A salt of gl-v8, its preparation method and application

Abstract

The application belongs to the technical field of medicine, and discloses a salt of GL-V8, a preparation method and application of the salt, wherein the salt of GL-V8 is a salt formed by 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butyloxy-8-methoxyflavone and a pharmaceutically acceptable acid, and the stoichiometric ratio of the salt is 1:1; the salt of GL-V8 or a pharmaceutical composition thereof can be used for preparing a medicine for preventing or treating liver fibrosis, sepsis, colitis or cancer; the salt of GL-V8 has strong in-vivo anti-tumor activity, and is expected to become a new medicine for treating leukemia.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a specific salt form of GL-V8, and a specific salt form of the pharmaceutically active compound 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone, including its amorphous and crystalline forms (including solvate and hydrate forms), its preparation method and uses. Background Technology

[0002] GL-V8 is a derivative of the flavonoid compound baicalein, with the molecular formula C0.05. 24 H 29 NO7, chemically named 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone, has the structure shown in (Ⅰ):

[0003] GL-V8 is a derivative of the flavonoid baicalin, an active ingredient in the traditional Chinese herbal medicine Scutellaria baicalensis. GL-V8 is one of the derivatives of baicalin and has the structural characteristics of a lysosomal molecule.

[0004] Studies have shown that GL-V8 possesses broad pharmacological activity, exhibiting significant inhibitory effects on various tumor cells, including T-lymphoblastic leukemia, colorectal cancer, and pancreatic cancer, both in vivo and in vitro. Its preliminary mechanism of action involves lysosomal damage-mediated lysosome-dependent cell death, thereby selectively killing malignant tumor cells without significant toxic side effects on normal tissues and cells—a characteristic not found in conventional chemotherapy drugs. GL-V8 is a derivative of baicalin and possesses lysosomal affinity. LC-MS analysis indicates that GL-V8 can accumulate at high concentrations in lysosomes within a short period. Experimental results show that GL-V8 initially induces lysosomal damage in tumor cells, increasing the accumulation of sphingomyelin in the lysosomal membrane lipid component and reducing the activity of acid sphingomyelinase (ASM), effectively inducing lysosome-dependent cell death in tumor cells and exerting its anti-tumor effect. We screened the target proteins of CPU-008 in lysosomes using a DARTS-based proteomics approach, initially identifying HSP70 as the binding target of GL-V8. We then used the CETSA method to verify the binding ability of the compound to the target molecule, suggesting that GL-V8 interferes with lysosomal stability by targeting and inhibiting HSP70.

[0005] Existing technologies disclose various pharmacological effects of GL-V8 and its applications in various pathological models, including anti-glioma (for example, Chinese invention patent with publication number CN116999430A discloses a QL-H008 preparation for treating glioma and its preparation method and application patent), and anti-melanoma (for example, Chinese invention patent with publication number CN117243941A discloses the application of the flavonoid derivative CPU-008 in the preparation of melanoma treatment drugs), etc.

[0006] Generally, the salts, solvates, hydrates, polymorphs, crystalline and amorphous forms of a given substance typically differ in their crystalline behavior and / or crystalline solid properties. Therefore, they may have different physical and pharmaceutical properties, such as shape, density, hardness, deformability, stability, purity, hygroscopicity, flowability, compactness, solubility and / or dissolution properties. These properties can, for example, affect: their manufacturability, processability, pharmacokinetic properties (e.g., bioavailability), drug stability (shelf life), dosability and / or reconstitutionability, such as their suitability as solid, semi-solid or liquid drug dosage forms (e.g., tablets, capsules, suspensions, solutions, suppositories or other drug dosage forms).

