A urea or thiourea compound and its preparation and application

Abstract

This invention discloses a urea or thiourea compound, its preparation method and application. The general structural formula of this compound is shown in formulas (I), (II) and (III). It has excellent β-glucuronidase (βGUS) inhibitory activity and can be used as a βGUS activity inhibitor. Clinically, it can be used as a treatment drug for serious gastrointestinal adverse reactions.

Description

(I) Technical Field

[0001] This invention belongs to the field of medicinal chemistry, and specifically relates to urea or thiourea compounds, their preparation methods, and applications. (II) Background Technology

[0002] Gastrointestinal adverse events (GIAEs) have always been a serious problem for human survival and development. Studies have found that excessive activity of β-glucuronidase (βGUS) is the main cause of these problems. Inhibiting its excessive activity can effectively alleviate serious adverse events (SAEs) such as diarrhea, vomiting, and loss of appetite during drug use. Therefore, the use of βGUS inhibitors can effectively address these issues. However, currently known βGUS inhibitors suffer from poor efficacy and significant side effects. Therefore, finding highly effective and safe βGUS inhibitors should be a key focus of current research. (III) Summary of the Invention

[0003] The purpose of this invention is to provide a urea or thiourea compound, its preparation method and application. Using urea or thiourea as a lead skeleton, a series of novel urea or thiourea compounds have been designed and synthesized, exhibiting significant βGUS inhibitory activity and potential as βGUS inhibitors. These compounds can be used to develop and utilize drugs for treating severe gastrointestinal adverse reactions caused by β-glucuronidase.

[0004] The technical solution adopted in this invention is:

[0005] This invention provides a urea or thiourea compound, wherein the urea compound has the structure shown in formula (I), and the thiourea compound has the structures shown in formulas (II) and (III):

[0006]

[0007] In formula (I), the R1 group is one or more substitutions, and the R1 group is one of methoxy, ethoxy, propoxy, butoxy, phenoxy, trifluoromethoxy, methylenedioxy, fluorine, chlorine, bromine, iodine, trifluoromethyl, difluoromethyl, methyl, ethyl, propyl, butyl, tert-butyl, nitro or cyano; the R2 group in formula (II) and the R3 group in formula (III) are the same as the R1 group in formula (I).

[0008] Furthermore, the R1 group is 2-fluoro, 3-fluoro, 4-fluoro, 2-chloro, 3-chloro, 4-chloro, 2-bromo, 3-bromo, 4-bromo, 2-methyl, 3-methyl, 4-methyl, 3,5-dimethyl, 2-methoxy, 3-methoxy, 4-methoxy, 2-ethoxy, 3-ethoxy, 4-ethoxy, 2-trifluoromethyl, 3-trifluoromethyl, 4-trifluoromethyl, 2-trifluoromethoxy, 3-trifluoromethoxy, or 4-trifluoromethoxy.

[0009] Furthermore, the R1 group is 2-fluoro, 3-fluoro, 4-fluoro, 2-chloro, 3-chloro, 4-chloro, 2-trifluoromethyl, 3-trifluoromethyl, 4-trifluoromethyl, 3,5-di(trifluoromethyl) or 4-methoxy.

[0010] Furthermore, the R2 group is 2-fluoro, 3-fluoro, 4-fluoro, 2-chloro, 3-chloro, 4-chloro, 2-trifluoromethyl, 3-trifluoromethyl, 4-trifluoromethyl, 3,5-di(trifluoromethyl), 2-methoxy, 3-methoxy, or 4-methoxy.

[0011] Furthermore, the R3 group is 2-fluoro, 3-fluoro, 4-fluoro, 2-chloro, 3-chloro, 4-chloro, 2-trifluoromethyl, 3-trifluoromethyl, 4-trifluoromethyl, 3,5-di(trifluoromethyl), 2-methoxy, 3-methoxy, or 4-methoxy.

