1. A thiotriazole compound is characterized by having a structural formula as follows:

wherein R is₁Any one selected from the group consisting of a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted phenyl group, and a substituted or unsubstituted heteroaliphatic ring group; r₂Is a substituted aromatic radical, R₃Is a substituted phenyl group.

2. The thiotriazole compound of claim 1, wherein R is₁Any one of functional groups formed by C1-C10 substituted or unsubstituted alkyl, C3-C15 substituted or unsubstituted cycloalkyl, C6-C20 substituted or unsubstituted phenyl and C2-C10 substituted or unsubstituted heteroaliphatic ring groups;

preferably, R₁Is a group with 4-6 carbon atoms and hetero atoms;

preferably, R₁The substituent of the substituted phenyl group of (1) is selected from any one of the functional groups formed by halogen, alkyl and acyl;

preferably, R₁The substituents of the substituted phenyl group of (1) are selected from chlorine, iodine, formyl and C1-C5 alkyl;

preferably, R₁Selected from methyl, ethyl, propyl, cyclohexane, cyclopropane, monosubstituted phenyl,Any one of the formed functional group groups;

preferably, R₁The mono-substituted phenyl is ortho-substituted phenyl or para-substituted phenyl.

3. The thiotriazole compound of claim 1, wherein R is₂Is a substituted phenyl group;

preferably, R₂The substituent of the substituted phenyl group of (2) is selected from any one of the functional groups formed by halogen, amine group, alkyl group, amino group and ether group;

preferably, R₂The substituent of the substituted phenyl is selected from F, dimethylamino and NH₂-, tertiary amino, methyl ether and C1-C5 alkyl;

preferably, R₂The substituted phenyl group of (a) is a monosubstituted phenyl group;

preferably, R₂The mono-substituted phenyl group is a para-substituted mono-substituted phenyl group.

4. The thiotriazole compound of claim 1, wherein R is₃The substituted phenyl is monosubstituted phenyl or disubstituted phenyl;

preferably, R₃The substituted phenyl is any one of para-substituted phenyl, meta-substituted phenyl, ortho-para-phenyl and meta-para-phenyl;

preferably, R₃The substituent of the substituted phenyl group is selected from any one of the functional groups formed by halogen, cyano, substituted alkyl, substituted ether group, ester group, borate and phenyl;

preferably, R₃Any one selected from the following groups:

5. the thiotriazole compound of any one of claims 1-4, which is selected from any one of the compounds represented by the following structural formula:

6. a process for the preparation of the thiotriazole compound as claimed in any one of claims 1 to 5, which comprises synthesizing the thiotriazole compound with reference to the following synthetic route:

7. the method according to claim 6, wherein the conditions of step i are as follows: containing R₁And a compound containing R₂The molar ratio of the raw material compounds is 1: 1-1.2; the reaction temperature is 80-100 ℃, and the reaction time is 30-60 minutes;

the operation of step ii is: adding an alkaline substance into the reaction system, and then reacting for 30-60 minutes at the temperature of 60-110 ℃;

the operation of step iii is: when the reaction system is cooled to 5-25 ℃, adding an acidic substance into the reaction system to ensure that the pH of the reaction system is 1-2;

the conditions of step iv are: the reaction temperature is 60-100 ℃, and the reaction time is 30-60 minutes.

8. Use of the thiotriazole compound of any one of claims 1 to 5 for the preparation of an antitumor medicament.

9. The use according to claim 8, wherein the tumor is gastric cancer or lung adenocarcinoma.

10. Use of the thiotriazole compound of any one of claims 1 to 5 for the preparation of an inhibitor for inhibiting the activity of histone demethylase or cyclin-dependent kinase 8.

Background

Cancer (cancer) refers to malignant tumor originated from epithelial tissue, which is the most common type of malignant tumor, and has many kinds of malignant tumors, different nature types, different involved tissues and organs, different disease stages, and different responses to various treatments, so that most patients need to be treated comprehensively. The comprehensive treatment is to comprehensively adopt measures such as surgery, chemotherapy, radiotherapy, immunotherapy, traditional Chinese medicine treatment, interventional therapy, microwave treatment and the like according to the physical condition of a patient, the pathological type of tumors, the invasion range and the like so as to greatly improve the cure rate and improve the life quality of the patient. Targeting small molecules of drugs for treating malignant tumors in the prior art are hot research points, while small molecules containing heteroatoms are the key point of research, but any small molecule containing heteroatoms does not have related performance or treatment effect.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a thiotriazole compound, a preparation method and application thereof. The thiotriazole compound effectively inhibits the activity of histone demethylase, and then has good treatment effect on tumors such as gastric cancer or lung adenocarcinoma.

The invention is realized by the following steps:

in a first aspect, the present invention provides a thiotriazole compound, which has a structural formula shown as follows:wherein R is₁Any one selected from the group consisting of a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted phenyl group, and a substituted or unsubstituted heteroaliphatic ring group; r₂Is a substituted aromatic radical, R₃Is a substituted phenyl group.

In a second aspect, the present invention provides a process for the preparation of a thiotriazole compound according to any one of the preceding embodiments, comprising synthesizing the thiotriazole compound with reference to the following synthetic pathway:

in a third aspect, the present invention provides a thiotriazole compound according to any one of the preceding embodiments, for use in the preparation of an anti-tumor medicament.

In a fourth aspect, the present invention provides a use of a thiotriazole compound according to any one of the preceding embodiments in the preparation of an inhibitor for inhibiting the activity of histone demethylase (LSD1) or cyclin-dependent kinase 8(CDK 8).

The invention has the following beneficial effects: the thiotriazole compound has a good antiproliferative effect on a gastric cancer cell line HGC27 or a lung adenocarcinoma cell line H1975, can effectively inhibit the activity of histone demethylase, and further has a good treatment effect on tumors such as gastric cancer or lung adenocarcinoma.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The embodiment of the invention provides a thiotriazole compound, which has the following structural formula:

wherein R is₁Any one selected from the group consisting of a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted phenyl group, and a substituted or unsubstituted heteroaliphatic ring group; r₂Is a substituted aromatic radical, R₃Is a substituted phenyl group. The thiotriazoleThe compounds can effectively inhibit the activity of histone demethylase, and have good treatment effect on tumors such as gastric cancer or lung adenocarcinoma.

Wherein R is₁Any one of functional groups formed by C1-C10 substituted or unsubstituted alkyl, C3-C15 substituted or unsubstituted cycloalkyl, C6-C20 substituted or unsubstituted phenyl and C2-C10 substituted or unsubstituted heteroaliphatic ring groups; for example, R₁The substituent of the substituted phenyl group of (1) is selected from any one of the functional groups formed by halogen, alkyl and acyl; r₁The substituents of the substituted phenyl groups of (a) are selected from chlorine, iodine, formyl and C1-C5 alkyl. R1 is selected from methyl, ethyl, propyl, cyclohexane, cyclopropane, monosubstituted phenyl (e.g., R₁The mono-substituted phenyl is ortho-substituted phenyl or para-substituted phenyl),Any one of the formed functional group groups; preferably, R₁Is a group with 4-6 carbon atoms and hetero atoms; specifically, R₁Is selected from

Any one of them.

Further, R₂Is a substituted phenyl group; r₂The substituent of the substituted phenyl group of (2) is selected from any one of the functional groups formed by halogen, amine group, alkyl group, amino group and ether group; for example, R₂The substituent of the substituted phenyl group is selected from F, NH₂-, tertiary amino, methyl ether and C1-C5 alkyl; and R is₂The substituted phenyl group of (a) is a monosubstituted phenyl group; r₂The mono-substituted phenyl group is a para-substituted mono-substituted phenyl group.

