EGFR-dependent small molecule conjugate-1, 3, 4 and preparation method and application thereof

Abstract

The application discloses EGFR-dependent small molecule conjugates-1, 3 and 4, a preparation method and application thereof, and belongs to the technical field of biological medicines. The EGFR-dependent small molecule conjugate-1 disclosed by the application is obtained by introducing an IDO inhibitor D-MT into a lapatinib structure through reasonable structural modification. A glutathione S-transferase inhibitor NBDHEX is introduced into the above structural fragment of the lapatinib through different connectors to obtain target conjugates-3 and 4. The results show that the conjugates all retain the inhibitory effect of the parent compounds on EGFR, IDO or GST enzyme activity, have a strong inhibitory effect on the proliferation of various types of triple-negative breast cancer cells, can effectively reverse the lapatinib-mediated drug resistance of triple-negative breast cancer cells, and can be used for preparing antitumor drugs.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, more particularly relates to EGFR-dependent small molecule conjugate-1, 3, 4 and a preparation method and application thereof. BACKGROUND

[0002] Targeted therapy as an important part of precision treatment has made breakthrough progress in recent years. With the success of several antibody-drug conjugates (ADC) drugs on the market, polymer-drug conjugates (PDC), immune-stimulating-antibody conjugates (ISAC) and small-molecule-drug conjugates (SMDC) have emerged, which together constitute a new blueprint for targeted drug research and development. Among them, SMDC shows better therapeutic effect than ADC in some solid tumors due to its excellent cell penetration ability, non-immunogenicity and relatively small molecular weight. SMDC is a new type of drug that couples small molecules as targeting ligands with other small molecule drugs with different mechanisms of action to achieve precise treatment of diseases and produce 1+1>2 effect. Its chemical structure consists of three parts: small molecule ligand targeting receptor, linker and other drug 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 / Sailbio, 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, and only AST-3424 (clinical phase II) independently developed by Aishindawae and PEN-866 (clinical phase II) obtained by Sailbio from Tarveda in March 2020. Although the research on SMDC drug molecules is relatively few at present, the existing clinical data shows that the existing SMDC drug molecules all show good clinical efficacy. In addition, global pharmaceutical giants such as Merck and Novartis have shown interest in SMDC, which further confirms that SMDC drug molecules have potential to be explored.

[0003] It is well known that triple-negative breast cancer (TNBC) has become one of the malignant tumors with extremely high mortality in women due to its strong tumor heterogeneity, lack of effective therapeutic targets, insensitivity to common endocrine therapy and targeted therapy, and easy to develop acquired resistance to traditional chemotherapy drugs. Early TNBC patients can achieve better clinical efficacy through surgery, radiotherapy and chemotherapy, and the 5-year survival rate can reach about 77%, but for patients with advanced or metastatic cancer cells, there is currently a lack of effective treatment methods, and the 5-year survival rate is only about 14%, and effective treatment methods are urgently needed. Through a large amount of data analysis and literature research, it is found that about 60% of basal cell-like TNBC patients have high expression of epidermal growth factor receptor (EGFR) in cancer cells, and about 89% of TNBC patients are sensitive to EGFR therapy, especially for EGFR-overexpressing basal-like-2 tumors. However, the use of EGFR inhibitors alone is difficult to achieve satisfactory clinical efficacy. Therefore, using EGFR inhibitors as a targeting ligand, coupling with other drugs with different mechanisms to obtain new SMDC drug molecules may be an effective way to obtain TNBC treatment drugs.

[0004] Indoleamine 2,3-dioxygenase (IDO) is the only enzyme outside the liver that catalyzes the metabolism of tryptophan, breaking it down along the kynurenine pathway to produce a series of metabolites, including quinolinic acid. Studies have shown that IDO, by consuming tryptophan to produce kynurenine, increases tissue IL-6 expression, which in turn increases VEGF expression and promotes tumor angiogenesis. Furthermore, persistently high expression of IDO in tumor cells leads to the continuous depletion of tryptophan in the tumor microenvironment, converting it to kynurenine. This metabolic regulation can inhibit the functional activity of tryptophan-sensitive T cells, including affecting T cell proliferation and regulatory T cell differentiation. However, clinical studies of existing IDO inhibitors have shown that monotherapy with IDO inhibitors is unlikely to achieve satisfactory clinical efficacy, and the anti-tumor effects of selective IDO inhibitors may be negated by alternative mechanisms that bypass IDO function. Therefore, effectively utilizing IDO as a target and developing more effective therapeutics remain urgent challenges. Glutathione S-transferase (GST) is a multifunctional Phase II metabolic enzyme that catalyzes the binding of glutathione to electrophilic substances and plays a crucial role in protecting organisms from toxicants. This enzyme has a dual role: on the one hand, it enhances the body's ability to detoxify carcinogens, preventing tumor development; on the other hand, it increases cancer cells' ability to metabolize chemotherapeutic drugs, leading to drug resistance. Clinical statistics show that the rate of GST expression in patients with triple-negative breast cancer is significantly higher than that in patients with non-triple-negative breast cancer, and GST expression is positively correlated with tumor malignancy and prognosis in these patients. Therefore, inhibiting GST may be an effective approach for treating triple-negative breast cancer and / or overcoming drug resistance in 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 in the clinical treatment of epidermal growth factor receptor overexpression, and advanced or metastatic breast cancer that has previously received treatment including anthracyclines, paclitaxel, and trastuzumab. By analyzing the structure-activity relationship of lapatinib, this application found that the secondary amine group on the fatty chain in the lapatinib structure 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 are currently no reports of SMDC medicinal molecules that can simultaneously inhibit EGFR and IDO or GST, and are used to treat triple-negative breast cancer and reverse the drug resistance of triple-negative breast cancer cells. Summary of the Invention

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

[0007] In order to solve the above technical problems, the technical solutions adopted by the present application are as follows:

[0008] A kind of small molecule conjugate for inhibiting EGFR and IDO enzyme activity, its structural formula is as shown in the following conjugate-1 or conjugate-2:

[0009] .

