Naphthalene cyclomycin pentacyclic aromatic polyketone compound and preparation method thereof

Abstract

The invention discloses a naphthocycline type pentacyclic aromatic polyketone compound. The structural formula of the naphthocycline type pentacyclic aromatic polyketone compound is as shown in a formula (I): # imgabs0 #. According to the invention, six naphthocycline type pentacyclic aromatic polyketone compounds are separated from a rice fermentation product of actinomycetes Streptomyces sp.NAK5495 for the first time, and the six naphthocycline type pentacyclic aromatic polyketone compounds are separated from rice fermentation products of actinomycetes Streptomyces sp.NAK5495; according to the present invention, with the medium-pressure liquid chromatography (MPLC) system and the semi-preparative HPLC separation and purification technology, the efficient separation and purification of the naphthocycline pentacyclic aromatic polyketones are achieved, and the recovery rate and the purity of the target compound are improved.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a naphthycycline pentacyclic aromatic polyketide compound produced by an actinomycete Streptomyces strain Streptomyces p.NAK5495 and a preparation method thereof. Background Art

[0002] Aromatic polyketides are a class of natural products with unique chemical structures and a wide range of biological activities. Their molecular structures are typically composed of multiple aromatic rings and polyketide chains. This structural diversity endows them with a rich variety of chemical properties and biological functions. The backbone structure of aromatic polyketides typically contains aromatic rings such as benzene and naphthalene rings, and polyketide chains composed of functional groups such as keto, hydroxyl, and methoxy groups. The different types and arrangements of these functional groups give aromatic polyketides a wide range of chemical properties and biological activities.

[0003] Aromatic polyketides have attracted considerable attention from researchers due to their remarkable medicinal properties. They demonstrate significant application value in a variety of fields, including: Antibiotic activity: Many aromatic polyketides exhibit broad-spectrum antibacterial activity, effectively inhibiting the growth of a wide range of bacteria, including some drug-resistant strains. For example, some aromatic polyketides exhibit potent inhibitory effects against both Gram-positive and Gram-negative bacteria, providing an important foundation for the development of new antibiotics. Antifungal activity: Aromatic polyketides also demonstrate significant potential in the prevention and treatment of fungal infections. They can inhibit the growth and reproduction of fungi, particularly common pathogenic fungi such as Candida albicans and Aspergillus. Antiviral activity: Some aromatic polyketides also possess antiviral activity, interfering with viral replication and inhibiting viral proliferation. This provides new insights and directions for the development of antiviral drugs. Anticancer activity: Recent studies have revealed that some aromatic polyketides exhibit significant inhibitory effects against various cancer cells. These anticancer effects can be achieved by inducing apoptosis, inhibiting cancer cell proliferation, and blocking the cell cycle, offering new hope for cancer treatment. Immunomodulatory activity: Aromatic polyketide compounds also have immunomodulatory effects, can regulate the body's immune response, enhance the body's immune function, and have potential therapeutic value for some immune-related diseases.

[0004] The application areas of aromatic polyketides include agriculture, pharmaceuticals, and environmental remediation. Agriculture: In agriculture, the antibacterial and antifungal activities of aromatic polyketides can be used to develop new pesticides for the prevention and control of plant diseases, reducing the use of pesticides, reducing environmental pollution, and improving the yield and quality of crops. Pharmaceuticals: The multiple medicinal activities of aromatic polyketides make them an important resource for the development of new drugs. They can be used to develop new antibiotics, antifungal drugs, antiviral drugs, anticancer drugs, and immunomodulators, etc., providing new solutions to the current drug resistance problems and cancer treatment difficulties faced by the medical field. Environmental remediation: Some aromatic polyketides are biodegradable and can be decomposed into harmless substances by microorganisms. Therefore, they have potential application value in environmental remediation. They can be used to treat organic pollutants, degrade harmful substances in the environment, and reduce environmental pollution.

[0005] Actinomycetes are an important source of aromatic polyketides. Actinomycetes are a class of microorganisms widely found in soil and possess a rich capacity for synthesizing secondary metabolites. They are able to synthesize a variety of biologically active natural products through complex metabolic pathways, of which aromatic polyketides are one of their key metabolites. During their growth, actinomycetes convert simple precursors into complex aromatic polyketides through a series of biosynthetic pathways. These compounds not only aid the growth and survival of the actinomycetes themselves but also possess a variety of biological activities, providing humanity with a rich resource of natural products.

