Application of 2-methyl-4 (3H)-quinazolinone in preparation of anti-angiogenesis drugs

Abstract

The invention relates to an application of 2-methyl-4 (3H)-quinazolinone in preparation of an anti-angiogenesis medicine. The invention discloses the application of the 2-methyl-4 (3H)-quinazolinone compound as an angiogenesis inhibitor in anti-angiogenesis drugs for the first time, and the 2-methyl-4 (3H)-quinazolinone compound has a remarkable inhibition effect on internode blood vessels and dorsal longitudinal anastomosis angiogenesis of a Tg (flk: mCherry) fluorescent transgenic zebrafish strain. Meanwhile, the growth of the zebrafish embryos of the strain is not obviously influenced, and the toxicity is extremely low. The compound not only can be extracted from streptomyces, but also can be obtained through an artificial synthesis process, has a reliable and stable source, and has a good application prospect in the field of anti-angiogenesis drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical formulations, in particular to the application of 2-methyl-4(3H)-quinazolinone in the preparation of anti-angiogenic drugs. BACKGROUND

[0002] Angiogenesis is a normal physiological process that occurs during embryonic development, but abnormal angiogenesis is also associated with certain disease states, including neovascular eye diseases, psoriasis, and tumorigenesis. Pathological angiogenesis is usually persistent and has been identified as a signal for cancer development, playing a key role in the proliferation and metastasis of tumors, and therefore, anti-angiogenic strategies are considered a potentially new cancer treatment method.

[0003] 2-methyl-4(3H)-quinazolinone (chemical formula: C9H8N2O, CAS number: 1769-24-0) is an alkaloid compound that can be produced by microorganisms such as bacteria and fungi or synthesized by chemical methods. Literature (The Journal of Antibiotics, 1991, 44: 111-112) discloses the isolation of 2-methyl-4(3H)-quinazolinone from bacteria. Literature (The Journal of Organic Chemistry, 2025, 90: 385-393) discloses the one-step synthesis of 2-methyl-4(3H)-quinazolinone using solid calcium carbide as a substitute for gaseous acetylene. According to existing information, 2-methyl-4(3H)-quinazolinone is a highly effective competitive poly(ADP-ribose) synthetase inhibitor with an inhibition strength (Ki value) of 1.1 μmmol / L. At the same time, it is also an inhibitor of mammalian aspartate transcarbamoylase (ATCase) with a half-inhibitory concentration (IC 50 value) of 0.20 mmol / L. It is not usually used as a direct drug itself, but the quinazolinone structure is a recognized pharmacophore, so it is produced and sold as a pharmaceutical intermediate, a pesticide intermediate, or an industrial raw material for further chemical synthesis or industrial mass production. SUMMARY

[0004] The present application aims to disclose the application of 2-methyl-4(3H)-quinazolinone in the preparation of anti-angiogenic drugs to solve one or more technical problems existing in the prior art and provide at least one beneficial option or create conditions.

[0005] To achieve the above-mentioned purpose, the present application realizes the following technical solutions: The first aspect of the present application is to provide a new application of 2-methyl-4(3H)-quinazolinone.

[0006] The second aspect of the present application is to provide a method for preparing 2-methyl-4(3H)-quinazolinone by biological method.

[0007] The application in the first aspect of the present application refers to the direct use of 2-methyl-4(3H)-quinazolinone as an effective ingredient for preparing anti-angiogenic drugs. The structure of 2-methyl-4(3H)-quinazolinone is shown as follows: .

[0008] In some embodiments of the first aspect of the present application, the effective amount of 2-methyl-4(3H)-quinazolinone is 20 μmol / L.

[0009] In some embodiments of the first aspect of the present application, the anti-angiogenesis is achieved by down-regulating the gene expression of VEGF alpha 、 VEGFR2 、 Ang1 、and Tie2 , which inhibits two core pathways of angiogenesis VEGF alpha - VEGFR2 pathway and Ang1 - Tie2 .

[0010] In some embodiments of the first aspect of the present application, the drug further comprises a pharmaceutically acceptable excipient. As a preferred embodiment, the pharmaceutically acceptable excipient comprises at least one of diluent, binding agent, wetting agent, lubricant, disintegrating agent, solvent, emulsifying agent, co-solvent, solubilizing agent, preservative, pH adjusting agent, osmotic pressure adjusting agent, surfactant, coating material, antioxidant, bacteriostatic agent or buffer.