[0010] The preparation method of the conjugate-1 is specifically: the amino group in the structure of IDO inhibitor 1-methyl-D-tryptophan is protected by using (Boc)2O to obtain intermediate 1-methyl-D-tryptophan-Boc;Then, the intermediate 1-methyl-D-tryptophan-Boc is subjected to amide condensation reaction with lapatinib, and then 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 shown below:

[0011] .

[0012] The preparation method of the conjugate-2 is specifically: lapatinib is reacted with succinic anhydride to obtain intermediate compound lapatinib-COOH;Then, amide condensation reaction is carried out between the intermediate compound and IDO inhibitor NLG919, to obtain the conjugate-2;The reaction process is as shown below:

[0013] .

[0014] A kind of small molecule conjugate for inhibiting EGFR and GST enzyme activity, its structural formula is as shown in the following conjugate-3 or conjugate-4:

[0015] .

[0016] The preparation method of the conjugate-3 is specifically: lapatinib is reacted with succinic anhydride to obtain intermediate compound lapatinib-COOH;Then, esterification reaction is carried out between GST inhibitor NBDHEX and the intermediate compound lapatinib-COOH, to obtain the target conjugate-3;The reaction process is as shown below:

[0017] .

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

[0019]

[0020]

[0021] .

[0022] The conjugate 1 or / and 2 is used for preparing a drug for reversing lapatinib-mediated drug resistance of cancer cells.

[0023] The conjugate 1 or / and 2 is used for preparing a drug for inhibiting EGFR enzyme activity in cancer cells and / or promoting immune T cell secretion in cancer cells.

[0024] The conjugate 3 or / and 4 is used for preparing a drug for reversing lapatinib-mediated drug resistance of cancer cells.

[0025] The conjugate 3 or / and 4 is used for preparing a drug for inhibiting EGFR enzyme activity in cancer cells and / or inhibiting GST P1-1 enzyme activity in cancer cells.

[0026] Compared with the prior art, the present application has the following beneficial effects:

[0027] 1) The present application 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 application 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.

[0028] 2) The results of the present application show that the conjugate-1 can effectively reverse lapatinib-mediated drug resistance of MDA-MB-231 cells.

[0029] 3) The GST inhibitor NBDHEX of the present application is introduced into the structure of lapatinib through different linkers to obtain target conjugate-3 and conjugate-4. The results of the present application show that the compounds have strong inhibitory effect on the expression of pEGFR(Y1068) protein in triple-negative breast cancer MDA-MB-231 cells, and the compounds can also strongly inhibit the activity of GST P1-1 enzyme, and the inhibitory effect of conjugate-3 on the activity of GST P1-1 enzyme is slightly better than that of NBDHEX.

[0030] 4) The results of the present application show that conjugate-3 and conjugate-4 exhibit very strong cytotoxicity to the five cancer cells tested. Among them, the cytotoxicity of conjugate-3 to the five cancer cells is 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 drug resistance of MDA-MB-231 cells mediated by lapatinib, and the resistance index (RI) reaches 1.36 and 1.27, respectively. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 Figure is a graph of the inhibition of pEGFR(Y1068) and EGFR protein levels by lapatinib and target compounds conjugate-1-4;

[0032] Figure 2 Figure is a graph of 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);

[0033] Figure 3 Figure is a graph of the expression level of CD4+ T cells in cancer cells after treatment with different conjugates (A) and a single analysis graph (B). DETAILED DESCRIPTION

[0034] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application will be further described below in combination with specific examples. In the following examples, if not specified in detail, the technical means used are conventional means well known to those skilled in the art.

[0035] 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 breast epithelial cell line MCF-12A used in the present application were purchased from Jiangsu Keygen Biotech Co., Ltd. (Nanjing, Jiangsu, China). The culture medium used for each cell culture was the corresponding conventional culture medium in the art, wherein DMEM medium was used for MCF-7, MDA-MB-231, HS-578T and MCF-12A cell lines, and RPMI-1640 medium was used for HCC1187 and BT20 cells.

[0036] Example 1

[0037] 1. Preparation of intermediate D-MT-Boc

[0038] 0.25 g of D-MT and 0.29 g of NaHCO3 were weighed and dissolved in a mixed solution of 30 mL of tetrahydrofuran (THF) and water (V:V = 1:1). After the reaction solution was cooled to 0°C, 0.3 g of (Boc)2O (1.37 mmol) was slowly added to the reaction solution and stirred for 10 minutes. The temperature of the reaction solution was slowly raised to room temperature and the reaction was continued to stir for 24 hours. After the reaction was completed, THF was removed under reduced pressure, and the solution pH was adjusted to 1.0 with 1 mol / L hydrochloric acid. Then, the aqueous layer was extracted with ethyl acetate (15 mL x 3), washed with saturated brine (15 mL x 1), dried over anhydrous Na2SO4, and finally concentrated under vacuum to obtain yellow intermediate compound 1-methyl-D-tryptophan-Boc (0.27 g, yield: 74.1%). The reaction process is shown below:

[0039] .