[0006] Currently, a pentacyclic aromatic polyketone compound and a preparation method thereof have not been reported. Summary of the Invention

[0007] The object of the present invention is to provide a naphthycycline pentacyclic aromatic polyketide compound and a preparation method thereof.

[0008] In order to solve the problems of the prior art, the present invention provides the following technical solutions: In a first aspect, the present application provides an antibiotic compound.

[0009] In a second aspect, the present application provides a naphthcycline pentacyclic aromatic polyketide compound.

[0010] In a third aspect, the present application provides a method for preparing a naphthycycline pentacyclic aromatic polyketide compound.

[0011] In a first aspect, the present application provides a naphthycycline pentacyclic aromatic polyketone compound, the structural formula of which is shown in formula (I):

[0012]

[0013] The second aspect of the present application provides a method for preparing a naphthcycline pentacyclic aromatic polyketone compound, comprising the following steps:

[0014] (1) Strain culture: The actinomycete strain Streptomyces p.NAK5495 was cultured on ISP2 plate medium and placed in a 30°C incubator for 2-3 days to prepare an actinomycete culture;

[0015] (2) Seed solution preparation: The actinomycete culture obtained in step (1) was cut into pieces, inoculated into a TSB liquid shake flask, and cultured at 30°C and 220 rpm for 1-3 days to prepare a seed solution;

[0016] (3) solid fermentation: the seed liquid prepared in step (2) was inoculated into a rice solid culture medium and cultured at 30° C. for 6-8 days;

[0017] (4) Extraction: extracting the culture medium obtained in step (3) with ethyl acetate as an organic solvent and concentrating the extract to obtain a crude extract;

[0018] (5) Medium pressure liquid chromatography (MPLC) separation: Gradient elution was performed on the crude extract obtained in step (4) using a reverse phase C18 chromatographic column to obtain 6 components. Each medium pressure sample was subjected to liquid chromatography HPLC analysis to obtain the target component;

[0019] (6) High performance liquid chromatography (HPLC) purification: The target component obtained in step (5) is separated by HPLC to obtain the naphthycycline pentacyclic aromatic polyketide compound.

[0020] Furthermore, in step (1), the ISP2 plate culture medium includes 4.0 g of yeast extract powder, 10.0 g of malt extract powder, 4.0 g of glucose, 20.0 g of agar, 1 L of distilled water, and a pH of 7.4-7.6.

[0021] Furthermore, in step (3), the rice solid culture medium is composed of rice and ME liquid culture medium, and the rice solid culture medium is prepared by adding 45 ml of ME liquid culture medium to 33 g of rice and sterilizing by high pressure at 115° C. for 30 minutes to prepare the rice solid culture medium;

[0022] The ME liquid culture medium consists of malt, sucrose and peptone. The preparation method of the ME liquid culture medium is as follows: 20g of malt is steamed for 25-30 minutes, filtered, and then 20g of sucrose and 1g of peptone are added, and the volume is fixed to 1000ml with distilled water to prepare the liquid culture medium.

[0023] Furthermore, in step (5), the MPLC chromatographic conditions are as follows: using a 60 g reverse phase C18 chromatographic column, mobile phase A: water, mobile phase B: methanol, and the gradient elution program: the volume percentage of mobile phase B increases linearly from 10% to 100%, the flow rate is 50 mL / min, and after elution for 13 column volumes, elution is maintained at 100% mobile phase B for 3 column volumes.

[0024] Furthermore, in step (6), in the HPLC separation, the elution conditions for compound 1 were 44% acetonitrile, a flow rate of 2.0 mL / min, and an elution time of 32 minutes; and the elution conditions for compound 2 were 65% acetonitrile, a flow rate of 2.0 mL / min, and an elution time of 15 minutes.

[0025] Furthermore, in HPLC separation, the elution conditions for compound 3 were 65% acetonitrile, a flow rate of 2.0 mL / min, and elution for 12 minutes; the elution conditions for compound 4 were 58% acetonitrile, a flow rate of 2.0 mL / min, and elution for 30 minutes.

[0026] Furthermore, in step (6), in the HPLC separation, the separation and purification conditions for compound 5 are 54% acetonitrile eluted at a flow rate of 2.0 mL / min for 13 minutes; the separation and purification conditions for compound 6 are 65% acetonitrile eluted at a flow rate of 2.0 mL / min for 40 minutes.

[0027] Beneficial effects: The present invention realizes the efficient separation and purification of naphthcycline pentacyclic aromatic polyketones through medium pressure liquid chromatography (MPLC) system and semi-preparative HPLC separation and purification technology, thereby improving the recovery rate and purity of the target compound.

