CN111116698B

CN111116698B - Dammarane type triterpene compound, preparation and application

Info

Publication number: CN111116698B
Application number: CN202010012312.XA
Authority: CN
Inventors: 付宏征; 吴琼
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2020-01-07
Filing date: 2020-01-07
Publication date: 2021-03-02
Anticipated expiration: 2040-01-07
Also published as: CN111116698A

Abstract

Dammarane type triterpene compounds, and preparation and application thereof are provided. The invention belongs to the fields of medicinal chemistry, pharmacology and preparations, and relates to application of dammarane type triterpene compounds in treating or preventing heart failure, myocardial infarction, coronary heart disease, coronary arteriosclerosis, myocardial ischemia and myocardial ischemia reperfusion.

Description

Dammarane type triterpene compound, preparation and application

Technical Field

The invention belongs to the fields of medicinal chemistry, pharmacology and preparations, and particularly relates to a dammarane type triterpene compound, preparation, research on pharmacological action mechanism, application and preparation, and application of the dammarane type triterpene compound in treating or preventing heart failure, myocardial infarction, coronary heart disease, coronary arteriosclerosis, myocardial ischemia and myocardial ischemia reperfusion.

Background

Currently, cardiovascular diseases have become one of the leading causes of human death. Heart failure is an overload heart disease, the terminal stage of a variety of cardiovascular and non-cardiovascular system diseases, with about 260 million people worldwide suffering from heart failure. The primary pathogenesis of the disease is various, and the pathogenesis of the disease is complicated.

Positive inotropic drugs are drugs which can achieve the effect of treating heart failure by strengthening myocardial contractility, and mainly comprise the following classes: cardiac glycosides, a class of Na⁺/K⁺-ATPase inhibitors such as digoxin and the like; beta adrenoceptor agonists such as dobutamine and the like; phosphodiesterase inhibitors such as milrinone and the like. However, it has been clinically found that beta adrenoreceptor agonists are prone to drug tolerance and that PDE inhibitors tend to accelerate disease progression and increase mortality.

Na⁺/K⁺ATPase is a membrane protein present on the cell membrane that pumps 3 sodium ions out of the cell while pumping 2 potassium ions into the cell. When Na is present⁺/K⁺After the ATP-ase is inhibited, the sodium ion in the cell is gradually increased and passes through Na on the cell membrane⁺-Ca²⁺The exchange system raises the calcium ion concentration in the cell to reach the positive inotropic effect. Thus, Na⁺/K⁺The ATPATP enzyme is a very important and well-defined target of action against heart failure.

Dammarane type pentacyclic triterpene compounds such as panaxatriol and the like have wide biological activities in various aspects such as blood sugar reduction, tumor resistance, inflammation resistance, cholesterol reduction and the like, and have moderate-intensity myocardial ischemia reperfusion activity. Therefore, structural modification of panaxatriol to improve its pharmacological activity is of great significance in finding lead compounds that can be applied to the market.

The invention mainly carries out structural modification on the C-2, C-3, C-20 and C-22 positions of panaxatriol through oxidation, ester condensation and O₃A series of reactions such as oxidation, hydrolysis and the like are carried out to carry out structural modification, and a compound with remarkable anti-heart failure activity is discovered through in vitro molecular level, cell level and in vivo animal level researches.

Technical scheme

The invention relates to dammarane type triterpene compounds, which have a structural general formula as follows:

R₁when is H, R₂，R₃Is selected from

Any one of the above-mentioned (a) and (b),

or R₂Any one selected from H and NOH,

or: r₃Is selected from ═ O, -OH, -OBn, ═ NOH, and,

Any one of the above;

R₁，R₂is composed of

When R is₃Selected from ═ O;

or: r₄Is selected from ═ O, -OH, -OAc,-any one of the OBn's,

or: r₅Is selected from

Any one of them.

The invention relates to a dammarane type triterpene compound, the structure of which is any one or more of the following:

the dammarane type triterpene compound is used for preparing a composition for treating or preventing myocardial infarction.

The dammarane type triterpene compound is used for preparing a composition for treating or preventing heart failure.

The dammarane type triterpene compound is used for preparing a composition for treating or preventing myocardial ischemia.

The dammarane type triterpene compound is used for preparing a composition for treating or preventing coronary heart disease.

The dammarane type triterpene compound is used for preparing a composition for treating or preventing coronary arteriosclerosis.

The dammarane type triterpene compound is used for preparing a composition for treating or preventing diseases related to myocardial ischemia-reperfusion.

The composition of the dammarane type triterpene compound comprises 0.01-500mg of the dammarane type triterpene compound.

The dammarane-type triterpene compound composition contains 0.1-200mg of dammarane-type triterpene compound per preparation unit.

The invention relates to a composition of dammarane-type triterpene compounds, which contains 1-100mg of dammarane-type triterpene compounds per preparation unit.

The dammarane type triterpene compound of the invention: the method is characterized in that: the auxiliary materials in the field are adopted to prepare the medicine and the health care product.

The dammarane type triterpene compound composition comprises capsules, tablets, granules, pellets, sustained-release capsules, freeze-dried powder, injection and the like.

