CN112694441B

CN112694441B - C 20 Diterpenoid alkaloids, their preparation and use for treating pain related diseases

Info

Publication number: CN112694441B
Application number: CN201911016321.XA
Authority: CN
Inventors: 石建功; 郭庆兰; 夏欢; 张天泰; 武玉卓; 徐成博; 邵帅; 朱承根
Original assignee: Institute of Materia Medica of CAMS
Current assignee: Institute of Materia Medica of CAMS
Priority date: 2019-10-22
Filing date: 2019-10-22
Publication date: 2023-03-21
Anticipated expiration: 2039-10-22
Also published as: CN112694441A

Abstract

The invention discloses a ₂₀ Diterpenoid alkaloids, their preparation and use for treating pain related diseases, in particular the invention discloses C ₂₀ The diterpene alkaloid compound can play a dose-dependent analgesic effect activity under a safe dose.

Description

C 20 Diterpenoid alkaloids, their preparation and use for treating pain related diseases

Technical Field

The invention relates to C with a novel framework, which is extracted, separated and purified from traditional Chinese medicine monkshood ₂₀ Diterpene alkaloid and C obtained by extraction and separation ₂₀ The diterpene alkaloid-Songcoline is obtained by simple reaction, and its derivatives and medicinal salts can be used for preventing and treating acute inflammation, acute or chronic pain, etc. Belongs to the technical field of medicine.

Background

Radix Aconiti lateralis is lateral root of Aconitum carmichaeli Debx of Aconitum of Ranunculaceae, collecting, removing mother root, eliminating fibrous root and silt, air dryingThe processed radix Aconiti lateralis Preparata includes radix Aconiti lateralis Preparata, and radix Aconiti lateralis Preparata ^[1] . The monkshood has the effects of restoring yang and rescuing collapse, tonifying fire and supporting yang, and dispelling cold and relieving pain, and is used for treating symptoms such as yang exhaustion and collapse, cold limbs and pulse, deficiency of heart yang, chest obstruction and cardiodynia, deficiency cold vomiting and diarrhea, abdominal cold pain, deficiency and weakness of kidney yang, impotence and cold uterus, yin cold edema, yang deficiency and external contraction, cold-dampness arthralgia and the like in traditional Chinese medicine. In Huangdi's classic, radix Aconiti lateralis Preparata, radix Ginseng, radix rehmanniae Preparata, and radix Et rhizoma Rhei are listed as "four-vitamin in medicine"; "Fu Zi" recorded in Shen nong Ben Cao Jing "means wind cold, cough, evil qi, warming middle energizer, incised wound, mass, blood obstruction, cold-dampness, prolapsus knee pain, 36484m, spasm knee pain, unable to walk; "Fu Zi from Ming Yi Bie Lu" means pain and weakness of feet, wind-cold in lumbar and back, psychroalgia of heart and abdomen, cholera, spasm of tendons, dysentery with reddish white color, muscle and bone stiffness, yin tonification, abortion, and long for hundreds of herbs; in the treatise on Cold-induced diseases and the treatise on the lack of gold, 23 and 29 prescriptions of Fu Zi are used ^[2,3] . According to statistics, 960 of 3074 Chinese herbal compositions containing radix Aconiti lateralis can treat various pains; of the 224 Chinese patent medicines containing aconite, 134 ones with analgesic effect were found (https:// db. Yaozh. Com /). It can be seen that analgesia is one of the most important basic effects of aconite, the status of aconite in traditional Chinese medicine and its wide application in pain treatment.

The research of the modern pharmacology and toxicity and chemical components of monkshood respectively begins in 30 and 60 years of the 20 th century, and related research results show that the pharmacology activities of monkshood extract and chemical components thereof mainly comprise strengthening heart, easing pain, calming, resisting inflammation, resisting arrhythmia, resisting tumor, enhancing immunity, reducing blood sugar, blood fat, resisting aging, protecting kidney and the like; the toxicity mainly comprises cardiotoxicity, neurotoxicity, embryotoxicity, nephrotoxicity and the like; the analgesic active ingredient and toxic ingredient are aconitine type C ₁₉ Diterpene alkaloid, and effective components with cardiotonic effect such as higenamine (radix Aconiti lateralis), norsalsoline, methyldopamine chloride and C ₁₉ -diterpene alkaloids. The method comprises the following specific steps:

efficacy and toxicity of the water-decocted material: decocting radix Aconiti lateralis Preparata with water, extracting with water, precipitating with ethanol, and making into powderThe rat acetic acid writhing experiment shows that the compound has good analgesic activity ^[4,5] (ii) a The water decoction of salted aconite, black shun tablet, white aconite tablet, steamed aconite tablet, fried aconite tablet and bland aconite tablet can obviously reduce the times of mouse writhing reaction caused by acetic acid, and can prolong the latent period of mouse reaction to heat pain in tail pressure test and mouse tail flick test ^[6-10] . The decoction of radix Aconiti lateralis and processed radix Aconiti lateralis has obvious cardiotonic effect on isolated heart, in vivo heart and experimental heart failure, etc., and has different cardiotonic effect strength and cardiotoxicity in different models, and the processed radix Aconiti lateralis has obviously reduced cardiotoxicity ^[11,12] . In addition, the aconite decoction has obvious inhibiting effect on ear inflammation of mice caused by croton oil and foot seed expansion of mice caused by formaldehyde ^[13] It can be used for preventing ventricular fibrillation of mouse caused by chloroform ^[14] And endothelium-dependent effects on aortic relaxation ^[15] It is suggested that Fu Zi decoction also has anti-inflammatory and anti-arrhythmic pharmacological activities.

