Isoquinoline alkaloid derivatives for efficiently inhibiting autophagy and reversing tumor multidrug resistance, preparation method and application thereof

Abstract

The present application relates to a kind of isoquinoline alkaloid derivatives for reversing tumor multidrug resistance by inhibiting autophagy and preparation method and application.The derivative can efficiently reverse the multidrug resistance of various solid tumor cells such as gastric cancer, lung cancer and esophageal cancer, the derivative can inhibit the autophagy flow of tumor cells, it is combined with vincristine, mitoxantrone, colchicine, docetaxel and various chemotherapeutic drugs, can efficiently reverse tumor multidrug resistance in vivo and in vitro, this kind of derivative has excellent oral bioavailability and lower toxic side effects, provides a kind of alternative strategy for autophagy inhibitor as antitumor chemosensitizer, can be used as lead compound for the research and development of new drug-resistant tumor reversing agent.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical chemistry, and in particular to an isoquinoline alkaloid derivative that effectively inhibits autophagy and reverses multidrug resistance in tumors, as well as its preparation method and application. Background Technology

[0002] Multidrug resistance (MDR) is the development of resistance in malignant tumors to multiple anticancer drugs with different structures and mechanisms of action after initial exposure to one anticancer drug. MDR frequently occurs during long-term chemotherapy, significantly reducing its effectiveness and being a major cause of chemotherapy failure in most metastatic tumors. Increased autophagy in tumor cells is a significant contributor to MDR. Once a tumor forms, autophagy regulates cellular homeostasis, providing essential survival environments for cancer cells, resisting nutritional stress after chemotherapy, and weakening the cell-killing effects of chemotherapeutic drugs. This promotes tumorigenesis and protects tumor cells, ultimately leading to chemotherapy resistance, refractory tumors, and cancer recurrence. In this situation, inhibiting autophagy can resensitize drug-resistant cancer cells, enhancing the efficacy of chemotherapeutic drugs. Therefore, the discovery of highly effective autophagy inhibitors and their co-administration with traditional chemotherapeutic drugs has long been considered an effective strategy for reversing clinical multidrug resistance.

[0003] Currently, only chloroquine (CQ) and its derivative hydroxychloroquine (HCQ) are used as autophagy inhibitors for patients. However, the side effects of CQ or HCQ greatly limit their clinical application. Therefore, developing a novel autophagy inhibitor for cancer treatment is of significant clinical importance. Natural isoquinoline alkaloids possess a wide range of biological activities, including anti-inflammatory, antitumor, and anti-oxidative stress effects, and have been reported as a major scaffold for MDR reversal drugs. However, like other natural products, isoquinoline alkaloids have drawbacks such as limited efficacy, poor solubility, metabolic instability, and unfavorable toxicological properties. Summary of the Invention

[0004] The purpose of this invention is to provide a powdered isoquinoline alkaloid derivative that specifically inhibits autophagy and reverses multidrug resistance in tumors, along with its preparation method and applications. This derivative can efficiently reverse multidrug resistance in various solid tumor cells, including gastric cancer, lung cancer, and esophageal cancer. It exhibits low cytotoxicity and specifically inhibits autophagic flux in tumor cells. When used in combination with various chemotherapeutic drugs such as vincristine, it can efficiently reverse multidrug resistance in tumors both in vivo and in vitro. This type of specific autophagy inhibitor provides an alternative strategy as an antitumor chemosensitizer and can serve as a lead compound for the development of novel drug-resistant tumor reversal agents.

[0005] The technical solution of the present invention:

[0006] An isoquinoline alkaloid derivative that effectively reverses multidrug resistance in tumors has the following general formula:

[0007]

[0008] Where: n = 0 or 1, R is H, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heteroaryl, heteroarylalkyl, heterocyclic, heterocyclic alkyl, acyl, or has the following structure. Any of the substituted phenyl groups.

[0009] The R in 1 R 2 R 3 R 4 or R 5 Each can be independently any one of H, F, Cl, Br, I, trifluoromethyl, ester, cyano, sulfone, nitro, or hydroxyl.

[0010] The compound includes its stereoisomers.

[0011] This invention employs a process in which substituted phenethylamine and substituted phenylacetic acid are refluxed and condensed in an organic solvent under boric acid catalysis to synthesize amide intermediate I; then, under the action of phosphorus oxychloride, reflux is used to dehydrate and cyclize to synthesize imine intermediate II; subsequently, it is reduced by sodium borohydride to secondary amine intermediate III; secondary amine intermediate III is reductively amination to obtain intermediate IV; intermediate IV undergoes a palladium-catalyzed Buchwald–Hartwig cross-coupling reaction to obtain intermediate V; intermediate V undergoes deprotection with trifluoroacetic acid to obtain intermediate VI; intermediate VI is then subjected to a palladium-catalyzed Buchwald–Hartwig cross-coupling or substitution reaction to obtain the final product.

[0012] The preparation method of the above-mentioned highly effective isoquinoline alkaloid derivatives for reversing multidrug resistance in tumors includes the following steps:

[0013]

[0014] 1) 3,4-Dimethoxyphenethylamine and p-bromophenylacetic acid were added to a toluene solution and stirred at 110-150°C for 12-20 hours under boric acid catalysis. The solvent was removed by concentration to obtain the reaction product. Ethyl acetate was added to the reaction product and washed 2-3 times. The product was then filtered to obtain intermediate product I.

[0015] 2) Dissolve intermediate I in dichloromethane, then add phosphorus oxychloride to obtain a reaction solution, heat to 70-78°C, stir for 5-8 hours, slowly add saturated sodium bicarbonate aqueous solution to quench it until no more bubbles are generated in the reaction solution, extract to obtain an organic solvent layer, dry with anhydrous sodium sulfate to obtain intermediate II;

[0016] 3) Dissolve intermediate II in methanol, cool to room temperature in an ice bath, slowly add sodium borohydride, stir at room temperature for 5 hours, and remove excess anhydrous methanol solution to obtain intermediate III;

[0017] 4) Intermediate III was reductively amination to introduce a methyl group onto NH-, and then purified by column chromatography to obtain intermediate IV;

[0018] 5) In an argon atmosphere, N-Boc-piperazine or N-Boc-periperazine was added to a toluene (0.1M) solution of intermediate IV, Pd2(dba)3, BINAP and K2CO3. The mixture was refluxed at 110°C for 24 hours. After the reaction solution was cooled to room temperature, it was filtered with diatomaceous earth, concentrated, and purified by column chromatography to obtain intermediate V.

[0019] 6) Dissolve intermediate V in 0.1M DMC, slowly add trifluoroacetic acid, stir overnight at room temperature, remove the solvent by evaporation under reduced pressure, and purify by column chromatography to obtain intermediate VI;

[0020] 7) Intermediate VI undergoes a Buchwald–Hartwig cross-coupling reaction or substitution reaction, introducing different groups onto the NH group to obtain the final product.

[0021] In step 1), the molar ratio of 3,4-dimethoxyphenethylamine: p-bromophenylacetic acid: boric acid is 11:11:0.88.

[0022] The molar ratio of intermediate I to phosphorus oxychloride in step 2) is 10-11:33;

[0023] The molar ratio of intermediate II to sodium borohydride in step 3) is 10.36:51.8;

[0024] The extraction in steps 2) and 3) uses dichloromethane.

