A highly water-soluble bisaconitic acid molecule chemically modified paclitaxel compound, synthesis method and application

Abstract

The present application discloses a highly water-soluble paclitaxel compound chemically modified with a bisaconitic acid molecule, a synthesis method, and applications. The compound comprises paclitaxel and two aconitic acid molecules covalently bonded via an ester bond. The preparation method of the water-soluble paclitaxel compound is simple and suitable for industrial production. The paclitaxel drug molecule, after structural modification by the method of the present application, possesses amphiphilic properties, i.e., both hydrophilic and lipophilic properties. The improved water solubility and permeability provide the possibility of developing various oral and aerosol inhalation drug formulations. The compound has high water solubility at room temperature, dissolves without foaming, and can reach a concentration of more than 8 mg / ml without requiring any cosolvent. The compound has lower toxicity than the original drug paclitaxel. The ester bond of the compound undergoes accelerated hydrolysis and cleavage under the weakly acidic conditions of tumor tissue in vivo, thereby exhibiting certain tumor tissue targeting properties, good bioavailability, and anti-tumor effects.

Description

Technical Field

[0001] The present invention belongs to the field of organic synthesis and medicine, and particularly relates to a highly water-soluble paclitaxel compound for treating malignant tumors, a preparation method thereof, and pharmaceutical use thereof as an anticancer drug. Background Art

[0002] Paclitaxel, traded as Taxol, has a unique mechanism of action for treating cancer and is now widely used as an anti-tumor drug for the treatment of lung, breast, and ovarian cancers. However, paclitaxel has a very low solubility in water, less than 0.03 mg / mL. To improve its water solubility, paclitaxel is currently used clinically as an injectable solution, using a colorless, viscous solution of polyoxyethylene castor oil / anhydrous ethanol (1:1 ratio) as a cosolvent. While polyoxyethylene castor oil increases paclitaxel's water solubility, it can also cause various adverse reactions, including allergic reactions, nephrotoxicity, neurotoxicity, and cardiovascular toxicity. To prevent severe allergic reactions, pre-administration of paclitaxel with medications such as corticosteroids and diphenhydramine is often used clinically, but this still results in some patients experiencing allergic reactions after paclitaxel injection. Abraxane, a solvent-free paclitaxel albumin nanoparticle injectable suspension, was approved by the FDA for marketing in the United States in 2005. This albumin-bound paclitaxel nanoparticle suspension is made by combining paclitaxel and human albumin using high-pressure vibration technology to create nanoparticles. Each vial contains 100 mg of paclitaxel and approximately 900 mg of human albumin. Paclitaxel is the active ingredient, and human albumin serves as an excipient to disperse and stabilize the particles and transport the active ingredient. Paclitaxel albumin nanoparticles reduce toxic side effects, eliminating the toxicity associated with polyoxyethylene castor oil. Pre-infusion treatment with antiallergic medication and specialized infusion equipment are unnecessary, significantly shortening the infusion time. However, during the preparation of albumin-bound paclitaxel, paclitaxel and human albumin are formed into nanoparticles using high-pressure vibration technology. This process involves physical mixing, not covalently linking paclitaxel to albumin, resulting in a small amount of drug leakage. Prior to administration, the albumin-bound paclitaxel particles are dissolved into a soluble solution because the albumin in the system is prone to foaming. Some patients have allergic reactions to albumin while using this drug. In addition, cancer patients with hypertension, heart disease, hypervolemic heart failure, severe anemia, etc. will have adverse reactions to the solubilizer albumin in this drug.

