WO2020181686A1 - 一种用于抗体药物偶联物的药物-连接子mc-mmaf的制备方法及其中间体 - Google Patents
一种用于抗体药物偶联物的药物-连接子mc-mmaf的制备方法及其中间体 Download PDFInfo
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- WO2020181686A1 WO2020181686A1 PCT/CN2019/092948 CN2019092948W WO2020181686A1 WO 2020181686 A1 WO2020181686 A1 WO 2020181686A1 CN 2019092948 W CN2019092948 W CN 2019092948W WO 2020181686 A1 WO2020181686 A1 WO 2020181686A1
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- CAJDDHDKAIAROQ-PSPOEUPBSA-N CC[C@H](C)[C@@H]([C@@H](CC(C[C@@H](CCC1)C[C@@H]1[C@@H]([C@@H](C)C(O)=O)OC)=O)OC)N(C)C([C@H](C(C)C)NC([C@H](C(C)C)NC)=O)=O Chemical compound CC[C@H](C)[C@@H]([C@@H](CC(C[C@@H](CCC1)C[C@@H]1[C@@H]([C@@H](C)C(O)=O)OC)=O)OC)N(C)C([C@H](C(C)C)NC([C@H](C(C)C)NC)=O)=O CAJDDHDKAIAROQ-PSPOEUPBSA-N 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N N[C@@H](Cc1ccccc1)C(O)=O Chemical compound N[C@@H](Cc1ccccc1)C(O)=O COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- IFTWVTAUEXLCHB-QMMMGPOBSA-N N[C@@H](Cc1ccccc1)C(O)O Chemical compound N[C@@H](Cc1ccccc1)C(O)O IFTWVTAUEXLCHB-QMMMGPOBSA-N 0.000 description 1
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
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- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- the invention relates to the field of organic synthesis, in particular to a preparation method and intermediates of a drug-linker MC-MMAF used for antibody drug conjugates.
- Antibody drug conjugate is a new type of anti-tumor drug. Its principle is to link cytotoxin to antibody. Through antibody recognition of specific antigen on the surface of cancer cell, it enters the cancer through endocytosis. Cells, thereby transporting cytotoxins to the target, achieve the purpose of targeted treatment of malignant tumors. Compared with traditional small-molecule anti-tumor drugs, ADC is more specific and effective because it can rely on the target recognition of antibodies and the high activity of toxins.
- ADC includes three different components, namely antibody, linker and cytotoxin.
- the antibody achieves targeting, and the linker ensures the stability of the ADC during blood transport. After reaching the target point, the toxin exerts a killing effect on cancer cells.
- toxins suitable for ADC are classified into microtubule inhibitors, DNA damaging agents, RNA polymerase inhibitors, and so on.
- the toxins used by ADCs sold in the market and in clinical trials are mainly microtubule inhibitors, mainly including dolastatin-based compounds, such as MMAE, MMAF and MMAD, and maytansine-based (Maytansine-based) designed compounds, such as DM1 and DM4.
- the main applications are non-cleavable types, such as valine-citrulline (Valine-Citriline) and cyclohexyl carboxylic acid (MCC).
- Valine-Citriline valine-citrulline
- MCC cyclohexyl carboxylic acid
- antibody-drug conjugates There are many ways to form antibody-drug conjugates. Either the amino or sulfhydryl group on the antibody and the drug linker can be chemically coupled, or the antibody can be modified. After a specific functional group is introduced on the antibody, it can be coupled with the drug linker for chemical reaction or enzymatic reaction coupling. United. The structure of the antibody drug conjugate MC-MMAF involved in the present invention is shown below.
- MC-MMAF The synthesis route of MC-MMAF currently reported in the literature is to use the toxin MMAF and MC-hex-Acid (1-maleimido n-hexanoic acid) to undergo a dehydration reaction to obtain MC-MMAF.
- the structure of MMAF is:
- the N-terminal valine of this route of MMAF has a methyl group on the N, which is sterically hindered. In this case, the reaction speed of connecting 1-maleimido n-hexanoic acid to MMAF will be slower. Even if different amide condensing agents are used, it will cause racemization of the chiral carbon linked to the phenylpropionamide group of MMAF.
- This route is used for the synthesis of MC-MMAF of less than 1g, and finally high-pressure reverse phase preparation is used to remove isomeric impurities, and the yield is less than 50%.
