CN114533719B - Application of abietane diterpenoid compound in preparation of anti-inflammatory drugs - Google Patents

Application of abietane diterpenoid compound in preparation of anti-inflammatory drugs Download PDF

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CN114533719B
CN114533719B CN202011291033.8A CN202011291033A CN114533719B CN 114533719 B CN114533719 B CN 114533719B CN 202011291033 A CN202011291033 A CN 202011291033A CN 114533719 B CN114533719 B CN 114533719B
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compound
abietane
cells
delta
abietane diterpenoid
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CN114533719A (en
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胡晓
聂岚岚
颜仁杰
黄春跃
王玲玲
马梦洁
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Shanghai Institute of Pharmaceutical Industry
China State Institute of Pharmaceutical Industry
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Shanghai Institute of Pharmaceutical Industry
China State Institute of Pharmaceutical Industry
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/04—Drugs for disorders of the respiratory system for throat disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/16—Otologicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/07—Optical isomers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention discloses application of abietyl diterpenoid compounds in preparing anti-inflammatory medicaments, which are shown in a formula I, can effectively inhibit the content of TNF-alpha, IL-1 beta and/or NO in RAW264.7 cells and BV2 cells, have obvious anti-inflammatory activity, and can be used for preparing medicaments for preventing, treating and/or relieving inflammatory diseases.

Description

Application of abietane diterpenoid compound in preparation of anti-inflammatory drugs
Technical Field
The invention relates to application of abietane diterpenoid compounds in preparation of anti-inflammatory drugs.
Background
Inflammation refers to the defensive response of living tissue with vascular system to damaging factors. The central link in the inflammatory process is the vascular response, which is mainly manifested as redness, swelling, heat, pain and dysfunction. Inflammation is associated with the pathogenesis of a variety of diseases including cardiovascular and cerebrovascular diseases, diabetes, rheumatoid arthritis, and the like.
The herb Dracocephalum moldavica (Dracocephalum moldavica L.) is a plant of the genus Dracocephalum of the family Labiatae, also known as Badirk Ji Buya, which is pungent, bitter and cool in taste. Has effects in clearing lung-heat, relieving exterior syndrome, cooling liver, stopping bleeding, inducing resuscitation, relieving pain, and removing toxic substances. The abietane diterpenoid components are firstly separated from the moldavica dragonhead, the biological activity of the compound is mainly divided into two types, and the compound is used as a defensive component in a plant body, so that the compound has the effects of preventing insects from feeding, resisting plant pathogenic bacteria and the like; the other group is used as medicinal active ingredient, and has antiulcer, antitumor, antimalarial, antibacterial and antiviral activities.
Disclosure of Invention
The invention aims to solve the technical problem of providing application of abietane diterpenoid compounds in preparation of drugs for preventing, treating and/or relieving inflammatory diseases. The abietane diterpenoid compound has anti-inflammatory activity.
The technical problems are solved by the following technical scheme.
The invention provides an application of abietane diterpenoid compound shown in formula I or pharmaceutically acceptable salt thereof in preparing medicines for preventing, treating and/or relieving inflammatory diseases,
wherein R is 1 Methyl or methoxy;
R 2 is hydroxyl;
alternatively, R 1 And R is 2 Together forming a carbonyl group;
is a single bond or a double bond.
In some embodiments, the abietane diterpenoid compound represented by formula I has any one of the following structures:
wherein R is 1 Methyl or methoxy;
R 2 is hydroxyl;
alternatively, R 1 And R is 2 Together forming a carbonyl group.
In some embodiments, the abietane diterpenoid compound represented by formula I has any one of the following structures:
wherein R is 1 Methyl or methoxy;
R 2 is hydroxyl;
alternatively, R 1 And R is 2 Together forming a carbonyl group.
In some embodiments, a rosin alkane diterpenoid compound as described in any one of the preceding embodiments, R 1 Is methyl.
In some embodiments, a rosin alkane diterpenoid compound as described in any one of the preceding embodiments, R 1 Is methoxy.
