A staurosporine compound and its preparation method and use

Abstract

The invention discloses a staurosporine compound and a preparation method and use thereof. The compound is characterized in that the structural formula is shown in I. The preparation method comprises the steps of obtaining a fermentation product of the staurosporine compound by fermenting and culturing an actinomycete with a preservation number of CCTCC No: M2020953, then soaking the fermentation product in ethyl acetate to extract a crude extract, and then subjecting the crude extract to normal phase silica gel column chromatography, reverse medium pressure column chromatography and reverse phase semi-preparative high performance liquid chromatography separation and purification. The compound is used in the preparation of a drug-resistant breast cancer cell line inhibitor, and has the advantage that the staurosporine has activity against drug-resistant breast cancer cell lines and can be used to develop drugs against drug-resistant breast cancer.

Description

Technical Field

[0001] The present invention relates to staurosporine compounds, in particular to a staurosporine compound extracted from marine actinomycetes and a preparation method and application thereof. Background Art

[0002] Staurosporine antibiotics are a class of natural products consisting of six-membered chain amine compounds with indolecarbazole as the core. It was first isolated from soil Streptomyces by Satoshi Omura and his colleagues in 1977. Consistent with its high structural specificity, staurosporine has a wide spectrum of biological activities, including anti-tumor, antiviral, insecticidal, antibacterial and anti-cancer. Staurosporine compounds have attracted the attention of chemical or biosynthetic researchers due to their significant activity and diverse structures.

[0003] The inventors studied marine actinomycetes Steptomyces In the chemical investigation of the ethyl acetate extract of sp. 4-7 fermented in medium A, a new natural product of the staurosporine type was discovered. So far, the chemical structure of the compound and its activity against drug-resistant breast cancer cell lines have not been reported, so there are no related drugs on the market. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a new staurosporine compound having an inhibitory effect on drug-resistant breast cancer cell lines, and a preparation method and use thereof.

[0005] The technical solution adopted by the present invention to solve the above technical problems is:

[0006] 1. A staurosporine compound, the structural formula of which is shown in (I):

[0007] (I).

[0008] 2. A method for preparing a staurosporine compound, comprising the following steps:

[0009] (1) Fermentation production

[0010] The marine actinomycetes with the deposit number of CCTCC NO: M2020953 ( Steptomyces sp. 4-7) streaked on a plate of Gao's solid medium No. 1, cultured and activated in an incubator at 28°C for 7 days, picked a single colony and inoculated it into Gao's liquid medium No. 1, cultured and increased the bacteria on a shaker at 28°C and 180 rpm / min, collected the seed liquid after 3 days of culture, and then inoculated the seed liquid into medium A at a volume ratio of 10%, cultured at 28°C for 11 days, and obtained the fermentation product;

[0011] (2) Extraction of extract

[0012] Add an equal amount of ethyl acetate to the fermented product obtained in step (1), extract repeatedly for 3 times, and then evaporate the ethyl acetate extract under reduced pressure to dryness to obtain a crude extract;

[0013] (3) Isolation and preparation of compounds

[0014] The crude extract obtained in step (2) was dissolved in a mixed solvent of dichloromethane and methanol in a volume ratio of 1:1, and 200-300 mesh silica gel was added to mix the sample, and normal phase medium pressure column chromatography was performed, using a petroleum ether-ethyl acetate solution in a volume ratio of (100:1) to (0:1) as an eluent for gradient elution, and the eluted fractions were collected and arranged in descending order according to the polarity of the fractions, and 11 components were obtained by merging; the collected 10th component was subjected to reverse phase medium pressure column chromatography gradient elution, using acetonitrile-water in a volume ratio of 30-100% as an eluent, and the eluted fractions were collected and arranged in descending order according to the polarity of the fractions, and 10 components were obtained by merging; the obtained 7th component was separated and purified by semi-preparative reverse phase high performance liquid chromatography, using a solution of acetonitrile and water in a volume ratio of 43:57 as a mobile phase, to obtain a compound, whose structure is shown in (I)

[0015] (I).

