Polyoxopregnane aromatic acid ester as well as preparation method and application thereof

Abstract

The invention discloses polyoxopregnane aromatic acid ester as well as a preparation method and application thereof. A polyoxopregnane aglycone compound 1 is obtained by separating and purifying metaplexis japonica C21 steroid total aglycone, a nicotinate derivative 2 of the polyoxopregnane aglycone compound 1 is synthesized, and multidrug resistance hepatoma carcinoma cell model research finds that the compound 2 can remarkably reverse the drug resistance of a cell model to P-gp substrate chemotherapeutic drugs; when the compound 2 is combined with paclitaxel, the paclitaxel-induced HepG2 / Dox cell apoptosis can be obviously enhanced. The compound 2 serving as a non-substrate inhibitor is combined with P-gp, efflux of P-gp-mediated substrate anticancer drugs is remarkably inhibited, and therefore multi-drug resistance tumor cells caused by P-gp high expression can obtain sensitivity to P-gp substrate chemotherapeutic drugs again. The compound 2 disclosed by the invention has an application prospect of being developed into a specific adjuvant of taxane or vinblastine anticancer drugs, and the curative effect on P-gp mediated multidrug resistance tumors is improved.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular discloses a polyoxypregnane aromatic ester, a preparation method and an application thereof. Background Art

[0002] After exposure to a single anticancer drug, tumor cells develop resistance not only to the drug but also to multiple other anticancer drugs with different structures and mechanisms of action. This cross-resistance is known as multidrug resistance (MDR). MDR is the primary cause of tumor recurrence, metastasis, chemotherapy failure, and mortality in over 90% of cancer patients. Tumor cells can develop MDR through various pathways, with increased drug efflux being one of the key factors contributing to MDR. Overexpression of a class of energy-dependent efflux pumps, called ATP-binding cassette (ABC) transporters, in tumor cells is considered a major mechanism of MDR. ABC transporters belong to the transmembrane protein superfamily, of which P-glycoprotein (P-gp) is the most extensively studied. This transporter utilizes the energy from ATP hydrolysis to mediate the efflux of a wide variety of drugs with diverse structures. P-gp is known to be involved in the efflux of over 200 drugs, reducing their intracellular accumulation and thus contributing to cellular resistance. Overcoming MDR in tumor cells and improving chemotherapy efficacy are key challenges in anticancer drug development. MDR reversal agents can enhance or restore the sensitivity of multidrug-resistant tumor cells to chemotherapeutic drugs. Developing tumor cell MDR reversal agents or modulators is an important strategy for addressing multidrug resistance in tumors. To date, a variety of MDR reversal agents or modulators have been developed, most of which are small molecule compounds with different chemical structures but exhibit P-gp inhibitory effects. These include early first-generation P-gp inhibitors such as verapamil and cyclosporine A, and second-generation dexverapamil, as well as third-generation P-gp inhibitors discovered in the past decade or so, such as Zosuquidar and Tariquidar. The former has not been used clinically due to significant toxic side effects, while the latter has entered clinical trials but has not been approved for marketing.

[0003] Traditional Chinese medicine and natural products are important resources for discovering MDR reversal agents with high activity and low toxicity. 21 Steroids are a class of compounds that have the effect of reversing MDR. 21Steroids are a class of steroidal compounds with a core structure composed of 21 carbon atoms linked together in a carbon-carbon pattern, including pregnane and its ester derivatives. Patent CN1911234A discloses the use of a class of steroidal compounds in the preparation of agents for reversing multidrug resistance in tumor cells. These steroidal compounds are derivatives of tongguangsan aglycone B, dihydrocarbendazole, or tongguangsan aglycone C. Their in vitro activity in reversing tumor MDR is similar to that of verapamil. Therefore, providing a new compound with lower inherent toxicity and side effects but more potent efficacy in reversing multidrug resistance in tumor cells as a tumor MDR reversal agent has significant application prospects. Summary of the Invention

[0004] The object of the present invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide a polyoxypregnane aromatic ester, wherein the polyoxypregnane aromatic ester is 3β, 12β, 14β, 17β, 20(S)-pentahydroxy-5α-pregnane-3β-O-nicotinate-12β-O-(E)-cinnamate.

[0005] The second object of the present invention is to provide a method for preparing the above-mentioned polyoxypregnane aromatic ester.

[0006] The third object of the present invention is to provide the use of the above-mentioned polyoxypregnane aromatic ester in the preparation of drugs for reversing tumor multidrug resistance.

[0007] The fourth object of the present invention is to provide the use of the above-mentioned combination of polyoxypregnane aromatic ester and tumor chemotherapy drug in the preparation of drugs for reversing tumor multidrug resistance or drugs for treating tumors.

[0008] The fifth object of the present invention is to provide a drug for reversing tumor multidrug resistance containing the above-mentioned polyoxypregnane aromatic ester.

[0009] The sixth object of the present invention is to provide a pharmaceutical composition for reversing tumor multidrug resistance, comprising the above-mentioned polyoxypregnane aromatic ester and tumor chemotherapy drugs.

[0010] The above-mentioned object of the present invention is achieved through the following technical solutions: The present invention provides a polyoxypregnane aromatic ester, wherein the polyoxypregnane aromatic ester is 3β, 12β, 14β, 17β, 20(S)-pentahydroxy-5α-pregnane-3β-O-nicotinate-12β-O-(E)-cinnamate; the structural formula thereof is shown in the following formula (II): .

