Preparation and application of cephalomycin derivative with gram-positive bacterium activity resistance

Abstract

The invention discloses preparation and application of a hand mycin derivative with gram-positive bacterium activity resistance, and belongs to the technical field of medicine preparation. According to the present invention, the ctd gene cluster is subjected to heterologous expression in the strain Streptomyces lividans TK64, such that the obtained hand mycin derivatives Carpatamide N and Carpatamide O show excellent biological activity on the gram-positive bacteria; the MIC (minimal inhibitory concentration) values of the Carpamide N and the Carpamide O on streptococcus faecium and enterococcus faecium are 4 mu g / mL and 1 mu g / mL respectively; the MIC (minimal inhibitory concentration) values for staphylococcus aureus and bacillus subtilis are respectively 0.25 mu g / mL and 0.125 mu g / mL, the MIC values for MRSA (Methicillin-Resistant Staphylococcus Aureus) are respectively 1 mu g / mL and 0.5 mu g / mL, and the Gram-positive bacterium resisting activity of the compound is equivalent to or better than that of vancomycin.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparation, and particularly relates to the preparation and application of chromomycin derivatives with anti-Gram-positive bacteria activity. Background Art

[0002] In recent years, antibiotic resistance has become a major challenge in the global public health field. The abuse of antibiotics and the resistance of antibacterial drugs have made originally curable infections difficult to control and even fatal. Among them, methicillin-resistant Staphylococcus aureus (MRSA) is one of the most typical drug-resistant bacteria. MRSA carries the mecA gene, which changes the structure of penicillin-binding protein (PBP2a), thereby developing resistance to β-lactam antibiotics. This strain often causes skin and soft tissue infections and pneumonia, and spreads rapidly in hospitals and communities. Although drugs such as vancomycin, linezolid, and daptomycin are still the main choices for treating MRSA infections, some strains have developed resistance to vancomycin, and these drugs all have their own limitations. Therefore, it is necessary to develop new anti-MRSA drugs to expand clinical treatment options and provide more clinically valuable treatment strategies for MRSA infections. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide the preparation and application of chromomycin derivatives with anti-Gram-positive bacteria activity to solve the technical problem of poor effects of anti-Gram-positive bacteria drugs.

[0004] To achieve the above object, the technical solution adopted by the present invention is to provide a chromomycin derivative, characterized in that the chromomycin derivative is Carpatamides N and Carpatamides O, and their chemical structural formulas are shown as follows:

[0005]

[0006] The present invention also discloses a preparation method of the chromomycin derivative, comprising the following steps:

[0007] S1. Prepare the fermentation culture of the strain S. lividans TK64 / pHZAUFXJ3E20, extract to obtain a crude extract paste;

[0008] S2. Subject the crude extract paste to normal-phase silica gel column chromatography, and perform gradient elution with methylene chloride / methanol as the eluent from a volume ratio of 100:0 to 90:10, and collect the fractions eluted at a volume ratio of methylene chloride and methanol of 97:3 and 96:4 to obtain fraction A;

[0009] S3. Elute the component A successively by medium-pressure liquid chromatography and high-performance liquid chromatography to obtain compounds Carpatamides N and Carpatamides O.

[0010] Based on the above technical solution, the present invention can also be improved as follows:

[0011] Furthermore, the preparation steps of the fermentation culture in S1 include:

[0012] S101. Heterologously express the ctd gene cluster in the strain Streptomyces lividans TK64, inoculate the successfully heterologously expressed bacterial mass into the seed medium, and culture it at 25 - 30 °C and 200 - 250 rpm for 46 - 50 h to obtain the seed liquid;

[0013] S102. Inoculate the seed liquid into the fermentation medium at an inoculation amount of 1.5% - 2.5%, and culture it by shaking at 25 - 30 °C and 200 - 250 rpm for 6 - 8 d to obtain the fermentation culture.

[0014] The heterologous expression strain Streptomyces lividans TK64 involved in the present invention is a model strain, and the biosynthetic gene cluster ctd of Carpatamide has been published in the literature: Genome-based mining of carpatamides I-M and their candidate biosynthetic gene cluster. Mar Drugs. 2024;22(11):521. doi:10.3390 / md22110521. The source strain of its genome is Streptomyces parvus 1268, which is published in the literature: Genome-based mining of carpatamides I-M and their candidate biosynthetic gene cluster. Mar Drugs. 2024;22(11):521. doi:10.3390 / md22110521.