In some embodiments, a rosin alkane diterpenoid compound as described in any one of the preceding embodiments, R 1 And R is 2 Together forming a carbonyl group.
Preferably, the compounds of formula I include, but are not limited to, the following:
the invention also provides application of the abietane diterpenoid compound shown in the formula I or pharmaceutically acceptable salt thereof in preparation of medicines.
The present invention also provides a pharmaceutical composition comprising:
(i) The abietane diterpenoid compound shown in the formula I or pharmaceutically acceptable salt thereof; and
(ii) At least one pharmaceutical excipient.
The present invention also provides a pharmaceutical composition comprising:
(i) The abietane diterpenoid compound shown in the formula I or pharmaceutically acceptable salt thereof;
(ii) A non-steroidal anti-inflammatory component; and
(iii) At least one pharmaceutical excipient.
The pharmaceutical composition is useful for preventing, treating and/or alleviating inflammatory disorders.
The non-steroidal anti-inflammatory component is an anti-inflammatory component which does not contain a steroidal structure, and is preferably one or more of indomethacin, aspirin and quercetin. The concentration ratio of the abietyl diterpenoid compound shown in the formula I or pharmaceutically acceptable salt and the nonsteroidal anti-inflammatory component is preferably (10-30): 1. The choice of the pharmaceutical excipients varies depending on the route of administration and the nature of the action.
The pharmaceutical excipients are generally one or more compatible solid or liquid filler or gel substances and are suitable for human use and must be of sufficient purity and sufficiently low toxicity. By compatibility is meant that the components of the composition are able to blend with the compound and with each other without significantly reducing the efficacy of the compound. The pharmaceutical adjuvant can be cellulose and its derivatives (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate), gelatin, pulvis Talci, solid lubricant (such as stearic acid, magnesium stearate), calcium sulfate, and plantVegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerin, mannitol, sorbitol), emulsifiers (e.g.) Wetting agents (e.g., sodium lauryl sulfate), coloring agents, flavoring agents, stabilizing agents, antioxidants, preservatives, pyrogen-free water.
The invention also provides a method for inhibiting the secretion of TNF-alpha, IL-1 beta and/or NO by a cell under in vitro or ex vivo conditions, comprising the steps of: contacting the abietane diterpenoid compound shown in the formula I or pharmaceutically acceptable salt thereof with cells.
In a certain embodiment, the cells may be conventional in the art, preferably RAW264.7 cells and BV2 cells.
The pharmaceutical compositions of the present invention may be prepared in accordance with the disclosure using any method known to those of skill in the art. For example, conventional mixing, dissolving, granulating, emulsifying, levigating, encapsulating, entrapping or lyophilizing processes.
The mode of administration of the pharmaceutical composition of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration, with the preferred mode of administration being oral administration.
Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is admixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) Fillers or compatibilizers, such as starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) Binders such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, such as glycerin; (d) Disintegrants, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) a slow solvent, such as paraffin; (f) an absorption accelerator, such as a quaternary amine compound; (g) Wetting agents, such as cetyl alcohol and glycerol monostearate; (h) an adsorbent, such as kaolin; and (i) a lubricant, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.
Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds in such compositions may be released in a delayed manner in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. The active compound may also be in the form of microcapsules with one or more of the above excipients, if desired.
Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils which can be one or more of cottonseed, groundnut, corn germ, olive, castor and sesame oils. In addition to the inert diluents, the pharmaceutical compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar-agar or mixtures of these substances.
Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.
Dosage forms of the compounds of the present invention for topical administration include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants which may be required if necessary.
Unless otherwise specified, all technical and scientific terms used herein have the standard meaning of the art to which the claimed subject matter belongs. In case there are multiple definitions for a term, the definitions herein control.
