CN109206429B - Isoquinoline alkaloid compound and preparation method and application thereof - Google Patents
Isoquinoline alkaloid compound and preparation method and application thereof Download PDFInfo
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- CN109206429B CN109206429B CN201710553004.6A CN201710553004A CN109206429B CN 109206429 B CN109206429 B CN 109206429B CN 201710553004 A CN201710553004 A CN 201710553004A CN 109206429 B CN109206429 B CN 109206429B
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
- C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
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Abstract
The invention discloses an isoquinoline alkaloid compound and a preparation method and application thereof. The invention discloses an isoquinoline alkaloid compound shown as a formula (I) and a preparation method and application thereof. The invention also discloses the application of the hypecoum leptocarpum. The compound is separated from the fine fruit fennel, alcohol extraction is adopted to dry whole herb coarse powder of the fine fruit fennel, decompression concentration is carried out, MCI column chromatography elution is carried out, silica gel column chromatography elution is carried out, TLC detection is carried out, and the compound is prepared through two-dimensional liquid chromatography. Pharmacodynamic tests prove that the compound and the fennel fruit have good antitumor activity.
Description
Technical Field
The invention belongs to the technical field of novel alkaloids, and particularly relates to an isoquinoline alkaloid compound and a preparation method and application thereof.
Background
Hypecoum leptocarpum hook.f. et Thoms.) is a plant of Carum of Papaveraceae, is one of the authentic species of the traditional Tibetan medicine, and has the effects of treating cold and fever, pneumonia and cough, fever of febrile infectious diseases, hepatitis, cholecystitis, arthralgia, sore throat, conjunctival congestion, food poisoning and the like.
The chemical composition research of the illicium tenuipes which can be consulted in the literature at present reports that twenty alkaloid compounds have less research on the pharmacological activity of monomer compounds thereof.
No report that the hypecoum leptocarpum and alkaloid components thereof have anticancer effect is found.
Disclosure of Invention
The invention discloses an isoquinoline alkaloid compound shown in the formula (I) or pharmaceutically acceptable salt or solvate thereof:
wherein R is1、R2Each independently selected from hydrogen, hydroxy, C1～C4An alkoxy group; r3Is selected from- (CH)2)mNH(CH2)nCH3And m and n are independently selected from integers of 0-3.
Further, R1、R2Are all methoxy groups.
Further, m is 2 and n is 0.
Further, the compound is
A process for the preparation of a compound of the invention comprising the steps of: taking dried whole plants of the hypecoum leptocarpum, crushing and sieving, extracting for 1-3 times by using a solvent which is miscible with water, combining extracting solutions, dispersing by water after concentration, extracting, concentrating a water phase, performing column chromatography by using macroporous resin or microporous resin, eluting by using a solvent, performing silica gel column chromatography, performing gradient elution by using a mixed solvent, detecting by TLC, performing column preparation by using C18, and eluting by using the solution to obtain the hypecoum leptocarpum.
Further, the water-miscible solvent is ethanol.
Further, the number of extraction times was 3, and each extraction time was 12 hours.
Furthermore, the mass ratio of the volume of the extraction solvent used to the dry whole plant of the illicium henryi per time is 4-20L: 1kg, preferably 10L:1kg.
Further, the extraction solvent is petroleum ether.
Further, the volume ratio of the extraction solvent to the aqueous solution is 0.5-10: 1, preferably 1.5: 1.
Further, the macroporous resin is D101 and AB-8 type macroporous resin, and the microporous resin is MCI resin;
furthermore, the solvents used for the macroporous resin or microporous resin column chromatography elution are water and methanol in sequence.
Further, the solvent used for silica gel column chromatography is dichloromethane-methanol solvent; and the elution gradient is: dichloromethane: the volume ratio of methanol is 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3: 7.
The elution solution of the C18 column is a mixed solution of acetonitrile and phosphoric acid aqueous solution with the volume ratio of 15: 85.
Preferably, the mass concentration of phosphoric acid in the phosphoric acid aqueous solution is 0.1%.
Use of a compound of the invention, or a pharmaceutically acceptable salt thereof, or a solvate thereof, characterized in that: can be used for preventing and/or treating tumor.
