阅读说明：本技术 一种具有抗前列腺癌活性的四氢喹啉生物碱及其制备方法和应用 (Tetrahydroquinoline alkaloid with anti-prostate cancer activity and preparation method and application thereof ) 是由 王俊锋 王军舰 黄小龙 刘永宏 于 2020-12-07 设计创作，主要内容包括：本发明公开了一种具有抗前列腺癌活性的四氢喹啉生物碱及其制备方法和应用。四氢喹啉生物碱,如式(I)所示。本发明从海洋链霉菌Streptomyces sp.HNM0561中制备得到一个具有抗前列腺肿瘤活性的化合物1,可用于开发抗前列腺肿瘤药物,因此本发明不仅为开发新的抗前列腺肿瘤药物提供了备选化合物,同时对开发中国海洋微生物药物资源具有重要的意义。(The invention discloses tetrahydroquinoline alkaloid with anti-prostate cancer activity and a preparation method and application thereof. The tetrahydroquinoline alkaloid is shown as a formula (I). The compound 1 with the anti-prostate tumor activity is prepared from Streptomyces sp.)

1. Tetrahydroquinoline alkaloid or medicinal salt thereof shown in formula (I).

2. The application of tetrahydroquinoline alkaloid shown as a formula (I) or medicinal salt thereof in preparing an anti-prostate cancer medicament.

3. An anti-prostate cancer drug comprising an effective amount of the tetrahydroquinoline alkaloid represented by the formula (I) in claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable carrier.

4. A process for the preparation of tetrahydroquinoline alkaloid according to claim 1, characterized in that it is isolated from the fermentation culture of Streptomyces sp.

5. The method of claim 4, comprising the steps of:

A. preparing a fermentation culture of Streptomyces sp.HNM0561;

B. separating fermentation liquor and mycelium, extracting the fermentation liquor by using ethyl acetate, and concentrating ethyl acetate extract liquor to obtain fermentation liquor extract; extracting mycelium with acetone water solution, concentrating the extractive solution, extracting with ethyl acetate, and concentrating the ethyl acetate extractive solution to obtain mycelium extract; mixing the fermented liquid extract and mycelium extract, separating with reverse phase silica gel, and separating with methanol: water elution from 10:90,20:80,30:70,50:50,70:30,80:20,90:10,100:0, v/v gradient sequence gives 8 components fr1-fr8, methanol was collected: 80 parts of water: and (3) carrying out Sephadex LH-20 column chromatography on the component fr6 eluted at 20v/v, eluting the purified product by using methanol as an eluent, and purifying by using a high-efficiency liquid phase to obtain the tetrahydroquinoline alkaloid.

6. The preparation method according to claim 5, wherein the fermentation culture for preparing Streptomyces sp.HNM0561 is obtained by inoculating Streptomyces sp.HNM0561 into a fermentation medium containing per 1000 mL: 20g of glucose, 10g of yeast extract, 3g of beef extract, 3g of corn steep liquor, 10g of soluble starch, 0.5g of dipotassium hydrogen phosphate, 0.5g of magnesium sulfate, 2g of calcium carbonate and the balance of water.

7. The process according to claim 5, wherein the purification by high performance liquid chromatography is carried out at a wavelength of 210 and 330nm, using a flow rate of 4ml/min, in acetonitrile: semi-preparative HPLC (YMC-pack ODS-A, 10X 250mm,5 μm) using an isocratic elution with water at 75:25, v/v, retention time t_RObtaining tetrahydroquinoline alkaloid in 33.9 min.

8. Use of Streptomyces sp. hnm0561 from marine species for the preparation of tetrahydroquinoline alkaloid according to claim 1.

The technical field is as follows:

the invention belongs to the field of natural products, and particularly relates to separation and purification of a microbial natural product and application of the microbial natural product in preparation of an anti-tumor drug.

