CN113912658A - 4' -N-demethyl-vicenistatin, and preparation method and application thereof - Google Patents

4' -N-demethyl-vicenistatin, and preparation method and application thereof Download PDF

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CN113912658A
CN113912658A CN202111322195.8A CN202111322195A CN113912658A CN 113912658 A CN113912658 A CN 113912658A CN 202111322195 A CN202111322195 A CN 202111322195A CN 113912658 A CN113912658 A CN 113912658A
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vicenistatin
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李青连
鞠建华
梁智铖
李骏
凌春耀
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South China Sea Institute of Oceanology of CAS
Southern Marine Science and Engineering Guangdong Laboratory Guangzhou
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Abstract

The invention discloses 4' -N-methyl-vicenistatin and a preparation method and application thereof, belonging to the technical field of natural products. The N-demethyl vicenin derivative 4 ' -N-demethyl-vicenin is separated from an N-methyltransferase inactivated mutant strain SCSIO Mla-L010/delta vicG of S.parvus SCSIO Mla-L010, and test results show that the 4 ' -N-demethyl-vicenin has obvious inhibitory activity on various fungi and bacteria and shows the important value of the derivative in the development of anti-infective medicaments, wherein the antibacterial activity of the 4 ' -N-demethyl-vicenin on fungi candida albicans is 2-4 times stronger than that of the vicenin, and the structural derivative has better medicament forming potential. In addition, 4' -N-demethyl-vicenistatin also shows stronger cytotoxic activity to various cancer cells, and has the potential of being developed into antitumor drugs.

Description

4' -N-demethyl-vicenistatin, and preparation method and application thereof
The technical field is as follows:
the invention belongs to the technical field of natural products, and particularly relates to 4' -N-demethyl-vicenistatin, and a preparation method and application thereof.
Background art:
broad-spectrum, highly effective, low-toxicity antibiotics have been considered as one of the greatest inventions in humans in the 20 th century. Many secondary metabolites produced by actinomycetes are developed and utilized as main antibiotic-producing bacteria, and are clinically useful antibacterial drugs in medical and health fields, for example, erythromycin (erythromycin), vancomycin (vancomycin), daptomycin (daptomycin), and the like, which are clinically used against infection. However, due to the phenomenon of antibiotic abuse and misuse which widely exist in the world, the occurrence of antibiotic resistance is more frequent, pathogenic microorganisms can easily obtain drug resistance genes from the environment, and the effectiveness of clinically used antibiotics on pathogenic bacteria is greatly reduced. Therefore, the problem of antibiotic resistance has become a serious challenge for clinical anti-infective drug treatment, the clinically available antibiotics are not enough to cope with pathogenic "superbacteria" with broad-spectrum drug resistance, and the development of antibiotic drugs with novel antibacterial mechanisms becomes an important means for solving the current bacterial drug resistance dilemma.
Tumors are serious diseases which threaten human lives and affect human health, and the incidence and the death rate of the tumors are in a rapidly increasing trend according to the report of the world health organization. Chemotherapy is an important means for tumor treatment, various chemotherapeutic drugs are widely used in the process of tumor treatment, however, the main reasons for chemotherapy failure are that tumor cells generate multidrug resistance to chemotherapeutic drugs, the toxic and side effects of chemotherapeutic drugs and the insensitivity of some patients to chemotherapeutic drugs. Therefore, in the field of tumor treatment, there is an urgent and great need for innovative drugs with new mechanisms and new targets.
Vicenistatin is a macrocyclic lactam antibiotic with a structure shown in formula a, and was first isolated by Shindo et al in 1993 from the actinomycete Streptomyces halstedii HC 34. Structurally, the vicenistatin consists of a specific aminosugar vicenisamine and a macrocyclic lactam nucleus, viceniactam. The research on the biological activity of the vicenitatin shows that the compound has remarkable cytotoxic activity on a human blood cancer cell line HL-60(IC50 is 0.12 mu g/mL) and a human colon cancer cell line COLO205(IC50 is 0.19 mu g/mL) under in vitro conditions, and the vicenitatin has remarkable in vivo anti-tumor activity in a nude mouse model implanted with a human colon cancer cell line Co-3. Therefore, the large ring lactam natural product vicenistatin is a lead compound with drug-forming potential, and the drug-forming property of the compound is worthy of deep research.
Figure BDA0003345912830000021
In recent years, the research and development of microbial-derived drugs are gradually difficult, and terrestrial microbial resources mainly comprising soil actinomycetes are screened in a large range with high intensity for a long time of nearly 80 years, so that the repeated discovery probability of strains and secondary metabolites is greatly increased. Therefore, the strategy of 'new use of old drugs' is taken as guidance, the treatment application of the existing effective clinical drugs is widened, the patent drug potential of the known lead compound is fully excavated, and the urgent requirements of new mechanisms and new target drugs in the research and development of new antibacterial and antitumor drugs can be met. On the other hand, a drug derivative having a better therapeutic effect is often obtained by physiologically modifying and modifying the structure of the original drug, and a structural derivative obtained by modifying the structure of a known lead compound is an important means for developing a candidate drug having a good pharmacological effect. Therefore, the biological activity evaluation and the structural derivatization research of the lead compound vicenistatin have important significance for the development of new anti-infection and anti-tumor drugs.
