CN114437155A - Two macrolide compounds and application thereof in preparation of antibacterial and anticancer drugs - Google Patents
Two macrolide compounds and application thereof in preparation of antibacterial and anticancer drugs Download PDFInfo
- Publication number
- CN114437155A CN114437155A CN202210128899.XA CN202210128899A CN114437155A CN 114437155 A CN114437155 A CN 114437155A CN 202210128899 A CN202210128899 A CN 202210128899A CN 114437155 A CN114437155 A CN 114437155A
- Authority
- CN
- China
- Prior art keywords
- compound
- scsio
- antibacterial
- cancer
- gdmcc
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
-
- 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
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
-
- 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
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
- C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Engineering & Computer Science (AREA)
- General Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicinal Chemistry (AREA)
- Oncology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Communicable Diseases (AREA)
- Hematology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses two macrolide compounds and application thereof in preparing antibacterial and anticancer medicaments. MacrolidesThe structure of the compound is shown as a formula (I). The macrolide compound-compound 1 and compound 2 are novel deep sea rare actinomycete source compounds, have obvious inhibition effect on tested pathogenic bacteria and cancer cells, and can be used for preparing bacteriostatic drugs or anti-cancer drugs, so that the macrolide compound-compound 1 and compound 2 provide new alternative compounds for developing clinical treatment of bacterial infection or cancer, and have important significance for developing marine drug resources in China.
Description
The technical field is as follows:
the invention belongs to the field of natural products, and particularly relates to two macrolide compounds and application thereof in preparation of antibacterial and antimicrobial medicaments.
Background art:
pathogenic drug-resistant bacteria such as vancomycin-resistant enterococci (VRE) and methicillin-resistant Staphylococcus aureus (MRSA) are found in the middle of the last century and spread all over the world. Drug-resistant bacteria infection has the characteristics of high morbidity and high mortality, and occupies and loses a large amount of social medical resources, so that the demand of novel antibiotics for resisting the drug-resistant bacteria by human is increasingly urgent. Meanwhile, cancer is a serious threat to human health and life.
The extreme living environment of the deep-sea actinomycetes promotes the deep-sea actinomycetes to evolve a unique metabolic pathway adapting to the extreme environment, has the potential of generating compounds with novel structures and unique activity mechanisms, and is always an important resource for drug development. Based on the secondary metabolic pathway in actinomycetes, the active lead compound is modified by a synthetic biology technology, and the method has great significance for developing microbial drugs. The secondary metabolites of the actinomycetes have macrolide compounds, have wide biological activities including various biological activities such as anti-tumor, antibacterial and antiviral activities, and have good drug forming potential.
The marine actinomyces from deep sea is taken as a research object, the macrolide active secondary metabolite pathway is researched and modified, and a positive propulsion effect is generated for relieving the current predicament that human beings are lack of drug-resistant bacteria and anti-cancer drugs.
The invention content is as follows:
it is a first object of the present invention to provide two macrolide compounds having antibacterial and anticancer activities and pharmaceutically acceptable salts thereof.
The structure of two macrolide compounds or pharmaceutical salts thereof of the invention is shown in formula (I):
the inventor discovers that the secondary metabolite component with the characteristic absorption peak of the macrolide compound at the ultraviolet spectrum of 254nm has obvious bacteriostatic activity by carrying out bacteriostatic activity screening and HPLC-DAD spectrum analysis on a fermentation extract of rare actinomycetes Pseudonocardia sp. Further, amplifying fermentation, extraction, separation and purification by a shaking table to obtain a macrolide compound 1; and further knocking out a glucosyltransferase gene orf292 by a gene knocking-out technology, and carrying out amplified fermentation, extraction, separation and purification on the obtained knocked-out mutant strain Pseudonocardia sp.SCSIO 07407-delta 292 to obtain another deglucosylated macrolide compound 2. Through HRESIMS (+), 1D, 2D NMR and other technologies, two monomers are determined to be novel macrolide compounds, and the specific structure is shown as a formula (I).
The antibacterial activity evaluation of the compound 1 and the compound 2 shows that the compound has obvious inhibitory activity on staphylococcus aureus, enterococcus faecium, enterococcus faecalis, lysostaphin or bacillus subtilis, and has the potential of developing antibacterial drug lead compounds.
The compound 1 is tested for tumor cytotoxic activity, and is found to have good activity on selected human colorectal cancer cells (RKO), liver cancer cells (HepG2), cervical cancer cells (Hela), leukemia cells (HL60), non-small cell lung cancer (A549), breast cancer (MCF-7) and the like, is superior to an anti-tumor medicament cisplatin, and has the potential of being developed into an anti-cancer medicament lead compound.
Therefore, the second purpose of the invention is to provide the application of the compound 1 or the compound 2 in preparing antibacterial or anticancer drugs.
