CN116854653A - Diphenyl heptane compound and its preparation method and application - Google Patents

Diphenyl heptane compound and its preparation method and application Download PDF

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CN116854653A
CN116854653A CN202310703671.3A CN202310703671A CN116854653A CN 116854653 A CN116854653 A CN 116854653A CN 202310703671 A CN202310703671 A CN 202310703671A CN 116854653 A CN116854653 A CN 116854653A
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ethanol
compound
water
gradient elution
volume ratio
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肖伟
李海波
董洁
董雪红
胡晗绯
曹亮
王振中
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Jiangsu Kanion Pharmaceutical Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D309/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings
    • C07D309/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
    • C07D309/08—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D309/10—Oxygen atoms
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    • 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]

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Abstract

The application discloses a diphenyl heptane compound which is a novel chemical component found in fructus alpiniae oxyphyllae. The application also carries out structural identification on the compounds separated by the method through physicochemical properties and modern spectroscopy means. The application also uses LPS to induce RAW264.7 cell inflammation model and other active screening systems to evaluate the activity, and discovers that the compound has a certain protection effect on the RAW264.7 of the mouse macrophage line, can obviously inhibit the release of NO, and shows stronger anti-inflammatory effect.

Description

Diphenyl heptane compound and its preparation method and application
Technical Field
The application relates to the technical field of medicines, in particular to a diphenyl heptane compound, a preparation method and application thereof.
Background
Intelligence development (Alpinia oxyphylla Miq.) is a perennial herb, growing in the shade wet area under forests, mainly distributed in Guangdong, hainan and other provinces. The fruit is dried mature fruit of Zingiberaceae plant, which is generally spindle-shaped or elliptic, and has a length of about 1.2-2 cm and a diameter of about 1-1.3 cm. The surface of the seed is brown, 13-20 irregular intermittent raised lines are arranged, and the peel is thin and hard and clings to the seeds. The seed ball has a light brown membrane dividing it into three chambers, each chamber containing 6-11 seeds. The seeds are in irregular polyhedral shape, have the diameter of 3-4 mm and have yellowish false seed coats.
Intelligence development is used as one of four south-greater drugs, and the history of drug administration is long, and the beginning of drug administration is carried out in Ben Cao Shi Yi (herbal medicine pickup), and the store is as follows: vomit-stopping and vomit-stopping … … contains saliva-controlling substances. The "Zhi Yi Zhi is used for treating" spermatorrhea, deficiency and leak, dribbling urine ", which is described in the" Zhi Ben Cao ", highlights the actions of improving intelligence and warming kidney. The "Ben Cao gang mu" describes that it has the functions of promoting intelligence and curing frequent urination, heart deficiency, urination, turbid urine, abdominal pain, diarrhea, halitosis, metrorrhagia and metrostaxis. The traditional Chinese medicine named ' Jiquan ' pill, yizhi powder has the functions of invigorating kidney, reducing urination, and the compound medicine ' Yizhi ' pill, yizhi Qi granule and Yizhi Xingnao decoction ' is used clinically in treating hypomnesis, neurasthenia, etc. Modern pharmacological research shows that the alpinia oxyphylla has extensive pharmacological effects such as neuroprotection, kidney protection, antidiuretic, anti-inflammatory and antioxidant. As a plant resource with high safety and dual purposes of medicine and food, the development of the product of the fructus alpiniae oxyphyllae is little at present, so that the application of the fructus alpiniae oxyphyllae is limited.
Therefore, the composition of the fructus alpiniae oxyphyllae is further studied to obtain the fructus alpiniae oxyphyllae active ingredient with the medicinal effect, which is not only beneficial to understanding the composition of the compounds in the fructus alpiniae oxyphyllae, but also has important significance for deep development and application of the fructus alpiniae oxyphyllae.
Disclosure of Invention
The application aims at carrying out more intensive research on anti-inflammatory active ingredients in fructus alpiniae oxyphyllae and finding out the active ingredients.
