PL244020B1 - Method for simultaneous preparation of 3β,7α,17β-trihydroxyandrost-5-ene and 3β,7β,17β-trihydroxyandrost-5-ene - Google Patents
Method for simultaneous preparation of 3β,7α,17β-trihydroxyandrost-5-ene and 3β,7β,17β-trihydroxyandrost-5-ene Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 13
- 238000002360 preparation method Methods 0.000 title claims description 4
- OEVZKEVBDIDVOI-YSZCXEEOSA-N (3s,7r,8r,9s,10r,13s,14s,17s)-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1h-cyclopenta[a]phenanthrene-3,7,17-triol Chemical compound C1[C@@H](O)CC[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3[C@@H](O)C=C21 OEVZKEVBDIDVOI-YSZCXEEOSA-N 0.000 title abstract description 3
- OEVZKEVBDIDVOI-FMKNUBLKSA-N androst-5-ene-3beta,7alpha,17beta-triol Chemical compound C1[C@@H](O)CC[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3[C@H](O)C=C21 OEVZKEVBDIDVOI-FMKNUBLKSA-N 0.000 title abstract 2
- KPRGOTLNGIBVFL-UHFFFAOYSA-N 7-Oxodehydroepiandrosterone Natural products C1C(O)CCC2(C)C3CCC(C)(C(CC4)=O)C4C3C(=O)C=C21 KPRGOTLNGIBVFL-UHFFFAOYSA-N 0.000 claims abstract description 6
- KPRGOTLNGIBVFL-GINZOMEDSA-N 7-ketodehydroepiandrosterone Chemical group C1[C@@H](O)CC[C@]2(C)[C@H]3CC[C@](C)(C(CC4)=O)[C@@H]4[C@@H]3C(=O)C=C21 KPRGOTLNGIBVFL-GINZOMEDSA-N 0.000 claims abstract description 6
- 241000123196 Piptoporus betulinus Species 0.000 claims abstract description 5
- 239000000758 substrate Substances 0.000 claims abstract description 5
- 230000009466 transformation Effects 0.000 claims description 10
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 9
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 239000003960 organic solvent Substances 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 239000003480 eluent Substances 0.000 claims description 3
- 241000233866 Fungi Species 0.000 claims description 2
- 238000004587 chromatography analysis Methods 0.000 claims description 2
- 238000000605 extraction Methods 0.000 claims 1
- 230000002906 microbiologic effect Effects 0.000 abstract description 3
- 230000009467 reduction Effects 0.000 abstract description 2
- 102000004190 Enzymes Human genes 0.000 abstract 1
- 108090000790 Enzymes Proteins 0.000 abstract 1
- OKKJLVBELUTLKV-MZCSYVLQSA-N Deuterated methanol Chemical compound [2H]OC([2H])([2H])[2H] OKKJLVBELUTLKV-MZCSYVLQSA-N 0.000 description 8
- 150000003431 steroids Chemical class 0.000 description 8
- 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 6
- 239000000047 product Substances 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 4
- FMGSKLZLMKYGDP-UHFFFAOYSA-N Dehydroepiandrosterone Natural products C1C(O)CCC2(C)C3CCC(C)(C(CC4)=O)C4C3CC=C21 FMGSKLZLMKYGDP-UHFFFAOYSA-N 0.000 description 3
- 230000003110 anti-inflammatory effect Effects 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 230000002519 immonomodulatory effect Effects 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 229960002847 prasterone Drugs 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- QADHLRWLCPCEKT-UHFFFAOYSA-N Androstenediol Natural products C1C(O)CCC2(C)C3CCC(C)(C(CC4)O)C4C3CC=C21 QADHLRWLCPCEKT-UHFFFAOYSA-N 0.000 description 2
- 241000010214 Mucor hiemalis f. silvaticus Species 0.000 description 2
- 241000306282 Umbelopsis isabellina Species 0.000 description 2
- QADHLRWLCPCEKT-LOVVWNRFSA-N androst-5-ene-3beta,17beta-diol Chemical compound C1[C@@H](O)CC[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CC=C21 QADHLRWLCPCEKT-LOVVWNRFSA-N 0.000 description 2
- 229950009148 androstenediol Drugs 0.000 description 2
- 230000036983 biotransformation 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
- 230000000694 effects Effects 0.000 description 2
- 230000002124 endocrine Effects 0.000 description 2
- 208000030159 metabolic disease Diseases 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 241000235389 Absidia Species 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
- 208000002705 Glucose Intolerance Diseases 0.000 description 1
- 206010018429 Glucose tolerance impaired Diseases 0.000 description 1
- 208000012659 Joint disease 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
- 206010027940 Mood altered Diseases 0.000 description 1
- 208000019022 Mood disease Diseases 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 208000027626 Neurocognitive disease Diseases 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000011208 chromatographic data Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229930182851 human metabolite Natural products 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000033444 hydroxylation Effects 0.000 description 1
- 238000005805 hydroxylation reaction Methods 0.000 description 1
