THK01 and its derivatives, methods of preparation and use in the preparation of anti-inflammatory drugs

Abstract

The application discloses THK01 and derivatives thereof, a preparation method and application of the THK01 and the derivatives in preparation of anti-inflammatory drugs, and relates to the following steps: a series of chemical reactions, including Wittig reaction, Diels-Alder reaction, Cadogan reaction, Fischer indole synthesis reaction, demethylation reaction, reduction reaction and click chemistry reaction, are carried out on ortho-nitrobenzaldehyde containing specific substituents, so that THK01 structural modification derivatives with different substituents are obtained. The THK01 structural modification derivatives are synthesized in various routes, and the reaction success rate is high. In vitro and in vivo experiments show that the compounds can significantly inhibit the production of NO and inflammatory reactions at a low concentration, and can effectively treat inflammatory bowel disease and H1N1 virus-induced acute pneumonia in an animal model. The findings provide valuable candidate compounds and new treatment strategies for developing new, safe and efficient anti-inflammatory drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to THK01 and its derivatives, preparation methods, and their application in the preparation of anti-inflammatory drugs. Background Technology

[0002] Inflammation is a protective immune response of the body to harmful stimuli (such as pathogen invasion, tissue damage, or foreign bodies). Under normal physiological conditions, the inflammatory response helps eliminate pathogens and promotes tissue repair; however, excessive or chronic inflammation can lead to the occurrence and development of various diseases, including rheumatoid arthritis, inflammatory bowel disease, atherosclerosis, and neurodegenerative diseases. At the molecular level, the inflammatory process involves the release of various mediators, such as cytokines (TNF-α, IL-1, IL-6, etc.), chemokines, prostaglandins, and reactive oxygen species / nitrogen compounds. These factors regulate the expression of inflammatory genes by activating key signaling pathways such as NFκB and MAPK. Although many anti-inflammatory drugs are currently used clinically, such as nonsteroidal anti-inflammatory drugs (NSAIDs) and glucocorticoids, their long-term use is limited by adverse reactions and drug resistance. Therefore, the development of novel, highly effective, and low-toxicity anti-inflammatory drugs is of significant clinical importance.

[0003] THK01 is an indolecarbazole natural product isolated from a secondary metabolite of marine Streptomyces. Studies have confirmed its significant effect in inhibiting breast cancer metastasis. However, the potential of THK01 in the anti-inflammatory field has not been systematically explored, and the structure-activity relationship between specific sites in its molecular structure and its anti-inflammatory activity remains to be elucidated. By modifying the THK01 molecular skeleton, particularly by introducing functional substituents such as halogens, hydroxyl groups, and alkoxy groups at different sites, it is hoped that novel derivatives with stronger anti-inflammatory activity and better selectivity can be developed, and the impact of key structural modifications on biological activity can be revealed.

[0004] Given that there are currently no systematic reports on the use of THK01 and its derivatives in the treatment of inflammation-related diseases, studying the structurally modified derivatives of THK01 and their anti-inflammatory activities is not only of significant academic value, but also provides potential molecular entities and technical pathways for the development of novel anti-inflammatory drugs. Summary of the Invention

[0005] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0006] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0007] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide THK01 and its derivatives, preparation method and application in the preparation of anti-inflammatory drugs.

[0008] The chemical structure of THK01 is shown in formula (A), and the structural formula of the THK01 derivative is shown in formula (I):

[0009]

[0010] Wherein, X is selected from H or O; Y is selected from hydrogen or propynyl; R1 is selected from hydrogen, fluorine, methoxy, hydroxyl, or thiomethyl; R2 is selected from hydrogen, hydroxyl, or methoxy; R3 is selected from hydrogen, hydroxyl, or methoxy; R4 is selected from hydrogen, methyl, propynyl, propynyl derivatives, or triazole derivatives; and R5 is selected from hydrogen, methyl, propynyl, amide derivatives, or triazole derivatives.

[0011] Another object of the present invention is to overcome the deficiencies in the prior art and provide an application of THK01 derivatives in the preparation of anti-inflammatory drugs, characterized in that: the application in the preparation of drugs for treating and / or preventing diseases or conditions, wherein the diseases or conditions are selected from one of the following: inflammatory diseases, autoimmune diseases, fibrotic diseases, transplant rejection, diseases associated with excessive secretion of IL-17, IL-21 and / or IL-23, eye diseases, cardiovascular and cerebrovascular diseases, tumor metastasis, central nervous system diseases, diabetic nephropathy, and dermatological diseases.

[0012] As a preferred embodiment of the application described in this invention, the disease or condition is an inflammatory disease or an autoimmune disease, including inflammatory bowel disease, rheumatoid arthritis, osteoarthritis, juvenile idiopathic arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, allergic airway disease, chronic obstructive pulmonary disease, asthma, bronchitis, systemic lupus erythematosus, cutaneous lupus erythematosus, lupus nephritis, dermatomyositis, autoimmune liver disease, Sjögren's syndrome, multiple sclerosis, dry eye disease, type I diabetes and its complications, atopic eczema, thyroiditis, contact dermatitis, Sjögren's syndrome, or amyotrophic lateral sclerosis.

[0013] In a preferred embodiment of the application described in this invention, the disease or condition is a fibrotic disease including pulmonary fibrosis, liver fibrosis, kidney fibrosis, myocardial fibrosis, cutaneous fibrosis, or systemic sclerosis.

[0014] Another object of the present invention is to overcome the deficiencies in the prior art and provide a pharmaceutical composition characterized in that: the pharmaceutical composition contains the THK01 derivative or its pharmaceutical salt, and one or more pharmaceutically acceptable carriers, diluents and / or excipients.

[0015] Another objective of this invention is to overcome the shortcomings of the prior art and provide a method for preparing THK01 derivatives, characterized in that:

[0016] o-nitrobenzaldehyde containing the R3 substituent was subjected to Wittig reaction, Diels-Alder reaction, Cadogan reaction, Fischer indole synthesis reaction and reduction reaction to obtain the THK01 structure-modified derivative shown in general formula (IA).