In view of this, the present application provides a diphenyl heptane compound or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers, prodrug molecules and metabolites thereof, wherein the structure of the compound is shown in formula I:
further, the above-mentioned compound may be,
another object of the present application is to provide a method for preparing the above compound, comprising the steps of:
a) Reflux extracting fructus Alpinae Oxyphyllae with 50-70% ethanol, and removing solvent to obtain total extract;
b) Dissolving the total extract in water, separating by macroporous adsorption resin column chromatography, eluting with water, 45-55% ethanol, 65-75% ethanol and 90-100% ethanol in sequence, respectively collecting each eluent, concentrating under reduced pressure to obtain water eluting part, 45-55% ethanol, 65-75% ethanol and 90-100% ethanol; 4 column volumes per gradient elution (same below);
c) Separating the 65-75% ethanol elution part by silica gel column chromatography, collecting 10 fractions of 3A-3J by cyclohexane-ethyl acetate gradient elution, eluting 3F by ODS column chromatography methanol-water gradient elution to obtain 5 fractions of 3F1-3F5, separating 3F5 by preparative liquid chromatography to obtain 11 fractions of 3F5A-3F5K, and separating 3F5E by preparative liquid phase to obtain a compound 1; further, compound 1 can be separated from compound 1 by preparative liquid phase to obtain compounds 1a and 1b.
In particular, the fructus Alpinae Oxyphyllae may be dried mature fruits of fructus Alpinae Oxyphyllae.
Further, the step a) includes: reflux extracting dried fructus Alpinae Oxyphyllae with 3-5 times of 50-70% ethanol for 1-3 times each for 1-3 hr, mixing extractive solutions, and removing solvent under reduced pressure to obtain the total extract.
Preferably, the step B) includes: sequentially eluting with water, 50% ethanol, 70% ethanol and 95% ethanol, collecting the eluates, and concentrating under reduced pressure until no ethanol smell is present to obtain water eluate, 50% ethanol eluate, 70% ethanol eluate and 95% ethanol eluate.
The cyclohexane-ethyl acetate gradient elution of the step C) is carried out according to the volume ratio of 100-90:0-10 to 0:100; the methanol-water gradient elution is carried out according to the volume ratio of 30-50:70-50 to 100:0.
Specifically, the macroporous adsorption resin comprises one or more of D101 type macroporous adsorption resin, HP-20 type macroporous adsorption resin, HPD-100A type macroporous adsorption resin or HPD-300 type macroporous adsorption resin.
Further, the cyclohexane-ethyl acetate gradient elution of step C) is a gradient elution at a volume ratio of 98:2, 95:5, 9:1, 85:15, 8:2, 7:3, 6:4, 1:1, 0:1; the methanol-water gradient elution is performed at a volume ratio of 40:60, 45:55, 50:40, 55:45, 65:35, 80:20, 100:0.
Further, the step A) is to reflux extract with 60% ethanol 2 times for 2 hours each.
Specifically, the conditions for preparing the liquid chromatograph include: specification is C 18 5 μm, 10X 250mm Phenomenex Gemini; the mobile phase of the separated fraction 3F5 is methanol-water with the volume ratio of 55:45; separating acetonitrile-water with the volume ratio of 3F5E mobile phase of 35:65; the detection wavelength was 223nm, and the flow rate was 3mL/min. .
Further, the method further comprises: subjecting said compound 1 to preparative liquid phase separation on a column of size EnantinPak Y3,5 μm, C18, 250X 4.60 mm; wherein, the volume ratio of the mobile phase to acetonitrile-water is 40:60, the detection wavelength is 223nm, and the flow rate is 1mL/min.
The application aims to provide application of the compound or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers, prodrug molecules and metabolites thereof in preparing anti-inflammatory drugs.
The application also provides a medicament comprising the diphenyl heptane compounds or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers, prodrug molecules and metabolites thereof.
