A sialic acid-camptothecin prodrug Sia-ss-CPT and a preparation method and application thereof

Abstract

The application discloses a sialic acid-camptothecin prodrug Sia-ss-CPT, and a structural formula of the sialic acid-camptothecin prodrug Sia-ss-CPT is shown in the following formula: The sialic acid-camptothecin prodrug Sia-ss-CPT is prepared by coupling sialic acid and camptothecin, and the water solubility and tumor targeting of free camptothecin are enhanced. Meanwhile, sialic acid is an endogenous substance in the human body, and the bio-compatibility of free camptothecin is enhanced by coupling with sialic acid, and the toxic and side effects on the organism are reduced. The sialic acid and camptothecin are connected by a disulfide bond, and the prodrug can release CPT under glutathione in a tumor site, and has a certain slow-release effect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmaceutical preparations, and particularly relates to a sialic acid-camptothecin prodrug Sia-ss-CPT and a preparation method and application thereof. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing an understanding of the general background of the application and does not necessarily constitute an admission or a recognition that the information forms part of the prior art already known to a person of ordinary skill in the art.

[0003] Cancer is one of the leading causes of human death worldwide, and a huge number of cancer cases and cancer death cases impose a serious social and economic burden on each country. Chemotherapy is an important part of cancer treatment, and many drugs have been approved for clinical chemotherapy of cancer. Unfortunately, cancer chemotherapy is often accompanied by serious adverse reactions, and in most cases, the prognosis of patients is poor. The low tumor targeting of chemotherapeutic drugs, poor water solubility, low bioavailability, large toxic side effects, and even induction of multi-drug resistance in the body greatly limit their application in clinical practice.

[0004] In order to overcome the above-mentioned shortcomings, many researchers have focused on nanocarriers, which can transport anticancer drugs to tumor cells, enhance the accumulation of drugs in tumors, and prevent adverse side effects. However, the biological safety of nanocarriers is still controversial, and their effectiveness is also affected by physical and chemical properties. The characterization, safety issues, regulation, and large-scale manufacturing of nanomedicines further hinder the research and application of cancer nanomedicines. Therefore, it is of great significance to develop new types of chemotherapeutic drug formulations.

[0005] Camptothecin (20-(S)-Camptothecin, CPT) is a pyrroloquinoline cytotoxic alkaloid, and is one of the most studied natural antitumor drugs other than paclitaxel. It has strong antitumor activity against a variety of tumors. Although CPT has excellent antitumor effect, its water insolubility and serious side effects, including myelosuppression, vomiting, diarrhea, and severe hemorrhagic cystitis, greatly limit its clinical application. Further research is needed to solve the water solubility problem of CPT and its derivatives, to improve the lack of targeting of traditional anticancer drugs and the poor bioavailability of oral drugs, and to reduce the side effects of CPT and its derivatives, increase drug utilization, and have better efficacy and drug safety, so as to achieve more remarkable results in treatment and promote the further application of CPT and its derivatives to clinical practice. SUMMARY

[0006] In order to overcome the above problems, the application provides a sialic acid- camptothecin prodrug Sia-ss-CPT and a preparation method and application thereof. The sialic acid- camptothecin conjugate Sia-ss-CPT which can target tumors is synthesized by taking the glycoconjugate on the surface of tumor cells as a target, the water solubility and targeting ability of CPT are enhanced by using sialic acid, so that better tumor inhibition efficacy is generated, and the toxic and side effects of drugs on the body are reduced.

[0007] In order to achieve the above technical purposes, the application adopts the following technical solutions:

[0008] In a first aspect of the application, a sialic acid-camptothecin prodrug Sia-ss-CPT is provided, and the structural formula is as follows:

[0009]

[0010] In a second aspect of the application, a preparation method of the above sialic acid-camptothecin prodrug Sia-ss-CPT is provided, and the method comprises the following steps:

[0011] S1, triphenylphosphine is dissolved in a THF solution, diethyl azodicarboxylate is added dropwise under the condition of 0 DEG C, reaction is carried out for 10 min, solution A is prepared; after sialic acid triethylamine salt is dissolved in DMF, mercaptoacetic acid is added dropwise, solution B is prepared; solution B is added to solution A, stirring reaction is carried out at room temperature for 3-4 h, methanol is added to terminate the reaction, and 9-SAc-Sia is obtained after purification;