Further, the tumor is lung adenocarcinoma and/or gastric cancer.
A pharmaceutical composition for treating tumor is a preparation prepared by taking the compound, or the solvate or the pharmaceutically acceptable salt thereof as an active ingredient and adding pharmaceutically acceptable auxiliary materials.
Use of Hypecoum leptocarpum for preventing and/or treating tumor is provided.
Further, the tumor is lung adenocarcinoma.
The preparation of the invention comprises injection, powder injection, tablets, capsules, granules, capsules, pills, dripping pills, oral solution and the like.
The invention shows that the illicium henryi medicinal material has an inhibition effect on the proliferation of human lung adenocarcinoma cells (A549), shows that the medicinal material has a certain antitumor activity, and can be used for developing antitumor medicaments. The compound extracted from the star anise has obvious inhibition effect on the proliferation of human lung adenocarcinoma cells (A549) and human gastric cancer cells (MGC-803), and shows that the compound has better anti-tumor activity.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
Detailed Description
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
The experimental materials used in the following examples were all commercially available.
Example 1
(1) Extraction of compound leptin acaramine
Taking 1.5Kg of dried whole plant of the fine Hypecoum leptocarpum, crushing and sieving by a 40-mesh sieve, respectively leaching 3 times by 15L of 95% ethanol, each time for 12 hours, combining ethanol extract, dispersing by 1L of water after decompression concentration, extracting by 1.5L of petroleum ether, discarding a petroleum ether layer, performing MCI column chromatography after decompression concentration of a water layer, respectively eluting by water and methanol, collecting methanol eluent, performing silica gel column chromatography after concentration, and performing dichloromethane-methanol gradient elution with the gradient of dichloromethane: methanol in a 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7 by TLC assay of the pooled fractions, combining the same fractions to give 6 fractions numbered fr.1, fr.2, fr.3, fr.4, fr.5, fr.6 in that order, passing the fraction containing the compound leptin ocaramine (fr.4) through a C18 preparative column, passing acetonitrile: pure compound leptin ocaramine17mg was prepared by elution with water (containing 0.1% phosphoric acid) ═ 15: 85.
(2) Structure identification of compound leptin ocaramine
The compound of the invention is light yellow powder, is dissolved in d-DMSO and is subjected to determination of the chemical structure of the compoundThe following map detection: (1) high resolution mass spectrometry; (2) nuclear magnetic resonance one-dimensional spectrum1H、13C; (3) nuclear magnetic resonance two-dimensional spectra HSQC and HMBC; (4) ultraviolet and infrared spectra.
And (3) structure identification process: according to one-dimensional nuclear magnetism1H、13C NMR spectrum, and preliminarily judging to be alkaloid compounds. Molecular ion Peak 367.1656[ M + H ] obtained according to ESI-MS]+And nuclear magnetic data to obtain molecular formula C21H22N2O4. The unsaturation degree was 10. 3431cm in IR Spectroscopy-1Absorption peaks for secondary amino groups 2956,2870,2922 and 2820cm-1Methylene and methyl absorption peaks. Further confirmation by two-dimensional nuclear magnetic spectrum data, especially in HMBC spectra, -OCH2O- (δ 6.37, s,1H) is associated with C-7(δ 144.8) and C-8(δ 0141.5), OMe (δ 13.84, s,3H) is associated with C-4 '(δ 2149.0), OMe (δ 33.80, s,3H) is associated with C-5' (δ 147.5), NMe (δ 2.55, s,3H) is associated with C-8 '(δ 49.6), H-7' (δ 2.92, t, J ═ 7.3Hz,2H) is associated with C-5 '(δ 113.8), C-6' (δ 127.6), C-1 '(δ 131.9), and C-4' (δ 149.0). These conclusions are consistent with functional groups as indicated by infrared spectroscopy. By means of further1H、13C NMR, HSQC and HMBC spectrogram analysis (Table 1) finally confirms that the structural formula of the compound is as follows:
the compound structure data are as follows: molecular formula C21H22N2O4Molecular weight is 366; UV (MeOH) λ max (log ε) 208,244,310,378 nm; IR (KBr) v max 3431,2956,2870,2922,2820,1631,1521,1460,1377,1128,1048,614,582cm–1；positive HRESIMS:m/z 367.1656[M+H]+(calcd for C21H23N2O4[M+H]+,367.1658)；1H、13Nuclear magnetic data such as C are shown in table 1.