[0025] Step 4) The specific method for the reductive amination is to pre-stir intermediate III with 37% formaldehyde solution for half an hour, and then add sodium borohydride for reduction;

[0026] Preferably, the molar ratio of intermediate III: formaldehyde solution: sodium borohydride is 10.00–11.00: 30.00–31.00: 40;

[0027] In step 5), the molar ratio of N-Boc-piperazine or N-Boc-hyperpiperazine: intermediate IV: Pd2(dba)3: BINAP: K2CO3 is 9.0–11.0: 9.0–10.0: 0.99: 1.98: 19.8.

[0028] In step 6), the molar ratio of intermediate V to trifluoroacetic acid is 6.00–8.00: 20.00–50.00.

[0029] In step 7), the substitution reaction involved dissolving intermediate VI and potassium fluoride in acetonitrile (75 mL).

[0030] 0.1M), then iodide was added, and the reaction was carried out at room temperature for 12 hours. After filtration and concentration, the product was purified by column chromatography. Preferably, the molar ratio of intermediate VI: potassium fluoride: iodide was 7.52:75.2:9.02.

[0031] The Buchwald–Hartwig cross-coupling reaction is carried out by adding an aryl iodide to a toluene solution of intermediate VI, Pd2(dba)3, BINAP, and K2CO3 in an argon atmosphere, refluxing at 110°C for 24 hours, cooling the reaction solution to room temperature, filtering with diatomaceous earth, concentrating, and purifying by column chromatography; preferably, the molar ratio of aryl iodide: intermediate VI: Pd2(dba)3: BINAP: K2CO3 is 6.86–8.27: 6.24–7.25: 0.62–0.75: 1.24–1.5: 12.4–15.04;

[0032] Step 7) The different groups are alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heteroaryl, heteroarylalkyl, heterocyclic, heterocyclic alkyl, acyl, or have the following structure Any of the substituted phenyl groups.

[0033] The application of the above-mentioned isoquinoline alkaloid derivatives in the preparation of cancer treatment drugs.

[0034] Preferably, the cancer is stomach cancer, lung cancer, breast cancer, pancreatic cancer, prostate cancer, leukemia, or esophageal cancer;

[0035] Preferably, the drug is a specific autophagy inhibitor; more preferably, the inhibitor is used as an antitumor chemotherapy sensitizer.

[0036] The application of the above-mentioned isoquinoline alkaloid derivatives in combination with vincristine in the preparation of cancer treatment drugs.

[0037] Preferably, the cancer is stomach cancer, lung cancer, breast cancer, pancreatic cancer, prostate cancer, leukemia, or esophageal cancer.

[0038] The drug is a specific autophagy inhibitor; preferably, the inhibitor is used as a sensitizer for antitumor chemotherapy.

[0039] A series of isoquinoline alkaloid derivatives were synthesized using chemical methods and applied to the screening of highly efficient autophagy inhibitors to reverse tumor MDR, which is of great significance for the combination therapy of multidrug-resistant tumors.

[0040] The method described in this invention achieves structural simplification and optimization of natural isoquinoline alkaloids. The novel isoquinoline alkaloid derivatives prepared have advantages such as simple operation, good safety, short reaction time (averaging only two to three days to obtain the final product), wide applicability of reaction substrates, diverse product structures, good atom economy, environmental friendliness, and high yield. The isoquinoline alkaloid derivatives prepared using the method described in this invention significantly enhance the limited MDR reversal activity of natural isoquinoline alkaloids (such as tebufenozide and tetrandrine), improve pharmacokinetic properties, and show no significant toxicity in in vivo experiments.

[0041] The isoquinoline alkaloid derivatives prepared by the method described in this invention have the advantages of high autophagy inhibition activity, high oral bioavailability, and excellent activity in reversing tumor multidrug resistance. They are a good lead compound with potential for further drug development.

[0042] The applicant's experimental verification: The compound was shown to inhibit autophagic flux by Western blotting experiments, and showed excellent reversal of multidrug resistance in tumors both in vivo and in vitro by tumor cytotoxicity experiments and animal tumor-bearing experiments. Attached Figure Description

[0043] Figure 1 IC for reversing drug resistance to OY-102 50 Value determination.

[0044] Figure 2 This is a diagram of a plate colony formation experiment.

[0045] Figure 3 The results of animal experiments with OY-102.

[0046] Figure 4 To observe the increase in autophagosomes in OY-102-treated cells using laser confocal microscopy.

[0047] Figure 5 The image shows the ultrastructure of cells treated with 5 μM MOY-102 for 24 hours, as observed by transmission electron microscopy. N represents the cell nucleus, M represents the mitochondria, and the scale bar is 1 μm or 0.5 μm.

[0048] Figure 6 To detect the effects of different concentrations of OY-102 on autophagy-related proteins using Western blotting.

[0049] Figure 7 To detect the effects of OY-102 at different treatment times on autophagy-related proteins using Western blotting.

[0050] Figure 8 To detect the effect of OY-102 on autophagy-related proteins in other drug-resistant cells using Western blotting.

[0051] Figure 9 To detect autophagy-related proteins by Western blotting (cross-comparison of OY-102 with typical autophagy inducer RAPA and autophagy inhibitor Baf A1).

[0052] Figure 10 To detect the effects of different stereoconfigurations of OY-102 on autophagy-related proteins using Western blotting.

[0053] Figure 11 The image shows the HPLC chromatogram of the racemic OY-102.

[0054] Figure 12 The HPLC chromatogram is for the isomer (R)-OY-102.

[0055] Figure 13 The HPLC chromatogram is for the isomer (S)-OY-102. Detailed Implementation

[0056] The present invention will be described below with reference to specific embodiments. Those skilled in the art will understand that these embodiments are for illustrative purposes only and do not limit the scope of the invention in any way.

[0057] Reagents:

[0058] Phenethylamine, acetic acid derivatives, boric acid, phosphorus oxychloride, sodium borohydride, BINAP, Pd2(dba)3 formaldehyde (Beijing Inokai Biotechnology Co., Ltd.)

[0059] The others are commercially available analytical grade.

[0060] The water mentioned in this invention is distilled water, and the organic solvents are all commercially available analytical grade polar or non-polar solvents, such as benzene, toluene, dichloromethane, chloroform, acetonitrile, methanol, tetrahydrofuran, petroleum ether, ethyl acetate, etc.

[0061] Example 1

[0062] Take a 250 ml round-bottom flask and add 11 mmol of 3,4-dimethoxyphenethylamine, 11 mmol of p-bromophenylacetic acid, and 0.88 mmol of boric acid sequentially. Then dissolve them in 120 ml of toluene and stir the reaction under reflux at 150 °C for 12 hours. Afterward, concentrate to remove the solvent, wash the mixture 2-3 times with 30 ml of ethyl acetate, and then filter off the washings to obtain intermediate I (10.91 mmol crude product).

[0063] 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 saturated sodium bicarbonate aqueous solution was slowly added to quench it until bubbles were no longer generated in large quantities 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 with anhydrous sodium sulfate, and dichloromethane was removed by rotary evaporation to obtain intermediate II (10.36 mmol crude product).

[0064] Take a 250 ml round-bottom flask, dissolve 10.36 mmol of intermediate II in 100 ml of anhydrous methanol, and then slowly add 51.8 mmol of sodium borohydride under ice bath conditions. Seal the round-bottom flask and attach a balloon to the seal. Stir at room temperature for 5 hours, and then remove excess anhydrous methanol solution to obtain intermediate III (10.2 mmol crude product).

