CN116041370A - Staurosporine compound and preparation method and application thereof - Google Patents
Staurosporine compound and preparation method and application thereof Download PDFInfo
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- CN116041370A CN116041370A CN202310019142.1A CN202310019142A CN116041370A CN 116041370 A CN116041370 A CN 116041370A CN 202310019142 A CN202310019142 A CN 202310019142A CN 116041370 A CN116041370 A CN 116041370A
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- HKSZLNNOFSGOKW-UHFFFAOYSA-N ent-staurosporine Natural products C12=C3N4C5=CC=CC=C5C3=C3CNC(=O)C3=C2C2=CC=CC=C2N1C1CC(NC)C(OC)C4(C)O1 HKSZLNNOFSGOKW-UHFFFAOYSA-N 0.000 title claims abstract description 25
- CGPUWJWCVCFERF-UHFFFAOYSA-N staurosporine Natural products C12=C3N4C5=CC=CC=C5C3=C3CNC(=O)C3=C2C2=CC=CC=C2N1C1CC(NC)C(OC)C4(OC)O1 CGPUWJWCVCFERF-UHFFFAOYSA-N 0.000 title claims abstract description 25
- 238000002360 preparation method Methods 0.000 title claims abstract description 22
- -1 Staurosporine compound Chemical class 0.000 title claims abstract description 21
- 150000001875 compounds Chemical class 0.000 claims abstract description 32
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 24
- 238000012258 culturing Methods 0.000 claims abstract description 13
- 239000000287 crude extract Substances 0.000 claims abstract description 11
- 238000000855 fermentation Methods 0.000 claims abstract description 10
- 230000004151 fermentation Effects 0.000 claims abstract description 10
- 238000004440 column chromatography Methods 0.000 claims abstract description 8
- 238000004321 preservation Methods 0.000 claims abstract description 5
- 238000010828 elution Methods 0.000 claims description 20
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 15
- 239000001963 growth medium Substances 0.000 claims description 12
- 239000003814 drug Substances 0.000 claims description 11
- 229940079593 drug Drugs 0.000 claims description 11
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- 230000001580 bacterial effect Effects 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 9
- 206010006187 Breast cancer Diseases 0.000 claims description 8
- 208000026310 Breast neoplasm Diseases 0.000 claims description 8
- 239000003480 eluent Substances 0.000 claims description 6
- SRCZQMGIVIYBBJ-UHFFFAOYSA-N ethoxyethane;ethyl acetate Chemical compound CCOCC.CCOC(C)=O SRCZQMGIVIYBBJ-UHFFFAOYSA-N 0.000 claims description 6
- 239000003208 petroleum Substances 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 5
- 238000004007 reversed phase HPLC Methods 0.000 claims description 5
- 238000000926 separation method Methods 0.000 claims description 5
- 239000007787 solid Substances 0.000 claims description 4
- 239000001888 Peptone Substances 0.000 claims description 3
- 108010080698 Peptones Proteins 0.000 claims description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 3
- 229920002472 Starch Polymers 0.000 claims description 3
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 claims description 3
- 230000003213 activating effect Effects 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
- 238000001704 evaporation Methods 0.000 claims description 3
- 238000000605 extraction Methods 0.000 claims description 3
- 239000002054 inoculum Substances 0.000 claims description 3
- 238000002386 leaching Methods 0.000 claims description 3
- 238000009630 liquid culture Methods 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 239000012046 mixed solvent Substances 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- 239000000741 silica gel Substances 0.000 claims description 3
- 229910002027 silica gel Inorganic materials 0.000 claims description 3
- 235000002639 sodium chloride Nutrition 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 235000019698 starch Nutrition 0.000 claims description 3
- 239000008107 starch Substances 0.000 claims description 3
- 230000001954 sterilising effect Effects 0.000 claims description 3
- 238000005303 weighing Methods 0.000 claims description 3
- 229940041514 candida albicans extract Drugs 0.000 claims description 2
- 239000003112 inhibitor Substances 0.000 claims description 2
- 239000002609 medium Substances 0.000 claims description 2
- 239000012138 yeast extract Substances 0.000 claims description 2
- 238000000034 method Methods 0.000 claims 5
- 241000186361 Actinobacteria <class> Species 0.000 abstract description 3
- 238000010898 silica gel chromatography Methods 0.000 abstract description 2
