An indometacin derivative containing 2-amino-1,3,4-thiadiazole, its preparation method and application

Abstract

The application belongs to the technical field of anti-inflammatory drug development, and discloses an indometacin derivative containing 2-amino-1,3,4-thiadiazole and preparation and application thereof. The derivative is a compound with the structure shown in formula 2 or a pharmaceutically acceptable salt thereof. The application first designs and successfully synthesizes a novel indometacin derivative containing 2-amino-1,3,4-thiadiazole, and the structure is characterized. The preparation method of the indometacin derivative containing 2-amino-1,3,4-thiadiazole is simple, convenient to operate, and can quickly synthesize the compounds. Research shows that the indometacin derivative containing 2-amino-1,3,4-thiadiazole has good anti-inflammatory activity and good application prospect in inflammatory diseases.

Description

Technical Field

[0001] This invention belongs to the field of anti-inflammatory drug development technology, specifically relating to an indomethacin derivative containing 2-amino-1,3,4-thiadiazole and its preparation and application. Background Technology

[0002] Indomethacin, chemically named 1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indole-3-acetic acid, CAS number: 53-86-1.

[0003]

[0004] Inflammation is a common clinical disease worldwide, seriously threatening human health. It has become a major challenge for public health systems. Although many scientists are dedicated to developing new treatments for various types of inflammation, it remains a global clinical focus and a difficult problem.

[0005] Indomethacin was approved for marketing in the United States in June 1965 for the treatment of inflammation and fever. As a nonsteroidal anti-inflammatory drug, indomethacin can reduce the synthesis of prostaglandins by inhibiting cyclooxygenase 2 (COX-2) produced by tissues in response to inflammation, thereby playing an anti-inflammatory, antipyretic, and analgesic role.

[0006] Indomethacin belongs to the arylaceous acid class of nonsteroidal anti-inflammatory drugs (NSAIDs) and possesses analgesic and antipyretic properties. Its pharmacological effects are achieved by inhibiting the synthesis of pain-, fever-, and inflammation-related factors. It effectively inhibits the synthesis of prostaglandins (PGs) in the body, producing an antipyretic effect 10–20 times stronger than acetaminophen (paracetamol). In clinical trials, indomethacin has been shown to effectively relieve pain and reduce fever, swelling, redness, and tenderness in acute gouty arthritis. Due to its pharmacological effects, the use of indomethacin is associated with an increased risk of serious cardiovascular thrombotic events, including myocardial infarction and stroke, as well as gastrointestinal effects such as bleeding, ulceration, and gastric or intestinal perforation. Summary of the Invention

[0007] In order to overcome the shortcomings and deficiencies of the prior art, the primary objective of this invention is to provide an indomethacin derivative containing 2-amino-1,3,4-thiadiazole, which can enhance the anti-inflammatory activity of indomethacin and reduce gastrointestinal side effects, that is, it has excellent anti-inflammatory activity and few side effects.

[0008] Another objective of this invention is to provide a method for preparing the above-mentioned indomethacin derivative containing 2-amino-1,3,4-thiadiazole. This method modifies the structure of indomethacin and utilizes the amide condensation of the terminal free carboxyl group of indomethacin with a series of active small molecules of thiadiazole to form a novel pharmaceutical compound. It is expected that the adverse reactions can be reduced without affecting its anti-inflammatory activity or even enhancing its anti-inflammatory activity.

[0009] Another object of the present invention is to provide the use of the above-mentioned indomethacin derivatives containing 2-amino-1,3,4-thiadiazole.

[0010] The objective of this invention is achieved through the following technical solution:

[0011] An indomethacin derivative containing 2-amino-1,3,4-thiadiazole, said derivative being a compound with the structure shown in Formula 2 or a pharmaceutically acceptable salt thereof:

[0012]

[0013] Wherein, R is H, 2-CH3, 3-CH3, 4-CH3, 2-CF3, 3-CF3, 4-CF3, 3-NO2, 4-NO2, 2-OCH3, 3-OCH3, 4-OCH3, 3-F, 4-F, 2-Cl, 3-Cl, or 4-Cl.

[0014] The specific functional groups of the above compounds are summarized in Table 1:

[0015] Table 1. Compound Numbers and Specific Functions

[0016]

[0017] The pharmaceutically acceptable salt is a salt formed by a compound with the structure shown in Formula 2 and hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, fumaric acid, maleic acid, oxalic acid, malonic acid, succinic acid, citric acid, malic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, glutamic acid, or aspartic acid.

[0018] The above-mentioned indomethacin derivatives containing 2-amino-1,3,4-thiadiazole are characterized in that:

[0019] The pharmaceutically acceptable salt has the following structural formula:

[0020]

[0021] The above-mentioned method for preparing indomethacin derivatives containing 2-amino-1,3,4-thiadiazole includes the following steps:

[0022] (1) Thiourea is reacted with benzoic acid with different substituents to obtain an intermediate; the structural formula of the benzoic acid with different substituents is shown in Formula 3, and the structural formula of the intermediate is shown in Formula 4.

[0023]

[0024] Wherein, R is H, 2-CH3, 3-CH3, 4-CH3, 2-CF3, 3-CF3, 4-CF3, 3-NO2, 4-NO2, 2-OCH3, 3-OCH3, 4-OCH3, 3-F, 4-F, 2-Cl, 3-Cl or 4-Cl;

[0025] (2) The intermediate obtained in step (1) was used as a reaction raw material with indomethacin, and N,N-dimethylformamide was used as a solvent. 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) and N,N-diisopropylethylamine (DIPEA) were added to carry out a condensation reaction to obtain an indomethacin derivative containing 2-amino-1,3,4-thiadiazole with the structure shown in Formula 2.

[0026] The above preparation method is carried out according to the following equation:

[0027]

[0028] The molar ratio of thiourea to benzoic acid with different substituents in step (1) is 1:1.1;

[0029] The specific operation of the reaction is as follows: thiourea and benzoic acid with different substituents are dissolved in phosphorus oxychloride, and the reaction is stirred at 75°C for 0.5 h. The starting material is monitored by LCMS. The reaction is terminated when the starting material is ≤1%. After cooling to room temperature, ice water is added dropwise to the reaction solution, and the reaction is carried out at 110°C for 4 h. The reaction is terminated and cooled to room temperature. The pH is then adjusted to 8-9 with NaOH solution, and a solid is precipitated to obtain the intermediate.

