EGFR-dependent micromolecular conjugates 1, 3 and 4, and preparation method and application of EGFR-dependent micromolecular conjugates 1, 3 and 4

Abstract

The invention discloses EGFR (epidermal growth factor receptor) dependent micromolecular conjugates 1, 3 and 4 as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. According to the EGFR-dependent micromolecular conjugine-1 disclosed by the invention, an IDO inhibitor D-MT is introduced into a lapatinib structure through reasonable structural modification, so that the target conjugine-1 is obtained. A glutathione S-transferase inhibitor NBDHEX is introduced into the structural fragment of the lapatinib through different connectors, and target conjugates 3 and 4 are obtained. Results show that the conjugate retains the inhibition effect of a parent compound on the activity of EGFR, IDO or GST and the powerful inhibition effect on proliferation of multiple different types of triple-negative breast cancer cells, can effectively reverse drug resistance of lapatinib-mediated triple-negative breast cancer cells, and can be used for preparing antitumor drugs.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and more specifically, relates to EGFR-dependent small molecule conjugates-1, 3, 4 and preparation methods and applications thereof. Background Art

[0002] As an important part of precision treatment, targeted therapy has made breakthrough progress in recent years. With the successful launch of a number of antibody-drug conjugates (ADCs), polymer-drug conjugates (PDCs), immunostimulating-antibody conjugates (ISACs) and small-molecule-drug conjugates (SMDCs) have emerged, which together have built a new blueprint for the development of targeted drugs. Among them, SMDC has shown better therapeutic effects than ADC in some solid tumors due to its excellent cell penetration ability, non-immunogenicity and relatively small molecular weight. SMDC is an emerging drug that uses small molecules as targeting ligands to couple with other small molecule drugs with different mechanisms of action to achieve precise treatment of diseases and produce a 1+1>2 effect. Its chemical structure consists of three parts: a small molecule binder targeting the receptor, a linker, and other medicinal molecules (payload) with different mechanisms of action. At present. There are about 10 products in clinical research worldwide, including Vintafolide (clinical phase III) developed by Merck & Endocyte, PEN-866 (clinical phase II) developed by Tarveda / Saisheng Pharmaceuticals, 177Lu-PSMA-617 (clinical phase II) developed by Novartis / Endocyte, and Epofolate (clinical phase II) developed by BMS@Endocyte. At present, there are relatively few SMDC drug molecules in clinical research in China. Currently, there are only AST-3424 (clinical phase II) independently developed by Aixindawei and PEN-866 (clinical phase II) obtained by Saisheng Pharmaceuticals from Tarveda in March 2020. Although there are relatively few studies on SMDC drug molecules, the existing clinical data show that the existing SMDC drug molecules have shown good clinical efficacy. In addition, global pharmaceutical giants such as Merck and Novartis have extended olive branches to SMDC, further confirming that SMDC's medicinal molecules have potential worth exploring.

[0003] As we all know, triple-negative breast cancer (TNBC) has become one of the most deadly female malignant tumors due to its strong tumor heterogeneity, lack of effective therapeutic targets, insensitivity to conventional endocrine therapy and targeted therapy, and easy acquired resistance to traditional chemotherapy drugs. Early TNBC patients can achieve good clinical efficacy through surgery, radiotherapy and chemotherapy, and the 5-year survival rate can reach about 77%. However, due to the lack of effective treatment methods, the 5-year survival rate of patients with advanced or metastatic cancer cells is only about 14%, and effective treatment methods are urgently needed. Through a large amount of data analysis and literature research, it was found that about 60% of basal cell-like TNBC patients have highly expressed epidermal growth factor receptor (EGFR) in their cancer cells, and about 89% of TNBC patients are sensitive to EGFR treatment, especially for basal-like type-2 tumors with overexpression of EGFR. However, it is difficult to achieve satisfactory clinical efficacy using EGFR inhibitors alone. Therefore, using EGFR inhibitors as targeting ligands and coupling them with other drugs with different mechanisms of action to obtain new SMDC drug molecules may be an effective way to obtain drugs for the treatment of TNBC.

[0004] Indoleamine 2,3-dioxygenase (IDO) is the only rate-limiting enzyme outside the liver that can catalyze the metabolism of tryptophan and decompose it along the kynurenine pathway to produce a series of metabolites including quinolinic acid. Studies have shown that IDO can increase the expression of IL-6 in tissues by consuming tryptophan to produce kynurenine, thereby increasing the expression of VEGF and promoting tumor angiogenesis. In addition, the continuous high expression of IDO in tumor cells will promote the continuous consumption of tryptophan in the tumor microenvironment and convert it into kynurenine. This metabolic regulation can inhibit the functional activity of tryptophan-sensitive T cells, including affecting the proliferation of T cells and the differentiation of regulatory T cells. However, the results of clinical studies on existing IDO inhibitors show that it is difficult to achieve satisfactory clinical efficacy using IDO inhibitors alone, and the anti-tumor effect of selective IDO inhibitors may also fail due to alternative mechanisms that bypass the function of IDO. Therefore, how to effectively utilize the IDO target and develop more effective therapeutic drugs is also an urgent problem that needs to be solved for the IDO target. Glutathione S-transferase (GST) is a multifunctional phase II metabolic enzyme that catalyzes the binding of glutathione to electrophilic substances and plays an important role in protecting organisms from toxic damage. This type of enzyme system has a dual role, namely, on the one hand, it improves the body's ability to detoxify carcinogens and prevent tumor occurrence; on the other hand, it also increases the metabolic capacity of cancer cells to chemotherapy drugs and produces drug resistance. Clinical statistical data show that the GST positive expression rate in patients with triple-negative breast cancer is significantly higher than that in non-triple-negative breast cancer, and the expression level of GST is positively correlated with the tumor malignancy and prognosis of patients with triple-negative breast cancer. Therefore, inhibiting GST may be one of the effective ways to treat triple-negative breast cancer and / or overcome the drug resistance of triple-negative breast cancer cells.