[0017]

[0018] Among them, R1 is selected from hydrogen, fluorine, methoxy, hydroxyl, and thiomethyl; R2 is selected from hydrogen, hydroxyl, and methoxy; and R3 is selected from hydrogen, hydroxyl, and methoxy.

[0019] A method for preparing a THK01 derivative, characterized in that it includes: obtaining a THK01 structure-modified derivative of general formula (IC) by substituting a compound of general formula (IB);

[0020]

[0021] Beneficial effects of this invention:

[0022] This invention provides a method for preparing THK01 structurally modified derivatives. By subjecting o-nitrobenzaldehyde containing specific substituents to a series of chemical reactions, including Wittig reaction, Diels-Alder reaction, Cadogan reaction, Fischer indole synthesis reaction, demethylation reaction, reduction reaction and click chemistry reaction, THK01 structurally modified derivatives with different substituents are obtained.

[0023] Specifically, it is shown in formula (B):

[0024]

[0025] This invention further provides the use of the compound as defined in claim 6 in the preparation of medicaments for treating or preventing inflammatory diseases (the diseases listed in claim 7). The inventors have discovered that THK01 itself and a series of newly synthesized derivatives exhibit in vitro anti-inflammatory activity. For example, in an LPS-induced RAW264.7 cell model, THK01 and some derivatives of this invention significantly inhibited NO production, and some derivatives showed superior activity to the positive control drug even at lower concentrations. Preliminary in vivo pharmacodynamic studies have confirmed that THK01 has certain therapeutic effects in a DSS-induced mouse colitis model and an H1N1 virus-induced acute pneumonia model.

[0026] This invention provides the use of the THK01 derivatives or their pharmaceutical salts in the preparation of medicaments for the treatment and / or prevention of diseases or conditions, wherein the diseases or conditions are selected from inflammatory diseases, autoimmune diseases, fibrotic diseases, transplant rejection, diseases associated with excessive secretion of IL-17, IL-21 and / or IL-23, eye diseases, cardiovascular and cerebrovascular diseases, tumor metastases, central nervous system diseases, diabetic nephropathy, and dermatological diseases.

[0027] Among these, inflammatory diseases or autoimmune diseases are preferably selected from inflammatory bowel disease (e.g., Crohn's disease or ulcerative colitis), rheumatoid arthritis, osteoarthritis, juvenile idiopathic arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, allergic airway disease, chronic obstructive pulmonary disease, asthma, bronchitis, systemic lupus erythematosus, cutaneous lupus erythematosus, lupus nephritis, dermatomyositis, autoimmune liver disease, Sjögren's syndrome, multiple sclerosis, dry eye disease, type I diabetes and its complications, atopic eczema, thyroiditis, contact dermatitis, Sjögren's syndrome, or amyotrophic lateral sclerosis. Fibrotic diseases are preferably selected from pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis), liver fibrosis, kidney fibrosis, myocardial fibrosis, skin fibrosis (e.g., scleroderma), or systemic sclerosis.

[0028] Therefore, the THK01 derivatives provided by this invention (including new compounds and known compounds THK01 with new uses) have definite anti-inflammatory activities, providing new candidate compounds and treatment strategies for the development of novel anti-inflammatory drugs. Attached Figure Description

[0029] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0030] Figure 1 The bar chart shows the effect of representative compounds of the present invention at different concentrations on NO production in LPS-induced RAW264.7 cells.

[0031] Figure 2 This is a schematic diagram illustrating the therapeutic effect of THK01 on a DSS-induced mouse acute colitis model according to an embodiment of the present invention. (A) Comparison of colon morphology and length in mice of different treatment groups. (B) Hematoxylin-eosin (HE) stained pathological sections of colon tissue in mice of different treatment groups.

[0032] Figure 3 This is a schematic diagram illustrating the therapeutic effect of THK01 on an H1N1 virus-induced mouse acute pneumonia model according to an embodiment of the present invention.Figure 3 (A) is a comparison of lung morphology in mice from different treatment groups. Figure 3 (B) shows the results of TGF-1 immunohistochemical staining in lung tissue sections from mice in different treatment groups. Figure 3 (C) shows the SMA immunohistochemical staining results of lung tissue sections from mice in different treatment groups.

[0033] Figure 4 The chemical structural formula of THK01-50 in this embodiment of the invention is shown below. Detailed Implementation

[0034] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0035] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0036] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0037] Unless otherwise specified, all raw materials used in the embodiments of this invention are commercially available.

[0038] Example 1

[0039] The THK01 structure-modified derivative of this invention adopts a synthetic route similar to that of the original THK01, as shown in Formula 1:

[0040]

[0041] (1) Step 1: Wittig reaction, compound a (1 mmol) and phosphorus ylide (1.2 mmol) were dissolved in anhydrous acetonitrile and stirred under reflux at 60 °C for 12 h. The reaction progress was monitored by TLC until the starting material disappeared. The reaction product was concentrated under reduced pressure and separated by silica gel column chromatography (mobile phase was dichloromethane) to obtain a pale yellow solid b (yield 100%).

[0042] (2) Second step: Product b (1 mmol) was dissolved in 10 mL of anhydrous dichloromethane in a three-necked reaction flask. The apparatus was connected to fill the reaction system with N2, and triethylamine (5 mmol) and tert-butyldimethylsilyltrifluoromethane sulfonate (TBDMSOTf, 2 mmol) were added dropwise. The mixture was stirred and refluxed at 50 °C for 4 h to terminate the reaction. The product was extracted with saturated sodium carbonate, washed with water, and dehydrated with anhydrous sodium sulfate. The product was then separated by silica gel column chromatography (mobile phase: petroleum ether: dichloromethane = 8:1) to give a pale yellow solid c (yield 85%).

