CN113149905A - Low-toxicity antiarrhythmic compound and pharmaceutical application thereof - Google Patents

Abstract

The invention provides a low-toxicity antiarrhythmic compound and a pharmaceutical application thereof. The structure of the compound is shown as a formula I. Experimental results show that the compound provided by the invention has excellent antiarrhythmic activity, can delay VPB latency of aconitine-induced arrhythmia rats and effectively prevent further development of VPB, and the antiarrhythmic effect of the compound is even better than that of a positive medicament lidocaine under the dosage of 0.60 mg/kg. More importantly, compared with bulleyaconitine A, the cardiotoxicity of the compound is obviously reduced; with the known product delta of the margarine stir-frying^15,16Compared with the 16-demethoxyindaconitine, the compound of the invention has the advantages of obviously lower acute toxicity, higher safety and wider dosage range. The invention relates to a method for preparing low-toxicity antiarrhythmic drugProvides a new choice and provides a new method for controlling the quality of the sand-fried aconitum yunnanense or the sand-fried aconitum crassimum.

Description

Low-toxicity antiarrhythmic compound and pharmaceutical application thereof

Technical Field

The invention belongs to the field of pharmacy, and particularly relates to a low-toxicity antiarrhythmic compound and pharmaceutical application thereof.

Background

Ranunculaceae Ranunculus Aconitum L. plants have more than 300 species all over the world, are widely distributed in northern temperate regions, have important medicinal value, and China has more than 200 species of Aconitum plants, all provinces are distributed, mainly distributed in western Sichuan, northwest Yunnan and eastern mountain regions of Tibetan, 76 species can be used as medicines, and the application is wide in traditional Chinese medicine and minority medicine clinic. For example, radix aconiti kusnezoffii and radix aconiti lateralis are common varieties in traditional Chinese medicine, and aconitum pendulum and radix aconiti kusnezoffii are common varieties in Tibetan medicine, Qiang medicine and Yi medicine, have the effects of dispelling wind and removing dampness, warming meridians and relieving pain and relieving fever, and are used for treating anemofrigid-damp arthralgia, arthralgia and other symptoms.

Diterpenoid alkaloids are active ingredients of Aconitum plants with analgesic and anti-inflammatory effects, but also have cardiotoxicity and neurotoxicity. For example, the mechanism of toxicity caused by aconitine is that aconitine can be specifically combined with voltage sensitive sodium channels on the cell membranes of cardiac muscle cells and nerve cells, so that the sodium channels are continuously activated, sodium ions continuously flow in, and homeostasis is damaged, so that cell functions are influenced, numbness of limbs, respiratory depression and arrhythmia occur, and severe patients can cause damage to multi-organ functions and even death.

Bulleyaconitine A is the main ingredient of Aconitum Bulleyanum Diels and Aconitum crassifolium (A. crassifolium W. T. Wang), and has been developed into various dosage forms such as injection and tablet for treating rheumatic and rheumatoid arthritis, scapulohumeral periarthritis, etc. Some scholars perform statistical analysis on the adverse reaction reports of bulleyaconitine A tablets from 2009 to 2017 of the national drug adverse reaction monitoring center, and find that 28.78% of 403 patients with single bulleyaconitine A tablets have palpitation and 17.62% of patients have taste decline, which indicates that the bulleyaconitine A still has strong toxicity. Arrhythmia refers to an abnormality in the frequency, rhythm, site of origin, conduction velocity, or activation order of cardiac impulses. Arrhythmia is an important group of cardiovascular diseases, and can be caused independently or together with other cardiovascular diseases. The prognosis is related to the cause, induction and evolution trend of arrhythmia, whether it causes severe hemodynamic disturbance, sudden death due to sudden onset, or failure due to continuous involvement of heart.

Most of the commonly used antiarrhythmic drugs in the current market are accompanied by considerable adverse reactions, for example, lidocaine is often manifested as vertigo, abnormal sensation, confusion and other nervous system adverse reactions, amiodarone has adverse reactions such as hypothyroidism or hyperthyroidism, lung injury and other adverse reactions, if the dosage of verapamil is improperly adjusted, adverse reactions in the aspects of cardiovascular, nervous and endocrine are also caused, and the ideal antiarrhythmic drugs in all aspects are difficult to find. How to overcome the problem of adverse reaction of antiarrhythmic drugs in the prior art is the problem which needs to be solved urgently at present.

