Acyl thiourea compound as well as preparation method and application thereof

Abstract

The invention discloses an acylthiourea compound as well as a preparation method and application thereof. The preparation method comprises the following steps: firstly, preparing an acyl isothiocyanate intermediate from caffeic acid serving as a raw material, protecting hydroxyl on a benzene ring of the raw material by using TBS, then reacting with primary amine to prepare an acylthiourea intermediate, and finally, removing the TBS protective agent under the action of TBAF, thereby obtaining the acylthiourea compound. Experiments show that the acylthiourea compound has a medicinal value, can be used for treating inflammatory bowel diseases, and can be applied to development of related drugs.

Description

Technical Field

[0001] The invention belongs to the field of medical technology and relates to preparing acylthiourea compounds by chemical synthesis using caffeic acid as a starting material and verifying the pharmacological effects thereof on an established IBD model. Background Art

[0002] Inflammatory bowel disease (IBD) is a chronic, nonspecific intestinal inflammatory disease with two clinical subtypes: ulcerative colitis (UC) and Crohn's disease (CD). When the disease is prolonged, patients may experience complications such as intestinal perforation, obstruction, abdominal abscess, and even toxic megacolon, which seriously affect the quality of life of patients and cause great pain to patients. IBD is prone to occur in young and middle-aged people. The number of IBD patients in Europe is 2.6 to 3.7 million, and there are more than 1 million IBD patients in North America. In recent years, the incidence of IBD has been on the rise, and it has become another major threat to intestinal health after colorectal cancer. At present, the clinical drugs used to treat IBD mainly include steroidal anti-inflammatory drugs such as glucocorticoids, non-steroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen, and immunosuppressants such as azathioprine. The advantage of these drugs is that they can control inflammation quickly. However, some side effects will occur after long-term exposure. For example, the use of hormone drugs is prone to various adverse reactions such as hypertension, diabetes, osteoporosis, etc. The use of purine drugs may increase the risk of malignant tumors such as blood diseases and skin cancer. For patients who are ineffective with hormone and immunosuppressant treatment or are hormone-dependent and cannot tolerate the treatment of the above drugs, although biological agents such as TNF-α monoclonal antibodies can also be used for treatment, the long-term use effect is not ideal and is prone to cause adverse reactions such as headaches, nausea, and upper respiratory tract infections. For patients with IBD who cannot be controlled by drug treatment, only surgery can be performed, and postoperative fistulas and complications will seriously reduce the patient's quality of life. Therefore, the development of new drugs for the treatment of inflammatory bowel disease is imminent.

[0003] Natural products are of great significance for the discovery, design and synthesis of new drugs, and are also an important source of bioactive substances and effective ingredients of drugs. The advantage of natural products in drug development is that biosynthesized natural product small molecules (SMNPs) have better biocompatibility with enzymes and receptors, making them easier or more suitable as lead compounds for drugs.

[0004] Caffeic acid (3,4-dihydroxycinnamic acid) is a polyphenolic natural product widely found in fruits, grains and some mint family herbs. It has a wide range of biological activities such as antibacterial, anti-inflammatory, cancer prevention and neuroprotection. The structure of caffeic acid is shown below:

[0005] In view of the fact that caffeic acid compounds, such as phenyl acrylic acid, also have multiple effects such as anti-inflammatory, caffeic acid derivatives with targeting groups or other functional groups have been designed and synthesized in the development of many drugs (see CN103922936A, CN117304033A, etc.), but the experiments and verification of the anti-inflammatory activity and related pharmacological effects of the modified molecules are often ignored. In addition, some compounds with obvious structural differences from caffeic acid have also been reported to be used in the development of drugs for the treatment of inflammation and related diseases. For example, CN1615295A provides acyl, sulfur, carbamate, thiocarbamate and related compounds, and verifies their activity in inhibiting TNF-α, etc., but they are mainly used to treat diabetes and arthritis; CN1705642A provides N-hydroxythiourea, urea and amide compounds, but they are mainly used to play an analgesic effect in the treatment of inflammatory diseases. At present, there are no research reports on acylthiourea compounds based on the caffeic acid parent core structure and their anti-inflammatory activity. Summary of the invention

[0006] The object of the present invention is to provide an acylthiourea compound and a preparation method and use thereof, so as to broaden the application scope of the compound obtained by modifying caffeic acid (including the treatment of inflammatory bowel disease).

[0007] In order to achieve the above object, the present invention adopts the following technical solutions: In a first aspect, an acylthiourea compound is provided, wherein the compound is a compound having a structure as shown in Formula I or any one of the pharmaceutically acceptable salts of the compound:

[0008] In formula I, R is selected from any one of an alkyl group, a cycloalkyl group, a heterocycloalkyl group, a substituted alkyl group, an aryl group, a substituted aryl group, and a heterocycloaryl group.

[0009] Preferably, the R is selected from an alkyl group or a substituted alkyl group, wherein the number of carbon (C) atoms of the alkyl group is 1 to 10.

[0010] Preferably, R is selected from cycloalkyl, heteroaryl or heterocycloalkyl, wherein the number of ring atoms (i.e., atoms constituting the ring) is 3 to 7 (i.e., between a three-membered ring and a seven-membered ring), and the heteroatom is N or O; the heteroaryl or heterocycloalkyl may be substituted on the ring, for example, with alkyl, alkoxy and other substituents.

[0011] Preferably, the R is selected from an aryl group, specifically a phenyl group, a naphthyl group, and the like.

[0012] Preferably, R is selected from substituted aromatic groups, wherein the substituent groups on the aromatic ring are one or more of halogen, alkyl, alkoxy, haloalkyl, dioxa, mercapto, and nitro.

[0013] Preferably, the acylthiourea compound is any one of the compounds shown in formulas Ⅰ-1 to Ⅰ-37 or the pharmaceutically acceptable salts of the compounds: .

[0014] In a second aspect, a method for preparing an acylthiourea compound is provided, comprising the following steps: The invention uses caffeic acid as a raw material, utilizes the phenyl acryloyl group in the structure of the raw material and obtains the acyl thiourea compound modified by caffeic acid through multi-step chemical synthesis.

[0015] Preferably, the preparation method of the acylthiourea compound, for example, the compound having a structure as shown in the above formula I, specifically comprises the following steps: 1) Prepare acyl isothiocyanate intermediates when the phenolic hydroxyl group in the caffeic acid structure is protected, and then prepare acyl thiourea intermediates through nucleophilic addition reaction; 2) preparing the target acyl thiourea compound by deprotecting the acyl thiourea intermediate prepared in step 1.

[0016] Preferably, in step 1, the acyl isothiocyanate intermediate is a 2 The organic amine undergoes the nucleophilic addition reaction.

[0017] Preferably, in step 1, the solvent used in the reaction is tetrahydrofuran (THF), acetonitrile, benzene, dichloromethane (DCM) or chloroform; the reaction temperature is 25 to 35 °C, the reaction time is 1 to 3 h, and the acyl isothiocyanate intermediate and the structure R-NH 2 The molar ratio of the organic amine is 1:1-2; after the reaction is completed, the solid product in the system is dissolved and chromatographed on a silica gel column (specifically, mixing the dissolved solid product with the chromatography column filler, evaporating the solvent under reduced pressure, loading the filler into the column, and eluting) to obtain an acyl thiourea intermediate.

[0018] Preferably, the dissolution uses dichloromethane, acetonitrile, tetrahydrofuran or the like as a solvent.

[0019] Preferably, the mobile phase used in the silica gel column chromatography for purifying the acylthiourea intermediate is a petroleum ether (PE) / ethyl acetate (EA) mixed solvent (volume ratio of PE:EA=10-20:1).

[0020] Preferably, in step 2, the solvent used in the reaction is tetrahydrofuran, dichloromethane, acetonitrile, N,N-dimethylformamide (DMF) or 18-crown-6 ether; the reaction temperature is -5 to 5 °C, the reaction time is 1 to 3 h, and the molar ratio of the acyl thiourea intermediate to the deprotection reagent (specifically the reaction reagent) is 1:1.5 to 3; after the reaction is completed, extraction, washing, drying, vacuum concentration, and silica gel column chromatography (specifically including packing, vacuum concentration, sample loading, and elution) are sequentially performed to obtain the compound shown in formula I.

