A dimeric aromatic polyketone compound and its preparation method and application

Abstract

The present invention discloses a dimeric aromatic polyketide compound, a preparation method and an application thereof. The structural formula of the dimeric aromatic polyketide compound is shown in formula (I): The present invention is the first to isolate two novel dimeric aromatic polyketide compounds, spirocyclines A and B, from a rice fermentation product of the actinomycete Streptomyces spectabilis NA07643. These compounds have a novel ten-membered oxaspiro dimer skeleton, providing novel chemical entities for drug research and development.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to novel dimeric aromatic polyketide compounds (named by the inventors as spirocycline A and spirocycline B) produced by actinomycete Streptomyces spectabilis NA07643 and other strains, as well as preparation and application thereof. Background Art

[0002] Natural products are an important source of innovative drugs. More than 70% of antibiotics in clinical practice are directly or indirectly derived from natural products. With the long-term and widespread use of anti-infective drugs and the slow progress in the development of new antibiotics, the problem of bacterial resistance has become increasingly prominent, posing a serious threat to public health. Therefore, it is urgent to discover drugs with new skeletons and new activities. Bacterial aromatic polyketides (also known as type II polyketides) are an important family of natural products. They exhibit multiple biological activities such as antibacterial, antitumor, antiviral, and antiparasitic activities, and therefore have important clinical medicinal value. They have always been considered an important source of new drug discovery.

[0003] Actinomycetes are an important source of aromatic polyketide drugs. Through gene mining and culture screening, the inventors isolated two novel dimeric aromatic polyketide compounds from S. spectabilis NA07643, named spirocyclines A and B. These compounds possess a rare and novel ten-membered oxaspirocyclic dimer backbone. This structural discovery may suggest novel biological activities and synthetic mechanisms, providing potential new drug-derived molecules for clinical use. Patent protection for the sources, preparations, and uses of these compounds and their analogs is urgently needed. Summary of the Invention

[0004] The purpose of the present invention is to solve the problems of the prior art and provide a dimer aromatic polyketone compound and a preparation method and application thereof.

[0005] To achieve the above objectives, the present invention employs the following technical solutions: First, the present application provides a dimeric aromatic polyketide compound. Second, the present application provides a method for preparing the dimeric aromatic polyketide compound. Third, the present application provides a Streptomyces spectabilis NA07643 strain agent prepared from the actinomycete Streptomyces spectabilis NA07643 strain. Fourth, the present invention provides the use of the dimeric aromatic polyketide compound in the preparation of an oncology drug.

[0006] A dimer aromatic polyketone compound of the present invention has a structural formula as shown in formula (I):

[0007]

[0008] The preparation method of the dimer aromatic polyketone compound of the present invention comprises the following steps:

[0009] (1) The actinomycete Streptomyces spectabilis NA07643 strain was cultured on Streptomyces medium No. 2 ISP2 plates at 30°C for 5-7 days;

[0010] (2) cutting the culture obtained in step (1) into pieces, inoculating the pieces into a TSB liquid shake flask, and culturing at 30° C. and 220 rpm for 1-2 days to prepare a seed solution;

[0011] (3) The seed liquid obtained in step (2) is inoculated into a rice ME solid medium consisting of rice and ME liquid medium, wherein the formula of the rice ME solid medium is 33g of rice added to 45ml of ME liquid medium, and sterilized at high temperature and high pressure; the prepared rice ME solid medium is statically cultured at 30±5°C for 10-14 days; the ME liquid medium formula is 20g of malt cooked for half an hour, and the resulting filtrate is added with 20g of sucrose, 1g of peptone, and 1000ml of distilled water;

[0012] (4) extracting the culture medium obtained in step (3) with ethyl acetate as an organic solvent and concentrating the extract to obtain a crude extract F1;

[0013] (5) The crude extract F1 was first separated using a medium pressure liquid chromatography (MPLC) system with a Biotage SNAP Ultra C18 column to obtain 14 components. Each component was analyzed by HPLC to obtain the target molecular component for HPLC preparation.

[0014] (6) These components were then separated and purified by gel column chromatography and HPLC to obtain dimeric aromatic polyketide compounds, named Spectacycline A and Spectacycline B.

[0015] Furthermore, in step (3), the solid culture medium consisting of rice and ME is inoculated and statically cultured at 30±5°C for 10-14 days.

[0016] Furthermore, in step (4), the HPLC chromatographic conditions are as follows: using a reverse phase chromatographic column, mobile phase A: water, mobile phase B: acetonitrile, and an isocratic elution program: the mass percentage of mobile phase A is 10%, the mass percentage of mobile phase B is 90%, and the time is 18 minutes; the HPLC separation and purification process is as follows: HPLC semi-preparative reverse phase high performance liquid chromatography: ODS-2 Hypersil column, mobile phase: acetonitrile-water volume ratio = 2:3, isocratic elution at a flow rate of 2.5 mL / min for 25 minutes; the pump model is Hitachipump L-7100, and the ultraviolet lamp model is UV detector L-7400.