Actually, in the clinical practice of traditional Chinese medicine and national medicine, most aconite herbs are not directly used, but processed and then used. However, the rule of conversion of the components in aconite herbs in processing is still unclear. If the processing conversion product which not only has the effect of resisting arrhythmia but also can reduce toxicity can be researched, the method has important significance for developing new antiarrhythmic drugs.

Disclosure of Invention

An object of the present invention is to provide an antiarrhythmic compound with low toxicity and pharmaceutical use thereof.

The invention also aims to provide the application of the compound in preparing the marked components of the aconitum yunnanense or the aconitum crassimum which is stir-fried.

The invention provides a compound shown as a formula I, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof:

in the formula I, R₁、R₂、R₃、R₄Each independently selected from C_1～4An alkyl group.

Further, the structure of the compound is shown as formula II:

further, the structure of the compound is shown as follows:

the present invention also provides a method for producing the aforementioned compound 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: the method comprises the following steps:

(1) preparing a bulleyaconitine A processing product: dissolving bulleyaconitine A in organic solvent, evaporating to remove organic solvent, and oil-bath to obtain bulleyaconitine A processed product;

(2) separation and purification: dissolving bulleyaconitine A processed product with organic solvent, separating and purifying by silica gel column chromatography, gradient eluting, collecting fraction containing the compound of claim 3, and separating R_fThe eluate, having a value of 0.41, purified by preparative liquid chromatography to give the compound of claim 3.

Further, in the step (1), the organic solvent is dichloromethane, the temperature of the oil bath is 150-170 ℃, and the time is 20-40 min.

Further, in the step (1), the organic solvent is dichloromethane, and the temperature of the oil bath is 160 ℃ for 30 min.

Further, in the step (2), when the silica gel column chromatography is used for separation and purification, an eluant for gradient elution is a petroleum ether-acetone-triethylamine mixed solution, and the volume ratios of the petroleum ether-acetone-triethylamine are 8:1:0.01, 6:1:0.01 and 3:1:0.01 in sequence;

the R is_fThe eluate with a value of 0.41 was subjected to the following thin layer chromatography conditionsAnd (3) detection: the developing solvent is petroleum ether: acetone: mixed solution of triethylamine with volume ratio of 3:3: 0.01; the eluent in the preparation of liquid chromatography purification is as follows: acetonitrile-0.03 moL/L ammonium bicarbonate solution with the volume ratio of 42:58 and the pH value of 9.5.

The invention also provides a low-toxicity antiarrhythmic, which is a preparation prepared by taking the compound, or pharmaceutically acceptable salt thereof, or stereoisomer thereof as an active ingredient and adding pharmaceutically acceptable auxiliary materials.

Further, the preparation is an oral preparation, an injection preparation, a suppository or a transdermal preparation.

The invention also provides application of the compound, or pharmaceutically acceptable salt thereof, or stereoisomer thereof in preparing low-toxicity antiarrhythmic drugs.

Further, the anti-arrhythmic agent causes low cardiotoxicity; and/or the antiarrhythmic agent has low acute toxicity.

The invention also provides a sand-fried aconitum yunnanense or crude stem aconitum kusnezoffii which is characterized in that: it contains the above-mentioned compound.

The invention also provides application of the compound in preparing a marked component of the aconitum yunnanense or the aconitum crassimum prepared by sand frying.

The Aconitum carmichaeli (Aconitum Bulleyanum Diels) and Aconitum crassifolium (A. crassifolium W. T. Wang) are dried root tubers of Aconitum carmichaeli (Aconitum Bulleyanum Diels) and Aconitum crassifolium (Aconitum Bulleyanum Diels) of Ranunculaceae.

Experimental results show that the bulleyaconitine A processing product compound 1 provided by the invention has excellent antiarrhythmic activity, can delay the Ventricular Premature Beat (VPB) incubation period of aconitine-induced arrhythmia rats, effectively prevents the further development of VPB, and has antiarrhythmic effect even superior to that of a positive medicament lidocaine under the dosage of 0.60 mg/kg.

More importantly, the compound 1 does not cause arrhythmia phenomena such as VPB, Ventricular Tachycardia (VT) and Ventricular Fibrillation (VF) within 30min after administration, and compared with bulleyaconitine A, the cardiotoxicity of the compound 1 is obviously reduced; with the known product delta of the margarine stir-frying^15,16-16-dCompared with emethoxindaconitine, the compound 1 has the advantages of obviously lower acute toxicity, higher safety and wider dosage range. The invention provides a new choice for preparing the antiarrhythmic drug with low toxicity.

