Preparation and Use of Neolignan Compounds in Angelica sinensis

Abstract

The present invention belongs to the field of pharmaceutical technology, and discloses the preparation and use of neolignans in Angelica sinensis, specifically relating to the significant sedative and hypnotic effects of new neolignan compounds. The results of animal experiments prove that compounds (+)-1 and (-)-1 have obvious sedative and hypnotic effects and are expected to become therapeutic drugs for mental diseases related to insomnia, convulsion, epilepsy / anxiety and depression.

Description

Technical Field

[0001] The present invention relates to the extraction, separation and purification of new 2,7'-epoxy-8,9'-neolignan enantiomers from Chinese medicine Angelica sinensis, and the application of its derivatives and medicinal salts in the preparation of prevention and treatment of insomnia, convulsions, epilepsy and other diseases, belonging to the field of medical technology. Background Art

[0002] At present, the sedative and hypnotic drugs used in clinical practice mainly include GABAa receptor agonists represented by diazepam, oxazepam, zolpidem, eszopiclone, zopiclone, etc., melatonin receptor agonists represented by ramelteon and tasimelteon, orexin receptor antagonists represented by suvorexant developed by Merck and launched in the United States in 2014, and antidepressants with sedative and hypnotic effects. However, most of the drugs currently used in clinical practice have certain side effects, such as benzodiazepines. This type of drug is prone to tolerance, dependence and withdrawal symptoms, etc. Therefore, there is a lack of effective sedatives and hypnotic drugs with few side effects, and the market demand is urgent.

[0003] The diversity of natural product structures and biological activities and their biological adaptability have long been the research focus and hotspot of medicinal chemistry, biology, organic chemistry and other disciplines. In particular, the research on active ingredients in traditional Chinese herbal medicines and folk medicinal biological resources has received much attention. Recent studies have found that many natural products have potential sedative and hypnotic effects. For example, matrine and oxymatrine can exert sedative effects by increasing neurotransmitters in the brain, rhynchophylline can enhance the sedative and hypnotic effects of sodium pentobarbital, scopolamine can enhance the hypnotic effects of ketamine hydrochloride, the volatile oil in Acorus tatarinowii is its main sedative and hypnotic active ingredient, and paeoniflorin can prolong the slow-wave sleep time of normal rats and restore caffeine-induced insomnia rats to normal [1]. The discovery of sedative and hypnotic active ingredients from natural sources provides a lead compound for the development of clinical drugs for the treatment of insomnia. The compound in this application is a new natural product with hypnotic effect isolated from the traditional Chinese medicine Angelica sinensis.

[0004] The traditional Chinese medicine Angelica sinensis is the dried root of the plant Angelica sinensis (Oliv.) Diels of the Umbelliferae family, and has the effects of enriching blood and promoting blood circulation, regulating menstruation and relieving pain, and moistening the intestines and promoting defecation [2]. The whole of Angelica sinensis is also called "complete Angelica sinensis", the root head is called "Angelica head", the main root is called "Angelica body", and the lateral roots and the tips of the lateral roots are called "Angelica tail". Different parts have different effects [2]. In China, "complete Angelica sinensis" is often used as a tonic medicine to be eaten with meat or cooked in soup; in Europe and North America, it is usually used as a food additive [3, 4]. The main producing area of Angelica sinensis in China is Gansu. In addition to meeting the applications such as food and medicine, it is also a local cash crop.

[0005] Pharmacological studies have shown that Angelica sinensis extracts and their chemical components have a variety of pharmacological activities, including anti-inflammatory, antispasmodic, antioxidant, antifibrotic, neuroprotective, and prevention and treatment of cardiovascular and cerebrovascular diseases, etc. [2-4]. At present, about 100 chemical components have been identified from Angelica sinensis extracts, including phthalides, phenylpropanoids, lignans, coumarins, alkynes, terpenes, steroids, alkaloids, fatty acids, and polysaccharides, etc. [3-11]. The main components ligustilide, ferulic acid, and polysaccharides are considered to be the main active components, however, the contents of these components are significantly affected by the processing and extraction methods [3-5]. At present, there are few studies on "Angelica head" in the literature, and the research on chemical components mainly focuses on ethanol or methanol extracts, which is inconsistent with the traditional Chinese medicine method of using water decoction [6-13]. Therefore, the project team carried out the study on the chemical components of the water extract of "Angelica head", and cooperated with the pharmacological activity tracking evaluation to obtain the new lignan compounds with significant sedative and hypnotic effects in this application.

