Human serum albumin covalent binding type targeted folate receptor micromolecule drug conjugate prodrug and application thereof

Abstract

The invention belongs to the field of biological medicine, and relates to a human serum albumin covalent binding type targeted folate receptor micromolecule drug conjugate prodrug and application thereof. The prodrug disclosed by the invention has a structure as shown in a formula I, six-carbon maleimide is used as a site combined with endogenous albumin, and albumin is used as a carrier to deliver the drug. The prodrug can increase accumulation of the drug in tumors under the EPR effect. A dipeptide chain composed of valine and alanine is used as a cleavage site of cathepsin B, an adapter composed of p-aminobenzyl ether and N-methylamino-4-nitrophenol releases drugs in an acidic environment, an azidohexanoic acid structure is added to a side chain of p-aminobenzyl ether, so that the site of a targeting ligand can be increased, and SN38 is used as a small molecule cytotoxic load. The micromolecular prodrug has dual functions of active targeting and passive targeting, and can effectively improve the concentration in solid tumors, reduce toxic and side effects and improve the anti-tumor effect under the mediation of related receptors.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a human serum albumin covalently bound folate receptor targeted small molecule drug conjugate prodrug and application thereof. Background Art

[0002] Cancer is one of the malignant diseases that threaten human health and survival. Traditional chemotherapy drugs are not selective for tumors, do not preferentially target tumor sites, and are highly toxic to healthy tissues and organs of the human body. With the development of science and technology, targeted delivery of highly effective chemotherapy drugs has been established as an efficient method for treating tumors. Antibody-drug conjugates (ADCs) are a very promising new class of anticancer drugs. Currently, two ADCs, Trastuzumab Emtansine and Brentuximab vedotin, have achieved great success on the market. SMDC (small molecule drug conjugates) are an alternative to ADCs for targeted delivery of cytotoxic drugs to malignant tissues, allowing better enrichment of drugs in solid tumors.

[0003] In recent years, some new drug carriers have been developed as transport carriers for chemotherapy drugs, which can significantly improve the physical and chemical properties of drugs, reduce drug toxicity, improve tumor targeting, and increase the anti-tumor efficacy of drugs. In addition to exogenous drug carriers such as liposomes and chitosan that can form micelles and nanoparticles, human serum albumin (HSA) has been found to be a class of drug carriers with great potential.

[0004] Human serum albumin (HSA) is the most abundant protein in the human body, with a concentration of 35-50g / L. It is a good endogenous protein with reduced immunogenicity and a half-life of up to 19 days. In addition, HSA is a natural carrier of many substances in the body, and can transport some fatty acids, amino acids, metabolites, etc. HSA has cysteine ​​residues and can react with maleimide to form a covalent conjugate by Michael addition reaction. This albumin drug delivery system can increase tumor accumulation through the tumor EPR effect and some albumin-specific receptors.

[0005] Folic acid (FA) (vitamin B9) is a vitamin essential for the proliferation and maintenance of all cells. It is essential for the de novo production of RNA and DNA. Since FA is a limiting nutrient, upregulation of FR-α on the surface of cancer cells can compete more actively for folate. FR-α has been found to be overexpressed in breast cancer, head and neck cancer, ovarian cancer, lung cancer, endometrial cancer, bladder cancer, pancreatic cancer, colon cancer and kidney cancer. The overexpression of FR-α on cancer cells may be the result of their increased demand for folate. Oxidized folate (FA) shows the highest affinity for membrane-bound FR. This property remains unchanged even when drug payloads are immobilized on it to form FA-SMDC.

[0006] 7-Ethyl-10-hydroxycamptothecin (SN38) is an active derivative of the natural drug camptothecin and an active metabolite of the marketed drug irinotecan hydrochloride. Its mechanism of action is consistent with camptothecin, inhibiting topoisomerase I, thereby inhibiting DNA replication and leading to cell apoptosis. However, SN38 is highly toxic and has a higher anti-tumor efficacy, but SN38 has extremely low solubility and no targeting, which greatly limits its clinical application.

