Novel ansa-mycin compounds, methods of making and using the same

Abstract

The application discloses a novel ansa mycin compound and a preparation method and application thereof, and belongs to the compound and preparation technical field. The strain of Amycolatopsis sp YINM00005 is an endophytic fungus of stone grass in Kunming, Yunnan, and can metabolize a novel ansa mycin compound after fermentation. The fermentation steps mainly include the following steps: fermenting the strain under suitable culture conditions to obtain a fermentation liquor, and then extracting and concentrating the fermentation liquor, and then combining column chromatography to prepare 10 novel compounds, the total concentration of the 10 novel compounds in the fermentation liquor can reach 3.5-6.8 mg / L, and the purity of the 10 novel compounds after extraction and separation can reach more than 99%. The 10 compounds all have strong antitumor cytotoxicity. The method has low cost, simple process and environmental friendliness.

Description

Technical Field

[0001] This invention relates to novel ansamycin-like compounds, their preparation methods, and applications, belonging to the field of compound and preparation technology. Background Technology

[0002] In recent years, due to the characteristics of microorganisms, such as short metabolic cycles, mild reaction conditions, few byproducts, and strong stereoselectivity, the method of preparing target active compounds through microbial fermentation has received increasing attention. In actual production, there are already numerous applications of using microbial fermentation to produce compounds with medicinal or economic value.

[0003] Ansamycins are a class of type I polyketide compounds. These antibiotics are characterized by low toxicity, high efficacy, and a broad antibacterial spectrum. They specifically interact with DNA-dependent RNA polymerases, inhibiting bacterial DNA transcription; they also inhibit RNA-dependent DNA reverse transcriptases of tumor RNA viruses. They are widely used to treat Gram-positive bacteria, tuberculosis, leprosy, and HIV-related mycobacterial infections. In conclusion, ansamycins have significant research and development potential as drug lead compounds.

[0004] Amycolatopsis sp. YINM00005 is an endophytic actinomycete obtained from the medicinal plant *Amycolatopsis pubescens* in Kunming, Yunnan. Analysis of its secondary metabolites revealed 10 new compounds, indicating that this strain has the potential to be developed as an engineered bacterium for the production of novel compounds. Summary of the Invention

[0005] One objective of this invention is to provide 10 novel ansamycin-like compounds, the structures of which are shown in Formulas I to X:

[0006]

[0007]

[0008]

[0009]

[0010] The application of the 10 novel ansamycin compounds described in this invention in the preparation of drugs for treating leukemia, lung cancer, liver cancer, breast cancer, or colon cancer.

[0011] The second object of the present application is to provide a preparation method of the 10 novel ansa mycin compounds, which comprises the following steps: culturing Amycolatopsis sp. YINM00005 in an activation culture, seed culture and fermentation culture to obtain a bacterial liquid, and then separating 10 novel ansa mycin compounds from the bacterial liquid.

[0012] Preferably, the Amycolatopsis sp. YINM00005 has been preserved in the Guangdong Microbial Culture Collection Center on July 19, 2023, with a preservation number of GDMCC No. 63669 and a taxonomic name of Amycolatopsis sp.

[0013] Preferably, the culture medium for the activation culture comprises 8-12 g / L of soluble starch, 0.8-1.2 g / L of K2HPO4, 0.8-1.2 g / L of MgSO4·7H2O, 0.8-1.2 / L of NaCl, 1.6-2.4 g / L of (NH4)2SO4, 1.6-2.4 g / L of CaCO3, 0.8-1.2 mL of trace salt solution, 15.0-20 g / L of agar and 1.0 L of distilled water, and has a pH of 7.0.

[0014] The culture medium for the seed culture comprises 8-12 g / L of soluble starch, 0.8-1.2 g / L of K2HPO4, 0.8-1.2 g / L of MgSO4·7H2O, 0.8-1.2 / L of NaCl, 1.6-2.4 g / L of (NH4)2SO4, 1.6-2.4 g / L of CaCO3, 0.8-1.2 mL of trace salt solution, 1.0 L of distilled water, and has a pH of 7.0.

[0015] The culture medium for the fermentation culture comprises 90-110 g / L of sucrose, 9-11 g / L of glucose, 0.1-0.15 g / L of acid hydrolysis casein, 4-6 g / L of yeast extract, 18-24 g / L of MOPS (3-(N-morpholine) propanesulfonic acid), 0.8-1.2 mL of trace elements, 0.2-0.3 g / L of K2SO4, 8-12 g / L of MgCl2·6H2O, and has a pH of 7.0.

[0016] Preferably, the preparation steps of the 10 novel ansa mycin compounds are as follows:

[0017] (1) Bacterial activation: inoculating Amycolatopsis sp. YINM00005 into an activation culture medium for activation and standby;

[0018] (2) Preparation of bacterial seed: inoculating the activated bacterial body in step (1) into a seed culture medium for shaking culture to obtain a bacterial seed;

[0019] (3) fermentation process: inoculate the strain prepared in step (2) into a fermentation medium and shake culture to obtain a fermentation liquor;

[0020] (4) after the fermentation is completed, the fermentation liquor is extracted with an equal volume of ethyl acetate, concentrated to obtain a crude extract, the crude extract is dissolved in a mixed solution of dichloromethane and methanol, and then mixed with silica gel, and gradient elution is performed on a normal silica gel column with dichloromethane and methanol as eluents to obtain an eluate;

[0021] (5) the eluate obtained in step (4) is mixed with silica gel, gradient elution is performed on a reverse silica gel column with water and methanol as eluents, and finally semi-preparative high performance liquid chromatography is used for purification to obtain the compounds shown in formula I to formula X.

[0022] Preferably, in the gradient elution of step (4), the volume ratio of dichloromethane to methanol in the dichloromethane-methanol eluent is 100:0, 80:1, 50:1, 30:1, 10:1 and 0:100, respectively, and the eluate with a volume ratio of dichloromethane to methanol of 30:1 is selected for the operation of step (5).

[0023] Preferably, in the gradient elution of step (5), the volume ratio of methanol to water is 10:90, 20:80, 30:70, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20 and 100:0, respectively, wherein the eluate with a volume ratio of 75:25 contains the compound shown in formula I, the eluate with a volume ratio of 80:20 contains the compound shown in formula II, the eluate with a volume ratio of 55:45 contains the compounds shown in formula III and formula VIII, the eluate with a volume ratio of 65:35 contains the compounds shown in formula IV and formula X, the eluate with a volume ratio of 70:30 contains the compound shown in formula V, the eluate with a volume ratio of 50:50 contains the compound shown in formula VI, and the eluate with a volume ratio of 60:40 contains the compounds shown in formula VII and formula IX.

[0024] Advantages of the present application

[0025] (1) The present application separates and purifies 10 kinds of compounds from the culture solution by special fermentation culture of Amycolatopsis sp YINM00005, and identifies that the 10 kinds of compounds are new ansamycin compounds, and experiments have verified that the 10 kinds of compounds have the effects of inhibiting leukemia cells, lung cancer cells, liver cancer cells, breast cancer cells and colon cancer cells, and thus can be used for preparing drugs for treating these diseases, providing more choices for preparing drugs for treating these diseases.

