A tetrandrine derivative for specifically inhibiting P-gp to reverse tumor multi-drug resistance, and preparation method and application thereof

Abstract

The present invention relates to a simplified tetrandrine compound that specifically inhibits P-gp and reverses tumor multidrug resistance, as well as a preparation method and application. The simplified tetrandrine compound that specifically inhibits P-gp and reverses tumor multidrug resistance has the following general structure: #imgabs0# The simplified compound can efficiently reverse the multidrug resistance of various solid tumor cells such as gastric cancer, lung cancer, and esophageal cancer. The compound has low cytotoxicity and can specifically inhibit the key efflux pump protein P-gp (P-glycoprotein) that mediates tumor cell multidrug resistance. The simplified compound can be used in combination with various chemotherapy drugs such as vincristine, mitoxantrone, colchicine, and docetaxel to efficiently reverse tumor multidrug resistance both in vivo and in vitro. This type of specific P-gp inhibitor provides an alternative strategy as an anti-tumor chemotherapy sensitizer and can be used as a lead compound for the research and development of new drug-resistant tumor reversal agents.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical chemistry, and in particular to a simplified tetrandrine capable of specifically inhibiting P-gp and reversing tumor multidrug resistance, as well as a preparation method and application thereof. Background Art

[0002] Multidrug resistance (MDR) is the development of resistance in malignant tumors to multiple other anticancer drugs with different structures and mechanisms of action after exposure to one anticancer drug. MDR often occurs during long-term chemotherapy, greatly reducing the efficacy of chemotherapy and being the main reason for the failure of chemotherapy in most metastatic tumors. Increased drug efflux caused by ABC transporters is an important cause of MDR. P-glycoprotein (P-gp; ABCB1; MDR1) is the most typical and common ABC transporter, often overexpressed in cancer cells, and is used to transport anticancer drugs to reduce intracellular therapeutic concentrations. Inhibiting P-gp function increases the accumulation of anticancer drugs in cells and restores the effective therapeutic concentration for killing cancer cells. Therefore, the discovery of highly effective P-gp inhibitors and their co-administration with traditional chemotherapy drugs have long been considered an effective strategy for reversing clinical multidrug resistance.

[0003] To date, medicinal chemists have developed dozens of P-gp inhibitors, but none have been approved for clinical use due to limitations in efficacy, toxicity, specificity, and adverse drug-drug interactions. Natural isoquinoline alkaloids, such as tetrandrine, exhibit a wide range of biological activities, including anti-inflammatory, anti-tumor, and anti-oxidative stress activities, and have been reported as a primary scaffold for MDR-reversing drugs. However, like other natural products, isoquinoline alkaloids suffer from limited efficacy, poor solubility, metabolic instability, and poor toxicological properties. Furthermore, their structural complexity complicates their synthesis and optimization to meet drugability requirements. Previous structural modifications of tetrandrine have involved simple modifications to natural tetrandrine, resulting in relatively simple products with limited activity improvements and remaining unchanged from the difficulty of achieving total chemical synthesis. Furthermore, traditional screening and evaluation systems for reversing tumor resistance often rely on cell viability assays, which are time-consuming and labor-intensive. There is an urgent need for more efficient screening and evaluation systems.

[0004] Therefore, it is of great significance for the combination therapy of multidrug resistance of tumors to rationally simplify the structure of isoquinoline alkaloids represented by tetrandrine, establish an effective synthesis method of isoquinoline alkaloid derivatives and a screening and evaluation system for reversing tumor resistance, and obtain highly efficient, low-toxic and highly selective P-gp inhibitors from them. Summary of the Invention

[0005] The present invention aims to provide a simplified tetrandrine compound that specifically inhibits P-gp and reverses tumor multidrug resistance, as well as a preparation method and application. The simplified compound is highly effective in reversing multidrug resistance in various solid tumor cells, including gastric cancer, lung cancer, and esophageal cancer. The compound exhibits low cytotoxicity and specifically inhibits P-gp (P-glycoprotein), a key efflux pump protein that mediates multidrug resistance in tumor cells. When used in combination with various chemotherapy drugs, such as vincristine, mitoxantrone, colchicine, and docetaxel, the simplified compound is highly effective in reversing tumor multidrug resistance both in vitro and in vivo. This specific P-gp inhibitor offers an alternative strategy as an anti-tumor chemotherapy sensitizer and can serve as a lead compound for the development of novel agents for reversing drug-resistant tumors.

[0006] The simplified product has been shown to be able to stabilize P-gp protein through thermal migration experiments, and has been shown to have excellent activity in reversing tumor multidrug resistance both in vivo and in vitro through tumor cell toxicity experiments and animal tumor-bearing experiments, and has low cytotoxicity itself.

[0007] The simplified tetrandrine compound of the present invention, which specifically inhibits P-gp and reverses tumor multidrug resistance, has the following general structure:

[0008]

[0009] Where: R 1 、 R 2 Each independently represents any one of H, hydroxyl, alkoxy or oxybenzyl, or R 1 and R 2 Is 1,2-dioxyethylene;

[0010] R 3 is any one of H, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heteroaryl, heteroarylalkyl, heterocyclyl, heterocyclylalkyl, or acyl;

[0011] When n=0, R 4 is H, C1-C16 alkyl, aryl, heteroaryl, cyano, carbonyl, trifluoromethyl, or vinyl;

[0012] When n=1, R 4 is H, C1-C15 alkyl, cyano, trifluoromethyl, nitro, amino, alkenyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heteroaryl, or a structure of Any one of substituted phenyl groups.

