WO2021253794A1 - 一种美登木素衍生物及其合成方法和应用 - Google Patents

一种美登木素衍生物及其合成方法和应用 Download PDF

Info

Publication number
WO2021253794A1
WO2021253794A1 PCT/CN2020/141335 CN2020141335W WO2021253794A1 WO 2021253794 A1 WO2021253794 A1 WO 2021253794A1 CN 2020141335 W CN2020141335 W CN 2020141335W WO 2021253794 A1 WO2021253794 A1 WO 2021253794A1
Authority
WO
WIPO (PCT)
Prior art keywords
astc
methanol
psbt11
extract
maytansinoid
Prior art date
Application number
PCT/CN2020/141335
Other languages
English (en)
French (fr)
Inventor
沈月毛
鲁春华
李小曼
Original Assignee
山东大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 山东大学 filed Critical 山东大学
Publication of WO2021253794A1 publication Critical patent/WO2021253794A1/zh

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D498/08—Bridged systems
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/188—Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms

Definitions

  • the invention provides a maytansinoid derivative with tumor inhibitory activity, and specifically relates to a maytansinoid derivative and a preparation method and application thereof, and belongs to the technical field of natural medicinal chemistry and medicine application.
  • Maytansinoids belong to the Ansa macrocyclic lactams. According to their sources, they can be divided into two categories: plant maytansinoids and bacterial maytansinoids. They have strong antibacterial and antitumor activities. Studies have found that maytansinoid derivatives can bind to tubulin ⁇ subunits, prevent the formation of microtubule bundles, disrupt the mitotic process, and thereby inhibit tumor cell growth. Due to the neurotoxicity, maytansinoid derivatives cannot be directly used in clinics, but they can be used as "warheads" coupled with specific immune proteins to exert anti-cancer effects.
  • bacterial maytansinoids Compared with plant maytansinoids, bacterial maytansinoids have high yield and simple preparation process, which can provide low-cost raw materials for the production of maytansinoids antibody conjugate drugs.
  • there is no suitable site for coupling with the linker in the original structure of the bacterial maytansinoid that is, ansamicin. It is necessary to remove the C-3 ester group through a reduction reaction, and then select C3. Aminoacylation.
  • This method not only has high cost and low yield, but also has by-products that are not easy to separate and remove, which seriously hinders the research, production and clinical application of maytansinoid antibody conjugate drugs.
  • the study on the anti-tumor structure-activity relationship of maytansin derivatives has found that the C-3 ester side chain plays a key role in its anti-tumor activity (Chem Pharm Bull 2004, 52, 1-26), which is used to prepare maytansine derivatives.
  • the lignin antibody conjugate is an alanyl maytansinol derivative containing a disulfide bond or a sulfhydryl group at the C-3 position.
  • the present invention provides a maytansinoid derivative, which is produced by actinomycetes and has a C-3 alanyl group in the structure.
  • the invention also provides synthetic methods and applications of maytansinoid derivatives.
  • the objective of the present invention is to efficiently prepare alanylated maytansinoid derivatives with coupling sites through biosynthesis, which will greatly accelerate the development process of maytansinoid antibody conjugate drugs. Highly effective new anti-tumor drugs are of great significance.
  • a maytansin derivative its chemical structure is as follows:
  • the EA extract is dissolved in methanol, and then extracted with petroleum ether several times.
  • the methanol phase is reduced under reduced pressure and concentrated to dryness at a temperature of 38-40°C to obtain a methanol extract;
  • the preferred preparation conditions are as follows:
  • step (1) of the above preparation method the mutant strain HGF052+pJTU824-asm18+pSBT11-astC of Actinosynnema pretiosum ssp.autantium ATCC 31565 expressing alanylase is subjected to solid fermentation, Incubate at 28°C for 10 days.
  • step (4) of the above preparation method the methanol used in the extraction is 95% methanol.