In view of the toxicity of aconite, the method of processing, decocting for a long time and strictly controlling the dosage is usually adopted in clinical application to reduce the toxic and side effects of aconite, and the method of decocting aconite with water has been widely used ^[16] . Research shows that the toxicity of the water decocted extract of aconite is less than that of the soaked extract, the total alkaloid content tends to be stable after the water decocted extract of aconite is decocted for 3-6 hours, and the content of toxic component-ester alkaloid is obviously reduced ^[17-21] (ii) a The fat-soluble alkaloid has higher toxicity than the original medicinal material, and the toxicity similar to the original medicinal material is not found in the water-soluble part ^[22] . Meanwhile, it is reported that residents from Shaanxi Zhou to Hu county, one of the major production areas of monkshood, always have a habit of eating large dose of monkshood, and the residents boil monkshood with water for more than 4 hours and are combined with liquorice, so that the daily food intake can reach 200-400g and far exceeds the medicinal dose of monkshood ^[23] . In a word, the toxicity and activity of the unprocessed radix aconiti lateralis preparata and the radix aconiti lateralis preparata decocted in water at different time are compared, and the application mode of the traditional water decoction of the radix aconiti lateralis preparata can effectively reduce the toxicity of the radix aconiti lateralis preparata and simultaneously keep the remarkable activities of analgesia and the like ^[24] 。

Therefore, the analgesic effect of the aconite water extract is definite, and the scientificity of processing, water decoction and toxicity reduction of aconite is confirmed by modern experimental research. The exact functional ingredients are not fully understood.

Chemical components: at present, more than 120 compounds are reported to be separated and identified from aconite, and the structure types comprise aconitane type C ₁₉ Diterpene alkaloids, C of various skeleton types ₂₀ Diterpene alkaloids, benzylisoquinoline alkaloids and other alkaloids, and flavones, malic acid, ceramides, fatty acids, glycerol bisglycosides and polysaccharides ^[30-40] Wherein aconitane type C ₁₉ Diterpene alkaloids as main components.

Analgesic components: aconitane type C ₁₉ The analgesic activity intensity of diterpenoid alkaloids is related to the types, the number and the positions of ester groups in the structure, the substitution of N atoms of an A ring, the saturation degree of a D ring and the like ^[41,42] The analgesic activity intensity of sinoaconitine is higher than aconitine is higher than hypaconitine; at 2mg/kg, the inhibition rate of lipomeconinine (content 0.0062%) on the acetic acid writhing frequency of mice is equivalent to aconitine (0.2 mg/kg) and mesaconine (0.1 mg/kg), but LD is the same ₅₀ (10-40 mg/kg) is 20 and 40 times of aconitine and mesaconitine respectively ^[43] . Their analgesic action is exerted mainly by activating or inhibiting Na ⁺ Channel, activation of downstream inhibitor alpha ₂ -epinephrine and 5-hydroxytryptaminergic neurons and the like ^[44-46] 。

Toxic components: the poisoning symptoms of aconite are similar to those caused by aconitine alone, and the analysis results of the relative contents of chemical components and toxic value confirm that 3-acetyl aconitine, neoaconitine, aconitine, hypaconitine, deoxyaconitine, 10-hydroxy neoaconitine and 10-hydroxy aconitine are the main toxicity related substances of raw aconite and processed aconite, so that it is considered that aconitane type C ₁₉ Diterpenoid alkaloids are the key toxic components of aconite. The toxicity includes cardiotoxicity, neurotoxicity, embryotoxicity and nephrotoxicity ^[47] . The alkaloid structure contains two organic acid ester groups, and the diester-type C is obtained during processing and long-time decoction of radix Aconiti lateralis ₁₉ The diterpene alkaloid can be hydrolyzed, and the content is remarkably reduced along with the decoction timeTherefore, the toxicity reduction or detoxification of processed and decocted aconite for a long time is considered to be caused by the reduction of the content of diester-type alkaloids.

However, the results of the studies have shown that the analgesic and toxic components of aconite overlap, and the vast majority of the analgesic components are diester C ₁₉ -diterpene alkaloids.

In conclusion, through decades of research, scientists in China have obtained a great deal of research results in the research aspect of the classical traditional Chinese medicine monkshood. However, although the diester form C was found ₁₉ Diterpene alkaloids have a significant analgesic effect, but are also key toxic substances of aconite, and can be degraded by processing and prolonged decoction, and thus their content in the decoction is very low or no longer present. However, the monoester type C19-diterpenoid alkaloids with low toxicity or no toxicity in the hydrolysate have no analgesic effect or the effect strength is far from reaching the effect of the water decoction. Therefore, the key components and action mechanism of the analgesic efficacy of aconite are the key scientific problems that need to be deeply researched and clarified.