[0007] Nearly half of drug molecules exist in the form of salts. Drug formulation can improve certain undesirable physicochemical or biopharmaceutical properties of drugs, such as altering solubility or dissolution rate, reducing hygroscopicity, improving stability, changing melting point, facilitating preparation and purification, and improving permeability. Selecting a suitable salt form for drug development is essential. Furthermore, a single salt form may exist in different crystal forms. Different crystal forms have different melting points, solubilities, dissolution properties, and chemical stability, and these physicochemical properties can directly affect drug efficacy. Therefore, salt form screening and crystal form screening are crucial steps in drug development. For a specific compound, the advantages and disadvantages of its free state, various salt forms, and corresponding crystal forms are unknown. Based on further considerations of its druggability, finding a suitable salt form to provide a variety of intermediate products and / or active pharmaceutical ingredients for subsequent drug development is of great significance.

[0008] However, there are currently no reports on GL-V8 salt forms. Therefore, it is necessary to conduct a comprehensive and systematic multi-salt screening of GL-V8 to select the most suitable salt form for drug development. Furthermore, during the development of the GL-V8 manufacturing process, its strong free base electrostatic interaction hinders the large-scale production of formulations. Summary of the Invention

[0009] The purpose of this invention is to overcome the shortcomings of the prior art and provide an acid addition salt (especially hydrochloride salt) of 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone with good pharmacological activity, and its preparation method.

[0010] To achieve the above objectives, the present invention is implemented through the following technical solution: In a first aspect, the present invention provides a salt of GL-V8, wherein the salt of GL-V8 is a salt formed by 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone and a pharmaceutically acceptable acid in a stoichiometric ratio of 1:1.

[0011] Inorganic acids that form pharmaceutically acceptable acid addition salts include (by way of example and not limitation) hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, etc.

[0012] Organic acids that form pharmaceutically acceptable acid addition salts include (by way of example and not limitation) acetic acid, 2,2-dichloroacetic acid, adipic acid, ascorbic acid (in its D- or L- form, especially its L- form), aspartic acid (in its D- or L- form, especially its L- form), benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid (in its (+)- or (mon)- form, especially its (+) form), camphor-10-sulfonic acid (in its (+)- or (mon)- form, especially its (+) form), capric acid (capric acid), caprylic acid (capric acid), carbonic acid, cinnamic acid, citric acid, cyclohexane, dodecyl sulfate, ethane-1, 2-Disulfonic acid, ethanesulfonic acid, 2-hydroxy-ethanesulfonic acid, formic acid, galactobionic acid, gentianic acid, glucoheponic acid (in D- or L-form, especially the D-form), gluconic acid (in D- or L-form, especially the D-form), glucuronic acid (in D- or L-form, especially the D-form), glutamic acid, glutamate, 2-oxoglutamate, glycerophosphate, glycolic acid, hippuric acid, isobutyric acid, lactic acid (in D- or L-form), lactobionic acid, lauric acid, maleic acid, malic acid (in D- or L-form) (Forms), naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, palmitic acid, dihydroxynaphthoic acid (purine), propionic acid, pyroglutamic acid (in its D- or L- form, especially its L- form), salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, thiocyanate, and undecenoic acid, etc.

[0013] The salts of GL-V8 exist in the form of organic solvates, hydrates, or mixtures of organic solvates and hydrates; in the form of organic solvates, for example, in the form of ethanol compounds; in the form of mixtures of hydrates and organic solvates, for example, in the form of mixtures of hydrates and ethanol compounds.

[0014] The salts of GL-V8 exist in the form of crystals, partial crystals, amorphous or polymorphic crystals.

[0015] The structure of the salts in GL-V8 is mainly selected from the following:

[0016] Secondly, the present invention provides a method for preparing a salt of GL-V8, wherein 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone is dissolved in a solvent, and then a pharmaceutically acceptable acid is added dropwise or added one or more times, stirred to precipitate crystals, and the crystals are recovered to obtain the salt.

[0017] Furthermore, seed crystals are added during crystallization to initiate the reaction.

[0018] According to a preferred embodiment of the present invention, the solvent is selected from one or more of the following: alcohols, ethers, esters, and haloalkanes.

