Glucose-targeted GSH-responsive maytansine derivative as well as preparation method and application thereof

Abstract

The invention discloses a sugar-targeted GSH (glutathione)-responsive maytansine derivative, the structural general formula of which is as shown in the specification: # imgabs0. The sugar-targeted GSH-responsive maytansine derivative provided by the invention has excellent sugar targeting property and GSH responsiveness, and can improve the solubility and enhance the anti-tumor curative effect at the same time.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug preparation, and in particular, relates to a sugar-targeted glutathione (GSH)-responsive maytansine derivative and a preparation method and application thereof. Background Art

[0002] Maytansine is a natural alkaloid, which was first isolated from Maytansine in 1972 and exists in the genus Maytansine and its relatives in the family Celastraceae. Maytansine has a clear mechanism, acts on microtubules and tubulin, blocks the formation of spindles in the process of cell mitosis by inhibiting the depolymerization of cell microtubules, and has a strong ability to inhibit tumor cell proliferation. Maytansine is the first ansa macrolide antibiotic with antitumor activity. It has strong killing activity for nasopharyngeal carcinoma tumor cells KB cells (EC50 = 8pM), murine lymphocytic leukemia P-388 cells (EC50 = 0.6pM) and murine leukemia L1210 cells (EC50 = 2pM), and has good stability and solubility. However, maytansine has a narrow treatment window and lacks selectivity, and has strong toxic and side effects, such as neurotoxicity and gastrointestinal reactions. It cannot be used alone and is not a true tumor chemotherapy drug. It has been clinically prohibited from being directly used for human treatment. Therefore, it is necessary to conduct structure-activity relationship research and structural modification of maytansine to improve the anti-cancer activity and targeting of maytansine analogs, so as to save and prolong the lives of more patients and benefit mankind.

[0003] In recent years, antibody-drug conjugates (ADCs) formed by connecting antibodies and cytotoxic drugs through appropriate chemical groups have been used for targeted tumor therapy. By coupling cytotoxic molecules to antibodies of tumor-specific antigens, drug molecules are given excellent tumor specificity, which has become a research hotspot in the field of targeted tumor therapy. Among them, ADC drugs with maytansine derivatives as cytotoxic components have been favored by researchers and have made a series of breakthroughs. In 2013, the U.S. Food and Drug Administration (FDA) approved Kadcyla (Ado-trastuzumab Emtansine, T-DM1) through the priority review process for the treatment of HER2-positive advanced (metastatic) breast cancer patients. This is the first ADC drug based on maytansine derivatives approved for marketing. DM-1 is the most commonly used maytansine ADC effector molecule in clinical practice. It has a reactive thiol site at the end, which can form an uncleavable bond with maleimide, and can also react with thiol to form a disulfide bond, which is then reduced and cleaved by endogenous glutathione to form the maytansine DM1 prototype drug. Abnormal glycosylation on the surface of tumor cells leads to overexpression of lectin-like receptors, which have a high affinity for mannose, galactose, fructose and lactose. Targeting these highly expressed sugar transporters and sugar receptors, new tumor imaging and treatment strategies with sugars as targeting ligands can be designed. Direct structural modification of maytansine DM1 by glycochemistry can greatly improve its water solubility and sugar targeting ability, and also improve its anti-tumor activity. The cleavable disulfide bond can ensure its release at the tumor site, thereby reducing systemic toxic side effects. Summary of the invention

[0004] The first object of the present invention is to provide a sugar-targeted GSH-responsive maytansine derivative.

[0005] The second object of the present invention is to provide a method for preparing the sugar-targeted GSH-responsive maytansine derivative.

[0006] The third object of the present invention is to provide a use of the sugar-targeted GSH-responsive maytansine derivative in the preparation of anti-tumor drugs.

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

[0008] In a first aspect of the present invention, a sugar-targeted GSH-responsive maytansine derivative is provided, the general structural formula of which is shown below:

[0009]

[0010] R1 is selected from hydrogen,

[0011]

[0012]

[0013] # indicates the connection position;

[0014] n is selected from an integer of 0-2000 (e.g., 0, 1, 2, 3, 4, 5, 6, 10, 100, 200, 500, 1000).