[0007] Cathepsin B is a lysosomal cysteine ​​protease that is widely present in mammalian cells. It is mainly involved in the degradation and metabolism of proteins in cells, and also plays an important role in processes such as extracellular matrix remodeling, inflammatory response, and tumorigenesis. Cathepsin B is highly active in acidic environments (such as lysosomes and tumor microenvironments), so it is widely used in the design of drug delivery systems. Tumor tissues usually present an acidic microenvironment, and cathepsin B is overexpressed in tumor cells. Taking advantage of this property, delivery systems sensitive to cathepsin B can be designed to achieve tumor-specific drug release. Cathepsin B can specifically cleave certain chemical bonds (such as peptide bonds) and convert prodrugs into active drugs, thereby increasing drug concentrations at the tumor site and reducing systemic toxicity.

[0008] Therefore, it is particularly important to develop a new anti-tumor drug system that improves the physicochemical properties of drugs and reduces toxicity, thereby improving drug application. Summary of the invention

[0009] The purpose of the present invention is to improve the drug properties of small molecule drug conjugates and enhance tumor efficacy, and to propose a human serum albumin covalently bound small molecule drug conjugate prodrug targeting folate receptor and its application. The small molecule drug conjugate of the present invention is covalently bound to human serum albumin via maleimide. After intravenous injection, it reacts with the residual thiol group of albumin to form a macromolecular drug-carrying system, which increases the half-life of the drug, that is, prolongs the circulation in the body, and improves the anti-tumor efficacy.

[0010] The second object of the present invention is to provide a pharmaceutical composition containing the above-mentioned prodrug.

[0011] The third object of the present invention is to provide the use of the above-mentioned medicine.

[0012] The present invention achieves the above object through the following technical solutions:

[0013] In the first aspect of the present invention, a human serum albumin covalently bound small molecule drug conjugate prodrug targeting folate receptor is provided, which has a structure as shown in Formula I:

[0014]

[0015] The small molecule drug conjugate prodrug of the present invention is Mal-VA-FA-SN38, which has a structure as shown in Formula I, and is composed of a VA dipeptide chain composed of maleimide, valine and alanine, an aptamer, an SN38 cytotoxic drug, folic acid, and a linker for connecting folic acid to other parts. The functions of each part are described as follows:

[0016] 1. Maleimide: used for Michael addition to human serum albumin;

[0017] 2. The VA dipeptide chain composed of valine and alanine acts as a trigger;

[0018] 3. Aptamers: used to connect drug triggers and targeting ligands and improve the stability of drugs in plasma;

[0019] 4. SN38: is a cytotoxic drug;

[0020] 5. Folic acid (FA): acts as a targeting ligand and binds to cell surface receptors;

[0021] 6. Linker: used to connect folic acid to other parts of the compound and make folic acid flexible.

[0022] The hexacarbon maleimide is used as a binding site for endogenous albumin, and albumin is used as a carrier to deliver the drug. The prodrug can increase the accumulation of the drug in the tumor under the EPR effect. The dipeptide chain composed of valine and alanine serves as the cleavage site of cathepsin B. The adapter composed of p-aminobenzyl ether and N-methylamino-4-nitrophenol releases the drug in an acidic environment. The addition of azidohexanoic acid structure to the side chain of p-aminobenzyl ether can increase the site of the targeting ligand, and SN38 serves as a small molecule cytotoxic payload. The small molecule prodrug has the dual functions of active targeting and passive targeting. Under the mediation of relevant receptors, it can effectively increase the concentration in solid tumors while reducing toxic side effects and improving the anti-tumor effect.

[0023] In the second aspect of the present invention, a pharmaceutical composition is provided, comprising the human serum albumin covalently bound folate receptor targeted small molecule drug conjugate prodrug or a pharmaceutically acceptable salt thereof as described in the first aspect, and one or more pharmaceutical carriers. The macromolecular carrier may be human serum albumin and liposome.

[0024] In the third aspect of the present invention, there is provided a use of the human serum albumin covalently bound folate receptor targeted small molecule drug conjugate prodrug or a pharmaceutically acceptable salt thereof as described in the first aspect in the preparation of a human serum albumin covalently bound anti-tumor drug. The tumors include ovarian cancer, oral cancer, breast cancer, lung cancer, endometrial cancer, kidney cancer, gastric cancer, lung cancer, colorectal cancer, etc.

[0025] In the fourth aspect of the present invention, there is provided a use of the human serum albumin covalently bound folate receptor targeting small molecule drug conjugate prodrug or a pharmaceutically acceptable salt thereof as described in the first aspect in the preparation of an anti-tumor drug. The tumors include ovarian cancer, oral cancer, breast cancer, lung cancer, endometrial cancer, kidney cancer, gastric cancer, lung cancer, colorectal cancer, etc.