Further, the medicament contains a therapeutically effective amount of a compound of formula (I) above or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, prodrug molecule, metabolite, and one or more pharmaceutically acceptable carriers thereof.
Specifically, the medicine can be any dosage form in pharmacy, including tablets, capsules, soft capsules, gels, oral preparations, suspension, medicinal granules, patches, ointments, pills, powder, injection, infusion solution, freeze-dried injection, intravenous emulsion, liposome injection, suppositories, sustained-release preparations or controlled-release preparations.
Further, the pharmaceutically acceptable carrier refers to a conventional pharmaceutical carrier in the pharmaceutical field, for example: diluents, excipients, water and the like, fillers such as starch, sucrose, lactose, microcrystalline cellulose and the like; binders such as cellulose derivatives, alginates, gelatin and polyvinylpyrrolidone; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, hydroxypropyl cellulose, croscarmellose, agar, calcium carbonate and sodium bicarbonate; absorption promoters such as quaternary ammonium compounds; surfactants such as cetyl alcohol, sodium lauryl sulfate; adsorption carriers such as kaolin and bentonite; lubricants such as talc, calcium and magnesium stearate, silica gel micropowder, polyethylene glycol, etc. Other adjuvants such as flavoring agent, sweetener, etc. can also be added into the composition.
The diphenyl heptane compound disclosed by the application is a novel chemical component found by researchers in fructus alpiniae oxyphyllae, and the compound is found to exist stably in fructus alpiniae oxyphyllae in each batch. The inventors have determined by physicochemical properties and modern spectroscopy means (MS, 1 H-NMR、 13 C-NMR, etc.), the compound isolated by the above method is subjected to structural identification, and the structure is confirmed as formula(I) Novel compounds are shown. The application also uses LPS to induce RAW264.7 cell inflammation model and other active screening systems to evaluate the activity, and discovers that the compound has a certain protection effect on the RAW264.7 of the mouse macrophage line, can obviously inhibit the release of NO, and shows stronger anti-inflammatory effect. Has good research and development prospect.
Drawings
FIG. 1 is a spectrum of HR-ESI-Q-TOF-MS of compound 1 of the present application;
FIG. 2 shows Compound 1 of the present application 1 H-NMR spectrum
FIG. 3 shows Compound 1 of the present application 13 C-NMR spectrum;
FIG. 4 shows Compound 1 of the present application 13 C-NMR and DEPT-135 spectra;
FIG. 5 is H of Compound 1 of the present application 1 -H 1 COSY spectrogram;
FIG. 6 is a HSQC spectrum of Compound 1 of the present application;
FIG. 7 is a HMBC spectrum of Compound 1 of the present application;
FIG. 8 is a NOESY pattern of Compound 1 of the present application;
FIG. 9 is a UV spectrum of compound 1 of the present application;
FIG. 10 is an IR spectrum of compound No. 1 of the present application;
FIG. 11 is an HPLC chart of chiral column resolution compound 1a/1b of compound 1 of the present application;
FIG. 12 is an X-ray pattern of compounds 1a and 1b of the present application;
FIG. 13 shows the NO release inhibiting IC of the compounds 1a and 1b of the present application 50 A curve.
Detailed Description
The following detailed description of specific embodiments of the application is provided in connection with the accompanying drawings and examples in order to provide a better understanding of the aspects of the application and advantages thereof. However, the following description of specific embodiments and examples is for illustrative purposes only and is not intended to be limiting of the application.
The following will specifically describe the contents of experimental examples.
It is particularly pointed out that similar substitutions and modifications to the application will be apparent to those skilled in the art, which are all deemed to be included in the application. It will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, or in the appropriate variations and combinations, without departing from the spirit and scope of the application. It will be apparent that the described embodiments are only some, but not all, embodiments of the application.