Pharmacodynamic experiments of the invention

Pharmacodynamic experiment I: compound pair Na⁺/K⁺In vitro study of the inhibition of ATPase

Materials and methods

1. Experimental Material

The new synthetic compound was provided by the department of medicine of Beijing university. Human Na⁺/K⁺-ATPase(Yanjing Co.,Ltd，Shanghai,China)；ATP(MP Biomedicals,Irvine,CA,USA)。

2. Laboratory apparatus

A FORMA 700 model ultra-low temperature refrigerator, Thermo corporation; YC-300L type medicine storage cabinet, Mitsubishi low temperature science and technology, Inc., of China; Direct-Q with pump type ultrapure water instrument, Millopore corporation; SW-CJ-2FD type superclean bench: suzhou clarification plant, Inc.; forma 3111 type water-jacketed CO2 incubator: thermo Electron company; berthold LB941 microplate type multifunctional microplate reader, Berthold Co.

3. Experimental methods

(1) Experimental grouping and drug concentration selection

All compound concentrations were selected: 0. mu.M, 0.01. mu.M, 0.1. mu.M, 0.5. mu.M, 1. mu.M, 5. mu.M. All compounds were solubilized with DMSO at 0. mu.M in an equal volume of DMSO as a blank, at a final DMSO concentration of 1% (v/v).

(2) Enzyme activity detection under Cell free system

The concentrations (0. mu.M, 0.01. mu.M, 0.1. mu.M, 0.5. mu.M, 1. mu.M, 5. mu.M) of the different compounds were mixed with 0.03U/ml Na⁺/K⁺Addition of ATPase to Na⁺125mM，K⁺2.8mM，Mg²⁺4.5mM, EDTA 0.5mM Tris (24mM, pH 7.8) buffer, mixed and incubated at 37 ℃ for 15 minutes, followed by addition of 5mM ATP to measure the amount of inorganic phosphate released by ATP within 15 minutes. After the Taussky-Shorr staining, the enzyme-linked immunosorbent assay kit is detected by an enzyme-linked immunosorbent assay instrument. Immediately after setting the blank well, the absorbance at 655nm was recorded. GraphPad Prism software fitted the inhibition curves and calculated IC50 values. Inhibition rate (control group OD value-Experimental group OD value)) OD value of control group X100%.

Second, experimental results

TABLE 1 in vitro IC50 values for different compounds on Na- + K- + atp enzymes

The experimental results show that: the invention carries out in-vitro human Na on a series of dammarane type triterpene derivatives⁺/K⁺Evaluation of ATPase inhibitory Activity, results show that Compounds 1-10 are vs. Na⁺/K⁺ATPase has significant inhibitory activity, comparable to that of the positive control compound Digoxin.

And a second pharmacodynamic experiment: myocardial cell injury experiment mediated by compound to hypoxia reoxygenation model

Materials and methods

1. Experimental Material

The new synthetic compound was provided by the department of medicine of Beijing university. Sodium dithionite (Sigma-Aldrich, st. louis, MO); sugar-free DMEM medium (Gibco, Grand Island, NY, USA); glucose (Glu) (national drug group chemical agents limited); penicillin (Sigma-Aldrich, st. louis, MO); streptomycin (Sigma-Aldrich, st. louis, MO); trypsin (Gibco, Grand Island, NY, USA); fetal Bovine Serum (FBS) (Hyclone, Logan, UT, USA). Butylphthalide (Sigma-Aldrich, St. Louis, Mo.).

Clean SD pregnant rat with weight of 200-.

2. Laboratory apparatus

A three gas incubator, Thermo corporation; SHZ-III model circulating water pump, tokyo kol instruments ltd; Direct-Q with pump type ultrapure water instrument, Millopore corporation; low temperature high speed centrifuge model 3K15, Sigma company; model TCL-16G-a high speed refrigerated centrifuge: shanghai' an pavilion scientific instrument factory; forma 3111 type water jacket CO₂An incubator: theThe rmo Electron company; YXQ-LS-50 SII type vertical pressure steam sterilizer; berthold LB941 microplate type multifunctional microplate reader, Berthold Co.

3. Experimental methods

(1) Setting and grouping of drug concentration:

the ginsengenin derivative and butylphthalide (NBP) are prepared into mother liquor according to corresponding requirements, and the mother liquor is diluted according to multiple times and then used for subsequent experiments. Blank control group (primary myocardial cells + complete DMEM medium for 50 h); OGD/R experimental group (primary myocardial cells + derivatives with concentration of (0. mu.M, 0.1. mu.M, 1. mu.M, 10. mu.M) are cultured for 24h, then anoxic is carried out for 2h, and reoxygenation is carried out for 24 h); NBP positive drug group (primary cardiomyocytes + NBP at concentration (0. mu.M, 0.1. mu.M, 1. mu.M, 10. mu.M) after 24h of culture, hypoxia was performed for 2h, and reoxygenation was performed for 24 h).

(2) The experimental steps are as follows:

primary culture of rat cardiomyocytes: sterilizing SD rat suckling mouse (0-1d) with 75% ethanol solution, shearing myocardial tissue of suckling mouse in sterile table, adding mixed solution of 0.125% trypsin and 1% collagenase II, sterilizing in 37 deg.C water bath oscillator for 10min, stopping digestion with DMEM containing 10% Fetal Bovine Serum (FBS), and sucking the mixed solution. Repeating digestion for 5 times, collecting digestive juice, filtering with 200 mesh sieve, centrifuging the filtrate (800rpm,10min), discarding supernatant, adding appropriate amount of culture solution, suspending and precipitating, inoculating to 6-hole plastic culture plate, standing at 37 deg.C and 5% CO₂Culturing in an incubator.