[0026] (2) The present application prepares new ansamycin compounds by microbial fermentation, raw materials are cheap and easy to obtain, reaction conditions are mild, process is simple, equipment requirement is simple, environment is pollution-free, and it is suitable for large-scale production. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The H-NMR spectrum of the compound I provided by the present application 1 H-NMR spectrum.

[0028] Figure 2 The H-NMR spectrum of the compound I provided by the present application 13 C-NMR spectrum.

[0029] Figure 3 The HR-ESI-MS spectrum of the compound I provided by the present application

[0030] Figure 4 The H-NMR spectrum of the compound II provided by the present application 1 H-NMR spectrum.

[0031] Figure 5 The H-NMR spectrum of the compound II provided by the present application 13 C-NMR spectrum.

[0032] Figure 6 The HR-ESI-MS spectrum of the compound II provided by the present application

[0033] Figure 7 The H-NMR spectrum of the compound III provided by the present application 1 H-NMR spectrum.

[0034] Figure 8 The H-NMR spectrum of the compound III provided by the present application 13 C-NMR spectrum.

[0035] Figure 9 The HR-ESI-MS spectrum of the compound III provided by the present application

[0036] Figure 10 The H-NMR spectrum of the compound IV provided by the present application 1 H-NMR spectrum.

[0037] Figure 11 The H-NMR spectrum of the compound IV provided by the present application 13 C-NMR spectrum.

[0038] Figure 12 The HR-ESI-MS spectrum of the compound IV provided by the present application

[0039] Figure 13 The H-NMR spectrum of the compound V provided by the present application 1 H-NMR spectrum.

[0040] Figure 14 H-NMR spectrum of the compound V provided by the present application. 13 C-NMR spectrum.

[0041] Figure 15 HR-ESI-MS spectrum of the compound V provided by the present application.

[0042] Figure 16 H-NMR spectrum of the compound VI provided by the present application. 1 H-NMR spectrum of the compound VI provided by the present application.

[0043] Figure 17 C-NMR spectrum of the compound VI provided by the present application. 13 C-NMR spectrum of the compound VI provided by the present application.

[0044] Figure 18 HR-ESI-MS spectrum of the compound VI provided by the present application.

[0045] Figure 19 H-NMR spectrum of the compound VII provided by the present application. 1 H-NMR spectrum of the compound VII provided by the present application.

[0046] Figure 20 C-NMR spectrum of the compound VII provided by the present application. 13 C-NMR spectrum of the compound VII provided by the present application.

[0047] Figure 21 HR-ESI-MS spectrum of the compound VII provided by the present application.

[0048] Figure 22 H-NMR spectrum of the compound VIII provided by the present application. 1 H-NMR spectrum of the compound VIII provided by the present application.

[0049] Figure 23 C-NMR spectrum of the compound VIII provided by the present application. 13 C-NMR spectrum of the compound VIII provided by the present application.

[0050] Figure 24 HR-ESI-MS spectrum of the compound VIII provided by the present application.

[0051] Figure 25 H-NMR spectrum of the compound IX provided by the present application. 1 H-NMR spectrum of the compound IX provided by the present application.

[0052] Figure 26 C-NMR spectrum of the compound IX provided by the present application. 13 C-NMR spectrum of the compound IX provided by the present application.

[0053] Figure 27 HR-ESI-MS spectrum of the compound IX provided by the present application.

[0054] Figure 28 H-NMR spectrum of the compound X provided by the present application. 1 H-NMR spectrum of the compound X provided by the present application.

[0055] Figure 29 HR-ESI-MS spectrum of compound X provided in the present application 13 C-NMR spectrum.

[0056] Figure 30 HR-ESI-MS spectrum of compound X provided in the present application

[0057] Figure 31 Crystal structure of compound V provided in the present application DETAILED DESCRIPTION

[0058] The technical solutions of the present application are further illustrated below in combination with the drawings and through specific embodiments. However, the following embodiments are merely simple examples of the present application and do not represent or limit the protection scope of the present application, and the protection scope of the present application is subject to the claims.

[0059] The formula of the culture medium used in the embodiments is described as follows, and all the culture media need to be sterilized at 121°C for 30 min before use.

[0060] (1) The components of the activation culture medium are: soluble starch 10.0 g / L, K2HPO4 1.0 g / L, MgSO4·7H2O 1.0 g / L, NaCl 1.0 g / L, (NH4)2SO4 2.0 g / L, CaCO3 2.0 g / L, trace salt solution 1.0 mL, agar 15.0 g / L, distilled water 1.0 L, pH 7.0;

[0061] (2) The components of the seed culture medium are: soluble starch 10.0 g / L, K2HPO4 1.0 g / L, MgSO4·7H2O 1.0 g / L, NaCl 1.0 g / L, (NH4)2SO4 2.0 g / L, CaCO3 2.0 g / L, trace salt solution 1.0 mL, distilled water 1.0 L, pH 7.0;

[0062] (3) The components of the fermentation culture medium are: sucrose 100 g / L, glucose 10 g / L, acid hydrolysis casein 0.1 g / L, yeast extract 5.0 g / L, MOPS (3-(N-morpholine)propanesulfonic acid) 21.0 g / L, trace elements 1.0 mL, K2SO4 0.25 g / L, MgCl2·6H2O 10.0 g / L, pH 7.0.

[0063] Example 1

[0064] The new compound is prepared by using Amycolatopsis sp. YINM00005, and specifically includes the following steps:

[0065] (1) Activation of bacteria: inoculate Amycolatopsis sp. YINM00005 into the activation medium, and place it in a constant temperature incubator at 28°C for 4 days, and then place it in a refrigerator at 4°C for standby.

[0066] (2) Preparation of bacteria: inoculate the activated bacteria into the seed culture medium, and perform fermentation at 28°C and 200 rpm for 3 days.

[0067] (3) Fermentation process: inoculate the bacteria prepared in step (2) into 30 L of fermentation medium (sucrose 100 g / L, glucose 10 g / L, acid hydrolysis casein 0.1 g / L, yeast extract 5.0 g / L, MOPS (3-(N-morpholine) propanesulfonic acid) 21.0 g / L, trace elements 1.0 mL, K2SO4 0.25 g / L, MgCl2·6H2O 10.0 g / L, pH 7.0) at a volume fraction of 5%, and perform fermentation at 28°C and 200 rpm for 15 days to obtain a fermentation broth.