[0012] S2, 9-SAc-Sia prepared in S1 is added with sodium methoxide methanol solution under the protection of ice bath and inert gas, neutralization is carried out after a period of reaction, the resin is removed after filtration, and 9-SH-Sia (compound 2) is obtained after the mixed solution is concentrated with water and freeze-dried;

[0013] S3, CTP (compound 3) and dimethylaminopyridine (DMAP) are dissolved in anhydrous dichloromethane under the protection of ice bath and inert gas, solution C is prepared; triphosgene is dissolved in a small amount of anhydrous dichloromethane to prepare solution D; then solution D is added dropwise into solution C, 2-[2-(pyridyl) disulfide]ethanol is added after a period of reaction, and the reaction is carried out overnight at room temperature; after the reaction is completed, compound 4 is obtained after the product is purified;

[0014] S4, 9-SH-Sia prepared in step S2 is dissolved in super-dry methanol to prepare solution E; compound 4 obtained in step S3 is dissolved in anhydrous dichloromethane to prepare solution F, solution F is added dropwise into solution E, stirring reaction is carried out for 10-14 h, and the sialic acid-camptothecin prodrug Sia-ss-CPT is prepared after the product is purified after the reaction is completed;

[0015]

[0016] In a third aspect of the present application, a medicine for treating tumor is provided, which comprises the sialic acid-camptothecin prodrug Sia-ss-CPT described above.

[0017] The present application has the following advantages:

[0018] (1) The present application uses the glycoconjugate on the surface of tumor cells as a target, and synthesizes the sialic acid-camptothecin conjugate Sia-ss-CPT which can target tumor. The sialic acid enhances the water solubility and targeting ability of CPT, thereby producing better tumor inhibition efficacy and reducing the toxic side effects of the drug on the body.

[0019] (2) The present application connects sialic acid and camptothecin through a disulfide bond, so that the prodrug can release CPT in response to glutathione at the tumor site, and has a certain sustained-release effect.

[0020] (3) Sialic acid is an endogenous substance in the human body, and the coupling with sialic acid enhances the biocompatibility of free camptothecin and reduces the toxic side effects on the body. BRIEF DESCRIPTION OF DRAWINGS

[0021] The accompanying drawings, which form a part of this description, are included to provide further understanding of the application and are incorporated in and constitute a part of this description. The illustrative embodiments of the present application and their description serve to explain the present application. They do not, however, limit the present application solely thereto.

[0022] Figure 1 a nuclear magnetic resonance hydrogen spectrum of 9-SH-Sia (compound 2);

[0023] Figure 2 a nuclear magnetic resonance carbon spectrum of 9-SH-Sia (compound 2);

[0024] Figure 3 a nuclear magnetic resonance hydrogen spectrum of compound 4;

[0025] Figure 4 a nuclear magnetic resonance carbon spectrum of compound 4;

[0026] Figure 5 a nuclear magnetic resonance hydrogen spectrum of compound Sia-ss-CPT;

[0027] Figure 6 a nuclear magnetic resonance carbon spectrum of compound Sia-ss-CPT;

[0028] Figure 7 a comparison chart of the stability of CPT and Sia-ss-CPT lactone in Example 1 of the present application;

[0029] Figure 8Figure for the GSH-responsive release of Sia-ss-CPT in Experimental Example 2 of the present invention;

[0030] Figure 9 Figure for the killing efficacy of CPT and Sia-ss-CPT against 4T1 cells in Experimental Example 3 of the present invention;

[0031] Figure 10 Figure for the killing efficacy of CPT and Sia-ss-CPT against EL4 cells in Experimental Example 3 of the present invention;

[0032] Figure 11 Figure for the tumor growth curve of CPT and Sia-ss-CPT treatment in Experimental Example 4 of the present invention;

[0033] Figure 12 Figure for the survival rate of the treatment group of CPT and Sia-ss-CPT in 4T1 tumor model in Experimental Example 4 of the present invention;

[0034] Figure 13 Figure for the actual comparison of the group tumor of CPT and Sia-ss-CPT in 4T1 tumor model in Experimental Example 4 of the present invention;

[0035] Figure 14 Figure for the tumor growth curve of CPT and Sia-ss-CPT treatment in Experimental Example 5 of the present invention;

[0036] Figure 15 Figure for the body weight change of the treatment group of CPT and Sia-ss-CPT in H22 tumor model in Experimental Example 5 of the present invention;

[0037] Figure 16 Figure for the actual comparison of the group tumor of CPT and Sia-ss-CPT in H22 tumor model in Experimental Example 5 of the present invention. DETAILED DESCRIPTION

[0038] It should be noted that the following detailed description is merely exemplary and is intended to provide further description of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0039] It is also important to note that the terms used herein are not intended to limit the exemplary embodiments to the specific embodiments described herein. Rather, the terms are used only to describe specific embodiments and are intended to be interpreted broadly. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof.

