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

Abstract

The present invention discloses a hirsutinolide-type sesquiterpene lactone having anti-prostate cancer activity, a preparation method and an application thereof, wherein the hirsutinolide-type sesquiterpene lactone has a compound represented by formula (I); wherein R1 is selected from C 1～10 The hirsutinolide-type sesquiterpene lactone provided by the present invention has significant anti-prostate cancer cell proliferation activity, and its IC 50 Low value.

Description

Technical Field

[0001] The present invention relates to the technical field of natural medicines, and in particular to a hirsutinolide-type sesquiterpene lactone with anti-prostate cancer activity, and a preparation method and application thereof. Background Art

[0002] Prostate cancer is an epithelial malignancy that arises in the prostate gland. It is one of the most common male cancers worldwide, with 1.6 million new cases annually. Treatment options include surgical resection and a combination of medications. Commonly used clinical treatments include steroidal and nonsteroidal antiandrogens, such as cyproterone acetate, bicalutamide, and enzalutamide. However, these drugs have limited efficacy and are associated with varying 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, in a first aspect, the present invention provides a hirsutinolide-type sesquiterpene lactone, which has high anti-prostate cancer activity.

[0005] The second aspect of the present invention further provides a method for preparing hirsutinolide-type sesquiterpene lactone.

[0006] The third aspect of the present invention further provides an application of a hirsutinolide-type sesquiterpene lactone.

[0007] According to the first aspect of the present invention, a hirsutinolide-type sesquiterpene lactone or a pharmaceutically acceptable salt thereof is provided, which has the compound represented by formula (I);

[0008]

[0009] Wherein, R1 is selected from C 1～10 of alkyl.

[0010] The hirsutinolide-type sesquiterpene lactone or a pharmaceutically acceptable salt thereof according to the embodiments of the present invention has at least the following beneficial effects:

[0011] The hirsutinolide-type sesquiterpene lactone provided by the present invention has significant anti-prostate cancer cell proliferation activity, and its IC 50 Low value.

[0012] According to some embodiments of the present invention, the hirsutinolide-type sesquiterpene lactone is selected from one of the following structural formulas:

[0013]

[0014] According to a second aspect of the present invention, an embodiment provides a method for preparing the hirsutinolide-type sesquiterpene lactone described above, comprising the following steps:

[0015] S1. Grinding the night-blooming sedge, extracting it by soaking it in an organic solvent, filtering and concentrating the extract to obtain an extract;

[0016] S2. After dispersing the extract with water, extracting with chloroform, ethyl acetate, and n-butanol in sequence, removing the extraction solvent to obtain the chloroform fraction, ethyl acetate fraction, and n-butanol fraction, respectively;

[0017] S3, using normal phase silica gel column chromatography to separate the chloroform fraction to obtain 9 groups of fractions, namely Fr1-Fr9;

[0018] S4, using Sephadex LH-20 chromatography to remove the pigment in Fr4 and obtain three fractions, namely Fr4A-Fr4C;

[0019] S5. Purify the Fr4B portion using reverse-phase silica gel and semi-preparative HPLC to obtain two compounds of formula (I-1) and formula (I-2), respectively.

[0020] 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.

[0021] According to some embodiments of the present invention, in step S3, the solvent system of the column chromatography method is n-hexane:ethyl acetate (100:1 to 1:0, v / v) and methanol.

[0022] According to some embodiments of the present invention, in step S3, the particle size of the normal phase silica gel is 200-300 mesh.

[0023] According to some embodiments of the present invention, in step S4, the particle size of the Sephadex LH-20 is 100-200 mesh.

[0024] According to some embodiments of the present invention, in step S5, the conditions of the semi-preparative HPLC are as follows:

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

[0026] ii. Mobile phase 70%-100% acetonitrile;

[0027] iii. Elution time: 30 min to 90 min;

[0028] IVC 18 Chromatographic column.

[0029] According to some embodiments of the present invention, in step S1, the organic solvent is 95% ethanol.

[0030] According to some embodiments of the present invention, in step S2, the extraction is performed 3 to 10 times.

[0031] The third aspect of the present invention provides the use of the hirsutinolide-type sesquiterpene lactone described above in the preparation of a drug for treating and / or preventing prostate cancer.