[0003] Existing approaches to addressing paclitaxel's water solubility include dosage form modification and chemical structure modification. In recent years, the main approaches include liposomes, water-soluble polymer carriers, and albumin carriers. For example, Chinese Patent No. CN104758250A discloses a paclitaxel liposome, its preparation method, and use. The paclitaxel liposomes prepared in this patent have a small particle size and a narrow particle distribution, significantly improving the drug's penetration rate, allowing the drug to more quickly penetrate the lesion site for administration, thereby enhancing efficacy. However, this liposome delivery system suffers from issues such as low encapsulation efficiency, drug leakage, and stability. Another approach using water-soluble polymer carriers, such as Chinese Patent No. CN106554329B, discloses a water-soluble paclitaxel anticancer drug compound, its preparation method, and use. This patented technology covalently bonds the anticancer drug active moiety, paclitaxel, to a polyethylene glycol monoalkyl ether via a linker group (carbonylmethoxyacetyl) to form the water-soluble paclitaxel anticancer drug compound. Chinese Patent CN104231047B discloses water-soluble, targeted, activated paclitaxel derivatives, their preparation, and uses, using water-soluble polyamino acids as carriers to improve the water solubility of the original paclitaxel drug. However, these patents suffer from complex structural modification and synthesis methods, uneven molecular weight of the water-soluble polymer carrier, and demanding separation and purification methods. In particular, Patent CN109675047B utilizes a single cis-aconitic anhydride (CA) to modify various original drugs. This is synthesized through a ring-opening reaction between the original drug and CA, using triethylamine as a catalyst, and further ligated with N,N'-di-dodecyl-L-glutamic acid diamide, which in turn increases the drug's hydrophobicity. Chinese Patent CN101658516B discloses a pharmaceutical composition of a paclitaxel drug and a pharmaceutically acceptable biological carrier, and a method for its preparation. This pharmaceutical composition comprises a nanoparticle suspension prepared using the carrier protein, an organic phase, a stabilizer, a lyoprotectant, and the paclitaxel drug. However, some patients are allergic to albumin, and albumin preparations lack the diffusivity and penetration of small molecule drugs into tumor tissue, limiting the clinical application of pharmaceutical compositions prepared using this method. Chinese Patent CN111281979A discloses a cationic polyphosphazene compound, a polyphosphazene-drug conjugate, and a method for preparing the same. The polyphosphazene-drug conjugate is obtained by chemically linking a hydrophobic drug to the compound, such as by linking a spacer group to the cis-aconitic anhydride linker on the drug molecule docetaxel. However, this method suffers from issues such as molecular weight heterogeneity, poor drug loading reproducibility, and difficulty in fully determining the structure of the conjugate molecule. Therefore, providing a small molecule paclitaxel compound has become a pressing technical challenge in the field. Summary of the Invention

[0004] The purpose of the present invention is to provide a highly water-soluble small molecule drug, a synthesis method and pharmaceutical use of a bisaconitic acid-modified paclitaxel compound, and to solve the problems of stability, drug leakage and difficulty in purification in existing liposome drug systems.

[0005] Specifically, this application provides the following solutions:

[0006] First, the present application provides a highly water-soluble bisaconitic acid molecule chemically modified paclitaxel compound, the structure of which is shown in the following formula (I):

[0007]

[0008] Secondly, the present application provides a method for synthesizing the highly water-soluble bisaconitic acid molecule chemically modified paclitaxel compound (I), the specific steps of which are as follows:

[0009] 1) mixing aconitic anhydride and paclitaxel in an anhydrous solvent, and subjecting the mixture to an esterification reaction at 90° C. for 8 hours to obtain a reaction product;

[0010] The molar mass ratio of the above-mentioned paclitaxel to aconitic anhydride is 1:5;

[0011] The above-mentioned anhydrous solvent is preferably pyridine;

[0012] 2) The reaction product obtained in step 1) was evaporated at 60° C. to remove as much anhydrous solvent as possible, yielding a dark brown, sticky product, which was the evaporation product. After adding laboratory pure water and stirring (the sticky product partially dissolved and partially precipitated), the mixture was centrifuged at 5000 rpm for 5 minutes, and the supernatant was collected.

[0013] The volume ratio of the viscous product to the added water is preferably 1:10;

[0014] In a specific implementation, the precipitate obtained by centrifugation can also be added with water solvent again and centrifuged to obtain the supernatant;

[0015] 3) placing the supernatant obtained in step 2) into a 300 molecular weight dialysis bag and dialyzing it with deionized water for 24 hours to remove impurities, thereby obtaining a dialyzed product; and then freeze-drying the product at -20°C to obtain a powdery product.

[0016] 4) Recrystallization: The powdery product obtained in step 3) was dissolved in 60°C water (experimental grade), allowed to stand at 4°C for 2 hours, filtered, and the solid was dried to obtain the highly water-soluble bisaconitic acid molecule chemically modified paclitaxel compound.