[0019] According to the present invention, the alcohol is preferably a C1-C10 alcohol, more preferably a C1-C8 alcohol, and even more preferably a C1-C5 alcohol.

[0020] According to a preferred embodiment of the present invention, the alcohol is selected from methanol, ethanol, n-propanol, isopropanol, n-butanol, or combinations thereof.

[0021] According to the present invention, the ether is preferably a C2-C8 ether, and more preferably a C2-C5 ether.

[0022] According to a preferred embodiment of the present invention, the ether is selected from: diethyl ether, tetrahydrofuran, or a combination thereof.

[0023] According to the present invention, the ester is preferably a C1-C10 ester, more preferably a C1-C7 ester, and even more preferably a C1-C5 ester.

[0024] According to a preferred embodiment of the present invention, the ester is selected from: methyl formate, ethyl acetate, isobutyl formate, isopropyl acetate, or combinations thereof.

[0025] According to a preferred embodiment of the present invention, the halogenated alkanes are C2-C8, and more preferably C2-C5 alkanes.

[0026] According to a preferred embodiment of the present invention, the haloalkanes are selected from: dichloromethane, chloroform, and carbon tetrachloride.

[0027] According to the present invention, the molar ratio of 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone to a pharmaceutically acceptable acid is preferably 1:0.8-1:1.5, more preferably 1:0.9-1:1.3, and even more preferably 1:1.0-1:1.1.

[0028] According to a preferred embodiment of the present invention, the temperature range during crystallization is 10-80℃, more preferably 25-30℃; the reaction time is 0.1-10h, more preferably 0.5-6h.

[0029] According to the present invention, the drying temperature during the recovery crystallization process is preferably 10-90℃, more preferably 20-80℃, and even more preferably 40-70℃; the drying time is 5-150h, more preferably 20-100h, and even more preferably 24-60h.

[0030] According to the present invention, the purity of the product obtained by the method is preferably 50%-99.9%, more preferably 75%-99.9%, and even more preferably 85%-99.9%.

[0031] Thirdly, the present invention provides a pharmaceutical composition comprising a salt of the GL-V9 and a pharmaceutically acceptable carrier and / or diluent.

[0032] Fourthly, the present invention provides the use of the salt of said GL-V9 or a pharmaceutical composition thereof for use in the preparation of medicaments for the prevention or treatment of liver fibrosis, sepsis, colitis or cancer.

[0033] The cancers mentioned include stomach cancer, lung cancer, breast cancer, prostate cancer, leukemia, ovarian cancer, esophageal cancer, liver cancer, colon cancer, and brain cancer.

[0034] The GL-V8 salt form, especially the hydrochloride form, prepared by this invention can exist stably and has advantages in terms of physicochemical properties, formulation processing performance, and bioavailability. For example, it has advantages in terms of melting point, solubility, hygroscopicity, stability, adhesion, compressibility, flowability, in vivo and in vitro dissolution, in vitro efficacy, and bioavailability.

[0035] The present invention has the following beneficial effects: (1) The GL-V8 salt type (hydrochloride) prepared by the present invention has low hygroscopicity. Low hygroscopicity can ensure that the sample can maintain low hygroscopic weight gain and not deliquescence during the later production, processing and storage transportation process, thereby ensuring the stability of drug quality; at the same time, the water solubility of hydrochloride is higher than that of base, which is beneficial to subsequent drug preparation.

[0036] (2) The GL-V8 salt form prepared by this invention has high chemical stability under high temperature and high humidity conditions.

[0037] (3) This invention provides a method for preparing GL-V8 salt form. In the prior art, during the development of the production process of GL-V8, the free alkali has a strong electrostatic effect, which is not conducive to the large-scale production of the formulation. This invention uses the salt formation method, and the electrostatic effect of GL-V8 after salt formation is weak, which is conducive to large-scale industrial production. Attached Figure Description

[0038] Figure 1 The growth inhibition curves of different GL-V8 salts on human hepatocellular carcinoma cells MHCC-97H are shown.