[0021] Preferably, the deprotection reagent is TBAF / AcOH, 4-methoxyhydrogen aldehyde·BF 3 TBAF / NH 4 F. TBAF·(t-BuOH) 4 、(Me 2 N) 3 S + F 2 SiMe 3 - (TAS-F), KF, KF·Al 2 O 3 、Bu 4 NCl / KF·H 2 O, Aq·HF, Pyridine·HF or AcOH / H 2 O / THF (3:1:1).

[0022] Preferably, the solvent added in the extraction is ethyl acetate, dichloromethane or petroleum ether.

[0023] Preferably, the mobile phase used in the silica gel column chromatography for purifying the compound represented by formula I is a DCM / methanol (MeOH) mixed solvent (the volume ratio of DCM:MeOH = 10 to 20:1) or an EA / MeOH mixed solvent (the volume ratio of EA:MeOH = 10 to 20:1).

[0024] In a third aspect, an anti-inflammatory drug is provided, wherein the main active ingredient of the drug is the above-mentioned acylthiourea compound.

[0025] Preferably, in addition to the main active ingredient, the drug also contains a small amount of secondary ingredients and / or formulation excipients that do not affect the efficacy of the main active ingredient. For example, anti-inflammatory drugs may contain sweeteners to improve the taste, antioxidants to prevent oxidation, and pharmaceutically acceptable carriers.

[0026] Preferably, the dosage form of the anti-inflammatory drug is not limited, as long as it is a dosage form that can effectively allow the main active ingredients to reach the body. For example, the anti-inflammatory drug can be in a common dosage form such as an injection or a sustained-release dosage form such as a nanoformulation.

[0027] In a fourth aspect, the invention provides the use of the above-mentioned acylthiourea compounds in the preparation of anti-inflammatory drugs.

[0028] Preferably, the anti-inflammatory drug has the effect of treating non-specific intestinal inflammatory diseases (such as inflammatory bowel disease).

[0029] In a fifth aspect, the invention provides the use of the above-mentioned acylthiourea compounds in the preparation of a method for preventing and / or treating NLRP3 inflammasome-related diseases.

[0030] Some of the terms that appear in the technical solutions of the present invention are defined as follows: The term "pharmaceutically acceptable salt" refers to a salt (e.g., sodium salt, potassium salt, etc.) formed by the acylthiourea compound of the present invention and a pharmaceutically acceptable base (e.g., inorganic base), wherein the inorganic base includes sodium hydroxide, potassium hydroxide, sodium hydride, etc.; wherein "pharmaceutically acceptable" means suitable for use in humans without excessive adverse side effects (e.g., toxicity, irritation, and allergic reactions), that is, having a reasonable benefit / risk ratio.

[0031] The beneficial effects of the present invention are embodied in: The present invention screens the drug activity of the synthesized acylthiourea compounds of the general formula I structure (involving a red blood cell hemolysis model) and selects the selected small molecules (such as compound I-15) for pharmacological experimental verification (involving an inflammatory bowel disease mouse model). The results show that the acylthiourea compounds can make the clinical scores of inflammatory bowel disease tend to normal, improve the shortening of the colon, increase the number of goblet cells, arrange cells neatly, and reduce the histopathological score. At the same time, the compounds have the effect of inhibiting the hemolysis of red blood cells caused by nigericin stimulation. The present invention reveals for the first time through experiments the therapeutic effect of acylthiourea compounds on inflammatory bowel disease; at the same time, they can be used as active ingredients to prepare drugs for anti-inflammatory and prevention and / or treatment of NLRP3 inflammasome-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The inflammatory bowel disease (IBD) mouse model was established; (A) mouse body weight changes; (B) clinical score analysis; (C) mouse colon anatomical appearance; (D) colon length analysis (N = 7, ****p < 0.0001); (E) H&E staining of colon sections; Ctrl represents mice drinking normal water; DSS represents mice drinking 3% DSS solution.

[0033] Figure 2The therapeutic effect of compound Ⅰ-15 on inflammatory bowel disease; wherein: (A) changes in mouse body weight; (B) clinical score analysis; (C) anatomical appearance of mouse colon; (D) colon length analysis (N = 6, ns means no statistical difference, ***p < 0.001, ****p < 0.0001); (E) H&E staining of colon sections; Ctrl represents normal drinking water mice injected with PBS; PBS represents IBD mice injected with PBS; Ⅰ-15 represents IBD mice injected with compound Ⅰ-15.

[0034] Figure 3 The following is the overall synthetic route of the acylthiourea compounds in the examples.

[0035] Figure 4 This is the synthetic route of compound Ⅰ-15 in the example.

[0036] Figure 5 The toxicity test results of compound I-15 in the examples are as follows: (A) analysis of lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA) and urea nitrogen (UREA) in the peripheral blood of mice after intraperitoneal injection of I-15 for 4 days (N = 8, ns indicates no statistical difference); (B) H&E staining of liver and kidney sections of mice after intraperitoneal injection of compound I-15 for 4 days; Ctrl represents the control group mice injected with PBS for 4 days, and I-15 represents the experimental group mice injected with compound I-15 for 4 days. DETAILED DESCRIPTION

[0037] The present invention is further described below in conjunction with the accompanying drawings and examples. It should be understood that the examples are only used to illustrate the present invention or understand the present invention and are not used to limit the scope of protection of the present invention.

[0038] 1. Design and synthesis of acylthiourea compounds See also Figure 3 , using caffeic acid as the raw material, the phenolic hydroxyl group on caffeic acid (i.e., the hydroxyl group on the benzene ring in caffeic acid) is protected by TBS, and then the carboxyl group is converted into an acyl chloride, and then a three-step reaction is carried out with ammonium thiocyanate for nucleophilic substitution to obtain an acyl isothiocyanate intermediate (see compound 1), which is then subjected to a nucleophilic addition reaction with an organic (primary) amine to obtain an acyl thiourea intermediate (see compound 2), and finally the TBS protective agent is removed under the action of tetrabutylammonium fluoride (TBAF) to obtain an acyl thiourea compound with a structure as shown in formula I.

[0039] The synthesis of compound 1 can be carried out with reference to existing literature.

[0040] 2. Synthesis examples of acylthiourea compounds 2.1 Synthesis of compounds Ⅰ-1 to Ⅰ-22 Step ① Add the compound 1 (449.8 mg, 1 mmol) and 5 mL of acetonitrile to a 20 mL reaction bottle containing a magnetic electron, add p-bromoaniline and other organic amines with different structures (1.0 mmol) to the reaction bottle under argon protection and stirring conditions, and stir the resulting mixture for reaction at room temperature for 1.0 h. A large amount of white solid will be produced during the stirring process, and TLC shows the reaction end point; after the reaction is completed, the precipitated solid is separated from the reaction solution by filtration and directly dissolved in 15 mL of DCM, and an appropriate amount (10 to 20 g) of 200 to 300 mesh silica gel is added and then dried under reduced pressure (35° C.), so that the sample after the solvent is removed is evenly attached to the silica gel, and column chromatography is performed under the development condition of PE:EA=20:1 (referring to isocratic elution) to obtain the compound 2.

[0041] Step ② In an ice-water bath, add the compound 2 (0.6 mmol, 1.0 eq.) and 6 mL THF to a 20 mL reaction bottle containing a magnetic electron, and then add glacial acetic acid (30 mmol, 50.0 eq.) and 1.32 mL TBAF (1.32 mmol, 2.2 eq.) in sequence under stirring. Continue stirring and react at 0 °C for 2.0 h. When TLC shows the end point of the reaction, add 60 mL saturated NaHCO 3 The reaction was quenched with EA (60 mL × 3) and the organic phases were combined and washed with saturated brine. 2 SO 4 The mixture was dried and concentrated under reduced pressure (35° C.), and column chromatography (filler: 200-300 mesh silica gel) was performed under the developing condition of DCM:MeOH = 20:1 (referring to isocratic elution) to obtain the acyl thiourea compound.

