CN116621765B - Synthesis method and application of 2-indolone analogue containing alpha, beta-unsaturated ketone - Google Patents

Synthesis method and application of 2-indolone analogue containing alpha, beta-unsaturated ketone Download PDF

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CN116621765B
CN116621765B CN202310579725.XA CN202310579725A CN116621765B CN 116621765 B CN116621765 B CN 116621765B CN 202310579725 A CN202310579725 A CN 202310579725A CN 116621765 B CN116621765 B CN 116621765B
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indolone
trifluoromethyl
cox
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CN116621765A (en
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穆淑珍
何茂飞
邓璐璐
李江
潘桃花
范艳华
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Guizhou Natural Products Research Center
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/30—Indoles; Hydrogenated indoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to carbon atoms of the hetero ring
    • C07D209/32—Oxygen atoms
    • C07D209/34—Oxygen atoms in position 2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

The invention aims to provide a synthesis method and application of a 2-indolone analogue containing alpha, beta-unsaturated ketone, and the technical scheme is as follows: the preparation method is characterized by comprising the step of carrying out a condensation reaction on aromatic aldehyde and 2-indolone derivatives Knoevenagel, wherein the structure of the aromatic aldehyde is shown as a formula I. The preparation method has the characteristics of simplicity and easiness in operation, and can be realized through one or more steps of limited reactions, and the reaction raw materials are simple and easily obtained. And the prepared compound is tested for in vitro anti-inflammatory activity and COX-2 inhibitory activity. The test result shows that the prepared compound has obvious anti-inflammatory activity, particularly, the compounds e and h have better inhibition activity on the release of NO by RAW264.7 cells induced by LPS than the positive control PDTC, and the compound d, e, f, h has obvious inhibition effect on COX-2. The application of the compound in anti-inflammatory and COX-2 inhibition activities is not reported, and the compound has wide application prospect in the field of anti-inflammatory drugs.

Description

Synthesis method and application of 2-indolone analogue containing alpha, beta-unsaturated ketone
Technical Field
The invention relates to the fields of organic chemistry and pharmaceutical chemistry, in particular to a preparation method and application of a 2-indolone analogue containing alpha, beta-unsaturated ketone.
Background
Inflammation is a defense mechanism against the invasion of harmful substances into life, allowing damaged tissues to repair and heal. At the same time, however, inflammation may cause a series of injuries to the human body, and serious persons may even be life threatening. Arachidonic acid, which is directly related to inflammatory reaction, is a precursor of various bioactive substances, and is one of main metabolic pathways in which cyclooxygenase catalyzes and produces Prostaglandin (PG) and thromboxane, cyclooxygenase has two types, COX-1 and COX-2, COX-1 exists in most tissues such as intestines, stomach and kidneys, and the like, and by promoting synthesis of PG and A2, gastrointestinal mucosa is protected, kidney restriction is regulated, platelet aggregation is promoted, and other internal environmental stability is promoted, COX-1 inhibitor can cause gastrointestinal adverse reaction, while COX-2 is not usually detected in most normal tissues, is mainly induced to generate activity by inflammatory mediators at the inflammatory sites, and mediates pain, fever, inflammation and other reactions through promotion of PG synthesis, so selective COX-2 inhibitor can be studied to avoid adverse reaction of drugs on gastrointestinal tract.
2-indolone compounds widely exist in natural products and cause general attention of scientific researchers due to the wide biological activity of the compounds, but the structure of the derivatives is complicated due to the fact that the anti-inflammatory activity of the 2-indolone derivatives is freshly reported, so that the synthesis steps are long, and the application of the 2-indolone derivatives in the field of medicines is limited. If the structure modification and optimization are carried out on the 2-indolone compounds, candidate compounds which have high curative effect, simple structure, fewer synthesis steps, and are convenient for mass production and are safe and effective are searched for as clinically useful proto-drugs, and the method has important research significance.
The invention designs and synthesizes the 2-indolone derivative containing alpha, beta-unsaturated ketone, and the obtained target compound has simple structure, few synthesis steps and convenient mass production. And the inhibition activity test of inflammatory cells and the test of COX-2 inhibition activity are carried out in vitro, and the activity test result shows that the prepared 2-indolone analogue has obvious anti-inflammatory activity, especially the compounds e and h have better inhibition activity on NO released by RAW264.7 cells induced by LPS than a positive control PDTC, and the prepared 2-indolone analogue d, e, f, g has obvious inhibition effect on COX-2 in the COX-2 inhibition activity test.