[0016] Furthermore, the preparation method of medium A described in step (1) is as follows: weigh 20 g soluble starch, 6 g peptone, 2 g yeast extract, 30 g sea salt, 100 mg KBr, 40 mg Fe2(SO4)3·4H2O and 1 g CaCO3, add them to 1000 mL distilled water, and sterilize them by high pressure at 121°C for 20 min.

[0017] Furthermore, the elution gradient volume ratios of the petroleum ether-ethyl acetate solution in step (3) are 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1 and 0:1, respectively.

[0018] Furthermore, in the reverse phase medium pressure column chromatography gradient elution described in step (3), the volume ratio of acetonitrile is from 30 to 100%, and the elution time is 150 min.

[0019] Furthermore, the flow rate of the semi-preparative reversed-phase HPLC compound separation preparation in step (3) is 2.0 mL / min.

[0020] 3. Use of the above-mentioned staurosporine compound in the preparation of drug-resistant breast cancer cell line inhibitors.

[0021] Compared with the prior art, the advantages of the present invention are: a staurosporine compound and its preparation method and use are obtained by microbial fermentation culture to obtain a fermentation product of staurosporine, and then the fermentation product is soaked and extracted with ethyl acetate to obtain a crude extract, and then the crude extract is separated and purified by medium-pressure forward silica gel column chromatography, medium-pressure reverse column chromatography, and reversed-phase semi-preparative high-performance liquid chromatography. The compound has significant anti-resistant breast cancer cell line MCF / ADR activity and can be used to develop drugs with anti-resistant breast cancer activity. The biological activity evaluation shows that compound 1 has an effective inhibitory effect on drug-resistant breast cancer cell lines, IC 50 The value is 4.47 μ M.

[0022] The above marine actinomycetes ( Steptomyces sp . 4-7), the deposit number is CCTCC NO: M2020953, and it was deposited in the China Center for Type Culture Collection on December 21, 2020. The deposit address is Wuhan University, Wuhan, China. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the hydrogen nuclear magnetic resonance spectrum of the compound of the present invention;

[0024] Figure 2 The carbon NMR spectrum of the compound of the present invention is

[0025] Figure 3 is the nuclear magnetic resonance DEPT-135 spectrum of the compound of the present invention;

[0026] Figure 4 is the COSY nuclear magnetic resonance spectrum of the compound of the present invention;

[0027] Figure 5 is the nuclear magnetic resonance HSQC spectrum of the compound of the present invention;

[0028] Figure 6 is the nuclear magnetic resonance HMBC spectrum of the compound of the present invention;

[0029] Figure 7 is the nuclear magnetic resonance NOESY spectrum of the compound of the present invention;

[0030] Figure 8 is the mass spectrum of the compound of the present invention;

[0031] Fig. 9 is the CD spectrum of the compound of the present invention;

[0032] Fig.10 This is the CD spectrum of the known compound staurosporine M1. DETAILED DESCRIPTION

[0033] The present invention is further described in detail below with reference to the accompanying drawings.

[0034] Example 1

[0035] The structural formula of a staurosporine compound is shown in (I):

[0036] (I).

[0037] Example 2

[0038] A method for preparing a staurosporine compound comprises the following steps:

[0039] (1) Fermentation production

[0040] The marine actinomycetes with the deposit number of CCTCC NO: M2020953 ( Steptomyces sp . 4-7) Streak on a plate of Gao's solid medium No. 1, incubate in a 28 ℃ incubator for 7 days, pick a single colony and inoculate it into Gao's liquid medium No. 1, and place it on a shaker at 28 ℃ and 180 rpm / min for bacterial growth. Collect the seed liquid after 3 days of incubation, and then inoculate the seed liquid into medium A at a volume ratio of 10%, and incubate at 28 ℃ for 11 days to obtain the fermentation product; the preparation method of medium A is as follows: weigh 20 g soluble starch, 6 g peptone, 2 g yeast extract, 30 g sea salt, 100 mg KBr, 40 mg Fe2(SO4)3·4H2O and 1 g CaCO3, add them to 1000 mL distilled water, and sterilize them at 121 ℃ for 20 min;