[0065] 10.2 mmol of intermediate III and 30.6 mmol of 37% formaldehyde aqueous solution were added to 100 ml of anhydrous methanol. Then, under ice bath conditions, 40 mmol of sodium borohydride was slowly added. The round-bottom flask was then sealed, and a balloon was attached to the seal. The mixture was stirred at room temperature for 5 hours. Excess anhydrous methanol solution was removed by vortexing. The solution was purified by silica gel column chromatography (eluent: dichloromethane:methanol = 80:1) to obtain 9.9 mmol of intermediate IV.

[0066] In an argon-atmospheric glove box, N-Boc-periperazine (10.89 mmol, 1.1 eq.) was added to a toluene (99 mL, 0.1 M) solution of intermediate IV (9.9 mmol, 1.0 eq.), Pd2(dba)3 (0.99 mmol, 0.1 eq.), BINAP (1.98 mmol, 0.2 eq.), and K2CO3 (19.8 mmol, 2.0 eq.), and the mixture was refluxed at 110 °C for 24 hours. After cooling to room temperature, the reaction solution was filtered through diatomaceous earth. The concentrate was then purified by column chromatography to give intermediate V (7.92 mmol).

[0067] The above intermediate V (7.92 mmol) was dissolved in DMC (79 mL, 0.1 M), and trifluoroacetic acid (39.6 mL, 5.0 eq.) was slowly added. The mixture was stirred overnight at room temperature, and the solvent was removed by evaporation under reduced pressure. The product was then purified by column chromatography to obtain intermediate VI (7.52 mmol).

[0068] In an argon-atmospheric glove box, p-bromoiodobenzene (8.27 mmol, 1.1 eq.) was added to a toluene (75 mL, 0.1 M) solution of intermediate VI (7.52 mmol, 1.0 eq.), Pd2(dba)3 (0.75 mmol, 0.1 eq.), BINAP (1.5 mmol, 0.2 eq.), and K2CO3 (15.04 mmol, 2.0 eq.), and the mixture was refluxed at 110 °C for 24 h. After cooling to room temperature, the reaction solution was filtered through diatomaceous earth. The concentrate was purified by column chromatography to give the final product (3.85 mmol), a brown solid, in a total yield of 35%. The structural formula is shown below:

[0069]

[0070] 1 H NMR (600MHz, CDCl3) δ7.31–7.27(m,2H),6.95(d,J=8.0Hz,2H),6.67–6.61(m,2H),6.61 –6.58(m,2H),6.57(s,1H),6.01(s,1H),3.84(d,J=1.7Hz,3H),3.70(dd,J=8.3,4.6Hz,1 H),3.61(s,4H),3.52(d,J=1.7Hz,3H),3.44–3.34(m,4H),3.28–3.16(m,2H),2.84(dtd ,J=17.9,10.6,10.1,4.0Hz,2H),2.73–2.62(m,2H),2.57(s,3H),2.08(p,J=6.4Hz,2H). 13 C NMR (150MHz, CDCl3) δ147.18,146.12,146.06,145.36,132.12,130.93,128.70,126.93,125.18,113.14,111.40,111.09,110 .92,107.65,64.97,55.67,55.33,48.70,48.37,47.38,47.28,46.54,42.34,40.22,25.05,23.45.HRMS(ESI)m / z:calculated forC 30 H 36 79 BrN3O2[M+H] + :550.2064,found550.2055,calculated for C 30 H 36 81 BrN3O2[M+H] +:552.2043,found 552.2033.

[0071] IC50 of SGC7901 / VCR cells after treatment with the above compounds for 48 hours 50 The value was 7.05±0.40 μM, and the IC50 value of SGC7901 / VCR cells treated with VCR for 48 hours was 7.05±0.40 μM. 50 The IC50 value of the above compound in combination with VCR at 2.0 μM was 22960.00 ± 3500.00 nM, and the 48-hour IC50 of SGC7901 / VCR cells was 22960.00 ± 3500.00 nM. 50 The value is 40.26 ± 12.51 nM, and the corresponding reversal factor is 570.29.

[0072] 48-hour IC50 treatment of Eca109 / VCR cells with VCR 50 The IC50 value of the above compound in combination with VCR at 2.0 μM was 6830.00 ± 407.30 nM, and the 48-hour IC50 of Eca109 / VCR cells was 6830.00 ± 407.30 nM. 50 The value was 49.64 ± 4.34 nM, and the corresponding reversal factor was 137.59.

[0073] Example 2

[0074] Take a 250 mL round-bottom flask and add 11 mmol of 3,4-dimethoxyphenethylamine, 11 mmol of p-bromophenylacetic acid, and 0.88 mmol of boric acid sequentially. Then dissolve them in 120 mL of toluene and stir the mixture under reflux at 150 °C for 12 hours. Afterward, concentrate the solution to remove the solvent, wash the mixture 2-3 times with 30 mL of ethyl acetate, and then filter off the washings to obtain intermediate I (10.91 mmol crude product).

[0075] 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 saturated sodium bicarbonate aqueous solution was slowly added to quench it until bubbles were no longer generated in large quantities 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 with anhydrous sodium sulfate, and dichloromethane was removed by rotary evaporation to obtain intermediate II (10.36 mmol crude product).

[0076] Take a 250 ml round-bottom flask, dissolve 10.36 mmol of intermediate II in 100 ml of anhydrous methanol, and then slowly add 51.8 mmol of sodium borohydride under ice bath conditions. Seal the round-bottom flask and attach a balloon to the seal. Stir at room temperature for 5 hours, and then remove excess anhydrous methanol solution to obtain intermediate III (10.2 mmol crude product).

[0077] 10.2 mmol of intermediate III and 30.6 mmol of 37% formaldehyde aqueous solution were added to 100 ml of anhydrous methanol. Then, under ice bath conditions, 40 mmol of sodium borohydride was slowly added. The round-bottom flask was then sealed, and a balloon was attached to the seal. The mixture was stirred at room temperature for 5 hours. Excess anhydrous methanol solution was removed by vortexing. The solution was purified by silica gel column chromatography (eluent: dichloromethane:methanol = 80:1) to obtain 9.9 mmol of intermediate IV.

[0078] In an argon-atmospheric glove box, N-Boc-periperazine (10.89 mmol, 1.1 eq.) was added to a toluene (99 mL, 0.1 M) solution of intermediate IV (9.9 mmol, 1.0 eq.), Pd2(dba)3 (0.99 mmol, 0.1 eq.), BINAP (1.98 mmol, 0.2 eq.), and K2CO3 (19.8 mmol, 2.0 eq.), and the mixture was refluxed at 110 °C for 24 hours. After cooling to room temperature, the reaction solution was filtered through diatomaceous earth. The concentrate was then purified by column chromatography to give intermediate V (7.92 mmol).

[0079] The above intermediate V (7.92 mmol) was dissolved in DMC (79 mL, 0.1 M), and trifluoroacetic acid (39.6 mL, 5.0 eq.) was slowly added. The mixture was stirred overnight at room temperature, and the solvent was removed by evaporation under reduced pressure. The product was then purified by column chromatography to obtain intermediate VI (7.52 mmol).

