CN102146057B - C19-diterpene alkaloids, preparation method thereof, pharmaceutical composition and application using the compound as an active ingredient - Google Patents

Abstract

式I中R¹是氢，羟基或甲氧基；R²是氢或甲基；R³，R⁴和R⁵是氢或羟基。This project relates to C ₁₉ -diterpene alkaloids of the formula I and their pharmaceutically acceptable salts having cardiotonic and anti-heart failure activity, processes for their preparation, medicaments containing them and their use as cardiotonic and anti-heart failure Drug use.

In formula I, R ¹ is hydrogen, hydroxyl or methoxy; R ² is hydrogen or methyl; R ³ , R ⁴ and R ⁵ are hydrogen or hydroxyl.

Description

C19-diterpene alkaloids, preparation method thereof, pharmaceutical composition and application using the compound as an active ingredient

Technical field:

The present invention relates to C ₁₉ -diterpene alkaloids N-desethylaconitine, bucurine, N-desethylaconitine and isodelnine and their homologues with medicinal value. They are isolated from aconitine or prepared semi-synthetically from aconitine.

The present invention also relates to two C ₁₉ -diterpene alkaloids, unine and subunine. They are prepared by semi-synthesis from aconitine which is isolated from the plant aconitum.

At the same time, a preparation method of the above compound and a pharmaceutical composition using the compound as an active ingredient are disclosed. The invention belongs to the technical field of extraction and preparation of active ingredients of natural medicines.

Background technique:

Aconitum carmichaeli Debx., a traditional Chinese medicine, is a processed product of the root of Aconitum carmichaeli Debx. It has the function of recovering yang and rescuing adversity, tonifying fire and supporting yang, expelling wind-cold-damp evil, warming meridian and relieving pain. For yang deficiency, cold extremities, weak pulse, impotence, cold palace, cold pain in trusted subordinates, vomiting and diarrhea due to deficiency and cold, edema due to yin and cold, exogenous pathogenesis due to yang deficiency, arthralgia due to cold and dampness (Chinese Pharmacopoeia, 2000 edition, p. 150) . In my country, aconite is rich in resources and widely distributed. It is widely used in clinical practice as a famous traditional Chinese medicine.

Studies have found that the cardiotonic components in aconite include dl-demethylcoclaurine (T.Kosuge et al, Chem.Pharm.Bull, 1976, 24:176-178), methyl dopamine chloride (coryneine chloride) (C.Lammp, PlataMedica, 1979, 35: 150-), salsoline (Chen Dihua et al., Acta Pharmaceutica Sinica, 1982, 17: 792-794), uracil (Han Gongyu et al., Research and Development of Natural Products, 1997, 9(3):30-34) and fuzinoside (Xu Dongming et al., Chinese Herbal Medicine, 2004, 35:964-966; Chinese Invention Patent, 2006, CN100386338C). However, there is no report that diterpene alkaloid compounds isolated from aconite have cardiotonic effects. In recent years, we have used the method of cardiac activity tracking, and found that the compound C ₁₉ -diterpene alkaloid N-desethylaconitine, northern Unine, N-desethylunine, isodermine, mesounine and subunine. However, due to the low content of these alkaloids in aconite, we have also studied the semi-synthetic preparation of N-desethylhypounine, mesounine and The method of subunine.

Invention content:

The invention provides a C ₁₉ -diterpene alkaloid N-desethylaconitine, beritunine, N-desethylaconine, isodermine and homologues thereof with medicinal value.

The invention also provides a method for extracting the above-mentioned substances from the aconite and aconitum.

The invention also provides a method for preparing N-desethylaconitine, mesounine and subunine by using aconitine as a raw material, which is suitable for industrial production.

The present invention further provides a pharmaceutical composition with the above-mentioned compound as an active ingredient, which is used for the preparation of drugs for strengthening heart and preventing heart failure.

The compound of the present invention has the following general structural formula (I):

wherein R ¹ is hydrogen, hydroxyl or methoxy; R ² is hydrogen or methyl; R ³ , R ⁴ and R ⁵ are hydrogen or hydroxyl.

The C ₁₉ -diterpene alkaloid compound with cardiotonic and anti-heart failure effects of the present invention includes mesounine and subunine.

The present invention provides following technical method:

(1) Extract and separate N-desethylaconitine (XII) and isodermine (XIII) from aconite or aconitum.

(2) Prepare Beiwuning (XIV) by hydrolysis with Beiwuting as raw material.

(3) Using aconitine as a raw material, through peracetylation, N-deethylation, N-methylation and hydrolysis, to prepare mesounine (II).

(4) Using aconitine as a raw material, undergo chlorination, elimination, hydrogenation, peracetylation, N-deethylation, N-methylation and hydrolysis to prepare subunine (III).

(5) Using aconitine as a raw material, prepare N-desethylhine (XV) through chlorination, elimination, hydrogenation, peracetylation, N-deethylation and hydrolysis.

The present invention extracts and separates the method for N-desethylaconitine and isodermine from aconite or aconitum, comprising the following steps:

(1) After pulverizing the aconite or aconitum, decocting with water to obtain the extract;

(2) alcohol precipitation of the extract, filtration, and concentration of the filtrate under reduced pressure to obtain a solid extract;

(3) After the extract is dissolved in water and alkalized, it is extracted with n-butanol, and the solvent is recovered to obtain the n-butanol extract;

(4) The n-butanol extract was separated by silica gel column chromatography and eluted with a mixed solvent of chloroform-methanol in different proportions to obtain the desired compound.

The method used in the invention is simple and easy to implement, is suitable for expanding the scale, and is easy to realize the purpose of industrialized production.

The present invention prepares the method for Wu Ning base, comprises the following steps:

1) Under the catalysis of p-toluenesulfonic acid, react aconitine with acetyl chloride or acetic anhydride at room temperature for 6-12 hours, and obtain a solid after conventional treatment, crystallized in 95% ethanol to obtain compound 3, 13, 15-triacetyl aconitum Base (V);

2) Mix 3,13,15-triacetylaconitine with 3 equivalents of N-bromosuccinimide and 20-40 times glacial acetic acid, react at room temperature for 2-6 hours, concentrate under reduced pressure to obtain a white solid . After silica gel column chromatography (petroleum ether: acetone = 2:1), N-desethyl-3,13,15-triacetylaconitine (VI) was obtained;

3) N-desethyl 3,13,15-triacetylaconitine was mixed with an equivalent amount of methyl iodide, tetrahydrofuran was used as a solvent, reacted at room temperature for 2-4 hours, and dried under reduced pressure to obtain a white solid. After silica gel column chromatography (petroleum ether: ethyl acetate (3:1), 3,13,15-triacetyl mesoconitine (VII);

4) 3,13,15-Triacetyl mesoconitine was hydrolyzed with 5% sodium hydroxide, left at room temperature overnight, and dried under reduced pressure to obtain a white solid. Through silica gel column chromatography (chloroform-methanol saturated with ammonia water, 95:1), the compound Wuning base (II) was obtained.

