Oleanolic acid derivative and lapatinib conjugate as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of chemical medicines, and discloses an oleanolic acid derivative and lapatinib conjugate as well as a preparation method and application thereof. The oleanolic acid derivative and the lapatinib conjugate improve the selectivity and activity of oleanolic acid by modifying the structure of an oleanolic acid mother nucleus, and are connected with lapatinib through a disulfide bond connecting bridge. The oleanolic acid derivative and lapatinib conjugate can be subjected to targeted breakage by glutathione to release free compounds. The invention provides a synthetic route and a purification method of the derivative.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceuticals and relates to a class of oleanolic acid derivatives and lapatinib conjugates, and their preparation methods and applications. Background Art

[0002] Breast cancer is the second most common type of cancer. Clinically, breast cancer can be divided into three subtypes according to the hormone receptor status: luminal A and luminal B types, HER2-positive type, and triple-negative breast cancer. Among them, the proportion of patients with HER2-positive breast cancer is about 15% to 20%. At present, HER2 has become a suitable target for anti-breast cancer drug discovery and research. Although the application of trastuzumab has significantly improved the overall survival of patients with HER2-positive breast cancer, about 30% of patients have drug resistance, resulting in ineffective treatment or tumor recurrence. Therefore, improving multi-drug resistance is very necessary for breast cancer treatment.

[0003] Pentacyclic triterpenoids (PTs) are a class of plant natural products composed of three terpene units, with anti-tumor, anti-inflammatory, and anti-viral activities. The biological functions of pentacyclic triterpenoid compounds are very extensive, but their action intensity is weak and their bioavailability is low. Therefore, their clinical applications are very limited. To solve this problem, the structure of pentacyclic triterpenoids is modified, and pentacyclic triterpenoid conjugates synthesized by connecting with another modified molecule through an appropriate linker: such as oleanolic acid derivatives and rhodamine conjugates, betulinic acid and aromatic amine conjugates. The conjugates have better activity and selectivity than the parent pentacyclic triterpenoids, thereby being able to improve the anti-cancer activity of the compounds.

[0004] Currently, studies have confirmed that the combined use of oleanolic acid derivatives and the HER2 inhibitor trastuzumab can produce a synergistic effect, reverse the drug resistance of trastuzumab, and increase the drug efficacy (Menendez J A, Vellon L, Colomer R, et al. [J]. ANNALS OF ONCOLOGY, 2005, 16(3): 359 - 371.). However, this method of combining two single drugs has more side effects, higher toxicity, low bioavailability, and rapid metabolism. Therefore, we chose to design and synthesize conjugates of oleanolic acid derivatives and the HER2 inhibitor lapatinib. The conjugates can deliver the two drugs to the same cell (such as tumor cells) simultaneously, reduce multi-drug resistance, and improve bioavailability. In addition, since disulfide bonds can be targeted and cleaved by GSH in tumor cells to release two free compounds, disulfide compounds were selected as the linker of the conjugate to synthesize the conjugate of the two as a prodrug for targeted release in anti-tumor cells. Summary of the Invention

[0005] The purpose of the present invention is to provide a class of novel oleanolic acid derivatives and lapatinib conjugates.

[0006] Another object of the present invention is to provide the method for preparing the above conjugate.

[0007] Another object of the present invention is to provide the application of the above conjugate in preparing anti-tumor drugs.

[0008] Technical solution of the present invention:

[0009] An oleanolic acid derivative and lapatinib conjugate, the structural formula (I) thereof is shown as follows:

[0010]

[0011] Wherein, the oleanolic acid derivative and lapatinib conjugate is composed of the following structures:

[0012] (a) An oleanolic acid derivative, the structural formula thereof is shown as N-4;

[0013] (b) HER2 inhibitor lapatinib, the structural formula thereof is shown as Lp-1;

[0014] (c) A linking bridge of a disulfide compound, the structural formula thereof is shown as S-1;

[0015]

[0016] A method for preparing an oleanolic acid derivative and lapatinib conjugate, comprising the following steps:

[0017] (1) Synthesis of intermediate N-1:

[0018] At low temperature, oleanolic acid (OA) and 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) are dissolved in N,N-dimethylformamide (DMF) solvent, N,N-diisopropylethylamine (DIPEA) is added, after stirring at room temperature, N-BOC ethylenediamine is added, wherein the molar ratio of compound OA to HATU is 1:1 to 1:2; the molar ratio of OA to N-BOC ethylenediamine is 1:2 to 1:3, and the molar ratio of compound oleanolic acid OA to N,N-diisopropylethylamine is 1:0.25 to 1:0.5, and the reaction is stirred overnight at room temperature under a nitrogen atmosphere; after extraction with saturated sodium chloride solution and ethyl acetate, drying and concentration with anhydrous sodium sulfate, purification by column chromatography of petroleum ether and ethyl acetate at a ratio of 3:1 gives white powdery intermediate N-1;

