Flavone-dihydropyrazole derivative as well as preparation method and application thereof

Abstract

The invention discloses a flavone-dihydropyrazole derivative as well as a preparation method and application thereof. The structural formula of the flavone-dihydropyrazole derivative is as follows: # imgabs0 #; wherein R is selected from 2-benzyl, 3-benzyl, 4-benzyl, 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, 2-chlorphenyl, 3-chlorphenyl, 4-chlorphenyl, 2-bromophenyl, 3-bromophenyl, 4-bromophenyl, 2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl, phenyl, p-ethyl phenyl, 4-methoxy-3-methylphenyl and p-trifluoromethylphenyl, and R is selected from the group consisting of 2-benzyl, 3-benzyl, 4-benzyl, 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, 2-chlorphenyl, 3-chlorphenyl, 4-chlorphenyl, 2-bromophenyl, 3-bromophenyl, 4-bromophenyl, 2-bromophenyl, 3-bromophenyl, 4-bromophenyl, 2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl, phenyl, p-ethyl and 4, 4-trifluoromethoxy phenyl. A five-membered heterocyclic structure is introduced to a flavone molecular skeleton, the flavone-dihydropyrazole derivative is obtained, and a biological activity test result shows that the flavone-dihydropyrazole compound has good activity for treating vascular calcification.

Description

Technical Field

[0001] The invention belongs to the field of organic synthesis, and specifically relates to a flavonoid-dihydropyrazole derivative and a preparation method and application thereof. Background Art

[0002] Flavonoids are natural products with C6-C3-C6 (two benzene rings connected by a propane bridge) in their structure. They are widely found in certain plants and berries in nature, such as rutin, hesperidin, green tea polyphenols, mint, etc. Natural and synthetic flavonoids have always attracted attention due to their rich biological activities. They have anti-anxiety, anti-inflammatory, anti-diabetic, antiviral, antiprotozoal, anti-tuberculosis, anti-tumor, antioxidant and antibacterial activities. Compared with coumarin, another natural product with similar structure, they have different effects on different diseases, thanks to their different skeletons. In addition, as a natural product, flavonoids have low toxicity and are not easy to cause adverse reactions. Therefore, flavonoids can be used as a parent core structure in the design and development of new drugs for the treatment and prevention of related diseases.

[0003] Aromatic heterocycles are the dominant structures for drug design and discovery. Pyrazole and its derivatives are the most important members of five-membered nitrogen-containing heterocycles. 4,5-dihydro-1H-pyrazole (also known as 2-pyrazoline) has a cyclic hydrazine part and an intracyclic double bond, and is also a reduced form of pyrazole. Research on the dihydropyrazole structure in recent years has proved that the structure has potential anti-tumor, antibacterial, anti-inflammatory, and antiviral activities. Dihydropyrazole compounds have very broad and rich biological and pharmacological properties. At the same time, the structure is increasingly used in organic synthesis and other fields, providing great research and development space for future drug development research. Therefore, it is of great significance to establish a heterocyclic system with a dihydropyrazole structure.

[0004] Vascular calcification is a systemic vascular disease that is common in people with atherosclerosis, hypertension, diabetes, the elderly, and chronic kidney disease. Vascular calcification is mainly a pathological process caused by the ectopic deposition of hydroxyapatite on the vascular wall. It is generally believed that vascular smooth muscle cells (VSMCs) are the key factor in vascular calcification. The key to vascular calcification lies in the transformation of vascular cells, especially vascular smooth muscle cells, to an osteoblast-like phenotype. During the differentiation of smooth muscle cells into osteoblast-like cells, the expression of contractile marker proteins decreases, while the expression of osteoblast-specific proteins increases. Runt-related transcription factor 2 (RUNX2) is a key transcription factor in the osteogenic differentiation process. Alkaline phosphatase (ALP) is an enzyme that catalyzes the hydrolysis of inorganic pyrophosphate, an inhibitor of vascular calcification. Bone morphogenetic protein 2 (BMP2) promotes the occurrence of vascular calcification by upregulating RUNX2, Osterix, etc., which participate in the transdifferentiation of VSMCs. Currently, there is no drug that can effectively prevent or treat vascular calcification in clinical practice, and anticoagulants such as statins and warfarin have the side effect of promoting calcification. Therefore, it is of great significance to find and develop drugs to treat vascular calcification. Summary of the invention

[0005] The purpose of the present invention is to provide a flavonoid-dihydropyrazole derivative and its preparation method and use. The present invention adopts a design strategy of combined pharmacophores, introduces the active pharmacophore-dihydropyrazole ring into a flavonoid skeleton with good activity, and screens and synthesizes a novel structure with high efficiency and low toxicity as a candidate compound for treating vascular calcification. The flavonoid-dihydropyrazole compounds screened by the present invention have good activity in alleviating the osteogenic differentiation of vascular smooth muscle cells induced by high phosphate, and have potential application prospects in the treatment of vascular calcification.

[0006] The flavonoid-dihydropyrazole derivatives of the present invention have the following general structural formula:

[0007] ;

[0008] wherein R is selected from 2-benzyl, 3-benzyl, 4-benzyl, 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, 2-chlorophenyl, 3-chlorophenyl, 4-chlorophenyl, 2-bromophenyl, 3-bromophenyl, 4-bromophenyl, 2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl, phenyl, p-ethylphenyl, 4-methoxy-3-methylphenyl, p-trifluoromethylphenyl or p-trifluoromethoxyphenyl.

[0009] The preparation method of the flavonoid-dihydropyrazole derivatives of the present invention comprises the following steps:

[0010] Step 1: Weigh a small amount of NaOH solid and add it to a 100 mL round-bottom flask, add 20 mL of ethanol dropwise, add a magnetic stirrer, and stir to dissolve; weigh m-benzaldehyde (30 mmol) and 2-hydroxyacetophenone (50 mmol) and add them to the system, stir at room temperature for 5 h, and monitor the reaction by TLC; after the reaction, add dilute hydrochloric acid dropwise to the reaction system, adjust the pH to neutral, concentrate using a rotary evaporator, and separate and purify by silica gel column chromatography to obtain the intermediate chalcone A.

[0011] The structural formula of the intermediate A is:

[0012] .

