Preparation method and application of a caffeic acid derivative from Scutellaria baicalensis

Abstract

The present invention discloses the use of dibenzo[b,f]oxepin ring compounds or pharmaceutical salts thereof shown in formula II in the preparation of drugs for upregulating KLF2 expression. The present invention also discloses the use of dibenzo[b,f]oxepin ring compounds or pharmaceutical salts thereof shown in formula II in the preparation of drugs for treating cardiovascular diseases or anti-inflammatory drugs. The inventors used the KLF2 screening model to evaluate its cardiovascular activity: using KLF2‑COS7 cells as experimental subjects, it was first discovered that dibenzo[b,f]oxepin ring compounds or pharmaceutical salts thereof have the activity of significantly upregulating the expression of target KLF2 related to atherosclerosis, anti-inflammation, etc., and have good application prospects in the treatment of atherosclerosis-related diseases. The present invention also discloses a method for preparing the dibenzo[b,f]oxepin ring compounds. #imgabs0#

Description

Technical Field

[0001] The present invention belongs to the technical field of natural medicines, and relates to a method for preparing a caffeic acid derivative, a dibenzo[b,f]oxepin ring compound moldavica acid A, from plants, especially Lamiaceae plants, including, but not limited to, cynanchum cynanchum, and use of the dibenzo[b,f]oxepin ring compound moldavica acid A or a pharmaceutically acceptable salt thereof in preparing drugs for cardiovascular diseases such as atherosclerosis, thrombosis, coronary heart disease, angina pectoris, myocardial infarction or stroke. Background Art

[0002] In recent years, with the improvement of people's material living standards, the incidence of cardiovascular and cerebrovascular diseases has shown a clear upward trend. The number of deaths from cardiovascular and cerebrovascular diseases accounts for 1 / 3 of the global population deaths. Cardiovascular diseases have become the leading cause of death among urban and rural residents in my country, bringing a great economic burden to society and residents. Among them, atherosclerotic diseases have become the number one "killer" of humans. Atherosclerosis (AS) is a chronic inflammatory disease and the common pathophysiological basis of ischemic cardiovascular and cerebrovascular diseases (such as coronary heart disease, angina pectoris, myocardial infarction, stroke, etc.). At present, atherosclerosis in my country shows a high incidence and a trend of younger age.

[0003] Although there are many kinds of drugs for AS treatment, each drug has certain limitations, which limits the clinical application of drugs to a certain extent. KLF2 (Krueppel-like factor 2) is a member of the zinc finger family of DNA binding transcription factors. Because it is highly expressed in the lung, it is also called lung kruupel-like transcription factor 2 (LKLF2). Its C-terminus is a highly conserved zinc finger domain, which is convenient for binding to specific DNA sequences and nuclear localization. The N-terminus is a highly dispersed non-DNA binding region, which plays a transcriptional activation or transcriptional inhibition role. KLF2 plays a very important regulatory role as a key "molecular switch" in vascular function and disease. KLF2 has multiple functions to negatively regulate the expression of inflammatory factors and thus play an anti-inflammatory role. It also has the functions of maintaining the structure and function of endothelial cells, preventing thrombosis and anti-oxidation, and is closely related to atherosclerosis.

[0004] Natural products have always been an important source of innovative drugs. Dracocephalum Moldavica L., a plant of the genus Dracocephalum in the Lamiaceae family, is a traditional medicine used by the Uygur ethnic group. It has the effects of benefiting the heart and brain, protecting the liver and stomach, and opening up blockages in the brain. It has a long history of being used to treat diseases such as heart disease, hypertension, and headaches, and has a definite effect. In recent years, with the continuous advancement of research on Dracocephalum Moldavica, its effects on coronary heart disease, atherosclerosis, myocardial ischemia, and other diseases have gradually attracted attention. Summary of the invention

[0005] The purpose of the present invention is to separate and purify dibenzo[b,f]oxepin ring compounds from plants, especially the Lamiaceae plant, the KLF2 screening model, to broaden the channels for obtaining the dibenzo[b,f]oxepin ring compounds. The inventors use the KLF2 screening model to evaluate the cardiovascular activity of the compounds: using KLF2-COS7 cells as experimental objects, the KLF2 extracts, extraction fractions and dibenzo[b,f]oxepin ring compounds are screened for up-regulating the expression activity of KLF2. It is found for the first time that the dibenzo[b,f]oxepin ring compounds or their pharmaceutical salts have the activity of significantly up-regulating the expression of target KLF2 related to atherosclerosis, anti-inflammation and the like, and have good application prospects in the treatment of atherosclerosis-related diseases, and are expected to become a new generation of clinical drugs for the treatment of cardiovascular diseases.