The antibacterial drug is used for resisting gram-positive bacteria, preferably staphylococcus aureus and drug-resistant bacteria thereof, enterococcus faecium and drug-resistant bacteria thereof, streptococcus faecalis, hemolytic staphylococcus or bacillus subtilis.
The anti-cancer drug is a drug for resisting colorectal cancer, liver cancer, cervical cancer, leukemia, non-small cell lung cancer or breast cancer.
The third purpose of the invention is to provide an antibacterial and anticancer drug, which is characterized by comprising an effective amount of compound 1 or compound 2 shown as formula (I) as an active ingredient, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
A fourth object of the present invention is to provide a process for producing the above macrolide compound, wherein the compound 1 is a compound obtained from Pseudonocardia sp.scsio 07407 GDMCC No: 62195, and compound 2 is isolated from a fermentation culture of pseudomonadacea sp.scsio 07407- Δ 292, wherein said pseudomonadacea sp.scsio 07407- Δ 292 is prepared by contacting pseudomonadacea sp.scsio 07407 GDMCC No: 62195, the nucleotide sequence of the glucosyltransferase is shown in SEQ ID NO.1(NCBI access number: OM 632674).
The fifth purpose of the invention is to provide Pseudonocardia sp.SCSIO 07407 GDMCC No: 62195 in the preparation of Compound 1.
A sixth object of the present invention is to provide the use of Pseudonocardia sp.scsio 07407- Δ 292, wherein Pseudonocardia sp.scsio 07407- Δ 292 is obtained by contacting Pseudonocardia sp.scsio 07407 GDMCC No: 62195, the nucleotide sequence of the glucosyltransferase is shown in SEQ ID NO.1(NCBI access number: OM 632674).
A seventh object of the present invention is to provide Pseudonocardia sp.scsio 07407, accession No.: GDMCC No: 62195.
The macrolide compound-compound 1 and compound 2 are novel compounds of marine origin, and have obvious inhibition effect on tested pathogenic bacteria and cancer cells, so that the invention provides an alternative compound for developing new antibacterial and anticancer drugs, and has great significance for building a 'blue drug library' in China.
The rare actinomycetes pseudomona sp.SCSIO 07407 from deep sea sediment is preserved in Guangdong province microbial culture collection center (GDMCC) at 1 month and 10 days in 2022, and the address is as follows: building No. 59, building No. 5 of the institute of microbiology, department of science, guangdong province, 100 of the first furious middle school, guangdong province, zip code: 510070, accession number: GDMCC No: 62195.
description of the drawings:
FIG. 1 is a drawing of Compound 11H NMR (500MHz) spectrum, solvent: deuterated MeOD;
FIG. 2 is a drawing of Compound 113C NMR (125MHz) spectrum, solvent: a deuterated MeOD;
FIG. 3 is a schematic representation of Compound 21H NMR (700MHz) spectrum, solvent: a deuterated MeOD;
FIG. 4 is a drawing of Compound 213C NMR (175MHz) spectrum, solvent: deuterated MeOD.
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.
Example 1:
construction of the mutant Strain Pseudonocardia sp.SCSIO 07407-Delta 292
Construction of Pseudonocardia sp.SCSIO 07407 genetic manipulation System
1. Construction of genomic libraries
The genomic library was constructed by reference to SuperCos1 Cosmid Vector Kit and Gigapck III XL packing Extract operating manual. Culturing Pseudomonas sp.SCSIO 07407 in TSB liquid culture medium for 2 days, collecting thallus, extracting genome DNA by mature phenol chloroform extraction method, taking appropriate amount of high purity DNA, performing enzyme digestion treatment with different enzyme digestion time and different Sau3AI enzyme concentration, determining optimal reaction time and enzyme reaction concentration, performing multitubular enzyme digestion of the same system, and performing dephosphorylation treatment; meanwhile, the SuperCosI vector is treated by XbaI restriction endonuclease, and dephosphorylation treatment is also carried out after enzyme digestion; then carrying out BamHI enzyme digestion on the dephosphorylated SuperCos1 vector. For fragment recovery, mature phenol chloroform extraction was used. Finally, carrying out enzyme digestion treatment on the double-enzyme digested SuperCosI vector and Sau3AI, dephosphorylating the genomic DNA, connecting the genomic DNA with T4 ligase at room temperature, carrying out in-vitro phage packaging, and infecting a host strain E.coli LE 392. The infected strain was spread on a resistant plate containing 100. mu.g/mL kanamycin, and about 2000 clones were randomly picked and stored in a 96-well plate to complete the construction of a genomic library of the strain.