  • step (5) of the above preparation method the reverse-phase silica gel column and the preparative HPLC column packing are C-18, and the gel column model is Sephadex LH-20.
  • step (5) of the above preparation method the step of separating the methanol extract is:
  • step (1) of the above-mentioned preparation method the preparation method of the A. alanylase-expressing Actinomyces precious orange mutant strain HGF052+pJTU824-asm18+pSBT11-astC is as follows:
  • NCBI gene bank registration number of the alanylase gene astC described in step (a) KF813023.1.
  • HeLa human cervical cancer cells
  • HCT116 human colon cancer cells
  • MDA-MB-231 human breast cancer cells
  • the invention also provides the pharmaceutical use of the maytansinoid derivatives, which can be used to prepare anti-tumor drugs.
  • the tumor is cervical cancer, colon cancer or breast cancer.
  • An anti-tumor pharmaceutical composition comprising the above maytansinoid derivative and one or more pharmaceutically acceptable carriers or excipients.
  • the maytansin derivative with tumor suppressive activity obtained by the biosynthesis method of the present invention avoids the waste of raw materials and difficult-to-remove by-products caused by traditional chemical synthesis methods.
  • the present invention constructs the integrated expression vector pSBT11-astC containing the alanylase gene astC, and realizes the heterologous expression of the alanylase gene in the mutant strain of Actinomyces aurantiacus.
  • a modified maytansinoid derivative after alanylation was isolated and purified from the crude extract of the precious A. orange mutant strain expressing alanylase.
  • In vitro anti-tumor activity tests showed that the compound has IC50 values of 3.1, 9.4 and 10.4 nM for human cervical cancer cells (HeLa), human colon cancer cells (HCT116) and human breast cancer cells (MDA-MB-231), respectively. Therefore, it can be used to prepare anti-tumor drugs, and can be combined with different antibodies and linkers to form antibody conjugates.
  • the Escherichia coli ET12567/pUZ8002 was transformed with pSBT11-astC to obtain the Escherichia coli-actinomycete conjugative transfer donor strain ET12567/pUZ8002/pSBT11-astC.
  • the ET12567/pUZ8002/pSBT11-astC and the mycelium of the mutant strain HGF052+pJTU824-asm18 of Actinomyces orange ATCC 31565 were conjugatively transferred to obtain the mutant strain HGF052+pJTU824-asm18 expressing alanylase. +pSBT11-astC.
  • Example 2 In vitro anti-tumor activity test of the compound 1
  • Ansamicin AP-3* purchased from MCE (MedChemExpress).
  • HeLa human cervical cancer cells
  • HCT116 human colon cancer cells
  • MDA-MB-231 human breast cancer cells
  • Test method The sulforhodamine B (SRB) protein staining method was used to determine the cell growth inhibition rate. Specifically:
  • the tumor cells are cultured to the logarithmic growth phase, trypsinized, and the cell density is adjusted to 30,000-70,000 cells/mL with DMEM medium.
  • DMEM medium a medium that was adjusted to 30,000-70,000 cells/mL with DMEM medium.
  • the compound concentration was diluted to 2 times the detection concentration with DMEM medium, 100 ⁇ L was added to a 96-well plate, and the culture was continued for 72 hours.
  • Test results The results are shown in Table 2.
  • the maytansinoid derivative 1 of the present invention has shown effects on human cervical cancer cells (HeLa), human colon cancer cells (HCT116) and human breast cancer cells (MDA-MB-231) Obvious cytotoxic activity, with IC 50 values of 3.1, 9.4 and 10.4 nM, respectively.
  • the compound of the present invention can be used to prepare anti-tumor drugs, can be combined with other drugs to make anti-tumor drug compositions, and can also be coupled with different antibodies and linkers to make antibody conjugates.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

本发明涉及一种美登木素衍生物及其合成方法和应用。本发明构建了包含丙氨酰化酶基因astC的整合型表达载体pSBT11-astC，并实现了丙氨酰化酶基因在珍贵橙色束丝放线菌突变株中的异源表达。从表达有丙氨酰化酶的珍贵橙色束丝放线菌突变株的粗提物分离纯化一个丙氨酰化后修饰的美登木素衍生物。体外抗肿瘤活性试验表明，该化合物对人***细胞(HeLa)、人结肠癌细胞(HCT116)和人乳腺癌细胞(MDA-MB-231)的IC 50值分别为3.1、9.4和10.4nM，因此可用于制备抗肿瘤药物，可与不同的抗体和连接子组成制抗体偶联物。