[0015] Furthermore, the seed medium is TSBY medium, and its components are as follows: sucrose 10.5 wt%, peptone soy broth 3 wt%, and yeast extract 0.5 wt%.

[0016] Furthermore, the components of the fermentation medium are as follows: 2 wt% soybean powder, 2 wt% mannitol, and 4 wt% macroporous adsorption resin.

[0017] Further, the extraction step in S1 includes: centrifuging the fermentation culture to obtain the fermentation broth and mycelium; extracting the fermentation broth and mycelium with ethyl acetate respectively, then combining the obtained extracts and performing vacuum distillation to obtain a crude extract paste.

[0018] Further, the methyl chloride is dichloromethane or chloroform.

[0019] Further, the steps of medium pressure liquid chromatography in S3 are: performing gradient elution on component A with a volume ratio of MeOH:H 2 O = 0:100 to 100:0 to obtain component B.

[0020] Further, the chromatographic packing material for high performance liquid chromatography in S3 is ODS-A, and the steps are: using CH 3 CN:H 2 O = 0:100 to 50:50 in volume ratio to perform gradient elution on component B at a flow rate of 2 - 4 mL / min to obtain compounds Carpatamides N and Carpatamides O.

[0021] The present invention also discloses the application of handamycin derivatives in the preparation of antibacterial drugs.

[0022] Further, the bacterium is a Gram-positive bacterium.

[0023] Further, the bacterium is Streptococcus faecalis, Staphylococcus aureus, Bacillus subtilis, Enterococcus faecium or methicillin-resistant Staphylococcus aureus.

[0024] The beneficial effects of the present invention are as follows: By heterologously expressing the ctd gene cluster in the strain Streptomyces lividans TK64, two new Carpatamide compounds were isolated from the positive strains with successful heterologous expression. In the screening of their antibacterial activities, it was found that Carpatamide N and Carpatamide O showed excellent biological activities against Gram-positive bacteria. Among them, the MIC values for Streptococcus faecalis and Enterococcus faecium were 4 μg / mL and 1 μg / mL respectively; the MIC values for Staphylococcus aureus and Bacillus subtilis were 0.25 μg / mL and 0.125 μg / mL respectively, and the MIC values for MRSA were 1 μg / mL and 0.5 μg / mL respectively, and their anti-MRSA activities were comparable to or more excellent than the clinical drug vancomycin. Description of the Drawings

[0025] Figure 1 It is a schematic diagram for the construction of the BAC plasmid pHZAUFXJ3E20;

[0026] Figure 2PCR verification results of the heterologous expression strain S. lividans TK64 / pHZAUFXJ3E20;

[0027] Figure 3 Construction of the heterologous expression strain S. lividans TK64 / pHZAUFXJ3E20 and sequencing verification results of the upstream and downstream of the gene cluster;

[0028] Figure 4 Low-resolution mass spectrometry detection results of Carpatamide N;

[0029] Figure 5 High-resolution mass spectrometry detection results of Carpatamide N;

[0030] Figure 6 For Carpatamide N's 1 1H nuclear magnetic resonance detection results (solvent CD 3 OD);

[0031] Figure 7 For Carpatamide N's 13 13C nuclear magnetic resonance detection results (solvent CD 3 OD);

[0032] Figure 8 For Carpatamide N's 1 1H- 1 1H COSY nuclear magnetic resonance detection results (solvent CD 3 OD);

[0033] Figure 9 HSQC nuclear magnetic resonance detection results of Carpatamide N (solvent CD 3 OD);

[0034] Figure 10 HMBC nuclear magnetic resonance detection results of Carpatamide N (solvent CD 3 OD);

[0035] Figure 11 NOESY nuclear magnetic resonance detection results of Carpatamide N (solvent CD 3 OD);

[0036] Figure 12 For Carpatamide N's 1 1H nuclear magnetic resonance detection results (solvent DMSO-d 6 );

[0037] Figure 13 For Carpatamide N's13 13C NMR test results (solvent DMSO-d 6 );

[0038] Figure 14 for Carpatamide N's 1 1H- 1 1H COSY NMR test results (solvent DMSO-d 6 );

[0039] Figure 15 for Carpatamide N's HSQC NMR test results (solvent DMSO-d 6 );

[0040] Figure 16 for Carpatamide N's HMBC NMR test results (solvent DMSO-d 6 );