[0040] 1 H NMR (600 MHz, DMSO-d6) δ 12.53 (s, 1H), 7.55 (d, J = 7.7 Hz, 1H),7.37 (d, J = 7.9 Hz, 1H), 7.13 (dd, J = 15.4, 7.5 Hz, 2H), 7.02 (t, J = 7.2Hz, 1H), 6.97 (d, J = 7.8 Hz, 1H), 4.16 (d, J = 4.2 Hz, 1H), 3.71 (s, 3H),3.13 (dd, J = 14.2, 3.8 Hz, 1H), 2.99 (dd, J = 14.1, 9.5 Hz, 1H), 1.33 (s,8H), 1.21 (s, 1H) ppm. 13C NMR (150 MHz, DMSO-d6) δ 174.32, 155.90, 137.01, 128.42, 128.03, 121.51, 118.92, 110.03, 78.51, 55.07, 32.73, 28.64, 27.20 ppm. ESI-MS: m / z [M+Na] + = 341.1477; found 341.1489.

[0041] 2. Preparation of intermediate Lapatinib-COOH

[0042] Lapatinib (0.58 g, 1 mmol), succinic anhydride (0.15 g, 1.5 mmol) and triethylamine (0.2 g, 2 mmol) were weighed and dissolved in 20 mL of dichloromethane, and stirred at room temperature overnight. After the reaction was completed, the solvent was removed by concentration under reduced pressure, 30 mL of water was added, heated to 60°C, stirred for 2 hours, and then cooled to room temperature. The solvent was removed by filtration to obtain the crude product, which was recrystallized with 10 mL of methanol to obtain the intermediate compound Lapatinib-COOH (0.64 g, yield: 94.0%). The reaction process is shown below:

[0043] .

[0044] 1H NMR (600 MHz, DMSO-d6) δ 12.24 (s, 1H), 10.01 (d, J = 23.4 Hz, 1H), 8.77 (d, J = 23.3 Hz, 1H), 8.57 (s, 1H), 8.21-8.13 (m, 1H), 8.04 (dd, J = 12.2, 2.0 Hz, 1H), 7.80 (dd, J = 8.6, 4.4 Hz, 1H), 7.76 (dd, J = 8.6, 2.4 Hz, 1H), 7.48 (dd, J = 14.2, 7.8 Hz, 1H), 7.34 (t, J = 9.2 Hz, 2H), 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 = 6.2 Hz, 1H), 3.74 (t, J = 4.6 Hz, 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, 4.8 Hz, 2H) ppm.

[0045] 3. Preparation of intermediate Lapatinib-alkyne

[0046] Weigh 0.68 g of Lapatinib-COOH (1 mmol) and 0.39 g of O-benzotriazol-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU, 1.2 mmol) and dissolve them in 10 mL of N,N-dimethylformamide (DMF), stir for 15 minutes at room temperature, add 0.15 g of triethylamine (TEA, 1.5 mmol) and continue stirring for 15 minutes, add 0.07 g of 3-butyn-1-ol (1 mmol) and continue stirring for 12-24 hours. Monitor the reaction process by thin layer chromatography (TLC) until the raw material is completely reacted. After the reaction is completed, spin dry and dissolve, add 10 mL of water, extract with dichloromethane (10 mL x 3), combine the organic phases, and concentrate under reduced pressure to obtain the crude product. Then, use a mixture of dichloromethane and methanol (V 二氯甲烷 :V 甲醇 = 50:1) as the mobile phase to purify by column chromatography to obtain the intermediate compound Lapatinib-alkyne (0.57 g, yield: 78.3%). The reaction process is as follows:

[0047] .

[0048] 1 H NMR (600 MHz, DMSO-d6) δ 10.87 (s, 1H), 9.05 (d, J = 21.0 Hz, 1H),8.73 (s, 1H), 8.26 (dd, J = 25.6, 8.6 Hz, 1H), 8.01 (dd, J = 10.2, 2.0 Hz,1H), 7.87 (d, J = 8.6 Hz, 1H), 7.75 (t, J = 8.2 Hz, 1H), 7.48 (dd, J = 14.0,7.8 Hz, 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 = 6.4 Hz, 1H), 3.75 (t, J = 5.8 Hz,1H), 3.58 (t, J = 7.4 Hz, 1H), 3.35 (t, J = 5.6 Hz, 1H), 3.07 (s, 1H), 3.03(s, 2H), 2.90-2.86 (m, 2H), 2.61 (dd, J = 12.4, 6.4 Hz, 2H), 2.47 (td, J=6.6, 2.6 Hz, 1H), 1.23 (s, 1H), 1.19 (t, J = 7.4 Hz, 1H) ppm. 13C NMR (150 MHz, DMSO-d6) δ 172.82, 171.95, 171.47, 163.49, 161.87, 158.87, 153.15, 152.46, 151.98, 151.15, 140.03, 131.10, 131.05, 129.34, 125.65, 123.82, 121.56, 118.30, 115.30, 115.16, 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. 24.53 ppm. ESI-MS: m / z [M+H]+ = 733.1894; found 733.1899.

[0049] 4. Preparation of intermediate NBDHEX-azido

[0050] The intermediate NBDHEX-azido was prepared from NBDHEX and 4-(azidomethyl)benzoic acid according to the procedure for the synthesis of intermediate Lapatinib-alkynyl (yield: 83.1%).

