CN108530518A - 10 analog of aplysiatoxin and its preparation method and application - Google Patents
10 analog of aplysiatoxin and its preparation method and application Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
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- C07K—PEPTIDES
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- C07K7/02—Linear peptides containing at least one abnormal peptide link
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- A—HUMAN NECESSITIES
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
技术领域technical field
本发明涉及生物医药领域，涉及一类五肽化合物，特别涉及一类具有抗癌活性的海兔毒素10类似物及其制备方法和应用。The invention relates to the field of biomedicine, relates to a class of pentapeptide compounds, in particular to a class of dolastatin 10 analogues with anticancer activity and its preparation method and application.
背景技术Background technique
海兔毒素10(dolastatin 10)是美国亚利桑那州立大学的Pettit教授课题组从印度洋的海洋软体动物Dolabella auricularia里分离提取出来的一种天然线性五肽。海兔毒素10对于多种肿瘤细胞如黑素瘤、小细胞肺癌、卵巢癌等都具有极强的抑制能力。但由于其太强的细胞毒性，在临床研究中出现了各种各样的副反应，故未能通过临床实验。人们通过对海兔毒素10的构效关系研究来进行结构修饰，期望能找到一个毒性更小的抗癌药物。Dolastatin 10 is a natural linear pentapeptide isolated from the marine mollusk Dolabella auricularia in the Indian Ocean by the research group of Professor Pettit of Arizona State University. Dolastatin 10 has a strong inhibitory ability to various tumor cells such as melanoma, small cell lung cancer, and ovarian cancer. However, due to its too strong cytotoxicity, various side effects appeared in clinical research, so it failed to pass clinical trials. People carry out structural modification through the study of the structure-activity relationship of dolastatin 10, hoping to find a less toxic anticancer drug.
被称为“Auristatins”的一系列海兔毒素10的类似物相继被设计合成出来，其中，Miyazaki发现可以将海兔毒素10的N-端的甲基移除一个而不影响其活性，该化合物被命名为Auristatin D(MMAD)；Doronina报道了将MMAD结构里C-端的噻唑环用羧基代替，得到的化合物命名为Auristatin F(MMAF)，该化合物被应用到抗体药物偶联物(ADCs)中，目前正处于临床试验阶段。A series of analogues of dolastatin 10 called "Auristatins" have been designed and synthesized successively. Among them, Miyazaki found that one methyl group at the N-terminal of dolastatin 10 can be removed without affecting its activity. Named Auristatin D (MMAD); Doronina reported that the C-terminal thiazole ring in the MMAD structure was replaced with a carboxyl group, and the resulting compound was named Auristatin F (MMAF), which was applied to antibody-drug conjugates (ADCs), It is currently in clinical trials.
海兔毒素10的N-端为伯胺时，则更有利于作为ADCs中药物分子与抗体的连接点。目前为止，一系列关于海兔毒素10的N-端结构修饰都只局限于移除其中一个甲基，同时移除两个甲基使得化合物的N-端为伯胺后，会发现其活性大大降低。When the N-terminal of dolastatin 10 is a primary amine, it is more favorable to serve as the connection point between the drug molecule and the antibody in ADCs. So far, a series of N-terminal structural modifications of dolastatin 10 are limited to removing one of the methyl groups. After removing two methyl groups at the same time to make the N-terminal of the compound a primary amine, it will be found that its activity is greatly improved. reduce.
发明内容Contents of the invention
本发明的目的是在MMAF的结构基础上，对化合物MMAD的N-端进行修饰，提供一系列新的海兔毒素10类似物，所述海兔毒素10类似物具有良好的抗肿瘤活性，尤其对HCT-116人结肠癌细胞具有较好的抑制活性，并且毒性小，稳定性高。本发明还提供了所述海兔毒素10类似物的制备方法，以苯丙氨酸盐酸盐为原料，分别经过四次重复的脱保护和酰胺缩合反应，得到五肽片段化合物，五肽片段化合物再经甲酯水解、保护基脱除，制备得到海兔毒素10类似物。该方法成本低，操作方便，效率高。The purpose of the present invention is to modify the N-terminus of the compound MMAD on the basis of the structure of MMAF to provide a series of new dolastatin 10 analogs, which have good antitumor activity, especially It has good inhibitory activity against HCT-116 human colon cancer cells, and has low toxicity and high stability. The present invention also provides a preparation method of the dolastatin 10 analogue, using phenylalanine hydrochloride as a raw material, and undergoing four repeated deprotection and amide condensation reactions respectively to obtain a pentapeptide fragment compound, a pentapeptide fragment The compound is hydrolyzed with methyl ester and the protective group is removed to prepare the dolastatin 10 analogue. The method has the advantages of low cost, convenient operation and high efficiency.
本发明提出的海兔毒素10类似物，由五个氨基酸组成，五个氨基酸的N-端氨基酸均为为L-缬氨酸，如L-缬氨酸、L-丝氨酸、L-丙氨酸、L-苯丙氨酸、L-亮氨酸、L-天冬氨酸，其结构如下式(1)所示：The dolastatin 10 analogue proposed by the present invention is composed of five amino acids, and the N-terminal amino acids of the five amino acids are all L-valine, such as L-valine, L-serine, and L-alanine , L-phenylalanine, L-leucine, L-aspartic acid, its structure is shown in the following formula (1):
其中，R为C1～C10的烷基，羟甲基、苯基、C1～C10取代的苯基，乙酰氨基、羧基、氨基、氨甲基。Wherein, R is C1-C10 alkyl, hydroxymethyl, phenyl, C1-C10 substituted phenyl, acetamido, carboxyl, amino, aminomethyl.
优选地，R为异丙基、羟甲基、甲基、苯甲基、异丁基、乙酰氨基、羧基、氨基、正丁基、苯基、氨甲基。Preferably, R is isopropyl, hydroxymethyl, methyl, benzyl, isobutyl, acetamido, carboxyl, amino, n-butyl, phenyl, aminomethyl.
进一步优选地，R为异丙基、羟甲基、甲基、苯甲基、异丁基、乙酰氨基。More preferably, R is isopropyl, hydroxymethyl, methyl, benzyl, isobutyl, acetamido.
进一步优选地，所述海兔毒素10类似物包括如下结构的化合物1a、1b、1c、1d、1e和1f：Further preferably, the dolastatin 10 analogs include compounds 1a, 1b, 1c, 1d, 1e and 1f of the following structures:
本发明提出的海兔毒素10类似物的制备方法，包括以下步骤：The preparation method of the dolastatin 10 analog proposed by the present invention comprises the following steps:
(1)四肽片段化合物(6)N-Boc-Val-Dil-Dap-Phe-OMe的合成(1) Synthesis of tetrapeptide fragment compound (6) N-Boc-Val-Dil-Dap-Phe-OMe
i)二肽片段化合物(3)N-Boc-Dap-Phe-OMe的合成i) Synthesis of dipeptide fragment compound (3) N-Boc-Dap-Phe-OMe
在有机溶剂中，化合物(2)N-Boc-Dap、苯丙氨酸甲酯盐酸盐、DIPEA和DEPC进行酰胺缩合反应，得到化合物(3)。In an organic solvent, compound (2) N-Boc-Dap, phenylalanine methyl ester hydrochloride, DIPEA and DEPC undergo amide condensation reaction to obtain compound (3).
步骤i)中，所述有机溶剂选自DMF、二氯甲烷、四氢呋喃；优选地，为DMF。In step i), the organic solvent is selected from DMF, dichloromethane, tetrahydrofuran; preferably, it is DMF.
步骤i)中，所述酰胺缩合反应的温度为-5℃-10℃；优选地，为0℃。In step i), the temperature of the amide condensation reaction is -5°C-10°C; preferably, it is 0°C.
步骤i)中，所述酰胺缩合反应的时间为3-5h；优选地，为4h。In step i), the time for the amide condensation reaction is 3-5 hours; preferably, 4 hours.
步骤i)中，化合物(2)、苯丙氨酸甲酯盐酸盐、DIPEA、DEPC的摩尔比为1：(1.0-1.5)：(4.5-6.0)：(1.25-1.75)；优选地，化合物(2)、苯丙氨酸甲酯盐酸盐、DIPEA、DEPC的摩尔比为1：1.2：5.0：1.5。In step i), the molar ratio of compound (2), phenylalanine methyl ester hydrochloride, DIPEA, and DEPC is 1: (1.0-1.5): (4.5-6.0): (1.25-1.75); preferably, The molar ratio of compound (2), phenylalanine methyl ester hydrochloride, DIPEA, and DEPC is 1:1.2:5.0:1.5.
步骤i)中，所述有机溶剂的用量为化合物(2)的8-13倍体积；优选地，为化合物(2)的10倍体积。In step i), the amount of the organic solvent used is 8-13 times the volume of the compound (2); preferably, 10 times the volume of the compound (2).
步骤i)中，DIPEA为碱，DEPC为酰胺缩合反应所需的缩合剂。In step i), DIPEA is the base, and DEPC is the condensing agent required for the amide condensation reaction.
ii)三肽片段化合物(5)N-Boc-Dil-Dap-Phe-OMe的合成：ii) Synthesis of tripeptide fragment compound (5) N-Boc-Dil-Dap-Phe-OMe:
在第一有机溶剂中，化合物(3)和三氟乙酸(TFA)进行Boc保护基脱除反应，反应完全后，再加入第二有机溶剂、化合物(4)、DIPEA和DEPC，进行酰胺缩合反应，得到化合物(5)。In the first organic solvent, compound (3) and trifluoroacetic acid (TFA) carry out Boc protecting group removal reaction, after the reaction is complete, then add the second organic solvent, compound (4), DIPEA and DEPC, and carry out amide condensation reaction , to obtain compound (5).
步骤ii)中，所述第一有机溶剂选自二氯甲烷、四氢呋喃、乙酸乙酯；优选地，为二氯甲烷。In step ii), the first organic solvent is selected from dichloromethane, tetrahydrofuran, ethyl acetate; preferably, it is dichloromethane.
步骤ii)中，所述第二有机溶剂选自DMF、二氯甲烷、四氢呋喃；优选地，为DMF。In step ii), the second organic solvent is selected from DMF, dichloromethane, tetrahydrofuran; preferably, it is DMF.
步骤ii)中，所述Boc保护基脱除反应的温度为0℃-25℃(室温)；优选地，为25℃(室温)。In step ii), the temperature of the Boc protecting group removal reaction is 0°C-25°C (room temperature); preferably, 25°C (room temperature).
步骤ii)中，所述Boc保护基脱除反应的时间为5-8h；优选地，为6h。In step ii), the time for removing the Boc protecting group is 5-8 hours; preferably, 6 hours.
步骤ii)中，所述酰胺缩合的温度为-5℃-10℃；优选地，为0℃。In step ii), the amide condensation temperature is -5°C-10°C; preferably, 0°C.
步骤ii)中，所述酰胺缩合的时间为3-5h；优选地，为4h。In step ii), the amide condensation time is 3-5h; preferably, 4h.
步骤ii)中，在Boc保护基脱除反应中，所述化合物(3)、TFA的摩尔比为1：(7.5-12)，优选地，化合物(3)、TFA的摩尔比为1：10。In step ii), in the Boc protecting group removal reaction, the molar ratio of compound (3) and TFA is 1: (7.5-12), preferably, the molar ratio of compound (3) and TFA is 1:10 .
步骤ii)中，所述第一有机溶剂的用量为化合物(3)的4-7倍体积；优选地，为化合物(3)的5倍体积。In step ii), the amount of the first organic solvent used is 4-7 times the volume of the compound (3); preferably, 5 times the volume of the compound (3).
步骤ii)中，在酰胺缩合反应中，当Boc保护基脱除反应中使用的化合物(3)摩尔数为1时，所述DIPEA、DEPC、化合物(4)的摩尔数分别为(4.5-6.0)、(1.25-1.75)、(1.0-1.25)；优选地，所述DIPEA、DEPC、化合物(4)的摩尔数分别为5.0、1.5、1.1。In step ii), in the amide condensation reaction, when the molar number of compound (3) used in the Boc protecting group removal reaction is 1, the molar numbers of DIPEA, DEPC, and compound (4) are respectively (4.5-6.0 ), (1.25-1.75), (1.0-1.25); preferably, the moles of DIPEA, DEPC, and compound (4) are 5.0, 1.5, and 1.1, respectively.