[0028] Compared with the existing technology, the present invention has the following advantages: (1) The present invention successfully discovered, separated and identified new naphthcycline-type pentacyclic aromatic polyketide compounds through an innovative preparation method, enriched the natural product structure library, and provided a new material basis for research in related fields.

[0029] (2) The present invention utilizes medium-pressure liquid chromatography (MPLC) and semi-preparative high-performance liquid chromatography (HPLC) separation and purification techniques to achieve efficient separation and purification of naphthycycline pentacyclic aromatic polyketones. Compared to traditional methods, this technology significantly improves the recovery and purity of the target compound, reduces impurity content, and enhances product quality and performance, providing a purer raw material for subsequent pharmacological research and drug development.

[0030] (3) The preparation method of the present invention encompasses detailed steps from culturing actinomycetes, fermentation, extraction, to chromatographic separation and purification, forming a complete and highly operable process flow. This process flow has been verified by test examples and examples and has good feasibility and repeatability. It provides reliable technical support for the large-scale production of pentacyclic aromatic polyketide compounds, reduces production costs, improves production efficiency, and facilitates the industrial production of such compounds.

[0031] (4) This invention provides an in-depth structural analysis of the newly discovered naphthycycline pentacyclic aromatic polyketones, providing detailed data such as high-resolution electrospray ionization mass spectrometry (HRESI-MS) and nuclear magnetic resonance spectroscopy (NMR). These data not only clarify the structural characteristics of the compounds but also provide a solid scientific basis for further research on their physicochemical properties, biological activities, and structure-activity relationships, helping to accelerate the drug development process and improve the success rate of R&D. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments or descriptions of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0033] Figure 1-Figure 42 The figures are the nuclear magnetic resonance data and mass spectrometry data of the naphthycycline pentacyclic aromatic polyketone 1-6 of the present invention. DETAILED DESCRIPTION

[0034] In order to make the technical problems, technical solutions and beneficial effects to be solved by this application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not intended to limit this application.

[0035] In this application, the term "and / or" describes the relationship between related objects, indicating the existence of three relationships. For example, A and / or B means: A exists alone, A and B exist simultaneously, and B exists alone. A and B are singular or plural. The character " / " generally indicates that the related objects are in an "or" relationship.

[0036] In this application, "at least one" means one or more, and "plurality" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c are single or plural, respectively.

[0037] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps are executed in parallel or one after another, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0038] The terms used in the embodiments of the present application are for the purpose of describing specific embodiments only and are not intended to limit the present application. The singular forms "a", "an", "the" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms unless the context clearly indicates otherwise.

[0039] The Chinese translation of Naphthacemycin is naphthacemycin.

[0040] In a first aspect, the present invention provides a naphthycycline pentacyclic aromatic polyketone compound. The chemical structure of the naphthycycline pentacyclic aromatic polyketone compound is shown in Formula (I):

[0041]

[0042] A second aspect of the present invention provides a method for preparing a naphthcycline pentacyclic aromatic polyketide compound, comprising the following steps:

[0043] (1) Strain culture: The actinomycete strain Streptomyces p.NAK5495 was cultured on ISP2 plate medium and placed in a 30°C incubator for 2-3 days to prepare an actinomycete culture;

[0044] (2) Seed solution preparation: The actinomycete culture obtained in step (1) was cut into pieces, inoculated into a TSB liquid shake flask, and cultured at 30°C and 220 rpm for 1-3 days to prepare a seed solution;

[0045] (3) solid fermentation: the seed liquid prepared in step (2) was inoculated into a rice solid culture medium and cultured at 30° C. for 6-8 days;

[0046] (4) Extraction: extracting the culture medium obtained in step (3) with ethyl acetate as an organic solvent and concentrating the extract to obtain a crude extract;

[0047] (5) Medium pressure liquid chromatography (MPLC) separation: gradient elution was performed on the crude extract obtained in step (4) using a reverse phase C18 chromatographic column to obtain 6 components. Each medium pressure sample was subjected to liquid chromatography (HPLC) analysis to obtain the target component.

[0048] (6) High performance liquid chromatography (HPLC) purification: The target component obtained in step (5) is separated by HPLC to obtain the naphthycycline pentacyclic aromatic polyketide compound.

[0049] In some embodiments, in step (1), the ISP2 plate culture medium includes 4.0 g yeast extract powder, 10.0 g malt extract powder, 4.0 g glucose, 20.0 g agar, 1 L distilled water, and a pH of 7.4-7.6.