[0040] The first typical embodiment of the present application provides a sialic acid-camptothecin prodrug Sia-ss-CPT, which has the following structural formula:

[0041]

[0042] The second typical embodiment of the present application provides a preparation method of the sialic acid-camptothecin prodrug Sia-ss-CPT, which comprises the following steps:

[0043] S1, triphenylphosphine is dissolved in THF solution, diethyl azodicarboxylate is added dropwise at 0°C, and the reaction is carried out for 10 min to prepare solution A; sialic acid triethylamine salt is dissolved in DMF, and then mercaptoacetic acid is added dropwise to prepare solution B; solution B is added to solution A, and the reaction is carried out under stirring at room temperature for 3-4 h, methanol is added to terminate the reaction, and 9-SAc-Sia is obtained after purification;

[0044] S2, 9-SAc-Sia prepared in S1 is added with sodium methoxide methanol solution under ice bath and inert gas protection, and after a period of reaction, DOEWX resin is added for neutralization, the resin is removed by filtration, and after the mixed solution is concentrated with water and freeze-dried, 9-SH-Sia (compound 2) is obtained;

[0045] S3, CTP (compound 3) and dimethylaminopyridine (DMAP) are dissolved in anhydrous dichloromethane under ice bath and inert gas protection to prepare solution C; triphosgene is dissolved in a small amount of anhydrous dichloromethane to prepare solution D; then solution D is added dropwise into solution C, 2-[2-(pyridyl) disulfide]ethanol is added after a period of reaction, and the reaction is carried out overnight at room temperature; after the reaction is completed, compound 4 is obtained after the product is purified;

[0046] S4, 9-SH-Sia prepared in step S2 is dissolved in super-dry methanol to prepare solution E; compound 4 obtained in step S3 is dissolved in anhydrous dichloromethane to prepare solution F, solution F is added dropwise into solution E, and the reaction is carried out under stirring for 10-14 h; after the reaction is completed, the sialic acid-camptothecin prodrug Sia-ss-CPT is prepared after the product is purified;

[0047]

[0048] In one or more embodiments, in step S1, the molar ratio of triphenylphosphine, diethyl azodicarboxylate, sialic acid triethylamine salt and mercaptoacetic acid is 1-2:1-2:0.8-1.2:3-5, and preferably 1.8:1.8:1:4.

[0049] In one or more embodiments, in step S1, the concentration of triphenylphosphine in THF solution is 0.07-0.25 mol / L; preferably 0.18 mol / L.

[0050] In one or more embodiments, in step S1, the concentration of sialic acid triethylamine salt in DMF is 0.05-0.125 mol / L; preferably 0.1 mol / L.

[0051] In one or more embodiments, in step S1, the stirring reaction time is 4 h.

[0052] In one or more embodiments, in step S1, the purification conditions are: the solvent is removed by distillation under reduced pressure, and the product is purified by silica gel column chromatography.

[0053] In one or more embodiments, in step S2, the concentration of sodium methoxide methanol solution is 0.8-1.2 mol / L, preferably 1 mol / L.

[0054] In one or more embodiments, in step S2, the mass of 9-SAc-Sia to the volume of sodium methoxide methanol solution is 0.4-0.6 g: 1 mL, preferably 0.5: 1 mL.

[0055] In one or more embodiments, in step S2, the volume ratio of sodium methoxide methanol solution to water is 1:5-8, preferably 1:6.

[0056] In one or more embodiments, in step S2, the reaction time is 1.5-2.5 h, preferably 2 h.

[0057] In one or more embodiments, in step S3, the molar ratio of CTP, dimethylamino pyridine, triphosgene, 2-[2-(pyridyl) disulfide] ethanol is 0.8-1.2: 1.5-2.5: 0.3-0.5: 0.8-1.2, preferably 1:2:0.35:1.