[0011] The present invention obtains a polyoxypregnane aglycone 3β, 12β, 14β, 17β, 20(S)-pentahydroxy-5α-pregnane-12β-O-(E)-cinnamate (Compound 1, with a structural formula shown in Formula (I) below) extracted from the Apocynaceae plant Asclepias ciliata. Based on the possibility of changing the biological activity by esterifying the natural product with nicotinate, Compound 1 having a monoaryl pregnane ester structure is further prepared into its nicotinate ester derivative (Compound 2), namely 3β, 12β, 14β, 17β, 20(S)-pentahydroxy-5α-pregnane-3β-O-nicotinate-12β-O-(E)-cinnamate (Compound 2, with a structural formula shown in Formula (II)).

[0012] The present invention provides a method for preparing the above-mentioned polyoxypregnane aromatic ester, wherein the compound 1 represented by formula (I) is subjected to nicotinoylation reaction and purified to obtain the above-mentioned polyoxypregnane aromatic ester (compound 2). .

[0013] Furthermore, the nicotinoylation reaction is a reaction of compound 1 with nicotinoyl chloride.

[0014] Furthermore, the molar ratio of compound 1 to nicotinoyl chloride is 1:2-4.

[0015] Furthermore, the catalyst for the reaction is 4-dimethylaminopyridine or anhydrous pyridine.

[0016] Furthermore, the purification method is silica gel column chromatography.

[0017] Specifically, the chemical reaction equation of the polyoxypregnane aromatic ester is:

[0018] Furthermore, the preparation method of compound 1 is: S1. The radish powder was extracted with ethanol, ethyl acetate, and hydrolyzed with sulfuric acid to obtain radish total aglycones; S2. The total aglycone of Asclepias rutaecarpa was subjected to column chromatography and recrystallized from acetone to obtain Compound 1.

[0019] Furthermore, in step S2, the mobile phase of the column chromatography is petroleum ether:acetone = 80:20 (volume ratio).

[0020] Furthermore, the stationary phase of the column chromatography in step S2 is 200-300 mesh silica gel.

[0021] The present invention further used a P-glycoprotein (P-gp) overexpression MDR liver cancer cell (HepG2 / Dox) model to study and found that compound 2 at a non-cytotoxic concentration (10 μM) can significantly reverse the cell resistance to P-gp substrate chemotherapy drugs paclitaxel (PTX) and vinblastine, with reversal factors of 335.8 and 140.0 times, respectively, which are generally better than 118.5 and 198.3 times of compound 1 and 103.3 and 66.1 times of the positive control verapamil. The present invention found that 500 nM PTX could not induce apoptosis of HepG2 / Dox cells, but the combination of 10 μM compound 2 and 500 nM PTX can significantly enhance PTX-induced apoptosis of HepG2 / Dox cells ( P <0.001). Further studies found that compound 2, used alone or in combination with PTX, did not affect the expression of P-gp in HepG2 / Dox cells, but could significantly inhibit the efflux of the fluorescent substrate Rhodamine 123 mediated by P-gp ( P <0.001); in the Caco-2 cell monolayer transport experiment, compound 2 behaved as a non-efflux low permeability compound [Papp value (AP-BL) was 0.27×10 -6 cm·s -1 , ER value 0.89]; molecular docking results showed that compound 2 has the potential to form a stable complex with P-gp (binding energy -8.4 kcal / mol). These data indicate that compound 2 does not affect P-gp expression but can bind to P-gp as a non-substrate, inhibiting P-gp's substrate efflux function, thereby restoring sensitivity to substrate-based chemotherapeutic drugs in tumor cells with high P-gp expression. Compound 2 specifically enhances the sensitivity of P-gp-overexpressing MDR liver cancer cells to paclitaxel and vinblastine, demonstrating promising prospects for the development of MDR-reversing agents for the treatment of drug-resistant tumors.

[0022] Therefore, the present invention provides the use of polyoxypregnane aromatic esters in preparing drugs for reversing tumor multidrug resistance.

[0023] The present invention also provides the use of the composition of the polyoxypregnane aromatic ester and tumor chemotherapy drugs in the preparation of drugs for reversing tumor multidrug resistance or drugs for treating tumors.

[0024] Furthermore, the tumor chemotherapy drug is a P-glycoprotein substrate drug.

[0025] Furthermore, the P-glycoprotein substrate drugs are taxanes and vinblastines.

[0026] Preferably, the taxane drug is paclitaxel; and the vinca alkaloid drug is vinblastine.

[0027] Furthermore, the tumor is a tumor that overexpresses P-glycoprotein.

[0028] Furthermore, the drug achieves treatment by increasing the accumulation of tumor chemotherapy drugs in tumor cells.

[0029] The present invention also provides a drug for reversing multidrug resistance of tumors, wherein the drug contains the polyoxypregnane aromatic ester.

[0030] The present invention also provides a pharmaceutical composition for reversing multidrug resistance of tumors, wherein the pharmaceutical composition contains the polyoxypregnane aromatic ester and tumor chemotherapy drugs.

[0031] Furthermore, the medicine also includes other pharmaceutically acceptable excipients.

[0032] Furthermore, the auxiliary material is an excipient or a drug carrier.

[0033] Furthermore, the dosage form of the drug is a pharmaceutically acceptable dosage form, including tablets, capsules, granules, oral liquids, sustained-release preparations, controlled-release preparations, nanoformulations, or injections.

[0034] Furthermore, the tumor is liver cancer.