[0019]

[0020] (2) Synthesis of intermediate N-2:

[0021] Dissolve the intermediate N-1 obtained in step (1) in a mixed solvent of dichloromethane and acetone with a volume ratio of 1:1, add Jones reagent, where the molar ratio of N-1 to chromium trioxide in Jones reagent is 1:1 to 1:1.5, and react at room temperature for 0.5 to 1 hour; after terminating the reaction with isopropanol, spin-dry the reaction solution, extract with dichloromethane and water, dry and concentrate with anhydrous sodium sulfate, and purify by column chromatography with petroleum ether and ethyl acetate at a ratio of 3:1 to obtain the white powdery intermediate N-2;

[0022]

[0023] (3) Synthesis of intermediate N-3:

[0024] Dissolve the intermediate N-2 obtained in step (2) in DMF solvent, add potassium carbonate and paraformaldehyde; where the molar ratio of intermediate N-2, potassium carbonate and paraformaldehyde is 1:7.5:20 to 1:10:30, react at 110 °C for 5 hours, extract with saturated sodium chloride solution and ethyl acetate, dry and concentrate with anhydrous sodium sulfate, and purify by column chromatography with petroleum ether and ethyl acetate at a ratio of 3:1 to obtain the white solid intermediate N-3;

[0025]

[0026] (4) Synthesis of oleanolic acid derivative N-4:

[0027] Dissolve the intermediate N-3 obtained in step (3) in a mixed solvent of dichloromethane and trifluoroacetic acid with a volume ratio of 1:1, where the molar ratio of intermediate N-3 to trifluoroacetic acid is 1:15 to 1:20, stir at room temperature for 0.5 to 1 hour; spin-dry the reaction solution, redissolve with dichloromethane and add saturated sodium bicarbonate to adjust the pH to 12, extract and dry and concentrate with anhydrous sodium sulfate to obtain the white solid compound N-4;

[0028]

[0029] (5) Synthesis of intermediate S-2:

[0030] Dissolve compound S-1 in anhydrous tetrahydrofuran, add acetic anhydride and 4-dimethylaminopyridine (DMAP), where the molar ratio of compound S-1, acetic anhydride and DMAP is 1:0.5:0.25 to 1:1:0.5, react at room temperature overnight, spin-dry the reaction solution; extract with ethyl acetate and dilute hydrochloric acid (1 mol / L), dry and concentrate with anhydrous sodium sulfate, and purify by column chromatography with petroleum ether and ethyl acetate at a ratio of 3:1 to obtain the pale yellow oily intermediate S-2;

[0031]

[0032] (6) Synthesis of intermediate Lp-s-1:

[0033] Dissolve the intermediate S-2 obtained in step (5) and DIPEA in anhydrous dichloromethane, and dropwise add a solution of triphosgene in anhydrous dichloromethane at low temperature. The molar ratio of intermediate S-2, DIPEA and triphosgene is 1:0.35:2.0 to 1:0.5:2.0. React for 5 hours under a nitrogen atmosphere. After rotary evaporation of the reaction solution and redissolving it in dichloromethane, add a mixed solution of lapatinib, DIPEA, and DMAP in anhydrous dichloromethane at 0 °C. The molar ratio of intermediate S-2, lapatinib, DIPEA, and DMAP is 1:1:2:0.5 to 1:1:2:1. React overnight at room temperature. After rotary evaporation of the reaction solution, extract with water and ethyl acetate, and purify by column chromatography on a column of dichloromethane and methanol at a ratio of 70:1 to obtain a yellow solid Lp-s-1.

[0034]

[0035] (7) Synthesis of intermediate Lp-s-2:

[0036] Dissolve the intermediate Lp-s-2 obtained in step (6) in a mixed solution of methanol and tetrahydrofuran with a volume ratio of 3:1. Add an aqueous solution of potassium carbonate with a concentration of 0.5 mol / L to it at low temperature. The molar ratio of intermediate Lp-s-2 and potassium carbonate is 1:1.5. Stir at room temperature for 1 hour, then rotary evaporate the reaction solution, extract with saturated sodium chloride solution and ethyl acetate, dry and concentrate with anhydrous sodium sulfate, and purify by column chromatography on a column of dichloromethane and methanol at a ratio of 50:1 to obtain a yellow solid Lp-s-2.

