A c-19 modified isorisantamarin derivative and its preparation method and application

Abstract

The application discloses a C-19 modified isosteviol derivative and a preparation method and application thereof, aims to provide an isosteviol derivative with a heart protection activity, transforms a C-19 position of isosteviol, converts a C-19 position carboxyl into a primary amine or isocyanic acid by using a Curtius rearrangement, and further modifies the primary amine to introduce a fatty acid, aromatic acid fragment to convert into an amide fragment, or converts the isocyanic acid into a urea fragment, obtains a series of novel nitrogen-containing isosteviol derivatives by a strategy of reducing one carbon atom, and the nitrogen-containing isosteviol derivatives show good heart protection activity on a zebra fish model of myocardial injury, and can be used for preparing heart protection drugs, and the application relates to the technical field of medicines.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to a isosteviol derivative, and the present application also relates to a preparation method and application of the C-19 modified isosteviol derivative. BACKGROUND

[0002] In recent years, with the aggravation of population aging and the widespread prevalence of cardiovascular risk factors, cardiovascular disease, as the leading cause of global mortality, has become a severe public health burden and a major medical problem to be solved in China. Drug research and development for this disease has great scientific significance and clinical application value in reducing social medical burden and improving patient prognosis. Under this background, it is particularly urgent to actively promote the research and development of heart protection drugs, and natural products, as an important source of drug discovery, have shown great potential in the development of cardiovascular disease treatment drugs due to their unique chemical structure and physiological activity, providing a rich material basis for the discovery of innovative drugs.

[0003] Isosteviol belongs to the tetracyclic diterpenoid of the betulane type, which can be obtained by hydrolysis of cheap steviol glycosides under acidic conditions and high yield by Wagner-Meerwein rearrangement. Isosteviol has a wide range of biological activities, such as anti-tumor, anti-viral, anti-bacterial, anti-thrombus, hypoglycemic, cardiovascular protection, etc. At present, the structural modification of isosteviol for heart protection activity is still very limited, so the development of new isosteviol derivatives with heart protection activity has important scientific significance and research value. SUMMARY

[0004] One of the purposes of the present application is to provide an isosteviol derivative with heart protection activity.

[0005] The second purpose of the present application is to provide a preparation method of the C-19 modified isosteviol derivative.

[0006] The third purpose of the present application is to provide the application of the isosteviol derivative in the preparation of heart protection drugs.

[0007] To this end, the first technical solution provided by the present application is as follows:

[0008] A C-19 modified isosteviol derivative has the structure of formula 1:

[0009] ;

[0010] Formula 1;

[0011] In which:

[0012] R1 is one of methyl, 3,4-(methylenedioxy)benzyl, 3-pyridyl, 4-(1,2,3-trimethoxybenzyl), N-ethyl, N-isopropyl, tetrahydropyrrolyl, N-thiazol-2-yl, N-cyclohexyl, N-p-tolyl, N-p-fluorophenyl, N-p-methoxyphenyl, N-(3,4,5-trimethoxybenzyl), N-(3,4-methylenedioxybenzyl), N-(4-pyridyl), N-(3-pyridyl), N-benzyl.

[0013] Further, the above-mentioned isosteviol derivative, the specific structural formula is as follows:

[0014] .

[0015] The application also has a technical scheme to provide the preparation method of the above-mentioned C-19 modified isosteviol derivative, which comprises the following steps in sequence:

[0016] 1) Dissolve isosteviol, diphenyl phosphoryl azide and catalyst A in solvent A, heat to 70-90 DEG C reflux reaction for 8-10 h, cool the reaction liquid to room temperature, heat the dilute hydrochloric acid solution to 40-60 DEG C reflux reaction for 6-8 h, and obtain compound 2 after post-treatment;

[0017] 2) Dissolve compound 2 prepared in step 1), 1-ethyl-3-(3-dimethylammonium propyl) carbon ammonium, catalyst B, 1-hydroxybenzotriazole and carboxylic acid compound in solvent B, stir at room temperature for 1.5-2.5 h, and obtain one of compounds 3a-3d after post-treatment;

[0018] The molar ratio of isosteviol and diphenyl phosphoryl azide in step 1) is 1.2:2.4;

[0019] The molar ratio of compound 2, 1-ethyl-3-(3-dimethylammonium propyl) carbon ammonium, 1-hydroxybenzotriazole and carboxylic acid compound in step 2) is 1.73:2.6:1.73:2.1.

[0020] The synthetic route is as follows:

[0021] .

[0022] Further, the above-mentioned preparation method of C-19 modified isosteviol derivative, the solvent A is tert-butyl alcohol; the solvent B is dichloromethane; the catalyst A is triethylamine; the catalyst B is 4-dimethylaminopyridine; the concentration of the dilute hydrochloric acid solution is 5 N; the carboxylic acid compound is one of acetic acid, 3,4-(methylenedioxy)benzyl, 3-pyridyl, 4-(1,2,3-trimethoxybenzyl), N-ethyl, N-isopropyl, tetrahydropyrrolyl, N-thiazol-2-yl, N-cyclohexyl, N-p-tolyl, N-p-fluorophenyl, N-p-methoxyphenyl, N-(3,4,5-trimethoxybenzyl), N-(3,4-methylenedioxybenzyl), N-(4-pyridyl), N-(3-pyridyl), N-benzyl.

[0023] Furthermore, in the preparation method of the above-mentioned C-19-modified isosteviol derivative, the post-treatment described in step 1) is to add an equivalent amount of sodium hydroxide solution to the system after the reaction in step 1), extract with dichloromethane, then wash with saturated brine to remove inorganic matter, dry with anhydrous sodium sulfate and concentrate, and then use a silica gel column to separate and purify the mixture to obtain compound 2; the post-treatment described in step 2) is to add a mixed solution of dichloromethane and saturated brine in a volume ratio of 10:1 to the system after the reaction for extraction, collect the organic layer, dry over anhydrous sodium sulfate and concentrate, and then use a silica gel column to separate and purify the mixture to obtain one of compounds 3a-3d.