Further, R₃The substituted phenyl is monosubstituted phenyl or disubstituted phenyl; r₃The substituted phenyl is any one of para-substituted phenyl, meta-substituted phenyl, ortho-para-phenyl and meta-para-phenyl; wherein R is₃The substituent of the substituted phenyl group is selected from halogen, cyano, substituted alkyl, substituted ether group, esterAny one of the group of functional groups formed by a phenyl group and a phenyl group; r₃Any one selected from the following groups:

it should be noted that: (1) the dotted line or wavy line in the group shown in the structural formula represents the position of bond breaking, namely the position of the group connected with the parent nucleus of the thiotriazole compound.

(2) The mono-substituted phenyl and the di-substituted phenyl are 1 substituent and 2 substituents besides the position connected with the parent nucleus of the thiotriazole compound.

(3) On the basis of meeting the chemical valence bond rules, the substituted phenyl can be mono-substituted or di-substituted, and can also be multi-substituted such as tri-substituted or 4-substituted.

(4) The mono-substituted phenyl can be any one of ortho-meta-para substitution, and the di-substitution can be the combination of any two of ortho-meta-para substitution, and can be both ortho-position or both meta-position.

(5) The alkyl can be methyl, ethyl, propyl, isopropyl and tert-butyl, and can also be other C1-C10 alkyl or C1-C5 alkyl;

the cycloalkyl group may be an aliphatic cycloalkane such as a cyclopentyl group, in addition to a cyclohexyl group and a cyclopropyl group;

the acyl group may be an acyl group such as acetyl or propionyl, in addition to the formyl group;

the amine group may be a tertiary amine, a primary amine or a secondary amine;

the ether group may be a methyl ether group, an ethyl ether group, a propyl ether group, or the like;

the alkyl group, substituted phenyl group, acyl group, amine group, ether group, cycloalkyl group, cyano group, ester group and the like may be further substituted, and for example, the corresponding substituent group may be further substituted with halogen, nitro group, alkyl group and the like.

The thiotriazole compound is selected from any one of the compounds shown in the following structural formula:

this embodiment provides a method for preparing the above thiotriazole compound, including synthesizing the thiotriazole compound by referring to the following synthetic route:

specifically, the operation of step i includes: to a 25ml round bottom flask was added 1mmol of a different compound containing R₁And 1-1.2mmol of a compound containing R₂The starting compound (e.g., 1mmol, 1.1mmol, 1.2mmol, or any value between 1 and 1.2 mmol) of (A), dissolving the starting compound in an alcohol solvent (e.g., 10mL of ethanol), and heating the resulting mixture at 80 to 100 deg.C (80 deg.C, 85 deg.C, 90 deg.C, 95 deg.C, 100 deg.C, or any value between 80 and 100 deg.C) under reflux for 30 to 60 minutes (e.g., 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, or any value between 30 and 60 minutes). And (5) TCL monitoring. After the reaction is finished, putting the round-bottom flask into ice water for cooling, filtering and collecting the obtained solid, washing with water, and drying in vacuum to obtain the intermediate.

The operation of step ii comprises: adding the intermediate obtained in step i to a 25mL round-bottom flask, adding a basic substance (for example, 10mL of 2mol/L aqueous NaOH solution) to dissolve the intermediate, heating under reflux at 60-110 deg.C (for example, 100 deg.C, 101 deg.C, 102 deg.C, 103 deg.C, 104 deg.C, 105 deg.C, 106 deg.C, 107 deg.C, 108 deg.C, 109 deg.C, 110 deg.C, etc. at any value between 60-110 deg.C) for 30-60 minutes (for example, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, and any value between 30-60 minutes), and monitoring by TCL.

The operation of step iii comprises: after completion of the reaction in step ii, the reaction mixture is cooled to 5 to 25 ℃, an acidic substance (e.g., dilute hydrochloric acid) is slowly added to the reaction system while stirring until the PH becomes 1 to 2, for example, a value between 1 and 2 such as PH 1, 1.5 or 2, the obtained solid is collected by filtration, washed with an acidic substance (e.g., dilute hydrochloric acid solution) several times, and dried under vacuum to obtain an intermediate.

The operation of step iv comprises: adding 1mmol of raw material compound containing R3 and 3-5eq of basic substance (such as KOH) into a 25ml round bottom flask, adding an alcohol solution (such as ethanol) to dissolve, adding 1-1.2mmol of intermediate obtained in step iii (such as 1-1.2mmol, 1.1mmol or 1.2 mmol), 60-100 deg.C (such as 80 deg.C, 85 deg.C, 90 deg.C, 95 deg.C and 100 deg.C) under heating and refluxing for 30-60 min (such as 30 min, 35 min, 40 min, 45 min, 50 min, 55 min and 30-60 min), TCL monitoring, spinning off the reaction solution after the reaction is completed, and purifying by silica gel column chromatography to obtain the compound of thiotriazole.

The embodiment also provides an application of the thiotriazole compound in preparing an anti-tumor drug, wherein the tumor is gastric cancer or lung adenocarcinoma, and the thiotriazole compound is applied in preparing an inhibitor for inhibiting the activity of histone demethylase.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

The embodiment of the invention provides a thiotriazole compound (marked as g1), which has the following structural formula:

the embodiment of the invention also provides a preparation method of the thiotriazole compound, which comprises the following steps:

a25 mL round bottom flask was charged with 1mmol of 4-carboxyphenylisothiocyanate and 1mmol of 4-methoxybenzoyl hydrazine, dissolved by addition of 10mL of ethanol, and the reaction was stirred at 90 ℃ under reflux for 1 hour. And (5) TCL monitoring. After the reaction was complete, the round bottom flask was placed in ice water to cool for 30 minutes, the resulting solid was collected by filtration, washed 2 times with water and dried under vacuum to give an intermediate.

Adding the intermediate obtained in the previous step into a 25mL round-bottom flask, adding 10mL of 2mol/L NaOH aqueous solution to dissolve the intermediate, heating at 105 ℃, refluxing and stirring for 1 hour, monitoring by TCL, placing the reaction solution into ice water after the reaction is completed, slowly adding concentrated hydrochloric acid into the reaction system while stirring until the pH is 1, filtering and collecting the obtained solid, washing for 2 times by using dilute hydrochloric acid solution, and drying in vacuum to obtain the intermediate.

Adding 1mmol of 4-fluorobenzyl bromide and 5eq KOH into a 25mL round-bottom flask, adding 10mL of ethanol for dissolving, adding the 1mmol of the intermediate obtained in the previous step, heating, refluxing and stirring at 90 ℃ for 1 hour, monitoring by TCL, after the reaction is finished, spin-drying the reaction liquid, and purifying by silica gel column chromatography to obtain the final product g 1.

The compound was characterized by the following data: white solid, yield 66.6%;¹H NMR(400MHz,DMSO-d6)δ8.03(d,J＝8.5Hz,2H),7.42–7.37(m,4H),7.27(d,J＝8.9Hz,2H),7.16–7.11(m,2H),6.91(d,J＝8.9Hz,2H),4.39(s,2H),3.73(s,3H).HRMS(ESI):m/z calcd for C₂₃H₁₉FN₃O₃S⁺(M+H)⁺436.1126；found:436.0952.

example 2

The embodiment of the invention provides a thiotriazole compound (marked as g2), which has the following structural formula:

the synthesis method is the same as that of example 1, except that the raw material compound containing R3 is different, and the characterization data of the thiotriazole compound are as follows: white solid, yield 57.6%;¹H NMR(400MHz,DMSO-d6)δ8.04(d,J＝8.5Hz,2H),7.43(s,3H),7.40–7.32(m,1H),7.27(d,J＝8.9Hz,2H),7.21(ddd,J＝8.8,4.3,2.0Hz,1H),6.92(d,J＝8.9Hz,2H),4.38(s,2H),3.73(s,3H).HRMS(ESI)m/z:calcd for C₂₃H₁₈F₂N₃O₃S⁺(M+H)⁺:454.103；found:454.0965.