[0080] Intermediate product VI (7.52 mmol) and potassium fluoride (75.2 mmol, 10 eq.) were dissolved in acetonitrile (75 mL, 0.1 M), followed by the addition of 6-iodo-1-hexyne (9.02 mmol, 1.2 eq.), and the reaction was carried out at room temperature for 12 hours. The mixture was filtered, concentrated, and purified by column chromatography to give the final product (5.64 mmol), a colorless oil, with an overall yield of 51%. The structural formula is shown below:

[0081]

[0082] 1H NMR (600MHz, CDCl3) δ6.92(d,J=8.6Hz,2H),6.59(d,J=8.7Hz,2H),6.56(s,1H),6.00(s,1H),3.83( s,3H),3.67(dd,J=8.2,4.8Hz,1H),3.53(s,5H),3.45(t,J=6.3Hz,2H),3.26–3.19(m,1H),3.16(dd ,J=13.6,4.6Hz,1H),2.86(s,1H),2.79(s,1H),2.75(s,2H),2.71–2.57(m,4H),2.55(s,3H),2.53– 2.46(m,2H),2.21(t,J=8.2Hz,2H),2.00–1.93(m,3H),1.60(p,J=6.9Hz,2H),1.53(p,J=6.8Hz,2H). 13 C NMR (150Hz, CDCl3) δ147.50,147.19,146.10,130.63,111.45,111.25,110.98,84.30,68.46,65.05,5 7.21,55.71,55.42,54.49,47.99,46.59,40.18,27.58,26.28,18.28.HRMS(ESI)m / z:calculatedfor C 30 H 41 N3O2[M+H] + :476.3272,found 476.3261. Example 3

[0083] Take a 250 mL round-bottom flask and add 11 mmol of 3,4-dimethoxyphenethylamine, 11 mmol of p-bromophenylacetic acid, and 0.88 mmol of boric acid sequentially. Then dissolve them in 120 mL of toluene and stir the mixture under reflux at 150 °C for 12 hours. Afterward, concentrate the solution to remove the solvent, wash the mixture 2-3 times with 30 mL of ethyl acetate, and then filter off the washings to obtain intermediate I (10.91 mmol crude product).

[0084] 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 saturated sodium bicarbonate aqueous solution was slowly added to quench it until bubbles were no longer generated in large quantities 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 with anhydrous sodium sulfate, and dichloromethane was removed by rotary evaporation to obtain intermediate II (10.36 mmol crude product).

[0085] Take a 250 ml round-bottom flask, dissolve 10.36 mmol of intermediate II in 100 ml of anhydrous methanol, and then slowly add 51.8 mmol of sodium borohydride under ice bath conditions. Seal the round-bottom flask and attach a balloon to the seal. Stir at room temperature for 5 hours, and then remove excess anhydrous methanol solution to obtain intermediate III (10.2 mmol crude product).

[0086] 10.2 mmol of intermediate III and 30.6 mmol of 37% formaldehyde aqueous solution were added to 100 ml of anhydrous methanol. Then, under ice bath conditions, 40 mmol of sodium borohydride was slowly added. The round-bottom flask was then sealed, and a balloon was attached to the seal. The mixture was stirred at room temperature for 5 hours. Excess anhydrous methanol solution was removed by vortexing. The solution was purified by silica gel column chromatography (eluent: dichloromethane:methanol = 80:1) to obtain 9.9 mmol of intermediate IV.

[0087] In an argon-atmospheric glove box, N-Boc-piperazine (10.89 mmol, 1.1 eq.) was added to a toluene (99 mL, 0.1 M) solution of intermediate IV (9.9 mmol, 1.0 eq.), Pd2(dba)3 (0.99 mmol, 0.1 eq.), BINAP (1.98 mmol, 0.2 eq.), and K2CO3 (19.8 mmol, 2.0 eq.), and the mixture was refluxed at 110 °C for 24 hours. After cooling to room temperature, the reaction solution was filtered through diatomaceous earth. The concentrate was then purified by column chromatography to give intermediate V (6.93 mmol).

[0088] Intermediate V (6.93 mmol) was dissolved in DMC (69 mL, 0.1 M), and trifluoroacetic acid (34.7 mL, 5.0 eq.) was slowly added. The mixture was stirred overnight at room temperature, and the solvent was removed by evaporation under reduced pressure. The product was then purified by column chromatography to obtain intermediate VI (6.24 mmol).

[0089] In an argon-atmospheric glove box, 3,5-dichloroiodobenzene (6.86 mmol, 1.1 eq.) was added to a toluene (68 mL, 0.1 M) solution of intermediate VI (6.24 mmol, 1.0 eq.), Pd2(dba)3 (0.62 mmol, 0.1 eq.), BINAP (1.24 mmol, 0.2 eq.), and K2CO3 (12.4 mmol, 2.0 eq.), and the mixture was refluxed at 110 °C for 24 h. After cooling to room temperature, the reaction solution was filtered through diatomaceous earth. The concentrate was purified by column chromatography to give the final product (3.85 mmol), a brown solid, in a total yield of 35%. The structural formula is shown below:

[0090]

[0091] 1 H NMR(600MHz, CDCl3) δ7.29(d,J=8.9Hz,1H),7.05–6.97(m,3H),6.87(d,J=8.2Hz,2H),6.78(dd ,J=9.0,2.8Hz,1H),6.56(s,1H),6.00(s,1H),3.83(s,3H),3.70(dd,J=8.0,5.0Hz,1H),3.54( s,3H),3.33–3.27(m,4H),3.25(dd,J=7.3,3.9Hz,4H),3.23–3.14(m,2H),2.85(ddd,J=15.3,9 .0,5.8Hz,1H),2.77(ddt,J=21.8,13.6,6.2Hz,2H),2.61(dt,J=15.9,4.5Hz,1H),2.55(s,3H). 13 C NMR (150MHz, CDCl3) δ150.49,149.25,147.12,146.06,132.71,131.62,130.48,130.39,128.84,125.54,122.35,117.35,1 16.39,115.47,110.96,110.95,64.76,55.63,55.39,49.48,48.76,46.58,42.46,40.18,25.19.HRMS(ESI)m / z:calculated for C 29 H 33 35 Cl2N3O2[M+H] + :526.2023,found 526.2015,calculated forC 29 H 33 37 Cl2N3O2[M+H] + :528.1993,found 528.1984.

[0092] IC50 of SGC7901 / VCR cells after treatment with the above compounds for 48 hours 50 The value was 7.99±0.56μM, and the IC50 value of SGC7901 / VCR cells treated with VCR for 48 hours was 7.99±0.56μM. 50 The IC50 value of the above compound in combination with VCR at 2.0 μM was 22960.00 ± 3500.00 nM, and the 48-hour IC50 of SGC7901 / VCR cells was 22960.00 ± 3500.00 nM. 50The value was 22.70 ± 9.49 nM, and the corresponding reversal factor was 1011.45.

[0093] 48-hour IC50 treatment of Eca109 / VCR cells with VCR 50 The IC50 value of the above compound in combination with VCR at 2.0 μM was 6830.00 ± 407.30 nM, and the 48-hour IC50 of Eca109 / VCR cells was 6830.00 ± 407.30 nM. 50 The value is 213.70 ± 11.08 nM, and the corresponding reversal factor is 31.96.

[0094] Example 4

[0095] Take a 250 mL round-bottom flask and add 11 mmol of 3,4-dimethoxyphenethylamine, 11 mmol of p-bromophenylacetic acid, and 0.88 mmol of boric acid sequentially. Then dissolve them in 120 mL of toluene and stir the mixture under reflux at 150 °C for 12 hours. Afterward, concentrate the solution to remove the solvent, wash the mixture 2-3 times with 30 mL of ethyl acetate, and then filter off the washings to obtain intermediate I (10.91 mmol crude product).