[0024] Another technical solution of the present invention is to provide another method for preparing the above-mentioned C-19 modified isosteviol derivative, which comprises the following steps in sequence:

[0025] 1) Dissolve isosteviol, diphenylphosphoryl azide, and catalyst A in solvent C, heat to 80-100°C and reflux for 8-10 hours, cool the reaction solution to room temperature, and post-treat to obtain compound 4;

[0026] 2) Compound 4 prepared in step 1), catalyst A, and an amine compound are added to solvent D, stirred at room temperature for 3-5 hours, and after completion of the reaction, treated to obtain one of compounds 5a-5m;

[0027] The molar ratio of isosteviol and diphenylphosphoryl azide in step 1) is 0.39:0.79;

[0028] The molar ratio of compound 4 and the amine compound in step 2) is 0.36:0.54.

[0029] Its synthetic route is as follows:

[0030]

[0031] .

[0032] Furthermore, in the preparation method of the above-mentioned C-19-modified isosteviol derivative, the amine compound is one of ethylamine, isopropylamine, tetrahydropyrrole, 2-aminothiazole, cyclohexylamine, p-toluidine, p-fluoroaniline, p-methoxyaniline, 3,4,5-trimethoxyaniline, 3,4-methylenedioxyaniline, 4-aminopyridine, 3-aminopyridine, and benzylamine; the solvent C is anisole; and the solvent D is tetrahydrofuran.

[0033] Furthermore, in the preparation method of the above-mentioned C-19-modified isosteviol derivative, the post-treatment described in step 1) is to add ethyl acetate to the reaction system to extract the product, then wash it with saturated brine to remove inorganic matter, dry it with anhydrous sodium sulfate, and then concentrate it; then use a silica gel column to separate and purify the product to obtain compound 4; the post-treatment described in step 2) is to add ethyl acetate to the reaction system to extract the product, then wash it with saturated brine to remove inorganic matter, dry it with anhydrous sodium sulfate, and then concentrate it, and then use a silica gel column to separate and purify the mixture to obtain one of compounds 5a-5m.

[0034] Another technical solution of the present invention is to provide a cardioprotective active drug, including the C-19 modified isosteviol derivative described in the first technical solution.

[0035] Furthermore, the above-mentioned cardioprotective active drug also includes pharmaceutically acceptable excipients; the excipients are at least one of a binder, a disintegrant, a lubricant, a filler, a surfactant, an antioxidant or a pH regulator; the dosage form of the drug is an injection, a tablet or an oral solution, a granule or a capsule.

[0036] Compared to existing technologies, the present invention modifies the C-19 position of isosteviol, utilizing Curtis rearrangement to convert the C-19 carboxyl group into a primary amine or isocyanate. The primary amine is then further modified to introduce fatty acid or aromatic acid fragments, or the isocyanate is converted into a urea fragment. This strategy, by reducing one carbon atom, yields a series of novel nitrogen-containing isosteviol derivatives. This series of novel structural derivatives significantly improved the survival rate of zebrafish with myocardial injury in a doxorubicin-induced myocardial injury model, improved myocardial injury, restored cardiac aberrations, enhanced systolic and diastolic function in damaged hearts, and effectively reduced mRNA levels of the myocardial injury markers ANP, BNP, and cTnT. Compared to the parent compound, isosteviol, these derivatives exhibited significantly enhanced anti-myocardial injury activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Compound 5j 1 H NMR spectrum;

[0038] Figure 2 Compound 5j 13 C-NMR spectrum;

[0039] Figure 3 For compound 5l 1 H NMR and spectra;

[0040] Figure 4 For compound 5l 13 C-NMR spectrum;

[0041] Figure 5This figure shows the effect of isosteviol derivatives on the survival rate of zebrafish embryos induced by doxorubicin;

[0042] Figure 6 The figure shows the effects of different concentrations of isosteviol derivatives on the survival rate of zebrafish embryos induced by doxorubicin and the toxicity of zebrafish embryos;

[0043] Figure 7 This figure shows the evaluation of isosteviol derivatives with excellent cardioprotective activity on doxorubicin-induced zebrafish cardiac function;

[0044] Among them: (A) Representative optical microscope image of zebrafish; (B) Representative fluorescence microscope image of zebrafish heart; (C) Heart rate statistics; (D) Fractional shortening statistics; (E) Stroke volume statistics; (F) Cardiac output statistics.

[0045] Figure 8 This figure shows the effects of isosteviol derivatives with excellent cardioprotective activity on the relative mRNA levels of adriamycin-induced cardiomyopathy biomarkers ANP, BNP and cTnT in zebrafish embryos. DETAILED DESCRIPTION

[0046] The present invention will be further explained below in conjunction with implementation and accompanying drawings, but implementation does not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the present invention are commercially available.

[0047] Example 1 Synthesis of Compounds 3a-3d

[0048] As shown in Synthesis Route 1, isosteviol is used as a starting material, isosteviol is dissolved in tert-butanol, diphenylphosphoryl azide and triethylamine are added, and the mixture is heated under reflux to react to produce compound 2. Compound 2 is then reacted with 1-ethyl-3-(3-dimethylammoniumpropyl)carbonium, 4-dimethylaminopyridine, 1-hydroxybenzotriazole, and a carboxylic acid compound in a dichloromethane solvent to produce compounds 3a-3d.

[0049] Reaction conditions and reagents in Synthesis Route 1: (a) diphenylphosphoryl azide, triethylamine, tert-butanol, heating under reflux; (b) carboxylic acid compound, 1-ethyl-3-(3-dimethylammoniumpropyl)carbonate, 4-dimethylaminopyridine, 1-hydroxybenzotriazole, anhydrous dichloromethane.

[0050]

[0051] Synthesis route 1.