The application is carried out according to the conventional conditions or the conditions suggested by manufacturers if the specific conditions are not noted, and the raw materials or auxiliary materials and the reagents or instruments are conventional products which can be obtained commercially if the manufacturers are not noted. Intelligence development (ALPINIAE OXYPHYLLAE FRUCTUS) is obtained from the company of Shiyuan commerce, inc. of Annational City in 2020, hainan province at the place of production.
The application is further illustrated by the following examples:
EXAMPLE 1 preparation of the Compounds of the application
Step (1): reflux-extracting dried mature fruit of fructus Alpinae Oxyphyllae with 50% ethanol for 2 times each for 2 hr, mixing extractive solutions, and removing solvent under reduced pressure to obtain total extract. Dissolving the total extract in water, separating by HP-20 macroporous adsorbent resin column chromatography, eluting with water, 50% ethanol, 70% ethanol, and 95% ethanol sequentially, eluting with 4 column volumes (same below) each gradient, collecting eluate, concentrating under reduced pressure until no alcohol smell, to obtain water eluting part, 50% ethanol eluting part, 70% ethanol eluting part, and 95% ethanol eluting part;
step (2): separating the 70% ethanol elution part in the step (1) by silica gel column chromatography, performing gradient elution by using cyclohexane-ethyl acetate 98:2, 95:5, 9:1, 85:15, 8:2, 7:3, 6:4, 1:1 and 0:1 volume ratio, collecting 10 fractions (3A-3J), performing gradient elution on the fraction 3F by using ODS column chromatography methanol-water (40:60, 45:55, 50:40, 55:45, 65:35, 80:20, 100:0 and v/v) to obtain 5 fractions in total, separating the fraction 3F5 by using preparative liquid chromatography I to obtain 11 fractions in total of 3F5A-3F5K, separating the 3F5E by using preparative liquid chromatography II to obtain a compound 1, and separating the compound 1 by using preparative liquid chromatography III to obtain the compounds 1a and 1b.
Wherein, the conditions for preparing the liquid chromatography I and II in the step (2) are that: phenomenex Gemini, C 18 5 μm, 10X 250mm, the preparative high performance liquid chromatograph is Shimadzu, pump: LC-6AD (SHIMADZU, LIQUID CHROMATOGRAH); a detector: SPD-20A (Prominence UV/VIS DETECTOR); work station: LC solution. The conditions for preparing the liquid chromatograph III are as follows: enantinPak Y3 (C18, 5 μm, 4.60deg.C.250 mm) column, prepared as a high performance liquid chromatograph, shimadzu, pump: LC-6AD (SHIMADZU, LIQUID CHROMATOGRAH); a detector: SPD-20A (Prominence UV/VIS DETECTOR); work station: LC solution. The mobile phase of fraction 3F5 is methanol-water with the volume ratio of 55:45, the detection wavelength is 223nm, and the flow rate is 3mL/min; the mobile phase of the compound 1 is acetonitrile-water with the volume ratio of 35:65, the detection wavelength is 223nm, and the flow rate is 3mL/min. The mobile phases of the compound 1a and the compound 1b are in volume proportion: acetonitrile-water at a detection wavelength of 223nm at a flow rate of 1mL/min at a ratio of 40:60.
EXAMPLE 2 preparation of the Compounds of the application
(1) Reflux-extracting dried mature fruit of fructus Alpinae Oxyphyllae with 60% ethanol for 2 times and 2 hr each time, mixing extractive solutions, and removing solvent under reduced pressure to obtain total extract. Dissolving the total extract in water, separating by HP-20 macroporous adsorbent resin column chromatography, eluting with water, 45% ethanol, 65% ethanol, and 90% ethanol sequentially, collecting eluates, concentrating under reduced pressure until no ethanol smell is present, and collecting water eluate, 45% ethanol eluate, 65% ethanol eluate, and 90% ethanol eluate;
(2) Separating the 65% ethanol elution part in the step (1) by silica gel column chromatography, performing gradient elution by cyclohexane-ethyl acetate (98:2, 95:5, 9:1, 85:15, 8:2, 7:3, 6:4, 1:1, 0:1 v/v), collecting 10 fractions (3A-3J), performing gradient elution on the fraction 3F by ODS column chromatography methanol-water (40:60, 45:55, 50:40, 55:45, 65:35, 80:20, 100:0, v/v) to obtain 3F1-3F5 total 5 fractions, performing preparative liquid chromatography on the fraction 3F5 to obtain 11 fractions of the fraction 3F5A-3F5K, performing preparative liquid phase separation on the 3F5E to obtain a compound 1, and performing preparative liquid phase separation on the compound 1 to obtain a compound 1a and a compound 1b.