The invention content is as follows:
the first purpose of the invention is to provide an anti-infection and anti-tumor N-demethyl vicenistatin derivative 4 '-N-demethyl-vicenistatin, wherein the structure of the 4' -N-demethyl-vicenistatin is shown as formula (I):
Figure BDA0003345912830000031
the second purpose of the invention is to provide a preparation method of 4 '-N-demethyl-vicenitatin shown in formula (I), wherein the 4' -N-demethyl-vicenitatin is separated from an N-methyltransferase inactivated mutant SCSIO Mla-L010/delta vicG of streptomyces parvus SCSIO Mla-L010, the SCSIO Mla-L010/delta vicG is obtained by replacing an N-methyltransferase gene vicG in a vicenitatin gene cluster of an S.parvus SCSIO Mla-L010 genome by a resistance marker gene by using a homologous recombination technology, and the vicG sequence is shown in SEQ ID NO. 1.
The marine-derived vicenistatin-producing strain S.parvus SCSIO Mla-L010 is separated from a marine invertebrate monodentate snail (Monodonata labio) sample, and the sample collection place is the high intertidal zone of the great Asian gulf of south China sea. Replacing an N-methyltransferase gene vicG in a vicenistatin gene cluster of an S.parvus SCSIO Mla-L010 genome with a resistance marker gene by utilizing a homologous recombination technology to obtain an N-methyltransferase inactivated mutant SCSIO Mla-L010/delta vicG.
In a preferred embodiment, the preparation method of the 4' -N-methyl-vicenistatin comprises the following steps:
(1) preparing a fermentation culture of an N-methyltransferase inactivated mutant strain SCSIO Mla-L010/delta vicG, centrifugally separating fermentation supernatant and mycelia, extracting the mycelia with acetone, extracting the supernatant with butanone to obtain acetone extract and butanone extract, concentrating the extract under reduced pressure to obtain mycelia extract and supernatant extract, and mixing the two extracts to obtain a crude extract;
(2) mixing the crude extract obtained in the step (1) with silica gel, carrying out normal phase silica gel column chromatography, and carrying out gradient elution by adopting a chloroform/methanol system according to the following volume ratio: 100: 0; 98: 2; 96: 4; 94: 6; 92: 8; 9: 1; 8: 2; 6: 4; 5: 5; 0:100, sequentially obtaining 10 components Fr.A1-10; the components chloroform/methanol 92: 8; 9: 1; 8: 2; and (3) combining Fr.A5-8 eluted at the ratio of 6:4, and purifying by HPLC to obtain the 4' -N-demethyl-vicenistatin.
Further, the step of extracting the mycelium and the supernatant is to add acetone with the volume of 3 times to the mycelium precipitate for extraction, repeat the extraction for 3 times, concentrate the extract into an extract under reduced pressure, add butanone with the same volume to the fermented supernatant for extraction for 3 times, and concentrate the extract into a concentrated extract under reduced pressure.
Further, the purification by HPLC described in the step (2) is carried out by using methanol as a mobile phase, performing chromatography by using Sephadex LH-20 hydroxypropyl Sephadex column chromatography and collecting a target component, and preparing a target peak by using semi-preparative high performance liquid chromatography and ODS column chromatography (250X 10mm,5 μm) under the following conditions: CH (CH)3CN/H2Isocratic elution in O (32:68, V/V, containing 0.1% acetic acid) system for 25min at flow rate of 2.5mL/min to obtain the 4' -N-demethyl-vicenistatin (t)R＝14.1min)。
The third purpose of the invention is to provide the application of the 4' -N-demethyl-vicenistatin shown in the formula (I) in preparing anti-infective drugs or anti-tumor drugs.
In a preferred embodiment, the anti-infective drug is an antibacterial drug or an antifungal drug; further, the anti-infective drug is an anti-Staphylococcus aureus (Staphylococcus aureus), Methicillin-resistant Staphylococcus aureus (Methicillin-resistant Staphylococcus aureus), Micrococcus luteus (Micrococcus luteus), Bacillus subtilis (Bacillus subtilis), cryptococcus neoformans (cryptococcus neoformans), Helicobacter pylori (Helicobacter pylori) or Candida albicans (Candida albicans) drug.
In another preferred embodiment, the anti-tumor drug is a drug for resisting breast cancer, liver cancer, cervical cancer, non-small cell lung cancer, colon adenocarcinoma or leukemia; furthermore, the anti-tumor drug is a drug for resisting human breast cancer cells MCF-7, human liver cancer cells HepG2, human cervical cancer cells HeLa, human non-small cell lung cancer cells A549, human colon adenocarcinoma cells RKO or human acute promyelocytic leukemia cells HL-60.
The fourth object of the present invention is to provide an anti-infective drug containing 4' -N-demethyl-vicenistatin represented by the formula (I) as an active ingredient.