[0052] The specific steps are as follows:

[0053] (1) Synthesis of compound 2

[0054] Isosteviol (374 mg, 1.2 mmol), diphenylphosphoryl azide (0.52 mL, 2.4 mmol), and triethylamine (0.17 mL, 1.2 mmol) were added to tert-butanol (4 mL) and heated under reflux at 80°C for 8-10 h. The reaction was monitored by TLC until completion. The mixture was cooled to room temperature, and 1 mL of 5 N dilute hydrochloric acid was added. The mixture was heated under reflux at 50°C for 6-8 h. The reaction was monitored by TLC until completion. An equivalent amount of sodium hydroxide solution was added, and the mixture was extracted with an appropriate amount of dichloromethane. The mixture was then washed with saturated brine to remove inorganic matter, dried over anhydrous sodium sulfate, and concentrated. The mixture was separated and purified using a silica gel column (n-hexane:acetone = 7:3) to obtain Compound 2 (49 mg, 15%) as a white solid. The NMR spectrum of Compound 2 is shown below:

[0055] Compound 2: white solid: mp 122.5–123.9 °C; [α] 25 D -70 ( c 1.0, CH3OH); 1 H NMR (CDCl3, 400 MHz) δ 2.72 (1H, dd, J = 3.8, 18.7 Hz, H-15), 1.08 (3H, s, H-18),1.01 (3H, s, H-17), 0.98 (3H, s, H-20), 1.57-0.87 (21H, m, CH, CH2 in beylene skeleton); 13 C NMR (CDCl3, 100 MHz) δ 222.7, 55.2, 54.6, 51.3, 48.9, 48.8, 42.3,41.0, 39.3, 39.1, 37.5, 37.3, 33.3, 31.5, 30.1, 19.9, 19.9, 19.9, 19.4, 17.7,15.0.

[0056] (2) Synthesis of compounds 3a-3d

[0057] Compound 2 (500 mg, 1.73 mmol), 1-ethyl-3-(3-dimethylammoniumpropyl)carbonium (497 mg, 2.6 mmol), 4-dimethylaminopyridine (106.14 mg, 0.87 mmol), 1-hydroxybenzotriazole (234 mg, 1.73 mmol) and a carboxylic acid compound (2.1 mmol) were dissolved in 10 ml of dichloromethane and stirred at room temperature for 2 h. The reaction progress was monitored by TLC. A mixed solution of dichloromethane and saturated brine in a volume ratio of 10:1 was added for extraction. The organic layer was collected, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain compounds 3a-3d.

[0058] Among them: the carboxylic acid compound used in the synthesis of 3a is acetic acid, the carboxylic acid compound used in 3b is 3,4-(methylenedioxy)phenylacetic acid, the carboxylic acid compound used in 3c is 3-pyridinecarboxylic acid, and the carboxylic acid compound used in 3d is 3,4,5-trimethoxybenzoic acid.

[0059] The NMR spectra of compounds 3a-3d are shown below:

[0060] Compound 3a: 1 H NMR (CDCl3, 400 MHz) δ 5.16 (1H, s, CO-NH), 2.80 (1H, d, J =14.0 Hz, H-3), 2.65 (1H, dd, J = 3.8, 18.5 Hz, H-15), 1.92 (3H, s, CO-CH3), 1.42 (3H, s, H-18), 0.99 (6H, d, J = 4.0 Hz, H-17, H-20), 1.66-0.99 (18H, m,CH, CH2 in beylene skeleton); 13 C NMR (CDCl3, 100 MHz) δ 222.1, 169.2, 56.2, 55.9,55.2, 54.3, 48.8, 40.8, 39.2, 39.0, 37.5, 37.1, 35.7, 31.4, 26.9, 25.0, 19.8,19.4, 17.6, 14.8, 14.1.

[0061] Compound 3b: 1H NMR (CDCl3, 400 MHz) δ 6.74 (3H, overlapping, Ph-H), 5.99 (2H, m, O-CH2-O), 5.24 (1H, s, NH), 3.44 (2H, s, CO-CH2), 2.74 (1H, dq, J = 2.7, 15.2Hz, H-3), 2.48 (1H, dd, J = 3.8, 18.5 Hz, H-15), 1.41 (3H, s, H-18), 0.98 (3H,s, H-17), 0.47 (3H, s, H-20), 1.60-0.47 (18H, m, CH, CH2 in beylene skeleton); 13 C NMR (CDCl3, 100 MHz) δ 222.2, 170.2, 148.3, 147.0, 128.8, 122.9, 122.5, 109.9,108.8, 101.3, 101.0, 56.3, 55.8, 55.0, 54.3, 48.7, 48.6, 44.9, 40.8, 39.1, 38.8, 37.2, 37.1, 35.5, 27.0, 20.0, 19.1, 17.6, 14.0.

[0062] Compound 3c: 1 H NMR (CDCl3, 400 MHz) δ 8.90 (1H, d, J = 2.3 Hz, 2-Pyr),8.72 (1H, dd, J = 1.7, 4.9 Hz, 4-Pyr), 8.08 (1H, dt, J = 2.0, 8.0 Hz, 6-Pyr),7.42 (1H, dd, J = 4.8, 8.0 Hz, 5-Pyr), 5.96 (1H, s, NH), 2.95 (1H, dq, J = 2.8,14.3 Hz, H-3), 2.69 (1H, dd, J = 3.8, 18.5 Hz, H-15), 1.56 (3H, s, H-18), 1.10 (3H, s, H-17), 1.01 (3H, s, H-20), 1.74-1.01 (18H, m, CH, CH2 in beylene skeleton); 13C NMR (CDCl3, 100 MHz) δ 221.9, 164.2, 151.7, 147.1, 135.1, 131.7, 123.8,56.8, 56.3, 55.2, 54.3, 48.7, 48.7, 40.8, 39.2, 38.8, 37.6, 37.1, 35.7, 27.0,19.9, 19.8, 19.5, 17.7, 15.3.

[0063] Compound 3d: 1H NMR (CDCl3, 400 MHz) δ 7.83 (2H, overlapping, NH, 6-Ph), 6.77 (1H, d, J = 9.0 Hz, 5-Ph), 3.91 (9H, overlapping, 3×OCH3), 3.00 (1H, dq, J = 2.8,13.8 Hz, H-3), 2.74 (1H, dd, J = 3.7, 18.5 Hz, H-15), 1.54 (3H, s, H-18),1.00 (6H, overlapping, H-17, H-20), 1.73-0.93 (18H, m, CH, CH2 in beylene skeleton); 13CNMR (CDCl3, 100 MHz) δ 222.3, .8, 37.2, 36.4, 27.2, 19.9, 19.8, 19.6, 17.8, 14.9.

[0064] Table 1 Structural formulas of compounds 3a-3d

[0065]

[0066] Example 2 Synthesis of Compounds 5a-5m

[0067] As shown in Synthesis Route 2, isosteviol is used as a starting material, isosteviol is dissolved in anisole, diphenylphosphoryl azide and triethylamine are added, and the mixture is heated under reflux to react to produce compound 4. Compound 4 is then reacted with triethylamine and an amine compound in tetrahydrofuran solvent at room temperature to produce compounds 5a-5m.