The bulleyaconitine A is the main component of Aconitum yunnanense and Aconitum crassimum. The compound 1 of the invention is taken as a marker component, and the compound 1 can be used as a standard substance for controlling the quality of the sand-fried aconitum yunnanense or the sand-fried aconitum crassimum when the sand-fried aconitum yunnanense or the aconitum crassimum are fried. The invention provides a new method for controlling the quality of the sand-fried aconitum yunnanense or the sand-fried aconitum crassimum.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 is a drawing of Compound 1¹H-¹H COSY (-), HMBC (H → C) and NOESY

A correlation spectrum.

FIG. 2 is a characteristic electrocardiogram of SD rats; in the figure, A: normal electrocardiogram; b: ventricular premature beat; c: ventricular tachycardia; d: the ventricles flutter.

FIG. 3 shows Compound 1 group (16-demethoxy-. DELTA.¹⁵⁽¹⁶⁾-effect of 8-deacetylcrassiculine a), model control (Ctrl), positive control lidocaine (Lid), positive control propafenone (Pro) on SD rat VPB latency; in the figure, compared to the model control group:^*p is less than 0.05; compared with lidocaine of a positive control group:⁺p is less than 0.05; comparison with the positive control propafenone:^#P＜0.05。

figure 4 is a graph of the effect of different doses of compound 1 on the incidence of VT in SD rats.

FIG. 5 shows Compound 1 (16-demethoxy-. DELTA.¹⁵⁽¹⁶⁾-8-deacetylcrassiculine A) and lidocaine (Lid) arrhythmia complete inhibition rate results.

FIG. 6 is Compound Δ^15,16-16-demethoxyindosonine structure.

Fig. 7 is a high resolution mass spectrum of compound 1.

FIG. 8 is a drawing of Compound 1¹H-NMR spectrum.

FIG. 9 is a drawing of Compound 1¹³C-NMR spectrum.

Fig. 10 is a representative map of HMBC for compound 1.

FIG. 11 is a drawing of Compound 1¹H-¹H COSY representative map.

FIG. 12 is a NOESY representative map of Compound 1.

Detailed Description

The raw materials and equipment used in the invention are known products and are obtained by purchasing commercial products.

EXAMPLE 1 preparation of Compound 1 of the present invention

1. Preparation of column chromatography separation sample

Taking 1.83g of bulleyaconitine A, placing into a 250mL round-bottom flask, adding 25mL of dichloromethane for dissolving, evaporating the solvent by using a rotary evaporator at 40 ℃ to dryness to ensure that the bulleyaconitine A is uniformly attached to the inner wall of the round-bottom flask, carrying out oil bath at 160 ℃ for 30min, taking out, cooling to obtain a bulleyaconitine A processed product (1.65g), and feeding the processed product onto a column.

2. Separating and purifying

Dissolving bulleyaconitine A processed product with dichloromethane, separating and purifying by silica gel column chromatography (200 meshes, 300 meshes, 120g), gradient eluting with petroleum ether-acetone-triethylamine 8:1:0.01(0.4L), 6:1:0.01(3L), 3:1:0.01(1.2L), thin layer plate inspecting, and mixing similar fractions to obtain A, B, C three components. Component B (1g) was further purified by preparative thin layer chromatography using petroleum ether: acetone: developing triethylamine (3:3:0.01), air drying at room temperature, developing iodine vapor at the edge to find three spots, R_fThe values were 0.41, 0.51 and 0.77, respectively, and the silica gel on the corresponding bands was scraped off, eluted with methanol, and after recovering the solvent, three eluates were obtained. R_fPurifying the eluate (200mg) with a chromatographic condition of acetonitrile-0.03 moL/L ammonium bicarbonate solution 42:58 (pH 9.5 adjusted with concentrated ammonia water and detection wavelength of 260nm) by using a preparative liquid phase to obtain a product, recovering the collected fraction under reduced pressure until no acetonitrile smell exists, extracting the water phase with dichloromethane twice, combining dichloromethane extracts, adding anhydrous sodium sulfate to dehydrate, filtering, and concentrating the filtrate to obtain the compound 16-demethoxy-delta¹⁵⁽¹⁶⁾8-deacetylcrassiculine A (i.e. Compound 1, 110 mg).

3. Structural characterization of Compound 1

The structural characterization maps of compound 1 are shown in fig. 7-12.