[0005] Lapatinib is an oral EGFR / Her2 dual-target tyrosine kinase inhibitor, which is mainly used in combination with capecitabine to treat epidermal growth factor receptor overexpression, and advanced or metastatic breast cancer that has been treated with anthracyclines, paclitaxel, and trastuzumab in the past. This application analyzes the structure-activity relationship of lapatinib and finds that the secondary amine group on the fatty chain in the structure of lapatinib is mainly inserted into the solvent region and can be used as a modifiable site. Based on this, this application introduces IDO inhibitors and GST inhibitors into its structure through reasonable modification of lapatinib to obtain a new type of EGFR-dependent SMDC medicinal molecules, and studies their application in the preparation of anti-tumor drugs. However, there is no report on SMDC medicinal molecules that can simultaneously inhibit EGFR and IDO or GST, and are used to treat triple-negative breast cancer and reverse triple-negative breast cancer cell resistance. Summary of the invention

[0006] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide EGFR-dependent small molecule conjugates-1, 3, and 4. Another technical problem to be solved by the present invention is to provide a preparation method of EGFR-dependent small molecule conjugates-1, 3, and 4. Another technical problem to be solved by the present invention is to provide the application of EGFR-dependent small molecule conjugates-1, 3, and 4 for preparing drugs against triple-negative breast cancer.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0008] A class of small molecule conjugates that inhibit EGFR and IDO enzyme activity, whose structural formula is shown in the following conjugate-1 or conjugate-2:

[0009]

[0010] The preparation method of conjugate-1 is specifically as follows: (Boc)2O is used to protect the amino group in the structure of the IDO inhibitor 1-methyl-D-tryptophan to obtain the intermediate 1-methyl-D-tryptophan-Boc; the intermediate 1-methyl-D-tryptophan-Boc is then subjected to an amide condensation reaction with lapatinib, and the original amino protecting group Boc is removed under the action of trifluoroacetic acid to obtain the target conjugate-1; the reaction process is as follows:

[0011]

[0012] The preparation method of conjugate-2 is specifically as follows: lapatinib is reacted with succinic anhydride to obtain an intermediate compound lapatinib-COOH; then the intermediate compound is reacted with the IDO inhibitor NLG919 to undergo an amide condensation reaction to obtain conjugate-2; the reaction process is as follows:

[0013]

[0014] A class of small molecule conjugates that inhibit EGFR and GST enzyme activity, whose structural formula is shown in the following conjugate-3 or conjugate-4:

[0015]

[0016] The preparation method of conjugate-3 is specifically as follows: lapatinib is reacted with succinic anhydride to obtain an intermediate compound lapatinib-COOH; then the GST inhibitor NBDHEX is esterified with the intermediate compound lapatinib-COOH to obtain the target conjugate-3; the reaction process is as follows:

[0017]

[0018] The preparation method of conjugate-4 is specifically as follows: lapatinib is reacted with succinic anhydride to obtain an intermediate compound lapatinib-COOH; then the intermediate compound lapatinib-COOH is subjected to an esterification reaction with 3-butyn-1-ol to obtain an intermediate lapatinib-alkynyl; then NBDHEX is subjected to an esterification reaction with 4-(azidomethyl)benzoic acid to obtain an intermediate NBDHEX-azido; finally, the intermediate lapatinib-alkynyl and the intermediate NBDHEX-azido are subjected to a click reaction under the catalysis of CuSO4·5H2O and vitamin C to obtain conjugate-4; the reaction process is as follows:

[0019]

[0020] Use of conjugate 1 or / and 2 in the preparation of a drug for reversing lapatinib-mediated cancer cell resistance.

[0021] Use of conjugate 1 or / and 2 in the preparation of a drug for inhibiting EGFR enzyme activity in cancer cells and / or promoting immune T cell secretion in cancer cells.

[0022] Use of conjugate 3 or / and 4 in the preparation of a drug for reversing lapatinib-mediated cancer cell resistance.

[0023] Use of conjugate 3 or / and 4 in the preparation of a drug for inhibiting EGFR enzyme activity in cancer cells and / or inhibiting GST P1-1 enzyme activity in cancer cells.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1) The present invention introduces the IDO inhibitor D-MT into the structure of lapatinib through reasonable structural modification to obtain the target conjugate-1. The results of the present invention show that the compound has a strong inhibitory effect on the expression of pEGFR (Y1068) protein in triple-negative breast cancer MDA-MB-231 cells, and the compound can also promote the differentiation of CD4+T cells by inhibiting kynurenine in MDA-MB-231 cells.

[0026] 2) The results of the present invention show that conjugate-1 can effectively reverse the lapatinib-mediated drug resistance of MDA-MB-231 cells.

[0027] 3) The GST inhibitor NBDHEX of the present invention is introduced into the structure of lapatinib through different linkers to obtain the target conjugate-3 and conjugate-4. The results of the present invention show that this type of compound has a strong inhibitory effect on the expression of pEGFR (Y1068) protein in triple-negative breast cancer MDA-MB-231 cells, and this type of compound can also strongly inhibit the activity of GST P1-1 enzyme, and the inhibitory effect of conjugate-3 on GST P1-1 enzyme activity is slightly better than NBDHEX.

[0028] 4) The results of the present invention show that conjugate-3 and conjugate-4 exhibit very potent cytotoxicity to the five cancer cells tested. Among them, the cytotoxicity of conjugate-3 to the five cancer cells was significantly better than that of lapatinib, NBDHEX, and the physical mixture of lapatinib and NBDHEX. In addition, conjugate-3 and conjugate-4 can also strongly reverse the lapatinib-mediated MDA-MB-231 cell resistance, with the resistance index (RI) reaching 1.36 and 1.27, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The graphs show the inhibition of pEGFR (Y1068) and EGFR protein levels by lapatinib and target compound conjugates-1 to 4;

[0030] Figure 2 The relative expression level of kynurenine in MDA-MB-231 cells after treatment with different conjugates (A is the blank group; B is the D-MT group; C is the NLG919 group; D is the conjugate-1 group; E is the conjugate-2 group; F is the mixture-1 group; G is the mixture-2 group);

[0031] Figure 3 The expression level of CD4+T cells in cancer cells after treatment with different conjugates (A) and a single analysis graph (B). DETAILED DESCRIPTION

[0032] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art.

[0033] The normal breast cancer cell line MCF-7, four triple-negative breast cancer cell lines (MDA-MB-231, HCC1187, BT20, HS-578T) and normal human mammary epithelial cell line MCF-12A used in this application were all purchased from Jiangsu Keygene Biotechnology Co., Ltd. (Nanjing, Jiangsu, China). The culture medium used for each cell culture was a conventional culture medium corresponding to the art, wherein MCF-7, MDA-MB-231, HS-578T and MCF-12A cell lines used DMEM culture medium, and HCC1187 and BT20 cells used RPMI-1640 culture medium.