2. Screening and obtaining of Cosmid containing target glucosyltransferase gene orf292
The sequencing work of the pseudomonad sp.SCSIO 07407 genome completion map was performed by Shanghai Linn biology, Inc. According to the complete sequence of the strain genome provided by Linn bioinformatics, Inc., combined with the online bioinformatics software and the structural characteristics of the compound 1, we obtained the biosynthetic gene cluster of the compound 1, in which orf292 gene (the nucleotide sequence of which is shown in SEQ ID NO.1, NCBI access number: OM632674) is the target glucosyltransferase gene. To determine the function of orf292 gene in the biosynthesis process of compound 1, we performed gene knock-outs on it separately. According to the requirement of PCR-targeting operation, in order to obtain cosmid capable of knocking out orf292, two pairs of screening library primers are designed at the upstream and the downstream of the orf292 gene sequence, namely upstream 292-F1 (5'-GAAATCGACCTCAAAGCGGC-3') and 292-R1 (5'-AGGTCGTCGTCGATATCAAC-3') and downstream 292-F2 (5'-CCGCTTCGTCGTCGGCTACA-3') and 292-R2 (5'-GGTGGTGGTCATGCGCTCCC-3'). Using these two pairs of primers, positive clones were screened from the constructed genomic library using PCR technology. The PCR procedure was as follows: 5min at 95 ℃; 45s at 95 ℃, 45s at 58 ℃, 60s at 72 ℃ and 30 cycles; 10min at 72 ℃. Multiple positive clones were selected, and by end sequencing, we selected the clone (named Cosmid214G) at the corresponding (4, G) position in 96-well plate No. 21 for the next step of orf292 gene knock-out.
Construction of orf292 Gene deletion mutant Strain Pseudonocardia sp.SCSIO 07407
The orf292 gene is inactivated by adopting a lambda-RED mediated PCR-Targeting technology. The plasmid 214G containing orf292 gene was transformed into E.coli BW25113/pIJ790 to obtain E.coli BW25113/pIJ 790/plasmid 214G. PCR amplification reaction of Apr resistant fragment aa c (3) IV-oriT resistant fragment with wild type 39bp homology arm using pIJ773 plasmid fragment digested by EcoRI and HindIII as template and knock-out primer with 39bp homology arm respectively with orf292 gene (delF:5'-CGCCACCATCGGGACCCGGGCCCGCGCGGCCTCCAGGACattccggggatccgtcg acc-3', delR:5'-GTGGTGGCGGACATGGTCGGGCGCGGGCACCGCGTCACCtgtaggctggagct gcttc-3' capital letter part represents homology with orf292 gene). The amplified aac (3) IV-oriT resistant fragment is electrically transformed into E.coli BW25113/pIJ790/Cosmid214G competent cells, and modified recombinant c osmid is obtained through homologous recombination mediated by lambda-RED, and the plasmid is named pcosmid 216C-delta orf 292. The correctness of the recombinant cosmid is determined by PCR amplification (95 ℃ for 5 min; 95 ℃ for 45 s; 58 ℃ for 45 s; 72 ℃ for 2 min; 30 cycles; 72 ℃ for 10min) with PCR-targeting verification primers (ID-orf 292-F:5'-CTCGCCCAGGTCCAGCTCAG-3', ID-orf292-R: 5'-CGGTCACATCAACCCCACCC-3') of orf292 gene. Subsequently, the recombinant pCosmid214G- Δ orf292 was transferred into E.coli ET12567/pUZ8002 for conjugal transfer, thereby obtaining E.coli ET12567/pUZ8002, namely E.coli ET12567/pUZ8002/pORF292, into which recombinant plasmids were transferred, which were designated as E.coli ET12567/pUZ8002/pORF292, that was used for mediating the conjugal transfer of wild-type strains.
The process of bond transfer is as follows: culturing spores of wild type pseudomona sp.SCSIO 07407 in TSB liquid medium at 28 deg.C under shaking for 1-2 hr to make the spores germinate; coli ET12567/pUZ8002/pORF292 was cultured in LB medium supplemented with kanamycin (Kan, final concentration 50 μ g/mL), chloramphenicol (Cml, final concentration 25 μ g/mL) and adriamycin (Apr, final concentration 50 μ g/mL) to an optical density OD of 0.6, cells were collected by centrifugation, washed twice with sterile LB liquid medium, resuspended in 500 μ L of liquid sterile LB, and mixed with the germinated wild type. The mixture was spread on MS solid medium with 10mM MgSO4 and 3% sea salt added; after 18 hours of growth at 28 ℃, each solid media plate was overlaid with 1mL of sterile water supplemented with trimethoprim and antibiotic drugs of apramycin to kill e.coli ET12567/pUZ8002/porf292, which contained 30 μ Ι _, of trimethoprim (Tmp,50mg/mL), 30 μ Ι, of apramycin (Apr, 50mg/mL), and the overlaid plates continued to grow for 7-9 days at 28 ℃ incubator until zygotes appeared; the double crossover mutant strain Pseudonocardia sp.SCSIO 07407-delta 292 was selected for its resistance phenotype to apramycin, and PCR amplification using the same validation primers (ID-orf292-F, ID-orf292-R) as previously constructed for pcosmid-delta orf292 confirmed that the selected mutant was a double crossover mutant, thereby obtaining Pseudonocardia sp.SCSIO 07407 with deletion mutation of orf292 glucosyltransferase gene, which was designated as double crossover mutant strain Pseudonocardia sp.SCSIO 07407-delta 292. This strain was further verified by fermentation using M-ISP3 medium (consistent with the seed medium formulation in example 2) confirming that it no longer produces compound 1 with glucose groups but accumulates compound 2 without glucose groups.
