WO2024027521A1 - Methylophiopogonone a derivative, and preparation and use thereof - Google Patents

Abstract

Provided are a methylophiopogonone A derivative, and the preparation and the use thereof. The present invention specifically relates to three derivatives of methylophiopogonone A, which derivatives have the chemical structures of formula I, formula II and formula III; and a method for preparing the derivatives, and the use of the derivatives in the preparation of a drug for preventing and/or treating cardiovascular diseases, etc.

Description

Methyl Ophiopogon japonicus flavonoid A derivatives and their preparation and application Technical field

The invention belongs to the field of medicinal chemistry and specifically relates to three methyl Ophiopogon japonicus flavonoid A derivatives of formula I, formula II and formula III and their preparation and application, especially in the treatment and/or prevention of cardiovascular diseases and related diseases. use.

Background technique

Cardiovascular disease (CVD) is the leading cause of death worldwide, accounting for 31.5% of deaths worldwide and 45.0% of deaths from non-communicable diseases. It is higher than tumors and other diseases, and its prevalence continues to rise. According to the "China Cardiovascular Health and Disease Report 2021", it is estimated that the number of patients with cardiovascular disease in my country is 330 million, accounting for about 23% of the total population, including 11.39 million coronary heart disease, 8.9 million heart failure, 245 million hypertension, and pulmonary 5 million for heart disease, 4.87 million for atrial fibrillation, and 2.5 million for rheumatic heart disease. At the same time, the age of onset is gradually getting younger. The total hospitalization expenses for cardiovascular and cerebrovascular diseases are increasing year by year, and the average growth rate is much higher than the growth rate of gross national product.

In the past 40 years, there has been great progress in the treatment of cardiovascular disease drugs, including beta blockers, calcium channel blockers, diuretics, renin-angiotensin-aldosterone system drugs, lipid-lowering drugs, and vasodilators. , sinus node channel blockers, drugs to improve myocardial metabolism, and Chinese patent medicines such as Salvia miltiorrhiza tablets, Suxiao Jiuxin Pills, and Ginkgo leaf preparations. However, in recent years, the research and development of therapeutic drugs in this field has encountered bottlenecks. In addition to new uses of old drugs (such as sodium-glucose cotransporter 2 inhibitors empagliflozin and dapagliflozin) and a few new target drug candidates ( Vericiguat and Omecamtiv Mecarbil) have made certain progress, but overall research and development clearly lags behind tumor drugs and autoimmune disease drugs. Existing drugs cannot meet the needs of clinical treatment, and there is an urgent need to develop new drug therapies. At the same time, current standard treatments still involve cardiovascular risks, and patients receiving secondary prevention will still have considerable residual cardiovascular risks, with the risk of major adverse cardiovascular events still ranging from 4 to 12%.

The discovery and development of innovative drugs using traditional Chinese medicine and natural products as resources has always been an important way to create new drugs. China’s original and internationally recognized innovative drugs include artemisinin, dihydroartemisinin, huperzine A, bicyclol, butyrate Phthalide, etc. are all derived from traditional Chinese medicine. Methyl Ophiopogon flavonoid A is an ingredient contained in the traditional Chinese medicine Ophiopogon japonicus. Studies have shown that it has anti-non-alcoholic fatty liver and hepatoprotective activity. Our research found that it has cardiovascular disease prevention and treatment activity. In the present invention, we used methyl Ophiopogon flavonoid A as the lead compound, discovered a class of methyl Ophiopogon flavonoid A derivatives with better activity, and conducted pharmacodynamic evaluation.

Contents of the invention

The technical problem solved by the present invention is to provide three methyl Ophiopogon japonicus flavonoid A derivatives, their preparation methods and their application in preparing drugs for preventing and/or treating cardiovascular-related diseases.

In order to solve the technical problems of the present invention, the present invention provides the following technical solutions:

The first aspect of the technical solution of the present invention is to provide three methyl Ophiopogon japonicus flavonoid A derivatives of formula I, formula II, formula III, and pharmaceutically acceptable salts thereof, with the following structural formulas:

The second aspect of the technical solution of the present invention is to provide the preparation method of the three methyl Ophiopogon japonicus flavonoid A derivatives of formula I, formula II, formula III and their pharmaceutically acceptable salts described in the first aspect.

Methyl Ophiopogon japonicus flavonoid A derivative I of the present invention can be obtained by the following method, which method includes the following steps:

Under alkaline conditions, the compound of formula (2) is prepared by reacting 2,4,6-trihydroxyacetophenone (1) with halomethoxymethyl ether; under alkaline conditions, the formula The compound of formula (2) structure is reacted with piperonal to prepare the compound of formula (3) structure; the double bond of the compound of formula (3) structure is reduced to prepare the compound of formula (4) structure; the compound of formula (4) structure is reacted with The compound of formula I is prepared by reacting phosphorus oxychloride and N,N-dimethylformamide:

Preferably, in step one, the base is triethylamine or N,N-diisopropylethylamine, and the halomethoxymethyl ether is bromomethoxymethyl ether. In step two, the base is sodium hydride or sodium alkoxide, KOH/EtOH, etc. In step three, the reducing agent is palladium carbon/hydrogen or stannous chloride, etc.

More preferably, in step one, the base is N,N-diisopropylethylamine. In step 2, the base is sodium hydride, and the reaction is carried out in the presence of a solvent such as tetrahydrofuran, N,N-dimethylformamide, etc. In step three, the reducing agent is palladium carbon/hydrogen.

Methyl Ophiopogon japonicus flavonoid A derivatives II and III of the present invention can be obtained by the following method, which method includes the following steps:

Reducing 2,4,6-trihydroxybenzaldehyde to 2,4,6-trihydroxytoluene and reacting with acetic anhydride to prepare a compound of formula (5); under alkaline conditions, formula (5) reacts with halomethyl The compound of formula (6) structure is prepared by the reaction of oxymethyl ether; under alkaline conditions, the compound of formula (6) structure is reacted with piperonal to prepare the compound of formula (7) structure; the compound of formula (7) structure is The compound of formula (8) structure is prepared by the double bond reduction of the compound; the compound of formula (8) structure is reacted with phosphorus oxychloride and N,N-dimethylformamide to prepare the compounds of formula II and formula III structure:

Preferably, in step one, the reducing agent is a metal reducing agent or metal borohydride. In step three, the base is triethylamine or N,N-diisopropylethylamine. In step four, the base is sodium hydride or sodium alkoxide, KOH/EtOH. In step five, the reducing agent is palladium carbon/hydrogen or stannous chloride, etc.

More preferably, in step one, the reducing agent is a metal reducing agent. In step three, the base is N,N-diisopropylethylamine. In step four, the base is sodium hydride, and the reaction is carried out in the presence of tetrahydrofuran. In step five, the reducing agent is palladium carbon/hydrogen.

The third aspect of the technical solution of the present invention provides a pharmaceutical composition, which contains a therapeutically and/or preventively effective amount of the methyl Ophiopogon flavonoid A derivatives and their pharmaceutically acceptable salts described in the first aspect of the present invention. , and optionally one or more pharmaceutically acceptable carriers or excipients.

The pharmaceutical composition can be prepared according to methods well known in the art. Any dosage form suitable for human or animal use may be prepared by combining the compounds of the present invention with one or more pharmaceutically acceptable solid or liquid excipients and/or auxiliaries. The content of the compound of the invention in its pharmaceutical composition is usually 0.1-95% by weight.

The compound of the present invention or the pharmaceutical composition containing it can be administered in unit dosage form, and the administration route can be intestinal or parenteral, such as oral, intravenous injection, intramuscular injection, subcutaneous injection, nasal cavity, oral mucosa, eye, lung and Respiratory tract, skin, vagina, rectum, etc.

The dosage form for administration may be a liquid, solid or semi-solid dosage form. Liquid dosage forms can be solutions (including true solutions and colloidal solutions), emulsions (including o/w type, w/o type and double emulsion), suspensions, injections (including water injections, powder injections and infusions), eye drops agents, nose drops, lotions and liniments, etc.; solid dosage forms can be tablets (including ordinary tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules ( Including hard capsules, soft capsules, enteric-coated capsules), granules, powders, pellets, dripping pills, suppositories, films, patches, aerosols (powder), sprays, etc.; semi-solid dosage forms can be ointments, Gels, pastes, etc.

The compounds of the present invention can be made into ordinary preparations, sustained-release preparations, controlled-release preparations, targeted preparations and various particulate drug delivery systems.

