WO2020177568A1 - 新型LL-D49194 α1类似物，及其制备方法和应用 - Google Patents

新型LL-D49194 α1类似物，及其制备方法和应用 Download PDF

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WO2020177568A1
WO2020177568A1 PCT/CN2020/076478 CN2020076478W WO2020177568A1 WO 2020177568 A1 WO2020177568 A1 WO 2020177568A1 CN 2020076478 W CN2020076478 W CN 2020076478W WO 2020177568 A1 WO2020177568 A1 WO 2020177568A1
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pharmaceutically acceptable
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唐功利
董雷
侯现锋
潘海学
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Shanghai Institute of Organic Chemistry of CAS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/22—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains four or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/26—Acyclic or carbocyclic radicals, substituted by hetero rings
    • 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
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/465—Streptomyces

Definitions

  • the present invention belongs to the field of biotechnology engineering and the field of medicine. Specifically, the present invention relates to a new type of LL-D49194 ⁇ 1 analogue and its preparation method and application.
  • Anthracycline antibiotics have an anthracycline skeleton and also include methylation, glycosyl modification, hydroxylation and other special modifications.
  • Anthracycline antibiotics are mainly used clinically to treat leukemia, melanoma, lymphoma, lung cancer, breast cancer, uterine cancer and ovarian cancer.
  • LL-D49194s is a type of polyketone compound with an anthraquinone skeleton.
  • the oxygen-containing three-membered spiro ring is the active unit in its structure, which can inhibit the synthesis of DNA and RNA.
  • other LL-D49194 ⁇ 1 analogues were obtained, including LL-D49194 ⁇ 1, LL-D49194 ⁇ 2, etc., especially LL-D49194 ⁇ 1 has the highest yield and activity, and there are many early pharmaceutical studies Take LL-D49194 ⁇ 1 as the main research target.
  • Most of the LL-D49194 compounds have good anti-Gram-positive bacteria activity, the cancer cell inhibition ability is equivalent to or better than cisplatin, and the relative cytotoxicity is also lower.
  • the purpose of the present invention is to provide a new class of LL-D49194 ⁇ 1 analogs with higher activity.
  • Another object of the present invention is to provide a preparation method and application of the LL-D49194 ⁇ 1 analog.
  • R 1 is selected from: H, hydroxyl, halogen, C 1-4 alkyl, and C 1-4 alkoxy;
  • R 2 is selected from: H, hydroxyl, C 1-4 alkoxy,
  • R 3 is selected from the group consisting of H, C 1-4 alkyl.
  • said R 1 is selected from: H, hydroxyl, C 1-4 alkoxy; said R 2 is selected from: H, hydroxyl,
  • the compound has a structure selected from the following group of formula Ia, Ib or Ic:
  • the second aspect of the present invention provides a pharmaceutical composition, the pharmaceutical composition comprising: the LL-D49194 ⁇ 1 analogue of any one of the first aspect of the present invention, or a pharmaceutically acceptable salt thereof; and Acceptable carrier.
  • the pharmaceutical composition is used to treat tumors; preferably, the tumors are selected from the following group: leukemia, melanoma, lymphoma, lung cancer, breast cancer, uterine cancer, and ovarian cancer.
  • the third aspect of the present invention provides a mutant strain of Streptomyces vinaceusdrappus NRRL15735 that can be used to produce the LL-D49194 ⁇ 1 analogue as described in the first aspect of the present invention, characterized in that the In the strain, a gene selected from the group of LL-D49194 ⁇ 1 biosynthetic gene cluster is inactivated and knocked out: cytochrome P450 oxidoreductase gene (lldO10), glycosyltransferase gene (lldB3), or cytochrome P450 Oxidoreductase gene (lldO2).
  • the mutant strain is a recombinant strain with lldO10, lldB3 or lldO2 deleted in the same frame.
  • the fourth aspect of the present invention is a method for preparing the LL-D49194 ⁇ 1 analog or a pharmaceutically acceptable salt thereof as described in the first aspect of the present invention, the method comprising the steps:
  • step (b) Separating the LL-D49194 ⁇ 1 analog from the fermentation product, and optional step (c)
  • step (b) includes:
  • the mutant strain is a recombinant strain lacking lldO10
  • the LL-D49194 ⁇ 1 analog is a compound of formula Ia.
  • the mutant strain is a recombinant strain with a deletion of 11dB3
  • the LL-D49194 ⁇ 1 analog is a compound of formula Ib.
  • the mutant strain is a recombinant strain lacking lldO2
  • the LL-D49194 ⁇ 1 analog is a compound of formula Ic.
  • the fermentation is liquid shake flask fermentation.
  • the fermentation includes: first culturing the strain to obtain a fermentation seed culture solution, and then inoculating it into a fermentation medium for secondary culture.
  • the inoculation amount is 5%-10%, based on the volume of the fermentation medium.
  • the secondary culture further includes: after the strain enters the stable growth phase, adding 2%-3% macroporous resin HP20 for co-cultivation and further fermentation to obtain the LL-D49194 ⁇ 1 analog.
  • the primary culture is carried out in a TSB culture flask without resistance.
  • the fifth aspect of the present invention provides an application of the LL-D49194 ⁇ 1 analogue or a pharmaceutically acceptable salt thereof as an antitumor drug as described in the first aspect of the present invention.
  • Figure 1 shows the results of electrophoresis of Streptomyces vinaceusdrappus NRRL15735 in-frame knockout mutants.
  • Figure 1i shows the sDL05030 knockout mutant;
  • Figure 1ii shows the sDL05021 knockout mutant;
  • Figure 1iii shows the sDL05027 knockout mutant.
  • Figure 2 shows the 1 H NMR (600 MHz, CDCl 3 ) of LL-D49194 ⁇ 2.
  • Figure 3 shows the 13 C NMR (600 MHz, CDCl 3 ) of LL-D49194 ⁇ 2.
  • Figure 4 shows the COSY (600MHz, CDCl 3 ) of LL-D49194 ⁇ 2.
  • Figure 5 shows the HSQC (600MHz, CDCl 3 ) of LL-D49194 ⁇ 2.
  • Figure 6 shows the HMBC (600MHz, CDCl 3 ) of LL-D49194 ⁇ 2.
  • Figure 7 shows the NOESY (600MHz, CDCl 3 ) of LL-D49194 ⁇ 2.
  • Figure 8 shows the 1 H NMR (600 MHz, CDCl 3 ) of LL-D49194 ⁇ 3.
  • Figure 9 shows the 13 C NMR (600 MHz, CDCl 3 ) of LL-D49194 ⁇ 3.
  • Figure 10 shows the COSY (600MHz, CDCl 3 ) of LL-D49194 ⁇ 3.
  • Figure 11 shows the HSQC (600MHz, CDCl 3 ) of LL-D49194 ⁇ 3.
  • Figure 12 shows the HMBC (600MHz, CDCl 3 ) of LL-D49194 ⁇ 3.
  • Figure 13 shows the NOESY (600MHz, CDCl 3 ) of LL-D49194 ⁇ 3.
  • Figure 14 shows the 1 H NMR (600 MHz, CDCl 3 ) of LL-D49194 ⁇ 4.
  • Figure 15 shows the 13 C NMR (600 MHz, CDCl 3 ) of LL-D49194 ⁇ 4.
