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

Abstract

The invention discloses a THK01 ester prodrug as well as a preparation method and application thereof in preparation of an anti-inflammatory drug, and the preparation method comprises the step of reacting THK01 with acyl chloride, anhydride or carboxylic acid in an organic solvent in the presence of a catalyst to obtain a THK01 esterified derivative. The THK01 esterified derivative disclosed by the invention is simple in synthetic route, high in reaction yield and easy to operate and implement; as a prodrug, the THK01 esterified derivative can effectively adjust the physicochemical properties (such as lipid-water partition coefficient) and transmembrane permeability of the compound, and shows typical ester prodrug characteristics in an in-vitro liver microsome experiment; in-vitro and in-vivo experiments show that the compounds can significantly inhibit NO generation and inflammatory reaction at low concentration, and the activity of the compounds is superior to that of positive control drugs, so that the compounds are expected to be developed into novel drugs for treating various inflammation-related diseases.

Description

Technical Field

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

[0002] Inflammation is a complex biological response to harmful stimuli such as injury or infection, and is a crucial defense mechanism. However, when the inflammatory response becomes dysregulated, excessive, or prolonged, it can lead to tissue damage and trigger a variety of diseases, including chronic inflammatory diseases such as arthritis, inflammatory bowel disease (IBD), asthma, psoriasis, and rheumatoid arthritis, severely impacting patients' quality of life.

[0003] THK01 is a natural product with an indolecarbazole skeleton discovered from the secondary metabolites of marine microorganisms (such as Streptomyces). However, the natural product THK01 itself may have defects in drugability such as poor solubility and low bioavailability, which limits its further clinical development potential. The prodrug strategy is a common and effective method to improve the properties of drugs. By reversibly chemically modifying the parent drug molecule, a prodrug molecule is formed that can release the active parent drug through chemical or enzymatic processes in the body, in order to improve the solubility, stability, permeability, targeting or reduce its toxic side effects. Ester bonds are a commonly used connection method in prodrug design. The ester prodrugs formed usually have higher lipid solubility, help improve the transmembrane absorption of the drug, and can be hydrolyzed by widely present esterases in the body to release the active parent drug.

[0004] There are currently no reports on the use of THK01 esterified derivatives as prodrugs for the treatment of inflammation-related diseases. Therefore, the development of THK01 esterified prodrugs and their application in the preparation of anti-inflammatory drugs have important research value and application prospects. 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 ester prodrug and a preparation method thereof and use thereof in the preparation of anti-inflammatory drugs.

[0008] To solve the above technical problems, the present invention provides the following technical solution: a THK01-based esterified derivative, characterized in that the chemical structure of the THK01-based esterified derivative is as shown in formula (I):

[0009]

[0010] wherein R1 is selected from H or O; R2 is selected from C1-C6 straight or branched alkyl, C3-C7 cycloalkyl, heterocyclic group, aryl, heteroaryl, -(CH2)nCOOH, amino acid derivatives

[0011] Another object of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing a THK01-type esterified derivative, characterized in that: the method comprises: obtaining the compound having a structure as shown in formula (II) by condensing it with an acyl chloride derivative as shown in formula (II), a carboxylic acid derivative as shown in formula (II), or an acid anhydride derivative as shown in formula (II);

[0012]

[0013] Another object of the present invention is to overcome the deficiencies in the prior art and provide a use of THK01-based esterified derivatives, characterized in that the derivatives are used 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 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.

[0014] 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.

[0015] 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.

[0016] Another object of the present invention is to overcome the deficiencies in the prior art and provide a drug characterized in that the active ingredient is the THK01 esterified derivative.

[0017] Another object of the present invention is to overcome the deficiencies in the prior art and provide a pharmaceutical composition, characterized in that it comprises a therapeutically effective amount of the compound according to claim 1 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers or excipients.

[0018] As a preferred embodiment of the pharmaceutical composition of the present invention, the pharmaceutically acceptable carrier or excipient is selected from fillers, diluents, binders, wetting agents, disintegrants, lubricants, solubilizers, buffers, preservatives, sweeteners, flavors, colorants, thickeners, stabilizers and combinations thereof.

[0019] As a preferred embodiment of the pharmaceutical composition of the present invention, the dosage form of the pharmaceutical composition is selected from tablets, capsules, granules, powders, injections, solutions, syrups, suspensions or suppositories.

[0020] Beneficial effects of the present invention:

[0021] (1) 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, wherein the disease or condition is 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. 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.

[0022] (2) The present invention found that this type of esterified derivatives exhibited significant in vitro anti-inflammatory activity, for example, they could effectively inhibit the production of nitric oxide (NO) by macrophages induced by lipopolysaccharide (LPS), and the activity of some compounds was better than that of the positive control drug.

[0023] (3) The present invention study confirmed that this type of esterified derivatives has good drug potential. In vitro experiments showed that esterification modification can effectively regulate the physicochemical properties (such as lipid-water partition coefficient) and transmembrane permeation characteristics of this type of compound. Representative compounds (such as THKE04) showed typical ester prodrug characteristics in in vitro liver microsome experiments: chemical properties are stable under simulated body fluid pH conditions, but can be effectively cleaved and released by liver metabolic enzymes to release the parent drug THK01, and THK01 itself has high metabolic stability. More importantly, in vivo pharmacodynamic studies (for example, in a DSS-induced mouse colitis model) confirmed that representative compounds (such as THKE28) can significantly improve inflammatory symptoms and reduce tissue damage, and their therapeutic effects are better than those of the parent compound THK01 and the positive control drug.

[0024] (3) The THK01 esterified derivatives provided by the present invention have the advantages of novel structure, feasible preparation, significant in vitro anti-inflammatory activity, good prodrug characteristics and definite in vivo anti-inflammatory effect, etc., and provide valuable candidate compounds and technical solutions for the development of new drugs for the treatment of inflammatory diseases (such as rheumatoid arthritis, inflammatory bowel disease, psoriasis, etc.), autoimmune diseases and other diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] 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:

[0026] Figure 1 These are the results of in vitro liver microsome testing of THK01 and THKE04 in the present invention;

[0027] Figure 2 The results of the anti-inflammatory bowel disease effects of THK01 and THKE28 in vivo according to the present invention are as follows: Figure 2 (A) Colon length of mice in different treatment groups, Figure 2 (B) Pathological sections of colon tissues of mice in different treatment groups stained with hematoxylin-eosin (HE). Figure 2 (C) Expression of inflammatory factors in mice in different treatment groups.

[0028] Figure 3 is the chemical structural formula of Example THK01-33 of the present invention. DETAILED DESCRIPTION

[0029] 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.

[0030] 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.

[0031] 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.

[0032] Unless otherwise specified, all raw materials used in the examples of the present invention are commercially available.

[0033] Example 1

[0034] Compound IA (1 mmol) was dissolved in pyridine (5 mL), 4-dimethylaminopyridine (DMAP, 0.1 mmol) and triethylamine (2 mmol) were added, and the corresponding acid chloride (1.5 mmol) was added dropwise. The mixture was stirred at 0°C for 1 hour, and then the mixture was warmed to room temperature and reacted for 3-12 hours. After TLC monitoring, water (10 mL) was added to quench 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 THKE01 / 02 / 03 / 04 / 08 / 09 / 10 / 11 / 13 / 15 / 16 / 17 / 18 / 19 / 27 / 28 / 29 / 30 / 31 in a yield of approximately 50-90%.