步骤ii)中，所述第二有机溶剂的用量为化合物(3)的8-13倍体积；优选地，为化合物(3)的10倍体积。In step ii), the amount of the second organic solvent is 8-13 times the volume of the compound (3); preferably, 10 times the volume of the compound (3).
步骤ii)中，TFA用于Boc保护基的脱除，DIPEA为碱，DEPC为酰胺缩合反应所需的缩合剂。In step ii), TFA is used to remove the Boc protecting group, DIPEA is the base, and DEPC is the condensing agent required for the amide condensation reaction.
iii)四肽片段化合物(6)N-Boc-Val-Dil-Dap-Phe-OMe的合成iii) Synthesis of tetrapeptide fragment compound (6) N-Boc-Val-Dil-Dap-Phe-OMe
在第一有机溶剂中，化合物(5)和三氟乙酸(TFA)进行Boc保护基的脱除反应，反应完全后，再加入第二有机溶剂、N-Boc-L-缬氨酸、DIPEA和溴代三(二甲基氨基)磷六氟磷酸盐(Brop)，进行酰胺缩合反应，得到化合物(6)。In the first organic solvent, compound (5) and trifluoroacetic acid (TFA) carry out the removal reaction of Boc protecting group, after the reaction is complete, then add the second organic solvent, N-Boc-L-valine, DIPEA and Bromotris(dimethylamino)phosphorus hexafluorophosphate (Brop) was subjected to amide condensation reaction to obtain compound (6).
步骤iii)中，所述第一有机溶剂选自二氯甲烷、四氢呋喃、乙酸乙酯；优选地，为二氯甲烷。In step iii), the first organic solvent is selected from dichloromethane, tetrahydrofuran, ethyl acetate; preferably, it is dichloromethane.
步骤iii)中，所述第二有机溶剂选自干燥二氯甲烷、乙腈、四氢呋喃；优选地，为干燥二氯甲烷。In step iii), the second organic solvent is selected from dry dichloromethane, acetonitrile, tetrahydrofuran; preferably, dry dichloromethane.
步骤iii)中，所述Boc保护基脱除反应的温度为0℃-25℃；优选地，为25℃(室温)。In step iii), the temperature of the Boc protecting group removal reaction is 0°C-25°C; preferably, 25°C (room temperature).
步骤iii)中，所述Boc保护基脱除反应的时间为5-8h；优选地，为6h。In step iii), the Boc protecting group removal reaction time is 5-8 hours; preferably, 6 hours.
步骤iii)中，所述酰胺缩合反应的温度为0℃-25℃；优选地，为25℃(室温)。In step iii), the temperature of the amide condensation reaction is 0°C-25°C; preferably, 25°C (room temperature).
步骤iii)中，所述酰胺缩合反应的时间为15-48h；优选地，为避光反应24小时。In step iii), the time for the amide condensation reaction is 15-48 hours; preferably, the reaction is protected from light for 24 hours.
步骤iii)中，优选地，在进行酰胺缩合反应前，在冰水浴中加入各物质，然后升温至合适温度再进行酰胺缩合反应。In step iii), preferably, before carrying out the amide condensation reaction, each substance is added in an ice-water bath, and then the temperature is raised to a suitable temperature before the amide condensation reaction is carried out.
步骤iii)中，在Boc保护基的脱除反应中，所述化合物(5)、TFA的摩尔比为1：(7.5-12)；优选地，所述化合物(5)、TFA的摩尔比为1：10。In step iii), in the removal reaction of the Boc protecting group, the molar ratio of the compound (5) to TFA is 1: (7.5-12); preferably, the molar ratio of the compound (5) to TFA is 1:10.
步骤iii)中，所述第一有机溶剂的用量为化合物(5)的4-7倍体积；优选地，为化合物(5)的5倍体积。In step iii), the amount of the first organic solvent used is 4-7 times the volume of the compound (5); preferably, 5 times the volume of the compound (5).
步骤iii)中，在酰胺缩合反应中，所述化合物(5)、DIPEA、Brop、N-Boc-L-缬氨酸的摩尔比为1：(2.5-4.5)：(1.25-1.75)：(1.2-3.0)；优选地，所述化合物(5)、DIPEA、Brop、N-Boc-L-缬氨酸的摩尔比为1：3.6：1.5：2.5。In step iii), in the amide condensation reaction, the molar ratio of the compound (5), DIPEA, Brop, and N-Boc-L-valine is 1: (2.5-4.5): (1.25-1.75): ( 1.2-3.0); Preferably, the molar ratio of the compound (5), DIPEA, Brop, and N-Boc-L-valine is 1:3.6:1.5:2.5.
步骤iii)中，所述第二有机溶剂的用量为化合物(5)的8-13倍体积；优选地，为化合物(5)的10倍体积。In step iii), the amount of the second organic solvent is 8-13 times the volume of the compound (5); preferably, 10 times the volume of the compound (5).
步骤iii)中，TFA用于Boc保护基的脱除，DIPEA为碱，Brop为酰胺缩合反应所需的缩合剂。In step iii), TFA is used to remove the Boc protecting group, DIPEA is the base, and Brop is the condensing agent required for the amide condensation reaction.
反应过程如下反应式(I)所示：The reaction process is shown in the following reaction formula (I):
(2)五肽片段化合物(7)的合成。(2) Synthesis of pentapeptide fragment compound (7).
在第一有机溶剂中，化合物(6)和三氟乙酸(TFA)进行Boc保护基的脱除反应，反应完全后，再加入第二有机溶剂、N-Boc-L-缬氨酸、DIPEA和DEPC，进行酰胺缩合反应，得到化合物(7)；In the first organic solvent, compound (6) and trifluoroacetic acid (TFA) carry out the removal reaction of Boc protecting group, after the reaction is complete, add the second organic solvent, N-Boc-L-valine, DIPEA and DEPC, carry out amide condensation reaction, obtain compound (7);
反应过程如下反应式(II)所示：The reaction process is shown in the following reaction formula (II):
步骤(2)中，所述第一有机溶剂选自二氯甲烷、四氢呋喃、乙酸乙酯；优选地，为二氯甲烷。In step (2), the first organic solvent is selected from dichloromethane, tetrahydrofuran, ethyl acetate; preferably, it is dichloromethane.
步骤(2)中，所述第二有机溶剂选自干燥DMF、乙腈、四氢呋喃；优选地，为干燥DMF。In step (2), the second organic solvent is selected from dry DMF, acetonitrile, tetrahydrofuran; preferably, dry DMF.
步骤(2)中，所述Boc保护基脱除反应的温度为0℃-25℃；优选地，为25℃(室温)。In step (2), the temperature of the Boc protecting group removal reaction is 0°C-25°C; preferably, 25°C (room temperature).
步骤(2)中，所述Boc保护基脱除反应的时间为5-8h；优选地，为6h。In step (2), the Boc protecting group removal reaction time is 5-8 hours; preferably, 6 hours.
步骤(2)中，所述酰胺缩合的温度为-5℃-10℃；优选地，为0℃。In step (2), the amide condensation temperature is -5°C-10°C; preferably, 0°C.
步骤(2)中，所述酰胺缩合的时间为3-5h；优选地，为4h。In step (2), the amide condensation time is 3-5 h; preferably, 4 h.
步骤(2)中，优选地，在冰水浴中加入各物质，然后再进行酰胺缩合反应。In step (2), preferably, each substance is added in an ice-water bath, and then the amide condensation reaction is carried out.
步骤(2)中，在Boc保护基的脱除反应中，所述化合物(6)、TFA的摩尔比为1：(7.5-12)，优选地，投料量的摩尔比为化合物(6)、TFA的摩尔比为＝1：10。In step (2), in the removal reaction of the Boc protecting group, the molar ratio of the compound (6) and TFA is 1: (7.5-12), preferably, the molar ratio of the feeding amount is compound (6), The molar ratio of TFA is =1:10.
步骤(2)中，所述第一有机溶剂的用量为化合物(6)的4-7倍体积；优选地，为化合物(6)的5倍体积。In step (2), the amount of the first organic solvent is 4-7 times the volume of the compound (6); preferably, 5 times the volume of the compound (6).
步骤(2)中，在酰胺缩合反应中，所述化合物(6)、DIPEA、DEPC、N-Boc-L-缬氨酸的摩尔比为1：(4.5-6.0)：(1.25-1.75)：(2.0-4.5)；优选地，所述化合物(6)、DIPEA、DEPC、N-Boc-L-缬氨酸的摩尔比为1：5.0：1.5：3.0。In step (2), in the amide condensation reaction, the molar ratio of the compound (6), DIPEA, DEPC, and N-Boc-L-valine is 1: (4.5-6.0): (1.25-1.75): (2.0-4.5); Preferably, the molar ratio of the compound (6), DIPEA, DEPC, and N-Boc-L-valine is 1:5.0:1.5:3.0.
步骤(2)中，所述第二有机溶剂的用量为化合物(6)的8-13倍体积；优选地，为化合物(6)的10倍体积。In step (2), the amount of the second organic solvent is 8-13 times the volume of the compound (6); preferably, 10 times the volume of the compound (6).
步骤(2)中，TFA用于Boc保护基的脱除，DIPEA为碱，DEPC为酰胺缩合反应所需的缩合剂。In step (2), TFA is used to remove the Boc protecting group, DIPEA is the base, and DEPC is the condensing agent required for the amide condensation reaction.
(3)五肽片段化合物(7)的酯基水解和N-Boc保护基脱除反应(3) Ester group hydrolysis and N-Boc protecting group removal reaction of pentapeptide fragment compound (7)
在第一有机溶剂中，化合物(7)和LiOH.H2O进行酯基水解反应，得到化合物(7)甲酯水解后的羧酸盐；在第二有机溶剂中，所述化合物(7)甲酯水解后的羧酸盐和三氟乙酸进行Boc保护基脱除反应，得到所述式(1)海兔毒素10类似物化合物。In the first organic solvent, compound (7) and LiOH.H 2 O carry out ester group hydrolysis reaction, obtain compound (7) carboxylate after methyl ester hydrolysis; In the second organic solvent, the compound (7) Carboxylate after methyl ester hydrolysis and trifluoroacetic acid are subjected to Boc protecting group removal reaction to obtain the dolastatin 10 analog compound of formula (1).
具体地，在第一有机溶剂中，化合物(7)和LiOH.H2O进行酯基水解反应(甲酯水解)，再将溶剂减压旋干，得到白色固体1(为化合物(7)甲酯水解后的羧酸盐)；将所述白色固体1用溶液溶解，调节pH，有白色固体2(为化合物(7)甲酯水解之后的游离羧酸)析出，过滤，干燥，得到化合物(7)甲酯水解后的羧酸盐；将化合物(7)甲酯水解后的羧酸盐溶于第二有机溶剂中，和三氟乙酸进行Boc保护基脱除反应，得到所述式(1)海兔毒素10类似物化合物。Specifically, in the first organic solvent, compound (7) and LiOH.H 2 O undergo ester group hydrolysis reaction (methyl ester hydrolysis), and then the solvent is spin-dried under reduced pressure to obtain a white solid 1 (compound (7) A The carboxylate after hydrolysis of the ester); the white solid 1 is dissolved in a solution, and the pH is adjusted, and the white solid 2 (free carboxylic acid after the hydrolysis of the compound (7) methyl ester) is precipitated, filtered, and dried to obtain the compound ( 7) carboxylate after methyl ester hydrolysis; the carboxylate after compound (7) methyl ester hydrolysis is dissolved in the second organic solvent, and trifluoroacetic acid carries out Boc protecting group removal reaction, obtains described formula (1 ) dolastatin 10 analog compound.