[0068] (4) After the fermentation is completed, extract the fermentation broth with an equal volume of ethyl acetate three times, and vacuum concentrate to obtain a crude extract (22.50 g). Detect the crude extract by high performance liquid chromatography, dissolve the concentrated crude extract in a dichloromethane-methanol (volume ratio of dichloromethane to methanol is 1:1) mixed solution to obtain a crude extract solution; stir the prepared crude extract solution and 200-300 mesh silica gel with a glass rod until the solvent is completely volatilized. Gradient elution is performed on a forward silica gel (200-300 mesh) column with dichloromethane-methanol solutions with volume ratios of 100:0, 80:1, 50:1, 30:1, 10:1, and 0:100, respectively, to obtain 6 eluates.

[0069] (5) Take an appropriate amount of the 6 eluates for TLC spotting and HPLC analysis. According to the results of TLC spotting and HPLC analysis, select the eluate with a dichloromethane-methanol volume ratio of 30:1, and slowly drop the eluate onto prepared defatted cotton at room temperature until the solvent is completely volatilized. After loading, gradient elution is performed on a reverse silica gel column (40-63 μm) with methanol-water elution solvents with volume ratios of 10:90, 20:80, 30:70, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, and 100:0, respectively, to obtain 13 eluates.

[0070] Based on the TLC and HPLC analysis results, a methanol-water volume ratio of 75:25 was selected as the eluent for semi-preparative HPLC purification (semi-preparative gradient elution method: 0 min, 80% methanol; 15 min, 88% methanol) to obtain compound I (76.0 mg). HPLC analysis of compound I (retention time t...) R The purity of the sample (=25.5 min) reached over 98% (analytical gradient elution method: 0 min, 30% methanol; 30 min, 100% methanol).

[0071] Based on the TLC and HPLC analysis results, an eluent with a methanol-water volume ratio of 80:20 was selected for semi-preparative HPLC purification (semi-preparative gradient elution method: 0 min, 80% methanol; 15 min, 90% methanol) to obtain compound II (84.0 mg). HPLC analysis of compound II (retention time t) yielded compound II. R =26.9 min) The purity reached over 98% (analytical gradient elution method: 0 min, 30% methanol; 30 min, 100% methanol).

[0072] Based on the TLC and HPLC analysis results, a methanol-water volume ratio of 55:45 was selected as the eluent for semi-preparative HPLC purification (semi-preparative gradient elution method: 0 min, 75% methanol; 15 min, 88% methanol) to obtain compound III (110.0 mg). HPLC analysis of compound III (retention time t) was performed. R =23.3min) The purity reached over 98% (analytical gradient elution method: 0min, 30% methanol; 30min, 100% methanol).

[0073] Based on the TLC and HPLC analysis results, an eluent with a methanol-water volume ratio of 65:35 was selected for semi-preparative HPLC purification (semi-preparative gradient elution method: 0 min, 78% methanol; 15 min, 88% methanol) to obtain compound IV (72.0 mg). HPLC analysis of compound IV (retention time t) was performed. R The purity of the sample (=24.3 min) reached over 98% (analytical gradient elution method: 0 min, 30% methanol; 30 min, 100% methanol).

[0074] According to the results of TLC and HPLC analysis, the eluent of methanol-water (volume ratio 70:30) was selected to purify the compound V (70.0 mg) by semi-preparative HPLC (semi-preparative gradient elution method: 0 min, 80% methanol; 15 min, 88% methanol). The purity of compound V (retention time t R = 25.2 min) reached more than 98% by HPLC analysis (analytical gradient elution method: 0 min, 30% methanol; 30 min, 100% methanol).

[0075] According to the results of TLC and HPLC analysis, the eluent of methanol-water (volume ratio 50:50) was selected to purify the compound VI (92.0 mg) by semi-preparative HPLC (semi-preparative gradient elution method: 0 min, 70% methanol; 15 min, 82% methanol). The purity of compound VI (retention time t R = 20.4 min) reached more than 98% by HPLC analysis (analytical gradient elution method: 0 min, 30% methanol; 30 min, 100% methanol).

[0076] According to the results of TLC and HPLC analysis, the eluent of methanol-water (volume ratio 60:40) was selected to purify the compound VII (136.0 mg) by semi-preparative HPLC (semi-preparative gradient elution method: 0 min, 75% methanol; 15 min, 88% methanol). The purity of compound VII (retention time t R = 23.4 min) reached more than 98% by HPLC analysis (analytical gradient elution method: 0 min, 30% methanol; 30 min, 100% methanol).

[0077] According to the results of TLC and HPLC analysis, the eluent of methanol-water (volume ratio 55:45) was selected to purify the compound VIII (70.0 mg) by semi-preparative HPLC (semi-preparative gradient elution method: 0 min, 76% methanol; 15 min, 86% methanol). The purity of compound VIII (retention time t R = 22.9 min) reached more than 98% by HPLC analysis (analytical gradient elution method: 0 min, 30% methanol; 30 min, 100% methanol).

[0078] According to the results of TLC and HPLC analysis, the eluent of methanol-water with the volume ratio of 60:40 was selected to carry out semi-preparative HPLC purification (semi-preparative gradient elution method: 0 min, 78% methanol; 15 min, 90% methanol) to obtain compound IX (104.0 mg); the purity of compound IX (retention time t = 24.0 min) reached more than 98% by HPLC analysis (analytical gradient elution method: 0 min, 30% methanol; 30 min, 100% methanol). R R According to the results of TLC and HPLC analysis, the eluent of methanol-water with the volume ratio of 60:40 was selected to carry out semi-preparative HPLC purification (semi-preparative gradient elution method: 0 min, 78% methanol; 15 min, 90% methanol) to obtain compound IX (104.0 mg); the purity of compound IX (retention time t = 24.0 min) reached more than 98% by HPLC analysis (analytical gradient elution method: 0 min, 30% methanol; 30 min, 100% methanol).

[0079] According to the results of TLC and HPLC analysis, the eluent of methanol-water with the volume ratio of 60:40 was selected to carry out semi-preparative HPLC purification (semi-preparative gradient elution method: 0 min, 78% methanol; 15 min, 90% methanol) to obtain compound IX (104.0 mg); the purity of compound IX (retention time t = 24.0 min) reached more than 98% by HPLC analysis (analytical gradient elution method: 0 min, 30% methanol; 30 min, 100% methanol). R R According to the results of TLC and HPLC analysis, the eluent of methanol-water with the volume ratio of 60:40 was selected to carry out semi-preparative HPLC purification (semi-preparative gradient elution method: 0 min, 78% methanol; 15 min, 90% methanol) to obtain compound IX (104.0 mg); the purity of compound IX (retention time t = 24.0 min) reached more than 98% by HPLC analysis (analytical gradient elution method: 0 min, 30% methanol; 30 min, 100% methanol).

[0080] Finally, the concentrations of compounds I, II, III, IV, V, VI, VII, VIII, IX and X in the fermentation broth were 3.8 mg / L, 4.2 mg / L, 5.5 mg / L, 3.6 mg / L, 3.5 mg / L, 4.6 mg / L, 6.8 mg / L, 3.5 mg / L, 5.2 mg / L and 3.6 mg / L, respectively, as analyzed by HPLC.