[0035] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a polyoxypregnane aromatic ester and its preparation method and application. 21 The steroidal aglycones 3β, 12β, 14β, 17β, 20(S)-pentahydroxy-5α-pregnane-12β-O-(E)-cinnamate (Compound 1) were isolated and purified, and its nicotinoyl derivative, 3β, 12β, 14β, 17β, 20(S)-pentahydroxy-5α-pregnane-3β-O-nicotinate-12β-O-(E)-cinnamate (Compound 2), was synthesized. Using a P-glycoprotein (P-gp)-overexpressing MDR hepatocellular carcinoma cell model (HepG2 / Dox), the present invention demonstrated that Compound 2 significantly reversed the cell resistance to the P-gp substrate chemotherapy drugs doxorubicin, paclitaxel, and vinblastine at non-cytotoxic concentrations. The present invention found that 500 nM PTX could not induce apoptosis in HepG2 / Dox cells, but the combination of 10 μM compound 2 and 500 nM PTX could significantly enhance PTX-induced apoptosis in HepG2 / Dox cells ( P<0.001). Further studies revealed that compound 2, used alone or in combination with PTX, did not affect P-gp expression in HepG2 / Dox cells. Instead, it acted as a non-substrate inhibitor, binding to P-gp and significantly inhibiting the efflux of P-gp-mediated substrate anticancer drugs. This restored sensitivity to P-gp substrate chemotherapy drugs in multidrug-resistant tumor cells caused by overexpression of P-gp. Therefore, compound 2 has the potential to be developed as a specific adjuvant (modulator) for taxanes or vinca alkaloids, potentially improving the efficacy of P-gp-mediated multidrug-resistant tumors in clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The results of the detection of P-gp expression in HepG2 and HepG2 / Dox cells are shown in Table 1. Figure 1 A in the figure is the protein expression observed by Western blot, and B is the quantitative analysis of protein bands. Note: The data are the mean ± standard deviation of the results of three repeated experiments. Compared with HepG2, ** P <0.01.

[0037] Figure 2 The results show that compound 2 significantly enhances paclitaxel-induced apoptosis in HepG2 / Dox cells. Note: a. Cell control group, b. Paclitaxel (500 nM) group, c. (Paclitaxel 500 nM + Verapamil 10 μM) group, d. Compound 1 (10 μM) group, e. Compound 2 (10 μM) group, f. (Paclitaxel 500 nM + Compound 1 10 μM) group, g. (Paclitaxel 500 nM + Compound 2 10 μM) group. Note: Compared with the paclitaxel (500 nM) group, * P <0.05,** P <0.01,*** P <0.001.

[0038] Figure 3 Compound 2 has no significant effect on P-gp expression in HepG2 / Dox cells. Figure 3 A and C are Western Blot observations of protein expression; B and D are quantitative analyses of protein bands.

[0039] Figure 4 The combination of compound 2 and paclitaxel had no significant effect on P-gp expression in HepG2 / Dox cells. Figure 4 A is the Western Blot method to observe protein expression, and B is the quantitative analysis of protein bands.

[0040] Figure 5 Compound 2 significantly inhibited the efflux of Rh-123 by HepG2 / Dox cells. Figure 5 Figure A shows the intracellular fluorescence intensity observed by flow cytometry, and Figure B shows the quantification of intracellular fluorescence intensity. Note: Negative: cells without Rh-123; Control: cells with Rh-123. # P <0.05, compared with the negative control group; *** P <0.001, compared with the paclitaxel (500 nM) group.

[0041] Figure 6 The visualization results of the molecular docking between P-gp and compounds 1 and 2 (left: three-dimensional image, right: two-dimensional docking image). Figure 6 A in FIG1 represents the docking of compound 1 with P-gp; B represents the docking of compound 2 with P-gp. DETAILED DESCRIPTION

[0042] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0043] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0044] 1. Materials and Reagents The plant was collected from Jianshui County, Honghe Prefecture, Yunnan Province, and was identified by Peng Hua, a researcher at the Kunming Institute of Botany, Chinese Academy of Sciences, as Asclepias spp. (Apocynaceae). Metaplexis japonica(Thunb.) Makino.) specimens (#LM2021-0704) are stored in the Phytochemistry Laboratory, Science and Technology Innovation Center, Guangzhou University of Chinese Medicine. All organic solvents and chemical reagents used in the extraction and preparation procedures were of domestic analytical grade. Paclitaxel (PTX) (lot number 0000211019), doxorubicin (Dox) (lot number BCCG1786), and vinblastine (VBL) (lot number T1668) were purchased from Sigma, USA; verapamil (VRP) (lot number L14098) was purchased from MP Biomedicals, USA; RPMI1640 (lot number 2251341), trypsin (lot number 2756239), and PBS (lot number 2314093) were purchased from Biological Industries, USA; fetal bovine serum (FBS) (lot number 2534396) was purchased from Gibco, USA; CCK-8 cell counting kit (lot number GK10001) was purchased from GLPBIO, USA; and Prestain protein Maker (lot number MPC2403011), Wuhan Sevier Biotechnology Co., Ltd.; P-gp antibody (lot number H681201059), Hangzhou Huaan Biotechnology Co., Ltd.; GAPDH antibody (lot number AF7021), Affinity Biotechnology Co., Ltd.; rabbit secondary antibody (lot number 8715), SAB Biotechnology Co., Ltd.; BCA protein concentration assay kit (lot number P1002), Rhodamine 123 (Rh-123) (lot number C2007), and Annexin-V / PI double staining kit (lot number 103123240530), Shanghai Beyotime Biotechnology Co., Ltd.

[0045] The test compound was prepared by the inventors, and the purity was ≥98.0% as determined by HPLC. The specific method is as shown in Example 1.

[0046] 2. Main instruments AVANCE NEO Ascend 600 and AVANCE NEO 400 nuclear magnetic resonance spectrometers, Bruker, Germany; 1290 Infinity II-6546 ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometer and LC1260 high-performance liquid chromatograph, Agilent, USA; Nicolet iS20 infrared chromatograph, Thermo Scientific, USA; HF90 CO2 constant-temperature incubator, Shanghai Likang Medical Equipment Co., Ltd.; CK40 inverted optical microscope, Olympus, Japan; OptiClean-1300 vertical flow clean bench, Shanghai Zhicheng Analytical Instrument Co., Ltd.; MK3 microplate reader, Thermometer Fisher, USA; 041BR159108 electrophoresis instrument and ChemiDoc chemiluminescence scanner, Bio-Rad, USA; CytoFLEX flow cytometer, Beckman Coulter, USA.