  • Figure 16 shows the COSY (600MHz, CDCl 3 ) of LL-D49194 ⁇ 4.
  • Figure 17 shows the HSQC (600MHz, CDCl 3 ) of LL-D49194 ⁇ 4.
  • Figure 18 shows the HMBC (600MHz, CDCl 3 ) of LL-D49194 ⁇ 4.
  • Figure 19 shows the NOESY (600 MHz, CDCl 3 ) of LL-D49194 ⁇ 4.
  • Figure 20 shows the HPLC analysis results of the novel LL-D49194 ⁇ 1 analogue obtained by optimizing the fermentation conditions of the mutant.
  • Figure 20i is the fermentation result of the wild-type Streptomyces vinaceusdrappus NRRL15735;
  • Figure 20ii is the fermentation result of the mutant sDL05030, and sDL05030 is the production LL-D49194 ⁇ 2 strain;
  • Figure 20iii is the fermentation result of mutant sDL05021, sDL05021 is the strain producing LL-D49194 ⁇ 3;
  • Figure 20iv is the fermentation result of mutant sDL05027, sDL05027 is the strain that produces LL-D49194 ⁇ 4.
  • A, B, C, D, E, F represent the number of the ring
  • the C ring represents the ring numbered C
  • the numbers 1, 2, 3, 4 represent the numbering sequence of the carbon skeleton of the compound
  • the C-2 position is Represents the carbon atom numbered 2.
  • the present inventors studied the biosynthetic mechanism of LL-D49194 ⁇ 1 and used the method of constructing gene-in-frame deletion mutants, and unexpectedly discovered that when the cytochrome P450 oxidoreductase gene (lldO10) or glycosyltransferase is deleted
  • the mutant strains of the gene (lldB3) or the cytochrome P450 oxidoreductase gene (lldO2) are subjected to liquid fermentation, the LL-D49194 ⁇ 1 analogue LL- dehydroxylated at the C-2 position of the C ring and reduced at the C-4 position are prepared respectively.
  • the terms "compounds of the present invention”, “active ingredients of the present invention”, “analogs of the present invention” and “LL-D49194 ⁇ 1 analogs of the present invention” can be used interchangeably, and all refer to LL-D49194 ⁇ 1 analogs Such as LL-D49194 ⁇ 2 shown in formula Ia, LL-D49194 ⁇ 3 shown in Ib and LL-D49194 ⁇ 4 shown in Ic.
  • C 1-4 alkyl refers to a straight or branched chain alkyl group having 1 to 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl Group, isobutyl, sec-butyl, tert-butyl, or similar groups; the term C 1-4 alkoxy methoxy, ethoxy, propoxy, isopropyloxy, or similar groups;
  • halogen refers to F, Cl, Br and I.
  • a preferred class of LL-D49194 ⁇ 1 analogs has the chemical structural formula shown in the following formula I:
  • LL-D 49194 ⁇ 2 has the chemical structural formula shown in Formula Ia.
  • LL-D 49194 ⁇ 3 has a chemical structure as shown in Formula Ib.
  • LL-D 49194 ⁇ 4 has a chemical structural formula shown in Formula Ic.
  • pharmaceutically acceptable salt refers to a salt formed by a compound of the present invention and an acid or base suitable for use as a drug.
  • Pharmaceutically acceptable salts include inorganic salts and organic salts.
  • the inventors not only confirmed the structure of the compound through fermentation, separation and purification, and structure analysis of the novel LL-D49194 ⁇ 1 analog. In addition, it was also confirmed through tumor cell suppression experiments that the LL-D49194 ⁇ 1 analog of the present invention has a significantly higher tumor cell suppression ability than the LL-D49194 ⁇ 1 natural product.
  • the term "the starting strain of the present invention” refers to Streptomyces vinaceusdrappus NRRL 15735 (purchased from the American Agricultural Research Institute (ARS)), which was obtained from the Microorganism Collection of the United States Department of Agriculture. Streptomyces vinaceusdrappus NRRL15735 in Maiese W M. et al. "LL-D49194 Antibiotics, New Anti-tumor Drug Family: Taxonomy, Fermentation and Biological Characteristics[J]. The Journal of Antibiotics, 1990 , 43(3):253-258.” and US4626503. It should be understood that the starting strain not only includes the strain of Streptomyces vinaceusdrappus NRRL15735, but also its derivative strains and other strains that produce LL-D49194 ⁇ 1.
  • a mutant strain of Streptomyces vinaceusdrappus NRRL15735 which produces the LL-D49194 ⁇ 1 analog of the present invention, wherein the LL-D49194 ⁇ 1 biosynthetic gene cluster is in the strain
  • the cytochrome P450 oxidoreductase gene (lldO10), or glycosyltransferase gene (lldB3) or cytochrome P450 oxidoreductase gene (lldO2) in the cytochrome P450 oxidoreductase gene is inactivated and knocked out.
  • the present invention also provides a method for constructing mutant strains that can produce LL-D49194 ⁇ 1 analogues, which includes the construction of in-frame knockout plasmids, the plasmids with screening resistance are introduced into wild-type strains, and the plasmids carry the same
  • the source arm segment is recombined with the homologous segment of the wild-type strain genome, thereby replacing the corresponding gene from the genome, thereby obtaining mutant strains with genes knocked out or deleted in frame.
  • a method for constructing a mutant strain and fermenting the mutant strain to prepare an analog of LL-D49194 ⁇ 1 which includes:
  • PCR was used to amplify the left arm and right arm fragments used for gene knockout of lldO10, lldB3 and lldO2 genes, and the two fragments of the left and right arms were connected into the temperature-sensitive shuttle plasmid pKC1139 (US5,955,319) to obtain the recombinant plasmid.
  • the recombinant plasmids were transformed into E. coli DH5 ⁇ , and the monoclonal colonies were selected for amplification and culture, and then the verified plasmids were transformed into E. coli S17-1 respectively.
  • Fresh spores of wild-type Streptomyces vinaceusdrappus NRRL15735 were collected and washed three times with TES buffer solution. The washed spores were resuspended in 500uL TES buffer and placed in a 50°C water bath for 10 minutes. The heat-shocked spores were transferred to 37°C to germinate for about 4 hours, mixed and smeared with E.coli S17-1 containing recombinant plasmid on the IWL-4 medium plate according to a certain ratio, and incubated at 30°C for 16 hours before using Anpu Cover the plate with mycin.
  • the zygote grows after 5 to 7 days of incubation, and the monoclonal zygote is selected and cultured on an apramycin resistant plate at 37°C for 2 to 3 days. Select the well-growing zygote to passage more than 8 times in the anti-TSB culture solution, and then streak a single colony on the IWL-4 medium plate. Through apramycin resistance screening, strains without apramycin resistance were selected for genotype PCR verification to obtain recombinant strains with lldO10, lldB3 and lldO2 deleted in frame.
  • the present invention provides a culturing plan for strains producing LL-D49194 ⁇ 1 and its analogues, which adopts the method of liquid shake flask for fermentation, and at the same time optimizes the amount of oxygen, temperature, inoculum, etc., and adopts the first-level seed fermentation method , Firstly incubate in a TSB culture flask without resistance for 36 hours to prepare a fermentation seed culture solution, and then inoculate 5%-10% (based on the volume of the fermentation medium) into the fermentation medium, and the strain will stabilize in 2-3 days After the growth period, 2%-3% macroporous resin HP20 is added for co-cultivation, and LL-D49194 ⁇ 1 and its analogs can be obtained stably after 6 days of fermentation.