[0081] Example 2

[0082] Example 2

[0083] The structure of the compound obtained in Example 1 was identified by one-dimensional nuclear magnetic resonance spectroscopy (1D-NMR), two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS).

[0084] According to the HSQC spectrum combined with the carbon spectrum, the nuclear magnetic resonance chemical shift δ of compound I was assigned as shown in Table 1.

[0085] Table 1 Assignment of the nuclear magnetic resonance chemical shift δ of compound I obtained in Example 1 1 H (600 MHz) and 13 C (150 MHz) NMR data (methanol-d4)

[0086] Table 1 Assignment of the nuclear magnetic resonance chemical shift δ of compound I obtained in Example 1

[0087] As Figure 3 shown, HR-ESI-MS m / z of compound I: [M+H] + The quasi-molecular ion peak is 458.2901 (C 27 H 40 NO5[M+H] + , calc m / z: 458.2901) indicates that the molecular formula of compound I is C 27 H 39 NO5, containing 9 degrees of unsaturation. Through the analysis of the 1 H-NMR( Figure 1 ) of the compound, 13 C-NMR( Figure 2 ) and HSQC spectrum, it is known that compound I contains 5 methyl groups [δ H 0.73 (H3-22), δ H 0.90 (H3-23), δ H 3.33 (H3-24), δ H 1.11 (H3-25), δ H 0.75 (H3-27) respectively corresponding to δ C 17.7 (C-22), δ C 20.8 (C-23), δ C 58.4 (C-24), δ C 17.7 (C-25), δ C 14.2 (C-27)], 6 methylene groups [δ H 2.70 (H a -8), 2.46 (H b -8), δ H 2.52 (H a -10), 2.10 (H b -10), δ H 1.61 (H a -11), 1.22 (H b -11), δ H 1.39 (H a -13), 1.16 (H b -13), δ H 2.90 (H a -15), 1.81 (H b -15), δ H 2.02 (H a -26), 1.82 (H b -26) respectively corresponding to δ C 43.5 (C-8), δC 42.4 (C-10), δ C 30.2 (C-11), δ C 46.5 (C-13), δ C 41.8 (C-15), δ C 21.1 (C-26)], 9 methine [δ H 5.83 (H-2), δ H 7.05 (H-3), δ H 5.64 (H-5), δ H 2.77 (H-6), δ H 3.60 (H-7), δ H 1.56 (H-12), δ H 1.71 (H-14), δ H 6.45 (H-19), δ H 6.51 (H-21) correspond to δ C 118.0 (C-2), δ C 145.9 (C-3), δ C 141.5 (C-5), δ C 37.2 (C-6), δ C 81.8 (C-7), δ C 31.4 (C-12), δ C 31.7 (C-14), δ C 112.4 (C-19), δ C 118.8 (C-21)] and 7 quaternary carbons [δ C 171.6 (C-1), δ C 141.4 (C-4), δ C 213.4 (C-9), δ C 126.5 (C-16), δ C 132.9 (C-17), δ C 145.2 (C-18), δ C 150.8 (C-20)].

[0088] Analysis 1 H- 1 H COSY spectrum, H-5 / H-6 / H-7 / H a / b -8 had clear correlations, H a / b -10 / H a / b -11 / H-12 / H a / b -13 / H-14 / H a / b-15 shows a clear correlation, belonging to two fragments from C-5 to C-8 and C-10 to C-15 respectively. Further analysis of the HMBC spectrum reveals that H3-22 has a significant correlation signal with C-13 / C-14 / C-15, and H3-23 has a significant correlation signal with C-11 / C-12 / C-13. a / b -8 and H a / b -10 shows a significant correlation signal to C-9, and H3-25 shows a significant correlation signal to C-5 / C-6 / C-7. These correlations indicate a continuous chain from C-5 to C-15 with three methyl groups attached to the carbon chain. Furthermore, based on the significant HMBC correlation signal of H3-24 to C-7, it can be inferred that the methoxy group is attached at the C-7 position. 1 H-NMR shows that δ H 6.45 and δ H Both 6.51 and 6.51 are doublets with relatively small coupling constants (J = 3.0 Hz). Furthermore, according to HMBC correlations, H-19 is correlated with C-18 / C-20 / C-21, and H-21 is correlated with C-16 / C-20 / C-19. These correlations suggest the possible presence of a benzene ring chromophore. Based on the HMBC correlation... a / b -15 shows a significant correlation with C-21, thus proving that the C-5 to C-15 segments are attached to the chromophore of the benzene ring. Based on the chemical shift values ​​and coupling constants of H-2, H-3, and H-5, it can be determined that C-2 / C-3 may be a double bond, and C-4 / C-5 may be a double bond with C-4 being a quaternary carbon. These assumptions are supported by... 1 H- 1 Confirmed by H COSY (H-2 / H-3 and H-5 / H-6) correlation and HMBC (H-3 to C-2 / C-4 / C-5) correlation. According to 13 C-NMR shows that δ C 213.4(C-9) and δ C 171.6 (C-1) could be the resonances of the carbonyl carbon and the amide carbon, respectively, according to HMBC(H a / b -8 / H a / b The correlation between -10 and C-9 (a clear correlation signal is observed between -NH- and C-1 / C-2) confirms the presence of carbonyl and amide carbons. HMBC (-NH- to C-17 / C-18 / C-19) correlations indicate that this compound is a macrocyclic lactam, and a literature review confirms that this molecule is a previously unreported novel compound.

[0089] To determine the relative configuration, the NOESY spectrum and related literature analysis were combined to identify the relative configuration of compound I as 6R*, 7S*, 12S*, 14R*. To determine the absolute stereochemistry of compound I, conformational search was performed on the two possible stereoisomers [(6R, 7S, 12S, 14R) and (6S, 7R, 12R, 14S)] of compound I, and the conformations with a distribution ratio of more than 1% were subjected to ECD calculation at the B3LYP / 6-31G(d) level using the calculation software. By comparing with the experimental ECD spectrum, it was found that the calculation results of the isomer (6R, 7S, 12S, 14R)-compound I and the experimental results were highly matched, both having the same Cottons effect. Therefore, the absolute configuration of compound I was identified as 6R, 7S, 12S, 14R.

[0090] Based on the above, it can be determined that the structural formula of compound I separated in Example 1 is:

[0091]

[0092] Compound II separated in Example 1 was subjected to one-dimensional nuclear magnetic resonance spectroscopy (1D-NMR), two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) to identify its structure.

