CN113105348B - Isosteviol derivative and preparation method and application thereof - Google Patents

Isosteviol derivative and preparation method and application thereof Download PDF

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CN113105348B
CN113105348B CN202110316315.7A CN202110316315A CN113105348B CN 113105348 B CN113105348 B CN 113105348B CN 202110316315 A CN202110316315 A CN 202110316315A CN 113105348 B CN113105348 B CN 113105348B
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isosteviol
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nmr
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赵昱
张汉源
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Guangdong University of Technology
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Abstract

The invention relates to an isosteviol derivative and a preparation method and application thereof. The isosteviol derivative has a structure shown as a formula (I), wherein R in the formula (I) 1 Selected from hydroxy or carbonyl; r 2 Selected from H, C 1～5 Saturated straight-chain alkyl radical, C 3～6 Saturated cyclic alkyl, pyridine ring, phenyl, benzyl/substituted benzyl, phenethyl/substituted phenethyl, phenylpropyl/substituted phenylpropyl; r 3 Selected from carboxyl or primary amine. The derivative obtained by specifically modifying the specific active site of isosteviol has good heart protection activity, and the isosteviol derivative is mainly used for protecting the death and the injury of myocardial cells by inhibiting the excessive generation of active oxygen, recovering the mitochondrial membrane potential and maintaining the mitochondrial morphology.
Figure DDA0002991438940000011

Description

Isosteviol derivative and preparation method and application thereof
Technical Field
The invention relates to the field of drug design and pharmaceutical chemistry, and in particular relates to an isosteviol derivative and a preparation method and application thereof.
Background
Cardiovascular diseases (CVDs) are the first mortality disease worldwide, with about 1790 million deaths annually due to cardiovascular disease accounting for 31% of all deaths worldwide. Cardioprotective agents that protect the metabolism, structure and function of the heart and blood vessels and reduce their damage in primary or secondary prevention are of great importance in the treatment of cardiovascular diseases or patients at high risk for cardiovascular diseases. Although several groups of cardioprotective drugs including statins, angiotensin Converting Enzyme Inhibitors (ACEIs), beta-blockers (BBs), angiotensin ii type 1 receptor blockers (ARBs) and aldosterone receptor blockers (AIRBs) have proven effective in achieving a hard endpoint, treatment of hypertension by these drugs reduces mortality by no more than 30% and residual cardiovascular risk remains quite high. Thus, there has been an increase in research findings for new cardioprotective agents.
Natural products are an important source of pharmaceuticals and have long been used to treat cardiovascular disease. Stevioside is a natural sweetener and is the main component of stevia leaves. Isosteviol is prepared by acid hydrolysis of stevioside, and has a structure shown in the following formula after Wagner-Meerwein rearrangement:
Figure BDA0002991438920000011
researches show that the compound has broad-spectrum pharmacological effects including anti-diabetes, anti-cancer, anti-inflammation, anti-hyperglycemia, anti-hypertension, anti-diarrhea, diuretic and immunoregulatory activity and cardiovascular protection effects, and the cardioprotection effect in the form of sodium salt thereof is widely evaluated in various in-vivo models, the activity of the compound is remarkably improved through chemical modification of reactive active sites of the compound, different modifications are performed on different active sites, and the compound can be applied to different medicine fields, for example, chinese patents CN108456240A, CN103099805A and CN102718658A modify the active sites of isosteviol to enable the compound to be used for preparing antitumor drugs.
Therefore, further research on the modification of the active site of isosteviol is still needed to further improve the activity of isosteviol and expand the application of isosteviol.
Disclosure of Invention
The invention aims to provide an isosteviol derivative with heart protection activity, which is obtained by taking isosteviol as a parent and carrying out specific chemical modification on a specific active site.
Another object of the invention is to provide a preparation method of the isosteviol derivative.
Another object of the present invention is to provide the use of the isosteviol derivative in the treatment of cardiovascular diseases.
In order to realize the purpose, the invention adopts the following technical scheme:
an isosteviol derivative having the structure of formula (I):
Figure BDA0002991438920000021
in the formula (I), R 1 Independently selected from hydroxy or carbonyl;
R 2 selected from H, C 1～5 Saturated straight-chain alkyl radical, C 3～6 Saturated cyclic alkyl, pyridine ring, phenyl, benzyl/substituted benzyl, phenethyl/substituted phenethyl, phenylpropyl/substituted phenylpropyl;
R 3 independently selected from carboxyl or primary amine.
Isosteviol as a pharmaceutical compositionThe compound has a plurality of reactive sites, and can be used for treating different diseases by modifying the reactive sites. The research of the invention finds that the specific modification (such as R) is carried out on the specific active site of isosteviol 1 、R 2 And R 3 ) The obtained derivative has good heart protection activity; further, through the research on the molecular mechanism of H9c2 cells, the isosteviol derivative is found to protect the death and damage of myocardial cells mainly by inhibiting the overproduction of active oxygen, restoring mitochondrial membrane potential and maintaining mitochondrial morphology.
Preferably, said R is 2 Is a substituted benzyl, substituted phenethyl or substituted phenylpropyl group.
Preferably, the substituted group is one or more of a halogen atom, a methyl group, a methoxy group, a hydroxyl group or a methyleneoxy group.
Further preferably, the substituted group is methoxy, and the number of the substituted groups is more than or equal to 2.
Further preferably, said R 2 Is any one of the following groups:
Figure BDA0002991438920000031
preferably, R 1 Is a carbonyl group; r 2 Is substituted benzyl, substituted phenethyl or substituted phenylpropyl; r3 is primary amine.
Further preferably, the isosteviol derivative is any one of the following compounds:
Figure BDA0002991438920000032
the preparation method of the isosteviol derivative comprises the following steps:
s1, carrying out hydroxymethylation reaction on isosteviol and polyformaldehyde under an alkaline condition at the temperature of 60-100 ℃ to obtain a compound 2 (the compound 2 has a 15 beta-hydroxymethyl structure) with a structural formula shown in a formula (II):
Figure BDA0002991438920000033
s2, acylating the 15-beta-hydroxymethyl group of the compound 2 obtained in the step S1 to obtain a compound 3 shown in a formula (III);
or reacting compound 2 obtained in S1 with diphenylphosphoryl azide and Et 3 N is rearranged by Curtius to convert 19-COOH into primary amine to obtain a compound 5 shown as a formula (V), and further acylation is carried out on 15-beta-hydroxymethyl of the compound 5 to obtain a compound 6 shown as a formula (VI);
Figure BDA0002991438920000034
s3, oxidizing the 16-alpha hydroxyl of the compound 3 obtained in the S2 to obtain a 16-position ketone derivative 4 shown in a formula (IV);
or, oxidizing the 16-alpha hydroxyl of the compound 6 obtained from S2 to obtain a 16-position ketone derivative 7 thereof, as shown in formula (VII):
Figure BDA0002991438920000041
it should be noted that, the isosteviol and the aldehyde can have a hydroxymethylation reaction, and formaldehyde is usually selected, but has high toxicity, so that the polyformaldehyde is selected to replace the formaldehyde, and the polyformaldehyde is depolymerized when being heated under an alkaline condition to release the formaldehyde to react with the isosteviol.
Preferably, the molar ratio of the isosteviol to the aldehyde group in the polyformaldehyde in the step S1 is 1:5-8; more preferably 1:6. In the invention, the aldehyde group in the polyformaldehyde is the total mole number of the aldehyde group after depolymerization.
Preferably, the isosteviol and polyoxymethylene are dissolved in an organic solvent in step S1, and the organic solvent is further preferably absolute ethanol.
Preferably, the alkaline condition in step S1 is one or a combination of sodium hydroxide and potassium hydroxide.
Preferably, the acylation process in the step S2 is esterification of 15-beta-hydroxymethyl and acid compound.
Preferably, the acid compound is one or a combination of more of acetic anhydride, cyclohexanecarboxylic acid, nicotinic acid, isonicotinic acid and benzoic acid.
The application of the isosteviol derivative in treating cardiovascular diseases is also within the protection scope of the invention.
Preferably, the isosteviol derivative is used for preparing a medicament for treating cardiovascular diseases.
Compared with the prior art, the invention has the following beneficial effects:
the isosteviol is used as a matrix, specific chemical modification is carried out on specific active sites of the isosteviol to obtain isosteviol derivatives with heart protection activity, experimental studies show that the derivatives can be used for treating morphological aberration and cardiac insufficiency of zebra fish caused by DOX, and the death and injury of myocardial cells are protected by inhibiting excessive generation of active oxygen, recovering mitochondrial membrane potential and maintaining mitochondrial morphology, so that the isosteviol derivatives have good heart protection activity.
Drawings
FIG. 1 is a graph of the effect of isosteviol derivatives on DOX-induced survival of zebrafish embryos;
FIG. 2 is a graph of the effect of different concentrations of isosteviol derivatives on DOX-induced zebrafish embryo survival and zebrafish embryo toxicity tests;
FIG. 3 is a DOX-induced assessment of cardiac function of zebrafish embryos by isosteviol derivatives;
FIG. 4 is a DOX-induced assessment of heart function of zebrafish embryos with different concentrations of compound 7 d;
FIG. 5 is a graph of the effect of Compound 7d on DOX-induced relative mRNA levels of the biomarkers ANP and cTnT of embryonic cardiomyopathy in zebrafish;
FIG. 6 is a graph of the effect of Compound 7d on DOX-induced H9c2 cytotoxicity and ROS;
FIG. 7 is a graph showing the effect of compound 7d on the mitochondrial membrane potential and morphology of H9c2 cells under DOX.