Example 2:
preparation and structural identification of Compound 1 and Compound 2 of formula (I)
Preparation of compound 1 and compound 2 shown in formula (I)
1. Seed culture:
(1) the seed culture medium formula comprises: calculated by the mass fraction of 100 percent, comprises 2 percent of soluble starch, 0.5 percent of corn flour, 1 percent of malt extract, 1 percent of maltose, 1 percent of glucose and trace elements (ZnCl)2:0.8g/L,FeCl3·6H2O:4g/L,CuCl2·H2O: 0.2g/L,NaB4O7·10H2O:0.2g/L,MnCl2·4H20.2g/L of O, water as solvent), 0.01 percent of crude sea salt, pH7.2-7.4, CaCO30.2 percent and the balance of water. The weighed substances are dissolved and mixed evenly according to the formula, 50mL of the mixture is subpackaged in 250mL conical bottles, and the conical bottles are sterilized at 115 ℃ for 30 minutes to serve as seed culture media.
(2) The seed solid culture medium formula comprises: calculated by 100 percent of mass fraction, the agar comprises 0.4 percent of glucose, 1 percent of malt extract, 0.5 percent of yeast extract, 0.3 percent of crude sea salt, pH7.2-7.4, 1.5 percent of technical agar powder and the balance of water. Dissolving the weighed substances according to the formula, uniformly mixing, sterilizing at 115 ℃ for 30 minutes, pouring the mixture into a flat plate to be used as a seed solid culture medium, and standing and culturing at 28 ℃.
(3) Culturing seeds: mycelia of strains Pseudonocardia sp.SCSIO 07407 and Pseudonocardia sp.SCSIO 07407-Delta 292, which were cultured on seed solid media for 7 days, were inoculated into the seed media, and shake-cultured at 28 ℃ for 36 hours at 200rpm to obtain seed culture solutions.
2. Amplification fermentation culture:
(1) the formula of the amplified fermentation medium is as follows: comprises (by total mass fraction 100%) soluble starch 2%, corn flour 0.5%, malt extract 1%, maltose 1%, glucose 1%, trace elements (same seed culture medium) 0.01%, crude sea salt 0.3%, pH7.2-7.4, CaCO30.2 percent, 2 percent of macroporous resin (XAD-16) and the balance of water. The weighed substances are dissolved and mixed uniformly according to the formula to prepare about 20L of total volume, and then 200mL of the total volume is subpackaged into 1000mL conical flasks, and the conical flasks are sterilized at 115 ℃ for 30 minutes to serve as an amplification fermentation culture medium.
(2) Fermentation culture:
respectively inoculating the cultured seed culture solution into an amplification fermentation culture medium under sterile conditions, inoculating half flask of seed culture solution (about 25mL) into each 1000mL conical flask (containing about 200mL amplification fermentation culture medium), and performing shake culture at the rotation speed of 200rpm and the temperature of 28 ℃ for 7 days to obtain the fermentation products of the marine rare actinomyces pseudomonad sp.SCSIO 07407 and the mutant strain pseudomonad sp.SCSIO 07407-delta 292.
3. Extraction and separation:
centrifuging the fermentation product of strain Pseudomonas sp.SCSIO 07407 at 3900rpm for 10min to obtain a mixture of precipitated mycelium and macroporous resin. Extracting the mixture of the precipitated mycelium and macroporous resin with 2L food grade ethanol for 4 times, and concentrating the ethanol extract at 35 deg.C under reduced pressure to obtain crude extract. The extract is separated by 100-mesh silica gel of 200 meshes, and is subjected to sample mixing and column filling by a dry method, and a chloroform/methanol system (C/M,100/0,98/2,96/4,94/6,92/8,90/10, 80) is adopted/20,70/30,50/50 v/v) to give 9 fractions (A1-A9). Fractions A5-A9(C/M,92/8,90/10,80/20,70/30,50/50, v/v elution fraction) were combined and further eluted with a gradient (CH) using reversed-phase ODS medium-pressure MPLC at a detection wavelength of 254nm at a flow rate of 15mL/min3CN/H2O is 20/80-100/0, v/v) within 120min, and each 10min is divided into 12 fractions (B1-B12). Fractions B4-B7(30-70min fractions eluted) were prepared using A reverse phase chromatography column YMC-PackODS-A (250X 20mm,5 μm), respectively, at A flow rate of 2.5mL/min, A detection wavelength of 254nm, and A volume ratio of A/B: 20/80% -80/20% v/v was eluted with a linear gradient (A: water, B: acetonitrile). Final retention time 15.6min gave Compound 1(20.3 mg).