The compound 1 is white powder, and the white powder is white powder,

(c＝0.92,CH₃OH),C₃₂H₄₃NO₈。HR-ESI-MS:m/z 570.3052[M+H]⁺(calcd.570.2989), from the NMR spectrum (Table 1) it can be seen that Compound 1 has a-N-CH₂-CH₃(δ_H 1.06,3H,t,J＝7.3Hz；δ_H2.51,2.59,each 1H,m；δ_C13.3q,47.0t), three methoxy groups (. delta.),_H3.24,3.30,3.32,each 3H,s；δ_C56.2q,57.5q,59.2q), a p-methoxybenzoyl group (. delta.) (S)_H7.90,2H,t,J＝8.8Hz；3.85,3H,s；6.92,2H,d,J＝8.8Hz；δ_C167.1s,121.6s,131.7d (2C),114.0d (2C),163.9s,55.5q), these characteristics indicate that Compound 1 is an aconitine type diterpenoid alkaloid. Delta_H5.17(1H, d, J ═ 3.0Hz) assigned to H-14 β, indicating that p-methoxybenzoyl is attached at the C-14 position, and in the HMBC spectrum, 1-OCH₃(δ_H 3.24,s),6-OCH₃(δ_H 3.30,s),18-OCH₃(δ_H3.32, s) are each independently related to C-1 (. delta.)_C 85.1,d),C-6(δ_C81.7, d) and C-18 (. delta.) (d)_C80.6, t) are related, indicating that the three methoxy groups are located at the C-1, C-6 and C-18 positions, respectively (FIG. 1); compound 1 has eight oxocarbon signals, lacking the acetyl signal at the C-8 position compared to bulleyaconitine a. Thus, in addition to the p-methoxybenzoyl group and the three methoxy groups, Compound 1 should also have two hydroxyl groups, and the HMBC spectrum shows C-8 (. delta.) (Δ_C73.9, s) and H-5 (. delta.))_H 2.07),H-6(δ_H 4.19),H-7(δ_H 2.11),H-9(δ_H 2.55),H-10(δ_H2.17),H-14(δ_H 5.17),H-16(δ_H 5.91),H-17(δ_H3.07) correlation; c-13 (delta)_C76.9, s) and H-9 (. delta.) (_H 2.55),H-12(δ_H 3.02,1.96),H-14(δ_H 5.17),H-15(δ_H5.61) are correlated. These two hydroxyl groups are located at the C-8 and C-13 positions, respectively (Table 1).

The chemical shifts of C-15 and C-16 of bulleyaconitine A are 39.3 and 83.7 respectively, so that¹³In C-NMR, it can be seen that Compound 1 lacks the methoxy signal at the C-16 position and that the chemical shifts of C-15 and C-16 shift to 130.1 and 131.8, respectively, low fields. Thus, in¹In H-NMR,. delta._H5.61(1H, d, J ═ 9.5Hz) and δ_H5.91(1H, d, J. 9.5Hz) were assigned H-15 and H-16, respectively, indicating the formation of a double bond between C-15 and C-16, and the long-range correlation between H-15 and C-13, and H-16 and C-8 in the HMBC spectra also confirmed the presence of a double bond structure between C-15 and C-16 (FIG. 1), based on the fact that¹H-NMR,¹³C-NMR and 2D-NMR (HMBC,¹H-¹h COSY, NOESY) data (Table 1), it can be confirmed that Compound 1 has the structure of 16-demethoxy-. DELTA.¹⁵⁽¹⁶⁾-8-deacetylcrassiculine A (N-ethyl-. DELTA.¹⁵⁽¹⁶⁾-14 α - [ (4' -methoxy) benzoyloxy group]-1 α,6 α, 18-trimethoxy-aconitane-8 β,13 β -diol).

TABLE 1 NMR data for Compound 1

The beneficial effects of the present invention are demonstrated by the following experimental examples.

Reagents, reagents and animals adopted in the experimental examples of the present invention:

bulleyaconitine A (DST190704-057, purity more than or equal to 98%, Chengdu Desite biotechnology limited); aconitine (WTZ10013, purity 99%, shanxi hao chen biotechnology ltd); propafenone hydrochloride (101190-; uratan (2017090501, Chengduo chemical reagent factory).

SPF Kunming mice, weight (18 plus or minus 2) g, male and female; SPF SD rats, male and female, weight (200 + -20) g, all provided by Woods laboratory animals GmbH, production license number: SCXK (Sichuan) -2020-.