[0058] In one or more embodiments, in step S3, the concentration of CTP in anhydrous dichloromethane is 0.05-0.12 mol / L, preferably 0.1 mol / L.

[0059] In one or more embodiments, in step S3, anhydrous dichloromethane is added dropwise to triphosgene until triphosgene is just completely dissolved.

[0060] In one or more embodiments, in step S3, solution D is added dropwise to solution C, and the reaction time is 15-25 min, preferably 20 min.

[0061] In one or more embodiments, in step S3, the solution D is added dropwise into the solution C, and the reaction is carried out under ice-bath and inert gas protection.

[0062] In one or more embodiments, in step S3, the method for purification is as follows: the solvent is removed by distillation under reduced pressure, and the product is purified by silica gel column chromatography.

[0063] In one or more embodiments, in step S4, the molar ratio of 9-SH-Sia to compound 4 is 1.5-2.5:0.8-1.2, preferably 2:1.

[0064] In one or more embodiments, in step S4, the concentration of 9-SH-Sia in the super-dry methanol is 0.5-2.5 mol / L, preferably 1 mol / L.

[0065] In one or more embodiments, in step S4, the concentration of compound 4 in the anhydrous dichloromethane is 0.16-0.6 mol / L, preferably 0.5 mol / L.

[0066] In one or more embodiments, in step S4, the reaction is stirred for 12 h.

[0067] In one or more embodiments, in step S4, the method for purification is as follows: the solvent is removed by distillation under reduced pressure, and the product is purified by silica gel column chromatography, then purified by BioGel P-2 gel column, eluted and purified by deionized water to remove silica gel, and then freeze-dried.

[0068] In a third typical embodiment of the present application, a pharmaceutical preparation for resisting tumors is provided, which comprises the above-mentioned sialic acid-camptothecin prodrug Sia-ss-CPT.

[0069] In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in conjunction with specific examples.

[0070] Example 1

[0071] Preparation of compound 2

[0072] To prepare solution A, 1.8 mmol of triphenylphosphine was dissolved in 10 mL of THF solution, and 1.8 mmol of diethyl azodicarboxylate was added dropwise at 0°C, and the reaction was allowed to proceed for 10 min. To prepare solution B, 1 mmol of sialic acid triethylamine salt was dissolved in 10 mL of DMF, and 4 mmol of mercaptoacetic acid was added dropwise. Solution B was added to solution A, and the reaction was allowed to proceed at room temperature for 4 h. The reaction was terminated by adding 0.5 mL of methanol, and the solvent was removed by distillation under reduced pressure. Purification was performed by silica gel column chromatography to obtain 9-SAc-Sia (yield: 54.71%).

[0073] To prepare solution A, 1.8 mmol of triphenylphosphine was dissolved in 10 mL of THF solution, and 1.8 mmol of diethyl azodicarboxylate was added dropwise at 0°C, and the reaction was allowed to proceed for 10 min. To prepare solution B, 1 mmol of sialic acid triethylamine salt was dissolved in 10 mL of DMF, and 4 mmol of mercaptoacetic acid was added dropwise. Solution B was added to solution A, and the reaction was allowed to proceed at room temperature for 4 h. The reaction was terminated by adding 0.5 mL of methanol, and the solvent was removed by distillation under reduced pressure. Purification was performed by silica gel column chromatography to obtain 9-SAc-Sia (yield: 54.71%).

[0074] Figure 1 and Figure 2 are the hydrogen spectrum and carbon spectrum of compound 2, respectively. 1 H NMR (600 MHz, D2O) δ 4.05-3.95 (m, 3H), 3.91 (t, J = 10.2 Hz, 1H), 3.55 (dd, J = 8.2, 1.2 Hz, 1H), 3.25 (dd, J = 14.2, 2.9 Hz, 1H), 2.86 (dd, J = 14.2, 8.6 Hz, 1H), 2.20 (dd, J = 12.9, 4.9 Hz, 1H), 2.06 (s, 3H), 1.82 (dd, J = 12.9, 11.5 Hz, 1H).

[0075] Carbon spectrum 13 C NMR (150 MHz, D2O) δ 187.01, 185.07, 106.80, 80.98, 79.50, 77.65, 70.49, 62.73, 55.45, 49.71, 34.84.