The fermentation product of mutant Pseudonocardia sp.SCSIO 07407-delta 292 was centrifuged at 3900rpm for 10min to obtain a mixture of precipitated mycelium and macroporous resin. Extracting the mixture of the precipitated mycelium and macroporous resin with 2L food grade ethanol for 4 times, and concentrating the ethanol extract at 35 deg.C under reduced pressure to obtain crude extract. The extract is separated by 100-mesh silica gel with 200 meshes, and 9 components (A1-A9) are obtained by adopting a chloroform/methanol system (C/M,100/0,98/2,96/4,94/6,92/8,90/10,80/20,70/30 and 50/50 v/v) gradient elution sequence after sample mixing and column filling by a dry method. Fractions A4-A7(C/M,94/6, 92/8,90/10,80/20, v/v elution fractions) were pooled by HPLC-DAD detection and further gradient eluted using reversed-phase ODS medium-pressure MPLC at a detection wavelength of 254nm at a flow rate of 15mL/min (CH)3CN/H2O is 20/80-100/0, v/v) within 120min, and is divided into one fraction every 10min, so that 12 fractions (B1-B12) are obtained. Fractions B4-B6(30-60min fractions eluted) were prepared separately using A reversed-phase column YMC-Pack ODS-A (250X 20mm,5 μm) at A flow rate of 2.5mL/min and A detection wavelength of 254nm, and the ratio of A/B: 20/80% -80/20% v/v was eluted with a linear gradient (A: water, B: acetonitrile). Compound 2(4.3mg) was finally obtained at a retention time of 16.7 min.
Second, physicochemical data of Compound 1 and Compound 2
The following physicochemical property data were obtained by performing structural analysis tests on compound 1 and compound 2:
compound 1: brown colourAmorphous powder, [ alpha ]]25 D-12.21(c0.01，MeOH)；UV(MeOH)λmax(logε)254 (3.80)nm；IR(ATR)νmax3393,2974,2359,1654,1624,1458,1375,1205,984,679cm-1；1H and13the CNMR spectrogram is shown in figure 1-2; (+) -HRESIMSm/z1185.6910[ M + H]+(calcdforC60H100N2O21, 1184.68).
Compound 2: brown amorphous powder, [ alpha ]]25 D-12.11(c0.01，MeOH)；UV(MeOH)λmax(logε)254 (3.80)nm；IR(ATR)νmax3391,2976,2359,1654,1614,1459,1365,1201,984,681cm-1；1H and13the CNMR spectrogram is shown in FIGS. 3-4; (+) -HRESIMSm/z [ M + H]+1023.6385(calcdforC54H90N2O16, 1022.63).
According to the analysis of the physicochemical data, the structures of the compound 1 and the compound 2 are shown as the formula (I).
Example 3:
bacteriostatic experiments on macrolide compound-compound 1 and compound 2 of example 1.
Staphylococcus aureus and its resistant bacterium (Staphylococcus aureus), Enterococcus faecium and its resistant bacterium (Enterococcus faecium), Streptococcus faecalis (Enterococcus faecium), Staphylococcus haemolyticus (Staphylococcus haemolyticus) or Bacillus subtilis (Bacillus subtilis) are used as test bacteria, and 100 μ l system test activity is performed on the compounds 1-2 by referring to CLSI microplate method. The method comprises the following specific steps:
1) and (3) bacterial culture: the experimental bacteria were cultured in LB liquid medium when they grew for 8-12h to about 0.5 Mcfarland concentration (1X 10)8CFU) is ready for use. And preparing a sample solution and a positive control solution with certain concentration, wherein the positive control is ampicillin or vancomycin hydrochloride (water soluble).
2) And preparing a sample and diluted bacteria liquid. The sample (Compound 1 or 2)) Configured into 3200. mu.g/mL, all dissolved in DMSO. The bacterial liquid is reasonably diluted to ensure that the final test concentration is about 5 multiplied by 104CFU/mL。
3) LB liquid culture medium is added. A96-well plate was inoculated with LB liquid medium using a line gun, 92. mu.l of sterile LB liquid medium was added to column 1, 100. mu.l of sterile LB liquid medium was added to column 12, and 50. mu.l of sterile LB liquid medium was added to each of the remaining columns, and column 11 and column 12 were used as positive and negative controls, respectively.