Rat myocardial cell hypoxia-hypoglycaemia injury model: and selecting cells growing to about 7d for carrying out in-vitro OGD/R model. Regulating cell density at 4X 10⁴The cell density of (2) was seeded in 48-well culture plates. Except for the blank control group, the OGD/R model was prepared by culturing 24 hours in a DMEM medium containing wq type drugs or NBP and 10% FBS sugar at concentrations of (0, 0.1. mu.M, 1. mu.M, 10. mu.M). First, the medium was changed to DMEM medium without glucose and serum, and the cells were placed in a DMEM medium containing 5% CO₂、95％N₂Culturing in a three-air culture box for 2h to complete the process of oxygen deficiency and sugar; the cell culture medium was then changed to DMEM medium completely containing 10% FBS and sugars, while the cells were placed in a medium containing 5% CO₂、20％O₂The culture is carried out in an incubator for 24h, the reoxygenation process is completed, and 20 mu L of MTT solution (5mg/mL) is added into each well 4h before the end of reoxygenation. After incubation is finished, discarding supernatant of each well, adding 150 mu L DMSO into each well, oscillating on a cell oscillator for 10min, and measuring OD (optical density) by using an enzyme-labeling instrument after crystals are fully dissolved₅₇₀。

(3) Data statistics

The survival rate of primary myocardial cells of different groups is calculated by setting a model control group, and the result is expressed in a mean plus or minus SD form. Statistical differences between data groups using the two-way ANOVA and Tukey's test, P values less than 0.05 were considered significant differences. The formula is survival rate ═ (OD value in experimental group/OD value in control group) × 100%.

Second, experimental results

Table 2 effect of compounds on rat cardiomyocyte survival.

The experimental results show that: the invention performs an in vitro rat myocardial cell hypoxia reoxygenation model-mediated damage experiment on a series of dammarane type triterpene derivatives, and the result shows that the compounds 1-10 have obvious protective effect on hypoxia reoxygenation myocardial cell damage.

And (3) pharmacodynamic experiment III: protective effect of compound 3 on myocardial ischemia reperfusion in rat body

Materials and methods

1. Experimental Material

SPF grade SD rat, 220-240 g. Purchased from laboratory animal technology, Inc. of Wei Tong Li Hua, Beijing. An aspartate aminotransferase (AST/GOT) kit and a creatine kinase isoenzyme (CK-MB) kit are purchased from Nanjing to build a bioengineering research institute.

2. Laboratory apparatus

GZX-9140MBE type forced air drying cabinet, Shanghai Boxun industries Co., Ltd medical equipment factory; Direct-Q with pump type ultrapure water instrument, Millopore corporation; BS224 type electronic balance: beijing Saedodes Instrument systems, Inc.; low temperature high speed centrifuge model 3K15, Sigma company; model TCL-16G-a high speed refrigerated centrifuge: shanghai' an pavilion scientific instrument factory; SW-CJ-2FD type superclean bench: suzhou clarification plant, Inc.; YXQ-LS-50 SII type vertical pressure steam sterilizer; berthold LB941 microplate type multifunctional microplate reader, Berthold Co.

3. Experimental methods

(1) Drug dose setting and grouping:

SD rats were divided into 8 groups of 5 (n-5): 1) sham group (Sham): the chest is opened, but the coronary artery is not ligated, and 40mg/kg physiological saline is administered by intragastric administration for three days before the model is made, once in the morning and at night. 2) Single drug addition group (D): the chest was opened, but the coronary artery was not ligated, and 40mg/kg of compound was administered by gavage for 3 days before molding, once in the morning and evening. 3) Model set (IR): the left anterior descending branch of the coronary artery was ligated and then reperfusion was carried out for 24 h. The 40mg/kg normal saline is administrated by stomach irrigation for three days before the model building, and is taken once in the morning and at night. 4) High dose group (IR +40 mg/kg): the left anterior descending branch of the coronary artery was ligated and then reperfusion was carried out for 24 h. The compound 40mg/kg is administered by intragastric administration for 3 days before molding, once in the morning and evening. 5) Middle dose group (IR +20 mg/kg): the left anterior descending branch of the coronary artery was ligated and then reperfusion was carried out for 24 h. The compound 20mg/kg is administered by gavage before molding for 3 days, once in the morning and evening. 6) Low dose group (IR +10 mg/kg): the left anterior descending branch of the coronary artery was ligated and then reperfusion was carried out for 24 h. The compound 10mg/kg is administered by gavage before molding for 3 days, once in the morning and evening. 7) Positive control group (IR + captopril): the left anterior descending branch of the coronary artery was ligated and then reperfusion was carried out for 24 h. The administration of captopril 20mg/kg by intragastric administration before the model building is carried out for three days, once in the morning and at night. 8) Panaxatriol control group (IR + panaxatriol): the left anterior descending branch of the coronary artery was ligated and then reperfusion was carried out for 24 h. The panaxatriol 40mg/kg is administrated by intragastric administration for three days before the model building, and is taken once in the morning and at night.

