Sugar-targeted GSH-responsive paclitaxel derivatives and preparation methods and applications thereof

Abstract

The present invention discloses a paclitaxel derivative, the general structural formula of which is shown below: #imgabs0#R1 is selected from #imgabs1# or #imgabs2#. The paclitaxel derivative of the present invention has a glycosyl target head, which can interact with the glycosyl receptor overexpressed on the surface of tumor cells with high affinity, enter the cells through receptor-mediated internalization, and be reduced and cleaved by glutathione overexpressed in the tumor cells to release the prototype drug, thereby exerting the anti-tumor effect of the drug.

Description

Technical Field

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

[0002] Paclitaxel was first isolated from the bark of Pacific yew (Taxus brevifolia) by American chemists MC Wani and Monre E. Wall. It has been clinically proven to have good anti-tumor effects, especially for ovarian cancer, uterine cancer and breast cancer, which have a high incidence of cancer. On December 29, 1992, the US FDA officially approved paclitaxel as a new anticancer drug for the treatment of advanced ovarian cancer. Paclitaxel is the most popular anticancer drug in the international market in recent years and is considered to be one of the most effective anticancer drugs for humans in the next 20 years. Paclitaxel acts on the microtubule / tubulin system. It can promote the assembly of tubulin into microtubules and inhibit the depolymerization of microtubules, thereby causing abnormal arrangement of vascular bundles, forming astrocytes, causing the hypophysis to lose its normal function, and causing cancer cells to die. Although paclitaxel and its analogs have become new anticancer drugs that are popular all over the world, there are still some problems that cannot be ignored: poor water solubility, oral ineffectiveness and easy to cause allergies. Therefore, it is necessary to conduct structure-activity relationship research and structural modification of paclitaxel to improve the anticancer activity and water solubility of paclitaxel analogs, so as to save and prolong the lives of more patients and benefit mankind.

[0003] Currently, paclitaxel has a variety of dosage forms, including paclitaxel for injection (albumin-bound), paclitaxel microemulsion injection, paclitaxel liposome injection, paclitaxel lipid microsphere injection, and paclitaxel polymer micelle for injection. Although the emergence of these preparations has greatly improved the safety of paclitaxel, they are more expensive than the prototype drug and are not targeted. Direct modification of the paclitaxel structure can greatly improve its water solubility, targeting and anti-tumor activity. At the same time, the introduction of activation-responsive linkers can better release the paclitaxel prototype drug at the tumor site, thereby better exerting the drug activity and reducing the damage of the drug to normal cells. 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. Summary of the invention

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

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

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

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

[0008] The first aspect of the present invention provides a paclitaxel derivative, the general structural formula of which is shown below:

[0009]

[0010] R1 is selected from

[0011] Wherein, n is selected from an integer of 0-2000 (preferably 4);

[0012] R2 is selected from one of the following structures:

[0013]

[0014]

[0015] represents a connecting bond; selected from α or β configuration.

[0016] Most preferably, the paclitaxel derivative is selected from one of the following structures:

[0017]

[0018] The disulfide chain in the paclitaxel derivative of the present invention is broken in the presence of GSH and converted into a paclitaxel derivative with a terminal thiol group, and then a six-membered cyclic compound is formed in the molecule to release the active paclitaxel prototype drug. The specific drug release process is as follows:

[0019]

[0020] The paclitaxel derivative of the present invention is connected with mannose with tumor targeting to form a GSH-responsive anti-tumor prodrug. Triple-negative breast cancer is used as a research object. The paclitaxel derivative targets the mannose receptor on the surface of tumor cells and then is internalized into the cells to exert anti-tumor effects.

[0021] The second aspect of the present invention provides a method for preparing the paclitaxel derivative, comprising the following steps:

[0022]

[0023] The first step is to dissolve compound 1, compound 2 and a base in a solvent at a molar ratio of (1-3):1:(1-3), and react at a temperature of 0-100° C. (preferably 0° C.) to obtain compound 3;

[0024] In the first step, the base is selected from sodium carbonate, potassium carbonate, sodium hydride, etc., preferably sodium hydride.

[0025] In the first step, the solvent is selected from dichloromethane, N,N-dimethylformamide, tetrahydrofuran, etc., preferably tetrahydrofuran.

[0026] The second step is to dissolve compound 3 and phosgene in a molar ratio of (2-5):1 in a solvent, dropwise add triethylamine, the molar ratio of triethylamine to compound 3 is 1:1, and react under ice bath conditions to obtain a newly prepared compound solution;

[0027] Paclitaxel, DMAP, triethylamine and the above-prepared compound solution in a molar ratio of (5-15):1:(5-20):(10-30) were mixed, reacted in an ice bath, and stirred at room temperature overnight to obtain compound 4;

[0028] The solvent in the second step is selected from dichloromethane, N,N-dimethylformamide, tetrahydrofuran, etc., preferably dichloromethane.