[0017] Third, the present application provides the use of the highly water-soluble bisaconitic acid-modified paclitaxel compound (I) in the preparation of an anticancer drug. This anticancer drug is used to prevent or treat solid tumor cancers (such as breast cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, colon cancer, rectal cancer, and bladder cancer), sarcomas, gliomas, and hematologic cancers (such as leukemia, lymphoma, and myeloma). The dosage forms of this anticancer drug include oral administration and aerosol inhalation preparations.

[0018] Fourth, the present application provides a pharmaceutical composition, which is obtained by dissolving the highly water-soluble bisaconitic acid molecule chemically modified paclitaxel compound (I) in a pharmaceutically acceptable carrier or excipient, and the dosage form can be tablets (oral), aqueous solution, or spray; the carrier is preferably a liquid carrier, such as water, PBS buffer, physiological saline or glucose solution, etc., to prepare injections and various oral and aerosol inhalation pharmaceutical preparations.

[0019] The highly water-soluble bisaconitic acid chemically modified paclitaxel compound (I) prepared in this application contains a lipophilic anticancer active moiety and a hydrophilic moiety (carboxyl and hydroxyl groups). The lipophilic anticancer active moiety is the anticancer drug compound paclitaxel, and the hydrophilic moiety is formed by covalently ester-bonding two molecules of water-soluble aconitic acid and the anticancer drug compound paclitaxel. The structural modification of the paclitaxel drug molecule by this method imparts amphiphilic properties, namely, hydrophilicity and lipophilicity. Its improved water solubility and permeability suggest the compound may be used in the development of oral and aerosol inhalation drug formulations.

[0020] Furthermore, the present application utilizes water-soluble groups, including various organic acids and water-soluble amino acids, to enhance the water solubility of paclitaxel. These carriers, after forming esters with the hydroxyl groups of the original drug, can be further converted into salts, such as carboxylates, sulfonates, phosphates, or ammonium salts, significantly improving its water solubility. Due to the large molecular weight of paclitaxel and its predominantly hydrophobic groups, the addition of one or two hydrophilic groups, such as amino or carboxyl groups, is generally difficult to significantly improve its solubility. The present application uses aconitic anhydride to esterify the hydroxyl groups at positions 2 and 7 of paclitaxel, adding four water-soluble carboxyl groups to the paclitaxel molecule. These four carboxyl groups can then be converted into sodium salts, further enhancing water solubility. Furthermore, in the esterification process of taxane compounds, the esterification reaction conditions are similar to the acid chloride method. Conventional methods involve direct esterification of the acid with the alcohol in the presence of a carbodiimide dehydrating agent, such as dicyclohexylcarbodiimide (DCC) or diisopropylcarbodiimide (DIC), and the catalyst 4-dimethylaminopyridine (DMAP). However, these methods will bring problems in subsequent product separation and purification. Therefore, the present invention uses aconitic anhydride to react with paclitaxel in pyridine at 90-95°C to esterify the two hydroxyl groups of paclitaxel. The reaction is simple and the product purification process is simple.

[0021] The method for preparing a highly water-soluble bisaconitic acid-modified paclitaxel compound provided by the present invention is simple, rapid, and high-yield, making it suitable for large-scale industrial production. In vivo, cis-aconitic acid is an intermediate product of the isomerization of citric acid to isocitric acid under the action of aconitase (located in the mitochondria), and can be degraded and absorbed in the body. Compared with existing technologies (such as Chinese patent CN101658516B), the bisaconitic acid-modified paclitaxel compound prepared by this method is a single small molecule compound that does not contain a carrier protein, an organic phase, a stabilizer, or a lyoprotectant. The drug's purity and quality indicators are controllable, and its physical and chemical properties are more stable. The preparation process omits the high-pressure homogenization technology of the aforementioned patent, making it simpler and more feasible, making it more suitable for large-scale preparation and industrial production. The compound has a water solubility of over 8 mg / ml (saturated dissolution) at room temperature, achieving an effective drug concentration far exceeding the 0.075-1.0% concentration in patent CN101658516B. Furthermore, the compound dissolves without any cosolvent. When dissolved in an aqueous solution, it forms a clear aqueous solution. Because tumor cells produce large amounts of lactic acid through anaerobic glycolysis, the pH of the tumor tissue microenvironment is 5.7-7.2, significantly lower than the pH of 7.4 in normal tissue (Webb BA, Chimenti M, Jacobson MP, et al. Dysregulated pH: a perfect storm for cancer progression. Nat Rev Cancer, 2011, 11: 671-677). The bisaconitic acid-modified paclitaxel compound prepared by this method undergoes accelerated hydrolysis and cleavage of the ester bond under the weakly acidic conditions of tumor tissue in vivo, releasing the original paclitaxel molecule, which is then accumulated in the tumor tissue. Consequently, the compound exhibits certain tumor tissue targeting properties, good bioavailability, and anti-tumor efficacy.

