Fluorodeoxyuridine monophosphate prodrug and application thereof in preparation of medicine for treating liver cancer

Abstract

The invention discloses a fluorodeoxyuridine monophosphate prodrug as shown in a formula I, application of the prodrug in preparation of a medicine for treating liver cancer, a pharmaceutical composition containing the prodrug and pharmaceutically acceptable auxiliary materials or taraxasterol and application of the prodrug in preparation of the medicine for treating liver cancer, and belongs to the field of pharmacy. The compound is modified by specific natural amino acid residues and can be used for preparing medicines for treating liver cancer. The in-vitro and in-vivo anti-tumor activity of the compound is remarkably superior to that of the prior art, the compound has a synergistic effect when being combined with taraxasterol, the highest tumor inhibition rate reaches 91.72%, the compound is high in selectivity, good in safety and high in liver targeting performance, and a more effective new scheme is provided for liver cancer treatment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pharmacy, and particularly relates to a fluorodeoxyuridine monophosphate prodrug, a pharmaceutical composition comprising the compound, and the use thereof in the preparation of a medicament for treating liver cancer. BACKGROUND

[0002] Liver cancer is a malignant tumor with high morbidity and mortality worldwide, and is showing an increasing trend year by year. According to the report of the National Cancer Center (NCC) in 2024, among all cancers, the incidence of liver cancer has risen to the fourth place, reaching a total of 367,700 cases per year; the mortality rate ranks second, with an average of 316,500 deaths per year. Liver cancer has characteristics such as high malignancy, strong invasiveness, easy metastasis, and diagnosis in the middle and late stages. So far, the effective treatment options are very limited, and the effective rate of drugs such as immune checkpoint inhibitors and targeted kinase inhibitors is less than 10%.

[0003] Fluorouracil (5-FU) has been a basic drug for treating digestive system tumors (such as colorectal cancer, gastric cancer, liver cancer, etc.), head and neck cancer, breast cancer, etc. since it was synthesized in 1957 and used clinically in 1960. However, 5-FU has low oral bioavailability, and is mostly used for intravenous or arterial infusion in clinical practice. It has a short half-life, high toxicity, poor tumor targeting, easy induction of gastrointestinal reactions, myelosuppression, and drug resistance, which limits its efficacy. In the non-surgical treatment of liver cancer, the only effective treatment option so far is to continuously inject high-dose 5-FU through the hepatic artery to maintain a high local drug concentration in the liver, and its effective rate can reach about 60%. When 5-FU enters the body, more than 85% of it is first degraded by the liver cells dihydropyrimidine dehydrogenase DPD into alpha-fluoro-beta-alanine (FBAL), which is the main source of cardiotoxicity and neurotoxicity. The rapid metabolism of 5-FU by liver DPD leads to a sharp decrease in the concentration of 5-FU in the blood, which is the main reason for the reduced efficacy of 5-FU.

[0004] In fact, 5-FU must undergo a series of metabolic processes in cells before it can release its final active product, 5-fluoro-2'-deoxyuridine monophosphate (FdUMP). FdUMP mainly inhibits thymidylate synthase (TS) to inhibit DNA synthesis in cancer cells. In addition, 5-FU can also be converted into 5-fluorouridine triphosphate (FUTP) through the orotate phosphoribosyltransferase OPRT metabolic pathway to inhibit RNA synthesis in cancer cells.

[0005] To improve the in vivo activity and targeting of nucleoside drugs and reduce the above-mentioned toxic side effects due to the degradation products of 5-FU in the liver and blood, researchers have developed phosphoramidate prodrugs (ProTide strategy). This technology masks the phosphate group of nucleoside monophosphate through chemical modification, improves the drug's passage through the liver cell membrane, bypasses the metabolic process of dihydropyrimidine DPD in the liver, and improves the efficiency of the drug's phosphorylation activation in target cells. 5-Fluoro-2'-deoxyuridine (FdUR) is modified as a prodrug through a phosphoramidate structure, which utilizes the specific hydrolysis of the phosphoramidate bond function of the highly expressed histidine triad nucleotide-binding protein (Histidine triad nucleotide-binding protein 1, Hint1) in the liver to first release free nucleoside monophosphate / monophosphonate catalyzed by enzymes, achieve the "in situ activation" of the prodrug in liver cells, and then exert biological activity, thus achieving specific targeting. Therefore, the prodrug designed by the ProTide strategy will not have similar toxic side effects as 5-FU. The applicant's previous research (CN 118772216 A) shows that esterification modification of the 3'-OH group of 2'-deoxy-5-fluorouridine (FdUR) based on the ProTide strategy can significantly improve the anti-liver cancer activity. However, the activity, selectivity, and mechanism of action of the compounds in the prior art still need to be further optimized and clarified. SUMMARY

[0006] The purpose of the present application is to provide fluorodeoxyuridine monophosphate prodrugs, pharmaceutical compositions containing the compounds, and their use in the preparation of drugs for the treatment of liver cancer. Based on the foregoing prior art, the inventors have conducted in-depth research and structural optimization, and have designed and synthesized a series of novel fluorodeoxyuridine monophosphate prodrugs. Surprisingly, it was found that the specific compounds (such as DL-2, DL-5, DL-14, etc. as described below) provided by the present application are significantly superior to the compounds disclosed in the prior art (CN 118772216 A) and 5-FU itself in terms of in vitro anti-liver cancer cell activity. More importantly, the present application first elucidates through systematic in vitro and in vivo experiments that the use of such prodrugs in combination with Taraxasterol can produce a significant synergistic anti-tumor effect. The synergistic mechanism is related to the upregulation of the expression of Histidine Triad Nucleotide Binding Protein 1 (HINT1) in tumor cells by Taraxasterol. This new combination therapy strategy and its mechanism discovery provide a more effective solution for the treatment of liver cancer.

[0007] In a first aspect, the present application provides a compound as shown in general formula I, or a pharmaceutically acceptable salt, hydrate, solvate, polymorph or stereoisomer thereof:

[0008] wherein: R1 is an amino acid residue; the amino acid residue refers to the rest of a single natural amino acid after the alpha-carboxyl of the amino acid forms an ester bond with the oxygen atom at the 3' position of the sugar ring shown in general formula I; the natural amino acid is selected from valine, proline, tyrosine, cysteine, methionine, serine, lysine, arginine, histidine, glutamic acid, tryptophan, threonine, asparagine or glutamine.

[0009] In some specific embodiments, in the amino acid residue, the alpha-amino of the natural amino acid is protected by a protecting group selected from C 1-6 alkoxycarbonyl, C 1-6 alkylacyl or aryl C 1-6 alkoxycarbonyl.

[0010] In some specific embodiments, the natural amino acid is selected from proline, lysine, tryptophan, methionine, tyrosine, threonine, valine or glutamic acid.

