CN113214157A - Application of pyrrolidone compound in preparation of medicine for treating inflammatory diseases - Google Patents
Application of pyrrolidone compound in preparation of medicine for treating inflammatory diseases Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 239000003814 drug Substances 0.000 title claims description 7
- 208000027866 inflammatory disease Diseases 0.000 title description 3
- -1 pyrrolidone compound Chemical class 0.000 title description 3
- 150000001875 compounds Chemical class 0.000 claims abstract description 37
- 150000003839 salts Chemical class 0.000 claims abstract description 5
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- 238000010828 elution Methods 0.000 claims description 60
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- OMFXVFTZEKFJBZ-HJTSIMOOSA-N corticosterone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@H](CC4)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 OMFXVFTZEKFJBZ-HJTSIMOOSA-N 0.000 abstract description 7
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D223/00—Heterocyclic compounds containing seven-membered rings having one nitrogen atom as the only ring hetero atom
- C07D223/14—Heterocyclic compounds containing seven-membered rings having one nitrogen atom as the only ring hetero atom condensed with carbocyclic rings or ring systems
- C07D223/18—Dibenzazepines; Hydrogenated dibenzazepines
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
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- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Neurology (AREA)
- Neurosurgery (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
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Abstract
The invention aims to provide a compound shown in a formula I and a pharmaceutically acceptable salt thereof, and a preparation method and application of the compound shown in the formula I. Cell level experiments show that the compound shown in the formula I has an obvious protective effect on PC12 cells damaged by corticosterone, and is a novel preparation for preparing nerve cell protective drugs.
Description
Technical Field
The invention relates to the field of biological medicines, in particular to application of pyrrolidone compounds in preparation of medicines for treating inflammatory diseases.
Background
Herba Duchesneae Indicae is fresh or dried whole plant of Emilia sonchifolia (Linnaeus) de Candole of Compositae (Compositae). The product is 10-50 cm long. The roots are thin and curved with fibrous roots. The stem is thin and cylindrical, the surface is dark green, and the lower part is hairy. The leaves are shrunk more, the flattened basal raw leaves are split in a piano shape, the length is 5-10 cm, the width is 2.5-5 cm, the leaves are gray green or dark green, the front end split is large and is approximately triangular, the base part embraces the stem, and the edge is provided with sparse blunt teeth; cauline leaves are progressively narrowed. 2-3 head inflorescences are arranged into an umbrella shape, the total bracts are cylindrical, 1 layer of bracts are in a strip shape and are in a needle shape or a nearly strip shape, and the length is about 1 cm; the tubular flower is brownish yellow and the crown hair is white. The thin fruit is round, about 3mm long and has edges.
China is wild in south China, southwest China and the like. The chrysanthemum tea is slightly red, has mild nature, is bitter and slightly pungent in taste, cools blood and detoxifies, is mainly prepared from tender tips and leaves of food, can be fried to eat, used as soup or used as a hot pot material, is crisp and tasty in texture, is similar to the taste of crowndaisy chrysanthemum, has the effects of clearing heat and detoxicating, promoting blood circulation and dissipating blood stasis and the like, and can also treat urinary system infection, sphagitis, sore and the like.
A neuroprotective agent is a neuromodulator that protects vulnerable neurons, reducing or preventing disease progression. Can reduce neuronal death, and the commonly used neuroprotective agents include calcium channel blockers, free radical scavengers, glutamate antagonists, cell membrane stabilizers, and the like. Currently, neuroprotective agents are one of the therapeutic approaches for acute stroke. Neuroprotective agents can inhibit cell death and prevent tissue reperfusion injury in ischemic areas, thereby prolonging treatment time with fibrinolytic therapy after stroke onset.