"inflammatory disease" according to the present invention refers to the defensive response of living tissue with blood vessels to damaging factors, usually manifested as redness, swelling, heat, pain and dysfunction. Such as rheumatoid arthritis, pharyngolaryngitis, otitis media, gastritis or periodontitis. In the inflammatory process, on the one hand, the damaged factor directly or indirectly causes destruction of tissue cells, and on the other hand, the damaged factor is diluted, killed and surrounded by inflammatory reaction and exudation reaction, and damaged tissue is repaired and healed by regeneration of parenchyma and interstitial cells, so that inflammation can be said to be a unified process of damage and damage resistance.
On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.
The reagents and materials used in the present invention are commercially available.
The invention has the positive progress effects that: the abietane diterpenoid compound shown in the formula I or pharmaceutically acceptable salt thereof has anti-inflammatory activity.
Drawings
FIG. 1 shows the DEPT 135℃spectrum (CDCl) of the abietane diterpenoid 1 obtained in example 1 3 ，150MHz)。
FIG. 2 shows the abietane diterpenoid 1 obtained in example 1 1 H- 1 H COSY spectrum (CDCl) 3 ，600MHz)。
FIG. 3 shows the HSQC spectrum (CDCl) of abietane diterpenoid 1 obtained in example 1 3 ，600MHz)。
FIG. 4 shows the rosin obtained in example 1HMBC spectra (CDCl) of alkane diterpenoid 1 3 ，600MHz)。
FIG. 5 shows NOESY spectra (CDCl) of abietane diterpenoid 1 obtained in example 1 3 ，600MHz)。
FIG. 6 shows NOESY spectra (CDCl) of abietane diterpenoid 1 obtained in example 1 3 600 MHz) partial enlargement.
FIG. 7 shows the measured ECD of the abietane diterpenoid 1 obtained in example 1 and the calculated ECD of its absolute configuration 3R,5R,10S,16S and 3S,5S,10R,16R respectively (JASCO Corp., J-810. Used solvent: chromatographic grade acetonitrile; test concentration: 0.02 mg/mL).
FIG. 8 shows the CD pattern of abietane diterpenoid 2 obtained in example 1. ((JASCO Corp., J-810. Solvent used: chromatographic grade acetonitrile; test concentration: 0.02 mg/mL).
Detailed Description
The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
Example 1 preparation of abietane diterpenoids 1 and 2
1) Extracting: reflux extracting herba Dracocephali with 70% ethanol, wherein 70% ethanol is 10 (v/m) times of the raw materials, extracting for 2 times each for 1 hr, filtering the extractive solutions, mixing, and drying to obtain herba Dracocephali dry extract. Suspending the extract in 2 times of water, and sequentially extracting with petroleum ether, dichloromethane, ethyl acetate and n-butanol to obtain ethyl acetate extract.
2) Column chromatography: subjecting the ethyl acetate extract to silica gel column chromatography, respectively eluting with petroleum ether and acetone solutions (100:1→50:1→20:1→10:1→5:1→2:1→1:1) in gradient, wherein the amount of mixed solvent used in each gradient is 3 times of column volume (v/v) (i.e. the used volume of the solvent is 3 times of the packed column volume of the silica gel), and sequentially collecting according to each column volume to obtain 21 components.
Collecting component 17 (i.e. the amount of eluent is 17 thThe eluent collected when the column volume is multiplied, namely the mixed solvent (petroleum ether: acetone solution=2:1) is collected when the volume of the column is multiplied by 2, and the developing agent is methylene dichloride after detection by silica gel thin layer chromatography, wherein the Rf value=0.60 when the methanol=100:1; concentrating under reduced pressure, subjecting to gel column (Sephadex LH-20), GE Healthcare Bio-Sciences AB, uppsala, sweden) chromatography, eluting with dichloromethane: methanol at 1:1, sequentially collecting with 10ml test tubes, identifying with silica gel thin layer plate, combining test tubes according to Rf value to obtain 4 components, collecting components 17-3 with Rf=0.53-0.54 (silica gel thin layer chromatography detection, developing solvent is dichloromethane: methanol=50:1); concentrating and drying, and subjecting to reverse phase silica gel ODS-C 18 (ODS-se:Sup>A-HG, YMC co., ltd., japan) gradient elution with methanol/water (60% → 80% → 100%) respectively, the amount of mixed solvent used for each gradient was 3 column volumes, and the total of 9 fractions were collected for each column volume, fractions 17-3-5 (i.e., the eluent collected when the amount of eluent was 5 column volumes, i.e., the eluent collected when the amount of mixed solvent (methanol/water=80%) was 2 column volumes) were collected, and fraction I was obtained after concentration and drying by detecting that the developing solvent was methylene chloride: methanol=50:1, rf=0.53 to 0.54 by silicse:Sup>A gel thin layer chromatography.