4) Add sample (drug product): mu.l of the previously prepared sample solution or positive control solution (also dissolved in DMSO) was pipetted and added to column 1. The gun volume was set to 50 μ l and the test drug in column 1 was carefully pipetted 4-5 times up and down to mix well, while preventing over-forceful spillage.
5) The sample (drug) was mixed well. Sucking 50 mul from the first row with a row gun, adding into the corresponding second row, sucking carefully 4-5 times up and down, mixing well, sucking 50 mul, and adding into the third row. This was followed until dilution to column 10, and 50. mu.l was removed from column 10 and discarded.
6) And (4) testing the activity. 50. mu.l of diluted test bacterial solution was added to each well of 1-11 columns. At this time, the concentrations of the drugs in the 1 st to 10 th columns were 128, 64, 32, 16, 8, 4, 2, 1, 0.5 and 0.25. mu.g/ml, respectively, and the test concentration of the test bacteria was about 5X 104CFU/mL. Covering a cover, slightly shaking, placing in an incubator at 37 ℃ for 12-18 hours, performing drug-free bacteria liquid control in the 11 th column, adding 8 mu l of prepared bacteria liquid in advance, and performing blank control in the 12 th column to determine the MIC value of each sample.
Each sample was done in 3 replicates. The results are shown in Table 1.
As can be seen from Table 1, the compounds 1 and 2 have significant antibacterial activity on 24 tested pathogenic bacteria, are superior to ampicillin as a positive control and have activity equivalent to vancomycin hydrochloride, and provide support for research and development of drug-resistant antibiotics based on the positive control.
Table 1: MIC values (μ g/mL) of Compounds 1-2 against 24 test bacteria
Example 3:
anticancer cell assay of macrolide Compound 1 in example 1
The cytotoxic activity of compound 1 was determined by the CCK8 method. The tumor cells used in this experiment were human colorectal cancer cells (RKO), liver cancer cells (HepG2, LO2, A549), cervical cancer cells (Hela), leukemia cells (HL60), non-small cell lung cancer cells (A549), breast cancer cells (MCF-7) and normal lung epithelial cells (BEAS-2B). The compound 1 is dissolved by dimethyl sulfoxide to obtain mother liquor with the concentration of 20mmol/L, and then the mother liquor is diluted to the required concentration by a culture medium corresponding to a cell strain. Taking each cell in logarithmic growth phase, inoculating the cell into 96-well plate at 3000/well, making 5 auxiliary wells for each concentration gradient, setting 3 blank zero-setting, and adjusting at 37 deg.C and 5% CO2The culture was carried out in an incubator for 24 hours. After the cells adhere to the wall, the prepared compound 1 solution is added into each hole according to the required concentration gradient, the culture medium with the same volume is added into the negative control, and the total volume of the liquid in each hole is 100 mu l. Placing at 37 deg.C and 5% CO2After culturing in an incubator for 48 hours, 10. mu.L of CCK8 was added to each well, and after 3 hours in the incubator, the absorbance (A) at 450nm was measured using a microplate reader, the cell activity and the inhibition rate of cell growth after drug action were calculated using the following formulas, and the half-lethal concentration (IC50 value) of tumor cells was calculated using GraphPad Prism 5.0 software.
Cell activity (%). A sample group/A negative control group X100%
The experimental results are shown in table 2, and after the compound 1 is used for treating cells for 48 hours, the compound 1 is found to have obvious inhibitory activity on 8 tested cancer cells, is partially superior to the anticancer drug cisplatin, and has the potential of being further developed into an anticancer drug lead compound.
Table 2: cytotoxic Activity (IC) of Compound 150,μM)
In conclusion, the invention provides a new lead compound for developing novel antibacterial and anti-cancer drugs, and has important significance for further developing marine drug resources in China on the basis.
Sequence listing
<110> Nanhai ocean institute of Chinese academy of sciences
<120> two macrolide compounds and their use in preparing antibacterial and anticancer drugs
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1206
<212> DNA
<213> actinomycetes SCSIO 07407(Pseudonocardia sp.)