Disclosure of Invention
The invention discloses compounds of formula I and pharmaceutically acceptable salts thereof;
the preparation method of the compound of the formula I comprises the following steps:
A. reflux-extracting herba Duchesneae Indicae with 80-95 vol% ethanol for 1-3 times, mixing extractive solutions, concentrating, and drying to obtain extract;
B. mixing the extract and diatomite at a weight ratio of 1:1-3, sequentially eluting with petroleum ether, dichloromethane and ethyl acetate under reflux, collecting ethyl acetate reflux extract, and recovering solvent to obtain ethyl acetate extract;
C. separating the ethyl acetate extract with silica gel column chromatography, gradient eluting with chloroform-methanol solution system at volume ratio of 20:1-0:1, and collecting eluate of 11:1-9: 1;
D. separating the elution residue obtained in the step C by silica gel column chromatography, performing gradient elution by adopting a chloroform-methanol solution system in a volume ratio of 50:1-0:1, and eluting the elution residue of the collection section by chloroform-methanol 29:1-20: 1;
E. d, continuously carrying out silica gel column chromatographic separation on the elution fraction obtained in the step D, carrying out gradient elution by adopting a chloroform-methanol solution system according to the volume ratio of 100:0-0:100, and taking the elution fraction eluted from the collection section by chloroform-methanol of 40-21: 60-79;
F. and E, separating the elution residue obtained in the step E by MCIgel column chromatography, performing gradient elution by adopting a methanol-water solution system according to the volume ratio of 10:90-100:0, and recovering the solvent from the elution residue of 30:70 sections of methanol-water.
The invention also discloses application of the compound shown in the formula I in preparing a nerve cell protection medicament.
The invention also discloses a nerve cell protection pharmaceutical composition which contains the compound shown in the formula I and/or pharmaceutically acceptable salt thereof.
The pharmaceutical composition also contains one or more of herba Duchesneae Indicae, herba Hedyotidis Diffusae, caulis Spatholobi, radix Rhodomyrti, radix Malloti Apeltae, radix Urenae Lobatae, and herba Thlaspis.
The medicinal composition also contains one or more extracts of herba Duchesneae Indicae, herba Hedyotidis Diffusae, caulis Spatholobi, radix Rhodomyrti, radix Malloti Apeltae, radix Urenae Lobatae, and herba Thlaspis.
The dosage form of the pharmaceutical composition comprises tablets, capsules, granules, powder, pills, solutions, suspensions, syrups, injections, ointments, suppositories or sprays.
The compound of the formula I is a compound newly found in emilia sonchifolia, and the cell level experiment of the experimental example 2 shows that the compound has an obvious protective effect on PC12 cells damaged by corticosterone, embodies a better nerve cell protective effect, and is a novel nerve cell protective medicament.
Drawings
The invention is shown in the following drawings:
FIG. 1 is a graph showing the results of the effect of various concentrations of a compound of formula I on the viability of corticosterone-damaged PC12 cells;
FIG. 2 is a formula I of the compound of the present invention.
Detailed Description
Example 1
The preparation method of the compound comprises the following steps:
A. taking 30kg of emilia sonchifolia, drying, cutting into pieces, performing reflux extraction for 2 times with 6 times of 80% ethanol by volume for 2 hours each time, combining the extracting solutions, concentrating and drying to obtain an extract for later use; mixing the extract with diatomite at a weight ratio of 1:1
B. B, performing reflux elution on the mixture of the extract and the diatomite in the step A by using petroleum ether, taking the residue after the reflux elution of the petroleum ether, performing reflux elution by using dichloromethane, taking the residue after the reflux elution of the dichloromethane, performing reflux elution by using ethyl acetate, and collecting eluent, wherein the name of the eluent is ZF-3;