[0076] Example 2

[0077] Preparation of compound 4:

[0078] In an ice bath and under inert gas protection, 1 mmol of CTP (compound 3) was dissolved in 10 mL of anhydrous dichloromethane with 2 mmol of dimethylaminopyridine (DMAP) to prepare solution C; 0.35 mmol of triphosgene was dissolved in a small amount of anhydrous dichloromethane to prepare solution D; then solution D was added dropwise into solution C, and after a period of reaction, 1 mmol of 2-[2-(pyridyl)disulfanyl]ethanol was added, and the reaction was carried out at room temperature overnight; after the reaction was completed, the solvent was removed by distillation under reduced pressure, and the product was purified by silica gel column chromatography to obtain compound 4 with a yield of 65.36%.

[0079] Figure 3 and Figure 4 are the hydrogen spectrum and carbon spectrum of compound 2, respectively, and the hydrogen spectrum 1 H NMR (600 MHz, CDC13) δ 8.42 (dd, J = 4.6, 1.6 Hz, 1H), 8.40 (s, 1H), 8.22 (d, J = 8.5 Hz, 1H), 7.93 (dd, 1H), 7.83 (ddd, J = 8.3, 6.7, 1.4 Hz, 1H), 7.70-7.65 (m, 1H), 7.63-7.61 (m, 2H), 7.33 (s, 1H), 7.03 (td, J = 5.2, 2.9 Hz, 1H), 5.69 (d, J = 17.1 Hz, 1H), 5.39 (d, J = 17.1 Hz, 1H), 5.31-5.27 (m, 2H), 4.41-4.28 (m, 2H), 3.06 (t, J = 6.6 Hz, 2H), 2.29 (dq, J = 14.8, 7.4 Hz, 1H), 2.15 (dq, J = 14.8, 7.5 Hz, 1H), 1.00 (t, J = 7.5 Hz, 3H).

[0080] carbon spectrum 13 C NMR (150 MHz, CDC13) δ 167.43, 159.44, 157.42, 153.52, 152.40, 149.83, 149.01, 146.62, 145.66, 137.30, 131.33, 130.87, 129.81, 128.56, 128.32, 128.31, 128.24, 121.04, 120.42, 119.96, 96.13, 78.14, 67.22, 66.52, 50.13, 36.98, 32.03, 7.76.

[0081] Example 3

[0082] Preparation of sialic acid-camptothecin prodrug Sia-ss-CPT:

[0083] The 2 mmol 9-SH-Sia prepared in step S2 was dissolved in 2 mL of super dry methanol at room temperature to prepare solution E; compound 4 (1 mmol) obtained in step S3 was dissolved in 2 mL of anhydrous dichloromethane to prepare solution F, and solution F was added dropwise into solution E, and the reaction was stirred for 12 h. After the reaction was completed, the solvent was removed by distillation under reduced pressure, and the product was purified by silica gel column chromatography, and then purified by BioGel P-2 gel column eluted with deionized water to remove silica gel, and then freeze-dried.

[0084] Figure 5 and Figure 6 are the hydrogen spectrum and carbon spectrum of sialyl-camptothecin prodrug Sia-ss-CPT, respectively. 1 HNMR (600 MHz, DMSO-d6) δ 8.70 (s, 1H), 8.20 (d, J = 8.5 Hz, 1H), 8.13 (d, J = 8.1 Hz, 1H), 8.04 (d, J = 8.3 Hz, 1H), 7.90-7.84 (m, 1H), 7.72 (t, J = 7..5 Hz, 1H), 5.52 (d, J = 4.1 Hz, 2H), 5.31 (d, J = 2.7 Hz, 2H), 4.36-4.29 (m, 2H), 3.81 (m, 1H), 3.73 (d, J = 10.5 Hz, 1H), 3.69-3.63 (m, 1H), 3.54-3.41 (m, 1H), 3.24-3.20 (m, 1H), 3.15 (dd, J = 13.4, 2.4 Hz, 1H), 2.97 (td, J = 6.1, 3.1 Hz, 2H), 2.79 (dd, J = 13.2, 9.3 Hz, 1H), 2.24-2.12 (m, 2H), 1.96 (dd, J = 13.1, 5.1 Hz, 1H), 1.74 (s, 3H), 1.70 (d, J = 12.2 Hz, 1H), 0.92 (t, J = 7.4 Hz, 3H).