[0050] In some embodiments, in step (3), the rice solid culture medium is composed of rice and ME liquid culture medium, and the rice solid culture medium is prepared by adding 45 ml of ME liquid culture medium to 33 g of rice and sterilizing by high pressure at 115° C. for 30 minutes to prepare the rice solid culture medium;

[0051] ME liquid culture medium is composed of malt, sucrose and peptone. The preparation method of ME liquid culture medium is as follows: 20g of malt is steamed for 25-30 minutes, filtered, and then 20g of sucrose and 1g of peptone are added, and the volume is adjusted to 1000ml with distilled water to prepare the liquid culture medium.

[0052] In some embodiments, in step (5), the MPLC chromatographic conditions are: using a 60 g reverse phase C18 chromatographic column, mobile phase A: water, mobile phase B: methanol, and the gradient elution program is: the volume percentage of mobile phase B increases linearly from 10% to 100%, the flow rate is 50 mL / min, and after elution for 13 column volumes, 100% mobile phase B is maintained for elution for 3 column volumes.

[0053] In some embodiments, in step (6), the elution conditions for compound 1 are 44% acetonitrile, a flow rate of 2.0 mL / min, and elution for 32 minutes.

[0054] In some embodiments, the elution conditions for Compound 2 are 65% acetonitrile, a flow rate of 2.0 mL / min, and elution for 15 minutes.

[0055] In some embodiments, in HPLC separation, the elution conditions for compound 3 are 65% acetonitrile, a flow rate of 2.0 mL / min, and elution for 12 minutes.

[0056] In some embodiments, the elution conditions for compound 4 are 58% acetonitrile, a flow rate of 2.0 mL / min, and elution for 30 minutes.

[0057] In some embodiments, in step (6), in the HPLC separation, the separation and purification conditions of compound 5 are 54% acetonitrile eluted at a flow rate of 2.0 mL / min for 13 minutes.

[0058] In some embodiments, the separation and purification conditions for compound 6 are 65% acetonitrile and elution at a flow rate of 2.0 mL / min for 40 minutes.

[0059] Example 1

[0060] like Figure 1-Figure 42 As shown, Figure 1-Figure 42 The figures are the nuclear magnetic resonance data and mass spectrometry data of the naphthycycline pentacyclic aromatic polyketone 1-6 of the present invention.

[0061] The naphthcycline pentacyclic aromatic polyketide compound of the present invention has a chemical structural formula as shown in formula (I):

[0062]

[0063] Example 2

[0064] The preparation method of the naphthcycline pentacyclic aromatic polyketide compound of the present invention comprises the following steps:

[0065] (1) Strain Culture: A glycerol seed tube of the actinomycete Streptomyces sp. NAK5495 was spread onto ISP2 plate culture medium and incubated in a 30°C incubator for 2 days to prepare an actinomycete culture. On the ISP2 plate, mycelium was white at the beginning of the culture and then turned gray. The ISP2 plate culture medium consisted of 4.0 g yeast extract, 10.0 g malt extract, 4.0 g glucose, 20.0 g agar, and 1 L distilled water, pH 7.5.

[0066] (2) Preparation of seed solution: The actinomycete culture obtained in step (1) was cut into pieces, inoculated into TSB (tryptone soy broth) liquid shake flask, and cultured at 30°C and 220 rpm for 3 days to prepare seed solution;

[0067] (3) solid fermentation: the seed solution prepared in step (2) was inoculated into a rice solid culture medium and cultured at 30° C. for 6 days;

[0068] The rice solid culture medium comprises the following steps: adding 45 ml of ME liquid culture medium to 33 g of rice, and sterilizing the mixture by autoclaving at 115° C. for 30 minutes to obtain the rice solid culture medium; the ME liquid culture medium comprises malt, sucrose, and peptone; and preparing the malt extract comprises steaming 20 g of malt for 28 minutes, filtering, adding 20 g of sucrose and 1 g of peptone, and distilling the mixture to 1000 ml with distilled water to obtain the malt extract.

[0069] (4) Extraction: extracting the culture medium obtained in step (3) with ethyl acetate as an organic solvent and concentrating the extract to obtain a crude extract;

[0070] (5) Medium pressure liquid chromatography (MPLC) separation: The crude extract obtained in step (4) was subjected to gradient elution using a reversed-phase C18 chromatographic column to obtain 6 components. Each medium pressure sample was subjected to liquid chromatography HPLC analysis to obtain the target component. The MPLC chromatographic conditions were: a 60 g reversed-phase C18 chromatographic column was used, mobile phase A: water, mobile phase B: methanol, and the gradient elution program was: the volume percentage of mobile phase B increased linearly from 10% to 100%, the flow rate was 50 mL / min, and after elution for 13 column volumes, 100% mobile phase B was maintained for elution for 3 column volumes.