[0006] References:

[0007] [1] Zhang Ying; Ma Mixia; Wang Gang; et al. Comparative Chemistry 2018, 2, 54–79.

[0008] [2] Pharmacopoeia Commission of the People's Republic of China. Pharmacopoeia of the People's Republic of China (Volume I) 2020 Edition [M]. Beijing: China Medical Science and Technology Press, 2020: 139.

[0009] [3] I.L.I.Hook, J Ethnopharm, 2014, 152, 1-13.

[0010] [4] W.L.Wei, R.Zeng, C.M.Gu, Y.Qu, L.F.Hunag, J Ethnopharm, 2016, 190, 116-141.

[0011] [5] J.P.Ma, Z.B.Guo, L.Jin, Y.D.Li, Chin J Nat Med, 2015, 13, 241-149.

[0012] [6]L.B.Zhang,J.L.Lv,J.W.Liu,J Nat Prod,2016,79,1857-1861.

[0013] [7]W.Gong,Y.Zhou,X.Li,et al.,Molecules,2016,21,549.

[0014] [8]L.Zhang,J.Lv,Chem Nat Comp,2018,54,13-17.

[0015] [9]J.L.Lv,L.B.Zhang,L.M.Gao,Fitoterapia,2018,129,102-107.

[0016]

[10] J.Zou,G.D.Chen,H.Zhao,et al.,Org Lett,2018,20,884-887.

[0017]

[11] J.Zou,G.D.Chen,H.Zhao,et al.,Chem Commun,2019,55,6221-6224.

[0018]

[12] B.Sheng,Y.Vo,P.Lan,et al.,Org Lett,2019,21,6295-6299.

[0019]

[13] K.Duric,Y.Liu,S.N.Chen,J Nat Prod,2019,82,2400-2408. Summary of the Invention

[0020] The technical problem to be solved by the present invention is to provide a new class of drugs with sedative and hypnotic effects.

[0021] To solve the technical problems of the present invention, the present invention provides the following technical solutions:

[0022] The first aspect of the technical solution of the present invention is to provide a new lignan and its derivatives represented by the general formula (I).

[0023] Specifically, compounds represented by the general formula (I) and pharmaceutically acceptable salts thereof are provided:

[0024]

[0025] Among them, the chiral bonds of the compound can be R or S or racemic.,

[0026] R1 is selected from H, OH, OCH3, OCH2CH3, NH2, NHCH3;

[0027] R2 is selected from H, CH3, CH2CH3;

[0028] R3 is selected from H, CH3, CH2CH3.

[0029] The more preferred compounds of the present invention are selected from the following group:

[0030]

[0031] The second aspect of the technical solution of the present invention is to provide a preparation method of the compound described in the first aspect.

[0032] After 97 kg of dried Angelica sinensis roots are pulverized, they are extracted with distilled water, 300 L of water each time, and heated for 30 min after boiling. The extraction is carried out three times, and the filtrates are combined. The solvent is recovered under reduced pressure to obtain an extract, which is separated by macroporous resin column chromatography, and eluted successively with water: ethanol at a gradient of 1:0 to 0:1. TLC or HPLC is used for monitoring, and the solvent is recovered under reduced pressure to obtain the corresponding elution fractions. The 50% ethanol fraction is separated by MCI resin, and eluted successively with water: ethanol at a gradient of 1:0 to 0:1. TLC or HPLC is used for monitoring and the same components are combined; among them, the 95% ethanol elution fraction of the MCI column chromatography is further separated by silica gel column chromatography, and eluted with petroleum ether-ethyl acetate at a gradient of 1:0 to 0:1. TLC or HPLC is used for monitoring and the same components are combined to obtain components C1-C20; sub-component C18 is eluted with Sephadex LH-20 with an isocratic eluent of petroleum ether-chloroform-methanol at 5:5:1, and then separated by reversed-phase HPLC with 45% methanol-water to obtain compound 1; compound 1 is further separated by chiral HPLC with an IG chiral column and n-hexane-ethanol at 3:1 to obtain (+)-1 and (-)-1.

