An antitumor drug and a preparation method thereof

Abstract

The application discloses an antitumor drug and a preparation method thereof. The antitumor drug is a genistein modified drug, which is prepared by combining genistein with RGDV tumor targeting polypeptide. The drug improves the toxicity of genistein to lung cancer A549 and 95D cells, the introduced RGDV peptide is non-toxic to organisms, and the drug also has high antitumor activity.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an antitumor drug using VDGR polypeptide to modify genistein and its preparation method. Background Technology

[0002] Genistein (abbreviated as Gen), also known as genistein flavonoid, is a natural isoflavone compound extracted from legumes. It is chemically named 4',5,7-trihydroxyisoflavone. Genistein is also an epidermal growth factor receptor (EGF-R) tyrosine kinase inhibitor. Tyrosine kinases are the intracellular domains of many growth factor receptors. Approximately half of the known oncogene products encode growth factor receptors containing tyrosine kinases. High-level expression of tyrosine kinases is closely related to cell transformation because receptor tyrosine kinases are important proteins controlling cell growth and differentiation.

[0003] The antitumor mechanism of genistein involves seven pathways: inhibition of protein tyrosinase phosphorylation; inhibition of topoisomerase activity; inhibition of cell cycle; induction of cell differentiation; induction of apoptosis; inhibition of angiogenesis; and antioxidant effects. In vivo, in vitro, and epidemiological studies have shown that genistein has inhibitory effects on various tumors, such as breast cancer, prostate cancer, kidney cancer, gastric cancer, colorectal cancer, bladder cancer, lung cancer, uterine cancer, skin cancer, leukemia, lymphoma, neuroblastoma, and head and neck cancer.

[0004] Gentian isoflavones have limited applications in the food and pharmaceutical fields due to their bitter taste, poor water solubility, and low bioavailability. Improving their biological activity and expanding their application range through structural modification has become a current research hotspot.

[0005] Cancer is a leading cause of death and a significant obstacle to extending life expectancy worldwide. According to data released online by the IARC in 2022, lung cancer was the most common cancer globally, accounting for 12.4% of all cancer cases, followed by breast cancer (11.5%), colorectal cancer (9.6%), prostate cancer (7.3%), and stomach cancer (4.9%) (Cao Mengdi, Chen Wanqing. Interpretation of Global Cancer Statistics in 2022. Chinese Journal of Medical Frontiers, 2024, (06): 1-5 [2024-06-21]). Prostate cancer, lung cancer, and colorectal cancer are the most common in men, while breast cancer, lung cancer, and colorectal cancer are the top three most common cancers in women. Therefore, strengthening targeted cancer prevention and control measures is particularly necessary.

[0006] The RGD sequences of the α and γ chains of integrin receptors on the surface of tumor cells can be specifically recognized by GPIIb / IIIa receptors, leading to binding to relevant cells or platelets and exerting infiltration or promoting tumor metastasis. Because of the specific binding of RGD sequences to GPIIb / IIIa receptors, RGD-containing peptides possess excellent tumor-targeting properties, making them potential target peptides for antitumor agents, and also possessing the potential to expand antitumor or anti-metastatic activity. Therefore, studying the therapeutic effects of genistein on lung cancer is of great significance for exploring new methods of treating lung cancer and expanding the application scope of genistein. Summary of the Invention

[0007] To overcome the shortcomings of the prior art, the present invention provides an antitumor drug or a salt thereof, the structure of which is as follows:

[0008]

[0009] in,

[0010] R1 is selected from: substituted or unsubstituted C 1-6 Alkyl, C 3-6 Alkyl groups, hydroxyl groups;

[0011] VDGR is a polypeptide of -Arg-Gly-Asp-Val.

[0012] Specifically, R1 may be selected from at least one of the following: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, cyclopentyl, cyclohexyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, and tert-butoxy.

[0013] Specifically, R1 is a hydroxyl group.

[0014] Specifically, the genistein-modified drug finally prepared by the present invention can be pharmacologically permissible by adding acid or base, such as reacting with trifluoroacetic acid or hydrochloric acid in an optional suitable solvent to obtain trifluoroacetic acid salt and hydrochloride salt of genistein-modified drug, respectively.

[0015] In another aspect of the present invention, a method for preparing a genistein-modified drug is provided, wherein the preparation method involves combining genistein with an RGDV tumor-targeting polypeptide, and the synthetic route of the method is as follows:

[0016]

[0017] The specific preparation method includes the following steps:

[0018] (i) Compound 7 was dissolved, and an alkaline reagent and an esterification reagent were added. The reaction was carried out, and after post-treatment, compound 8 was obtained.

[0019] (ii) Compound 8 was dissolved, reacted with an alkaline reagent, and post-treated to obtain compound 9;

[0020] (iii) Compound 9 was dissolved, and a catalyst, condensing agent, basic reagent and Arg(NO2)-Gly-Asp(OBzl)-Val-Obzl were added in sequence. After reaction and post-treatment, GEN-(Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl)2 was obtained.

[0021] (ⅳ) GEN-(Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl)2 was dissolved, a deprotecting agent was added, and the reaction was carried out. After post-treatment, GEN-(Arg-Gly-Asp-Val)2 was obtained.

[0022] Specifically, the structure of compound 7 described in step (i) is as follows:

[0023] Specifically, R1 may be selected from at least one of the following: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, cyclopentyl, cyclohexyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, and tert-butoxy.

[0024] Specifically, R1 is a hydroxyl group.

[0025] In some embodiments of the present invention, compound 7 is genistein.

[0026] Specifically, the alkaline reagent used in the reaction described in step (i) is at least one of pyridine, sodium methoxide, sodium ethoxide, sodium bicarbonate, potassium bicarbonate, ammonium bicarbonate, sodium carbonate, potassium carbonate, lithium carbonate, cesium carbonate, ammonium carbonate, sodium hydroxide, lithium hydroxide, potassium hydroxide, cesium hydroxide, and barium hydroxide.