得到的白色固体1为化合物(7)的羧酸盐形式，白色固体2为游离的羧酸。这样操作的目的是，通过调节pH直接析出固体，再通过过滤就可实现产物与杂质的分离，避免了柱层析的使用。The obtained white solid 1 was the carboxylate salt form of compound (7), and the white solid 2 was the free carboxylic acid. The purpose of this operation is to directly precipitate solids by adjusting the pH, and then to separate the product from impurities by filtering, avoiding the use of column chromatography.
反应过程如下反应式(III)所示：The reaction process is shown in the following reaction formula (III):
步骤(3)中，所述第一有机溶剂选自甲醇、乙醇、异丙醇；优选地，为甲醇。In step (3), the first organic solvent is selected from methanol, ethanol, and isopropanol; preferably, it is methanol.
步骤(3)中，所述第二有机溶剂选自二氯甲烷、四氢呋喃、乙酸乙酯；优选地，为二氯甲烷。In step (3), the second organic solvent is selected from dichloromethane, tetrahydrofuran, and ethyl acetate; preferably, it is dichloromethane.
步骤(3)中，所述使白色固体1溶解的溶液选自2N的盐酸、甲醇；优选地，为2N的盐酸。In step (3), the solution for dissolving the white solid 1 is selected from 2N hydrochloric acid and methanol; preferably, it is 2N hydrochloric acid.
步骤(3)中，所述pH为3-7，优选地，为5-6。In step (3), the pH is 3-7, preferably 5-6.
步骤(3)中，所述酯基水解反应的温度为15℃-25℃；优选地，为25℃(室温)。In step (3), the temperature of the ester group hydrolysis reaction is 15°C-25°C; preferably, 25°C (room temperature).
步骤(3)中，所述酯基水解反应的时间为2-5h；优选地，为3h。In step (3), the time for the ester group hydrolysis reaction is 2-5 hours; preferably, it is 3 hours.
步骤(3)中，所述Boc保护基脱除反应的温度为0℃-25℃；优选地，为25℃(室温)。In step (3), the temperature of the Boc protecting group removal reaction is 0°C-25°C; preferably, 25°C (room temperature).
步骤(3)中，所述Boc保护基脱除反应的时间为3-7h；优选地，为5小时。In step (3), the time for the Boc protecting group removal reaction is 3-7 hours; preferably, 5 hours.
步骤(3)中，化合物(7)、LiOH·H2O、三氟乙酸的摩尔比为1：(1.5-4.0)：(8.0-12.0)；优选地，化合物(7)、LiOH·H2O、三氟乙酸的摩尔比为1：3.0：10.0。In step (3), the molar ratio of compound (7), LiOH·H 2 O and trifluoroacetic acid is 1:(1.5-4.0):(8.0-12.0); preferably, compound (7), LiOH·H 2 O. The molar ratio of trifluoroacetic acid is 1:3.0:10.0.
步骤(3)中，所述第一有机溶剂的用量为化合物(7)的4-7倍体积；优选地，为化合物(7)的5倍体积。In step (3), the amount of the first organic solvent is 4-7 times the volume of the compound (7); preferably, 5 times the volume of the compound (7).
步骤(3)中，所述第二有机溶剂的用量为化合物(7)的4-7倍体积；优选地，为化合物(7)的5倍体积。In step (3), the amount of the second organic solvent is 4-7 times the volume of the compound (7); preferably, 5 times the volume of the compound (7).
步骤(3)中，TFA用于Boc保护基的脱除，LiOH·H2O用于甲酯水解。In step (3), TFA was used to remove the Boc protecting group, and LiOH·H 2 O was used to hydrolyze the methyl ester.
在一个具体的实施例方式中，本发明海兔毒素10类似物的制备方法，其合成步骤如下：In a specific embodiment, the preparation method of the dolastatin 10 analog of the present invention, the synthesis steps are as follows:
(1)四肽片段化合物(6)N-Boc-Val-Dil-Dap-Phe-OMe的合成。(1) Synthesis of tetrapeptide fragment compound (6) N-Boc-Val-Dil-Dap-Phe-OMe.
i)二肽片段化合物(3)N-Boc-Dap-Phe-OMe的合成i) Synthesis of dipeptide fragment compound (3) N-Boc-Dap-Phe-OMe
将化合物(2)溶解于8-10倍体积的DMF中，再依次加入苯丙氨酸甲酯盐酸盐和DIPEA，冰水浴冷却。接着，称取DEPC溶于3倍体积的DMF，于0℃滴加入上述反应液中，加完使体系维持冰水浴反应3-5小时。加水淬灭反应，乙酸乙酯萃取，洗涤有机相，干燥，过滤，滤液减压浓缩，得到化合物(3)的粗产品，粗产物通过柱层析分离纯化(乙酸乙酯：石油醚＝1：1)，得到化合物(3)。Dissolve compound (2) in 8-10 times the volume of DMF, then add phenylalanine methyl ester hydrochloride and DIPEA in sequence, and cool in an ice-water bath. Next, weigh DEPC dissolved in 3 times the volume of DMF, add dropwise to the above reaction solution at 0°C, and keep the system in an ice-water bath for 3-5 hours after the addition is complete. Add water to quench the reaction, extract with ethyl acetate, wash the organic phase, dry, filter, and concentrate the filtrate under reduced pressure to obtain the crude product of compound (3), which is separated and purified by column chromatography (ethyl acetate:petroleum ether=1: 1), to obtain compound (3).
上述投料量的摩尔比为化合物(2)：苯丙氨酸甲酯盐酸盐：DIPEA：DEPC＝1：(1.0-1.5)：(4.5-6.0)：(1.25-1.75)，优选地，投料量的摩尔比为化合物(2)：苯丙氨酸甲酯盐酸盐：DIPEA：DEPC＝1：1.2：5.0：1.5。The molar ratio of the above-mentioned feeding amount is compound (2): phenylalanine methyl ester hydrochloride: DIPEA: DEPC=1: (1.0-1.5): (4.5-6.0): (1.25-1.75), preferably, feeding The molar ratio of the amount is compound (2): phenylalanine methyl ester hydrochloride: DIPEA: DEPC = 1: 1.2: 5.0: 1.5.
ii)三肽片段化合物(5)N-Boc-Dil-Dap-Phe-OMe的合成：ii) Synthesis of tripeptide fragment compound (5) N-Boc-Dil-Dap-Phe-OMe:
将化合物(3)溶解于5-6倍体积的二氯甲烷中，再加入三氟乙酸(TFA)，于室温搅拌，直至TLC显示反应完成。减压浓缩，将得到的残余物溶解于8-10倍体积的干燥DMF中，再依次加入化合物(4)和DIPEA，冰水浴冷却。接着，称取DEPC溶于3倍体积的DMF，于0℃滴加入上述反应液中，加完使体系维持冰水浴反应2-5小时。加水淬灭反应，乙酸乙酯萃取，洗涤有机相，干燥，过滤，滤液减压浓缩，得到化合物(5)的粗产品，粗产物通过柱层析分离纯化(乙酸乙酯：石油醚＝1：1)，得到化合物(5)。Compound (3) was dissolved in 5-6 volumes of dichloromethane, then trifluoroacetic acid (TFA) was added, and stirred at room temperature until TLC showed that the reaction was complete. Concentrate under reduced pressure, dissolve the obtained residue in 8-10 times the volume of dry DMF, then add compound (4) and DIPEA in sequence, and cool in an ice-water bath. Next, weigh DEPC and dissolve it in 3 times the volume of DMF, add it dropwise to the above reaction solution at 0°C, and keep the system in an ice-water bath for 2-5 hours after the addition is complete. Add water to quench the reaction, extract with ethyl acetate, wash the organic phase, dry, filter, and concentrate the filtrate under reduced pressure to obtain the crude product of compound (5), which is separated and purified by column chromatography (ethyl acetate:petroleum ether=1: 1), to obtain compound (5).
在Boc保护基脱除反应中，上述投料量的摩尔比为化合物(3)：TFA＝1：(7.5-12)，优选地，投料量的摩尔比为化合物(3)：TFA＝1：10。In the Boc protecting group removal reaction, the molar ratio of the above-mentioned feeding amount is compound (3): TFA=1: (7.5-12), preferably, the molar ratio of feeding amount is compound (3): TFA=1: 10 .
在酰胺缩合反应中，当Boc保护基脱除反应中使用的化合物(3)摩尔数为1时，所述DIPEA、DEPC、化合物(4)的摩尔数分别为(4.5-6.0)：(1.25-1.75)：(1.0-1.25)；优选地，所述DIPEA、DEPC、化合物(4)的摩尔数分别为5.0：1.5：1.1。In the amide condensation reaction, when the molar number of compound (3) used in the Boc protecting group removal reaction is 1, the molar numbers of DIPEA, DEPC, and compound (4) are respectively (4.5-6.0): (1.25- 1.75): (1.0-1.25); preferably, the molar numbers of DIPEA, DEPC, and compound (4) are 5.0:1.5:1.1, respectively.
iii)四肽片段化合物(6)N-Boc-Val-Dil-Dap-Phe-OMe的合成：iii) Synthesis of tetrapeptide fragment compound (6) N-Boc-Val-Dil-Dap-Phe-OMe:
将化合物(5)溶解于5-6倍体积的二氯甲烷中，再加入三氟乙酸(TFA)，于室温搅拌，直至TLC显示反应完成。减压浓缩，将得到的残余物溶解于8-10倍体积的干燥二氯甲烷中，再依次加入N-Boc-L-缬氨酸和DIPEA，冰水浴冷却。接着，称取溴代三(二甲基氨基)磷六氟磷酸盐(Brop)直接加入反应液，于0℃直接加入上述反应液中，加完使体系升至室温避光反应24小时。加水，二氯甲烷萃取，洗涤有机相，干燥，过滤，滤液减压浓缩，得到化合物(6)的粗产品，粗产物通过柱层析分离纯化(乙酸乙酯：石油醚＝2：1)，得到化合物(6)。Compound (5) was dissolved in 5-6 volumes of dichloromethane, then trifluoroacetic acid (TFA) was added, and stirred at room temperature until TLC showed that the reaction was complete. Concentrate under reduced pressure, dissolve the obtained residue in 8-10 times the volume of dry dichloromethane, then add N-Boc-L-valine and DIPEA in sequence, and cool in an ice-water bath. Next, bromotris(dimethylamino)phosphorus hexafluorophosphate (Brop) was weighed and directly added to the reaction solution, and directly added to the above reaction solution at 0° C., after the addition, the system was warmed to room temperature and reacted in the dark for 24 hours. Add water, extract with dichloromethane, wash the organic phase, dry, filter, and concentrate the filtrate under reduced pressure to obtain a crude product of compound (6), which is separated and purified by column chromatography (ethyl acetate:petroleum ether=2:1), Compound (6) was obtained.
上述投料量的摩尔比为化合物(5)：TFA＝1：(7.5-12)，优选地，投料量的摩尔比为化合物(5)：TFA＝1：10。The molar ratio of the above feeding amount is compound (5):TFA=1:(7.5-12), preferably, the molar ratio of feeding amount is compound (5):TFA=1:10.
上述投料量的摩尔比为化合物(5)：DIPEA：Brop：N-Boc-L-缬氨酸＝1：(2.5-4.5)：(1.25-1.75)：(1.2-3.0)，优选地，投料量的摩尔比为化合物(5)：DIPEA：Brop：N-Boc-L-缬氨酸＝1：3.6：1.5：2.5。The molar ratio of the above-mentioned feeding amount is compound (5): DIPEA: Brop: N-Boc-L-valine=1: (2.5-4.5): (1.25-1.75): (1.2-3.0), preferably, feeding The molar ratio of the amount is compound (5):DIPEA:Brop:N-Boc-L-valine=1:3.6:1.5:2.5.
反应过程如反应式(I’)所示：Reaction process is shown in reaction formula (I'):
(2)五肽片段化合物(7)的合成。(2) Synthesis of pentapeptide fragment compound (7).