  • the present invention also provides a method for preparing the compound of formula I, which includes the steps of: centrifuging and discarding the supernatant to obtain a mixture of bacteria and HP20 adsorption resin; after immersing the bacteria and HP20 adsorption resin in 2 times the volume of acetone, centrifugation to take the supernatant; After the supernatant was distilled and drained under reduced pressure, the obtained paste was crudely fractionated with a pre-installed 200-300 mesh silica gel column, and then further prepared and purified by HPLC. The effluent containing the compound of formula I was collected and drained. Finally get the target product.
  • the present inventors not only confirmed the structure of the compound through a large amount of fermentation and separation and purification of the compound of formula I, but also confirmed that the active product of the present invention is significantly improved compared with the tumor cell suppression ability of LL-D49194 ⁇ 1 through tumor cell suppression experiments.
  • Example 1 Construction of mutant strains sDL05030, sDL05021 and sDL05027
  • the primer sequence of the left arm of clone lldO10 in-frame deletion is as follows:
  • the primer sequence of the right arm of clone lldO10 in-frame deletion is as follows:
  • the primer sequence of the left arm of clone lldB3 in-frame deletion is as follows:
  • the primer sequence of the right arm of clone lldB3 in-frame deletion is as follows:
  • the primer sequence of the left arm of clone lldO2 in-frame deletion is as follows:
  • the primer sequence of the right arm of clone lldO2 in-frame deletion is as follows:
  • the cloned left and right arm fragments were separated by gel electrophoresis, gel cut, recovered and purified, and the restriction enzymes EcoRI and XbaI and XbaI and HindIII were added to digest the recovered fragments, which were connected to the restriction enzymes EcoRI and HindIII digested vector plasmid pKC1139, and then the ligation system was transformed into E.coli DH5 ⁇ , and a single colony was picked and cultured in an LB culture tube (containing apramycin antibiotic) overnight with shaking. The extracted plasmid is verified by restriction digestion and sent to sequencing for further verification. Transform the verified plasmid into E.coli S17-1.
  • Fresh spores of wild-type Streptomyces vinaceusdrappus NRRL15735 were collected and washed three times with TES buffer solution. The washed spores were resuspended in 500uL TES buffer and placed in a 50°C water bath for 10 minutes. The heat-shocked spores were transferred to 37°C to germinate for about 4 hours, mixed and smeared with E.coli S17-1 containing the recombinant plasmid on the IWL-4 medium plate in a certain proportion, and cultured at 30°C for 16 hours. Cover the plate with pramycin (50 ⁇ g/mL).
  • the zygote grows after 5 to 7 days of incubation, and the monoclonal zygote is selected and cultured on an apramycin-resistant plate at 37°C for 2 to 3 days. Select the well-growing zygote to passage more than 8 times in the non-resistant TSB culture solution, and then streak a single colony on the plate. Through apramycin resistance screening, the strains without apramycin resistance were selected for genotype PCR verification to obtain recombinant strains sDL05030, sDL05021 and sDL05027 with the same frame deletion of lldO10, lldB3 and lldO2 genes. The experimental results are shown in Figure 1.
  • Figure 1i shows the verification result of the sDL05030 knockout mutant. From the electropherogram in Figure 1i, it can be seen that the sDL05030 gene in the sDL05030 knockout mutant is deleted by about 1.15kb, indicating that the sDL05030 knockout mutation was successfully constructed in this example. Strain.
  • Figure 1ii shows the verification result of the sDL05021 knockout mutant. From the electropherogram in Figure 1ii, it can be seen that the sDL05021 gene in the sDL05021 knockout mutant is missing about 1.16 kb, indicating that the sDL05021 knockout mutation was successfully constructed in this example. Strain.
  • Figure 1iii shows the verification result of the sDL05027 knockout mutant. From the electropherogram of Figure 1iii, it can be seen that the sDL05027 gene in the sDL05027 knockout mutant is deleted by approximately 1.18kb, indicating that the sDL05027 knockout mutation was successfully constructed in this example Strain.
  • mutant strains sDL05030, sDL05021, and sDL05027 were respectively inoculated into 100mL seed culture medium and cultured with shaking (including TSB 30g/L), rotating at 220 rpm and temperature at 30°C for 36 hours.
  • LL-D49194 ⁇ 1 analogue LL-D49194 ⁇ 2 (structure shown in formula Ia) was obtained by separating the fermentation product of sDL05030, and appeared in the elution fraction of 75:1 and 50:1 (dichloromethane: methanol).
  • LL-D49194 ⁇ 3 (structure shown in formula Ib) was obtained by separating the fermentation product of sDL05021, and it appeared in the elution fraction of 20:1 and 10:1 (dichloromethane: methanol).
  • LL-D49194 ⁇ 4 (structure shown in formula Ic) is obtained by separating the fermentation product of sDL05027, which appears in the elution part of 30:1 and 20:1 (dichloromethane: methanol), and collects containing LL-D49194 ⁇ 2, LL-D49194 The eluents of ⁇ 3 and LL-D49194 ⁇ 4 were drained under reduced pressure, dissolved in 3ml methanol, and then purified by HPLC.
  • the target product LL-D49194 ⁇ 2 was identified, and the NMR assignment results are shown in Table 3 (CD 3 Cl, 600MHz); the target product LL-D49194 ⁇ 3 was identified, and the NMR assignment results were shown in Table 4 (CD 3 Cl, 600MHz); The product LL-D49194 ⁇ 4 was identified, and the NMR assignment results are shown in Table 5 (CD 3 Cl, 600MHz).
  • Figure 2 Figure 3, Figure 4, Figure 5, Figure 6 and Figure 7 are the 1 H NMR (CD 3 Cl, 600MHz), 13 C NMR (CD 3 Cl, LL-D49194 ⁇ 2 (structure shown in formula Ia), 600MHz), COSY (CD 3 Cl, 600MHz), HSQC (CD 3 Cl, 600MHz), HMBC (CD 3 Cl, 600MHz) and NOESY (CD 3 OD, 600MHz) spectra. Structure identification showed that the inventors successfully prepared LL-D49194 ⁇ 2 (structure shown in Ia).
  • Figure 8, Figure 9, Figure 10, Figure 11, Figure 12 and Figure 13 are the 1 H NMR (CD 3 Cl, 600MHz), 13 C NMR (CD 3 Cl, LL-D49194 ⁇ 3 (structure shown in formula Ib) 600MHz), COSY (CD 3 Cl, 600MHz), HSQC (CD 3 Cl, 600MHz), HMBC (CD 3 Cl, 600MHz) and NOESY (CD 3 OD, 600MHz) spectra. Structure identification showed that the inventors successfully prepared LL-D49194 ⁇ 3 (structure shown in Ib).