[0039] Figure 2 The growth inhibition curves of different GL-V8 salts on human colon cancer cells HCT 116 are shown.

[0040] Figure 3 The growth inhibition curves of different GL-V8 salt types on human acute monocytic leukemia cells THP-1 are shown.

[0041] Figure 4 The growth inhibition curves of different GL-V8 salts on human non-small cell lung cancer cells H460 are shown.

[0042] Figure 5 The growth inhibition curves of different GL-V8 salts on human gastric cancer cells BGC-823 are shown.

[0043] Figure 6 The growth inhibition curves of different GL-V8 salt types on human glioma cells U87 are shown. Detailed Implementation

[0044] The present invention will be described in detail below with reference to the embodiments, which describe in detail the preparation and use methods of the salt forms of the present invention, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. It will be apparent to those skilled in the art that many modifications to both materials and methods can be made without departing from the scope of the present invention.

[0045] Synthesis of Formula I compounds The compound of formula I described in this invention is 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone.

[0046]

[0047] The free base 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone was prepared by the preparation method described in patent application number CN115160279.

[0048] Example 1: Preparation of hydrochloride of 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone 2.0 g of 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone was dissolved in 20 ml of ethanol. The solution was cooled to 5 ± 5 °C, and 1.2 equivalents of hydrochloric acid (2 mol / L ethanol solution) were slowly added. The solution changed from turbid to clear. The mixture was then stirred at 20 ± 5 °C for 1 h, resulting in the precipitation of a large amount of yellow solid. The solid was filtered to obtain the hydrochloride salt. The obtained solid was placed in a vacuum drying oven and dried under vacuum at 50 °C for 24 h to obtain 2.0 g of 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone hydrochloride, a yellow solid powder.

[0049] Example 2: Preparation of sulfate of 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone 2.0 g of 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone was dissolved in 20 ml of ethanol. The solution was cooled to 5 ± 5 °C, and 1.2 equivalents of sulfuric acid (2 mol / L ethanol solution) were slowly added. The solution changed from turbid to clear. The mixture was then stirred at 20 ± 5 °C for 1 h, resulting in the precipitation of a large amount of yellow solid. The solid was filtered to obtain the sulfate. The obtained solid was placed in a vacuum drying oven and dried under vacuum at 50 °C for 24 h to obtain 2.1 g of yellow solid powder, 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone sulfate.

[0050] Example 3: Preparation of hydrobromide of 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone 2.0 g of 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone was dissolved in 20 ml of ethanol, cooled to 5 ± 5 °C, and 1.2 equivalents of hydrobromic acid (48% aqueous solution) were slowly added. The solution changed from turbid to clear. Then, it was stirred at 20 ± 5 °C for 1 h, and a large amount of yellow solid precipitated out. The solid was filtered to obtain hydrobromide. The obtained solid was placed in a vacuum drying oven and dried under vacuum at 50 °C for 24 h to obtain 2.1 g of hydrobromide of 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone as a yellow solid powder.

[0051] Example 4: Preparation of acetate of 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone 2.0 g of 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone was dissolved in 20 ml of ethanol, cooled to 5 ± 5 °C, and 1.2 equivalents of acetic acid were slowly added. The solution changed from turbid to clear. Then, it was stirred at 20 ± 5 °C for 1 h, and a large amount of yellow solid precipitated out. The solid was filtered to obtain acetate. The obtained solid was placed in a vacuum drying oven and dried under vacuum at 50 °C for 24 h to obtain 1.8 g of 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone acetate, a yellow solid powder.