C. performing chromatographic separation on the eluent ZF-3 by using column chromatography silica gel of 60-180 meshes, wherein the elution solvent is chloroform-methanol, and the volume ratio of the chloroform to the methanol is gradually changed from 20:1to 0: 1; it sequentially collects the elution fractions, wherein the elution fraction is collected as Fr1 with chloroform-methanol (20:1, 19:1, 18: 1); eluting with chloroform-methanol (17:1, 16:1, 15:1) to obtain Fr 2; eluting with chloroform-methanol (14:1, 13:1, 12:1) to obtain Fr 3; eluting with chloroform-methanol (11:1, 10:1, 9:1) to obtain Fr 4; eluting with chloroform-methanol (8:1, 7:1, 6:1) to obtain Fr 5; eluting with chloroform-methanol (5:1, 4:1, 3:1) to obtain Fr 6; eluting with chloroform-methanol (2:1, 1:1, 0:1) to obtain Fr 7;
D. separating the eluate obtained in step C by silica gel column chromatography, performing gradient elution with chloroform-methanol solution system at volume ratio of 50:1-0:1, wherein chloroform-methanol (50:1-45:1) is used for eluting and collecting as Fr4.1; eluting with chloroform-methanol (44:1-40:1) to obtain Fr4.2; eluting with chloroform-methanol (39:1-35:1) to obtain Fr4.3; eluting with chloroform-methanol (34:1-30:1) to obtain Fr4.4; eluting with chloroform-methanol (29:1-20:1) to obtain Fr4.5; eluting with chloroform-methanol (19:1-10:1) to obtain Fr4.6; eluting with chloroform-methanol (9:1-0:1) to collect as Fr4.7. Collecting eluate fraction Fr4.5 of the eluate collection section with chloroform-methanol (29:1-20: 1);
E. d, continuously carrying out silica gel column chromatographic separation on the elution residue obtained in the step D, and carrying out gradient elution by adopting a chloroform-methanol solution system according to the volume ratio of 100:0-0:100, wherein the elution is collected as Fr4.5.1 by using chloroform-methanol (100-81: 0-19); eluting with chloroform-methanol (80-61:20-39) to obtain Fr4.5.2; eluting with chloroform-methanol (60-41:40-59) to obtain Fr4.5.3; eluting with chloroform-methanol (40-21:60-79) to obtain Fr4.5.4; eluting with chloroform-methanol (20-100:80-0) to collect as Fr4.5.5. Collecting eluate Fr4.5.4 of the eluate collection section with chloroform-methanol (40-21: 60-79);
F. and E, separating the elution residue obtained in the step E by MCIgel column chromatography, performing gradient elution by adopting a methanol-water solution system according to the volume ratio of 10:90-100:0, and recovering the solvent from the elution residue of a methanol-water section of 30:70 to obtain the compound shown in the formula 1.
The prepared compound is detected by mass spectrum, nuclear magnetic resonance hydrogen spectrum and nuclear magnetic resonance carbon spectrum, and the result proves that the obtained compound is the compound shown in the formula 1.
Example 2
The preparation method of the compound comprises the following steps:
A. taking 30kg of emilia sonchifolia, drying, cutting into pieces, carrying out reflux extraction for 2 times with 9 times of 95% ethanol by volume for 2 hours each time, combining the extracting solutions, concentrating and drying to obtain an extract for later use; mixing the extract with diatomite at a weight ratio of 1:3
B. B, performing reflux elution on the mixture of the extract and the diatomite in the step A by using petroleum ether, taking the residue after the reflux elution of the petroleum ether, performing reflux elution by using dichloromethane, taking the residue after the reflux elution of the dichloromethane, performing reflux elution by using ethyl acetate, and collecting eluent, wherein the name of the eluent is ZF-3;