[0085] Carbon spectrum 13 C NMR (150 MHz, DMSO-d6) δ 172.26, 171.77, 167.56, 156.95, 153.26, 152.70, 148.38, 146.68, 145.23, 132.04, 130.91, 130.26, 129.48, 128.96, 128.46, 128.21, 119.57, 95.21, 94.99, 78.35, 71.74, 70.53, 68.32, 66.89, 66.48, 66.07, 53.49, 50.78, 45.81, 36.16, 30.69, 29.46, 22.82, 8.01.

[0086] The reaction equations of Examples 1-3 are as follows:

[0087]

[0088] Experimental Example 1

[0089] Lactone stability assay

[0090] CPT and Sia-ss-CPT were dissolved in DMSO, and the concentration of CPT was indicated. The concentration of CPT and Sia-ss-CPT in DMSO was 1 mg / mL CPT, and they were diluted with pH 7.4 PBS (to 5 μg / mL). After being placed at room temperature for different times, 10 μL of each solution was injected into high performance liquid chromatography (HPLC). The peak area trends of the carboxyl peak and the lactone peak of the CPT moiety were compared over time. The results are shown in Figure 7 Within 0-8 hours, 46.34% of the lactone structure of CPT was hydrolyzed into a carboxylic acid form, while only 4.43% of Sia-ss-CPT was destroyed into a carboxylic acid. After 16 h, the lactone structure of CPT was only 11.57% of the initial content, while the lactone structure of Sia-ss-CPT was still 56.28%. These results show that Sia-ss-CPT can significantly increase the lactone stability of CPT.

[0091] Experimental Example 2

[0092] Responsive release of Sia-ss-CPT

[0093] To evaluate the responsiveness of Sia-ss-CPT to GSH, Sia-ss-CPT was dissolved in a 1 mM glutathione PBS solution to a concentration of 100 μg / mL, and the cleavage of the disulfide bond in Sia-ss-CPT and the responsiveness of CPT release were evaluated by HPLC analysis of the reaction solution at different reaction time points. As shown in Figure 8 In a 1 mM GSH solution, the disulfide bond of Sia-ss-CPT was gradually cleaved, and CPT was slowly released. Without GSH, the cleavage of the disulfide bond of Sia-ss-CPT was reduced. This indicates that Sia-ss-CPT can respond to the reducing properties of GSH at the tumor site, thereby slowly releasing CPT, and has a certain slow-release effect.

[0094] Experimental Example 3

[0095] Evaluation of the toxicity of Sia-ss-CPT to tumor cells

[0096] 4T1 or EL4 tumor cells were added to 96-well plates overnight, 100 μL medium per well. After overnight incubation, 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.4, 0.8, 1, 2, 4 μg of CPT was added to each well, respectively, and 0.002, 0.011, 0.022, 0.111, 0.223, 0.445, 0.891, 1.782, 2.227, 4.454, 8.908 μg of Sia-ss-CPT was added to each well, respectively. After 18 h incubation, blank wells and zero wells were set. After incubation, the medium was removed, and the cells were washed twice with PBS (pH 7.4) preheated at 37 °C to remove CPT or Sia-ss-CPT in the medium. Then 100 μL of medium containing 10 μL of MTT solution (5 mg / mL) was added for 4 h incubation. After 4 h, the medium was removed, 100 μL of DMSO was added, and the mixture was shaken at 37 °C for 10 min to dissolve the formazan completely. The OD value was measured at 570 nm (OD: 0-0.7, >0.7, linear inaccuracy). The results are shown in Figures Figure 9 (4T1), Figure 10 (EL4), which shows that Sia-ss-CPT has an effective killing effect on tumor cells.

[0097] Experimental Example 4

[0098] Tumor inhibition effect of Sia-ss-CPT in 4T1-BALB / c tumor model

[0099] 4T1 tumor cells were inoculated subcutaneously into female BALB / c mice at a dose of 1 x 10 6 cells / 100 μL PBS. When the tumor volume reached 80-100 mm 3 , the tumor-bearing mice were randomly divided into 3 groups, 6 mice in each group. PBS, CPT and Sia-ss-CPT (70 μg CPT / mouse) were injected into the tail vein of the mice on the 1st day and the 3rd day after grouping, respectively. The tumor size was measured every day. After treatment, the mice were euthanized, and the tumors were dissected, weighed, and photographed. The results are shown in Figures Figure 11 , Figure 12 and Figure 13 , which shows that Sia-ss-CPT can significantly inhibit the growth of tumors in mice and significantly improve the survival rate of treated mice.