[0034] Example 1

[0035] 1. Preparation of intermediate D-MT-Boc

[0036] Weigh 0.25g of D-MT and 0.29g of NaHCO3 and dissolve them in a mixed solution of 30mL of tetrahydrofuran (THF) and water (V:V=1:1). After cooling the reaction solution to 0°C, slowly add 0.3g of (Boc)2O (1.37mmol) to the reaction solution and stir for 10 minutes. Slowly raise the temperature of the reaction solution to room temperature and continue stirring the reaction for 24 hours. After the reaction is completed, concentrate under reduced pressure to remove THF, and adjust the pH value of the solution to 1.0 with 1mol / L hydrochloric acid. Subsequently, extract the water layer with ethyl acetate (15mL×3), wash with saturated brine (15mL×1), dry with anhydrous Na2SO4, and finally concentrate in vacuo to obtain the yellow intermediate compound 1-methyl-D-tryptophan-Boc (0.27g, yield: 74.1%). The reaction process is as follows:

[0037]

[0038] 1 H NMR (600MHz, DMSO-d6) δ12.53(s,1H),7.55(d,J=7.7Hz,1H),7.37(d,J=7.9Hz,1H),7.13(dd,J=15.4,7.5Hz,2H),7.02(t,J=7.2Hz,1H),6.97( d,J=7.8Hz,1H),4.16(d,J=4.2Hz,1H),3.71(s,3H),3.13(dd,J=14.2,3.8Hz,1H),2.99(dd,J=14.1,9.5Hz,1H),1.33(s,8H),1.21(s,1H)ppm. 13C NMR (150MHz, DMSO-d6) δ174.32,155.90,137.01,128.42,128.03,121.51,118.92,110.03,78.51,55.07,32.73,28.64,27.20ppm.ESI-MS:m / z[M+Na] + =341.1477; found 341.1489.

[0039] 2. Preparation of intermediate lapatinib-COOH

[0040] Weigh 0.58g of lapatinib (1mmol), 0.15g of succinic anhydride (1.5mmol) and 0.2g of triethylamine (2mmol) and dissolve them in 20mL of dichloromethane, and stir overnight at room temperature for 1 hour. After the reaction is completed, the solvent is removed by concentration under reduced pressure, 30mL of water is added, heated to 60°C, stirred for 2 hours, and then cooled to room temperature. The solvent is filtered to obtain a crude product, which is then recrystallized from 10mL of methanol to obtain the intermediate compound lapatinib-COOH (0.64g, yield: 94.0%). The reaction process is as follows:

[0041]

[0042] 1 H NMR (600MHz, DMSO-d6) δ12.24(s,1H),10.01(d,J=23.4Hz,1H),8.77(d,J=23.3Hz,1H),8.57(s,1H),8.21-8.13(m,1H),8.04(dd ,J=12.2,2.0Hz,1H),7.80(dd,J=8.6,4.4Hz,1H),7.76(dd,J=8.6,2.4Hz,1H),7.48(dd,J=14.2,7.8Hz,1H),7.34(t,J=9.2Hz,2 H),7.30-7.27(m,1H),7.21-7.18(m,1H),7.12-7.06(m,1H),6.60-6.50(m,1H),5.27(s,2H),4.76(s,1H),4.69(s,1H),3.87(t, J=6.2Hz,1H),3.74(t,J=4.6Hz,1H),3.59(s,2H),3.07(s,1H),3.03(s,2H),2.86-2.81(m,2H),2.47(dd,J=6.8,4.8Hz,2H)ppm.

[0043] 3. Preparation of intermediate lapatinib-alkyne

[0044] Weigh 0.68g of lapatinib-COOH (1mmol) and 0.39g of O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU, 1.2mmol) and dissolve them in 10mL of N,N-dimethylformamide (DMF), stir and react for 15 minutes at room temperature, add 0.15g of triethylamine (TEA, 1.5mmol) and continue stirring for 15 minutes, add 0.07g of 3-butyn-1-ol (1mmol) and continue stirring and react for 12-24 hours. Monitor the reaction process by thin layer chromatography (TLC) until the raw materials are completely reacted. After the reaction is completed, spin dry the solution, add 10mL of water, extract with dichloromethane (10mL×3), combine the organic phases, and concentrate under reduced pressure to obtain a crude product. Afterwards, use a mixed solution of dichloromethane and methanol (V 二氯甲烷 :V 甲醇 =50:1) as the mobile phase, and the intermediate compound lapatinib-alkynyl (0.57 g, yield: 78.3%) was obtained by column chromatography. The reaction process is as follows:

[0045]

[0046] 1 H NMR (600MHz, DMSO-d6) δ10.87(s,1H),9.05(d,J=21.0Hz,1H),8.73(s,1H),8.26(dd,J=25.6,8.6Hz,1H),8.01(dd,J=10.2,2.0Hz,1H),7.87(d,J=8.6 Hz,1H),7.75(t,J=8.2Hz,1H),7.48(dd,J=14.0,7.8Hz,1H),7.34(t,J=6.6 Hz,2H),7.32(s,1H),7.27(s,1H),7.22-7.18(m,1H),6.60(dd,J=22.8,3.1 Hz,1H),5.29(s,2H),4.76(s,1H),4.69(s,1H),4.09-4.03(m,2H),3.86(t, J=6.4Hz,1H),3.75(t,J=5.8Hz,1H),3.58(t,J=7.4Hz,1H),3.35(t,J=5.6H z,1H),3.07(s,1H),3.03(s,2H),2.90-2.86(m,2H),2.61(dd,J=12.4,6.4H z,2H),2.47(td,J=6.6,2.6Hz,1H),1.23(s,1H),1.19(t,J=7.4Hz,1H)ppm. 13C NMR(150MHz,DMSO-d6)δ172.82,171.95,171.47,163.49,161.87,158.87,153.15,152.46,1 51.98,151.15,140.03,131.10,131.05,129.34,125.65,123.82,121.56,118.30,115.30,1 15.16,114.68,114.61,114.46,111.50,111.28,109.19,81.28,72.95,69.87,62.30,51.72 ,41.25,41.10,29.36,29.22,28.19,18.75.24.53ppm.ESI-MS:m / z[M+H]+=733.1894; found 733.1899.

[0047] 4. Preparation of intermediate NBDHEX-azido

[0048] Using NBDHEX and 4-(azidomethyl)benzoic acid as raw materials, referring to the synthesis steps of the intermediate lapatinib-alkyne, the intermediate NBDHEX-azido was obtained (yield: 83.1%).

[0049]

[0050] 1 H NMR(600MHz, CDCl3)δ8.30(d,J=7.8Hz,1H),7.90(d,J=7.7Hz,1H),7.88(s, 1H),7.43(d,J=7.6Hz,1H),7.37(t,J=7.6Hz,1H),7.07(d,J=7.8Hz,1H),4.3 3(s,1H),4.26(t,J=6.6Hz,2H),3.21(t,J=7.2Hz,2H),1.85-1.79(m,2H),1. 77-1.72(m,2H),1.55(dt,J=15.0,7.2Hz,2H),1.47(dt,J=15.0,7.4Hz,2H). 13C NMR (150MHz, CDCl3) δ165.10,148.16,141.45,140.86,134.89,131.53,131.41,129.91,129.80,128.34,128.0 8,127.96,119.35,63.89,53.27,30.66,27.47,27.45,26.74,24.53ppm.ESI-MS: m / z[M+Na]+=479.1114; found 479.1113.

