RU2825632C2 - Antiarrhythmic agent, method for preparation thereof and pharmaceutical composition based on antiarrhythmic agent - Google Patents

Abstract

FIELD: medicine; pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry, namely to an antiarrhythmic agent, a method for its preparation and a pharmaceutical composition based on the antiarrhythmic agent. Antiarrhythmic agent is a substance isolated from plants of the genus Aconitum (wrestler) of the family Ranunculaceae (buttercup), which contains alkaloids: lappaconitine, septefine, N-acetylanthranoyl-isolappaconidine, isoranaconitine, N-deacetylranaconitine, N-deacetylappaconitine, N-acetylsepaconitine, ranaconitine, isolappaconitine, 9-deoxylappaconitine or their pharmaceutically acceptable salts. Pharmaceutical composition for oral administration, having antiarrhythmic action, contains an antiarrhythmic agent in an effective amount and a pharmaceutically acceptable carrier. Method of producing antiarrhythmic agent extraction is carried out using an extractant selected from the group consisting of ethyl alcohol, isopropyl alcohol, acetone and a mixture of ethyl and isopropyl alcohol, adding to the extract an adsorbing additive selected from the group consisting of silica gel, aluminum oxide and activated carbon and the obtained solution is evaporated to obtain a dry residue of the end product containing a mixture of lappaconitine hydrobromide and salts of hydrobromides of accompanying alkaloids, including septefin, N-acetylanthranoyl-isolappaconidine, isoranaconitine, sepaconitine, N-deacetylranaconitine, N-deacetylappaconitine, N-acetylsepaconitine, ranaconitine, isoranaconitine, 9-deoxylappaconitine. Said antiarrhythmic agent is an effective and safe antiarrhythmic agent and has high antiarrhythmic activity in various forms of arrhythmia.

EFFECT: method for preparing this antiarrhythmic agent enables improving the quality of the finished product by reducing the time of thermal exposure, which enables to avoid adding additional impurities.

13 cl, 5 tbl, 9 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

The claimed group of inventions relates to the chemical-pharmaceutical industry, namely to means and methods for producing lappaconitine hydrobromide, in particular to methods for producing a mixture of lappaconitine hydrobromide with accompanying alkaloids, as well as to an antiarrhythmic agent, which is a mixture of lappaconitine hydrobromide with accompanying alkaloids, obtained by this method.

LEVEL OF TECHNOLOGY

The prior art contains an antiarrhythmic agent, which is a hydrobromide of the natural alkaloid lappaconitine (SU 1335293), from which the medicinal product “allapinin” is currently produced.

This drug belongs to class IC membrane stabilizing drugs. The pharmacological action of lappaconitine hydrobromide is based on its ability to block fast transmembrane potential-dependent Na+ channels built into the outer cell membrane of cardiomyocytes and thereby prevent the entry of Na+ ions into the cytosol of cardiomyocytes (Valeev A.E. et al. Neurophysiology. 1990. No. 2. pp. 201-206). It is known that drugs based on lappaconitine hydrobromide slow down the conduction of excitation and shorten the refractory period in the atria, atrioventricular node, bundle of His and Purkinje fibers (Mashkovsky M.D. Medicines. Moscow: Novaya Volna, 2002. Vol. 1. p. 371). In clinical practice, the drugs are used for supraventricular and ventricular extrasystoles (hereinafter referred to as VES); paroxysms of atrial fibrillation and flutter; paroxysmal supraventricular tachycardia (including WPW syndrome); paroxysmal ventricular tachycardia (in the absence of organic heart disease).

The closest to the claimed agent is an antiarrhythmic agent (RU 2624240, published 03.07.2017), which is a substance isolated from plants of the genus Aconitum (wrestler) of the family Ranunculaceae (buttercup), which contains lappaconitine in combination with six other alkaloids: N-acetylsepaconitine, 1-desmethyllappaconitine, ranaconitine, N-deacetyllappaconitine, isolappaconitine, 9-deoxylappaconitine, present in the original raw material, or their pharmaceutically acceptable salts, including hydrobromides.

However, this product contains only seven associated alkaloids, while the original raw material may contain a wide range of alkaloids that also have an antiarrhythmic effect.

Thus, from the roots with rhizomes or grass of plants of the genus Aconitum (wrestler) of the family Ranunculaceae (buttercup) - white-mouthed aconite (Aconitum leucostomum) or the roots with rhizomes or grass of northern aconite (Aconitum septentrionale) it is potentially possible to obtain antiarrhythmic agents with various accompanying alkaloids.

Given the existing need for such agents, their production remains a highly relevant technical task aimed at expanding the arsenal of cardioprotective agents based on lappaconitine and related alkaloids.

Known methods for obtaining antiarrhythmic drugs are based on the extraction of the roots or herbs of plants of the genus Aconitum (wrestler) of the Ranunculaceae family (buttercup) - white-mouthed aconite (Aconitum leucostomum) or the roots or herbs of northern aconite (Aconitum septentrionale) with various solvents: methanol (Marion L., Fonzer L., Wilkins C.K., Boca Jr and J.P., Sandberg F., Thorsen R., Linden E. // Can J. Chem. 1967. vol. 45, pp. 969-973); 80% ethanol (Ross A., Pelletier S.W. // Tetrahedron. 1992, vol. 48, No. 7, pp. 1183-1192); dichloroethane (Platonova T.F., Kuzovkov A.D., Massagetov P.S.//ZHOKH. - 1958, pp. 258-261); chloroform (Usmanova S.K., Telnov V.A., Yunusov M.S., Abdullaev N.D., Shreter A.I., Filippova G.B. // KhPS. - 1987, pp. 879-883).

The generally accepted sequence for obtaining lappaconitine and associated alkaloids, as well as their salts, with minor variations in extraction conditions and extractants, known from the prior art, includes the following stages:

- extraction of crushed dry plant material with an organic solvent,

- evaporation of the obtained extract until the solvent is completely removed,

- acidification of the residue to an acidic reaction,

- extraction of the resulting acidic solution with an organic solvent that is immiscible with water to remove non-basic impurities,

- alkalization of an aqueous solution to an alkaline reaction,

- separation of the released lappaconitine (by filtration or extraction),

- conversion of the lappaconitine base into hydrobromide by the action of hydrobromic acid,

- purification to pharmacopoeial purity of lappaconitine hydrobromide by crystallization from alcohol.

In some cases, column chromatography on aluminum oxide is additionally used for additional purification of the bulk of the lappaconitine base (Marion L., Fonzer L., Wilkins C.K., Boca Jr and J.P., Sandberg F., Thorsen R., Linden E. // Can J. Chem. 1967. vol. 45, pp. 969-973) or combined mother liquors (Ross A., Pelletier S.W. // Tetrahedron. 1992, vol. 48, No. 7, pp. 1183-1192).

The structure of lappaconitine contains a fragment of N-acetyl anthranilic (o-aminobenzoic) acid ester. Due to this, this molecule is very labile. It was found that both the ester and N-acetyl fragments can be hydrolyzed quite quickly in both acidic and basic media. Thus, the process of obtaining lappaconitine from plant materials according to known methods required great care in maintaining the pH of the solution and the temperature regime at the stage of acidification of the evaporated extract, at the stage of alkalization after extraction of non-basic impurities, and also when obtaining hydrobromide by acidification of the lappaconitine base with hydrobromic acid. In addition, in order to avoid hydrolysis, all these methods had to be carried out relatively quickly and not take much time to minimize the side processes of hydrolysis. Failure to comply with the above conditions can lead to a significant decrease in the yield of the target product due to the hydrolysis process at one or more stages of the method.

A method for solving the said problem is disclosed in patent RU 2123347 C1 (published 20.12.1998), in which lappaconitine hydrobromide is obtained by 3-5-fold extraction of the roots or herb of northern aconite or the roots or herb of white-mouthed aconite with a 50-80% by volume aqueous solution of acetone, followed by distillation of acetone from the combined aqueous-acetone extract, removal of non-alkaloid impurities from the aqueous residue acidified with sulfuric acid by a single treatment with dichloroethane, isolation of alkaloids from the alkalized solution by 2-3-fold extraction with dichloroethane or ethyl acetate, treatment of alkaloids with an alcoholic solution of hydrobromic acid and a single recrystallization of lappaconitine hydrobromide from aqueous alcohol.

However, the production cycle of this method is very long - about 60 hours: 21-28 hours are spent on the extraction of plant material by means of 3-5-fold extraction, a certain time is spent on evaporation of the extractant, 4 extractions with dichloroethane are carried out, about 24 hours are spent on the crystallization of the lappaconitine base and about 10 hours on the crystallization of lappaconitine hydrobromide. In addition, this method is very resource-intensive. When implementing it, there is a high probability of side reactions of hydrolysis. Finally, the method under consideration is characterized by an insufficiently high yield of the product - the average yield of the target product is about 0.595% based on the initial dry raw material.