[0041] (2) Extraction of extract

[0042] Add an equal amount of ethyl acetate to the fermented product obtained in step (1), extract repeatedly for 3 times, and then evaporate the ethyl acetate extract under reduced pressure to dryness to obtain a crude extract;

[0043] (3) Isolation and preparation of compounds

[0044] The crude extract obtained in step (2) was dissolved in a mixed solvent of dichloromethane and methanol in a volume ratio of 1:1, and 200-300 mesh silica gel was added to mix the sample, and normal phase medium pressure column chromatography was performed, using a petroleum ether-ethyl acetate solution in a volume ratio of (100:1) to (0:1) as an eluent for gradient elution, and the eluted fractions were collected and arranged in order of polarity from small to large, and 11 components were obtained by merging; the collected 10th component was subjected to reverse phase medium pressure column chromatography gradient elution, using acetonitrile-water in a volume ratio of 30-100% as an eluent, the elution time was 150 min, the eluted fractions were collected, and the polarity of the fractions were arranged in order from large to small, and 10 components were obtained by merging; the obtained 7th component was separated and purified by semi-preparative reverse phase high performance liquid chromatography, using a solution of acetonitrile and water in a volume ratio of 43:57 as a mobile phase, and the flow rate was 2.0 mL / min, a compound having a structure as shown in (I) was obtained, and the elution gradient volume ratios of the petroleum ether-ethyl acetate solution were 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1 and 0:1, respectively.

[0045] (I).

[0046] Example 3

[0047] Structural identification of compounds and attribution of NMR signals

[0048] The compound is a yellow powder. High-resolution mass spectrometry (HR-ESI-MS) in positive ion mode gives its quasi-molecular ion peak m / z 525.2139 [M + H] + , combined with 13 C NMR spectrum confirmed that its molecular formula is C 30 H 29 N4O5. 1 H and 13 CNMR data can be found at Figure 1-7 And as shown in Table 1.

[0049] Table 1. 1D NMR data of compound 1 (DMSO- d 6)

[0050]

[0051] Note 1: s—singlet, d—doublet, t—triplet, q—quartet, m—multiplet;

[0052] Note 2: 1 H was obtained at 600 MHz NMR; 13 C was obtained at 150 MHz NMR.

[0053] Figure 3-7is the main two-dimensional correlation of the compounds of the present invention, Figure 3 It can be seen that the compound contains 30 carbon atoms, including 13 quaternary carbon atoms, 4 methyl carbon atoms, 2 methylene carbon atoms, and 11 methine carbon atoms. Figure 4 It can be seen that compounds H-8 / H-9 / H-10 / H-11, H-1 / H-2 / H-3 / H-4 have COSY correlation. Figure 5 It can be seen that the carbon and hydrogen of the compound are directly related, Figure 6 It can be seen that the carbon and hydrogen in the compound are remotely related, and the carbons can be linked together; Figure 7 NOE correlations for the compounds of the present invention. NOESY correlations exist between H3-2′ and H-4′, indicating that they are located on the same face of the sugar moiety. Coupling constant ( 3 J H-3′ , H-4′ =4.27 Hz) indicating that H-3′ and H-4′ are located in the same orientation. Figure 8 The mass spectrum of the compound of the present invention shows that the molecular weight of the compound of the present invention is 525.2139 and the molecular formula is C 30 H 29 N4O5 Fig. 9 and 10 The CD spectra of the compound of the present invention and the CD spectra of the known compound staurosporine M1 are shown in Figure 1. It can be seen that the CD spectrum of compound 1 is almost the same as that of staurosporine M1. This shows that the absolute configuration of compound 1 is consistent with that of staurosporine M1, which is classified as 2′S, 3′R, 4′R, 6′R.

