CN115974832A

CN115974832A - Disulfide bond-containing N-acetyl-L-cysteine derivative and preparation method and application thereof

Info

Publication number: CN115974832A
Application number: CN202310167525.3A
Authority: CN
Inventors: 赵忠熙; 李根举; 李楠
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2023-02-27
Filing date: 2023-02-27
Publication date: 2023-04-18
Anticipated expiration: 2043-02-27
Also published as: CN115974832B

Abstract

The invention belongs to the field of biological medicines, and relates to an N-acetyl-L-cysteine derivative containing a disulfide bond, and a preparation method and application thereof. The general formula is (I),

formula I, wherein: r is thiophen-2-ylmethyl, furan-2-ylmethyl, pyridin-4-ylmethyl, benzyl, 4-methylbenzyl, 4-ethylbenzyl, 4-fluorobenzyl, 4-chlorobenzyl, 4-bromobenzyl, 4-tert-butylbenzyl, 2-methylbenzyl, 2-chlorobenzyl, 2-bromobenzyl, 3-fluorobenzyl, 3- (trifluoromethyl) benzyl, 2,4,6-trimethylbenzyl, 2,4-dichlorobenzyl, naphthalen-2-ylmethyl, phenethyl, 1-phenylethyl, 4-methoxyphenyl, cyclohexyl, isopropyl, n-butyl. The compounds have good antioxidant and anti-inflammatory activities, and can be used as potential drugs for preventing and treating oxidative stress and inflammatory stress related diseases such as Chronic Obstructive Pulmonary Disease (COPD), pulmonary fibrosis, acute lung injury/acute respiratory distress syndrome (ALI/ARDS) and the like.

Description

Disulfide bond-containing N-acetyl-L-cysteine derivative and preparation method and application thereof

Technical Field

The invention belongs to the field of organic synthesis and biomedicine, and particularly relates to a disulfide bond-containing N-acetyl-L-cysteine (NAC) derivative, and a preparation method and application thereof.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

N-acetyl-L-cysteine (NAC), a basic drug approved and approved by the world health organization, is commonly used clinically as a mucolytic agent and to treat acetaminophen overdose. Its antioxidant and anti-inflammatory capabilities are the biochemical basis for the treatment of diseases associated with oxidative stress and inflammation. NAC has direct antioxidant effect and indirect antioxidant effect. The direct antioxidant effect is manifested by the action of free sulfhydryl and Reactive Oxygen Species (ROS) and the elimination thereof. Indirect antioxidant action results from its ability to increase intracellular Glutathione (GSH) concentration, the most critical biological thiol responsible for cellular redox imbalance. NAC acts as an anti-inflammatory compound that can reduce the levels of tumor necrosis factor and interleukins by inhibiting the activity of nuclear factor (NF- κ B).

For lung diseases related to oxidative stress and inflammatory stress, NAC can not only improve airway mucus secretion, regulate immune system and other effects, but also reduce lung epithelial cell apoptosis, promote apoptotic cell clearance and reduce collagen deposition by resisting oxidation and regulating cytokine production, and is beneficial to protecting lung tissue cells and promoting inflammation absorption.

Despite the relevant therapeutic potential of NAC, its effectiveness in clinical trials for different pathological conditions remains limited in a number of experimental studies. In addition, NAC has a free thiol group, is not stable, and is easily oxidized, resulting in a short half-life and further affecting its pharmacological effects. Second, the non-hydrophobicity of NAC results in limited cell and tissue penetration, resulting in high doses administered. NAC has been shown to have limited utility in the treatment of inflammation and in immunomodulation.

The paper "synthesis of L-cysteine series derivatives and their medical use" discloses various L-cysteine derivatives containing a single sulfur bond and their medical uses such as relieving cough, eliminating phlegm, diminishing inflammation, reducing fever, relieving pain, inhibiting bacteria, resisting tumor, etc. However, this document does not refer to antioxidant drugs necessary for the prevention and treatment of respiratory diseases, and its antioxidant and anti-inflammatory activities are greatly restricted due to the L-cysteine derivative of its monosulfur bond.

Patent CN101232877a discloses the use of N-acetylcysteine amide (NAC amide) for the treatment of diseases and disorders associated with oxidative stress, but the related compounds are limited to NAC amide compounds, and the effect of the compounds in the prevention and treatment of respiratory diseases is limited.

For the prevention and treatment of oxidative stress and inflammatory stress related intractable diseases such as chronic obstructive pulmonary disease (chronic obstructive pulmonary disease, COPD), pulmonary fibrosis, acute lung injury/acute respiratory distress syndrome (ALI/ARDS), no N-acetyl-L-cysteine (NAC) derivative with good antioxidant and anti-inflammatory activities has been found at present.

Disclosure of Invention

In order to solve the above problems, the present invention provides a disulfide bond-containing N-acetyl-L-cysteine (NAC) derivative, a method for preparing the same, and use thereof. The invention designs and synthesizes a series of N-acetyl-L-cysteine derivatives by carrying out structural modification on NAC to further improve the activity of the NAC. Meanwhile, the synthesis route is simple and efficient, the operation is simple and safe, and the method has good practical application value.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect of the present invention, there is provided a disulfide bond-containing N-acetyl-L-cysteine derivative selected from the group consisting of compounds represented by the following formula I,

formula I

Wherein R is selected from: thien-2-ylmethyl, furan-2-ylmethyl, pyridin-4-ylmethyl, benzyl, 4-methylbenzyl, 4-ethylbenzyl, 4-fluorobenzyl, 4-chlorobenzyl, 4-bromobenzyl, 4-tert-butylbenzyl, 2-methylbenzyl, 2-chlorobenzyl, 2-bromobenzyl, 3-fluorobenzyl, 3- (trifluoromethyl) benzyl, 2,4,6-trimethylbenzyl, 2,4-dichlorobenzyl, naphthalen-2-ylmethyl, phenethyl, 1-phenylethyl, 4-methoxyphenyl, cyclohexyl, isopropyl, n-butyl.

The invention obtains the derivative containing the disulfide bond structure by properly derivatizing NAC, and the derivative can release active sulfhydryl substances in vivo on the basis of improving the stability of the derivative, thereby achieving the aim of prolonging the half-life period. Secondly, on the basis of enhancing the permeability of cells and tissues and reducing the administration dosage, the composition has better effects of antioxidation, anti-inflammation and immunoregulation, and further improves the pharmacological activity of the composition.

The above compounds also include pharmaceutically acceptable salts or esters or solvates, tautomers, mesomers, racemates, stereoisomers, metabolites or prodrugs thereof;

as understood by those of ordinary skill in the art, the above pharmaceutically acceptable salts include alkali metal salt forms (specific examples are calcium salts or sodium salts or potassium salts) of the above compounds, or salts of the above compounds with inorganic salts such as hydrochloric acid, sulfuric acid, nitric acid or hydrobromic acid, and salts with organic acids such as methanesulfonic acid, toluenesulfonic acid or trifluoroacetic acid. The term "pharmaceutically acceptable" or "pharmaceutically acceptable" used interchangeably therewith, such as in the description of "pharmaceutically acceptable salts", means that the salt is not only physiologically acceptable to the subject, but also refers to synthetic materials of pharmaceutical value, such as salts formed as intermediates in the performance of chiral resolution, which salts may function in order to obtain the end products of the invention, although such intermediate salts may not be directly administered to the subject.