[0041] Figure 17 for Carpatamide N's 1 1H- 1 1H NOESY NMR test results (solvent DMSO-d 6 );

[0042] Figure 18 for Carpatamide O's high-resolution mass spectrometry test results;

[0043] Figure 19 for Carpatamide O's 1 1H NMR test results (solvent CD 3 OD);

[0044] Figure 20 for Carpatamide O's 13 13C NMR test results (solvent CD 3 OD);

[0045] Figure 21 for Carpatamide O's 1 1H- 1 1H COSY NMR test results (solvent CD 3 OD);

[0046] Figure 22 for Carpatamide O's HSQC NMR test results (solvent CD 3 OD);

[0047] Figure 23HMBC nuclear magnetic resonance detection results of Carpatamide O (solvent CD 3 OD);

[0048] Figure 24 For Carpatamide O 1 H- 1 H NOESY nuclear magnetic resonance detection results (solvent CD 3 OD). Specific embodiments

[0049] The following describes the specific embodiments of the present invention to facilitate those skilled in the art of the present technology to understand the present invention. For those not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments not specified for the manufacturer, they are all conventional products that can be obtained through commercial purchase. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art of the present technology, as long as various changes are within the spirit and scope of the present invention defined and determined by the appended claims, these changes are obvious, and all inventions and creations using the concept of the present invention are within the scope of protection.

[0050] The sequence information of the primers used in the following experiments is shown in Table 1.

[0051] Table 1 Sequence information table

[0052]

[0053] Example 1 Heterologous expression of the Carpatamides biosynthetic gene cluster ctd

[0054] (1) Screen the BAC library of Streptomyces parvus 1268 with primers containing the upstream and downstream genes of the ctd biosynthetic gene cluster. The positive BAC plasmid pHZAUFXJ3E20 was obtained by screening from the genomic library of Streptomyces parvus 1268 strain constructed by Wuhan Eight Stars Company. Screen the BAC library of Streptomyces parvus 1268 with three pairs of primers: ctd-SU-F / R, ctd-SM-F / R, and ctd-SD-F / R. The primer sequences are shown in Table 1. Finally, find the biosynthetic gene cluster containing ctd and complete the sequencing. The results are as Figures 1 - 3 shown, and it is determined that the pHZAUFXJ3E20 plasmid contains the entire ctd biosynthetic gene cluster.

[0055] (2) The verified positive BAC clone E. coli DH10B / pBAC-3E20 and the helper strain E. coli ET12567 / pUB307 were inoculated into 5 mL of LB liquid medium containing the antibiotic apramycin and cultured overnight at 37 °C. Then, the overnight culture was inoculated into 5 mL of LB culture medium containing the antibiotic apramycin at an inoculation amount of 10% and cultured at 37 °C for 3.5 h. The Streptomyces mycelium cultured in TSBY liquid medium (10.5 wt% sucrose, 3 wt% peptone soy broth, and 0.5 wt% yeast extract) for 48 h was washed twice with LB liquid medium. Then, 100 μL of the young mycelium solution was mixed with 100 μL of the positive BAC clone strain E. coli DH10B / pBAC-3E20 and the helper strain E. coli ET12567 / pUB307 that had been washed twice with LB liquid medium, and cultured at 28 °C and 200 rpm for 45 min. Then, the culture solution was taken out and spread on an SFM solid plate containing 20 μM Mg 2+ and cultured in a biochemical incubator at 28 °C for 15 h. Then, 1 mL of sterile water containing the antibiotics trimethoprim (100 μg / mL) and apramycin (50 μg / mL) was added for covering. After the surface of the medium was dried, it was cultured in an inverted manner in an incubator at 28 °C. After about 5 - 7 days, the growth of conjugative transposons could be seen. The grown conjugative transposons were spread on an SFM solid medium plate containing the antibiotics trimethoprim (100 μg / mL) and apramycin (50 μg / mL). Finally, for the monoclonal strains with correct phenotypes, their genomic DNA was extracted and verified by PCR and sequencing to obtain the correct transformants.