Wherein, the conditions for preparing the liquid chromatography I and II in the step (2) are that: phenomenex Gemini, C 18 5 μm, 10X 250mm, the preparative high performance liquid chromatograph is Shimadzu, pump: LC-6AD (SHIMADZU, LIQUID CHROMATOGRAH); a detector: SPD-20A (Prominence UV/VIS DETECTOR); work station: LC solution. The conditions for preparing the liquid chromatograph III are as follows: enantinPak Y3 (C18, 5 μm, 4.60deg.C.250 mm) column, prepared as a high performance liquid chromatograph, shimadzu, pump: LC-6AD (SHIMADZU, LIQUID CHROMATOGRAH); a detector: SPD-20A (Prominence UV/VIS DETECTOR); work station: LC solution. The mobile phase of fraction 3F5 is methanol-water with the volume ratio of 55:45, the detection wavelength is 223nm, and the flow rate is 3mL/min; the mobile phase of the compound 1 is acetonitrile-water with the volume ratio of 35:65, the detection wavelength is 223nm, and the flow rate is 3mL/min. The mobile phases of the compound 1a and the compound 1b are acetonitrile-water with the volume ratio of 40:60, the detection wavelength is 223nm, and the flow rate is 1mL/min.
EXAMPLE 3 preparation of the Compounds of the application
(1) Collecting dried mature fruit with intelligence improving effect, using70% ethanolReflux-extracting for 2 times and 2 hours each time, mixing the extractive solutions, and removing solvent under reduced pressure to obtain total extract. Dissolving the total extract in water, separating by HP-20 macroporous adsorbent resin column chromatography, eluting with water, 55% ethanol, 75% ethanol and 100% ethanol sequentially, collecting each eluent, concentrating under reduced pressure until no ethanol smell exists, and obtaining water eluting part, 55% ethanol eluting part, 75% ethanol eluting part and 100% ethanol eluting part;
(2) Separating the 75% ethanol elution part in the step (1) by silica gel column chromatography, performing gradient elution by cyclohexane-ethyl acetate (98:2, 95:5, 9:1, 85:15, 8:2, 7:3, 6:4, 1:1, 0:1 v/v), collecting 10 fractions (3A-3J), performing gradient elution on the fraction 3F by ODS column chromatography methanol-water (40:60, 45:55, 50:40, 55:45, 65:35, 80:20, 100:0, v/v) to obtain 3F1-3F5 total 5 fractions, performing preparative liquid chromatography on the fraction 3F5 to obtain 11 fractions of 3F5A-3F5K, performing preparative liquid phase separation on the 3F5E to obtain a compound 1, and performing preparative liquid phase separation on the compound 1 to obtain a compound 1a and a compound 1b.