The anti-inflammatory analgesic drugs are widely used in clinic, and have very large usage amount regardless of prescription drugs or non-prescription drugs. At present, the anti-inflammatory analgesic drugs with the widest clinical application range are traditional non-steroidal drugs, and the representative drugs mainly comprise aspirin, acetaminophen and ibuprofen. However, in the treatment process of the traditional nonsteroidal anti-inflammatory analgesic drugs, the anti-inflammatory and analgesic effects are realized by inhibiting the exertion of the effect of cyclooxygenase II and further inhibiting the synthesis of inflammatory prostaglandin. However, in the process of exerting the drug action, cyclooxygenase I is also inhibited, so that the synthesis of physiological prostaglandin is greatly reduced, gastrointestinal side effects such as gastrointestinal mucosal erosion, ulcer and the like are caused to the patients, and in severe cases, the patients have the symptoms of blood coagulation dysfunction, renal toxicity and the like. In addition, the blood system symptoms, the cardiovascular system symptoms and the like are also adverse reactions frequently occurring in the taking process of the anti-inflammatory analgesic, and the clinical application range of the traditional anti-inflammatory analgesic is greatly limited by the safety venereal disease seriously influencing the medication of patients. Therefore, there is an urgent need to develop new anti-inflammatory analgesic drugs with less side effects and wide clinical application range.

The search for anti-inflammatory analgesic active ingredients from natural products is becoming a research focus in recent years. Researches find that natural products such as saponin, polysaccharide, alkaloid, flavone, coumarin and the like have certain anti-inflammatory and analgesic effects, and the effects are mainly shown in that inflammatory swelling or granuloma caused by chemical and physical stimulation can be remarkably inhibited, the permeability of abdominal cavity capillaries after inflammation can be reduced, and the expression of inflammatory factors and genes thereof can be inhibited; the analgesic effect of natural products is usually manifested by an increase in pain threshold for a variety of pain-causing factors such as thermal pain, tenderness, and chemical stimulation. The compound in the application is a natural product with anti-inflammatory and analgesic effects, which is obtained by separating from traditional Chinese medicine monkshood.

Reference documents:

[1] national pharmacopoeia committee the pharmacopoeia of the people's republic of china 2015 edition one, beijing, chinese medical science and technology publishing house 2015.

[2] Li Shizhen, ben Cao gang mu, business brochure 1955, 17.

[3] Chen Su Sheng, shang Han and Shi nan, fujian science and technology publishing house 2005.

[4] The pharmacological research on warming the middle and relieving pain of Zhu Zi, shen Yao Qin and Fu Zi, chinese materia medica journal, 1992,17 (4): 238-241.

[5] Dungjiagang, vanlili, poplar, duzhencai, lantai, experimental study of the analgesic effect and dose-effect relationship of radix Aconiti lateralis, chinese materia medica journal, 2009, (11): 2249-2251.

[6] Paniculate swallowwort root, evaluation and control research on the quality of the toxic traditional Chinese medicine monkshood based on biological toxicity value detection and chemical component analysis, chengdu university of traditional Chinese medicine, 2013.

[7] The pain relieving effect of the paniculate swallowwort rhizome, the common swallowwort root and the monkshood on rats with neuropathic pain, the journal of Chinese anesthesiology, 2005,25 (5), 381-384.

[8] Zhang Ming, shen Yaqin, the pharmacological research of warming the channels, alleviating pain and removing arthralgia by Wen Liao medicine, chinese journal of information and pharmacy, 2000, 7.

[9] Zhang Shi Jun, quality and pharmacological research of radix Aconiti lateralis Preparata, international J.TCM. 1999, (5): 12-15.

[10] The research on the anti-inflammatory, analgesic and immunity-improving effects of different processed products of aconite carmichaeli (radix aconiti carmichaeli) can be conducted on the basis of the research on the effects of anti-inflammation, analgesia and immunity-improving effects of different processed products of aconite carmichaeli (radix aconiti carmichaeli) on autumn rhyme, plum-dream-mings, mullulin, sea-lode, xiexiang and Pengzheng (2017), (1): 123-127).

[11] Zhongyuan Peng, liu culture, zeng Gui Yun, chen Di Hua, li Hui Ying, song Wei Liang Aconitine and its analogues have toxicity and influence on cardiac contractile function, reports of pharmacy 1984,19 (9): 641-646.

[12] Lyric, experimental study of aconite water-soluble alkaloid for treatment of acute heart failure, chengdu university of traditional Chinese medicine 2015.

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[15]Li H.,Liu X.,Niu C.,Zhang T.Tension effect of water decoction of Aconitum carmichaeli Debx on rabbit aorta in vitro.Chin.J.Tiss.Eng.Res.2015,19(33):5312-5317.