[0056] Example 2

[0057] A method for preparing a chromomycin - like derivative with anti - Gram - positive bacteria activity, comprising the following steps:

[0058] S1. Prepare the fermentation culture of the strain S. lividans TK64 / pHZAUFXJ3E20, extract, and obtain a crude extract; specifically:

[0059] S101. Heterologously express the ctd gene cluster in the strain Streptomyces lividans TK64, and inoculate the successfully heterologously expressed bacterial mass into 100 mL of TSBY liquid medium (10.5 wt% sucrose, 3 wt% peptone soy broth, and 0.5 wt% yeast extract), and culture at 28 °C and 220 rpm for 48 h to obtain a seed solution;

[0060] S102. Inoculate the seed solution into a 15 L fermentation medium (2 wt% soybean powder, 2 wt% mannitol, and 4 wt% macroporous adsorption resin) at an inoculation amount of 2%, and shake culture at 28 °C and 220 rpm for 7 d to obtain a fermentation culture;

[0061] S103. Centrifuge the fermentation culture to obtain a fermentation broth and mycelia; extract the fermentation broth and mycelia with ethyl acetate respectively. The mycelia are extracted 3 times with 1 L of ethyl acetate, and the fermentation broth is extracted 3 times with 3 L of ethyl acetate. Then, combine the ethyl acetate phases and perform vacuum distillation to obtain a crude extract;

[0062] S2. Subject the crude extract to normal-phase silica gel column chromatography, and perform gradient elution with dichloromethane / methanol as the eluent from a volume ratio of 100:0 to 90:10, and collect the fractions eluted at volume ratios of dichloromethane and methanol of 97:3 and 96:4 to obtain fraction A;

[0063] S3. Further purify fraction A by medium-pressure liquid chromatography (MPLC) on an ODS column, and perform gradient elution on fraction A with a volume ratio of MeOH:H 2 O of 0:100 to 100:0 to obtain fraction B; then perform elution using high-performance liquid chromatography (the packing material is ODS-A, 250×10 mm, 5 μm, YMC, the flow rate is 3 mL / min), and perform gradient elution on fraction B with a volume ratio of CH 3 CN:H 2 O of 0:100 to 50:50 (0 - 29 min, 50:50; 29 - 33 min, 0:100; 33 - 35 min, 50:50, v / v) to obtain compound Carpatamides N (5.7 mg) and Carpatamides O (4.4 mg), and their retention times are 15.0 min and 15.2 min respectively.

[0064] Experimental Example 1 Structure Identification

[0065] Perform structure characterization on Carpatamide N and Carpatamide O prepared in Example 2, and their 1 H NMR and 13 CNMR data are shown in Table 2.

[0066] Table 2 13 C NMR and 1 H NMR data of Carpatamide N and Carpatamide O

[0067]

[0068]

[0069] Note: a: The solvent is CD 3 OD; b: The solvent is DMSO-d 6 .

