CN111471050B - Staurosporine derivatives and preparation method and application thereof - Google Patents
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Abstract
The invention discloses staurosporine derivatives which can be used for developing anti-tumor related medicaments and medicaments for inhibiting related diseases with kinases such as protein kinase, tyrosine kinase and the like. The invention also provides a preparation method of the staurosporine derivatives, which is obtained by separating and purifying a fermentation crude extract obtained by fermenting the actinomycete rice solid culture medium and extracting the fermentation crude extract by ethyl acetate by adopting gel column chromatography, silica gel column chromatography, medium-pressure preparative chromatography and high performance liquid chromatography, and is easy to operate and implement. The structural type of the staurosporine derivative is found in natural products for the first time; the invention has simple experiment operation, easy expanded production and better application prospect.
Description
The application is a divisional application of 'a class of staurosporine derivatives, and a preparation method and application thereof', wherein the application date of the original application is 9/19/2019, and the application number of the original application is 201910887005.3.
Technical Field
The invention relates to the field of preparation of active compounds from actinomycete fermentation products, and in particular relates to staurosporine derivatives and a preparation method and application thereof.
Background
Malignant tumor is a disease seriously threatening human health, and along with the gradual aggravation of the aging population in China, the acceleration of the industrialized and urbanized process, the accumulation of dangerous factors such as unhealthy life style and environment and the like, the prevention and control situation of the malignant tumor is extremely severe.
Cancer statistical data published by national cancer center in 1 month in 2019 show that malignant tumor death accounts for 23.91% of all the causes of death of residents, and the morbidity and mortality of malignant tumors are in a continuously rising situation in recent decades. Lung cancer, liver cancer, upper digestive system tumors, colorectal cancer, male prostate cancer, female breast cancer and the like still remain main malignant tumors in China.
At present, the relative survival rate of malignant tumor of China for 5 years is about 40.5%, which is improved by about 10% compared with that before 10 years, but has a great gap with developed countries. Surgery, radiotherapy, chemotherapy and molecular targeted drugs are still the major means for treating cancer, and therefore finding more efficient antitumor drugs is the most effective way to reduce the mortality of malignant tumors.
Staurosporine (STA), which was first discovered in 1977, is a broad-spectrum kinase inhibitor,especially has strong inhibition effect (IC) on Protein Kinase C (PKC)501-20 nM), and therefore, is widely used to study the role of PKC in various cell signaling processes. However, STA is a non-selective kinase inhibitor, limiting its use as a clinical drug.
A plurality of STA derivatives including UCN-01 of natural source, enzastaurin obtained by structural modification, edotecarin, CEP-2563, CEP-1347 and the like enter clinical research or complete clinical research. The derivative, lestautinib, was approved by the FDA in 2006 as an orphan drug for the treatment of acute myeloid leukemia; the semisynthetic derivative, midostaurin, was a multi-target kinase inhibitor and was approved by the FDA in 2017 for the treatment of acute myeloid leukemia and systemic cell proliferative hypertrophy. This shows that the mother nucleus with staurosporine structure is a compound mother nucleus with great development prospect.
However, these compounds are generally less selective and have inhibitory effects on both cancer and normal cells. Therefore, the search for more staurosporine derivatives with novel structures and high selectivity is an effective way for finding compounds with antitumor effect.
The patent specification with the publication number of CN 107569491A discloses the application of staurosporine compounds, 5 staurosporine compounds are obtained by research and separation and purification of actinomycetes, and the obtained staurosporine compounds have higher activity on prostate cancer cells and higher inhibition effect on Brd4 protein, and can be used for preparing medicines for treating cancer, inflammation or AIDS and Brd4 protein inhibitors.
Disclosure of Invention
Aiming at the defects in the field, the invention provides a staurosporine derivative which is obtained by solid fermentation of actinomycetes, has obvious antitumor activity and protein kinase inhibition activity, and can be used for developing medicaments for preventing and treating malignant tumors and diseases related to kinase expression abnormality.