[0006] The purpose of the present invention is to provide a dibenzo[b,f]oxepin ring compound or a pharmaceutically acceptable salt thereof as shown in formula II for use in preparing a drug for upregulating KLF2 expression.

[0007]

[0008] The molecular formula of the compound moldavica acid A is: 17 H 12 O6, molecular weight: 312.

[0009] Another object of the present invention is to provide the use of the dibenzo[b,f]oxepin ring compound (denoted as moldavica acid A) or its pharmaceutically acceptable salt shown in formula II in the preparation of drugs for treating cardiovascular diseases or anti-inflammatory drugs.

[0010] The cardiovascular diseases include atherosclerosis, thrombosis, coronary heart disease, angina pectoris, myocardial infarction or stroke.

[0011] The pharmaceutically acceptable salts of the dibenzo[b,f]oxepin ring compounds include salts formed by dibenzo[b,f]oxepin ring compounds and inorganic acids, organic acids, amino acids or sulfonic acids.

[0012] The inorganic acid is hydrochloric acid or sulfuric acid; the organic acid is acetic acid, trifluoroacetic acid, citric acid, maleic acid, oxalic acid, succinic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, ascorbic acid or malic acid; the amino acid is alanine, aspartic acid or lysine; and the sulfonic acid is methanesulfonic acid or p-toluenesulfonic acid.

[0013] Another object of the present invention is to provide a pharmaceutical composition, which is prepared by using the dibenzo[b,f]oxepin ring compound or its pharmaceutically acceptable salt as the active ingredient or main effective ingredient and one or more pharmaceutically acceptable carriers.

[0014] Another object of the present invention is to provide use of the pharmaceutical composition in preparing a drug for up-regulating KLF2 expression.

[0015] Another object of the present invention is to provide the use of the pharmaceutical composition in preparing drugs for treating cardiovascular diseases or anti-inflammatory drugs.

[0016] The dibenzo[b,f]oxepin ring compound or its pharmaceutically acceptable salt shown in formula II can also be combined with a drug known to upregulate KLF2 expression or treat cardiovascular diseases to form a compound preparation.

[0017] The pharmaceutical composition contains 0.1 to 99.9% by weight of the dibenzo[b,f]oxepin ring compound or its pharmaceutically acceptable salt represented by formula II, and 0.1 to 99.9% by weight of the pharmaceutically acceptable carrier.

[0018] The pharmaceutical composition is in the form of a preparation suitable for pharmaceutical use.

[0019] The preparation is a tablet, capsule, granule, pill, powder, ointment, suspension, injection, powder injection, suppository, cream, drop or patch. The tablet is a sugar-coated tablet, a film-coated tablet, an enteric-coated tablet or a sustained-release tablet; the capsule is a hard capsule, a soft capsule or a sustained-release capsule; and the powder injection is a freeze-dried powder injection.

[0020] The pharmaceutical composition of the present invention, as a preparation form, contains an effective amount of 0.1 to 1000 mg of the dibenzo[b,f]oxepin ring compound or a pharmaceutically acceptable salt thereof shown in Formula II in each dose. Although the amount of active ingredient contained in the dosage unit form can vary, it is generally adjusted within the range of 1 to 800 mg according to the efficacy of the selected active ingredient. Those skilled in the art can determine the preferred dose suitable for a certain situation by conventional methods. Generally, the initial treatment amount is lower than the optimal dose of the active ingredient, and then the dosage is gradually increased until the optimal therapeutic effect is achieved. For treatment needs, the total daily dose can be administered once or in divided doses.

[0021] Each dose refers to each unit of preparation, such as each tablet, each capsule, or it can also refer to the dosage taken each time, such as 100 mg per dose.

[0022] The pharmaceutical composition of the present invention may be prepared into solid or semisolid pharmaceutical preparations in the form of powders, tablets, dispersible powders, capsules, cachets, suppositories and ointments, and may use a solid carrier. The solid carrier is selected from one or more substances selected from diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, expanders, etc., or may be an encapsulating material. In powdered preparations, 5 to 70% of the micronized active ingredient is contained in the carrier. The solid carrier includes magnesium carbonate, magnesium stearate, talc, sucrose, lactose, pectin, dextrin, starch, gelatin, methylcellulose, sodium carboxymethylcellulose, low boiling wax, cocoa butter, etc. Tablets, powders, cachets and capsules represent the most advantageous oral solid preparations due to their ease of administration.

