Streptomyces secondary metabolites, methods for their production and medical uses thereof

Abstract

The application belongs to the technical field of medicine, and relates to a Streptomyces secondary metabolite and a preparation method and application thereof. The Streptomyces secondary metabolite is a polycyclic aromatic polyketone compound, and is specifically as shown below, and the application is prepared from Streptomyces Streptomyces rosealbus A series of new compounds with polycyclic aromatic polyketone nucleus structure are separated and extracted from the fermentation culture, and the obtained compounds all have good activities of resisting multiple tumor cells and pathogenic bacteria.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to Streptomyces secondary metabolites (polycyclic aromatic polyketides) and their preparation methods and applications, specifically the application of polycyclic aromatic polyketides in the preparation of antitumor and antibacterial drugs. Background Technology

[0002] Malignant tumors are a serious threat to human health, and their incidence is increasingly affecting younger people, posing a significant threat to human well-being. As a large developing country, my country faces an increasingly severe situation regarding malignant tumors due to rapid industrialization, urbanization, population aging, unhealthy lifestyles, and environmental pollution. Therefore, finding safer and more effective anti-tumor drugs is of paramount importance. Besides malignant tumors, we also face the enormous challenge of increasingly prevalent bacteria and drug-resistant bacteria. Continuing to develop new drugs against pathogenic microorganisms and researching effective dosing regimens remains a challenging task. Meanwhile, as chemically synthesized drugs increasingly reveal their toxic side effects, the long growth cycles and scarcity of rare medicinal plant resources of natural drugs, primarily derived from plants, increasingly highlight the superiority of products derived from microbial secondary metabolites. Microbial resources are a treasure trove bestowed upon humanity by nature, and the extraction of bioactive substances from the secondary metabolites of microbial strains is receiving increasing attention from technical personnel in related fields.

[0003] Streptomyces rosealbus can be obtained from various soils. Its morphological characteristics, physiological and biochemical features, and phylogenetic analysis have been reported in the literature. As early as 2005, the species was first discovered in soil samples from secondary forests in Yongsheng County, Yunnan Province, and its identification was completed for the first time (LHXu et al., Antonie Van Leeuwenhoek, 2005, 87(3), 189-194). However, to date, there are no reports on the secondary metabolites of Streptomyces rosealbus. Summary of the Invention

[0004] The purpose of this invention is to provide a series of Streptomyces secondary metabolites (polycyclic aromatic polyketides).

[0005] A second objective of this invention is to provide a method for the extraction, separation, and purification of the aforementioned polycyclic aromatic polyketide compounds.

[0006] A third object of the present invention is to provide a pharmaceutical composition containing the aforementioned polycyclic aromatic polyketide compound.

[0007] A fourth object of the present invention is to provide the use of the aforementioned polycyclic aromatic polyketide compounds or pharmaceutical compositions in the preparation of antitumor and antibacterial drugs.

[0008] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0009] A type of Streptomyces secondary metabolite: Streptomyces secondary metabolites are polycyclic aromatic polyketides, specifically those represented by general formulas I, II, III, IV, V, VI, or VII.

[0010]

[0011] In the above formula,

[0012] In general formulas I, II, III, IV, V, VI, or VII, R1 and R2 may be the same or different and are selected from H, C1-C4 carbonyl, hydroxyl, C1-C6 alkyl, ... Halogen or cyano;

[0013] Or pharmaceutically acceptable salts, solvates, or isomers of the polycyclic aromatic ketone compounds shown in the above general formulas.

[0014] Preferably, in formulas I, II, III, IV, V, VI, or VII...

[0015] R1 and R2 can be the same or different selected from... C1-C4 carbonyl, hydroxyl or C1-C4 alkyl;

[0016] Or pharmaceutically acceptable salts, solvates, or isomers of the polycyclic aromatic ketone compounds shown in the above general formulas.

[0017] A more preferred compound is:

[0018]

[0019] A method for preparing secondary metabolites of the genus Streptomyces involves fermentation and purification of Streptomyces rosealbus to obtain polycyclic aromatic polyketides represented by general formulas I, II, III, IV, V, VI, or VII.

[0020] Specifically:

[0021] (1) Fermentation production of strain: The activated strain Streptomyces rosealbus was inoculated into the primary seed culture medium and cultured at 28-30℃ with shaking for 24-36h. The bacterial solution was collected as the primary seed solution. The primary seed solution was then inoculated into the secondary seed culture medium and cultured at 28-32℃ with shaking for 24-36h to obtain the secondary seed solution. The secondary seed solution was then inoculated into the fermentation medium for fermentation. The fermentation temperature was 28-30℃, the aeration condition was 0.2-0.4 vvm, and the fermentation cycle was 7-10 days to obtain the fermentation broth.

[0022] (2) Preparation of extract: Take the fermentation broth from step (1), centrifuge at 8000-10000 rpm for 10-15 min to remove the cells, soak the supernatant in ethyl acetate, sonicate, and then distill under reduced pressure to obtain the extract;

[0023] (3) Isolation and purification of compounds: Fermentation yields extracts, which are then subjected to silica gel column chromatography with a dichloromethane / methanol mixed solution (dichloromethane / methanol (v / v) is 1:100-50:50) to obtain the secondary metabolites of Streptomyces shown in general formula I, II, III, IV, V, VI or VII respectively.

[0024] The fermented extract was dissolved and purified by silica gel column chromatography using a dichloromethane / methanol mixture. The crude fraction (fraction B) with a dichloromethane / methanol (V / V) ratio of 90:10-85:15 was collected. Then, the fraction was separated by Sephadex LH-20 column chromatography using pure methanol as the mobile phase (flow rate 0.1 ml / min). The obtained fraction was then separated and purified by preparative liquid chromatography (UV 220 nm) using an acetonitrile-water mixture (acetonitrile-water (v / v) ratio of 80:20-60:40) as the mobile phase (flow rate 3 ml / min) to obtain the compounds shown in general formulas I and II.