Description

一种美登木素衍生物及其合成方法和应用 技术领域
本发明提供一种具有肿瘤抑制活性的美登木素衍生物，具体涉及美登木素衍生物及其制备方法和应用，属于天然药物化学与医药应用技术领域。
背景技术
美登木素属于安莎类大环内酰胺，根据其来源可分为植物美登木素和细菌美登木素两大类，都有极强的抗菌和抗肿瘤活性。研究发现美登木素衍生物可以结合微管蛋白β亚基，阻止微管束聚集形成，破坏有丝***过程，从而抑制肿瘤细胞生长。由于存在神经毒性，美登木素衍生物不能直接用于临床，但可以作为“弹头”与特异免疫蛋白偶联发挥抗癌作用。2013年，首个针对实体瘤的抗体偶联物药物ado-trastuzumab entansine(商品名Kadcyla)被FDA批准用于治疗HER2阳性乳腺癌，美登木素抗体偶联物药物的研发再次引起了广泛关注。
与植物美登木素相比，细菌美登木素的产量高、制备流程简单，可以为美登木素抗体偶联物药物的生产提供价格低廉的原料。然而，细菌美登木素(即安丝菌素)原始结构中并没有合适的、与连接子进行偶联的位点，需要通过还原反应脱去C-3位酯基，然后再选择性丙氨酰化。该方法不但成本高、产率低，还有不易分离除去的副产物，严重阻碍美登木素抗体偶联物药物的研究、生产和临床应用。
对美登木素衍生物抗肿瘤构效关系的研究发现，C-3位酯基侧链对其抗肿瘤活性起关键作用(Chem Pharm Bull 2004,52,1-26)，用于制备美登木素抗体偶联物的是C-3位含有二硫键或巯基的丙氨酰美登醇衍生物。虽然放线菌中美登木素衍生物的产量较高，但无法产生C-3位为丙氨酰基的化合物。
发明内容
针对现有技术的不足，本发明提供一种美登木素衍生物，该化合物由放线菌产生、在结构中具有C-3位丙氨酰基。本发明还提供美登木素衍生物的合成方法和应用。
本发明的目标是通过生物合成高效制备具有偶联位点的丙氨酰化美登木素衍生物，将可能极大加快美登木素抗体偶联物药物的研发进程，因此对于发现广谱高效的新型抗肿瘤药物具有重要意义。
一、美登木素衍生物
一种美登木素衍生物，其化学结构如下所示：
Figure PCTCN2020141335-appb-000001
二、美登木素衍生物的合成方法
本发明所述美登木素衍生物的合成方法，其特征在于，包括以下步骤：
(1)用YMG培养基对表达有丙氨酰化酶的珍贵橙色束丝放线菌(Actinosynnema pretiosum ssp.autantium ATCC 31565)的突变株HGF052+pJTU824-asm18+pSBT11-astC进行15L固体发酵，28-30℃培养7-10天；
(2)将培养物切成小块，室温下用体积比80:20的乙酸乙酯/甲醇浸泡提取三次，合并提取液，减压及38-40℃的温度下浓缩至干，得粗提物；
(3)用水溶解粗提物，使用乙酸乙酯萃取，乙酸乙酯相减压及38-40℃的温度下浓缩至干得EA提取物；
(4)EA提取物溶于甲醇，再经石油醚多次萃取，甲醇相减压及38-40℃的温度下浓缩至干得甲醇提取物；
(5)甲醇提取物依次经反相硅胶柱层析分离，凝胶柱层析分离，薄层层析和制备型HPLC分离将组分相同的洗脱液进行合并得到美登木素衍生物。
根据本发明的合成方法，优选的制备条件如下：
上述制备方法步骤(1)中，对表达有丙氨酰化酶的珍贵橙色束丝放线菌(Actinosynnema pretiosum ssp.autantium ATCC 31565)的突变株HGF052+pJTU824-asm18+pSBT11-astC进行固体发酵，28℃培养10天。