- This reaction route shows certain defects during scale-up production, such as: 1. Because the condensing agent will activate the carboxyl group on MMAF at the same time, this method will cause 30-50% racemization, forming difficult to remove isomer impurities, and affect Yield; 2. Due to the aforementioned steric hindrance, the reaction time is long, and the impurities are many, which cause difficulties in the post-treatment and purification of the reaction; 3. The final product needs to be prepared by high-pressure reverse phase to remove isomers, which increases operating costs; 4. Direct Using the toxin MMAF as a raw material, it is necessary to do a good job of protection in a large number of synthesis operations, and the selection of protective equipment will bring obstacles to production operations.
- the present invention provides a method for synthesizing MC-MMAF.
- the key to the reaction is to use the structural formula as Compound with L-phenylalanine
- the condensation reaction directly obtains MC-MMAF or its salt, and R is selected from one or more of hydrogen, succinimidyl, pentafluorophenyl, p-nitrophenyl, and phthalamide.
- the synthesis method includes the following steps: 1) The compound Dissolve in a suitable solvent, and The amide condensation reaction takes place to obtain MC-MMAF.
- the suitable solvent is selected from dichloromethane, dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, tetrahydrofuran, 1, One or more of 4-dioxane and 2-methyltetrahydrofuran; more preferably, the suitable solvent is selected from one or more of dichloromethane and N,N-dimethylformamide Kind.
- reagent N is added under the action of reagent M, which is selected from the group consisting of DCC, DCEP, EDC, DIC, HATU, HBTU, HBPIPU, HBPyU, HSPyU, HCTU, HOTU, HOTT, HSTU, HDMA, TATU, TBTU, TCTU, TCFH, TDBTU, TOTU, TOTT, TPTU, TFFH, BTFFH, TNTU, TSTU, COMU, T3P, BOP, PyBOP, PyBrOP, PyClOP, BroP, PyAOP, One or more of PyCIU, CDI, TPSI, TSTU, DEPBT, DMTMM, EEDQ, CIP, CIB, DMC, HOBt and EDCI; more preferably, the reagent M is selected from EDCI, EDC, DIC, HOAt and HOBt One or more of them; further preferably,
- the reagent N is selected from triethylamine, diisopropylethylamine (DIEA), pyridine, N,N-dimethyl-4-pyridine, preferably diisopropylethylamine (DIEA).
- the reaction temperature of the reaction is -20°C to 40°C, preferably -10°C to 25°C.
- step 1) if R is one or more of perimidyl, pentafluorophenyl, p-nitrophenyl, and phthalamide, under the action of reagent P, versus A reaction occurs and MC-MMAF is obtained.
- the reagent P is selected from triethylamine, diisopropylethylamine (DIEA), pyridine, N,N-dimethyl-4-pyridine, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, lithium carbonate And one or more of lithium bicarbonate, preferably sodium carbonate, or diisopropylethylamine (DIEA).
- the reaction temperature is 0°C to 100°C, preferably 15°C to 50°C.
- step 1) it further includes a step of separating MC-MMAF from the reaction liquid after the reaction is completed.
- the separation includes evaporating the solvent under reduced pressure, and then purifying or recrystallization by medium pressure chromatography to obtain MC-MMAF.
- the preparation method of the present invention improves the reactivity of the N terminal, thereby effectively controlling the occurrence of racemization; instead of directly using the toxin MMAF, it uses the less toxic L-phenylalanine, which reduces the operation difficulty in mass production ; No need for reverse phase preparation, simple operation. As mentioned above, this method reduces the difficulty of operation, makes the quality standard easier to control, and can be applied to the preparation of one hundred grams.
- this patent also provides an intermediate compound for the synthesis of MC-MMAF, the structural formula of which is Wherein, R is selected from one of hydrogen, succinimidyl, pentafluorophenyl, p-nitrophenyl, and phthalamide. It is preferably the following compound, as shown in Table 1:
- the present invention also provides a synthetic The method of synthesis is as follows,
- This route contains the important intermediate H for synthesis, and there is no report on the synthesis method of this compound before.
- the carboxyl group of compound F will also participate in the reaction during the condensation process, resulting in a complicated reaction and many by-products.
- the compound G, HATU and DIEA are first stirred and reacted at room temperature under nitrogen protection for 30 minutes, and then compound F is added and reacted under nitrogen protection at room temperature and stirred for 4 hours.
- the carboxyl group of compound F does not participate in the reaction, and compound H can be obtained in a high yield.
- the yield of compound H is greatly improved, and the route has the possibility of being applied to production.
- the present invention abandons the existing MMAF synthesis route and regards MC-MMAF as a whole for synthesis.
- the biggest problem is that MC linkers are fragments with relatively high reactivity. Connecting MC in advance will increase the difficulty of synthesis. Those skilled in the art would not think of this route. Through many studies, we have solved the problem of instability in the synthesis of the MC fragment compound introduced in advance, so that this overall synthetic route can be realized.