[0013] described R in 5 、R 6 、R 7 、R 8 or R9 Each is independently H, F, Cl, Br, I, trifluoromethyl, ester, cyano, sulfone, nitro, hydroxyl, -OR 10 Any one of R 6 and R 7 is methylenedioxy, R 7 and R 8 It is methylenedioxy.

[0014] Said-OR 10 R in 10 is any one of H, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heteroaryl, heteroarylalkyl, heterocyclyl, heterocyclylalkyl, or acyl.

[0015] The present invention adopts a method in which substituted phenylethylamine and substituted phenylacetic acid are subjected to reflux condensation in an organic solvent under the catalysis of boric acid to prepare an amide intermediate I; then, under the action of phosphorus oxychloride, reflux dehydration cyclization is carried out to prepare an imine intermediate II; then, sodium borohydride is used to reduce the secondary amine intermediate III; and the secondary amine intermediate III is subjected to reductive amination or NH- substitution with a halogenated hydrocarbon to obtain the final product IV.

[0016] The method for preparing the simplified material of the present invention comprises the following steps:

[0017]

[0018] 1) 11 mmol of substituted phenylethylamine and 11 mmol of substituted phenylacetic acid were added to a toluene solution, stirred and reacted at 110° C. for 20 hours in the presence of 0.88 mmol of boric acid, and the solvent was concentrated to obtain a reaction product. Ethyl acetate was added to the reaction product and washed 2 to 3 times, and the washed solution was filtered to obtain an intermediate product I;

[0019] 2) adding the intermediate I obtained in 1) to dichloromethane, and then adding 33 mmol of phosphorus oxychloride to obtain a reaction solution; heating the reaction solution to 78° C. and stirring for 7-8 hours, and then quenching the reaction solution with an aqueous sodium bicarbonate solution until no large bubbles are generated in the reaction solution; then extracting the reactant with dichloromethane to obtain an organic solvent layer, and then drying the organic solvent layer over anhydrous sodium sulfate to obtain intermediate II;

[0020] 3) The intermediate II obtained in 2) was added to methanol, cooled in an ice bath, and 50 mmol of sodium borohydride was added, followed by stirring at room temperature for 5 hours, and the solvent was removed by concentration. Dichloromethane was added and extracted to obtain an organic solvent layer, which was then dried over anhydrous sodium sulfate to obtain intermediate III;

[0021] 4) After reductive amination or substitution with a halogenated hydrocarbon, the intermediate III can be introduced into the NH- group of tetrahydroisoquinoline with any one of alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heteroaryl, heteroarylalkyl, heterocyclyl, heterocyclylalkyl, and acyl groups to obtain the final product IV.

[0022] The method of the present invention achieves a rational simplification and optimization of the tetrandrine structure, changing the traditional method of simply making derivative modifications on the natural tetrandrine structure. The simplified tetrandrine prepared has the advantages of simple operation, no dangerous operations, a short reaction time, and an average of only two to three days to obtain the final product. It also has the advantages of a wide range of reaction substrate applications, diverse product structures, good atom economy, environmental friendliness, and a relatively good yield. The simplified tetrandrine prepared by the method of the present invention has low cytotoxicity, a simple synthesis method, good water solubility, and excellent activity in reversing tumor multidrug resistance. It is a good lead compound with potential for further drug development.

[0023] The simplified tetrandrine is used in combination with one or more of vincristine, mitoxantrone, colchicine and docetaxel in the preparation of a drug for treating cancer.

[0024] The cancer is lung cancer, stomach cancer, breast cancer, pancreatic cancer, prostate cancer, leukemia or esophageal cancer.

[0025] The simplified substance specifically inhibits P-gp.

[0026] The present invention rationally simplifies the structure of natural tetrandrine (TET) based on molecular dynamics simulation and fragment growth, and obtains a new simplified compound that is easy to prepare, has high reversal activity and low cytotoxicity, has higher binding free energy, and contains stereoisomers of the compound.

[0027] The present invention provides another strategy for designing new specific P-gp inhibitors as anti-tumor chemotherapy sensitizers.

[0028] The simplified tetrandrine of the present invention is used to measure the intracellular accumulation of P-gp fluorescent substrate by laser confocal microscopy and flow cytometry, and the activity of the simplified tetrandrine in reversing tumor drug resistance is quickly screened. Figure 11 and 13 , and quickly screen the activity of simplified tetrandrine in reversing tumor resistance through fluorescence accumulation.

