A compound for treating pulmonary fibrosis and a preparation method and application thereof

Abstract

The present application relates to the technical field of medicine, in particular to a kind of compound for treating pulmonary fibrosis and its preparation method and application.2-amino-4,6-dimethoxybenzoic acid is used as starting material in the present application, and the compound product 2-benzoyl amino-6-hydroxy-4-methoxybenzoic acid of formula I is prepared;The 2-benzoyl amino-6-hydroxy-4-methoxybenzoic acid can effectively treat pulmonary fibrosis, and has good application prospect in the treatment of pulmonary fibrosis drugs.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a compound for the treatment of pulmonary fibrosis, its preparation method, and its application. Background Technology

[0002] Fibrosis refers to the abnormal and excessive repair and replacement of normal, functional cells and tissues in the lungs, forming hardened scar tissue. This scar tissue is called "fiber," which thickens and hardens the lung walls, causing them to lose elasticity. Pulmonary fibrosis prevents the lungs from expanding effectively, making it difficult for oxygen to enter the bloodstream from the alveoli, leading to persistent hypoxia. It is a progressive and irreversible disease, meaning that lung scarring will continue to worsen and spread over time.

[0003] Invention patent CN118955521B discloses a tetrandrine ferulic acid salt and its application in the preparation of drugs for treating pulmonary fibrosis. The pharmacological effects of tetrandrine and ferulic acid, as well as after salt formation, were studied. It was found that both have anti-pulmonary fibrosis effects, and the combination of the two can synergistically exert a better anti-pulmonary fibrosis effect. At the same time, it can also increase the solubility of tetrandrine.

[0004] Invention patent CN120118023A discloses an inhibitor for idiopathic pulmonary fibrosis (IPF), comprising compounds HO11553 and / or HO11552. This IPF molecular inhibitor targets the G protein-coupled receptor CCR2 and is a dual-pocket inhibitor capable of binding to both the ortho- and posterior binding sites of CCR2. By inhibiting CCR2, the molecular inhibitor slows the progression of pulmonary fibrosis, providing an effective treatment for IPF and demonstrating promising application prospects.

[0005] Chinese patent CN118324775B discloses a polyketide compound and its application in the preparation of drugs for treating pulmonary fibrosis. The polyketide compound provided by this invention, or its pharmaceutically acceptable salts, esters, or solvates, can effectively inhibit fibrosis in the respiratory system and other tissues and organs.

[0006] There are few reports in the prior art regarding the synthesis of a drug for the treatment of pulmonary fibrosis using 2-amino-4,6-dimethoxybenzoic acid as a starting material. This invention addresses this technical problem and provides a novel compound drug for the treatment of pulmonary fibrosis. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a compound for the treatment of pulmonary fibrosis, its preparation method, and its application. This invention is the first to synthesize a drug for the treatment of pulmonary fibrosis using 2-amino-4,6-dimethoxybenzoic acid as a starting material. This drug can effectively treat pulmonary fibrosis, expanding the types and categories of such drugs.

[0008] This invention discloses a compound having therapeutic activity for pulmonary fibrosis, named 2-benzoylamino-6-hydroxy-4-methoxybenzoic acid, with the following structural formula:

[0009] , Formula I.

[0010] The present invention also provides the use of the compound in the preparation of a medicament for treating pulmonary fibrosis.

[0011] This invention also provides a drug for treating pulmonary fibrosis, the chemical structural formula of which is:

[0012] , Formula I.

[0013] The present invention also provides a method for preparing the above-mentioned mixture or drug, comprising the following steps:

[0014] Step 1: Methyl 2-amino-4,6-dimethoxybenzoate is prepared by reacting a solvent, 2-amino-4,6-dimethoxybenzoic acid, a basic reagent, and iodomethane.

[0015] Step 2: Methyl 2-benzoamide-4,6-dimethoxybenzoate was prepared by reacting a solvent, methyl 2-amino-4,6-dimethoxybenzoate, benzoic acid, N,N,N',N'-tetramethylchloromethamidine hexafluorophosphate and N-methylimidazolium.