Detailed Description
The present invention will be further described with reference to the following specific examples and drawings, which are not intended to limit the invention in any manner. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the present invention are commercially available.
Example 1
This example provides a series of isosteviol derivatives prepared according to the following two synthetic routes:
the first synthetic route is as follows:
reaction conditions and reagents: (a) NaOH (HCHO) n ,EtOH,reflux；(b)Ac 2 O,Pyridine,DMAP for 3a；(c)Acid,DMAP,EDCI,DCM for 3b-3n；(d)PDC,DMF,rt.)
Figure BDA0002991438920000051
The second synthetic route is as follows:
reaction conditions and reagents: (a) DPPA, et 3 N, t BuOH,reflux；(b)Acid,DMAP,EDCI, DCM；(c)PDC,DMF.
Figure BDA0002991438920000061
Specifically, the preparation method of each compound comprises the following steps:
(1) Synthesis of Compound 2
Isosteviol (50mg, 0.2mmol), sodium hydroxide (31.4mg, 0.8mmol) and paraformaldehyde (37.8mg, 1.3mmol) were dissolved in absolute ethanol (2 mL), and the mixture was stirred at 80 ℃ to react for 5 hours. The mixture was diluted with ethyl acetate, neutralized with hydrochloric acid (1N), the solution was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated, and the residue was chromatographed using a silica gel column to give compound 2 (138 mg, yield 58 wt%) as a white solid.
(2) Synthesis of Compound 3a
Intermediate 2 (200mg, 0.6 mmol), acetic anhydride (0.07mL, 0.7 mmol) and 4-dimethylaminopyridine (73.3mg, 0.6 mmol) were dissolved in anhydrous pyridine (5 mL) and the reaction was stirred at room temperature for 2h. The mixture was diluted with ethyl acetate, neutralized with hydrochloric acid (1N), the solution was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated, and the residue was chromatographed using a silica gel column to give 3a (78 mg, yield 35 wt%) as a white solid.
The structure, appearance, melting point, specific optical rotation, nuclear magnetic resonance spectrum data and high resolution mass spectrum of the compound 3a are as follows:
compound 3a:
Figure BDA0002991438920000062
3a: white solid (35%), mp 157.0-158.4 ℃; [ alpha ] to] 25 D -87(c 0.6,CH 3 OH)； 1 H NMR(CDCl 3 ,400MHz) δ4.22(1H,dd,J＝6.2,10.8Hz,H-1'a),4.05(1H,dd,J＝8.9,10.8Hz,H-1'b),3.45(1H,d,J＝ 4.6Hz,H-16),2.20(1H,m,H-15),2.16–2.06(4H,overlap,H-3,COCH 3 ),1.21(3H,s,H-18), 0.91(3H,s,H-17),0.84(3H,s,H-20),1.85–0.80(17H,m,CH,CH 2 in ent-beyerane skeleton)； 13 C NMR(CDCl 3 ,100MHz)δ183.67,171.56,85.27,66.32,57.48,56.97,54.01,47.36,43.56,42.87, 40.81,39.46,38.36,37.72,34.72,33.22,29.70,28.80,24.98,21.91,19.49,18.77,12.93；HRMS (ESI,m/z)calcd for C 23 H 36 O 5 Na,415.2461[M+Na + ]；found,415.2455.
(3) Synthesis of Compound 3b-3n
The intermediate 2 (1 eq), 1-ethyl-3 (3-dimethylpropylamine) carbodiimide (5 eq), 4-dimethylaminopyridine (2 eq) and the corresponding acid derivative (1.5 eq) were dissolved in anhydrous dichloromethane. The mixture was stirred at room temperature and reacted for 1h. The solution was then diluted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated in vacuo, and the residue was chromatographed using a silica gel column to give the pure product 3b-3n.
The structure, appearance, melting point, specific optical rotation, nuclear magnetic resonance spectrogram data and high-resolution mass spectrum of the compound 3b-3n are as follows:
compound 3b:
Figure BDA0002991438920000071
3b: white solid (35%), mp 91.1-91.5 ℃; [ alpha ] to] 25 D -57(c 1.0,CH 3 OH)； 1 H NMR(CDCl 3 ,400MHz) δ4.21(1H,dd,J＝4.8,10.8Hz,H-1'a),4.00(1H,m,H-1'b),3.52(1H,d,J＝4.6Hz,H-16),2.33 (1H,m,COCH),2.24–2.11(2H,overlap,H-15,H-3),1.25(3H,s,H-18),0.93(3H,s,H-17),0.87 (3H,s,H-20),1.90–0.80(27H,m,CH,CH 2 in ent-beyerane skeleton or carbon chain)； 13 C NMR (CDCl 3 ,100MHz)δ176.23,173.13,85.69,66.21,57.46,57.18,54.13,47.50,45.25,43.28,42.76, 40.93,39.40,38.32,38.01,34.85,33.14,30.21,29.22,29.09,28.13,25.74,25.48,25.42,25.07, 19.61,18.76,13.98；HRMS(ESI,m/z)calcd for C 28 H 44 O 5 Na,483.3087[M+Na + ]；found, 483.3091.
Compound 3c:
Figure BDA0002991438920000072
3c: white solid (33%), mp 113.5-114.0 deg.C; [ alpha ] of] 25 D -55(c 1.4,CH 2 Cl 2 )； 1 H NMR(CDCl 3 ,400MHz) δ9.21(1H,s,2-pyrimidine),8.75(1H,d,J＝4.9Hz,4-pyrimidine),8.30(1H,d,J＝8.0Hz, 6-pyrimidine),7.38(1H,dd,J＝4.9,8.0Hz,5-pyrimidine),4.52(1H,dd,J＝4.9,10.9Hz,H-1'a), 4.27(1H,m,H-1'b),3.64(1H,d,J＝4.8Hz,H-16),2.34(1H,m,H-15),1.27(3H,s,H-18),0.94 (3H,s,H-17),0.92(3H,s,H-20)；2.25–0.80(18H,m,CH,CH 2 in ent-beyerane skeleton)；13C NMR(CDCl 3 ,100MHz)δ173.22,165.52,153.38,150.88,137.28,126.13,123.51,85.40,67.34, 57.46,57.16,54.19,47.41,45.30,42.81,41.06,39.35,38.35,37.99,34.93,33.13,28.11,25.06, 22.00,19.59,18.80,14.04；HRMS(ESI,m/z)calcd for C 27 H 38 NO 5 ,456.2750[M+H + ]；found, 456.2740.
Compound 3d:
Figure BDA0002991438920000081
3d: white solid (32%), mp 139.2-140.4 ℃; [ alpha ] to] 25 D -44(c 1.6,CH 3 OH)； 1 H NMR(CDCl 3 ,400MHz) δ8.74(2H,d,J＝5.8Hz,3,5-pyrimidine),7.85(2H,d,J＝5.8Hz,2,6-pyrimidine),4.50(1H,dd, J＝5.4,11.0Hz,H-1'a),4.25(1H,dd,J＝8.7,11.0Hz,H-1'b),3.58(1H,d,J＝4.7Hz,H-16), 2.35(1H,m,H-15),2.17(1H,d,J＝13.2Hz,H-3),1.27(3H,s,H-18),0.97–0.91(6H,overlap, H-17,H-20),2.00–0.85(17H,m,CH,CH 2 in ent-beyerane skeleton)； 13 C NMR(CDCl 3 ,100MHz) δ173.11,165.41,150.61,137.35,122.99,85.56,67.73,57.43,57.19,54.14,53.44,47.34,45.29, 42.77,41.02,39.34,38.33,37.98,34.89,33.07,29.71,28.20,25.02,21.98,19.54,18.84,14.09； HRMS(ESI,m/z)calcd for C 27 H 38 NO 5 ,456.2750[M+H + ]；found,456.2738.
Compound 3e:
Figure BDA0002991438920000082
3e: white solid (42%), mp 96.7-96.9 ℃; [ alpha ] to] 25 D -54(c1.1,CH 3 OH)； 1 H NMR(CDCl 3 ,400MHz) δ8.03(2H,d,J＝7.4Hz,2,6-Ph),7.56(1H,t,J＝7.4Hz,4-Ph),7.43(2H,t,J＝7.4Hz,3,5-Ph), 4.45(1H,dd,J＝4.9,10.9Hz,H-1'a),4.22(1H,m,H-1'b),3.63(1H,d,J＝4.7Hz,H-16),2.32 (1H,m,H-15),2.17(1H,d,J＝13.0Hz,H-3),1.27(3H,s,H-18),0.96–0.90(6H,overlap,H-17, H-20)；2.0–0.80(17H,m,CH,CH 2 in ent-beyerane skeleton)； 13 C NMR(CDCl 3 ,150MHz)δ 173.21,166.64,148.84,125.57,124.28,122.98,116.55,86.48,67.95,61.66,57.60,57.21,56.22, 54.01,47.82,45.30,42.72,41.02,39.48,38.33,34.99,33.12,28.15,25.12,22.71,22.18,18.78, 13.93；HRMS(ESI,m/z)calcd for C 28 H 37 O 5 ,453.2641[M-H + ]；found,453.2650.