上述制备方法步骤(4)中，所述提取采用的甲醇为95％甲醇。
上述制备方法步骤(5)中，所述反相硅胶柱、制备型HPLC柱填料为C-18、凝胶柱型号为Sephadex LH-20。
上述制备方法步骤(5)中，对甲醇提取物进行分离的步骤为：
甲醇提取物首先经反相硅胶柱层析分离，分别用水，30％，50％，70％，100％甲醇依次洗脱，每个组分洗脱1L，200mL/份接收，TLC检测，用CH 2Cl 2：MeOH＝10:1v/v展开，碘化铋钾显色，合并70％洗脱组分；继续用凝胶柱层析分离，甲醇洗脱，5mL/管,合并25-29管；继续用制备型HPLC分离，流动相为体积百分比38％乙腈，收集目标峰，得到美登木素衍生物1。
上述制备方法步骤(1)中，所述表达有丙氨酰化酶的珍贵橙色束丝放线菌突变株HGF052+pJTU824-asm18+pSBT11-astC的制备方法按如下步骤所述：
(a)将丙氨酰化酶基因astC的开放阅读框***到质粒pSBT11上的Ned I和EcoR I限制性酶切位点之间，使该核苷酸序列位于ermE*启动子的下游并受其调控；
(b)用整合型表达载体pSBT11-astC转化大肠杆菌ET12567/pUZ8002，获得大肠杆菌-放线菌接合转移供体菌ET12567/pUZ8002/pSBT11-astC；
(c)将ET12567/pUZ8002/pSBT11-astC与HGF052+pJTU824-asm18的菌丝体进行接合转移，获得表达有丙氨酰化酶的突变株HGF052+pJTU824-asm18+pSBT11-astC。
步骤(a)所述的丙氨酰化酶基因astC的NCBI基因库登记号：KF813023.1。
步骤(c)所述的HGF052+pJTU824-asm18菌株，按照如下文献报道的方法获得：
Appl Microbiol Biotechnol 2016，100，2641-2649。
三、美登木素衍生物的应用
经药理试验研究表明，该化合物对人***细胞(HeLa)、人结肠癌细胞(HCT116)和人乳腺癌细胞(MDA-MB-231)显示了明显的细胞毒活性，IC 50值分别为3.1、9.4和10.4nM，
本发明还提供美登木素衍生物的制药用途，可用于制备抗肿瘤药物。优选的，所述的肿瘤为***、结肠癌或乳腺癌。
一种抗肿瘤的药物组合物，包括上述美登木素衍生物和一种或多种药学上可接受载体或赋形剂。
有益效果：
1、本发明通过生物合成方法得到的肿瘤抑制活性的美登木素衍生物，避免了传统的化学合成方法带来的原料浪费和难以去除的副产物。
2、本发明构建了包含丙氨酰化酶基因astC的整合型表达载体pSBT11-astC，并实现了丙氨酰化酶基因在珍贵橙色束丝放线菌突变株中的异源表达。从表达有丙氨酰化酶的珍贵橙 色束丝放线菌突变株的粗提物分离纯化一个丙氨酰化后修饰的美登木素衍生物。体外抗肿瘤活性试验表明，该化合物对人***细胞(HeLa))、人结肠癌细胞(HCT116)和人乳腺癌细胞(MDA-MB-231)的IC50值分别为3.1、9.4和10.4 nM，因此可用于制备抗肿瘤药物，可与不同的抗体和连接子组成制抗体偶联物。
具体实施方式
以下实施例仅用于说明本发明的技术效果，但所述的实施例不用于限制本发明。
在如下的实施例中所指的美登木素衍生物1的化学结构式(结构式中的***数字是化学结构中碳原子的标位)：