- 230000029849 luteinization Effects 0.000 description 1
- 230000007510 mood change Effects 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 230000000324 neuroprotective effect Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 230000027758 ovulation cycle Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- -1 steroid triols Chemical class 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- RINCXYDBBGOEEQ-UHFFFAOYSA-N succinic anhydride Chemical class O=C1CCC(=O)O1 RINCXYDBBGOEEQ-UHFFFAOYSA-N 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000000844 transformation Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P33/00—Preparation of steroids
- C12P33/06—Hydroxylating
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J1/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, not substituted in position 17 beta by a carbon atom, e.g. estrane, androstane
- C07J1/0003—Androstane derivatives
- C07J1/0018—Androstane derivatives substituted in position 17 beta, not substituted in position 17 alfa
- C07J1/0022—Androstane derivatives substituted in position 17 beta, not substituted in position 17 alfa the substituent being an OH group free esterified or etherified
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P33/00—Preparation of steroids
- C12P33/12—Acting on D ring
- C12P33/16—Acting at 17 position
- C12P33/18—Hydroxylating at 17 position
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
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- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Steroid Compounds (AREA)
Abstract
Wynalazek dotyczy jednoczesnego sposobu otrzymywania 3β,7α, 17β-trihydroksy-androst-5-enu i 3β,7β,17β-trihydroksy-androst-5-enu, o wzorach 2 i 3, na drodze mikrobiologicznej redukcji, w której jako substrat stosuje się 7-okso-DHEA, o wzorze 1, przy użyciu systemu enzymatycznego grzyba strzępkowego Piptoporus betulinus AM39.The invention concerns a simultaneous method of obtaining 3β,7α, 17β-trihydroxy-androst-5-ene and 3β,7β,17β-trihydroxy-androst-5-ene, of formulas 2 and 3, by microbiological reduction, in which the substrate is 7-oxo-DHEA, formula 1, using the enzyme system of the filamentous fungus Piptoporus betulinus AM39.
Description
Opis wynalazkuDescription of the invention
Przedmiotem wynalazku jest sposób jednoczesnego otrzymywania 3β,7α, 17e-trihydroksy-androst-5-enu i 3e,7e,17e-trihydroksy-androst-5-enu, o wzorach 2 i 3, przedstawionych na rysunku.The subject of the invention is a method for the simultaneous preparation of 3β,7α, 17e-trihydroxy-androst-5-ene and 3e,7e,17e-trihydroxy-androst-5-ene, of formulas 2 and 3, shown in the figure.
Wynalazek może znaleźć zastosowanie do otrzymywania trójwodorotlenowych naturalnych metabolitów dehydroepiandrosteronu (DHEA) o wysokim potencjale terapeutycznym.The invention may be used to obtain trihydric natural metabolites of dehydroepiandrosterone (DHEA) with high therapeutic potential.
Wprowadzenie grupy hydroksylowej do cząsteczki steroidowej może skutkować wzmocnieniem lub zmianą aktywności biologicznej związku (Donova M.V., Egorova O.V., App. Microbiol. Biotechnol., 2012, 94, 1423-1447; El Kihel L, Steroids, 2012, 77, 10-26). Hydroksylowane metabolity DHEA wykazują szereg różnorodnych właściwości biologicznych, wcześniej częściowo błędnie przypisywanych samemu DHEA. 7α- i 7e-hydroksy-DHEA mają działanie neuroprotekcyjne, przeciwutleniające, przeciwnowotworowe, immunomodulacyjne i przeciwzapalne. Z powodu tych aktywności związki te mogą być użyteczne w terapiach wielu schorzeń, m. in. chorób autoimmunologicznych, neurodegeneracyjnych czy zaburzeń metabolizmu (El Kihel L, Steroids, 2012, 77, 10-26; Kancheva R. et al., Neuroscience, 2011,191,22-27;Pellissier M. et al., Steroids, 2011,69,137-144; Starka L, Physiol. Res., 2011, 66, 285-294).The introduction of a hydroxyl group into a steroid molecule may result in an enhancement or change in the biological activity of the compound (Donova M.V., Egorova O.V., App. Microbiol. Biotechnol., 2012, 94, 1423-1447; El Kihel L, Steroids, 2012, 77, 10-26) . Hydroxylated DHEA metabolites exhibit a number of diverse biological properties, previously partly erroneously attributed to DHEA itself. 7α- and 7e-hydroxy-DHEA have neuroprotective, antioxidant, anticancer, immunomodulatory and anti-inflammatory effects. Due to these activities, these compounds may be useful in the treatment of many diseases, including: autoimmune, neurodegenerative diseases or metabolic disorders (El Kihel L, Steroids, 2012, 77, 10-26; Kancheva R. et al., Neuroscience, 2011, 191, 22-27; Pellissier M. et al., Steroids, 2011, 69,137-144; Starka L, Physiol. Res., 2011, 66, 285-294).
