Micromolecular agonist of bitter receptor 38 subtype as well as preparation method and application of micromolecular agonist

Abstract

The invention discloses a bitterness receptor 38 subtype micromolecule agonist as well as a preparation method and application thereof, and particularly discloses a compound as shown in a formula (I) or pharmaceutically acceptable salt thereof. The compound has relatively good TAS2R38 receptor agonistic activity and good druggability. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the fields of biology and medicinal chemistry, and relates to a small molecule agonist of bitter taste receptor 38 subtype, a preparation method and an application thereof. Background Art

[0002] Bitter taste receptors belong to the T subfamily of the G protein-coupled receptor superfamily. Their primary physiological function is to recognize and bind to bitter ligands. They are key proteins in the bitter taste signaling pathway and have played a crucial evolutionary role in the avoidance of naturally toxic foods in humans and animals. As taste receptors, bitter taste receptors are primarily located in taste buds; in addition, they are widely expressed ectopically in various organs and tissues throughout the human body and are believed to be involved in several physiological processes and disease treatment. Bitter taste receptor subtype 38 (TAS2R38) is a bitter taste receptor that specifically recognizes bitter thioglycosides found in vegetables. It also specifically recognizes isothiocyanates and thioureas from both natural and artificial sources. TAS2R38 is ectopically expressed in organs and tissues such as the sinuses, respiratory tract, colon, and endocrine cells. Recent studies have linked it to several important physiological processes and disease treatment. So far, only more than 20 TAS2R38 agonists have been reported (Meyerhof W, et al. ChemSenses, 2010, 35: 157-170). Among them, the most active compounds have an EC 50 It is only 1-2 micromolar (Wooding S, et al. Chem Senses, 2010, 35, 685-692). Among the currently known agonists, only three can reach the micromolar level (PTC, 1.1uM; PROP, 2.1uM; DPT, 2.3uM). Summary of the Invention

[0003] In response to the problem that TAS2R38 receptor agonists in the prior art have weak activity, the present invention provides a small molecule agonist of bitter taste receptor 38 subtype and its preparation method and application. Such compounds have good TAS2R38 receptor agonist activity and good drugability.

[0004] The present invention provides a compound as shown in formula (I) or a pharmaceutically acceptable salt thereof,

[0005]

[0006] in,

[0007] R 1 、R 2 、R 3 、R 4 、R 5 and R 6 are independently hydrogen, halogen, C1-10 Alkyl or C 6-10 aryl; and R 1 、R 2 、R 3 、R 4 、R 5 and R 6 Not hydrogen at the same time.

[0008] In some embodiments, R 1 is hydrogen, halogen or C 6-10 Aryl.

[0009] In some embodiments, R 2 is hydrogen, halogen or C 6-10 Aryl.

[0010] In some embodiments, R 3 is hydrogen, halogen or C 6-10 Aryl.

[0011] In some embodiments, R 5 is hydrogen, halogen or C 6-10 Aryl.

[0012] In some embodiments, the compound represented by formula (I) is a compound represented by formula (I-1), R 1 Halogen or C 6-10 Aryl.

[0013] In some embodiments, the compound represented by formula (I) is a compound represented by formula (I-2), R 2 Halogen or C 6-10 Aryl.

[0014] In some embodiments, the compound represented by formula (I) is a compound represented by formula (I-3), R 3 Halogen or C 6-10 Aryl.

[0015] In some embodiments, the compound represented by formula (I) is a compound represented by formula (I-4), R 4 Halogen, C 1-10 Alkyl or C 6-10 Aryl.

[0016] In some embodiments, the compound represented by formula (I) is a compound represented by formula (I-5), R 5 Halogen or C 6-10 Aryl.

[0017] In some embodiments, the compound represented by formula (I) is a compound represented by formula (I-6), R 6 Halogen, C 1-10 Alkyl or C 6-10 Aryl.

[0018] In some embodiments, the compound represented by formula (I) is a compound represented by formula (I-7), R 3 and R 5 are independently halogen.

[0019] In some embodiments, the compound represented by formula (I) is a compound represented by formula (I-8), R 3 、R 4 and R 5 are independently halogen.

[0020] In some embodiments, each halogen is independently fluoro, chloro, bromo, or iodo.

[0021] In some embodiments, each C 1-10 The alkyl groups are independently C 1-6 Alkyl is preferably methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl or tert-butyl; for example methyl, isopropyl or tert-butyl.

[0022] In some embodiments, each C 6-10 Aryl is independently phenyl or naphthyl, for example phenyl.

[0023] In some embodiments, R 1 is hydrogen, chlorine, bromine, iodine or phenyl.

[0024] In some embodiments, R 2 is hydrogen, fluorine, chlorine, bromine, iodine or phenyl.

[0025] In some embodiments, R 3 is hydrogen, fluorine, chlorine, bromine, iodine or phenyl.

[0026] In some embodiments, R 4 is hydrogen, fluorine, chlorine, bromine, iodine, methyl, isopropyl, tert-butyl or phenyl.

[0027] In some embodiments, R 5 is hydrogen, fluorine, chlorine, bromine, iodine or phenyl.

[0028] In some embodiments, R 6 is hydrogen, fluorine, chlorine, bromine, iodine, methyl or phenyl. In some embodiments, the compound shown in formula (I) is any of the following structures:

[0029]

[0030]

[0031] The present invention also provides a method for preparing the compound represented by formula (I), which is method A or method B:

[0032] Method A includes the following steps:

[0033] In an organic solvent, the compound represented by formula (II) is first reacted with 1,1-thiocarbonyldiimidazole; after the reaction, the resulting reaction solution is further reacted with a methanol solution of ammonia to obtain the compound represented by formula (I);

[0034]

[0035] In method A, R 1 、R 2 、R 3 、R 4 、R 5 and R 6 The definition of is the same as that of any one of the embodiments of the compound represented by formula (I);

[0036] Method B comprises the following steps:

[0037] In an organic solvent, the compound represented by formula (II) is first reacted with benzoyl isothiocyanate; after the reaction, the resulting reaction solution is further reacted with an alkaline aqueous solution to obtain a compound represented by formula (I);

[0038]

[0039] In method B, R 1 、R 2 、R 3 、R 4 、R 5 and R 6 The definition of is the same as that described in any scheme of the compound represented by formula (I).

[0040] In the method A, the organic solvent may be a halogenated alkane solvent, such as dichloromethane.

[0041] In the method A, the molar volume ratio of the compound represented by formula (II) to the organic solvent may be 0.05-0.5 mol / L, for example 0.1 mol / L.

[0042] In the method A, the molar ratio of the compound represented by formula (II) to the 1,1-thiocarbonyldiimidazole can be 1:(1-3), for example, 1:1.7.

[0043] In the method A, the concentration of the methanol solution of ammonia may be 6-8 mol / L, for example 7 mol / L.

[0044] In the method A, the molar volume ratio of the compound represented by formula (II) to the methanol solution of ammonia can be 0.3-0.7 mol / L, for example 0.5 mol / L.

[0045] In the method A, the reaction temperature is 20-40°C, for example 25°C.

[0046] In the method B, the organic solvent may be a ketone solvent, preferably acetone.

[0047] In the method B, the molar volume ratio of the compound represented by formula (II) to the organic solvent may be 0.1-0.5 mol / L, for example 0.17 mol / L.

[0048] In the method B, the molar ratio of the compound represented by formula (II) to the benzoyl isothiocyanate can be 1:(1-2), for example, 1:1.1.

[0049] In the method B, the alkaline aqueous solution is a NaOH aqueous solution.

[0050] In the method B, the concentration of the alkaline aqueous solution may be 0.5-2 mol / L, for example 1 mol / L.