[0025] Alternatively, the fermented extract can be dissolved and purified by silica gel column chromatography using a dichloromethane / methanol mixture. The crude fraction (fraction C) with a dichloromethane / methanol (V / V) ratio of 85:15-80:20 can be collected and then separated by Sephadex LH-20 column chromatography using pure methanol as the mobile phase (flow rate 0.1 ml / min). The obtained fraction can be further purified by preparative liquid chromatography (UV 220 nm) using an acetonitrile-water mixture (acetonitrile-water (v / v) ratio of 80:20-60:40) as the mobile phase (flow rate 3 ml / min) to obtain the compound represented by general formula III.

[0026] Alternatively, the fermented extract can be dissolved and purified by silica gel column chromatography using a dichloromethane / methanol mixture. The crude fraction (fraction D) with a dichloromethane / methanol (V / V) ratio of 80:20-60:40 can be collected and then separated by Sephadex LH-20 column chromatography using pure methanol as the mobile phase (flow rate 0.1 ml / min). The obtained fraction can be further purified by preparative liquid chromatography (UV 220 nm) using an acetonitrile-water mixture (acetonitrile-water (v / v) ratio of 60:40-20:80) as the mobile phase (flow rate 3 ml / min) to obtain compounds represented by general formulas IV, V, and VI.

[0027] Alternatively, the fermented extract can be dissolved and purified by silica gel column chromatography using a dichloromethane / methanol mixture. The crude fraction (fraction E) with a dichloromethane / methanol (V / V) ratio of 60:40-50:50 can be collected and then separated by Sephadex LH-20 column chromatography using pure methanol as the mobile phase (flow rate 0.1 ml / min). The obtained fraction can be further purified by preparative liquid chromatography (UV 220 nm) using an acetonitrile-water mixture (acetonitrile-water (V / V) ratio of 40:60-20:80) as the mobile phase (flow rate 3 ml / min) to obtain the compound represented by general formula VII.

[0028] The primary and secondary seed culture media (1000ml) are prepared as follows: add 4.0g yeast powder, 4.0g glucose, 2.0g malt powder, and 1.0ml trace salt to every 1000ml of water, adjust the pH to 7.2, and sterilize at 121℃ for 30min.

[0029] The fermentation medium (1000ml) is as follows: add 20.0ml of glycerol, 4.0g of malt powder, 8.0g of yeast extract, and 1.0ml of trace salt solution to every 1000ml of water, adjust the pH to 7.2, and sterilize at 121℃ for 30min.

[0030] The trace salt formula is: 0.1g FeSO4·7H2O, 0.1g MnCl2·4H2O, 0.1g ZnSO4·7H2O, and 100ml water.

[0031] The use of a polycyclic aromatic polyketide compound, the use of the compounds represented by the general formulas, their pharmaceutically acceptable salts, solvates or isomers in the preparation of antitumor or antibacterial drugs.

[0032] The compounds represented by the general formulas, their pharmaceutically acceptable salts, solvates, or isomers may be used in the preparation of MMP2 protein in antitumor drugs.

[0033] An antitumor or antibacterial composition comprising compounds of the general formulas shown.

[0034] The compounds isolated and identified in this invention have strong antitumor effects against human lung cancer cells A549, human liver cancer cells HepG2, human breast cancer cells MCF-7, human gastric cancer cells (MGC-803), mouse breast cancer cells (4T-1), and human triple-negative breast cancer cells (MDA-MB-231), with good activity, especially compounds A1 and A4, which have more obvious effects.

[0035] The compounds isolated and identified in this invention exhibit varying degrees of antibacterial activity against Staphylococcus aureus, Klebsiella pneumoniae, and Enterococcus faecium, with compounds A3, A4, and A5 showing particularly significant activity.

[0036] Advantages of this invention:

[0037] This invention isolates and extracts a series of novel compounds with a polycyclic aromatic polyketide core structure from the fermentation culture of *Streptomyces rosealbus*. All the obtained compounds exhibit good activity against various tumor cells and pathogenic bacteria. Pharmacological experiments have demonstrated that the compounds isolated and identified in this invention have strong antitumor effects against human lung cancer cells A549, human liver cancer cells HepG2, human breast cancer cells MCF-7, human gastric cancer cells (MGC-803), mouse breast cancer cells (4T-1), and human triple-negative breast cancer cells (MDA-MB-231). Their activity is better than that of the positive control drug 5-fluorouracil, especially compounds A1 in formula I and A4 in formula III. Pharmacological experiments have also demonstrated that the compounds isolated and identified in this invention have good antibacterial activity against *Staphylococcus aureus*, *Klebsiella pneumoniae*, and *Enterococcus faecalis*, especially compounds A3 in formula II, A4 in formula III, and A5 in formula IV. Attached Figure Description

[0038] Figure 1 The strain-compound-target network (A) and the protein-protein interaction network (PPI) (B) are shown.

[0039] Figure 2 The diagram shows the molecular docking 3D interaction effect between the obtained compound and the MMP2 protein provided in the embodiments of the present invention, wherein A is compound A4 and B is compound A5.

[0040] Figure 3 The diagram shows the BLI interaction between compounds A4 and A5 obtained in this embodiment of the invention and the MMP2 protein, where A is compound A4 and B is compound A5. Detailed Implementation

[0041] The following examples further illustrate specific embodiments of the present invention. It should be noted that the specific embodiments described herein are merely for illustration and explanation and are not intended to limit the scope of the present invention.