[0012] The present invention also provides a method for preparing the urea compound shown in formula (I), the method comprising the following steps:

[0013]

[0014] In the presence of a solvent, after the reaction of compound (A) and compound (B) is complete at room temperature and under magnetic stirring (preferably 2 hours), no column chromatography separation and purification is required; simply evaporate the solvent to obtain compound (I). The solvent is dichloromethane (CH2Cl2). In compound (B), the R1 group is one or more substituted groups, and the R1 group is one of methoxy, ethoxy, propoxy, butoxy, phenoxy, trifluoromethoxy, methylenedioxy, fluorine, chlorine, bromine, iodine, trifluoromethyl, difluoromethyl, methyl, ethyl, propyl, butyl, tert-butyl, nitro, or cyano.

[0015] That is, experiencing the following reaction:

[0016]

[0017] Furthermore, the preparation method of the urea compound shown in formula (I) is carried out according to the following steps: Compound (A) is added to a solvent, stirred and dissolved, and then compound (B) is slowly added dropwise at a rate of 2-4 mL / min. The mixture is reacted at room temperature on a magnetic stirrer for 2 hours. During the reaction, thin-layer chromatography is performed using petroleum ether:ethyl acetate (PE:EA) at a volume ratio of 3:1 as the developing solvent. The product exhibits a single absorption point under ultraviolet light, and the specific gravity (R0) is [value missing]. fWhen the solvent concentration (CA) is between 0.3 and 0.5, the reaction is complete. The reaction solution does not require column chromatography for separation and purification. The compound of formula (I) is obtained by evaporating the solvent using a rotary evaporator, or it can be left open in a fume hood and allowed to evaporate naturally overnight. The solvent is dichloromethane (CH2Cl2). The molar ratio of the compound of formula (A) to the compound of formula (B) is 1:1-3 (preferably 1:1). The volume of solvent used is 5-15 mL / mmol (preferably 10 mL / mmol) based on the amount of substance of the compound of formula (A).

[0018] The present invention also provides a method for preparing the thiourea compound shown in formula (II), the method comprising the following steps:

[0019]

[0020] In the presence of a solvent, compound (C) and compound (D) are heated to 80°C and reacted completely under argon (Ar) protection and magnetic stirring (preferably 4 hours). The reaction solution does not require column chromatography separation and purification; simply evaporating the solvent yields compound (II). The solvent is anhydrous acetonitrile (CH3CN). In compound (D), the R2 group is one or more substituted groups, and the R2 group is one of methoxy, ethoxy, propoxy, butoxy, phenoxy, trifluoromethoxy, methylenedioxy, fluorine, chlorine, bromine, iodine, trifluoromethyl, difluoromethyl, methyl, ethyl, propyl, butyl, tert-butyl, nitro, or cyano.

[0021] That is, experiencing the following reaction:

[0022]

[0023] Further, the preparation method of the thiourea compound shown in formula (II) is carried out according to the following steps: Compound (C) is added to a solvent and stirred to dissolve. Compound (D) is then slowly added dropwise at a rate of 2-4 mL / min. The mixture is heated to 80°C under argon (Ar) protection using a magnetic stirrer and stirred for 4 hours. Thin-layer chromatography is performed during the reaction using petroleum ether:ethyl acetate (PE:EA) at a volume ratio of 3:1 as the developing solvent. The product exhibits a single absorption point under ultraviolet light, and the specific gravity (R0) is [value missing]. f When the solvent concentration (C) is between 0.4 and 0.6, the reaction is complete. The reaction solution does not require column chromatography for separation and purification. The solvent is evaporated to dryness using a rotary evaporator to obtain compound (II). Alternatively, the bottle can be left open in a fume hood and allowed to evaporate naturally overnight. The solvent is anhydrous acetonitrile (CH3CN). The molar ratio of compound (C) to compound (D) is 1:1-3 (preferably 1:1). The volume of solvent used is 5-15 mL / mol (preferably 10 mL / mol) based on the amount of compound (C).