[0093] From the HSQC spectrum combined with the carbon spectrum, the chemical shifts δ of H and C connected thereto of compound II were assigned as shown in Table 2:

[0094] Table 2 H and C NMR data of compound II separated in Example 1 1 H (600 MHz) and 13 C (150 MHz) NMR data, methanol-d4 as solvent

[0095]

[0096] As shown in Figure 6 , the HR-ESI-MS m / z of compound II: [M+H] + quasi-molecular ion peak is 458.2901 (C 27 H 40 NO5[M+H] + , calc m / z: 458.2901) indicates that its molecular formula is C 27 H 39 NO5, containing 9 degrees of unsaturation. According to the HR-ESI-MS, 1 H-NMR and 13 C-NMR and comparison of compound I, it can be known that compound II has one less carbon-carbon double bond (Δ 2), and possibly form a six-membered or seven-membered ring connected by an ether bond, according to 1 H-NMR and HMBC (H a / b -2 to C-1 / C-3 / C-4; H-3 to C-1 / C-2 / C-4 / C-5 / C-17 / C-26) related to the formation of a new seven-membered ring connected by an ether bond. To determine its relative configuration, combined with NOESY spectrum and related literature analysis, the relative configuration of compound II was identified as 3S*, 6R*, 7S*, 12S*, 14R*. In order to determine the absolute stereochemical configuration of compound II, conformational search was performed on two possible stereoisomers [(3S, 6R, 7S, 12S, 14R) and (3R, 6S, 7R, 12R, 14S)], and the conformations with a distribution ratio greater than 1% were calculated by ECD using TD-DFT theory at the B3LYP / 6-31G(d) level using the calculation software. By comparing with the experimental ECD spectrum, it was found that the calculation results of the isomer (3S, 6R, 7S, 12S, 14R)-compound II and the experimental results were highly matched, both had the same Cottons effect. Therefore, the absolute configuration of compound II was identified as 3S, 6R, 7S, 12S, 14R.

[0097] Based on the above, it can be determined that the structural formula of compound II separated in Example 1 is:

[0098]

[0099] Taking the compound III separated in Example 1, its structure was identified by one-dimensional nuclear magnetic resonance spectrum (1D-NMR), two-dimensional nuclear magnetic resonance spectrum (2D-NMR) and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS).

[0100] From the HSQC spectrum combined with the carbon spectrum, the chemical shifts δ of H and C connected to compound III were assigned as shown in Table 3:

[0101] Table 3 H and C chemical shifts δ of compound III separated in Example 1 1 H (600 MHz) and 13 C (150 MHz) NMR data, methanol-d4 as solvent

[0102]

[0103] As shown in Figure 9 , the HR-ESI-MS m / z of compound III is [M+H] + quasimolecular ion peak is 412.2486 (C 25 H 34 NO4[M+H] +, calc m / z: 412.2482) indicated that the molecular formula of compound III was C 25 H 33 NO4, containing 10 unsaturations. According to HR-ESI-MS, 1 H-NMR and 13 C-NMR and compound III can be compared, compound III C-7 position missing a-OCH3 fragment, may be formed between C-7 and C-8 a carbon-carbon double bond (Delta 7 ), according to 1 H-NMR and HMBC (H3-24 to C-5 / C-6 / C-7, H-7 to C-9 / C-C-8 / C-6 / C-5 and H-8 to C-9 / C-10 / C-5 / C-6 / C-7) correlation can be known, to confirm the formation of a carbon-carbon double bond between C-7 and C-8 (Delta 7 ), in addition, according to 1 H-NMR and HMBC (H3-25 to C-3 / C-4 / C-5) correlation can be known, the-CH2CH3 fragment connected to C-4 in compound I becomes-CH3 fragment. To determine its relative configuration, combined with NOESY spectrum and related literature analysis, the relative configuration of compound III is identified as 6R*, 12S*, 14R. In order to determine the absolute stereochemical configuration of compound III, conformational search was performed on two possible stereoisomers [(6R, 12S, 14R) and (6S, 12R, 14S)], and the conformations with a distribution ratio of more than 1% were subjected to ECD calculation using TD-DFT theory at the B3LYP / 6-31G(d) level by using the calculation software. By comparing with the experimental ECD spectrum, it was found that the calculation results of the isomer (6R, 12S, 14R)-compound III and the experimental results were highly matched, both had the same Cottons effect. Therefore, the absolute configuration of compound III was identified as 6R, 12S, 14R.

[0104] In summary, it can be determined that the structural formula of compound III separated in example 1 is:

[0105]

[0106] Take the compound IV separated in example 1, and identify its structure by one-dimensional nuclear magnetic resonance spectrum (1D-NMR), two-dimensional nuclear magnetic resonance spectrum (2D-NMR) and high resolution electrospray ionization mass spectrometry (HR-ESI-MS).

[0107] From HSQC spectrum combined with carbon spectrum, the chemical shift δ of H and C connected to it of compound IV can be obtained as shown in table 4:

[0108] Table 4 Chemical shift δ of H and C connected to it of compound IV separated in example 1 1H (600MHz) and 13 C150MHz NMR data, methanol-d4 as solvent

[0109]

[0110] like Figure 12 As shown, the HR-ESI-MS m / z of compound IV is: [M+H] + The quasi-molecular ion peak is at 412.2481 (C). 25 H 34 NO4[M+H] + The value of calc m / z (412.2482) indicates that its molecular formula is C. 25 H 33 NO4 contains 10 degrees of unsaturation. (Based on HR-ESI-MS) 1 H-NMR and 13 A comparison of C-NMR and compound III reveals that compound IV is missing one carbon-carbon double bond (ΔC-NMR). 2 And may form a six- or seven-membered ring linked by an ether bond, depending on 1 H-NMR and HMBC (H a / b As can be seen from the relationship between H-2 and C-1 / C-3 / C-4; and H-3 and C-1 / C-2 / C-4 / C-5 / C-17 / C-25, a new seven-membered ring linked by an ether bond is formed. To determine its relative configuration, based on NOESY spectroscopy and relevant literature analysis, the relative configuration of compound IV is identified as 3S*, 6R*, 12S*, 14R*. To determine the absolute stereoconfiguration of compound IV, a conformational search was conducted for its two possible stereoisomers [(3S, 6R, 12S, 14R) and (3R, 6S, 12R, 14S)]. For conformations with a distribution ratio greater than 1%, ECD calculations were performed using TD-DFT theory at the B3LYP / 6-31G(d) level. Comparison with experimental ECD spectra revealed a high degree of agreement between the calculated and experimental results for the isomers (3S, 6R, 12S, 14R) and compound IV, both exhibiting the same Cottons effect. Therefore, the absolute configuration of compound IV was identified as 3S, 6R, 12S, 14R.

[0111] In summary, the structural formula of compound IV isolated in Example 1 can be determined as follows:

[0112]

[0113] Compound V obtained in Example 1 was used to identify its structure by one-dimensional nuclear magnetic resonance spectroscopy (1D-NMR), two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR), and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS).