The present invention prepares the method for subunine base, comprises the following steps:

(1) Using tetrahydrofuran as a solvent, mix aconitine with an equivalent amount of SOCl ₂ , react overnight at room temperature for 1-3 hours, and dry under reduced pressure to obtain a white solid. After adding ethyl acetate-95% ethanol-glacial acetic acid (3:3:1, w/w) mixed solution, under the catalysis of palladium carbon, it was hydrogenated under normal pressure for 1-3 days. Drying under reduced pressure gave a white solid. Crystallized by 95% ethanol to obtain deoxyaconitine (VIII);

(2) Under the catalysis of p-toluenesulfonic acid, hexaconitine was mixed with 1.5 equivalents of acetyl chloride or acetic anhydride, stirred, reacted at room temperature overnight, and dried under reduced pressure to obtain a white solid. Then crystallized with 95% ethanol to obtain compound 13,15-diacetyldeoxyaconitine) (IX);

(3) Mix 13,15-diacetyldeoxyaconitine with 3 equivalents of N-bromosuccinimide and 20-40 times glacial acetic acid, react at room temperature for 2-6 hours, concentrate under reduced pressure to obtain a white solid . After silica gel column chromatography (petroleum ether-acetone, 2:1), the compound N-desethyl-13,15-diacetyldeoxyaconitine (X) was obtained;

(4) N-deethyl-13,15-diacetyldeoxyaconitine was mixed with an equivalent amount of methyl iodide, tetrahydrofuran was used as a solvent, and reacted at room temperature for 2-4 hours, and then dried under reduced pressure to obtain a white solid. After silica gel column chromatography (petroleum ether-ethyl acetate, 3:1), the compound 13,15-diacetylcontoconine (XI) was obtained;

(5) Add 5% sodium hydroxide solution to 13,15-diacetylcontoconine, leave it overnight at room temperature, and treat it in the usual way to obtain a white solid. Silica gel column chromatography (chloroform-methanol saturated with ammonia water, 95:1) gave the compound subunine (III).

The present invention prepares the method for N-deethylthine, comprises the following steps:

(1) Using aconitine as a raw material, prepare N-deethyl-13,15-diacetyldeoxyaconitine (X) according to the steps ①-③ in the above-mentioned method for preparing hypounine;

(2) N-desethyl-13,15-diacetyldeoxyaconitine was added to 5% NaOH methanol solution, room temperature overnight, and treated by conventional methods to obtain a white solid. Silica gel column chromatography (ammonia saturated chloroform-methanol, 95:1) eluted to obtain the desired compound N-desethylhine (XV).

The above-mentioned mesounine, subunine and N-deethyl-unine have the following physical and chemical properties respectively through spectral analysis:

分子式为C₂₄H₃₉NO₉；易溶于水，不溶于丙醇，氯仿。通过充分的包括二维核磁等光谱分析，确定其结构式为(II)。中乌宁碱的光谱数据如下：Mesounine: white amorphous powder with a specific rotation of

Molecular formula is C ₂₄ H ₃₉ NO ₉ ; easily soluble in water, insoluble in propanol and chloroform. Through sufficient spectral analysis including two-dimensional NMR, it is determined that its structural formula is (II). The spectral data of wuningine are as follows:

IR(KBr)cm ^-1 : 3424, 2932, 2892, 2821, 1639, 1453, 1106;

¹ H NMR (CDCl ₃ ) δ: 3.07 (1H, dd, J=8.4, 6.0Hz, H-1), 3.75 (1H, m, H-3), 4.15 (1H, d, J=6.8Hz, H -6), 3.93 (1H, d, J=5.2Hz, H-14β), 4.49 (1H, t, J=5.6Hz, H-15), 3.19 (1H, d, J=6.0Hz, H-16 ), 2.91 (1H, s, H-17), 3.65, 3.74 (each 1H, ABq, J=9.6Hz, H ₂ -18), 2.48, 2.79 (each 1H, ABq, J=11.2Hz, H ₂ - 19), 2.34 (3H, s, NCH ₃ ), 3.26 (3H, s, OCH ₃ -1), 3.34 (3H, s, OCH ₃ -6), 3.64 (3H, s, OCH ₃ -16), 3.32 (3H, s, OCH ₃ -18);

¹³ C NMR (CDCl ₃ ) δ: 82.6 (C-1), 33.8 (C-2), 71.5 (C-3), 43.4 (C-4), 46.5 (C-5), 83.2 (C-6) , 46.4(C-7), 78.8(C-8), 48.8(C-9), 41.6(C-10), 50.1(C-11), 36.9(C-12), 76.3(C-13), 78.7(C-14), 81.5(C-15), 90.7(C-16), 62.5(C-17), 76.8(C-18), 49.8(C-19), 42.5(NCH ₃ ), 56.3( OCH ₃ -1), 57.9 (OCH ₃ -6), 61.3 (OCH ₃ -16), 59.1 (OCH ₃ -18);

ESI-MS m/z(%): 486[M+H](100);

HR-MS: The measured value of quasi-molecular weight is 486.2699, and the calculated value is 486.2624.

分子式为C₂₄H₃₉NO₈；易溶于水，不溶于丙酮，氯仿。通过充分的包括二维核磁等光谱方法，确定其结构式为(III)。Mesounine: white amorphous powder with a specific rotation of

Molecular formula is C ₂₄ H ₃₉ NO ₈ ; easily soluble in water, insoluble in acetone and chloroform. Through sufficient spectroscopic methods including two-dimensional NMR, it is determined that its structural formula is (III).

The spectral data of subunine base are as follows:

IR(KBr)cm ^-1 : 3442, 2925, 2820, 1635, 1456, 1111;

¹ H NMR (CDCl ₃ ) δ: 3.12 (1H, t, J=7.2Hz, H-1), 4.12 (1H, d, J=7.2Hz, H-6), 3.82 (1H, d, J=4.8 Hz, H-14β), 4.42 (1H, d, J=6.0Hz, H-15), 3.05 (1H, d, J=6.0Hz, H-16), 2.93 (1H, s, H-17), 3.65(1H, ABq, J=8.4Hz, H-18), 2.76(1H, ABq, J=10.8Hz, H-19), 2.35(3H, s, NCH ₃ ), 3.25(3H, s, OCH ₃ -1), 3.33 (3H, s, OCH ₃ -6), 3.59 (3H, s, OCH ₃ -16), 3.28 (3H, s, OCH ₃ -18);

¹³ C NMR (CDCl ₃ ) δ: 84.3 (C-1), 25.1 (C-2), 32.5 (C-3), 38.8 (C-4), 46.9 (C-5), 83.1 (C-6) , 46.7(C-7), 78.0(C-8), 48.9(C-9), 41.9(C-10), 50.0(C-11), 37.3(C-12), 76.4(C-13), 78.5(C-14), 81.1(C-15), 91.7(C-16), 62.9(C-17), 80.2(C-18), 56.7(C-19), 41.6(NCH ₃ ), 54.8( OCH ₃ -1), 56.7 (OCH ₃ -6), 60.1 (OCH ₃ -16), 58.1 (OCH ₃ -18);

ESI-MS m/z(%): 470[M+H] ⁺ (100);

HR-MS m/z: The measured value of the quasi-molecular weight is 470.2749, and the calculated value is: 470.2675.

(c＝0.52，甲醇)；分子式为C₂₃H₃₇NO₈；易溶于水，不溶于氯仿，乙醚。通过光谱方法确定其结构式为(XV)。N-去乙基次乌宁碱的光谱数据如下：N-desethylmethine: white amorphous powder with a specific rotation of

(c=0.52, methanol); molecular formula is C ₂₃ H ₃₇ NO ₈ ; easily soluble in water, insoluble in chloroform and ether. Its structural formula is determined as (XV) by spectroscopic method. The spectral data of N-desethylunine are as follows:

IR(KBr)cm ^-1 : 3396, 2936, 2825, 1451, 1098;

¹ H NMR (CDCl ₃ ) δ: 4.60 (1H, d, J = 6.4Hz), 4.15 (1H, d, J = 6.4Hz), 3.87 (1H, d, J = 4.4Hz), 3.70 (3H, s ), 3.35(3H, s), 3.31(3H, s), 3.20(3H, s), 3.12(1H, d, J=8.0Hz);

¹³ C NMR (CDCl ₃ )δ: 91.0(d), 83.5(d), 82.4(d), 81.1(d), 79.9(t), 78.5(s), 78.4(d), 76.2(s), 61.9 (d), 59.1(q), 58.1(q), 56.3(q), 55.3(q), 51.5(d), 50.2(s), 49.0(t), 47.7(d), 42.8(d), 41.1 (d), 38.7(s), 36.8(t), 28.5(t), 23.0(t);

ESI-MS m/z (%): 456 [M+H] ⁺ (100).