[0042] 2.2 Synthesis results and identification of compounds (1) Compound Ⅰ-1 The compound 1 was reacted with p-fluoroaniline (refer to the above step ①) and then the TBS protective agent was removed (refer to the above step ②), and finally 160.8 mg of a yellow solid was obtained with a yield of 81%. HPLC purity (96.42%). R = 0.40 (DCM / MeOH = 15:1). mp: 188.6 ~ 190.4 ℃. 1 H NMR (400 MHz, DMSO- d 6 ) δ 12.70(s, 1H), 11.44 (s, 1H), 9.61 (s, 1H), 9.29 (s, 1H), 7.67 (dd, J= 8.8, 5.1 Hz,2H), 7.60 (d, J = 15.5 Hz, 1H), 7.23 (t, J = 8.8 Hz, 2H), 7.05 (d, J = 2.1 Hz, 1H),6.96 (dd, J = 8.2, 2.0 Hz, 1H), 6.83 – 6.75 (m, 2H). 13 C NMR (101 MHz, DMSO- d 6 ) δ179.55, 166.83, 159.87 (d, J = 244.4 Hz), 148.95, 145.69, 145.52, 134.23 (d, J =3.3 Hz), 126.49 (d, J = 8.1 Hz), 125.60, 122.10, 115.66 (d, J = 29.3 Hz), 115.41, 115.18, 114.34. 19 F NMR (377 MHz, DMSO- d 6 ) δ -115.63. IR (KBr, 400-4000 cm -1 ):3324, 3222, 2359, 1683, 1573, 1552, 1504, 1326, 1286, 1154, 1109, 970, 811,711. HRMS(ESI) m / z: [M+H] + calculated for (based on the molecular formula below) C 16 H 13 FN 2 O 3 S (the structure is shown below) 333.07037; found (the actual measured value) 333.07022.

[0043]

[0044] It was thus determined that the target acyl thiourea compound (i.e., compound Ⅰ-1) was obtained.

[0045] (2) Compound Ⅰ-2 The compound 1 was reacted with cyclopentylamine (refer to step ① above) and then the TBS protective agent was removed (refer to step ② above), and finally 153.5 mg of a yellow solid was obtained with a yield of 84%. HPLC purity (98.39%). R = 0.40 (DCM / MeOH = 15:1). mp: 175.9 ~ 177.2 ℃. 1 H NMR (600 MHz, DMSO- d 6 ) δ 11.14 (s, 1H), 11.01 (d, J = 7.4 Hz, 1H), 9.63 (s, 1H), 9.29 (s, 1H), 7.51 (d, J = 15.5Hz, 1H), 6.99 (d, J = 2.0 Hz, 1H), 6.91 (dd, J = 8.2, 2.1 Hz, 1H), 6.78 (d, J = 8.1Hz, 1H), 6.69 (d, J = 15.5 Hz, 1H), 4.50 (dp, J = 13.1, 6.7, 6.2 Hz, 1H), 1.99(dq, J = 12.5, 5.9 Hz, 2H), 1.69 – 1.56 (m, 4H), 1.55 – 1.48 (m, 2H). 13 C NMR (151 MHz, DMSO- d 6 ) δ 179.44, 166.85, 148.78, 145.67, 144.92, 125.63, 121.92,115.80, 115.76, 114.27, 55.93, 31.92, 23.28. IR (KBr, 400-4000 cm -1 ): 3221, 2955, 2862, 1646, 1586, 1510, 1445, 1347, 1239, 1102, 981, 850, 700. HRMS(ESI) m / z: [M+H] + calculated for C 15 H 18 N 2 O 3S (structure shown below) 307.11109; found307.11096.

[0046]

[0047] It was thus determined that the target acyl thiourea compound (i.e., compound Ⅰ-2) was obtained.

[0048] (3) Compound I-3 The compound 1 was reacted with tert-butylamine (refer to step ① above) and then the TBS protective agent was removed (refer to step ② above), and finally 158.9 mg of a yellow solid was obtained with a yield of 90%. HPLC purity (97.22%). R = 0.50(DCM / MeOH = 15:1). mp: 179.2 ~ 180.1 ℃. 1 H NMR (600 MHz, DMSO- d 6 ) δ 11.19(s, 1H), 10.90 (s, 1H), 9.60 (s, 1H), 9.30 (s, 1H), 7.50 (d, J = 15.5 Hz, 1H),6.99 (d, J = 2.1 Hz, 1H), 6.90 (dd, J = 8.2, 2.1 Hz, 1H), 6.78 (d, J = 8.1 Hz, 1H),6.70 (d, J = 15.6 Hz, 1H), 1.50 (s, 9H). 13 C NMR (151 MHz, DMSO- d 6 ) δ 178.57,166.86, 148.74, 145.66, 144.81, 125.67, 121.88, 115.90, 115.80, 114.30,53.46, 27.55. IR (KBr, 400-4000 cm -1 ): 3399, 3229, 2966, 1656, 1607, 1537,1507, 1287, 1157, 1126, 970, 813, 697. HRMS(ESI) m / z: [M+H] + calculated for C 14 H 18 N 2O 3 S (structure shown below) 295.11109; found 295.11105.

[0049]

[0050] It was thus determined that the target acyl thiourea compound (i.e., compound Ⅰ-3) was obtained.

[0051] (4) Compound Ⅰ-4 The compound 1 was reacted with adamantane methylamine (refer to the above step ①) and then the TBS protective agent was removed (refer to the above step ②), and finally 193.3 mg of a yellow solid was obtained with a yield of 87%. HPLC purity (98.45%). R =0.50 (DCM / MeOH = 15:1). mp: 201.1 ~ 202.7 ℃. 1 H NMR (600 MHz, DMSO- d 6 ) δ11.10 (s, 1H), 10.84 (s, 1H), 9.45 (s, 2H), 7.49 (d, J = 15.5 Hz, 1H), 6.99 (d, J = 2.1 Hz, 1H), 6.90 (dd, J = 8.2, 2.1 Hz, 1H), 6.78 (d, J = 8.1 Hz, 1H), 6.69(d, J = 15.6 Hz, 1H), 2.25 – 2.21 (m, 6H), 2.07 (s, 3H), 1.65 (s, 6H). 13 C NMR (151 MHz, DMSO- d 6 ) δ 177.81, 166.87, 148.76, 145.66, 144.80, 125.65, 121.88,115.90, 115.78, 114.27, 53.98, 39.82, 35.77, 28.81. IR (KBr, 400-4000 cm -1 ):3528, 3348, 2904, 2847, 1675, 1608, 1547, 1508, 1292, 1183, 969, 806, 691. HRMS(ESI) m / z: [M+H] +calculated for C 20 H 24 N 2 O 3 S (structure shown below) 373.15804; found373.15790.

[0052]

[0053] It was thus determined that the target acyl thiourea compound (i.e., compound Ⅰ-4) was obtained.

[0054] (5) Compound Ⅰ-5 The compound 1 was reacted with benzylamine (refer to the above step ①) and then the TBS protective agent was removed (refer to the above step ②), and finally 173.8 mg of a yellow solid was obtained with a yield of 88%. HPLC purity (97.65%). R = 0.50(DCM / MeOH = 15:1). mp: 190.7 ~ 192.5 ℃. 1 H NMR (600 MHz, DMSO- d 6 ) δ 11.27(s, 1H), 11.24 (t, J = 5.7 Hz, 1H), 9.65 (s, 1H), 9.29 (s, 1H), 7.51 (d, J = 15.6Hz, 1H), 7.38 – 7.34 (m, 4H), 7.31 – 7.27 (m, 1H), 7.01 (d, J = 2.1 Hz, 1H),6.92 (dd, J = 8.2, 2.2 Hz, 1H), 6.79 (d, J = 8.1 Hz, 1H), 6.72 (d, J = 15.6 Hz,1H), 4.83 (d, J = 5.7 Hz, 2H). 13 C NMR (151 MHz, DMSO- d 6) δ 180.62, 166.57,148.81, 145.68, 145.00, 137.41, 128.51, 127.51, 127.29, 125.64, 121.97,115.81, 115.76, 114.27, 47.88. IR (KBr, 400-4000 cm -1 ): 3509, 3378, 3211,1652, 1581, 1545, 1502, 1441, 1299, 1270, 1169, 968, 809, 699. HRMS(ESI) m / z:[M+H] + calculated for C 17 H 16 N 2 O 3 S (structure shown below) 329.09544; found 329.09528.

[0055]

[0056] It was thus determined that the target acyl thiourea compound (i.e., compound Ⅰ-5) was obtained.