[0011] Further, the compound is selected from one of the following ten compounds:

[0012] In a second aspect, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of the compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt, hydrate, solvate, polymorph or stereoisomer thereof, and a pharmaceutically acceptable excipient.

[0013] In a third aspect, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of the compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt, hydrate, solvate, polymorph or stereoisomer thereof, taraxasterol, and a pharmaceutically acceptable excipient.

[0014] Further, the mass ratio of the compound, or a pharmaceutically acceptable salt, hydrate, solvate, polymorph or stereoisomer thereof, to taraxasterol and the pharmaceutically acceptable excipient is 80:1 to 10:1.

[0015] In a third aspect, the present application provides use of the above-mentioned compound, or a pharmaceutically acceptable salt, hydrate, solvate, polymorph or stereoisomer thereof, or the above-mentioned pharmaceutical composition in the preparation of a medicament for treating liver cancer.

[0016] Further, the liver cancer is primary liver cancer.

[0017] Compared with the prior art, the present application has the following beneficial technical effects. (1) The specific fluorodeoxyuridine monophosphate prodrug compound provided by the present application shows a significant increase in in vitro anti-liver cancer cell (such as HepG2) activity compared with the compound disclosed in the closest prior art (CN 118772216 A), has a lower IC 50 value, and means stronger inhibition effect. (2) The specific fluorodeoxyuridine monophosphate prodrug compound provided by the present application shows strong inhibition activity on various liver cancer cell lines (such as HepG2, HUH7) in vitro, and has very low toxicity to normal liver cells (such as HL-7702), which indicates that it has high selectivity. (3) In the nude mouse liver orthotopic tumor model, the specific fluorodeoxyuridine monophosphate prodrug compound provided by the present application shows strong antitumor effect, and its activity is better than that of 5-FU, and does not cause obvious toxic side effects to major organs, which indicates that it has good safety. (4) The fluorodeoxyuridine monophosphate prodrug of the present application is first proposed to be used in combination with taraxasterol, and it is confirmed that the two have a synergistic anti-liver cancer effect. The synergistic mechanism is related to the up-regulation of HINT1 protein expression in tumor cells by taraxasterol, thereby promoting the hydrolysis and activation of the prodrug. This combination strategy provides a new treatment idea for the field and greatly enhances the treatment effect. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is the PNMR data recorded in the metabolic hydrolysis process of the compound DL-2 synthesized by the present application under the action of carboxypeptidase Y. 31 PNMR data.

[0019] Figure 2 is the metabolic stability result graph of the compound DL-2 synthesized by the present application in rat plasma.

[0020] Figure 3 is the comparison of the liver orthotopic cancer tumor volume of nude mice treated with the compound DL-2, DL-5 synthesized by the present application and the control group and 5-FU treatment group, and the HE staining section of the liver of nude mice.

[0021] Figure 4 is the original graph of the inhibition effect of the compound DL-2 synthesized by the present application and the compound 11b disclosed in the prior art (CN 118772216 A) on the proliferation of human liver cancer cells inoculated in the liver of nude mice.

[0022] Figure 5 is the comparison of the liver orthotopic cancer tumor volume of nude mice treated with different dosages of the compound DL-2 synthesized by the present application and the HE staining graph of the liver of nude mice.

[0023] Figure 6 is the result graph of the WB experiment for detecting the up-regulation of HINT1 protein expression in liver cancer cells (HepG2 / HUH7) induced by taraxasterol.

[0024] Figure 7 This is a graph showing the synergistic inhibitory effect of the compound DL-2 synthesized in this invention in combination with taraxasterol on liver cancer cells (HepG2 / HUH7).

[0025] Figure 8 This figure shows the effect of the compound DL-2 synthesized in this invention, in combination with taraxasterol, on the expression of apoptosis-related proteins in tumor tissue.

[0026] Figure 9 These are H&E stained sections of the heart, liver, spleen, lungs, and kidneys of mice treated with compounds DL-2 and DL-5 synthesized in this invention.

[0027] Figure 10 This is a graph showing the effect of the compound DL-2 synthesized in this invention on the proliferation of knockdown cell line (HINT1-KD) and wild-type (WT) HUH7 cells.

[0028] Figure 11 The study compared the tumor volume of in situ hepatic carcinoma in nude mice treated with compound DL-2 with that in the sorafenib treatment group after the nude mice were inoculated in situ with knockdown cell lines (HINT1-KD) and wild-type (WT) HUH7 cells.

[0029] Figure 12 The present invention describes the inhibitory effect of the compound DL-2 synthesized in combination with taraxasterol on hepatic orthotopic tumors and subcutaneous xenografts. Detailed Implementation

[0030] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the protection scope of the present invention. Example 1

[0031] Synthesis of compounds.

[0032] The synthetic routes for two specific compounds, DL-5 and DL-12, are shown below. All compounds represented by general formula I can be prepared by referring to this synthetic route. The molar ratios of various reactants are based on stoichiometry, and the reaction temperature is room temperature.

[0033] (1) Using 5-fluoro-2'-deoxyuridine as raw material and N,N-dimethylformamide as solvent, under an anhydrous and oxygen-free argon atmosphere, it reacts with tert-butyldimethylsilane chloride to obtain 5-fluoro-2'-deoxyuridine with tert-butyldimethylsilyl group protection at the 5' position.

[0034] (2) Using 5'-O-tert-butyldimethylsilyl-5-fluoro-2'-deoxyuridine as the raw material and 1,4-dioxane as the solvent, 3'-O-tert-butyloxycarbonyl-5'-O-tert-butyldimethylsilyl-5-fluoro-2'-deoxyuridine was reacted with ditert-butyl dicarbonate in the presence of DMAP to obtain 3'-O-tert-butyloxycarbonyl-5'-O-tert-butyldimethylsilyl-5-fluoro-2'-deoxyuridine.

[0035] (3) Using 3'-O-tert-butyloxycarbonyl-5'-O-tert-butyldimethylsilyl-5-fluoro-2'-deoxyuridine as the raw material and tetrahydrofuran as the solvent, it reacts with 1 M tetra-n-butylammonium fluoride to obtain 3'-O-tert-butyloxycarbonyl-5-fluoro-2'-deoxyuridine.

[0036] (4) Using 3'-O-tert-butyloxycarbonyl-5-fluoro-2'-deoxyuridine as a raw material, under anhydrous and oxygen-free conditions and in an argon atmosphere, with 1 M tert-butylmagnesium chloride solution, it reacts with N-[(S)-(2,3,4,5,6-pentafluorophenoxy)phenoxyphosphoryl]-L-alanine isopropyl ester to obtain 3'-O-tert-butyloxycarbonyl-5-fluoro-2'-deoxyuridine-5'-O-[phenyl(isopropoxy-L-alanyl)] phosphate.