[0030] The molar ratio of indomethacin to the intermediate in step (2) is 1:1.1; the molar ratio of indomethacin to N,N-diisopropylethylamine is 1:1.5; the molar ratio of indomethacin to 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate is 1:2; and the condensation reaction is carried out at 25°C for 1-2 hours.

[0031] The specific functional groups of the above intermediates (A1-A17) are summarized in Table 1:

[0032] Table 2. Intermediate (A1-A17) Numbers and Specific Groups

[0033]

[0034]

[0035] The present invention has the following advantages and beneficial effects:

[0036] (1) The indomethacin derivatives provided by this invention are a new type of compound that has not been reported before. This invention designs and successfully synthesizes these compounds for the first time, and at the same time characterizes their structures.

[0037] (2) The preparation method of the indomethacin derivative containing 2-amino-1,3,4-thiadiazole of the present invention is simple and convenient to operate, and can rapidly synthesize these compounds.

[0038] (3) Through extensive and in-depth research, this invention has synthesized a large number of novel indomethacin derivatives containing 2-amino-1,3,4-thiadiazole with anti-inflammatory activity and screened for anti-inflammatory activity. For the first time, it was found that this type of compound has good in vitro anti-inflammatory activity and is suitable as a novel anti-inflammatory drug for the prevention and treatment of inflammatory diseases. Attached Figure Description

[0039] Figure 1 This is the NMR spectrum of compound 1.

[0040] Figure 2 This is the NMR spectrum of compound 2.

[0041] Figure 3 This is the NMR spectrum of compound 3.

[0042] Figure 4 This is the NMR spectrum of compound 4.

[0043] Figure 5 This is the NMR spectrum of compound 5.

[0044] Figure 6 This is the NMR spectrum of compound 6.

[0045] Figure 7 This is the NMR spectrum of compound 7.

[0046] Figure 8 This is the NMR spectrum of compound 8.

[0047] Figure 9 This is the NMR spectrum of compound 9.

[0048] Figure 10 This is the NMR spectrum of compound 10.

[0049] Figure 11 This is the NMR spectrum of compound 13.

[0050] Figure 12This is the NMR spectrum of compound 14.

[0051] Figure 13 This is the NMR spectrum of compound 15.

[0052] Figure 14 This is the NMR spectrum of compound 16.

[0053] Figure 15 This is the NMR spectrum of compound 17.

[0054] Figure 16 The MTT assay was used to determine the cytotoxicity of indomethacin and its derivatives on RAW264.7 cells.

[0055] Figure 17 The inhibitory effect of indomethacin and its derivatives on NO content in RAW264.7 cells was determined using the Griess method.

[0056] Figure 18 The inhibitory effect of indomethacin and its derivatives on COX-2 protein expression in RAW264.7 cells was analyzed using Western blot. Detailed Implementation

[0057] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0058] The method for preparing the indomethacin derivative containing 2-amino-1,3,4-thiadiazole according to the present invention comprises the following steps:

[0059] (1) Thiourea was reacted with benzoic acid with different substituents as shown in Formula 3 to obtain intermediate I with the structure shown in Formula 4.

[0060] (2) The intermediate I obtained in step (1) was reacted with indomethacin to obtain an indomethacin derivative containing 2-amino-1,3,4-thiadiazole as shown in Formula 2.

[0061] The intermediate I has the structural formula 4:

[0062]

[0063] The preferred ratio of thiourea to benzoic acid with different substituents in step (1) is 1:1.1. The preferred specific operation of the reaction is as follows: thiourea and benzoic acid with different substituents (structure shown in Formula 3) are dissolved in phosphorus oxychloride, stirred at 75°C for 0.5 h, the starting material is monitored by LCMS, the reaction is terminated when the starting material is ≤1%, cooled to room temperature, and 3V ice water is slowly added dropwise to the reaction solution, and the reaction is carried out at 110°C for 4 h; the reaction is terminated and cooled to room temperature, and the pH is adjusted to 8-9 with NaOH solution, and a solid is precipitated to obtain intermediate I with the structure shown in Formula 4.

[0064] The preferred specific operation for the reaction described in step (2) is as follows:

[0065] Under ice bath conditions, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) and N,N-diisopropylethylamine (DIPEA) were added dropwise to indomethacin. After 0.5 h, intermediate I was added dropwise to the reaction solution. The reaction conditions were 25 °C for 1–2 h.

[0066] The above reaction preferably uses N,N-dimethylformamide as a solvent, the molar ratio of indomethacin to intermediate I is preferably 1:1.1, the molar ratio of indomethacin to DIPEA is preferably 1:1.5, the molar ratio of indomethacin to HATU is preferably 1:2, and the reaction conditions are preferably 25°C for 1 to 2 hours.

[0067] The specific synthesis route is shown in the following formula:

[0068]

[0069] In the examples, all benzoic acid derivatives and other reagents were commercially available.

[0070] Example 1: Preparation of 1,3,4-thiadiazole intermediate A1:

[0071] 1.0 g (10.9 mmol) of thiourea was dissolved in 10 mL of phosphorus oxychloride, and then benzoyl chloride was slowly added. The mixture was stirred at 75 °C for 0.5 h, and the starting material was monitored by LC-MS. The reaction was terminated when the starting material concentration was ≤1%. After cooling to room temperature, 30 mL of ice water was slowly added dropwise to the reaction solution, and the temperature was raised to 110 °C for 4 h. After 4 h, the reaction was terminated and cooled to room temperature. The pH was then adjusted to 8–9 with NaOH solution, and the precipitated solid was 2-amino-5-phenyl-1,3,4-thiadiazole (intermediate A1).

[0072] Following the method of Example 1 (with identical molar amounts of reactants, reaction conditions, purification, etc.), intermediates A2-A17 were obtained, and the structures of each intermediate are shown in Table 2.

