CN112368266B - 一种制备普瑞巴林的方法 - Google Patents
一种制备普瑞巴林的方法 Download PDFInfo
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- CN112368266B CN112368266B CN201880093413.5A CN201880093413A CN112368266B CN 112368266 B CN112368266 B CN 112368266B CN 201880093413 A CN201880093413 A CN 201880093413A CN 112368266 B CN112368266 B CN 112368266B
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- lipase
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- isomerase
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- 238000000034 method Methods 0.000 title claims abstract description 35
- AYXYPKUFHZROOJ-ZETCQYMHSA-N pregabalin Chemical compound CC(C)C[C@H](CN)CC(O)=O AYXYPKUFHZROOJ-ZETCQYMHSA-N 0.000 title abstract description 30
- 229960001233 pregabalin Drugs 0.000 title abstract description 29
- 150000001875 compounds Chemical class 0.000 claims abstract description 70
- 102000004190 Enzymes Human genes 0.000 claims abstract description 59
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- 108090000769 Isomerases Proteins 0.000 claims abstract description 23
- 108090001060 Lipase Proteins 0.000 claims description 58
- 239000004367 Lipase Substances 0.000 claims description 58
- 102000004882 Lipase Human genes 0.000 claims description 58
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- 238000006243 chemical reaction Methods 0.000 claims description 53
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- 239000002904 solvent Substances 0.000 claims description 20
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 18
- 239000003960 organic solvent Substances 0.000 claims description 17
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 16
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 14
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 9
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 8
- 241001661345 Moesziomyces antarcticus Species 0.000 claims description 8
- DKGAVHZHDRPRBM-UHFFFAOYSA-N Tert-Butanol Chemical compound CC(C)(C)O DKGAVHZHDRPRBM-UHFFFAOYSA-N 0.000 claims description 8
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- JHIVVAPYMSGYDF-UHFFFAOYSA-N cyclohexanone Chemical compound O=C1CCCCC1 JHIVVAPYMSGYDF-UHFFFAOYSA-N 0.000 claims description 6
- 108010047540 sucrose isomerase Proteins 0.000 claims description 6
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- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 4
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 claims description 3
