THK01 and derivatives thereof, preparation method and application of THK01 and derivatives thereof in preparation of anti-inflammatory drugs

Abstract

The invention discloses THK01 and derivatives thereof, a preparation method and application of THK01 and derivatives thereof in preparation of anti-inflammatory drugs, and the THK01 and derivatives thereof are prepared by performing a series of chemical reactions on o-nitrobenzaldehyde containing a specific substituent group, including Wittig reaction, Diels-Alder reaction, Cadolan reaction, Fischer indole synthesis reaction, demethylation reaction, reduction reaction, click chemical reaction and the like. The THK01 structure modified derivatives with different substituent groups are obtained. The THK01 structure modified derivative disclosed by the invention is diversified in synthetic route and high in reaction success rate; in-vitro and in-vivo experiments show that the compounds can significantly inhibit NO generation and inflammatory response at low concentration, and can effectively treat inflammatory bowel disease and acute pneumonia induced by H1N1 virus in an animal model. These findings provide valuable candidate compounds and new treatment strategies for developing novel, safe and efficient anti-inflammatory drugs.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to THK01 and its derivatives, a preparation method and application thereof in the preparation of anti-inflammatory drugs. Background Art

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

[0003] THK01, an indolecarbazole natural product isolated from a secondary metabolite of Streptomyces marineus, has been shown to significantly inhibit 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 structurally modifying the THK01 molecular backbone, particularly by introducing functional substituents such as halogens, hydroxyls, and alkoxy groups at various sites, it is hoped that novel derivatives with enhanced anti-inflammatory activity and improved selectivity will be developed, and the impact of key structural modifications on biological activity will be revealed.

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

[0005] The purpose of this section is to summarize some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of this application to avoid obscuring the purpose of this section, the abstract and the title of the invention, and such simplifications or omissions should not be used to limit the scope of the present invention.

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

[0007] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a THK01 and its derivatives, a preparation method and use thereof 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, thiomethyl; R2 is selected from hydrogen, hydroxyl, methoxyl; R3 is selected from hydrogen, hydroxyl, methoxyl; R4 is selected from hydrogen, methyl, propynyl, propenyl derivatives, triazole derivatives; R5 is selected from hydrogen, methyl, propynyl, amide derivatives, triazole derivatives.

[0011] Another object of the present invention is to overcome the deficiencies in the prior art and provide a use of a THK01 derivative in the preparation of an anti-inflammatory drug, characterized in that the use is in the preparation of a drug for treating and / or preventing a disease or condition selected from one of inflammatory diseases, autoimmune diseases, fibrotic diseases, transplant rejection, diseases related to 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 of the present invention, the disease or condition is an inflammatory disease or 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 1 diabetes and its complications, atopic eczema, thyroiditis, contact dermatitis, Sjögren's syndrome or amyotrophic lateral sclerosis.

[0013] As a preferred embodiment of the application of the present invention, the disease or condition is a fibrotic disease including pulmonary fibrosis, liver fibrosis, renal fibrosis, myocardial fibrosis, skin 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 a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents and / or excipients.

[0015] Another object of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing a THK01 derivative, characterized in that:

[0016] The o-nitrobenzaldehyde containing an R3 substituent is subjected to Wittig reaction, Diels-Alder reaction, Cadogan reaction, Fischer indole synthesis reaction and reduction reaction to obtain a THK01 structurally modified derivative represented by general formula (IA);

[0017]

[0018] Wherein, R1 is selected from hydrogen, fluorine, methoxy, hydroxy, and thiomethyl; R2 is selected from hydrogen, hydroxy, and methoxy; and R3 is selected from hydrogen, hydroxy, and methoxy.

[0019] A method for preparing a THK01 derivative, characterized by comprising: subjecting a compound represented by general formula (IB) to a substitution reaction to obtain a THK01 structurally modified derivative represented by general formula (IC);

[0020]

[0021] Beneficial effects of the present invention:

[0022] The present invention provides a method for preparing structurally modified derivatives of THK01, wherein structurally modified derivatives of THK01 with different substituents are obtained 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.