[0043] (3) Third step: Diels-Alder reaction. Product c (1 mmol) and maleimide (1 mmol) were dissolved in 10 mL of toluene and refluxed at 100 °C in the dark for 9 h. The reaction product was concentrated under reduced pressure and separated by silica gel column chromatography (mobile phases were dichloromethane:petroleum ether = 8:1, 1:0; dichloromethane:ethyl acetate = 10:1, 5:1) to give a pale yellow solid d (yield 80%).

[0044] (4) Fourth step: Cadogan reaction. Weigh product d (1 mmol) into a 50 mL reaction flask, add triethyl phosphite (5 mmol), and stir and reflux at 160 °C for 4 h. After removing the solvent by vacuum distillation, the product is extracted with saturated sodium bicarbonate, washed with water, dehydrated with anhydrous sodium sulfate, and then separated by silica gel column chromatography (mobile phase: dichloromethane:ethyl acetate = 6:1, 4:1) to obtain a pale yellow solid e (yield 55%).

[0045] (5) Step 5: Fischer indole synthesis reaction. Product e (1 mmol) and phenylhydrazine hydrochloride derivative (1.5 mmol) were added to a 50 mL reaction flask, along with 10 mL of solvent (V / V ratio AcOH:EtOH = 1:1). The mixture was stirred and refluxed at 80 °C for 12 h. After removing the solvent by vacuum distillation, the product was separated by silica gel column chromatography (mobile phase: petroleum ether:ethyl acetate = 1:1) to obtain a yellow solid THK29 or THK30 (yield 63%).

[0046] THK29: Yellow solid. HRESIMS gives it as a quasi-molecular ion peak at [M+H]. + m / z 372.0803 (calculated value 372.0801), molecular formula C 21 H 13 N3O2S.

[0047] 1H NMR (400MHz, DMSO-d6): δ11.77(1H,s),11.75(1H,s),11.01(1H,s),8.98(2H,overlap),7.80(2H,overlap),7.55(2H,overlap),7.35(1H,m),2.59(3H,s).

[0048] 13 C NMR (151MHz, DMSO-d6): δ171.40,171.37,140.38,138.76,129.41,129.12,128.39,127.34,126.91 ,124.39,123.44,122.42,121.64,120.40,120.14,120.01,115.72,114.91,112.93,112.18,17.34.

[0049] THK30: Yellow solid. HRESIMS gives it as a quasi-molecular ion peak at [M+H]. + m / z 374.0935 (calculated value 374.0935), molecular formula C 21 H 12 FN3O3.

[0050] 1 H NMR (600MHz, DMSO-d6): δ11.81(1H,s),11.62(1H,s),10.98(1H,s),8.97(1H,d,J=8.5Hz) ,8.66(1H,d,11.5),7.80(1H,d,J=8.5Hz),7.56(2H,overlap),7.34(1H,m),4.01(3H,s).

[0051] 13 C NMR (151MHz, DMSO-d6): δ171.56,171.37,147.90,140.31,137.40,129.13,129.09,126.79,124.28 ,121.65,120.31,119.88,118.94,115.86,115.19,113.33,112.05,109.77,109.62,96.67,56.34.

[0052] Example 2

[0053] The synthesis route is shown in Equation 2:

[0054]

[0055] Dissolve 1 mmol of THK30 in 10 mL of anhydrous dichloromethane. Connect the apparatus to fill the reaction system with N2, and add 10 mmol of boron tribromide dropwise at 0 °C. React at room temperature for 24 h. After the reaction is complete, add water to terminate the reaction. The reaction mixture is extracted with saturated sodium carbonate, washed with water, and dehydrated with anhydrous sodium sulfate. Then, it is separated by silica gel column chromatography (mobile phase: dichloromethane:methanol = 20:1) to obtain an orange-yellow solid THK31 (yield 80%).

[0056] THK31: Orange-yellow solid. HRESIMS gives it a quasi-molecular ion peak as [M+H]. + m / z 360.0778 (calculated value 360.0779), molecular formula C 20 H 10 FN3O3.

[0057] 1 H NMR (600MHz, DMSO-d6): δ11.71(1H,s),11.52(1H,s),10.95(1H,s),10.25(1H,s),8.96(1 H,d,J=8.2Hz),8.61(1H,d,11.2),7.78(1H,d,J=8.2Hz),7.53(1H,m),7.33(2H,overlap).

[0058] 13 C NMR (151MHz, DMSO-d6): δ172.05,171.85,140.89,138.21,129.62,129.47,127.20,124.72,122 .16,120.71,119.98,119.25,116.66,115.68,113.56,113.49,112.51,110.45,110.30,100.03.

[0059] Example 3

[0060] The synthesis route is shown in Equation 3:

[0061]

[0062] The procedure was the same as in Example 1 to obtain compound IA-1. IA-1 (1 mmol) was dissolved in DMF, and potassium carbonate (5 mmol) was added. After reacting at room temperature for about 15 minutes, bromopropyne (1.5 mmol) was added, and the reaction was continued at room temperature for 3-6 hours. The reaction was terminated by adding water, and the mixture was extracted with ethyl acetate (3 × 10 mL). The organic layers were combined, and the solvent was evaporated under reduced pressure. The residue was separated by high-performance liquid chromatography (HPLC) (separation conditions: preparative column, Welch). XB-C18, 21.2×250mm, 5μm; elution system: methanol / water (volume ratio) = 50%-100%, containing 0.5‰ trifluoroacetic acid (volume percentage), flow rate: 10ml / min, elution time: 40min) purification, to obtain the corresponding compounds THK32 / THK34 as yellow solids, with a yield of approximately 30-50%.

[0063] THK32: Yellow solid. HRESIMS gives it as a quasi-molecular ion peak at [M+H]. + m / z 424.1292 (calculated value 424.1292), molecular formula C 25 H 17 N3O4.

