CN110372532A - 六氢呋喃并呋喃醇衍生物的制备方法、其中间体及其制备方法 - Google Patents

六氢呋喃并呋喃醇衍生物的制备方法、其中间体及其制备方法 Download PDF

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CN110372532A
CN110372532A CN201810326250.2A CN201810326250A CN110372532A CN 110372532 A CN110372532 A CN 110372532A CN 201810326250 A CN201810326250 A CN 201810326250A CN 110372532 A CN110372532 A CN 110372532A
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CN110372532B (zh
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高照波
陈建华
万志东
何大伟
周增乐
马晓东
向韦
林荆鑫
梅义将
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Jiangsu Ruike Medicine Science And Technology Co Ltd
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Abstract

本发明涉及医药合成领域，具体涉及六氢呋喃并呋喃醇衍生物的制备方法、其中间体及其制备方法。该制备方法以双羰基化合物或式D0化合物为起始原料，其中，X1，X2相同或不同地为卤素，烷氧基或者构建成环。在六氢呋喃并呋喃醇衍生物的制备过程中，通过酶法来构建手性，采用这样的技术手段能够非常高光学纯度制备得到产物。该制备方法能商业化生产制备达芦那韦关键中间体(3R,3aS,6aR)‑六氢呋喃并[2,3‑b]‑3‑醇，是一条非常经济，适合于工业化生产的路线。

Description

六氢呋喃并呋喃醇衍生物的制备方法、其中间体及其制备 方法
技术领域
本发明涉及医药合成领域，具体涉及六氢呋喃并呋喃醇衍生物的制备方法、其中间体及其制备方法。
背景技术
具有下列式Z结构的化合物为化学名称为(3R,3aS,6aR)-六氢呋喃并[2,3-b]-3-醇：
属于六氢呋喃并呋喃醇衍生物的一种，是抗艾滋病药物达芦那韦的中间体。
达芦那韦原研厂家泰博特克药品有限公司的中国专利申请号分别为02817639.1(申请日：2002-9-6)和200580010400.X提供了上述(3R,3aS,6aR)-六氢呋喃并[2,3-b]-3-醇的制备方法，其中原料为如下的式(3)化合物，
式(3)化合物由起始原料式(1)化合物制备。
达芦那韦仿制药厂家日本住友化学株式会社的中国专利申请号为200380109926.4提供了上述(3R,3aS,6aR)-六氢呋喃并[2,3-b]-3-醇的制备方法，其中起始原料为如下的式VⅢ化合物，
日本住友化学株式会社在手性构型的产生上，选择了使用手性配体催化剂，虽然这的确是构建手性构型的方法，但是，不太适合产业化。
达芦那韦仿制药厂家瑞士Lonza Ltd公司的欧洲专利申请EP2634180A1(申请日：2012-1-3)涉及使用羰基还原酶多肽或含有羰基还原酶多肽的微生物实现羰基还原为羟基，来构建达芦那韦中间体的手性，其说明书中列举了许多种可以商业化购买的酶如酿酒酵母YNL331C等，并且提及如下式Ia所示的化合物是比较合适的构型，
考虑到(3R,3aS,6aR)-六氢呋喃并[2,3-b]-3-醇是制备达芦那韦药物的关键中间体，有必要开发出更多的该关键中间体的制备方法，该方法不仅能获得高收率和高DE值的该关键中间体，而且成本低，反应条件温和，适合产业化。
发明内容
本发明的制备(3R,3aS,6aR)-六氢呋喃并[2,3-b]-3-醇的方法从起始原料的选择和手性构型的构建上着手，研究开发出了不同于上述已有专利申请中的起始原料制备达芦那韦关键中间体的制备方法。本发明的制备方法采用酶法来构建手性，成本低，反应条件温和，为该达芦那韦关键中间体的制备提供了另一条适合产业化的路线。
为实现本发明的技术目的，本发明提供了如下的技术方案：
本发明第一方面提供了制备(3R,3aS,6aR)-六氢呋喃并[2,3-b]-3-醇的中间体化合物，结构式如下：
其中，RZ为烷氧基，-NR4R5；RP为氢，-C-C-O-Bn，-C-C-OH,-C-CO2R6，-C-CO2Ar；RL为-OH或羟基保护基。所述羟基保护基可以为-O-Bn,-O-Bz,-OCOR6，-OR6，-OAr,-OCOAr等，Ar可以为芳基，杂芳基，取代芳基或取代杂芳基，所述杂芳基可以为呋喃、吡啶、噻吩、吲哚或萘等；RP,RL并构成环；R4，R5相同或不同地为苯基，烷基或取代苯基；R6为烷基；*表示手性。
较优选地，结构式如下：
其中，RP为氢，-C-C-O-Bn，-C-C-OH,-C-CO2R6，-C-CO2Ar；RL为-OH，-O-Bn,-O-Bz,-OCOR6，-OR6，-OAr，-OCOAr等；R4，R5相同或不同地为苯基，烷基或取代苯基；R6为烷基；*表示手性。
更优选地，结构式如下：
本发明第二方面提供了上述中间体化合物的制备方法，由式B化合物经酶法还原反应构建手性制备，
其中，RZ,RP,RL的定义与上述定义相同。