[0032] 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

[0033] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0034] Figure 1 This is the HRESIMS spectrum of the compound of formula (I-1) of sesquiterpene lactone according to Example 1 of the present invention;

[0035] Figure 2 This is the IR spectrum of the compound of formula (I-1) of sesquiterpene lactone according to Example 1 of the present invention;

[0036] Figure 3 This is the UV spectrum of the compound of formula (I-1) of sesquiterpene lactone according to Example 1 of the present invention;

[0037] Figure 4 The sesquiterpene lactone compound of formula (I-1) prepared in Example 1 of the present invention 1 H NMR spectrum;

[0038] Figure 5 The compound of the sesquiterpene lactone formula (I-1) of the embodiment of the present invention is 13 C NMR spectrum;

[0039] Figure 6 This is the DEPT 135 spectrum of the compound of formula (I-1) of sesquiterpene lactone according to Example 1 of the present invention;

[0040] Figure 7 This is the HSQC spectrum of the compound of formula (I-1) of Example 1 of the present invention;

[0041] Figure 8The compound of the sesquiterpene lactone formula (I-1) of the embodiment of the present invention is 1 H- 1 H COSY spectrum;

[0042] Figure 9 This is the HMBC spectrum of the compound of formula (I-1) of sesquiterpene lactone according to Example 1 of the present invention;

[0043] Figure 10 This is the ROESY spectrum of the compound of formula (I-1) of sesquiterpene lactone according to Example 1 of the present invention;

[0044] Figure 11 This is the ECD spectrum of the compound of formula (I-1) of 1-sesquiterpene lactone according to the present invention;

[0045] Figure 12 This is the HRESIMS spectrum of the compound of formula (I-2) of 1-sesquiterpene lactone in the present invention;

[0046] Figure 13 This is the IR spectrum of the compound of formula (I-2) of 1-sesquiterpene lactone according to the present invention;

[0047] Figure 14 This is the UV spectrum of the compound of formula (I-2) of 1-sesquiterpene lactone according to the present invention;

[0048] Figure 15 The compound of the sesquiterpene lactone formula (I-2) of the embodiment of the present invention is 1 H NMR spectrum;

[0049] Figure 16 The compound of the sesquiterpene lactone formula (I-2) of the embodiment of the present invention is 13 C NMR spectrum;

[0050] Figure 17 This is the DEPT 135 spectrum of the compound of formula (I-2) of the sesquiterpene lactone of the present invention;

[0051] Figure 18 The compound of the sesquiterpene lactone formula (I-2) of the embodiment of the present invention is 1 H- 1 H COSY spectrum;

[0052] Figure 19 This is the HMBC spectrum of the compound of formula (I-2) of 1-sesquiterpene lactone according to the present invention;

[0053] Figure 20 This is the HSQC spectrum of the compound of formula (I-2) of 1-sesquiterpene lactone according to the present invention;

[0054] Figure 21This is the ECD spectrum of the compound of formula (I-2) of 1-sesquiterpene lactone according to the present invention;

[0055] Figure 22 Graph showing the effects of the sesquiterpene lactone compound of the present invention at different concentrations and paclitaxel (2 nM) on the cell viability of prostate cancer cells (PC-3). DETAILED DESCRIPTION

[0056] 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.

[0057] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0058] 1. Experimental instruments

[0059] Ultrapure water was prepared using an ultrapure water machine IQ7000. Semi-preparative HPLC was performed on a Waters 1500 series (Waters, Milford, USA), with a Waters 2489 UV / visible detector (Waters, Milford, USA) and a chromatographic column. OBD column (250 × 10 mm, 5 μm, Milford, USA). IR data were obtained using a Thermo Fisher Scientific IS5 Fourier transform infrared spectrometer (Thermo, Waltham, USA). UV data were obtained using a Shimadzu UV-2600 UV-visible spectrophotometer (Shimadzu, Kyoto, Japan). Mass spectral data were obtained on an AB SCIEXX500R QTOF mass spectrometer (AB SCIEX, Redwood, USA). NMR data were recorded on a Bruker AVANCE NEO 500 spectrometer (Bruker, Bremen, Germany) using deuterated chloroform as the solvent. Chemical shifts are expressed as δ (ppm) of chloroform in deuterated chloroform. Optical rotations were determined using chromatographic methanol at 20°C using an MCP 200 automated polarimeter (Anton Paar, Graz, Austria). CD data were obtained using a Chirascan spectrometer (Applied Photophysics Ltd, Surrey, UK) with methanol as the solvent. The Series 8000WJ CO2 incubator, 37°C water bath, microplate reader, and biosafety cabinet were all Thermo Scientific products from the United States. The MS205DU electronic balance was from METTLER TOLEDO, USA. The TD5A-WS low-speed centrifuge was from Xiangyi Instruments, China.

