CN111732579B - Polyether polyketone compound polydecaminmycin and preparation method and application thereof - Google Patents
Polyether polyketone compound polydecaminmycin and preparation method and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D407/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00
- C07D407/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings
- C07D407/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
-
- 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
- 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, e.g. protozoa; 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
-
- 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
- C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
- C12P17/162—Heterorings having oxygen atoms as the only ring heteroatoms, e.g. Lasalocid
Abstract
The invention discloses a polyether polyketone compound polydecaminmycin and a preparation method and application thereof. The compound polydecaminmycin has a structure shown in a formula (I). The polydecalinmycin compound is obtained by separating and purifying a fermentation culture of a spmO gene deletion mutant strain SPM257 of Streptomyces (Streptomyces sp.) SCSIO03032, and is a polyether polyketone compound with a novel framework structure. The polyether polyketide is a complex molecule with various excellent biological activities, and is expected to develop a novel drug lead compound by further screening and researching the biological activities of polydecaminmycin.
Description
Technical Field
The invention relates to the field of industrial microorganisms, in particular to a polyether polyketone compound polydecaminmycin, a preparation method thereof and application thereof in preparing antitumor drugs.
Background
Polyether polyketones (Polyether polyketides) compounds are complex polyketones containing carboxylic acid groups and a plurality of five-membered or six-membered ether rings, and the compounds of the family have various structures, generally have long chains and a plurality of continuous chiral centers, and have various biological activities such as antibiosis, antitumor, antioxidation and the like. Polydecalinmycins are polyether polyketides with a decalin (decalin) backbone structure that were first discovered. We found that Streptomyces sp.SCSIO03032 mainly produces secondary metabolites of spironoidimicins and identified a biosynthetic gene cluster SPM of the compounds, wherein a spmO (tryptophan oxidase) gene is a key gene for synthesizing the spironoidimicins, and a spmO gene deletion mutant SPM257 which does not produce the spironoidimicins can be obtained by knocking out the spmO gene. Polydecalinmycins are novel compounds discovered by research strategies that activate other metabolic pathways in the strain by knocking out the biosynthesis genes of the main products of spironoideimicins, and thereby discover compounds that are absent or present in very small amounts in the original wild-type strain.
Disclosure of Invention
The first object of the present invention is to provide a novel polyether polyketide, polydecalinmycin, or a pharmaceutically acceptable salt thereof.
The novel polyether polyketone compound polydecaminmycin of the invention has the structure shown in the formula (I):
a compound polydecaminmycin with the molecular formula C43H70O8The compound is characterized by comprising a decalin (decalin) ring, a tetrahydrofuran connected tetrahydropyran ring and a terminal carboxyl, wherein cis double bond enol form is formed between C-8 and C-9, trans double bond is formed between C-20 and C-21, C-2 is S configuration, C-3 is R configuration, C-6 is S configuration, C-22 is S configuration, C-23 is S configuration, C-25 is S configuration, C-28 is S configuration, C-29 is R configuration, C-32 is R configuration, and C-33 is R configuration.
The second purpose of the invention is to provide a preparation method of novel polyether polyketone compound polydecalinmycin. The compound polydecaminmycin is obtained by separating and purifying from a fermentation culture of a SPM gene deletion mutant strain SPM257 of Streptomyces (Streptomyces sp.) SCSIO 03032.
Specifically, the preparation method comprises the following steps:
a. preparing a fermentation culture of a spmO gene deletion mutant strain SPM257, inoculating the activated mutant strain SPM257 into a seed culture medium, culturing at 28 ℃ and 200rpm for 72h to obtain a seed solution, inoculating the seed solution into the fermentation culture medium with the inoculation amount of 10% v/v, performing shaking culture at 28 ℃ and 200rpm for 5 days, adding sterilized Amberlite XAD-16 macroporous resin into the culture medium, continuously culturing for 2 days, separating the macroporous resin adsorbed with fermentation metabolites from fermentation liquor and mycelia, eluting the macroporous resin with acetone, distilling and concentrating eluent to obtain a water mixed solution, extracting the water mixed solution with butanone, and concentrating a butanone extraction layer to obtain a total extract. The seed culture medium comprises 10g of soluble starch, 4g of yeast extract powder, 2g of bacteriological peptone and 30g of sea salt, and water is added to the seed culture medium to ensure that the volume is 1L and the pH value is 7.0; the fermentation medium comprises 10g of soluble starch and K2HPO41g，MgSO4·7H2O 1g，(NH4)2SO4 2g，CaCO32g of trace elements, and adding water to a constant volume of 1L and a pH value of 7.0.