A method is known that solves the problem of simplifying the technological process of producing lappaconitine hydrobromide, reducing its duration and increasing the yield of the target product of pharmacopoeial purity. Thus, patent RU 2545799 C2 (published on 10.04.2015) discloses a method that includes extracting the roots or herb of northern aconite (Aconitum septentrioale) or white-mouthed aconite (Aconitum leucostomum) with an organic solvent, purifying the extract, treating lappaconitine with a solution of hydrobromic acid and crystallizing the target product. In this case, the extraction of the said plant material was carried out with methylene chloride in a continuous extraction apparatus, followed by purification of the resulting extract by flash chromatography. The duration of the production cycle in this method was reduced to 30 hours, and the average yield of the target product reached 0.73% with a maximum yield of 0.95%.

However, the production cycle according to this method is still very long and resource-intensive, while providing a relatively low yield. This can be explained by the fact that this method uses flash chromatography, which involves the use of significant amounts of organic solvent to elute the sorbed alkaloids and subsequent evaporation of the resulting eluate, as well as re-dissolution of the material obtained during evaporation to convert the crude lappaconitine base into lappaconitine hydrobromide in the liquid phase using hydrobromic acid solutions.

In addition, this method produces precisely lappaconitine hydrobromide, while patent RU2545799 does not disclose its production in a mixture with other alkaloids. The above-mentioned methods are aimed at obtaining precisely lappaconitine or its pharmaceutically acceptable salts, in particular, lappaconitine hydrobromide.

The prior art also includes effective methods for producing antiarrhythmic agents that include alkaloids other than lappaconitine.

Patent RU 2039568 C1 (published 20.07.1995) discloses a method for obtaining a product with an antiarrhythmic effect, including eightfold extraction of northern aconitum rhizomes/Aconitum Septentrionale Koell/ with an organic solvent, evaporation of the combined extract, dissolution of the alkaloid precipitate in a sulfuric acid solution, washing them with an organic solvent, alkalization to pH 9-10, extraction of the alkaloids with chloroform, repeated evaporation, purification of the sum of the alkaloids, precipitation of the target product with a 5% solution of hydrobromic acid, while the extraction is carried out with a 78-80% solution of ethanol in a ratio of 1 2.2 2.6, evaporation is carried out to 10% of the initial volume, the alkaloids are precipitated by alkalization to pH 9-10 and dissolve them in a 10% solution of sulfuric acid, the sulfuric acid solution is washed with chloroform, and the purification of the sum of alkaloids is carried out by treating the evaporated concentrate with ethyl alcohol and the hydrobromide salt of the alkaloids is recrystallized from methanol.

The antiarrhythmic agent obtained by this method contains such alkaloids as lappaconitine, lyconine, N-deacetyllappaconitine, N-acetylsepaconitine and ranocanitine.

The disadvantages of this method include the high duration and resource intensity of the method, due to the fact that multiple and long-term extraction of alkaloids from plant raw materials with 80% ethyl alcohol takes 56 hours; low yield of the target product due to the formation of significant amounts of long-term non-stratifying emulsions (up to several days), which complicate the technological process and reduce the yield of the finished product, as well as due to the loss of the product with the mother liquor formed after filtration of the technical amount of alkaloids (up to 10% of the direct yield of the finished product), an increased risk of toxic damage associated with multiple phase separation and with the isolation of the technical amount of alkaloids, the purification of which is carried out in a heterogeneous medium; obtaining a product containing only five alkaloids present in the original raw materials.

The closest to the claimed method is the method disclosed in patent RU 2624240 C1 (published 03.07.2017).

The isolation of the technical amount of alkaloids according to this method is carried out by fourfold extraction of plant material from plants of the genus Aconitum (wrestler) of the family Ranunculaceae (buttercup) using ethyl alcohol (ethanol) on a battery of three extractors to obtain aqueous-alcoholic extracts and use the first extract from each load of each extractor for evaporation, and the second, third and fourth extracts of each load - as an extractant for the first, second and third extractions of the next loads, wherein the fourth extraction of each load is carried out with alcohol. During the starting period, the first, second and third loads of raw materials are carried out in the first, second and third extractors, respectively; the first extraction of the first load is carried out with alcohol and sent for evaporation, the second extraction of the first load is carried out with alcohol and sent to the second extractor to obtain the first extraction of the second load, which is sent for evaporation, the third extraction of the first load is carried out with ethyl alcohol and sent to the second extractor to obtain the second extraction of the second load, which is sent to the third extractor to obtain the first extraction of the third load, which is sent for evaporation, the fourth extraction of the first load is carried out with ethyl alcohol and sent to the second extractor to obtain the third extraction of the second load, which is sent to the third extractor to obtain the second extraction of the third load, which is sent to the working period, the fourth extraction of the second load is carried out with ethyl alcohol and sent to the third extractor to obtain the third extraction of the third load, which is sent to the working period, the fourth extraction of the third load is carried out with ethyl alcohol and sent to the working period, during which the fourth, fifth, sixth and so on loadings of raw materials are carried out in the first, second and third extractors, respectively; wherein the first extraction of each batch is sent for evaporation, and the second, third and fourth - to obtain the first, second and third extractions of each subsequent batch. After this, the first aqueous-alcoholic extraction is evaporated under vacuum and the resulting aqueous still residue is purified from ballast substances with ethyl acetate, with acidification using mineral or organic acids of the aqueous still residue saturated with ethyl acetate, and repeated extraction with chloroform while monitoring the pH of the medium, which should not exceed a value of 2, evaporation of the chloroform extracts under vacuum and displacement of chloroform with ethyl alcohol to obtain a suspension of the finished product, which is filtered, washed with ethyl alcohol and dried.

Moreover, for the first extraction of alkaloids of the first loading of the starting period, 80% ethyl alcohol is fed into the first extractor at a ratio of raw material and extractant of 1:8, and the first extraction is carried out for 3 hours, at room temperature and stirring, with subsequent regeneration of alcohol from the spent raw material, purification with ethyl acetate of the first extract from ballast substances after evaporation is carried out with ethyl acetate saturated with water four times for 30 minutes with stirring, an aqueous solution of the extract saturated with ethyl acetate is evaporated under vacuum, cooled to room temperature, acidified with mineral or organic acids until the pH of the medium does not exceed 2, holding is carried out with a working stirrer, and at the end of the holding, treatment with chloroform is carried out. The chloroform treatment is carried out four times to obtain four chloroform extracts, which are evaporated under vacuum, and to displace the chloroform, rectified ethyl alcohol is added and evaporated again until the chloroform is completely removed.

When purifying a technical product, it is dissolved, recrystallized, filtered, washed and dried to obtain a finished product, wherein said recrystallization is performed from a solution of the technical product in a mixture of two monohydric aliphatic saturated alcohols, one of which is taken from the group: methanol, ethanol, and the second from the group of monohydric aliphatic saturated alcohols with a number of carbon atoms from 3 to 5, wherein the dissolution of the technical product is initially carried out in one of the monohydric aliphatic saturated alcohols from the group: methanol, ethanol, then a monohydric aliphatic saturated alcohol from the group of monohydric aliphatic saturated alcohols with a number of carbon atoms from 3 to 5 is added to the resulting solution in an amount of 4-20 vol. % of the obtained solution, evaporate the obtained solution under vacuum pressure until the monohydric aliphatic saturated alcohol from the group of methanol, ethanol is completely removed, and the final crystallization of the target product is carried out in a monohydric aliphatic saturated alcohol from the group of monohydric aliphatic saturated alcohols with a number of carbon atoms from 3 to 5, after which the aforementioned filtration, washing and drying are carried out to obtain the target purified product.

General features of the claimed method with the prototype: extraction of a mixture of alkaloids with an organic solvent or a mixture of alcohol and water; acidification of the product to obtain a hydrobromide salt is carried out in an organic solvent; in some variants, recrystallization is carried out from aliphatic alcohols.

The disadvantages of this method include its multi-stage nature, duration and resource-intensiveness, since it provides for many stages of extraction, evaporation, drying and subsequent dissolution of the obtained intermediate products for their recrystallization, repeated evaporation and drying, which, as is well known to specialists in this field of technology, are very lengthy and resource-intensive, as well as the fact that this method allows for the isolation of only seven alkaloids present in the original raw material that have antiarrhythmic activity.

The above confirms the existing problem in obtaining antiarrhythmic agents based on lappaconitine hydrobromide, the solution of which would ensure a shorter and more resource-intensive method, while achieving a comparable or higher yield of the target product isolated from the initial raw material of plants of the genus Aconitum (wrestler) of the family Ranunculaceae (buttercup) and containing a greater number of alkaloids with antiarrhythmic activity.

ESSENCE OF THE INVENTION

The main objective of the claimed group of inventions is to expand the arsenal of cardioprotective agents with antiarrhythmic activity and methods for obtaining them.

The technical result is the implementation of this purpose.

At the same time, the claimed group of inventions allows:

- increase the speed of the technological process for obtaining the claimed antiarrhythmic agent by eliminating the resource- and energy-intensive stages of evaporation, drying and subsequent dissolution in solvents different from those used at the extraction stage;

- improve the quality of the finished product by reducing the time of thermal exposure, which allows avoiding the introduction of additional impurities;

- to obtain a cardioprotective agent with antiarrhythmic properties and including 11 alkaloid hydrobromides.