In order to formulate the compounds of the present invention into tablets, various excipients known in the art can be widely used, including diluents, binders, wetting agents, disintegrants, lubricants, and co-solvents. The diluent can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; the wetting agent can be water, ethanol, isopropyl Propanol, etc.; the binder can be starch slurry, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, acacia glue, gelatin slurry, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl Cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.; the disintegrant can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyethylene glycol, etc. Vinyl pyrrolidone, croscarmellose sodium, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitol fatty acid ester, sodium lauryl sulfonate, etc.; lubricants and co-solvents It can be talc, silica, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

Tablets can also be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bi-layer and multi-layer tablets.

In order to make the administration unit into a capsule, the active ingredient compound of the present invention can be mixed with a diluent and a co-solvent, and the mixture can be placed directly into a hard capsule or soft capsule. The active ingredient compound of the present invention can also be first made into granules or pellets with a diluent, binder, and disintegrant, and then placed in a hard capsule or soft capsule. Various diluents, binders, wetting agents, disintegrants, and co-solvents used to prepare tablets of the compound of the present invention can also be used to prepare capsules of the compound of the present invention.

To prepare the compounds of the present invention into injections, water, ethanol, isopropyl alcohol, propylene glycol or their mixtures can be used as solvents and an appropriate amount of solubilizers, co-solvents, pH regulators and osmotic pressure regulators commonly used in this field can be added. The solubilizer or co-solvent can be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; the pH regulator can be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc.; the osmotic pressure regulator can be It is sodium chloride, mannitol, glucose, phosphate, acetate, etc. If preparing freeze-dried powder for injection, mannitol, glucose, etc. can also be added as supporting agents.

In addition, if necessary, colorants, preservatives, fragrances, flavoring agents or other additives can also be added to the pharmaceutical preparations.

In order to achieve the purpose of medication and enhance the therapeutic effect, the medicine or pharmaceutical composition of the present invention can be administered by any known administration method.

The dosage of the pharmaceutical composition of the present invention can vary widely depending on the nature and severity of the disease to be prevented or treated, the individual condition of the patient or animal, the route of administration and dosage form, etc. Generally speaking, a suitable daily dosage range of the compounds of the present invention is 0.001-5 mg/Kg body weight. The above dosage may be administered as a single dosage unit or divided into several dosage units, depending on the physician's clinical experience and the dosage regimen including the use of other therapeutic modalities.

The compounds or compositions of the present invention can be taken alone or in combination with other therapeutic or symptomatic drugs. When the compound of the present invention has a synergistic effect with other therapeutic drugs, its dosage should be adjusted according to the actual situation.

The fourth aspect of the technical solution of the present invention provides the methyl Ophiopogon japonicus flavonoid A derivatives and their pharmaceutically acceptable salts described in the first aspect and the pharmaceutical compositions described in the third aspect in the preparation of prevention and/or treatment and Application in drugs for cardiovascular related diseases. The cardiovascular-related diseases described therein are selected from one or more of the following: myocardial ischemia, myocardial injury, myocardial hypertrophy, coronary heart disease, hypertension, heart failure, cardiomyopathy, arrhythmia, myocardial infarction, Angina pectoris.

Beneficial technical effects

The methyl Ophiopogon japonicus flavonoid A derivatives of the present invention all perform significantly in the isoproterenol-induced cardiomyocyte injury model, the hypoxia-reoxygenation-induced cardiomyocyte injury model, and the angiotensin II-induced cardiomyocyte injury model. It has cardiomyocyte injury protection activity and can significantly improve myocardial cell survival rate. 0.1μM shows good efficacy. In the cardiomyocyte hypertrophy model induced by angiotensin II, it also showed significant activity in inhibiting cardiomyocyte hypertrophy, with 0.1 μM showing good efficacy. Myocardial damage accompanies the occurrence and development of cardiovascular diseases, and myocardial hypertrophy is an important pathological process leading to heart failure such as hypertension. Multiple experimental results show that the compound of the present invention has good activity in protecting myocardial cells and inhibiting myocardial hypertrophy, and under the experimental conditions, most of the activity results are better than those of methyl Ophiopogon flavonoid A and diltiazem.

The methyl Ophiopogon japonicus flavonoid A derivatives of formula I and formula II of the present invention have significant activity in the following aspects in in vivo models: clear myocardial deficiency in the classic isoprenaline-induced ischemic heart disease mouse model The therapeutic effect of blood and cardiac hypertrophy can reduce the levels of heart failure/cardiac hypertrophy biomarkers NT-proBNP and ANP, reduce the level of serum cardiac enzyme CK-MB, reduce myocardial tissue calcium overload, and significantly improve the pathological damage of myocardial tissue; in angiotensin The mouse model of hypertensive heart disease induced by II has clear blood pressure lowering and prevention and treatment effects on related heart disease. It can significantly reduce the progressive increase in systolic blood pressure induced by angiotensin II and reduce the level of the heart failure/cardiac hypertrophy biomarker BNP. , reduce the levels of serum myocardial injury biomarkers troponin cTn-T and cardiac enzyme CK-MB, reduce the production and release of inflammatory factor TNF-α, and reduce the damage of the renin angiotensin aldosterone system (RAAS) system and sympathetic nervous system. Overactivation effect, improve myocardial hypertrophy, improve left ventricular systolic and diastolic function, and increase left ventricular compliance. In the in vivo animal model, the activity of the methyl Ophiopogon flavonoid A derivatives of the present invention is better than that of methyl Ophiopogon flavonoid A, diltiazem and spironolactone, and is equivalent to telmisartan, and has no hypotension caused by telmisartan. adverse effects.

Based on the above in vitro and in vivo experimental results, the methyl Ophiopogon japonicus flavonoid A derivatives of the present invention have potential value in the development of anti-cardiovascular disease drugs.

Description of the drawings

Figure 1. Diagram showing the effect of methyl Ophiopogon flavone A derivatives, Formula I and Formula II, on the improvement of cardiomyocyte hypertrophy and damage caused by angiotensin II (AngII).

Figure 2. The ameliorative effect of methyl Ophiopogon flavone A derivatives Formula I and Formula II on the pathological changes of cardiac tissue in isoproterenol (ISO)-induced myocardial ischemia/cardiac hypertrophy mice (H.E.200×).

Figure 3. Effects of methyl Ophiopogon flavonoids A derivatives Formula I and Formula II on M-mode echocardiography in mice with angiotensin II (AngII)-induced hypertensive heart disease.

Detailed ways

The invention provides three methyl Ophiopogon japonicus flavonoid A derivatives for treating cardiovascular diseases and their preparation and application. The following examples are enumerated to further illustrate the present invention. The examples are only for explanation and illustration and are in no way meant to limit the scope of the present invention in any way.

The structure of the compound is determined by hydrogen nuclear magnetic resonance spectroscopy ( ^1H NMR) and/or mass spectrometry (MS). NMR shifts (δ) are given in units of 10-6 (ppm). NMR was measured using a Bruker AV400 nuclear magnetic resonance spectrometer. MS measurements were performed using a Thermo scientific (ESI) mass spectrometer.

Column chromatography generally uses 200 to 300 mesh silica gel as the carrier.

The known starting materials of the present invention can be synthesized by methods known in the art, or can be purchased from companies such as Coupling Technology, Bailingwei Technology, and Anaiji Chemical.

The hydrogen atmosphere refers to the reaction bottle connected to a hydrogen balloon with a volume of about 1L.

In the following pharmacological research examples, experimental methods without specifying specific conditions should be selected according to conventional methods and conditions, or according to product instructions.

In the following examples, the corresponding Chinese names of some substances or abbreviations are as follows:

Control: blank control

Model: model comparison

ISO: Isoproterenol

H/R: Hypoxia-reoxygenation

AngII: angiotensin II

DMSO: dimethyl sulfoxide

NaH: sodium hydride

Na ₂ SO ₄ : sodium sulfate

NaCl: sodium chloride

SFC: supercritical liquid chromatography

DEA: Diethylamine

Example 1 Preparation of methyl Ophiopogon japonicus flavonoid A derivatives of formula I

1. Preparation of compounds with the structure of formula (2) (1-(2-hydroxy-4,6-bis(methoxymethoxy)phenyl)ethan-1-one)

Dissolve 2,4,6-trihydroxyacetophenone (10.0g, 59.5mmol) and N,N-diisopropylethylamine (23.1g, 178.5mmol) in 200mL of methylene chloride, slowly in an ice bath Add bromomethoxymethyl ether (10.7 mL, 130.9 mmol) and complete the addition within 2 hours. Monitor the reaction progress with UPLC-MS. After the raw materials disappear, add methanol to quench the reaction. The reaction solution is concentrated under reduced pressure and the residue is filtered through silica gel. Purification by column chromatography, eluent petroleum ether/ethyl acetate = 10:1, yielded 8.0 g of the title compound, yield 52%.