Preferably, the anti-infective drug is an antibacterial drug or an antifungal drug; more preferably, the anti-infective agent is an anti-Staphylococcus aureus (Staphylococcus aureus), Methicillin-resistant Staphylococcus aureus (Methicillin-resistant Staphylococcus aureus), Micrococcus luteus (Micrococcus luteus), Bacillus subtilis (Bacillus subtilis), cryptococcus neoformans (cryptococcus neoformans), Helicobacter pylori (Helicobacter pylori), or Candida albicans (Candida albicans) agent.
The fifth object of the present invention is to provide an antitumor agent comprising 4' -N-demethyl-vicenistatin represented by formula (I) as an active ingredient.
Preferably, the anti-tumor drug is a drug for resisting breast cancer, liver cancer, cervical cancer, non-small cell lung cancer, colon adenocarcinoma or leukemia; more preferably, the anti-tumor drug is a drug for resisting human breast cancer cells MCF-7, human liver cancer cells HepG2, human cervical cancer cells HeLa, human non-small cell lung cancer cells A549, human colon adenocarcinoma cells RKO or human acute promyelocytic leukemia cells HL-60.
The sixth purpose of the invention is to provide the application of the N-methyltransferase inactivated mutant SCSIO Mla-L010/delta vicG in the preparation of 4' -N-demethyl-vicenistatin.
Compared with the prior art, the invention has the following beneficial effects:
(1) test results show that 4' -N-demethyl-vicenistatin has remarkable inhibitory activity on bacteria and fungi, has a Minimum Inhibitory Concentration (MIC) of 0.125-2 mu g/mL on Staphylococcus aureus ATCC 29213, 6 Methicillin-resistant Staphylococcus aureus (MRSA) shhs-E1, MRSA 16339, MRSA745524, MRSA 16162, MRSA718306 and MRSA 6917, Micrococcus luteus, Bacillus subtilis, Cryptococcus neoformans, Helicobacter pylori and Candida albicans, and shows an important value in anti-infection drug development;
(2) the antibacterial activity of the N-demethyl vicenistatin derivative 4' -N-demethyl-vicenistatin on Candida albicans is 2-4 times stronger than that of the vicenistatin, and the structural derivative has better medicament forming potential.
(3) The 4' -N-demethyl-vicenistatin also shows stronger cytotoxic activity to various cancer cells, and has the potential of being developed into antitumor drugs.
The actinomycete strain Streptomyces parvus SCSIO Mla-L010 is disclosed in http:// hyw.journal system.net/ch/reader/view _ abstrate.aspxfile _ No. 202104020000001, and the strain is also held by the applicant and is guaranteed to be released to the public within 20 years from the application date.
Description of the drawings:
FIG. 1 shows the construction of a double-crossover mutant of the SCSIO Mla-L010/Δ vicG gene;
FIG. 2 is an HPLC analysis of fermentation extracts of wild type strain S.parvus SCSIO Mla-L010 and vicG gene mutant SCSIO Mla-L010/Δ vicG;
FIG. 3 is the information related to the high resolution mass spectrum of 4' -N-methyl-vicenistatin;
FIG. 4 shows 4' -N-demethyl-vicenistatin1H-NMR related information;
FIG. 5 shows 4' -N-demethyl-vicenistatin13C-NMR-related information.
The specific implementation mode is as follows:
the following examples are further illustrative of the present invention and are not intended to be limiting thereof. The experimental methods used are well known to those skilled in the art and the experimental materials used are commercially available, unless otherwise specified.
General description of the invention
The marine strain Streptomyces parvus SCSIO Mla-L010 related by the invention is separated from a marine invertebrate single-tooth snail (Monodonata labio) sample, and the sample collection place is the high intertidal zone of the Bay of the great Asia in south China sea. The resistance marker gene is used for replacing an N-methyltransferase gene vicG in a vicenistatin gene cluster of the S.parvus SCSIO Mla-L010 genome by a homologous recombination technology, the nucleotide sequence of the gene is shown as SEQ ID NO.1, the length is 711bp, and an N-methyltransferase inactivated mutant SCSIO Mla-L010/delta vicG is obtained.
(1) Preparing a spore-forming culture medium and a fermentation culture medium:
an MS solid culture medium is used as a spore-producing plate of the two strains, and the spore-producing plate comprises the following components: 10g/L of soybean slices, 10g/L of soybean powder and 20g/L of D-mannitol, adjusting the pH value to 7.3, subpackaging, adding technical agar powder with the mass fraction of 2.5%, and sterilizing at 121 ℃ for 30min for later use.
The modified AM3 liquid culture medium is used as a culture medium for vial fermentation and large-scale fermentation of the two strains, and the composition of the culture medium is as follows: 2.5g/L of soybean meal, 15g/L of starch, 15g/L of glycerol, 7.5g/L of bacteriological peptone, 1g/L of ammonium sulfate and 30g/L of sea salt, adjusting the pH value to 7.3, adding 5g/L of calcium carbonate, and sterilizing at 121 ℃ for 30min for later use.