The claimed technical result is achieved by the fact that the agent possessing antiarrhythmic action is a substance isolated from the plants of northern aconite (high aconite) - Aconitum septentrionale Koelle, of the buttercup family - Ranunculaceae or white-mouthed aconite - Aconitum leucostomum, of the buttercup family - Ranunculaceae, including hydrobromides of the alkaloids lappaconitine, septefine,

alkaloid hydrobromide of structural formula

alkaloid hydrobromide of structural formula

sepaconitine, N-deacetylranaconitine, N-deacetyllappaconitine, N-acetylsepaconitine, ranaconitine, isolappaconitine, 9-deoxylapaconitine in the following mass ratio %:

lappaconitine hydrobromide - 79.36-92.06,

septefin hydrobromide - 0.02-0.14,

alkaloid structural formula

hydrobromide - 0.10-0.86,

alkaloid structural formula

hydrobromide 0.02-0.11,

sepaconitine hydrobromide 0.03-0.12,

N-deacetylranaconitine hydrobromide 0.01-0.16,

N-deacetylapaconitin hydrobromide 0.84-3.56,

N-acetyl sepaconitine hydrobromide 1.75-4.86,

ranaconitine hydrobromide 0.79-9.75,

isolappaconitine hydrobromide 0.86-3.26,

9-deoxylappaconitine hydrobromide 0.42-0.76.

The antiarrhythmic agent can be isolated from plant materials, which are the rhizomes with roots of the northern aconite (tall aconite) - Aconitum septentrionale Koelle, of the buttercup family - Ranunculaceae, or the rhizomes and roots of the white-mouthed aconite plant - Aconitum leucostomum, of the buttercup family - Ranunculaceae.

An antiarrhythmic agent can be isolated from the herb of the plant Aconitum leucostomum, of the buttercup family Ranunculaceae.

The claimed technical result is also achieved by the fact that, unlike the known method for obtaining an antiarrhythmic agent based on grinding the plant material of northern aconite (high aconite) - Aconitum septentrionale Koelle, of the buttercup family - Ranunculaceae or white-mouthed aconite - Aconitum leucostomum, of the buttercup family - Ranunculaceae, of the genus Aconitum (aconite) of the Ranunculaceae family (buttercup), extracting the plant material using an extractant selected from the group of organic solvents, filtering, washing and drying to obtain the target purified product in the form of salts formed by a mineral acid, and purifying it, in the claimed method, the extraction is carried out at a ratio of the mass of the raw material in grams and the volume of the extractant in ml of 1:1, using an extractant selected from the group consisting of ethyl alcohol, isopropyl alcohol, acetone and a mixture of ethyl and isopropyl alcohol, an adsorbent additive selected from the group consisting of silica gel, aluminum oxide and activated carbon is added to the extract, and the amount of the adsorbent additive is selected in a volume of from 0.1/100 to 5/100 to the weight of the loaded raw material, after mixing, the adsorbent additive with the impurities and resins adsorbed on it is separated by filtration, the resulting filtrate is acidified with a solution of hydrobromic acid, and the resulting solution is evaporated until a dry residue of the target product is obtained, containing a mixture of lappaconitine hydrobromide and hydrobromides of accompanying alkaloids, including septefin, sepaconitine, N-deacetylranaconitine, N-deacetyllappaconitine, N-acetylsepaconitine, ranaconitine, isolappaconitine, 9-deoxylappaconitine,

alkaloid structural formula

alkaloid structural formula

It is preferable to choose plant materials from the group consisting of the grass of white-mouthed aconite, rhizomes and roots of white-mouthed aconite - Aconitum leucostomum Worosch, rhizomes and roots of northern aconite (high aconite) - Aconitum septentrionale Koelle (A. excelsum Reichenb.) of the buttercup family - Ranunculaceae).

It is preferable to select the extractant from the group consisting of ethyl alcohol with a concentration of 80-95 vol.%, isopropyl alcohol, acetone and a mixture of ethyl and isopropyl alcohol in a ratio of 1/9 to 9/1 (vol/vol).

It is optimal to carry out extraction for 2-4 hours.

It is preferable to set the mixing time of the additive with the extract within 5 - 60 minutes.

It is optimal to mix the adsorbent additive and extract for 30 minutes at a mixing speed of 100-300 rpm.

It is preferable to separate the adsorbent additive by filtration under vacuum or gravity through a porous glass filter, or a paper filter, or a woven filter or a synthetic fiber, such as polyamide.

The hydrobromic acid solution can be selected from the ratio of 0.12 ml of 48% hydrobromic acid to 100 g of raw material.

It is optimal to acidify the resulting filtrate with a solution of hydrobromic acid in the solvent that was used as an extractant.

In some variants of the method, the resulting target product is additionally dissolved in alcohol and recrystallized.

The claimed method allows to significantly reduce the production time of an antiarrhythmic agent (up to 5-10 hours) and the resource intensity of this process by extracting the target components from plant materials in a simple and effective manner in a Soxhlet extractor (Jensen W. B. The Origin of the Soxhlet Extractor (English) // J. Chem. Educ. - 2007. - Vol. 84, no. 12. - P. 1913-1914), which does not require the use of significant quantities of extractant, purifying the resulting extract by adding an adsorbing additive with its subsequent separation with the resins and impurities adsorbed on it by simple filtration, which does not require the very resource- and energy-intensive stages of evaporation, drying and subsequent dissolution in solvents other than those used at the extraction stage, i.e. stages, usually included in similar methods known from the state of the art, and used, among other things, in the method selected as the closest analogue. In some embodiments, the resource intensity is also increased by converting the obtained alkaloids into the corresponding hydrobromides by simply acidifying the filtrate obtained after separating the adsorbing additive with a solution of hydrobromic acid in the same solvent that was used in the extraction stage, which also additionally eliminates the very long, as well as resource- and energy-intensive stages of obtaining the product in a solid state and its subsequent dissolution in other solvents. In addition, the elimination of a change of solvent in the claimed method makes it possible to avoid excessive thermal effects on the obtained intermediate and final products and thereby reduce the likelihood of hydrolysis of the target components, as well as to avoid introducing additional impurities into the final product. Without wishing to be limited by any particular theory, the authors of the present invention believe that this also allows the method according to the invention to provide a high yield of the product from various types of raw materials (from 0.7% to 1.0%), a yield that is comparable to those provided by the known prior art methods for obtaining similar agents, and often exceeds them. The method according to the invention also allows for the production of an agent that contains a greater number of alkaloids present in the original raw material, possessing similar antiarrhythmic activity. Due to this, the effectiveness of the antiarrhythmic agent according to the invention is higher than that of the known prior art analogs.

IMPLEMENTATION OF THE INVENTION

The claimed method includes the following stages of obtaining an antiarrhythmic agent:

- crush the plant material selected from the group consisting of the grass of white-mouthed aconite, the rhizomes and roots of white-mouthed aconite - Aconitum leucostomum Worosch, the rhizomes and roots of northern aconite (high aconite) - Aconitum septentrionale Koelle (A. excelsum Reichenb.) of the buttercup family - Ranunculaceae)

- the crushed plant material is reacted with the extractant in a soxhlet extractor at a ratio of the mass of the raw material in grams: the volume of the extractant in ml of 1:1, with the extractant selected from the group consisting of ethyl alcohol with a concentration of 80-95 vol. %, isopropyl alcohol, acetone and a mixture of ethyl and isopropyl alcohol in a ratio of 1/9 to 9/1 (vol/vol). The optimal extraction time is 2-4 hours, longer extraction does not lead to an increase in the yield;

- an adsorbent additive selected from the group consisting of silica gel, aluminum oxide and activated carbon is added to the obtained extract and the obtained mixture is stirred for 30 minutes at a stirring speed of 100-300 rev/min. The amount of the adsorbent additive is taken in a ratio of 1/100 to the mass of the loaded raw material, it is possible to decrease and increase the amount of the adsorbent additive in the range from 0.1/100 to 5/100 to the mass of the loaded raw material. The stirring time can be varied from 5 min to 60 min;

- then the adsorbent additive is separated from the impurities and resins adsorbed on it by filtration under vacuum or gravity through a porous glass filter, or a paper filter, or a woven filter or synthetic fiber (for example, polyamide);

- acidify the obtained filtrate with a solution of hydrobromic acid (in an optimal ratio of 0.12 ml of 48% hydrobromic acid to 100 g of the raw material taken) in the same solvent that was used as an extractant, and evaporate the resulting solution until a dry residue containing pure lappaconitine with associated alkaloids is obtained; An increase in the amount of hydrobromic acid leads to contamination of the product, a decrease leads to incomplete formation of alkaloid hydrobromides;

- in some embodiments, the obtained product is dissolved in alcohol and recrystallized. Recrystallization is used to increase the proportion of the main component - lappaconitine in the alkaloid mixture in the form of hydrobromides.

The alkaloids included in the declared antiarrhythmic agent have the following structural formulas, chemical names and characteristics:

Lappaconitine

IUPAC: (1S,4S,5S,7S,8S,9S,10S,11S,13R,14S,16S,17R)-20-ethyl-8,9-dihydroxy-1,14,16-trimethoxy aconitan-4-yl 2-(acetylamino)benzoate.

Chemical formula: C ₃₂ H ₄₄ N ₂ O ₈ .

