Cytidine precursor compound as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicine synthesis, and particularly relates to a cytidine precursor compound as well as a preparation method and application thereof. The cytidine precursor compound provided by the invention is a compound with a structure as shown in a formula (I) or a pharmaceutically acceptable salt thereof, in the formula (I), R2 is selected from one of hydrogen, formyl, acetyl, propionyl, n-butyryl and isobutyryl; r1, R3, R4, R5 and R6 are respectively and independently selected from two or three of hydrogen, hydroxyl and fluorine. The cytidine precursor compound provided by the invention is convenient to prepare oral and intravenous injection preparations, can be effectively converted into a raw drug in vivo, has bioavailability and tumor aggregation which are obviously higher than those of a cytidine raw drug, has an excellent drug treatment effect, has obvious clinical advantages, and greatly meets the clinical application requirements of cytidine antitumor drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical synthesis technology, specifically relating to a cytidine precursor compound, its preparation method, and its application. Background Technology

[0002] Prodrugs are a class of compounds obtained through chemical structural modification. They are typically inactive or have very low activity in vitro, but can release their active ingredients and exert their therapeutic effects in vivo through enzymatic or non-enzymatic conversion. The transformation of prodrugs into the active drug through in vivo metabolism not only improves drug bioavailability, enhances drug stability, and strengthens drug targeting, but also reduces toxicity and side effects, and prolongs the duration of drug action.

[0003] With advancements in medicine, the design of prodrugs has garnered increasing attention in new drug development. These designs aim to improve the physicochemical properties and pharmacokinetic characteristics of the parent drug, addressing common problems such as easy enzymatic inactivation, poor lipid solubility, poor oral absorption, and low membrane permeability. Prodrugs are obtained by modifying the structure of existing drugs; such modifications often offer advantages such as relatively low risk, lower investment, and rapid results.

[0004] Currently, cytidine drugs have become the most widely used anticancer drugs in clinical practice. Among them, gemcitabine (GEM) and cytarabine (Ara-C), as typical cytidine anticancer drugs, are used as first-line treatments for pancreatic cancer and acute myeloid leukemia (AML), respectively. They mainly act on the DNA synthesis phase, killing cancer cells by inhibiting metabolism.

[0005] However, the aforementioned cytidine drugs are difficult to absorb orally due to their high polarity and poor intestinal permeability. Even after absorption, the 4-amino group of these drugs is easily deaminated by cytidine deaminases in the body, resulting in inactive metabolites and extremely low oral bioavailability of gemcitabine and cytarabine. Furthermore, oral gemcitabine also exhibits severe gastrointestinal toxicity. Therefore, the clinical use of gemcitabine and cytarabine faces certain challenges, while the development of prodrugs offers new possibilities for overcoming these problems.

[0006] To address the shortcomings of gemcitabine and cytarabine in the clinical treatment of cancer, researchers have explored prodrug design strategies. In existing studies, the modification sites for gemcitabine and cytarabine are generally based on the two functional groups 4'-amino and 5'-hydroxyl. However, current reports indicate that existing prodrugs modified at the 5'-hydroxyl group are at risk of rapid metabolic inactivation by cytidine deaminases in vivo, while prodrugs modified at the 4'-amino group cannot be effectively converted to the parent drug in vivo, thus failing to exert a therapeutic effect and limiting their further clinical application.

[0007] Therefore, finding a suitable oral prodrug compound that can be effectively degraded into the original drug in vivo, thereby improving the bioavailability and therapeutic effect of cytidine drugs, is of practical clinical significance for improving the safety and anti-tumor effects of cytidine drugs. Summary of the Invention

[0008] To overcome the shortcomings of the prior art, the first objective of this invention is to provide a cytidine precursor compound that is suitable for oral administration and can be effectively converted into the original drug in vivo, while significantly improving the therapeutic effect of cancer.

[0009] The second objective of this invention is to provide a method for preparing the above-mentioned cytidine precursor compounds, which has mild conditions, simple steps, and is easy to implement for the preparation of precursor drugs.

[0010] A third objective of this invention is to provide applications of the aforementioned cytidine precursor compounds.

[0011] One of the objectives of this invention is achieved through the following technical solution: A cytidine precursor compound, which is a compound of formula (I) or a pharmaceutically acceptable salt thereof: ; In formula (I), R2 is selected from one of hydrogen, formyl, acetyl, propionyl, n-butyryl, and isobutyryl; R1, R3, R4, R5, and R6 are each independently selected from two or three of hydrogen, hydroxyl, and fluorine.

[0012] To improve the bioavailability and antitumor effect of the prodrug, more preferably, the cytidine prodrug compound is a compound with the following structure or a pharmaceutically acceptable salt thereof: , , , .

[0013] This invention does not impose specific limitations on the types of pharmaceutically acceptable salts, and those skilled in the art can make reasonable selections based on drug requirements. More preferably, the pharmaceutically acceptable salt is one of the following: hydrochloride, sulfate, phosphate, nitrate, acetate, lactate, and citrate.