[0037]

[0038] (8) Synthesis of the target compound Lp-ss-N4:

[0039] Dissolve the intermediate Lp-s-2 obtained in step (7) and DIPEA in anhydrous dichloromethane, and dropwise add a solution of triphosgene in anhydrous dichloromethane at low temperature. The molar ratio of Lp-s-2, DIPEA and triphosgene is 1:0.35:2.0 to 1:0.5:2.0. React for 5 hours under a nitrogen atmosphere. After rotary evaporation of the reaction solution and redissolving it in dichloromethane, add a mixed solution of intermediate N-4, DIPEA, and DMAP in anhydrous dichloromethane at low temperature. The molar ratio of intermediate Lp-s-2, intermediate N-4, DIPEA, and DMAP is 1:1:2:0.5 to 1:1:2:1. React overnight at room temperature. After rotary evaporation of the reaction solution, extract with water and ethyl acetate, and purify by column chromatography on a column of dichloromethane and methanol at a ratio of 70:1 to obtain a yellow solid Lp-ss-N4.

[0040]

[0041] A drug conjugate, which comprises the oleanolic acid derivative N-4, lapatinib Lp-1 and the disulfide compound S-1 described in claim 1.

[0042] The conjugate contains a therapeutically effective amount of the oleanolic acid derivative N-4 and the HER2 inhibitor lapatinib Lp-1.

[0043] Use of a conjugate in the preparation of a drug for treating tumor diseases.

[0044] The tumor disease is breast cancer tumor.

[0045] Advantages of the present invention: The present invention provides a conjugate of an oleanolic acid derivative and lapatinib. Through chemical synthesis, a novel conjugate that can be targeted and cleaved by glutathione can be obtained. This compound can be cleaved by glutathione in tumor cells, thereby simultaneously releasing two free drugs. By utilizing the potential synergistic or targeted effects of the two drugs, the pharmacokinetics can be improved and the toxicity can be reduced, and the anti-tumor effect can be better exerted. Description of the Drawings

[0046] Figure 1 It is the MS detection chart of the compound Lp-ss-N4 of the present application;

[0047] Figure 2 It is of the compound Lp-SS-N4 of the present application 1 1H NMR detection chart;

[0048] Figure 3 It is the inhibitory effect of Lp-1 and N-4 on MDA-MB-231 cells at different concentrations. Among them, (a) is the dose-effect curve of Lp-1 on MDA-MB-231 cells, and (b) is the dose-effect curve of N-4 on MDA-MB-231 cells. Detailed Embodiments

[0049] The following further illustrates the detailed embodiments of the present invention in conjunction with the drawings and technical solutions.

[0050] Example 1

[0051] (a) Synthesis of intermediate N-1

[0052] Weigh compound OA (100 g, 0.22 mmol, 1.0 eq) into a 100 ml conical flask, add DMF (5 mL) to dissolve it, and then add DIPEA (192 μL, 1.10 mmol, 5.0 eq). Cool the reaction mixture to 0 °C, add HATU (167 mg, 0.44 mmol, 2.0 eq) and stir for 10 min, then add N-Boc-ethylenediamine (105.40 g, 0.44 mmol, 3.0 eq), and stir the solution overnight under a nitrogen atmosphere. After the substrate is completely consumed (monitored by TLC analysis), pour the reaction mixture into saturated sodium chloride solution and extract with ethyl acetate. Wash the combined organic layers successively with saturated NaHCO 3 solution, HCl solution (1 N) and saturated brine, and dry over Na 2 SO 4 and concentrate by evaporation. Separate by column chromatography with petroleum ether:ethyl acetate = 3:1 to obtain 100 mg of a white solid with a yield of 77%.

[0053] 1H NMR (400 MHz, Chloroform-d) δ 6.35 (s, 1H), 5.39 (t, J = 3.6 Hz, 1H), 4.12 (q, J = 7.1 Hz, 1H), 3.44 (dt, J = 12.4, 6.3 Hz, 1H), 3.30–3.11 (m, 4H), 2.60–2.51 (m, 1H), 2.06–1.88 (m, 4H), 1.68–1.49 (m, 10H), 1.44 (s, 12H), 1.29–1.23 (m, 3H), 1.16 (s, 4H), 0.99 (s, 4H), 0.94–0.88 (m, 9H), 0.81–0.71 (m, 7H).