[0029] The product prepared by the present invention can be separated by column chromatography, using a polar solvent as the developing solvent and a mixed solvent of a non-polar solvent as the eluent, with the recommended solvent being dichloromethane / methanol. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 IC values of human normal cells (LX-2, HK-2, HEK293T, GES-1), cancer cells (Eca109), and drug-resistant cancer cells (Eca109 / VCR) treated with test compounds (OY-101, VRP, TET, NEF, CEP, and TQ) for 48 h 50 value;

[0031] Figure 2 Comparison of water solubility between natural tetrandrine and its simplified compound OY-101;

[0032] Figure 3 To test the activity of tetrandrine simplified compound OY-101 in reversing tumor drug resistance;

[0033] Figure 4 Flow cytometry was used to detect the apoptosis of drug-resistant strains induced by the simplified tetrandrine OY-101 combined with VCR;

[0034] Figure 5 This is a bar graph showing the statistical results of three flow cytometry measurements;

[0035] Figure 6 For plate colony formation experiments;

[0036] Figure 7 The differences in efflux pump protein expression between the parental strain and the drug-resistant strain and the effect of the simplified compound OY-101 on efflux pump protein expression were analyzed.

[0037] Figure 8 To observe the effect of simplified compound OY-101 on the intracellular accumulation of P-gp substrates by laser confocal microscopy;

[0038] Figure 9 To simplify, the intracellular accumulation of OY-101 on RH123 was concentration- and time-dependent;

[0039] Figure 10 To simplify the effect of OY-101 on the intracellular accumulation of four fluorescent substrates;

[0040] Figure 11 is the mapping between the fluorescent substrate and the efflux pump protein;

[0041] Figure 12 Flow cytometry was used to observe the intracellular accumulation of the simplified substance OY-101 to the fluorescent substrate;

[0042] Figure 13 Statistical graph of the data from three flow cytometry experiments;

[0043] Figure 14 The thermal stability test confirmed that the simplified substance OY-101 has a direct binding effect with P-gp;

[0044] Figure 15 is the reversal fold of P-gp-specific and nonspecific chemotherapeutic drugs by the simplified substance OY-101;

[0045] Figure 16 To evaluate the in vivo efficacy and toxicity of the simplified compound OY-101.

[0046] Figure 17 To observe the apoptosis-inducing effect of simplified OY-10 combined with VCR on breast cancer cells by flow cytometry;

[0047] Figure 18 To observe the apoptosis-inducing effect of simplified OY-10 combined with VCR on gastric cancer cells by flow cytometry;

[0048] Figure 19 To observe the apoptosis-inducing effect of simplified OY-10 combined with VCR on lung cancer cells by flow cytometry;

[0049] Figure 20 The figure is the HPLC chromatogram of the stereoisomers of the simplified product OY-101. DETAILED DESCRIPTION

[0050] The present invention is described below with reference to specific examples. Those skilled in the art will appreciate that these examples are only for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention in any way.

[0051] Reagents:

[0052] Phenylethylamine, acetic acid derivatives, boric acid, phosphorus oxychloride, sodium borohydride, BINAP, formaldehyde (Beijing Inokai Biotechnology Co., Ltd.)

[0053] The acidic additives were commercially available of analytical grade.

[0054] The water in the present invention is distilled water, and the organic solvents are commercially available analytically pure polar solvents or non-polar solvents, such as benzene, toluene, dichloromethane, chloroform, acetonitrile, methanol, tetrahydrofuran, petroleum ether, ethyl acetate, etc.

[0055] Example 1

[0056] To a 250ml round-bottom flask, 11mmol of 3,4-dimethoxyphenylethylamine, 11mmol of 4-trifluoromethylphenylacetic acid, and 0.88mmol of boric acid were added sequentially. The mixture was then dissolved in 120ml of toluene and stirred at reflux at 150°C for 12 hours. The solvent was then concentrated to remove the residue, and the residue was washed 2-3 times with 30ml of ethyl acetate. The washings were then filtered to obtain Intermediate Product I (10.91mmol crude product).

[0057] 10.91 mmol of intermediate I and 33 mmol of phosphorus oxychloride were added to 120 ml of dichloromethane to obtain a reaction solution. The reaction solution was heated to 70°C and stirred for 5 hours, then cooled. The reaction solution was poured into a 250 ml beaker and quenched by slowly adding a saturated aqueous sodium bicarbonate solution until bubbles in the beaker were no longer generated in large quantities. 30 ml of dichloromethane was added to extract the quenched reaction solution to obtain an organic phase. The organic phase was dried over anhydrous sodium sulfate, and the dichloromethane was removed by vortexing to obtain intermediate II (9.97 mmol crude product).

[0058] Take a 250ml round-bottom flask and dissolve 9.97mmol of intermediate II in 100ml of anhydrous methanol. Then, slowly add 50mmol of sodium borohydride under ice bath conditions. Then seal the round-bottom flask and tie a balloon at the seal. Then stir at room temperature for 5 hours, spin off the excess anhydrous methanol solution to obtain intermediate III (9.02mmol crude product).