[0024] The present invention also provides a method for preparing the thiourea compound shown in formula (III), the method comprising the following steps:

[0025]

[0026] In the presence of a solvent, after the reaction of compound (E) and compound (F) is complete at room temperature and under magnetic stirring, the reaction solution does not require column chromatography for separation and purification. Simply evaporate the solvent to obtain compound (III). The solvent is dichloromethane (CH2Cl2). In compound (F), the R3 group is one or more substituted groups, and the R3 group is one of methoxy, ethoxy, propoxy, butoxy, phenoxy, trifluoromethoxy, methylenedioxy, fluorine, chlorine, bromine, iodine, trifluoromethyl, difluoromethyl, methyl, ethyl, propyl, butyl, tert-butyl, nitro, or cyano.

[0027] That is, experiencing the following reaction:

[0028]

[0029] Furthermore, the preparation method of the thiourea compound shown in formula (III) is carried out according to the following steps: Compound (E) is added to a solvent, stirred and dissolved, and then compound (F) is slowly added dropwise at a rate of 2-4 mL / min. The reaction is carried out at room temperature on a magnetic stirrer until complete (preferably 2 hours). During the reaction, thin-layer chromatography is performed using ethyl acetate (EA) as the developing solvent. The product exhibits a single absorption point under ultraviolet light, and the specific gravity (R0) is [value missing]. f When the solvent concentration (CE) is between 0.5 and 0.7, the reaction is complete. The reaction solution does not require column chromatography for separation and purification. The solvent is evaporated to dryness using a rotary evaporator to obtain compound (III). Alternatively, the bottle can be left open in a fume hood and allowed to evaporate naturally overnight. The solvent is dichloromethane (CH2Cl2). The molar ratio of compound (E) to compound (F) is 1:1-3 (preferably 1:1). The volume of solvent used is 5-15 mL / mmol (preferably 10 mL / mmol) based on the amount of compound (E).

[0030] In this invention, the reaction temperature can be carried out over a wide range, preferably at room temperature. If the reaction limit is small at room temperature, different solvents need to be selected according to the actual situation to increase the temperature of the reaction system (to approach the boiling point of the corresponding solvent to achieve reflux). All the target compounds mentioned above do not require column chromatography separation and purification.

[0031] This invention also provides the use of the urea or thiourea compound in the preparation of β-glucuronidase activity inhibitors. The activity inhibitor is a compound of formula (I), (II), or (III), or a pharmaceutically acceptable salt.

[0032] Furthermore, the β-glucuronidase is Escherichia coli β-glucuronidase.

[0033] The present invention also provides the use of the urea or thiourea compound in the preparation of a drug for treating gastrointestinal adverse reactions, the drug including drugs for treating diarrhea, vomiting or loss of appetite.

[0034] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0035] The urea or thiourea compounds provided by this invention have a simple preparation method and high yield; the compounds exhibit significant inhibitory activity against βGUS and have good biological activity, and can be used as βGUS inhibitors; this invention expands the application prospects of benzo[a]azole heterocyclic and substituted benzene structures in βGUS inhibitors. (iv) Description of the attached drawings

[0036] Figure 1 The proton nuclear magnetic resonance spectrum of the compound shown in formula (Ⅰ-1) 1 H NMR spectrum.

[0037] Figure 2 The carbon NMR spectrum of the compound shown in formula (Ⅰ-1) 13 C NMR (C100NMR) plot.

[0038] Figure 3 The proton nuclear magnetic resonance spectrum of the compound shown in formula (Ⅱ-1) 1 H NMR spectrum.

[0039] Figure 4 The carbon NMR spectrum of the compound shown in formula (Ⅱ-1) 13 C NMR (C100NMR) plot.

[0040] Figure 5 The proton nuclear magnetic resonance spectrum of the compound shown in formula (Ⅲ-1) 1 H NMR spectrum.

[0041] Figure 6 The carbon NMR spectrum of the compound shown in formula (Ⅲ-1) 13 C NMR (C100NMR) plot. (V) Detailed Implementation

[0042] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0043] The room temperature mentioned in this invention refers to 25°C. The temperature set during constant-temperature heating is determined according to the boiling point of the selected solvent (slightly below the solvent boiling point for reflux). Unless otherwise specified, the reagents and methods involved in the examples are all commonly used reagents and methods in the art.