C. performing chromatographic separation on the eluent ZF-3 by using column chromatography silica gel of 60-180 meshes, wherein the elution solvent is chloroform-methanol, and the volume ratio of the chloroform to the methanol is gradually changed from 20:1to 0: 1; it sequentially collects the elution fractions, wherein the elution fraction is collected as Fr1 with chloroform-methanol (20:1, 19:1, 18: 1); eluting with chloroform-methanol (17:1, 16:1, 15:1) to obtain Fr 2; eluting with chloroform-methanol (14:1, 13:1, 12:1) to obtain Fr 3; eluting with chloroform-methanol (11:1, 10:1, 9:1) to obtain Fr 4; eluting with chloroform-methanol (8:1, 7:1, 6:1) to obtain Fr 5; eluting with chloroform-methanol (5:1, 4:1, 3:1) to obtain Fr 6; eluting with chloroform-methanol (2:1, 1:1, 0:1) to obtain Fr 7;
D. separating the eluate obtained in step C by silica gel column chromatography, performing gradient elution with chloroform-methanol solution system at volume ratio of 50:1-0:1, wherein chloroform-methanol (50:1-45:1) is used for eluting and collecting as Fr4.1; eluting with chloroform-methanol (44:1-40:1) to obtain Fr4.2; eluting with chloroform-methanol (39:1-35:1) to obtain Fr4.3; eluting with chloroform-methanol (34:1-30:1) to obtain Fr4.4; eluting with chloroform-methanol (29:1-20:1) to obtain Fr4.5; eluting with chloroform-methanol (19:1-10:1) to obtain Fr4.6; eluting with chloroform-methanol (9:1-0:1) to collect as Fr4.7. Collecting eluate fraction Fr4.5 of the eluate collection section with chloroform-methanol (29:1-20: 1);
E. d, continuously carrying out silica gel column chromatographic separation on the elution residue obtained in the step D, and carrying out gradient elution by adopting a chloroform-methanol solution system according to the volume ratio of 100:0-0:100, wherein the elution is collected as Fr4.5.1 by using chloroform-methanol (100-81: 0-19); eluting with chloroform-methanol (80-61:20-39) to obtain Fr4.5.2; eluting with chloroform-methanol (60-41:40-59) to obtain Fr4.5.3; eluting with chloroform-methanol (40-21:60-79) to obtain Fr4.5.4; eluting with chloroform-methanol (20-100:80-0) to collect as Fr4.5.5. Collecting eluate Fr4.5.4 of the eluate collection section with chloroform-methanol (40-21: 60-79);
F. and E, separating the elution residue obtained in the step E by MCIgel column chromatography, performing gradient elution by adopting a methanol-water solution system according to the volume ratio of 10:90-100:0, and recovering the solvent from the elution residue of a methanol-water section of 30:70 to obtain the compound shown in the formula 1.
The prepared compound is detected by mass spectrum, nuclear magnetic resonance hydrogen spectrum and nuclear magnetic resonance carbon spectrum, and the result proves that the obtained compound is the compound shown in the formula 1.
Example 3
The preparation method of the compound comprises the following steps:
A. taking 30kg of emilia sonchifolia, drying, cutting into pieces, performing reflux extraction for 2 times with 7 times of 90% ethanol by volume for 2 hours each time, combining the extracting solutions, concentrating and drying to obtain an extract for later use; mixing the extract with diatomite at a weight ratio of 1:2
B. B, performing reflux elution on the mixture of the extract and the diatomite in the step A by using petroleum ether, taking the residue after the reflux elution of the petroleum ether, performing reflux elution by using dichloromethane, taking the residue after the reflux elution of the dichloromethane, performing reflux elution by using ethyl acetate, and collecting eluent, wherein the name of the eluent is ZF-3;
C. performing chromatographic separation on the eluent ZF-3 by using column chromatography silica gel of 60-180 meshes, wherein the elution solvent is chloroform-methanol, and the volume ratio of the chloroform to the methanol is gradually changed from 20:1to 0: 1; it sequentially collects the elution fractions, wherein the elution fraction is collected as Fr1 with chloroform-methanol (20:1, 19:1, 18: 1); eluting with chloroform-methanol (17:1, 16:1, 15:1) to obtain Fr 2; eluting with chloroform-methanol (14:1, 13:1, 12:1) to obtain Fr 3; eluting with chloroform-methanol (11:1, 10:1, 9:1) to obtain Fr 4; eluting with chloroform-methanol (8:1, 7:1, 6:1) to obtain Fr 5; eluting with chloroform-methanol (5:1, 4:1, 3:1) to obtain Fr 6; eluting with chloroform-methanol (2:1, 1:1, 0:1) to obtain Fr 7;
