Multisubstituted thiazole derivatives and their applications in disease treatment

Abstract

This application belongs to the pharmaceutical field and relates to a multisubstituted thiazole derivative and its use in the treatment of diseases. Specifically, it relates to a compound of formula I or a pharmaceutically acceptable salt thereof, a pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof, and its use in the treatment or prevention of dihydroorotate dehydrogenase-mediated diseases, including tumors, viral infections, and immune diseases.

Description

Technical Field

[0001] This application belongs to the pharmaceutical field and relates to a multisubstituted thiazole derivative and its use in the treatment of diseases. Specifically, it relates to a compound of formula I or a pharmaceutically acceptable salt thereof, a pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof, and its use in the treatment or prevention of dihydroorotate dehydrogenase-mediated diseases, including tumors, viral infections, and immune diseases. Background Technology

[0002] Dihydroorotate dehydrogenase (DHODH) is an iron-containing flavin-dependent enzyme located in the inner mitochondrial membrane that catalyzes the fourth step of the de novo synthesis pathway of pyrimidine nucleotides. Pyrimidine nucleotides are used in the biosynthesis of DNA, RNA, glycoproteins, and phospholipids, and are essential for cell metabolism and cell proliferation.

[0003] DHODH is closely related to the occurrence and development of various tumors. Therefore, inhibiting or downregulating DHODH can reduce tumor cell proliferation, induce apoptosis, or enhance the anti-tumor effects of other targeted drugs. The small-molecule DHODH inhibitor, buquina, is clinically used for breast cancer, head and neck cancer, colon cancer, gastrointestinal cancer, lung cancer, and ovarian cancer. There are also reports that DHODH inhibitors can induce leukemia cell differentiation and are used to treat acute myeloid leukemia (AML). Furthermore, several drugs, such as ASLAN003, BAY 2402234, PTC299, RP7214, JNJ-74856665, and WO2018192535A1, have been reported for the treatment of AML.

[0004] In the human body, resting lymphocytes primarily obtain pyrimidine nucleotides needed for cellular metabolism through the salvage pathway. However, under immune activation, lymphocytes' demand for pyrimidine nucleotides can reach more than eight times that of the resting state. At this time, lymphocyte proliferation and the completion of various immune functions require the initiation of the de novo synthesis pathway to replenish pyrimidine nucleotides. Inhibition of DHODH can block the synthesis of new pyrimidines, leading to impaired DNA synthesis and inhibiting activated T lymphocytes and B lymphocytes, thus playing an important role in immunosuppression. Leflunomide was the first marketed DHODH inhibitor used to treat rheumatoid arthritis. Teriflunomide, the active metabolite of leflunomide, was approved for marketing in 2012 for the treatment of multiple sclerosis. In addition, IMU-838 and PP-001 are in clinical trials for the treatment of ulcerative colitis and non-infectious uveitis, respectively.

[0005] DHODH inhibitors can prevent viral replication. This study simplifies the de novo pyrimidine synthesis pathway and its role in viral replication. DHODH inhibitors can inhibit the replication of various viruses, including negative-sense RNA viruses (such as influenza A and B viruses), positive-sense RNA viruses (hepatitis C virus and dengue virus), and DNA viruses (vaccinia virus and adenovirus). Currently, the only commercially available DHODH inhibitors are leflunomide and teriflunomide, both of which have significant toxic side effects, such as diarrhea, abnormal liver enzymes, rash, and hypertension. Therefore, developing highly effective and low-toxicity DHODH inhibitors has significant commercial value and practical implications. Summary of the Invention

[0006] This application provides, in one respect, a compound of formula I as a novel DHODH inhibitor or a pharmaceutically acceptable salt thereof:

[0007]

[0008] in,

[0009] R1 or R2 is independently selected from hydrogen, halogen, C. 1-6 Alkyl, C 1-6 Either alkoxy or cyano;

[0010] R3 is -(NR7) p -O-R5; R5 or R7 is independently selected from hydrogen or C. 1-6 alkyl;

[0011] R4 can be randomly selected from C 1-6 Alkyl or C 3-6 cycloalkyl;

[0012] M is a nitrogen atom or CR6; R6 is arbitrarily selected from hydrogen, halogens, and C. 1-6 Any of the alkyl groups;

[0013] m or n can be any integer between 0 and 5; and p is 0 or 1.

[0014] As a preferred embodiment of this application, this application provides a compound of formula I or a pharmaceutically acceptable salt thereof.

[0015]

[0016] in,

[0017] R1 or R2 can be arbitrarily selected from hydrogen, halogen, C. 1-3 Alkyl, C 1-3 alkoxy or cyano groups;

[0018] R3 is -(NR7)pO-R5; R5 and R7 are arbitrarily selected from hydrogen or C. 1-3 alkyl;

[0019] R4 can be randomly selected from C 1-4 Alkyl or C 3-6 cycloalkyl;

[0020] M is a nitrogen atom or CR6; R6 is arbitrarily selected from hydrogen, halogens, and C. 1-3 Any of the alkyl groups;

[0021] m or n is any integer from 0 to 3; and p is 0 or 1;

[0022] As a preferred embodiment of this application, the compound of formula I of this application has the structure of formula Ia as follows:

[0023]

[0024] in,

[0025] X is a halogen;

[0026] R1 or R2 is independently selected from hydrogen, halogen, C. 1-6 Alkyl, C 1-6 Either alkoxy or cyano;

[0027] R3 is -(NR7) p -O-R5; R5 or R7 is independently selected from hydrogen or C. 1-6 alkyl;

[0028] R4 can be randomly selected from C 1-6 Alkyl or C 3-6 cycloalkyl;

[0029] M is a nitrogen atom or CR6; R6 is arbitrarily selected from hydrogen, halogens, and C. 1-6 Any of the alkyl groups;

[0030] m or n is any integer from 0 to 5; and p is 0 or 1.

[0031] As a preferred embodiment of this application, the compound of formula I of this application has the following structure:

[0032]

[0033] in,

[0034] R1 or R2 is independently selected from hydrogen, halogen, C. 1-6 Alkyl, C 1-6 Either alkoxy or cyano;

[0035] R3 is -(NR7) p -O-R5; R5 or R7 is independently selected from hydrogen or C. 1-6 alkyl;

[0036] R4 is C3-6 cycloalkyl;

[0037] R6 can be arbitrarily selected from hydrogen or C. 1-6 alkyl;

[0038] m or n is any integer from 0 to 5; and p is 0 or 1.

[0039] As a preferred embodiment of this application, the compound of formula I of this application has the following Ic structure.

[0040]

[0041] in,

[0042] R1 or R2 is independently selected from hydrogen, halogen, C. 1-6 Alkyl, C 1-6 Either alkoxy or cyano;

[0043] R3 is -(NR7) p -O-R5; R5 or R7 is independently selected from hydrogen or C. 1-6 alkyl;

[0044] R4 can be arbitrarily selected from hydrogen, C 1-6 Alkyl and C 3-6 Any of the cycloalkyl groups;

[0045] m or n is any integer from 0 to 5; and p is 0 or 1.

[0046] As a preferred embodiment of this application, in compound formula I, R1 or R2 is any one of hydrogen, halogen, methyl, methoxy, trifluoromethyl, trifluoromethoxy, and cyano.

[0047] X is fluorine, chlorine, or bromine;

[0048] R3 is a hydroxyl group or -NR7-O-R5;

[0049] R5 is hydrogen, methyl, ethyl, n-propyl, or isopropyl; preferably, R5 is hydrogen, methyl, or ethyl.

[0050] R7 is selected from hydrogen, methyl, or ethyl;

[0051] R4 is methyl, ethyl, propyl, unsubstituted or halogenated cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl; preferably, R4 is methyl, ethyl, propyl, 2-fluorocyclopropyl or 2,2-difluorocyclopropyl; wherein when the cycloalkyl group of R4 has a chiral center, the present application further includes its corresponding stereoisomers.

[0052] R6 is hydrogen, fluorine, chlorine, methyl, or ethyl; preferably, R6 is hydrogen, fluorine, or methyl.

[0053] m or n is 0, 1, or 2; and

[0054] p is 0 or 1.

[0055] The compounds of Formula I in this application include, but are not limited to, the following compounds:

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063] The preferred compounds of Formula I in this application include the following compounds:

[0064]

[0065] This application also provides a method for preparing a compound of formula I, the method comprising: reacting formula II with formula III to prepare the compound shown in formula I.