- VBUYCZFBVCCYFD-JJYYJPOSSA-N 2-dehydro-D-gluconic acid Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C(=O)C(O)=O VBUYCZFBVCCYFD-JJYYJPOSSA-N 0.000 claims description 3
- 241000228245 Aspergillus niger Species 0.000 claims description 3
- 108030002100 D-tagatose 3-epimerases Proteins 0.000 claims description 3
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- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 claims description 3
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- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 2
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 2
- 125000000959 isobutyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])* 0.000 claims description 2
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 claims description 2
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- IWUCXVSUMQZMFG-AFCXAGJDSA-N Ribavirin Chemical compound N1=C(C(=O)N)N=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 IWUCXVSUMQZMFG-AFCXAGJDSA-N 0.000 abstract 1
- 229960000329 ribavirin Drugs 0.000 abstract 1
- HZCAHMRRMINHDJ-DBRKOABJSA-N ribavirin Natural products O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1N=CN=C1 HZCAHMRRMINHDJ-DBRKOABJSA-N 0.000 abstract 1
- 238000001914 filtration Methods 0.000 description 31
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- 239000000243 solution Substances 0.000 description 25
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 20
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 13
- 229910000029 sodium carbonate Inorganic materials 0.000 description 12
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 11
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- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 5
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 5
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- 208000004296 neuralgia Diseases 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- 241000589513 Burkholderia cepacia Species 0.000 description 2
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 206010036376 Postherpetic Neuralgia Diseases 0.000 description 2
- 241000235403 Rhizomucor miehei Species 0.000 description 2
- 241000303962 Rhizopus delemar Species 0.000 description 2
- 229910052783 alkali metal Inorganic materials 0.000 description 2
- 229910000288 alkali metal carbonate Inorganic materials 0.000 description 2