Further, the NAC derivatives of formula i include:

a1: N-acetyl-S- ((thien-2-ylmethyl) thio) -L-cysteine;

a2: N-acetyl-S- ((furan-2-ylmethyl) thio) -L-cysteine;

a3: N-acetyl-S- ((pyridin-4-ylmethyl) thio) -L-cysteine;

a4: N-acetyl-S- (benzylsulfanyl) -L-cysteine;

a5: N-acetyl-S- ((4-methylbenzyl) thio) -L-cysteine;

a6: N-acetyl-S- ((4-ethylbenzyl) thio) -L-cysteine;

a7: N-acetyl-S- ((4-fluorobenzyl) thio) -L-cysteine;

a8: N-acetyl-S- ((4-chlorobenzyl) thio) -L-cysteine;

a9: N-acetyl-S- ((4-bromobenzyl) thio) -L-cysteine;

a10: N-acetyl-S- ((4- (tert-butyl) benzyl) thio) -L-cysteine;

a11: N-acetyl-S- ((2-methylbenzyl) thio) -L-cysteine;

a12: N-acetyl-S- ((2-chlorobenzyl) thio) -L-cysteine;

a13: N-acetyl-S- ((2-bromobenzyl) thio) -L-cysteine;

a14: N-acetyl-S- ((3-fluorobenzyl) thio) -L-cysteine;

a15: N-acetyl-S- ((3- (trifluoromethyl) benzyl) thio) -L-cysteine;

a16: N-acetyl-S- ((2,4,6-trimethylbenzyl) thio) -L-cysteine;

a17: N-acetyl-S- ((2,4-dichlorobenzyl) thio) -L-cysteine;

a18: N-acetyl-S- ((naphthalen-2-ylmethyl) thio) -L-cysteine;

a19: N-acetyl-S- (phenethylthio) -L-cysteine;

a20: N-acetyl-S- ((1-phenylethyl) thio) -L-cysteine;

a21: N-acetyl-S- ((4-methoxyphenyl) thio) -L-cysteine;

a22: N-acetyl-S- (cyclohexylthio) -L-cysteine;

a23: N-acetyl-S- (isopropylthio) -L-cysteine;

a24: N-acetyl-S- (butylthiol) -L-cysteine;

in a second aspect of the present invention, there is provided a method for synthesizing the disulfide bond-containing N-acetyl-L-cysteine derivative, comprising:

synthesizing the N-acetyl-L-cysteine derivative containing the disulfide bond by adopting a first route or a second route;

further, in the above preparation method, the synthetic route of the compound represented by the formula I is as follows:

synthetic route 1

Further, the synthetic route of the compound shown in the formula I specifically comprises the following steps:

(1) Dissolving a compound II in a methanol solution of hydrochloric acid, adding the solution into a methanol solution of methoxycarbonylsulfonyl chloride at a low temperature, continuously stirring at a low temperature for reaction, removing the solvent and reaction raw materials to obtain a solid, and purifying to obtain a compound III;

(2) And dissolving the compound containing the compound corresponding to the general formula IV or the salt thereof in methanol, adding a methanol solution containing the compound corresponding to the general formula III under the condition of stirring, stirring overnight, adding a mixed solution of triethylamine and methanol, continuously stirring, and purifying to obtain the compound corresponding to the general formula V.

(3) Dissolving the compound containing the general formula V in acetic acid, adding acetic anhydride under stirring, reacting at 40 ℃, stirring overnight, removing the solvent, and purifying the obtained oily crude product to obtain the compound I

Further, in the step (1), the low temperature condition is about 0 ℃, and the low temperature condition can be realized by adopting an ice-water bath mode, the molar ratio of the compound II with the general formula to the methoxycarbonyl sulfonyl chloride is 1:1-3, and in one specific embodiment of the invention, the molar ratio of the compound II with the general formula to the methoxycarbonyl sulfonyl chloride is 1.36; after the solvent and the reaction raw materials are removed, the solvent and the excessive methoxycarbonyl sulfonyl chloride can be removed by rotary evaporation, and the water bath temperature is controlled to be less than 30 ℃;

further, the step of purifying the solid comprises: washed repeatedly with diethyl ether and filtered.

Further, in step (2), the molar ratio of the compound of formula iv to the compound of formula iii or a salt thereof is 1 to 0.1, preferably 1;

further, the molar volume ratio of triethylamine to methanol is 1:1-3 (mmol/mL), preferably 1:2.

Further, the specific steps of purification include: the precipitate was filtered, washed with methanol and ether, respectively, and dried.

Further, the reaction of the step (2) can be carried out at room temperature.

Further, in the step (3), the molar ratio of the compound corresponding to the general formula V to the acetic anhydride is 1:1-8, preferably 1:4; the molar volume ratio of the compound corresponding to the general formula V to the solvent acetic acid is 1:1-4 (mmol/mL), preferably 1:2;

further, the solvent removal can be specifically realized by rotary evaporation, and the water bath temperature is controlled at 50-55 ℃;

further, the purification step comprises: normal phase column chromatography, 100-200 mesh silica gel filling, mobile phase dichloromethane: methanol (20-50; and/or reverse phase medium pressure preparative chromatography with methanol as the mobile phase: water (0.4-1:1) in volume ratio.

Further, the preparation method comprises the following steps:

step (1): dissolving the compound (II, 1.36 mmol) in the general formula in 1.7 mL methanol hydrochloride solution (0.19 mol/L hydrochloric acid), slowly adding the methanol solution of the compound (II, 1.36 mmol) in the methanol solution of methoxycarbonylsulfonyl chloride (2.04 mmol) in the methanol solution of 2 mL) in the ice-water bath for 20 min, and continuously stirring the mixture to react for 1h in the ice-water bath. The solvent and excess methoxycarbonylsulfonyl chloride were removed by rotary evaporation (bath temperature < 30 ℃). The obtained solid was repeatedly washed with diethyl ether, filtered to obtain colorless, odorless and white crystalline solid intermediate (III), dried and stored under sealed conditions at-20 deg.C.

Step (2): dissolving a compound containing a corresponding compound of a general formula (IV) or a salt thereof (0.5 mmol) in 0.4 mL methanol, slowly dropwise adding a methanol solution (0.25 mmol, dissolved in 0.25 mL methanol) containing a corresponding compound of a general formula (III) at room temperature under stirring, dropwise adding the solution after 45 min, continuously stirring at room temperature overnight, dropwise adding a mixed solution of triethylamine (0.25 mmol) and 0.5 mL methanol, continuously stirring at room temperature for 30min, filtering white precipitates, washing the precipitates with methanol and ether respectively, and drying to obtain a corresponding compound of a general formula (V).

And (3): the compound (2 mmol) containing the corresponding compound of formula (V) was dissolved in 4mL of glacial acetic acid, acetic anhydride (8 mmol) was added under stirring at room temperature, and the system was warmed to 40 ℃ and stirred overnight. Rotary evaporation to remove solvent (water bath temperature 50-55 deg.C) to obtain yellow oily crude product, and subjecting to normal phase column chromatography, packing with 100-200 mesh silica gel, mobile phase is dichloromethane: methanol (20-50; and/or reverse phase medium pressure preparative chromatography with methanol as the mobile phase: water (0.4-1:1) in volume ratio; to obtain the corresponding compound of the general formula (I).

Further, the second synthetic route of the compound shown in the formula I is as follows:

synthetic route two

Further, the second synthetic route of the compound shown in the formula I specifically comprises:

(4) And dissolving the compound VI in the general formula in dry dichloromethane, and stirring at low temperature for 5min under the protection of nitrogen. Dissolving m-CPBA (m-chloroperoxybenzoic acid) in dry dichloromethane, slowly (for more than 30 min) dripping the solution into the dichloromethane, continuously stirring the solution for 2h at low temperature, washing the reaction solution for four times by using a sodium bicarbonate solution, drying the organic phase, removing the solvent to obtain a solid, and purifying the solid to obtain VII;

(5) Adding a compound containing a compound corresponding to the general formula VII and triethylamine into acetonitrile containing the compound corresponding to the general formula VII under the condition of stirring, removing the solvent after stirring overnight, diluting with water, extracting with diethyl ether, drying with an organic phase, removing the solvent, and purifying to obtain the compound corresponding to the general formula I.