Staurosporine derivatives have a structural formula shown as any one of the following structures:
the staurosporine derivative can be produced by solid fermentation of actinomycetes and is obtained by separation and purification, and is easy to operate and implement.
The invention also provides a preparation method of the staurosporine derivative, which comprises the following steps:
(1) inoculating actinomycetes into a Gao's first culture medium, and performing shake culture to obtain a seed solution;
(2) inoculating the obtained seed liquid into a rice culture medium, standing for culture, and extracting to obtain a crude extract of a fermentation product;
(3) separating and purifying the crude extract of the obtained fermentation product to obtain the staurosporine derivative.
In the step (1), the actinomycetes can adopt a commercially available product, such as Streptomyces sp.CICC 11031 sold by China Industrial microorganism culture Collection management center, order a website: http:// www.china-cic.
Preferably, in step (1), the conditions of shake culture are as follows: culturing at 28 ℃ and 180rpm for 4-6 days.
Preferably, in the step (2), the rice culture medium is prepared from rice and sea brine, and the ratio of the mass of the rice to the volume of the sea brine is 40 g: 60mL, namely the rice is prepared by high-pressure damp-heat sterilization after the weight of rice is 40g and the volume of sea brine is 60 mL;
the inoculation amount of the seed liquid is as follows: inoculating 10mL of seed liquid to every 40g of rice;
the conditions of the static culture are as follows: standing and culturing at 28 deg.C for 70 days.
Preferably, the sea brine mass concentration is 25 per mill.
Preferably, in step (3), the separation and purification method comprises: extraction, gel column chromatography, medium pressure preparative chromatography, silica gel column chromatography and high performance preparative liquid chromatography.
Preferably, the extraction conditions are: soaking in ethyl acetate solvent for 3 days;
the gel column chromatographic conditions are as follows: the adopted filler is hydroxypropyl sephadex (LH-20), the adopted eluent is methanol-water solution, and the methanol-water solution is eluted according to the proportion of 20 percent, 40 percent, 60 percent, 80 percent and 100 percent of methanol volume percentage;
the medium pressure preparative chromatography conditions are as follows: the filler is octadecylsilane chemically bonded silica, and the mobile phase is methanol with the volume percentage of 40-100 percent and 0.05 percent TFA (trifluoroacetic acid) -water solution for gradient elution;
the silica gel column chromatography conditions are as follows: adopting 300-400-mesh refined silica gel, wherein an eluent is dichloromethane-methanol solution, and an elution system is dichloromethane, methanol is 100:1,80:1,60:1,40:1,20:1,10:1 and 1: 0;
the high performance preparative liquid chromatography conditions are as follows: the adopted filler is octadecylsilane chemically bonded silica, the adopted mobile phases are methanol-water and acetonitrile-water solution, and the adopted mobile phases are 40-100% methanol-water solution and 40-60% acetonitrile-water solution.
The compound of the invention is evaluated for the tumor cytotoxic activity by adopting a human colon cancer cell line HCT116 and a prostate cancer cell line PC 3; the protein kinase PKC theta and ROCK2 are adopted to test the inhibition activity of the compounds on protein kinase, and the results show that the staurosporine compounds can effectively inhibit the growth of HCT116 and PC3 cells, and have better PKC theta and ROCK2 kinase inhibition activity, which indicates that the compounds have related protein kinase inhibition effect and tumor cytotoxicity effect, so that the staurosporine compounds can be further used for researching and developing protein kinase inhibitors and antitumor drugs.
The invention also provides application of the staurosporine derivatives in preparing medicaments for treating colon cancer and prostatic cancer.
The invention also provides application of the staurosporine derivatives in preparation of protein kinase inhibitors, wherein the protein kinase is PKC theta and ROCK 2.
The compound provided by the invention can be used for treating various malignant tumors related to inhibition of protein kinase, tyrosine kinase and the like, HIV, leukemia, Alzheimer's disease and other related diseases, thereby having good application prospect.