- Figure 1 is a high performance liquid chromatogram of DMT-3 synthesized in the present invention.
- Figure 2 is a liquid chromatogram of compound A synthesized in the present invention.
- Figure 3 is a mass spectrum of compound A synthesized in the present invention.
- Figure 4 is a liquid chromatogram of compound C synthesized in the present invention.
- Figure 5 is a mass spectrum of compound C synthesized in the present invention.
- Figure 6 is a liquid chromatogram of compound E synthesized in the present invention.
- Figure 7 is a mass spectrum of compound E synthesized in the present invention.
- Figure 8 is a liquid chromatogram of compound F synthesized in the present invention.
- Figure 9 is a mass spectrum of compound F synthesized in the present invention.
- Figure 10 is a liquid chromatogram of compound H synthesized in the present invention.
- Figure 11 is a mass spectrum of compound H synthesized in the present invention.
- Figure 12 is a liquid chromatogram of the target product MC-MMAF synthesized in Condensation Scheme 1 of the present invention.
- Figure 13 is a mass spectrum of the target product MC-MMAF synthesized in Condensation Scheme 1 of the present invention.
- Figure 14 is the NMR spectrum of the target product MC-MMAF synthesized in Condensation Scheme 1 of the present invention.
- Figure 15 is a high performance liquid chromatogram of the target product MC-MMAF synthesized by the condensation scheme 1 of the present invention.
- Figure 16 is a liquid chromatogram of compound J synthesized in the present invention.
- Figure 17 is a mass spectrum of compound J synthesized in the present invention.
- Figure 18 is a liquid chromatogram of the target product MC-MMAF synthesized in Condensation Scheme 1 of the present invention.
- Figure 19 is a mass spectrum of the target product MC-MMAF synthesized in Condensation Scheme 1 of the present invention.
- LCMS means liquid-mass spectrometry detection method
- HPLC means high-performance liquid chromatography detection.
- the raw materials and reagents for each step of the reaction involved in the present invention can be purchased from the market or prepared according to the method of the present invention.
- the present invention provides a method for synthesizing MC-MMAF, which includes the following steps:
- the suitable solvent is selected from dichloromethane, dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, tetrahydrofuran, 1,4- One or more of dioxane and 2-methyltetrahydrofuran, and The amide condensation reaction takes place to obtain MC-MMAF.
- step 1) if R is hydrogen, under the action of reagent M, add reagent N, which is selected from DCC, DCEP, EDC, DIC, HATU, HBTU, HBPIPU, HBPyU, HSPyU, HCTU, HOTU , HOTT, HSTU, HDMA, TATU, TBTU, TCTU, TCFH, TDBTU, TOTU, TOTT, TPTU, TFFH, BTFFH, TNTU, TSTU, COMU, T3P, BOP, PyBOP, PyBrOP, PyClOP, Brop, PyAOP, PyCIU, CDI , TPSI, TSTU, DEPBT, DMTMM, EEDQ, CIP, CIB, DMC, HOBt and EDCI; more preferably, the reagent M is selected from one of EDCI, EDC, DIC, HOAt and HOBt One or more; further preferably, the reagent M is a mixture of
- the reagent N is selected from triethylamine, diisopropylethylamine (DIEA), pyridine, N,N-dimethyl-4-pyridine, preferably diisopropylethylamine (DIEA).
- the reaction temperature is -20°C to 40°C, preferably -10°C to 25°C.
- step 1) if R is one or more of perimido group, pentafluorophenyl group, p-nitrophenyl group, and phthalamide group, under the action of reagent P, and A reaction occurs and MC-MMAF is obtained.
- the reagent P is selected from triethylamine, diisopropylethylamine (DIEA), pyridine, N,N-dimethyl-4-pyridine, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, lithium carbonate And one or more of lithium bicarbonate, preferably sodium carbonate, or diisopropylethylamine (DIEA).
- the reaction temperature is 0°C to 100°C, preferably 15°C to 50°C.
- step 1) it also includes the step of separating MC-MMAF from the reaction solution after the reaction is completed.
- the separation includes evaporating the solvent under reduced pressure, and then purifying or recrystallization by medium pressure chromatography to Get MC-MMAF.
- the invention also provides a synthetic Method, first put the compound HATU and DIEA are stirred at room temperature under nitrogen protection, and then the compound The reaction was carried out by stirring at room temperature under nitrogen protection.
- reaction route of this example is as follows:
- the reaction of the raw material Dil.HCl was completed and the reaction was completed.