Further, in the step (4), the low temperature condition is about 0 ℃ in particular, and can be realized by adopting an ice-water bath mode, the molar ratio of the compound VI with the general formula to m-CPBA (m-chloroperoxybenzoic acid) is 1:1-1.5, and in one embodiment of the invention, the molar ratio of the compound VI with the general formula to m-CPBA is 1.05; the sodium bicarbonate solution can be 5% water solution, the organic phase can be dried by anhydrous sodium sulfate or magnesium sulfate, the solvent can be removed by rotary evaporation, and the water bath temperature is controlled to be less than 40 deg.C;

further, the step of purifying the solid comprises: normal phase column chromatography, 100-200 mesh silica gel filling, and petroleum ether as mobile phase: ethyl acetate (20-50.

Further, in the step (5), the molar ratio of the compound corresponding to the general formula VII to the compound corresponding to the general formula VIII is 1:2-8, preferably 1:4; the molar ratio of the corresponding compound in the general formula VII to triethylamine is 1:2-10, preferably 1:5; the molar volume ratio of the corresponding compound in the general formula VII to the solvent acetonitrile is 1;

Further, the preparation method comprises the following steps:

and (4): the compound (VI, 1.51 mmol) in the general formula is dissolved in 6 mL dry dichloromethane and stirred for 5min under the protection of nitrogen and ice-water bath. The compound m-CPBA (1.59 mmol) was dissolved in 5mL of dry dichloromethane and slowly added dropwise to the above solution, and the reaction was continued with stirring in an ice-water bath to 2 h. The reaction solution was washed four times with 5% sodium bicarbonate solution, the organic phase was washed once with water, dried over anhydrous sodium sulfate and the solvent was removed by rotary evaporation (water bath temperature < 40 ℃). The obtained solid is subjected to normal phase column chromatography and is filled with 100-200 meshes of silica gel, and the mobile phase is petroleum ether: ethyl acetate (20-50.

Further, step (5): dissolving the corresponding compound (0.29 mmol) containing the general formula (VII) in 14.5 mL acetonitrile, adding triethylamine (1.44 mmol) corresponding to the general formula (VIII, 1.16 mmol), stirring overnight at room temperature under nitrogen protection, and rotary evaporating to remove the solvent (water bath temperature 50-55 deg.C). Diluting the obtained solid with water (15 mL), extracting with diethyl ether for three times, combining organic phases, drying with anhydrous sodium sulfate, removing solvent by rotary evaporation, subjecting the obtained solid to normal phase column chromatography, filling with 100-200 mesh silica gel, and using dichloromethane as mobile phase: methanol (20-50; and/or reverse phase medium pressure preparative chromatography with methanol as the mobile phase: water (0.4-1:1) according to the volume ratio to obtain the corresponding compound of the general formula (I).

Further, the present invention provides a pharmaceutical composition comprising a compound of the first aspect. More specifically, it functions as an active ingredient, and may include other components having antioxidant and anti-inflammatory effects in addition to the compound of the first aspect, and is not particularly limited herein.

And a pharmaceutical formulation comprising a compound as described in the first aspect above, and at least one pharmaceutically acceptable adjuvant and/or carrier.

The auxiliary material refers to components except for active ingredients in the pharmaceutical composition or the pharmaceutical preparation, and is non-toxic to a subject. Adjuvants commonly used in the art such as buffers, stabilizers, preservatives or excipients, commonly used excipients such as binders, fillers, wetting agents, disintegrants and the like.

By way of example, optional excipients in the formulations of the present invention include, but are not limited to: the excipient is selected from calcium phosphate, magnesium stearate, talc, dextrin, starch, gelatin cellulose, methyl cellulose, sodium carboxymethyl cellulose and polyvinylpyrrolidone.

The pharmaceutical carrier of the present invention may be a pharmaceutically acceptable solvent, suspending agent, vesicle, nanomaterial, etc., for delivering the compound of the above first aspect of the present invention into the animal or human body. The carrier may be a liquid or solid and is selected according to the intended mode of administration. Proteins and liposomes are also drug carriers.

The compounds of the present invention may be formulated into pharmaceutical compositions or formulations using well known techniques by those skilled in the art. For example, any of the compounds (at least one compound) disclosed in the above first aspect of the present invention may be mixed with a pharmaceutically acceptable excipient, and then, if necessary, the resulting mixture may be formed into a desired shape. The preparation of pharmaceutical preparations can also be carried out according to known pharmaceutical preparations, except as mentioned in the present invention. And, in addition to those mentioned herein, suitable pharmaceutical excipients are known in the art, see for example the 2005 edition handbook of pharmaceutical excipients (fourth edition original), author (en) r.c. lo (Raymond c. Rowe) (usa) p.j. Sertoli (Paul j. Sheskey).

In a third aspect of the present invention, there is provided the use of the disulfide bond-containing N-acetyl-L-cysteine derivative described above in the manufacture of a pharmaceutical product for combating respiratory disorders, said respiratory disorders comprising: chronic obstructive pulmonary disease, pulmonary fibrosis, acute lung injury/acute respiratory distress syndrome.

Further, the product is specifically a drug which has pharmacological effects of oxidation resistance, anti-inflammation and the like, and is used for various indications, including dissolving phlegm, relieving acetaminophen drug poisoning, chronic Obstructive Pulmonary Disease (COPD), pulmonary fibrosis, acute lung injury/acute respiratory distress syndrome (ALI/ARDS), hyperoxia-induced lung injury, human Immunodeficiency Virus (HIV) infection, benign tumor or malignant tumor.

In a fourth aspect of the invention, there is provided an antioxidant, anti-inflammatory method comprising administering to a subject a therapeutically effective amount of a compound according to the first aspect of the invention or a pharmaceutical composition or formulation according to the third aspect of the invention.

The subject of the present invention refers to an animal, preferably a mammal, most preferably a human, who has been the object of treatment, observation or experiment.

The therapeutically effective amount of the present invention is that amount of active compound or pharmaceutical agent, including a compound of the present invention, which elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other medical professional, which includes alleviation or partial alleviation of the symptoms of the disease, syndrome, condition, or disorder being treated.

The range of therapeutically effective amounts that can be used will be known to the researcher, veterinarian, medical doctor or other medical professional in the art based on clinical trials or other means known in the art.

The disulfide bond-containing N-acetyl-L-cysteine (NAC) derivative of the present invention has superior antioxidant and anti-inflammatory activities. The antioxidant activity result shows that the derivatives such as A2, A3, A9, A10, A11, A16, A17, A18, A20, A21 and A22 enable the expression increasing trend of Nrf2 protein to be obvious, the expression increasing amount of Nrf2 protein exceeds NAC, and the significant difference exists. The anti-inflammatory activity result shows that the derivatives such as A3, A6, A7, A8, A9, A10, A11, A12, A14, A15, A16, A17, A18, A19, A21 and the like can obviously inhibit NO secretion of RAW264.7 cells and relieve inflammatory reaction generated by stimulation of LPS, and the anti-inflammatory activity of the derivatives is superior to that of a positive control drug fudosteine, and the derivatives have high anti-inflammatory activity.

The disulfide bond-containing N-acetyl-L-cysteine (NAC) derivative of the present invention can be used for the prevention and treatment of oxidative stress and inflammatory stress-related diseases such as Chronic Obstructive Pulmonary Disease (COPD), pulmonary fibrosis, acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Therefore, the N-acetyl-L-cysteine (NAC) derivative has the potential of being developed into antioxidant and anti-inflammatory medicaments.

The invention has the advantages of

(1) The invention provides a series of disulfide bond-containing N-acetyl-L-cysteine derivatives, and provides potential active compounds for the development of N-acetyl-L-cysteine derivative drugs. Tests in the technical scheme prove that the prepared compound has higher antioxidant and anti-inflammatory activities, so that the compound can be used for preparing medicines with better effect than NAC. In addition, the compound provided by the technical scheme has a simple and efficient preparation process, is suitable for industrial scale-up production, and has a good application prospect.

