Novel ansamycin compound as well as preparation method and application thereof

Abstract

The invention discloses a novel ansamycin compound as well as a preparation method and application thereof, and belongs to the technical field of compounds and preparation thereof. The amycolatopsis sp. YINM00005 is endophyte of peperomia dindygulensis in Yunnan, and a large amount of novel ansamycin compounds can be metabolized after fermentation; the fermentation method mainly comprises the following steps: fermenting the strain under proper culture conditions to obtain fermentation liquor, extracting and concentrating by a solvent, and combining with column chromatography to prepare 10 new compounds, the total concentration of the 10 new compounds in the fermentation liquor can reach 3.5-6.8 mg / L, the purity after extraction and separation can reach more than 99%, and the 10 compounds have relatively strong anti-tumor cytotoxic activity. The method is low in cost, simple in process and environment-friendly.

Description

Technical Field

[0001] The invention relates to a novel ansamycin compound and a preparation method and application thereof, belonging to the technical field of compounds and preparation thereof. Background Art

[0002] In recent years, the method of preparing target active compounds through microbial fermentation has attracted more and more attention because of the characteristics of microorganisms such as short metabolic cycle, mild reaction conditions, few by-products, and strong stereoselectivity. In actual production, there are already a large number of application examples of producing some compounds with medicinal or economic value through microbial fermentation.

[0003] Ansamycins are a type I polyketide compound. This type of antibiotic has low toxicity, high efficacy and a broad antibacterial spectrum. It can specifically act on DNA-dependent RNA polymerase to inhibit the transcription process of bacterial DNA; and it has an inhibitory effect on RNA-dependent DNA reverse transcriptase of tumor RNA viruses. It is widely used in the treatment of G+ (Gram-positive bacteria), tuberculosis, leprosy and mycobacterial infections related to AIDS. In summary, ansamycin compounds have good research and development potential as drug lead compounds.

[0004] Amycolatopsis sp. YINM00005 is an endophytic actinomycete obtained from the traditional Chinese medicinal plant Amycolatopsis sp. in Kunming, Yunnan. Analysis of its secondary metabolites revealed 10 new compounds, so this strain has the potential to be developed as an engineered bacterium to produce new compounds. Summary of the invention

[0005] One of the purposes of the present invention is to provide 10 novel ansamycin compounds, the structures of which are shown in Formula I to Formula X:

[0006]

[0007]

[0008]

[0009]

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

[0011] The second object of the present invention is to provide a method for preparing the 10 novel ansamycin compounds: performing activation culture, seed culture and fermentation culture on Amycolatopsis spp. YINM00005 to obtain a bacterial solution, and separating and obtaining 10 novel ansamycin compounds from the bacterial solution;

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

[0013] Preferably, the components of the culture medium for activation culture are: 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;

[0014] The composition of the culture medium 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;

[0015] The culture medium for fermentation culture is: sucrose 90-110 g / L, glucose 9-11 g / L, acid hydrolyzed casein 0.1-0.15 g / L, yeast extract 4-6 g / L, MOPS (3-(N-morpholino)propanesulfonic acid) 18-24 g / L, trace elements 0.8-1.2 mL, K2SO4 0.2-0.3 g / L, MgCl2·6H2O 8-12 g / L, pH=7.0.

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

[0017] (1) Bacterial activation: Amycolatopsis sp. YINM00005 was inoculated into the activation medium for activation and standby use;

[0018] (2) strain preparation: inoculating the activated bacteria in step (1) into a seed culture medium and culturing on a shaking table to obtain strains;

[0019] (3) Fermentation process: inoculating the strain prepared in step (2) into a fermentation medium and fermenting it on a shaking platform to obtain a fermentation liquid;

[0020] (4) After the fermentation is completed, the fermentation broth is extracted with an equal volume of ethyl acetate and concentrated to obtain a crude extract, the crude extract is dissolved with a mixed solution of dichloromethane and methanol and then mixed evenly with silica gel, and a forward silica gel column chromatography gradient elution is performed using dichloromethane and methanol eluents to obtain an eluent;

[0021] (5) The eluent obtained in step (4) is selected and mixed evenly with silica gel, and reverse silica gel column chromatography is performed with water-methanol eluent for gradient elution, and finally semi-preparative high performance liquid chromatography is used for purification to obtain compounds represented by Formula I to Formula X.

[0022] Preferably, the volume ratio of dichloromethane to methanol in the dichloromethane-methanol eluent used in the forward silica gel column chromatography gradient elution 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 to perform the operation of step (5).

[0023] Preferably, the volume ratio of methanol to water used in the reverse silica gel column chromatography gradient elution 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, 100:0, wherein the 75:25 eluent contains the compound of formula I, the 80:20 eluent contains the compound of formula II, the 55:45 eluent contains the compounds of formula III and formula VIII, the 65:35 eluent contains the compounds of formula IV and formula X, the 70:30 eluent contains the compound of formula V, the 50:50 eluent contains the compound of formula VI, and the 60:40 eluent contains the compounds of formula VII and formula IX.