[0054] (b) Synthesis of intermediate N-2

[0055] Weigh compound N-1 (500 g, 0.835 mmol, 1.0 eq.) into a 100 ml reaction flask, add a mixed solvent of 10 ml of acetone and 10 ml of dichloromethane, and stir to dissolve. Protect with nitrogen, slowly add freshly prepared Jones reagent (100 mg of chromium trioxide, 1.0 mmol, 1.2 eq.) dropwise at 0 °C, then raise the reaction to room temperature and continue the reaction. Monitor the reaction by TLC until it is complete. Concentrate the reaction solution, add dichloromethane to dissolve the solid, and transfer it to a separatory funnel. Extract with dichloromethane and combine the organic phases. Wash the organic phase with saturated NaHCO 3 solution, wash the organic phase with saturated sodium chloride solution, and dry over anhydrous sodium sulfate. Filter, rotary evaporate and concentrate the organic phase, and separate by column chromatography with petroleum ether:ethyl acetate = 3:1 to obtain 132 g of a white solid with a yield of 30%.

[0056] 1H NMR (400 MHz, Chloroform-d) δ 6.36 (s, 1H), 5.41 (t, J = 3.6 Hz, 1H), 4.12 (q, J = 7.2 Hz, 1H), 3.48–3.10 (m, 4H), 2.56 (ddt, J = 18.5, 11.2, 5.7 Hz, 2H), 2.36 (ddd, J = 15.9, 6.7, 3.6 Hz, 1H), 2.08–1.84 (m, 5H), 1.75 (t, J = 13.4 Hz, 1H), 1.69–1.61 (m, 3H), 1.61–1.50 (m, 2H), 1.49 (d, J = 6.1 Hz, 2H), 1.46–1.37 (m, 9H), 1.32 (ddd, J = 7.6, 5.1, 2.0 Hz, 2H), 1.26 (t, J = 7.1 Hz, 2H), 1.22–1.19 (m, 1H), 1.17 (s, 3H), 1.10–0.96 (m, 9H), 0.95–0.73 (m, 8H).

[0057] (c) Synthesis of Intermediate N-3

[0058] Weigh compound N-3 (123 mg, 0.21 mmol, 1.0 eq) into a 25 ml reaction flask, add DMF (5 ml) and stir to dissolve. Add K 2 CO 3 (213.76 mg, 1.55 mmol, 7.5 eq) and paraformaldehyde particles (197.97 mg, 6.60 mmol, 30 eq) to the mixed solution. Heat the mixed solution at 110 °C for 5 hours; after the substrate is completely consumed (monitored by TLC analysis), add saturated sodium chloride solution to the reaction mixture and extract with ethyl acetate. The combined organic layers are washed with saturated sodium chloride solution and dried over anhydrous Na 2 SO 4 After drying, concentrate under vacuum. Purify the residue by flash column chromatography on silica gel (petroleum ether: ethyl acetate = 3:1 to 2:1) to obtain white solid N-3 (54 mg, 44%).

[0059] 1H NMR (500 MHz, Chloroform-d) δ 6.39 (s, 1H), 5.99 (t, J = 2.3 Hz, 1H), 5.44 (t, J = 3.7 Hz, 1H), 5.16 (t, J = 2.3 Hz, 1H), 5.01 (s, 1H), 4.12 (q, J = 7.1 Hz, 1H), 3.43 (d, J = 13.3 Hz, 1H), 3.24 (s, 3H), 2.74–2.51 (m, 2H), 2.14 (d, J = 15.1 Hz, 1H), 2.02 (d, J = 23.7 Hz, 4H), 1.80–1.51 (m, 7H), 1.44 (s, 11H), 1.34 (dq, J = 7.8, 5.2, 4.3 Hz, 2H), 1.26 (t, J = 7.1 Hz, 2H), 1.19 (s, 4H), 1.13 (s, 3H), 1.06 (s, 3H), 0.96–0.88 (m, 8H), 0.82 (s, 3H).

[0060] (d) Synthesis of Intermediate N-4

[0061] Weigh compound N-3 (105 mg, 0.164 mmol, 1.0 eq) into a 25 ml conical flask, add 5 ml of dichloromethane to dissolve it. Dropwise add TFA (244 μl, 3.29 mmol, 20.0 eq) to the mixed solution at 0 °C, then warm it up to room temperature and stir for 30 min. After the substrate is completely consumed (detected by TLC analysis), concentrate the reaction solution to obtain a red-brown oil. The remaining TFA is removed by rotary evaporation with dichloromethane several times. Then adjust the pH to 12 with NaOH solution (0.5 mol / L), extract with ethyl acetate, wash the organic phase with saturated sodium chloride solution, dry over anhydrous Na2SO4, and concentrate to obtain compound N-4 (74 mg, 74%).

