CN113816970A

CN113816970A - Selective copper ion chelating agent, preparation method thereof and application thereof in pulmonary fibrosis

Info

Publication number: CN113816970A
Application number: CN202111111063.0A
Authority: CN
Inventors: 刘艳荣; 石小龙; 贾孟亭; 李莹; 孙涛; 张苗苗
Original assignee: AFFILIATED HOSPITAL OF JINING MEDICAL UNIVERSITY
Current assignee: AFFILIATED HOSPITAL OF JINING MEDICAL UNIVERSITY
Priority date: 2021-09-18
Filing date: 2021-09-18
Publication date: 2021-12-21
Anticipated expiration: 2041-09-18
Also published as: CN113816970B

Abstract

The invention discloses a selective copper ion chelating agent, a preparation method thereof and application thereof in pulmonary fibrosis, wherein the chelating agent is N- (2- (pyridine-3-yl) thieno [3,2-c ] pyridine-4-yl) acetamide. The JYFY-001 can improve and inhibit the level of pulmonary fibrosis of an organism, inhibit excessive deposition of extracellular matrix of lung, reduce inflammatory cells, inhibit proliferation of fibroblasts, and improve blood supply of lung so as to relieve dyspnea, has a good treatment effect on the pulmonary fibrosis, and has the advantages of easy acceptance, small toxic and side effects and low price. In addition, JYFY-001 changes the market pattern of the existing medicine for treating pulmonary fibrosis, and becomes a clinical medicine which can be taken for a long time, and effectively inhibits pulmonary fibrosis and improves the pulmonary function condition.

Description

Selective copper ion chelating agent, preparation method thereof and application thereof in pulmonary fibrosis

Technical Field

The invention belongs to the field of medicines, and particularly relates to a selective copper ion chelating agent, a preparation method thereof and application thereof in pulmonary fibrosis.

Background

Pulmonary fibrosis is the terminal change of a large group of lung diseases characterized by fibroblast proliferation and accumulation of large amounts of extracellular matrix with inflammatory lesions and destruction of tissue architecture, i.e. structural abnormalities resulting from abnormal repair after damage to normal alveolar tissue (karapitsakos T, et al, Eur J pharmacol.2017aug 5; 808: 35-43). Early stages are often overlooked or misdiagnosed as other diseases due to their non-specific manifestations, usually exertional dyspnea with or without dry cough. Severe pulmonary fibrosis is characterized by a change in normal lung tissue structure, loss of function, and replacement of alveoli by fibrotic tissues without gas exchange function, resulting in failure of oxygen to enter the blood. So that patients suffer from unsmooth breathing, hypoxia, acidosis, loss of labor force and death caused by serious patients. Pulmonary fibrosis incidence and mortality increase year by year, with an average survival period of only 3-5 years after diagnosis, with mortality rates higher than that of most tumors (Hutchinson J, et al, Eur Respir J.2015Sep; 46(3): 795-. Clinically, the disease progression is mainly delayed by a series of comprehensive treatment means such as pulmonary rehabilitation exercise, oxygen therapy, lung transplantation and drug therapy, and the current long-term anti-fibrosis drugs are mainly pirfenidone and nintedanib, which can significantly slow down the decline of lung function and possibly prolong the life cycle (Wijsenbeek M, et al, Lancet Respir med.2020may; 8(5): 424-. Pirfenidone, however, causes photosensitivity and nerve damage, while nintedanib causes myocardial infarction and severe acute bronchitis. Therefore, the development of novel high-efficiency and low-toxicity anti-fibrosis drugs is urgently needed.