[0057] (6) Compound I-6 The compound 1 was reacted with aniline (refer to step ① above) and then the TBS protective agent was removed (refer to step ② above), and finally 172.3 mg of a yellow solid was obtained with a yield of 91%. HPLC purity (97.25%). R = 0.40 (DCM / MeOH = 15:1). mp: 172.4 ~ 173.8 ℃. 1 H NMR (600 MHz, DMSO- d 6 ) δ 12.83(s, 1H), 11.45 (s, 1H), 7.68 (d, J = 7.9 Hz, 2H), 7.60 (d, J = 15.6 Hz, 1H), 7.41(t, J = 7.8 Hz, 2H), 7.26 (t, J = 7.4 Hz, 1H), 7.04 (d, J = 2.1 Hz, 1H), 6.96 (dd, J = 8.1, 2.0 Hz, 1H), 6.81 (d, J= 8.1 Hz, 1H), 6.78 (d, J = 15.6 Hz, 1H). 13 C NMR (151 MHz, DMSO- d 6 ) δ 179.05, 166.96, 149.05, 145.75, 145.61, 137.91, 128.71,126.21, 125.62, 124.01, 122.20, 115.87, 115.54, 114.37. IR (KBr, 400-4000 cm-1): 3233, 3030, 1660, 1586, 1563, 1515, 1360, 1235, 1154, 1109, 996, 809,752. HRMS(ESI) m / z: [M+H] + calculated for C 16 H 14 N 2 O 3 S (structure shown below) 315.07979; found 315.07962.

[0058]

[0059] It was thus determined that the target acyl thiourea compound (i.e., compound Ⅰ-6) was obtained.

[0060] (7) Compound Ⅰ-7 The compound 1 was reacted with 2-chloroaniline (refer to step ① above) and then the TBS protective agent was removed (refer to step ② above), and finally 185.4 mg of a yellow solid was obtained with a yield of 89%. HPLC purity (96.32%). R = 0.50(DCM / MeOH = 15:1). mp: 164.8 ~ 166.2 ℃. 1 H NMR (600 MHz, DMSO- d 6 ) δ 12.89(s, 1H), 11.64 (s, 1H), 9.71 (s, 1H), 9.32 (s, 1H), 8.15 (dd, J = 8.2, 1.5 Hz,1H), 7.64 (d, J = 15.5 Hz, 1H), 7.58 (dd, J= 8.1, 1.3 Hz, 1H), 7.40 (td, J = 7.8, 1.4 Hz, 1H), 7.31 (td, J = 7.7, 1.5 Hz, 1H), 7.05 (d, J = 2.0 Hz, 1H), 6.97 (dd, J = 8.0, 2.1 Hz, 1H), 6.81 (d, J = 8.4 Hz, 1H), 6.78 (d, J = 15.8 Hz, 1H). 13 C NMR(151 MHz, DMSO - d 6 ) δ 180.02, 167.00, 149.09, 145.97, 145.73, 135.23, 129.48, 127.91, 127.69, 127.42, 127.20, 125.63, 122.26, 115.86, 115.26, 114.40. IR(KBr, 400 - 4000 cm-1): 3347, 3157, 1666, 1580, 1512, 1442, 1297, 1242, 1165, 1112, 975, 801, 725. HRMS(ESI) m / z: [M + H] + calculated for C 16 H 13 ClN 2 O 3 S (structure shown below) 349.04082; found 349.04086.

[0061]

[0062] Thus, the target acylthiourea compound (i.e., Compound I - 7) was determined to be prepared.

[0063] (8) Compound I - 8 After reacting the said Compound 1 with 2 - bromoaniline (refer to Step ① above) and then removing the TBS protecting agent (refer to Step ② above), a yellow solid of 206.3 mg was finally obtained with a yield of 87%. HPLC purity (95.77%). R = 0.40 (DCM / MeOH = 15:1). m.p.: 166.2 - 168.1 °C. 11H NMR (600 MHz, DMSO- d 6 ) δ 12.74 (s, 1H), 11.63 (s, 1H), 9.71 (s, 1H), 9.33 (s, 1H), 7.98 (dd, J J = 8.1, 1.6 Hz, 1H), 7.73 (dd, J J = 8.1, 1.4 Hz, 1H), 7.64 (d, J J = 15.6 Hz, 1H), 7.44 (td, J J = 7.7, 1.4 Hz, 1H), 7.24 (td, J J = 7.7, 1.6 Hz, 1H), 7.05 (d, J J = 2.1 Hz, 1H), 6.97 (dd, J J = 8.2, 2.2 Hz, 1H), 6.81 (d, J J = 8.1 Hz, 1H), 6.78 (d, J J = 15.6 Hz, 1H). 13 13C NMR (101 MHz, DMSO- d 6 ) δ 180.20, 166.88, 149.00, 145.85, 145.67, 136.65, 132.58, 128.35, 128.28, 127.68, 125.61, 122.14, 118.74, 115.82, 115.30, 114.41. IR (KBr, 400 - 4000 cm-1): 3344, 1669, 1623, 1607, 1576, 1509, 1440, 1289, 1149, 1108, 1015, 978, 756, 713. HRMS (ESI) m / z: [M + H] + calculated for C 16 H 13 BrN 2 O 3 S (structure shown below) 392.99030; found 392.99039.

[0064]

[0065] Thus, the target acylthiourea compound (i.e., Compound I-8) was confirmed to be prepared.

[0066] (9) Compound I-9 After reacting the said Compound 1 with 2-iodoaniline (refer to Step ① above) and then removing the TBS protecting agent (refer to Step ② above), 207.0 mg of a yellow solid was finally obtained with a yield of 78%. HPLC purity (96.95%). R = 0.30 (DCM / MeOH = 15:1). m.p.: 175.8 - 177.3 °C. 1 H NMR (600 MHz, DMSO- d 6 ) δ 12.54(s, 1H), 11.64 (s, 1H), 9.89 (s, 1H), 9.46 (s, 1H), 7.92 (dd, J = 7.9, 1.2 Hz,1H), 7.69 (dd, J = 8.0, 1.4 Hz, 1H), 7.61 (d, J = 15.5 Hz, 1H), 7.47 – 7.40 (m,1H), 7.09 (d, J = 2.1 Hz, 1H), 7.07 (td, J = 7.6, 1.5 Hz, 1H), 6.96 (dd, J = 8.2,2.1 Hz, 1H), 6.86 (d, J = 8.1 Hz, 1H), 6.82 (d, J = 15.6 Hz, 1H). 13 C NMR (151 MHz,DMSO- d 6 ) δ 180.54, 167.01, 149.19, 145.92, 145.82, 140.17, 138.88, 128.84,128.64, 128.50, 125.59, 122.15, 116.07, 115.37, 114.68, 97.22. IR (KBr, 400 - 4000 cm -1 ): 3500, 3333, 1681, 1607, 1572, 1515, 1435, 1242, 1169, 1109, 982,849, 792. HRMS(ESI) m / z: [M+H] + calculated for C 16 H13 IN 2 O 3 S (structure as shown below) 440.97643; found 440.97662.

[0067]

[0068] Thus, it was determined that the target acylthiourea compound (i.e., compound I-9) was prepared.

[0069] (10) Compound I-10 After reacting the said compound 1 with 2-trifluoromethylaniline (refer to step ① above) and then removing the TBS protecting agent (refer to step ② above), finally 188.4 mg of a yellow solid was obtained with a yield of 82%. HPLC purity (98.01%). R = 0.50 (DCM / MeOH = 15:1). m.p.: 176.4 - 178.1 °C. 1 H NMR (600 MHz, DMSO- d 6 ) δ12.78 (s, 1H), 11.71 (s, 1H), 9.72 (s, 1H), 9.33 (s, 1H), 7.85 (d, J = 8.0 Hz,1H), 7.80 (d, J = 7.8 Hz, 1H), 7.74 (t, J = 7.9 Hz, 1H), 7.65 (d, J = 15.6 Hz, 1H),7.53 (t, J = 7.7 Hz, 1H), 7.05 (d, J = 1.9 Hz, 1H), 6.98 (dd, J = 8.1, 2.1 Hz, 1H),6.81 (d, J = 8.2 Hz, 1H), 6.77 (d, J = 15.6 Hz, 1H). 13 C NMR (101 MHz, DMSO- d 6 ) δ181.48, 167.19, 149.05, 146.05, 145.68, 135.70, 132.66, 130.52, 127.50,126.05 (q, J= 5.2 Hz), 125.58, 124.15 (q, J = 29.3 Hz), 123.39 (q, J = 273.7 Hz),122.19, 115.82, 115.13, 114.40. 19 F NMR (377 MHz, DMSO- d 6 ) δ -59.94。 IR (KBr,400 - 4000 cm-1): 3336, 2359, 1682, 1607, 1541, 1510, 1468, 1320, 1287, 1146,1105, 815, 761。 HRMS(ESI) m / z: [M+H] + calculated for C 17 H 13 F 3 N 2 O 3 S (as shown in the structure below) 383.06717; found 383.06741。

[0070]

[0071] Thus, it was determined that the target acylthiourea compound (i.e., Compound I-10) was prepared.