b. And (2) carrying out silica gel column chromatography on the total extract, using chloroform/methanol as an eluent, carrying out gradient elution from the volume ratio of 100: 0-0: 100, collecting fraction Fr.1 obtained by gradient elution from the chloroform/methanol volume ratio of 92:8, then carrying out SephadexLH-20 through a Sephadex column, using the chloroform/methanol volume ratio of 1:1 as a mobile phase for elution and concentration, obtaining fraction Fr.1-1 containing polydecamycin, separating and purifying the fraction Fr.1-1 through a Sephadex LH-20 column, and using methanol as a mobile phase for elution, thus obtaining the compound polydecamycin.
The third purpose of the invention is to provide the application of the compound polydecaminmycin or the medicinal salt thereof in preparing the antitumor medicament.
Preferably, the application of the compound polydecaminomycin in preparing the medicine for treating human neural cancer.
Preferably, the application of the compound polydecaminomycin in preparing the medicine for treating human breast cancer cells.
Preferably, the application of the compound polydecaminomycin in preparing the medicine for treating human liver cancer.
Preferably, the application of the compound polydecaminomycin in preparing the medicine for treating human lung cancer.
Preferably, the application of the compound polydecaminomycin in preparing the medicine for treating human colorectal cancer is provided.
Preferably, the application of the compound polydecaminomycin in preparing the medicine for treating human acute lymphoblastic leukemia.
The fourth object of the present invention is to provide an antitumor agent comprising an effective amount of the above-mentioned compound polydecamycin or a pharmaceutically acceptable salt thereof as an active ingredient.
The fifth object of the present invention is to provide the use of the spmO gene deletion mutant SPM257 of the above-mentioned Streptomyces (Streptomyces sp.) SCSIO03032 for the preparation of the above-mentioned compound polydecaminomycin.
The invention has the beneficial effects that:
the invention provides a novel polyether polyketone compound polydecaminmycin and a preparation method thereof. Experiments prove that the compound polydecaminomycin has better cytotoxic activity on various human tumor cells, and the activity on partial tumor cell strains is better than that of clinically used positive drugs of azithromycin (adriamycin) and 5-fluorouracil (5-FU). The compound polydecaminmycin is expected to be developed into a novel antitumor drug.
The Streptomyces (Streptomyces sp.) SCSIO03032 is preserved in China Center for Type Culture Collection (CCTCC) at 7-18 months in 2011, and the address is as follows: the preservation number of Wuhan university in Wuhan City of China is CCTCC NO. M2011258, and the invention name is disclosed in the patent application number CN 201210087537.7: streptomycete, an anti-tumor compound Spiro-Indomycin A-D, a preparation method and an application thereof.
The spmO gene deletion mutant SPM257 of the present invention is obtained by knocking out the spmO (tryptophan oxidase) gene of Streptomyces sp SCSIO03032, and the specific method is disclosed in the literature: liang Ma, Wenjun Zhang, Yiguang Zhu, Guangtao Zhang, Haibo Zhang, Qingbo Zhang, Liping Zhang, Chengshan Yuan, Changsheng Zhang, Identification and charaterization of a Biosynthetic Gene Cluster for Trypophan Dimers in Deep Sea-Derived Streptomyces sp.SIO 03032 Applied Microbiology and Biotechnology 2017,101(15) 6123 6136. The strain the applicant also holds, warranting supply to the public since 20 years.
Drawings
FIG. 1 is a high performance liquid chromatogram of a spmO gene deletion mutant SPM257 total extract and a Wild Type (WT) total extract of Streptomyces sp.scSIO 03032.