[16]Liu S.,Li F.,Li Y.,Li W,Xu J,Du H.A review of traditional and current methods used to potentially reduce toxicity of Aconitum roots in Traditional Chinese Medicine.J.Ethnopharmacol. 2017,207:237-250.

[17] Zhang Ying (open silver), wu Runyu (Wu Runyu), liu Tian Bei (Liu Tian), fuzi toxicity study, proc. Pharmacopeia, 1966,13 (5): 350-355.

[18]Bisset N.G.Arrow poisons in China.Part II.Aconitum-Botany,chemistry,and pharmacology.J Ethnopharmacol 1981,4(3):247–336.

[19]Chan T.Y.K.Aconite poisoning.Clin.Toxicol 2009,47(4):279–285.

[20]Gao F.,Li Y.Y.,Wang D.,Huang X,Liu Q.Diterpenoid alkaloids from the Chinese traditional herbal“Fuzi”and their cytotoxic activity,Molecules.2012,17(5):5187-5194.

[21] Zhou Zi, yangming, these bolt(s) and Huang Qin Lian, the toxicity transmission rule of Fu Zi, the third international conference on science and technology of modern Chinese medicine 2015,245-253.

[22] Shao Lu, zhou Cheng Peng, the effect of water-soluble part of Fu Zi on arrhythmia, chinese medicine Notification, 1988,13 (6): 42-44,64.

[23] Toxicity research of Zhulinping and prepared aconite root general Jiangxi traditional Chinese medicine, 2004,35 (6): 53-55.

[24] Xue Jie, li Yong Du, the method of supervising and safely applying the toxicity of monkshood by preferring monkshood from Zhou of Shaanxi province to county, beijing TCM, 2016,25 (12): 743-744.

Although the literature reports that various chemical components as described above are obtained from monkshood and the pharmacological activities of the chemical components in monkshood extract and part of monkshood, the present invention has been directed to C ₂₀ Diterpenoid alkaloids and derivatives thereof, and the compounds or the derivatives thereof have no report on the analgesic function; meanwhile, the compound with the novel framework has no related structural report.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a novel drug with analgesic effect.

In order to solve the technical problem, the invention provides the following technical scheme:

the first aspect of the technical scheme of the invention provides C shown as general formulas (I) to (IV) ₂₀ Diterpene alkaloids and their derivatives.

Specifically, compounds represented by general formula (I) and pharmaceutically acceptable salts thereof are provided:

wherein R is ₁ Selected from H, CH ₃ 、CH ₂ CH ₃ ；R ₂ Is selected from CH ₃ 、OH、OCH ₃ 、OCH ₂ CH ₃ 。

A compound of formula (II):

wherein R is ₁ Selected from H, CH ₃ 、CH ₂ CH ₃ ；R ₂ Selected from H, CH ₃ 、OH、OCH ₃ 、OCH ₂ CH ₃ (ii) a X is selected from SO ₃ 、HSO ₄ 、OH、CF ₃ COO、CH ₃ COO、HCOO、Cl、Br、I。

A compound of formula (III) and pharmaceutically acceptable salts thereof:

wherein R is ₁ Selected from H, CH ₃ 、CH ₂ CH ₃ ；R ₂ Selected from H, OH, OCH ₃ 、OCH ₂ CH ₃ 、SO ₃ H、 SO ₃ ^- ；R ₃ Selected from H, CH ₃ 、CH ₂ CH ₃ 、OH、OCH ₃ 、OCH ₂ CH ₃ 。

A compound of formula (IV) and pharmaceutically acceptable salts thereof:

Further preferred compounds of the invention are selected from the group consisting of:

in a second aspect of the present invention, there is provided a process for the preparation of a compound according to the first aspect.

Drying monkshood, crushing, extracting for 2-4 times by using distilled water at 35-50 ℃ for 4-8 hours each time, merging extracting solutions, recovering a solvent under reduced pressure to obtain an extract, separating by using a macroporous resin column chromatography, and sequentially using water: ethanol 1, gradient elution from 0 to 0, TLC or HPLC monitoring, eluting each fraction until no significant elution of the sample, recovering the solvent under reduced pressure to give the corresponding eluted fractions, wherein the 50% ethanol fraction is separated on MCI resin, and sequentially using water: gradient elution with ethanol 1; wherein, the ratio of water: ethanol 7: eluting with ethanol 1 in a gradient manner from 0 to 0, and monitoring by TLC or HPLC to combine the same components to obtain corresponding eluted fractions C2-1-C2-4; adjusting pH of water elution part C2-1 to 10 with concentrated ammonia water, and extracting with ethyl acetate to obtain organic phase C2-1-A and water phase C2-1-C; separating C2-1-A through basic pH 8-9 silica gel column chromatography, eluting with petroleum ether-acetone-diethylamine 15; separating C2-1-A-3 by reversed phase silica gel MPLC chromatography, and eluting with methanol-water gradient to obtain C2-1-A-3-1-C2-1-A-3-5; recrystallizing C2-1-A-3-3 with methanol to obtain Sonogoling and compounds (I) and (II); heating Songcoline and sodium sulfite in 50% methanol water solution for 8 hr, and performing reverse phase HPLC to obtain compounds 1 and 2; adjusting the pH value of the water phase C2-1-C to 4 by using 6N HCl, extracting by using N-butanol, performing normal phase silica gel column chromatography on the obtained water phase C2-1-C, performing gradient elution by using chloroform-methanol 50; separating C2-1-C-5 by Sephadex LH-20 column chromatography with water as mobile phase to obtain component C2-1-C-5-1-C2-1-C-5-6, wherein C2-1-C-5-1 is separated by HW-40C column chromatography with water as mobile phase to obtain C2-1-C-5-1-6; and (3) performing semi-preparative phenyl column on the C2-1-C-5-1-4, and performing reverse phase HPLC to obtain compounds (III) and (IV).