[0044] Example 1: Preparation of compound (Ⅰ-1)

[0045]

[0046] In a 25 mL round-bottom flask, 0.067 g of 2-aminobenzoxazole A (0.5 mmol, 1.0 eqv) dry powder was added to 5 mL of dichloromethane (CH2Cl2) and stirred. Then, 0.061 mL of 2-fluoroisocyanate phenyl ester B-1 (0.5 mmol, 1.0 eqv) was slowly added dropwise at a rate of 2-4 mL / min. The reaction vessel was placed on a magnetic stirrer at room temperature and stirred automatically for 2 h. During the reaction, petroleum ether:ethyl acetate (PE:EA) at a volume ratio of 3:1 was used as the developing solvent. Thin-layer chromatography (TLC) was performed under UV light at a wavelength of 254 nm using a GF-254 silica gel plate. The product exhibited a single absorption point under UV light, and the specific gravity (R0) was [not specified]. f The reaction is complete when the solvent concentration (CI) is 0.43. The reaction solution does not require column chromatography purification; the solvent is evaporated using a rotary evaporator to obtain compound (I-1); alternatively, the bottle can be left open in a fume hood and allowed to evaporate naturally overnight to obtain 0.130 g of a yellow solid, which is 1-(2-benzoxazolyl)-3-(2-fluorophenyl)urea (I-1). The overall yield of this one-step reaction is 96%. 1 See H NMR spectrum Figure 1 , 13 See the C NMR spectrum. Figure 2 .

[0047] Examples 2-11: Preparation of compounds (Ⅰ-2) to (Ⅰ-11)

[0048] Using the method of Example 1, compound B-1 was substituted with the substituents shown in Table 1, with other operations remaining the same, to obtain compounds of formulas (I-2) to (I-11). The appearance, yield, and high-resolution mass spectrometry (HRMS) data of the above compounds are listed in Table 1, and the nuclear magnetic resonance (NMR) data are listed in Table 2. It can be seen from the above that the structures of the above compounds are correct, and they are all compounds of formula (I).

[0049] Example 12, Preparation of compound (Ⅱ-1)

[0050]

[0051] In a 25 mL round-bottom flask, 0.067 g of 2-aminobenzimidazole C (0.5 mmol, 1.0 eqv) dry powder was added to 5 mL of anhydrous acetonitrile (CH3CN) and stirred. Then, 0.067 mL of phenyl 2-fluoroisothiocyanate D-1 (0.5 mmol, 1.0 eqv) was slowly added dropwise at a rate of 2-4 mL / min. Under argon (Ar) protection, the reaction vessel was placed on a magnetic stirrer and heated to 80 °C for 4 h with automatic stirring. During the reaction, petroleum ether:ethyl acetate (PE:EA) at a volume ratio of 3:1 was used as the developing solvent. Thin-layer chromatography (TLC) was performed under UV light at a wavelength of 254 nm using a GF-254 silica gel plate. The product exhibited a single absorption point under UV light, and the specific gravity (R0) was [not specified]. f The reaction is complete when the solvent concentration (V) is 0.56. The reaction solution does not require column chromatography purification; the solvent is evaporated using a rotary evaporator to obtain compound (Ⅱ-1); alternatively, the bottle can be left open in a fume hood and allowed to evaporate naturally overnight to obtain 0.133 g of a pale yellow solid, which is 1-(2-benzimidazolyl)-3-(2-fluorophenyl)thiourea (Ⅱ-1). The overall yield of this one-step reaction is 93%. 1 See H NMR spectrum Figure 3 , 13 See the C NMR spectrum. Figure 4 .

[0052] Examples 13-24: Preparation of compounds (II-2) to (II-11)

[0053] Using the method of Example 12, compound D-1 was substituted with the substituents shown in Table 1, with all other operations remaining the same, to obtain compounds of formulas (II-2) to (II-11). The appearance, yield, and high-resolution mass spectrometry (HRMS) data of the above compounds are listed in Table 1, and the nuclear magnetic resonance (NMR) data are listed in Table 2. It can be seen from the above that the structures of the above compounds are correct, and they are all compounds of formula (II).