[0072] (11) Compound I-11 After reacting the said Compound 1 with 2-chloro-6-methylaniline (refer to Step ① above) and then removing the TBS protecting agent (refer to Step ② above), a yellow solid of 162.6 mg was finally obtained with a yield of 75%. HPLC purity (97.74%). R = 0.50 (DCM / MeOH = 15:1). m.p.: 133.5 - 135.2 °C. 1 H NMR (600 MHz, DMSO- d 6 ) δ12.10 (s, 1H), 11.59 (s, 1H), 9.49 (s, 2H), 7.61 (d, J = 15.5 Hz, 1H), 7.39(dd, J = 5.7, 3.8 Hz, 1H), 7.32 – 7.22 (m, 2H), 7.04 (d, J = 2.1 Hz, 1H), 6.96(dd,J = 8.2, 2.1 Hz, 1H), 6.81 (d, J = 8.1 Hz, 1H), 6.77 (d, J = 15.6 Hz, 1H), 2.25 (s, 3H). 13 C NMR (151 MHz, DMSO- d 6 ) δ 180.88, 166.78, 149.00, 145.73,145.66, 138.45, 134.75, 131.56, 129.14, 128.76, 127.04, 125.66, 122.16,115.86, 115.48, 114.37, 18.19. IR (KBr, 400-4000 cm -1 ): 3154, 1674, 1593, 1506, 1441, 1280, 1231, 1160, 1108, 974, 814, 768. HRMS(ESI) m / z: [M+H] + calculated for C 17 H 15 C1N 2 O 3 S (structure shown below) 363.05647; found 363.05649.

[0073]

[0074] It was thus determined that the target acyl thiourea compound (i.e., compound Ⅰ-11) was obtained.

[0075] (12) Compound Ⅰ-12 The compound 1 was reacted with 2-methylthioaniline (refer to the above step ①) and then the TBS protective agent was removed (refer to the above step ②), and finally 158.0 mg of a yellow solid was obtained with a yield of 73%. HPLC purity (96.25%). R =0.35 (DCM / MeOH = 15:1). mp: 166.5 ~ 167.4 ℃. 1 H NMR (600 MHz, DMSO- d 6 ) δ12.61 (s, 1H), 11.55 (s, 1H), 9.68 (s, 1H), 9.32 (s, 1H), 7.76 (dd, J= 7.8,1.3 Hz, 1H), 7.63 (d, J = 15.6 Hz, 1H), 7.39 (dd, J = 8.0, 1.4 Hz, 1H), 7.30 (td, J = 7.6, 1.3 Hz, 1H), 7.24 (td, J = 7.6, 1.4 Hz, 1H), 7.05 (d, J = 2.0 Hz, 1H),6.97 (dd, J = 8.2, 2.0 Hz, 1H), 6.83 – 6.76 (m, 2H), 2.45 (s, 3H). 13 C NMR (151MHz, DMSO- d 6 ) δ 180.34, 166.93, 149.00, 145.74, 145.70, 135.56, 133.83,127.44, 127.08, 126.77, 125.64, 125.05, 122.18, 115.84, 115.37, 114.36,14.88. IR (KBr, 400-4000 cm -1 ): 3336, 1675, 1604, 1539, 1508, 1287, 1241,1160, 1105, 976, 838, 804, 728. HRMS(ESI) m / z: [M+H] + calculated for C 17 H 16 N 2 O 3 S 2 (Structure shown below) 361.06751; found 361.06760.

[0076]

[0077] It was thus determined that the target acyl thiourea compound (i.e., compound Ⅰ-12) was obtained.

[0078] (13) Compound Ⅰ-13 The compound 1 was reacted with 3-nitroaniline (refer to step ① above) and then the TBS protective agent was removed (refer to step ② above), and finally 199.2 mg of a yellow solid was obtained with a yield of 92%. HPLC purity (95.60%). R=0.50 (DCM / MeOH = 15:1)。 m.p.: 214.5 ~ 215.8 ℃。 1 H NMR (600 MHz, DMSO- d 6 ) δ12.98 (s, 1H), 11.64 (s, 1H), 9.71 (s, 1H), 9.35 (s, 1H), 8.82 (s, 1H), 8.11(dd, J = 8.3, 2.0 Hz, 1H), 7.99 (dd, J = 7.8, 1.6 Hz, 1H), 7.69 (t, J = 8.1 Hz,1H), 7.62 (d, J = 15.5 Hz, 1H), 7.05 (d, J = 1.9 Hz, 1H), 6.97 (dd, J = 8.2, 1.9Hz, 1H), 6.81 (d, J = 8.1 Hz, 1H), 6.78 (d, J = 15.6 Hz, 1H)。 13 C NMR (151 MHz,DMSO- d 6 ) δ 179.67, 166.84, 149.12, 147.53, 145.91, 145.75, 139.09, 130.75,130.01, 125.58, 122.30, 120.73, 118.64, 115.87, 115.35, 114.36。 IR (KBr, 400-4000 cm -1 ): 3529, 3417, 3089, 1662, 1585, 1519, 1345, 1302, 1271, 1161, 970,834, 713。 HRMS(ESI) m / z: [M+H] + calculated for C 16 H 13 N 3 O 5 S(结构如下所示)360.06487; found 360.06473。

[0079]

[0080] It was thus determined that the target acyl thiourea compound (i.e., compound Ⅰ-13) was obtained.

[0081] (14) Compound Ⅰ-14 The compound 1 was reacted with 3-bromoaniline (refer to step ① above) and then the TBS protective agent was removed (refer to step ② above), and finally 211.1 mg of a yellow solid was obtained with a yield of 90%. HPLC purity (95.50%). R = 0.50(DCM / MeOH=15:1). mp: 186.2 ~ 187.8 ℃. 1 H NMR (600 MHz, DMSO- d 6 ) δ 12.83 (s,1H), 11.55 (s, 1H), 9.76 (s, 1H), 9.36 (s, 1H), 8.08 (s, 1H), 7.60 (d, J = 15.6Hz, 1H), 7.57 (d, J = 8.1 Hz, 1H), 7.45 (d, J = 8.0 Hz, 1H), 7.37 (t, J = 8.0 Hz,1H), 7.05 (s, 1H), 6.96 (d, J = 8.1 Hz, 1H), 6.81 (d, J = 8.1 Hz, 1H), 6.77 (d, J =15.6 Hz, 1H). 13 C NMR (151 MHz, DMSO- d 6 ) δ 179.35, 166.89, 145.78, 145.76,139.47, 130.60, 128.91, 126.58, 125.58, 123.28, 122.23, 115.90, 115.41,114.41. IR (KBr, 400-4000 cm -1 ): 3532, 3153, 2359, 1653, 1574, 1506, 1475, 1284, 1159, 1114, 974, 848, 791. HRMS(ESI) m / z: [M+H] + calculated for C 16 H 13 Bn 2O 3 S (structure shown below) 392.99030; found 392.99023.

[0082]

[0083] It was thus determined that the target acyl thiourea compound (i.e., compound Ⅰ-14) was obtained.