(2) The N-acetyl-L-cysteine (NAC) derivative can obviously increase the expression trend of Nrf2 protein, can also obviously inhibit RAW264.7 cells from secreting NO, and relieve inflammatory response generated by LPS stimulation, has better effect on preventing and treating oxidative stress and inflammatory stress related diseases such as chronic obstructive pulmonary disease (chronic obstructive pulmonary disease, COPD), pulmonary fibrosis, acute lung injury/acute respiratory distress syndrome (ALI/ARDS) and the like, and has the potential of being developed into related antioxidant and anti-inflammatory drugs.

(3) The invention obtains the derivative containing the disulfide bond structure by properly derivatizing NAC, and the derivative can release active sulfhydryl substances in vivo on the basis of improving the stability of the derivative, thereby achieving the aim of prolonging the half-life period. Secondly, on the basis of enhancing the permeability of cells and tissues and reducing the administration dosage, the composition has better effects of antioxidation, anti-inflammation and immunoregulation, and further improves the pharmacological activity of the composition.

(4) The preparation method is simple, strong in practicability and easy to popularize.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

Figure 1 is a graph of the effect of NAC derivative a10 on lung tissue injury index in COPD mice;

FIG. 2 is a graph of the effect of NAC derivative A10 on total cell number (a) and neutrophil number (b) in lung lavage (BALF) from COPD mice;

FIG. 3 is a graph showing the effect of NAC derivative A10 on the expression of SOD1 (a) and GPX2 (b) mRNA in lung tissue of COPD mice;

figure 4 is a graph of the effect of NAC derivative a10 on the area of pulmonary fibrosis in fibrotic mice;

FIG. 5 is a graph of the effect of NAC derivative A10 on the lung injury index in fibrotic mice;

FIG. 6 is a graph of the effect of NAC derivative A10 on the dry-to-wet ratio of pulmonary edema in fibrotic mice;

FIG. 7 is a graph of the effect of NAC derivative A10 on the total number of cells (a) and the number of neutrophils (b) in lung lavage fluid (BALF) from fibrotic mice;

FIG. 8 is a graph of the effect of NAC derivative A10 on the inflammatory factor IL-1 β (a) and transforming growth factor TGF- β (b) in pulmonary fibrosis mouse lung homogenates;

FIG. 9 is a graph of the effect of NAC derivative A10 on the lung injury index in ALI mice;

FIG. 10 is a graph of the effect of NAC derivative A10 on the dry-to-wet ratio of pulmonary edema in ALI mice;

FIG. 11 is a graph of the effect of NAC derivative A10 on the total number of cells (a) and the number of neutrophils (b) in lung lavage fluid (BALF) from ALI mice;

FIG. 12 is a graph showing the effect of NAC derivative A10 on the inflammatory factor IL-1 β (a) and transforming growth factor TNF- α (b) in mouse lung homogenates;

fig. 13 shows the effect of NAC derivative a10 on the antioxidant factors GSH (a) and SOD (b) in mouse lung homogenates.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all 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.

The present invention is described in further detail below with reference to specific examples, which are intended to be illustrative of the invention and not limiting.

Example 1:

preparation of N-acetyl-S- ((thien-2-ylmethyl) thio) -L-cysteine (A1)

0.215 g (1.36 mmol) L-anhydrous cysteine is dissolved in 1.7 mL methanol hydrochloride solution (hydrochloric acid is about 0.19 mol/L), the L-anhydrous cysteine solution is slowly and dropwise added into a solution of methoxycarbonylsulfonyl chloride (2.04 mmol) dissolved in 2 mL methanol within 20 min under the condition of an ice-water bath, and the reaction is continuously stirred under the condition of the ice-water bath, namely 1 h. The solvent and excess methoxycarbonylsulfonyl chloride were removed by rotary evaporation (bath temperature < 30 ℃). The solid obtained was washed repeatedly with diethyl ether and then filtered to obtain intermediate iii as a white crystalline solid. Dissolving raw material 2-thiophenemethylmercaptan (65 mg,0.5 mmol) in 0.4 zxft 3236 methanol, slowly dropwise adding a methanol solution of intermediate M1 (0.25 mmol,62.5 mg in 0.25 zxft 5262 methanol) at room temperature under stirring, dropwise adding a mixed solution of triethylamine (35 uL, 0.25 mmol) and 0.5 zxft 3763 methanol after completing dropwise adding for 45 min, continuously stirring overnight at room temperature, dropwise adding a mixed solution of triethylamine (35 uL, 0.25 mmol) and 0.5 zxft 3763 methanol, continuously stirring at room temperature for 30min, filtering white precipitate, washing the precipitate with methanol and ether respectively, and drying to obtain a white powder intermediate V. Intermediate V (2 mmol) was dissolved in 4mL glacial acetic acid, acetic anhydride (8 mmol) was added at room temperature with stirring, and the system was warmed to 40 ℃ and stirred overnight. The solvent was removed by rotary evaporation (water bath temperature 50-55 ℃) to give a crude yellow oil which was purified by normal phase column chromatography on 100-200 mesh silica gel with the mobile phase dichloromethane: methanol (20-50; and/or reverse phase medium pressure preparative chromatography with methanol as the mobile phase: water (0.4-1:1) in volume ratio to obtain white powder compound A1. ¹ H NMR (400 MHz, MeOD) δ7.31 (d, J = 4.5 Hz, 1H), 7.03 (d, J = 2.8 Hz, 1H), 6.94 (dd, J = 4.9, 3.6 Hz,1H), 4.65 (dd, J = 9.0, 4.3 Hz, 1H), 4.22 – 4.11 (m, 2H), 2.98 (dd, J = 13.8,4.4 Hz, 1H), 2.78 (dd, J = 13.8, 9.1 Hz, 1H), 2.00 (d, J = 5.9 Hz, 3H). HRMS:calculated for C ₁₀ H ₁₄ NO ₃ S ₃ (M+H ⁺ ): 292.0058, found: 292.0642.

Example 2:

preparation of N-acetyl-S- ((furan-2-ylmethyl) thio) -L-cysteine (A2)

The preparation method refers to example 1. So as to obtain the white powder, ¹ H NMR (400 MHz, MeOD) δ7.44 (s, 1H), 6.40 – 6.25 (m, 2H), 4.63 (dd, J = 9.0, 4.3 Hz, 1H), 4.01 – 3.88(m, 2H), 2.91 (dd, J = 13.8, 4.4 Hz, 1H), 2.75 (dd, J = 13.8, 9.1 Hz, 1H), 2.00(d, J = 7.4 Hz, 3H). HRMS: calculated for C ₁₀ H ₁₄ NO ₄ S ₂ (M+H ⁺ ): 275.0286, found: 275.1022.

example 3:

preparation of N-acetyl-S- ((pyridin-4-ylmethyl) thio) -L-cysteine (A3)

The preparation process is referred to example 1. So as to obtain the white powder, ¹ H NMR (400 MHz, MeOD) δ8.46 – 8.38 (m, 2H), 7.34 – 7.22 (m, 2H), 4.64 – 4.57 (m, 1H), 4.39 – 4.30 (m,2H), 2.92 – 2.81 (m, 1H), 2.74 – 2.58 (m, 1H), 1.95 (s, 3H). HRMS: calculatedfor C ₁₁ H ₁₅ N ₂ O ₃ S ₂ (M+H ⁺ ):287.0446, found: 287.0520.

example 4:

preparation of N-acetyl-S- (benzylthio) -L-cysteine (A4)

The preparation method refers to example 1. So as to obtain the white powder, ¹ HNMR (600 MHz, MeOD) δ 7.44 – 7.15 (m, 5H), 4.68 – 4.60 (m, 1H), 3.98 – 3.88 (m,2H), 2.95 – 2.85 (m, 1H), 2.77 – 2.65 (m, 1H), 1.98 (s, 3H). HRMS: calculatedfor C ₁₂ H ₁₆ NO ₃ S ₂ (M+H ⁺ ):285.0493, found: 285.1105.

example 5:

preparation of N-acetyl-S- ((4-methylbenzyl) thio) -L-cysteine (A5)