[0054] Example 25: Preparation of compound (Ⅲ-1)

[0055]

[0056] In a 25 mL round-bottom flask, 0.067 g of 6-aminobenzimidazole E (0.5 mmol, 1.0 eqv) dry powder was added to 5 mL of dichloromethane (CH2Cl2) and stirred. Then, 0.067 mL of phenyl 2-fluoroisothiocyanate F-1 (0.5 mmol, 1.0 eqv) was slowly added dropwise at a rate of 2-4 mL / min. The reaction vessel was placed on a magnetic stirrer at room temperature and stirred automatically for 2 h. Ethyl acetate (EA) was used as the developing solvent, and the reaction was monitored by thin-layer chromatography (TLC) using a GF-254 silica gel plate under UV light at a wavelength of 254 nm. The product exhibited a single absorption point under UV light. f The reaction is complete when the piezoresistive value is 0.59. The reaction solution does not require column chromatography purification; the solvent is evaporated to dryness using a rotary evaporator to obtain compound (Ⅲ-1); alternatively, the bottle can be left open in a fume hood and allowed to evaporate naturally overnight to obtain 0.140 g of a white solid, which is 1-(6-benzimidazolyl)-3-(2-fluorophenyl)thiourea (Ⅲ-1). The overall yield of this one-step reaction is 98%. 1 See H NMR spectrum Figure 5 , 13 See the C NMR spectrum. Figure 6 .

[0057] Examples 26-37, Preparation of compounds (Ⅲ-2) to (Ⅲ-11)

[0058] Using the method of Example 25, compound F-1 was substituted with the substituents shown in Table 1, with other operations remaining the same, to obtain compounds of formulas (Ⅲ-2) to (Ⅲ-11). The appearance, yield, and high-resolution mass spectrometry (HRMS) data of the above compounds are listed in Table 1, and the nuclear magnetic resonance (NMR) data are listed in Table 2. It can be seen from the above that the structures of the above compounds are correct, and they are all compounds of formula (Ⅲ).

[0059] Table 1. Appearance, yield, and HRMS data of the compounds.

[0060]

[0061] Table 2 NMR data of the compounds

[0062]

[0063]

[0064]

[0065]

[0066]

[0067] Example 38: Screening of the inhibitory activity of urea or thiourea compounds shown in formulas (I), (II), and (III) against βGUS.

[0068] Enzyme solution: Escherichia coli β-glucuronidase (EcGUS) was dissolved in PBS buffer to prepare a 2 mg / mL solution. The final concentration added to the wells was 10. -4 mg / mL.

[0069] Substrate: p-Nitrophenyl-β-D-glucuronide (PNPG) was dissolved in PBS buffer to prepare a 10 mM solution, and then diluted with PBS buffer to a concentration of 2.5 mM. The final concentration added to the well plate was 250 μM.

[0070] Inhibitors in the experimental group: The test compounds prepared in Examples 1-37 were dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mM solution, and then diluted to 1 mM with PBS buffer. The final concentration added to the well plate was 100 μM.

[0071] Positive control: D-glucono-1,4-lactone (DSL) was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mM solution, and then diluted to 1 mM with PBS buffer. The final concentration added to the well plate was 100 μM.

[0072] Experimental method: The in vitro enzyme inhibitory activity of the compounds was tested using colorimetric analysis. The specific procedures are as follows:

[0073] Prepare a 1 mM p-nitrophenol (PNP) solution and add 0, 10, 20, 40, 60, and 80 μL to each well of a 96-well plate, respectively. Make up the volume to 100 μL with PBS buffer, incubate at 37 °C for 30 min, and measure the absorbance (OD) at 405 nm. 405nm A standard curve for PNP was created using Excel software, with PNP concentration on the x-axis and absorbance on the y-axis. The curve equation is y = 3.262x.