[0051] In the method B, the molar ratio of the compound represented by formula (II) to the alkaline aqueous solution can be 1:(2-4), for example 1:3.

[0052] In the method B, when the compound represented by formula (II) is first reacted with benzoyl isothiocyanate, the reaction temperature is 50-80°C, for example 65°C.

[0053] In the method B, when the reaction solution reacts with the alkaline aqueous solution, the reaction temperature is 40-60°C, for example 50°C.

[0054] The present invention also provides a compound as shown below:

[0055]

[0056] The present invention provides a pharmaceutical composition comprising the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, and at least one pharmaceutical excipient.

[0057] The present invention also provides the use of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition described above, in the preparation of a TAS2R38 receptor agonist. In such applications, the TAS2R38 receptor agonist can be used in mammalian organisms; it can also be used in vitro, primarily for experimental purposes, such as providing a standard or control sample for comparison, or preparing a kit according to conventional methods in the art to provide rapid detection of the effect of stimulating the TAS2R38 receptor protein.

[0058] The present invention also provides the use of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition described above, in the preparation of a drug for treating and / or preventing chronic sinusitis.

[0059] The present invention provides the use of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition described above, in the preparation of a medicament for treating and / or preventing a disease associated with or mediated by the TAS2R38 receptor. The disease may be chronic sinusitis.

[0060] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0061] The term "alkyl" refers to a straight or branched chain alkyl group having a specified number of carbon atoms (e.g., C1 to C6). Alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl.

[0062] The term "aryl" refers to a group having a specified number of carbon atoms (e.g., C6 to C 10 ) cyclic groups consisting only of carbon atoms, which are monocyclic or polycyclic, and at least one ring is aromatic (in accordance with Huckel's rule). Aryl groups are connected to other fragments in the molecule through aromatic or non-aromatic rings. Aryl groups include but are not limited to phenyl, naphthyl, etc.

[0063] The term "pharmaceutically acceptable salt" refers to a salt obtained by reacting a compound with a pharmaceutically acceptable (relatively non-toxic, safe, and suitable for use by patients) acid or base. When the compound contains a relatively acidic functional group, a base addition salt can be obtained by contacting the free form of the compound with a sufficient amount of a pharmaceutically acceptable base in a suitable inert solvent. Pharmaceutically acceptable base addition salts include, but are not limited to, sodium salts, potassium salts, calcium salts, aluminum salts, magnesium salts, bismuth salts, ammonium salts, and the like. When the compound contains a relatively basic functional group, an acid addition salt can be obtained by contacting the free form of the compound with a sufficient amount of a pharmaceutically acceptable acid in a suitable inert solvent. Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochlorides, sulfates, methanesulfonates, and the like.

[0064] The term "therapeutically effective amount" refers to the amount of a compound administered to a patient that is sufficient to effectively treat a disease. The therapeutically effective amount will vary depending on the compound, the type of disease, the severity of the disease, the age of the patient, etc., but can be adjusted by those skilled in the art as appropriate.

[0065] The term "pharmaceutical excipients" refers to excipients and additives used in the production of medicines and the preparation of prescriptions. It is all substances contained in pharmaceutical preparations in addition to the active ingredients.

[0066] The term "treat" refers to any of the following: (1) alleviating one or more biological manifestations of a disease; (2) interfering with one or more points in the biological cascade that leads to a disease; or (3) slowing the progression of one or more biological manifestations of a disease.

[0067] The term "prevent" refers to reducing the risk of developing a disease.

[0068] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0069] The reagents and raw materials used in the present invention are commercially available.

[0070] The positive advances of this invention lie in the fact that by incorporating an isoquinoline core into the molecular structure and using a thiourea group as the key functional group for recognizing and activating TAS2R38, a series of novel small molecule compounds have been designed. These compounds exhibit significantly enhanced activity compared to known agonists. The vast majority of the compounds exhibit activity below 1 μM, with approximately one-third reaching below 100 nM, and the most active compound exhibiting activity of approximately 10 nM. DETAILED DESCRIPTION

[0071] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0072] Example 1

[0073] Synthesis of compounds

[0074] 1. The general synthesis formula of partially alkyl- or halogen-substituted 3-aminoisoquinoline intermediates is as follows:

[0075]

[0076] In a reaction flask, dissolve 4.4 mmol of 2,2-diethoxyacetonitrile in 20 mL of methanol. To this solution, add dropwise 0.4 mmol of a 30% sodium methoxide / methanol solution and stir at room temperature for 16 hours. Add 0.4 mmol of glacial acetic acid to adjust the pH to 7-8, then add 4.0 mmol of a substituted benzylamine. Heat to 40°C and stir for 4 hours. Remove the solvent using a rotary evaporator, add 4 mL of concentrated sulfuric acid, and heat to 40°C and stir for 16 hours. After the reaction is complete, neutralize with aqueous ammonia in an ice bath and concentrate to obtain the crude product. The crude product is separated by column chromatography (200-300 mesh silica gel, eluent: n-hexane:ethyl acetate = 3:1) to obtain the pure intermediate. This synthetic method can obtain 6-alkyl / halogenated-3-aminoisoquinoline from 4-alkyl / halogenated benzylamine, or simultaneously obtain 5-halogenated-3-aminoisoquinoline and 7-halogenated-3-aminoisoquinoline from 3-halogenated benzylamine, or obtain 8-alkyl / halogenated-3-aminoisoquinoline from 2-alkyl / halogenated benzylamine, or obtain 5,7-dihalogenated-3-aminoisoquinoline from 3,5-dihalogenated benzylamine, or obtain 5,6,7-trihalogenated-3-aminoisoquinoline from 3,4,5-trihalogenated benzylamine.

[0077]

[0078] 5,6,7-Trifluoro-3-aminoisoquinoline, isolated yield 63%. 1 H NMR (600MHz, CDCl3), δ (ppm) 7.72 (s, 1H, CH), 8.00 (t, J = 8.4Hz, 1H, CH), 8.95 (s, 1H, CH); 13 C NMR (150MHz, CDCl3), δ (ppm) 96.5 (dd, J = 6.0, 2.7Hz), 105.4 (dd, J = 17.6, 4.5Hz), 117.3 (dd, J = 8.2, 5.1Hz), 123.8 (d, J = 13.4H z), 126.6 (d, J = 14.4Hz), 141.7 (d, J = 235.0Hz), 142.5 (d, J = 241.6Hz), 148.0 (dd, J = 246.9, 12.0Hz), 147.2 (d, J = 38.0Hz); HRMS calcd for C9H5F3N2[M+H] + :199.0478; found:199.0475.

[0079] 2. The synthesis of some 4-halogenated-3-aminoisoquinoline intermediates is as follows:

[0080]

[0081] Dissolve 1 mmol of 3-aminoisoquinoline in 5 mL of DMF in a reaction flask. Add 1.5 mmol of SelectFluor® to this solution and stir at room temperature for 1 hour. After the reaction, concentrate the reaction solution and separate it using column chromatography (200-300 mesh silica gel, eluent: dichloromethane:methanol = 25:1) to obtain the pure intermediate 4-fluoro-3-aminoisoquinoline in a 30% yield. 1 HNMR (600MHz, CDCl3), δ (ppm) 7.67 (ddd, J=7.8, 7.2, 0.6Hz, 1H, CH), 7.84 (ddd, J=7.8, 7.2, 0.6Hz,1H,CH),8.04(dd,J=7.8,0.6Hz,1H,CH),8.19(d,J=7.8Hz,1H,CH),8.95(s,1H,CH); 13 C NMR (125MHz, CDCl3), δ (ppm) 116.4 (d, J = 3.4Hz), 124.1 (J = 248.3Hz), 126.6 (d, J = 13.9 Hz),127.2,127.4,127.7,129.5,130.2(d,J=10.9Hz),138.6(d,J=6.5Hz); HRMScalcd for C9H7FN2[M+H] + :163.0666;found:163.0668.