The preparation process is referred to example 1. So as to obtain the white powder, ¹ H NMR (400 MHz, MeOD) δ 7.22 (d, J =7.9 Hz, 2H), 7.12 (d, J = 7.8 Hz, 2H), 4.62 (dd, J = 9.0, 4.4 Hz, 1H), 3.95 –3.82 (m, 2H), 2.87 (dd, J = 13.8, 4.4 Hz, 1H), 2.70 (dd, J = 13.8, 9.1 Hz, 1H),2.31 (s, 3H), 1.98 (s, 3H).HRMS: calculated for C ₁₃ H ₁₆ NO ₃ S ₂ (M-H ⁺ ): 298.0650, found: 298.0579.

example 6:

preparation of N-acetyl-S- ((4-ethylbenzyl) thio) -L-cysteine (A6)

The preparation process is referred to example 1. So as to obtain the white powder, ¹ H NMR (600 MHz, MeOD) δ7.24 (d, J = 8.0 Hz, 2H), 7.15 (d, J = 8.1 Hz, 2H), 4.61 (dd, J = 9.1, 4.4 Hz,1H), 3.94 – 3.87 (m, 2H), 2.85 (dd, J = 13.8, 4.5 Hz, 1H), 2.71 – 2.66 (m, 1H),2.62 (q, J = 7.6 Hz, 2H), 1.98 (s, 3H), 1.21 (t, J = 7.6 Hz, 3H). HRMS:calculated for C ₁₄ H ₂₀ NO ₃ S ₂ (M+H ⁺ ):314.0806, found: 314.1511.

example 7:

preparation of N-acetyl-S- ((4-fluorobenzyl) thio) -L-cysteine (A7)

The preparation process is referred to example 1. So as to obtain the white powder, ¹ H NMR (400 MHz, MeOD) δ 7.36 (dd, J =8.4, 5.5 Hz, 2H), 7.04 (t, J = 8.7 Hz, 2H), 4.64 (dd, J = 9.0, 4.4 Hz, 1H),3.99 – 3.83 (m, 2H), 3.00 – 2.80 (m, 1H), 2.73 (dd, J = 13.8, 9.0 Hz, 1H), 1.99(s, 3H). HRMS: calculated for C ₁₂ H ₁₅ FNO ₃ S ₂ (M+H ⁺ ): 304.0399, found: 304.0471.

example 8:

preparation of N-acetyl-S- ((4-chlorobenzyl) thio) -L-cysteine (A8)

The preparation process is referred to example 1. So as to obtain the white powder, ¹ H NMR (400 MHz, MeOD) δ 7.36 – 7.23(m, 4H), 4.64 (dd, J = 8.9, 4.5 Hz, 1H), 4.00 – 3.81 (m, 2H), 2.90 (dt, J =31.0, 15.5 Hz, 1H), 2.73 (dd, J = 13.8, 9.0 Hz, 1H), 1.99 (s, 3H). HRMS:calculated for C ₁₂ H ₁₅ ClNO ₃ S ₂ (M+H ⁺ ):320.0104, found: 320.0181.

example 9:

preparation of N-acetyl-S- ((4-bromobenzyl) thio) -L-cysteine (A9)

0.607 g (1.51 mmol) 1,2-bis (4-bromobenzyl) disulfide was dissolved in 6 mL dry dichloromethane and stirred for 5min under nitrogen protection and ice water bath conditions. The compound m-CPBA (85%, 321mg, 1.59mmol) was dissolved in 5mL of dry dichloromethane, slowly added dropwise to the above solution, and the reaction was continued with stirring in an ice-water bath to 2 h. The reaction solution was washed four times with 5% sodium bicarbonate solution, the organic phase was washed once with water, dried over anhydrous sodium sulfate and the solvent was removed by rotary evaporation (water bath temperature < 40 ℃). Carrying out normal phase column chromatography on the obtained solid, filling 100-200 mesh silica gel, and carrying out petroleum ether as a mobile phase: ethyl acetate (20-50. 0.121g (0.29 mmol) of raw material S- (4-bromobenzyl) (4-bromophenyl) methanesulfonate is dissolved in 14.5 mL acetonitrile, N-acetylcysteine (VIII, 1.16 mmol) and triethylamine 0.145g (1.44 mmol) are added, and the mixture is protected by nitrogen and at room temperatureStirring overnight and rotary evaporation to remove the solvent (bath temperature 50-55 ℃). Diluting the obtained solid with water (15 mL), extracting with diethyl ether for three times, combining organic phases, drying with anhydrous sodium sulfate, removing solvent by rotary evaporation, subjecting the obtained solid to normal phase column chromatography, filling with 100-200 mesh silica gel, and using dichloromethane as mobile phase: methanol (20-50; and/or reverse phase medium pressure preparative chromatography with methanol as the mobile phase: water (0.4-1:1) to obtain white powder compound A9. ¹ H NMR (400 MHz, MeOD) δ7.77 (dd, J = 8.4, 5.5 Hz, 2H), 7.05 (t, J = 8.7 Hz, 2H), 4.64 (dd, J = 9.0,4.4 Hz, 1H), 3.97 – 3.80 (m, 2H), 2.99 – 2.78 (m, 1H), 2.73 (dd, J = 13.8, 9.0Hz, 1H), 1.99 (s, 3H). HRMS: calculated for C ₁₂ H ₁₅ BrNO ₃ S ₂ (M+H ⁺ ): 363.9598, found: 363.9756.

Example 10:

preparation of N-acetyl-S- ((4- (tert-butyl) benzyl) thio) -L-cysteine (A10)

The preparation method refers to example 9. So as to obtain the white powder, ¹ H NMR (400 MHz, MeOD) δ7.30 (dd, J = 35.6, 8.0 Hz, 4H), 4.60 (dd, J = 9.1, 4.3 Hz, 1H), 3.96 – 3.84(m, 2H), 2.80 (dd, J = 13.8, 4.3 Hz, 1H), 2.65 (dd, J = 13.7, 9.3 Hz, 1H), 1.98(s, 3H), 1.30 (s, 9H). HRMS: calculated for C ₁₆ H ₂₄ NO ₃ S ₂ (M+H ⁺ ): 342.1119, found: 342.1190.

example 11:

preparation of N-acetyl-S- ((2-methylbenzyl) thio) -L-cysteine (A11)

The preparation method refers to example 1. So as to obtain the white powder, ¹ H NMR (400 MHz, MeOD) δ7.28 – 7.05 (m, 4H), 4.63 (dd, J = 8.9, 4.3 Hz, 1H), 4.05 – 3.90 (m, 2H), 2.83(dd, J = 13.8, 4.4 Hz, 1H), 2.67 (dd, J = 13.8, 9.0 Hz, 1H), 2.41 (s, 3H), 1.98(s, 3H). HRMS: calculated for C ₁₃ H ₁₇ NO ₃ S ₂ (M+H ⁺ ): 300.0650, found: 300.0725.

example 12:

preparation of N-acetyl-S- ((2-chlorobenzyl) thio) -L-cysteine (A12)

The preparation process is referred to example 1. So as to obtain the white powder, ¹ H NMR (600 MHz, MeOD) δ7.46 – 7.35 (m, 2H), 7.31 – 7.21 (m, 2H), 4.65 (dd, J = 9.0, 4.4 Hz, 1H), 4.06(q, J = 12.7 Hz, 2H), 2.97 – 2.87 (m, 1H), 2.76 (dt, J = 16.4, 8.2 Hz, 1H),1.99 (s, 3H). HRMS: calculated for C ₁₂ H ₁₅ ClNO ₃ S ₂ (M+H ⁺ ): 320.0104, found: 320.0185.

example 13:

preparation of N-acetyl-S- ((2-bromobenzyl) thio) -L-cysteine (A13)