[0084] (15) Compound Ⅰ-15 See also Figure 4 , using the compound 1 and p-bromoaniline, a yellow solid of 198.4 mg was finally obtained with a yield of 84 %. HPLC purity (95.49 %). R = 0.40 (DCM / MeOH = 15:1). mp: 210.7 ~ 212.4 ℃. 1 HNMR (600 MHz, DMSO- d 6 ) δ 12.80 (s, 1H), 11.51 (s, 1H), 9.71 (s, 0H), 9.33 (s,0H), 7.66 (d, J = 8.9 Hz, 1H), 7.60 (dd, J = 9.1, 6.8 Hz, 2H), 7.04 (d, J = 2.1 Hz,1H), 6.96 (dd, J = 8.1, 2.1 Hz, 1H), 6.80 (d, J = 8.1 Hz, 1H), 6.77 (d, J = 15.5 Hz, 1H). 13 C NMR (151 MHz, DMSO- d 6 ) δ 179.19, 166.88, 145.74, 145.70, 137.30,131.51, 126.17, 125.60, 122.22, 118.37, 115.86, 115.46, 114.35 (one carbon disappeared due to overlap). IR (KBr, 400-4000 cm -1): 3326, 3236, 2926, 1682,1592, 1583, 1545, 1506, 1283, 1238, 1107, 977, 807, 690. HRMS(ESI) m / z: [M+H] + calculated for C 16 H 13 Bn 2 O 3 S (structure shown below) 392.99030; found 392.99030.

[0085]

[0086] It was thus determined that the target acyl thiourea compound (i.e., compound Ⅰ-15) was obtained.

[0087] (16) Compound I-16 The compound 1 was reacted with 4-methoxyaniline (refer to step ① above) and then the TBS protective agent was removed (refer to step ② above), and finally 139.6 mg of a yellow solid was obtained with a yield of 68%. HPLC purity (95.58%). R =0.50 (DCM / MeOH = 15:1). mp: 193.3 ~ 194.9 ℃. 1 H NMR (600 MHz, DMSO- d 6 ) δ12.65 (s, 1H), 11.39 (s, 1H), 9.68 (s, 1H), 9.31 (s, 1H), 7.59 (d, J = 15.5 Hz,1H), 7.54 (d, J = 9.0 Hz, 2H), 7.04 (d, J = 2.1 Hz, 1H), 6.98 – 6.94 (m, 3H), 6.80 (d, J = 8.1 Hz, 1H), 6.78 (d, J = 15.6 Hz, 1H), 3.77 (s, 3H). 13 C NMR (151MHz, DMSO- d 6) δ 179.07, 157.36, 148.94, 145.71, 145.39, 130.77, 125.67,125.63, 122.12, 115.83, 115.61, 114.31, 113.80, 55.29. IR (KBr, 400-4000 cm -1 ): 3335, 2359, 1660, 1587, 1547, 1506, 1285, 1248, 1160, 1114, 972, 809,708. HRMS(ESI) m / z: [M+H] + calculated for C 17 H 16 N 2 O 4 S (structure shown below) 345.09035; found 345.09036.

[0088]

[0089] It was thus determined that the target acyl thiourea compound (i.e., compound Ⅰ-16) was obtained.

[0090] (17) Compound Ⅰ-17 The compound 1 was reacted with 2-methoxy-5-methylaniline (refer to step ① above) and then the TBS protective agent was removed (refer to step ② above), and finally 143.1 mg of a yellow solid was obtained with a yield of 67%. HPLC purity (97.97%). R = 0.40 (DCM / MeOH = 15:1). mp: 175.7 ~ 177.6 ℃. 1 H NMR (600 MHz, DMSO- d 6 ) δ 13.04 (s, 1H), 11.38 (s, 1H), 9.49 (s, 2H), 8.46 (d, J = 1.7 Hz, 1H),7.61 (d, J = 15.5 Hz, 1H), 7.04 (d, J = 2.1 Hz, 1H), 7.01 (d, J = 2.6 Hz, 2H), 6.97(dd, J = 8.1, 2.1 Hz, 1H), 6.80 (d, J= 8.1 Hz, 1H), 6.77 (d, J = 15.6 Hz, 1H), 3.83 (s, 3H), 2.26 (s, 3H). 13 C NMR (151 MHz, DMSO- d 6 ) δ 177.74, 166.66,148.94, 148.42, 145.71, 145.47, 128.58, 126.68, 126.61, 125.70, 123.33,122.11, 115.84, 115.55, 114.36, 111.16, 56.08, 20.50. IR (KBr, 400-4000 cm -1 ):3522, 3173, 2831, 1657, 1587, 1540, 1525, 1493, 1363, 1234, 1180, 997, 796. HRMS(ESI) m / z: [M+H] + calculated for C 18 H 18 N 2 O 4 S (structure shown below) 359.10600; found359.10559.

[0091]

[0092] It was thus determined that the target acyl thiourea compound (i.e., compound Ⅰ-17) was obtained.

[0093] (18) Compound Ⅰ-18 The compound 1 was reacted with 3,4-methylenedioxyaniline (refer to step ① above) and then the TBS protective agent was removed (refer to step ② above), and finally 141.9 mg of a yellow solid was obtained with a yield of 66%. HPLC purity (96.18%). R = 0.50 (DCM / MeOH = 15:1). mp: 207.4 ~ 208.8 ℃. 1 H NMR (600 MHz, DMSO- d 6 ) δ 12.64 (s, 1H), 11.42 (s, 1H), 9.69 (s, 1H), 9.31 (s, 1H), 7.59 (d, J =15.5 Hz, 1H), 7.35 (d,J = 2.1 Hz, 1H), 7.03 (d, J = 2.1 Hz, 1H), 7.00 – 6.91 (m,3H), 6.80 (d, J = 8.1 Hz, 1H), 6.77 (d, J = 15.6 Hz, 1H), 6.06 (s, 2H). 13 C NMR (151 MHz, DMSO- d 6 ) δ 179.26, 166.88, 148.97, 146.97, 145.72, 145.47, 145.38,131.88, 125.62, 122.15, 117.76, 115.84, 115.55, 114.32, 107.88, 106.04, 101.50. IR (KBr, 400-4000 cm -1 ): 3541, 3403, 2893, 1662, 1597, 1578, 1527,1491, 1304, 1273, 1166, 971, 805, 707. HRMS(ESI) m / z: [M+H] + calculated for C 17 H 14 N 2 O 5 S (structure shown below) 359.06962; found 359.06961.

[0094]

[0095] It was thus determined that the target acyl thiourea compound (i.e., compound Ⅰ-18) was obtained.

[0096] (19) Compound Ⅰ-19 The compound 1 was reacted with 3,5-dimethoxyaniline (refer to step ① above) and then the TBS protective agent was removed (refer to step ② above), and finally 144.2 mg of a yellow solid was obtained with a yield of 64%. HPLC purity (98.20%). R = 0.40 (DCM / MeOH = 15:1). mp: 198.1 ~ 199.2 ℃. 1 H NMR (600 MHz, DMSO- d 6)δ 12.89 (s, 1H), 11.45 (s, 1H), 9.69 (s, 1H), 9.33 (s, 1H), 7.60 (d, J = 15.5Hz, 1H), 7.04 (d, J = 2.2 Hz, 1H), 6.97 (d, J = 2.2 Hz, 2H), 6.95 (dd, J = 8.6 Hz, J = 2.6 Hz, 1H), 6.80 (d, J = 8.1 Hz, 1H), 6.77 (d, J = 15.6 Hz, 1H), 6.41 (t, J =2.2 Hz, 1H), 3.75 (s, 6H). 13 C NMR (151 MHz, DMSO- d 6 ) δ 178.57, 166.93, 160.30,149.02, 145.72, 145.63, 139.44, 125.60, 122.18, 115.84, 115.47, 114.34,101.74, 98.10, 55.34. IR (KBr, 400-4000 cm -1 ): 3555, 3370, 3224, 1606, 1564,1525, 1481, 1301, 1272, 1146, 828, 673. HRMS(ESI) m / z: [M+H] + calculated for C 18 H 18 N 2 O 5 S (structure shown below) 375.10092; found 375.10126.

[0097]

[0098] It was thus determined that the target acyl thiourea compound (i.e., compound Ⅰ-19) was obtained.