Molecular weight: 584.71.

Septefin

IUPAC: (1S,4S,5S,7S,8S,9S,10S,11S,13R,14S,16S,17R)-20-ethyl-8,9-dihydroxy-1,14,16-trimethoxy aconitan-4-yl 2-(methylamino)benzoate.

Chemical formula: C ₃₁ H ₄₄ N ₂ O ₇ .

Molecular weight: 556.70.

IUPAC: (1S,4S,5S,7S,8S,9S,10S,11S,13R,14S,16S,17R)-20-ethyl-1,8,10-trihydroxy-14,16-dimethoxyaconitan-4-yl 2 -(acetylamino)benzoate.

Chemical formula: C ₃₁ H ₄₂ N ₂ O ₈ .

Molecular weight: 570.68.

IUPAC: (1S,4S,5S,7S,8S,9S,10S,11S,13R,14S,16S,17R)-20-ethyl-7,8,10-trihydroxy-1,14,16-trimethoxyaconitan-4-yl 2-(acetylamino)benzoate.

Chemical formula: C ₃₂ H ₄₄ N ₂ O ₉ .

Molecular weight: 600.71.

Sepaconitine

IUPAC: (1S,4S,5S,7S,8S,9S,10S,11S,13R,14S,16S,17R)-20-ethyl-8,9,10-trihydroxy-1,14,16-trimethoxyaconitan-4-yl 2-aminobenzoate.

Chemical formula: C ₃₀ H ₄₂ N ₂ O ₈ .

Molecular weight: 558.67.

N-deacetylranaconitine

IUPAC: (1S,4S,5S,7S,8S,9S,10S,11S,13R,14S,16S,17R)-20-ethyl-7,8,9-trihydroxy-1,14,16-trimethoxyaconitan-4-yl 2-aminobenzoate.

Chemical formula: C ₃₀ H ₄₂ N ₂ O ₈ .

Molecular weight: 558.67.

N-deacetylapaconitine

IUPAC: (1S,4S,5S,7S,8S,9S,10S,11S,13R,14S,16S,17R)-20-ethyl-8,9-dihydroxy-1,14,16-trimethoxyaconitan-4-yl 2-aminobenzoate.

Chemical formula: C ₃₀ H ₄₂ N ₂ O ₇ .

Molecular weight: 542.67.

N-acetylsepakonitine

IUPAC: (1S,4S,5S,7S,8S,9S,10S,11S,13R,14S,16S,17R)-20-ethyl-8,9,10-trihydroxy-1,14,16-trimethoxy aconitan-4-yl 2-(acetylamino)benzoate.

Chemical formula: C ₃₂ H ₄₄ N ₂ O ₉ .

Molecular weight: 600.71.

Ranaconitine

IUPAC: (1S,4S,5S,7S,8S,9S,10S,11S,13R,14S,16S,17R)-20-ethyl-7,8,9-trihydroxy-1,14,16-trimethoxyaconitan-4-yl 2-(acetylamino)benzoate.

Chemical formula: C ₃₂ H ₄₄ N ₂ O ₉ .

Molecular weight: 600.71.

Isolappaconitine

IUPAC: (1S,4S,5S,7S,8S,9S,10S,11S,13R,14S,16S,17R)-20-ethyl-7,8-dihydroxy-1,14,16-trimethoxyaconitan-4-yl 2 -(acetylamino)benzoate.

Chemical formula: C ₃₂ H ₄₄ N ₂ O ₈ .

Molecular weight: 584.71.

9-deoxylappaconitine

IUPAC: (1S,4S,5S,7S,8S,9S,10S,11S,13R,14S,16S,17R)-20-ethyl-8-hydroxy-1,14,16-trimethoxyaconitan-4-yl 2-(acetoamino)benzoate.

Chemical formula: C ₃₂ H ₄₄ N ₂ O ₇ .

Molecular weight: 568.71.

The implementation of the claimed inventions is further demonstrated by means of the following illustrative non-limiting examples. These examples are presented solely to show the possibility of implementing the claimed inventions with the achievement of the above-mentioned technical results, and they should not be used to limit the scope of claims under this application in any way, since it is obvious to a specialist in this field that the claimed inventions can be implemented with the achievement of the claimed technical results in other particular cases not directly shown in the examples.

Example 1

A 500 ml continuous extraction apparatus of the Soxhlet type was loaded with 250 g of dry plant material (Agrimony spp.), ground to a fine powder (passing through a sieve with a pore diameter of 0.5 mm). Extraction was carried out using 250 ml of ethyl alcohol with a concentration of 80 vol. % for 2 hours. 2.5 g of silica gel (70-230 mesh) were added to the resulting extract and mixed for 30 min. The precipitate was filtered under vacuum through a porous glass filter with a filter porosity characteristic of POR40 for 15 min, and ~0.3 ml of 48 % (mass) hydrobromic acid in 2 ml of ethyl alcohol with a concentration of 80 vol. % was added to the resulting solution and mixed for 15 min. The solution was evaporated to dryness under vacuum and the resulting lappaconitine hydrobromide with accompanying alkaloids of about 90% purity (mass) based on lappaconitine hydrobromide was recrystallized from ethyl alcohol with a concentration of 95 vol.% at the rate of 2 ml of alcohol per 850 mg of lappaconitine hydrobromide.

We obtained about 1.9 g of lappaconitine hydrobromide with hydrobromides of accompanying alkaloids with a purity of more than 97% in terms of lappaconitine hydrobromide.

The total yield of lappaconitine hydrobromide extraction from plant material by this method is about 0.76%. The yield of hydrobromides of accompanying alkaloids for this example is given in Table 3.

Example 2

A 500 ml Soxhlet continuous extraction apparatus was loaded with 250 g of dry plant material (rhizomes and roots of northern agnus) ground to a fine powder (passing through a sieve with a pore diameter of 0.5 mm). Extraction was carried out using 250 ml of isopropyl alcohol for 2.5 hours. 2.5 g of activated carbon (BAU-MF) were added to the resulting extract and mixed for 30 min. The precipitate was filtered under vacuum through a blue ribbon paper filter (dense, fine-grained filter paper), and ~0.3 ml of 48% hydrobromic acid in 2 ml of isopropyl alcohol was added to the resulting solution and mixed for 15 min. The solution was evaporated to dryness under vacuum and about 2.5 g of lappaconitine hydrobromide with hydrobromides of accompanying alkaloids of greater than 97% purity in terms of lappaconitine hydrobromide were obtained.

The total yield of lappaconitine hydrobromide extraction from plant material by this method was about 1.0%. The yield of hydrobromides of accompanying alkaloids according to this example is given in Table 3.

Example 3

A 500 ml Soxhlet continuous extraction apparatus was loaded with 250 g of dry plant material (rhizomes and roots of white-mouthed agrimony), ground to a fine powder (passing through a sieve with a pore diameter of 0.5 mm). Extraction was carried out using 250 ml of a mixture of isopropyl and ethyl alcohol (30/70 (v/v)) for 2.5 hours. 2.5 g of silica gel (60-120 mesh) were added to the resulting extract and mixed for 30 min. The precipitate was filtered by gravity through a blue ribbon paper filter, and ~0.3 ml of 48% hydrobromic acid in 2 ml of a mixture of isopropyl and ethyl alcohol (30/70 (v/v)) was added to the resulting solution and mixed for 15 min. The solution was evaporated to dryness under vacuum and about 2.2 g of lappaconitine hydrobromide with hydrobromides of accompanying alkaloids of greater than 97% purity in terms of lappaconitine hydrobromide were obtained.

The total yield of lappaconitine hydrobromide extraction from plant material by this method was about 0.88%. The yield of hydrobromides of accompanying alkaloids for this example is given in Table 3.

Example 4

A 500 ml continuous extraction apparatus of the Soxhlet type was loaded with 250 g of dry plant material (rhizomes and roots of white-mouthed agrimony), ground to a fine powder (passing through a sieve with a pore diameter of 0.5 mm). Extraction was carried out using 250 ml of a mixture of isopropyl and ethyl alcohol (60/40 (vol/vol)) for 2.5 hours. 2.5 g of activated carbon (BAU-A) were added to the resulting extract and mixed for 30 min. The precipitate was filtered under vacuum through a fabric filter, and ~0.3 ml of 48% hydrobromic acid in 2 ml of a mixture of isopropyl and ethyl alcohol (60/40 (vol/vol)) was added to the resulting solution and mixed for 15 min. The solution was evaporated to dryness under vacuum and the resulting lappaconitine hydrobromide with accompanying alkaloids of about 90% purity in terms of lappaconitine hydrobromide was recrystallized from isopropyl alcohol at a rate of 3 ml of alcohol per 1 g of lappaconitine hydrobromide.

We obtained about 2.2 g of lappaconitine hydrobromide with hydrobromides of accompanying alkaloids with a purity of more than 97% in terms of lappaconitine hydrobromide.

The total yield of lappaconitine hydrobromide extraction from plant material by this method is about 0.88%. The yield of hydrobromides of accompanying alkaloids for this example is given in Table 3.