[0066]

[0067] Wherein, R1, R2, R3, R4, M, m, and n have the same definition range as Formula I, and R8 is an amino protecting group or hydrogen. The protecting group includes, but is not limited to, tert-butoxycarbonyl, benzyloxycarbonyl, etc. When R8 is a protecting group, Formula II is first deprotected before reacting with Formula III. When R3 is a hydroxyl group, it can be further reacted with NH2OH, NH2OR5, and NHR7OR5 or their corresponding salts to prepare the compound of Formula I.

[0068] When M is CR6, compound II can be obtained by coupling reaction of formula IV and formula V under metal catalysis. The preferred metal catalysts are tris(dibenzylidene indeneacetone)dipalladium and 2-dicyclohexylphosphine-2′,6′-diisopropoxy-1,1′-biphenyl as ligands, and alkali metal carbonates as bases.

[0069]

[0070] Among them, R2, R4, R6, R8, and n have the same definition range as the aforementioned structure.

[0071] When M is CR6 and R6 is a halogen, such as fluorine-substituted, formula IV can be prepared by reacting formula VI with formula VII.

[0072]

[0073] When M is a nitrogen atom, Formula II is prepared by the following method:

[0074] Formula XX is obtained by reacting R2-substituted benzonitrile with bis(trimethylsilylamine)-lithium catalysis; then, it is reacted with R4-substituted isocyanate XXI under alkaline conditions to prepare formula XXII, wherein the alkaline base is, for example, an organic base, including but not limited to triethylamine, diisopropylethylamine, etc. Formula XXII is reacted with elemental iodine and an inorganic base to obtain formula XXIII, wherein the inorganic base includes, but is not limited to, potassium carbonate, cesium carbonate, etc. Formula XXIII is reacted with nitrite, such as sodium nitrite, to prepare formula XXIV; then, it is reduced by a metal reducing agent to obtain formula II, wherein the metal reducing agent includes, but is not limited to, iron powder, zinc powder, etc. The process is described as follows:

[0075]

[0076] This application further provides a compound of formula II,

[0077]

[0078] in,

[0079] M is nitrogen or CR6, and R6 is arbitrarily selected from hydrogen or C. 1-3 Either alkyl or fluorine atom;

[0080] R2 can be selected from hydrogen, halogen, or C. 1-3 Alkyl, C 1-3 Any one of alkoxy or cyano groups;

[0081] R4 can be randomly selected from C 1-4 Alkyl or C 3-6 cycloalkyl;

[0082] R8 is a protecting group of an amino group or a hydrogen atom, and the protecting group includes, but is not limited to, tert-butoxycarbonyl, benzyloxycarbonyl, etc.; and

[0083] m can be any value of 0, 1, 2, or 3.

[0084] The compounds of Formula I in this application also include stereoisomers and optical isomers. Pharmaceutically acceptable salts of the compounds in this application can be salts formed with inorganic bases or inorganic acids, or salts formed with organic bases, and can be converted into the corresponding salts by known conventional methods. The salts formed with inorganic bases include, but are not limited to: sodium salts, potassium salts, calcium salts, hydrochloride salts, sulfates, phosphates, etc.; the salts formed with organic bases include, but are not limited to: ethanolamine salts, ammonium salts, and salts formed with various amino acids, etc.; as a preferred embodiment, the pharmaceutically acceptable salts of Formula I in this application are sodium salts, potassium salts, calcium salts, and ethanolamine salts.

[0085] In another aspect, this application provides a pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof, the composition being administered by inhalation in the form of a solution, suspension, inhaler, etc.; or orally in the form of tablets, capsules, granules, etc.; or rectally or transdermally in the form of a suppository; or in the form of an injection.

[0086] When the pharmaceutical composition of this application is prepared into an oral dosage form, the composition may further contain a pharmaceutically acceptable diluent, including but not limited to excipients, binders, disintegrants, and lubricants. The excipients include lactose, starch, glucose, mannitol, microcrystalline cellulose, hydroxypropyl cellulose, etc.; the binders include polyvinyl alcohol, ethyl cellulose, methyl cellulose, hydroxypropyl cellulose, etc.; the disintegrants include starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, croscarmellose sodium, etc.; and the lubricants include magnesium stearate, talc, polyethylene glycol, etc. After mixing the above diluents with the pharmaceutical composition, it can be compressed into tablets using conventional methods. If coating is required, the tablet core can be coated with a sugar solution such as gelatin or titanium dioxide. Alternatively, the compound of this application can be thoroughly mixed with the diluents and then directly filled into capsules to prepare a capsule dosage form.

[0087] The compounds of Formula I described in this application, or pharmaceutically acceptable salts thereof, and drug combinations containing compounds of Formula I described in this application or pharmaceutically acceptable salts thereof, may be used to treat or prevent DHODH-mediated diseases, including but not limited to: autoimmune diseases, tumors, viral infections, etc. Autoimmune diseases include rheumatoid arthritis, psoriasis, etc.; viral infections include diseases caused by influenza viruses, coronaviruses, etc.

[0088] Terminology Explanation:

[0089] In this application, "alkyl" refers to a saturated alkane having 1 to 6 carbon atoms, such as a straight-chain alkane or a branched-chain alkane; "alkoxy" refers to a saturated alkyloxy group having 1 to 6 carbon atoms, such as a straight-chain alkoxy group or a branched-chain alkoxy group. Specific alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, etc. Specific alkoxy groups include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butyloxy, etc.

[0090] In this application, "halogen" refers to fluorine, chlorine, bromine, and iodine; preferably, the halogen in this application is fluorine, chlorine, or bromine.

[0091] In this application, "cycloalkyl" refers to a saturated cycloalkyl group containing 3 to 6 carbon atoms, including cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0092] The alkyl, alkoxy, and cycloalkyl groups described in this application may be unsubstituted or optionally substituted by one or more substituents; as a preferred embodiment of this application, the substituents are halogens, such as fluorine, chlorine, and bromine; as a preferred embodiment of this application, the alkyl, alkoxy, and cycloalkyl groups are trifluoromethyl, trifluoromethoxy, 2-fluorocyclopropyl, 2,2-difluorocyclopropyl, etc. Attached Figure Description

[0093] Figure 1 Two embodiments of this application are shown to demonstrate the efficacy of human monocytic leukemia THP-1 cells in the mouse xenograft tumor model CB17 SCID.

[0094] Figure 2 Two embodiments of this application demonstrate the efficacy of human promyelocytic leukemia HL-60 cells in a mouse xenograft tumor model.

[0095] Figure 3 Two embodiments of this application demonstrate the efficacy of human small cell lung cancer cells NCI-H82 in a mouse xenograft tumor model. Detailed Implementation

[0096] To better understand this application, the specific technical solutions of this application will be described below in conjunction with specific embodiments and accompanying drawings. However, it should be understood that this application is not limited thereto. Unless otherwise specified, the compounds, reagents, laboratory animals, etc. used in the embodiments of this application are commercially available.

[0097] This application incorporates, in its entirety, the prior patent application CN107382902A of the inventors. Specifically, the synthesis of some compounds may refer to the methods described in CN107382902A, for example, the preparation of the reference example compound (E)-4-(2-chlorophenyl)-2-[1-methyl-2-(2-carboxybenzyl)hydrazyl]thiazole.

[0098] Example 1:

[0099] (E)-4-(2-chlorophenyl)-2-[1-cyclopropyl-2-(2-carboxy-4-fluorobenzyl)hydrazyl]thiazole(1)

[0100]

[0101] Step (a) tert-butyl (p-toluenesulfonyl) carbamate (1b)

[0102] 20 g (0.15 mol) of tert-butyl hydroxycarbamate 1a was weighed and placed in a 500 mL three-necked flask. 30 mL of dichloromethane was added, and 20 mL of a dichloromethane solution of 32.5 g (0.165 mol) of p-toluenesulfonyl chloride was added while stirring at 0 °C. After stirring for 5 minutes, 15.2 g (0.15 mol) of N-methylmorpholine was added. The reaction mixture was heated to room temperature and reacted for 16 hours. The reaction mixture was quenched with water (10 mL) at 0 °C. The mixture was extracted three times with dichloromethane (15 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a white powdery solid, title compound 1b (34 g), with a yield of 79.1%.