[0033] The third aspect of the technical solution of the present invention is to provide a pharmaceutical composition, which includes a neolignan represented by the general formula (I) as an active ingredient and carriers commonly used in the pharmaceutical field.

[0034] Generally, the pharmaceutical composition of the present invention contains 0.1-95% by weight of the compound of the present invention.

[0035] The pharmaceutical composition of the compound of the present invention can be prepared according to methods well known in the art. For this purpose, if necessary, the compound of the present invention can be combined with one or more solid or liquid pharmaceutical excipients and / or adjuvants to form a suitable administration form or dosage form for use as a human or veterinary drug.

[0036] The compounds of the present invention or pharmaceutical compositions containing the same can be administered in unit dosage forms, and the administration routes can be enteral or parenteral, such as oral, intramuscular, subcutaneous, nasal, buccal mucosa, dermal, peritoneal or rectal, etc., with oral administration being preferred.

[0037] The administration route of the compounds of the present invention or pharmaceutical compositions containing the same can be injection. Injection includes intravenous injection, intramuscular injection, subcutaneous injection and intradermal injection, etc.

[0038] The dosage forms can be liquid dosage forms or solid dosage forms. For example, liquid dosage forms can be true solution types, colloid types, particulate dosage forms, emulsion dosage forms, suspension dosage forms. Other dosage forms such as tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, suppositories, freeze-dried powder injections, etc.

[0039] The extracts or compounds of the present invention can be made into conventional preparations, or sustained-release preparations, controlled-release preparations, targeted preparations and various particulate drug delivery systems.

[0040] In order to make the unit dosage forms into tablets, various carriers well-known in the art can be widely used. Examples of carriers 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, etc.; wetting agents and binders, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, acacia mucilage, gelatin mucilage, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone, etc.; disintegrants, such as dry starch, alginate, agar powder, laminarin, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene sorbitan fatty acid ester, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors, such as sucrose, glyceryl tristearate, cocoa butter, hydrogenated oil, etc.; absorption promoters, such as quaternary ammonium salts, sodium dodecyl sulfate, etc.; lubricants, such as talc, silica, corn starch, stearates, boric acid, liquid paraffin, polyethylene glycol, etc. Tablets can also be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets.

[0041] For example, in order to make the dosage units into pills, various carriers well-known in the art can be widely used. Examples of carriers are, for example, diluents and absorbents, such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Gelucire, kaolin, talc, etc.; binders, such as acacia, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or batter, etc.; disintegrants, such as agar powder, dry starch, alginate, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, etc.

[0042] For example, in order to make the administration unit into a capsule, the active ingredient, the extract or compound of the present invention, is mixed with the various carriers described above, and the resulting mixture is placed in a hard gelatin capsule or a soft capsule. The active ingredient, the compound of the present invention, can also be made into a microcapsule, suspended in an aqueous medium to form a suspension, and can also be filled into a hard capsule or made into an injection for use.

[0043] For example, the extract or compound of the present invention is made into a preparation for injection, such as a solution, a suspension solution, an emulsion, a freeze-dried powder injection. Such a preparation can be aqueous or non-aqueous, and can contain one and / or more pharmacodynamically acceptable carriers, diluents, binders, lubricants, preservatives, surfactants or dispersants. For example, the diluent can be selected from water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxidized isostearyl alcohol, polyoxyethylene sorbitol fatty acid ester, etc. In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerol can be added to the injection preparation. In addition, conventional solubilizers, buffers, pH regulators, etc. can also be added. These excipients are commonly used in the art.

[0044] In addition, if necessary, coloring agents, preservatives, fragrances, flavoring agents, sweetening agents or other materials can also be added to the pharmaceutical preparation.

[0045] To achieve the purpose of medication and enhance the therapeutic effect, the drugs or pharmaceutical compositions of the present invention can be administered by any known administration method.