[0035] As shown in formula 1:

[0036]

[0037] The characterization data of H NMR spectrum and C NMR spectrum are as follows:

[0038] THKE01: Yellow solid. HRESIMS gives the quasi-molecular ion peak as [M+Na] + m / z 406.0796 (calculated value 406.0798), molecular formula is C 22 H 13 N3O4.

[0039] 1 H NMR (600MHz, DMSO-d6): δ11.90(1H,s),11.88(1H,s),11.03(1H,s),8.99(1H,d,J=7.9Hz),8.65(1H,d,J=2.2H z),7.85(2H,overlap),7.56(1H,t,J=7.4Hz),7.36(1H,t,J=7.4Hz),7.33(1H,dd,J=2.2,8.6Hz),2.37(3H,s).

[0040] 13 C NMR (151MHz, DMSO-d6): δ171.42,171.31,170.03,144.17,140.36,138.07,129.90,129.16,126.92,124 .37,121.81,121.62,121.23,120.41,120.11,119.94,116.53,115.73,115.29,112.65,112.19,20.99.

[0041] THKE02: Yellow solid. HRESIMS gives the quasi-molecular ion peak as [M+Na] + m / z 420.0955 (calculated value 420.0955), molecular formula is C 23 H 15 N3O4.

[0042] 1 H NMR (600MHz, DMSO-d6): δ11.84(2H,overlap),11.02(1H,s),8.99(1H,d,J=7.9Hz),8.65(1H,d,J=2.3Hz),7.83(2 H,overlap),7.56(1H,m),7.36(1H,m),7.33(1H,dd,J=2.3,8.6Hz),2.72(2H,q,J=7.5Hz),1.21(3H,t,J=7.5Hz).

[0043] 13C NMR (151MHz, DMSO-d6): δ173.41,171.42,171.32,144.22,140.36,138.07,129.89,129.15,126.93,124.3 8,121.83,121.62,121.25,120.42,120.12,119.95,116.51,115.73,115.31,112.67,112.21,27.01,9.05.

[0044] THKE03: Yellow solid. HRESIMS gives the quasi-molecular ion peak as [M+Na] + m / z 434.1112 (calculated value 434.1111), molecular formula is C 24 H 17 N3O4.

[0045] 1 H NMR (600MHz, DMSO-d6): δ11.84(2H,overlap),11.01(1H,s),9.00(1H,d,J=7.9Hz),8.65(1H,d,J=2.3Hz),7.85(2H,ove rlap),7.56(1H,m),7.36(1H,m),7.32(1H,dd,J=2.3,8.6Hz),2.67(2H,t,J=7.3Hz),1.73(2H,m),1.04(3H,t,J=7.3Hz).

[0046] 13 C NMR (151MHz, DMSO-d6): δ172.54,171.42,171.33,144.17,140.36,138.08,129.90,129.15,126.94,124.38,12 1.85,121.62,121.23,120.43,120.12,119.95,116.53,115.74,115.30,112.70,112.21,35.43,18.09,13.51.

[0047] THKE04: Yellow solid. HRESIMS gives the quasi-molecular ion peak as [M+Na] + m / z 448.1269 (calculated value 448.1268), molecular formula is C 25 H 19 N3O4.

[0048] 1H NMR (600MHz, DMSO-d6): δ11.86 (2H, overlap), 11.02 (1H, s), 8.98 (1H, d, J = 7.9 Hz), 8.65 (1H, d, J = 2.3 Hz), 7.85 (2H, overlap), 7.56(1H,m),7.36(1H,m),7.31(1H,dd,J=2.3,8.6Hz),2.69(2H,t,J=7.3Hz),1.70(1H,m),1.46(1H,m),0.97(3H,t,J=7.3Hz).

[0049] 13 C NMR (151MHz, DMSO-d6): δ172.69,171.42,171.33,144.18,140.37,138.08,129.90,129.16,126.94,124.38,121.8 5,121.62,121.21,120.42,120.12,119.95,116.52,115.74,115.30,112.68,112.20,33.34,26.68,21.73,13.76.

[0050] THKE08: Yellow solid. HRESIMS gives the quasi-molecular ion peak as [M+Na] + m / z 432.0953 (calculated value 432.0955), molecular formula is C 24 H 15 N3O4.

[0051] 1 H NMR (600MHz, DMSO-d6): δ11.82 (2H, overlap), 10.99 (1H, s), 8.96 (1H, d, J = 8.0Hz), 8.60 (1H, d, J = 2.3Hz), 7.7 9(2H,overlap),7.53(1H,m),7.32(1H,m),7.29(1H,dd,J=2.3,8.6Hz),1.97(1H,m),1.08(2H,m),1.04(2H,m).

[0052] 13C NMR (151MHz, DMSO-d6): δ173.76,171.44,171.32,144.14,140.37,138.09,129.91,129.15,126.94,124.3 8,121.82,121.62,121.16,120.43,120.13,119.95,116.48,115.75,115.29,112.68,112.21,12.70,9.09.

[0053] THKE09: Yellow solid. HRESIMS gives the quasi-molecular ion peak as [M+Na] + m / z 446.1115 (calculated value 446.1111), molecular formula is C 25 H 17 N3O4.

[0054] 1 H NMR (600MHz, DMSO-d6): δ11.93(2H,overlap),11.02(1H,s),8.99(1H,d,J=8.0Hz),8.63(1H,d,J=2.3Hz),7.83(2H,over lap),7.56(1H,m),7.36(1H,m),7.33(1H,dd,J=2.3,8.6Hz),3.56(1H,m),2.38(4H,overlap),2.05(1H,m),1.95(1H,m).

[0055] 13 C NMR (151MHz, DMSO-d6): δ174.23,171.44,171.36,144.17,140.35,138.08,129.93,129.19,127.63,124.46,12 1.84,121.63,120.82,120.12,119.95,116.68,116.20,115.72,115.27,112.68,112.20,37.71,36.94,24.93.

[0056] THKE10: Yellow solid. HRESIMS gives the quasi-molecular ion peak as [M+Na] + m / z 476.1217 (calculated value 476.1217), molecular formula is C 26 H 19 N3O5.

[0057] 1H NMR (600MHz, DMSO-d6): δ11.96(2H,overlap),11.03(1H,s),8.99(1H,d,J=8.0Hz),8.65(1H,d,J=2.3Hz),7.84(2H,overlap),7.56(1H,m),7.36(1H,m) ,7.33(1H,dd,J=2.3,8.6Hz),4.43(1H,dd,J=2.8,10.0Hz),4.02(1H,m),3. 58(1H,m),2.07(1H,m),1.88(1H,m),1.77(1H,m),1.68(1H,m),1.58(2H,m).