- the reaction solution was washed with citric acid aqueous solution (2L*1), saturated sodium bicarbonate solution (2L*1), saturated brine (2L*1), the organic layer was dried with anhydrous sodium sulfate, filtered with suction, and desolventized 531g of crude product was obtained.
- reaction solution was washed with citric acid aqueous solution (20mL), saturated brine (20mL), dried over anhydrous sodium sulfate, spin-dried, and the crude product was purified by medium pressure reverse phase (80g industrial pre-packed C18 reverse phase column), purification gradient Water/acetonitrile (90/10-10/90, v/v), time 1 hour.
- the pure product was collected and lyophilized to obtain compound MC-MMAF (white solid, 1.11 g, yield 75%, HPLC purity 99% by UV 220nm).
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Abstract
本发明提供一种用于抗体偶联药物的药物-连接子MC-MMAF的制备方法及其中间体。本发明的制备方法提高了N端的反应活性,从而有效的控制了消旋反应的发生;不直接使用毒素MMAF,而采用毒性较低的化合物,降低了大量生产时的操作难度;无需反相制备,操作简便。
Description
本发明涉及有机合成领域,特别涉及一种用于抗体药物偶联物的药物-连接子MC-MMAF的制备方法及其中间体。
抗体药物偶联物(Antibody drug conjugate,简称ADC)是一类新型的抗肿瘤药物,其原理是将细胞毒素连接在抗体上,通过抗体对癌细胞表面特定抗原的识别,通过内吞作用进入癌细胞,从而将细胞毒素运输到靶点,达到靶向性治疗恶性肿瘤的目的。ADC与传统的小分子抗肿瘤药物相比,因能借助抗体的靶向识别性与毒素的高活性,故更具备特异性和有效性。
ADC包括三个不同的组成部分,即抗体、连接子和细胞毒素。抗体实现靶向性,连接子保证在ADC在血液转运过程中的稳定性,而到达作用靶点后,毒素发挥对癌细胞的杀伤作用。根据作用机制的不同,适用于ADC的毒素分为微管类抑制剂(Microtubule inhibitors),DNA损伤剂(DNA damaging agents),RNA聚合酶抑制剂(RNA polymerase inhibitors)等。目前,市场上销售和临床试验中的ADC所采用的毒素主要为微管类抑制剂,主要包括基于海兔毒素(Dolastatin-based)设计的化合物,比如MMAE、MMAF和MMAD,以及基于美登素(Maytansine-based)设计的化合物,比如DM1和DM4。连接子方面,主要应用的为不可裂解型,如缬氨酸-瓜氨酸(Valine-Citriline)和环己基甲酸(MCC),经过溶酶体水解后,药物仍然具有活性,并通过连接区与某个氨基酸残基结合在一起。
抗体药物偶联物的形成方式有多种。既可以通过抗体上的氨基或巯基和药物连接子进行化学反应偶联,也可以对抗体进行修饰,在抗体上引入特定功能基后,再和药物连接子进行化学反应偶联或酶催化反应偶联。本发明涉及的抗体药物偶联物MC-MMAF结构如下所示。
目前文献报道的MC-MMAF的合成路线为使用毒素MMAF与MC-hex-Acid(1-马来酰亚胺基正己酸)发生脱水反应,得到MC-MMAF。MMAF的结构为:
文献报道的合成方案为:
该路线MMAF的N终端缬氨酸的N上带有一个甲基,位阻较大,在这种情况下,将1-马来酰亚氨基正己酸接到MMAF上反应速度就会较慢,即使采用不同的酰胺缩合剂,也会造成MMAF的苯丙酰胺基连接的手性碳消旋。该路线用于小于1g的MC-MMAF合成,最后要应用高压反相制备除去异构杂质,产率低于50%。