[0027] Specifically, the esterification reagent in step (i) is at least one of ethyl bromoacetate, propyl bromoacetate, butyl bromoacetate, isopropyl bromoacetate, isobutyl bromoacetate, ethyl iodoacetate, ethyl chloroacetate, and isobutyl chloroacetate.

[0028] In some embodiments of the present invention, the esterification reagent for the reaction is ethyl bromoacetate.

[0029] Specifically, the reaction temperature in step (i) is 15 to 35°C, for example, 15°C, 20°C, 25°C, 30°C, or 35°C.

[0030] Specifically, the reaction time described in step (i) is 1 to 8 days, for example, 1 day, 2 days, 3 days, 5 days, 7 days, and 8 days.

[0031] In some embodiments of the present invention, the reaction temperature is room temperature; the reaction time is 5 days.

[0032] Specifically, the post-processing steps described in step (i) include: filtration, concentration, and recrystallization.

[0033] Specifically, the alkaline reagent used in the reaction described in step (ii) is at least one of sodium hydroxide, lithium hydroxide, potassium hydroxide, cesium hydroxide, barium hydroxide, sodium methoxide, sodium ethoxide, sodium carbonate, potassium carbonate, lithium carbonate, and cesium carbonate.

[0034] In some embodiments of the present invention, the alkaline reagent for the reaction is sodium hydroxide.

[0035] Specifically, the reaction temperature in step (ii) is 15 to 35°C, for example, 15°C, 20°C, 25°C, 30°C, or 35°C.

[0036] Specifically, the reaction time described in step (ii) is 1 to 6 hours, for example, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours.

[0037] In some embodiments of the present invention, the reaction temperature is room temperature; the reaction time is 1 hour.

[0038] Specifically, the post-processing steps described in step (ii) include: pH adjustment, water washing, and drying.

[0039] Specifically, the acidic reagent used to adjust the pH in step (ii) is at least one of potassium bisulfate, sodium bisulfate, trifluoroacetic acid, citric acid, oxalic acid, maleic acid, salicylic acid, and fumaric acid.

[0040] Specifically, the reagent used for dissolution in step (iv) is at least one of trifluoroacetic acid, tetrahydrofuran, N,N-dimethylformamide, and acetonitrile.

[0041] Specifically, the deprotecting agent mentioned in step (iv) is one of trifluoromethanesulfonic acid (TFMSA) and / or hydrofluoric acid (HF).

[0042] In some embodiments of the present invention, the deprotecting agent is trifluoromethanesulfonic acid (TFMSA).

[0043] Specifically, the reaction temperature in step (iv) is -5 to 15°C, for example -5°C, 0°C, 5°C, 10°C, or 15°C.

[0044] Specifically, the reaction time described in step (iv) is 0.5 to 4 hours, for example, 0.5 hours, 1 hour, 1.5 hours, 2 hours, 3 hours, or 4 hours.

[0045] In some embodiments of the present invention, the reaction temperature is an ice bath; the reaction time is 1 hour.

[0046] Specifically, the post-processing steps described in step (iv) include: quenching reaction, crystallization, pH adjustment, column chromatography, and lyophilization.

[0047] Specifically, the preparation method of Arg(NO2)-Gly-Asp(OBzl)-Val-Obzl includes the following steps:

[0048] (1) Dissolve Boc-Arg(NO2), add catalyst, condensing agent, basic reagent and HCl·Gly-OBzl in sequence, react, and post-process to obtain Boc-Arg(NO2)-Gly-Obzl;

[0049] (2) Dissolve Boc-Arg(NO2)-Gly-Obzl, add alkaline reagent to react, and then treat to obtain Boc-Arg(NO2)-Gly-OH;

[0050] (3) Dissolve Boc-Asp(OBzl), add catalyst, condensing agent, alkaline reagent and HCl·Val-OBzl in sequence, react, and post-process to obtain Boc-Asp(OBzl)-Val-OBzl;

[0051] (4) Dissolve Boc-Asp(OBzl)-Val-OBzl, add deprotecting reagent to react, and then perform post-treatment to obtain Asp(OBzl)-Val-OBzl;

[0052] (5) Dissolve Boc-Arg(NO2)-Gly-OH, add catalyst, condensing agent, basic reagent and Asp(OBzl)-Val-OBzl in sequence, react, and post-process to obtain Boc-Arg(NO2)-Gly-Asp(OBzl)-Val-Obzl;

[0053] (6) Dissolve Boc-Arg(NO2)-Gly-Asp(OBzl)-Val-Obzl, add deprotecting reagent to react, and then process to obtain Arg(NO2)-Gly-Asp(OBzl)-Val-Obzl.

[0054] Specifically, the reagents used for dissolution in steps (1), (3), (5), and (ⅲ) are at least one of tetrahydrofuran, 2-methyltetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, isopropyl acetate, acetonitrile, dichloromethane, toluene, and thionyl chloride.

[0055] In some embodiments of the present invention, the reagent used for dissolution is tetrahydrofuran.

[0056] Specifically, the catalyst mentioned in steps (1), (3), (5), and (ⅲ) is at least one of N-hydroxysuccinimide (HOSu), N-hydroxy-7-azabenzotriazole (HOAt), 1-hydroxybenzotriazole (HOBt), and 4-dimethylaminopyridine (DMAP).

[0057] In some embodiments of the present invention, the catalyst is 1-hydroxybenzotriazole (HOBt).

[0058] Specifically, the condensing agent mentioned in steps (1), (3), (5), and (iii) is at least one of N,N'-dicyclohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), N,N'-carbonyldiimidazole (CDI), N,N'-diisopropylcarbodiimide (DIC), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU), propyl tricyclic phosphate anhydride (T3P), n-butyl phosphate anhydride (T4P), diphenyl azidophosphate (DPPA), and bis(2-oxo-3-oxazolyl)hypophosphine chloride (BOP-Cl).