Experimental example 1 cardiotoxicity test of bulleyaconitine A and its Sand-parched product Compound 1 test method

20 SPF SD rats, half male and half female, were randomly divided into bulleyaconitine A group and Compound 1 group (n is 10, fasted without water prohibition 12 hours before experiment). Injecting 20% urethane (dosage 1.2g/kg) into abdominal cavity for anesthesia, fixing in supine position, inserting needle electrode into four limbs subcutaneously, using BL-420F multifunctional physiological recorder to observe rat II-lead electrocardiogram for 20min, and observing electrocardiogram change within 30min after administration. Through preliminary experiments, 0.10mg/kg of bulleyaconitine A is found to cause arrhythmia phenomena such as Ventricular Premature Beat (VPB), Ventricular Tachycardia (VT) and Ventricular Fibrillation (VF) of rats, and whether the arrhythmia phenomena are caused by the compound 1 under the same dosage can be visually reflected by whether the toxicity of the bulleyaconitine A is reduced due to the structural change of the bulleyaconitine A after processing.

2. Cardiotoxicity test results

The results are shown in Table 2 and FIG. 2. The bulleyaconitine A0.10 mg/kg can cause arrhythmia such as VPB, VT and VF and the like of normal rats, the arrhythmia latency period is (182.8 +/-84.6) s, and meanwhile, the arrhythmia latency period is accompanied with the reactions of chest swelling, convulsion, uncomfortable throat sound and the like; but compound 1 did not show arrhythmic phenomena and behavioral responses at the same dose. Therefore, compared with bulleyaconitine A, the cardiotoxicity of the compound 1 is obviously reduced.

TABLE 2 comparison of cardiotoxicity of bulleyaconitine A and its stir-baked products: (

n＝10)

"-" indicates that no arrhythmia occurred within 30min of administration.

The experimental results show that compared with bulleyaconitine A, the cardiotoxicity of the sand-fried product compound 1 is obviously reduced.

Experimental example 2 study of antiarrhythmic Activity of Compound 1

1. Test method

111 SD rats were selected for male and female use and randomly divided into 8 groups, namely a blank solvent group (preparation method: 4mL of 1% ethanol hydrochloride, adding physiological saline to a constant volume of 100mL), a model control group (aconitine; dose 0.03mg/kg), a positive control group (Lidocaine group, dose 5 mg/kg; propafenone group, dose 3.2mg/kg), and compound 1 different dose groups (0.05mg/kg, 0.20mg/kg, 0.40mg/kg, and 0.60 mg/kg). The aconitine, the positive medicine and each tested medicine are prepared by adopting a blank solvent preparation method. The blank solvent group was given only an equal volume of blank solvent and was observed to see if the solvent had an effect on the electrocardiogram of SD rats.

Before the experiment is started, SD rats are subjected to intraperitoneal injection of 20% urethane (the dose is 1.2g/kg) for anesthesia, the rats are fixed in a supine position, a needle electrode is inserted under the skin of four limbs, a BL-420F multifunctional physiological recorder is used for recording normal II-lead electrocardiograms of the rats for 20min, femoral veins are exposed, test drugs, positive drugs and physiological saline with the same volume are respectively injected into the femoral veins at corresponding doses, aconitine (0.03mg/kg) is injected into the femoral veins after 10min to establish an arrhythmia model, and the incidence rate of arrhythmia of each group of rats, the time for first VPB appearance and the presence or absence of tachycardia are recorded within 30 min.

2. Data processing

The experimental data were statistically analyzed by using SPSS 20.0 software, and the data were used

And (4) showing. If the data are in accordance with normal distribution and the variances are uniform, adopting single-factor variance analysis; if the data are in accordance with normal distribution but the variance is not uniform, a test of Tamhane' T2 is adopted. When analyzing the data of the tachycardia rate and the arrhythmia complete inhibition rate, adopting Chi²And (5) checking and judging the significance of the occurrence frequency. The above data are all expressed as P<0.05 indicates that the difference is statistically significant.

3. Results of the experiment

3.1 Effect of Compound 1 on Aconitine-induced VPB latency in rats

VPB is the initial manifestation of arrhythmic rats with an electrocardiogram showing a premature appearance of QRS complexes, a wide malformation of morphology, a reversal of the T wave direction from the QRS main wave direction, and a disappearance of the P wave. The VPB latency is the time from the injection of aconitine to the appearance of VPB for the first time, the length of the VPB latency can measure the magnitude of the arrhythmia caused by the aconitine resistance of the tested medicine, and the longer the latency is, the stronger the arrhythmia resistance of the tested medicine is.