[0042] Example 1

[0043] The enrichment and preparation of polycyclic aromatic polyketides includes the following steps:

[0044] (1) Fermentation production of microbial strains:

[0045] After activation using conventional methods, the Streptomyces rosealbus strain was 10 2 Inoculate with CFU / ml into primary seed culture medium, incubate at 28℃ for 24 hours, collect the bacterial suspension as primary inoculum, and then inoculate at 10... 3 Inoculate the primary seed culture into the secondary seed culture medium at an inoculation rate of CFU / 100ml, and incubate at 28℃ with shaking for 24 hours to obtain the secondary seed culture. Then, divide the secondary seed culture into 10... 4 The inoculum was inoculated into the fermentation medium at a rate of CFU / 100ml. The fermentation temperature was 28℃, the aeration condition was 0.2vvm, and the fermentation cycle was 7 days to obtain the fermentation broth.

[0046] The primary and secondary seed culture media (1000ml) are prepared as follows: add 4.0g yeast powder, 4.0g glucose, 2.0g malt powder, and 1.0ml trace salt to every 1000ml of water, adjust the pH to 7.2, and sterilize at 121℃ for 30min.

[0047] The fermentation medium (1000ml) is as follows: add 20.0ml of glycerol, 4.0g of malt powder, 8.0g of yeast extract, and 1.0ml of trace salt solution to every 1000ml of water, adjust the pH to 7.2, and sterilize at 121℃ for 30min.

[0048] The trace salt formula is: 0.1g FeSO4·7H2O, 0.1g MnCl2·4H2O, 0.1g ZnSO4·7H2O, and 100ml water.

[0049] (2) Preparation of extract:

[0050] Take 3L of fermentation broth, centrifuge at 8000rpm for 10min to remove the cells, extract the supernatant three times with an equal volume of ethyl acetate, and then concentrate by rotary evaporation and vacuum to obtain 10.5g of dark red extract.

[0051] (3) Examples of compound isolation, purification, and preparation:

[0052] The extract obtained in step (2) was dissolved in 10 ml of methanol and then mixed with silica gel (100-200 mesh) at a mass equal to the sample weight. After the methanol in the mixed sample had evaporated completely, the sample was loaded onto a silica gel (200-300 mesh) column at a mass equal to 10 times the sample weight using a dry loading method. Eleven gradients were performed using dichloromethane / methanol (100:1, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 65:45, 50:50) solution as the mobile phase, with each gradient consisting of two column volumes. The eluted fractions were collected, and after thin-layer chromatography analysis, six fractions (A, F, and F) were obtained. Fraction A had lower polarity and higher fatty acid content, while fraction F had higher polarity and was therefore discarded.

[0053] Fraction B (crude fraction with a dichloromethane / methanol volume ratio of 90:10-85:15) was collected and separated using Sephadex LH-20 gel chromatography with 100% pure methanol as the mobile phase (flow rate 0.1 ml / min). All eluents were collected and then prepared by preparative high performance liquid chromatography (UV 220 nm) with 73% acetonitrile-water as the mobile phase at 220 nm. Compound A1 was obtained in about 20-25 min, compound A2 was obtained in about 30-35 min, and compound A3 was prepared with 70% acetonitrile-water as the mobile phase at 220 nm.

[0054] The collected fraction C (crude fraction with a dichloromethane / methanol volume ratio of 85:15-80:20) was separated by Sephadex LH-20 gel chromatography using 100% pure methanol as the mobile phase (flow rate 0.1 ml / min). All eluents were collected and then prepared by preparative high performance liquid chromatography (UV 220 nm) using 70% acetonitrile-water as the mobile phase at 220 nm to obtain compound A4.

[0055] Fraction D (crude fraction with a dichloromethane / methanol volume ratio of 80:20-60:40) was separated by ODS reversed-phase column chromatography using methanol-water as the mobile phase (flow rate 2 ml / min). Six gradients (40%, 50%, 60%, 70%, 85%, and 100%) were eluted sequentially, with two column volumes for each gradient, yielding six by-fractions D1-D6. Fraction D2 (eluted with 50% methanol) was then prepared by preparative high-performance liquid chromatography (UV 220 nm) using 40% acetonitrile-water as the mobile phase at 220 nm to obtain compound A5.

[0056] Fraction D3 (eluted with 60% methanol) was prepared by preparative high performance liquid chromatography (UV 220 nm) with 25% acetonitrile-water as the mobile phase at 220 nm. Compound A6 was obtained in about 34-44 min and compound A7 was obtained in about 48-58 min.

[0057] Fraction D4 (eluted with 70% methanol) was prepared by preparative high performance liquid chromatography (UV 220 nm) with 33% acetonitrile-water as the mobile phase at 220 nm. Compound A8 was obtained in about 20-25 min and compound A9 was obtained in about 40-50 min.

[0058] The collected fraction E (crude fraction with a dichloromethane / methanol volume ratio of 60:40-50:50) was separated by Sephadex LH-20 gel chromatography using 100% pure methanol as the eluent (flow rate 0.1 ml / min). All eluents were collected and then prepared by preparative high performance liquid chromatography (UV 220 nm) with 15% acetonitrile-water as the mobile phase at 220 nm. Compound A10 was obtained in about 30-40 min and compound A11 was obtained in about 60-70 min.

[0059] The mobile phase rate for all the above-mentioned preparative high-performance liquid chromatography (HPLC) processes was 3 ml / min.

[0060] The strain described above, *Streptomyces rosealbus*, can be isolated from soil samples. This strain is a common strain, for example, as described in *Streptomyces roseoalbus sp.nov., an actinomycete isolated from soil in Yunnan, China*.