Figure PCTCN2020141335-appb-000002
实施例1：所述化合物1的生物合成和制备
1.构建表达有丙氨酰化酶的突变株
1)构建表达载体
扩增丙氨酰化酶基因astC(Genbank accession No.KF813023.1)的DNA片段，***到质粒pSBT11的Ned I和EcoR I限制性酶切位点之间，使该核苷酸序列位于ermE*启动子的下游并受其调控，得到整合型表达载体pSBT11-astC。
2)构建突变株
用pSBT11-astC转化大肠杆菌ET12567/pUZ8002，获得大肠杆菌-放线菌接合转移供体菌ET12567/pUZ8002/pSBT11-astC。将ET12567/pUZ8002/pSBT11-astC与珍贵橙色束丝放线菌ATCC 31565的突变株HGF052+pJTU824-asm18的菌丝体进行接合转移，获得表达有丙氨酰化酶的突变株HGF052+pJTU824-asm18+pSBT11-astC。
2.化合物1的制备
1)用YMG培养基对珍贵橙色束丝放线菌突变株HGF052+pJTU824-asm18+pSBT11-astC 进行固体发酵(15L)，28℃培养10天。
2)培养物切成小块后室温用乙酸乙酯/甲醇(80:20)混合溶剂浸泡提取三次，合并提取液，减压及38-40℃的温度下浓缩至干，得粗提物。
3)粗提物溶于水，用乙酸乙酯多次萃取，乙酸乙酯相减压及38-40℃的温度下浓缩至干得EA提取物(3.3g)。
4)EA提取物溶于95％甲醇，再经石油醚多次萃取，甲醇相减压及38-40℃的温度下浓缩至干得甲醇提取物(2.9g)。
5)甲醇提取物首先经反相硅胶柱层析(RP-18，180g柱)分离，分别用水，30％，50％，70％，100％甲醇依次洗脱，每个组分洗脱1L，200mL/份接收，TLC检测，用CH 2Cl 2：MeOH＝10:1展开，碘化铋钾显色，合并70％洗脱组分；继续用凝胶(Sephadex LH-20)柱层析分离，甲醇洗脱，5mL/管,合并25-29管(39.7mg)。继续用制备型HPLC分离，流动相为38％乙腈，收集目标峰，得到化合物1(5.5mg)。
3.化合物的结构鉴定
ESI-MS测得化合物1的准分子离子峰为m/z 648.27[M+H] +。 1H和 13C NMR显示，化合物1共含有32个碳原子(表1)，包括5个甲基，2个甲氧基，3个亚甲基，11个次甲基和11个季碳。根据HMQC和HMBC的信号，确定了该化合物为美登木素衍生物。根据C-3位质子与C-1’的远程相关关系，确定了丙氨酰基的取代位置是在C-3位的O上，对全部的NMR光谱数据进行指定，确定为一新化合物。
表1、化合物1的核磁共振数据
No 13C 1H HMBC
1 173.2s / /
2 30.1t 1.28(m),2.07(m) /
3 74.7d 5.45(br s) C-1'
4 134.3s   /
5 126.7d 5.35(d,8.0) C-6-Me
6 38.5d 2.55(m) C-5,C-7
7 78.0d 4.24(br s) /
8 35.8t 1.31(m),1.90(m) /
9 81.7s / /
10 88.9d 3.61(d,9.0) C-9,C-10-OMe,C-12
11 128.1d 5.56(dd,9.3,14.4) /
