Amycolatopsis YINM00005 and application thereof in preparation of ansamycin compounds

Abstract

The invention discloses amycolatopsis sp. YINM00005 and application of the amycolatopsis sp. YINM00005 in preparation of ansamycin compounds, and belongs to the technical field of microorganisms. 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 step 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 the four ansamycin compounds, the total concentration of the four ansamycin compounds in the fermentation liquor can reach 4.5-8.6 mg / L, the purity can reach 98% or above after extraction and separation, and the purity of the ansamycin compounds can reach 98% or above after extraction and separation. The four compounds all have very strong anti-tumor cytotoxic activity. The method is relatively low in cost, simple in process and environment-friendly, can be used for mass production and preparation of the ansamycin compounds, not only meets the requirements of low-carbon economy, but also lays a foundation for later production of the ansamycin compounds and development of related products.

Description

Technical Field

[0001] The invention relates to a strain of Amycolatopsis YINM00005 and application thereof in preparing ansamycin compounds, belonging to the technical field of microorganisms. 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 class of polyketide compounds. This class of antibiotics has low toxicity, high efficacy and a broad antibacterial spectrum. They can specifically act on DNA-dependent RNA polymerases to inhibit bacterial DNA transcription; and they also have an inhibitory effect on RNA-dependent DNA reverse transcriptases of tumor RNA viruses. They are widely used in the treatment of G+ (Gram-positive bacteria), tuberculosis, leprosy and mycobacterial infections associated with AIDS. In summary, ansamycin compounds have good research and development potential as drug lead compounds.

[0004] At present, ansamycin compounds are mainly obtained through the separation and purification of microbial fermentation metabolites, and the strains that can metabolize and produce ansamycin compounds are mostly plant or soil-derived actinomycetes. The yield of ansamycin compounds metabolized by them is low, and the low yield restricts the development and utilization of ansamycin compounds.

[0005] Amycolatopsis Amycolatopsis sp .) YINM00005 is an endophytic actinomycete obtained from the Herba Lycopodii in Kunming, Yunnan. Analysis of its secondary metabolites revealed that the yield of novel ansamycin compounds was large and easy to separate and purify. Therefore, this strain has the potential to be developed as an engineered bacterium to produce novel ansamycin compounds. Summary of the invention

[0006] One of the objects of the present invention is to provide a strain of Amycolatopsis spp. YINM00005, which has been deposited in Guangdong Microbial Strain Collection Center (GDMCC for short) on July 19, 2023. The deposit address is: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, with a deposit number of GDMCC No. 63669 and a taxonomic name of: Amycolatopsis sp. .

[0007] The second object of the present invention is to provide an application of Amycolatopsis YINM00005 in the preparation of novel ansamycin compounds.

[0008] There are four novel ansamycin compounds prepared by the present invention, wherein the structure of the first compound is shown in Formula I;

[0009] Formula Ⅰ.

[0010] The structure of the second compound is shown in Formula II;

[0011] Formula II.

[0012] The structure of the third compound is shown in Formula III;

[0013] Formula III.

[0014] The structure of the fourth compound is shown in Formula IV;

[0015] Formula IV.

[0016] The third object of the present invention is to provide a method for preparing novel ansamycin compounds by using Amycolatopsis spp. YINM00005, which mainly comprises the following steps:

[0017] (1) Bacterial activation: Inoculate Amycolatopsis spp. YINM00005 into the strain activation medium for activation and set aside.

[0018] (2) Preparation of bacterial strains: The activated bacterial cells in step (1) are inoculated into a seed culture medium for cultivation to obtain bacterial strains.

[0019] (3) Fermentation process: The strain prepared in step (2) is inoculated into a fermentation medium for fermentation to obtain a fermentation broth.

[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 dichloromethane-methanol and mixed evenly with silica gel. The dichloromethane-methanol solution is used for forward silica gel column chromatography gradient elution to obtain an eluate.