[0061] The spectral data of the obtained compound are as follows:

[0062] A1: Yellowish-brown amorphous powder; HRESIMS: m / z 309.071 6 [M+H] + (The calculated value is C) 18 H 12 O5,309.075 7); UV(MeOH)λ max (logε)209(4.04), (logε)253(3.86), (logε)262(3.90), (logε)385(3.19)nm; 1 H-NMR (600MHz, DMSO-d6): δ H6.80(1H,d,J=1.45Hz,H-2), 7.15(1H,br s,H-4), 7.67(1H,s,H-5), 7.43(1H,overlap,H-9), 7.42(1H,overlap,H-11), 7.73(1H,m,H-10), 2.39(3H,s,H-13), 13.00(1H,s,1-OH); 13 C-NMR (150MHz, DMSO-d6): δ C 155.3(C-1), 115.7(C-2), 137.7(C-3), 118.4(C-4), 133.5(C-4a), 119.2(C-5), 150.2(C-6), 144.9(C-6a), 143.2(C-7a), 146 .7(C-8), 120.5(C-9), 115.4(C-10), 125.6(C-11), 112.3(C-11a), 180.4(C-12), 116.2(C-12a), 112.5(C-12b), 20.6(C-13).

[0063] A2: Orange amorphous powder; HRESIMS: m / z 423.143 5 [M+H] + (The calculated value is C) 24 H 22 O7,423.143 8); UV(MeOH)λ max 207, 245, 250, 261, and 366 nm; 1H-NMR (600MHz, DMSO-d6): δ7.01(1H,d,J=1.9Hz,H-2), 7.34(1H,d,J=1.9Hz,H-4), 7.65(1H,d,J=9.1Hz,H-5), 7.76(1H,d ,J=8.5Hz,H-6), 8.30(1H,d,J=9.1Hz,H-10), 7.50(1H,d,J=8.5Hz,H-11), 2.42(3H,s,H-13), 3.57(1H,ddd,J=4.9,8.5,11 .2Hz,H-1′), 2.23(1H,ddd,J=2.0,4.9,12.8Hz,H-2′a), 1.36(1H,dt,J=11.4,12.9Hz,H-2′b), 4.94(1H,dd,J=2.0,11.4Hz ,H-3′), 2.92(1H,t,J=8.8Hz,H-4′), 3.38(1H,dq,J=6.1,9.0Hz,H-5′), 1.28(3H,d,J=6.1Hz,6′-H), 13.31(1H,s,1-OH).; 13 C-NMR (150MHz, DMSO-d6): δ155.6(C-1), 120.5(C-2), 137.8(C-3), 118.8(C-4), 133.0(C -4a)139.6(C-5), 118.2(C-6), 158.1(C-6a), 143.8(C-7a), 142.0(C-8), 135.3(C-9), 122 .3(C-10), 115.2(C-11), 120.7(C-11a), 180.1(C-12), 115.0(C-12a), 117.4(C-12b), 20 .4(C-13), 71.8(C-1′), 40.3(C-2′), 71.2(C-3′), 77.1(C-4′), 76.1(C-5′), 18.4(C-6′).

[0064] A3: Orange amorphous powder; HRESIMS: m / z 386.0716 [M+H] + (The calculated value is C) 20 H 18 O6,386.0757); UV(MeOH)λ max 209, 253 (3.86), 262, and 385 nm; 1 H-NMR (600MHz, DMSO-d6): δ H 7.42 (1H, s, H-5), δ H7.41(1H,dd,J=7.5,1.1Hz,H-9), 7.84(1H,dd,J=7.8,7.6Hz,H-10), 7.81(1H,dd,J=7.5,1.0Hz,H-11), 1.20(3H,s,H-13), 12.11(1H,s,6-OH), 12.09(1H,s,8-OH), 13.53(1H,s,12b-OH), 3.06(3H,s,1-OCH3); 13 C-NMR (150MHz, DMSO-d6): δ C 170.9(C-1), 46.3(C-2), 71.0(C-3), 39.5(C-4), 140.5(C-4a), 131.5( C-5), 155.8(C-6), 111.2(C-6a), 189.9(C-7), 116.1(C-7a), 161.5(C- 8), 124.6(C-9), 137.3(C-10), 119.2(C-11), 133.2(C-11a), 186.2(C- 12), 111.8(C-12a), 157.1(C-12b), 26.4(C-3-CH3), 51.51(C-1-OCH3).

[0065] A4: Red amorphous powder; HRESIMS: m / z 434.0716 [M+H] + (The calculated value is C) 25 H 22 O7,434.0757); UV(MeOH)λ max 195, 253, and 263 nm; 1 H-NMR (600MHz, DMSO-d6): δ H 7.82(1H,br s,H-3), 7.77(1H,br s,H-4), 7.55(1H,s,H-7), 6.97(1H,s,H-9), 8.43(1H,s,H-11), 2.25(1H,m,H-12), 2.42(1H,s,H-13), 4.68(1H,d,J=10.7,H-1′), 2.09(1H,br d,J=11.7,H-2′a), 1.37(1H,br d,J=11.7,H-2′b), 2.02(1H,br d,J=12.2,H-3′a), 1.54(1H,br d,J=12.2,H-3′b), 3.12,(1H,br s,H-4′), 3.34(1H,m,H-5′), 1.26(1H,d,J=5.9), 13.40(1H,s,1-OH);13 C-NMR (150MHz, DMSO-d6): δ C 158.5(C-1), 138.2(C-2), 132.9(C-3), 118.6(C-4), 132.1(C-4a), 186.5(C-5), 108. 9(C-5a), 162.2(C-6), 128.2(C-6a), 114.3(C-7), 141.9(C-8), 116.1(C-9), 156.0(C- 10), 124.2(C-10a), 116.7(C-11), 125.2(C-11a), 187.4(C-12), 116.3(C-12a), 21.9( C-13), 72.5(C-1′), 31.5(C-2′), 32.9(C-3′), 70.5(C-4′), 78.7(C-5′), 18.6(C-6′).