Example 5

A 500 ml continuous extraction apparatus of the Soxhlet type was loaded with 250 g of dry plant material (rhizomes and roots of white-mouthed agrimony), ground to a fine powder (passing through a sieve with a pore diameter of 0.5 mm). Extraction was carried out using 250 ml of acetone for 4 hours. 2.5 g of activated carbon BAU-MF (birch activated carbon grade MF) were added to the resulting extract and mixed for 30 min. The precipitate was filtered under vacuum through a nylon filter, and ~0.3 ml of 48% hydrobromic acid in 2 ml of acetone was added to the resulting solution and mixed for 15 min. The solution was evaporated to dryness under vacuum and the resulting lappaconitine hydrobromide with hydrobromides of accompanying alkaloids of about 90% purity in terms of lappaconitine hydrobromide was recrystallized from isopropyl alcohol at a rate of 3 ml of alcohol per 1 g of lappaconitine hydrobromide.

We obtained about 2.1 g of lappaconitine hydrobromide with hydrobromides of accompanying alkaloids with a purity of more than 97% in terms of lappaconitine hydrobromide.

The total yield of lappaconitine hydrobromide extraction from plant material by this method was about 0.84%. The yield of hydrobromides of accompanying alkaloids for this example is given in Table 3.

Example 6

A 500 ml continuous extraction apparatus of the Soxhlet type was loaded with 250 g of dry plant material (rhizomes with roots of northern agnus) ground to a fine powder (passing through a sieve with a pore diameter of 0.5 mm). Extraction was carried out using 250 ml of ethyl alcohol with a concentration of 95 vol. % for 2 hours. 2.5 g of aluminum oxide (grade AOA-1) were added to the resulting extract and mixed for 30 min. The precipitate was filtered under vacuum through a POR 40 porous glass filter, and ~0.3 ml of 48% hydrobromic acid in 2 ml of ethyl alcohol with a concentration of 95 vol. % was added to the resulting solution and mixed for 15 min. The solution was evaporated to dryness under vacuum and the resulting lappaconitine hydrobromide with accompanying alkaloids of about 90% purity based on lappaconitine hydrobromide was recrystallized from ethyl alcohol with a concentration of 95 vol.% at the rate of 2 ml of alcohol per 850 mg of lappaconitine hydrobromide.

About 2.0 g of lappaconitine hydrobromide with hydrobromides of accompanying alkaloids with a purity of more than 97% in terms of lappaconitine hydrobromide were obtained.

The total yield of lappaconitine hydrobromide extraction from plant material by this method was about 0.8%. The yield of hydrobromides of accompanying alkaloids for this example is given in Table 3.

Example 7

A 500 ml Soxhlet continuous extraction apparatus was loaded with 250 g of dry plant material (Agrimony spp.), ground to a fine powder (passing through a sieve with a pore diameter of 0.5 mm). Extraction was carried out using 250 ml of a mixture of isopropyl and ethyl alcohol (80/20 (vol/vol)) for 2.5 hours. 2.5 g of aluminum oxide (grade AOA-2) were added to the resulting extract and the mixture was stirred for 30 min. The precipitate was filtered under vacuum through a POR 40 porous glass filter, and ~0.3 ml of 48% hydrobromic acid in 2 ml of a mixture of isopropyl and ethyl alcohol (80/20 (vol/vol)) was added to the resulting solution and the mixture was stirred for 15 min. The solution was evaporated to dryness under vacuum and the resulting lappaconitine hydrobromide with accompanying alkaloids of about 90% purity in terms of lappaconitine hydrobromide was recrystallized from isopropyl alcohol at a rate of 3 ml of alcohol per 1 g of lappaconitine hydrobromide.

About 1.8 g of lappaconitine hydrobromide with hydrobromides of accompanying alkaloids of greater than 97% purity in terms of lappaconitine hydrobromide were obtained.

The total yield of lappaconitine hydrobromide extraction from plant material by this method was about 0.72%. The yield of hydrobromides of accompanying alkaloids for this example is given in Table 3.

Example 8

A 500 ml continuous extraction apparatus of the Soxhlet type was loaded with 250 g of dry plant material (Acrocarpus balm) ground to a fine powder (passing through a sieve with a pore diameter of 0.5 mm). Extraction was carried out using 250 ml of ethyl alcohol with a concentration of 80 vol. % for 2 hours. 2.5 g of aluminum oxide were added to the resulting extract and mixed for 30 min. The precipitate was filtered under vacuum through a blue ribbon paper filter, and ~0.3 ml of 48% hydrobromic acid in 2 ml of ethyl alcohol with a concentration of 80 vol. % was added to the resulting solution and mixed for 15 min. The solution was evaporated to dryness under vacuum and the resulting lappaconitine hydrobromide with accompanying alkaloids of about 90% purity based on lappaconitine hydrobromide was recrystallized from ethyl alcohol with a concentration of 95 vol.% at the rate of 2 ml of alcohol per 850 mg of lappaconitine hydrobromide.

We obtained about 1.7 g of lappaconitine hydrobromide with hydrobromides of accompanying alkaloids with a purity of more than 97% in terms of lappaconitine hydrobromide.

The total yield of lappaconitine hydrobromide extraction from plant material by this method was about 0.68%. The yield of hydrobromides of accompanying alkaloids for this example is given in Table 3.

Example 9

A 500 ml continuous extraction apparatus of the Soxhlet type was loaded with 250 g of dry plant material (rhizomes with roots of northern agnathus) ground to a fine powder (passing through a sieve with a pore diameter of 0.5 mm). Extraction was carried out using 250 ml of acetone for 4 hours. 2.5 g of silica gel (100-200 mesh) were added to the resulting extract and mixed for 30 min. The precipitate was filtered under vacuum through a POR 40 porous glass filter, and ~0.3 ml of 48% hydrobromic acid in 2 ml of acetone was added to the resulting solution and mixed for 15 min. The solution was evaporated to dryness under vacuum and the resulting lappaconitine hydrobromide with accompanying alkaloids of about 90% purity, calculated as lappaconitine hydrobromide, was recrystallized from isopropyl alcohol at a rate of 3 ml of alcohol per 1 g of lappaconitine hydrobromide.

About 2.3 g of lappaconitine hydrobromide with hydrobromides of accompanying alkaloids of greater than 97% purity in terms of lappaconitine hydrobromide were obtained.

The total yield of lappaconitine hydrobromide extraction from plant materials using this method was approximately 0.92%.

The yield of hydrobromides of accompanying alkaloids for this example is given in Table 3.

Below is a summary table 1 of the components used in the isolation of Lappaconitine and associated alkaloids (in the form of hydrobromides) from plant materials.

Table 1

No. Extraction agent Sorbent Lappaconitine yield,
% of the dry weight of the original raw material Raw materials Production time 1 80% ethyl alcohol silica gel 0.76% grass of white-mouthed wrestler 6.5 h 2 isopropyl alcohol activated carbon 1.0% rhizomes with roots of northern wrestler 6 h 3 mixture of isopropyl and ethyl alcohol (30/70 (vol/vol) silica gel 0.88% rhizomes and roots of white-mouthed aconite 9.5 hours 4 mixture of isopropyl and ethyl alcohol (60/40 (vol/vol) activated carbon 0.88% rhizomes and roots of white-mouthed aconite 6 h 5 acetone activated carbon 0.84% rhizomes and roots of white-mouthed aconite 5 h 6 95% ethyl alcohol aluminum oxide 0.8% rhizomes with roots of northern wrestler 7 h 7 mixture of isopropyl and ethyl alcohol (80/20 (vol/vol) aluminum oxide 0.72% grass of white-mouthed wrestler 5.5 h 8 80% ethyl alcohol aluminum oxide 0.68% grass of white-mouthed wrestler 7 h 9 acetone silica gel 0.92% rhizomes with roots of northern wrestler 5.5 h

The obtained products were analyzed using the following analytical procedure.

Analysis method:

The detection of specific alkaloids present in the product and determination of their quantity were carried out by high-performance liquid chromatography (hereinafter referred to as HPLC), carried out under the following conditions.

Preparation of solutions.

Solvent (mobile phase A): Methanol: water: chloroform: triethylamine (70: 30: 2: 0.1).

Test solution. About 100 mg of the analyzed substance was placed in a 10 ml measuring flask, dissolved in 9 ml of solvent in an ultrasonic bath (USB) for 15-20 min, cooled to room temperature, the volume of the solution was brought up to the mark with solvent and mixed. The resulting solution was filtered through a nylon membrane filter with a pore size of 0.45 μm.

Chromatography

Chromatography conditions and equipment:

- liquid chromatograph equipped with a spectrophotometric detector;

- Phenomenex Luna C18(2) column, 250 mm × 4.6 mm × 5 µm with a pre-column of the same phase Phenomenex C18 3 × 4 mm or similar;

- flow rate 1.0 ml/min;

- column temperature 20-25°C;

- 250 nm detector;

- the volume of the injected sample is 25 µl (it is permissible to increase the volume of the injected sample to 100 µl if necessary, for better identification of minor peaks of alkaloids);

- mobile phase A: methanol: water: chloroform: triethylamine (70: 30: 2: 0.1);

- mobile phase B: methanol;

- elution mode: gradient.

Table 2 shows the ratio of mobile phases A (PFA) and B (PFV) during gradient elution.

