CN114075161B - Preparation method of alpha-pyrone compound for treating respiratory diseases - Google Patents
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- C07D309/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings
- C07D309/34—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
- C07D309/36—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with oxygen atoms directly attached to ring carbon atoms
- C07D309/38—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with oxygen atoms directly attached to ring carbon atoms one oxygen atom in position 2 or 4, e.g. pyrones
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
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Abstract
The invention relates to an alpha-pyrone compound, a preparation method thereof and application thereof in treating respiratory diseases, wherein the alpha-pyrone compound has a structure shown in a formula I:
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to an alpha-pyrone compound, a preparation method thereof and application thereof in treating respiratory diseases.
Background
Baikal skullcap root, also called camellia root and native gold tea root, is the dry root of Scutellaria baicalensis Georgi of Labiatae, and has bitter and cold properties, enters lung, gallbladder, spleen, small intestine and large intestine channels, has the effects of clearing heat and drying dampness, purging fire and removing toxicity, stopping bleeding and the like, and is clinically used for treating damp-warm disease, summer-heat and dampness, chest distress and nausea, lung heat and cough and other symptoms. The inventor previously isolated a compound with strong inhibitory activity to Respiratory Syncytial Virus (RSV) from scutellaria baicalensis (201710929445.1), and isolated an alpha-pyrone compound with strong inhibitory activity to RSV from scutellaria baicalensis in order to further study an antiviral compound with a novel structure in scutellaria baicalensis.
Disclosure of Invention
The invention provides an alpha-pyrone compound, a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof, which is separated from scutellaria baicalensis, and is characterized in that the alpha-pyrone compound has a structure shown in a formula I:
Another embodiment of the present invention provides a method for preparing the α -pyrones of the structure of formula I, characterized by comprising the steps of:
(1) Crushing: pulverizing Scutellariae radix to 20-80 mesh to obtain fine powder;
(2) Extraction: reflux-extracting the fine powder obtained in the step (1) with ethyl acetate for 2-3 times, each time for 0.5-1.0 hour, mixing the extracts, and concentrating to obtain an extract for later use;
(3) And (3) purification: sequentially carrying out normal phase silica gel column chromatographic separation and high performance liquid chromatographic separation on the extract obtained in the step (2) to obtain an alpha-pyrone compound with the structure shown in the formula I, wherein R is H;
(4) Optionally, the alpha-pyrone compound with the structure shown in the formula I obtained in the step (3) is dissolved in an organic solvent, and the alpha-pyrone compound with the structure shown in the formula I, wherein R is C1-C3 alkyl or C1-C3 alkyl acyl, is obtained through alkylation reaction or acylation reaction.
In the step (2) of the preparation method, 4-6L of ethyl acetate is used for each kilogram of scutellaria; in the chromatographic separation of the normal phase silica gel column in the step (3), the stationary phase is 200-300 meshes of silica gel, the mobile phase is 30-40% of ethyl acetate/petroleum ether mixed solvent, and the elution volume is 4-5 column volumes; semi-preparative C is used as chromatographic column in high performance liquid chromatography 18 Chromatography column, kromasil,7 μm, 10X 250mm, flow rate 2mL/min, with 90% methanol/water mixed solvent (containing 0.1% acetic acid) as the mobile phase; the percentages of the mixed solvents are volume percentages; in the step (4), the organic solvent is preferably one or a mixture of more of dichloromethane, chloroform, acetone, toluene, THF, diethyl ether and DMF.
Another embodiment of the present invention provides a pharmaceutical composition, which is characterized in that the pharmaceutical composition comprises an α -pyrone compound having the structure of formula I, a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof, as an active ingredient.
Another embodiment of the present invention provides a pharmaceutical composition, wherein the pharmaceutical composition comprises α -pyrone compounds, stereoisomers, tautomers or pharmaceutically acceptable salts thereof having the structure of formula I as an active ingredient; the pharmaceutical composition also comprises other antiviral drugs. Further comprises pharmaceutical excipients (such as pharmaceutically acceptable carriers, excipients and diluents).
Another embodiment of the present invention provides the use of the α -pyrone compounds, stereoisomers, tautomers or pharmaceutically acceptable salts thereof having the structure of formula I above for the preparation of antiviral medicaments. The antiviral agent is used for preventing and/or treating diseases caused by Respiratory Syncytial Virus (RSV).