<400> 1
atggcccaca tcctcgtggt cgccccgccg gcggccggtc acatcaaccc caccctcgga 60
gtggtggcgg acatggtcgg gcgcgggcac cgcgtcacct acgccaccac cgcccgctac 120
cgcgaccgga tcgccgagac cggcgcgacg ctgctggcgc acgagtcgac catgccgccc 180
ccggcgacga agccgtacac gctcaccggg accgacctca cccgcggcct gctcgccggg 240
ctgcgcgagt cccgggtccg cccggccgag ctgcgccgcc gcctcgccgg cgaccgcccg 300
gacctcgtgg tgttcgacgg cctcaccgcc tggtggggcc ggctgctcgc gcgcgagctc 360
gacgtgccgg tggtgccgtg ctggccgaac ttcgtcgcca acgagcactg gtcgctcatg 420
ggcacctaca tccgctccaa ccgcctcgac ccgaggctgt ggtggttcgc cctgcggatg 480
cagcggctcg cgtccgcggc cgggctaacc ctcgccgagc tggtcgacag cgtcgggacg 540
ggggtccgcg cgcagctggt gttcctgccc cgcagcttcc agttcgcgca cgagaccttc 600
gacgacaccc accacttcgt cggtccctgt ccgggtgcgc gcacattcca ggggacctgg 660
tcgccgcccg cgtccgggcg gccggtctgc ctggtctcgc tgggcacggt ctactccgcg 720
gaccgcgggt tctacgagct ggcgctcgcg gcgttgcggg agtccgggct gcacgtggtg 780
ctggtcgtcg gcgagcacgt cgacccctac tccttcggcg cgcacggccc gcacgtcgag 840
atccacagct cggtgccgca gctgcaggtc ctcgagcacg ccgcggtgtt cgtgacccac 900
ggcggcatga acagcgtcct ggaggccgcg cgggcccggg tcccgatggt ggcggtgccg 960
cagatggccg agcagcgggc gaacgccgac cggctcgctg agctggacct gggcgagcgg 1020
ctcgacccgg ccgggctgtc cgcgggcgcg ctgcgcgccg cggtcgaccg ggtcaccggg 1080
gaccggcggc tcgccgccgg gctggaccgc atcgcggccg agatggagcg cgccggcggg 1140
acgtccgcgg cggcggacct gctggaggcc gcgctgcacg ggccggtcac cgccgcccgg 1200
acctga 1206
Claims (10)
1. A macrolide compound or a pharmaceutically acceptable salt thereof, having the structure shown in any one of the following formulas (i):
2. use of compound 1 or compound 2 of claim 1 for the preparation of an antibacterial or anticancer agent.
3. The use according to claim 2, wherein the antibacterial agent is an anti-gram-positive agent.
4. The use of claim 3, wherein said antibacterial agent is an agent against Staphylococcus aureus or resistant bacteria thereof, enterococcus faecium or resistant bacteria thereof, Streptococcus faecalis, Staphylococcus haemolyticus, or Bacillus subtilis.
5. The use of claim 2, wherein the anticancer agent is an anti-colorectal cancer, liver cancer, cervical cancer, leukemia, non-small cell lung cancer or breast cancer.
6. An antibacterial or anticancer agent comprising an effective amount of compound 1 or compound 2 of claim 1, or a pharmaceutically acceptable salt thereof, as an active ingredient, and a pharmaceutically acceptable carrier.
7. A process for the preparation of a macrolide compound according to claim 1, wherein compound 1 is prepared from Pseudonocardia sp.scsio 07407 GDMCC No: 62195, and compound 2 is isolated from a fermentation culture of pseudomonadacea sp.scsio 07407- Δ 292, wherein said pseudomonadacea sp.scsio 07407- Δ 292 is prepared by contacting pseudomonadacea sp.scsio 07407 GDMCC No: 62195, the nucleotide sequence of said glucosyltransferase is shown in SEQ ID NO. 1.
Pseudomonaddia sp.scsio 07407 GDMCC No: 62195 for the preparation of compound 1 of claim 1.
Use of pseudomonad sp.scsio 07407- Δ 292, wherein pseudomonad sp.scsio 07407- Δ 292 is prepared by contacting pseudomonad sp.scsio 07407 GDMCC No: 62195, the nucleotide sequence of said glucosyltransferase is shown in SEQ ID NO. 1.