[0047] Example 1 Preparation of test compounds 1. Experimental Methods 1. 3.56 kg of dried coarse powder of Asclepias rutaecarpa (A. rutaecarpa) was extracted with 8 times the volume of 90% ethanol by heating and refluxing four times for 2 hours each time. The combined extracts were then removed by evaporation under reduced pressure, and the extracts were extracted five times with ethyl acetate. The layers were separated, the ethyl acetate was combined, and the extract was evaporated to dryness under reduced pressure (208.0 g). The ethyl acetate extract (104.0 g) was dissolved in 95% ethanol (1000 mL), and 1000 mL of 0.20 mol / L aqueous H₂SO₄ was added. The extract was heated under reflux for 1 hour, cooled to 40°C, and 30% aqueous NaOH was added to a pH of 7. The ethanol was removed by evaporation under reduced pressure, and the extracts were extracted five times with ethyl acetate. The layers were separated, the ethyl acetate was combined, and the extract was evaporated to dryness under reduced pressure (92.1 g). This solvent fraction tested positive for the Liebermann-Burchard reaction and was designated as Asclepias rutaecarpa total aglycones. Total aglycones from Asclepias ciliata (60.0 g) were separated by column chromatography (3000 g of 200-300 mesh silica gel as the stationary phase and petroleum ether (boiling range 60°C-90°C; the same below)-acetone (95:5-70:30, v / v) as the mobile phase). 200 mL of each fraction was collected and analyzed for chemical composition by silica gel thin-layer chromatography (developed with 3% vanillin-6% perchloric acid). Identical fractions were combined. The petroleum ether-acetone (80:20) eluate (fractions 17-19) were combined and repeated for silica gel column chromatography. Recrystallization from acetone yielded the monomer compound 1 (3.40 g).

[0048] 2. Compound 1 (50 mg; 0.10 mmol), 4-dimethylaminopyridine (25 mg; 0.30 mmol), and nicotinoyl chloride (35 mg; 0.25 mmol) were placed in a 25 mL round-bottom flask. Anhydrous pyridine (0.5 mL) was added and mixed thoroughly. The mixture was stirred magnetically at room temperature (27°C) for 3 h. Silica gel thin-layer chromatography (TLC) confirmed the disappearance of the reaction spot. 2 mL of ethanol was added, the mixture was allowed to stand at room temperature for 0.5 h, and 1 mL of water was added. The mixture was evaporated to dryness under reduced pressure. Compound 2 (44.5 mg) was isolated and purified by silica gel column chromatography (CC) with a yield of 74%. The solvents for TLC and CC were dichloromethane-methanol (98:2, v / v).

[0049]

[0050] 2. Experimental Results Compound 1, white powder. UV (MeOH) λ max (log ε ): 205 (2.65), 217 (2.64), 280 (2.79)nm. IR (KBr) ν max 3388 (broad and strong), 2930, 2859, 1780 (broad and strong), 1636, 1449, 1283, 1204, 1171, 1034, 984, 957, 770, 713, 685 cm -1 High-resolution electrospray ionization mass spectrometry (HR-ESIMS) (positive ionization mode) m / z 521.2860 (C 30 H 42 O6+ Na) + (Calculated value 521.2874). 1 H-and 13 C-NMR data: δ H 7.752 (1H, d, J = 16.2 Hz), 7.547 ~ 7.565 (2H, m), 7.417 ~7.444 (3H, m), 6.446 (1H, d, J = 16.2 Hz) and δ C 166.0 (C-1′), 146.3 (C3′), 133.9 (C-4′), 130.8 (C-7′), 129.0 (C-6′, C-8′), 128.3 (C-5′, C-9′), 117.3 (C-2′) showed trans-cinnamoyl (Cin; C9 fragment). This is consistent with cinnamate substituted pregnane (C 21+C9) structure. H 4.679 (1H, dd , J = 9.6, 4.8 Hz) is the characteristic of 12α-H of pregnane, which is in the lower field, indicating that the 12β-hydroxyl group is esterified; the heteronuclear multiple bond correlation (HMBC) spectrum indicates that the 12α-H and the cinnamoyl carbonyl (δ C 166.1) remotely related, proving that cinnamoyl and 12 β -oxygen ester. A characteristic quartet proton signal in the hydrogen spectrum (δ H 3.641, J = 6.0 H Z ) and a doublet methyl signal (δ H 1.119, J = 6.0 H Z , 21-CH3) indicates the presence of 20-hydroxyl and the absence of a proton at C-17 to couple with 20-hydrogen; δ H The septet proton at 3.623 is consistent with the 3α-hydrogen signal characteristics. Therefore, the structure of compound 1 is determined to be 3β, 12β, 14β, 17β, 20( S )-pentahydroxy-5α-pregnant-12β- O -( E )-cinnamate. Compound 1 was previously used as a C 21 Steroidal aglycone tomentodin [12β- O -( E )-cinnamoyl-20(S)- O The saponification product of [-acetyl-tomentogenin] has been reported before, but this is the first time that compound 1 has been isolated and identified from plants as a natural product. 1 H-and 13 C-NMR data are shown in Table 1.