[0058] 13 C NMR (151MHz, DMSO-d6): δ171.89,171.80,171.12,144.19,140.81,138.64,130.40,129.63,127.42,124.85,122.31,1 22.06,121.37,120.88,120.62,120.40,116.81,116.21,115.67,113.25,112.65,75.61,67.44,28.94,25.58,22.67.

[0059] THKE11: Yellow solid. HRESIMS gives the quasi-molecular ion peak as [M+H] + m / z 536.2290 (calculated value 536.2292), molecular formula is C 31 H 29 N5O4.

[0060] 1 H NMR (600MHz, DMSO-d6): δ11.93(2H,overlap),11.03(1H,s),8.98(1H,d,J=8.0Hz),8.65(1H,d,J=2.3Hz),7.82(2H,d,J=8.4Hz),7.57(1H,m),7.3 6(1H,m),7.31(1H,dd,J=2.3,8.6Hz),3.48(3H,overlap),3.17(1H,m),2.98(3H,overlap),2.13(2H,overlap),1.87(9H,overlap),1.44(1H,m).

[0061] 13C NMR (151MHz, DMSO-d6): δ171.46,171.35,153.61,144.79,140.39,129.93,129.19,126.97,124.38,121.74,121. 61,121.53,120.42,120.08,119.90,116.74,115.71,115.32,112.44,112.19,95.52,62.45,49.30,23.00,21.53.

[0062] THKE15: Yellow solid. HRESIMS gives the quasi-molecular ion peak as [M+H] + m / z 468.1661 (calculated value 468.1666), molecular formula is C 26 H 21 N5O4.

[0063] 1 H NMR (600MHz, DMSO-d6): δ12.06 (2H, overlap), 11.02 (1H, s), 8.99 (1H, d, J = 7.9Hz), 8.69 (1H, d, J = 2.3Hz), 7.81 (2H,overlap),7.56(1H,t,J=7.9Hz),7.36(2H,overlap),3.45(6H,overlap),3.27(2H,overlap),2.89(3H,s).

[0064] 13 C NMR (151MHz, DMSO-d6): δ171.45,171.33,153.48,144.57,140.41,138.03,129.96,129.21,126.96,124.39,12 1.74,121.61,121.41,120.42,120.12,119.90,116.73,115.73,115.30,112.51,112.18,52.16,48.67,42.42.

[0065] THKE16: Yellow solid. HRESIMS gives the quasi-molecular ion peak as [M+H] + m / z 490.1032 (calculated value 490.1034), molecular formula is C 28 H 15 N3O6.

[0066] 1H NMR (600MHz, DMSO-d6): δ11.87(1H,s),11.83(1H,s),11.03(1H,s),9.00(1H,d,J=7.8Hz),8.75(1H,d,J=2.3Hz),7.90(1H,d,J=8.6Hz) ,7.85(2H,overlap),7.65(1H,d,J=1.7Hz),7.57(1H,m),7.46(1H,dd,J=2.3,8.6Hz),7.37(1H,m),7.17(1H,d,J=8.0Hz),6.22(2H,s).

[0067] 13 C NMR (151MHz, DMSO-d6): δ171.43,171.31,164.74,152.23,147.97,144.31,140.37,138.18,129.93,129.17,126.94,126.09,124 .39,122.87,121.87,121.62,121.26,120.43,120.16,119.96,116.63,115.77,115.31,112.77,112.21,109.24,108.61,102.41.

[0068] THKE17: Yellow solid. HRESIMS gives the quasi-molecular ion peak as [M+Na] + m / z 474.0520 (calculated value 474.0519), molecular formula is C 25 H 13 N3O4S.

[0069] 1 H NMR (600MHz, DMSO-d6): δ11.90(1H,s),11.84(1H,s),11.04(1H,s),9.00(1H,d,J=7.8Hz),8.77(1H,d,J=2.3Hz),8.14(1H,dd,J=1.1,5.1H z), 8.12 (1H, dd, J = 1.1, 3.7Hz), 7.91 (1H, d, J = 8.7Hz), 7.84 (1H, d, J = 8.1Hz), 7.58 (1H, m), 7.49 (1H, dd, J = 2.3, 8.7Hz), 7.36 (2H, overlap).

[0070] 13C NMR (151MHz, DMSO-d6): δ171.44,171.30,160.96,143.85,140.37,138.28,135.36,135.26,132.13,129.95,129.17, 128.89,126.96,124.38,121.87,121.61,121.16,120.44,120.20,119.97,116.56,115.81,115.28,112.87,112.22.

[0071] THKE18: Yellow solid. HRESIMS gives the quasi-molecular ion peak as [M+Na] + m / z 520.0472 (calculated value 520.0471), molecular formula is C 27 H 13 ClFN3O4.

[0072] 1 H NMR (600MHz, DMSO-d6): δ11.95(1H,s),11.89(1H,s),11.03(1H,s),9.00(1H,d,J=8.0Hz),8.79(1H,s),8.38(1H,dd,J=1.7,7.0Hz),8 .27(1H,m),7.91(1H,dd,J=3.7,8.7Hz),7.83(1H,d,J=8.0Hz),7.71(1H,m),7.57(1H,t,J=7.5Hz),7.51(1H,m),7.37(1H,t,J=7.5Hz).

[0073] 13 C NMR (151MHz, DMSO-d6): δ171.45,171.32,167.84,163.60,144.08,140.38,138.33,132.44,129.97,129.19,127.14,126.98, 124.56,124.40,121.86,121.62,121.09,120.69,120.22,119.96,117.92,117.78,116.54,115.80,115.28,112.86,112.23.

[0074] THKE19: Yellow solid. HRESIMS gives the quasi-molecular ion peak as [M+H] + m / z 465.0991 (calculated value 465.0994), molecular formula is C 26 H 13 N4O4.

[0075] 1 H NMR (600MHz, DMSO-d6): δ11.99(1H,s),11.92(1H,s),11.04(1H,s),8.98(1H,d,J=7.9Hz),8.87(1H,d,J=2.7Hz),8.80(1H,d,J=1.9Hz),8.43 (1H,m),8.03(1H,m),7.92(1H,d,J=8.9Hz),7.82(1H,d,J=8.2Hz),7.57(1H,t,J=7.5Hz),7.52(1H,dd,J=2.0,8.6Hz),7.37(1H,t,J=7.5Hz).

[0076] 13 C NMR (151MHz, DMSO-d6): δ171.46,171.32,163.25,144.20,143.61,140.39,138.92,138.75,138.32,129.98,129.19,128 .13,126.97,124.57,124.40,121.91,121.62,121.03,120.44,120.20,119.98,116.41,115.81,115.28,112.89,112.21.

[0077] THKE27: white solid. HRESIMS gives the quasi-molecular ion peak as [M+H] + m / z 522.2500 (calculated value 522.2500), molecular formula is C 31 H 31 N5O3.

[0078] 1H NMR (600MHz, DMSO-d6): δ11.72(1H,s),11.57(1H,s),8.88(1H,d,J=2.4Hz),8.52(1H,s),8.04 (1H,d,J=7.7Hz),7.79(1H,d,J=8.2Hz),7.70(1H,d,J=8.7Hz),7.48(1H,m),7.31(1H,m),7.18( 1H,dd,J=2.4,8.7Hz),4.97(2H,s),4.45(1H,m),4.23(1H,m),3.48(3H,overlap),3.14(1H,m) ,2.99(3H,overlap),2.12(2H,overlap),1.89(2H,overlap),1.71(5H,overlap),1.44(1H,m).