Disclosure of Invention
In order to solve the problems of the prior art, the invention aims to provide a synthesis method and application of a 2-indolone analogue containing alpha, beta-unsaturated ketone. The compounds have remarkable inhibition effect on inflammatory cells and COX-2.
In order to achieve the above purpose, the invention adopts the following technical scheme: a2-indolone compound containing alpha, beta-unsaturated ketone shown in formula I,
wherein R is selected from hydrogen, 3- (trifluoromethyl) -3-vinylbenzene, 2- (trifluoromethyl) -3-vinylbenzene, 4- (trifluoromethyl) -3-vinylbenzene; r is R 1 Selected from the group consisting of hydrogen, halogen, trifluoromethyl, amino, 3- (trifluoromethyl) phenylmethylimide, 3,5- (trifluoromethyl) phenylmethylimide, p-nitrobenzenesulfonamide; r is R 2 Selected from hydrogen, acetyl.
As a preferred mode of the invention, the compound is selected from the following compounds:
the invention also provides a preparation method of the compound, which can be selected from the synthetic route: consists of the steps Scheme 1-3:
(1) Preparation of Compounds a-e: adding raw material 2-indolone and benzaldehyde with different substituents in the presence of piperidine as a catalyst, ethanol as a solvent and at the temperature of 80 ℃ to prepare intermediate 8-12 of alpha, beta-unsaturated ketone with different substituents, and then acetylating the intermediate 8-12 and acetic anhydride as raw materials in the presence of sodium carbonate as a catalyst in the presence of tetrahydrofuran as a solvent to prepare 2-indolone analogues a-e containing alpha, beta-unsaturated ketone.
Scheme 1：
Wherein R' is hydrogen, chlorine, trifluoromethyl and amino; r' is trifluoromethyl (2-trifluoromethyl; 3-trifluoromethyl; 4-trifluoromethyl) at different substitution positions.
(2) Preparation of Compounds f-g: takes 5-nitroindole, iron powder and ammonium chloride as raw materials, V Ethanol :V Water and its preparation method =4: 1 is solvent, and the intermediate 5-nitroindole is prepared. Adding an intermediate 5-aminoindole and benzaldehyde with different substituents under the catalysis of Lewis acid and under the condition of taking ethanol as a solvent to prepare compounds f-g with different substituents;
Scheme 2：
wherein R is 1 Trifluoromethyl (3-trifluoromethyl, 3, 5-bis (trifluoromethyl)) in different substitution positions.
(3) Takes 5-nitroindole, iron powder and ammonium chloride as raw materials, V Ethanol :V Water and its preparation method =4: 1 is solvent, preparing intermediate 5-nitroindole; then, taking the intermediate 14 (5-aminoindole) and the compound 17 (p-nitrobenzenesulfonyl chloride) as raw materials, and carrying out condensation reaction under the condition that pyridine is taken as a solvent to prepare an intermediate 18; finally, the intermediate 18 and 3- (trifluoromethyl) benzaldehyde are used as raw materials, piperidine is used as a catalyst, and methanol is used as a solvent to react, so that the alpha, beta-unsaturated ketone analogue h is prepared;
Scheme 3：
wherein R is selected from hydrogen, 3- (trifluoromethyl) -3-vinylbenzene, 2- (trifluoromethyl) -3-vinylbenzene, 4- (trifluoromethyl) -3-vinylbenzene; r is R 1 Selected from the group consisting of hydrogen, halogen, trifluoromethyl, amino, 3- (trifluoromethyl) phenylmethylimide, 3,5- (trifluoromethyl) phenylmethylimide, p-nitrobenzenesulfonamide; r is R 2 Selected from hydrogen, acetyl.