[0037] (5) 3'-O-tert-butoxycarbonyl-5-fluoro-2'-deoxyuridine-5'-O-[phenyl(isopropoxy-L-alanyl)] phosphate reacts with trifluoroacetic acid to give 5-fluoro-2'-deoxyuridine-5'-O-[phenyl(isopropoxy-L-alanyl)] phosphate.

[0038] (6) 5-Fluoro-2'-deoxyuridine-5'-O-[phenyl(isopropoxy-L-alanyl)] phosphate, in anhydrous dichloromethane as solvent, reacted with Boc-L-methionine in the presence of EDCI and DMAP to give 3'-O-(tert-butoxycarbonyl-L-methionyl)-5-fluoro-2'-deoxyuridine-5'-O-[phenyl(isopropoxy-L-alanyl)] phosphate. This yielded compound DL-5.

[0039] (7) 3'-O-(tert-butoxycarbonyl-L-methionyl)-5-fluoro-2'-deoxyuridine-5'-O-[phenyl(isopropoxy-L-alanyl)] phosphate was reacted with trifluoroacetic acid to give 3'-O-(L-methionyl)-5-fluoro-2'-deoxyuridine-5'-O-[phenyl(isopropoxy-L-alanyl)] phosphate. This yielded compound DL-12. Example 2

[0040] Preparation and characterization of specific compounds.

[0041] The following fluorodeoxyuridine monophosphate prodrugs were prepared according to the synthetic route in Example 1, and were named and characterized. All compounds were characterized by proton and carbon NMR spectra on a JNM-EPC 600 MHz NMR spectrometer. All compounds were characterized by high-resolution mass spectrometry (HRMS) on a QTOF Ultima GLOBAL spectrometer.

[0042]

[0043] DL-2 (2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2- ((((S)-(((S)-1-isopropoxy-1-oxopropan-2-yl)amino)(phenoxy)phosphoryl)oxy) methyl)tetrahydrofuran-3-yl N 2 ,N 6 -bis(tert-butoxycarbonyl)-L-lysinate 1 H NMR (400 MHz, Chloroform-d) δ 7.82 (d, J = 6.3 Hz, 1H), 7.33 –7.28 (m, 2H), 7.21 (d, J = 7.9 Hz, 2H), 7.15 (t, J = 7.3 Hz, 1H), 6.18 (ddd,J = 8.9, 5.2, 1.6 Hz, 1H), 5.29 (d, J = 7.2 Hz, 1H), 5.19 (d, J = 7.4 Hz,1H), 5.01 (hept, J = 6.3 Hz, 1H), 4.69 (t, J = 6.1 Hz, 1H), 4.36 (dh, J =6.0, 2.6 Hz, 2H), 4.14 (p, J = 2.5 Hz, 1H), 4.02 – 3.76 (m, 2H), 3.10 (d, J =6.4 Hz, 2H), 2.42 (dd, J = 14.2, 5.3 Hz, 1H), 1.94 – 1.72 (m, 2H), 1.49 (dd,J = 14.4, 7.3 Hz, 3H), 1.43 (d, J = 1.5 Hz, 18H), 1.39 (s, 2H), 1.36 (d, J =6.9 Hz, 3H), 1.22 (dd, J = 6.2, 1.8 Hz, 6H). 13C NMR (100 MHz, Chloroform-d) δ172.95, 157.06, 156.80, 150.43, 148.81, 129.91, 125.46, 120.40 (d, J = 4.8Hz), 85.20, 83.25, 80.26, 79.33, 74.96, 69.60, 66.22, 53.74, 50.52, 39.78,37.33, 29.75, 28.44 (d, J = 15.2 Hz), 22.65, 21.72 (d, J = 5.6 Hz), 21.06.HRESIMS m / z 866.3323 [M+Na] + (calcd for C 37 H 55 FN5O 14 P + 866.3359) DL-3 tert-butyl 3-((S)-2-((tert-butoxycarbonyl)amino)-3-(((2R,3S,5R)-5- fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-((((S)-(((S)-1-isopropoxy- 1-oxopropan-2-yl)amino)(phenoxy)phosphoryl)oxy)methyl)tetrahydrofuran-3-yl) oxy)-3-oxopropyl)-1H-indole-1-carboxylate

[0044] 1 H NMR (400 MHz, Chloroform-d) δ 7.78 (d, J = 6.2 Hz, 1H), 7.51 –7.43 (m, 2H), 7.34 – 7.27 (m, 3H), 7.25 (dd, J = 7.6, 1.1 Hz, 2H), 7.23 –7.12 (m, 3H), 6.06 – 6.00 (m, 1H), 5.23 – 5.20 (m, 1H), 5.08 (d, J = 7.5 Hz,1H), 5.00 (p, J = 6.2 Hz, 1H), 4.58 (q, J = 6.8 Hz, 1H), 4.30 – 4.16 (m, 2H),3.99 – 3.92 (m, 1H), 3.88 (s, 1H), 3.82 – 3.74 (m, 1H), 3.18 (d, J = 6.5 Hz,2H), 2.29 (dd, J = 14.1, 5.3 Hz, 1H), 1.65 (s, 8H), 1.42 (s, 9H), 1.36 (d, J= 7.0 Hz, 4H), 1.21 (d, J = 6.3 Hz, 6H).13 C NMR (100 MHz, Chloroform-d) δ172.87 (d, J = 7.6 Hz), 171.87, 156.97, 150.38 (d, J = 6.6 Hz), 149.60,148.65, 135.45, 130.23, 129.91, 125.46, 124.88, 124.30, 123.84, 122.81,120.38 (d, J = 4.6 Hz), 118.88, 115.53, 114.74, 85.25, 84.06, 83.03, 80.51,75.44, 69.62, 66.16, 53.84, 50.52, 37.33, 28.30 (d, J = 6.5 Hz), 27.75, 21.71(d, J = 4.6 Hz), 21.11. HRESIMS m / z 924.3189 [M+Na] + (calcd for C 42 H 53 FN5NaO 14 P + 924.3203) DL-4 5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-((((S)-(((S)- 1-isopropoxy-1-oxopropan-2-yl)amino)(phenoxy)phosphoryl)oxy)methyl) tetrahydrofuran-3-yl (2S)-2-((tert-butoxycarbonyl)amino)-3-(4-((tert- butoxycarbonyl)oxy)phenyl)propanoate

