wodnej kultury mikroorganizmu, znanym sposobem, przez ekstrakcję rozpuszczalnikiem organicznym niemieszającym się z wodą (chloroform).The application concerns a method of producing 6β,11α-dihydroxyprogesterone of the formula 2. The method consists in the fact that as a result of the activity of the enzyme system contained in the cells of the Isaria farinosa KCh KW1.1 strain with the sequence 1, hydroxylation occurs at the non-activated carbon atoms C-6 and C- 11. The product thus obtained is isolated from the aqueous culture of the micro-organism in a known manner by extraction with a water-immiscible organic solvent (chloroform).
Description
Opis wynalazkuDescription of the invention
Przedmiotem wynalazku jest sposób wytwarzania 6β ,11 α-dihydroksyprogesteronu.The present invention relates to a process for the production of 6β, 11α-dihydroxyprogesterone.
Metoda, według wynalazku może znaleźć zastosowanie w przemyśle farmaceutycznym do wytwarzania leków o właściwościach antyzapalnych i regulujących prawidłowy rozwój ciąży.The method according to the invention may find application in the pharmaceutical industry for the production of drugs with anti-inflammatory properties and regulating the proper development of pregnancy.
11a-hydroksyprogesteron jest ważnym związkiem farmaceutycznym. Ma działanie antyandrogenne i słabe właściwości estrogenne (J. Tamm, M. Seckelmann, U. Volkwein, E. Ludwig, The effect of the antiandrogen 11a-hydroxyprogesterone on sebum production and cholesterol concentration of sebum, Br. J. Dermatol. 107 (1982) 63-70). Aktywuje procesy ułatwiające zagnieżdżeniu się zapłodnionej komórce jajowej w macicy (A.H. van der Willigen, J.D. Peereboom-Wynia, T. van Joost, E. Stolz, A preliminary study of the effect of 11a-hydroxyprogesterone on the hair growth in men suffering from androgenetic alopecia, Acta Derm. Venereol 67(1987) 82-85; J. Nikolaus, K.T. Nguyen, C. Virus, J.L. Riehm, M. Hutter, R. Bernhardt, Engineering of CYP106A2 for steroid 9α- and 63-hydroxylation. Steroids 120 (2017) 41-48). Ponadto, 11a-hydroksyprogesteron wpływa na regulację ciśnienia krwi, powodując selektywne hamowanie dehydrogenazy 11 β-hydroksysteroidowej typu 2. Ten enzym metabolizuje kortyzol do kortyzonu u ludzi, a zatem związek ten jest ważny w regulacji równowagi elektrolitowej nerek (H. Morita, M. Zhou, M.F. Foecking, E.P. Gomez-Sanchez, E.N. Cozza, C.E. Gomez-Sanchez, 11 βHydroxysteroid dehydrogenase type 2 complementary deoxyribonucleic acid stably transfected into Chinese hamster ovary cells: specific inhibition by 11 a-hydroxyprogesterone, Endocrinol 137 (1996) 23082314; G.W. Souness, D.J. Morris, 11α- and 11β-hydroxyprogesterone, potent inhibitors of 11β-hydroxysteroid dehydrogenase, possess hypertensinogenic activity in the rat, Hypertension 27 (1996) 421 -425). Ponadto jako substrat można stosować 11a-hydroksyprogesteron do wytwarzania kortyzonu i hydrokortyzonu (J.A. Hogg, P.F. Beal, A.H. Nathan, F.H. Lincoln, W.P. Schneider, B.J. Magerlein, A. R. Hanze, R.W. Jackson, The adrenal hormones and related compounds. I. A “direct” synthesis of hydrocortisone acetate and cortisone acetate from 11a-hydroxyprogesterone, J. Am. Chem. Soc. 77 (16) (1955) 4436-4438; J. Fried, R.W. Thoma, J.R. Gerke, J.E. Herz, M.N. Donin, D. Perlman, Oxidation of steroids by microorganisms. II. hydroxylations in position 11 and synthesis of cortisone from Reichenstein’s compound S, J. Am. Chem. Soc. 74 (15) (1952) 3962-3963). 