Wherein, the step (2) The conditions for preparing the liquid chromatography I and II are as follows: phenomenex Gemini, C 18 5 μm, 10X 250mm, the preparative high performance liquid chromatograph is Shimadzu, pump: LC-6AD (SHIMADZU, LIQUID CHROMATOGRAH); a detector: SPD-20A (Prominence UV/VIS DETECTOR); work station: LC solution. The conditions for preparing the liquid chromatograph III are as follows: enantinPak Y3 (C18, 5 μm, 4.60deg.C.250 mm) column, prepared as a high performance liquid chromatograph, shimadzu, pump: LC-6AD (SHIMADZU, LIQUID CHROMATOGRAH); a detector: SPD-20A (Prominence UV/VIS DETECTOR); work station: LC solution. The mobile phase of fraction 3F5 is methanol-water with the volume ratio of 55:45, the detection wavelength is 223nm, and the flow rate is 3mL/min; the mobile phase of the compound 1 is acetonitrile-water with the volume ratio of 35:65, the detection wavelength is 223nm, and the flow rate is 3mL/min. The mobile phases of the compound 1a and the compound 1b are in volume proportion: acetonitrile-water at a detection wavelength of 223nm at a flow rate of 1mL/min at a ratio of 40:60.
EXAMPLE 4 structural identification of the Compounds of the application
Yellow oily body. HR-ESI-MS gives m/z 329.1758[ M+H ]] + (calculated as 329.1753), the molecular formula of the compound was determined as C 20 H 24 O 4 The calculated unsaturation was 9.
As shown in Table 1 and FIGS. 1 to 12, compound 1 1 H-NMR(600MHz,in CDCl 3 ) In the spectrum, a mono-substituted benzene ring hydrogen signal [ delta ] was observed H 7.28(2H,m,H-3″,5″),7.19(3H,m,H-2″,4″,6″)]1,2, 4-substituted benzene ring hydrogen signal [ delta ] H 6.95(1H,br s,H-2′),6.88(2H,d,J＝7.7Hz,H-5′,6′)]3 SPs 3 Hybrid oxygen-methyl hydrogen signal [ delta ] H 4.77(1H,dd,J＝11.8,2.0Hz,H-1),4.35(1H,m,H-3),3.96(1H,m,H-5)]1 methoxy hydrogen signal [ delta ] H 3.91(3H,s,3′-OCH 3 )]And a plurality of SPs 3 Hybrid methylene hydrogen signal. 13 C-NMR(150MHz,in CDCl 3 ) The spectrum combined with the DETP 135 spectrum showed a total of 20 carbon signals, including: 1 methoxy signal (delta) C 56.0 4 methylene signals (. Delta.) C 40.6,38.6,38.0,31.8), 11 methine signals (. Delta C 128.6×2,128.5×2,125.8,118.9,114.2,108.9,73.4,71.3,65.2), 4 quaternary carbon signals (delta) C 146.5,144.9,142.5,135.2). The compound 1 was a diphenylheptane compound as estimated by 1D-NMR.
H-1 was observed to be associated with C-2,3,5,1',2',6' and H-7 was observed to be associated with C-5,6,1 ", 2", 6 "in the HMBC spectra; comprehensive synthesis 1 H- 1 H-1/H in the H COSY Spectrum 2 -2/H-3/H 2 -4/H-5, thereby determining that the two benzene rings are linked by the pyran ring.
In the NOESY pattern, H-1 was observed to be associated with H-5, indicating that both are on the same side. Compound 1 was separated into a pair of enantiomers 1a and 1b by chiral HPLC. In combination with the collection of Cu K alpha diffraction data in the X single crystal diffraction test, the absolute configurations of 1a and 1b are determined to be 1S,3S,5S and 1R,3R,5R respectively, and are identified as (1S, 3S, 5S) -3-hydroxy-1,5-epoxy-1- (3-hydroxy-4-methoxyphenyl) -7-phenyl-hept an-3-ol and (1R, 3R, 5R) -3-hydroxy-1,5-epoxy-1- (3-hydroxy-4-methoxyphenyl) -7-phenyl-hept an-3-ol respectively, and the structures are as follows:
TABLE 1 Compound 1 1 H and 13 c NMR data
Multiplets and or overlapped signals are reported without designating multiplicity
The same procedure was used to identify the compounds of each example.