[0014] The second objective of this invention is achieved by the following technical solution: A method for preparing the cytidine precursor compound as described above, the method comprising the following steps: heating and reacting a cytidine drug and a hydroxylamine compound in a mixture of water and a first solvent, followed by purification, to obtain the cytidine precursor compound; Alternatively, the method for preparing the cytidine precursor compound includes the following steps: heating and reacting a cytidine drug and a hydroxylamine compound in a mixture of water and a first solvent, followed by purification to obtain an intermediate; reacting the intermediate with an acyl chloride compound in a second solvent in the presence of a catalyst and an organic base at room temperature, followed by purification to obtain the cytidine precursor compound. The cytidine drug in question is gemcitabine or cytarabine.

[0015] More preferably, the first solvent is selected from one or more of methanol, ethanol, propanol, isopropanol, and acetonitrile; the second solvent is selected from one or more of acetonitrile, N,N-dimethylformamide, tetrahydrofuran, and acetone.

[0016] More preferably, the hydroxylamine compound is hydroxylamine sulfate or hydroxylamine hydrochloride; the catalyst is 4-dimethylaminopyridine or pyridine; the organic base is triethylamine or trimethylamine; and the acyl chloride compound is one of formyl chloride, acetyl chloride, propionyl chloride, n-butyryl chloride, and isobutyryl chloride.

[0017] To improve reaction efficiency and product selectivity, the molar ratio of the cytidine drug to the hydroxylamine compound is further preferably 1:(2.5~3.5), more preferably 1:3. The molar ratio of the intermediate to the acyl chloride compound is 1:(0.8~1.2), more preferably 1:1. The heating reaction is carried out at a temperature of 70~85℃ for 20~30h; the room temperature reaction is carried out at a temperature of 20~30℃ for 8~16h; and the purification is performed by column chromatography.

[0018] The third objective of this invention is achieved by the following technical solution: The use of a cytidine precursor compound as described above in the preparation of a medicament for treating cancer, wherein the cancer is one of pancreatic cancer, breast cancer, lung cancer, prostate cancer, colorectal cancer, ovarian cancer, liver cancer, skin cancer, cervical cancer, or leukemia, more preferably pancreatic cancer and leukemia.

[0019] More preferably, the leukemia is acute myeloid leukemia, acute lymphoblastic leukemia, or chronic myeloid leukemia.

[0020] More preferably, the drug is an intravenous injection drug or an oral drug.

[0021] More preferably, the oral medication is one or more of tablets, capsules, solutions, and suspensions.

[0022] More preferably, the drug also includes pharmaceutically acceptable excipients, such as fillers, binders, disintegrants, solubilizers, lubricants, etc., which can be conventionally selected by technicians according to the drug dosage form.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows: The cytidine precursor compound provided by this invention has a novel structure. After administration into the bloodstream, the 4-amino group is protected, preventing rapid metabolism by cytidine deaminase and prolonging the drug's circulation time in the blood. Upon reaching the liver, it effectively releases the original drug, thereby exerting its therapeutic effect on tumors. Furthermore, the cytidine precursor compound of this invention is a white solid with excellent stability, facilitating the preparation of oral and intravenous formulations.

[0024] The method for preparing cytidine precursor compounds of the present invention involves first modifying the N4 position of the cytidine drug with hydroxylamine to prevent the amino group from being rapidly degraded by cytidine deaminase in vivo, and then modifying the C5 position with esterification to increase the lipid solubility of the precursor drug and improve the membrane permeability of the compound. Therefore, the precursor compounds prepared by this method can both protect the amino group from metabolic inactivation and be effectively converted into the original drug in the liver, thus exerting their anti-tumor therapeutic effect.

[0025] In summary, the cytidine precursor compounds of the present invention can be effectively converted into the original drug in vivo, exhibiting significantly higher bioavailability, tumor aggregation, and superior tumor treatment effects than the original cytidine drugs, demonstrating significant clinical advantages and greatly satisfying the clinical application needs of cytidine antitumor drugs. Attached Figure Description