¹ H NMR (400MHz, CDCl ₃ ) δ13.72 (s, 1H), 6.26 (dd, J = 9.3, 2.3Hz, 2H), 5.26 (s, 2H), 5.17 (s, 2H), 3.52 (s, 3H),3.47(s,3H),2.66(s,3H).

2. Preparation of compounds with the structure of formula (3) (3-(benzo[d][1,3]dioxol-5-yl)-1-(2-hydroxy-4,6-bis(methoxymethoxy)phenyl)prop- 2-en-1-one)

Dissolve the compound of formula (2) (8.0g, 31.2mmol) in N,N-dimethylformamide, slowly add NaH (1.1g, 46.8mmol) under ice bath, and then add piperonal (4.7g, 31.2 mmol), the reaction solution was continued to stir for 30 minutes in an ice bath, and the reaction progress was monitored by UPLC-MS. After the raw materials disappeared, water was added, and then extracted with ethyl acetate. The organic phase was washed with water and saturated NaCl aqueous solution, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, and the eluent was petroleum ether/ethyl acetate = 5:1 to obtain 9.0 g of the title compound, with a yield of 74%.

¹ H NMR (400MHz, CDCl ₃ ) δ13.89 (s, 1H), 7.82–7.69 (m, 2H), 7.12–7.07 (m, 2H), 6.84 (d, J = 7.9Hz, 1H), 6.28 ( dd,J＝28.0,2.3Hz,2H),6.03(s,2H),5.29(s,2H),5.19(s,2H),3.54(s,3H),3.49(s,3H).

3. Preparation of compounds with the structure of formula (4) (3-(benzo[d][1,3]dioxol-5-yl)-1-(2-hydroxy-4,6-bis(methoxymethoxy)phenyl)propan- 1-one)

Dissolve the compound of formula (3) (9g, 23.2mmol) in 150ml tetrahydrofuran, add Pd/C (0.9g), and pass hydrogen gas at room temperature for 16h. Monitor the reaction progress with UPLC-MS. After the raw materials disappear, filter The solid was removed, and the liquid was concentrated under reduced pressure to obtain 7.2 g of the title compound, yield: 79%.

¹ H NMR (400MHz, CDCl ₃ ) δ 13.68 (s, 1H), 7.26 (s, 1H), 6.77–6.65 (m, 3H), 6.27 (dd, J = 11.6, 2.3Hz, 2H), 5.93 ( s,2H),5.24(s,2H),5.17(s,2H),3.48(d,J＝3.1Hz,7H),3.35–3.29(m,2H),2.94(t,J＝7.6Hz,2H ).

4. Preparation of formula I (3-(benzo[d][1,3]dioxol-5-yl)-1-(2-hydroxy-4,6-bis(methoxymethoxy)phenyl)propan-1-one)

Dissolve the compound of formula (4) (300 mg, 0.76 mmol) in 5 mL of ethyl acetate. Under a nitrogen atmosphere, slowly add N, N-dimethylformamide (224 mg, 3.06 mmol) and phosphorus oxychloride (470 mg). , 3.06mmol), the reaction solution continued to stir at room temperature for 5h, UPLC-MS monitored the reaction progress, after the raw materials disappeared, add water to quench the reaction, then add ethyl acetate extraction (3 × 25mL), the organic phase was water and saturated NaCl aqueous solution Wash, dry over anhydrous Na ₂ SO ₄ , filter, and concentrate the filtrate under reduced pressure. The residue is purified by silica gel column chromatography. The eluent is n-hexane/ethyl acetate = 1:1 to obtain 34 mg of the title compound, with a yield of 24.5%.

¹ H NMR (400MHz, DMSO-d ₆ ) δ12.70(s,1H),11.36–11.08(m,1H),8.19(s,1H),6.90–6.73(m,3H),6.31-6.30(m ,1H),6.16-6.15(m,1H),5.95(s,2H),3.57(s,2H).

MS(ESI-):[(MH) ^- ]311.0.

HPLC purity: 99.34% (254nm), 99.13% (214nm).

Example 2: Preparation of methyl Ophiopogon japonicus flavonoid A derivatives of formula II and formula III

1. Preparation of compounds with the structure of formula (5) (1-(2-hydroxy-4,6-bis(methoxymethoxy)phenyl)ethan-1-one)

Dissolve 2,4,6-trihydroxytoluene (5g, 35.70mmol) in 100mL glacial acetic acid, add boron trifluoride ether (6.08g, 42.80mmol) and acetic anhydride (4.37g, 42.80mmol) at room temperature, N React _at 100°C for 5 hours. After cooling, remove acetic acid under reduced pressure and add ethyl acetate. The organic phase is washed with water and saturated NaCl aqueous solution, dried over anhydrous Na ₂ SO ₄ , filtered, and the filtrate is concentrated under reduced pressure. The residue is filtered through silica gel. Purify by column chromatography, the eluent is petroleum ether/ethyl acetate = 5:1, and 4.0 g of the title compound is obtained, with a yield of 62%. MS(ESI+):[(M+H) ⁺ ]183.1.

2. Preparation of compounds with the structure of formula (6) (1-(2,4,6-tris(methoxymethoxy)-3-methylphenyl)ethan-1-one)

Dissolve the compound of formula (5) (4.0g, 22.0mmol) in 60mL THF, slowly add NaH (3.70g, 154.0mmol) in an ice bath, and then add bromomethoxymethyl ether (16.50g) dropwise , 132.0mmol), the reaction solution continued to stir for 3 hours in an ice bath, slowly added water and ethyl acetate to quench the reaction, and then extracted with ethyl acetate (3x 20mL). The organic phase was washed with water and saturated NaCl aqueous solution, and concentrated under reduced pressure. , the residue was purified by silica gel column chromatography, the eluent was petroleum ether/ethyl acetate = 5:1, and 1.13g of the title compound was obtained, with a yield of 16%.

¹ H NMR (400MHz, DMSO-d ₆ ) δ6.72(s,1H),5.23(s,2H),5.18(s,2H),4.86(s,2H),3.40(s,3H),3.39( s,3H),3.36(s,3H),2.42(s,3H),2.04(s,3H).

MS(ESI+):[(M+H)+]315.1.

3. Preparation of compounds with the structure of formula (7) (E)-4-(benzo[d][1,3]dioxol-5-yl)-1-(2,4,6-tris(methoxymethoxy)-3- methylphenyl)but-2-en-1-one

Dissolve the compound of formula (6) (1.13g, 3.6mmol) in 20mL tetrahydrofuran. NaH (130mg, 5.4mmol) and piperonal (0.54g, 3.6mmol) are slowly added under ice bath. The reaction solution is kept under ice bath. Continue to stir the reaction for 1 hour, slowly add water and ethyl acetate to quench the reaction, add ethyl acetate for extraction (3x 20mL), wash the organic phase with water and saturated NaCl aqueous solution, concentrate under reduced pressure, and the residue is purified by silica gel column chromatography. The removal agent was PE/EA=5:1, and 1.1g of the title compound was obtained with a yield of 58%.

¹ H NMR (400MHz, DMSO-d ₆ ) δ7.41 (s, 1H), 7.22 (d, J = 16.0Hz, 1H), 7.16 (d, J = 8.1Hz, 1H), 6.94 (dd, J = 11.9,9.6Hz,2H),6.76(s,1H),6.08(s,2H),5.26(s,2H),5.13(s,2H),4.85(s,2H),3.48(s,1H), 3.43(s,3H),3.39(d,J＝5.3Hz,1H),3.33(s,3H),3.27(s,3H),2.08(s,3H).

MS(ESI+):[(M-13)+]447.3.

4. Preparation of compounds with the structure of formula (8) (4-(benzo[d][1,3]dioxol-5-yl)-1-(2,4,6-tris(methoxymethoxy)-3-methylphenyl)butan -1-one)

Dissolve the compound of formula (7) (400 mg, 0.87 mmol) in 10 mL of tetrahydrofuran, add Pd/C (462 mg, 4.34 mmol), pass hydrogen gas at room temperature for 8 hours, monitor the reaction progress with UPLC-MS, and filter after the raw materials disappear. The solid was removed, the liquid was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography. The eluent was petroleum ether/ethyl acetate = 10:1 to obtain 200 mg of the title compound, with a yield of 50%.