[0103] 1 H NMR (400MHz, DMSO-d6) δ 11.23 (s, 1H), 7.79 (d, J = 8.4Hz, 2H), 7.49 (d, J = 8.1Hz, 2H), 2.43 (s, 3H), 1.23 (s, 9H).

[0104] Step (b) tert-butyl-2-cyclopropylhydrazine carbonate (1c)

[0105] 9.93 g (0.17 mol) of cyclopropylamine was weighed and placed in a 500 mL three-necked flask. 20 mL of tetrahydrofuran was added, followed by 3.52 g (34.8 mmol) of N-methylmorpholine under stirring at -5 °C. Then, 20 mL of a tetrahydrofuran solution containing 10 g (34.8 mmol) of intermediate 1b was added under stirring at -5 °C. The reaction mixture was heated to room temperature and reacted for 16 hours. The reaction mixture was quenched with water (10 mL) at 0 °C. The mixture was extracted three times with dichloromethane (15 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give 2.5 g (yellow oil) of the title compound 1c, with a yield of 41.7%.

[0106] 1H NMR (400MHz, DMSO-d6) δ8.54 (s, 1H), 6.44 (d, J=2.8Hz, 1H), 1.12 (s, 9H), 0.57 (td, J=7.0, 4.6Hz, 2H), 0.47-0.40 (m, 2H).

[0107] Step (c) 1-(2-chlorophenyl)-2-thiocyanate ethane-1-one (1e)

[0108] 4 g (17.1 mmol) of 1d (2-bromo-1-(2-chlorophenyl)ethyl ketone) was weighed into a 100 mL single-necked flask, and 50 mL of anhydrous ethanol was added. 4.0 g (41.2 mmol) of potassium thiocyanate was added while stirring at room temperature. The reaction mixture was refluxed at 85 °C for 3 hours. The reaction mixture was concentrated under reduced pressure, and column chromatography was used to separate the product into a yellow oil, 1e (3.8 g), with a yield of 97%. LCMS: 211.9, 213.9 ([M+H]+).

[0109] Step (d) 2-Bromo-4-(2-chlorophenyl)thiazole (1f)

[0110] 3.8 g (18.0 mmol) of intermediate 1e was weighed and placed in a 100 mL single-necked flask. 50 mL of glacial acetic acid was added, followed by 40 mL of a hydrobromic acid solution of acetic acid while stirring at room temperature. The reaction mixture was refluxed at 140 °C for 3 hours. The reaction mixture was poured into 100 mL of ice water, and the mixture was extracted three times with 50 mL of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude product. The crude product was separated by column chromatography to yield a yellow oily product (2.5 g), with a yield of 51%. LCMS: 273.9, 275.9 ([M+H]+).

[0111] Step (e) 2-(4-(2-chlorophenyl)thiazol-2-yl)-2-cyclopropylhydrazine-1-carboxylic acid tert-butyl ester (1g)

[0112] 3 g (11 mmol) of intermediate 2-bromo-4-(2-chlorophenyl)thiazole 1f was weighed and placed in a 500 mL single-necked flask. 1.9 g (11 mmol) of intermediate 1c, 0.51 g (0.55 mmol) of tris(dibenzylindenacetone)dipalladium, 0.51 g (1.10 mmol) of 2-dicyclohexylphosphine-2′,6′-diisopropoxy-1,1′-biphenyl, and 7.19 g (22 mmol) of cesium carbonate were added. The reaction mixture was stirred at room temperature and purged three times with nitrogen. The reaction mixture was then stirred at 100 °C for 16 hours. The reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure. The crude product was separated by column chromatography to obtain 1 g (100 mg, yield: 3%) of a yellow solid intermediate. LCMS: 366.1, 368.1 ([M+H]+).

[0113] Step (f) 4-(2-chlorophenyl)-2-(1-cyclopropylhydrazine)thiazole (1h)

[0114] 100 mg (0.27 mmol) of the intermediate tert-butyl 2-(4-(2-chlorophenyl)thiazolyl-2-yl)-2-cyclopropylhydrazine-1-carboxylic acid was weighed into a 100 mL single-necked flask, 10 mL of dichloromethane was added, and 2 mL of trifluoroacetic acid was added while stirring at room temperature. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure to give a yellow oily intermediate (70 mg) in 1 hour (97.2%). LCMS: 266.0, 268.0 ([M+H]+).

[0115] Step (g)(E)-4-(2-chlorophenyl)-2-[1-cyclopropyl-2-(2-carboxy-4-fluorobenzyl)hydrazyl]thiazole (1)

[0116] 70 mg (0.26 mmol) of the intermediate 4-(2-chlorophenyl)-2-(1-cyclopropylhydrazine)thiazole was weighed and placed in a 100 mL single-necked flask. 5 mL of dichloromethane and 44 mg (0.26 mmol) of 3-fluoro-6-aldehyde benzoic acid were added, and the reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure and purified by preparative high-performance liquid chromatography to give a white solid, target compound 1 (27 mg, yield: 25%).

[0117] 1 H NMR (400MHz, DMSO-d6) δ13.67 (s, 1H), 9.09 (s, 1H), 8.05 (dd, J=8.8, 5.7Hz, 1H), 7.94 (dd, J=7.7, 1.7Hz, 1H), 7.68 (dd, J=9.5, 2.8H z, 1H), 7.60-7.50 (m, 2H), 7.50-7.39 (m, 2H), 7.36 (td, J=7.6, 1.8Hz, 1H), 2.91-2.83 (m, 1H), 1.27-1.17 (m, 2H), 1.08-0.98 (m, 2H).

[0118] Example 2:

[0119] (E)-4-(2-chlorophenyl)-2-[1-cyclopropyl-2-(2-carboxy-4-chlorobenzyl)hydrazyl]thiazole(2)

[0120]

[0121] Similar to the method in Example 1, 2-chloro-6-aldehyde benzoic acid was used instead of 2-fluoro-6-aldehyde benzoic acid to react with the intermediate for 1 hour to obtain 42 mg of title compound 2.

[0122] 1 H NMR (400MHz, DMSO-d6) δ13.72 (s, 1H), 9.09 (s, 1H), 8.02 (d, J = 8.6Hz, 1H), 7.98-7.86 (m, 2H), 7.75 ( dd, J=8.5, 2.3Hz, 1H), 7.62-7.27(m, 4H), 2.98-2.78(m, 1H), 1.31-1.12(m, 2H), 1.10-0.95(m, 2H).

[0123] Example 3:

[0124] (E)-4-(2-chlorophenyl)-2-[1-cyclobutyl-2-(2-carboxy-4-fluorobenzyl)hydrazyl]thiazole (3)

[0125]

[0126] Similar to the method for intermediate 1c, cyclobutylamine was used instead of cyclopropylamine to react with intermediate 1b to obtain 3c; similar to the method in Example 1, 27 mg of the title compound 3 was obtained.

[0127] 1 H NMR (400MHz, DMSO-d6) δ13.66 (s, 1H), 8.76 (s, 1H), 8.02 (dd, J=8.7, 5.8Hz, 1H), 7.96-7.88 (m, 1H), 7.66 (d, J=7.2Hz, 1H), 7. 57-7.46(m, 3H), 7.47-7.38(m, 1H), 7.40-7.31(m, 1H), 4.83(s, 1H), 2.89-2.74(m, 2H), 2.61-2.52(m, 2H), 1.90-1.75(m, 2H).

[0128] Example 4:

[0129] (E)-4-(3-chlorophenyl)-2-[1-cyclopropyl-2-(2-carboxy-4-fluorobenzyl)hydrazyl]thiazole (4)

[0130]

[0131] Similar to the method in Example 1, 2-bromo-1-(3-chlorophenyl)ethyl ketone was used instead of 1d to obtain 29 mg of title compound 4.

[0132] 1H NMR (400MHz, DMSO-d6) δ13.71 (s, 1H), 9.13 (s, 1H), 8.04 (dd, J=8.8, 5.7Hz, 1H), 7.95 (t, J=1.7Hz, 1H), 7.88 (d, J=7.8Hz, 1H), 7.67 (dd, J=9.5, 2.8Hz, 1H ), 7.62 (s, 1H), 7.53 (td, J=8.5, 2.8Hz, 1H), 7.46 (t, J=7.9Hz, 1H), 7.40-7.3 3(m, 1H), 2.91(tt, J=6.8, 3.8Hz, 1H), 1.31-1.21(m, 2H), 1.10-0.99(m, 2H).