[0024] Beneficial effects of the present invention

[0025] (1) The present invention performs special fermentation culture on Amycolatopsis sp. YINM00005, separates and purifies 10 compounds obtained in the culture solution, and identifies the 10 compounds as novel ansamycin compounds. Experiments have verified that the 10 compounds have the effect of inhibiting leukemia cells, lung cancer cells, liver cancer cells, breast cancer cells and colon cancer cells. Therefore, the 10 compounds can be used to prepare drugs for treating these diseases, providing more options for preparing drugs for treating these diseases.

[0026] (2) The present invention prepares new ansamycin compounds by a microbial fermentation method. The raw materials are cheap and readily available, the reaction conditions are mild, the process is simple, the equipment requirements are simple, the environment is pollution-free, and it is suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0028] Figure 2 The compound I provided by the present invention is 13 C-NMR spectrum.

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

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

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

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

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

[0034] Figure 8 The compound III provided by the present invention is 13 C-NMR spectrum.

[0035] Fig. 9 The HR-ESI-MS spectrum of compound III provided by the present invention.

[0036] Fig.10 The compound IV provided by the present invention 1 H-NMR spectrum.

[0037] Fig.11 The compound IV provided by the present invention 13 C-NMR spectrum.

[0038] Fig.12 The HR-ESI-MS spectrum of compound IV provided by the present invention.

[0039] Fig.13 The compound V provided by the present invention is 1 H-NMR spectrum.

[0040] Fig.14 The compound V provided by the present invention is 13 C-NMR spectrum.

[0041] Fig.15 The HR-ESI-MS spectrum of compound V provided by the present invention.

[0042] Fig.16 The compound VI provided by the present invention is 1 H-NMR spectrum.

[0043] Fig.17 The compound VI provided by the present invention is 13 C-NMR spectrum.

[0044] Fig.18 The HR-ESI-MS spectrum of the compound VI provided by the present invention.

[0045] Fig.19 The compound VII provided by the present invention 1 H-NMR spectrum.

[0046] Fig. 20 The compound VII provided by the present invention 13 C-NMR spectrum.

[0047] Fig.21 The HR-ESI-MS spectrum of compound VII provided by the present invention.

[0048] Fig. 22 The compound VIII provided by the present invention 1 H-NMR spectrum.

[0049] Fig.23 The compound VIII provided by the present invention 13 C-NMR spectrum.

[0050] Fig.24 The HR-ESI-MS spectrum of compound VIII provided by the present invention.

[0051] Fig.25 The compound IX provided by the present invention 1 H-NMR spectrum.

[0052] Fig.26 The compound IX provided by the present invention 13 C-NMR spectrum.

[0053] Fig. 27 The HR-ESI-MS spectrum of compound IX provided by the present invention.

[0054] Fig.28 The compound X provided by the present invention 1 H-NMR spectrum.

[0055] Fig.29 The compound X provided by the present invention 13 C-NMR spectrum.

[0056] Fig.30 The HR-ESI-MS spectrum of the compound X provided by the present invention.

[0057] Fig.31 The crystal structure of compound V provided by the present invention. DETAILED DESCRIPTION

[0058] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following embodiments are only simple examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

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

[0060] (1) The components of the activation 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 composition of the seed culture medium is: 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 composition of the fermentation medium was as follows: sucrose 100 g / L, glucose 10 g / L, acid hydrolyzed casein 0.1 g / L, yeast extract 5.0 g / L, MOPS (3-(N-morpholino)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 novel compound is prepared by using Amycolatopsis sp. YINM00005, which specifically comprises the following steps:

[0065] (1) Bacterial activation: Amycolatopsis sp. YINM00005 was inoculated into the activation medium, cultured in a 28°C constant temperature incubator for 4 days, and then placed in a 4°C refrigerator for later use.

[0066] (2) Preparation of bacterial strains: The activated bacterial cells were inoculated into the seed culture medium and fermented at 28°C and 200 rpm for 3 days.

[0067] (3) Fermentation process: The strain prepared in step (2) was added into 30 L fermentation medium (sucrose 100 g / L, glucose 10 g / L, acid hydrolyzed casein 0.1 g / L, yeast extract 5.0 g / L, MOPS (3-(N-morpholino)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 fermented at 28°C and 200 rpm for 15 days to obtain a fermentation broth.

[0068] (4) After the fermentation, the fermentation liquid was extracted three times with an equal volume of ethyl acetate, and vacuum concentrated to obtain a crude extract (22.50 g). The crude extract was detected by high performance liquid chromatography, and the concentrated crude extract was dissolved in a mixed solution of dichloromethane-methanol (the volume ratio of dichloromethane to methanol was 1:1) to obtain a crude extract solution; the prepared crude extract solution and 200-300 mesh silica gel were stirred and mixed with a glass rod, and the solvent was slowly stirred until it was completely evaporated, and the dichloromethane-methanol solution with a volume ratio of 100:0, 80:1, 50:1, 30:1, 10:1, and 0:100 was used in sequence for forward silica gel (200-300 mesh) column chromatography gradient elution to obtain 6 eluates respectively.