[0046] The dosage of the compound and pharmaceutical composition of the present invention depends on many factors, such as the nature and severity of the disease to be prevented or treated, the gender, age, weight, personality and individual response of the patient or animal, the administration route, the number of administrations, the treatment purpose. Therefore, the therapeutic dose of the present invention can vary within a wide range. Generally speaking, the dosage of the Chinese medicine components in the present invention is well known to those skilled in the art. It can be appropriately adjusted according to the actual amount of the drug contained in the final preparation of the compound composition of the present invention to meet the requirement of its therapeutically effective amount and achieve the preventive or therapeutic purpose of the present invention. The appropriate daily dosage range of the compound of the present invention, the dosage of the extract or compound of the present invention is 0.001 - 150 mg / kg body weight, preferably 0.01 - 100 mg / kg body weight, more preferably 0.01 - 60 mg / kg body weight, and most preferably 0.1 - 10 mg / kg body weight. The above dosage can be administered in a single dose form or divided into several, such as two, three or four dose forms, which is limited by the clinical experience of the administering doctor and the dosing regimen including the use of other treatment means.

[0047] The total dose required for each treatment can be divided into multiple doses or administered as a single dose. The compounds and compositions of the present invention can be taken alone, or used in combination with other therapeutic drugs or symptomatic drugs and the dosage can be adjusted.

[0048] The fourth aspect of the technical solution of the present invention is to provide the application of the new lignan compounds shown in the general formula (I) in the preparation of drugs for treating diseases such as insomnia, convulsion, epilepsy, etc.

[0049] The present invention also relates to the application of angelica extract in the preparation of drugs for preventing or treating mental diseases such as insomnia, convulsion, epilepsy, anxiety, depression, etc.

[0050] The inventors found that the compound (I) of the present invention and its pharmaceutically acceptable salts have a certain sedative and hypnotic effect. Therefore, the compound (I) of the present invention and its pharmaceutically acceptable salts on the other hand relate to a method for treating and improving sleep-related diseases. The method includes administering a therapeutically effective amount of the compound of formula I or its pharmaceutically acceptable salt or its pharmaceutical composition to a patient in need of treatment.

[0051] The present invention shows that the compound (I) has a good sedative and hypnotic effect at the whole animal level. The compound (I) or its pharmaceutically acceptable salt has not been reported in public.

[0052] Beneficial technical effects

[0053] During the research on the active ingredients of the traditional Chinese medicine angelica, the inventors of the present invention isolated new lignan compounds 1, (+)-1 and (-)-1 from angelica by an activity-tracking method. Among them, compound 1 is a racemate of (+)-1 and (-)-1. Through the combined subthreshold dose of sodium pentobarbital-induced sleep experiment in mice, the activity of these compounds was evaluated. The results showed that the compounds (+)-1 and (-)-1 have significant sedative and hypnotic effects at the whole animal level. They are new leading compounds with value in the research and development of sedative and hypnotic drugs. Description of the drawings

[0054] Figure 1 Flow chart for the separation of compounds 1, (+)-1 and (-)-1

[0055] Figure 2 Structural formula diagram of the positive drug YZG-331 Detailed implementation manners

[0056] The following experimental examples can further illustrate the present invention, but do not limit the present invention in any way.

[0057] Example 1. Compounds 1, (+)-1 and (-)-1 are new lignan compounds extracted, separated and purified from angelica. The separation and purification process is as follows:

[0058] After pulverizing 97 kg of dried Angelica sinensis heads, they were extracted with distilled water. Each time, 300 L of water was used, and after boiling, heating was continued for 30 min. Extraction was carried out three times, and the filtrates were combined. The solvent was recovered under reduced pressure to obtain an extract, which was separated by HP20 macroporous resin column chromatography. It was eluted successively with water (650 L), 50% ethanol (500 L), and 95% ethanol (250 L), and the solvent was recovered under reduced pressure to obtain the corresponding elution fractions (A - C). Among them, component B (1.6 kg) was suspended in water and treated with MCI resin of model CHP 20. It was eluted successively with water, 30% ethanol, 50% ethanol, and 95% ethanol to obtain subcomponents B1 - B4. B4 (10 g) was combined with C (135 g), and then separated by normal-phase silica gel column chromatography with a gradient elution of petroleum ether - ethyl acetate from 1:0 to 0:1. The same components were monitored by TLC or HPLC and combined to obtain components C1 - C20. Subcomponent C18 (2.4 g) was eluted isocratically with Sephadex LH-20, petroleum ether - chloroform - methanol at 5:5:1. The same components were monitored by TLC or HPLC and combined to obtain components C18-1 - C18-7. C18-7 (114 mg) was further separated by reverse-phase HPLC with 45% methanol - water as the mobile phase to prepare subcomponents C18-7-1 - C18-7-2. Among them, C18-7-2 (25.6 mg) was further separated by reverse-phase HPLC with 30% acetonitrile - water as the mobile phase to obtain compound 1 (20.3 mg). Compound 1 was further separated by chiral HPLC with an IG chiral column and n-hexane - ethanol at 3:1 as the mobile phase to obtain (+)-1 (7.8 mg) and (-)-1 (7.8 mg)

[0059] Compound 1: White amorphous powder; 0 (c 0.10, CH3OH); IRν max 3348, 2931, 2850, 1673, 1611, 1575, 1514, 1449, 1427, 1374, 1328, 1276, 1126, 1034, 933, 876, 853, 822, 775, 678, 600, 570 cm -1 ; (+)-HR-ESIMS m / z 355.1182 [M–H] – (calcd. for C 20 H 19 O6, 355.1187).

[0060] Compound (+)-1: +105.1 (c 0.78, MeOH); CD(CH3OH)λ max(Δε) 213.0 (–0.52), 222.5 (–1.74), 235.0 (+1.06), 251.5 (–2.01), 293.5 (+2.85), 300.0 (+2.70), 321.5 (+4.93) nm.

[0061] Compound (–)-1: –102.6 (c 0.78, MeOH); CD(CH3OH) λ max (Δε) 206.0 (+6.31), 216.0 (+3.50), 222.0 (+3.85), 235.5 (–0.40), 251.0 (+3.00), 293.0 (–3.33), 300.5 (–3.04), 321.5 (–6.00) nm.

[0062] Experimental Example 1, Sedative and Hypnotic Effects, Effects of Compounds (+)-1 and (–)-1 on Synergistic Pentobarbital Sodium-Induced Sleep in Mice

[0063] Test Animals: ICR mice, male, weighing 18 - 22 g, purchased from the Experimental Animal Center of the Chinese Academy of Medical Sciences. Animals were acclimated to the environment for at least 3 days before the experiment, and the room temperature was maintained at 25 ± 1 °C, with free access to food and water. All treatments of animals were in accordance with the requirements of the Ethics Committee of the International Association for the Study of Pain.

[0064] Test Compounds: The test substances were compounds (+)-1 and (–)-1, and the positive drug was YZG-331.

[0065] Method: The test substances or solvent control were administered to mice by gavage or intraperitoneal injection. After 15 min, pentobarbital sodium (32 mg / kg) was injected intraperitoneally and timing was started. The disappearance time and recovery time of the righting reflex in mice were observed and recorded. In this experiment, the disappearance of the righting reflex > 1 min was used as the sleep onset criterion, N = 8.

[0066] Experimental Results: YZG-331 (10 mg / kg, ig) could significantly increase the sleep onset rate of mice induced by subthreshold dose of pentobarbital sodium, indicating the reliability of this experimental system. Compounds (+)-1 and (–)-1 could both significantly increase the sleep onset rate of mice induced by subthreshold pentobarbital sodium (the sleep onset rate was 87.5% for both). The specific results are shown in Table 1.

[0067] Table 1. Evaluation Results of Compounds (+)-1 and (–)-1

[0068]

[0069] Fisher's exact test (compared with the solvent control group).

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof:

2. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the compound of claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient.

3. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 2 in the preparation of a medicament for preventing or treating insomnia.

4. The application according to claim 3, characterized in that, The medicament for preventing or treating sleep-related diseases has an effective sedative and hypnotic effect.

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