[0051] .

[0052] 1 H NMR (600 MHz, CDCl3) δ 8.30 (d, J = 7.8 Hz, 1H), 7.90 (d, J = 7.7Hz, 1H), 7.88 (s, 1H), 7.43 (d, J = 7.6 Hz, 1H), 7.37 (t, J = 7.6 Hz, 1H),7.07 (d, J = 7.8 Hz, 1H), 4.33 (s, 1H), 4.26 (t, J = 6.6 Hz, 2H), 3.21 (t, J= 7.2 Hz, 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, 7.4 Hz, 2H). 13C NMR (150 MHz, CDCl3) δ 165.10,148.16, 141.45, 140.86, 134.89, 131.53, 131.41, 129.91, 129.80, 128.34,128.08, 127.96, 119.35, 63.89, 53.27, 30.66, 27.47, 27.45, 26.74, 24.53 ppm.ESI-MS: m / z [M+Na]+ = 479.1114; found 479.1113.

[0053] 5. Preparation of target compound conjugate-1

[0054] 0.48 g of the intermediate compound D-MT-Boc (1.5 mmol), 0.87 g of lapatinib (1.5 mmol), 0.68 g of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 1.8 mmol), and 0.29 g of N,N-diisopropylethylamine (DIPEA, 2.25 mmol) were dissolved in 20 mL of DMF. The mixture was stirred at room temperature for 12-24 hours, and the reaction progress was monitored by TLC. After the reaction, the solvent was evaporated, 10 mL of dichloromethane was added, and the mixture was sonicated for 10 minutes. The mixture was filtered to obtain a filter cake, which was then redissolved in 10 mL of dichloromethane. This process was repeated 2-3 times. The final filter cake was redissolved in 10 mL of dichloromethane, and 3 mL of trifluoroacetic acid (TFA) was added dropwise to the solution. The mixture was 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%).

[0055] .

[0056] 1H NMR (600 MHz, DMSO-d6) δ 10.60 (s, 1H), 9.24 (d, J = 7.6 Hz, 1H), 8.68 (s, 1H), 8.56 (d, J = 6.2 Hz, 1H), 8.16 (d, J = 2.0 Hz, 1H), 8.13 (d, J = 8.9 Hz, 1H), 7.89 (d, J = 8.6 Hz, 1H), 7.82-7.77 (m, 1H), 7.63-7.56 (m, 1H), 7.47 (dd, J = 14.0, 7.4 Hz, 1H), 7.42-7.36 (m, 1H), 7.34 (s, 1H), 7.32 (d, J = 9.4 Hz, 3H), 7.28 (d, J = 1.8 Hz, 1H), 7.26 (d, J = 8.8 Hz, 1H), 7.18 (t, J = 7.6 Hz, 1H), 7.13 (t, J = 7.4 Hz, 1H), 7.03-6.97 (m, 2H), 6.51 (dd, J = 18.6, 2.6 Hz, 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 = 11.2 Hz, 2H) ppm. 13 C NMR (150MHz, DMSO-d6) δ 169.60, 169.55, 163.48, 161.87, 158.29, 153.32, 150.22, 140.22, 137.03, 135.07, 131.08, 131.03, 129.87, 129.20, 127.87, 124.99, 123.81, 123.32, 121.85, 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, 27.25 ppm. ESI-MS: m / z [M+H] + = 781.2283; found 781.2370.

[0057] 6. Preparation of the target compound conjugate-2

[0058] 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 is obtained (yield: 78.6%).

[0059] .

[0060] 1H NMR (600 MHz, DMSO-d6) δ 10.02 (s, 1H), 8.99 (d, J = 12.2 Hz, 1H), 8.84 (d, J = 4.8 Hz, 1H), 8.77 (s, 1H), 8.60-8.57 (m, 1H), 8.20 (t, J = 8.0 Hz, 1H), 8.02 (d, J = 11.2 Hz, 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 = 8.8 Hz, 1H), 7.21-7.17 (m, 1H), 6.65 (dd, J = 6.4, 2.6 Hz, 1H), 5.65 (d, J = 13.8 Hz, 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 = 6.8 Hz, 2H), 3.07 (d, J = 3.8 Hz, 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 = 3.4 Hz, 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 = 11.2 Hz, 2H), 1.12-1.01 (m, 3H), 0.94-0.84 (m, 2H) ppm. 13C NMR (150 MHz, DMSO-d6) δ 172.14, 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, 120.73, 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, 44.94, 41.83, 41.72, 41.34,41.01, 35.81, 29.49, 29.19, 28.49, 28.35, 27.48, 26.25, 25.95 ppm. ESI-MS: m / z [M+H] + = 945.3207; found 945.3117.

[0061] 7. Preparation of the target compound conjugate-3

[0062] The target compound conjugate-3 (yield: 81.4%) was obtained by using NBDHEX and intermediate compound lapatinib-COOH as raw materials, and referring to the synthesis steps of intermediate compound lapatinib-alkynyl.

[0063] .