[0045] 1 H NMR (400 MHz, DMSO- d 6) δ 7.88 (d, J = 6.8 Hz, 1H), 7.44 (d, J = 7.7Hz, 1H), 7.34 – 7.29 (m, 2H), 7.25 (d, J = 8.5 Hz, 2H), 7.17 (d, J = 8.5 Hz,2H), 7.06 (d, J = 8.6 Hz, 2H), 6.05 (d, J = 3.2 Hz, 1H), 5.18 – 5.12 (m, 1H),4.83 (p, J = 6.3 Hz, 1H), 4.15 (dt, J= 14.8, 7.1 Hz, 3H), 3.96 (s, 1H), 3.77(h, J = 7.0 Hz, 1H), 2.99 – 2.84 (m, 2H), 2.18 (t, J = 6.6 Hz, 2H), 1.43 (s,9H), 1.31 (s, 8H), 1.18 (d, J = 7.3 Hz, 3H), 1.11 (d, J = 6.3 Hz, 6H). 13 C NMR(100 MHz, Chloroform- d ) δ 172.91, 171.67, 156.99, 156.73, 151.92, 150.39,148.68, 133.26, 130.19, 129.91, 125.45, 121.74, 120.36 (d, J = 4.5 Hz),85.34, 83.73, 83.01, 80.55, 75.32, 69.61, 66.24, 54.71, 50.52, 37.61 (d, J =31.3 Hz), 28.34, 27.75, 21.72 (d, J = 5.2 Hz), 21.15, 1.09. HRESIMS m / z901.3031 [M+Na] + (calcd for C 40 H 52 FN4NaO 15 P + 901.3043) DL-5 (2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2- ((((S)-(((S)-1-isopropoxy-1-oxopropan-2-yl)amino)(phenoxy)phosphoryl)oxy) methyl)tetrahydrofuran-3-yl (tert-butoxycarbonyl)-L-methioninate

[0046] 1 H NMR (400 MHz, Chloroform- d ) δ 7.82 (d, J = 6.2 Hz, 1H), 7.31 (dd, J = 8.6, 7.2 Hz, 2H), 7.21 (d, J= 8.8 Hz, 2H), 7.17 – 7.13 (m, 1H), 6.19(ddd, J = 9.1, 5.3, 1.7 Hz, 1H), 5.34 – 5.29 (m, 1H), 5.14 (d, J = 8.0 Hz,1H), 5.04 – 4.97 (m, 1H), 4.41 – 4.31 (m, 3H), 4.19 (t, J = 2.4 Hz, 1H), 4.01– 3.91 (m, 1H), 3.88 – 3.79 (m, 1H), 2.56 (td, J = 7.5, 2.6 Hz, 2H), 2.44(dd, J = 14.3, 5.2 Hz, 1H), 2.09 (s, 4H), 1.92 (ddt, J = 22.9, 15.2, 8.0 Hz,2H), 1.44 (s, 9H), 1.36 (d, J = 7.0 Hz, 3H), 1.22 (dd, J = 6.3, 1.5 Hz, 6H). 13 C NMR (100 MHz, Chloroform- d ) δ 172.92 (d, J = 7.4 Hz), 172.11, 157.07,156.80, 155.51, 150.38 (d, J = 6.6 Hz), 148.83, 141.92, 139.55, 129.92,125.47, 124.15, 123.80, 120.37 (d, J = 4.6 Hz), 85.24, 83.18 (d, J = 7.4 Hz),80.49, 75.35, 69.62, 66.21, 53.01, 50.52, 37.39, 31.36, 30.14, 28.35, 21.72(d, J = 5.6 Hz), 21.08 (d, J = 5.2 Hz), 15.65. HRESIMS m / z 769.2300 [M+Na] + (calcd for C31 H 44 FN4NaO 12 PS + 769.2290) DL-6 1-(tert-butyl) 2-((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4- dihydropyrimidin-1(2H)-yl)-2-((((S)-(((S)-1-isopropoxy-1-oxopropan-2-yl) amino)(phenoxy)phosphoryl)oxy)methyl)tetrahydrofuran-3-yl) (S)-pyrrolidine-1, 2-dicarboxylate

[0047] 1 H NMR (400 MHz, Chloroform-d) δ 7.84 (t, J = 6.0 Hz, 1H), 7.33 –7.27 (m, 2H), 7.21 (d, J = 8.0 Hz, 2H), 7.17 – 7.12 (m, 1H), 6.19 (td, J =9.5, 5.0 Hz, 1H), 5.31 – 5.26 (m, 1H), 5.01 (p, J = 6.3 Hz, 1H), 4.36 (dd, J= 6.2, 2.6 Hz, 2H), 4.26 (ddd, J = 18.2, 8.9, 4.1 Hz, 1H), 4.14 (dt, J =13.1, 3.0 Hz, 1H), 4.02 – 3.91 (m, 1H), 3.78 (q, J = 10.2 Hz, 1H), 3.53 –3.45 (m, 1H), 3.45 – 3.35 (m, 1H), 2.40 (dd, J = 14.1, 5.3 Hz, 1H), 2.24 (td,J = 12.8, 10.5, 6.2 Hz, 1H), 1.98 – 1.85 (m, 4H), 1.44 (s, 5H), 1.40 (s, 4H),1.36 (d, J = 7.1 Hz, 3H), 1.22 (dd, J = 6.3, 1.9 Hz, 6H). 13C NMR (100 MHz,Chloroform-d) δ 173.13 – 172.56 (m), 157.00, 154.50, 148.84, 129.90, 125.46,120.42 (d, J = 8.8 Hz), 85.15, 83.44, 80.26, 74.77, 69.60, 66.31, 58.88 (d, J= 14.1 Hz), 50.52, 46.58 (d, J = 21.1 Hz), 37.31, 30.98, 29.93, 28.44, 24.61,23.70, 21.72 (d, J = 5.3 Hz), 21.12. HRESIMS m / z 735.2431 [M+Na] + (calcd forC 31 H 42 FN4NaO 12 P + 735.2413 ) DL-11 (2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2- ((((S)-(((S)-1-isopropoxy-1-oxopropan-2-yl)amino)(phenoxy)phosphoryl)oxy) methyl)tetrahydrofuran-3-yl L-valinate