- 238000004128 high performance liquid chromatography Methods 0.000 abstract 1
- 238000002791 soaking Methods 0.000 abstract 1
- HKSZLNNOFSGOKW-FYTWVXJKSA-N staurosporine Chemical class C12=C3N4C5=CC=CC=C5C3=C3CNC(=O)C3=C2C2=CC=CC=C2N1[C@H]1C[C@@H](NC)[C@@H](OC)[C@]4(C)O1 HKSZLNNOFSGOKW-FYTWVXJKSA-N 0.000 description 12
- 238000005481 NMR spectroscopy Methods 0.000 description 10
- 210000004027 cell Anatomy 0.000 description 9
- 229940125904 compound 1 Drugs 0.000 description 8
- 238000001142 circular dichroism spectrum Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 238000001228 spectrum Methods 0.000 description 5
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 230000000259 anti-tumor effect Effects 0.000 description 3
- 230000002596 correlated effect Effects 0.000 description 3
- 238000001819 mass spectrum Methods 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 239000004215 Carbon black (E152) Substances 0.000 description 2
- 230000001093 anti-cancer Effects 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 238000005100 correlation spectroscopy Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 229930195733 hydrocarbon Natural products 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 238000001460 carbon-13 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 239000002024 ethyl acetate extract Substances 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 125000003147 glycosyl group Chemical group 0.000 description 1
- 238000001052 heteronuclear multiple bond coherence spectrum Methods 0.000 description 1
- 238000000990 heteronuclear single quantum coherence spectrum Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 125000005462 imide group Chemical group 0.000 description 1
- VVVPGLRKXQSQSZ-UHFFFAOYSA-N indolo[3,2-c]carbazole Chemical compound C1=CC=CC2=NC3=C4C5=CC=CC=C5N=C4C=CC3=C21 VVVPGLRKXQSQSZ-UHFFFAOYSA-N 0.000 description 1
- 229960005544 indolocarbazole Drugs 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000000749 insecticidal effect Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 125000001434 methanylylidene group Chemical group [H]C#[*] 0.000 description 1
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 239000003592 new natural product Substances 0.000 description 1
- 238000000238 one-dimensional nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
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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
- C07D498/22—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 four or more hetero rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/14—Drugs for genital or sexual disorders; Contraceptives for lactation disorders, e.g. galactorrhoea
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- 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
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- 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
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention discloses a staurosporine compound and a preparation method and application thereof, which are characterized in that the structural formula of the compound is shown as I, the preparation method comprises the steps of fermenting and culturing actinomycetes with the preservation number of CCTCC No. M2020953 to obtain a fermentation product of the staurosporine compound, soaking and extracting the fermentation product with ethyl acetate to obtain a crude extract, and separating and purifying the crude extract by normal phase silica gel column chromatography, reverse medium pressure column chromatography and reverse phase semi-preparation high performance liquid chromatography on the basis of the crude extract.
Description
Technical Field
The invention relates to staurosporine compounds, in particular to staurosporine compounds extracted from marine actinomycetes, and a preparation method and application thereof.
Background
Staurosporine antibiotics are a group of natural products consisting of six-membered chain amine compounds with indolocarbazole as a core. It was originally isolated from Streptomyces soil by Japanese Korea and his colleagues in 1977, and consistent with its high structural specificity, stellate sporins have a broad spectrum of biological activities including anti-tumor, antiviral, insecticidal, antibacterial and anticancer. Staurosporine compounds have attracted attention from chemical or biological synthesis researchers due to their remarkable activity and diverse structures.