Compared with the prior art, the invention has the main advantages that:
the staurosporine compound can be used for developing anti-tumor related medicaments and medicaments for inhibiting related diseases with kinases such as protein kinase, tyrosine kinase and the like; the structural type of the compound is found in natural products for the first time; the invention has simple experiment operation, easy expanded production and better application prospect.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The following examples are conducted under conditions not specified, usually according to conventional conditions, or according to conditions recommended by the manufacturer.
In this example, staurosporine compounds of the structural formulas 1 to 14 were obtained by the following method.
Fermentation of Actinomycetes
The actinomycetes adopt Streptomyces sp.CICC 11031 sold by China general microbiological culture collection management center;
1) inoculating actinomycetes into a Gao's first culture medium, and carrying out shake culture at the temperature of 28 ℃ and the speed of 180rpm for 4-6 days to obtain a seed solution;
the Gao's first culture medium is: soluble starch 20g, KNO3 1g，K2HPO4 0.5g，MgSO4·7H2O 0.5g，FeSO4·7H2O0.01 g and sea salt 25g, adding water to 1L, and adjusting pH to 7.2.
2) Inoculating the seed liquid obtained in the step 1) into a solid culture medium (solid culture medium, which is prepared from the following components: the mass of the rice is 40 g; 60mL of seawater is put into a 500mL conical flask and then is subjected to high-pressure damp-heat sterilization), the inoculation amount of each flask is 10mL, the flask is kept still and cultured for 70 days at the temperature of 28 ℃, and the culture is soaked, extracted and concentrated by ethyl acetate to obtain a crude extract of a fermentation product containing the compound.
Preparation of the Compounds
Subjecting the obtained crude product to gel column chromatography (filler is hydroxypropyl sephadex LH-20), eluting with 20%, 40%, 60%, 80%, 100% methanol-water system by volume percentage, and collecting each fraction by 1/4 column volume, and mixing the fractions by TLC analysis to obtain Fr.A-Fr.X components, wherein Fr.N-Fr.X contains target type compounds. The Fr.N-Fr.X components are respectively subjected to gel column chromatography (mobile phase: dichloromethane: methanol: 1) to obtain corresponding small components, and then medium-pressure or high-pressure liquid phase preparation is carried out. The compound is obtained by separating the compound with a high-pressure chromatographic column of Agilent Pursuit C-18(10 mu m, 21.2 multiplied by 250mm), a detection wavelength of 292 or 316nm and a filler of octadecylsilane chemically bonded silica.
Preparation of fr.q-4 using medium pressure liquid chromatography (40% methanol-water-0.05% TFA solution) gave Fr. Q-4-1 to fr.q-4-5 fractions, and separation of fr.q-4-4 using silica gel column chromatography (methanol: dichloromethane: 100:1,80:1,60:1,40:1,20:1,10:1,1: 0) gave fractions fr.q-4-4-1 to fr.q-4-4-14. Q-4-4-7 is prepared by high pressure liquid phase (42% acetonitrile-water solution, detection wavelength 292nm), and collecting 28min and 30min peaks to obtain compounds 6 and 7; q-4-4-8 is prepared by high pressure liquid phase (40% acetonitrile-water solution, detection wavelength 292nm), and the peak of 20min is collected to obtain compound 2;
separating Fr.Q-7 by silica gel column chromatography (methanol: dichloromethane: 40:1,30:1,20:1,10:1,5:1,1:1,1: 0) to obtain components Fr.Q-7-1 to Fr.Q-7-8, and collecting the peak of Fr.Q-7-1 by high pressure liquid phase preparation (40% acetonitrile-water solution) for 50min and 54min to obtain compounds 4 and 5;
q-9 was prepared using high pressure liquid phase (70% methanol-water solution) and the peaks were collected for 7min and 25min to give compounds 9 and 8.
