CN113105348A - Isosteviol derivative and preparation method and application thereof - Google Patents

Isosteviol derivative and preparation method and application thereof Download PDF

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CN113105348A
CN113105348A CN202110316315.7A CN202110316315A CN113105348A CN 113105348 A CN113105348 A CN 113105348A CN 202110316315 A CN202110316315 A CN 202110316315A CN 113105348 A CN113105348 A CN 113105348A
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isosteviol
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CN113105348B (en
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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)1Selected from hydroxy or carbonyl; r2Selected from H, C1～5Saturated straight-chain alkyl radical, C3～6Saturated cyclic alkyl, pyridine ring, phenyl, benzyl/substituted benzyl, phenethyl/substituted phenethyl, phenylpropyl/substituted phenylpropyl; r3Selected 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 each year from 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 acidic hydrolysis of stevioside through Wagner-Meerwein rearrangement, and has the following structure:
Figure BDA0002991438920000011
researches show that the compound has broad-spectrum pharmacological effects including antidiabetic, anticancer, anti-inflammatory, antihyperglycemic, antihypertensive, antidiarrheal, 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 carried out 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 medicines.
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 present invention is to provide a method for preparing 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 achieve the purpose, the invention adopts the following technical scheme:
an isosteviol derivative having the structure shown in formula (I):
Figure BDA0002991438920000021
in the formula (I), R1Independently selected from hydroxy or carbonyl;
R2selected from H, C1～5Saturated straight-chain alkyl radical, C3～6Saturated cyclic alkyl, pyridine ring, phenyl, benzyl/substituted benzyl, phenethyl/substituted phenethyl, phenylpropyl/substituted phenylpropyl;
R3independently selected from carboxyl or primary amine.
Isosteviol, a pharmaceutical compound, has multiple reactive active sites, and can be used for treating different diseases by modifying the reactive active sites. The research of the invention finds that the specific modification (such as R) is carried out on the specific active site of isosteviol1、R2And R3) The obtained derivative has good heart protection activity; further, through the molecular mechanism research of H9c2 cells, the isosteviol derivative is found to protect the death and damage of myocardial cells mainly through inhibiting the overproduction of active oxygen, restoring mitochondrial membrane potential and maintaining mitochondrial morphology.
Preferably, said R is2Is substituted benzyl, substituted phenethyl or substituted phenylpropyl.
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.
Go toPreferably, said R2Is any one of the following groups:
Figure BDA0002991438920000031
preferably, R1Is a carbonyl group; r2Is substituted benzyl, substituted phenethyl or substituted phenylpropyl; r3 is a 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 at 60-100 ℃ under an alkaline condition to obtain a compound 2 (the compound 2 has a 15 beta-hydroxymethyl structure) with a structural formula shown as 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 Et3N 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 step 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 in S2 to obtain a 16-position ketone derivative 7 thereof, as shown in the 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 aldehyde groups in the polyformaldehyde in the step S1 is 1: 5-8; further 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 that 15-beta-hydroxymethyl is esterified with 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)Ac2O,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, Et3N,tBuOH,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 anhydrous ethanol (2mL), 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(138mg, yield 58 wt%) as a white solid.
(2) Synthesis of Compound 3a
Intermediate 2(200mg, 0.6mmol), acetic anhydride (0.07mL, 0.7mmol) and 4-dimethylaminopyridine (73.3mg, 0.6mmol) were dissolved in anhydrous pyridine (5mL) and the reaction was stirred at room temperature for 2 h. 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 (78mg, 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 3 a:
Figure BDA0002991438920000062
3 a: white solid (35%), mp 157.0-158.4 ℃; [ alpha ] to]25 D-87(c 0.6,CH3OH)；1H NMR(CDCl3,400MHz) 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,COCH3),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,CH2 in ent-beyerane skeleton)；13C NMR(CDCl3,100MHz)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 C23H36O5Na,415.2461[M+Na+]；found,415.2455.
(3) Synthesis of Compound 3b-3n
The intermediate 2(1eq), 1-ethyl-3 (3-dimethylpropylamine) carbodiimide (5eq), 4-dimethylaminopyridine (2eq) and the corresponding acid derivative (1.5eq) were dissolved in anhydrous dichloromethane. The mixture was stirred at room temperature and reacted for 1 h. 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-3 n.