[0059] 9.02 mmol of Intermediate III and 27 mmol of a 37% aqueous formaldehyde solution were added to 100 ml of anhydrous methanol. Then, in an ice bath, 40 mmol of sodium borohydride was slowly added. The round-bottom flask was sealed and a balloon was inserted into the seal. The mixture was stirred at room temperature for 5 hours. The excess anhydrous methanol solution was removed by swirl, and the mixture was purified on a silica gel column (eluent: dichloromethane:methanol = 80:1) to obtain 5.68 mmol of the final product, a simplified tetrandrine. The total yield was 60%. The structural formula is shown below:

[0060]

[0061] 6,7-dimethoxy-2-methyl-1-(4-(trifluoromethyl)benzyl)-1,2,3,4-tetrahydroisoquinoline(16):

[0062] Yellow oil,yield:60%. 1 H NMR (600MHz, CDCl3) δ7.49(d,J=8.0Hz,2H),7.19(d,J=7.9Hz,2H),6.55(s,1H),6.00(s,1H),3.83(s,3H),3.74(t,J=6.5 Hz,1H),3.56(s,3H),3.23–3.15(m,2H),2.91(dd,J=13.6,7.3Hz,1H),2.85–2.73(m,2H),2.58–2.53(m,1H),2.52(s,3H). 13C NMR (150MHz, CDCl3) δ147.3,146.4,144.1,130.1,128.4,128.2(q,J C-F =32.1Hz),126.1,124.8,124.3(q,J C-F =270.3Hz),111.1,110.6,64.4,55.6,55.4,46.7,42.6,40.8,25.3.HRMS(ESI)m / z:calculated for C 20 H 22 F3NO2[M+H] + :366.1676,found366.1674.

[0063] Example 2

[0064] To a 250ml round-bottom flask, 11mmol of 3,4-dimethoxyphenylethylamine, 11mmol of 4-nitrophenylacetic acid, and 0.88mmol of boric acid were added sequentially. The mixture was then dissolved in 120ml of toluene and stirred at reflux at 150°C for 12 hours. The solvent was then concentrated to remove the residue, and the mixture was washed 2-3 times with 30ml of ethyl acetate. The washings were then filtered to obtain Intermediate Product I (11.05mmol crude product).

[0065] 11.05 mmol of intermediate I and 33 mmol of phosphorus oxychloride were added to 120 ml of dichloromethane to obtain a reaction solution. The reaction solution was heated to 70°C and stirred for 5 hours, then cooled. The reaction solution was poured into a 250 ml beaker and quenched by slowly adding a saturated aqueous sodium bicarbonate solution until no more bubbles were generated in the beaker. 30 ml of dichloromethane was added to extract the quenched reaction solution to obtain an organic phase. The organic phase was dried over anhydrous sodium sulfate, and the dichloromethane was removed by vortexing to obtain intermediate II (10.55 mmol crude product).

[0066] Take a 250ml round-bottom flask and dissolve 10.55mmol of intermediate II in 100ml of anhydrous methanol. Then, slowly add 50mmol of sodium borohydride under ice bath conditions. Then seal the round-bottom flask and tie a balloon on the seal. Then stir at room temperature for 5 hours, spin off the excess anhydrous methanol solution to obtain intermediate III (9.97mmol crude product).

[0067] 9.97 mmol of Intermediate III and 27 mmol of a 37% aqueous formaldehyde solution were added to 100 ml of anhydrous methanol. Then, in an ice bath, 40 mmol of sodium borohydride was slowly added. The round-bottom flask was sealed and a balloon was inserted into the seal. The mixture was stirred at room temperature for 5 hours. The excess anhydrous methanol solution was removed by swirl, and the mixture was purified on a silica gel column (eluent: dichloromethane:methanol = 80:1) to obtain 6.68 mmol of the final product. The total yield was 68%. The structural formula is shown below:

[0068]

[0069] 6,7-dimethoxy-2-methyl-1-(4-nitrobenzyl)-1,2,3,4-tetrahydroisoquinoline:

[0070] Yellow oil,yield:68%. 1 H NMR (600MHz, CDCl3) δ8.07(d,J=8.7Hz,2H),7.23(d,J=8.7Hz,2H),6.53(s ,1H),6.26(s,1H),3.84(s,3H),3.77(t,J=6.0Hz,1H),3.69(s,3H),3.17(d d,J=13.8,6.1Hz,1H),3.12(ddd,J=12.5,8.8,4.9Hz,1H),3.05(dd,J=13.9 ,6.0Hz,1H),2.77–2.68(m,2H),2.49(s,3H),2.46(dt,J=17.4,5.4Hz,1H). 13 C NMR (150MHz, CDCl3) δ147.8,147.5,146.8,146.3,130.5,128.3,126.8,123.0,11 1.2,110.3,64.3,55.8,55.7,47.3,42.7,41.0,25.5.HRMS(ESI)m / z:calculated for C 19 H 22 N2O4[M+H] + :343.1653,found 343.1652.

[0071] Example 3

[0072] To a 250ml round-bottom flask, 27mmol of 3,4-dimethoxyphenylethylamine, 27mmol of 3-hydroxy-4-methoxyphenylacetic acid, and 2.1mmol of boric acid were added sequentially. The mixture was then dissolved in 150ml of toluene and stirred at reflux at 150°C for 12 hours. The solvent was then concentrated to remove the residue, and the residue was washed 2-3 times with 60ml of ethyl acetate. The washings were then filtered to obtain Intermediate Product I (27mmol crude product).