[0068] Reaction conditions and reagents in synthetic route 2: (a) diphenylphosphoryl azide, triethylamine, anisole, heat reflux; (b) amine compound, triethylamine, tetrahydrofuran.

[0069]

[0070] Synthetic route 2.

[0071] The specific steps are as follows:

[0072] (1) Synthesis of compound 4

[0073] Diphenylphosphoryl azide (0.17 mL, 0.79 mmol) and triethylamine (0.54 mL, 0.39 mmol) were added to anisole (4 mL), and heated to reflux at 90°C for 8-10 h. TLC plate was used to monitor the progress until the reaction was completed. After cooling to room temperature, an appropriate amount of ethyl acetate was added to extract, and then washed with saturated brine to remove inorganic substances. After drying with anhydrous sodium sulfate, the mixture was concentrated and purified by silica gel column (n-hexane: ethyl acetate = 9:1) to obtain white solid compound 4 (93 mg, 79%). The nuclear magnetic resonance spectral data of compound 4 are as follows:

[0074] Compound 4: white solid; mp 72.4-74.6 °C; [a] 25 D -48.0 (c 0.3, CH3OH); 1 H NMR (CDCI3, 400 MHz) δ 2.73 (1H, dd, J = 3.9, 18.7 Hz, H-15), 1.38 (3H, s, H-18),1.01 (3H, s, H-17), 0.98 (3H, s, H-20), 1.71-0.87 (19H, m, CH, CH2in the bea skeleton); 13 C NMR (CDCI3, 100 MHz) δ 222.2, 121.8, 59.1, 55.1, 54.7, 54.5, 48.9,48.7, 41.5, 40.3, 39.2, 38.5, 37.5, 37.1, 31.9, 19.9, 19.9, 19.8, 18.0, 14.1.

[0075] (2) Synthesis of compounds 5a-5m

[0076] Compound 4 (114 mg, 0.36 mmol), triethylamine (0.15 mL, 1.08 mmol), and an amine compound (0.54 mmol) were added to tetrahydrofuran and stirred at room temperature for 4 hours. TLC plate was used to monitor the reaction progress until completion. An appropriate amount of ethyl acetate was added for extraction, followed by washing with saturated brine to remove inorganic matter, drying over anhydrous sodium sulfate, and concentration. The mixture was separated and purified using a silica gel column to yield compounds 5a-5m.

[0077] Among them: the amine compound used in the synthesis of 5a is ethylamine, the amine compound used in 5b is isopropylamine, the amine compound used in 5c is tetrahydropyrrole, the amine compound used in 5d is 2-aminothiazole, the amine compound used in 5e is cyclohexylamine, the amine compound used in 5f is p-toluidine, the amine compound used in 5g is p-fluoroaniline, the amine compound used in 5h is p-methoxyaniline, the amine compound used in 5i is 3,4,5-trimethoxyaniline, the amine compound used in 5j is 3,4-methylenedioxyaniline, the amine compound used in 5k is 4-aminopyridine, the amine compound used in 5l is 3-aminopyridine, and the amine compound used in 5m is benzylamine.

[0078] The structures and NMR spectra of compounds 5a-5m are shown below:

[0079] Compound 5a: 1 H NMR (CDCl3, 400 MHz) δ 4.77 (1H, s, NH-CH2CH3), 4.32 (1H,s, NH-CO), 3.15 (2H, p, J = 7.1 Hz, CH2-CH3), 2.86 (1H, dq, J = 2.8, 14.2 Hz, H-3), 2.71 (1H, dd, J = 3.7, 18.4 Hz, H-15), 1.41 (3H, s, H-18), 1.11 (3H, t, J =7.2 Hz, CH3-CH2), 1.02 (3H, s, H-17), 0.99 (3H, s, H-20), 1.63-0.92 (18H, m,CH, CH2 in beylene skeleton); 13C NMR (CDCl3, 100 MHz) δ 223.1, 157.5, 56.8, 55.1,54.9, 54.2, 49.0, 48.9, 41.0, 39.3, 39.3, 37.6, 37.2, 36.6, 35.1, 27.9, 19.9,19.8, 19.4, 17.7, 15.5, 15.1.

[0080] Compound 5b: 1 H NMR (CDCl3, 400 MHz) δ 4.30 (1H, d, J = 8.0 Hz, NH-(CH)CH3),4.11 (1H, s, NH-CO), 3.77 (1H, m, CH-(CH3)2), 2.83 (1H, dq, J = 2.7, 14.0 Hz, H-3), 2.69 (1H, dd, J = 3.7, 18.5 Hz, H-15), 1.41 (3H, s, H-18), 1.12 (6H, dd, J =1.7, 6.5 Hz, (CH3)2-CH), 1.01 (3H, s, H-17), 0.99 (3H, s, H-20), 1.63-0.99 (18H, m, CH, CH2 in beylene skeleton); 13 C NMR (CDCl3, 100 MHz) δ 222.7, 156.7, 56.7,55.2, 55.0, 54.3, 48.9, 48.8, 42.1, 41.0, 39.3, 39.3, 37.6, 37.2, 36.7, 27.9,23.7, 23.6, 19.9, 19.9, 19.4, 17.7, 15.2.

[0081] Compound 5c: 1 H NMR (CDCl3, 400 MHz) δ 4.09 (1H, s, NH-CO), 3.28 (4H, m,N-(CH2)2), 2.81 (1H, m, H-3), 2.66 (1H, dd, J= 3.8, 18.5 Hz, H-15), 1.43 (3H,s, H-18), 1.03 (3H, s, H-17), 0.98 (3H, s, H-20), 2.37-0.98 (22H, m, CH, CH2 in beylene skeleton); 13 C NMR (CDCl3, 100 MHz) δ 222.3, 156.2, 56.7, 55.2, 55.1, 55.0,54.3, 48.8, 48.7, 45.4, 40.9, 39.2, 39.2, 37.5, 37.1, 36.3, 27.9, 27.2, 25.6,19.9, 19.8, 19.3, 17.7, 15.3.