The present inventors have found that for marine actinomycetesSteptomyces sp.4-7 in the chemical investigation of ethyl acetate extracts under fermentation in medium A, a new natural product of the staurosporine type was found. At present, the chemical structure of the compound and the activity of the drug-resistant breast cancer cell strain are not reported, so that the related drugs are not yet available in the market.
Disclosure of Invention
The invention aims to solve the technical problem of providing a novel staurosporine compound with an inhibiting effect on drug-resistant breast cancer cell strains, and a preparation method and application thereof.
The technical scheme adopted for solving the technical problems is as follows:
1. a staurosporine compound, the structural formula of which is shown as (I):
2. a preparation method of staurosporine compounds comprises the following steps:
(1) Fermentation production
The preservation number is CCTCC NO: m2020953 Marine actinomycetesSteptomyces sp, 4-7) streaking on a flat plate of a solid culture medium of Gaoshi No. 1, culturing and activating the solid culture medium in an incubator at 28 ℃ for 7 days, picking single bacterial colonies, inoculating the single bacterial colonies in a liquid culture medium of Gaoshi No. 1, culturing and sterilizing the single bacterial colonies on a shaking table at the temperature of 28 ℃ and the rotating speed of 180 rpm/min, collecting seed liquid after culturing for 3 days, inoculating the seed liquid into a culture medium A according to the inoculum size of 10% by volume, and culturing the seed liquid at the temperature of 28 ℃ for 11 days to obtain a fermentation product;
(2) Extraction of extractum
Adding equal amount of ethyl acetate into the fermented product obtained in the step (1), repeatedly extracting for 3 times, and then evaporating ethyl acetate leaching liquor under reduced pressure to obtain crude extract;
(3) Separation and preparation of compounds
The volume ratio of the crude extract obtained in the step (2) is 1:1, after dissolving a dichloromethane and methanol mixed solvent, adding 200-300 meshes of silica gel, mixing, performing positive phase column chromatography, performing gradient elution by using petroleum ether-ethyl acetate solution with volume ratio of (100:1) - (0:1) as an eluent, collecting elution fractions, arranging according to the polarity of the fractions from small to large, and combining to obtain 11 components; performing reverse phase pressure column chromatography gradient elution on the 10 th component, adopting acetonitrile-water with the volume ratio of 30-100% as an eluent, collecting elution fractions, arranging the elution fractions according to the polarity of the fractions from large to small, and combining to obtain 10 components; separating and purifying the 7 th component by semi-preparative reverse phase high performance liquid chromatography, and mixing acetonitrile and water at a volume ratio of 43:57 to obtain a compound with a structure shown as (I)
Further, the preparation method of the A culture medium in the step (1) comprises the following steps: weighing 20 g soluble starch, 6 g peptone, 2 gYeast extract, 30 g sea salt, 100 mg KBr,40 mg Fe 2 (SO 4 ) 3 ·4H 2 O and 1 g CaCO 3 Adding into 1000 mL distilled water, and autoclaving at 121deg.C for 20 min.
Further, the elution gradient volume ratio of the petroleum ether-ethyl acetate solution in the step (3) is sequentially 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1 and 0:1.
And (3) performing gradient elution on the reversed-phase medium-pressure column by using acetonitrile with the volume ratio of 30-100% for 150 min.
Further, the flow rate of the compound separation preparation of the semi-preparative reverse phase high performance liquid chromatography described in the step (3) was 2.0 mL/min.
3. The application of the staurosporine compound in preparing drug-resistant breast cancer cell line inhibitors.
Compared with the prior art, the invention has the advantages that: the invention relates to a staurosporine compound and a preparation method and application thereof, which are characterized in that fermentation products of staurosporine are obtained through microbial fermentation culture, then the fermentation products are soaked and extracted by ethyl acetate to obtain crude extract, and the crude extract is separated and purified by medium-pressure forward silica gel column chromatography, medium-pressure reverse column chromatography and reversed-phase semi-preparation high-efficiency liquid chromatography. Biological activity evaluation shows that the compound 1 has effective inhibition effect on drug-resistant and drug-resistant breast cancer cell lines and IC 50 A value of 4.47μM。
The marine actinomycetes are preparedSteptomyces sp. 4-7), the preservation number is CCTCC NO: m2020953, deposited with the China center for type culture Collection, with a deposition address of university of Wuhan, china, 12 months, 21 days of 2020.