The rats of the model control group show typical VPB and VT after being injected with aconitine through femoral vein, and a part of rats can show VF and last for more than 30min, which indicates that the arrhythmia model is successfully replicated.

In the aconitine arrhythmic model control group, the VPB latency of SD rats is (116.5 +/-36.4) s; the positive drug Lidocaine group (Lid) is (280.3 + -128.7) s, the propafenone group (Pro) is (193.3 + -39.9) s; compared with a model control group, the two positive drugs have significant difference (P is less than 0.05).

Compared with the model control group, the other dosage groups (0.20mg/kg, 0.40mg/kg and 0.60mg/kg) of the compound 1 have significant difference (P is less than 0.05) except for the 0.05mg/kg group; the VPB latency periods for the 0.40mg/kg and 0.60mg/kg dose groups were (493.7 + -148.7) s and (547.3 + -241.8) s, respectively; compared with the lidocaine group, the group with 0.40mg/kg dose has significant difference (P is less than 0.05); compared with the propafenone group, the 0.40mg/kg and 0.60mg/kg dose groups have significant difference (P < 0.05), as shown in figure 3.

The above results indicate that compound 1 can delay the time of appearance of aconitine-induced arrhythmia (VPB latency) dose-dependently, and that the antiarrhythmic effect of the 0.40mg/kg and 0.60mg/kg dose groups is significantly superior to that of the positive drugs.

3.2 Effect of Compound 1 on incidence of Aconitine-induced rat VT

VT is the result of further development of VPB, and the electrocardiogram characteristics of VT are as follows: premature ventricular contractions occurred 3 or more times in succession, with a wide and abnormal QRS complex and no constant P wave. The incidence of VT may assess whether the test drug is able to prevent further development of VPB, with lower incidence of VT indicating that the test drug is able to prevent further development of VPB and the better the efficacy.

Dose groups with similar VT incidence need to be combined as per the chi-square test data, so compound 1 changed from the original 4 groups to 3 groups: 0.05-0.20mg/kg, 0.40mg/kg and 0.60 mg/kg. The chi-square test result shows: the difference in VT incidence (χ) between the 3 dose groups²14.704, P ═ 0.001), as shown in table 3; the results of pairwise comparison show: the incidence of VT was significantly reduced in the 0.60mg/kg dose group (χ) compared to the 0.05-0.20mg/kg dose group²11.313, P0.001), only 18.2%, as shown in fig. 4.

The above results indicate that compound 1 is able to dose-dependently reduce VT incidence, effectively preventing further VPB development.

Table 3 overall chi-square analysis of VT incidence between different dose groups of compound 1

3.3 Effect of Compound 1 on the complete inhibition Rate of Aconitine-induced arrhythmia in rats

The arrhythmia incidence rate is the rate of arrhythmia in 30min after rat injects tested medicine in advance via vein, and establishes arrhythmia model with aconitine (any electrocardiogram change of VPB, VT or VF is counted as arrhythmia). The arrhythmia complete inhibition rate (%) is 100% -arrhythmia incidence rate (%), that is, the rate of no arrhythmia within 30min, and it can be evaluated whether the tested drug can completely inhibit the proarrhythmic effect of aconitine. The complete arrhythmia inhibition rate is the index which can most visually reflect the strength of the drug effect of the tested drug, and the higher the complete arrhythmia inhibition rate is, the better the drug effect is, the more the tested drug can resist the arrhythmogenic effect of aconitine.

As the number of rats which do not have arrhythmia in the 0.05mg/kg and 0.20mg/kg dose groups is small, the requirement of chi-square test cannot be met according to the original grouping, therefore, chi-square analysis is carried out by combining the 0.05mg/kg and 0.20mg/kg dose groups into the 0.05-0.20mg/kg dose group.

The chi-square test result shows: the arrhythmia complete inhibition rates of different dose groups (0.05-0.20mg/kg, 0.40mg/kg and 0.60mg/kg) and lidocaine (5.0mg/kg) of the compound 1 are different²15.457, P ═ 0.001), and, in the dose range of 0.05mg/kg to 0.60mg/kg, compound 1 exhibited a tendency to gradually increase in the rate of complete inhibition of arrhythmia as the dose increased, as shown in table 4, fig. 5. The results of two-two comparison show that the arrhythmia complete inhibition rate of the 0.60mg/kg dose group and the lidocaine is obviously improved (Chi) compared with the 0.05-0.20mg/kg dose group²＝14.258，P＝0.0002；χ²6.820, P0.009). And the complete arrhythmia inhibition rate of the 0.60mg/kg dose group was 56%, and lidocaine was 42.9%, at which dose the antiarrhythmic effect of compound 1 was better.

