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WO2021253794A1 - Dérivé de maytansine, son procédé de synthèse et son utilisation - Google Patents

Dérivé de maytansine, son procédé de synthèse et son utilisation Download PDF

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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
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WO
WIPO (PCT)
Prior art keywords
astc
methanol
psbt11
extract
maytansinoid
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/CN2020/141335
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English (en)
Chinese (zh)
Inventor
沈月毛
鲁春华
李小曼
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Shandong University
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Shandong University
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Publication date
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Publication of WO2021253794A1 publication Critical patent/WO2021253794A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • 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)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne un dérivé de maytansine, son procédé de synthèse et son utilisation. L'invention construit un vecteur d'expression intégrative pSBT11-litC constitué d'un gène d'enzyme alanyl astC, réalisant une expression hétérologue du gène d'enzyme alanyl dans une souche mutante d'actinosynnema pretiosum. Un dérivé de maytansine modifié alanylé est séparé et purifié à partir d'un extrait brut de la souche mutante d'actinosynnema prétiosum qui exprime l'enzyme alanyl. Selon un test d'activité anti-tumorale in vitro, les valeurs IC50 du composé à la cellule du cancer du col de l'utérus humain (HeLa), la cellule du cancer du côlon humain (HCT116) et la cellule du cancer du sein humain (MDA-MB-231) sont 3,1, 9,4 et 10,4 nM respectivement, ainsi, le composé peut être utilisé pour préparer des médicaments antinéoplasiques et pour former des conjugués anticorps-médicament avec différents anticorps et connexons.
PCT/CN2020/141335 2020-06-17 2020-12-30 Dérivé de maytansine, son procédé de synthèse et son utilisation Ceased WO2021253794A1 (fr)

Applications Claiming Priority (2)

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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
CN202010551651.5 2020-06-17

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Cited By (1)

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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

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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

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WO1982001188A1 (fr) * 1980-10-08 1982-04-15 Takeda Chemical Industries Ltd Composes 4,5-deoxymaytansinoide et leur procede de preparation
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
CN107417701A (zh) * 2016-03-03 2017-12-01 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

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Non-Patent Citations (2)

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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 *
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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

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