3e,7e,17e-trihydroksy-androst-5-en jest występującym u ludzi metabolitem DHEA o aktywności przeciwzapalnej wielokrotnie silniejszej od działania dehydroepiandrosteronu (Morfin R., Starka, L, Int. Rev. Neurobiol., 2001, 46, 79-95; Loria R. M., Steroids, 2002, 67, 953-966). Wykazuje on również aktywność antyglukokortykoidową, dzięki czemu może być wykorzystywany do łagodzenia zaburzeń nastroju (Loria R. M., 1997, Psychoneuroendocrinology 22, 103-108; Duskova M. et al., 2011, Endocrine Regulations 45, 131-137). Ze względu na właściwości immunomodulacyjne rozważane jest jego stosowanie w terapii schorzeń stawów oraz zaburzeń metabolizmu, m. in. otyłości i nietolerancji glukozy (Ahlem C. N. et al., J. Steroid Biochem. Mol. Biol., 2011, 126, 87-94). W badaniach klinicznych wykazano, że 3e,7e,17e-trihydroksy-androst-5-en wpływa na obniżenie poziomu cholesterolu u zdrowych mężczyzn (stickney D. M. et al., Am. J. Transl. Res., 2011,3, 275-283).3e,7e,17e-trihydroxy-androst-5-ene is a human metabolite of DHEA with anti-inflammatory activity many times stronger than that of dehydroepiandrosterone (Morfin R., Starka, L, Int. Rev. Neurobiol., 2001, 46, 79-95 ; Loria R. M., Steroids, 2002, 67, 953-966). It also has antiglucocorticoid activity, so it can be used to alleviate mood disorders (Loria R. M., 1997, Psychoneuroendocrinology 22, 103-108; Duskova M. et al., 2011, Endocrine Regulations 45, 131-137). Due to its immunomodulatory properties, its use is considered in the treatment of joint diseases and metabolic disorders, including: obesity and glucose intolerance (Ahlem C. N. et al., J. Steroid Biochem. Mol. Biol., 2011, 126, 87-94). Clinical studies have shown that 3e,7e,17e-trihydroxy-androst-5-ene reduces cholesterol levels in healthy men (Stickney D. M. et al., Am. J. Transl. Res., 2011, 3, 275-283 ).
Podobnie jak jego 7β izomer, 3e,7a,17e-trihydroksy-androst-5-en jest immunomodulacyjnym związkiem o silnym działaniu przeciwzapalnym (Loria R. M.,1997, Psychoneuroendocrinology 22, 103-108). Obniżony poziom tego steroidu skorelowany został ze zmianami nastroju i zaburzeniami neurokognitywnymi występującymi u kobiet w fazie lutealnej cyklu menstruacyjnego (Duskova M. et al., 2011, Endocrine Regulations 45, 131-137).Like its 7β isomer, 3e,7a,17e-trihydroxy-androst-5-ene is an immunomodulatory compound with a strong anti-inflammatory effect (Loria R. M., 1997, Psychoneuroendocrinology 22, 103-108). Reduced levels of this steroid have been correlated with mood changes and neurocognitive disorders occurring in women in the luteal phase of the menstrual cycle (Duskova M. et al., 2011, Endocrine Regulations 45, 131-137).