[0015] The structure of the sugar-targeted GSH-responsive maytansine derivative is shown below:

[0016]

[0017] The second aspect of the present invention provides a method for preparing the sugar-targeted GSH-responsive maytansine derivative, comprising the following steps:

[0018]

[0019] The first step is to dissolve compound 1 and triethylene glycol in a solvent at a molar ratio of 1:(1-4) (preferably 1:3), and add an acidic reagent dropwise under an ice bath condition, wherein the molar ratio of the acidic reagent to compound 1 is 1:1. After the addition is complete, the ice bath is removed and the reaction is carried out at low temperature to obtain compound 2;

[0020] In the first step, the solvent is selected from dichloromethane, N,N-dimethylformamide, and tetrahydrofuran;

[0021] In the first step, the acidic reagent is selected from boron trifluoride etherate and ferric bromide.

[0022]

[0023] The second step is to dissolve compound 2 in a solvent, and add a bromination reagent dropwise under an ice bath condition, wherein the molar ratio of the bromination reagent to compound 2 is (1-2):1 (preferably 1.2:1). After the addition is complete, the ice bath is removed and the reaction is carried out at low temperature to obtain compound 3;

[0024] In the second step, the bromination reagent is selected from phosphorus tribromide and carbon tetrabromide.

[0025] The solvent in the second step is selected from dichloromethane, N,N-dimethylformamide, and tetrahydrofuran.

[0026]

[0027] The third step is to dissolve compound 3 and potassium thioacetate in a solvent at a molar ratio of 1:(1-2) (preferably 1:1.5), and react to obtain compound 4;

[0028] In the third step, the solvent is selected from dichloromethane, N,N-dimethylformamide, and tetrahydrofuran.

[0029]

[0030] Step 4, dissolving compound 4 in a solvent, adding a base, the molar ratio of the base to compound 4 is (5-10):1 (preferably 5:1), and the reaction solution is kept at room temperature overnight to obtain compound 5;

[0031] In the fourth step, the base is selected from potassium hydroxide and sodium methoxide.

[0032] In the fourth step, the solvent is selected from dichloromethane, methanol, and tetrahydrofuran.

[0033]

[0034] Step 5: under argon protection, compound 5 and dithiopyridine in a molar ratio of 1:1 were added to the solvent and stirred overnight to obtain compound 6;

[0035] In the fifth step, the solvent is selected from water, methanol and dichloromethane.

[0036]

[0037] In the sixth step, compound 6 and maytansine are dissolved in a solvent at a molar ratio of 1:(1-20) (preferably 1:10), and the reaction system is stirred at room temperature overnight under argon protection. After the reaction is completed, compound 7 is obtained.

[0038] In the sixth step, the solvent is selected from dichloromethane, methanol, and tetrahydrofuran.

[0039] The third aspect of the present invention provides a use of the sugar-targeted GSH-responsive maytansine derivative in the preparation of an anti-tumor drug.

[0040] The tumor is selected from lung cancer, colon cancer, ovarian cancer, breast cancer, cervical cancer, melanoma, head and neck cancer, brain tumor, lymphoma, leukemia, nasopharyngeal carcinoma, and uterine cancer.

[0041] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects:

[0042] The disulfide bond in the sugar-targeted GSH-responsive maytansine derivative provided by the present invention is broken in the presence of GSH to release the active maytansine DM1 prototype drug.

[0043] The sugar-targeted GSH-responsive maytansine derivative provided by the present invention is simple to synthesize, has excellent sugar targeting and GSH responsiveness, and can improve solubility and enhance anti-tumor efficacy.

[0044] The maytansine derivative provided by the present invention contains a glycosyl target head, which can interact with the glycosyl receptor overexpressed on the surface of tumor cells with high affinity, has a certain targeting, and can enter the cell through receptor-mediated endocytosis, and be reduced and cleaved by glutathione overexpressed in tumor cells, thereby exerting the anti-tumor effect of the drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is a schematic diagram of the cytotoxicity test results of the prototype drug of maytansine DM1 in MB231 and HeLa cells.