[0133] Example 5:

[0134]

[0135] 2-Bromo-1-(2-trifluoromethylphenyl)ethyl ketone (5d)

[0136] 10 g (53.2 mmol) of 2-trifluoromethylacetophenone (5c) was weighed and placed in a 500 mL single-necked flask. 50 mL of methanol was added, followed by 5.68 g (58.5 mmol) of ammonium bromide and 35.98 g (58.5 mmol) of potassium peroxymonosulfonate under stirring at room temperature. The reaction mixture was purged with argon three times under stirring at room temperature and reacted at 65 °C for 16 hours. The reaction mixture was filtered, and the filtrate was quenched with water (10 mL). The mixture was extracted with dichloromethane (15 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give an oily intermediate 5d (15 g) with a yield of 71%.

[0137] (E)-4-(2-trifluoromethylphenyl)-2-[1-cyclopropyl-2-(2-carboxy-4-fluorobenzyl)hydrazyl]thiazole (5)

[0138] Similar to the method in Example 1, 2-bromo-1-(2-trifluoromethylphenyl)ethyl ketone 5d was used instead of 1d to obtain 38 mg of title compound 5.

[0139] 1 H NMR (400MHz, DMSO-d6) δ13.66 (s, 1H), 9.08 (s, 1H), 8.04 (dd, J=8.8, 5.7Hz, 1H), 7.83 (d, J=7.8Hz, 1H), 7.76-7 .64 (m, 3H), 7.65-7.50 (m, 2H), 7.11 (s, 1H), 2.91-2.75 (m, 1H), 1.22-1.09 (m, 2H), 1.10-0.90 (m, J=3.8Hz, 2H).

[0140] Example 6:

[0141] (E)-4-(2-Tolyl)-2-[1-Cyclopropyl-2-(2-Carboxy-4-Fluorobenzylidene)hydrazyl]thiazole (6)

[0142]

[0143] Similar to the method in Example 5, 2-methylacetophenone (6c) was used instead of 2-trifluoromethylacetophenone (5c) to obtain title compound 6.

[0144] 1 H NMR (400MHz, DMSO-d6) δ13.56 (s, 1H), 9.08 (s, 1H), 8.05 (dd, J=8.8, 5.7Hz, 1H), 7.68 (dd, J=9.5, 2.8Hz, 1H), 7.64-7 .51 (m, 2H), 7.31-7.19 (m, 3H), 7.10 (s, 1H), 2.91-2.79 (m, 1H), 2.48 (s, 14H), 1.26-1.16 (m, 2H), 1.09-0.94 (m, 2H).

[0145] Example 7:

[0146] (E)-4-(4-chlorophenyl)-2-[1-cyclopropyl-2-(2-carboxy-4-fluorobenzyl)hydrazyl]thiazole (7)

[0147]

[0148] Similar to the method in Example 5, 4-chloroacetophenone (7c) was used instead of 2-trifluoromethylacetophenone (5c) to obtain title compound 7.

[0149] 1 H NMR (400MHz, DMSO-d6) δ13.68 (s, 1H), 9.11 (s, 1H), 8.04 (dd, J=8.8, 5.7Hz, 1H), 7.93 (d, J=8.5Hz, 2H), 7.67 (dd, J=9.5, 2.8Hz, 1H), 7.58-7.52 (m, 2H), 7.48 (d, J=8.6Hz, 2H), 2.99-2.81 (m, 1H), 1.27-1.22 (m, 2H), 1.04 (q, J=6.5, 5.5Hz, 2H).

[0150] Example 8:

[0151] (E)-4-(3-methylphenyl)-2-[1-cyclopropyl-2-(2-carboxy-4-fluorobenzyl)hydrazino]thiazole (8)

[0152]

[0153] Similar to the method in Example 5, 3-methylacetophenone (8c) was used instead of 2-trifluoromethylacetophenone (5c) to obtain title compound 8.

[0154] 1 H NMR (400MHz, DMSO-d6) δ9.10 (s, 1H), 8.04 (s, 1H), 7.75-7.63 (m, 2H), 7.61-7.48 (m, 2H), 7.43 (s, 1H), 7.3 5-7.25(m, 1H), 7.16-7.08(m, 1H), 2.92-2.85(m, 1H), 2.36(s, 3H), 1.28-1.21(m, 2H), 1.10-0.98(m, 2H).

[0155] Example 9:

[0156] (E)-4-(2-methoxyphenyl)-2-[1-cyclopropyl-2-(2-carboxy-4-fluorobenzyl)hydrazyl]thiazole (9)

[0157]

[0158] Similar to the method in Example 5, 2-trifluoromethylacetophenone was replaced with 2-methoxyacetophenone to obtain title compound 9.

[0159] 1 H NMR (400MHz, DMSO-d6) δ9.09 (s, 1H), 8.15 (dd, J=7.6, 1.8Hz, 1H), 8.01 (dd, J=8.9, 5.7Hz, 1H), 7.76-7.60 (m, 2H), 7.55-7.45 (m, 2H) , 7.33-7.23 (m, 1H), 7.09 (d, J=8.3Hz, 1H), 7.06-6.98 (m, 1H), 3.90 (s, 3H), 2.91-2.82 (m, 1H), 1.26-1.17 (m, 2H), 0.95-0.86 (m, 2H).

[0160] Example 10:

[0161] (E)-4-(2-trifluoromethoxyphenyl)-2-[1-cyclopropyl-2-(2-carboxy-4-fluorobenzyl)hydrazyl]thiazole (10)

[0162]

[0163] Similar to the method in Example 5, 2-trifluoromethylacetophenone was substituted with 2-trifluoromethoxyacetophenone to obtain title compound 10.

[0164] 1 H NMR (400MHz, DMSO-d6) δ9.09 (s, 1H), 8.14-8.06 (m, 1H), 8.06-7.97 (m, 1H), 7.70-7.61 (m, 1H) ), 7.57-7.39(m, 4H), 7.30(s, 1H), 2.91-2.80(m, 1H), 1.27-1.16(m, 2H), 1.05-0.95(m, 2H).

[0165] Example 11:

[0166] (E)-4-(3-methoxyphenyl)-2-[1-cyclopropyl-2-(2-carboxy-4-fluorobenzyl)hydrazyl]thiazole (11)

[0167]

[0168] Similar to the method in Example 5, 3-methoxyacetophenone was used instead of 2-trifluoromethylacetophenone to obtain title compound 11.

[0169] 1 H NMR (400MHz, DMSO-d6) δ9.11 (s, 1H), 8.11-8.00 (m, 1H), 7.82-7.60 (m, 2H), 7.60-7.44 (m, 3H), 7.41-7. 29 (m, 1H), 6.89 (d, J=8.2Hz, 1H), 3.81 (s, 3H), 2.94-2.83 (m, 1H), 1.30-1.20 (m, 2H), 1.08-1.01 (m, 2H).

[0170] Example 12:

[0171] (E)-4-(3-trifluoromethoxyphenyl)-2-[1-cyclopropyl-2-(2-carboxy-4-fluorobenzyl)hydrazyl]thiazole (12)

[0172]

[0173] Similar to the method in Example 5, 3-trifluoromethoxyacetophenone was used instead of 2-trifluoromethylacetophenone to obtain title compound 12.

[0174] 1H NMR (400MHz, DMSO-d6) δ9.12 (s, 1H), 8.04 (dd, J=8.7, 5.6Hz, 1H), 7.95 (d, J=7.5Hz, 1H), 7.87 (s, 1H), 7.74-7.62 (m, 3H), 7.60-7.50 (m, 2H), 7.30 (d, J=7.9Hz, 1H), 2.95- 2.87(m, 1H), 1.27-1.20(m, 4H), 1.08-1.01(m, 2H).

[0175] Example 13:

[0176] (E)-4-(2-fluorophenyl)-2-[1-cyclopropyl-2-(2-carboxy-4-fluorobenzyl)hydrazyl]thiazole (13)

[0177]

[0178] Similar to the method in Example 5, 2-fluoroacetophenone was used instead of 2-trifluoromethylacetophenone to obtain title compound 13.

[0179] 1 H NMR (400MHz, DMSO-d6) δ9.12 (s, 1H), 8.19-8.07 (m, 1H), 8.05-7.95 (m, 1H), 7.68-7.59 (m, 1H), 7.57-7.44(m, 2H), 7.40-7.15(m, 3H), 2.92-2.83(m, 1H), 1.28-1.17(m, 2H), 1.09-0.98(m, 2H).