- 150000008041 alkali metal carbonates Chemical class 0.000 description 2
- -1 alkali metal hydrogencarbonates Chemical class 0.000 description 2
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- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 2
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- 238000003786 synthesis reaction Methods 0.000 description 2
- HTSGKJQDMSTCGS-UHFFFAOYSA-N 1,4-bis(4-chlorophenyl)-2-(4-methylphenyl)sulfonylbutane-1,4-dione Chemical compound C1=CC(C)=CC=C1S(=O)(=O)C(C(=O)C=1C=CC(Cl)=CC=1)CC(=O)C1=CC=C(Cl)C=C1 HTSGKJQDMSTCGS-UHFFFAOYSA-N 0.000 description 1
- GUGXRXLTTHFKHC-UHFFFAOYSA-N 4-(2-methylpropyl)pyrrolidin-2-one Chemical compound CC(C)CC1CNC(=O)C1 GUGXRXLTTHFKHC-UHFFFAOYSA-N 0.000 description 1
- 101710137647 Cellobiose 2-epimerase Proteins 0.000 description 1
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- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 1
- 229910000388 diammonium phosphate Inorganic materials 0.000 description 1
- 235000019838 diammonium phosphate Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
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- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 150000002170 ethers Chemical class 0.000 description 1
- NLFBCYMMUAKCPC-KQQUZDAGSA-N ethyl (e)-3-[3-amino-2-cyano-1-[(e)-3-ethoxy-3-oxoprop-1-enyl]sulfanyl-3-oxoprop-1-enyl]sulfanylprop-2-enoate Chemical compound CCOC(=O)\C=C\SC(=C(C#N)C(N)=O)S\C=C\C(=O)OCC NLFBCYMMUAKCPC-KQQUZDAGSA-N 0.000 description 1
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- 239000003446 ligand Substances 0.000 description 1
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- 229910000028 potassium bicarbonate Inorganic materials 0.000 description 1
- 235000015497 potassium bicarbonate Nutrition 0.000 description 1
- 239000011736 potassium bicarbonate Substances 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
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- TYJJADVDDVDEDZ-UHFFFAOYSA-M potassium hydrogencarbonate Chemical compound [K+].OC([O-])=O TYJJADVDDVDEDZ-UHFFFAOYSA-M 0.000 description 1
- 229940086066 potassium hydrogencarbonate Drugs 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
- C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
- C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
- C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