[0013] Step 2: Add intermediate chalcone A to a round-bottom flask, add N, N-dimethylformamide and stir to dissolve, add iodine, set the reaction temperature to 110°C, reflux the obtained reaction mixture overnight, and monitor the reaction by TLC. After the reaction is completed, the reaction mixture is cooled to room temperature and poured into ice water, a large amount of light yellow solid is precipitated, washed with sodium thiosulfate, filtered, the filter cake is collected, and dried in an oven to obtain intermediate B.

[0014] The structural formula of the intermediate B is:

[0015] .

[0016] Step 3: Add a small amount of sodium hydroxide solid and ethanol into the reactor, stir and dissolve; weigh intermediate B and ketone compounds with different substituents, add them into the above system, stir and react at room temperature to precipitate solid; after the reaction is completed by TLC monitoring, pour the reaction mixture into a Buchner funnel for suction filtration, collect the filter cake, dissolve it in dichloromethane, then add Na2SO4 to dry to remove moisture, dehydrate, and concentrate by reduced pressure distillation to obtain intermediate C.

[0017] Wherein, the structural formula of the ketone compounds with different substituents is: .

[0018] The structural formula of intermediate C is: .

[0019] wherein R is selected from 2-benzyl, 3-benzyl, 4-benzyl, 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, 2-chlorophenyl, 3-chlorophenyl, 4-chlorophenyl, 2-bromophenyl, 3-bromophenyl, 4-bromophenyl, 2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl, phenyl, p-ethylphenyl, 4-methoxy-3-methylphenyl, p-trifluoromethylphenyl, and p-trifluoromethoxyphenyl.

[0020] Step 4: Add intermediate C to the reactor, add acetic acid and stir to dissolve, weigh hydrazine hydrate (2 mL) and add to the above system, reflux at 100°C for 6 h; after the reaction, cool the reaction solution to room temperature, pour the reaction mixture into ice water, precipitate a large amount of solid, filter, collect the filter cake, dissolve in dichloromethane, then add Na2SO4 to dry to remove moisture, dehydrate, and concentrate by reduced pressure distillation to obtain a crude product, separate and purify by silica gel column chromatography, and recrystallize with petroleum ether to obtain a flavonoid-dihydropyrazole derivative FPY1-20.

[0021] In step 1, the molar ratio of 2-hydroxyacetophenone to m-benzaldehyde is 5:3.

[0022] In step 1, the eluent for column chromatography separation is petroleum ether:ethyl acetate=3:1, v / v.

[0023] In step 2, 4 to 6 grains of iodine are added to the system.

[0024] In step 3, the molar ratio of the intermediate B and the ketone compounds with different substituents is 1:1.

[0025] In step 4, the amount of hydrazine hydrate used is 2 mL.

[0026] In step 4, the eluent for column chromatography separation is petroleum ether:ethyl acetate=2:1, v / v.

[0027] The invention discloses an application of the flavonoid-dihydropyrazole derivatives in preparing a medicine for treating vascular calcification.

[0028] The drug for treating vascular calcification has good activity in relieving high phosphate-induced osteogenic differentiation of vascular smooth muscle cells on HASMC cells. HASMC cells are often used in in vitro studies of diseases, drug metabolism, in vitro toxicity experiments, etc., in order to confirm the activity of flavonoid-dihydropyrazole compounds in relieving high phosphate-induced osteogenic differentiation of vascular smooth muscle cells.

[0029] Furthermore, the drug for treating vascular calcification can alleviate the osteogenic differentiation of vascular smooth muscle cells induced by high phosphate.

[0030] The present invention introduces a five-membered heterocyclic structure into the flavonoid molecular skeleton of flavonoids based on the principle of drug combination and the concept of drug molecule design based on structure, thereby obtaining flavonoid-dihydropyrazole derivatives. The biological activity test results show that the flavonoid-dihydropyrazole compounds of the present invention have good activity in treating vascular calcification. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1The cytotoxicity of the flavonoid-dihydropyrazole compound FPY1-20 (5 μM) on HASMC cells. ns means no significant difference, ***p<0.001 means very significant difference.

[0032] Figure 2 The results of Alizarin red staining of osteogenic differentiation of smooth muscle cells treated with flavonoid-dihydropyrazole compounds FPY1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 16, 17, 18, 19, 20 (5 μM).

[0033] Figure 3 The most significant effect at the molecular level was the effect of FPY19 compound on the expression levels of ALPL, BMP2 and RUNX2 proteins in smooth muscle cell calcification. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further described in detail by the following specific examples, but it should be noted that the scope of the present invention is not limited by these examples.

[0035] Example 1: Preparation of 2-(3-(1-acetyl-3-(o-methylphenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenyl)-4H-coumarin-4-one (Compound FPY1)

[0036]

[0037] 1. Weigh a small amount of NaOH solid and add it to a 100 mL round-bottom flask, add 20 mL of ethanol, add a magnetic stirrer, and stir to dissolve; weigh m-benzaldehyde (30 mmol) and o-hydroxyacetophenone (50 mmol), stir at room temperature for 5 h, and monitor the reaction by TLC. After the reaction is completed, add dilute hydrochloric acid to the above reaction system, adjust the pH to neutral, use a rotary evaporator to concentrate, and separate and purify by silica gel column chromatography to obtain intermediate chalcone A.

[0038] 2. Add the intermediate chalcone A obtained above to a round-bottom flask, add N, N-dimethylformamide and stir to dissolve, add 4-6 grains of I2, set the reaction temperature to 80°C, reflux the obtained reaction mixture overnight, and monitor the reaction by TLC. After the reaction is completed, the reaction mixture is cooled to room temperature and poured into ice water, a large amount of light yellow solid is precipitated, washed with sodium thiosulfate, filtered, the filter cake is collected, and dried in an oven to obtain intermediate B.

[0039] 3. Add a small amount of sodium hydroxide solid and ethanol to the reactor, stir and dissolve; weigh intermediate B (2.0 equiv) and 2-methylacetophenone (2.0 equiv) and add them to the above system, stir and react at room temperature to precipitate solid. After the reaction is completed by TLC monitoring, pour the reaction mixture into a Buchner funnel for suction filtration, collect the filter cake, dissolve it in dichloromethane, then add Na2SO4 to dry to remove moisture, dehydrate, and concentrate by vacuum distillation to obtain intermediate C.