TABLE 4 Total Cacurative inhibition of arrhythmia for Compound 1 and Lidocaine assay

The experimental results show that the compound 1 has excellent antiarrhythmic activity, can delay the VPB latency of aconitine-induced arrhythmia rats and effectively prevent the further development of VPB, and the antiarrhythmic effect of the compound 1 is superior to that of a positive medicament lidocaine under the dosage of 0.60 mg/kg.

EXAMPLE 3 Compounds 1 and. DELTA.^15,16Comparison of acute toxicity of-16-demethoxyindaconitine

1. Experimental methods

Acute toxicity is an important component of toxicological studies and safety evaluation of drugs, and refers to a toxic reaction that occurs in the body (human or animal) after one or more administrations of the drug to be tested within 24h for a short period of time (generally no more than 14 d). Acute toxicity is the first step of understanding and researching the toxic effect of the drug on the body, and meanwhile, the toxic action target organ can be preliminarily judged by observing the toxic symptoms of the body (human or animal). The information obtained from acute toxicity has important reference value for the selection of the initial dose of phase I clinical trials of certain drugs, and can provide some information about acute toxicity of excessive drugs in human beings.

There are many assays for acute toxicity testing, half-lethal dose method (LD)₅₀) Is a relatively classical method, and the LD of the tested medicament is calculated by adopting a Bliss method in the experiment₅₀The Bliss method is the most accurate determination method recognized at present and is also the LD which is specified as a new drug by the Ministry of health of China at present₅₀The method that must be used for the assessment is determined. LD₅₀Refers to the dose, LD, that causes half of the deaths of a group of test animals₅₀A smaller number indicates greater toxicity of the test agent; LD₅₀The larger the number, the lower the toxicity.

1.1 preparation of Experimental drugs

Δ^15,16The-16-demethoxyindaconitine is a diterpene alkaloid separated from the product of parching with sand of lappaconitine, and has a structure shown in FIG. 6. Taking Delta^15,16-16-demethoxyindosonitine (obtained by the method described in Chinese patent application having application No. CN 201911107946.7), Compound 1 (obtained by the method of example 1 and having purity)>95 percent), dissolving the mixture by using a small amount of 1 percent hydrochloric acid ethanol, preparing the mixture into different concentrations by using normal saline, fully and uniformly mixing the mixture, and putting the mixture into a refrigerator for cold storage and standby.

1.2 preliminary experiments

32 mice, each half of male and female, were selected, stratified by body weight and randomly grouped into 4 mice per group. A group of mice are selected for each sample according to different concentrations to carry out a pre-experiment, each mouse is administrated once according to 0.15mL/10g tail vein (12 h before the experiment is not deprived of food or water), the toxic reaction and death condition of the mice after administration are closely observed respectively, and finally the highest lethal dose (Dm, which refers to the dose just causing the whole death of experimental animals) and the lowest lethal dose (Dn, which refers to the dose just not causing the death of the experimental animals) of each drug are calculated to be used for the reference of the formal experiment.

Conjecture of Delta from the results of preliminary experiments^15,16The highest lethal dose (Dm) and the lowest lethal dose (Dn) of 16-demethoxyindaconitine and Compound 1 were about 20mg/kg, 26.78mg/kg and 8.192mg/kg, 10.04mg/kg, respectively.

1.3 official experiment

110 SPF-level Kunming mice are selected, the body mass (18 +/-2) g and the sex is half, the mice are randomly divided into 11 groups after being layered according to the body weight, and the mice are fasted for 12 hours before administration without water prohibition. According to the results of preliminary experiments, the equal ratio of 0.8 between groups is selected to design each dose group, and the dosage is delta^15,1616-demethoxyindonitine 6.55, 8.19, 10.24, 12.8, 16mg/kg group, compound 110.31, 12.89, 16.12, 20.15, 25.18mg/kg group, together with a solvent control group. Each mouse was administered 1 time in a tail vein of 0.15mL/10g, and the symptoms of intoxication, death and death time were observed within 7 days after administration, and the mice that had expired or died after intoxication were dissected and the changes in the major organs were visually observed. half-Lethal Dose (LD) of animals was calculated by Bliss method₅₀) And a 95% confidence limit.