example 3-example 48

Examples 3 to 48 each provide a thio groupTriazole compound, its preparation method is the same as example 1, except that R is contained₁A raw material compound of (1), containing R₂Or contains R₃The starting compounds of (a) are different. The structural formula and the characterization of the finally formed thiotriazole compound are shown as follows:

example 3: structural formula (xvi):4- (3- ((4-trifluoromethylbenzyl) thio) -5- (4-methoxyphenyl) -4H-1,2, 4-triazol-4-yl) benzoic acid (g 3): white solid, yield 59.8%;¹H NMR(400MHz,DMSO-d6)δ8.03(d,J＝8.5Hz,2H),7.67(d,J＝8.2Hz,2H),7.59(d,J＝8.1Hz,2H),7.43(d,J＝8.3Hz,2H),7.28(d,J＝8.9Hz,2H),6.92(d,J＝8.9Hz,2H),4.49(s,2H),3.73(s,3H).HRMS(ESI)m/z:calcd for C₂₄H₁₉F₃N₃O₃S⁺(M+H)⁺:486.1094；found:486.1116.

example 4: structural formula (xvi):4- (3- ((4-chloro-3-fluorobenzyl) thio) -5- (4-methoxyphenyl) -4H-1,2, 4-triazol-4-yl) benzoic acid (g 4): white solid, yield 60.4%;¹H NMR(400MHz,DMSO-d6)δ13.33(s,1H),8.04(d,J＝8.4Hz,2H),7.57(d,J＝8.9Hz,1H),7.42(s,2H),7.38–7.31(m,2H),7.27(d,J＝8.8Hz,2H),6.91(d,J＝8.9Hz,2H),4.37(s,2H),3.73(s,3H).HRMS(ESI)m/z:calcd for C₂₃H₁₈ClFN₃O₃S⁺(M+H)+:470.0736；found:470.0776.

example 5: structural formula (xvi):4- (3- ((3,4-, dichlorobenzyl) thio) -5- (4-methoxyphenyl) -4H-1,2, 4-triazol-4-yl) benzoic acid (g 5): pale yellow solid, yield 70.2%;¹H NMR(400MHz,DMSO-d6)δ8.04(d,J＝8.5Hz,2H),7.61(d,J＝1.9Hz,1H),7.56(d,J＝8.3Hz,1H),7.41(d,J＝8.5Hz,2H),7.35(dd,J＝8.3,2.0Hz,1H),7.27(d,J＝8.9Hz,2H),6.91(d,J＝8.9Hz,2H),4.37(s,2H),3.73(s,3H).HRMS(ESI)m/z:calcd for C₂₃H₁₈Cl₂N₃O₃S⁺(M+H)⁺:486.0440；found:486.0427.

example 6: structural formula (xvi):4- (3- ((4-bromo-2-fluorobenzyl) thio) -5- (4-methoxyphenyl) -4H-1,2, 4-triazol-4-yl) benzoic acid (g 6): white solid, yield 65.6%;¹H NMR(400MHz,DMSO-d6)δ8.04(d,J＝8.5Hz,2H),7.52(dd,J＝9.7,1.4Hz,1H),7.44(d,J＝8.4Hz,2H),7.41–7.35(m,2H),7.27(d,J＝8.9Hz,2H),6.92(d,J＝8.9Hz,2H),4.34(s,2H),3.74(s,3H).HRMS(ESI)m/z:calcd for C₂₃H₁₈BrFN₃O₃S⁺(M+H)⁺:514.0231found:514.0224.

example 7: structural formula (xvi):4- (3- ((2-chloro-4-fluorobenzyl) thio) -5- (4-methoxyphenyl) -4H-1,2, 4-triazol-4-yl) benzoic acid (g 7): white solid, yield 66.1%;¹H NMR(400MHz,DMSO-d6)δ8.04(d,J＝8.5Hz,2H),7.59–7.53(m,1H),7.46–7.40(m,3H),7.27(d,J＝8.8Hz,2H),7.19(td,J＝8.5,2.6Hz,1H),6.92(d,J＝8.8Hz,2H),4.43(s,2H),3.74(s,3H).HRMS(ESI)m/z:calcd for C₂₃H₁₈ClFN₃O₃S⁺(M+H)⁺:470.0736；found:470.0760.

example 8: structural formula (xvi):4- (3- ((4-trifluoromethoxybenzyl) thio) -5- (4-methoxyphenyl) -4H-1,2, 4-triazol-4-yl) benzoic acid (g 8): white solid, yield 58.8%;¹H NMR(400MHz,DMSO-d6)δ8.03(d,J＝8.5Hz,2H),7.49(d,J＝8.7Hz,2H),7.41(d,J＝8.5Hz,2H),7.31(d,J＝8.0Hz,2H),7.27(d,J＝8.9Hz,2H),6.92(d,J＝8.9Hz,2H),4.43(s,2H),3.74(s,3H).HRMS(ESI)m/z:calcd for C₂₄H₁₈F₃N₃O₄S⁺(M+H)⁺:502.1043；found:502.1096.

example 9: structural formula (xvi):3- ((2-chloro-4-fluorobenzyl) thio) -5- (4-fluorophenyl) -4- (4-iodophenyl) -4H-1,2, 4-triazole (g 9): yellow solid, yield 70.4%;¹H NMR(400MHz,DMSO-d6)δ7.88(d,J＝8.6Hz,2H),7.58(dd,J＝8.6,6.2Hz,1H),7.44–7.38(m,3H),7.28–7.20(m,3H),7.14(d,J＝8.6Hz,2H),4.44(s,2H).HRMS(ESI)m/z:calcd for C₂₁H₁₄ClF₂IN₃S⁺(M+H)⁺:539.9604；found:539.9578.

example 10: structural formula (xvi): 3- ((3, 4-fluorobenzyl) thio) -5- (4-fluorophenyl) -4- (4-iodophenyl) -4H-1,2, 4-triazole (g 10): white solid, yield 69.7%;¹H NMR(400MHz,DMSO-d6)δ7.89(d,J＝8.6Hz,2H),7.41(dd,J＝8.9,5.4Hz,2H),7.39–7.32(m,2H),7.24(t,J＝8.9Hz,3H),7.15(d,J＝8.6Hz,2H),4.40(s,2H).HRMS(ESI)m/z:calcd for C₂₁H₁₃F₃IN₃NaS⁺(M+Na)⁺:545.9719；found:545.9661.

example 11: structural formula (xvi):3- ((3-chloro-4-fluorobenzyl) thio) -5- (4-fluorophenyl) -4- (4-iodophenyl) -4H-1,2, 4-triazole (g 11): white solid, yield 62.1%;¹H NMR(400MHz,DMSO-d6)δ7.88(d,J＝8.6Hz,2H),7.59(dd,J＝7.2,2.0Hz,1H),7.43–7.35(m,4H),7.24(t,J＝8.9Hz,2H),7.13(d,J＝8.6Hz,2H),4.38(s,2H).HRMS(ESI)m/z:calcd for C₂₁H₁₄ClF₂IN₃S⁺(M+H)⁺:539.9604；found:539.9566.

example 12: structural formula (xvi):3- (4-fluorophenyl) -4- (4-iodophenyl) -5- ((4- (trifluoromethyl) benzyl) thio) -4H-1,2, 4-trisAzole (g 12): white solid, yield 71.1%;¹H NMR(400MHz,DMSO-d6)δ7.88(d,J＝8.6Hz,2H),7.68(d,J＝8.2Hz,2H),7.61(d,J＝8.2Hz,2H),7.42(dd,J＝8.9,5.4Hz,2H),7.24(t,J＝8.9Hz,2H),7.14(d,J＝8.6Hz,2H),4.50(s,2H).HRMS(ESI)m/z:calcd for C₂₂H₁₅F₄IN₃S⁺(M+H)⁺:555.9962；found:555.9882.