12 133.4d 6.66(dd,11.0,15.1) /
13 133.4d 6.03(d,10.2) C-14-Me
14 139.2s / /
15 45.7t 3.28(d,14.5),3.45(d,14.7) C-14-Me,C-16
16 140.8s / /
17 125.5d 7.05(s) C-21
18 135.1s / /
19 121.6s / /
20 156.0s / /
21 119.1d 6.97(s) C-17
1' 172.7s / /
2' 49.3d 4.35(m) C-1'
3' 170.4s / /
4' 22.5q 1.89(s) C-4'
4-Me 15.2q 1.65(s) C-4
6-Me 15.5q 1.05(d,6.5) C-5,C-6,C-7
3'-Me 15.4q 1.35(d,5.3) C-1',C-2'
7-carbam 152.7s / /
10-OMe 56.4q 3.32(s) C-10
14-Me 15.2q 1.79(s) C-13,C-15
20-OMe 56.7q 3.92(s) C-20
2'-NH / 5.56(d,5.4) C-4'
实施例2：所述化合物1的体外抗肿瘤活性试验
安丝菌素AP-3*：购自MCE(MedChemExpress)。
细胞株：人***细胞(HeLa))、人结肠癌细胞(HCT116)和人乳腺癌细胞(MDA-MB-231)购自中科院上海细胞库。
试验方法：采用硫酰罗丹明B(sulforhodamine B,SRB)蛋白染色法测定细胞生长抑制率。具体为：
1)将肿瘤细胞培养至对数生长期，胰酶消化，用DMEM培养基调整细胞密度至3-7万个/mL。在96孔板中，每孔加入100μL细胞，置于37℃、5.0％CO 2培养过夜。用DMEM培养基将化合物浓度稀释至2倍检测浓度，取100μL加入96孔板，继续培养72h。
2)小心吸干培养基，缓慢加入预冷的10％TCA溶液100μL，4℃静置1h以上。弃去TCA固定液，用流水冲洗五次，用吸水纸吸干水分。
3)加入100μL SRB染色液，孵育30min。弃去SRB染色液，用1％的冰醋酸冲洗五遍，去除游离的SRB染料。室温空干，加入100μL Tris溶液(10mM，pH 10.0)，过夜溶解SRB染料。
4)使用酶标仪在波长570nm下测定给药孔和空白孔OD值。细胞生长抑制率＝(1－用药组平均OD值/对照组孔OD值)×100％。
试验结果的评判与解释：细胞半数生长抑制时的药物浓度IC 50按照量效数据进行换算。每个实验重复三次，吸收值差异小于5％，IC 50差异小于30％。以IC 50≤100nM为有效标准。
试验结果：结果如表2所示，本发明美登木素衍生物1对人***细胞(HeLa))、人结肠癌细胞(HCT116)和人乳腺癌细胞(MDA-MB-231)显示了明显的细胞毒活性，其IC 50值分别为3.1、9.4和10.4nM。
表2、化合物1对3株肿瘤细胞的细胞毒试验结果(IC 50,nM)
Figure PCTCN2020141335-appb-000003
*阳性对照药
试验结论：通过药理学试验，可以看出化合物1对人***细胞(HeLa)、人结肠癌细胞(HCT116)和人乳腺癌细胞(MDA-MB-231)显示了明显的细胞毒活性。因此，本发明的化合物可用于制备抗肿瘤药物，可与其他药物制成抗肿瘤药物组合物，还可以与不同的抗体和连接子偶联制成抗体偶联物。