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

[0022] Preferably, the components of the activation medium in step (1) are: 4.0 g / L yeast extract, 4.0 g / L glucose, 10.0 g / L malt extract, 15.0 g / L agar, pH 7.0, the culture temperature is 28° C., and the culture time is 3 days.

[0023] Preferably, the composition of the seed culture medium in step (2) is: 4.0 g / L yeast extract, 4.0 g / L glucose, 10.0 g / L malt extract, pH 7.0; the culture temperature is 28° C., and the culture time is 3 days.

[0024] Preferably, the composition of the fermentation medium in step (3) is: 50 g / L glycerol, 25 g / L corn flour, 5 g / L yeast powder, pH 7.0; the inoculation amount of the strain is 5-10% of the volume of the fermentation medium; the culture temperature is 28°C, and the culture time is 10-15 days.

[0025] Preferably, in step (4), ethyl acetate extraction is performed three times; in the dichloromethane-methanol solution used to dissolve the crude extract, the volume ratio of dichloromethane to methane is 1:1; and the silica gel used is 200-300 mesh silica gel.

[0026] Preferably, in the dichloromethane-methanol eluent in the forward silica gel column chromatography gradient elution in step (4), the volume ratio of dichloromethane to methanol is 100:0, 50:1, 30:1, 10:1, 5:1, 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).

[0027] Preferably, the particle size of the silica gel used in step (5) is 40-60 μm.

[0028] Preferably, in the reverse silica gel column chromatography gradient elution in step (5), the volume ratio of water to methanol in the water-methanol eluate is 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100, respectively, wherein the water-methanol eluate with a volume ratio of 60:40 contains the compound of formula I; the water-methanol eluate with a volume ratio of 50:50 contains the compound of formula II; the water-methanol eluate with a volume ratio of 40:60 contains the compound of formula III; and the water-methanol eluate with a volume ratio of 30:70 contains the compound of formula IV.

[0029] The fourth object of the present invention is to provide a novel ansamycin compound for use in the preparation of drugs for treating leukemia, lung cancer, liver cancer, breast cancer or colon cancer.

[0030] Beneficial effects of the present invention

[0031] (1) The present invention provides a strain of Amycolatopsis that can produce a large amount of ansamycins ( Amycolatopsis sp.) YINM00005, which can produce 4 new ansamycins with high yield.

[0032] (2) This invention synthesized four new ansamycins for the first time, enriching the types of ansamycins.

[0033] (3) The raw materials used in the method of the present invention are cheap and easily 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 the large-scale production of ansamycin compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The compound amycolansamycin A provided by the present invention 1 H-NMR spectrum.

[0035] Figure 2 The compound amycolansamycin A provided by the present invention 13 C-NMR spectrum.

[0036] Figure 3 The compound amycolansamycin A provided by the present invention 1 H- 1 H COSY spectrum.

[0037] Figure 4 The HMBC spectrum of the compound amycolansamycin A provided by the present invention.

[0038] Figure 5 The HSQC spectrum of the compound amycolansamycin A provided by the present invention.

[0039] Figure 6 The ROESY spectrum of the compound amycolansamycin A provided by the present invention.

[0040] Figure 7 The present invention provides a HR-ESI-MS spectrum of the compound amycolansamycin A.

[0041] Figure 8 The compound amycolansamycin B provided by the present invention 1 H-NMR spectrum.

[0042] Fig. 9 The compound amycolansamycin B provided by the present invention 13 C-NMR spectrum.

[0043] Fig.10 The compound amycolansamycin B provided by the present invention 1 H- 1 H COSY spectrum.

[0044] Fig.11 The HMBC spectrum of the compound amycolansamycin B provided by the present invention.

[0045] Fig.12 The HSQC spectrum of the compound amycolansamycin B provided by the present invention.

[0046] Fig.13 The ROESY spectrum of the compound amycolansamycin B provided by the present invention.

[0047] Fig.14 The present invention provides a HR-ESI-MS spectrum of the compound amycolansamycin B.

[0048] Fig.15 The compound amycolansamycin C provided by the present invention 1 H-NMR spectrum.