[0180] Example 14:

[0181] (E)-4-(2-cyanophenyl)-2-[1-cyclopropyl-2-(2-carboxy-4-fluorobenzyl)hydrazyl]thiazole (14)

[0182]

[0183] Similar to the method in Example 5, 2-cyanoacetophenone was used instead of 2-trifluoromethylacetophenone to obtain title compound 14.

[0184] 1H NMR (400MHz, DMSO-d6) δ9.14 (s, 1H), 8.09-7.97 (m, 2H), 7.90 (dd, J=7.3, 2.0Hz, 1H), 7.81-7.73 (m, 1H), 7.71-7 .64 (m, 1H), 7.61 (d, J=5.2Hz, 1H), 7.59-7.50 (m, 2H), 2.92-2.82 (m, 2H), 1.26-1.17 (m, 2H), 1.11-1.02 (m, 2H).

[0185] Example 15:

[0186] (E)-4-(2-bromophenyl)-2-[1-cyclopropyl-2-(2-carboxy-4-fluorobenzyl)hydrazyl]thiazole (15)

[0187]

[0188] Similar to the method in Example 5, 2-bromoacetophenone was used instead of 2-trifluoromethylacetophenone to obtain title compound 15.

[0189] Example 16:

[0190] (E)-4-(3-trifluoromethylphenyl)-2-[1-cyclopropyl-2-(2-carboxy-4-fluorobenzyl)hydrazyl]thiazole (16)

[0191]

[0192] Similar to the method in Example 5, 3-trifluoromethylacetophenone was used instead of 2-trifluoromethylacetophenone to obtain title compound 16.

[0193] 1 H NMR (400MHz, DMSO-d6) δ9.11 (s, 1H), 8.07-7.99 (m, 1H), 7.81-7.74 (m, 1H), 7.72 (d, J=7.9Hz, 1H), 7.70-7.63 (m, 1H), 7 .59-7.51 (m, 1H), 7.51-7.42 (m, 1H), 7.37 (s, 1H), 7.34-7.25 (m, 1H), 2.85 (s, 1H), 1.21 (d, J=14.2Hz, 2H), 1.03 (s, 2H).

[0194] Example 17:

[0195] (E)-4-(3-bromophenyl)-2-[1-cyclopropyl-2-(2-carboxy-4-fluorobenzyl)hydrazyl]thiazole (17)

[0196]

[0197] Similar to the method in Example 5, 3-bromoacetophenone was used instead of 2-trifluoromethylacetophenone to obtain title compound 17.

[0198] 1 H NMR (400MHz, DMSO-d6) δ9.13 (s, 1H), 8.11-8.07 (m, 1H), 8.07-7.98 (m, 1H), 7.96-7.88 (m, 1H), 7.70-7.6 0(m, 2H), 7.56-7.46(m, 2H), 7.43-7.34(m, 1H), 2.95-2.85(m, 2H), 1.29-1.20(m, 4H), 1.08-0.98(m, 2H).

[0199] Example 18:

[0200] (E)-4-(4-methylphenyl)-2-[1-cyclopropyl-2-(2-carboxy-4-fluorobenzyl)hydrazyl]thiazole (18)

[0201]

[0202] Similar to the method in Example 5, 2-trifluoromethylacetophenone was replaced with 4-methylacetophenone to obtain title compound 18.

[0203] 1 H NMR (400MHz, DMSO-d6) δ9.10 (s, 1H), 8.03-7.96 (m, 1H), 7.78 (d, J=7.9Hz, 2H), 7.63 (dd, J=12.4, 3.0Hz, 1H), 7.54-7. 44 (m, 1H), 7.35 (s, 1H), 7.20 (d, J=7.9Hz, 2H), 2.91-2.81 (m, 1H), 2.30 (s, 3H), 1.27-1.17 (m, 2H), 1.07-0.97 (m, 2H).

[0204] Example 19:

[0205] (E)-4-(4-trifluoromethylphenyl)-2-[1-cyclopropyl-2-(2-carboxy-4-fluorobenzyl)hydrazyl]thiazole (19)

[0206]

[0207] Similar to the method in Example 5, 4-trifluoromethylacetophenone was used instead of 2-trifluoromethylacetophenone to obtain title compound 19.

[0208] 1H NMR (400MHz, DMSO-d6) δ9.11 (s, 1H), 8.10 (d, J = 8.2Hz, 2H), 8.01 (dd, J = 8.8, 5.5Hz, 1H), 7.76 (d, J = 8.2Hz, 2H), 7.69 (s, 1H), 7.65 (dd, J=9.4, 3.0Hz, 2H), 7.52 (s, 1H), 2.94-2.84 (m, 1H), 1.27-1.18 (m, 4H), 1.06-0.99 (m, 2H).

[0209] Example 20:

[0210] (E)-5-fluoro-4-(2-chlorophenyl)-2-[1-methyl-2-(2-carboxybenzyl)hydrazyl]thiazole (20)

[0211]

[0212] 2-Chloro-2-fluoro-1-(2-chlorophenyl)ethyl ketone

[0213] Weigh 10.0 g (41.9 mmol) of o-chloroiodobenzene into a 250 mL three-necked flask, add 100 mL of ultradry THF, and stir until dissolved. Under argon protection, cool to -78 °C, and add 20.1 mL of n-butyllithium solution (2.5 M in hexane). After the addition is complete, react at -78 °C for 40 min, then add 10.0 g (71.2 mmol) of ethyl chlorofluorocarbonate. After the addition is complete, react at -78 °C for another 40 min. Quench with saturated ammonium chloride (200 mL), extract three times with ethyl acetate (120 mL * 3), combine the organic phases, dry to anhydrous sodium sulfate, filter, concentrate, and separate by column chromatography (EA / PE, 0%–10%) to give 5.8 g of colorless liquid, yield 67.0%.

[0214] 1 H NMR (400MHz, CDCl3) δ7.66 (dd, J=8.0, 1.0Hz, 1H), 7.54-7.46 (m, 2H), 7.43-7.37 (m, 1H), 7.00 (d, J=50.5Hz, 1H).

[0215] 4-(2-Chlorophenyl)-5-fluoro-2-(1-methylhydrazinyl)thiazole

[0216] 500 mg (2.4 mmol) of the intermediate 2-chloro-2-fluoro-1-(2-chlorophenyl)ethyl ketone was weighed into a 25 mL round-bottom flask, and 380 mg (3.6 mmol) of 2-methylaminothiourea and 10 mL of ultra-dry tetrahydrofuran were added. The reaction was carried out at 80 °C for 25 hours under nitrogen protection. The reaction was cooled to room temperature, the solvent was removed by concentration, 100 mL of water was added, and the mixture was extracted three times with ethyl acetate (50 mL * 3). The organic phases were combined, concentrated, and separated by column chromatography to give 220 mg of gray solid, with a yield of 35.3%.

[0217] (E)-5-fluoro-4-(2-chlorophenyl)-2-[1-methyl-2-(2-carboxybenzyl)hydrazinyl]thiazole

[0218] 100 mg (0.39 mmol) of the intermediate 4-(2-chlorophenyl)-5-fluoro-2-(1-methylhydrazyl)thiazole was weighed into a 25 mL round-bottom flask, and 87.5 mg (0.58 mmol) of 2-aldehyde benzoic acid, 64 mg (0.78 mmol) of sodium acetate, and 10 mL of methanol were added. The reaction was carried out at room temperature for 16 hours under nitrogen protection. The solvent was removed by concentration, and 50 mL of water was added. The mixture was extracted three times with ethyl acetate (50 mL * 3). The organic phases were combined and concentrated, and separated by column chromatography to give 50 mg of the title compound as a yellow solid, in 32.3% yield.

[0219] 1 H NMR (400MHz, DMSO-d6) δ8.59 (s, 1H), 7.94 (d, J=7.9Hz, 1H), 7.89 (dd, J=7.9, 1.4H z, 1H), 7.63 (t, J=7.6Hz, 1H), 7.59-7.52 (m, 2H), 7.51-7.39 (m, 3H), 3.51 (s, 3H).

[0220] Example 21:

[0221] (E)-5-fluoro-4-(2-chlorophenyl)-2-[1-methyl-2-(2-carboxy-4-fluorobenzyl)hydrazyl]thiazole (21)

[0222]

[0223] Similar to the method in Example 20, 2-aldehyde-4-fluorobenzoic acid was substituted for 2-aldehyde-benzoic acid to obtain title compound 21.