  • Figure 14, Figure 15, Figure 16, Figure 17, Figure 18 and Figure 19 are the 1 H NMR (CD 3 Cl, 600MHz), 13 C NMR (CD 3 Cl, LL-D49194 ⁇ 4 (structure shown in formula Ic) 600MHz), COSY (CD 3 Cl, 600MHz), HSQC (CD 3 Cl, 600MHz), HMBC (CD 3 Cl, 600MHz) and NOESY (CD 3 OD, 600MHz) spectra. Structural identification showed that the inventors successfully prepared LL-D49194 ⁇ 4 (structure shown in Ic).
  • Figure 20 shows the HPLC analysis results of the novel LL-D49194 ⁇ 1 analogue obtained by optimizing the fermentation conditions of the mutant.
  • Figure 20i is the fermentation result of the wild-type Streptomyces vinaceusdrappus NRRL15735;
  • Figure 20ii is the HPLC analysis of the mutant strain sDL05030, the fermentation results indicate The mutant strain sDL05030 can produce LL-D49194 ⁇ 2 (shown in formula Ia);
  • Figure 20iii is the HPLC analysis of the mutant strain sDL05021, and the fermentation results show that the mutant strain sDL05021 can produce LL-D49194 ⁇ 3 (shown in formula Ib);
  • Figure 20iv is the mutant strain HPLC analysis of sDL05027, fermentation results showed that the mutant strain sDL05027 can produce LL-D49194 ⁇ 4 (shown in formula Ic).
  • the anti-tumor activity of LL-D49194 ⁇ 1 and its analogues was determined by the following method: adjust the concentration of cells to 3 ⁇ 10 4 cells/mL, and inoculate 100 ⁇ L/well in 96-well flat-bottomed clear cell culture plate, respectively after 24 hours Add drugs containing different concentrations for an appropriate period of time (for example: 24 or 48 hours). After adding 10 ⁇ l of CCK-8 reagent to each well for 2 hours, the absorbance OD value at 450nm (reference wavelength 630nm) was measured with a microplate reader.
  • the blank group is a cell-free medium
  • the control group is added with the same volume of DMSO as the drug
  • HL-60 and B16-F10 cells were selected for tumor cell inhibitory activity experiments.
  • the half-inhibitory concentration (IC 50 ) test results showed that the IC 50 value of LL-D49194 ⁇ 1 analogue was lower than the corresponding original natural product LL-D49194 ⁇ 1 -4 times (Table 7), which shows that the C-ring C-2 position is dehydroxylated and the C-4 position reduced LL-D49194 ⁇ 1 analog has higher anti-tumor activity, which inhibits HL-60 and B16-F10
  • the cell activity is 2-4 times higher than that of the corresponding original LL-D49194 ⁇ 1 natural product.

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Abstract

本发明涉及一种LL-D49194α1类似物，及其制法制备方法和应用。具体地，本发明提供了去糖基化和去甲基化的LL-D49194α1类似物。所获得的LL-D49194α1类似物具有相较于LL-D49194α1更好的抗肿瘤活性。

Description

新型LL-D49194 α1类似物，及其制备方法和应用 技术领域
本发明属于生物技术工程领域和药物领域，具体地，本发明涉及一类新型LL-D49194 α1类似物及其制法和应用。
背景技术
蒽环类抗生素拥有一个蒽环骨架，还包括甲基化、糖基修饰、羟化和其他一些特殊修饰。蒽环类抗生素临床上主要被用于治疗白血病、黑色素瘤、淋巴瘤、肺癌、乳腺癌、子宫癌和卵巢癌等。
LL-D49194s，是一类具有蒽醌骨架的聚酮类化合物，含氧三元螺环是其结构中的活性单元，能抑制DNA和RNA的合成。早期在对LL-D49194 α1分离时，还得到了其余LL-D49194 α1类似物，包括LL-D49194 β1、LL-D49194 β2等，尤以LL-D49194α1产量最高，活性最好，早期药学研究也多以LL-D49194 α1为主要研究目标。LL-D49194类化合物多具有良好的抗革兰氏阳性菌活性，癌细胞抑制能力与顺铂相当或更好，相对细胞毒性也更低。
大多数蒽环类抗生素主要是边链糖基结构存在差别，而针对蒽环骨架的改造研究则较少。为了提高LL-D49194 α1的抗肿瘤活性以及其他特性，本领域一直在尝试改造蒽环骨架从而得到LL-D49194 α1的类似物或衍生物，然而迄今为止尚未开发出具有更高活性等优点的LL-D49194 α1类似物。因此，本领域迫切需要开发各类新型的LL-D49194 α1类似物。
发明内容
本发明的目的是提供一类具有更高活性的新型LL-D49194 α1类似物。
本发明的另一目的是提供所述的LL-D49194 α1类似物的制法和应用。
在本发明的第一方面，提供了一种新型LL-D49194 α1类似物，或其药学上可接受的盐，其特征在于，所述化合物的结构如式I所示：
Figure PCTCN2020076478-appb-000001
式I中，R 1选自：H、羟基、卤素、C 1-4烷基、C 1-4烷氧基；
R 2选自：H、羟基、C 1-4烷氧基、
Figure PCTCN2020076478-appb-000002
R 3选自下组：H、C 1-4烷基。
在另一优选例中，所述R 1选自：H、羟基、C 1-4烷氧基；所述R 2选自：H、羟基、
Figure PCTCN2020076478-appb-000003
在另一优选例中，所述化合物具有选自下组式Ia、Ib或Ic所示的结构：