[0051] 5. Preparation of target compound conjugate-1

[0052] Weigh 0.48g of the intermediate compound D-MT-Boc (1.5mmol), 0.87g of lapatinib (1.5mmol), 0.68g of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 1.8mmol) and 0.29g of N,N-diisopropylethylamine (DIPEA, 2.25mmol) and dissolve them in 20mL of DMF solvent, stir and react at room temperature for 12-24 hours, and detect the reaction process by TLC. After the reaction is completed, spin dry the solvent, add 10mL of dichloromethane, ultrasonicate for 10 minutes, filter by suction, obtain a filter cake, and dissolve the filter cake again with 10mL of dichloromethane, and repeat the above process 2-3 times. The filter cake obtained by the final suction filtration is dissolved again with 10mL of dichloromethane, and 3mL of trifluoroacetic acid (TFA) is added dropwise to the solution, and stirred at room temperature for 2-3 hours. After the reaction was completed, the mixture was dried and dissolved in dichloromethane and methanol (V 二氯甲烷 :V 甲醇 =20:1) as the mobile phase and purified by column chromatography to obtain the target compound conjugate-1 (0.51 g, yield: 43.7%).

[0053]

[0054] 1H NMR (600MHz, DMSO-d6) δ10.60(s,1H),9.24(d,J=7.6Hz,1H),8.68(s,1H),8.56(d,J=6.2Hz,1H),8.16(d,J=2.0Hz,1H),8.13(d,J=8.9H z,1H),7.89(d,J=8.6Hz,1H),7.82-7.77(m,1H),7.63-7.56(m,1H),7.47(dd,J=14.0,7.4Hz,1H),7.42-7.36(m,1H),7.34(s,1H),7.32 (d,J=9.4Hz,3H),7.28(d,J=1.8Hz,1H),7.26(d,J=8.8Hz,1H),7.18(t,J=7.6Hz,1H),7.13(t,J=7.4Hz,1H),7.03-6.97(m,2H),6.51(d d,J=18.6,2.6Hz,1H),5.26(s,2H),3.73(s,2H),3.59(s,1H),3.42-3.35(m,7H),3.00(s,2H),2.88(s,1H),2.71(d,J=11.2Hz,2H)ppm. 13 C NMR(150MHz,DMSO-d6)δ169.60,169.55,163.48,161.87,158.29,153.32,150.22,140.2 2,137.03,135.07,131.08,131.03,129.87,129.20,127.87,124.99,123.81,123.32,12 1.85,121.28,121.08,119.26,118.75,115.24,115.10,114.57,114.43,112.39,110.32 ,106.15,69.84,51.05,50.30,36.27,32.91,32.76,31.25,27.25ppm.ESI-MS:m / z[M+H] + =781.2283; found 781.2370.

[0055] 6. Preparation of target compound conjugate-2

[0056] Using NLG919 and the intermediate compound lapatinib-COOH as raw materials, and referring to the synthesis steps of the intermediate compound lapatinib-alkynyl, the target compound conjugate-2 was obtained (yield: 78.6%).

[0057]

[0058] 1 H NMR(600MHz,DMSO-d6)δ10.02(s,1H),8.99(d,J=12.2Hz,1H),8.84(d,J=4.8Hz,1H),8.77(s,1H),8.60-8.57(m,1H),8.20(t,J=8.0Hz,1H),8.02(d,J=11.2Hz,1H),7.84-7.79(m,1H),7.79-7.76(m,1H),7.75-7.71(m,1H),7.67(s,1H),7.63-7.59(m,1H),7.49-7.46(m,2H),7.44-7.40(m,1H),7.35-7.31(m,2H),7.29(d,J=8.8Hz,1H),7.21-7.17(m,1H),6.65(dd,J=6.4,2.6Hz,1H),5.65(d,J=13.8Hz,1H),5.27(s,2H),4.80(s,1H),4.76-4.70(m,1H),4.61(s,1H),3.87-3.78(m,1H),3.73-3.64(m,2H),3.59-3.53(m,1H),3.26(d,J=6.8Hz,2H),3.07(d,J=3.8Hz,1H),2.94(s,1H),2.92(s,1H),2.87-2.84(m,1H),2.78-2.72(m,1H),2.67(d,J=3.4Hz,1H),2.40-2.32(m,1H),2.28-2.23(m,1H),2.19-2.09(m,1H),1.64-1.55(m,3H),1.52(d,J=11.2Hz,2H),1.12-1.01(m,3H),0.94-0.84(m,2H)ppm. 13C NMR (150MHz, DMSO-d6) δ172.14,171.97,171.47,163.49,161.88,158.06,154.93,152.98,152.35,152.01,151. 66,150.29,144.89,140.15,133.50,131.06,131.01,129.03,128.73,124.70,123.80,122.99,122.88,121.56,1 20.73,117.48,115.80,115.24,115.10,114.78,114.57,114.43,111.34,110.98,108.34,72.77,69.89,51.71,4 4.94,41.83,41.72,41.34,41.01,35.81,29.49,29.19,28.49,28.35,27.48,26.25,25.95ppm.ESI-MS:m / z[M+H] + =945.3207; found 945.3117.

[0059] 7. Preparation of target compound conjugate-3

[0060] Using NBDHEX and the intermediate compound lapatinib-COOH as raw materials, the target compound conjugate-3 (yield: 81.4%) was obtained by referring to the synthesis steps of the intermediate compound lapatinib-alkynyl.

[0061]

[0062] 1H NMR (600MHz, DMSO-d6) δ10.25 (s, 1H), 9.09 (s, 1H), 8.52 (d, J = 5.4Hz, 1H), 8.48 (t,J=7.2Hz,1H),8.18-8.07(m,2H),7.87(s,1H),7.78-7.73(m,1H),7.50-7.4 5(m,1H),7.42(t,J=7.2Hz,1H),7.33(t,J=9.4Hz,2H),7.23(s,1H),7.18(t,J= 8.6Hz,1H),6.65(d,J=3.0Hz,1H),5.25(s,2H),4.74(s,1H),4.67(s,1H),4.01( t,J=6.4Hz,1H),3.94(t,J=6.4Hz,1H),3.89-3.83(m,1H),3.76-3.72(m,1H),3 .60-3.55(m,1H),3.29-3.22(m,2H),3.06(s,1H),3.02(s,2H),2.93-2.89(m,1H ),2.79-2.75(m,1H),2.61-2.57(m,2H),1.71-1.68(m,1H),1.66-1.62(m,1H), 1.56-1.52(m,1H),1.51-1.46(m,1H),1.44-1.31(m,3H),1.29-1.21(m,3H)ppm. 13 C NMR(150MHz,DMSO-d6)δ172.80,172.03,163.48,161.86,158.01,154.76,153.06,151.36,149.96,149.50,1 49.35,143.02,140.50,140.20,133.84,132.74,132.36,131.05,131.00,128.54,128.36,124.46,123.79,1 22.72,122.51,121.29,115.94,115.21,115.08,114.55,114.52,111.54,69.84,64.11,51.72,44.72,41.10 ,31.01,29.51,28.43,28.37,28.32,28.28,27.68,25.29ppm.ESI-MS: m / z[M+H]+=960.2331; found960.2258.