Component 18 (i.e., eluent collected when the amount of eluent was 18 times the column volume, i.e., eluent collected when the amount of mixed solvent (petroleum ether: acetone solution=2:1) was 3 times the column volume) was collected, and the developing agent was methylene chloride by silica gel thin layer chromatography detection, wherein the Rf value=0.55 when methanol=50:1; after concentration under reduced pressure, the mixture was chromatographed on a gel column (hydroxypropyl Sephadex LH-20 (Sephadex LH-20), GE Healthcare Bio-Sciences AB, uppsala, sweden) using methylene chloride: eluting with methanol=1:1 to obtain 7 fractions, and collecting fraction 18-5 with rf=0.55 (silica gel thin layer chromatography detection, developing solvent dichloromethane: methanol=50:1); concentrating and drying, and subjecting to reverse phase silica gel ODS-C 18 (ODS-A-HG, YMC Co., td., japan) by gradient elution with methanol/water (60%. Fwdarw.100%) to obtain 2 fractions. Component 18-5-1 was collected, rf=0.55 (silica gel thin layer chromatography assay, developing solvent dichloromethane: methanol=50:1), and concentrated under reduced pressure to give eluted component II.
3) HPLC preparation: preparing the above component I by HPLC (chromatographic column type: XBIdge BEH C) 18 5 μm, 10X 250mm; sample injection amount: 200. Mu.L; column temperature: 25 ℃; the stationary phase is C18 bonded silica gel; the mobile phase is 40% acetonitrile water solution, the detection wavelength is 210nm, and the flow rate is 15mL/min; preparing a liquid phase of LC 3050N-type high performance liquid chromatograph) to obtain the abietane diterpenoid compound 1:12, 16-epoxy-3,11,14-trihydroxy-17 (15- > 16), 18 (4- > 3) -trans-4 (19), 8,11, 13-abietyl tetraen-7-one (t) R =13.5 min); preparing the above component II by HPLC (chromatographic column type: XBIdge BEH C) 18 5 μm, 10X 250mm; sample injection amount: 200. Mu.L; column temperature: 25 ℃; the stationary phase is C18 bonded silica gel; the mobile phase is 30% acetonitrile water solution, the detection wavelength is 210nm, and the flow rate is 15mL/min; preparing a liquid phase of LC 3050N-type high performance liquid chromatograph) to obtain the abietane diterpenoid compound 2:12, 16-epoxy-3,11,14-trihydroxy-17 (15- > 16), 18 (4- > 3) -trans-4 (19), 5,8,11,13-abietyl-penten-7-one (t) R ＝29.4min)。
Example 2 structural identification of abietane diterpenoids 1 and 2
Structural identification of abietane diterpenoid 1:
ESI-MS m/z 343.1542[M-H] - the molecular weight of the compound was determined to be 344.