[0073] Example 2 Synthesis of 2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-(5-phenyl-1,3,4-thiadiazol-2-yl)acetamide (Compound 1)

[0074] In a single-bore round-bottom flask, indomethacin (1.0 g, 2.79 mmol) was dissolved in 10 mL of N,N-dimethylformamide. DIPEA (0.72 g, 5.58 mmol) was added to the flask, followed by HATU (1.59 g, 4.19 mmol) under ice bath conditions. After 30 minutes, 5-phenyl-1,3,4-thiadiazole-2-amino (intermediate A1) (0.54 g, 3.07 mmol) was added to the system. The mixture was then transferred to room temperature and stirred for 2 hours. The reaction was monitored by TLC. Once the reaction was complete, the system was... 100 ml each of water and ethyl acetate were added sequentially, then transferred to a separatory funnel and shaken. The mixture was allowed to stand until separation. The organic phase was washed twice with saturated sodium chloride aqueous solution (15% w / v) and dried over anhydrous sodium sulfate. The organic phase was collected and evaporated to dryness to obtain the crude product. The crude product was redissolved in dichloromethane, and 2 g of 100-200 mesh silica gel was added and thoroughly mixed. The mixture was purified by column chromatography to obtain the product 2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-(5-phenyl-1,3,4-thiadiazol-2-yl)acetamide, with a yield of 51.3%. The NMR spectrum of the product is shown below. Figure 1 As shown, 1 H NMR(600MHz,DMSO-d6)δ12.99(s,1H),7.98–7.86(m,2H),7.73–7.68(m,2H),7.67–7.62(m,2H),7.56–7.46(m,3H) ,7.20(d,J=2.6Hz,1H),6.94(d,J=9.0Hz,1H),6.73(dd,J=9.0,2.6Hz,1H),3.98(s,2H),3.77(s,3H),2.31(s,3H).

[0075] Example 3 Synthesis of 2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-(5-(o-tolyl)-1,3,4-thiadiazol-2-yl)acetamide (Compound 2)

[0076] In a single-bore round-bottom flask, indomethacin (1.0 g, 2.79 mmol) was dissolved in 10 mL of N,N-dimethylformamide. DIPEA (0.72 g, 5.58 mmol) was added to the flask, followed by HATU (1.59 g, 4.19 mmol) under ice bath conditions. After 30 minutes, 2-amino-5-(o-toluene)-1,3,4-thiadiazole (intermediate A2) (0.59 g, 3.07 mmol) was added to the system. The mixture was then transferred to room temperature and stirred for 2 hours. The reaction was monitored by TLC. After the reaction was complete, the solution was added to the system... Add 100 ml each of water and ethyl acetate sequentially, then transfer to a separatory funnel and shake. Allow to stand until separation. Wash the organic phase twice with saturated sodium chloride aqueous solution (15% w / v) and dry with anhydrous sodium sulfate. Collect the organic phase and evaporate to dryness to obtain the crude product. Redissolve in dichloromethane, add 2 g of 100-200 mesh silica gel, mix thoroughly, and purify by column chromatography to obtain the product 2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-(5-(o-tolyl)-1,3,4-thiadiazol-2-yl)acetamide, yield 47.7%. The NMR spectrum of the product is shown below. Figure 2 As shown, 1 H NMR(600MHz,DMSO-d6)δ12.97(s,1H),7.72–7.68(m,2H),7.65(dd,J=8.4,1.9Hz,3H),7.45–7.36(m,2H),7.34(td,J=7.3,2.0Hz,1 H),7.21(d,J=2.5Hz,1H),6.94(d,J=9.0Hz,1H),6.73(dd,J=9.0,2.6Hz,1H),3.99(s,2H),3.77(s,3H),2.49(s,3H),2.31(s,3H).

[0077] Example 4 Synthesis of 2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-(5-(m-tolyl)-1,3,4-thiadiazol-2-yl)acetamide (Compound 3)

[0078] In a single-bore round-bottom flask, indomethacin (1.0 g, 2.79 mmol) was dissolved in 10 mL of N,N-dimethylformamide. DIPEA (0.72 g, 5.58 mmol) was added to the flask, followed by the partial addition of HATU (1.59 g, 4.19 mmol) under ice bath conditions. After 30 minutes, 2-amino-5-(m-toluene)-1,3,4-thiadiazole (intermediate A3) (0.59 g, 3.07 mmol) was added to the system. The mixture was then transferred to room temperature and stirred for 2 hours. The reaction was monitored by TLC. Once the reaction was complete, the system was... 100 ml each of water and ethyl acetate were added sequentially, then transferred to a separatory funnel and shaken. The mixture was allowed to stand until separation. The organic phase was washed twice with saturated sodium chloride aqueous solution (15% w / v) and dried over anhydrous sodium sulfate. The organic phase was collected and evaporated to dryness to obtain the crude product. The crude product was redissolved in dichloromethane, and 2 g of 100-200 mesh silica gel was added and thoroughly mixed. The mixture was purified by column chromatography to obtain the product 2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-(5-(m-tolyl)-1,3,4-thiadiazol-2-yl)acetamide, with a yield of 64.5%. The NMR spectrum of the product is shown below. Figure 3 As shown, 1 H NMR (600MHz, DMSO-d6) δ13.0(s,1H),7.8(d,J=1.7Hz,1H),7.7–7.7(m,2H),7.7–7.6(m,2H),7.4(t,J=7.7Hz,1H),7.3(d,J=7.5 Hz,1H),7.2(d,J=2.5Hz,1H),6.9(d,J=9.0Hz,1H),6.7(dd,J=9.0,2.6Hz,1H),4.0(s,2H),3.8(s,3H),2.4(s,3H),2.3(s,3H).

[0079] Example 5 Synthesis of 2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-(5-(p-tolyl)-1,3,4-thiadiazol-2-yl)acetamide (Compound 4)

[0080] In a single-bore round-bottom flask, indomethacin (1.0 g, 2.79 mmol) was dissolved in 10 mL of N,N-dimethylformamide. DIPEA (0.72 g, 5.58 mmol) was added to the flask, followed by the partial addition of HATU (1.59 g, 4.19 mmol) under ice bath conditions. After 30 minutes, 2-amino-5-(p-toluene)-1,3,4-thiadiazole (intermediate A4) (0.59 g, 3.07 mmol) was added to the system. The mixture was then transferred to room temperature and stirred for 2 hours. The reaction was monitored by TLC. Once the reaction was complete, the system was... 100 ml each of water and ethyl acetate were added sequentially, then transferred to a separatory funnel and shaken. The mixture was allowed to stand until separation. The organic phase was washed twice with saturated sodium chloride aqueous solution (15% w / v) and dried over anhydrous sodium sulfate. The organic phase was collected and evaporated to dryness to obtain the crude product. The crude product was redissolved in dichloromethane, and 2 g of 100-200 mesh silica gel was added and thoroughly mixed. The mixture was purified by column chromatography to obtain the product 2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-(5-(p-tolyl)-1,3,4-thiadiazol-2-yl)acetamide, with a yield of 64.1%. The NMR spectrum of the product is shown below. Figure 4 As shown, 1 H NMR (600MHz, DMSO-d6) δ12.94(s,1H),7.88–7.75(m,2H),7.73–7.59(m,4H),7.33(d,J=8.0Hz,2H),7.19(d,J=2. 5Hz,1H),6.93(d,J=8.9Hz,1H),6.73(dd,J=9.0,2.6Hz,1H),3.97(s,2H),3.76(s,3H),2.36(s,3H),2.30(s,3H).