Figure PCTCN2020076478-appb-000004
本发明第二方面，提供了一种药物组合物，所述的药物组合物包括：本发明第一方面任一所述的LL-D49194 α1类似物，或其药学上可接受的盐；以及药学上可接受的载体。
在另一优选例中，所述的药物组合物用于治疗肿瘤；较佳地，所述的肿瘤选自下组：白血病、黑色素瘤、淋巴瘤、肺癌、乳腺癌、子宫癌、卵巢癌。
本发明的第三方面，提供了一种可用于产生如本发明第一方面所述的LL-D49194 α1类似物的酒红土褐链霉菌(Streptomyces vinaceusdrappus NRRL15735)突变菌株，其特征在于，所述的菌种中，LL-D49194 α1生物合成基因簇中选自下组的一个基因被失活敲除：细胞色素P450氧化还原酶基因(lldO10)、糖基转移酶基因(lldB3)，或细胞色素P450氧化还原酶基因(lldO2)。
在另一优选例中，所述的突变菌株为lldO10、lldB3或lldO2同框缺失的重组菌株。
本发明第四方面，一种制备如本发明第一方面所述的LL-D49194 α1类似物或其药学上可接受的盐的方法，所述方法包括步骤：
(a)对本发明第三方面所述菌株进行发酵，得到发酵产物；和
(b)从发酵产物中分离出所述的LL-D49194 α1类似物，和任选的步骤(c)
(c)将所述化合物转化为其药学上可接受的盐。
在另一优选例中，所述的步骤(b)包括：
(b1)对发酵产物离心弃上清，得到菌体和HP20吸附树脂混合物；
(b2)用有机溶剂浸泡所述的混合物，离心取上清；
(b3)对所述的上清液经减压蒸馏抽干，获得膏状物；
(b4)对所述的膏状物用预装的200-300目硅胶柱进行柱层析，收集包含目标化合物的流出液，然后用HPLC纯化得到目标产物。
在另一优选例中，所述柱层析的洗脱液选自下组：二氯甲烷：甲醇＝75-50：1(v/v)、二氯甲烷：甲醇＝20-10：1(v/v)、二氯甲烷：甲醇＝30-20：1(v/v)。
在另一优选例中，当所述的突变菌株为lldO10缺失的重组菌株时，所述的LL-D49194 α1类似物为式Ia化合物。
在另一优选例中，当所述的突变菌株为lldB3缺失的重组菌株时，所述的LL-D49194 α1类似物为式Ib化合物。
在另一优选例中，当所述的突变菌株为lldO2缺失的重组菌株时，所述的LL-D49194 α1类似物为式Ic化合物。
在另一优选例中，所述的发酵为液体摇瓶发酵。
在另一优选例中，所述的发酵包括：对所述菌株进行一级培养，得到发酵种子培养液，然后接种至发酵培养基中进行二级培养。
在另一优选例中，所述的接种量为5％-10％，以发酵培养基的体积计。
在另一优选例中，所述的二级培养还包括：菌株进入稳定生长期后，添加2％-3％大孔树脂HP20共培养并进行进一步发酵，从而获得LL-D49194 α1类似物。
在另一优选例中，所述的一级培养在无抗的TSB培养瓶中进行。
本发明的第五方面，提供了一种如本发明第一方面所述的LL-D49194 α1类似物或其药学上可接受的盐作为抗肿瘤药物的应用。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
下列附图用于说明本发明的具体实施方案，而不用于限定由权利要求书所界定的本发明范围。
图1为酒红土褐链霉菌Streptomyces vinaceusdrappus NRRL15735同框敲除突变株的电泳结果。图1i为sDL05030敲除突变株；图1ii中为sDL05021敲除突变株；图1iii中为sDL05027敲除突变株。
图2为LL-D49194 α2的 1H NMR(600MHz，CDCl 3)。
图3为LL-D49194 α2的 13C NMR(600MHz，CDCl 3)。
图4为LL-D49194 α2的COSY(600MHz，CDCl 3)。
图5为LL-D49194 α2的HSQC(600MHz，CDCl 3)。
图6为LL-D49194 α2的HMBC(600MHz，CDCl 3)。
图7为LL-D49194 α2的NOESY(600MHz，CDCl 3)。
图8为LL-D49194 α3的 1H NMR(600MHz，CDCl 3)。
图9为LL-D49194 α3的 13C NMR(600MHz，CDCl 3)。
图10为LL-D49194 α3的COSY(600MHz，CDCl 3)。
图11为LL-D49194 α3的HSQC(600MHz，CDCl 3)。
图12为LL-D49194 α3的HMBC(600MHz，CDCl 3)。
图13为LL-D49194 α3的NOESY(600MHz，CDCl 3)。
图14为LL-D49194 α4的 1H NMR(600MHz，CDCl 3)。
图15为LL-D49194 α4的 13C NMR(600MHz，CDCl 3)。
图16为LL-D49194 α4的COSY(600MHz，CDCl 3)。
图17为LL-D49194 α4的HSQC(600MHz，CDCl 3)。
图18为LL-D49194 α4的HMBC(600MHz，CDCl 3)。
图19为LL-D49194 α4的NOESY(600MHz，CDCl 3)。
图20表示对突变株进行发酵条件优化获得新型LL-D49194 α1类似物的HPLC分析结果，其中，图20i为野生型Streptomyces vinaceusdrappus NRRL15735的发酵结果；图20ii为突变株sDL05030的发酵结果，sDL05030是产生LL-D49194 α2的菌株；图20iii为突变株sDL05021的发酵结果，sDL05021是产生LL-D49194 α3的菌株；图20iv为突变株sDL05027的发酵结果，sDL05027是产生LL-D49194 α4的菌株。
符号说明
式Ia中A、B、C、D、E、F代表环的编号，C环即代表编号为C的环，数字1、2、3、4代表化合物碳骨架的编号顺序，C-2位即代表编号为2的碳原子。
具体实施方法
本发明人通过对LL-D49194 α1生物合成机制进行研究，利用构建基因同框缺失突变株的方法，意外地发现，当对缺失了细胞色素P450氧化还原酶基因(lldO10)、或糖基转移酶基因(lldB3)或细胞色素P450氧化还原酶基因(lldO2)的突变株进行液体发酵时，分别制备得到C环C-2位去羟基化，C-4位还原的LL-D49194 α1类似物LL-D49194 α2、LL-D49194 α3、LL-D49194 α4，通过抗HL-60和B16-F10肿瘤细胞活性实验发现抑制活性提高了2-4倍。在此基础上完成了本发明。
活性成分
如本文所用，术语“本发明化合物”、“本发明的活性成分”、“本发明的类似物”和“本发明的LL-D49194 α1类似物”可以互换使用，都指LL-D49194 α1类似物，如式Ia所示的LL-D49194 α2、Ib所示的LL-D49194 α3和Ic所示LL-D49194α4。
应理解，所述术语还意在包括本发明化合物的各种晶型形式、药学上可接受的盐、水合物或溶剂合物。
在本发明优选的实施方式中，术语“C 1-4烷基”指具有1-4个碳原子的直链或支链烷基，例如甲基、乙基、丙基、异丙基、丁基、异丁基、仲丁基、叔丁基、或类似基团；术语C 1-4烷氧基指甲氧基、乙氧基、丙氧基、异丙基氧基、或类似基团；术语“卤素”指F、Cl、Br和I。
一类优选的LL-D49194 α1类似物具有下式I所示的化学结构式：
Figure PCTCN2020076478-appb-000005
如本文所用，术语“LL-D 49194α2”，其化学结构式如式Ia所示。
如本文所用，术语“LL-D 49194α3”，其化学结构式如式Ib所示。
如本文所用，术语“LL-D 49194α4”，其化学结构式如式Ic所示。
如本文所用，术语“药学上可接受的盐”指本发明中化合物与酸或碱所形成的适合用作药物的盐。药学上可接受的盐包括无机盐和有机盐。
本发明人不仅通过对新型LL-D49194 α1类似物的发酵、分离纯化和结构解析，确证了化合物的结构。此外，还通过肿瘤细胞抑制实验证实了，本发明的LL-D49194 α1类似物比LL-D49194 α1天然产物的肿瘤细胞抑制能力有显著提高。
菌株