Drawings
FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of a compound of the present invention;
FIG. 2 is a nuclear magnetic resonance carbon spectrum of a compound of the present invention
FIG. 3 is a nuclear magnetic resonance DEPT-135 spectrum of a compound of the present invention;
FIG. 4 is a nuclear magnetic resonance COSY spectrum of a compound of the present invention;
FIG. 5 is a nuclear magnetic resonance HSQC spectrum of a compound of the present invention;
FIG. 6 is a nuclear magnetic resonance HMBC spectra of a compound of the present invention;
FIG. 7 is a nuclear magnetic resonance NOESY spectrum of a compound of the present invention;
FIG. 8 is a mass spectrum of a compound of the present invention;
FIG. 9 is a CD spectrum of a compound of the invention;
FIG. 10 is a CD spectrum of the known compound staurosporine M1.
Detailed Description
The invention is described in further detail below with reference to the embodiments of the drawings.
Example 1
The structural formula of the staurosporine compound is shown as (I):
example 2
A preparation method of staurosporine compounds comprises the following steps:
(1) Fermentation production
The preservation number is CCTCC NO: m2020953 Marine actinomycetesSteptomyces sp.4-7) streaking on a plate of a solid culture medium of Gaoshi No. 1, culturing and activating the plate in an incubator at 28 ℃ for 7 days, picking single bacterial colonies, inoculating the single bacterial colonies into a liquid culture medium of Gaoshi No. 1, culturing and sterilizing the single bacterial colonies on a shaking table at the temperature of 28 ℃ and the rotating speed of 180 rpm/min, collecting seed liquid after culturing for 3 days, inoculating the seed liquid into a culture medium A according to the inoculum size with the volume ratio of 10%, and culturing the seed liquid at the temperature of 28 ℃ for 11 days to obtain a ferment; the preparation method of the culture medium A comprises the following steps: weighing 20 g soluble starch, 6 g peptone, 2 g yeast extract, 30 g sea salt, 100 mg KBr,40 mg Fe 2 (SO 4 ) 3 ·4H 2 O and 1 g CaCO 3 Adding into 1000 mL distilled water, and autoclaving at 121deg.C for 20 min;
(2) Extraction of extractum
Adding equal amount of ethyl acetate into the fermented product obtained in the step (1), repeatedly extracting for 3 times, and then evaporating ethyl acetate leaching liquor under reduced pressure to obtain crude extract;
(3) Separation and preparation of compounds
The volume ratio of the crude extract obtained in the step (2) is 1:1, after dissolving a dichloromethane and methanol mixed solvent, adding 200-300 meshes of silica gel, mixing, performing positive phase column chromatography, performing gradient elution by using petroleum ether-ethyl acetate solution with volume ratio of (100:1) - (0:1) as an eluent, collecting elution fractions, arranging according to the polarity of the fractions from small to large, and combining to obtain 11 components; performing reverse phase pressure column chromatography gradient elution on the 10 th component, adopting acetonitrile-water with the volume ratio of 30-100% as an eluent, eluting for 150 min, collecting elution fractions, arranging the fractions according to the polarity from large to small, and combining to obtain 10 components; separating and purifying the 7 th component by semi-preparative reverse phase high performance liquid chromatography, taking acetonitrile and water mixed solution as mobile phase according to the volume ratio of 43:57, wherein the flow rate is 2.0 mL/min, obtaining the compound, the structure is shown as (I), the elution gradient volume ratio of the petroleum ether-ethyl acetate solution is sequentially 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1 and 0:1,
example 3
Structure identification and nuclear magnetic signal attribution of compound
The compound is yellow powder, and high resolution mass spectrum (HR-ESI-MS) in positive ion mode gives its excimer ion peak M/z 525.2139 [ M+H ]] + Is combined with 13 C NMR spectrum to confirm its molecular formula as C 30 H 29 N 4 O 5 . The compound is 1 H and 13 the C NMR data are shown in FIGS. 1 to 7 and Table 1.