将化合物(6)溶解于5-6倍体积的二氯甲烷中，再加入三氟乙酸(TFA)，于室温搅拌，直至TLC显示反应完成。减压浓缩，将得到的残余物溶解于8-10倍体积的干燥DMF中，再依次加入N-Boc-L-缬氨酸和DIPEA，冰水浴冷却。接着，称取DEPC溶于3倍体积的DMF，于0℃滴加入上述反应液中，加完使体系维持冰水浴反应3-6小时。加水淬灭反应，乙酸乙酯萃取，洗涤有机相，干燥，过滤，滤液减压浓缩，得到化合物(7)的粗产品，粗产物通过柱层析分离纯化(乙酸乙酯：石油醚＝1：1)，得到化合物(7)。Compound (6) was dissolved in 5-6 volumes of dichloromethane, then trifluoroacetic acid (TFA) was added, and stirred at room temperature until TLC showed that the reaction was complete. Concentrate under reduced pressure, dissolve the obtained residue in 8-10 times the volume of dry DMF, then add N-Boc-L-valine and DIPEA in sequence, and cool in an ice-water bath. Next, weigh DEPC and dissolve it in 3 times the volume of DMF, add it dropwise to the above reaction solution at 0°C, and keep the system in an ice-water bath for 3-6 hours after the addition is complete. Add water to quench the reaction, extract with ethyl acetate, wash the organic phase, dry, filter, and concentrate the filtrate under reduced pressure to obtain the crude product of compound (7), which is separated and purified by column chromatography (ethyl acetate:petroleum ether=1: 1), to obtain compound (7).
上述投料量的摩尔比为化合物(6)：TFA＝1：(7.5-12)，优选地，投料量的摩尔比为化合物(6)：TFA＝1：10。The molar ratio of the above feeding amount is compound (6):TFA=1:(7.5-12), preferably, the molar ratio of feeding amount is compound (6):TFA=1:10.
上述投料量的摩尔比为化合物(6)：DIPEA：DEPC：N-Boc-L-缬氨酸＝1：(4.5-6.0)：(1.25-1.75)：(2.0-4.5)，优选地，投料量的摩尔比为化合物(6)：DIPEA：DEPC：N-Boc-L-缬氨酸＝1：5.0：1.5：3.0。The molar ratio of the above-mentioned feeding amount is compound (6): DIPEA: DEPC: N-Boc-L-valine=1: (4.5-6.0): (1.25-1.75): (2.0-4.5), preferably, feeding The molar ratio of the amount is compound (6):DIPEA:DEPC:N-Boc-L-valine=1:5.0:1.5:3.0.
反应过程如反应式(II’)所示：Reaction process is shown in reaction formula (II'):
(3)五肽片段化合物(7)的酯基水解和N-Boc保护基脱除反应(3) Ester group hydrolysis and N-Boc protecting group removal reaction of pentapeptide fragment compound (7)
将化合物(7)溶于5-6倍体积甲醇中，再称取LiOH.H2O加入上述溶液中，室温搅拌3小时。然后减压旋除溶剂，得到白色固体，加水使固体溶解，再用2N的盐酸调节溶液PH为5-6，有白色固体析出，过滤，烘干固体。将得到的固体溶于5-6倍体积二氯甲烷中，再加入三氟乙酸，于室温搅拌5小时，减压旋除溶剂，得到的粗产品用制备HPLC(SB-C18柱,5μm,9.4×250mm,40％CH3CN/60％H2O)分离纯化，得到白色粉末状终产物1。Compound (7) was dissolved in 5-6 volumes of methanol, and then LiOH.H 2 O was weighed and added to the above solution, and stirred at room temperature for 3 hours. Then the solvent was spinned off under reduced pressure to obtain a white solid, which was dissolved by adding water, and the pH of the solution was adjusted to 5-6 with 2N hydrochloric acid, and a white solid was precipitated, filtered, and dried. The obtained solid was dissolved in 5-6 times the volume of dichloromethane, then trifluoroacetic acid was added, stirred at room temperature for 5 hours, the solvent was removed under reduced pressure, and the obtained crude product was prepared by preparative HPLC (SB-C18 column, 5 μm, 9.4 ×250mm, 40% CH 3 CN/60% H 2 O) separation and purification, the final product 1 was obtained as a white powder.
上述投料量的摩尔比为化合物(7)：LiOH·H2O：三氟乙酸＝1：(1.5-4.0)：(8.0-12.0)，优选地，投料量的摩尔比为化合物(7)：LiOH·H2O：三氟乙酸＝1：3.0：10.0。The molar ratio of the above-mentioned feeding amount is compound (7): LiOH·H 2 O: trifluoroacetic acid=1: (1.5-4.0): (8.0-12.0), preferably, the molar ratio of feeding amount is compound (7): LiOH·H 2 O:trifluoroacetic acid=1:3.0:10.0.
反应过程如反应式(III’)所示：Reaction process is shown in reaction formula (III'):
本发明的反应机理如式(a)所示，DEPC作为酰胺缩合剂，首先DEPC与底物羧酸形成混合酸酐，将羧酸活化得到活化中间体，然后亲核性的胺再去进攻该活化中间体，最后磷酸酯离去形成酰胺键，从而制备得到式(1)海兔毒素10类似物。The reaction mechanism of the present invention is shown in formula (a), DEPC is as amide condensing agent, first DEPC and substrate carboxylic acid form mixed acid anhydride, carboxylic acid is activated to obtain activated intermediate, then nucleophilic amine goes to attack this activation again intermediate, and finally the phosphate ester leaves to form an amide bond, thereby preparing the dolastatin 10 analogue of formula (1).
本发明还提出了式(1)所示海兔毒素10类似物在制备抗肿瘤药物中的应用。其中，所述肿瘤包括结肠癌、乳腺癌、小细胞肺癌；优选地，所述肿瘤为结肠癌；进一步优选地，结肠癌细胞为HCT-116。The present invention also proposes the application of the dolastatin 10 analog shown in formula (1) in the preparation of antitumor drugs. Wherein, the tumor includes colon cancer, breast cancer, and small cell lung cancer; preferably, the tumor is colon cancer; more preferably, the colon cancer cell is HCT-116.
本发明的有益效果在于，本发明在MMAF的结构基础上，采用不同的氨基酸取代原有的N,N-二甲基缬氨酸结构单元，合成一系列得到新的海兔毒素10类似物。本发明设计的这类新的海兔毒素10类似物，对海兔毒素10的N-端进行了修饰，由原来的仲胺或叔胺拓展到了伯胺。式(1)海兔毒素10类似物具有良好的抗肿瘤活性，尤其是对于HCT-116人结肠癌细胞具有较好的抑制能力，IC50在0.25～11.13μM之间，其中的四个化合物1a，1c，1d以及1e比对照化合物MMAF显示出更好的抑制能力；式(1)海兔毒素10类似物还具有毒性小，稳定性高的特点。本发明海兔毒素10类似物的制备方法，成本低，操作方便，效率高。本发明的海兔毒素10类似物的N-端结构为伯胺，更有利于作为抗体药物偶联物(ADCs)中药物分子与接头的连接位点，为更多其他类型的ADCs研究提供有利条件。The beneficial effect of the present invention is that, on the basis of the structure of MMAF, the present invention uses different amino acids to replace the original N,N-dimethylvaline structural unit, and synthesizes a series of new dolastatin 10 analogs. The novel dolastatin 10 analogue designed in the present invention modifies the N-terminal of dolastatin 10, extending from the original secondary or tertiary amines to primary amines. The dolastatin 10 analogs of formula (1) have good antitumor activity, especially for HCT-116 human colon cancer cells, with IC 50 between 0.25 and 11.13 μM, and four compounds 1a , 1c, 1d and 1e show better inhibitory ability than the control compound MMAF; the dolastatin 10 analogue of formula (1) also has the characteristics of low toxicity and high stability. The preparation method of the dolastatin 10 analogue of the present invention has low cost, convenient operation and high efficiency. The N-terminal structure of the dolastatin 10 analogue of the present invention is a primary amine, which is more conducive to the connection site between the drug molecule and the linker in antibody drug conjugates (ADCs), and provides advantages for more other types of ADCs research. condition.
具体实施方式Detailed ways
结合以下具体实施例，对本发明作进一步的详细说明，本发明的保护内容不局限于以下实施例。在不背离发明构思的精神和范围下，本领域技术人员能够想到的变化和优点都被包括在本发明中，并且以所附的权利要求书为保护范围。实施本发明的过程、条件、试剂、实验方法等，除以下专门提及的内容之外，均为本领域的普遍知识和公知常识，本发明没有特别限制内容。In conjunction with the following specific examples, the present invention will be further described in detail, and the protection content of the present invention is not limited to the following examples. Without departing from the spirit and scope of the inventive concept, changes and advantages conceivable by those skilled in the art are all included in the present invention, and the appended claims are the protection scope. The process, conditions, reagents, experimental methods, etc. for implementing the present invention are general knowledge and common knowledge in the art except for the content specifically mentioned below, and the present invention has no special limitation content.
实施例1海兔毒素10类似物的制备The preparation of embodiment 1 dolastatin 10 analog
(1)四肽片段化合物(6)N-Boc-Val-Dil-Dap-Phe-OMe的合成(1) Synthesis of tetrapeptide fragment compound (6) N-Boc-Val-Dil-Dap-Phe-OMe
i)二肽片段化合物(3)的合成i) Synthesis of dipeptide fragment compound (3)
将化合物(2)(2.87g,10mmol)溶解于DMF(30mL)中，再依次加入苯丙氨酸甲酯盐酸盐(2.58g,12mmol)和DIPEA(5.16g,40mmol).，冰水浴冷却。接着，称取DEPC(2.45g,15mmol)溶于DMF(5mL)，于0℃滴加入上述反应液中，加完使体系维持冰水浴反应5小时。加水淬灭反应，乙酸乙酯萃取，洗涤有机相，干燥，过滤，滤液减压浓缩，得到化合物(3)的粗产品，粗产物通过柱层析分离纯化(乙酸乙酯：石油醚＝1：1)，得到化合物(3)(4.03g,90％)。Compound (2) (2.87g, 10mmol) was dissolved in DMF (30mL), and phenylalanine methyl ester hydrochloride (2.58g, 12mmol) and DIPEA (5.16g, 40mmol) were added successively. Cooled in an ice-water bath . Next, DEPC (2.45 g, 15 mmol) was weighed and dissolved in DMF (5 mL), and added dropwise to the above reaction solution at 0° C., and the system was maintained in an ice-water bath for 5 hours after the addition was complete. Add water to quench the reaction, extract with ethyl acetate, wash the organic phase, dry, filter, and concentrate the filtrate under reduced pressure to obtain the crude product of compound (3), which is separated and purified by column chromatography (ethyl acetate:petroleum ether=1: 1) to obtain compound (3) (4.03g, 90%).
1H NMR(400MHz,CDCl3)δ7.36–7.03(m,5H),4.93–4.66(m,1H),3.77(t,J＝31.2Hz,4H),3.55(d,J＝5.0Hz,1H),3.37(s,3H),3.17(dd,J＝14.0,5.2Hz,2H),3.10–2.97(m,1H),2.34(dd,J＝31.4,6.3Hz,1H),1.76(s,2H),1.61(d,J＝25.7Hz,2H),1.49(s,9H),1.16(d,J＝7.0Hz,3H)。 1 H NMR (400MHz, CDCl 3 ) δ7.36–7.03(m,5H), 4.93–4.66(m,1H), 3.77(t,J=31.2Hz,4H), 3.55(d,J=5.0Hz, 1H), 3.37(s, 3H), 3.17(dd, J＝14.0, 5.2Hz, 2H), 3.10–2.97(m, 1H), 2.34(dd, J＝31.4, 6.3Hz, 1H), 1.76(s , 2H), 1.61 (d, J = 25.7Hz, 2H), 1.49 (s, 9H), 1.16 (d, J = 7.0Hz, 3H).
ii)三肽片段化合物(5)的合成ii) Synthesis of tripeptide fragment compound (5)
将化合物(3)(3.58g,8mmol)溶解于二氯甲烷(30mL)中，再加入三氟乙酸(9.12g,80mmol)，于室温搅拌，直至TLC显示反应完成。减压浓缩，将得到的残余物溶解于干燥DMF(35mL)中，再依次加入化合物(4)(2.67g,8.8mmol)和DIPEA(5.16g,40mmol)，冰水浴冷却。接着，称取DEPC(1.96g,12mmol)溶于(5mL)DMF，于0℃滴加入上述反应液中，加完使体系维持冰水浴反应3小时。加水淬灭反应，乙酸乙酯萃取，洗涤有机相，干燥，过滤，滤液减压浓缩，得到化合物(5)的粗产品，粗产物通过柱层析分离纯化(乙酸乙酯：石油醚＝1：1)，得到化合物(5)(4.31g,85％)。Compound (3) (3.58g, 8mmol) was dissolved in dichloromethane (30mL), and trifluoroacetic acid (9.12g, 80mmol) was added, and stirred at room temperature until TLC showed that the reaction was complete. Concentrate under reduced pressure, and dissolve the obtained residue in dry DMF (35 mL), then add compound (4) (2.67 g, 8.8 mmol) and DIPEA (5.16 g, 40 mmol) in sequence, and cool in an ice-water bath. Next, DEPC (1.96 g, 12 mmol) was weighed and dissolved in (5 mL) DMF, and added dropwise to the above reaction solution at 0° C., and the system was maintained in an ice-water bath for 3 hours after the addition was complete. Add water to quench the reaction, extract with ethyl acetate, wash the organic phase, dry, filter, and concentrate the filtrate under reduced pressure to obtain the crude product of compound (5), which is separated and purified by column chromatography (ethyl acetate:petroleum ether=1: 1) to obtain compound (5) (4.31 g, 85%).