EXAMPLE 5 in vitro anti-PGE of the Compounds of the application 2 Experiment
1. Material
1.1 pharmaceutical compounds 1a and 1b;
1.2 cell model mouse macrophage line RAW264.7, which is derived from the academy of sciences of traditional Chinese medicine; culture conditions: dmem+10% Fetal Bovine Serum (FBS), 37 ℃,5% CO 2 。
2. Principle and method
2.1 principle of experiment
Lipopolysaccharide (LPS) of the outer membrane of gram-negative bacteria (Sigma Co., USA, lot number 114M 4009) is one of the most prominent pathogenic molecules mediating infectious inflammatory lesions, and many diseases are closely related to LPS-induced persistent subclinical inflammation. In animal and cellular experiments, LPS is widely used to induce the onset of inflammation.
Macrophages play a critical role in the inflammatory response, and upon stimulation, macrophage production of inflammatory factors and mediator activation are key processes of inflammation, and their inhibition is often an important indicator for evaluating the anti-inflammatory activity of drugs.
2.2 inhibition test of NO secretion by drug
1. Experimental procedure
The cells were blown down with a pipette and the cell density was adjusted to 2X 10 in DMEM medium containing 10% FBS 5 individual/mL; uniformly inoculating to a 96-well plate, placing 100 mu L of each well into an incubator for culturing for 24 hours after the plates are placed. Subsequently, the 96-well plate was removed, the supernatant was aspirated, and a drug-containing medium prepared by adding serum-free DMEM medium was added, and the experimental setup was specifically as follows:
(1) Solvent control group (-group): 100. Mu.L of serum-free DMEM medium containing one thousandth of DMSO was added to each well;
(2) Model group (+group): 100. Mu.L of LPS-free DMEM medium with a final concentration of 0.5. Mu.g/ml was added to each well;
(3) Administration sample group (including compounds 1a and 1b, positive control hydrocortisone): mu.L of medium containing LPS at a corresponding concentration and 0.5. Mu.g/ml was added to each well;
after the drug addition is finished, the 96-well plate is put into CO 2 The cells were cultured in an incubator for 24 hours.
2. Experimental results
(1) Nitrite concentration in the medium was measured as an indicator of NO production according to Griess method (Beyotime Biotechnology, S0021). The cell-free supernatant and Griess reagent (nitrate reduction assay reagent) were thoroughly mixed in the same amount of 50. Mu.L; the absorbance of the final product was measured at 540nm on a microplate reader. The nitrite concentration and inhibition rate were calculated based on a standard calibration curve, and the results of the nitrite concentrations in each of the experimental group and the control group are shown in FIG. 13;
(2) The inhibition of LPS-induced NO production by test compounds is described as IC 50 Values, the results of which are detailed in Table 2.
Inhibition of LPS-induced NO production by BV-2 cells by the compounds of Table 2
Experimental data show that the diphenyl heptane compounds 1a and 1b obtained by separating the fructus alpiniae oxyphyllae in the scheme have inhibition effect on NO production in LPS-induced RAW264.7 cells (see FIG. 13 for details). IC by detecting inhibition of LPS-induced NO in RAW264.7 cells by a fraction of compounds 50 The values found that compound 1a has IC that induces inhibition of NO in RAW264.7 cells by LPS 50 IC with a value of 31.84. Mu.M, which has a stronger anti-inflammatory effect than 1b, and compound 1b has an inhibitory effect on LPS-induced NO in RAW264.7 cells 50 The value was 44.63. Mu.M.
The foregoing is merely exemplary of the present application, and specific technical solutions and/or features that are well known in the art have not been described in detail herein. It should be noted that, for those skilled in the art, several variations and modifications can be made without departing from the technical solution of the present application, and these should also be regarded as the protection scope of the present application, which does not affect the effect of the implementation of the present application and the practical applicability of the patent. The scope of the application is to be determined by the appended claims, the description of the specific embodiments and the like in the description should be construed as merely illustrative of the preferred embodiments of the application, and it should be understood by those skilled in the art that various modifications and adaptations to the application may be made without departing from the principles of the application.