Chemiczna synteza leków steroidowych wymaga zwykle przeprowadzania licznych operacji technologicznych, związanych m. in. z koniecznością osłaniania niektórych grup funkcyjnych przed niespecyficznymi co do miejsca i konformacji reakcjami chemicznymi. W przeciwieństwie do metod chemicznych, transformacje mikrobiologiczne pozwalają modyfikować strukturę cząsteczek jednoetapowo, umożliwiając wysoką regio- oraz stereoselektywność reakcji przy zachowaniu łagodnych warunków procesu.The chemical synthesis of steroid drugs usually requires numerous technological operations, including: with the need to protect some functional groups against chemical reactions that are non-specific in terms of location and conformation. Unlike chemical methods, microbiological transformations allow the structure of molecules to be modified in one step, enabling high regio- and stereoselectivity of the reaction while maintaining mild process conditions.
Znany jest sposób otrzymywania mieszaniny 3e,7a,17e-trihydroksy-androst-5-enu i 3β,7β,17βtrihydroksy-androst-5-enu jako produktu biotransformacji androstendiolu lub DHEA przeprowadzanych w kulturach grzybów strzępkowych Mortierella isabellina AM212 (Kołek T. et al., Org. Biomol. Chem., 2011, 9, 5414-5422), Absidia coreulea AM93 (Milecka-Tronina N. et al., Bioorg. Med. Chem., 2013, 22, 883-891) i Mucor silvaticus (Wang Y. et al., Biocatal. Biotransformation, 2013, 31, 168-174). W transformacji androstendiolu przez A. coreulea AM93 otrzymano odpowiednio 9,5% i 19% 3e,7a,17e-trihydroksy-androst-5-enu i 3e,7e,17e-trihydroksy-androst-5-enu. W reakcji z udziałem M. isabellina AM212 a także M. silvaticus mieszanina produktów zawierała kilkukrotnie więcej 3e,7a,17e-trihydroksy-androst-5-enu w porównaniu do izomeru 7β.There is a known method of obtaining a mixture of 3e,7a,17e-trihydroxy-androst-5-ene and 3β,7β,17βtrihydroxy-androst-5-ene as a product of the biotransformation of androstenediol or DHEA carried out in cultures of the filamentous fungi Mortierella isabellina AM212 (Kołek T. et al. ., Org. Biomol. Chem., 2011, 9, 5414-5422), Absidia coreulea AM93 (Milecka-Tronina N. et al., Bioorg. Med. Chem., 2013, 22, 883-891) and Mucor silvaticus ( Wang Y. et al., Biocatal. Biotransformation, 2013, 31, 168-174). In the transformation of androstenediol by A. coreulea AM93, 9.5% and 19% of 3e,7a,17e-trihydroxy-androst-5-ene and 3e,7e,17e-trihydroxy-androst-5-ene were obtained, respectively. In the reaction involving M. isabellina AM212 and M. silvaticus, the product mixture contained several times more 3e,7a,17e-trihydroxy-androst-5-ene compared to the 7β isomer.
Szczep Piptoporus betulinus AM39 był wcześniej ujawniony w literaturze jako mikroorganizm zdolny do mikrobiologicznej hydoksylacji pochodnej bezwodnika kwasu bursztynowego (Olejniczak T., World J. Bicrob. Biot., 2010, 26, 2053-2060).The Piptoporus betulinus AM39 strain was previously disclosed in the literature as a microorganism capable of microbiological hydroxylation of a succinic anhydride derivative (Olejniczak T., World J. Bicrob. Biot., 2010, 26, 2053-2060).
Istota wynalazku polega na tym, że do podłoża odpowiedniego dla wzrostu grzybów strzępkowych wprowadza się zawiesinę komórek Piptoporus betulinus AM39. Hodowlę prowadzi się przy ciągłym wstrząsaniu w temperaturze 22-26°C. Po upływie co najmniej 4 dni do hodowli dodaje się substrat, którym jest 7-okso-DHEA, rozpuszczony w rozpuszczalniku organicznym mieszającym się z wodą. Transformację prowadzi się w temperaturze od 22°C do 26°C, przy ciągłym wstrząsaniu, co najmniej 24 godziny. Produkty ekstrahuje się rozpuszczalnikiem organicznym niemieszającym się z wodą i oczyszcza chromatograficznie, w wyniku czego otrzymuje się 3e,7a,17e-trihydroksy-androst-5-en oraz 3 β ,7β ,17e-tri hyd roksy-androst-5-en.The essence of the invention is that a suspension of Piptoporus betulinus AM39 cells is introduced into a medium suitable for the growth of filamentous fungi. The cultivation is carried out with constant shaking at a temperature of 22-26°C. After at least 4 days, the substrate, which is 7-oxo-DHEA, dissolved in an organic solvent miscible with water, is added to the culture. The transformation is carried out at a temperature of 22°C to 26°C, with constant shaking, for at least 24 hours. The products are extracted with an organic solvent immiscible with water and purified by chromatography to obtain 3e,7a,17e-trihydroxy-androst-5-ene and 3β,7β,17e-trihydroxy-androst-5-ene.