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

[0024]

[0025] The present invention further provides the use of a compound as defined in claim 6 in the preparation of a medicament for treating or preventing inflammatory diseases (such as those listed in claim 7). The present inventors have discovered through research 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 of its derivatives can significantly inhibit NO production, and some of these derivatives exhibit superior activity compared to the positive control drug at lower concentrations. In vivo pharmacodynamic studies have preliminarily confirmed that THK01 has a certain therapeutic effect in both a DSS-induced mouse colitis model and an H1N1 virus-induced acute pneumonia model.

[0026] The present invention provides the use of the THK01 derivatives or pharmaceutically acceptable salts thereof in the preparation of a medicament for treating and / or preventing a disease or condition selected from inflammatory diseases, autoimmune diseases, fibrotic diseases, transplant rejection, diseases related to 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.

[0027] Among them, inflammatory diseases or autoimmune diseases are preferably selected from inflammatory bowel disease (such as 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 (such as idiopathic pulmonary fibrosis), liver fibrosis, kidney fibrosis, myocardial fibrosis, skin fibrosis (such as scleroderma) or systemic sclerosis.

[0028] Therefore, the THK01 derivatives provided by the present invention (including new compounds and known compounds THK01 with new uses) have clear anti-inflammatory activity, and provide new candidate compounds and treatment strategies for the development of new anti-inflammatory drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort. Among them:

[0030] Figure 1 The figure is a bar graph showing the NO production in LPS-induced RAW264.7 cells at different concentrations of representative compounds of the present invention.

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

[0032] Figure 3 This is a schematic diagram showing the therapeutic effect of THK01 on the H1N1 virus-induced acute pneumonia model in mice. Figure 3 (A) is a comparison of lung morphology of mice in different treatment groups. Figure 3 (B) is the result of TGF-1 immunohistochemical staining of lung tissue sections of mice in different treatment groups. Figure 3 (C) Shows the results of SMA immunohistochemical staining of lung tissue sections of mice in different treatment groups.

[0033] Figure 4 is the chemical structural formula of Example THK01-50 of the present invention. DETAILED DESCRIPTION

[0034] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0035] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0036] Secondly, the term "one embodiment" or "embodiment" 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 various places throughout this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive of other embodiments.

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

[0038] Example 1

[0039] The structurally modified derivatives of THK01 of the present invention adopt a similar synthetic route to 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 at 60°C under reflux 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: dichloromethane) to obtain a pale yellow solid b (yield 100%).

[0042] (2) Step 2: Product b (1 mmol) was dissolved in 10 mL of anhydrous dichloromethane in a three-necked reaction flask. The apparatus was connected so that the reaction system was filled with N2, and triethylamine (5 mmol) and tert-butyldimethylsilyl trifluoromethanesulfonate (TBDMSOTf, 2 mmol) were added dropwise in sequence. 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. It was then separated by silica gel column chromatography (mobile phase: petroleum ether: dichloromethane = 8:1) to obtain a light yellow solid c (yield 85%).

[0043] (3) Step 3: 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 phase: dichloromethane:petroleum ether = 8:1, 1:0; dichloromethane:ethyl acetate = 10:1, 5:1) to obtain a light yellow solid d (yield 80%).

[0044] (4) Step 4: Cadogan reaction. Product d (1 mmol) was weighed into a 50 mL reaction flask, triethyl phosphite (5 mmol) was added, and the mixture was stirred and refluxed at 160°C for 4 h. The solvent was removed by vacuum distillation, and the product was extracted with saturated sodium bicarbonate, washed with water, and dehydrated with anhydrous sodium sulfate. The product was then separated by silica gel column chromatography (mobile phase: dichloromethane:ethyl acetate = 6:1, 4:1) to obtain a light yellow solid e (yield 55%).

[0045] (5) Step 5: Fischer indole synthesis reaction: Product e (1 mmol) and a phenylhydrazine hydrochloride derivative (1.5 mmol) were placed in a 50 mL reaction flask, 10 mL of solvent (AcOH:EtOH = 1:1 by volume) was added, and the mixture was stirred and refluxed at 80°C for 12 h. The solvent was removed by vacuum distillation and 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 the quasi-molecular ion peak as [M+H] + m / z 372.0803 (calculated value 372.0801), molecular formula is 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 the quasi-molecular ion peak as [M+H] + m / z 374.0935 (calculated value 374.0935), molecular formula is 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 synthetic route is shown in Formula 2:

[0054]

[0055] THK30 (1 mmol) was dissolved in 10 mL of anhydrous dichloromethane. The reaction system was then filled with nitrogen. Boron tribromide (10 mmol) was added dropwise at 0°C and allowed to react for 24 h at room temperature. After completion, water was added to terminate the reaction. The reaction mixture was extracted with saturated sodium carbonate, washed with water, and dehydrated with anhydrous sodium sulfate. The mixture was then separated by silica gel column chromatography (mobile phase: dichloromethane:methanol = 20:1) to yield THK31 as an orange-yellow solid (80% yield).