所述酶是一种生物酶，如醛酮还原酶，其中，醛酮还原酶来源于Saccharomyceskudriavzevii菌株。其氨基酸序列为SEQ ID NO：1所示的蛋白质，或SEQ ID NO：1经过一个或几个氨基酸残基取代、缺失或添加后具有醛酮还原酶活性的蛋白质，或与SEQ ID NO：1所示的氨基酸序列具有80％以上同源性且具有醛酮还原酶活性的蛋白质；其核苷酸序列如序列表中SEQ ID NO：2所示。醛酮还原酶可以来自于基因工程菌全细胞、破碎酶液、冻干粉或固定化酶或固定化细胞。
所述酶的投料量可以为50-100g/L，所述反应温度可以为25-37℃。
所述酶法还原反应中可以选择性加入辅酶，所述辅酶为NADP+或NADPH。
所述酶法还原反应中可以选择性加入葡萄糖脱氢酶。
所述酶法还原反应在溶剂存在的条件下进行，所述溶剂为水或缓冲溶液与有机溶剂组成的混合溶剂。
所述缓冲溶液选自磷酸盐缓冲溶液、碳酸盐缓冲溶液、Tri-HCl缓冲溶液、柠檬酸盐缓冲溶液或MOPS缓冲溶液中的一种或多种。
所述有机溶剂选自DMSO、乙酸乙酯、乙酸丁酯、异丙醇、DMF、TBME、二氯甲烷、乙酸乙烯酯中的一种或几种。
本发明酶法还原反应的生物转化过程中利用HPLC-MS和HPLC进行监控，直至底物完全利用。
较优选地，由式B2化合物经酶法还原反应构建手性制备，
其中，RP,RL的定义与上述相同。
所述还原反应构建手性的方法为酶法，所述酶的定义与上述相同。
本发明上述中间体化合物用于制备(3R,3aS,6aR)-六氢呋喃并[2,3-b]-3-醇的中间体化合物。
本发明第三方面提供了制备(3R,3aS,6aR)-六氢呋喃并[2,3-b]-3-醇的方法，由式C化合物经还原反应制备式C-i化合物，
然后，式C-i化合物经关环反应制备(3R,3aS,6aR)-六氢呋喃并[2,3-b]-3-醇，
其中，RZ,RP,RL的定义与上述相同。
较优选地，由式C2化合物经还原反应用于制备式C-i 1化合物，
所述还原反应中还原剂可以为硼类还原剂，铝类还原剂或硅锂类还原剂。如硼氢化钠，氰基硼氢化钠，四氢铝锂，红铝，氢化铝锂，二异丁基氢化铝，二异丙基氨基锂，六甲基二硅基氨基锂。
进一步，式C-i1化合物经关环反应制备(3R,3aS,6aR)-六氢呋喃并[2,3-b]-3-醇，
其中，RP,RL的定义与上述定义相同。
所述关环反应的试剂为本领域常见的酸或碱。
经还原反应制备得到的式C-i1化合物可不经分离，经关环反应制备(3R,3aS,6aR)-六氢呋喃并[2,3-b]-3-醇。
本发明第四方面提供了式B2化合物的制备方法，由二羰基化合物与胺类化合物反应制备，
其中，X1为卤素，烷氧基或离去基，RP，RL的定义与上述相同。
较优选地，由二羰基化合物与N-甲基苯胺反应后经进一步地取代反应制备，
其中，X1或X2相同或不同地为卤素，烷氧基，离去基或并构为环，RP，RL的定义与上述相同。
更优选地，由二羰基化合物与苯甲醇反应后，进一步与N-甲基苯胺反应后，进一步与卤酸酯化合物如溴乙酸乙酯反应制备，反应式分别为：
X为卤素，R6为烷基；
本发明第五方面提供了式B2化合物的另一种制备方法，由二羰基化合物与RLH化合物反应后，进一步与卤代化合物反应制备，
其中，X为卤素，RL为-O-Ar，-O-CO-Ar，-O-CO-Ar，-O-Bn,-O-Bz，-OR6或-OCOR6。
较优选地，由二羰基化合物与苯甲醇反应后，进一步与溴代化合物反应制备，
本发明第六方面提供了式B2化合物的不同的另一种制备方法，由式D化合物与卤代化合物如氯代化合物反应后，进一步与N-甲基苯胺反应制备，
当Ar为苄基时，反应式如下：
本发明第七方面提供了式B2化合物的以式D0化合物为原料的另一种制备方法，由式D0化合物在卤素如Br2的作用下与N-甲基苯胺反应后经进一步取代反应制备，
其中，RL为-O-Ar，-O-CO-Ar，-O-Bz,-OR6,-OCOR6或-OR6，X为卤素，较优选地，为溴。
本发明上述(3R,3aS,6aR)-六氢呋喃并[2,3-b]-3-醇的制备方法，较优选地实施方式为：由式D0化合物在卤素的作用下与N-甲基苯胺反应后经两步取代反应，环合反应，酶法还原反应和还原关环反应制备，
其中，RL为-O-Ar，-O-CO-Ar，-O-Bz,-OR6,-OCOR6或-OR6，X为卤素，较优选地，为溴，R6为烷基，较优选地，为乙基。
本发明上述(3R,3aS,6aR)-六氢呋喃并[2,3-b]-3-醇的制备方法，较优选地另一种实施方式为：由式D0化合物在卤素的作用下与N-甲基苯胺反应后经两步取代反应，酶法还原反应和还原关环反应制备，
其中，RL为-O-Ar，-O-CO-Ar，-O-Bz,-OCOR6或-OR6。优选地，RL为乙酸基(-OCOCH3)或叔丁氧基，X为卤素，较优选地，为溴，R6为烷基，较优选地，为乙基。
本发明上述(3R,3aS,6aR)-六氢呋喃并[2,3-b]-3-醇的制备方法，较优选地其他另一种实施方式为：由二羰基化合物与苯甲醇反应后，进一步与N-甲基苯胺反应后，进一步与卤酸酯化合物如卤乙酸乙酯反应，后经酶法还原反应，还原关环反应制备，
其中，X为卤素，较优选地，为溴，R6为烷基，较优选地，为乙基。
本发明上述(3R,3aS,6aR)-六氢呋喃并[2,3-b]-3-醇的制备方法，较优选地其他另一种实施方式为：由式D化合物与卤代化合物反应后，与N-甲基苯胺反应，与卤酸酯如卤乙酸乙酯反应，后经酶法还原反应，还原关环反应制备，
其中，X为卤素，较优选地，为溴，R6为烷基，较优选地，为乙基。
本发明上述(3R,3aS,6aR)-六氢呋喃并[2,3-b]-3-醇的制备方法，较优选地其他另一种实施方式为：由二羰基化合物与苯甲醇反应后，进一步与卤代化合物反应，与N-甲基苯胺化合物反应，后经酶法还原反应，还原关环反应制备，