[0082] Compound 5d: 1 H NMR (CDCl3, 400 MHz) δ 7.31 (1H, d, J = 3.7 Hz, CH-N), 6.78(1H, d, J = 3.7 Hz, CH-S), 2.92 (1H, d, J = 16.2 Hz, H-3), 2.75 (1H, dd, J = 3.6,18.5 Hz, H-15), 1.49 (3H, s, H-18), 1.06 (3H, s, H-17), 1.00 (3H, s, H-20),1.68-0.94 (18H, m, CH, CH2 in beylene skeleton); 13 C NMR (CDCl3, 100 MHz) δ 222.5,162.8, 153.8, 136.9, 110.5, 56.7, 55.5, 55.2, 54.4, 49.0, 48.8, 41.0, 39.3,39.1, 37.6, 37.2, 36.5, 27.6, 19.9, 19.8, 19.6, 17.7, 15.4.

[0083] Compound 5e: 1 H NMR (CDCl3, 400 MHz) δ 4.05 (1H, d, J = 8.2 Hz, CH-NH), 4.00(1H, s, NH-CH), 2.77 (1H, m, H-3), 2.67 (1H, dd, J= 3.8, 18.5 Hz, H-15), 1.41(3H, s, H-18), 1.00 (3H, s, H-17), 0.99 (3H, s, H-20), 1.75-0.99 (29H, m, CH,CH2 in beylene skeleton); 13 C NMR (CDCl3, 100 MHz) δ 222.6, 156.7, 56.7, 55.2, 55.0,54.3, 49.5, 48.8, 41.0, 39.3, 37.6, 37.2, 36.7, 34.2, 34.1, 31.4, 30.2, 29.7,27.9, 25.6, 25.1, 19.8, 19.5, 17.7, 15.2, 14.1.

[0084] Compound 5f: 1 H NMR (CDCl3, 400 MHz) δ 7.13 (4H, overlapping, Ph-H), 6.14 (1H, s, NH-Ph), 4.55 (1H, s, NH-CO), 2.82 (1H, d, J = 13.8 Hz, H-3), 2.55 (1H, dd, J =3.8, 18.5 Hz, H-15), 2.34 (3H, s, CH3-Ph), 1.45 (3H, s, H-18), 0.98 (3H, s,H-17), 0.69 (3H, s, H-20), 1.72 -0.69 (18H, m, CH, CH2 in beylene skeleton); 13 C NMR (CDCl3, 100 MHz) δ 222.8, 155.4, 135.6, 134.9, 130.0, 130.0, 123.6, 123.6,56.7, 55.2, 55.1, 54.3, 48.8, 48.8, 40.9, 39.2, 39.1, 37.4, 37.2, 36.2, 27.7,20.9, 19.8, 19.3, 17.7, 14.7, 2.0.

[0085] Compound 5g: 1H NMR (CDCl3, 400 MHz) δ 7.57 (1H, m, NH-Ph), 7.30 (2H, overlapping, 2,6-Ph), 6.95 (2H, overlapping, 3,5-Ph), 4.86 (1H, m, NH-CO), 2.96 (1H, m, H-3), 2.72 (1H, dd, J = 3.6, 18.3 Hz, H-15), 1.44 (3H, s, H-18), 1.00 (3H, s, H-17),0.95 (3H, s, H-20), 1.64-0.95 (18H, m, CH, CH2 in beylene skeleton); 13 C NMR (CDCl3,100 MHz) δ 224.3, 155.2, 133.0, 122.2, 115.6, 115.6, 115.3, 115.3, 56.7,55.3, 54.9, 54.1, 49.2, 49.2, 41.0, 39.4, 39.3, 37.6, 37.3, 36.2, 34.5, 27.8,19.9, 19.3, 17.7, 14.9.

[0086] Compound 5h: 1 H NMR (CDCl3, 400 MHz) δ 7.21 (2H, s, 2,6-Ph), 6.85 (2H, s,3,5-Ph), 6.73 (1H, s, NH-Ph), 4.66 (1H, s, NH-CO), 3.79 (3H, s, OCH3-Ph), 2.84 (1H, d, J = 14.0 Hz, H-3), 2.58 (1H, dd, J = 3.6, 18.5 Hz, H-15), 1.44 (3H,s, H-18), 0.98 (3H, s, H-17), 0.75 (3H, s, H-20), 1.62-0.89 (18H, m, CH, CH2 in beylene skeleton); 13C NMR (CDCl3, 100 MHz) δ 223.2, 156.9, 156.0, 131.4, 125.0,114.5, 114.5, 114.5, 56.7, 55.6, 55.1, 55.0, 54.2, 48.9, 48.9, 40.9, 39.3,39.2, 37.4, 37.2, 36.3, 29.7, 27.8, 19.8, 19.2, 17.7, 14.7.

[0087] Compound 5i: 1 H NMR (CDCl3, 400 MHz) δ 6.80 (1H, s, NH-Ph), 6.61 (2H, overlapping, 2,6-Ph), 4.67 (1H, s, NH-CO), 3.83 (6H, overlapping, 2×OCH3), 3.80 (3H, s, OCH3-4-Ph), 2.89 (1H, d, J = 13.8 Hz, H-3), 2.64 (1H, dd, J = 3.6, 18.4 Hz, H-15),1.46 (3H, s, H-18), 1.01 (3H, s, H-17), 0.87 (3H, s, H-20), 1.76 (18H, m, CH,CH2 in beylene skeleton); 13 C NMR (CDCl3, 100 MHz) δ 223.2, 155.1, 153.5, 153.5, 135.1,134.6, 99.32, 99.3, 61.0, 56.6, 56.2, 56.2, 55.3, 55.0, 54.2, 48.9, 40.9,39.3, 39.2, 37.5, 37.2, 36.3, 30.1, 27.7, 19.9, 19.9, 19.3, 17.7, 14.8.

[0088] Compound 5j: 1 H NMR (CDCl3, 400 MHz) δ 6.81 (1H, d, J = 2.1 Hz, 2-Ph), 6.75(1H, d, J = 8.2 Hz, 6-Ph), 6.66 (1H, dd, J= 2.1, 8.2 Hz, 5-Ph), 6.11 (1H, s, NH-Ph), 5.98 (2H, q, J = 1.4 Hz, O-CH2-O), 4.54 (1H, s, NH-CO), 2.78 (1H, m, H-3), 2.56 (1H, dd, J = 3.8, 18.5 Hz, H-15), 1.45 (3H, s, H-18), 0.98 (3H, s, H-17),0.70 (3H, s, H-20), 1.69-0.87 (18H, m, CH, CH2 in beylene skeleton); 13 C NMR (CDCl3, 100 MHz) δ 222.8, 155.6, 148.3, 145.3, 132.2, 117.5, 108.3, 106.2, 101.5,56.6, 55.2, 55.1, 54.3, 48.8, 48.8, 40.9, 39.2, 39.1, 37.4, 37.2, 36.2, 27.7,19.8, 19.8, 19.3, 17.7, 14.6; 1 H NMR spectrum reference Figure 1 ;That 13 C-NMR spectrum reference Figure 2 .