Korzystnie jest, gdy stosunek masy dodawanego substratu do objętości hodowli wynosi 0,3 g : 1 L.It is preferable when the ratio of the mass of the added substrate to the culture volume is 0.3 g: 1 L.
Korzystnie także jest, gdy transformację prowadzi się w temperaturze 25°C.It is also preferred that the transformation is carried out at a temperature of 25°C.
Korzystnie jest, gdy transformację prowadzi się przez 24 godziny.It is preferable for the transformation to be carried out for 24 hours.
Korzystne jest, gdy jako ekstrahent stosuje się chloroform.It is preferred when chloroform is used as the extractant.
Korzystne jest także, gdy jako eluent stosuje się mieszaninę octan etylu : chlorek metylenu : metanol w proporcji objętościowej składników 3 : 2 : 0,2.It is also advantageous when a mixture of ethyl acetate: methylene chloride: methanol is used as the eluent in a volume ratio of 3: 2: 0.2.
Sposobem według wynalazku, w wyniku redukcji 7-okso-DHEA (7-okso-dehydroepiandrosteronu) można otrzymać steroidowe triole, które ze względu na swoją aktywność biologiczną mają wysoki potencjał terapeutyczny.Using the method according to the invention, as a result of the reduction of 7-oxo-DHEA (7-oxo-dehydroepiandrosterone), steroid triols can be obtained, which due to their biological activity have a high therapeutic potential.
Zasadniczą zaletą wynalazku jest otrzymanie mieszaniny 3e,7a,17e-trihydroksy-androst-5-enu i 3e,7e,17e-trihydroksy-androst-5-enu z wydajnością 27% i 47% w łagodnych warunkach reakcji i w pH bliskim obojętnemu.The main advantage of the invention is the preparation of a mixture of 3e,7a,17e-trihydroxy-androst-5-ene and 3e,7e,17e-trihydroxy-androst-5-ene with a yield of 27% and 47% under mild reaction conditions and at a pH close to neutral.
Wynalazek jest bliżej objaśniony na przykładzie wykonania.The invention is explained in more detail using an example embodiment.
Do kolby Erlenmeyera o pojemności 300 mL, w której znajduje się 100 mL sterylnej pożywki zawierającej 3 g glukozy i 1 g aminobaku, wprowadza się 2 cm3 zawiesiny komórek Piptoporus betulinus AM39. Hodowlę prowadzi się przez kolejne cztery dni przy stałym wstrząsaniu w temperaturze 25°C. Następnie do hodowli mikroorganizmu dodaje się 30 mg 7-okso-DHEA, o wzorze 1, w postaci zawiesiny w 0,6 cm3 acetonu. Transformację prowadzi się przy ciągłym wstrząsaniu przez 24 godziny w warunkach, w których prowadzona była hodowla mikroorganizmu. Następnie uzyskany roztwór transformacyjny ekstrahuje się trzykrotnie chloroformem, osusza bezwodnym siarczanem magnezu i odparowuje rozpuszczalnik. Wydajność otrzymania produktów według danych chromatograficznych (GC) wynosi odpowiednio 30% i 49%. Po oczyszczeniu surowego produktu za pomocą chromatografii kolumnowej, z zastosowaniem jako eluentu mieszaniny octan etylu : chlorek metylenu : metanol w proporcji objętościowej składników 3 : 2 : 0,2 otrzymuje się 8 mg 3e,7a,17e-trihydroksy-androst-5-enu oraz 14 mg 3e,7e,17e-trihydroksy-androst-5-enu (łączna wydajność izolowana 74% mol.). 2 cm3 of Piptoporus betulinus AM39 cell suspension is introduced into a 300 mL Erlenmeyer flask containing 100 mL of sterile medium containing 3 g of glucose and 1 g of aminobac. The culture is continued for another four days with constant shaking at a temperature of 25°C. Then, 30 mg of 7-oxo-DHEA, formula 1, as a suspension in 0.6 cm3 of acetone is added to the microorganism culture. The transformation is carried out with continuous shaking for 24 hours in the conditions in which the microorganism was cultured. The resulting transformation solution is then extracted three times with chloroform, dried over anhydrous magnesium sulfate, and the solvent is evaporated. The yield of products obtained according to chromatographic data (GC) is 30% and 49%, respectively. After purifying the crude product by column chromatography, using a mixture of ethyl acetate: methylene chloride: methanol as an eluent in a volume ratio of 3: 2: 0.2, 8 mg of 3e,7a,17e-trihydroxy-androst-5-ene and 14 mg of 3e,7e,17e-trihydroxy-androst-5-ene (total isolated yield 74% mol.).