(2) The strain culture and fermentation conditions are as follows:
the 4' -N-demethyl-vicenitatin-producing strain SCSIO Mla-L010/deltavicG strain was cultured in an incubator at 28 ℃ for 7 days until spores mature using MS solid medium (containing 50. mu.g/mL of opromycin) as a spore-producing plate.
Small bottle fermentation: mature spores on MS solid medium were inoculated into modified AM3 liquid medium (50mL), shake-cultured for 7d with shaking table set at 28 deg.C and 200 r/min. Adding butanone 2 times the volume of the fermentation broth, concentrating under reduced pressure to obtain extract, adding methanol to dissolve, and metering to 1mL to obtain strain fermentation concentrated solution, and injecting 20 μ L of concentrated solution into HPLC for analysis, wherein the HPLC analysis results of the fermentation conditions of the two strains are shown in FIG. 2.
Large-scale fermentation: inoculating mature spores on MS solid culture medium into modified AM3 liquid culture medium (50mL) as seed liquid, shaking and culturing for 2d, transferring the seed liquid to 200mL modified AM3 liquid culture medium, fermenting for 7d, and setting shaking table conditions at 28 deg.C and 200 r/min.
Example 1 construction of methyltransferase Gene mutant SCSIO Mla-L010/Δ vicG
Construction of S.parvus SCSIO Mla-L010 genomic library and screening of cosmid containing methyltransferase Gene
Construction of the S.parvus SCSIO Mla-L010 genomic library
The S.parvus SCSIO Mla-L010 genomic library was constructed by referring to SuperCos1 Cosmid Vector Kit (Agilent) and Gigapck III XL packing Extract (Epicentre) handbook of operation. Liquid submerged culture of S.parvus SCSIO Mla-L010 strain for 2 days, centrifugation to collect thallus, and phenol-chloroform extraction to extract genome DNA. Adding a high-purity DNA with a proper concentration into a Sau3AI enzyme with a certain concentration, reacting for a certain time to cut the genome DNA into a proper fragment size, detecting by running nucleic acid gel, extracting and purifying the cut product by using phenol-chloroform, and carrying out dephosphorylation treatment. Meanwhile, the SuperCos1 vector was treated with XbaI restriction enzyme, and after phenol-chloroform extraction, dephosphorylation was performed in the same manner, and after dephosphorylation, BamHI restriction enzyme treatment was performed again, followed by extraction and purification. The genomic DNA treated by the method is randomly connected with the SuperCos1 vector under the action of T4 ligase, and the connection efficiency is verified by running glue. The ligation products were then packaged with packaging protein and infected with library strain e.coli LE392, which was then spread on kanamycin-containing resistant plates. After the single clone grows out, randomly selecting about 2000 clones to inoculate in a 96-well plate, culturing at 37 ℃ to a certain concentration, and then storing in a refrigerator at-80 ℃ to complete the construction of the S.parvus SCSIO Mla-L010 genome library.
1.2 screening of cosmid plasmid 1-F11 containing the methyltransferase gene vicG
And (3) carrying out whole genome sequencing on S.parvus SCSIO Mla-L010 by adopting a second-generation sequencing technology Oxford Nanopore and splicing to obtain a whole genome map and a whole genome sequence. The sequence is uploaded to online bioinformatics analysis software anti SMASH (https:// anti major. second major. peptides. org/#. |/start), and a secondary metabolite gene cluster which may encode polyether compounds is searched and verified by a gene knockout experiment. Further analyzing the functions of each gene in the gene cluster, screening out the gene which codes methyl transferase as vicG (the nucleotide sequence of the gene is shown in SEQ ID NO. 1), and determining the distribution position of the gene. Since cosmid can carry DNA fragments with the size of 30-40kb, in order to screen cosmid plasmids containing the methyltransferase gene vicG, three pairs of screening library primers are designed at proper positions on a gene cluster, and the sequences are as follows:
vic-upstream-F：5’-CGCTTCGTCCACATCTCCAC-3’，
vic-upstream-R：5’-ACTTCGTCGGTTCCCACCAC-3’；
vic-midstream-F：5’-GACACCTACCGTCTGCACATTG-3’，
vic-midstream-R：5’-GTCACTCAGCCAGGTCTGCAC-3’；
vic-downstream-F：5’-CGGAAGAGGCCACCGACCGG-3’，
vic-downstream-R：5’-GGTGTCGACGAGCAGAAAGC-3’。
by using the three pairs of primers, cosmid plasmids 1 to F11 containing the methyltransferase gene vicG are obtained by PCR screening, and the PCR program is as follows: 5min at 95 ℃; 45s at 95 ℃, 45s at 58 ℃ and 1min at 72 ℃ for 30 cycles; 10min at 72 ℃.