[0089] Compound 5k: 1 H NMR (CD3OD, 400 MHz) δ 8.24 (2H, d, J = 5.6 Hz, 3,5-Py),7.58 (1H, s, NH-Py), 7.43 (2H, d, J = 5.8 Hz, 2,6-Py), 5.43 (1H, d, J = 8.7 Hz,NH-CO), 2.79 (1H, d, J = 13.8 Hz, H-3), 2.68 (1H, dd, J = 3.7, 18.6 Hz, H-15),1.41 (3H, s, H-18), 1.04 (3H, s, H-17), 0.95 (3H, s, H-20), 1.68-0.95 (18H,m, CH, CH2 in beylene skeleton); 13C NMR (CD3OD, 100 MHz) δ 223.7, 154.1, 149.3, 147.8,147.8, 112.3, 112.3, 56.4, 55.2, 54.8, 53.9, 40.6, 39.2, 39.0, 37.4, 37.0,36.3, 29.6, 29.4, 26.7, 19.7, 19.0, 19.0, 17.4, 14.3.

[0090] Compound 5l: 1 H NMR (CDCl3, 400 MHz) δ 8.39 (1H, d, J = 2.6 Hz, 2-Py), 8.13 (2H, overlapping, 4,6-Py), 7.20 (1H, dd, J = 4.7, 8.4 Hz, 5-Py), 5.30 (1H, s, NH-Py), 5.08 (1H, s, NH-CO), 2.95 (1H, dt, J = 3.7, 13.4 Hz, H-3), 2.71 (1H, m, H-15), 1.44 (3H, s, H-18), 1.00 (6H, overlapping, H-17, H-20), 1.69-0.94 (18H, m, CH, CH2 in beylene skeleton); 13 C NMR (CDCl3, 100 MHz) δ 224.5, 154.8, 142.6, 140.0, 137.2,126.5, 123.8, 56.6, 55.4, 54.8, 54.1, 49.1, 48.7, 40.9, 39.4, 38.8, 37.6,37.3, 36.3, 27.7, 19.9, 19.3, 17.7, 17.5, 15.0; 1 H NMR spectrum reference Figure 3 ;That 13 C-NMR spectrum reference Figure 4 .

[0091] Compound 5m: 1 H NMR (CDCl3, 400 MHz) δ 7.30-7.23 (5H, overlapping, 2,3,4,5,6-Ph), 5.24 (1H, s, NH-CH2), 4.37 (1H, s, NH-CO), 4.30 (2H, m, CH2-Ph), 2.87 (1H, dq,J = 2.7, 14.3 Hz, H-3), 2.63 (1H, dd, J = 3.7, 18.5 Hz, H-15), 1.39 (3H, s, H-18), 0.91 (3H, s, H-17), 0.85 (3H, s, H-20), 1.57-0.85 (18H, m, CH, CH2 in beylene skeleton); 13 C NMR (CDCl3, 100 MHz) δ 223.3, 157.5, 139.5, 128.6, 128.6, 127.4,127.4, 127.2, 56.7, 55.0, 54.9, 54.2, 48.9, 48.8, 44.5, 41.0, 39.3, 39.3,37.6, 37.1, 36.5, 27.9, 19.9, 19.7, 19.1, 17.7, 15.0.

[0092] Table 2 Structural formulas of compounds 5a-5m

[0093]

[0094] In order to demonstrate the advantages of the technical solution provided by this application, an embodiment of the technical solution provided by this application is given below.

[0095] Experimental Example 1: Screening Experiment of Cardioprotective Activity of Isosteviol Derivatives and Evaluation Experiment of Cardiac Function in Zebrafish

[0096] (1) Zebrafish culture and embryo collection: Wild-type AB strain and cardiac fluorescent marker (Tg [cmlc2:GFP]) zebrafish (3-12 months old) were purchased from the National Zebrafish Resource Center. They were cultured in a fish room with 14 h light / 10 h dark and fed with live brine shrimp twice a day. A recirculating aquaculture system was used, equipped with a filtration and impurity removal device, a chlorine removal and pure water device, an oxygenation device, an ultraviolet sterilization and disinfection device, and a temperature control device. The system water maintained a conductivity of 500-550 μS / cm, a pH range of 7.0 ± 0.5, a water temperature range of 28 ± 1 °C, a dissolved oxygen concentration of 6.0-8.0 mg / L, and an ammonia nitrogen concentration of less than 0.02 mg / L. Zebrafish (female:male = 1:1) were mated, and embryos were collected and placed in a 0.0002% (w / v) methylene blue disinfectant solution in a constant temperature incubator (28 ± 0.5°C) for 24 h. Deformed or dead embryos were selected using a stereomicroscope and set aside for later use.

[0097] (2) Drug screening using a doxorubicin-induced zebrafish embryo myocardial injury model: 24 hpf (24 hours after fertilization) wild-type zebrafish embryos were distributed into 24-well plates (20 embryos / well), and 1 mL of different solutions (wherein the final concentration of doxorubicin was 90 μM and the final concentrations of the test compound were 5, 15, and 40 μM) were added to each well. Technical replicates were performed on four wells in each group. After the addition of samples, the well plates were placed in a constant temperature incubator (28 ± 0.5 ℃) and cultured for 72 h. The number of surviving embryos was observed under an inverted fluorescence microscope (Zeiss, Germany). The zebrafish atria and ventricles lost their contractile force and were considered dead. The survival rate was used as the evaluation standard for the activity of the compound.