[0100] Experimental Example 5

[0101] Tumor inhibition effect of Sia-ss-CPT in H22-KM tumor model

[0102] H22 tumor cells were inoculated subcutaneously into female KM mice at a dose of 1 x 10 6cells / 100 μL PBS was inoculated subcutaneously into female H22 mice. 3 Tumor-bearing mice were randomly divided into three groups, with six mice in each group. PBS, CPT, and Sia-ss-CPT (70 μg CPT / mouse) were injected via the tail vein on the first and third days after grouping, respectively. Tumor size and mouse body weight were measured daily, and growth curves were plotted. After treatment, the mice were euthanized, and the tumors were dissected, weighed, and photographed. Figure 14 、 Figure 15 and Figure 16 As shown, Sia-ss-CPT can significantly inhibit the growth of mouse tumors and reduce the toxic side effects of CPT. Therefore, the tumor inhibitory effect of Sia-ss-CPT has a broad spectrum.

[0103] Research has shown that the sialic acid-camptothecin conjugate, Sia-ss-CPT, exhibits superior water solubility and greater lactone stability than free CPT. Furthermore, Sia-ss-CPT exhibits excellent tumor cell-killing potency, significantly inhibiting tumor growth in mice and reducing the toxic side effects of free CPT.

Claims

1. A sialic acid-camptothecin prodrug Sia-ss-CPT, characterized in that Its structural formula is shown below: 。 2. A method for preparing the sialic acid-camptothecin prodrug Sia-ss-CPT according to claim 1, characterized in that: The method comprises the following steps: S1. Dissolve triphenylphosphine in THF solution, add diethyl azodicarboxylate dropwise at 0°C, and react for 10 min to obtain solution A. Dissolve sialic acid triethylamine salt in DMF and add thioglycolic acid dropwise to obtain solution B. Add solution B to solution A, stir at room temperature for 3-4 h, and terminate the reaction by adding methanol dropwise. Purify and obtain 9-SAc-Sia. S2. Add sodium methoxide methanol solution to the 9-SAc-Sia prepared in S1 in an ice bath under inert gas protection. After a period of reaction, add DOEWX resin for neutralization. After filtering to remove the resin, the mixed solution is concentrated with water and then freeze-dried to obtain 9-SH-Sia (compound 2). S3. In an ice bath and under inert gas protection, CTP (Compound 3) and dimethylaminopyridine (DMAP) were dissolved in anhydrous dichloromethane to obtain Solution C. Triphosgene was dissolved in anhydrous dichloromethane to obtain Solution D. Solution D was then added dropwise to Solution C. After a period of reaction, 2-[2-(pyridyl)disulfanyl]ethanol was added and the reaction was allowed to proceed overnight at room temperature. After completion of the reaction, the product was purified to obtain Compound 4. S4. At room temperature, 9-SH-Sia prepared in step S2 was dissolved in ultra-dry methanol to obtain solution E; compound 4 obtained in step S3 was dissolved in anhydrous dichloromethane to obtain solution F, which was added dropwise to solution E, and the mixture was stirred for 10 to 14 hours. After the reaction, the product was purified to obtain sialic acid-camptothecin prodrug Sia-ss-CPT; 。 3. The preparation method according to claim 2, wherein In step S1, the molar ratio of triphenylphosphine, diethyl azodicarboxylate, triethylamine sialic acid and thioglycolic acid is 1-2:1-2:0.8-1.2:3-5.

4. The preparation method according to claim 3, wherein In step S1, the molar ratio of triphenylphosphine, diethyl azodicarboxylate, triethylamine sialic acid and thioglycolic acid is 1.8:1.8:1:

4.

5. The preparation method according to claim 2, wherein The concentration of triphenylphosphine in THF solution is 0.07~0.25 mol / L.

6. The preparation method according to claim 5, wherein The concentration of triphenylphosphine in the THF solution was 0.18 mol / L.

7. The preparation method according to claim 2, wherein The concentration of sialic acid triethylamine salt in DMF is 0.05~0.125mol / L.

8. The preparation method according to claim 7, wherein The concentration of sialic acid triethylamine salt in DMF was 0.1 mol / L.