[0059] In some embodiments of the present invention, the condensing agent is N,N'-dicyclohexylcarbodiimide (DCC).

[0060] Specifically, the alkaline reagent mentioned in steps (1), (3), (5), and (iii) is at least one of triethylamine (TEA), N-methylmorpholine (NMM), N,N-diisopropylethylamine (DIPEA), 8-diazabicycloundec-7-ene (DBU), pyridine (Py), imidazole, N-methylimidazolium (NMI), and 2,6-dimethylpyridine.

[0061] In some embodiments of the present invention, the alkaline reagent is N-methylmorpholine (NMM).

[0062] Specifically, the molar ratio of catalyst to condensing agent in steps (1), (3), (5), and (iii) is 1:0.5 to 1:2.0, for example, 1:0.5, 1:1.0, 1:1.2, 1:1.5, or 1:2.0.

[0063] Specifically, in steps (1), (3), (5), and (iii), the molar ratio of the condensing agent to any one of Boc-Arg(NO2), Boc-Asp(OBzl), Boc-Arg(NO2)-Gly-OH, and / or compound 9 is 0.8:1 to 3.0:1, for example, 0.8:1, 1.0:1, 1.2:1, 1.4:1, 1.8:1, 2.0:1, 2.4:1, 2.8:1, or 3.0:1.

[0064] Specifically, in steps (1), (3), (5), and (ⅲ), the molar ratio of the additive to any one of Boc-Arg(NO2), Boc-Asp(OBzl), Boc-Arg(NO2)-Gly-OH, and / or compound 9 is 0.5:1 to 2.0:1, for example, 0.5:1, 0.8:1, 1.0:1, 1.2:1, 1.4:1, 1.6:1, or 2.0:1.

[0065] Specifically, the reaction temperature described in steps (1), (3), (5), and (ⅲ) is 15 to 35°C, for example, 15°C, 20°C, 25°C, 30°C, and 35°C.

[0066] Specifically, the reaction time described in steps (1), (3), (5), and (iii) is 4 to 18 hours, for example, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, and 18 hours.

[0067] In some embodiments of the present invention, the reaction temperature is a greenhouse temperature; the reaction time is 8 hours.

[0068] Specifically, the post-processing steps described in steps (1), (3), (5), and (iii) include: quenching reaction, concentration, extraction, washing, drying, concentration, and column chromatography.

[0069] Specifically, the solvent used for extraction in steps (1), (3), (5), and (iii) is at least one of dichloromethane, ethyl acetate, tetrahydrofuran, and 2-methyltetrahydrofuran.

[0070] Specifically, the drying agent used in steps (1), (3), (5), and (ⅲ) is at least one of anhydrous sodium sulfate, anhydrous magnesium sulfate, anhydrous calcium sulfate, calcium chloride, and molecular sieve.

[0071] In some embodiments of the present invention, the drying agent used is anhydrous sodium sulfate.

[0072] Specifically, the solvent used to dissolve compound 2 in step (2) is at least one of methanol, ethanol, isopropanol, tetrahydrofuran, and acetonitrile.

[0073] Specifically, the alkaline reagent mentioned in step (2) is at least one of triethylamine, butylamine, pentylamine, hexylamine, diethylamine, dibutylamine, dipentylamine, tributylamine, aniline, N-methylaniline, N,N-diisopropylethylamine, imidazole, pyrrole, piperazine, pyrazine, pyridine, sodium methoxide, sodium ethoxide, sodium bicarbonate, potassium bicarbonate, ammonium bicarbonate, sodium carbonate, potassium carbonate, cesium carbonate, lithium carbonate, cesium carbonate, ammonium carbonate, sodium hydroxide, lithium hydroxide, potassium hydroxide, cesium hydroxide, and barium hydroxide.

[0074] Specifically, the temperature of the reaction in step (2) is -5 to 15°C, for example -5°C, 0°C, 5°C, 10°C, 15°C.

[0075] Specifically, the reaction time in step (2) is 4 to 12 hours, for example, 4 hours, 6 hours, 8 hours, 10 hours, or 12 hours.

[0076] In some embodiments of the present invention, the reaction temperature is an ice bath; the reaction time is 6 hours.

[0077] Specifically, the post-processing steps in step (2) include: quenching reaction, concentration, pH adjustment, extraction, washing, drying, and concentration.

[0078] Specifically, the acidic reagent used to adjust the pH in step (2) is at least one of potassium bisulfate, sodium bisulfate, trifluoroacetic acid, citric acid, oxalic acid, maleic acid, salicylic acid and fumaric acid.

[0079] In some embodiments of the present invention, the acidic reagent used for adjusting the pH is potassium bisulfate.

[0080] Specifically, the reagents used for dissolution in steps (4) and (6) are at least one of anhydrous ethyl acetate, anhydrous methanol, anhydrous ethanol and anhydrous 1,4-dioxane.

[0081] Specifically, the deprotecting agent mentioned in steps (4) and (6) is at least one of the following: ethyl acetate solution of hydrogen chloride (4M HCl / EtOAc), methanol solution of hydrogen chloride (4M HCl / EtOAc), ethanol solution of hydrogen chloride (4M HCl / EtOAc), and 1,4-dioxane solution of hydrogen chloride (4M HCl / EtOAc).

[0082] In some embodiments of the present invention, the deprotecting agent is an ethyl acetate solution of hydrogen chloride (4MHCl / EtOAc).

[0083] Specifically, the reaction temperature in steps (4) and (6) is -5 to 15°C, for example -5°C, 0°C, 5°C, 10°C, and 15°C.