In a third aspect of the present invention, there is provided a pharmaceutical composition comprising C represented by the general formulae (I) to (IV) as an active ingredient ₂₀ Diterpenoid alkaloids, and

products

1 and 2 obtained by reaction with major component songorine in radix Aconiti lateralis as raw material, and conventional carrier in pharmaceutical field.

Typically, the pharmaceutical compositions of the present invention contain 0.1 to 95% by weight of a compound of the present invention.

Pharmaceutical compositions of the compounds of the invention may be prepared according to methods well known in the art. For this purpose, the compounds of the invention can, if desired, be combined with one or more solid or liquid pharmaceutical excipients and/or adjuvants, in a suitable administration form or dosage form for use as human or veterinary medicine.

The compounds of the present invention or pharmaceutical compositions containing them may be administered in unit dosage form, either enterally or parenterally, such as orally, intramuscularly, subcutaneously, nasally, oromucosally, dermally, peritoneally or rectally, preferably orally.

The route of administration of the compounds of the invention or the pharmaceutical compositions containing them may be by injection. Injections include intravenous, intramuscular, subcutaneous, intradermal, and the like.

The administration dosage form can be liquid dosage form or solid dosage form. For example, the liquid dosage form can be true solution, colloid, microparticle, emulsion, or suspension. Other dosage forms such as tablet, capsule, dripping pill, aerosol, pill, powder, solution, suspension, emulsion, granule, suppository, lyophilized powder for injection, etc.

The extract or the compound can be prepared into common preparations, sustained release preparations, controlled release preparations, targeting preparations and various microparticle drug delivery systems.

In order to prepare the unit dosage form into tablets, various carriers well known in the art can be widely used. Examples of the carrier are, for example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate and the like; wetting agents and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone and the like; disintegrating agents such as dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene sorbitol fatty acid ester, sodium dodecylsulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors such as sucrose, glyceryl tristearate, cacao butter, hydrogenated oil and the like; absorption accelerators such as quaternary ammonium salts, sodium lauryl sulfate and the like; lubricants, for example, talc, silica, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, and the like. The tablets may be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer and multi-layer tablets.

For example, to form the dosage unit into a pill, a wide variety of carriers known in the art are used. Examples of the carrier are, for example, diluents and absorbents such as glucose, lactose, starch, cacao butter, hydrogenated vegetable oil, polyvinylpyrrolidone, gelucire, kaolin, talc and the like; binders such as acacia, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or batter, etc.; disintegrating agents, such as agar powder, dried starch, alginate, sodium dodecylsulfate, methylcellulose, ethylcellulose, etc.

For example, in order to encapsulate the administration unit, the active ingredient of the extract or compound of the present invention is mixed with the above-mentioned various carriers, and the thus-obtained mixture is placed in a hard gelatin capsule or soft capsule. The effective component of the compound can also be prepared into microcapsules, suspended in an aqueous medium to form a suspension, and also can be filled into hard capsules or prepared into injections for application.

For example, the extract or compound of the present invention may be formulated into injectable preparations such as solutions, suspensions, emulsions, lyophilized powders, which may be aqueous or non-aqueous, and may contain one or more pharmaceutically acceptable carriers, diluents, binders, lubricants, preservatives, surfactants or dispersants. For example, the diluent may be selected from water, ethanol, polyethylene glycol, 1, 3-propanediol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitol fatty acid ester, and the like. In addition, for the preparation of isotonic injection, sodium chloride, glucose or glycerol may be added in an appropriate amount to the preparation for injection, and conventional cosolvents, buffers, pH adjusters and the like may also be added. These adjuvants are commonly used in the art.

In addition, colorants, preservatives, flavors, flavorings, sweeteners, or other materials may also be added to the pharmaceutical preparation, if desired.

For the purpose of administration and enhancing the therapeutic effect, the drug or pharmaceutical composition of the present invention can be administered by any known administration method.