The beneficial technical effects of the invention are as follows: the invention designs and synthesizes the 2-indolone derivative containing alpha, beta-unsaturated ketone, and the obtained target compound has simple structure, few synthesis steps and convenient mass production. And the inhibition activity test of inflammatory cells and the test of COX-2 inhibition activity are carried out in vitro, and the activity test result shows that the prepared 2-indolone analogue has obvious anti-inflammatory activity, especially the compounds e and h have better inhibition activity on NO released by RAW264.7 cells induced by LPS than a positive control PDTC, and the prepared 2-indolone analogue d, e, f, g has obvious inhibition effect on COX-2 in the COX-2 inhibition activity test.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention, and that other drawings can be obtained according to these drawings without inventive faculty for a person skilled in the art.
FIG. 1 is a graph showing the results of cell viability of the compound prepared in example 9 of the present invention.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
The preparation method of the a:
experimental procedure As shown in Scheme 1, first, 2-indolone (399 mg,3.0 mmol), 3- (trifluoromethyl) benzaldehyde (514 mg,3.3 mmol) and 8mL absolute ethanol were added to a round bottom flask, piperidine (52 mg,0.6 mmol) was slowly added dropwise, and the mixture was reacted at 80℃for 3-4h. A precipitate is generated and is generated,the precipitate was filtered and washed with ethanol to give (E) -3- (3- (trifluoromethyl) benzylidene) indol-2-one. (E) -3- (3- (trifluoromethyl) benzylidene) indol-2-one was dissolved in 5mL of tetrahydrofuran, acetic anhydride (4.5 mL,15.0 mmol) and sodium carbonate (156 mg,15.0 mmol) were added, stirred at room temperature for 12h, after completion of the reaction by thin layer chromatography monitoring, diluted with water, extracted with ethyl acetate (3X 50 mL), the organic layer was washed with saturated brine (2X 100 mL) and then dried over anhydrous MgSO 4 Drying, and purifying the crude product on a silica gel column (petroleum ether: ethyl acetate=60:1, V/V) to obtain a target compound a as a yellow solid with a yield of 30.2%, m.p.121.5-123.3 ℃; 1 H NMR(600MHz,CDCl 3 )δ(ppm)8.36(d,J＝8.4Hz,1H),7.92(s,1H),7.87(s,1H),7.84(d,J＝7.8Hz,1H),7.74(d,J＝7.8Hz,1H),7.65(dd,J＝18.0Hz,8.4Hz,1H),7.56(d,J＝7.8Hz,1H),7.38(t,J＝7.8Hz,1H),7.07(t,J＝7.8Hz,1H),2.80(s,3H)； 13 C NMR(150MHz,CDCl 3 )δ(ppm)170.78,168.21,140.66,136.05,135.27,132.19,130.93,129.45,127.61,126.44,125.90,124.68,122.08,121.28,117.00,26.92ppm；ESI-HRMS m/z calculated for C 18 H 12 NO 2 F 3 Na[M+Na] + 354.0712,found354.0705。
example 2
The preparation method of the b:
preparation method As in example 1, 6-chloro-2-indolone (500 mg,3.0 mmol), 3- (trifluoromethyl) benzaldehyde (514 mg,3.3 mmol) and 8mL of absolute ethanol were added to a round bottom flask, piperidine (52 mg,0.6 mmol) was slowly added dropwise and reacted at 80℃for 3-4h. Precipitate is generated, and the precipitate is filtered and washed with ethanol to obtain (E) -6-chloro-3- (3 (trifluoromethyl) benzylidene) indol-2-one. (E) -6-chloro-3- (3 (trifluoromethyl) benzylidene) indol-2-one was dissolved in 5mL of tetrahydrofuran, acetic anhydride (4.5 mL,15.0 mmol) and sodium carbonate (156 mg,15.0 mmol) were added, stirred at room temperature for 12h, diluted with water and extracted with ethyl acetate (3X 50 mL). The organic layer was washed