Claims (10)

  1. 一种美登木素衍生物，其特征在于，化学结构如下所示：
    Figure PCTCN2020141335-appb-100001
  2. 如权利要求1所述美登木素衍生物的合成方法，其特征在于，包括以下步骤：
    (1)用YMG培养基对表达有丙氨酰化酶的珍贵橙色束丝放线菌(Actinosynnema pretiosum ssp.autantium ATCC 31565)的突变株HGF052+pJTU824-asm18+pSBT11-astC进行15L固体发酵，28-30℃培养7-10天；
    (2)将培养物切成小块，室温下用体积比80:20的乙酸乙酯/甲醇浸泡提取三次，合并提取液，减压及38-40℃的温度下浓缩至干，得粗提物；
    (3)用水溶解粗提物，使用乙酸乙酯萃取，乙酸乙酯相减压及38-40℃的温度下浓缩至干得EA提取物；
    (4)EA提取物溶于甲醇，再经石油醚多次萃取，甲醇相减压及38-40℃的温度下浓缩至干得甲醇提取物；
    (5)甲醇提取物依次经反相硅胶柱层析分离，凝胶柱层析分离，薄层层析和制备型HPLC分离将组分相同的洗脱液进行合并得到美登木素衍生物。
  3. 如权利要求2所述美登木素衍生物的合成方法，其特征在于，步骤(1)中，所述表达有丙氨酰化酶的珍贵橙色束丝放线菌突变株HGF052+pJTU824-asm18+pSBT11-astC的制备方法按如下步骤所述：
    (a)将丙氨酰化酶基因astC的开放阅读框***到质粒pSBT11上的Ned I和EcoR I限制性酶切位点之间，使该核苷酸序列位于ermE*启动子的下游并受其调控；
    (b)用整合型表达载体pSBT11-astC转化大肠杆菌ET12567/pUZ8002，获得大肠杆菌-放线菌接合转移供体菌ET12567/pUZ8002/pSBT11-astC；
    (c)将ET12567/pUZ8002/pSBT11-astC与HGF052+pJTU824-asm18的菌丝体进行接合转移，获得表达有丙氨酰化酶的突变株HGF052+pJTU824-asm18+pSBT11-astC。
  4. 如权利要求2所述美登木素衍生物的合成方法，其特征在于，步骤(1)中，对表达有丙氨酰化酶的珍贵橙色束丝放线菌(Actinosynnema pretiosum ssp.autantium ATCC 31565)的突变株HGF052+pJTU824-asm18+pSBT11-astC进行固体发酵，28℃培养10天。
  5. 如权利要求2所述美登木素衍生物的合成方法，其特征在于，步骤(4)中，所述提取采用的甲醇为95％甲醇。
  6. 如权利要求2所述美登木素衍生物的合成方法，其特征在于，步骤(5)中，所述反相硅胶柱、制备型HPLC柱填料为C-18、凝胶柱型号为Sephadex LH-20。
  7. 如权利要求2所述美登木素衍生物的合成方法，其特征在于，步骤(5)中，对甲醇提取物进行分离的步骤为：
    甲醇提取物首先经反相硅胶柱层析分离，分别用水，30％，50％，70％，100％甲醇依次洗脱，每个组分洗脱1L，200mL/份接收，TLC检测，用CH 2Cl 2：MeOH＝10:1v/v展开，碘化铋钾显色，合并70％洗脱组分；继续用凝胶柱层析分离，甲醇洗脱，5mL/管,合并25-29管；继续用制备型HPLC分离，流动相为体积百分比38％乙腈，收集目标峰，得到美登木素衍生物1。
  8. 权利要求1所述美登木素衍生物在制备抗肿瘤药物中的应用。
  9. 如权利要求8所述美登木素衍生物的应用，其特征在于，所述的肿瘤为***、结肠癌或乳腺癌。
  10. 一种抗肿瘤的药物组合物，其特征在于，包括权利要求1所述美登木素衍生物和一种或多种药学上可接受载体或赋形剂。
PCT/CN2020/141335 2020-06-17 2020-12-30 一种美登木素衍生物及其合成方法和应用 WO2021253794A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010551651.5 2020-06-17
CN202010551651.5A CN111635418A (zh) 2020-06-17 2020-06-17 一种美登木素衍生物及其合成方法和应用