[0048] 1 H NMR (400 MHz, DMSO- d 6) δ 7.92 (d, J = 6.8 Hz, 1H), 7.32 (t, J = 7.8Hz, 2H), 7.16 (t, J = 7.7 Hz, 4H), 6.14 – 6.06 (m, 1H), 6.03 (d, J = 11.6 Hz,1H), 4.83 (p, J = 6.5 Hz, 1H), 4.26 – 4.09 (m, 4H), 3.82 – 3.70 (m, 1H), 3.12(d, J = 5.3 Hz, 1H), 2.25 (d, J = 6.0 Hz, 2H), 1.83 (h, J = 6.9 Hz, 1H), 1.18(d, J = 7.8 Hz, 4H), 1.12 (d, J = 5.7 Hz, 6H), 0.85 (d,J = 6.7 Hz, 3H), 0.81(d, J = 6.8 Hz, 3H). 13 C NMR (100 MHz, Methanol- d 4) δ 173.88, 172.97, 150.68(d, J = 7.0 Hz), 129.53, 125.04, 124.30, 123.96, 120.20 (d, J = 4.7 Hz),85.62, 83.09 (d, J = 8.1 Hz), 74.84, 68.90, 66.28, 59.36, 50.41, 36.79,31.87, 29.41, 20.61 (d, J = 7.8 Hz), 19.18 (d, J = 6.7 Hz), 17.96, 16.73.HRESIMS m / z 637.2029 [M+ Na] + (calcd for C 26 H 36 FN4NaO 10 P + 637.2045). DL-12 (2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2- ((((S)-(((S)-1-isopropoxy-1-oxopropan-2-yl)amino)(phenoxy)phosphoryl)oxy) methyl)tetrahydrofuran-3-yl L-methioninate 1 H NMR (400 MHz, DMSO- d 6) δ 7.93 (dd, J = 6.9, 2.2 Hz, 1H), 7.36 –7.30 (m, 2H), 7.15 (dd, J = 14.4, 7.5 Hz, 3H), 6.17 (t, J = 7.4 Hz, 1H), 6.06(t, J = 11.4 Hz, 1H), 5.30 (d, J = 5.1 Hz, 1H), 4.83 (p, J = 6.3 Hz, 1H),4.25 (d, J = 4.8 Hz, 2H), 4.20 (dd, J= 10.4, 3.8 Hz, 1H), 3.77 (q, J = 8.3,7.8 Hz, 1H), 2.57 (tq, J = 14.1, 7.0, 6.5 Hz, 2H), 2.31 (t, J = 7.0 Hz, 2H),2.07 (dd, J = 14.9, 7.7 Hz, 2H), 2.02 (s, 3H), 1.19 (d, J = 7.3 Hz, 3H), 1.12(dd, J = 6.3, 2.2 Hz, 6H). 13 C NMR (100 MHz, Methanol- d 4) δ 168.69, 149.30,129.55, 125.09, 124.41, 124.06, 120.15 (d, J = 4.6 Hz), 114.87, 85.67, 82.61,76.76, 68.94, 51.42, 36.62, 29.12, 28.73, 20.60 (d, J = 7.9 Hz), 19.17,13.70. HRESIMS m / z 669.1751 [M+H] + (calcd for C 26 H 36 FN4NaO 10 PS + 669.1766). DL-14 (2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2- ((((S)-(((S)-1-isopropoxy-1-oxopropan-2-yl)amino)(phenoxy)phosphoryl)oxy) methyl)tetrahydrofuran-3-yl L-tyrosinate

[0049] 1 H NMR (400 MHz, Methanol- d 4) δ 7.79 (d, J = 6.5 Hz, 1H), 7.37 – 7.30(m, 2H), 7.25 – 7.20 (m, 2H), 7.17 (t, J = 7.9 Hz, 1H), 7.09 – 7.05 (m, 2H),6.80 – 6.76 (m, 2H), 6.00 (ddd,J = 8.8, 5.4, 1.6 Hz, 1H), 5.32 (dt, J = 6.6,1.8 Hz, 1H), 4.95 (p, J = 6.3 Hz, 1H), 4.32 – 4.23 (m, 3H), 3.97 (dq, J =6.9, 4.1, 3.5 Hz, 1H), 3.92 – 3.85 (m, 1H), 3.32 (s, 1H), 3.11 (qd, J = 14.2,7.3 Hz, 2H), 2.38 (ddd, J = 14.5, 5.6, 1.6 Hz, 1H), 2.14 (ddd, J = 14.8, 8.8,6.5 Hz, 1H), 1.32 (dd, J = 7.2, 1.1 Hz, 3H), 1.20 (d, J = 6.2 Hz, 6H). 13 C NMR(100 MHz, Methanol- d 4) δ 172.97, 168.64, 157.15, 150.67, 149.22, 141.73,139.39 (d, J = 2.0 Hz), 130.23, 129.55, 125.09, 124.31, 124.02, 120.16 (d, J = 4.7 Hz), 115.65, 85.58, 82.54 (d, J = 8.1 Hz), 76.42, 68.94, 66.12, 53.98,36.63, 35.57, 20.60 (d, J = 8.0 Hz), 19.15 (d, J = 6.7 Hz). HRESIMS m / z 677.2020 [M-H] - (calcd for C 30 H 35 FN4O 11 P - 677.2029). DL-17 (2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2- ((((S)-(((S)-1-isopropoxy-1-oxopropan-2-yl)amino)(phenoxy)phosphoryl)oxy)methyl)tetrahydrofuran-3-yl L-threoninate 1 H NMR (400 MHz, Methanol- d 4) δ 7.84 (d, J = 6.5 Hz, 1H), 7.33 (dd, J = 8.6, 7.2 Hz, 2H), 7.25 – 7.21 (m, 2H), 7.20 – 7.15 (m, 1H), 6.19 (ddd, J =8.8, 5.5, 1.6 Hz, 1H), 5.46 (dt, J = 6.7, 1.9 Hz, 1H), 4.95 (p, J = 6.3 Hz,1H), 4.36 (dd, J = 6.5, 3.3 Hz, 2H), 4.34 – 4.30 (m, 2H), 4.02 (d, J = 3.7Hz, 1H), 3.89 (dd, J = 10.2, 7.1 Hz, 1H), 2.49 (ddd, J = 14.4, 5.6, 1.7 Hz,1H), 2.22 (ddd, J = 14.8, 8.7, 6.6 Hz, 1H), 1.32 (d, J = 6.7 Hz, 6H), 1.20(dd, J = 6.3, 1.2 Hz, 6H). 13 C NMR (100 MHz, Methanol- d 4) δ 172.96, 167.51,149.29, 134.81, 129.55, 127.43, 125.11, 124.33, 123.98, 120.15 (d, J = 4.6Hz), 118.25, 85.43, 82.66, 77.15, 68.95, 66.16, 51.57, 36.33, 25.26, 20.58(d, J = 7.6 Hz), 19.12 (d, J = 6.4 Hz). HRESIMS m / z 639.1821 [M+ Na] +(calcdfor C 25 H 34 FN4NaO 11 P + 639.1838). DL-18 (S)-2-amino-5-(((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4- dihydropyrimidin-1(2H)-yl)-2-((((S)-(((S)-1-isopropoxy-1-oxopropan-2-yl) amino)(phenoxy)phosphoryl)oxy)methyl)tetrahydrofuran-3-yl)oxy)-5-oxopentanoic acid