[0099] (20) Compound Ⅰ-20 The compound 1 was reacted with 2,6-diisopropylaniline (refer to step ① above) and then the TBS protective agent was removed (refer to step ② above), and finally 189.8 mg of a yellow solid was obtained with a yield of 79%. HPLC purity (96.94%). R = 0.50 (DCM / MeOH = 15:1). mp: 154.2 ~ 155.6 ℃. 1 H NMR (400 MHz, DMSO- d 6 )δ 12.09 (s, 1H), 11.48 (s, 1H), 9v.63 (s, 1H), 9.26 (s, 1H), 7.63 (d, J = 15.6Hz, 1H), 7.33 – 7.29 (m, 1H), 7.20 (d, J = 7.7 Hz, 2H), 7.05 (d, J = 2.1 Hz, 1H),6.97 (dd, J = 8.2, 2.0 Hz, 1H), 6.82 (d, J = 3.3 Hz, 1H), 6.79 (d, J = 10.8 Hz,1H), 2.99 (p, J = 6.9 Hz, 2H), 1.21 (d, J = 6.8 Hz, 6H), 1.11 (d, J = 6.9 Hz, 6H). 13 C NMR (101 MHz, DMSO- d 6 ) δ 181.87, 145.58, 145.09, 133.14, 128.25, 123.28,122.01, 115.81, 115.55, 114.37, 28.27, 24.00, 22.75. IR (KBr, 400-4000 cm -1 ):3199, 2961, 2867, 1681, 1598, 1504, 1303, 1251, 1203, 1140, 1108, 802, 704. HRMS(ESI) m / z: [M+H] + calculated for C 22 H 26 N 2 O3 S (structure shown below) 399.17369; found399.17389.

[0100]

[0101] It was thus determined that the target acyl thiourea compound (i.e., compound Ⅰ-20) was obtained.

[0102] (21) Compound Ⅰ-21 The compound 1 was reacted with 2-iodo-4-trifluoromethylaniline (refer to step ① above) and then the TBS protective agent was removed (refer to step ② above), and finally 232.4 mg of yellow solid was obtained with a yield of 76%. HPLC purity (95.43%). R = 0.40 (DCM / MeOH = 15:1). mp: 201.6 ~ 203.2 ℃. 1 H NMR (400 MHz, DMSO- d 6 ) δ 12.71 (s, 1H), 11.69 (s, 1H), 9.66 (s, 1H), 9.26 (s, 1H), 8.25 (s, 1H),8.01 (d, J = 8.4 Hz, 1H), 7.81 (d, J = 8.4 Hz, 1H), 7.65 (d, J = 15.5 Hz, 1H), 7.06(d, J = 2.0 Hz, 1H), 6.98 (dd, J = 8.2, 2.0 Hz, 1H), 6.82 (d, J = 4.2 Hz, 1H), 6.79(d, J = 11.7 Hz, 1H). 13 C NMR (101 MHz, DMSO- d 6 ) δ 180.40, 166.82, 149.07,146.02, 145.69, 144.08, 135.41 (q, J = 4.4 Hz), 128.68, 128.25 (q, J = 32.3 Hz),125.57, 125.24 (q, J = 3.3 Hz), 122.87 (q, J= 273.7 Hz), 122.22, 115.82, 115.18,114.39, 97.29. 19 F NMR (377 MHz, DMSO- d 6 ) δ -60.92. IR (KBr, 400-4000 cm -1 ):3599, 3516, 3010, 1669, 1602, 1505, 1314, 1248, 1167, 1090, 984, 850, 795,703. HRMS(ESI) m / z: [M+H] + calculated for C 17 H 12 F 3 IN 2 O 3 S (structure shown below) 508.96382; found 508.96414.

[0103]

[0104] It was thus determined that the target acyl thiourea compound (i.e., compound Ⅰ-21) was obtained.

[0105] (22) Compound I-22 The compound 1 was reacted with 1-naphthylamine (refer to step ① above) and then the TBS protective agent was removed (refer to step ② above), and finally 211.0 mg of a yellow solid was obtained with a yield of 97%. HPLC purity (96.07%). R = 0.50(DCM / MeOH = 15:1). mp: 191.5 ~ 192.8 ℃. 1 H NMR (600 MHz, DMSO- d 6 ) δ 12.97(s, 1H), 11.64 (s, 1H), 9.70 (s, 1H), 9.35 (s, 1H), 8.01 (d, J = 7.9 Hz, 1H),7.97 – 7.89 (m, 3H), 7.69 (d, J = 15.6 Hz, 1H), 7.64 – 7.53 (m, 3H), 7.08 (s,1H), 7.00 (d, J = 8.1 Hz, 1H), 6.85 (d, J= 15.7 Hz, 1H), 6.82 (d, J = 8.1 Hz, 1H). 13 CNMR (151 MHz, DMSO- d 6 ) δ 180.80, 167.19, 149.01, 145.73, 133.91, 133.69,128.43, 128.34, 127.13, 126.83, 126.34, 125.68, 125.40, 123.98, 122.20,121.75, 115.86, 115.55, 114.36. IR (KBr, 400-4000 cm -1 ): 3348, 1674, 1605, 1540, 1505, 1465, 1288, 1191, 1149, 968, 763, 702. HRMS(ESI) m / z: [M+H] + calculated for C 20 H 16 N 2 O 3 S (structure shown below) 365.09544; found 365.09573.

[0106]

[0107] It was thus determined that the target acyl thiourea compound (i.e., compound Ⅰ-22) was obtained.

[0108] 2.3 Synthesis of other acylthiourea compounds In addition to synthesizing the above compounds Ⅰ-1 to Ⅰ-22, refer to Figure 3 The synthetic route and reaction conditions shown in the figure are also used to synthesize other acyl thiourea compounds having structures as shown in Formula I and in which R is a cycloalkyl group, a substituted aryl group, or a heterocyclic aromatic group or a heterocyclic alkyl group containing 1 to 2 nitrogen atoms or oxygen atoms.

[0109] 3. Example of preparation of powder injection containing acylthiourea compounds Dissolve 150 mg of compound Ⅰ-5, Ⅰ-9, Ⅰ-10, Ⅰ-11, Ⅰ-15 or Ⅰ-21 and 750 mg of mannitol in 17 mL of water for injection, mix for the first time and adjust the volume to 30 mL, filter the resulting solution, put it into vials (1 mL per vial), freeze-dry, seal and sterilize to obtain a freeze-dried powder injection with a content of 5 mg / vial of compound Ⅰ-5, Ⅰ-9, Ⅰ-10, Ⅰ-11, Ⅰ-15 or Ⅰ-21.

[0110] 4. Establishment of Inflammatory Bowel Disease (IBD) Model (In Vivo Modeling Experiment) 4.1 Experimental methods (1) Fourteen C57 / BL6 male mice aged 8 to 12 weeks were divided into two groups, namely, the dextran sulfate sodium group (DSS group) and the control group (Ctrl group).

[0111] (2) Dissolve 3 g of dextran sulfate sodium salt (DSS) powder purchased from Sigma-Aldrich in 100 mL of ddHO. 2 O, 3% (g / mL) DSS was prepared and provided to mice in the dextran sulfate sodium salt group (DSS group) for free drinking for 7 days (Day), during which new DSS solution of the same concentration was replaced every other day (to ensure that DSS was sufficient for mice to drink freely); mice in the control group (Ctrl group) were allowed to drink tap water freely for 7 days. The two groups of mice were observed every day for diarrhea and bloody stools, and their body weight and activity were monitored.

[0112] (3) Clinical scores were performed based on daily observation and recording of the general condition, body weight, feces (including hidden or obvious bleeding), and activity of the two groups of mice (see Table 1).

[0113] Table 1. Clinical scores

[0114] Note: In Table 1, semi-formed stool refers to loose stool that does not adhere to the anus and is in a paste-like state; watery stool refers to loose stool that can adhere to the anus; the clinical score is the sum of the scores of each item; the “-” in the row of score 1 indicates that no abnormality was found.

[0115] (4) Seven days later, the mice were killed and their colons were dissected and separated. The appearance of the colon was observed and the length of the colon was recorded. Part of the colon was removed to remove feces, cleaned, folded into a Swiss roll, fixed with 4% neutral formalin, and paraffin sections were prepared and stained with H&E. The sections were observed and photographed under a microscope, and histological pathology analysis was performed.

[0116] (5) Experimental data statistics: The experimental results of each group were statistically analyzed using the t-test analysis method.

