A sesquiterpene lactone with anti-prostate cancer activity and its preparation method and application

Abstract

The present invention discloses a sesquiterpene lactone having anti-prostate cancer activity, a preparation method and an application thereof. The sesquiterpene lactone has a structure shown in formula (1): #imgabs0# The sesquiterpene lactone provided by the present invention has significant anti-prostate cancer activity, and its IC 50 The value is 6.4±0.34μM. It is expected to be used in the preparation of anti-prostate cancer drugs.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical biotechnology, and in particular to a sesquiterpene lactone having anti-prostate cancer activity and a preparation method and application thereof. Background Art

[0002] Prostate cancer is a common malignant tumor and the leading cause of cancer death in men. It is the second most deadly malignant tumor in men after skin cancer. In 2020, there were 1,414,259 new cases of prostate cancer and 375,304 deaths worldwide, accounting for 14.1% and 6.8% of the total incidence and mortality of cancer in men, respectively. According to the American Cancer Society, the risk of a man being diagnosed with cancer in his lifetime is one in nine, and about 1 in 41 will die from prostate cancer. The cause of prostate cancer is not yet clear, and the identified risk factors are age, family history of prostate cancer, and certain genetic mutations. In addition, its early clinical symptoms are not obvious, and the diagnostic method is prostate-specific antigen detection (PSA), so the coverage of its diagnostic reagents is also an important factor in the incidence of prostate cancer, resulting in most patients being diagnosed in the middle and late stages. The first choice of treatment for patients with advanced prostate cancer is androgen deprivation therapy (ADT). After 1 to 2 years of treatment, almost all patients will progress to castration resistant prostate cancer (CRPC), with a short survival time and high mortality rate. It is a complex heterogeneous disease in which androgens and their receptors play a vital role in progression and development. The current treatment for prostate cancer is a combination of surgery, hormone therapy, radiation and chemotherapy. Commonly used therapeutic drugs in clinical practice include steroidal and nonsteroidal antiandrogens, such as cyproterone acetate, bicalutamide and enzalutamide, but these drugs all have different side effects.

[0003] Therefore, it is necessary to develop a new anti-prostate cancer compound. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the first aspect of the present invention provides a sesquiterpene lactone, which has significant anti-prostate cancer cell proliferation activity and IC 50 The value is low.

[0005] The second aspect of the present invention also provides a method for preparing sesquiterpene lactone.

[0006] The third aspect of the present invention also provides an application of sesquiterpene lactone.

[0007] According to the first aspect of the present invention, a sesquiterpene lactone or a pharmaceutically acceptable salt thereof is provided, which has a structure shown in formula (1):

[0008]

[0009] The sesquiterpene lactone according to the embodiment of the present invention has at least the following beneficial effects:

[0010] The sesquiterpene lactone provided by the present invention has significant anti-prostate cancer activity, and its IC 50 The value is 6.4±0.34μM. It is expected to be used in the preparation of anti-prostate cancer drugs.

[0011] According to the second aspect of the present invention, a method for preparing sesquiterpene lactone is provided, comprising the following steps:

[0012] S1, crushing the night-scented sedge, soaking and extracting with an organic solvent, filtering and concentrating the extract to obtain an extract;

[0013] S2, extracting the extract with chloroform, ethyl acetate, and n-butanol in sequence, removing the extraction solvent, and obtaining a chloroform fraction, an ethyl acetate fraction, and an n-butanol fraction, respectively;

[0014] S3, separating the chloroform part by column chromatography to obtain 9 parts Fr1 to Fr9;

[0015] S4, removing the pigment from Fr7 through a small-pore resin and dividing it into 9 parts to obtain Fr7A-I;

[0016] S5, Fr7F was separated by reverse phase silica gel in a methanol water-methanol elution system to obtain five parts, namely Fr7F1-5;

[0017] S6 and Fr7F5 were separated by reverse phase silica gel in a methanol water-methanol elution system to obtain five parts, namely Fr7F5a~Fr7F5e;

[0018] S7 and Fr7F5b were separated by several semi-preparative HPLC to obtain sesquiterpene lactones.