Copper is an essential trace element in humans and there is increasing evidence that disturbances in copper ion levels are associated with the formation of pulmonary fibrosis. Research shows that the content of copper ions in sputum of patients with pulmonary fibrosis is obviously higher than that of normal patients (Forte et al, chemisphere 2021,271,129514); an increase in copper ion content accelerates the development of pulmonary fibrosis (He C, et al, J Biol chem.2013,288, (28), 20745-57); copper mediated H₂O₂Production results in increased collagen deposition, promoting the formation of pulmonary fibrosis (He C, et al, J Biol chem.2011,286(17): 15597-607); copper ions in serum as a main monoamine oxidase play a role in catalyzing collagen covalent bonding in the process of pulmonary collagen fibrosis formation, and are closely related to the pathogenesis process of pulmonary fibrosis (Loke thrifty, significance of serum sialic acid and ceruloplasmin on pulmonary fibrosis in the early stage of pneumoconiosis [ J]Journal of China labor health occupational disease, 2006,24(2): 119-; in addition, it has been found that tetrathiomolybdate, as a copper chelator, acts as an anti-fibrotic agent by inhibiting the Transforming Growth Factor (TGF) -beta, a key factor in fibrosis formation (Medici V, et al, IDrugs.2008Aug; 11(8): 592-606); the above evidence all suggest that targeting copper ions can be a therapeutic strategy for pulmonary fibrosis. Currently, the commonly used copper ion chelating agents in clinic comprise penicillamine, trientine, dimercaptosuccinic acid, sodium dimercaptopropanesulfonate and tetrathiomolybdate, but all of the chelating agents have the defect of poor specificity, and when copper is complexed, the chelating agents are also complexed with iron, zinc, calcium and the like in vivo, so that side effects such as gastrointestinal reaction, lupus erythematosus, myasthenia gravis, polymyositis and the like are easily caused. The invention provides a specific copper ion chelating agent JYFY-001 which can inhibit the progress of pulmonary fibrosis by specific chelating copper ions; and has less side effects.

Disclosure of Invention

The invention aims to provide a selective copper ion chelating agent JYFY-001, a preparation method thereof and application thereof in preparing a medicament for treating pulmonary fibrosis.

In order to achieve the above object, the present invention adopts the following technical route:

in one aspect, the present application provides a compound that is N- (2- (pyridin-3-yl) thieno [3,2-c ] pyridin-4-yl) acetamide (compound JYFY-001).

It has a molecular structure shown in formula JYFY-001:

in another aspect, the present application provides a process for preparing the above compound, which process proceeds according to the following reaction scheme:

further, the method comprises the following steps:

(1) 2-bromothieno [3,2-c ] pyridine-4-amine is taken as a raw material and condensed with acetic acid to generate an intermediate N- (2-bromothieno [3,2-c ] pyridine-4-yl) acetamide.

(2) And coupling the intermediate with 3-pyridine boronic acid to generate a target compound JYFY-001.

Further, the method comprises the steps of:

(1) acetic acid, DMAP (4-dimethylaminopyridine) and EDCI (1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride) were added to dichloromethane and stirred at room temperature. 2-bromothieno [3,2-c ] pyridin-4-amine was added and stirring was continued at room temperature. The dichloromethane was spin dried, ethyl acetate was added, and washed with water. Drying the product with anhydrous sodium sulfate, filtering, decompressing, concentrating, and carrying out silica gel column chromatography to obtain the intermediate N- (2-bromothieno [3,2-c ] pyridin-4-yl) acetamide.

(2) The intermediate, 3-pyridine boric acid and Pd (PPh)₃)₄、K₂CO₃(Potassium carbonate) was added to a mixed solvent of DMF (N, N-dimethylformamide) and water. The reaction was stirred at 100 ℃ under nitrogen protection. Ethyl acetate was added, washed with water, and dried over anhydrous sodium sulfate. Filtering, concentrating under reduced pressure, and performing silica gel column chromatography to obtain the target compound JYFY-001.

Further, in step (1), the molar ratio of 2-bromothieno [3,2-c ] pyridin-4-amine, acetic acid, DMAP, and EDCI is 1:2:2: 2.

Further, in step (1), acetic acid, DMAP and EDCI were added to dichloromethane and stirred at room temperature for 10 min.

Further, in the step (1), after 2-bromothieno [3,2-c ] pyridin-4-amine is added, the reaction is continued to be stirred at room temperature for 6 hours.

Further, in step (1), the eluent ratio in column chromatography is dichloromethane: methanol-100: 1.

Further, in the step (2), the intermediate, 3-pyridine boronic acid and Pd (PPh)₃)₄、K₂CO₃The molar ratio is 1:1.2:0.07: 3.

Further, in the step (2), nitrogen protection is required immediately after the feeding is finished.