[0082] Dissolve 1 mmol of 3-aminoisoquinoline in 5 mL of methanol in a reaction flask. Add 1.2 mmol of N-chlorosuccinimide to this solution and stir at room temperature for 3 hours. After the reaction, concentrate the reaction mixture and separate it using column chromatography (200-300 mesh silica gel, eluent: n-hexane:ethyl acetate = 7:1) to obtain the pure intermediate 4-chloro-3-aminoisoquinoline.

[0083] 3. The synthesis of 1-iodo-3-aminoisoquinoline intermediate is as follows:

[0084]

[0085] In a reaction flask, 1 mmol of 1-bromo-3-aminoisoquinoline, 0.2 mmol of cuprous iodide, 0.4 mmol of N,N'-dimethylethylenediamine (DMEDA), and 2 mmol of sodium iodide were dissolved in 6 mL of 1,4-dioxane and heated to 110°C with stirring for 16 hours. After the reaction, the reaction mixture was concentrated and separated by column chromatography (200-300 mesh silica gel, eluent: n-hexane:ethyl acetate = 5:1) to obtain the pure intermediate 1-iodo-3-aminoisoquinoline.

[0086] 4. The general synthesis formula of phenyl-3-aminoisoquinoline intermediate is as follows:

[0087]

[0088] To a reaction flask, add 0.5 mmol of bromo-3-aminoisoquinoline, 1.0 mmol of phenylboronic acid, 0.025 mmol of tetrakis(triphenylphosphine)palladium, and 1.5 mmol of potassium carbonate. Evacuate and purge with nitrogen three times. Add 2 mL of 1,4-dioxane, 2 mL of toluene, and 0.8 mL of water, and heat to 110°C with stirring for 12 hours. After the reaction is complete, add 3 mL of water, separate the organic phase, extract the aqueous phase three times with ethyl acetate, combine the combined organic phases, dry over anhydrous sodium sulfate, filter, and concentrate to obtain the crude product. The crude product is separated by column chromatography (200-300 mesh silica gel, eluent: dichloromethane:methanol = 50:1) to obtain the pure intermediate phenyl-3-aminoisoquinoline.

[0089]

[0090] 5-Phenyl-3-aminoisoquinoline, isolated yield 95%. 1 H NMR (600MHz, CDCl3), δ (ppm) 7.36 (s, 1H, CH), 7.45-7.51 (m, 3H, CH), 7.50 (d, J = 8.4Hz, 2H, CH), 7.57 ( dd,J=8.4,7.2Hz,1H,CH),7.62(dd,J=7.2,1.2Hz,1H,CH),8.01(d,J=7.8Hz,1H,CH),8.97(s,1H,CH); 13 HRMS calcd for C 15 H 12 N2[M+H] + :221.1073; found:221.1072.

[0091]

[0092] 8-Phenyl-3-aminoisoquinoline, isolated yield 95%. 1H NMR (600MHz, CDCl3), δ (ppm) 7.25 (d, J = 7.2Hz, 1H, CH), 7.44-7.54 (m, 5H, CH), 7.60 (s ,1H,CH),7.71(dd,J=8.4,7.2Hz,1H,CH),7.79(d,J=8.4Hz,1H,CH),8.89(s,1H,CH); 13 HRMS calcdfor C 15 H 12 N2[M+H] + :221.1073;found:221.1071.

[0093] 5. Synthesis of 1-(4-fluoroisoquinolin-3-yl)thiourea (Compound 1)

[0094]

[0095] In a reaction flask, 0.5 mmol of 4-fluoro-3-aminoisoquinoline was dissolved in 3 mL of acetone. To this solution was added 0.55 mmol of benzoyl isothiocyanate, and the mixture was heated to 65°C and stirred for 12 hours. The reaction solution was concentrated, redissolved in 4 mL of ethanol, and 1.5 mL of 1 M aqueous NaOH was added dropwise. The mixture was heated to 50°C and stirred for 6 hours. After the reaction, saturated ammonium chloride solution was added to quench the reaction. The mixture was extracted three times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was separated by column chromatography (200-300 mesh silica gel, eluent: dichloromethane:methanol = 50:1) to obtain the pure target product, 1-(4-fluoroisoquinolin-3-yl)thiourea. 1-(4-fluoroisoquinolin-3-yl)thiourea (Compound 1) was isolated in an 80% yield. 1 H NMR (800MHz, DMSO-d6), δ (ppm) 7.69 (ddd, J=8.0, 7.2, 0.8Hz, 1H, CH), 7.87 (ddd, J=8.0, 6.4, 1.6Hz, 1H, CH), 8.06 (dd ,J=8.0,0.8Hz,1H,CH),8.20(d,J=8.0Hz,1H,CH),8.66(s,1H,NH),9.05(s,1H,CH),9.21(s,1H,NH),9.86(s,1H,NH); 13C NMR (200MHz, DMSO-d6), δ (ppm) 118.8 (d, J = 3.4Hz), 126.6 (d, J = 13.8Hz), 127.0, 127.1 ( HRMS calcd for C 10 H8FN3S[M+H] + :222.0496;found:222.0494.

[0096] 6. Synthesis of 1-(5-fluoroisoquinolin-3-yl)thiourea (Compound 2):

[0097]

[0098] In a reaction flask, 0.5 mmol of 5-fluoroisoquinolin-3-amine was dissolved in 5 mL of dichloromethane. To this solution was added 0.85 mmol of 1,1-thiocarbonyldiimidazole (TCDI, 85% purity). After stirring at room temperature for 12 hours, 1 mL of a 7M ammonia solution in methanol was added and stirring continued at room temperature for 1 hour. After completion of the reaction, the reaction solution was concentrated and isolated by column chromatography (200-300 mesh silica gel, eluent: n-hexane:ethyl acetate = 3:1 or dichloromethane:methanol = 50:1) to obtain the pure target product, 1-(5-fluoroisoquinolin-3-yl)thiourea (Compound 2), in a 36% yield. 1 H NMR (800MHz, DMSO-d6), δ (ppm) 7.47-7.49 (m, 1H, CH), 7.54 (dd, J = 10.4, 8.0Hz, 1H, CH), 7.73 (s, 1H, CH),7.92(d,J=8.0Hz,1H,CH),8.81(s,1H,NH),9.21(s,1H,CH),10.15(s,1H,NH),10.70(s,1H,NH); 13 C NMR (200MHz, DMSO-d6), δ (ppm) 98.2 (d, J = 3.4Hz), 114.6 (d, J = 18.2Hz), 124.3 (d, J = 4.0Hz), 125.4 (d, J = 7.4 Hz), 125.7 (d, J = 4.8Hz), 127.9 (d, J = 17.8Hz), 149.4, 150.2 (d, J = 2.6Hz), 156.4 (d, J = 249.6Hz), 180.5; HRMS calcd for C 10 H8FN3S[M+H] + :222.0496;found:222.0496.

[0099] 7. Synthesis of other compounds:

[0100]

[0101] 1-(6-Fluoroisoquinolin-3-yl)thiourea (Compound 3) was prepared by referring to the preparation method of Compound 2 with an isolated yield of 54%. 1 H NMR(600MHz, DMSO-d6), δ(ppm)7.40(td,J=9.0,2.4Hz,1H,CH),7.54(s,1H,CH),7.62(dd,J=10.2,2.4Hz,1H ,CH),8.17(dd,J=9.0,5.4Hz,1H,CH),8.76(s,1H,NH),9.13(s,1H,CH),10.23(s,1H,NH),10.64(s,1H,NH); 13 C NMR (150MHz, DMSO-d6), δ (ppm) 105.0 (d, J = 5.8Hz), 109.1 (d, J = 21.6Hz), 116.3 (d, J = 26.4Hz), 12 2.2,131.8(d,J=10.4Hz),139.0(d,J=11.6Hz),149.6,150.2,163.3(d,J=249.2Hz),180.5; HRMS calcd forC 10 H8FN3S[M+H] + :222.0496; found:222.0499.