[0026] In the fifth aspect of the present invention, there is provided a use of the pharmaceutical composition as described in the second aspect in the preparation of a human serum albumin covalently bound anti-tumor drug. The tumors include ovarian cancer, oral cancer, breast cancer, lung cancer, endometrial cancer, kidney cancer, gastric cancer, lung cancer, colorectal cancer, etc.

[0027] In the sixth aspect of the present invention, the use of the pharmaceutical composition according to the second aspect in the preparation of an anti-tumor drug is provided. The tumors include ovarian cancer, oral cancer, breast cancer, lung cancer, endometrial cancer, kidney cancer, gastric cancer, lung cancer, colorectal cancer, etc.

[0028] The present invention has the following beneficial effects:

[0029] The present invention provides a new small molecule drug conjugate prodrug, which is composed of SN38, an aptamer, a trigger, a folic acid ligand, and a hexacarbon maleimide. The maleimide in the prodrug can be covalently bound to human serum albumin, enter the tumor interstitium through the tumor EPR effect and the related receptors of albumin, and achieve drug accumulation. The half-life in vivo is extended to a certain extent, and the physicochemical properties are also improved to a certain extent compared with SN38, and the anti-tumor effect is more effective and the side effects are smaller. It can be seen from the results of Examples 2 and 3 of the present invention that the prodrug compound of the present invention has high stability in PBS solution and plasma at pH 7.4. It can be seen from the results of Example 4 of the present invention that the prodrug is very stable and has little cytotoxicity without drug release; after adding cathepsin B, the cytotoxic drug SN38 can be effectively released, and the anti-tumor effect is much greater than that of the SN38 and Irinotecan groups. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a stability chart of the prodrug compound prepared in Example 1 in PBS and mouse plasma; wherein “ / ” represents that SN38 was not detected.

[0031] Figure 2 This is the enzymatic test curve of the prodrug compound prepared in Example 1.

[0032] Figure 3 This is a schematic diagram of the structure of the human serum albumin covalently bound folate receptor targeted small molecule drug conjugate prodrug of the present invention, wherein the red part is maleimide; the cyan part is the VA dipeptide chain composed of valine and alanine; the blue part is the aptamer; the green part is SN38; the pink part is folic acid (FA); and the black part is a linker for connecting folic acid to other parts. DETAILED DESCRIPTION

[0033] The present invention is further described in detail with reference to the following specific embodiments and drawings, which do not limit the present invention.

[0034] Example 1 Preparation of compound Mal-VA-FA-SN38

[0035] The synthetic route is as follows:

[0036]

[0037]

[0038] The specific synthesis steps are as follows:

[0039] Preparation of compound 2: Compound 1 (15 g, 83.8 mmol) was dissolved in 30% methylamine ethanol solution (69.3 g, 669.7 mmol) and reacted at room temperature overnight. The reaction was complete after monitoring by TLC plate. The solid was filtered and washed twice with a small amount of ethanol. The white solid was dried under vacuum to obtain 16 g (yield 91%). LC-MS (m / z) (ESI), calculated for C9H 10 N2O4:210.06; found [M+H] + :211.1.

[0040] Preparation of compound 3: Compound 2 (16 g, 76.2 mmol) was dissolved in 200 mL of anhydrous tetrahydrofuran, and borane dimethyl sulfide (2.0 M in THF) (76.2 mL, 152.4 mmol) was slowly added under nitrogen protection and ice bath conditions, and the ice bath was removed, and the reaction was heated to 70°C. After 5 hours, the reaction was monitored by TLC plate. Methanol was slowly added at 0°C to quench the unreacted borane dimethyl sulfide, and 4M HCl (34.2 mL, 136 mmol) was added. The reaction was carried out at 60°C for 12 hours, and the reaction mixture was cooled to 0 degrees Celsius, cooled and crystallized, filtered, and dried under vacuum to obtain 13 g of a white solid (yield 88%). LC-MS (m / z) (ESI), calculated for C9H 12 N2O3:196.08; found [M+H] + :197.08.