[0060] 2. Experimental Reagents and Fillers

[0061] Acetonitrile for mass spectrometry and chromatography was purchased from Shanghai Titan Reagent Co., Ltd.; ultrapure water was used for HPLC; 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.

[0062] Column chromatography packing materials included 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; and Sephadex LH-20 (100-200 mesh) purchased from Beijing Solai Baoneng Biotechnology Co., Ltd. GF254 silica gel plates were purchased from Yantai Institute of Chemical Industry. Deuterated chloroform and chromatography-grade acetonitrile were purchased from Anage. Cell lines were purchased from ATCC.

[0063] 3. Plant sources

[0064] The night-scented cow plant was collected in Guangxi Zhuang Autonomous Region in June 2022, and the specimen is preserved in Room 204, Ma Lanfang Building, Wuyi University.

[0065] Example 1

[0066] This embodiment provides a hirsutinolide-type sesquiterpene lactone, the structural formula of which is as follows and the preparation method is as follows:

[0067]

[0068] A total of 30.15 kg of the aerial parts of S1 and Herba Lycoris were crushed in a pulverizer to yield 29.4 kg, representing a crushing loss of 0.75 kg. The first soaking was performed using 160 L of 95% ethanol. The second and third soakings were then followed by 64 L of 95% ethanol. Each soaking lasted for 7 days at 25°C. After filtration, the filtrates from the three soakings were collected and the solvent evaporated to dryness using a rotary evaporator at 45°C, yielding 2262 g of an extract with an extraction yield of 7.69%.

[0069] S2. Suspend and disperse the extract in 10L of pure water, then extract with equal amounts of chloroform, ethyl acetate, and n-butanol for 5 times respectively. Remove the extraction solvent to obtain chloroform, ethyl acetate, and n-butanol fractions.

[0070] S3. The chloroform fraction was distilled under reduced pressure and weighed to 777.5 g. This fraction was crudely separated using 4000 g dry-packed column chromatography on normal phase silica gel (200-300 mesh) using a solvent system of n-hexane:ethyl acetate (100:1-1:0, v / v) and methanol, resulting in nine fractions, Fr1-Fr9.

[0071] S4 and Fr4 (30.5 g) contained a lot of pigments, and some of the pigments were removed using Sephadex LH-20 gel. They were then initially segmented based on the molecular sieve principle using a dichloromethane:methanol (1:1, v / v) system, and finally divided into three parts, namely Fr4A-Fr4C.

[0072] The S5 and Fr4B samples were purified and identified using reverse phase silica gel and semi-preparative HPLC to obtain compounds of formula (I-1) and formula (I-2).

[0073] Structural confirmation:

[0074] Compound of formula (I-1). Colorless oil; (c 0.1,CH3OH); IR(KBr)ν max 3678,2971,1763,1055cm -1 ;UV(CH3OH)λ max (logε):217(4.32),285nm(4.03); ECD(c 0.1,CH3OH)λ max (Δε)220(+6.72),292(+3.95); 1 H NMR (500MHz, CDCl3) δ7.05(d,J=7.2Hz,1H),6.25(d,J=8.0Hz,1H),5.89(s,1H),5.11(d,J=12.9H z,1H),5.04(d,J=12.9Hz,1H),3.63(dd,J=8.8,7.1Hz,1H),3.43(dd,J=8.8,7.1Hz,1H),2.45(dd, J=15.8,11.7Hz,1H),2.14(m,1H),2.11(m,1H),2.10(s,3H),1.98(m,1H),1.90(m,1H),1.87(m,1 H),1.84(s,3H),1.80(d,J=7.2Hz,3H),1.48(s,3H),1.20(t,J=7.1Hz,3H),0.91(d,J=6.9Hz,3H); 13C NMR (126MHz, CDCl3) δ170.3,167.8,167.3,150.6,146.5,138.5,129.7,128.5,127.1,111.2 ,80.2,68.4,56.5,55.7,42.6,39.9,36.0,33.2,27.6,20.9,17.0,15.6,14.5,12.0; HRESIMS m / z471.1987[M+Na] + (calcd for C 24 H 32 O8Na,471.1989).