[0114] From HSQC spectrum combined with carbon spectrum, the chemical shifts of H and C connected with them of compound V were assigned as shown in Table 5:

[0115] Table 5 Chemical shifts of H and C of compound V isolated from Example 1 1 H (600 MHz) and 13 C (150 MHz) NMR data, methanol-d4 as solvent

[0116]

[0117] As shown in Figure 15 HR-ESI-MS m / z: [M+H] + quasi-molecular ion peak at 426.2635 (C 26 H 36 NO4[M+H] + , calc m / z: 426.2639) indicated that the molecular formula of compound V was C 26 H 35 NO4, containing 10 unsaturations. According to HR-ESI-MS, 1 H-NMR and 13 C-NMR and comparison with compound IV, it was known that compound V had one more methylene group (δ H 1.90, 2.21), according to 1 H-NMR and HMBC (H a / b -25 to C-3 / C-4 / C-5 / C-26; H3-26 to C-4 / C-25) correlation, it was known that in compound V, a -CH2CH3 fragment was connected to C-4. In order to determine the relative configuration, combined with NOESY spectrum and literature analysis, the relative configuration of compound V was identified as 3S*, 6R*, 12S*, 14R. In order to determine the absolute stereochemical configuration of compound V, conformational search was performed on two possible stereoisomers [(3S, 6R, 12S, 14R) and (3R, 6S, 12R, 14S)], and ECD calculation was performed on conformations with a distribution ratio of more than 1% using TD-DFT theory at the B3LYP / 6-31G(d) level by using calculation software. By comparing with the experimental ECD spectrum, it was found that the calculation results of the isomer (3S, 6R, 12S, 14R)-compound V and the experimental results were highly matched, both had the same Cottons effect, and finally the crystal structure of compound V was successfully obtained ( Figure 31 ), therefore, the absolute configuration of compound V was identified as 3S, 6R, 12S, 14R.

[0118] Based on the above, it can be determined that the structural formula of compound V isolated from Example 1 is:

[0119]

[0120] Compound VI, isolated in Example 1, was used to identify its structure by one-dimensional nuclear magnetic resonance spectroscopy (1D-NMR), two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR), and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS).

[0121] The chemical shifts δ of H and C atoms in compound VI, obtained from HSQC spectroscopy combined with carbon spectroscopy, are shown in Table 6.

[0122] Table 6 shows the compound VI isolated in Example 1. 1 H (600MHz) and 13 C150MHz NMR data, methanol-d4 as solvent

[0123]

[0124] like Figure 18 As shown, the HR-ESI-MS m / z of compound VI is: [M+H] + The quasi-molecular ion peak is at 473.2648 (C). 26 H 37 N₂O₆[M+H] + The value of calc m / z (473.2646) indicates that its molecular formula is C. 26 H 36 N₂O₆ contains 10 degrees of unsaturation. According to HR-ESI-MS... 1 H-NMR and 13 A comparison of C-NMR with compound III reveals that compound VI lacks one double bond between C-7 and C-8 and has a missing carbonyl signal at C-9. Based on literature reports and chemical shift values ​​from C-NMR and H-NMR spectra, it is inferred that a carbamate fragment has formed. 1 H-NMR and HMBC (H a / b-8 to C-6 / C-7 / C-10; H-7 to C-8 / C-9) related to the fact that a six-membered closed ring system with a carbamate linkage was formed and C-9 was connected with both an amino and a hydroxyl group. To determine the relative configuration, the NOESY spectrum and related literature were analyzed, and the relative configuration of compound VI was identified as 6R*, 7S*, 9R*, 12S*, 14R*. To determine the absolute stereochemistry of compound VI, conformational search was performed on two possible stereoisomers [(6R, 7S, 9R, 12S, 14R) and (6S, 7R, 9S, 12R, 14S)], and ECD calculation was performed on conformations with a distribution ratio greater than 1% using TD-DFT theory at the B3LYP / 6-31G(d) level using the calculation software. By comparing with the experimental ECD spectrum, it was found that the calculation results of the isomer (6R, 7S, 9R, 12S, 14R)-compound VI and the experimental results were highly matched, both having the same Cottons effect. Therefore, the absolute configuration of compound VI was identified as 6R, 7S, 9R, 12S, 14R.

[0125] Based on the above, it can be determined that the structural formula of compound VI separated in Example 1 is:

[0126]

[0127] Taking the compound VII separated in Example 1, its structure was identified by one-dimensional nuclear magnetic resonance spectroscopy (1D-NMR), two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS).

[0128] From the HSQC spectrum combined with the carbon spectrum, the chemical shifts δ of H and C connected thereto of compound VII were assigned as shown in Table 7:

[0129] Table 7 H and C chemical shift δ assignment of compound VII separated in Example 1 1 H (600 MHz) and 13 C (150 MHz) NMR data, methanol-d4 as solvent

[0130]

[0131] As shown in Figure 21 , the HR-ESI-MS m / z of compound VII: [M+H] + quasi-molecular ion peak is 501.2952 (C 28 H 41 N2O6[M+H] + , calc m / z: 501.2959) indicates that its molecular formula is C 28 H 40N2O6, containing 10 unsaturations. According to HR-ESI-MS, 1 H-NMR and 13 C-NMR and comparing compound VI, it can be known that compound VII connects a methoxy group at C-9 position, according to 1 H-NMR and HMBC (H3-24 to C-9) correlation can be confirmed, in addition, there is a double bond between C-2 and C-3, and it can form a six-membered ring or a seven-membered ring connected by an ether bond, according to 1 H-NMR and HMBC (H a / b -2 to C-1 / C-3 / C-4; H-3 to C-1 / C-2 / C-4 / C-5 / C-17 / C-27) correlation can be known that a seven-membered ring connected by an ether bond is newly formed. In order to determine the relative configuration thereof, combined with NOESY spectrum and related literature analysis, it is identified that the relative configuration of compound VII is 3S*, 6R*, 7S*, 9R*, 12S*, 14R*. In order to determine the absolute stereochemical configuration of compound VII, conformational search is performed on two possible stereoisomers [(3S, 6R, 7S, 9R, 12S, 14R) and (3R, 6S, 7R, 9S, 12R, 14S)], and ECD calculation is performed on the conformations with a distribution ratio greater than 1% by using TD-DFT theory at B3LYP / 6-31G(d) level and calculation software. By comparing with the experimental ECD spectrum, it is found that the calculation results of the isomer (3S, 6R, 7S, 9R, 12S, 14R)-compound VII and the experimental results are highly matched, and both have the same Cottons effect. Therefore, it is identified that the absolute configuration of compound VII is 3S, 6R, 7S, 9R, 12S, 14R.

[0132] Based on the above, it can be determined that the structural formula of compound VII separated in Example 1 is:

[0133]

[0134] Taking the compound VIII separated in Example 1, its structure is identified by one-dimensional nuclear magnetic resonance spectrum (1D-NMR), two-dimensional nuclear magnetic resonance spectrum (2D-NMR) and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS).