1 H-NMR(CDCl 3 600 MHz) shows absorption peaks for 3 methyl hydrogen atoms in the spectrum: delta H 1.50(3H,d,J＝6Hz,Me-17),δ H 1.45(3H,s,Me-18),δ H 1.15 (3H, s, me-20); hydrogen atoms on 4 methylene groups: delta H 1.87(1H,m,H-1α)，δ H 3.09(1H,m,H-1β)，δ H 1.79(1H,m,H-2α),δ H 1.71(1H,m,H-2β)，δ H 2.69(1H,t,J＝14.4Hz,H-6α),δ H 2.43(1H,dd,J＝3.0,16.2Hz,H-6β)，δ H 3.36(1H,dd,J＝9.0,15.6Hz,H-15α),δ H 2.84 (1 h, dd, j=7.2, 15.0hz, h-15 β); hydrogen atoms on 1 methine group: delta H 3.31 (1H, dt, H-5); 1 hydrogen atom delta on oxygen-methyl group H 5.12 (1H, m, H-16); hydrogen atoms on the 2 terminal double bonds: delta H 5.18(1H,d,J＝1.2Hz,H-19α),δ H 4.71 (1 h, d, j=1.8 hz, h-19 a). And the assignment of the hydrogen spectrum is completed with the help of HSQC correlation.
13 C-NMR Spectroscopy (CDCl) 3 150 MHz) shows that the molecule contains 20 carbon atoms, and the binding of the DEPT spectrum shows that the molecule contains 3 methyl groups, 5 methylene groups, 2 methine groups and 10 quaternary carbons. The carbon spectrum shows that the compound contains 3 methyl carbon atoms, and Me-18 (delta) is found by combining with the HSQC spectrum H 1.45,3H, s) and delta C 28.0，Me-17(δ H 1.50,3h, d, j=6 Hz) and δ C 22.2，Me-20(δ H 1.15,3H, s) and delta C 14.6, further attributed to carbon spectrum data as follows: 22.2 (C-17), delta C 28.0(C-18)，δ C 14.6 (C-20); discovery of H-1 and delta in combination with HSQC Profile C 31.0, H-2 and delta C 36.9, H-6 and delta C 37.5, H-15 and delta C 34.5, H-19 and delta C 108.1 are related, and further ascribed to 5 methylene carbon atoms as follows: delta C 31.0(C-1)，δ C 36.9(C-2)，37.5(C-6)，δ C 34.5 (C-15) and 1 double bond carbon atom delta C 108.1 (C-19); 2 methine carbon atoms delta C 42.2(C-5)，δ C 83.5 (C-16); the 10 quaternary carbon atoms include 1 carbonyl carbon atom delta C203.8 (C-7); delta of 6 benzene ring carbon atoms C 111.1(C-8)，δ C 137.7(C-9)，δ C 132.0(C-11)，δ C 156.0(C-12、14)，δ C 110.6 (C-13); a double bond carbon atom delta C 152.0 (C-4) and delta C 71.2(C-3)，δ C 41.0 (C-10), the compound has a abietane-type diterpenoid skeleton. And the nuclear magnetic data of this compound is similar to that of compound caryopteroisoid C except that the unsaturated double bonds of C-5 and C-6 are saturated. Combining HMBC spectrograms 1 H- 1 H COSY spectra found H-16 and H-15, H-17; h-5, H-6, H-19; h-1 and H-2 are related; h-1 and C-2, C-3, C-10; h-2 and C-1, C-3, C-10; h-6 and C-5, C-7; h-15 and C-13, C-14, C-16, C-17 are related, so the planar structure of the compound is determined.