[0069] (5) Take an appropriate amount of 6 portions of the eluent for TLC spot plate and HPLC analysis. According to the results of TLC spot plate and HPLC analysis, select an eluent with a volume ratio of dichloromethane to methanol of 30:1, and slowly add the eluent to the prepared absorbent cotton with a dropper. Leave it at room temperature until the solvent is completely evaporated. After loading, use 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 in sequence for reverse silica gel column (40-63 μm) chromatography gradient elution to obtain 13 portions of eluent.

[0070] According to the results of TLC spot plate and HPLC analysis, an eluent with a methanol-water volume ratio of 75:25 was selected 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 =25.5min) reached a purity of more than 98% (analytical gradient elution method: 0min, 30% methanol; 30min, 100% methanol).

[0071] According to the results of TLC spot plate and HPLC analysis, 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 R =26.9min) reached a purity of more than 98% (analytical gradient elution method: 0min, 30% methanol; 30min, 100% methanol).

[0072] According to the results of TLC spot plate and HPLC analysis, an eluent with a methanol-water volume ratio of 55:45 was selected 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 R =23.3min) reached a purity of more than 98% (analytical gradient elution method: 0min, 30% methanol; 30min, 100% methanol).

[0073] According to the results of TLC spot plate and HPLC analysis, 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 R =24.3min) reached a purity of more than 98% (analytical gradient elution method: 0min, 30% methanol; 30min, 100% methanol).

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

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

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

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

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

[0079] According to the results of TLC spot plate and HPLC analysis, 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, 90% methanol) to obtain compound X (72.0 mg); HPLC analysis of compound X (retention time t R =24.7min) reached a purity of more than 98% (analytical gradient elution method: 0min, 30% methanol; 30min, 100% methanol).

[0080] Finally, after high performance liquid chromatography analysis, the concentration of compound I in the fermentation broth was 3.8 mg / L; the concentration of compound II was 4.2 mg / L; the concentration of compound III was 5.5 mg / L; the concentration of compound IV was 3.6 mg / L; the concentration of compound V was 3.5 mg / L; the concentration of compound VI was 4.6 mg / L; the concentration of compound VII was 6.8 mg / L; the concentration of compound VIII was 3.5 mg / L; the concentration of compound IX was 5.2 mg / L; and the concentration of compound X was 3.6 mg / L.

[0081] Example 2

[0082] Example Identification of the structure of the compound you obtained

[0083] The structure of the compound I separated 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] From the HSQC spectrum combined with the carbon spectrum, it can be obtained that the NMR chemical shift δ of compound I is assigned as shown in Table 1.

[0085] Table 1 Compound I isolated from Example 1 1 H(600MHz) and 13 C(150MHz)NMR data(methanol-d4)

[0086]

[0087] like Figure 3 As 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 its molecular formula is C 27 H 39 NO5, contains 9 unsaturated degrees. 1 H-NMR( Figure 1 ), 13 C-NMR( Figure 2 ) and HSQC spectrum analysis showed that compound Ⅰ contained 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) corresponds 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 correspond 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 groups [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) corresponds 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] analyze 1 H- 1 H COSY spectrum correlation shows that H-5 / H-6 / H-7 / H a / b -8 has a clear correlation, H a / b -10 / H a / b -11 / H-12 / H a / b -13 / H-14 / H a / b-15 has a clear correlation, which can be attributed to two fragments from C-5 to C-8 and C-10 to C-15 respectively. Further analysis of the HMBC spectrum shows that H3-22 has obvious correlation signals for C-13 / C-14 / C-15, H3-23 has obvious correlation signals for C-11 / C-12 / C-13, and H a / b -8 and H a / b -10 has obvious correlation signals for C-9, and H3-25 has obvious correlation signals for C-5 / C-6 / C-7. The above correlations indicate that C-5 to C-15 are continuous and there are three methyl groups connected to the carbon chain. In addition, according to the obvious HMBC correlation signal of H3-24 for C-7, it can be known that the methoxy fragment is connected to the C-7 position. 1 H-NMR shows that δ H 6.45 and δ H 6.51 are doublets with a small coupling constant (J = 3.0 Hz). According to the HMBC correlation, H-19 is correlated with C-18 / C-20 / C-21, and H-21 is correlated with C-16 / C-20 / C-19. The above correlations indicate that there may be a benzene ring chromophore. a / b -15 has a significant correlation with C-21, thus proving that the C-5 to C-15 fragment is connected to the benzene ring chromophore. Based on the chemical shift values ​​and coupling constants of H-2, H-3, and H-5, it can be judged that C-2 / C-3 may be double bonded, C-4 / C-5 may be double bonded, and C-4 is a quaternary carbon. The above assumptions are 1 H- 1 H COSY (H-2 / H-3 and H-5 / H-6) correlation and HMBC (H-3 to C-2 / C-4 / C-5) correlation confirmed. 13 C-NMR shows that δ C 213.4(C-9) and δ C 171.6 (C-1) may be the resonance of carbonyl carbon and amide carbon, respectively. a / b -8 / H a / b -10 has obvious correlation signals to C-9, -NH- to C-1 / C-2 has obvious correlation signals) correlation can prove the existence of carbonyl carbon and amide carbon. According to HMBC (-NH- to C-17 / C-18 / C-19) correlation, it is proved that the compound type is macrolide. According to the relevant literature, the molecule is a new compound that has not been reported.