[0049] Fig.16 The compound amycolansamycin C provided by the present invention 13 C-NMR spectrum.

[0050] Fig.17 The compound amycolansamycin C provided by the present invention 1 H- 1 H COSY spectrum.

[0051] Fig.18 This is the HMBC spectrum of the compound amycolansamycin C provided by the present invention.

[0052] Fig.19 The HSQC spectrum of the compound amycolansamycin C provided by the present invention.

[0053] Fig. 20 The present invention provides a ROESY spectrum of the compound amycolansamycin C.

[0054] Fig.21 The present invention provides a HR-ESI-MS spectrum of the compound amycolansamycin C.

[0055] Fig. 22 The compound amycolansamycin D provided by the present invention 1 H-NMR spectrum.

[0056] Fig.23The compound amycolansamycin D provided by the present invention 13 C-NMR spectrum.

[0057] Fig.24 The compound amycolansamycin D provided by the present invention 1 H- 1 H COSY spectrum.

[0058] Fig.25 The HMBC spectrum of the compound amycolansamycin D provided by the present invention.

[0059] Fig.26 The HSQC spectrum of the compound amycolansamycin D provided by the present invention.

[0060] Fig. 27 The ROESY spectrum of the compound amycolansamycin D provided by the present invention.

[0061] Fig.28 The present invention provides a HR-ESI-MS spectrum of the compound amycolansamycin D. DETAILED DESCRIPTION

[0062] 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 is subject to the claims, and technical operations not specifically described are operations well known to those skilled in the art.

[0063] The surface of the scutellaria baicalensis collected from Kunming, Yunnan was sterilized by sodium hypochlorite solution (effective chlorine concentration 5.0%), 75% ethanol immersion and sterile water washing. Then the plant tissue was processed into small pieces and implanted into an activation medium sterilized at 121°C. After culturing at 28°C for 7 days, the endophytic bacteria were continuously purified using the activation medium sterilized at 121°C. Amycolatopsis sp. YINM00005, the deposit number is GDMCC No.63669.

[0064] The Amycoticola sp. YINM00005 of the present invention is a rare actinomycete, and its surface is smooth on MS medium, with a small amount of gray spores and a large amount of white mycelium. The 16s RNA sequence of Amycoticola sp. YINM00005 is shown in SEQ ID No. 1:

[0065]

[0066] The formula of the culture medium used in the examples is as follows, and all culture media need to be sterilized at 121°C for 20 min before use.

[0067] (1) The composition of the activation medium is: yeast extract 4.0 g / L, glucose 4.0 g / L, malt extract 10.0 g / L, agar 15.0 g / L, pH 7.0;

[0068] (2) The composition of the seed culture medium is: yeast extract 4.0 g / L, glucose 4.0 g / L, malt extract 10.0 g / L, pH 7.0;

[0069] (3) The composition of the fermentation medium is: 50 g / L glycerol, 25 g / L corn flour, 5 g / L yeast powder, pH 7.0.

[0070] Example 1

[0071] The novel ansamycin compounds are prepared by using Amycolatopsis YINM00005, which specifically comprises the following steps:

[0072] (1) Bacterial activation: Inoculate Amycolatopsis spp. YINM00005 into the activation medium, culture it in a constant temperature incubator at 28°C for 3 days, and then place it in a refrigerator at 4°C for later use.

[0073] (2) Preparation of bacterial strains: The activated bacterial cells were inoculated into the seed culture medium and fermented at 28°C and 180 rpm for 3 days.

[0074] (3) Fermentation process: The strain prepared in step (2) was added into 20 L fermentation medium (50 g / L glycerol, 25 g / L corn flour, 5 g / L yeast powder, pH 7.0) at a volume fraction of 5%, and fermented at 28°C and 180 rpm for 15 days to obtain a fermentation liquid.