¹ H NMR (400MHz, DMSO-d ₆ ) δ6.83–6.79(m,2H),6.71-6.68(m,2H),5.95(s,2H),5.23(s,2H),5.13(s,2H ),4.80(s,2H),3.40-3.39(m,5H),3.36(s,3H),3.32(s,3H),3.02(t,J＝7.4Hz,2H),2.82(t,J＝ 7.4Hz,2H),2.03(s,3H).MS(ESI+):[(M-13) ⁺ ]449.2.

5. Preparation of Formula II and Formula III

(3-(benzo[d][1,3]dioxol-5-ylmethyl)-5,7-dihydroxy-8-methyl-4H-chromen-4-one(Ⅱ)and 3-(benzo[d][1 ,3]dioxol-5-ylmethyl)-5,7-dihydroxy-6-methyl-4H-chromen-4-one(Ⅲ))

Dissolve the compound of formula (8) (200 mg, 0.43 mmol) in 10 mL of ethyl acetate. Under a nitrogen atmosphere, slowly add N, N-dimethylformamide (70 mg, 0.95 mmol) and trichloride in an ice bath. Oxonophosphorus (663 mg, 4.32 mmol) was raised to room temperature and the reaction was continued to stir for 5 hours. UPLC-MS was used to monitor the reaction progress. After the raw materials disappeared, water was added to quench the reaction, and ethyl acetate was added for extraction (3x 10 mL). The filtrate was decompressed. After concentration, the residue was purified by preparative HPLC to obtain a mixture of formula II and formula III, which was further purified by SFC (mobile phase: CO2/MeOH (0.1% DEA) = 65/35) to obtain formula II (t _R = 1.073). min, 20 mg, yield 14%) and the compound of formula III (t _R = 0.903 min, 9 mg, yield 6%).

Ⅱ: ¹ H NMR (400MHz, DMSO-d ₆ ) δ12.62 (s, 1H), 10.79 (s, 1H), 8.27 (s, 1H), 6.87 (d, J = 1.2Hz, 1H), 6.82- 6.80(m,1H),6.77–6.73(m,1H),6.29(s,1H),5.95(s,2H),3.58(s,2H),2.06(s,3H).

MS(ESI+):[(M+H) ⁺ ]327.0.

HPLC purity: 99.71% (254nm), 99.33% (214nm).

III: ¹ H NMR (400MHz, DMSO-d ₆ ) δ12.96 (s, 1H), 10.86 (s, 1H), 8.19 (s, 1H), 6.87 (d, J = 1.4Hz, 1H), 6.82- 6.80(m,1H),6.76-6.74(m,1H),6.42(s,1H),5.95(s,2H),3.59(s,2H),1.96(s,3H).

MS(ESI+):[(M+H) ⁺ ]327.0.

HPLC purity: 99.48% (254nm), 98.77% (214nm).

Pharmacological experiments

Experimental Example 1: Detection of Example Compounds Using Isoproterenol (ISO)-Induced Cardiomyocyte Injury Model

experimental method:

Rat H9C2 cardiomyocytes in the logarithmic growth phase were inoculated into 96-well plates at 7 × 10 ⁴ cells/mL, 100 μL per well, and divided into blank control group, ISO model group, ISO+diltiazem 10 μM group, ISO+Formula I 0.1 μM group, ISO+Formula I 0.3μM group, ISO+Formula I 1μM group, ISO+Formula I 3μM group, ISO+Formula I 5μM group, ISO+Formula I 10μM group, ISO+Formula II 0.1μM group, ISO+Formula II 0.3μM group, ISO+type II 1μM group, ISO+Formula II 3μM group, ISO+Formula II 5μM group, ISO+Formula II 10μM group, ISO+Formula III 1μM group, ISO+Formula III 5μM group, ISO+Formula III 10μM group, ISO+Methyl Ophiopogon flavonoid A 1μM group, ISO+Methyl Ophiopogon flavonoid A 5μM group, ISO+Methyl Ophiopogon flavonoid A 10μM group. After 24 hours of culture, 800 μM ISO and corresponding concentrations of drugs were added to the drug group, 800 μM ISO and an equal volume of DMSO were added to the ISO model group, and an equal volume of DMSO was added to the blank control group, and the culture was continued for 24 hours. After removing the supernatant, add 100 μL MTT solution (0.5 mg/mL) to each well, and place it in a cell culture incubator for 4 hours. After removing the supernatant, add 150 μL DMSO to each well, shake and mix for 10 min, and place it on a microplate reader at 570 nm. Measure the absorbance and calculate the cell viability. Cell viability (%) = [(As-Ab)/(Ac-Ab)] × 100%. As: Experimental well (culture medium containing cells, MTT, ISO); Ac: Control well (culture medium containing cells, MTT, without ISO); Ab: Blank well (culture medium without cells, MTT, without ISO) ISO).

Experimental results:

The results are shown in Table 1. Compared with the blank control group, when ISO was used to treat H9C2 cardiomyocytes for 24 hours, the survival rate of cardiomyocytes was significantly reduced (54.71%), and the OD value was statistically different from the blank control group. Compared with the ISO model group, the doses of 0.1-10 μM of formula I compound, 0.1-10 μM of formula II compound and 1-10 μM of formula III compound all showed significant protective activity against ISO-induced cardiomyocyte damage and could significantly improve cell survival rate and OD value. There is a statistical difference compared with the model group. Methyl Ophiopogon flavone A also showed significant protective effects on ISO-damaged cardiomyocytes at doses of 1 μM and 5 μM. Compared with the same dose of methyl Ophiopogon flavonoid A, the compounds of Formula I and Formula II showed better activity, and the activity of the compound of Formula III was equivalent to that of methyl Ophiopogon flavonoid A. Diltiazem 10 μM also has protective activity against ISO-induced cardiomyocyte damage. The protective activity of compounds of formula I and formula II against ISO-damaged cardiomyocytes is better than that of diltiazem. The activity of compound of formula III at 1 μM is better than that of diltiazem 10 μM.

Table 1 The protective effect of the compounds of the examples on myocardial cell damage caused by isoproterenol (ISO)

^*** P<0.001 compared with the blank control group; ^# P<0.05, ^## P<0.01, ^### P<0.001 compared with the model group; ^& P<0.05, ^&& P<0.01 compared with the same dose of methyl Ophiopogon japonicus Comparison of flavonoid group A.

Note: The dosage of the drug is not toxic to H9C2 cardiomyocytes, the data is not shown, the same below.

Experimental Example 2: Detection of Example Compounds Using the Hypoxia-Reoxygenation (H/R)-Induced Cardiomyocyte Injury Model

experimental method:

H9C2 cardiomyocytes in the logarithmic growth phase were inoculated into a 96-well plate at 7 × ¹⁰ cells/mL, 100 μL per well, and divided into blank control group, H/R model group, H/R+diltiazem 10 μM group, and H/R+ formula I 0.1μM group, H/R+Formula I 0.3μM group, H/R+Formula I 1μM group, H/R+Formula I 3μM group, H/R+Formula I 5μM group, H/R+Formula I 10μM group, H/R+Formula II 0.1μM group, H/R+Formula II 0.3μM group, H/R+Formula II 1μM group, H/R+Formula II 3μM group, H/R+Formula II 5μM group, H/R+Formula II 10μM group, H/R+type III 0.1μM group, H/R+Formula III 0.3μM group, H/R+Formula III 1μM group, H/R+Formula III 3μM group, H/R+Formula III 5μM group, H/R+Formula III 10μM group, H/R+A Basic Ophiopogon flavonoid A 1μM group, H/R+Methyl Ophiopogon flavonoid A 5μM group, H/R+Methyl Ophiopogon flavonoid A 10μM group. After 24 hours of culture, the culture medium of the H9C2 cardiomyocytes in the H/R group and the drug administration group was replaced with serum-free sugar-free DMEM medium, 100 μL per well, and placed in a hypoxic incubator, followed by hypoxia in a 37°C cell culture incubator. nourish. After 6 hours, discard the supernatants of the H/R group and the administration group, add high-glucose medium containing 10% fetal calf serum, 100 μL per well, and add corresponding concentrations of drugs to each administration group, H/R An equal volume of DMSO was added to the model group and blank control group, and the cells were reoxygenated and cultured in a 37°C, 95% O ₂ , 5% CO ₂ cell culture incubator for 12 hours. After removing the supernatant, add 100 μL of CCK solution to each well (the volume of CCK accounts for 10% of the volume of DMEM culture medium), place it in a cell culture incubator and culture it for 1 hour, place it in a microplate reader to measure the absorbance at 450 nm, and calculate the cell survival rate. Cell viability = (experimental well-blank well)/(control well-blank well)×100%.