After in vitro cardiotonic activity screening of isolated frog hearts and isolated rat hearts, as well as cardiotonic activity tests on normal rat hearts and rat hearts with heart failure, the compound has cardiotonic and anti-heart failure effects.

The following pharmacological tests have confirmed the cardiotonic and anti-heart failure pharmacological activities of the compounds of the present invention.

Experimental example 1: Protective effect of mesounine and subunine on ischemic reperfusion injury in isolated rat hearts

1. Materials and methods

Experimental animals: SD rats, male, provided by the Experimental Animal Center of the Academy of Military Medical Sciences of the Chinese People's Liberation Army, certificate number, SCXK (Army) 2007-0004.

Tested products: Mesounine base, molecular weight 485, easily soluble in water; sub-unine base, molecular weight 469, easily soluble in water, prepared with KH solution to the corresponding concentration before use.

Positive control drug: deacetyllanatoside injection, batch number 090409, produced by Shanghai Xudong Haipu Pharmaceutical Co., Ltd., prepared to the corresponding concentration with KH solution before use.

Experimental liquid: KH liquid (in 2000ml distilled water, add NaCl: 13.7918g; KCl: 0.7008g; CaCl ₂ : 0.56605g; anhydrous MgSO ₄ : 0.2841g; KH ₂ PO ₄ : 0.3212g; NaHCO ₃ : 4.1803g; Glucose: 4.3994g. Magnetic stirring while adding sample, spare).

laboratory apparatus:

1. Langendorff isolated heart perfusion device, American BIOPAC company.

2. DC100A multi-channel physiological recorder, American BIOPAC company.

Experimental steps:

1. Rats were anesthetized, and the chest was quickly opened. The left hand gently lifted the heart with tweezers, and the right hand cut off the superior and inferior vena cava, pulmonary artery, aorta and the tissues around the heart, and removed the heart (about 0.5-1.0 cm length, for cardiac intubation), immediately transferred to a petri dish containing 4°C KH solution, gently squeezed the heart to remove the remaining blood in the ventricle, and cleaned the residual blood in the heart.

2. Quickly connect the ascending branch of the cardiac aorta to the perfusion cannula, and fix it with a thread ligation. Immediately perfuse with oxygenated KH solution. The perfusion pressure is 70 mm Hg, the perfusion temperature is (38±0.5)°C, and the flow rate is (11.5±0.5) ml/min.

3. Make a small cut on the side wall of the left atrium, and insert the small latex water sac into the left ventricle through the atrioventricular opening. The latex water bladder and the connected catheter are filled with ultrapure water, and the other end of the catheter is connected to the pressure transducer through a three-way pipe to completely remove the internal air bubbles. The left ventricular pressure was stabilized for 30 minutes before the test was performed.

4. Use a graduated cylinder to record the coronary flow of the heart every minute; the pressure transducer signal is input into the multi-conductor recording system through the bridge amplifier. Through the computer Acq373 software, complete the real-time signal acquisition and processing.

5. Grouping and test drugs: model group: perfuse with oxygen-containing KH solution for 10 minutes, stop perfusion for 20 minutes, and then resume perfusion with oxygen-containing KH solution for 60 minutes; positive control group and test drug group: the positive drug dose is 10 ^-12 mol/L, the doses of unine and subunine in the test substance were both 10 ^-9 mol/L, and the corresponding amount of medicine was given through the test drug hole immediately when the perfusion was just resumed, and the rest of the operations were the same as those of the model group. The volume of the test drug was 1ml.

6. Each group recorded the coronary flow rate (CBF), left ventricular end-diastolic pressure (LVEDP), left ventricular pressure maximum rate of rise (dp/dtmax) and Heart rate, n=3.

7. The data are expressed as mean ± standard deviation, and the data analysis adopts T-test test.

2. The results are shown in Table 1-4.

Table 1. Effects of mesounine and subunine on CBF (coronary flow) (ml/min, n=3)

Compared with the model group, ^* P<0.05, ^** P<0.01

Table 2. Effects of mesounine and subunine on dpdtmax (maximum rise rate of left ventricular pressure) (mmHg/s, n=3)

Compared with the model group, ^* P<0.05, ^** P<0.01

Table 3. Effects of Zhongwuningine and Diwuningine on LVEDP (left ventricular end-diastolic pressure) (mmHg, n=3)

Compared with the model group, ^* P<0.05, ^** P<0.01

Table 4. Effects of mesounine and subunine on heart rate (times/min, n=3)

Compared with the model group, ^* P<0.05, ^** P<0.01

In this experiment, a rat isolated heart ischemia/reperfusion model was used to observe the effects of unine and subunine in the test product on coronary flow, the maximum rate of rise in left ventricular pressure, left ventricular end-diastolic pressure and heart rate. Impact.

Coronary flow index can reflect the blood flow of myocardial coronary artery. From the results of this experiment, it can be seen that after administration of the model group, positive drug control group, Zhongwuningine and Hewuningine groups, they all showed a trend of increasing first and then decreasing, but the statistical results showed that each administration group was comparable to the model group. No significant difference was found.

The index of the maximum rise rate of left ventricular pressure reflects the systolic function of the myocardium during diastole. Compared with the model, the positive drug control group significantly increased at the 10min and 20min time points after administration, and there was a significant difference in statistics; There was no significant difference between the subunine group and the model group.

The left ventricular end-diastolic pressure index indirectly reflects the diastolic compliance of the ventricle. Compared with the model group, the positive drug control group was significantly lower at each time point from 30 min to 60 min, and there was a significant difference; the left ventricular end-diastolic pressure index at the five time points from 20 min to 60 min was compared with the model group There were significant differences, but there was no significant difference in this index in the subunine group at the above time points.

In terms of heart rate, compared with the model group, the positive drug control group was significantly slower at each time point after administration, and there was a significant difference in statistical analysis; there was a significant difference in the 10min and 20min time points of the Zhongwuningine group, There was a significant difference at the time point of 10 minutes in the subunine group.

The above experimental results suggest that wuningine in the test product can directly increase myocardial contractility and improve myocardial diastolic function on rat ischemia/reperfusion isolated hearts at a dose of 10 ^-9 mol/L.

Experimental Example 2: The Cardiotonic Effect of Zhongwuningine on the Heart of Heart Failure Rats

1 Materials and methods

1.1 Drugs and reagents

Zhongwuning base: molecular weight 485, easily soluble in water.

1.2 Animals and test equipment

Experimental animals: SD rats, weighing 200-250g, both male and female, provided by the West China Medical Experimental Animal Center of Sichuan University.

Test equipment: BL-420F biological signal acquisition and processing system: produced and provided by Chengdu Taimeng Technology Co., Ltd.

1.3 Test method

The SD rats were randomly divided into 5 groups, 4 in each group, which were respectively the normal control group, the model group (normal saline 10ml/kg), the large (0.05mg/kg) and medium (0.0375mgml/kg) , small (0.025ml/kg) dose group. The normal control group only underwent left ventricular intubation to record the left ventricular pressure, without thoracotomy and ligation of the left coronary artery, and was given normal saline 10ml/kg after the operation.