[0063] 8. Preparation of target compound conjugate-4

[0064] 0.27 g of the intermediate compound NBDHEX-azido (0.6 mmol) and 0.44 g of the intermediate compound lapatinib-alkynyl were dissolved in a mixed solution of 10 mL tetrahydrofuran (THF, 5 mL) and water (5 mL), stirred at room temperature for 5 minutes, 0.0075 g of copper sulfate pentahydrate (0.03 mmol) and 0.0053 g of ascorbic acid (0.03 mmol) were added, nitrogen was protected, stirred overnight at room temperature, and the reaction process was monitored by TLC. After the reaction was completed, the solution was dried and a mixed solution of dichloromethane and methanol was used as the mobile phase (V 二氯甲烷 :V 甲醇 =10:1), and purified by column chromatography to obtain the target compound conjugate-4 (0.38 g, yield: 52.6%).

[0065]

[0066] 1 H NMR (600MHz, DMSO-d6) δ9.85(d,J=18.8Hz,1H),8.70(d,J=11.4Hz,1H),8.55(s,1H),8.47(d,J=7 .8Hz,1H),8.11(dd,J=26.2,8.4Hz,1H),8.04(d,J=16.8Hz,2H),7.87(d,J=10.2Hz,2H),7.75(t, J=8.2Hz,2H),7.54(d,J=7.6Hz,1H),7.50-7.44(m,2H),7.39(d,J=8.0Hz,1H),7.32(t,J=9.4Hz, 2H),7.27(t,J=7.6Hz,1H),7.17(t,J=8.4Hz,1H),7.10-7.03(m,1H),6.65-6.49(m,1H),5.70(d, J=44.6Hz,3H),5.24(d,J=5.4Hz,2H),4.75(s,1H),4.68(s,1H),4.22(dt,J=19.0,6.4Hz,4H),3. 76(t,J=6.0Hz,1H),3.36(t,J=6.8Hz,2H),3.27(t,J=7.0Hz,2H),3.07(s,1H),3.03(s,2H),2.94 (dd,J=18.4,10.2Hz,2H),2.89-2.86(t,J=6.4Hz,1H),2.78(t,J=6.2Hz,1H),2.8(t,J=6.8Hz,2H ),1.73(t,J=6.0Hz,2H),1.68(t,J=6.2Hz,2H),1.47(t,J=5.8Hz,2H),1.41(t,J=6.2Hz,2H)ppm.13 CNMR(150MHz,DMSO-d6)δ172.93,172.00,171.53,165.81,163.47,161.86,157.93,152.95,152.43,150.21,149.47,144.00, 142.98,140.53,140.10,137.37,133.46,133.05,132.66,132.36,131.03,130.70,129.68,129.20,128.84,128.34,124.56,1 23.76,123.49,122.71,122.38,121.52,117.31,115.71,115.22,115.08,114.66,114.54,114.40,111.36,108.29,69.83,65 .16,63.24,55.37,52.65,52.02,51.70,44.87,41.24,41.08,31.02,29.26,28.41,28.30,27.69,25.39ppm.ESI-MS:m / z[M+H] + =1189.3109; found 1189.3102.

[0067] Example 2

[0068] 1. The target compounds lapatinib, D-MT, NLG919, NBDHEX, conjugate-1, conjugate-2, conjugate-3 and conjugate-4 were prepared into 2 mmol / L solutions with DMSO solution, and the target compounds were diluted into a series of solutions with concentrations of 0.156, 0.625, 2.5, 10 and 40 μmol / L with cell culture medium before use, wherein the final concentration of DMSO did not exceed 4‰ (the same below). Lapatinib, D-MT, NLG919, NBDHEX, the physical mixture of lapatinib and D-MT (mixture-1, v:v=1:1), the physical mixture of lapatinib and NLG919 (mixture-2, v:v=1:1) and the physical mixture of lapatinib and NBDHEX (mixture-3, v:v=1:1) were used as positive controls, and each group of experiments was tested in parallel three times.

[0069] 2. Cytotoxicity of the target compound to five breast cancer cell lines and normal human breast epithelial cell lines

[0070] Cells in the logarithmic growth phase (MCF-7, MDA-MB-231, HCC1187, BT20, HS-578T and MCF-12A) were counted, and the above 6 types of cells were inoculated in 96-well culture plates, with 8000-10000 cells per well, and 200 μL of culture medium containing different concentrations of the test compound was added respectively; cultured overnight, and after the cells adhered to the wall, the old culture medium in each well was aspirated with a pipette and discarded, and 200 μL of culture medium containing different concentrations of the test compound was added again. The test compound groups refer to: ① Lapatinib positive control group; ② D-MT positive control group; ③ NLG919 positive control group; ④ NBDHEX positive control group; ⑤ Conjugate-1 treatment group; ⑥ Conjugate-2 treatment group; ⑦ Conjugate-3 treatment group; ⑧ Conjugate-4 treatment group; ⑨ Mixture-1 positive control group; ⑩ Mixture-2 positive control group and Mixture-3 positive control group, set up 3 replicates for each concentration. After adding the drug, culture for 72 hours, add 20μL of 5mg / mL MTT, incubate at 37℃ for 4 hours, remove the supernatant, and add 150μL of DMSO to dissolve. Use an enzyme reader to measure the OD value of each well at a wavelength of 490nM, and calculate the inhibition rate. Make a concentration-inhibition rate curve to calculate the IC 50 value.