Furthermore, in the step (2), stirring and reacting for 6 hours at 100 ℃ after the feeding is finished.

Further, in step (2), the eluent ratio in column chromatography is 50:1 dichloromethane to methanol.

In yet another aspect, the present application provides a composition comprising an effective amount of the above compound or an isomer or solvate or pharmaceutically acceptable salt thereof.

In yet another aspect, the present application provides a pharmaceutical formulation comprising an effective amount of a compound as described above or an isomer or solvate or pharmaceutically acceptable salt thereof or a composition as described above.

Further, the pharmaceutical preparation is an oral preparation selected from tablets, pills and capsules, and further comprises one or more pharmaceutically acceptable excipients and/or carriers.

Further, the excipient is selected from calcium phosphate, magnesium stearate, talc, dextrin, starch, gelatin cellulose, methyl cellulose, sodium carboxymethyl cellulose, and polyvinylpyrrolidone.

In addition, the application also provides the application of the compound or the isomer or the solvate or the medicinal salt or the composition in the preparation of Cu⁺/Cu²⁺Use of a chelating agent in medicine.

In addition, the application also provides the application of the compound or the isomer or the solvate or the pharmaceutically acceptable salt thereof or the composition in preparing anti-pulmonary fibrosis drugs.

The application is in inhibiting the level of pulmonary fibrosis of the body, inhibiting excessive deposition of extracellular matrix of the lung and inhibiting excessive proliferation of fibroblast cells of the lung.

The medicine for treating pulmonary fibrosis provided by the invention has the advantages that: the JYFY-001 can improve and inhibit the level of pulmonary fibrosis of an organism, inhibit excessive deposition of extracellular matrix of lung, reduce inflammatory cells, inhibit proliferation of fibroblasts, improve blood supply of lung and increase blood supply of lung so as to relieve dyspnea, has good treatment effect on the pulmonary fibrosis, and is easy to accept by patients with the medicine, small in toxic and side effects and low in price. In addition, JYFY-001 changes the market pattern of the existing medicine for treating pulmonary fibrosis, and becomes a clinical medicine which can be taken for a long time, and effectively inhibits pulmonary fibrosis and improves the pulmonary function condition.

Drawings

FIG. 1 is a schematic representation of JYFY-001 compound of example 1¹H NMR spectrum;

FIG. 2 is a schematic representation of JYFY-001 compound of example 1¹³A C NMR spectrum;

FIG. 3 is a HRMS spectrum of JYFY-001 of the compound in example 1;

FIG. 4 is the effect of JYFY-001 on HE histopathological changes in pulmonary fibrosis mice;

FIG. 5 is the effect of JYFY-001 on Masson histopathological changes in pulmonary fibrosis mice;

FIG. 6 is the effect of JYFY-001 on the change in pulmonary fibrosis mouse lung coefficients;

FIG. 7 is the effect of JYFY-001 on the percentage of hydroxyproline in lung tissue of mice with pulmonary fibrosis;

FIG. 8 is the effect of JYFY-001 on MDA content in pulmonary tissue of mice with pulmonary fibrosis.

For a person skilled in the art, other relevant figures can be obtained from the above figures without inventive effort.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments that can be derived by one of ordinary skill in the art from the embodiments given herein are intended to be within the scope of the present invention.

The test materials and sources thereof used in the present invention include:

(1) mice: the experimental animal is an SPF-level Kunming mouse purchased from Jinanpunyue experimental animal breeding Limited company, and the license number of the experimental animal is as follows: SCXK (lu) 20190003.

(2) Test article

JYFY-001 salt: white powder, preservation of test article: and (5) normal temperature.

(3) The preparation method of the used medicines and reagents comprises the following steps:

preparing a JYFY-001 solution: weighing JYFY-001 powder 1mg, and dissolving in 290ul ddH₂O, the mixture was dissolved sufficiently to prepare a 10mmol/l solution, which was used after being sterilized by filtration through a 0.22 μm filter, and the solution was prepared as it is each time. Both the preparation and use of the solution should be conducted in a sterile biosafety cabinet.