Compound 3f:
Figure BDA0002991438920000083
3f: white solid (35%), mp 108.1-109.1 ℃; [ alpha ] to] 25 D -59(c1.4,CH 3 OH)； 1 H NMR(CDCl 3 ,400MHz) δ8.05(2H,m,2,6-Ph),7.10(2H,m,3,5-Ph),4.44(1H,m,H-1'a),4.20(1H,m,H-1'b),3.59(1H, d,J＝4.7Hz,H-16),2.32(1H,m,H-15),2.17(1H,d,J＝13.4Hz,H-3),1.27(3H,s,H-18), 0.96–0.90(6H,overlap,H-17,H-20),1.90–0.80(17H,m,CH,CH 2 in ent-beyerane skeleton)； 13 C NMR(CDCl 3 ,100MHz)δ173.14,165.90,132.23,132.14,126.38,126.35,115.78,115.56,85.71, 67.03,57.47,57.21,54.18,47.53,45.29,42.77,40.98,39.37,38.34,38.01,34.90,33.10,28.17, 25.05,22.03,19.60,18.81,14.06；HRMS(ESI,m/z)calcd for C 28 H 37 FO 5 Na,495.2523[M+Na + ]； found,495.2539.
Compound 3g:
Figure BDA0002991438920000091
3g: white solid (27%), mp 201.7-202.5 ℃; [ alpha ] to] 25 D -53(c 0.3,CH 3 OH)； 1 H NMR(CDCl 3 ,400MHz) δ7.98(2H,d,J＝8.9Hz,3,5-Ph),6.91(2H,d,J＝8.9Hz,2,6-Ph),4.40(1H,dd,J＝5.0,10.9Hz, H-1'a),4.18(1H,dd,J＝8.9,10.9Hz,H-1'b),3.86(3H,s,OCH 3 ),3.61(1H,d,J＝4.7Hz,H-16), 2.34(1H,m,H-15),2.17(1H,d,J＝13.5Hz,H-3),1.26(3H,s,H-18),0.96–0.91(6H,overlap, H-17,H-20),1.80–0.80(17H,m,CH,CH 2 in ent-beyerane skeleton)； 13 C NMR(CDCl 3 ,100MHz) δ173.13,166.59,163.42,131.64,131.64,122.54,113.77,113.77,85.63,66.57,57.51,57.19, 55.44,54.21,47.63,45.30,42.80,40.95,39.40,38.34,38.03,34.91,33.16,28.11,25.09,22.07, 19.63,18.80,14.07；HRMS(ESI,m/z)calcd for C 29 H 40 O 6 Na,507.2723[M+Na + ]；found, 507.2711.
Compound 3h:
Figure BDA0002991438920000092
3h: white solid (35%), mp 112.0-113.5 ℃; [ alpha ] of] 25 D -52(c 1.1,CH 3 OH)； 1 H NMR(CDCl 3 ,400MHz) δ7.41(1H,dd,J＝1.8,7.6Hz,6-Ph),7.18–7.00(2H,overlap,3,4-Ph),4.49(1H,dd,J＝4.5,10.3 Hz,H-1'a),4.20(1H,dd,J＝10.3,12.1Hz,H-1'b),3.86(6H,s,2×OCH 3 ),3.57(1H,d,J＝4.4 Hz,H-16),2.30(1H,m,H-15),2.21(1H,d,J＝3.5Hz,H-3),1.28(3H,s,H-18),0.95(3H,s, H-17),0.90(3H,s,H-20),1.90–0.80(17H,m,CH,CH 2 in ent-beyerane skeleton)； 13 C NMR (CDCl 3 ,150MHz)δ173.16,166.84,133.03,130.14,129.93,129.59,129.59,128.51,128.51, 85.64,66.90,57.48,57.18,54.19,47.55,45.28,42.80,41.00,39.39,38.34,38.01,34.91,33.14, 28.11,25.07,24.88,22.07,19.62,18.78,14.04；HRMS(ESI,m/z)calcd for C 30 H 42 O 7 Na, 537.2829[M+Na + ]；found,537.2843.
Compound 3i:
Figure BDA0002991438920000101
3i: white solid (40%), mp 169.3-170.8 ℃; [ alpha ] to] 25 D -65(c 1.0,CH 3 OH)； 1 H NMR(CDCl 3 ,400MHz) δ7.68(1H,d,J＝8.4Hz,6-Ph),7.55(1H,s,2-Ph),6.89(1H,d,J＝8.4Hz,5-Ph),4.41(1H,dd,J ＝4.7,10.9Hz,H-1'a),4.22(1H,dd,J＝8.6,10.9Hz,H-1'b),3.93(3H,s,OCH 3 ),3.92(3H,s, OCH 3 ),3.64(1H,d,J＝4.6Hz,H-16),2.32(1H,m,H-15),2.17(1H,d,J＝13.5Hz,H-3),1.27 (3H,s,H-18),0.96–0.90(6H,overlap,H-17,H-20),1.90–0.80(17H,m,CH,CH 2 in ent-beyerane skeleton)； 13 C NMR(CDCl 3 ,100MHz)δ173.15,166.63,153.06,148.69,123.64,122.65,112.01, 110.44,85.47,66.56,57.51,57.19,56.04,56.04,54.26,47.59,45.29,42.79,40.97,39.39,38.34, 38.01,34.92,33.17,28.12,25.11,22.08,19.64,18.79,14.06；HRMS(ESI,m/z)calcd for C 30 H 42 O 7 K,553.2568[M+K + ]；found,553.2562.
Compound 3j:
Figure BDA0002991438920000102
3j: white solid (23%), mp 96.6-97.2 ℃; [ alpha ] to] 25 D -54(c2.1,CH 3 OH)； 1 H NMR(CDCl 3 ,400MHz) δ7.62(1H,d,J＝9.0Hz,6-Ph),6.68(1H,d,J＝9.0Hz,5-Ph),4.40(1H,dd,J＝4.6,10.2Hz, H-1'a),4.09(1H,m,H-1'b),3.84(3H,s,OCH 3 ),3.82(3H,s,OCH 3 ),3.77(3H,s,OCH 3 ),3.49(1H, d,J＝4.5Hz,H-16),3.21(1H,s,16-OH),2.23(1H,m,H-15),2.12(1H,d,J＝13.7Hz,H-3), 1.21(3H,s,H-18),0.89(3H,s,H-17),0.83(3H,s,H-20),1.85–0.75(17H,m,CH,CH 2 in ent-beyerane skeleton)； 13 C NMR(CDCl 3 ,100MHz)δ172.13,165.07,156.57,153.08,141.89, 126.68,116.36,106.47,85.73,66.72,60.99,60.04,56.58,56.18,55.11,53.03,46.88,44.28,41.61, 39.94,38.44,37.31,33.95,32.11,28.68,27.15,24.11,21.16,18.67,17.77,12.92；HRMS(ESI, m/z)calcd for C 31 H 44 O 8 Na,567.2934[M+Na + ]；found,567.2909.
Compound 3k:
Figure BDA0002991438920000103
3k is as follows: white solid (31%), mp 114.4-115.8 ℃; [ alpha ] to] 25 D -35(c 1.0,CH 3 OH)； 1 H NMR(CDCl 3 ,400MHz) δ7.32(2H,s,2,6-Ph),4.43(1H,dd,J＝4.3,10.9Hz,H-1'a),4.27(1H,dd,J＝8.8,10.9Hz, H-1'b),3.90(3H,s,OCH 3 ),3.90(6H,s,2×OCH 3 ),3.69(1H,d,J＝4.7Hz,H-16),2.35(1H,m, H-15),2.17(1H,d,J＝13.4Hz,H-3),1.28(3H,s,H-18),0.94(3H,s,H-17),0.92(3H,s,H-20), 1.90–0.80(17H,m,CH,CH 2 in ent-beyerane skeleton)； 13 C NMR(CDCl 3 ,100MHz)δ173.18, 166.43,153.02,153.02,142.28,125.21,106.83,106.83,85.36,66.66,60.94,57.48,57.18,56.28, 56.28,54.31,47.47,45.25,42.80,41.04,39.36,34.93,33.16,31.45,30.20,28.13,25.12,22.11, 19.65,18.73,14.04；HRMS(ESI,m/z)calcd for C 31 H 44 O 8 K,583.2673[M+K + ](ii) a found,583.2665 compound 3l:
Figure BDA0002991438920000111
3l: white solid (39%), mp 127.3-128.5 ℃; [ alpha ] to] 25 D -56(c 1.9,CH 2 Cl 2 )； 1 HNMR(CDCl 3 ,400MHz) δ7.61(1H,d,J＝16.0Hz,CH 2 ＝),6.68(2H,d,J＝2.3Hz,2,6-Ph),6.49(1H,t,J＝2.3Hz,4-Ph), 6.44(1H,d,J＝16.0Hz,CH 2 ＝),4.33(1H,dd,J＝5.0,10.8Hz,H-1'a),4.14(1H,m,H-1'b),3.81 (6H,s,2×OCH 3 ),3.56(1H,d,J＝4.6Hz,H-16),2.26(1H,m,H-15),2.16(1H,d,J＝13.1Hz, H-3),1.26(3H,s,H-18),0.93(3H,s,H-17),0.89(3H,s,H-20),1.85–0.80(17H,m,CH,CH 2 in ent-beyerane skeleton)； 13 C NMR(CDCl 3 ,100MHz)δ173.14,167.16,161.02,161.02,145.18, 136.27,118.40,106.07,106.07,102.82,85.76,66.72,57.45,57.17,55.47,55.47,54.08,47.44, 45.28,42.81,40.99,39.38,38.32,34.88,33.16,29.72,28.08,25.04,22.05,19.62,18.77,13.94； HRMS(ESI,m/z)calcd for C 32 H 44 O 7 Na,563.2985[M+Na + ]；found,563.2996.