Another embodiment of the present invention provides the use of an α -pyrone compound of the structure of formula I, a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof, as described above, for the preparation of an anti-RSV medicament.
Another embodiment of the present invention provides the use of the α -pyrone compounds of the structure of formula I, stereoisomers, tautomers or pharmaceutically acceptable salts thereof as described above for the preparation of a medicament for the prevention and/or treatment of respiratory diseases. The respiratory disease is preferably caused by Respiratory Syncytial Virus (RSV).
The term "pharmaceutically acceptable salts" as used herein refers to non-toxic inorganic or organic acid and/or base addition salts, as described in "Salt selection for basic drugs", int.j.pharm. (1986), 33,201-217.
The alkylation reaction conditions described in the present invention are conventional in the art: under the action of alkali and alkylating reagent, wherein the alkylating reagent is preferably R 2 X, wherein X is halogen, R 2 The alkylating agent is preferably methyl, ethyl, n-propyl, isopropyl or cyclopropyl, the alkylating agent is preferably methyl iodide or ethyl bromide, and the base is preferably alkali metal hydride, alkali metal alkyl, alkali metal hydroxide or alkali metal carbonate.
The acylation reaction conditions described in the present invention are also conventional in the art: reacting under the action of alkali and acylating agent, wherein the acylating agent is selected from R 2 X (acyl halide), R 2 OR 2 (acid anhydride), R 2 OH (acid), wherein X is halogen, R 2 As the C1-3 alkyl acyl group, acetyl chloride, acetyl bromide, acetic anhydride, acetic acid and the like are preferable as the acylating agent, and alkali metal hydroxide, triethylamine, DCC/DMAP, pyridine and the like are preferable as the base.
The invention has the advantages that: the alpha-pyrone compound with a brand-new structure and a structure shown in formula I is separated from the scutellaria baicalensis for the first time, and an activity test shows that the alpha-pyrone compound with modified hydroxyl groups has a toxicity inhibiting index (TI) = TC for Respiratory Syncytial Virus (RSV) 50 /EC 50 And higher.
Detailed Description
In order to facilitate a further understanding of the invention, the following examples are provided to illustrate it in more detail. However, these examples are only for better understanding of the present invention and are not intended to limit the scope or the principle of the present invention, and the embodiments of the present invention are not limited to the following. The scutellaria baicalensis used in the embodiment of the invention is scutellaria baicalensis decoction pieces.
Example 1
(1) Crushing: pulverizing Scutellariae radix (1 kg) to 20-80 mesh to obtain fine powder;
(2) Extraction: refluxing and extracting the fine powder obtained in the step (1) with ethyl acetate (4L) for 3 times, wherein the refluxing and extracting time is 0.5h each time, combining the extracting solutions and concentrating to obtain an extract for later use;
(3) And (3) purification: taking the extract obtained in the step (2), firstly carrying out normal phase silica gel column chromatographic separation, wherein the stationary phase is as follows: 200-300 mesh silica gel, mobile phase is 30% ethyl acetate/petroleum ether mixed solvent, 5 column volumes are eluted, high performance liquid chromatography separation is carried out after eluent is concentrated, and the fixed phase: semi-preparation of C 18 Chromatography column (Kromasil, 7 μm, 10X 250 mm) at a flow rate of 2mL/min with 90% methanol/water, 0.1% AcOH as mobile phase; the percentages of the mixed solvents are volume percentages, and the alpha-pyrone compound (41.2 mg, R is H, white powder) with the structure of the formula I is obtained. Structure confirmation data:
formula I (R = H):
(c,0.12,MeOH), 1 H NMR(CDCl 3 400 MHz) and 13 C NMR(CDCl 3 100 MHz), see table 1; HRESIMS m/z 267.1960[ M ] +H] + (calcd for C 16 H 27 O 3 ,267.1955).