example 13: structural formula (xvi):3- ((4-fluorobenzyl) thio) -5- (4-fluorophenyl) -4- (4-iodophenyl) -4H-1,2, 4-triazole (g 13): white solid, yield 58.6%;¹H NMR(400MHz,DMSO-d6)δ7.88(d,J＝8.6Hz,2H),7.44–7.38(m,4H),7.24(t,J＝8.9Hz,2H),7.13(t,J＝8.3Hz,4H),4.40(s,2H).HRMS(ESI)m/z:calcd for C₂₁H₁₅F₂IN₃NaS⁺(M+Na)⁺:505.9994；found:505.9964.

example 14: structural formula (xvi):3- ((3-chloro-4-fluorobenzyl) thio) -5- (4-fluorophenyl) -4- (2-chlorophenyl) -4H-1,2, 4-triazole (g 14): white solid, yield 64.9%;¹H NMR(400MHz,DMSO-d6)δ7.70(ddd,J＝15.1,7.9,1.5Hz,2H),7.61(td,J＝8.9,8.4,6.1Hz,2H),7.57(dd,J＝7.6,1.5Hz,1H),7.47–7.40(m,3H),7.26–7.17(m,3H),4.49(s,2H).HRMS(ESI)m/z:calcd for C₂₁H₁₄Cl₂F₂N₃S⁺(M+H)⁺:448.0248；found:448.0222.

example 15: structural formula (xvi):3- ((4-bromo-2-fluorobenzyl) thio) -5- (4-fluorophenyl) -4- (2-chlorophenyl) -4H-1,2, 4-triazole (g 15): white solid, yield 56.8%;¹H NMR(400MHz,DMSO-d6)δ7.70(ddd,J＝12.5,7.9,1.4Hz,2H),7.63(td,J＝7.8,1.7Hz,1H),7.58–7.51(m,2H),7.44–7.37(m,4H),7.23(t,J＝8.9Hz,2H),4.41(d,J＝2.7Hz,2H).HRMS(ESI)m/z:calcd for C₂₃H₁₈ClFN₃O₃S⁺(M+H)⁺:491.9743；found:491.9710.

example 16: structural formula (xvi):3- (4- (tert-butyl) phenyl) -5- ((4-fluorobenzyl) thio) -4- (4-iodophenyl) -4H-1,2, 4-triazole (g 16): white solid, yield 65.2%;¹H NMR(400MHz,DMSO-d6)δ7.88(d,J＝8.6Hz,2H),7.39(d,J＝8.3Hz,4H),7.30(d,J＝8.6Hz,2H),7.13(dt,J＝8.9,4.6Hz,4H),4.39(s,2H),1.24(s,10H).HRMS(ESI)m/z:calcd for C₂₅H₂₄FIN₃S⁺(M+H)⁺:544.0714；found:544.0662.

example 17: structural formula (xvi):4- (((5- (4-aminophenyl) -4-phenethyl-4H-1, 2, 4-triazol-3-yl) thio) methyl) benzonitrile (g 17): white solid, yield 68.0%;¹H NMR(400MHz,Methanol-d₄)δ7.72(d,J＝8.4Hz,2H),7.64(d,J＝8.3Hz,2H),7.16(dt,J＝18.8,7.7Hz,5H),6.93(d,J＝8.6Hz,2H),6.75(d,J＝6.7Hz,2H),4.57(s,2H),4.36(t,J＝6.8Hz,2H),2.92(t,J＝6.7Hz,2H).HRMS(ESI)m/z:calcd for C₂₄H₂₁KN₅S⁺(M+K)⁺:450.1155；found:450.1137.

example 18: structural formula (xvi):2- (4- (((5- (4-aminophenyl) -4-phenethyl-4H-1, 2, 4-triazol-3-yl) thio) methyl) phenyl) propanoic acid (g 18): white solid, yield 58.9%;¹H NMR(400MHz,Methanol-d₄)δ7.24(d,J＝8.1Hz,2H),7.17–7.11(m,5H),7.04(d,J＝8.5Hz,2H),6.74(d,J＝8.4Hz,4H),4.20(s,2H),3.88(t,J＝7.1Hz,2H),2.59(t,J＝7.1Hz,2H),1.39(d,J＝7.2Hz,3H).HRMS(ESI)m/z:calcd for C₂₆H₂₇N₄O₂S⁺(M+H)⁺:459.1849；found:459.1849.

example 19: structural formula (xvi):4- (((5- (4-aminophenyl) -4- (3-morpholinopropyl) -4H-1,2, 4-triazol-3-yl) thio) methyl) benzonitrile (g 19): white solid, yield 69.6%;¹H NMR(400MHz,Methanol-d4)δ7.66(d,J＝8.2Hz,2H),7.49(d,J＝8.2Hz,2H),7.26(d,J＝8.5Hz,2H),6.78(d,J＝8.5Hz,2H),4.43(s,2H),4.01–3.90(m,2H),3.59–3.52(m,4H),2.18(s,4H),2.12(t,J＝6.6Hz,2H),1.56(p,J＝6.7Hz,2H).HRMS(ESI)m/z:calcd for C₂₃H₂₇N₆OS⁺(M+H)⁺:435.1962；found:435.1967.

example 20: structural formula (xvi):4- (((5- (4-aminophenyl) -4- (3-morpholinopropyl) -4H-1,2, 4-triazol-3-yl) thio) methyl) benzonitrile (g 19): pale yellow solid, yield 74.7%;¹H NMR(400MHz,Methanol-d4)δ7.35(d,J＝8.5Hz,2H),7.14(d,J＝8.4Hz,2H),7.08(d,J＝8.4Hz,2H),6.78(d,J＝8.5Hz,2H),4.43(s,2H),1.94(q,J＝12.4Hz,2H),1.76(d,J＝11.5Hz,2H),1.57(s,1H),1.29(s,3H),1.17(s,1H),0.93–0.79(m,3H).HRMS(ESI)m/z:calcd for C₂₂H₂₅F₂N₄OS⁺(M+H)⁺:431.1712；found:431.1719.

example 21: structural formula (xvi):methyl 4- (((5- (4-aminophenyl) -4-cyclohexyl-4H-1, 2, 4-triazol-3-yl) thio) benzoate (g 21): white solid, yield 58.0%;¹H NMR(400MHz,DMSO-d6)δ7.98(s,1H),7.86(d,J＝7.8Hz,1H),7.68(d,J＝7.7Hz,1H),7.48(t,J＝7.7Hz,1H),7.08(d,J＝8.4Hz,2H),6.65(d,J＝8.4Hz,2H),4.56(s,2H),3.91(dt,J＝11.9,3.3Hz,1H),3.85(s,3H),1.87(dd,J＝21.2,8.8Hz,2H),1.70(d,J＝12.4Hz,2H),1.59(d,J＝13.5Hz,2H),1.53(s,1H),1.17–1.00(m,3H).HRMS(ESI)m/z:calcd for C₂₃H₂₇N₄O₂S⁺(M+H)⁺:423.1849；found:423.1849.

example 22: structural formula (xvi):4- (5- (([1,1' -biphenyl) phenyl)]-4-ylmethyl) thio) -4-cyclohexyl-4H-1, 2, 4-triazol-3-yl) aniline (g 22): white solid, yield 66.9%;¹H NMR(400MHz,Methanol-d4)δ7.52(t,J＝7.1Hz,3H),7.44–7.31(m,5H),7.28(d,J＝7.6Hz,1H),7.00(d,J＝8.5Hz,2H),6.72(d,J＝8.5Hz,2H),4.45(s,2H),3.93(ddt,J＝12.6,7.6,4.0Hz,1H),1.80(q,J＝11.9Hz,2H),1.67(d,J＝12.8Hz,2H),1.39(d,J＝11.2Hz,2H),1.07(dq,J＝23.2,12.9Hz,4H).HRMS(ESI)m/z:calcd for C₂₇H₂₉N₄S⁺(M+H)⁺:441.2107；found:441.2110.