[0064] 1H NMR (600 MHz, DMSO-d6) δ 10.25 (s, 1H), 9.09 (s, 1H), 8.52 (d, J =5.4 Hz, 1H), 8.48 (t, J = 7.2 Hz, 1H), 8.18-8.07 (m, 2H), 7.87 (s, 1H), 7.78-7.73 (m, 1H), 7.50-7.45 (m, 1H), 7.42 (t, J = 7.2 Hz, 1H), 7.33 (t, J = 9.4Hz, 2H), 7.23 (s, 1H), 7.18 (t, J = 8.6 Hz, 1H), 6.65 (d, J = 3.0 Hz, 1H),5.25 (s, 2H), 4.74 (s, 1H), 4.67 (s, 1H), 4.01 (t, J = 6.4 Hz, 1H), 3.94 (t,J = 6.4 Hz, 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. 13C NMR (150 MHz, DMSO-d6) δ 172.80, 172.03, 163.48, 161.86, 158.01, 154.76, 153.06, 151.36, 149.96, 149.50, 149.35, 143.02, 140.50, 140.20, 133.84, 132.74, 132.36, 131.05, 131.00, 128.54, 128.36, 124.46, 123.79, 122.72, 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, 25.29 ppm. ESI-MS: m / z [M+H]+ = 960.2331; found 960.2258.

[0065] 8. Preparation of the target compound conjugate-4

[0066] The intermediate compound NBDHEX-azido (0.6 mmol) 0.27 g and the intermediate compound Lapatinib-alkyne 0.44 g were weighed out and dissolved in a mixed solution of tetrahydrofuran (THF, 5 mL) and water (5 mL), stirred for 5 min at room temperature, and then 0.0075 g of copper sulfate pentahydrate (0.03 mmol) and 0.0053 g of ascorbic acid (0.03 mmol) were added. The reaction was stirred overnight at room temperature under nitrogen protection, and the reaction process was monitored by TLC. After the reaction was completed, the solution was rotary evaporated, and purified by column chromatography using a mixed solution of dichloromethane and methanol (V 二氯甲烷 :V 甲醇 =10:1) as the mobile phase, to obtain the target compound conjugate-4 (0.38 g, yield: 52.6%).

[0067] .

[0068] 1H NMR (600 MHz, DMSO-d6) δ 9.85 (d, J = 18.8 Hz, 1H), 8.70 (d, J =11.4 Hz, 1H), 8.55 (s, 1H), 8.47 (d, J = 7.8 Hz, 1H), 8.11 (dd, J = 26.2, 8.4Hz, 1H), 8.04 (d, J = 16.8 Hz, 2H), 7.87 (d, J = 10.2 Hz, 2H), 7.75 (t, J =8.2 Hz, 2H), 7.54 (d, J = 7.6 Hz, 1H), 7.50-7.44 (m, 2H), 7.39 (d, J = 8.0Hz, 1H), 7.32 (t, J = 9.4 Hz, 2H), 7.27 (t, J = 7.6 Hz, 1H), 7.17 (t, J = 8.4Hz, 1H), 7.10-7.03 (m, 1H), 6.65-6.49 (m, 1H), 5.70 (d, J = 44.6 Hz, 3H),5.24 (d, J = 5.4 Hz, 2H), 4.75 (s, 1H), 4.68 (s, 1H), 4.22 (dt, J = 19.0, 6.4Hz, 4H), 3.76 (t, J = 6.0 Hz, 1H), 3.36(t, J = 6.8 Hz, 2H), 3.27 (t, J = 7.0Hz, 2H), 3.07 (s, 1H), 3.03 (s, 2H), 2.94 (dd, J = 18.4, 10.2 Hz, 2H), 2.89-2.86 (t, J = 6.4 Hz, 1H), 2.78 (t, J = 6.2 Hz, 1H), 2.8 (t, J = 6.8 Hz, 2H),1.73 (t, J = 6.0 Hz, 2H), 1.68 (t, J = 6.2 Hz, 2H), 1.47 (t, J = 5.8 Hz, 2H),1.41 (t, J = 6.2 Hz, 2H) ppm. 13C NMR (150 MHz, DMSO-d6) δ 172.93, 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, 123.76, 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, 25.39 ppm. ESI-MS: m / z [M+H] + = 1189.3109; found 1189.3102.

[0069] Example 2

[0070] 1. The target compounds Lapatinib, D-MT, NLG919, NBDHEX, Conjugate-1, Conjugate-2, Conjugate-3 and Conjugate-4 were respectively prepared into 2 mmol / L solution with DMSO solution, and before use, the target compounds were respectively diluted into series of solutions with the concentration of 0.156, 0.625, 2.5, 10 and 40 μmol / L with cell culture medium, wherein the final concentration of DMSO was not more than 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 detected in triplicate.

[0071] 2. Cytotoxicity of the target compounds to five breast cancer cell lines and normal human breast epithelial cell line

[0072] The cells in logarithmic growth phase (MCF-7, MDA-MB-231, HCC1187, BT20, HS-578T and MCF-12A) were counted, and 8000-10000 cells of each of the above six kinds of cells were inoculated into 96-well culture plates, and 200 μL of culture medium containing different concentrations of the test compound was added. After overnight culture, the old culture medium in each well was removed with a pipette and discarded, and 200 μL of culture medium containing different concentrations of the test compound was added. The test compound groups were as follows: 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 ⑪ mixture-3 positive control group, with three replicate wells for each concentration. After 72 hours of drug addition, 20 μL of MTT with a concentration of 5 mg / mL was added, and incubation was carried out at 37°C for 4 hours. The supernatant was removed, and 150 μL of DMSO was added for dissolution. The OD value of each well was determined by an enzyme-labeled instrument at a wavelength of 490 nM, and the inhibition rate was calculated to draw a concentration-inhibition rate curve to calculate the IC 50 value.