[0040] 4. Add intermediate C (1.0 equiv) into the reactor, add acetic acid and stir to dissolve, weigh hydrazine hydrate (2 mL) and add into the above system, reflux at 100°C for 6 h. After the reaction is completed, cool to room temperature and pour the reaction mixture into ice water to precipitate a large amount of solid. Filter and collect the filter cake, dissolve it in dichloromethane, then add Na2SO4 to dry to remove moisture, dehydrate, and concentrate by vacuum distillation to obtain a crude product, which is separated and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 3:1, v / v gradient elution), and recrystallized from petroleum ether to obtain a flavonoid-dihydropyrazole derivative FPY1 as a white solid with a yield of 71.3% and a melting point of 91-95°C.

[0041] 1 H NMR (600 MHz, Chloroform-d) δ 8.21 (s, 1H), 7.82 (d, J = 8.6 Hz,2H), 7.69 (s, 1H), 7.55 (s, 1H), 7.49 (s, 1H), 7.41 (d, J = 8.0 Hz, 2H), 7.37(d, J = 7.6 Hz, 1H), 7.32 (d, J = 4.2 Hz, 2H), 6.80 (s, 1H), 5.62 (s, 1H), 3.89 (s, 1H), 3.27 (s, 1H), 2.71 (s, 3H), 2.44 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ 178.45, 169.18, 163.06, 156.23, 154.66, 143.09, 138.29, 133.87,132.45, 132.05, 129.82, 129.80, 129.74, 128.98, 128.75, 126.05, 125.70,125.68, 125.31, 123.93, 123.68, 118.14, 107.83, 58.86, 44.58, 23.56, 22.08.HR-MS(ESI): calcd for C27 H 22 N2O3, [M+H] + , 423.1709; found 423.1701.

[0042] Example 2: Preparation of 2-(3-(1-acetyl-3-(m-methylphenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenyl)-4H-coumarin-4-one (Compound FPY2)

[0043]

[0044] The preparation method is the same as that of Example 1. 3-methylacetophenone is used instead of 2-methylacetophenone to obtain a light yellow solid with a yield of 70.2% and a melting point of 171.2-173°C.

[0045] 1 H NMR (600 MHz, Chloroform-d) δ 8.20 (s, 1H), 7.79 (d, J = 11.5 Hz,2H), 7.68 (s, 1H), 7.60 (s, 1H), 7.54 (d, J = 9.4 Hz, 2H), 7.48 (s, 1H), 7.40(d, J = 7.9 Hz, 2H), 7.32 (s, 1H), 7.26 (s, 1H), 6.79 (s, 1H), 5.69 (s, 1H), 3.82 (s, 1H), 3.21 (s, 1H), 2.47 (s, 3H), 2.40 (s, 3H). 13 C NMR (151 MHz, CDCl3) δ 178.41, 169.04, 163.06, 156.23, 154.07, 143.10, 138.57, 133.83,132.45, 131.40, 131.04, 129.78, 128.78, 128.73, 127.22, 125.69, 125.65,125.28, 123.94, 123.91, 123.64, 118.16, 107.84, 59.75, 42.44, 22.00, 21.42.HR-MS(ESI): calcd for C 27 H 22 N2O3, [M+H] + , 423.1709; found 423.1702.

[0046] Example 3: Preparation of 2-(3-(1-acetyl-3-(p-methylphenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenyl)-4H-coumarin-4-one (Compound FPY3)

[0047]

[0048] The preparation method is the same as that of Example 1. 4-methylacetophenone is used instead of 2-methylacetophenone to obtain a yellow solid with a yield of 72.3% and a melting point of 201-203.5°C.

[0049] 1 H NMR (600 MHz, Chloroform-d) δ 8.22 (s, 1H), 7.82 (s, 1H), 7.79 (s,1H), 7.70 (s, 1H), 7.65 (d, J = 8.2 Hz, 2H), 7.56 (s, 1H), 7.49 (s, 1H), 7.42(d, J = 7.2 Hz, 2H), 7.25 (s, 2H), 6.81 (s, 1H), 5.68 (s, 1H), 3.82 (s, 1H), 3.20 (s, 1H), 2.46 (s, 3H), 2.40 (s, 3H). 13 C NMR (151 MHz, CDCl3) δ 178.42,168.99, 163.07, 156.22, 153.99, 143.14, 140.95, 133.83, 132.42, 129.77,129.53, 128.79, 128.35, 126.64, 125.67, 125.63, 125.27, 123.93, 123.67,118.16, 107.82, 59.72, 42.42, 21.98, 21.53 . HR-MS(ESI): calcd for C 27 H 22 N2O3,[M+H] + , 423.1709; found 423.1705.

[0050] Example 4: Preparation of 2-(3-(1-acetyl-3-(2-chlorophenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenyl)-4H-coumarin-4-one (Compound FPY4)

[0051]

[0052] The preparation method is the same as that of Example 1. 2-Chloroacetophenone is used instead of 2-methylacetophenone to obtain a light yellow solid with a yield of 67.3% and a melting point of 164-167°C.

[0053] 1 H NMR (600 MHz, Chloroform-d) δ 8.20 (s, 1H), 7.81 (d, J = 8.5 Hz,2H), 7.77 (s, 1H), 7.68 (s, 1H), 7.55 (s, 1H), 7.49 (s, 1H), 7.46 – 7.38 (m,3H), 7.35 (d, J = 1.9 Hz, 2H), 6.80 (s, 1H), 5.66 (s, 1H), 4.00 (s, 1H), 3.40(s, 1H), 2.43 (s, 3H). . 13 C NMR (101 MHz, CDCl3) δ 178.43, 169.23, 163.04,156.24, 153.52, 142.80, 133.85, 133.08, 132.51, 131.09, 131.01, 130.40,129.78, 128.92, 127.11, 125.76, 125.71, 125.29, 123.96, 123.65, 118.15,107.87, 60.14, 45.15, 22.02. HR-MS(ESI): calcd for C 26 H 19 ClN2O3, [M+H] + ,443.1662; found 443.1658.