[0084] Specifically, the reaction time in steps (4) and (6) is 1 to 8 hours, for example, 1 hour, 2 hours, 3 hours, 5 hours, 7 hours, or 8 hours.

[0085] In some embodiments of the present invention, the reaction temperature is an ice bath; the reaction time is 2 hours.

[0086] Specifically, the post-processing steps described in steps (4) and (6) include: quenching reaction, concentration, washing, and concentration.

[0087] In a third aspect of the invention, the method for modifying genistein according to the invention is provided for use in the preparation of a medicament for antitumor purposes.

[0088] Specifically, anti-tumor diseases include, but are not limited to: breast cancer, cervical cancer, ovarian cancer, prostate cancer, bladder cancer, stomach cancer, liver cancer, colon cancer, rectal cancer, lung cancer, skin cancer, and malignant tumors such as leukemia and lymphoma.

[0089] Specifically, the genistein-modified drug prepared by this invention can enhance the toxicity of genistein to lung cancer A549 and 95D cells, and the introduced RGDV peptide is non-toxic to organisms and has high anti-tumor activity.

[0090] The chemical names corresponding to some of the abbreviations used in this invention are as follows:

[0091] Boc-Arg(NO2): tert-Butyloxycarbonyl-nitro-arginine

[0092] HCl·Gly-OBzl: Glycine benzyl ester hydrochloride

[0093] Boc-Arg(NO2)-Gly-Obzl: tert-Butyloxycarbonyl-nitro-arginine-glycine benzyl ester

[0094] Boc-Arg(NO2)-Gly-OH: tert-Butyloxycarbonyl-nitro-arginine-glycine

[0095] Boc-Asp(OBzl): tert-Butoxycarbonyl-aspartic acid benzyl ester

[0096] HCl·Val-OBzl: Benzyl valine hydrochloride

[0097] Boc-Asp(OBzl)-Val-OBzl: tert-Butyloxycarbonyl-aspartic benzyl ester-valine benzyl ester

[0098] Asp(OBzl)-Val-OBzl: Aspartate benzyl ester-valine benzyl ester

[0099] Boc-Arg(NO2)-Gly-Asp(OBzl)-Val-Obzl: tert-Butyloxycarbonyl-nitro-arginine-glycine-aspartic acid benzyl ester-valine benzyl ester

[0100] Arg(NO2)-Gly-Asp(OBzl)-Val-Obzl: Nitro-Arginine-Glycine-Aspartic Acid Benzyl Ester-Valine Benzyl Ester

[0101] GEN: Gentian Isoflavone

[0102] GEN-(Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl)2: GEN-nitro-arginine-glycine-aspartic acid benzyl ester-valine benzyl ester

[0103] RGDV: Arg-Gly-Asp-Val (arginine-glycine-aspartic acid-valine)

[0104] GEN-(Arg-Gly-Asp-Val)2: GEN-arginine-glycine-aspartic acid-valine Attached Figure Description

[0105] Figure 1 The results of RGDV assay on CCK-8 in lung cancer cells A549 and 95D are shown.

[0106] Figure 2 The figure shows the apoptosis induced in A549 cells by treatment with different concentrations of drugs;

[0107] Note: A is the control group, B is the 50 μmol / L genistein group, C is the 100 μmol / L genistein group, D is the 150 μmol / L genistein group, E is the 50 μmol / L genistein modified drug group, F is the 100 μmol / L genistein modified drug group, and G is the 150 μmol / L genistein modified drug group.

[0108] Figure 3 The figure shows the apoptosis induced by different concentrations of drugs in 95D cells;

[0109] Note: A is the control group, B is the 50 μmol / L genistein group, C is the 100 μmol / L genistein group, D is the 150 μmol / L genistein group, E is the 50 μmol / L genistein modified drug group, F is the 100 μmol / L genistein modified drug group, and G is the 150 μmol / L genistein modified drug group.

[0110] Figure 4 The results of CCK-8 assays on lung cancer cells A549 and 95D are shown in the control group, genistein, and RGDV.

[0111] Note: Significant differences between the genistein and genistein-modified drug groups and the control group are indicated by * (*P < 0.05, **P < 0.01, ***P < 0.001); significant differences between the genistein-modified drug group and the genistein group are indicated by ▲ (▲▲P < 0.01, ▲▲▲P < 0.001); (X ± SD, n = 3)

[0112] A is the control group, B is the 50 μmol / L genistein group, C is the 100 μmol / L genistein group, D is the 150 μmol / L genistein group, E is the 50 μmol / L genistein-modified drug group, F is the 100 μmol / L genistein-modified drug group, and G is the 150 μmol / L genistein-modified drug group. Detailed Implementation

[0113] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0114] Example 1: Preparation of the genistein-modified drug RGDV-GEN

[0115] Preparation of 1.0Arg(NO2)-Gly-Asp(OBzl)-Val-Obzl

[0116] 1.1 Preparation of compound 1 (Boc-Arg(NO2)-Gly-OBzl)

[0117] 0.500 g (1.50 mmol) of Boc-Arg(NO2) was dissolved in 20 mL of anhydrous tetrahydrofuran. Under ice bath and stirring, 0.200 g (1.50 mmol) of N-hydroxybenzotriazole (HOBt) and 0.310 g (1.50 mmol) of N,N-dicyclohexylcarbodiimide (DCC) solution dissolved in anhydrous THF were added sequentially. The mixture was stirred thoroughly for 30 minutes to obtain solution A. Then, the pH was adjusted to 8-9 with 10 mL of anhydrous THF and 0.286 g (1.42 mmol) of HCl·Gly-Obzl (glycine benzyl ester hydrochloride), and added to reaction solution A. The ice bath was removed, and the mixture was stirred at room temperature for 8 hours. TLC under UV (254 nm) (developing solvent: dichloromethane:methanol = 40:1) showed that the starting material HCl·Lys(Boc)-OBzl completely disappeared. The filtrate was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate. The filtrate was transferred to a 250 mL separatory funnel, and the ethyl acetate layer was successively extracted with saturated sodium bicarbonate aqueous solution (n=3), saturated sodium chloride aqueous solution (n=3), 5% potassium bisulfate aqueous solution (n=3), saturated sodium chloride aqueous solution (n=3), saturated sodium bicarbonate aqueous solution (n=3), and saturated sodium chloride aqueous solution (n=3). The upper ethyl acetate phases were combined, dried over anhydrous sodium sulfate for 4 hours, filtered, and the resulting filtrate was concentrated to dryness under reduced pressure. After silica gel column chromatography, 0.556 g (84%) Boc-Arg(NO2)-Gly-OBzl was obtained.