FIG. 2 is key to the compound polydecaminomycin1H-1H COSY and HMBC have related signals and chemical structures.
FIG. 3 is a drawing of the compound polydecaminomycin1H NMR spectrum.
FIG. 4 shows DEPT135 and13c NMR spectrum.
FIG. 5 is of the compound polydecaminomycin1H-1H COSY spectrum.
FIG. 6 is an HSQC spectrum of the compound polydecaminmycin.
FIG. 7 is an HMBC chromatogram of the compound polydecaminmycin.
FIG. 8 is a ROESY spectrum of the compound polydecaminmycin.
FIG. 9 is an HRESIMS spectrum of the compound polydecamycin.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1: separation, preparation, structure identification and antitumor activity test of Polydecalinmycins
1. Preparation of SPmO Gene-deleted mutant SPM257 from Streptomyces (Streptomyces sp.) SCSIO03032
The applicant obtains the whole genome information of Streptomyces sp SCSIO03032 through team earlier-stage sequencing, establishes a genetic operation system of the strain, identifies a tryptophan oxidase gene spmO from the genetic operation system, and obtains a spmO gene deletion mutant strain SPM257 by applying a PCR-targeting technology. Specific methods are disclosed in the literature: liang Ma, Wenjun Zhang, Yiguang Zhu, Guangtao Zhang, Haibo Zhang, Qingbo Zhang, Liping Zhang, Chengshan Yuan, Changsheng Zhang, Identification and charaterization of a Biosynthetic Gene Cluster for Trypophan Dimers in Deep Sea-Derived Streptomyces sp.SIO 03032 Applied Microbiology and Biotechnology 2017,101(15) 6123 6136.
2. Preparation of culture Medium
Preparing a seed culture medium: 10g of soluble starch, 4g of yeast extract powder, 2g of bacteriological peptone and 30g of sea salt, adding water to a constant volume of 1L, adjusting the pH value to 7.0, sterilizing at 115 ℃ for 30min for later use;
preparing a fermentation culture medium: soluble starch 10g, K2HPO4 1g，MgSO4·7H2O 1g，(NH4)2SO4 2g，CaCO32g of trace elements, adding water to a constant volume of 1L, pH 7.0, 115 DEG CSterilizing for 30 min.
2 wt% agar powder was added to the corresponding solid medium.
3. Fermentation culture
Seed culture: inoculating a spmO gene deletion mutant strain SPM257 activated on a plate culture medium into a liquid seed culture medium (1000mL), and culturing at 28 ℃ and 200rpm for 72h to prepare a seed solution;
large-scale fermentation culture: inoculating the seed solution into a fermentation medium (10L) at the inoculation amount of 10% v/v, performing shaking culture at 28 deg.C and 200rpm for 5 days, adding sterilized Amberlite XAD-16 macroporous resin, fermenting for 2 days, and collecting macroporous resin with secondary metabolite adsorbed in the fermentation liquid.
4. Extracting the total extract
Filtering the fermentation liquid to obtain a macroporous resin part for adsorbing secondary metabolites, washing residual thalli and bacteria liquid by clear water, eluting the macroporous resin part by acetone after drying, distilling and concentrating eluent to obtain a water mixed liquid, extracting the water mixed liquid by butanone, and concentrating a butanone extraction layer to obtain a total extract (about 5 g).