Separating Fr.T-4 by silica gel column chromatography (methanol: dichloromethane: 100:1,80:1,60:1,40:1,20:1,10:1,1: 0) to obtain components Fr.T-4-1 to Fr.T-4-12, and collecting 47min peak of Fr.T-4-1 by high pressure liquid phase preparation (40-100% methanol-water solution, 60min, detection wavelength 317nm) to obtain compound 11; t-4-2 adopts high pressure liquid phase preparation (70% isocratic, 0-40 min; 70% -100% gradient methanol-water solution, 40-70min, detection wavelength 292nm) to collect peaks of 59min and 67min to obtain compounds 13 and 14; t-4-3 adopts high pressure liquid phase preparation (50-65% acetonitrile-water solution) to collect peak for 12min to obtain compound 12; t-4-4 adopts high pressure liquid phase preparation (68% methanol-water solution, detection wavelength 292nm) to collect the peak of 31min to obtain compound 3;
Fr.U-3 adopts high pressure liquid phase preparation (50% -80% gradient methanol-water solution, 55min, detection wavelength 292nm) to collect peak of 24min to obtain compound 1;
v-3 high pressure liquid phase preparation (60-80% gradient methanol-water solution, 40min, detection wavelength 317nm) is adopted, and the peak of 33min is collected to obtain the compound 10.
Identification of Compounds
The compound 1 is a white solid, and the high-resolution mass spectrum HR-ESI-MS gives an excimer ion peak M/z 673.2325[2M + Na ]]+, calcd 673.2322, suggesting a molecular formula of C21H15N3O, named 7-methyl-K252c, and the specific nuclear magnetic data are shown in tables 1 and 2.
The compound 2 is a colorless crystal, and the high-resolution mass spectrum HR-ESI-MS gives an excimer ion peak M/z 559.1957[ M + Na ]]+Calcd 559.1957, for the molecular formula C31H28N4O5Named 4' -N-demethyl- (3 ' -hydroxy-2 ' -pyrrolidinone) staurosporine, and the specific nuclear magnetic data are shown in tables 1 and 2.
The compound 3 is a colorless crystal, and the high-resolution mass spectrum HR-ESI-MS gives an excimer ion peak M/z 646.2065[ M + Na ]]+(calculated 646.2066) indicating a formula of C37H29N5O5Named 4'-N-demethyl- (4' -indolyl-2 ',3' -propanoedione) staurosporine, and the specific nuclear magnetic data are shown in tables 1 and 2.
The compound 4 is brown crystal, and the high resolution mass spectrum HR-ESI-MS gives an excimer ion peak M/z 522.1639[ M + Na ]]+Calcd 522.1641, for the molecular formula C28H25N3O6Named 7(S) -method-MLR-52, and the specific nuclear magnetic data are shown in tables 1 and 2.
The compound 5 is light yellow powder, and the high resolution mass spectrum HR-ESI-MS gives an excimer ion peak M/z 522.1636[ M + Na ]]+Calcd 522.1641, for the molecular formula C28H25N3O6The structure is similar to that of Compound 4, with the difference that OCH 3Chemical shifts of-7, suggesting that compounds 5 and 4 are the 7-methoxy isomers. Therefore, the compound is named as 7(R) -methoxy-MLR-52, and specific nuclear magnetic data are shown in tables 1 and 2.
The compound 6 is a light yellow solid, and the high resolution mass spectrum HR-ESI-MS gives an excimer ion peak M/z 508.1479[ M + Na ]]+Calcd 508.1485, for the molecular formula C27H23N3O6. The difference between this compound and 4 is OCH 3-7 is replaced by OH-7 substitution, thus named 7(S) -hydroxy-MLR-52, with specific nuclear magnetic data as shown in tables 1, 2.
Compound 7 is similar to compound 6, and the high resolution mass spectrum HR-ESI-MS shows that the peak of the excimer ion is M/z 508.1479[ M + Na ]]+Calcd 508.1485, for the molecular formula C27H23N3O6. The difference between the two is the chemical shift of the hydrogen at position 7, suggesting that compounds 7 and 6 are 7 hydroxy isomers. Therefore, the compound is named as 7(R) -hydroxyl-MLR-52, and specific nuclear magnetic data are shown in tables 1 and 2.
The compound 8 is a white solid, and the high-resolution mass spectrum HR-ESI-MS gives an excimer ion peak M/z 510.1639[ M + Na ]]+Calcd 510.1641, for the molecular formula C27H25N3O6. Therefore, the product is named as 7(R) -methoxy-K252d, and the specific nuclear magnetic data are shown in tables 1 and 2.