[0074] Add 10 μL of enzyme solution, 70 μL of PBS buffer, 10 μL of PBS (negative control group) / DSL (positive control group) / inhibitor (experimental group) to a 96-well plate sequentially, and 10 μL of substrate. Immediately after adding the substrate, read the absorbance at 405 nm on a microplate reader; record this as 0 min (OD). 0min The PNP concentration at 0 min (c) was obtained based on the PNP standard curve. 0min Incubate at 37°C for 30 minutes, then measure the absorbance and record it as 30 min (OD). 30min The PNP concentration (c) at 30 min was obtained based on the PNP standard curve. 30min ).

[0075] The absorbance difference (x) is obtained by subtracting the absorbance at two time points according to formula (1). The PNP concentration difference (y) is calculated according to formula (2), the relative activity (RA) of the test compound is calculated according to formula (3), and the inhibition rate (IR) is calculated according to formula (4). EG refers to the experimental group (containing the target compound or positive control drug), and CG refers to the blank control group (containing no inhibitor).

[0076] The calculation formula is as follows:

[0077] Absorbance difference x = ΔOD 405nm =OD 30min -OD 0min Formula (1)

[0078] PNP concentration difference y=Δc PNP =c 30min -c 0min Formula (2)

[0079]

[0080] Inhibition rate IR = 1 - RA (Formula 4)

[0081] The inhibitory activity of the test compounds against βGUS was determined by colorimetric analysis, and the results are shown in Table 3. Table 3 shows that the target compounds exhibited different βGUS inhibitory activities, among which compounds (Ⅲ-6), (Ⅲ-9), and (Ⅲ-13) had good βGUS inhibitory activities, especially compound (Ⅲ-9), which was superior to the positive control DSL and has great development potential.

[0082] Table 3. Inhibition rate of compounds against βGUS (final concentration 100 μM)

[0083] Ⅰ-1 46.9±5.5 Ⅱ-1 30.3±0.8 Ⅲ-1 35.6±1.6 Ⅰ-2 47.3±5.2 Ⅱ-2 26.6±3.7 Ⅲ-2 44.4±4.3 Ⅰ-3 44.3±3.9 Ⅱ-3 21.5±2.0 Ⅲ-3 40.1±2.2 Ⅰ-4 40.3±4.3 Ⅱ-4 19.0±7.3 Ⅲ-4 44.2±6.9 Ⅰ-5 31.1±4.3 Ⅱ-5 33.7±1.9 Ⅲ-5 44.7±2.1 Ⅰ-6 36.9±3.3 Ⅱ-6 31.5±3.1 Ⅲ-6 57.8±2.3 Ⅰ-7 32.1±3.0 Ⅱ-7 21.7±8.0 Ⅲ-7 40.0±5.6 Ⅰ-8 28.2±1.6 Ⅱ-8 18.0±7.1 Ⅲ-8 33.7±7.6 Ⅰ-9 25.4±1.6 Ⅱ-9 13.2±7.5 Ⅲ-9 94.3±1.5 Ⅰ-10 30.2±3.5 Ⅱ-10 18.2±5.7 Ⅲ-10 30.8±6.0 Ⅰ-11 17.7±9.5 Ⅱ-11 19.3±1.7 Ⅲ-11 37.0±2.8 DSL 76.2±3.0 Ⅱ-12 20.9±0.3 Ⅲ-12 30.9±1.4 - - Ⅱ-13 37.5±3.4 Ⅲ-13 63.8±1.6

[0084] Example 39, IC50 of compound (Ⅲ-9) on βGUS 50 Value Research

[0085] Enzyme solution: Escherichia coli β-glucuronidase (EcGUS) was dissolved in PBS buffer to prepare a 2 mg / mL solution. The final concentration added to the wells was 10. -4 mg / mL.

[0086] Substrate: p-Nitrophenyl-β-D-glucuronide (PNPG) was dissolved in PBS buffer to prepare a 10 mM solution, and then diluted with PBS buffer to a concentration of 2.5 mM. The final concentration added to the well plate was 250 μM.

[0087] Inhibitors in the experimental group: The test compound (III-9) was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mM solution, and then diluted with PBS buffer to different concentrations. The final concentrations added to the well plates were 50 μM, 30 μM, 10 μM, 5 μM, 3 μM, 1 μM, 0.5 μM, 0.3 μM, and 0.1 μM.