[0017] Furthermore, Streptomyces spectabilis strain; deposit name: Streptomyces spectabilis NA07643 strain; deposit number: CGMCC 30990; deposit location: China General Microbiological Culture Collection Center (CGMCC), Beijing, China; deposit date: June 18, 2024.

[0018] The invention provides an actinomycete Streptomyces spectabilis NA07643 strain and a Streptomyces spectabilis NA07643 strain agent prepared therefrom.

[0019] The active ingredient of the actinomycete Streptomyces spectabilis NA07643 agent of the present invention is any one of the following (a), (b) and (c):

[0020] (a) Culture of the actinomycete Streptomyces spectabilis strain NA07643;

[0021] (b) Ultrasonic lysis supernatant of Streptomyces spectabilis NA07643 cells;

[0022] (c) Ultrasonic lysis pellet of Streptomyces spectabilis NA07643 cells.

[0023] The invention relates to an application of the dimeric aromatic polyketone compound in the preparation of drugs for treating tumors.

[0024] Furthermore, the tumor is colon cancer.

[0025] Furthermore, the dimeric aromatic polyketide compound serves as a lead molecule for anti-tumor drugs.

[0026] Beneficial effects: The present invention is the first to isolate two new dimeric aromatic polyketide compounds, Spirocycline A and B, from the rice fermentation product of actinomycete Streptomyces spectabilis NA07643. These compounds have a new ten-membered oxaspirocyclic dimer skeleton, providing a new chemical entity for drug research and development.

[0027] Compared with the prior art, the present invention has the following advantages:

[0028] (1) Biological activity: Spirocycline A and Spirocycline B showed good anti-tumor effects in biological activity tests, especially Spirocycline B, which had a more significant inhibitory effect on colon cancer cell lines, which provides a strong candidate molecule for the development of new therapeutic drugs for colon cancer.

[0029] (2) Potential applications: These two new dimeric aromatic polyketide compounds can not only be further studied and developed as anti-tumor lead drugs, but are also expected to play a role in other medical fields such as antibacterial, antiviral, and antiparasitic, thus broadening their scope of application.

[0030] (3) Social value: With the increasing severity of bacterial resistance, the demand for new antibiotics and anti-tumor drugs is becoming increasingly urgent. The compounds of this invention are expected to become a new option to solve this problem and have important public health significance.

[0031] (4) Economic value: The development of new drugs is often accompanied by huge market potential. The discovery and patent protection of Spirocycline A and B provide related companies and research institutions with opportunities to develop new drugs, which is expected to bring significant economic benefits. 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 any creative work.

[0033] Figures 1-16 The figures are the nuclear magnetic resonance data and mass spectrometry data spectra of spirocycline A and B of the present invention.

[0034] Figure 17The figure shows the results of the activity experiments of spirocycline A and B in colon cancer cell lines of the present invention, wherein spirocycline B has a more significant effect in killing cancer cells. DETAILED DESCRIPTION

[0035] 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.

[0036] The following examples will help to further understand the present invention, but the present invention is not limited thereto.

[0037] The first aspect of the present invention provides a dimer aromatic polyketone compound, the structural formula of the dimer aromatic polyketone compound is shown in formula (I):

[0038]

[0039] The second aspect of the present invention provides a method for preparing a dimer aromatic polyketone compound, comprising the following steps:

[0040] (1) The actinomycete Streptomyces spectabilis NA07643 strain was cultured on ISP2 plates at 30°C for 5-7 days;

[0041] (2) Cut the culture obtained in step (1) into pieces, inoculate into TSB liquid shake flasks, and culture at 30°C and 220 rpm for 1-2 days to prepare seed solution;

[0042] (3) The seed liquid obtained in step (2) is inoculated into a rice ME solid medium consisting of rice and ME liquid medium, wherein the formula of the rice ME solid medium is 33g of rice added to 45ml of ME liquid medium, and sterilized at high temperature and high pressure; the prepared rice ME solid medium is statically cultured at 30±5°C for 10-14 days; the ME liquid medium formula is 20g of malt cooked for half an hour, and the resulting filtrate is added with 20g of sucrose, 1g of peptone, and 1000ml of distilled water;

[0043] (4) extracting the culture medium obtained in step (3) with ethyl acetate as an organic solvent and concentrating the extract to obtain a crude extract F1;

[0044] (5) The crude extract F1 was first separated using a medium pressure liquid chromatography (MPLC) system with a Biotage SNAP Ultra C18 column to obtain 14 components. Each component was analyzed by HPLC to obtain the target molecular component for HPLC preparation.