[0089] In order to determine its relative configuration, combined with NOESY spectrum and related literature analysis, the relative configuration of compound I was identified as 6R*, 7S*, 12S*, 14R*. In order to determine the absolute stereo configuration of compound I, its two possible stereoisomers [(6R, 7S, 12S, 14R) and (6S, 7R, 12R, 14S)] were searched for conformation, and the conformations with a distribution ratio greater than 1% were calculated using TD-DFT theory at the B3LYP / 6-31G(d) level using 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 were highly matched with the experimental results, and both had the same Cottons effect. Therefore, the absolute configuration of compound I was identified as 6R, 7S, 12S, 14R.

[0090] In summary, it can be determined that the structural formula of the compound I isolated in Example 1 is:

[0091]

[0092] The structure of compound II separated 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).

[0093] From the HSQC spectrum combined with the carbon spectrum, it can be obtained that the chemical shift δ assignments of H and the C connected to compound II are shown in Table 2:

[0094] Table 2 Compound II isolated from Example 1 1 H(600MHz) and 13 C (150 MHz) NMR data, methanol-d4 as solvent

[0095]

[0096] like Figure 6 As shown, HR-ESI-MS m / z of compound II: [M+H] + The 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, contains 9 unsaturated degrees. According to HR-ESI-MS, 1 H-NMR and 13 Comparison of C-NMR and compound I shows that compound II lacks one carbon-carbon double bond (Δ 2), and may form a six-membered or seven-membered ring connected by ether bonds, 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), it can be seen that a new seven-membered ring connected by an ether bond is formed. In order 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 stereo configuration of compound II, two possible stereoisomers [(3S, 6R, 7S, 12S, 14R) and (3R, 6S, 7R, 12R, 14S)] were searched for conformation, and the conformations with a distribution ratio greater than 1% were calculated using TD-DFT theory at the B3LYP / 6-31G (d) level using 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 were highly matched with the experimental results, and both had the same Cottons effect. Therefore, the absolute configuration of compound II was identified as 3S, 6R, 7S, 12S, 14R.

[0097] In summary, it can be determined that the structural formula of compound II isolated in Example 1 is:

[0098]

[0099] The structure of compound III separated 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).

[0100] From the HSQC spectrum combined with the carbon spectrum, it can be obtained that the chemical shift δ assignments of H and the C connected to compound III are shown in Table 3:

[0101] Table 3 Compound III isolated from Example 1 1 H(600MHz) and 13 C (150 MHz) NMR data, methanol-d4 as solvent

[0102]

[0103] like Fig. 9 As shown, HR-ESI-MS m / z of compound III: [M+H] + The quasi-molecular ion peak is 412.2486 (C 25 H 34 NO4[M+H] +, calc m / z: 412.2482) indicates that its molecular formula is C 25 H 33 NO4, contains 10 unsaturated degrees. According to HR-ESI-MS, 1 H-NMR and 13 Comparison of C-NMR and compound III shows that a -OCH3 fragment is missing at the C-7 position of compound III, which may form a carbon-carbon double bond (Δ 7 ),according to 1 H-NMR and HMBC (H3-24 to C-5 / C-6 / C-7, H-7 to C-9 / C-8 / C-6 / C-5 and H-8 to C-9 / C-10 / C-5 / C-6 / C-7) showed that a carbon-carbon double bond (Δ 7 ), in addition, according to 1 H-NMR and HMBC (H3-25 to C-3 / C-4 / C-5) correlations show that the -CH2CH3 fragment connected to the C-4 position in compound I has become a -CH3 fragment. In order to determine its relative configuration, combined with NOESY spectrum and related literature analysis, the relative configuration of compound III was identified as 6R*, 12S*, 14R*. In order to determine the absolute stereo configuration of compound III, its two possible stereoisomers [(6R, 12S, 14R) and (6S, 12R, 14S)] were searched for conformations, and the conformations with a distribution ratio greater than 1% were calculated using TD-DFT theory at the B3LYP / 6-31G(d) level using calculation software. By comparing with the experimental ECD spectrum, it was found that the calculation results of the isomer (6R, 12S, 14R)-compound III were highly consistent with the experimental results, and 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 isolated in Example 1 is:

[0105]

[0106] The structure of compound IV separated 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).