[0054] Example 5: Preparation of 2-(3-(1-acetyl-3-(3-chlorophenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenyl)-4H-coumarin-4-one (Compound FPY5)

[0055]

[0056] The preparation method is the same as that of Example 1. 3-Chloroacetophenone is used instead of 2-methylacetophenone to obtain a white solid with a yield of 69.4% and a melting point of 165.2-167.5°C.

[0057] 1H NMR (600 MHz, Chloroform-d) δ 8.18 (s, 1H), 7.79 (s, 1H), 7.75 (d,J = 1.8 Hz, 2H), 7.67 (s, 1H), 7.59 (s, 1H), 7.53 (s, 1H), 7.47 (s, 1H), 7.37(dd, J = 18.4, 7.6 Hz, 4H), 6.77 (s, 1H), 5.69 (s, 1H), 3.79 (s, 1H), 3.17(s, 1H), 2.45 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ 178.39, 169.12, 162.96,156.20, 152.49, 142.80, 134.92, 133.88, 132.91, 132.49, 130.42, 130.11,129.85, 128.72, 126.61, 125.77, 125.67, 125.32, 124.78, 123.89, 123.59,118.16, 107.85, 59.98, 42.24, 22.00. HR-MS(ESI): calcd for C 26 H 19 ClN2O3, [M+H] + ,443.1662; found 443.1657.

[0058] Example 6: Preparation of 2-(3-(1-acetyl-3-(4-chlorophenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenyl)-4H-coumarin-4-one (Compound FPY6)

[0059]

[0060] The preparation method is the same as that of Example 1. 4-Chloroacetophenone is used instead of 2-methylacetophenone to obtain a light yellow solid with a yield of 62.5% and a melting point of 220-222.7°C.

[0061] 1H NMR (600 MHz, Chloroform-d) δ 8.20 (s, 1H), 7.82 (s, 1H), 7.77 (s,1H), 7.70 (t, J = 8.1 Hz, 3H), 7.55 (s, 1H), 7.49 (s, 1H), 7.44 – 7.36 (m,4H), 6.80 (s, 1H), 5.69 (s, 1H), 3.81 (s, 1H), 3.17 (s, 1H), 2.45 (s, 3H). 13 CNMR (151 MHz, CDCl3) δ 178.44, 169.08, 163.05, 156.25, 152.67, 142.88,136.57, 133.89, 132.55, 129.85, 129.65, 129.12, 128.77, 127.90, 125.79,125.74, 125.34, 123.93, 123.64, 118.14, 107.91, 59.98, 42.30, 21.97. HR-MS(ESI): calcd for C 26 H 19 ClN2O3, [M+H] + , 443.1662; found 443.1655.

[0062] Example 7: Preparation of 2-(3-(1-acetyl-3-(2-methoxyphenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenyl)-4H-coumarin-4-one (Compound FPY7)

[0063]

[0064] The preparation method is the same as that of Example 1. 2-Methoxyacetophenone is used instead of 2-methylacetophenone to obtain a yellow solid with a yield of 71.3% and a melting point of 150.2-154.8°C.

[0065] 1H NMR (600 MHz, Chloroform-d) δ 8.22 (s, 1H), 7.97 (s, 1H), 7.82 (d,J = 8.6 Hz, 2H), 7.70 (s, 1H), 7.56 (s, 1H), 7.49 (s, 1H), 7.45 – 7.37 (m,3H), 7.04 (s, 1H), 6.95 (s, 1H), 6.81 (s, 1H), 5.62 (s, 1H), 3.96 (s, 1H), 3.83 (s, 3H), 3.38 (s, 1H), 2.44 (s, 3H). 13 C NMR (151 MHz, CDCl3) δ 178.40,169.01, 163.18, 158.31, 156.23, 154.22, 143.41, 133.81, 132.31, 131.81,129.66, 129.14, 128.96, 125.66, 125.48, 125.25, 123.94, 123.75, 120.97,120.27, 118.16, 111.65, 107.77, 59.83, 55.49, 45.65, 22.01. HR-MS(ESI): calcdfor C 27 H 22 N2O4, [M+H] + , 439.1658; found 439.1655.

[0066] Example 8: Preparation of 2-(3-(1-acetyl-3-(3-methoxyphenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenyl)-4H-coumarin-4-one (Compound FPY8)

[0067]

[0068] The preparation method is the same as that of Example 1. 3-methoxyacetophenone is used instead of 2-methylacetophenone to obtain a yellow solid with a yield of 79.6% and a melting point of 157-159.9°C.

[0069] 1H NMR (400 MHz, Chloroform-d) δ 8.18 (s, 1H), 7.78 (d, J = 7.1 Hz,2H), 7.68 (s, 1H), 7.54 (s, 1H), 7.48 (s, 1H), 7.40 (d, J = 5.9 Hz, 2H), 7.37– 7.28 (m, 3H), 7.00 (s, 1H), 6.78 (s, 1H), 5.67 (s, 1H), 3.86 (s, 3H), 3.81 (s, 1H), 3.21 (s, 1H), 2.47 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ 178.36,169.02, 162.98, 159.82, 156.19, 153.79, 143.03, 133.82, 132.41, 129.86,129.78, 128.77, 125.74, 125.64, 125.54, 125.26, 123.90, 123.61, 119.33,118.15, 116.43, 111.62, 107.80, 59.83, 55.40, 42.43, 22.00. HR-MS(ESI): calcdfor C 27 H 22 N2O4, [M+H] + , 439.1658; found 439.1650.

[0070] Example 9: Preparation of 2-(3-(1-acetyl-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenyl)-4H-coumarin-4-one (Compound FPY9)

[0071]

[0072] The preparation method is the same as that of Example 1. 4-methoxyacetophenone is used instead of 2-methylacetophenone to obtain a white solid with a yield of 55.4% and a melting point of 185-188.2°C.