[0022] Compared with the above-mentioned prior art, the method for preparing the highly water-soluble paclitaxel compound chemically modified with aconitic acid molecule obtained in the present invention has the advantages of being simple, rapid and having a high yield. After the structure of paclitaxel is modified by this method, the chemical properties are stable and the water solubility is greatly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 H NMR spectrum of the water-soluble paclitaxel anticancer drug compound.

[0024] Figure 2 Photograph of an 8 mg / mL solution of the water-soluble paclitaxel anticancer drug compound.

[0025] Figure 3 HPLC analysis results of purified water-soluble paclitaxel anticancer drug compound.

[0026] Figure 4 Experimental diagram of water-soluble paclitaxel anticancer drug compound on human breast cancer MCF7 cells.

[0027] Figure 5 Experimental diagram of water-soluble paclitaxel anticancer drug compound on human non-small cell lung cancer A549 cells.

[0028] Figure 6 Experimental diagram of water-soluble paclitaxel anticancer drug compound on human umbilical vein endothelial HUVEC cells.

[0029] Figure 7 Experimental diagram of water-soluble paclitaxel anticancer drug compound on osteogenic MC3T3 cells.

[0030] Figure 8 Photographs of human breast cancer cell MCF-7 xenograft tumor growth in nude mice without drug treatment.

[0031] Figure 9 Photo of the inhibitory effect of the common paclitaxel anticancer drug compound on the growth of human breast cancer cell MCF-7 xenograft tumors in nude mice.

[0032] Figure 10 Photo showing the inhibitory effect of water-soluble paclitaxel anticancer drug compound on the growth of human breast cancer cell MCF-7 xenograft tumors in nude mice.

[0033] Figure 11 Photo of the inhibitory effect of water-soluble paclitaxel anticancer drug compound on breast cancer cell 4T1 xenograft tumor growth in mice.

[0034] Figure 12 Inhibitory effect of water-soluble paclitaxel anticancer drug compound on the growth of breast cancer cell 4T1 and MCF-7 xenograft tumors in mice (tumor volume versus time). DETAILED DESCRIPTION

[0035] To more fully describe the features of the present invention, the present invention is further described below with reference to specific examples. Water-soluble paclitaxel anticancer drug compounds within the scope of the present invention can be synthesized using the same or similar methods and have been shown to produce the same or similar results. The examples described herein are intended to facilitate understanding and implementation of the present invention and are not intended to limit the present invention.

[0036] Example 1 Preparation of highly water-soluble bisaconitic acid-modified paclitaxel compounds

[0037] 1. Take 0.2135g of paclitaxel (purchased from Dalian Meilun Company, the same below) and 0.1951g of cis-aconitic anhydride (purchased from Aladdin Reagent Company, the same below), place them in a 60°C oven and dry for half an hour. In this example, the molar mass ratio of paclitaxel to aconitic anhydride is 1:5.

[0038] 2. Take 1g of anhydrous calcium chloride and put it in a 90℃ oven to dry for half an hour (try to ensure it is dry) and set aside.

[0039] 3. Take 5 ml of pyridine and place the anhydrous calcium chloride in step 2 in pyridine (C5H5N) for water absorption treatment.

[0040] 4. Centrifuge the pyridine treated with water in step 3 at 5000 rpm for 5 minutes, take the supernatant and put it into a penicillin bottle, add a dry magnet and set aside.

[0041] 5. Dissolve the paclitaxel and aconitic anhydride dried in step 1 in the pyridine in step 4 and allow to react.

[0042] 6. Place the pyridine to be reacted and the raw materials in step 5 in a 90°C magnetic stirring heating mantle for reaction for 8 hours.