The preparation process is referred to example 1. So as to obtain the white powder, ¹ H NMR (400 MHz, MeOD) δ7.58 (d, J = 7.3 Hz, 1H), 7.43 (dd, J = 7.6, 1.3 Hz, 1H), 7.31 (t, J = 7.0 Hz,1H), 7.22 – 7.15 (m, 1H), 4.66 (dd, J = 8.9, 4.3 Hz, 1H), 4.14 – 4.02 (m, 2H),2.94 (dd, J = 13.8, 4.4 Hz, 1H), 2.77 (dd, J = 13.8, 9.0 Hz, 1H), 1.99 (s, 3H).HRMS: calculated for C ₁₂ H ₁₅ BrNO ₃ S ₂ (M+H ⁺ ): 363.9598, found: 363.9673.

example 14:

preparation of N-acetyl-S- ((3-fluorobenzyl) thio) -L-cysteine (A14)

The preparation process is referred to example 1. So as to obtain the white powder, ¹ H NMR (400 MHz, MeOD) δ7.32 (dd, J = 14.0, 7.8 Hz, 1H), 7.13 (dd, J = 24.2, 8.7 Hz, 2H), 6.99 (td, J =8.6, 2.2 Hz, 1H), 4.65 (dd, J = 9.0, 4.4 Hz, 1H), 4.00 – 3.88 (m, 2H), 2.92(dd, J = 13.8, 4.4 Hz, 1H), 2.74 (dd, J = 13.8, 9.1 Hz, 1H), 1.99 (s, 3H).HRMS: calculated for C ₁₂ H ₁₅ FNO ₃ S ₂ (M+H ⁺ ): 304.0399, found: 304.0474.

example 15:

preparation of N-acetyl-S- ((3- (trifluoromethyl) benzyl) thio) -L-cysteine (A15)

The preparation process is referred to example 1. So as to obtain the white powder, ¹ H NMR (400 MHz, MeOD) δ7.57 (ddd, J = 26.8, 16.1, 8.5 Hz, 4H), 4.65 (dd, J = 9.1, 4.4 Hz, 1H), 4.09 –3.92 (m, 2H), 2.91 (dd, J = 13.9, 4.4 Hz, 1H), 2.73 (dd, J = 13.8, 9.2 Hz, 1H),1.99 (s, 3H). HRMS: calculated for C ₁₃ H ₁₅ F ₃ NO ₃ S ₂ (M+H ⁺ ): 354.0367, found: 354.0439.

example 16:

preparation of N-acetyl-S- ((2,4,6-trimethylbenzyl) thio) -L-cysteine (A16)

The preparation method refers to example 9. So as to obtain the white powder, ¹ H NMR (400 MHz, MeOD) δ6.82 (s, 2H), 4.71 (dd, J = 9.0, 4.2 Hz, 1H), 4.08 (s, 2H), 3.06 (dd, J = 13.8,4.3 Hz, 1H), 2.83 (dd, J = 13.8, 9.2 Hz, 1H), 2.38 (s, 6H), 2.22 (s, 3H), 1.99(s, 3H). HRMS: calculated for C ₁₅ H ₂₂ NO ₃ S ₂ (M+H ⁺ ): 328.0963, found: 328.1041.

example 17:

preparation of N-acetyl-S- ((2,4-dichlorobenzyl) thio) -L-cysteine (A17)

The preparation method refers to example 1. So as to obtain white powder, and then the white powder is obtained, ¹ H NMR (400 MHz, MeOD) δ7.45 (dd, J = 20.7, 5.0 Hz, 2H), 7.30 (dd, J = 8.2, 1.9 Hz, 1H), 4.66 (dd, J =8.9, 4.3 Hz, 1H), 4.08 – 3.97 (m, 2H), 2.94 (dd, J = 13.8, 4.4 Hz, 1H), 2.78(dd, J = 13.8, 9.0 Hz, 1H), 1.99 (s, 3H).HRMS: calculated for C ₁₂ H ₁₄ Cl ₂ NO ₃ S ₂ (M+H ⁺ ): 353.9714, found: 353.2670.

example 18:

preparation of N-acetyl-S- ((naphthalen-2-ylmethyl) thio) -L-cysteine (A18)

The preparation method refers to example 1. So as to obtain the white powder, ¹ H NMR (400 MHz, MeOD) δ7.89 – 7.75 (m, 4H), 7.53 – 7.40 (m, 3H), 4.65 (dd, J = 8.7, 4.4 Hz, 1H), 4.14– 4.06 (m, 2H), 2.89 (dd, J = 13.8, 4.5 Hz, 1H), 2.69 (dd, J = 13.8, 9.0 Hz,1H), 1.99 (d, J = 13.0 Hz, 3H). HRMS: calculated for C ₁₆ H ₁₈ NO ₃ S ₂ (M+H ⁺ ): 336.0650, found: 336.0725.

example 19:

preparation of N-acetyl-S- (phenethylthio) -L-cysteine (A19)

The preparation process is referred to example 1. So as to obtain the white powder, ¹ H NMR (400 MHz, MeOD) δ7.34 – 7.09 (m, 5H), 4.71 (dd, J = 9.0, 4.3 Hz, 1H), 3.23 (dd, J = 13.9, 4.4Hz, 1H), 3.03 – 2.88 (m, 5H), 1.99 (s, 3H). HRMS: calculated for C ₁₃ H ₁₈ NO ₃ S ₂ (M+H ⁺ ): 300.0650, found: 300.0727.

example 20:

preparation of N-acetyl-S- ((1-phenylethyl) thio) -L-cysteine (A20)

The preparation process is referred to example 1. So as to obtain the white powder, ¹ H NMR (400 MHz, CDCl3) δ7.42 – 7.13 (m, 5H), 4.65 – 4.55 (m, 1H), 4.19 – 4.05 (m, 1H), 2.87 – 2.57 (m,2H), 1.98 (d, J = 2.3 Hz, 3H), 1.65 (dd, J = 7.0, 1.7 Hz, 3H). HRMS: calculatedfor C ₁₃ H ₁₈ NO ₃ S ₂ (M+H ⁺ ):300.0650, found: 300.0728.

example 21:

preparation of N-acetyl-S- ((4-methoxyphenyl) thio) -L-cysteine (A21)

The preparation method refers to example 9. So as to obtain the white powder, ¹ H NMR (400 MHz, MeOD) δ7.50 (d, J = 8.8 Hz, 2H), 6.92 (d, J = 8.8 Hz, 2H), 4.74 (dd, J = 8.9, 4.3 Hz,1H), 3.80 (s, 3H), 3.21 (dd, J = 13.9, 4.4 Hz, 1H), 2.98 (dd, J = 13.9, 9.0 Hz,1H), 1.95 (s, 3H). HRMS: calculated for C ₁₂ H ₁₆ NO ₄ S ₂ (M+H ⁺ ): 302.0442, found: 302.0516.

example 22:

preparation of N-acetyl-S- (cyclohexylthio) -L-cysteine (A22)

/>

The preparation method refers to example 9. So as to obtain white powder, and then the white powder is obtained, ¹ H NMR (400 MHz, MeOD) δ4.68 (dd, J = 9.0, 4.2 Hz, 1H), 3.89 (s, 1H), 3.18 (dt, J = 15.3, 6.5 Hz, 1H),2.93 (dd, J = 13.7, 9.2 Hz, 1H), 2.78 (s, 1H), 2.12 – 1.88 (m, 5H), 1.78 (s,2H), 1.63 (d, J = 10.9 Hz, 1H), 1.46 – 1.11 (m, 5H). HRMS: calculated for C ₁₁ H ₂₀ NO ₃ S ₂ (M+H ⁺ ): 278.0806, found: 278.0881.

example 23:

preparation of N-acetyl-S- (isopropylthio) -L-cysteine (A23)

The preparation method refers to example 1. White powder was obtained by 1H NMR (400 MHz, meOD) delta 4.69 (dd, J = 9.0, 4.3 Hz, 1H), 3.21 (dd, J = 13.7, 4.4 Hz, 1H), 3.09-2.88 (m, 2H), 2.00 (s, 3H), 1.29 (d, J = 6.7 Hz, 6H), HRMS calcaulated for C ₈ H ₁₆ NO ₃ S ₂ (M+H ⁺ ): 238.0493, found: 238.0566.