[0056] THK31: Orange-yellow solid. HRESIMS gives the quasi-molecular ion peak as [M+H] + m / z 360.0778 (calculated value 360.0779), molecular formula is 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 synthetic route is shown in Formula 3:

[0061]

[0062] The steps were consistent with Example 1 to obtain compound IA-1. IA-1 (1 mmol) was dissolved in DMF, potassium carbonate (5 mmol) was added, and the reaction was carried out at room temperature for about 15 minutes. Propylene bromide (1.5 mmol) was added and the reaction was carried out at room temperature for 3-6 hours. After the reaction was completed, water was added to terminate the reaction, 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 purified by high performance liquid chromatography (separation conditions: preparative column Welch XB-C18, 21.2×250 mm, 5 μm; elution system: methanol / water (volume ratio) = 50%-100%, containing 0.5‰ trifluoroacetic acid by volume, flow rate: 10 ml / min, elution time: 40 min) to obtain the corresponding compounds THK32 / THK34 as yellow solids in a yield of approximately 30-50%.

[0063] THK32: Yellow solid. HRESIMS gives the quasi-molecular ion peak as [M+H] + m / z 424.1292 (calculated value 424.1292), molecular formula is 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 the quasi-molecular ion peak as [M+H] + m / z 424.1292 (calculated value 424.1292), molecular formula is 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] IA-1 (1 mmol) was subjected to the same experiment as in Example 2, and the reaction was carried out for 24 h. The mixture was separated by high performance liquid chromatography (Separation conditions: Welch column XB-C18, 21.2×250mm, 5μm; elution system: methanol / water (volume ratio) = 50%-100%, containing 0.5‰ trifluoroacetic acid by volume, flow rate: 10ml / min, elution time: 40min) to obtain the product THK35 as an orange-yellow solid (yield 70%); after 48h of reaction, the product was purified by high performance liquid chromatography (separation conditions: preparative column Welch XB-C18, 21.2×250 mm, 5 μm; elution system: methanol / water (volume ratio) = 50%-100%, containing 0.5‰ trifluoroacetic acid by volume, flow rate: 10 ml / min, elution time: 40 min) to obtain the product THK33 as an orange-yellow solid (yield 62%).

[0070] THK33: Orange-yellow solid. HRESIMS gives the quasi-molecular ion peak as [M+H] + m / z 476.0236 (calculated value 476.0240), molecular formula is 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 the quasi-molecular ion peak as [M+H] + m / z 396.0981 (calculated value 396.0979), molecular formula is 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] The reaction raw material bromopropyne was replaced with bromoacetonitrile and the steps were the same as above to obtain the product THK36 as a yellow solid with a yield of about 30%.

[0077] THK36: Yellow solid. HRESIMS gives the quasi-molecular ion peak as [M+H] +m / z 425.1243 (calculated value 425.1244), molecular formula is 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 synthetic route is shown in Formula 4:

[0082]

[0083] The steps were the same as those in Example 3. When X was H and the compound was THK01, THK01 (1 mmol) was added with propargyl bromide (2 mmol) to give THK37 (white solid, 40% yield) and THK38 (white solid, 35% yield). Iodomethane (2 mmol) was added to give THK41 (white solid, 53% yield) and THK42 (gray solid, 25%). Iodoacetamide (1.5 mmol) was added to give THK43 (off-white solid, 55% yield).

[0084] When X is O, the compound is THK03. THK03 (1 mmol) is used as a raw material, and propargyl bromide (2 mmol) is added to react to obtain the product THK39 (yellow solid, yield 70%).