(2) Preparation of rat myocardial ischemia reperfusion model

A rat myocardial ischemia reperfusion injury model is prepared by carrying out abdominal anesthesia on a rat by using a 3% sodium pentobarbital solution (0.2ml/100g), fixing limbs on an operating table in a supine position, connecting limb electrocardiogram electrodes and tracing a standard 16-lead electrocardiogram. The trachea cannula is inserted through the mouth, the small animal respirator is connected, the tidal volume is adjusted to 15mL/kg, the respiratory frequency is 70 times/min, and the respiratory ratio is 1: 1.5. Performing antethoracic depilation, performing blunt fascia muscle layer by opening chest at 3-4 times, exposing heart, tearing pericardium, inserting needle 1-2mm below left auricle, crossing 3-4mm, inserting needle depth of 1.5-2mm, and ligating coronary artery left anterior descending branch by 6-0 suture push tube method. Real-time monitoring and recording electrocardiogram, continuously raising by ST segment, gradually cyanotizing far-end blood-supply cardiac muscle of ligation point, and clarifying to show that the myocardial ischemia molding is successful; removing ligature after 30min, suturing, taking ST segment descent and ischemia pallor myocardial reddening as successful mark of reperfusion model, taking blood from abdominal aorta after reperfusion for 24h, separating plasma at 3500rpm for 10min, and storing at-80 deg.C; the heart was removed and stored at-80 ℃ until use.

(3) Evans Blue/TTC detection of myocardial infarction area

After 24h of reperfusion, rats were anesthetized (same model as the method), blood was taken from the abdominal aorta, the heart was taken out, washed with physiological saline, placed at-80 ℃ for 7min and taken out, and the heart was transected into 5-7 slices with a diameter of 1-2mm from the apex of the heart along the ligature direction. Placing into 1% TTC solution, heating in water bath at 37 deg.C for 12min, fixing in neutral formalin, standing overnight at room temperature, photographing the next day, and measuring myocardial infarction area (myocardial infarction ratio: area of infarcted area/left heart area) of each slice with Image-Pro plus5.0 Image analysis software. After 24h of reperfusion, all animals were bled from abdominal aorta and blood samples were centrifuged in centrifuge tubes at 3500rpm for 10min to separate plasma, which was stored at-80 ℃ for further use. The detection method is carried out according to the instruction of the corresponding determination kit.

Second, experimental results

The experimental results show that: the compound 3 can obviously reduce the infarct size at high, medium and low doses, presents concentration dependence, and has stronger effect of reducing the myocardial infarct size at high, medium and low doses than the positive medicine captopril. In addition, when the dosage is 40mg/kg, the effect of the compound 3 for reducing the myocardial infarction area is stronger than that of the parent compound panaxatriol.

The experimental results show that: compared with a pseudo-operation group and a single-dosing group, the AST and CK activities in the serum of a reperfusion model group are obviously increased; compared with the ischemia reperfusion model group, the AST and CK activities in serum can be obviously reduced by the compound 3 in the high, medium and low dose groups.

Drawings

FIG. 1: effect of Compound 3 on the area of ischemia-reperfusion myocardial infarction in rats

FIG. 2: effect of Compound 3 on plasma Creatine Kinase (CK) and aspartate Aminotransferase (AST) levels in ischemia-reperfused rats

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

Example 1

Preparation of Compounds 12, 13

476mg of Compound 11, namely 20(R) -panaxatriol, was dissolved in 30mL of methylene chloride, and 424mg of dessimutan oxidant was added thereto to conduct a reaction at room temperature for 3 hours. After the reaction was completed, the reaction was quenched with saturated sodium bicarbonate and sodium bisulfite. Extracting with dichloromethane, and recovering solvent under reduced pressure to obtain crude product. Silica gel column chromatography (petroleum ether/ethyl acetate 5:3) gave compound 12 and compound 13. Compound 12:¹³C NMR(100MHz,CDCl₃):δ211.90,78.51,76.63,73.30,69.43,65.62,54.83,52.65,51.08,50.20,48.95,46.29,43.65,39.94,37.58,36.45,35.71,33.06,31.14,30.85,27.56,27.16,27.05,24.90,19.44,17.33,17.05,16.27,15.68,15.16.ESI-MS:m/z 473.68[M-H]^-. Compound 13:¹³C NMR(100MHz,CDCl₃):δ219.33,76.73,73.35,69.75,67.87,59.07,54.66,51.18,48.97,48.37,47.29,45.21,40.50,39.78,38.14,36.52,35.81,33.12(2C),32.05,31.35,31.25,27.23,25.18,19.64,19.52,17.68,17.04,16.35,16.03.ESI-MS:m/z 473.68[M-H]^-.

example 2

Preparation of Compound 1

284mg of Compound 13 was dissolved in 20mL of tetrahydrofuran, and 120mg of NaH was added to the solution to conduct a reaction for 1 hour. 205mg of benzyl bromide was added thereto, and the mixture was heated to 70 ℃ for 8 hours. After the reaction is finished, extracting by ethyl acetate, and evaporating the solvent to dryness under reduced pressure to obtain a crude product. Silica gel column chromatography (petroleum ether/ethyl acetate 6:1) gave compound 1.