[0062] 1H NMR (400 MHz, Chloroform-d) δ 6.47–6.28 (m, 2H), 5.99 (t, J = 2.3 Hz, 1H), 5.40 (dd, J = 14.1, 10.5 Hz, 2H), 5.30 (s, 1H), 5.17 (d, J = 7.2 Hz, 1H), 3.41 (dt, J = 14.3, 7.2 Hz, 2H), 2.87–2.78 (m, 3H), 2.67–2.58 (m, 3H), 2.04–1.94 (m, 5H), 1.19 (t, J = 2.9 Hz, 6H), 1.14 (s, 2H), 1.07–1.05 (m, 3H), 1.04 (d, J = 4.1 Hz, 3H), 0.94–0.91 (m, 10H), 0.83 (d, J = 5.5 Hz, 2H), 0.79–0.71 (m, 6H).

[0063] (e) Synthesis of Intermediate S-2

[0064] Weigh compound S-1 (100 mg, 0.65 mmol, 1.0 eq) and DMAP (4 mg, 0.033 mmol, 0.05 eq) into a 50 ml conical flask, add anhydrous THF (5 ml) to dissolve, dropwise add acetic anhydride (31 μl, 0.33 mmol, 0.51 eq) to the solution, stir at room temperature for 12 h. After the substrate reaction is complete (detected by TLC analysis), rotary evaporate the reaction solution, dissolve it with ethyl acetate and wash it with dilute hydrochloric acid (1 mol / L). The organic phase is washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate and then concentrated. Column chromatography (petroleum ether: ethyl acetate = 2:1) gives the oily compound S-2 (32 mg, 25%).

[0065] 1H NMR (500 MHz, Chloroform-d) δ 4.35 (t, J = 6.7 Hz, 2H), 3.89 (t, J = 5.8 Hz, 2H), 2.91 (dt, J = 20.6, 6.2 Hz, 4H), 2.08 (s, 4H).

[0066] (f) Synthesis of Intermediate Lp-s-1

[0067] Weigh compound S-2 (43 mg, 0.22 mmol, 1.0 eq) into a 25 ml conical flask, dissolve it with anhydrous dichloromethane (5 ml) and then dropwise add DIPEA (115 μl, 0.66 mol, 3.0 eq). At 0 °C, dropwise add an anhydrous dichloromethane solution of BTC (21 mg, 0.07 mmol, 1.0 eq) to the mixture. Under nitrogen protection, react at 0 °C for 5 h. After the substrate reaction is complete (detected by TLC analysis), rotary evaporate the reaction solution and redissolve it in anhydrous dichloromethane. At 0 °C, add a mixed solution of lapatinib (127 mg, 0.22 mmol, 1.0 eq), DIPEA (77 μl, 0.44 mmol, 2.0 eq) and DMAP (13.40 mg, 0.11 mmol, 0.5 eq) in anhydrous dichloromethane to it. Stir at room temperature overnight under nitrogen protection. After the substrate reaction is complete (detected by TLC analysis), rotary evaporate the reaction solution. Extract with water and ethyl acetate, and then extract the organic phase with saturated sodium chloride solution. Then dry and concentrate over anhydrous Na 2 SO 4 Dry and concentrate, and purify by column chromatography (dichloromethane: methanol = 100:1 - 70:1) to obtain the yellow solid Lp-s-1 (65 mg, 40%).

[0068] 1H NMR (400 MHz, Chloroform-d) δ 8.65 (d, J = 23.7 Hz, 2H), 8.44 (s, 1H), 7.98–7.84 (m, 3H), 7.75–7.67 (m, 1H), 7.36 (td, J = 8.1, 5.9 Hz, 1H), 7.28–7.19 (m, 4H), 7.05–6.94 (m, 2H), 6.75 (d, J = 3.3 Hz, 1H), 6.51–6.40 (m, 1H), 5.16 (s, 2H), 4.62 (d, J = 14.4 Hz, 2H), 4.42 (t, J = 6.1 Hz, 2H), 4.07 (d, J = 8.8 Hz, 2H), 3.46 (d, J = 13.2 Hz, 2H), 2.97 (d, J = 6.5 Hz, 2H), 2.93 (s, 5H), 2.06 (s, 3H).