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 3 b:
Figure BDA0002991438920000071
3 b: white solid (35%), mp 91.1-91.5 ℃; [ alpha ] to]25 D-57(c 1.0,CH3OH)；1H NMR(CDCl3,400MHz) 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,CH2 in ent-beyerane skeleton or carbon chain)；13C NMR (CDCl3,100MHz)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 C28H44O5Na,483.3087[M+Na+]；found, 483.3091.
Compound 3 c:
Figure BDA0002991438920000072
3 c: white solid (33%), mp 113.5-114.0 deg.C; [ alpha ] to]25 D-55(c 1.4,CH2Cl2)；1H NMR(CDCl3,400MHz) 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,CH2in ent-beyerane skeleton)；13C NMR(CDCl3,100MHz)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 C27H38NO5,456.2750[M+H+]；found, 456.2740.
Compound 3 d:
Figure BDA0002991438920000081
3 d: white solid (32%), mp 139.2-140.4 ℃; [ alpha ] to]25 D-44(c 1.6,CH3OH)；1H NMR(CDCl3,400MHz) 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,CH2 in ent-beyerane skeleton)；13C NMR(CDCl3,100MHz) 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 C27H38NO5,456.2750[M+H+]；found,456.2738.
Compound 3 e:
Figure BDA0002991438920000082
3 e: white solid (42%), mp 96.7-96.9 ℃; [ alpha ] to]25 D-54(c1.1,CH3OH)；1H NMR(CDCl3,400MHz) 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,CH2 in ent-beyerane skeleton)；13C NMR(CDCl3,150MHz)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 C28H37O5,453.2641[M-H+]；found,453.2650.
Compound 3 f:
Figure BDA0002991438920000083
3 f: white solid (35%), mp 108.1-109.1 ℃; [ alpha ] to]25 D-59(c1.4,CH3OH)；1H NMR(CDCl3,400MHz) 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,CH2 in ent-beyerane skeleton)；13C NMR(CDCl3,100MHz)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 C28H37FO5Na,495.2523[M+Na+]； found,495.2539.
Compound 3 g:
Figure BDA0002991438920000091
3 g: white solid (27%), mp 201.7-202.5 ℃; [ alpha ] to]25 D-53(c 0.3,CH3OH)；1H NMR(CDCl3,400MHz) 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,OCH3),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,CH2 in ent-beyerane skeleton)；13C NMR(CDCl3,100MHz) 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 C29H40O6Na,507.2723[M+Na+]；found, 507.2711.
Compound 3 h:
Figure BDA0002991438920000092
3 h: white solid (35%), mp 112.0-113.5 ℃; [ alpha ] to]25 D-52(c 1.1,CH3OH)；1H NMR(CDCl3,400MHz) 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×OCH3),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,CH2 in ent-beyerane skeleton)；13C NMR (CDCl3,150MHz)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 C30H42O7Na, 537.2829[M+Na+]；found,537.2843.
Compound 3 i:
Figure BDA0002991438920000101
3 i: white solid (40%), mp 169.3-170.8 ℃; [ alpha ] to]25 D-65(c 1.0,CH3OH)；1H NMR(CDCl3,400MHz) 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,OCH3),3.92(3H,s, OCH3),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,CH2 in ent-beyerane skeleton)；13C NMR(CDCl3,100MHz)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 C30H42O7K,553.2568[M+K+]；found,553.2562.
Compound 3 j:
Figure BDA0002991438920000102
3 j: white solid (23%), mp 96.6-97.2 ℃; [ alpha ] to]25 D-54(c2.1,CH3OH)；1H NMR(CDCl3,400MHz) 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,OCH3),3.82(3H,s,OCH3),3.77(3H,s,OCH3),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,CH2 in ent-beyerane skeleton)；13C NMR(CDCl3,100MHz)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 C31H44O8Na,567.2934[M+Na+]；found,567.2909.