[0071] The results are shown in Table 1. Lapatinib showed moderate inhibitory effects on the proliferation of five different breast cancer cells. Its IC 50 The values ​​were between 9.56 and 16.29 μM, and the inhibitory effect on the proliferation of triple-negative breast cancer MDA-MB-231 cells was the strongest, with an IC 50 The value was 9.56 μM. The two IDO inhibitors were relatively weak in cytotoxicity to the breast cancer cells and normal breast cells tested, among which D-MT had an IC of 2.37 μM for the cells tested. 50 The values ​​were all over 100 μM; NLG919 was relatively cytotoxic, but its IC 50 The values ​​of NBDHEX were all greater than 68.0 μM. The GST inhibitor NBDHEX was highly cytotoxic, with an IC 50 The values ​​were all lower than 6.5 μM, and the toxicity to MDA-MB-231 cells was the strongest, with IC 50 The value reached 2.72μM. However, it is worth noting that NBDHEX is also relatively toxic to normal breast cells MCF-12A. Its IC 50 The value also reached 3.67μM. Conjugate-1 and conjugate-2 obtained by conjugating two IDO inhibitors with lapatinib, both of which retain or exceed the cytotoxicity of the parent compound lapatinib against breast cancer cells. Conjugate-1, IC 50The values ​​are comparable to those of lapatinib and mixture-1; the toxicity of conjugate-2 to breast cancer cells is significantly improved compared with that of lapatinib and mixture-2. IC values ​​of lapatinib and mixture-1 on MDAIMB-231 cells 50 The values ​​were 9.56 and 8.73 μM, respectively, while the IC of conjugate-2 against MDAMB-231 cells was 50 The value reached 4.31μM, and its cytotoxicity was increased by 2.21 and 2.02 times compared with lapatinib and mixture-2, respectively. In addition, the IC value of conjugate-2 against normal breast cancer cells MCF-12A was 50 The value was only 65.73 μM. NBDHEX was conjugated with lapatinib through two different linkers to obtain conjugates-3 and conjugates-4, among which conjugate-3 showed more potent cytotoxicity than the parent compound and mixture-3, and its IC 50 The values ​​were 0.22 and 0.63 μM, respectively, and the IC values ​​of other three breast cancer cells MCF-7, HCC1187 and BT20 were 50 The values ​​also reached 1.08, 1.72 and 3.47 μM respectively. In addition, the IC values ​​of conjugate-3 against normal breast cells MCF-12A 50 The value was 12.48 μM, which was significantly higher than NBDHEX (IC 50 =3.67 μM) and mixture-3 (IC 50 =4.53 μM) 50 The value further highlights the selectivity of conjugate-3 for cancer cells. Due to its larger molecular weight, conjugate-4 has significantly lower cytotoxicity to the tested cancer cells compared with the parent compound. For example, conjugate-4 has the highest cytotoxicity to HS-578T, with an IC 50 The value was only 17.81 μM; the cytotoxicity to BT20 was the weakest, IC 50 The value reached 31.71μM. However, it is worth noting that the IC 50 The value was also relatively high, reaching 33.59 μM, which was much higher than that of NBDHEX and the physical mixture of NBDHEX and lapatinib.

[0072] Table 1 Cytotoxicity of target compounds to five breast cancer cell lines and normal human breast epithelial cell lines

[0073]

[0074] a IC 50 The values ​​are expressed as mean ± SD of three parallel experiments (n = 3).

[0075] Example 3

[0076] MDA-MB-231 / Lapatinib cells in the logarithmic growth phase (the cells are disclosed in the literature https: / / doi.org / 10.1021 / acs.jmedchem.1c01013) were inoculated into a 96-well culture plate, and 200 μL of culture medium containing 8000-10000 cells was added to each well. After culture overnight, after the cells adhered to the wall, the old culture medium in each well was aspirated with a pipette and discarded, and 200 μL of fresh culture medium containing different concentrations of the test compound was added again. The test compound groups refer to: ① Lapatinib positive control group; ② D-MT positive control group; ③ NLG919 positive control group; ④ NBDHEX positive control group; ⑤ Conjugate-1 treatment group; ⑥ Conjugate-2 treatment group; ⑦ Conjugate-3 treatment group; ⑧ Conjugate-4 treatment group; ⑨ Mixture-1 positive control group; ⑩ Mixture-2 positive control group and Mixture-3 positive control group, set up 3 replicates for each concentration. After adding the drug, culture for 72 hours, add 20μL of 5mg / mL MTT, incubate at 37℃ for 4 hours, remove the supernatant, and add 150μL of DMSO to dissolve. Use an enzyme reader to measure the OD value of each well at a wavelength of 490nM, and calculate the inhibition rate. Make a concentration-inhibition rate curve to calculate IC 50 value.

[0077] The results are shown in Table 2. Lapatinib is insensitive to MDA-MB-231 / Lapatinib-resistant cell lines. 50 The value was only 42.85 μM, and the resistance index RI value was only 4.48. In contrast, the sensitivity of D-MT, NLG-919 and NBDHEX to MDA-MB-231 / Lapatinib cells was almost the same as that to MDA-MB-231 cells. The cytotoxicity of conjugate-1 and conjugate-2 to MDA-MB-231 / Lapatinib was significantly lower than that of lapatinib, and their IC 50 The values ​​were 22.37 and 14.85 μM, respectively, and the RI values ​​were 2.83 and 3.44, respectively, which were significantly lower than lapatinib, indicating that they can reverse lapatinib-mediated MDA-MB-231 resistance to a certain extent. In contrast, both conjugate-3 and conjugate-4 showed a strong ability to reverse lapatinib-mediated MDA-MB-231 resistance. For example, the IC 50 The value reached 0.31 μM, and the RI value was 1.36, which was 0.30 times that of lapatinib; the IC value of conjugate-4 for MDA-MB-231 / Lapatinib cells 50The value was 26.14 μM, and the RI value reached 1.27. The above results show that the introduction of IDO inhibitors or GST inhibitors into the structure of lapatinib can effectively inhibit lapatinib-mediated MDA-MB-231 cell resistance.

[0078] Table 2 Cytotoxicity of target compounds to MDA-MB-231 / Lapatinib cells

[0079] Compound <![CDATA[IC 50 (μM) a ]]> <![CDATA[RI b ]]> Compound <![CDATA[IC 50 (μM) a ]]> <![CDATA[RI b ]]> Lapatinib 42.85±3.87 4.48 Conjugate-3 0.31±0.091 1.36 D-MT >100 -- Conjugate-4 26.14±2.87 1.27 NLG-919 74.41±3.49 0.98 Mixture-1 28.77±3.66 3.29 NBDHEX 5.64±0.58 2.07 Mixture-2 25.57±2.89 4.01 Conjugate-1 22.37±4.21 2.83 Mixture-3 4.81±0.74 2.95 Conjugate-2 14.85±1.36 3.44

[0080] a IC 50 Values ​​are expressed as mean ± SD of three parallel experiments (n = 3); b Resistance index (RI) = IC 50 (MDA-MB-231 / Lapatinib) / IC 50 (MDA-MB-231).