[0066] A5: Pale yellow amorphous powder; m / z 369.097 9 [MH] - (The calculated value is C) 20 H 18 O7,369.098 0); UV(MeOH)λ max 223, 271, and 414nm; 1 H-NMR (600MHz, DMSO-d6): δ7.48(1H,d,J=2.5Hz,H-1), 7.25(1H,dd,J=2.6,8.6Hz,H-2), 8.13(1H,d,J=8.6Hz,H-3), 7.82 (1H,d,J=7.8Hz,H-7), 7.72(1H,d,J=7.8Hz,H-8), 3.54(1H,ddt,J=4.6,8.9,11.3Hz,H-1′), 2.25(1H,ddd,J=2.0,4.9,12. 8Hz,H-2′a), 1.33-1.28(1H,m,H-2′b), 4.80(1H,dd,J=2.0,11.4Hz,H-3′), 2.89(1H,td,J=4.8,8.8Hz,H-4′), 3.41-3.34( 1H,m,H-5′), 1.27(3H,d,J=6.0Hz,H-6′), 13.24(1H,s,5-OH), 4.93(1H,d,J=4.7Hz,3′-OH), 5.02(1H,d,J=5.1Hz,4′-OH); 13C-NMR (150MHz, DMSO-d6): δ157.8(C-1), 137.6(C-2), 132.5(C-3), 118.8( C-4), 131.9(C-4a), 112.6(C-5), 121.6(C-6), 129.9(C-7), 164.1(C-8), 1 35.6(C-8a), 187.7(C-9), 115.1(C-9a), 181.9(C-10), 124.4(C-10a), 71. 7(C-1′), 40.0(C-2′), 70.6(C-3′), 77.0(C-4′), 76.1(C-5′), 18.4(C-6′).

[0067] A6: Yellow amorphous powder; HRESIMS: m / z 483.128 8[M+H] + (The calculated value is C) 25 H 22 O 10 ,483.128 6); UV(MeOH)λ max 240 and 427nm; 1 H NMR (600MHz, DMSO-d6): δ8.13(1H,d,J=7.6Hz,H-6), 7.96(1H,d,J=7.5Hz,H-7), 7.87(1H,d,J=7.5Hz,H-11), 7.66(1H,d,J=7.6Hz,H-12), 3.17(1H,d,J=17.5Hz,H-3), 3.10(1H,d,J=17.6Hz,H-3), 1.43(3H,s,H-14), 3.5 6(1H,d,J=5.5Hz,H-1′), 2.26(1H,dd,J=4.8,13.1Hz,H-2′), 1.37-1.30(1H,m,H-2′), 4.79(1H,d,J=11.2Hz, H-3′), 2.90(1H,d,J=8.8Hz,H-4′), 3.41-3.35(1H,m,H-5′), 1.27(3H,d,J=6.0Hz,H-6′)12.67(1H,s,9-OH); 13C-NMR (150MHz, DMSO-d6): δ170.5(C-2), 39.9(C-3), 89.4(C-4), 201.5(C-5), 128.4(C-5a), 129.4(C-6), 119.7(C-7), 138.7(C-7a), 187.9(C-8), 115.3(C-8a), 157.8(C-9), 136.9(C-1 0), 133.6(C-11), 118.4(C-12), 132.4(C-12a), 179.8(C-13), 118.4(C-13a), 168.8(C-13b) , 22.2(C-14), 71.7(C-1′), 40.0(C-2′), 70.6(C-3′), 77.0(C-4′), 76.1(C-5′), 18.4(C-6′).

[0068] A7: Yellow amorphous powder; HRESIMS: m / z 483.1283 [M+H] + (The calculated value is C) 25 H 22 O 10 ,483.128 6); UV(MeOH)λ max 240 and 427nm; 1 H-NMR (600MHz, DMSO-d6): δ8.16(1H,d,J=7.8Hz,H-6), 8.00(1H,d,J=7.8Hz,H-7), 7.89(1H,d,J=7.8Hz,H-11), 7.70(1H,d ,J=7.9Hz,H-12), 1.44(3H,s,H-14), 3.56(1H,d,J=7.2Hz,H-1′), 2.25(1H,ddd,J=2.0,4.9,12.7Hz,H-2′), 1.39-1.31(1H m,H-2′), 4.81(1H,dd,J=2.0,11.4Hz,H-3′), 2.90(1H,t,J=8.9Hz,H-4′), 3.38(dd,J=4.5,7.7Hz ,H-5′), 1.26(3H,d,J=6.1Hz,H-6′), 12.69(1H,s,9-OH), 4.95(1H,s,3′-OH), 5.03(1H,s,4′-OH); 13C-NMR (150MHz, DMSO-d6): δ170.1(C-2), 39.97(C-3), 88.8(C-4), 201.3(C-5), 127.5(C-5a) , 129.7(C-6), 120.0(C-7), 139.1(C-7a), 187.0(C-8), 115.3(C-8a), 157.6(C-9), 136.9(C-1 0), 133.7(C-11), 118.6(C-12), 132.3(C-12a), 179.8(C-13), 118.7(C-13a), 168.9(C-13b) , 22.2(C-14), 71.7(C-1′), 40.0(C-2′), 70.5(C-3′), 77.0(C-4′), 76.1(C-5′), 18.4(C-6′).