Compound 3m:
Figure BDA0002991438920000112
3m: white solid (30%), mp 75.0-76.4 ℃; [ alpha ] to] 25 D -60(c 0.9,CH 3 OH)； 1 HNMR(CDCl 3 ,400MHz) δ7.18(2H,d,J＝8.6Hz,2,6-Ph),6.85(2H,d,J＝8.6Hz,3,5-Ph),4.21(1H,dd,J＝5.1,10.7Hz, H-1'a),3.96(1H,t,J＝10.2Hz,H-1'b),3.78(3H,s,OCH 3 ),3.59(2H,s,COCH 2 ),3.33(1H,d,J ＝4.6Hz,H-16),2.20–2.08(2H,overlap,H-15,H-3),1.23(3H,s,H-18),0.87(3H,s,H-17),0.86 (3H,s,H-20),1.85–0.80(17H,m,CH,CH 2 in ent-beyerane skeleton)； 13 C NMR(CDCl 3 ,100 MHz)δ173.22,172.09,158.76,130.30,130.30,126.20,114.15,114.15,85.49,66.88,57.41, 57.15,55.31,53.95,47.39,45.24,42.70,40.85,40.71,39.34,38.29,37.98,34.88,34.78,33.10, 28.09,24.94,22.02,19.58,13.92；HRMS(ESI,m/z)calcd for C 30 H 43 O 6 ,499.3059[M+H + ]；found, 499.3080.
Compound 3n:
Figure BDA0002991438920000121
3n: white solid (34%), mp 174.0-175.4 deg.C; [ alpha ] to] 25 D -51(c2.6,CH 2 Cl 2 )； 1 H NMR(CDCl 3 ,400MHz) δ6.51(2H,s,2,6-Ph),4.25(1H,dd,J＝5.1,10.8Hz,H-1'a),3.99(1H,t,J＝10.0Hz,H-1'b), 3.85(6H,s,2×OCH 3 ),3.82(3H,s,OCH 3 ),3.59(2H,s,COCH 2 ),3.37(1H,d,J＝4.7Hz,H-16), 2.23–2.07(2H,overlap,H-15,H-3),1.24(3H,s,H-18),0.90–0.81(6H,overlap,H-17,H-20), 1.85–0.80(17H,m,CH,CH 2 in ent-beyerane skeleton)； 13 C NMR(CDCl 3 ,100MHz)δ173.20, 171.69,153.34,137.19,129.63,106.33,106.33,85.39,66.87,60.87,57.37,57.14,56.16,56.16, 53.94,47.45,45.24,42.70,41.85,40.82,39.30,38.29,37.96,34.78,33.02,31.45,28.15,24.93, 22.01,19.55,18.74,13.94；HRMS(ESI,m/z)calcd for C 32 H 45 O 8 ,557.3115[M-H + ]；found, 557.3111.
(4) Synthesis of Compounds 4a-4n
Compounds 3a-3N (1 eq) and pyridinium dichromate (3 eq) were dissolved in anhydrous N, N-dimethylformamide. The mixture was stirred at room temperature and reacted for 24h. The mixture was diluted with ethyl acetate, the solution was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated, and the residue was chromatographed using a silica gel column to give the pure product 4a-4n.
The structure, appearance, melting point, specific optical rotation, nuclear magnetic resonance spectrogram data and high-resolution mass spectrum of the compound 4a-4n are as follows:
compound 4a:
Figure BDA0002991438920000122
4a: white solid (47%), mp 159.6-160.3 ℃; [ alpha ] of] 25 D -30(c 1.4,CH 2 Cl 2 )； 1 H NMR(CDCl 3 ,400MHz) δ4.30(2H,m,H-1'),2.66(1H,m,H-15),2.16(1H,d,J＝13.3Hz,H-3),2.03(3H,s,COCH 3 ), 1.25(3H,s,H-18),0.97(3H,s,H-17),0.78(3H,s,H-20),1.90–0.80(17H,m,CH,CH 2 in ent-beyerane skeleton)； 13 C NMR(CDCl 3 ,100MHz,)δ221.74,183.53,170.67,62.28,56.93, 53.00,51.21,48.11,43.59,40.65,39.51,38.45,35.47,31.52,30.15,29.71,28.92,21.46,20.85, 19.79,19.60,18.75,13.17；HRMS(ESI,m/z)calcd for C 23 H 34 O 5 Na,413.2304[M+Na + ]；found, 413.2301.
Compound 4b:
Figure BDA0002991438920000131
4b: white colourSolids (45%), mp 145.3-146.6 ℃; [ alpha ] to] 25 D -68(c 2.4,CH 2 Cl 2 )； 1 H NMR(CDCl 3 ,400MHz) δ4.34(1H,dd,J＝4.5,11.4Hz,H-1'a),4.23(1H,dd,J＝3.2,11.4Hz,H-1'b),2.58(1H,m, H-15),2.32–2.13(2H,overlap,COCH,H-3),1.26(3H,s,H-18),0.98(3H,s,H-17),0.82(3H,s, H-20),1.90–0.80(27H,m,CH,CH 2 in ent-beyerane skeleton or carbon chain)； 13 C NMR(CDCl 3 , 100MHz)δ221.47,175.54,172.85,61.81,57.19,56.98,53.09,50.70,48.14,45.26,43.14,40.55, 39.42,38.40,37.93,37.13,35.33,29.71,28.99,28.91,28.16,25.73,25.43,25.37,21.57,19.79, 18.74,14.19；HRMS(ESI,m/z)calcd for C 28 H 43 O 5 ,459.3110[M+H + ]；found,459.3090.
Compound 4c:
Figure BDA0002991438920000132
4c: white solid (66%), mp 108.9-109.6 ℃; [ alpha ] to] 25 D -40(c 1.1,CH 3 OH)； 1 H NMR(CDCl 3 ,400MHz) δ9.15(1H,s,2-pyrimidine),8.77(1H,d,J＝3.7Hz,4-pyrimidine),8.27(1H,m,6-pyrimidine), 7.39(1H,dd,J＝4.8,8.0Hz,5-pyrimidine),4.66(1H,dd,J＝4.9,11.5Hz,H-1'a),4.51(1H,dd, J＝3.6,11.5Hz,H-1'b),2.75(1H,m,H-15),2.21(1H,d,J＝12.8Hz,H-3),1.26(3H,s,H-18), 1.03(3H,s,H-17),0.90(3H,s,H-20),2.00–0.80(17H,m,CH,CH 2 in ent-beyerane skeleton)； 13 C NMR(CDCl 3 ,100MHz)δ221.01,172.91,164.93,153.53,150.75,137.27,125.76,123.51,62.95, 57.13,56.86,53.23,50.72,48.28,45.31,40.60,39.34,38.44,37.08,35.45,31.45,28.19,21.57, 19.96,19.70,18.80,14.28；HRMS(ESI,m/z)calcd for C 27 H 35 NO 5 Na,476.2413[M+Na + ]；found, 476.2382.
Compound 4d:
Figure BDA0002991438920000133
4d: white solid (35%), mp 72.3-72.9 ℃; [ alpha ] of] 25 D -56(c 2.0,CH 3 OH)； 1 H NMR(CDCl 3 ,400MHz) δ8.76(2H,d,J＝5.5Hz,3,5-pyrimidine),7.81(2H,d,J＝5.5Hz,2,6-pyrimidine),4.61(1H,dd, J＝5.6,11.5Hz,H-1'a),4.50(1H,dd,J＝3.8,11.5Hz,H-1'b),2.79(1H,m,H-15),2.20(1H,d,J ＝13.6Hz,H-3),1.26(3H,s,H-18),1.03(3H,s,H-17),0.92(3H,s,H-20),2.00–0.85(17H,m, CH,CH 2 in ent-beyerane skeleton)； 13 C NMR(CDCl 3 ,150MHz)δ220.82,172.93,164.88,150.69, 150.69,136.97,124.00,122.91,63.34,57.15,56.81,53.18,50.84,48.26,45.32,40.64,39.31, 38.45,37.05,35.52,31.45,30.21,28.23,21.55,19.99,18.84,14.33；HRMS(ESI,m/z)calcd for C 27 H 35 NO 5 K,492.2152[M+K + ]；found,492.2167.
Compound 4e:
Figure BDA0002991438920000141
4e: white solid (58%), mp 98.0-98.9 ℃; [ alpha ] of] 25 D -79(c 1.7,CH 3 OH)； 1 H NMR(CDCl 3 ,400MHz) δ7.97(2H,d,J＝7.7Hz,2,6-Ph),7.54(1H,t,J＝7.7Hz,4-Ph),7.42(2H,t,J＝7.7Hz,3,5Ph), 4.60(1H,dd,J＝4.8,11.4Hz,H-1'a),4.49(1H,dd,J＝3.3,11.4Hz,H-1'b),2.73(1H,m,H-15), 2.21(1H,d,J＝13.1Hz,H-3),1.30(3H,s,H-18),1.04(3H,s,H-17),0.90(3H,s,H-20), 2.0–0.80(17H,m,CH,CH 2 in ent-beyerane skeleton)； 13 C NMR(CDCl 3 ,100MHz)δ221.32, 172.87,166.27,133.08,129.61,128.47,129.61,128.47,62.57,57.18,56.96,53.22,50.93,48.24, 45.31,40.63,39.40,38.45,37.94,37.17,35.44,28.18,22.70,21.60,19.93,19.73,18.81,14.29； HRMS(ESI,m/z)calcd for C 28 H 36 O 5 Na,475.2461[M+Na + ]；found,475.2465.