The compound 9 is brown solid, and the high-resolution mass spectrum HR-ESI-MS shows that the peak of the excimer ion is M/z 496.1481[ M + Na ]]+Calcd 496.1485, for the molecular formula C26H23N3O6. Therefore, the name is 7(S) -hydroxy-K252 d, and specific nuclear magnetic data are shown in tables 1 and 2.
The compound 10 is yellow powder, and the high resolution mass spectrum HR-ESI-MS shows that the peak of the excimer ion is M/z 519.1634[ M + Na ]]+Calcd 519.1644, for the molecular formula C28H24N4O5Named as 7-oxo-4' -N-Acetyl-holyrine A, and the specific nuclear magnetic data are shown in tables 1 and 2.
The compound 11 is a yellow solid, and the high-resolution mass spectrum HR-ESI-MS gives an excimer ion peak M/z 488.1219[ M + Na ]]+Calcd 488.1222, hint moleculeFormula is C27H19N3O5. The compound is similar to the known compound 3'-epi-4' -oxo-TAN-1030A, except that the compound 4 has carbonyl groups at both the 5-and 7-positions, and is named 3'-epi-7,4' -dioxo-TAN-1030A, and specific nuclear magnetic data are shown in tables 1 and 2.
The compound 12 is a light yellow solid, and the high-resolution mass spectrum HR-ESI-MS gives an excimer ion peak M/z [ M + Na ]]+440.1611 calcd 440.1605, suggesting a molecular formula of C26H21N3O4. This compound is similar to the known compound streptocarbazoles C and is therefore named 2',3' -epi-streptocarbazoles C, and the specific nuclear magnetic data are shown in tables 1 and 2.
The compound 13 is a white solid, and the high-resolution mass spectrum HR-ESI-MS gives an excimer ion peak M/z 506.1687[ M + Na ]]+Calcd 506.1692, for the molecular formula C28H25N3O5. The compound is similar to the known compound streptocarbazoles B, except that the double bond between C-4 'and C-5' is changed into a single bond, and the 5 'position is substituted by hydroxyl, so the compound is named as 5' -hydroxy-streptocarbazoles B, and the specific nuclear magnetic data are shown in tables 1 and 2.
The compound 14 is a white solid, and the high-resolution mass spectrum HR-ESI-MS shows that the peak M/z of the excimer ion is 607.1592[ M + Na ]]+Calcd 607.1594, for the molecular formula C34H24N4O6. Specific nuclear magnetic data are shown in tables 1 and 2.
TABLE 1 Compound Hydrogen Spectroscopy data
Note:a，b，crepresenting the measurement at 600,500,400MHz respectively;d，e，frespectively indicates that the solvent is CD3OD,DMSO-d6,CDCl3。
TABLE 2 carbon spectra data of compounds
Note:a，b，crespectively, at 150,125,100 MHz;d，e，frespectively represents that the solvent is CD3OD,DMSO-d6,CDCl3. Data was obtained by BC spectroscopy.
Antitumor Activity test of Compounds
The proliferation inhibition effect of the compound on human colon cancer cell strain HCT116 cells is detected by adopting Sulforhodamine B (SRB) colorimetric method. Taking cells in logarithmic growth phase, configuring into 5 × 104one/mL, 100. mu.L/well in 96-well culture plates, CO2Culturing for 24 hr in incubator, taking out culture plate, adding samples to be tested with different concentrations into each hole, setting 3 multiple holes for each concentration, adding into CO after adding medicine2After the culture in the incubator is continued for 72 hours, the culture plate is taken out, the culture solution is discarded, 100 mu L of trichloroacetic acid (TCA) with the mass percentage of 10% precooled in a 4 ℃ refrigerator is added into each hole for fixation, and after the fixation is kept still for 5 minutes, the culture plate is moved to the 4 ℃ refrigerator for overnight. And pouring out the fixing liquid, washing each hole for 5 times by using deionized water, drying by drying, and drying by air. Add 70. mu.L of SRB solution to each well, leave at 25 ℃ for 20 minutes, remove the supernatant, wash 5 times with 1% by mass acetic acid, and air dry. Bound SRB was dissolved with 100 μ L/well 10mmoL/L Tris base (pH 10.5) with shaking. And (3) placing the sample in a microplate reader to measure the light absorption of each hole, wherein the measurement wavelength is 515 nm. And (3) calculating the inhibition rate of the drugs on cell proliferation according to the OD value of each well: inhibition rate＝[1-(OD515 medicine feeding hole/OD515 control well)]X 100%, calculating the half inhibitory concentration IC from the inhibitory rate of each concentration50. The results are shown in Table 3.