[0045] (6) These components were then separated and purified by gel column chromatography and HPLC to obtain dimeric aromatic polyketide compounds, which were named spirocycline A and spirocycline B.

[0046] In some embodiments, in step (3), the solid culture medium composed of rice and ME is inoculated and statically cultured at 30±5°C for 10-14 days.

[0047] In some embodiments, in step (4), the HPLC chromatography conditions are: using a reverse phase chromatography column, mobile phase A: water, mobile phase B: acetonitrile, and the isocratic elution program is: the mass percentage of mobile phase A is 10%, the mass percentage of mobile phase B is 90%, and the time is 18 minutes; the HPLC separation and purification process is as follows: HPLC semi-preparative reverse phase high performance liquid chromatography: ODS-2 Hypersil column, mobile phase: acetonitrile-water volume ratio = 2:3, isocratic elution at a flow rate of 2.5 mL / min for 25 minutes; the pump model is Hitachi pumpL-7100, and the ultraviolet lamp model is UV detector L-7400.

[0048] The third aspect of the present invention provides a Streptomyces spectabilis strain; deposit name: Streptomyces spectabilis NA07643 strain; deposit number: CGMCC 30990; deposit location: China General Microbiological Culture Collection Center (CGMCC), Beijing, China; deposit date: June 18, 2024.

[0049] The invention provides an actinomycete Streptomyces spectabilis NA07643 strain and a prepared Streptomyces spectabilis spectabilis NA07643 strain agent.

[0050] The active ingredient of the actinomycete Streptomyces spectabilis NA07643 agent of the present invention is any one of the following (a), (b) and (c):

[0051] (a) Culture of the actinomycete Streptomyces spectabilis strain NA07643;

[0052] (b) Ultrasonic lysis supernatant of Streptomyces spectabilis NA07643 cells;

[0053] (c) Ultrasonic lysis pellet of Streptomyces spectabilis NA07643 cells.

[0054] A fourth aspect of the present invention provides a use of a dimeric aromatic polyketone compound in the preparation of a drug for treating tumors.

[0055] In some embodiments, the tumor is colon cancer.

[0056] In some embodiments, the dimeric aromatic polyketide compound is used as a lead molecule for anti-tumor drugs.

[0057] Example 1

[0058] Activation of Streptomyces spectabilis NA07643 strain.

[0059] Lyophilized powder of the endophytic fungus Streptomyces spectabilis NA07643 was spread onto ISP2 plates (4.0 g yeast extract, 10.0 g malt extract, 4.0 g glucose, 20.0 g agar, 1 L distilled water, pH 7.4-7.6) and incubated in a 30°C incubator for 3-7 days to obtain the actinomycete. Initially, the hyphae on the ISP2 plates were white, then turned orange-red. Morphological analysis and 16S sequencing identified the strain as Streptomyces spectabilis, a member of the genus Streptomyces. The strain was named Streptomyces spectabilis NA07643.

[0060] A Streptomyces spectabilis strain of the present invention; deposited name: Streptomyces spectabilis NA07643 strain; deposit number: CGMCC 30990; deposit location: China General Microbiological Culture Collection Center (CGMCC), Beijing, China; deposit date: June 18, 2024.

[0061] Example 2

[0062] Fermentation of Streptomyces spectabilis NA07643 in rice culture medium

[0063] The Streptomyces spectabilis NA07643 strain was transferred to a plate of ISP2 medium and cultured in a 30°C incubator for 5-7 days. Once the mycelium completely covered the plate, it was cut into small cubes and transferred to a TSB liquid shake flask. Incubated at 30°C and 220 rpm for 1-2 days to grow into a seed liquid. The mycelium was then transferred to rice ME solid medium and cultured at 30°C for 30 days.

[0064] Example 3

[0065] Extraction and Isolation of Spirocycline AB

[0066] The rice culture medium obtained in Example 2 was extracted with ethyl acetate and concentrated to obtain extract F1. The crude extract F1 was first separated using a medium pressure liquid chromatography (MPLC) system with a Biotage SNAP Ultra C18 column (60g), and a total of 14 components were obtained. These components were then subjected to gel column chromatography and semi-preparative HPLC (chromatographic column: Allsphere ODS-2.5mm column) with a mobile phase of acetonitrile-water volume ratio of 2:3, and isocratic elution at a flow rate of 2.5mL / min for 25 minutes to obtain spirocycline A (25mg) and spirocycline B (10mg) described in the present invention. The pump model was Hitachi pump L-7100, and the UV lamp model was UVdetector L-7400.