with saturated brine (2X 100 mL) and then dried over anhydrous MgSO 4 Drying, crude productPurification on silica gel column (V Petroleum ether :V Acetic acid ethyl ester =60:1) to give the target compound b as a yellow solid with a yield of 39%, m.p.142.9-144.1 ℃; 1 H NMR(600MHz,CDCl 3 )δ(ppm)8.43(d,J＝1.8Hz,1H),7.84(s,1H),7.59-7.51(m,3H),7.49(s,1H),7.35(d,J＝6.6Hz,1H),7.06(dd,J＝8.4Hz,1.8Hz,1H),2.78(s,3H)； 13 C NMR(150MHz,CDCl 3 )δ(ppm)168.22,159.84,140.92,139.07,135.88,135.38,130.05,125.32,124.64,123.10,121.49,117.28,116.10,114.23,77.25,76.83,55.42,26.82.ESI-HRMS m/z calculated for C 18 H 11 NO 2 F 3 ClNa[M+Na] + 388.0323,found 388.0311。
example 3
The preparation method of the invention c comprises the following steps:
preparation method As in example 2, 6-chloro-2-indolone (500 mg,3.0 mmol), 2- (trifluoromethyl) benzaldehyde (514 mg,3.3 mmol) and 8mL absolute ethanol were added to a round bottom flask, piperidine (52 mg,0.6 mmol) was slowly added dropwise and reacted at 80℃for 3-4h. Precipitate is generated, and the precipitate is filtered and washed with ethanol to obtain (E) -6-chloro-3- (2-fluoro-3-chlorobenzyl) indol-2-one. (E) -6-chloro-3- (2-fluoro-3-chlorobenzyl) indol-2-one was dissolved in 5mL of tetrahydrofuran, acetic anhydride (4.5 mL,15.0 mmol) and sodium carbonate (156 mg,15.0 mmol) were added, stirred at room temperature for 12h, diluted with water, extracted with ethyl acetate (3X 50 mL), the organic layer was washed with saturated brine (2X 100 mL) and then with anhydrous MgSO 4 Drying, and purifying the crude product on silica gel column (V Petroleum ether :V Acetic acid ethyl ester =60:1), to give the objective compound 1c. Yellow solid, yield 25%, m.p.131.0-133.6 ℃; 1 H NMR(600MHz,CDCl 3 )δ(ppm)8.66(d,J＝6.0Hz,1H),7.96(s,1H),7.56-7.60(m,3H),7.40(d,J＝8.4Hz,1H),7.04(dd,J＝8.4Hz,1.8Hz,2H),2.98(s,3H)ppm； 13 C NMR(150MHz,CDCl 3 )δ(ppm)170.59,167.51,141.34,136.73,129.50,127.91,126.56,125.25,124.94,124.70,124.50,124.47,123.26,120.31,119.72,119.05,117.47,26.79；ESI-HRMS m/z calculated for C 18 H 11 NO 2 FCl 3 Na[M+Na] + 388.0323,found 388.0311。
example 4
The preparation method of the d:
preparation method As in example 3, 6-trifluoromethyl-2-indolone (603 mg,3.0 mmol), 3- (trifluoromethyl) benzaldehyde (514 mg,3.3 mmol) and 8mL absolute ethanol were added to a round bottom flask and piperidine (52 mg,0.6 mmol) was slowly added dropwise and heated at 80℃for 3-4h. Precipitate is generated, and the precipitate is filtered and washed with ethanol to obtain (E) -1-acetyl-6-trifluoromethyl-3- (3-trifluoromethyl benzylidene) indol-2-one. (E) -1-acetyl-6-trifluoromethyl-3- (3-trifluoromethylbenzylidene) indol-2-one was dissolved in 10mL of tetrahydrofuran, acetic anhydride (4.5 mL,15.0 mmol) and sodium carbonate (156 mg,15.0 mmol) were added, stirred at room temperature for 12h, diluted with water and extracted with ethyl acetate (3X 50 mL). The organic layer was washed with saturated brine (2X 100 mL) and then dried over anhydrous MgSO 4 Drying, and purifying the crude product on silica gel column (V Petroleum ether :V Acetic acid ethyl ester =60:1) to give the target compound d. Yellow solid, yield 35.2%, m.p.131.2-132.1 ℃, 1 HNMR(600MHz,CDCl 3 )δ(ppm)8.61(s,1H),8.41(s,1H),8.39(d,J＝8.0Hz,1H),7.76(s,1H),7.74(s,1H),7.71(s,1H),7.65(s,1H),7.55(d,J＝0.8Hz,1H),2.77(s,3H)ppm； 13 C NMR(150MHz,CDCl 3 )δ(ppm)170.92,165.44,139.10,138.95,134.91,133.37,131.60,130.95,129.02,129.02,128.79,127.87,127.32,125.31,121.90,119.08,119.08,114.05,26.88；ESI-HRMS m/z calculated for C 19 H 11 F 6 NO 2 Na[M+Na]+422.05927,found 422.05862。