其中，X为卤素。
本发明提供的(3R,3aS,6aR)-六氢呋喃并[2,3-b]-3-醇的制备方法，采用酶法构建手性，能够高收率，高光学纯度的制备得到产物。虽然现有技术如上述欧洲申请中已公开羰基还原酶多肽或含有羰基还原酶多肽的微生物实现羰基还原为羟基，然而，本发明使用的酶具备更显著地优势，体现在更高光学纯度的得到产物和更适宜的反应条件上。本发明的(3R,3aS,6aR)-六氢呋喃并[2,3-b]-3-醇的制备方法适宜于工业化生产。
具体实施方式
为了进一步理解本发明，下面结合实施例对本发明提供的六氢呋喃并呋喃醇衍生物的制备方法、其中间体及其制备方法进行详细说明。需要理解的是，这些实施例描述只是为进一步详细说明本发明的特征，而不是对本发明范围或本发明权利要求范围的限制。
实施例1：
于500ml四口瓶中依次加入式D0化合物(30.00g，1.0eq)，二氯甲烷(150ml)，内温冷却至-20℃后，滴加溴素(57.00g溶于90mlDCM中)，滴毕；于另一500ml四口瓶中依次加入N-甲基苯胺(38.20g，1.0eq)，二氯甲烷(120ml)，三乙胺(36.10g，1.0eq)，内温冷却至-20℃后，将前一反应瓶中的反应液滴加至该反应液中，滴毕，缓慢升温至室温并保温搅拌，反应完成后，依次采用水，饱和食盐水洗涤，无水硫酸钠干燥，40℃减压浓缩干后得红棕色油状物81.29g，收率84.33％。
实施例2：
于氮气氛围中，反应瓶内依次加入N-甲基苯胺(7.01g，1.1eq)，甲苯(60ml)，式D0化合物(5.00g)，加热使内温升至100℃，保温搅拌，停止反应，冷却至室温后，加入8％稀盐酸(60.00ml)搅拌洗涤，分层后，水层加入甲苯反萃，合并有机层，加入水洗涤，取甲苯层加入适量无水硫酸钠干燥，45℃减压浓缩干后得油状物9.80g，收率86.19％。
实施例3：
于反应瓶中依次加入实施例1制备得到的二羰基化合物(2.00g，1.0eq)，DMF(16.00ml)，乙酸钾(0.73g，1.0eq)，搅拌。反应完成后，依次加入乙酸乙酯和水，搅拌分层后，取EA层，水层加入EA反萃，合并EA层，用水洗涤，饱和食盐水洗涤后，有机相加入适量无水硫酸钠干燥，40℃减压浓缩干后得1.58g棕色油状物，收率85.59％。
实施例4：
于反应瓶中依次加入实施例2制备得到的棕色油状物(6.00g，1.0eq)，DCM(24.00ml)，内温冷却，缓慢滴加溴素(5.01g溶于12ml DCM，1.0eq)，滴毕，缓慢升至室温(20-25℃)保温搅拌。停止反应，30℃减压浓缩干得产物粗品6.96g，收率82.05％。
实施例5：
于反应瓶中依次加入式B2化合物(RP为氢，RL为乙酸基)(1.30g，1.0eq)，DMF(13.00ml)，溴乙酸乙酯(0.91g，1.05eq)，碳酸钾(0.79g，1.1eq)，室温搅拌。反应完成后，依次加入乙酸乙酯和水搅拌分层后，取EA层，水层加入EA反萃，合并EA层，水洗，饱和食盐水洗涤后，有机相加入适量无水硫酸钠干燥，40℃减压浓缩干后得1.40g棕色油状物，收率94.05％。1HNMR(CDCl3):1.24(t,J＝6.8,3H),2.10(s,3H),2.68(dd,J1＝17.2,J2＝5.6,1H),2.97(dd,J1＝17.2,J2＝8.8,1H),3.31(s,3H),3.99～4.03(m,1H),4.09(dd,J1＝14.4,J2＝7.2,1H),4.17(d,J＝17.2,1H),4.56(d,J＝17.2,1H),7.29～7.48(m,5H).
实施例6：
醛酮还原酶基因工程菌全细胞的制备
重组醛酮还原酶基因工程菌，具体制备方法是：选择来源于Saccharomyceskudriavzevii的醛酮还原酶的基因序列，进行人工设计，将人工设计后的序列通过全基因合成(委托金斯瑞生物科技有限公司合成)，克隆入表达载体pET28a的Nde I和Xho I酶切位点，转化宿主菌E.coli BL21(DE3)感受态细胞；挑取阳性转化子并测序鉴定后，得到重组表达载体；将重组表达载体转入E.coli BL21(DE3)菌株中，获得可以诱导表达重组醛酮还原酶的重组醛酮还原酶基因工程菌。
将重组醛酮还原酶基因工程菌接种到含有卡那霉素的LB培养基中，于37℃过夜培养，得到种子培养液；将种子培养液接种到含卡那霉素的TB培养基中，接种量为含卡那霉素的TB培养基体积的1％；然后置于37℃下培养2-5h，加入无菌的IPTG诱导，使IPTG终浓度达到0.1mM，置于25℃下继续培养20h。最后通过高速离心得到来源于Saccharomyceskudriavzevii的醛酮还原酶基因工程菌全细胞。采用超声破碎方法对得到的基因工程菌全细胞进行超声破碎，得到来源于Saccharomyces kudriavzevii的醛酮还原酶基因工程菌全细胞的破碎酶液。醛酮还原酶为氨基酸序列为SEQ ID NO：1所示的蛋白质，醛酮还原酶基因的核苷酸序列如序列表中SEQ ID NO：2所示。
经诱导表达后在45kDa处有明显的蛋白条带，表明醛酮还原酶在重组菌中得到了高效表达。测定醛酮还原酶纯蛋白的酶活性，反应体系为0.25ml，包括Tris-HCl，pH为8.0，2mmol/L NADPH，0.1mmol/L底物以及适量酶，测定340nm处吸光度的减少，酶活力单位(U)定义为：在上述条件下，每分钟催化1umol NADPH氧化所需的酶量。
结果表明，重组的基因工程醛酮还原酶较欧洲专利(EP2634180A1)序列的醛酮还原酶活性提高了20％以上，较未突变的醛酮还原酶活性提高了50％以上。