[0023] Liquid preparations of the present invention include solutions, suspensions and emulsions. For example, parenteral injections can be in the form of water or water-propylene glycol solutions, and their isotonicity, pH, etc. are adjusted to be suitable for the physiological conditions of the living body. Liquid preparations can also be made into solutions in polyethylene glycol and aqueous solutions. Oral aqueous solutions can be prepared by dissolving the active ingredient in water and then adding an appropriate amount of coloring agent, flavoring agent, stabilizer and thickening agent. The micronized active ingredient can be dispersed in viscous materials such as natural and synthetic gums, methylcellulose, sodium carboxymethylcellulose and other known suspending agents to prepare aqueous suspensions suitable for oral administration.

[0024] For ease of administration and uniformity of dosage, it is particularly advantageous to formulate the above-mentioned pharmaceutical preparations in dosage unit form. The dosage unit form of the preparation refers to physically separate units suitable for use as single doses, each unit containing a predetermined amount of active ingredient calculated to produce the desired therapeutic effect. Such dosage unit forms can be packaged forms, such as tablets, capsules, or powders in small tubes or vials, or ointments, gels, or creams in tubes or bottles.

[0025] Another object of the present invention is to provide a method for preparing the dibenzo[b,f]oxepin ring compounds, comprising: taking dried whole herb of Scutellaria baicalensis, adding 0-60% ethanol according to a weight-to-volume ratio of 1:3 (kg / L or g / mL) of dried whole herb of Scutellaria baicalensis to 0-60% ethanol, and reflux extraction for 2-3 times to obtain an extract; concentrating the extract until there is no alcohol taste to obtain a suspension, and adding distilled water to obtain a clear solution; subjecting the clear solution to macroporous adsorption resin column chromatography, using water, 30% ethanol, 50% ethanol and 95% ethanol gradient elution to obtain 4 elution sites: a water elution site, a 30% ethanol elution site, a 50% ethanol elution site, and a 95% ethanol elution site; taking the 50% ethanol elution site, extracting it with ethyl acetate 3 times to obtain an ethyl acetate site; subjecting the ethyl acetate extraction site to silica gel column chromatography, Sephadex LH-20 column chromatography, and semi-preparative HPLC to purify the compound shown in formula II.

[0026] Preferably, dry whole herb of Scutellaria baicalensis is taken, 40% ethanol is added according to a weight volume ratio of dry whole herb of Scutellaria baicalensis to 40% ethanol of 1:3 (kg / L or g / mL), and reflux extraction is performed for 3 times.

[0027] The volume ratio of the suspension to distilled water is 1:2.

[0028] The macroporous adsorption resin is HP20 macroporous adsorption resin.

[0029] The filler of the silica gel column chromatography is 200-300 mesh silica gel, and the eluent is dichloromethane:methanol=100:0-0:100 V:V, and 15 fractions are obtained, which are recorded as: XQL-1-XQL-15. Fraction XQL-8 is subjected to Sephadex LH-20 column chromatography.

[0030] The eluent of the Sephadex LH-20 column chromatography was dichloromethane:methanol = 1:1 V:V, and 11 sub-fractions were obtained, which were recorded as XQL-8-1 to XQL-8-11. Sub-fraction XQL-8-10 was subjected to semi-preparative HPLC.

[0031] The chromatographic column of the semi-preparative HPLC is: CAPCELL PAK PFP (particle size 5 μm, inner diameter 10 mm×length 250 mm), the mobile phase is acetonitrile: 0.1% trifluoroacetic acid aqueous solution = 33:67 V:V, and the flow rate is 1.5 ml / min. DETAILED DESCRIPTION

[0032] The embodiments of the present invention are applicable to the preparation of the compound of formula II from plants, but not limited to Scutellaria baicalensis. The following examples are used to help those skilled in the art better understand the technical scheme of the present invention, but do not limit the present invention in any way.