[0051] Compound 2, white amorphous powder. HR-ESIMS (positive ion mode) m / z 604.3271 (C 36 H 45 O7N + H) + (Calculated value 604.3269) shows the molecular formula is C 36 H 45 O7N, namely the nicotinic acid ester (Nic) of compound 1 (C 30 H 41 O6+ C6H4ON). Compound 2 13 C-NMR showed 36 carbon signals, supporting that it was the mononicotinate of 1.1 The H-NMR spectrum shows three groups of methyl signals of steroidal aglycones in the high field region. δ H 1.341 ( s )、1.122( d , 6.4 Hz) and 0.908 ( s ),in δ H The doublet proton at 1.122 is located at δ H 3.647 protons ( d , 6.4 Hz) have a coupling relationship, indicating that the latter should be attributed to the 20-hydroxy carbon-hydrogen group, the 20-hydroxyl group is not esterified, and the 17-carbon group is connected to a tertiary hydroxyl group, which is consistent with the characteristics of compound 1; the NMR spectrum of compound 2 shows a cinnamoyl fragment: δ H 7.755 and 6.444 (1H each, dd , J = 15.6, 2.0 Hz, 3'-H and 2'-H), hydrogen-hydrogen correlation spectrum ( 1 H- 1 H Correlated spectroscopy, 1 H- 1 H COSY) showed that the two were coupled to each other; J = 2.0 Hz, indicating that the two undergo long-range coupling with the 5'-H and 9'-H of the benzene ring), 7.54 ~ 7.56 (2H, m , 5'-H and 9'-H), 7.37 ~ 7.41 (4H, m , 4''-H, 6'-H, 7'-H and 8'-H); Nicotinate fragment: δ H 9.202 (1H, s , 2''-H), 8.756 (1H, d , J = 4.8Hz, 6''-H), 8.293 (1H, dd , J = 8.0, 4.8 Hz, 5''-H), 7.42 ~ 7.37 (4H, m , 4''-H, 6'-H, 7'-H and 8'-H; COSY spectrum shows that δ H 7.390 is present with nicotinic acid 5''-H signal δ H 8.756 coupled 4''-H); δ C150.8 (C-2''), 126.7 (C-3''), 123.3 (C-4''), 137.1 (C-5''), 153.2 (C-6''), 164.7 (C-7'')]. Since both the 3β-hydroxyl group and the 12β-hydroxyl group of compound 2 are esterified, the electron-withdrawing effect of the ester group makes the two characteristic protons δ 4.993 ( hept , 4.8 Hz), 4. 692 ( dd , 11.6, 4.4 Hz) are in low field and therefore designated as 3α-H and 12α-H, respectively; δ H The proton signal at 4.692 is consistent with the carbon and hydrogen of 12β-cinnamate of 1, and the lower-field septet proton should be the carbon and hydrogen of 3β-nicotinate. δ H The singlet protons at 4.491 and 3.442 1 H- 1 There are no relevant protons and carbon cores in the H COSY spectrum and HMBC spectrum, which should be hydroxyl or water peak signals. δ H 1.122 21-methyl proton not only with δ H 3.647 20-H signal (COSY spectrum); also related to δ C The carbon signals at 71.4 and 88.1 are correlated (HMBC spectrum), indicating that the two carbons are C-20 and C-17 respectively. Therefore, the structure of compound 2 is confirmed to be 3β, 12β, 14β, 17β, 20( S )-pentahydroxy-5α-pregnant-3β- O -nicotinate-12β- O -( E )-cinnamate. Compound 2 1 H-and 13 C-NMR data are shown in Table 1.

[0052] Table 1 Compounds 1 and 2 1 H-NMR and 13 C-NMR data attribution

[0053] Note: Compound 1 1 H-NMR or 13 C-NMR chemical shift ( δ , ppm) were measured in CDCl3 (with TMS as internal standard) at 600 / 150 MHz. The NMR data of compound 2 were measured in CDCl3 (with TMS as internal standard) at 400 / 100 MHz. The attribution of proton signals is referenced to1 H- 1 H coupling spectrum ( 1 H- 1 The carbon multiplicity is assigned according to broadband decoupling spectroscopy, distortionless enhanced polarization transfer (DEPT) spectroscopy, and heteronuclear multiple bond correlation (HMBC) spectroscopy ( 1 H→ 13 C) Feature confirmation.

[0054] Example 2 Study on the effect of test compounds on reversing tumor MDR 1. Experimental Methods 1. Cells and cell culture Human hepatocellular carcinoma cells (HepG2) were purchased from the Cell Bank of the Chinese Academy of Sciences. HepG2 / Dox, an MDR substrain that overexpresses P-glycoprotein induced by doxorubicin, was kindly provided by Professor Fang Hongxun of the City University of Hong Kong. Cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% anti-antibody. HepG2 / Dox cells were cultured at 1.2 μM doxorubicin to maintain drug resistance. Cell culture was maintained in a 37°C, humidified, 5% CO2 incubator. HepG2 / Dox cells were cultured in doxorubicin-free medium for one week before use in experiments.

[0055] 2. Detection of tumor cell viability by CCK-8 assay The cells were collected by trypsin digestion and made into a density of 5×10 4 mL -1 The single cell suspension was seeded into 96-well plates at a rate of 100 μL per well and incubated in an incubator until the confluence reached about 60%. The culture medium was replaced with 100 μL of fresh culture medium containing different concentrations of P-gp substrate anti-tumor compounds (paclitaxel (PTX), doxorubicin (Dox), vinblastine (VBL)) and test compounds (Compound 1, Compound 2) at a certain concentration. Three replicate wells were set for each drug concentration, and a cell-free culture medium blank group, a drug-free cell control group, and a positive drug (VRP) control group were also set up. After 48 hours of drug action, the cell viability of each group was detected according to the instructions of the CCK-8 kit, and the inhibitory ability of PTX, Dox, and VBL on cell proliferation was analyzed. The half inhibitory concentration (IC50) was used as the control group. 50 ) is expressed as follows. According to the formula, drug resistance multiple = IC 50(HepG2 / Dox） / IC 50(HepG2) The drug resistance of HepG2 / Dox cells to drugs was calculated based on the IC values ​​of PTX, Dox and VBL alone or in the presence of a certain concentration of the test compound on cell proliferation. 50 The value is to evaluate whether the test compound can enhance the drug sensitivity of cells and reverse drug resistance.