[0224] 1H NMR (400MHz, DMSO-d6) δ13.64 (s, 2H), 8.54-8.50 (m, 1H), 7.98 (dd, J=8.8, 5.6Hz, 1 H), 7.64 (dd, J=9.4, 2.9Hz, 1H), 7.59-7.48 (m, 3H), 7.47-7.39 (m, 2H), 3.51 (s, 3H).

[0225] Example 22:

[0226] (E)-2-((2-(4-(2-chlorophenyl)thiazolyl-2-yl)-2-methylhydrazineyl)methyl)-N-hydroxybenzamide (22)

[0227]

[0228] Following the synthetic method reported in the literature, the intermediate compound (E)-4-(2-chlorophenyl)-2-[1-methyl-2-(2-carboxybenzyl)hydrazyl]thiazole was obtained.

[0229] Weigh 1.0 g (2.6 mmol) of the above intermediate compound into a 100 mL round-bottom flask, add 30 mL of tetrahydrofuran, and stir at room temperature until the solid is completely dissolved. Add 484 mg (3.0 mmol) of carbonyl diimidazole, and react at room temperature for 16 hours. Then, under an ice-water bath, add the reaction mixture dropwise to 20 mL of 50% hydroxylamine aqueous solution and stir for 1 hour. Add 50 mL of saturated saline solution and extract three times with ethyl acetate (50 mL * 3). Combine the organic phases, concentrate, and separate by column chromatography to obtain 135 mg of the title compound as a yellow solid, in 13% yield.

[0230] 1 H NMR (400MHz, Methanol-d4, DMSO-d6) δ8.46 (s, 1H), 8.11 (d, J=8.0Hz, 1H), 7.99 (dd, J=7. 8, 1.8Hz, 1H), 7.91-7.84 (m, 1H), 7.67 (t, J=7.6Hz, 1H), 7.56-7.29 (m, 5H), 3.73 (s, 3H).

[0231] Example 23:

[0232] (E)-2-((2-(4-(2-chlorophenyl)thiazolyl-2-yl)-2-methylhydrazineyl)methyl)-N-methoxybenzamide (23)

[0233]

[0234] Weigh 500 mg (1.3 mmol) of (E)-4-(2-chlorophenyl)-2-[1-methyl-2-(2-carboxybenzyl)hydrazino]thiazole into a 100 mL round-bottom flask, add 25 mL of tetrahydrofuran, and stir at room temperature until the solid is completely dissolved. Add 512 mg (1.3 mmol) of HATU, 271 mg (2.6 mmol) of triethylamine, and 167 mg (2.0 mmol) of O-methylhydroxylamine hydrochloride, and react at room temperature for 16 hours. Add 50 mL of water and extract three times with ethyl acetate (50 mL * 3). Combine the organic phases, concentrate, and separate by column chromatography to give 167 mg of the title compound as a white solid, yield 30%.

[0235] 1 H NMR (400MHz, DMSO-d6) δ7.81 (dd, J=7.7, 2.0Hz, 1H), 7.77 (d, J=7.3Hz, 1H), 7.72-7.62 (m, 2H), 7.60-7.52 (m, 1H), 7. 47 (dd, J=7.8, 1.5Hz, 1H), 7.39-7.23 (m, 3H), 6.94 (d, J=3.4Hz, 1H), 5.97 (d, J=3.3Hz, 1H), 3.91 (s, 3H), 3.21 (s, 3H).

[0236] Example 24:

[0237] (E)-2-((2-(3-(2-chlorophenyl)-1,2,4-thiadiazol-5-yl)-2-methylhydrazine)methyl)benzoic acid (133)

[0238]

[0239] Step (a) 2-Chlorophenylmethylamidine

[0240] 73.0 mL of a 2.0 M, 146.0 mmol tetrahydrofuran solution of bis(trimethylsilylamino)lithium in tetrahydrofuran was placed in a 500 mL reaction flask, and 150 mL of tetrahydrofuran was added. Under nitrogen protection and temperature control (0-10 °C), 10.0 g (73.0 mmol) of 2-chlorobenzonitrile in 50 mL tetrahydrofuran solution was slowly added dropwise. TLC analysis showed complete consumption of the initial material. The reaction solvent was removed by concentration, and 200 mL of purified water was added. The mixture was then extracted three times with dichloromethane (200 mL x 3). The organic phases were combined and washed once each with 2 M hydrochloric acid (200 mL) and 300 mL saturated brine. The solvent was removed by concentration to obtain 9.5 g of a yellow solid, which was directly added to the next reaction step. LCMS: 155.1 ([M+H) + ).

[0241] Step (b) 2-Chloro-N-(methylthiourea)phenylformamidin

[0242] 9.5 g (61.3 mmol) of 2-chlorophenylformamidinium and 6.8 g (92.5 mmol) of methyl isocyanate were weighed and placed in a 500 mL reaction flask, and 170 mL of acetonitrile was added. The temperature was controlled below 20 °C, and triethylamine (18.7 g, 185.1 mmol) was slowly added dropwise. After the addition was complete, the reaction mixture was allowed to react at room temperature for 3 hours. After the starting material was completely consumed by LCMS, the reaction solvent was concentrated and purified by column chromatography (petroleum ether:ethyl acetate = 20:1) to give 10.0 g of a yellow solid. The overall yield of this two-step reaction was 60.4%.

[0243] 1 H NMR (300MHz, DMSO-d6): δ9.03 (d, J=2.3Hz, 1H), 8.60-8.56 (m, 1H), 7.52-7.32 (m, 5H), 2.84 (d, J=2.3Hz, 3H). LCMS: 228.1 ([M+H]+).

[0244] Step (c) 3-(2-chlorophenyl)-N-methyl-1,2,4-thiadiazole-5-amine

[0245] 10.0 g (44.1 mmol) of 2-chloro-N-(methylthiourea)phenylformamidinium and 13.4 g (52.9 mmol) of elemental iodine were weighed and placed in a 250 mL reaction flask. Acetonitrile (100 mL) and potassium carbonate (9.1 g, 66.1 mmol) were added. After stirring at room temperature for 0.5 hours, the reactants were monitored by LC-MS to ensure complete consumption. Water (200 mL) was added to the reaction solution, and the mixture was extracted three times (200 mL x 3) with ethyl acetate. The organic phases were combined and washed twice with saturated brine (200 mL x 2), dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (petroleum ether:ethyl acetate = 5:1) to give 5.0 g of a yellow solid, yield 50.4%.

[0246] 1 H NMR (300MHz, DMSO-d6): δ8.49 (brs, 1H), 7.78-7.75 (m, 1H), 7.55-7.53 (m, 1H), 7.46-7.41 (m, 2H), 2.94 (d, J=4.8Hz, 3H). LCMS: 226.0 ([M+H] + ).

[0247] Step (d) N-(3-(2-chlorophenyl)-1,2,4-thiadiazol-5-yl)-N-methyldiazepine

[0248] Weigh 500 mg (2.2 mmol) of 3-(2-chlorophenyl)-N-methyl-1,2,4-thiadiazol-5-amine into a 50 mL reaction flask. Add 5 mL of 2 M hydrochloric acid at 0 °C, followed by dropwise addition of 2 mL of an aqueous solution of sodium nitrite (150 mg, 2.2 mmol). Incubate the mixture at 0–5 °C for 1 hour. After complete consumption of the reactants by LC-MS, pour the reaction mixture into 2 mL of water and extract three times (5 mL x 3) with ethyl acetate. Combine the organic phases and wash once with saturated brine (10 mL). Dry the mixture with anhydrous sodium sulfate and then perform column chromatography (petroleum ether:ethyl acetate = 1:1) to obtain 200 mg of a white solid, with a yield of 35.4%.

[0249] 1 H NMR (300MHz, DMSO-d6): δ8.02-7.99 (m, 1H), 7.73-7.70 (m, 1H), 7.66-7.56 (m, 2H), 4.62 (s, 3H). LCMS: 255.0 ([M+H] + ).