Kinase inhibitory Activity assay of Compounds
The experiment adopts a 384-well plate, the inhibitory activity of the obtained compound on PKCtheta and ROCK2 kinase is measured, and the inhibitory effect is measured based on a real-time resolution fluorescence technology. Preparing a compound to be detected into a required concentration, diluting the compound by using a kinase buffer solution, and preparing corresponding concentrations of the kinase, STK substrate biotin, ATP, a termination marker and the like according to a kit instruction. An enzyme reaction stage, adding 4 mu L of a compound to be detected, 2 mu L of kinase, 2 mu L of LSTL substrate biotin and 2 mu LATP, incubating for 30min at room temperature or 37 ℃, a detection stage, adding 5 mu L of Sa-XL665 and 5 mu L of STK Antibody-Eu (K), taking Ethylene Diamine Tetraacetic Acid (EDTA) as a termination solution, incubating for 1h at room temperature, measuring fluorescence intensity at lambda-665 and 620nm by adopting HRTF, calculating inhibition rate under each sample concentration according to corresponding signal intensity ratio, and calculating half inhibition concentration IC (integrated circuit) of each kinase according to each concentration inhibition rate50(μ M). The results are shown in Table 3.
Anti-tumor and kinase inhibitory Activity (IC) of the Compounds of Table 350)
Note: STA (staurosporine) was a positive control. The primary screening concentration of the HCT116/PC3 cell strain is 10 mug/mL; the primary screening concentration of PK theta is 2.5 mu g/mL; the primary screening concentration of ROCK2 was 0.5. mu.g/mL.
The activity evaluation of the compound is carried out by adopting human colon cancer cell strains HCT116 and prostate cancer cell strains PC3, and the results show that the compounds 2-7, 10 and 11 can obviously inhibit the growth of the cancer cell strains HCT116, wherein the compound 2 has the best activity, and IC (integrated Circuit) is500.146. mu.M; the compounds 2-8 and 10-12 can obviously inhibit the growth of a prostate cancer cell line PC3, wherein the compound 5 has good effect and IC50It was 0.76. mu.M. The result shows that the compounds have stronger tumor cell toxic activity and can be further researchedCan be used for preparing anti-tumor medicines.
The protein kinase PKC theta and ROCK2 are adopted to test the inhibition activity of the compounds on the protein kinase, and the results show that the compounds 2, 3, 5, 6 and 11 have stronger PKC theta inhibition activity, particularly the compound 2 has the best effect and IC500.17. mu.M; the compounds 2, 3, 10 and 11 have strong ROCK2 inhibition activity, wherein the compound 2 has the best effect and IC50It was 0.26. mu.M.
The compounds 4 and 5 are isomers of each other, and the compounds 6 and 7 are isomers of each other, and the difference can be found only by comparing R with each other1The structural orientation of the groups is different, but the activities of the compounds 4 and 5 and the compounds 6 and 7 on the human colon cancer cell line HCT116 and the prostate cancer cell line PC3 are obviously different, which shows that R is different from the other groups in the prior art1The antitumor activity can be significantly influenced by the change of the configuration of the group. In addition, comparison of compounds 5 and 7 reveals that R1Has more excellent antitumor activity than that of a hydroxyl group when the group is an oxymethyl group, and when R is1When the group is an oxymethyl group, compound 5 shows significantly more excellent inhibitory activity against PKC θ than compound 7, which are the first findings of the present invention.