[0073] 27 mmol of intermediate I and 54 mmol of potassium carbonate were dissolved in 150 ml of acetonitrile, and then 28 mmol of benzyl bromide solution was added. The mixture was stirred at reflux at 85°C for 8 hours. After the solution was cooled, the solvent was dried by rotary evaporation and 60 ml of dichloromethane was added for extraction to obtain an organic phase. The organic phase was dried over anhydrous sodium sulfate, and the dichloromethane was removed by rotary evaporation to obtain intermediate II (27 mmol of crude product).

[0074] 27 mmol of intermediate II and 81 mmol of phosphorus oxychloride were added to 150 ml of dichloromethane to obtain a reaction solution. The reaction solution was heated to 70°C and stirred for 5 hours, then cooled. The reaction solution was poured into a 250 ml beaker and quenched by slowly adding a saturated sodium bicarbonate aqueous solution until bubbles in the beaker were no longer generated in large quantities. 60 ml of dichloromethane was added to extract the quenched reaction solution to obtain an organic phase. The organic phase was dried over anhydrous sodium sulfate, and the dichloromethane was removed by vortexing to obtain intermediate III (27 mmol crude product).

[0075] Take a 250ml round-bottom flask and dissolve 7.35mmol of intermediate III in 100ml of anhydrous methanol. Then, slowly add 38mmol of sodium borohydride under ice bath conditions. Then seal the round-bottom flask and tie a balloon at the seal. Then stir at room temperature for 5 hours, spin off the excess anhydrous methanol solution to obtain intermediate IV (7.0mmol crude product).

[0076] 0.2 mmol of Intermediate IV, 0.2 mmol of benzyl bromide solution, and 0.4 mmol of cesium carbonate were added to 50 ml of acetonitrile and stirred at 85°C for 8 hours. After cooling, unreacted solids were filtered off, the solution was concentrated to dryness, and purified on a silica gel column (eluent: dichloromethane:methanol = 80:1) to obtain 27.1 mg of the final product. The total yield was 60%. The structural formula is shown below:

[0077]

[0078] 2-benzyl-1-(3-(benzyloxy)-4-methoxybenzyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline:

[0079] Pale yellow solid, yield: 60%. 1 H NMR (600 MHz, CDCl3) δ 7.41–7.36 (m, 2H), 7.31 (dd, J = 8.3, 6.7 Hz, 2H), 7.26 (ddd, J = 7.6, 3.5, 1.5 Hz, 3H), 7.24–7.19 (m, 3H), 6.76 (d, J = 8.1 Hz, 1H), 6.60 (d, J = 2.0 Hz, 1H), 6.58–6.55 (m, 2H), 6.00 (s, 1H), 5.01 (d, J = 2.3 Hz, 2H), 3.86 (s, 3H), 3.85 (s, 3H), 3.75 (s, 2H), 3.68 (t, J = 6.9 Hz, 1H), 3.58 (s, 3H), 3.24 (ddd, J = 13.0, 10.8, 4.9 Hz, 1H), 3.04 (dd, J = 13.6, 6.6 Hz, 1H), 2.89 (ddd, J = 16.5, 10.7, 6.1 Hz, 1H), 2.82 (ddd, J = 13.1, 6.3, 2.3 Hz, 1H), 2.74 (td, J = 13.6, 12.8, 5.6 Hz, 1H), 2.49–2.43 (m, 1H). 13 C NMR (150 MHz, CDCl3) δ 148.0, 147.7, 147.3, 146.4, 139.5, 137.2, 132.7, 129.5, 128.9, 128.7, 128.5, 128.1, 127.7, 127.4, 126.8, 126.1, 125.5, 122.6, 115.7, 111.5, 111.3, 111.1, 70.8, 62.1, 57.9, 56.2, 55.8, 55.6, 43.5, 41.3, 24.5. HRMS (ESI) m / z: calculated for C 33 H 35 NO4 [M + H] + : 510.2639, found 510.2639.

[0080] Example 4

[0081] This example is based on a specific P-gp inhibitor with great medicinal value obtained using the research method of the present invention. This study rationally simplified the structure of natural tetrandrine (TET) based on molecular dynamics simulation and fragment growth, and obtained a new simplified compound OY-101 that is easy to prepare, has high reversal activity and low cytotoxicity. The excellent synergistic anticancer effect (IC) of OY-101 with vincristine (VCR) on drug-resistant Eca109 / VCR cells was confirmed by reversal activity assay, flow cytometry, plate colony formation assay and drug synergy analysis. 50 =9.9nM, RF=690). Mechanistic exploration revealed that the accumulation of the fluorescent substrate within cells after OY-101 treatment and its enhanced sensitization to antitumor drugs were both P-gp-dependent. Furthermore, cellular thermal shift analysis revealed that OY-101 increased the thermal stability of P-gp, indicating direct binding of OY-101 to P-gp. Importantly, OY-101 enhanced VCR sensitization in vivo without significant toxicity. The following are the steps for the synthesis of OY-101:

[0082] To a 250ml round-bottom flask, 27mmol of 3,4-dimethoxyphenylethylamine, 27mmol of 3-hydroxy-4-methoxyphenylacetic acid, and 2.1mmol of boric acid were added sequentially. The mixture was then dissolved in 150ml of toluene and stirred at reflux at 150°C for 12 hours. The solvent was then concentrated to remove the residue, and the residue was washed 2-3 times with 60ml of ethyl acetate. The washings were then filtered to obtain Intermediate Product I (26.5mmol crude product).