[0041] Preparation of compound 4: Compound 3 (13 g, 66.3 mmol) was dissolved in anhydrous dichloromethane, triethylamine (11 mL, 79.2 mmol) was added, and Boc anhydride (15.9 g, 72.9 mmol) was slowly added under ice bath. After 5 h, the reaction was complete as monitored by TLC plate. The reaction solution was concentrated under reduced pressure, washed with anhydrous ammonium chloride, extracted with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 18.7 g of yellow viscous solid (yield 95%). LC-MS (m / z) (ESI), calculation for C 14 H 20 N2O5:296.14; found [M+H] + :297.14.

[0042] Preparation of compound 5: Compound 4 (18 g, 60.8 mmol) was dissolved in anhydrous dichloromethane, triethylamine (11 mL, 79.2 mmol) and dimethylaminopyridine (11.1 g, 90.9 mmol) were added, and tert-butyldimethylsilyl chloride (20 g, 72.8 mmol) was added under ice bath. After 6 h, the reaction was complete as monitored by TLC plate, and column chromatography PE:EA=100:1 to 20:1 was used for purification to obtain 23.7 g of white solid (yield 95%). LC-MS (m / z) (ESI), calculated for C 20 H 34 N2O5Si:410.22; found [M+H] + :411.22.

[0043] Preparation of compound 6: Compound 5 (20 g, 48.8 mmol) was dissolved in methanol, 10% palladium carbon (2 g) was added, and the mixture was reacted for 3 h under hydrogen atmosphere. The reaction was complete after monitoring by TLC plate, and the mixture was filtered and concentrated under reduced pressure to obtain 16.7 g of yellow oil (yield 90%). LC-MS (m / z) (ESI), calculated for C 20 H 36 N2O3Si:380.25; found [M+H] + :381.25.

[0044] Preparation of compound 7: Alanine (2.24 g, 25 mmol) was dissolved in 20 mL of tetrahydrofuran and water, sodium bicarbonate (2.16 g, 25 mmol) was added and stirred for 20 minutes, compound 7a (10 g, 2.3 mmol) was added and reacted for 12 hours, after the reaction, part of the solvent was dried, 1 M hydrochloric acid was added, the solid was filtered out, the solid was slurried with PE:EA=2:1, slurried twice, slurried for 3 hours each time, the solid was filtered, and the solvent was removed under reduced pressure to obtain 9 g of white solid (yield 95%). LC-MS (m / z) (ESI), calculated for C 23 H 26 N2O5:410.18; found [M+H] + :411.18.

[0045] Preparation of compound 8: Compound 6 (15 g, 39.5 mmol) and compound 7, i.e., Fmoc-Val-Ala-OH (17.8 g, 43.4 mmol) were dissolved in DMF, tetramethylchloroformamidine hexafluorophosphate (16.6 g, 59.2 mmol) and N-methylimidazole (12.6 mL, 158.3 mmol) were added, and the mixture was reacted at room temperature for 12 h. The reaction was complete after TLC monitoring, and the mixture was purified by column chromatography with DCM:MeOH = 100:1 to 80:1 and dried to obtain 12.2 g of a yellow-white solid (yield 40%). LC-MS (m / z) (ESI), calculated for C 43 H 60 N4O7Si:772.42; found [M+H] + :773.42.

[0046] Preparation of compound 9: Compound 8 (12 g, 15.6 mmol) was dissolved in anhydrous dichloromethane, and pyridine hydrogen fluoride solution (3 mL, 31.3 mmol) was added at 0°C. After 30 minutes, the reaction was monitored by spot plate. Column chromatography DCM:MeOH = 100:1 to 50:1 was used for purification to obtain 6.1 g of a white solid (yield 60%). LC-MS (m / z) (ESI), calculated for C 37 H 46 N4O7:658.34; found [M+H] + :659.34.

[0047] Preparation of compound 10: N-bromosuccinimide (2.1 g, 11.8 mmol) and triphenylphosphine (3.1 g, 11.8 mmol) were dissolved in anhydrous tetrahydrofuran and stirred for 20 min, compound 9 (6 g, 9.1 mmol) was added, and the reaction was complete after 3 h of TLC plate monitoring, and column chromatography DCM to DCM: EA = 80: 1 was used for purification to obtain 5.3 g of yellow solid (yield 80%). LC-MS (m / z) (ESI), calculation for C 37 H 45 BrN4O6:720.25; found [M+H] + :721.25.