[0026] Figure 1 These are the stability test results of different glycoside compounds in plasma and liver tissue homogenates in this invention; Figure 1 In the table, A represents the stability of GEM, compound 1, and compound 2 in plasma; B represents the stability of GEM, compound 1, and compound 2 in liver tissue homogenate; C represents the stability of Ara-C, compound 3, and compound 4 in plasma; and D represents the stability of Ara-C, compound 3, and compound 4 in liver tissue homogenate. Figure 2These are the test results of the in vitro toxicity of different glycoside compounds to tumor cells in this invention; Figure 2 In the diagram, A represents the IC50 concentration of PANC-02 cells after treatment with GEM, compound 1, and compound 2. 50 Value; B represents the IC50 value of C1498 cells after treatment with Ara-C, compound 3, and compound 4. 50 value; Figure 3 These are the pharmacokinetic results of different glycoside compounds in rats according to the present invention; wherein, A is the time-concentration curve of the drug in SD rats after intravenous injection of GEM, intravenous injection of compound 1, oral administration of GEM, oral administration of compound 1, and oral administration of compound 2; B is the oral bioavailability of the oral GEM group, oral administration of compound 1 group, and oral administration of compound 2 group; C is the time-concentration curve of the drug in SD rats after intravenous injection of Ara-C, intravenous injection of compound 3, oral administration of Ara-C, oral administration of compound 3, and oral administration of compound 4; D is the oral bioavailability of the oral Ara-C group, oral administration of compound 3 group, and oral administration of compound 4 group. Figure 4 The results show the drug distribution of different glycoside compounds in mice in this invention; Figure 4 In the table, A represents the amount of drug accumulation in the tumors of mice at various time points after oral administration of GEM and compound 2; B represents the amount of drug accumulation in the pancreas of mice at various time points after oral administration of GEM and compound 2; C represents the amount of drug accumulation in the tumors of mice at various time points after intravenous administration of GEM and compound 1; and D represents the amount of drug accumulation in the pancreas of mice at various time points after intravenous administration of GEM and compound 1. Figure 5 The results of the pharmacodynamic evaluation of cytidine precursor compound 2 in mice in this invention are shown. Figure 5 In the table, A represents the change in body weight of pancreatic cancer-bearing mice after treatment with the negative control group, oral GEM group, oral compound 2 group, and intravenous GEM group; B represents the change in tumor volume of pancreatic cancer-bearing mice after treatment with the negative control group, oral GEM group, oral compound 2 group, and intravenous GEM group; C represents the change in tumor wet weight after treatment with the negative control group, oral GEM group, oral compound 2 group, and intravenous GEM group; and D represents the change in tumor volume after treatment with the negative control group, oral GEM group, oral compound 2 group, and intravenous GEM group. Figure 6 The results of the pharmacodynamic evaluation of cytidine precursor compound 4 in mice in this invention are shown. Figure 6In the table, A represents the changes in body weight of mice after treatment with the negative control group, the oral Ara-C group, the oral compound 4 group, and the intravenous Ara-C group; B represents the semi-quantitative comparison of fluorescence signals of leukemia cells in mice of each group on day 7 after inoculation; and C represents the semi-quantitative comparison of fluorescence signals of leukemia cells in mice of each group on day 14 after inoculation. Detailed Implementation

[0027] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. It should be noted that the following embodiments are merely illustrative of the invention and not intended to limit it. Without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments. Unless otherwise specified, the reagents or raw materials used in the following embodiments are commercially available.

[0028] Example 1

[0029] A cytidine precursor compound, with the structural formula shown below, is designated as compound 1: .

[0030] The preparation method of the above-mentioned cytidine precursor compound (compound 1) includes the following steps: 1 g of gemcitabine (3.8 mmol) was placed in a 100 mL round-bottom flask. Separately, 1.869 g of hydroxylamine sulfate (11.4 mmol) was placed in a 100 mL beaker. First, 12 mL of pure water was added to the beaker, followed by 28 mL of isopropanol. The mixture was stirred to obtain a hydroxylamine sulfate solution. This hydroxylamine sulfate solution was added to the round-bottom flask containing gemcitabine. The mixture was stirred at 78 °C for 24 h. The organic solvent and water were then removed by rotary evaporation. The resulting product was purified by column chromatography (dichloromethane:methanol = 10:1, v / v), and finally dried. The resulting white powder was compound 1, with a yield of 36.7%.

[0031] The structure of compound 1 was analyzed using nuclear magnetic resonance (NMR) technology, and the characterization results are as follows: 1 H NMR (400 MHz, DMSO)δ 10.11 (s, 1H), 9.80 (s, 1H), 6.95 (d, J = 8.2 Hz, 1H), 6.24 (d, J = 6.4 Hz,1H), 6.06 – 5.93 (m, 1H), 5.61 (d, J = 8.2 Hz, 1H), 5.19 (t, J = 5.4 Hz, 1H), 4.13 (hept, J = 7.6 Hz, 1H), 3.79 – 3.69 (m, 2H), 3.59 (dt, J = 12.9, 4.4 Hz, 1H).

[0032] Example 2

[0033] A cytidine precursor compound, with the structural formula shown below, is designated as compound 2: .

[0034] The preparation method of the above-mentioned cytidine precursor compound (compound 2) includes the following steps: 0.2 g of compound 1 (0.72 mmol) obtained in Example 1 was dissolved in 8 mL of N,N-dimethylformamide (DMF), followed by the addition of 0.2 mL of triethylamine, 5 mg of 4-dimethylaminopyridine (DMAP), and 0.076 g of isobutyryl chloride (0.72 mmol). The mixture was stirred at room temperature (25 ± 5 °C) for 12 h. The resulting product was then purified by column chromatography (dichloromethane:methanol = 15:1) and dried. The resulting white powder was compound 2, with a yield of 41.1%.

[0035] The structure of compound 2 was analyzed using nuclear magnetic resonance (NMR) technology, and the characterization results are as follows: 1 H NMR (400 MHz, DMSO)δ 11.14 (s, 1H), 7.35 (d, J = 8.3 Hz, 1H), 6.30 (d, J = 6.5 Hz, 1H), 6.03 (t,J = 8.0 Hz, 1H), 5.84 – 5.74 (m, 1H), 5.30 – 5.21 (m, 1H), 4.16 (ddd, J =19.2, 9.5, 5.7 Hz, 1H), 3.85 – 3.72 (m, 2H), 3.61 (ddt, J = 12.7, 6.9, 3.5Hz, 1H), 2.88 – 2.62 (m, 1H), 1.13 (dd, J = 9.7, 6.9 Hz, 6H).

[0036] Example 3

[0037] A cytidine precursor compound, with the structural formula shown below, is designated as compound 3: .

[0038] The preparation method of the above-mentioned cytidine precursor compound (compound 3) includes the following steps: 1 g of cytarabine (4.11 mmol) was placed in a 100 mL round-bottom flask. Separately, 2 g of hydroxylamine sulfate (12.2 mmol) was placed in a 100 mL beaker. First, 12 mL of pure water was added to the beaker, followed by 28 mL of isopropanol. The mixture was stirred to obtain a hydroxylamine sulfate solution. This hydroxylamine sulfate solution was added to the round-bottom flask containing cytarabine. The mixture was stirred at 78 °C for 24 h. The organic solvent and water were then removed by rotary evaporation. The resulting product was purified by column chromatography (dichloromethane:methanol = 6:1, v / v), and finally dried. The resulting white powder was compound 3, with a yield of 49.7%.

[0039] The structure of compound 3 was analyzed using nuclear magnetic resonance (NMR) technology, and the characterization results are as follows: 1 H NMR (400 MHz, DMSO)δ 9.97 (s, 1H), 9.47 (s, 1H), 7.05 (d, J = 8.2 Hz, 1H), 5.73 (d, J = 6.3 Hz, 1H), 5.57 (d, J = 8.2 Hz, 1H), 5.23 (s, 1H), 5.01 (s, 2H), 3.97 (t, J = 5.8Hz, 1H), 3.92 (dd, J = 5.3, 3.1 Hz, 1H), 3.77 (q, J = 3.4 Hz, 1H), 3.53 (qd,J = 11.9, 3.6 Hz, 2H).

[0040] Example 4

[0041] A cytidine precursor compound, with the structural formula shown below, is designated as compound 4: .

[0042] The preparation method of the above-mentioned cytidine precursor compound (compound 4) includes the following steps: 0.2 g of compound 3 (0.77 mmol) obtained in Example 3 was dissolved in 8 mL of DMF, followed by the addition of 0.2 mL of triethylamine, 5 mg of DMAP and 0.082 g of isobutyryl chloride (0.77 mmol). The mixture was stirred at room temperature for 12 h. The resulting product was then purified by column chromatography (dichloromethane:methanol = 12:1) and dried. The resulting white powder was compound 4, with a yield of 43.4%.

[0043] The structure of compound 4 was analyzed using nuclear magnetic resonance (NMR) technology, and the characterization results are as follows: 1H NMR (400 MHz, DMSO)δ 10.82 (d, J = 2.3 Hz, 1H), 9.72 (s, 1H), 7.22 (d, J = 8.2 Hz, 1H), 5.98–5.89 (m, 1H), 5.66–5.49 (m, 1H), 5.44 (d, J = 4.4 Hz, 1H), 5.02 (dt, J =10.0, 5.4 Hz, 1H), 3.98 (tt, J = 8.1, 3.9 Hz, 1H), 3.88 (p, J = 3.7 Hz, 1H), 3.70 (q, J = 4.6 Hz, 1H), 3.66–3.50 (m, 2H), 2.81 (hept, J = 6.6 Hz, 1H),1.21–1.13 (m, 6H).

[0044] The technical effects of this invention are illustrated below with specific test examples. The data from this invention were tested using one-way ANOVA to examine differences between groups. P <0.05,** P <0.01, *** P <0.001, **** P <0.0001 indicates that the difference between groups is statistically significant.

[0045] Test Example 1: Stability Test in Plasma and Liver Tissue Homogenate This invention first investigates the stability of compounds 1-4 prepared in the above embodiments, as well as gemcitabine (GEM) and cytarabine (Ara-C), in rat plasma and liver tissue homogenates, and preliminarily determines the degradation process of GEM, compound 1, compound 2, Ara-C, compound 3, and compound 4 in vivo.

[0046] The test method was as follows: Multiple rat plasma and liver tissue homogenates were placed in EP tubes. Standard solutions of GEM, compound 1, compound 2, Ara-C, compound 3, and compound 4 were added to the plasma and liver tissue homogenates, respectively, to achieve a final drug concentration of 20 µg / mL. Each EP tube was placed in a 37°C constant-temperature shaker, and 100 µL samples were taken at 0, 1, 2, 4, 6, 8, and 12 h. 100 µL of methanol and 200 µL of acetonitrile were added to each sample, and the mixture was vortexed for 10 min. The mixture was then centrifuged (12000 rpm, 10 min), and the supernatant was collected. The supernatant was dried in air at 45°C, and the residue was reconstituted with 200 µL of pure water. The mixture was vortexed for 10 min, centrifuged (12000 rpm, 10 min), and the supernatant was collected. The drug concentration in the samples was detected by high-performance liquid chromatography (HPLC), and the remaining drug content was calculated to assess the drug stability in plasma and liver tissue homogenates.

[0047] The parameters for determining the drug content using high-performance liquid chromatography (HPLC) were set as follows: mobile phase a was acetonitrile-0.5wt% acetic acid aqueous solution (3:97, v / v), and mobile phase b was acetonitrile-0.5wt% acetic acid aqueous solution (20:80, v / v). The injection volume was 10 μL, gradient elution was used, the flow rate was 1.0 mL / min, and the detection wavelength was 268 nm.

[0048] Figure 1 The results of stability tests on different glycosides in plasma and liver tissue homogenates; Figure 1 In the table, A represents the stability of GEM, compound 1, and compound 2 in plasma; B represents the stability of GEM, compound 1, and compound 2 in liver tissue homogenate; C represents the stability of Ara-C, compound 3, and compound 4 in plasma; and D represents the stability of Ara-C, compound 3, and compound 4 in liver tissue homogenate.

[0049] like Figure 1 As shown in A and B, GEM is rapidly degraded in plasma and liver tissue homogenates, and compound 2 is rapidly degraded into compound 1. Furthermore, compound 1 remains relatively stable in plasma and is effectively degraded into the original GEM drug in liver tissue homogenates.

[0050] like Figure 1 As shown in C and D, Ara-C is rapidly degraded in plasma and liver tissue homogenates, and compound 4 is rapidly degraded into compound 3. Furthermore, compound 3 remains relatively stable in plasma and is effectively degraded into the original Ara-C drug in liver tissue homogenates.

[0051] Test Example 2: Tumor Cytotoxicity Test To verify the anti-tumor application potential of the compounds, this invention conducted a comparative study on the in vitro toxicity of compounds 1, 2, 3, and 4, as well as GEM and Ara-C, to tumor cells. The test method was as follows: PANC-02 cells (mouse pancreatic cancer cells) and C1498 cells (mouse leukemia cells) in logarithmic growth phase were collected and evenly seeded into 96-well plates at 10,000 cells per well. Different concentrations of each drug (compounds 1-4) were added to the 96-well plates, resulting in a final concentration gradient of 50, 100, 150, 250, 500, 1000, and 2000 μM. After incubating the 96-well plates at 37°C and 5% CO2 for 24 h, 10 μL of CCK-8 solution was added to each well using a pipette, and the plates were incubated for another 2 h in a shaker. The absorbance of each well was measured at 450 nm using a microplate reader. Based on the absorbance, the half-maximal inhibitory concentration (IC50) of each drug against cancer cells was calculated. 50 ).

[0052] Figure 2 The results show the in vitro toxicity of different glycoside compounds to tumor cells. Figure 2 In the diagram, A represents the IC50 concentration of PANC-02 cells after treatment with GEM, compound 1, and compound 2. 50 Value; B represents the IC50 value of C1498 cells after treatment with Ara-C, compound 3, and compound 4. 50 value.

[0053] like Figure 2 As shown in A, GEM incubation of PANC-02 cells for 24 h resulted in an IC50 concentration of 1,000 mg / L. 50 Significantly higher than compound 2 ( P <0.001), indicating that compound 2 is significantly more toxic to tumor cells than GEM. For example... Figure 2 As shown in B, the IC50 of Ara-C after 24 hours of in vitro incubation of C1498 cells... 50 Significantly higher than compound 4 ( P <0.001), indicating that compound 4 is significantly more toxic to tumor cells than Ara-C.

[0054] Test Example 3: Pharmacokinetic Study The present invention will next investigate the pharmacokinetics of compounds 1, 2, 3, and 4 in rats. The testing method was as follows: Male SD rats, weighing 220±20g, meeting the experimental requirements, were randomly divided into 10 groups of 5 rats each. The specific groupings were as follows: (i) The five groups were: intravenous GEM group, intravenous compound 1 group, oral GEM group, oral compound 1 group, and oral compound 2 group. The dosages for these five groups were 50 mg / kg for GEM, 53.1 mg / kg for compound 1, and 66.4 mg / kg for compound 2. GEM and compounds 1 and 2 had equimolar doses.

[0055] (ii) Intravenous Ara-C group, intravenous compound 3 group, oral Ara-C group, oral compound 3 group, and oral compound 4 group. The dosages for these five groups were 50 mg / kg for Ara-C, 53.3 mg / kg for compound 3, and 66.4 mg / kg for compound 4. Ara-C and compounds 1 and 2 had equimolar doses.

[0056] All experimental groups were treated with intravenous injection or oral administration of the corresponding drugs according to the grouping requirements. Blood samples were collected from the orbital cavity at 0.083h, 0.167h, 0.25h, 0.5h, 1h, 2h, 4h, 6h, 8h, 10h, and 12h after drug administration. The whole blood was centrifuged at 4000rpm for 15min, and 100μL of the supernatant plasma was collected to precipitate the protein. The drug content of each group was determined by high performance liquid chromatography (analytical parameters are the same as in test example 1).

[0057] Figure 3 The results represent the pharmacokinetic assays of different glycoside compounds in rats; where A represents the time-concentration curves of the drugs in SD rats after intravenous injection of GEM, intravenous injection of compound 1, oral administration of GEM, oral administration of compound 1, and oral administration of compound 2; B represents the oral bioavailability of the oral GEM group, oral administration of compound 1 group, and oral administration of compound 2 group; C represents the time-concentration curves of the drugs in SD rats after intravenous injection of Ara-C, intravenous injection of compound 3, oral administration of Ara-C, oral administration of compound 3, and oral administration of compound 4 group; and D represents the oral bioavailability of the oral Ara-C group, oral administration of compound 3 group, and oral administration of compound 4 group.

[0058] like Figure 3 As shown in Figure A, the initial concentrations of GEM and compound 1 injected intravenously in SD rats were the highest, and the concentrations decreased over time. The concentration of compound 1 injected intravenously was significantly higher than that of GEM injected intravenously within 2 hours. P <0.05), and intravenous injection of compound 1 C max This is 1.56 times that of intravenously administered GEM. This indicates that gemcitabine's N... 4 After modification, the rate of degradation by cytidine deaminase in vivo can be slowed down. Compared with oral GEM, the AUC and C of oral compounds 1 and 2 were significantly lower. maxSignificant improvement. This indicates that although compound 1 also has high water solubility, after oral absorption, the 4-amino group is protected, preventing rapid metabolic inactivation by cytidine deaminase in the body and slowing down drug clearance. Compound 2 not only has a protected 4-amino group but also increased lipid solubility, enabling rapid absorption and thus improving drug distribution in the bloodstream.

[0059] like Figure 3 As shown in B, compared with oral GEM, the bioavailability of both compound 1 and compound 2 was significantly improved by oral administration. Among them, compound 2 had the highest oral bioavailability compared with oral GEM and oral compound 1, showing a significant difference ( ). P <0.05). Therefore, this invention selected compound 2 for subsequent experimental studies on its in vivo distribution and efficacy.

[0060] like Figure 3 As shown in Figure C, the initial concentrations of intravenously injected Ara-C and compound 3 were the highest, and the concentrations decreased over time. The concentration of intravenously injected compound 3 was significantly higher than that of intravenously injected Ara-C within 6 hours. P <0.05), and intravenous injection of compound 3 C max It is 1.22 times that of intravenously injected Ara-C. This indicates the N... 4 After modification, the rate of degradation by cytidine deaminase in vivo can be slowed down. Furthermore, compared with oral Ara-C, the AUC and C of oral compounds 3 and 4 were significantly lower. max Significant improvement. This indicates that although compound 3 also has high water solubility, after partial oral absorption, the 4-amino group is protected, preventing rapid metabolic inactivation by cytidine deaminases in the body and slowing down drug clearance. Compound 4 not only has a protected 4-amino group but also increased lipid solubility, enabling rapid absorption and thus improving drug distribution in the bloodstream.

[0061] like Figure 3 As shown in D, the bioavailability of compounds 3 and 4 was significantly improved when taken orally compared with that of Ara-C. P <0.05%. Among them, the oral compound 4 had the highest bioavailability, showing a significant difference compared to the oral GEM ( P <0.05). Therefore, this invention selected compound 4 for subsequent experimental studies on its efficacy.

[0062] Test Example 4: In vivo drug distribution study The present invention will next investigate the drug distribution of compounds 1 and 2 in mice. The testing method was as follows: mouse pancreatic cancer PANC-02 cells were resuscitated, and the PANC-02 cells in good growth condition were digested, centrifuged, and resuspended in PBS (cell density 2 × 10⁻⁶). 7The cell suspension was stored in an icebox (cells / mL). Then, the PANC-02 cell suspension was inoculated into the right forelimb axilla of 7-week-old C57 mice (0.1 mL per mouse). The inoculation continued until the tumor volume reached 80 mm². 3 Subsequently, the successfully modeled PANC-02 pancreatic cancer-bearing mice were randomly divided into four groups (n=5): an oral GEM group, an oral compound 2 group, an intravenous GEM group, and an intravenous compound 1 group. The dosages for each group were: GEM 60 mg / kg, compound 1 63.6 mg / kg, and compound 2 79.6 mg / kg. GEM, compound 1, and compound 2 were administered at equimolar doses.

[0063] Mice were sacrificed at 0.25, 0.5, 1, 2, 4, and 6 hours after oral administration. Mice were also sacrificed at 0.083, 0.25, 0.5, 1, 2, 4, and 6 hours after intravenous administration. The pancreas and tumor were removed. Adhering bloodstains and tissue fluid were washed away with physiological saline, and the mice were dried with absorbent paper. Adhering mucosa and blood vessels were trimmed away, and the tissue was weighed. Twice the volume of physiological saline was added, and the homogenate was automatically prepared using an instrument to obtain a tissue homogenate. 100 μL of the tissue homogenate was precisely pipetted, and after protein precipitation, the drug content was quantitatively determined using high-performance liquid chromatography (HPLC) with the same analytical parameters as in Test Example 1.

[0064] Figure 4 The results show the drug distribution of different glycoside compounds in mice; Figure 4 In the table, A represents the amount of drug accumulation in the tumor of mice at various time points after oral administration of GEM and compound 2; B represents the amount of drug accumulation in the pancreas of mice at various time points after oral administration of GEM and compound 2; C represents the amount of drug accumulation in the tumor of mice at various time points after intravenous administration of GEM and compound 1; and D represents the amount of drug accumulation in the pancreas of mice at various time points after intravenous administration of GEM and compound 1.

[0065] Figure 4 The comparison of drug accumulation in mouse tumors and pancreas at various time points in the oral GEM group, oral compound 2 group, intravenous GEM group, and intravenous compound 1 group shows that, compared with oral GEM, oral compound 2 achieved higher drug concentrations in tumors and pancreas at all time points. Compared with intravenous GEM, intravenous compound 1 achieved higher drug concentrations in tumors and pancreas at almost all time points. This indicates that, compared with intravenous and oral administration of the original GEM, oral compound 2 and intravenous compound 1 can effectively improve drug distribution at tumor sites and have good tumor accumulation capabilities.

[0066] Test Example 5: In vivo pharmacodynamics study (1) In vivo pharmacodynamic studies of compound 2 This invention further investigates the pharmacodynamics of compound 2 in mice. The testing method was as follows: a PANC-02 pancreatic cancer mouse model was constructed according to the modeling method described in Test Example 4, and the tumor volume was increased to 80 mm². 3 Pharmacodynamic studies were initiated by randomly assigning five mice to each successfully modeled mouse population. The groups were as follows: negative control (saline); oral GEM group; oral compound 1 group; and intravenous GEM group. Dosage was as follows: the oral and intravenous GEM groups received 20 mg / kg, the oral compound 1 group received 26.54 mg / kg (equimolar amount with the oral GEM group), and the negative control group received the same volume of compound 1 as the oral GEM group. Administered the medication every other day. After administration, the mice were sacrificed, dissected, and tumor tissue was extracted. Changes in mouse body weight, tumor volume, tumor wet weight, and final tumor volume were measured.

[0067] Figure 5 The results of the pharmacodynamic evaluation of the cytidine precursor compound 2 of the present invention in mice; Figure 5 In the table, A represents the change in body weight of pancreatic cancer-bearing mice after treatment with the negative control group, oral GEM group, oral compound 2 group, and intravenous GEM group; B represents the change in tumor volume of pancreatic cancer-bearing mice after treatment with the negative control group, oral GEM group, oral compound 2 group, and intravenous GEM group; C represents the change in tumor wet weight after treatment with the negative control group, oral GEM group, oral compound 2 group, and intravenous GEM group; and D represents the change in tumor volume after treatment with the negative control group, oral GEM group, oral compound 2 group, and intravenous GEM group.

[0068] like Figure 5 The comparison of mouse body weight changes, tumor volume changes, tumor wet weight, and final tumor volume shown in Figures A, B, C, and D indicates that the tumor volume in the control group grew rapidly, and the tumor volume remained at 250 mm² after the drug administration was completed. 3 Around 175 mmHg. After the oral GEM administration was completed, the tumor volume in the group was approximately 175 mmHg. 3 The results indicate that oral GEM does not effectively inhibit tumor growth. The tumor volume in the intravenous GEM group and the oral compound group was approximately 110 mm. 3 Around 100 mm, the tumor volume was significantly smaller than that of the oral GEM group ( P <0.05), and the tumor wet weight in the oral compound 2 group was significantly smaller than that in the oral GEM group ( P <0.0001), indicating that the oral compound 2 has a good antitumor effect.

[0069] (2) In vivo pharmacodynamic studies of compound 4 This invention further investigates the pharmacodynamics of compound 4 in mice. The testing method was as follows: mouse leukemia C1498 cells labeled with luciferase were resuscitated, and C1498 cells in the logarithmic growth phase were collected, washed, centrifuged, and resuspended in PBS (cell density 2 × 10⁻⁶). 7 Cells / mL were used to store the cell suspension in an ice box. Then, these cell suspensions were injected intravenously into mice (4 × 10⁶ cells / mL per mouse). 6 (cells). Mice were randomly assigned to groups of 5 mice each after inoculation. The groups were as follows: negative control (saline); oral Ara-C group; oral compound 4 group; and intravenous Ara-C group. The dosages were as follows: the oral and intravenous Ara-C groups received 20 mg / kg per dose; the oral compound 4 group received 27.08 mg / kg per dose (equimolar amount with the oral Ara-C group); and the negative control group received the same volume of medication as the oral Ara-C group. Administration began two days after inoculation and was repeated every other day. Post-inoculation weight changes were assessed, and fluorescence images were acquired in mice on days 7 and 14 using a small animal in vivo imaging system.

[0070] Figure 6 The results of the pharmacodynamic evaluation of the cytidine precursor compound 4 of the present invention in mice; Figure 6 In the table, A represents the changes in body weight of mice after treatment with the negative control group, the oral Ara-C group, the oral compound 4 group, and the intravenous Ara-C group; B represents the semi-quantitative comparison of fluorescence signals of leukemia cells in mice of each group on day 7 after inoculation; and C represents the semi-quantitative comparison of fluorescence signals of leukemia cells in mice of each group on day 14 after inoculation.

[0071] pass Figure 6 The comparison of mouse body weight and in vivo fluorescence intensity shown in Figures A, B, and C reveals a significant decrease in body weight in the control group and the oral Ara-C group. No significant decrease in body weight was observed in the oral compound 4 group and the intravenous Ara-C group during treatment. Simultaneously, the fluorescence intensity of leukemia cells in the control group and the oral Ara-C group increased rapidly, with significantly higher fluorescence intensities on days 7 and 14 after inoculation compared to the oral compound 4 group and the intravenous Ara-C group. P <0.0001). This indicates that oral Ara-C does not effectively inhibit the growth of leukemia cells, while oral compound 4 has an excellent inhibitory effect on the growth of leukemia cells.

[0072] In summary, the cytidine precursor compounds provided by this invention have the potential to be used as oral drugs, and can significantly increase the accumulation of cytidine drugs in the body, effectively enhancing the anti-tumor effect of the drugs, and have obvious clinical advantages.

[0073] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. A cytidine precursor compound, characterized in that, Compounds of the structure shown in formula (I) or pharmaceutically acceptable salts thereof: ； In formula (I), R2 is selected from one of hydrogen, formyl, acetyl, propionyl, n-butyryl, and isobutyryl; R1, R3, R4, R5, and R6 are each independently selected from two or three of hydrogen, hydroxyl, and fluorine.

2. The cytidine precursor compound according to claim 1, characterized in that, The cytidine precursor compound is a compound with the following structure or a pharmaceutically acceptable salt thereof: 、 、 、 。 3. The cytidine precursor compound according to claim 1 or 2, characterized in that, The pharmaceutically acceptable salt is one of the following: hydrochloride, sulfate, phosphate, nitrate, acetate, lactate, and citrate.

4. A method for preparing the cytidine precursor compound as described in claim 1, characterized in that, The method for preparing the cytidine precursor compound includes the following steps: heating and reacting a cytidine drug and a hydroxylamine compound in a mixture of water and a first solvent, followed by purification to obtain the cytidine precursor compound; Alternatively, the method for preparing the cytidine precursor compound includes the following steps: heating and reacting a cytidine drug and a hydroxylamine compound in a mixture of water and a first solvent, followed by purification to obtain an intermediate; reacting the intermediate with an acyl chloride compound in a second solvent in the presence of a catalyst and an organic base at room temperature, followed by purification to obtain the cytidine precursor compound. The cytidine drug in question is gemcitabine or cytarabine.

5. The method for preparing the cytidine precursor compound according to claim 4, characterized in that, The first solvent is selected from one or more of methanol, ethanol, propanol, isopropanol, and acetonitrile; the second solvent is selected from one or more of acetonitrile, N,N-dimethylformamide, tetrahydrofuran, and acetone.

6. The method for preparing the cytidine precursor compound according to claim 4, characterized in that, The hydroxylamine compound is hydroxylamine sulfate or hydroxylamine hydrochloride; the catalyst is 4-dimethylaminopyridine or pyridine; the organic base is triethylamine or trimethylamine; and the acyl chloride compound is one of formyl chloride, acetyl chloride, propionyl chloride, n-butyryl chloride, and isobutyryl chloride.

7. The method for preparing the cytidine precursor compound according to claim 4, characterized in that, The molar ratio of the cytidine drug to the hydroxylamine compound is 1:(2.5~3.5); the molar ratio of the intermediate to the acyl chloride compound is 1:(0.8~1.2); the heating reaction temperature is 70~85℃ and the time is 20~30h; the room temperature reaction temperature is 20~30℃ and the time is 8~16h; the purification is column chromatography purification.

8. The use of a cytidine precursor compound as described in claim 1 or 2 in the preparation of a medicament for treating cancer, characterized in that, The cancer mentioned is one of the following: pancreatic cancer, breast cancer, lung cancer, prostate cancer, colorectal cancer, ovarian cancer, liver cancer, skin cancer, cervical cancer, or leukemia.

9. The use of the cytidine precursor compound according to claim 8 in the preparation of a medicament for treating cancer, characterized in that, The drug is either an intravenous injection drug or an oral drug.

10. The application of the cytidine precursor compound according to claim 9, characterized in that, The oral medication is one or more of the following: tablets, capsules, solutions, and suspensions.

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