TABLE 1
Example 2
(1) Crushing: pulverizing Scutellariae radix (1 kg) to 20-80 mesh to obtain fine powder;
(2) Extraction: ultrasonically extracting the fine powder obtained in the step (1) with ethyl acetate (6L) for 2 times, performing reflux extraction for 1h each time, combining the extracting solutions, and concentrating to obtain an extract for later use;
(3) And (3) purification: taking the extract obtained in the step (2), carrying out normal phase silica gel column chromatographic separation, wherein the stationary phase is as follows: 200-300 mesh silica gel, the mobile phase is 40% ethyl acetate/petroleum ether mixed solvent, 4 column volumes are eluted, the high performance liquid chromatography separation is carried out after the eluent is concentrated, the stationary phase: semi-preparation of C 18 Chromatography column (Kromasil, 7 μm, 10X 250 mm) with 90% methanol/water, 0.1% AcOH as mobile phase; the percentages of the mixed solvents are volume percentages, and the alpha-pyrone compound (38.3mg, R is H, white powder) with the structure of the formula I is obtained, and the structure confirmation data is consistent with that of the example 1.
Example 3
5mg of the alpha-pyrone compound (R is H) with the structure of formula I obtained in example 1 was dissolved in 3mL of acetone, 0.3mL of methyl iodide and 6mg of potassium carbonate were added at room temperature, after 3 hours of reaction, TLC detected that the reaction was complete, the reaction solution was extracted with dichloromethane and purified according to the methodThe solution is treated with 1M HCl and saturated NaHCO 3 Washing with saturated NaCl, drying with anhydrous sodium sulfate, concentrating, and performing silica gel column chromatography (ethyl acetate/petroleum ether = 1/12-1/8) to obtain α -pyrone compounds (R is Me,4.1mg, colorless oil) with structure confirmed data: formula I (R = Me):
-51.0(c,0.12,MeOH),HRESIMS m/z 281.2110[M+H] + (calcd for C 17 H 29 O 3 ,281.2111). 1 H NMR(CDCl 3 ,400MHz):δ5.97(s,1H,H-5),3.90(s, 3H,4-OCH 3 ),2.62-2.59(m,1H,H-7),1.76(s,3H,CH 3 -14),1.47-1.43(m,2H,H-8-1, H-9),1.31-1.27(m,2H,H-8-2,H-11),1.21-1.17(m,1H,H-12-1),1.14(d,6.8Hz,3H, CH 3 -15),1.09-1.06(m,1H,H-12-2),1.02-1.00(m,2H,H-10),0.80(t,7.1Hz,3H, CH 3 -13),0.76(d,6.0Hz,3H,CH 3 -16),0.71(d,6.4Hz,3H,CH 3 -17).
example 4
5mg of the alpha-pyrone compound (R is H) with the structure of formula I obtained in example 1 is dissolved in 3mL of dichloromethane, 0.5mL of acetic anhydride and 1.0mL of pyridine are added at room temperature, after 2 hours of reaction, TLC detection shows that the reaction is complete, the reaction solution is extracted with dichloromethane and sequentially 1M HCl and saturated NaHCO 3 Washing with saturated NaCl, drying with anhydrous sodium sulfate, concentrating, and performing silica gel column chromatography (ethyl acetate/petroleum ether = 1/10-1/6) to obtain an alpha-pyrone compound (R is Ac,4.4 mg) with a structure shown in formula I, wherein the data of the structure is confirmed: formula I (R = Ac):
-36.7(c,0.12,MeOH),HRESIMS m/z 309.2062[M+H] + (calcd for C 18 H 29 O 4 , 309.2060).
example 5
The antiviral activity of the α -pyrones of the structure of formula i of the present invention was tested according to the method described in the applicant's prior patent (201710929445.1).