[0046] Figure 2 Schematic diagram of the cytotoxicity test results of compound 7 in MB231 and HeLa cells. DETAILED DESCRIPTION

[0047] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. It should be understood by those skilled in the art that the following specific description is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0048] Example 1

[0049] Preparation of compound 2:

[0050]

[0051] Add 20 mL of dichloromethane to a 100 mL round-bottom flask, add compound 1 (3.0 g, 7.69 mmol) and triethylene glycol (3.46 g, 23.06 mmol), add boron trifluoride ether solution (1.09 g, 7.68 mmol) dropwise to the reaction system under ice bath conditions, remove the ice bath after the addition is complete, react at room temperature, and track the reaction progress by TLC. After the reaction is complete, add sodium bicarbonate to neutralize the reaction system and continue stirring for 30 min. Stop the reaction and add water, extract with dichloromethane (3×50 mL), wash the organic phase with water and salt, dry it with anhydrous Na2SO4, filter, and vacuum dry the filtrate. Mix the residue with silica gel for column chromatography separation and purification (petroleum ether: ethyl acetate = 2:1) to obtain a colorless viscous liquid (2.8 g, 5.83 mmol, 76%). 1HNMR(600MHz, CDCl3)δ5.37(dd,J=10.1,3.4Hz,1H),5.30(d,J=10.0Hz,1H),5.28–5.26 (m,1H),4.88(s,1H),4.28(dd,J=12.2,4.9Hz,1H),4.11(dd,J=12.1,2.3Hz,1H),4.09–4 .06(m,1H),3.85–3.80(m,1H),3.73(t,J=4.5Hz,2H),3.67(d,J=6.2Hz,7H),3.61(t,J= 4.5Hz,2H),2.15(s,3H),2.10(s,3H),2.04(s,3H),1.99(s,3H).HRMS(ESI,m / z):[M+Na] + Calculate for C 20 H 32 O 13 Na + ,503.1746,found 503.1741.

[0052] Preparation of compound 3:

[0053]

[0054] Add 20mL of dichloromethane to a 50ml round-bottom flask, add compound 2 (1.8g, 3.75mmol), and dropwise add PBr3 (1.22g, 4.5mmol) dissolved in 10mL of dichloromethane under ice bath conditions. Remove the ice bath after the addition is complete, react at room temperature, and track the reaction progress by TLC. After the reaction is complete, add sodium bicarbonate to neutralize the reaction system and continue stirring for 30min. Stop the reaction and add water, extract with dichloromethane (3×50mL), wash the organic phase with water, wash with salt, dry with anhydrous Na2SO4, filter, and vacuum dry the filtrate. Mix the residue with silica gel for column chromatography separation and purification (petroleum ether: ethyl acetate = 2:1) to obtain a colorless viscous liquid (1.59g, 2.95mmol, 78%). 1H NMR (400MHz, CDCl3) δ5.36(dd,J=10.0,3.4Hz,1H),5.30(d,J=9.9Hz,1H),5.26(dd,J=3.3,1.7Hz,1H),4.87(d,J=1.7Hz,1H),4.33–4.25(m,1H ),4.12–4.03(m,2H),3.82(t,J=6.3Hz,3H),3.67(d,J=4.7Hz,7H),3.48(t,J=6.2Hz,2H),2.15(s,3H),2.10(s,3H),2.04(s,3H),1.99(s,3H). HRMS(ESI,m / z):[M+Na] + Calculate for C 20 H 31 O 12 Br + ,565.0905,found 565.0897. Preparation of compound 4:

[0055]

[0056] Compound 3 (660 mg, 1.22 mmol) and potassium thioacetate (208.0 mg, 1.82 mmol) were dissolved in 25 mL DMF. After the reaction was completed, the reaction solution was extracted with ethyl acetate (3×50 mL). The organic phase was washed with water and salt, dried over anhydrous Na2SO4, filtered, and the filtrate was vacuum dried. The residue was mixed with silica gel for column chromatography separation and purification (petroleum ether: ethyl acetate = 2:1) to obtain a light yellow viscous liquid (578 mg, 1.07 mmol, 88%). 1 H NMR (400MHz, CDCl3) δ5.34(dd,J=10.0,3.4Hz,1H),5.28(d,J=9.9Hz,1H),5.25(dd,J=3.3,1.7Hz,1H),4.86(d,J=1.8Hz,1H),4.28(dd,J=12.4,5.1Hz, 1H),4.11–4.02(m,2H),3.84–3.76(m,1H),3.68–3.57(m,9H),3.08(t,J=6. 4Hz,2H),2.32(s,3H),2.14(s,3H),2.09(s,3H),2.02(s,3H),1.97(s,3H).