Background art:

the prostatic cancer is the most common malignant tumor of men, is the second cause of male cancer death worldwide, and is the tumor with the highest morbidity in urinary surgery in China. The most diseased tumors. Endocrine therapy is the primary treatment for prostate cancer, but after a median time of 14-30 months, almost all patients will turn to Castration Resistant Prostate Cancer (CRPC). Although the second generation Androgen Receptor (AR) targeting drugs such as enzalutamide and the like make a breakthrough in the treatment of CRPC, the patients almost inevitably develop drug resistance to them after receiving the treatment, which results in the failure of clinical treatment of CRPC. The natural products are important sources for innovative medicine research, two thirds of 1394 small molecule new medicines which are promoted all over the world in thirty-nine years of 1981-2019 can be traced back to the natural products or are inspired by the natural products, and 84% of 185 anticancer small molecule medicines are related to the natural products. Microbial metabolites are important components of natural products, and are also one of important sources of anticancer drugs, such as adriamycin, actinomycin D, bleomycin and the like, and are secondary metabolites derived from microorganisms and derivatives thereof. The special environment of the ocean creates the particularity and diversity of the marine microorganisms, endows the marine microorganisms with the diversity and complexity of natural product structures, and provides an important biological resource and a compound source for the research of marine medicines.

Therefore, the screening and discovery of new natural products with antitumor activity in marine microorganisms are of great significance to the development of anti-prostate cancer drugs.

The invention content is as follows:

the first purpose of the invention is to provide a tetrahydroquinoline alkaloid compound with anti-prostate cancer activity.

The tetrahydroquinoline alkaloid compound has a structure shown in a formula (I):

the invention obtains the compound 1 from a strain of Streptomyces sp. Through structural analysis, the compounds are determined to be chlorine-containing tetrahydroquinoline alkaloid compounds, and the specific structure is shown as the formula (I). Through the evaluation of the antitumor activity of the compound 1, the compound 1 is found to have remarkable inhibitory effect (IC) on two human prostate cancer cells C4-2B and 22RV1₅₀Are all made of<0.28. mu.M), especially the IC of Compound 1 on human prostate cancer cells C4-2B₅₀Reaches 0.067 mu M, and can be used as a lead compound for developing anti-prostate cancer drugs.

Therefore, the second object of the present invention is to provide the use of tetrahydroquinoline alkaloid represented by formula (I) or its pharmaceutically acceptable salt in the preparation of anti-prostate cancer drugs.

The third purpose of the invention is to provide an anti-prostate cancer medicament, which comprises an effective amount of tetrahydroquinoline alkaloid shown as a formula (I) or medicinal salt thereof as an active ingredient, and a pharmaceutically acceptable carrier.

The fourth purpose of the invention is to provide a preparation method of the tetrahydroquinoline alkaloid, which is obtained by separating from a fermentation culture of Streptomyces sp.

Preferably, the method comprises the following steps:

A. preparing a fermentation culture of Streptomyces sp.HNM0561;

B. separating fermentation liquor and mycelium, extracting the fermentation liquor by using ethyl acetate, and concentrating ethyl acetate extract liquor to obtain fermentation liquor extract; extracting mycelium with acetone water solution, concentrating the extractive solution, extracting with ethyl acetate, and concentrating the ethyl acetate extractive solution to obtain mycelium extract; mixing the fermented liquid extract and mycelium extract, separating with reverse phase silica gel, and separating with methanol: water elution from 10:90,20:80,30:70,50:50,70:30,80:20,90:10,100:0, v/v gradient sequence gives 8 components fr1-fr8, methanol was collected: 80 parts of water: and (3) carrying out Sephadex LH-20 column chromatography on the component fr6 eluted at 20v/v, eluting the purified product by using methanol as an eluent, and purifying by using a high-efficiency liquid phase to obtain the tetrahydroquinoline alkaloid.

Preferably, the fermentation culture for preparing the Streptomyces maritima sp.HNM0561 is obtained by inoculating the Streptomyces maritima sp.HNM0561 into a fermentation culture medium for culture, wherein each 1000mL of the fermentation culture medium contains: 20g of glucose, 10g of yeast extract, 3g of beef extract, 3g of corn steep liquor, 10g of soluble starch, 0.5g of dipotassium hydrogen phosphate, 0.5g of magnesium sulfate, 2g of calcium carbonate and the balance of water.