[0054] Example 4

[0055] Activity of staurosporine compounds

[0056] Activity and application of staurosporine compounds described in Example 1

[0057] (1) Experimental samples

[0058] Preparation of the sample solution: The test sample is the pure compound 1 separated and purified in Example 1 above. Accurately weigh an appropriate amount of the sample and use DMSO to prepare a solution of the required concentration for testing anti-tumor activity. The tumor cells used in this experiment are drug-resistant breast cancer cell lines;

[0059] (2) Experimental methods

[0060] Drug-resistant breast cancer cells were seeded in 96-well plates, allowed to adhere overnight, and treated with the indicated concentrations of compound 1 for 72 h. Cell viability was determined using a cell counting kit. The absorbance was then measured at a wavelength of 450 nm on a microplate reader;

[0061] (3) Experimental results

[0062] In the anti-tumor test, compound 1 was 4.47 μ Compared with the case where there is no methyl group on the 4'-imide group, it exhibits stronger anticancer activity in drug-resistant and parental cells. The IC 50 = 4.47 μM.

[0063] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. A staurosporine compound, characterized in that The structural formula of the staurosporine compound is shown in (I):

2. A method for preparing the staurosporine compound according to claim 1, characterized in that The steps include: (1) Fermentation production The marine actinomycetes with the deposit number of CCTCC NO: M2020953 were streaked on a plate of Gao's solid medium No. 1, and after being cultured and activated in an incubator at 28°C for 7 days, a single colony was picked and inoculated into Gao's liquid medium No. 1, and the culture was placed on a shaker at a temperature of 28°C and a speed of 180 rpm / min to increase the bacteria, and the seed liquid was collected after culturing for 3 days, and then the seed liquid was inoculated into medium A at an inoculum amount of 10% by volume, and the culture was carried out at a temperature of 28°C for 11 days to obtain a fermentation product; (2) Extraction Add an equal amount of ethyl acetate to the fermented product obtained in step (1), extract repeatedly for 3 times, and then evaporate the ethyl acetate extract under reduced pressure to obtain a crude extract; (3) Isolation and preparation of compounds The crude extract obtained in step (2) is dissolved in a mixed solvent of dichloromethane and methanol in a volume ratio of 1:1, and 200-300 mesh silica gel is added to mix the sample, and normal phase medium pressure column chromatography is performed, and petroleum ether-ethyl acetate solution in a volume ratio of (100:1) to (0:1) is used as an eluent for gradient elution, and the eluted fractions are collected and arranged from small to large according to the polarity of the fractions, and 11 components are obtained by merging; the collected 10th component is subjected to reverse phase medium pressure column chromatography gradient elution, and acetonitrile-water in a volume ratio of 30-100% is used as an eluent, and the eluted fractions are collected and arranged from large to small according to the polarity of the fractions, and 10 components are obtained by merging; the obtained 7th component is separated and purified by semi-preparative reverse phase high performance liquid chromatography, and a solution of acetonitrile and water mixed in a volume ratio of 43:57 is used as a mobile phase to obtain a compound, whose structure is shown in (I) 3. The method for preparing a staurosporine compound according to claim 2, characterized in that The preparation method of medium A described in step (1) is as follows: weigh 20 g soluble starch, 6 g peptone, 2 g yeast extract, 30 g sea salt, 100 mg KBr, 40 mg Fe2(SO4)3·4H2O and 1 g CaCO3, add them to 1000 mL distilled water, and sterilize them under high pressure at 121°C for 20 min.

4. The method for preparing a staurosporine compound according to claim 2, characterized in that: The elution gradient volume ratios of the petroleum ether-ethyl acetate solution in step (3) are 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1 and 0:1, respectively.

5. The method for preparing a staurosporine compound according to claim 2, characterized in that: In the reverse phase medium pressure column chromatography gradient elution described in step (3), the volume ratio of acetonitrile is from 30 to 100%, and the elution time is 150 minutes.

6. The method for preparing a staurosporine compound according to claim 2, characterized in that: The flow rate of the semi-preparative reversed-phase HPLC compound separation preparation described in step (3) is 2.0 mL / min.

7. Use of the staurosporine compound according to claim 1 in the preparation of a drug-resistant breast cancer cell line inhibitor.

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