[0081] Example 4

[0082] 1. Protein extraction

[0083] 1) Preparation of MDA-MB-231 cell suspension

[0084] Preparation of cell suspension: Into the cell flasks (cell flask size: 25cm 2 The number of cells is about 1×10 6 Add 2mL of EDTA-free trypsin digestion solution to each tube and digest for 1-2min. Use a 1.0mL pipette to suck up the trypsin digestion solution and slowly rinse the bottom of the cell bottle to completely remove the cancer cells from the bottom of the cell bottle. Transfer the trypsin digestion solution containing cancer cells to a 15mL sterile centrifuge tube and centrifuge at 1500 rpm for 5min at room temperature. Slowly pour out the trypsin digestion solution in the centrifuge tube and add 2mL of fresh culture medium. Use a pipette to suck up the culture medium and repeatedly blow the cancer cells to completely disperse them. Then add 8mL of fresh culture medium to the centrifuge tube and slowly shake the centrifuge tube to evenly distribute the cancer cells in 10mL of fresh culture medium. The final concentration of cancer cells in the culture medium is 1×10 5 cells / mL.

[0085] 2) Add 2 mL of 1×10 5cells / mL of MDA-MB-231 cell suspension, and incubate in a 37°C cell culture incubator containing 5% CO2 for 12h. After that, remove the old culture medium in each well of the 6-well plate, and add ①2mL of fresh culture medium containing 10μM lapatinib, ②2mL of fresh culture medium containing 10μM conjugate-1, ③2mL of fresh culture medium containing 10μM conjugate-1, ④2mL of fresh culture medium containing 10μM conjugate-2, ⑤2mL of fresh culture medium containing 10μM conjugate-3, and ⑥2mL of fresh culture medium containing 10μM conjugate-4, respectively, and continue to incubate in a cell culture incubator for 12h. After the incubation, collect the cells into a 15mL centrifuge tube, centrifuge at 1500 rpm for 5min, remove the culture medium, and slowly wash twice with 2mL PBS. Place the 15 mL centrifuge tube containing the collected cells on crushed ice and add 80 μL of cell lysis buffer. Lyse for 30 min. After lysis, transfer the cells to a 2 mL centrifuge tube and place it in a refrigerated centrifuge (4°C). Centrifuge at 15,000 rpm for 15 min. Take out the supernatant and store it in a -20°C refrigerator.

[0086] 2. Protein quantification and sample preparation

[0087] The protein content was determined on a Varioskan multimode microplate spectrophotometer using the Coomassie Brilliant Blue G250 method, and then Loading Buffer was added to the extracted protein and the sample was boiled at 100°C for 15 min.

[0088] 3. Preparation and loading of SDS-PAGE gel

[0089] Add TEMED to the 12% separation gel prepared in advance, blow and mix well, transfer to the concave-convex glass plate of the electrophoresis instrument, and immediately seal with water. After the gel is completely solidified, remove the water, add 6% concentrated gel, and insert the groove teeth. After the gel is completely solidified, slowly pull out the groove teeth, add the above sample diluted with SDS to 10μL in the groove teeth of the gel, and add Marker as a reference. Adjust the voltage of the electrophoresis instrument to 100V and run the gel for 30min; then adjust it to 150V and stop when the Marker is completely separated and the Loading Buffer runs to the bottom of the gel.

[0090] 4. Transfer

[0091] Soak the PVDF membrane completely with methanol and soak it in the transfer solution together with the filter paper. Take out the gel and soak the part containing the target protein in the transfer solution for 10 minutes. Then put it in the semi-dry transfer apparatus together with the PVDF and transfer it at 350mA for 45-60 minutes. After that, put the PVDF membrane in 5% skim milk powder and block it on a shaker for 90 minutes.

[0092] 5. Immunity and Exposure

[0093] Place the blocked PVDF membrane on a shaker and wash it with TBST. Incubate the primary antibody overnight at 4°C, then wash it with TBST on a shaker, repeat five times, 30 minutes each time. Incubate the secondary antibody at 37°C for 1 hour, then wash it with TBST on a shaker, repeat five times, 10 minutes each time. Finally, image using the Odyssey scanning system;

[0094] 6. Inhibition of pEGFR (Y1068) and EGFR protein levels by lapatinib and target compound conjugates-1 to 4

[0095] The results are as follows Figure 1 As shown in the figure, lapatinib as an EGFR inhibitor can effectively inhibit the expression of p-EGFR (Y1068) protein in MDA-MB-231 and has no significant effect on EGFR protein expression, while conjugates-1 to 4 can also exert the strong inhibitory effect of the parent compound lapatinib on p-EGFR (Y1068) protein expression. Except for conjugate-4, the inhibitory effects of the other conjugates are almost equivalent to lapatinib, and have no significant effect on EGFR protein expression. These results show that conjugates 1 to 4 can effectively inhibit the expression of p-EGFR (Y1068) protein.

[0096] Example 5

[0097] MDA-MB-231 cells were cultured at 3 × 10 4 The cells were inoculated at a density of 100 μL / well in a 48-well plate and incubated in a 37°C cell culture incubator containing 5% CO2 for 12 hours. The culture medium of each well was removed and a total volume of 200 μL of cell culture medium containing 100 μL of L-tryptophan, hIFN-γ (final concentration of 25 ng / mL) and 10 μM of the test compound (conjugate-1 or conjugate-2) was added. After incubation in the cell culture incubator for 48 hours, 140 μL of supernatant in each well was transferred to a 96-well plate and 15 μL of culture medium containing 30% trifluoroacetic acid was added. The 96-well plate was incubated at 50°C for 30 minutes, and then placed in a plate centrifuge at 3000 rpm for 10 minutes to remove the precipitate. 100 μL of supernatant was taken from each well and mixed with 100 μL of 2% (w / v) p-dimethylaminobenzaldehyde acetic acid solution. Then, the 96-well plate was placed in an ELISA instrument, and the absorbance value in each well was detected at a wavelength of 480 nM. Each group was detected three times in parallel, and then the relative content of kynurenine in each well was calculated compared with that in the control group.

[0098] The results are as follows Figure 2As shown in the figure, both IDO inhibitors D-MT and NLG919 effectively inhibited the level of kynurenine in MDA-MB-231 cells, and the conjugates-1 and -2 obtained by grafting the D-MT and NLG919 structures into the lapatinib structure had significantly better inhibitory effects on kynurenine levels than the parent compound. The inhibitory effects of conjugate-1 on kynurenine were 1.84 and 1.25 times that of the D-MT and mixture-1 groups, respectively; the inhibitory effects of conjugate-2 on kynurenine were 1.64 and 1.48 times that of the NLG919 and mixture-2 groups, respectively. In summary, both conjugate-1 and conjugate-2 can block the degradation of tryptophan to produce kynurenine, and their abilities are significantly better than those of the parent compound.