该反应路线在放大生产时表现出一定的缺陷,比如:1.该方法因为缩合剂会同时活化MMAF上的羧基,而造成30-50%的消旋,形成难以除去的异构体杂质,影响产率;2.由于前述的位阻原因,反应时间长,杂质多,给反应的后处理和纯化造成困难;3.最终产物需要高压反相制备除去异构体,增加操作成本;4.直接应用毒素MMAF为原料,在大量合成操作上需做好防护,选择好防护设备,为生产操作带来障碍。
发明内容
一方面,针对现有技术存在的缺陷,本发明提供一种MC-MMAF的合成方法,该反应的关键是用结构式为
的化合 物与L-苯丙氨酸
发生缩合反应直接得到MC-MMAF或其盐,R选自氢、琥珀酰亚胺基、五氟苯基、对硝基苯基、邻苯二甲酰胺基中的一种或多种。
本发明的上述目的采用以下技术方案来实现。
优选地,在步骤1)中,所述合适的溶剂选自二氯甲烷、二甲基亚砜、N,N-二甲基甲酰胺、N,N-二甲基乙酰胺、四氢呋喃、1,4-二氧六环和2-甲基四氢呋喃中的一种或多种;更优选地,所述合适的溶剂选自二氯甲烷和N,N-二甲基甲酰胺中的一种或多种。
优选地,在步骤1)中,如R为氢,在试剂M的作用下加入试剂N,所述试剂M选自选自DCC、DCEP、EDC、DIC、HATU、HBTU、HBPIPU、HBPyU、HSPyU、HCTU、HOTU、HOTT、HSTU、HDMA、TATU、TBTU、TCTU、TCFH、TDBTU、TOTU、TOTT、TPTU、TFFH、BTFFH、TNTU、TSTU、COMU、T3P、BOP、PyBOP、PyBrOP、PyClOP、BrOP、PyAOP、PyCIU、CDI、TPSI、TSTU、DEPBT、DMTMM、EEDQ、CIP、CIB、DMC、HOBt和EDCI中的一种或多种;更优选地,所述试剂M选自EDCI、EDC、DIC、HOAt和HOBt中的一种或多种;进一步优选地,所述试剂M为EDCI、EDC或DIC与HOAt或HOBt的混合物;最优选地,所述试剂M为EDCI和HOBt的混合物。所述试剂 N选自三乙胺、二异丙基乙胺(DIEA)、吡啶、N,N-二甲基-4-吡啶,优选为二异丙基乙胺(DIEA)。反应的反应温度为-20℃~40℃,优选为-10℃~25℃。
优选地,在步骤1)中,如R为珀酰亚胺基、五氟苯基、对硝基苯基、邻苯二甲酰胺基中的一种或多种,在试剂P的作用下,与
发生反应,得到MC-MMAF。所述试剂P选自三乙胺、二异丙基乙胺(DIEA)、吡啶、N,N-二甲基-4-吡啶、碳酸钠、碳酸氢钠、碳酸钾、碳酸氢钾、碳酸锂和碳酸氢锂中的一种或多种,优选为碳酸钠,或二异丙基乙胺(DIEA)。反应温度为0℃~100℃,优选为15℃~50℃。
优选地,在步骤1)中,还包括在反应完成后,将MC-MMAF从反应液中分离的步骤。
优选地,所述分离包括通过减压蒸干溶剂,然后用中压色谱纯化或重结晶,以得到MC-MMAF。
本发明的制备方法提高了N端的反应活性,从而有效的控制了消旋反应的发生;不直接使用毒素MMAF,而采用毒性较低的L-苯丙氨酸,降低了大量生产时的操作难度;无需反相制备,操作简便。如上所述,该方法降低了操作难度,使得质量标准较易控制,制备百克级别都可以应用。
表1
本路线中包含合成重要中间体H,该化合物之前没有合成方法的报道。
合成化合物H,通常思路是用如下路线:
然而实验发现H1在脱叔丁基过程中会分解,收率很低。
而如果不保护羧基,缩合过程中化合物F的羧基也会参与反应,导致反应很复杂,副产物很多。
本发明采用的方法先将化合物G、HATU和DIEA氮气保护下室温搅拌反应30分钟,再加入化合物F在氮气保护下室温搅拌进行反应4小时。通过反复尝试改进加料顺序和投料比率,使化合物F的羧基不参与反应,能够以很高的收率得到化合物H。通过改变反应条件,大大提高了化合物H的产率,使该路线具有应用于生产的可能。
本发明放弃已有的MMAF合成路线,把MC-MMAF看做一个整体来合成。其中最大的问题是MC连接子是反应活性比较高的片段,提前把MC接上去会使合成难度增加,本领域技术人员不会想到这一路线。我们通过很多研究,解决了提前引入MC片段化合物在合成中不稳定的问题,使这条整体合成路线能够实现。
如本文所用,常用的有机物缩写的定义及其相应的CAS号如表2所示:
表2
图1是本发明合成的DMT-3的高效液相色谱图。
图2是本发明合成的化合物A的液相色谱图。
图3是本发明合成的化合物A的质谱图。
图4是本发明合成的化合物C的液相色谱图。
图5是本发明合成的化合物C的质谱图。
图6是本发明合成的化合物E的液相色谱图。
图7是本发明合成的化合物E的质谱图。
图8是本发明合成的化合物F的液相色谱图。
图9是本发明合成的化合物F的质谱图。
图10是本发明合成的化合物H的液相色谱图。
图11是本发明合成的化合物H的质谱图。
图12是本发明缩合方案1合成的目标产物MC-MMAF的液相色谱图。
图13是本发明缩合方案1合成的目标产物MC-MMAF的质谱图。
图14是本发明缩合方案1合成的目标产物MC-MMAF的核磁共振谱图。
图15是本发明缩合方案1合成的目标产物MC-MMAF的高效液相色谱图.