[0064] 1 H NMR (600MHz, DMSO-d6): δ11.98(1H,s),11.06(1H,s),8.70(1H,d,J=2.5Hz),8.67(1H,d,J=2.5Hz),7.83(1H,d,J=8.8Hz),7.70(1H,d, J=8.8Hz),7.29(1H,dd,J=2.5,8.8Hz),7.24(1H,dd,J=2.5,8.8Hz),5.79(1H,d,J=1.9Hz),3.92(2H,overlap),3.30(1H,t,J=2.2Hz).

[0065] 13 C NMR (151MHz, DMSO-d6): δ171.25,171.21,154.50,154.02,135.98,135.90,129.90,129.06,122.36,121.42,120. 39,119.64,116.92,116.65,116.29,116.04,112.82,111.23,107.17,106.47,79.37,75.98,55.67,55.58,34.58.

[0066] THK34: Yellow solid. HRESIMS gives it as a quasi-molecular ion peak at [M+H]. + m / z 424.1292 (calculated value 424.1292), molecular formula C 25 H 17 N3O4.

[0067] 1H NMR (600MHz, DMSO-d6): δ11.58(2H,overlap),8.49(2H,overlap),7.71(2H,overlap),7.19(2H,overlap),4.46(2H,overlap),3.90(6H,overlap),3.27(1H,m).

[0068] 13 C NMR (151MHz, DMSO-d6): δ168.77,154.01,135.20,129.72,121.97,118.17,116.52,115.56,112.97,105.94,79.23,73.59,55.48,26.50.

[0069] The experiment with IA-1 (1 mmol) was performed as in Example 2, and the reaction was repeated after 24 h by high performance liquid chromatography (separation conditions: preparative column Welch). XB-C18 column, 21.2×250mm, 5μm; elution system: methanol / water (v / v) = 50%-100%, containing 0.5‰ trifluoroacetic acid (v / v), flow rate: 10ml / min, elution time: 40min) purification yielded orange-yellow solid THK35 (70% yield); after reacting for 48h, the product was purified by high performance liquid chromatography (separation conditions: preparative column Welch). XB-C18, 21.2×250mm, 5μm; elution system: methanol / water (volume ratio) = 50%-100%, containing 0.5‰ trifluoroacetic acid (volume percentage), flow rate: 10ml / min, elution time: 40min) purification yielded orange-yellow solid THK33 (yield 62%).

[0070] THK33: Orange-yellow solid. HRESIMS gives it a quasi-molecular ion peak as [M+H]. + m / z 476.0236 (calculated value 476.0240), molecular formula C 23 H 14 BrN3O4.

[0071] 1 H NMR (600MHz, DMSO-d6): δ11.70(1H,s),10.95(1H,s),9.33(1H,s),9.23(1H,s),8.55(1H,d,J=2.2Hz), 8.49(1H,d,J=2.2Hz),7.56(2H,overlap),7.06(2H,overlap),5.80(2H,s),5.76(1H,s),5.61(1H,s).

[0072] 13 C NMR (151MHz, DMSO-d6): δ171.20,171.17,152.18,151.64,135.11,134.99,130.35,129.02,128.64,122.61 ,121.77,120.08,119.41,119.33,116.96,116.91,116.41,115.80,112.39,111.15,109.36,108.78,51.52.

[0073] THK35: Orange-yellow solid. HRESIMS gives it as a quasi-molecular ion peak at [M+H]. + m / z 396.0981 (calculated value 396.0979), molecular formula C 23 H 13 N3O4

[0074] 1 H NMR (600MHz, DMSO-d6): δ11.84(1H,s),10.96(1H,s),9.35(1H,s),9.24(1H,s),8.53(1H,d,J=2.5Hz),8.49(1H,d,J=2.5Hz),7.69(1H,d ,J=8.6Hz),7.59(1H,d,J=8.6Hz),7.11(1H,dd,J=2.5,8.6Hz),7.08(1H,dd,J=2.5,8.6Hz),5.74(1H,d,J=1.7Hz),3.27(1H,t,J=2.3Hz).

[0075] 13 C NMR (151MHz, DMSO-d6): δ171.18,171.14,152.20,151.63,135.20,135.15,129.87,129.08,122.68,121.7 3,120.20,119.37,116.95,116.72,116.46,116.17,112.42,110.83,109.35,108.80,79.51,75.75,34.47.

[0076] By replacing the reactant bromopropyne with bromoacetonitrile and following the same steps as above, the product THK36, a yellow solid, can be obtained with a yield of approximately 30%.

[0077] THK36: Yellow solid. HRESIMS gives it a quasi-molecular ion peak as [M+H]. +m / z 425.1243 (calculated value 425.1244), molecular formula C 24 H 16 N4O4.

[0078] 1 H NMR (600MHz, DMSO-d6): δ12.16(1H,s),11.15(1H,s),8.72(1H,d,J=2.5Hz), 8.68(1H,d,J=2.5Hz), 7.89(1H,d,J=8. 9Hz),7.71(1H,d,J=8.9Hz),7.35(1H,d,J=2.5,8.9Hz),7.27(1H,d,J=2.5,8.9Hz),6.21(2H,s),3.92(6H,overlap).

[0079] 13 C NMR (151MHz, DMSO-d6): δ171.10,154.98,154.16,136.11,135.53,129.48,129.00,122.64,121.41,121.17,11 9.61,117.50,117.44,116.98,116.68,116.54,116.31,112.80,110.99,107.48,106.54,55.69,55.58,33.82.

[0080] Example 4

[0081] The synthesis route is shown in Equation 4:

[0082]

[0083] The steps are the same as in Example 3. When X is H, the compound is THK01. Using THK01 (1 mmol) as a starting material, adding bromopropyne (2 mmol) yields THK37 (white solid, 40% yield) and THK38 (white solid, 35% yield). Adding iodomethane (2 mmol) yields THK41 (white solid, 53% yield) and THK42 (gray solid, 25%). Adding iodoacetamide (1.5 mmol) yields product THK43 (grayish-white solid, 55% yield).