D. separating the eluate obtained in step C by silica gel column chromatography, performing gradient elution with chloroform-methanol solution system at volume ratio of 50:1-0:1, wherein chloroform-methanol (50:1-45:1) is used for eluting and collecting as Fr4.1; eluting with chloroform-methanol (44:1-40:1) to obtain Fr4.2; eluting with chloroform-methanol (39:1-35:1) to obtain Fr4.3; eluting with chloroform-methanol (34:1-30:1) to obtain Fr4.4; eluting with chloroform-methanol (29:1-20:1) to obtain Fr4.5; eluting with chloroform-methanol (19:1-10:1) to obtain Fr4.6; eluting with chloroform-methanol (9:1-0:1) to collect as Fr4.7. Collecting eluate fraction Fr4.5 of the eluate collection section with chloroform-methanol (29:1-20: 1);
E. d, continuously carrying out silica gel column chromatographic separation on the elution residue obtained in the step D, and carrying out gradient elution by adopting a chloroform-methanol solution system according to the volume ratio of 100:0-0:100, wherein the elution is collected as Fr4.5.1 by using chloroform-methanol (100-81: 0-19); eluting with chloroform-methanol (80-61:20-39) to obtain Fr4.5.2; eluting with chloroform-methanol (60-41:40-59) to obtain Fr4.5.3; eluting with chloroform-methanol (40-21:60-79) to obtain Fr4.5.4; eluting with chloroform-methanol (20-100:80-0) to collect as Fr4.5.5. Collecting eluate Fr4.5.4 of the eluate collection section with chloroform-methanol (40-21: 60-79);
F. and E, separating the elution residue obtained in the step E by MCIgel column chromatography, performing gradient elution by adopting a methanol-water solution system according to the volume ratio of 10:90-100:0, and recovering the solvent from the elution residue of a methanol-water section of 30:70 to obtain the compound shown in the formula 1.
The prepared compound is detected by mass spectrum, nuclear magnetic resonance hydrogen spectrum and nuclear magnetic resonance carbon spectrum, and the result proves that the obtained compound is the compound shown in the formula 1.
Experimental example 1
And (3) structural identification:
a compound of formula I
A white powder; UV (MeOH) lambdamax(logε)239.9(5.16)nm,290.1(4.20)nm；IRνmax 3360,3205,2920,1698,1645,1499,1208,808cm-1；1H and 13C NMR data,see Table 1；HR-ESI-MS m/z282.0768[M–H]–(calcd for C16H12NO4 –,282.0772).
Carbon and hydrogen spectra of the compounds of formula I
Experimental example 3
Study of the neuroprotective effect of the alkaloid compounds of the general formula I:
protective effect of Emilia alkaloids on corticosterone-induced PC12 cell injury
The compound of formula I is used as a research object to investigate the protective effect on nerve cells. The PC12 cell line is a rat adrenal pheochromocyte clone cell line, and the differentiated PC12 cell has typical nerve cell characteristics and is rich in glucocorticoid receptor expression on the cell membrane. The induction of PC12 cell damage with high concentrations of corticosterone has become a common in vitro model for the study of depression. In the research, high-concentration corticosterone and PC12 cells are incubated together to simulate the cerebral neuron damage state of depression patients, so that the protective effect of a bit of red alkaloid on nerve cells is observed.
1 materials and reagents
1.1 pharmaceutical compounds of formula I.
1.2 reagent rat pheochromocytoma PC12 cells (Shanghai cell bank); refined fetal bovine serum, horse serum, penicillin sodium, streptomycin (Gibco); corticosterone, DMEM, MTT, dimethylsulfoxide (Sigma).
1.3 Instrument Water purification apparatus (Millipore, USA); carbon dioxide incubator (NAPCO 6500TC france); continuous wavelength plate reader (BIO-TEK, USA).
2 method
2.1PC12 cell culture
Placing the PC12 cell fluid in a 15mL centrifuge tube, centrifuging at 1000rpm for 5min, discarding the supernatant, suspending the cells in high-sugar DMEM medium containing 10% fetal calf serum and 5% horse serum, subpackaging in 25mL culture bottles, and culturing in a carbon dioxide incubator at 37 ℃ for 1 time every 2-3 days.