[0102]

[0103] 1-(7-Fluoroisoquinolin-3-yl)thiourea (Compound 4) was prepared by referring to the preparation method of Compound 2 with an isolated yield of 44%. 1 H NMR (800MHz, DMSO-d6), δ (ppm) 7.62-7.65 (m, 2H, CH), 7.87 (dd, J = 9.6, 2.4Hz, 1H, CH), 7.92 (d d,J=9.6,5.6Hz,1H,CH),8.72(s,1H,NH),9.10(s,1H,CH),10.12(s,1H,NH),10.61(s,1H,NH); 13C NMR (200MHz, DMSO-d6), δ (ppm) 105.7, 110.6 (d, J = 20.8Hz), 122.2 (d, J = 26.0Hz), 125.0 (d, J = 9.0Hz), 129.3(d,J=8.4Hz),134.8,148.8(d,J=2.6Hz),149.3(d,J=6.0Hz),159.1(d,J=243.8Hz),180.4; HRMS calcd for C 10 H8FN3S[M+H] + :222.0496; found:222.0499.

[0104]

[0105] 1-(8-Fluoroisoquinolin-3-yl)thiourea (Compound 5) was prepared by referring to the preparation method of Compound 2 with an isolated yield of 61%. 1 H NMR (600MHz, DMSO-d6), δ (ppm) 7.27 (dd, J=10.8, 7.2, 1.2Hz, 1H, CH), 7.63-7.69 (m,3H,CH),8.82(s,1H,NH),9.24(s,1H,CH),10.16(s,1H,NH),10.70(s,1H,NH); 13 C NMR (150MHz, DMSO-d6), δ (ppm) 105.1, 109.2 (d, J = 18.3Hz), 115.1 (d, J = 16.2Hz), 122.5 (d, J = 4.0Hz), 132.1(d,J=9.0Hz), 138.9(d,J=3.2Hz), 143.7(d,J=4.5Hz), 149.9, 158.6(d,J=253.0Hz), 180.5; HRMS calcd for C 10 H8FN3S[M+H] + :222.0496;found:222.0498.

[0106]

[0107] 1-(1-Chloroisoquinolin-3-yl)thiourea (Compound 6) was prepared by referring to the preparation method of Compound 2 with an isolated yield of 34%. 1H NMR (800MHz, DMSO-d6), δ (ppm) 7.47 (ddd, J=8.0, 7.2, 0.8Hz, 1H, CH), 7.78-7.80 (m, 2H, CH), 7.91 ( d,J=8.0Hz,1H,CH),8.16(d,J=8.8Hz,1H,CH),8.77(s,1H,NH),9.18(s,1H,NH),10.73(s,1H,NH); 13 C NMR(200MHz, DMSO-d6), δ(ppm)106.2,122.4,125.6,127.0,127.4,132.3,139.4,146.8,147.6,180.3; HRMS calcd for C 10 H8ClN3S[M+H] + :238.0200;found:238.0195.

[0108]

[0109] 1-(4-Chloroisoquinolin-3-yl)thiourea (Compound 7) was prepared by referring to the preparation method of Compound 1 with an isolated yield of 95%. 1 H NMR (800MHz, DMSO-d6), δ (ppm) 7.69 (t, J = 8.0Hz, 1H, CH), 7.93 (t, J = 8.0Hz, 1H, CH), 8.08 (d, J = 8.8Hz, 1H,CH),8.21(d,J=8.0Hz,1H,CH),8.97(s,1H,NH),9.02(s,1H,NH),9.17(s,1H,CH),9.56(s,1H,NH); 13 C NMR(200MHz, DMSO-d6), δ(ppm)111.9,122.4,126.2,127.0,128.6,133.0,134.2,143.4,149.4,180.4; HRMS calcd for C 10 H8ClN3S[M+H] + :238.0200;found:238.0197.

[0110]

[0111] 1-(5-Chloroisoquinolin-3-yl)thiourea (Compound 8) was prepared by referring to the preparation method of Compound 2 with an isolated yield of 28%. 1H NMR (800MHz, DMSO-d6), δ (ppm) 7.49 (t, J=8.0Hz, 1H, CH), 7.89 (dd, J=8.0, 0.8Hz, 1H, CH), 7.94 (s, 1H ,CH),8.08(d,J=8.8Hz,1H,CH),8.82(s,1H,NH),9.20(s,1H,CH),10.14(s,1H,NH),10.75(s,1H,NH); 13 C NMR(200MHz, DMSO-d6), δ(ppm)101.8,125.5,125.7,127.6,128.9,131.3,135.1,150.1,150.9,180.5; HRMS calcd for C 10 H8ClN3S[M+H] + :238.0200;found:238.1999.

[0112]

[0113] 1-(6-Chloroisoquinolin-3-yl)thiourea (Compound 9) was prepared by referring to the preparation method of Compound 2 with an isolated yield of 57%. 1 H NMR(600MHz, DMSO-d6), δ(ppm)7.50(dd,J=9.0,1.8Hz,1H,CH),7.54(s,1H,CH),7.96(d,J=1.2Hz,1H ,CH),8.10(d,J=9.0Hz,1H,CH),8.77(s,1H,NH),9.14(s,1H,CH),10.18(s,1H,NH),10.67(s,1H,NH); 13 C NMR(150MHz, DMSO-d6), δ(ppm)104.7,123.0,124.7,126.3,130.4,136.4,138.2,149.8,150.3,180.5; HRMS calcd for C 10 H8ClN3S[M+H] + :238.0200;found:238.0201.

[0114]

[0115] 1-(7-Chloroisoquinolin-3-yl)thiourea (Compound 10) was prepared by referring to the preparation method of Compound 2 with an isolated yield of 26%. 1H NMR (800MHz, DMSO-d6), δ (ppm) 7.60 (s, 1H, CH), 7.69 (dd, J=8.8, 1.6Hz, 1H, CH), 7.87 (d, J=8.8Hz, 1H ,CH),8.20(d,J=2.4Hz,1H,CH),8.76(s,1H,NH),9.11(s,1H,CH),10.12(s,1H,NH),10.66(s,1H,NH); 13 C NMR(200MHz, DMSO-d6), δ(ppm)105.5,125.1,126.6,128.4,129.6,131.9,135.9,149.3,149.4,180.4; HRMS calcd for C 10 H8ClN3S[M+H] + :238.0200;found:238.0205.

[0116]

[0117] 1-(8-Chloroisoquinolin-3-yl)thiourea (Compound 11) was prepared by referring to the preparation method of Compound 2 with an isolated yield of 43%. 1 H NMR (600MHz, DMSO-d6), δ (ppm) 7.62-7.67 (m, 3H, CH), 7.81 (d, J = 8.4Hz, 1H, CH), 8.82 (s, 1H, NH), 9.31 (s, 1H, CH), 10.15 (s, 1H, NH), 10.72 (s, 1H, NH); 13 C NMR(150MHz, DMSO-d6), δ(ppm)105.6,121.6,125.92,125.94,131.2,131.7,139.0,146.6,149.8,180.5; HRMScalcd for C 10 H8ClN3S[M+H] + :238.0200;found:238.0195.