6β-hydroksyprogesteron może być stosowany jako substrat do syntezy 6b,14a-dihydroksyandrost-4-eno-3,17-dionu - inhibitora wzrostu komórek raka piersi (H. Yoshioka, S. Asada, S. Fujita (1994), Process for production of 6β, 14a-hydroxyandrost-4-ene-3,17-dione, European Patent 5: 99, 658). Stwierdzono również, że 6β-hydroksyprogesteron hamuje aktywność 5a-reduktazy steroidowej u samców szczurów. Dlatego jest on interesującym związkiem pod kątem wytwarzania nowych przeciw nowotworom gruczołu krokowego (A. Yamada, M. Yamada, Y. Fujita, T. Nishigami, K. Nakasho, K. Uematsu, Selfaugmentation effect of male-specific products on sexually differentiated progesterone metabolism in adult male rat liver microsomes, J. Biol. Chem. 276 (2001) 4604-S4610).11α-hydroxyprogesterone is an important pharmaceutical compound. It has antiandrogenic activity and weak estrogenic properties (J. Tamm, M. Seckelmann, U. Volkwein, E. Ludwig, The effect of the antiandrogen 11a-hydroxyprogesterone on sebum production and cholesterol concentration of sebum, Br. J. Dermatol. 107 (1982) ) 63-70). Activates processes that facilitate the implantation of the fertilized egg in the uterus (AH van der Willigen, JD Peereboom-Wynia, T. van Joost, E. Stolz, A preliminary study of the effect of 11a-hydroxyprogesterone on the hair growth in men suffering from androgenetic alopecia , Acta Derm. Venereol 67 (1987) 82-85; J. Nikolaus, KT Nguyen, C. Virus, JL Riehm, M. Hutter, R. Bernhardt, Engineering of CYP106A2 for steroid 9α- and 63-hydroxylation. Steroids 120 ( 2017) 41-48). In addition, 11a-hydroxyprogesterone influences the regulation of blood pressure, causing the selective inhibition of type 2 β-hydroxysteroid dehydrogenase. This enzyme metabolizes cortisol to cortisone in humans, so this compound is important in regulating the electrolyte balance of the kidneys (H. Morita, M. Zhou , MF Foecking, EP Gomez-Sanchez, EN Cozza, CE Gomez-Sanchez, 11 βHydroxysteroid dehydrogenase type 2 complementary deoxyribonucleic acid stably transfected into Chinese hamster ovary cells: specific inhibition by 11 a-hydroxyprogesterone, Endocrinol 137 (1996) 23082314; , DJ Morris, 11α- and 11β-hydroxyprogesterone, potent inhibitors of 11β-hydroxysteroid dehydrogenase, possess hypertensinogenic activity in the rat, Hypertension 27 (1996) 421-425). In addition, 11α-hydroxyprogesterone can be used as a substrate for the production of cortisone and hydrocortisone (JA Hogg, PF Beal, AH Nathan, FH Lincoln, WP Schneider, BJ Magerlein, AR Hanze, RW Jackson, The adrenal hormones and related compounds. I. A "direct "Synthesis of hydrocortisone acetate and cortisone acetate from 11a-hydroxyprogesterone, J. Am. Chem. Soc. 77 (16) (1955) 4436-4438; J. Fried, RW Thoma, JR Gerke, JE Herz, MN Donin, D. Perlman, Oxidation of steroids by microorganisms. II. Hydroxylations in position 11 and synthesis of cortisone from Reichenstein's compound S, J. Am. Chem. Soc. 74 (15) (1952) 3962-3963). 6β-hydroxyprogesterone can be used as a substrate for the synthesis of 6b, 14a-dihydroxyandrost-4-ene-3,17-dione - an inhibitor of breast cancer cell growth (H. Yoshioka, S. Asada, S. Fujita (1994), Process for production of 6β, 14a-hydroxyandrost-4-ene-3,17-dione, European Patent 5: 99, 658). 6β-hydroxyprogesterone has also been found to inhibit steroid 5α-reductase activity in male rats. Therefore, it is an interesting compound in terms of the production of new ones against prostate neoplasms (A. Yamada, M. Yamada, Y. Fujita, T. Nishigami, K. Nakasho, K. Uematsu, Selfaugmentation effect of male-specific products on sexually differentiated progesterone metabolism). in adult male rat liver microsomes, J. Biol. Chem. 276 (2001) 4604-S4610).