Rats in each group were anesthetized by intraperitoneal injection of 0.03ml/kg 10% chloral hydrate. Fix it on the mouse board, insert the electrocardiogram electrode into the subcutaneous of the limbs, and monitor the animal's electrocardiogram with the standard II lead; surgically separate the trachea and bilateral common carotid arteries; perform tracheal intubation and connect a ventilator to keep breathing unobstructed; perform the left common carotid artery Intubate, connect with BL-420F biological signal acquisition and processing system through a pressure transducer, record the blood pressure curve; cut the third or fourth rib close to the left edge of the sternum, and bluntly separate the upper and lower intercostal muscles longitudinally, exposing the mediastinum and left Lateral thorax, the heart can be seen; bluntly divide the pericardium, 2 mm below the junction of the conus pulmonary artery and the left atrial appendage, near the opening of the left anterior descending branch of the left coronary artery, insert a suture needle (6/0) from the side of the left ventricle Penetrate and pass out from the side of the conus pulmonary artery for ligation; intubate the right common carotid artery to the left ventricle to record the left ventricular pressure, and then ligate and fix the cannula; ligate the left anterior descending coronary artery, and ligate the coronary artery at the above separation site. Whether the ST-segment elevation is greater than 0.1mv, combined with changes in hemodynamic indicators to determine whether the heart failure model is successfully established; the coronary artery is ligated, and intraperitoneal administration is administered after stabilization. Heart rate (HR), systolic blood pressure (SP), diastolic blood pressure (DP), left ventricular systolic pressure (LVSP), left Ventricular end-diastolic pressure (LVEDP) and the maximum rate of rise and fall of left ventricular pressure (±dp/dtmax).

表示，与造模前作差处理后采用SPSS13.0统计软件进行均数比较t检验，检验水准α＝0.05。All data are presented as mean ± standard deviation after modeling

Mean comparison t-test was carried out with SPSS13.0 statistical software after the difference treatment with before modeling, and the test level α=0.05.

2 test results

2.1 See Table 5 for the effect of Wuningine on the heart rate (HR) of rats with heart failure.

In Table 5, the effect of unine base on the heart rate (HR) of heart failure rats (x ± s, n=4)

Compared with the normal group, #: P<0.05, ##: P<0.01; compared with the model group, ^* : P<0.05, ^** : P<0.01;

Compared with the high-dose group, ▲: P<0.05, ▲▲: P<0.01; compared with the medium-dose group: △: P<0.05, △△: P<0.01.

The results in Table 5 show that the ratio between each dose group of wuningine, the stable HR of the middle dose group does not make it significantly lower than that of other dose groups, especially at 20 min, the effect of rising HR is obvious in the larger dose group (P＜0.05), There was no significant difference in HR among the other groups at each time period. It shows that wuningine has the effect of stabilizing the heart rate of rats with heart failure.

2.2 See Table 6 for the effect of Wuningine on the systolic blood pressure (SBP) of rats with heart failure.

In table 6, the influence of unine base on systolic blood pressure (SBP) of rats with heart failure (x ± s, n=4)

Compared with the normal group, #: P<0.05, ##: P<0.01; compared with the model group, ^* : P<0.05, ^** : P<0.01;

Compared with the high-dose group, ▲: P<0.05, ▲▲: P<0.01; compared with the medium-dose group: △: P<0.05, △△: P<0.01.

The results in Table 6 show that there is no significant difference in the ratio of each dosage group of Zhongwuningine, and the SBP of each dosage and each time period. It shows that Zhongwuningine can significantly increase the systolic blood pressure in rats with heart failure.

2.3 See Table 7 for the effect of wuningine on the diastolic blood pressure (DBP) of rats with heart failure.

In table 7, the influence of unine base on diastolic blood pressure (DBP) of rats with heart failure (x±s, n=4)

Compared with the normal group, #: P<0.05, ##: P<0.01; compared with the model group, ^* : P<0.05, ^** : P<0.01;

Compared with the high-dose group, ▲: P<0.05, ▲▲: P<0.01; compared with the medium-dose group: △: P<0.05, △△: P<0.01.

The results in Table 7 show that among the groups of different dosages of wuningine, the reduction of DBP in the middle dosage is stronger than that of the low dosage group (P<0.05) at 50 minutes, and there is no significant difference in DBP between the other groups in each time period. It shows that wuningine can significantly resist the decrease of diastolic blood pressure in rats with heart failure.

2.4 The effect of Wuningine on the left ventricular systolic pressure (LVSP) in rats with heart failure is shown in Table 8.

In table 8, the influence of unine base on the left ventricular systolic pressure (LVSP) of heart failure rats (x±s, n=4)

Compared with the normal group, #: P<0.05, ##: P<0.01; compared with the model group, ^* : P<0.05, ^** : P<0.01;

Compared with the high-dose group, ▲: P<0.05, ▲▲: P<0.01; compared with the medium-dose group: △: P<0.05, △△: P<0.01.

Table 8 shows that there is no significant difference in LVSP between groups of different dosages of Zhongwuningine, and LVSP of each dosage and each time period. It shows that wuningine can significantly improve the left ventricular systolic function of rats with heart failure.

2.5 In Table 9, the effect of unine base on left ventricular end-diastolic pressure (LVEDP) in rats with heart failure.

In Table 9, the effect of unine base on left ventricular end-diastolic pressure (LVEDP) in rats with heart failure (x±s, n=4)

Compared with the normal group, #: P<0.05, ##: P<0.01; compared with the model group, ^* : P<0.05, ^** : P<0.01;

Compared with the high-dose group, ▲: P<0.05, ▲▲: P<0.01; compared with the medium-dose group: △: P<0.05, △△: P<0.01.

Table 9 shows that the inter-group ratios of various doses of wuningine were more obvious (P < 0.05-0.01) for the increase of LVEDP in the medium dose at 20 and 60 min, and at the small dose at 30-40 min (P < 0.05-0.01). There was no significant difference in LVEDP among the dose groups. It shows that wuningine has the effect of reducing left ventricular end-diastolic pressure in rats with heart failure.

2.6 In Table 10, the effect of Wuningine on the maximum increase rate (+dp/dtmax) of left ventricular pressure in the isovolumic phase of heart failure rats is shown in Table 10.

In Table 10, the influence of unine base on the maximum rate of rise (+dp/dtmax) of left ventricular pressure in the isovolumic phase of heart failure rats (x ± s, n = 8)

Compared with the normal group, #: P<0.05, ##: P<0.01; compared with the model group, ^* : P<0.05, ^** : P<0.01;

Compared with the high-dose group, ▲: P<0.05, ▲▲: P<0.01; compared with the medium-dose group: △: P<0.05, △△: P<0.01.

Table 10 shows that the ratio between the doses of Wuningine, middle dose+dp/dtmax is significantly higher than high dose group (P＜0.05) when 50-60min, and +dp/dtmax is significantly higher than small dose group (P＜0.05) when 60min P<0.05). It shows that wuningine can significantly improve the maximum contraction rate of left ventricle in rats with heart failure.

Table 11 shows the effect of 2.7 Wuningine on the maximum pressure drop rate (-dp/dtmax) of the left ventricle in the isovolumic phase of heart failure rats.

In Table 11, the influence of unine base on the maximum drop rate (-dp/dtmax) of left ventricular isovolumic phase pressure in heart failure rats (x±s, n=4)

Compared with the normal group #: P<0.05, ##: P<0.01; compared with the model group, ^* : P<0.05, ^** : P<0.01;

Compared with the high-dose group, ▲: P<0.05, ▲▲: P<0.01; compared with the medium-dose group: △: P<0.05, △△: P<0.01.