The high performance liquid purification is carried out by detecting at 210 and 330nm wavelength, adopting flow rate of 4ml/min, and adding acetonitrile: semi-preparative HPLC (YMC-pack ODS-A, 10X 250mm,5 μm) using an isocratic elution with water at 75:25, v/v, retention time t_RObtaining tetrahydroquinoline alkaloid in 33.9 min.

The fifth purpose of the invention is to provide the application of Streptomyces sp.

The compound 1 with the anti-prostate tumor activity is prepared from Streptomyces sp.

Streptomyces sp.HNM0561 is preserved in China Center for Type Culture Collection (CCTCC) in 2020, 06 and 30 days, and the address is as follows: eight-way Lojia mountain in Wuchang district, Wuhan university, Wuhan province, Hubei province, preservation number: CCTCC No: m2020255.

Description of the drawings:

FIG. 1: malaymycin (1) Main¹H-¹H COSY, HMBC, and NOESY information;

FIG. 2: ECD map of Malaymycin (1)

FIG. 3 proliferation inhibitory Activity of Malaymycin (1) on two prostate cancer cell lines (C24B and 22RV1) at different concentrations

FIG. 4 Malaymycin (1) action on C42B cell cycle arrest

FIG. 5 Malaymycin (1) significantly inhibits the expression of the AR gene in prostate tumor cells

The specific implementation mode is as follows:

the following examples are further illustrative of the present invention and are not intended to be limiting thereof.

Example 1: preparation and structural characterization of Compound 1

Preparation of compound 1 shown as formula (I)

1. And (3) microorganism culture conditions:

each 1000mL of the medium was prepared by: taking 20g of glucose, 10g of yeast extract, 3g of beef extract, 3g of corn steep liquor, 10g of soluble starch, 0.5g of dipotassium hydrogen phosphate, 0.5g of magnesium sulfate and 2g of calcium carbonate, dissolving in a proper amount of water, diluting to 1000mL with water, and sterilizing at 121 ℃ for 20min for later use.

Inoculating Streptomyces sp.HNM0561 into the culture medium, carrying out shake culture at 28 ℃ for 3 days to obtain a seed culture solution, inoculating the seed culture solution into the culture medium according to the volume ratio of 1%, and carrying out shake culture at 28 ℃ for 11 days to obtain a fermentation product of Streptomyces sp.HNM0561.

2. Extraction and separation:

and centrifuging the fermentation product of the Streptomyces sp.HNM0561 at 3600rpm to obtain supernatant fermentation liquid and precipitated mycelium. Extracting the supernatant fermentation liquor with ethyl acetate for 3 times in the same volume, and concentrating ethyl acetate extract at a temperature lower than 40 deg.C under reduced pressure to obtain fermentation liquor extract; ultrasonically extracting the precipitated mycelium with 85% acetone aqueous solution, distilling the extract under reduced pressure, repeatedly extracting with ethyl acetate for three times, and concentrating the ethyl acetate extract at a temperature lower than 40 deg.C under reduced pressure to obtain mycelium extract; the fermentation liquid extract and the mycelium extract are combined to obtain about 27.6g of extract. The extract is separated by reverse phase silica gel, and after sample mixing and column filling by a dry method, methanol is adopted: water (10:90,20:80,30:70,50:50,70:30,80:20,90:10,100:0, v/v) was used in a gradient elution sequence to obtain 8 fractions (fr1-fr 8). Fraction fr6 (methanol: water 80:20 v/v fraction) is chromatographed on Sephadex LH-20 column using methanolThe purified product was eluted as eluent at wavelengths of 210 and 330nm, using a flow rate of 4ml/min, in acetonitrile: water (75:25, v/v) was subjected to isocratic elution for semi-preparative high performance liquid chromatography (HPLC (YMC-pack ODS-A, 10X 250mm,5 μm) to give novel Compound 1(3.3mg, retention time t;)_R33.9min)。

II, structural identification of Compound 1

The chemical structure of compound 1 was determined by data testing such as Mass Spectrometry (MS), Nuclear Magnetic Resonance (NMR), Optical Rotation (OR), and Circular Dichroism (CD).