[0069] A8: Yellowish-brown amorphous powder; HRESIMS: m / z 483.1280 [M+H] + (The calculated value is C) 25 H 22 O 10 ,483.128 6); UV(MeOH)λ max 209, 250, 338, and 419 nm; 1 H-NMR (600MHz, DMSO-d6): δ8.32(1H,d,J=7.9Hz,H-4), 8.51(1H,d,J=7.9Hz,H-5), 7.89(1H,d,J=7.9Hz,H-9), 7.79( 1H,d,J=7.9Hz,H-10), 3.25(1H,d,J=17.1Hz,H-12), 3.80(1H,d,J=17.0Hz,H-12), 1.85(3H,s,H-14), 3.55(1H,ddd, J=4.9,8.5,11.3Hz,1′-H), 3.25(1H,d,J=17.0Hz,2′-H), 1.27-1.22(1H,m,2′-H), 4.80(1H,dd,J=2.0,11.3Hz,3′-H ), 2.89(1H,t,J=8.8Hz,4′-H), 3.39(1H,dd,J=6.0,9.1Hz,,5′-H), 1.28(3H,d,J=6.0Hz,6′-H), 12.58(1H,s,7-OH); 13C-NMR (150MHz, DMSO-d6): δ167.4(C-1), 88.1(C-3), 152.0(C-3a), 129.6(C-4), 128.8(C-5) , 128.4(C-5a), 187.6(C-6), 115.1(C-6a), 157.5(C-7), 137.9(C-8), 133.5(C-9), 119.6(C- 10), 132.0(C-10a), 182.0(C-11), 133.2(C-11a), 137.8(C-11b), 40.4(C-12), 170.3(C-13) , 24.1(C-14), 71.6(C-1′), 40.0(C-2′), 70.6(C-3′), 77.0(C-4′), 76.1(C-5′), 18.4(C-6′).

[0070] A9: Yellowish-brown amorphous powder; HRESIMS: m / z 483.1270 [M+H] + (The calculated value is C) 25 H 22 O 10 ,483.128 6); UV(MeOH)λ max 208, 250, 340, and 419nm; 1 H-NMR (600MHz, DMSO-d6): δ8.30(1H,d,H-4), 8.50(1H,d,J=7.9Hz,H-5), 7.90(1H,d,J=7.1H z,H-9), 7.79(1H,d,J=7.8Hz,H-10), 3.21(1H,d,J=17.1Hz,H-12), 3.81(1H,d,J=17.0Hz,H- 12), 1.82(3H,s,H-14), 3.59-3.49(1H,m,1′-H), 2.32-2.22(1H,m,1H,2′-H), 4.82(1H,d,J= 11.2Hz, 3′-H), 2.90 (1H, t, J = 8.8Hz, 4′-H), 3.39 (1H, m, 5′-H), 1.28 (3H, d, J = 6.2Hz, 6′-H); 13C-NMR (150MHz, DMSO-d6): δ167.4(C-1), 88.3(C-3), 152.4(C-3a), 129.6(C-4), 128.7(C-5) , 128.3(C-5a), 187.5(C-6), 115.1(C-6a), 157.7(C-7), 137.9(C-8), 133.4(C-9), 119.5(C- 10), 132.0(C-10a), 182.0(C-11), 133.4(C-11a), 138.0(C-11b), 40.9(C-12), 170.5(C-13) , 24.1(C-14), 71.6(C-1′), 40.0(C-2′), 70.6(C-3′), 77.0(C-4′), 76.1(C-5′), 18.4(C-6′).

[0071] A10: Light blue gel-like substance; HRESIMS: m / z 390.0716 [M+H] + (The calculated value is C) 19 H 18 O9,390.0757); UV(MeOH)λ max 195, 235, and 385nm; 1 H NMR (600MHz, DMSO-d6): δ H 2.46(1H,d,J=18.6Hz,H-2a), 2.35(1H,dd,J=18.6,3.0Hz,H-2b), 1.83(1H,d,J=14.4Hz,H-4a ), 2.30(1H,dd,J=14.4,3.0Hz,H-4b), 2.07(1H,m,H-5a), 1.76(1H,m,H-5b), 4.56(1H,m,H-6) , 7.34(1H,m,H-9), 7.70(1H,m,H-10), 7.48(1H,m,H-11), 1.07(3H,s,H-13), 5.05(1H,s,OH-4 a), 4.62(1H,d,J=6.7Hz,OH-6), 5.72(1H,s,OH-6a), 10.86(1H,s,OH-8), 5.51(1H,s,OH-12b); 13 C NMR (150MHz, DMSO-d6): δ C204.5(C-1),47.8(C-2),75.3(C-3),47.6(C-4),77.7(C-4a),38.8(C-5),63.4(C-6),78.7(C-6a),198(C-7),117.4(C-7a),1 59.5(C-8), 123.9(C-9), 135.9(C-10), 118.5(C-11), 133.1(C-11a), 191.0(C-12), 81.3(C-12a), 70.5(C-12b), 24.6(C-13).

[0072] A11: Yellowish-brown amorphous powder; HRESIMS: m / z 372.0716 [M+H] + (The calculated value is C) 19 H 16 O8,372.0757); UV(MeOH)λ max 195, 208, 243, 251, and 374 nm; 1 H NMR (600MHz, DMSO-d6): δ H 2.54(1H,d,J=18.7Hz,H-2a), 2.50(1H,dd,J=18.7,2.3Hz,H-2b), 2.12(1H,d,J=14.9Hz,H- 4a), 2.15(1H,dd,J=14.9,3.0Hz,H-4b), 2.86(1H,d,J=18.2Hz,H-5a), 2.67(1H,d,J=18.2Hz ,H-5b), 7.36(1H,d,J=8.3Hz,H-9), 7.75(1H,m,H-10), 7.48(1H,m,H-11), 1.10(3H,s,H-13) , 5.48(1H,s,OH-4a), 15.05(1H,d,J=6.7Hz,OH-6), 11.82(1H,s,OH-8), 5.6(1H,s,OH-12b); 13 C NMR (150MHz, DMSO-d6): δ C204.9(C-1), 47.8(C-2), 72.2(C-3), 49.9(C-4), 72.2(C-4a), 42.2(C-5), 183.2(C-6), 107.2(C-6a), 189.9(C-7), 115.5(C-7a) , 161.0(C-8), 124.9(C-9), 137.0(C-10), 119.5(C-11), 131.8(C-11a), 189.8(C-12), 77.8(C-12a), 76.6(C-12b), 24.9(C-13).