[0096] 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 saturated sodium bicarbonate aqueous solution was slowly added to quench it until bubbles were no longer generated in large quantities 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 with anhydrous sodium sulfate, and dichloromethane was removed by rotary evaporation to obtain intermediate II (10.36 mmol crude product).

[0097] Take a 250 ml round-bottom flask, dissolve 10.36 mmol of intermediate II in 100 ml of anhydrous methanol, and then slowly add 51.8 mmol of sodium borohydride under ice bath conditions. Seal the round-bottom flask and attach a balloon to the seal. Stir at room temperature for 5 hours, and then remove excess anhydrous methanol solution to obtain intermediate III (10.2 mmol crude product).

[0098] 10.2 mmol of intermediate III and 30.6 mmol of 37% formaldehyde aqueous solution were added to 100 ml of anhydrous methanol. Then, under ice bath conditions, 40 mmol of sodium borohydride was slowly added. The round-bottom flask was then sealed, and a balloon was attached to the seal. The mixture was stirred at room temperature for 5 hours. Excess anhydrous methanol solution was removed by vortexing. The solution was purified by silica gel column chromatography (eluent: dichloromethane:methanol = 80:1) to obtain 9.9 mmol of intermediate IV.

[0099] In an argon-atmospheric glove box, N-Boc-piperazine (10.89 mmol, 1.1 eq.) was added to a toluene (99 mL, 0.1 M) solution of intermediate IV (9.9 mmol, 1.0 eq.), Pd2(dba)3 (0.99 mmol, 0.1 eq.), BINAP (1.98 mmol, 0.2 eq.), and K2CO3 (19.8 mmol, 2.0 eq.), and the mixture was refluxed at 110 °C for 24 hours. After cooling to room temperature, the reaction solution was filtered through diatomaceous earth. The concentrate was then purified by column chromatography to give intermediate V (6.93 mmol).

[0100] Intermediate V (6.93 mmol) was dissolved in DMC (69 mL, 0.1 M), and trifluoroacetic acid (34.7 mL, 5.0 eq.) was slowly added. The mixture was stirred overnight at room temperature, and the solvent was removed by evaporation under reduced pressure. The product was then purified by column chromatography to obtain intermediate VI (6.24 mmol).

[0101] In an argon-atmospheric glove box, p-bromoiodobenzene (6.86 mmol, 1.1 eq.) was added to a toluene (68 mL, 0.1 M) solution of intermediate VI (6.24 mmol, 1.0 eq.), Pd2(dba)3 (0.62 mmol, 0.1 eq.), BINAP (1.24 mmol, 0.2 eq.), and K2CO3 (12.4 mmol, 2.0 eq.), and the mixture was refluxed at 110 °C for 24 h. After cooling to room temperature, the reaction solution was filtered through diatomaceous earth. The concentrate was purified by column chromatography to give the final product (4.73 mmol), a brown solid, in a total yield of 43%. The structural formula is shown below:

[0102]

[0103] 1 H NMR (600MHz, CDCl3) δ7.36(d,J=8.5Hz,2H),7.02(d,J=8.1Hz,2H),6.88(d,J=8.1Hz,2H ),6.84(d,J=8.6Hz,2H),6.56(s,1H),5.99(s,1H),3.84(s,3H),3.71(dd,J=8.1,5.0Hz ,1H),3.54(s,3H),3.27(hept,J=3.9Hz,8H),3.21(ddd,J=22.9,13.2,6.7Hz,2H),2.90 –2.78(m,2H),2.75(dd,J=13.6,8.1Hz,1H),2.63(dt,J=15.9,4.5Hz,1H),2.56(s,3H). 13C NMR (150MHz, CDCl3) δ150.16,149.41,147.17,146.09,131.85,131.43,130.49,128.76,125.46,117.82,11 6.36,112.09,110.98,64.81,55.66,55.42,49.58,49.14,42.45,40.21,25.17.HRMS(ESI)m / z:calculated for C 29 H 34 79 BrN3O2[M+H] + :536.1907,found 536.1899,calculated for C 29 H 34 81 BrN3O2[M+H] + :538.1887,found 538.1878.

[0104] IC50 of SGC7901 / VCR cells after treatment with the above compounds for 48 hours 50 The value was 17.29±2.16 μM, and the IC50 value of SGC7901 / VCR cells treated with VCR for 48 hours was 17.29±2.16 μM. 50 The IC50 value of the above compound in combination with VCR at 2.0 μM was 22960.00 ± 3500.00 nM, and the 48-hour IC50 of SGC7901 / VCR cells was 22960.00 ± 3500.00 nM. 50 The value is 373.10 ± 104.28 nM, and the corresponding reversal factor is 61.54.

[0105] 48-hour IC50 treatment of Eca109 / VCR cells with VCR 50 The IC50 value of the above compound in combination with VCR at 2.0 μM was 6830.00 ± 407.30 nM, and the 48-hour IC50 of Eca109 / VCR cells was 6830.00 ± 407.30 nM. 50 The value is 658.30 ± 21.45 nM, and the corresponding reversal factor is 10.38.

[0106] Example 5

[0107] This example is based on an autophagy inhibitor with significant medicinal value obtained using the research method of this invention, named OY-102. It can effectively reverse multidrug resistance in tumors and has advantages such as simple synthesis, high oral bioavailability, and high resistance reversal activity. Reversal activity assays, plate colony formation assays, and drug synergy analysis confirmed its excellent synergistic anticancer effect with vincristine (VCR) on drug-resistant SGC7901 / VCR cells (combined with 2 μM OY-102, IC50). 50=15.77 nM, RF = 1455.93). It also demonstrated excellent drug resistance reversal activity in nude mouse xenograft tumor models, while exhibiting low in vivo toxicity. Mechanistic investigation revealed that OY-102 can inhibit autophagic flux in various drug-resistant tumor cells. The following are the synthetic steps of OY-102:

[0108] Take a 250 ml round-bottom flask and add 11 mmol of 3,4-dimethoxyphenethylamine, 11 mmol of p-bromophenylacetic acid, and 0.88 mmol of boric acid sequentially. Then dissolve them in 120 ml of toluene and stir the reaction under reflux at 150 °C for 12 hours. Afterward, concentrate to remove the solvent, wash the mixture 2-3 times with 30 ml of ethyl acetate, and then filter off the washings to obtain intermediate I (10.91 mmol crude product).

[0109] 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 saturated sodium bicarbonate aqueous solution was slowly added to quench it until bubbles were no longer generated in large quantities 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 with anhydrous sodium sulfate, and dichloromethane was removed by rotary evaporation to obtain intermediate II (10.36 mmol crude product).

[0110] Take a 250 ml round-bottom flask, dissolve 10.36 mmol of intermediate II in 100 ml of anhydrous methanol, and then slowly add 51.8 mmol of sodium borohydride under ice bath conditions. Seal the round-bottom flask and attach a balloon to the seal. Stir at room temperature for 5 hours, and then remove excess anhydrous methanol solution to obtain intermediate III (10.2 mmol crude product).