- C07C229/08—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to hydrogen atoms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/90—Isomerases (5.)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
- C07C227/22—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton from lactams, cyclic ketones or cyclic oximes, e.g. by reactions involving Beckmann rearrangement
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
- C07C227/30—Preparation of optical isomers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
- C12N9/60—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi from yeast
-
- 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
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/005—Amino acids other than alpha- or beta amino acids, e.g. gamma amino acids
-
- 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
- C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
- C12P41/006—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y501/00—Racemaces and epimerases (5.1)
- C12Y501/03—Racemaces and epimerases (5.1) acting on carbohydrates and derivatives (5.1.3)
- C12Y501/03011—Cellobiose epimerase (5.1.3.11)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y504/00—Intramolecular transferases (5.4)
- C12Y504/99—Intramolecular transferases (5.4) transferring other groups (5.4.99)
- C12Y504/99011—Isomaltulose synthase (5.4.99.11)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D207/18—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having one double bond between ring members or between a ring member and a non-ring member
- C07D207/22—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having one double bond between ring members or between a ring member and a non-ring member with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D207/24—Oxygen or sulfur atoms
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Abstract
涉及一种生物酶法制备普瑞巴林的方法。具体是以化合物A为原料在生物酶的作用下,生成普瑞巴林B和R‑构型化合物C;经过分离、回收得到的R‑构型化合物C在异构酶的作用下,发生构型翻转生成S‑构型化合物D;化合物D又在生物酶的作用下生成普瑞巴林B。
Description
技术领域
本发明涉及一种新型生物酶法制备普瑞巴林,应用于药物合成技术领域。
背景技术
普瑞巴林(Pregabalin)化学名为(3S)-(+)-3-氨甲基-5-甲基己酸,结构式如下:
是由美国Pfizer公司开发的氨基丁酸(GABA)受体拮抗剂,是一种服用次数少、剂量低、副作用小的抗惊厥、抗癫痫及抗焦虑药物,用于治疗中枢神经疼痛及脊髓损伤,也可治疗糖尿病性外周神经痛、带状疱疹后遗神经痛、中枢性神经痛和纤维肌痛,是被美国和欧洲认证用于治疗带状疱疹后遗神经痛和糖尿病性外周神经痛的首选药物,具有广阔的市场前景。
目前,文献报道中关于化学法合成普瑞巴林的研究十分广泛,合成路线繁多,主要有使用手性拆分试剂的化学拆分方法和使用不对称催化剂、手性配体的不对称合成法。
此类方法或步骤繁杂,或反应条件苛刻,且普遍存在环境污染问题,很大程度上制约了工业化应用。
鉴于普瑞巴林良好的药物前景,因此需要开发一种反应条件温和、反应步骤少、环境友好,所得产物普瑞巴林光学纯度高、易于工业化生产的工艺路线。
发明内容