[0052] Example 5: Preparation of methanesulfonate of 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone 2.0 g of 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone was dissolved in 20 ml of ethanol, cooled to 5 ± 5 °C, and 1.2 equivalents of methanesulfonic acid were slowly added. The solution changed from turbid to clear, and then stirred at 20 ± 5 °C for 1 h. A large amount of yellow solid precipitated out, which was filtered to obtain methanesulfonate. The obtained solid was placed in a vacuum drying oven and dried under vacuum at 50 °C for 24 h to obtain 1.5 g of methanesulfonate of 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone as a yellow solid powder.

[0053] Example 6: Preparation of p-toluenesulfonate of 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone 2.0 g of 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone was dissolved in 20 ml of ethanol, cooled to 5 ± 5 °C, and 1.2 equivalents of p-toluenesulfonic acid were slowly added. The solution changed from turbid to clear, and then stirred at 20 ± 5 °C for 1 h. A large amount of yellow solid precipitated out, which was filtered to obtain p-toluenesulfonate. The obtained solid was placed in a vacuum drying oven and dried under vacuum at 50 °C for 24 h to obtain 1.6 g of p-toluenesulfonate of 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone as a yellow solid powder.

[0054] Experimental Example 1: Investigation of the Hygroscopicity of GL-V8 Salt Approximately 1000 mg of the salt from GL-V8 prepared in Examples 1-6 was weighed and placed at 25±5℃ and 80% relative humidity for 24 hours. The sample mass before and after the treatment was compared. The details are shown in Table 1 below.

[0055] Regarding the description of hygroscopic characteristics and the definition of hygroscopic weight gain (Chinese Pharmacopoeia 2020 Edition, General Chapter 9103, Guidelines for Hygroscopicity Tests of Drugs, Experimental conditions: 25±1℃, 80% relative humidity, 24 hours): Deliquescence: Absorbs sufficient water to form a liquid. Extremely hygroscopic: Moisture absorption increases weight by at least 15.0%. It is hygroscopic: its weight gain due to moisture absorption is less than 15.0% but not less than 2.0%. Slightly hygroscopic: Moisture absorption weight gain is less than 2.0% but not less than 0.2%. None or almost none hygroscopicity: moisture absorption weight gain less than 0.2% Table 1 Hygroscopicity of GL-V8 Salt GL-V8 base 925.66 25±5℃ 80%RH 24 hours 942.53 16.87 1.82% hydrochloride 896.54 25±5℃ 80%RH 24 hours 898.08 1.54 0.17% sulfates 899.05 25±5℃ 80%RH 24 hours 904.70 5.65 0.63% hydrobromide 922.55 25±5℃ 80%RH 24 hours 934.89 12.34 1.34% acetate 993.54 25±5℃ 80%RH 24 hours 1011.83 18.29 1.84% Methane sulfonate 993.77 25±5℃ 80%RH 24 hours 1043.48 49.17 5.00% p-Toluenesulfonate 963.56 25±5℃ 80%RH 24 hours 1056.46 92.90 9.64% The results showed that among the GL-V8 salt types, hydrochloride, sulfate, hydrobromide, and acetate had low hygroscopicity. The hydrochloride type had a weight gain of less than 0.2%, indicating no or almost no hygroscopicity. The sulfate, hydrobromide, and acetate types had a weight gain of less than 2.0% but not less than 0.2%, indicating slight hygroscopicity. The methanesulfonate and p-toluenesulfonate types had a weight gain of less than 15% but not less than 2%, indicating hygroscopicity; these two salt types were not studied in subsequent research. The salt types of this invention (hydrochloride, sulfate, hydrobromide, and acetate) have low hygroscopicity, especially the hydrochloride type, indicating that these salt types are less prone to deliquescence during pharmaceutical production and storage.

[0056] Experimental Example 2: Salt Stability Study of GL-V8 A certain amount of the sample to be tested was weighed and placed in a petri dish, and then placed at 25℃ and 60%RH and 40℃ and 75%RH, respectively. After 30 days, the purity was determined by HPLC. The experimental results are shown in Table 2 below, and the HPLC purity test method is shown in Table 3.

