WO2021042723A1

WO2021042723A1 - Glutaminase gls1 inhibitor containing triazole structure or pharmaceutically acceptable salt thereof, preparation method therefor, and application thereof

Info

Publication number: WO2021042723A1
Application number: PCT/CN2020/084163
Authority: WO
Inventors: 李志裕; 卞金磊; 徐熙; 孟颖; 王举波
Original assignee: 中国药科大学
Priority date: 2019-09-05
Filing date: 2020-04-10
Publication date: 2021-03-11
Also published as: CN110746416A

Abstract

Disclosed are a glutaminase GLS1 inhibitor containing a triazole structure or a pharmaceutically acceptable salt thereof, a preparation method therefor, and an application thereof. A series of compounds containing a triazole structure of the present invention have glutaminase inhibitory activity, and can be used for the treatment of diseases and illnesses related to glutaminase dysfunction or elevated glutaminase activity. These compounds can effectively bind to an allosteric site of glutaminase, leading to changes in the conformation of the glutaminase and blocking the fulfilment of the biological functions thereof. In vitro experiments showed that the compounds of the present invention have good inhibitory activity on various glutamine-dependent cancer cells, such as colon cancer, triple-negative breast cancer, and lung cancer.

Description

Glutaminase GLS1 inhibitor containing triazole structure or its pharmaceutically acceptable salt, preparation method and application thereof

Technical field

The present invention relates to a glutaminase GLS1 inhibitor or a pharmaceutically acceptable salt thereof, a preparation method and application thereof, and particularly to a glutaminase GLS1 inhibitor containing a triazole structure or a pharmaceutically acceptable salt thereof, and a preparation method thereof And uses.

Background technique

Metabolic reprogramming is an important sign of tumors. Tumors undergo metabolic reprogramming to meet the energy requirements and material synthesis requirements of rapid proliferation. Classical tumor metabolic reprogramming has two characteristics, one is the aerobic glycolysis of glucose; the other is relying on glutamine to replenish the tricarboxylic acid cycle. Glutamine can not only be used as a carbon source to replenish the tricarboxylic acid cycle, but also provide a nitrogen source for the synthesis of protein, hexose and nucleotides and other biological macromolecules. In addition, it is also one of the precursors of glutathione. An important way for the body to resist oxidative stress and maintain redox homeostasis. Glutaminase (GLS) catalyzes the reaction of glutamine deamination to produce glutamate. It is the rate-limiting enzyme of glutamine glycolysis. It controls the entrance of glutamine catabolism and the body’s influence on glutamine metabolism. The regulation is mainly achieved through GLS.

High dependence on glutamine is an important metabolic feature of tumor cells, also known as "glutamine addiction". Glutaminease (GLS) catalyzes the reaction of glutamine to glutamate, which is the result of glutamine glycolysis The first metabolic enzyme. GLS can be divided into renal type glutaminase (GLS1) and liver type glutaminase (GLS2). GLS1 is highly expressed in most tumors, and GLS2 is low. GLS1 has a "pro-cancer effect", while GLS2 has an "anti-cancer effect". GLS plays an important role in the occurrence and development of a variety of tumors. GLS is of great significance to the diagnosis, progression and prognosis evaluation of tumors. GLS1 is a potential target of tumor metabolism therapy, and its specific inhibitor is expected to become a new type of anti-tumor metabolism drug.

Summary of the invention

Object of the invention: The object of the invention is to provide a glutaminase GLS1 inhibitor containing a triazole structure or a pharmaceutically acceptable salt thereof.

Another object of the present invention is to provide a pharmaceutical composition containing a therapeutically effective amount of one or more glutaminase GLS1 inhibitors of general formula (I) containing triazole structure or their pharmaceutically acceptable Salt, and a pharmaceutically acceptable carrier.

Another object of the present invention is to provide a pharmaceutical composition containing a therapeutically effective amount of one or more of the triazole-containing glutaminase GLS1 inhibitors of general formula (I) or Medicinal salts and pharmaceutically acceptable excipients.

Another object of the present invention is to provide a method for preparing the glutaminase GLS1 inhibitor containing a triazole structure or a pharmaceutically acceptable salt thereof.

The last objective of the present invention is to provide the use of the glutaminase GLS1 inhibitor containing the triazole structure or its pharmaceutically acceptable salt in the preparation of drugs for the treatment of diseases mediated by GLS1.