¹H NMR(400MHz,CDCl₃):7.48-7.16(m,5H),6.48(s,1H),4.02(m,1H),3.49(m,1H),3.19(m,1H),3.06(m,1H),2.40(m,1H),1.35(s,3H),1.33(s,3H),1.28(s,3H),1.23(s,3H),1.19(s,3H),1.02(s,3H),0.90(s,3H),0.78(s,3H).¹³C NMR(100MHz,CDCl₃):δ219.12,140.53,129.04(2C),128.55(2C),126.05,76.84,73.45,69.75,67.97,58.35,54.66,51.18,49.00,48.91,48.73,47.52,45.12,42.92,40.47,38.17,36.87,36.52,35.82,33.13,32.15,31.26(2C),27.26,25.19,19.53,19.21,19.18,17.02,16.36,16.00.ESI-MS:m/z 565.84[M+H]⁺。

Example 3

Preparation of Compound 2

474mg of Compound 2 was dissolved in 30mL of tetrahydrofuran, and 200mg of NaH was added to the solution to react for 1 hour. 0.24mL of benzyl bromide was added and the reaction was carried out at 70 ℃ for 8 hours. After the reaction is finished, extracting by ethyl acetate, and evaporating the solvent to dryness under reduced pressure to obtain a crude product. Silica gel column chromatography (petroleum ether/ethyl acetate 6:1) gave compound 2.¹H NMR(400MHz,CDCl₃):δ7.33-7.21(m,5H),6.32(s,1H),4.63(d,J＝12.0Hz,1H),4.40(d,J＝12.0Hz,1H),3.60(td,J＝10.3Hz,4.8Hz,1H),2.82(dd,J＝11.5Hz,3.6Hz,1H),2.53(d,J＝11.6Hz,1H),2.09(s,1H),1.25(s,6H),1.21(s,3H),1.16(s,3H),1.03(s,3H),0.97(s,6H),0.91(s,3H).¹³C NMR(100MHz,CDCl₃):δ211.58,139.17,128.17(2C),127.39(2C),127.25,85.71,76.55,73.19,71.25,69.35,65.91,54.77,52.60,50.98,50.13,48.90,46.18,43.48,39.79,37.74,36.37,35.63,33.01,31.05,30.85,27.75,27.09,24.83,22.69,19.37,17.26,17.00,16.23,16.21,15.60.ESI-HRMS(m/z)[M+H]⁺calcd for C₃₇H₅₇O₄,565.4257；found,565.4246.

Example 4

Preparation of Compound 14

379mg of Compound 13 was dissolved in 30mL of anhydrous tetrahydrofuran, and 346mg of anhydrous sodium methoxide and 0.87mL of diethyl oxalate were added thereto to conduct a reaction at room temperature for 6 hours. After the reaction is finished, extracting by ethyl acetate, and evaporating the solvent to dryness under reduced pressure to obtain a crude product. The crude product was dissolved in 30mL of ethanol, 110mg of hydroxylamine hydrochloride was added, and the mixture was heated under reflux for 4 hours. After the reaction was stopped, ethyl acetate was extracted, and the crude product was evaporated to dryness under reduced pressure, and purified by column chromatography (petroleum ether/ethyl acetate 2/1) to obtain compound 14.¹H NMR(400MHz,CDCl₃):δ6.50(s,1H),4.41(q,J＝7.1Hz,2H),4.14(m,1H),3.61(td,J＝10.0Hz,4.7Hz),2.89(d,J＝16.0Hz,1H),2.13(d,J＝16.0Hz,1H),1.58(s,3H),1.51(s,3H),1.39(t,J＝7.2Hz,3H),1.28(s,3H),1.23(s,3H),1.20(s,3H),1.09(s,3H),0.93(s,3H),0.85(s,3H).¹³C NMR(100MHz,CDCl₃):δ177.15,160.78(2C),110.26,76.76,73.36,69.68,67.80,61.76,59.13,54.76,51.20,48.98,47.85,46.00,40.74,40.57,36.57(2C),36.22,35.86,33.12,32.56,31.32,31.00,27.35,25.22,20.68,19.57,17.12,17.10,16.70,16.37,14.39.ESI-HRMS(m/z)[M+H]⁺calcd for C₃₄H₅₄NO₆,572.3951；found,572.3952。

Example 5

Preparation of Compound 3

Compound 258mg of Compound 14 was dissolved in 20mL of ethanol, and 34mg of lithium hydroxide was added thereto, followed by reaction at room temperature for 12 hours. After the reaction is finished, 1M hydrochloric acid solution is added, ethyl acetate is used for extraction, and the solvent is evaporated to dryness under reduced pressure to obtain a crude product. Silica gel column chromatography (dichloromethane/methanol-20/1) afforded compound 3.¹H NMR(400MHz,CD₃OD):δ4.08(m,1H),3.62(m,1H),2.92(d,J＝15.8Hz,1H),2.18(d,J＝15.8Hz,1H),2.03-1.96(m,2H),1.59(s,3H),1.52(s,3H),1.29(s,3H),1.22(s,6H),1.11(s,3H),0.98(s,3H),0.88(s,3H).¹³C NMR(100MHz,CD₃OD):δ178.60,163.08,155.82,111.57,78.17,74.61,71.39,67.85,59.73,55.93,52.31,49.84,48.73,46.01,41.55,41.39,37.41(2C),37.29,36.75,33.45,32.79,32.23,31.62,27.66,26.08,20.94,19.89,17.49,17.33,17.12,16.86.ESI-HRMS(m/z)[M+H]⁺calcd forC₃₂H₅₀NO₆,544.3638；found,544.3639。