Publications (1)

Publication Number Publication Date
WO2021253794A1 true WO2021253794A1 (zh) 2021-12-23

Family

ID=72325729

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/141335 WO2021253794A1 (zh) 2020-06-17 2020-12-30 一种美登木素衍生物及其合成方法和应用

Country Status (2)

Country Link
CN (1) CN111635418A (zh)
WO (1) WO2021253794A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116199695A (zh) * 2022-08-30 2023-06-02 浙江大学 一类桔霉素衍生物及其制备方法和应用

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111635418A (zh) * 2020-06-17 2020-09-08 山东大学 一种美登木素衍生物及其合成方法和应用
CN114805391B (zh) * 2021-01-18 2024-03-29 复旦大学 天然美登素类化合物及其在制药中的用途
CN113461708A (zh) * 2021-07-22 2021-10-01 深圳市泰尔康生物医药科技有限公司 安丝菌素p-3衍生物及其在制备治疗胃癌药物中的应用
CN114875095B (zh) * 2022-05-30 2024-05-10 山东大学 一种丙氨酰美登醇及其合成方法和应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111635418A (zh) * 2020-06-17 2020-09-08 山东大学 一种美登木素衍生物及其合成方法和应用

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1982001188A1 (en) * 1980-10-08 1982-04-15 Takeda Chemical Industries Ltd 4,5-deoxymaytansinoide compounds and process for preparing same
JPH0662632B2 (ja) * 1986-07-23 1994-08-17 明治製菓株式会社 新規抗生物質ａ１−ｒ２３９７物質及びその製造法
WO2017148416A1 (zh) * 2016-03-03 2017-09-08 凯惠科技发展(上海)有限公司 一种美登素酯的制备方法及其中间体
GB201615725D0 (en) * 2016-09-15 2016-11-02 Polytherics Ltd Novel cytotoxic agents and conjugates thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111635418A (zh) * 2020-06-17 2020-09-08 山东大学 一种美登木素衍生物及其合成方法和应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LI SHANREN; LU CHUNHUA; CHANG XIAOYAN; SHEN YUEMAO: "Constitutive overexpression ofasm18increases the production and diversity of maytansinoids inActinosynnema pretiosum", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, SPRINGER BERLIN HEIDELBERG, BERLIN/HEIDELBERG, vol. 100, no. 6, 17 November 2015 (2015-11-17), Berlin/Heidelberg, pages 2641 - 2649, XP035870718, ISSN: 0175-7598, DOI: 10.1007/s00253-015-7127-7 *
LI XIAOMAN: "Function-orientated Structure Optimizations of Bacterial Maytansinoids via Synthetic Biology", CHINA DOCTORAL DISSERTATIONS FULL-TEXT DATABASE, MEDICINE AND HEALTH SCIENCES, no. 2, 15 February 2020 (2020-02-15), XP055881374 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116199695A (zh) * 2022-08-30 2023-06-02 浙江大学 一类桔霉素衍生物及其制备方法和应用
CN116199695B (zh) * 2022-08-30 2024-05-28 浙江大学 一类桔霉素衍生物及其制备方法和应用

Also Published As

Publication number Publication date
CN111635418A (zh) 2020-09-08

Similar Documents

Publication Publication Date Title
WO2021253794A1 (zh) 一种美登木素衍生物及其合成方法和应用
CN108484699B (zh) 联吡啶类生物碱、其制备方法和用途
Dramae et al. Antimalarial 20-membered macrolides from Streptomyces sp. BCC33756
CN109134574B (zh) 甾体化合物及其制备方法与应用和抗肿瘤的药物
CN112939865A (zh) 一种大环内酰胺化合物FW05328-d及其高效发酵方法
CN109810919B (zh) 一类安莎全碳环聚酮类抗生素及其在制备抗菌药物或抗肿瘤药物中的应用
CN109942658B (zh) 一类杂萜化合物及其制备方法与应用和抗肿瘤的药物
CN109985044B (zh) 白桦醇及其衍生物在制备抗肿瘤药物中的应用
CN100591673C (zh) 一种除莠霉素衍生物及其制备方法和抗肿瘤应用
CN114920721B (zh) 具有抗肿瘤活性的聚酮类化合物及其制备方法和应用
CN112500340B (zh) 一种具有抗***癌活性的四氢喹啉生物碱及其制备方法和应用
CN114875095B (zh) 一种丙氨酰美登醇及其合成方法和应用
CN109384823B (zh) 两个粉蝶霉素葡萄糖苷及其在抗肾癌药物中的应用
US7470714B2 (en) GM-95-containing antitumor effect potentiator, combined antitumor preparation and antitumor agent
Siyu-Mao et al. Two novel ansamitocin analogs from Actinosynnema pretiosum
CN116082362B (zh) 一种可用于合成靶向抗肿瘤药物的氨甲酰美登醇及其合成方法与应用
AU618790B2 (en) Bu-3862t antitumor antibiotic
CN115124543B (zh) 具有抗病毒活性的三环吡啶酮类化合物及其应用
CN115536616B (zh) 一种珊瑚球菌来源的重排甾体化合物及其制备方法与应用
CN116041305B (zh) 青霉菌（Penicillium mali）的发酵化合物及其制备方法和抗肿瘤应用
CN114605430B (zh) 一种大环双内酯类化合物及其制备方法和应用
CN109384710B (zh) 三个粉蝶霉素类天然产物及其在制备抗肾癌药物中的应用
CN117551034A (zh) 天赐霉素类似物及其制备方法和应用
CN116445350A (zh) 链霉菌l15a-01390及其应用、色霉素类化合物的制备方法及应用、抗肿瘤药物
CN117417241A (zh) 卷柏中的化合物及其用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20940523

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20940523

Country of ref document: EP

Kind code of ref document: A1