[0075] Compound of formula (I-2). Colorless oil; (c 0.1,CH3OH); IR(KBr)ν max 3649,2921,1763,1225cm -1 ;UV(CH3OH)λ max (logε):218(4.32),285nm(4.05); ECD(c 0.1,CH3OH)λ max (Δε)221(+7.76),292(+6.71); 1 H NMR (500MHz, CDCl3) δ7.03(d,J=6.8Hz,1H),6.23(d,J=8.0Hz,1H),5.86(s,1H),5.07(d,J=12.9Hz,1H),5.01(d,J =12.9Hz,1H),3.55(dt,J=8.8,6.0Hz,1H),3.34(dt,J=8.8,6.0Hz,1H),2.43(dd,J=15.8,11.7Hz,1H),2.13(m,1H ),2.07(s,3H),2.06(m,1H),1.94(m,1H),1.89(m,1H),1.85(m,1H),1.82(s,3H),1.77(d,J=6.8Hz,3H),1.73(ddd ,J=15.8,8.0,1.8),1.53(m,2H),1.46(s,3H),1.39(d,J=7.3Hz,2H),0.92(t,J=7.3Hz,3H),0.88(d,J=6.8Hz,3H); 13C NMR (126MHz, CDCl3) δ170.2,167.9,167.3,150.7,146.6,138.5,129.7,128.5,127.1,111.0,80.3 ,68.4,60.5,55.7,42.9,40.0,36.0,33.1,32.2,27.5,20.9,19.7,17.0,14.5,14.0,12.0; HRESIMS m / z 499.2299[M+Na] + (calcd for C 26 H 36 O8Na,499.2302).

[0076] The compound of formula (I-1) was analyzed by HRESIMS (eg Figure 1 ) molecular ion peak 471.1987[M+Na] + (C 24 H 32 The calculated value of O8Na is 471.1989), and its molecular formula is speculated to be C 24 H 32 O8, contains 9 degrees of unsaturation. Infrared spectrum (such as Figure 2 ) indicates the presence of hydroxyl groups (3678 cm -1 ), carbonyl (1763cm -1 ) and ester groups (1055cm -1 ) functional groups. By UV spectroscopy at about 217, 285 nm (such as Figure 3 ) confirmed the presence of unsaturated carbonyl groups and conjugated lactones.

[0077] 1 H (such as Figure 4 )and 13 C NMR (such as Figure 5 ) is attributed through DEPT (such as Figure 6 ), HSQC (such as Figure 7 ), 1 H- 1 H COSY (such as Figure 8 ), and HMBC (such as Figure 9 ) was determined by spectral analysis. H The characteristic resonance proton signals at 7.05 (1H, d, J = 7.2 Hz), 1.80 (3H, d, J = 7.2 Hz) and 1.84 (3H, s) indicate the presence of tiglate (Tig) groups (δ C 167.8, 128.6, 138.5, 14.5 and 12.0). CThe signals of 167.3, 150.6, 146.5, and 129.7 indicate the presence of an α,β-unsaturated γ-lactone ring. 1 H and 13 C NMR data showed that compound 1 is a hirsutinolide-type sesquiterpene lactone, which is similar to 8β-(2-Methylacry-loyloxy)-hirsubinolide-13(O)-acetate. 1 H and 13 Comparison of C NMR data confirmed that the compound of formula (I-1) is hirsutinolide substituted by ethoxy at C-1. This can be inferred from the H-2″[δ H 1.20 (3H, t, J=7.1Hz)] and H-1″α[δ H 3.43 (1H, dd, J=8.8, 7.1Hz)] and H-1″β[δ H 3.63 (1H, dd, J = 8.8, 7.1 Hz)] 1 H- 1 H COSY correlation and H-1α″ and C-2″ (δ C 15.6), C-1(δ C 111.2) were confirmed by HMBC correlation.

[0078] H-3α[δ H 1.98 (1H, m)] and H-1″α[δ H 3.43 (1H, dd, J=8.8, 7.1Hz)], H3-15[δ H 1.48

[0079] (3H, s)] between ROESY correlation (such as Figure 10 ); H-8[δ H 6.25(1H,d,J=8.0Hz)] and H-9α[δ H 1.75 (1H, ddd, J=15.8, 8.0, 1.7Hz)]; H-9β[δ H 2.45 (1H, dd, J = 15.8, 11.7 Hz) and H3-14 [δ H 0.91 (3H, d, J = 6.9 Hz)] indicates that H-1″α and H3-15 are α-oriented, H3-14 is in β orientation, and H-8 and H-10 are in α orientation, which is consistent with the hirsutinolide-type sesquiterpene lactone naturally occurring in the genus Thunbergia. The ECD spectrum of the compound of formula (I-1) (such as Figure 11) showed positive Cotton effect at 220 and 292 nm, which is consistent with the literature. Therefore, the relative configuration of the compound of formula (I-1) was determined to be 1S, 4R, 8S, 10R.