如本文所用，术语“本发明出发菌株”指的是酒红土褐链霉菌Streptomyces vinaceusdrappus NRRL15735(购自美国农业研究院(ARS))是从美国农业部微生物保藏中心获取的。酒红土褐链霉菌Streptomyces vinaceusdrappus NRRL15735在Maiese W M.等人的“LL-D49194抗生素,新型抗肿瘤药物家族：分类学，发酵和生物学特性[J].抗生素杂志(The Journal of antibiotics),1990,43(3):253-258.”和US4626503均有记载。应理解，出发菌株不仅包括酒红土褐链霉菌Streptomyces vinaceusdrappus NRRL15735的菌株，还包括其衍生菌株及其他产LL-D49194 α1的菌株。
在本发明的一个优选例中，提供了一种生产本发明LL-D49194 α1类似物的酒红土褐链霉菌Streptomyces vinaceusdrappus NRRL15735的突变菌株，其中，所述的菌株中LL-D49194 α1生物合成基因簇中的细胞色素P450氧化还原酶基因(lldO10)、或糖基转移酶基因(lldB3)或细胞色素P450氧化还原酶基因(lldO2)被失活敲除。
本发明还提供了一种构建可产生LL-D49194 α1类似物突变株的方法，它包括同框敲除质粒的构建，将带筛选抗性的质粒导入野生型菌株，通过质粒上带有的同源臂片段与野生型菌株基因组同源片段发生重组，从而从基因组中替代相应基因，从而获得基因同框敲除或缺失的突变株。缺失LL-D49194 α1生物合成基因簇中的细胞色素P450氧化还原酶基因(lldO10)、或糖基转移酶基因(lldB3)或细胞色素P450氧化还原酶基因(lldO2)的突变株无法正常合成LL-D49194 α1。
在本发明的一个优选例中，提供了一种构建突变株，并发酵突变株制备LL-D49194 α1类似物的方法，其中包括：
1.构建lldO10、lldB3和lldO2同框缺失质粒
lldO10、lldB3和lldO2的基因序列及对应的蛋白序列
Figure PCTCN2020076478-appb-000006
Figure PCTCN2020076478-appb-000007
Figure PCTCN2020076478-appb-000008
Figure PCTCN2020076478-appb-000009
PCR分别扩增用于基因敲除lldO10、lldB3和lldO2基因的左臂和右臂片段，将左、右臂两个片段连入温敏型穿梭质粒pKC1139中(US5,955,319)，获得重组质粒。分别将重组质粒转化至E.coli DH5α中，挑取单克隆菌落扩增培养，然后将验证正确的质粒分别转化至E.coli S17-1中。
2.制备重组菌株
收集野生型的Streptomyces vinaceusdrappus NRRL15735的新鲜孢子，用TES缓冲溶液清洗三次。清洗后的孢子重悬于500uL TES缓冲液中，放置于50℃水浴锅中热激10分钟。热激后的孢子转移至37℃萌发4小时左右，与含有重组质粒的E.coli S17-1在IWL-4培养基平板上按照一定比例混合涂抹，并30℃中培养16小时后用安普霉素覆盖平板。接合子在温箱培养5到7天后长出，挑选单克隆接合子到安普霉素抗性平板上37℃培养2到3天。选取长势良好的接合子于无抗TSB培养溶液中传代8次以上，再于IWL-4培养基平板上划线筛选单菌落。通过安普霉素抗性筛选，选取不具有安普霉素抗性的菌株进行基因型PCR验证以 获得lldO10、lldB3和lldO2同框缺失的重组菌株。
3.优化液体发酵方案
本发明提供了一种针对LL-D49194α1及其类似物产生菌株的培养方案，采用液体摇瓶的方式进行发酵，同时从通氧量、温度、接种量等方面进行优化，采用一级种子发酵方式，先在无抗的TSB培养瓶中培养36小时制备得到发酵种子培养液，再接种5％-10％(以发酵培养基的体积计)至发酵培养基中，在2-3天菌株进行稳定生长期后，添加2％-3％大孔树脂HP20共培养，发酵6天可以稳定的获得LL-D49194 α1及其类似物。
4.活性成分的制备
本发明还提供了一种制备式I化合物的方法，包括步骤：离心弃去上清得到菌体和HP20吸附树脂混合物，2倍体积丙酮浸泡菌体和HP20吸附树脂后，离心取上清，将上清液经减压蒸馏抽干后，获得的膏状物先用预装的200-300目硅胶柱进行粗分，再用HPLC进一步制备纯化，收集包含式I化合物的流出液并抽干，最终得到目标产物。
本发明人不仅通过对式I化合物的大量发酵以及分离纯化确证了化合物的结构，而且通过肿瘤细胞抑制实验证实了本发明的活性产物比LL-D49194 α1的肿瘤细胞抑制能力有显著的提高。
本发明的主要优点包括：
(a)提供了一种具有式I结构的新颖的、具有更高活性等优点的LL-D49194 α1类似物。
(b)以本发明的LL-D49194 α1类似物为基础，有助于制备其他蒽环类抗生素衍生物。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的 条件。除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明中。文中所述的较佳实施方法与材料仅作示范之用，但不能限制本发明的内容。
实施例1.突变菌株sDL05030、sDL05021和sDL05027的构建
1.构建lldO10、lldB3和lldO2基因的缺失质粒
克隆lldO10同框缺失左臂的引物序列如下：
lldO10左臂正向引物(SEQ ID NO:1)
5’-ATAGAATTCACGAGGGTGCGGTCGTCT-3’
lldO10左臂反向引物(SEQ ID NO:2)
5’-ATATCTAGAATCGGCGACCAGGGCCTC-3’
克隆lldO10同框缺失右臂的引物序列如下：
lldO10右臂正向引物(SEQ ID NO:3)
5’-ATATCTAGACAACGCCTGCCGGTCCGGTTC-3’
lldO10右臂反向引物(SEQ ID NO:4)
5’-ATAAAGCTTACGTTCGCGCTGCCCCGGTTC-3’
克隆lldB3同框缺失左臂的引物序列如下：
lldB3左臂正向引物(SEQ ID NO:5)
5’-ATAGAATTCCCTGTTCCAGCTCCGCCTGG-3’
lldB3左臂反向引物(SEQ ID NO:6)
5’-ATATCTAGAACCCTGGAGAGGCTCACCGC-3’
克隆lldB3同框缺失右臂的引物序列如下：
lldB3右臂正向引物(SEQ ID NO:7)
5’-ATATCTAGAGTGAGCCTGGACGGGCCACA-3’
lldB3右臂反向引物(SEQ ID NO:8)
5’-ATAAAGCTTGGAGCAGCCGCCTCATCAAG-3’
克隆lldO2同框缺失左臂的引物序列如下：
lldO2左臂正向引物(SEQ ID NO:9)
5’-ATAGAATTCCGCCGGCGTCTGCGGCCA-3’
lldO2左臂反向引物(SEQ ID NO:10)
5’-ATATCTAGAGGTGTCGACCTCAGTGGCCAT-3’
克隆lldO2同框缺失右臂的引物序列如下：
lldO2右臂正向引物(SEQ ID NO:11)
5’-ATATCTAGACCGGTCCGGATCCGCTGAC-3’
lldO2右臂反向引物(SEQ ID NO:12)
5’-ATAAAGCTTCGTGCCGGACGGCCCCGA-3’
以Streptomyces vinaceusdrappus NRRL15735的总DNA为模板，以DMSO，dNTP，无酶水，正、反向引物，高保真的KOD DNA聚合酶及缓冲液构成PCR反应体系，分别扩增用于lldO10、lldB3和lldO2基因敲除的左臂和右臂片段。将克隆后的左、右臂片段进行凝胶电泳分离，切胶回收纯化，分别加入限制性内切酶EcoRI和XbaI以及XbaI和HindIII消化回收片段，将其连入用限制性内切酶EcoRI和HindIII酶切处理过的载体质粒pKC1139中，随后将连接体系转化至E.coli DH5α中，挑取单克隆菌落于LB培养试管(含有安普霉素抗生素)中过夜振荡培养。提取质粒经过酶切验证后送测序进一步验证。将验证正确的质粒转化至E.coli S17-1中。
2.重组菌株sDL05030、sDL05021和sDL05027的获得