[0071] (6) High-performance liquid chromatography (HPLC) purification: The target component obtained in step (5) was separated by HPLC. The elution conditions for compound 1 were 44% acetonitrile, a flow rate of 2.0 mL / min, and elution for 32 minutes to obtain compound 1 (5.4 mg).

[0072] The Streptomyces sp. NAK5495 strain of the present invention was purchased from China General Microbiological Culture Collection Center, with a collection number of CGMCC No. 15050; the collection location is China General Microbiological Culture Collection Center (CGMCC), Beijing, China.

[0073] Pentacyclic aromatic polyketides of the naphthacylin class are uniquely structured aromatic polyketides containing five aromatic rings. This complex structure confers unique chemical properties and biological activities. The discovery of this structure may indicate new biological activities and synthetic mechanisms, providing a potential new drug source molecule for clinical use.

[0074] Example 3

[0075] Example 3 differs from Example 2 in that, in step (1), strain culture: a glycerol seed tube of the actinomycete strain Streptomyces p. NAK5495 was spread onto ISP2 plate culture medium and cultured in a 30°C incubator for 2 days to produce an actinomycete culture. On the ISP2 plate, the mycelium was white at the beginning of the culture and then turned gray. The ISP2 plate culture medium consisted of 4.0 g yeast extract, 10.0 g malt extract, 4.0 g glucose, 20.0 g agar, and 1 L distilled water, with a pH of 7.4.

[0076] In step (2), seed solution preparation: the actinomycete culture obtained in step (1) was cut into pieces, inoculated into a TSB liquid shake flask, and cultured at 30°C and 220 rpm for 2 days to prepare seed solution;

[0077] In step (3), solid fermentation: the seed liquid prepared in step (2) is inoculated into a rice solid culture medium and cultured at 30° C. for 8 days; the formula of the rice solid culture medium is as follows: 45 ml of ME liquid culture medium is added to 33 g of rice, and the rice solid culture medium is sterilized by high pressure at 115° C. for 30 minutes to prepare the rice solid culture medium; the ME liquid culture medium is composed of malt, sucrose and peptone, and the preparation method of the ME liquid culture medium is as follows: 20 g of malt is boiled for 30 minutes, filtered, 20 g of sucrose and 1 g of peptone are added, and the volume is adjusted to 1000 ml with distilled water to prepare the ME liquid culture medium.

[0078] In step (6), the target component obtained in step (5) was separated by HPLC. The elution conditions for compound 2 were 65% acetonitrile, a flow rate of 2.0 mL / min, and elution for 15 minutes to obtain compound 2 (8.7 mg).

[0079] Example 4

[0080] Example 4 differs from Example 2 in that, in step (1), strain culture: a glycerol seed tube of the actinomycete strain Streptomyces p. NAK5495 was spread onto ISP2 plate culture medium and incubated in a 30°C incubator for 2 days to produce an actinomycete culture. On the ISP2 plate, the mycelium was white at the beginning of the culture and then turned gray. The ISP2 plate culture medium consisted of 4.0 g yeast extract, 10.0 g malt extract, 4.0 g glucose, 20.0 g agar, and 1 L distilled water, with a pH of 7.6.

[0081] In step (2), seed solution preparation: the actinomycete culture obtained in step (1) was cut into pieces, inoculated into a TSB liquid shake flask, and cultured at 30°C and 220 rpm for 1 day to prepare seed solution;

[0082] In step (3), solid fermentation: the seed liquid prepared in step (2) is inoculated into a rice solid culture medium and cultured at 30° C. for 7 days; the formula of the rice solid culture medium is as follows: 45 ml of ME liquid culture medium is added to 33 g of rice, and the rice solid culture medium is sterilized by high pressure at 115° C. for 30 minutes to prepare the rice solid culture medium; the ME liquid culture medium is composed of malt, sucrose and peptone, and the preparation method of the ME liquid culture medium is as follows: 20 g of malt is boiled for 25 minutes, filtered, and then 20 g of sucrose and 1 g of peptone are added, and the volume is adjusted to 1000 ml with distilled water to prepare the ME liquid culture medium.