TABLE 1D NMR data for Compound 1 (DMSO-d 6 )
Note 1: s-Shan Chongfeng, d-doublet, t-triplet, q-quartet and m-multiplet;
and (2) injection: 1 h was obtained at 600 MHz NMR; 13 c was obtained by NMR at 150 MHz.
FIGS. 3-7 show the major two-dimensional correlation of the compounds of the present invention, from FIG. 3, the compounds contain 30 carbons, 13 quaternary carbons, 4 methyl carbons, 2 methylene carbons, 11 methine carbons, from FIG. 4, the compounds H-8/H-9/H-10/H-11, H-1/H-2/H-3/H-4 are correlated with the presence of COSY, from FIG. 5, the compounds hydrocarbon are directly correlated, from FIG. 6, the compounds hydrocarbon are remotely correlated, and the carbons can be linked together; FIG. 7 is a NOE-related, H of a compound of the present invention 3 There is a NOESY correlation between 2 'and H-4', indicating that they are on the same side of the glycosyl. Coupling constant [ ] 3 J H-3′ ， H-4′ =4.27 Hz) indicates that H-3 'and H-4' are in the same orientation. FIG. 8 is a mass spectrum of a compound of the present invention showing that the molecular weight of the compound of the present invention is 525.2139 and the molecular formula is C 30 H 29 N 4 O 5 FIGS. 9 and 10 are, respectively, the CD spectra of the compound of the invention and the CD spectra of the known compound staurosporine M1; from this, it was found that the CD spectrum of Compound 1 was almost identical to that of staurosporine M1. The absolute configuration of compound 1 was shown to be consistent with staurosporine M1, ascribed to 2's,3' r,4'r,6' r.
Example 4
Activity of staurosporine compounds
Staurosporine compound Activity and application as described in example 1
(1) Experimental sample
Preparing a tested sample solution: the test sample is the pure product of the compound 1 separated and purified in the example 1, a proper amount of the sample is precisely weighed, and a solution with the required concentration is prepared by using DMSO for testing the anti-tumor activity. The tumor cells used in the experiment are drug-resistant breast cancer cell lines;
(2) Experimental method
Drug resistant breast cancer cells were seeded in 96-well plates, attached overnight, and treated with compound 1 at the indicated concentrations for 72h. Cell viability was determined using a cell counting kit. Absorbance was then measured at a wavelength of 450 nm on a miniature flat panel reader;
(3) Experimental results
In the antitumor test, compound 1 was found to be at 4.47μHas obvious cytotoxicity to drug-resistant breast cancer cell lines at the time of M. IC of Compound 1, which exhibits stronger anticancer activity in drug-resistant and parent cells than in the absence of methyl group at the imide group at the 4' position 50 = 4.47 μM。
The above description is not intended to limit the invention, nor is the invention limited to the examples described above. Variations, modifications, additions, or substitutions will occur to those skilled in the art and are therefore within the spirit and scope of the invention.