[0019] According to some embodiments of the present invention, in step S1, an organic solvent is used to perform soaking extraction several times, and the extracts from the several extractions are combined, filtered, and concentrated to obtain an extract.

[0020] According to some embodiments of the present invention, in step S3, the solvent system of the column chromatography method is n-hexane:ethyl acetate (100-0:1, v / v) and methanol.

[0021] According to some embodiments of the present invention, the elution system of steps S5 and S6 is 30% methanol water-100% methanol.

[0022] According to some embodiments of the present invention, in step S1, the organic solvent is selected from 95% ethanol.

[0023] According to some embodiments of the present invention, in step S1, the temperature of the soaking extraction is 20-40°C.

[0024] According to some embodiments of the present invention, in step S7, the chromatographic conditions of the semi-preparative HPLC at least satisfy one of the following:

[0025] iFlow rate 3~5mL / min;

[0026] ii mobile phase 25% acetonitrile;

[0027] iii. Elution time 30 to 90 minutes;

[0028] IVC 18 Chromatographic column.

[0029] The third aspect of the present invention provides the use of the above-mentioned sesquiterpene lactone or the sesquiterpene lactone prepared by the above-mentioned method in the preparation of a drug for treating and / or preventing cancer.

[0030] According to some embodiments of the invention, the cancer is prostate cancer.

[0031] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0033] Figure 1 is the HRESIMS spectrum of the sesquiterpene lactone compound of the present invention;

[0034] Figure 2 is the IR spectrum of the sesquiterpene lactone compound of the present invention;

[0035] Figure 3 is the UV spectrum of the sesquiterpene lactone compound of the present invention;

[0036] Figure 4 The sesquiterpene lactone compound of the present invention 1 H NMR spectrum;

[0037] Figure 5 The sesquiterpene lactone compound of the present invention 13 C NMR spectrum;

[0038] Figure 6 is the DEPT 135 spectrum of the sesquiterpene lactone compound of the present invention;

[0039] Figure 7 is the HSQC spectrum of the sesquiterpene lactone compound of the present invention;

[0040] Figure 8 The sesquiterpene lactone compound of the present invention 1 H- 1 H COSY spectrum;

[0041] Fig. 9 The HMBC spectrum of the sesquiterpene lactone compound of the present invention is

[0042] Fig.10 The calculated ECD and experimental ECD spectra of the sesquiterpene lactone compound of the present invention;

[0043] Fig.11 The figure is a graph showing the effect of different concentrations of the sesquiterpene lactone compound of the present invention and paclitaxel (2 nM) on the cell viability of prostate cancer cells (PC-3). DETAILED DESCRIPTION

[0044] The following are specific embodiments of the present invention, and the technical solutions of the present invention are further described in conjunction with the embodiments, but the present invention is not limited to these embodiments.

[0045] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0046] The night-scented cowberry plant was collected in Guangxi Zhuang Autonomous Region in June 2022, and the specimens are preserved in Room 204, Ma Lanfang Building, Wuyi University.

[0047] Semi-preparative HPLC was performed on a Waters 1500 (Waters, Milford, USA) series, the detector was a Waters 2489 UV / visible detector (Waters, Milford, USA), and the chromatographic column was OBD column (250×10 mm, 5 μm, Milford, USA). IR data were obtained by using a Thermo Fisher Fourier transform IS5 infrared spectrometer (Thermo, Waltham, USA). UV data were obtained by using a Shimadzu UV-visible spectrophotometer UV-2600 (Shimadzu, Kyoto, Japan). Mass spectrometry data were obtained by using an AB SCIEX X500R QTOF mass spectrometer (ABSCIEX, Redwood, USA). NMR data were recorded by a Bruker AVANCE NEO 500 spectrometer (Bruker, Bremen, Germany) using deuterated chloroform as solvent, and chemical shift values ​​were expressed as δ (ppm) of chloroform in deuterated chloroform. Optical rotation was measured using chromatographic methanol at 20°C using an MCP 200 automatic polarimeter (Anton Paar, Graz, Austria). CD data were obtained by using Chirascan Spectrometer (Applied Photophysics Ltd, Surrey, UK) with methanol as solvent. Series 8000WJ CO2 incubator, 37°C constant temperature water bath, microplate reader and biosafety cabinet were all from Thermo scientific brand in the United States. MS205DU electronic balance was from METTLER TOLEDO in the United States. TD5A-WS low-speed centrifuge was from Zhongxiang Instrument.