Therefore, the compound provided by the invention can be used for treating various malignant tumors related to inhibition of protein kinase, tyrosine kinase and the like, HIV, leukemia, Alzheimer's disease and other related diseases, and has good application prospect.
Furthermore, it should be understood that various changes and modifications can be made by one skilled in the art after reading the above description of the present invention, and equivalents also fall within the scope of the invention as defined by the appended claims.
Claims (4)
1. Staurosporine derivatives are characterized by the following structural formula:
2. a preparation method of staurosporine derivatives is characterized by comprising the following steps:
(1) inoculating actinomycetes into a Gao's first culture medium, and performing shake culture to obtain a seed solution; the actinomycetes adopt Streptomyces sp.CICC 11031 sold by China Industrial microorganism culture Collection management center; the conditions of shake culture are as follows: culturing at 28 ℃ and 180rpm for 4-6 days; the Gao's first culture medium is: soluble starch 20g, KNO3 1g，K2HPO40.5g，MgSO4·7H2O 0.5g，FeSO4·7H20.01g of O and 25g of sea salt, adding water to 1L, and adjusting the pH value to 7.2;
(2) inoculating the obtained seed liquid into a rice culture medium, standing for culture, and extracting to obtain a crude extract of a fermentation product; the rice culture medium is prepared from rice and sea brine, wherein the ratio of the mass of the rice to the volume of the sea brine is 40 g: 60 mL; the inoculation amount of the seed liquid is as follows: inoculating 10mL of seed liquid to every 40g of rice; the conditions of the static culture are as follows: standing and culturing at 28 deg.C for 70 days;
(3) separating and purifying the crude extract of the obtained fermentation product to obtain staurosporine derivatives with any structure shown as the following structural formula:
the separation and purification method comprises the following steps: extraction, gel column chromatography, medium pressure preparative chromatography, silica gel column chromatography and high performance preparative liquid chromatography;
the extraction conditions are as follows: soaking in ethyl acetate solvent for 3 days;
the gel column chromatographic conditions are as follows: the adopted filler is hydroxypropyl sephadex LH-20, the adopted eluent is methanol-water solution, the elution is carried out according to the proportion of 20 percent, 40 percent, 60 percent, 80 percent and 100 percent of methanol volume percentage, each 1/4 column volume is one fraction, the fractions are combined by TLC analysis to obtain Fr.A-Fr.X components, wherein Fr.N-Fr.X contain a target type compound, the Fr.N-Fr.X components are firstly respectively subjected to gel column chromatographic separation, and the mobile phase is as follows: dichloromethane and methanol are 1:1, and corresponding small components are obtained;
preparing Fr.Q-4 by medium-pressure liquid phase, and separating by 40% methanol-water-0.05% TFA solution to obtain Fr.Q-4-1-Fr.Q-4-5 components, wherein the Fr.Q-4-4 is subjected to silica gel column chromatography, and the methanol is dichloromethane which is 100:1,80:1,60:1,40:1,20:1,10:1 and 1:0 to obtain Fr.Q-4-4-1-Fr.Q-4-4-14 components; q-4-4-7 is prepared by high pressure liquid phase, 42% acetonitrile-water solution, detecting wavelength 292nm, collecting 28min and 30min peak to obtain compounds 6 and 7;
and Fr.Q-7 is subjected to silica gel column chromatography, methanol and dichloromethane are respectively 40:1,30:1,20:1,10:1,5:1,1:1 and 1:0, so that components Fr.Q-7-1 to Fr.Q-7-8 are obtained, Fr.Q-7-1 is prepared by adopting a high-pressure liquid phase, a 40% acetonitrile-water solution is adopted, and peaks of 50min and 54min are collected so as to obtain the compounds 4 and 5.
3. The use of staurosporine derivatives according to claim 1 in the preparation of a medicament for the treatment of colon and prostate cancer.
4. The use of staurosporine derivatives according to claim 1, wherein the protein kinase is PKC θ, ROCK 2.
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