[0083] 26.5 mmol of intermediate I and 53 mmol of potassium carbonate were dissolved in 150 ml of acetonitrile, and then 28 mmol of benzyl bromide solution was added. The mixture was stirred at reflux at 85°C for 8 hours. After the solution was cooled, the solvent was dried by rotary evaporation and 60 ml of dichloromethane was added for extraction to obtain an organic phase. The organic phase was dried over anhydrous sodium sulfate, and the dichloromethane was removed by rotary evaporation to obtain intermediate II (26.0 mmol of crude product).

[0084] 26.0 mmol of intermediate II and 78 mmol of phosphorus oxychloride were added to 150 ml of dichloromethane to obtain a reaction solution. The reaction solution was heated to 70°C and stirred for 5 hours, then cooled. The reaction solution was poured into a 250 ml beaker and quenched by slowly adding a saturated aqueous sodium bicarbonate solution until bubbles in the beaker were no longer generated in large quantities. 60 ml of dichloromethane was added to extract the quenched reaction solution to obtain an organic phase. The organic phase was dried over anhydrous sodium sulfate, and the dichloromethane was removed by vortexing to obtain intermediate III (25.0 mmol crude product).

[0085] Take a 250ml round-bottom flask and dissolve 7.35mmol of intermediate III in 100ml of anhydrous methanol. Then, slowly add 38mmol of sodium borohydride under ice bath conditions. Then seal the round-bottom flask and tie a balloon at the seal. Then stir at room temperature for 5 hours, spin off the excess anhydrous methanol solution to obtain intermediate IV (7.0mmol crude product).

[0086] 2 mmol of intermediate IV and 6 mmol of a 37% aqueous formaldehyde solution were added to 50 ml of anhydrous methanol. Then, under ice bath conditions, 6 mmol of sodium borohydride was slowly added. The round-bottom flask was sealed and a balloon was inserted into the seal. The mixture was stirred at room temperature for 5 hours. The excess anhydrous methanol solution was removed by swirl, and the mixture was purified on a silica gel column (eluent: dichloromethane:methanol = 80:1) to obtain 1.61 mmol of the final product. The total yield was 75%. The structural formula is shown below:

[0087]

[0088] 1-(3-(benzyloxy)-4-methoxybenzyl)-6,7-dimethoxy-2-methyl-1,2,3,4-tetrahydroisoquinoline(OY-101):

[0089] Pale yellow solid,yield:75%. 1 H NMR (600MHz, CDCl3) δ7.41(d,J=7.2Hz,2H),7.34(t,J=7.6Hz,2H),7.28(d,J=7.4Hz,1H),6.77(d ,J=8.1Hz,1H),6.66(d,J=2.0Hz,1H),6.60(dd,J=8.1,2.0Hz,1H),6.54(s,1H),5.98(s,1H),5.0 7(s,2H),3.85(s,3H),3.83(s,3H),3.60(dd,J=7.7,4.9Hz,1H),3.56(s,3H),3.16–3.04(m,2H), 2.79(ddd,J=15.2,8.9,5.6Hz,1H),2.75–2.69(m,2H),2.55(dt,J=16.0,4.7Hz,1H),2.48(s,3H). 13C NMR (150MHz, CDCl3) δ147.9,147.6,147.1,146.2,137.2,132.3,129.0,128.4,127.7,127.2,125.8,122.5,1 15.7,111.4,111.0,110.8,70.9,64.7,56.0,55.7,55.4,46.8,42.6,40.6,25.4.HRMS(ESI)m / z:calculated for C 27 H 31 NO4[M+K] + :472.1885,found 472.1881.

[0090] The structural formula of the stereoisomers that can be obtained by the method of the present invention is:

[0091]

[0092] 1-(4-bromobenzyl)-6,7-dimethoxy-2-methyl-1,2,3,4-

[0093] tetrahydroisoquinoline, its stereoisomer HPLC chromatogram is shown in Figure 20 .

[0094] Example 5 Biological activity test part of OY-101:

[0095] 1. Experiments on the cytotoxicity and water solubility of OY-101

[0096] OY-101, like the positive drug verapamil (VRP), has low cytotoxicity against human esophageal cancer cell line Eca109 and drug-resistant cell line Eca109 / VCR. 50 The values were all greater than 100 μM ( Figure 1 ). In sharp contrast, the cytotoxicity of natural isoquinoline alkaloids (TET, NEF, and CEP) and the third-generation P-gp inhibitor (TQ) was much higher. Similar cytotoxicity tests were applied to normal human cells, including human hepatic stellate cells (LX-2), human renal epithelial cells (HEK293T), human gastric mucosal cells (GES-1), and human renal tubular epithelial cells (HK-2). The results showed that OY-101 also had low cytotoxicity, with an IC 50 The value is >50 μM. This means that OY-101 is an excellent MDR reversal agent candidate that can be used to sensitize drug-resistant cancer cells while causing less damage to normal cells.