[0084] When X is O, the compound is THK03. Using THK03 (1 mmol) as a raw material, adding bromopropyne (2 mmol) can produce product THK39 (yellow solid, yield 70%).

[0085] THK37: White solid. HRESIMS gives it as a quasi-molecular ion peak at [M+H].+ m / z 366.1237 (calculated value 366.1237), molecular formula C 23 H 15 N3O2.

[0086] 1 H NMR (600MHz, DMSO-d6): δ11.43(1H,s),8.97(1H,s),8.74(1H,d,J=2.5Hz),8.51(1H,s),8.05(1H,d,J=7.8Hz),7.86(1H,d,J=8.3Hz),7 .55(1H,m),7.52(1H,d,J=8.6Hz),7.37(1H,m),6.98(1H,dd,J=2.5,8.6Hz),5.80(2H,d,J=2.2Hz),4.94(2H,s),3.26(1H,t,J=2.3Hz).

[0087] 13 C NMR (151MHz, DMSO-d6): δ172.28,150.62,139.99,134.37,132.35,128.32,125.59,125.26,122.89,122.8 8,121.30,120.75,119.73,117.22,115.62,114.67,111.68,110.32,109.78,79.68,75.64,45.22,34.33.

[0088] THK38: White solid. HRESIMS gives it as a quasi-molecular ion peak at [M+H]. + m / z404.1399

[0089] (Calculated value is 404.1394), molecular formula is C 26 H 17 N3O2.

[0090] 1 H NMR (600MHz, DMSO-d6): δ9.23(1H,s),8.79(2H,d,J=2.6Hz),8.64(1H,s),8.06(2H,d,J=7.6Hz),7.88(2H,d,J=8.2Hz),7.63(2H,overlap ),7.43(1H,m),7.05(1H,dd,J=2.5,8.6Hz),5.43(1H,d,J=2.1Hz),5.26(1H,d,J=2.1Hz),4.96(1H,t,J=2.3Hz),3.48,3.37(1H,overlap).

[0091] 13 C NMR (151MHz, DMSO-d6): δ171.58,152.23,143.21,138.59,134.09,130.68,129.68,126.13,125.83,123.92,121.76, 121.49,120.84,120.57,117.60,115.83,112.50,112.08,110.69,79.10,78.90,76.58,76.12,45.15,38.90,37.89.

[0092] THK39: Yellow solid. HRESIMS gives it as a quasi-molecular ion peak at [M+H]. + m / z456.1344

[0093] (Calculated value is 456.1343), molecular formula is C 29 H 17 N3O3.

[0094] 1 H NMR (600MHz, DMSO-d6): δ9.54(1H,s),9.09(1H,d,J=7.9Hz),8.55(1H,d,J=2.4Hz),7.91(1H,d,J=8.3Hz),7.72(2H,overlap),7.49(1H,m),7.19(1H,dd ,J=2.5,8.7Hz),5.43(2H,d,J=2.1Hz),5.35(2H,d,J=2.1Hz),4.47(2H,d,J= 2.1Hz), 3.57(1H,t,J=2.3Hz), 3.52(1H,t,J=2.3Hz), 3.29(1H,t,J=2.3Hz).

[0095] 13 C NMR (151MHz, DMSO-d6): δ167.87,167.85,153.13,144.35,138.69,132.61,131.78,128.15,124.58,123.60,122.59,122.16,12 0.37,119.70,119.66,119.24,117.59,112.94,112.23,109.41,78.87,78.61,78.48,77.01,76.83,73.76,38.94,38.47,26.76.

[0096] THK41: White solid. HRESIMS gives a quasi-molecular ion peak as [M+Na]. + m / z342.1233

[0097] (Calculated value is 342.1237), molecular formula is C 21 H 15 N3O2.

[0098] 1 H NMR (600MHz, DMSO-d6): δ11.42(1H,s),8.95(1H,s),8.72(1H,d,J=2.5Hz),8.46(1H,s),8.03(1H,d,J=7.6Hz), 7.78(1H,d,J=8.3Hz),7.53(2H,overlap),7.32(1H,m),6.96(1H,dd,J=2.5,8.6Hz),4.93(2H,s),4.40(3H,s).

[0099] 13 C NMR (151MHz, DMSO-d6): δ172.47,150.55,140.48,134.35,132.59,129.45,125.89,124.95,123.07 ,122.10,121.10,119.91,118.95,116.83,115.35,113.63,111.72,109.75,109.71,45.21,32.00.

[0100] THK42: Gray solid. HRESIMS gives a quasi-molecular ion peak as [M+Na]. + m / z 356.1390 ​​(calculated value 356.1394), molecular formula C 22 H 17 N3O2.

[0101] 1 H NMR (600MHz, DMSO-d6): δ9.08(1H,s),8.81(1H,d,J=2.5Hz),8.54(1H,s),8.04(1H,d,J=7.7Hz),7.77(1H,d,J=8.2Hz) ,7.57(1H,m),7.50(1H,d,J=8.7Hz),7.37(1H,m),7.02(1H,dd,J=2.5,8.7Hz),4.93(1H,s),4.26(1H,s),4.14(1H,s).

[0102] 13C NMR (151MHz, DMSO-d6): δ172.05,151.19,143.06,138.15,133.10,131.12,129.84,125.58,123.98,12 2.75,121.08,120.62,119.48,118.48,116.08,115.53,110.95,110.91,110.25,48.67,37.10,36.40.

[0103] THK43: A grayish-white solid. HRESIMS gives it a quasi-molecular ion peak as [M+H]. + m / z 407.1116 (calculated value 407.1115), molecular formula C 22 H 16 N4O3.

[0104] 1 H NMR (600MHz, DMSO-d6): δ11.23(1H,s),8.95(1H,s),8.73(1H,d,J=2.4Hz),8.48(1H,s),8.04(1H,d,J=7.7Hz),7.79(1H,s), 7.63(1H,d,J=8.2Hz),7.51(1H,m),7.47(2H,overlap),7.34(1H,m),6.95(1H,dd,J=2.4,8.6Hz),5.46(2H,s),4.94(2H,s).