[0048] Preparation of compound 11: Compound 11a (2 g, 11.4 mmol) was dissolved in 10 mL of water and then adjusted to pH 3-4 with 1 M hydrochloric acid, extracted with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate. The organic phase was spin-dried to obtain 1.4 g of dark brown solid 11b. Formic acid (4.2 g, 91.3 mmol) and acetic anhydride (10 eq, 9.3 g, 91.1 mmol) were mixed and heated at 70°C for 2 hours, cooled to room temperature, and a tetrahydrofuran solution of compound 11b (1.4 g, 9.2 mmol) was added. The reaction was carried out at room temperature for 3 hours, and the reaction solvent was removed under reduced pressure. The product was dissolved with a small amount of dichloromethane, and the solution was precipitated in n-hexane. The product was filtered to obtain 1.2 g of a light yellow solid (yield 72%). LC-MS (m / z) (ESI), calcd for C7H6N2O4: 182.03; found [M+H] + :183.03.

[0049] Preparation of compound 12: Compound 10 (5 g, 6.9 mmol), silver oxide (1.9 g, 8.2 mmol), and compound 11 (1.3 g, 6.9 mmol) were dissolved in anhydrous tetrahydrofuran, and the reaction was carried out overnight in the dark. The reaction was monitored by TLC plate, and the column chromatography DCM to DCM:MeOH=80:1 was used for purification to obtain 3.4 g of yellow solid (yield 60%). LC-MS (m / z) (ESI), calculation for C 44 H 50 N6O 10 :822.36; found [M+H] + :823.36.

[0050] Preparation of compound 13: Under nitrogen protection, compound 12 (3 g, 3.6 mmol) was dissolved in anhydrous tetrahydrofuran, and borane dimethyl sulfide (3.6 mL, 7.3 mmol) was slowly added. After 4 h, the reaction was complete as monitored by TLC plate, and 2.4 g of yellow solid was purified by column chromatography from DCM to DCM: MeOH = 80:1 (yield 80%). LC-MS (m / z) (ESI), calculation for C 44 H 52 N6O9:808.38; found [M+H] + :809.38.

[0051] Preparation of compound 14: Compound 13 (2 g, 2.5 mmol) was dissolved in anhydrous DCM under nitrogen protection, N,N-diisopropylethylamine (1.8 mL, 9.9 mmol) and triphosgene (0.4 g, 1.5 mmol) were added, and the reaction was complete after 30 min of TLC monitoring. The reaction was directly carried out in the next step without any treatment. LC-MS (m / z) (ESI), calculation for C45 H 51 ClN6O 10 :870.34; found[M+H] + :871.34.

[0052] Preparation of compound 15: Dimethylaminopyridine (0.9 g, 7.4 mmol) and SN38 (1 g, 2.5 mmol) were added to the reaction system in the previous step and reacted overnight. The reaction was complete when monitored by TLC plate. The product was directly used for the next step without purification. LC-MS (m / z) (ESI), calculation for C 67 H 70 N8O 15 :1226.50; found[M+H] + :1227.50.

[0053] Preparation of compound 16: Compound 15 (2 g, 1.6 mmol) was dissolved in anhydrous dichloromethane with a concentration of 20% trifluoroacetic acid. After reacting for 3 h, the reaction was monitored by TLC plate. Column chromatography DCM to DCM:MeOH = 50:1 was used for purification to obtain 1.7 g of a yellow solid (yield 95%). LC-MS (m / z) (ESI), calculated for C 62 H 62 N8O 13 :1126.44; found[M+H] + :1127.44.

[0054] Preparation of compound 17: 6-aminohexanoic acid (2 g, 15.2 mmol), potassium carbonate (3.6 g, 25.9 mmol) and copper sulfate pentahydrate (38 mg, 0.2 mmol) were dissolved in methanol, 1H-imidazole-1-sulfonyl azide (3.8 g, 18.3 mmol) was added under ice bath, and the ice bath was removed and the reaction was allowed to react overnight at room temperature. The reaction was monitored by TLC plate, and the solvent was removed under reduced pressure. 75 mL of water was added to the reaction system, and 2.5 mL of concentrated hydrochloric acid was added to acidify the residue. The mixture was extracted with ether in small amounts several times. The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure to obtain 1.7 g of a transparent oily liquid (yield 70%). LC-MS (m / z) (ESI), calculated for C6H 11 N3O2:157.09; found [M+H] + :158.09.