Compound 3 k:
Figure BDA0002991438920000103
3 k: white solid (31%), mp 114.4-115.8 ℃; [ alpha ] to]25 D-35(c 1.0,CH3OH)；1H NMR(CDCl3,400MHz) 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,OCH3),3.90(6H,s,2×OCH3),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,CH2 in ent-beyerane skeleton)；13C NMR(CDCl3,100MHz)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 C31H44O8K,583.2673[M+K+](ii) a found,583.2665 compound 3 l:
Figure BDA0002991438920000111
3 l: white solid (39%), mp 127.3-128.5 ℃; [ alpha ] to]25 D-56(c 1.9,CH2Cl2)；1HNMR(CDCl3,400MHz) δ7.61(1H,d,J＝16.0Hz,CH2＝),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,CH2＝),4.33(1H,dd,J＝5.0,10.8Hz,H-1'a),4.14(1H,m,H-1'b),3.81 (6H,s,2×OCH3),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,CH2 in ent-beyerane skeleton)；13C NMR(CDCl3,100MHz)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 C32H44O7Na,563.2985[M+Na+]；found,563.2996.
Compound 3 m:
Figure BDA0002991438920000112
3 m: white solid (30%), mp 75.0-76.4 ℃; [ alpha ] to]25 D-60(c 0.9,CH3OH)；1HNMR(CDCl3,400MHz) 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,OCH3),3.59(2H,s,COCH2),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,CH2 in ent-beyerane skeleton)；13C NMR(CDCl3,100 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 C30H43O6,499.3059[M+H+]；found, 499.3080.
Compound 3 n:
Figure BDA0002991438920000121
3 n: white solid (34%), mp 174.0-175.4 deg.C; [ alpha ] to]25 D-51(c2.6,CH2Cl2)；1H NMR(CDCl3,400MHz) 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×OCH3),3.82(3H,s,OCH3),3.59(2H,s,COCH2),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,CH2 in ent-beyerane skeleton)；13C NMR(CDCl3,100MHz)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 C32H45O8,557.3115[M-H+]；found, 557.3111.
(4) Synthesis of Compounds 4a-4n
Compounds 3a-3N (1eq) and pyridinium dichromate (3eq) were dissolved in anhydrous N, N-dimethylformamide. The mixture was stirred at room temperature and reacted for 24 h. 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-4 n.
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 4 a:
Figure BDA0002991438920000122
4 a: white solid (47%), mp 159.6-160.3 ℃; [ alpha ] to]25 D-30(c 1.4,CH2Cl2)；1H NMR(CDCl3,400MHz) 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,COCH3), 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,CH2 in ent-beyerane skeleton)；13C NMR(CDCl3,100MHz,)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 C23H34O5Na,413.2304[M+Na+]；found, 413.2301.
Compound 4 b:
Figure BDA0002991438920000131
4 b: white solid (45%), mp 145.3-146.6 ℃; [ alpha ] to]25 D-68(c 2.4,CH2Cl2)；1H NMR(CDCl3,400MHz) 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,CH2 in ent-beyerane skeleton or carbon chain)；13C NMR(CDCl3, 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 C28H43O5,459.3110[M+H+]；found,459.3090.
Compound 4 c:
Figure BDA0002991438920000132
4 c: white solid (66%), mp 108.9-109.6 ℃; [ alpha ] to]25 D-40(c 1.1,CH3OH)；1H NMR(CDCl3,400MHz) 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,CH2 in ent-beyerane skeleton)；13C NMR(CDCl3,100MHz)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 C27H35NO5Na,476.2413[M+Na+]；found, 476.2382.
Compound 4 d:
Figure BDA0002991438920000133
4 d: white solid (35%), mp 72.3-72.9 ℃; [ alpha ] to]25 D-56(c 2.0,CH3OH)；1H NMR(CDCl3,400MHz) 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,CH2 in ent-beyerane skeleton)；13C NMR(CDCl3,150MHz)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 C27H35NO5K,492.2152[M+K+]；found,492.2167.
Compound 4 e:
Figure BDA0002991438920000141
4 e: white solid (58%), mp 98.0-98.9 ℃; [ alpha ] to]25 D-79(c 1.7,CH3OH)；1H NMR(CDCl3,400MHz) 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,CH2 in ent-beyerane skeleton)；13C NMR(CDCl3,100MHz)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 C28H36O5Na,475.2461[M+Na+]；found,475.2465.
Compound 4 f:
Figure BDA0002991438920000142
4 f: white solid (63%), mp 205.4-206.2 ℃; [ alpha ] to]25 D-90(c 1.1,CH2Cl2)；1H NMR(CDCl3,400MHz) 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,CH2in ent-beyerane skeleton)；13C NMR(CDCl3,100MHz)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 C28H35FO5Na,493.2367 [M+Na+]；found,493.2342.