9. The preparation method according to claim 2, wherein In step S1, the stirring reaction time is 4 hours.

10. The preparation method according to claim 2, wherein In step S1, the purification conditions are as follows: the solvent is removed by distillation under reduced pressure, and the product is purified by silica gel column chromatography.

11. The preparation method according to claim 2, wherein In step S2, the concentration of the sodium methoxide methanol solution is 0.8-1.2 mol / L.

12. The preparation method according to claim 11, characterized in that In step S2, the concentration of the sodium methoxide methanol solution is 1 mol / L.

13. The preparation method according to claim 2, wherein In step S2, the volume ratio of the mass of 9-SAc-Sia to the sodium methoxide methanol solution is 0.4-0.6 g:1 mL.

14. The preparation method according to claim 13, wherein In step S2, the volume ratio of the mass of 9-SAc-Sia to the sodium methoxide methanol solution is 0.5 g:1 mL.

15. The preparation method according to claim 2, wherein In step S2, the volume ratio of the sodium methoxide methanol solution to water is 1:5-8.

16. The preparation method according to claim 15, characterized in that In step S2, the volume ratio of the sodium methoxide methanol solution to water is 1:

6.

17. The preparation method according to claim 2, wherein In step S2, the reaction time is 1.5 to 2.5 hours.

18. The preparation method according to claim 17, wherein In step S2, the reaction time is 2 hours.

19. The preparation method according to claim 2, wherein In step S3, the molar ratio of CTP, dimethylaminopyridine, triphosgene, and 2-[2-(pyridyl)disulfide]ethanol is 0.8-1.2:1.5-2.5:0.3-0.5:0.8-1.

2.

20. The preparation method according to claim 19, wherein The molar ratio of the CTP, dimethylaminopyridine, triphosgene, and 2-[2-(pyridyl)disulfide]ethanol is 1:2:0.35:

1.

21. The preparation method according to claim 2, wherein In step S3, the concentration of CTP in anhydrous dichloromethane is 0.05-0.12 mol / L.

22. The preparation method according to claim 21, wherein In step S3, the concentration of CTP in anhydrous dichloromethane is: 0.1 mol / L.

23. The preparation method according to claim 2, wherein In step S3, the concentration of triphosgene in anhydrous dichloromethane is 0.02-0.05 mol / L.

24. The preparation method according to claim 2, wherein In step S3, solution D is added dropwise to solution C, and the reaction time is 15 to 25 minutes.

25. The preparation method according to claim 24, characterized in that In step S3, solution D is added dropwise to solution C, and the reaction time is 20 minutes.

26. The preparation method according to claim 2, wherein In step S3, solution D is added dropwise to solution C, and the reaction is carried out in an ice bath under the protection of an inert gas.

27. The preparation method according to claim 2, wherein In step S3, the purification method is: removing the solvent by distillation under reduced pressure, and purifying the product by silica gel column chromatography.

28. The preparation method according to claim 2, wherein The molar ratio of 9-SH-Sia to compound 4 is 1.5~2.5:0.8~1.

2.

29. The preparation method according to claim 28, characterized in that The molar ratio of 9-SH-Sia to compound 4 was 2:

1.

30. The preparation method according to claim 2, wherein The concentration of 9-SH-Sia in ultra-dry methanol is 0.5~2.5 mol / L.

31. The preparation method according to claim 30, wherein The concentration of 9-SH-Sia in ultra-dry methanol was 1 mol / L.

32. The preparation method according to claim 2, wherein The concentration of compound 4 in anhydrous dichloromethane is 0.16~0.6mol / L.

33. The preparation method according to claim 32, wherein The concentration of compound 4 in anhydrous dichloromethane was 0.5 mol / L.

34. The preparation method according to claim 2, wherein In step S4, the reaction was stirred for 12 hours.

35. The preparation method according to claim 2, wherein In step S4, the purification method is as follows: the solvent is removed by distillation under reduced pressure, the product is purified by silica gel column chromatography, then passed through a BioGel P-2 gel column, eluted with deionized water to purify the silica gel, and then freeze-dried.

36. An anti-tumor pharmaceutical preparation, characterized in that: The pharmaceutical preparation comprises the sialic acid-camptothecin prodrug Sia-ss-CPT according to claim 1 or the sialic acid-camptothecin prodrug Sia-ss-CPT prepared by the preparation method according to any one of claims 2 to 35.

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