图16是本发明合成的化合物J的液相色谱图。
图17是本发明合成的化合物J的质谱图。
图18是本发明缩合方案1合成的目标产物MC-MMAF的液相色谱图。
图19是本发明缩合方案1合成的目标产物MC-MMAF的质谱图。
下面结合具体实施方式对本发明的技术方案作进一步非限制性的详细说明。需要指出 的是,下述实施例仅为说明本发明的技术构思及特点,其目的在于让熟悉此项技术的人士能够了解本发明的内容并据以实施,并不能以此限制本发明的保护范围。凡根据本发明精神实质所作的等效变化或修饰,都应涵盖在本发明的保护范围之内。
LCMS表示液质联用检测方法;HPLC表示高效液相色谱检测。
本发明所涉及的每一步反应的原料和试剂可从市场购得或依本发明所述方法制得。
本发明提供了一种合成MC-MMAF的方法,该合成方法包括如下步骤:
1)将化合物
溶解于合适的溶剂中,所述合适的溶剂选自二氯甲烷、二甲基亚砜、N,N-二甲基甲酰胺、N,N-二甲基乙酰胺、四氢呋喃、1,4-二氧六环和2-甲基四氢呋喃中的一种或多种,与
发生酰胺缩合反应,得到MC-MMAF。
在步骤1)中,如R为氢,则在试剂M的作用下,加入试剂N,所述试剂M选自DCC、DCEP、EDC、DIC、HATU、HBTU、HBPIPU、HBPyU、HSPyU、HCTU、HOTU、HOTT、HSTU、HDMA、TATU、TBTU、TCTU、TCFH、TDBTU、TOTU、TOTT、TPTU、TFFH、BTFFH、TNTU、TSTU、COMU、T3P、BOP、PyBOP、PyBrOP、PyClOP、BrOP、PyAOP、PyCIU、CDI、TPSI、TSTU、DEPBT、DMTMM、EEDQ、CIP、CIB、DMC、HOBt和EDCI中的一种或多种;更优选地,所述试剂M选自EDCI、EDC、DIC、HOAt和HOBt中的一种或多种;进一步优选地,所述试剂M为EDCI、EDC或DIC与HOAt或HOBt的混合物;最优选地,所述试剂M为EDCI和HOBt的混合物。所述试剂N选自三乙胺、二异丙基乙胺(DIEA)、吡啶、N,N-二甲基-4-吡啶,优选为二异丙基乙胺(DIEA)。反应温度为-20℃~40℃,优选为-10℃~25℃。
在步骤1)中,如R为珀酰亚胺基、五氟苯基、对硝基苯基、邻苯二甲酰胺基中的一种或多种,则在试剂P的作用下,与
发生反应,得到MC-MMAF。所述试剂P选自三乙胺、二异丙基乙胺(DIEA)、吡啶、N,N-二甲基-4-吡啶、碳酸钠、碳酸氢钠、碳酸钾、碳酸氢钾、碳酸锂和碳酸氢锂中的一种或多种,优选为碳酸钠,或二异 丙基乙胺(DIEA)。反应温度为0℃~100℃,优选为15℃~50℃。
在步骤1)中,还包括在反应完成后,将MC-MMAF从反应液中分离的步骤,优选地,所述分离包括通过减压蒸干溶剂,然后用中压色谱纯化或重结晶,以得到MC-MMAF。
实施例1
本实施例的反应路线如下:
缩合方案1
缩合方案2
在3L三口瓶中,加入1.5L二氯甲烷和Dil.HCl(202.3g,0.683mol 1.0eq),磁力搅拌,氮气保护,依次加入Z-Val-OH(163.23g,0.65mol,0.95eq)和HATU(311.6g,0.82mol,1.20eq),室温搅拌30min钟后降温至冰浴,控温10度滴加DIEA(452.5ml,4.0eq),滴加完毕,冰浴下搅拌30min后,移至室温,反应16h,HPLC检测,主峰为产品峰(保留时间29.98min),原料Dil.HCl反应完全,反应结束。反应液以柠檬酸水溶液洗涤(2L*1),饱和碳酸氢钠溶液洗涤(2L*1),饱和食盐水洗涤(2L*1),有机层以无水硫酸钠干燥后,抽滤,脱溶得粗品531g.该粗品溶于800ml甲醇,冰浴搅拌下滴加1.1ml(1mol/L)稀盐酸(约1小时),室温搅拌12h,停止搅拌,分层,分离上层水层,下层产品以油泵拉干,得325g DMT-1,收率91%
在2L单口瓶中,加入800ml甲醇和DMT-1(LN114-38,325g,0.66mol)和110g Pd(OH)2/C,H2置换三次,室温反应5h,TLC监测原料DMT-1反应完全。于砂芯漏斗加入硅藻土,抽滤,并以1L甲醇洗涤滤饼,收集滤液,滤液蒸干,油泵抽至产品不起泡,得DMT-2 230.2g,纯度94%;收率:97%。