[0080] HRESIMS of the compound of formula (I-2) (such as Figure 12 ) shows a molecular ion peak [M+Na] at m / z 499.2299 + (calculated value is 499.2301), corresponding to the molecular formula C 26 H 36 O8, including nine degrees of unsaturation. Infrared spectrum (such as Figure 13 ) indicates the presence of hydroxyl groups (3649 cm -1 ), carbonyl (1763cm -1 ) and ester groups (1225cm -1 ) functional groups. UV spectrum (such as Figure 14 ) shows strong broad bands at about 218 and 285 nm, indicating the presence of unsaturated carbonyl groups and conjugated lactones. 1 H (such as Figure 15 ), 13 C NMR (such as Figure 16 ) is based on the DEPT of the compound (e.g. Figure 17 ), HSQC (such as Figure 18 ), 1 H- 1 H COSY (such as Figure 19 )、HMBC(such as Figure 20 ) spectrum was obtained by comprehensive analysis. The 1D NMR data of the compound of formula (I-2) are similar to those of the compound of formula (I-1), except that C-1″[δ H 3.34 (1H, dt, J=8.8, 6.0Hz), 3.55 (1H, dt, J=8.8, 6.0Hz)], C-2″[δ H 1.53(2H,m),δ C 32.2],C-3″[δ H 1.39 (2H, d, J = 7.3 Hz), δ C 19.7] and C-4″[δ H 0.92 (3H, t, J = 7.3 Hz), δ C 14.0]. It is inferred that the compound is a hirsutinolide-type sesquiterpene lactone substituted by butoxy at C-1, which can be inferred from the H-1″α[δ H 3.34 (1H, dt, J=8.8, 6.0Hz)] and H-1″β[δ H3.55 (1H, dt, J = 8.8, 6.0 Hz] and H2-2" [δ H 1.53 (2H, m)], H2-3″[δ H 1.39 (2H, d, J = 7.3 Hz)] and H3-4″ [δ H 0.92 (3H, t, J = 7.3 Hz)] 1 H- 1 H COSY correlation and H-1″ and C-1(δ C 111.0) was confirmed by the HMBC correlation between the compounds. Figure 21 ) data and NMR data are similar to those of the compound of formula (I-1). It is inferred that the relative configuration of the compound of formula (I-2) is the same as that of the compound of formula (I-1).

[0081] Example 2

[0082] The anti-prostate cancer activity experiment was conducted as follows:

[0083] (1) Experimental methods

[0084] PC-3 cells were cultured at a rate of 5 × 10 4 The 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. 200 μL of MTT solution (0.5 mg / mL) was then added to the 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.

[0085] (2) Experimental results

[0086] The hirsutinolide type sesquiterpene lactone of the present invention has significant anti-prostate cancer cell proliferation activity (such as Figure 22 ), IC of the compound of formula (I-1) and the compound of formula (I-2) 50 The values were 29.9±1.46μM and 8.5±0.68μM, respectively.

[0087] 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 of ordinary technicians in the relevant technical field without departing from the scope of the present invention.

Claims

1. A method for preparing hirsutinolide-type sesquiterpene lactones, characterized in that: The steps include: S1. Grinding the night-blooming sedge, extracting it by soaking it in an organic solvent, filtering and concentrating the extract to obtain an extract; S2. After dispersing the extract with water, extracting with chloroform, ethyl acetate, and n-butanol in sequence, removing the extraction solvent to obtain the chloroform fraction, ethyl acetate fraction, and n-butanol fraction, respectively; S3, using normal phase silica gel column chromatography to separate the chloroform fraction to obtain 9 groups of fractions, namely Fr1-Fr9; S4, removing the Fr4 pigment by Sephadex LH-20 chromatography and obtaining three fractions, namely Fr4A-Fr4C; S5. Purify the Fr4B portion using reverse phase silica gel and semi-preparative HPLC to obtain two compounds of formula (I-1) and formula (I-2), respectively; In step S1, the organic solvent is 95% ethanol; in step S3, the particle size of the normal phase silica gel is 200-300 mesh; in step S4, the pore size of the Sephadex LH-20 is 100-200 mesh; the solvent system of the Sephadex LH-20 chromatography method is dichloromethane:methanol 1:1, v / v; In step S3, the solvent system of the column chromatography method is n-hexane:ethyl acetate 100:1 to 1:0, v / v and methanol; In step S5, the conditions of the semi-preparative HPLC are as follows: iFlow rate 3-5 mL / min; ii. Mobile phase 70%-100% acetonitrile; iii. Elution time: 30 min to 90 min; IVC 18 Chromatographic columns; The hirsutinolide-type sesquiterpene lactone is selected from the following structural formula:

2. The preparation method according to claim 1, characterized in that 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.

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