Technical solution: The present invention provides a glutaminase GLS1 inhibitor containing a triazole structure or a pharmaceutically acceptable salt thereof,

Wherein, n is an integer of 1-4;

L is: CH ₂ SCH ₂ , CH ₂ CH ₂ , CH ₂ CH ₂ CH ₂ , CH ₂ , CH ₂ S, SCH ₂ , CH ₂ NHCH ₂ , CH=CH or

Wherein any hydrogen in CH or CH ₂ can be substituted by alkyl or alkoxy; hydrogen in -NH group can be substituted by alkyl; in -CH ₂ CH ₂ , CH ₂ CH ₂ CH ₂ group A single CH ₂ may be substituted by a hydroxyl group; the _{two groups R 1} and R ₂ and the atoms to which they are attached may optionally form a cycloalkane together;

X ₁ and X ₂ are respectively: S, O and CH=CH, wherein any hydrogen in CH can be substituted by an alkyl group;

Y is: H or CH ₂ O(CO) R ₅ , R ₅ is: H, substituted or unsubstituted alkyl, alkoxy, amino, heterocycloalkyl, aromatic cycloalkyl or heterocycloalkoxy ；

R ₁ and R ₂ are respectively: H, alkyl, alkoxy or hydroxyl;

R ₃ is: alkanes, substituted alkanes, aromatic hydrocarbons, aromatic alkanes, cyano groups, cycloalkanes, cycloaromatic alkanes, hydrogen, halogen, halogen-substituted alkanes, heteroatom aromatic hydrocarbons, heteroatom aromatic alkanes, heteroatom cycloalkanes, C(R ₆ )(R ₇ )(R ₈ ), N(R ₉ )(R ₁₀ ), OR ₁₁ , any hydroxyl group can be acetylated to C(O)R ₇ ;

R ₄ is: alkanes, substituted alkanes, cycloalkanes, aromatic hydrocarbons, aromatic alkanes, substituted aromatic hydrocarbons or substituted aromatic alkanes;

R ₆ , R ₇ , and R ₈ are respectively: hydrogen, substituted or unsubstituted alkyl, hydroxyl, hydroxyalkyl, amino, acetamido, alkene, alkyne, alkoxy, aryl, arylalkyl, cycloalkane , Heterocyclic or heteroatom aromatic hydrocarbons;

R ₉ and R ₁₀ are respectively: hydrogen, substituted or unsubstituted alkyl, hydroxyl, hydroxyalkyl, amino, acetamido, alkene, alkyne, alkoxy, aryl, arylalkyl, cycloalkane, heterocycle , Heteroatom aromatic hydrocarbons, any hydroxyl group can be acetylated to C(O)R ₇ ;

R ₁₁ is: hydrogen, substituted or unsubstituted alkyl, hydroxy, hydroxyalkyl, amino, acetamido, alkene, alkyne, alkoxy, aryl, arylalkyl, cycloalkane, heterocycle, heteroatom aromatic For hydrocarbons, any hydroxyl group can be acetylated to C(O)R ₇ .

Further, the L is CH ₂ CH ₂ .

Further, the glutaminase GLS1 inhibitor with the general formula (I) containing a triazole structure or a pharmaceutically acceptable salt thereof is any one of the following:

Among them, R is -CH ₂ CH ₂ OH, -C(CH ₃ ) ₂ OH, -C(CH ₃ )(OH)(C ₂ H ₅ ), -CH ₂ CH ₂ COOH, -COOC ₂ H ₅ ,

-Ph, 4'-CH ₃ -Ph, 4'-CF ₃ -Ph, 4'-F-Ph, 4'-Cl-Ph, 4'-Br-Ph, 4'-OCH ₃ -Ph, 3' -OCH ₃ -Ph, 3'-OH-Ph, 3'-NH ₂ -Ph, 4'-NH ₂ -Ph, 2'-Pyridine;

Where R is -Ph, 4'-CN-Ph, 4'-NO ₂ -Ph, 4'-F-Ph, 4'-Cl-Ph, 4'-Br-Ph, 4'-CH ₃ -Ph , 4'-OCH ₃ -Ph, 3'-OCH ₃ -4'-OCH ₃ -Ph, 2'-CH ₃ -4'-CH ₃ -Ph, 2'-CH ₃ -6'-CH ₃ -Ph ；

Among them, R is 4'-CH ₃ -Ph, 4'-CN-Ph, 4'-NO ₂ -Ph, 4'-F-Ph, 4'-Cl-Ph, 4'-Br-Ph, 4' -OCH ₃ -Ph, 3'-OCH ₃ -4'-OCH ₃ -Ph, 2'-CH ₃ -4'-CH ₃ -Ph, 2'-CH ₃ -6'-CH ₃ -Ph, 4' -CF ₃ -Ph, 4'-OCF ₃ -Ph;

Where R is -Ph, 4'-CN-Ph, 4'-NO ₂ -Ph, 4'-F-Ph, 4'-Cl-Ph, 4'-Br-Ph, 4'-CH ₃ -Ph , 4'-OCH ₃ -Ph, 3'-OCH ₃ -4'-OCH ₃ -Ph, 4'-OCF ₃ -Ph, 2'-CH ₃ -6'-CH ₃ -Ph, 4'-CF ₃ -Ph;

Where R is -Ph, 4'-CN-Ph, 4'-NO ₂ -Ph, 4'-F-Ph, 4'-Cl-Ph, 4'-Br-Ph, 4'-OCH ₃ -Ph , 3'-OCH ₃ -4'-OCH ₃ -Ph, 4'-CH ₃ -Ph, 2'-CH ₃ -6'-CH ₃ -Ph, 4'-CF ₃ -Ph, 4'-OCF ₃ -Ph;

Where R is -Ph, 4'-CN-Ph, 4'-NO ₂ -Ph, 4'-F-Ph, 4'-Cl-Ph, 4'-Br-Ph, 4'-CH ₃ -Ph , 4'-OCH ₃ -Ph, 3'-OCH ₃ -4'-OCH ₃ -Ph, 4'-OCF ₃ -Ph, 2'-CH ₃ -6'-CH ₃ -Ph, 4'-CF ₃ -Ph.