[0097] Moreover, compared with the original drug TET, the water solubility of the simplified OY-101 has been significantly improved ( Figure 2 ).

[0098] 2. Experiment on reversal of VCR (vincristine) resistance by OY-101

[0099] In this study, another more important indicator than toxicity and water solubility is the reversal activity against chemotherapeutic drugs. To this end, we conducted a series of validation experiments to confirm the effect of OY-101 on reversing VCR resistance.

[0100] The applicant tested the IC of VCR against the drug-resistant cell line Eca109 / VCR in the presence of different concentrations of the test compound. 50 It was found that VCR could effectively inhibit the parental Eca109 cells, but its inhibitory activity against drug-resistant Eca109 / VCR cells was relatively weak. Figure 3 As shown, the IC of VCR for the drug-resistant cell line Eca109 / VCR 50 The IC50 value was 6830 nM, far higher than the 65.70 nM of VCR against the parental strain Eca109 cells. OY-101 at 1, 2.5, and 5.0 μM significantly enhanced the killing effect of VCR against the drug-resistant strain Eca109 / VCR, achieving approximately 3.65-, 103.36-, and 690.60-fold reversal activity, respectively. OY-101's reversal activity was significantly superior to that of TET (RF = 232.55), NEF (RF = 1.8), CEP (RF = 15.64), and VRP (RF = 10.06).

[0101] The simplified tetrandrine OY-101 combined with VCR can significantly induce apoptosis of breast cancer cells. Figure 17 The simplified tetrandrine OY-101 combined with VCR can significantly induce apoptosis in gastric cancer cells, see Figure 18 The simplified tetrandrine OY-101 combined with VCR can significantly induce apoptosis in lung cancer cells, see Figure 19 .

[0102] Moreover, the new simplified tetrandrine OY-101 combined with VCR can significantly induce apoptosis of Eca109 / VCR cells. Flow cytometry was used to detect the effects of different concentrations of OY-101 combined with VCR on apoptosis of Eca109 / VCR cells. Figure 4As shown in Figure 2, 5 μM DMSO, 1 μM OY-101, 2.5 μM OY-101, 5 μM OY-101, 5 μM TET, 5 μM VRP, and 100 nM VCR had no significant cytotoxicity to Eca109 / VCR cells. However, once co-administered, OY-101, TET, and VRP significantly enhanced the cytotoxicity of VCR to Eca109 / VCR cells. The results of three repeated experiments are shown in Figure 2. Figure 5 As shown, OY-101 has the best reversal activity.

[0103] Furthermore, the combined use of OY-101 and VCR can significantly inhibit the clone formation of Eca109 / VCR. The plate clone formation experiment also confirmed that OY-101 can effectively reverse the drug resistance of VCR to Eca109 / VCR. Figure 6 As shown, co-administration of 2 μM OY-101 and 40 nM VCR almost completely inhibited the clonal growth of drug-resistant cells Eca109 / VCR.

[0104] 3. Experiments on the specific inhibition of P-gp efflux function by OY-101

[0105] After confirming that OY-101 can effectively reverse the drug resistance of Eca109 / VCR, the present invention explored the relevant mechanism. First, the effect of OY-101 on P-gp expression was studied. The expression of the main drug resistance-related proteins ABCG2, P-gp and MRP1 in the parental cells Eca109 and the drug-resistant cells Eca109 / VCR, as well as the effect of 2.5μM, 5μM and 10μM OY-101 on the expression of the above proteins were detected by western blotting. The results showed that the amount of P-gp in the drug-resistant cells Eca109 / VCR was significantly higher than that in the parental cells Eca109, while the difference in ABCG2 and MRP1 proteins between the two cell lines was not significant, and OY-101 had no effect on the expression of the above proteins ( Figure 7 ).

[0106] In order to more directly verify the inhibitory effect of OY-101 on the efflux function of P-gp, laser confocal microscopy was used to observe the effect of OY-101 on the intracellular accumulation of P-gp substrates within 3 hours. A dose-dependent increase in the intracellular accumulation of rhodamine 123 (RH123) and doxorubicin (DOX) was clearly observed in the OY-101-treated drug-resistant cells Eca109 / VCR. The increase in fluorescence intensity after OY-101 treatment was significantly higher than that after TET and VRP treatment. In addition to observing an increase in intracellular fluorescence intensity, we also observed an increase in the co-localization of DOX with the cell nucleus, which means that the toxicity of DOX to the cell nucleus is increased ( Figure 8 ).

[0107] The applicant further studied the intracellular accumulation of P-gp specific substrate RH123. The results showed that OY-101 can increase the intracellular fluorescence intensity of RH123 in a concentration- and time-dependent manner, and eventually tend to saturation ( Figure 9 ).

[0108] The present invention also investigated the intracellular accumulation of four different fluorescent substrates after OY-101 treatment ( Figure 10 ). RH123 is a specific substrate of P-gp. Calcein-AM is also a substrate of P-gp and has no fluorescence itself. After entering the cell, it is hydrolyzed by endogenous esterases to generate polar Calcein, which is a substrate of MRP1 and has strong green fluorescence. DOX can be excreted by P-gp and MRP1. H33342 can be excreted by ABCG2 and MRP1. Therefore, considering that OY-101 affects the intracellular accumulation of four fluorescent substrates in the order of RH123, Calcein-AM, DOX and H33342, we believe that OY-101 is a highly effective and specific P-gp inhibitor ( Figure 11 ).