收集野生型的Streptomyces vinaceusdrappus NRRL15735的新鲜孢子，用TES缓冲溶液清洗三次。清洗后的孢子重悬于500uL TES缓冲液中，放置于50℃水浴锅中热激10分钟。热激后的孢子转移至37℃萌发4小时左右，与含有重组质粒的E.coli S17-1在IWL-4培养基平板上按照一定比例混合涂抹，并于30℃中培养16小时后用安普霉素(50μg/mL)覆盖平板。接合子在温箱培养5到7天后长出，挑选单克隆接合子到安普霉素抗性平板上37℃培养2到3天。选取长势良好的接合子于无抗性TSB培养溶液中传代8次以上，再于平板上划线筛选单菌落。通过安普霉素抗性筛选，分别选取不具有安普霉素抗性的菌株进行基因型PCR验证以获得lldO10、lldB3和lldO2基因同框缺失的重组菌株sDL05030、sDL05021和sDL05027。实验结果如图1所示。
图1i中为sDL05030敲除突变株的验证结果，从图1i的电泳图可以看出，sDL05030敲除突变株中sDL05030基因缺失了大约1.15kb，说明本实施例中成功地构建了sDL05030敲除突变株。
图1ii中为sDL05021敲除突变株的验证结果，从图1ii的电泳图可以看出，sDL05021敲除突变株中sDL05021基因缺失了大约1.16kb，说明本实施例中成功地构建了sDL05021敲除突变株。
图1iii中为sDL05027敲除突变株的验证结果，从图1iii的电泳图可以看出，sDL05027敲除突变株中sDL05027基因缺失了大约1.18kb，说明本实施例中成功地构建了sDL05027敲除突变株。
实施例2.LL-D49194 α1类似物的发酵、检测、分离纯化和结构鉴定
分别将突变株sDL05030、sDL05021和sDL05027接种于100mL种子培养基中振荡培养(含TSB 30g/L)，转速220rpm，温度30℃培养36小时。将5-10mL种子摇瓶菌液接种于100mL发酵培养基中(含可溶性淀粉60g/L，葡萄糖10g/L，酵母提取物10g/L，氯化钠3g/L，磷酸氢二钾1g/L，七水硫酸镁1g/L，碳酸钙2g/L,微量盐溶液0.1mL(1000倍储存液：五水硫酸铜70g/L，七水硫酸亚铁10g/L，四水氯化锰8g/L，七水硫酸锌2g/L，七水氯化钴0.06g/L)，pH＝7.3±0.2)，30℃，220rpm培养，接种第2-3天后加入HP20树脂(3g/100mL)，继续培养至接种后第6天后处理发酵液。
分别对sDL05030、sDL05021和sDL05027发酵液离心收集沉淀，用2倍体积丙酮分两次浸泡沉淀，旋转蒸发仪旋去丙酮，用乙酸乙酯萃取四次，将乙酸乙酯经减压蒸馏抽干得到深褐色膏状物。分别对膏状物进行分离，用200-300目硅胶预装的正相柱进行粗分，梯度洗脱条件见表1。
表1
Figure PCTCN2020076478-appb-000010
Figure PCTCN2020076478-appb-000011
LL-D49194 α1类似物LL-D49194 α2(式Ia所示结构)通过分离sDL05030发酵产物得到，出现在75：1和50：1(二氯甲烷：甲醇)的洗脱部分中。
LL-D49194 α3(式Ib所示结构)通过分离sDL05021发酵产物得到，出现在20：1和10：1(二氯甲烷：甲醇)的洗脱部分中。
LL-D49194 α4(式Ic所示结构)通过分离sDL05027发酵产物得到，出现在30：1和20：1(二氯甲烷：甲醇)的洗脱部分中，收集含有LL-D49194 α2、LL-D49194 α3和LL-D49194 α4的洗脱液并进行减压抽干，溶于3ml甲醇，然后使用HPLC制备化合物纯品。
HPLC的半制备条件为：
仪器：岛津LC-20-AT(日本岛津公司)
检测波长：UV＝400nm
色谱柱：YMC-Pack ODS-AQ，C18column,10×250mm，5μM(日本YMC公司)
流动相：A＝H 2O；B＝CH 3CN
流速：2mL/min
流动相梯度配比见表2。
表2
时间(min) A％ B％
0 80 20
5 80 20
20 35 65
21 10 90
24 10 90
26 80 20
30 80 20
按上述的HPLC的洗脱条件，分别收集LL-D49194 α2、LL-D49194 α3和LL-D49194 α4的流出液，最终得到目标产物LL-D49194 α2、LL-D49194 α3和LL-D49194 α4。对目标产物LL-D49194 α2进行鉴定，核磁归属结果见表3(CD 3Cl,600MHz)；对目标产物LL-D49194 α3进行鉴定，核磁归属结果见表4(CD 3Cl,600MHz)；对目标产物LL-D49194 α4进行鉴定，核磁归属结果见表5(CD 3Cl,600MHz)。
表3
Figure PCTCN2020076478-appb-000012
HR-ESI-MS(m/z)：[M+H] +测量值为457.1129(C 23H 21O 10计算值为457.1129)。
图2、图3、图4、图5、图6和图7分别为LL-D49194 α2(式Ia所示结构)的 1H NMR(CD 3Cl,600MHz)、 13C NMR(CD 3Cl,600MHz)、COSY(CD 3Cl,600MHz)、HSQC(CD 3Cl,600MHz)、HMBC(CD 3Cl,600MHz)和NOESY(CD 3OD, 600MHz)图谱。结构鉴定表明，本发明人成功制备了LL-D49194 α2(如Ia所示结构)。
表4
Figure PCTCN2020076478-appb-000013
HR-ESI-MS(m/z)：[M+H] +测量值为487.1235(C 24H 23O 11计算值为487.1235)。
图8、图9、图10、图11、图12和图13分别为LL-D49194 α3(式Ib所示结构)的 1H NMR(CD 3Cl,600MHz)、 13C NMR(CD 3Cl,600MHz)、COSY(CD 3Cl,600MHz)、HSQC(CD 3Cl,600MHz)、HMBC(CD 3Cl,600MHz)和NOESY(CD 3OD,600MHz)图谱。结构鉴定表明，本发明人成功制备了LL-D49194 α3(如Ib所示结构)。
表5
Figure PCTCN2020076478-appb-000014
Figure PCTCN2020076478-appb-000015
HR-ESI-MS(m/z)：[M+Na] +测量值为811.2786(C 39H 48O 17计算值为811.2784)。
图14、图15、图16、图17、图18和图19分别为LL-D49194 α4(式Ic所示结构)的 1H NMR(CD 3Cl,600MHz)、 13C NMR(CD 3Cl,600MHz)、COSY(CD 3Cl,600MHz)、HSQC(CD 3Cl,600MHz)、HMBC(CD 3Cl,600MHz)和NOESY(CD 3OD,600MHz)图谱。结构鉴定表明，本发明人成功制备了LL-D49194 α4(如Ic所示结构)。
实施例3.出发菌株和改造菌株的发酵产物HPLC分析
HPLC(图20)的检测条件为：
仪器：Dionex Ultimate 3000系统
检测波长：UV＝400nm
色谱柱：Acclaim PolarAdvantage II,C18,
Figure PCTCN2020076478-appb-000016
4.6×250mm，5μm,(ThermoScientific公司)；
流动相：A＝H 2O(含1‰HCOOH)；B＝CH 3CN(含1‰HCOOH)
流速：1mL/min
流动相梯度配比见表6。
表6
时间(min) A％ B％
0 10 90
5 10 90
24 90 10
26 90 10
27 10 90
31 10 90
发酵产物分别用HPLC进行分析，结果如下：
图20表示对突变株进行发酵条件优化获得新型LL-D49194 α1类似物的HPLC分析结果，其中，图20i为野生型Streptomyces vinaceusdrappus NRRL15735的发酵结果；图20ii为突变株sDL05030的HPLC分析，发酵结果表明突变株sDL05030可以产生LL-D49194 α2(式Ia所示)；图20iii为突变株sDL05021的HPLC分析，发酵结果表明突变株sDL05021可以产生LL-D49194 α3(式Ib所示)；图20iv为突变株sDL05027的HPLC分析，发酵结果表明突变株sDL05027可以产生LL-D49194 α4(式Ic所示)。结果表明，野生型菌株能够正常产生LL-D49194 α1，突变株不具有合成产生LL-D49194 α1的能力，但具有产生LL-D49194 α1类似物的能力。