[0105] 1 H NMR (600MHz, DMSO-d6): δ172.42,169.68,150.52,140.51,134.24,132.45,129.69,125.79,125.03,12 3.01,122.59,121.16,120.34,119.10,117.14,115.40,114.03,111.55,109.75,109.59,47.42,45.24.

[0106] Example 5

[0107] The synthesis route is shown in Equation 5:

[0108]

[0109] When X is H, R1 is H, and R3 is a hydroxyl group, the compound is THK37. In a reaction vessel, 20 mM THK37 in DMSO solution (1 mmol), 40 mM sodium vitamin C aqueous solution (1 mmol), 40 mM copper sulfate pentahydrate aqueous solution (0.2 mmol), 10 mM TBTA in DMSO solution (0.2 mmol), and 20 mM azide derivative in DMSO solution (1.5 mmol) were added. The reaction was carried out at 60 °C for 24 h. The reaction product was separated by high performance liquid chromatography (separation conditions: preparative column Welch). XB-C18, 21.2×250mm, 5μm; elution system: acetonitrile / water (volume ratio) = 20%-50%, containing 0.5‰ trifluoroacetic acid (volume percentage), flow rate: 10ml / min, elution time: 40min) purification, to obtain product THK40 (white solid, yield 90%).

[0110] When X is O, R1 is a hydroxyl group, and R3 is a hydroxyl group, the compound is THK35. Using THK35 as a raw material, the steps are the same as described above. By adding different azide derivatives, products THK44 (orange-red solid, 90% yield), THK45 (brown solid, 80% yield), THK46 (yellow solid, 95% yield), and THK47 (yellow solid, 90% yield) can be obtained.

[0111] THK40: White solid. HRESIMS gives it as a quasi-molecular ion peak of [M+Na]. + m / z 876.3474 (calculated value 876.3474), molecular formula C 43 H 51 N9O8S.

[0112] 1H NMR (600MHz, DMSO-d6): δ11.48(1H,s),8.74(1H,d,J=2.4Hz),8.49(1H,s),8.01(1H,d,J=7.8Hz),7.88(1H,d,J=8.5Hz),7.87(1H,s),7.80(1H,t ,J=5.5Hz),7.51(2H,overlap),7.32(1H,t,J=7.4Hz),6.97(1H,dd,J=2.4,8.5Hz),6.41(1H,s),6.19(1H,s),4.93(2H,s),4.38(2H,t,J=5.2Hz), 4.27(1H,dd,J=5.2,7.7Hz),4.10(1H,dd,J=4.5,7.7Hz),3.65(2H,t,J=5.2Hz),3.57(2H,overlap),3.41(6H,overlap),3.33(2H,m),3.26(2H,m) ,3.21(2H,m),3.16(2H,m),3.06(1H,m),2.79(1H,dd,J=5.2,12.4Hz),2. 57(1H,m),2.04(2H,t,J=7.5Hz),1.58(1H,m),1.47(1H,m),1.28(1H,m).

[0113] 13 C NMR (151MHz, DMSO-d6): δ172.37,172.22,162.79,150.61,143.51,139.85,134.39 ,132.46,128.60,125.72,125.03,123.62,123.05,122.66,121.16,120.39,119.34 ,117.28,115.45,114.24,111.76,110.46,109.75,69.70,69.68,69.56,69.53,69.21,68.73,61.10,59.26,55.49,49.43,45.24,38.49,35.16,28.27,28.11,25.33.

[0114] THK44: Orange-red solid. HRESIMS gives it as a quasi-molecular ion peak at [M+H]. + m / z 665.1737 (calculated value 665.1739), molecular formula C 32 H 24 N8O9.

[0115] 1H NMR (600MHz, DMSO-d6): δ11.84(1H,s),11.34(1H,s),10.90(1H,s),9.26(2H,overlap),8.50(2H,ov erlap),8.13(1H,s),7.92(1H,d,J=8.2Hz),7.73(1H,d,J=8.2Hz),7.57(1H,d,J=8.2Hz),7.05(2H,o verlap),6.37(1H,d,J=5.5Hz),6.13(1H,s),5.73(1H,d,J=4.5Hz),5.64(1H,d,J=7.7Hz),5.32(1H, t,J=5.8Hz),5.21(1H,s),4.29(1H,m),4.00(1H,m),3.68(1H,d,J=11.4Hz),3.54(1H,d,J=11.4Hz).

[0116] 13 C NMR (151MHz, DMSO-d6): δ171.26,171.24,162.98,151.93,151.57,150.39,142.88,140.16,135.15,135.00,130.12,129.17,124.68,122.34, 121.83,119.68,119.44,116.79,116.70,116.36,115.53,112.51,111. 00,109.21,108.74,102.31,85.91,85.68,68.96,64.79,60.28,60.27.

[0117] THK45: Brown solid. HRESIMS gives it a quasi-molecular ion peak as [M+H]. + m / z 601.1680 (calculated value 601.1678), molecular formula C 29 H 24 N6O9.

[0118] 1H NMR (600MHz, DMSO-d6): δ11.91(1H,d,J=9.7Hz),10.91(1H,s),8.51(1H,m),8.04(1H,s),7.73(1H,dd,J=8.9,14.8 Hz),7.60(1H,dd,J=2.2,8.6Hz),7.05(2H,overlap),6.11(2H,overlap),5.03(1H,s),4.77(1H,d,J=8.1Hz),4.73( 1H,dd,J=3.3,11.1Hz),4.22(1H,dd,J=8.1,10.7Hz),4.15(1H,dd,J=3.3,11.1Hz),3.97(1H,dd,J=3.3,10.7Hz),3. 87(1H,t,J=6.6Hz), 3.79(1H,d,J=3.0Hz), 3.66(1H,d,J=3.3Hz), 3.53(3H,overlap), 3.42(1H,dd,J=6.6,10.7Hz).