The dose of the compound, pharmaceutical composition of the present invention to be administered depends on many factors such as the nature and severity of the disease to be prevented or treated, sex, age, body weight, character and individual response of the patient or animal, administration route, administration frequency, therapeutic purpose, and thus the therapeutic dose of the present invention can be widely varied. Generally, the dosage of the pharmaceutical ingredients of the present invention used is well known to those skilled in the art. The prophylactic or therapeutic objectives of the present invention can be accomplished by appropriate adjustment of the actual amount of drug contained in the final formulation of the compound composition of the present invention to achieve the desired therapeutically effective amount. A suitable daily dosage range of the compound of the invention is 0.001-150mg/kg body weight, preferably 0.01-100mg/kg body weight, more preferably 0.01-60mg/kg body weight, most preferably 0.1-10mg/kg body weight. The above-mentioned dosage may be administered in a single dosage form or divided into several, e.g., two, three or four dosage forms which is limited by the clinical experience of the administering physician and by dosage regimens which include the use of other therapeutic means.

The total dose required for each treatment can be divided into multiple doses or administered as a single dose. The compound and the composition of the invention can be taken alone or combined with other therapeutic drugs or symptomatic drugs and the dosage is adjusted.

The fourth aspect of the technical proposal of the invention provides C shown as general formulas (I) to (IV) ₂₀ The diterpenoid alkaloids and the

compounds

1 and 2 can be used for preparing medicines for treating diseases such as somatic pain, visceral pain, neuropathic pain or cancer pain.

The invention also relates to the application of the aconite extract in preparing medicines for preventing or treating acute or chronic pain and other diseases. The various pains include pains associated with the central nervous system or the peripheral nervous system, various acute or chronic pains, nociceptive pains, somatic pains, visceral pains, neuropathic pains, or cancer pains.

The inventor finds that the compounds (I) to (IV) as well as the

compounds

1 and 2 and pharmaceutically acceptable salts have certain analgesic effect. Thus, compounds (I) - (iv) and compounds 1 and 2 and pharmaceutically acceptable salts of the present invention are further directed to methods of treating, ameliorating or treating conditions associated with analgesia. The method comprises administering to a patient in need thereof a therapeutically effective amount of a compound of formulae (I) - (IV) and

Compounds

1 and 2 or a pharmaceutically acceptable salt or a pharmaceutical composition thereof.

The invention shows that the compounds (I) - (IV) and the compound 1 have good analgesic effect at the level of whole animals, and 2-4 have weak analgesic effect, but have dose dependence. Compounds (I) to (IV) or pharmaceutically acceptable salts have not been reported in the publication;

compounds

1 and 2 have not been reported publicly for their use with analgesic activity.

Advantageous technical effects

In the research process of the active ingredients of the traditional Chinese medicine monkshood, the inventor of the invention separates C with a novel skeleton from monkshood by an activity tracking method ₂₀ Diterpenoid alkaloids 3 and 4, and a major component songorine, and the

compounds

1 and 2 are prepared by simple reaction. The compounds are subjected to activity evaluation through an acetic acid writhing experiment, and the results show that the compound 1 has a good analgesic effect at the level of the whole animal, 2-4 has a weak analgesic effect, but has dose dependence, and belongs to a lead compound in the research and development process of analgesic drugs.

Drawings

FIG. 1, scheme for preparation of Compounds 1-4

Detailed Description

The following experimental examples further illustrate the invention but do not limit it in any way.

Example 1, compound 1 and2 is a novel C extracted, separated and purified from aconite ₂₀ The diterpenoid alkaloid is separated, purified and identified as follows:

drying radix Aconiti lateralis 50Kg, pulverizing, extracting with water at 40 deg.C for 3 times, each for 6 hr, mixing extractive solutions, recovering solvent under reduced pressure to 120L, separating with macroporous resin (HPD-110, 19kg) column chromatography (20 × 200 cm), sequentially eluting with water (50L), 30% ethanol (120L), 50% ethanol (120L), and 95% ethanol (100L), recovering solvent under reduced pressure to obtain corresponding eluate fractions A (water), B (30% ethanol), C (50% ethanol), and D (95% ethanol), wherein the fraction C is separated with MCI resin (CHP 20P), sequentially eluting with water (10L), 30% ethanol (30L), 50% ethanol (20L), and 95% ethanol (10L) to obtain corresponding eluate fractions C1-C4. The 30% ethanol elution fraction (C2, 750 g) was separated with MCI resin, and eluted sequentially with water (3L), 30% ethanol (4L), 50% ethanol (4L) and 95% ethanol (1L) to give the corresponding eluted fraction (C2-1-C2-4). C2-1 (200 g) was adjusted to pH 10 with concentrated aqueous ammonia, and extracted with ethyl acetate (500 ml. Times.4) to obtain an organic phase C2-1-A and an aqueous phase C2-1-C, respectively. The organic phase (C2-1-a, 60 g) was subjected to column chromatography on basic silica gel (pH 8-9) and eluted with a petroleum ether-acetone-diethylamine 15 gradient from 1. C2-1-A-3 (3.0 g) was separated by reversed phase silica gel MPLC chromatography, eluting with a gradient of methanol-water 1 from 0 to 0, to give C2-1-A-3-1-C2-1-A-3-5. Recrystallizing the C2-1-A-3-3 (2.5 g) with methanol to obtain the compound songgoline (1 g). Songcoline and sodium sulfite are dissolved in 50% methanol water, heated and reacted for 8 hours, and then subjected to HPLC (phenyl column, 20% methanol, 0.2% TFA, 2ml/min) to obtain Compound 1 (t-TFA, 2ml/min) _R =36 min) and 2 (t) _R =41 min). C2-1-C was adjusted to pH 4 with 6N HCl, extracted with N-butanol (500 ml. Times.4), and the aqueous phase C2-1-C (32 g) was subjected to silica gel column chromatography with chloroform-methanol 50 gradient elution to obtain C2-1-C-1-C2-1-C-6. Wherein C2-1-C-5 (13 g) is subjected to Sehpadex LH-20 column chromatography (water) to obtain C2-1-C-5-1-C2-1-C-5-6. Separating (water) C2-1-C-5-1 (800 mg) by HW-40C column chromatography to obtain C2-1-C-5-1-6. Separating C2-1-C-5-1-4 (28 mg) by semi-preparative phenyl column HPLC (20% methanol, 0.2% TFA, 2ml/min) to obtain Compound 3 (7 mg, t) _R =56 min) and 4 (3mg _R =51 min). The separation scheme is shown in FIG. 1.