Table 11 shows that among the doses of wuningine, the effect of increasing -dp/dtmax in the middle dose group at 20, 50-60 min was significantly higher than that in the high dose group (P<0.05).

3. Conclusion Based on the above results, Zhongwuningine can improve the cardiac function of rats with heart failure. It shows that wuningine in the compound has significant cardiotonic and anti-heart failure effects.

The present invention proposes a medicine containing the compound represented by the general formula (I) of the present invention or a pharmaceutically acceptable salt thereof.

The present invention also proposes medicines containing unine and hypounine or their pharmaceutically acceptable salts among the compounds of the present invention.

The compound is converted into a suitable pharmaceutical form, if necessary using inert auxiliaries and excipients to make a medicament.

At the same time, the present invention proposes to use the unine base and the sub-unine base or their pharmaceutically acceptable salts in the compound as cardiotonic and anti-heart failure drugs for clinical treatment.

The pharmaceutical compositions of the present invention may be ointments, gels, pastes, sprays, lotions, suspensions, solvents or emulsions of the active ingredients in aqueous and non-aqueous diluents, syrups, granules or powders.

Application of the medicine of unine base and sub-unine base or their pharmaceutically acceptable salts in the compound of the present invention, the dosage form that can adopt is the usual galenic administration dosage form, for example: ointment, tablet, pill, capsule, suppository, emulsion, Infusion and injection. These preparations are prepared by known methods using conventional additives and excipients. The medicine thus prepared can be administered locally, parenterally, orally, or by injection according to needs.

Diluents (e.g. granules) for pharmaceutical compositions suitable for forming tablets, dragees, capsules, pills and injections include:

(a) fillers such as cornstarch, sugar and silicic acid;

(b) binders such as cellulose derivatives, alginates, gelatin and polyvinylpyrrolidone;

(c) humectants, such as glycerin;

(d) disintegrants such as agar, calcium carbonate and sodium bicarbonate;

(e) absorption enhancers, such as quaternary ammonium compounds;

(f) surfactants, such as cetyl alcohol;

(g) adsorption carriers such as kaolin and bentonite;

(h) Lubricants such as talc, calcium stearate, magnesium stearate and solid polyethylene glycol;

(i) pH regulators, such as hydrochloric acid, sodium bicarbonate, sodium hydroxide;

(j) isotonic agents, such as citrate, sodium citrate, lactic acid and sodium lactate;

(k) cosolvents, such as methylparaben, ethylparaben, Tween-80;

(l) Antioxidants, such as sodium metabisulfite, sodium thiosulfate, sodium sulfite and vitamin C.

Tablets, dragees, and capsules formed from the pharmaceutical composition of the present invention can bear the usual coating, film and protective matrix, which may contain opacifying agents. They can be formulated so that they release the active ingredient only or preferably in specific parts of the body for a certain period of time. Coatings, membranes and protective matrices may be formed, for example, from polymeric substances or waxes.

The active ingredient may also be in microencapsulated form together with one or more of the above diluents.

The production of the above-mentioned pharmaceutical composition or medicine can be carried out by any known method in this technology, for example, one or several active ingredients are mixed with one or several diluents to form a pharmaceutical composition, and then the composition is made into drug.

According to the dose of the compound represented by the general formula (I) of the present invention and mesounine and hypounine as clinical medicines, it is preferably in the range of 0.0001 to 5 mg/kg body weight, preferably 0.01-5 mg / kg body weight range.

Through the pharmacological experiments done by the present invention, it is found that the compound represented by the general formula (I), mesounine and subunine or their pharmaceutically acceptable salts have cardiotonic and anti-heart failure effects. This effect can be used clinically as a cardiotonic and anti-heart failure drug.

Detailed ways:

Embodiment 1: the preparation of N-deethylaconitine base (XII)

Weigh 22 kg of aconite, extract 3 times with 3 times the amount of hot water, precipitate the extract with 75% ethanol, and concentrate the supernatant under reduced pressure to obtain 175 g of extract. Dissolve in water, adjust the pH to 10 with ammonia water, and degrease with ether. The aqueous layer was extracted with n-butanol, and the extract was dried to obtain 10 g of n-butanol extract. Silica gel (Qingdao Ocean Chemical Factory, the same below) column chromatography, eluting with chloroform-methanol (95:5-85:15) to obtain fraction D (3.2g), silica gel column chromatography, chloroform-methanol (15:1) eluting off to obtain 60 mg of the desired compound.

White amorphous powder. C ₂₅ H ₄₁ NO ₉ .

ESIMS m/z(%): 472[M+H] ⁺ (100);

¹ H NMR (CDCl ₃ ) δ: 4.51 (1H, d, J = 4.0Hz), 4.31 (1H, d, J = 5.2Hz), 4.17 (1H, d, J = 2.4Hz), 3.60 (3H, s ), 3.43(3H, s), 3.39(3H, s), 3.34(3H, s), 3.26(3H, s);

¹³ C NMR (CDCl ₃ ) δ: 91.6(d), 81.7(d), 80.2(d), 79.9(d), 77.9(s), 78.2(d), 76.8(t), 76.0(s), 69.1 (d), 66.9(d), 60.4(q), 59.0(q), 57.2(q), 54.1(q), 51.5(t), 50.3(s), 43.2(s), 42.3(d), 40.5 (d), 40.5(d), 40.4(d), 36.4(t), 29.0(t).

Embodiment 2: the preparation of isodelnine base (XIII)

Part B (2.1 g) obtained by silica gel column chromatography in Example 1 was eluted by silica gel column chromatography with chloroform-methanol (8:2→7:3) to obtain 25 mg of the desired compound.

White amorphous powder. C ₂₄ H ₃₉ NO ₇ .

ESI-MS m/z(%): 454[M+H] ⁺ (100);

¹ H NMR (CDCl ₃ ) δ: 4.38 (1H, d, J = 6.8Hz), 4.21 (1H, d, J = 7.0Hz), 4.07 (1H, d, J = 4.4Hz), 3.73 (1H, d , J=3.2Hz), 3.54(1H, d, J=8.4Hz), 3.41(3H, s), 3.34(3H, s), 3.29(6H, s), 2.40(3H, s);

¹³ C NMR (CDCl ₃ ) δ: 92.2(d), 84.5(t), 80.6(d), 79.4(d), 79.2(s), 76.3(d), 72.9(d), 63.6(d), 59.1 (q), 58.9(t), 58.1(q), 57.5(q), 57.2(q), 50.6(s), 49.8(d), 49.3(d), 48.4(d), 45.3(d), 44.4 (d), 41.6(q), 39.3(s), 31.7(t), 30.7(t), 29.8(t).

Embodiment 3: the preparation of North Uning (XIV)

Weigh 1.0 g of beiwutine, add 20 ml of 5% NaOH methanol solution, leave at room temperature overnight, and dry under reduced pressure to obtain 0.7 g of a white solid. Silica gel column chromatography, eluting with chloroform-methanol (7:3) saturated with ammonia water, gave the desired compound.

Yield: 92%. White amorphous powder. C ₂₄ H ₃₉ N0 ₁₀ .

ESI-MS m/z(%): 502[M+H] ⁺ (100);

¹ H NMR (CDCl ₃ ) δ: 4.45 (1H, d, J = 6.0Hz), 4.26 (1H, d, J = 5.2Hz), 3.16 (1H, d, J = 6.4Hz), 3.82 (1H, d , J=8.4Hz), 3.57(3H, s), 3.35(3H, s), 3.29(3H, s), 3.27(3H, s), 2.37(3H, s);

¹³ C NMR (CDCl ₃ )δ: 92.6(d), 84.4(d), 83.2(d), 79.4(s), 78.5(d), 78.4(d), 78.1(s), 77.7(s), 75.8 (t), 70.6(d), 61.5(q), 59.4(d), 59.1(q), 58.4(q), 56.8(s), 55.9(q), 51.1(t), 49.0(q), 49.0 (t), 47.8(d), 44.4(s), 42.8(d), 42.4(d), 34.7(t), 34.7(t).