[0081] Example 6 Synthesis of 2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-(5-(2-(trifluoromethyl)phenyl)-1,3,4-thiadiazol-2-yl)acetamide (Compound 5)

[0082] In a single-bore round-bottom flask, indomethacin (1.0 g, 2.79 mmol) was dissolved in 10 mL of N,N-dimethylformamide. DIPEA (0.72 g, 5.58 mmol) was added to the flask, followed by the partial addition of HATU (1.59 g, 4.19 mmol) under ice bath conditions. After 30 minutes, 5-(2-(trifluoromethyl)phenyl)-1,3,4-thiadiazol-2-amine (intermediate A5) (0.75 g, 3.07 mmol) was added to the system. The mixture was then transferred to room temperature and stirred for 2 hours. The reaction was monitored by TLC. After the reaction was complete, the system was... 100 ml each of water and ethyl acetate were added sequentially, then transferred to a separatory funnel and shaken. The mixture was allowed to stand until separation. The organic phase was washed twice with saturated sodium chloride aqueous solution (15% w / v) and dried over anhydrous sodium sulfate. The organic phase was collected and evaporated to dryness to obtain the crude product. The crude product was redissolved in dichloromethane, and 2 g of 100-200 mesh silica gel was added and thoroughly mixed. The mixture was purified by column chromatography to obtain the product 2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-(5-(2-(trifluoromethyl)phenyl)-1,3,4-thiadiazol-2-yl)acetamide, with a yield of 54.1%. The NMR spectrum of the product is shown below. Figure 5 As shown, 1 H NMR (600MHz, DMSO-d6) δ13.08(s,1H),7.96(dd,J=7.8,1.3Hz,1H),7.83(td,J=7.6,1.4Hz,1H),7.79(t,J=7.6Hz,1H),7.75(dd,J=7.6,1.5Hz,1H),7. 72–7.69(m,2H),7.67–7.63(m,2H),7.20(d,J=2.6Hz,1H),6.94(d,J=9.0H z,1H),6.73(dd,J=9.0,2.6Hz,1H),4.01(s,2H),3.77(s,3H),2.31(s,3H).

[0083] Example 7 Synthesis of 2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-(5-(3-(trifluoromethyl)phenyl)-1,3,4-thiadiazol-2-yl)acetamide (Compound 6)

[0084] In a single-bore round-bottom flask, indomethacin (1.0 g, 2.79 mmol) was dissolved in 10 mL of N,N-dimethylformamide. DIPEA (0.72 g, 5.58 mmol) was added to the flask, followed by the partial addition of HATU (1.59 g, 4.19 mmol) under ice bath conditions. After 30 minutes, 5-(3-(trifluoromethyl)phenyl)-1,3,4-thiadiazol-2-amine (intermediate A6) (0.75 g, 3.07 mmol) was added to the system. The mixture was then transferred to room temperature and stirred for 2 hours. The reaction was monitored by TLC. After the reaction was complete, the system was... 100 ml each of water and ethyl acetate were added sequentially, then transferred to a separatory funnel and shaken. The mixture was allowed to stand until separation. The organic phase was washed twice with saturated sodium chloride aqueous solution (15% w / v) and dried over anhydrous sodium sulfate. The organic phase was collected and evaporated to dryness to obtain the crude product. The crude product was redissolved in dichloromethane, and 2 g of 100-200 mesh silica gel was added and thoroughly mixed. The mixture was purified by column chromatography to obtain the product 2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-(5-(3-(trifluoromethyl)phenyl)-1,3,4-thiadiazol-2-yl)acetamide, with a yield of 57.3%. The NMR spectrum of the product is shown below. Figure 6 As shown, 1 H NMR(600MHz,DMSO-d6)δ13.1(s,1H),8.3–8.1(m,2H),7.9–7.8(m,1H),7.8–7.7(m,1H),7.7–7.7(m,2H),7.7 –7.6(m,2H),7.2(d,J=2.6Hz,1H),6.9(d,J=9.0Hz,1H),6.7(dd,J=9.0,2.6Hz,1H),4.0(s,2H),3.8(s,3H).

[0085] Example 8 Synthesis of 2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-(5-(4-(trifluoromethyl)phenyl)-1,3,4-thiadiazol-2-yl)acetamide (Compound 7)

[0086] In a single-bore round-bottom flask, indomethacin (1.0 g, 2.79 mmol) was dissolved in 10 mL of N,N-dimethylformamide. DIPEA (0.72 g, 5.58 mmol) was added to the flask, followed by the partial addition of HATU (1.59 g, 4.19 mmol) under ice bath conditions. After 30 minutes, 5-(4-(trifluoromethyl)phenyl)-1,3,4-thiadiazol-2-amine (intermediate A7) (0.75 g, 3.07 mmol) was added to the system. The mixture was then transferred to room temperature and stirred for 2 hours. The reaction was monitored by TLC. Once the reaction was complete, the system was... 100 ml each of water and ethyl acetate were added sequentially, then transferred to a separatory funnel and shaken. The mixture was allowed to stand until separation. The organic phase was washed twice with saturated sodium chloride aqueous solution (15% w / v) and dried over anhydrous sodium sulfate. The organic phase was collected and evaporated to dryness to obtain the crude product. The crude product was redissolved in dichloromethane, and 2 g of 100-200 mesh silica gel was added and thoroughly mixed. The mixture was purified by column chromatography to obtain the product 2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-(5-(4-(trifluoromethyl)phenyl)-1,3,4-thiadiazol-2-yl)acetamide, with a yield of 72.3%. The NMR spectrum of the product is shown below. Figure 7 As shown, 1 H NMR (600MHz, DMSO-d6) δ13.1(s,1H),8.2(d,J=8.2Hz,2H),7.9(d,J=8.3Hz,2H),7.7–7.7(m,2H),7.7–7.6(m,2 H),7.2(d,J=2.5Hz,1H),6.9(d,J=9.0Hz,1H),6.7(dd,J=9.0,2.5Hz,1H),4.0(s,2H),3.8(s,3H),2.3(s,3H).