[0029] Step 3: Under argon protection, compound 5 and compound 6 in a molar ratio of 1:1 are dissolved in a solvent and mixed, and a boron trifluoride·ethyl ether solution is added under ice bath conditions, wherein the molar ratio of boron trifluoride to compound 5 is 1:(0.5-1). After the addition is complete, the ice bath is removed to obtain compound 7;

[0030] In the third step, the solvent is selected from dichloromethane, N,N-dimethylformamide, tetrahydrofuran, etc., preferably dichloromethane.

[0031] The fourth step is to dissolve a catalytic amount of a base and compound 7 in a solvent and react at a temperature of 0 to 60° C. (preferably 25° C.) to obtain compound 8;

[0032] In the fourth step, the base is selected from potassium hydroxide, sodium methoxide, etc., preferably sodium methoxide.

[0033] In the fourth step, the solvent is selected from dichloromethane, methanol, tetrahydrofuran, etc., preferably methanol.

[0034] The fifth step is to dissolve compound 4, a divalent copper catalyst, compound 8 and sodium ascorbate in a molar ratio of 1:(2-5):(1-3):(5-10) in a solvent and react at a temperature of 0-60° C. (preferably 25° C.) to obtain compound 9.

[0035] The solvent in the fifth step is a mixture of water, methanol and dichloromethane, with water:methanol:dichloromethane=1:5:1.

[0036] In the fifth step, the divalent copper catalyst is selected from copper sulfate pentahydrate.

[0037] The third aspect of the present invention provides a use of the sugar-targeted GSH-responsive paclitaxel derivative in the preparation of a sugar-targeted anti-tumor drug.

[0038] The tumor is selected from lung cancer, colon cancer, ovarian cancer, cervical cancer, triple-negative breast cancer, melanoma, head and neck cancer, brain tumor, lymphoma, leukemia, nasopharyngeal carcinoma or uterine cancer.

[0039] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects:

[0040] The paclitaxel derivative of the present invention has a glycosyl target head, which can interact with the glycosyl receptor overexpressed on the surface of tumor cells with high affinity, enter the cells through receptor-mediated internalization, and is reduced and cleaved by the glutathione overexpressed in the tumor cells to release the prototype drug, thereby exerting the anti-tumor effect of the drug.

[0041] The paclitaxel derivative of the present invention has excellent sugar targeting and GSH responsiveness, and can improve solubility and enhance anti-tumor efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 These are the cytotoxicity test results of the paclitaxel prototype drug in MB231 and HeLa cells.

[0043] Figure 2 The cytotoxicity test results of compound 9 in MB231 and HeLa cells. DETAILED DESCRIPTION

[0044] 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.

[0045] Example 1

[0046] Preparation of compound 3

[0047]

[0048] Add 50 mL of tetrahydrofuran to a 100 mL round-bottom flask, add compound 1 (3.85 g, 25 mmol) and compound 2 (1 mL, 12.5 mmol), then add NaH powder (60 wt%, 1.0 g, 25 mmol) to the reaction system at 0 ° C, and the solution color turns orange. Stir at room temperature for 8 hours, TLC shows that the raw material has almost reacted completely. Add water to quench the reaction, 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 = 4:1) to obtain a light yellow transparent oil, namely compound 3 (2.81 g, 60%). 1 HNMR (600MHz, CDCl3) δ4.19 (d, J=2.3Hz, 2H), 3.90 (t, J=5.8Hz, 2H), 3.80 (t, J= 6.4Hz, 2H), 2.93 (t, J = 6.4Hz, 2H), 2.88 (t, J = 5.8Hz, 2H), 2.46 (t, J = 2.2Hz, 1H).

[0049]

[0050] To a 100 mL round-bottom flask, add 50 mL of anhydrous dichloromethane, compound 3 (270 mg, 1.4 mmol) and phosgene (139 mg, 0.47 mmol), stir for 30 min in an ice bath, add triethylamine (TEA) (141.67 mg, 1.4 mmol) dropwise, and stir for 2 h to obtain a fresh compound solution for use.