[0073] 1H NMR (600 MHz, Chloroform-d) δ 8.17 (s, 1H), 7.77 (d, J = 5.4 Hz,2H), 7.68 (t, J = 8.3 Hz, 3H), 7.53 (s, 1H), 7.46 (s, 1H), 7.39 (d, J = 3.6Hz, 2H), 6.92 (d, J = 8.8 Hz, 2H), 6.78 (s, 1H), 5.65 (s, 1H), 3.82 (s, 3H), 3.78 (s, 1H), 3.15 (d, J = 17.6 Hz, 1H), 2.44 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ 178.43, 168.86, 163.10, 161.50, 156.22, 153.66, 143.17, 133.85,132.40, 129.77, 128.82, 128.29, 125.67, 125.62, 125.29, 123.90, 123.72,123.65, 118.17, 114.23, 107.80, 59.66, 55.44, 42.46, 22.54. HR-MS(ESI): calcdfor C 27 H 22 N2O4, [M+H] + , 439.1658; found 439.1651.

[0074] Example 10: Preparation of 2-(3-(1-acetyl-3-phenyl-4,5-dihydro-1H-pyrazol-5-yl)phenyl)-4H-coumarin-4-one (Compound FPY10)

[0075]

[0076] The preparation method is the same as that of Example 1. Acetophenone is used instead of 2-methylacetophenone to obtain a light yellow solid with a yield of 79.6% and a melting point of 118-120°C.

[0077] 1H NMR (600 MHz, Chloroform-d) δ 8.20 (s, 1H), 7.85 – 7.74 (m, 4H), 7.69 (s, 1H), 7.57 (s, 1H), 7.49 (s, 1H), 7.46 – 7.37 (m, 5H), 6.80 (s, 1H), 5.70 (s, 1H), 3.84 (s, 1H), 3.22 (s, 1H), 2.47 (s, 3H). 13 C NMR (151 MHz, CDCl3) δ 178.41, 169.07, 163.06, 156.24, 153.86, 143.06, 133.84, 132.47,131.13, 130.56, 129.80, 128.84, 128.78, 126.68, 125.69, 125.29, 123.94,123.67, 107.86, 59.82, 42.38, 21.99. HR-MS(ESI): calcd for C 26 H 20 N2O3, [M+H] + ,409.1552; found 409.1547.

[0078] Example 11: Preparation of 2-(3-(1-acetyl-3-(2-bromophenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenyl)-4H-coumarin-4-one (Compound FPY11)

[0079]

[0080] The preparation method is the same as that of Example 1. 2-Bromoacetophenone is used instead of 2-methylacetophenone to obtain a light yellow solid with a yield of 62.1% and a melting point of 115.5-117°C.

[0081] 1H NMR (600 MHz, Chloroform-d) δ 8.21 (s, 1H), 7.85 (s, 1H), 7.82 (s,1H), 7.70 (s, 1H), 7.66 (s, 1H), 7.62 (s, 1H), 7.57 (s, 1H), 7.50 (s, 1H),7.46 (s, 1H), 7.41 (d, J = 7.0 Hz, 2H), 7.28 (s, 1H), 6.82 (s, 1H), 5.67 (s,1H), 3.98 (s, 1H), 3.41 (s, 1H), 2.43 (s, 3H). 13 C NMR (151 MHz, CDCl3) δ178.42, 169.29, 163.07, 156.26, 154.39, 142.78, 134.29, 133.86, 132.51,132.49, 131.15, 130.70, HR-MS(ESI):calcd for C 26 H 19 BrN2O3, [M+H] + , 487.0657; found 487.0652.

[0082] Example 12: Preparation of 2-(3-(1-acetyl-3-(3-bromophenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenyl)-4H-coumarin-4-one (Compound FPY12)

[0083]

[0084] The preparation method is the same as that of Example 1. 3-bromoacetophenone is used instead of 2-methylacetophenone to obtain a white solid with a yield of 73.5% and a melting point of 180.3-182.6°C.

[0085] 1H NMR (600 MHz, Chloroform-d) δ 8.19 (s, 1H), 7.91 (s, 1H), 7.80 (s,1H), 7.76 (s, 1H), 7.66 (d, J = 23.0 Hz, 2H), 7.54 (d, J = 8.1 Hz, 2H), 7.48(s, 1H), 7.39 (d, J = 11.7 Hz, 2H), 7.29 (s, 1H), 6.78 (s, 1H), 5.68 (dd, J =12.0, 4.8 Hz, 1H), 3.80 (dd, J = 17.8, 12.0 Hz, 1H), 3.16 (dd, J = 17.8, 4.9Hz, 1H), 2.45 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ 178.33, 169.09, 162.91,156.17, 152.38, 142.79, 133.86, 133.30, 133.17, 132.45, 130.35, 129.84,129.50, 128.70, 125.74, 125.63, 125.30, 125.22, 123.89, 123.58, 122.99,118.16, 107.82, 59.99, 42.19, 22.02. HR-MS(ESI): calcd for C 26 H 19 BrN2O3, [M+H] + ,487.0657; found 487.0650.

[0086] Example 13: Preparation of 2-(3-(1-acetyl-3-(4-bromophenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenyl)-4H-coumarin-4-one (Compound FPY13)

[0087]

[0088] The preparation method is the same as that of Example 1. 4-bromoacetophenone is used instead of 2-methylacetophenone to obtain a light yellow solid with a yield of 69.4% and a melting point of 218.2-220.4°C.

[0089] 1H NMR (400 MHz, Chloroform-d) δ 8.20 (s, 1H), 7.81 (s, 1H), 7.77 (s,1H), 7.69 (s, 1H), 7.62 (d, J = 8.3 Hz, 2H), 7.59 – 7.47 (m, 4H), 7.41 (d, J= 3.9 Hz, 2H), 6.78 (s, 1H), 5.68 (s, 1H), 3.81 (s, 1H), 3.19 (s, 1H), 2.45(s, 3H). 13 C NMR (101 MHz, CDCl3) δ 178.38, 169.04, 162.95, 156.21, 152.76,142.86, 133.86, 132.51, 132.05, 130.07, 129.84, 128.73, 128.09, 125.74,125.69, 125.31, 124.88, 123.92, 123.59, 118.14, 107.87, 59.97, 42.64, 21.99.HR-MS(ESI): calcd for C 26 H 19 BrN2O3, [M+H] + , 487.0657; found 487.0651.

[0090] Example 14: 2-(3-(1-acetyl-3-(2-fluorophenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenyl)-4H-coumarin-4-one

[0091] Preparation of (Compound FPY14)

[0092]

[0093] The preparation method is the same as that of Example 1. 2-Fluoroacetophenone is used instead of 2-methylacetophenone to obtain a light yellow solid with a yield of 75.2% and a melting point of 172.2-175°C.