Experimental results:

The results are shown in Table 2. Compared with the blank control group, reoxygenation after glucose and oxygen deprivation caused significant damage to myocardial cells, and the cell survival rate was only 50.80%. Compounds of formula I (0.1, 0.3, 1, 3, 10 μM), compounds of formula II (0.1, 0.3, 3 μM) and compounds of formula III (3 μM) show significant protective activity against cardiomyocyte injury induced by hypoxia and reoxygenation, and significantly improve The survival rate and OD value of myocardial cells injured by oxygen and reoxygenation were statistically different from those in the model group. Methyl Ophiopogon flavone A at a concentration of 10 μM also showed significant protective activity against myocardial injury induced by hypoxia and reoxygenation. Methyl Ophiopogon flavone A at 1 μM and 5 μM did not show significant protective activity against myocardial injury induced by hypoxia and reoxygenation. Diltiazem 10 μM did not show significant protective effect on myocardial injury induced by hypoxia-reoxygenation. In this model, compounds of Formula I and Formula II are more active than methyl Ophiopogon flavonoid A, and are effective at low doses. Compounds of formula I, formula II and formula III are more active than diltiazem. Compounds of Formula I, Formula II and Formula III exhibit anti-ischemia-reperfusion-induced cardiomyocyte injury activity, and the activity of the compounds of Formula I and Formula II is better than that of the compound of Formula III.

Table 2 Example compounds protect against myocardial cell damage caused by hypoxia-reoxygenation (H/R)

^*** P<0.001 compared with the blank control group; ^# P<0.05, ^## P<0.01, ^### P<0.001 compared with the model group.

Experimental Example 3: Detection of Example Compounds Using angiotensin II (Ang II)-induced cardiomyocyte injury model

experimental method:

Rat H9C2 cardiomyocytes in the logarithmic growth phase were inoculated into 96-well plates at 7 × 10 ⁴ cells/mL, 100 μL per well, and divided into blank control group, AngⅡ model group, AngⅡ+diltiazem 10 μM group, and AngⅡ+ Formula I 0.1μM group, AngⅡ+Formula I 0.3μM group, AngⅡ+Formula I 1μM group, AngⅡ+Formula I 3μM group, AngⅡ+Formula I 10μM group, AngⅡ+Formula II 0.1μM group, AngⅡ+Formula II 0.3μM group , AngⅡ+Formula II 1μM group, AngⅡ+Formula II 3μM group, AngⅡ+Formula II 10μM group, AngⅡ+Methyl Ophiopogon flavonoid A 10μM group. After 24 hours of culture, 50 μM AngⅡ and corresponding concentrations of drugs were added to the drug group, 50 μM AngⅡ and an equal volume of DMSO were added to the AngⅡ model group, and an equal volume of DMSO was added to the blank control group, and the culture was continued for 24 hours. After removing the supernatant, add 100 μL MTT solution (0.5 mg/mL) to each well, and place it in a cell culture incubator for 4 hours. After removing the supernatant, add 150 μL DMSO to each well, shake and mix for 10 min, and place it on the microplate reader at 570 nm. Measure the absorbance and calculate the cell viability. Cell viability (%) = [(As-Ab)/(Ac-Ab)] × 100%. As: Experimental well (medium containing cells, MTT, AngⅡ); Ac: Control well (medium containing cells, MTT, without AngⅡ); Ab: Blank well (medium without cells, MTT, without AngⅡ) AngII).

Experimental results:

The results are shown in Table 3. Compared with the blank control group, 50 μM Ang II caused significant damage to myocardial cells, with a cell survival rate of only 70.81%. The OD value was significantly different from the blank control group. Compared with the model group, the compound of formula I (3, 10 μM) and the compound of formula II (0.1, 1, 10 μM) both showed significant protective activity against cardiomyocyte damage caused by Ang II, significantly improved the cell survival rate, and the OD value was higher than that of the model group. Statistical difference. Other dosage groups of the compounds of Formula I and Formula II also had a tendency to improve cardiomyocyte damage caused by Ang II. 10 μM methyl Ophiopogon flavone A and 10 μM diltiazem also showed significant protective activity against cardiomyocyte damage caused by Ang II. In the cardiomyocyte injury model caused by Ang II, the activity of the compounds of Formula I and Formula II was comparable to that of methyl Ophiopogon flavonoid A and diltiazem, and the activity of the compound of Formula II at 0.1 μM was slightly better than that of methyl Ophiopogon flavonoid A 10 μM.

Table 3 The protective effects of the compounds of Examples on cardiomyocyte damage caused by angiotensin II (Ang II)

^*** P<0.001 compared with the blank control group; ^# P<0.05, ^## P<0.01, ^### P<0.001 compared with the model group.

Experimental Example 4: Detection of Example Compounds Using angiotensin II (Ang II)-induced cardiomyocyte hypertrophy model

experimental method:

H9C2 cardiomyocytes in the logarithmic growth phase were adjusted to a density of 1.2×10 ⁵ /well and seeded in six-well plates, and divided into blank control group, AngⅡ model group, AngⅡ+diltiazem 10μM group, AngⅡ+Formula I 0.1μM group, AngⅡ +Formula I 0.5μM group, AngⅡ+Formula I 1μM group, AngⅡ+Formula II 0.1μM group, AngⅡ+Formula II 0.5μM group, AngⅡ+Formula II 1μM group. After 24 hours of culture, 40 μM AngⅡ and corresponding concentrations of drugs were added to each administration group. The AngⅡ+diltiazem 10 μM group was added with 40 μM AngⅡ and 10 μM diltiazem. The AngⅡ model group was added with 40 μM AngⅡ and an equal volume of DMSO. The blank control group was added with an equal volume of DMSO. Continue culturing for 24 hours. Discard the culture medium, wash 3 times with PBS, add 1 mL of methanol to each well, fix at -20°C for 20 minutes, and then place it at room temperature. After discarding the methanol, add 1 mL of 0.5% crystal violet dye to each well for staining for 10 minutes, wash with water, and then Take pictures under an inverted microscope and randomly select the field of view to take pictures. Image J software was used to measure the sum of the surface areas of all cardiomyocytes within the field of view, and at the same time count the number of cells within the field of view to obtain the surface area of each cardiomyocyte.

The surface area of a single cardiomyocyte = the surface area of all cardiomyocytes within the field of view and/the number of cardiomyocytes within the field of view.

Experimental results:

The results are shown in Figure 1 and Table 4. AngⅡ40μM acted on H9C2 cells for 24 hours. Compared with the blank control group, the number of cardiomyocytes in the model group was significantly reduced, and the volume of cardiomyocytes was significantly increased. The compound of formula I was 0.1μM, 0.5μM, and 1μM, and the compound of formula II was 0.1μM, 0.5μM, and 1μM. All of them can significantly improve the decrease in cell number caused by Ang II, and the number of cells increases significantly. The results of the surface area of single cardiomyocytes are shown in Table 4. The surface area of single cardiomyocytes in the model group increased significantly compared with the blank control group, and there was a statistical difference. Compounds of formula I at 0.1 μM, 0.5 μM, and 1 μM and compounds of formula II at 0.1 μM, 0.5 μM, and 1 μM can significantly inhibit the hypertrophy of cardiomyocytes caused by Ang II, and the surface area of individual cardiomyocytes is significantly reduced compared with the model group. Diltiazem 10 μM also significantly reduced Ang II-induced cardiomyocyte hypertrophy. The inhibitory activity of the compounds of formula I and formula II on cardiomyocyte hypertrophy at doses of 0.1 μM, 0.5 μM, and 1 μM was better than that of the positive drug diltiazem 10 μM.

Table 4 Effect of Example Compounds on Cardiomyocyte Hypertrophy - Single Cardiomyocyte Surface Area (μM ² )

^*** P<0.01 compared with the blank control group; ^### P<0.01 compared with the model group.