[0073] The results are shown in Table 1. Lapatinib exhibited a moderate inhibitory effect on the proliferation of five different breast cancer cells, with an IC 50 value of 9.56-16.29 μM. The inhibitory effect on the proliferation of triple-negative breast cancer MDA-MB-231 cells was the strongest, with an IC 50 value of 9.56 μM. The two IDO inhibitors had relatively weak cytotoxicity on the tested breast cancer cells and normal breast cells, with an IC 50 value of more than 100 μM for D-MT on the tested cells. NLG919 had relatively strong cytotoxicity, but its IC 50 value on the tested cancer cells was also greater than 68.0 μM. The GST inhibitor NBDHEX had strong cytotoxicity, with an IC 50 value of less than 6.5 μM for the five tested breast cancer cells, of which the toxicity on MDA-MB-231 cells was the strongest, with an IC 50 value of 2.72 μM. However, it is worth noting that NBDHEX also had relatively strong toxicity on normal breast cells MCF-12A, with an IC 50 value of 3.67 μM. The conjugates-1 and conjugates-2 obtained by coupling the two IDO inhibitors with lapatinib both retained or surpassed the cytotoxicity of the parent compound lapatinib on breast cancer cells. The IC 50values were comparable to lapatinib and mixture-1; conjugate-2 showed significantly higher cytotoxicity against breast cancer cells than lapatinib and mixture-2. The IC 50 values were 9.56 and 8.73 μΜ, respectively, while conjugate-2 showed an IC 50 value of 4.31 μΜ, which was 2.21 and 2.02 times higher than lapatinib and mixture-2, respectively. In addition, conjugate-2 showed an IC 50 value of 65.73 μΜ. NBDHEX was conjugated to lapatinib via two different linkers to obtain conjugate-3 and conjugate-4. Conjugate-3 exhibited more potent cytotoxicity than the parent compound and mixture-3, with IC 50 values of 0.22 and 0.63 μΜ, respectively, against MDA-MB-231 and HS-578T cells. The IC 50 values against the other three breast cancer cell lines, MCF-7, HCC1187 and BT20, were 1.08, 1.72 and 3.47 μΜ, respectively. In addition, conjugate-3 showed an IC 50 value of 12.48 μΜ against normal breast cell MCF-12A, which was significantly higher than NBDHEX (IC 50 = 3.67 μΜ) and mixture-3 (IC 50 = 4.53 μΜ) 50 values, further highlighting the selectivity of conjugate-3 against cancer cells. Conjugate-4 showed significantly reduced cytotoxicity against the tested cancer cells compared to the parent compound due to its larger molecular weight. For example, conjugate-4 showed the highest cytotoxicity against HS-578T cells with an IC 50 value of 17.81 μΜ; the weakest cytotoxicity against BT20 cells with an IC 50 value of 31.71 μΜ. However, it is worth noting that conjugate-4 showed an IC 50 value of 33.59 μΜ against MCF-12A, which was relatively high compared to NBDHEX and the physical mixture of NBDHEX and lapatinib.

[0074] Table 1. Cytotoxicity of target compounds against five breast cancer cell lines and a normal human breast epithelial cell line

[0075]

[0076] a IC 50 values are presented as the mean ± SD of triplicate experiments (n = 3).

[0077] Example 3

[0078] MDA-MB-231 / Lapatinib cells (the cells are disclosed in the literature https: / / doi.org / 10.1021 / acs.jmedchem.1c01013) in logarithmic growth phase were inoculated in 96-well culture plates, 200 μL of culture medium containing 8000-10000 cells was added to each well. After overnight culture, the old culture medium in each well was aspirated with a pipette and discarded after the cells adhered, and 200 μL of fresh culture medium containing different concentrations of test compounds was added. The test compound groups are respectively: 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 ⑪ mixture-3 positive control group, 3 replicate wells were set for each concentration. After drug addition, culture for 72 h, add 20 μL of MTT with a concentration of 5 mg / mL, incubate at 37 ℃ for 4 h, remove the supernatant, add 150 μL of DMSO for dissolution. The OD value of each well was determined by a microplate reader at a wavelength of 490 nM, and the inhibition rate was calculated, and the concentration-inhibition rate curve was calculated IC 50 value.

[0079] The results are shown in Table 2, lapatinib is not sensitive to MDA-MB-231 / Lapatinib drug-resistant cell lines, the IC 50 value is only 42.85 μM, and the resistance index RI value is only 4.48. In contrast, D-MT, NLG-919 and NBDHEX have almost the same sensitivity to MDA-MB-231 / Lapatinib cells as to MDA-MB-231 cells. The cytotoxicity of conjugate-1 and conjugate-2 to MDA-MB-231 / Lapatinib is significantly lower than that of lapatinib, and the IC 50 values are 22.37 and 14.85 μM, respectively, and the RI values are 2.83 and 3.44, respectively, which are significantly lower than those of lapatinib, indicating that they can reverse the lapatinib-mediated MDA-MB-231 resistance to some extent. In contrast, conjugate-3 and conjugate-4 both show strong ability to reverse lapatinib-mediated MDA-MB-231 resistance. For example, the IC 50 value of conjugate-3 to MDA-MB-231 / Lapatinib cells is 0.31 μM, and the RI value is 1.36, which is 0.30 times that of lapatinib; the IC 50The value is 26.14 μΜ, and the RI value also reaches 1.27. The above results show that the introduction of IDO inhibitors or GST inhibitors in the structure of lapatinib can effectively inhibit lapatinib-mediated drug resistance of MDA-MB-231 cells.