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

[0108] Table 4 Compound IV isolated from Example 1 1H(600MHz) and 13 C (150 MHz) NMR data, methanol-d4 as solvent

[0109]

[0110] like Fig.12 As shown, HR-ESI-MS m / z of compound IV: [M+H] + The quasi-molecular ion peak is 412.2481 (C 25 H 34 NO4[M+H] + , calc m / z: 412.2482) indicates that its molecular formula is C 25 H 33 NO4, contains 10 unsaturated degrees. According to HR-ESI-MS, 1 H-NMR and 13 Comparison of C-NMR and compound III shows that compound IV lacks one carbon-carbon double bond (Δ 2 ), and may form a six-membered or seven-membered ring connected by ether bonds, 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-25), a new seven-membered ring connected by an ether bond was formed. In order to determine its relative configuration, combined with NOESY spectrum and related literature analysis, the relative configuration of compound IV was identified as 3S*, 6R*, 12S*, 14R*. In order to determine the absolute stereo configuration of compound IV, two possible stereoisomers [(3S, 6R, 12S, 14R) and (3R, 6S, 12R, 14S)] were searched for conformation, and the conformations with a distribution ratio greater than 1% were calculated using TD-DFT theory at the B3LYP / 6-31G (d) level 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 IV were highly matched with the experimental results, and both had the same Cottons effect. Therefore, the absolute configuration of compound IV was identified as 3S, 6R, 12S, 14R.

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

[0112]

[0113] The structure of the compound V isolated 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).

[0114] From the HSQC spectrum combined with the carbon spectrum, it can be obtained that the chemical shift δ assignments of H of compound V and the C connected thereto are shown in Table 5:

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

[0116]

[0117] like Fig.15 As shown, HR-ESI-MS m / z of compound V: [M+H] + The quasi-molecular ion peak is 426.2635 (C 26 H 36 NO4[M+H] + , calc m / z: 426.2639) indicates that its molecular formula is C 26 H 35 NO4, contains 10 unsaturated degrees. According to HR-ESI-MS, 1 H-NMR and 13 C-NMR comparison with compound IV shows that compound V has an additional 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) shows that a -CH2CH3 fragment is connected to the C-4 position in compound V. In order to determine its relative configuration, combined with NOESY spectrum and related literature analysis, the relative configuration of compound V was identified as 3S*, 6R*, 12S*, 14R*. In order to determine the absolute stereo configuration of compound V, a conformational search was performed for its two possible stereoisomers [(3S, 6R, 12S, 14R) and (3R, 6S, 12R, 14S)], and the ECD calculations were performed using TD-DFT theory at the B3LYP / 6-31G(d) level using calculation software for conformations with a distribution ratio greater than 1%. By comparing the ECD spectra with the experimental ones, it was found that the calculated results of the isomer (3S, 6R, 12S, 14R)-compound V were highly consistent with the experimental results and both had the same Cottons effect. Finally, the crystal structure of compound V was successfully obtained ( Fig.31 ), therefore the absolute configuration of compound V was identified as 3S, 6R, 12S, 14R.

[0118] In summary, it can be determined that the structural formula of the compound V isolated in Example 1 is:

[0119]

[0120] The structure of the compound VI isolated 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).

[0121] From the HSQC spectrum combined with the carbon spectrum, it can be obtained that the chemical shift δ assignments of H and the C connected to compound VI are shown in Table 6:

[0122] Table 6 Compound VI isolated from Example 1 1 H(600MHz) and 13 C (150 MHz) NMR data, methanol-d4 as solvent

[0123]

[0124] like Fig.18 As shown, HR-ESI-MS m / z of compound VI: [M+H] + The quasi-molecular ion peak is 473.2648 (C 26 H 37 N2O6[M+H] + , calc m / z: 473.2646) indicates that its molecular formula is C 26 H 36 N2O6, contains 10 unsaturated degrees. According to HR-ESI-MS, 1 H-NMR and 13 Comparison of C-NMR with compound III shows that compound VI lacks a double bond between C-7 and C-8 and the carbonyl signal at C-9 is missing. Based on literature reports and the chemical shift values ​​of carbon and hydrogen spectra, it is inferred that a carbamate fragment has been formed. 1 H-NMR and HMBC (H a / b-8 to C-6 / C-7 / C-10; H-7 to C-8 / C-9) shows that a six-membered closed ring system connected by carbamate is formed and the C-9 position is connected by both amino and hydroxyl groups. In order to determine its relative configuration, combined with NOESY spectrum and related literature analysis, the relative configuration of compound VI was identified as 6R*, 7S*, 9R*, 12S*, 14R*. In order to determine the absolute stereo configuration of compound VI, two possible stereoisomers [(6R, 7S, 9R, 12S, 14R) and (6S, 7R, 9S, 12R, 14S)] were searched for conformation, and the conformations with a distribution ratio greater than 1% were calculated using TD-DFT theory at the B3LYP / 6-31G (d) level using 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 were highly matched with the experimental results, and both had the same Cottons effect. Therefore, the absolute configuration of compound VI was identified as 6R, 7S, 9R, 12S, 14R.