[0051] Paclitaxel (600 mg, 0.7 mmol), DMAP (8.55 mg, 0.07 mmol) and triethylamine (99.5 mg, 0.98 mmol) were added to a 100 mL round-bottom flask in sequence, and the above-prepared compound solution was added dropwise under ice bath conditions, the ice bath was removed, and the mixture was stirred at room temperature overnight. The reaction was stopped, water was added, and the mixture was extracted with dichloromethane (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 and purified by column chromatography (dichloromethane: methanol = 50: 1) to obtain a white solid, namely compound 4 (510 mg, 68%). 1HNMR(400MHz,DMSO)δ9.30(d,J=8.5Hz,1H),8.00–7.96(m,2H),7.87–7.83(m,2H),7.72(d,J=7.3Hz,1H),7.65(dd,J=8.2,6.7Hz,2H),7.58–7.53(m,1H),7.52–7.47(m,2H),7.47–7.43(m,4H),7.19(ddd,J=8.8,5.3,3.3Hz,1H),6.29(s,1H),5.82(s,1H),5.54(t,J=8.6Hz,1H),5.41(d,J=7.2Hz,1H),5.36(d,J=8.9Hz,1H),4.99–4.83(m,3H),4.64(s,1H),4.37(td,J=6.0,4.0Hz,2H),4.15(d,J=2.4Hz,1H),4.13(d,J=2.4Hz,2H),4.04–3.97(m,2H),3.67(t,J=6.4Hz,1H),3.65–3.60(m,3H),3.58(d,J=6.8Hz,1H),3.44–3.41(m,1H),2.99(t,J=6.2Hz,2H),2.90(t,J=5.8Hz,3H),2.79(t,J=6.5Hz,1H),2.25(s,2H),2.10(s,3H),1.80–1.77(m,3H),1.51–1.48(m,3H),1.02(s,3H),0.99(s,3H).HRMS(ESI,m / z):[M+Na] + calcdforC 55 H 61 NaNO 17 S2 + 1094.3279;found,1094.3281.

[0052]

[0053] Add 20 mL of anhydrous dichloromethane to a 100 mL round-bottom flask, add compound 5 (1.78 g, 4.56 mmol) and tetraethylene glycol hydroxyl group, i.e. compound 6 (1 g, 4.56 mmol) under argon protection, add boron trifluoride ether solution (426 mg, 3 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 0.5 g of potassium carbonate and continue stirring for 30 min. Stop the reaction, 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, i.e. compound 7 (1.02 g, 40%). 1 HNMR (400MHz, CDCl3) δ5.35(dd,J=10.0,3.4Hz,1H),5.31–5.24(m,2H),4.86(d,J=1.8Hz,1H),4.28(dd,J=12.5,5.2Hz,1H),4.07(td,J=11.4,10.1 ,3.7Hz,2H),3.80(ddt,J=10.4,7.7,3.5Hz,1H),3.66(t,J=3.9Hz,13H),3 .38(t,J=5.0Hz,2H),2.14(s,3H),2.09(s,3H),2.03(s,3H),1.98(s,3H).

[0054] Add 20 mL of methanol to a 100 mL round-bottom flask, add compound 7 (93 mg, 0.10 mmol), and then add a catalytic amount of freshly prepared sodium methoxide solution 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 colorless product, namely compound 8 (37 mg, 98%). 1 HNMR (400MHz, DMSO) δ4.71(dd,J=4.8,3.8Hz,2H),4.61(d,J=1.6Hz,1H),4.56(d,J=5.9Hz,1H),4.43(t,J=6.0Hz,1H),3.69–3.61(m,2H) ,3.61–3.57(m,3H),3.54(q,J=1.9Hz,5H),3.52–3.51(m,4H),3.49–3.45(m,1H),3.45–3.36(m,5H),3.29(ddd,J=11.4,6.0,2.9Hz,2H).

[0055]