[0075] (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 (32.90 g). The crude extract was detected by high performance liquid chromatography, and the concentrated crude extract was dissolved in an appropriate amount of a dichloromethane-methanol mixed solution 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. The dichloromethane-methanol solution with a volume ratio of 100:0, 50:1, 30:1, 10:1, 5:1, and 0:100 was used in sequence for normal silica gel (200-300 mesh) column chromatography gradient elution to obtain 6 eluates respectively.

[0076] (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. Use a dropper to slowly add the eluent to the prepared absorbent cotton and leave it at room temperature until the solvent is completely evaporated. After loading, use water-methanol solutions with volume ratios of 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100 in sequence for reverse silica gel column (40-60 μm) chromatography gradient elution to obtain 10 portions of eluent.

[0077] According to the TLC spot plate and HPLC analysis results, an eluent with a water-methanol volume ratio of 60:40 was selected for semi-preparative HPLC purification to obtain compound A (172.0 mg). The purity of compound A reached more than 98% according to HPLC analysis.

[0078] According to the TLC spot plate and HPLC analysis results, an eluent with a water-methanol volume ratio of 50:50 was selected, and semi-preparative high performance liquid chromatography (Semi-preparative HPLC) was performed to purify compound B (132.0 mg); HPLC analysis showed that the purity of compound B reached more than 98%.

[0079] According to the TLC spot plate and HPLC analysis results, an eluent with a water-methanol volume ratio of 40:60 was selected, and semi-preparative high performance liquid chromatography (Semi-preparative HPLC) was performed to purify compound C (116.0 mg); HPLC analysis showed that the purity of compound C reached more than 98%.

[0080] According to the TLC spot plate and HPLC analysis results, an eluent with a water-methanol volume ratio of 30:70 was selected, and semi-preparative high performance liquid chromatography (Semi-preparative HPLC) was performed to purify the compound D (90.0 mg); the purity of the compound D was more than 98% according to HPLC analysis.

[0081] Finally, after high performance liquid chromatography analysis, the concentration of compound A in the fermentation broth was 8.6 mg / L; the concentration of compound B was 6.6 mg / L; the concentration of compound C was 5.8 mg / L; and the concentration of compound D was 4.5 mg / L.

[0082] Example 2

[0083] The novel ansamycin compounds are prepared by using Amycolatopsis YINM00005, which specifically comprises the following steps:

[0084] (1) Bacterial activation: Inoculate Amycolatopsis spp. YINM00005 into the activation medium, culture it in a constant temperature incubator at 28°C for 3 days, and then place it in a refrigerator at 4°C for later use.

[0085] (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.

[0086] (3) Fermentation process: The strain prepared in step (2) was added into 20 L fermentation medium (50 g / L glycerol, 25 g / L corn flour, 5 g / L yeast powder, pH 7.0) at a volume fraction of 10%, and fermented at 28°C and 200 rpm for 10 days to obtain a fermentation liquid.

[0087] (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 (18.90 g). The crude extract was detected by high performance liquid chromatography, and the concentrated crude extract was dissolved in an appropriate amount of a dichloromethane-methanol mixed solution 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 mixture was slowly stirred until the solvent was completely evaporated. The dichloromethane-methanol solution with a volume ratio of 100:0, 50:1, 30:1, 10:1, 5:1, and 0:100 was used in sequence for gradient elution on a silica gel (200-300 mesh) column to obtain 6 eluates respectively.

[0088] (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. Use a dropper to slowly add the eluent to the prepared absorbent cotton and leave it at room temperature until the solvent is completely evaporated. After loading, use water-methanol solutions with volume ratios of 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100 in sequence for reverse silica gel column (40-60 μm) chromatography gradient elution to obtain 10 portions of eluent.

[0089] According to the results of TLC spot plate and HPLC analysis, an eluent with a water-methanol volume ratio of 60:40 was selected for semi-preparative HPLC purification to obtain the metabolite ansamycin compound A (148.0 mg). The purity of compound A reached more than 98% by HPLC analysis.