[0043] 7. The reaction product was treated on a rotary evaporator at 60°C for 1 hour to remove as much pyridine as possible, obtaining a dark brown viscous product. 30 ml of laboratory pure water (purchased from Millipore) was added and stirred to dissolve, obtaining a partially dissolved and partially precipitated product.

[0044] 8. Centrifuge the partially dissolved and partially precipitated product in step 7 at 5000 rpm for 5 min, filter, and take the supernatant for later use; the separated precipitate is recovered and reused; the supernatant is placed in a 300 molecular weight dialysis bag (300 molecular weight grade, Spectrum, USA) and dialyzed with deionized water.

[0045] 9. In step 8, the deionized water used for dialysis should be changed every two hours for 24 hours.

[0046] 10. The dialyzed product in step 9 was freeze-dried at -20°C for 24 hours to obtain a light yellow powder (i.e., a water-soluble paclitaxel compound) having the structural formula shown in formula (I) above. The yield was calculated to be 60% and the purity was 99%.

[0047] 11. Recrystallization: Add small amounts of laboratory pure water (60° C.) gradually to the light yellow powder obtained in step 10. After the powder is completely dissolved, cool naturally to room temperature, place in a 4° C. refrigerator for 2 hours, filter, and dry the solid (freeze-dry at -20° C. for 24 hours) to obtain a purified sample, which is the purified highly water-soluble bisaconitic acid molecule chemically modified paclitaxel compound (purified water-soluble paclitaxel anticancer drug compound).

[0048] Figure 1 This is the H NMR spectrum of the yellow powder obtained in step 10, where the y-axis represents intensity.

[0049] Figure 2The light yellow powder obtained in step 10 above was dissolved in water to obtain a light yellow clear solution. The powder was rapidly dissolved in water without foaming. The concentration of the obtained solution was 8 mg / ml, indicating that the compound had good water solubility.

[0050] Figure 3 This is the HPLC analysis chart of the purified water-soluble paclitaxel anticancer drug compound obtained in step 11 above. The sharp peaks in the chart prove that the synthesized compound has a single structure and high purity.

[0051] Example 2 Preparation of water-soluble paclitaxel anticancer drug compound

[0052] This embodiment includes an injection made of water, physiological saline (0.9% sodium chloride) or glucose (5%) injection, and the preparation formula contains the water-soluble paclitaxel anticancer drug compound of the present invention, and the content of each component in the formula is calculated by weight percentage.

[0053] 1) Aqueous injection of water-soluble paclitaxel anticancer drug compound

[0054] 100 mg of the purified water-soluble paclitaxel anticancer drug compound prepared in Example 1 (Step 11) was added to a 20 mL volumetric flask and diluted to 20 mL with deionized water. The prepared injection solution was filtered through a 0.2 μm filter (Maiborui Biomembrane Technology (Nantong) Co., Ltd., model 0.2 μm, the same below) and then placed in a sterile glass bottle.

[0055] 2) 1.0% water-soluble paclitaxel anticancer drug compound in normal saline (0.9% sodium chloride) injection

[0056] 50 mg of the purified water-soluble paclitaxel anticancer drug compound prepared in Example 1 (Step 11) was added to a 10 mL volumetric flask, and the volume was adjusted to 100 mL with normal saline. After shaking, the prepared injection solution was filtered through a 0.2 μm filter and then placed in a sterile glass bottle.

[0057] 3) 5% glucose injection of water-soluble paclitaxel anticancer drug compound

[0058] 50 mg of the purified water-soluble paclitaxel anticancer drug compound prepared in Example 1 (Step 11) was placed in a 10 mL volumetric flask, and the volume was adjusted to 10 mL with 5% glucose injection. After shaking, the prepared injection solution was filtered through a 0.2 μm filter and then placed in a sterile glass bottle.