[0088] Positive control drug: D-glucono-1,4-lactone (DSL) was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mM solution, and then diluted with PBS buffer to different concentrations. The final concentrations added to the well plates were 500 μM, 300 μM, 100 μM, 50 μM, 30 μM, 10 μM, 5 μM, 3 μM, and 1 μM.

[0089] Experimental method: The in vitro enzyme inhibitory activity of the compounds was tested using colorimetric analysis. The specific procedures are as follows:

[0090] Add 10 μL of enzyme solution, 70 μL of PBS buffer, 10 μL of PBS (negative control group) / DSL (positive control group) / inhibitor (experimental group) to a 96-well plate sequentially, and 10 μL of substrate. Immediately after adding the substrate, read the absorbance at 405 nm on a microplate reader; record this as 0 min (OD). 0min The PNP concentration at 0 min (c) was obtained according to the PNP standard curve of Example 38. 0min Incubate at 37°C for 30 minutes, then measure the absorbance and record it as 30 min (OD). 30min The PNP concentration (c) at 30 min was obtained according to the PNP standard curve of Example 38. 30min ).

[0091] The absorbance difference (x) is obtained by subtracting the absorbance at two time points according to formula (1). The PNP concentration difference (y) is calculated according to formula (2), the relative activity (RA) of the test compound is calculated according to formula (3), and the inhibition rate (IR) is calculated according to formula (4). EG refers to the experimental group (containing the target compound or positive control drug), and CG refers to the blank control group (containing no inhibitor). The calculation formula is the same as in Example 38.

[0092] IC50 was plotted in GraphPad Prism software using the PNP concentration difference and relative activity at different inhibitor concentrations. 50 Value curve, to obtain IC 50 The results are shown in Table 4. Table 4 shows that compound (Ⅲ-9) has good βGUS inhibitory activity, which is 17 times that of the positive control inhibitor DSL, and has great development potential.

[0093] Table 4. IC50 of compound (Ⅲ-9) against βGUS50 value

[0094] Ⅲ-9 2.7 DSL 45.8

[0095] The above embodiments describe the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention should not be limited to the above embodiments. The contents described in the above embodiments and the specification are only illustrative of the principles of the present invention. Various optimizations and improvements can be made to the present invention without departing from the scope of the principles of the present invention, and all such optimizations and improvements fall within the protection scope of the present invention.

Claims

1. The application of a thiourea compound in the preparation of a β-glucuronidase inhibitor, characterized in that, The inhibitor is a drug for treating gastrointestinal adverse reactions, and the structure of the thiourea compound is shown in formula (III): Ⅲ In formula (Ⅲ), the R3 group is 4-Cl, 4-CF3, or 4-OCH3.

2. The application as described in claim 1, characterized in that, The preparation method of the thiourea compound shown in formula (Ⅲ) includes the following steps: EF In the presence of a solvent, after the reaction of compound (E) and compound (F) is complete at room temperature and under magnetic stirring, the solvent in the reaction solution is evaporated to obtain compound (III); the solvent is dichloromethane; the R3 group in compound (F) is 4-Cl, 4-CF3, or 4-OCH3.

3. The application as described in claim 2, characterized in that, The preparation method of the thiourea compound shown in formula (III) is carried out according to the following steps: Compound (E) is added to a solvent and stirred to dissolve. Compound (F) is then slowly added dropwise at a rate of 2-4 mL / min. The mixture is reacted at room temperature on a magnetic stirrer for 2 h. Thin-layer chromatography is performed using ethyl acetate as the developing solvent during the reaction. The product exhibits a single absorption point under ultraviolet light. f The reaction is complete when the value is between 0.5 and 0.

7. The reaction solution is dried by evaporating the solvent in a rotary evaporator to obtain compound (III), or it can be left open in a fume hood and allowed to dry naturally overnight. The molar ratio of compound (E) to compound (F) is 1:1-3, and the volume of solvent used is 5-15 mL / mmol based on the amount of substance of compound (E).

4. The application as described in claim 1, characterized in that, The β-glucuronidase is Escherichia coli β-glucuronidase.

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