[0098] like Figure 5 As shown, 90 μM DOX treatment for 72 hours induced 50% zebrafish mortality. Compared to the doxorubicin (DOX) model group, treatment with the clinically active drug 40 μM levosimendan (LSD) restored zebrafish survival to 68%. Treatment with 5 μM of the parent isosteviol 1 restored zebrafish survival to 57%. Compounds 2, 3c, 3d, 5h, 5i, 5j, and 5l effectively enhanced zebrafish survival in doxorubicin-induced myocardial injury to 85%, 93%, 86%, 86%, 86%, 96%, and 88% at concentrations of 5, 40, 5, 40, 15, 40, and 40 μM, respectively.

[0099] (3) Research on effective dose of compounds: Based on Figure 5 As a result, seven compounds with excellent activity were found: 2, 3c, 3d, 5h, 5i, 5j, and 5l. Next, the transgenic zebrafish Tg (cmlc2:GFP) that specifically expresses green fluorescent protein in the heart was further used to evaluate the effect of these five compounds on the recovery of zebrafish cardiac function. First, the modeling concentration and optimal dose concentration of the new model were explored. It was found that 90 μM DOX exposure for 48 hours would cause 50% of zebrafish death. The optimal dosages of the parent compound isosteviol 1 and LSD were 10 and 60 μM, respectively. The optimal dosages of isosteviol derivatives 2, 3c, 3d, 5h, 5i, 5j, and 5l were 0.8, 30, 5, 40, 20, 5, and 50 μM, respectively. Figure 6 , #### p < 0.0001 compared with the control group, **p < 0.01, ***p < 0.001, **** p < 0.0001 compared with the model group).

[0100] (4) Zebrafish embryo toxicity evaluation experiment: 24 hpf wild-type zebrafish embryos were distributed into 96-well plates (1 embryo / well), 200 mL of the test compound of the required concentration was added, 20 wells per group, and cultured in an incubator for 72 h. The embryo survival rate was observed under an inverted fluorescence microscope (Zeiss, Germany). In order to test whether the compounds (1, 2, 3c, 3d, 5h, 5i, 5j, 5l and LSD) would cause any toxicity, the embryos were treated with different concentrations of the test compounds alone. Figure 6 As shown in the data (#### p < 0.0001 compared with the control group, **p < 0.01, ***p < 0.001, **** p < 0.0001 compared with the model group), all the test compounds did not cause the death of zebrafish even when the administration concentration was much higher than the effective dose, and the zebrafish developed normally, the same as the control group.

[0101] (5) Evaluation of zebrafish cardiac function: 24 hpf (24 hours post fertilization) transgenic zebrafish embryos with green fluorescent markers of the heart were distributed into 24-well plates (20 embryos / well). 1 mL of different solutions (where the final concentration of doxorubicin was 90 μM and the final concentration of the test compound was the optimal concentration) was added to each well. Four wells in each group were used as technical replicates and cultured in an incubator for 48 hours. The zebrafish larvae were then washed with Holt buffer and fixed in 4% methylcellulose fixative. The zebrafish were adjusted to lie on their side with their eyes overlapping and their bodies at the same level, maintaining their original shape. The zebrafish were photographed under a 50x upright fluorescence microscope (Zeiss, Germany). The EGFP fluorescence channel was then used. The heart was located at 100x magnification and the heart was photographed and recorded for 40 seconds. Using Adobe Premiere video software, we played the video frame by frame and captured photos of the ventricular diastole and systole during a cycle. We then measured the major axis (a) and minor axis (b) using Photoshop. The contraction fraction (%) was calculated using the formula: (diastolic minor axis - contracted minor axis) / (diastolic minor axis) × 100%. Then, we used v = 4 / 3πab 2 Calculate the ventricular volume respectively. The stroke volume is the difference between the ventricular volume at end-diastole and the ventricular volume at end-systole. The stroke volume is the product of the stroke volume and the heart rate. The final processed heart rate, contraction fraction, stroke volume and stroke volume are as follows. Figure 7 As shown, #### p < 0.0001 compared with the control group, ***p < 0.001, **** p < 0.0001 compared with the model group. Figure 7The middle image (A) is a representative optical microscopy image of zebrafish (scale bar = 1000 μm); the middle image (B) is a representative fluorescence microscopy image of zebrafish heart (scale bar = 1000 μm). μm); Figure (C) heart rate statistics; Figure (D) shortening fraction statistics; Figure (E) stroke volume statistics; Figure (F) cardiac output statistics; It can be seen that zebrafish treated with doxorubicin alone showed distorted body shape, severe pericardial edema and an elongated heart, and each value was significantly reduced, indicating abnormal ventricular contraction and dysfunction in zebrafish; after treatment with levosimendan LSD and the parent compound isosteviol 1, the heart morphology and pericardial edema were only slightly restored; while the test compounds 2, 3c, 3d, 5h, 5i, 5j and 5l had better cardiac repair effects than the positive control drug (LSD) and the parent compound isosteviol 1 at the optimal dose, among which compounds 2, 3c, 3d, 5h, and 5i showed better recovery of zebrafish morphology and heart morphology after treatment, but still had slight pericardial edema; 5j and 5l restored the zebrafish morphology, pericardial edema, and heart morphology to a level close to that of the blank control group. By statistically analyzing the zebrafish cardiac function indicators such as heart rate, shortening fraction, stroke volume and cardiac output, the cardioprotective activity in the zebrafish myocardial injury model was: 5j > 5l > 3c > 3d > 5i > 5h > 2 >LSD > 1, among which 5j had the best effect and could significantly restore the zebrafish cardiac function to normal levels, which deserves further study.