[0056] 3. Detection of cell apoptosis by Annexin V / PI double staining 2 mL was added to each well to obtain 2 × 10 5 mL -1 HepG2 / Dox cell suspensions were seeded into 6-well plates and incubated for 24 h. The culture medium was replaced with fresh culture medium containing different concentrations of drugs and test compounds. A control group, an Annexin V-FITC single staining group, and a PI single staining group were set up and incubated for 24 h. All culture medium and cells were collected (digested with EDTA-free trypsin), centrifuged (1000 rpm, 5 min), and the supernatant was removed. The cells were resuspended in PBS and counted. 2 × 10 5 The cells were placed in a 1.5 mL EP tube and stained according to the instructions of the Annexin V / PI kit. Cell apoptosis was detected by flow cytometry.

[0057] 2. Experimental Methods 1. Compound 2 significantly reversed the MDR of HepG2 / Dox cells Many clinically used cancer chemotherapy drugs are transport substrates of P-gp. Therefore, overexpression of P-gp can lead to drug resistance of tumor cells to a variety of substrate drugs. Compared with sensitive HepG2 cells, the sensitivity of HepG2 / Dox cells to P-gp transport substrate drugs Dox, PTX and VBL is significantly reduced, with resistance multiples as high as 35.5, 130 and 140 times, respectively. Overexpression of P-gp in HepG2 / Dox cells leads to its significant drug resistance ( Figure 1 and Table 2).

[0058] The present invention investigates the effect of the diarylpregnantate compound 2 on P-gp-mediated MDR. The results (Table 2) show that compounds 1 and 2, at their non-cytotoxic concentrations of 5 μM and 10 μM (cell viability >90%, data not shown), can significantly reduce the resistance of HepG2 / Dox cells to the three drugs in a dose-dependent manner; compounds 1 and 2 even completely reverse the cell resistance to PTX and VBL at 10 μM, with sensitization factors reaching 118.5-fold and 198.3-fold (1) or 335.8-fold and 140-fold (2), respectively, and the sensitization effect is stronger than that of the positive control VRP).

[0059] Table 2 Reversal effects of compounds 1 and 2 on P-gp-mediated MDR in HepG2 / Dox cells

[0060] Note: a Data are expressed as mean ± SD (n = 3); b Fold of resistanceres ) =IC 50 (HepG2 / Dox） / IC 50 (HepG2) ; c Fold increase in drugsensitivity (F sen ) = Drug IC 50 / IC of drug and modulator combinations 50 . With drug IC 50 compared to, *** P <0.001, and **** P <0.0001. F res ≤1.0 indicates that drug resistance is completely reversed.

[0061] 2. Compound 2 can significantly enhance paclitaxel-induced apoptosis in HepG2 / Dox cells The effects of compound 1 or 2 on paclitaxel-induced MDR cell apoptosis were further investigated. Figure 2 As shown in the figure, 500 nM paclitaxel alone could not induce apoptosis in drug-resistant HepG2 / Dox cells. However, the combination of non-cytotoxic 1 or 2 (10 μM) with paclitaxel significantly increased the apoptosis rate (*** P <0.001), with a stronger effect than the combination of VRP and paclitaxel. Compounds 1 and 2 significantly enhanced paclitaxel-induced P-gp-mediated apoptosis in MDR tumor cells, with compound 2 showing a significantly greater effect than compound 1.

[0062] Example 3 Study on the Mechanism of Reversal of Tumor MDR by Test Compounds 1. Experimental Methods 1. Western blot analysis of intracellular P-gp expression Cells were incubated in 100 mm culture dishes in growth medium containing drugs or test compounds for 24 hours and then lysed on ice using RIPA cell lysis buffer (RIPA:PMSF = 100:1) containing PMSF. The cell lysate was collected and centrifuged (4°C, 12,000 rpm, 30 min) to remove the pellet. The supernatant (total protein) was assayed for protein concentration using a BCA assay kit. Protein samples were then separated by SDS-PAGE electrophoresis and electrotransferred to a PVDF membrane. The PVDF membrane was blocked in 5% skim milk for 1 hour at room temperature and incubated with the primary antibody against the target protein overnight at 4°C, followed by incubation with the secondary antibody for 1 hour at room temperature. Protein bands were visualized using a chemiluminescence imager and scanned. Protein grayscale values ​​were analyzed using Image J.

[0063] 2. P-gp functional test in HepG2 / Dox cells HepG2 / Dox cells were collected by trypsin digestion and made into a cell density of 5 × 10 5 mL -1 Prepare a single-cell suspension. Add 1 mL of cell suspension to a 1.5 mL EP tube and centrifuge (6000 rpm, 5 min). Discard the supernatant. Resuspend the cells in 1 mL of fresh culture medium containing 5 μM Rh-123 with or without the test compound or VRP. Place the EP tube in a 37°C, 5% CO2 incubator for 40 min. Place the EP tube on ice to terminate the reaction. After complete cooling, centrifuge (4°C, 6000 rpm, 5 min) and discard the supernatant. Wash the cells twice with ice-cold PBS and resuspend in 300 μL of PBS. Filter through a 300-mesh filter into a flow cytometer tube, store on ice, and immediately load the tube onto the flow cytometer to measure intracellular fluorescence intensity using the FITC channel.