[0109] To further verify the above speculation, the intracellular fluorescence intensity after co-incubation of the test compound and the fluorescent substrate was measured by flow cytometry ( Figure 12 After three repetitions, the DMSO group was used as the control and a statistical graph of the relative average intracellular fluorescence intensity was drawn ( Figure 13 Similar results were observed: 1) OY-101 had the most significant effect on intracellular accumulation in RH123 cells, followed by Calcein-AM. 2) OY-101 had similar effects to the third-generation P-gp inhibitor TQ, both significantly increasing the intracellular accumulation of RH123 and Calcein-AM, and both were superior to TET and VRP. 3) Accumulation of the fluorescent substrate was lower in the resistant Eca109 / VCR cells than in the parental Eca109 cells.

[0110] Subsequently, the present invention used a cell thermal shift assay (CETSA) to confirm the direct binding affinity of OY-101 to P-gp. Eca109 / VCR cell lysate was incubated with OY-101, DMSO (negative control), and the third-generation P-gp inhibitor TQ (positive control). Then, as the solution temperature continued to rise, unbound proteins denatured and precipitated at the elevated temperature, while proteins bound to the compound remained in the solution. Figure 14 As shown, OY-101 and TQ significantly stabilized P-gp protein, especially at high temperature (67°C), while P-gp protein in the solvent control group was significantly degraded at this temperature. The results show that OY-101, like the third-generation inhibitor TQ, can directly bind to and stabilize P-gp.

[0111] The applicants determined the reversal activity of OY-101 against certain chemotherapeutic drugs that are typically transported by ABC efflux pumps. The IC values of different anti-tumor drugs were determined for parental Eca109 cells and drug-resistant Eca109 / VCR cells when co-administered with 5 μM OY-101. 50 Value. Figure 15 As shown, the resistant cells Eca109 / VCR exhibited significant multidrug resistance to P-gp substrates, including any of the anticancer drugs mitoxantrone, colchicine, docetaxel, and doxorubicin, but had no resistance to the non-P-gp transported substrate lapatinib. OY-101 could significantly reverse the resistance to P-gp substrate chemotherapy drugs.

[0112] 4. Experiments on animals using OY-101

[0113] A xenograft tumor model carrying Eca109 / VCR cells overexpressing P-gp was established in nude mice to test the reversal activity of OY-101 in vivo. Nude mice were treated with solvent control (10 ml / kg / 2 days, intravenous injection), VCR (0.2 mg / kg / 2 days, intravenous injection), OY-101 (30 mg / kg / 2 days, intravenous injection), and the OY-101 / VCR combination. Figure 16 As shown, only co-administration of OY-101 and VCR could effectively inhibit tumor proliferation in vivo (P<0.001) and significantly reduce tumor weight. After 3 weeks of treatment, the tumor growth inhibition rate of the OY-101 / VCR combination was 79.13%, which was significantly lower than that of the single treatment group and the solvent control group ( Figure 16 During the treatment period of the experimental dose, there was no significant change in body weight in each group ( Figure 16 D). Pathological and TUNEL analysis of the tumors showed that treatment, especially the OY-101 / VCR combination, increased the proportion of cells with pyknosis, vacuoles, and apoptosis in tumor tissue. HE staining analysis showed that the combined administration of OY-101 and VCR did not increase hepatotoxicity ( Figure 16 These results indicate that OY-101 is a potent P-gp inhibitor that can reverse VCR resistance and inhibit tumor growth in Eca109 / VCR xenografts with low toxicity.

[0114] in conclusion:

[0115] The present invention relates to a novel class of simplified tetrandrine compounds. Through computer-assisted drug design and rational structural simplification, we discovered that these novel simplified tetrandrine compounds can specifically inhibit P-gp and effectively reverse multidrug resistance in tumor cells. Among these simplified compounds, represented by OY-101, exhibited excellent reversal activity both in vivo and in vitro. Importantly, OY-101 can be efficiently synthesized via a five-step reaction with a total yield of 75%, and exhibits low cytotoxicity and good water solubility. Further mechanistic studies confirmed that OY-101 is a specific and highly effective P-gp inhibitor. It effectively inhibits the growth of drug-resistant tumors both in vivo and in vitro. Therefore, due to its low cytotoxicity, simple synthesizability, good water solubility, and excellent reversal activity for drug-resistant tumors, OY-101 has the potential to be further developed into a drug-resistant tumor sensitizer.

Claims

1. Use of a simplified tetrandrine as a specific P-gp inhibitor in combination with one or more of vincristine, mitoxantrone, colchicine, and docetaxel in the preparation of a drug for treating cancer; The structure of the simplified tetrandrine is: .

2. The application according to claim 1, characterized in that: The cancer is lung cancer, stomach cancer, breast cancer, pancreatic cancer, prostate cancer, leukemia or esophageal cancer.

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