[0016] Step 3: Prepare 2-benzoamide-6-hydroxy-4-methoxybenzoic acid by reacting a mixed solvent, methyl 2-benzoamide-4,6-dimethoxybenzoate and HBr solution.

[0017] The synthetic route in the preparation method of this invention is as follows:

[0018] In the above synthetic route, 1-1 is 2-amino-4,6-dimethoxybenzoic acid; 1-2 is methyl 2-amino-4,6-dimethoxybenzoate; 1-3 is methyl 2-benzoamide-4,6-dimethoxybenzoate; and 1 is the target product 2-benzoamide-6-hydroxy-4-methoxybenzoic acid.

[0019] Preferably, the solvent in step 1 includes at least one of dimethylformamide and dimethylacetamide.

[0020] Preferably, the alkaline reagent in step 1 includes at least one of sodium carbonate and potassium carbonate.

[0021] Preferably, the solvent in step 2 includes acetonitrile.

[0022] Preferably, the mixed solvent in step 3 is a mixture of methanol and water.

[0023] Preferably, in step 1, the weight ratio of 2-amino-4,6-dimethoxybenzoic acid, alkaline reagent and iodomethane is (100-200):(150-250):(80-130).

[0024] Preferably, in step 2, the weight ratio of methyl 2-amino-4,6-dimethoxybenzoate, benzoic acid, N,N,N',N'-tetramethylchloromethacin hexafluorophosphate and N-methylimidazole is (80-130):(70-95):(180-260):(90-150).

[0025] Preferably, in step 3, the ratio of methyl 2-benzoamide-4,6-dimethoxybenzoate to HBr solution is (70-120) g : (200-400) mL. The concentration of HBr solution is 0.5-1.5 mol / L.

[0026] Preferably, in step 1, the reaction temperature and time are 20-35℃ and 0.3-2 hours.

[0027] Preferably, in step 2, the reaction temperature and time are 20-35℃ and 0.5-2.5 hours.

[0028] Preferably, in step 3, the reaction temperature and time are 20-40℃ and 5-12 hours.

[0029] Preferably, the above preparation method specifically includes the following steps:

[0030] Step 1: Add 2-amino-4,6-dimethoxybenzoic acid and alkaline reagent to the solvent and mix well. Add iodomethane and stir to react (reaction temperature and time: 20-35℃, 0.3-2 hours). Dilute with water and extract with ethyl acetate. Concentrate and dry the extract to obtain methyl 2-amino-4,6-dimethoxybenzoate.

[0031] Step 2: Add methyl 2-amino-4,6-dimethoxybenzoate and benzoic acid to the solvent and mix well. Add N,N,N',N'-tetramethylchloromethamide hexafluorophosphate and N-methylimidazole and stir to react (reaction temperature and time: 20-35℃, 0.5-2.5 hours). Recover the solvent under reduced pressure, dilute with water to obtain a precipitate, wash the precipitate (e.g., with water), and dry to obtain methyl 2-benzoamide-4,6-dimethoxybenzoate.

[0032] Step 3: Add methyl 2-benzoamide-4,6-dimethoxybenzoate to the mixed solvent and stir to mix. Add HBr solution in batches (e.g., 2-5 times) and stir to react (reaction temperature and time: 20-40℃, 5-12 hours). Dilute with water, precipitate, filter, wash (e.g., with water), dry, and purify to obtain 2-benzoamide-6-hydroxy-4-methoxybenzoic acid.

[0033] Preferably, in step 3, purification includes: silica gel column chromatography followed by elution with a petroleum ether / ethyl acetate mixed solvent, followed by concentration and drying of the elution product to obtain the 2-benzoamide-6-hydroxy-4-methoxybenzoic acid.

[0034] The advantages of this invention compared to the prior art are:

[0035] The present invention provides a novel mixture 1 for treating pulmonary fibrosis, which is synthesized from 2-amino-4,6-dimethoxybenzoic acid as a starting material, and the compound has highly effective activity in treating pulmonary fibrosis.