[0090] According to the TLC spot plate and HPLC analysis results, an eluent with a water-methanol volume ratio of 50:50 was selected, and semi-preparative high performance liquid chromatography (Semi-preparative HPLC) was performed to purify compound B (104.0 mg); HPLC analysis showed that the purity of compound B reached more than 98%.

[0091] According to the TLC spot plate and HPLC analysis results, an eluent with a water-methanol volume ratio of 40:60 was selected, and semi-preparative high performance liquid chromatography (Semi-preparative HPLC) was performed to purify compound C (92.0 mg); HPLC analysis showed that the purity of compound C reached more than 98%.

[0092] According to the TLC spot plate and HPLC analysis results, an eluent with a water-methanol volume ratio of 30:70 was selected, and semi-preparative high performance liquid chromatography (Semi-preparative HPLC) was performed to purify the compound D (96.0 mg); the purity of the compound D was more than 98% according to HPLC analysis.

[0093] Finally, after high performance liquid chromatography analysis, the concentration of compound A in the fermentation broth was 7.4 mg / L; the concentration of compound B was 5.2 mg / L; the concentration of compound C was 4.6 mg / L; and the concentration of compound D was 4.8 mg / L.

[0094] Example 3

[0095] Identification of the structures of the obtained novel ansamycin compounds

[0096] Compound A isolated in Example 1 was analyzed 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) ( Figure 1-7 ) to identify its structure.

[0097] From the HSQC spectrum combined with the carbon spectrum, it can be obtained that the chemical shift δ assignments of H of compound A and the C connected thereto are shown in Table 1:

[0098] Table 1 is the separation of compound A obtained in Example 1 1 H (500MHz) and 13 C (125 MHz) NMR data, methanol-d4 was the solvent.

[0099] Table 1 Compound A isolated from Example 1 1 H (500MHz) and 13 C (125 MHz) NMR data

[0100]

[0101] like Figure 7 As shown, HR-ESI-MS m / z of compound A: [M+H] + The quasi-molecular ion peak is 487.2802 (C 27 H 39 N2O6[M+H] + , calc m / z: 487.2803) indicates that its molecular formula is C 27 H 38 N2O6, contains 9 degrees of unsaturation. 1 H-NMR( Figure 1 ), 13 C-NMR( Figure 2 ) and HSQC ( Figure 5 ) Spectral analysis shows that compound A contains 4 methyl groups [δ H 0.68 (H3-22), δ H 0.92 (H3-23), δ H 1.23 (H3-25), δ H 0.81 (H3-27) corresponds to δ C 17.2 (C-22), δ C 21.4 (C-23), δ C 17.5 (C-25), δ C 14.1 (C-27)], 6 methylene groups [δ H 2.05(H a -8), 1.25 (H b -8), δ H 2.03(H a -10), 1.41 (H b -10), δ H 1.58(H a -11), 0.88 (H b -11), δ H 1.31(H a -13), 1.14 (H b -13), δ H 2.89(H a -15), 1.80 (H b -15), δ H 2.26(H a -26), 1.96 (H b -26) respectively correspond to δ C 41.7 (C-8), δ C 38.1 (C-10), δ C 29.2 (C-11), δC 47.9 (C-13), δ C 42.3 (C-15), δ C 21.6 (C-26)], 9 methine groups [δ H 5.86 (H-2), δ H 7.06 (H-3), δ H 5.47 (H-5), δ H 2.72 (H-6), δ H 4.11 (H-7), δ H 1.49 (H-12), δ H 1.89 (H-14), δ H 6.49 (H-19), δ H 6.53 (H-21) corresponds to δ C 119.2 (C-2), δ C 146.0 (C-3), δ C 140.1 (C-5), δ C 40.0 (C-6), δ C 78.7 (C-7), δ C 32.0 (C-12), δ C 30.7 (C-14), δ C 113.3 (C-19), δ C 119.7 (C-21)] and 8 quaternary carbons [δ C 171.9 (C-1), δ C 142.6 (C-4), δ C 81.6 (C-9), δ C 126.4 (C-16), δ C 132.9 (C-17), δ C 146.3 (C-18), δ C 150.9 (C-20), δ C 156.0 (C-24)].