2. The construction of the gene knockout mutant strain of the methyltransferase gene mutant strain by utilizing the PCR-targeting technology mainly adopts the PCR-targeting technology, and the concrete operation steps are as follows: first, cosmid plasmid 1-F11 carrying the methyltransferase gene vicG was introduced into E.coli BW25113/pIJ790 and made competent. Meanwhile, knockout primers for each methyltransferase gene (forward primer 5'-GCGGGGGAGGCCGACCGCATCCGCGAACTGGCCCTCGAAATTCCGGGGATCCGTCGACC-3' and reverse primer 5'-CACCGCGCACCCGGCCGACCGGAACGCCGCCTCGTACTCTGTAGGCTGGAGCTGCTTC-3') were designed to amplify an apramycin (Apr) resistant gene fragment having homologous arms at both ends, using a PCR program of: 2min at 95 ℃; 30 cycles of 95 ℃ for 20s, 60 ℃ for 20s, 72 ℃ for 90 s; 10min at 72 ℃. The fragment is introduced into an E.coli BW25113/pIJ790 competent strain carrying cosmid 1-F11, and a homologous recombination system of the E.coli BW25113/pIJ790 is utilized to cause the homologous recombination of an apramycin (Apr) resistance gene fragment and a key functional region of a target gene vicG on the cosmid, thereby causing the mutation inactivation of the target gene and the failure of normal expression. The mutated cosmid plasmid 1-F11 was then extracted and introduced into E.coli ET12567/pUZ 8002. And finally, inducing the cosmid 1-F11 carrying the mutant gene to be transferred into wild streptomyces S.parvus SCSIO Mla-L010 by utilizing non-shuttle type pUZ8002 plasmid in E.coli ET through conjugal transfer, mutating the target gene in the genome through homologous recombination to obtain the methyltransferase gene mutant strain Mla-L010/delta vicG, and verifying whether the constructed mutant strain is correct through resistance verification and PCR (a forward primer 5'-CGACATCTACCGACGGCACAAG-3' and a reverse primer 5'-AGAACAGCGACAGCTCCTGCTC-3'). Mutant cosmid 1-F11 E.coli ET12567/pUZ8002 bacteria were obtained, and finally, the correctly verified mutants were stored in a-80 ℃ refrigerator. The specific construction process is shown in fig. 1.
The experimental operation flow of the joint transfer is as follows:
the constructed bacteria harboring the mutation cosmid 1-F11 E.coli ET12567/pUZ8002 were inoculated into 3-5mL LB liquid medium supplemented with 50. mu.g/mL chloramphenicol (Cm), 50. mu.g/mL apramycin (Apr), and 50. mu.g/mL kanamycin (Kan), and cultured overnight in a shaker at 200rpm at 37 ℃. 500-1000ul of overnight culture broth (the inoculum size is determined according to the broth concentration) was transferred to a 250mL flask containing 50mL LB (50. mu.g/mL chloramphenicol, 50. mu.g/mL adriamycin, and 50. mu.g/mL kanamycin were added), and cultured at 37 ℃ for 2-3 hours in a shaker at 200rpm to obtain OD600The value reaches 0.6-0.8And (4) finishing. And (3) putting 45ml of bacterial liquid into a 50ml centrifuge tube, centrifuging at normal temperature and 4000rpm for 10min, removing supernatant and absorbing residual liquid. The cells were washed with 40ml of non-antibiotic LB, antibiotics were washed off, centrifuged at 4000rpm for 10min at room temperature, the supernatant was discarded and the residual liquid was aspirated off, and the washing was repeated once. According to the concentration of the thallus, an appropriate amount of LB is added to fully suspend the thallus and subpackaged into a sterile EP tube of 1.5 ml.
The streptomyces s SCSIO Mla-L010 spores were scraped into a 50ml centrifuge tube with an appropriate amount of LB, and vortexed to disperse the spores sufficiently. Subpackaging into 1.5ml EP tube, sealing with sealing film, placing in 50 deg.C constant temperature water bath, thermally shocking for 10min, cooling with tap water for 2-3min, and stimulating spore pregermination due to external temperature change. The EP tube was placed on a shaker at 28 ℃ and 200rpm for approximately 5 h.
Adding the subpackaged E.coli ET into the subpackaged spores, uniformly mixing, and coating the mixture to a position with 10mM Mg2+Or 20mM Mg2+Uniformly spreading the bacterial liquid on a flat plate on the MS solid culture medium, airing, placing at the constant temperature of 28 ℃ for culturing for 16-20h, and then covering, wherein the covering step is as follows: each plate was supplemented with apramycin (Apr) and trimethoprim (Tmp) at final concentrations of 50. mu.g/mL, and for ease of coverage, antibiotics were added to 1mL of double distilled water and mixed well before application to the plate and spread evenly on a glass coating rod, the plate was allowed to air dry and incubated at 28 ℃ for 3-4 days after which zygotes were observed to grow out. Thus obtaining the N-methyltransferase inactivation mutant SCSIO Mla-L010/delta vicG.