[0083] In step (6), the target component obtained in step (5) was separated by HPLC. The elution conditions for compound 3 were 65% acetonitrile, a flow rate of 2.0 mL / min, and elution for 12 minutes to obtain compound 3 (8.6 mg).

[0084] Example 5

[0085] The difference between Example 5 and Example 2 is that in step (6), the target component obtained in step (5) was separated by HPLC, and the elution conditions for compound 4 were 58% acetonitrile, a flow rate of 2.0 mL / min, and elution for 30 minutes to obtain compound 4 (4.3 mg).

[0086] Example 6

[0087] The difference between Example 6 and Example 2 is that in step (6), the target component obtained in step (5) is separated by HPLC, and the separation and purification conditions of compound 5 are 54% acetonitrile at a flow rate of 2.0 mL / min for 13 minutes to obtain compound 5 (2.1 mg).

[0088] Example 7

[0089] The difference between Example 7 and Example 2 is that in step (6), the target component obtained in step (5) is separated by HPLC, and the separation and purification conditions of compound 6 are 65% acetonitrile at a flow rate of 2.0 mL / min for 40 minutes to obtain compound 6 (1.9 mg).

[0090] Test Example 1

[0091] Compound 1 in Example 2 was analyzed. Compound 1, a yellow powder, had a molecular ion peak m / z of 611.1233 [M+H] as determined by high-resolution electrospray ionization mass spectrometry (HRESI-MS). + The molecular formula is estimated to be C 32 H 28Cl2O8 (calculated value: 611.1234), with a degree of unsaturation of 18. H-3 shows HMBC correlations with C-2 and C-4, H-1 and methoxy H-22 show HMBC correlations with C-2, and methoxy H-23 shows HMBC correlations with C-4, indicating that methoxy groups 22 and 23 are located at C-2 and C-4 of the benzene ring, respectively. Methyl groups 13 and 14 also show HMBC correlations with C-12, indicating that two methyl groups, H-13 and H-14, are attached to C-12. H-3 shows HMBC correlations with C-4a and C-5, suggesting that C-5 is a carbonyl carbon based on chemical shifts. Aromatic hydrogen H-8 shows HMBC correlations with C-9 and C-10, while methoxy H-24 shows HMBC correlations only with C-9, suggesting that this group is located at C-9. H-8 also shows HMBC correlations with carbonyl C-6, suggesting that it is a carbonyl based on chemical shifts. H-8 is related to C-7 and C-15, so it is believed that C-7 and C-15 connect two benzene rings, and the basic structure of compound 1 is determined. 1 H and 13 The data assignments of C NMR and two-dimensional spectra are shown in Table 1.

[0092] The structure of compound 1 is as follows:

[0093]

[0094] Table 1

[0095]

[0096]

[0097] Test Example 2

[0098] Compound 2 in Example 3 was analyzed. Compound 2 was a yellow powder. High-resolution electrospray ionization mass spectrometry (HRESI-MS) determined its molecular ion peak m / z to be 563.1472 [M+H] + The molecular formula is estimated to be C 31 H 27ClO8 (calculated value is 563.1467), with an unsaturation degree of 18; the chemical shifts of C-22 and C-23 indicate that they are both methoxy groups, and based on the HMBC correlation signals, it is determined that the two are connected to C-2 and C-4 respectively; H-8 and H-10 have HMBC correlation with C-9, and based on the chemical shift of C-9, it is determined that C-9 is connected to a hydroxyl group and is located between C-8 and C-10; aromatic hydrogen H-19 and methyl H-21 have HMBC correlation with C-20, indicating that the methyl group is connected to C-20; two methoxy groups H-24 and H-25 have HMBC correlation with C-16 and C-18 respectively, and H-19 also has HMBC correlation with C-17 and C-18. The position of the substituents on the benzene ring is determined by these correlation signals and the molecular formula inferred by the mass spectrum. 1 H and 13 The data attribution of C NMR and two-dimensional spectra are shown in Table 2:

[0099] The structure of compound 2 is as follows:

[0100]

[0101] Table 2

[0102]

[0103]