Example 6

Preparation of Compound 4

474mg of Compound 13 was dissolved in ethanol, and 139mg of hydroxylamine hydrochloride and 168mg of sodium hydrogencarbonate were added thereto and heated under reflux for 2 hours. After the reaction was completed and cooled to room temperature, the mixture was extracted with dichloromethane. Decompressing and evaporating the solvent to obtain a crude product. Silica gel column chromatography (petroleum ether/ethyl acetate 2:1) gave compound 4.¹H NMR(400MHz,CDCl₃):δ6.35(s,1H),4.02(m,1H),3.50(m,1H),3.15(m,1H),2.10-2.01(m,1H),1.96-1.89(m,1H),1.39(s,3H),1.37(s,3H),1.24(s,3H),1.19(s,3H),1.16(s,3H),1.00(s,3H),0.88(s,3H),0.82(s,3H).¹³C NMR(100MHz,CDCl₃):δ167.82,76.72,73.30,69.85,67.75,58.36,54.62,51.16,48.94,48.79,45.49,40.40,40.39,38.75,38.47,36.52,35.80,33.78,33.11,31.31,31.21,27.21,25.17,21.03,19.49,18.02,17.02,16.96,16.34,16.12.ESI-HRMS(m/z)[M+H]⁺calcd for C₃₀H₅₂NO₄,490.3896；found,490.3894。

Example 7

Preparation of Compound 5

474mg of Compound 13 was dissolved in 20mL of t-butanol, 672mg of potassium t-butoxide was added, and 0.4mL of isoamyl nitrite was slowly added to react at room temperature for 3 hours. After the reaction was completed, dichloromethane was extracted. The organic layer is decompressed and the solvent is evaporated to dryness to obtain a crude product.Silica gel column chromatography (petroleum ether/ethyl formate 1:1) gave compound 5.¹H NMR(400MHz,CD₃OD):δ3.96(m,1H),3.61(m,1H),2.90(d,J＝18.6Hz,1H),2.32(d,J＝18.6Hz,1H),2.04-1.97(m,1H),1.92-1.82(m,3H),1.45(s,3H),1.29(s,6H),1.22(s,6H),1.09(s,3H),0.98(s,3H),0.87(s,3H).¹³C NMR(100MHz,CD₃OD):δ207.00,155.18,78.18,74.63,71.31,67.63,59.29,55.90,52.29,49.88,47.92,47.85,45.26,43.35,41.12,37.69,37.40,36.74,33.46,32.25,32.16,31.99,27.64,26.06,20.67,19.88,17.87,17.26,17.13,16.12.ESI-HRMS(m/z)[M+H]⁺calcd for C₃₀H₅₀NO₅,504.3689；found,504.3676。

Example 8

Preparation of Compound 6

503mg of Compound 5 was dissolved in 10mL of anhydrous pyridine, and 0.21m of acetyl chloride was added thereto, followed by heating and refluxing for 2 hours. After the reaction, 1M diluted hydrochloric acid was added, and extraction was performed with ethyl acetate. The organic layer is decompressed and the solvent is evaporated to dryness to obtain a crude product. Silica gel column chromatography (petroleum ether/ethyl formate 2:1) gave compound 6.¹H NMR(400MHz,CDCl₃):δ6.34(s,1H),5.34(m,1H),3.68(m,1H),2.79(m,1H),2.49(s,3H),2.21-2.14(m,2H),2.09(s,3H),1.44(s,3H),1.35(s,3H),1.27(s,3H),1.23(s,3H),1.22(s,3H),1.20(s,6H),0.91(s,3H).¹³C NMR(100MHz,CDCl₃):δ195.73,171.60,170.08,161.48,131.07,76.75,73.38,69.76,69.46,57.19,54.68,51.08,48.63,47.12,42.96,42.48,41.72,41.16,36.53,35.80,33.21,32.68,31.33,31.26,27.29,25.02,21.90,21.57,20.72,19.53,17.39,16.92,16.32,14.30.ESI-HRMS(m/z)[M+H]⁺calcd for C₃₄H₅₂NO₆,570.3795；found,570.3802。

Example 9

Preparation of Compound 15

238mg of panaxatriol compound 11 was dissolved in 30mL of dichloromethane, and 324mg of pyridinium chlorochromate salt was added thereto to carry out a reaction at room temperature for 3 hours. And after the reaction is finished, filtering, and evaporating the solvent from the filtrate under reduced pressure to obtain a crude product. Silica gel column chromatography (petroleum ether/ethyl acetate ═ 6:1) gave compound 15.¹³C NMR(100MHz,CDCl₃):δ214.44,210.97,76.57,73.30,69.20,65.30,54.79,52.45,51.01,49.78,48.94,46.95,46.16,43.38,41.14,36.38,35.64,33.90,33.02,31.12,30.93,27.13,24.83,24.18,21.63,19.40,17.25,16.42,16.22,15.75.ESI-MS:m/z 471.65[M-H]^-.

Example 10

Preparation of Compound 7

236mg of Compound 7 was dissolved in 10mL of glacial acetic acid, and 131mg of 4-methoxyphenylhydrazine hydrochloride was added thereto, followed by heating and refluxing for 4 hours. After the reaction is finished, excessive acid is removed, ethyl acetate is used for extraction, and the organic layer is decompressed and evaporated to dryness to obtain a crude product. Silica gel column chromatography (petroleum ether/ethyl acetate 8:1) gave compound 7.¹HNMR(400MHz,CDCl₃):δ7.78(s,1H),7.19(d,J＝8.7Hz,1H),6.84(br s,1H),6.78(dd,J＝8.7Hz,1.8Hz,1H),6.49(s,1H),3.84(s,3H),3.74(td,J＝10.3Hz,4.8Hz,1H),2.96(d,J＝15.0Hz,1H),2.67(s,1H),2.64(d,J＝12.3Hz,1H),2.57(d,J＝15.0Hz,1H),2.34-2.24(m,2H),2.01(d,J＝12.3Hz,1H),1.72(s,3H),1.39(s,3H),1.29(s,3H),1.25(s,3H),1.22(s,3H),1.07(s,3H),1.06(s,3H),1.03(s,3H).¹³C NMR(100MHz,CDCl₃):δ212.06,153.99,142.14,131.34,128.29,111.35,111.28,105.38,100.14,76.73,73.45,69.55,64.15,55.97,54.82,52.62,51.17,49.25,49.22,45.18,44.24,39.53,36.53,35.81,34.37,33.09,31.77,31.35,30.77,27.26,24.98,24.59,19.57,18.65,17.39,16.37,15.50.ESI-HRMS(m/z)[M+H]⁺calcd for C₃₇H₅₄NO₄,576.4053；found,576.4055。