Embodiment 4: the extraction separation of aconitine (IV)

Weigh 10kg of rhizome powder of Aconitum carchaeli Debx. produced in Xinjiang, put it into a percolation cartridge, infiltrate with 10-20 times of 0.3% HCl aqueous solution, alkalinize the leachate with concentrated ammonia water, extract with ethyl acetate, extract The liquid was dried under reduced pressure to obtain a brown solid.

Silica gel column chromatography (ethyl acetate-petroleum ether-diethylamine, 60:40:2, w/w) separated to obtain crude aconitine. Then crystallized by 95% ethanol to obtain the desired compound.

Yield: 0.7%. Colorless needle crystal. C ₃₄ H ₄₇ NO ₁₁ .

ESI-MS m/z(%): 646[M+H] ⁺ (100);

¹ H NMR (CDCl ₃ ) δ: 8.03 (1H, d, J = 7.2Hz), 7.58 (1H, t, J = 7.2Hz), 7.46 (1H, t, J = 7.2Hz), 4.87 (1H, d , J=5.2Hz), 4.47(1H, dd, J=5.2, 2.8Hz), 4.39(1H, d, J=2.8Hz), 4.03(1H, d, J=6.4Hz), 3.96(3H, s ), 3.75(3H, s), 3.62(1H, d, J=9.2Hz), 3.50(1H, d, J=8.8Hz), 3.34(1H, d, J=5.2Hz), 3.30(3H, s ), 3.26(3H, s), 3.16(3H, s), 1.39(3H, s), 1.10(3H, d, J=7.2Hz);

¹³ C NMR (CDCl ₃ )δ: 172.4(s), 166.0(s), 133.3(d), 129.6(s), 129.5(d), 128.6(d), 91.9(s), 89.9(d), 83.3 (d), 82.3(d), 82.3(d), 78.8(d), 78.8(d), 76.6(t), 74.0(s), 71.3(d), 61.1(q), 61.0(d), 59.1 (q), 57.9(q), 55.9(q), 49.9(s), 48.9(t), 46.9(d), 46.7(d), 44.6(d), 44.1(d), 43.0(s), 40.8 (d), 35.7(t), 33.5(t), 21.4(q), 13.3(q).

Embodiment 5: the preparation of wuning base

(1) Preparation of 3,13,15-triacetylaconitine (V)

Weigh 18.0 mol of aconitine, put it into a three-necked flask, add 6 g of p-toluenesulfonic acid and 72 ml of acetic anhydride, stir, react at room temperature for 72 hours, and treat it according to a conventional method to obtain a white solid. After silica gel column chromatography (or acetone-ether crystallization), the desired compound was obtained.

Yield: 95%. Colorless needle crystal. C ₄₀ H ₅₃ NO ₁₄ .

ESI-MS m/z(%): 772[M+H] ⁺ (100);

¹ H NMR (CDCl ₃ ) δ: 8.16 (1H, d, J = 7.2Hz), 7.56 (1H, t, J = 7.2Hz), 7.48 (1H, t, J = 7.2Hz), 6.09 (1H, d , J = 5.6Hz), 5.13 (1H, d, J = 5.2Hz), 4.91 (1H, dd, J = 8.4, 5.2Hz), 4.11 (1H, m), 4.05 (1H, d, J = 5.6Hz ), 3.75 (1H, d, J = 8.8Hz), 3.68 (1H, d, J = 5.2Hz), 3.64 (1H, d, J = 5.2Hz), 3.40 (3H, s), 3.24 (3H, s ), 3.19(6H,s), 3.11(1H,dd, J=10.0,7.2Hz), 2.14(3H,s), 2.07(3H,s), 2.06(3H,s), 2.01(3H,s) , 1.23(3H, s), 1.16(3H, t, J=7.2Hz);

¹³ C NMR (CDCl ₃ ) δ: 170.3(s), 170.3(s), 169.7(s), 168.8(s), 166.2(s), 133.3(d), 129.9(d), 129.5(s), 128.7 (d), 88.7(d), 83.6(d), 81.5(d), 81.0(d), 78.8(d), 76.7(d), 71.3(t), 61.4(q), 60.4(d), 58.7 (q), 58.6(q), 56.1(q), 49.8(s), 48.9(t), 47.0(t), 45.5(d), 45.1(d), 43.9(d), 42.1(s), 41.2 (d), 35.9(t), 31.9(t), 21.2(q), 21.2(q), 21.1(q), 21.1(q), 13.6(q).

(2) Preparation of N-deethyl-3,13,15-triacetylaconitine (VI)

Weigh 17.0 moles of 3,13,15-triacetylaconitine, add it to a three-necked flask, add 420 ml of glacial acetic acid and 54.5 moles of N-bromosuccinimide, stir, react at room temperature for 4 hours, and The method is processed to obtain a white solid. After silica gel column chromatography (petroleum ether-acetone, 2:1), the desired compound was obtained.

Yield: 75%. White amorphous powder. C ₃₈ H ₄₉ NO ₁₄ .

ESI-MS m/z(%): 744[M+H] ⁺ (100);

¹ H NMR (CDCl ₃ ) δ: 7.48-8.18 (5H, m, Ar-H), 6.10 (1H, d, J=6.0Hz, H-15), 5.16 (1H, d, J=5.2Hz, H -14β), 5.04 (1H, dd, J=10.0, 5.6Hz, H-3), 4.07 (1H, d, J=6.8Hz, H-6), 3.86 (1H, d, J=5.6Hz, H -16), 3.75, 2.97 (each 1H, ABq, J=8.8Hz, H ₂ -18), 3.21, 3.23, 3.30, 3.40 (each 3H, s, OAc×4), 2.06, 2.07, 2.18 (each 3H , s, OAc×3), 1.25 (3H, s, OAc-8);

¹³ C NMR (CDCl ₃ ) δ: 170.5(s), 170.4(s), 169.9(s), 168.7(s), 166.1(s), 133.4(d), 129.9(d), 129.3(s), 128.7 (d), 88.3(d), 88.3(s), 83.7(d), 80.8(s), 80.6(d), 78.7(d), 76.7(d), 72.9(t), 71.8(d), 61.4 (d), 58.8(q), 58.5(q), 55.8(q), 55.7(q), 51.4(d), 49.9(s), 44.6(d), 42.9(d), 42.7(s), 41.3 (d), 41.1(t), 34.6(t), 31.7(t), 21.3(q), 21.3(q), 21.1(q), 21.1(q).

(3) Preparation of 3,13,15-triacetyl mesoconitine (VII)

Weigh 13 moles of N-desethyl-3,13,15-triacetylaconitine, put it into a three-necked flask, add 13 moles of sodium hydride and 300 ml of tetrahydrofuran, stir for 10 minutes under argon flow, and then add methyl iodide 15 mmoles, stirred, reacted at room temperature for 3 hours, drained the solvent, and processed according to conventional methods to obtain a white solid. After silica gel column chromatography (chloroform-methanol=95:1), the desired compound was obtained.

Yield: 90%. Colorless prism. C ₃₉ H ₅₁ NO ₁₄ .