[0250] Step (f) 3-(2-chlorophenyl)-5-(1-methylhydrazine)-1,2,4-thiadiazole

[0251] 100 mg (1.57 mmol) of zinc powder and 2 mL of glacial acetic acid were weighed and placed in a 25 mL reaction flask. The mixture was heated to 45 °C, and 2 mL of a glacial acetic acid solution of N-(3-(2-chlorophenyl)-1,2,4-thiadiazol-5-yl)-N-methyldiazamide (200 mg, 0.79 mmol) was slowly added dropwise. The mixture was stirred at 45-50 °C for 12 hours. After the starting material was completely consumed under LCMS monitoring, the zinc powder was removed by filtration and the solution was washed twice with ethyl acetate (20 mL * 2). The mother liquor was washed three times with saturated sodium carbonate solution (10 mL * 3) and three times with saturated brine (10 mL * 3). After drying with anhydrous sodium sulfate, the solution was purified by column chromatography (petroleum ether:ethyl acetate = 1:1) to obtain 64 mg of white solid, with a yield of 67.7%.

[0252] 1 H NMR (300MHz, DMSO-d6): δ7.74 (dd, J=7.4, 2.0Hz, 1H), 7.53 (dd, J=7.8, 1.4Hz, 1H), 7.47-7.38 (m, 2H), 5.65 (s, 2H). LCMS: 241.1 ([M+H] + ).

[0253] Step (g) (E)-2-((2-(3-(2-chlorophenyl)-1,2,4-thiadiazol-5-yl)-2-methylhydrazine)methyl)benzoic acid

[0254] 60 mg (0.25 mmol) of 3-(2-chlorophenyl)-5-(1-methylhydrazyl)-1,2,4-thiadiazole, 45 mg (0.3 mmol) of o-aldehyde benzoic acid, and 3 mL of tetrahydrofuran were weighed and placed in a 10 mL reaction flask. The mixture was heated to 60 °C and reacted for 6 hours. After the starting materials were completely consumed by LCMS monitoring, the reaction solvent was concentrated and removed. The remaining solid was slurried with 5 mL of methanol to give 41 mg of the title compound as a white solid.

[0255] 1 H NMR (400MHz, DMSO-d6): δ13.39 (brs, 1H), 8.78 (s, 1H), 7.98-7.94 (m, 2H), 7.88 (dd, J=7. 6, 2.0Hz, 1H), 7.71 (t, J=7.5Hz, 1H), 7.62-7.43 (m, 4H), 3.73 (s, 3H). LCMS: 373.0 ([M+H] + ).

[0256] Example 25: Salt Preparation

[0257] Compound 21(E)-5-fluoro-4-(2-chlorophenyl)-2-[1-methyl-2-(2-carboxylic acid-4-fluorobenzylidene)hydrazino]thiazole (300 mg, 0.74 mmol) was weighed and dissolved in tetrahydrofuran (5 mL), and sodium hydroxide (29.5 mg, 0.74 mmol) was added. The mixture was stirred at room temperature for 2 hours, and the solvent was removed by concentration to give 328 mg of the pale yellow solid sodium salt of compound 21 (yield 99%). 1 H NMR (400MHz, DMSO-d6) δ9.09 (s, 1H), 7.84 (dd, J=8.8, 5.9Hz, 1H), 7.64-7.38 (m, 5H), 7.17 (td, J=8.5, 2.8Hz, 1H), 3.47 (s, 3H).

[0258] Referring to the above method, the compounds of this application can be reacted with different bases to convert them into pharmaceutically acceptable salts.

[0259] Activity Test 1: Inhibitory Activity Test of the Compounds in this Application against Human DHODH Enzyme

[0260] This method links the activity of the DHODH enzyme to the colorimetric effect of the dye 2,6-dichlorophenolindophenol (DCIP). Specifically, purified recombinant human DHODH is diluted to 0.4 μg / mL with test buffer (containing 50 mM tris(hydroxymethyl)aminomethane, 150 mM potassium chloride, 0.1% Triton X-100, pH 8.0); a substrate solution containing 2 mM L-dihydroorotic acid, 0.2 mM decyl ubiquinone, and 0.12 mM DCIP is prepared using the same test buffer; 50 μL of the DHODH enzyme solution and 50 μL of the substrate solution are added to each well of a 96-well plate, mixed thoroughly for 5 seconds, and then the absorbance is measured at 600 nM using a Spectramax spectrophotometer. Enzyme activity can be calculated by adding different test compounds and observing the different absorbance values. Purified recombinant human DHODH enzyme was purchased from R&D Systems (catalog number: 10062-DD), and other reagents were purchased from Sigma-Aldrich. Brequinar (purchased from Sigma-Aldrich) was used as a positive control in the experiment. The inhibition rate of cell proliferation by the compounds was calculated based on cell viability; and the half-maximal inhibitory concentration (IC50) was fitted based on the inhibition rates of different compound concentrations. 50 Values ​​(see Table 1 for details).

[0261] Table 1

[0262]

[0263] Activity Test 2: Anti-proliferative activity test of the compound of this application against human monocytic leukemia cells THP-1

[0264] THP-1 cells at 5000 cells / well were seeded in 96-well plates with 90% RPMI 1640 (Gibco, catalog number 22400-089), 10% fetal bovine serum (Coming, catalog number 35-076-CV), and 0.05 mM mercaptoethanol (Sigma-Aldrich, catalog number 63689) and cultured overnight. Cells were then incubated with different concentrations of the test compounds for 72 hours. Cell viability was analyzed using the Promega Cell Titer-Glo luminescence assay kit (Promega-G7573) according to the supplier's instructions. The inhibition rate of cell proliferation by the compounds was calculated based on cell viability; and the half-maximal inhibitory concentration (IC50) was fitted based on the inhibition rates of different compound concentrations. 50 Values ​​(see Table 2 for details). In the experiment, Staurosporine was selected as the positive control compound.

[0265] Table 2

[0266]

[0267] Activity Test 3: Anti-proliferative activity test of the compound of this application against human promyelocytic leukemia cells HL-60

[0268] HL-60 cells at 2500 cells / well were seeded in 96-well plates with 80% IMDM (Gibco, catalog number 12440053) and 20% fetal bovine serum (Corning, catalog number 35-076-CV) and cultured overnight. Cells were then incubated with different concentrations of the test compound for 72 hours. Cell viability was analyzed using the Promega CellTiter-Glo luminescence assay kit (Promega-G7573) according to the supplier's instructions. The inhibition rate of cell proliferation by the compound was calculated based on cell viability; and the half-maximal inhibitory concentration (IC50) was fitted based on the inhibition rates of different compound concentrations. 50 Values ​​(see Table 3 for details). In the experiment, Staurosporine was selected as the positive control compound.

[0269] Table 3

[0270]

[0271] Activity Test 4: Anti-proliferative activity test of the compound of this application against human small cell lung cancer cells NCI-H82

[0272] 5000 NCI-H82 cells / well were seeded in 96-well plates with 90% RPMI 1640 (Gibco, catalog number 22400-089) and 10% fetal bovine serum (Corning, catalog number 35-076-CV) and cultured overnight. Cells were then incubated with different concentrations of the test compound for 72 hours. Cell viability was analyzed using the Promega CellTiter-Glo luminescence assay kit (Promega-G7573) according to the supplier's instructions. The inhibition rate of cell proliferation by the compound was calculated based on cell viability; and the half-maximal inhibitory concentration (IC50) was fitted based on the inhibition rates of different compound concentrations. 50 Values ​​(see Table 4 for details). In the experiment, Staurosporine was selected as the positive control compound.

[0273] Table 4

[0274]

[0275] Based on enzyme activity data, the compounds in this application exhibit good DHOHD enzyme inhibitory activity, with some compounds showing enzyme activity ≤5 nM. The compounds in this application also demonstrate good anti-proliferative activity against tumor cells, with tested IC50 values... 50 Below approximately 1 μM.

[0276] Activity Test 5: The efficacy of the compound in this application against a THP-1 cell xenograft model

[0277] Female CB17 SCID mice, 6-8 weeks old and weighing 18-22 grams, were selected. THP-1 cells (10 × 10^6 cells / 0.2 mL with Matrigel) were subcutaneously injected into each mouse. After tumor formation, the average tumor size reached 100-150 mm. 3 At that time, medication should be started.

[0278] To investigate whether tumor growth has been inhibited, slowed, or cured, the tumor diameter was measured twice weekly using calipers. The formula for calculating tumor volume is: V = 0.5a × b 2 a and b represent the long and short diameters of the tumor, respectively. Examples 20 and 21, and the positive control compound cytarabine, show the results for this animal model as follows: Figure 1 .