Example 11

Preparation of Compound 8

472mg of compound 15 was dissolved in 250mL of tetrahydrofuran, and 480mg of pyridinium tribromide was slowly added in an ice-water bath to react at room temperature for 12 hours. The reaction was quenched with sodium thiosulfate and extracted with ethyl acetate. Decompressing and evaporating the organic solvent to obtain a crude product. The crude product was dissolved in 30mL of anhydrous ethanol, 532mg of thiourea was added, and the reaction was performed under reflux with heating for 4 hours. After the reaction is stopped, cooling to room temperature, adding water for dilution, extracting by ethyl acetate, and evaporating to dryness under reduced pressure by the organic phase to obtain a crude product. Silica gel column chromatography (petroleum ether/ethyl acetate 8/1) gave compound 8.¹H NMR(400MHz,CDCl₃):δ6.38(s,1H),4.38(q,J＝6.8Hz,2H),3.61(m,1H),3.01(d,J＝16.6Hz,1H),2.70(d,J＝16.6Hz,1H),2.58-2.56(m,2H),2.20(br d,1H),2.00-1.90(m,3H),1.68(s,3H),1.40(s,3H),1.22(s,3H),1.18(s,3H),1.14(s,3H),0.99(s,3H),0.97(s,3H),0.95(s,3H).¹³C NMR(100MHz,CDCl₃):δ210.37,160.67,160.13,155.14,132.22,76.57,73.34,69.15,63.09,62.23,54.69,52.25,50.96,48.97,48.73,44.89,43.98,41.12,37.40,36.39,35.66,33.01,31.51,31.22,30.63,27.12,24.84,23.99,19.44,18.40,17.19,16.22,15.31,14.33.ESI-HRMS(m/z)[M+H]⁺calcd for C₃₄H₅₂NO₅S,586.3566；found,586.3570。

Example 12:

synthesis of Compound 16

472mg of Compound 7 was dissolved in 30mL of ethanol, and 152mg of hydroxylamine hydrochloride and 202mg of sodium hydrogencarbonate were added to the solution and the mixture was refluxed for 2 hours. After the reaction, the mixture was diluted with water and extracted with dichloromethane. The organic layer was evaporated to dryness under reduced pressure to give a crude product, which was chromatographed on silica gel (petroleum ether/ethyl acetate 4:1) to give compound 16.¹H NMR(400MHz,CDCl₃):δ6.38(s,1H),3.57(m,1H),3.26(m,1H),2.51(d,J＝11.5Hz,1H),2.31(s,1H),2.02-1.87(m,5H),1.81(d,J＝11.5Hz,1H),1.77-1.69(m,2H),1.41(s,3H),1.22(s,3H),1.17(s,3H),1.13(s,6H),1.02(s,3H),0.96(s,3H),0.92(s,3H).¹³C NMR(100MHz,CDCl₃):δ211.77,165.21,76.62,73.33,69.37,66.19,54.83,52.64,51.04,50.03,48.92,46.33,43.79,40.27,39.98,36.43,35.69,33.06,31.10,30.84,27.15,25.35,24.86,22.84,19.41,17.21,16.69,16.44,16.25,15.69.ESI-MS:m/z 488.41[M+H]⁺。

Example 13

Preparation of Compound 9

487mg of Compound 16 was dissolved in THF, and 80mg of NaH was added thereto, followed by reaction at ordinary temperature for 1 hour. 0.24mL of 2-chloro-5- (chloromethyl) thiophene was added and the reaction was carried out at 60 ℃ for 8 hours. Adding water to quench the reaction, extracting with ethyl acetate, and evaporating the solvent from the organic layer under reduced pressure to obtain a crude product. Silica gel column chromatography (petroleum ether/ethyl acetate 10:1) afforded compound 9.¹H NMR(400MHz,CDCl₃):δ6.77-6.74(m,2H),6.38(s,1H),5.06-5.4.98(m,2H),3.56(m,1H),2.98(m,1H),2.50(d,J＝11.9Hz,1H),2.22(s,1H),2.14-2.07(m,1H),1.28(s,3H),1.26(s,3H),1.22(s,3H),1.16(s,3H),1.15(s,3H),0.96(s,3H),0.93(s,3H),0.76(s,3H).¹³C NMR(100MHz,CDCl₃):δ213.68,163.45,139.42,130.71,126.20,125.42,76.66,73.38,70.16,69.45,68.14,54.73,51.42,49.72,49.46,43.50,40.13,38.09,37.78,36.50,35.74,33.09,32.70,32.64,31.95,31.29,27.22,26.30,25.00,24.28,20.32,19.54,16.46,16.33,15.78.