Detecting the total extract of the spmO gene deletion mutant strain SPM257 and the total extract of the Wild Type (WT) of Streptomyces (Streptomyces sp.) SCSIO03032 (i.e. the total extract obtained by fermenting Streptomyces (Streptomyces sp.) SCSIO03032 according to the above steps) by using a High Performance Liquid Chromatography (HPLC), wherein the HPLC detection conditions are as follows: the chromatographic column is Phenomenex kinetex C18 (250X 4.6mm,5 μm), the mobile phase comprises A phase and B phase, and the mobile phase comprises A phase: acetonitrile 10% (volume fraction) and trifluoroacetic acid 0.08% (volume fraction), the solvent is water, mobile phase B: 90% (volume fraction) acetonitrile, and water as solvent; sample introduction procedure: 0-20min, the mobile phase proportion is A phase/B phase (volume ratio): 95:5-0: 100; 20-25min, the mobile phase proportion is A phase/B phase (volume ratio): 0: 100; 25-26min, the mobile phase proportion is A phase/B phase (volume ratio): 0:100-95: 5; 26-30min, the mobile phase proportion is A phase/B phase (volume ratio): 95:5, detection wavelength of 280nm, flow rate of 1mL/min, wherein peak 1 appeared at 24.8min and is the compound polydecaminmycin, and the result is shown in FIG. 1.
5. Isolation and purification of Compounds
Subjecting the total extract to silica gel column chromatography, performing gradient elution from a volume ratio of 100: 0-0: 100 by using chloroform/methanol as an eluent, tracking a target compound by using a high performance liquid chromatography, collecting fraction Fr.1 subjected to gradient elution from a chloroform/methanol volume ratio of 92:8, subjecting the fraction Fr.1 to Sephadex LH-20 column chromatography, eluting by using a chloroform/methanol volume ratio of 1:1 as a mobile phase, concentrating, obtaining a fraction Fr.1-1 containing polydecamycin, subjecting the fraction Fr.1-1 to Sephadex LH-20 column separation and purification, and eluting by using methanol as a mobile phase, thus obtaining a pure monomer compound polydecamycin (30 mg).
6. Identification of Compounds
Nuclear Magnetic Resonance (NMR) and high resolution mass spectrometry (HRESIMS) were performed on the polydecamycin (formula (I)) prepared from SPM gene deletion mutant SPM257 fermentation culture of streptomyces SCSIO03032, and data analysis and structure identification were performed, with the following results:
the compound polydecaminomycin: pale yellow gum, UV (CH)3OH)λmax(logε)282nm(4.99)；
109.16(c 0.8,CH3OH)；1H NMR(500MHz,CDCl3) And13C NMR(125MHz,CDCl3) The data are shown in Table 1, HRMS (ESI-TOF) M/z:715.5141[ M + H ]]+,Calcd for C43H71O8715.5143.1D and 2D NMR and HRESIMS are shown in FIGS. 3-9;
TABLE 1 preparation of the compound polydecaminomycin1H and13c NMR data (delta in ppm, J in Hz)a
aThe test solvent was CDCl3，1The H NMR was measured at 500MHz,13c NMR measurements at 125MHz showed both peak splitting and coupling constants for the overlapping signals.
Chemical combinationThe UV of the polydecalinmycin compound shows a characteristic maximum absorption peak at 282nm, and HRESIMS shows that the peak of its excimer ion is M/z 715.5141[ M + H ]]+(calcd for C43H71O8715.5143), the compound is obtained according to formula C43H70O8The unsaturation was calculated to be 9. Analyze it1H and13c NMR data showed that the compound had 43 carbons in total, including 7 methyl groups, 14 methylene groups, 12 sp3Hybridized methine, 5 sp2Hybridized methine, 2 carbonyl, 1 sp2Hybridized quaternary carbon and 2 sp3A hybrid quaternary carbon. By 2D NMR (1H-1H COSY, HSQC, HMBC and ROESY) (figure 2), the plane structure and partial spatial configuration of the compound can be deduced, and the compound can be determined to be a polyether polyketone compound with a decalin skeleton, which is discovered for the first time, by combining with characteristic analysis of biosynthesis of the compound.