The test method comprises the following steps: digesting the cultured monolayer Hep-2 cells by pancreatin, and inoculating the cells into a 96-well plate to grow a monolayer for later use; inoculating RSV virus into Hep-2 cells, adding serum-free 1640 culture medium, and making the mixture at 37 deg.C and 5% CO 2 Culturing under the condition, repeatedly freezing and thawing for 3 times after more than 90% of pathological changes appear, blowing, beating, centrifuging, quantitatively packaging, and freezing and storing in a refrigerator at-80 ℃ for later use. After each test sample was dissolved in 10. Mu.L of methanol per tube, 200. Mu.L of 2-th 1640 culture solution was added, and the mixture was diluted 10 times by 2-fold ratio and 10 dilutions were made, and then inoculated transversely to a monolayer of cells in a 96-well, with 11 columns being virus control and 12 columns being cell control, at 37 ℃ and 5% CO 2 Culturing, observing lesions every hour, and continuously observing for 24h (RSV); after more than 90% of pathological changes appear in the virus control, the liquid in the plate hole is sucked and discarded, 1% neutral red is added for staining, the OD value is measured at the wavelength of 540nm, and the half-poisoning concentration (TC) of the medicine is calculated by a Reed-Muench method 50 ) Half the effective concentration (IC) 50 ) And observing the virus inhibiting and killing effect of the medicine. Dividing the half cytotoxic concentration by the half effective concentration TC 50 /IC 50 The toxicity-inhibiting index is shown. The results are detailed in the table below.
| Test compounds | IC 50 (μM) | TC 50 (μM) | TC 50 /IC 50 |
| Formula I (R = H) | 1.23 | 2.85 | 2.3 |
| Formula I (R = Me) | 0.85 | 7.80 | 9.2 |
| Formula I (R = Ac) | 0.42 | 15.8 | 37.6 |
| Ribavirin | 20.35 | >2000 | _ |
The result shows that the alpha-pyrone compound with the structure of the formula I has the inhibition effect on RSV, and particularly, when the hydroxyl is modified, the toxicity inhibition index (TI) = TC 50 /EC 50 Is remarkably improved, and is expected to be developed for treating respiratory diseases.
Claims (2)
1. A preparation method of alpha-pyrone compounds with a structure shown in formula I is characterized by comprising the following steps:
(1) Crushing: pulverizing Scutellariae radix to 20-80 mesh to obtain fine powder;
(2) Extraction: refluxing and extracting the fine powder obtained in the step (1) with ethyl acetate for 2-3 times, wherein each extraction is carried out for 0.5-1.0 hour, combining the extracting solutions and concentrating to obtain an extract for later use;
(3) And (3) purification: sequentially carrying out normal phase silica gel column chromatographic separation and high performance liquid chromatographic separation on the extract obtained in the step (2) to obtain an alpha-pyrone compound with the structure shown in the formula I, wherein R is H;
(4) Dissolving the alpha-pyrone compound with the structure shown in the formula I, wherein R is H, obtained in the step (3) in an organic solvent, and performing alkylation reaction or acylation reaction to obtain the alpha-pyrone compound with the structure shown in the formula I, wherein R is C1-C3 alkyl or C1-C3 alkyl acyl;
the alpha-pyrone compound with the structure of the formula I is
R is selected from H, C1-C3 alkyl, C1-C3 alkanoyl.
2. The process according to claim 1, wherein the amount of ethyl acetate used in the step (2) is 4 to 6L per kg of Scutellaria baicalensis; in the chromatographic separation of the normal phase silica gel column in the step (3), the stationary phase is 200-300 meshes of silica gel, the mobile phase is 30-40% of ethyl acetate/petroleum ether mixed solvent, and the elution volume is 4-5 column volumes; the chromatographic column adopted in the high performance liquid chromatography separation is semi-preparative C 18 Chromatography column, kromasil,7 μm, 10X 250mm, with 90% methanol/water mixed solvent, containing 0.1% acetic acid as mobile phase; the percentages of the mixed solvents are volume percentages; in the step (4), the organic solvent is one or a mixture of more of dichloromethane, chloroform, acetone, toluene, THF, diethyl ether and DMF.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5808062A (en) * | 1993-11-19 | 1998-09-15 | Warner-Lambert Company | Pyrone derivatives as protease inhibitors and antiviral agents |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| ES2191035T3 (en) * | 1993-11-19 | 2003-09-01 | Parke Davis & Co | DERIVATIVES OF PIRONA AS INHIBITORS OF PROTEASA AND ANTIVIRICAL AGENTS. |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5808062A (en) * | 1993-11-19 | 1998-09-15 | Warner-Lambert Company | Pyrone derivatives as protease inhibitors and antiviral agents |
Non-Patent Citations (1)
| Title |
|---|
| Fupyrones A and B, two new α-pyrones from an endophytic fungus, Fusarium sp. F20;Gao, Han等;《Natural Product Research》;20190103;第34卷(第3期);全文 * |
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