ESI-MS m/z(%): 758[M+H] ⁺ (100);

¹ H NMR (CDCl ₃ ) δ: 7.49-8.17 (5H, m), 6.07 (1H, d, J = 5.6Hz), 5.14 (1H, d, J = 5.2Hz), 4.91 (1H, dd, J = 12.8, 6.0Hz), 4.05 (1H, d, J = 6.8Hz), 3.85 (1H, d, J = 5.2Hz), 3.40, 3.27, 3.27, 3.19 (each 3H, s), 3.76, 2.95 (each 1H , ABq, J=8.8Hz), 2.66(1H, ABq, J=11.2Hz), 2.45(3H, s), 2.16, 2.07, 2.06 (each 3H, s), 1.24(3H, s);

¹³ C NMR (CDCl ₃ ) δ: 170.3(s), 170.1(s), 169.5(s), 168.7(s), 166.2(s), 133.3(d), 130.0(d), 129.6(s), 128.6 (d), 88.8(d), 88.6(s), 83.5(d), 81.6(d), 81.1(s), 78.9(d), 76.7(d), 71.3(d), 71.2(t), 62.0 (d), 61.2(q), 58.7(q), 58.6(q), 56.3(q), 49.9(s), 49.6(t), 45.0(d), 44.3(d), 43.9(d), 42.6 (q), 42.4(s), 41.3(d), 35.7(t), 31.9(t), 21.2(q), 21.1(q), 21.1(q), 21.1(q).

(4) Preparation of Wuning base (II)

Weigh 10.0 moles of 3,13,15-triacetyl mesoconitine, put it into a three-necked bottle, add 250 ml of 5% sodium hydroxide in methanol, stir at 60°C for 30 minutes, drain the solvent, and treat it according to a conventional method to obtain white solid. After silica gel column chromatography (chloroform-methanol, 95:5), the desired compound was obtained.

Yield: 95%. White amorphous powder. C ₂₄ H ₃₉ NO ₉ .

Spectral (IR, ¹ HNMR, ¹³ CNMR, ESI-MS) data are described above.

Embodiment 6: the preparation of subunine base

(1) Preparation of deoxyaconitine (VIII)

Weigh 2.0 moles of aconitine, put it into a three-necked flask, add 100 ml of tetrahydrofuran and 7 ml of thionyl chloride, stir, react at room temperature for 2 hours, and treat it according to the conventional method to obtain a yellow-brown solid. Put it into a three-necked flask, add 80 ml each of ethyl acetate and 95% ethanol, add 2 ml of glacial acetic acid, 1 g of palladium carbon, and pass in hydrogen gas (about 0.1 kg/cm ² ) while stirring, and react for 72 hours , according to conventional methods to obtain a white solid. After silica gel column chromatography (chloroform-methanol, 95:5), the desired compound was obtained.

Yield: 90%. Colorless needle crystal. C ₃₄ H ₄₇ NO ₁₀ .

ESI-MS m/z(%): 630[M+H](100);

¹ H NMR (CDCl ₃ ) δ: 8.02 (1H, d, J = 7.2Hz), 7.57 (1H, t, J = 7.2Hz), 7.45 (1H, t, J = 7.2Hz), 4.87 (1H, d , J=4.8Hz), 4.45(1H, dd, J=5.2, 2.8Hz), 4.37(1H, d, J=2.8Hz), 3.97(1H, d, J=6.0Hz), 3.89(1H, s ), 3.73(3H, s), 3.63(1H, d, J=8.4Hz), 3.28(3H, s), 3.26(3H, s), 3.15(3H, s), 3.10(1H, d, J= 8.4Hz), 1.36(3H, s), 1.06(3H, t, J=7.2Hz);

¹³ C NMR (CDCl ₃ )δ: 172.4(s), 166.1(s), 133.2(d), 129.7(d), 129.6(s), 128.5(d), 92.0(s), 90.0(d), 84.9 (d), 83.1(d), 80.2(t), 78.9(d), 78.8(d), 74.0(s), 61.4(d), 61.0(q), 58.6(q), 57.9(q), 56.3 (q), 53.0(t), 49.8(s), 49.2(t), 49.0(d), 45.0(d), 44.5(d), 40.9(d), 39.0(s), 36.6(t), 35.2 (t), 26.3(t), 21.4(q), 13.4(q).

(2) Preparation of 13,15-diacetyldeoxyaconitine (IX)

Weigh 3.0 mol of hexaconitine and put it into a three-necked flask, add 2 g of p-toluenesulfonic acid and 20 ml of acetic anhydride, stir, react at 45°C for 48 hours, and treat according to conventional methods to obtain a white solid. Crystallization from acetone-ether gave the desired compound.

Yield: 90%. White amorphous powder. C ₃₈ H ₅₁ NO ₁₂ .

ESI-MS m/z(%): 714[M+H] ⁺ (100);

¹ H NMR (CDCl ₃ ) δ: 8.16 (1H, d, J = 7.2Hz), 7.56 (1H, t, J = 7.2Hz), 7.48 (1H, t, J = 7.2Hz), 6.07 (1H, d , J=5.6Hz), 5.14(1H, d, J=5.2Hz), 3.94(1H, d, J=6.4Hz), 3.84(1H, d, J=5.6Hz), 3.40(3H, s), 3.25(3H, s), 3.22(3H, s), 3.15(3H, s), 2.13(3H, s), 2.04(3H, s), 1.25(3H, s), 1.13(3H, t, J = 7.2Hz);

¹³ C NMR (CDCl ₃ ) δ: 170.3(s), 169.8(s), 168.8(s), 166.3(s), 133.3(d), 129.9(d), 129.5(s), 128.7(d), 88.9 (s), 88.7(d), 84.7(d), 83.4(d), 81.1(s), 80.0(t), 78.9(d), 76.9(d), 61.4(q), 61.2(d), 59.6 (q), 58.4(q), 53.0(q), 50.1(s), 49.1(t), 49.1(t), 48.7(d), 45.1(d), 43.9(d), 41.7(d), 38.8 (s), 36.2(t), 35.2(t), 26.3(t), 21.3(q), 21.2(q), 21.1(q), 13.6(q).

(3) Preparation of N-deethyl-13,15-diacetyldeoxyaconitine (X)

Weigh 5.0 moles of 13,15-diacetyldeoxyaconitine, put it into a three-necked bottle, add 90 ml of glacial acetic acid and 15 moles of N-bromosuccinimide, stir and react at room temperature for 3 hours, and then method to obtain a white solid. After silica gel column chromatography (ammonia saturated chloroform-methanol, 8:2), the desired compound was obtained.

Yield: 75%. White amorphous powder. C ₃₂ H ₄₃ NO ₁₀ .

ESI-MS m/z(%): 602[M+H] ⁺ (100);

¹ H NMR (CDCl ₃ ) δ: 8.17 (1H, d, J = 7.2Hz), 7.57 (1H, t, J = 7.2Hz), 7.49 (1H, t, J = 7.2Hz), 6.16 (1H, d , J=6.5Hz), 5.17(1H, d, J=4.8Hz), 3.93(1H, dd, J=9.6, 6.4Hz), 3.41(3H, s), 3.29(3H, s), 3.27(3H , s), 3.18(3H, s), 2.21(3H, s), 2.08(3H, s), 1.26(3H, s);

¹³ C NMR (CDCl ₃ )δ: 170.6(s), 170.5(s), 168.7(s), 166.1(s), 133.4(d), 130.0(d), 129.3(s), 128.7(d), 88.7 (s), 87.9(d), 83.7(d), 82.5(d), 80.7(s), 79.8(t), 78.6(d), 77.0(d), 61.5(d), 59.1(q), 58.4 (q), 56.8(q), 55.5(q), 50.1(s), 49.2(d), 49.0(t), 43.4(d), 42.7(d), 41.1(d), 39.0(s), 35.4 (t), 29.7(t), 29.6(t), 24.0(t).