Znane są mikrobiologiczne metody otrzymywania 6β,11 α-dihydroksyprogesteronu z progesteronu: - z wydajnością 65.5% w wyniku zastosowania szczepu Rhizomucor pusillus (Habibi Z, Yousefi M, Ghanian S, Mohammadi M, Ghasemi S. Biotransformation of progesterone by Absidia griseolla var. igachii and Rhizomucor pusillus. Steroids 77 (2012) 1446-1449.); - w wyniku zastosowania 10 mg progesteronu w kulturze szczepu Acremonium chrysogenum oraz Absidia griseolla uzyskano odpowiednio 2,72 i 3,73 mg (Yousefi M, Mohammadi M, Habibi Z, Shafiee A. Biotransformation of Progesterone by Acremonium chrysogenum and Absidia griseolla var. igachii. Biocatalysis and Biotransformation, 2010; 28(4): 254-258.); - z wydajnością 21% w wyniku zastosowania szczepu Mucor M881 (Kolet SP, Niloferjahan S, Haldar S, Gonnade R, Thulasiram HV. Biocatalyst mediated production of 6b, 11a-dihydroxy derivatives of 4-ene-3-one steroids. Steroids 78 (2013) 1152-1158); - w kulturach czterech szczepów z rodzaju Trichoderma (T. aureoviride, T. hamatum, T. koningii, T. pseudokoningii) (El-Kadi I.A., Eman Mostafa M. Hydroxylation of Progesterone by Some Trichoderma Species. Folia Microbiol. 49 (3), (2004) 285-290); - z wydajnością 17% w wyniku zastosowania szczepu Beauveria bassiana (Świzdor A, Kołek T, Panek A, Białońska A Microbial Baeyer-Villiger oxidation of steroidal ketones using Beauveria bassiana: Presence of an 11a-hydroxyl group essential to generation of D-homo lactones. Biochimica et Biophysica Acta 1811 (2011) 253-262).There are known microbiological methods of obtaining 6β, 11 α-dihydroxyprogesterone from progesterone: - with an efficiency of 65.5% as a result of the use of the Rhizomucor pusillus strain (Habibi Z, Yousefi M, Ghanian S, Mohammadi M, Ghasemi S. Biotransformation of progesterone by Absidia griseolla var. and Rhizomucor pusillus. Steroids 77 (2012) 1446-1449.); - as a result of using 10 mg of progesterone in the culture of Acremonium chrysogenum and Absidia griseolla strains, 2.72 and 3.73 mg were obtained (Yousefi M, Mohammadi M, Habibi Z, Shafiee A. Biotransformation of Progesterone by Acremonium chrysogenum and Absidia griseolla var. igachii Biocatalysis and Biotransformation, 2010; 28 (4): 254-258.); - with an efficiency of 21% due to the use of the Mucor M881 strain (Kolet SP, Niloferjahan S, Haldar S, Gonnade R, Thulasiram HV. Biocatalyst mediated production of 6b, 11a-dihydroxy derivatives of 4-ene-3-one steroids. Steroids 78 ( 2013) 1152-1158); - in cultures of four strains of the genus Trichoderma (T. aureoviride, T. hamatum, T. koningii, T. pseudokoningii) (El-Kadi IA, Eman Mostafa M. Hydroxylation of Progesterone by Some Trichoderma Species. Folia Microbiol. 49 (3) , (2004) 285-290); - with an efficiency of 17% as a result of the use of the Beauveria bassiana strain (Świzdor A, Kołek T, Panek A, Białońska A Microbial Baeyer-Villiger oxidation of steroidal ketones using Beauveria bassiana: Presence of an 11a-hydroxyl group essential to generation of D-homo lactones Biochimica et Biophysica Acta 1811 (2011) 253-262).
Istota wynalazku polega na tym, że regioselektywne wprowadzenie dwóch grup hydroksylowych w cząsteczce substratu, którym jest progesteron, w wyniku którego otrzymuje się 6β,11α-dihydroksyprogesteron, a reakcję prowadzi się w wodnej kulturze szczepu Isaria farinosa KCh KW1.1.The essence of the invention consists in the regioselective introduction of two hydroxyl groups in the substrate molecule, which is progesterone, as a result of which 6β, 11α-dihydroxyprogesterone is obtained, and the reaction is carried out in an aqueous culture of the strain Isaria farinosa KCh KW1.1.