[0079] 13 C NMR (151MHz, DMSO-d6): δ172.44,158.38,153.84,143.90,139.34,136.78,133.14,127.95,126.42,122.87,122.59,121.27,12 0.04,119.80,118.81,117.52,115.44,114.40,112.04,111.60,62.48,49.29,45.43,42.86,42.54,26.10,25.70,22.99,21.54.

[0080] THKE28: white solid. HRESIMS gives the quasi-molecular ion peak as [M+H] + m / z 441.1554 (calculated value 441.1557), molecular formula is C 25 H 20 N4O4.

[0081] 1 H NMR (600MHz, DMSO-d6): δ11.55(1H,s),11.39(1H,s),8.89(1H,d,J=2.4Hz),8.54(1H,s),8.04(1H,d,J=7.8Hz),7.79(1H,d,J =8.2Hz),7.71(1H,d,J=8.7Hz),7.48(1H,m),7.31(1H,m),7.20(1H,dd,J=2.5,8.7Hz),4.97(2H,s),3.70(6H,s),3.47(2H,s).

[0082] 13C NMR (151MHz, DMSO-d6): δ172.42,154.16,143.92,139.31,136.75,133.11,127.90,126.36,125.17,122.8 8,122.60,121.27,120.04,119.87,118.86,117.55,115.49,114.40,112.06,111.64,65.95,45.42,44.64.

[0083] THKE29: white solid. HRESIMS gives the quasi-molecular ion peak as [M+H] + m / z 454.1870 (calculated value 454.1874), molecular formula is C 26 H 23 N5O3.

[0084] 1 H NMR (600MHz, DMSO-d6): δ11.77(1H,s),11.64(1H,s),8.92(1H,d,J=2.3Hz),8.52(1H,s),8.06(1H,d,J=7.9Hz),7.79(1H,d,J=8.1Hz),7.73(1H, d,J=8.7Hz),7.48(1H,m),7.31(1H,t,J=7.5Hz),7.20(1H,dd,J=2.3,8.7Hz),4.97(2H,s),4.26(2H,overlap),3.50(6H,overlap),2.88(3H,s).

[0085] 13 C NMR (151MHz, DMSO-d6): δ172.43,153.69,143.68,139.35,136.87,133.17,127.94,126.45,125.21,122.8 7,122.58,121.28,120.04,119.68,118.82,117.50,115.42,114.43,112.04,111.67,52.16,45.43,42.41.

[0086] THKE30: white solid. HRESIMS gives the quasi-molecular ion peak as [M+H] + m / z 370.1185 (calculated value 370.1186), molecular formula is C 22 H 15 N3O3.

[0087] 1H NMR (600MHz, DMSO-d6): δ11.57(1H,s),11.44(1H,s),8.89(1H,d,J=2.3Hz),8.54(1H,s),8.05(1H,d,J=7.8Hz),7.80(1H,d, J=8.1Hz),7.73(1H,d,J=8.8Hz),7.48(1H,m),7.31(1H,t,J=7.6Hz),7.20(1H,dd,J=2.3,8.8Hz),4.97(2H,s),2.35(3H,s).

[0088] 13 C NMR (151MHz, DMSO-d6): δ172.42,170.15,143.32,139.32,136.92,133.15,127.89,126.37,125.20,12 2.96,122.60,121.28,120.06,119.50,118.88,117.27,115.46,114.46,112.08,111.84,45.44,21.02.

[0089] THKE31: white solid. HRESIMS gives the quasi-molecular ion peak as [M+Na] + m / z 396.1342 (calculated value 396.1343), molecular formula is C 24 H 17 N3O3.

[0090] 1 H NMR (600MHz, DMSO-d6): δ11.58(1H,s),11.44(1H,s),8.88(1H,d,J=2.4Hz),8.54(1H,s),8.04(1H,d,J=7.8Hz),7.80(1H,d,J=8.2Hz),7.73 (1H,d,J=8.6Hz),7.48(1H,m),7.31(1H,m),7.17(1H,dd,J=2.4,8.6Hz),4.97(2H,s),1.99(1H,m),1.10(2H,overlap),1.05(2H,overlap).

[0091] 13C NMR (151MHz, DMSO-d6): δ173.87,172.41,143.28,139.32,136.93,133.15,127.89,126.38,125.20,122.9 6,122.59,121.28,120.06,119.42,118.87,117.24,115.44,114.47,112.07,111.84,45.45,12.70,8.99.

[0092] Example 2

[0093] Compound IA (1 mmol) was dissolved in acetonitrile (5 mL), TCFH (1.2 mmol) and NMI (3.5 mmol) were added, and the corresponding carboxylic acid (1.5 mmol) was added dropwise. The reaction was allowed to react at room temperature for 12-24 hours. After TLC monitoring, water (10 mL) was added to quench 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 THKE12 / 14 / 20 / 21 / 22 / 23 / 24 / 25 / 26 / 33 in yields of approximately 40-90%.

[0094] As shown in formula 2:

[0095]

[0096] The characterization data of H NMR spectrum and C NMR spectrum are as follows:

[0097] THKE12: Yellow solid. HRESIMS gives the quasi-molecular ion peak as [M+H] + m / z 469.1511

[0098] (calculated value is 469.1506), molecular formula is C 26 H 20 N4O5.

[0099] 1H NMR (600MHz, DMSO-d6): δ12.10(2H,overlap),11.03(1H,s),8.99(1H,d,J=8.0Hz),8.75(1H,s),7.88(1H,d,J=8.8Hz),7.81(1H,d,J=8 .0Hz),7.57(1H,t,J=7.4Hz),7.42(1H,d,J=8.8Hz),7.36(1H,t,J=7.4Hz),4.44(2H,overlap),3.83(4H,overlap),3.24(4H,overlap).

[0100] 13 C NMR (151MHz, DMSO-d6): δ171.48,171.42,171.32,143.32,140.42,138.40,130.02,129.21,127.00,124.39 ,121.85,121.59,120.74,120.44,120.25,119.91,116.33,115.82,115.18,112.86,112.19,52.31,48.66.

[0101] THKE14: Yellow solid. HRESIMS gives the quasi-molecular ion peak as [M+H] + m / z 482.1825

[0102] (calculated value is 482.1823), molecular formula is C 27 H 23 N5O4.

[0103] 1 H NMR (600MHz, DMSO-d6): δ12.05 (2H, overlap), 11.01 (1H, s), 9.00 (1H, d, J = 7.9Hz), 8.68 (1H, d, J = 2.3Hz), 7.85 (1H, d, J = 8.7Hz), 7.80 (1 H,d,J=8.2Hz),7.57(1H,m),7.36(2H,overlap),3.83(2H,s),3.43(2H,overlap),3.14(4H,overlap),2.82(3H,s),2.79(2H,overlap).