[0102] analyze 1 H- 1 H COSY( Figure 3 ) 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 ( Figure 4 ) It can be seen that H3-22 has obvious correlation signals with C-13 / C-14 / C-15, H3-23 has obvious correlation signals with 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 have three methyl groups connected to the carbon chain. 1 H-NMR shows that δ H 6.49 and δ H 6.53 are doublets. 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 156.0 (C-24) and δ C 171.9 (C-1) may be the resonance of amide carbon. The presence of amide carbon can be proved by HMBC (-NH- to C-24, -NH- to C-1). The HMBC (-NH- to C-17 / C-18 / C-19) correlation proves that the compound type is macrolide. The relevant literature search shows that the molecule is an unreported new ansamycin compound, named amycolansamycin A.

[0103] In order to determine its relative configuration, combined with NOESY spectrum and related literature analysis, the relative configuration of amycolansamycin A was identified as 6R*, 7S*, 9R*, 12R*, 14S*. In order to determine the absolute stereo configuration of amycolansamycin A, conformational searches were performed on its two possible stereoisomers [(6R, 7S, 9R, 12R, 14S) and (6S, 7R, 9S, 12S, 14R)], and the ECD calculations were performed on the conformations with a distribution ratio greater than 1% using TD-DFT theory at the B3LYP / 6-31G(d) level using calculation software. By comparing with the experimental ECD spectra, it was found that the calculated results of the isomer (6S, 7S, 9R, 12R, 14S)-amycolansamycin A were highly consistent with the experimental results, and both had the same Cottons effect. Therefore, the absolute configuration of compound amycolansamycin A was identified as 6R, 7S, 9R, 12R, 14S.

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

[0105]

[0106] Compound B isolated in Example 1 was analyzed 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) ( Figure 8-14 ) to identify its structure.

[0107] From the HSQC spectrum combined with the carbon spectrum, it can be obtained that the chemical shift δ assignments of H of compound B and the C connected to it are shown in Table 2:

[0108] Table 2 Compound B isolated from Example 1 1 H (500MHz) and 13 C (125 MHz) NMR data, methanol-d4 was the solvent.

[0109] Table 2 Compound B isolated from Example 1 1 H (500MHz) and 13 C (125 MHz) NMR data

[0110]

[0111] like Fig.14 As shown, HR-ESI-MS m / z of compound B: [M+H] + The quasi-molecular ion peak is 501.2964 (C 28 H 41N2O6[M+H] + , calc m / z: 501.2959) indicates that its molecular formula is C 28 H 40 N2O6, contains 9 unsaturated degrees. According to HR-ESI-MS, 1 H-NMR and 13 Comparison of C-NMR and amycolansamycin A shows that compound B has an extra methoxy group. 1 H-NMR and HMBC (H3-28 to C-9) correlation showed that the hydroxyl group at the C-9 position was replaced by -OCH3, and compound B was named amycolansamycin B. In order to determine its relative configuration, combined with NOESY spectrum and relevant literature analysis, the relative configuration of amycolansamycin B was identified as 6R*, 7S*, 9R*, 12R*, 14S*. In order to determine the absolute stereo configuration of amycolansamycin B, a conformation search was performed for its two possible stereoisomers [(6R, 7S, 9R, 12R, 14S) and (6S, 7R, 9S, 12S, 14R)], and the ECD calculations were 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 spectra, it was found that the calculated results of the isomer (6R, 7S, 9R, 12R, 14S)-amycolansamycin B were highly consistent with the experimental results, and both had the same Cottons effect. Therefore, the absolute configuration of the compound amycolansamycin B was identified as 6R, 7S, 9R, 12R, 14S.

[0112] In summary, it can be determined that the structural formula of the compound isolated in Example 1 is:

[0113]

[0114] Compound C isolated in Example 1 was analyzed 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) ( Figure 15-21 ) to identify its structure.