[0104] Test Example 3

[0105] Compound 3 in Example 4 was analyzed. Compound 3 was a yellow powder. High-resolution electrospray ionization mass spectrometry (HRESI-MS) determined its molecular ion peak m / z to be 539.0672 [M+H] + The molecular formula is estimated to be C 28 H 21 Cl2O7 (calculated value is 539.0670), with an unsaturation degree of 17; based on the splitting and coupling constants of the hydrogen spectrum of compound 3, it was determined that H-1 and H-3 were in the same spin system and had meta-coupling, and both H-1 and H-3 had HMBC correlations with C-2. Based on the chemical shifts of the carbon spectra of C-2 and C-4, it was inferred that each of them was connected to a hydroxyl group; H-1, two methyl groups H-13, and H-14 had HMBC correlations with C-12, indicating that there were also two methyl groups on the second ring; aromatic hydrogen H-11 had HMBC correlations with C-12, C-11a, and carbonyl C-5, which could determine the positions of the substituents on the second and third rings; H-8 had HMBC correlations with C-15, and H-19 and methyl group H-21 on the other benzene ring also had HMBC correlation signals with C-15, so it was believed that the two benzene rings were connected by a CC bond; the skeletal structure of compound 3 was determined. 1 H and 13The data assignments of C NMR and two-dimensional spectra are shown in Table 3.

[0106] The structure of compound 3 is as follows:

[0107]

[0108] Table 3

[0109]

[0110] Test Example 4

[0111] Compound 4 in Example 5 was analyzed. Compound 4 was a yellow powder. High-resolution electrospray ionization mass spectrometry (HRESI-MS) determined its molecular ion peak m / z to be 547.1171 [M+H] + The molecular formula is estimated to be C 30 H 24 ClO8 (calculated value is 547.1165), unsaturation is 18; according to 1 H spectrum and HMBC spectrum, H-1, H-3 are HMBC correlated with C-2, H-3 is also HMBC correlated with C-4, chemical shift confirmed that C-2 and C-4 are connected with hydroxyl groups, and methyl hydrogen H-22 is also HMBC correlated with C-2, indicating that position 2 is connected with methoxy substitution; H-10 is HMBC correlated with carbonyl C-11, and coupling constants confirm that H-10 and H-8 are located in the meta position of the ring system, H-8 is HMBC correlated with C-15, methyl H-21, benzene ring hydrogen H-19 is also correlated with C-15, thus inferring that the two benzene rings are connected through CC, and the basic skeleton of compound 4 is determined. 1 H and 13 The data assignments of C NMR and two-dimensional spectra are shown in Table 4.

[0112] The structure of compound 4 is as follows:

[0113]

[0114] Table 4

[0115]

[0116]

[0117] Test Example 5

[0118] Compound 5 in Example 6 was analyzed. Compound 5 was a brown powder. High-resolution electrospray ionization mass spectrometry (HRESI-MS) determined its molecular ion peak m / z to be 535.1153 [M+H] + The molecular formula is estimated to be C 29 H 23ClO8 (calculated value is 535.1154), the degree of unsaturation is 18; according to the HMBC spectrum, H-1, H-13, and H-14 all have HMBC correlations with C-12, indicating that two methyl groups are connected to C-12; based on the coupling constants of H-1 and H-3, it is speculated that the two are in the meta position of the benzene ring; H-1, H-3, and methoxy H-22 all have HMBC correlations with C-2, indicating that C-2 is located between C-1 and C-3 and is connected to a methoxy group; H-10 has HMBC correlation signals with carbonyl C-11, H-8 is correlated with C-15, methyl H-21 and H-19 are also correlated with C-15, and H-19 is also correlated with C-17 and C-18. Based on the above correlations, the basic structure of compound 5 was determined; 1 H and 13 The data assignments of C NMR and two-dimensional spectra are shown in Table 5.

[0119] The structure of compound 5 is as follows:

[0120]

[0121] Table 5

[0122]

[0123]

[0124] Test Example 6

[0125] Compound 6 in Example 7 was analyzed. Compound 6 was a yellow powder. High-resolution electrospray ionization mass spectrometry (HRESI-MS) determined its molecular ion peak m / z to be 583.0915 [M+H] + The molecular formula is estimated to be C 30 H 24 Cl2O8 (calculated value is 583.0921), with an unsaturation degree of 18. Based on the coupling constants of H-1 and H-3, it is speculated that the two are in the meta position of the benzene ring; aromatic hydrogen H-10 has an HMBC correlation with C-9 connected to a hydroxyl group; H-8 and H-10 both have an HMBC correlation with C-6a; H-8 has an HMBC correlation with C-15, indicating that it is connected to a benzene ring via a CC bond; methoxy groups H-23 and H-24 have HMBC correlations with C-16 and C-18, respectively, and methyl group H-21 has an HMBC correlation with C-15, C-19, and C-20. Combined with the molecular formula and chemical shifts inferred by HRESIMS, it is inferred that C-17 and C-19 are connected to two chlorine atoms, and the structure of compound 6 is determined. 1 H and 13 The data assignments of C NMR and two-dimensional spectra are shown in Table 6.