[0111] 10.2 mmol of intermediate III and 30.6 mmol of 37% formaldehyde aqueous solution were added to 100 ml of anhydrous methanol. Then, under ice bath conditions, 40 mmol of sodium borohydride was slowly added. The round-bottom flask was then sealed, and a balloon was attached to the seal. The mixture was stirred at room temperature for 5 hours. Excess anhydrous methanol solution was removed by vortexing. The solution was purified by silica gel column chromatography (eluent: dichloromethane:methanol = 80:1) to obtain 9.9 mmol of intermediate IV.

[0112] In an argon-atmospheric glove box, N-Boc-periperazine (10.89 mmol, 1.1 eq.) was added to a toluene (99 mL, 0.1 M) solution of intermediate IV (9.9 mmol, 1.0 eq.), Pd2(dba)3 (0.99 mmol, 0.1 eq.), BINAP (1.98 mmol, 0.2 eq.), and K2CO3 (19.8 mmol, 2.0 eq.), and the mixture was refluxed at 110 °C for 24 hours. After cooling to room temperature, the reaction solution was filtered through diatomaceous earth. The concentrate was then purified by column chromatography to give intermediate V (7.92 mmol).

[0113] The above intermediate V (7.92 mmol) was dissolved in DMC (79 mL, 0.1 M), and trifluoroacetic acid (39.6 mL, 5.0 eq.) was slowly added. The mixture was stirred overnight at room temperature, and the solvent was removed by evaporation under reduced pressure. The product was then purified by column chromatography to obtain intermediate VI (7.52 mmol).

[0114] In an argon-atmospheric glove box, 3,5-dichloroiodobenzene (8.27 mmol, 1.1 eq.) was added to a toluene (75 mL, 0.1 M) solution of intermediate VI (7.52 mmol, 1.0 eq.), Pd2(dba)3 (0.75 mmol, 0.1 eq.), BINAP (1.5 mmol, 0.2 eq.), and K2CO3 (15.04 mmol, 2.0 eq.), and the mixture was refluxed at 110 °C for 24 hours. After cooling to room temperature, the reaction solution was filtered through diatomaceous earth. The concentrate was purified by column chromatography to give the final product (3.3 mmol), a brown solid, in a total yield of 30%. The structural formula is shown below:

[0115]

[0116] 1H NMR (600MHz, CDCl3) δ7.22(d,J=9.0Hz,1H),6.96(d,J=8.3Hz,2H),6.75(d,J=2.6Hz,1H),6.63(d,J=8.4Hz, 2H),6.58–6.52(m,2H),6.05(s,1H),3.84(s,3H),3.69–3.64(m,1H),3.61(s,4H),3.54(s,3H),3.46–3.35(m ,4H),3.20(ddd,J=13.2,9.2,5.1Hz,1H),3.11(dd,J=13.7,4.7Hz,1H),2.84(dt,J=15.3,7.3Hz,1H),2.77( dt,J=10.0,4.6Hz,1H),2.69(dd,J=13.7,8.1Hz,1H),2.63–2.58(m,1H),2.54(s,3H),2.08(p,J=6.0Hz,2H). 13 C NMR (150MHz, CDCl3) δ147.06,146.77,146.03,145.20,133.14,130.94,130.79,129.43,127.60,125.71,118.51,112.84,111.47,111 .13,111.11,110.99,64.95,55.70,55.39,48.87,48.46,47.54,47.38,46.75,42.62,40.28,25.39,23.44.HRMS(ESI)m / z:calculated for C 30 H 35 35 Cl2N3O2[M+H] + :540.2179,found 540.2173,calculated forC 30 H 35 37 Cl2N3O2[M+H] + :542.2150,found 542.2142.

[0117] The method described in this invention yields the following structural formula for the stereoisomer:

[0118]

[0119] See the HPLC chromatogram of its stereoisomers. Figure 11-13 .

[0120] IC50 of SGC7901 / VCR cells after treatment with the above compounds for 48 hours 50 The value was 4.64 ± 0.39 μM, and the IC50 value of SGC7901 / VCR cells treated with VCR for 48 hours was 4.64 ± 0.39 μM. 50 The IC50 value of the above compound in combination with VCR at 2.0 μM was 22960.00 ± 3500.00 nM, and the 48-hour IC50 of SGC7901 / VCR cells was 22960.00 ± 3500.00 nM. 50 The value is 15.77 ± 4.22 nM, and the corresponding reversal factor is 1455.93.

[0121] 48-hour IC50 treatment of Eca109 / VCR cells with VCR 50 The IC50 value of the above compound in combination with VCR at 2.0 μM was 6830.00 ± 407.30 nM, and the 48-hour IC50 of Eca109 / VCR cells was 6830.00 ± 407.30 nM. 50 The value is 24.39 ± 2.28 nM, and the corresponding reversal factor is 280.03.

[0122] Example 6: Bioactivity testing of OY-102:

[0123] 1. OY-102 Antidrug Resistance Reversal Activity Test

[0124] like Figure 1 A. The 48-hour IC50 effect of VCR on human gastric cancer cells SGC7901 and its drug-resistant cell line SGC7901 / VCR was determined by the CCK8 assay for both cell lines. 50 Values, and the 48-hour IC50 values ​​of VCR on SGC7901 / VCR cells after VCR was combined with 1 μM LIEN, 2 μM LIEN, and 2 μM TET, respectively. 50 Value. Furthermore, using the same experimental conditions, the 48-hour IC50 of doxorubicin (ADR) on human breast cancer cells MCF7 and its drug-resistant cell line MCF / ADR was determined. 50 value( Figure 1 B); The 48-hour IC50 of VCR on human esophageal cancer cells Eca109 and its drug-resistant cell line Eca109 / VCR was determined. 50 value( Figure 1 C) The 48-hour IC50 of paclitaxel (TAX) on human lung cancer cells A549 and its drug-resistant cell line A549 / TAX was determined. 50 value( Figure 1 D); and the 48-hour IC50 values ​​of the corresponding resistance drugs after combined administration of 1 μM MIY-102, 2 μM MIY-102, 2 μM LIEN, and 2 μM MTET in the above-mentioned drug-resistant cell lines. 50 Value change. From Figure 1 A- Figure 1 As shown in Figure D, the drug-resistant cell lines exhibited higher resistance compared to their corresponding parental cells, especially A549 / TAX, followed by SGC7901 / VCR and MCF / ADR. Compound OY-102 reversed the drug resistance of the drug-resistant cell lines in a dose-dependent manner, particularly SGC7901 / VCR, with the highest reversal fold reaching 1455.9, followed by Eca109 / VCR and MCF / ADR. OY-102 demonstrated a stronger drug resistance reversal effect compared to the same concentrations of the natural isoquinoline alkaloids LIEN and TET.

[0125] 2. Plate colony formation assay to investigate the drug resistance reversal activity of OY-102.

[0126] Cell colony formation was observed in SGC7901 / VCR, MCF / ADR, and Eca109 / VCR cells after treatment with 0.5 μM OY-102 and 0.5 μM or 1 μM of the corresponding antibiotic resistance drug (VCR or DOX) for 72 hours. Figure 2 0.5 μM OY-102 and 0.5 μM anticancer drug have almost no toxicity to cells, but the combination of the two almost completely killed drug-resistant cell lines.

