CN114437155B - 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 PDF

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CN114437155B
CN114437155B CN202210128899.XA CN202210128899A CN114437155B CN 114437155 B CN114437155 B CN 114437155B CN 202210128899 A CN202210128899 A CN 202210128899A CN 114437155 B CN114437155 B CN 114437155B
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鞠建华
周镇槟
杨佳凡
宋永相
田新朋
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South China Sea Institute of Oceanology of CAS
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Abstract

The invention discloses two macrolide compounds and application thereof in preparing antibacterial and anticancer medicaments. The structure of the macrolide 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.
Figure DDA0003501785750000011

Description

Two macrolide compounds and application thereof in preparation of antibacterial and anticancer drugs
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. The method is based on the secondary metabolic pathway in actinomycetes, and has great significance for developing microbial drugs by modifying active lead compounds through a synthetic biology technology. 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):
Figure BDA0003501785730000021
the inventor discovers that the secondary metabolite component with the characteristic absorption peak of the macrolide compound at 254nm of an ultraviolet spectrum has obvious antibacterial activity by carrying out antibacterial activity screening and HPLC-DAD spectrum analysis on a fermentation extract of rare actinomycetes pseudomonad sp.SCSIO 07407 from deep sea bottom mud. 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 preferably a drug for resisting staphylococcus aureus and drug-resistant bacteria thereof, enterococcus faecium and drug-resistant bacteria thereof, streptococcus faecalis, staphylococcus haemolyticus 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).
A fifth object of the present 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 a use of Pseudonocardia sp.scsio 07407- Δ 292 for preparing compound 2, wherein said Pseudonocardia sp.scsio 07407- Δ 292 is obtained by contacting Pseudonocardia sp.scsio 07407 GDMCC No: 62195, the nucleotide sequence of said 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 diagram of Compound 1 1 H NMR(500MHz) Map, solvent is: a deuterated MeOD;
FIG. 2 is a drawing of Compound 1 13 C NMR (125MHz) spectrum, solvent: a deuterated MeOD;
FIG. 3 is a schematic representation of Compound 2 1 H NMR (700MHz) spectrum, solvent: a deuterated MeOD;
FIG. 4 is a drawing of Compound 2 13 C 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 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 multi-tube 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, the double digested SuperCosI vector and Sau3AI were used for digestion treatment and dephosphorylation of genomic DNA using T4 ligase at room temperature ligation, in vitro phage packaging and infection of 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 pseudomonarda sp.SCSIO 07407 genome completion diagram 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-out 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 inactivation of orf292 gene adopts lambda-RED mediated PCR-Targeting technology. The Cosmid214G containing orf292 gene was transformed into E.coli BW25113/pIJ790 to obtain E.coli BW25113/pIJ790/Cosmid 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, modified recombinant c osmid is obtained through lambda-RED mediated homologous recombination, and the plasmid is named pcosmid 216C-delta orf 292. The correctness of the recombinant cosmid was 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 for orf292 gene (ID-orf 292-F:5'-CTCGCCCAGGTCCAGCTCAG-3', ID-orf292-R: 5'-CGGTCACATCAACCCCACCC-3'). 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 by 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. The 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,FeCl 3 ·6H 2 O:4g/L,CuCl 2 ·H 2 O: 0.2g/L,NaB 4 O 7 ·10H 2 O:0.2g/L,MnCl 2 ·4H 2 0.2g/L of O, water as solvent), 0.01 percent of crude sea salt, pH7.2-7.4, CaCO 3 0.2 percent and the balance of water. The weighed substances are dissolved and mixed uniformly according to the formula, 50mL of the solution is subpackaged into 250mL conical flasks per flask, and the conical flasks 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: in total mass fraction100% of the total content of the components comprises 2% of soluble starch, 0.5% of corn flour, 1% of malt extract, 1% of maltose, 1% of glucose, 0.01% of trace elements (same seed culture medium), 0.3% of crude sea salt, pH7.2-7.4, CaCO 3 0.2 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 mixture is subpackaged into 1000mL conical flasks per flask, and the conical flasks are sterilized at 115 ℃ for 30 minutes to serve as an amplification fermentation medium.