[0085] THK37: white solid. HRESIMS gives the quasi-molecular ion peak as [M+H]+ m / z 366.1237 (calculated value 366.1237), molecular formula is 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 the quasi-molecular ion peak as [M+H] + m / z 404.1399

[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 the quasi-molecular ion peak as [M+H] + m / z 456.1344

[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 the quasi-molecular ion peak as [M+Na] + m / z 342.1233

[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 the quasi-molecular ion peak as [M+Na] + m / z 356.1390 ​​(calculated value 356.1394), molecular formula is 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: Off-white solid. HRESIMS gives the quasi-molecular ion peak as [M+H] + m / z 407.1116 (calculated value 407.1115), molecular formula is 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 synthetic route is shown in Formula 5:

[0108]

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

[0110] When X is O, R1 is hydroxyl, and R3 is hydroxyl, the compound is THK35. Using THK35 as the starting material, the steps are the same as above, and different azide derivatives are added to obtain the products THK44 (orange-red solid, yield 90%), THK45 (brown solid, yield 80%), THK46 (yellow solid, yield 95%), and THK47 (yellow solid, yield 90%).

[0111] THK40: white solid. HRESIMS gives the quasi-molecular ion peak as [M+Na] + m / z 876.3474 (calculated value 876.3474), molecular formula is 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 the quasi-molecular ion peak as [M+H] + m / z 665.1737 (calculated value 665.1739), molecular formula is 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 the quasi-molecular ion peak as [M+H] + m / z 601.1680 (calculated value 601.1678), molecular formula is 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 the quasi-molecular ion peak as [M+Na] + m / z 563.1286 (calculated value 563.1286), molecular formula is 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 the quasi-molecular ion peak as [M+Na] + m / z 505.1235 (calculated value 505.1231), molecular formula is 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] Evaluation of the anti-inflammatory activity of THK01 and its derivatives in vitro

[0128] NO production inhibitory activity assay

[0129] This example aimed 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, all test compounds underwent a preliminary cytotoxicity assessment using the sulforhodamine B (SRB) assay to ensure cell viability exceeding 85% at the concentrations used in subsequent activity assays. For the activity assay, RAW264.7 cells in the logarithmic growth phase were cultured in DMEM medium supplemented with 10% fetal bovine serum under 5% CO and 37°C. Cells were seeded at a density of 3×104 cells / mL in 96-well plates, with 100 μL per well, and cultured overnight. 50 μL of test compound at various concentrations was added. One hour later, lipopolysaccharide (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 (Biyuntian) based on the Griess reaction. The NO concentration was calculated according to the sodium nitrite standard curve, and the inhibition rate was calculated. The positive control was indomethacin (50 μM).

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

[0131] The compounds with better activity were further tested in multiple concentration gradients. The results showed that (e.g. Figure 1 As shown, these compounds exhibited typical concentration-dependent inhibition of NO production. Comprehensive analysis revealed that compound THK01 exhibited significant in vitro anti-inflammatory activity (measured by inhibition of NO production), while some derivatives of the present invention, such as THK02, THK10, and THK33, exhibited comparable or even superior inhibitory activity to THK01. These results successfully identified a group of candidate compounds with promising anti-inflammatory activity and potential for further development.

[0132] Table 1

[0133]

[0134]

[0135]

[0136] Example 7

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

[0138] Anti-inflammatory bowel disease effect

[0139] This example aims to evaluate the in vivo therapeutic efficacy of THK01 in a dextran sodium sulfate (DSS)-induced acute colitis mouse model. Male C57BL / 6 mice of appropriate age (e.g., 6-8 weeks old) 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 remaining groups were allowed to freely drink a 3% DSS-containing aqueous solution for 7 consecutive days to establish a colitis model. The drug-treated group received continuous gavage for 7 consecutive days starting on day 1 of modeling, while the normal control and model groups received an equal volume of solvent. During the experiment, the mice were observed and recorded for indicators such as body weight change, disease activity index (DAI) score, and colon length. After the experiment, the mice were sacrificed, colon length was measured, and colon tissue was obtained 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 significantly reduced colon length compared to the control group. Histopathological analysis further confirmed that the degree of inflammatory cell infiltration and mucosal damage in the colonic tissue of THK01-treated mice were significantly reduced compared to the control group. Under these experimental conditions, the improvement effect of THK01 (15 mg / kg) on ​​these indicators was comparable to that of the active agent dexamethasone. These results fully demonstrate that the compound THK01 has significant in vivo anti-inflammatory activity and has a clear therapeutic effect on inflammatory bowel disease.