[0118]

[0119] 1-(1-Bromoisoquinolin-3-yl)thiourea (Compound 12) was prepared by referring to the preparation method of Compound 2 with an isolated yield of 57%. 1H NMR (600MHz, DMSO-d6), δ (ppm) 7.64 (ddd, J=8.4, 6.6, 1.2Hz, 1H, CH), 7.78 (ddd, J=7.8, 6.6, 1.2Hz, 1H, CH), 7.83 (s, 1 H,CH),7.89(d,J=8.4Hz,1H,CH),8.09(dd,J=9.0,0.6Hz,1H,CH),8.77(s,1H,NH),9.13(s,1H,NH),10.73(s,1H,NH); 13 C NMR(150MHz, DMSO-d6), δ(ppm)106.5,124.6,127.1,127.7,127.8,132.2,138.9,141.2,147.2,180.2; HRMScalcd for C 10 H8BrN3S[M+H] + :281.9695;found:281.9698.

[0120]

[0121] 1-(4-Bromoisoquinolin-3-yl)thiourea (Compound 13) was prepared by referring to the preparation method of Compound 1 with an isolated yield of 91%. 1 H NMR (600MHz, DMSO-d6), δ (ppm) 7.66 (ddd, J=7.8, 7.2, 0.6Hz, 1H, CH), 7.90 (ddd, J=7.8, 6.6, 0.6Hz, 1H, CH), 8.01 ( d,J=8.4Hz,1H,CH),8.17(d,J=8.4Hz,1H,CH),8.86(s,1H,NH),9.07(s,1H,NH),9.16(s,1H,CH),9.65(s,1H,NH); 13 C NMR (150MHz, DMSO-d6), δ (ppm) 104.0, 125.0, 126.3, 126.9, 128.7, 133.2, 135.8, 144.7, 150.2, 180.4; HRMS calcdfor C 10 H8BrN3S[M+H] + :281.9695;found:281.9693.

[0122]

[0123] 1-(5-Bromoisoquinolin-3-yl)thiourea (Compound 14) was prepared by referring to the preparation method of Compound 2 with an isolated yield of 48%. 1H NMR (600MHz, DMSO-d6), δ (ppm) 7.42 (dd, J=7.8, 7.2Hz, 1H, CH), 7.93 (s, 1H, CH), 8.06 (dd, J=7.8, 1.2Hz, 1H,CH),8.11(d,J=8.4Hz,1H,CH),8.81(s,1H,NH),9.16(s,1H,CH),10.12(s,1H,NH),10.76(s,1H,NH); 13 C NMR (150MHz, DMSO-d6), δ (ppm) 104.4,119.8,125.7,126.2,128.2,135.0,136.4,150.4,151.0,180.5; HRMS calcd for C 10 H8BrN3S[M+H] + :281.9695;found:281.9690.

[0124]

[0125] 1-(6-Bromoisoquinolin-3-yl)thiourea (Compound 15) was prepared by referring to the preparation method of Compound 2 with an isolated yield of 48%. 1 H NMR (600MHz, DMSO-d6), δ (ppm) 7.53 (s, 1H, CH), 7.60 (dd, J=9.0, 1.8Hz, 1H, CH), 8.00 (d, J=8.4Hz, 1H ,CH),8.11(d,J=1.8Hz,1H,CH),8.77(s,1H,NH),9.13(s,1H,CH),10.17(s,1H,NH),10.67(s,1H,NH); 13 C NMR(150MHz, DMSO-d6), δ(ppm)104.5,123.1,125.6,127.9,128.8,130.2,138.5,149.7,150.4,180.5; HRMS calcd for C 10 H8BrN3S[M+H] + :281.9695;found:281.9692.

[0126]

[0127] 1-(7-Bromoisoquinolin-3-yl)thiourea (Compound 16) was prepared by referring to the preparation method of Compound 2 with an isolated yield of 46%. 1H NMR(600MHz,DMSO-d6), δ(ppm)7.58(s,1H,CH),7.76-7.80(m,2H,CH),8.35(s,1H,CH),8.76(s,1H,NH),9.10(s,1H,CH),10.12(s,1H,NH),10.66(s,1H,NH); 13 C NMR (150MHz, DMSO-d6), δ (ppm) 105.4,118.0,125.6,128.4,129.9,134.3,136.0,149.3,149.4,180.4; HRMS calcd for C 10 H8BrN3S[M+H] + :281.9695;found:281.9602.

[0128]

[0129] 1-(8-Bromoisoquinolin-3-yl)thiourea (Compound 17) was prepared by referring to the preparation method of Compound 2 with an isolated yield of 48%. 1 H NMR(600MHz, DMSO-d6), δ(ppm)7.57(dd,J=7.8,7.2Hz,1H,CH),7.61(s,1H,CH),7.79(d,J=7.8Hz,1H ,CH),7.83(d,J=8.4Hz,1H,CH),8.82(s,1H,NH),9.22(s,1H,CH),10.14(s,1H,NH),10.72(s,1H,NH); 13 C NMR(150MHz, DMSO-d6), δ(ppm)105.5,121.6,122.6,126.5,129.6,132.0,139.2,149.0,149.8,180.5; HRMS calcd for C 10 H8BrN3S[M+H] + :281.9695;found:281.9695.

[0130]

[0131] 1-(1-iodoisoquinolin-3-yl)thiourea (Compound 18) was prepared by referring to the preparation method of Compound 2 with an isolated yield of 44%. 1H NMR (800MHz, DMSO-d6), δ (ppm) 7.61 (ddd, J=8.0, 7.2, 0.8Hz, 1H, CH), 7.74 (ddd, J=8.0, 7.2, 0.8Hz, 2H, CH ),7.79(d,J=8.0Hz,1H,CH),7.92(d,J=8.0Hz,1H,CH),8.79(s,1H,NH),9.32(s,1H,NH),10.69(s,1H,NH); 13 C NMR(200MHz, DMSO-d6), δ(ppm)106.3,124.2,127.2,127.8,128.1,131.9,132.0,137.4,147.9,180.2; HRMS calcd for C 10 H8IN3S[M+H] + :329.9556; found:329.9551.

[0132]

[0133] 1-(4-iodoisoquinolin-3-yl)thiourea (Compound 19) was prepared by referring to the preparation method of Compound 1 with an isolated yield of 82%. 1 H NMR (800MHz, DMSO-d6), δ (ppm) 7.65 (t, J=8.0Hz, 1H, CH), 7.88 (td, J=8.0, 1.6Hz, 1H, CH), 7.95 (d, J=8.8 Hz,1H,CH),8.12(d,J=8.0Hz,1H,CH),8.91(s,1H,NH),9.02(s,1H,NH),9.10(s,1H,CH),9.64(s,1H,NH); 13 C NMR (200MHz, DMSO-d6), δ (ppm)85.0,126.0,126.9,128.8,130.2,133.4,138.9,148.0,151.1,180.4; HRMS calcd for C 10 H8IN3S[M+H] + :329.9556; found:329.9555.

[0134]

[0135] 1-(5-iodoisoquinolin-3-yl)thiourea (Compound 20) was prepared by referring to the preparation method of Compound 2 with an isolated yield of 30%. 1H NMR(800MHz, DMSO-d6), δ(ppm)7.28(dd,J=8.0,7.2Hz,1H,CH),7.86(s,1H,CH),8.11(d,J=8.8Hz,1H,CH ),8.30(dd,J=7.2,0.8Hz,1H,CH),8.78(s,1H,NH),9.06(s,1H,CH),10.12(s,1H,NH),10.78(s,1H,NH); 13 C NMR(200MHz, DMSO-d6), δ(ppm)97.1,109.3,125.4,126.8,128.8,139.2,142.2,150.6,151.3,180.5; HRMS calcd for C 10 H8IN3S[M+H] + :329.9556; found:329.9551.