[0118] 1.2 Preparation of compound 2 (Boc-Arg(NO2)-Gly-OH)

[0119] 4.660 g (1 mmol) of Boc-Arg(NO2)-Gly-OBzl was dissolved in 50 mL of methanol. 2N NaOH aqueous solution was slowly added dropwise to the resulting solution under ice bath and stirring for 6 hours. UV (254 nm) TLC (developing solvent: dichloromethane:methanol = 40:1) showed complete disappearance of Boc-Arg(NO2)-Gly-OBzl. The pH was adjusted to 7 by slow addition of saturated KHSO4 aqueous solution under ice bath and stirring, and the methanol was removed by concentration under reduced pressure. The pH was adjusted to 2 with saturated potassium bisulfate aqueous solution, and the aqueous solution was repeatedly extracted three times with ethyl acetate (40 mL × 3), washed three times with saturated sodium chloride aqueous solution, and the ethyl acetate layers were combined. The solution was dried over anhydrous sodium sulfate for 2 hours, filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain 2.850 g (76%) of (Boc-Arg(NO2)-Gly-OH).

[0120] 1.3 Preparation of compound 3 (Boc-Asp(OBzl)-Val-OBzl)

[0121] 0.323 g (1.0 mmol) of Boc-Asp (OBzl) was dissolved in 20 mL of anhydrous tetrahydrofuran. Under ice bath and stirring, 0.200 g (1.50 mmol) of N-hydroxybenzotriazole (HOBt) and 0.310 g (1.50 mmol) of N,N-dicyclohexylcarbodiimide (DCC) solution dissolved in anhydrous THF were added sequentially. The mixture was stirred thoroughly for 30 minutes to obtain solution A. Then, the pH was adjusted to 8-9 with 10 mL of anhydrous THF and 0.345 g (0.90 mmol) of HCl·Val-OBzl, and added to reaction solution A. The mixture was stirred at room temperature for 8 hours after removing the ice bath. TLC under UV (254 nm) (developing solvent: dichloromethane:methanol = 40:1) showed complete disappearance of the starting material HCl·Val-OBzl. The filtrate was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate. The filtrate was transferred to a 250 mL separatory funnel. The ethyl acetate layer was successively extracted with saturated sodium bicarbonate aqueous solution (n=3), saturated sodium chloride aqueous solution (n=3), 5% potassium bisulfate aqueous solution (n=3), saturated sodium chloride aqueous solution (n=3), saturated sodium bicarbonate aqueous solution (n=3), and saturated sodium chloride aqueous solution (n=3). The upper ethyl acetate phases were combined and dried with anhydrous sodium sulfate for 4 hours. The mixture was filtered, and the filtrate was concentrated to dryness under reduced pressure. The filtrate was then subjected to silica gel column chromatography to obtain 0.390 g (84%) Boc-Asp(OBzl)-Val-OBzl.

[0122] 1.4 Preparation of compound 4 (Asp(OBzl)-Val-OBzl)

[0123] Weigh 1.000 g of Boc-Asp(OBzl)-Val-OBzl into a 250 mL flask, and add 10 mL of anhydrous ethyl acetate to dissolve it. Add 100 mL of an ethyl acetate solution of hydrogen chloride (4 M HCl / EtOAc) to the solution while stirring in an ice bath, and stir for 2 h. TLC (dichloromethane / methanol, 40:1) under UV (254 nm) showed complete disappearance of the starting material Boc-Asp(OBzl)-Val-OBzl. Concentrate the reaction mixture to dryness under reduced pressure. Dilute the residue with 30 mL of anhydrous ethyl acetate, and then concentrate to dryness under reduced pressure. Repeat this operation three times. Dilute the resulting residue with 30 mL of anhydrous diethyl ether, and then concentrate to dryness under reduced pressure. Repeat this operation three times to obtain Boc-Arg(NO2)-Gly.

[0124] 1.5 Preparation of compound 5 (Boc-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl)

[0125] 0.374 g (1.0 mmol) of Boc-Arg(NO2)-Gly was dissolved in 20 mL of anhydrous tetrahydrofuran. Under ice bath and stirring, 0.200 g (1.50 mmol) of N-hydroxybenzotriazole (HOBt) and 0.310 g (1.50 mmol) of N,N-dicyclohexylcarbodiimide (DCC) solution dissolved in anhydrous THF were added sequentially. The mixture was stirred thoroughly for 30 minutes to obtain solution A. Then, the pH was adjusted to 8-9 with 0.410 g (0.90 mmol) of HCl·Asp(OBzl)-Val-OBzl dissolved in 10 mL of anhydrous THF and N-methylmorpholine. This solution was then added to reaction solution A. The mixture was stirred at room temperature for 8 hours after removing the ice bath. TLC under UV (254 nm) (developing solvent: dichloromethane:methanol = 40:1) showed that the starting material HCl·Asp(OBzl)-Val-OBzl completely disappeared. The filtrate was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate. The filtrate was transferred to a 250 mL separatory funnel, and the ethyl acetate layer was successively extracted with saturated sodium bicarbonate aqueous solution (n=3), saturated sodium chloride aqueous solution (n=3), 5% potassium bisulfate aqueous solution (n=3), saturated sodium chloride aqueous solution (n=3), saturated sodium bicarbonate aqueous solution (n=3), and saturated sodium chloride aqueous solution (n=3). The upper ethyl acetate phases were combined, dried over anhydrous sodium sulfate for 4 hours, filtered, and the resulting filtrate was concentrated to dryness under reduced pressure. The filtrate was then subjected to silica gel column chromatography to give 0.519 g (74%) Boc-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl.