1H NMR(400MHz,CDCl3)δ7.33–7.17(m,5H),4.96–4.71(m,1H),4.17–4.05(m,4H),3.89(s,1H),3.78–3.67(m,3H),3.47–3.40(m,1H),3.40–3.31(m,6H),[3.19(d,J＝5.3Hz)and3.16(d,J＝4.9Hz),1H],3.12–3.00(m,1H),2.78–2.63(m,2H),2.49–2.30(m,2H),2.23(s,1H),1.93(d,J＝7.5Hz,1H),1.87–1.60(m,4H),1.46(d,J＝6.6Hz,9H),1.34(td,J＝7.1,1.0Hz,4H),1.17(d,J＝7.0Hz,3H),1.01–0.85(m,6H)。 1 H NMR (400MHz, CDCl 3 ) δ7.33–7.17(m,5H),4.96–4.71(m,1H),4.17–4.05(m,4H),3.89(s,1H),3.78–3.67(m ,3H),3.47–3.40(m,1H),3.40–3.31(m,6H),[3.19(d,J＝5.3Hz)and3.16(d,J＝4.9Hz),1H],3.12–3.00 (m,1H),2.78–2.63(m,2H),2.49–2.30(m,2H),2.23(s,1H),1.93(d,J＝7.5Hz,1H),1.87–1.60(m,4H ), 1.46 (d, J=6.6Hz, 9H), 1.34 (td, J=7.1, 1.0Hz, 4H), 1.17 (d, J=7.0Hz, 3H), 1.01–0.85 (m, 6H).
iii)四肽片段化合物(6)的合成iii) Synthesis of tetrapeptide fragment compound (6)
将化合物(5)(4.11g,6.5mmol)溶解于二氯甲烷(30mL)中，再加入三氟乙酸(7.41g,65mmol)，于室温搅拌，直至TLC显示反应完成。减压浓缩，将得到的残余物溶解于干燥二氯甲烷(40mL)中，再依次加入N-Boc-L-缬氨酸(3.47g,16mmol)和DIPEA(3.02g,23.4mmol)，冰水浴冷却。接着，称取溴代三(二甲基氨基)磷六氟磷酸盐(Brop)(3.78g,9.75mmol)直接加入反应液，于0℃直接加入上述反应液中，加完使体系升至室温避光反应24小时。加水，二氯甲烷萃取，洗涤有机相，干燥，过滤，滤液减压浓缩，得到化合物(6)的粗产品，粗产物通过柱层析分离纯化(乙酸乙酯：石油醚＝2：1)，得到化合物(6)(3.38g,71％)。Compound (5) (4.11 g, 6.5 mmol) was dissolved in dichloromethane (30 mL), and trifluoroacetic acid (7.41 g, 65 mmol) was added, and stirred at room temperature until TLC showed that the reaction was complete. Concentrate under reduced pressure, dissolve the obtained residue in dry dichloromethane (40mL), then add N-Boc-L-valine (3.47g, 16mmol) and DIPEA (3.02g, 23.4mmol) in turn, and ice-water bath cool down. Next, weigh bromotris(dimethylamino)phosphorus hexafluorophosphate (Brop) (3.78g, 9.75mmol) and add it directly to the reaction solution, and directly add it to the above reaction solution at 0°C, and let the system rise to room temperature after adding Protect from light for 24 hours. Add water, extract with dichloromethane, wash the organic phase, dry, filter, and concentrate the filtrate under reduced pressure to obtain a crude product of compound (6), which is separated and purified by column chromatography (ethyl acetate:petroleum ether=2:1), Compound (6) (3.38 g, 71%) was obtained.
1H NMR(400MHz,CDCl3)δ7.25–7.09(m,5H),4.91–4.52(m,2H),4.33(dd,J＝9.2,6.8Hz,1H),4.25–3.93(m,2H),3.89–3.76(m,1H),[3.69(d,J＝13.2Hz)and3.63(d,J＝6.3Hz),3H],3.40–3.33(m,1H),3.30–3.24(m,5H),3.14–2.98(m,2H),2.95(d,J＝12.2Hz,2H),2.44–2.20(m,3H),2.00–1.80(m,3H),1.79–1.54(m,4H),[1.35(s)and1.34(s),9H],1.13–1.08(m,3H),1.01–0.77(m,15H)； 1 H NMR (400MHz, CDCl 3 ) δ7.25–7.09(m,5H), 4.91–4.52(m,2H), 4.33(dd,J=9.2,6.8Hz,1H), 4.25–3.93(m,2H ),3.89–3.76(m,1H),[3.69(d,J＝13.2Hz)and3.63(d,J＝6.3Hz),3H],3.40–3.33(m,1H),3.30–3.24(m ,5H),3.14–2.98(m,2H),2.95(d,J＝12.2Hz,2H),2.44–2.20(m,3H),2.00–1.80(m,3H),1.79–1.54(m,4H ),[1.35(s)and1.34(s),9H],1.13–1.08(m,3H),1.01–0.77(m,15H);
(2)五肽片段化合物(7)的合成(2) Synthesis of pentapeptide fragment compound (7)
(2-1)化合物(7)a的合成(2-1) Synthesis of Compound (7)a
将化合物(6)(146mg,0.2mmol)溶解于二氯甲烷(1.5mL)中，再加入三氟乙酸(TFA)(228mg,2mmol)，于室温搅拌，直至TLC显示反应完成。减压浓缩，将得到的残余物溶解于干燥DMF(2.5mL)中，再依次加入N-Boc-L-缬氨酸(43.4mg,0.6mmol)和DIPEA(129mg,1mmol)，冰水浴冷却。接着，称取DEPC(19mg,0.3mmol)溶于DMF(0.5mL)，于0℃滴加入上述反应液中，加完使体系维持冰水浴反应3小时。加水淬灭反应，乙酸乙酯萃取，洗涤有机相，干燥，过滤，滤液减压浓缩，得到化合物(7)a的粗产品，粗产物通过柱层析分离纯化(二氯甲烷：甲醇＝30：1)，得到化合物(7)a(135mg,81％)。Compound (6) (146 mg, 0.2 mmol) was dissolved in dichloromethane (1.5 mL), and trifluoroacetic acid (TFA) (228 mg, 2 mmol) was added, and stirred at room temperature until TLC showed that the reaction was complete. Concentrate under reduced pressure, dissolve the obtained residue in dry DMF (2.5 mL), add N-Boc-L-valine (43.4 mg, 0.6 mmol) and DIPEA (129 mg, 1 mmol) in sequence, and cool in an ice-water bath. Next, DEPC (19 mg, 0.3 mmol) was weighed and dissolved in DMF (0.5 mL), and added dropwise to the above reaction solution at 0° C., and the system was kept in an ice-water bath for 3 hours after the addition was complete. Add water to quench the reaction, extract with ethyl acetate, wash the organic phase, dry, filter, and concentrate the filtrate under reduced pressure to obtain the crude product of compound (7)a, which is separated and purified by column chromatography (dichloromethane:methanol=30: 1) to obtain compound (7)a (135 mg, 81%).
1H NMR(400MHz,CDCl3)δ7.33–7.15(m,5H),7.09(d,J＝6.8Hz,1H),7.01(d,J＝7.1Hz,1H),6.72–6.63(m,1H),5.18–5.01(m,1H),4.94–4.85(m,1H),4.80–4.68(m,2H),4.23–4.09(m,2H),4.02–3.91(m,1H),3.87(ddd,J＝13.8,7.4,2.2Hz,1H),3.81–3.75(m,1H),3.70(d,J＝5.7Hz,2H),3.47–3.38(m,1H),3.35(dd,J＝13.4,7.5Hz,6H),3.21–3.07(m,2H),3.02(d,J＝8.5Hz,2H),2.51–2.30(m,3H),2.14–1.66(m,8H),1.44(s,9H),1.38–1.32(m,2H),1.20–1.15(m,3H),1.04–0.78(m,20H)； 1 H NMR (400MHz, CDCl 3 ) δ7.33–7.15 (m, 5H), 7.09 (d, J=6.8Hz, 1H), 7.01 (d, J=7.1Hz, 1H), 6.72–6.63 (m, 1H),5.18–5.01(m,1H),4.94–4.85(m,1H),4.80–4.68(m,2H),4.23–4.09(m,2H),4.02–3.91(m,1H),3.87( ddd,J＝13.8,7.4,2.2Hz,1H),3.81–3.75(m,1H),3.70(d,J＝5.7Hz,2H),3.47–3.38(m,1H),3.35(dd,J＝ 13.4,7.5Hz,6H),3.21–3.07(m,2H),3.02(d,J＝8.5Hz,2H),2.51–2.30(m,3H),2.14–1.66(m,8H),1.44(s ,9H),1.38–1.32(m,2H),1.20–1.15(m,3H),1.04–0.78(m,20H);
(2-2)化合物(7)b的合成(2-2) Synthesis of compound (7)b
化合物(7)b，与化合物(7)a的合成操作步骤一样，将化合物(7)a中的N-Boc-L-缬氨酸用N-Boc-L-丝氨酸代替即可得到，收率87％。Compound (7)b, the same as the synthetic operation steps of compound (7)a, can be obtained by replacing N-Boc-L-valine in compound (7)a with N-Boc-L-serine, and the yield 87%.
1H NMR(400MHz,CDCl3)δ7.33–7.14(m,5H),4.90–4.60(m,3H),4.23–4.11(m,3H),3.90(dd,J＝21.0,7.4Hz,2H),3.79–3.69(m,3H),3.62–3.52(m,1H),3.48–3.26(m,8H),3.23–3.10(m,2H),3.04(dt,J＝30.4,13.3Hz,3H),2.60–2.25(m,5H),2.10–1.88(m,3H),1.87–1.62(m,4H),1.44(s,9H),1.16(d,J＝7.0Hz,3H),1.07–0.80(m,14H)。 1 H NMR (400MHz, CDCl 3 ) δ7.33–7.14(m,5H),4.90–4.60(m,3H),4.23–4.11(m,3H),3.90(dd,J＝21.0,7.4Hz,2H ),3.79–3.69(m,3H),3.62–3.52(m,1H),3.48–3.26(m,8H),3.23–3.10(m,2H),3.04(dt,J＝30.4,13.3Hz,3H ),2.60–2.25(m,5H),2.10–1.88(m,3H),1.87–1.62(m,4H),1.44(s,9H),1.16(d,J＝7.0Hz,3H),1.07– 0.80(m,14H).
(2-3)化合物(7)c的合成(2-3) Synthesis of compound (7)c
化合物(7)c，与化合物(7)a的合成操作步骤一样，将化合物(7)a中的N-Boc-L-缬氨酸用N-Boc-L-丙氨酸代替即可得到，收率46％。Compound (7)c can be obtained by replacing N-Boc-L-valine in compound (7)a with N-Boc-L-alanine in the same synthesis steps as compound (7)a, Yield 46%.