[0055] Preparation of compound 18: Compound 17 (0.3 g, 2.0 mmol), 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (0.4 g, 2 mmol), N-hydroxysuccinimide (0.2 g, 2 mmol) were dissolved in anhydrous dichloromethane, and compound 16 (1.5 g, 1.3 mmol) and triethylamine (0.2 mL, 1.7 mmol) were added after 4 h, and the reaction was allowed to proceed overnight. The reaction was monitored by TLC plate, and the column chromatography DCM to DCM: MeOH = 50:1 was used for purification to obtain 1.2 g of a yellow solid (yield 70%). LC-MS (m / z) (ESI), calculation for C 68 H 71 N 11 O 14 :1265.52; found[M+H] + :1266.52.

[0056] Preparation of compound 19: Compound 18 (1 g, 0.8 mmol) was dissolved in a 20% diethylamine solution in anhydrous tetrahydrofuran. After 3 h, the reaction was complete as monitored by TLC plate. Column chromatography DCM to DCM:MeOH = 50:1 was used for purification to obtain 0.8 g of a yellow solid (yield 95%). LC-MS (m / z) (ESI), calculated for C 53 H 61 N 11 O 12 :1043.45; found[M+H] + :1044.45.

[0057] Preparation of compound 23: Compound 22 (5.0 g, 33.7 mmol) was dissolved in 100 mL of anhydrous DCM, triethylamine (4.7 mL, 33.7 mmol) was added, and Boc anhydride (6.1 g, 27.9 mmol) was slowly added dropwise to the reaction system under ice bath conditions. After 6 h, the reaction was complete as monitored by TLC plate, and column chromatography DCM to DCM: MeOH = 20:1 was used for purification to obtain 3.5 g of colorless transparent liquid (yield 50%). LC-MS (m / z) (ESI), calculation for C 11 H 24 N2O4:248.17; found [M+H] + :249.17.

[0058] Preparation of compound 24: 5-hexynoic acid (1.5 g, 13.3 mmol) was dissolved in 50 mL of anhydrous DCM, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.8 g, 14.5 mmol) and N-hydroxysuccinimide (1.7 g, 14.5 mmol) were added, and the mixture was reacted at room temperature for 30 min. After the reaction of the raw materials was completed as monitored by TLC plate, compound 23 (3.0 g, 12.1 mmol) and N, N-diisopropylethylamine (3.2 mL, 18.1 mmol) were added, and the mixture was reacted at room temperature overnight. The reaction of compound 23 was completed as monitored by TLC plate. Column chromatography DCM to DCM: MeOH = 40: 1 was used for purification to obtain 2.5 g of a colorless transparent liquid (yield 60%). LC-MS (m / z) (ESI), calculation for C 17 H 30 N2O5:342.22; found [M+H] + :343.22.

[0059] Preparation of compound 25: Compound 24 (2.0 g, 5.8 mmol) was dissolved in 20 mL of anhydrous DCM, 10 mL of 4 M HCl (dissolved in 1,4-dioxane) was added, and the reaction was carried out at room temperature for 2 h. The reaction was completed after monitoring by TLC plate. The solvent was removed under vacuum to obtain 1 g of colorless transparent liquid (yield 70%), which was directly used for the next step without purification. LC-MS (m / z) (ESI), calculated for C 12 H 22 N2O3:242.16; found [M+H] + :243.16.

[0060] Preparation of compound 26: Fmoc-L-glutamic acid 1-tert-butyl ester (1.8 g, 4.1 mmol) was dissolved in anhydrous DCM, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.0 g, 5.0 mmol) and N-hydroxysuccinimide (0.6 g, 5.0 mmol) were added, and the mixture was reacted at room temperature for 30 min. After the reaction was completed as monitored by TLC plate, compound 25 (1.0 g, 4.1 mmol) and N, N-diisopropylethylamine (0.9 mL, 5.0 mmol) were added, and the mixture was reacted at room temperature overnight. The reaction was completed as shown by TLC plate. Column chromatography DCM to DCM: MeOH = 60: 1 was used for purification to obtain 1.3 g of a light yellow liquid (yield 50%). LC-MS (m / z) (ESI), calculated for C 36 H 47 N3O8:649.34; found [M+H] + :650.34.