PL 237 128 B1PL 237 128 B1
Istota wynalazku polega na tym, że do podłoża odpowiedniego dla grzybów strzępkowych wprowadza się szczep Isaria farinosa KCh KW1.1. Po upływie co najmniej 48 godzin do hodowli wprowadza się substrat, którym jest progesteron o wzorze 1, rozpuszczony w rozpuszczalniku organicznym mieszającym się z wodą. Transformację prowadzi się w temperaturze od 20 do 30 stopni Celsjusza, przy ciągłym wstrząsaniu, co najmniej 12 godzin. Kolejno produkt ekstrahuje się rozpuszczalnikiem organicznym niemieszającym się z wodą i oczyszcza chromatograficznie.The essence of the invention consists in introducing the Isaria farinosa KCh KW1.1 strain into a substrate suitable for filamentous fungi. After at least 48 hours, the substrate is introduced into the culture, which is progesterone I, dissolved in a water-miscible organic solvent. The transformation is carried out at a temperature of 20 to 30 degrees Celsius with continuous shaking for at least 12 hours. Subsequently, the product is extracted with a water-immiscible organic solvent and purified by chromatography.
Korzystnie jest, gdy stosunek masy dodawanego substratu do objętości hodowli wynosi 0,1 mg : 1 mL.Preferably, the ratio of weight of the substrate added to the culture volume to be added is 0.1 mg: 1 mL.
Korzystnie także jest, gdy proces prowadzi się w temperaturze 25 stopni Celsjusza.It is also preferred that the process is carried out at a temperature of 25 degrees Celsius.
Dodatkowo, korzystnie jest, gdy transformację prowadzi się przez 24 godziny.Additionally, it is preferred that the transformation is carried out for 24 hours.
Postępując zgodnie z wynalazkiem, w wyniku działania układu enzymatycznego zawartego w komórkach szczepu Isaria farinosa KCh KW1.1, następuje steroselektywna hydroksylacja przy atomie węgla C-6 i C-11. Uzyskany w ten sposób produkt wydziela się z wodnej kultury mikroorganizmu, znanym sposobem, przez ekstrakcję rozpuszczalnikiem organicznym niemieszającym się z wodą (chlorof orm).Following the invention, as a result of the action of the enzyme system contained in the cells of the strain Isaria farinosa KCh KW1.1, steroselective hydroxylation takes place at carbon C-6 and C-11. The product obtained in this way is separated from the aqueous culture of the microorganism in a known manner by extraction with a water-immiscible organic solvent (chloroform).
Zasadniczą zaletą wynalazku jest otrzymanie 63,11a-dihydroksyprogesteronu z wydajnością izolowaną na poziomie 20% (konwersja substratu według GC = 100%; udział procentowy w mieszaninie reakcyjnej > 80%), w temperaturze pokojowej i przy pH naturalnym dla szczepu.The main advantage of the invention is the obtaining of 63,11a-dihydroxyprogesterone with an isolated yield of 20% (substrate conversion according to GC = 100%; percentage of the reaction mixture> 80%), at room temperature and pH natural for the strain.
Wynalazek jest bliżej objaśniony na przykładzie wykonania.The invention is explained in more detail using an exemplary embodiment.
P r z y k ł a d. Do kolby Erlenmajera o pojemności 2000 cm3, w której znajduje się 500 cm3 sterylnej pożywki zawierającej 5 g aminobaku i 15 g glukozy, wprowadza się szczep Isaria farinosa KCh KW1.1. Po 72 godzinach jego wzrostu dodaje się 100 mg 33-hydroksyandrost-5-en-17-onu o wzorze 1, rozpuszczonego w 1 cm3 DMSO. Transformację prowadzi się w 25 stopniach Celsjusza przy ciągłym wstrząsaniu przez 24 godziny. Następnie mieszaninę poreakcyjną ekstrahuje się trzykrotnie chloroformem, osusza bezwodnym siarczanem magnezu i odparowuje rozpuszczalnik. Otrzymany ekstrakt oczyszcza się chromatograficznie, używając jako eluentu mieszaniny heksanu i acetonu w stosunku objętościowym 2:1.Example d. To the Erlenmeyer flask with a capacity of 2,000 cm 3, which is 500 cm 3 of a sterile medium containing 5 g aminobaku and 15 g of glucose, the strain is introduced Isaria farinosa SDS KW1.1. After 72 hours the growth was added 100 mg of 33-hydroxyandrost-5-en-17-one of formula 1, dissolved in 1 cm 3 of DMSO. The transformation is carried out at 25 degrees Celsius with continuous shaking for 24 hours. The reaction mixture was then extracted three times with chloroform, dried with anhydrous magnesium sulfate, and the solvent was evaporated. The extract obtained is purified by chromatography using a 2: 1 v / v mixture of hexane and acetone as eluent.
