WO2020177568A1 - Nouvel analogue de ll-d49194 α1, procédé de préparation correspondant et utilisation associée - Google Patents

Nouvel analogue de ll-d49194 α1, procédé de préparation correspondant et utilisation associée 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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fermentation
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

La présente invention concerne un analogue de LL-D49194α1, un procédé de préparation correspondant et une utilisation associée. Plus particulièrement, la présente invention concerne un analogue de LL-D49194α1 déglycosylé et déméthylé. L'analogue de LL-D49194α1 obtenu présente une meilleure activité antitumorale que LL-D49194α1.
PCT/CN2020/076478 2019-03-01 2020-02-24 Nouvel analogue de ll-d49194 α1, procédé de préparation correspondant et utilisation associée Ceased WO2020177568A1 (fr)

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CN201910156416.5A CN109836433B (zh) 2019-03-01 2019-03-01 新型LL-D49194α1类似物，及其制备方法和应用

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WO2023096904A3 (fr) * 2021-11-24 2023-07-06 President And Fellows Of Harvard College Trioxacarcines modifiées en c-16, conjugués anticorps-médicament, et leurs utilisations

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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 (fr) * 2012-11-26 2014-05-30 President And Fellows Of Harvard College Trioxacarcines, conjugués trioxacarcine-anticorps, et leurs utilisations

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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 (fr) * 2012-11-26 2014-05-30 President And Fellows Of Harvard College Trioxacarcines, conjugués trioxacarcine-anticorps, et leurs utilisations

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WO2023096904A3 (fr) * 2021-11-24 2023-07-06 President And Fellows Of Harvard College Trioxacarcines modifiées en c-16, conjugués anticorps-médicament, et leurs utilisations

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