Example 24:

preparation of N-acetyl-S- (butylthiol) -L-cysteine (A24)

The preparation process is referred to example 1. So as to obtain the white powder, ¹ H NMR (400 MHz, MeOD) δ4.70 (dd, J = 9.2, 4.2 Hz, 1H), 3.21 (dd, J = 13.8, 4.3 Hz, 1H), 2.93 (dd, J =13.8, 9.3 Hz, 1H), 2.72 (t, J = 7.3 Hz, 2H), 2.00 (s, 3H), 1.72 – 1.59 (m, 2H),1.48 – 1.36 (m, 2H), 0.94 (t, J = 7.4 Hz, 3H). HRMS: calculated for C ₉ H ₁₈ NO ₃ S ₂ (M+H ⁺ ): 252.0650, found: 252.0726.

example 25:

the antioxidant activity of the compounds of the above examples was measured.

The Western Blot technology is adopted to examine the capability of NAC series derivatives on the expression of Nrf2 protein in 16HBE of human bronchial epithelial cells, NAC is taken as a positive drug, and potential lead compounds are screened by comparison.

16HBE cells are adherent cells, and the culture environment is as follows: 10 cm sterile Petri dishes containing 8 mL DMEM complete Medium, culture temperature of 37 degrees C, CO ₂ The concentration is 5%, the humidity is saturated, and the fresh culture medium is replaced at the right time to meet the requirement of cell growth. When the 16HBE cells were in logarithmic growth phase and in good condition, they were passaged to finally obtain 16HBE single cell suspensions, which were counted. Sterile 6-well plates were removed and complete DMEM medium was added at 500. Mu.L/well. Cells were then transferred to 6-well plates at a density of 1X 10 ⁶ One/well, 500 μ L per well, was mixed well with the previously added medium. And adding NAC series derivatives with different concentrations (2.5. Mu.M and 5. Mu.M) and NAC as positive drug, 24 h in the next day, and extracting protein from the cells.

Protein content was determined by BCA method. And preparing a BCA working solution (BCA: cu is 50. A small amount of Bovine Serum Albumin (BSA) 5 mg/mL standard was removed and diluted to 1 mg/mL with PBS and a BSA standard solution was prepared in a 96-well plate. And adding 20 mu L of diluted sample into the other row, adding 200 mu L of BCA working solution, mixing, reacting at 37 ℃ for 30min, and measuring the OD value at 562 nm after the reaction is finished. The protein content in the measurement sample was determined by fitting the OD value of the BSA standard solution. And finally, detecting the Nrf2 protein expression condition by Western Blotting (protein immunoblotting technology). As shown in table 1, the Nrf2 protein expression promoting amounts of the compounds including derivatives A2, A3, A9, a10, a11, a16, a17, a18, a20, a21, and a22 exceeded the positive control NAC, and the compounds had good antioxidant activity.

Table 1: N-acetyl-L-cysteine (NAC) derivatives affect Nrf2 protein expression ability

The experiment was repeated three times, and the data are the fold of the dosed group relative to the blank group and are expressed as mean ± SD (mean plus minus standard deviation).

Example 26:

the anti-inflammatory activity of the compounds of examples 1 to 24 was determined.

LPS induction is adopted in the experiment to construct an in vitro cell inflammation model, and the in vitro anti-inflammatory effect of the NAC series derivatives is evaluated. Firstly, selecting the NO level of cell culture supernatant as a detection index, co-culturing LPS (0-5 mu g/mL) with different concentrations and RAW264.7 cells, detecting the level change by an NO detection kit, and selecting proper LPS stimulation concentration and time. The results show that when the concentration of LPS is 4 mug/mL, the relative level of NO is obviously improved under the conditions of 12 h and 24 h which are co-cultured with the cells. Therefore, an inflammation model in the anti-inflammatory activity screening experiment is established by using 12 h of RAW264.7 cells induced by LPS with the concentration of 4 mu g/mL. After modeling, 2.5 and 5 mu M NAC series derivatives are given, fudosteine clinically used for treating diseases such as bronchitis and the like is selected as a positive drug, and the concentration of NO in the supernatant is detected to be used as an anti-inflammatory activity screening result. As shown in Table 2, the anti-inflammatory activity of the series of compounds, such as A3, A6, A7, A8, A9, A10, A11, A12, A14, A15, A16, A17, A18, A19, A21 and the like, is superior to that of the positive control fudosteine, and the anti-inflammatory activity is good.

Table 2: N-acetyl-L-cysteine (NAC) derivatives affect LPS stimulation of NO levels in the supernatant of RAW264.7 cells

* The experiment was repeated three times, with data as a fold of each group relative to the Ctrl group (control), fudostein.

Example 27:

has better antioxidant and anti-inflammatory activity, and compound A10 has the evaluation of anti-chronic obstructive pulmonary activity.

Selecting active smoking and trachea infusion LPS to establish a slow obstructive pulmonary disease (COPD) mouse model, selecting a C57BL/6 mouse as a research object, and taking N-acetyl-L-cysteine (NAC) as a positive control drug; in order to increase the solubility of a10, a10 was prepared as a methyl- β -cyclodextrin inclusion compound, and the anti-chronic obstructive pulmonary efficacy was evaluated after intraperitoneal injection in mice.

The C57BL/6 experimental mice (total 42) were randomly divided into 6 groups of 7 mice per group: (1) blank Control (Control): mice were given the same amount of PBS at the time of tracheal instillation and drug treatment; (2) COPD model group (CS + LPS): mice in COPD group and treatment group received tracheal instillation of LPS (10 μ g-50 μ L) on days 1 and 14, and then except for days 1 and 14, mice were exposed to smoke of 8 cigarettes each day for 2h each day for 30 days; (3) treatment group: the low, medium and high doses are respectively 20 mg/kg (L), 40 mg/kg (M) and 80mg/kg (H), and the A10 inclusion compound is injected into the abdominal cavity once a day from the 2 nd day; (4) NAC group: 80 The mg/kg dose was the same as the A10 dose.

The results of the determination of the pathological changes of lung tissues, the changes of the number of inflammatory cells in the airways and alveoli and the antioxidant related factors of the lungs of mice in the COPD model after A10 treatment are shown in FIG. 1, FIG. 2 and FIG. 3, respectively. As shown in fig. 1 to fig. 3, the therapeutic effects of a high dose (80 mg/kg, H) of a10 on COPD, such as lung injury index (fig. 1), total cell number in lung lavage fluid (a in fig. 2), neutrophil number (b in fig. 2), and SOD1 and GPX2 mRNA in lung tissue (fig. 3), are significantly better than those of NAC in the same dose, and the antioxidant activity (e.g., SOD1 and GPX2 mRNA) of a10 in a medium dose (40 mg/kg, M) is also significantly better than that of NAC, showing that the preferred NAC derivative (e.g., a 10) can significantly improve the therapeutic effect on chronic obstructive lung.

Example 28:

the compound A10 has better antioxidant and anti-inflammatory activity and can be used for evaluating the pulmonary fibrosis resistance.

A high-pressure spray needle is used for instilling bleomycin sulfate (BLM) into the trachea of a C57BL/6 mouse to establish a mouse pulmonary fibrosis model, A10 and positive control NAC are administered through gastric lavage, and the therapeutic effects of A10 groups on pulmonary fibrosis resistance, lung tissue damage, pulmonary edema, cascade inflammation and the like are investigated by recording weight change. Mice were randomly divided into blank control group (Ctrl), a10 control group (80 mg/kg, a 10), BLM model group (BLM), BLM + a10 low dose group (20 mg/kg, L), BLM + a10 medium dose group (40 mg/kg, M), BLM + a10 high dose group (80 mg/kg, H), BLM + NAC positive drug group (500 mg/kg, NAC), total 7 groups. After the mice are anesthetized, a BLM (5 mg/kg) sulfate solution is dripped into the lungs of the mice from the trachea of the mice through a high-pressure spray needle, the mice are kept in a sitting state after injection, the mice are slightly shaken left and right to enable the BLM and normal saline to be uniformly distributed in the lungs, and a mouse pulmonary fibrosis model is established. 24 After h, saline, a10 (L, M, H) and NAC were separately gavaged, and after a day of uninterrupted dosing, all animals were sacrificed painlessly and Bronchoalveolar lavage fluid (BALF) and lung tissue were collected as samples for subsequent experimental studies.