[0080] Table 2 Cytotoxicity of target compounds on MDA-MB-231 / Lapatinib cells

[0081]

[0082] a IC 50 The value is expressed as the average value ± SD of three parallel experiments (n = 3); b Resistance index (RI) = IC 50 (MDA-MB-231 / Lapatinib) / IC 50 (MDA-MB-231).

[0083] Example 4

[0084] 1. Protein extraction

[0085] 1) Preparation of MDA-MB-231 cell suspension

[0086] Preparation of cell suspension: 2 mL of trypsin digestion solution without EDTA was added to each cell bottle (cell bottle specifications: 25 cm 2 , and the number of cells was about 1 x 10 6 cells / mL) full of cancer cells (MDA-MB-231 cells), and the cells were digested for 1-2 min. The trypsin digestion solution was slowly washed with a 1.0 mL pipette to completely detach the cancer cells from the bottom of the cell bottle. The trypsin digestion solution containing cancer cells was transferred to a 15 mL sterile centrifuge tube, and centrifuged at 1500 rpm for 5 min at room temperature. The trypsin digestion solution in the centrifuge tube was slowly poured out, and 2 mL of fresh culture medium was added. The cancer cells were completely dispersed by repeatedly blowing the culture medium with a pipette, and then 8 mL of fresh culture medium was added to the centrifuge tube. The cancer cells were evenly distributed in 10 mL of fresh culture medium by slowly shaking the centrifuge tube. The final concentration of cancer cells in the culture medium was 1 x 10 5 cells / mL.

[0087] 2) 2 mL of 1 x 10 5Cells were suspended in an MDA-MB-231 suspension at 100 cells / mL and incubated in a 37°C cell culture incubator with 5% CO2 for 12 hours. The old culture medium was then removed from each well of the 6-well plate. Fresh culture medium containing ①2 mL of 10 μM lapatinib, ②2 mL of 10 μM conjugate-1, ③2 mL of 10 μM conjugate-1, ④2 mL of 10 μM conjugate-2, ⑤2 mL of 10 μM conjugate-3, and ⑥2 mL of 10 μM conjugate-4 were then added, followed by a further 12 hours of incubation in the cell culture incubator. Following incubation, cells were harvested into a 15 mL centrifuge tube, centrifuged at 1500 rpm for 5 minutes, the culture medium removed, and the tubes gently washed twice with 2 mL of 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 minutes. 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 minutes. Remove the supernatant and store it in a -20°C refrigerator.

[0088] 2. Protein quantification and sample preparation

[0089] The protein content was determined using the Coomassie Brilliant Blue G250 method on a Varioskan multimode microplate spectrophotometer. Loading Buffer was then added to the extracted protein and the sample was boiled at 100°C for 15 min.

[0090] 3. Preparation and loading of SDS-PAGE gel

[0091] Add TEMED to the pre-prepared 12% separating gel, pipette to mix thoroughly, and transfer to the concave-convex glass plate of the electrophoresis apparatus. Immediately seal with water. After the gel has completely solidified, remove the water, add 6% stacking gel, and insert the grooves. After the gel has completely solidified, slowly remove the grooves and add 10 μL of the above sample diluted with SDS to the grooves of the gel. Add a marker as a reference. Adjust the electrophoresis apparatus voltage to 100 V and run the gel for 30 minutes. Then adjust it to 150 V and stop when the marker is completely separated and the loading buffer has run to the bottom of the gel.

[0092] 4. Transfer

[0093] Thoroughly wet the PVDF membrane with methanol and immerse it, along with the filter paper, in transfer buffer. Remove the gel and completely soak the portion containing the target protein in transfer buffer for 10 minutes. Transfer the membrane along with the PVDF membrane onto a semi-dry transfer apparatus and transfer at 350 mA for 45-60 minutes. Afterward, place the PVDF membrane in 5% skim milk powder and block on a shaker for 90 minutes.

[0094] 5. Immunization and exposure

[0095] The closed PVDF membrane was washed with TBST on a shaker. The primary antibody was incubated at 4°C overnight, and after the end, the PVDF membrane was washed with TBST on a shaker, repeated five times, 30 min each time. The secondary antibody was incubated at 37°C for 1 h, and after the end, the PVDF membrane was washed with TBST on a shaker, repeated five times, 10 min each time. Finally, the Odyssey scanning system was used for imaging;

[0096] 6. Inhibition of pEGFR (Y1068) and EGFR protein levels by lapatinib and conjugates 1-4

[0097] The results are shown in Figure 1 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 the expression of EGFR protein, and conjugates 1-4 can also exert the strong inhibitory effect of the parent compound lapatinib on the expression of p-EGFR (Y1068) protein. Except for conjugate 4, the inhibitory effect of the rest of the conjugates is almost equivalent to that of lapatinib, and has no significant effect on the expression of EGFR protein. These results show that conjugates 1-4 can effectively inhibit the expression of p-EGFR (Y1068) protein.