[0069] (g) Synthesis of Intermediate Lp-s-2

[0070] Weigh compound Lp-s-1 (28 mg, 0.035 mmol, 1.0 eq) and dissolve it in a mixed solution of methanol:tetrahydrofuran = 3:1. Add K 2 CO 3 aqueous solution (0.5 mol / L, 105 ul, 1.5 eq) thereto at 0 °C. Stir for 1 h. After the substrate reaction is complete (detected by TLC analysis), concentrate the reaction solution. Extract with ethyl acetate and water, combine the organic phases, wash with saturated brine, and then dry over anhydrous Na 2 SO 4 and concentrate. Purify by column chromatography (anhydrous dichloromethane:methanol = 50:1) to obtain a yellow compound (16 mg, 60%).

[0071] 1H NMR (400 MHz, Chloroform-d) δ 8.66 (s, 2H), 8.42 (s, 1H), 8.00–7.76 (m, 3H), 7.77–7.64 (m, 1H), 7.41–7.32 (m, 1H), 7.23 (t, J = 8.8 Hz, 2H), 7.01 (ddd, J = 21.2, 10.9, 5.8 Hz, 2H), 6.74 (d, J = 3.3 Hz, 1H), 6.56–6.35 (m, 1H), 5.30 (s, 1H), 5.15 (s, 2H), 4.61 (d, J = 15.7 Hz, 2H), 4.44 (t, J = 6.3 Hz, 2H), 4.05 (dq, J = 14.6, 7.5, 6.2 Hz, 2H), 3.87 (dt, J = 11.8, 5.9 Hz, 2H), 3.41 (d, J = 13.6 Hz, 2H), 3.01–2.80 (m, 7H).

[0072] (h) Synthesis of Intermediate Lp-s-N4

[0073] Weigh compound Lp-s-2 (30 mg, 0.04 mmol, 1.0 eq) into a 25 ml conical flask. After dissolving it with anhydrous dichloromethane (5 ml), add DIPEA (20 μl, 0.12 mol, 3.0 eq) dropwise. At 0 °C, add a solution of BTC (5 mg, 0.02 mmol, 0.4 eq) in anhydrous dichloromethane dropwise to the mixture. Under nitrogen protection, react at 0 °C for 5 h. After the substrate reaction is complete (detected by TLC analysis), spin-dry the reaction solution and redissolve it in anhydrous dichloromethane. At 0 °C, add a mixed solution of N-5 (21 mg, 0.04 mmol, 1.0 eq), DIPEA (20 μl, 0.02 mmol, 2.0 eq) and DMAP (5 mg, 0.02 mmol, 0.5 eq) in anhydrous dichloromethane to it. Stir overnight at room temperature under nitrogen protection. After the substrate reaction is complete (detected by TLC analysis), spin-dry the reaction solution. Extract with water and ethyl acetate, then extract the organic phase with saturated sodium chloride solution, and then dry and concentrate with anhydrous Na 2 SO 4 Dry and concentrate, and purify by column chromatography (dichloromethane: methanol = 100:1 - 70:1) to obtain yellow solid Lp-s-N4 (5 mg, 15%).

[0074] 11H NMR (400 MHz, Chloroform-d) δ 8.66 (d, J = 23.0 Hz, 2H), 8.45 (s, 1H), 8.00–7.82 (m, 3H), 7.73 (dd, J = 9.1, 2.7 Hz, 1H), 7.36 (td, J = 8.0, 5.7 Hz, 1H), 7.23 (t, J = 8.8 Hz, 5H), 7.02 (dt, J = 15.1, 5.7 Hz, 2H), 6.74 (d, J = 3.4 Hz, 1H), 6.46 (d, J = 11.9 Hz, 1H), 6.29 (d, J = 5.4 Hz, 1H), 5.99 (d, J = 2.4 Hz, 1H), 5.62 (s, 1H), 5.41 (t, J = 3.6 Hz, 1H), 5.30 (s, 2H), 5.16 (s, 3H), 4.62 (d, J = 12.8 Hz, 2H), 4.43 (t, J = 6.4 Hz, 2H), 4.29 (d, J = 7.1 Hz, 2H), 4.04 (d, J = 10.4 Hz, 2H), 3.46 (d, J = 7.8 Hz, 4H), 2.96 (q, J = 14.4, 10.3 Hz, 8H), 2.02–1.89 (m, 3H), 1.27 (d, J = 11.1 Hz, 8H), 1.17 (d, J = 10.4 Hz, 5H), 1.12 (s, 3H), 1.05 (s, 4H), 0.94–0.83 (m, 12H), 0.77 (s, 3H).