1H NMR(400MHz,CDCl3)δ7.25–7.09(m,5H),5.12–5.02(m,1H),4.85–4.79(m,1H),4.67(dt,J＝20.9,9.9Hz,2H),4.18–4.07(m,3H),3.81(d,J＝7.6Hz,1H),[3.70(d,J＝8.1Hz)and3.63(d,J＝5.9Hz),3H],3.40–3.24(m,8H),3.12–2.87(m,5H),2.43–2.18(m,4H),2.01–1.83(m,3H),1.80–1.72(m,1H),1.70–1.55(m,3H),1.36(s,10H),1.09(d,J＝7.0Hz,3H),0.98–0.72(m,16H)。 1 H NMR (400MHz, CDCl 3 ) δ7.25–7.09(m,5H),5.12–5.02(m,1H),4.85–4.79(m,1H),4.67(dt,J＝20.9,9.9Hz,2H ),4.18–4.07(m,3H),3.81(d,J=7.6Hz,1H),[3.70(d,J=8.1Hz)and3.63(d,J=5.9Hz),3H],3.40– 3.24(m,8H),3.12–2.87(m,5H),2.43–2.18(m,4H),2.01–1.83(m,3H),1.80–1.72(m,1H),1.70–1.55(m,3H ), 1.36 (s, 10H), 1.09 (d, J=7.0Hz, 3H), 0.98–0.72 (m, 16H).
(2-4)化合物(7)d的合成(2-4) Synthesis of compound (7)d
化合物(7)d，与化合物(7)a的合成操作步骤一样，将化合物(7)a中的N-Boc-L-缬氨酸用N-Boc-L-苯丙氨酸代替即可得到，收率64％。Compound (7)d, the same as the synthetic operation steps of compound (7)a, can be obtained by replacing N-Boc-L-valine in compound (7)a with N-Boc-L-phenylalanine , yield 64%.
1HNMR(400MHz,CDCl3)δ7.31–7.13(m,10H),5.04–4.83(m,2H),4.79–4.68(m,2H),4.38(s,1H),4.16(s,1H),3.88(dd,J＝7.6,1.7Hz,1H),3.77(d,J＝14.9,1H),3.70(d,J＝8.9,2H),3.50–3.22(m,8H),3.20–2.93(m,6H),2.48–2.25(m,3H),2.16(ddd,J＝20.2,15.0,7.4Hz,2H),1.99(ddd,J＝19.0,12.3,7.4Hz,3H),1.91–1.54(m,5H),1.39(s,9H),1.16(d,J＝7.0Hz,3H),1.03–0.93(m,6H),0.93–0.81(m,8H)。 1 HNMR (400MHz, CDCl 3 ) δ7.31–7.13(m,10H),5.04–4.83(m,2H),4.79–4.68(m,2H),4.38(s,1H),4.16(s,1H) ,3.88(dd,J＝7.6,1.7Hz,1H),3.77(d,J＝14.9,1H),3.70(d,J＝8.9,2H),3.50–3.22(m,8H),3.20–2.93( m,6H),2.48–2.25(m,3H),2.16(ddd,J＝20.2,15.0,7.4Hz,2H),1.99(ddd,J＝19.0,12.3,7.4Hz,3H),1.91–1.54( m, 5H), 1.39 (s, 9H), 1.16 (d, J=7.0Hz, 3H), 1.03–0.93 (m, 6H), 0.93–0.81 (m, 8H).
(2-5)化合物(7)e的合成(2-5) Synthesis of Compound (7)e
化合物(7)e，与化合物(7)a的合成操作步骤一样，将化合物(7)a中的N-Boc-L-缬氨酸用N-Boc-L-亮氨酸代替即可得到，收率65％。Compound (7)e can be obtained by replacing N-Boc-L-valine in compound (7)a with N-Boc-L-leucine in the same synthesis steps as compound (7)a, Yield 65%.
1H NMR(400MHz,CDCl3)δ7.26–7.10(m,5H),5.09–4.94(m,1H),4.90–4.57(m,3H),4.09(d,J＝13.8Hz,2H),3.91(d,J＝6.7Hz,1H),3.84–3.77(m,1H),3.71(d,J＝9.4Hz,1H),3.63(d,J＝6.2Hz,2H),3.40–3.20(m,8H),3.16–2.89(m,5H),2.46–2.14(m,4H),2.03–1.80(m,3H),1.80–1.56(m,5H),1.36(s,9H),1.09(d,J＝7.0Hz,3H),0.99–0.69(m,22H)。 1 H NMR (400MHz, CDCl 3 )δ7.26–7.10(m,5H),5.09–4.94(m,1H),4.90–4.57(m,3H),4.09(d,J=13.8Hz,2H), 3.91(d,J=6.7Hz,1H),3.84–3.77(m,1H),3.71(d,J=9.4Hz,1H),3.63(d,J=6.2Hz,2H),3.40–3.20(m ,8H),3.16–2.89(m,5H),2.46–2.14(m,4H),2.03–1.80(m,3H),1.80–1.56(m,5H),1.36(s,9H),1.09(d , J=7.0Hz, 3H), 0.99–0.69 (m, 22H).
(2-6)化合物(7)f的合成(2-6) Synthesis of Compound (7)f
化合物(7)f，与化合物(7)a的合成操作步骤一样，将化合物(7)a中的N-Boc-L-缬氨酸用N-Boc-L-天冬氨酸代替即可得到，收率65％。Compound (7) f is the same as the synthetic operation steps of compound (7) a, and N-Boc-L-valine in compound (7) a is replaced by N-Boc-L-aspartic acid to obtain , yield 65%.
1HNMR(400MHz,CDCl3)δ7.45(d,J＝8.1Hz,1H),7.33–7.16(m,5H),6.09(d,J＝7.6Hz,1H),5.90(s,1H),4.81–4.64(m,2H),4.47(s,1H),4.15(s,2H),3.87(dd,J＝7.5,1.6Hz,1H),3.77(d,J＝10.3Hz,1H),3.70(d,J＝4.8Hz,2H),3.49–3.25(m,8H),3.22–2.92(m,5H),2.64–2.30(m,5H),2.10–1.88(m,3H),1.88–1.60(m,4H),1.43(s,9H),1.16(d,J＝7.0Hz,3H),1.05–0.77(m,15H)。 1 HNMR (400MHz, CDCl 3 )δ7.45(d, J＝8.1Hz, 1H), 7.33–7.16(m, 5H), 6.09(d, J＝7.6Hz, 1H), 5.90(s, 1H), 4.81–4.64(m,2H),4.47(s,1H),4.15(s,2H),3.87(dd,J=7.5,1.6Hz,1H),3.77(d,J=10.3Hz,1H),3.70 (d,J=4.8Hz,2H),3.49–3.25(m,8H),3.22–2.92(m,5H),2.64–2.30(m,5H),2.10–1.88(m,3H),1.88–1.60 (m, 4H), 1.43 (s, 9H), 1.16 (d, J=7.0Hz, 3H), 1.05–0.77 (m, 15H).
(3)五肽片段化合物(7)的酯基水解和N-Boc保护基的脱除(3) Ester hydrolysis of pentapeptide fragment compound (7) and removal of N-Boc protecting group
(3-1)化合物(1)a的合成(3-1) Synthesis of Compound (1)a
将化合物(7)a(30mg,0.06mmol)溶于甲醇(2mL)中，再称取LiOH.H2O(5mg,0.12mmol)加入上述溶液中，室温搅拌3小时。然后减压旋除溶剂，得到白色固体，加水使固体溶解，再用2N的盐酸调节溶液PH为5-6，有白色固体析出，过滤，烘干固体。将得到的固体溶于二氯甲烷(2.5mL)中，再加入三氟乙酸(68mg,0.6mmol)，于室温搅拌5小时，减压旋除溶剂，得到的粗产品用制备HPLC(SB-C18柱,5μm,9.4×250mm,40％CH3CN/60％H2O)分离纯化，得到白色粉末状终产物1a(24mg,93％)。Compound (7)a (30 mg, 0.06 mmol) was dissolved in methanol (2 mL), and then LiOH.H 2 O (5 mg, 0.12 mmol) was weighed and added to the above solution, and stirred at room temperature for 3 hours. Then the solvent was spinned off under reduced pressure to obtain a white solid, which was dissolved by adding water, and the pH of the solution was adjusted to 5-6 with 2N hydrochloric acid, and a white solid was precipitated, filtered, and dried. The obtained solid was dissolved in dichloromethane (2.5mL), then trifluoroacetic acid (68mg, 0.6mmol) was added, stirred at room temperature for 5 hours, the solvent was removed under reduced pressure, and the obtained crude product was analyzed by preparative HPLC (SB-C18 Column, 5 μm, 9.4×250 mm, 40% CH 3 CN/60% H 2 O) for separation and purification to obtain the final product 1a (24 mg, 93%) in the form of white powder.
1H NMR(400MHz,CD3OD)δ7.19–7.09(m,5H),4.71–4.51(m,2H),4.10–3.92(m,1H),3.73–3.64(m,1H),3.60–3.52(m,1H),3.46–3.38(m,1H),3.34–3.15(m,10H),2.83(dd,J＝24.8,11.5Hz,1H),2.39–2.33(m,2H),2.23–1.94(m,4H),1.84–1.59(m,3H),1.58–1.37(m,2H),1.37–1.15(m,4H),1.09(dd,J＝11.1,6.8Hz,3H),1.02(d,J＝6.4Hz,1H),0.98–0.66(m,18H)。 1 H NMR (400MHz, CD 3 OD) δ7.19–7.09(m,5H),4.71–4.51(m,2H),4.10–3.92(m,1H),3.73–3.64(m,1H),3.60– 3.52(m,1H),3.46–3.38(m,1H),3.34–3.15(m,10H),2.83(dd,J＝24.8,11.5Hz,1H),2.39–2.33(m,2H),2.23– 1.94(m,4H),1.84–1.59(m,3H),1.58–1.37(m,2H),1.37–1.15(m,4H),1.09(dd,J＝11.1,6.8Hz,3H),1.02( d, J = 6.4 Hz, 1H), 0.98–0.66 (m, 18H).
(3-2)化合物(1)b的合成(3-2) Synthesis of Compound (1)b
化合物(1)b，与化合物(1)a的合成操作步骤一样，收率87％。Compound (1)b, the synthetic procedure is the same as that of compound (1)a, and the yield is 87%.
1H NMR(400MHz,CD3OD)δ7.34–7.09(m,5H),4.81–4.66(m,2H),4.17–3.86(m,3H),3.78–3.62(m,2H),3.60–3.39(m,2H),3.36–3.28(m,8H),3.21(s,1H),3.18–3.05(m,2H),2.95(dd,J＝25.3,14.9Hz,1H),2.57–2.09(m,4H),1.96–1.78(m,3H),1.73–1.38(m,3H),1.32(d,J＝17.0Hz,3H),1.20(d,J＝8.7Hz,3H),1.13–0.84(m,13H)。 1 H NMR (400MHz, CD 3 OD) δ7.34–7.09(m,5H),4.81–4.66(m,2H),4.17–3.86(m,3H),3.78–3.62(m,2H),3.60– 3.39(m,2H),3.36–3.28(m,8H),3.21(s,1H),3.18–3.05(m,2H),2.95(dd,J＝25.3,14.9Hz,1H),2.57–2.09( m,4H),1.96–1.78(m,3H),1.73–1.38(m,3H),1.32(d,J＝17.0Hz,3H),1.20(d,J＝8.7Hz,3H),1.13–0.84 (m,13H).
(3-3)化合物(1)c的合成(3-3) Synthesis of compound (1)c
化合物(1)c，与化合物(1)a的合成操作步骤一样，收率94％。Compound (1)c, the synthetic procedure is the same as that of compound (1)a, and the yield is 94%.