[0115] From the HSQC spectrum combined with the carbon spectrum, it can be obtained that the chemical shift δ assignments of H of compound C and the C connected to it are shown in Table 3:

[0116] Table 3 Compound C isolated from Example 1 1 H (600MHz) and 13 C (150 MHz) NMR data, methanol-d4 was the solvent.

[0117] Table 3 Compound C isolated from Example 1 1 H (600MHz) and 13 C (150 MHz) NMR data

[0118]

[0119] like Fig.21 As shown, HR-ESI-MS m / z of compound C: [M+H] + The quasi-molecular ion peak is 487.2804 (C 27 H 39 N2O6[M+H] + , calc m / z: 487.2803) indicates that its molecular formula is C 27 H 38 N2O6, contains 9 unsaturated degrees. According to HR-ESI-MS, 1 H-NMR and 13 Comparison of C-NMR and amycolansamycin B shows that the -CH2CH3 at the C-4 position of compound C is replaced by a methyl group. 1 H-NMR and HMBC (H3-26 to C-3 / C-4 / C-5, -OCH3-9 to C-9) correlation showed that the -CH2CH3 connected to the C-4 position was replaced by a -CH3, and compound C was named amycolansamycin C. In order to determine its relative configuration, combined with NOESY spectrum and related literature analysis, the relative configuration of amycolansamycin C was identified as 6R*, 7S*, 9R*, 12R*, 14S*. In order to determine the absolute stereo configuration of amycolansamycin C, a conformation search was performed for its two possible stereoisomers [(6R, 7S, 9R, 12R, 14S) and (6S, 7R, 9S, 12S, 14R)], and the ECD calculations were 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 spectra, it was found that the calculated results of the isomer (6R, 7S, 9R, 12R, 14S)-amycolansamycin C were highly consistent with the experimental results, and both had the same Cottons effect. Therefore, the absolute configuration of the compound amycolansamycin C was identified as 6R, 7S, 9R, 12R, 14S.

[0120] In summary, it can be determined that the structural formula of the compound isolated in Example 1 is:

[0121]

[0122] Compound D isolated in Example 1 was analyzed 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) ( Figure 22-28 ) to identify its structure.

[0123] From the HSQC spectrum combined with the carbon spectrum, it can be obtained that the chemical shift δ assignments of the H and the C connected to the compound amycolansamycin D are shown in Table 4:

[0124] Table 4 Compound D isolated from Example 1 1 H (600MHz) and 13 C (150 MHz) NMR data, methanol-d4 was the solvent.

[0125] Table 4 Compound D isolated from Example 1 1 H (600MHz) and 13 C (150 MHz) NMR data

[0126]

[0127] like Fig.28 As shown, HR-ESI-MS m / z of compound D: [M+H] + The quasi-molecular ion peak is 426.2637 (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 amycolansamycin A shows that amycolansamycin D lacks a carbamate fragment at C-7 / C-8 / C-9. C 205.0) becomes a carbonyl carbon and has an additional double bond between C-7 / C-8. 1 H-NMR and HMBC (H-7 / H-8 / H a / b-10 to C-9) can prove the above inference, and compound D is named amycolansamycin D. In order to determine its relative configuration, combined with NOESY spectrum and related literature analysis, the relative configuration of amycolansamycin D was identified as 6R*, 12R*, 14S*. In order to determine the absolute stereo configuration of amycolansamycin D, its two possible stereoisomers [(6R, 12R, 14S) and (6S, 12S, 14R)] 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 calculated results of the isomer (6R, 12R, 14S)-amycolansamycin D were highly consistent with the experimental results, and both had the same Cottons effect. Therefore, the absolute configuration of amycolansamycin D was identified as 6R, 12R, 14S.

[0128] In summary, it can be determined that the structural formula of the compound isolated in Example 1 is:

[0129]

[0130] Example 4

[0131] Example 1 Screening of anti-tumor cytotoxic activity of Amycolansamycins AD prepared

[0132] Principle of MTS method for detecting cell activity: MTS is a new MTT analogue, the 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-carboxymethylphenoxy)-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 soluble formazan compounds. 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.