[0094] 1H NMR (400 MHz, Chloroform-d) δ 8.21 (s, 1H), 8.04 (s, 1H), 7.81 (d,J = 12.6 Hz, 2H), 7.70 (s, 1H), 7.57 (s, 1H), 7.50 (s, 1H), 7.46 – 7.37 (m,3H), 7.24 (s, 1H), 7.11 (s, 1H), 6.81 (s, 1H), 5.66 (s, 1H), 3.94 (s, 1H), 3.34 (s, 1H), 2.46 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ 178.43, 169.16, 163.09,156.24, 150.71, 142.99, 133.85, 132.47, 132.19, 132.10, 129.78, 128.84,125.70, 125.29, 124.60, 124.57, 123.93, 123.62, 119.26, 119.15, 118.16,116.74, 116.52, 107.85, 59.84, 44.77, 21.99. HR-MS(ESI): calcd for C 26 H 19 FN2O3,[M+H] + , 427.1458; found 427.1455.

[0095] Example 15: Preparation of 2-(3-(1-acetyl-3-(3-fluorophenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenyl)-4H-coumarin-4-one (Compound FPY15)

[0096]

[0097] The preparation method is the same as that of Example 1. 3-Fluoroacetophenone is used instead of 2-methylacetophenone to obtain a yellow solid with a yield of 73.5% and a melting point of 136.3-139°C.

[0098] 1H NMR (600 MHz, Chloroform-d) δ 8.19 (s, 1H), 7.81 (s, 1H), 7.77 (s,1H), 7.68 (s, 1H), 7.54 (s, 1H), 7.48 (t, J = 7.8 Hz, 3H), 7.43 – 7.37 (m,3H), 7.13 (s, 1H), 6.79 (s, 1H), 5.68 (s, 1H), 3.81 (s, 1H), 3.18 (s, 1H),2.45 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ 178.39, 169.12, 162.97, 156.21,152.68, 142.83, 133.87, 133.34, 133.26, 132.50, 130.52, 130.44, 129.85,128.73, 125.76, 125.68, 125.31, 123.91, 123.60, 122.50, 122.47, 118.15,117.58, 117.37, 113.48, 113.25, 107.85, 59.99, 42.30, 21.98. HR-MS(ESI):calcd for C 26 H 19 FN2O3, [M+H] + , 427.1458; found 427.1452.

[0099] Example 16: Preparation of 2-(3-(1-acetyl-3-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenyl)-4H-coumarin-4-one (Compound FPY16)

[0100]

[0101] The preparation method is the same as that of Example 1. 4-Fluoroacetophenone is used instead of 2-methylacetophenone to obtain a white solid with a yield of 78.3% and a melting point of 110.5-112.3°C.

[0102] 1H NMR (600 MHz, Chloroform-d) δ 8.20 (s, 1H), 7.82 (s, 1H), 7.78 –7.73 (m, 3H), 7.70 (s, 1H), 7.55 (s, 1H), 7.49 (s, 1H), 7.41 (d, J = 7.1 Hz,2H), 7.13 (d, J = 8.6 Hz, 2H), 6.81 (s, 1H), 5.67 (s, 1H), 3.81 (s, 1H), 3.19(s, 1H), 2.45 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ 178.38, 168.99, 162.99,156.19, 152.81, 142.94, 133.87, 132.44, 129.82, 128.76, 128.73, 128.64,127.43, 127.40, 125.69, 125.65, 125.30, 123.89, 123.59, 118.14, 116.11,115.89, 107.81, 59.89, 42.41, 21.97. HR-MS(ESI): calcd for C 26 H 19 FN2O3, [M+H] + ,427.1458; found 427.1450.

[0103] Example 17: Preparation of 2-(3-(1-acetyl-3-(4-ethylphenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenyl)-4H-coumarin-4-one (Compound FPY17)

[0104]

[0105] The preparation method is the same as that of Example 1. 4-ethylacetophenone is used instead of 2-methylacetophenone to obtain a white solid with a yield of 69.3% and a melting point of 113-117°C.

[0106] 1H NMR (600 MHz, Chloroform-d) δ 8.21 (s, 1H), 7.81 (s, 1H), 7.79 (s,1H), 7.72 – 7.66 (m, 3H), 7.56 (s, 1H), 7.48 (s, 1H), 7.41 (d, J = 7.0 Hz,2H), 7.27 (s, 1H), 7.26 (s, 1H), 6.81 (s, 1H), 5.66 (s, 1H), 3.82 (s, 1H),3.20 (s, 1H), 2.69 (d, J = 7.6 Hz, 2H), 2.46 (s, 3H), 1.25 (t, J = 7.6 Hz,3H). 13 C NMR (101 MHz, CDCl3) δ 178.44, 169.00, 163.09, 156.24, 153.97,147.28, 143.14, 133.84, 132.45, 129.78, 128.80, 128.58, 128.37, 126.75,125.70, 125.65, 125.29, 123.93, 123.65, 118.17, 107.85, 59.71, 42.44, 28.87,21.99, 16.22. HR-MS(ESI): calcd for C 28 H 24 N2O3, [M+H] + ,437.1865; found437.1860.

[0107] Example 18: Preparation of 2-(3-(1-acetyl-3-(4-methoxy-3-methylphenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenyl)-4H-coumarin-4-one (Compound FPY18)

[0108]

[0109] The preparation method is the same as that of Example 1. 4-methoxy-3-methylacetophenone is used instead of 2-methylacetophenone to obtain a yellow solid with a yield of 70.1% and a melting point of 119-122.3°C.

[0110] 1H NMR (600 MHz, Chloroform-d) δ 8.21 (s, 1H), 7.81 (s, 1H), 7.78 (s,1H), 7.69 (s, 1H), 7.61 (s, 1H), 7.56 (s, 1H), 7.47 (d, J = 7.7 Hz, 2H), 7.41(d, J = 7.0 Hz, 2H), 6.83 (s, 1H), 6.81 (s, 1H), 5.66 (s, 1H), 3.87 (s, 3H), 3.79 (s, 1H), 3.18 (s, 1H), 2.46 (s, 3H), 2.25 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ 178.42, 168.83, 163.09, 159.80, 156.20, 154.02, 143.23, 133.82,132.35, 129.73, 128.86, 128.81, 127.30, 126.02, 125.63, 125.57, 125.26,123.89, 123.63, 123.14, 118.16, 109.76, 107.77, 59.99, 55.47, 42.47, 21.99,16.29. HR-MS(ESI): calcd for C 28 H 24 N2O4, [M+H] + , 453.1814; found 453.1810.