Na tej drodze otrzymuje się 20 mg 63,11a-dihydroksyprogesteronu (wydajność 20%, konwersja substratu według GC = 100%; udział procentowy w mieszaninie reakcyjnej > 80%).In this way, 20 mg of 63,11a-dihydroxyprogesterone are obtained (yield 20%, substrate conversion according to GC = 100%; percentage of the reaction mixture> 80%).
Uzyskany produkt charakteryzuje się następującymi danymi spektralnymi:The obtained product is characterized by the following spectral data:
1H NMR (600MHz) (ppm) (CDCI3) δ: 0.73 (s, 3H, 18-H); 1.10 (t, 1H, J = 10.3 Hz, 9-H); 1.27-1.32 (m, 2H, 14-H, 15-Κβ); 1.50 (s, 3H, 19-H); 1.51 (t, 1H, J = 11.4 Hz, 12-Ha); 1.67-1.78 (m, 2H, 15-Ha, 16-Ha); 1.92 (td, 1H, J = 14.4, 4.4 Hz, 1-Ha); 1.99 (dt, 1H, J = 13.9, 3.0 Hz, 7-Ha); 2.01-2.07 (qd, 1H, J = 11.9, 2.7 Hz, 8-H); 2.13 (s, 3H, 21-H); 2.16-2.23 (m, 1H, 16-Hβ); 2.28-2.40 (m, 3H, 2-Ha, 7-Hβ, 12-Hβ); 2.51-2.58 (m, 2H, 2-Hβ, 17-Ha); 2.79 (ddd, 1H, J = 13.7, 4.8, 3.0 Hz, 1-Hβ); 4.09 (td, 1H, J = 10.6, 4.8 Hz, 11 -Hβ); 4.34 (t, 1H, J = 2.8 Hz, 6-Ha); 5.81 (s, 1H, 4-H). 1 H NMR (600MHz) (ppm) (CDCl3) δ: 0.73 (s, 3H, 18-H); 1.10 (t, 1H, J = 10.3Hz, 9-H); 1.27-1.32 (m, 2H, 14-H, 15-Κβ); 1.50 (s, 3H, 19-H); 1.51 (t, 1H, J = 11.4Hz, 12-Ha); 1.67-1.78 (m, 2H, 15-Ha, 16-Ha); 1.92 (td, 1H, J = 14.4, 4.4 Hz, 1-Ha); 1.99 (dt, 1H, J = 13.9, 3.0 Hz, 7-Ha); 2.01-2.07 (qd, 1H, J = 11.9, 2.7 Hz, 8-H); 2.13 (s, 3H, 21-H); 2.16-2.23 (m, 1H, 16-Hβ); 2.28-2.40 (m, 3H, 2-Ha, 7-Hβ, 12-Hβ); 2.51-2.58 (m, 2H, 2-Hβ, 17-Ha); 2.79 (ddd, 1H, J = 13.7, 4.8, 3.0 Hz, 1-Hβ); 4.09 (td, 1H, J = 10.6, 4.8 Hz, 11 -Hβ); 4.34 (t, 1H, J = 2.8 Hz, 6-Ha); 5.81 (s, 1H, 4-H).
13 C NMR (151 MHz) (ppm) (CDCI3) δ: 14,67 (C-18); 20,33 (C-19); 23,14 (C-16); 24,32 (C-15); 28,50 (C-8); 31,45 (C-21); 34,55 (C-2); 37,66 (C-7); 39,11 (C-1); 39,39 (C-10); 44,40 (C-13); 50,52 (C-12); 55,44 (C-14); 59,10 (C-9); 63,27 (C-17); 69,02 (C-11); 73,19 (C-6); 127,17 (C-4); 168,41 (C-5); 201,14 (C-3); 209,06 (C-20). 13 C NMR (151 MHz) (ppm) (CDCl 3) δ: 14.67 (C-18); 20.33 (C-19); 23.14 (C-16); 24.32 (C-15); 28.50 (C-8); 31.45 (C-21); 34.55 (C-2); 37.66 (C-7); 39.11 (C-1); 39.39 (C-10); 44.40 (C-13); 50.52 (C-12); 55.44 (C-14); 59.10 (C-9); 63.27 (C-17); 69.02 (C-11); 73.19 (C-6); 127.17 (C-4); 168.41 (C-5); 201.14 (C-3); 209.06 (C-20).
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