[0048] Fillers and reagents

[0049] Column chromatography fillers include normal phase silica gel (200-300, 300-400 mesh) purchased from Qingdao Ocean Chemical Plant; small pore resin (100-20 / 45) purchased from Fu Silysia Chemical Ltd in the United States; Sephadex LH-20 (100-200 mesh) purchased from Beijing Solai Baoneng Biotechnology Co., Ltd.; reversed phase silica gel purchased from Fu Silysia Chemical Co., Ltd. in the United States. GF ​​silica gel plate 254 Purchased from Yantai Institute of Chemical Industry. Deuterated chloroform and chromatography grade acetonitrile were purchased from Anaiji. Cell lines were purchased from ATCC, USA. Molecular biology grade DMSO and thiazolyl blue (MTT) were purchased from Sigma-Aldrich, USA. DMEM culture medium, PBS buffer, penicillin-streptomycin solution (double antibody) and 0.4% trypan blue solution were purchased from Gibco, USA. Fetal bovine serum FBS was purchased from BI, USA.

[0050] Example 1

[0051] Example 1 provides a sesquiterpene lactone having a structure shown in formula (1):

[0052]

[0053] The preparation method is as follows:

[0054] S1, 30.15kg of the whole herb of night-scented cowpea was crushed by a pulverizer and weighed to obtain 29.4kg, with a crushing loss of 0.75kg. 160L of 95% ethanol was used to immerse the medicinal material for the first time, and 64L of 95% ethanol was added for the second and third soakings. Each soaking lasted for 7 days, and the soaking temperature was 25°C. After filtering, the filtrate of the three soakings was collected, and the solvent was evaporated at 45°C using a rotary evaporator to obtain 2262g of extract, with an extraction rate of 7.69%.

[0055] S2. The extract was suspended and dispersed with 10 L of pure water, and then extracted with equal amounts of chloroform, ethyl acetate, and n-butanol, respectively, for 5 times, and the extraction solvent was removed to obtain chloroform, ethyl acetate, and n-butanol fractions.

[0056] S3, the chloroform fraction was taken and distilled under reduced pressure to weigh 777.5 g. The fraction was roughly separated by normal phase silica gel (200-300 mesh) 4000 g dry column chromatography, with the solvent system of n-hexane: ethyl acetate (100:1-1:0, v / v) and methanol, and divided into 9 fractions, Fr1-Fr9.

[0057] S4 and Fr7 (55.8 g) were initially depigmented by a small-pore resin with a solvent system of 30% methanol water-100% methanol and then divided into 9 parts, namely Fr7A-I.

[0058] S5 and Fr7F (55.8 g) were separated using 700 g reverse phase silica gel in a 30% methanol water-100% methanol elution system to obtain five parts, namely Fr7F1-5.

[0059] S6 and Fr7F5 (1.88 g) were separated using 50 g reverse phase silica gel in a 30% methanol water-100% methanol elution system to obtain five parts, namely Fr7F5a-Fr7F5e.

[0060] S7 and Fr7F5b (710.5 mg) were separated and prepared by repeated semi-preparative HPLC. The final purification chromatographic conditions were 5 ml / min, 25% acetonitrile, and 60 min to obtain sesquiterpene lactone (4.3 mg).

[0061] Structural identification of the prepared compounds:

[0062] Colorless oil, (c 0.1,CH3OH); IR(KBr)ν max 3454,1722,1224cm-1 ; UV(CH3OH)λ max (logε)223(4.49),288(4.32)nm; ECD(c 0.0021,CH3OH)λ max (Δε)236(+3.47),275(-8.88)nm; 1 H NMR data (500MHz, CDCl3) δ6.12 (d, J = 8.4Hz, 1H), 6.09 (s, 1H), 5.71 (m, 1H), 5.59 (s, 1H), 5.4 5(d,J=11.5,1H),5.00(d,J=12.6Hz,1H),4.92(d,J=12.6Hz,1H),4.09(dd,J=16.2,13.4H z,1H),3.56(d,J=10.9Hz,1H),3.49(d,J=10.9Hz,3H),2.62(dd,J=16.3,8.4Hz,1H),2.46 (m,2H),2.15(d,J=1.0Hz,3H),2.06(d,J=2.9Hz,3H),1.91(d,J=1.0Hz,3H),1.75(s,3H); 13 C NMR data (126 MHz, CDCl3) δ 170.6, 167.6, 165.0, 160.6, 154.5, 145.2, 132.1, 129.9, 128.3, 126.8, 114.5, 74.0, 73.3, 63.9, 56.1, 55.0, 46.3, 43.4, 31.3, 27.7, 20.9, 20.6; HRESIMS m / z 477.1279 [M+Na] + (calcd for C 22 H 27 ClO8Na,477.1286).

[0063] Based on HRESIMS (such as Figure 1 ) has a molecular ion peak of m / z 477.1279[m+Na] + Its molecular formula was determined to be C 22 H 27 ClO8(C 22 H 27 Calculated value of ClO8Na, 477.1286). Infrared spectrum (such as Figure 2 ) absorption bands indicate the presence of hydroxyl groups (3420, 2928 cm -1 ), carbonyl (1722cm -1 ) and ester groups (1224cm -1 ) functional groups. UV spectra (such as Figure 3) has a strong band at about 288 nm, indicating the presence of conjugated lactones, and a strong absorption band at 236 nm, indicating the presence of unsaturated carbonyl groups.

[0064] 1 H and 13 C NMR (such as Figure 4 and 5 ), DEPT (such as Figure 6 ) and HSQC (such as Figure 7 ) data indicate the presence of seneciate (Sen) groups [δ H 5.59 (1H, s), 2.15 (3H, d, J = 1.0 Hz) and 1.91 (3H, d, J = 1.0 Hz); δ C 165.0, 160.6, 114.5, 27.7 and 20.6]; α, β-unsaturated γ-lactone ring [δ C 167.6, 154.5, 145.2, 128.3]; one acetyl group [δ H 2.06(3Hs);δ C 170.6, 20.9], one chloromethylene [δ H 3.56 (1H, d, J = 10.9Hz), 3.49 (1H, d, J = 10.9Hz); δ C 55.0]; one oxygen-containing methylene group [δ H 4.92 (1H, d, J = 12.6Hz), 5.00 (1H, d, J = 12.6Hz); δ C 56.1]; three double bond carbons [δ H 5.44 (1H, d, J=11.5Hz), 5.71 (1H, m), 6.09 (1H, s); δ C 129.9, 132.1, 126.8]; a quaternary carbon with oxygen (δ C 74.0). The above data suggest that the compound is a hirsutinolide-type sesquiterpene lactone. These data are consistent with (1S * ,4R * ,8S * ,10R * )-1,4-epoxy-8-senecioyloxy-10-hydroxy-13-acetoxy-5Z,7(11)-germacradien-6,12-olide has similar data, except that C-1(δ C 129.9), C-2(δ C 132.1), C-4(δ C 74.0), and C-14 (δ C55.0). These findings indicate that the C-1,4 ether ring in the structure of this compound is broken, a double bond is formed between C-1 and C-2, and a chloromethylene group is located at C-10, which can be confirmed by the H-1[δ H 5.45(1H,d,J=11.5Hz)],H-2[δ H 5.71(1H,m)] and H-3[δ H 2.62 (1H, dd, J = 16.2, 8.4 Hz); 4.09 (1H, dd, J = 16.2, 13.4 Hz)] 1 H- 1 H COSY related (such as Figure 8 ); and HMBC related properties (such as Fig. 9 )H-1[δ H 5.45(1H,d,J=11.5Hz)] / C-14(δ C 55.0); H2-9[δ H 2.46(2H,m)] / C-10(δ C 74.0) proves that the planar structure of the compound is obtained.