Example 24 isolation and Structure identification of N-demethyl-vicenistatin
A fermentation culture of the 4' -N-demethyl-vicenitatin-producing strain SCSIO Mla-L010/Δ vicG was prepared using the scale fermentation conditions described in example 1, and the mycelium precipitate and fermentation supernatant were obtained by centrifugation (4000r/min, 15 min). Adding 3 times volume of acetone into the mycelium precipitate for extraction, repeatedly extracting for 3 times, and concentrating the extract under reduced pressure to obtain extract. Adding isovolumetric butanone into the fermentation supernatant for extraction for 3 times, and concentrating the extract under reduced pressure to obtain concentrated extract. Mixing the above two concentrated extracts, adding silica gel, stirring, performing normal phase silica gel column chromatography, and performing gradient chromatography with chloroform/methanol system at a volume ratioAnd (4) degree elution: 100: 0; 98: 2; 96: 4; 94: 6; 92: 8; 9: 1; 8: 2; 6: 4; 5: 5; 0:100, and sequentially obtaining 10 components Fr.A1-10. Combining the components Fr.A5-8 (namely fractions eluted at 92: 8; 9: 1; 8: 2; 6: 4), performing chromatography by using Sephadex LH-20 hydroxypropyl Sephadex chromatographic column by using methanol as a mobile phase, collecting target components, and preparing target peaks by using semi-preparative high performance liquid chromatography and ODS chromatographic column (250 × 10mm,5 μm) according to the following conditions: CH (CH)3CN/H2Isocratic elution in O (32:68, V/V, containing 0.1% acetic acid) system for 25min at flow rate of 2.5mL/min to obtain 4' -N-demethyl-vicenistatin (t)R＝14.1min)
Identification of 4' -N-demethyl-vicenistatin: is white amorphous powder; (+) HRESI-MS (FIG. 3) presents [ M + H]+(M/z 487.3544) Peak (Cal.487.3536[ M + H)]+) Is combined with1H NMR (FIG. 4) and13c NMR (FIG. 5) confirmed the formula to be C29H46N2O4. The nuclear magnetic data are as follows:1H NMR(700MHz,Pyr)δ8.52(d,J＝7.7Hz,NH-19),7.63(dd,J＝14.7,11.2Hz,H-3),6.82(dd,J＝14.7,11.2Hz,H-14),6.29(d,J＝14.7Hz,H-2),6.22(dd,J＝15.4,11.2Hz,H-4),5.98(d,J＝11.2Hz,H-13),5.88(dd,J＝15.4,9.8Hz,H-5),5.71(m,H-15),5.36(d,J＝11.2Hz,H-1’),5.22(t,J＝7.0Hz,H-9),4.35(d,J＝1.4,H-3’),4.10(m,H-5’),4.05(dt,J＝13.3,9.8Hz,H-19a),3.40(t,J＝8.4Hz,H-7),3.10(dd,J＝15.4,8.4Hz,H-8a),3.05(ddd,J＝13.3,4.2,2.8Hz,H-19b),2.76(d,J＝15.4Hz,H-11a),2.68(dd,J＝9.8,1.4Hz,H-4’)2.65(d,J＝15.4Hz,H-11b),2.42(overlapped,H-6,H-16a,H-2’a),2.29(m,H-8b),2.09(overlapped,H-16b),2.00(overlapped,H-2’b),1.97(s,H-22),1.88(overlapped,H-18),1.72(s,H-21),1.58(overlapped,H-17a),1.52(d,J＝5.6Hz,H-6’),1.48(m,H-17b),1.10(d,J＝6.3Hz,H-20),0.86(d,J＝7.0Hz,H-23)。13C NMR(176MHz,Pyr)δ166.79(C-1),143.82(C-5),140.78(C-3),135.53(C-10),134.50(C-12),133.05(C-15),128.89(C-4,C-14),128.57(C-13),125.02(C-2),122.45(C-9),100.40(C-1’),86.50(C-7),72.12(C-5’),69.12(C-3’),57.88(C-4’),49.71(C-11),46.87(C-6),43.50(C-19),40.89(C-2’),37.08(C-8),33.98(C-18),33.15(C-17),28.06(C-16),20.00(C-6’),19.70(C-20),18.44(C-21),18.44(C-23),17.86(C-22)。
in conclusion, the structure of the 4' -N-methyl-vicenistatin is shown as the formula (I),
Figure BDA0003345912830000131
example 34 determination of minimum inhibitory concentration of N-methyl-vicenistatin for a series of Activity-indicating strains
The evaluation of the antibacterial activity of the vicenistatin and 4' -N-demethyl-vicenistatin was performed by the broth dilution method. The vicenistatin, 4' -N-demethyl-vicenistatin and positive reference Vancomycin (Vancomycin), Ampicillin (ampicilin) and amphotericin B (Amphotericin B) were dissolved in dimethyl sulfoxide (DMSO) and prepared into a final concentration of 1.6mg/mL, and stored in a refrigerator at-20 ℃ for further use.
Selecting Mueller-Hinton (MH) broth liquid culture medium to culture activity test strain, wherein the formula of the strain comprises the following beef extract powder 300g/L, soluble starch 15g/L and casein acid hydrolysate 17.5g/L, adjusting pH value to 7.3, and sterilizing at 121 ℃ for 30min for later use.