[0057] Table 2 Salt stability of GL-V8

[0058] Table 3. HPLC methods for detecting related substances

[0059] Solution preparation: Dissolve 1 mg of sample in 1 ml of methanol by sonication to obtain the solution.

[0060] The results show that the salts of GL-V8 described in this invention have high chemical stability and can remain physicochemically stable for at least one month.

[0061] Experimental Example 3: Study on the solubility of GL-V8 salt in water 1 mL of pure water was added to a 1.5 mL glass vial using a pipette. An appropriate amount of the salt forms prepared in Examples 1-4 was weighed and placed in the glass vial to prepare a supersaturated aqueous solution. The solution was sonicated at room temperature for 30 min. The supernatant was collected, and the content was determined using a standard curve. The water solubility results of each salt form were shown in Table 4 below. Table 4. Solubility test results of GL-V8 salt form in water GL-V8 base 10.05 hydrochloride 16.78 sulfates 8.66 hydrobromide 12.23 acetate 20.45 The results showed that hydrochloride and acetate had high water solubility, which was significantly improved compared to the free state.

[0062] Experiment Example 4: Test of growth inhibitory activity against human tumor cells Experimental materials 1. Cells Human hepatocellular carcinoma cell line MHCC-97H, human colon cancer cell line HCT 116, human acute monocytic leukemia cell line THP-1, human non-small cell lung cancer cell line H460, human pancreatic cancer cell line BXPC3, human gastric cancer cell line BGC-823, and human glioma cell line U87 were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. They were cultured in MEM, DMEM, and RPMI 1640 medium containing 10% fetal bovine serum at 37°C in a humid cell culture incubator containing 5% CO2.

[0063] 2. Reagents (1) Cell culture medium: GIBCO, USA. Store at 4°C in the dark.

[0064] (2) Fetal bovine serum: Vicente Biotech Ltd., Canada. Inactivate in a 56°C water bath for 30 min before use to remove endotoxins, etc., and store at 4°C after aliquoting.

[0065] (3) Dimethyl sulfoxide (DMSO): Sigma-Aldrich, USA. Store at room temperature away from light.

[0066] (4) Methylthiazolyl tetrazolium (MTT) solution: Fluka, USA. Weigh 250 mg MTT and add it to 50 mL of 0.01 M PBS (pH 7.2) buffer. Sonicate at room temperature for 1 h until clear and transparent to prepare a 5 mg / mL MTT solution. Filter the solution through a 0.22 μm microporous membrane for sterilization and store at -80℃ protected from light.

[0067] Experimental instruments 1. Model 3111 water-jacketed CO2 incubator: Thermo Fisher Scientific, USA.

[0068] 2. YJ-875 Medical Clean Workbench: Suzhou Clean Equipment Factory.

[0069] 3. Varioskan full-wavelength microplate reader: Thermo Scientific, USA.

[0070] Experimental methods In vitro cell culture Human tumor cells were seeded into cell culture flasks using cell culture medium containing 10% fetal bovine serum and cultured in a humid cell culture incubator at 37°C with 5% CO2. After the cells reached the adherent state, the culture medium was discarded, and the cells were rinsed twice with 2-3 mL of 0.25% EDTA solution each time. After discarding the excess EDTA, 1 mL of 0.05% trypsin-EDTA solution was added, rinsed again, and then discarded. The flasks were then placed in a cell culture incubator for digestion. For this process, a new culture flask could be prepared, with 4.5 mL of fresh culture medium added. Digestion continued until most cells detached, and then 2 mL of fresh culture medium was added to stop the digestion. The cells were then seeded into new cell culture flasks according to the required cell density for the experiment.