[0033] Example 1

[0034] Extraction: Take 40 kg of dried whole herb of Scutellaria baicalensis, extract it three times with 3 times the amount (120 L) of 40% ethanol under reflux to obtain an extract, concentrate the extract until there is no alcohol taste, and obtain 20 L of suspension; add 40 L of distilled water to the suspension to obtain 60 L of clear solution;

[0035] Isolation and purification of the compound of formula II: the clarified solution was subjected to an HP-20 macroporous adsorption resin column, the unabsorbed solution was discarded, and gradient elution was performed using water, 30% ethanol, 50% ethanol and 95% ethanol to obtain 4 elution portions: a water elution portion, a 30% ethanol elution portion, a 50% ethanol elution portion, and a 95% ethanol elution portion; the 50% ethanol elution portion was extracted three times with ethyl acetate to obtain an ethyl acetate portion (41.1 g); the ethyl acetate extraction portion was subjected to a silica gel (200-300 mesh) column, and gradient elution was performed using dichloromethane:methanol=100:0-0:100 (V:V) as an eluent to obtain 15 fractions (referred to as XQL-1 to XQL-15); XQL-8 was subjected to a Sephadex LH-20 column, and dichloromethane:methanol=1:1 (V:V) as an eluent to obtain 11 Subfractions (denoted as XQL-8-1 to XQL-8-11); subfraction XQL-8-10 was purified by semi-preparative HPLC (chromatographic column: CAPCELL PAK PFP (5 μm, 10 mm×250 mm); flow rate: 1.5 ml / min) with acetonitrile: 0.1% trifluoroacetic acid aqueous solution = 33:67 (V:V) as the mobile phase to obtain compound 2 as a yellow amorphous powder.

[0036] ESI-MS gave a quasi-molecular ion peak of m / z 311.1 [M–H] – , HRESIMS m / z 311.0568, indicating that its molecular composition is C 17 H 12 O6 (calcd for C 17 H 11 O6 311.0556), and the degree of unsaturation is 12. IR spectrum indicates that compound 2 contains hydroxyl group (3250 cm -1 ), carboxylic acid (3000-2500cm -1 , strong and broad absorption peak), conjugated carbonyl (1670cm -1 ), carbon-carbon double bond (1650cm -1 ), benzene ring (1600, 1494, 1451cm -1 ) characteristic absorption peak. 1 The H NMR spectrum showed a 1,3,4,5-tetrasubstituted benzene ring [δ H 7.08 (d, J = 1.8 Hz, H-5) and 6.96 (d, J = 1.8 Hz, H-9)], a 1,2,4,5-tetrasubstituted benzene ring [δ H 6.83 (s, H-7') and 6.96 (d, J = 1.8 Hz, H-4')], a disubstituted trans double bond [δ H6.30 (d, J = 16.2 Hz, H-2) and 7.39 (d, J = 16.2 Hz, H-3)] and a disubstituted cis double bond [δ H 6.58 (d, J = 11.4 Hz, H-2') and 6.50 (d, J = 11.4 Hz, H-1')] proton resonance signals; in addition, there are 3 active [δ H 9.56 (s, OH-6), 9.39 (s, OH-6') and 8.89 (s, OH-7')] proton resonance signals. 13 The C NMR spectrum shows 17 carbon atoms. In addition to the carbon data of the four units shown in the above hydrogen spectrum, there is also a carbonyl resonance signal (δ C 167.6). In order to accurately determine the chemical structure of compound 2, a 2D NMR experiment was performed. 1 H and its corresponding 13 The C resonance signals were assigned one by one (see Table 1). 1 H- 1 The H COSY spectrum shows H-2 / H-3 double bond proton coupling related signals, and the HMBC spectrum shows heteronuclear long-range correlation signals between H-2 and C-1 and C-4, H-3 and C-4, C-5 and C-9, H-5 and C-3, C-6, C-7 and C-9, H-9 and C-3, C-5 and C-7, and OH-6 and C-5, C-6 and C-7. Combined with the chemical shifts of these protons and carbons and the molecular composition, it is shown that the compound contains a 6-OH,7-O,8-disubstituted caffeic acid unit. 1 H- 1The H COSY spectrum shows the double bond proton coupling related signals of H-1' / H-2', and the HMBC spectrum shows the heteronuclear long-range correlation signals of H-2' with C-3', C-4' and C-8', H-5' with C-3', C-4', C-6' and C-7', H-8' with C-2', C-4', C-6' and C-7', OH-6' with C-5', C-6' and C-7', and OH-7' with C-6'. Combined with the chemical shifts of these carbons and protons, it is shown that the compound contains a 4'-O,6',7'-dihydroxy-substituted styrene unit. In addition, in the HMBC spectrum, the correlation signals of H-1' with C-7, C-8 and C-9, H-2' with C-8, and H-9 with C-1' show that the above two units are connected through the C-8–C-1' bond. Finally, combined with the fact that there are only 6 oxygen atoms and 12 unsaturation degrees in the molecular composition of compound 2, it is shown that the compound has another ring. Therefore, the 7-O and 4'-O substitutions in compound 2 should share an oxygen atom, and an ether bond is formed between C-7 and C-4', which confirms that the compound contains a dibenzo[b,f]oxepin ring system, thereby determining that compound 2 is (E)-3-(4,7,8-trihydroxydibenzo[b,f]oxepin ring-2)-acrylic acid, and it is named moldavica acidA. According to the scifinder database search, compound 2 has been synthesized in the Chinese patent CN107936034A, but the compound has not been analyzed and attributed, nor has the pharmacological activity of the compound been evaluated. Therefore, compound 2 is a new natural product, obtained from nature for the first time, and its structure is shown in formula II:

[0037]

[0038] Table 1. Compound 2 1 H NMR and 13 C NMR data (DMSO-d 6, 600MHz)

[0039]

[0040] Example 2 Test of compound 2's ability to upregulate KLF2 expression

[0041] The KLF2 screening model was used, COS7 cells transfected with KLF2 plasmid (KLF2-COS7) were used as experimental objects, luciferase (Bright-Lite Luciferase Assay System, Nanjing Novogene Biotech Co., Ltd., batch number: 7E512L1) was used as the reporter gene, and the luminescence intensity of the reaction between luciferase and substrate was detected using the EnVision multifunctional microplate reader, which was expressed in relative luminescence units (RLU). During the experiment, a blank control well (without adding test sample) was set, and the ratio of the fluorescence intensity of the drug-added well to the blank control well was the multiple value of the drug upregulating KLF2.

[0042] The specific experimental method is as follows: COS7 cells in the logarithmic growth phase were digested, diluted with 10% fetal bovine serum DMEM medium, and the cells were counted to 5 × 10 5 / ml, and transiently transfected with KLF2 plasmid, inoculated in 96-well plates, 150μL / well, after 4-6h of cell attachment, replaced with 5% fetal bovine serum DMEM medium 200μL / well, and then added 1μL of compound 2 dissolved in DMSO, the concentration of compound 2 in the drug-dosing well was 100μM, and tested after 18-24h. Compounds with an upregulation factor greater than 1.5 were identified as having the activity of upregulating KLF2, and compound 2 had an upregulation factor greater than 2. Continue to determine its EC 50 : Diluted to 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78 and 0.078 μM gradient concentrations with 5% fetal bovine serum DMEM medium, 200 μL / well, and EC was prepared using GraphPadPrism 8 software after detection. 50 curve.

[0043] Experimental results: In the KLF2 screening model, compound 2 has a significant upregulation effect on KLF2. At a concentration of 100 μM, it can upregulate the expression of KLF2 by 10.6 times. Its EC 50 It is 23.0μM.

Claims

1. A method for preparing a dibenzo[b,f]oxepin ring compound of formula II, characterized in that: include: Take the dried whole herb of Scutellaria baicalensis, add 40% ethanol according to the weight volume ratio of the dried whole herb of Scutellaria baicalensis to 40% ethanol of 1:3 kg / L or g / mL, reflux and extract 3 times to obtain an extract; The extract is concentrated until there is no alcohol taste to obtain a suspension, and distilled water is added to obtain a clear solution; the clear solution is subjected to macroporous adsorption resin column chromatography, and water, 30% ethanol, 50% ethanol and 95% ethanol are used for gradient elution to obtain 4 elution parts: a water elution part, a 30% ethanol elution part, a 50% ethanol elution part, and a 95% ethanol elution part; the 50% ethanol elution part is extracted with ethyl acetate three times to obtain an ethyl acetate part; the ethyl acetate extraction part is subjected to silica gel column chromatography, Sephadex LH-20 column chromatography, and semi-preparative HPLC to purify the compound shown in formula II; The eluent of the silica gel column chromatography is dichloromethane:methanol=100:0-0:100V:V; the eluent of the Sephadex LH-20 column chromatography is dichloromethane:methanol=1:1V:V; the chromatographic column of the semi-preparative HPLC is: CAPCELL PAKPFP, 5μm, 10mm×250mm, the mobile phase is acetonitrile:0.1% trifluoroacetic acid water=33:67V:V, and the flow rate is 1.5ml / min;

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