[0102] Quantitative polymerase chain reaction of zebrafish embryos: 24 hpf (24 hours post-fertilization) wild-type zebrafish embryos were plated into 24-well plates (20 embryos / well). 1 mL of the different solutions (containing 90 µM doxorubicin and the desired test compound concentration) was added to each well and incubated in an incubator for 48 hours. The zebrafish larvae were then washed with Holt buffer. Total RNA was extracted from 20 zebrafish larvae using the standard TRIzol protocol (Novagen, Nanjing) according to the kit's instructions. The purity and concentration of the extracted RNA were then determined using a Nanodrop 2000 S spectrophotometer (Thermo Fisher Scientific, Germany). mRNA levels of specific genes were determined by PCR amplification using a real-time quantitative PCR kit (Novagen, Nanjing). The qPCR primers are as follows:

[0103] ANP-F, 5′-ATG GCC GGG GGA CTAATT CT-3′ (nucleotide sequence shown in SEQ ID NO: 1);

[0104] ANP-R, 5′-AGA GTT GCA ACC GAG GGT GC-3′ (nucleotide sequence shown in SEQ ID NO: 2);

[0105] BNP-F, 5′-AAG AGC AGC CCG ATA CTT ACC T-3′ (nucleotide sequence shown in SEQ ID NO: 3);

[0106] BNP-R, 5′-TCC CAAAGA CGA CAT TGAACC-3′ (nucleotide sequence shown in SEQ ID NO: 4);

[0107] cTnT-F, 5′-GTC TGC ACT TCG GCG GTT ACA-3′ (nucleotide sequence shown in SEQ ID NO: 5);

[0108] cTnT-R, 5′-AGG TAA AAT CTA TAT TGT TCA GTG AAA TCT AAC CG-3′ (nucleotide sequence shown in SEQ ID NO: 6);

[0109] β-actin-F 5'-CCT ACT AAT ACA CAG CCA TGG ATG A-3' (the nucleotide sequence is shown in SEQ ID NO: 7); β-actin-R, 5'-TC CCA TGC CAA CCA TCA C-3' (the nucleotide sequence is shown in SEQ ID NO: 8).

[0110] like Figure 8 As shown in the data (#### p < 0.0001 compared with the control group, **p < 0.01, **** p < 0.0001 compared with the model group), the mRNA levels of ANP, BNP, and cTnT, the myocardial injury markers, were significantly increased in zebrafish after doxorubicin treatment. Compounds 5j and 5l significantly reduced the levels of ANP, BNP, and cTnT, with the most significant effects at 5 and 50 μM, respectively, in a dose-dependent manner. These results further demonstrate the cardioprotective activity of compounds 5j and 5l.

Claims

1. A C-19 modified isosteviol derivative, characterized in that: The specific structural formula is as follows: 。 2. The method for preparing the C-19 modified isosteviol derivative according to claim 1, characterized in that: The method includes the following steps in sequence: 1) Dissolve isosteviol, diphenylphosphoryl azide, and catalyst A in solvent A, heat to 70-90°C and reflux for 8-10 hours, cool the reaction solution to room temperature, heat a dilute hydrochloric acid solution to 40-60°C and reflux for 6-8 hours, and post-treat to obtain compound 2; 2) Dissolving compound 2 prepared in step 1), 1-ethyl-3-(3-dimethylammoniumpropyl)carbonium, catalyst B, 1-hydroxybenzotriazole, and a carboxylic acid compound in solvent B, stirring at room temperature for 1.5-2.5 hours, and post-processing to obtain one of compounds 3c-3d; The molar ratio of isosteviol to diphenylphosphoryl azide in step 1) is 1.2:2.4; The molar ratio of compound 2, 1-ethyl-3-(3-dimethylammoniumpropyl)carbonyl ammonium, 1-hydroxybenzotriazole, and carboxylic acid compound in step 2) is 1.73:2.6:1.73:2.1; The structural formula of the compound 2 is as follows: ； The carboxylic acid compound is one of 3-pyridinecarboxylic acid and 3,4,5-trimethoxybenzoic acid.

3. The method for preparing a C-19 modified isosteviol derivative according to claim 2, wherein: The solvent A is tert-butanol; the solvent B is dichloromethane; the catalyst A is triethylamine; the catalyst B is 4-dimethylaminopyridine; and the concentration of the dilute hydrochloric acid solution is 5 N.

4. The method for preparing a C-19 modified isosteviol derivative according to claim 2, wherein: The post-treatment in step 1) is to add an equivalent amount of sodium hydroxide solution to the reaction system in step 1), extract with dichloromethane, wash with saturated brine to remove inorganic matter, dry with anhydrous sodium sulfate and concentrate, and then separate and purify the mixture using a silica gel column to obtain compound 2; the post-treatment in step 2) is to add a mixed solution of dichloromethane and saturated brine in a volume ratio of 10:1 to the reaction system for extraction, collect the organic layer, dry over anhydrous sodium sulfate and concentrate, and then separate and purify the mixture using a silica gel column to obtain one of compounds 3c-3d.

5. The method for preparing the C-19 modified isosteviol derivative according to claim 1, characterized in that: The method includes the following steps in sequence: 1) Dissolve isosteviol, diphenylphosphoryl azide, and catalyst A in solvent C, heat to 80-100°C and reflux for 8-10 hours, cool the reaction solution to room temperature, and post-treat to obtain compound 4; 2) Compound 4 prepared in step 1), catalyst A, and an amine compound are added to solvent D, stirred at room temperature for 3-5 hours, and after completion of the reaction, treated to obtain one of compounds 5j, 5l, and 5i; The molar ratio of isosteviol and diphenylphosphoryl azide in step 1) is 0.39:0.79; The molar ratio of compound 4 and amine compound in step 2) is 0.36:0.54; The structural formula of the compound 4 is as follows: ； The amine compound is one of 3,4,5-trimethoxyaniline, 3,4-methylenedioxyaniline and 3-aminopyridine.

6. The method for preparing a C-19 modified isosteviol derivative according to claim 5, wherein: The catalyst A is triethylamine; the solvent C is anisole; and the solvent D is tetrahydrofuran.

7. The method for preparing a C-19 modified isosteviol derivative according to claim 5, wherein: The post-treatment in step 1) is to add ethyl acetate to the reaction system to extract the product, then wash it with saturated brine to remove inorganic matter, dry it with anhydrous sodium sulfate, and concentrate it; then separate and purify the product using a silica gel column to obtain compound 4; the post-treatment in step 2) is to add ethyl acetate to the reaction system to extract the product, then wash it with saturated brine to remove inorganic matter, dry it with anhydrous sodium sulfate, and concentrate it; then separate and purify the mixture using a silica gel column to obtain one of compounds 5j, 5l, and 5i.

8. A cardioprotective drug, characterized in that Including the C-19 modified isosteviol derivative according to claim 1.

9. A cardioprotective drug according to claim 8, characterized in that: Also includes pharmaceutically acceptable excipients; The auxiliary material is at least one of a binder, a disintegrant, a lubricant, a filler, a surfactant, an antioxidant or a pH adjuster; The dosage form of the medicine is injection, tablet, oral solution, granule or capsule.

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