[0059] Example 3. Cytological study of the inhibitory effect of water-soluble paclitaxel on the growth of human breast cancer cell MCF-7 xenograft tumors in nude mice

[0060] Tumor cells in the logarithmic growth phase (human non-small cell lung cancer A549 cells, human breast cancer MCF-7 cells, human umbilical cord endothelial HUVEC normal cells, and MC3T3 osteoblasts purchased from the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, all commercial cells) were taken. After the cells covered the bottom of the culture flask, they were digested with 0.25% trypsin and the cells were pipetted into a single cell suspension with culture medium. The cells were counted using a cell counting plate, and the number of cells per well was approximately 10 5 / mL was inoculated into a 96-well plate, and 200 μL of cell culture fluid (Gibco cell culture medium, the main components of which are amino acids, glucose, inorganic salts, vitamins, and trace elements, etc.) was added to each well. After the 96-well plate was cultured in a cell culture incubator for 24 hours, it was observed under an inverted microscope. After the cells were evenly spread across the bottom of the plate, different concentrations of purified water-soluble paclitaxel (20 nM, 100 nM, 200 nM, 500 nM, 1 μM, 5 μM, 10 μM, 15 μM, 20 μM) prepared in Example 1 (Step 11) were prepared with PBS solution. The concentrations were sequentially added to each well from high to low. To increase the accuracy of the experiment, four wells were provided for each concentration of the drug. The positive control group was also treated with the original drug, paclitaxel (dissolved in 1 μL of dimethyl sulfoxide (DMSO)) at concentrations of 20 nM, 100 nM, 200 nM, 500 nM, 1 μM, 5 μM, 10 μM, 15 μM, and 20 μM. A blank control group received no drug but an equal amount of cell culture medium. After 24 hours, 100 μL of DMEM (RPMI) (containing 10% CCK-8) was added to each well and the cells were cultured in an incubator for another 4 hours. The absorbance (OD) of each well was measured using a microplate reader (Thermo Fisher Scientific Multiskan FC) at a detection wavelength of 492 nm and a reference wavelength of 630 nm. Data were recorded and repeated three times.

[0061] Calculation of tumor cell growth inhibition rate (IR) value:

[0062] IR (%) = (1 - average OD value of the experimental group / average OD value of the normal control group) * 100%, with drug concentration as the horizontal axis and cell inhibition rate as the vertical axis.

[0063] EXCEL data analysis software was used for intergroup T-test and analysis of variance. P < 0.05 was considered statistically significant.

[0064] Results and Discussion

[0065] The inhibitory effect of the drug on tumor cells was evaluated using human non-small cell lung cancer A549 cells and human breast cancer MCF-7 cells.

[0066] like Figure 4As shown, water-soluble paclitaxel has a good inhibitory effect on human non-small cell lung cancer A549 cells, but the inhibitory effect on A549 tumor cells is slightly lower than that of the positive control drug paclitaxel.

[0067] like Figure 5 As shown in the figure, water-soluble paclitaxel has a high inhibition rate at each concentration. The positive control drug paclitaxel (20nM~20μM concentration range) has a stronger inhibitory effect on human breast cancer MCF-7 cells than water-soluble paclitaxel.

[0068] The inhibitory effect of the drug on normal tissue cells was evaluated using human umbilical cord endothelial HUVEC normal cells and MC3T3 osteoblasts.

[0069] like Figure 6 As shown, the positive control drug paclitaxel (within the concentration range of 20 nM to 20 μM) has a stronger inhibitory effect on normal human umbilical cord endothelial HUVEC cells than water-soluble paclitaxel. The drug toxicity of water-soluble paclitaxel is weaker than that of the original drug paclitaxel.

[0070] like Figure 7 As shown in the results, at a concentration of 5 μM, water-soluble paclitaxel had an inhibitory effect on MC3T3 osteoblasts that was essentially equivalent to that of the positive control drug, paclitaxel. However, at other drug concentrations, the positive control drug, paclitaxel (ranging from 20 nM to 15 μM), was more toxic to osteoblasts than water-soluble paclitaxel.

[0071] In summary, compared with the original drug paclitaxel, the anti-tumor efficacy of water-soluble paclitaxel is slightly reduced, but it still has a good anti-tumor effect, but the toxicity of the drug to normal tissue cells is reduced.

[0072] Example 4 Pharmacodynamic Study on the Inhibitory Effect of Water-Soluble Paclitaxel on the Growth of Human Breast Cancer Cell MCF-7 Xenografts in Nude Mice

[0073] 1) Preparation of drug solution

[0074] The purified water-soluble paclitaxel prepared in Example 1 (Step 11) was added to 0.9% sodium chloride solution to a concentration of 5 mg / ml. The dosage volume was 0.2 ml / 20 g, i.e., a 10 mg / kg dose. The solution was then diluted with 0.9% sodium chloride solution to other desired doses. A control drug, paclitaxel injection (30 mg x 5 ml / box, Haikou Pharmaceutical Factory), was fully diluted with normal saline to 1 mg / ml. The dosage volume was 0.2 ml / 20 g, i.e., a 10 mg / kg dose.