[0125] In summary, it can be determined that the structural formula of the compound VI isolated in Example 1 is:

[0126]

[0127] The structure of compound VII isolated 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).

[0128] From the HSQC spectrum combined with the carbon spectrum, it can be obtained that the chemical shift δ assignments of H and the C connected to compound VII are shown in Table 7:

[0129] Table 7 Compound VII isolated from Example 1 1 H(600MHz) and 13 C (150 MHz) NMR data, methanol-d4 as solvent

[0130]

[0131] like Fig.21 As shown, HR-ESI-MS m / z of compound VII: [M+H] + The 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, contains 10 unsaturated degrees. According to HR-ESI-MS, 1 H-NMR and 13 Comparison of C-NMR and compound VI shows that compound VII has a methoxy group connected to the C-9 position. 1 H-NMR and HMBC (H3-24 to C-9) correlations confirmed that there was one less double bond between C-2 and C-3, and a six-membered or seven-membered ring connected by an ether bond may be formed. 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), a new seven-membered ring connected by an ether bond was formed. In order to determine its relative configuration, combined with NOESY spectrum and related literature analysis, the relative configuration of compound VII was identified as 3S*, 6R*, 7S*, 9R*, 12S*, 14R*. In order to determine the absolute stereo configuration of compound VII, two possible stereoisomers [(3S, 6R, 7S, 9R, 12S, 14R) and (3R, 6S, 7R, 9S, 12R, 14S)] were searched for conformation, and the conformations with a distribution ratio greater than 1% were calculated using TD-DFT theory at the B3LYP / 6-31G(d) level using calculation software. By comparing with the experimental ECD spectrum, it was found that the calculation results of the isomer (3S, 6R, 7S, 9R, 12S, 14R)-compound VII were highly matched with the experimental results, and both had the same Cottons effect. Therefore, the absolute configuration of compound VII was identified as 3S, 6R, 7S, 9R, 12S, 14R.

[0132] In summary, it can be determined that the structural formula of compound VII isolated in Example 1 is:

[0133]

[0134] The structure of compound VIII isolated 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).

[0135] From the HSQC spectrum combined with the carbon spectrum, it can be obtained that the chemical shift δ assignments of H and the C connected to compound VIII are shown in Table 8:

[0136] Table 8 Compound VIII isolated from Example 1 1 H(600MHz) and 13 C (150 MHz) NMR data, methanol-d4 as solvent

[0137]

[0138] like Fig.24 As shown, HR-ESI-MS m / z of compound VIII: [M+H] + The quasi-molecular ion peak is 487.2807 (C 27 H 39 N2O6[M+H] + , calc m / z: 487.2803) indicates that its molecular formula is C 27 H 38 N2O6, contains 10 unsaturated degrees. According to HR-ESI-MS, 1 H-NMR and 13 Comparison of C-NMR and compound VII shows that compound VIII has a methyl group attached to the C-4 position. 1 H-NMR and HMBC (H3-27 to C-3 / C-4 / C-5) correlation can confirm. In order to determine its relative configuration, combined with NOESY spectrum and related literature analysis, the relative configuration of compound VIII was identified as 3S*, 6R*, 7S*, 9R*, 12S*, 14R*. In order to determine the absolute stereo configuration of compound VIII, two possible stereoisomers [(3S, 6R, 7S, 9R, 12S, 14R) and (3R, 6S, 7R, 9S, 12R, 14S)] were searched for conformation, and the conformations with a distribution ratio greater than 1% were calculated using TD-DFT theory at the B3LYP / 6-31G (d) level using calculation software. By comparing with the experimental ECD spectrum, it was found that the calculation results of the isomer (3S, 6R, 7S, 9R, 12S, 14R)-compound VIII were highly matched with the experimental results, and both had the same Cottons effect. Therefore, the absolute configuration of compound VIII was identified as 3S, 6R, 7S, 9R, 12S, 14R.