[0071] 2. Cell viability assay (MTT assay) The MTT assay was used to evaluate the inhibitory effect of the test compound on the in vitro growth of human tumor cell lines. Mitochondria in living cells can reduce MTT to water-insoluble purple formazan crystals, which are deposited in the cells. DMSO can dissolve these crystals; therefore, after dissolution with DMSO, the absorbance value can be measured at 570 nm using a microplate reader. The number of living cells is directly proportional to the absorbance value within a certain range.

[0072] Seven types of human tumor cells in logarithmic growth phase (human liver cancer cell line MHCC-97H, human colon cancer cell line HCT116, human acute myeloid leukemia cell line THP-1, human lung cancer cell line H460, human pancreatic cancer cell line BXPC3, human gastric cancer cell line BGC-823, and human glioma cell line U87) were seeded in 96-well plates at a density of 2500-4000 cells / well, with 5 accessory wells per group. Drug administration began 24 h after seeding. The cells were treated with the target compound at gradient concentrations (intervention concentration range of 0.75 μM-30 μM) for 24 h. Then, 20 μL of MTT (5 mg / mL) solution was added to each well, and the cells were incubated in a cell culture incubator at 37°C in the dark for 4 h. The supernatant was discarded with a 1 mL syringe, and 100 μL of DMSO was added to each well to dissolve the bottom crystals. The absorbance was measured at 570 nm.

[0073] Cell growth inhibition rate % = (1 - (x μM absorbance - absorbance of blank group) / (0 μM absorbance - absorbance of blank group)) × 100% 3. Data statistics and analysis Experimental data are expressed as Mean ± SD. Statistical analysis was performed using GraphPad Prism 9.5.

[0074] Experimental results 1. IC50 (μM) against 7 human tumor cell lines Table 5 IC50 (μM) of GL-V8 salt form against human tumor cell lines GL-V8 base 8.635 0.61 2.749 13.61 10.19 18.13 21.33 hydrochloride 21.38 3.864 9.221 5.56 9.882 27.91 12.77 acetate 30.55 21.72 12.46 8.62 6.53 19.19 14.69 hydrobromic acid 21.62 1.472 8.506 5.09 6.679 17.80 11.03 sulfates 26.17 5.67 9.181 9.95 5.134 29.30 15.26 2. The growth inhibition curves of some of the above-mentioned human tumor cell lines are as follows: Figures 1-6 As shown.

[0075] The results showed that the salts of GL-V8 exhibited strong inhibitory activity against the aforementioned human tumor cell models. Among them, GL-V8 hydrochloride showed significantly better inhibitory activity than GL-V8 bases in human pancreatic cancer cells BXPC3 and human glioma cells U87.

[0076] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. However, the above description is merely a specific embodiment of the present invention, and the technical features of the present invention are not limited thereto. Any other embodiments derived by those skilled in the art without departing from the technical solution of the present invention should be covered within the patent scope of the present invention.

Claims

1. A GL-V8 salt, characterized in that, The salt of GL-V8 is a stoichiometric salt formed by 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone and hydrochloric acid in a 1:1 ratio.

2. The method for preparing the salt of GL-V8 according to claim 1, characterized in that, The method includes the following steps: Dissolve 5-hydroxy-7-(4-(bis(2-hydroxyethyl)amino)-1-yl)butoxy-8-methoxyflavone in a solvent, then add hydrochloric acid dropwise or repeatedly while stirring to induce crystallization. The crystals are then recovered to obtain the final product.

3. The method for preparing the salt of GL-V8 according to claim 2, characterized in that, Seed crystals are added during crystallization to initiate the reaction.

4. The method for preparing the salt of GL-V8 according to claim 2, characterized in that, The solvent is selected from one or more of alcohols, ethers, esters, and haloalkanes.

5. A pharmaceutical composition, characterized in that, It contains a salt of GL-V8 as described in claim 1 and a pharmaceutically acceptable carrier and / or diluent.

6. Use of the salt of GL-V8 of claim 1 or the pharmaceutical composition of claim 5 in the preparation of a medicament for treating pancreatic cancer and / or glioma.

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