example 23: structural formula (xvi):4- (5- ((4- (benzyloxy) benzyl) thio) -4-cyclohexyl-4H-1, 2, 4-triazol-3-yl) aniline (g 23): white solid, yield 79.6%;¹H NMR(400MHz,Methanol-d4)δ7.35(dt,J＝21.7,7.3Hz,5H),7.21(t,J＝7.9Hz,1H),7.12(d,J＝8.5Hz,2H),6.88(t,J＝6.0Hz,2H),6.82(s,1H),6.76(d,J＝8.5Hz,2H),4.58(s,2H),4.36(s,2H),3.97(dddd,J＝13.2,7.3,5.8,3.4Hz,1H),1.90–1.81(m,2H),1.71(d,J＝13.1Hz,2H),1.46(d,J＝11.3Hz,2H),1.11(dd,J＝25.8,13.3Hz,4H).HRMS(ESI)m/z:calcd for C₂₈H₃₁N₄OS⁺(M+H)⁺:471.2213；found:471.2220.

example 24: structural formula (xvi):4- (((5- (4- (dimethylamino) phenyl) -4-phenethyl-4H-1, 2, 4-triazol-3-yl) thio) methyl) benzonitrile (g 24): white solid, yield 69.7%;¹H NMR(400MHz,Methanol-d4)δ7.64(d,J＝8.3Hz,2H),7.47(d,J＝8.3Hz,2H),7.20(d,J＝8.9Hz,2H),7.16–7.12(m,3H),6.79–6.74(m,3H),4.32(s,2H),4.12(t,J＝7.0Hz,2H),3.01(s,6H),2.71(t,J＝7.0Hz,2H).HRMS(ESI)m/z:calcd for C₂₆H₂₅KN₅S⁺(M+K)⁺:478.1462；found:478.1466.

example 25: structural formula (xvi):2- (4- (((5- (4- (dimethylamino) phenyl) -4-phenethyl-4H-1, 2, 4-triazol-3-yl) thio) methyl) phenyl) propanoic acid (g 25): white solid, yield 54.8%;¹H NMR(400MHz,Methanol-d4)δ7.24(d,J＝8.0Hz,2H),7.20–7.16(m,3H),7.13(dd,J＝5.5,2.9Hz,4H),6.80(d,J＝8.8Hz,2H),6.74(dd,J＝6.3,2.7Hz,2H),4.20(s,2H),3.90(t,J＝7.1Hz,2H),3.02(s,6H),2.58(t,J＝7.0Hz,2H),1.39(d,J＝7.1Hz,3H).HRMS(ESI)m/z:calcd for C₂₈H₃₁N₄O₂S⁺(M+H)⁺:487.2157；found:487.2172.

example 26: structural formula (xvi):4- (5- ((4- (difluoromethoxy) benzyl) thio) -4-phenethyl-4H-1, 2, 4-triazol-3-yl) -N, N-dimethylaniline (g 26): white solid, yield 74.2%;¹H NMR(400MHz,DMSO-d6)δ7.45(d,J＝8.6Hz,2H),7.41(d,J＝9.0Hz,2H),7.22–7.19(m,3H),7.16(d,J＝8.5Hz,2H),6.93–6.89(m,2H),6.87(d,J＝8.9Hz,2H),4.44(s,2H),4.24(t,J＝7.2Hz,2H),3.03(s,6H),2.83(d,J＝4.5Hz,2H).HRMS(ESI)m/z:calcd for C₂₆H₂₇F₂N₄OS⁺(M+H)⁺:481.1868；found:481.1869.

example 27: structural formula (xvi):4- (5- (([1,1' -biphenyl) phenyl)]-4-ylmethyl) thio) -4-phenethyl-4H-1, 2, 4-triazol-3-yl) -N, N-dimethylaniline (g 27): white solid, yield 58.5%; 1H NMR (400MHz, Methanol-d4) δ 7.60(s,1H), 7.57-7.53 (m,3H),7.42(t, J ═ 7.6Hz,4H),7.34(t, J ═ 7.3Hz,1H),7.17(d, J ═ 9.0Hz,2H),7.08(dt, J ═ 14.2,6.9Hz,3H),6.80(d, J ═ 9.0Hz,2H),6.71(d, J ═ 6.9Hz,2H),4.51(s,2H),4.24(t,J＝7.0Hz,2H),3.05(s,6H),2.81(t,J＝7.0Hz,2H).HRMS(ESI)m/z:calcd for C₃₁H₃₁N₄S⁺(M+H)⁺:491.2264；found:491.2266.

Example 28: structural formula (xvi):4- (5- ((4- (benzyloxy) benzyl) thio) -4-phenethyl-4H-1, 2, 4-triazol-3-yl) -N, N-dimethylaniline (g 28): white solid, yield 76.9%;¹H NMR(400MHz,Methanol-d4)δ7.36(t,J＝8.0Hz,2H),7.32(t,J＝7.2Hz,2H),7.29–7.26(m,1H),7.24(d,J＝8.8Hz,3H),7.13(t,J＝8.2Hz,3H),7.03–6.96(m,2H),6.94(dd,J＝8.1,2.2Hz,1H),6.84(d,J＝9.0Hz,2H),6.75(d,J＝6.4Hz,2H),5.03(s,2H),4.42(s,2H),4.25(t,J＝7.0Hz,2H),3.06(s,6H),2.84(t,J＝6.9Hz,2H).HRMS(ESI)m/z:calcd for C₃₂H₃₃KN₄OS⁺(M+K)⁺:559.1928；found:559.1926.

example 29: structural formula (xvi):4- (((5- (4- (dimethylamino) phenyl) -4- (3-morpholinopropyl) -4H-1,2, 4-triazol-3-yl) thio) methyl) benzonitrile (g 29): white solid, yield 75.8%;¹H NMR(400MHz,Methanol-d4)δ7.45(d,J＝8.9Hz,2H),7.21(t,J＝7.9Hz,1H),6.92–6.83(m,5H),4.66(s,2H),4.37(s,2H),4.00–3.92(m,2H),3.85(s,3H),3.13(s,3H),3.04(s,6H),3.00–2.94(m,2H),1.97–1.84(m,2H).HRMS(ESI)m/z:calcd for C₂₅H₃₁N₆OS⁺(M+H)⁺:463.2275；found:463.2279.

example 30: structural formula (xvi):2- (4- (((5- (4- (dimethylamino) phenyl) -4- (3-morpholinopropyl) -4H-1,2, 4-triazol-3-yl) thio) methyl) propanoic acid (g 30): white solid, yield 67.4%;¹H NMR(400MHz,Methanol-d4)δ7.35(d,J＝8.9Hz,2H),7.26(d,J＝8.1Hz,2H),7.10(d,J＝8.1Hz,2H),6.85(d,J＝8.9Hz,2H),4.25(s,2H),3.70–3.62(m,4H),3.60(t,J＝4.7Hz,4H),3.02(s,6H),2.37–2.29(m,4H),2.23(t,J＝6.8Hz,2H),1.43(d,J＝7.1Hz,3H).HRMS(ESI)m/z:calcd for C₂₇H₃₆N₅O₃S⁺(M+H)⁺:510.2533；found:510.2541.

example 31: structural formula (xvi):4- (5- (([1,1' -biphenyl) phenyl)]-4-ylmethyl) thio) -4- (3-morpholinopropyl) -4H-1,2, 4-triazol-3-yl) -N, N-dimethylaniline (g 31): white solid, yield 67.0%;¹HNMR(400MHz,Methanol-d4)δ7.53(d,J＝8.4Hz,3H),7.38(d,J＝8.3Hz,3H),7.33(t,J＝7.3Hz,2H),7.24(d,J＝8.9Hz,3H),6.78(d,J＝8.9Hz,2H),4.38(s,2H),3.82–3.77(m,2H),3.50–3.46(m,4H),3.00(s,6H),2.09(s,4H),2.02(d,J＝6.5Hz,2H),1.45–1.38(m,2H).HRMS(ESI)m/z:calcd for C₃₀H₃₄N₅OS⁺(M+H)⁺:514.2635；found:514.2637.