[0117] 4.2 Experimental Results See also Figure 1 In this study, dextran sulfate sodium salt (DSS) was used to construct an inducible (specifically induced UC clinical subtype) inflammatory bowel disease (IBD) mouse model, and the mice in the control group that drank tap water for 7 days were compared with the mice in the experimental group (i.e., dextran sulfate sodium salt group) that drank 3% DSS solution for 7 days. The results showed that the mice that drank 3% DSS solution had a significant decrease in body weight ( Figure 1 A), clinical scores improved day by day ( Figure 1B) The colon is lesional and significantly shortened ( Figure 1 C. Figure 1 D). The results of H&E staining of the colons of the mice after further dissection showed that compared with the mice drinking tap water (i.e., normal drinking water), the colon epithelial cells of the mice that drank 3% DSS solution were severely damaged, the crypts were destroyed, and the number of goblet cells was reduced ( Figure 1 E), the corresponding histopathological scores increased, which is consistent with the characteristics of acute inflammation. These results show that the IBD model of the experimental group was successfully constructed (the DSS-induced model in this experiment mainly affects the colon, and its pathophysiological characteristics are more consistent with ulcerative colitis: this model damages colon epithelial cells through DSS, triggers nonspecific immune responses, and leads to the occurrence of colon inflammation. Its clinical symptoms and pathological characteristics are very similar to those of human ulcerative colitis; from the specific experimental results, the DSS-induced model shows diffuse inflammation of the colon mucosa, ulcer formation, crypt abscesses and other characteristics, which are also significantly different from the inflammatory manifestations and pathological characteristics of Crohn's disease in other parts of the intestine). 5. Toxicity Test To detect the effects of acylthiourea compounds on liver and kidney function in mice, 16 C57B / L6 male mice aged 8 to 12 weeks were divided into two groups. The mice in the experimental group were intraperitoneally injected with 20 mg / kg of the synthesized compound Ⅰ-15 ( Figure 4 ), and the control group (Ctrl group) was injected intraperitoneally with an equal volume of phosphate buffered saline (PBS) for 4 days. After 4 days, peripheral blood samples were collected from the two groups of mice to test liver and kidney function indicators. The results are as follows Figure 5 As shown in A, there was no statistical difference in liver function and kidney function indicators between the experimental group mice injected with acylthiourea compounds and the control group mice injected with PBS, and there was no difference in normal indicators with healthy mice.

[0118] At the same time, the livers and kidneys of the two groups of mice were dissected and sliced ​​for H&E staining. Figure 5 As shown in B, there was no obvious damage to the liver and kidney of the experimental group mice injected with acylthiourea compounds and the control group mice injected with PBS, and there was no difference between the normal liver and kidney tissues of healthy mice.

[0119] These results suggest that the acylthiourea compound ( Figure 3 ) has no toxic side effects on the liver and kidney functions of mice.

[0120] 6. Drug activity screening using the red blood cell hemolysis model For the RBC hemolysis model, hemolysis refers to the rupture or destruction of red blood cells (RBCs), resulting in the release of hemoglobin and other cellular contents into the surrounding environment. Experimental treatment: MCC950 was used as a positive control; the cells were first incubated with drugs (acylthiourea compounds with a structure as shown in Formula I) at different concentrations, and then stimulated with the induction activation reagent Nigericin; in the model control group, Nigericin could induce cell pyroptosis, leading to hemoglobin outflow.

[0121] After the treatment, a free hemoglobin detection kit is used to quantitatively detect hemoglobin. Based on the fact that the hemoglobin content is proportional to the absorbance value (OD505), small molecules with inhibitory effects are screened out from acylthiourea compounds with structures shown in Formula I.

[0122] The preliminary screening results after using the red blood cell hemolysis model and testing showed that the acylthiourea compound ( Figure 3 ) The 22 acylthiourea compounds, including compounds Ⅰ-1 to Ⅰ-22, have the effect of inhibiting erythrocyte hemolysis to varying degrees, and can be used as candidate drug molecules for preventing and / or treating NLRP3 inflammasome-related diseases, among which Ⅰ-15 has a stronger activity.

[0123] 7. The therapeutic effect of compound Ⅰ-15 on IBD (in vivo pharmacological experiment) 7.1 Experimental Methods (1) Twelve IBD mice were prepared using the above method for establishing an induced IBD model. After the IBD model was successfully established, the corresponding IBD mice were divided into two groups, namely, a model control group (referred to as the PBS group) and a compound Ⅰ-15 injection group (referred to as the drug administration group or Ⅰ-15 group). A normal drinking water group (referred to as the Ctrl group) was also set up, and the mice in this group were injected with an equal volume of PBS.

[0124] (2) Experimental treatment: The model control group mice were intraperitoneally injected with an equal volume (100 μL) of PBS, and the drug group mice were intraperitoneally injected with 20 mg / kg of the synthesized compound Ⅰ-15 ( Figure 4 ), I-15 was dissolved with DMSO before injection, and then diluted with PBS, and the drug was administered for 4 consecutive days (injected once a day). During the continuous injection of PBS or drug solution, the mice in each group were observed every day for diarrhea and bloody stool, and their body weight, activity and clinical scores were monitored; after the 4th day, the colon of the mice was dissected, the colon was observed and the length of the colon was recorded, and then the colon pathology section and H&E staining were performed.

[0125] 7.2 Experimental Results like Figure 2 As shown, the body weight of IBD mice injected with compound Ⅰ-15 increased, while the body weight of IBD mice injected with PBS decreased ( Figure 2A); At the same time, compared with IBD mice injected with PBS, the clinical scores of IBD mice injected with compound Ⅰ-15 were reduced ( Figure 2 B) The colon length increased significantly and was not significantly different from that of mice that drank normal water and were injected with PBS ( Figure 2 C. Figure 2 D). Further pathological analysis of the colon tissues of experimental mice showed that compared with IBD mice injected with PBS, IBD mice injected with compound Ⅰ-15 had more colon goblet cells and the cells were arranged neatly ( Figure 2 E), and the corresponding histopathological scores were reduced. These results indicate that compound Ⅰ-15 has the effect of alleviating inflammatory bowel disease.

[0126] In summary, the present invention uses caffeic acid to prepare acylthiourea compounds for the first time. And according to the experimental results, this type of compound (referring to compound I-15 and a class of compounds having the same acylthiourea structure as compound I-15) has significant clinical application value in the treatment of inflammatory bowel disease. At the same time, since the acylthiourea compound (such as compound I-15) can produce a clear inhibitory effect on inflammation caused by inflammatory bowel disease, the acylthiourea compound can be used as an active ingredient to prepare anti-inflammatory drugs, especially for the preparation of anti-inflammatory drugs for the treatment of inflammatory bowel disease. In addition, the acylthiourea compound is expected to be used in the development of drugs for the prevention and / or treatment of NLRP3 inflammasome-related diseases.

Claims

1. An acylthiourea compound, characterized in that: The compound is a compound having a structure as shown in Formula I or any one of the pharmaceutically acceptable salts of the compound: Wherein, R is selected from any one of an alkyl group, a cycloalkyl group, a heterocycloalkyl group, a substituted alkyl group, an aryl group, a substituted aryl group, and a heterocycloaryl group.

2. An acylthiourea compound according to claim 1, characterized in that: The R is selected from any one of C1-C10 alkyl groups or substituted alkyl groups.

3. An acylthiourea compound according to claim 1, characterized in that: The R is selected from any one of a cycloalkyl group, a heteroaryl group or a heterocycloalkyl group having 3 to 7 ring atoms.

4. An acylthiourea compound according to claim 1, characterized in that: The substituent groups on the aromatic ring of the substituted aromatic group are one or more of halogen, alkyl, alkoxy, alkyl halide, dioxa, mercapto, and nitro.

5. A method for preparing an acylthiourea compound, characterized in that: The following steps are involved: Acyl thiourea compounds are prepared by chemical synthesis using caffeic acid as a raw material and the phenyl acryloyl group of caffeic acid.

6. A method for preparing an acylthiourea compound, characterized in that: The following steps are involved: 1) Prepare acyl isothiocyanate intermediates under the condition that the phenolic hydroxyl group of caffeic acid is protected, and then prepare acyl thiourea intermediates through nucleophilic addition reaction; 2) preparing the target acyl thiourea compound by deprotecting the acyl thiourea intermediate prepared in step 1.

7. An anti-inflammatory drug, characterized in that: The drug comprises the acylthiourea compound as claimed in any one of claims 1 to 4.

8. Use of the acylthiourea compound according to any one of claims 1 to 4 in the preparation of anti-inflammatory drugs.

9. Use of the acylthiourea compound according to any one of claims 1 to 4 in the preparation of a medicament for treating inflammatory bowel disease.

10. Use of the acylthiourea compound according to any one of claims 1 to 4 in the preparation of a drug for preventing and / or treating NLRP3 inflammasome-related diseases.

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