Compound 4 g:
Figure BDA0002991438920000143
4 g: white solid (41%), mp 202.2-202.9 ℃; [ alpha ] to]25 D-80(c 1.5,CH2Cl2)；1H NMR(CDCl3,400MHz) 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,OCH3),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,CH2 in ent-beyerane skeleton)；13C NMR(CDCl3,100MHz)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 C29H38O6Na,505.2566[M+Na+]；found,505.2575.
Compound 4 h:
Figure BDA0002991438920000151
4 h: white solid (30%), mp 117.3-118.6 ℃; [ alpha ] to]25 D-67(c 2.2,CH3OH)；1H NMR(CDCl3,400MHz) 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×OCH3),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,CH2 in ent-beyerane skeleton)；13C NMR(CDCl3,100MHz)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 C30H39O7,511.2696[M-H+]；found,511.2647.
Compound 4 i:
Figure BDA0002991438920000152
4 i: white solid (58%), mp 105.3-106.0 ℃; [ alpha ] to]25 D-72(c 0.7,CH3OH)；1H NMR(CDCl3,400MHz) 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, OCH3),3.90(3H,s,OCH3),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,CH2 in ent-beyerane skeleton)；13C NMR(CDCl3,150MHz)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 C30H40O7K,551.2411[M+K+]；found,551.2438.
Compound 4 j:
Figure BDA0002991438920000161
4 j: white solid (70%), mp 96.7-97.6 ℃; [ alpha ] to]25 D-54(c1.1,CH2Cl2)；1H NMR(CDCl3,400MHz) 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,OCH3),3.89(3H,s,OCH3),3.86(3H,s, OCH3),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,CH2 in ent-beyerane skeleton)；13C NMR(CDCl3,100 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 C31H42O8K,581.2517 [M+K+]；found,581.2511.
Compound 4 k:
Figure BDA0002991438920000162
4 k: white solid (58%), mp 109.9-110.8 ℃; [ alpha ] to]25 D-50(c 1.0,CH3OH)；1H NMR(CDCl3,400MHz) 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×OCH3),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,CH2 in ent-beyerane skeleton)；13C NMR(CDCl3,100MHz)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 C31H42O8Na,565.2778[M+Na+]；found,565.2752.
Compound 4 l:
Figure BDA0002991438920000163
4 l: white solid (53%), mp 149.3-150.0 ℃; [ alpha ] to]25 D-99(c 1.1,CH2Cl2)；1H NMR(CDCl3,400 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,CH2 in ent-beyerane skeleton)；13C NMR(CDCl3,150MHz)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 C32H43O7,539.3009[M+H+]； found,539.3019.
Compound 4 m:
Figure BDA0002991438920000171
4 m: white solid (56%), mp 87.1-87.9 ℃; [ alpha ] to]25 D-84(c 1.3,CH3OH)；1H NMR(CDCl3,400MHz) 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,OCH3),3.52(2H,d,J＝3.5Hz, COCH2),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,CH2 in ent-beyerane skeleton)；13C NMR (CDCl3,100MHz)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 C30H40O6Na, 519.2723[M+Na+]；found,519.2747.
Compound 4 n:
Figure BDA0002991438920000172
4 n: white solid (50%), mp 85.1-86.0 deg.C; [ alpha ] to]25 D-56(c11.9,CH3OH)；1H NMR(CDCl3,400MHz) 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×OCH3),3.82(3H,s,OCH3),3.60–3.43(2H,m,COCH2),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,CH2 in ent-beyerane skeleton)；13C NMR(CDCl3,100MHz)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 C32H44O8K,595.2673[M+K+]；found, 595.2688.
(5) Synthesis of Compound 5
Intermediate 2(280mg, 0.8mmol), diphenylphosphorylazide (0.2mL, 0.9mmol) and Et3N (0.25mL, 1.8mmol) 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(231mg, 90 wt% yield) as a white solid.
(6) Synthesis of Compounds 6a-6e
The compound 5(1eq), 1-ethyl-3 (3-dimethylpropylamine) carbodiimide (5eq), 4-dimethylaminopyridine (2eq) and the corresponding acid derivative (1.5eq) were dissolved in anhydrous dichloromethane. The mixture was stirred at room temperature and reacted for 1 h. 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 products 6a-6 e.