Compound 1 is a white amorphous powder; high resolution mass spectrum HRESIMSm/z445.1906[ M-H ]]^–(calcd for C₂₄H₃₀ClN₂O₄) The suggested molecular formula is C₂₄H₃₁ClN₂O₄Unsaturation degree is 10;¹h and¹³c NMR data are shown in Table 1; one-dimensional nuclear magnetic data shows that the molecule contains 24 carbon atoms, including 3 methyl groups, 1 methoxy group, 6 methylene groups, 4 methine groups, and 10 quaternary carbons. Aromatic proton H-5 (. delta.)_H6.56,d,J＝9.0Hz)、H-6(δ_H7.55, d, J ═ 9.0Hz) and H-2(δ)_H7.56, s) presuming the presence of a 1,3, 4-trisubstituted benzene ring segment in the molecule;¹H-¹the HCOSY spectrum shows an adjacent methylene 8-CH₂And a methine 9-CH fragment, with H-5 observed in combination with C-1, C-3, H in HMBC spectra₂8 and C-2, C-3, C-4, C-9 and C-10, and 4-NH and C-3, C-4, C-5 and C-9 hydrocarbons, it can be presumed that a tetrahydroquinoline fragment is present in the molecule; from two methylene groups H₂-11[δ_H1.81(m,H_a-11),1.62(m,H_b-11)]And H₂-12[δ_H2.08(m,H_a-12),2.00(m,H_b-12)]And three allylmethyl groups H₃-15(δ_H1.61,s)，H₃-16(δ_H1.63,s)，H₃-18(δ_H1.61, s) to two olefin quaternary carbons C-13 (. delta.)_C126.6) and C-14 (. delta.))_C125.0) revealed a 2, 3-dimethylpent-2-ene fragment; h was observed₂11 with C-9 and C-10 and H₂-17 HMBC correlation signal with C-10, indicating2, 3-dimethylpent-2-ene and methylene 17-CH with attached methoxy groups₂Are all connected at the C-10 position of the tetrahydroquinoline; methylene hydrogen H is observed in a two-dimensional nuclear magnetic spectrum₂3' and C-1', C-5', H₂HMBC cross peaks of 4' with C-1', C-2', C-5' and of the active hydrogen 2' -OH with C-1', C-2', C-3', indicating the presence of a cyclopent-2-enone fragment containing a hydroxyl substitution at C-3 '; combining high resolution mass spectrometry with the chemical shift of C-9, it is presumed that the Cl atom is attached at the C-9 position. The relative configuration of compound 1, H-9 and H, can be determined by NOESY spectrum₂NOE correlation between-11, indicating that they are on the same side of the molecular plane. Further we determined by ECD calculations that the absolute configurations of compound 1 are 9S and 10S. Through SciFinder search, the compound 1 is a novel compound with a novel structure and is named Malaymycin. Compound 1 is mainly¹H-¹The HCOSY, HMBC and NOESY information are shown in FIG. 1, and the ECD profile of Compound 1 is shown in FIG. 2.

TABLE 1 NMR spectra data (700MHz, CDCl) of Malaymycin (1)₃,ppm)

The structural formula of the compound 1 is shown as a formula (I), and the compound is named as Malaymycin:

example 2: inhibitory Activity of Malaymycin (1) on two human prostate cancer cells

Human prostate cancer cells C4-2B were ordered from eurocor inc. Human prostate cancer cells 22Rv1 were ordered from the American Type Culture Collection (Manassas, VA, USA).