Uzyskane produkty charakteryzują się następującymi danymi spektralnymi:The obtained products are characterized by the following spectral data:
3e,7a,17e-trihydroksy-androst-5-en:3e,7a,17e-trihydroxy-androst-5-ene:
1H NMR (CD3OD) δ (ppm): 0.75 (3H, s, 18-Me); 1.02 (3H, s, 19-Me); 3.44-3.55 (1H, m, 3a-H); 3.61 (1H, t, J = 8.5 Hz, 17a-H); 3.76 (1H, t, J = 3.6 Hz, 7p-H); 5.54 (1H, d, J = 5.0 Hz, 6-H) 13C-NMR (CD3OD): 11.4 (18-C), 18.7 (19-C), 21.5 (11-C), 24.2 (15-C), 30.7 (16-C), 32.2 (2-C), 37.5 (12-C), 38.2 (C-1), 38.5 (10-C), 39.1 (8-C), 42.9 (4-C), 43.6 (13-C), 43.7 (9-C), 45.4 (14-C), 65.5 (7-C), 72.0 (3-C), 82.5 (17-C), 125.0 (6-C), 146.7 (5-C)1H NMR (CD3OD) δ (ppm): 0.75 (3H, s, 18-Me); 1.02 (3H, s, 19-Me); 3.44-3.55 (1H, m, 3a-H); 3.61 (1H, t, J = 8.5 Hz, 17a-H); 3.76 (1H, t, J = 3.6 Hz, 7p-H); 5.54 (1H, d, J = 5.0 Hz, 6-H) 13 C-NMR (CD3OD): 11.4 (18-C), 18.7 (19-C), 21.5 (11-C), 24.2 (15-C) , 30.7 (16-C), 32.2 (2-C), 37.5 (12-C), 38.2 (C-1), 38.5 (10-C), 39.1 (8-C), 42.9 (4-C), 43.6 (13-C), 43.7 (9-C), 45.4 (14-C), 65.5 (7-C), 72.0 (3-C), 82.5 (17-C), 125.0 (6-C), 146.7 (5-C)
3e,7e,17e-trihydroksy-androst-5-en:3e,7e,17e-trihydroxy-androst-5-ene:
1H NMR (CD3OD) δ (ppm): 0.76 (3H, s, 18-Me); 1.09 (3H, s, 19-Me); 3.37-3.48 (1H, m, 3a-H); 3.56 (1H, t, J = 8.5 Hz, 17a-H); 3.72 (1H, dt, J = 2.1 Hz, J = 7.8 Hz, 7a-H); 5.25 (1H, t, J = 1.8 Hz, 6-H).1H NMR (CD3OD) δ (ppm): 0.76 (3H, s, 18-Me); 1/09 (3H, s, 19-Me); 3.37-3.48 (1H, m, 3a-H); 3.56 (1H, t, J = 8.5 Hz, 17a-H); 3.72 (1H, dt, J = 2.1 Hz, J = 7.8 Hz, 7a-H); 5.25 (1H, t, J = 1.8 Hz, 6-H).
13C NMR (CD3OD): 11.6 (18-C), 19.6 (19-C), 21.9 (11-C), 26.6 (15-C), 30.9 (16-C), 32.3 (2-C), 37.7 (10-C), 37.8 (12-C), 38.3 (1-C), 41.3 (8-C), 42.6 (4-C), 44.3 (13-C), 50.1 (9-C), 52.3 (14-C), 72.2 (3-C), 74.0 (7-C), 82.3 (17-C), 127.4 (6-C), 144.1 (5-C). 13 C NMR (CD3OD): 11.6 (18-C), 19.6 (19-C), 21.9 (11-C), 26.6 (15-C), 30.9 (16-C), 32.3 (2-C), 37.7 (10-C), 37.8 (12-C), 38.3 (1-C), 41.3 (8-C), 42.6 (4-C), 44.3 (13-C), 50.1 (9-C), 52.3 ( 14-C), 72.2 (3-C), 74.0 (7-C), 82.3 (17-C), 127.4 (6-C), 144.1 (5-C).
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