EXAMPLE 1 Synthesis of JYFY-001 Compound

Acetic acid (0.24g, 4.0mmol), DMAP (0.49g, 4.0mmol) and EDCI (0.77g, 4.0mmol) were added to 6mL CH₂Cl₂And stirring for 10min at room temperature. Adding 2-bromothieno [3,2-c ]]After pyridin-4-amine (Compound 1) (0.46g, 2.0mmol) the reaction was continued stirring at room temperature for 6 h. Spin-drying CH₂Cl₂Ethyl acetate was added, washed 2 times with water and 1 time with saturated NaCl solution. Anhydrous Na₂SO₄Drying, filtering, concentrating under reduced pressure, and performing silica gel column chromatography (dichloromethane: methanol: 100:1) to obtain white powdery solid N- (2-bromothieno [3, 2-c)]Pyridin-4-yl) acetamide (intermediate 2)0.43g, yield 79.3%. Melting point 178-;¹H NMR(400MHz,DMSO)δ8.42(d,J＝5.5Hz,1H),8.16(d,J＝5.5Hz,1H),7.84(s,1H),2.19(s,3H)；¹³C NMR(100MHz,DMSO)δ172.32,150.56,146.42,142.87,134.16,124.78,118.94,118.72,26.53；HRMS(ESI):Calcd.for C₉H₇BrN₂OS[M+H]⁺:270.9535,found:270.9538。

2(81.34mg, 0.30mmol), 3-pyridineboronic acid (44.25mg, 0.36mmol), Pd (PPh)₃)₄(17.33mg，0.02mmol)、K₂CO₃(124.39mg, 0.90mmol) was added to a mixed solvent of DMF (6mL) and water (0.4mL). The reaction is stirred for 6 hours at 100 ℃ under the protection of nitrogen. Ethyl acetate was added, washed 2 times with water and 1 time with saturated NaCl solution. Drying with anhydrous sodium sulfate, filtering, concentrating under reduced pressure, and performing silica gel column chromatography (dichloromethane: methanol: 50:1) to obtain white powdery solid N- (2- (pyridin-3-yl) thieno [3, 2-c)]Pyridin-4-yl) acetamide (compound JYFY-001)34mg in 42.1% yield. The melting point is 237 and 239 ℃;¹H NMR(400MHz,DMSO)δ10.57(s,1H),9.01(d,J＝2.0Hz,1H),8.62(d,J＝4.6Hz,1H),8.25(d,J＝5.5Hz,1H),8.18(d,J＝8.0Hz,1H),7.93(m,2H),7.55(dd,J＝7.9,4.8Hz,1H),2.22(s,3H)；¹³C NMR(100MHz,DMSO)δ169.83,150.18,148.52,147.37,147.32,142.21,139.29,134.14,130.09,129.48,124.71,121.30,115.52,24.00；HRMS(ESI):Calcd.for C₁₄H₁₁N₃OS[M+H]⁺:270.0696,found:270.0702。

example 2 establishment of mouse pulmonary fibrosis model and drug administration treatment

In the research, a mouse pulmonary fibrosis model is established, and the inducer is Bleomycin (BLM). In the experiment, 30 mice are randomly divided into 3 groups, 10 blank groups and 20 model groups. The pulmonary fibrosis model of the mice is induced by bleomycin, and the mice in the model group are fasted for 16h in advance before modeling. Mice in the model group were administered aqueous BLM (5 mg. kg-1) via tracheal perfusion, and the blank group was perfused with the same volume of saline in the same manner.

On day 7, 20 mice from the model group were randomly assigned to 2 groups. Respectively as follows: 10 models and 10 male and female halves of a JYFY-001 group. On day 8, the JYFY-001 group was started to administer a 50mg/kg dose of tail vein administration treatment (weighed before administration), and the blank group and the model group were administered with a corresponding volume of physiological saline for 14 days. From the day of administration, the body weight of each group of mice was measured every day, and the state of the mice was observed and recorded. After the administration, the lung tissue of the mice is weighed, and the lung coefficient is calculated as follows: lung coefficient ═ lung weight (mg) ÷ body weight (g).