本发明实施例中所使用的醛酮还原酶基因工程菌全细胞均采用此方法制备。
本发明实施例及对比例中所使用的葡萄糖脱氢酶均为购自于sigma-aldrich的商品酶。
对映异构体过量表达的ee值的算法为:
ee(syn)＝([R,R]-[S,S])/([R,R]+[S,S])
ee(anti)＝([R,S]-[S,R])/([R,S]+[S,R])
de＝{([R,S]+[S,R])-([R,R]+[S,S])}/{([R,S]+[S,R])+([R,R]+[S,S])}。
酶法还原反应，反应式：
步骤1：反应在1L摇瓶中进行，反应体系控制为300mL，在摇瓶中用250mL灭菌后的去离子水悬浮醛酮还原酶基因工程菌全细胞，投入葡萄糖脱氢酶，加入2.5mol/L的葡萄糖10mL和0.26g的NADP+，再称取10g的底物，溶于30mL乙酸丁酯中，将溶有底物的乙酸丁酯溶液缓慢倒入摇瓶中，反应1h后补加2.5mol/L的葡萄糖10mL，投入醛酮还原酶基因工程菌全细胞的量为75g/L，投入葡萄糖脱氢酶的量为25mg/L，控制转化体系的温度为37℃；转化反应在摇床中进行，摇床的转速控制为200r/min，转化时间为12h，得到含目标化合物的转化液，转化率为97.8％。
步骤2：对步骤1得到的目标化合物的转化液进行纯化，向反应体系中加入等体积的乙酸乙酯，37℃下萃取15min，重复3次，离心收集乙酸乙酯层，向收集的乙酸乙酯层中加入5％的无水硫酸镁振荡15min后过滤除去硫酸镁，再将除水后的乙酸乙酯层进行高温减压浓缩，得到目标化合物9.32g，所制备的目标化合物的de值为97.1％，ee(anti)值为99.5％。
实施例7：
于冰浴中，反应瓶内依次加入式B2化合物(RP为RL为乙酸基)(4.40g，1.0eq)，THF(44.00ml)，甲醇(4.40ml)，内温冷却，分批缓慢加入硼氢化钠(0.4g，0.8eq)，加毕保温搅拌。反应完成后，缓慢滴加10％稀硫酸调节pH，滴毕缓慢升至室温搅拌，依次加入乙酸乙酯和水搅拌分层后，取EA层，水层加入EA反萃，合并EA层，水洗，饱和食盐水洗涤后，有机相加入适量无水硫酸钠干燥，40℃减压浓缩干后得3.98g无色油状物，收率86.43％。
实施例8：
于反应瓶中依次加入式B2化合物(RP为RL为乙酸基)(1.50g，1.0eq)，THF(20.00ml)，内温冷却，分批缓慢加入LiAlH4(0.68g，4.0eq)，加毕缓慢升温至室温(20-25℃)保温搅拌。反应完成后，内温冷却，缓慢滴加10％稀硫酸调节pH，滴毕缓慢升至室温搅拌。内温冷却，缓慢滴加4％NaOH水溶液调节反应液pH，调毕，加入甲苯搅拌萃取，合并甲苯层，取水层，加入DCM搅拌萃取，合并DCM层，取DCM层加入适量无水硫酸钠干燥，40℃减压浓缩干后得无色油状物0.43g，收率74.55％。
实施例9：
于反应瓶中依次加入式D化合物(25.55g，1.0eq)，DCM(350.00ml)，内温冷却，缓慢滴入吡啶(29.33g，2.15eq)，滴毕，保温搅拌，缓慢滴加氯代化合物(35.00g，1.07eq)，滴毕后缓慢升至室温(20-25℃)保温搅拌，加入稀盐酸溶液，搅拌静置分层后，取有机层，水层加入DCM反萃，合并DCM层，依次加入水，饱和食盐水洗涤，加入适量无水硫酸钠干燥，40℃减压浓缩干后得产物53.56g，收率96.73％。
实施例10：
于反应瓶中依次加入实施例9中的产物(2.00g，1.0eq),甲苯(16.00ml)N-甲基苯胺(1.05eq)，氮气保护下升温至回流保温搅拌，冷却至室温后，加入5％稀盐酸(40.00ml)搅拌洗涤，取甲苯层，依次加入水,饱和食盐水洗涤分液后，加入适量无水硫酸钠干燥，45℃减压浓缩的产物粗品1.83g，收率90.15％。
实施例11：
于500ml四口瓶中依次加入苯甲醇(20.76g，1.0eq)，THF(180.00ml)，内温冷却，加入NaH(16.11g，2.1eq)，加毕保温搅拌，缓慢滴加二羰基化合物(30.00g，0.95eq)，加毕于室温下自然升温搅拌反应。降温，滴加5％的盐酸溶液调节pH，调毕，加入乙酸乙酯萃取分液，其中水层用乙酸乙酯萃取分液，合并有机相后用水洗涤分液，有机层继续用饱和食盐水洗涤分液，有机相加入适量无水硫酸钠干燥，45℃减压旋蒸后得油状物40.65g，收率89.62％。
实施例12：
于反应瓶中依次加入二羰基化合物(5.00g，1.0eq),甲苯(40.00ml),N-甲基苯胺(1.2eq)，氮气保护下升温至回流保温搅拌，冷却至室温后，加入5％稀盐酸搅拌洗涤，取甲苯层，依次加入水,饱和食盐水洗涤分液后，加入适量无水硫酸钠干燥，45℃减压浓缩的产物粗品3.27g，收率52.02％。
实施例13：
于反应瓶中依次加入式B2化合物(RP为氢，RL为-OBn)(2.00g，1.0eq)，DMF(20.00ml)，溴乙酸乙酯(1.24g，1.1eq)，碳酸钾(1.12g，1.2eq)，室温(20-25℃)搅拌，停止反应，加入EA和水搅拌静置分层，取EA层，水层加入EA反萃，合并EA层，将EA层以水洗涤后，加入适量无水硫酸钠干燥除水，40℃减压干燥后得产物粗品2.21g，收率85.59％。
1HNMR(CDCl3):1.18(t,J＝7.2,3H),2.62(dd,J1＝16.8,J2＝6.4,1H),2.78(dd,J1＝17.2,J2＝7.6,1H),3.24(s,3H),3.81(dd,J1＝48.0,J2＝16.8,2H),4.01～4.06(m,2H),4.13(t,J＝6.8,1H),7.09～7.32(m,10H).