Compound 4f:
Figure BDA0002991438920000142
4f: white solid (63%), mp 205.4-206.2 ℃; [ alpha ] to] 25 D -90(c 1.1,CH 2 Cl 2 )； 1 H NMR(CDCl 3 ,400MHz) δ8.00(2H,m,2,6-Ph),7.09(2H,m,3,5-Ph),4.56(1H,dd,J＝5.5,11.4Hz,H-1'a),4.46(1H,dd, J＝3.6,11.4Hz,H-1'b),2.76(1H,m,H-15),2.20(1H,d,J＝13.4Hz,H-3),1.30(3H,s,H-18), 1.02(3H,s,H-17),0.91(3H,s,H-20),1.90–0.80(17H,m,CH,CH 2 in ent-beyerane skeleton)； 13 C NMR(CDCl 3 ,100MHz)δ221.13,172.88,165.34,132.26,132.26,132.17,115.78,115.78, 115.56,62.71,57.21,56.89,53.21,51.04,48.21,45.32,40.65,39.36,38.44,37.92,37.11,35.49, 28.27,21.58,19.96,19.69,18.84,14.32；HRMS(ESI,m/z)calcd for C 28 H 35 FO 5 Na,493.2367 [M+Na + ]；found,493.2342.
Compound 4g:
Figure BDA0002991438920000143
4g: white solid (41%), mp 202.2-202.9 ℃; [ alpha ] to] 25 D -80(c 1.5,CH 2 Cl 2 )； 1 H NMR(CDCl 3 ,400MHz) δ7.92(2H,d,J＝8.9Hz,3,5-Ph),6.89(2H,d,J＝8.9Hz,2,6-Ph),4.54(1H,dd,J＝5.1,11.5Hz, H-1'a),4.46(1H,dd,J＝3.3,11.5Hz,H-1'b),3.84(3H,s,OCH 3 ),2.73(1H,m,H-15),2.20(1H, d,J＝13.4Hz,H-3),1.26(3H,s,H-18),1.03(3H,s,H-17),0.90(3H,s,H-20),1.80–0.80(17H, m,CH,CH 2 in ent-beyerane skeleton)； 13 C NMR(CDCl 3 ,100MHz)δ221.42,172.81,166.01, 163.46,131.68,131.68,122.18,113.74,113.74,62.32,57.22,56.96,55.42,53.23,51.12,48.20, 45.31,40.65,39.41,38.44,35.45,31.45,30.21,28.24,21.61,19.95,19.72,18.83,14.33；HRMS (ESI,m/z)calcd for C 29 H 38 O 6 Na,505.2566[M+Na + ]；found,505.2575.
Compound 4h:
Figure BDA0002991438920000151
4h: white solid (30%), mp 117.3-118.6 ℃; [ alpha ] to] 25 D -67(c 2.2,CH 3 OH)； 1 H NMR(CDCl 3 ,400MHz) δ7.25(1H,d,J＝2.0Hz,6-Ph),7.15–6.98(2H,overlap,3,4-Ph),4.58(1H,dd,J＝5.6,11.5Hz, H-1'a),4.50(1H,dd,J＝3.4,11.5Hz,H-1'b),3.87(6H,s,2×OCH 3 ),2.74(1H,m,H-15),2.17 (1H,d,J＝13.2Hz,H-3),1.24(3H,s,H-18),0.98(3H,s,H-17),0.82(3H,s,H-20),2.00–0.80 (17H,m,CH,CH 2 in ent-beyerane skeleton)； 13 C NMR(CDCl 3 ,100MHz)δ221.42,172.94, 165.44,153.48,149.40,125.70,123.76,122.23,115.95,62.47,61.67,57.20,57.02,56.10,52.88, 50.66,48.21,45.28,40.59,39.46,38.43,37.98,37.22,35.34,28.15,21.64,19.80,19.73,18.73, 14.23；HRMS(ESI,m/z)calcd for C 30 H 39 O 7 ,511.2696[M-H + ]；found,511.2647.
Compound 4i:
Figure BDA0002991438920000152
4i: white solid (58%), mp 105.3-106.0 ℃; [ alpha ] of] 25 D -72(c 0.7,CH 3 OH)； 1 H NMR(CDCl 3 ,400MHz) δ7.61(1H,dd,J＝2.0,8.5Hz,6-Ph),7.47(1H,d,J＝2.0Hz,2-Ph),6.88(1H,d,J＝8.5Hz, 5-Ph),4.57(1H,dd,J＝4.8,11.4Hz,H-1'a),4.50(1H,dd,J＝3.3,11.4Hz,H-1'b),3.92(3H,s, OCH 3 ),3.90(3H,s,OCH 3 ),2.72(1H,m,H-15),2.20(1H,d,J＝13.4Hz,H-3),1.26(3H,s, H-18),1.04(3H,s,H-17),0.89(3H,s,H-20),1.90–0.80(17H,m,CH,CH 2 in ent-beyerane skeleton)； 13 C NMR(CDCl 3 ,150MHz)δ221.36,172.86,166.01,153.10,148.66,122.28,111.92, 110.37,62.40,57.17,56.99,56.02,56.02,53.24,50.97,48.20,45.31,39.43,38.45,37.95,37.16, 35.43,31.45,30.20,28.15,21.61,19.96,19.73,18.78,14.28；HRMS(ESI,m/z)calcd for C 30 H 40 O 7 K,551.2411[M+K + ]；found,551.2438.
Compound 4j:
Figure BDA0002991438920000161
4j: white solid (70%), mp 96.7-97.6 ℃; [ alpha ] to] 25 D -54(c1.1,CH 2 Cl 2 )； 1 H NMR(CDCl 3 ,400MHz) δ7.53(1H,d,J＝9.0Hz,6-Ph),6.68(1H,d,J＝9.0Hz,5-Ph),4.52(1H,dd,J＝4.7,11.4Hz, H-1'a),4.46(1H,dd,J＝3.6,11.4Hz,H-1'b),3.90(3H,s,OCH 3 ),3.89(3H,s,OCH 3 ),3.86(3H,s, OCH 3 ),2.69(1H,m,H-15),2.20(1H,d,J＝13.4Hz,H-3),1.25(3H,s,H-18),1.01(3H,s,H-17), 0.88(3H,s,H-20),2.00–0.75(17H,m,CH,CH 2 in ent-beyerane skeleton)； 13 C NMR(CDCl 3 ,100 MHz)δ221.44,172.88,164.65,157.22,155.03,142.90,126.91,117.40,106.83,62.15,61.87, 61.05,57.19,57.01,56.05,52.90,50.83,48.21,45.30,40.61,39.46,38.44,37.99,37.22,35.39, 28.16,21.64,19.82,19.73,18.75,14.26；HRMS(ESI,m/z)calcd for C 31 H 42 O 8 K,581.2517 [M+K + ]；found,581.2511.
Compound 4k:
Figure BDA0002991438920000162
4k: white solid (58%), mp 109.9-110.8 ℃; [ alpha ] of] 25 D -50(c 1.0,CH 3 OH)； 1 H NMR(CDCl 3 ,400MHz) δ7.22(2H,s,2,6-Ph),4.60(1H,dd,J＝4.4,11.4Hz,H-1'a),4.53(1H,dd,J＝3.4,11.4Hz, H-1'b),3.78–3.95(9H,overlap,3×OCH 3 ),2.71(1H,d,J＝4.1Hz,H-15),2.20(1H,d,J＝13.2 Hz,H-3),1.30(3H,s,H-18),1.05(3H,s,H-17),0.87(3H,s,H-20),1.90–0.80(17H,m,CH,CH 2 in ent-beyerane skeleton)； 13 C NMR(CDCl 3 ,100MHz)δ221.26,172.89,165.84,152.97,152.97, 124.78,107.64,106.79,106.79,60.94,57.18,57.01,56.63,56.25,53.25,50.76,48.21,48.18, 45.28,43.48,40.65,38.45,37.15,35.42,28.12,21.59,19.98,19.75,18.76,14.26,13.33；HRMS (ESI,m/z)calcd for C 31 H 42 O 8 Na,565.2778[M+Na + ]；found,565.2752.
Compound 4l:
Figure BDA0002991438920000163
4l: white solid (53%), mp 149.3-150.0 ℃; [ alpha ] to] 25 D -99(c 1.1,CH 2 Cl 2 )；1H NMR(CDCl 3 ,400 MHz)δ7.57(1H,d,J＝16.0Hz,CH2＝),6.66(2H,d,J＝2.3Hz,2,6-Ph),6.49(1H,t,J＝2.3Hz, 4-Ph),6.34(1H,d,J＝16.0Hz,CH2＝),4.45(2H,m,H-1'),3.80(6H,s,2×OCH3),2.71(1H, m,H-15),2.15(1H,d,J＝13.4Hz,H-3),1.24(3H,s,H-18),1.01(3H,s,H-17),0.81(3H,s, H-20),1.85–0.80(17H,m,CH,CH 2 in ent-beyerane skeleton)；13C NMR(CDCl 3 ,150MHz)δ 221.78,182.02,166.46,161.01,161.01,145.16,136.21,118.22,106.10,106.10,102.53,62.43, 56.98,56.98,55.43,55.43,53.05,51.24,48.18,43.52,40.76,39.55,38.46,37.69,37.22,35.51, 28.84,21.55,19.90,19.64,18.76,13.26；HRMS(ESI,m/z)calcd for C 32 H 43 O 7 ,539.3009[M+H + ]； found,539.3019.