在3L三口瓶中,将DMT-2(LN114-40-01,230.2g,实际0.60mol,1.0eq)溶于500ml DCM中,充分搅拌,加入Fmoc-Me-val(202.6g,0.57mol,0.95eq)和HATU(292.9g,0.77mol,1.20eq),再加入1L DCM,室温搅拌30min后降温至冰浴,控温10度滴加DIEA(212.7ml,2.0eq),滴加完毕,冰浴下搅拌30min后,移至室温,反应16.0h,HPLC检测,主峰为产品峰(保留时间为36.00min),原料DMT-2反应完全,反应结束。反应液以水洗涤(2.0L*1),柠檬酸水溶液洗涤(2L*1),饱和碳酸氢钠溶液洗涤(2L*1),饱和食盐水洗涤(1L*1),有机层以无水硫酸钠干燥后,抽滤,脱溶得粗品655g.该粗品溶于650ml甲醇,搅拌下滴加360ml(1mol/L)稀盐酸,室温搅拌12h,停止搅拌,分层,分离上层水层,如此两遍。下层产品以油泵拉干,得373g DMT-3,HPLC纯度为96.7%,收率90%。
将化合物DMT-3(173g,0.25mol)加入二氯甲烷(500mL)充分搅拌,在冰浴下加 入三氟乙酸(170mL),氮气保护下缓慢升至室温,搅拌反应16小时,LCMS显示反应液中化合物DMT-3少于1%视为反应结束。将反应液用水(500mL×3)洗涤,再用饱和食盐水(500mL)洗涤,有机相用无水Na
2SO
4干燥,旋转蒸发仪浓缩得淡黄色粗品165g;将粗品加入1.1L(石油醚:乙酸乙酯=10:1)混合溶剂中机械搅拌,约1小时后会析出白色固体,过滤,滤饼用DCM(500mL)洗两次,真空干燥得到化合物A(136g,收率85%,)。MS:638.27(M+H
+)
将化合物A(2.38g,3.71mmol),化合物B(456mg,3.96mmol)和化合物EDCI(760mg,3.96mmol)溶于二氯甲烷(20mL),氮气保护下室温搅拌反应2小时,LCMS显示反应液中化合物A少于5%视为反应结束。反应液用饱和食盐水(20mL)洗涤,无水硫酸钠干燥,旋干,粗品用中压反相纯化(选用80g的工业预填充C18反相柱),纯化梯度水/乙腈(90/10-10/90,v/v),时间1小时。收集纯的产品冻干,得到化合物C(白色固体,2.63g,收率97%)。MS:735.43(M+H
+)
将化合物C(2.63g,3.60mmol),化合物D(0.81g,3.60mmol)和DIEA(0.93g,7.20mmol)溶于二氯甲烷(20mL),氮气保护下室温搅拌反应18小时,LCMS显示反应液中化合物C少于3%视为反应结束。反应液依次用柠檬酸水溶液(20mL)和饱和食盐水(20mL)洗涤,无水硫酸钠干燥,旋干,粗品用中压反相纯化(选用120g的工业预填充C18反相柱),纯化梯度水/乙腈(90/10-10/90,v/v),时间1小时。收集纯的产品冻干,得到化合物E(白色固体,2.49g,收率86%)。MS:807.56(M+H
+)
将化合物E(2.49g,3.08mmol)和二乙胺(5mL)加入到二氯甲烷(20mL),氮气保护下室温搅拌反应4小时,LCMS显示反应液中化合物E少于3%视为反应结束。反应液旋干,粗品用中压反相纯化(选用120g的工业预填充C18反相柱),纯化梯度水/乙腈(90/10-10/90,v/v),时间1小时。收集纯的产品冻干,得到化合物F(白色固体,1.67g,收率93%)。MS:585.36(M+H
+)
将化合物G(405mg,1.92mmol)、HATU(730mg,1.92mmol)和DIEA(495mg,3.84mmol)加入到二氯甲烷(20mL),氮气保护下室温搅拌反应30分钟,再加入化合物F(1.12g,1.92mmol),氮气保护下室温搅拌反应4小时,LCMS显示反应液中化合物F少于3%视为反应结束。反应液依次用柠檬酸水溶液(20mL),饱和食盐水(20mL)洗涤, 无水硫酸钠干燥,旋干,粗品用中压反相纯化(选用80g的工业预填充C18反相柱),纯化梯度水/乙腈(90/10-10/90,v/v),时间1小时。收集纯的产品冻干,得到化合物H(白色固体,1.30g,收率87%)。MS:778.43(M+H