[0070] Carpatamide N is a light yellow powder. According to the signals m / z 413.1688 ([M+Na] + )( Figure 4 and Figure 5 ) that appear in ESI-HRMS, its molecular formula is determined to be C 20 H 26 N 2 O 6 , indicating that it has 9 degrees of unsaturation ( Figure 6 and Figure 7 ). Through 13 C NMR and HSQC spectrum analysis, it is found that its structure contains 3 methyl proton signals, 3 methylene proton signals, 8 methine proton signals, and 6 quaternary carbons, including four carbonyl carbons ( Figure 7 and Figure 9 ). 1 In the H- 1 H COSY spectrum, the correlation signals of H-11 / H-12, H-12 / H-13, H-13 / H-14, H-14 / H-15, H-15 / H-16, H-16 / H-17, H-16 / H-18, and the correlations in the HMBC spectrum of H-11 / C-10, C-12; H-12 / C-10, C-11, C-13, C-14; H-13 / C-11, C-12, C-14, C-15; H-14 / C-12, C-13, C-15, C-16; H-15 / C-13, C-14, C-16, C-17, C-18; H-16 / C-14, C-15, C-17, C-18; H-17 / C-15, C-16, C-18; H-18 / C-15, C-16, C-17 indicate the presence of a fatty chain fragment ("upper chain") ( Figure 8 and Figure 10 ). The conjugated E-configured diene contained in the "upper chain" is further confirmed by the J values between H-11 / H-12 and H-13 / H-14, both of which are 15.2 Hz (Table 2). In the 1 H NMR spectrum, the signals of H-7 (δ H 2.38, 2.58, m), H-8 (δ H 2.32, m) and the 1 H- 1 H COSY correlation of H-7 / H-8 indicate the presence of a C-7-C-8 fragment ( Figure 6 andFigure 8 ). Through the correlations of H-7 with C-8 and C-9 and H-8 with C-7 and C-9 in HMBC, it was determined that this fragment was connected to C-9, forming the "lower chain"( Figure 10 ). C-19 (δ C 171.6) and C-20 (δ H 2.12, δ C 21.8) signals revealed the presence of an acetyl group through the HMBC correlation of H-20 with C-19( Figure 10 ). In addition to the three double bonds of C-2 / C-3, C-11 / C-12 and C-13 / C-14, and the four carbonyl signals of δ C 168.1 (C-10), δ C 171.6 (C-19), δ C 177.1 (C-9), δ C 189.8 (C-1), there are also two degrees of unsaturation in compound 1. Therefore, this compound may contain two rings. Through the 1 H- 1 H COSY correlations of H-5 / H-6, the long-range coupling between H-5 and H-3 (J = 2.8 Hz), and the correlations of H-3 with C-1, C-2, C-4, C-5; H-5 with C-1, C-3, C-4, C-6; H-6 with C-1, C-2, C-4, C-5 in HMBC, a cyclohexenone structural unit may be established( Figures 6 - 10 ). In the 13 C NMR spectrum, the chemical shifts of C-5 (δ C 55.8) and C-6 (δ C 53.9) and their proton signals δ H 4.31 (H-5, dd, J = 4.0, 2.8 Hz) and δ H 3.61 (H-6, d, J = 4.0 Hz) indicate that they may form an epoxy group, which is consistent with the speculation of the last degree of unsaturation in compound 1. The HMBC correlations of the exchangeable proton signal δ H 9.29 (2-NH) with C-1, C-2, C-11 indicate that the "upper chain" is connected to C-2 through 2-NH substitution( Figures 12 - 16 ). The "lower chain" is connected to compound 1 through C-4 of the epoxycyclohexenone unit, which is confirmed by the HMBC correlations: the correlations of H-3 with C-4, C-5, C-7; H-5 with C-3, C-4, C-7; H-7 with C-3, C-4, C-5; H-8 with C-4 all support this conclusion( Figure 15 and Figure 16 ). H-9-NH2 (δ H 6.80, 7.34) The HMBC correlations between C-8, C-9 and the acetyl group at C-19 / C-20 suggest that the acetyl group at C-19 / C-20 may not be connected to C-9 through NH, but rather through an oxygen atom to C-4, which is also consistent with the 1 H- 1 H COSY spectrum correlations of the two exchanging proton signals (H-9-NH2, δ H 6.80, 7.34) ( Figures 12 - 16 ). 1 H- 1 H NOESY spectrum correlations of H-5 (δ H 4.31) with H-6 (δ H 3.61), and H-5 (δ H 4.31) with H-7 (δ H 2.38, 2.58) indicate that they are on the same side ( Figure 17 ). By comparing the experimental ECD with the calculated theoretical ECD results, the absolute configuration of compound 1 was determined to be 4R, 5S, 6R. Finally, this compound was identified and named carpatamide N.

[0071] Based on the ESI-HRMS signal m / z 385.1729 ([M+Na] + ), the molecular formula of Carpatamide O was determined to be C 19 H 26 N 2 O 5 ( Figure 18 ). 1 1H NMR and 13 13C NMR data are similar to those of Carpatamide N, but the acetyl group signals of C-19 (δ C 171.6) and C-20 (δ H 2.12, δ C 21.8) in Carpatamide N are absent in Carpatamide O, and a new methoxy signal at C-4-OCH 3 appears (δ H 3.21, δ C 51.7) ( Figures 19 - 23 ). Through the C-4-OCH 3 / C-4 correlation in HMBC, it was confirmed that this methoxy group is substituted at the C-4 position ( Figure 23 ). Therefore, this compound was finally identified as 4-OCH 3-novodarylamide, named Carpatamide O. Its absolute configuration was determined to be 4R, 5S, 6R by ECD calculation, consistent with Carpatamide N( Figure 24 ).