Compound 4m:
Figure BDA0002991438920000171
4m is as follows: white solid (56%), mp 87.1-87.9 ℃; [ alpha ] to] 25 D -84(c 1.3,CH 3 OH)； 1 H NMR(CDCl 3 ,400MHz) δ7.14(2H,d,J＝8.6Hz,2,6-Ph),6.84(2H,d,J＝8.6Hz,3,5-Ph),4.34(1H,dd,J＝4.9,11.4Hz, H-1'a),4.24(1H,dd,J＝3.2,11.4Hz,H-1'b),3.78(3H,s,OCH 3 ),3.52(2H,d,J＝3.5Hz, COCH 2 ),2.55(1H,m,H-15),2.16(1H,d,J＝13.5Hz,H-3),1.27(3H,s,H-18),0.88(3H,s, H-17),0.80(3H,s,H-20)；1.85–0.80(17H,m,CH,CH 2 in ent-beyerane skeleton)； 13 C NMR (CDCl 3 ,100MHz)δ221.45,172.90,171.40,158.74,130.45,130.45,125.81,114.07,114.07, 62.38,57.14,56.91,55.32,52.65,50.74,48.01,45.26,40.56,40.44,39.38,38.37,37.94,37.07, 35.23,28.14,21.53,19.66,19.63,18.74,14.17；HRMS(ESI,m/z)calcd for C 30 H 40 O 6 Na, 519.2723[M+Na + ]；found,519.2747.
Compound 4n:
Figure BDA0002991438920000172
4n: white solid (50%), mp 85.1-86.0 ℃; [ alpha ] to] 25 D -56(c11.9,CH 3 OH)； 1 H NMR(CDCl 3 ,400MHz) δ6.44(2H,s,2,6-Ph),4.38(1H,dd,J＝4.8,11.4Hz,H-1'a),4.25(1H,dd,J＝3.2,11.4Hz, H-1'b),3.84(6H,s,2×OCH 3 ),3.82(3H,s,OCH 3 ),3.60–3.43(2H,m,COCH 2 ),2.56(1H,m, H-15),2.16(1H,d,J＝13.6Hz,H-3),1.27(3H,s,H-18),0.86(3H,s,H-17),0.80(3H,s,H-20), 1.85–0.80(17H,m,CH,CH 2 in ent-beyerane skeleton)； 13 C NMR(CDCl 3 ,100MHz)δ221.35, 172.89,170.99,153.29,153.29,137.07,129.31,106.35,106.35,62.50,60.84,57.10,56.85,56.11, 56.11,52.62,50.66,48.00,45.27,41.79,40.43,39.33,38.38,37.91,37.00,35.26,28.15,21.50, 19.63,19.59,18.75,14.20；HRMS(ESI,m/z)calcd for C 32 H 44 O 8 K,595.2673[M+K + ]；found, 595.2688.
(5) Synthesis of Compound 5
Intermediate 2 (280mg, 0.8mmol), diphenylphosphorylazide (0.2mL, 0.9mmol) and Et 3 N (0.25mL, 1.8 mmol) was dissolved in dry tert-butanol (6 mL). The mixture was heated to reflux and stirred for 7 hours. After concentration in vacuo, the residue was chromatographed using a silica gel column to give 5 (231 mg, 90wt% yield) as a white solid.
(6) Synthesis of Compounds 6a-6e
The compound 5 (1 eq), 1-ethyl-3 (3-dimethylpropylamine) carbodiimide (5 eq), 4-dimethylaminopyridine (2 eq) and the corresponding acid derivative (1.5 eq) were dissolved in anhydrous dichloromethane. The mixture was stirred at room temperature and reacted for 1h. The solution was then diluted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated in vacuo, and the residue was chromatographed using a silica gel column to give pure products 6a-6e.
The structure, appearance, melting point, specific optical rotation, nuclear magnetic resonance spectrogram data and high-resolution mass spectrum of the compounds 6a-6e are shown as follows:
compound 6a:
Figure BDA0002991438920000181
6a: white solid (43%), mp 76.9-77.3 ℃; [ alpha ] to] 25 D -45(c1.6,CH 3 OH)； 1 H NMR(CDCl 3 ,400MHz) δ9.25(1H,s,2-pyrimidine),8.77(1H,dd,J＝1.8,4.9Hz,4-pyrimidine),8.32(1H,m, 6-pyrimidine),7.41(1H,dd,J＝4.9,7.9Hz,5-pyrimidine),4.59(1H,dd,J＝6.1,11.0Hz,H-1'a), 4.29(1H,dd,J＝8.2,11.0Hz,H-1'b),3.59(1H,d,J＝5.0Hz,H-16),2.47(1H,m,H-15),1.33 (3H,s,H-18),1.11(3H,s,H-17),0.94(3H,s,H-20),1.95–0.80(18H,m,CH,CH 2 in ent-beyerane skeleton)； 13 C NMR(CDCl 3 ,100MHz)δ165.56,153.48,150.94,137.08,126.13, 123.48,85.32,67.20,59.08,57.45,54.98,54.44,47.44,42.53,41.68,40.93,38.34,37.68,33.73, 32.96,31.84,24.95,20.38,19.12,17.87,13.76；HRMS(ESI,m/z)calcd for C 26 H 39 N 2 O 3 , 427.2960[M+H + ]；found,427.2951.
Compound 6b:
Figure BDA0002991438920000182
6b: white solid (33%), mp 173.4-175.2 ℃; [ alpha ] to] 25 D -54(c 0.4,CH 3 OH)； 1 H NMR(CDCl 3 ,400MHz) δ7.95(2H,d,J＝8.2Hz,3,5-Ph),6.87(2H,d,J＝8.2Hz,2,6-Ph),4.43(1H,dd,J＝6.5,11.0Hz, H-1'a),4.16(1H,dd,J＝7.7,11.0Hz,H-1'b),3.79(3H,s,OCH 3 ),3.48(1H,d,J＝5.0Hz,H-16), 2.37(1H,m,H-15),1.25(3H,s,H-18),1.05(3H,s,H-17),0.86(3H,s,H-20),1.95–0.80(18H,m, CH,CH 2 in ent-beyerane skeleton)； 13 C NMR(CDCl 3 ,100MHz)δ165.60,162.45,130.58, 130.58,121.51,112.78,112.78,84.41,65.41,58.07,56.48,54.43,53.94,53.46,46.69,41.49, 40.78,39.77,37.36,36.67,32.70,31.96,30.84,23.95,19.38,18.13,16.88,12.75；HRMS(ESI, m/z)calcd for C 28 H 42 NO 4 ,456.3114[M+H + ]；found,456.3079.
Compound 6c:
Figure BDA0002991438920000191
6c: white solid (36%), mp 164.4-165.2 ℃; [ alpha ] to] 25 D -67(c 0.7,CH 3 OH)； 1 H NMR(CDCl 3 ,400MHz) δ7.40(1H,dd,J＝1.8,7.7Hz,6-Ph),7.12(2H,m,3,4-Ph),4.63(1H,dd,J＝5.1,10.4Hz,H-1'a), 4.13(1H,t,J＝10.9Hz,H-1'b),3.91(3H,s,OCH 3 ),3.87(3H,s,OCH 3 ),3.60(1H,m,H-16), 3.08(1H,s,16-OH),2.35(1H,m,H-15),1.86(1H,dd,J＝5.2,13.8Hz,H-3),1.37(3H,s,H-18), 1.03(3H,s,H-17),0.96(3H,s,H-20),1.95–0.80(17H,m,CH,CH 2 in ent-beyerane skeleton)； 13 C NMR(CDCl 3 ,100MHz)δ166.85,154.52,153.52,125.70,124.37,122.70,116.49,86.34,67.83, 61.68,57.92,56.22,56.08,55.86,54.12,47.77,42.53,41.00,38.69,37.68,35.70,34.22,32.90, 26.95,25.04,19.70,19.14,17.43,14.29；HRMS(ESI,m/z)calcd for C 29 H 43 NO 5 Na,508.3039 [M+Na + ]；found,508.3063.
Compound 6d:
Figure BDA0002991438920000192
6d: white solid (55%), mp 51.7-52.9 ℃; [ alpha ] to] 25 D -70(c0.4,CH 3 OH)； 1 H NMR(CDCl 3 ,400MHz) δ7.71(1H,d,J＝8.9Hz,2-Ph),6.76(1H,d,J＝8.9Hz,3-Ph),4.56(1H,dd,J＝5.0,10.3Hz, H-1'a),4.18(1H,dd,J＝10.3,11.9Hz,H-1'b),3.94(3H,s,OCH 3 ),3.91(3H,s,OCH 3 ),3.85(3H, s,OCH 3 ),3.58(1H,d,J＝4.7Hz,H-16),2.40(1H,m,H-15),1.87(1H,m,H-3),1.35(3H,s, H-18),1.09(3H,s,H-17),0.97(3H,s,H-20),1.95–0.80(17H,m,CH,CH 2 in ent-beyerane skeleton)；13C NMR(CDCl 3 ,100MHz)δ166.24,157.59,154.08,142.90,127.64,117.48,107.49, 86.61,67.69,62.00,61.07,59.07,57.53,56.13,55.05,54.35,47.91,42.40,41.72,40.92,38.36, 37.68,33.78,32.98,31.88,25.08,20.48,19.22,17.87,13.64；HRMS(ESI,m/z)calcd for C 30 H 46 NO 6 ,516.3325[M+H + ]；found,516.3315.