[0104] 13C NMR (151MHz, DMSO-d6): δ171.44,171.35,169.43,143.69,140.42,138.21,129.96,129.21,126.99,124.39,121.85,1 21.60,121.07,120.43,120.18,119.91,116.47,115.77,115.23,112.72,112.19,56.92,52.78,48.63,42.34,40.02.

[0105] THKE20: Yellow solid. HRESIMS gives the quasi-molecular ion peak as [M+Na] + m / z 535.1591 (calculated value 535.1588), molecular formula is C 26 H 13 N4O4.

[0106] 1 H NMR (600MHz, DMSO-d6): δ11.85(2H,overlap),11.02(1H,s),8.98(1H,d,J=7.9Hz),8.65(1H,s),7.82(2H,overlap ),7.55(1H,t,J=7.7Hz),7.35(2H,overlap),7.06(1H,m),3.35(2H,overlap),2.80(1H,t,J=6.7Hz),1.40(9H,s).

[0107] 13 C NMR (151MHz, DMSO-d6): δ171.42,171.37,171.01,155.72,144.16,140.40,138.14,129.91,129.19,126.99,124.42, 121.81,121.36,120.47,120.16,119.98,116.56,115.76,115.35,112.24,77.94,48.71,40.02,36.26,34.49,28.37.

[0108] THKE21: Yellow solid. HRESIMS gives the quasi-molecular ion peak as [M+Na] + m / z 683.1904 (calculated value 683.1901), molecular formula is C 40 H 28 N4O6.

[0109] 1H NMR (600MHz, DMSO-d6): δ11.85(2H,overlap),11.03(1H,s),8.99(1H,d,J=7.7Hz),7.90(2H,overlap),7.83(2H,overlap),7.71(2H,overlap) ,7.56(1H,t,J=7.5Hz),7.35(7H,overlap),4.62(1H,m),4.41(3H,overlap),3.50(2H,overlap),2.47(1H,m),2.26(1H,m),2.01(2H,overlap).

[0110] 13 C NMR (151MHz, DMSO-d6): δ172.06,171.87,171.36,143.98,143.87,143.53,140.85, 140.37,138.19,129.93,129.15,127.78,127.30,127.26,126.96,125.26,125.23, 124.39,121.81,121.61,120.86,120.44,120.25,120.23,120.18,119.95,116.27,116.19,115.77,115.26,112.79,112.21,66.90,59.14,46.88,46.69,29.66,24.29.

[0111] THKE22: Yellow solid. HRESIMS gives the quasi-molecular ion peak as [M+Na] + m / z 733.2056 (calculated value 733.2058), molecular formula is C 44 H 30 N4O6.

[0112] 1H NMR (600MHz, DMSO-d6): δ11.86(1H,s),11.83(1H,s),11.04(1H,s),8.98(1H,d,J=7.9Hz),8. 67(1H,d,J=2.2Hz),8.17(1H,d,J=7.9Hz),7.89(2H,overlap),7.83(2H,overlap),7.69(2H, overlap),7.57(1H,m),7.40(4H,overlap),7.36(3H,overlap),7.30(3H,overlap),7.17(1H ,dd,J=2.3,8.7Hz),4.60(1H,m),4.37(1H,m),4.32(1H,m),4.24(1H,m),3.28(2H,overlap).

[0113] 13 C NMR (151MHz, DMSO-d6): δ171.63,171.41,171.37,156.19,144.06,143.84,14 0.84,140.40,138.22,137.40,129.95,129.46,129.18,128.50,127.76,127.2 0,127.01,126.79,125.34,124.43,121.84,121.64,120.84,120.48,120.23,119.99,116.48,115.80,115.30,112.80,112.25,65.84,55.87,46.72,36.60.

[0114] THKE23: Yellow solid. HRESIMS gives the quasi-molecular ion peak as [M+Na] + m / z 675.1749 (calculated value 675.1745), molecular formula is C 38 H 26 N4O6.

[0115] 1H NMR (600MHz, DMSO-d6): δ11.84(1H,s),11.82(1H,s),11.04(1H,s),8.99(1H,d, J=7.8Hz),8.08(1H,d,J=7.3Hz),7.90(2H,overlap),7.86(2H,overlap),7.75(2 H,overlap),7.57(1H,m),7.41(2H,overlap),7.33(4H,overlap),7.28(1H,dd,J=2.4,8.7Hz),4.41(3H,overlap),4.28(1H,t,J=6.8Hz),1.54(3H,d,J=7.1Hz).

[0116] 13 C NMR (151MHz, DMSO-d6): δ171.48,171.40,171.36,156.15,149.67,144.15 ,143.94,143.88,140.86,138.19,129.94,129.18,127.75,127.21,125.3 4,125.31,124.48,124.41,121.85,121.64,120.92,120.48,120.24,119. 98,116.38,115.81,115.30,112.83,112.25,65.83,49.68,46.75,17.02.

[0117] THKE24: white solid. HRESIMS gives the quasi-molecular ion peak as [M+Na] + m / z 719.2591 (calculated value 719.2589), molecular formula is C 40 H 36 N6O6.

[0118] 1H NMR (600MHz, DMSO-d6): δ11.61(1H,s),11.48(1H,s),8.91(1H,dd,J=2.4,8.8Hz),8.53(1H,m),8.39(1H,m),8.0 4(1H,d,J=7.8Hz),7.80(1H,d,J=8.0Hz),7.76(1H,dd,J=2.8,8.6Hz),7.56(2H,overlap),7.49(1H,m),7.40(5H, overlap),7.31(1H,m),7.15(1H,m),6.75(1H,t,J=15.4Hz),4.97(2H,s),4.72(1H,m),4.56(2H,overlap),4.44( 1H,m),3.70(2H,overlap),2.09(2H,overlap),1.92(2H,overlap),1.50(1H,d,J=7.4Hz),1.30(1H,d,J=7.1Hz).

[0119] 13 C NMR (151MHz, DMSO-d6): δ172.84,172.11,171.46,171.10,164.96,143.76,139.79 ,139.55,137.44,135.40,133.62,130.03,129.48,128.35,128.06,126.86,125.66 ,123.38,123.04,122.43,121.74,120.52,119.69,119.33,117.57,115.90,114.93,112.53,112.35,60.19,48.45,47.34,46.76,45.90,29.71,24.93,17.75,17.35.

[0120] THKE25: Yellow solid. HRESIMS gives the quasi-molecular ion peak as [M+Na] + m / z 691.2275 (calculated value 691.2276), molecular formula is C 38 H 32 N6O6.

[0121] 1H NMR (600MHz, DMSO-d6): δ11.61(1H,s),11.49(1H,s),8.94(1H,d,J=2.3Hz),8.54(1H,s),8.49(1H,t, J=5.7Hz),8.45(1H,t,J=5.7Hz),8.04(1H,d,J=7.8Hz),7.80(1H,d,J=8.2Hz),7.72(3H,overlap),7. 53(2H,overlap),7.35(4H,overlap),7.18(1H,dd,J=2.3,8.6Hz),7.04(1H,d,J=15.6Hz),4.97(2H,s ),4.41(1H,m),4.29(2H,overlap),3.78(4H,overlap),2.12(1H,m),2.02(1H,m),1.93(2H,overlap).