[0135] From the HSQC spectrum combined with the carbon spectrum, the chemical shifts δ of H and C connected therewith of compound VIII are attributed as shown in Table 8:

[0136] Table 8 H and C NMR data of compound VIII separated in Example 1 1 H (600 MHz) and 13 C (150 MHz) NMR data, methanol-d4 as solvent

[0137]

[0138] like Figure 24 As shown, the HR-ESI-MS m / z of compound VIII is: [M+H] + The quasi-molecular ion peak is at 487.2807 (C). 27 H 39 N₂O₆[M+H] + The value of calc m / z (487.2803) indicates that its molecular formula is C. 27 H 38 N₂O₆ contains 10 degrees of unsaturation. According to HR-ESI-MS... 1 H-NMR and 13 A comparison of C-NMR and compound VII reveals that compound VIII has a methyl group attached at the C-4 position. 1 H-NMR and HMBC (H3-27 to C-3 / C-4 / C-5) correlations confirm the presence of the compound. To determine its relative configuration, based on NOESY spectroscopy and relevant literature analysis, the relative configuration of compound VIII was identified as 3S*, 6R*, 7S*, 9R*, 12S*, 14R*. To determine the absolute stereoconfiguration of compound VIII, a conformational search was performed on its two possible stereoisomers [(3S, 6R, 7S, 9R, 12S, 14R) and (3R, 6S, 7R, 9S, 12R, 14S)]. For conformations with a distribution ratio greater than 1%, ECD calculations were performed using TD-DFT theory at the B3LYP / 6-31G(d) level. Comparison with experimental ECD spectra revealed a high degree of agreement between the calculated and experimental results for the isomers (3S, 6R, 7S, 9R, 12S, 14R) – compound VIII – exhibiting the same Cottons effect. Therefore, the absolute configuration of compound VIII was identified as 3S, 6R, 7S, 9R, 12S, 14R.

[0139] In summary, the structural formula of compound VIII isolated in Example 1 can be determined as follows:

[0140]

[0141] The structure of compound IX obtained in Example 1 was identified by one-dimensional nuclear magnetic resonance spectroscopy (1D-NMR), two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR), and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS).

[0142] The chemical shifts δ of H and C atoms in compound IX, obtained from HSQC spectroscopy combined with carbon spectroscopy, are shown in Table 9.

[0143] Table 9 shows the compound IX isolated in Example 1. 1 H (600MHz) and13 C150MHz NMR data, methanol-d4 as solvent

[0144]

[0145] like Figure 27 As shown, the HR-ESI-MS m / z of compound IX is: [M+H] + The quasi-molecular ion peak is at 560.3215 (C). 31 H 46 NO8[M+H] + The calc m / z: 560.3218 indicates that its molecular formula is C. 31 H 45 NO8 contains 10 degrees of unsaturation. (Based on HR-ESI-MS) 1 H-NMR and 13 A comparison of C-NMR with compound I reveals that compound IX has different substituents at the C-7 position, potentially indicating the presence of glycerol and acetate fragments. 1 H- 1 H COSY related (H a / b -24 / H-28 / H a / b -29) and HMBC related (H a / b The presence of the glycerol ethyl ester fragment (H-29 to C-24 / C-28 / C-30, H3-31 to C-30) was confirmed. According to the HMBC correlation (H-7 to C-24), the glycerol ethyl ester is linked to the C-7 position via an ether bond. To determine its relative configuration, based on NOESY spectroscopy and relevant literature analysis, the relative configuration of compound IX was identified as 6R*, 7S*, 12S*, 14R*, 28R*. To determine the absolute stereoconfiguration of compound IX, a conformational search was performed on its two possible stereoisomers [(6R, 7S, 12S, 14R, 28R) and (6S, 7R, 12R, 14S, 28S)]. For conformations with a distribution ratio greater than 1%, ECD calculations were performed using TD-DFT theory at the B3LYP / 6-31G(d) level. Comparison with experimental ECD spectra revealed a high degree of agreement between the calculated and experimental results for the isomers (6R, 7S, 12S, 14R, 28R) – compound IX – exhibiting the same Cottons effect. Therefore, the absolute configuration of compound IX was identified as 6R, 7S, 12S, 14R, 28R.

[0146] In summary, the structural formula of compound IX isolated in Example 1 can be determined as follows:

[0147]

[0148] The structure of compound X obtained in Example 1 was identified by one-dimensional nuclear magnetic resonance spectroscopy (1D-NMR), two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS).

[0149] The chemical shifts δ of H and C atoms in compound X can be obtained from HSQC spectroscopy combined with carbon spectroscopy, as shown in Table 10:

[0150] Table 10 shows the compound X isolated in Example 1. 1 H (600MHz) and 13 C150MHz NMR data, methanol-d4 as solvent

[0151]

[0152] like Figure 30 As shown, the HR-ESI-MS m / z of compound X is: [M+H] + The quasi-molecular ion peak is at 560.3215 (C). 31 H 46 NO8[M+H] + The calc m / z: 560.3218 indicates that its molecular formula is C. 31 H 45 NO8 contains 10 degrees of unsaturation. (Based on HR-ESI-MS) 1 H-NMR and 13 A comparison of C-NMR and compound IX reveals that compound X lacks a double bond between C-2 and C-3, and may form a six- or seven-membered ring linked by an ether bond. 1 H-NMR and HMBC (H a / b-2 to C-1 / C-3; H-3 to C-1 / C-2 / C-4 / C-5 / C-17 / C-27) can be known that a seven-membered ring connected by an ether bond is newly formed, to determine the relative configuration thereof, the relative configuration of compound X is identified as 3S*, 6R*, 7S*, 12S*, 14R*, 28R* in combination with the NOESY spectrum and related literature analysis. In order to determine the absolute stereochemical configuration of compound X, conformational search is performed on two possible stereoisomers [(3S, 6R, 7S, 12S, 14R, 28R) and (3R, 6S, 7R, 12R, 14S, 28S)] of compound X, and ECD calculation is performed on the conformations with a distribution ratio of greater than 1% by using the TD-DFT theory at the B3LYP / 6-31G(d) level and the calculation software. By comparison with the experimental ECD spectrum, it is found that the calculation results of the isomer (3S, 6R, 7S, 12S, 14R, 28R)-compound X and the experimental results are highly matched, and both have the same Cottons effect. Therefore, the absolute configuration of compound X is identified as 3S, 6R, 7S, 12S, 14R, 28R.

[0153] Based on the above, it can be determined that the structural formula of compound X separated in Example 1 is as follows:

[0154]

[0155] Example 4

[0156] Screening of antitumor cytotoxic activity of compounds I-X prepared in Example 1

[0157] Principle of MTS method for detecting cell activity: MTS is a brand-new MTT analog, and its full name is 3-(4, 5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), which is a yellow dye. Succinate dehydrogenase in the mitochondria of living cells can metabolize and reduce MTS to generate a soluble formazan compound, and the content of formazan can be determined at 490 nm by using an enzyme marker. Under normal circumstances, the amount of formazan generated is proportional to the number of living cells, so the number of living cells can be inferred according to the optical density OD value.

[0158] The cells used in this example are leukemia HL-60 cells, lung cancer A549 cells, liver cancer HepG2 cells, breast cancer MDA-MB-231 cells and colon cancer SW480 cells.