[0050] 1 H NMR (400 MHz, Methanol- d 4) δ 7.82 (d, J = 6.5 Hz, 1H), 7.33 (dd, J = 8.4, 7.3 Hz, 2H), 7.26 – 7.20 (m, 2H), 7.17 (td, J = 7.4, 1.1 Hz, 1H), 6.15(ddd, J = 8.8, 5.4, 1.6 Hz, 1H), 5.32 (dt, J = 6.7, 1.9 Hz, 1H), 4.95 (p, J =6.3 Hz, 1H), 4.34 (dd, J = 6.2, 3.2 Hz, 2H), 4.26 (q, J = 2.7 Hz, 1H), 4.03(t, J = 6.8 Hz, 1H), 3.89 (dd, J = 10.2, 7.1 Hz, 1H), 2.68 – 2.60 (m, 2H),2.41 (ddd, J = 14.3, 5.6, 1.7 Hz, 1H), 2.24 (dt, J = 14.5, 7.2 Hz, 1H), 2.19– 2.09 (m, 2H), 1.32 (dd, J = 7.1, 1.1 Hz, 3H), 1.20 (dd, J = 6.3, 1.0 Hz,6H). 13 C NMR (100 MHz, Methanol- d4) δ 172.98, 171.82, 170.08, 150.71, 149.32,141.75, 139.42, 129.53, 125.06, 124.37, 124.03, 120.19 (d, J = 4.8 Hz), 85.52, 83.10 (d, J = 7.8 Hz), 74.80, 68.92, 66.27, 51.71, 50.42, 36.72,29.10, 25.09, 20.59 (d, J = 7.0 Hz), 19.17 (d, J = 6.4 Hz). HRESIMS m / z 643.1811 [MH] - (calcd for C 26 H 33 FN4O 12 P - 643.1822). Example 3

[0051] In vitro anti-proliferative activity of the compound against liver cancer cells and comparison with normal cells (IC50 by CCK-8 assay) 50 value).

[0052] (1) Experimental methods HepG2 and HUH7 human liver cancer cells, as well as HL-7702 normal human liver cells, were collected and placed in 96-well plates. Compounds of various concentration gradients were added, resulting in a final volume of 200 mL per well. Cells were incubated at 37°C for 48 hours, followed by the addition of 20 mL of CCK-8 to each well, and incubation at 37°C for another 4 hours. The supernatant was removed, and the residue was dissolved in DMSO. Absorbance was recorded at 490 nm using SpectraMax to calculate the cell proliferation inhibition rate and IC50 of the compounds. 50 value.

[0053] IR (%) = [1 - (OD) Drug -OD Blank ) / (OD Control -OD Blank )]×100.

[0054] In this experiment, compound IC 50 The values ​​were calculated and analyzed using the software GraphPad Prism 7.0.

[0055] Table 1

[0056] The experimental results are shown in Table 1. The results indicate that the tested compounds exhibited superior inhibitory activity against HepG2 cells compared to the 5-FU group. Among them, compounds DL-2, DL-5, and DL-14 showed superior inhibitory activity against liver cancer cells compared to compound 11b disclosed in the prior art (CN 118772216 A). In the HepG2 cell line, compound DL-5 showed an activity more than 100-fold higher than the 5-FU group. These findings suggest that compounds DL-2, DL-5, DL-14, and this series of prodrugs have broad application prospects in the preparation of drugs for treating liver cancer-related diseases. Example 4

[0057] Carboxypeptidase Y (EC 3.4.16.1) assay.

[0058] First, dissolve compound DL-2 (5 mg) in acetone-d6 (0.15 mL), then add 0.30 mL of Trizma buffer (pH 7.6). Use at 25°C. 31 P NMR data were used as a control. Then, carboxypeptidase Y (0.1 mg dissolved in 0.15 mL Trizma) was added to the sample solution, and the NMR data were analyzed at specific time points. 31 The P NMR was measured at 25°C, and the spectrum was recorded by 64 scans. 31 Data recorded by pNMR were analyzed using the MNova program. Carboxypeptidase Y and Trizma buffer were purchased from Beijing Jinming Biotechnology Co., Ltd.

[0059] The study used carboxypeptidase Y. Compound DL-2 and carboxypeptidase Y were dissolved in acetone-d6 and Trizma buffer (pH 7.6) and used at 25°C. 31 P NMR data was recorded continuously for 80 minutes, with scans acquired every 10 minutes. The recorded spectrum is as follows: Figure 1 As shown, compound DL-2 was rapidly hydrolyzed into intermediate metabolites a (δ 5.23 ppm) and b (7.34 ppm) lacking ester motifs. After 80 minutes, compound DL-2 was mainly metabolized into intermediate B. This indicates that compound DL-2 can undergo initial activation under the action of carboxypeptidase Y, releasing intermediate metabolite B to participate in subsequent biological metabolic activation, thus enabling the final delivery of the effective form of 5-fluorodeoxyuridine monophosphate in tumor cells. Example 5

[0060] In vitro metabolic stability assay.

[0061] Compound DL-2 (1 mg / mL in acetonitrile) was incubated in rat plasma at 37°C for 0 min, 0.5 h, 1 h, 2 h, 4 h, 8 h, 24 h, and 48 h. At each time point, each sample solution was diluted and quenched with 600 μL of acetonitrile, shaken for 30 seconds, and centrifuged at 12000 rpm for 10 min. The solution was then incubated in a container equipped with C... 18 Samples were analyzed by HPLC on an Agilent 1260 HPLC instrument with a column (2.1 mm × 100 mm, 3.5 μm). Compounds were eluted with MeOH / phosphate buffered saline over 20 minutes. Absorbance was measured at 268 nm at a flow rate of 1 mL / min.

[0062] The in vitro metabolic stability of compound DL-2 was studied, and the results are as follows: Figure 2 As shown, compound DL-2 is stable in rat plasma, and more than 50% of the original drug residue was observed after 48 hours of culture. Example 6

[0063] The inhibitory effects of compounds DL-2 and DL-5 on the proliferation of human hepatocellular carcinoma cells in nude mice in situ.

[0064] Twenty-five Balb / c nude mice were used. After laparotomy, HepG2 cells (2 × 10⁻⁶) were extracted. 6 50 μL of DL-2 (cells / mL) was seeded into the right lobe of the liver. Three days later, mice were randomly divided into four groups and injected with the drug via the tail vein. DL-2 and DL-5 were administered at a dose of 45 mg / kg / 2 days, every two days, with 5-FU as a control. The vector consisted of 8% Tween 80, 2% dimethyl sulfoxide, and 90% saline. On day 21, mice were euthanized, and the liver was dissected. The length, width, and height of the hepatocellular carcinoma tumor were measured. The tumor data were analyzed using the DRAP package software in R, and the inhibition rate was calculated. The liver tissue was further fixed, stained with hematoxylin and eosin (HE), and the infiltration and proliferation of intrahepatic cancer cells were observed to evaluate the inhibitory activity of the compound (see [link to relevant documentation]). Figure 3 The results showed that the inhibition rates of DL-2 and DL-5 were 68.73% and 75.61%, respectively. The control group showed no significant inhibitory activity from 5-FU. Example 7

[0065] The inhibitory effects of compounds DL-2 and 11b on the proliferation of human hepatocellular carcinoma cells in nude mice in situ were compared.