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

[0140]

[0141] The structure of compound IX isolated 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] From the HSQC spectrum combined with the carbon spectrum, it can be obtained that the chemical shift δ assignments of H and the C connected to compound IX are shown in Table 9:

[0143] Table 9 Compound IX isolated from Example 1 1 H(600MHz) and13 C (150 MHz) NMR data, methanol-d4 as solvent

[0144]

[0145] like Fig. 27 As shown, HR-ESI-MS m / z of compound IX: [M+H] + The quasi-molecular ion peak is 560.3215 (C 31 H 46 NO8[M+H] + , calc m / z: 560.3218) indicates that its molecular formula is C 31 H 45 NO8, contains 10 unsaturated degrees. According to HR-ESI-MS, 1 H-NMR and 13 Comparison of C-NMR with compound I shows that compound IX has different substituent groups at C-7, and there may be propylene glycol fragments and acetate fragments. 1 H- 1 H COSY correlation (H a / b -24 / H-28 / H a / b -29) and HMBC related (H a / b The presence of glycerol ethyl ester fragments can be confirmed by HMBC correlation (H-7 to C-24), and glycerol ethyl ester is connected to C-7 through an ether bond. In order to determine its relative configuration, combined with NOESY spectrum and related literature analysis, the relative configuration of compound IX was identified as 6R*, 7S*, 12S*, 14R*, 28R*. In order to determine the absolute stereo configuration of compound IX, a conformation search was performed for its two possible stereoisomers [(6R, 7S, 12S, 14R, 28R) and (6S, 7R, 12R, 14S, 28S)], and the ECD calculation was performed using TD-DFT theory at the B3LYP / 6-31G(d) level using calculation software for the conformations with a distribution ratio greater than 1%. By comparing with the experimental ECD spectrum, it was found that the calculation results of the isomer (6R, 7S, 12S, 14R, 28R)-compound IX were highly matched with the experimental results, and both had the same Cottons effect. Therefore, the absolute configuration of compound IX was identified as 6R, 7S, 12S, 14R, 28R.

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

[0147]

[0148] The structure of the compound X separated 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] From the HSQC spectrum combined with the carbon spectrum, it can be obtained that the chemical shift δ assignments of H and the C connected to compound X are shown in Table 10:

[0150] Table 10 Compound X isolated from Example 1 1 H(600MHz) and 13 C (150 MHz) NMR data, methanol-d4 as solvent

[0151]

[0152] like Fig.30 As shown, HR-ESI-MS m / z of compound X: [M+H] + The quasi-molecular ion peak is 560.3215 (C 31 H 46 NO8[M+H] + , calc m / z: 560.3218) indicates that its molecular formula is C 31 H 45 NO8, contains 10 unsaturated degrees. According to HR-ESI-MS, 1 H-NMR and 13 Comparison of C-NMR and compound IX shows that compound X lacks a double bond between C-2 and C-3, and may form a six-membered ring or a seven-membered ring connected 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), it can be seen that a new seven-membered ring connected by an ether bond is formed. In order to determine its relative configuration, combined with NOESY spectrum and related literature analysis, the relative configuration of compound Ⅹ was identified as 3S*, 6R*, 7S*, 12S*, 14R*, 28R*. In order to determine the absolute stereo configuration of compound X, two possible stereoisomers [(3S, 6R, 7S, 12S, 14R, 28R) and (3R, 6S, 7R, 12R, 14S, 28S)] were searched for conformation, and the conformations with a distribution ratio greater than 1% were calculated using TD-DFT theory at the B3LYP / 6-31G(d) level using calculation software. By comparing with the experimental ECD spectrum, it was found that the calculation results of the isomer (3S, 6R, 7S, 12S, 14R, 28R)-compound X were highly consistent with the experimental results, and both had the same Cottons effect. Therefore, the absolute configuration of compound X was identified as 3S, 6R, 7S, 12S, 14R, 28R.

[0153] In summary, it can be determined that the structural formula of the compound X isolated in Example 1 is:

[0154]

[0155] Example 4

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

[0157] Principle of MTS method for detecting cell activity: MTS is a new MTT analogue, the full name of which is 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazoliu m (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethylphenoxy)-2-(4-sulfophenyl)-2H-tetrazoliu m), which is a yellow dye. Succinate dehydrogenase in the mitochondria of living cells can metabolize and reduce MTS to generate soluble formazan compounds, and the content of formazan can be measured at 490nm 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 based on the optical density OD value.

[0158] The cells used in this example include 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] Inoculation of cells: Prepare a single cell suspension with culture medium (DMEM or RMPI1640) containing 10% fetal bovine serum, inoculate 3000 to 15000 cells per well into a 96-well plate, with a volume of 100 μL per well. The cells were inoculated and cultured 24 hours in advance.

[0161] Add the solution of the compound to be tested: the compound is dissolved in DMSO, the compound is initially screened at a concentration of 40 μM, the final volume of each well is 200 μL, and 3 replicate wells are set for each treatment.

[0162] Color development: After culturing at 37 degrees Celsius for 48 hours, discard the culture medium in the wells of adherent cells, add 20μL of MTS solution and 100μL of DMEM culture medium to each well; discard 100μL of culture supernatant of suspended cells, add 20μL of MTS solution to each well; set up 3 blank replicate wells (a mixture of 20μL MTS solution and 100μL culture medium), continue incubation for 2-4 hours, allow the reaction to proceed fully, and then measure the light absorbance value.