[0057] Preparation of compound 5:

[0058]

[0059] Add 20 mL of methanol to a 50 mL round-bottom flask, add compound 4 (275.0 mg, 0.51 mmol), and then add sodium methoxide (137.75 mg, 2.55 mmol) to the reaction system. Under good stirring conditions, the reaction solution was kept at room temperature overnight. The reaction was stopped after TLC showed that the raw materials had reacted completely. The pH of the solution was adjusted to neutral, and the filtrate was dried to obtain a light yellow viscous liquid (164.0 mg, 0.50 mmol, 98%). 1 H NMR(400MHz,MeOD)δ4.80(d,J=1.9Hz,1H),3.87–3.80(m,3H),3.78–3.70(m,2H),3.69–3.66 (m,3H),3.65–3.62(m,5H),3.61–3.56(m,3H),2.66(t,J=6.4Hz,2H).HRMS(ESI,m / z):[M+Na] + Calculate for C 12 H 24 oeLh + ,351.1084,found351.1091.

[0060] Preparation of compound 6

[0061]

[0062] Add 20 mL of anhydrous methanol to a 100 mL round-bottom flask, add compound 5 (91.0 mg, 0.28 mmol) and dithiopyridine (61.0 mg, 0.28 mmol) under argon protection, and stir overnight. After the reaction stops, the solution is vacuum-dried, and the residue is mixed with silica gel for column chromatography separation and purification (petroleum ether: ethyl acetate = 2:1) to obtain a light yellow viscous liquid (578 mg, 1.07 mmol, 88%). 1 H NMR(400MHz,MeOD)δ8.41–8.37(m,1H),7.95(d,J=8.1Hz,1H),7.87–7.81(m ,1H),7.26–7.20(m,1H),4.80(d,J=1.7Hz,1H),3.87–3.83(m,1H),3.82(dd ,J=3.9,1.8Hz,2H),3.71(t,J=6.0Hz,4H),3.67(t,J=4.5Hz,2H),3.64–3.5 9(m,4H),3.58–3.54(m,3H),3.02(t,J=6.0Hz,2H).HRMS(ESI,m / z):[M+Na] + Calculate for C 17 H 28 NO8S2+ ,438.1251,found 438.1257.

[0063] Preparation of compound 7:

[0064]

[0065] Compound 6 (15 mg, 0.03 mmol) and maytansine DM1 (25 mg, 0.3 mmol) were added to a 100 mL round-bottom flask, and 5 mL of anhydrous methanol was added. The reaction system was stirred at room temperature overnight under argon protection. TLC showed that the reaction of the raw materials was complete, the reaction was stopped, and the solution was vacuum dried to obtain a white solid, namely compound 7 (16 mg, 0.01 mmol, 40%). 1 H NMR (400MHz, MeOD) δ7.51–7.33(m,1H),7.13(d,J=1.8Hz,1H),6.69(d,J=1.8Hz,1 H),6.61(dd,J=15.1,11.2Hz,1H),5.70(dd,J=15.1,9.1Hz,1H),5.49(q,J=6.8Hz ,1H),4.79(d,J=1.7Hz,1H),4.67(dd,J=12.1,3.0Hz,1H),4.62(s,1H),4.20(td, J=10.5,4.0Hz,1H),3.98(s,3H),3.86–3.83(m,1H),3.82–3.80(m,2H),3.73–3.6 8(m,3H),3.66(dd,J=5.7,3.5Hz,3H),3.64–3.60(m,5H),3.60–3.53(m,6H),3.37 (s,3H),3.27(s,3H),3.23(d,J=12.4Hz,1H),2.96(q,J=4.9,4.2Hz,3H),2.91(d, J=6.5Hz,1H),2.87(s,3H),2.84–2.67(m,4H),2.15(dd,J=14.3,3.0Hz,1H),1.69 (s,3H),1.57–1.46(m,3H),1.36–1.28(m,6H),1.24(d,J=6.4Hz,3H),0.85(s,3H). HRMS(ESI,m / z):[M+Na] + Calculate for C 47 H 70 N3O 18 S2ClNa + ,1086.3677,found 1086.3683.

[0066] Cytotoxicity test

[0067] The experimental cells were triple-negative breast cancer cells MDA-MB-231 with high expression of mannose receptor and cervical cancer cell line Hela with low expression of mannose receptor as control.

[0068] Experimental method: After the cells were seeded in a transparent 96-well plate (the density of MDA-MB-231 cells was 5000 cells / well, and the density of Hela cells was 4000 cells / well), they were placed in a 37°C incubator containing 5% CO2 and 95% air and cultured overnight until they adhered to the wall. After the growth state was appropriate, the drugs were administered and different concentrations of drugs (DM1 and compound 7: 0, 10, 20, 40, 80, 160, 320 nM) were added to the well plates respectively. After incubation for 72 hours, the cell viability was detected using the CCK8 cell proliferation and activity detection kit.