[0087] Example 9 Synthesis of 2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-(5-(3-nitrophenyl)-1,3,4-thiadiazol-2-yl)acetamide (Compound 8)

[0088] In a single-bore round-bottom flask, indomethacin (1.0 g, 2.79 mmol) was dissolved in 10 mL of N,N-dimethylformamide. DIPEA (0.72 g, 5.58 mmol) was added to the flask, followed by the partial addition of HATU (1.59 g, 4.19 mmol) under ice bath conditions. After 30 minutes, 5-(3-nitrophenyl)-1,3,4-thiadiazole-2-amine (intermediate A8) (0.68 g, 3.07 mmol) was added to the system. The mixture was then transferred to room temperature and stirred for 2 hours. The reaction was monitored by TLC. Once the reaction was complete, the system was... 100 ml each of water and ethyl acetate were added sequentially, then transferred to a separatory funnel and shaken. The mixture was allowed to stand until separation. The organic phase was washed twice with saturated sodium chloride aqueous solution (15% w / v) and dried over anhydrous sodium sulfate. The organic phase was collected and evaporated to dryness to obtain the crude product. The crude product was redissolved in dichloromethane, and 2 g of 100-200 mesh silica gel was added and thoroughly mixed. The mixture was purified by column chromatography to obtain the product 2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-(5-(3-nitrophenyl)-1,3,4-thiadiazol-2-yl)acetamide, with a yield of 62.3%. The NMR spectrum of the product is shown below. Figure 8 As shown, 1 H NMR (600MHz, DMSO-d6) δ13.13(s,1H),8.68(t,J=2.0Hz,1H),8.34(dd,J=8.0,2.0Hz,2H),7.82(t,J=8.0Hz,1H),7.72–7.68(m,2H), 7.67–7.62(m,2H),7.19(d,J=2.5Hz,1H),6.93(d,J=9.0Hz,1H),6.73(dd,J=9.0,2.6Hz,1H),3.99(s,2H),3.76(s,3H),2.31(s,3H).

[0089] Example 10 Synthesis of 2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-(5-(4-nitrophenyl)-1,3,4-thiadiazol-2-yl)acetamide (Compound 9)

[0090] In a single-bore round-bottom flask, indomethacin (1.0 g, 2.79 mmol) was dissolved in 10 mL of N,N-dimethylformamide. DIPEA (0.72 g, 5.58 mmol) was added to the flask, followed by the partial addition of HATU (1.59 g, 4.19 mmol) under ice bath conditions. After 30 minutes, 5-(4-nitrophenyl)-1,3,4-thiadiazol-2-amine (intermediate A9) (0.68 g, 3.07 mmol) was added to the system. The mixture was then transferred to room temperature and stirred for 2 hours. The reaction was monitored by TLC. Once the reaction was complete, the system was... 100 ml each of water and ethyl acetate were added sequentially, then transferred to a separatory funnel and shaken. The mixture was allowed to stand until separation. The organic phase was washed twice with saturated sodium chloride aqueous solution (15% w / v) and dried over anhydrous sodium sulfate. The organic phase was collected and evaporated to dryness to obtain the crude product. The crude product was redissolved in dichloromethane, and 2 g of 100-200 mesh silica gel was added and thoroughly mixed. The mixture was purified by column chromatography to obtain the product 2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-(5-(4-nitrophenyl)-1,3,4-thiadiazol-2-yl)acetamide, with a yield of 74.1%. The NMR spectrum of the product is shown below. Figure 9 As shown, 1 H NMR(600MHz,DMSO-d6)δ13.16(s,1H),8.41–8.31(m,2H),8.28–8.12(m,2H),7.72–7.68(m,2H),7.68–7.63(m,2H) ,7.19(d,J=2.5Hz,1H),6.93(d,J=9.0Hz,1H),6.73(dd,J=9.0,2.5Hz,1H),4.00(s,2H),3.76(s,3H),2.31(s,3H).

[0091] Example 11 Synthesis of 2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-(5-(2-methoxyphenyl)-1,3,4-thiadiazol-2-yl)acetamide (Compound 10)

[0092] In a single-bore round-bottom flask, indomethacin (1.0 g, 2.79 mmol) was dissolved in 10 mL of N,N-dimethylformamide. DIPEA (0.72 g, 5.58 mmol) was added to the flask, followed by the partial addition of HATU (1.59 g, 4.19 mmol) under ice bath conditions. After 30 minutes, 5-(2-methoxyphenyl)-1,3,4-thiadiazol-2-amine (intermediate A10) (0.64 g, 3.07 mmol) was added to the system. The mixture was then transferred to room temperature and stirred for 2 hours. The reaction was monitored by TLC. Once the reaction was complete, the contents were... 100 ml each of water and ethyl acetate were added sequentially to the mixture, which was then transferred to a separatory funnel and shaken. The mixture was allowed to stand until it separated into layers. The organic phase was washed twice with a saturated sodium chloride aqueous solution (15% w / v) and dried over anhydrous sodium sulfate. The organic phase was collected and evaporated to dryness to obtain the crude product. The crude product was redissolved in dichloromethane, and 2 g of 100-200 mesh silica gel was added and thoroughly mixed. The mixture was purified by column chromatography to obtain the product 2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-(5-(2-methoxyphenyl)-1,3,4-thiadiazol-2-yl)acetamide, with a yield of 75.8%. The NMR spectrum of the product is shown below. Figure 10 As shown, 1 H NMR (600MHz, DMSO-d6) δ12.8(s,1H),8.3(dd,J=7.9,1.7Hz,1H),7.7–7.7(m,2H),7.7–7.6(m,2H),7.5(ddd,J=8.9,7.3,1.8Hz,1H),7.3(dd,J=8.4,1.0 Hz,1H),7.2(d,J=2.6Hz,1H),7.1(td,J=7.5,1.0Hz,1H),6.9(d,J=9.0Hz,1 H),6.7(dd,J=9.0,2.6Hz,1H),4.0(s,5H),3.8(s,3H),2.3(d,J=5.8Hz,3H).