2. Results of the experiment

TABLE 5 death, intoxication symptoms and autopsy results for different compounds

TABLE 6. DELTA.^15,16-16-demethoxyindaconitine and Compound 1 acute toxicity test results

The symptoms of poisoning of the mice are shown in table 5, the surviving mice gradually return to normal within 24h, no abnormal behavior occurs thereafter, all animals are killed 7 days after observation, and no obvious visceral abnormality is seen in animals killed by anatomical poisoning and observation. The results show that^15,16The toxic target organs of-16-demethoxyindaconitine and Compound 1 were both likely to be central nervous system.

Calculating LD by Bliss method₅₀The results are shown in Table 6. Compound delta^15,16LD of-16-demethoxyindaconitine₅₀9.017mg/kg, and the 95% confidence interval is 7.990-10.031 mg/kg; LD of Compound 1₅₀12.108mg/kg, and the 95% confidence interval is 10.646-13.358 mg/kg. And delta^15,16Compound 1 is significantly less toxic (LD) than 16-demethoxyindaconitine₅₀＝12.108mg/kg>9.017mg/kg), i.e. higher safety and wider dosage range (0.05 mg/kg-0.60 mg/kg).

The above experimental results show that^15,16Compared with the 16-demethoxyindaconitine, the compound 1 has the advantages of obviously lower acute toxicity, higher safety and wider dosage range.

In conclusion, the invention provides a low-toxicity antiarrhythmic compound shown in formula I and a pharmaceutical application thereof. Experimental results show that the compound provided by the invention has excellent antiarrhythmic activity, can delay VPB latency of aconitine-induced arrhythmia rats and effectively prevent further development of VPB, and the antiarrhythmic effect of the compound is even better than that of a positive medicament lidocaine under the dosage of 0.60 mg/kg. More importantly, compared with bulleyaconitine A, the cardiotoxicity of the compound is obviously reduced; with the known product delta of the margarine stir-frying^15,16Compared with the 16-demethoxyindaconitine, the compound of the invention has the advantages of obviously lower acute toxicity, higher safety and wider dosage range. The invention provides a new choice for preparing low-toxicity antiarrhythmic drugs, and provides a new method for preparing the antiarrhythmic drugsA new method for controlling the quality of radix Aconiti Yunnanensis parched with sand or radix Aconiti Kusnezoffii parched with sand is provided.

Claims (10)

1. A compound of formula I, or a pharmaceutically acceptable salt, or stereoisomer thereof:

in the formula I, R₁、R₂、R₃、R₄Each independently selected from C_1～4An alkyl group.

2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein: the structure of the compound is shown as formula II:

3. the compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein: the structure of the compound is shown as follows:

4. a process for preparing a compound of claim 3, or a pharmaceutically acceptable salt, or a stereoisomer thereof, wherein: the method comprises the following steps:

(1) preparing a bulleyaconitine A processing product: dissolving bulleyaconitine A in organic solvent, evaporating to remove organic solvent, and oil-bath to obtain bulleyaconitine A processed product;

(2) separation and purification: dissolving bulleyaconitine A processed product with organic solventSeparating and purifying by silica gel column chromatography, collecting fraction containing the compound of claim 3 after gradient elution, and separating R_fThe eluate, having a value of 0.41, purified by preparative liquid chromatography to give the compound of claim 3.

5. The method of claim 4, wherein: in the step (1), the organic solvent is dichloromethane, the temperature of the oil bath is 150-170 ℃, and the time is 20-40 min.

6. A low-toxicity antiarrhythmic drug, which is a preparation prepared by taking the compound of any one of claims 1 to 3, or a pharmaceutically acceptable salt or a stereoisomer thereof as an active ingredient and adding pharmaceutically acceptable auxiliary materials.

7. The low toxicity antiarrhythmic agent of claim 6, wherein: the preparation is oral preparation, injection preparation, suppository or transdermal preparation.

8. Use of a compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, for the manufacture of a low toxicity antiarrhythmic agent.

9. A stir-fried aconitum yunnanense or crude stem aconitum kusnezoffii is characterized in that: comprising a compound according to any one of claims 1 to 3.

10. Use of the compound according to any one of claims 1 to 3 for preparing a marked component of aconitum yunnanense or aconitum crassimum prepared by sand frying.

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