[0098] Example 5

[0099] MDA-MB-231 cells were seeded in a 48-well plate at a density of 3 x 10 4 cells / well and incubated in a 37°C cell culture incubator containing 5% CO2for 12 hours. The medium in 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 medium containing 30% trifluoroacetic acid was added. The 96-well plate was incubated at 50°C for 30 minutes, and then it was placed in a plate centrifuge at 3000 rpm per minute for 10 minutes to remove the precipitate. 100 μL of supernatant was aspirated from each well and mixed with 100 μL of a solution containing 2% (w / v) p-dimethylaminobenzaldehyde acetic acid. Then the 96-well plate was placed in a microplate reader, and the absorbance value of each well was detected at a wavelength of 480 nM, with three parallel tests for each group. The relative content of kynurenine in each well was then calculated compared to the control group.

[0100] The results are shown in Figure 2As shown, the IDO inhibitors D-MT and NLG919 both effectively inhibited kynurenine levels in MDA-MB-231 cells. Conjugates 1 and 2, obtained by grafting the D-MT and NLG919 structures onto the lapatinib structure, exhibited significantly greater inhibitory effects on kynurenine levels than the parent compound. Conjugate 1 exhibited 1.84- and 1.25-fold greater inhibitory effects on kynurenine than the D-MT and mixture 1 groups, respectively; while Conjugate 2 exhibited 1.64- and 1.48-fold greater inhibitory effects on kynurenine than the NLG919 and mixture 2 groups, respectively. In summary, both Conjugate 1 and Conjugate 2 were able to block the degradation of tryptophan to produce kynurenine, with significantly greater potency than the parent compound.

[0101] Example 6

[0102] 1×10 3 MDA-MB-231 cells were seeded in 6-well plates at a density of 100 cells / well and incubated 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 incubated 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 PBMCs were collected from each well, suspended in PBS, and labeled with a FITC-conjugated CD4 antibody. Finally, the expression of CD4+ T cells in each group was determined using a CytoFLEX S flow cytometer (Beckman Coulter, USA).

[0103] 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 NLG919 is more capable of promoting CD4+ T cell secretion than 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 outstanding. In summary, conjugate-1 and conjugate-2 reduce the level of kynurenine by inhibiting IDO enzyme activity, thereby promoting the secretion of T cells in cancer cells.

[0104] Example 7

[0105] The inhibitory activities of conjugate-3 and conjugate-4 against GST P1-1 enzymatic activity were determined using commercial services provided by Shanghai Fusheng Industrial Co., Ltd. (Shanghai, China), and NBDHEX was used as a positive control.

[0106] The results are shown in Table 3. NBDHEX showed a strong inhibitory effect on GST P1-1 enzyme activity, IC 50 The value reached 0.86 μM. Conjugate-3 retained the inhibitory effect of the target compound on GST P1-1, IC 50 The value also reached 0.79 μM; probably due to its large molecular weight, conjugate-4 had a relatively weak inhibitory effect on GST P1-1 enzyme activity, only 16.71 μM.

[0107] Table 3 Inhibitory effects of conjugate-3 and conjugate-4 on GST P1-1 enzyme activity

[0108]

[0109] a IC 50 The values ​​are expressed as the mean ± SD of three parallel experiments (n = 3).

[0110] Based on the SMDC design concept, this application uses lapatinib as the parent compound and conjugates two different IDO inhibitors (D-MT and NLG919) and a GST inhibitor (NBDHEX) via different linkers to obtain four target conjugates 1-4. In summary, target conjugates 1-4 all retain the parent compound's inhibitory activity against EGFR and exhibit potent antiproliferative effects against various types of triple-negative breast cancer cells. Furthermore, conjugates-1 and -2 effectively reverse lapatinib-mediated MDA-MB-231 cell resistance by effectively inhibiting IDO activity within cancer cells and promoting intracellular CD4+ T cell secretion. 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, making it suitable for the preparation of anti-tumor drugs.

[0111] The above description is illustrative and non-limiting to the present invention. Those skilled in the art will appreciate that many modifications, variations, or equivalents may be made without departing from the spirit and scope of the appended claims, and all of these modifications, variations, or equivalents will fall within the scope of protection of the present invention. The above description is illustrative and non-limiting to the present invention. Those skilled in the art will appreciate that many modifications, variations, or equivalents may be made without departing from the spirit and scope of the appended claims, and all of these modifications, variations, or equivalents 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 then undergoes 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 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: ； 。 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: Lapatinib 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; NBDHEX is then esterified with 4-(azidomethyl)benzoic acid to obtain the intermediate NBDHEX-azido; finally, the intermediate lapatinib-alkynyl and the intermediate NBDHEX-azido undergo a click reaction under the catalysis of CuSO4·5H2O and vitamin C to obtain conjugate-4; the reaction process is as follows: ； ； ； 。 7. Use of the conjugate-1 according to claim 1 in the preparation of a medicament for reversing lapatinib-mediated cancer cell resistance, wherein the cancer is triple-negative breast cancer.

8. Use of the conjugate-1 according to claim 1 in the preparation of a medicament for inhibiting EGFR enzyme activity in cancer cells and / or promoting immune T cell secretion in cancer cells, wherein the cancer is triple-negative breast cancer.

9. Use of the conjugate-3 according to claim 3 and / or the conjugate-4 according to claim 4 in the preparation of a medicament for reversing lapatinib-mediated drug resistance in cancer cells, wherein the cancer is triple-negative breast cancer.

10. Use of the conjugate-3 according to claim 3 and / or the conjugate-4 according to claim 4 in the preparation of a medicament for inhibiting EGFR enzyme activity and / or inhibiting GST P1-1 enzyme activity in cancer cells, wherein the cancer is triple-negative breast cancer.

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