example 32: structural formula (xvi):4- (4-cyclohexyl-5- ((4- (difluoromethoxy) benzyl) thio) -4H-1,2, 4-triazol-3-yl) -N, N-dimethylaniline (g 32): white solid, yield 62.9%;¹H NMR(400MHz,Methanol-d4)δ7.53(d,J＝8.5Hz,2H),7.48(d,J＝8.9Hz,2H),7.13(d,J＝8.5Hz,2H),6.98(d,J＝8.9Hz,2H),4.62(s,2H),4.29(tt,J＝12.6,3.5Hz,1H),3.11(s,6H),2.22–2.10(m,2H),1.91(t,J＝12.3Hz,4H),1.69(d,J＝11.9Hz,1H),1.35–1.24(m,3H).HRMS(ESI)m/z:calcd for C₂₄H₂₉F₂N₄OS⁺(M+H)⁺:459.2025；found:459.2029.

example 33: structural formula (xvi):methyl 4- (((4-cyclohexyl-5- (4- (dimethylaminophenyl) -4H-1,2, 4-triazol-3-yl) thio) methyl) benzoate (g33) as a pale yellow solid in 72.8% yield;¹HNMR(400MHz,Methanol-d4)δ7.92(d,J＝9.5Hz,2H),7.58(d,J＝7.7Hz,1H),7.43(t,J＝7.7Hz,1H),7.24(d,J＝8.9Hz,2H),6.84(d,J＝8.9Hz,2H),4.49(s,2H),3.99(tt,J＝12.6,3.7Hz,1H),3.88(s,3H),3.02(s,6H),1.95–1.86(m,2H),1.74(d,J＝13.1Hz,2H),1.51(d,J＝11.1Hz,2H),1.20–1.07(m,4H).HRMS(ESI)m/z:calcd for C₂₅H₃₁N₄O₂S⁺(M+H)⁺:451.2126；found:451.2183.

example 34: structural formula (xvi):2- (4- (((4-cyclohexyl-5- (4- (dimethylamino) phenyl) -4H-1,2, 4-triazol-3-yl) thio) methyl) phenoxy) acetic acid (g 34): white solid, yield 79.3%;¹H NMR(400MHz,Methanol-d4)δ7.72(s,4H),7.49(d,J＝9.0Hz,2H),7.00(d,J＝9.0Hz,2H),4.69(s,2H),4.30(tt,J＝12.6,3.5Hz,1H),3.35(s,2H),3.11(s,6H),2.17(q,J＝10.9,9.4Hz,2H),1.98–1.87(m,4H),1.38–1.20(m,4H).HRMS(ESI)m/z:calcd for C₂₅H₃₀NaN₄O₃S⁺(M+Na)⁺:489.1931；found:489.2175.

example 35: structural formula (xvi):4- (5- (([1,1' -biphenyl) phenyl)]-4-ylmethyl) thio) -4-cyclohexyl-4H-1, 2, 4-triazol-3-yl) -N, N-dimethylaniline (g 35): white solid, yield 68.3%;¹H NMR(400MHz,Methanol-d4)δ7.72(s,1H),7.59(dd,J＝14.0,8.0Hz,5H),7.48–7.42(m,4H),7.35(t,J＝7.4Hz,1H),7.28(d,J＝8.8Hz,2H),4.69(s,2H),4.24(ddd,J＝12.5,9.1,3.6Hz,1H),3.18(s,6H),2.16–2.07(m,2H),1.86(t,J＝13.4Hz,4H),1.33–1.15(m,4H).HRMS(ESI)m/z:calcd for C₂₉H₃₃N₄S⁺(M+H)⁺:460.2420；found:469.2409.

example 36: structural formula (xvi):4- (((5- (4- (dimethylamino) phenyl) -4-methyl-4H-1, 2, 4-tris)Azol-3-yl) thio) methyl) benzonitrile (g 36): white solid, yield 61.9%;¹H NMR(400MHz,Methanol-d4)δ7.66(d,J＝8.3Hz,2H),7.46(dd,J＝12.8,8.6Hz,4H),6.85(d,J＝8.9Hz,2H),4.38(s,2H),3.48(s,3H),3.03(s,6H).HRMS(ESI)m/z:calcd for C₁₉H₁₉N₅NaS⁺(M+Na)⁺:372.1253；found:372.1255.

example 37: structural formula (xvi):(4- (((5- (4- (dimethylamino) phenyl) -4-methyl-4H-1, 2, 4-triazol-3-yl) thio) methyl) phenyl) propanoic acid (g 37): white solid, yield 64.0%;¹H NMR(400MHz,Methanol-d4)δ7.70(s,2H),7.59(d,J＝9.0Hz,2H),7.40(d,J＝7.5Hz,2H),6.95(d,J＝9.1Hz,2H),4.52(s,2H),3.66(s,3H),3.10(s,6H).HRMS(ESI)m/z:calcd for C₂₁H₂₅N₄O₂S⁺(M+H)⁺:397.1693；found:397.1702.

example 38: structural formula (xvi):4- (5- ((4- (difluoromethoxy) benzyl) thio) -4-methyl-4H-1, 2, 4-triazol-3-yl) -N, N-dimethylaniline (g 38): white solid, yield 68.4%;¹H NMR(400MHz,DMSO-d6)δ7.59(d,J＝8.9Hz,2H),7.49(d,J＝8.6Hz,2H),7.16(d,J＝8.6Hz,2H),6.90(d,J＝9.0Hz,2H),4.49(s,2H),3.61(s,3H),3.04(s,6H).HRMS(ESI)m/z:calcd for C₁₉H₂₁F₂N₄OS⁺(M+H)⁺:391.1399；found:391.1398.

example 39: structural formula (xvi):methyl 4- (((5- (4- (dimethylamino) phenyl) -4-methyl-4H-1, 2, 4-triazol-3-yl) thio) methyl) benzoate (g 39): white solid, yield 74.7%;¹H NMR(400MHz,Methanol-d4)δ7.91(d,J＝7.8Hz,1H),7.79(s,1H),7.53(d,J＝7.7Hz,1H),7.45–7.38(m,3H),6.84(d,J＝8.9Hz,2H),4.34(s,2H),3.84(s,3H),3.33(s,3H),3.02(s,6H).HRMS(ESI)m/z:calcd for C₂₃H₁₈ClFN₃O₃S⁺(M+H)⁺:383.1536；found:383.1517.

example 40: structural formula (xvi):4- (5- (([1,1' -biphenyl) phenyl)]-4-ylmethyl) thio) -4-methyl-4H-1, 2, 4-triazol-3-yl) -N, N-dimethylaniline (g 40): white solid, yield 52.9%;¹H NMR(400MHz,Methanol-d4)δ7.53–7.47(m,3H),7.40–7.35(m,3H),7.30(dd,J＝11.5,8.2Hz,5H),6.76(d,J＝8.9Hz,2H),4.30(s,2H),3.23(s,3H),2.99(s,6H).HRMS(ESI)m/z:calcd for C₂₄H₂₄N₄NaS⁺(M+Na)⁺:423.1614；found:423.1621.

example 41: structural formula (xvi):4- (5- ((4- (benzyloxy) benzyl) thio) -4-methyl-4H-1, 2, 4-triazol-3-ene) -N, N-dimethylaniline (g 41): white solid, yield 70.3%, purity 95%;¹H NMR(400MHz,Methanol-d4)δ7.38(d,J＝8.9Hz,2H),7.33–7.25(m,5H),7.19(t,J＝7.9Hz,1H),6.89(dd,J＝8.2,2.2Hz,1H),6.80(t,J＝8.5Hz,3H),6.67(s,1H),4.94(s,2H),4.18(s,2H),3.17(s,3H),3.00(s,6H).HRMS(ESI)m/z:calcd for C₂₃H₁₈ClFN₃O₃S⁺(M+H)⁺:431.1900；found:431.1904.