The test bacterial liquid cultured overnight is diluted to the light absorption value of about 1 at the wavelength of 600nm by using MH culture medium, and then diluted to the original 1 per mill by using MH culture medium for standby. Line 1, column 1 of a 96 well plate was filled with 100. mu.L of sterile MH medium, and the remaining wells of line 1 were filled with 50. mu.L of sterile MH medium as a blank control. Starting from row 2, column 1 of each row was added 92 μ L of sterile MH medium and the remaining columns were added 50 μ L of sterile MH medium and marked for backup. In column 1 (except row 1), 8. mu.L of test compound solution and positive control were added to give an initial concentration of 128. mu.g/mL, and 3 parallel controls were made for each sample. Setting the volume of the row gun to be 50 mu L, carefully sucking the test liquid medicine in the 1 st column up and down for 4-5 times, and uniformly mixing. Draw 50 μ L from it and add to column 2, repeat the previous steps to mix, and so on until diluting to the last column, take 50 μ L and discard it, so that the final concentration of test drug solution from well 1 to well 12 is 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625 μ g/mL. Adding 50 mu L of diluted experimental bacteria liquid into each micropore, mixing uniformly, placing the mixture in an incubator at 37 ℃ for culturing for 16h, observing the growth condition of the test bacteria, and obtaining the minimum sample concentration capable of effectively inhibiting the growth of the strain, namely the Minimum Inhibitory Concentration (MIC) value.
Test results show that 4' -N-demethyl-vicenitatin has remarkable inhibitory activity on Staphylococcus aureus Staphylococcus aureus ATCC 29213, 6 Methicillin-resistant Staphylococcus aureus (MRSA) shhs-E1, MRSA 16339, MRSA 7424, MRSA 16162, MRSA718306 and MRSA 6917, Micrococcus luteus, Bacillus subtilis, Cryptococcus neoformans, Helicobacter pylori and Candida albicans, and the Minimum Inhibitory Concentration (MIC) is 0.125-2 mu g/mL, and shows important value in drug development against infection. It is worth noting that the N-demethylation vicenistatin derivative 4' -N-demethyl-vicenistatin has 2-4 times stronger antibacterial activity than the vicenistatin, and shows that the structural derivative has better drug forming potential. The results are shown in Table 1.
TABLE 1 MIC (. mu.g/mL) values for Vincinistatin and 4' -N-methyl-vicenistatin
Figure BDA0003345912830000141
Figure BDA0003345912830000151
Example 44 semi-Inhibitory Concentration (IC) of N-demethyl-vicenistatin on Serial cell lines50μ M) assay
Adopting an international universal tumor cell strain, namely: human breast cancer cell MCF-7, human liver cancer cell HepG2, human cervical cancer cell HeLa, human non-small cell lung cancer cell A549, human colon adenocarcinoma cell RKO and human acute promyelocytic leukemia cell HL-60. The test method is MTT method:
1) cell culture: tumor cells in logarithmic growth phase were seeded at 100 μ L/well in 96-well plates and grown adherently for 24 hours, depending on the cell growth rate.
2) Add sample (drug product): mu.L of 4' -N-demethyl-vicenistatin culture medium dilution solution with different concentrations is added to each well, and culture medium solvent control and cell-free withered wells with corresponding concentrations are set.
3) Adding medicine to culture cells: tumor cells 5% CO at 37 ℃2Incubated under conditions for 68 hours.
4) And (3) activity test: after culturing, 20 μ L (5mg/mL) of MTT solution is added to each well of the cultured cells, the culture is continued for 4 hours, the supernatant medium is washed away, and 150 μ L of dimethyl sulfoxide (DMSO) is added to dissolve the formazan crystals converted from MTT.
5) Measuring OD value with 540nm wavelength of a microplate reader.
6) And (6) reporting the activity. 3 parallel experiments were performed for each cell line, and the results are shown in Table 2:
TABLE 24 inhibitory Activity of N-demethyl-vicenistatin on a series of tumor cells (IC)50，μM)
Figure BDA0003345912830000161
Sequence listing
<110> Nanhai ocean institute of Chinese academy of sciences
Guangdong Laboratory of Southern Marine Science and Engineering (Guangzhou)
<120> 4' -N-demethyl-vicenistatin, preparation method and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 711
<212> DNA
<213> Marine actinomycetes (Streptomyces parvus SCSIO Mla-L010)
<400> 1
gtgtacgaag aggatttcgc ccgcgtctac gacgacatct accgacggca caagaactat 60
gcgggggagg ccgaccgcat ccgcgaactg gccctcgaat accgacccga cgcgtcgagt 120
ctgctggacg tcggctgcgg cacgggcgag catctggccc gtctgcggca gcatttcgac 180
gtggccggtg tggatctcgc cccgcccatg atccgtattg ccacagccaa gcttcccggg 240
gtgcccctgc tccaggacga catgcgcacc ttctcgctgg accggacctt cgacgtcgtc 300
tgttcgatgt acagctcggt gggctatctg gcgacggccg acgacctgtc caccgccgtc 360
aagaacatga ccggtcatct gcgtccgggc ggtgtgctga tcgtcgagcc gtggatcctc 420
cgggaggact ggaacggcgg cgacctcgtg caggccgact tcgagaacga ggagggcaag 480
gtcgtccgga tgggccgctg gaccaccagg aacgggcgca gccgcgtcga gatgcactat 540
ctggtcgcca cggactcggg accggtgggt cacttcgtgg acgagcagga gctgtcgctg 600
ttctcccgcg aggagtacga ggcggcgttc cggtcggccg ggtgcgcggt ggagtaccgc 660
ccggacggct acgcggaccg cggaatcttc gtgggcgtac ggcaggactg a 711

Claims (10)

1.4' -N-methyl-vicenistatin, which is characterized in that the structure is shown as formula (I),
Figure FDA0003345912820000011
2. the method for preparing 4 '-N-demethyl-vicenitatin according to claim 1, wherein the 4' -N-demethyl-vicenitatin is isolated from an N-methyltransferase inactivated mutant of Streptomyces parvus SCSIO Mla-L010/Δ vicG, wherein Mla-L010/Δ vicG is obtained by inactivating an N-methyltransferase gene vicG in a vicenitatin gene cluster of S.parvus SCSIO Mla-L010 genome, and wherein the sequence of the vicG is shown in SEQ ID No. 1.