[0064] 3. Caco2 cell monolayer permeability test Caco-2 cells (ATCC source, grown in MEM containing 10% FBS and 1% non-essential amino acids (NEFA)) were cultured at a rate of 3.5 × 10 cells per cm 4 Cells were seeded into the top chamber (AP side) of a 96-well Transwell plate for culture, while 250 μL of culture medium was added to the bottom chamber (BL side). Culture medium was refreshed every 4–5 days until a confluent cell monolayer formed between days 21–28. Before transport experiments, the cell monolayer was rinsed three times with D-Hanks buffer. For AP-BL transport, 75 μL of D-Hanks buffer containing the test drug (1 and 2: 2 μM; digoxin: 10 μM) was added to the AP side, and 250 μL of drug-free D-Hanks buffer was added to the BL side. For BL-AP transport, 250 μL of drug-containing D-Hanks buffer was added to the BL side, and 75 μL of drug-free D-Hanks buffer was added to the AP side. The drug-loaded Transwell plate was incubated in a cell culture incubator for 2 hours. The concentrations of 1, 2, and digoxin in the receiving buffer were determined by LC-MS / MS using tolbutamide as the internal standard. The apparent permeability coefficient (Papp), efflux rate (ER), and solution recovery (% solution recovery) were calculated. After the transport experiment, the integrity of the cell monolayer was evaluated by lucifer yellow rejection assay. Only when the Papp of lucifer yellow was verified to be ≤2.5×10 -6 cm·s -1 , only the data measured by the transport experiment can be used.

[0065] Papp (cm·s -1)=(dCr / dt)×Vr / (A×C0) Efflux Ratio (ER)=Papp (BA) / Papp (AB) Solution Recovery %=100×[(Vr×Cr) + (Vd×Cd)] / (Vd×C0) Where dCr / dt is the ratio of the concentration of the compound on the receiving side to the time, Vr is the volume of the receiving side (750 μL on the AP side and 250 μL on the BL side), and A is the transport surface area, which is 0.143 cm 2 , C0 is the initial concentration on the dosing side, Cd and Cr are the final concentrations measured on the dosing side and the receiving side, respectively, and Vd is the volume on the dosing side.

[0066] When the solution recovery rate of the test compound during AP-BL transport is ≥50%, if Papp < ​​0.6 × 10 -6 cm·s -1 It is indicated as a low permeability compound if Papp ≥ 6.0 × 10 -6 cm·s -1 When the ER of the test compound is greater than 2, it indicates that the compound may be a substrate of P-gp, otherwise it is not.

[0067] 4. Statistical analysis GraphPad Prism 8.0.2 software was used for statistical analysis and plotting of the experimental data. Statistical variables were expressed as mean ± standard deviation (mean ± SD), and one-way analysis of variance was used for comparison among multiple groups. P A difference of <0.05 was considered statistically significant.

[0068] 2. Experimental Results 1. Compound 2 did not affect the expression of P-gp in HepG2 / Dox cells, but significantly inhibited its transport function In the above experiments, compounds 1 and 2 significantly reversed the resistance of HepG2 / Dox cells to P-gp substrate drugs, which are associated with P-gp. P-gp is an energy-dependent transmembrane transporter that mediates drug resistance in tumor cells by expelling substrate drugs that enter the cell. Reversal of P-gp-mediated MDR can be achieved by downregulating P-gp expression or inhibiting its transport function.

[0069] First, the effects of 1 and 2 on P-gp expression were tested. The results showed that 1 and 2, at their effective concentrations for reversing MDR, did not affect the expression level of P-gp in HepG2 / Dox cells, either alone or in combination with PTX. Their effects were similar to those of VRP ( Figure 3 and Figure 4 ). This indicates that the reversal effect of compounds 1 and 2 on HepG2 / Dox cell MDR is not achieved by downregulating the expression of P-gp.

[0070] 2. Then, the P-gp fluorescent substrate Rh-123 was used as a probe to test the effects of compounds 1 and 2 on the P-gp transport function in HepG2 / Dox cells. Figure 5 As shown in Figure 2, compounds 1 and 2 can significantly enhance the fluorescence intensity of Rh-123 in cells at their effective concentration of reversing MDR (10 μM) (*** P <0.001). This indicates that compounds 1 and 2 can significantly inhibit the transport function of P-gp to substrates, which should be the main reason for their reversal of MDR in HepG2 / Dox cells.

[0071] 3. Compound 2 behaves as a non-P-gp substrate in the Caco-2 cell monolayer model In the aforementioned studies, compounds 1 and 2 acted as modulators of P-gp function, but it is unclear whether their interaction with P-gp is substrate-like or non-substrate-like. Caco-2 cell monolayers (which highly express P-gp and other transport proteins) mimic the barrier function and drug transport processes of the human small intestinal epithelium and are commonly used to evaluate intestinal drug absorption, transport, efflux, or uptake. Therefore, whether compounds 1 and 2 are P-gp substrates can be determined through bidirectional transport experiments in the Caco-2 cell monolayer model: an efflux ratio (ER) greater than 2 indicates a potential P-gp substrate, whereas a negative ER indicates a negative P-gp substrate.

[0072] The experimental results are shown in Table 3. Digoxin, used as a P-gp substrate control, had an efflux rate as high as 181, indicating that Caco-2 cells are mature and stably express P-gp protein. The measured ER values ​​for 1 and 2 were 0.83 and 0.89, respectively, indicating that they are not effluxed and are not P-gp transport substrates. Their interaction with P-gp is in a non-substrate mode. Furthermore, the measured apparent permeability Papp (AP→BL) values ​​for 1 and 2 were 9.28×10 -6 cm·s -1 and 0.27×10 -6 cm·s -1, indicating that compound 1 is a high permeability compound and compound 2 is a low permeability compound. The solution recovery rates of both compounds were less than 55%, suggesting that these two compounds may have obvious specific adsorption or cellular metabolism effects. The fluorescent yellow permeability test at the end of the bidirectional transport experiment showed that the Papp value of fluorescent yellow in all test wells was less than 2.5×10 -6 cm·s -1 The results indicate that the constructed Caco-2 cell monolayer is intact and the measured data are reliable. This suggests that compounds 1 and 2 may have the potential to enhance the oral bioavailability of P-gp substrate drugs such as paclitaxel or vinblastine and develop oral formulations.