The configuration of C-10 and C-16 is S by analysis of biogenic pathway of abietane diterpenoid compounds. From NOESY patterns, H-5 and H-1α, H-20 and H-1β are related, H-18 and H-2β, and H-20 and H-2β are related; the ECD map shows that absolute configurations of the abietane diterpenoid 1 are 3R,5R,10S and 16S, corresponding ECD curves show positive cotton effect at about 195nm, 220nm and 300nm, and show negative cotton effect at 270 nm; the absolute configuration of the abietane diterpenoid compound 1 is 3S,5S,10R and 16R, the corresponding ECD curves show negative cotton effect at about 195nm, 220nm and 300nm, and show positive cotton effect at 270 nm; the configuration of C-3, C-5 was determined to be R, R by NOESY profile in combination with ECD profile. The three-dimensional structure of the abietane diterpenoid 1 is as follows:
structural identification of abietane diterpenoid 2:
from the following components 1 H-NMR(CDCl 3 600 MHz) shows absorption peaks for 3 methyl hydrogen atoms in the spectrum: delta H 1.53(3H,d,J＝6.0Hz,H-17)，δ H 1.52(3H,s,H-18)，δ H 1.50 (3H, s, H-20); hydrogen atoms on 3 methylene groups: delta H 1.93(1H,m,H-1α)，δ H 3.08(1H,m,H-1β)，δ H 1.87(2H,m,H-2)，δ H 3.40(1H,dd,J＝9.0,15.6Hz,H-15α)，δ H 2.89 (1 h, dd, j=7.2, 15.2hz, h-15 β); 1 hydrogen atom on the oxygen-linked methylene group: delta H 5.14 (1H, m, H-16); one double bond hydrogen atom: delta H 6.22 (1H, s, H-6); hydrogen atoms on the 2 terminal double bonds: delta H 5.28(1H,s,H-19α)；δ H 5.20(1H,s,H-19β)。
13 C-NMR Spectroscopy (CDCl) 3 150 MHz) shows that the molecule contains 20 carbon atoms, and literature [1] The data in (a) are compared and attributed as follows: delta C 29.15(C-1)；δ C 35.04(C-2)；δ C 72.53(C-3)；δ C 151.55(C-4)；δ C 167.52(C-5)；δ C 123.39(C-6)；δ C 189.63(C-7)；δ C 109.31(C-8)；δ C 136.13(C-9)；δ C 42.38(C-10)；δ C 131.09(C-11)；δ C 154.45(C-12)；δ C 111.24(C-13)；δ C 154.11(C-14)；δ C 34.41(C-15)；δ C 83.15(C-16)；δ C 21.97(C-17)；δ C 26.74(C-18)；δ C 112.21(C-19)；δ C 21.40 (C-20). And determining the planar structure of the compound.
The configuration of C-10 and C-16 is S by analysis of biogenic pathway of abietane diterpenoid compounds. And literature [1] The CD data and nuclear magnetic data of the compound 15 are compared, and the three-dimensional structure of the abietane diterpenoid compound 2 is determined as follows:
[1] the data for compound 15 in Table 3 are presented in the reference "J.Nat.Prod.2016,79, 886-893".
Example 3 anti-inflammatory Activity of abietane diterpenoids 1 and 2
1. Instrument for measuring and controlling the intensity of light
Microplate reader (BioTek, EPOCH); CO 2 Incubator, (Thermo 3111); an adjustable pipette (eppendorf); microscope (olympus CX 23); biosafety cabinet (Heal Force, HFsafe-1200 LC).
2. Material
Cell culture dish: coning,430167; 96-well cell culture plate: corning,3599;75cm 2 Cell culture flask: corning,43063; DMEM medium (Gibco, C11995500 CP), RPMI1640 medium (Gibco, C11875500 BT); fetal bovine serum (FBS, gibco,2110875 CP); antibacterial-antifungal agents (antibacterial-antibacterial, lifetechnologies, 15240-112); phosphate Buffered Saline (PBS), pH7.4 (Gibco, 10010-500 BT); pancreatin-EDTA (Trypsin-EDTA, 0.25%) (Gibco, 25200-056); bovine serum albumin (Lifetechnologies, 15561012); mouse TNFα ELISA kit, multisciences, cat. EK282/3; mouse IL-1. Beta. ELISA kit, multisciences, cat# EK201B/3; LPS (bi yun tian, S1732); total nitric oxide detection kit (Nanjing built, A013-2).
3. Method of
1) Cells were routinely cultured in DMEM medium with 10% fbs, and inoculated in 96-well cell culture plates at 20000/100 μl/well, serum concentration was 10%;
2) The following day, adding the medicine prepared by the culture medium and negative control according to the experimental setting, and allowing the cells to act for 2 hours; LPS was prepared to 10. Mu.g/mL with DMEM containing 10% FBS, and 96-well cell culture plates were added at 10. Mu.L/well, and culture was continued for 8 hours;
3) According to the requirements of the kit, a multifunctional enzyme-labeled instrument is used for measuring the absorbance value, detecting the concentration of each index, calculating the concentration of each index, and calculating the inhibition rate of drugs with different concentrations on the generation of each index of LPS stimulated cells, thereby obtaining the IC of different compounds 50 。
4) The sample to be tested and indomethacin are accurately weighed, and the sample to be tested and indomethacin are diluted to the working concentration by using a detection buffer solution before an experiment.