TABLE 1 Nuclear magnetic data (Ind-DMSO) for the Compounds of the invention
The advantageous effects of the present invention are described below by experiments.
Experiment 1 Ex vivo antitumor Activity study of Hypecoum leptocarpum medicinal Material
1. Sample pretreatment
Taking 200g of dried whole plant of the fine fructus Hypecoi Levoniwii, crushing, sieving with a 40-mesh sieve, respectively extracting with 2L of 95% ethanol for 3 times, each time for 12 hours, combining ethanol extract, concentrating under reduced pressure, and freeze-drying to obtain an alcohol extract sample of the fine fructus Hypecoi Levoniwii.
2. Experimental study on in vitro antitumor activity of alcohol extract of Hypecoum leptocarpum
(1) Experimental cell lines
Human lung adenocarcinoma (a549) cells, purchased from the chinese academy of sciences cell bank.
(2) Experimental drugs and reagents
Experimental drugs: the alcohol extract of the fine fruit caraway is obtained by the pretreatment.
Reagent: DMEM cell complete medium (Gibco); fetal bovine serum (Hyclone corporation); pancreatin cell digest (ilex purpurea Hassk); penicillin streptomycin mixed solution double antibody (zilu pharmaceuticals ltd).
(3) Experimental methods
a cell culture
A549 cells (human lung adenocarcinoma) were cultured in DMEM medium containing 10% fetal bovine serum at 37 deg.C and 5% CO2In the incubator, cells in logarithmic growth phase are taken for experiment.
b MTT method for determining influence of compound on activity of different tumor cells
Introducing CO2Tumor cells cultured in an incubator were digested with 0.25% trypsin, blown into a single cell suspension, and the cell suspension was seeded in a 96-well plate (1X 10)4Perwell), cultured for 24 h. The compound was dissolved in DMSO, added to a 96-well plate, and cultured for 24 hours, followed by addition of 20. mu.L of MTT per well and culture at 37 ℃ for 4 hours. The supernatant was discarded, 150. mu.L of DMSO was added to each well to completely dissolve formazan, and the absorbance value was measured at a wavelength of 490nm using a microplate reader, and the cell death rate was calculated as IC50Values represent the cytotoxic activity of the compound.
c statistical method
All data are shown as (X + -SD), the data are analyzed and processed by GraphPad Prism 5 software, and whether significant difference exists between each group and a blank group is observed by using t test, wherein P < 0.05 is significant difference.
The half inhibitory rate IC50 value of human lung adenocarcinoma cells (a549) was 21.07 ± 3.72 μ g/mL as a result of measuring the cell inhibitory rate by the d MTT method (n ═ 5).
The result shows that the hypecoum leptocarpum medicinal material has an inhibiting effect on the proliferation of human lung adenocarcinoma cells (A549), and the medicinal material has anti-tumor activity and can be used for developing anti-tumor medicaments.
Experiment 2 Experimental study of in vitro antitumor Activity of Compounds of the present invention
(1) Experimental cell lines
Human lung adenocarcinoma (A549) cells and human gastric carcinoma (MGC-803) cells were purchased from the cell bank of Chinese academy of sciences.
(2) Experimental drugs and reagents
Experimental drugs: the compound leptin ocaramine isolated in example 1 was used;
positive control drug: doxorubicin (Doxorubicin), purchased from shenzhen kale pharmaceutical limited, lot No.: 1612E 1;
reagent: DMEM cell complete medium (Gibco); fetal bovine serum (Hyclone corporation); pancreatin cell digest (ilex purpurea Hassk); penicillin streptomycin mixed solution double antibody (zilu pharmaceuticals ltd).