[0066] Twenty-five Balb / c nude mice were used. After laparotomy, HUH7 cells (2×10⁻⁶) were extracted. 650 μL of DL-2 (cells / mL) was seeded into the right lobe of the liver. Three days later, mice were randomly divided into four groups and injected with the drug via the tail vein. DL-2 and 11b were administered at a dose of 45 mg / kg / 2d every two days, with a blank control group serving as the standard. The vector consisted of 8% Tween 80, 2% dimethyl sulfoxide, and 90% saline. On day 21, mice were euthanized, and the liver was dissected. The length, width, and height of the hepatocellular carcinoma tumor were measured. The tumor data were analyzed using the DRAP package software in R, and the inhibition rate was calculated. The liver tissue was further fixed, stained with hematoxylin and eosin (HE), and the infiltration and proliferation of intrahepatic cancer cells were observed to evaluate the inhibitory activity of the compound (see [link to relevant documentation]). Figure 4 The results showed that the inhibition rates of 11b and DL-2 were 41.1% and 68.5%, respectively. At the same dose, DL-2 showed significantly better in vivo anti-hepatotum activity than compound 11b disclosed in the prior art (CN 118772216 A). Example 8

[0067] Dose-effect relationship of compound DL-2 was investigated.

[0068] Twenty-five Balb / c nude mice were used. After laparotomy, HepG2 cells (2 × 10⁻⁶) were extracted. 6 50 μL of DL-2 (cells / mL) was seeded into the right lobe of the liver. Three days later, mice were randomly divided into four groups and injected with the drug via the tail vein. DL-2 was administered at three doses: low (15 mg / kg / 2d), medium (45 mg / kg / 2d), and high (135 mg / kg / 2d). The carrier consisted of 8% Tween 80, 2% dimethyl sulfoxide, and 90% saline. On day 21, the mice were euthanized, and the liver was dissected. The length, width, and height of the hepatocellular carcinoma tumor were measured. The tumor data were analyzed using the DRAP package software in R, and the inhibition rate was calculated. The liver tissue was further fixed, stained with hematoxylin and eosin (HE), and the infiltration and proliferation of intrahepatic cancer cells were observed to evaluate the inhibitory activity of the compound (see [link to relevant documentation]). Figure 5 The results showed that the antitumor activity of DL-2 in vivo gradually increased with increasing dose, and the inhibition rate reached almost 100% at a dose of 135 mg / kg (the statistical data had ±5% SD). Example 9

[0069] Study on the synergistic antitumor effects and mechanisms of the compound and taraxasterol.

[0070] (1) Dandelion sterol upregulates HINT1 expression in liver cancer cells Experimental methods: HepG2 and HUH7 cells were treated with different concentrations (0, 5, 10, 20 μM) of taraxasterol for 24 or 48 hours, and the expression level of HINT1 in the cells was detected by Western blotting.

[0071] Experimental results: Taraxacum sterol dose-dependently upregulated HINT1 expression levels in two types of hepatocellular carcinoma cells, indicating that taraxasterol can affect the metabolic activation of phosphoramide ester prodrugs by regulating HINT1 expression (see details). Figure 6 ).

[0072] (2) Taraxasterol enhances the anti-hepatocellular carcinoma effect of compound DL-2. Experimental Methods: A human hepatocellular carcinoma (HCC) cell line (HUH7) was established by inoculating nude mice with hepatic orthotopic tumors. The mice were divided into the following groups: solvent control, DL-2 monotherapy (45 mg / kg), taraxasterol monotherapy (1 mg / kg), and a combination of DL-2 (45 mg / kg) and taraxasterol (1 mg / kg). The treatment regimen was the same as in Example 6. After treatment, the volume and weight of the hepatocellular carcinoma were assessed. The expression levels of HINT1 and apoptosis-related proteins in the tumor tissue were analyzed by Western blotting and immunohistochemistry (IHC).

[0073] Experimental results: as shown in Table 2, Figure 7 and Figure 8 As shown.

[0074] 1. Combined anticancer effect: DL-2 alone showed an inhibition rate of 68.56% against liver cancer, while taraxasterol alone had no significant inhibitory activity. The control drug sorafenib showed an inhibition rate of 41.78%. However, DL-2 combined with taraxasterol at doses of 2.5 mg / kg, 5 mg / kg, and 10 mg / kg significantly improved the inhibition rate of DL-2, reaching 69.63%, 71.74%, and 91.72%, respectively. The mean tumor volume and weight in the combined treatment group were significantly smaller than those in the DL-2 monotherapy group and the taraxasterol monotherapy group. P <0.0001), the tumor inhibition rate was the highest, showing a significant synergistic anti-tumor effect (see details). Figure 7 (and Table 2).

[0075] 2. Mechanism Validation: Western blot and IHC analyses showed that the expression level of HINT1 protein in tumor tissues of the combination therapy group was higher than that of the control group and the single-drug group. Mechanistic studies showed that the apoptosis marker protein Cleaved Caspase-3 and the DNA damage marker protein γ-H2AX were most significantly expressed in the combination therapy group (see details). Figure 8 ).

[0076] Table 2

[0077] (3) Safety studies of compounds DL-2 and DL-5 After the experiment in Example 6 above was completed, major organs and serum were collected to show H&E stained sections of the heart, liver, spleen, lungs, and kidneys. Figure 9The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea (BUN), serum creatinine (CREA), and creatine kinase isoenzyme (CK-MB) in the experimental groups were not significantly different from those in the negative control group, indicating that there were no obvious toxic side effects at effective doses. However, compared with the 5-FU control group, the levels of the above indicators in mice were significantly increased after the administration of 5-FU, indicating that 5-FU has a certain toxic effect on mice, while compound DL-2 had no obvious toxic effect on mice.

[0078] Table 3

[0079] (4) Knocking down the HINT1 gene significantly weakened the anticancer effect of compound DL-2 (reverse verification experiment) Experimental Methods: HINT1 expression in HUH7 cells was knocked down using siRNA technology, and a knockdown cell line (HINT1-KD) was established. After stable passage of HINT1-KD cells, HINT1-KD cells and wild-type (WT) HUH7 cells were inoculated into nude mouse livers in situ and treated with DL-2. The anticancer efficacy of DL-2 on HINT1-KD cells and wild-type (WT) HUH7 cells was compared.

[0080] Experimental results: In vitro experiments showed that in HINT1-KD cells, DL-2 had an IC50 value of [missing information]. 50 The significantly increased value indicates that cells knocked down HINT1 have reduced sensitivity to DL-2; while DL-2 has a significantly higher IC50 value in wild-type (WT) HUH7 cells. 50 The value is very low ( Figure 10 ).