[0075] 2) Experimental Animal Source, Species, Strain, and Grouping: BALB / c-null nude mice (Shanghai Institute of Materia Medica, Chinese Academy of Sciences), weight: 18-24 g, sex: female. Experimental animal groups: blank control (n=6), paclitaxel control (n=6), water-soluble paclitaxel treatment (n=6).

[0076] 3) Implanted tumor and experimental methods

[0077] Human breast cancer cell line MCF-7 was inoculated subcutaneously in the axilla of nude mice at a cell inoculation rate of 2*10 6 Human breast cancer cell line MCF-7 in logarithmic growth phase was prepared into 1×10 8 / ml cell suspension, 0.1ml was inoculated into the right axilla of nude mice. The diameter of the transplanted tumor in nude mice was measured with a vernier caliper. When the tumor grew to 100-300mm 3 The animals were then randomly divided into groups. The anti-tumor effects of the test substance were dynamically observed by measuring tumor diameter. Tumor diameter was measured every three days. The dosing volume was 0.5 ml / 20 g. After 28 days, the mice were sacrificed, and the tumors were surgically removed and weighed.

[0078] 4) Results and Discussion

[0079] Test results such as Figure 8 、 Figure 9 and Figure 10 As shown in the figure, the drug was administered via tail vein injection at a dose of 10 mg / kg every two days for a total of 14 doses. The experimental group achieved an inhibition rate of 81.2% ± 0.8% against MCF-7 nude mouse xenografts. Paclitaxel stock drug, administered via tail vein injection at a dose of 10 mg / kg every two days for a total of 14 doses, achieved an inhibition rate of 72.6% ± 0.8% against MCF-7 nude mouse xenografts. The results showed that the water-soluble paclitaxel, at a dose of 10 mg / kg, had an 8.6% higher tumor inhibition rate than the positive control group, with a smaller effect on mouse body weight, indicating that water-soluble paclitaxel is more effective and less toxic than paclitaxel.

[0080] Example 5 Pharmacodynamics and Histological Analysis of the Inhibitory Effect of Water-Soluble Paclitaxel on the Growth of Breast Cancer Cell 4T1 Xenografts in Mice

[0081] 1) Preparation of drug solution

[0082] The purified water-soluble paclitaxel prepared in Example 1 (Step 11) was added to 0.9% sodium chloride solution to a concentration of 5 mg / ml. The dosage volume was 0.2 ml / 20 g, i.e., a 10 mg / kg dose. The solution was then diluted with 0.9% sodium chloride solution to other desired doses. A control drug, paclitaxel injection (30 mg x 5 ml / box, Haikou Pharmaceutical Factory), was fully diluted with normal saline to 1 mg / ml. The dosage volume was 0.2 ml / 20 g, i.e., a 10 mg / kg dose.

[0083] 2) Source, species, strain and grouping of experimental animals

[0084] BALB / c mice (Shanghai Institute of Materia Medica, Chinese Academy of Sciences), weight: 18-24 g, sex: female. Experimental animal groups: 6 blank control mice, 6 paclitaxel control mice, and 6 water-soluble paclitaxel treatment mice. 3) Tumor Implantation and Experimental Methods

[0085] Human breast cancer cell line 4T1 was inoculated subcutaneously in the axilla of nude mice, with a cell inoculation volume of 2*10 6 Human breast cancer cell line MCF-7 in logarithmic growth phase was prepared into 1×10 8 / ml cell suspension, 0.1ml was inoculated into the right axilla of nude mice. The diameter of the transplanted tumor in nude mice was measured with a vernier caliper. When the tumor grew to 100-300mm 3 The animals were then randomly divided into groups. The anti-tumor effects of the test substance were dynamically observed by measuring tumor diameter. Tumor diameter was measured every three days. The dosing volume was 0.5 ml / 20 g. After 28 days, the mice were sacrificed, and the tumors were surgically removed and weighed.