[0065] Calculate based on 13 The relative configurations of the compounds were analyzed by DP4+ possibilities of the CNMR data. Conformational searches were performed in the Spartan program for the four possible diastereomers, including 4R * , 8S * , 10R * (a) 4R * , 8S * , 10S * (b) 4R * ,8R * , 10S * (c) and 4R * ,8R * , 10R * (d) 13 The C NMR experimental data are shown in Table 1. In all calculations, the probability of existence of isomers a and b is 99.06% and 0.94% respectively, and the probability of existence of isomers c and d is 0, as shown in Table 2. Therefore, it is inferred that the relative configuration of the compound is consistent with a, which is 4R * , 8S * , 10R * .

[0066] Table 1 Calculation of four enantiomers (ad) of the compound 13 C NMR values

[0067]

[0068]

[0069] By comparing the calculated and experimental ECD spectral data (e.g. Fig.10 ), the absolute configuration of the compound was deduced to be 4R, 8S, 10R, and it was named cyanolide A.

[0070] Example 2

[0071] Anti-prostate cancer activity test

[0072] 1. Experimental Methods

[0073] PC-3 cells were cultured at 5×10 4 Cells were seeded at a density of 10 cells / well in a 96-well cell plate and incubated at 37°C and 5% CO2 for 24 hours. Fresh culture medium containing different compounds with a series of concentration gradients was added to each well and cultured in an incubator for 72 hours. Then 200 μL of MTT solution (0.5 mg / mL) was added to PC-3 cells for 4 hours. After that, the old culture medium was removed and 150 μL of dimethyl sulfoxide was added to each well. After shaking in a microplate reader for 15 minutes, the optical density was measured at 570 nm. The cell viability and IC of each compound were calculated by GraphPad Prism 8.0 software. 50 value.

[0074] 2. Experimental results

[0075] result Fig.11 As shown in Figure 2, the compound has significant antiproliferative activity against prostate cancer cells, and its IC 50 The value was 6.4±0.34μM.

[0076] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge scope of ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. A sesquiterpene lactone or a pharmaceutically acceptable salt thereof, characterized in that: A compound having a structure represented by formula (1): 。 2. The method for preparing sesquiterpene lactone according to claim 1, characterized in that: The steps include: S1, crushing the night-scented sedge, soaking and extracting with an organic solvent, filtering and concentrating the extract to obtain an extract; S2, extracting the extract with chloroform, ethyl acetate, and n-butanol in sequence, removing the extraction solvent, and obtaining a chloroform fraction, an ethyl acetate fraction, and an n-butanol fraction, respectively; S3, separating the chloroform part by column chromatography to obtain 9 parts Fr1~Fr9; S4, removing the pigment from Fr7 through a small-pore resin and dividing it into 9 parts to obtain Fr7A~I; S5, Fr7F was separated by reverse phase silica gel in a methanol water-methanol elution system to obtain five parts, namely Fr7F1~5; S6 and Fr7F5 were separated by reverse phase silica gel in a methanol water-methanol elution system to obtain five parts, namely Fr7F5a~Fr7F5e; S7 and Fr7F5b were separated by several semi-preparative HPLC to obtain sesquiterpene lactones; In step S3, the solvent system of the column chromatography method is n-hexane: ethyl acetate and methanol in a volume ratio of 100 to 0:1; the elution system of steps S5 and S6 is 30% methanol water-100% methanol; In step S7, the chromatographic conditions of the semi-preparative HPLC at least meet one of the following: iFlow rate 3~5 mL / min; ii mobile phase 25% acetonitrile; iii. Elution time: 30-90 min; IV C 18 Chromatographic column.

3. The method for preparing sesquiterpene lactone according to claim 2, characterized in that: In step S1, an organic solvent is used to perform soaking extraction for several times, and the extracts from the several extractions are combined, filtered, and concentrated to obtain an extract.

4. The method for preparing sesquiterpene lactone according to claim 2, characterized in that: In step S1, the organic solvent is selected from 95% ethanol.

5. The method for preparing sesquiterpene lactone according to claim 2, characterized in that: In step S1, the temperature of the soaking extraction is 20-40°C.

6. Use of the sesquiterpene lactone according to claim 1 or the sesquiterpene lactone prepared by the preparation method according to any one of claims 2 to 5 in preparing a drug for treating and / or preventing prostate cancer.

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