[0093] Example 12 Synthesis of 2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-(5-(3-methoxyphenyl)-1,3,4-thiadiazol-2-yl)acetamide (Compound 11)

[0094] In a single-bore round-bottom flask, indomethacin (1.0 g, 2.79 mmol) was dissolved in 10 mL of N,N-dimethylformamide. DIPEA (0.72 g, 5.58 mmol) was added to the flask, followed by the partial addition of HATU (1.59 g, 4.19 mmol) under ice bath conditions. After 30 minutes, 5-(3-methoxyphenyl)-1,3,4-thiadiazole-2-amine (intermediate A11) (0.64 g, 3.07 mmol) was added to the system. The mixture was then transferred to room temperature and stirred for 2 hours. The reaction was monitored by TLC. Once the reaction was complete, the contents were... 100 ml each of water and ethyl acetate were added sequentially to the mixture, which was then transferred to a separatory funnel and shaken. The mixture was allowed to stand until it separated into layers. The organic phase was washed twice with a saturated sodium chloride aqueous solution (15% w / v) and dried over anhydrous sodium sulfate. The organic phase was collected and evaporated to dryness to obtain the crude product. The crude product was redissolved in dichloromethane, and 2 g of 100-200 mesh silica gel was added and mixed thoroughly. The product 2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-(5-(3-methoxyphenyl)-1,3,4-thiadiazol-2-yl)acetamide was purified by column chromatography with a yield of 66.8%.

[0095] Example 13 Synthesis of 2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-(5-(4-methoxyphenyl)-1,3,4-thiadiazol-2-yl)acetamide (Compound 12)

[0096] In a single-bore round-bottom flask, indomethacin (1.0 g, 2.79 mmol) was dissolved in 10 mL of N,N-dimethylformamide. DIPEA (0.72 g, 5.58 mmol) was added to the flask, followed by the partial addition of HATU (1.59 g, 4.19 mmol) under ice bath conditions. After 30 minutes, 5-(4-methoxyphenyl)-1,3,4-thiadiazol-2-amine (intermediate A12) (0.64 g, 3.07 mmol) was added to the system. The mixture was then transferred to room temperature and stirred for 2 hours. The reaction was monitored by TLC. Once the reaction was complete, the contents were... 100 ml each of water and ethyl acetate were added sequentially to the mixture, which was then transferred to a separatory funnel and shaken. The mixture was allowed to stand until it separated into layers. The organic phase was washed twice with a saturated sodium chloride aqueous solution (15% w / v) and dried over anhydrous sodium sulfate. The organic phase was collected and evaporated to dryness to obtain the crude product. The crude product was redissolved in dichloromethane, and 2 g of 100-200 mesh silica gel was added and mixed thoroughly. The product 2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-(5-(4-methoxyphenyl)-1,3,4-thiadiazol-2-yl)acetamide was purified by column chromatography with a yield of 81.7%.

[0097] Example 14 Synthesis of 2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-(5-(3-fluorophenyl)-1,3,4-thiadiazol-2-yl)acetamide (Compound 13)

[0098] In a single-bore round-bottom flask, indomethacin (1.0 g, 2.79 mmol) was dissolved in 10 mL of N,N-dimethylformamide. DIPEA (0.72 g, 5.58 mmol) was added to the flask, followed by the partial addition of HATU (1.59 g, 4.19 mmol) under ice bath conditions. After 30 minutes, 5-m-fluorophenyl-2-amino-1,3,4-thiadiazole (intermediate A13) (0.60 g, 3.07 mmol) was added to the system. The mixture was then transferred to room temperature and stirred for 2 hours. The reaction was monitored by TLC. Once the reaction was complete, the system was... 100 ml each of water and ethyl acetate were added sequentially, then transferred to a separatory funnel and shaken. The mixture was allowed to stand until separation. The organic phase was washed twice with saturated sodium chloride aqueous solution (15% w / v) and dried over anhydrous sodium sulfate. The organic phase was collected and evaporated to dryness to obtain the crude product. The crude product was redissolved in dichloromethane, and 2 g of 100-200 mesh silica gel was added and thoroughly mixed. The mixture was purified by column chromatography to obtain the product 2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-(5-(3-fluorophenyl)-1,3,4-thiadiazol-2-yl)acetamide, with a yield of 68.7%. The NMR spectrum of the product is shown below. Figure 11 As shown, 1 H NMR(400MHz, DMSO-d6)δ13.1(s,1H),7.8–7.7(m,2H),7.7–7.7(m,2H),7.7–7.6(m,2H),7.6(td,J=8.2,6.0Hz,1H),7.4(tdd,J= 8.5,2.6,1.0Hz,1H),7.2(d,J=2.6Hz,1H),6.9(d,J=9.0Hz,1H),6.7(dd,J=9.0,2.6Hz,1H),4.0(s,2H),3.8(s,3H),2.3(s,3H).

[0099] Example 15 Synthesis of 2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-(5-(4-fluorophenyl)-1,3,4-thiadiazol-2-yl)acetamide (Compound 14)

[0100] In a single-bore round-bottom flask, indomethacin (1.0 g, 2.79 mmol) was dissolved in 10 mL of N,N-dimethylformamide. DIPEA (0.72 g, 5.58 mmol) was added to the flask, followed by the partial addition of HATU (1.59 g, 4.19 mmol) under ice bath conditions. After 30 minutes, 5-p-fluorophenyl-2-amino-1,3,4-thiadiazole (intermediate A14) (0.60 g, 3.07 mmol) was added to the system. The mixture was then transferred to room temperature and stirred for 2 hours. The reaction was monitored by TLC. Once the reaction was complete, the system was... 100 ml each of water and ethyl acetate were added sequentially, then transferred to a separatory funnel and shaken. The mixture was allowed to stand until separation. The organic phase was washed twice with saturated sodium chloride aqueous solution (15% w / v) and dried over anhydrous sodium sulfate. The organic phase was collected and evaporated to dryness to obtain the crude product. The crude product was redissolved in dichloromethane, and 2 g of 100-200 mesh silica gel was added and thoroughly mixed. The mixture was purified by column chromatography to obtain the product 2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-(5-(4-fluorophenyl)-1,3,4-thiadiazol-2-yl)acetamide, with a yield of 66.6%. The NMR spectrum of the product is shown below. Figure 12 As shown, 1 H NMR (400MHz, DMSO-d6) δ13.0(s,1H),8.0(ddd,J=8.7,5.3,2.7Hz,2H),7.7(d,J=8.6Hz,2H),7.7(d,J=8.6Hz,2H),7.4(t, J=8.8Hz,2H),7.2(d,J=2.5Hz,1H),6.9(d,J=9.0Hz,1H),6.7(dd,J=9.0,2.6Hz,1H),3.9(s,2H),3.8(s,3H),2.3(s,3H).