[0127] 3. Pharmacokinetic parameters of OY-102 after intravenous injection and oral administration in SD rats

[0128] The applicant determined the pharmacokinetic parameters of OY-102 in SD rats after intravenous injection (1 mg / kg) and oral administration (10 mg / kg). The pharmacokinetic parameters calculated using a non-compartmental model are shown in Table 1. Peak plasma concentrations (Tc) were reached 0.10 and 4.0 hours after administration in the intravenous (iv) and oral (op) groups, respectively. max The corresponding peak concentrations (C) in group IV. max The rate of elimination in the first group was nearly 2.6 times that of the second group. Both groups had low terminal elimination rate constants (λz) and low half-lives (t). 1 / 2 The average length of stay (MRT) is relatively long (around 9 hours) and the average length of stay is also relatively long. 0-inf The time under the concentration-time curve (AUC) was also relatively long, indicating that OY-102 had a relatively longer duration of action in both intravenous and opioid (IV) treatments. 0-t and AUC 0-inf The results showed that OY-102 had a higher oral bioavailability, and based on this, the oral bioavailability of OY-102 was calculated to be 62.29%. This indicates that OY-102 has a high oral bioavailability, and thus the effective doses for animal studies were determined: OY-102 monotherapy (10 mg / kg / 2d); and low-dose OY-102 (5 mg / kg / 2d) in combination with VCR (0.5 mg / kg / 2d).

[0129] Table 1. Pharmacokinetic parameters of OY-102 after oral and intravenous administration.

[0130]

[0131] 4. Animal Experiment Results

[0132] A nude mouse xenograft tumor model was established using SGC7901 / VCR cells to detect the tumor MDR reversal activity of OY-102 in vivo. Patients were divided into four treatment groups: solvent control group (10 mL / kg / 2d, gavage), VCR group (0.5 mg / kg / 2d, tail vein), OY-102 group (10 mg / kg / 2d, gavage), and a combined treatment group of VCR (0.5 mg / kg / 2d) / OY-102 (5 mg / kg / 2d). After 3 weeks of treatment, the combined treatment of OY-102 and VCR effectively inhibited tumor proliferation in vivo. Figure 3 A and Figure 3 B); OY-102 monotherapy at 10 mg / kg also showed an anti-tumor proliferation effect (B); Figure 3 A and Figure 3 B); There was no significant change in the body weight of nude mice in each group during the treatment period. Figure 3 C). Tumor pathology and TUNEL analysis showed that OY-102 / VCR combination therapy and OY-102 monotherapy increased the proportion of pyknotted, vacuolated, and apoptotic cells in tumor tissue, while Ki-67 proliferation staining was significantly reduced. HE staining of liver tissue showed that OY-102 combined with VCR did not have significant hepatotoxicity. Figure 3 D). These results indicate that OY-102 is an effective anticancer agent and MDR reversal agent that can reverse VCR resistance in tumors and inhibit the growth of SGC7901 / VCR xenografts without significant hepatotoxicity or systemic toxicity.

[0133] 5. OY-102 increases the number of intracellular autophagosomes.

[0134] To determine whether compound OY-102 affects autophagy in human cells, the applicant labeled the autophagy marker protein LC3 with the fluorescent protein EGFP, constructed the EGFP-LC3 plasmid, and observed the expression of EGFP-LC3 protein in cells using laser confocal scanning microscopy to reflect the accumulation of autophagosomes. First, SGC7901 / VCR and MCF / ADR cells were transfected with the EGFP-LC3 plasmid for 24 hours, then treated with 5 μM OY-102 or without for 24 hours. Subsequently, the accumulation of autophagosomes was observed using laser confocal scanning microscopy. Figure 4Treatment of cells with compound OY-102 resulted in a significant increase in the formation of EGFP-LC3 fluorescent spots in SGC7901 / VCR and MCF / ADR cells, indicating an increase in autophagosomes in the cells.

[0135] 6. Transmission electron microscopy observation of the effect of OY-102 on autophagy in tumor cells

[0136] To further determine the effect of compound OY-102 on autophagy in SGC7901 / VCR and MCF7 / ADR cells, transmission electron microscopy (TEM) was used to directly observe the accumulation of autophagosomes within the cells. Figure 5 Compared with the control group, the number of autophagosomes in cancer cells treated with 5 μM OY-102 for 24 hours was increased. Swelling mitochondria and mitophagy were also significantly increased in the treatment group.

[0137] 7. Western blot analysis of the effects of OY-102 on autophagy-related proteins in tumor cells.

[0138] To verify whether the increased number of autophagosomes was due to drug-induced increased autophagosome production or to increased accumulation caused by inhibited autophagosome degradation, the applicant used Western blotting to simultaneously detect LC3B-I and LC3B-II with LC3B antibody and p62 with SQSTM1 antibody. The aim was to verify whether the increased number of autophagosomes induced by OY-102 was due to induction or inhibition of autophagy. Figure 6 A- Figure 6 C and Figure 7 A- Figure 7 C. The applicant studied the effects of compound OY-102 on LC3B transformation and p62 expression in SGC7901 / VCR and MCF / ADR cells, and found that LC3B-II and p62 accumulated in a dose- and time-dependent manner after OY-102 treatment. Figure 6 B. Figure 6 C is Figure 6 A statistical graph of the ratios of LC3B-II / β-Actin and p62 / β-Actin relative to the negative control after three replicates of A; Figure 7 B. Figure 7 C is Figure 7 A statistical plot of the ratios of LC3B-II / β-Actin and p62 / β-Actin relative to the negative control after three replicates (mean ± SD, n = 3; ns indicates P > 0.05, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001)). This suggests that OY-102 is an autophagy inhibitor, leading to the inhibition of LC3B-II and p62 degradation, rather than an autophagy inducer (increased LC3B-II and decreased p62).

[0139] In addition, the applicant also determined the effects of compound OY-102 on LC3B and p62 levels in drug-resistant cell lines A549 / TAX, SGC7901 / DDP, and Eca109 / VCR, respectively. Figure 8 After treatment with OY-102, the above-mentioned drug-resistant cell lines all showed accumulation of LC3B-II and p62, indicating that OY-102 can inhibit autophagy in different drug-resistant cell lines.

[0140] 8. Comparison of OY-102 with classic autophagy inhibitors and autophagy inducers

[0141] The applicant introduced the classic autophagy inducer rapamycin (RAPA), which induces autophagy by inhibiting autophagy inhibition mediated by the mTOR-ULK pathway, and the autophagy inhibitor bafilomycin A1 (Baf A1), which inhibits autophagosome degradation by inhibiting lysosomal acidification. Figure 9 A- Figure 9 As shown in C, OY-102 treatment alone, similar to Baf A1, promoted an increase in both LC3B-II and p62, unlike RAPA treatment alone (which slightly increased LC3B-II while decreasing p62). Simultaneously, OY-102 inhibited RAPA-induced autophagy; that is, the combined use of OY-102 and RAPA further increased LC3B-II expression and reversed the increase in p62 expression, with the effect being more pronounced after 24 hours of treatment. Figure 9 B and C represent the percentage values ​​of LC3B-II / β-Actin and p62 / β-Actin relative to the negative control after three replicates of 9A. (Mean ± SD, n = 3; ns indicates P > 0.05, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001)). This indicates that compound OY-102 is an autophagy inhibitor similar to BafA1, unlike RAPA which promotes autophagy degradation. Furthermore, OY-102 inhibits RAPA-induced autophagy.

[0142] 9. The S configuration of OY-102 exhibits better activity than the R configuration.