Compound 6e:
Figure BDA0002991438920000201
6e: white solid (38%), mp 75.4-76.6 ℃; [ alpha ] to] 25 D -24(c 1.7,CH 3 OH)； 1 H NMR(CDCl 3 ,400MHz) δ7.33(2H,s,2,6-Ph),4.55(1H,dd,J＝5.5,10.9Hz,H-1'a),4.26(1H,dd,J＝8.3,10.9Hz, H-1'b),3.91(6H,s,2×OCH 3 ),3.90(3H,s,OCH 3 ),3.64(1H,d,J＝5.0Hz,H-16),2.44(1H,m, H-15),1.34(3H,s,H-18),1.10(3H,s,H-17),0.94(3H,s,H-20),1.95–0.80(18H,m,CH,CH 2 in ent-beyerane skeleton)； 13 C NMR(CDCl 3 ,100MHz)δ166.45,153.03,153.03,142.31,125.20, 106.81,106.81,85.38,66.69,60.93,59.08,57.55,56.25,56.25,55.04,54.55,47.59,42.57,41.73, 40.92,38.38,37.69,33.75,32.98,31.86,25.01,20.38,19.18,17.88,13.78；HRMS(ESI,m/z) calcd for C 30 H 46 NO 6 ,516.3325[M+H + ]；found,516.3319.
(7) Synthesis of Compounds 7a-7e
Compounds 6a-6e (1 eq) and pyridinium dichromate (3 eq) were dissolved in anhydrous N, N-dimethylformamide. The mixture was stirred at room temperature and reacted for 24h. The mixture was diluted with ethyl acetate, the solution was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated, and the residue was chromatographed using a silica gel column to give pure products 7a-7e.
The structures, appearances, melting points, specific optical rotations, nuclear magnetic resonance spectrogram data and high-resolution mass spectrums of the compounds 7a to 7e are as follows:
compound 7a:
Figure BDA0002991438920000202
7a: white solid (57%), mp 124.8-125.6 ℃; [ alpha ] to] 25 D -48(c 0.9,CH 3 OH)； 1 H NMR(CDCl 3 ,400MHz) δ9.18(1H,s,2-pyrimidine),8.78(1H,d,J＝4.9Hz,4-pyrimidine),8.26(1H,d,J＝8.0Hz, 6-pyrimidine),7.42(1H,dd,J＝4.9,8.0Hz,5-pyrimidine),4.67(1H,dd,J＝6.6,11.6Hz,H-1'a), 4.50(1H,dd,J＝3.3,11.6Hz,H-1'b),2.91(1H,m,H-15),1.96(1H,d,J＝13.8Hz,H-3),1.35 (3H,s,H-18),1.07(3H,s,H-17),1.02(3H,s,H-20),1.95–0.80(17H,m,CH,CH 2 in ent-beyerane skeleton)； 13 C NMR(CDCl 3 ,100MHz)δ220.92,165.03,153.65,150.83,137.00, 125.69,123.54,63.28,58.97,56.74,55.12,53.39,51.39,48.19,41.43,40.48,38.31,37.79,36.88, 34.40,31.81,19.93,19.93,19.23,17.86,14.14；HRMS(ESI,m/z)calcd for C 26 H 37 N 2 O 3 , 425.2804[M+H + ]；found,425.2797.
Compound 7b:
Figure BDA0002991438920000211
7b: white solid (52%), mp 118.1-119.5 ℃; [ alpha ] to] 25 D -48(c 2.7,CH 3 OH)； 1 H NMR(CDCl 3 ,400MHz) δ7.95(2H,d,J＝8.7Hz,3,5-Ph),6.93(2H,d,J＝8.7Hz,2,6-Ph),4.56(1H,dd,J＝7.1,11.5Hz, H-1'a),4.42(1H,dd,J＝2.9,11.5Hz,H-1'b),3.85(3H,s,OCH 3 ),2.92(1H,m,H-15),1.96(1H, m,H-3),1.34(3H,s,H-18),1.09(3H,s,H-17),1.01(3H,s,H-20),1.95–0.80(17H,m,CH,CH 2 in ent-beyerane skeleton)； 13 C NMR(CDCl 3 ,100MHz)δ221.28,166.10,163.55,131.59,131.59, 122.12,113.84,113.84,62.70,58.98,56.78,55.45,55.11,53.36,51.85,48.11,41.53,40.55,38.34, 37.80,36.93,34.44,31.83,19.97,19.93,19.24,17.90,14.16；HRMS(ESI,m/z)calcd for C 28 H 40 NO 4 ,454.2957[M+H + ]；found,454.2945.
Compound 7c:
Figure BDA0002991438920000212
7c: white solid (53%), mp 152.3-153.2 ℃; [ alpha ] to] 25 D -89(c 0.8,CH 3 OH)； 1 H NMR(CDCl 3 ,400MHz) δ7.28(1H,m,6-Ph),7.13–7.05(2H,overlap,3,4-Ph),4.55(1H,dd,J＝6.9,11.5Hz,H-1'a),4.44 (1H,dd,J＝3.4,11.5Hz,H-1'b),3.89(3H,s,OCH 3 ),3.88(3H,s,OCH 3 ),2.82(1H,m,H-15), 1.34(3H,s,H-18),0.99(3H,s,H-17),0.99(3H,s,H-20),2.00–0.75(18H,m,CH,CH 2 in ent-beyerane skeleton)； 13 C NMR(CDCl 3 ,100MHz)δ221.04,165.68,154.55,153.65,125.53, 123.91,122.04,116.02,62.74,61.74,57.10,56.09,56.00,53.44,52.90,51.47,48.14,40.52,38.69, 37.79,36.91,35.66,34.84,26.95,19.81,19.19,19.05,17.44,14.66；HRMS(ESI,m/z)calcd for C 29 H 42 NO 5 ,484.3063[M+H + ]；found,484.3082.
Compound 7d:
Figure BDA0002991438920000213
7d: white solid (57%), mp 109.2-109.9 ℃; [ alpha ] to] 25 D -70(c1.0,CH 3 OH)； 1 H NMR(CDCl 3 ,400MHz) δ7.55(1H,d,J＝8.9Hz,6-Ph),6.71(1H,d,J＝8.9Hz,5-Ph),4.52(1H,dd,J＝6.7,11.5Hz, H-1'a),4.43(1H,dd,J＝3.3,11.5Hz,H-1'b),3.92(3H,s,OCH 3 ),3.89(3H,s,OCH 3 ),3.87(3H,s, OCH 3 ),2.88(1H,m,H-15),1.26(3H,s,H-18),1.06(3H,s,H-17),1.00(3H,s,H-20),2.00–0.75 (18H,m,CH,CH 2 in ent-beyerane skeleton)； 13 C NMR(CDCl 3 ,100MHz)δ221.35,164.71, 157.31,155.10,142.96,126.72,117.28,106.92,62.46,61.88,61.04,58.97,56.82,56.06,55.11, 53.15,51.63,48.11,41.60,40.51,38.35,37.79,36.97,34.41,31.85,29.71,19.84,19.24,17.89, 14.12；HRMS(ESI,m/z)calcd for C 30 H 44 NO 6 ,514.3168[M+H + ]；found,514.3148.
Compound 7e:
Figure BDA0002991438920000221
7e: white solid (57%), mp 64.0-64.8 ℃; [ alpha ] to] 25 D -66(c 1.1,CH 3 OH)； 1 H NMR(CDCl 3 ,400MHz) δ7.24(2H,s,2,6-Ph),4.62(1H,m,H-1'a),4.50(1H,m,H-1'b),3.89(9H,overlap,3×OCH 3 ), 2.86(1H,m,H-15),1.35(3H,s,H-18),1.05(3H,s,H-17),1.03(3H,s,H-20),2.0–0.80(18H,m, CH,CH 2 in ent-beyerane skeleton)； 13 C NMR(CDCl 3 ,100MHz)δ221.20,165.92,153.00, 153.00,142.37,124.73,106.75,106.75,62.91,60.93,59.00,56.94,56.19,56.19,55.09,53.44, 51.39,48.14,41.73,40.52,38.38,37.80,36.94,34.36,31.81,19.95,19.95,19.27,17.85,14.09； HRMS(ESI,m/z)calcd for C 30 H 44 NO 6 ,514.3168[M+H + ]；found,514.3158.
Example 2
This example is a zebrafish experiment of a series of isosteviol derivatives prepared in the examples, and cardiac activity of isosteviol derivatives of the present invention was evaluated through the zebrafish experiment.
(1) Zebra fish culture and embryo collection: zebra fish (3-12 months old) are purchased from the national zebra fish resource center, and are cultured in a mobile breeding box with the photoperiod of 14 h (illumination: dark illumination) and the temperature of 28.5 +/-1 ℃, and live saturated salt water shrimps are fed twice a day; zebrafish (male: 1:1) were mated, embryos were collected, washed with Holt Buffer, cultured in an incubator (28.5 ± 1 ℃) for 24h, and screened by microscopic examination.