[0086] 4) Anatomical Experiment Design and Tumor Histopathological Examination

[0087] At the end of the experiment, all mice were killed by cervical dislocation. The tumors were removed and weighed to calculate the tumor growth inhibition percentage (TGI%), and then stored in 4% paraformaldehyde. For subsequent tumor tissue staining. TGI% = (W negative control group - W experimental group) / W negative control group × 100% W negative control group is the average tumor weight of mice in the negative control group; W experimental group is the average tumor weight of mice in the drug-treated group. The number of days of death of the remaining mice was recorded for survival studies. The "Guidelines for Tumor Induction in Mice and Rats (Updated in May 2013)" states that "the size of tumors in adult mice cannot exceed 20 mm (2.0 cm) in any direction", so any animal with a tumor size exceeding 20 mm is defined as terminated from the study.

[0088] Antitumor efficacy studies were conducted when mouse tumors reached a size of 62.50 ± 10 mm³ (5 mm x 5 mm x 5 mm). Groups 1 and 2 received intraperitoneal injections of either the original paclitaxel or a water-soluble paclitaxel solution (8 mg kg⁻¹ according to the instructions for treating solid tumors), respectively. Group 3 served as a negative control group and received no treatment. Three weeks later, mice were sacrificed by cervical dislocation, and tumors from the heart, liver, spleen, lung, kidney, and stomach were harvested and weighed. Dissected tissues were fixed histologically and sectioned.

[0089] 5) Results and Discussion

[0090] Test results such as Figure 11 As shown, Figure 11 In the experiment, AC were control group, group injected with original paclitaxel and group injected with water-soluble paclitaxel. Figure 11 It can be seen that the tumors of the mice in the control group that did not receive the drug were larger. In comparison, the tumors of the mice in the group injected with the original paclitaxel agent and the group injected with the water-soluble paclitaxel agent were smaller than those in the control group, but the tumors in the group injected with the water-soluble paclitaxel agent were relatively smaller, indicating that both the injection of the original paclitaxel agent and the water-soluble paclitaxel agent can inhibit the growth of the tumor, among which the injection of the water-soluble paclitaxel agent has a better inhibitory effect.

[0091] Figure 12 Figure 3 is the curve of tumor volume change. It can be seen that the tumor volume of mice in the group injected with original paclitaxel and the group injected with water-soluble paclitaxel was smaller than that of the control group, and the tumor in the group injected with water-soluble paclitaxel was relatively smaller, indicating that both the injection of original paclitaxel and water-soluble paclitaxel can inhibit tumor growth, but the inhibitory effect of injection of water-soluble paclitaxel is better.

Claims

1. A highly water-soluble bisaconitic acid molecule chemically modified paclitaxel compound, the structural formula of which is shown in formula (I): (I).

2. The method for synthesizing a highly water-soluble bisaconitic acid molecule chemically modified paclitaxel compound according to claim 1, wherein: The specific steps are as follows: 1) mixing aconitic anhydride and paclitaxel in an anhydrous solvent, and performing an esterification reaction at 90° C. to obtain an esterification reaction product; the molar mass ratio of paclitaxel to aconitic anhydride is 1:5; and the anhydrous solvent is pyridine; 2) The esterification reaction product obtained in step 1) was evaporated at 60°C to obtain a viscous product, which was stirred with water and then centrifuged to obtain the supernatant; 3) placing the supernatant obtained in step 2) into a 300 molecular weight dialysis bag and dialyzing it against deionized water for 24 hours, followed by freeze-drying at -20°C to obtain a powdery product; 4) dissolving the powdered product obtained in step 3) in water at 60° C., allowing to stand at 4° C., filtering, and drying the solid to obtain the highly water-soluble bisaconitic acid molecule chemically modified paclitaxel compound.

3. Use of the highly water-soluble bisaconitic acid molecule chemically modified paclitaxel compound according to claim 1 in the preparation of anticancer drugs for prevention or treatment.

4. A pharmaceutical composition, characterized in that The pharmaceutical composition is obtained by dissolving the highly water-soluble bisaconitic acid molecule chemically modified paclitaxel compound as claimed in claim 1 in a pharmaceutically acceptable carrier or excipient.

5. The pharmaceutical composition according to claim 4, wherein The pharmaceutically acceptable carrier or excipient includes water, PBS buffer, physiological saline or glucose solution.

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