[0075] Application Example 1

[0076] Evaluation of the synergistic effect of the two drugs in the conjugate of the present invention on tumor cells:

[0077] MTT assay:

[0078] MDA-MB-231 cells in the logarithmic growth phase were prepared into a single-cell suspension with a cell density of 5×10 4 / mL. It was inoculated into a 96-well plate at a volume of 100 μL / well. After inoculation, it was placed in an incubator and cultured for another 24 h. After the cells adhered to the wall, the cell state was observed under a microscope. If the cells grew normally and were evenly distributed in the wells, they were placed in a laminar flow hood. The old medium was aspirated with a pipette, and fresh complete medium containing different concentrations of drugs was added and cultured for another 48 h. After the drug treatment was completed, the old medium was aspirated, and 100 μL of serum-free medium with a final concentration of 0.05 mg / mL of MTT was added to each well. It was incubated in the dark for 4 h. After incubation, the liquid in the wells was aspirated, and 100 μL of DMSO was added to dissolve the formed formazan. The absorbance at 37 °C and 490 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader, and the cell viability was calculated according to the following formula:

[0079] Cell survival rate (%) = (A 实验 - A 空白 ) / (A 对照 - A 空白 ) × 100%

[0080] where A 实验 is the absorbance of the drug-treated group; A 对照 is the absorbance of the control group without drug treatment; A 空白 is the absorbance of the background.

[0081] The biological effects of Lp-1 and N-4 in MDA-MB-231 cells were determined by the above MTT method, and the dose-effect curves were plotted as Figure 3 shown.

[0082] The following are the concentrations corresponding to a 50% inhibition rate of the drug and the combination index (CI) of the combined drug:

[0083] <![CDATA[IC 50 > Lp-1 20 (uM) N-4 44 (uM) Lp-1 + N-4 10 (uM) CI 0.79

[0084] The above results show that Lp-1 and N-4 have a concentration-dependent anti-proliferative effect and exhibit a synergistic effect in inhibiting tumors.