Claims (10)

1. A diphenyl heptane compound or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, prodrug molecule, metabolite thereof, the structure of the compound is shown in formula I:
2. a compound according to claim 1, having the structure:
3. a process for the preparation of a compound as claimed in claim 1, comprising the steps of:
a) Reflux extracting fructus Alpinae Oxyphyllae with 50-70% ethanol, and removing solvent to obtain total extract;
b) Dissolving the total extract in water, separating by macroporous adsorption resin column chromatography, eluting with water, 45-55% ethanol, 65-75% ethanol and 90-100% ethanol in sequence, respectively collecting each eluent, concentrating under reduced pressure to obtain water eluting part, 45-55% ethanol, 65-75% ethanol and 90-100% ethanol;
c) And separating the 65-75% ethanol elution part by silica gel column chromatography, collecting 10 fractions of 3A-3J by cyclohexane-ethyl acetate gradient elution, obtaining 5 fractions of 3F1-3F5 by ODS column chromatography methanol-water gradient elution, obtaining 11 fractions of 3F5A-3F5K by preparative liquid chromatography separation of the fraction 3F5, and separating the 3F5E by preparative liquid phase separation to obtain the compound 1.
4. A process according to claim 3, wherein,
the step A) comprises the following steps: reflux extracting fructus Alpinae Oxyphyllae with 3-5 times of 60% ethanol for 1-3 times each for 1-3 hr, mixing extractive solutions, and removing solvent under reduced pressure to obtain total extract;
the step B) comprises the following steps: sequentially eluting with water, 50% ethanol, 70% ethanol and 95% ethanol, respectively collecting each eluent, concentrating under reduced pressure until no ethanol smell exists, and obtaining water eluting part, 50% ethanol eluting part, 70% ethanol eluting part and 95% ethanol eluting part;
the cyclohexane-ethyl acetate gradient elution of step C) is a gradient elution at a volume ratio of 100:0 to 0:100; the methanol-water gradient elution is performed at a volume ratio of 40:60 to 100:0.
5. The method according to claim 3, wherein the macroporous adsorbent resin comprises one or more of D101 type macroporous adsorbent resin, HP-20 type macroporous adsorbent resin, HPD-100A type macroporous adsorbent resin and HPD-300 type macroporous adsorbent resin.
6. The method of claim 3, wherein the cyclohexane-ethyl acetate gradient elution of step C) is a gradient elution at a volume ratio of 98:2, 95:5, 9:1, 85:15, 8:2, 7:3, 6:4, 1:1, 0:1; the methanol-water gradient elution is performed at a volume ratio of 40:60, 45:55, 50:40, 55:45, 65:35, 80:20, 100:0.
7. The method according to claim 3, wherein the step A) is a reflux extraction with 60% ethanol for 2 times each for 2 hours; the conditions for the preparation of liquid chromatography of fraction 3F5 include: specification is C 18 5 μm, 10X 250mm Phenomenex Gemini; the mobile phase of the separated fraction 3F5 is methanol-water with the volume ratio of 55:45; separating acetonitrile-water with the volume ratio of 3F5E mobile phase of 35:65; the detection wavelength was 223nm, and the flow rate was 3mL/min.
8. The method of manufacturing according to claim 7, further comprising: the compound 1 is subjected to preparative liquid phase separation, wherein the specification is EnantinPak Y3,5 μm, C18,250×4.60mm column, the volume ratio of mobile phase is acetonitrile-water of 40:60, the detection wavelength is 223nm, and the flow rate is 1mL/min.
9. The use of a diphenyl heptane-based compound or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, prodrug molecule, metabolite thereof according to claim 1 in the preparation of an anti-inflammatory medicament.
10. A medicament comprising the diphenylheptane compound of claim 1 or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers, prodrug molecules, metabolites thereof.
CN202310703671.3A 2023-06-13 2023-06-13 Diphenyl heptane compound and its preparation method and application Pending CN116854653A (en)

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