[0136]

[0137] 1-(6-iodoisoquinolin-3-yl)thiourea (Compound 21) was prepared by referring to the preparation method of Compound 2 with an isolated yield of 29%. 1 H NMR (600MHz, DMSO-d6), δ (ppm) 7.48 (s, 1H, CH), 7.76 (dd, J = 8.4, 1.2Hz, 1H, CH), 7.83 (d, J = 8.4H z,1H,CH),8.31(s,1H,CH),8.76(s,1H,NH),9.10(s,1H,CH),10.17(s,1H,NH),10.65(s,1H,NH); 13 CNMR (150MHz, DMSO-d6), δ (ppm) 100.1, 104.1, 123.3, 129.6, 134.0, 134.4, 138.7, 149.5, 150.4, 180.4; HRMS calcd for C 10 H8IN3S[M+H] + :329.9556; found:329.9559.

[0138]

[0139] 1-(7-iodoisoquinolin-3-yl)thiourea (Compound 22) was prepared by referring to the preparation method of Compound 2 with an isolated yield of 13%. 1H NMR (800MHz, DMSO-d6), δ (ppm) 7.55 (s, 1H, CH), 7.62 (d, J=8.8Hz, 1H, CH), 7.90 (dd, J=8.8, 1.6H z,1H,CH),8.53(s,1H,CH),8.75(s,1H,NH),9.07(s,1H,CH),10.15(s,1H,NH),10.64(s,1H,NH); 13 C NMR(200MHz, DMSO-d6), δ(ppm)90.7,105.4,126.2,128.0,136.1,136.4,139.3,149.2,149.3,180.4; HRMS calcd for C 10 H8IN3S[M+H] + :329.9556; found:329.9560.

[0140]

[0141] 1-(8-Iodoisoquinolin-3-yl)thiourea (Compound 23) was prepared by referring to the preparation method of Compound 2 with an isolated yield of 26%. 1 H NMR(600MHz, DMSO-d6), δ(ppm)7.39(dd,J=8.4,7.2Hz,1H,CH),7.51(s,1H,CH),7.82(d,J=9.0Hz,1H,CH ),8.04(dd,J=7.2,0.6Hz,1H,CH),8.80(s,1H,NH),9.05(s,1H,CH),10.15(s,1H,NH),10.70(s,1H,NH); 13 C NMR (150MHz, DMSO-d6), δ (ppm)98.3,105.4,124.8,127.2,132.3,137.0,138.7,149.8,153.7,180.4; HRMS calcd for C 10 H8IN3S[M+H] + :329.9556; found:329.9550.

[0142]

[0143] 1-(5,7-Difluoroisoquinolin-3-yl)thiourea (Compound 24) was prepared by referring to the preparation method of Compound 2 with an isolated yield of 47%. 1H NMR (600MHz, DMSO-d6), δ (ppm) 7.68-7.72 (m, 1H, CH), 7.74 (s, 1H, CH), 7.78 (dd, J = 9.0 ,1.8Hz,1H,CH),8.80(s,1H,NH),9.16(s,1H,CH),10.01(s,1H,NH),10.69(s,1H,NH); 13 C NMR (150MHz, DMSO-d6), δ (ppm) 98.6, 107.1 (dd, J = 30.4, 22.8Hz), 107.3 (dd, J = 21.4, 5.0Hz), 124.7 (dd, J = 10.6, 6.2Hz), 125.6 HRMScalcd for C 10 H7F2N3S[M+H] + :240.0402;found:240.0406.

[0144]

[0145] 1-(5,7-Dichloroisoquinolin-3-yl)thiourea (Compound 25) was prepared by referring to the preparation method of Compound 2 with an isolated yield of 25%. 1 H NMR (600MHz, DMSO-d6), δ (ppm) 7.96 (s, 1H, CH), 8.01 (d, J=2.4Hz, 1H, CH), 8.24 (d, J= 1.2Hz,1H,CH),8.85(s,1H,NH),9.17(s,1H,CH),10.01(s,1H,NH),10.78(s,1H,NH); 13 C NMR(150MHz, DMSO-d6), δ(ppm)101.9,125.4,126.3,128.8,130.4,131.3,133.9,150.2,150.3,180.5; HRMS calcd for C 10 H7Cl2N3S[M+H] + :271.9810;found:271.9805.

[0146]

[0147] 1-(5,7-Dibromoisoquinolin-3-yl)thiourea (Compound 26) was prepared by referring to the preparation method of Compound 2 with an isolated yield of 33%. 1 H NMR (600MHz, DMSO-d6), δ (ppm) 7.94 (s, 1H, CH), 8.22 (d, J=1.8Hz, 1H, CH), 8.42 (d, J= 0.6Hz,1H,CH),8.84(s,1H,NH),9.13(s,1H,CH),10.00(s,1H,NH),10.80(s,1H,NH); 13 C NMR(150MHz, DMSO-d6), δ(ppm)104.5,117.1,121.1,126.0,130.1,135.3,136.8,150.3,150.6,180.5; HRMS calcd for C 10 H7Br2N3S[M+H] + :359.8800; found:359.8804.

[0148]

[0149] 1-(5,6,7-Trifluoroisoquinolin-3-yl)thiourea (Compound 27) was prepared by referring to the preparation method of Compound 2 with an isolated yield of 25%. 1 H NMR (600MHz, DMSO-d6), δ (ppm) 7.75 (s, 1H, CH), 8.07 (t, J = 8.4Hz, 1H, CH), 8.85 (s, 1H, NH), 9.14 (s, 1H, CH), 9.98 (s, 1H, NH), 10.72 (s, 1H, NH); 13 C NMR (150MHz, DMSO-d6), δ (ppm) 97.9 (dd, J = 6.0, 2.7Hz), 109.6 (dd, J = 17.6, 4.5Hz), 119.4 (dd, J = 8.2, 5.1Hz), 124.3 (d, J = 13.4Hz) ,126.4(d,J=14.4Hz),140.5(d,J=235.0Hz),143.8(d,J=241.6Hz),148.2(dd,J=246.9,12.0Hz),149.6(d,J=38.0Hz),180.5; HRMS calcd forC 10 H6F3N3S[M+H] + :258.0307;found:258.0305.

[0150]

[0151] 1-(6-Methylisoquinolin-3-yl)thiourea (Compound 28) was prepared by referring to the preparation method of Compound 2 with an isolated yield of 74%. 1 H NMR (600MHz, DMSO-d6), δ (ppm) 2.45 (s, 3H, CH3), 7.32 (dd, J = 9.0, 1.8Hz, 1H, CH), 7.44 (s, 1H, CH), 7.54 (s ,1H,CH),7.92(d,J=8.4Hz,1H,CH),8.70(s,1H,NH),9.01(s,1H,CH),10.31(s,1H,NH),10.55(s,1H,NH); 13 C NMR(150MHz, DMSO-d6), δ(ppm)21.6,104.9,123.3,124.6,127.7,128.0,137.7,141.6,149.2,149.5,180.4; HRMS calcd for C 11 H 11 N3S[M+H] + :218.0746; found:218.0744.

[0152]

[0153] 1-(8-Methylisoquinolin-3-yl)thiourea (Compound 29) was prepared by referring to the preparation method of Compound 2 with an isolated yield of 68%. 1 H NMR (600MHz, DMSO-d6), δ (ppm) 2.69 (s, 3H, CH3), 7.29 (d, J = 6.6Hz, 1H, CH), 7.53-7.57 (m, 2H, CH) ,7.62(d,J=8.4Hz,1H,CH),8.74(s,1H,NH),9.21(s,1H,CH),10.35(s,1H,NH),10.60(s,1H,NH); 13 CNMR (150MHz, DMSO-d6), δ (ppm) 18.0, 105.8, 123.8, 124.3, 126.2, 131.3, 135.7, 137.9, 147.1, 149.0, 180.4; HRMS calcd for C 11 H 11 N3S[M+H] + :218.0746;found:218.0740.