Table 2

Time, min PFA, % PFV, % 0 100 0 40 65 35 45 100 0 50 100 0

After completion of the analysis, the column was washed with isopropyl alcohol for 60 minutes at a flow rate of 0.5 ml/min to clean and prepare the column for subsequent analyses.

When chromatographed under the specified conditions, the retention time of the lappaconitine peak, which is the most intense peak on the chromatogram, was about 20-25 min.

The relative retention times of the associated alkaloid peaks were:

Septefin - 0.33-0.43;

alkaloid with structural formula - 0.35-0.45;

alkaloid with structural formula - 0.45-0.55;

sepaconitine - 0.55-0.65;

N-deacetylranaconitine - 0.62-0.72;

N-deacetylapaconitine - 0.70-0.80;

N-acetylsepakonitine - 0.78-0.88;

ranaconitine - 0.88-0.98;

lappaconitine - 1.0;

isolappaconitine - 1.04-1.14;

9-deoxylappaconitine - 1.50-1.70.

Peaks with relative retention times shorter than the relative retention time of the septefin peak are systemic or belong to an impurity (a compound with a structure different from aconitane) and are not taken into account.

The results of the analysis are considered reliable if the requirements of the "Chromatographic System Suitability Test" are met.

Checking the suitability of the chromatographic system

A chromatographic system is considered suitable (State Pharmacopoeia of the Russian Federation (XIV edition), OFS.1.2.1.2.0005.15 "High-performance liquid chromatography") if the following conditions are met on the chromatogram of the test solution:

- resolution between the peaks of N-deacetyllappaconitine and N-acetylsepaconitine and the peaks of ranaconitine and N-acetylsepaconitine - not less than 1.5;

- resolution between the peaks of ranaconitine and lappaconitine and lappaconitine and isolappaconitine - not less than 1.0;

- the efficiency of the chromatographic column, calculated based on the peaks of ranaconitine, N-acetyl sepaconitin and N-deacetyllasepaconitin, is not less than 2000 theoretical plates;

- the asymmetry coefficient of the peaks of ranaconitine, N-acetylsepaconitine and N-deacetylsepaconitine is no more than 2.0.

The results obtained on the content of lappaconitine hydrobromides and associated alkaloids according to the described methods of production (examples 1-9) are given in Table 3, where their relative content by weight is indicated.

Table 3

Alkaloid hydrobromides Example #1 Example #2 Example #3 Example #4 Example #5 Example #6 Example #7 Example #8 Example #9 Septefina hydrobromide 0.06 0.01 0.03 0.10 0.07 0.14 0.02 0.02 0.27 Sepaconitine hydrobromide 0.08 0.05 0.05 0.12 0.02 0.08 0.05 0.04 0.03 N-deacetylranaconitine hydrobromide 0.04 0.01 0.01 0.09 0.06 0.16 0.01 0.01 0.02 N-deacetylapaconitin hydrobromide 2.61 2.68 2.63 2.63 1.78 3.56 2.63 0.84 1.35 N-acetyl sepaconitine hydrobromide 3.58 2.06 2.03 4.86 4.39 3.50 2.01 1.75 2.44 Ranaconitine hydrobromide 5.27 0.82 0.81 9.75 7.43 0.71 0.79 4.31 0.79 Lappaconitine hydrobromide 85.44 91.59 91.73 79.36 83.52 87.08 91.85 91.05 92.06 Isolappaconitine hydrobromide 2.03 2.11 2.04 1.76 1.86 3.26 1.99 0.86 1.97 9-deoxylappaconitine hydrobromide 0.47 0.56 0.56 0.44 0.47 0.42 0.54 0.52 0.76

In order to identify the alkaloids contained in the substance, 11 fractions were obtained from the substance using semi-preparative chromatography, each containing one individual compound. Each of the 11 fractions was collected in a volume of 50 ml and lyophilized. The dry lyophilisate was then analyzed to identify the alkaloids.

Two identification methods were selected: tandem mass spectrometry and nuclear magnetic resonance (NMR). Mass spectrometric analysis was performed under electrospray ionization conditions in the positive ion registration mode.

Mass spectrometric determination of alkaloids allows us to determine their mass, and also, using fragmentation of parent ions, their gross formula. Similar fragmentation of parent ions with the same fragmentation energy indicates the uniform nature of the substances under study. In combination with chromatographic separation of alkaloids, the tandem mass spectrometry method allows us to speak about evidence of detection of 11 alkaloids of the aconite series. (Table 4.)

Table 4. Mass spectrometric analysis data

Identification of the isolated alkaloids was also carried out using the nuclear magnetic resonance method. When recording the NMR spectra, a standard single-pulse sequence with subsequent Fourier transform was used (in the case of 13C spectra, broadband proton decoupling was additionally used). Chemical shifts of the NMR signals were determined relative to tetramethylsilane. The signals were assigned based on theoretical calculations and literature data, taking into account the signal intensity and spin-spin interactions. The NMR results for each identified alkaloid are presented in Table 5.

