CN114656393B

CN114656393B - Pyrrole-2-aldehyde compound and preparation method and application thereof

Info

Publication number: CN114656393B
Application number: CN202210326291.8A
Authority: CN
Inventors: 蒋跃平; 刘韶; 雷光华; 贺佳; 刘蓉
Original assignee: Xiangya Hospital of Central South University
Current assignee: Xiangya Hospital of Central South University
Priority date: 2021-04-19
Filing date: 2022-03-30
Publication date: 2023-06-06
Anticipated expiration: 2042-03-30
Also published as: CN114656393A

Abstract

The invention relates to a pyrrole-2-aldehyde compound and a derivative thereof, wherein the structural general formula of the compound is shown as (I):

wherein R is ₁ Is glycosylbenzyl, substituted glycosylbenzyl, C1-C10 alkyl, C1-C10 substituted alkyl, C1-C12 aryl, C1-C12 substituted aryl, C1-C6 fatty alcohol or its ester with C1-C6 carboxylic acid, C1-C6 carboxylic acid or its ester with C1-C6 fatty alcohol, C1-C10 oxyalkyl, C1-C10 substituted oxyalkyl, nitrogen-containing heterocycle or sulfur-containing heterocycle; the invention also relates to application of the compounds in neuroprotection and health care foods. Experiments show that the compound has remarkable antioxidation injury effect. The compound has the characteristics of clear active mechanism, low toxicity, safety and the like, and has wide application prospect.

Description

Pyrrole-2-aldehyde compound and preparation method and application thereof

Technical Field

The invention belongs to the field of pharmacy, and in particular relates to pyrrole-2-aldehyde compounds, a preparation method and application thereof.

Background

Moringa oleifera (Moringa oleifeera lam.) is a plant of the genus Moringa of the family Moringaceae, and is native to tropical and subtropical regions such as India, africa, etc. (Guo Liqun, etc. Tropical agricultural sciences 2015, 35 (6): 11-17; ren Anxu, etc. food research and development 2016,37 (16): 219-222). After 60 s of the last century, there were large-area plants in Yunnan, hainan, guangdong, guangxi and other places in China (Dong Xiaoying, et al Guangdong feed 2008,17 (9): 39-41). The roots, stems, leaves, flowers and seeds of Moringa oleifera are pharmaceutically acceptable (Chinese Phytophyta, 1984:34 (1): 6). The moringa seed is moringa seed, and the main chemical components of the moringa seed are phenols, alkaloids, amides, flavone, polysaccharide and the like. Modern pharmacological studies have shown that Moringa seed has hypoglycemic, hypolipidemic, hypotensive, hepatoprotective, antioxidant, nervous system protective, antitumor activities (Qu Zhenli, etc., clinical studies in TCM 2017,9 (12): 18-19; liu Bing, etc., university of vinca, journal of vinca, 2010, 26 (4): 179-180; muhammad, et al Asian Pac J Trop Biomed 2016;6 (10): 896-902; xu Min, et al, food science 2016,37 (23): 291-301). However, the pharmacological activities are all researches on water extracts or alcohol extracts of moringa seeds, and related activities of monomer components are not reported.

According to the invention, a series of novel pyrrole-2-aldehydes compounds are discovered from moringa seeds, a series of novel derivatives are synthesized, and the pyrrole-2-aldehydes have antioxidant injury activity and have potential brain protection and nerve protection effects through researches for the first time.

Disclosure of Invention

The invention aims to provide a novel pyrrole-2-aldehyde compound and application thereof in medicines or foods for preventing or treating nerve cell injury.

In order to achieve the above object, the technical scheme of the present invention is as follows:

a pyrrole-2-aldehyde compound has a structural formula:

wherein R is ₁ Is glycosylbenzyl, substituted glycosylbenzyl, C1-C10 alkyl, C1-C10 substituted alkyl, C1-C12 aryl, C1-C12 substituted aryl, C1-C6 fatty alcohol or its ester with C1-C6 carboxylic acid, C1-C6 carboxylic acid or its ester with C1-C6 fatty alcohol, C1-C10 oxyalkyl, C1-C10 substituted oxyalkyl, nitrogen-containing heterocycle or sulfur-containing heterocycle;

R ₂ is hydrogen, C1-C10 alkyl, C1-C10 substituted alkyl, C1-C6 fatty alcohol or its ester with C1-C6 carboxylic acid, C1-C6 carboxylic acid or its ester with C1-C6 fatty alcohol, nitrogen-containing heterocycle, sulfur-containing heterocycle, C1-C12 aromatic hydrocarbon or C1-C12 substituted aromatic hydrocarbon;

R ₃ is hydrogen, C1-C10 alkyl, C1-C10 substituted alkyl, C1-C12 aryl, C1-C12 substituted aryl, nitrogen-containing heterocycle or sulfur-containing heterocycle.

Preferably, the glycosyl attached to the glycosyl benzyl is hexacarbopyranose.

Preferably, the hexacarbopyranose is glucopyranose, mannopyranose or rhamnose.

Preferably, the substituent on the substituted glycosylbenzyl is C1-C6 alkyl, C1-C6 alkoxy, hydroxyl, carboxyl, halogen, sulfonic acid group, amino or aldehyde group, and more preferably, the C1-C6 alkyl is methyl, ethyl, propyl, butyl or amyl; C1-C6 alkoxy is methoxy, ethoxy, propoxy, butoxy or pentoxy.

Preferably, the C1-C10 alkyl is methyl, ethyl, propyl, butyl or pentyl.

Preferably, the C1-C10 substituted alkyl is a C1-C6 substituted alkyl.

Preferably, the substituent on the C1-C6 substituted alkyl is hydroxyl, carboxyl, halogen, sulfonic acid, amino or aldehyde; further preferably, the halogen is fluorine, chlorine, bromine or iodine.

Preferably, the C1-C12 aromatic hydrocarbon group is phenyl or naphthyl.

Preferably, the C1-C12 substituted aryl is a substituted phenyl or substituted naphthyl.

Preferably, the substituents on the substituted phenyl and substituted naphthyl groups are C1-C6 alkyl, C1-C6 alkoxy, hydroxy, carboxyl, halogen, sulfo, amino or aldehyde groups, and more preferably, the C1-C6 alkyl groups are methyl, ethyl, propyl, butyl or pentyl groups; C1-C6 alkoxy is methoxy, ethoxy, propoxy, butoxy or pentoxy; further preferably, the halogen is fluorine, chlorine, bromine or iodine.

Preferably, the C1-C6 fatty alcohol is methanol, ethanol, propanol, butanol, pentanol, propylene glycol, glycerol or butanediol.

Preferably, the C1-C6 carboxylic acid is formic acid, acetic acid, propionic acid, butyric acid or valeric acid.

Preferably, the C1-C10-oxo-alkyl is a C1-C6-oxo-alkyl; preferably, the C1-C6-oxo alkyl is methoxy, ethoxy, propoxy, butoxy or pentoxy.

Preferably, the C1-C10 substituted oxy alkyl is a C1-C6 substituted oxy alkyl; preferably, the C1-C6 substituted oxy alkyl is substituted methoxy, substituted ethoxy, substituted propoxy, substituted butoxy or substituted pentoxy; further preferably, the substituent on the substituted methoxy, substituted ethoxy, substituted propoxy or substituted butoxy, substituted pentoxy is hydroxy, carboxy, halogen, sulfo, amino or aldehyde; further preferably, the halogen is fluorine, chlorine, bromine or iodine.

Preferably, the nitrogen-containing heterocycle is a pyridine ring, a pyrrole ring, a pyrazole ring, an imidazole ring, a piperidine ring, a pyrimidine ring, a pyrazine ring or a piperazine ring.

Preferably, the sulfur-containing heterocycle is a thiophene ring or a thiazole ring.

Preferably, the structure and substituents of the pyrrole-2-aldehyde compound are shown in table 1:

TABLE 1 substituents and structures of pyrrole-2-aldehydes compounds of the present invention

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Preferably, the method for extracting the pyrrole-2-aldehyde compound comprises the following steps:

s1, crushing moringa seeds, extracting, concentrating, filtering and adsorbing by macroporous resin; then eluting with water, 50% ethanol and 90% ethanol in sequence; collecting water eluent, concentrating to obtain fluid extract;

s2, loading the fluid extract on resin, eluting with water, 50% ethanol and 90% ethanol in sequence, collecting the eluent of 50% ethanol, and concentrating to obtain extract; loading the extract on reverse phase C18 silica gel column, eluting with 30% ethanol, 50% ethanol and methanol sequentially, collecting 50% ethanol eluate, concentrating, and drying to obtain 50% ethanol part (B);

s3, loading the B part on a normal phase silica gel column, performing gradient elution by using mixed solutions of dichloromethane and methanol with two column volumes respectively, tracking a point plate by thin layer chromatography, and obtaining different fractions according to retention time; mixing the similar fractions, drying, and mixing to obtain fractions B-1 to B-13; concentrating B-1 to obtain pale yellow blocky crystals which are compound XY-4; b-9 is processed by reversed phase ODS semi-preparative HPLC with 30% acetonitrile-water as mobile phase to obtain compound XY-5 (t) _R =30 min) and XY-24 (t _R =13.8 min); b-5 was subjected to reversed-phase ODS semi-preparative HPLC using 35% acetonitrile-water as the mobile phase to give fraction B-5-3 (t) _R =13.6 min). B-5-3 is continuously processed by phenyl semi-preparative HPLC, 44% methanol-water is taken as a mobile phase, and the compound XY-12 (t) _R ＝22.3min),XY-29(t _R =27.5 min); b-3 was prepared by phenyl semi-prep HPLC using 32% acetonitrile-water as mobile phase to give compound XY-19 (t) _R ＝33.8min),XY-25(t _R =23.3 min); b-6 preparation of Compound XY-20 (t) by phenyl semi-preparative HPLC using 45% methanol-water as mobile phase _R ＝12.0min)、XY-26(t _R =18.8 min). B-4 semi-preparative HPLC on ODS with 45% acetonitrile-water (containing thousands ofOne-half formic acid) as mobile phase to obtain compound XY-30 (t) _R =8.3 min). B-10 Compound XY-56 (t) was prepared by ODS semi-preparative HPLC using 45% methanol-water (containing thousandth of formic acid) as the mobile phase _R ＝29.6min)。

Preferably, in step S3, fraction B is eluted with a gradient of methylene chloride to methanol (mass ratio) (100:0; 100:1;50:1;25:1;10:1;5:1;1:1; 0:1) system.

Preferably, the method for extracting the pyrrole-2-aldehyde compound specifically comprises the following steps:

pulverizing 10kg of moringa seed, adding 8 times of purified water, ultrasonically extracting for three times each for 1h, mixing the extracting solutions, concentrating, filtering, adsorbing the filtrate with PHD-300 macroporous resin column, eluting with water, 50% ethanol and 90 ethanol sequentially, respectively collecting water, 50% ethanol and 90 ethanol eluents, and concentrating to obtain fluid extract of three elution parts. Wherein, the liquid extract of the water washing part is about 1.0kg, MCI resin is added, water, 50 percent ethanol and 90 percent ethanol are sequentially used for eluting, water, 50 percent ethanol and 90 percent ethanol eluent are respectively collected, eluent is collected, concentrated and dried under reduced pressure, thus obtaining the extract of three elution parts of the MCI column. Wherein 104g of MCI50% ethanol elution part extract is put on a reversed phase C18 silica gel column, and is eluted by 30% ethanol, 50% ethanol and methanol in turn, three eluents are respectively collected, concentrated and dried under reduced pressure, and 30% ethanol elution part (A), 50% ethanol part (B) and methanol elution part (C) are respectively obtained. 12.0g of the B part is put on a normal phase silica gel column to be eluted by a system gradient of two column volumes of dichloromethane and methanol (100:0; 100:1;50:1;25:1;10:1;5:1;1:1; 0:1), a thin layer chromatography is used for tracking a point plate, similar fractions are combined, and the fractions are dried under reduced pressure and combined to obtain fractions B-1 to B-13. The pale yellow bulk crystals (60 mg) were eluted during concentration of B-1 to give Compound XY-4.B-9 (about 0.5 g) was subjected to reversed-phase semi-preparative HPLC using 30% acetonitrile-water as the mobile phase to give compound XY-5 (8 mg), XY-24 (4 mg). B-5 (about 0.2 g) was subjected to ODS semi-preparative HPLC using 35% acetonitrile-water as the mobile phase to give fraction B-5-3 (about 50 mg). B-5-3 Compound XY-12 (2 mg), XY-29 (1.5 mg) was prepared by further phenyl semi-preparative HPLC using 44% methanol-water as the mobile phase and 35% acetonitrile-water as the mobile phase. B-3 (about 0.3 g) was subjected to reversed-phase semi-preparative HPLC using 32% acetonitrile-water as the mobile phase to give compound XY-19 (4 mg), XY-25 (3 mg). B-6 (about 0.4 g) was subjected to reversed-phase semi-preparative HPLC using 45% methanol-water as the mobile phase to give compound XY-20 (6 mg), XY-26 (2 mg). B-4 (about 0.3 g) was prepared by ODS semi-preparative HPLC using 45% acetonitrile-water (containing thousandth of formic acid) as the mobile phase to give compound XY-30 (10 mg). B-10 (about 0.3 g) was prepared by ODS semi-preparative HPLC using 45% methanol-water (containing thousandth of formic acid) as the mobile phase to give compound XY-56 (3 mg).

The invention also provides a synthetic route of the pyrrole-2-aldehyde compound, which comprises the following steps:

wherein R is ₁ 、R ₂ 、R ₃ As above.

Preferably, the synthesis route of the pyrrole-2-aldehyde compound is as follows:

wherein R is ₁ 、R ₂ 、R ₃ As above.

The invention also provides application of the pyrrole-2-aldehyde compound in preparing medicines or foods for resisting oxidative stress induced nerve cell injury.

Preferably, when the compounds of the present invention are used as medicaments, they may be used as such or in the form of pharmaceutical compositions. Comprising as active ingredient at least one compound of formula (I) in combination with one or more pharmaceutically acceptable carriers and excipients which are non-toxic and inert to humans.

Preferably, the carriers and excipients used are one or more solid, semi-solid and liquid diluents, fillers and pharmaceutical formulation adjuvants. The pharmaceutical composition of the invention is prepared into various dosage forms, such as liquid preparations (suspension, syrup, oral liquid, injection, etc.), solid preparations (tablets, capsules, granules, etc.), sprays, etc., by adopting a method accepted in the pharmaceutical field. The above drugs can be administered orally, sublingually or by injection (intravenous injection, intramuscular injection or subcutaneous injection, etc.).

Preferably, the nerve cell damage disease comprises a cerebral ischemia reperfusion injury disease.

The invention is further explained below:

the phenolic hydroxyl groups contained in part of the structure can react with free radicals to generate relatively stable conjugated semi-quinoid free radicals, so that the in-vivo free radical reaction can be interrupted, in addition, aldehyde groups in the structure have reducibility, can generate oxidation-reduction reaction with the free radicals, eliminate the free radicals, and can also interrupt the in-vivo free radical reaction to play a role in resisting oxidative damage. In addition, nuclear factor-kB (NF-kB) and transcription factor NF-E2-related factor 2 (Nrf 2) are important transcription factors that regulate cellular oxidative stress, which can reduce cellular damage caused by reactive oxygen species and electrophiles by inducing and regulating a range of antioxidant proteins. The pyrrole-2-aldehyde compound can overactivate Nrf2 and inhibit Nrf2, so that downstream signals are regulated and controlled to inhibit oxidative damage, thereby treating nerve cell injury diseases and treating cerebral ischemia reperfusion injury.

The beneficial effects of the invention are as follows: the invention extracts and separates active compounds from natural medicinal plants, has simple synthesis and preparation flow of partial compounds, rich natural resources, low preparation cost, simple and convenient operation, small toxicity of most organic solvents, recycling and high treatment efficiency, and not only increases benefits, but also avoids pollution.

Drawings

FIG. 1 is a HRESI-MS of compound XY-4;

FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of compound XY-4;

FIG. 3 is a nuclear magnetic resonance carbon spectrum of compound XY-4;

FIG. 4 is a HRESI-MS plot of compound XY-5;

FIG. 5 is a nuclear magnetic resonance hydrogen spectrum of compound XY-5;

FIG. 6 is a nuclear magnetic resonance carbon spectrum of compound XY-5;

FIG. 7 is a HRESI-MS plot of compound XY-10;

FIG. 8 is a nuclear magnetic resonance hydrogen spectrum of compound XY-10;

FIG. 9 is a nuclear magnetic resonance carbon spectrum of compound XY-10;

FIG. 10 is a HRESI-MS plot of compound XY-12;

FIG. 11 is a nuclear magnetic resonance hydrogen spectrum of compound XY-12;

FIG. 12 is a nuclear magnetic resonance carbon spectrum of compound XY-12;

FIG. 13 is a HRESI-MS plot of compound XY-19;

FIG. 14 is a nuclear magnetic resonance hydrogen spectrum of compound XY-19;

FIG. 15 is a nuclear magnetic resonance carbon spectrum of compound XY-19;

FIG. 16 is a HRESI-MS plot of compound XY-20;

FIG. 17 is a nuclear magnetic resonance hydrogen spectrum of compound XY-20;

FIG. 18 is a nuclear magnetic resonance carbon spectrum of compound XY-20;

FIG. 19 is a HRESI-MS plot of compound XY-24;

FIG. 20 is a nuclear magnetic resonance hydrogen spectrum of compound XY-24;

FIG. 21 is a nuclear magnetic resonance carbon spectrum of compound XY-24;

FIG. 22 is a HRESI-MS plot of compound XY-25;

FIG. 23 is a nuclear magnetic resonance hydrogen spectrum of compound XY-25;

FIG. 24 is a nuclear magnetic resonance carbon spectrum of compound XY-25;

FIG. 25 is a HRESI-MS plot of compound XY-26;

FIG. 26 is a nuclear magnetic resonance hydrogen spectrum of compound XY-26;

FIG. 27 is a nuclear magnetic resonance carbon spectrum of compound XY-26;

FIG. 28 is a HRESI-MS plot of compound XY-29;

FIG. 29 is a nuclear magnetic resonance hydrogen spectrum of compound XY-29;

FIG. 30 is a nuclear magnetic resonance carbon spectrum of compound XY-29;

FIG. 31 is a HRESI-MS plot of compound XY-30;

FIG. 32 is a nuclear magnetic resonance hydrogen spectrum of compound XY-30;

FIG. 33 is a nuclear magnetic resonance carbon spectrum of compound XY-30;

FIG. 34 is a nuclear magnetic resonance hydrogen spectrum of compound XY-56;

FIG. 35 is a nuclear magnetic resonance carbon spectrum of compound XY-56;

FIG. 36 is a HRESI-MS plot of compound XYHC-1;

FIG. 37 is a nuclear magnetic resonance hydrogen spectrum of compound XYHC-1;

FIG. 38 is a nuclear magnetic resonance carbon spectrum of compound XYHC-1;

FIG. 39 is a HRESI-MS plot of compound XYHC-2;

FIG. 40 is a nuclear magnetic resonance hydrogen spectrum of compound XYHC-2;

FIG. 41 is a nuclear magnetic resonance carbon spectrum of compound XYHC-2;

FIG. 42 is a graph of the compounds XY-4, XY-5 and XY-20 viability of neuronal-like PC12 cells at various concentrations;

FIG. 43 is a graph of half maximal inhibitory concentration (IC 50) of compounds XY-4, XY-5 and XY-20 (a: XY-4; b: XY-5; c: XY-20);

FIG. 44 is the protective effect of oxidative damage in hypoxia-induced neuronal-like PC12 cells of compounds XY-4, XY-5, XY-20;

FIG. 45 is a graph of protein signaling mediated by compounds XY-4, XY-5 and XY-20 for oxidative damage in hypoxia-induced neuronal-like PC12 cells (a: NF-kB and Nrf2 Western felt graph; b: NF-kB protein relative expression; c: nrf2 protein relative expression; OGD is a low-glucose hypoxia model; EDA is edaravone; P < 0.05; P < 0.01; P <0.001vs.OGD group; #P > 0.05; P < 0.05; and P <0.05vs.

FIG. 46 is a graph of the results of a flow assay for compound XY-30;

FIG. 47 is a graph showing that compound XY-30 has protective effects on apoptosis induced by hypoxia inducible model;

FIG. 48 is a graph of the modulation of inflammatory signals of compound XY-30 on oxidative damage in hypoxia-induced neuronal-like PC12 cells.

Detailed Description

The present invention will be described in detail with reference to examples. The invention will be more readily understood by reference to the following examples, which are given to illustrate the invention and are not intended to limit the scope thereof.

Materials and methods

Example 1

Extraction separation and structure identification of pyrrole-2-aldehyde compound

Pulverizing 10kg of moringa seed, adding 8 times of purified water, ultrasonically extracting for three times each for 1h, mixing the extracting solutions, concentrating, filtering, adsorbing the filtrate with PHD-300 macroporous resin column, eluting with water, 50% ethanol and 90 ethanol sequentially, respectively collecting water, 50% ethanol and 90 ethanol eluents, and concentrating to obtain fluid extract of three elution parts. Wherein, the liquid extract of the water washing part is about 1.0kg, MCI resin is added, water, 50 percent ethanol and 90 percent ethanol are sequentially used for eluting, water, 50 percent ethanol and 90 percent ethanol eluent are respectively collected, eluent is collected, concentrated and dried under reduced pressure, thus obtaining the extract of three elution parts of the MCI column. Wherein 104g of MCI50% ethanol elution part extract is put on a reversed phase C18 silica gel column, and is eluted by 30% ethanol, 50% ethanol and methanol in turn, three eluents are respectively collected, concentrated and dried under reduced pressure, and 30% ethanol elution part (A), 50% ethanol part (B) and methanol elution part (C) are respectively obtained. 12.0g of the B part is put on a normal phase silica gel column, the mixture is eluted in a gradient way by a methylene dichloride-methanol system (the volume ratio of chloroform to methanol is from 100:0 to 0:1), a point plate is tracked by thin layer chromatography, similar fractions are combined, and the fractions B-1 to B-13 are obtained by decompression and drying. The pale yellow bulk crystals (60 mg) were eluted during concentration of B-1 to give Compound XY-4.B-9 (about 0.5 g) was subjected to reversed-phase semi-preparative HPLC using 30% acetonitrile-water as the mobile phase to give compound XY-5 (8 mg), XY-24 (4 mg). B-5 (about 0.2 g) was subjected to reversed-phase semi-preparative HPLC using 35% acetonitrile-water as the mobile phase to give compound XY-12 (2 mg), XY-29 (1.5 mg). B-3 (about 0.3 g) was subjected to reversed-phase semi-preparative HPLC using 32% acetonitrile-water as the mobile phase to give compound XY-19 (4 mg), XY-25 (3 mg). B-6 (about 0.4 g) was subjected to reversed-phase semi-preparative HPLC using 45% methanol-water as the mobile phase to give compound XY-20 (6 mg), XY-26 (2 mg). B-4 (about 0.3 g) was subjected to reversed-phase semi-preparative HPLC using 45% methanol-water as the mobile phase to give compound XY-30 (10 mg).

The structure of each compound was identified as follows:

the compound XY-4 is a light yellow blocky crystal and has a chemical structural formula as follows:

the spectral data are as follows: ESI-MS m/z 216.1075[ M+H ]] ⁺ ,238.0846[M+Na] ⁺ ,HR-ESIMS m/z 238.0846[M+Na]+(calcd for C ₁₃ H ₁₃ NO ₂ ). HRESI-MS is shown in FIG. 1. ¹ H-NMR(CD ₃ OD，500MHz)δ：9.33(1H,s,H-7),6.99(1H,d,J＝4.0Hz,H-3),6.84(2H,d,J＝9.0Hz,H-2′,6′),6.69(2H,d,J＝9.0Hz,H-3′,5′),6.11(1H,d,J＝4.0Hz,H-4),5.53(2H,s,H-7′),2.20(3H,s,H-6)。 ¹³ C NMR(CD ₃ OD，125MHz)δ：180.1(C-7),157.8(C-4′),143.5(C-5),132.9(C-2),129.9(C-1′),128.7(C-2′,6′),127.4(C-3),116.3(C-3′,5′),111.8(C-4),48.6(C-7′),12.3(C-6)。

Compound XY-5 is a yellow oil of the formula:

the spectral data are as follows: ESI-MSm/z 362.1717[ M+H ]] ⁺ ,384.1146[M+Na] ⁺ ,400.1163[M+K] ⁺ ,HR-ESIMS m/z 400.1163[M+K]+(calcd for C ₁₉ H ₂₃ NO ₆ ). HRESI-MS is shown in FIG. 4. ¹ H-NMR(CD ₃ OD,500MHz)δ:9.34(1H,s,H-7),7.02(1H,d,J＝4.0Hz,H-3),6.99(2H,d,J＝9.0Hz,H-2′,6′),6.94(2H,d,J＝9.0Hz,H-3′,5′),6.14(1H,d,J＝4.0Hz,H-4),5.59(2H,s,H-7′),5.37(1H,d,J＝2.0Hz,H-1″),3.96(1H,dd,J＝3.5,2.0Hz,H-2″),3.81(1H,dd,J＝9.5,3.5Hz,H-3″),3.61(1H,dq,J＝9.5,6.0Hz,H-5″),3.43(1H,t,J＝9.5Hz,H-4″),2.22(3H,s,H-6),1.20(3H,d,J＝6.0Hz,H-6″)。 ¹³ C-NMR(CD ₃ OD，125MHz)δ:180.0(C-7),157.1(C-4′),143.2(C-2),132.9(C-5,C-1′),128.6(C-2′,C-6′),127.5(C-3),117.7(C-3′,5′),111.8(C-4),99.9(C-1″),73.8(C-4″),72.2(C-3″),72.1(C-2″),70.7(C-5″),48.5(C-7′),18.0(C-6″),12.1(C-6)。

Compound XY-10 is a brown oil of the formula:

the spectral data are as follows: the spectral data are as follows: HR-ESIMS m/z 260.1304[ M+H ]] ⁺ (calcd for C ₁₅ H ₁₇ NO ₃ ). HRESI-MS is shown in FIG. 7. ¹ H-NMR(CD ₃ OD,500MHz)δ:9.47(1H,s,H-7),7.03(1H,d,J＝4.0Hz,H-3),6.84(2H,d,J＝8.5Hz,H-2′,6′),6.68(2H,d,J＝8.5Hz,H-3′,5′),6.32(1H,d,J＝4.0Hz,H-4),5.60(2H,s,H-7′),4.42(2H,s,H-6),3.46(2H,q,J＝6.0Hz,H-1″),1.12(3H,t,J＝6.0Hz,H-2″)。 ¹³ C-NMR(CD ₃ OD，125MHz)δ:181.4(C-7),157.8(C-4′),141.9(C-5),134.1(C-1′),130.1(C-1′),128.7(C-2′,6′),125.7(C-3),116.3(C-3′,5′),112.9(C-4),66.9(C-1″),64.8(C-6),48.5(C-7′),15.3(C-2″)。

The compound XY-12 is mauve powder and has a chemical structural formula:

the spectral data are as follows: the spectral data are as follows: HR-ESIMS m/z 232.0995[ M+H ]] ⁺ (calcd for C ₁₃ H ₁₃ NO ₃ ). HRESI-MS is shown in FIG. 10. ¹ H-NMR(CD ₃ OD,500MHz)δ:9.34(1H,s,H-7),7.00(1H,d,J＝4.0Hz,H-3),6.66(1H,d,J＝8.0Hz,H-5′),6.43(H,d,J＝2.0Hz,H-2′),6.36(1H,dd,J＝8.0,2.0Hz,H-6′),6.12(1H,d,J＝4.0Hz,H-4),5.50(2H,s,H-7′),2.22(3H,s,H-6)。 ¹³ C-NMR(CD ₃ OD,125MHz)δ:180.1(C-7),146.6(C-3′),145.6(C-4′),143.6(C-5),133.0(C-2),130.7(C-1′),127.5(C-3),118.9(C-6′),116.3(C-5′),114.6(C-2′),111.8(C-4),48.5(C-7′),12.3(C-6)。

The compound XY-19 is colorless powder and has a chemical structural formula:

the spectral data are as follows: the spectral data are as follows: HR-ESIMS m/z 228.1042[ M-H ]] ^- (calcd for C ₁₄ H ₁₅ NO ₂ ). HRESI-MS is shown in FIG. 13. ¹ H-NMR(CD ₃ OD,500MHz)δ：9.32(1H,s,H-7),7.00(1H,d,J＝4.0Hz,H-3),6.83(2H,dd,J＝8.0,2.0Hz,H-2′,6′),6.64(2H,dd,J＝8.0,2.0Hz,H-3′,5′),5.98(1H,d,J＝4.0Hz,H-4),4.40(2H,t,J＝7.0Hz,H-8′),2.86(2H,t,J＝7.0Hz,H-7′),1.92(3H,s,H-6)。 ¹³ C-NMR(CD ₃ OD,125MHz)δ:179.6(C-7),157.3(C-4′),143.5(C-5),132.3(C-2),131.1(C-2′,C-6′),130.5(C-1′),127.6(C-3),116.2(C-3′,5′),111.2(C-4),48.5(C-8′),37.5(C-7),11.9(C-6)。

Compound XY-20 was a brown oil of the formula:

the spectral data are as follows: the spectral data are as follows: HR-ESIMS m/z 232.0996[ M+H ]] ⁺ (calcd for C ₁₃ H ₁₃ NO ₃ ). HRESI-MS is shown in FIG. 16. ¹ H-NMR(CD ₃ OD,500MHz)δ:9.44(1H,s,H-7),7.03(1H,d,J＝4.0Hz,H-3),6.85(2H,d,J＝8.5Hz,H-2′,6′),6.68(2H,d,J＝8.5Hz,H-3′,5′),6.32(1H,d,J＝4.0Hz,H-4),5.61(2H,s,H-7′),4.52(2H,s,H-6)。 ¹³ C-NMR(CD ₃ OD,125MHz)δ:181.2(C-7),157.8(C-4′),145.1(C-5),133.8(C-2),130.1(C-1′),128.7(C-2′,6′),126.2(C-3),116.3(C-3′,5′),111.6(C-4),56.8(C-6),48.6(C-7′)。

Compound XY-24 is a yellow oil of the formula:

the spectral data are as follows: ESI-MSm/z 348.1462[ M+H ]] ⁺ ,(calcd for C ₁₈ H ₂₁ NO ₆ ). HRESI-MS is shown in FIG. 19. ¹ H-NMR(CD ₃ OD,500MHz)δ：9.46(1H,s,H-6),7.23(1H,brs,H-5),7.11(2H,dd,J＝8.0,2.0Hz,H-2′,6′),7.06(1H,dd,J＝4.0,2.0Hz,H-3),6.99(2H,dd,J＝8.0,2.0Hz,H-3′,5′),6.29(1H,dd,J＝4.0,2.0Hz,H-4),5.50(2H,s,H-7′),5.38(1H,d,J＝2.0Hz,H-1″),3.96(1H,dd,J＝3.5,2.0Hz,H-2″),3.81(1H,dd,J＝9.5,3.5Hz,H-3″),3.61(1H,dq,J＝9.5,6.0Hz,H-5″),3.43(1H,t,J＝9.5Hz,H-4″),1.20(3H,d,J＝6.0Hz,H-6″)。 ¹³ C-NMR(CD ₃ OD,125MHz)δ：181.1(C-6),157.3(C-4′),133.6(C-5),133.2(C-2),132.7(C-1′),129.6(C-2′,C-6′),126.7(C-3),117.5(C-3′,5′),111.3(C-4),99.8(C-1″),73.8(C-4″),72.2(C-3″),72.0(C-2″),70.6(C-5″),52.1(C-7′),18.0(C-6″)。

Compound XY-25 is a yellow oil of the formula:

the spectral data are as follows: ESI-MS m/z 202.0887[ M+H ]] ⁺ (calcd for C ₁₂ H ₁₁ NO ₂ ). HRESI-MS is shown in FIG. 22. ¹ H-NMR(CD ₃ OD,500MHz)δ：9.46(1H,s,H-6),7.19(1H,brs,H-5),7.04(1H,dd,J＝4.0,2.0Hz,H-3),7.03(2H,dd,J＝8.0,2.0Hz,H-2′,6′),6.70(2H,dd,J＝8.0,2.0Hz,H-3′,5′),6.27(1H,dd,J＝4.0,2.0Hz,H-4),5.44(2H,s,H-7′)。 ¹³ C-NMR(CD ₃ OD,125MHz)δ：181.1(C-6),158.1(C-4′),133.5(C-5),133.7(C-2),130.2(C-1′),129.8(C-2′,C-6′),126.6(C-3),116.3(C-3′,5′),111.2(C-4),52.2(C-7′)。

Compound XY-26 is a brown oil of the formula:

the spectral data are as follows: the spectral data are as follows: HR-ESIMS m/z 246.1151[ M+H ]] ⁺ (calcd for C ₁₄ H ₁₅ NO ₃ ). HRESI-MS is shown in FIG. 25. ¹ H-NMR(CD ₃ OD,500MHz)δ：9.45(1H,s,H-7),7.00(1H,d,J＝4.0Hz,H-3),6.90(2H,dd,J＝6.5,2.0Hz,H-2′,6′),6.66(2H,dd,J＝6.5,2.0Hz,H-3′,5′),6.19(1H,d,J＝4.0Hz,H-4),4.48(2H,t,J＝7.5Hz,H-8′),4.26(2H,s,H-6),2.90(2H,t,J＝7.5Hz,H-7′),1.92(3H,s,H-6)。 ¹³ C NMR(CD ₃ OD,125MHz)δ:180.9(C-7),157.2(C-4′),144.9(C-5),133.3(C-2),131.0(C-2′,C-6′),130.6(C-1′),126.5(C-3),116.2(C-3′,5′),111.2(C-4),56.4(C-6),48.5(C-8′),37.8(C-7)。

Compound XY-29 was a colorless oil having the chemical formula:

the spectral data are as follows: the spectral data are as follows: HR-ESIMS m/z 304.1572[ M+H ]] ⁺ (calcd for C ₁₇ H ₂₁ NO ₄ ). HRESI-MS is shown in FIG. 28. ¹ H-NMR(CD ₃ OD,500MHz)δ：9.46(1H,s,H-7),7.03(1H,d,J＝4.0Hz,H-3),6.84(2H,brd,J＝8.0Hz,H-2′,6′),6.69(2H,dd,J＝8.0,2.0Hz,H-3′,5′),6.34(1H,d,J＝4.0Hz,H-4),5.63(2H,s,H-7′),4.52(H,d,J＝18.0Hz,H-6a),4.47(H,d,J＝18.0Hz,H-6b),3.64(1H,dq,J＝6.5,4.0Hz,H-3″),3.35(1H,dq,J＝6.5,4.0Hz,H-2″),1.08(6H,d,J＝6.5Hz,H-1″,6′)。 ¹³ C-NMR(CD ₃ OD,125MHz)δ：181.3(C-7),157.7(C-4′),142.2(C-5),134.0(C-2),130.2(C-1′),128.6(C-2′,6′),125.7(C-3),116.3(C-3′,5′),112.9(C-4),80.3(C-2″),70.9(C-3″),63.3(C-6),48.5(C-7′),18.5(C-4″),14.9(C-1″)。

Compound XY-30 is a brown oil of the formula:

the spectral data are as follows: the spectral data are as follows: HR-ESIMS m/z 196.0995[ M+H ]] ⁺ (calcd for C ₁₀ H ₁₃ NO ₃ ). HRESI-MS is shown in FIG. 31. ¹ H-NMR(CD ₃ OD,600MHz)δ：9.28(1H,s,H-7),6.95(1H,d,J＝4.2Hz,H-3),6.07(H,d,J＝4.20Hz,H-4),4.33(2H,t,J＝7.8Hz,H-1′),4.26(2H,s,H-6),2.32(3H,s,H-6),2.31(2H,t,J＝7.8Hz,H-3′),1.96(2H,quin,H-2′)。 ¹³ C-NMR(CD ₃ OD,150MHz)δ：179.7(C-7),176.6(C-4′),142.9(C-5),132.6(C-2),127.4(C-3),111.5(C-4),45.3(C-1′),31.6(C-3′),27.1(C-2′),12.1(C-6)。

Compound XY-56 is a yellow oil of the formula:

the spectral data are as follows: the spectral data are as follows: HR-ESIMS m/z 450.1572[ M+H ]] ⁺ (calcd for C ₂₃ H ₃₁ NO ₈ )。 ¹ H-NMR(CD ₃ OD,500MHz)δ：9.46(1H,s,H-7),7.05(1H,d,J＝4.0Hz,H-3),6.98(2H,brd,J＝9.0Hz,H-3′,5′),6.93(2H,dd,J＝9.0,2.0Hz,H-2′,6′),6.36(1H,d,J＝4.0Hz,H-4),5.68(2H,s,H-7′),5.37(1H,d,J＝1.5Hz,H-1″′),4.53(H,d,J＝12.5Hz,H-6a),4.47(H,d,J＝12.5Hz,H-6b),3.96(1H,dd,J＝3.0,2.0Hz,H-2″′),3.81(1H,dd,J＝9.0,3.0Hz,H-3″′),3.63(1H,m,H-3″),3.59(1H,dq,J＝9.0,6.5Hz,H-5″′),3.43(1H,t,J＝9.0Hz,H-4″′),3.36(1H,dq,J＝6.5,5.5Hz,H-2″),1.20(3H,d,J＝6.0Hz,H-4″),1.06(6H,d,J＝6.5Hz,H-1″,6″′)。 ¹³ C-NMR(CD ₃ OD,125MHz)δ：181.4(C-7),157.0(C-4′),142.2(C-5),134.0(C-2),133.2(C-1′),128.5(C-2′,6′),125.9(C-3),117.6(C-3′,5′),113.0(C-4),99.9(C-1″),80.4(C-2″),73.8(C-4″′),72.2(C-3″′),72.0(C-2″′),70.9(C-3″),70.6(C-5″′),63.3(C-6),48.5(C-7′),18.5(C-6″′),18.1(C-4″),14.9(C-1″)。

Example 2

Synthesis of pyrrole-2-aldehydes

Firstly, KOH, pyrrole-2-aldehyde components and benzyl chloride with the molar ratio of 1:1:1 are taken, 5mL of DMSO and oil bath at 65 ℃ are added, heated and stirred for 24 hours, after the reaction is finished, the silica gel is stirred, silica gel chromatographic column separation is carried out, petroleum ether with the molar ratio of 1:1 is used: eluting with ethyl acetate, collecting the fractions, combining the fractions with a spot plate, purifying to obtain a reaction product, and synthesizing to obtain the products XYHC-1 and XYHC-2 in a yield of 60% -80%. The reaction general formula is as follows:

wherein R is ₂ Is hydrogen, methyl, hydroxymethyl, aliphatic ether group, nitrogen-containing heterocycle, sulfur-containing heterocycle, halogenated benzene ring or halogenated benzyl; r is R ₃ Is hydrogen, methyl, ethyl, benzene ring, benzyl, halogenated benzene ring, halogenated benzyl, N-containing heterocycle or sulfur-containing heterocycle. The structure of the derivative of pyrrole-2-aldehyde compound is identified as follows:

compound XYHC-1 is a brown oil of the formula:

the spectral data are as follows: ESI-MS m/z 186.0933[ M+H ]] ⁺ (calcd for C ₁₂ H ₁₁ NO). HRESI-MS is shown in FIG. 36. ¹ H-NMR(CD ₃ OD,400MHz)δ：9.40(1H,s,H-6),7.20(2H,dd,J＝7.2,1.6Hz,H-2′,6′),7.18(1H,dd,J＝7.2,1.6,H-4′),7.11(1H,brd,J＝4.0Hz,H-3),7.08(2H,dd,J＝7.2,1.6Hz,H-3′,5′),6.99(1H,dd,J＝4.0,1.6Hz,H-5),6.24(1H,dd,J＝4.0,1.6Hz),5.46(2H,s,H-7′)。 ¹³ C-NMR(CD ₃ OD，100MHz)δ：180.8(C-6),139.3(C-1′),133.5(C-5),132.5(C-2),129.5(C-2′,C-6′),128.5(C-3),128.0(C-3′,5′),126.4(C-4′),111.2(C-4),52.5(C-7′)。

XYHC-2 is brown oily matter, and has a chemical structural formula:

the spectral data are as follows: ESI-MS m/z 236.1351[ M+Na ]] ⁺ (calcd for C ₁₄ H ₁₅ NO). HRESI-MS is shown in FIG. 39. ¹ H-NMR(CD ₃ OD,400MHz)δ：9.49(1H,s,H-6),7.22(2H,brd,J＝7.6,Hz,H-2′,6′),7.18(1H,brd,J＝7.6H-4′),6.94(2H,d,J＝7.6Hz,H-3′,5′),5.94(1H,s,H-4),5.57(2H,s,H-7′),2.31(3H,s,H-8),2.11(3H,s,H-7)。 ¹³ C-NMR(CD ₃ OD，100MHz)δ：178.1(C-6),142.2(C-5),139.4(C-1′),138.1(C-2),129.6(C-2′,C-6′),128.8(C-4′),128.1(C-3),127.1(C-3′,5′),113.4(C-4),48.9(C-7′),12.0(C-7),11.2(C-8)。

Example 3

Effect of pyrrole-2-aldehydes on free radical scavenging Rate of oxidative stress

Dpph (1, 1-Diphenyl-2-picrylhydrazyl radical), i.e. 1,1-Diphenyl-2-picrylhydrazyl radical. In the molecule, due to the presence of multiple electron-withdrawing-NO ₂ And a large pi bond of the benzene ring, so that the nitrogen radical can exist stably. As DPPH radicals are scavenged, the absorbance A at 519nm of their absorption maxima decreases.

Preparation and treatment of DPPH solvent

Preparation of DPPH test solution: dissolving DPPH 1mg in about 20mL of solvent (ethanol, 95% ethanol or methanol), and performing ultrasonic treatment for 5min, and shaking thoroughly to homogenize the upper and lower parts. 1mL of DPPH solution was taken and A was measured at 519nm to optimize A=1.2-1.3. The DPPH solution is preferably stored in the absence of light and used up within 3.5 hours.

Determination of the test sample

Pyrrole-2-aldehydes samples were dissolved in DMSO to prepare a range of concentrations. Another 0.1mL sample was added to 3.5mL DPPH methanol solution (0.06 mmol/L) and sonicated for 5min, and the mixture was thoroughly shaken to mix the upper and lower portions well. The decrease in absorbance at 517nm was measured after 30min of dark setting at 25 ℃. Each sample was run 3 times with the following formula:

clearance (%) = [ (Acontrol-Asample)/Acontrol ] ×100%

Wherein Acontrol is absorbance of 3.5mL DPPH solution mixed with 0.1mL LDMSO, and Asmple 3.5mL DPPH solution mixed with 0.1mL sample.

By measuring the clearance rate of DPPH free radicals, the pyrrole-2-aldehyde compound has better scavenging capacity of oxidative stress free radicals.

2. The total antioxidant capacity (ABTS) method is the most widely used indirect assay method, and can be used for measuring the antioxidant capacity of hydrophilic and lipophilic substances. The ABTS generates stable blue-green cation free radical ABTS+ after oxidation, can be dissolved in water phase or acid ethanol medium, and has maximum absorption at 734 nm. After the measured substance is added into the ABTS+ solution, the contained antioxidant component can react with the ABTS+ to fade the reaction system. The change in absorbance was detected at 734nm and the antioxidant capacity of the antioxidant substances was quantified using Trolox as a control system.

The ABTS free radical working fluid was formulated as described with reference to the kit. Pyrrole samples were dissolved in DMSO to prepare a range of concentrations. And adding 2.85mL of ABTS free radical working solution into 0.15mL of sample, and fully shaking to uniformly and fully mix the upper part and the lower part. The absorbance of the sample was measured at 734nm after 10min at 25 ℃. Each sample was run 3 times with the following formula:

clearance (%) = [ (Acontrol-Asample)/Acontrol ] ×100%

Wherein Acontrol is the absorbance of 2.85mL of ABTS solution mixed with 0.15mL of DMSO, and Asmple 2.85mL of ABTS solution mixed with 0.15mL of sample. The results are shown in Table 2.

TABLE 2 Total antioxidant Capacity results

The results (Table 2) show that the SOD value of pyrrole-2-aldehyde compound at low concentration (5. Mu.M) was higher than that of model group, and it was possible to be stress reaction. However, as the concentration increases, the SOD value decreases significantly, indicating that the compound has better total antioxidant capacity, and the concentration of the compound cannot be too high (40. Mu.M of SOD value also increases).

3. Superoxide radical (O) ^2- ) Determination of clearance

Under weakly alkaline conditions, pyrogallol undergoes autoxidation to form a superoxide anion, and a colored intermediate product having a characteristic absorption peak at 320 nm. When added, the superoxide anion scavenger reacts rapidly with superoxide anions, thereby preventing accumulation of intermediate products and weakening light absorption of the solution at 320 nm. The scavenging effect of the scavenger on superoxide anions can be evaluated by measuring the a320 value.

Pyrogallol solution: 30mmol/L, accurately weighing 0.1892g of pyrogallol, dissolving with 10mmol/L of hydrochloric acid solution, and fixing the volume in a 50mL volumetric flask, wherein the solution needs to be prepared in situ; tris-HCl buffer (pH 9.0): 50mL of 0.1mol/L Tris solution was mixed with 7.0mL of 0.1mol/L hydrochloric acid solution, and diluted to 100mL with water; the reagents are all analytically pure, and the experimental water is quartz subaqueous boiling water.

Taking 4.5mL of 50mmol/L Tris-HCl buffer solution (pH 8.2), placing in a water bath at 25 ℃ for heat preservation for 20min, respectively adding 1mL of pyrrole sample solution and 0.4mL of 25mmol/L pyrogallol solution, uniformly mixing, reacting for 5min in the water bath at 25 ℃, adding 1mL of 8mmol/L HCl to terminate the reaction, measuring absorbance (Ax) at 320nm, and replacing the sample with distilled water with the same volume in a blank control group. The O2-clearance was calculated as follows:

a0—absorbance of blank control; ax—absorbance of sample solution.

TABLE 3 superoxide radical (O ^2- ) Clearance (%) results

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The results (Table 3) show that the better scavenging effect of pyrrole-2-aldehydes on superoxide anions can be seen by measuring the A320 value. Most pyrrole-2-aldehydes have the same superoxide anion clearance rate as the positive control drug, wherein XY-4, XY-10 and XY-30 have the same superoxide radical (O ^2- ) The clearance rate is more remarkable. The pyrrole-2-aldehyde compound has better antioxidation capability.

Example 4

Protection effect of pyrrole-2-aldehyde compound on oxidative stress induced nerve cell injury

Oxidative stress is a series of adaptive reactions caused by imbalance between the active oxygen components of the body and the antioxidant system. The experiment induces PC12 cell oxidative damage by constructing an anoxic culture model. NF-kB and Nrf2 are important transcription factors for regulating and controlling cell oxidative stress, and can reduce cell damage caused by active oxygen and electrophiles by inducing and regulating a series of antioxidant proteins. Therefore, whether the pyrrole-2-aldehyde compound in the moringa seed can inhibit oxidative damage or not is verified by detecting biological indexes such as Nrf2, NF-kB and the like, so that the nerve cells are protected.

1. Experimental materials

PC12 cells; pyrrole-2-aldehydes and derivatives thereof; edaravone; caffeic acid; cocl2; an anoxic incubator; normal incubator, etc

2. Experimental grouping

Normal group; hypoxia model group (OGD group); positive control [ Edaravone (EDA), caffeic Acid (CAF) ] group; pyrrole-2-aldehyde compound and derivative experimental group thereof

3. Preparation and processing of models

The hypoxia model group (OGD group) was cultured by changing the complete medium to a low-sugar serum-free medium, incubating in a normal incubator for 30 minutes, and culturing in a hypoxia incubator for 6 hours. Positive control group low sugar serum-free culture medium is added with positive control medicament edaravone (0.1 mu M), and caffeic acid (1 mu M) is incubated in a normal incubator for 30 minutes and then placed in a low oxygen incubator for 6 hours. Pyrrole-2-aldehyde compound and derivative thereof are respectively added into low-sugar serum-free culture medium, and then are incubated for 30 minutes in normal culture boxes of pyrrole-2-aldehyde compound and derivative thereof, namely XY-4, XY-5, XY-10, XY-12, XY-19, XY-20, XY-24, XY-25, XY-26, XY-29, XY-30, XY-56, XYHC-1 and XYHC-2, and then are placed in a low-oxygen culture box for 6 hours. After hypoxia culture, the culture medium is replaced by a complete culture medium containing the corresponding medicine.

(1) Detailed description of the invention

Experiment one: PC12 cells were incubated in complete DMEM medium (10% foetal calf serum, 100. Mu.g/mL penicillin, 100. Mu.g/mL streptomycin) at 37℃with 5% CO ₂ Culturing under saturated humidity. The primary liquid is changed every 2-3 times. Taking logarithmic growth cells, adding 0.25% trypsin for digestion, diluting the cell concentration to 10% with complete medium ⁵ Each mL was inoculated into 96-well cell culture plates at 100. Mu.L per well. After overnight incubation, different concentrations of compound (0.01. Mu.M-100. Mu.M) were added to each well and incubation continued for 24 hours. Detecting absorbance value of CCK8 reagent by enzyme-linked quarantine instrument, calculating cell survival rate and IC ₅₀ Values, the experiment was repeated three times.

The compounds have certain toxicity. Cell viability IC of Compounds XY-4, XY-5, XY-20 ₅₀ The results of the values are shown in Table 4 and FIGS. 42-43.

TABLE 4 cell survival rate

As shown in Table 4, the toxicity of the different compounds was inconsistent, with the greater toxicity being XY-4, XY-5, XY-10, XY-20, XY-25, XY-19, XY-30, and XYHC-2.

As shown in CCK8 results (FIG. 42), a finer concentration study of XY-4, XY-5, XY-20 revealed that cell viability decreased with increasing drug concentration.

IC of three compounds ₅₀ XY-4 IC as shown in FIG. 43a ₅₀ IC of XY-5 as shown in FIG. 43b at 2.603. Mu.M ₅₀ An IC of XY-20 as shown in FIG. 43c at 3.488. Mu.M ₅₀ 7.041. Mu.M.

Experiment II: PC12 cells were incubated in complete DMEM medium (10% foetal calf serum, 100. Mu.g/mL penicillin, 100. Mu.g/mL streptomycin) at 37℃with 5% CO ₂ Culturing under saturated humidity. The primary liquid is changed every 2-3 times. Taking logarithmic growth cells, adding 0.25% trypsin for digestion, diluting the cell concentration to 10% with complete medium ⁵ Each mL was inoculated into 96-well cell culture plates at 100. Mu.L per well. After overnight culture, the supernatant medium was aspirated, and a low-sugar serum-free medium of pyrrole-2-aldehyde compound and its derivatives was added at a suitable concentration to each well and placed in a low-oxygen incubator. After 6 hours of culture, the culture is changed to continuous culture with complete culture medium for 24 hours, the CCK8 method is adopted to measure the cell activity, and normal group is added with physiological saline with the same metering.

The CCK8 detection results are shown in table 5 and fig. 44. The cell viability of the normal group was 100% and that of the model group was 75.45%. Cell viability at various concentrations of the remaining compounds is shown in table 5. It can be seen that pyrrole-2-aldehydes and derivatives thereof can significantly improve the viability of cells. Wherein the effects of XY-4, XY-5, XY-24, XY-19 and XY-30 are optimal. The pyrrole-2-aldehyde compound and the derivative thereof have the protection effect on cell injury of hypoxia culture.

TABLE 5 cell viability in anaerobic culture

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Cell viability for XY-4, XY-5, XY-20 are shown in FIGS. 44a,44b,44c, respectively. The results show that compared with the OGD group, the edaravone, pyrrole-2-aldehyde compounds XY-4 and XY-5, which are positive medicines, can obviously improve the reduction of the cell viability caused by the low-sugar hypoxia treatment; whereas XY-20 did not change significantly compared to the OGD group. (OGD is a low-sugar hypoxia model; EDA is edaravone; P < 0.05; P < 0.01; P < 0.001; P >0.05vs. OGD group; P <0.05vs. EDA group).

Similarly, XY-25, XY-19 and XY-30 significantly improved the decrease in cell viability caused by the low-sugar hypoxia treatment, and protected against hypoxia-cultured cell damage. The compounds are predicted to treat diseases caused by hypoxia such as ischemia reperfusion.

Experiment III: PC12 cells in the logarithmic growth phase are inoculated into a 6-well plate at 25 ten thousand per well, after the culture is carried out overnight, the supernatant culture medium is sucked, and 0.1 mu M of pyrrole-2-aldehyde compounds XY-4, XY-5, XY-10, XY-12, XY-19, XY-20, XY-24, XY-25, XY-26, XY-29, XY-30, XY-56, XYHC-1 and XYHC-2 low-sugar serum-free culture mediums are respectively added into each well and placed into a low-oxygen incubator. After 6 hours of incubation, the incubation was changed to continued with complete medium addition for 24 hours. And adding cell lysate to extract protein, and detecting the influence of the hypoxia model on cell damage through Western blot experiment.

The Western blot experiment result is shown in FIG. 45, and the low-sugar hypoxia model can induce the increase of key proteins Nrf2 and NF-kB of cell oxidative stress reaction. Compared with a model group, the positive control medicine caffeic acid is added, and the rising of Nrf2 after edaravone and pyrrole-2-aldehyde compounds is more obvious, so that the action mechanism of the positive control medicine caffeic acid is that oxidation damage is inhibited by activating Nrf2 to regulate and control downstream signals; in addition, NF-kB was significantly inhibited with the positive control drug caffeic acid compared to the model group. Compared with edaravone which is a positive control drug, the active compound of the pyrrole-2-aldehyde compound is more obviously inhibited by XY-4 and XY-20 NF-KB, and XY-5 has obvious inhibition effect. In addition, XY-25, XY-19 and XY-30 have obvious inhibition effects, and the pyrrole-2-aldehyde compound has a protective effect on nerve cell oxidative damage induced by an anoxic model, and can be a potential medicament for preventing ischemia and hypoxia damage. (OGD is a low-sugar hypoxia model; EDA is edaravone, P <0.05, P <0.01, P <0.001vs.OGD group, #P0.05, & P <0.05vs. EDA group).

Experiment IV: PC12 cells in the logarithmic growth phase are inoculated into a 6-pore plate culture flask at a rate of 35 ten thousand per pore, after the culture is carried out overnight, a supernatant culture medium is sucked, and pyrrole-2-aldehyde compound XY-30 is respectively added into each pore of a dosing group: 15 mu M,30 mu M and 40 mu M of 1% high sugar culture medium, 0.1 mu M edaravone is added to a positive control group, 1 mu M of 1% high sugar culture medium is added to a model group, 1% high sugar culture medium without liquid medicine is added to a normal group, 1% high sugar culture medium is added to a normal group, after pre-incubation for one hour, the supernatant culture medium is sucked, the model group is replaced by 600 mu M of cobalt dichloride 1% high sugar culture medium, the positive control group and the dosing group are replaced by 600 mu M of cobalt dichloride 1% high sugar culture medium with the drug concentration, after 12 hours of culture, the supernatant culture medium is sucked, PBS is washed twice every hole, and pyrrole-2-aldehyde compound XY-30 is respectively added to every hole of the dosing group: 15 mu M,30 mu M and 40 mu M of 10% high sugar culture medium, adding 0.1 mu M of 10% high sugar culture medium of edaravone into a positive control group, adding 10% high sugar culture medium into a model group and a normal group, continuously culturing for 24 hours, sucking the supernatant culture solution to a 15mL centrifuge tube, adding 0.5mL pancreatin into a 37 ℃ incubator for 1min into each hole, adding 1mL complete culture medium to stop digestion and lightly blow, transferring the digestion solution into a 15mL centrifuge tube, centrifuging at 1000rpm for 5min, sucking the supernatant, adding 1mLPBS into each tube for cleaning, centrifuging, removing the supernatant, and repeating the steps twice. And (3) using a Biyun Annexin V-FITC apoptosis kit to perform a flow type and QPCR method to detect the influence of the hypoxia model on the cell damage.

The flow experimental result shows that the pyrrole-2-aldehyde compound XY-30 has obvious protection effect on cells of the hypoxia and glucose-deficient model group as shown in figure 46. And calculating the apoptosis rate according to the flow experimental result, wherein the obtained result is shown in figure 47, and the apoptosis rate of the hypoxia and sugar-deficient model group is obviously higher than that of the positive control drug edaravone, namely the pyrrole-2-aldehyde compound XY-30. Further verifies the protection effect of pyrrole compounds on nerve cell oxidative damage induced by the hypoxia model, and can be a potential medicament for preventing ischemia hypoxia damage.

The QPCR method is used for detecting the hypoxia model by adopting a kit as a Biomarker 2X SYBR Green Fast qPCR Mix, and the cell damage process is as follows:

1. extraction of Total RNA

1. The supernatant was aspirated from each well, and 200. Mu.l of lysate was added thereto, followed by complete lysis. Immediately add 1ml of Trizol and continue grinding until the sample turns from pink ice cream to liquid and aspirate into EP tube (inside the ice box).

2. 200 μl of chloroform was added to each EP tube, mixed well upside down, and allowed to stand at room temperature for 5min with the aqueous and organic phases in full contact. Centrifugation was carried out at 12000rpm for 10min at 4℃and three layers were seen, RNA in the upper aqueous phase, DNA in the middle layer and protein in the lower organic phase. A gun was used to penetrate into the lower layer of the EP tube and a portion of Trizol was sucked off. After centrifugation at 12000rpm for 10min at 4℃the supernatant was aspirated into another EP tube (less supernatant was aspirated).

3. And the supernatant is added with about 0.5ml of equal amount of isopropanol, the mixture is gently and fully mixed, the mixture is inverted for several times, and the mixture is left at room temperature for 20min, so that the standing time is good. Centrifugation was performed at 12000rpm for 10min at 4℃and the supernatant was decanted, and RNA pellet was collected.

4. To the RNA pellet, 1ml of 75% ethanol was added. Centrifuge at 12000rpm for 10min at 4 ℃. Pouring out the ethanol in the EP pipe, reversely buckling on the water absorbing paper, and air drying. The precipitate cannot be overdry or overdry, the sediment is not easy to dissolve when overdry and the ethanol remains when overdry.

5. An appropriate amount of DEPC water was added according to the amount of the precipitate to dissolve the precipitate (the amount of DEPC water added may be adjusted according to the cell density, and 20. Mu.l of DEPC water may be added when the cell density is generally 80% or more). This time 10. Mu.l of DEPC water was added and after the addition was completed, the mixture was blown up. RNA concentration can be determined after 30min of dissolution at 4 ℃.

2. Reverse transcription

Experiment preparation: RNA samples were placed on ice boxes for use, 5X gDNA Eraser Buffer, gDNA Eraser, RNase Free dH2O, prime script RT Enzyrne Mix I, RT Prime Mix, 5X Prime script Buffer 2 were placed on ice boxes for use.

The experimental steps are as follows:

1. genomic DNA removal reaction

a. The above reagents were placed on ice for use. 5X gDNA Eraser Buffer. Mu.l, gDNA Eraser 1. Mu.l were fixed. Total RNA and RNase Free dH20 volumes were 7. Mu.l. Total RNA was loaded at 1. Mu.g, and the concentration divided by the sample was the Total RNA loading volume.

b. The qPCR tubes were well grouped, and a large volume of RNase Free dH20 was added before the calculated sample volume was added. 5X gDNA Eraser Buffer. Mu.l of gDNA Eraser 1. Mu.l was prepared in advance as Mix, (13 samples in this case were added 5X gDNA Eraser Buffer. Mu.l of gDNA Eraser 15. Mu.l by 15 calculation), vortexed, centrifuged and added 3. Mu.l to a qPCR tube. After vortexing and centrifugation. Then left to stand at room temperature for 5min (or 42 ℃,2min,4 ℃ hold requires a PCR set-up procedure).

c. Reverse transcription reaction

The following reagents were added to the above system:

the system is placed in a PCR instrument for reaction, and the reaction process is as follows: 37 ℃,15min,85 ℃,5s,4 ℃ hold.

After completion of the reaction, cDNA was obtained, and the whole system was 20. Mu.l, and diluted 10-fold with DEPC water to obtain 200. Mu.l of cDNA sample. If long-term preservation is required, the RNA should be preserved at-20deg.C, and the rest RNA should be preserved at-80deg.C.

3. PCR amplification

Experiment preparation: TB Green DE, DEPC water, cDNA, EP tube, PCR tube, 8-tube, EP tube rack, PCR tube rack, three gun heads, gun, tweezers.

1. Preparing fluorescent dye Mix-I:

this experiment measured two genes, nf-kb and IL-6, three duplicate wells per gene. The amount of Mix-i was calculated for the 7, 7×3=21 cDNA samples, and 24 wells, as shown in Table 6.

TABLE 6 fluorescent dye Mix-I

a. The EP tube was removed, 240. Mu.l of TB Green DE, 24. Mu. l m-Nf-kb F, 24. Mu. l m-Nf-kb R were added, mixed, vortexed, and centrifuged to give Mix Nf-kb.

b. The EP tube was removed, 240. Mu.l of TB Green DE, 24. Mu. l m-IL-6F, 24. Mu. l m-IL-6R were added, mixed, vortexed, and centrifuged to give Mix IL-6.

2. Preparing cDNA samples Mix-II:

each well requires 6. Mu.l of cDNA sample, 3 multiplex wells, 2 genes, 6 wells total, calculated as 8 wells. Each sample required 48 μl. As shown in table 7.

TABLE 7 cDNA sample Mix-II

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A row of PCR tubes are taken, marked according to the sample grouping of cDNA, and correspondingly arranged on a PCR tube frame. Mu.l of cDNA sample was pipetted into the PCR tube and Mix-II was prepared for each sample by adding 16. Mu.l of DEPC water (with what solvent was used to solubilize RNA, what solvent was added to Mix-II, and some ddH2O was used).

3. The eight-joint tube is put on a tube frame, 12 mu l of Mix-I is compacted and added into each hole, the gun head does not need to be deep to the bottom of the tube, the gun head does not need to be replaced, and in the process of adding Mix-II, the same gun is used for two times of sample adding, so that the sample adding error can be reduced. And (3) after sample addition, replacing PE gloves, covering each group of 8 connecting pipes with a cover, compacting the cover, tilting each connecting pipe after compacting, putting the connecting pipes in a sample plate of a PCR amplification instrument in sequence, and starting program amplification. The samples for QPCR performed after configuration and then set up are detailed in table 8.

TABLE 8 PCR amplified samples

The results are shown in FIG. 48. The QPCR detects the inflammatory factor IL-6 and the apoptosis protein NK-KB, and the result shown in the figure 48 shows that the expression of the inflammatory factor of XY-3040 mu M is obviously stronger than that of edaravone, and the expression of the apoptosis protein NK-KB can be obviously inhibited by the edaravone of XY-30 mu M and 30 mu M relative to a positive medicament. The pyrrole-2-aldehyde compound XY-30 can inhibit the expression of inflammatory factors IL-6 and apoptosis proteins NK-KB, and can play a good role in protecting nerve cell oxidative damage induced by a damage hypoxia model. XY-4, XY-5, XY-24, XY-19 also showed significant protection. Further illustrates the protection effect of pyrrole-2-aldehyde compounds on nerve cell oxidative damage induced by an anoxic model, and is a potential medicament for preventing ischemia and hypoxia damage.

Claims

1. Pyrrole-2-aldehyde compound is characterized in that the structure and substituent of the pyrrole-2-aldehyde compound are shown as follows:

XY-4：

XY-19：

XY-24：

2. the extraction method of the pyrrole-2-aldehyde compound is characterized by comprising the following steps of:

s2, loading the fluid extract on resin, eluting with water, 50% ethanol and 90% ethanol in sequence, collecting the eluent of 50% ethanol, and concentrating to obtain extract; loading the extract on reverse phase C18 silica gel column, eluting with 30% ethanol, 50% ethanol and methanol sequentially, collecting 50% ethanol eluate, concentrating, and drying to obtain 50% ethanol part B;

s3, loading the B part on a normal phase silica gel column, respectively carrying out gradient elution by using mixed solution of dichloromethane and methanol with two column volumes, tracking a point plate by thin layer chromatography, and obtaining different fractions according to retention time; mixing the similar fractions, drying, and mixing to obtain fractions B-1 to B-13; concentrating B-1 to obtain pale yellow blocky crystals which are compound XY-4; b-9 was prepared by reverse phase ODS semi-preparative HPLC using 30% acetonitrile-water as mobile phase, t _R Compound XY-5, t is obtained by =30min _R Compound XY-24 was obtained=13.8 min; b-5 was subjected to reversed-phase ODS semi-preparative HPLC with 35% acetonitrile-water as mobile phase, t _R =13.6 min to give fraction B-5-3; b-5-3 was continued by phenyl semi-prep HPLC with 44% methanol-water as mobile phase, t _R Compound XY-12, t is obtained =22.3 min _R Compound XY-29 was obtained=27.5 min; b-3 by phenyl semi-prep HPLC with 32% acetonitrile-water as mobile phase, t _R Compound XY-19, t was obtained by =33.8 min _R Compound XY-25 was obtained=23.3 min; b-6 by phenyl semi-prep HPLC with 45% methanol-water as mobile phase, t _R Preparation of Compound XY-20, t=12.0 min _R Compound XY-26 was prepared =18.8 min; b-4 by ODS semi-preparative HPLC with 45% acetonitrile-water containing thousandth of formic acid as mobile phase, t _R Compound XY-30 was prepared =8.3 min; b-10 by ODS semi-preparative HPLC with 45% methanol-water containing thousandth of formic acid as mobile phase, t _R Compound XY-56 was prepared = 29.6 min;

wherein the pyrrole-2-aldehyde compound has the structure as follows:

XY-4：

XY-5：

XY-12：

XY-19：

XY-20：

XY-24：

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XY-25：

XY-26：

XY-29：

XY-30：

XY-56：

in the step S3, the mass ratio of the part B is 100:0;100:1;50:1;25:1;10:1;5:1;1:1; gradient elution was performed with a 0:1 dichloromethane-methanol system.

3. The synthetic route of pyrrole-2-aldehydes compounds according to claim 1, wherein the following is specific:

the R is ₁ 、R ₂ 、R ₃ The respective groups shown in the corresponding positions of the pyrrole-2-aldehyde compound according to claim 1.

4. The application of pyrrole-2-aldehyde compounds in preparing medicaments for resisting oxidative stress induced nerve cell injury is characterized in that the structure and substituent of the pyrrole-2-aldehyde compounds are as follows:

XY-4：

XY-19：

XY-24：

XY-30：

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