Experimental Example 5: Detection of Example Compounds Using Isoproterenol (ISO)-Induced Ischemic Heart Disease Mouse Model

experimental method:

After adapting to the environment, male C57BL/6 mice were randomly divided into 9 groups, namely, blank control group, ISO model group, ISO+diltiazem 20mg/kg positive control drug group, ISO+Formula I (0.1, 0.5, 2mg/kg) group, ISO+ Formula II (0.1, 0.5, 2mg/kg) group. The mice in the administration group were intragastrically administered with corresponding doses of compounds of formula I and formula II once a day at the same time every day. The positive drug diltiazem group was intragastrically administered with corresponding doses of diltiazem once a day. The mice in the blank control group and model group were administered The same amount of solvent (0.5% CMC-Na) was administered at a dose of 10 mL/kg for 11 consecutive days. ISO modeling was carried out on the 4th to 10th day after administration. The blank control group was subcutaneously injected with normal saline, and the mice in the other groups were injected subcutaneously with ISO 40 mg/kg (all models were established 2 hours after administration), a total of 7 times.

Detection of cardiac hypertrophy/heart failure biomarkers: After the administration, the animals' eyeballs were removed to collect blood. The blood was allowed to stand at room temperature for 2 hours, and the serum was separated by centrifugation at 3500 rpm for 20 minutes. The serum ANP and NT-proBNP contents were measured according to the instructions of the ELISA kit.

Cardiac enzyme CK-MB detection: After the administration, the animals' eyes were removed to collect blood. The blood was allowed to stand at room temperature for 2 hours, and the serum was separated by centrifugation at 3500 rpm for 20 minutes. The serum CK-MB content was measured according to the instructions of the ELISA kit.

Detection of calcium ions in myocardial tissue: After the experiment, take part of the myocardial tissue and prepare 10% myocardial tissue homogenate with ultrapure water, centrifuge at 4°C and 1200 rpm for 20 min, and separate the supernatant of the myocardial tissue homogenate. Determine the calcium ion content according to the instructions of the kit; use the BCA protein detection method for protein quantification.

Pathological examination of myocardial tissue: After the experiment, the heart was washed with ice-cold saline and dried with absorbent paper, cut into two halves transversely, and the one-third part near the apex was fixed in 4% paraformaldehyde solution and dehydrated through gradient ethanol. , xylene is transparent, and then soaked in dissolved paraffin three times for embedding, 30 minutes each time, and then serially sectioned (thickness is about 4 μm) after embedding. Conventional hematoxylin-eosin (H.E.) staining. Observe the pathological status of myocardial tissue under an optical microscope, take pictures, and analyze.

Experimental results:

Cardiac hypertrophy/heart failure serum biomarker results:

Serum N-terminal B-type natriuretic peptide precursor (NT-proBNP) is a biomarker for early diagnosis of heart failure, and its reduced level is also strong evidence that drug treatment is effective. Atrial natriuretic peptide (ANP) is another serum-sensitive biomarker of heart failure. Current studies also show that NT-proBNP and ANP levels are positively correlated with the degree of myocardial hypertrophy and can be used as biomarkers of myocardial hypertrophy. ELISA kits were used to detect serum NT-proBNP and ANP levels, and the results are shown in Table 5. The serum NT-proBNP and ANP levels of the animals in the model group were significantly higher than those in the blank control group, indicating that the mice had serious heart damage, the possibility of heart failure, and the presence of cardiac hypertrophy symptoms. Formula II 0.1mg/kg, 0.5mg/kg, and 2mg/kg can significantly reduce serum NT-proBNP and ANP levels. Formula I 0.1mg/kg, 0.5mg/kg, and 2mg/kg can also significantly reduce serum ANP levels, and There are statistical differences between model groups. The three dosage groups of Formula I and Formula II have a better reducing effect on ANP than the positive control drug diltiazem 20 mg/kg. The three dosage groups of Formula II have a lowering effect on NT-proBNP than the positive control drug diltiazem 20 mg/kg.

Table 5 Effects of formula I and formula II on serum myocardial hypertrophy/heart failure biomarker NT-proBNP and ANP contents in mice with isoprenaline (ISO)-induced ischemic heart disease (n=8-9)

^* P<0.05, ^*** P<0.001 compared with the blank control group; ^# P<0.05, ^## P<0.01, ^### P<0.001 compared with the model group.

Myocardial injury serum biomarker cardiac enzyme CK-MB results:

Serum creatine kinase-isoenzyme (CK-MB) is another clinically specific and sensitive indicator for judging myocardial necrosis. The results are shown in Table 6. The serum cardiac enzyme CK-MB levels of mice in the ISO model group were significantly higher than those in the blank control group, further suggesting the existence of cardiac damage. Formula I 0.5mg/kg, Formula II 0.5mg/kg and 2mg/kg can significantly reduce serum CK-MB levels, with statistical differences compared with the model group. Formula I 0.1mg/kg, 2mg/kg, and Formula II 0.1mg/kg also tend to reduce serum CK-MB levels. The reducing effects of Formula I and Formula II on CK-MB are better than the positive control drug diltiazem 20mg/kg.

Table 6 Effects of formula I and formula II on serum cardiac enzyme CK-MB content in mice with isoprenaline (ISO)-induced ischemic heart disease (n=8-9)

^* P<0.05 compared with the blank control group; ^# P<0.05, ^## P<0.01 compared with the model group.

Myocardial tissue calcium ion concentration results:

Cardiomyocyte calcium ions (Ca ²⁺ ) play an important role in cardiomyocyte excitation-contraction coupling, and calcium overload is one of the important mechanisms in ISO-induced cardiac hypertrophy injury. The results are shown in Table 7. The Ca ²⁺ content in the myocardial tissue of mice in the ISO model group was significantly higher than that in the blank control group, and the calcium overload in the myocardial tissue was obvious. The three dose groups of Formula I and Formula II can significantly reduce the Ca ²⁺ content in myocardial tissue. The positive control drug diltiazem 20 mg/kg can also significantly reduce the Ca ²⁺ content in myocardial tissue. The improvement effect of tissue calcium overload is better than that of the positive control drug diltiazem 20mg/kg.

Table 7 Effects of Formula I and Formula II on Ca ²⁺ content in myocardial tissue of mice with isoproterenol (ISO)-induced myocardial ischemia (n=8-9)

^*** P<0.001, compared with the blank control group; ^## P<0.01, ^### P<0.001, compared with the model group.

Myocardial tissue pathological examination results:

The H&E staining results of myocardial tissue are shown in Figure 2. The myocardial tissue fibers of the mice in the blank control group were neatly arranged, the structure was clear and regular lines could be seen, the nuclei were clear, the cytoplasm was rich and uniform, and inflammatory cell infiltration was rare. The ISO model group of mice showed significant hypertrophy of the ventricular septum and papillary muscle cardiomyocytes, and myocardial cell edema and degeneration; local ventricular septum and papillary muscle myocardial tissue damage (myocardial cell necrosis) was severe, and the damage (necrosis) lesions were distributed in patches. They are connected to each other, and transmural myocardial tissue damage to the ventricular septum and papillary muscles can be seen locally; inflammation is significant at the lesion site, and significant proliferation of fibrous tissue can be seen at the injury site. Intragastric administration of three doses of formula I and formula II can significantly improve the pathological damage of mouse myocardial tissue caused by ISO, reduce myocardial cell edema and hypertrophy, reduce the degree of myocardial cell necrosis, and reduce the degree of fibrous tissue proliferation. The results of pathological scoring of cardiac tissue are shown in Table 8. The three doses of Formula I and Formula II significantly reduced the number of animals with severe myocardial tissue lesions and reduced the average lesion severity score. The positive control drug diltiazem 20mg/kg can also significantly reduce the degree of myocardial tissue lesions. Formula I 2mg/kg and formula II 2mg/kg are better than the positive control drug diltiazem 20mg/kg in improving the degree of myocardial tissue lesions.

Table 8 Effects of Formula I and Formula II on the degree of myocardial tissue lesions in mice with isoproterenol (ISO)-induced myocardial ischemia (H&E 200×)

^*** P<0.001, compared with the blank control group; ^### P<0.001, compared with the model group.

Attached are pathological scoring standards:

-:No abnormalities.

+: No damage to myocardial tissue, local ventricular septum and papillary muscle cardiomyocyte hypertrophy, local myocardial cell edema and degeneration, local muscle gap widening at the lesion site, and mild non-specific inflammation in the interstitium.

++: No obvious myocardial tissue damage (myocardial cell necrosis), local ventricular septum and papillary muscle cardiomyocyte hypertrophy, local myocardial cell edema and degeneration; local myofibrillar dissolution (striations disappear) can be seen, and the lesions are first localized. , distributed in a small focus shape; non-specific inflammation and local interstitial fibrous tissue hyperplasia were seen in the interstitium.