Example 3 histopathological assessment of pulmonary fibrosis degree observations in Lung tissue

Lung tissues of 3 groups of mice treated for 2 weeks were collected, fixed, dehydrated by a conventional method, and paraffin tissue sections were prepared, stained with hematoxylin-eosin (HE) and Masson, and morphological changes of the lung tissues were observed under a microscope.

Example 4 determination of the percentage of hydroxyproline in Lung tissue

Weighing the wet weight of the lung tissue, adding the hydrolysate, and mixing uniformly. Hydrolyzing with boiling water for 20min, mixing for 1 time every 10min, and detecting hydroxyproline content in lung tissue according to the operation of kit instruction. Calculating the formula: percent hydroxyproline/% (sample absorbance ÷ standard solution absorbance) × standard solution concentration × dilution factor × sample volume ÷ lung tissue dry powder weight × 100%; percent collagen/% ═ HYP (%) × 7.46.

Example 5 determination of lung tissue MDA content

5% lung tissue homogenate is prepared, supernatant is taken after centrifugation, MDA content is measured by a TBA method, the operation is carried out according to the instruction of the kit, then OD value is measured at 532nm wavelength by an enzyme-labeling instrument, and the MDA content in the lung tissue of each sample is calculated.

Experimental results and evaluation:

1. mouse life status observation and weight change

In the experimental process, the living states of the mice in each group are observed: the blank group of mice has sensitive reaction, and the fur is bright and glossy; compared with the blank group, the model group mice are listless, have disordered fur and lackluster and have emaciation; the mental, fur color and activity conditions of the JYFY-001 group of mice are improved compared with those of the model group.

Effect of JYFY-001 on pathological changes of pulmonary tissue in pulmonary fibrosis model mice

HE staining and histopathological scoring results (fig. 4) show: the lung tissue of the blank group has no tissue edema, inflammatory cell infiltration and fibrosis characteristics, and the alveolar cavity has no exudation. Compared with the blank group, the lung tissue structure of the model group is damaged, inflammatory cells are infiltrated in a large amount, alveolar cavities are obviously reduced, collagen fibers and fibroblasts are increased in a large amount, and obvious pulmonary interstitial fibrosis can be seen; the lung tissue inflammatory cell infiltration area of the JYFY-001 group is reduced compared with that of the model group, the alveolar inflammatory cell infiltration and the pulmonary fibrosis degree are reduced compared with that of the model group, the fibroblast proliferation is reduced, and the lung tissue structure is more complete compared with that of the model group. Therefore, JYFY-001 can improve the degree of pulmonary fibrosis in a dose-dependent manner.

Masson staining results show (fig. 5): the collagen layer of the bronchial wall of the lung tissue of the blank group of mice is thinner, and a small amount of collagen is distributed in the alveolar region; the bronchial wall and pulmonary interstitial collagen fibers of the lung tissue of the model group mouse are obviously increased, the wall of the small blood vessel and the periphery thereof have more collagen distribution than that of the blank group, the collagen around the bronchus is proliferated and extends to the pulmonary interstitial space and is deposited in the interstitial space, and the pulmonary tissue is locally hyperemia and edema and visible and obvious red blood cell aggregation; compared with the model group, the collagen staining of the wall of the bronchus in the pulmonary interstitium, the blood vessel wall and the alveolar septum and the local congestion and edema phenomenon of the lung tissue of the JYFY-001 group are obviously reduced compared with the model group.

Effect of JYFY-001 on pulmonary fibrosis model mouse Lung coefficient variation

Lung coefficient statistics show (fig. 6): compared with a blank group of mice, the lung coefficient of the model group of mice is obviously increased; compared with the model group, the lung coefficient of the JYFY-001 group mice is obviously reduced.

Influence of JYFY-001 on percentage content of hydroxyproline in lung tissue of pulmonary fibrosis model mouse

The results of the percentage content of hydroxyproline in lung tissue of the pulmonary fibrosis model mice show (fig. 7): compared with a blank group, the hydroxyproline percentage content of the lung tissue of the model group mouse is obviously increased; compared with the model group, the lung tissue hydroxyproline content of the JYFY-001 group mice is obviously reduced.