[0126] 1.6 Preparation of compound 6Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl

[0127] Weigh 5.000 g (6.5 mmol) of Boc-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl and dissolve it in 10 mL of anhydrous ethyl acetate in a flask. Add 100 mL of an ethyl acetate solution of hydrogen chloride (4 mol HCl / EtOAc) to the solution while stirring in an ice bath, and stir for 2 h. Concentrate the reaction mixture to dryness under reduced pressure. Dilute the residue with 30 mL of anhydrous ethyl acetate and concentrate to dryness under reduced pressure again. 4.27 g (98%) of compound 6 is given.

[0128] 2.0 Preparation of RGDV-GEN

[0129] The specific synthetic route for preparing RGDV-GEN is as follows:

[0130]

[0131] 2.1 Preparation of compound 8'

[0132] Gentian isoflavone, a derivative of soy isoflavones, was dissolved in anhydrous tetrahydrofuran. Anhydrous potassium carbonate was added, and after activation for 20 minutes, ethyl bromoacetate was added. The mixture was reacted at room temperature for 5 days, filtered, and evaporated to dryness. The solution was dissolved in methanol, and a pale yellow insoluble substance was obtained. The pale yellow solid was then filtered to obtain a pale yellow solid. The solid was then subjected to hot and cold recrystallization under the conditions of methanol as solvent and oil bath stirring and heating at 60°C for 1 hour. An insoluble solid was still present. The solid was then hot filtered, and the white solid was collected. The liquid was left at room temperature and then transferred to a refrigerator overnight. A large amount of white suspension precipitated the next day. The solid was filtered and collected as compound 8'.

[0133] 2.2 Preparation of compound 9'

[0134] Compound 8 was dissolved in methanol (which is sparingly soluble), and 2N NaOH was added. The solution dissolved and turned yellow. After reacting at room temperature for 1 hour, saturated potassium hydrogen sulfate solution was added to adjust the pH to 2. After adding excess water, the mixture was filtered and repeatedly rinsed with water to ensure that the salt was completely removed. Finally, the filter cake was rinsed with ether, and the solid was dried in an oven at 37°C to obtain compound 9'.

[0135] 2.3 Preparation of compound 10'(GEN-(Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl)2)

[0136] 0.160 g (0.41 mmol) of compound 9 was dissolved in 20 mL of anhydrous tetrahydrofuran. Under ice bath and stirring, 0.200 g (1.50 mmol) of N-hydroxybenzotriazole (HOBt) and 0.310 g (1.50 mmol) of N,N-dicyclohexylcarbodiimide (DCC) solution dissolved in anhydrous THF were added sequentially. The mixture was stirred thoroughly for 30 minutes to obtain solution A. Then, the pH was adjusted to 8-9 using 10 mL of anhydrous THF dissolved in 0.640 g (0.90 mmol) of HCl·Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl, and N-methylmorpholine was added to reaction solution A. After removing the ice bath and stirring at room temperature for 8 hours, TLC under UV (254 nm) (developing solvent: dichloromethane: methanol = 40:1) showed that the starting material HCl·Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl completely disappeared. The filtrate was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate. The filtrate was transferred to a 250 mL separatory funnel, and the ethyl acetate layer was successively extracted with saturated sodium bicarbonate aqueous solution (n=3), saturated sodium chloride aqueous solution (n=3), 5% potassium bisulfate aqueous solution (n=3), saturated sodium chloride aqueous solution (n=3), saturated sodium bicarbonate aqueous solution (n=3), and saturated sodium chloride aqueous solution (n=3). The upper ethyl acetate phases were combined, dried with anhydrous sodium sulfate for 4 hours, filtered, and the resulting filtrate was concentrated to dryness under reduced pressure and subjected to silica gel column chromatography to give 0.499 g (72%) GEN-(Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl)2.

[0137] 2.4 Preparation of compound 11'(GEN-(Arg-Gly-Asp-Val)2)

[0138] 0.1700 g (0.1 mmol) of GEN-(Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl)2 was added to a flask. 1.5 mL of trifluoroacetic acid (TFA) was added under an ice-salt bath to completely dissolve the compound. Then, 0.50 mL of trifluoromethanesulfonic acid (TFMSA) was added and the flask was covered with a drying tube. After 1 hour, the reaction was stopped by UV (254 nm) TLC (developing solvent: n-butanol:water:glacial acetic acid = 2:1:1) showing the disappearance of the starting spot. Anhydrous ether was added to the reaction mixture and stirred. The solid precipitated, and after standing, the supernatant was discarded. Anhydrous ether was added again, and the mixture was allowed to stand. The supernatant was discarded. This process was repeated 3 times. The mixture was then dried under reduced pressure. The solution was dissolved in a small amount of water, and the pH was adjusted to 7 with 15% ammonia. The solution was desalted using a Sephadex G15 column, purified using a C18 column, and lyophilized to obtain 28 mg of compound 11' (19%) GEN-(Arg-Gly-Asp-Val)2.