[0101] Example 16 Synthesis of 2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-(5-(2-chlorophenyl)-1,3,4-thiadiazol-2-yl)acetamide (Compound 15)

[0102] In a single-bore round-bottom flask, indomethacin (1.0 g, 2.79 mmol) was dissolved in 10 mL of N,N-dimethylformamide. DIPEA (0.72 g, 5.58 mmol) was added to the flask, followed by the partial addition of HATU (1.59 g, 4.19 mmol) under ice bath conditions. After 30 minutes, 2-amino-5-(2-chlorophenyl)-1,3,4-thiadiazole (intermediate A15) (0.65 g, 3.07 mmol) was added to the system. The mixture was then transferred to room temperature and stirred for 2 hours. The reaction was monitored by TLC. Once the reaction was complete, the contents were... 100 ml each of water and ethyl acetate were added sequentially to the mixture, which was then transferred to a separatory funnel and shaken. The mixture was allowed to stand until it separated into layers. The organic phase was washed twice with a saturated sodium chloride aqueous solution (15% w / v) and dried over anhydrous sodium sulfate. The organic phase was collected and evaporated to dryness to obtain the crude product. The crude product was redissolved in dichloromethane, and 2 g of 100-200 mesh silica gel was added and thoroughly mixed. The mixture was purified by column chromatography to obtain the product 2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-(5-(2-chlorophenyl)-1,3,4-thiadiazol-2-yl)acetamide, with a yield of 40.1%. The NMR spectrum of the product is shown below. Figure 13 As shown, 1 H NMR (400MHz, DMSO-d6) δ13.04(s,1H),7.97(t,J=1.9Hz,1H),7.86(dt,J=6.9,1.8Hz,1H),7.73–7.67(m,2H),7.66–7.61(m,2H),7. 59–7.52(m,2H),7.20(d,J=2.5Hz,1H),6.93(d,J=9.0Hz,1H),6.72(dd,J=9.0,2.6Hz,1H),3.99(s,2H),3.77(s,3H),2.31(s,3H).

[0103] Example 17 Synthesis of 2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-(5-(3-chlorophenyl)-1,3,4-thiadiazol-2-yl)acetamide (Compound 16)

[0104] In a single-bore round-bottom flask, indomethacin (1.0 g, 2.79 mmol) was dissolved in 10 mL of N,N-dimethylformamide. DIPEA (0.72 g, 5.58 mmol) was added to the flask, followed by the partial addition of HATU (1.59 g, 4.19 mmol) under ice bath conditions. After 30 minutes, 2-amino-5-(3-chlorophenyl)-1,3,4-thiadiazole (intermediate A16) (0.65 g, 3.07 mmol) was added to the system. The mixture was then transferred to room temperature and stirred for 2 hours. The reaction was monitored by TLC. Once the reaction was complete, the contents were... 100 ml each of water and ethyl acetate were added sequentially to the mixture, which was then transferred to a separatory funnel and shaken. The mixture was allowed to stand until it separated into layers. The organic phase was washed twice with a saturated sodium chloride aqueous solution (15% w / v) and dried over anhydrous sodium sulfate. The organic phase was collected and evaporated to dryness to obtain the crude product. The crude product was redissolved in dichloromethane, and 2 g of 100-200 mesh silica gel was added and thoroughly mixed. The mixture was purified by column chromatography to obtain the product 2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-(5-(3-chlorophenyl)-1,3,4-thiadiazol-2-yl)acetamide, with a yield of 67.1%. The NMR spectrum of the product is shown below. Figure 14 As shown, 1 H NMR (600MHz, DMSO-d6) δ13.1(s,1H),8.1(dd,J=7.7,1.9Hz,1H),7.7–7.7(m,2H),7.7–7.6(m,3H),7.5(dtd,J=20.2,7.4,1.6Hz,2H ),7.2(d,J=2.5Hz,1H),6.9(d,J=9.0Hz,1H),6.7(dd,J=9.1,2.5Hz,1H),4.0(d,J=7.2Hz,0H),4.0(s,2H),3.8(s,3H),2.3(s,3H).

[0105] Example 18 Synthesis of 2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-(5-(4-chlorophenyl)-1,3,4-thiadiazol-2-yl)acetamide (Compound 17)

[0106] In a single-bore round-bottom flask, indomethacin (1.0 g, 2.79 mmol) was dissolved in 10 mL of N,N-dimethylformamide. DIPEA (0.72 g, 5.58 mmol) was added to the flask, followed by the partial addition of HATU (1.59 g, 4.19 mmol) under ice bath conditions. After 30 minutes, 2-amino-5-(4-chlorophenyl)-1,3,4-thiadiazole (intermediate A17) (0.65 g, 3.07 mmol) was added to the system. The mixture was then transferred to room temperature and stirred for 2 hours. The reaction was monitored by TLC. Once the reaction was complete, the contents were... 100 ml each of water and ethyl acetate were added sequentially to the mixture, which was then transferred to a separatory funnel and shaken. The mixture was allowed to stand until it separated into layers. The organic phase was washed twice with a saturated sodium chloride aqueous solution (15% w / v) and dried over anhydrous sodium sulfate. The organic phase was collected and evaporated to dryness to obtain the crude product. The crude product was redissolved in dichloromethane, and 2 g of 100-200 mesh silica gel was added and thoroughly mixed. The mixture was purified by column chromatography to obtain the product 2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-(5-(4-chlorophenyl)-1,3,4-thiadiazol-2-yl)acetamide, with a yield of 81.8%. The NMR spectrum of the product is shown below. Figure 15 As shown, 1 H NMR (600MHz, DMSO-d6) δ13.0(s,1H),7.9(dd,J=8.6,1.9Hz,2H),7.7–7.7(m,2H),7.7(d,J=8.5Hz,2H),7.6(dd,J=8.8, 2.3Hz,2H),7.2(d,J=2.5Hz,1H),6.9(d,J=9.0Hz,1H),6.7(dd,J=9.0,2.6Hz,1H),4.0(s,2H),3.8(s,3H),2.3(s,3H).