[0133] The cells used in this example are leukemia HL-60 cells, lung cancer A549 cells, liver cancer SMMC-7721 cells, breast cancer MDA-MB-231 cells and colon cancer SW480 cells.

[0134] The experimental method is as follows:

[0135] Inoculation of cells: Prepare a single cell suspension with culture medium (DMEM or RMPI1640) containing 10% fetal bovine serum, inoculate 3000-15000 cells per well into a 96-well plate, with a volume of 100 μL per well. Cells should be inoculated and cultured 24 hours in advance.

[0136] 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.

[0137] 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 absorbance value.

[0138] 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.

[0139] Positive control compounds: Two positive compounds, cisplatin (DDP) and paclitaxel (Taxol), 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 5.

[0140] Table 5 Preliminary screening results of cell inhibition rate (%) of compound amycolansamycins AD at 40 μM concentration

[0141]

[0142] At a concentration of 40 μM, the compound amycolansamycins AD has good inhibitory activity on the in vitro tumor growth of leukemia HL-60, lung cancer A549, liver cancer SMMC-7721, breast cancer MDA-MB-231 and colon cancer SW480. Next, we will continue to screen for tumor cytotoxic activity (IC 50 detection).

[0143] 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 cisplatin as the positive control. The rescreening results are shown in Table 6.

[0144] Table 6 Half inhibitory concentration values ​​(μM) of compound Amycolansamycins AD on 5 tumor cell lines

[0145]

[0146] The half-maximal inhibitory concentrations of amycolansamycins AD on in vitro tumor growth of leukemia HL-60, lung cancer A549, liver cancer SMMC-7721, breast cancer MDA-MB-231, and colon cancer SW480 are shown in Table 2. IC 50 The values ​​ranged from 0.149 ± 0.017 to 25.11 ± 0.68 μM.

Claims

1. A strain of Amycolatopsis Amycolatopsis sp. )YINM00005, the deposit number is GDMCC No.63669.

2. Use of the Amycolatopsis spp. YINM00005 according to claim 1 in the preparation of ansamycin compounds.

3. The use according to claim 2, characterized in that: The prepared ansamycin compounds are as follows: Formula I; Formula II; Formula III; Formula IV.

4. The use according to claim 3, characterized in that: The specific preparation steps of the ansamycin compounds are as follows: (1) Bacterial activation: inoculate Amycolatopsis spp. YINM00005 into the activation medium for activation and standby use; (2) Preparation of bacterial strains: inoculating the activated bacterial cells in step (1) into a seed culture medium and culturing on a shaking table to obtain bacterial strains; (3) Fermentation process: inoculating the strain prepared in step (2) into the fermentation medium and fermenting it on a shaking table 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 dichloromethane-methanol and then mixed evenly with silica gel. The dichloromethane-methanol eluent is used for forward silica gel column chromatography gradient elution 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 of Formula I to Formula IV.

5. The application according to claim 4, characterized in that: The volume ratios of dichloromethane to methanol in the dichloromethane-methanol eluent used in the forward silica gel column chromatography gradient elution in step (4) are 100:0, 50:1, 30:1, 10:1, 5:1, 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).

6. The application according to claim 4, characterized in that: The volume ratio of water to methanol in the water-methanol eluate used in the reverse silica gel column chromatography gradient elution in step (5) is 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100, respectively, wherein the water-methanol eluate with a volume ratio of 60:40 contains the compound of formula I; the water-methanol eluate with a volume ratio of 50:50 contains the compound of formula II; the water-methanol eluate with a volume ratio of 40:60 contains the compound of formula III; and the water-methanol eluate with a volume ratio of 30:70 contains the compound of formula IV.

7. Use of the ansamycin compounds obtained by the use according to claim 3 in the preparation of drugs for treating leukemia, lung cancer, liver cancer, breast cancer or colon cancer.

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