[0122] 13 C NMR (151MHz, DMSO-d6): δ172.41,172.35,169.84,169.57,164.59,143.12,141 .43,139.35,137.02,135.02,133.20,129.78,128.87,128.24,128.13,127.91, 126.43,125.25,122.97,122.60,121.31,120.09,119.62,118.92,117.20,115.46,114.51,112.10,111.93,60.40,47.23,45.47,42.05,41.11,29.24,24.52.

[0123] THKE26: white solid. HRESIMS gives the quasi-molecular ion peak as [M+Na] + m / z 771.2905 (calculated value 771.2902), molecular formula is C 44 H 40 N6O6.

[0124] 1H NMR (600MHz, DMSO-d6): δ11.61(1H,s),11.48(1H,s),8.87(1H,d,J=2.4Hz),8.52(1H,s),8.06 (1H,d,J=7.8Hz),7.82(1H,d,J=8.4Hz),7.76(1H,d,J=8.7Hz),7.71(1H,d,J=7.3Hz),7.42(7H, overlap),7.14(1H,dd,J=2.4,8.4Hz),7.06(1H,d,J=15.5Hz),4.97(2H,s),4.68(3H,overlap) ,3.77(6H,overlap),2.41(6H,m),2.15(7H,overlap),1.93(2H,overlap),1.85(2H,overlap).

[0125] 13 C NMR (151MHz, DMSO-d6): δ172.37,171.59,170.41,169.84,163.37,143.24,140.89,139.3 5,136.99,135.10,133.18,129.73,128.92,128.14,127.91,126.42,125.22,122.91,122. 60,121.30,120.07,119.83,119.14,118.89,116.95,115.43,114.49,112.08,111.91,58.80,57.72,57.52,47.02,46.72,46.52,45.46,28.72,28.03,27.76,24.95,24.53,24.39.

[0126] THKE33: white solid. HRESIMS gives the quasi-molecular ion peak as [M+Na] + m / z 779.2833 (calculated value 779.2834), molecular formula is C 39 H 44 N6O8S.

[0127] 11H NMR (600 MHz, DMSO-d6): δ 11.66 (1H, s), 11.52 (1H, s), 8.89 (1H, d, J = 2.3 Hz), 8.53 (1H, s), 8.06 (1H, d, J = 7.7 Hz), 7.83 (1H, t, J = 5.5 Hz), 7.79 (1H, d, J = 8.1 Hz), 7.74 (1H, d, J = 8.7 Hz), 7.48 (1H, m), 7.31 (1H, t, J = 7.5 Hz), 7.17 (1H, dd, J = 2.3, 8.7 Hz), 6.42 (1H, s), 6.35 (1H, s), 4.97 (2H, s), 4.25 (1H, m), 4.08 (1H, m), 3.82 (2H, t, J = 6.2 Hz), 3.61 (4H, overlap), 3.56 (2H, m), 3.54 (2H, m), 3.41 (2H, t, J = 6.2 Hz), 3.18 (2H, q, J = 5.7 Hz), 3.05 (1H, m), 2.91 (2H, t, J = 6.2 Hz), 2.77 (1H, dd, J = 5.1, 12.5 Hz), 2.56 (1H, d, J = 12.8 Hz), 2.05 (2H, t, J = 7.4 Hz), 1.58 (1H, m), 1.50 (3H, overlap), 1.26 (2H, m).

[0128] 13 13C NMR (151 MHz, DMSO-d6): δ 172.87, 172.66, 171.44, 163.24, 143.70, 139.78, 137.39, 133.60, 128.36, 126.85, 125.65, 123.40, 123.04, 121.73, 120.50, 119.89, 119.32, 117.72, 115.90, 114.90, 112.51, 112.28, 70.35, 70.31, 70.14, 69.73, 66.64, 61.54, 59.69, 55.94, 45.90, 39.00, 35.62, 35.35, 28.72, 28.55, 25.79.

[0129] Example 3

[0130] Compound IA (1 mmol) was dissolved in pyridine (5 mL), 4-dimethylaminopyridine (DMAP, 0.1 mmol) and triethylamine (2 mmol) were added, and the corresponding anhydride (1.5 mmol) was added dropwise. The mixture was stirred at room temperature for 3-12 hours. After TLC monitoring, the reaction was completed, and water (10 mL) was added to quench the reaction. 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: Welch column) 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 THKE05 / 06 / 07 / 32 in a yield of approximately 40-70%.

[0131] As shown in formula 3:

[0132]

[0133] The characterization data of H NMR spectrum and C NMR spectrum are as follows:

[0134] THKE05: yellow solid. HRESIMS gives the quasi-molecular ion peak as [M+H] + m / z 422.1031 (calculated value 442.1034), molecular formula is C 24 H 15 N3O6.

[0135] 1 H NMR (600MHz, DMSO-d6): δ12.36(1H,s),11.85(2H,overlap),11.02(1H,s),8.99(1H,d,J=8.0Hz), 8.66(1H,d,J=2.3Hz), 7.85(2H,overlap),7.56(1H,m),7.36(1H,m),7.28(1H,dd,J=2.3,8.7Hz),2.89(2H,t,J=6.6Hz),2.66(2H,t,J=6.6Hz).

[0136] 13C NMR (151MHz, DMSO-d6): δ173.52,171.94,171.45,171.34,144.17,140.37,138.09,129.90,129.16,126.95,12 4.39,121.82,121.63,121.14,120.43,120.14,119.96,116.51,115.74,115.31,112.69,112.22,29.06,28.90.

[0137] THKE06: Yellow solid. HRESIMS gives the quasi-molecular ion peak as [M+Na] + m / z 478.1010 (calculated value 478.1010), molecular formula is C 25 H 17 N3O6.

[0138] 1 H NMR (600MHz, DMSO-d6): δ12.18(1H,s),11.90(2H,overlap),11.03(1H,s),8.98(1H,d,J=8.0Hz),8.65(1H,d,J=2.3Hz),7.84( 2H,overlap),7.56(1H,m),7.36(1H,m),7.34(1H,dd,J=2.3,8.6Hz),2.74(2H,t,J=7.4Hz),2.40(2H,t,J=7.4Hz),1.92(1H,m).

[0139] 13 C NMR (151MHz, DMSO-d6): δ174.14,172.32,171.43,171.35,144.12,140.36,138.09,129.92,129.18,126.94,124.3 9,121.83,121.62,121.25,120.42,120.12,119.95,116.51,115.73,115.29,112.66,112.19,32.81,32.75,20.04.

[0140] THKE07: Yellow solid. HRESIMS gives the quasi-molecular ion peak as [M+Na] + m / z 492.1166 (calculated value 492.1166), molecular formula is C 26 H 19 N3O6.

[0141] 1H NMR (600MHz, DMSO-d6): δ12.09(1H,s),11.95(2H,overlap),11.03(1H,s),8.99(1H,d,J=8.0Hz),8.64(1H,d,J=2.3Hz),7.82(2H,ove rlap),7.56(1H,m),7.36(1H,m),7.30(1H,dd,J=2.3,8.6Hz),2.70(2H,t,J=7.4Hz),2.32(2H,t,J=7.4Hz),1.73(2H,m),1.65(2H,m).