[0036] The tests of this invention show that, compared with the model group, the mice in the drug group treated with the active compound 1 of this invention have a slow increase in body weight, a significant reduction in the damage to lung tissue, a gradual decrease in the deposition of blue collagen fibers, a decrease in the fluorescence intensity of TGF-β and a weakening of TGF-β expression in the lung tissue, and a significant decrease in the mRNA expression of TNF-α and α-SMA in the lung tissue.

[0037] The compound 2-benzoamide-6-hydroxy-4-methoxybenzoic acid prepared in this invention can effectively improve inflammatory infiltration, alveolar space collapse, and collagen deposition in the lung tissue of mice with pulmonary fibrosis. It can also reduce the expression levels of pulmonary fibrosis-related genes TNF-α, α-SMA, and TGF-β, and is effective in mouse pulmonary fibrosis models, showing good therapeutic effects on pulmonary fibrosis. Attached Figure Description

[0038] Figure 1 : Proton NMR spectrum of compound 1.

[0039] Figure 2 Mass spectrum of compound 1.

[0040] Figure 3 Hydrogen spectra of compounds 1-2.

[0041] Figure 4 Mass spectra of compounds 1-2.

[0042] Figure 5 Hydrogen spectra of compounds 1-3.

[0043] Figure 6Mass spectra of compounds 1-3.

[0044] Figure 7 : Graph showing changes in mouse body weight.

[0045] Figure 8 HE staining image of mouse lung tissue.

[0046] Figure 9 Masson staining image of mouse lung tissue.

[0047] Figure 10 Immunofluorescence staining of mouse lung tissue (100×); (A) TGF-β fluorescence expression and (B) quantification.

[0048] Figure 11 : Expression of TNF-α and α-SMA mRNA in lung tissue. Detailed Implementation

[0049] The present invention will be described below through examples and experimental cases. However, the scope of the present invention is not limited to these examples and experimental cases.

[0050] I. Synthesis of 2-benzoamide-6-hydroxy-4-methoxybenzoic acid

[0051] Step 1: In 3 L of dimethylformamide solvent, add 2-amino-4,6-dimethoxybenzoic acid (150 g, compound 1-1) and potassium carbonate (210.2 g), and mix well; then add iodomethane (107.9 g) dropwise at room temperature and stir for 30 minutes. Then dilute with water, extract with ethyl acetate, concentrate under reduced pressure, and dry to obtain intermediate methyl 2-amino-4,6-dimethoxybenzoate (107 g, compound 1-2).

[0052] Step 2: In 3 L of acetonitrile solvent, methyl 2-amino-4,6-dimethoxybenzoate (107 g, compounds 1-2) and benzoic acid (86.6 g) were added and mixed well; N,N,N',N'-tetramethylchloromethamidine hexafluorophosphate (213.2 g) and N-methylimidazole (124.7 g) were added at room temperature, and the mixture was stirred for 60 minutes. After the solvent was recovered under reduced pressure, water was added to dilute the mixture and a precipitate was obtained. The precipitate was washed with pure water and dried to obtain the intermediate methyl 2-benzoamide-4,6-dimethoxybenzoate (96 g, compounds 1-3).

[0053] Step 3: In a 2 L mixture of methanol and water (4:1 v / v), methyl 2-benzoamide-4,6-dimethoxybenzoate (96 g, compounds 1-3) was added and mixed. The mixture was then added in three equal portions of HBr solution (1 mol / L, 300 mL total) and stirred at room temperature for 8 hours. The reaction mixture was then diluted with water to precipitate the product. The precipitate was filtered, washed with pure water, dried, and subjected to silica gel column chromatography. Elution was performed using 4 column volumes of a petroleum ether / ethyl acetate mixture (2:1 v / v). The eluted product was concentrated and dried to obtain 2-benzoamide-6-hydroxy-4-methoxybenzoic acid (1.6 g, compound 1).

[0054] The proton and mass spectra of compounds 1-2, 1-3, and 1 are shown below. Figure 1-6 .