[0111] Example 19: Preparation of 2-(3-(1-acetyl-3-(4-(trifluoromethyl)phenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenyl)-4H-coumarin-4-one (Compound FPY19)

[0112]

[0113] The preparation method is the same as that of Example 1. 4-trifluoromethylacetophenone is used instead of 2-methylacetophenone to obtain a white solid with a yield of 81.6% and a melting point of 110-112°C.

[0114] 1H NMR (400 MHz, Chloroform-d) δ 8.21 (s, 1H), 7.91- 7.82 (m, 3H), 7.78 (s, 1H), 7.73- 7.67 (m, 3H), 7.54 (d, J = 20.6 Hz, 2H), 7.41 (d, J = 8.1Hz, 2H), 6.80 (s, 1H), 5.72 (s, 1H), 3.86 (s, 1H), 3.25 (s, 1H), 2.48 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ 178.41, 169.21, 162.96, 156.20, 152.33,142.73, 134.50, 133.91, 132.52, 132.14, 131.81, 129.88, 128.74, 126.91,125.82, 125.79, 125.76, 125.72, 125.67, 125.35, 123.87, 123.59, 122.45,118.13, 107.84, 60.12, 42.20, 21.98. HR-MS(ESI):calcd for C 27 H 19 F3N2O3, [M+H] + ,477.1426; found 477.1420.

[0115] Example 20: Preparation of 2-(3-(1-acetyl-3-(4-(trifluoromethoxy)phenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenyl)-4H-coumarin-4-one (Compound FPY20)

[0116]

[0117] The preparation method is the same as that of Example 1. 4-trifluoromethoxyacetophenone is used instead of 2-methylacetophenone to obtain a yellow solid with a yield of 74.4% and a melting point of 165.2-166.8°C.

[0118] 1H NMR (600 MHz, Chloroform-d) δ 8.21 (s, 1H), 7.84 (s, 1H), 7.80 (d,J = 8.7 Hz, 2H), 7.77 (s, 1H), 7.70 (s, 1H), 7.56 (s, 1H), 7.50 (s, 1H), 7.41(d, J = 9.8 Hz, 2H), 7.28 (d, J = 8.3 Hz, 2H), 6.81 (s, 1H), 5.70 (s, 1H), 3.83 (s, 1H), 3.19 (s, 1H), 2.45 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ 178.41,169.08, 162.99, 156.22, 152.40, 142.84, 133.89, 132.53, 129.85, 129.79,128.76, 128.25, 125.78, 125.70, 125.33, 123.90, 123.57, 121.65, 121.14,119.08, 118.13, 107.86, 60.00, 42.32, 21.97. HR-MS(ESI): calcd forC 27 H 19 F3N2O3, [M+H] + , 493.1375; found 493.1371.

[0119] Example 21: Preparation of HASMC cell culture and calcification medium

[0120] We selected HASMC cells for culture. HASMC cells were cultured in DMEM high-glucose culture medium containing 10% fetal bovine serum and 100 U / mL penicillin and streptomycin. The incubator culture conditions were set to 5% CO2, 37°C, and the medium was changed daily to observe the growth of cells. When HASMC cells grew to 70-80% confluence, the old cell culture medium was discarded, and the cells were washed twice with PBS. 0.25% trypsin was added, and the cell morphology was observed under an inverted microscope. When the cells showed cytoplasm retraction, the cell body became round, and the intercellular space became larger, the digestion solution was discarded, PBS was added, and the attached cells were repeatedly and gently blown with a pipette to make them fall off and suspend. After adjusting the cells to the appropriate density, they were inoculated in a new culture dish and placed in a 5% CO2, 37°C incubator for culture. Adding 4 mM inorganic phosphate (Na2HPO4 and NaH2PO4 ratio of 1:2) to DMEM induced calcification in smooth muscle cells. The induction process lasted for 4 days, and fresh medium was replaced every two days.

[0121] Example 22: Evaluation of anti-vascular calcification activity of HASMC cells

[0122] We used the MTT method to determine the cytotoxicity of the compound FPY1-20 on HASMC cells. HASMC cells in the logarithmic growth phase were seeded into 96-well plates, with approximately 10,000 cells per well and a cell suspension volume of 200 μL per well. 22 groups were set up, namely: normal group, calcification group, and calcification-5 μM FPY1-20 group. Six replicate wells were set up for each drug group. Culture was carried out under the same culture conditions for 4 days. After the culture was completed, 20 μL of MTT dye solution (5 mg / mL, prepared with double-free culture medium) was added to each well. After incubation for 4 hours, the culture was terminated, the culture medium in the well was carefully aspirated, 150 μL of DMSO was added to each well, and the well was shaken at room temperature for 15 min to fully dissolve the crystals. Finally, a wavelength of 550 nm was selected, and the light absorbance of each well was detected on an enzyme-linked immunosorbent assay monitor, and the results were recorded. MTT results ( Figure 1 ) showed that compounds FPY1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 16, 17, 18, 19, and 20 at a concentration of 5 μM had no obvious toxicity to cells.

[0123] HASMC cells in the logarithmic growth phase were seeded into 12-well plates, and 500 cells were added to each well. After the cells were attached to the wall and stabilized overnight, the calcification medium was prepared according to the above-mentioned calcification medium preparation method. In the calcification medium environment, FPY1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 16, 17, 18, 19, and 20 compounds were added at a concentration of 5 μM to treat the cells. Cultured in an incubator, the culture medium was changed every two days, and the culture was cultured for a total of 4 days. After culture, the culture medium was aspirated, 4% paraformaldehyde was added to fix the cells for 30 min, and then washed 3 times with PBS for 5 min each time. Then, Alizarin red dye was added for staining for 30 min, and then washed 3 times with acidic PBS for 5 min each time. Take pictures. By Figure 2 It can be seen that high phosphate can significantly induce osteogenic differentiation of smooth muscle cells and produce calcium deposition, while the FPY19 compound can significantly inhibit this situation and has a good inhibitory effect on osteogenic differentiation of smooth muscle cells at a concentration of 5 μM.