[0069] Figure 1 This is a schematic diagram of the cytotoxicity test results of the prototype drug of maytansine DM1 in MB231 and HeLa cells. Figure 2 Schematic diagram of the cytotoxicity test results of compound 7 in MB231 and HeLa cells. Figure 1 and Figure 2 The results showed that the overall cytotoxicity of compound 7 was reduced compared with the prototype drug of maytansine. When the concentration was low, compound 7 had a better killing effect on breast cancer cells MDA-MB-231, but when the concentration was greater than or equal to 40nM, the prototype drug DM1 had better toxicity.

[0070] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with this patent can make some changes or modify the technical contents suggested above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the solution of the present invention.

Claims

1. A sugar-targeted GSH-responsive maytansine derivative, characterized in that: The general structural formula is shown below: R1 is selected from hydrogen, # indicates the connection position; n is selected from integers of 0-2000.

2. The sugar-targeted GSH-responsive maytansine derivative according to claim 1, characterized in that The structure of the sugar-targeted GSH-responsive maytansine derivative is shown below:

3. A method for preparing a sugar-targeted GSH-responsive maytansine derivative according to claim 2, characterized in that: The following steps are involved: The first step is to dissolve compound 1 and triethylene glycol in a solvent at a molar ratio of 1:(1-4), and add an acidic reagent dropwise under an ice bath condition, wherein the molar ratio of the acidic reagent to compound 1 is 1:

1. After the addition is complete, the ice bath is removed and the reaction is carried out at low temperature to obtain compound 2; In the first step, the acidic reagent is selected from boron trifluoride etherate and ferric bromide; The second step is to dissolve compound 2 in a solvent, add a bromination reagent dropwise under an ice bath condition, and the molar ratio of the bromination reagent to compound 2 is (1-2):

1. After the addition is complete, remove the ice bath and react at low temperature to obtain compound 3; In the second step, the bromination reagent is selected from phosphorus tribromide and carbon tetrabromide; The third step is to dissolve compound 3 and potassium thioacetate in a solvent at a molar ratio of 1:(1-2), and complete the reaction to obtain compound 4; Step 4, dissolving compound 4 in a solvent, adding a base, the molar ratio of the base to compound 4 being (5-10):1, and allowing the reaction solution to react at room temperature overnight to obtain compound 5; In the fourth step, the base is selected from potassium hydroxide and sodium methoxide; Step 5: under argon protection, compound 5 and dithiopyridine in a molar ratio of 1:1 were added to the solvent and stirred overnight to obtain compound 6; In the sixth step, compound 6 and maytansine in a molar ratio of 1:(1-20) are dissolved in a solvent, and the reaction system is stirred at room temperature overnight under argon protection. After the reaction is completed, compound 7 is obtained.

4. The method for preparing a sugar-targeted GSH-responsive maytansine derivative according to claim 3, characterized in that: In the first step, the solvent is selected from dichloromethane, N,N-dimethylformamide, and tetrahydrofuran.

5. The method for preparing a sugar-targeted GSH-responsive maytansine derivative according to claim 3, characterized in that: The solvent in the second step is selected from dichloromethane, N,N-dimethylformamide, and tetrahydrofuran.

6. The method for preparing a sugar-targeted GSH-responsive maytansine derivative according to claim 3, characterized in that: In the third step, the solvent is selected from dichloromethane, N,N-dimethylformamide, and tetrahydrofuran.

7. The method for preparing a sugar-targeted GSH-responsive maytansine derivative according to claim 3, characterized in that: In the fourth step, the solvent is selected from dichloromethane, methanol, and tetrahydrofuran.

8. The method for preparing a sugar-targeted GSH-responsive maytansine derivative according to claim 3, characterized in that: In the fifth step, the solvent is selected from water, methanol, and dichloromethane; In the sixth step, the solvent is selected from dichloromethane, methanol, and tetrahydrofuran.

9. Use of the sugar-targeted GSH-responsive maytansine derivative according to claim 1 or 2 in the preparation of anti-tumor drugs.

10. The use according to claim 9, characterized in that: The tumor is selected from lung cancer, colon cancer, ovarian cancer, breast cancer, cervical cancer, melanoma, head and neck cancer, brain tumor, lymphoma, leukemia, nasopharyngeal carcinoma, and uterine cancer.

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