[0073] Table 3 Efflux rate and apparent permeability of compounds 1 and 2 (×10 -6 cm·s -1 ) and solution recovery rate

[0074] Example 4 Molecular docking simulation of the interaction between the test compound and P-gp Log in to the PBD database (https: / / www.rcsb.org / ) and search for the target protein P-gp (PBD code: 6QEX). Download and save the 3D crystal structure (file format: PDB). Export the file to PDB format, manually add hydrogen using Autodock tools, select the "Receptor" (Macromolecule), and export it to PDBQT format. Draw the 2D structure of the test compound using ChemDraw software. Optimize the structure and convert the file format using Chem3D, Openbable, and PyMol software. Manually remove water and add hydrogen using Autodock tools, select the "Ligand" (Ligand), and export it to PDBQT format. Import the two processed PDBQT files into AutoDock Vina software for docking, and record the receptor-ligand binding mode with the lowest binding energy. Visualize the docking results and binding mode using PyMOL and Discovery Studio (2019 version).

[0075] In order to reveal the mode of action of compounds 1 and 2 with P-gp, molecular docking simulations were performed on 1 or 2 with P-gp. The obtained visualization results are shown in Figure 2. Figure 6 As shown: 1 has hydrogen bonding with amino acid residues GLN-824, THR-240, and SER-993 of P-gp, -Pi stacking with amino acid residue PHE-239, and hydrophobic interaction with PRO-996. The calculated binding energy is -8.2 kcal / mol ( Figure 6A); 2 has hydrogen bonding with the GLU-486 amino acid residue of P-gp, hydrophobic interactions with the ARG-489, TYR-490, LYS-915, and ARG-464 amino acid residues, and π-cation interaction with LYS-380. The calculated binding energy is -8.4 kcal / mol ( Figure 6 B). This indicates that compound 1 or 2 has a strong potential to bind to specific spatial regions of the protein through various forms of intermolecular forces with P-gp, thereby interfering with the function of the target protein P-gp and inhibiting the efflux function of P-gp.

[0076] In summary, the present invention is to extract C from Apocynaceae plant Rhizoma Coptidis 21 The steroidal aglycones isolated and purified from the total steroidal aglycones were identified as 3β, 12β, 14β, 17β, 20(S)-pentahydroxy-5α-pregnane-12β-O-(E)-cinnamate (Compound 1) through mass spectrometry and nuclear magnetic resonance analysis. A nicotinoyl derivative was synthesized and identified as 3β, 12β, 14β, 17β, 20(S)-pentahydroxy-5α-pregnane-3β-O-nicotinate-12β-O-(E)-cinnamate (Compound 2) through mass spectrometry and nuclear magnetic resonance analysis. This study revealed that Compound 2 has multidrug resistance-reversing activity in P-gp-overexpressing liver cancer cells. At non-cytotoxic concentrations, compound 2 highly sensitized the cytotoxicity of paclitaxel and vinblastine in multidrug-resistant HepG2 / Dox cells, completely reversing the cellular resistance. Combination administration with paclitaxel, a P-gp substrate anticancer drug, induced significant apoptosis in HepG2 / Dox cells, demonstrating significantly superior efficacy compared to compound 1. Mechanistic studies revealed that compound 2 did not alter P-gp expression in MDR cells, but instead acted as a non-substrate inhibitor, significantly inhibiting P-gp's substrate transport function. Compound 2 has the potential to be developed as a specific adjuvant (modulator) for taxane or vinblastine anticancer drugs, potentially improving clinical efficacy against P-gp-mediated multidrug-resistant tumors.

Claims

1. A polyoxypregnane aromatic ester, characterized in that: The polyoxypregnane aromatic ester is 3β, 12β, 14β, 17β, 20(S)-pentahydroxy-5α-pregnane-3β-O-nicotinate-12β-O-(E)-cinnamate; its structural formula is shown in the following formula (II): 。 2. The method for preparing the polyoxypregnane aromatic ester according to claim 1, characterized in that: The compound represented by formula (I) is subjected to nicotinoylation reaction and purified to obtain the polyoxypregnane aromatic ester according to claim 1. 。 3. Use of the polyoxypregnane aromatic ester according to claim 1 in the preparation of a drug for reversing multidrug resistance of tumors.

4. Use of the composition of the polyoxypregnane aromatic ester and a tumor chemotherapy drug according to claim 1 in the preparation of a drug for reversing tumor multidrug resistance or a drug for treating tumors.

5. The application according to claim 4, characterized in that: The tumor chemotherapy drug is a P-glycoprotein substrate drug.

6. The use according to claim 3 or 4, characterized in that: The tumor is a tumor that overexpresses P-glycoprotein.

7. The use according to claim 3 or 4, characterized in that: The drug achieves treatment by increasing the accumulation of tumor chemotherapy drugs in tumor cells.

8. A drug for reversing multidrug resistance of tumors, characterized in that: The medicine contains the polyoxypregnane aromatic ester according to claim 1.

9. A pharmaceutical composition for reversing multidrug resistance of tumors, characterized in that: The pharmaceutical composition contains the polyoxypregnane aromatic ester according to claim 1 and a tumor chemotherapy drug.

10. The drug according to claim 8 or the pharmaceutical composition according to claim 9, characterized in that: The medicine also includes other pharmaceutically acceptable excipients.

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