实施例14：
步骤1：反应在1L摇瓶中进行，反应体系控制为300mL，在摇瓶中用260mL灭菌后的磷酸钾缓冲溶液悬浮醛酮还原酶基因工程菌全细胞破碎酶液，投入葡萄糖脱氢酶，超声50min破碎细胞，再向摇瓶中加入25g的葡萄糖，0.42g的NADP+，再称取8g的反应物，溶于40mL的DMSO中，将溶有底物的DMSO溶液缓慢倒入摇瓶中，反应2h后补加12g的葡萄糖，投入醛酮还原酶基因工程菌全细胞的量为75g/L，投入葡萄糖脱氢酶的量为25mg/L，控制转化体系的温度为37℃；转化反应在摇床中进行，摇床的转速控制为200r/min，转化时间为12h，得到目标产物的转化液，转化率为97.8％。
步骤2：对步骤1得到的目标产物的转化液进行纯化，向反应体系中加入等体积的乙酸乙酯，37℃下萃取15min，重复3次，离心收集乙酸乙酯层，向收集的乙酸乙酯层中加入5％的无水硫酸镁振荡15min后过滤除去硫酸镁，再将除水后的乙酸乙酯层进行高温减压浓缩，得到目标产物7.39g，de值为96.2％，ee(anti)值为99.5％。
实施例15：
于氢化釜中依次加入式B2化合物(RP为RL为-OBn)(2.00g)，乙醇(20.00ml)，10％Pd/C(0.2g)，氢气置换三次，保持氢气压力(1.0MPa)，室温(20-25℃)搅拌8.0h后，抽滤，滤层采用约5ml乙醇洗涤后，合并洗涤液与滤液，40℃减压浓缩得产物1.41g，收率92.00％。
实施例16：
于反应瓶中依次加入式B2化合物(RP为RL为-OH)(1.50g，1.0eq)，THF(20.00ml)，内温冷却，分批缓慢加入LiAlH4(0.77g，4.0eq)，加毕缓慢升温至室温(20-25℃)保温搅拌。反应完成后，内温冷却，缓慢滴加10％稀盐酸调节pH，滴毕缓慢升至室温(20-25℃)搅拌。内温冷却，缓慢滴加4％NaOH水溶液调节反应液pH，调毕，加入甲苯搅拌萃取，合并甲苯层，取水层，加入DCM搅拌萃取，合并DCM层，取DCM层加入适量无水硫酸钠干燥，40℃减压浓缩干后得无色油状物0.48g，收率72.40％。
实施例17：
步骤1：反应在1L摇瓶中进行，反应体系控制为300mL，在摇瓶中用250mL灭菌后的去离子水悬浮醛酮还原酶基因工程菌全细胞，投入葡萄糖脱氢酶，加入2.5mol/L的葡萄糖10mL，0.26g的NADP+，再称取10g的底物，溶于30mL乙酸丁酯中，将溶有底物的乙酸丁酯溶液缓慢倒入摇瓶中，反应1h后补加2.5mol/L的葡萄糖10mL，投入醛酮还原酶基因工程菌全细胞的量为75g/L，投入葡萄糖脱氢酶的量为25mg/L，控制转化体系的温度为37℃；转化反应在摇床中进行，摇床的转速控制为200r/min，转化时间为12h，得到目标化合物的转化液，转化率为97.8％。
步骤2：对步骤1得到的目标化合物的转化液进行纯化，向反应体系中加入等体积的乙酸乙酯，37℃下萃取15min，重复3次，离心收集乙酸乙酯层，向收集的乙酸乙酯层中加入5％的无水硫酸镁振荡15min后过滤除去硫酸镁，再将除水后的乙酸乙酯层进行高温减压浓缩，得到目标化合物9.51g，所制备的目标化合物的de值为99.1％，对映异构体的ee(anti)值为99.7％。
实施例18：对照实施例
步骤1：反应在1L摇瓶中进行，反应体系控制为300mL，在摇瓶中用250mL灭菌后的去离子水悬浮醛酮还原酶基因工程菌全细胞，所用的醛酮还原酶基因工程菌全细胞的醛酮还原酶基因的编码序列如欧洲专利EP2634180中公布序列(即专利EP2634180中的SEQ IDNO12所示)，序列通过全基因合成(委托金斯瑞生物科技有限公司合成)，制备方法如实施例3，再加入葡萄糖脱氢酶，加入2.5mol/L的葡萄糖10mL，0.26g的NADP+，再称取10g的底物，溶于30mL乙酸丁酯中，将溶有底物的乙酸丁酯溶液缓慢倒入摇瓶中，反应1h后补加2.5mol/L的葡萄糖10mL，投入醛酮还原酶基因工程菌全细胞的量为75g/L，投入葡萄糖脱氢酶的量为25mg/L，控制转化体系的温度为37℃；转化反应在摇床中进行，摇床的转速控制为200r/min，转化时间为12h。
纯化步骤如实施例3，得到目标化合物8.07g，所制备的目标化合物的de值为85.1％，对映异构体的ee(anti)值为93.3％。
实施例19：对照实施例
步骤1：反应在1L摇瓶中进行，反应体系控制为300mL，在摇瓶中用250mL灭菌后的去离子水悬浮以Saccharomyces kudriavzevii的醛酮还原酶基因工程菌全细胞，所用的醛酮还原酶基因工程菌全细胞的醛酮还原酶基因的编码序列如SEQ ID NO 3所示，(醛酮还原酶基因的编码序列未经人工设计)，序列通过全基因合成(委托金斯瑞生物科技有限公司合成)，制备方法如实施例3，加入葡萄糖脱氢酶，加入2.5mol/L的葡萄糖10mL，0.26g的NADP+，再称取10g的底物，溶于30mL乙酸丁酯中，将溶有底物的乙酸丁酯溶液缓慢倒入摇瓶中，反应1h后补加2.5mol/L的葡萄糖10mL，投入醛酮还原酶基因工程菌全细胞的量为75g/L，投入葡萄糖脱氢酶为25mg/L，控制转化体系的温度为37℃；转化反应在摇床中进行，摇床的转速控制为200r/min，转化时间为12h。
纯化步骤如实施例3，得到目标化合物7.70g，所制备的目标化合物的de值为79.6％，对映异构体的ee(anti)值为88.7。
SEQ ID NO 1序列号为：
Met Ser Asp Leu Phe Lys Pro Ala Pro Glu Pro Pro Thr Glu Leu Gly
Arg Leu Arg Val Leu Ser Lys Thr Ala Gly Ile Arg Val Ser Pro Leu
Ile Leu Gly Gly Ala Ser Ile Gly Asp Ala Trp Ser Gly Phe Met Gly
Ser Met Asn Lys Glu Gln Ala Phe Glu Leu Leu Asp Ala Phe Tyr Glu
Ala Gly Gly Asn Cys Val Asp Thr Ala Asn Ser Tyr Gln Asn Glu Glu
Ser Glu Ile Trp Ile Gly Glu Trp Met Lys Ser Arg Lys Leu Arg Asp
Gln Ile Val Ile Ala Thr Lys Phe Thr Gly Asp Tyr Lys Lys Tyr Glu
Val Gly Gly Gly Lys Ser Ala Asn Tyr Cys Gly Asn His Lys His Ser
Leu His Val Ser Val Arg Asp Ser Leu Arg Lys Leu Gln Thr Asp Trp