Influence of JYFY-001 on MDA content of lung tissue of pulmonary fibrosis model mouse

Pulmonary fibrosis model mice lung tissue MDA content results show (fig. 8): compared with the blank group, the MDA content of the model group is obviously increased; compared with the model group, the lung tissue MDA content of the JYFY-001 group mice is obviously reduced.

The above examples show that: the JYFY-001 can inhibit excessive deposition of lung tissue extracellular matrix in a pulmonary fibrosis model, improve pulmonary fibrosis lesion, and has a therapeutic effect on pulmonary fibrosis.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A selective copper ion chelating agent characterized by: its name is N- (2- (pyridin-3-yl) thieno [3,2-c ] pyridin-4-yl) acetamide;

it has a structure shown in formula JYFY-001:

2. a preparation method of a selective copper ion chelating agent is characterized by comprising the following steps:

(1) condensing 2-bromothieno [3,2-c ] pyridine-4-amine serving as a raw material with acetic acid to generate an intermediate N- (2-bromothieno [3,2-c ] pyridine-4-yl) acetamide;

3. The method of claim 2, comprising the steps of:

(1) adding acetic acid, DMAP and EDCI into dichloromethane, and stirring at room temperature; adding 2-bromothieno [3,2-c ] pyridine-4-amine, and continuing stirring; spin-drying dichloromethane, adding ethyl acetate, and washing with water; drying with anhydrous sodium sulfate, filtering, concentrating under reduced pressure, and performing silica gel column chromatography to obtain intermediate N- (2-bromothieno [3,2-c ] pyridin-4-yl) acetamide;

(2) the intermediate, 3-pyridine boric acid and Pd (PPh)₃)₄、K₂CO₃Adding into a mixed solvent of DMF and water; stirring and reacting at 100 ℃ under the protection of nitrogen; adding ethyl acetate, washing with water, and drying with anhydrous sodium sulfate; filtering, concentrating under reduced pressure, and performing silica gel column chromatography to obtain the target compound.

4. The method of claim 3, wherein the selective copper ion chelating agent is prepared by: in the step (1), the molar ratio of the 2-bromothieno [3,2-c ] pyridin-4-amine, acetic acid, DMAP and EDCI is 1:2:2: 2; adding acetic acid, DMAP and EDCI into dichloromethane, and stirring at room temperature for 10 min; adding 2-bromothieno [3,2-c ] pyridine-4-amine, and then continuing stirring at room temperature for reaction for 6 hours; the eluent ratio in column chromatography dichloromethane to methanol is 100: 1.

5. The method of claim 3, wherein the selective copper ion chelating agent is prepared by: in the step (2), the intermediate, 3-pyridine boric acid and Pd (PPh)₃)₄、K₂CO₃The molar ratio is 1:1.2:0.07: 3; after the feeding is finished, nitrogen protection is required immediately; stirring and reacting for 6 hours at the temperature of 100 ℃ after the feeding is finished; the eluent ratio in column chromatography dichloromethane to methanol is 50: 1.

6. A composition comprising an effective amount of a compound of claim 1 or an isomer or solvate or pharmaceutically acceptable salt thereof.

7. A pharmaceutical formulation comprising an effective amount of a compound of claim 1 or an isomer or solvate or pharmaceutically acceptable salt thereof or a combination thereof.

8. A pharmaceutical formulation according to claim 7, which is an oral formulation selected from a tablet, pill or capsule, further comprising one or more pharmaceutically acceptable excipients and or carriers; the excipient is selected from calcium phosphate, magnesium stearate, pulvis Talci, dextrin, starch, gel cellulose, methylcellulose, sodium carboxymethyl cellulose or polyvinylpyrrolidone.

9. Use of the selective copper ion chelating agent of claim 1, or an isomer or solvate or pharmaceutically acceptable salt thereof, or a combination thereof, in the preparation of Cu⁺/Cu²⁺Use of a chelating agent in medicine.

10. Use of the selective copper ion chelating agent of claim 1, or an isomer or solvate or pharmaceutically acceptable salt thereof, or a combination thereof, for the manufacture of a medicament for the treatment of pulmonary fibrosis, wherein: inhibiting the level of pulmonary fibrosis of the body, inhibiting the excessive deposition of extracellular matrix in the lung and inhibiting the excessive proliferation of fibroblast in the lung.