[0011] In some embodiments of the first aspect of the present application, the dosage form of the drug comprises at least one of suspension, granule, capsule, powder, tablet, emulsion, solution, dripping pill, injection, oral agent, suppository, enema, aerosol, patch or drop.

[0012] In some embodiments of the first aspect of the present application, the administration route of the drug comprises at least one of intranasal administration, transdermal administration, intraspinal administration, intrarectal administration, intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration.

[0013] The method in the second aspect of the present application comprises the following steps: 1) large-scale fermentation culture of Streptomyces; 2) extraction of the culture with methanol to obtain a primary extract; 3) extracting the primary extract with ethyl acetate to obtain a secondary extract; 4) separating and purifying by silica gel column chromatography to obtain 2-methyl-4(3H)-quinazolinone.

[0014] In some embodiments of the second aspect of the present application, the culture medium required for the model fermentation culture in step 1) comprises the following components: peptone 5.0 g / L, yeast extract 1.0 g / L, ferric citrate 0.1 g / L, sodium chloride 19.45 g / L, magnesium chloride 5.98 g / L, sodium sulfate 3.24 g / L, calcium chloride 1.8 g / L, potassium chloride 0.55 g / L, sodium carbonate 0.16 g / L, potassium bromide 0.08 g / L, strontium chloride 0.034 g / L, boric acid 0.022 g / L, sodium silicate 0.004 g / L, sodium fluoride 0.0024 g / L, ammonium nitrate 0.0016 g / L, disodium hydrogen phosphate 0.008 g / L, and agar 15.0 g / L, with a pH value of 7.6±0.2.

[0015] In some embodiments of the second aspect of the present application, the model fermentation culture in step 1) is to culture the Streptomyces in the above-mentioned culture medium at 28℃ under static conditions for 7 days.

[0016] In some embodiments of the second aspect of the present application, the extraction in step 2) is to immerse the culture with methanol, repeat the immersion for 3 times, each time for 24 hours, combine the methanol immersion solutions of the 3 times, and concentrate under reduced pressure at 50℃.

[0017] In some embodiments of the second aspect of the present application, the extraction in step 3) is to extract the primary extract with an equal volume of ethyl acetate for 3 times, and concentrate the ethyl acetate extract under reduced pressure at 50℃.

[0018] In some embodiments of the second aspect of the present application, the separation and purification in step 4) comprises the following steps: a) separating the secondary extract by silica gel column chromatography, collecting the eluent of ethyl acetate / petroleum ether with a volume ratio of 1:9, 1:4, 3:7, 2:3, 1:1, and 3:2, and dividing the primary purification products into 6 groups; b) purifying the primary purification product of the 6th group by silica gel column chromatography, and eluting with methanol-dichloromethane with a volume ratio of 1:50 as the eluent.

[0019] Compared with the prior art, the present application has the following beneficial effects: The present application discloses for the first time that 2-methyl-4(3H)-quinazolinone is directly used as an effective component for the preparation of a drug. Experiments show that the prepared drug can down-regulate the expression of the genes related to the development of the zebrafish embryo VEGF alpha 、 VEGFR2 ,Ang1 ,and Tie2 Gene expression was inhibited, suppressing the two core pathways of angiogenesis ( VEGF alpha - VEGFR2 Pathways and Ang1 - Tie2 (The pathway) indicates that it has an anti-angiogenic effect, which can inhibit pathological angiogenesis, tumor proliferation and metastasis, and is a potential new cancer treatment method. Attached Figure Description

[0020] Figure 1 This is a fluorescence control image showing the effect on angiogenesis in an animal model in Example 2; Figure 2 This refers to the toxic effects and angiogenesis inhibition in the animal model described in Example 2; Figure 3 This refers to the effect of gene expression on angiogenesis in the animal model in Example 3. Detailed Implementation

[0021] Unless otherwise specified, the molecular biology experimental methods described in the following examples were performed in accordance with Molecular Cloning: A Laboratory Manual (3rd Edition) or the method and product instructions. Unless otherwise specified, the biological materials used in these methods are commercially available.