本发明的目的是克服化学拆分技术中拆分试剂毒性大、价格昂贵,操作步骤繁琐、拆分困难、转化率低,拆分产物光学纯度低,不易工业化等缺点,提供一种原子利用率高、立体选择性好、对环境污染小的制备普瑞巴林的新型方法,克服了化学拆分方法中产率只能在50%以内的限制,理论上原料可以100%转化为终产品。为实现上述目的,本发明采用如下技术方案:
一种制备普瑞巴林的方法,包括以下步骤:
步骤1:
化合物(I)在生物酶的作用下,在溶剂中生成目标化合物(III)和副产物(II);
步骤2:
步骤1中所得化合物(II)在异构酶的作用下,在溶剂中发生构型翻转生成化合物(IV);
步骤3:
步骤2中所得化合物(IV)在步骤1中所述生物酶的作用下,在溶剂中生成目标化合物(III);
其中R1和R2为氢原子或烷基,所述烷基优选为C1-C4的支链或直链烷基;所述烷基具体可以是甲基、乙基、丙基、异丙基、正丁基、叔丁基、异丁基。
当R1为H、R2为甲基时,对应化合物III为普瑞巴林。
具体反应过程如下:
步骤1:
以外消旋化合物(A)为酶解底物,在生物酶的选择性作用下,其中的S-构型被水解为目标化合物(B)(普瑞巴林);R-构型化合物(C)不被水解而得以保留;
步骤2:
步骤1中经过分离、回收得到的R-构型化合物(C)在异构酶的作用下发生构型翻转生成S-构型化合物(D);
步骤3:
步骤2中得到的S-构型化合物(D)又可以被步骤1中所述生物酶定向水解为目标化合物(B)(普瑞巴林)。
在本申请的方法中,其中步骤1和3中,所述生物酶为水解酶,具体为特定种类的脂肪酶,优选霉菌脂肪酶,如德氏根酶(Lipase from rhizopus delemar)、黑曲霉(Lipasefrom aspergillus niger)、米黑根毛酶(Lipase from rhizomucor miehei)、白地酶(Lipase from gertrichum candidum);酵母脂肪酶,如假丝酵母脂肪酶(Lipase fromcandida)(包括南极假丝酵母脂肪酶(Lipase from candida antarctica B)和柱状假丝酵母脂肪酶(Lipase from candidacylindracea))、粘红酵母酶(Lipase from rhodoaorulaglutinis);细菌脂肪酶,如伯克霍尔德菌脂肪酶(Lipase from burkholderia cepacia)、假单孢菌脂肪酶(Lipase from pseudomonas)、葡萄球菌脂肪酶(Lipase fromstaphylococcus epidermidis)。在所述脂肪酶中,更优选霉菌脂肪酶类的德氏根酶(Lipase from rhizopus delemar)和白地酶脂肪酶(Lipase from gertrichumcandidum),酵母脂肪酶类的南极假丝酵母脂肪酶(Lipase from candida antarctica B)和柱状假丝酵母脂肪酶(Lipase from candidacylindracea),以及细菌脂肪酶类的伯克霍尔德菌脂肪酶(Lipase from burkholderia cepacia)。
步骤1和3中,所述生物酶的形态可以是经过冷冻干燥后的固定化酶颗粒或酶粉末,也可以是经过提取、浓缩、脱水处理的含酶细胞或细胞器。所用酶可以是商品酶,也可以是通过培养产酶微生物所制备的粗酶。
步骤2中,所述异构酶具体为特定种类的差向异构酶(即变旋酶),优选D-木糖异构酶(glucose isomerase)、蔗糖异构酶(sucrose isomerase)、D-塔格糖3-差向异构酶(D-tagatose 3-epimerase)、D-阿洛酮糖3-差向异构酶(D-psicose 3-epimerase)、纤维二糖差向异构酶(cellobiose 2-epimerase)、2-酮基葡萄糖酸差向异构酶(2-ketogluconateepimerase)。在所述差向异构酶中,更优选D-木糖异构酶、蔗糖异构酶和纤维二糖差向异构酶。
步骤2中,所述异构酶其形态可以是经过冷冻干燥后的固定化酶颗粒或酶粉末,或经过提取、浓缩、脱水处理的含酶细胞或细胞器。所用酶可以是商品酶,也可以是通过培养产酶微生物所制备的粗酶。
步骤1中,所述生物酶和化合物(I)或(A)的质量比为1∶2-1∶20,优选为1∶5-1∶10。
步骤2中,所述异构酶和步骤1中所得化合物(II)或(C)的质量比为1∶1-1∶20,优选为1∶3-1∶9。
步骤3中,所述生物酶和步骤2中所得化合物(IV)或(D)的质量比为1∶2-1∶20,优选为1∶5-1∶10。
步骤1和3中,所述溶剂为水或水和有机溶剂的互溶体系,优选为水和有机溶剂的互溶体系。优选地,所述有机溶剂为醇类、醚类或酮类,更优选异丙醇、正丁醇、叔丁醇、四氢呋喃、1,4-二氧六环、丙酮和环己酮中的一种或多种(本申请文件中的多种指两种和两种以上),进一步优选为丙酮和异丙醇;优选地,所述水和有机溶剂的互溶体系中水和有机溶剂的混合比例为水和有机溶剂体积比2∶1-50∶1,更优选为10∶1。化合物I(或(A))或IV(或(D))和所述溶剂的质量体积比为1g∶10mL-1g∶50mL,优选为1g∶10mL-1g∶20mL。
步骤1和3中,酶解反应的溶剂pH值为7.0-10.0,优选为9.0-10.0。控制pH采用的是碱金属碳酸盐、碱金属碳酸氢盐和碱金属氢氧化物的水溶液或不同种类的缓冲液。碱金属碳酸盐优选碳酸钠和碳酸钾,碱金属碳酸氢盐优选碳酸氢钠和碳酸氢钾,碱金属氢氧化物优选氢氧化钠和氢氧化钾;缓冲液优选Gly-NaOH缓冲液(100mmol/L,pH9.0-10.0)。
步骤2中,所用溶剂为有机溶剂,优选为醇类或酮类,更优选异丙醇、正丁醇、叔丁醇、丙酮等。化合物II或(C)和所述溶剂的质量体积比为1g∶10mL-1g∶50mL,优选为1g∶10mL-1g∶20mL。
步骤1、2和3中,反应时间为5-20h,优选5-10h。