Claims (7)
1. The staurosporine compound is characterized in that the structural formula of the staurosporine compound is shown as (I):
（I）。
2. a process for the preparation of staurosporine-like compounds according to claim 1, characterized by the steps of:
(1) Fermentation production
The preservation number is CCTCC NO: m2020953 Marine actinomycetesSteptomycessp. 4-7) streaking on a plate of a solid culture medium of Gaoshi No. 1, culturing and activating the plate in an incubator at 28 ℃ for 7 days, picking single bacterial colonies, inoculating the single bacterial colonies into a liquid culture medium of Gaoshi No. 1, culturing and sterilizing the single bacterial colonies on a shaking table at the temperature of 28 ℃ and the rotating speed of 180 rpm/min, collecting seed liquid after culturing for 3 days, inoculating the seed liquid into a culture medium A according to the inoculum size with the volume ratio of 10%, and culturing the seed liquid at the temperature of 28 ℃ for 11 days to obtain a ferment;
(2) Extraction of extractum
Adding equal amount of ethyl acetate into the fermented product obtained in the step (1), repeatedly extracting for 3 times, and then evaporating ethyl acetate leaching liquor under reduced pressure to obtain crude extract;
(3) Separation and preparation of compounds
The volume ratio of the crude extract obtained in the step (2) is 1:1, after dissolving a dichloromethane and methanol mixed solvent, adding 200-300 meshes of silica gel, mixing, performing positive phase column chromatography, performing gradient elution by using petroleum ether-ethyl acetate solution with volume ratio of (100:1) - (0:1) as an eluent, collecting elution fractions, arranging according to the polarity of the fractions from small to large, and combining to obtain 11 components; performing reverse phase pressure column chromatography gradient elution on the 10 th component, adopting acetonitrile-water with the volume ratio of 30-100% as an eluent, collecting elution fractions, arranging the elution fractions according to the polarity of the fractions from large to small, and combining to obtain 10 components; separating and purifying the 7 th component by semi-preparative reverse phase high performance liquid chromatography, and mixing acetonitrile and water at a volume ratio of 43:57 to obtain a compound with a structure shown as (I)
（I）。
3. The method for preparing staurosporine compound according to claim 2, wherein the preparation method of the medium a in the step (1) is as follows: weighing 20 g soluble starch, 6 g peptone, 2 g yeast extract, 30 g sea salt, 100 mg KBr,40 mg Fe 2 (SO 4 ) 3 ·4H 2 O and 1 g CaCO 3 Adding into 1000 mL distilled water, and autoclaving at 121deg.C for 20 min.
4. The method for preparing staurosporine compound according to claim 2, wherein: the elution gradient volume ratio of the petroleum ether-ethyl acetate solution in the step (3) is sequentially 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1 and 0:1.
5. The method for preparing staurosporine compound according to claim 2, wherein: and (3) performing gradient elution on the reversed-phase medium-pressure column by using acetonitrile with the volume ratio of 30-100% and the elution time of 150 min.
6. The method for preparing staurosporine compound according to claim 2, wherein: the flow rate of the compound separation preparation of the semi-preparation reversed-phase high performance liquid chromatography in the step (3) is 2.0 mL/min.
7. Use of a staurosporine compound of claim 1 for the preparation of a drug resistant breast cancer cell line inhibitor.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5093330A (en) * | 1987-06-15 | 1992-03-03 | Ciba-Geigy Corporation | Staurosporine derivatives substituted at methylamino nitrogen |
| WO2017181149A1 (en) * | 2016-04-15 | 2017-10-19 | Blaze Bioscience, Inc. | Methods of treating breast cancer |
| WO2020261293A1 (en) * | 2019-06-24 | 2020-12-30 | Dr. Reddy's Laboratories Limited | Process for preparation of midostaurin |
| CN114437109A (en) * | 2022-03-08 | 2022-05-06 | 贵州省中国科学院天然产物化学重点实验室(贵州医科大学天然产物化学重点实验室) | Halogenated derivative of staurosporine, preparation method and application thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5093330A (en) * | 1987-06-15 | 1992-03-03 | Ciba-Geigy Corporation | Staurosporine derivatives substituted at methylamino nitrogen |
| WO2017181149A1 (en) * | 2016-04-15 | 2017-10-19 | Blaze Bioscience, Inc. | Methods of treating breast cancer |
| WO2020261293A1 (en) * | 2019-06-24 | 2020-12-30 | Dr. Reddy's Laboratories Limited | Process for preparation of midostaurin |
| CN114437109A (en) * | 2022-03-08 | 2022-05-06 | 贵州省中国科学院天然产物化学重点实验室(贵州医科大学天然产物化学重点实验室) | Halogenated derivative of staurosporine, preparation method and application thereof |
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