[0119] 13 C NMR (151MHz, DMSO-d6): δ171.27,151.93,151.58,135.15,135.01,130.13,129.21,124.41,122.78,122.30,121.89,119.65, 119.49,116.78,116.72,116.34,115.49,112.51,110.98,109.22,108.77,75.18,70.89,68.41,67.59,66.39,65.14,60.45.

[0120] THK46: Yellow solid. HRESIMS gives a quasi-molecular ion peak as [M+Na]. + m / z 563.1286 (calculated value 563.1286), molecular formula C 27 H 20 N6O7.

[0121] 1H NMR (600MHz, DMSO-d6): δ12.64(1H,s),11.84(1H,s),10.91(1H,s),9.27(1H,s),9.21(1H,s),8.51(1H,d,J=2.5Hz),8.50(1H,d,J=2.5Hz),8.02 (1H,s),7.70(1H,d,J=8.7Hz),7.57(1H,d,J=8.7Hz),7.05(2H,overlap),6.12(2H,s),4.42(2H,t,J=5.1Hz),3.91(2H,s),3.77(2H,t,J=5.1Hz).

[0122] 13 C NMR (151MHz, DMSO-d6): δ171.47,171.27,151.95,151.57,143.21,135.15,135.03,130.15,129.17,123.78,122.44,122.4 1,121.86,119.70,119.47,116.78,116.72,116.33,115.61,112.49,110.95,109.23,108.76,68.76,67.22,49.40,48.68.

[0123] THK47: Yellow solid. HRESIMS gives it as a quasi-molecular ion peak of [M+Na]. + m / z 505.1235 (calculated value 505.1231), molecular formula C 25 H 18 N6O5.

[0124] 1 H NMR (600MHz, DMSO-d6): δ11.84(1H,s),10.91(1H,s),9.28(1H,s),9.22(1H,s),8.51(1H,d,J=2.5Hz),8.50(1H,d,J=2.5Hz),7.93(1H,s) ,7.72(1H,d,J=8.6Hz),7.58(1H,d,J=8.6Hz),7.07(2H,overlap),6.12(2H,s),4.90(1H,t,J=5.2Hz),4.26(2H,t,J=5.2Hz),3.64(1H,m).

[0125] 13C NMR (151MHz, DMSO-d6): δ171.26,171.26,151.95,151.59,143.12,135.15,135.05,130.13,129.19,123.68,122.4 0,121.88,119.70,119.48,116.78,116.73,116.34,115.62,112.50,110.97,109.23,108.76,59.78,52.18,48.68.

[0126] Example 6

[0127] In vitro anti-inflammatory activity evaluation of THK01 and its derivatives

[0128] NO production inhibition activity assay

[0129] This embodiment aims to evaluate the ability of a series of THK01 derivatives to inhibit lipopolysaccharide (LPS)-induced nitric oxide (NO) production in mouse macrophage RAW264.7 cells, thereby assessing their in vitro anti-inflammatory activity. First, the cytotoxicity of all test compounds was pre-assessed using the sulfonylrhodamine B (SRB) method to ensure cell viability was higher than 85% at the concentrations used in subsequent activity assays. For the activity assay, logarithmically growing RAW264.7 cells were cultured in DMEM medium containing 10% fetal bovine serum at 5% CO2 and 37°C. Cells were seeded at a density of 3 × 10⁴ cells / mL in 96-well plates, 100 μL per well, and cultured overnight. 50 μL of different concentrations of test compounds were added. One hour later, LPS (0.5 μg / mL) was added to stimulate NO production. After 24 hours, 50 μL of the supernatant was collected, and the absorbance was measured at 540 nm using a commercially available NO detection kit (Beyotime) based on the Griess reaction principle. The NO concentration was calculated based on the sodium nitrite standard curve, and the inhibition rate was also calculated. The positive control was indomethacin (50 μM).

[0130] The inhibitory activity of THK01 and its derivatives (a total of 50 compounds) involved in this invention on LPS-induced NO production in RAW264.7 cells was screened and evaluated. The results showed (some representative data are shown in the table below) that 15 compounds exhibited significant NO inhibitory activity. The positive control, indomethacin, showed a NO inhibition rate of 34.69% at a concentration of 50 μM. Compared with the positive control, some compounds of this invention showed stronger inhibitory effects at lower concentrations. At a concentration of 5 μM, compounds THK02, THK04, THK06, THK24, THK27, THK30, THK36, THK48, and THK49 all showed NO production inhibition rates greater than 50%. At an even lower concentration of 2.5 μM, compounds THK01, THK03, THK10, THK31, and THK33 showed NO production inhibition rates greater than 50%, demonstrating good activity, with THK33 showing an inhibition rate close to 90%, exhibiting the most significant increase in activity.

[0131] Further analysis of the compounds with superior activity at multiple concentration gradients revealed the following results (e.g.) Figure 1 As shown in the figure, the inhibitory effect of these compounds on NO production exhibits a typical concentration-dependent effect. Comprehensive analysis revealed that compound THK01 possesses significant in vitro anti-inflammatory activity (indicating NO production inhibition), while some derivatives of this invention, such as THK02, THK10, and THK33, showed inhibitory activities comparable to or even superior to THK01. These results successfully screened a group of candidate compounds with good anti-inflammatory activity and further development potential.