2.2 Effect of test samples on the viability of corticosterone-damaged PC12 cells
PC12 cell culture medium is cultured to logarithmic growth phase, and cell is resuspended in DMEM medium (containing penicillin sodium 100U/ml and streptomycin 100. mu.g/ml) containing 10% fetal calf serum and 5% horse serum, and cell concentration is adjusted to 1 × 10 per ml5And (4) respectively. Cells are inoculated in a 96-well plate, 100 mu L of cell sap is added into each well, the plate is placed in a CO 2 incubator at 37 ℃ for adherent culture for 2-4d, and the cells can be used for experiments after growing to the bottom of the well. Adding 250 mu mol/mL corticosterone, acting for 24h, adding different concentrations (5.0,10.0 and 20 mu g/mL) of the compound of the formula I, acting for 24h, detecting the activity of the cells by using an MTT method, and measuring the absorbance A value of each group of cells at 450nm by using a microplate reader. The average absorption value of the control group is taken as 100%, and the ratio of the absorption value of each treatment group to the control group and the model group is used for calculating the survival rate.
The results of the experiments are shown in FIG. 1, from which it can be seen that the compound of formula I shows a strong protective effect (P <0.05) at 5. mu.g/mL) and a cell viability of 87%. at higher concentrations (20. mu.g/mL) the cell viability is reduced.
Claims (7)
1. A compound of formula I:
or a pharmaceutically acceptable salt thereof.
2. The compound of claim 1, wherein the preparation method comprises the steps of:
A. reflux-extracting herba Duchesneae Indicae with 80-95 vol% ethanol for 1-3 times, mixing extractive solutions, concentrating, and drying to obtain extract;
B. mixing the extract and diatomite at a weight ratio of 1:1-3, sequentially eluting with petroleum ether, dichloromethane and ethyl acetate under reflux, collecting ethyl acetate reflux extract, and recovering solvent to obtain ethyl acetate extract;
C. separating the ethyl acetate extract with silica gel column chromatography, gradient eluting with chloroform-methanol solution system at volume ratio of 20:1-0:1, and collecting eluate of 11:1-9: 1;
D. separating the elution residue obtained in the step C by silica gel column chromatography, performing gradient elution by adopting a chloroform-methanol solution system in a volume ratio of 50:1-0:1, and eluting the elution residue of the collection section by chloroform-methanol 29:1-20: 1;
E. d, continuously carrying out silica gel column chromatographic separation on the elution fraction obtained in the step D, carrying out gradient elution by adopting a chloroform-methanol solution system according to the volume ratio of 100:0-0:100, and taking the elution fraction eluted from the collection section by chloroform-methanol of 40-21: 60-79;
F. and E, separating the elution residue obtained in the step E by MCIgel column chromatography, performing gradient elution by adopting a methanol-water solution system according to the volume ratio of 10:90-100:0, and recovering the solvent from the elution residue of 30:70 sections of methanol-water.
3. Use of a compound according to claim 1 or 2 for the preparation of a medicament for neuroprotection.
4. A neuroprotective pharmaceutical composition comprising a compound of claim 1 or 2 and/or a pharmaceutically acceptable salt thereof.
5. The pharmaceutical composition of claim 4, wherein: also contains one or more of herba Duchesneae Indicae, herba Hedyotidis Diffusae, caulis Spatholobi, radix Rhodomyrti, radix Malloti Apeltae, radix Urenae Lobatae, and herba Thlaspis.
6. The pharmaceutical composition of claim 4 or 5, wherein: also contains one or more extracts of herba Duchesneae Indicae, herba Hedyotidis Diffusae, caulis Spatholobi, radix Rhodomyrti, radix Malloti Apeltae, radix Urenae Lobatae, and herba Thlaspis.
7. The pharmaceutical composition of claim 4, wherein: the dosage form of the pharmaceutical composition comprises tablets, capsules, granules, powder, pills, solutions, suspensions, syrups, injections, ointments, suppositories or sprays.
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