[0055] Compound 1: Molecular formula: C 15 H 13 NO5; ESIMS: 287.08 [M+H] + .

[0056] 1 H NMR (DMSO-d6, 400 MHz): δ 7.97-7.89 (m, 2H), 7.82 (d, J = 2.6 Hz, 1H), 7.66-7.51 (m, 3H), 6.23 (d, J = 2.6 Hz, 1H), 3.79 (s, 3H).

[0057] II. Test

[0058] (I) Experimental Objective

[0059] To investigate the therapeutic effect of compound NB211 on a mouse model of pulmonary fibrosis.

[0060] (II) Experimental Methods

[0061] 1. Laboratory animals and grouping

[0062] C57 / BL6 mice, male, weighing 18-22g, were acclimatized for 3 days. Their general condition, including diet, body weight, and mental state, was observed.

[0063] The experiment was divided into a blank control group, a model group, an NB211 group (compound 1, dose 200 mg / kg), and a positive control group (nintedanib, dose 100 mg / kg), with 6 animals in each group.

[0064] 2. Modeling Method

[0065] Mice were anesthetized by intraperitoneal injection of 0.6% sodium pentobarbital. After anesthesia, mice in the blank control group were instilled with 50 μL of physiological saline via tracheal infusion, while mice in the model group and drug-treated group were instilled with 50 μL of bleomycin via tracheal infusion at a dose of 5 mg / kg. The drugs were administered by gavage on the day of model establishment and continued for 30 days. The weight changes of the mice were monitored, and the mice were dissected and harvested on day 31.

[0066] 3. Administration method

[0067] The treatment group received oral administration once a day for 30 days starting from the beginning of the formal experiment.

[0068] 4. Obtain materials

[0069] Mice died from spinal dislocation after intraperitoneal injection of 0.2 mL of 0.6% sodium pentobarbital followed by blood collection from the orbital cavity. Lung tissue was collected after cardiac lavage and fixed in paraformaldehyde.

[0070] 5. Experimental reagents and equipment

[0071] (1) Experimental consumables, see Table 1.

[0072] Table 1: Experimental Consumables

[0073] Bleomycin Beijing Solarbio Technology Co., Ltd. Nidanip Shijiazhuang Pharmaceutical Group Enbipu Pharmaceutical Co., Ltd. Hematoxylin-eosin staining solution Beijing Solarbio Technology Co., Ltd. Masson staining kit SouthernBiotech DAPI Fluoromount-G Nanjing Novizan Biotechnology Co., Ltd. HiScript II Q RT SuperMix for qPCR Nanjing Novizan Biotechnology Co., Ltd. ChamQ Universal SYBR qPCR Master Mix Nanjing Novizan Biotechnology Co., Ltd.

[0074] (2) Experimental instruments, see Table 2.

[0075] Table 2: Experimental Instruments

[0076] A balance with an accuracy of 0.01% Guangzhou Shouke Instrument Technology Co., Ltd. tissue dehydrator Hubei Kuohai Biotechnology Co., Ltd. Embedding machine Hubei Kuohai Biotechnology Co., Ltd. Tissue slicer Leica Instruments GmbH, Germany Histopathology microscope Carl Zeiss Optics Co., Ltd. Low temperature grinding machine Sewell Biotechnology Co., Ltd. Ultra-micro UV spectrophotometer IMPLEN GmbH, Germany

[0077] 6. Pathological section dehydration and embedding process:

[0078] After the tissues were fixed with paraformaldehyde for 48 hours, they were immersed in different reagents, with the following specific steps: 75% ethanol for 1.5 hours, 85% ethanol for 1.5 hours, 95% ethanol for 1.5 hours, 100% ethanol I for 1.0 hour, 100% ethanol II for 1.0 hour, 100% ethanol III for 1.0 hour, xylene I for 1.5 hours, xylene II for 1.5 hours, paraffin I for 2.0 hours, and paraffin II for 2.0 hours.