Ile Asp Ile Leu Tyr Val His Trp Trp Asp Tyr Met Ser Ser Ile Glu
Glu Val Met Asp Ser Leu His Ile Leu Ile Gln Gln Gly Lys Val Leu
Tyr Leu Gly Val Ser Asp Thr Pro Ala Trp Val Val Ser Ala Ala Asn
Asn Tyr Ala Thr Ser His Gly Lys Thr Pro Phe Ser Ile Tyr Gln Gly
Lys Trp Asn Val Leu Asn Arg Asp Phe Glu Arg Asp Ile Ile Pro Met
Ala Arg His Phe Gly Met Ala Leu Ala Pro Trp Asp Val Met Gly Gly
Gly Lys Phe Gln Ser Lys Lys Ala Met Glu Glu Trp Lys Lys Asn Gly
Glu Gly Leu Arg Thr Ala Val Gly Gly Pro Glu Gln Thr Glu Leu Glu
Val Lys Ile Ser Glu Ala Leu Asn Lys Ile Ala Glu Glu His Gly Thr
Glu Ser Val Thr Ala Ile Ala Ile Ala Tyr Val Arg Ser Lys Ala Lys
Asn Val Phe Pro Leu Val Gly Gly Arg Lys Ile Glu His Leu Lys Gln
Asn Ile Glu Ala Leu Ser Ile Lys Leu Thr Pro Glu Gln Ile Glu Tyr
Leu Glu Ser Ile Val Thr Phe Asp Val Gly Phe Pro Lys Ser Asn Ile
Gly Asp Asp Pro Ala Val Thr Lys Lys Leu Ser Pro Leu Thr Ser Met
Ser Ala Arg Ile Ser Phe Asp Asn
SEQ ID NO 2序列号为：
SEQ ID NO 3序列号为：
序列表
<110>江苏瑞科医药科技有限公司
<120>六氢呋喃并呋喃醇衍生物的制备方法、其中间体及其制备方法
<160>3
<170>SIPOSequenceListing 1.0
<210>1
<211>376
<212>PRT
<213>artifical sequence
<400>1
Met Ser Asp Leu Phe Lys Pro Ala Pro Glu Pro Pro Thr Glu Leu Gly
1 5 10 15
Arg Leu Arg Val Leu Ser Lys Thr Ala Gly Ile Arg Val Ser Pro Leu
20 25 30
Ile Leu Gly Gly Ala Ser Ile Gly Asp Ala Trp Ser Gly Phe Met Gly
35 40 45
Ser Met Asn Lys Glu Gln Ala Phe Glu Leu Leu Asp Ala Phe Tyr Glu
50 55 60
Ala Gly Gly Asn Cys Val Asp Thr Ala Asn Ser Tyr Gln Asn Glu Glu
65 70 75 80
Ser Glu Ile Trp Ile Gly Glu Trp Met Lys Ser Arg Lys Leu Arg Asp
85 90 95
Gln Ile Val Ile Ala Thr Lys Phe Thr Gly Asp Tyr Lys Lys Tyr Glu
100 105 110
Val Gly Gly Gly Lys Ser Ala Asn Tyr Cys Gly Asn His Lys His Ser
115 120 125
Leu His Val Ser Val Arg Asp Ser Leu Arg Lys Leu Gln Thr Asp Trp
130 135 140
Ile Asp Ile Leu Tyr Val His Trp Trp Asp Tyr Met Ser Ser Ile Glu
145 150 155 160
Glu Val Met Asp Ser Leu His Ile Leu Ile Gln Gln Gly Lys Val Leu
165 170 175
Tyr Leu Gly Val Ser Asp Thr Pro Ala Trp Val Val Ser Ala Ala Asn
180 185 190
Asn Tyr Ala Thr Ser His Gly Lys Thr Pro Phe Ser Ile Tyr Gln Gly
195 200 205
Lys Trp Asn Val Leu Asn Arg Asp Phe Glu Arg Asp Ile Ile Pro Met
210 215 220
Ala Arg His Phe Gly Met Ala Leu Ala Pro Trp Asp Val Met Gly Gly
225 230 235 240
Gly Lys Phe Gln Ser Lys Lys Ala Met Glu Glu Trp Lys Lys Asn Gly
245 250 255
Glu Gly Leu Arg Thr Ala Val Gly Gly Pro Glu Gln Thr Glu Leu Glu
260 265 270
Val Lys Ile Ser Glu Ala Leu Asn Lys Ile Ala Glu Glu His Gly Thr
275 280 285
Glu Ser Val Thr Ala Ile Ala Ile Ala Tyr Val Arg Ser Lys Ala Lys
290 295 300
Asn Val Phe Pro Leu Val Gly Gly Arg Lys Ile Glu His Leu Lys Gln
305 310 315 320
Asn Ile Glu Ala Leu Ser Ile Lys Leu Thr Pro Glu Gln Ile Glu Tyr
325 330 335
Leu Glu Ser Ile Val Thr Phe Asp Val Gly Phe Pro Lys Ser Asn Ile
340 345 350
Gly Asp Asp Pro Ala Val Thr Lys Lys Leu Ser Pro Leu Thr Ser Met
355 360 365
Ser Ala Arg Ile Ser Phe Asp Asn