[0022] Example 1: Preparation of 2-methyl-4(3H)-quinazolinone by biological method The specific steps are as follows: 1.1) Preparation of culture medium: peptone 5.0 g / L, yeast extract 1.0 g / L, ferric citrate 0.1 g / L, sodium chloride 19.45 g / L, magnesium chloride 5.98 g / L, sodium sulfate 3.24 g / L, calcium chloride 1.8 g / L, potassium chloride 0.55 g / L, sodium carbonate 0.16 g / L, potassium bromide 0.08 g / L, strontium chloride 0.034 g / L, boric acid 0.022 g / L, sodium silicate 0.004 g / L, sodium fluoride 0.0024 g / L, ammonium nitrate 0.0016 g / L, disodium hydrogen phosphate 0.008 g / L, agar 15.0 g / L, pH 7.6±0.2, sterilized at 121 ℃ for 20 min.

[0023] 1.2) Culture: Streptomyces ( ) was taken out of the -80 ℃ freezer. Streptomyces albogriseolus The bacterial strain was spread on 2216E solid medium for evolution and activation. Then, a single colony was picked and spread evenly on the 2216E solid medium prepared according to step 1.1), and cultured for 7 days at 28 ℃ under static conditions.

[0024] 1.3) Primary extraction: collect the culture, add an appropriate amount of methanol to cover the culture, repeat the soaking for 3 times, each time for 24 hours, combine the methanol soaking solutions of the 3 times, and concentrate under reduced pressure at 50°C to obtain a methanol extract.

[0025] 1.4) Secondary extraction: use an equal volume of ethyl acetate to extract the primary extract obtained in step 1.3) for 3 times, and concentrate the ethyl acetate extract under reduced pressure at 50°C to obtain an ethyl acetate extract.

[0026] 1.5) Primary purification: separate the secondary extract obtained in step 1.4) by silica gel column chromatography, collect the eluent of ethyl acetate / petroleum ether with a volume ratio of 1:9, 1:4, 3:7, 2:3, 1:1, and 3:2, and divide the primary purification products into 6 groups.

[0027] 1.6) Secondary purification: purify the 6th group of primary purification products obtained in step 1.5) by silica gel column chromatography, and elute with methanol-dichloromethane with a volume ratio of 1:50 as the eluent to obtain a 2-methyl-4(3H)-quinazolinone compound. The nuclear magnetic resonance data thereof are shown in Table 1, and the structural formula is .

[0028] Table 1, 2-methyl-4(3H)-quinazolinone compound 1 H (600 MHz) and 13 C (150 MHz) NMR data (Chloroform- d )

[0029] Example 2: Animal model experiment Zebrafish is a widely used vertebrate model organism for genetic, developmental, and drug research due to its reproductive capacity and genetic similarity to mammals. The vascular system of vascular-specific transgenic fluorescent zebrafish is easily observed under a microscope without harming the organism, and is widely used in the fields of angiogenesis, tumor formation, anti-tumor drug screening and evaluation, etc. The red fluorescent transgenic zebrafish model Tg ( flk : mCherry ) is one of the models for evaluating the anti-angiogenic activity of natural products, and flk the promoter directs the expression of a red fluorescent protein variant mCherry , and the development and physiological changes of blood vessels can be quickly observed under a fluorescence microscope. Therefore, the present application has scientific rationality and representative significance in evaluating the anti-angiogenic activity of natural products by using the red fluorescent transgenic zebrafish model Tg ( flk : mCherry ).

[0030] Using a red fluorescent transgenic zebrafish model Tg ( flk : mCherry An experiment was conducted to investigate the anti-angiogenic activity of the 2-methyl-4(3H)-quinazolinone compound in Example 1: The experimental group dissolved the 2-methyl-4(3H)-quinazolinone compound in dimethyl sulfoxide (DMSO) to prepare a solution with a concentration of 10 mmol / L.

[0031] Healthy, sexually mature zebrafish were placed in a mating tank at a 1:1 ratio of males to females, separated by a transparent baffle. The tank was then kept in darkness overnight using a light-blocking cloth. After the baffle was removed and the females laid eggs, the fertilized eggs were collected and transferred to fresh embryo culture water (containing 3.5 g / L NaCl, 0.05 g / L KCl, 0.2 g / L NaHCO3, and 0.1 g / L CaCl2) and cultured in an incubator at 28 ℃.