3. The method of claim 2, comprising the steps of:
(1) preparing a fermentation culture of an N-methyltransferase inactivated mutant strain SCSIO Mla-L010/delta vicG, centrifugally separating fermentation supernatant and mycelia, extracting the mycelia with acetone, extracting the supernatant with butanone to obtain acetone extract and butanone extract, concentrating the extract under reduced pressure to obtain mycelia extract and supernatant extract, and mixing the two extracts to obtain a crude extract;
(2) mixing the crude extract obtained in the step (1) with silica gel, carrying out normal phase silica gel column chromatography, and carrying out gradient elution by adopting a chloroform/methanol system according to the following volume ratio: 100: 0; 98: 2; 96: 4; 94: 6; 92: 8; 9: 1; 8: 2; 6: 4; 5: 5; 0:100, sequentially obtaining 10 components Fr.A1-10; the components chloroform/methanol 92: 8; 9: 1; 8: 2; and (3) combining Fr.A5-8 eluted at the ratio of 6:4, and purifying by HPLC to obtain the 4' -N-demethyl-vicenistatin.
4. The preparation method according to claim 3, wherein the purification by HPLC in the step (2) is performed by using methanol as a mobile phase, performing chromatography using Sephadex LH-20 hydroxypropyl Sephadex chromatography column and collecting a target component, and preparing a target peak by using semi-preparative high performance liquid chromatography and ODS chromatography column (250X 10mm,5 μm) under the following conditions: CH (CH)3CN/H2Isocratic elution in O (32:68, V/V, containing 0.1% acetic acid) system for 25min at flow rate of 2.5mL/min to obtain the 4' -N-demethyl-vicenistatin, tR＝14.1min。
5. Use of 4' -N-demethyl-vicenistatin according to claim 1 for the preparation of an anti-infective or anti-tumor medicament.
6. An anti-infective drug characterized by containing the 4' -N-demethyl-vicenistatin according to claim 1 as an active ingredient.
7. The anti-infective agent of claim 6, wherein the anti-infective agent is an anti-Staphylococcus aureus (Staphylococcus aureus), Methicillin-resistant Staphylococcus aureus (Methicillin-resistant Staphylococcus aureus), Micrococcus luteus (Micrococcus luteus), Bacillus subtilis (Bacillus subtilis), Cryptococcus neoformans (Cryptococcus neoformans), Helicobacter pylori (Helicobacter pylori) or Candida albicans (Candida albicans) agent.
8. An antitumor agent comprising the 4' -N-demethyl-vicenistatin according to claim 1 as an active ingredient.
9. The antitumor drug as claimed in claim 8, wherein the antitumor drug is a drug for resisting breast cancer, liver cancer, cervical cancer, non-small cell lung cancer, colon adenocarcinoma or leukemia.
Use of an N-methyltransferase inactivating mutant SCSIO Mla-L010/Δ vicG, the sequence of which is shown in SEQ ID No.1, for the preparation of 4' -N-methyl-vicenitatin according to claim 1, wherein SCSIO Mla-L010/Δ vicG is obtained by inactivating an N-methyltransferase gene vicG in the vicenitatin gene cluster of the S.parvus SCSIO Mla-L010 genome.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH05186493A (en) * 1992-01-08 1993-07-27 Kirin Brewery Co Ltd New compound hc34, its use and production

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH05186493A (en) * 1992-01-08 1993-07-27 Kirin Brewery Co Ltd New compound hc34, its use and production

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ATSUSHI MINAMI,ET AL.: "Substrate Flexibility of Vicenisaminyltransferase VinC Involved in the Biosynthesis of Vicenistatin", 《J. AM. CHEM. SOC.》 *
YI JIANG，ET AL.: "Diversity and Bioactivity of Cultivable Animal Fecal Actinobacteria，", 《ADVANCES IN MICROBIOLOGY》 *

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