[0126] The structure of compound 6 is as follows:

[0127]

[0128] Table 6

[0129]

[0130] The basic principles, main features and advantages of the present invention are shown and described above. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and improvements may be made to the present invention without departing from the spirit and scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims, the description and their equivalents.

Claims

1. A naphthcycline pentacyclic aromatic polyketide compound, characterized in that: The structural formula of the naphthycycline pentacyclic aromatic polyketone compound is shown in formula (I):

2. The method for preparing the naphthycycline pentacyclic aromatic polyketide compound according to claim 1, characterized in that The steps include: (1) Strain culture: The actinomycete strain Streptomyces sp. NAK5495 was cultured on ISP2 plate medium and placed in a 30°C incubator for 2-3 days to prepare an actinomycete culture; (2) Seed solution preparation: The actinomycete culture obtained in step (1) was cut into pieces, inoculated into a TSB liquid shake flask, and cultured at 30°C and 220 rpm for 1-3 days to prepare a seed solution; (3) solid fermentation: the seed liquid prepared in step (2) was inoculated into rice ME solid culture medium and cultured at 30°C for 6-8 days; (4) Extraction: extracting the culture medium obtained in step (3) with ethyl acetate as an organic solvent and concentrating the extract to obtain a crude extract; (5) Medium pressure liquid chromatography (MPLC) separation: gradient elution was performed on the crude extract obtained in step (4) using a reverse phase C18 chromatographic column to obtain 6 components. Each medium pressure sample was subjected to liquid chromatography (HPLC) analysis to obtain the target component. (6) High performance liquid chromatography (HPLC) purification: The target component obtained in step (5) is separated by HPLC to obtain the naphthycycline pentacyclic aromatic polyketide compound.

3. The method for preparing the naphthycycline pentacyclic aromatic polyketone compound according to claim 2, wherein: In step (1), the ISP2 plate culture medium includes 4.0 g of yeast extract powder, 10.0 g of malt extract powder, 4.0 g of glucose, 20.0 g of agar, 1 L of distilled water, and a pH of 7.4-7.

6.

4. The method for preparing the naphthycycline pentacyclic aromatic polyketone compound according to claim 2, wherein: In step (3), the rice solid culture medium is composed of rice and ME liquid culture medium. The rice solid culture medium is prepared by adding 45 ml of ME liquid culture medium to 33 g of rice and sterilizing by high pressure at 115° C. for 30 minutes to prepare the rice solid culture medium; The ME liquid culture medium consists of malt, sucrose and peptone. The preparation method of the ME liquid culture medium is as follows: 20g of malt is steamed for 25-30 minutes, filtered, and then 20g of sucrose and 1g of peptone are added, and the volume is fixed to 1000ml with distilled water to prepare the liquid culture medium.

5. The method for preparing the naphthycycline pentacyclic aromatic polyketone compound according to claim 2, wherein: In step (5), the MPLC chromatographic conditions are as follows: using a 60 g reverse phase C18 chromatographic column, mobile phase A: water, mobile phase B: methanol, and the gradient elution program: the volume percentage of mobile phase B increases linearly from 10% to 100%, the flow rate is 50 mL / min, and after elution for 13 column volumes, elution is maintained at 100% mobile phase B for 3 column volumes.

6. The method for preparing the naphthycycline pentacyclic aromatic polyketide compound according to claim 2, wherein: In step (6), the elution conditions for compound 1 are 44% acetonitrile, a flow rate of 2.0 mL / min, and an elution time of 32 minutes; the elution conditions for compound 2 are 65% acetonitrile, a flow rate of 2.0 mL / min, and an elution time of 15 minutes.

7. The method for preparing the naphthycycline pentacyclic aromatic polyketone compound according to claim 2, wherein: In step (6), in the HPLC separation, the elution conditions for compound 3 are 65% acetonitrile, a flow rate of 2.0 mL / min, and elution for 12 minutes; the elution conditions for compound 4 are 58% acetonitrile, a flow rate of 2.0 mL / min, and elution for 30 minutes.

8. The method for preparing the naphthycycline pentacyclic aromatic polyketone compound according to claim 2, wherein: In step (6), in the HPLC separation, the separation and purification conditions for compound 5 are 54% acetonitrile eluted at a flow rate of 2.0 mL / min for 13 minutes; the separation and purification conditions for compound 6 are 65% acetonitrile eluted at a flow rate of 2.0 mL / min for 40 minutes.

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