370 375
<210>2
<211>1131
<212>DNA
<213>artifical seauence
<400>2
atgagcgatc tgtttaaacc ggcgccggaa ccgccgaccg aactgggccg cctgcgcgtg 60
ctgagcaaaa ccgcgggcat tcgcgtgagc ccgctgattc tgggcggcgc gagcattggc 120
gatgcgtgga gcggctttat gggcagcatg aacaaagaac aggcgtttga actgctggat 180
gcgttttatg aagcgggcgg caactgcgtg gataccgcga acagctatca gaacgaagaa 240
agcgaaattt ggattggcga atggatgaaa agccgcaaac tgcgcgatca gattgtgatt 300
gcgaccaaat ttaccggcga ttataaaaaa tatgaagtgg gcggcggcaa aagcgcgaac 360
tattgcggca accataaaca tagcctgcat gtgagcgtgc gcgatagcct gcgcaaactg 420
cagaccgatt ggattgatat tctgtatgtg cattggtggg attatatgag cagcattgaa 480
gaagtgatgg atagcctgca tattctgatt cagcagggca aagtgctgta tctgggcgtg 540
agcgataccc cggcgtgggt ggtgagcgcg gcgaacaact atgcgaccag ccatggcaaa 600
accccgttta gcatttatca gggcaaatgg aacgtgctga accgcgattt tgaacgcgat 660
attattccga tggcgcgcca ttttggcatg gcgctggcgc cgtgggatgt gatgggcggc 720
ggcaaatttc agagcaaaaa agcgatggaa gaatggaaaa aaaacggcga aggcctgcgc 780
accgcggtgg gcggcccgga acagaccgaa ctggaagtga aaattagcga agcgctgaac 840
aaaattgcgg aagaacatgg caccgaaagc gtgaccgcga ttgcgattgc gtatgtgcgc 900
agcaaagcga aaaacgtgtt tccgctggtg ggcggccgca aaattgaaca tctgaaacag 960
aacattgaag cgctgagcat taaactgacc ccggaacaga ttgaatatct ggaaagcatt 1020
gtgacctttg atgtgggctt tccgaaaagc aacattggcg atgatccggc ggtgaccaaa 1080
aaactgagcc cgctgaccag catgagcgcg cgcattagct ttgataacta a 1131
<210>3
<211>1131
<212>DNA
<213>artifical seauence
<400>3
atgtctgatg tatttggacc tgcacctgaa ccacctaccg agttaggacg tctaagagtt 60
ctctctaaaa cagctggtat aagagtctct ccgctaatat tgggaggtat gtcgattggt 120
gacgcctggt caggattcat ggggtcaatg aacaaggagc gggcttttga gctgcttgat 180
gcctttttcg aggcaggtgg aaacttcatt gatactgcaa ataattacca aaatgaacag 240
tcagaggcat ggataggtga atggatggtt tcaagaaaat tgcgtgacca aattgttatt 300
gccaccaaat tcaccacaga ctataagaag tatgaagtgg gcaagggcag aagtgccaac 360
ttctgtggta atcacaagca tagtttacac gtaagtgtga gagattctct tcgcaaattg 420
cagactgatt ggattgacat tctctatgtt cactggtggg attatatgag ttcgatcgag 480
gaagttatgg atagtctgca tattcttgtg cagcagggca aggtcctcta cctgggagta 540
tctgatacac ctgcatgggt cgtgtctgct gcaaattact acgctacctc tcacgggaaa 600
actcccttca gcatctatca aggtaaatgg aatctgttga atagggactt tgagcgtgaa 660
attattccaa tggctaggca ttttggtatg gctctcgctc catgggatgt catgggaggg 720
ggaagatttc agagcaaaaa agctttagaa gaacggaaga agagtggaga gggcctgcgt 780
agctttgttg gtacatctga acagacggat gcagaggtta agatcagcga ggcattgtcg 840
aaggttgctg aggaacatgg cattgagtct gtcacagcta ttgccattgc ctatgtccgc 900
tccaaagcga agcatgtttt cccattggtt ggaggaagga aaattgagca cctcaaacaa 960
aatattgagg cgttgagtat taaattgaca ccaggacaga tagaatatct agaaagcatt 1020
gtcccattcg atgttgggtt ccctagcaat ttcatcggag atgatcctgc agttactaag 1080
aaacttgcat tccttccagc aatgtctgcc aagattgctt ttgacgatta g 1131

Claims (29)

1.一种(3R,3aS,6aR)-六氢呋喃并[2,3-b]-3-醇中间体式B，C或C-i化合物，其特征在于，结构式如下：