example 5
The preparation method of the invention is as follows:
5-amino-2-indolone (399 mg,3.0 mmol), 3- (trifluoromethyl) benzaldehyde (514 mg,3.3 mmol) and 8mL of absolute ethanol were added to a round bottom flask, piperidine (52 mg,0.6 mmol) was slowly added dropwise, and the mixture was heated at 80℃for 3-4h. Precipitate is generated, and the precipitate is filtered and washed with ethanol to obtain (E) -1-acetyl-5-amino-3- (3-trifluoromethyl benzylidene) indol-2-one. (E) -1-acetyl-5-amino-3- (3-trifluoromethylbenzylidene) indol-2-one was dissolved in 10mL of tetrahydrofuran, acetic anhydride (4.5 mL,15.0 mmol) and sodium carbonate (156 mg,15.0 mmol) were added, stirred at room temperature for 12h, diluted with water and extracted with ethyl acetate (3X 50 mL). The organic layer was washed with saturated brine (2×100 mL) and then dried over anhydrous MgSO4, and the crude product was purified on a silica gel column (petroleum ether: ethyl acetate=60:1, V/V) to give the target compound e as a red solid in 34.4% yield m.p.165.9-166.2 ℃. 1 H NMR(600MHz,CDCl 3 )δ(ppm)8.15(d,J＝8.7Hz,1H),7.92(dq,J＝1.0,2.0Hz,1H),7.81(d,J＝9.5Hz,2H),7.76-7.70(m,1H),7.64(t,J＝7.9Hz,1H),6.87(d,J＝2.4Hz,1H),6.70(dd,J＝2.5,8.7Hz,1H),3.57(s,2H),2.75(s,3H)； 13 C NMR(150MHz,DMSO)δ(ppm)165.56,163.61,138.53,130.86,130.60,128.52,127.53,126.64,126.43,124.65,123.30,121.51,121.15,117.40,113.21,112.66,103.70,21.95；ESI-HRMS m/z calculated for C 18 H 13 F 3 N 2 O 2 Na[M+Na]+456.02078,found 456.01965。
Example 6
The preparation method of the invention:
experimental procedure As shown in Scheme 1, 5-nitroindolin-2-one (500 mg,2.81 mmol) and ammonium chloride (88 mg,1.67 mmol) were first placed in a 25mL round bottom flask, then a mixed solution of ethanol and water (V C2H5OH :V H2O =4: 1). After dissolution, iron powder (784 mg,14 mmol) was added to the mixture and refluxed at 100 ℃ for 1.2 hours. After the reaction is completed, the solution is filtered while it is still hot andconcentrating under reduced pressure to obtain the product 5-aminoindoline-2-ketone. 5-amino-2-indolone (399 mg,3.0 mmol), 3- (trifluoromethyl) benzaldehyde (514 mg,3.3 mmol) and 8mL of absolute ethanol were added to a round bottom flask, and the mixture was refluxed at 80℃for 3-4h under nitrogen protection. Extraction with ethyl acetate (3X 50 mL) and washing of the organic layer with saturated brine (2X 100 mL) followed by anhydrous MgSO 4 Drying, and purifying the crude product on a silica gel column (petroleum ether: ethyl acetate=10:1, V/V) to obtain a target compound f as white powder with a yield of 40.3%, m.p.166.3-166.8 ℃;1H NMR (600 MHz, CD) 3 OD)δ(ppm)8.69(s,1H),8.25(s,1H),8.16(d,J＝7.8Hz,1H),7.80(d,J＝7.5Hz,1H),7.71(t,J＝7.8Hz,1H),7.33(d,J＝2.2Hz,1H),7.26(dd,J＝8.2,2.2Hz,1H),6.95(d,J＝8.2Hz,1H),6.79-6.61(m,1H).3.34(p,J＝1.7Hz,2H)；13C NMR(150MHz,CD3OD)δ(ppm)178.52,157.13,145.60,142.40,137.31,131.85,129.39,128.30,127.10,126.85,124.51,123.81,121.26,117.40,117.38,109.74；ESI-HRMS m/z calculated for C 16 H 11 F 3 N 2 ONa[M+Na]+327.07193,found 327.07157。
Example 7
The preparation method of the g disclosed by the invention comprises the following steps:
preparation method As in example 6, 5-nitroindolin-2-one (500 mg,2.81 mmol) and ammonium chloride (88 mg,1.67 mmol) were placed in a 25mL round bottom flask and then a mixed solution of ethanol and water (V C2H5OH :V H2O =4: 1). After dissolution, iron powder (784 mg,14 mmol) was added to the mixture and refluxed at 100 ℃ for 1.2 hours. After the reaction is completed, the solution is filtered while the solution is hot and concentrated under reduced pressure to obtain the product 5-aminoindolin-2-one. 5-amino-2-indolone (399 mg,3.0 mmol), 3, 5-bis (trifluoromethyl) benzaldehyde (800 mg,3.3 mmol) and 8mL of absolute ethanol were added to a round bottom flask, and the mixture was refluxed at 80℃for 3-4h under nitrogen protection. Extraction with ethyl acetate (3×50 mL), washing of the organic layer with saturated brine (2×100 mL) and drying over anhydrous MgSO4, purification of the crude product on a silica gel column (petroleum ether: ethyl acetate=10:1)V/V) to give the target compound g. Yellow powder with a yield of 41.3%, m.p.191.1-191.5 ℃; 1 H NMR(600MHz,DMSO-d6)δ(ppm)10.49(s,1H),8.76(s,1H),8.42(s,2H),8.09(s,1H),7.23(d,J＝3.23Hz,2H),6.84(s,1H)；13C NMR(150MHz,DMSO-d6)δ(ppm)176.87,154.87,144.05,143.71,139.10,131.31,131.09,128.59,128.59,127.33,124.48,124.01,122.67,122.47,117.95,109.88,36.28.ESI-HRMS m/z calculated for C 17 H 10 F 6 N 2 ONa[M+Na]+395.05930,found 395.05895。
example 8
The preparation method of the h comprises the following steps:
experimental procedure 5-nitroindol-2-one (1,500 mg,2.81 nmol) and ammonium chloride (88 mg,1.67 mmol) are placed in a 25mL round bottom flask as shown in Scheme 1, followed by addition of C 2 H 5 OH and H 2 O (v/v 4:1). After dissolution, iron powder (784 mg,14 mmol) was added to the mixture and refluxed at 100 ℃ for 1.2 hours. After the reaction is completed, the solution is filtered while the solution is hot and concentrated under reduced pressure to obtain the product 5-aminoindolin-2-one. 5-amino-2-indolone (399 mg,3.0 mmol) and p-nitrobenzenesulfonamide (600 mg,3.0 mmol) were added to a round bottom flask, dissolved in pyridine, and reacted for 12h. After completion of the reaction, pyridine was neutralized with aqueous hydrochloric acid (pH 1-2), the aqueous solution was extracted with ethyl acetate (3X 50 mL), and the organic layer was dried over anhydrous sodium sulfate, filtered, and the organic solvent was recovered to obtain intermediate 15. Intermediate 15, 3- (trifluoromethyl) benzaldehyde and piperidine were added and dissolved in methanol and reacted at room temperature for 4h. After the reaction is finished, purifying by silica gel column chromatography to obtain a target product h, wherein the yield is 38.4%, the red orange powder is m.p.226.5-226.9 ℃;1H NMR (600 MHz, CD) 3 OCD 3 )δ9.68(s,1H),9.06(s,1H),8.36(d,J＝2.0Hz,1H),8.35(d,J＝2.0Hz,1H),7.95(d,J＝2.0Hz,1H),7.94(d,J＝2.0Hz,1H),7.93(s,2H),7.83(d,J＝7.8Hz,1H),7.76-7.71(m,2H),7.39(d,J＝2.1Hz,1H),7.10(dd,J＝2.2,8.3Hz,1H),6.90(d,J＝8.3Hz,1H)ppm； 13 C NMR(150MHz,CD 3 OCD 3 )δ168.30,168.19,150.22,145.13,141.40,135.76,134.76,132.36,130.84,130.63,130.36,129.79,128.99,128.71,128.60,126.05,126.02,125.99,124.22,121.67,118.52,110.67ppm；ESI-HRMS,m/z calculated for C 22 H 14 F 3 N 3 O 5 SNa[M+Na]+512.05066,found 512.04985。
Example 9
Test of the cytotoxicity and anti-inflammatory Activity of the Compounds of the invention
Since overproduction of Nitric Oxide (NO) in biological systems may lead to various diseases, such as inflammation and atherosclerosis, immune cell production of NO has been used as a visual indicator of the presence and extent of inflammation. The in vitro cytotoxic activity of the prepared compounds against mouse mononuclear macrophage leukemia cells (RAW 264.7) was evaluated using the Cell Counting Kit (CCK-8) assay.