example 42: structural formula (xvi):(4- (((5- (4-aminophenyl) -4-phenethyl-4H-1, 2, 4-triazol-3-yl) thio) methyl) phenyl) boronic acid, white solid, yield 80.4%, purity 95%;¹H NMR(400MHz,Methanol-d₄)δ7.66(s,2H),7.20(d,J＝7.2Hz,2H),7.16–7.10(m,3H),7.02(d,J＝8.1Hz,2H),6.72(d,J＝8.1Hz,4H),4.23(s,2H),3.92(t,J＝6.9Hz,2H),3.31(s,2H),2.61(t,J＝6.9Hz,2H).¹³C NMR(101MHz,DMSO)δ172.49,156.21,150.75,149.15,139.50,137.45,134.75,129.73,129.01,128.94,128.45,127.14,114.34,113.97,45.70,38.28,35.19.MS(ESI)m/z:calcd for C₂₃H₂₄BN₄O₂S⁺(M+H)⁺:431.2；found:431.2.

example 43: structural formula (xvi):(4- (((5- (4-aminophenyl) -4- (3-morpholinopropyl) -4H-1,2, 4-triazol-3-yl) thio) methyl) phenyl) boronic acid, white solid, yield 79.5%, purity 95%;¹H NMR(400MHz,Methanol-d₄)δ7.63(d,J＝7.5Hz,2H),7.22(t,J＝8.3Hz,4H),6.78(d,J＝8.3Hz,2H),4.34(s,2H),3.83(t,J＝7.1Hz,2H),3.72–3.61(m,8H),2.64(s,4H),1.69–1.58(m,2H).¹³C NMR(101MHz,MeOD)δ173.90,156.58,150.84,149.95,133.87,129.59,127.85,114.27,113.63,70.01,64.73,53.94,52.19,24.45,19.41.MS(ESI)m/z:calcd for C₂₂H₂₉BN₅O₃S⁺(M+H)⁺:454.2；found:454.2.

example 44: structural formula (xvi):(4- (((5- (4-aminophenyl) -4-cyclopropyl-4H-1, 2, 4-triazol-3-yl) thio) methyl) phenyl) boronic acid, white solid, 69.3% yield, 95% purity;¹H NMR(400MHz,DMSO-d₆)δ7.77(d,J＝7.9Hz,2H),7.41(d,J＝7.8Hz,2H),7.38–7.23(m,2H),6.86(dd,J＝67.1,7.4Hz,2H),4.57(s,2H),4.03(q,J＝7.1Hz,1H),2.00–1.81(m,4H),1.75(d,J＝13.5Hz,2H),1.57(d,J＝11.8Hz,1H),1.17(t,J＝7.1Hz,3H).¹³C NMR(101MHz,DMSO)δ156.67,150.76,148.21,139.34,134.75,130.58,128.55,126.74,114.49,113.91,56.51,37.78,31.31,25.82,24.98.MS(ESI)m/z:calcd for C₂₁H₂₆BN₄O₂S⁺(M+H)⁺:409.2；found:409.1.

example 45: structural formula (xvi):(4- (((5- (4-amino)Phenyl) -4-methyl-4H-1, 2, 4-triazol-3-yl) thio) methyl) phenyl) boronic acid, white solid, yield 49.5% and purity 95%;¹H NMR(400MHz,Methanol-d₄)δ7.67(s,2H),7.54(d,J＝8.7Hz,2H),7.39(d,J＝7.6Hz,2H),7.01(d,J＝8.6Hz,2H),4.51(s,2H),3.62(s,3H).¹³C NMR(101MHz,DMSO)δ156.56,150.87,149.24,142.16,139.52,134.75,129.72,128.42,114.12,113.93,38.12,31.98.MS(ESI)m/z:calcd for C₁₆H₁₈BN₄O₂S⁺(M+H)⁺:341.1；found:341.2.

example 46: structural formula (xvi):(4- (((5- (4-dimethylaminophenyl) -4-phenethyl-4H-1, 2, 4-triazol-3-yl) thio) methyl) phenyl) boronic acid, white solid, yield 66.5%, purity 95%;¹H NMR(400MHz,Methanol-d₄)δ7.62(s,2H),7.16(d,J＝8.6Hz,4H),7.14–7.11(m,3H),6.77(d,J＝8.9Hz,2H),6.75–6.71(m,2H),4.22(s,2H),3.92(s,2H),3.01(s,6H),2.61(t,J＝7.0Hz,2H).¹³C NMR(101MHz,DMSO)δ155.93,151.46,149.41,139.45,137.37,134.77,129.58,128.95,128.44,114.57,112.23,45.72,45.59,38.33,35.18.MS(ESI)m/z:calcd for C₂₅H₂₈BN₄O₂S⁺(M+H)⁺:459.2；found:459.1.

example 47: structural formula (xvi):(4- (((5- (4-dimethylaminophenyl) -4-cyclopropyl-4H-1, 2, 4-triazol-3-yl) thio) methyl) phenyl) boronic acid, white solid, 69.3% yield, 95% purity;¹H NMR(400MHz,Methanol-d₄)δ7.62(d,J＝45.7Hz,2H),7.25(d,J＝8.8Hz,4H),6.84(d,J＝8.8Hz,2H),4.42(s,2H),3.99(t,J＝13.8Hz,1H),3.02(s,6H),1.91(q,J＝11.3,10.3Hz,2H),1.73(d,J＝13.2Hz,2H),1.51(d,J＝11.5Hz,2H),1.30(d,J＝10.8Hz,2H),1.12(d,J＝13.5Hz,2H).¹³C NMR(101MHz,DMSO)δ156.42,151.47,148.33,139.34,134.76,130.47,128.67,128.56,114.71,112.15,56.61,56.56,37.80,31.31,25.81,24.96.MS(ESI)m/z:calcd for C₂₃H₃₀BN₄O₂S⁺(M+H)⁺:437.2；found:437.2.

example 48: structural formula (xvi):(4- (((5- (4-dimethylaminophenyl) -4-methyl-4H-1, 2, 4-triazol-3-yl) thio) methyl) phenyl) boronic acid, white solid, yield 58.3%, purity 95%;¹H NMR(400MHz,Methanol-d₄)δ7.70(s,2H),7.59(d,J＝9.0Hz,2H),7.40(d,J＝7.5Hz,2H),6.95(d,J＝9.1Hz,2H),4.52(s,2H),3.66(s,3H),3.10(s,6H).¹³C NMR(101MHz,DMSO)δ154.41,152.83,139.70,138.45,134.85,130.67,130.48,128.25,46.65,37.20,33.43.MS(ESI)m/z:calcd for C₁₈H₂₂BN₄O₂S⁺(M+H)⁺:369.2；found:369.3.

experimental example 1 cell proliferation inhibition experiment

(1) Preparing an experimental reagent: adding 10% fetal calf serum and 1% double antibody reagent into DMEM medium, shaking fetal calf serum solution, double antibody reagent and culture medium sufficiently, mixing well to obtain complete culture medium, subpackaging, and storing in refrigerator at 4 deg.C for use. Mixing 1 part of dimethyl sulfoxide (DMSO) and 9 parts of fetal calf serum uniformly to prepare a cell freezing medium, and mixing 1 part of complete culture medium and 10 parts of CCK-8 reagent uniformly for later use.

(2) Preparing a mother solution: dissolving a sample to be tested by 2mg with DMSO to prepare 10mM mother liquor, subpackaging and storing in a refrigerator at-20 ℃ in a dark place to avoid repeated freeze thawing. Appropriate amount of mother liquid was diluted to 10. mu.M with complete medium, and then three-fold diluted from 10. mu.M to obtain 6 working solutions of different concentrations of drug, each well having final concentrations of 10. mu.M, 3.333. mu.M, 1.111. mu.M, 0.370. mu.M, 0.123. mu.M, and 0.041. mu.M, respectively.