[0163] Colorimetry: Select 492nm wavelength, use a multifunctional microplate reader (MULTISKAN FC) to read the light absorption value of each well, record the results, and after data processing, draw a cell inhibition rate graph with the compound number as the horizontal axis and the cell inhibition rate as the vertical axis.

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

[0165] Table 11 Preliminary screening results of cell inhibition rate (%) of compounds I-X at 40 μM concentration

[0166]

[0167] At a concentration of 40 μM, compounds I-X showed good inhibitory activity against the in vitro tumor growth of leukemia HL-60, lung cancer A549, liver cancer HepG2, breast cancer MDA-MB-231 or colon cancer SW480. Next, we will continue to screen for tumor cytotoxic activity compounds (IC 50 detection).

[0168] The same method as the initial screening was used to rescreen the compounds at concentrations of 40 μM, 8 μM, 1.6 μM, 0.32 μM, and 0.064 μM, respectively, with doxorubicin and paclitaxel as positive controls. The rescreening results are shown in Table 12.

[0169] Table 12 Half-maximal inhibitory concentration values ​​(μM) of compounds Ⅰ-Ⅹ on 5 tumor cell lines

[0170]

[0171] Note: “—” indicates no screening, doxorubicin and paclitaxel are used as positive controls

[0172] The half-inhibitory concentrations of compounds I-X on in vitro tumor growth of 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 The values ​​ranged from 1.937 ± 0.093 to 28.11 ± 0.76 μM.

Claims

1. A novel ansamycin compound, characterized in that: There are 10 novel ansamycin compounds, and their structures are shown in Formula I to Formula X:

2. Use of the novel ansamycin compound according to claim 1 in the preparation of drugs for treating leukemia, lung cancer, liver cancer, breast cancer or colon cancer.

3. The method for preparing the novel ansamycin compound according to claim 1, characterized in that: The bacterial liquid obtained by activation culture, seed culture and fermentation culture of Amycolatopsis spp. YINM00005 was isolated from the bacterial liquid to obtain 10 new ansamycin compounds. The Amycolatopsis sp. YINM00005 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on July 19, 2023, with the deposit number GDMCC No.63669.

4. The method for preparing the novel ansamycin compound according to claim 3, characterized in that: The components of the culture medium for activation culture are: 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 composition of the culture medium 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; The culture medium for fermentation culture is: sucrose 90-110 g / L, glucose 9-11 g / L, acid hydrolyzed casein 0.1-0.15 g / L, yeast extract 4-6 g / L, 3-(N-morpholine)propanesulfonic acid 18-24 g / L, trace elements 0.8-1.2 mL, K2SO4 0.2-0.3 g / L, MgCl2·6H2O 8-12 g / L, pH=7.

0.

5. The method for preparing the novel ansamycin compound according to claim 3, characterized in that: The specific steps are as follows: (1) Bacterial activation: Amycolatopsis sp. YINM00005 was inoculated into the activation medium for activation and standby use; (2) strain preparation: inoculating the activated bacteria in step (1) into a seed culture medium and culturing on a shaking table to obtain strains; (3) Fermentation process: inoculating the strain prepared in step (2) into the fermentation medium and fermenting it on a shaking platform to obtain a fermentation liquid; (4) After the fermentation is completed, the fermentation broth is extracted with an equal volume of ethyl acetate and concentrated to obtain a crude extract, the crude extract is dissolved with a mixed solution of dichloromethane and methanol and then mixed evenly with silica gel, and a forward silica gel column chromatography gradient elution is performed using dichloromethane and methanol eluents to obtain an eluent; (5) The eluent obtained in step (4) is selected and mixed evenly with silica gel, and reverse silica gel column chromatography is performed with water-methanol eluent for gradient elution, and finally semi-preparative high performance liquid chromatography is used for purification to obtain compounds represented by Formula I to Formula X.

6. The method for preparing the novel ansamycin compound according to claim 5, characterized in that: The volume ratio of dichloromethane to methanol in the dichloromethane-methanol eluent used in the forward silica gel column chromatography gradient elution in step (4) is 100:0, 80:1, 50:1, 30:1, 10:1, and 0:100, respectively. The eluent with a volume ratio of dichloromethane to methanol of 30:1 is selected to perform the operation in step (5).

7. The method for preparing the novel ansamycin compound according to claim 5, characterized in that: The volume ratio of methanol to water used in the reverse silica gel column chromatography gradient elution 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, 100:0, wherein the 75:25 eluent contains the compound of formula I, the 80:20 eluent contains the compound of formula II, the 55:45 eluent contains the compounds of formula III and formula VIII, the 65:35 eluent contains the compounds of formula IV and formula X, the 70:30 eluent contains the compound of formula V, the 50:50 eluent contains the compound of formula VI, and the 60:40 eluent contains the compounds of formula VII and formula IX.

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