[0132] Table 1

[0133]

[0134]

[0135]

[0136] Example 7

[0137] Evaluation of the in vivo anti-inflammatory activity of THK01

[0138] Anti-inflammatory bowel disease effect

[0139] This embodiment aims to evaluate the in vivo therapeutic effect of THK01 using a mouse model of acute colitis induced by dextran sulfate sodium (DSS). Age-appropriate (e.g., 6-8 weeks old) male C57BL / 6 mice were randomly divided into: a normal control group, a model control group (DSS-induced), a positive drug control group (dexamethasone, oral gavage, 5 mg / kg), and a THK01 group (tail vein injection, 15 mg / kg). Except for the normal control group, mice in the other groups were allowed free access to an aqueous solution containing 3% DSS for 7 consecutive days to establish a colitis model. The drug-treated groups were administered the drug via gavage for 7 consecutive days starting from day 1 of model establishment, while the normal control and model groups received an equal volume of the drug. During the experiment, changes in mouse body weight, disease activity index (DAI) scores, and colon length were observed and recorded. After the experiment, mice were sacrificed, colon length was measured, and colon tissue was collected for HE staining and immunohistochemical analysis to assess the degree of inflammatory cell infiltration and tissue damage.

[0140] In a DSS-induced acute colitis model, mice treated with THK01 (15 mg / kg) showed a significant reduction in colon length compared to the control group. Histopathological analysis further confirmed that the degree of inflammatory cell infiltration and mucosal damage in the colon tissue of the THK01-treated mice were significantly reduced compared to the control group. Under the experimental conditions, the improvement effect of THK01 (15 mg / kg) on ​​the above indicators was comparable to that of the positive control drug dexamethasone. These results fully demonstrate that the compound THK01 has significant in vivo anti-inflammatory activity and a clear therapeutic effect on inflammatory bowel disease.

[0141] Anti-acute pneumonia effect

[0142] This embodiment aims to verify the protective effect of compound THK01 on lung tissue injury induced by H1N1 virus infection. An H1N1 infection model in mice was used for evaluation, combined with histological and immunohistochemical detection methods. The specific experimental steps are as follows: SPF-grade healthy mice were randomly divided into four groups (n=6 per group): normal control group, H1N1 model group, THK01 treatment group, and oseltamivir treatment group. Except for the normal control group, the other three groups of mice were inoculated with a certain dose (e.g., 10⁵ TCID) of H1N1 virus solution via intravenous drip on day 0 to establish a viral-induced lung injury model. Mice in the THK01 group were administered THK01 via tail vein injection at a dose of 15 mg / kg, once daily for 5 consecutive days, starting on the day of virus inoculation (day 0). Mice in the oseltamivir group were administered oseltamivir by gavage at the commercially recommended dose. The normal group and the model group were treated with an equal volume of carrier solution. On days 3 and 5 after virus inoculation, mice in each group were randomly sacrificed, and lung tissue was collected for analysis. After recording the gross morphological changes in lung tissue, some tissue samples were fixed (e.g., in 4% paraformaldehyde) and embedded in paraffin. TGF-β1 and α-SMA immunohistochemical staining was performed on lung tissue sections. After staining, the sections were observed and images were acquired under 10× and 40× microscopes to analyze the expression levels of inflammatory fibrotic factors.

[0143] In a mouse model of acute lung injury induced by H1N1 virus infection, compared with the model control group, the macroscopic pathological condition of the lungs (such as lung color and degree of edema) in the THK01 (15 mg / kg) treatment group was significantly improved, approaching the level of the normal control group. Its overall protective effect was comparable to that of the positive control drug oseltamivir group. Immunohistochemical staining of lung tissue showed that the expression levels of TGF-β1, a key pro-fibrotic factor, and α-SMA, a marker of myofibroblast activation, were significantly upregulated in the lung tissue of the model control group; however, after THK01 treatment, the expression levels of both factors showed a significant downregulation trend. This indicates that THK01 can effectively inhibit the early fibrotic response of lung tissue after viral infection. In summary, the results confirm that the compound THK01 can effectively reduce the pulmonary inflammatory response and tissue damage induced by H1N1 virus infection and inhibit the early fibrotic process, demonstrating its good in vivo activity as a potential antiviral pneumonia treatment drug.

[0144] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. A THK01 derivative, characterized in that: The THK01 derivative is selected from one of compounds THK33, THK34, and THK39, and the structural formulas of compounds THK33, THK34, and THK39 are shown below: 、 、 。 2. The application of the THK01 derivative as described in claim 1 in the preparation of anti-inflammatory drugs, characterized in that: Use in the preparation of medicaments for the treatment and / or prevention of diseases or conditions, wherein the diseases or conditions are selected from one of the following: inflammatory diseases, autoimmune diseases, fibrotic diseases, transplant rejection, diseases associated with excessive secretion of IL-17, IL-21 and / or IL-23, eye diseases, cardiovascular and cerebrovascular diseases, tumor metastases, central nervous system diseases, diabetic nephropathy, and dermatological diseases.

3. The application according to claim 2, characterized in that: The disease or condition described is an inflammatory disease or an autoimmune disease selected from inflammatory bowel disease, rheumatoid arthritis, osteoarthritis, juvenile idiopathic arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, allergic airway disease, chronic obstructive pulmonary disease, asthma, bronchitis, systemic lupus erythematosus, cutaneous lupus erythematosus, lupus nephritis, dermatomyositis, autoimmune liver disease, Sjögren's syndrome, multiple sclerosis, dry eye disease, type I diabetes and its complications, atopic eczema, thyroiditis, contact dermatitis, Sjögren's syndrome, or amyotrophic lateral sclerosis.

4. The application according to claim 3, characterized in that: The disease or condition referred to is a fibrotic disease selected from pulmonary fibrosis, liver fibrosis, kidney fibrosis, myocardial fibrosis, cutaneous fibrosis, or systemic sclerosis.

5. A pharmaceutical composition, characterized in that: The pharmaceutical composition contains a THK01 derivative or a pharmaceutical salt thereof according to any one of claims 1-2, and one or more pharmaceutically acceptable carriers, diluents and / or excipients.

6. A method for preparing a THK01 derivative, characterized in that: include, The compounds shown in IA-I were subjected to substitution reactions to give compounds THK33 and THK34; 、 、 ； Compound THK03 was replaced by a substitution reaction to obtain compound THK39; 、 。

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