[0081] Further analysis was conducted on the effects of DL-2 on the proliferation of knockdown cell lines (HINT1-KD) and wild-type (WT) HUH7 cells in situ seeded from nude mouse livers. Figure 11 A. The results showed that DL-2 had a significantly stronger anti-cancer effect on wild-type HUH7 cells than the groups with knocked-out HINT1 or inhibited HINT1 expression. Example 10

[0082] Effects of compound DL-2 on the growth sites of liver cancer cells (liver targeting verification experiment).

[0083] Experimental methods: A HepG2 cell subcutaneous xenograft model was established, and the effect of tail vein administration of DL-2 on the proliferation of subcutaneous xenografts was compared.

[0084] Experimental results: such as Figure 12As shown in Figure A, a subcutaneous xenograft model was treated with DL-2 via tail vein injection. DL-2 alone had no significant effect. However, when DL-2 was combined with low, medium, and high doses of taraxasterol, the effect of DL-2 gradually increased with the increase of taraxasterol dose. Figure 12 B showed that DL-2 had the same effect on tumor volume as the negative control group. In the DL-2 combined with low, medium and high doses of taraxasterol treatment groups, the tumor volume gradually decreased with the increase of taraxasterol dose. Figure 12 C indicates that the inhibition rate of DL-2 was 0.31%, the inhibition rate of DL-2 combined with low-dose taraxasterol reached 51.7%, the inhibition rate of DL-2 combined with medium-dose taraxasterol was 65.13%, and the inhibition rate of DL-2 combined with high-dose taraxasterol was 74.3%. These results demonstrate that DL-2 has liver-targeting inhibition of liver cancer.

[0085] DL-2's inhibitory effect on hepatic orthotopic tumors (inhibition rate >70%) is far superior to its inhibitory effect on subcutaneous xenograft tumors (inhibition rate approximately 30%). This further confirms that the phosphoramide ester prodrug of the present invention has the characteristic of liver-targeted metabolism, indicating that DL-2 is more advantageous in the treatment of primary liver cancer. Example 11

[0086] Preparation of pharmaceutical compositions.

[0087] 1. Tablet preparation: Take 80 g of compound DL-2, 1 g of taraxasterol, 150 g of lactose, 50 g of microcrystalline cellulose, and 2 g of magnesium stearate, mix them evenly, pulverize them through an 80-mesh sieve, granulate, dry, and compress them into tablets to make 1000 tablets, each containing 80 mg of compound DL-2 and 1 mg of taraxasterol.

[0088] 2. Preparation of injection: Take 40 mg of DL-2, 0.5 mg of taraxasterol, and 10 mL of physiological saline, stir to dissolve, filter to remove bacteria, fill and seal, sterilize, and prepare an injection. Example 12

[0089] Preparation of pharmaceutical compositions.

[0090] 1. Tablet preparation: Take 45g of compound DL-2, 1g of taraxasterol, 150g of lactose, 50g of microcrystalline cellulose, and 2g of magnesium stearate, mix them evenly, pulverize them through an 80-mesh sieve, granulate, dry, and compress them into tablets to make 1000 tablets, each containing 45mg of compound DL-2 and 1mg of taraxasterol.

[0091] 2. Preparation of injection: Take 22.5 mg of DL-2, 0.5 mg of taraxasterol and 10 mL of physiological saline, stir to dissolve, filter to remove bacteria, fill and seal, sterilize, and prepare injection. Example 13

[0092] Preparation of pharmaceutical compositions.

[0093] 1. Tablet preparation: Take 10 g of compound DL-2, 1 g of taraxasterol, 150 g of lactose, 50 g of microcrystalline cellulose, and 2 g of magnesium stearate, mix them evenly, pulverize them through an 80-mesh sieve, granulate, dry, and compress them into tablets to make 1000 tablets, each containing 10 mg of compound DL-2 and 1 mg of taraxasterol.

[0094] 2. Preparation of injection: Take 5mg of DL-2, 0.5mg of taraxasterol, and 10mL of physiological saline, stir to dissolve, filter to remove bacteria, fill and seal, sterilize, and prepare an injection.

[0095] In summary, this invention provides fluorodeoxyuridine monophosphate prodrug compounds, pharmaceutical compositions comprising these compounds, and their use in the preparation of drugs for treating liver cancer. These compounds and pharmaceutical compositions effectively inhibit the growth of liver cancer and exhibit good antitumor activity and safety in animal models. In particular, this invention discloses and verifies for the first time that the combination of such prodrugs with taraxasterol can produce a synergistic anti-liver cancer effect based on the upregulation of HINT1 protein expression, which provides a novel and more promising combination therapy strategy for the field.

[0096] Although embodiments of the present invention have been shown and described above, it is understood that these embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions, and alterations to the above embodiments within the scope of the present invention without departing from its principles and spirit. The scope of protection of the present invention is defined by the claims and their equivalents.

Claims

1. A compound as shown in Formula I, or a pharmaceutically acceptable salt, hydrate, solvate, polymorph or stereoisomer thereof: wherein: R1 is an amino acid residue; said amino acid residue refers to the rest of a single natural amino acid after the ester bond is formed between the a-carboxyl of the amino acid and the oxygen atom at 3' position of the sugar ring shown in Formula I; said natural amino acid is selected from valine, proline, tyrosine, cysteine, methionine, serine, lysine, arginine, histidine, glutamic acid, tryptophan, threonine, asparagine or glutamine. ， said natural amino acid is selected from proline, lysine, tryptophan, methionine, tyrosine, threonine, valine or glutamic acid. said compound is selected from one of the following compounds:

2. The compound of claim 1, wherein In the amino acid residues, the alpha-amino group of the natural amino acid is protected by a protecting group selected from C 1-6 alkylcarbonyl, C 1-6 alkylcarbonyl, C 1-6 alkylcarbonyl, C 3. The compound of claim 1, wherein a pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt, hydrate, solvate, polymorph or stereoisomer thereof, and a pharmaceutically acceptable excipient.

4. The compound of claim 1, wherein a pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt, hydrate, solvate, polymorph or stereoisomer thereof, taraxasterol, and a pharmaceutically acceptable excipient. 。 5. A pharmaceutical composition, characterized by, the mass ratio of the compound, or a pharmaceutically acceptable salt, hydrate, solvate, polymorph or stereoisomer thereof, to taraxasterol is 80:1 to 10:

1.

6. A pharmaceutical composition, characterized by, 8. Use of a compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt, hydrate, solvate, polymorph or stereoisomer thereof, or a pharmaceutical composition according to any one of claims 5 to 7 in the manufacture of a medicament for treating liver cancer.

7. The pharmaceutical composition of claim 6, wherein, ​ ​

Images