[0072] Experimental Example 2 Antibacterial Activity Assay

[0073] Inoculate the test strain on a plate of LB solid medium and incubate overnight in a constant temperature incubator at 37 °C. The next day, pick a single colony from the culture plate and inoculate it into LB liquid medium, and continue to incubate overnight in a constant temperature incubator at 37 °C. Dilute the overnight culture with fresh LB liquid medium to 10 8 CFU / mL for standby. Weigh appropriate amounts of Carpatamide N, Carpatamide O, and vancomycin respectively, dissolve them with methanol to prepare a stock solution of 1 mg / mL for use. Under a sterile environment, use a 96-well plate for drug serial dilution: Add 174.4 μL of LB liquid medium and 25.6 μL of the drug stock solution to the first well (A1) to make the final drug concentration in this well reach 64 μg / mL; add 100 μL of LB liquid medium to each well from A2 to A12. Pipette 100 μL of the solution from well A1 into well A2, mix well and then pipette 100 μL of the solution from well A2 into well A3, and so on until well A12. Finally, discard the excess 100 μL of solution in well A12. After the serial dilution is completed, add 100 μL of the bacterial solution diluted to 10 8 CFU / mL to each drug dilution well (A1 to A12). Place the 96-well plate in a constant temperature incubator at 37 °C for 16 - 18 h. Set up three parallel experiments for each drug, with vancomycin as the positive control and methanol as the blank control. The MIC experimental results are shown in Table 3.

[0074] Table 3 Antibacterial Activity Test Data of Carpatamide N and Carpatamide O

[0075]

[0076]

[0077] As can be seen from Table 3, Carpatamide N and Carpatamide O showed strong inhibitory effects on Gram-positive bacteria. Among them, the MIC values against Enterococcus faecalis and Enterococcus faecium were 4 μg / mL and 1 μg / mL, respectively; the MIC values against Staphylococcus aureus and Bacillus subtilis were 0.25 μg / mL and 0.125 μg / mL, respectively, and the MIC values against MRSA were 2 μg / mL and 0.5 μg / mL, respectively. Their anti-MRSA activities were comparable to or better than that of the clinical drug vancomycin.

Claims

1. A chiromycin derivative having anti-Gram-positive bacterial activity, characterized in that: The manumycin derivatives are Carpatamides N and Carpatamides O, and their chemical structures are shown below:

2. The method for preparing the manumycin derivatives according to claim 1, characterized in that: The following steps are involved: S1. preparing a fermentation culture of strain S. lividans TK64 / pHZAUFXJ3E20, extracting and obtaining a crude extract; S2. The crude extract was subjected to normal phase silica gel column chromatography, and gradient elution was performed using methane chloride / methanol as an eluent in a volume ratio of 100:0 to 90:10, and fractions of gradient elution with a volume ratio of methane chloride to methanol of 97:3 and 96:4 were collected to obtain component A; S3. eluting component A by medium pressure liquid chromatography and high performance liquid chromatography in sequence to obtain compounds Carpatamides N and Carpatamides O.

3. The method for preparing the manomycin derivatives according to claim 2, characterized in that: The step of preparing the fermentation culture in S1 comprises: S101, heterologously expressing the ctd gene cluster in the strain Streptomyces lividans TK64, inoculating the bacterial blocks with successful heterologous expression into a seed liquid culture medium, and culturing at 25-30° C. and 200-250 rpm for 46-50 h to obtain a seed liquid; S102, inoculating the seed liquid into a fermentation medium, and shaking culture at 25-30° C. and 200-250 rpm for 6-8 days to obtain a fermentation culture.

4. The method for preparing the manumycin derivatives according to claim 3, characterized in that: The inoculation amount of the seed liquid is 1.5% to 2.5%.

5. The method for preparing the manumycin derivatives according to claim 2, characterized in that: The extraction step in S1 comprises: centrifuging the fermentation culture to obtain fermentation broth and mycelium; extracting the fermentation broth and mycelium with ethyl acetate respectively, and then combining the obtained extracts and performing reduced pressure distillation to obtain a crude extract.

6. The method for preparing the manumycin derivatives according to claim 2, characterized in that: The methane chloride is dichloromethane or chloroform.

7. The method for preparing the manumycin derivatives according to claim 2, characterized in that: The step of the S3 medium pressure liquid chromatography is: gradient elution of component A with a volume ratio of MeOH:H2O=0:100 to 100:0 to obtain component B.

8. The method for preparing the manumycin derivatives according to claim 2, characterized in that: The chromatographic filler of the high performance liquid chromatography in S3 is ODS-A, and the steps are: gradient elution of component B is performed using a volume ratio of CH3CN:H2O=0:100 to 50:50 at a flow rate of 2 to 4 mL / min to obtain compounds Carpatamides N and Carpatamides O.

9. Use of the manumycin derivatives according to claim 1 in the preparation of antibacterial drugs.

10. The use according to claim 9, characterized in that: The bacteria are Gram-positive.

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