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 6 a:
Figure BDA0002991438920000181
6 a: white solid (43%), mp 76.9-77.3 ℃; [ alpha ] to]25 D-45(c1.6,CH3OH)；1H NMR(CDCl3,400MHz) 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,CH2 in ent-beyerane skeleton)；13C NMR(CDCl3,100MHz)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 C26H39N2O3, 427.2960[M+H+]；found,427.2951.
Compound 6 b:
Figure BDA0002991438920000182
6 b: white solid (33%), mp 173.4-175.2 ℃; [ alpha ] to]25 D-54(c 0.4,CH3OH)；1H NMR(CDCl3,400MHz) 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,OCH3),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,CH2 in ent-beyerane skeleton)；13C NMR(CDCl3,100MHz)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 C28H42NO4,456.3114[M+H+]；found,456.3079.
Compound 6 c:
Figure BDA0002991438920000191
6 c: white solid(36％)，mp 164.4–165.2℃；[α]25 D-67(c 0.7,CH3OH)；1H NMR(CDCl3,400MHz) 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,OCH3),3.87(3H,s,OCH3),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,CH2 in ent-beyerane skeleton)；13C NMR(CDCl3,100MHz)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 C29H43NO5Na,508.3039 [M+Na+]；found,508.3063.
Compound 6 d:
Figure BDA0002991438920000192
6 d: white solid (55%), mp 51.7-52.9 ℃; [ alpha ] to]25 D-70(c0.4,CH3OH)；1H NMR(CDCl3,400MHz) 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,OCH3),3.91(3H,s,OCH3),3.85(3H, s,OCH3),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,CH2 in ent-beyerane skeleton)；13C NMR(CDCl3,100MHz)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 C30H46NO6,516.3325[M+H+]；found,516.3315.
Compound 6 e:
Figure BDA0002991438920000201
6 e: white solid (38%), mp 75.4-76.6 ℃; [ alpha ] to]25 D-24(c 1.7,CH3OH)；1H NMR(CDCl3,400MHz) 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×OCH3),3.90(3H,s,OCH3),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,CH2 in ent-beyerane skeleton)；13C NMR(CDCl3,100MHz)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 C30H46NO6,516.3325[M+H+]；found,516.3319.
(7) Synthesis of Compounds 7a-7e
Compounds 6a-6e (1eq) and pyridinium dichromate (3eq) were dissolved in anhydrous N, N-dimethylformamide. The mixture was stirred at room temperature and reacted for 24 h. 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-7 e.
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 7 a:
Figure BDA0002991438920000202
7 a: white solid (57%), mp 124.8-125.6 ℃; [ alpha ] to]25 D-48(c 0.9,CH3OH)；1H NMR(CDCl3,400MHz) 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,CH2 in ent-beyerane skeleton)；13C NMR(CDCl3,100MHz)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 C26H37N2O3, 425.2804[M+H+]；found,425.2797.
Compound 7 b:
Figure BDA0002991438920000211
7 b: white solid (52%), mp 118.1-119.5 ℃; [ alpha ] to]25 D-48(c 2.7,CH3OH)；1H NMR(CDCl3,400MHz) 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,OCH3),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,CH2 in ent-beyerane skeleton)；13C NMR(CDCl3,100MHz)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 C28H40NO4,454.2957[M+H+]；found,454.2945.
Compound 7 c:
Figure BDA0002991438920000212
7 c: white solid (53%), mp 152.3-153.2 ℃; [ alpha ] to]25 D-89(c 0.8,CH3OH)；1H NMR(CDCl3,400MHz) 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,OCH3),3.88(3H,s,OCH3),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,CH2 in ent-beyerane skeleton)；13C NMR(CDCl3,100MHz)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 C29H42NO5,484.3063[M+H+]；found,484.3082.
Compound 7 d:
Figure BDA0002991438920000213
7 d: white solid (57%), mp 109.2-109.9 ℃; [ alpha ] to]25 D-70(c1.0,CH3OH)；1H NMR(CDCl3,400MHz) 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,OCH3),3.89(3H,s,OCH3),3.87(3H,s, OCH3),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,CH2 in ent-beyerane skeleton)；13C NMR(CDCl3,100MHz)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 C30H44NO6,514.3168[M+H+]；found,514.3148.
Compound 7 e:
Figure BDA0002991438920000221
7 e: white solid (57%), mp 64.0-64.8 ℃; [ alpha ] to]25 D-66(c 1.1,CH3OH)；1H NMR(CDCl3,400MHz) 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×OCH3), 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,CH2 in ent-beyerane skeleton)；13C NMR(CDCl3,100MHz)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 C30H44NO6,514.3168[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 by the zebrafish experiment.