Table 5

SEPTEFIN SEPACONITINE N-DEACETYLRANACONITINE Location (C) δ,ppm (C) Location
(N) δ, ppm
(N) Location (C) δ,ppm (C) Location (H) δ, ppm (N) Location
carbon (C) content δ,ppm (C) Location
zhenie (N) δ, ppm (N) 1 84.2 H1 3.48 (dd) 2 26.6 H1 3.76 (dd) 1 83.6 H1 3.80 (m) 2 26.2 H2α 1.48 (d) 3 31.7 H2α 2.13 (d) 2 26.1 H2α 2.14 (s) 3 51.9 H2β 2.09 (d) 4 83.7 H2β 2.16 (d) 3 32.0 H2β 2.18(m) 4 84.7 H3α 1.87(m) 5 44.1 H3α 2.26m) 4 84.3 H3α 2.27(m) 5 48.6 H3β 1.57 (d) 6 24.5 H3β 2.54 (d) 5 50.9 H3β 2.50(m) 6 26.8 H4 - 7 47.3 H4 - 6 32.9 H4 - 7 47.6 H5 2.35 (m) 8 75.1 H5 2.34(m) 7 77.7 H5 2.35(m) 8 75.6 H6α 2.07(dd) 9 80.3 H6α 1.65(dd) 8 85.8 H6α 1.67 (dd) 9 78.6 H6β 1.76 (m) 10 79.1 H6β 2.57(m) 9 78.7 H6β 2.60 (d) 10 36.4 H7 2.55 (d) 11 56.0 H7 2.38(m) 10 36.5 H7 - 11 51.0 H8 - 12 37.2 H8 - 11 51.0 H8 - 12 24.2 H9 - 13 34.0 H9 - 12 25.1 H9 - 13 49.0 H10 2.13(m) 14 88.0 H10 - 13 49.7 H10 2.25 14 90.0 H11 - 15 44.9 H11 - 14 90.4 H11 - 15 44.9 H12α 2.28 (m) 16 83.2 H12α 2.13 (m) 15 37.3 H12α 1.96(m) 16 82.9 H12β 2.7(d) 17 61.4 H12β 2.11(d) 16 83.0 H12β 2.10(d) 17 61.5 H13 2.44 (d) 19 55.2 H13 2.55 (d) 17 62.7 H13 2.44(m) 19 55.5 H14 3.42 (s) NCH2CH3 49.5 H14 3.66 (s) 19 55.0 H14 3.60(dd) NCH2CH3 49.9 H15α 2.21 (d) NCH2CH3 13.2 H15α 2.21 (d) NCH2CH3 48.4 H15α 2.25(m) NCH2CH3 13.5 H15β 2.37(m) 1-OMe 56.1 H15β 2.36 (m) NCH2CH3 14.1 H15β 2.34(m) 1-OMe 56.5 H16 3.32 (s) 14-OMe 57.9 H16 3.28 (s) 1-OMe 56.6 H16 3.30(m) 14-OMe 57.9 H17 2.88 (s) 16-OMe 56.3 H17 3.32 (s) 14-OMe 58.3 H17 3.15(s) 16-OMe 56.1 H19α 2.89 (d) C=O 167.3 H19α 2.64 (d) 16-OMe 56.0 H19α 2.68(dd) C=O 167.5 H19β 3.14 (s) C1 aroma 112.8 H19β 3.10 (s) C=O 167.1 H19β 3.06 (d) C1 aroma 115.8 1-OMe 3.40 (s) C2 aroma 151.1 1-OMe 3.27 (s) C1 aroma 115.2 1-OMe 3.24 (s) C2 aroma 141.7 14-OMe 3.44 (s) C3 aroma 115.3 14-OMe 3.36 (s) C2 aroma 142.2 14-OMe 3.33 (s) C3 aroma 120.3 16-OMe 3.30 (s) C4 aroma 134.1 16-OMe 3.25 (s) C3 aroma 119.5 16-OMe 3.21(s) C4 aroma 134.4 NHCH3 2.81(d) C5 aroma 117.3 COCH3 - C4 aroma 134.3 COCH3 - C5 aroma 122.3 NCH2 2.38(m) C6 aroma 131.7 NCH2 2.44(m) C5 aroma 121.4 NCH2 2.40(m) C6 aroma 131.1 NCH2CH3 1,12(s) NCH2CH3 1.5 (s) C6 aroma 131.3 NCH2CH3 1.11 (s) NHCH3 47.3 NHCH3 7.48(q) NH2 5.75(brs) NH2 5.60(brs) H-3'arom 6.63(d) H-3'arom 7.70 (d) H-3'arom 7.68(d) H-4'arom 7.45(t) H-4'arom 7.55(dd) H-4'arom 7.47(dd) H-5'arom 6.57(t) H-5'arom 6.56(dd) H-5'arom 6.48(dd) H-6'arom 7.68(d) H-6'arom 7.60(d) -DEACETYL APPACONITINE N-ACETYL SEPACONITINE RANACONITINE Location
carbon (C) content δ,ppm (C) Location
zhenie (N) δ, ppm (N) Location
carbon dioxide
(WITH) δ,ppm
(WITH) Location
zhenie (N) δ, ppm
(N) Location
carbon (C) content δ,ppm
(WITH) Location
zhenie (N) δ, ppm
(N) 1 83.2 H1 3.56(dd) 1 77.8 H1 3.74 (dd) 1 83.5 H1 3.77 (m) 2 26.3 H2α 1.55 (d) 2 26.5 H2α 2.11 (d) 2 26.5 H2α 2.04 (s) 3 32.3 H2β 2.11 (d) 3 31.6 H2β 2.17 (d) 3 31.6 H2β 2.1(m) 4 84.5 H3α (2.27m) 4 81.7 H3α (2.27m) 4 84.4 H3α 2.27(m) 5 48.9 H3β 2.04 (d) 5 44.3 H3β 2.48 (d) 5 51.1 H3β 2.53(m) 6 26.9 H4 - 6 24.5 H4 - 6 32.5 H4 - 7 47.7 H5 2.24(m) 7 46.9 H5 2.31(m) 7 77.9 H5 2.32(m) 8 75.8 H6α 1.65(dd) 8 74.6 H6α 1.63(dd) 8 85.7 H6α 1.61 (dd) 9 78.7 H6β 2.57(m) 9 78.9 H6β 2.44(m) 9 78.4 H6β 2.66 (d) 10 36.5 H7 2.38(m) 10 79.6 H7 2.35(m) 10 36.6 H7 - 11 51.0 H8 - 11 56.4 H8 - 11 51.4 H8 - 12 24.1 H9 - 12 37.5 H9 - 12 25.9 H9 - 13 49.1 H10 - 13 34.7 H10 - 13 49.8 H10 2.15 14 90.4 H11 - 14 87.9 H11 - 14 90.0 H11 - 15 44.9 H12α 2.13 (m) 15 44.8 H12α 1.98 (m) 15 37.8 H12α 1.98(m) 16 83.1 H12β 2.11(d) 16 82.8 H12β 2.00 (d) 16 82.9 H12β 2.09(d) 17 61.7 H13 2.55 (d) 17 61.5 H13 2.43 (d) 17 63.1 H13 2.47(m) 19 55.8 H14 3.66 (s) 19 55.6 H14 3.58 (s) 19 55.2 H14 3.63(dd) NCH2CH3 48.6 H15α 2.21 (d) NCH2CH3 48.5 H15α 2.15 (d) NCH2CH3 48.7 H15α 2.20(m) NCH2CH3 13.6 H15β 2.36 (m) NCH2CH3 13.4 H15β 2.29(m) NCH2CH3 14.4 H15β 2.37(m) 1-OMe 56.5 H16 3.28 (s) 1-OMe 56.3 H16 3.30 (s) 1-OMe 56.3 H16 3.33(m) 14-OMe 58.0 H17 3.32 (s) 14-OMe 58.0 H17 3.18 (s) 14-OMe 58.0 H17 3.11(s) 16-OMe 56.2 H19α 2.64 (d) 16-OMe 56.2 H19α 2.67 (d) 16-OMe 56.3 H19α 2.66(dd) C=O 167.7 H19β 3.10 (s) C1- C=O 167.1 H19β 3.04 (s) C1- C=O 167.7 H19β 3.04 (d) C1 aroma 112.2 1-OMe 3.29 (s) C1 aroma 115.8 1-OMe 3.29 (s) C1 aroma 115.9 1-OMe 3.27 (s) C2 aroma 150.7 14-OMe 3.41 (s) C2 aroma 144.7 14-OMe 3.41 (s) C2 aroma 141.8 14-OMe 3.45 (s) C3 aroma 116.8 16-OMe 3.25 (s) C3 aroma 120.3 16-OMe 3.25 (s) C3 aroma 120.8 16-OMe 3.31 (s) C4 aroma 134.0 COCH3 2.40 (s) C4 aroma 134.4 COCH3 2.40 (s) C4 aroma 134.6 COCH3 2.31 (s) C5 aroma 116.4 NCH2 2.44(m) C5 aroma 122.8 NCH2 2.44(m) C5 aroma 122.6 NCH2 2.24 (m) C6 aroma 131.8 NCH2CH3 1.17 (s) C6 aroma 131.0 NCH2CH3 1.17 (s) C6 aroma 131.3 NCH2CH3 1.12 (t) NH-Ac 11.00(brs) N-C=O 169.1 NH-Ac 11.00(brs)) N-C=O 169.5 NH-Ac 10.92(brs) H-3'arom 7.68 (d) CH3-C=O 25.5 H-3'arom 7.68 (d) CH3-C=O 25.6 H-3'arom 8.70 (d) H-4'arom 7.15(dd) H-4'arom 7.15(dd) H-4'arom 7.54(dd) H-5'arom 6.53 (dd) H-5'arom 6.53 (dd) H-5'arom 7.00 (dd) H-6'arom 7.38(d) H-6'arom 7.38(d) H-6'arom 7.76(d)

Thus, the analytical methods used for the analysis of the substance of lappaconitine hydrobromide with accompanying alkaloids and the methods for identifying the 11 alkaloids included in the substance made it possible to prove the qualitative and quantitative composition of the substance based on lappaconitine hydrobromide obtained by the claimed method,

The claimed antiarrhythmic agent, which is the active ingredient of the drug Laporitmin, has undergone clinical trials in research centers:

State Budgetary Healthcare Institution of the City of Moscow “City Polyclinic No. 2 of the Moscow City Health Department (1st clinical base);

Branch "Clinical Pharmacology of the Federal State Budgetary Scientific Institution "Scientific Center for Biomedical Technologies of the Federal Medical and Biological Agency" (2nd clinical base).

The total number of patients included in the study, conducted at two clinical sites, was 100 people: 25 patients - the main group, 25 patients - the control group at each clinical site, respectively.

The aim of the study was to identify and compare the antiarrhythmic activity and safety of the drug Laporitmin (25 mg tablets) with the drug Allapinin (25 mg tablets) in ventricular extrasystole.

The following studies formed the basis of the comparison methodology:

1. Evaluation of the antiarrhythmic activity of the drug Laporitmin in patients with ventricular extrasystole.

2. Evaluation of the safety of using the drug Laporitmin in patients with ventricular extrasystole.

3. Comparison of the efficacy and safety of using the drug Laporitmin (25 mg tablets) in patients with ventricular extrasystole with the efficacy and safety of using the drug Allapinin (25 mg tablets) and assessment of the statistical significance of the differences.

The diagnosis and main inclusion criteria were as follows:

- men and women aged 30 to 65 years;

- ventricular extrasystole in patients without organic heart disease (IV - V class according to the classification of R.J. Myerburg et al., 1984) (number of extrasystoles more than 1 per min.);

- the ability and desire to carry out visits and procedures provided for by this study (laboratory tests, functional tests, etc.);

- the presence of informed written consent from the patient to participate in the study and the patient's ability to adequately cooperate during the study.

The study drugs - Allapinin (batch number 160315) and Laporitmin were prescribed orally at 25 mg (1 tablet) 3 times a day. The tablets were taken after meals, washed down with a small amount of water at room temperature, without crushing. In case of ineffectiveness of therapy, the dose of the drug could be increased to 50 mg (2 tablets) 3 times a day.

Evaluation criteria:

Efficiency:

The effectiveness of the drug was assessed based on the dynamics of the effectiveness criteria:

- from visit 1 (examination and inclusion in the study) to visit 2 (start of therapy 3 days +/- 1 day after visit 1);

- from visit 2 (start of therapy 3 days +/- 1 day after visit 1) to visit 3 (day 7+/-1 of therapy);

- from visit 3 visit 3 (day 7+/-1 therapy) to visit 4 (day 14+/-1 therapy);

- from visit 4 (day 14+/-1 therapy) to visit 5 (day 21+/-1 therapy, completion of the study).

The primary efficacy criterion was a reduction in the number of gastric extrasystoles (GES) from visit 2 (start of therapy) to visit 3 (1 week of therapy); from visit 3 (1 week of therapy) to visit 4 (2 weeks of therapy); from visit 4 (2 weeks of therapy) to visit 5 (3 weeks of therapy).

Effective treatment - a reduction in the number of VES by 75-80% during the period of therapy from visit 2 (beginning of therapy) to visit 5 (3 weeks of therapy).