[0142] 13 C NMR (151MHz, DMSO-d6): δ174.44,172.52,171.43,171.35,144.15,140.35,138.06,129.93,129.18,126.94,124.39,1 21.83,121.62,121.22,120.41,120.11,119.95,116.53,115.71,115.27,112.64,112.17,33.45,33.34,24.09,24.04.

[0143] THKE32: white solid. HRESIMS gives the quasi-molecular ion peak as [M+H] + m / z 456.1551 (calculated value 456.1554), molecular formula is C 26 H 21 N3O5.

[0144] 1 H NMR (600MHz, DMSO-d6): δ12.04(1H,s),11.54(1H,s),11.40(1H,s),8.84(1H, d,J=2.4Hz),8.49(1H,s),8.01(1H,d,J=8.0Hz),7.76(1H,d,J=8.1Hz),7.71( 1H,d,J=8.7Hz),7.44(1H,m),7.27(1H,m),7.14(1H,dd,J=2.4,8.7Hz),4.92( 2H,s),2.64(2H,t,J=7.2Hz),2.27(2H,t,J=7.2Hz),1.68(2H,m),1.61(2H,m).

[0145] 13C NMR (151MHz, DMSO-d6): δ174.48,172.62,172.41,143.29,139.32,136.92,133.15,127.90,126.38,125.20,122.97,1 22.60,121.28,120.06,119.48,118.88,117.26,115.46,114.46,112.08,111.86,45.44,33.48,33.37,24.12,24.08.

[0146] Example 4

[0147] Determination of LogD of THK01 esterified derivatives

[0148] This example evaluates the lipid-water partitioning properties of THK01 and its esterified derivatives (exemplified by THKE26, THKE28, THKE29, THKE31, and THK32). The shake flask method was used for the determination: 1 mL of octanol and 1 mL of pH 7.4 phosphate buffer were added to a 5 mL reagent vial. Then, 20 μL of a 10 mM compound stock solution was added to the mixture. The mixture was shaken at 300 rpm for 1 hour at room temperature. After the solution was allowed to separate completely for 30 minutes, the upper and lower layers were aspirated. The separated solutions were transferred to a 96-well deep-plate for liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis. The octanol and aqueous phases were serially diluted with methanol (MeOH) to obtain the final samples for LC-MS / MS analysis. The MS peak areas of these solutions were used to generate a relationship between log(peak area) and log(relative concentration) based on a relative concentration calibration line. The LogD value was obtained by calculating the relative concentration ratio of the compound in the octanol phase and the aqueous phase.

[0149] Figure 1 These are the results of in vitro liver microsome testing of THK01 and THKE04 in the present invention;

[0150] Figure 2 The results of the anti-inflammatory bowel disease effects of THK01 and THKE28 in vivo according to the present invention are as follows: Figure 2 (A) Colon length of mice in different treatment groups, Figure 2 (B) Pathological sections of colon tissues of mice in different treatment groups stained with hematoxylin-eosin (HE). Figure 2 (C) Expression of inflammatory factors in mice in different treatment groups.

[0151] Figure 3is the chemical formula of Example THK01-33 of the present invention. Measurement results showed that at physiological pH 7.4, the parent compound THK01 had a LogD value of 2.25. Its series of esterified derivatives exhibited varying lipid-water partitioning properties: THKE26 had a LogD value of 2.26, essentially comparable to the parent compound, maintaining good lipid solubility. The remaining derivatives, THKE28, THKE29, THKE31, and THK32, had LogD values ​​of 1.59, 1.78, 1.46, and 0.51, respectively, representing decreases compared to the parent compound. This demonstrates that selective esterification can effectively modulate the lipophilicity / hydrophilicity balance of THK01 derivatives. The reduced LogD values ​​of some derivatives (such as THKE28, THKE29, THKE31, and THK32) suggest a relative increase in their water solubility, which may be beneficial for improving drug dissolution characteristics, formulation performance, or in vivo distribution behavior. The controllability of this physicochemical property confirms the potential of the esterification strategy in optimizing the pharmacokinetic properties of THK01 compounds and provides an important experimental basis for the development of prodrugs with specific advantages.

[0152] Table 1

[0153]

[0154] Example 5

[0155] Caco-2 cell uptake assay of THK01 esterified derivatives

[0156] This example uses a Caco-2 cell monolayer model to evaluate the in vitro transmembrane transport capacity of THK01 and its esterified derivatives (using THKE26, THKE28, THKE29, THKE31 and THK32 as examples). The specific operation is as follows: Caco-2 cells are seeded at a specific density (e.g., 1×105 cells / cm2) on The culture medium was replaced every two days on the porous polymer membrane (e.g., PET membrane) of the culture plate until a complete cell monolayer was formed on the 21st day. HBSS with a pH of 7.4 was used as the standard transport buffer in the experiment. To ensure the integrity and barrier function of the cell monolayer, the transepithelial electrical resistance (TEER) was measured before and after the transport experiment. The experiment required a TEER value of no less than 200Ω·cm2 to ensure the reliability of the experimental results. In the bidirectional permeability experiment, the test compound was diluted from a 10mM DMSO stock solution to a working concentration of 2μM or 10μM, and then added to the apical (A) chamber or the basolateral (B) chamber of the cell monolayer, respectively, and incubated for 2 hours at 37°C and 5% CO. After incubation, the solution samples of the receptor chamber were collected and quantitatively analyzed using high-performance liquid chromatography-tandem mass spectrometry (LC-MS / MS). Based on the sample concentration data, the apparent permeability coefficient (Papp) of each compound was calculated, including the permeability coefficient from apical to basolateral, Papp(A→B), and the permeability coefficient from basolateral to apical, Papp(B→A). The efflux ratio (ER = Papp(B→A) / Papp(A→B)) was calculated to assess whether the test compound was a P-glycoprotein (P-gp) substrate, thereby predicting its absorption and transport properties in vivo.

[0157] Caco-2 cell permeability studies revealed that the parent compound, THK01, exhibited low transmembrane permeability, with a Papp(A→B) value of 1.04×10 cm / s and a Papp(B→A) value of 0.36×10 cm / s, resulting in a calculated ER value of approximately 0.34. Partially esterified derivatives, such as THKE28, exhibited permeability similar to that of the parent compound, with Papp(A→B) values ​​of 1.05×10 cm / s, Papp(B→A) values ​​of 0.42×10 cm / s, and ER values ​​of approximately 0.40, maintaining the original low permeability. Other partial ester derivatives, such as THKE26, THKE29, THKE31, and THK32, displayed even lower permeability in this model. All tested compounds exhibited ER values ​​significantly below 2, suggesting that they are not strong substrates for major efflux transporters, such as P-gp, and are not readily actively pumped out of the cell. Although some esterified derivatives do not show a significant improvement in direct transmembrane permeability, the advantages of esterification as a prodrug design strategy are also reflected in the possibility of changing the drug absorption pathway, improving in vivo stability or achieving targeted release. Combined with subsequent studies such as metabolic stability, its comprehensive potential as a prodrug can be comprehensively evaluated.