[0032] At 10 hpf (hours post-fertilization), embryos were transferred to 12-well plates, 40 embryos per well. The experimental group received 2-methyl-4(3H)-quinazolinone solution to a final concentration of 20 μmol / L, the blank control group received 0.2% DMSO, and the positive control group received 4 μmol / L PTK787 compound. Each group had three replicates. The culture water was changed every 12 hours, and the plates were placed in a light incubator for continued embryo development. At 36 hpf, zebrafish fertilized eggs were treated with 30 mg / mL protease for demetabolism. At 48 hpf, angiogenesis in the zebrafish was observed and photographed under a fluorescence microscope. The relative inhibition rate of angiogenesis in each group was calculated, and embryo mortality was observed. Finally, a one-way ANOVA analysis was used to determine if there were significant differences between the groups. The results are as follows: Figure 1 , Figure 2 As shown.

[0033] Figure 1 The study showed the angiogenesis of zebrafish in the blank group, experimental group, and control group. In the blank group, intact intersegmental vessels (ISVs), dorsal aorta (DA), and dorsal longitudinal anastomoses (DLAVs) were observed. In contrast, the intersegmental vessels and dorsal longitudinal anastomoses in the experimental group were incomplete and did not close, indicating that 2-methyl-4(3H)-quinazolinone has an inhibitory effect on angiogenesis in zebrafish.

[0034] Figure 2The survival rate and the inhibition rate of angiogenesis of zebrafish in the blank group, the experimental group and the control group are shown. Compared with the blank group, the survival rate of zebrafish in the experimental group has no significant difference, and secondly, the 2-methyl-4(3H)-quinazolinone significantly increases the inhibition rate of angiogenesis of zebrafish compared with the blank group P <0.05), indicating that the 2-methyl-4(3H)-quinazolinone has an inhibitory effect on the angiogenesis of zebrafish and low toxicity. In the control group, the compound PTK787 is a tyrosine kinase inhibitor that can specifically inhibit the expression of vascular endothelial growth factor receptor, thereby inhibiting angiogenesis, especially the generation of intestinal subvenous vessels and intersegmental vessels is significantly reduced.

[0035] Example 3: Transcriptional regulation of angiogenesis by 2-methyl-4(3H)-quinazolinone The zebrafish embryos developed to the phenotype that can be observed were transferred to a 1.5 mL enzyme-free centrifuge tube, the embryo culture water was aspirated, 1 mL Trizol lysis solution was added, and vortex was blown to fully lyse the cells. Centrifugation was performed at 12000xg for 10 minutes, and the supernatant was collected into a new centrifuge tube and placed at room temperature for 5 minutes. 0.2 mL chloroform was added, vortexed for 15-30 seconds, mixed thoroughly, and placed at room temperature for 5 minutes. Centrifugation was performed at 12000xg for 15 minutes, and the solution was layered, the supernatant was collected into a new centrifuge tube, and two times the volume of isopropanol was added and placed at -20 °C for overnight precipitation. After overnight precipitation, it was taken out, centrifuged at 12000xg for 15 minutes, and the liquid was poured out and slightly centrifuged to remove the residual liquid at the bottom of the tube. 1 mL of 75% ethanol (prepared with enzyme-free water, pre-cooled) was added, centrifuged at 7500xg for 5 minutes, the liquid was poured out and slightly centrifuged to remove the residual liquid at the bottom of the tube, and naturally dried for 5-10 minutes. 50 μL of enzyme-free water was added to dissolve the precipitate, and the RNA concentration of each sample was determined. The RNA of each sample was reversely transcribed into cDNA, the expression amount of the angiogenesis-related gene was determined by using the Roche LightCycler® 480 II real-time fluorescent quantitative qPCR system, and three repeats were performed. The real-time quantitative qPCR amplification reaction conditions were as follows: 95 °C pre-denaturation for 2 minutes; 95 °C denaturation for 10 seconds, 60 °C annealing for 30 seconds, and cycling for 40 times; finally, 95 °C for 15 seconds, 60 °C for 60 seconds, 95 °C for 15 seconds, 1 cycle, and fluorescence signal acquisition. The results were relatively quantitatively analyzed by taking Actin as the internal reference.2 -ΔΔCt The method was used to quantify the relative expression of the blood vessel-related gene, and T-test was used for significant difference analysis.