Secondary performance evaluation parameters:

Dynamics from visit 2 (beginning of therapy) to visit 3 (1 week of therapy); from visit 3 (1 week of therapy) to visit 4 (2 weeks of therapy); from visit 4 (2 weeks of therapy) to visit 5 (3 weeks of therapy):

1) the severity of clinical symptoms (frequency of occurrence of the sensation of heartbeat (“interruptions” in the heart area); heart rate; pulse, pulse deficit, blood pressure.

2) indicators of the quality of life questionnaire.

Safety:

The safety assessment of treatment was carried out on the basis of the registration of adverse events by analyzing complaints and subjective symptoms, evaluating and interpreting the results of instrumental examination methods, analyzing clinically significant changes in laboratory parameters, as well as the severity of these changes.

The study assessed:

- the number of patients (in percent) who required a dose reduction or discontinuation of the study drug due to the development of side effects;

- vital signs;

- physical examination data;

- ECG data;

- results of Holter ECG monitoring;

- laboratory data;

- frequency and severity of adverse events.

Statistical methods

The main statistical hypothesis, which was planned to be confirmed in the course of clinical trials, was to confirm as a result of the study that the effectiveness of treatment of patients using Laporitmin as an antiarrhythmic drug is equivalent to the effectiveness of treatment using Allapinin as an antiarrhythmic drug.

Statistical analysis calculates descriptive statistics of variables for both groups of patients and for all patients overall. For continuous variables, the number of cases, mean, standard deviation, minimum, maximum, median, and quartiles are reported.

To compare the parameters under study, testing for normality of distribution was first performed. If the law of normal distribution is met, the Student criterion (t-test) is used. If the distribution pattern differs from normal, nonparametric statistical methods (Wilcoxon criterion, etc.) are used. Intergroup comparison at visit points is performed using the X2 test and Fisher's exact criterion (two-sided option).

Results of the effectiveness evaluation:

According to the protocol of this clinical study, it was established that treatment with the study drug Laporitmin and the reference drug Allapinin is effective, since the reduction in the number of VES during the period of therapy from visit 2 (inclusion and start of therapy) to visit 5 (completion of the study) was, respectively, 86.5% and 86.2% (1 clinical base) and 89.8% and 88.9% (2 clinical base) for the main and control groups, which exceeded the value of 75-80% established as the primary efficacy criterion).

Safety assessment results

The safety of treatment was assessed over the course of three weeks of the patient's participation in the study based on the registration of adverse events by monitoring the patient's condition, analyzing complaints and subjective symptoms, evaluating and interpreting the results of instrumental examination methods, analyzing clinically significant changes in laboratory parameters, as well as the severity of these changes.

The research assessed:

- the number of patients (in percent) who required a dose reduction or discontinuation of the study drug due to the development of side effects;

- vital signs;

- physical examination data;

- ECG data;

- results of Holter ECG monitoring;

- laboratory data.

During the clinical study, one patient in the main group at clinical center 2 experienced an increase in blood glucose concentration to 6.9 mmol/L during visit 2 (blood glucose concentration was normal during visit 5) and an increase in the PQ interval to 0.21 in one patient in the main group and two patients in the control group at clinical center 1.

Conclusion

According to the conducted clinical study, specialists have established that treatment with the study drug Laporitmin based on the claimed antiarrhythmic agent and the reference drug Allapinin is effective, since the reduction in the number of VES during the period of therapy from visit 2 (inclusion at the beginning of therapy) to visit 5 (completion of the study) exceeds the value of 75-80% established as the criterion of primary effectiveness.

Analysis of the dynamics of secondary efficacy parameters did not reveal any significant differences in the pharmadynamics of the studied drug Laporitmin and the reference drug Allapinin in patients with ventricular extrasystole.

Analysis of the dynamics of the health indicators of patients included in the study, according to the quality of life questionnaire, showed that patients in the main and control groups noted positive dynamics in their health, tolerance of daily physical activity. No reliable differences in the assessment of the health status of patients receiving the study drug Laporitmin and the compared drug Allapinin were found.

The findings presented in the Conclusion allow us to consider that the claimed antiarrhythmic agent is aimed at realizing its purpose - to exhibit antiarrhythmic properties, which consist of improving cardiac activity by reducing ventricular extrasystole (VES).

At the same time, the claimed method makes it possible to increase the speed of the technological process for obtaining the claimed antiarrhythmic agent by eliminating the resource- and energy-intensive stages of evaporation, drying and subsequent dissolution in solvents other than those used at the extraction stage, and also to improve the quality of the finished product by reducing the time of thermal exposure, which makes it possible to avoid introducing additional impurities into it.

Claims (37)

1. An antiarrhythmic agent, which is a substance isolated from the plants of northern acorn (high acorn) - Aconitum septentrionale Koelle, of the buttercup family - Ranunculaceae or white-mouthed acorn - Aconitum leucostomum, of the buttercup family - Ranunculaceae, including hydrobromides of the alkaloids lappaconitine, septephin, sepaconitine, N-deacetylranaconitine, N-deacetyllappaconitine, N-acetylsepaconitine, ranaconitine, isolappaconitine, 9-deoxylappaconitine, alkaloid hydrobromide of the structural formula, alkaloid hydrobromide of structural formula with the following mass ratio %: lappaconitine hydrobromide - 79.36-92.06, septefin hydrobromide - 0.02-0.14, sepaconitine hydrobromide 0.03-0.12, N-deacetylranaconitine hydrobromide 0.01-0.16, N-deacetylapaconitine hydrobromide 0.84-3.56, N-acetyl sepaconitine hydrobromide 1.75-4.86, ranaconitine hydrobromide 0.79-9.75, isolappaconitine hydrobromide 0.86-3.26, 9-deoxylappaconitine hydrobromide 0.42-0.76 alkaloid structural formula hydrobromide - 0.10-0.86, alkaloid structural formula hydrobromide - 0.02-0.11. 2. An antiarrhythmic agent according to claim 1, obtained from the rhizomes and roots of the northern aconite (tall aconite) plant - Aconitum septentrionale Koelle, of the buttercup family - Ranunculaceae, or from the rhizomes and roots of the white-mouthed aconite plant - Aconitum leucostomum, of the buttercup family - Ranunculaceae. 3. An antiarrhythmic agent according to claim 1, obtained from the herb of the plant Aconitum leucostomum, of the buttercup family Ranunculaceae. 4. A method for producing an antiarrhythmic agent according to claim 1, based on grinding plant material, extracting the plant material using an extractant selected from the group of organic solvents, filtration, obtaining the target purified product in the form of salts formed by a mineral acid, characterized in that the extraction is carried out at a ratio of the mass of the raw material in grams and the volume of the extractant in ml of 1:1, using an extractant selected from the group consisting of ethyl alcohol, isopropyl alcohol, acetone and a mixture of ethyl and isopropyl alcohol, adding an adsorbent additive selected from the group consisting of silica gel, aluminum oxide and activated carbon to the extract, and the amount of the adsorbent additive is selected in a volume of from 0.1/100 to 5/100 of the mass of the loaded raw material, after mixing, separating the adsorbent additive with the impurities and resins adsorbed on it by filtration, the resulting filtrate acidified with a solution of hydrobromic acid and the resulting solution is evaporated until a dry residue of the target product is obtained, containing a mixture of lappaconitine hydrobromide and hydrobromides of accompanying alkaloids, including septefine, sepaconitine, N-deacetylranaconitine, N-deacetyllappaconitine, N-acetylsepaconitine, ranaconitine, isolappaconitine, 9-deoxylappaconitine, alkaloid hydrobromide of structural formula alkaloid hydrobromide of structural formula . 5. The method according to item 4, characterized in that the extractant is selected from the group consisting of ethyl alcohol with a concentration of 80-95 vol.%, isopropyl alcohol, acetone and a mixture of ethyl and isopropyl alcohol in a ratio of 1/9 to 9/1 (vol./vol.). 6. The method according to item 4, characterized in that the extraction is carried out for 2-4 hours. 7. The method according to item 4, characterized in that the time for mixing the additive with the extract is set within 5-60 minutes. 8. The method according to item 4, characterized in that the adsorbent additive and the extract are mixed for 5-60 minutes at a mixing speed of 100-300 rpm. 9. The method according to item 4, characterized in that the adsorbent additive is separated by filtration under vacuum or gravity through a porous glass filter, or a paper filter, or a woven filter, or a synthetic fiber, for example, polyamide. 10. The method according to item 4, characterized in that the hydrobromic acid solution is selected from a ratio of 0.12 ml of 48% hydrobromic acid to 100 g of raw material. 11. The method according to item 4, characterized in that the obtained filtrate is acidified with a solution of hydrobromic acid in a solvent that was used as an extractant. 12. The method according to item 4, characterized in that the obtained target product is additionally dissolved in alcohol and recrystallized. 13. The method according to claim 4, characterized in that the plant material is selected from the group consisting of the grass of white-mouthed aconite, the rhizomes and roots of white-mouthed aconite - Aconitum leucostomum Worosch, the rhizomes with roots of northern aconite (tall aconite) - Aconitum septentrionale Koelle (A. excelsum Reichenb), of the buttercup family - Ranunculaceae).