[0159] The experimental method is as follows:

[0160] Seeding cells: Single cell suspension was prepared with culture solution (DMEM or RMPI1640) containing 10% fetal bovine serum, 3000-15000 cells per well were seeded into 96-well plates, 100 μL per well, and the cells were seeded 24 hours in advance.

[0161] Adding test compound solution: The compound was dissolved in DMSO, and the compound was initially screened at a concentration of 40 μM, 200 μL per well, and three replicates were set for each treatment.

[0162] Color development: after 48 hours of culture at 37 degrees Celsius, the adherent cells were discarded, 20 μL of MTS solution and 100 μL of DMEM culture solution were added per well; the suspended cells were discarded 100 μL of culture supernatant, 20 μL of MTS solution was added per well; three blank replicates (20 μL of MTS solution and 100 μL of culture solution mixture) were set, and the incubation was continued for 2-4 hours to ensure that the reaction was complete before measuring the optical absorption value.

[0163] Colorimetry: a wavelength of 492 nm was selected, and the multifunctional enzyme marker (MULTISKAN FC) was used to read the optical absorption value of each well, and the results were recorded, and after data processing, the cell inhibition rate graph was drawn with the compound number as the horizontal coordinate and the cell inhibition rate as the vertical coordinate.

[0164] Positive control compound: two positive compounds, doxorubicin and paclitaxel, were set for each experiment, and the cell growth curve was drawn with the concentration as the horizontal coordinate and the cell survival rate as the vertical coordinate, and the IC 50 value of the compound was calculated by the two-point method (Reed and Muench method). The initial screening results are shown in Table 11.

[0165] Table 11 Cell inhibition rate (%) of compounds I-X at a concentration of 40 μM in initial screening

[0166]

[0167] At a concentration of 40 μM, compounds I-X have good inhibitory activity on the in vitro tumor growth of leukemia HL-60, lung cancer A549, liver cancer HepG2, breast cancer MDA-MB-231, or colon cancer SW480, and the next step is to continue the screening of tumor cell toxicity activity compounds (IC 50 detection).

[0168] In the same way as the initial screening, the compound was rescreened at a concentration of 40 μM, 8 μM, 1.6 μM, 0.32 μM, and 0.064 μM, and doxorubicin and paclitaxel were used as positive controls, and the rescreening results are shown in Table 12.

[0169] Table 12 Half-inhibitory concentration values (μM) of compounds I-X on five tumor cell lines

[0170]

[0171] Note: "-" means not screened, doxorubicin and paclitaxel as positive controls

[0172] The in vitro tumor growth half-inhibitory concentrations of compounds I-X against leukemia HL-60, lung cancer A549, liver cancer HepG2, breast cancer MDA-MB-231 or colon cancer SW480 are shown in Table 12, IC 50 values are between 1.937 ± 0.093 and 28.11 ± 0.76 μM.

Claims

1. An anthracycline compound characterized in that: The ansa mycin compounds are 10, and the structures are shown in formula I~X: Formula I; Formula II; Formula III; Formula IV; Formula V; Formula VI; Formula VII; Formula VIII; Formula IX; Formula X.

2. The use of the ansa mycin compound of claim 1 in the preparation of a drug for treating leukemia, lung cancer, liver cancer, breast cancer or colon cancer.

3. The process for the preparation of an anasamycin compound according to claim 1, characterized in that: After the activation culture, seed culture and fermentation culture of Amycolatopsis sp. YINM00005, the bacterial liquid is obtained, and 10 ansa mycin compounds are separated from the bacterial liquid; The fermentation culture medium is: sucrose 90~110g / L, glucose 9~11g / L, acid hydrolysis casein 0.1~0.15g / L, yeast extract 4~6g / L, 3-(N-morpholine) propyl sulfonic acid 18~24g / L, trace elements 0.8~1.2mL, K2SO40.2~0.3g / L, MgCl2·6H2O 8~12g / L, pH=7.0; The specific steps are as follows: (1) Bacterial activation: inoculate Amycolatopsis sp. YINM00005 into the activation medium for activation; (2) Seed preparation: inoculate the activated bacteria in step (1) into the seed culture medium for shaking culture to obtain the seed; (3) Fermentation process: inoculate the prepared seed in step (2) into the fermentation medium for shaking fermentation culture to obtain the fermentation liquid; (4) After the fermentation is completed, the fermentation liquid is extracted with an equal volume of ethyl acetate, and the concentrated crude extract is dissolved in a mixed solution of dichloromethane and methanol, then mixed with silica gel, and gradient eluted with dichloromethane and methanol eluent for forward silica gel column chromatography to obtain the eluent; (5) Mix the eluent obtained in step (4) with silica gel, and gradient elute with water-methanol eluent for reverse silica gel column chromatography, and finally purify with semi-preparative high performance liquid chromatography to obtain the compounds shown in formula I~X; The volume ratio of dichloromethane to methanol in the dichloromethane-methanol eluent used in step (4) is 100:0, 80:1, 50:1, 30:1, 10:1, and 0:100, respectively, and the eluent with a volume ratio of dichloromethane to methanol of 30:1 is selected for step (5); The volume ratio of methanol to water used in step (5) is 10:90, 20:80, 30:70, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, and 100:0, respectively, wherein the eluent with a volume ratio of 75:25 contains the compound shown in formula I, the eluent with a volume ratio of 80:20 contains the compound shown in formula II, the eluent with a volume ratio of 55:45 contains the compounds shown in formula III and formula VIII, the eluent with a volume ratio of 65:35 contains the compounds shown in formula IV and formula X, the eluent with a volume ratio of 70:30 contains the compound shown in formula V, the eluent with a volume ratio of 50:50 contains the compound shown in formula VI, and the eluent with a volume ratio of 60:40 contains the compounds shown in formula VII and formula IX; The Amycolatopsis sp. YINM00005 is preserved in Guangdong Microbial Culture Collection Center on July 19, 2023, and the preservation number is GDMCC No. 63669.

4. The process for the preparation of an anasamycin compound according to claim 3, characterized in that: The culture medium composition for activation culture is: soluble starch 8~12 g / L, K2HPO4 0.8~1.2 g / L, MgSO4·7H2O 0.8~1.2 g / L, NaCl 0.8~1.2 / L, (NH4)2SO4 1.6~2.4 g / L, CaCO3 1.6~2.4 g / L, trace salt solution 0.8~1.2 mL, agar 15.0~20 g / L, distilled water 1.0 L, pH=7.0; The culture medium composition for seed culture is: soluble starch 8~12 g / L, K2HPO4 0.8~1.2 g / L, MgSO4·7H2O 0.8~1.2 g / L, NaCl 0.8~1.2 / L, (NH4)2SO4 1.6~2.4 g / L, CaCO3 1.6~2.4 g / L, trace salt solution 0.8~1.2 mL, distilled water 1.0 L, pH=7.0.

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