+++: Obvious hypertrophy of ventricular septum and papillary muscle cardiomyocytes, local myocardial cell edema and degeneration; local ventricular septum or papillary muscle myocardial tissue damage (myocardial cell necrosis, only one part of the sample is found), and the disease is limited to the local ventricular septum. Under the membrane (or papillary muscle), the lesion is localized and the scope of involvement is small; non-specific inflammation and local interstitial fibrous tissue hyperplasia can be seen in the interstitium of the lesion.

++++: Significant hypertrophy of ventricular septum and papillary muscle myocardial cells, local myocardial cell edema and degeneration; obvious local damage to the ventricular septum and papillary muscle myocardial tissue (myocardial cell necrosis), and the lesions are distributed in a focal (or small patch) shape , the scope of involvement is large; the inflammation in the lesion is obvious, and the local fibrous tissue is obviously hyperplasia.

+++++: The ventricular septum and papillary muscle cardiomyocytes are significantly hypertrophied, with local myocardial cell edema and degeneration; the local ventricular septum and papillary muscle myocardial tissue damage (myocardial cell necrosis) is severe, and the damage (necrosis) lesions are distributed in patches, and They are connected to each other, and transmural myocardial tissue damage of the ventricular septum and papillary muscles can be seen locally; inflammation is significant at the lesion site, and significant proliferation of fibrous tissue can be seen at the injury site.

Experimental Example 6: Detection of Example Compounds Using Angiotensin II (AngII)-Induced Hypertensive Heart Disease Mouse Model

experimental method:

After adapting to the environment, male C57BL/6 mice were randomly divided into 10 groups, namely, blank control group, AngⅡ model group, AngⅡ+telmisartan 10 mg/kg positive control group, AngⅡ+spironolactone 20 mg/kg positive control group, AngⅡ+ Formula I (0.1, 0.5, 2 mg/kg) group and Ang II + Formula II (0.1, 0.5, 2 mg/kg) group. The mice in the administration group were intragastrically administered the corresponding doses of the compounds of formula I and formula II once a day at the same time every day. The positive control group was intragastrically administered the corresponding doses of telmisartan and spironolactone once a day. The blank control group and the model group were intragastrically administered once a day. Mice were given the same amount of solvent (0.5% CMC-Na solution), the dosage was 10 mL/kg, for 14 consecutive days. AngⅡ modeling was performed on days 1 to 13 after administration. The blank control group was subcutaneously injected with normal saline, and the mice in the other groups were injected subcutaneously with AngⅡ 1.44 mg/kg (all models were established 2 hours after administration), a total of 13 times.

Blood pressure monitoring: The blood pressure of mice was measured on days 0, 7, and 13 of drug administration. Three mice were randomly selected from each group. The mice were placed in a holder to expose their tails. The holder was placed on a heating pad to maintain the mice. At normal body temperature, put the root of the rat tail through the pressurized tail cuff and fix it, start the inflation and pressurization, and use a measuring instrument to measure SBP. The blood pressure index of each animal was measured five times continuously and the average value was taken.

Echocardiography detection: On the 14th day of administration (13 times of Ang II modeling), 5 animals from each group were randomly selected, fixed after anesthesia, shaved chest hair, and applied a small amount of coupling agent on the chest. Vevo 770 Imaging System small animal ultrasound real-time imaging system was used to perform echocardiographic examination. All animal cardiogram indicators were measured continuously for 3 cardiac cycles and the average value was taken.

Detection of cardiac hypertrophy/heart failure biomarkers: After the experiment, the animals' eyeballs were removed to collect blood. The blood was left at room temperature for 2 hours, centrifuged at 3500 rpm for 20 minutes to separate the serum, and the serum BNP content was measured according to the instructions of the ELISA kit.

Detection of serum biomarkers of myocardial injury: After the experiment, the animals' eyeballs were removed to collect blood. The blood was left at room temperature for 2 hours, centrifuged at 3500 rpm for 20 minutes to separate the serum, and the serum troponin cTn-T and cardiac enzyme CK-MB contents were measured according to the instructions of the ELISA kit. .

Detection of serum inflammatory factors: After the experiment, the animals' eyeballs were removed to collect blood. The blood was left at room temperature for 2 hours, centrifuged at 3500 rpm for 20 minutes to separate the serum, and the serum TNF-α content was measured according to the instructions of the ELISA kit.

Serum aldosterone (ALD) level detection: After the experiment, the animals' eyeballs were removed to collect blood. The blood was left at room temperature for 2 hours, centrifuged at 3500 rpm for 20 minutes to separate the serum, and the serum ALD content was measured according to the instructions of the ELISA kit.

Serum cAMP level detection: After the experiment, the animals' eyes were removed to collect blood. The blood was allowed to stand at room temperature for 2 hours, and the serum was separated by centrifugation at 3500 rpm for 20 minutes. The serum cAMP content was measured according to the instructions of the ELISA kit.

Experimental results:

Blood pressure monitoring results:

The results are shown in Table 9. There was no significant difference in the basal systolic blood pressure (SBP value) of mice in each group before administration; after continuous stimulation with angiotensin II (AngII) for 7 days and 14 days, the SBP of mice in the model group increased significantly, which was statistically significant compared with the blank control group. learning differences. The three dose groups of Formula I and Formula II all significantly reduced SBP values, and there were statistical differences compared with the model group. The positive control drugs of spironolactone and telmisartan can also significantly reduce SBP values. The activity of reducing SBP in each dose group of formula I and formula II is equivalent to that of spironolactone 20mg/kg; the SBP value of mice after administration of telmisartan 10mg/kg group Lower than the animals in the blank control group. The SBP value on the 7th day was significantly lower than that in the blank control group, indicating a state of hypotension. This is consistent with clinical reports. Taking ARB drugs such as telmisartan may induce transient hypotension. Side effects: Formula I and Formula II do not have such adverse effects.

Table 9 Effects of Formula I and Formula II administration on SBP in mice with angiotensin II (Ang II)-induced hypertensive heart disease (n=3)

^** P<0.01, compared with the blank control group; ^# P<0.05, ^## P<0.01, ^### P<0.001, compared with the model group;

^& P<0.05, compared with the blank control group.

Echocardiography results:

[Correction 07.09.2023 under Rule 91]
Small animal echocardiography is a simple and reproducible technique for evaluating cardiac function in mice. This technology was used to obtain M-mode echocardiogram, as shown in Figure 3, and the data were measured and calculated. Some data are shown in Table 10. Continuous administration of Ang II leads to significant left ventricular dysfunction, causing a significant increase in left ventricular wall thickness during systole and diastole, and manifesting obvious symptoms of cardiac hypertrophy. The three dosage groups of Formula I and Formula II can significantly reduce the thickness of the left ventricular posterior wall during systole and diastole, with statistical differences compared with the model group, and significantly improve cardiac hypertrophy. The positive control drugs spironolactone and telmisartan can also significantly reduce left ventricular posterior wall thickness during systole and diastole. The three dose groups of Formula I and Formula II have better improving activity on cardiac hypertrophy than spironolactone 20mg/kg.

Table 10 Effects of Formula I and Formula II administration on echocardiographic data in mice with angiotensin II (Ang II)-induced hypertensive heart disease (n=5)

^** P<0.01, ^*** P<0.001 compared with the blank control group; ^# P<0.05, ^## P<0.01, ^### P<0.001 compared with the model group.

Cardiac Hypertrophy/Heart Failure Biomarker Results:

Serum brain natriuretic peptide (BNP) is another important biomarker for the early diagnosis of heart failure. It is also positively correlated with the degree of myocardial hypertrophy and is one of the biomarkers of myocardial hypertrophy. The results are shown in Table 11. The serum BNP levels of mice in the AngII model group were significantly higher than those in the blank control group. The three doses of formula I and formula II all significantly reduced serum BNP levels, and there were statistical differences compared with the model group. Spironolactone and telmisartan positive control drugs can also significantly reduce serum BNP levels. The three dosage groups of Formula I and Formula II have better BNP-lowering effects than the positive control drug spironolactone 20 mg/kg.

Table 11 Effects of administration of formula I and formula II on serum myocardial hypertrophy/heart failure biomarker BNP content in mice with angiotensin II (Ang II)-induced hypertensive heart disease (n=7-9)

^*** P<0.001 compared with the blank control group; ^# P<0.05, ^### P<0.001 compared with the model group.