实施例4.LL-D49194 α1类似物的抗肿瘤活性
对LL-D49194 α1及其类似物进行抗肿瘤活性的测定，方法如下：将细胞调整浓度至3×10 4个/mL，100μL/well接种于96孔平底透明细胞培养板中，24小时后分别加入含有不同浓度的药物作用一段适当的时间(例如：24或48小时)。向每孔加入10μl的CCK-8试剂作用2小时后，用酶标仪测定在450nm处的吸光度OD值(参考波长630nm)。空白组为无细胞的培养基，对照组为加入与药物同体积的DMSO，计算细胞存活率＝(实验组OD值-空白组OD值)/(对照组OD值-空白组OD值)。之后计算出化合物对测定细胞的半抑制浓度IC 50。
选取HL-60和B16-F10细胞进行肿瘤细胞抑制活性实验，半抑制浓度(IC 50)测试结果显示，LL-D49194 α1类似物的IC 50值比对应的原天然产物LL-D49194 α1降低了2-4倍(表7)，这表明，C环C-2位去羟基化，C-4位还原的LL-D49194 α1类似物具有更高的抗肿瘤活性，其抑制HL-60和B16-F10细胞的活性比对应的原LL-D49194 α1天然产物的活性提高2-4倍。
表7
化合物 IC50(HL60，nM) IC50(B16-F10，nM)
LL-D49194 α1 5.45±0.01 23.88±0.09
LL-D49194 α2 2.84±0.01 10.05±0.08
LL-D49194 α3 3.93±0.01 6.35±0.01
LL-D49194 α4 6.32±0.01 8.19±0.02
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种新型LL-D49194 α1类似物，或其药学上可接受的盐，其特征在于，所述化合物的结构如式I所示：
    Figure PCTCN2020076478-appb-100001
    式I中，R 1选自：H、羟基、卤素、C 1-4烷基、C 1-4烷氧基；
    R 2选自：H、羟基、
    Figure PCTCN2020076478-appb-100002
    R 3选自下组：H、C 1-4烷基。
  2. 如权利要求1所述的LL-D49194α1类似物，或其药学上可接受的盐，其中，所述R 1选自：H、羟基、C 1-4烷氧基；所述R 2选自：H、羟基、
    Figure PCTCN2020076478-appb-100003
  3. 如权利要求1-2任一所述的LL-D49194α1类似物，或其药学上可接受的盐，其中，所述化合物具有选自下组式Ia、Ib或Ic所示的结构：
    Figure PCTCN2020076478-appb-100004
    Figure PCTCN2020076478-appb-100005
  4. 一种药物组合物，其特征在于，所述的药物组合物包括：权利要求1-3任一所述的LL-D49194 α1类似物，或其药学上可接受的盐；以及药学上可接受的载体。
  5. 一种可用于产生如权利要求1-3任一项所述的LL-D49194 α1类似物的酒红土褐链霉菌NRRL15735(Streptomyces vinaceusdrappus NRRL15735)突变菌株，其特征在于，所述的菌种中，LL-D49194 α1生物合成基因簇中选自下组的一个基因被失活敲除：细胞色素P450氧化还原酶基因(lldO10)、糖基转移酶基因(lldB3)，或细胞色素P450氧化还原酶基因(lldO2)。
  6. 一种制备如权利要求1-3任一所述的LL-D49194 α1类似物或其药学上可接受的盐的方法，其特征在于，包括步骤：
    (a)对权利要求5所述菌株进行发酵，得到发酵产物；和
    (b)从发酵产物中分离出所述的LL-D49194 α1类似物，和任选的步骤(c)
    (c)将所述化合物转化为其药学上可接受的盐。
  7. 如权利要求6所述的方法，其特征在于，所述的发酵为液体摇瓶发酵。
  8. 如权利要求6所述的方法，其特征在于，所述的发酵包括：对所述菌株进行一级培养，得到发酵种子培养液，然后接种至发酵培养基中进行二级培养。
  9. 如权利要求8所述的方法，其特征在于，所述的二级培养还包括：菌株进入稳定生长期后，添加2％-3％大孔树脂HP20共培养并进行进一步发酵，从而获得LL-D49194 α1类似物。
  10. 权利要求1-3所述的LL-D49194 α1类似物或其药学上可接受的盐作为抗肿瘤药物的应用。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023096904A3 (en) * 2021-11-24 2023-07-06 President And Fellows Of Harvard College C-16 modified trioxacarcins, antibody drug conjugates, and uses thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4626503A (en) * 1984-04-04 1986-12-02 American Cyanamid Company Antitumor agents LL-D49194α1, LL-D49194β1, LL-D49194β2, LL-D49194β3, LL-D49194γ, LL-D49194δ, LL-D49194ε, LL-D49194ξ, LL-D49194η, LL-D49194ω1, LL-D49194ω2, and LL-D49194ω3
WO2014082065A1 (en) * 2012-11-26 2014-05-30 President And Fellows Of Harvard College Trioxacarcins, trioxacarcin-antibody conjugates, and uses thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4626503A (en) * 1984-04-04 1986-12-02 American Cyanamid Company Antitumor agents LL-D49194α1, LL-D49194β1, LL-D49194β2, LL-D49194β3, LL-D49194γ, LL-D49194δ, LL-D49194ε, LL-D49194ξ, LL-D49194η, LL-D49194ω1, LL-D49194ω2, and LL-D49194ω3
WO2014082065A1 (en) * 2012-11-26 2014-05-30 President And Fellows Of Harvard College Trioxacarcins, trioxacarcin-antibody conjugates, and uses thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZHANG, MEI ET AL.: "Biosynthesis of trioxacarcin revealing a different starter unit and complex tailoring steps for type II polyketide synthase", CHEMICAL SCIENCE, vol. 6, no. 6, 7 April 2015 (2015-04-07), pages 3440 - 3447, XP055736183 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023096904A3 (en) * 2021-11-24 2023-07-06 President And Fellows Of Harvard College C-16 modified trioxacarcins, antibody drug conjugates, and uses thereof

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