[0124] WB experiment was used to explore the effect of FPY19 compound on osteogenic-related factors during osteogenic differentiation of smooth muscle cells. First, protein extraction was performed, and HASMC cells in the logarithmic growth phase were seeded in 6-well plates. Calcification medium was added to induce osteogenic differentiation of cells, and 5 μM concentration of FPY19 compound was added for treatment. Four days later, the cells were washed twice with PBS. After adding protein lysis buffer for full lysis, centrifuged at 12000 r / min at 4℃ for 10 min, the supernatant was taken, and transferred to a -20℃ refrigerator for storage. The total protein concentration was determined by BCA method, 30 μg of total protein was quantified, and the protein sample loading volume was calculated. SDS-polyacrylamide gel constant voltage 124 V electrophoresis was used for about 90 min. The protein blot was transferred to the NC membrane under constant voltage 100 V. The target band was obtained according to the protein molecular weight and blocked with 5% skim milk at room temperature for 1 h. After blocking, the corresponding primary antibody (ALPL, BMP2, RUNX2) was added and incubated overnight at 4℃. The next day, the primary antibody was recovered, the strips were washed 3 times with PBST, each time for 8 min, and the strips were placed in the secondary antibody for incubation at room temperature for 1 h. The secondary antibody was recovered, the strips were washed 3 times with PBST, each time for 8 min, and the immunoblot was detected using an ECL chemiluminescence kit and imaging equipment. Figure 3 It can be seen that high phosphate can significantly induce the expression of osteoblast-related factors ALPL, BMP2, and RUNX2 in vascular smooth muscle cells, while 5 μM concentration of FPY19 compound can significantly inhibit the expression of osteoblast-related factors, thereby inhibiting the osteogenic differentiation of vascular smooth muscle cells.

[0125] In summary, the FPY19 compound of the present invention can significantly treat the osteogenic differentiation of vascular cells induced by high phosphorus, indicating that flavonoid-dihydropyrazole compounds have potential application prospects in the prevention and treatment of vascular calcification.

Claims

1. A flavonoid-dihydropyrazole derivative, characterized in that Its general structure is shown below: ； wherein R is selected from 2-benzyl, 3-benzyl, 4-benzyl, 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, 2-chlorophenyl, 3-chlorophenyl, 4-chlorophenyl, 2-bromophenyl, 3-bromophenyl, 4-bromophenyl, 2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl, phenyl, p-ethylphenyl, 4-methoxy-3-methylphenyl, p-trifluoromethylphenyl or p-trifluoromethoxyphenyl.

2. The method for preparing the flavonoid-dihydropyrazole derivative according to claim 1, characterized in that The steps include: Step 1: weigh solid NaOH and ethanol, add them to a reactor, and stir to dissolve; weigh m-benzaldehyde and 2-hydroxyacetophenone, add them to the system, stir to react at room temperature, and monitor by TLC; after the reaction, add dilute hydrochloric acid dropwise to the reaction system, adjust the pH to neutral, concentrate using a rotary evaporator, and separate and purify by silica gel column chromatography to obtain intermediate chalcone A; The structural formula of the intermediate chalcone A is: ； Step 2: Add intermediate chalcone A to a round-bottom flask, add N, N-dimethylformamide and stir to dissolve, add iodine, set the reaction temperature to 110°C, and monitor by TLC; after the reaction, cool the reaction mixture to room temperature and pour it into ice water to precipitate a light yellow solid, wash it with sodium thiosulfate, filter it, collect the filter cake, and dry it to obtain intermediate B; The structural formula of the intermediate B is: ； Step 3: adding solid sodium hydroxide and ethanol to a reactor, stirring and dissolving; weighing intermediate B and ketone compounds with different substituents, adding them to the above system, stirring and reacting at room temperature, and precipitating solids; after the reaction is completed by TLC monitoring, filtering and collecting the filter cake, dissolving it in dichloromethane, and then adding Na2SO4 to dry to remove moisture, dehydrating, and concentrating by vacuum distillation to obtain intermediate C; The structural formula of the ketone compounds with different substituents is: ; The structural formula of the intermediate C is: ; wherein R is selected from 2-benzyl, 3-benzyl, 4-benzyl, 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, 2-chlorophenyl, 3-chlorophenyl, 4-chlorophenyl, 2-bromophenyl, 3-bromophenyl, 4-bromophenyl, 2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl, phenyl, p-ethylphenyl, 4-methoxy-3-methylphenyl, p-trifluoromethylphenyl, p-trifluoromethoxyphenyl; Step 4: Add intermediate C to a reactor, add acetic acid and stir to dissolve, weigh hydrazine hydrate and add to the above system, and reflux at 100°C for reaction; after the reaction is completed, cool the reaction solution to room temperature, pour the reaction mixture into ice water, precipitate solid, filter, collect the filter cake, dissolve it in dichloromethane, then add Na2SO4 to dry to remove moisture, dehydrate, and concentrate by reduced pressure distillation to obtain a crude product, separate and purify it by silica gel column chromatography, and obtain the target product after recrystallization.

3. The preparation method according to claim 2, characterized in that: In step 1, the eluent for column chromatography separation is petroleum ether:ethyl acetate=3:1, v / v.

4. The preparation method according to claim 2, characterized in that: In step 4, the eluent for column chromatography separation is petroleum ether:ethyl acetate=2:1, v / v.

5. The preparation method according to claim 2, characterized in that: In step 4, the solvent used for recrystallization is petroleum ether.

6. Use of the flavonoid-dihydropyrazole derivative according to claim 1 in the preparation of a drug for treating vascular calcification.

7. The use according to claim 6, characterized in that: The drug for treating vascular calcification can alleviate the osteogenic differentiation of vascular smooth muscle cells induced by high phosphate.

8. A pharmaceutical composition for treating vascular calcification, characterized in that: The pharmaceutical composition comprises the flavonoid-dihydropyrazole derivative according to claim 1, and a pharmaceutically acceptable excipient and carrier.

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