1H NMR(400MHz,CD3OD)δ7.33–7.13(m,5H),4.77(d,J＝7.2Hz,1H),4.66(dd,J＝17.4,8.2Hz,1H),4.18–3.96(m,2H),3.67(s,1H),3.59–3.40(m,2H),3.34(d,J＝14.7Hz,8H),3.23(s,2H),3.14(s,1H),3.02–2.83(m,1H),2.67–2.39(m,2H),2.38–2.00(m,4H),1.95–1.72(m,3H),1.71–1.50(m,3H),1.50–1.43(m,3H),1.31(s,3H),1.24–1.15(m,3H),1.14–0.84(m,14H)。 1 H NMR (400MHz, CD 3 OD) δ7.33–7.13 (m, 5H), 4.77 (d, J=7.2Hz, 1H), 4.66 (dd, J=17.4, 8.2Hz, 1H), 4.18–3.96 (m,2H),3.67(s,1H),3.59–3.40(m,2H),3.34(d,J＝14.7Hz,8H),3.23(s,2H),3.14(s,1H),3.02– 2.83(m,1H),2.67–2.39(m,2H),2.38–2.00(m,4H),1.95–1.72(m,3H),1.71–1.50(m,3H),1.50–1.43(m,3H ), 1.31(s,3H), 1.24–1.15(m,3H), 1.14–0.84(m,14H).
(3-4)化合物(1)d的合成(3-4) Synthesis of compound (1)d
化合物(1)d，与化合物(1)a的合成操作步骤一样，收率94％。Compound (1)d, the synthetic procedure is the same as that of compound (1)a, and the yield is 94%.
1H NMR(400MHz,CD3OD)δ7.45–6.96(m,10H),4.72–4.52(m,2H),4.13–3.91(m,2H),3.54(d,J＝18.5Hz,2H),3.40(d,J＝14.5Hz,1H),3.34–3.00(m,13H),2.88-2.73(m,2H),2.40(d,J＝14.4Hz,1H),2.28–1.89(m,3H),1.87–1.62(m,3H),1.58–1.38(m,2H),1.21(d,J＝16.7Hz,3H),1.15–0.69(m,16H)。 1 H NMR (400MHz, CD 3 OD) δ7.45–6.96(m,10H),4.72–4.52(m,2H),4.13–3.91(m,2H),3.54(d,J＝18.5Hz,2H) ,3.40(d,J＝14.5Hz,1H),3.34–3.00(m,13H),2.88-2.73(m,2H),2.40(d,J＝14.4Hz,1H),2.28–1.89(m,3H ), 1.87–1.62 (m, 3H), 1.58–1.38 (m, 2H), 1.21 (d, J=16.7Hz, 3H), 1.15–0.69 (m, 16H).
(3-5)化合物(1)e的合成(3-5) Synthesis of Compound (1)e
化合物(1)e，与化合物(1)a的合成操作步骤一样，收率93％。Compound (1)e, the synthetic procedure is the same as that of compound (1)a, and the yield is 93%.
1H NMR(400MHz,CD3OD)δ7.21–7.02(m,5H),4.67(d,J＝8.2Hz,1H),4.54(d,J＝8.9Hz,1H),4.10–3.92(m,1H),3.91–3.80(m,1H),3.76(d,J＝9.3Hz,1H),3.59–3.55(m,1H),3.48–3.38(m,1H),3.34–3.15(m,11H),3.11(s,2H),3.02(s,1H),2.89–2.76(m,1H),2.48–2.28(m,2H),2.24–1.90(m,3H),1.84–1.64(m,3H),1.63–1.47(m,4H),1.20(t,J＝7.0Hz,3H),1.09(dd,J＝13.9,6.7Hz,3H),1.03–0.72(m,20H)。 1 H NMR (400MHz, CD 3 OD) δ7.21–7.02(m, 5H), 4.67(d, J=8.2Hz, 1H), 4.54(d, J=8.9Hz, 1H), 4.10–3.92(m ,1H),3.91–3.80(m,1H),3.76(d,J＝9.3Hz,1H),3.59–3.55(m,1H),3.48–3.38(m,1H),3.34–3.15(m,11H ),3.11(s,2H),3.02(s,1H),2.89–2.76(m,1H),2.48–2.28(m,2H),2.24–1.90(m,3H),1.84–1.64(m,3H ), 1.63–1.47 (m, 4H), 1.20 (t, J=7.0Hz, 3H), 1.09 (dd, J=13.9, 6.7Hz, 3H), 1.03–0.72 (m, 20H).
(3-6)化合物(1)f的合成(3-6) Synthesis of Compound (1)f
化合物(1)f，与化合物(1)a的合成操作步骤一样，收率82％。Compound (1)f, the synthetic procedure is the same as that of compound (1)a, and the yield is 82%.
1H NMR(400MHz,CD3OD)δ7.21–6.99(m,5H),4.67(d,J＝6.5Hz,1H),4.53(d,J＝7.9Hz,1H),4.17(td,J＝9.4,3.8Hz,1H),4.01(d,J＝34.0Hz,1H),3.63–3.51(m,1H),3.46–3.37(m,1H),3.33–3.29(m,1H),3.23(d,J＝15.3Hz,10H),3.11(s,2H),3.01(s,1H),2.88–2.71(m,2H),2.63–2.52(m,1H),2.38(d,J＝6.1Hz,2H),2.23–2.07(m,2H),1.93(d,J＝5.3Hz,1H),1.83–1.62(m,3H),1.60–1.42(m,2H),1.36–1.30(m,1H),1.20(d,J＝7.2Hz,3H),1.08(dd,J＝17.1,6.6Hz,3H),1.01(d,J＝6.5Hz,2H),0.97–0.86(m,8H),0.79(dd,J＝13.7,6.8Hz,4H)。 1 H NMR (400MHz, CD 3 OD) δ7.21–6.99 (m, 5H), 4.67 (d, J = 6.5Hz, 1H), 4.53 (d, J = 7.9Hz, 1H), 4.17 (td, J ＝9.4,3.8Hz,1H),4.01(d,J＝34.0Hz,1H),3.63–3.51(m,1H),3.46–3.37(m,1H),3.33–3.29(m,1H),3.23( d,J=15.3Hz,10H),3.11(s,2H),3.01(s,1H),2.88–2.71(m,2H),2.63–2.52(m,1H),2.38(d,J=6.1Hz ,2H),2.23–2.07(m,2H),1.93(d,J＝5.3Hz,1H),1.83–1.62(m,3H),1.60–1.42(m,2H),1.36–1.30(m,1H ),1.20(d,J=7.2Hz,3H),1.08(dd,J=17.1,6.6Hz,3H),1.01(d,J=6.5Hz,2H),0.97–0.86(m,8H),0.79 (dd, J = 13.7, 6.8 Hz, 4H).
实施例2海兔毒素10类似物的制备The preparation of embodiment 2 dolastatin 10 analogs
(1)四肽片段化合物(6)N-Boc-Val-Dil-Dap-Phe-OMe的合成(1) Synthesis of tetrapeptide fragment compound (6) N-Boc-Val-Dil-Dap-Phe-OMe
i)二肽片段化合物(3)的合成i) Synthesis of dipeptide fragment compound (3)
将化合物(2)(5.74g,20mmol)溶解于DMF(60mL)中，再依次加入苯丙氨酸甲酯盐酸盐(5.17g,24mmol)和DIPEA(15.48g,120mmol)，冰水浴冷却。接着，称取DEPC(4.90g,30mmol)溶于DMF(15mL)，于0℃滴加入上述反应液中，加完使体系维持冰水浴反应5小时。加水淬灭反应，乙酸乙酯萃取，洗涤有机相，干燥，过滤，滤液减压浓缩，得到化合物(3)的粗产品，粗产物通过柱层析分离纯化(乙酸乙酯：石油醚＝1：1)，得到化合物(3)(8.24g,92％)。Compound (2) (5.74g, 20mmol) was dissolved in DMF (60mL), and phenylalanine methyl ester hydrochloride (5.17g, 24mmol) and DIPEA (15.48g, 120mmol) were added successively, and cooled in an ice-water bath. Next, DEPC (4.90 g, 30 mmol) was weighed and dissolved in DMF (15 mL), and added dropwise to the above reaction solution at 0° C., and the system was maintained in an ice-water bath for 5 hours after the addition was complete. Add water to quench the reaction, extract with ethyl acetate, wash the organic phase, dry, filter, and concentrate the filtrate under reduced pressure to obtain the crude product of compound (3), which is separated and purified by column chromatography (ethyl acetate:petroleum ether=1: 1) to obtain compound (3) (8.24g, 92%).
ii)三肽片段化合物(5)的合成ii) Synthesis of tripeptide fragment compound (5)
将化合物(3)(7.16g,16mmol)溶解于二氯甲烷(60mL)中，再加入三氟乙酸(18.24g,160mmol)，于室温搅拌，直至TLC显示反应完成。减压浓缩，将得到的残余物溶解于干燥DMF(70mL)中，再依次加入化合物(4)(6.31g,20.8mmol)和DIPEA(10.32g,80mmol)，冰水浴冷却。接着，称取DEPC(3.91g,24mmol)溶于(12mL)DMF，于0℃滴加入上述反应液中，加完使体系维持冰水浴反应3小时。加水淬灭反应，乙酸乙酯萃取，洗涤有机相，干燥，过滤，滤液减压浓缩，得到化合物(5)的粗产品，粗产物通过柱层析分离纯化(乙酸乙酯：石油醚＝1：1)，得到化合物(5)(8.11g,80％)。Compound (3) (7.16g, 16mmol) was dissolved in dichloromethane (60mL), and trifluoroacetic acid (18.24g, 160mmol) was added, and stirred at room temperature until TLC showed that the reaction was complete. Concentrate under reduced pressure, dissolve the obtained residue in dry DMF (70 mL), then add compound (4) (6.31 g, 20.8 mmol) and DIPEA (10.32 g, 80 mmol) in sequence, and cool in an ice-water bath. Next, DEPC (3.91 g, 24 mmol) was weighed and dissolved in (12 mL) DMF, and added dropwise to the above reaction solution at 0° C., and the system was kept in an ice-water bath for 3 hours after the addition was complete. Add water to quench the reaction, extract with ethyl acetate, wash the organic phase, dry, filter, and concentrate the filtrate under reduced pressure to obtain the crude product of compound (5), which is separated and purified by column chromatography (ethyl acetate:petroleum ether=1: 1) to obtain compound (5) (8.11 g, 80%).
iii)四肽片段化合物(6)的合成iii) Synthesis of tetrapeptide fragment compound (6)
将化合物(5)(6.32g,10mmol)溶解于二氯甲烷(50mL)中，再加入三氟乙酸(12.54g,110mmol)，于室温搅拌，直至TLC显示反应完成。减压浓缩，将得到的残余物溶解于干燥二氯甲烷(60mL)中，再依次加入N-Boc-L-缬氨酸(4.34g,20mmol)和DIPEA(6.45g,50mmol)，冰水浴冷却。接着，称取溴代三(二甲基氨基)磷六氟磷酸盐(Brop)(4.86g,12.5mmol)直接加入反应液，于0℃直接加入上述反应液中，加完使体系升至室温避光反应24小时。加水，二氯甲烷萃取，洗涤有机相，干燥，过滤，滤液减压浓缩，得到化合物(6)的粗产品，粗产物通过柱层析分离纯化(乙酸乙酯：石油醚＝2：1)，得到化合物(6)(4.1g,56％)。Compound (5) (6.32g, 10mmol) was dissolved in dichloromethane (50mL), and trifluoroacetic acid (12.54g, 110mmol) was added, and stirred at room temperature until TLC showed that the reaction was complete. Concentrate under reduced pressure, dissolve the obtained residue in dry dichloromethane (60mL), then add N-Boc-L-valine (4.34g, 20mmol) and DIPEA (6.45g, 50mmol) in turn, and cool in an ice-water bath . Next, weigh bromotris(dimethylamino)phosphorus hexafluorophosphate (Brop) (4.86g, 12.5mmol) and add it directly to the reaction solution, and directly add it to the above reaction solution at 0°C, and let the system rise to room temperature after adding Protect from light for 24 hours. Add water, extract with dichloromethane, wash the organic phase, dry, filter, and concentrate the filtrate under reduced pressure to obtain a crude product of compound (6), which is separated and purified by column chromatography (ethyl acetate:petroleum ether=2:1), Compound (6) (4.1 g, 56%) was obtained.