[0036] Figure 3The study showed the effects of 2-methyl-4(3H)-quinazolinone treatment on gene expression related to angiogenesis in zebrafish embryos. Compared to the control group, zebrafish embryos treated with 2-methyl-4(3H)-quinazolinone showed significantly increased gene expression related to angiogenesis. VEGF alpha , VEGFR2 , Ang1 ,and Tie2 The gene expression levels of all genes showed a significant decrease. P <0.0001), and Ang1 The most significant decrease indicates that 2-methyl-4(3H)-quinazolinone inhibits the two core pathways of angiogenesis ( VEGF alpha - VEGFR2 and Ang1 - Tie2 (Pathways), which may inhibit angiogenesis through multi-target synergistic effects.

[0037] Through extensive experimentation, this invention has discovered that the 2-methyl-4(3H)-quinazolinone has the effect of... Tg ( flk : mCherry Fluorescent transgenic zebrafish strains showed a significant inhibitory effect on intersegmental and dorsal longitudinal anastomotic angiogenesis. Pathological angiogenesis is associated with various diseases; for example, tumor growth is accompanied by the proliferation and disorder of intertumoral blood vessels. VEGF and VEGFR It can induce tumor angiogenesis and plays a key role in tumor growth, while Ang1 High expression of these substances can promote angiogenesis and support tumor growth and spread. Therefore, the use of anti-angiogenic agents is one of the important methods of tumor treatment.

[0038] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. The use of 2-methyl-4(3H)-quinazolinone in the preparation of anti-angiogenic drugs, wherein the structural formula of 2-methyl-4(3H)-quinazolinone is shown below: 。 2. The application according to claim 1, characterized in that, The direct active ingredient of the drug is 2-methyl-4(3H)-quinazolinone.

3. The application according to claim 1, characterized in that, The effective dosage of the 2-methyl-4(3H)-quinazolinone is 20 μmol / L.

4. The application according to claim 1, characterized in that, The anti-angiogenesis is achieved by downregulating... VEGFα , VEGFR2 , Ang1 ,and Tie2 Gene expression, inhibition VEGFα - VEGFR2 Pathways and Ang1 - Tie2 path.

5. The application according to claim 1, characterized in that, The drug further includes pharmaceutically acceptable excipients; preferably, the pharmaceutically acceptable excipients include at least one of the following: diluents, binders, wetting agents, lubricants, disintegrants, solvents, emulsifiers, cosolvents, solubilizers, preservatives, pH adjusters, osmotic pressure adjusters, surfactants, coating materials, antioxidants, antibacterial agents, or buffers.

6. The application according to claim 1, characterized in that, The dosage form of the drug includes at least one of the following: suspension, granules, capsules, powders, tablets, emulsions, solutions, pellets, injections, oral preparations, suppositories, enemas, aerosols, patches, or drops.

7. The application according to claim 1, characterized in that, The drug can be administered via at least one of the following routes: nasal administration, transdermal administration, intraspinal administration, rectal administration, intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, and sublingual administration.

8. A method for biologically extracting 2-methyl-4(3H)-quinazolinone, characterized in that, Including the following steps: 1) Large-scale fermentation culture of Streptomyces; 2) Extract the culture with methanol to obtain a primary extract; 3) The primary extract was extracted with ethyl acetate to obtain the secondary extract; 4) The 2-methyl-4(3H)-quinazolinone was obtained by separation and purification using silica gel column chromatography.

9. The method according to claim 8, characterized in that, The separation and purification process includes the following steps: a) The secondary extract was separated by silica gel column chromatography, and the ethyl acetate / petroleum ether eluent was collected and divided into 6 groups of primary purified products; b) The primary purified product of group 6 was purified by silica gel column chromatography, and eluted with methanol-dichloromethane as the eluent.

10. The method according to claim 9, characterized in that, The volume ratio of ethyl acetate / petroleum ether eluent in step a) is 1:9 to 3:2; the volume ratio of methanol to dichloromethane in step b) is 1:50.

Images