步骤1、2和3中,反应温度为25-55℃,优选35-45℃。
所述步骤1和2之间还包括萃取步骤1的产物溶液的步骤,萃取所用的有机溶剂选自甲苯、二氯甲烷、甲基叔丁基醚或乙酸乙酯等,优选乙酸乙酯。
所述步骤1、2和3中,产物的光学纯度用手性高效液相色谱检测。色谱条件如下:色谱仪使用Agilent HPLC 1260;检测器使用紫外可变波长检测器,检测波长210nm;色谱柱使用Inertsil ODS-3(250×4.6mm,5μm),柱温30℃;流动相为缓冲液∶乙腈∶甲醇=84∶5∶11(V/V/V),其中缓冲液为0.04M磷酸氢二铵水溶液,用磷酸调pH至6.6;流速:0.8mL/min;进样量:50μL。
本发明给出的制备普瑞巴林的方法,提供一种以生物酶作为催化剂,具有步骤少、操作简单、条件温和、环保、成本低、原子利用率高、产物光学纯度高等优点,相比传统的化学拆分法,本发明的方法可以更绿色、更简便、更高效的合成普瑞巴林,具有很高的工业应用价值。
附图说明
图1为实施例1制备的化合物(B)的核磁氢谱。
具体实施方式
为了更好的理解本发明的内容,以下结合具体实例来说明本发明的技术方案,但保护的范围并不仅限于此。
所述实施例中,步骤1所用化合物(A)(4-异丁基-2-吡洛烷酮)为市售化学品;步骤1和3所用生物酶,以及步骤2所用异构酶均为商品酶。
下面结合实施例,对本发明的具体实施方式作进一步详细描述。以下实施例仅用于说明本发明,但不用于限制本发明的范围。
实施例1:
步骤1:
向反应瓶中加入化合物(A)(厂家:南京博米尔生物科技有限公司,纯度98%)50g、水450mL、丙酮50mL,搅拌升温至35℃,用质量分数10%的碳酸钠溶液调节体系pH值为9-10,加入南极假丝酵母脂肪酶(厂家:杭州创科生物科技有限公司,纯度98%)10g,保温搅拌5h。反应1h后,每隔1h使用pH试纸检测反应液pH变化,并补加适量碳酸钠溶液以保持pH值范围9-10。反应完毕后,过滤除去生物酶,用150mL乙酸乙酯萃取反应液2次,有机层水洗至中性,减压浓缩至约1/3体积,降温至5-10℃,过滤、干燥后得到副产物R-构型化合物(C)21.6g,收率43.2%,产物对映体过量值e.e.:98.21%;水层用盐酸调pH值至6.5-7.5,降温至5-10℃,过滤、干燥后得到目标产物化合物(B)普瑞巴林25.5g,收率45.2%,产物对映体过量值e.e.:99.74%。产物的核磁氢谱见附图1,具体数据如下:1H-NMR(D2O):δ0.7(t,J=8.0Hz,6H,CH3),1.05(t,J=8.0Hz,2H,CH2),1.44-1.46(m,1H,CH),2.03-2.05(m,1H,CH),2.17-2.29(m,2H,CH2COOH),2.82(d,J=8.0Hz,2H,CH2NH2),由上述数据确证化学结构为普瑞巴林。
步骤2:
向反应瓶中加入步骤1中得到的R-构型化合物(C)20g、200mL异丙醇,搅拌升温至35℃,加入D-木糖异构酶(厂家:金锦乐化学有限公司,纯度98%)5g,保温搅拌10h,反应完毕后,过滤除去异构酶,滤液浓缩至约1/3体积,加入50mL水,降温至0-5℃,过滤、干燥后得到S-构型化合物(D)17.7g,收率88.5%,产物对映体过量值ee值98.76%。
步骤3:
向反应瓶中加入步骤2中得到的S-构型化合物(D)15g、200mL水、100mL异丙醇,搅拌升温至35℃,用质量分数10%碳酸钠溶液调节体系pH值为10,加入南极假丝酵母脂肪酶(厂家:上海源叶生物科技有限公司,纯度98%)3g,保温搅拌5h,反应1h后,每隔1h补加适量碳酸钠溶液,保持pH值范围9-10。反应完毕后,过滤除去生物酶,滤液减压浓缩至2/3体积,用盐酸调pH值至6.5-7.5,降温至5-10℃,过滤、干燥后得到目标产物化合物(B)普瑞巴林15.4g,收率91.3%,产物对映体过量值e.e.:99.22%,产物的核磁氢谱见附图1,具体数据如下:1H-NMR(D2O):δ0.7(t,J=8.0Hz,6H,CH3),1.05(t,J=8.0Hz,2H,CH2),1.44-1.46(m,1H,CH),2.03-2.05(m,1H,CH),2.17-2.29(m,2H,CH2COOH),2.82(d,J=8.0Hz,2H,CH2NH2),由上述数据确证化学结构为普瑞巴林。
实施例2:
步骤1:
向反应瓶中加入化合物(A)(厂家:南京博米尔生物科技有限公司,纯度98%)50g、水350mL、四氢呋喃50mL,100mL Gly-NaOH缓冲液(100mmol/L,pH 9.0-10.0),搅拌升温至35℃,加入德氏根酶(厂家:广州乐试生物科技有限公司,纯度98%)10g,保温搅拌9h。反应完毕后,过滤除去生物酶,用150mL甲基叔丁基醚分2次萃取反应液,有机层水洗至中性,浓缩溶剂至约1/3体积,降温至5-10℃,过滤,干燥后得到副产物R-构型化合物(C)23.1g,收率46.2%,产物对映体过量值e.e.:98.72%;水层用盐酸调pH值至6.5-7.5,降温至5-10℃,过滤、干燥后得到目标产物化合物(B)普瑞巴林25.9g,收率45.9%,产物对映体过量值e.e.:99.71%,产物的核磁氢谱同附图1。
步骤2:
向反应瓶中加入步骤1中得到的R-构型化合物(C)15g、150mL丙酮,搅拌升温至35℃,加入蔗糖异构酶(厂家:南通飞宇生物科技有限公司,纯度98%)5g,保温搅拌10h,反应完毕后,过滤除去异构酶,滤液浓缩至约1/3体积,降温至-10-0℃,抽滤、干燥后得到S-构型化合物(D)13.8g,收率92.0%,产物对映体过量值e.e.:99.02%。
步骤3:
向反应瓶中加入步骤2中得到的化合物(D)10g、300mL水,搅拌升温至35℃,用质量分数10%碳酸钠溶液调节体系pH值为10,加入德氏根酶(厂家:广州乐试生物科技有限公司,纯度98%)2g,保温搅拌5h,反应1h后,每隔1h补加碳酸钠溶液,保持pH值范围9-10。反应完毕后,过滤除去生物酶,滤液用盐酸调pH值至6.5-7.5,降温至5-10℃,过滤烘干得到目标产物化合物(B)普瑞巴林10.5g,收率93.2%,产物对映体过量值e.e.:99.22%,产物的核磁氢谱同附图1。
实施例3:
步骤1:
向反应瓶中加入化合物(A)(厂家:南京博米尔生物科技有限公司,纯度98%)50g、水750mL,搅拌升温至35℃,用质量分数5%氢氧化钠溶液调节体系pH值为9,加入伯克霍尔德菌脂肪酶(厂家:上海迈瑞尔化学技术有限公司,纯度98%)5g,保温搅拌6h。反应1h后,每隔1h使用pH试纸监测反应液pH变化,补加质量分数5%的氢氧化钠溶液,保持pH值范围9-10。反应完毕后,过滤除去生物酶,用250mL甲苯分2次萃取,有机层浓缩至1/3体积,降温至5-10℃,过滤,干燥后得到副产物R-构型化合物(C)23.5g,收率47.0%,产物对映体过量值e.e.:97.98%;水层用盐酸调pH值至6.5-7.5,降温至5-10℃,过滤烘干得到目标产物化合物(B)普瑞巴林26.7g,收率47.3%,产物对映体过量值e.e.:99.83%,产物的核磁氢谱同附图1。
步骤2:
向反应瓶中加入步骤1中得到的R-构型化合物(C)15g、150mL正丁醇,搅拌升温至35℃,加入纤维二糖差向异构酶(厂家:上海宝曼生物科技有限公司,纯度97%)6g,保温搅拌10h,反应完毕后,过滤除去异构酶,滤液浓缩至约1/3体积,加入50mL水,降温至0-5℃,抽滤、干燥后得到S-构型化合物(D)14.1g,收率94.0%,产物对映体过量值e.e.:99.72%。
步骤3:
向反应瓶中加入步骤2中得到的化合物(D)10g、200mL水、20mL环己酮,搅拌升温至35℃,用饱和碳酸钠溶液调节体系pH值为10,加入伯克霍尔德菌脂肪酶(厂家:上海迈瑞尔化学技术有限公司,纯度98%)4g,保温搅拌7h,反应1h后,每隔1h补加碳酸钠溶液,保持pH值范围8-10。反应完毕后,过滤除去生物酶,滤液用盐酸调pH值至6.5-7.5,降温至5-10℃,过滤、干燥后得到目标化合物(B)普瑞巴林10.2g,收率90.3%,产物对映体过量值e.e.:99.64%,产物的核磁氢谱同附图1。
实施例4:
步骤1:
向反应瓶中加入化合物(A)(厂家:南京博米尔生物科技有限公司,纯度98%)50g、水450mL,1,4-二氧六环50mL,搅拌升温至45℃,用质量分数5%氢氧化钾溶液调节体系pH值为10,加入白地酶(厂家:上海麦克林生化科技有限公司,纯度97%)5g,保温搅拌5h。反应1h后,每隔1h使用pH试纸监测反应液pH变化,并补加适量氢氧化钾溶液以保持pH值范围9-10。反应完毕后,过滤除去生物酶,用250mL二氯甲烷分2次萃取,有机层水洗至中性,浓缩滤液至1/3体积,降温至5-10℃,过滤、干燥后得到副产物R-构型化合物(C)23.3g,收率46.6%,产物对映体过量值e.e.:97.08%;水层用盐酸调pH值至6.5-7.5,降温至5-10℃,过滤、烘干后得到目标化合物(B)普瑞巴林25.8g,收率45.8%,产物对映体过量值e.e.:99.91%,产物的核磁氢谱同附图1。
步骤2:
向反应瓶中加入步骤1中得到的R-构型化合物(C)15g、150mL叔丁醇,搅拌升温至45℃,加入D-塔格糖3-差向异构酶(厂家:上海宝曼生物科技有限公司,纯度97%)6g,保温搅拌10h,反应完毕后,过滤除去异构酶,滤液浓缩至约1/4体积,加入200mL水,降温至5-10℃,抽滤、干燥后得到S-构型化合物(D)14.1g,收率94.0%,产物对映体过量值e.e.:99.72%。
步骤3:
向反应瓶中加入步骤2中得到的化合物(D)10g、200mL水、100mL叔丁醇,搅拌升温至40℃,用饱和碳酸氢钠溶液调节体系pH值为10,加入白地酶(厂家:上海麦克林生化科技有限公司,纯度97%)5g,保温搅拌5h,反应1h后,每隔1h补加碳酸氢钠溶液,保持pH值范围9-10。反应完毕后,过滤除去生物酶,滤液减压浓缩至约2/3体积,用盐酸调pH值至6.5-7.5,降温至5-10℃,过滤、干燥后得到目标产物化合物(B)普瑞巴林10.2g,收率90.3%,产物对映体过量值e.e.:99.64%,产物的核磁氢谱同附图1。
实施例5:
步骤1:
向反应瓶中加入化合物(A)(厂家:南京博米尔生物科技有限公司,纯度98%)50g、水450mL、正丁醇50mL,搅拌升温至35℃,用质量分数10%的碳酸钠溶液调节体系pH值为9-10,加入柱状假丝酵母脂肪酶(厂家:杭州创科生物科技有限公司,纯度98%)10g,保温搅拌5h。反应1h后,每隔1h使用pH试纸检测反应液pH变化,并补加适量碳酸钠溶液以保持pH值范围9-10。反应完毕后,过滤除去生物酶,用150mL乙酸乙酯萃取反应液2次,有机层水洗至中性,减压浓缩至约1/3体积,降温至5-10℃,过滤、干燥后得到副产物R-构型化合物(C)19.8g,收率39.6%,产物对映体过量值e.e.:98.91%;水层用盐酸调pH值至6.5-7.5,降温至5-10℃,过滤、干燥后得到目标产物化合物(B)普瑞巴林25.5g,收率45.2%,产物对映体过量值e.e.:99.12%。产物的核磁氢谱同附图1。
步骤2:
向反应瓶中加入步骤1中得到的R-构型化合物(C)15g、150mL正丁醇,搅拌升温至35℃,加入纤维二糖差向异构酶(厂家:上海宝曼生物科技有限公司,纯度97%)6g,保温搅拌15h,反应完毕后,过滤除去异构酶,滤液浓缩至约1/3体积,加入50mL水,降温至0-5℃,抽滤、干燥后得到S-构型化合物(D)13.8g,收率92.0%,产物对映体过量值e.e.:99.65%。