Myocardial injury serum biomarker results:

When myocardial cells are necrotic, some protein substances contained in the myocardium will be released from the myocardial tissue and appear in the peripheral circulation blood, which can be used as a judgment indicator of myocardial damage. Troponin (Tn) is a regulatory protein for cardiac tissue contraction. Cardiac troponin (cTn) is different in molecular structure and immunology from Tn in skeletal muscle. Therefore, it is unique to the myocardium and is the most effective way to judge myocardial necrosis. Specific and sensitive marker of choice. There are three subtypes of cTn, cTn-T, cTn-I, and cTn-C. cTn-T has high diagnostic value for both early and late myocardial necrosis. The results are shown in Table 12. The serum myocardial injury marker protein cTn-T of mice in the model group was significantly increased compared with the blank control group. Administration of three doses of Formula I and Formula II could significantly reduce serum cTn-T levels. There is a statistical difference compared with the model group. Spironolactone and telmisartan positive control drugs can also significantly reduce serum cTn-T levels. The cTn-T reducing effect of formula I is equivalent to that of spironolactone 20 mg/kg, and the cTn-T reducing effect of formula II is better than spironolactone 20 mg/kg.

Serum creatine kinase-isoenzyme (CK-MB) is another clinically specific and sensitive indicator for judging myocardial necrosis. The results are shown in Table 12. The serum cardiac enzyme CK-MB level of mice in the AngII model group was significantly higher than that of the blank control group. Three doses of Formula I and Formula II can significantly reduce serum CK-MB levels. Telmisartan positive control drug can also significantly reduce serum CK-MB levels. Formula I and Formula II have excellent effects on reducing CK-MB. To the positive control drug spironolactone 20mg/kg.

Table 12 Effects of administration of formula I and formula II on serum myocardial damage biomarker cTn-T and CK-MB contents in mice with angiotensin II (Ang II)-induced hypertensive heart disease (n=7-9)

^*** P<0.001 compared with the blank control group; ^# P<0.05, ^### P<0.001 compared with the model group.

Serum inflammatory factor results:

The generation of inflammation is a key factor in the occurrence of cardiovascular diseases. The release of inflammatory factors (such as TNF-α) will directly induce myocardial cell apoptosis and damage the cardiac contractility, and even lead to heart failure. The results are shown in Table 13. The serum inflammatory factor TNF-α level of mice in the model group was significantly higher than that in the blank control group, and the inflammatory response in the body was obvious. Administration of three doses of Formula I and Formula II can significantly reduce serum TNF-α levels, and the positive control drugs of spironolactone and telmisartan can also significantly reduce serum TNF-α levels. The three dose groups of Formula I and Formula II have a lower effect on TNF-α than the positive control drug spironolactone 20mg/kg.

Table 13 Effects of administration of formula I and formula II on serum inflammatory factor TNF-α content in mice with angiotensin II (Ang II)-induced hypertensive heart disease (n=7-9)

^*** P<0.001 compared with the blank control group; ^# P<0.05, ^## P<0.01, ^### P<0.001 compared with the model group.

Result of renin angiotensin aldosterone system (RAAS) effector aldosterone (ALD) content:

AngII binds to cardiac angiotensin receptor 1 (AT1R), stimulating downstream signaling pathways and promoting increased production of aldosterone (ALD). In addition to regulating sodium reabsorption and blood volume, ALD also directly promotes cardiac hypertrophy. . In addition, ALD can further enhance the sensitivity of AngII to the effects of cardiomyocytes by increasing cardiac AT1R density. The results are shown in Table 14. The serum ALD level of mice in the model group was significantly higher than that of the blank control group. The three doses of formula I and formula II could significantly reduce the serum ALD level. The positive control drugs of spironolactone and telmisartan could also significantly reduce the serum ALD level. The three dose groups of Formula I and Formula II have a better effect on reducing ALD than the positive control drug spironolactone 20mg/kg.

Table 14 Effects of formula I and formula II administration on serum ALD content in mice with angiotensin II (Ang II)-induced hypertensive heart disease (n=7-9)

^*** P<0.001 compared with the blank control group; ^# P<0.05, ^## P<0.01, ^### P<0.001 compared with the model group.

Sympathetic nervous system endocrine factor cyclic adenosine monophosphate (cAMP) results:

Overexcitation of the sympathetic nervous system can lead to an excessive increase in cyclic adenosine monophosphate (cAMP) content, induce arrhythmias or activate the signaling pathways of cardiomyocytes, causing cardiac hypertrophy, fibrosis, and even affecting cardiac function. The results are shown in Table 15. The serum cAMP level of mice in the model group was significantly higher than that of the blank control group, indicating excessive increase in the excitability of the sympathetic nervous system. Formula I 0.1mg/kg, 0.5mg/kg, Formula II 0.1mg/kg, 0.5mg/kg, 2mg/kg can significantly reduce serum cAMP levels. Telmisartan positive control drug can also significantly reduce serum cAMP levels. Spironolactone has no significant reducing effect on excessively increased cAMP levels. The reducing effect of the three dosage groups of Formula II on excessively increased cAMP is equivalent to that of the positive control drug Telmisartan 10 mg/kg.

Table 15 Effects of administration of Formula I and Formula II on serum cAMP content in mice with angiotensin II (Ang II)-induced hypertensive heart disease (n=7-9)

^*** P<0.001 compared with the blank control group; ^# P<0.05, ^### P<0.001 compared with the model group.

Claims (8)

1. Three types of methyl Ophiopogon flavonoids A derivatives or pharmaceutically acceptable salts thereof, characterized in that the three types of methyl Ophiopogon flavonoids A derivatives have the structural formulas described in Formula I, Formula II and Formula III:
   
2. The preparation method of methyl Ophiopogon japonicus flavonoid A derivative I according to claim 1, characterized in that it includes the following steps:

   Under alkaline conditions, the compound of formula (2) is prepared by reacting 2,4,6-trihydroxyacetophenone (1) with halomethoxymethyl ether; under alkaline conditions, the formula The compound of formula (2) structure is reacted with piperonal to prepare the compound of formula (3) structure; the double bond of the compound of formula (3) structure is reduced to prepare the compound of formula (4) structure; the compound of formula (4) structure is reacted with The compound of formula I is prepared by reacting phosphorus oxychloride and N,N-dimethylformamide:
   
3. The preparation method of methyl Ophiopogon japonicus flavonoid A derivatives I according to claim 2, characterized in that, in step one, the base is triethylamine or N,N-diisopropylethylamine, so The halomethoxymethyl ether is bromomethoxymethyl ether; in step two, the base is sodium hydride or sodium alkoxide, KOH/EtOH; in step three, the reducing agent is palladium Carbon/hydrogen or stannous chloride.
4. The preparation method of methyl Ophiopogon japonicus flavonoid A derivatives II and III according to claim 1, characterized in that it includes the following steps:

   Reducing 2,4,6-trihydroxybenzaldehyde to 2,4,6-trihydroxytoluene and reacting with acetic anhydride to obtain the complex of formula (5) Compounds with the structure of formula (5) and halogenated methoxymethyl ether are reacted under alkaline conditions to prepare compounds with the structure of formula (6); under alkaline conditions, compounds with the structure of formula (6) react with pepper The compound of formula (7) is prepared by the aldehyde reaction; the double bond of the compound of formula (7) is reduced to prepare the compound of formula (8); the compound of formula (8) is reacted with phosphorus oxychloride, N, N - Compounds of formula II and formula III structures are prepared by reaction of dimethylformamide;
   
5. The method for preparing methyl Ophiopogon japonicus flavonoid A derivatives II and III according to claim 4, characterized in that in step one, the reducing agent is a metal reducing agent or metal borohydride. In step three, the base is triethylamine or N,N-diisopropylethylamine, and the halomethoxymethyl ether is bromomethoxymethyl ether; in step four, The base is sodium hydride or sodium alkoxide, KOH/EtOH; in step five, the reducing agent is palladium carbon/hydrogen or stannous chloride, etc.
6. A pharmaceutical composition, characterized in that it contains a preventive and/or therapeutically effective amount of the methyl Ophiopogon flavonoid A derivative described in claim 1 or a pharmaceutically acceptable salt thereof, and optionally one or A variety of pharmaceutically acceptable carriers or excipients.
7. The application of the methyl Ophiopogon flavonoid A derivative of claim 1 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 6 in the preparation of drugs for preventing and/or treating cardiovascular-related diseases.
8. The application according to claim 7, characterized in that the cardiovascular-related diseases are selected from one or more of the following: myocardial ischemia, myocardial damage, myocardial hypertrophy, coronary heart disease, hypertension, heart failure, myocardial disease, arrhythmia, myocardial infarction, angina pectoris.