其中，RZ为烷氧基，-NR4R5；RP为氢，-C-C-O-Bn，-C-C-OH,-C-CO2R6，-C-CO2Ar；RL为-OH或羟基保护基；RP,RL并构成环；R4，R5相同或不同地为苯基，烷基或取代苯基；R6为烷基；*表示手性。
2.根据权利要求1中的化合物，其特征在于，结构式如下：
其中，RP为氢，-C-C-O-Bn，-C-C-OH,-C-CO2R6，-C-CO2Ar；RL为羟基或羟基保护基；R4，R5相同或不同地为苯基，烷基或取代苯基；R6为烷基；*表示手性。
3.根据权利要求1中的化合物，其特征在于，结构式如下：
4.一种(3R,3aS,6aR)-六氢呋喃并[2,3-b]-3-醇中间体式C化合物的制备方法，其特征在于，由式B化合物经酶法还原反应构建手性制备，
所述酶为醛酮还原酶，氨基酸序列为SEQ ID NO：1的蛋白质或SEQ ID NO：1经过一个或几个氨基酸残基取代、缺失或添加后具有醛酮还原酶活性的蛋白质，或与SEQ ID NO：1所示的氨基酸序列具有80％以上同源性且具有醛酮还原酶活性的蛋白质。
其中，RZ,RP,RL的定义与权利要求1中的相同。
5.一种(3R,3aS,6aR)-六氢呋喃并[2,3-b]-3-醇中间体式C2化合物的制备方法，其特征在于，由式B2化合物经酶法还原反应构建手性制备，
其中，RP,RL的定义与权利要求2中的相同，
所述还原反应构建手性的方法为酶法，所述酶与权利要求4中的相同。
6.一种(3R,3aS,6aR)-六氢呋喃并[2,3-b]-3-醇中间体式C-i化合物的制备方法，其特征在于，由式C化合物经还原反应制备，
其中，RZ,RP,RL的定义与权利要求1中的相同。
7.一种(3R,3aS,6aR)-六氢呋喃并[2,3-b]-3-醇的制备方法，其特征在于，由式C-i化合物经关环反应制备，
其中，RZ,RP,RL的定义与权利要求1中的相同。
8.根据权利要求7所述的制备方法，其特征在于，由式C2化合物经还原反应用于制备式C-i1化合物，
其中，RP,RL的定义与权利要求1中的相同。
9.一种(3R,3aS,6aR)-六氢呋喃并[2,3-b]-3-醇的制备方法，其特征在于，由式C-i1化合物经关环反应制备，
其中，RP,RL的定义与权利要求1中的相同。
10.一种(3R,3aS,6aR)-六氢呋喃并[2,3-b]-3-醇中间体式B2化合物的制备方法，其特征在于，由二羰基化合物与胺类化合物反应制备，
其中，X1为卤素，烷氧基或离去基，RP，RL的定义与权利要求1中的相同。
11.一种(3R,3aS,6aR)-六氢呋喃并[2,3-b]-3-醇中间体式B2化合物的制备方法，其特征在于，由二羰基化合物与N-甲基苯胺反应后进一步地取代反应制备，
其中，X1或X2相同或不同地为卤素，烷氧基，离去基或并构为环，RP，RL的定义与权利要求1中的相同。
12.根据权利要求11所述的制备方法，其特征在于，由二羰基化合物与苯甲醇反应后，进一步与N-甲基苯胺反应后，进一步与卤酸酯化合物反应制备，
其中，X为卤素，R6为烷基。
13.一种(3R,3aS,6aR)-六氢呋喃并[2,3-b]-3-醇中间体式B2化合物的制备方法，其特征在于，由二羰基化合物与RLH化合物反应后，进一步与卤代化合物反应制备，
其中，X为卤素，RL为羟基保护基。
14.根据权利要求13所述的制备方法，其特征在于，由二羰基化合物与苯甲醇反应后，进一步与溴代化合物反应制备，
15.一种(3R,3aS,6aR)-六氢呋喃并[2,3-b]-3-醇中间体式B2化合物的制备方法，其特征在于，由式D化合物与卤代化合物反应后，进一步与N-甲基苯胺反应制备，
其中，X为卤素，R6与权利要求1中定义相同。
16.一种(3R,3aS,6aR)-六氢呋喃并[2,3-b]-3-醇中间体式B2化合物的制备方法，其特征在于，由式D0化合物在卤素的作用下与N-甲基苯胺反应后经进一步取代反应制备，
其中，RL与权利要求13中定义相同。
17.一种(3R,3aS,6aR)-六氢呋喃并[2,3-b]-3-醇的制备方法，其特征在于，由式D0化合物在卤素的作用下与N-甲基苯胺反应后经两步取代反应，环合反应，酶法还原反应和还原关环反应制备，
其中，RL与权利要求13中的定义相同，X为卤素，R6为烷基。
18.一种(3R,3aS,6aR)-六氢呋喃并[2,3-b]-3-醇的制备方法，其特征在于，由式D0化合物在卤素的作用下与N-甲基苯胺反应后经两步取代反应，酶法还原反应和还原关环反应制备，
其中，RL与权利要求13中的定义相同，X为卤素，R6为烷基。
19.根据权利要求13所述的制备方法，其特征在于，所述RL为叔丁氧基,-OCOCH3,-OBn，-OBz，-OCOR6，-OAr，-OCOAr,Ar为芳基，杂芳基，取代芳基或取代杂芳基，所述杂芳基为呋喃、吡啶、噻吩、吲哚或萘。
20.一种(3R,3aS,6aR)-六氢呋喃并[2,3-b]-3-醇的制备方法，其特征在于，由二羰基化合物与苯甲醇反应后，进一步与N-甲基苯胺反应后，进一步与卤酸酯化合物反应，后经酶法还原反应，还原关环反应制备，
其中，X为卤素，R6为烷基。
21.一种(3R,3aS,6aR)-六氢呋喃并[2,3-b]-3-醇的制备方法，其特征在于，由式D化合物与卤代化合物反应后，与N-甲基苯胺反应，与卤酸酯化合物反应，后经酶法还原反应，还原关环反应制备，
其中，X为卤素，R6为烷基。
22.一种(3R,3aS,6aR)-六氢呋喃并[2,3-b]-3-醇的制备方法，其特征在于，由二羰基化合物与苯甲醇反应后，进一步与卤代化合物反应，与N-甲基苯胺化合物反应，后经酶法还原反应，还原关环反应制备，
其中，X为卤素。
23.根据权利要求4所述的制备方法，其特征在于，所述醛酮还原酶基因的核苷酸序列为SEQ ID NO：2。
24.根据权利要求4所述的制备方法，其特征在于，所述醛酮还原酶为基因工程菌全细胞、破碎酶液、冻干粉或固定化酶或固定化细胞。
25.根据权利要求4所述的制备方法，其特征在于，所述反应体系中所述醛酮还原酶基因工程菌全细胞的投入量为10-100g/L，转化温度为25-37℃。
26.根据权利要求4所述的制备方法，其特征在于，所述反应在溶剂存在的条件下进行。
27.根据权利要求26所述的制备方法，其特征在于，所述溶剂为水或缓冲溶液与有机溶剂组成的混合溶剂。
28.根据权利要求27所述的制备方法，其特征在于，所述缓冲溶液选自磷酸盐缓冲溶液、碳酸盐缓冲溶液、Tri-HCl缓冲溶液、柠檬酸盐缓冲溶液或MOPS缓冲溶液中的一种或多种。
29.根据权利要求27所述的制备方法，其特征在于，所述有机溶剂选自DMSO、乙酸乙酯、乙酸丁酯、异丙醇、DMF、TBME、二氯甲烷、乙酸乙烯酯中的一种或几种。
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