[0139] ESI-MS(m / e):1469[MH]-;mp:135.4-136.9℃;

[0140] 1H NMR(DMSO-d6,300MHz)δ / ppm=12.2(s,1H),11.09(s,1H),8.42(m,3H),8.32(m,4H),8.18(m,4H),8.05(m,2H),7 .93(m,2H),7.79(m,2H),7.58(m,2H),7.09(m,2H),6.92(s,1H),6.53(s,1H),4.83(m,2H),4.56(m,2H),4.44(m, 1H),4.35(m,2H),4.15(m,2H),4.08(m,1H),3.82(m,2H),3.65(m,2H),3.51(m,1H),3.06(m,7H),2.76(m,3H),2. 32(m,1H),2.06(m,1H),1.86(m,4H),1.66(m,6H),1.53(m,9H),1.37(m,5H),1.24(m,5H),0.88(d,J=6.0Hz,6H).

[0141] Example 2: Screening of the antitumor activity of the genistein-modified drug RGDV-GEN

[0142] a. The in vitro antitumor activity was evaluated using non-small cell lung cancer A549 and 95D.

[0143] The specific implementation is as follows:

[0144] Log-phase lung cancer cells (95 and A549) were collected, the original culture medium was discarded, and sterile PBS was added to wash away residual culture medium and dead cells. A suitable amount of trypsin solution was added to cover the bottom of the flask, and the cells were digested for 1-3 minutes. Immediately afterward, twice the volume of complete culture medium was added to terminate the trypsin reaction. The cell concentration was adjusted to 4 × 10⁴ cells / mL using DMEM. 100 μL of cell suspension was seeded into each well of a 96-well plate, and the outer ring of the 96-well plate was sealed with 100 μL of PBS. After cell adhesion, drug was administered according to the drug concentrations and groupings specified in Table 1, and the cells were cultured at 37℃ and 5% CO₂ for 48 h. After the experiment, 10 μL of CCK-8 solution was added to each well, and the cells were incubated at 37℃ for 2 h. The absorbance (OD) of each well was measured using a microplate reader at a wavelength of 450 nm to determine the cell inhibition rate. The experiment was repeated three times, and the average OD value was recorded.

[0145] Table 1 Dosage Concentration Settings

[0146] drug Dosage concentration (μmol / L) RGDV (Arg-Gly-Asp-Val) 5、10、20、40、80、100 GEN (Gentian Isoflavone) 3.125、6.25、12.5、25、50、100 RGDV-GEN (Compound 11) 3.125、6.25、12.5、25、50、100

[0147] Cell inhibition rate = (OD control group - OD treatment group) / OD control group × 100%. Results are as follows: Figure 1 As shown.

[0148] The inhibitory effects of genistein, RGDV, and genistein-modified drugs on lung cancer cell proliferation after 48 h were determined using a CCK-8 assay kit. Different concentrations (5, 10, 20, 40, 80, 100 μmol / L) of RGDV did not inhibit cell growth in lung cancer 95D and A549 cells after 48 h; in fact, they promoted cell proliferation, indicating that RGDV has no toxicity to lung cancer 95D and A549 cells. Tables 2 and 3 show that the genistein-modified drugs had a greater inhibitory effect on lung cancer cell proliferation than genistein itself.

[0149] Table 2. Inhibitory effect on A549 cells after 48 hours of drug administration ( n=3)

[0150]

[0151]

[0152] Note: Significant differences between the GEN and RGDV-GEN treatment groups and the control group are indicated by *, *P<0.05, **P<0.01, ***P<0.001;

[0153] The difference between the RGDV-GEN group and the GEN group was statistically significant (denoted by ▲, ▲P<0.05, ▲▲P<0.01).

[0154] Table 3 Inhibitory effect on 95D cells 48 h after administration ( n=3)

[0155]

[0156] Note: * indicates significant differences between GEN, RGDV-GEN and the control group, *P<0.05, **P<0.01, ***P<0.001;

[0157] The difference between RGDV-GEN and GEN was statistically significant (▲), where ▲P < 0.01.

[0158] b. The specific procedure for detecting changes in lung cancer cell apoptosis induced by genistein-modified drugs using the Annexin V-FITC / PI double staining method is as follows:

[0159] Log-phase lung cancer 95D and A549 cells were seeded in 6-well plates at a density of 1×10⁵ cells / well, with a final volume of 2 mL per well.

[0160] A blank control group and a genistein treatment group were set up with concentrations of 50, 100, and 150 μmol / L, respectively. A genistein-modified drug treatment group was set with concentrations of 50, 100, and 150 μmol / L. Each treatment group had three replicates. After cell seeding, the cells were cultured overnight at 37°C in a 5% CO2 cell culture incubator. The complete adhesion of the seeded cells was observed under a microscope. The drugs were then applied by diluting genistein and genistein-modified drugs to the above concentrations using DMEM, and 500 μL of the drug solution was added to each well.

[0161] The cells were cultured at 37℃ and 5% CO2 for 48 hours. After the drug treatment, the drug-containing culture medium was discarded, and an appropriate amount of pre-cooled PBS was added to wash away the residual culture medium and dead cells suspended on the surface. 1.5 mL of trypsin was added to digest the cell clusters into single cells. 3 mL of DMEM was added to stop the digestion and the single cells were collected into a 15 mL centrifuge tube. The tube was centrifuged at 300 g for 5 min, and the supernatant was discarded. The collected cell samples were washed once with pre-cooled PBS solution to wash away as much residual culture medium as possible.

[0162] Take 5 μL each of Annexin V-FITC and PI staining solutions and add them to 100 μL of PBS (this is the staining amount for one sample). Resuspend the cells and incubate them at room temperature in the dark for 25 min. After staining, divide the sample into two portions for later use.

[0163] Cell apoptosis was detected using FACS Calibur flow cytometry. A total of 10,000 cells were collected for each sample. Data were analyzed using Flowjo V10 software. Q1 represents the dead cell population, Q2 represents the late apoptotic population, Q3 represents the early apoptotic population, and Q4 represents the viable cell population. Specific results are as follows: Figure 2-4 As shown.