[0141] Anti-acute pneumonia effect

[0142] This example aims to validate the protective effect of compound THK01 against lung injury induced by H1N1 virus infection. Evaluation was conducted using a mouse H1N1 infection model and combined histological and immunohistochemical assays. The specific experimental procedures are as follows: SPF healthy mice were randomly divided into four groups (n=6 per group): a normal control group, an H1N1 model group, a THK01-treated group, and an oseltamivir-treated group. Except for the normal control group, the other three groups of mice were inoculated with a dose (e.g., 105 TCID) of H1N1 virus solution via intravenous instillation on day 0 to establish a viral lung injury model. Mice in the THK01 group received 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 received oseltamivir via oral gavage according to the commercially recommended dose. The normal and model groups were treated with equal volumes of vehicle solution. Mice in each group were randomly sacrificed on days 3 and 5 after virus inoculation, and lung tissue was collected for analysis. After recording macroscopic morphological changes in lung tissue, sections were fixed (e.g., with 4% paraformaldehyde) and embedded in paraffin. Lung tissue sections were immunohistochemically stained for TGF-β1 and α-SMA. After staining, images were collected and observed under 10× and 40× magnification microscopes for analysis of inflammatory and fibrotic factor expression levels.

[0143] In a mouse model of acute lung injury induced by H1N1 virus infection, macroscopic lung pathology (e.g., lung color and edema) was significantly improved in the THK01 (15 mg / kg) treatment group compared to the control group, approaching levels in the normal control group. The overall protective effect was comparable to that of the positive control drug, oseltamivir. Lung tissue immunohistochemical staining revealed that expression of TGF-β1, a key pro-fibrotic factor, and α-SMA, a marker of myofibroblast activation, were significantly upregulated in the lungs of the control group. However, after THK01 treatment, expression of both factors showed a significant downregulation, indicating that THK01 can effectively inhibit the early fibrotic response in lung tissue following viral infection. Taken together, these data confirm that THK01 effectively mitigates H1N1 virus-induced lung inflammation and tissue damage, and inhibits the early stages of fibrosis, demonstrating its promising in vivo activity as a potential antiviral pneumonia treatment.

[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 the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, and all of these should be included in the scope of the present invention.

Claims

1. A THK01 derivative, characterized in that: The chemical structure of THK01 is shown in formula (A), and the structural formula of the THK01 derivative is shown in formula (I): wherein X is selected from H or O; Y is selected from hydrogen or propynyl; R1 is selected from hydrogen, fluorine, methoxy, hydroxyl, thiomethyl; R2 is selected from hydrogen, hydroxyl, methoxyl; R3 is selected from hydrogen, hydroxyl, methoxyl; R4 is selected from hydrogen, methyl, propynyl, propenyl derivatives, triazole derivatives; R5 is selected from hydrogen, methyl, propynyl, amide derivatives, triazole derivatives.

2. The use of the THK01 derivatives according to claim 1 in the preparation of anti-inflammatory drugs, characterized in that: Use in the preparation of a medicament for treating and / or preventing a disease or condition, wherein the disease or condition is selected from one of inflammatory diseases, autoimmune diseases, fibrotic diseases, transplant rejection, diseases related to 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.

3. The use according to claim 2, characterized in that: The disease or condition is an inflammatory disease or 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 1 diabetes and its complications, atopic eczema, thyroiditis, contact dermatitis, Sjögren's syndrome or amyotrophic lateral sclerosis.

4. The use according to claim 3, characterized in that: The disease or disorder is a fibrotic disease including pulmonary fibrosis, liver fibrosis, kidney fibrosis, myocardial fibrosis, skin fibrosis or systemic sclerosis.

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

6. A method for preparing a THK01 derivative, characterized in that: The o-nitrobenzaldehyde containing an R3 substituent is subjected to Wittig reaction, Diels-Alder reaction, Cadogan reaction, Fischer indole synthesis reaction and reduction reaction to obtain a THK01 structurally modified derivative represented by general formula (IA); Wherein, R1 is selected from hydrogen, fluorine, methoxy, hydroxy, and thiomethyl; R2 is selected from hydrogen, hydroxy, and methoxy; and R3 is selected from hydrogen, hydroxy, and methoxy.

7. A method for preparing a THK01 derivative, characterized by: include, The compound represented by the general formula (IB) is subjected to a substitution reaction to obtain a THK01 structurally modified derivative represented by the general formula (IC);

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