(2) Screening the medicaments by a DOX-induced zebra fish embryonic heart failure model: 24hpf wild type zebrafish embryos are distributed into 24-well plates (20 embryos/well), 1mL of DOX (100. Mu.M) and various test compounds (5, 15, 40. Mu.M) are added, the embryos are treated for 72h, the survival rate and abnormal conditions of the embryos are observed under an inverted fluorescence microscope (Zeiss, germany), the zebrafish lack movement or the atria and ventricles lose contractility and are considered dead, and the survival rate is taken as the evaluation criterion of the activity of the compounds, as shown in figure 1 (the specific data are detailed in Table 1), and the compounds 7d and 7e can effectively improve the survival rate of the zebrafish in a DOX model, so the DOX-induced zebrafish embryo heart function evaluation study is carried out on the 24hpf wild type zebrafish embryos. In addition, compounds 1,4j,4k,7d,7e and LSD showed the best efficiency at 10, 70, 60, 40, 60 and 50 μ M, respectively (fig. 2).
TABLE 1 Effect of isosteviol derivatives on DOX-induced survival of Zebra fish embryos
Figure BDA0002991438920000231
(3) Zebra fish embryos were tested for drug toxicity: 24hpf wild type zebrafish embryos are distributed into 96 well plates (1 embryo/well), 200mL of the desired concentration of test compound is added, exposed for 72h, embryo viability is observed under an inverted fluorescence microscope (zeiss, germany), and in order to test whether compounds (1,4j, 4k,7d,7e, lsd) cause any toxicity, embryos are treated individually with different concentrations of the test compound. As shown in fig. 2, all test compounds did not cause death of zebrafish even at concentrations much higher than the effective dose.
(4) Evaluation of heart function of zebra fish embryos induced by DOX: the 24hpf transgene Tg [ myl: EGFP]Zebra fishEmbryos were distributed into 24-well plates (20 embryos/well), 1mL DOX (80 μ M) and various test compounds (1,4j, 4k,7d,7e, lsd) (corresponding to optimal concentrations), treated for 48h, cardiac status and various cardiac markers of zebrafish were recorded under an upright fluorescence microscope (zeiss, germany), zebrafish 20s were photographed and recorded, long axis (a) and short axis (b) at end systole and end diastole were measured and the formula v =4/3 pi ab was used 2 The ventricular volume is calculated. Stroke volume is the difference between the end diastolic volume and the end systolic volume. The stroke volume is the product of stroke volume and heart rate. The shrinkage fraction (%) is calculated as: (diastolic minor axis-systolic minor axis)/(diastolic minor axis) × 100%; as shown in fig. 3, zebrafish (model group) with DOX exposure alone had significantly reduced systolic fraction, stroke volume and stroke volume, with a moderate drop in heart rate, indicating abnormal ventricular filling and systolic dysfunction. The optimal dosage of the DOX and the test article (1,4j, 4k,7d,7e, LSD) is combined for application, so that the heart edema caused by the DOX can be obviously reduced, the normal heart shape is maintained, and the heart function damage caused by the DOX is improved. Of all compounds tested, 7d showed the most significant efficiency. Myocardial protective Activity ranked 7d>7e>LSD>4j>1>4k, respectively. In addition, cardioprotection was evaluated for 7d at 20, 40 and 60 μ M. As shown in figure 4, at 60 μ M concentration, 7d did not rescue the heart as effectively as at 40 μ M, confirming our dose response results, i.e. 7d performed the best therapeutic effect at 40 μ M.
TABLE 2 protective Effect of isosteviol derivatives on DOX-induced cardiac toxicity of Zebra fish
Figure BDA0002991438920000241
(5) Quantitative polymerase chain reaction 20 embryos treated for 7d were subjected to RNA extraction using standard TRIzol protocol (general Biotech) and RNA concentration and quality was determined using a Nanodrop2000S Spectrophotometer (Thermo-Scientific). qRT-PCR amplification of total RNA was performed with a two-step RT-PCR kit (Nanjing Novozam). The qRT-PCR analysis was repeated at least three times. The qPCR primers were as follows: beta-actin L, CCT ACT AAT ACA CAG CCA TGG ATG A; β -actin R, GTC CCA TGC CAA CCA TCA C; cTnT-L, GTC TGC ACT TCG GCG GTT ACA; cTnT-R, AGG TAA AAT CTA TAT TGT TCA GTG AAC AAC CG; ANP-L, ATG GCC GGG GGA CTA ATT CT; ANP-R, AGA GTT GCA ACC GAG GGT GC; as shown in figure 5, DOX exposure resulted in significant increases in mRNA levels of ANP and CTnT, and the 7d derivatives were able to significantly reduce ANP and CTnT levels, and were dose-dependent, these results provide strong molecular evidence for the cardioprotective efficacy of 7 d.
(6) Cell viability assay: h9c2 cells were cultured in DMEM medium containing 10% fetal bovine serum, 100U/mL penicillin and 100 mg/mL streptomycin in a humidified incubator at 37 ℃ and 5% CO2. Cells were used until 70% fusion was achieved. Cell viability was determined using cell counting kit-8 (CCK 8). Briefly, H9c2 cells were cultured in 96-well plates containing CCK-8 (0.5 mg/mL) medium for 4 hours after 48 hours of DOX treatment with or without test compound. The optical density was measured at 450nm with a microplate reader.
(7) Evaluation of intracellular Reactive Oxygen Species (ROS): DCFH-DA was used to measure intracellular reactive oxygen species. Briefly, H9c2 cells seeded in 24-well plates were treated with or without test compounds for 48 hours. DCFH-DA diluted with DMEM to a final concentration of 5. Mu. Mol/L was added to H9C2 cells. The mixture was incubated at 37 ℃ for 40 minutes. The dye was then removed and the wells were washed three times with phosphate buffered saturated saline (PBS). The fluorescence intensity was then measured with a confocal microscope (Carl-Zeiss) to determine the reactive oxygen levels. As shown in fig. 6, the fluorescence intensity of DOX-treated H9c2 cells was higher compared to the control group, indicating that DOX induced ROS overproduction. The combined treatment of 7d and DOX significantly reduced the fluorescence intensity and was dose dependent. These results clearly indicate that 7d can reduce the overproduction of ROS, thereby protecting cells from oxygen stress damage.
(8) Measurement of mitochondrial membrane potential (. DELTA.. Psi.m) mitochondrial membrane potential was measured using cationic JC-1 (Sigma-Aldrich) dye: h9c2 cells seeded in confocal culture dishes were treated with or without test compound for 48 hours. JC-1 working solution prepared to a final concentration of 1. Mu.g/mL was added to H9c2 cells at 500. Mu.L/well. The mixture was incubated at 37 ℃ for 20 minutes. The dye was then removed and the wells were washed three times with PBS. H9c2 cells were observed with a Carl Zeiss confocal microscope (Carl-Zeiss) and the ratio of the red and green fluorescence intensities was analyzed to determine. DELTA.psi. As shown in fig. 7, after DOX treatment, Δ ψ m of cells was significantly reduced, and 7d dose-dependent co-treatment restored Δ ψ m, and 7d was effective in alleviating DOX-induced oxidative stress, restoring mitochondrial membrane potential, and maintaining mitochondrial morphology, thereby protecting cells from damage.
The experimental results show that the compound has a remarkable heart protection effect, and the potential and the form of mitochondria are recovered by inhibiting the excessive accumulation of ROS, so that the death and the injury of myocardial cells are protected, and the compound is worthy of further development as a potential heart protection clinical test candidate.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are merely exemplary embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims (5)

1. An isosteviol derivative having the structure:
Figure 657618DEST_PATH_IMAGE001
、
Figure 25145DEST_PATH_IMAGE002
、
Figure 266770DEST_PATH_IMAGE003
or
Figure 479577DEST_PATH_IMAGE004
。
2. The isosteviol derivative according to claim 1, which is any one of the following compounds:
Figure 123048DEST_PATH_IMAGE003
or
Figure 395898DEST_PATH_IMAGE004
。
3. A process for the preparation of isosteviol derivative of claim 1~2 comprising the steps of:
s1, carrying out hydroxymethylation reaction on isosteviol and polyformaldehyde under an alkaline condition at the temperature of 60-100 ℃ to obtain a compound 2 with a structural formula shown as a formula (II):
Figure 124819DEST_PATH_IMAGE005
formula (II);
s2, acylating the 15-beta-hydroxymethyl group of the compound 2 obtained in the step S1 to obtain a compound 3 shown in a formula (III);
or reacting compound 2 obtained in S1 with diphenylphosphoryl azide and Et 3 N is rearranged by Curtius to convert 19-COOH into primary amine to obtain a compound 5 shown as a formula (V), and further acylation is carried out on 15-beta-hydroxymethyl of the compound 5 to obtain a compound 6 shown as a formula (VI);
Figure 203634DEST_PATH_IMAGE006
Figure 373715DEST_PATH_IMAGE007
Figure 145362DEST_PATH_IMAGE008
formula (III), formula (V), formula (VI);
s3, oxidizing the 16-alpha hydroxyl of the compound 3 obtained in the S2 to obtain a 16-position ketone derivative 4 shown in a formula (IV);
or, oxidizing the 16-alpha hydroxyl of the compound 6 obtained from S2 to obtain a 16-position ketone derivative 7 thereof, as shown in formula (VII):
Figure 299263DEST_PATH_IMAGE009
Figure 181768DEST_PATH_IMAGE010
formula (IV) formula (VII);
the acylation in step S2 is carried out by esterifying 15-beta-hydroxymethyl with an acid compound, wherein the acid compound is 2,3,4-trimethoxy benzoic acid or 3,4,5-trimethoxy benzoic acid.
4. The preparation method according to claim 3, wherein the molar ratio of the isosteviol to the aldehyde group in the polyoxymethylene in the step S1 is 1 to 5-8.
5. Use of the isosteviol derivative of claim 1~2 in the manufacture of a medicament for the treatment of cardiovascular disease.
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