[0073] Application Example 1

[0074] The activity of the following cancer cells was detected by the MTT reduction method: human lung cancer cells (A549), human liver cancer cells (HepG2), human breast cancer cells (MCF-7), human gastric cancer cells (MGC-803), mouse breast cancer cells (4T-1), and human triple-negative breast cancer cells (MDA-MB-231). All cancer cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA).

[0075] These cells are adherent cells, specifically adherent tumor cells in the logarithmic growth phase. After trypsin digestion, the digested A549 and 4T1 cells were cultured in 1640 medium containing 10% fetal bovine serum at concentrations of 5 × 10⁻⁶ cells / mL. 4 / ml of cell suspension;

[0076] Using DMEM medium containing 10% fetal bovine serum, the digested HepG2, MCF-7, MGC-803, and MDA-MB-231 were respectively prepared into 5×10⁻⁶ solutions. 4 / ml of cell suspension;

[0077] Each cell suspension was seeded into a 96-well culture plate, 100 μl per well, and cultured at 37°C with 5% CO2 for 24 h.

[0078] Both experimental group (compounds A1-A11) and control group samples were dissolved in DMSO as test samples, with an initial test concentration of 1 μM. Each sample was added to the culture plate as described above, with three parallel wells per group. The plates were incubated at 37°C and 5% CO2 for 48 h. The supernatant was discarded, and the plates were washed twice with PBS. 100 μl of freshly prepared medium containing 0.5 mg / ml MTT was added to each well, and the plates were incubated at 37°C for another 4 h. The supernatant was carefully discarded, and 150 μl of DMSO was added. The mixture was stirred using a micro-shaker for 10 min, and the optical density was measured at 492 nm using a microplate reader. 5-Fluorouracil (5-FU) was used as a positive control.

[0079] The tumor cell growth inhibition rate was calculated using the following formula, and the half-maximal inhibitory concentration (IC50) of the compound sample was determined using software. Tumor cell growth inhibition rate (%) = [A] 492 (Negative Control) - A (Drug-Added Group) / A 492 (Negative control) × 100%.

[0080] 1640 medium: 10% fetal bovine serum, 1640 medium base components plus 2% glutamine. DMEM medium: 10% fetal bovine serum, DMEM medium base components plus 2% glutamine.

[0081] Table 1 shows the in vitro antitumor activity results of the compounds.

[0082]

[0083] 5-Fu was used as a positive control.

[0084] In summary, compounds A1-A5, A8, A10, and A11 all exhibit varying degrees of antitumor activity. Compound A1 showed strong antitumor activity against A549 and 4T-1, with IC50 values ​​of 15.74 μM and 19.94 μM, respectively. Compound A4 also showed strong antitumor activity against A549, HepG2, and 4T-1, with IC50 values ​​of 10.83 μM, 16.44 μM, and 9.53 μM, respectively. These compounds provide new options for the further development and preparation of clinical drugs for the prevention and treatment of tumors.

[0085] Application Example 2

[0086] Further research was conducted on the antitumor activity targets of the aforementioned secondary metabolites. The specific experimental methods and results are as follows:

[0087] 1) The PharmMapper database (http: / / www.lilab-ecust.cn / pharmmapper / ) was used to screen potential targets based on pharmacophore features. Cancer-related targets were retrieved from the public Therapeutic Targets database (http: / / bidd.nus.edu.sg / group / ttd / ) using the keyword "Cancer". The intersection of metabolite targets and cancer-related targets was obtained and imported into Cytoscape 3.9.1 (https: / / cytoscape.org / ) to construct a "strain-compound-target" network (see figure). The intersection targets were analyzed for PPI using the String website (https: / / cn.string-db.org / ). The PPI analysis results were imported into Cytoscape 3.9.1 for visualization and to construct a PPI network. Based on the experimental results, the most likely anti-tumor target was inferred to be matrix metalloproteinase-2 (MMP2).

[0088] 2) Chem 3D 14.0 software was used to obtain the lowest energy state of the antitumor compounds. The MMP2 protein crystal structure was downloaded from the Protein Data Bank (PDB) (https: / / www.rcsb.org / ). Subsequently, AutoDockVina 1.1.2 software (http: / / autodock.scripps.edu / resources / adt) was used to perform molecular docking on compounds A4 and A5, which were active in six tumor cell types (see Table 1) (see Table 1). Figure 2 The docking results showed that compounds A4 and A5 formed hydrogen bonds with key amino acid residues at the active site of the target protein MMP2, with good docking performance and binding energies of less than -10 kcal / mol (see Table 2).

[0089] Table 2 Results of Molecular Docking Experiments

[0090]

[0091] 3) Using the Octet K2 system from ForteBio (USA) and recombinant protein MMP2 (#AAH02576) purchased from ACRO Biosystems, compounds A4 and A5 were subjected to BLI binding affinity tests to analyze the interaction between the active small molecules and protein MMP2. The results showed that the dissociation constants KD between compounds A4 and A5 and MMP2 were both less than 10. -6 M.

[0092] In summary, these data demonstrate that MMP2 is one of the antitumor targets of the polycyclic aromatic ketone compounds in this study. The molecular docking results and BLI binding affinity test results directly experimentally prove that the antitumor compounds A4 and A5 in this study can interact strongly with the target MMP2. This example represents the first discovery of polycyclic aromatic ketone compounds acting on the MMP2 protein in an antitumor study.

[0093] Application Example 3

[0094] The antibacterial activity of the following pathogenic bacteria was determined by 96-well plate turbidimetry. The pathogenic bacteria were divided into Staphylococcus aureus, Klebsiella pneumoniae, and Enterococcus faecium.