The therapeutic effects of a10 on pulmonary fibrosis, such as pulmonary fibrosis area, lung tissue damage, pulmonary edema, changes in the number of inflammatory cells in airways and alveoli, inflammation in lung homogenates, etc., are shown in fig. 4, fig. 5, fig. 6, fig. 7 and fig. 8, respectively. As shown in fig. 4-8, the treatment effect of orally administered high dose (80 mg/kg, H) a10 against pulmonary fibrosis, lung injury, pulmonary edema, inflammatory stress, etc. is significantly better than that of 500 mg/kg NAC; the preferable NAC derivative (such as A10) is shown to remarkably improve the oral bioavailability of the medicine and improve the curative effect of resisting pulmonary fibrosis.

Example 29:

has better antioxidant and anti-inflammatory activity, and compound A10 can be used for evaluating the acute lung injury resistance.

A high-pressure spray needle is used for dripping Lipopolysaccharide (LPS) into the trachea of a C57BL/6 mouse to establish a mouse Acute Lung Injury (ALI) model, A10 and positive control dexamethasone sodium phosphate (DEX) are intragastrically administered, and the treatment effects of resisting lung tissue injury, pulmonary edema, oxidative stress, inflammatory stress and the like of A10 groups are examined by recording the weight change. Mice were randomly divided into 7 groups, blank control group (C), LPS model group (M), LPS + a10 low dose group 20 mg/kg (20), LPS + a10 medium dose group 40 mg/kg (40), LPS + a10 high dose group 80mg/kg (80), LPS + DEX positive drug group (5 mg/kg, P). After the mice are anesthetized, LPS (5 mg/kg) solution is dripped into the lungs of the mice from the trachea of the mice through a high-pressure spray needle, the mice are kept in a sitting state after injection, and the mice are slightly shaken left and right to enable the LPS and normal saline to be uniformly distributed in the lungs, so that a mouse pulmonary fibrosis model is established. After 1 hour, saline, a10 (20, 40, 80) and DEX were separately gavaged, and 24 hours after the non-interrupted dosing, all animals were sacrificed painlessly and Bronchoalveolar lavage fluid (BALF) and lung tissue were collected as samples for subsequent experimental studies.

The therapeutic effects of A10 on lung tissue damage, pulmonary edema, changes in the number of inflammatory cells in the airways and alveoli, and acute lung injury resistance such as inflammatory factors and antioxidant factors in lung homogenate are shown in FIGS. 9, 10, 11, 12 and 13, respectively. As shown in fig. 9-13, the treatment effects of high-dose injection (80 mg/kg, 80) a10 on lung injury, pulmonary edema, inflammatory stress, oxidative stress and the like are obviously better than that of the positive control dexamethasone; the preferred NAC derivatives (e.g., a 10) are shown to have significant efficacy against acute lung injury.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An N-acetyl-L-cysteine derivative containing a disulfide bond, which is selected from the group consisting of compounds represented by the following formula I,

formula I

Wherein R is selected from: thien-2-ylmethyl, furan-2-ylmethyl, pyridin-4-ylmethyl, 4-methylbenzyl, 4-ethylbenzyl, 4-fluorobenzyl, 4-chlorobenzyl, 4-bromobenzyl, 4-tert-butylbenzyl, 2-methylbenzyl, 2-chlorobenzyl, 2-bromobenzyl, 3-fluorobenzyl, 3- (trifluoromethyl) benzyl, 2,4,6-trimethylbenzyl, 2,4-dichlorobenzyl, naphthalen-2-ylmethyl, phenethyl, 1-phenylethyl, 4-methoxyphenyl, cyclohexyl, isopropyl, n-butyl.

2. A method for synthesizing the disulfide bond-containing N-acetyl-L-cysteine derivative according to claim 1, comprising:

synthesizing the N-acetyl-L-cysteine derivative by adopting a first route or a second route;

the first synthetic route is as follows:

the specific steps of the route one include:

dissolving the compound corresponding to the general formula II in a methanol solution of hydrochloric acid, then adding the solution into a methanol solution of methoxycarbonylsulfonyl chloride for reaction, removing the solvent and reaction raw materials to obtain a solid, and purifying the solid to obtain the compound corresponding to the general formula III;

dissolving a compound containing a compound corresponding to a general formula IV or a salt thereof in methanol, adding a methanol solution containing a compound corresponding to a general formula III, adding a mixed solution of triethylamine and methanol after overnight, continuously stirring, and purifying to obtain a compound corresponding to a general formula V;

dissolving a compound containing a general formula V in acetic acid, adding acetic anhydride, reacting overnight, removing the solvent, and purifying an obtained oily crude product to obtain a compound containing a general formula I;

the second synthetic route is as follows:

the specific steps of the second route comprise:

dissolving a compound corresponding to the general formula VI in dichloromethane, and uniformly mixing;

dissolving m-CPBA in dichloromethane, dripping the solution into the dichloromethane for reaction, washing the reaction solution with sodium bicarbonate solution for multiple times, drying an organic phase, removing the solvent to obtain a solid, and purifying the solid to obtain a compound corresponding to the general formula VII;

dissolving a compound containing a compound corresponding to a general formula VII in acetonitrile, adding a compound containing a compound corresponding to a general formula VIII and triethylamine, reacting overnight, removing the solvent, diluting with water, extracting with diethyl ether, drying with an organic phase, removing the solvent, and purifying to obtain the compound corresponding to a general formula I.

3. The method for synthesizing N-acetyl-L-cysteine derivatives having disulfide bonds as claimed in claim 2, wherein the molar ratio of the compound corresponding to the general formula II to methoxycarbonylsulfonyl chloride is 1:1-3.

4. The method for synthesizing N-acetyl-L-cysteine derivatives having disulfide bonds according to claim 2, wherein the molar ratio of the compound corresponding to the general formula IV or a salt thereof to the compound corresponding to the general formula III is 1.1 to 1.

5. The method for synthesizing N-acetyl-L-cysteine derivatives having disulfide bonds according to claim 2, wherein the molar volume ratio of triethylamine to methanol is 1:1-3mmol/mL.

6. The method for synthesizing N-acetyl-L-cysteine derivatives having disulfide bonds according to claim 2, wherein the molar ratio of the compound corresponding to the general formula V to acetic anhydride is 1:1-8.

7. The method for synthesizing N-acetyl-L-cysteine derivatives containing disulfide bonds according to claim 2, wherein the molar volume ratio of the compound corresponding to formula V to acetic acid as a solvent is 1:1-4mmol/mL.

8. The method for synthesizing N-acetyl-L-cysteine derivatives containing disulfide bonds as claimed in claim 2, wherein the molar ratio of the compound VI of the general formula to m-CPBA is 1:1-1.5.

9. The method for synthesizing N-acetyl-L-cysteine derivatives containing disulfide bonds as claimed in claim 4, wherein the molar ratio of the compound corresponding to the general formula VII to the compound corresponding to the general formula VIII is 1:2-8;

or the molar ratio of the corresponding compound in the general formula VII to triethylamine is 1:2-10;

or, the molar volume ratio of the corresponding compound in the general formula VII to the solvent acetonitrile is 1 50mmol/mL.

10. Use of the disulfide-containing N-acetyl-L-cysteine derivative of claim 1 for the preparation of a pharmaceutical product for treating a respiratory disorder, wherein said respiratory disorder comprises: chronic obstructive pulmonary disease, pulmonary fibrosis, acute lung injury/acute respiratory distress syndrome.