Example 14

Preparation of Compound 18

20(S) -Protopanaxatriol compound 17(476mg) was dissolved in pyridine, and 0.5mL of acetic anhydride and 12mg of 4-dimethylaminopyridine were added to the solution to conduct a reaction at room temperature for 9 hours. After the reaction, the mixture was diluted with water and extracted with ethyl acetate. The organic layer is decompressed and evaporated to dryness to obtain a crude product. Silica gel column layerThe mixture was separated (petroleum ether/ethyl acetate 8:1) to obtain compound 18.¹H NMR(400MHz,CDCl₃):δ5.30(m,1H),5.05(t,J＝6.7Hz,1H),4.88(m,1H),4.42(dd,J＝11.6Hz,4.5Hz,1H),2.44(m,1H),2.00(s,6H),1.80(s,3H),1.52(s,3H),1.16(s,6H),1.12(s,3H),0.99(s,3H),0.96(s,3H),0.91(s,3H),0.87(s,3H).¹³C NMR(100MHz,CDCl₃):δ170.89,170.48,170.22,137.42,124.55,80.09,73.88,70.84,70.38,58.62,50.83,49.70,49.27,46.51,43.69,42.44,40.88,39.39,38.30,37.77,31.97,30.35,29.30,29.22,28.43,28.18,23.19,23.05,22.02,21.29,21.14,17.20,16.87,16.83,16.72,12.23。ESI-MS:m/z 625.56[M+Na]⁺.

Example 15

Preparation of Compound 19

200mg of Compound 18 was dissolved in 20mL of a mixed solvent of dichloromethane/methanol 1:1, and nitrogen was purged for 20 to 30 min. Ozone is introduced at-78 ℃ for reaction for 15 minutes until the solution turns blue, and the reaction is stopped. Removing ozone, introducing nitrogen gas to discharge excessive O₃After the reaction was quenched by addition of dimethyl sulfide, the reaction system was gradually raised to room temperature and stirred at room temperature for 1 hour. And (5) decompressing and drying the solvent to obtain a crude product. Silica gel column chromatography (petroleum ether/ethyl acetate 4:1) gave compound 19.¹H NMR(400MHz,CDCl₃):δ5.32(m,1H),4.74(m,1H),4.43(dd,J＝11.6,3.1Hz,1H),2.84(m,1H),2.28(t,J＝10.9Hz,1H),2.09(s,3H),2.02(s,6H),1.89(s,3H),1.15(s,3H),1.00(s,3H),0.99(s,3H),0.93(s,3H),0.89(s,3H).¹³C NMR(100MHz,CDCl₃):δ211.45,170.95,170.86,170.21,80.07,74.26,70.26,58.73,51.91,51.42,49.79,48.55,42.67,40.88,39.44,38.38,37.83,32.27,30.38,29.51,27.83,27.36,23.21,22.04,21.34,21.11,17.37,16.93,16.86,16.81.ESI-MS:m/z 541.41[M+Na]⁺.

Example 16

Preparation of Compound 10

100mg of Compound 19 was taken, 48mg of NaOH was added, and the mixture was dissolved in 14mL of a mixed solvent of dichloromethane/methanol/water (3: 3: 1). The reaction was carried out at room temperature for 4 hours. After the reaction, water was added to dilute the reaction solution, followed by extraction with ethyl acetate. The organic layer was evaporated to dryness under reduced pressure and the crude product was chromatographed (dichloromethane/methanol-60/1) to give compound 10.¹³C NMR(100MHz,CD₃OD):δ216.90,79.50,72.42,68.81,62.16,54.37,53.51,52.37,51.42,47.66,42.14,40.52,40.21(2C),33.54,32.83,31.42,30.39,28.48,27.78,17.85,17.53,17.15,16.10。ESI-MS:m/z 391.41[M-H]^-.

Example 17

Preparation of the formulations

Preparation of tablets

30.5 g of compound; 10g of microcrystalline cellulose; 10g of poloxamer; 50ml of 95% ethanol; 50g of hydroxypropyl cellulose; 100ml of 25 percent starch slurry; 2g of magnesium stearate, granulating, drying at 60 ℃, grading by using a 12-mesh sieve, and pressing into tablets.

Preparation of capsules

30.5 g of compound; 10g of microcrystalline cellulose; 10g of poloxamer; 50ml of 95% ethanol; 50g of hydroxypropyl cellulose; 100ml of 25 percent starch slurry; granulating with 100 mesh sieve, drying at 80 deg.C, grading with 100 mesh sieve, and filling into empty capsule.

Preparation of freeze dried powder for injection

30.5 g of compound; 5g of poloxamer; adding 5.0g arginine, adding 20.0g mannitol, adding water for injection, heating to dissolve, diluting to 1000ml, filtering, ultrafiltering, packaging, freeze drying, and capping. The freeze drying is divided into four stages: (1) pre-freezing for 5 hours at-30 ℃; (2) drying under reduced pressure for 12 hr at-30 deg.C; (3) heating and drying for 6 hours at the temperature of minus 10 ℃; (4) the temperature is raised twice and the drying is carried out for 4 hours, and the temperature is 35 ℃.

Claims

1. A dammarane type triterpene compound has a structure of any one of the following:

2. use of dammarane-type triterpene compounds according to claim 1 for the preparation of a medicament for the treatment or prevention of myocardial hypoxia-reoxygenation injury.