[0099] Example 6

[0100] 1×10 3 MDA-MB-231 cells were seeded in a 6-well plate at a density of 10 cells / well and the plate was placed in a 37°C cell culture incubator with 5% CO2 for 24 hours. The old culture medium in each well was removed and 2 mL of fresh cell culture medium containing 50 ng / mL IFN-γ, 0.1 mM L-tryptophan and 10 μM of the test compound (conjugate-1 or conjugate-2) was added and placed in a cell culture incubator for another 48 hours. PBMCs (2×10 cells / well) were labeled with the fluorescent dye CellTraceTM Far Red Cell Proliferation Kit (CFSE). 5 Cells were added to each well of a 6-well plate after stimulation with PHA-M, and then placed in a cell culture incubator for 6 days. PBMCs cells in each well were collected and suspended in PBS, and then labeled with FITC-labeled CD4 antibody. Finally, the expression level of CD4+T cells in each group of cells was detected using a CytoFLEX S flow cytometer (Beckman Coulter, USA).

[0101] The results are as follows Figure 3 As shown in the results, compared with the blank group, both D-MT and NLG919 can effectively promote the secretion of CD4+T cells in MDA-MB-231 cells, and the ability of NLG919 to promote CD4+T cell secretion is better than that of D-MT. Compared with the positive control group, conjugate-1 and conjugate-2 showed a superior ability to promote CD4+T cells, and the ability of conjugate-2 was particularly prominent. In summary, conjugate-1 and conjugate-2 reduce the level of kynurenine by inhibiting IDO enzyme activity and promote the secretion of T cells in cancer cells.

[0102] Example 7

[0103] The inhibitory activities of conjugate-3 and conjugate-4 against GST P1-1 enzymatic activity were determined by commercial services provided by Shanghai Fusheng Industrial Co., Ltd. (Shanghai, China), and NBDHEX was used as a positive control.

[0104] The results are shown in Table 3. NBDHEX showed a strong inhibitory effect on GST P1-1 enzyme activity, IC 50 The value reached 0.86μM. Conjugate-3 retained the inhibitory effect of the target compound on GST P1-1, IC 50 The value also reached 0.79μM; probably due to its large molecular weight, conjugate-4 had a relatively weak inhibitory effect on the GST P1-1 enzyme activity, only 16.71μM.

[0105] Table 3 Inhibitory effects of conjugate-3 and conjugate-4 on GST P1-1 enzyme activity

[0106]

[0107] a IC 50 The values ​​are expressed as mean ± SD of three parallel experiments (n = 3).

[0108] This application is based on the design concept of SMDC. Lapatinib is used as the parent compound to couple two different IDO inhibitors (D-MT and NLG919) and a GST inhibitor (NBDHEX) through different linkers to obtain four target conjugates 1 to 4. In summary, target conjugates 1 to 4 retain the inhibitory effect of the parent compound on EGFR, and exhibit potent anti-proliferative effects on various types of triple-negative breast cancer cells. In addition, conjugates-1 and -2 effectively reverse lapatinib-mediated MDA-MB-231 cell resistance by effectively inhibiting IDO activity in cancer cells and promoting the secretion of intracellular CD4+T cells; conjugates-3 and -4 reverse lapatinib-mediated MDA-MB-231 cell resistance by inhibiting GST activity. In particular, conjugate-3 exhibits potent cytotoxicity and the ability to reverse cancer cell resistance, and can be used to prepare anti-tumor drugs.

[0109] The above description is only illustrative and not restrictive of the present invention. Those skilled in the art will appreciate that many modifications, changes or equivalents may be made without departing from the spirit and scope defined by the attached claims, but all of them will fall within the scope of protection of the present invention. The above description is only illustrative and not restrictive of the present invention. Those skilled in the art will appreciate that many modifications, changes or equivalents may be made without departing from the spirit and scope defined by the attached claims, but all of them will fall within the scope of protection of the present invention.

Claims

1. EGFR-dependent small molecule conjugate-1, the structural formula of which is shown below:

2. The method for preparing the EGFR-dependent small molecule conjugate-1 according to claim 1, characterized in that: (Boc)2O is used to protect the amino group in the structure of the IDO inhibitor 1-methyl-D-tryptophan to obtain the intermediate 1-methyl-D-tryptophan-Boc; the intermediate 1-methyl-D-tryptophan-Boc is then subjected to an amide condensation reaction with lapatinib, and the original amino protecting group Boc is removed under the action of trifluoroacetic acid to obtain the target conjugate-1; the reaction process is as follows:

3. EGFR-dependent small molecule conjugate-3, the structural formula of which is shown below:

4. The method for preparing the EGFR-dependent small molecule conjugate-3 according to claim 3, characterized in that: Lapatinib is reacted with succinic anhydride to obtain the intermediate compound lapatinib-COOH; then the GST inhibitor NBDHEX is esterified with the intermediate compound lapatinib-COOH to obtain the target conjugate-3; the reaction process is as follows:

5. EGFR-dependent small molecule conjugate-4, the structural formula of which is shown below:

6. The method for preparing the EGFR-dependent small molecule conjugate-4 according to claim 5, characterized in that: The intermediate compound lapatinib-COOH is reacted with succinic anhydride to obtain the intermediate compound lapatinib-COOH; the intermediate compound lapatinib-COOH is then esterified with 3-butyn-1-ol to obtain the intermediate lapatinib-alkynyl; Then, NBDHEX and 4-(azidomethyl)benzoic acid were subjected to an esterification reaction to obtain an intermediate NBDHEX-azido; finally, the intermediate lapatinib-alkynyl and the intermediate NBDHEX-azido were subjected to a click reaction under the catalysis of CuSO4·5H2O and vitamin C to obtain conjugate-4; the reaction process is shown below:

7. Use of the conjugate-1 according to claim 1 in the preparation of a drug for reversing lapatinib-mediated cancer cell resistance.

8. Use of the conjugate-1 according to claim 1 in the preparation of a drug for inhibiting EGFR enzyme activity in cancer cells and / or promoting the secretion of immune T cells in cancer cells.

9. Use of the conjugate-3 according to claim 3 and / or the conjugate-4 according to claim 4 in the preparation of a drug for reversing lapatinib-mediated cancer cell resistance.

10. Use of the conjugate-3 according to claim 3 and / or the conjugate-4 according to claim 4 in the preparation of a drug for inhibiting EGFR enzyme activity in cancer cells and / or inhibiting GST P1-1 enzyme activity in cancer cells.

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