[0056] 2 mL of dichloromethane and 10 mL of methanol were added to a 50 mL round-bottom flask, and compound 4 (88 mg, 0.08 mmol), compound 8 (37 mg, 0.1 mmol), copper sulfate pentahydrate (80 mg, 0.32 mmol) and sodium ascorbate (126 mg, 0.64 mmol) were added in sequence, and 2 mL of water was added, and the mixture was stirred at room temperature overnight. TLC showed that the raw materials were complete, the reaction was stopped, water was added, and the liquid was extracted with dichloromethane (3×30 mL), the organic phases were combined, dried over anhydrous Na2SO4, filtered, and the filtrate was vacuum dried, the residue was mixed with silica gel for column chromatography separation and purification, the components were collected, and then vacuum concentrated to obtain a white solid, namely compound 9 (62 mg, 54%). 1 HNMR(400MHz,DMSO)δ9.33(d,J=8.4Hz,1H),8.06(s,1H),7.99(dd,J=7.0,1.6Hz,2H),7.88–7.84(m,2H) ,7.74(t,J=7.3Hz,1H),7.66(t,J=7.4Hz,2H),7.59–7.54(m,1H),7.50(d,J=7.7Hz,2H),7.46(d,J=4.4H z,4H),7.41(d,J=7.6Hz,1H),7.30(d,J=7.8Hz,1H),7.22–7.17(m,1H),6.30(s,1H),5.83(t,J=9.2Hz,1 H),5.76(s,1H),5.54(t,J=8.7Hz,1H),5.42(d,J=7.2Hz,1H),5.38(d,J=9.0Hz,1H),4.93(t,J=9.1Hz,2 H),4.73(dd,J=7.8,4.8Hz,2H),4.66(s,1H),4.63(d,J=1.6Hz,1H),4.58(d,J=5.9Hz,1H),4.51(d,J=5. 4Hz,4H),4.44(t,J=6.0Hz,1H),4.41–4.33(m,2H),4.15–4.07(m,1H),3.81(t,J=5.2Hz,2H),3.68–3.61 (m,4H),3.60–3.57(m,2H),3.56–3.38(m,16H),2.98(t,J=6.1Hz,2H),2.89(t,J=6.3Hz,2H),2.54(s,2H ),2.26(s,3H),2.11(s,3H),1.80(s,3H),1.50(s,3H),1.03(s,3H),1.00(s,3H).HRMS(ESI,m / z):[M+Na] + calcdforC 69 H88 NaNO 26 S2 + 1475.5026; found, 1475.5027.

[0057] Cytotoxicity test

[0058] 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.

[0059] 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 (PTX: 0, 10, 20, 40, 80, 160 nM) were added to the 96-well plate. After incubation for 72 hours, the cell viability was detected using the CCK8 cell proliferation and activity detection kit.

[0060] Figure 1 These are the cytotoxicity test results of the paclitaxel prototype drug in MB231 and HeLa cells. Figure 2 The cytotoxicity test results of compound 9 in MB231 and HeLa cells. Figure 1 and Figure 2 The results showed that the overall cytotoxicity of compound 9 was reduced compared with the paclitaxel prototype drug. The paclitaxel prototype drug was more toxic to Hela cells, and compound 9 was more toxic to MB231 cells, indicating that the mannose target head can effectively target the mannose receptors on the surface of MB231 cells, making compound 9 easier to enter cells than the paclitaxel prototype drug, thereby exerting a cytotoxic effect.

[0061] 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 paclitaxel derivative, characterized in that: The structure of the paclitaxel derivative is shown below:

2. A method for preparing a paclitaxel derivative according to claim 1, characterized in that: The following steps are involved: The first step is to dissolve compound 1, compound 2 and a base in a solvent at a molar ratio of (1-3):1:(1-3), and react at a temperature of 0-100° C. to obtain compound 3; The second step is to dissolve compound 3 and phosgene in a molar ratio of (2-5):1 in a solvent, dropwise add triethylamine, the molar ratio of triethylamine to compound 3 is 1:1, and react under ice bath conditions to obtain a newly prepared compound solution; Paclitaxel, DMAP, triethylamine and the above-prepared compound solution in a molar ratio of (5-15):1:(5-20):(10-30) were mixed, reacted in an ice bath, and stirred at room temperature overnight to obtain compound 4; Step 3: Under argon protection, compound 5 and compound 6 in a molar ratio of 1:1 are dissolved in a solvent and mixed, and a boron trifluoride·ethyl ether solution is added under ice bath conditions, wherein the molar ratio of boron trifluoride to compound 5 is 1:(0.5-1). After the addition is complete, the ice bath is removed to obtain compound 7; Step 4: dissolving a catalytic amount of a base and compound 7 in a solvent and reacting at a temperature of 0 to 60° C. to obtain compound 8; The fifth step is to dissolve compound 4, a divalent copper catalyst, compound 8 and sodium ascorbate in a molar ratio of 1:(2-5):(1-3):(5-10) in a solvent, and react at a temperature of 0-60° C. to obtain compound 9.

3. Use of the paclitaxel derivative according to claim 1 in the preparation of sugar-targeted anti-tumor drugs.

4. The use of the paclitaxel derivative according to claim 3 in the preparation of a sugar-targeted anti-tumor drug, characterized in that: The tumor is selected from lung cancer, colon cancer, ovarian cancer, cervical cancer, triple-negative breast cancer, melanoma, head and neck cancer, brain tumor, lymphoma, leukemia or uterine cancer.

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