[0279] The compounds prepared in Examples 20 and 21, after oral administration for 29 days, showed dose-dependent inhibition of tumor growth. Example 20 (dose 9 mg / kg) achieved a tumor inhibition rate (TG) of 97%; Example 21 (dose 15 mg / kg) achieved a tumor inhibition rate of 85%. The positive control compound cytarabine (10 mg / kg) showed a tumor inhibition rate of 45%. The tumor inhibition rates of both compounds exceeded those of cytarabine.

[0280] Activity Test 6: The efficacy of the compound of this application on an HL-60 cell xenograft model

[0281] Balb / c nude mice, female, 6-8 weeks old, weighing 18-22 grams, were selected. HL-60 cells (10×10^6 cells / 0.2 mL with Matrigel) were subcutaneously injected into each mouse. After tumor formation, the average tumor size reached 100-150 mm. 3 At that time, medication should be started.

[0282] To investigate whether tumor growth has been inhibited, slowed, or cured, the tumor diameter was measured twice weekly using calipers. The formula for calculating tumor volume is: V = 0.5a × b 2 a and b represent the long and short diameters of the tumor, respectively. Examples 20 and 21, and the positive control compound cytarabine, show the results for this animal model as follows: Figure 2 .

[0283] The compounds prepared in Examples 20 and 21, after oral administration for 18 days, showed dose-dependent inhibition of tumor growth. Example 20 (dose 22.5 mg / kg) achieved a tumor inhibition rate (TG) of 101.6%; Example 21 (dose 36 mg / kg) achieved a tumor inhibition rate of 102.4%. The positive control compound cytarabine (25 mg / kg) showed a tumor inhibition rate of 77.9%. The tumor inhibition rates of both compounds exceeded those of cytarabine.

[0284] Activity Test 7: The efficacy of the compound of this application on the NCI-H82 cell xenograft model

[0285] Balb / c nude mice, female, 6-8 weeks old, weighing 18-22 grams. NCI-H82 cells (5 × 10^6 cells / 0.2 mL with Matrigel) were subcutaneously injected into each mouse. After tumor formation, the average tumor size reached 100-150 mm. 3 At that time, medication should be started.

[0286] To investigate whether tumor growth has been inhibited, slowed, or cured, the tumor diameter was measured twice weekly using calipers. The formula for calculating tumor volume is: V = 0.5a × b 2 a and b represent the long and short diameters of the tumor, respectively. The results of Examples 20, 21, and the positive control compound cisplatin + etoposide in this animal model are as follows: Figure 3 .

[0287] The compounds prepared in Examples 20 and 21, after oral administration for 31 days, showed dose-dependent inhibition of tumor growth. Example 20 (dose 22.5 mg / kg) achieved a tumor inhibition rate (TG) of 96.2%; Example 21 (dose 36 mg / kg) achieved a tumor inhibition rate of 95.3%. The positive control compound cisplatin combined with etoposide (2 mg / kg + 4 mg / kg) achieved a tumor inhibition rate of 58.7%. The tumor inhibition rates of both compounds exceeded those of the cisplatin and etoposide combination group.

[0288] Based on data from a mouse xenograft tumor model, the compounds in this application demonstrated excellent antitumor proliferative activity in animal models.

[0289] Activity Test 8: Pharmacokinetics of the Compounds in this Application in Mice

[0290] The compound was dissolved in physiological saline containing 5% DMSO and 10% Solutol (all percentages are volume ratios) to prepare solutions of 0.2 mg / mL and 1 mg / mL, respectively. The 0.2 mg / mL solution was used for intravenous administration, and the 1 mg / mL solution was used for gavage administration.

[0291] Pharmacokinetic studies were conducted on ICR mice, with three mice per group. The intravenous dose was 5 mL / kg, and the oral dose was 10 mL / kg. Blood samples were collected at 0, 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 hours after intravenous administration, and at 0, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after oral administration. Blood was collected via the orbital venous plexus, with 0.03 mL samples taken at each time point. Samples were placed in tubes containing K2-EDTA and stored on ice until centrifugation. Within one hour of collection, samples were centrifuged at 4000 RPM for 6 minutes at 2–8°C, and then frozen at approximately -80°C. The supernatant was used for LC / MS / MS analysis. The LC / MS / MS system was LC-MS / MS-23 (TQ6500+Triplequad). Samples were separated on an Acquity UPLC BEH C18 column, 1.7 μM, 50 × 2.1 mm, using a linear gradient elution starting with 30% mobile phase B. Mobile phase A was an aqueous solution containing 0.1% formic acid, and mobile phase B was an acetonitrile solution containing 0.1% formic acid. LC / MS / MS data were obtained using positive ion electrospray ionization, and plasma concentrations of the compounds were quantified using standard curves. After obtaining the plasma drug concentrations at each time point, the T0 values ​​were fitted using the FDA-approved pharmacokinetic program PhoenixWinNonlin 7.0 (Pharsight, USA). 1 / 2 C max T max And pharmacokinetic parameters such as bioavailability (F%).

[0292] The pharmacokinetic results of some of the compounds in the examples are detailed in Table 5.

[0293] Table 5

[0294] F(％) 30.99±4.62 19.43±2.64 56.75±5.05 73.06±11.73

[0295] The metabolic data showed that, compared with the reference compound, the compounds prepared in Examples 20 and 21 had significantly improved bioavailability (F(%)) after oral administration.

[0296] As a specific embodiment of this application, referring to the above synthesis method, preferably, the compound of general formula Ib further includes the compounds prepared in the examples shown in Table 6:

[0297]

[0298] Table 6

[0299]

[0300]

[0301]

[0302]

[0303] As a specific embodiment of this application, referring to the above synthesis method, preferably, the compound of general formula Ia further includes the compounds prepared in the examples shown in Table 7:

[0304]

[0305] Table 7

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314] As a specific embodiment of this application, referring to the above synthesis method, preferably, the compound of general formula Ic further includes the compounds prepared in the examples shown in Table 8:

[0315]

[0316] Table 8

[0317]

[0318]

[0319] The activity test results above show that the compounds of this application possess excellent DHODH inhibitory activity, and some of these compounds inhibit the IC50 of DHODH. 50 The values ​​even reach levels less than 1 nM. Therefore, the compounds of this application are novel DHODH inhibitors with low toxicity and high safety, and can be used as anti-inflammatory agents, autoimmune disease treatment agents, immunosuppressants, anticancer agents, and viral infection treatment agents.

[0320] It should be understood that after reading the above disclosure of this application, those skilled in the art can make various modifications, adjustments, or combinations to the technical solution of this application without departing from the concept and spirit of this application, and these equivalent forms also fall within the scope of protection claimed in this application.

Claims

1. A compound represented by formula I or a pharmaceutically acceptable salt thereof, Equation I; in, R1 or R2 is independently selected from hydrogen, halogen, methyl, methoxy, trifluoromethyl, trifluoromethoxy, and cyano; R3 is NR7-O-R5; R4 can be any one of C. 1-3 Alkyl or unsubstituted cyclopropyl or cyclobutyl, fluorinated cyclopropyl or cyclobutyl; M is a nitrogen atom or CR6; R6 is selected from fluorine; R5 or R7 is independently selected from hydrogen, methyl, and ethyl; m or n can be any value of 0, 1, or 2.

2. A compound selected from the following compounds or their pharmaceutically acceptable salts:

3. A compound of formula II, Formula II; in, M is nitrogen or CR6, and R6 is selected from fluorine; R2 is arbitrarily selected from hydrogen, halogen, methyl, methoxy, trifluoromethyl, trifluoromethoxy, and cyano; R4 can be any one of C. 1-3 Alkyl or unsubstituted cyclopropyl or cyclobutyl, fluorinated cyclopropyl or cyclobutyl; R8 is an amino protecting group, which is selected from tert-butoxycarbonyl or benzyloxycarbonyl; and m is arbitrarily 0, 1 or 2.

4. A pharmaceutical composition comprising the compound of any one of claims 1 or 2, or a pharmaceutically acceptable salt or a pharmaceutically acceptable carrier thereof.

5. Use of the pharmaceutical composition of claim 4 in the preparation of a medicament for treating or preventing DHODH-mediated diseases.

6. The use as described in claim 5, wherein, The DHODH-mediated diseases are selected from autoimmune diseases, tumors, and viral infections.

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