[0061] Preparation of compound 27: Compound 26 (1 g, 1.5 mmol) was dissolved in 10 mL of THF, 1,8-diazabicyclo[5.4.0]undec-7-ene (0.1 g, 0.8 mmol) was added, and the mixture was reacted at room temperature for 1 h. The reaction was complete as shown by TLC plate. Column chromatography DCM to DCM:MeOH=20:1 was used for purification to obtain 0.6 g of a colorless liquid (yield 90%). LC-MS (m / z) (ESI), calculated for C 21 H 37 N3O6:427.27; found [M+H] + :428.27.

[0062] Preparation of compound 28: Compound 27 (0.5 g, 1.2 mmol) and pteroic acid (0.37 g, 1.2 mmol) were dissolved in 20 mL DMSO, and benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (0.7 g, 1.8 mmol), 1-hydroxybenzotriazole (0.2 g, 1.8 mmol), and N,N-diisopropylethylamine (0.3 mL, 1.8 mmol) were added. The mixture was reacted overnight at room temperature in the dark. The reaction was complete after LCMS monitoring. 200 mL of purified water was added to the reaction system. At this time, a yellow solid precipitated. The mixture was filtered and dried under vacuum to obtain 0.3 g of a crude brown solid (crude product yield 40%). The yellow pure product 0.17 g (yield 20%) was obtained by reverse column purification. LC-MS (m / z) (ESI), calcd for C 35 H 47 N9O8:721.35; found [M+H] + :722.35.

[0063] Preparation of compound 29: Compound 28 (0.2 g, 0.3 mmol) was placed in a round-bottom flask, 5 mL of DCM and 2 mL of trifluoroacetic acid were added, and the reaction was carried out at room temperature for 6 h. The reaction was completed after monitoring by LCMS, and the solvent was removed under vacuum to obtain 0.17 g of a yellow solid (yield 90%). LC-MS (m / z) (ESI), calculated for C 31 H 39 N9O8:665.29; found [M+H] + :666.29.

[0064] Preparation of compound 20: Compound 19 (0.3 g, 0.2 mmol), compound 29 (0.17 g, 0.3 mmol) and tetraacetonitrile copper hexafluorophosphate (0.18 g, 0.4 mmol) were placed in a three-necked flask under nitrogen protection, and 5 mL of anhydrous DMSO was injected. The reaction was carried out at room temperature in the dark for 2 h. The reaction was completed after LCMS monitoring. The yellow solid was purified by reverse column to obtain 0.24 g (yield 50%). LC-MS (m / z) (ESI), calculated for C 84 H 100 N 20 O 20 :1708.74; found[M+H] + :1709.74.

[0065] Preparation of compound 21: Compound 20 (0.2 g, 0.1 mmol) was dissolved in anhydrous DMF, and N,N-diisopropylethylamine (51 μL, 0.3 mmol) and 6-(maleimido)hexanoic acid succinimidyl ester (88.5 mg, 0.3 mmol) were added. After 3 h, the reaction was complete as monitored by LCMS, and the mixture was purified by reverse column to obtain 0.1 g of a yellow solid (yield 50%). LC-MS (m / z) (ESI), calculated for C 94 H 111 N 21 O 23 :1901.82; found[M+H] + :1902.82.

[0066] The schematic diagram of the structure of the human serum albumin covalently bound small molecule drug conjugate prodrug targeting folate receptor of compound 21 is shown in Figure 3 As shown, the red part is maleimide; the cyan part is the VA dipeptide chain composed of valine and alanine; the blue part is the aptamer; the green part is SN38; the pink part is folic acid (FA); and the black part is a linker used to connect folic acid to other parts.

[0067] Example 2 PBS stability test of the compound

[0068] The prodrug compound 21 prepared in Example 1 was prepared into a 5 mM stock solution, diluted with a pH 7.4 PBS buffer to a final concentration of 50 uM, and incubated at 37°C. The sampling time points were 1 day, 3 days, 5 days, and 7 days, and three samples were taken at each time point. The experimental results were determined by HPLC. Figure 1 .

[0069] from Figure 1It can be seen that the release of SN38 was not detected within 7 days, indicating that the prodrug of the compound of the present invention has high stability in PBS solution at pH 7.4.

[0070] Example 3 Mouse plasma stability test of the compound

[0071] The prodrug compound 21 prepared in Example 1 was formulated into a 5mM mother solution, and mouse plasma was preheated at 37°C for 30 minutes. The compound prodrug was diluted with mouse plasma to a concentration of 50uM and incubated at 37°C. Sampling time points: 1 day, 3 days, 5 days, and 7 days. Take 40uL of the above plasma, add 160uL of cold acetonitrile solution, vortex, centrifuge at 4°C, 9000rpm, 30min. Take the supernatant and detect it by HPLC. The experimental results are shown in Figure 1 .