(4) Preparation of 13,15-diacetylcontoconine (XI)

Weigh 3.8 moles of N-desmethyl-13,15-diacetyldeoxyaconitine, put it into a three-necked flask, add 3.8 moles of sodium hydride, 80 ml of tetrahydrofuran and 4 moles of methyl iodide, stir, and react at room temperature for 3 hours Afterwards, it was processed according to conventional methods to obtain a white solid. After silica gel column chromatography (chloroform-methanol, 95:5), the desired compound was obtained.

Yield: 80%. White amorphous powder. C ₃₃ H ₄₉ NO ₁₂ .

ESI-MS m/z(%): 698[M+H] ⁺ (100);

¹ H NMR (CDCl ₃ ) δ: 8.17 (1H, d, J = 7.2Hz), 7.52 (1H, t, J = 7.2Hz), 7.48 (1H, t, J = 7.2Hz), 6.07 (1H, d , J=5.6Hz), 5.13(1H, d, J=5.2Hz), 3.40(3H, s), 3.27(6H, s), 3.15(3H, s), 2.42(3H, s), 2.13(3H , s), 2.03(3H, s), 1.22(3H, s);

¹³ C NMR (CDCl ₃ ) δ: 170.3(s), 169.7(s), 168.8(s), 166.2(s), 133.3(d), 129.9(d), 129.5(s), 128.6(d), 88.7 (s), 88.7(d), 84.6(d), 83.3(d), 81.1(s), 79.8(t), 78.9(d), 76.8(d), 62.3(d), 61.3(q), 58.9 (q), 58.3(q), 56.3(q), 55.7(t), 50.1(s), 47.6(q), 44.0(d), 43.8(d), 42.8(d), 41.1(d), 39.0 (s), 35.9(t), 34.8(t), 26.4(t), 21.3(q), 21.2(q), 21.1(q).

(5) preparation of sub-unine base (III)

Weigh 5.0 moles of 13,15-diacetylhiconitine, put it into a three-necked bottle, add 40 ml of 5% sodium hydroxide in methanol, stir and react at 60°C for 30 minutes, and treat it according to the conventional method to obtain a white solid. The desired compound was obtained by separation through silica gel column chromatography with ammonia saturated chloroform-methanol 95:5.

Yield: 92%. White amorphous powder. C ₂₄ H ₃₉ NO ₈ .

Spectral (ESI-MS, ¹ H NMR, ¹³ C NMR HR-MS) data see supra.

Embodiment 7: the preparation of N-deethylthine (XV)

Weigh 0.5 g of N-desmethyl-13,15-diacetylcontoconine in Example 5, put it into a three-necked flask, add 15 ml of 5% NaOH methanol solution, stir at room temperature overnight, and drain to obtain a white solid things. Silica gel column chromatography (chloroform-methanol, 7:3) gave the desired compound.

Yield: 92%. White amorphous powder. C ₂₃ H ₃₇ NO ₈ .

Spectral ( ¹ H NMR, ¹³ C NMR, ESI-MS, HR-MS) data are given above.

The representative pharmaceutical dosage forms set forth below are suitable for taking the compound represented by the general formula (I) and mesounine and subunine or their pharmaceutically acceptable salts (hereinafter referred to as "compound X") during treatment.

Claims (3)

1. The purposes of wuningine (II) and hypougineine (III) or their medicinal salts in the preparation of cardiotonic and anti-heart failure agents in C _{19 -diterpene} alkaloids,

. 2. the preparation method of the Wuningine that is used to prepare cardiotonic agent and anti-heart failure agent, comprises the following steps: 1) Under the catalysis of p-toluenesulfonic acid, react aconitine (IV) with acetyl chloride or acetic anhydride at room temperature for 6-12 hours, and after conventional treatment, a solid is obtained, which is crystallized in 95% ethanol to obtain compound 3, 13 , 15-triacetylaconitine (3,13,15-triacetylaconitine) (V);

2) Mix 3,13,15-triacetylaconitine with 3 equivalents of N-bromosuccinimide and 20-40 times glacial acetic acid, react at room temperature for 2-6 hours, concentrate under reduced pressure to obtain a white solid, Silica gel chromatographic separation, with petroleum ether, acetone, chloroform, dichloromethane, methanol or their mixed solvents as the eluent, to obtain N-deethyl-3,13,15-triacetylaconitine (N-deethyl -3,13,15-triacetylaconitine) (VI);

3) N-desethyl-3,13,15-triacetylaconitine was mixed with an equivalent amount of methyl iodide, THF was used as solvent, reacted at room temperature for 2-4 hours, and dried under reduced pressure to obtain a white solid, which was subjected to silica gel chromatography Separation, using petroleum ether, ethyl acetate, chloroform, methanol or their mixed solvents as the eluent to obtain 3,13,15-triacetylmesaconitine (3,13,15-triacetylmesaconitine) (VII);

4) 3,13,15-Triacetyl mesoconitine was hydrolyzed with 5% sodium hydroxide, left overnight at room temperature, and dried under reduced pressure to obtain a white solid, which was separated by silica gel chromatography and purified with petroleum ether, chloroform, dichloromethane, methanol Or their mixed solution is eluent, obtains Wu Ning base (II) in the compound,

. 3. the preparation method for the hypounine base that is used to prepare cardiotonic agent and anti-heart failure agent, comprises the following steps: 1) Using tetrahydrofuran as a solvent, mix aconitine with an equivalent amount of thionyl chloride, react overnight at room temperature for 1-3 hours, and dry it under reduced pressure to obtain a white solid, then add ethyl acetate-95% ethanol-glacial acetic acid After the mixed solution was catalyzed by palladium carbon, it was hydrogenated under normal pressure for 1-3 days, and then dried under reduced pressure to obtain a white solid, which was crystallized by 95% ethanol to obtain deoxyaconitine (VIII); 2) Under the catalysis of p-toluenesulfonic acid, mix hexaconitine with 1-2 equivalents of acetyl chloride or acetic anhydride, stir, react at room temperature overnight, and dry under reduced pressure to obtain a white solid, which is then crystallized with 95% ethanol. Obtain compound 13,15-diacetyldeoxyaconitine) (13,15-diaacetyldeoxyaconitine) (IX);

3) Mix 13,15-diacetyldeoxyaconitine with 2-4 equivalents of N-bromosuccinimide and 20-40 times glacial acetic acid, react at room temperature for 2-6 hours, concentrate under reduced pressure to obtain a white solid material, separated by silica gel chromatography, using petroleum ether, acetone, chloroform, methanol or their mixed solvents as eluents, to obtain the compound N-deethyl-13,15-diacetyldeoxyaconitine (N-deethyl- 13,15-diaacetyl-deoxyaconitine) (X); 4) N-deethyl-13,15-diacetyldeoxyaconitine was mixed with an equivalent amount of methyl iodide, tetrahydrofuran was used as a solvent, reacted at room temperature for 2-4 hours, and dried under reduced pressure to obtain a white solid, which was subjected to silica gel chromatography Separation, using petroleum ether, ethyl acetate, chloroform, acetone, methanol or their mixed solvents as eluent, to obtain compound 13,15-diacetylhypaconitine (13,15-diaacetylhypaconitine) (XI);

5) 13,15-diacetylhypaconitine (13,15-diaacetylhypaconitine) was added to 5% sodium hydroxide solution, room temperature overnight, and treated by the usual method to obtain a white solid, which was separated by silica gel chromatography, and was purified with chloroform, ethyl acetate, Dichloromethane, acetone, methyl alcohol or their mixed solvents are eluents to obtain compound subunine base (III),

.