4. Calculation of
5. Experimental results
5.1 Primary screening
TABLE 1 inhibition of TNF-alpha, IL-1 beta, NO in RAW264.7 cells by abietane diterpenoid compounds 1 and 2
TABLE 2 inhibition of TNF-alpha, IL-1 beta, NO in BV2 cells by abietane diterpenoid compounds 1 and 2
5.2 IC 50 Value determination
TABLE 3 rosin alkane diterpenoid compounds 1 and 2, caryopteroisoid C [a] And indomethacin vs. TNF-alpha, IL-1 beta, NO IC in RAW264.7 cells 50 Watch (watch)
[a] Data results for compound 35 in table 6, cited document "j.nat.prod.2018,81, 1508-1516"; "-" indicates that the data was not tested.
TABLE 4 rosin-based diterpenoids 1 and 2 and indomethacin vs. TNF-alpha, IL-1 beta, NO IC in BV2 cells 50 Watch (watch)
The results are shown in tables 1-4, and the results show that the abietyl diterpenoid compounds 1 and 2 have stronger inhibition activity on TNF-alpha, IL-1 beta and NO and IC thereof 50 The value is less than 10 mu M in RAW264.7 cells and BV2 cells, which shows that the medicine has obvious inhibiting effect on inflammation. Therefore, the abietyl diterpenoid compounds 1 and 2 can provide a new way for preventing, treating and/or relieving inflammatory diseases, and can be developed into anti-inflammatory drugs.
Comparative example 1 anti-inflammatory Activity of abietane diterpenoid A-d
The apparatus, materials and methods for testing the anti-inflammatory activity of compounds a-d were as described in example 3.
Experimental results:
TABLE 5 inhibition of TNF-alpha, IL-1 beta, NO by Compounds a-d in RAW264.7 cells
TABLE 6 inhibition of TNF-alpha, IL-1 beta, NO in BV2 cells by Compounds a-d
The results are shown in tables 5-6, and demonstrate that when the compound C4 has NO terminal double bond, the monomer compound has weak or NO anti-inflammatory activity against TNF- α, IL-1β, NO in LPS-induced RAW264.7 cells and BV2 cells.
Comparative example 2 anti-inflammatory Activity of abietane diterpenoid e-g
The apparatus, materials and methods for testing the anti-inflammatory activity of compounds e-g were as described in example 3.
Experimental results:
TABLE 7 inhibition of TNF-alpha, IL-1 beta, NO by Compounds e-g in RAW264.7 cells
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TABLE 8 inhibition of TNF-alpha, IL-1 beta, NO in BV2 cells by Compounds e-g
The results are shown in tables 7-8, and the results show that when the five-membered ring in the abietane diterpenoid compound is opened (such as compounds e, f and g), the inhibition capability of the monomer compound on TNF-alpha, IL-1 beta and NO in RAW264.7 cells and BV2 cells induced by LPS is obviously reduced, and the anti-inflammatory activity is basically absent.

Claims (1)

1. An application of abietane diterpenoid compound shown in formula 1 or 2 or pharmaceutically acceptable salt thereof in preparing medicine for preventing, treating and/or relieving inflammatory diseases,
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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Hu, Hai-Jun 等..Abietane Diterpenoids from the Roots of Clerodendrum trichotomum and Their Nitric Oxide Inhibitory Activities. .Journal of Natural Products.2018,第81卷(第7期),1508-1516. *
LUO Guoyong 等.Iridoid Glucosides and Diterpenoids from Caryopteris glutinosa..Journal of Natural Products.2016,第79卷(第4期),886-893. *

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