[0107] Table 3. Compound Numbers and Yields

[0108]

[0109]

[0110] Example 19: Effect Experiment

[0111] 1. Experimental Methods

[0112] 1.1 Cytotoxicity assay

[0113] Mouse monocyte-macrophage RAW 264.7 cells were passaged in DMEM medium containing 10% fetal bovine serum, 100 mg / L penicillin, and 100 mg / L streptomycin at 37°C in a 5% CO2 incubator. RAW 264.7 cells in the logarithmic growth phase were harvested and the cell density was adjusted to 1 × 10⁻⁶ cells / year. 5Cells were seeded at a density of 100 μL / ml in 96-well plates. After 12 h of cell adhesion, the culture medium was replaced with DMEM containing or without 50 μmol / L compound 1-17, with three replicates for each concentration. After 24 h of incubation, 100 μL of MTT was added to each well, and the cells were cultured for another 4 h. The supernatant was discarded, and 150 μL of dimethyl sulfoxide (DMSO) was added to each well. The plates were shaken for 10 min to allow the crystals to dissolve completely, and the absorbance (A) was measured at 490 nm. The experiment was repeated three times. Cell viability (%) was calculated as (Adetector well / Ablank well) × 100%.

[0114] 1.2 Effect of compounds on NO content

[0115] Take a suspension of RAW264.7 cells in the logarithmic growth phase and adjust the cell density to 2 × 10⁻⁶. 5 NO was inoculated at a concentration of 100 μL / mL into a 96-well plate. After 12 h of adhesion, 100 μL of each of compounds 1-17 at a concentration of 50 μmol / L were added and incubated for 3 h. Then, 100 ng / LLPS was added and incubated for 12 h. A blank group, an LPS group, and an LPS+drug group were set up, with 3 replicates for each group. The NO content in the supernatant was detected by the Griess method.

[0116] 1.3 Effects of compounds on COX-2 protein expression

[0117] Take a suspension of RAW264.7 cells in the logarithmic growth phase and adjust the cell density to 2 × 10⁻⁶. 5 COX-2 protein expression was detected by Western blot analysis. 1 mL of the compound was seeded into each well of a 12-well plate. After 12 h of adhesion, 50 μmol / L compound 1-17 was added and incubated for 3 h. Then, 100 ng / L LPS was added and incubated for 12 h. There were three groups: blank group, LPS group, and LPS+ drug group. Each group had 3 replicates. The expression of COX-2 protein was detected by Western blot analysis.

[0118] 1.4 Data Analysis

[0119] Data analysis was performed using Prism software. The t-test was used for comparisons between groups, and P < 0.05 was considered statistically significant.

[0120] 2 Results

[0121] The structure of the compound was confirmed by proton nuclear magnetic resonance spectroscopy, and the results are as follows: Figures 1-15 As shown.

[0122] According to the MTT assay results, compounds 5 and 8 had no significant effect on the viability of RAW 264.7 cells. Figure 16As shown in the figure. According to Griess's experimental results, compounds 2, 3, 4, 5, 7, 8, 9, 11, 12, 14, and 15 exhibited significantly better inhibitory effects on NO production in RAW264.7 cells than indomethacin, as shown in the figure. Figure 17 As shown in the figure. Based on the good NO-inhibiting ability and lack of significant cytotoxicity of compounds 5 and 8, the effects of these two compounds on LPS-induced COX-2 expression in RAW264.7 cells were investigated using Western blotting (WB) experiments. The WB results are shown in the figure. Figure 18 As shown, compound 8 can significantly inhibit LPS-induced expression of COX-2 protein in RAW264.7 cells.

[0123] The results showed that compound 8 of the present invention did not have a significant inhibitory effect on RAW264.7 cells, but could significantly reduce the release level of the inflammatory factor NO and the expression level of the inflammatory protein COX-2 induced by LPS in RAW264.7 cells, and had good anti-inflammatory activity, and could be used as an active ingredient of anti-inflammatory drugs.

[0124] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An indomethacin derivative containing 2-amino-1,3,4-thiadiazole, characterized in that: The derivative is a compound with the structure shown in Formula 2 or a pharmaceutically acceptable salt thereof: Formula 2 Where R is 2-CF3 or 3-NO2.

2. The indomethacin derivative containing 2-amino-1,3,4-thiadiazole according to claim 1, characterized in that: The pharmaceutically acceptable salt is a salt formed by a compound with the structure shown in Formula 2 and hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, fumaric acid, maleic acid, oxalic acid, malonic acid, succinic acid, citric acid, malic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, glutamic acid, or aspartic acid.

3. The method for preparing the indomethacin derivative containing 2-amino-1,3,4-thiadiazole according to claim 1 or 2, characterized in that... It includes the following steps: (1) Thiourea is reacted with benzoic acid with different substituents to obtain an intermediate; the structural formula of the benzoic acid with different substituents is shown in Formula 3, and the structural formula of the intermediate is shown in Formula 4. Wherein, R is 2-CF3 or 3-NO2; (2) The intermediate obtained in step (1) and indomethacin were used as reaction raw materials, and N,N-dimethylformamide was used as solvent. 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate and N,N-diisopropylethylamine were added to carry out a condensation reaction to obtain an indomethacin derivative containing 2-amino-1,3,4-thiadiazole with the structure shown in Formula 2.

4. The preparation method according to claim 3, characterized in that: The molar ratio of thiourea to benzoic acid with different substituents in step (1) is 1:1.1; The specific operation of the reaction is as follows: thiourea and benzoic acid with different substituents are dissolved in phosphorus oxychloride, and the reaction is stirred at 75°C for 0.5 h. The starting material is monitored by LCMS. The reaction is terminated when the starting material is ≤1%. After cooling to room temperature, ice water is added dropwise to the reaction solution, and the reaction is carried out at 110°C for 4 h. The reaction is terminated and cooled to room temperature. The pH is then adjusted to 8-9 with NaOH solution, and a solid is precipitated to obtain the intermediate.

5. The preparation method according to claim 3, characterized in that: The molar ratio of indomethacin to the intermediate in step (2) is 1:1.1; the molar ratio of indomethacin to N,N-diisopropylethylamine is 1:1.5; the molar ratio of indomethacin to 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate is 1:2; and the condensation reaction is carried out at 25°C for 1-2 hours.

6. The use of the indomethacin derivative containing 2-amino-1,3,4-thiadiazole as described in claim 1 or 2 in the preparation of anti-inflammatory products.

7. The application according to claim 6, characterized in that: The anti-inflammatory product is a drug for treating inflammatory infectious diseases.

8. The application according to claim 7, characterized in that: The inflammatory infectious diseases mentioned above are inflammatory diseases in humans or animals caused by inflammatory stimuli.

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