步骤3:
向反应瓶中加入步骤2中得到的化合物(D)10g、300mL水,搅拌升温至35℃,用质量分数10%碳酸钠溶液调节体系pH值为10,加入柱状假丝酵母脂肪酶(厂家:杭州创科生物科技有限公司,纯度98%)2g,保温搅拌7h,反应1h后,每隔1h补加碳酸钠溶液,保持pH值范围9-10。反应完毕后,过滤除去生物酶,滤液用盐酸调pH值至6.5-7.5,降温至5-10℃,过滤烘干得到目标产物化合物(B)普瑞巴林10.2g,收率90.5%,产物对映体过量值e.e.:99.31%,产物的核磁氢谱同附图1。
以上实施例所述步骤1、2和3中,反应完毕后过滤所得生物酶和异构酶均可回收使用。
Claims (21)
1.一种制备式(III)化合物的方法,包括以下步骤:
步骤1:
化合物(I)在生物酶的作用下,在溶剂中生成目标化合物(III)和副产物(II);
步骤2:
步骤1中所得化合物(II)在异构酶的作用下,在溶剂中发生构型翻转生成化合物(IV);
步骤3:
步骤2中所得化合物(IV)在步骤1中所述生物酶的作用下,在溶剂中生成目标化合物(III);
其中R1和R2为氢原子或烷基,所述烷基为甲基、乙基、正丙基、异丙基、正丁基、叔丁基或异丁基;
其中,
所述生物酶选自霉菌脂肪酶、酵母脂肪酶和细菌脂肪酶,其中,
所述霉菌脂肪酶选自德氏根酶、黑曲霉、米黑根毛酶和白地酶;
所述酵母脂肪酶选自南极假丝酵母脂肪酶、柱状假丝酵母脂肪酶和粘红酵母酶;
所述细菌脂肪酶选自伯克霍尔德菌脂肪酶、假单孢菌脂肪酶和葡萄球菌脂肪酶;
所述异构酶为差向异构酶,所述差向异构酶选自D-木糖异构酶、蔗糖异构酶、D-塔格糖3-差向异构酶、D-阿洛酮糖3-差向异构酶、纤维二糖差向异构酶和2-酮基葡萄糖酸差向异构酶;以及
其中,
步骤1和3中,所述溶剂为水或水和有机溶剂的互溶体系,所述有机溶剂为异丙醇、正丁醇、叔丁醇、四氢呋喃、1,4-二氧六环、丙酮和环己酮中的一种或多种;
步骤2中,所述溶剂为异丙醇、正丁醇、叔丁醇和丙酮中的一种或多种。
2.根据权利要求1所述的方法,其特征在于,
所述霉菌脂肪酶选自德氏根酶和白地酶;
所述酵母脂肪酶选自南极假丝酵母脂肪酶和柱状假丝酵母脂肪酶;
所述细菌脂肪酶为伯克霍尔德菌脂肪酶。
3.根据权利要求1所述的方法,其特征在于,所述差向异构酶选自D-木糖异构酶、蔗糖异构酶和纤维二糖差向异构酶。
4.根据权利要求1至3任一项所述的方法,其特征在于,所述生物酶或异构酶的形态是经过冷冻干燥后的固定化酶颗粒或酶粉末,或经过提取、浓缩、脱水处理的含酶细胞或细胞器。
5.根据权利要求1至3任一项所述的方法,其特征在于,R1为H、R2为甲基。
6.根据权利要求1至3任一项所述的方法,其特征在于,
步骤1中,所述生物酶和化合物(I)的质量比为1:2-1:20;
步骤2中,所述异构酶和步骤1中所得化合物(Ⅱ)的质量比为1:1-1:20;
步骤3中,所述生物酶和步骤2中所得化合物(Ⅳ)的质量比为1:2-1:20。
7.根据权利要求6所述的方法,其特征在于,
步骤1中,所述生物酶和化合物(I)的质量比为1:5-1:10;
步骤2中,所述异构酶和步骤1中所得化合物(Ⅱ)的质量比为1:3-1:9;
步骤3中,所述生物酶和步骤2中所得化合物(Ⅳ)的质量比为1:5-1:10。
8.根据权利要求1至3任一项所述的方法,其特征在于,步骤1和3中,所述有机溶剂选自丙酮和异丙醇。
9.根据权利要求1至3任一项所述的方法,其特征在于,所述水和有机溶剂的互溶体系中水和有机溶剂的混合比例为水和有机溶剂体积比2:1-50:1;酶解反应的溶剂pH值为7.0-10.0。
10.根据权利要求9所述的方法,其特征在于,所述水和有机溶剂的互溶体系中水和有机溶剂的混合比例为水和有机溶剂体积比10:1;酶解反应的溶剂pH值为9.0-10.0。
11.根据权利要求1至3任一项所述的方法,其特征在于,化合物Ⅰ或Ⅳ和所述溶剂的质量体积比为1g:10mL-1g:50mL。
12.根据权利要求11所述的方法,其特征在于,化合物Ⅰ或Ⅳ和所述溶剂的质量体积比为1g:10mL-1g:20mL。
13.根据权利要求1至3任一项所述的方法,其特征在于,所述步骤2中,化合物Ⅱ和所述溶剂的质量体积比为1g:10mL-1g:50mL。
14.根据权利要求13所述的方法,其特征在于,所述步骤2中,化合物Ⅱ和所述溶剂的质量体积比为1g:10mL-1g:20mL。
15.根据权利要求1至3任一项所述的方法,其特征在于,所述步骤1、2和3中,反应温度为25-55℃。
16.根据权利要求15所述的方法,其特征在于,所述步骤1、2和3中,反应温度为35-45℃。
17.根据权利要求15所述的方法,其特征在于,所述步骤1、2和3中,反应时间为5h-20h。
18.根据权利要求15所述的方法,其特征在于,所述步骤1、2和3中,反应时间为5h-10h。
19.根据权利要求1至3任一项所述的方法,其特征在于,所述步骤1和2之间还包括萃取步骤1的产物溶液的步骤。
20.根据权利要求19所述的方法,其特征在于,萃取所用的有机溶剂选自甲苯、二氯甲烷、甲基叔丁基醚和乙酸乙酯。
21.根据权利要求20所述的方法,其特征在于,萃取所用的有机溶剂为乙酸乙酯。
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US5663329A (en) * | 1994-06-15 | 1997-09-02 | Basf Aktiengesellschaft | Preparation of enantiomerically pure lactams |
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