Compound 1 is a white amorphous powder; (+) -ESIMS m/z 330[ m + H ], [ solution of calcium chloride ]] ⁺ ,342[M+Na] ⁺ ； (+)-HR-ESIMS m/z 330.20697[M+H] ⁺ (calcd.for C ₂₀ H ₂₈ NO ₃ ,330.20637).

Compound 2 is a white amorphous powder; (+) -ESIMS m/z 356[ m ] +H] ⁺ ；(+)-HR-ESIMS m/z 356.22238[M+H] ⁺ (calcd.for C ₂₀ H ₂₈ NO ₃ ,356.22202)..

Compound 3: white amorphous powder; [ alpha ] of] ²⁰ _D -38.1(c 0.28,MeOH)；UV(MeOH)λ _max (logε): 202(2.24),223(sh,1.62),267(0.89)nm；CD(MeOH)200(Δε-1.18),290(Δε-0.43), 329(Δε+0.04)；IRν _max 3393,2970,2922,2850,1738,1678,1485,1466,1420,1321, 1202,1140,1049,957,879,838,801,722cm ^-1 ；(+)-ESIMS m/z 438[M+H] ⁺ ,460 [M+Na] ⁺ ,(-)-ESIMS m/z 436[M-H] ^- ；(+)-HR-ESIMS m/z 438.1954[M+H] ⁺ (calcd. for C ₂₂ H ₃₂ NO ₆ S,438.1945),(-)-HR-ESIMS m/z 436.1813[M-H] ^- (calcd.for C ₂₂ H ₃₀ NO ₆ S,436.1799).

Compound 4: white amorphous powder; [ alpha ] to] ²⁰ _D -37.4(c 0.14,MeOH)；UV(MeOH)λ _max (logε) 202(2.46),219(sh,1.65)nm；CD(MeOH)200.0(Δε–0.54),296(Δε–0.65)；IRν _max 3394,2920,2850,1736(sh),1680,1543,1448,1358,1203,1140,1053,983,956,907, 880,842,802,724cm ^-1 ；(+)-ESI-MS:m/z 438[M+H] ⁺ ,460[M+Na]；(-)-ESI-MS: m/z 436[M-H] ^- ；(+)-HR-ESI-MS:m/z 438.1945[M+H] ⁺ (calcd for C ₂₂ H ₃₂ NO ₆ S, 48.1945),(-)-HR-ESIMS m/z 436.1814[M-H] ^- (calcd.for C ₂₂ H ₃₀ NO ₆ S,436.1799).

Experimental example 2 analgesic action, effect of Compound 1-4 on acetic acid-induced pain in mice

The acetic acid writhing experiment is a classical model of peripheral analgesia, which is caused by mouse peritoneal inflammation caused by acetic acid stimulation and is one of methods for evaluating and screening analgesics. Acetic acid with a certain volume and concentration is injected into the abdominal cavity of a mouse to stimulate the visceral layer and parietal peritoneum to cause inflammatory pain with a large deep part and a long time, so that the mouse has behavioral reactions such as abdominal indent, trunk and hind limb stretch, hip rising and the like, which are called writhing reaction. The frequency of the reaction is high within 15min after injection, so the times of writhing or the number of mice which react within 15min after injection is taken as the quantitative index of pain. [ Weiwei, wu Ximei, liyuan construction, pharmacological experimental methodology, national public health Press, 4 th edition, p770 ]

Test animals: healthy adult female ICR mice, clean grade, weight 18-22g. The experimental animals are raised in an independent environment with 12h-12h day and night alternation, the room temperature is maintained at 24 +/-2 ℃, water is freely drunk and food is eaten, and the experiment is carried out after the experimental animals are adapted to the environment for 1 week. All treatments for animals were in compliance with the requirements of the ethical committee of the international society for pain research.