[0154]

[0155] 1-(6-Isopropylisoquinolin-3-yl)thiourea (Compound 30) was prepared by referring to the preparation method of Compound 2 with an isolated yield of 67%. 1 H NMR (600MHz, DMSO-d6), δ (ppm) 1.28 (d, J=7.2Hz, 6H, CH3), 3.06 (hept, J=7.2Hz, 1H, CH), 7.45 (dd, J=9.0, 1.8Hz, 1H, CH), 7.4 9(s,1H,CH),7.58(s,1H,CH),7.98(d,J=8.4Hz,1H,CH),8.70(s,1H,NH),9.04(s,1H,CH),10.31(s,1H,NH),10.57(s,1H,NH); 13 CNMR (150MHz, DMSO-d6), δ (ppm) 23.3, 33.8, 105.3, 121.9, 123.7, 125.8, 128.0, 137.8, 149.2, 149.4, 152.0, 180.4; HRMS calcd for C 13 H 15 N3S[M+H] + :246.1059; found:246.1063.

[0156]

[0157] 1-(6-tert-Butylisoquinolin-3-yl)thiourea (Compound 31) was prepared by referring to the preparation method of Compound 2 with an isolated yield of 68%. 1 H NMR(600MHz,DMSO-d6),δ(ppm)1.36(s,9H,CH3),7.50(s,1H,CH),7.63-7.64(m,2H,CH),7.9 9(d,J=9.6Hz,1H,CH),8.70(s,1H,NH),9.04(s,1H,CH),10.32(s,1H,NH),10.58(s,1H,NH); 13 HRMS calcd for C 14 H 17 N3S[M+H] + :260.1216;found:260.1211.

[0158]

[0159] 1-(1-Phenylisoquinolin-3-yl)thiourea (Compound 32) was prepared by referring to the preparation method of Compound 2 with an isolated yield of 95%. 1 H NMR (600MHz, methanol-d4), δ (ppm) 7.37 (s, 1H, CH), 7.47 (ddd, J = 8.4, 6.6, 1.2Hz, 1H, CH), 7.55- 7.61(m,3H,CH),7.67-7.72(m,3H,CH),7.87(d,J=7.8Hz,1H,CH),8.02(dd,J=9.0,0.6Hz,1H,CH); 13 CNMR (150MHz, methanol-d4), δ (ppm) 106.3, 124.5, 127.2, 127.9, 128.5, 129.7, 130.3, 130.8, 132.2, 140.0, 140.8, 149.3, 160.0, 182.4; HRMScalcd for C 16 H 13 N3S[M+H] + :280.0903;found:280.0905.

[0160]

[0161] 1-(4-Phenylisoquinolin-3-yl)thiourea (Compound 33) was prepared by referring to the preparation method of Compound 1 with an isolated yield of 55%. 1 H NMR (600MHz, DMSO-d6), δ (ppm) 7.31 (d, J = 8.4Hz, 1H, CH), 7.46 (d, J = 7.2Hz, 2H, CH), 7.58-7.63 (m, 2H ,CH),7.67-7.70(m,3H,CH),7.82(s,1H,NH),8.19(d,J=8.4Hz,1H,CH),9.00(br,1H,NH),9.25(s,1H CH),10.10(br,1H,NH); 13 HRMS calcd for C 16 H 13 N3S[M+H] + :280.0903;found:280.0901.

[0162]

[0163] 1-(5-Phenylisoquinolin-3-yl)thiourea (Compound 34) was prepared by referring to the preparation method of Compound 2 with an isolated yield of 62%. 1 H NMR (600MHz, methanol-d4), δ (ppm) 7.44 (s, 1H, CH), 7.47-7.50 (m, 3H, CH), 7.56 (d, J = 7.8Hz, 2H, CH), 7. 60(dd,J=8.4,7.8Hz,1H,CH),7.65(dd,J=7.2,1.2Hz,1H,CH),8.05(d,J=7.8Hz,1H,CH),9.14(s,1H,CH); 13 HRMS calcd for C 16 H 13 N3S[M+H] + :280.0903;found:280.0906.

[0164]

[0165] 1-(6-Phenylisoquinolin-3-yl)thiourea (Compound 35) was prepared by referring to the preparation method of Compound 2 with an isolated yield of 51%. 1 H NMR(800MHz, DMSO-d6), δ(ppm)7.45(t,J=8.0Hz,1H,CH),7.53(t,J=8.0Hz,2H,CH),7.62(br,1H,NH),7.83-7.86(m,3H, CH),8.07(s,1H,CH),8.14(d,J=8.0Hz,1H,CH),8.74(br,1H,NH),9.14(s,1H,CH),10.30(br,1H,NH),10.66(s,1H,CH); 13 HRMS calcd for C 16H 13 N3S[M+H] + :280.0903;found:280.0902.

[0166]

[0167] 1-(7-Phenylisoquinolin-3-yl)thiourea (Compound 36) was prepared by referring to the preparation method of Compound 2 with an isolated yield of 55%. 1 H NMR (600MHz, methanol-d4), δ (ppm) 7.40-7.43 (m, 2H, CH), 7.52 (t, J = 7.8Hz, 2H, CH), 7.78 (dd, J = 8.4, 1. 2Hz,2H,CH),7.90(d,J=8.4Hz,1H,CH),8.03(dd,J=8.4,1.8Hz,1H,CH),8.26(s,1H,CH),9.15(s,1H,CH); 13 HRMS calcd for C 16 H 13 N3S[M+H] + :280.0903;found:280.0903.

[0168]

[0169] 1-(8-Phenylisoquinolin-3-yl)thiourea (Compound 37) was prepared by referring to the preparation method of Compound 2 with an isolated yield of 45%. 1 H NMR (600MHz, DMSO-d6), δ (ppm) 7.39 (d, J = 6.6 Hz, 1H, CH), 7.48-7.56 (m, 5H, CH), 7.63 (s, 1H, CH), 7.74 (dd, J = 8.4, 7.2Hz,1H,CH),7.82(d,J=8.4Hz,1H,CH),8.70(br,1H,NH),8.91(s,1H,CH),10.20(br,1H,NH),10.64(s,1H,NH); 13HRMS calcd for C 16 H 13 N3S[M+H] + :280.0903;found:280.0905.

[0170] Example 2

[0171] Detection method and results of compound agonist activity on TAS2R38 receptor

[0172] 1. Cell Viability Assay

[0173] HEK 293T cells were cultured in a 10 cm culture dish to a confluence of about 95%, digested with trypsin, resuspended in 1 mL of complete culture medium, and 200 μL was transferred to a 10 cm culture dish, for a total of 2 transfers (complete culture medium DMEM + 10% FBS + 1% double antibody), and the culture dish was placed in a CO2 incubator for overnight culture. 16g44 Plasmids were transfected into cells. Transfection system: Experimental group A: Opti-MEM 500 μL + 2 μg pcDNA3.1-T2R38 + 2 μg G 16g44 Mix well and incubate at room temperature for 5 min; control group A: Opti-MEM 500 μL + 2 μg empty pcDNA3.1 + 2 μg G 16g44 Mix 500μL of Opti-MEM® B and 16μL of Lipo2000 at room temperature for 5 minutes. Slowly add A to B, gently pipette to mix, incubate at room temperature for 5 minutes, add cells dropwise, gently shake to mix, and return the cells to the CO2 incubator for transfection for 24 hours. Digest the cells with Versene, centrifuge, and resuspend in 13mL of DMEM medium supplemented with 1% dialyzed serum. Plate one 384-well plate per group, using 30μL / well. Incubate in a cell culture incubator for 4-6 hours.