(3) Cell culture: when the cells grow to 70% -80% of the bottom area of the culture dish, the cells are digested and collected by pancreatin, after centrifugation, the cells are added into a culture medium for resuspension and are inoculated into a disposable cell culture dish with the diameter of 100mm, the disposable cell culture dish is placed in a constant-temperature incubator with the temperature of 37 ℃ and 5% carbon dioxide for static culture, and operations such as liquid changing and the like are carried out according to the growth condition of the cells.

(4) Cell passage: after culturing for a period of time, starting cell passage work when the cells grow to 70% -80% of the bottom area of the culture dish, discarding the original culture solution, cleaning the bottom of the cell culture dish for 3 times by using PBS (phosphate buffer solution), adding pancreatin for digestion for 1-2min, adding a complete culture medium to stop digestion, collecting the cells, centrifuging, adding the cells suspended by the culture medium, and suspending the cells according to the proportion of 1: 3 in the proportion of the total amount of the components, inoculating the components into a disposable cell culture dish with the diameter of 100mm, placing the dish in a constant temperature incubator with the temperature of 37 ℃ and the carbon dioxide of 5 percent for static culture, and carrying out operations such as changing liquid according to the growth condition of cells.

4X 10 per hole³And inoculating the cells into a 96-well cell plate, allowing the cell strain to be tested to grow adherently for 24 hours, and then discarding the old culture medium. The experiment is provided with a control group, a blank group and a drug treatment group, wherein the control group is 100 mu L of culture medium containing DMSO, the blank group is 100 mu L of cells and complete culture medium containing DMSO, and the experiment group is 100 mu L of cells and culture medium containing drugs with different concentrations. Setting 3 multiple wells for each concentration, culturing for 24h, absorbing the old culture medium, adding 110 μ L diluted CCK-8 test solution, culturing for 2h, and detecting OD value at 450nm with microplate reader.

The experiment was repeated 3 times, and the average of the experimental results was taken as the final experimental result. The growth inhibition (% Inh) — [ (experimental OD-blank OD)/(control OD-blank OD) ] × 100% was calculated according to the formula. IC50 values were obtained by non-linear fitting of sample activity to sample concentration.

It should be noted that in the examples of the present invention, the inhibition rate of g1-g48 at a concentration of 10 μ M in 4 LSD1 high expression cell lines was first tested as a primary screening, and the inhibition rate of the primary screening is higher than 50%, and the inhibition rate of the primary screening is rescreened, and the inhibition rate of the anti-tumor cell proliferation and the IC are obtained₅₀The results are as follows: (see Table 1-Table 2 for results)

TABLE 1

TABLE 2

From the above results, it can be seen that the thiotriazole compound provided in the embodiment of the present invention can specifically inhibit HGC27 cell line, H1975 cell line, HCT116 cell line, MCF7 cell line, molm13 cell line and PC-3 cell line, indicating that it can effectively treat tumors such as gastric cancer, lung adenocarcinoma, colon cancer, breast cancer, acute myelogenous leukemia, and the like.

Experimental example 2 inhibition of enzyme Activity

1. AlphaLisa experiment of Histone demethylase (LSD1)

(1) Preparing a reaction system: 1 × modified Tris buffer (pH 7.5); serial dilution of the sample solution, final DMSO concentration of 1%; enzyme solution: adding LSD1 to the 1 Xenzyme buffer solution; substrate peptide solution: to a 1x enzyme buffer solution was added H3K4me 2.

(2) Using an Echo voice-controlled pipetting system, 5. mu.L of test compound solution or blank control solution were transferred to 384-well cell plates and then preincubated with enzyme (final concentration of 5nM) for 15 minutes at room temperature.

(3) mu.L of substrate peptide solution (final concentration 100nM) was added to the reaction mixture and the reaction was started by incubation at room temperature for 60 min.

(4) The reaction was stopped by adding 15. mu.L of acceptor and donor solutions and incubated for 60 minutes at room temperature in the dark.

(5) By usingThe Alpha mode of (1) reads data.

(6) Data processing: the inhibition rate (% Inh) (Max-signal)/(Max-Min) × 100, wherein Max adds histone demethylase protein solution and dimethyl sulfoxide solution, Min adds buffer solution and dimethyl sulfoxide solution only; the% Inh and compound concentrations were substituted into the following equation in GraphPad Prism 5 software to obtain IC₅₀The value: y ═ Bottom + (Top-Bottom)/(1+10^ ((LogIC)₅₀-X) Hill Slope)), Y is% Inh, X is the compound concentration.

2. Gel migration hysteresis experiment of glycogen synthase kinase 3 beta (GSK3 beta)

(1) Preparing a reaction system: 1x kinase reaction buffer; a reaction stop solution; 2.5 × substrate peptide solution: adding FAM labeled peptide and ATP into 1 Xkinase reaction buffer; serial dilution of the sample solution, final DMSO concentration of 1%; 2.5 × enzyme solution: GSK3 β was added to the 1x enzyme buffer.

(2) mu.L of test compound solution and 10. mu.L of 2.5 XGSK 3 beta enzyme solution were added to 384 well cell plates and incubated at room temperature for 10 minutes.

(3) mu.L of 2.5 Xsubstrate peptide solution was added, and 30. mu.L of enzyme reaction terminator was added after reaction at 28 ℃ for a certain period of time.

(4) Data were collected by testing on a Caliper with an inhibition of enzyme activity (% Inh) — (max-conversion)/(max-min) of 100, "max" for DMSO control without compound added, "min" for low control. The% Inh and compound concentrations were substituted into the following equation in GraphPad Prism 5 software to obtain IC₅₀The value: y ═ Bottom + (Top-Bottom)/(1+10^ ((LogIC)₅₀-X) Hill Slope)), Y is% Inh, X is the compound concentration. See tables 3-5 for results.

TABLE 3

TABLE 4

TABLE 5

3. ADP-Glo assay for cyclin-dependent kinase 8(CDK8)

(1) Preparation of 1 × kinase buffer: 50mM HEPES, 0.01% Brij-35, 10mM MgCl₂，1mM EGTA，50mM HEPES。

(2) Compound screening: dilutions of compounds were transferred to plates using Echo 550, plates were sealed, and centrifuged at 1000g for 1 min. A1.5 x Tracer236 solution was prepared in 1x binding buffer and 10. mu.l of the 1.5x Tracer236 solution was added to the 384 well plates. Prepare 3 Xenzyme solution in 1 Xbinding buffer, add 5. mu.l 3 Xenzyme solution to 384 well plates, centrifuge at 1000g for 30 seconds, RT 60 minutes. Fluorescence signals at 615nm and 665nm were read on Envision 2104.

(3) Data processing: % inh 100- (signalmpd-SignalAve _ PC)/(SignalAve _ VC-SignalAve _ PC) × 100. The% Inh and compound concentrations were substituted into the following equation in GraphPad Prism 5 software to obtain IC₅₀The value: y ═ Bottom + (Top-Bottom)/(1+10^ ((LogIC)₅₀-X) Hill Slope)), Y is% Inh, X is the compound concentration.

(4) The experimental results are as follows: IC for only 5 compounds which perform better in HGC27 and H1975₅₀The results of the tests are shown in Table 6(STS is a positive compound):

TABLE 6

As can be seen from tables 3 to 6, the thiotriazole compound provided in the examples of the present invention can effectively inhibit the activities of LSD1 and CDK8, but has no inhibitory effect on GSK3 β, which indicates that the thiotriazole compound can be used as an inhibitor for inhibiting the activity of LSD1 and has a good therapeutic effect on diseases caused by excessive activity of LSD 1.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.