Test compounds: the test substance is compound 1-4, and the positive drug is 3-acetyl aconitine

The method comprises the following steps: dividing ICR female mice into 5 groups of 10 mice, wherein the groups are respectively a model control group (normal saline) and a positive control group (morphine, 0.3 mg/kg); a compound high dose group (1.0 mg/kg), a compound medium dose group (0.3 mg/kg), and a compound low dose group (0.1 mg/kg). Except for the model group, the other physiological saline was distilled water. In each group, 1% (0.1 ml/10 g) of chemical stimulant acetic acid solution is injected into the abdominal cavity 30 minutes after administration, the number of writhing of mice within 15 minutes after acetic acid injection is recorded, the inhibition rate of the drug on writhing reaction is calculated according to the following formula, and the analgesic effect of the drug is judged:

inhibition% = [ (average number of twists of negative control group-average number of twists of experimental group)/(average number of twists of negative control group) ] × 100%.

The experimental results are as follows: compared with the model group, the twisting times of the high, medium and low dose groups of the compounds 1 to 4 are less than that of the model group, and the compounds have obvious dose-dependent effect. The positive medicine has obvious analgesic effect, and the analgesic inhibition rate reaches 71.28 percent. The results show that the compound 1 has obvious analgesic effect, and 2-4 has certain analgesic effect in a high-dose group and dose dependence. Specific results are shown in table 1.

TABLE 1 analgesic Effect of Compounds 1-4

Note: data are expressed as mean ± sem, # p <0.01, and # p <0.001 indicates significance compared to model groups.

Claims

1. A compound of formula (IV):

wherein R is ₁ Selected from H, CH ₃ 、CH ₂ CH ₃ ；R ₂ Selected from SO ₃ ^- ；R ₃ Selected from H, CH ₃ 、CH ₂ CH ₃ 。

2. A group of compounds and pharmaceutically acceptable salts thereof, wherein the compounds are selected from the group consisting of:

3. a compound according to any one of claims 1 and 2, and pharmaceutically acceptable salts thereof, wherein the pharmaceutically acceptable salt is selected from salts of the compound with organic or inorganic acids.

4. A process for the preparation of a compound according to any one of claims 1 to 3, and pharmaceutically acceptable salts thereof, wherein said compound is prepared by:

drying monkshood, crushing, extracting for 2-4 times by using distilled water at 35-50 ℃ for 4-8 hours each time, merging extracting solutions, recovering a solvent under reduced pressure to obtain an extract, separating by using a macroporous resin column chromatography, and sequentially using water: ethanol 1, gradient elution from 0 to 0, TLC or HPLC monitoring, eluting each fraction until no significant elution of the sample, recovering the solvent under reduced pressure to give the corresponding eluted fractions, wherein the 50% ethanol fraction is separated on MCI resin, and sequentially using water: eluting with ethanol 1 in a gradient manner from 0 to 0, and combining the same components by TLC or HPLC monitoring; wherein, the water: ethanol 7: eluting with ethanol 1 in a gradient manner from 0 to 0, monitoring by TLC or HPLC, and combining the same components to obtain corresponding eluted fractions C2-1-C2-4; adjusting pH of water elution part C2-1 to 10 with concentrated ammonia water, and extracting with ethyl acetate to obtain organic phase C2-1-A and water phase C2-1-C; separating C2-1-A through basic pH 8-9 silica gel column chromatography, eluting with petroleum ether-acetone-diethylamine 15; separating C2-1-A-3 by reversed phase silica gel MPLC chromatography, and eluting with methanol-water gradient to obtain C2-1-A-3-1-C2-1-A-3-5; recrystallizing the C2-1-A-3-3 with methanol to obtain songorine; heating Songcoline and sodium sulfite in 50% methanol water solution for 8 hr, and performing reverse phase HPLC to obtain compound 1; adjusting the pH value of the water phase C2-1-C to 4 by using 6N HCl, extracting by using N-butanol, carrying out normal phase silica gel column chromatography on the obtained water phase C2-1-C, carrying out gradient elution by using chloroform-methanol 50; separating the C2-1-C-5 by Sephadex LH-20 column chromatography with water as mobile phase to obtain component C2-1-C-5-1-C2-1-C-5-6, wherein the C2-1-C-5-1 is separated by HW-40C column chromatography with water as mobile phase to obtain C2-1-C-5-1-6; the compound 3 is obtained by the semi-preparative phenyl column and the reversed phase HPLC of C2-1-C-5-1-4,

5. a pharmaceutical composition comprising a compound of any one of claims 1 and 2, and a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

6. Use of a compound according to any one of claims 1 and 2, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 5, for the manufacture of a medicament for the prevention or treatment of pain.

7. Application of compound as shown in the following 1 and pharmaceutically acceptable salt thereof in preparing medicines for preventing or treating pain

8. Use according to any one of claims 6 to 7, wherein the pain is associated with the central nervous system or the peripheral nervous system.

9. Use according to any one of claims 6 to 7, wherein the pain comprises acute pain or chronic pain.

10. Use according to any one of claims 6 to 7, wherein the pain is somatic pain, visceral pain or neuropathic pain.