The specific experimental steps are as follows:
(1) Cell culture: mouse mononuclear macrophage leukemia cells (RAW 264.7) were placed in DMEM medium supplemented with 10% fetal calf serum and 1% penicillin/streptomycin, 37℃and 5% CO 2 Is cultured in a saturated humidified incubator.
(2) Toxicity screening:
RAW264.7 cells at 80% density were scraped with a cell scraper, cells were collected, centrifuged and the supernatant was discarded, and cells were resuspended in a defined amount of complete culture medium and counted using a cell counting plate. According to 5X 10 4 Cell/well density (5X 10) 4 Individual cells/mL), 100 μl/well, inoculated in 96-well plates, and placed at 37 ℃, CO 2 Cells were grown on the wall in an incubator with a concentration of 5%. Setting a normal control group and a compound group with different concentrations of 200, 100, 50, 25, 12.5 and 6.25 mu M respectively, setting parallel compound holes, adding the compound groups into a 96-well plate, adding 10 mu L of CCK-8 solution into each hole after 24 hours of action, continuously culturing the compound groups in an incubator for 2-4 hours, and measuring absorbance at 450nm of an enzyme-labeled instrument.
As can be seen from FIG. 1, several compounds of the present invention showed better cell viability in RAW264.7 cells at concentrations of 5-20. Mu.M, and cytotoxicity assays showed that the prepared compounds were not significantly toxic to RAW264.7 cells, wherein compounds e, f, and g were also not toxic at 100. Mu.M.
(2) Anti-inflammatory Activity test:
lipopolysaccharide (LPS) is involved in the production of pro-inflammatory cytokines and induces inflammatory responses. To assess anti-inflammatory activity in vitro, we assessed the anti-inflammatory ability of all compounds, with particular attention to their amount of Nitric Oxide (NO) produced in LPS-stimulated RAW264.7 cells. The effect of the compound of the invention on the release of NO by LPS-induced RAW264.7 cell lines in vitro was tested by CCK-8 method using pyrrolidine dithio-ammonium formate (Ammonium pyrrolidinedithiocarbamate, DPTC) as a positive control.
The specific experimental steps are as follows:
1. inoculating cells: RAW264.7 cells were treated as described above to adjust the cell density to 4X 10 5 Cells were seeded in 24-well plates at a volume of 500. Mu.L per well at 5% CO per mL 2 Preculture was performed at 37 ℃. The experiments were divided into blank (cells only), model (cells+lps), experimental (cells+compounds+lps).
2. After 24 hours, old broth was discarded, fresh broth was added directly to the blank and model groups, and compound solutions (500. Mu.L) containing different concentrations (80, 40, 20, 10 and 5. Mu.M) were added per well to the experimental group. After 2h the model and experimental groups were stimulated with LPS to give final LPS concentrations of 5. Mu.g/mL.
3. After 24h, the cell supernatant was extracted, and the NO concentration in the cell supernatant was detected and calculated using the NO detection kit.
4. Data processing and experimental results: and (3) performing primary data standardization processing by using Excel software, and performing primary screening to calculate the cell proliferation inhibition rate (formula= (ODcontrol-ODdrug)/(ODcontrol-ODBlank) ×100%) through the OD value of each hole, and counting the inhibition rate. IC (integrated circuit) 50 Calculation of IC for samples by GraphPad Prism 8 (version 8.0.2,GraphPad Software Inc) 50 Values, experimental results are expressed as + -SD. The experimental results are as follows:
anti-inflammatory test results of the compounds prepared in Table 1 in vitro
As shown in Table 1, the compounds of the invention have remarkable inhibitory activity on the release of NO by RAW264.7 cell strain induced by LPS in vitro, and the inhibitory activity of compounds e and h on the release of NO by RAW264.7 cell strain induced by LPS is higher than that of positive control PDTC. These compounds are expected to be developed as novel anti-inflammatory lead compounds.
Example 10
Cyclooxygenase-2 (COX-2) is closely related to the occurrence and development of inflammation, and is an important target for developing nonsteroidal drugs for treating inflammation. To investigate whether the anti-inflammatory activity of these compounds was associated with COX-2, the COX-2 inhibitory activity of the prepared compounds was evaluated in vitro using a cyclooxygenase 2 inhibitor screening kit (Beyotime Corp., shanghai, china).
The experimental method comprises the following steps: the COX-2 inhibitory activity of the prepared compounds was tested using the CCK-8 method. Briefly, human recombinant COX-2 enzyme, COX-2 cofactor and COX-2 assay buffer were pre-incubated with test compound for 10 minutes at 37℃and then COX-2 probe was added. The reaction was started by adding the COX-2 substrate and allowed to proceed for 5 minutes. Fluorescence intensity was measured using a microplate reader using an excitation wavelength of 560nm and an emission wavelength of 590 nm. Celecoxib, a selective COX-2 inhibitor, was used as a positive control drug and all tests were independently repeated three times.
COX-2 test results of the compounds prepared in Table 2 in vitro
The results in Table 2 show that compounds e, f, g have an inhibitory effect on cyclooxygenase-2 in vitro 50 Respectively 17.81 + -1.86. Mu.M, 20.41.+ -. 1.61. Mu.M, and 16.31.+ -. 0.35. Mu.M. The results indicate that the anti-inflammatory activity of compound e may be associated with COX-2.
Taken together, the results show that the compound e has remarkable inhibitory activity on the release of NO by RAW264.7 cell strain induced by LPS in vitro, and the in vitro cyclooxygenase-2 activity test result further verifies that the compound possibly generates anti-inflammatory activity through COX-2 inhibition, thus indicating that the compound has potential value in the development of COX-2 inhibitors.
It will be understood by those skilled in the art that, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the prior art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
Finally, what should be said is: the above embodiments are only for illustrating the technical aspects of the present invention, and although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that: modifications and equivalents may be made thereto without departing from the spirit and scope of the invention, which is intended to be encompassed by the claims.

Claims (3)

1. Comprisesα,β-2-indolone compounds of unsaturated ketone, characterized in that said compounds are:
2. a process for the preparation of a compound according to claim 1, characterized by comprising the steps of:
(1) Preparation of 5-aminoindole intermediate 14: taking 5-nitroindole, iron powder and ammonium chloride as raw materials,V ethanol :V Water and its preparation method =4: 1 is solvent, preparing intermediate 5-nitroindole;
(2) Preparation of alpha, beta-unsaturated ketone analogues f-g of different substituents: intermediate 5-amino indole and benzaldehyde with different substituents are added under the catalysis of Lewis acid and under the condition that ethanol is taken as a solvent to prepare different substituentsα,β-an unsaturated ketone intermediate;
wherein R is 1 3-trifluoromethyl or 3, 5-bis (trifluoromethyl);
(3) Preparation of sulfonamide intermediate 18: taking intermediate 14 (5-aminoindole) and compound 17 (p-nitrobenzenesulfonyl chloride) as raw materials, and carrying out condensation reaction under the condition that pyridine is taken as a solvent to prepare an intermediate 18;
(4) Preparationα,βUnsaturated ketone analogue h: reacting an intermediate 18 with 3- (trifluoromethyl) benzaldehyde serving as a raw material under the condition that piperidine is used as a catalyst and methanol is used as a solvent to prepare a compound h;
3. the composition according to claim 1 comprisingα,β-application of 2-indolone compounds of unsaturated ketone in preparing anti-inflammatory drugs.
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CN114057755A (en) * 2021-10-17 2022-02-18 贵州省中国科学院天然产物化学重点实验室(贵州医科大学天然产物化学重点实验室) Preparation method and application of iridoid aglycone-based spiro indolone compound

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