[0067] Test Example 1

[0068] Structural Elucidation of Spirocycline AB. 1 H and 13 The data attribution of C NMR and two-dimensional spectra are shown in Table 1-2:

[0069]

[0070] The structure of spirocycline A and its correlation with 1H and 13C NMR and 2D NMR

[0071] Table 1

[0072]

[0073]

[0074]

[0075]

[0076] spirocycline B 1 H and 13 The data attribution of C NMR and two-dimensional spectra are shown in Table 2:

[0077] Table 2

[0078]

[0079] Test Example 2

[0080] Antitumor activity test

[0081] Experimental Materials:

[0082] Experimental methods:

[0083] 1) Activity assay

[0084] MTT colorimetric assay was used to detect cytotoxicity: colon cancer cell lines SW620, SW480, MC38, CT26, and HCT116 were cultured at 5×10 3 The cells were seeded at a density of 100 μg / well in a 96-well plate, with 3 replicates per group. After incubation at 37°C for 24 hours, the cells were treated with 0, 5, 10, 20, and 40 μM compounds for 24 hours. 20 μL of MTT (5 mg / mL) was added to each well and incubated at 37°C for 4 hours. The cell supernatant was discarded, and 150 μL of DMSO was added to each well. The cells were shaken on a shaker for 10 minutes, and the absorbance at 570 nm was measured using a microplate reader. Experimental results Figure 17 As shown: Figure 17 The figure shows the antitumor activity of the novel dimeric aromatic polyketide compound of the present invention against colon cancer cell lines.

[0085] Activity experiments in colon cancer cell lines showed that both spirocycline A and spirocycline B exhibited varying degrees of antitumor activity, suggesting potential for further screening and development of novel antitumor lead compounds. Activity against colon cancer and Micrococcus luteus was also tested, with the MIC of spirocycline A at 4 μg / ml.

[0086] 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 dimer aromatic polyketone compound, characterized in that: The structural formula of the dimer aromatic polyketone compound is shown in formula (I):

2. The method for preparing the dimer aromatic polyketone compound according to claim 1, characterized in that The steps include: (1) The actinomycete Streptomyces spectabilis NA07643 strain was cultured on ISP2 plates at 30°C for 5-7 days; (2) Cut the culture obtained in step (1) into pieces, inoculate into TSB liquid shake flasks, and culture at 30°C and 220 rpm for 1-2 days to prepare seed solution; (3) inoculating the seed liquid obtained in step (2) into a rice ME solid medium consisting of rice and ME liquid medium, wherein the formula of the rice ME solid medium is 33 g of rice added to 45 ml of ME liquid medium, and sterilizing by high temperature and high pressure; preparing the rice ME solid medium, and culturing it at 30±5° C. for 10-14 days; The ME liquid medium formula is 20g of malt boiled for half an hour, and the resulting filtrate is added with 20g of sucrose, 1g of peptone, and 1000ml of distilled water; (4) extracting the culture medium obtained in step (3) with ethyl acetate as an organic solvent and concentrating the extract to obtain a crude extract F1; (5) The crude extract F1 was first separated using a medium pressure liquid chromatography (MPLC) system with a Biotage SNAP Ultra C18 column to obtain 14 components. Each component was subjected to HPLC analysis to obtain the target molecular component for HPLC preparation. The HPLC analysis conditions were as follows: a reverse phase column was used, mobile phase A: water, mobile phase B: acetonitrile, and the isocratic elution procedure was: the mass percentage of mobile phase A was 10%, the mass percentage of mobile phase B was 90%, and the elution time was 18 minutes. The HPLC preparation process is as follows: HPLC semi-preparative reversed-phase high performance liquid chromatography: ODS-2 Hypersil column, mobile phase: acetonitrile-water volume ratio = 2:3, isocratic elution at a flow rate of 2.5 mL / min for 25 minutes; pump model is Hitachi pump L-7100, UV lamp model is UV detector L-7400; (6) These components were then separated and purified by gel column chromatography and HPLC to obtain dimeric aromatic polyketide compounds, which were named spectacycline A and spectacycline B.

3. The method for preparing the dimer aromatic polyketone compound according to claim 2, wherein: In step (3), the mixture is inoculated into a rice ME solid culture medium consisting of rice and ME liquid culture medium, and cultured at 30±5° C. for 7-10 days.

4. An actinomycete Streptomyces spectabilis NA07643 strain, characterized by: Streptomyces spectabilis strain; deposit name: Streptomyces spectabilis NA07643 strain; deposit number: CGMCC30990; deposit location: China General Microbiological Culture Collection Center (CGMCC), Beijing, China; deposit date: June 19, 2024.

5. Use of the dimeric aromatic polyketide compound according to claim 1 in the preparation of drugs for treating tumors.

6. The use according to claim 5, characterized in that: The tumor is colon cancer.

7. Use of the dimeric aromatic polyketide compound according to claim 1 in preparing a lead molecule for an anti-tumor drug.

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