Claims

1. A conjugate of an oleanolic acid derivative and lapatinib, characterized in that: The structural formula (I) of the oleanolic acid derivative and lapatinib conjugate is shown below: Wherein, the oleanolic acid derivative and lapatinib conjugate consists of the following structure: (a) an oleanolic acid derivative, the structural formula of which is shown in N-4; (b) HER2 inhibitor lapatinib, whose structural formula is shown as Lp-1; (c) a connecting bridge of a disulfide bond compound, the structural formula of which is shown in S-1; 2. A method for preparing the conjugate of oleanolic acid derivative and lapatinib according to claim 11, characterized in that: The following steps are involved: (1) Synthesis of intermediate N-1: At low temperature, the compound oleanolic acid OA and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate are dissolved in N,N-dimethylformamide solvent, N,N-diisopropylethylamine is added, and after stirring at room temperature, N-BOC ethylenediamine is added, wherein the molar ratio of the compound oleanolic acid OA to 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate is 1:1-1:2; the molar ratio of the compound oleanolic acid OA to N-BOC ethylenediamine is 1:2-1:3, and the molar ratio of the compound oleanolic acid OA to N,N-diisopropylethylamine is 1:0.25-1:0.5, and the mixture is stirred at room temperature overnight under nitrogen atmosphere to react; the mixture is extracted with saturated sodium chloride solution and ethyl acetate, dried and concentrated over anhydrous sodium sulfate, and purified by column chromatography with petroleum ether and ethyl acetate in a ratio of 3:1 to obtain a white powdery intermediate N-1; (2) Synthesis of intermediate N-2: The intermediate N-1 obtained in step (1) is dissolved in a mixed solvent of dichloromethane and acetone in a volume ratio of 1:1, and Jones reagent is added, wherein the molar ratio of the intermediate N-1 to the chromium trioxide in the Jones reagent is 1:1 to 1:1.5, and the reaction is carried out at room temperature for 0.5 to 1 hour; after terminating the reaction with isopropanol, the reaction solution is spin-dried, extracted with dichloromethane and water, dried and concentrated with anhydrous sodium sulfate, and purified by column chromatography with petroleum ether and ethyl acetate in a ratio of 3:1 to obtain a white powdery intermediate N-2; (3) Synthesis of intermediate N-3: The intermediate N-2 obtained in step (2) is dissolved in DMF solvent, and potassium carbonate and paraformaldehyde are added; wherein the molar ratio of the intermediate N-2, potassium carbonate and paraformaldehyde is 1:7.5:20 to 1:10:30, and the mixture is reacted at 110° C. for 5 hours, extracted with saturated sodium chloride solution and ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography with petroleum ether and ethyl acetate in a ratio of 3:1 to obtain a white solid intermediate N-3; (4) Synthesis of oleanolic acid derivative N-4: The intermediate N-3 obtained in step (3) is dissolved in a mixed solvent of dichloromethane and trifluoroacetic acid in a volume ratio of 1:1, wherein the molar ratio of the intermediate N-3 to the trifluoroacetic acid is 1:15 to 1:20, and stirred at room temperature for 0.5 to 1 hour; the reaction solution is spin-dried, redissolved in dichloromethane, and saturated sodium bicarbonate is added to adjust the pH to 12, extracted, dried over anhydrous sodium sulfate, and concentrated to obtain a white solid compound N-4; (5) Synthesis of intermediate S-2: Compound S-1 was dissolved in anhydrous tetrahydrofuran, and acetic anhydride and 4-dimethylaminopyridine were added, wherein the molar ratio of compound S-1, acetic anhydride and 4-dimethylaminopyridine was 1:0.5:0.25 to 1:1:0.5, and the mixture was reacted at room temperature overnight, and the reaction liquid was spin-dried; the mixture was extracted with ethyl acetate and 1 mol / L dilute hydrochloric acid, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography with petroleum ether and ethyl acetate in a ratio of 3:1 to obtain a light yellow oily intermediate S-2; (6) Synthesis of intermediate Lp-s-1: The intermediate S-2 obtained in step (5) and N, N-diisopropylethylamine are dissolved in anhydrous dichloromethane, and an anhydrous dichloromethane solution of triphosgene is added dropwise at low temperature, wherein the molar ratio of the intermediate S-2, N, N-diisopropylethylamine and triphosgene is 1:0.35:2.0 to 1:0.5:2.0, and the mixture is reacted for 5 hours under a nitrogen atmosphere. The reaction solution is spin-dried and redissolved in dichloromethane, and an anhydrous dichloromethane mixed solution of lapatinib, N, N-diisopropylethylamine and 4-dimethylaminopyridine is added at 0°C, wherein the molar ratio of the intermediate S-2, lapatinib, N, N-diisopropylethylamine and 4-dimethylaminopyridine is 1:1:2:0.5 to 1:1:2:1, and the mixture is reacted at room temperature overnight; the reaction solution is spin-dried, extracted with water and ethyl acetate, and subjected to column chromatography using dichloromethane and methanol 70:1 to obtain a yellow solid Lp-s-1; (7) Synthesis of intermediate Lp-s-2: The intermediate Lp-s-2 obtained in step (6) is dissolved in a mixed solution of methanol and tetrahydrofuran in a volume ratio of 3:1, and a 0.5 mol / L potassium carbonate aqueous solution is added thereto at low temperature, wherein the molar ratio of the intermediate Lp-s-2 to potassium carbonate is 1:1.5, and after stirring at room temperature for 1 hour, the reaction solution is spin-dried, extracted with a saturated sodium chloride solution and ethyl acetate, dried and concentrated with anhydrous sodium sulfate, and purified by column chromatography with dichloromethane and methanol 50:1 to obtain a yellow solid Lp-s-2; (8) Synthesis of target compound Lp-ss-N4: The intermediate Lp-s-2 and N,N-diisopropylethylamine obtained in step (7) are dissolved in anhydrous dichloromethane, and an anhydrous dichloromethane solution of triphosgene is added dropwise at low temperature, wherein the molar ratio of Lp-s-2, N,N-diisopropylethylamine and triphosgene is 1:0.35:2.0 to 1:0.5:2.0, and the mixture is reacted for 5 hours under a nitrogen atmosphere. The reaction solution is spin-dried and redissolved in dichloromethane, and an anhydrous dichloromethane mixed solution of the intermediate N-4, DIPEA and DMAP is added at low temperature, wherein the molar ratio of the intermediate Lp-s-2, intermediate N-4, DIPEA and DMAP is 1:1:2:0.5 to 1:1:2:1, and the mixture is reacted at room temperature overnight; the reaction solution is spin-dried, extracted with water and ethyl acetate, and subjected to column chromatography using dichloromethane and methanol 70:1 to obtain a yellow solid Lp-ss-N4; 3. A drug conjugate, characterized in that: The drug conjugate comprises the oleanolic acid derivative described in claim 1 and lapatinib conjugate.

4. The drug conjugate according to claim 3, characterized in that: The drug conjugate contains a therapeutically effective amount of the oleanolic acid derivative N-4 and the HER2 inhibitor lapatinib Lp-1.

5. Use of the oleanolic acid derivative and lapatinib conjugate according to claim 1 in the preparation of a drug for treating tumor diseases.

6. The use according to claim 5, characterized in that: The tumor disease is breast cancer tumor.

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