According to the data, the structure of the compound polydecaminomycin is confirmed to be shown as the formula (I):
example 3: determination of antitumor Activity of Compound polydecamycin
The compound polydecalinmycin was evaluated for in vitro cytotoxic activity using human nerve cancer cell SF-268, human breast cancer cell MCF-7, human hepatoma cell HepG-2, human lung cancer cell A549 and human colorectal cancer cell line (HCT-116, DLD1, RKO, HT-29, SW480, LOVO, CACO2, SW620, NCI-H716, COLO205) (Table 2). The specific experimental method comprises the following steps: tumor cells in logarithmic growth phase were taken and added to 96-well cell culture plates at 37 ℃ with 5% CO at 5000 cells per well (100. mu.L)2After 24 hours of incubation under the conditions, the compound polydecaminmycin, positive control, DMSO (negative control) was added at different concentrations and incubated for 72 hours. Then 30. mu.L of MTT medium solution (5mg mL)-1PBS) at 37 ℃ for 4 hours, remove the MTT medium, add 100. mu.L of DMSO solution per well to dissolve the pellet andthe air bubbles are removed. Finally, measuring the absorbance value of the sample at 570nm by using a microplate reader and calculating the half Inhibition Concentration (IC)50) The value is obtained. The above experiments were independently repeated 3 times. The experimental result shows that the compound polydecaminomycin has better cytotoxic activity on various human tumor cells, and the activity on partial tumor cell lines is better than that of clinically used positive drugs of azithromycin (adriamycin) and 5-fluorouracil (5-FU).
TABLE 2 in vitro toxicity of the compound polydecaminomycin against different tumor cells
The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.
Claims (8)
1. The compound polydecaminomycin or the medicinal salt thereof has the structure shown as the formula (I):
2.a method for preparing the compound polydecaminmycin as claimed in claim 1, which comprises the following steps:
a. preparing a fermentation culture of the spmO gene deletion mutant strain SPM257, adding macroporous resin into a fermentation culture medium, separating the macroporous resin adsorbed with fermentation metabolites from fermentation liquor and mycelia after the culture is finished, eluting the macroporous resin with acetone, distilling and concentrating eluent to obtain water mixed liquor, extracting the water mixed liquor with butanone, and concentrating a butanone extraction layer to obtain a total extract;
b. and (2) carrying out silica gel column chromatography on the total extract, using chloroform/methanol as an eluent, carrying out gradient elution from the volume ratio of 100: 0-0: 100, collecting fraction Fr.1 obtained by gradient elution from the chloroform/methanol volume ratio of 92:8, then carrying out Sephadex LH-20 through a Sephadex column, using the chloroform/methanol volume ratio of 1:1 as a mobile phase for elution and concentration, obtaining fraction Fr.1-1 containing polydecamycin, separating and purifying the fraction Fr.1-1 through a Sephadex LH-20 column, and using methanol as a mobile phase for elution, thus obtaining the compound polydecamycin.
3. The method as claimed in claim 2, wherein the step of preparing the fermentation culture of the spmO gene deletion mutant SPM257 comprises: inoculating the activated mutant strain SPM257 into a seed culture medium, culturing at 28 ℃ and 200rpm for 72h to obtain a seed solution, inoculating the seed solution into a fermentation culture medium at the inoculum size of 10% v/v, and performing shake culture at 28 ℃ and 200 rpm.
4. The method according to claim 3, wherein the seed medium comprises 10g of soluble starch, 4g of yeast extract powder, 2g of bacteriological peptone and 30g of sea salt, and is added with water to a volume of 1L and pH 7.0.
5. The method according to claim 3, wherein the fermentation medium comprises 10g of soluble starch, K2HPO4 1g，MgSO4·7H2O 1g，(NH4)2SO4 2g，CaCO32g of trace elements, and adding water to a constant volume of 1L and a pH value of 7.0.
6. The use of the compound polydecaminmycin of claim 1 or a pharmaceutically acceptable salt thereof for the preparation of an anti-neoplastic agent against neural cancer, breast cancer, liver cancer, lung cancer, colorectal cancer or acute lymphoblastic leukemia.
7. An antitumor drug characterized by comprising an effective amount of the compound polydecaminmycin of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the antitumor drug is an antitumor drug against nerve cancer, breast cancer, liver cancer, lung cancer, colorectal cancer or acute lymphoblastic leukemia.
8. Use of SPmO gene-deleted mutant SPM257 of Streptomyces (Streptomyces sp.) SCSIO03032 for preparing the compound polydecaminomycin according to claim 1.
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