Human-derived prostate cancer cell inhibitory activityThe CCK-8 assay was used for the sexual experiments. The cells were cultured in RPMI1640 medium, collected in the logarithmic growth phase, counted, resuspended, adjusted to the appropriate concentration, and seeded in 96-well plates (500-. Cells were incubated at 37 ℃ and 100% relative humidity, 5% CO₂Incubate in incubator for 24 hours. The test compound was diluted with the medium to the appropriate working concentration and cells were added at 50. mu.l/well. For C4-2B and 22Rv1 cells, compound 1(Malaymycin) and the positive control (enzalutamide ) were applied at final concentrations starting at 40 μ M, diluted in a 2-fold gradient, and at 9 concentration points. Cells were incubated at 37 ℃ and 100% relative humidity, 5% CO₂Incubate in incubator for 72 hours. The medium was aspirated off, and fresh complete medium containing 10% CCK-8 was added and incubated in an incubator at 37 ℃ for 2-4 hours. After gentle shaking, the absorbance at 450nm was measured on a SpectraMax M5 Microplate Reader, and the inhibition was calculated using the absorbance at 650nm as a reference. 3 replicates per sample.

The inhibition of prostate cancer cell growth by the compound was calculated as follows:

the cancer cell growth inhibition rate [ (Ac-As)/(Ac-Ab) ]. times.100%

As absorbance OA of sample (cell + CCK-8+ test Compound)

Ac absorbance OA of negative control (cell + CCK-8+ DMSO)

Ab Absorbance OA of Positive control (Medium + CCK-8+ DMSO)

IC was performed using the software Graphpad Prism 5 and using the calculation formula log (inhibition) vs₅₀Curve fitting and calculating IC₅₀The value is obtained.

Proliferation inhibition by Malaymycin (1) on two prostate cancer cells (C24B and 22RV1) and one small cell lung cancer cell (H446) is shown in table 2:

TABLE 2 proliferation Inhibition (IC) of Malaymycin (1) on three tumor cell lines₅₀,μM)

Through the evaluation of the in vitro anti-prostate cancer activity of the tetrahydroquinoline alkaloid novel compound 1 (figure 3), the tetrahydroquinoline alkaloid novel compound 1 is found to have remarkable inhibitory activity (IC) on two human prostate cancer cells C4-2B and 22RV1₅₀0.067 and 0.028 muM), the in vitro activity is obviously higher than that of the positive drug Enzalutamide (IC)₅₀18.26 and 37.6 μ M).

Due to the remarkable inhibitory activity of the novel tetrahydroquinoline alkaloid compound 1 as a novel natural product on C4-2B and 22RV1 cells, the invention further researches the blocking effect of the novel tetrahydroquinoline alkaloid compound 1 on the cell cycle of prostate tumor C4-2B under low toxic dose (0.3 mu M). FIG. 4 shows that the ratio of G0/G1 phase of cells increased from 41.62% to 56.11% 72 hours after 0.3. mu.M of the novel compound 1 had acted on C4-2B cells. Therefore, the effect of the novel compound 1 on the C4-2B cell cycle of human prostate cancer is presumed to be blocked at the stage G0/G1.

Western Blotting and quantitative fluorescence qRT-PCR experimental results show that Malaymycin (1) significantly inhibits the expression of the second-generation Androgen Receptor (AR) in CRPC cells in a dose-dependent manner at both protein and RNA levels, and also inhibits the gene expression of target genes KLK3 and KLK2 of AR to inhibit the AR signaling pathway (fig. 5). Shows that the tetrahydroquinoline alkaloid new compound 1 can play a role in resisting prostate tumor by inhibiting AR gene expression and a signal path thereof.

Therefore, based on the remarkable inhibitory activity of the tetrahydroquinoline alkaloid new compound 1 on two human prostate cancer cells C24B and 22RV1, the tetrahydroquinoline alkaloid new compound can be used as a lead compound for developing anti-prostate cancer medicaments. In conclusion, the invention provides a new candidate compound for developing a new anti-cancer drug, and has important significance for the development of a new marine anti-cancer drug with independent intellectual property rights in China.