[0072] from Figure 1 It can be seen that the release of SN38 was not detected within 7 days, indicating that the prodrug of the compound of the present invention has high stability in plasma.

[0073] Example 4 Experiment of the proliferation inhibition activity of the compound on KB, A549 and MCF-3 cell lines, i.e., cytotoxicity experiment

[0074] In order to evaluate the effect of the prodrug compound 21 prepared in Example 1 on the proliferation ability of tumor cells, the MTT method was used to select KB, A549, and MCF-3 cell lines (cells were purchased from Wuhan Pronocell Life Science Technology Co., Ltd. KB is oral cancer cells, A549 is non-small cell lung cancer cells, and MCF-3 is human breast cancer cells). After 72 hours, the inhibition of tumor cell proliferation by the compound was observed and determined, and the IC50 was calculated. The results are shown in Table 1.

[0075] Table 1

[0076]

[0077] Table 1 shows the results of the proliferation inhibition activity of SN38, the marketed drug irinotecan, and the above-mentioned prodrug compounds on KB, A549, and MCF-3 cell lines. From the results in Table 1, it can be seen that the cytotoxicity of MAL-VA-FA-SN38 is less than that of the other three, indicating that the prodrug is very stable and has little cytotoxicity without drug release; and Mal-VA-FA-SN38 with cathepsin B added has enhanced cytotoxicity, which is much greater than that of the SN38 and Irinotecan groups, because the cytotoxic drug SN38 is released under the action of the enzyme, indicating that compound 21 can effectively release the cytotoxic drug SN38 after adding cathepsin B.

[0078] Example 5 Enzyme test

[0079] Experimental method: Use PBS to dissolve the final product (i.e. compound 21 in Example 1) in PBS (PBS was purchased from Leyan) solution to prepare a 5mM solution. Take 40 microliters, add 480 microliters of ABS (ABS was purchased from Leyan, the pH environment of the final product in vivo is about 5.0, ABS is used to simulate the pH environment in vivo) and 240 microliters of enzyme stock solution (the full name of the enzyme is cathepsin B, the content is 10 units, purchased from Sigma), take 40 microliters of sample at 15min, 30min, 1h, 2h, 4h, 6h, 18h, 24h, 2d, 4d, 6d, respectively, and add to 120 microliters of cold acetonitrile (the purpose of adding cold acetonitrile is to inactivate the enzyme and stop drug release). Vortex, centrifuge at 4°C (the purpose of centrifugation is to precipitate the enzyme protein, which will not interfere with the results when performing HPLC analysis of drug release), 9000rpm, 30min, take the supernatant and detect it with HPLC. Experimental results are shown in Figure 2 .

[0080] from Figure 2 It can be seen that compound 21 finally released the drug in 6 days. Figure 2 From the drug release curve, it can be seen that compound 21 can effectively release the drug, proving that compound 21 can release the drug under the action of tissue protease B at a pH of about 5.0 (the pH environment for drug release in vivo is about 5.0).

[0081] The above are only embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A human serum albumin covalently bound small molecule drug conjugate prodrug targeting folate receptor, characterized in that: It has a structure as shown in Formula I:

2. A pharmaceutical composition, characterized in that It comprises the human serum albumin covalently bound folate receptor targeted small molecule drug conjugate prodrug or a pharmaceutically acceptable salt thereof as claimed in claim 1, and one or more pharmaceutical carriers.

3. Use of the human serum albumin covalently bound small molecule drug conjugate prodrug targeting folate receptor or a pharmaceutically acceptable salt thereof as claimed in claim 1 in the preparation of human serum albumin covalently bound anti-tumor drugs.

4. Use of the human serum albumin covalently bound small molecule drug conjugate prodrug targeting folate receptor or a pharmaceutically acceptable salt thereof as claimed in claim 1 in the preparation of anti-tumor drugs.

5. Use of the pharmaceutical composition according to claim 2 in the preparation of human serum albumin covalently bound anti-tumor drugs.

6. Use of the pharmaceutical composition according to claim 2 in the preparation of anti-tumor drugs.

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