[0158] Table 2

[0159]

[0160] Example 6

[0161] Liver microsome testing of THK01 esterified derivatives

[0162] This example aims to evaluate the metabolic stability of THK01 and its representative esterified derivatives (e.g., THKE04) in vitro in human liver microsomes (HLM) and to verify the enzymatic degradation properties of ester prodrugs. A specific concentration of the test compound (THK01 or THKE04) was mixed with human liver microsomal protein in phosphate buffer (final pH 7.4). After preincubation at 37°C, the metabolic reaction was initiated by the addition of the cofactor NADPH. At designated time points (e.g., 0, 5, 15, and 40 minutes), samples were removed and immediately terminated with ice-cold acetonitrile containing an internal standard. After centrifugation, the supernatant was analyzed by LC-MS / MS to quantify the residual test compound (or resulting parent drug) at each time point to assess its metabolic rate and half-life in liver microsomes. A control experiment was also conducted to investigate the chemical stability of THKE04 in enzyme-free PBS buffer (pH 7.4).

[0163] In vitro liver microsomal metabolic stability experiments demonstrated that the parent compound, THK01, exhibited high metabolic stability under human liver microsomal incubation conditions. In contrast, the ester prodrug, THKE04, was effectively metabolized in human liver microsomes, with its ester bond cleaved and the parent drug, THK01, successfully released. Further control experiments confirmed that THKE04 was stable in vitro in PBS buffer (pH 7.4), with the ester bond resistant to spontaneous hydrolysis. These results strongly demonstrate that the designed ester compound possesses the essential characteristics of a prodrug: chemical stability in an aqueous solution simulating physiological pH, yet effective cleavage in the presence of metabolic enzymes (such as liver esterases) to release the biologically active parent drug. This suggests that the esterification strategy is feasible for developing THK01 prodrugs, potentially enhancing the overall pharmacodynamics or pharmacokinetic performance of the parent drug by improving certain properties of the parent drug, such as solubility, absorption, or distribution.

[0164] Example 7

[0165] Evaluation of the anti-inflammatory activity of THK01 esterified derivatives in vitro

[0166] This example aims to evaluate the ability of a series of THK01 esterified derivatives to inhibit the production of nitric oxide (NO) in mouse macrophages RAW264.7 induced by lipopolysaccharide (LPS) to evaluate their in vitro anti-inflammatory activity. First, all test compounds were pre-evaluated for cytotoxicity by the sulforhodamine B (SRB) method to ensure that the cell viability was higher than 85% at the concentration used in the subsequent activity test. In the activity assay, RAW264.7 in the logarithmic growth phase were cultured in DMEM medium containing 10% fetal bovine serum, 5% CO, and 37°C. The cells were cultured at 3×10 4 Cells were seeded at a density of 100 μL per well in a 96-well plate 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 the cells to produce NO. 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. NO concentration was calculated using a sodium nitrite standard curve, and the inhibition rate was calculated. Indomethacin (50 μM) was used as a positive control.

[0167] Thirty-three THKO1 esterified derivatives of the present invention were screened and evaluated for their NO production inhibitory activity. The results (representative data are shown in the table below) showed that 20 compounds exhibited significant NO inhibitory activity, with some significantly outperforming the positive control, indomethacin (44.41% inhibition at 50 μM). Compounds THKE05, THKE06, THKE07, THKE08, THKE14, THKE17, THKE19, and THKE23 all exhibited NO production inhibition rates exceeding 50% at a concentration of 5 μM. THKE12, THKE15, THKE16, THKE18, THKE20, THKE24, and THKE25 exhibited stronger NO production inhibition at a lower concentration of 2.5 μM, exceeding 60%. THKE18 and THKE25 exhibited inhibition rates approaching 80%. THKE04 and THKE09 exhibited superior inhibitory potential, inhibiting NO production by greater than 50% at a concentration of 1.25 μM. These results demonstrate that specific esterification modification of the parent compound THK01 significantly enhances its in vitro anti-inflammatory activity (measured by inhibition of NO production), thereby identifying a group of highly active candidate compounds with promising development potential.

[0168] Table 3

[0169]

[0170]

[0171] Example 8

[0172] Evaluation of the anti-inflammatory activity of THK01 esterified derivatives in vivo

[0173] This example evaluates the in vivo therapeutic efficacy of a representative THK01 esterified derivative (exemplified by THKE28) 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 (mesalazine, 100 mg / kg), a parent compound group (THK01, 40 mg / kg), and a test esterified derivative group (THKE28, 40 mg / kg). Except for the normal control group, mice in all other groups were given a 3% DSS-containing aqueous solution ad libitum for 7 consecutive days to establish a colitis model. The drug-treated group received continuous oral administration 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, 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 blood samples were collected for routine blood tests. Serum levels of inflammatory factors such as TNFα, IL-1b, and IL-6 were measured using ELISA. Colon tissue was also collected for HE staining and immunohistochemistry to assess the extent of inflammatory cell infiltration and tissue damage.

[0174] In a DSS-induced colitis model, compared with the control group, THK01 and THKE28 groups showed significantly reduced colon length shortening and significantly decreased levels of pro-inflammatory cytokines such as TNFa, IL-1b, and IL-6, demonstrating efficacy superior to that of the active agent mesalazine. The esterified prodrug THKE28 was more effective than the parent drug THK01. These in vivo efficacy data strongly demonstrate that the THK01 esterified derivative THKE28 possesses potent anti-inflammatory activity in vivo, demonstrating that the esterified prodrug strategy successfully enhances the compound's therapeutic potential in inflammatory bowel disease models.

[0175] 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 esterified derivative, characterized in that: The chemical structure of the THK01 esterified derivative is shown in formula (I): Wherein, R1 is selected from H or O; R2 is selected from C1-C6 straight chain or branched alkyl, C3-C7 cycloalkyl, heterocyclic group, aryl, heteroaryl, -(CH2)nCOOH, and amino acid derivative group.

2. The method for preparing the THK01 esterified derivative according to claim 1, wherein: include, The compound having a structure as shown in formula (II) is obtained by condensing with an acyl chloride derivative as shown in formula (II), a carboxylic acid derivative as shown in formula (II), or an acid anhydride derivative as shown in formula (II); 3. The use of the THK01 esterified derivative according to claim 1, 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.

4. The use according to claim 3, 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.

5. 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.

6. A drug, characterized in that: The active ingredient is the THK01 esterified derivative according to claim 1.

7. A pharmaceutical composition, characterized in that: The invention comprises a therapeutically effective amount of the compound according to claim 1 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers or excipients.

8. The pharmaceutical composition according to claim 8, characterized in that The pharmaceutically acceptable carrier or excipient is selected from fillers, diluents, binders, wetting agents, disintegrants, lubricants, solubilizers, buffers, preservatives, sweeteners, flavors, colorants, thickeners, stabilizers and combinations thereof.

9. The pharmaceutical composition according to claim 8 or 9, characterized in that The dosage form of the pharmaceutical composition is selected from tablets, capsules, granules, powders, injections, solutions, syrups, suspensions or suppositories.

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