Pseudomona sp.scsio 07407, accession number: GDMCC No: 62195.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210128899.XA CN114437155B (en) | 2022-02-11 | 2022-02-11 | Two macrolide compounds and application thereof in preparation of antibacterial and anticancer drugs |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210128899.XA CN114437155B (en) | 2022-02-11 | 2022-02-11 | Two macrolide compounds and application thereof in preparation of antibacterial and anticancer drugs |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114437155A true CN114437155A (en) | 2022-05-06 |
| CN114437155B CN114437155B (en) | 2022-09-20 |
Family
ID=81372089
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210128899.XA Active CN114437155B (en) | 2022-02-11 | 2022-02-11 | Two macrolide compounds and application thereof in preparation of antibacterial and anticancer drugs |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114437155B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH05286990A (en) * | 1992-04-08 | 1993-11-02 | Meiji Seika Kaisha Ltd | New macrolide antibiotic and production of macrolide antibiotic utilizing conversion of microorganism |
| CN101830884A (en) * | 2009-11-25 | 2010-09-15 | 中国科学院南海海洋研究所 | Macrolide as well as preparation method and antibacterial application thereof |
| CN103396476A (en) * | 2013-07-29 | 2013-11-20 | 中国科学院南海海洋研究所 | Two cyclic hexapeptide compounds and application thereof in preparation of antibacterial drug |
-
2022
- 2022-02-11 CN CN202210128899.XA patent/CN114437155B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH05286990A (en) * | 1992-04-08 | 1993-11-02 | Meiji Seika Kaisha Ltd | New macrolide antibiotic and production of macrolide antibiotic utilizing conversion of microorganism |
| CN101830884A (en) * | 2009-11-25 | 2010-09-15 | 中国科学院南海海洋研究所 | Macrolide as well as preparation method and antibacterial application thereof |
| CN103396476A (en) * | 2013-07-29 | 2013-11-20 | 中国科学院南海海洋研究所 | Two cyclic hexapeptide compounds and application thereof in preparation of antibacterial drug |
Non-Patent Citations (2)
| Title |
|---|
| HAMMANN PETER ET AL.: ""Secondary metabolites by chemical screening. I. Elaiophylin derivatives and their biological activities"", 《JOURNAL OF ANTIBIOTICS》 * |
| 凌春耀等: ""海鞘来源放线菌Streptomyces pratensis SCSIO LCY05中anthracimycin 类化合物及其抗菌活性研究"", 《中国海洋药物》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114437155B (en) | 2022-09-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Moebius et al. | Biosynthesis of the respiratory toxin bongkrekic acid in the pathogenic bacterium Burkholderia gladioli | |
| CN110218244B (en) | Compound ilamycin F and application thereof | |
| Guerrero-Garzón et al. | Streptomyces spp. from the marine sponge Antho dichotoma: Analyses of secondary metabolite biosynthesis gene clusters and some of their products | |
| Malla et al. | Improvement in doxorubicin productivity by overexpression of regulatory genes in Streptomyces peucetius | |
| CN103060248A (en) | Method for constructing gene engineering FK506 high-producing strain and streptomyces tsukubaensis high-producing strain | |
| Gao et al. | Modulation of kanamycin B and kanamycin A biosynthesis in Streptomyces kanamyceticus via metabolic engineering | |
| Myronovskyi et al. | Generation of new compounds through unbalanced transcription of landomycin A cluster | |
| Paulus et al. | New alpiniamides from Streptomyces sp. IB2014/011-12 assembled by an unusual hybrid non-ribosomal peptide synthetase trans-AT polyketide synthase enzyme | |
| Khushboo et al. | Biotechnological and industrial applications of Streptomyces metabolites | |
| Guan et al. | Important role of a LAL regulator StaR in the staurosporine biosynthesis and high-production of Streptomyces fradiae CGMCC 4.576 | |
| Molloy et al. | Biological characterization of the hygrobafilomycin antibiotic JBIR-100 and bioinformatic insights into the hygrolide family of natural products | |
| El-Hawary et al. | Elicitation for activation of the actinomycete genome's cryptic secondary metabolite gene clusters | |
| Kontou et al. | Discovery and characterization of epemicins A and B, new 30-membered macrolides from Kutzneria sp. CA-103260 | |
| CN101120084A (en) | Methods and compositions for ultra-high throughput screening of natural products | |
| Yang et al. | Streptomyces alleviate abiotic stress in plant by producing pteridic acids | |
| Wang et al. | Improved milbemycin production by engineering two Cytochromes P450 in Streptomyces bingchenggensis | |
| Wu et al. | Improving gentamicin B and gentamicin C1a production by engineering the glycosyltransferases that transfer primary metabolites into secondary metabolites biosynthesis | |
| CN114437155B (en) | Two macrolide compounds and application thereof in preparation of antibacterial and anticancer drugs | |
| CN102533896B (en) | Method for obtaining geldanamycin derivatives by knocking out o-methyl-transferase gene | |
| Liu et al. | Construction and application of a “superplasmid” for enhanced production of antibiotics | |
| CN100393867C (en) | Construction of geldanamycin gene engineering high yield strain | |
| CN105061537B (en) | A kind of nucleoside antibiotic and its application in antibacterials are prepared | |
| JP5283927B2 (en) | Novel compound amicolamycin, its production method and its use | |
| CN111269867B (en) | Gene engineering strain for directionally producing antiviral and antibacterial active compound and application thereof | |
| CN106188093B (en) | A kind of rapamycin structure analog and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |