CA4 dimer prodrug compounds, their preparation methods, and applications.

Abstract

This invention discloses a CA4 dimer prodrug compound, its preparation method, and its applications. The structural formula of the CA4 dimer prodrug compound is shown in general formula I. The compound of this invention exerts a synergistic antitumor effect by inhibiting microtubule polymerization and regulating the CYP7A1-related pathway. In vitro experiments show that the IC50 value of CA4-dimer1 against human hepatocellular carcinoma Hep3B cells is 1.48 ± 0.18 nM, significantly superior to that of CA4 monomer. In animal experiments, the tumor inhibition rate reached 73.2%, without causing significant hematologic or hepatotoxic and renal toxicity. Pharmacokinetics show that intravenous injection can effectively convert it into CA4 and exert its antitumor effect; oral administration can release CA4. This invention also effectively improves the pharmacokinetic defects and toxic side effects of CA4, providing a new strategy for the development of anti-hepatocellular carcinoma drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical chemistry, specifically relating to a CA4 dimer prodrug compound, its preparation method, and its application. Background Technology

[0002] Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide. Statistics show that HCC ranks sixth in incidence globally and third in cancer-related deaths. Patients with advanced-stage HCC often lose the opportunity for surgery due to tumor metastasis or liver failure. Current first-line treatments rely on multi-target tyrosine kinase inhibitors (such as sorafenib and lenvatinib) and immune checkpoint inhibitors (such as atezolizumab). Only about 30% of HCC patients benefit from sorafenib treatment, and most develop resistance within 6 months of treatment. Although combination therapy (anti-PD-L1 antibody + anti-CTLA-4 antibody) has improved the 4-year overall survival rate of advanced HCC patients to 25.2%, drug toxicity and high cost limit its widespread application. Therefore, the development of novel, highly effective, and low-toxicity anti-HCC drugs is urgently needed.

[0003] Inhibiting tumor angiogenesis and tubulin aggregation has become a key strategy in the systemic treatment of advanced hepatocellular carcinoma (HCC). Combretastatin A4 (CA4), a natural tubulin inhibitor, reversibly binds to tubulin. CA4 selectively disrupts the microtubule cytoskeleton of tumor vascular endothelial cells, inducing tumor blood supply interruption, providing a new direction for liver cancer treatment. In our previous studies, CA4 bound to and inhibited CYP7A1, affecting the expression of CTSB and CSTD proteins through the autophagy pathway, thereby achieving an anti-tumor effect. CA4 has been shown to have broad-spectrum anti-tumor effects, demonstrating excellent anti-tumor efficacy in cell or animal models of tumors such as liver cancer, colorectal cancer, glioma, and non-small cell lung cancer. However, the clinical application of CA4 faces two major bottlenecks: first, its high lipophilicity and low water solubility lead to poor pharmacokinetics, with oral bioavailability of less than 2%; second, the phenolic hydroxyl group is oxidized by cytochrome P450 in vivo to generate quinone metabolites, causing dose-limiting hepatotoxicity.

[0004] Existing prodrug strategies have led to the development of numerous CA4 prodrugs to enhance water solubility, therapeutic efficacy, and reduce side effects; however, a proper balance between the efficacy and safety of CA4 has yet to be achieved. One study (PMID: 14977848) disclosed a CA4 phosphate ester prodrug (CA4P), but its plasma Cmax reached 95.2 μg / mL after intravenous injection, indicating increased renal burden due to phosphate metabolism. Besides phosphate ester prodrugs, existing technologies (such as PMID: 40323152) disclose CA4 dimer prodrugs based on aliphatic carbon chains or disulfide bonds. However, both strategies suffer from significant drug-likeness defects: introducing aliphatic carbon chains further increases the lipophilicity of the molecule, leading to poorer water solubility, and the additional carbon chains introduce unnecessary metabolic load; while the disulfide bond linkage strategy is highly susceptible to disulfide bond exchange reactions during synthesis and storage, resulting in poor product homogeneity and significantly increasing the difficulty of synthesis and purification. These defects still limit their application.

[0005] Therefore, there is an urgent need to develop a CA4 dimer prodrug compound and its synthesis method that combines high-efficiency drug release, metabolic safety, and ease of synthesis. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, this invention provides a CA4 dimer prodrug compound, its preparation method, and its applications. This invention is the first to use carbonate or oxalate esters, whose metabolites are non-toxic (CO2 and formic acid), as linking bonds to construct a symmetrical CA4 dimer, ensuring efficient release of the active drug while also possessing metabolic safety and ease of synthesis.

[0007] A first aspect of the present invention is to provide a CA4 dimer prodrug compound and its stereoisomers, tautomers, solvates, or pharmaceutically acceptable salts thereof, the structural formula of which is shown in general formula I:

[0008] ;

[0009] In the formula, R is -C(O)- or -C(O)C(O)-.

[0010] As a preferred embodiment of this application, the CA4 dimer prodrug compound and its stereoisomers, tautomers, solvates, or pharmaceutically acceptable salts thereof are characterized by:

[0011] When R is -C(O)-, the compound is bis(2-methoxy-5-((Z)-3,4,5-trimethoxystyryl)phenyl)carbonate, abbreviated as CA4-dimer1, and its structural formula is shown in general formula II;

[0012] When R is -C(O)C(O)-, the compound is bis(2-methoxy-5-((Z)-3,4,5-trimethoxystyryl)phenyl) ester, abbreviated as CA4-dimer2, and its structural formula is shown in general formula III:

[0013] .

[0014] A second aspect of the present invention provides a method for preparing the CA4 dimer prodrug compound CA4-dimer1, comprising the following steps:

[0015] Step 1: (1,2,3-trimethoxyphenyl-5-yl)methanol was reacted with phosphorus tribromide in dichloromethane, and the mixture was purified by extraction and column chromatography to obtain 5-(bromomethyl)-1,2,3-trimethoxyphenyl.

[0016] Step 2: The product obtained in Step 1 is reacted with triphenylphosphine in tetrahydrofuran, and triphenyl(3,4,5-trimethoxybenzyl)phosphine is obtained by solid-liquid separation;

[0017] Step 3: The product obtained in Step 2, 3-hydroxy-4-methoxybenzyl-1-carboxaldehyde, and potassium carbonate are condensed in ethanol and purified by column chromatography to obtain (Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenol.

[0018] Step 4: (Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenol, 4-nitrophenyl chloroformate and pyridine are reacted in dichloromethane at 70-90℃ for 4-10 h to obtain the intermediate (Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenyl(4-nitrophenyl) carbonate;

[0019] Step 5: The product of step 4 is coupled with another (Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenol under alkaline conditions, and the target compound CA4-dimer1 is obtained after purification.

[0020] The preparation method of compound CA4-dimer1 is referred to as Method A. It involves first constructing (Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenol via the Wittig reaction, and then coupling it with an activated carbonate.

[0021] A third aspect of the present invention provides a method for preparing the CA4 dimer prodrug compound CA4-dimer2, comprising the following steps:

[0022] Step 1: (1,2,3-trimethoxyphenyl-5-yl)methanol was reacted with phosphorus tribromide in dichloromethane, and the mixture was purified by extraction and column chromatography to obtain 5-(bromomethyl)-1,2,3-trimethoxyphenyl.

[0023] Step 2: The product obtained in Step 1 is reacted with triphenylphosphine in tetrahydrofuran, and triphenyl(3,4,5-trimethoxybenzyl)phosphine is obtained by solid-liquid separation;

[0024] Step 3: The product obtained in Step 2, 3-hydroxy-4-methoxybenzyl-1-carboxaldehyde, and potassium carbonate are condensed in ethanol and purified by column chromatography to obtain (Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenol.

[0025] Step 4: (Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenol and oxalyl chloride were condensed under alkaline conditions, and the resulting purified compound was CA4-dimer2.

[0026] The preparation method of compound CA4-dimer2 is referred to as Method B. It involves first constructing (Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenol via the Wittig reaction, and then directly condensing two molecules of the (Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenol with oxalyl chloride.

[0027] Steps 1-3 of Method A are the same as those of Method B, both being steps in the preparation of (Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenol.

[0028] As a preferred embodiment of this application, in step 1 of methods A and B, the molar ratio of (1,2,3-trimethoxyphenyl-5-yl)methanol to phosphorus tribromide is 1:2.

[0029] As a preferred embodiment of this application, in step 2 of methods A and B, the molar ratio of 5-(bromomethyl)-1,2,3-trimethoxybenzene to triphenylphosphine is 1:1-2.

[0030] As a preferred embodiment of this application, in step 3 of methods A and B, the molar ratio of triphenyl(3,4,5-trimethoxybenzyl)phosphine, 3-hydroxy-4-methoxybenzyl-1-carboxaldehyde, and potassium carbonate is 6:1:5.

[0031] As a preferred embodiment of this application, in step 4 of method A, the molar ratio of (Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenol, 4-nitrophenyl chloroformate and pyridine is 1:2:(3-3.2).

[0032] As a preferred embodiment of this application, in step 4 of method A: the reaction temperature is 80°C and the reaction time is 6 hours.

[0033] As a preferred embodiment of this application, in step 5 of method A, the molar ratio of the second (Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenol to the intermediate carbonate is 1:(0.8-1). More preferably, it is 1:0.84.

[0034] As a preferred embodiment of this application, in step 4 of method B, the molar ratio of (Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenol to oxaloyl chloride is 2:1.

[0035] The core of this invention lies in overcoming current limitations through dimer prodrug design, linking two CA4 molecules via carbonate or oxalate bonds to form a symmetrical structure (molecular weights of 658 Da and 686 Da, respectively). This symmetrical structure has the following advantages:

[0036] 1. Toxicity shielding: The ester bond masks the phenolic hydroxyl group on the benzene ring, preventing it from being oxidized into quinone metabolites in the body and causing hepatotoxicity.

[0037] 2. Pharmacokinetic optimization: The blood drug concentration fluctuations after intravenous injection in rats are relatively stable, avoiding the stimulation caused by the release of a large amount of drug in a short period of time.

[0038] 3. Controllable metabolism: The esterase specifically hydrolyzes to release two molecules of CA4 and non-toxic byproducts (CO2, formic acid). No liver or kidney pathological damage was observed after 21 days of treatment in a nude mouse model.

[0039] 4. Dual-target synergy: The small molecule compound dimer can inhibit the expression of CYP7A1, a key enzyme in the synthesis of tubulin and bile acids, and exert a synergistic anti-tumor effect.

[0040] A fourth aspect of the invention is to provide a pharmaceutical composition comprising a therapeutically effective amount of a CA4 dimer prodrug compound and its stereoisomers, tautomers, solvates, or pharmaceutically acceptable salts thereof.

[0041] As a preferred embodiment of this application, the pharmaceutical composition further includes one or more pharmaceutically acceptable carriers or mediators.

[0042] The fifth aspect of the invention is to provide the use of a CA4 dimer prodrug compound and its stereoisomers, tautomers, solvates, or pharmaceutically acceptable salts thereof, or the pharmaceutical composition thereof, in the preparation of an antitumor drug.

[0043] As a preferred embodiment of this application, the antitumor drug exerts its antitumor effect by releasing CA4 and inhibiting the expression of CYP7A1, a key enzyme in microtubule polymerization and bile acid synthesis.

[0044] As a preferred embodiment of this application, the antitumor drug is a drug for treating solid tumors.

[0045] As a preferred embodiment of this application, the tumor is a solid tumor such as liver cancer, gastric cancer, colorectal cancer, or non-small cell lung cancer.

[0046] As a preferred embodiment of this application, the liver cancer is hepatocellular carcinoma (HCC).

[0047] The CA4 dimer compounds of the present invention can be prepared in various forms, such as liquid, semi-solid, and solid dosage forms, including but not limited to solid dosage forms, semi-solid dosage forms, liquid dosage forms, and gaseous dosage forms. Various routes of administration of the dimer compounds, pharmaceutical compositions, or drugs of the present invention are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, dermal, oral, topical, nasal, pulmonary, rectal, and topical administration; however, the present invention is not limited to these exemplified routes of administration.

[0048] The term "pharmaceutically acceptable" refers to a substance suitable for human and / or mammalian use without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier used for the administration of a therapeutic agent, including various excipients and diluents. More preferably, it includes, but is not limited to: water, saline, buffer solutions, glycerol, ethanol, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should be matched to the route of administration, as is well known to those skilled in the art.

[0049] The pharmaceutical compositions of the present invention contain a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier. Generally, pharmaceutical formulations should be matched to the route of administration; the dosage forms of the pharmaceutical compositions of the present invention are injections, oral formulations (tablets, capsules, oral liquids), transdermal formulations, and sustained-release formulations. They are prepared, for example, using physiological saline or an aqueous solution containing glucose and other excipients by conventional methods. The pharmaceutical compositions are preferably manufactured under aseptic conditions. More preferably, the dosage forms of the pharmaceutical compositions are injections (e.g., intravenous injection, intramuscular injection) or oral dosage forms (e.g., tablets, capsules, oral liquids).

[0050] The effective amount of the active ingredient described in this invention can vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: the pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration.

[0051] Compared with the prior art, the beneficial effects of this application are:

[0052] 1. The design of the CA4 symmetrical dimer prodrug is structurally innovative; the metabolites are non-toxic due to the connection between carbonate or oxalate bonds.

[0053] 2. In in vitro experiments, CA4-dimer1 showed significantly better inhibitory activity against various liver cancer cell lines (such as Hep3B, LM3, and HepG2) than CA4 monomer.

[0054] 3. In a nude mouse human liver cancer model, CA4-dimer1 (31.2 mg / kg) achieved a tumor inhibition rate of 73.2%, which was 34.0% higher than that of CA4, demonstrating improved efficacy in vivo and in vitro, with good safety.

[0055] 4. After intravenous injection in rats, the blood drug concentration fluctuates relatively steadily, avoiding the stimulation caused by the release of a large amount of drug in a short period of time, thus optimizing pharmacokinetics.

[0056] 5. The dimer of small molecule compounds can inhibit the expression of tubulin and bile acid synthase CYP7A1, and exert a synergistic anti-tumor effect.

[0057] 6. The CA4 dimer prodrug metabolites are CO2 and formic acid, which are FDA-recognized safe substances; blood routine tests, blood biochemistry, and HE staining have confirmed that there is no liver or kidney toxicity, providing a safety guarantee for the clinical use of the drug; the preparation process avoids the use of highly toxic reagents, and the high yield and simple synthesis steps are suitable for industrial production. Attached Figure Description

[0058] Figure 1 This is a synthesis route diagram of CA4-dimer1 in Embodiment 1 of the present invention.

[0059] Figure 2 This is a synthesis route diagram of CA4-dimer2 in Embodiment 1 of the present invention.

[0060] Figure 3 This is a liquid chromatogram of CA4-dimer1 at a detection wavelength of 220 nm in Embodiment 1 of the present invention.

[0061] Figure 4 This is a liquid chromatogram of CA4-dimer1 at a detection wavelength of 254 nm in Embodiment 1 of the present invention.

[0062] Figure 5 This is the mass spectrum of CA4-dimer1 in Embodiment 1 of the present invention.

[0063] Figure 6 This is the ¹H NMR spectrum of CA4-dimer1 in Embodiment 1 of the present invention.

[0064] Figure 7 This is a liquid chromatogram of CA4-dimer2 at a detection wavelength of 220 nm in Embodiment 1 of the present invention.

[0065] Figure 8 This is a liquid chromatogram of CA4-dimer2 at a detection wavelength of 254 nm in Embodiment 1 of the present invention.

[0066] Figure 9 This is the mass spectrum of CA4-dimer2 in Embodiment 1 of the present invention.

[0067] Figure 10 The image shows the ¹H NMR spectrum of CA4-dimer2 in Embodiment 1 of this invention.

[0068] Figure 11 This is one of the antitumor activity diagrams of CA4, CA4-dimer1, and CA4-dimer2 against LM3 liver cancer cells in Embodiment 3 of the present invention.

[0069] Figure 12 This is the second diagram showing the antitumor activity of CA4, CA4-dimer1, and CA4-dimer2 against LM3 liver cancer cells in Example 3 of the present invention.

[0070] Figure 13 This is the third diagram showing the antitumor activity of CA4, CA4-dimer1, and CA4-dimer2 against LM3 liver cancer cells in Example 3 of the present invention.

[0071] Figure 14 This illustrates the synergistic inhibitory effect of CA4 on β-tublin and CYP7A1 in Embodiment 4 of the present invention.

[0072] Figure 15 The indicators are subcutaneous tumor-related indicators in nude mice after treatment in Example 5 of this invention (A is tumor inhibition rate; B is tumor size).

[0073] Figure 16 The weight fluctuation of nude mice after treatment in Example 5 of this invention.

[0074] Figure 17 This is a routine blood test of nude mice after treatment in Embodiment 5 of the present invention.

[0075] Figure 18 This is a blood biochemical test of nude mice after treatment in Example 5 of the present invention.

[0076] Figure 19 This is a histopathological section of a nude mouse after treatment, as shown in Example 5 of the present invention.

[0077] Figure 20This is the drug-time curve of intravenous injection of CA4 in Example 6 of the present invention.

[0078] Figure 21 This is the pharmacokinetic curve of intravenous injection of CA4-dimer1 in Example 6 of the present invention.

[0079] Figure 22 This is the pharmacokinetic curve of intravenous injection of CA4-dimer2 in Example 6 of the present invention. Detailed Implementation

[0080] The present invention will be further described in detail below with reference to specific embodiments, but the present invention is not limited to the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to different requirements of specific use, and the implementation conditions not specified are those in conventional experiments. The following embodiments and experimental examples are used to further illustrate the present invention, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the reagents and instruments used in the present invention are all commercially available conventional products, and the experimental methods refer to "Organic Synthesis Experiment Handbook" (3rd edition, Chemical Industry Press).

[0081] Example 1: Preparation and identification of compound CA4-dimer1, including the following steps:

[0082] 1. Preparation of CA4-dimer-1:

[0083] Step 1, Synthesis of 5-(bromomethyl)-1,2,3-trimethoxybenzene:

[0084] (1,2,3-trimethoxyphenyl-5-yl)methanol (25.0 g, 126.1 mmol) was dissolved in dichloromethane (DCM, 300 mL), and phosphorus tribromide (68.3 g, 252.3 mmol) was slowly added. After stirring at room temperature for 2 hours, the reaction was quenched with saturated ammonium chloride solution, and the mixture was extracted with ethyl acetate (EA, 3 × 100 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by normal-phase silica gel column chromatography (petroleum ether / ethyl acetate = 7:3) to give 15.0 g of yellow solid (yield 45.6%). LC-MS (ESI): m / z 261.20 [M+H] + The retention time is 1.57 min.

[0085] Step 2, Preparation of triphenyl(3,4,5-trimethoxybenzyl)phosphine:

[0086] The product obtained in step 1 (15.0 g, 57.5 mmol) was dissolved in tetrahydrofuran (THF, 300 mL) with triphenylphosphine (22.6 g, 86.2 mmol) and stirred at room temperature for 2 hours under nitrogen protection. The precipitated solid was filtered, washed with THF, and dried under vacuum to give 28.0 g of a yellow solid (yield 93.1%). LC-MS (ESI): m / z 443.10 [M+H] + Retention time: 1.02 min.

[0087] Step 3: Synthesis of (Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenol

[0088] The product from step 2 (20.65 g, 39.5 mmol), 3-hydroxy-4-methoxybenzaldehyde (1.0 g, 6.6 mmol), and potassium carbonate (4.6 g, 32.9 mmol) were added to ethanol (50 mL), and the mixture was reacted at 80 °C for 6 hours under nitrogen protection. The reaction solution was concentrated, extracted with EA (3 × 100 mL), washed with saturated brine, dried, and concentrated. Purification was performed by silica gel column chromatography (petroleum ether / ethyl acetate = 8:2) to give 5.0 g of a yellow solid (yield 48.1%). LC-MS (ESI): m / z 317.20 [M+H] + Retention time: 1.55 min.

[0089] Step 4: Preparation of intermediate carbonate:

[0090] The product obtained in step 3 (2.0 g, 6.3 mmol), 4-nitrobenzene chloroformate (2.6 g, 12.6 mmol), and pyridine (1.5 mL, 19.0 mmol) were dissolved in DCM (50 mL) and reacted at 80 °C for 6 hours. The mixture was purified by normal-phase column chromatography (petroleum ether / ethyl acetate = 8:2) to give 2.5 g of a yellow solid (yield 82.1%). LC-MS (ESI): m / z 482.20 [M+H] + Retention time: 2.05 min.

[0091] Step 5, Synthesis of CA4-dimer1:

[0092] The product from step 3 (1.6 g, 5.0 mmol) and the product from step 4 (2.0 g, 4.2 mmol) were dissolved in DCM (20 mL), and 1,8-diazabicycloundec-7-ene (DBU, 1.3 g, 8.3 mmol) was added. The mixture was reacted at room temperature for 2 hours. The reaction solution was extracted with EA, washed with saturated sodium carbonate, dried, and purified by silica gel column chromatography (petroleum ether / EA = 3:7) and Prep-HPLC (column: Triart c18, 250 × 20.0 mm, d, S-5 μm, 12 nm; mobile phase A: 10 mmol NH4CO3 / water, mobile phase B: CH3CN; gradient: 0-95%B; detection wavelength 220 nm, flow rate: 17 mL / min, column temperature 25 °C). The purified product was lyophilized to give 2.0 g of white solid (yield 73.1%).

[0093] 2. Structural identification of CA4-dimer1:

[0094] (1) HPLC purity analysis:

[0095] Chromatographic column: YMC-Triart C18 (50*4.6mm, 5μm);

[0096] Mobile phase: A (water + 0.1% TFA) - B (acetonitrile + 0.1% TFA);

[0097] Gradient: Maintain 20% B for 0.4 min, increase the 20% B gradient to 95% B from 0.4 to 3.4 min, and maintain for 0.8 min; Flow rate: 2.5 mL / min;

[0098] Detection wavelength: 220 / 254 nm.

[0099] Results: When detected at a wavelength of 220 nm, the retention time of the main peak was 4.011 min, the peak area was 11,712,645, and the area percentage reached 99.842%. Figure 3 When detected at a wavelength of 254 nm, the retention time of the main peak was 4.013 min, the peak area was 4,430,822, and the area percentage was 99.811%. Figure 4 This indicates that under these conditions, the purity requirements for pharmaceutical compounds are met.

[0100] (2) Mass spectrometry and proton NMR characterization:

[0101] LC-MS (ESI+): m / z=676.1 [M+NH4] + The calculated value is 658.24, which is consistent with the theoretical value of 658.2414. Figure 5 ).

[0102] like Figure 6 As shown, the ¹H NMR (400 MHz, DMSO-d⁶) values ​​are: δ 7.22 (d, J = 2.0 Hz, ¹H), 7.19 (d, J = 2.1 Hz, ¹H), 7.13 (t, J = 5.5 Hz, 4H), 6.55 (s, 4H), 6.52 (s, 4H), 3.78 (s, 10H), 3.63 (s, 10H), 3.59 (s, 17H), confirming the target structure as shown in Formula II.

[0103] .

[0104] Physicochemical properties of CA4-dimer1:

[0105] Molecular formula: C 37 H 38 O 11 ;

[0106] Molecular weight: 658.2414;

[0107] Appearance: White powder.

[0108] Example 2: Preparation and identification of compound CA4-dimer2, including the following steps:

[0109] 1. The preparation method of CA4-dimer2 is as follows:

[0110] Steps 1-3: Following steps 1-3 of Example 1, intermediate (Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenol (5.0 g, yield 48.1%) was prepared using the same method.

[0111] Step 4: Synthesis of CA4-dimer2

[0112] The product from step 3 (5.0 g, 15.8 mmol) was dissolved in dichloromethane (DCM, 50 mL), and sodium hydride (NaH, 0.38 g, 15.76 mmol) was added under ice bath conditions. The mixture was stirred at room temperature for 0.5 hours. Subsequently, oxalyl chloride (1.0 g, 7.9 mmol) was added dropwise, and the reaction was carried out at room temperature for 2 hours. The reaction solution was quenched with saturated ammonium chloride solution, extracted with EA (2 × 50 mL), and the organic phases were combined, washed with saturated sodium bicarbonate, and dried over anhydrous sodium sulfate. The solution was purified by silica gel column chromatography (petroleum ether / EA = 3:7) and preparative HPLC (column: Triart C18, 250 × 20 mm; mobile phase: 10 mM ammonium carbonate aqueous solution-acetonitrile, gradient elution; flow rate 17 mL / min), and lyophilized to give 1.5 g of yellow solid (yield 27.7%).

[0113] 2. Structural identification of CA4-dimer2

[0114] (1) HPLC purity analysis:

[0115] Column: YMC Triart C18 (50×4.6 mm, 5μm);

[0116] Mobile phase: A (water + 0.1% TFA) - B (acetonitrile + 0.1% TFA);

[0117] Gradient: Maintain 20% B for 0.4 min, increase gradient by 20%-95% B from 0.4 to 3.4 min, then maintain for 0.8 min; Flow rate: 2.5 mL / min; Detection wavelength: 220 / 254 nm.

[0118] Results: The retention time of the main peak was 3.98 min, and the peak area accounted for ≥98.4% ( Figure 7 and Figure 8 It meets the purity requirements for pharmaceutical compounds.

[0119] (2) Mass spectrometry and nuclear magnetic resonance characterization

[0120] LC-MS (ESI+): m / z=687.24 [M+H] + m / z = 704.2 [M+NH4] + The calculated value is 686.24, which is consistent with the theoretical value of 686.2363. Figure 9 ).

[0121] like Figure 10 ¹H NMR (400 MHz, DMSO-d⁶): δ 7.24 (d, J = 8.6 Hz, 2H), 7.18 (d, J = 8.6 Hz, 4H), 6.54 (s, 8H), 3.81 (s, 6H), 3.63 (s, 6H), 3.61 (s, 12H). This confirms the target structure as shown in Formula III.

[0122] .

[0123] Physicochemical properties of CA4-dimer2:

[0124] Molecular formula: C 38 H 38 O 12 ;

[0125] Molecular weight: 686.2363;

[0126] Appearance: Yellow powder.

[0127] Example 3: Inhibitory effect of CA4 dimer compound on solid tumor cancer cells

[0128] 1. Experimental materials:

[0129] Cell lines: Hep3B, HCC-LM3, Huh7, HepG2 liver cancer cell lines; DDP lung cancer cell line; KATO III gastric cancer cell line; SW480 colorectal cancer cell line (Zhejiang Meisen Cell Technology Co., Ltd.)

[0130] Compounds: CA4 (purity ≥98%, Shanghai Taoshu Biotechnology, catalog number T0853); CA4-dimer1, CA4-dimer2 (prepared in Examples 1-2, HPLC purity ≥98%)

[0131] Reagents: DMEM high glucose medium (Wuhan Pronosai, catalog number PM150210), RPMI-1640 medium (Wuhan Pronosai, catalog number PM150110), fetal bovine serum (Bio-Channel, catalog number BC-SE-FBS01), CCK-8 kit (MedChemExpress, catalog number HY-K0301), and matrix gel (MedChemExpress, catalog number HY-K6001).

[0132] Instruments: CO2 incubator (Thermo Fisher, model 371), multi-functional microplate reader (BioTek SynergyNeo2).

[0133] 2. Experimental methods:

[0134] (1) Cell culture:

[0135] Hep3B, LM3, Huh7, HepG2, KATO Ⅲ, and SW480 cells were passaged in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2. Cells in the logarithmic growth phase were used for experiments.

[0136] DDP cells were passaged in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2. Cells in the logarithmic growth phase were used for experiments.

[0137] (2) Cytotoxicity assay (CCK-8 assay):

[0138] Hep3B, LM3, Huh7, and HepG2 cells were seeded at a density of 5 × 10³ cells / well in 96-well plates and cultured for 24 hours until 80% confluence. Medium containing CA4, CA4-dimer1, and CA4-dimer2 (concentration gradient: 0, 3.90625, 7.8125, 15.625, 31.25, 62.5, 125, 250, 500, 1000 nM) was added, with 6 replicates per concentration. The control group received an equal volume of DMSO, with a final concentration ≤0.1% (v / v). DDP, KATO III, and SW480 cells were seeded at a density of 5 × 10³ cells / well in 96-well plates and cultured for 24 hours until 80% confluence. Medium containing 10 nM CA4, CA4-dimer1, and CA4-dimer2 was added, with 6 replicates per concentration. The control group received an equal volume of DMSO, with a final concentration ≤0.1% (v / v). After incubation at 37°C for 48 hours, 10 μL of CCK-8 reagent was added to each well, and the cells were incubated in the dark for 2 hours. The absorbance (OD value) was measured at 450 nm. Cell viability (%) was calculated as: (Experimental group OD450 - Blank group OD450) / (Control group OD450 - Blank group OD450) × 100%. Dose-response curves were fitted using GraphPad Prism 8.0 software, and the IC50 value was calculated. Each experiment was repeated three times. Data are expressed as mean ± standard deviation (n=3), and intergroup comparisons were performed using the Two-way Anova test.

[0139] (3) Cell scratch and invasion experiments:

[0140] Scratch and invasion assays were used to evaluate the anti-metastasis and anti-invasion activities of CA4-dimer1 and CA4-dimer2. In the scratch assay, LM3 cells were seeded in 6-well plates and cultured for 24 hours until 100% confluence. Scratches were then made in the wells using a pipette tip. LM3 cells were treated with CA4, CA4-dimer1, and CA4-dimer2 at 1 / 2 IC50 concentrations in low-serum medium for 48 hours. Images of the scratches were taken at 0 h and 48 h. In the anti-invasion activity study, LM3 cells were exposed to CA4, CA4-dimer1, and CA4-dimer2 at 1 / 2 IC50 concentrations for 48 hours. 1 ml of medium containing 10% FBS was added to the lower chamber, and after layering with matrix gel, 200 μL of cell suspension containing serum-free medium was added to the upper chamber. The cells were incubated in a 5% CO2 incubator for 24 h. Remove the chamber, wash gently with PBS, fix with 4% paraformaldehyde for 30 minutes, stain with 0.1% crystal violet for 20 minutes, rinse three times with PBS, and wipe away any unmigrated cells from the upper chamber with a cotton swab. Place the chamber on a glass slide and randomly select three fields of view to count the cells on the lower chamber surface.

[0141] (4) Experimental results:

[0142] Referring to Table 1, the two CA4 dimer compounds, CA4-dimer1 and CA4-dimer2, synthesized in this invention significantly inhibited the proliferation of four hepatocellular carcinoma cell lines (Hep3B, LM3, Huh7, and HepG2) in a dose-dependent manner. Compared with CA4, the CA4 dimer compounds CA4-dimer1 and CA4-dimer2 exhibited better antitumor activity at the cellular level. Figure 11 As shown, CA4-dimer1 and CA4-dimer2 both exhibited potential inhibitory effects on the permeation of LM3 cells through the transwell chambers covered by matrix gel at a concentration of 1 / 2 IC50, and were more effective than the same doses of the compounds CA4-dimer1 and CA4-dimer2. Figure 12 As shown, after 48 hours, without drug treatment, the scratch area significantly narrowed, while incubation with CA4-dimer1 and CA4-dimer2 significantly inhibited scratch healing. In summary, the results of scratch and invasion assays indicate that CA4-dimer1 and CA4-dimer2 are superior to CA4 in terms of anti-metastasis and anti-invasive efficacy.

[0143] Table 1. IC50 values ​​of CA, CA4-dimer1, and CA4-dimer2 against hepatocellular carcinoma cell lines.

[0144]

[0145] Table 2. Cell viability (%) of other solid tumor cell lines at 10 nM after treatment with CA, CA4-dimer1, and CA4-dimer2.

[0146]

[0147] Conclusion: CA4-dimer1 showed significantly better antiproliferative activity than CA4 monomer in Hep3B, LM3, and HepG2 cell lines in vitro (P<0.001). CA4-dimer2 showed significantly better activity than CA4 monomer in Hep3B, LM3, and Huh7 cell lines, but its activity was comparable to that of CA4 in HepG2 cells.

[0148] Example 4: Synergistic effect of CA4 on tubulin and CYP7A1

[0149] 1. Experimental materials

[0150] Compounds: CA4 (purity ≥98%, Shanghai Taoshu Biotechnology, catalog number T0853), CA4-dimer1 (prepared in Example 1, purity ≥98%), colchicine (purity ≥98%, BioReagent, ST1173), TUBB-siRNA (sequence: GCGCCGAGCUGGUUGAUUCTT)

[0151] Reagents: DMEM high-glucose medium (Wuhan Pronosai, catalog number PM150210), fetal bovine serum (Bio-Channel, catalog number BC-SE-FBS01), CCK-8 kit (MedChemExpress, catalog number HY-K0301); jetPRIME® DNA / siRNA transfection reagent (sartorius, catalog number 101000001)

[0152] Instruments: CO2 incubator (Thermo Fisher, model 371), multi-functional microplate reader (BioTek SynergyNeo2)

[0153] 2. Experimental Methods

[0154] (1) Cell culture: The method is the same as in Example 3.

[0155] (2) Synergistic effect of tubulin and CYP7A1 site:

[0156] Hep3B cells were injected at a rate of 5 × 10 4 Cells were seeded at a density of 5 × 10³ cells / well in 6-well plates and cultured for 24 hours until 50% confluence. Cells were then transfected with siRNA containing the empty vector sequence and the TUBB sequence, respectively. After incubation at 37°C for 48 hours, cells were collected, and proteins were extracted using RIPA lysis buffer. The effect of siRNA treatment on β-tublin protein was analyzed by Western blot. Hep3B cells were seeded at a density of 5 × 10³ cells / well in 96-well plates and cultured for 12 hours until 50% confluence. Cells were transfected with siRNA containing the TUBB sequence in 6 replicates. After incubation at 37°C for 24 hours, 10 nM CA4 was added. The CCK8 assay was performed as in Example 3 to calculate cell viability, and intergroup comparisons were performed using the Two-way Anova test.

[0157] (3) Calculation of drug synergy index:

[0158] Hep3B cells were seeded at a density of 5 × 10³ cells / well in 96-well plates and cultured for 24 hours until 80% confluence. Medium containing CA4, colchicine, and CA + colchicine (1:1) (concentration gradients: 0, 3.90625, 7.8125, 15.625, 31.25, 62.5, 125, 250, 500, 1000 nM) was added to each well, with 6 replicates per concentration. An equal volume of DMSO was added to the control group, with a final concentration ≤0.1% (v / v). After incubation at 37°C for 48 hours, 10 μL of CCK-8 reagent was added to each well, and the cells were incubated in the dark for 2 hours. The absorbance (OD value) was measured at 450 nm. Cell viability (%) was calculated as: (Experimental group OD450 - Control group OD450) / (Control group OD450 - Control group OD450) × 100%. Dose-response curves were fitted using GraphPad Prism 8.0 software, and IC50 values ​​were calculated. The combination index (CI) was calculated using Compusyn software.

[0159] (4) CA4-dimer1 exerts a synergistic effect by inhibiting tubulin and regulating CYP7A1-related pathways:

[0160] Cells were loaded at a rate of 1×10 5 Hep3B cells were seeded at a density of cells / well in 6-well plates and cultured for 24 hours until 80% confluence. Hep3B cells were treated with 1.5 nM CA4-dimer1 for 24 hours, while the control group received no treatment. Proteins were extracted using RIPA lysis buffer, and the effects of CA4-dimer1 treatment on downstream proteins of the CYP7A1 / β-tublin pathway were analyzed by Western blot.

[0161] (5) Experimental results:

[0162] like Figure 14 As shown in Figure A, after transfection with siRNA containing the TUBB sequence, β-tublin protein expression was significantly inhibited. At this point, the addition of CA4 significantly reduced Hep3B cell viability, indicating that CA4 acts on the CYP7A1 site and exerts a synergistic anti-tumor effect with β-tublin. Figure 14 As shown in B and C, the synergistic index (CI) of the CA4 inhibitor combined with colchicine was less than 1 (CI < 1), indicating a strong synergistic effect. In our previous study, inhibition of CYP7A1 affected the expression of CTSB and CSTD proteins through the autophagy pathway. Figure 14As shown in D in [reference], since CA4-dimer1 can be enzymatically released in vivo to release CA4, to explore its possible mechanism of action, we detected the effects of CA4-dimer1 on related pathway proteins. After treatment with CA4-dimer1, western-blot technology was used to analyze the downstream pathway proteins of CYP7A1 / β-tublin. The protein expressions of CTSB and CSTD in the CYP7A1 pathway were inhibited, and the expression of p-PI3K / p-AKT in the downstream pathway of β-tublin was inhibited. Therefore, CA4-dimer1 plays an anti-tumor effect by regulating tubulin β-tublin and CYP7A1 and their downstream pathways, and has the same pharmacological effect as CA4.

[0163] Example 5 Inhibitory effect of CA4-dimer1 on nude mouse liver cancer model derived from liver cancer tissues of patients with liver cancer

[0164] 1. Experimental materials:

[0165] Compounds: CA4 (purity ≥ 98%, Shanghai TargetMol, catalog number T0853), CA4-dimer1 (prepared in Example 1, purity ≥ 98%), CA4P (Fosbretabulin, MedChemExpress, catalog number HY-13226), Lenvatinib (Selleck, catalog number S1164);

[0166] Experimental animals: 30 BALB / c-nu nude mice (male, 6 weeks old, body weight 18 - 22 g, Hangzhou Medical College, license number SCXK(Zhe)2024 - 0002), housed in a SPF-level environment;

[0167] The liver cancer tissue source was approved by the informed consent of the patient and the Ethics Committee of the First Affiliated Hospital of Zhejiang University School of Medicine (IIT20240651B-R1). HCC surgical resection tissues were obtained, pathologically confirmed as moderately differentiated hepatocellular carcinoma, frozen in liquid nitrogen for later use, digested into single-cell suspension with collagenase IV before transplantation, and the concentration was adjusted to 5×10 7 cells / mL. The animal experiment was approved by the Animal Experiment Ethics Committee of the First Affiliated Hospital of Zhejiang University (20241634).

[0168] 2. Reagent preparation:

[0169] Blank solvent: 5% DMSO + 15% castor oil (Kolliphor EL) + 80% normal saline;

[0170] CA4-dimer1: 31.2 mg / kg, dissolved in the blank solvent;

[0171] Positive controls: CA4P (40 mg / kg, dissolved in normal saline), lenvatinib (5 mg / kg, 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% normal saline).

[0172] In the experimental design, we used equivalent molar doses for calculation, and the 31.2 mg / kg of CA4-dimer1 was equal to the 30 mg / kg of the CA4 group.

[0173] 3. Grouping of experimental animals:

[0174] Construction of a nude mouse hepatocellular carcinoma (HCC) model derived from patient tissue: HCC tissue from patients was cut into 3 mm sections. 3 The fragment was implanted subcutaneously into the right back of nude mice until the tumor volume reached 100-150 mm. 3 (Approximately 3 weeks later) the tumors were randomly divided into 5 groups (n=6) as shown in Table 3 according to their tumor volume; they were given a tail vein injection (CA4, CA4-dimer1, CA4P) or a gavage (lenvatinib) once a day for 21 consecutive days.

[0175] Table 3. Experimental grouping of subcutaneous tumor model (n=6)

[0176]

[0177] 4. Testing indicators:

[0178] 1. Tumor volume: Measure the major diameter (a) and minor diameter (b) every 2 days, calculate the volume using the formula φ = 1 / 2 × a × b², and plot the growth curve;

[0179] 2. Tumor inhibition rate: The inhibition rate is calculated as follows: (1 − mean volume of the treatment group / mean volume of the control group) × 100%;

[0180] 3. Safety evaluation: During the administration period, observe the mental state, activity, weight, diet, and fur luster of nude mice daily and record any abnormalities; use an automated blood analyzer to detect blood routine indicators such as WBC, RBC, and PLT; use a biochemical analyzer to detect ALT, AST, BUN, CRE, GLU, TC, and TG; after euthanizing the nude mice, fix the heart, liver, spleen, lungs, and kidneys with paraformaldehyde, embed them in paraffin, section them, and observe and evaluate the toxicity of the drug to the organs by HE staining.

[0181] 5. Experimental Results:

[0182] (1) Tumor suppression effect:

[0183] Intravenous injection of CA4, CA4-dimer1, CA4P, and oral administration of lenvatinib can inhibit the growth of subcutaneous tumors in nude mice. Our results demonstrated that the CA4-dimer1 group (31.2 mg / kg) used a lower molar equivalent dose (tumor inhibition rate was 73.2%), but the therapeutic effect was better than that of the high-dose (40 mg / kg) CA4 monomer (54.2%, P<0.01); the CA4-dimer1 group was comparable to the CA4P group (77.9%) and the lenvatinib group (79.7%) ( Figure 15 ), and obvious large-area necrosis appeared in the tumor tissues of the CA4-dimer1 group ( Figure 19 ). CA4-dimer1 showed better tumor inhibition effect at a lower dose in the subcutaneous tumor model nude mice derived from liver cancer patient tissues. Recording the average body weight of mice within 21 days of treatment showed that the body weight fluctuations of nude mice in each group were <5%, without statistical differences ( Figure 16 ).

[0184] (2) Biosafety evaluation:

[0185] Blood routine tests showed that the indexes such as WBC, MCH, MCV, PCT, and PLT in the CA4-dimer1 group were all within the normal range, without significant differences compared with the blank control group ( Figure 17 ). Blood biochemical tests showed that there were no significant differences in ALT, AST, BUN, CRE, TC, and TG in the CA4-dimer1 group compared with the blank control group ( Figure 18 ); Histopathological sections showed that the organ structures of the heart, liver, spleen, lung, and kidney were intact, without necrosis, inflammation, or fibrosis ( Figure 19 ), indicating that CA4-dimer1 did not have obvious negative effects on the body of the subcutaneous tumor model nude mice derived from liver cancer patient tissues. <00资源标签专利文本翻译专家翻译内容无关解释额外换行​​​​​​​​

[0191] The animal experiments were approved by the Animal Experiment Ethics Committee of the First Affiliated Hospital of Zhejiang University (20241634).

[0192] 2. Dosing regimen:

[0193] Intravenous group: CA4 (10 mg / kg), CA4-dimer1 (10.4 mg / kg), CA4-dimer2 (5.2 mg / kg);

[0194] Oral administration group: CA4 (40 mg / kg), CA4-dimer1 (41.6 mg / kg), CA4-dimer2 (42.6 mg / kg).

[0195] CA4: Dissolve in 5% DMSO + 15% Kolliphor EL + 80% Saline;

[0196] CA4-dimer1: Use 5% DMSO+15% Kolliphor EL+80% Saline;

[0197] CA4-dimer2 (oral): Dissolve in 10% N-methylpyrrolidone (NMP) + 10% Kolliphor EL + 30% PEG400 + 50% Saline;

[0198] CA4-dimer2 (intravenous injection): dissolve with 5% NMP+10% Kolliphor EL+85% Saline.

[0199] 3. Blood sample collection and processing:

[0200] In the intravenous group (2 min, 5 min, 10 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h) and the oral group (5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h), 50 μL of whole blood was collected from each group (three males and three females) at each time point. Protein was precipitated by adding 0.1 M zinc sulfate solution, extracted with acetonitrile (containing 0.1% formic acid and 1.000 ng / mL LMTA), centrifuged (17000 g, 4℃, 10 min), and the supernatant was stored at -80℃. The supernatant was then analyzed by UPLC-MS / MS. Chromatographic conditions: ACQUITY UPLC® BEH C18 (3.0 × 50 mm, 1.7 µm), mobile phase: A (water, containing 0.1% FA + 5 mM YSA) - B (acetonitrile), gradient elution (0.3-0.7 min 60%-95%). B; 2.5-2.8 min (95%-60% B), flow rate 0.5 mL / min; mass spectrometry conditions: ESI+, MRM mode.

[0201] 4. Data Analysis:

[0202] Phoenix WinNonlin 8.1 calculates pharmacokinetic parameters (t) 1 / 2 T max C max AUC last AUC inf MRT last F abs (etc.), the data are expressed as mean and SD (n=3).

[0203] 5. Experimental Results:

[0204] (1) Intravenous administration:

[0205] We further characterized the pharmacokinetics of intravenously injected CA4-dimer1, CA4-dimer2, and CA4. The whole blood pharmacokinetic (PK) values ​​are shown in Table 4 below. After administration, we detected the active metabolite CA4 released from the prodrug in the blood of rats injected with both CA4-dimer1 and CA4-dimer2, and no toxic quinone byproducts were detected. This directly confirms that our designed esterase-dependent prodrug activation mechanism is effective in vivo. Taking CA4-dimer1 as an example, it can be effectively hydrolyzed in vivo and continuously releases CA4. CA4-dimer1:t 1 / 2=1.54±0.58 h (female mice), 0.96 h (male mice), AUC=2610.87±427.83 h·ng / mL (female mice), 1636.44 h·ng / mL (male mice), CA4 bioavailability (dose normalized) was 58.31±9.81% (female mice), 43.09% (male mice); CA4-dimer2:t 1 / 2 =1.02±0.12 h (female rats), 1.05±0.32 h (male rats); AUC=374.7±7.78 h·ng / mL (female rats), 447.83±69.58 h·ng / mL (male rats); CA4 bioavailability (dose-normalized) was 17.10±0.36% (female rats), 23.56±3.48% (male rats). This indicates that CA4-dimer1, as a prodrug, can act as a "reservoir" after intravenous injection. This sustained-release characteristic helps maintain a more stable and sustained blood drug concentration, avoiding the drastic fluctuations in blood drug concentration that may occur with direct injection of CA4, which is closely related to its excellent efficacy and improved safety observed in the PDX model. In contrast, intravenous injection of CA4-dimer2 also releases CA4, but the conversion efficiency is lower than that of CA4-dimer1.

[0206] (2) Oral administration:

[0207] We further characterized the pharmacokinetics of oral CA4-dimer1, CA4-dimer2, and CA4. The whole blood pharmacokinetic pharmacokinetic values ​​are shown in Table 5 below.

[0208] Following administration, we detected the active metabolite CA4 released from the prodrug in the blood of rats orally administered CA4-dimer1, and no toxic quinone byproducts were detected. CA4-dimer1: Fabs = 0.86 ± 0.47% (female rats), Fabs = 1.11 ± 0.48% (male rats). CA4-dimer2: No original form or metabolites were detected in plasma. The data show that the bioavailability of both oral CA4-dimer1 and CA4-dimer2 is low, clearly indicating that this dimeric prodrug strategy may not be suitable for oral administration.

[0209] Conclusion: Both oral and intravenous CA4-dimer1 can be converted into CA4 to exert their effects, while intravenous CA4-dimer2 can also release CA4, but the conversion efficiency is low. The metabolism of both is controllable and they have the potential to be developed into drugs.

[0210] Table 4. PK parameters of CA4 in whole blood of rats after single intravenous administration of CA4, CA4-dimer1, and CA4-dimer2.

[0211]

[0212] (Note: NA indicates not measured).

[0213] Table 5. PK parameters of CA4 in whole blood of rats after single oral administration of CA4 and CA4-dimer1.

[0214]

[0215] (Note: NA indicates not measured).

[0216] Conclusion: CA4 was detectable in the blood after oral or intravenous administration of CA4-dimer1 and CA4-dimer2, indicating that the dimer designed in this invention can be converted into CA4 to function as the active ingredient. Intravenous administration of CA4-dimer1 provided a stable, sustained-release reservoir for CA4, exhibiting good pharmacokinetic characteristics, while CA4-dimer2 showed relatively low conversion efficiency. However, the bioavailability of the dimeric prodrug was limited after oral administration.

[0217] In summary, the CA4 dimer prodrug compound of this invention can release two molecules of Combretastatin A4 (CA4) through hydrolysis by esterase, and exert a synergistic antitumor effect by inhibiting microtubule polymerization and regulating the CYP7A1-related pathway. In vitro experiments showed that the IC50 value of CA4-dimer1 against human hepatocellular carcinoma Hep3B cells was 1.48±0.18 nM, significantly superior to that of the CA4 monomer (10.86±1.2 nM). In a BALB / c nude mouse model of human hepatocellular carcinoma subcutaneous tumor, CA4-dimer1 (31.2 mg / kg) achieved a tumor inhibition rate of 73.2% without causing significant hematologic or hepatotoxic and renal toxicity. Pharmacokinetics showed that intravenous injection of CA4-dimer1 effectively converted it to CA4 and exerted its antitumor effect; although oral administration resulted in detectable CA4 release, its bioavailability was low. This invention provides a new solution to overcome the problems of poor pharmacokinetics and high toxicity of CA4, and has promising applications in the preparation of anti-hepatocellular carcinoma drugs.

[0218] Based on the above research, this invention provides a compound with significant hepatocellular carcinoma inhibitory activity and favorable in vivo pharmacokinetic properties, exhibiting excellent drug-likeness. This compound can be used to prepare CA4 dimer drugs for the treatment of hepatocellular carcinoma and related diseases, showing broad application prospects.

[0219] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications listed herein, as well as variations in the methods and compositions of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. A CA4 dimer prodrug compound or a pharmaceutically acceptable salt thereof, characterized in that: The structural formula of the compound is shown in general formula I: ； In the formula, R is -C(O)- or -C(O)C(O)-; When R is -C(O)-, the compound is bis(2-methoxy-5-((Z)-3,4,5-trimethoxystyryl)phenyl)carbonate, abbreviated as CA4-dimer1, and its structural formula is shown in general formula II; When R is -C(O)C(O)-, the compound is bis(2-methoxy-5-((Z)-3,4,5-trimethoxystyryl)phenyl)oxalate, abbreviated as CA4-dimer2, and its structural formula is shown in general formula III: 。 2. The method for preparing the CA4 dimer prodrug compound according to claim 1, characterized in that, The compound is CA4-dimer1, and its preparation method includes the following steps: Step 1: 3,4,5-trimethoxybenzyl alcohol was reacted with phosphorus tribromide in dichloromethane, and the mixture was purified by extraction and column chromatography to obtain 5-(bromomethyl)-1,2,3-trimethoxybenzene; Step 2: The product obtained in Step 1 is reacted with triphenylphosphine in tetrahydrofuran, and triphenyl(3,4,5-trimethoxybenzyl)phosphine bromide is obtained by solid-liquid separation; Step 3: The product obtained in Step 2, 3-hydroxy-4-methoxybenzyl-1-carboxaldehyde, and potassium carbonate are condensed in ethanol and purified by column chromatography to obtain (Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenol. Step 4: (Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenol, 4-nitrophenyl chloroformate and pyridine are reacted in dichloromethane at 70-90℃ for 4-10 h to obtain the intermediate (Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenyl(4-nitrophenyl) carbonate; Step 5: The product obtained in step 4 is coupled with another (Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenol under alkaline conditions, and after purification, the target compound CA4-dimer1 is obtained.

3. The method for preparing the CA4 dimer prodrug compound according to claim 1, characterized in that, The compound is CA4-dimer2, and its preparation method includes the following steps: Step 1: 3,4,5-trimethoxybenzyl alcohol was reacted with phosphorus tribromide in dichloromethane, and the mixture was purified by extraction and column chromatography to obtain 5-(bromomethyl)-1,2,3-trimethoxybenzene; Step 2: The product obtained in Step 1 is reacted with triphenylphosphine in tetrahydrofuran, and triphenyl(3,4,5-trimethoxybenzyl)phosphine bromide is obtained by solid-liquid separation; Step 3: The product obtained in Step 2, 3-hydroxy-4-methoxybenzyl-1-carboxaldehyde, and potassium carbonate are condensed in ethanol and purified by column chromatography to obtain (Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenol. Step 4: (Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenol and oxalyl chloride were condensed under alkaline conditions, and the resulting purified compound was CA4-dimer2.

4. A pharmaceutical composition, characterized in that, Includes the CA4 dimer prodrug compound of claim 1 and its pharmaceutically acceptable salt.

5. The pharmaceutical composition according to claim 4, characterized in that, It also includes one or more pharmaceutically acceptable carriers or mediators.

6. Use of the CA4 dimer prodrug compound of claim 1 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 5 in the preparation of a medicament for treating solid tumors.

7. The use according to claim 6, characterized in that, The CA4 dimer prodrug compound or its pharmaceutically acceptable salt exerts its antitumor effect by releasing CA4 and inhibiting the expression of CYP7A1, a key enzyme in microtubule polymerization and bile acid synthesis.

8. The use according to claim 6 or 7, characterized in that, The drug for treating solid tumors is used to treat liver cancer, stomach cancer, colorectal cancer, or non-small cell lung cancer.

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