(2) Fermentation culture:
respectively inoculating the cultured seed culture solution into an amplification fermentation culture medium under aseptic conditions, inoculating half flask of seed culture solution (about 25mL) into each 1000mL conical flask (containing about 200mL of amplification fermentation culture medium), and performing shake culture at 28 ℃ for 7 days at 200rpm to obtain the fermentation products of the marine rare actinomycetes pseudomonad sp.SCSIO 07407 and the mutant 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 200-mesh silica gel, 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 A5-A9(C/M,92/8,90/10,80/20,70/30,50/50, v/v elution fractions) 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/min 3 CN/H 2 O is 20/80-100/0 within 120min, v/v), 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 the mutant strain Pseudonocardia sp.SCSIO 07407-delta 292 was centrifuged at 3900rpm for 10min to obtain a mixture of precipitated mycelia 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-inch silica gel of 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 and further gradient eluted at a detection wavelength of 254nm at a flow rate of 15mL/min using reversed-phase ODS medium-pressure MPLC (CH-DAD assay) 3 CN/H 2 O is 20/80-100/0 within 120min, v/v), 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 amorphous powder, [ alpha ]] 25 D -12.21(c0.01，MeOH)；UV(MeOH)λ max (logε)254 (3.80)nm；IR(ATR)ν max 3393,2974,2359,1654,1624,1458,1375,1205,984,679cm -1 ； 1 H and 13 the CNMR spectrogram is shown in figure 1-2; (+) -HRESIMSm/z1185.6910[ M + H] + (calcdforC 60 H 100 N 2 O 21 , 1184.68).
Compound 2: brown amorphous powder, [ alpha ]] 25 D -12.11(c0.01，MeOH)；UV(MeOH)λ max (logε)254 (3.80)nm；IR(ATR)ν max 3391,2976,2359,1654,1614,1459,1365,1201,984,681cm -1 ； 1 H and 13 CNMR spectrogramFIGS. 3-4; (+) -HRESIMSm/z [ M + H] + 1023.6385(calcdforC 54 H 90 N 2 O 16 , 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).
Figure BDA0003501785730000101
Example 3:
bacteriostatic experiments on macrolide compound of example 1, compound 1 and compound 2.
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 specifically comprises the following 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) 8 CFU) 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) Preparing a sample and diluted bacteria liquid. Samples (Compound 1 or 2) were prepared at 3200. mu.g/mL, both dissolved in DMSO. The bacterial liquid is reasonably diluted to ensure that the final test concentration is about 5 multiplied by 10 4 CFU/mL。
3) Adding LB liquid culture medium. 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): 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 medium was added to each well of columns 1-11. 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 10 4 CFU/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
Figure DEST_PATH_IMAGE001
Figure DEST_PATH_IMAGE002
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% CO 2 The 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 ℃ and 5% CO 2 After 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 (%). gtoreq.A sample group/A negative control group X100%
The experimental result is shown in table 2, after the compound 1 treats the 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 anticancer drug lead compounds.
Table 2: cytotoxic Activity (IC) of Compound 1 50 ,μM)
Figure DEST_PATH_IMAGE003
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 (7)

1. A macrolide compound or a pharmaceutically acceptable salt thereof, having the structure shown below:
Figure FDA0003752536820000011
2. the use of compound 1 of claim 1 in the preparation of an antibacterial or anticancer agent against staphylococcus aureus or resistant bacteria thereof, enterococcus faecium or resistant bacteria thereof, streptococcus faecalis, staphylococcus haemolyticus, 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.
3. An antibacterial or anticancer agent comprising an effective amount of compound 1 or a pharmaceutically acceptable salt thereof as claimed in claim 1 as an active ingredient, and a pharmaceutically acceptable carrier; the antibacterial drug is a drug for resisting staphylococcus aureus or drug-resistant bacteria thereof, enterococcus faecium or 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.
4. A process for the preparation of a macrolide compound, 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 the glucosyltransferase is shown in SEQ ID NO. 1;
the structural formulas of the compound 1 and the compound 2 are shown as follows:
Figure FDA0003752536820000021
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.
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