(1) Zebra fish culture and embryo collection: purchasing zebra fish (3-12 months old) from the national zebra fish resource center, culturing in a mobile feeding box with the photoperiod of 14:10h (illumination: dark illumination) and the temperature of 28.5 +/-1 ℃, and feeding live saturated salt water shrimps twice a day; zebrafish (male: female ═ 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 tested 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 of movement or the atria and ventricles lose contractility and are considered dead, the survival rate is taken as an evaluation criterion of the activity of the compounds, as shown in figure 1 (the specific data are shown 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 embryos are subjected to heart function evaluation research. Furthermore, compounds 1,4j,4k,7d,7e and LSD showed the best efficiency at 10, 70, 60, 40, 60 and 50 μ M, respectively (figure 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 compounds tested. 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 [ myl7: EGFP]Zebrafish embryos are distributed into 24-well plates (20 embryos/well), 1mL of DOX (80 μ M) and various test compounds (1,4j,4k,7d,7e, LSD) (corresponding to the optimal concentration) are added, treated for 48h, the heart status and various heart indices of zebrafish are recorded under an upright fluorescence microscope (zeiss, germany), zebrafish 20s are photographed and recorded, the major axis (a) and the minor axis (b) of the end systole and end diastole are measured, and the formula v is 4/3 pi ab2The 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 combination of DOX and the test article (1,4j,4k,7d,7e, LSD) can significantly reduce cardiac edema caused by DOX, maintain normal cardiac morphology and improve cardiac function damage caused by DOX. Of all compounds tested, 7d showed the most significantAnd high 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: β -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 calf serum, 100U/mL penicillin and 100 mg/mL streptomycin in a humidified incubator with 5% CO2 at 37 ℃. 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(0.5mg/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 for 48 hours with or without test compounds. 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 compounds 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 (10)

1. An isosteviol derivative having a structure represented by the formula (I):
Figure FDA0002991438910000011
in the formula (I), R1Selected from hydroxy or carbonyl;
R2selected from H, C1～5Saturated straight-chain alkyl radical, C3～6Saturated cyclic alkyl, pyridine ring, phenyl, benzyl/substituted benzyl, phenethyl/substituted phenethyl, phenylpropyl/substituted phenylpropyl;
R3selected from carboxyl or primary amine.
2. The isosteviol derivative of claim 1, wherein R2Is substituted benzyl, substituted phenethyl or substituted phenylpropyl.
3. The isosteviol derivative of claim 2, wherein the substituted group is one or more of a halogen atom, a methyl group, a methoxy group, a hydroxyl group and a methyleneoxy group.
4. The isosteviol derivative of claim 2, wherein R1Is a carbonyl group; r2Is substituted benzyl, substituted phenethyl or substituted phenylpropyl; r3 is a primary amine.
5. The isosteviol derivative of claim 4, wherein R2Is any one of the following groups:
Figure FDA0002991438910000012
6. the isosteviol derivative according to claim 4, which is any one of the following compounds:
Figure FDA0002991438910000013
7. a process for the preparation of isosteviol derivative as claimed in any one of claims 1 to 6, which comprises the steps of:
s1, carrying out hydroxymethylation reaction on isosteviol and polyformaldehyde under an alkaline condition at 60-100 ℃ to obtain a compound 2 with a structural formula shown as a formula (II):
Figure FDA0002991438910000021
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 Et3N 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 FDA0002991438910000022
s3, oxidizing the 16-alpha hydroxyl of the compound 3 obtained in the step 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 in S2 to obtain a 16-position ketone derivative 7 thereof, as shown in the formula (VII):
Figure FDA0002991438910000023
8. the preparation method according to claim 7, wherein the molar ratio of the aldehyde groups in isosteviol to paraformaldehyde in step S1 is 1: 5-8.
9. The method according to claim 7, wherein the acylation in step S2 is carried out by esterifying the 15- β -hydroxymethyl group with an acid compound, wherein the acid compound is one or more of acetic anhydride, cyclohexanecarboxylic acid, nicotinic acid, isonicotinic acid and benzoic acid.
10. Use of isosteviol derivative as claimed in any one of claims 1 to 6 for the treatment of cardiovascular diseases.
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