[0079] 7. HE staining steps

[0080] (1) The slices were dewaxed in xylene I and II for 15 min each;

[0081] (2) Gradient ethanol dehydration: Soak in 100% ethanol I and II for 3 min each; soak in 95% ethanol for 3 min; soak in 80% ethanol for 3 min;

[0082] (3) Soak in double-distilled water for 1 min;

[0083] (4) Stain with hematoxylin for 5 min, then rinse with running water for 1 min;

[0084] (5) Soak the sections in 1% hydrochloric acid ethanol for 5 seconds, then rinse with running water for 15 minutes;

[0085] (6) Stain with eosin for 2 min, then rinse with running water for 1 min;

[0086] (7) Gradient ethanol dehydration: Soak the sections in 80% ethanol for 2 seconds, and then let them stand in 100% ethanol (pure ethanol) for 7 minutes;

[0087] (8) Treat the slices with xylene I and II for 5 min;

[0088] (9) Neutral resin sealing.

[0089] 8. Masson staining procedure.

[0090] (1) Sections are routinely dewaxed and rehydrated, with the same steps as 7(1)-7(3);

[0091] (2) Stain with the prepared Weigert iron hematoxylin staining solution for 8 minutes;

[0092] (3) Differentiate in acidic ethanol solution for 10 seconds, then wash with water;

[0093] (4) Bluing with Masson blue solution for 4 min, then washing with water and distilled water for 1 min;

[0094] (5) Stain with Pomfret and fuchsin solution for 8 minutes;

[0095] (6) In the above operation, prepare a weak acid working solution with a volume ratio of distilled water: weak acid solution = 2:1, and wash with the weak acid working solution for 1 min.

[0096] (7) Wash with phosphomolybdic acid solution for 1.5 min; then wash with the prepared weak acid working solution for 1 min;

[0097] (8) Immerse directly in aniline blue staining solution for 1.5 min. Wash with prepared weak acid working solution for 1 min;

[0098] (9) Rapid dehydration with 95% ethanol for 2 seconds, and dehydration with anhydrous ethanol 3 times for 8 seconds each time;

[0099] (10) Xylene clearing 3 times, 1.5 min each time, and then seal with neutral resin.

[0100] 9. Immunofluorescence staining procedure

[0101] (1) Dewax the paraffin sections to water.

[0102] (2) Antigen retrieval: Tissue slides were placed in a retrieval box filled with antigen retrieval buffer and microwaved for antigen retrieval. Microwave on medium heat for 8 minutes, turn off for 8 minutes, then microwave on medium-low heat for another 7 minutes. During this process, excessive evaporation of the buffer should be prevented, and the slides should not be allowed to dry. After natural cooling, the slides were placed in PBS and washed three times on a decolorizing shaker for 5 minutes each time.

[0103] (3) Permeabilization: Permeabilize with Beyotime immunostaining permeabilization solution for 10 min, wash with PBS 3 times, 5 min each time.

[0104] (4) Blocking: Block with Beyotime immunostaining blocking solution for 15 min, wash with PBS 3 times, 5 min each time.

[0105] (5) Primary antibody incubation: Dilute the primary antibody with primary antibody dilution buffer, add 100 μL of primary antibody to each tablet, and incubate overnight at 4°C.

[0106] (6) Secondary antibody incubation: Remove the slides from the refrigerator and allow them to warm to room temperature for 25 min. Wash three times with PBS. Dilute the secondary antibody with secondary antibody dilution buffer. Add 100 μL of secondary antibody to each slide and incubate at room temperature for 1 h. Observe quickly under a fluorescence microscope to determine whether staining has occurred and to determine the termination time. Wash three times with PBS for 5 min each time, and wipe off the PBS outside the specimen with filter paper.

[0107] (7) Mounting + DAPI staining: Add DAPI-containing anti-fluorescence quenching agent to the mounting slide.

[0108] 10. qPCR experimental procedure

[0109] (1) Extract Total RNA from lung tissue according to the kit instructions and measure its concentration using an ultra-micro UV-Vis spectrophotometer.