+)
MC-MMAF合成缩合方案1
将化合物H(1.25g,1.60mmol)、HATU(641mg,1.69mmol)和DIEA(437mg,3.38mmol)加入到二氯甲烷(20mL),氮气保护下室温搅拌反应30分钟,再加入化合物I(317mg,1.92mmol),氮气保护下室温搅拌反应4小时,LCMS显示反应液中化合物H少于3%视为反应结束。反应液依次用柠檬酸水溶液(20mL),饱和食盐水(20mL)洗涤,无水硫酸钠干燥,旋干,粗品用中压反相纯化(选用80g的工业预填充C18反相柱),纯化梯度水/乙腈(90/10-10/90,v/v),时间1小时。收集纯的产品冻干,得到化合物MC-MMAF(白色固体,1.11g,收率75%,HPLC纯度99%by UV 220nm)。MS:925.66(M+H
+)
MC-MMAF合成缩合方案2
将化合物H(1.12g,1.43mmol),化合物B(198mg,1.72mmol)和化合物EDCI(329mg,1.72mmol)溶于二氯甲烷(10mL),氮气保护下室温搅拌反应2小时,LCMS显示反应液中化合物H少于2%视为反应结束。反应液用饱和食盐水(10mL)洗涤,无水硫酸钠干燥,旋干,粗品用中压反相纯化(选用80g的工业预填充C18反相柱),纯化梯度水/乙腈(90/10-10/90,v/v),时间1小时。收集纯的产品冻干,得到化合物J(白色固体,1.17g,收率97%)。MS:(M+H
+)
将化合物J(1.17g,1.34mmol),化合物I(0.26g,1.60mmol)和DIEA(0.35g,2.70mmol)溶于二氯甲烷(10mL),氮气保护下室温搅拌反应4小时,LCMS显示反应液中化合物J少于3%视为反应结束。反应液依次用柠檬酸水溶液(20mL)和饱和食盐水(20mL)洗涤,无水硫酸钠干燥,旋干,粗品用中压反相纯化(选用80g的工业预填充C18反相柱),纯化梯度水/乙腈(90/10-10/90,v/v),时间1小时。收集纯的产品冻干,得到化合物MC-MMAF(白色固体,1.15g,收率93%)。MS:925.66(M+H
+)
Claims (10)
- R为氢,反应时在试剂M的作用下,加入试剂N,所述试剂M选自DCC、DCEP、EDC、DIC、HATU、HBTU、HBPIPU、HBPyU、HSPyU、HCTU、HOTU、HOTT、HSTU、HDMA、TATU、TBTU、TCTU、TCFH、TDBTU、TOTU、TOTT、TPTU、TFFH、BTFFH、TNTU、TSTU、COMU、T3P、BOP、PyBOP、PyBrOP、PyClOP、BrOP、PyAOP、PyCIU、CDI、TPSI、TSTU、DEPBT、DMTMM、EEDQ、CIP、CIB、DMC、HOAt、HOBt和EDCI中的一种或多种,所述试剂N选自三乙胺、二异丙基乙胺(DIEA)、吡啶、N,N-二甲基-4-吡啶中的一种或多种;所述溶剂选自二氯甲烷、二甲基亚砜、N,N-二甲基甲酰胺、N,N-二甲基乙酰胺、四氢呋喃、1,4-二氧六环和2-甲基四氢呋喃中的一种或多种;反应温度为-20℃~40℃。
- 根据权利要求2所述的方法,其特征在于,所述试剂M为EDCI和HOBt的混合物,所述试剂N为二异丙基乙胺(DIEA)。
- 根据权利要求2所述的方法,其特征在于,反应温度为-10℃~25℃。
- 根据权利要求5所述的方法,其特征在于,试剂P为碳酸钠或二异丙基乙胺(DIEA)。
- 根据权利要求5所述的方法,其特征在于,反应温度为15℃~50℃。
- 根据权利要求2-7任一项所述的方法,其特征在于,所述反应完成后,将MC-MMAF从反应液中分离。
- 根据权利要求8所述的方法,其特征在于,所述分离操作包括通过减压蒸干溶剂,然后用中压色谱纯化或重结晶。
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