(3) Experimental methods
a cell culture
Taking A549 cells (human lung adenocarcinoma) and MGC-803 cells (human gastric cancer), culturing at 37 deg.C with DMEM culture solution containing 10% fetal calf serum and 5% CO2In the incubator, cells in logarithmic growth phase are taken for experiment.
b MTT method for determining influence of compound on activity of different tumor cells
Introducing CO2Digesting two kinds of tumor cells cultured in incubator with 0.25% pancreatin, respectively, beating into single cell suspension, and pulverizingThe cell suspension was inoculated in 96-well plates (1X 10)4Perwell), cultured for 24 h. The compound was dissolved in DMSO, added to a 96-well plate, and cultured for 24 hours, followed by addition of 20. mu.L of MTT per well and culture at 37 ℃ for 4 hours. The supernatant was discarded, 150. mu.L of DMSO was added to each well to completely dissolve formazan, and the absorbance value was measured at a wavelength of 490nm using a microplate reader, and the cell death rate was calculated as IC50Values represent the cytotoxic activity of the compound.
c statistical method
All data are shown as (X + -SD), the data are analyzed and processed by GraphPad Prism 5 software, and whether significant difference exists between each group and a blank group is observed by using t test, wherein P < 0.05 is significant difference.
The results of measuring the cell inhibition rate by the d MTT method (n ═ 5) are shown in table 2.
TABLE 2 inhibitory Effect of the Compounds of the present invention on human Lung adenocarcinoma cells and human stomach cancer cells
The results show that the novel compound leptin ocaramine has obvious inhibition effect on the proliferation of human lung adenocarcinoma cells (A549) and human gastric cancer cells (MGC-803), and the compound has good antitumor activity and can be used for developing antitumor drugs.
In conclusion, the invention discovers that the hypecoum leptocarpum medicinal material can resist tumors, particularly has obvious inhibiting effect on lung adenocarcinoma, and simultaneously, the invention separates a compound leptin caraamine from the hypecoum leptocarpum medicinal material, has definite anti-tumor activity, particularly has definite inhibiting effect on lung adenocarcinoma and gastric cancer, and has excellent application prospect.
Claims (10)
1. The following compounds or pharmaceutically acceptable salts thereof:
2. a process for the preparation of a compound according to claim 1, characterized in that: the method comprises the following steps: taking dried whole plants of the hypecoum leptocarpum, crushing and sieving, extracting for 1-3 times by using a solvent which is miscible with water, combining extracting solutions, dispersing by water after concentration, extracting, concentrating a water phase, performing column chromatography by using macroporous resin or microporous resin, eluting by using a solvent, performing silica gel column chromatography, performing gradient elution by using a mixed solvent, detecting by TLC, performing column preparation by using C18, and eluting by using the solution to obtain the hypecoum leptocarpum.
3. The method of claim 2, wherein: the water-miscible solvent is ethanol, and/or the extraction times are 3 times, and/or the extraction time is 12 hours each time, and/or the mass ratio of the volume of the extraction solvent used each time to the dry whole plant of the hypecoum leptocarpum is 4-20L: 1kg.
4. The production method according to claim 3, characterized in that: the mass ratio of the volume of the extraction solvent used to the dry whole herb of the hypecoum leptocarpum is 10L:1kg.
5. The method of claim 2, wherein: the extraction solvent is petroleum ether, and/or the volume ratio of the extraction solvent to the aqueous solution is 0.5-10: 1.
6. The method of claim 5, wherein: the volume ratio of the extraction solvent to the aqueous solution was 1.5: 1.
7. The production method according to any one of claims 2 to 6, characterized in that: the macroporous resin is D101 and AB-8 type macroporous resin, and the microporous resin is MCI resin; and/or the solvents used for the macroporous resin or microporous resin column chromatography elution are water and methanol in sequence; and/or the solvent used for silica gel column chromatography is a dichloromethane-methanol solvent, and the elution gradient is dichloromethane: the volume ratio of methanol is 9:1, 8:2, 7:3, 6:4, 5:5, 4:6 and 3: 7; and/or the elution solution of the C18 column is a mixed solution of acetonitrile and a phosphoric acid aqueous solution in a volume ratio of 15: 85.
8. The method of claim 7, wherein: the mass concentration of phosphoric acid in the phosphoric acid aqueous solution is 0.1%.
9. Use of a compound of claim 1 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the prevention and/or treatment of lung adenocarcinoma.
10. A pharmaceutical composition for treating tumors, comprising: the compound or the pharmaceutically acceptable salt thereof as an active ingredient, and pharmaceutically acceptable auxiliary materials.
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