[0110] (2) RT-PCR (20 μL per system), including 4 μL of 5x BUFFER, X μL of Total RNA, and μL of DEPC-H2O (16-X). After placing them in 8-tube sets, label them with serial numbers, and place them in a PCR instrument set to 50℃ / 15min and 85℃ / 5s for reverse transcription.

[0111] (3) Take out the transcribed cDNA and add 76 μL DEPC-H2O to make the total volume 96 μL. Centrifuge and multiply by the number of samples (SYBR 10 μL + primer 0.4 μL) to 10.4 μL / well of a 96-well plate. Add 9.6 μL of cDNA to make a 20 μL system and centrifuge to mix.

[0112] (4) Place the sample plate into the CFX Connect Real-Time System (real-time fluorescence quantitative PCR instrument) for detection. Perform the detection according to the procedure of (95℃ / 2min, 95℃ / 20s, 57℃ / 20s, 72℃ / 20s) for a total of 39 cycles, and then perform the detection at 95℃ / 1min, 55℃ / 30s, and 95℃ / 30s.

[0113] (5) Using 2 -ΔΔCt The experimental results were analyzed using the following formulas: △Ct target gene = Ct target gene - Ct internal reference gene; △△Ct target gene = △Ct experimental group target gene - △Ct control group target gene. 2 -ΔΔCt This indicates the fold increase in the expression of the target gene in the experimental group compared to the control group.

[0114] (III) Experimental Results

[0115] 1. Changes in mouse body weight

[0116] like Figure 7 As shown, the weight of mice in the blank control group gradually increased, while the weight of mice in the model group and the positive drug group initially decreased and then increased. The weight of mice in the NB211 group increased slowly. The weight of mice in each drug-treated group was slightly higher than that of the model group.

[0117] 2. HE staining results

[0118] like Figure 8 As shown, HE results indicated that the lung tissue in the model group was damaged, with significant inflammatory infiltration and alveolar space collapse, while both the NB211 group and the positive drug group showed significant relief after administration.

[0119] 3. Masson staining

[0120] like Figure 9 As shown, the control group mice had a small amount of collagen fiber deposition around the trachea in their lung tissue, while the model group had a large amount of blue collagen fiber deposition, indicating more severe pulmonary fibrosis. In contrast, the blue collagen fiber deposition in both the NB211 group and the positive control group decreased to varying degrees after drug administration.

[0121] 4. Immunofluorescence staining

[0122] like Figure 10 As shown, compared with the blank group, the fluorescence intensity of TGF-β in the lung tissue of the model group was significantly enhanced; compared with the model group, the fluorescence intensity of TGF-β in both the BN211 and positive control groups was weakened after administration. Statistical analysis results showed that compared with the blank group, the expression of TGF-β in the lung tissue of the model group was slightly increased; compared with the model group, the expression in the positive control group showed a slight decreasing trend.

[0123] 5. qPCR results

[0124] like Figure 11 As shown, compared with the blank group, the mRNA expression of TNF-α and α-SMA in the lung tissue of the model group was significantly increased; compared with the model group, the mRNA expression of TNF-α and α-SMA in each drug-treated group was significantly decreased. Statistical analysis showed that the differences were statistically significant (P<0.05).

[0125] (IV) Experimental Conclusions

[0126] Both NB211 and the positive control drug effectively improved inflammatory infiltration, alveolar space collapse, and collagen deposition in the lung tissue of mice with pulmonary fibrosis. They also reduced the expression levels of pulmonary fibrosis-related genes TNF-α, α-SMA, and TGF-β, demonstrating their effectiveness in the mouse model of pulmonary fibrosis.

[0127] For those skilled in the art, various corresponding changes and modifications can be made based on the above technical solutions and concepts, and all such changes and modifications should be included within the protection scope of the claims of this invention.

Claims

1. A compound, characterized in that, The compound has therapeutic activity against pulmonary fibrosis. The compound is named 2-benzoylamino-6-hydroxy-4-methoxybenzoic acid, and its structural formula is Formula I. Equation I.

2. The use of the compound according to claim 1 in the preparation of a medicament for treating pulmonary fibrosis.

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