[0143] Chiral separation yielded the S configuration ((S)-OY-102) and the R configuration ((R)-OY-102). For example... Figure 10 A- Figure 10 C, 5 μM (S)-OY-102, (R)-OY-102, and OY-102 were used to detect the expression of autophagy-related proteins (p62 and LC3B) in SGC7901 / VCR and MCF / ADR cells, respectively. It was found that (S)-OY-102 had a stronger effect on autophagy-related proteins than (R)-OY-102. Figure 10 B and C are Figure 10 A statistical plot of LC3B-II / β-Actin and p62 / β-Actin after three replicates (mean ± SD, n = 3)).

[0144] In addition, the applicant determined the 48-hour IC50 of the isoform against SGC7901 / VCR cells using the CCK8 assay. 50 Value, and the 48-hour IC50 values ​​of 1 μM OY-102, (S)-OY-102 and (R)-OY-102 combined with different concentrations of VCR on SGC7901 / VCR cells. 50 As shown in Table 2, the cytotoxicity and MDR reversal activity of (S)-OY-102 were both higher than those of (R)-OY-102 when used alone or in combination. This indicates that (S)-OY-102 has superior cytotoxicity, MDR reversal activity, and autophagy regulation activity compared to the isoforms (R)-OY-102 and OY-102, and will be a key configuration to focus on for further drug development.

[0145] Table 2. 48-hour IC50 values ​​of OY-102 monotherapy and VCR combined with 1 μM OY-102 in different stereoconfigurations. 50 Value and Reversal Multiple

[0146]

[0147] in conclusion:

[0148] The isoquinoline alkaloid derivatives described in this invention can inhibit autophagy and efficiently reverse multidrug resistance in tumor cells. Among them, the derivative represented by OY-102 exhibits excellent reversal activity both in vitro and in vivo, and OY-102 also possesses excellent oral bioavailability. Further mechanistic studies have confirmed that OY-102 is a highly effective tumor cell autophagy inhibitor, capable of reversing drug resistance by inhibiting autophagic flux. The mature and stable synthetic route of the preparation method described in this invention, the potent reversal activity of the derivatives, and their excellent oral bioavailability demonstrate their potential for further development into drug-resistant tumor sensitizers.

Claims

1. A highly effective isoquinoline alkaloid derivative for reversing multidrug resistance in tumors, characterized in that: The derivative has the following general formula: ； Where: n = 0 or 1, R is The substituted phenyl, the In the structure of R 2 R 4 For Cl, R 1 R 3 and R 5 For H; or R 3 For Br, R 1 R 2 R 4 R 5 For H.

2. The isoquinoline alkaloid derivative according to claim 1, characterized in that: The derivatives include their stereoisomers.

3. The method for preparing the isoquinoline alkaloid derivative according to claim 1, characterized in that, The steps are as follows: ； 1) 3,4-Dimethoxyphenethylamine and p-bromophenylacetic acid were added to a toluene solution and stirred at 110-150°C for 12-20 hours under boric acid catalysis. The solvent was removed by concentration to obtain the reaction product. Ethyl acetate was added to the reaction product and washed 2-3 times. The mixture was then filtered to obtain intermediate product I. 2) Dissolve intermediate I in dichloromethane, then add phosphorus oxychloride to obtain a reaction solution, heat to 70-78°C, stir for 5-8 hours, slowly add saturated sodium bicarbonate aqueous solution to quench it until no more bubbles are generated in the reaction solution, extract to obtain an organic solvent layer, dry with anhydrous sodium sulfate to obtain intermediate II; 3) Dissolve intermediate II in methanol, cool to room temperature in an ice bath, slowly add sodium borohydride, stir at room temperature for 5 hours, and remove excess anhydrous methanol solution to obtain intermediate III; 4) Intermediate III was reductively amination to introduce a methyl group onto NH-, and then purified by column chromatography to obtain intermediate IV; 5) In an argon atmosphere, N-Boc-piperazine or N-Boc-periperazine was added to a toluene (0.1 M) solution of intermediate IV, Pd2(dba)3, BINAP and K2CO3. The mixture was refluxed at 110°C for 24 hours. After the reaction solution was cooled to room temperature, it was filtered with diatomaceous earth, concentrated, and purified by column chromatography to obtain intermediate V. 6) Dissolve intermediate V in 0.1M DMC, slowly add trifluoroacetic acid, stir overnight at room temperature, remove the solvent by evaporation under reduced pressure, and purify by column chromatography to obtain intermediate VI; 7) Intermediate VI undergoes a Buchwald–Hartwig cross-coupling reaction or substitution reaction, introducing different groups onto the NH group to obtain the final product.

4. The method according to claim 3, characterized in that: In step 1), the molar ratio of 3,4-dimethoxyphenethylamine: p-bromophenylacetic acid: boric acid is 11:11:0.

88. In step 2), the molar ratio of intermediate I to phosphorus oxychloride is 10-11:

33. In step 3), the molar ratio of intermediate II to sodium borohydride is 10.36:51.

8. The extraction in steps 2) and 3) uses dichloromethane.

5. The method according to claim 3, characterized in that: Step 4) The specific method for the reductive amination is to pre-stir intermediate III with 37% formaldehyde solution for half an hour, and then add sodium borohydride for reduction; In step 5), the molar ratio of N-Boc-piperazine or N-Boc-hyperpiperazine: intermediate IV: Pd2(dba)3: BINAP: K2CO3 is 9.0–11.0: 9.0–10.0: 0.99: 1.98: 19.

8. Step 6) The molar ratio of intermediate V to trifluoroacetic acid is 6.00-8.00: 20.00-50.

00.

6. The method according to claim 5, characterized in that: The molar ratio of intermediate III: formaldehyde solution: sodium borohydride is 10.00-11.00: 30.00-31.00:

40.

7. The method according to claim 3, characterized in that: Step 7) The substitution reaction is as follows: intermediate product VI and potassium fluoride are dissolved in acetonitrile (75 mL, 0.1 M), followed by the addition of iodide. The reaction is carried out at room temperature for 12 hours, filtered, concentrated, and purified by column chromatography. Preferably, the molar ratio of intermediate product VI: potassium fluoride: iodide is 7.52:75.2:9.

02. The Buchwald–Hartwig cross-coupling reaction involved adding aryl iodides to a toluene solution of intermediates VI, Pd2(dba)3, BINAP, and K2CO3 in an argon atmosphere. The reaction was refluxed at 110 °C for 24 hours. After the reaction solution was cooled to room temperature, it was filtered through diatomaceous earth, concentrated, and purified by column chromatography. Step 7) The structure is R in substituted phenyl 2 R 4 For Cl, R 1 R 3 and R 5 For H; or R 3 For Br, R 1 R 2 R 4 R 5 For H.

8. The method of claim 7, characterized in that, The molar ratio of the aryl iodide: intermediate VI: Pd2(dba)3: BINAP: K2CO3 is 6.86~8.27: 6.24~7.25: 0.62~0.75: 1.24~1.5: 12.4~15.

04.

9. The use of the isoquinoline alkaloid derivative of claim 1 in the preparation of a cancer treatment drug, wherein the cancer is gastric cancer, lung cancer, breast cancer, or esophageal cancer.

10. The application according to claim 9, characterized in that, The drug is a specific autophagy inhibitor, which is used as a sensitizer for antitumor chemotherapy.

11. The use of the isoquinoline alkaloid derivative of claim 1 in combination with vincristine in the preparation of a cancer treatment drug, wherein the cancer is gastric cancer or esophageal cancer.

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