[0164] 95D and A549 cells were treated with different concentrations of GEN and RGDV-GEN, respectively. After 48 hours, the effects of GEN and RGDV-GEN on apoptosis of lung cancer 95D and A549 cells were detected by flow cytometry. Compared with the genistein-treated group, the genistein-modified drug group induced a higher proportion of apoptosis in A549 and 95D cells at the same concentration.

[0165] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0166] The foregoing embodiments and methods described in this invention may vary based on the capabilities, experience, and preferences of those skilled in the art.

[0167] The fact that the steps of the method are listed in a certain order in this invention does not constitute any restriction on the order of the method steps.

Claims

1. An anti-lung cancer drug or a salt thereof, having the following structure: ; in, R1 is a hydroxyl group; VDGR is a polypeptide of -Arg-Gly-Asp-Val.

2. The method for preparing the drug as described in claim 1, characterized in that, The drug is obtained by combining genistein with RGDV tumor-targeting peptides. Includes the following steps: (i) Compound 7 was dissolved, reacted with a basic reagent and an esterification reagent, and post-treated to give compound 8. The structure of compound 7 is as follows: ; The structure of compound 8 is as follows: ; (ii) Compound 8 was dissolved, reacted with an alkaline reagent, and post-treated to obtain compound 9, the structure of which is: ; (iii) Compound 9 was dissolved, and a catalyst, condensing agent, basic reagent, and Arg(NO2)-Gly-Asp(OBzl)-Val-Obzl were added sequentially. After reaction, the product was post-treated to obtain GEN-(Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl)2. The structure of GEN-(Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl)2 is as follows: ; (ⅳ) GEN-(Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl)2 is dissolved, a deprotecting agent is added, the reaction is carried out, and post-treatment is performed to obtain the drug. The method for preparing Arg(NO2)-Gly-Asp(OBzl)-Val-Obzl includes the following steps: (1) Dissolve Boc-Arg(NO2), add catalyst, condensing agent, alkaline reagent and HCl·Gly-OBzl in sequence, react and then process to obtain Boc-Arg(NO2)-Gly-Obzl; (2) Dissolve Boc-Arg(NO2)-Gly-Obzl, add alkaline reagent to react, and then treat to obtain Boc-Arg(NO2)-Gly-OH; (3) Dissolve Boc-Asp(OBzl), add catalyst, condensing agent, alkaline reagent and HCl·Val-OBzl in sequence, react and then process to obtain Boc-Asp(OBzl)-Val-OBzl; (4) Dissolve Boc-Asp(OBzl)-Val-OBzl, add deprotecting agent, react, and post-process to obtain Asp(OBzl)-Val-OBzl; (5) Dissolve Boc-Arg(NO2)-Gly-OH, add catalyst, condensing agent, basic reagent and Asp(OBzl)-Val-OBzl in sequence, react, and then process to obtain Boc-Arg(NO2)-Gly-Asp(OBzl)-Val-Obzl; (6) Dissolve Boc-Arg(NO2)-Gly-Asp(OBzl)-Val-Obzl, add deprotecting reagent to react, and then perform post-treatment to obtain Arg(NO2)-Gly-Asp(OBzl)-Val-Obzl; The basic reagent used in the reaction described in step (i) is potassium carbonate, and the esterification reagent is ethyl bromoacetate; The alkaline reagent used in the reaction described in step (ii) is sodium hydroxide; The reaction time described in step (ii) is 1 hour; The catalyst mentioned in step (iii) is 1-hydroxybenzotriazole HOBt, the condensing agent is N,N'-dicyclohexylcarbodiimide DCC, and the basic reagent is N-methylmorpholine NMM; The deprotecting agent mentioned in step (iv) is one of trifluoromethanesulfonic acid (TFMSA) and / or hydrofluoric acid (HF); The catalyst mentioned in steps (1), (3), and (5) is 1-hydroxybenzotriazole HOBt, the condensing agent is N,N'-dicyclohexylcarbodiimide DCC, and the basic reagent is N-methylmorpholine NMM.

3. The preparation method according to claim 2, characterized in that, The reaction temperature described in step (i) is 15–35°C.

4. The preparation method according to claim 2, characterized in that, The reaction time described in step (i) is 1 to 8 days.

5. The preparation method according to claim 2, characterized in that, The reaction temperature described in step (ii) is 15–35°C.

6. The preparation method according to claim 2, characterized in that, The reaction temperature described in step (iii) is 15–35°C.

7. The preparation method according to claim 2, characterized in that, The reaction time described in step (iii) is 6 to 10 hours.

8. The preparation method according to claim 2, characterized in that, The reagent used for dissolution in step (iv) is at least one of trifluoroacetic acid, tetrahydrofuran, N,N-dimethylformamide and acetonitrile; The reaction temperature described in step (iv) is -5 to 15°C; The reaction time described in step (iv) is 0.5 to 4 hours.

9. The preparation method according to claim 2, characterized in that, The solvent used for dissolution in step (2) is at least one of methanol, ethanol, isopropanol, tetrahydrofuran, and acetonitrile.

10. The preparation method according to claim 2, characterized in that, The alkaline reagent mentioned in step (2) is sodium hydroxide.

11. The preparation method according to claim 2, characterized in that, The temperature of the reaction in step (2) is -5 to 15°C.

12. The preparation method according to claim 2, characterized in that, The reaction time in step (2) is 4 to 8 hours.

13. The preparation method according to claim 2, characterized in that, The reagent used for dissolution in steps (4) and (6) is anhydrous ethyl acetate.

14. The preparation method according to claim 2, characterized in that, The reaction temperature described in steps (4) and (6) is -5 to 15°C.

15. The preparation method according to claim 2, characterized in that, The reaction time described in steps (4) and (6) is 1 to 3 hours.

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