(2)五肽片段化合物(7)的合成(2) Synthesis of pentapeptide fragment compound (7)
(2-1)化合物(7)a的合成(2-1) Synthesis of Compound (7)a
将化合物(6)(146mg,0.2mmol)溶解于二氯甲烷(1.5mL)中，再加入三氟乙酸(TFA)(228mg,2mmol)，于室温搅拌，直至TLC显示反应完成。减压浓缩，将得到的残余物溶解于干燥DMF(2.5mL)中，再依次加入N-Boc-L-缬氨酸(36.2mg,0.5mmol)和DIPEA(129mg,1mmol)，冰水浴冷却。接着，称取DEPC(16.5mg,0.26mmol)溶于DMF(0.5mL)，于0℃滴加入上述反应液中，加完使体系维持冰水浴反应3小时。加水淬灭反应，乙酸乙酯萃取，洗涤有机相，干燥，过滤，滤液减压浓缩，得到化合物(7)a的粗产品，粗产物通过柱层析分离纯化(二氯甲烷：甲醇＝30：1)，得到化合物(7)a(128mg,77％)。Compound (6) (146 mg, 0.2 mmol) was dissolved in dichloromethane (1.5 mL), and trifluoroacetic acid (TFA) (228 mg, 2 mmol) was added, and stirred at room temperature until TLC showed that the reaction was complete. Concentrate under reduced pressure, dissolve the obtained residue in dry DMF (2.5 mL), add N-Boc-L-valine (36.2 mg, 0.5 mmol) and DIPEA (129 mg, 1 mmol) in sequence, and cool in an ice-water bath. Next, DEPC (16.5 mg, 0.26 mmol) was weighed and dissolved in DMF (0.5 mL), and added dropwise to the above reaction solution at 0° C., and the system was kept in an ice-water bath for 3 hours after the addition was complete. Add water to quench the reaction, extract with ethyl acetate, wash the organic phase, dry, filter, and concentrate the filtrate under reduced pressure to obtain the crude product of compound (7)a, which is separated and purified by column chromatography (dichloromethane:methanol=30: 1) to obtain compound (7)a (128 mg, 77%).
(2-2)化合物(7)b的合成(2-2) Synthesis of compound (7)b
化合物(7)b，与化合物(7)a的合成操作步骤一样，将化合物(7)a中的N-Boc-L-缬氨酸用N-Boc-L-丝氨酸代替即可得到，收率79％。Compound (7)b, the same as the synthetic operation steps of compound (7)a, can be obtained by replacing N-Boc-L-valine in compound (7)a with N-Boc-L-serine, and the yield 79%.
(2-3)化合物(7)c的合成(2-3) Synthesis of compound (7)c
化合物(7)c，与化合物(7)a的合成操作步骤一样，将化合物(7)a中的N-Boc-L-缬氨酸用N-Boc-L-丙氨酸代替即可得到，收率56％。Compound (7)c can be obtained by replacing N-Boc-L-valine in compound (7)a with N-Boc-L-alanine in the same synthesis steps as compound (7)a, Yield 56%.
(2-4)化合物(7)d的合成(2-4) Synthesis of compound (7)d
化合物(7)d，与化合物(7)a的合成操作步骤一样，将化合物(7)a中的N-Boc-L-缬氨酸用N-Boc-L-苯丙氨酸代替即可得到，收率58％。Compound (7)d, the same as the synthetic operation steps of compound (7)a, can be obtained by replacing N-Boc-L-valine in compound (7)a with N-Boc-L-phenylalanine , yield 58%.
(2-5)化合物(7)e的合成(2-5) Synthesis of Compound (7)e
化合物(7)e，与化合物(7)a的合成操作步骤一样，将化合物(7)a中的N-Boc-L-缬氨酸用N-Boc-L-亮氨酸代替即可得到，收率68％。Compound (7)e can be obtained by replacing N-Boc-L-valine in compound (7)a with N-Boc-L-leucine in the same synthesis steps as compound (7)a, Yield 68%.
(2-6)化合物(7)f的合成(2-6) Synthesis of Compound (7)f
化合物(7)f，与化合物(7)a的合成操作步骤一样，将化合物(7)a中的N-Boc-L-缬氨酸用N-Boc-L-天冬氨酸代替即可得到，收率51％。Compound (7) f is the same as the synthetic operation steps of compound (7) a, and N-Boc-L-valine in compound (7) a is replaced by N-Boc-L-aspartic acid to obtain , yield 51%.
(3)五肽片段化合物(7)的酯基水解和N-Boc保护基的脱除(3) Ester hydrolysis of pentapeptide fragment compound (7) and removal of N-Boc protecting group
(3-1)化合物(1)a的合成(3-1) Synthesis of Compound (1)a
将化合物(7)a(30mg,0.06mmol)溶于甲醇(2mL)中，再称取LiOH.H2O(4mg,0.09mmol)加入上述溶液中，室温搅拌3小时。然后减压旋除溶剂，得到白色固体，加水使固体溶解，再用2N的盐酸调节溶液PH为5-6，有白色固体析出，过滤，烘干固体。将得到的固体溶于二氯甲烷(2.5mL)中，再加入三氟乙酸(68mg,0.6mmol)，于室温搅拌5小时，减压旋除溶剂，得到的粗产品用制备HPLC(SB-C18柱,5μm,9.4×250mm,40％CH3CN/60％H2O)分离纯化，得到白色粉末状终产物1a(22.7mg,88％)。Compound (7)a (30 mg, 0.06 mmol) was dissolved in methanol (2 mL), and then LiOH.H 2 O (4 mg, 0.09 mmol) was weighed and added to the above solution, and stirred at room temperature for 3 hours. Then the solvent was spinned off under reduced pressure to obtain a white solid, which was dissolved by adding water, and the pH of the solution was adjusted to 5-6 with 2N hydrochloric acid, and a white solid was precipitated, filtered, and dried. The obtained solid was dissolved in dichloromethane (2.5mL), then trifluoroacetic acid (68mg, 0.6mmol) was added, stirred at room temperature for 5 hours, the solvent was removed under reduced pressure, and the obtained crude product was analyzed by preparative HPLC (SB-C18 Column, 5 μm, 9.4×250 mm, 40% CH 3 CN/60% H 2 O) for separation and purification to obtain the final product 1a (22.7 mg, 88%) in the form of white powder.
(3-2)化合物(1)b的合成(3-2) Synthesis of Compound (1)b
化合物(1)b，与化合物(1)a的合成操作步骤一样，收率81％。Compound (1)b, the synthetic procedure is the same as that of compound (1)a, and the yield is 81%.
(3-3)化合物(1)c的合成(3-3) Synthesis of compound (1)c
化合物(1)c，与化合物(1)a的合成操作步骤一样，收率92％。Compound (1)c, the synthetic procedure is the same as that of compound (1)a, and the yield is 92%.
(3-4)化合物(1)d的合成(3-4) Synthesis of compound (1)d
化合物(1)d，与化合物(1)a的合成操作步骤一样，收率98％。Compound (1)d, the synthetic procedure is the same as that of compound (1)a, and the yield is 98%.
(3-5)化合物(1)e的合成(3-5) Synthesis of Compound (1)e
化合物(1)e，与化合物(1)a的合成操作步骤一样，收率85％。Compound (1)e, the synthetic procedure is the same as that of compound (1)a, and the yield is 85%.
(3-6)化合物(1)f的合成(3-6) Synthesis of Compound (1)f
化合物(1)f，与化合物(1)a的合成操作步骤一样，收率87％。Compound (1)f, the synthetic procedure is the same as that of compound (1)a, and the yield is 87%.
实施例3抗肿瘤活性测试Embodiment 3 antitumor activity test
将HCT-116人结肠癌细胞接种于McCoy,s5A培养液(10％血清，1％青-链霉素)和1640培养液中。置于37℃，5％CO2培养箱中，每2-3天传代一次，试验取对数生长期细胞。CCK-8法测定化合物对HCT116细胞的生长抑制效果。HCT-116 human colon cancer cells were inoculated in McCoy, s5A medium (10% serum, 1% penicillin-streptomycin) and 1640 medium. Place in a 37°C, 5% CO 2 incubator, passage once every 2-3 days, and take cells in the logarithmic growth phase for the test. CCK-8 method was used to determine the growth inhibitory effect of compounds on HCT116 cells.
取对数生长期细胞，以配置好的新鲜培养液调整细胞悬液至2500～4000/ml，取100μl(2000细胞/孔)细胞悬液接种到96孔培养板。置于5％CO2，37℃培养箱中过夜孵育培养后，更换新鲜细胞培养液，每孔加入200μl DMSO等体积稀释的浓度梯度药物，与细胞共孵育72h，更换新鲜细胞培养液，每孔加100μl+10μl CCK-8溶液，继续孵育1-4小时，终止培养，用多功能酶标仪(Molecular Devices M5)检测450nm的吸光度，620nm的吸光度校正细胞数差异。Take the cells in the logarithmic growth phase, adjust the cell suspension to 2500-4000/ml with the prepared fresh culture medium, and inoculate 100 μl (2000 cells/well) of the cell suspension into a 96-well culture plate. Place in 5% CO 2 , incubate overnight at 37°C incubator, replace with fresh cell culture medium, add 200 μl DMSO equivalent volume diluted concentration gradient drug to each well, incubate with cells for 72 hours, replace with fresh cell culture medium, each well Add 100 μl+10 μl CCK-8 solution, continue to incubate for 1-4 hours, terminate the culture, detect the absorbance at 450 nm with a multifunctional microplate reader (Molecular Devices M5), and correct the difference in cell number with the absorbance at 620 nm.
将待测化合物(1a，1b，1c，1d，1e，1f)溶解在DMSO中，并在培养液中进一步稀释。DMSO最终浓度不超过0.1％(v/v)。对照组为加入等体积DMSO的肿瘤细胞；空白组无细胞，在加入培养液中加入等体积DMSO。在一次实验内，每个实验条件均设3个复孔。计算各浓度的化合物对细胞生长的抑制率，计算公式为：抑制率(％)＝{1-[(加药组)-(空白组)]/[(对照组)-(空白组)]}×100％，用GraphPad Prim6计算出IC50(IC50抑制50％细胞生长所需的药物浓度)，检测结果如下表1所示：Compounds to be tested (1a, 1b, 1c, 1d, 1e, 1f) were dissolved in DMSO and further diluted in culture broth. The final concentration of DMSO does not exceed 0.1% (v/v). The control group was tumor cells added with an equal volume of DMSO; the blank group had no cells, and an equal volume of DMSO was added to the culture medium. In one experiment, 3 replicate wells were set up for each experimental condition. Calculate the inhibition rate of the compound of each concentration to cell growth, and the calculation formula is: inhibition rate (%)={1-[(dosing group)-(blank group)]/[(control group)-(blank group)]} ×100%, IC50 (drug concentration required for IC50 to inhibit 50% of cell growth) was calculated with GraphPad Prim6, and the test results are shown in Table 1 below:
表1本发明海兔毒素10类似物的抗肿瘤活性Table 1 Antitumor activity of dolastatin 10 analogs of the present invention
综上，生物活性测试实验结果表明，化合物1a，1b，1c，1d，1e和1f对人结肠癌细胞均具有显著的抑制作用，IC50在0.25～11.13μM之间；与对照化合物MMAF相比，化合物1a，1c，1d和1e抑制活性更高，有发展成为潜在治疗药物的潜力。本发明为开发治疗结肠癌或其他类型癌症的抗癌药物提供了广阔的发展空间。In summary, the results of biological activity tests showed that compounds 1a, 1b, 1c, 1d, 1e and 1f all had significant inhibitory effects on human colon cancer cells, with IC50 ranging from 0.25 to 11.13 μM; compared with the control compound MMAF , compounds 1a, 1c, 1d and 1e have higher inhibitory activity and have the potential to be developed into potential therapeutic drugs. The invention provides a broad development space for the development of anticancer drugs for treating colon cancer or other types of cancer.
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