[0095] The specific operation of the 96-well plate turbidimetric method is as follows:

[0096] 1) Preparation of bacterial suspension:

[0097] The activated bacteria were inoculated into LB medium and incubated at 28°C for 24 hours. An appropriate amount of sterile 0.9% physiological saline was added to the colonies. The bacterial cells were scraped using a sterile inoculation loop and transferred to a sterile test tube. The mixture was shaken to form a turbid bacterial suspension. The OD value of the suspension was then diluted to 0.3 (approximately 10⁻⁶) using a spectrophotometer (600 nm). 8 (cfu / mL). Then take 0.5 ml and dilute 10 times (approximately 10). 7 A bacterial suspension of (cfu / mL) was inoculated at 1% onto 50 mL of liquid culture medium to prepare a bacterial culture suspension for 96-well plates (approximately 10 cfu / mL). 5 (cfu / mL).

[0098] 2) Testing:

[0099] In a sterile 96-well plate, add the prepared blank LB liquid medium, the test sample (working concentration 64-2 μg / ml, half-dilution method) and the positive control drug to the corresponding wells in the following order, place the plate upright in a 28℃ incubator, and read the OD600 value using a microplate reader after 24 hours.

[0100]

[0101] 3) Antibacterial rate (%) = (1 - A / B) * 100%

[0102] A: Mean OD value of the experimental group - OD value of the blank control group;

[0103] B: Average OD value of solvent control group - OD value of blank control group

[0104] Table 3 shows the antibacterial activity results of the compounds.

[0105]

[0106] In summary, compounds A3-A10 all exhibit varying degrees of antibacterial activity. Among them, compounds A3 and A4 demonstrate better antibacterial activity against Staphylococcus aureus, with their MICs being [missing information]. 50 Compound A5 exhibited strong antibacterial activity against Klebsiella pneumoniae at concentrations of 32 and 8 μg / ml, respectively, with a MIC of [missing value]. 50 The concentration was 8 μg / ml. A3, A4, A6, A7, and A8 showed certain antibacterial activity against Enterococcus faecalis, with a MIC of 8 μg / ml. 50 All were 64 μg / ml.

Claims

1. A Streptomyces secondary metabolite, characterized in that: The secondary metabolites of Streptomyces are polycyclic aromatic polyketides, as shown in A4. Or a pharmaceutically acceptable salt of the polycyclic aromatic polyketide compound shown in A4.

2. A method for preparing the Streptomyces secondary metabolite according to claim 1, characterized in that: via Streptomyces Streptomyces rosealbus Fermentation and purification yielded the polycyclic aromatic polyketide compound shown in A4; Specifically: (1) Fermentation production of strains: The activated strains are fermented... Streptomycesrosealbus Inoculate the culture medium into a primary seed culture medium and incubate at 28-30 ℃ with shaking for 24-36 h. Collect the bacterial solution as the primary seed culture. Inoculate the primary seed culture into a secondary seed culture medium and incubate at 28-32 ℃ with shaking for 24-36 h to obtain the secondary seed culture. Inoculate the secondary seed culture into a fermentation medium for fermentation at 28-30 ℃ with an aeration rate of 0.2-0.4 vvm for 7-10 days to obtain the fermentation broth. (2) Preparation of extract: Take the fermentation broth from step (1), centrifuge at 8000-10000 rpm for 10-15 min to remove the cells, soak the supernatant in ethyl acetate, sonicate, and then distill under reduced pressure to obtain the extract; (3) Isolation and purification of compounds: Fermentation yielded an extract, which was then subjected to silica gel column chromatography, Sephadex LH-20 column chromatography and preparative high performance liquid chromatography to finally obtain the Streptomyces secondary metabolites shown in A4. The fermented extract was dissolved and purified by silica gel column chromatography using a dichloromethane / methanol mixture. The crude fraction with a dichloromethane / methanol V / V ratio of 85:15-80:20 was collected. Then, the fraction was separated by Sephadex LH-20 column chromatography using pure methanol as the mobile phase at a flow rate of 0.1 ml / min. The obtained fraction was then separated and purified by preparative liquid chromatography at UV 220 nm using an acetonitrile-water mixture with a V / V ratio of 80:20-60:40 as the mobile phase to obtain the compound shown in A4 at a flow rate of 3 ml / min.

3. The method for preparing Streptomyces secondary metabolites according to claim 2, characterized in that: The primary and secondary seed culture media are prepared by adding 4.0 g yeast powder, 4.0 g glucose, 2.0 g malt powder, and 1.0 ml trace salt to every 1000 ml of water, adjusting the pH to 7.2, and sterilizing at 121°C for 30 min. The fermentation medium is as follows: add 20.0 ml of glycerol, 4.0 g of malt powder, 8.0 g of yeast extract, and 1.0 ml of trace salt solution to every 1000 ml of water, adjust the pH to 7.2, and sterilize at 121℃ for 30 min. The trace salt formula is: FeSO4•7H2O 0.1 g, MnCl2•4H2O 0.1 g, ZnSO4•7H2O 0.1 g, and water 100 ml.

4. The application of a Streptomyces secondary metabolite as described in claim 1, characterized in that: The use of the Streptomyces secondary metabolites of claim 1 and their pharmaceutically acceptable salts in the preparation of antitumor drugs or antipathogenic agents.

5. The application of the Streptomyces secondary metabolites according to claim 4, characterized in that: The application of the Streptomyces secondary metabolite of claim 1 and its pharmaceutically acceptable salt in the preparation of MMP2 protein as an antitumor drug.

6. An antitumor composition, characterized in that: The composition comprises secondary metabolites of the Streptomyces genus as described in claim 1.

7. An antipathogenic composition, characterized in that: The composition comprises secondary metabolites of the Streptomyces genus as described in claim 1.

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