[0174] Dissolve one vial of the Calcium 6 bulk kit in 100 mL of HBSS plus 20 mM HEPES (pH 7.5) to create a 2X Calcium 6 stock solution. Aliquot and store at -20°C until ready for use. Prepare a 1X Calcium 6 working solution by adding 8.5 mL of 2X Calcium 6 to 8.5 mL of HBSS. Discard the cell supernatant and add 20 μL of 1X Calcium 6 to each well of the 384-well plate. Incubate at 37°C for 1 hour.

[0175] Compound stock solution: PTC was used as a positive control, N,N-dimethylacetamide solvent (DMA) without compound was used as a negative control, and 200 mM of the test compound was dissolved in DMA and stored at -20 degrees.

[0176] Assay buffer configuration: 0.1% BSA (0.030 g BSA + 30 mL HBSS)

[0177] The FLIPR assay was programmed as follows: 10 readings (1 reading per second) were taken as a baseline before adding 10 μL of drug solution. Fluorescence intensity was recorded 2 minutes after drug addition to determine small molecule activity.

[0178] Agonist activity assay:

[0179] In a 96V well plate, prepare 150μL of 3X working solution at the starting and ending concentrations of 100μM and 300μM and mix well. Then perform a 10-fold gradient dilution on these two groups. For example, take 15μL of the previous gradient compound solution and add 135μL of detection buffer and mix well to prepare the next gradient compound solution. Taking this as an example, dilute 6-7 times in total, with 16 concentration points. Use an electric dispenser to dispense the liquid into a 384V bottom microplate, 30μL / well, 3 replicates for each gradient, and add 10μL of compound to each well on the FLIPR machine and detect. Each compound should be measured at least 3 times. The results were exported and the data were analyzed using Graphpad software. The curve was fitted according to the following formula to obtain the EC value of the compound. 50 value:

[0180] Y=Bottom+(Top-Bottom) / (1+10^((LogEC 50 -X)))

[0181] Wherein, X represents the concentration of the compound, Y represents the measured fluorescence reading, Top represents the upper platform value of the curve, and Bottom represents the lower platform value of the curve.

[0182] EC 50 result:

[0183]

[0184]

[0185]

[0186] Although the above describes specific embodiments of the present invention, it should be understood by those skilled in the art that these are merely illustrative and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A compound represented by formula (I) or a pharmaceutically acceptable salt thereof, in, R 1 、R 2 、R 3 、R 4 、R 5 and R 6 are independently hydrogen, halogen, C 1-10 Alkyl or C 6-10 aryl; and R 1 、R 2 、R 3 、R 4 、R 5 and R 6 Not hydrogen at the same time.

2. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein: The compound represented by formula (I) satisfies one or more of the following conditions: (1)R 1 is hydrogen, halogen or C 6-10 aryl; (2)R 2 is hydrogen, halogen or C 6-10 aryl; (3)R 3 is hydrogen, halogen or C 6-10 Aryl (4)R 5 is hydrogen, halogen or C 6-10 Aryl.

3. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein: The compound represented by formula (I) satisfies one or more of the following conditions: The compound represented by formula (I) described in (1) is a compound represented by formula (I-1), R 1 Halogen or C 6-10 aryl; (22) The compound represented by formula (I) is a compound represented by formula (I-2), R 2 Halogen or C 6-10 aryl; (3) The compound represented by formula (I) is a compound represented by formula (I-3), R 3 Halogen or C 6-10 aryl; (4) The compound represented by formula (I) is a compound represented by formula (I-4), R 4 Halogen, C 1-10 Alkyl or C 6-10 aryl; (5) The compound represented by formula (I) is a compound represented by formula (I-5), R 5 Halogen or C 6-10 aryl; (6) The compound represented by formula (I) is a compound represented by formula (I-6), R 6 Halogen, C 1-10 Alkyl or C 6-10 aryl; (7) The compound represented by formula (I) is a compound represented by formula (I-7), R 3 and R 5 are independently halogen; (8) The compound represented by formula (I) is a compound represented by formula (I-8), R 3 、R 4 and R 5 are independently halogen.

4. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein: The compound represented by formula (I) satisfies one or more of the following conditions: (1) Each halogen is independently fluorine, chlorine, bromine or iodine; (2) Each C 1-10 The alkyl groups are independently C 1-6 alkyl; (3) Each C 6-10 Aryl is independently phenyl or naphthyl; (4)R 1 is hydrogen, chlorine, bromine, iodine or phenyl; (5)R 2 is hydrogen, fluorine, chlorine, bromine, iodine or phenyl; (6)R 3 is hydrogen, fluorine, chlorine, bromine, iodine or phenyl; (7)R 4 is hydrogen, fluorine, chlorine, bromine, iodine, methyl, isopropyl, tert-butyl or phenyl; (8)R 5 is hydrogen, fluorine, chlorine, bromine, iodine or phenyl; (9)R 6 is hydrogen, fluorine, chlorine, bromine, iodine, methyl or phenyl.

5. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, characterized in that: The compound shown in formula (I) is any of the following structures:

6. A method for preparing the compound of formula (I) according to any one of claims 1 to 5, characterized in that: It is either Method A or Method B: Method A includes the following steps: In an organic solvent, the compound represented by formula (II) is first reacted with 1,1-thiocarbonyldiimidazole; after the reaction, the resulting reaction solution is further reacted with a methanol solution of ammonia to obtain the compound represented by formula (I); In method A, R 1 、R 2 、R 3 、R 4 、R 5 and R 6 The definition as described in any one of claims 1 to 5; Method B comprises the following steps: In an organic solvent, the compound represented by formula (II) is first reacted with benzoyl isothiocyanate; after the reaction, the resulting reaction solution is further reacted with an alkaline aqueous solution to obtain a compound represented by formula (I); In method B, R 1 、R 2 、R 3 、R 4 、R 5 and R 6 The definition as described in any one of claims 1 to 5; The method A meets one or more of the following conditions: (1) The organic solvent is a halogenated alkane solvent; (2) The molar volume ratio of the compound represented by formula (II) to the organic solvent is 0.05-0.5 mol / L; (3) The molar ratio of the compound represented by formula (II) to the 1,1-thiocarbonyldiimidazole is 1:(1-3); (4) The concentration of the ammonia methanol solution is 6-8 mol / L; (5) The molar volume ratio of the compound represented by formula (II) to the methanol solution of ammonia is 0.3-0.7 mol / L; (6) The reaction temperature is 20-40°C; Method B satisfies one or more of the following conditions: (1) The organic solvent is a ketone solvent; (2) The molar volume ratio of the compound represented by formula (II) to the organic solvent is 0.1-0.5 mol / L; (3) The molar ratio of the compound represented by formula (II) to the benzoyl isothiocyanate is 1:(1-2); (4) The alkaline aqueous solution is a NaOH aqueous solution; (5) The concentration of the alkaline aqueous solution is 0.5-2 mol / L; (6) The molar ratio of the compound represented by formula (II) to the alkaline aqueous solution is 1:(2-4); (7) When the compound represented by formula (II) is first reacted with benzoyl isothiocyanate, the reaction temperature is 50-80°C; (8) When the reaction solution reacts with the alkaline aqueous solution, the reaction temperature is 40-60°C.

7. A compound as shown below:

8. A pharmaceutical composition, characterized in that The invention comprises a compound represented by formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, and at least one pharmaceutical excipient.

9. Use of the compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, or the pharmaceutical composition according to claim 8 in the preparation of a TAS2R38 receptor agonist.

10. Use of a compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, or a pharmaceutical composition according to claim 8, in the preparation of a medicament for treating and / or preventing a disease associated with or mediated by the TAS2R38 receptor, preferably chronic sinusitis.

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