A pharmaceutical composition containing a therapeutically effective amount of one or more of the glutaminase GLS1 inhibitors with the general formula (I) containing the triazole structure or their pharmaceutically acceptable salts, and pharmacy Acceptable carrier.

A pharmaceutical composition containing a therapeutically effective amount of one or more of the glutaminase GLS1 inhibitors with the general formula (I) containing the triazole structure or their pharmaceutically acceptable salts, and pharmacy Acceptable excipients.

The preparation method of the glutaminase GLS1 inhibitor containing the triazole structure with the general formula (I) or a pharmaceutically acceptable salt thereof includes the following steps:

Compound II reacts with different alkynes or azides to obtain corresponding compounds III-1 or III-2. Compounds III-1 and III-2 undergo Click reactions with different azide compounds or alkynes, respectively, under the catalysis of CuI. The final product of the glutaminase GLS1 inhibitor containing the triazole structure with the general formula (I) is obtained.

The use of the glutaminase GLS1 inhibitor with the general formula (I) containing a triazole structure or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of diseases mediated by GLS1.

Further, the disease is colon cancer, triple negative breast cancer or lung cancer.

The present invention has discovered a new drug drug with high efficiency and low toxicity for the treatment of cancer. According to the crystal structure of glutaminase and the representative GLS1 inhibitor BPTES and CB839 compound, the triazole group is introduced into the structure and investigated The effect of different substituents on the triazole group on the activity, optimize the optimal configuration, and the overall structure-effect relationship. The designed and synthesized compounds are targeted, can significantly inhibit the activity of glutaminase, block the hydrolysis of glutamine into glutamate, thereby cutting off the energy supply of tumor cells, and have a strong ability to inhibit glutamine-dependent tumors. And good therapeutic effects can be achieved through the combination of drugs.

Beneficial effects: The present invention designs and synthesizes a series of novel glutaminase inhibitor compounds containing a triazole structure based on the crystal structure of the allosteric site of glutaminase, which can significantly inhibit the biological activity of glutaminase. Experimental results show that these compounds can significantly inhibit the activity of glutaminase at the molecular level, block the hydrolysis of glutamine to glutamate, and show good anti-tumor effects at the cellular level and animal levels, and can be used to prepare anti-tumor agents. drug.

Description of the drawings

Figure 1 shows the thermostable migration experiment of GLS1 inhibitor to GLS1 protein;

Figure 2 shows (A) surface plasmon resonance test to determine the affinity of compound CPU-210 and GLS1 protein; (B) surface plasmon resonance test to determine the affinity of compound CPU-301 to GLS1 protein;

Figure 3 shows the experiment of compound CPU-301 and CB839 on intracellular glutamate content;

Figure 4 shows the experiment of compound CPU-301 and CB839 inducing the increase of intracellular ROS level.

detailed description

Example 1

Preparation of general formula compound I (CPU101-CPU118)

Preparation of ethyl 5-(5-amino-1,3,4-thiadiazolyl)valerate (2)

Intermediate 1 (10 g, 57.4 mmol) was dissolved in 100 mL POCl ₃ , then thiosemicarbazide (5.23 g, 57.4 mmol) was added, and the reaction was carried out at 85°C for 4 hours. After the completion of the reaction was detected by TLC, the reaction solution was cooled to room temperature, diluted with water, and then adjusted to pH=7 with 6M NaOH, a solid was precipitated, filtered with suction, and the filter cake was dried to obtain a white solid with a yield of 41.85%. HRMS(ESI):m/z,calcd for C ₉ H ₁₅ N ₃ O ₂ S[M+H] ⁺ ,230.0963; found:230.0962.

Preparation of 5-(5-(2-phenylacetylamino)-1,3,4-thiadiazole) ethyl valerate (3)

Intermediate 2 (2.0g, 8.73mmol) was dissolved in 20mL THF, then triethylamine (1.3mL, 9.62mmol) was added and phenylacetyl chloride (1.35g, 8.73mmol) was added dropwise. The reaction mixture was stirred at room temperature for 24 hours. TLC detected that the reaction was completed and the solvent was removed under reduced pressure, and the mixture was beaten with water to obtain a white solid with a yield of 92.5%. HRMS(ESI):m/z,calcd for C ₁₇ H ₂₁ N ₃ O ₃ S ₂ [M+H] ⁺ ,348.1382; found:348.1371.

Preparation of 5-(5-(2-phenylacetylamino)-1,3,4-thiadiazole)pentanoic acid (4)

Compound 3 (2.0 g, 5.76 mmol) was dissolved in 4N NaOH (15 mL) and MeOH (10 mL), and stirred at room temperature for 3 h. After the completion of the reaction, the solvent was removed under reduced pressure, pH was adjusted to 7 with 4N HCl, and a white solid was precipitated, which was dried by suction and dried with a yield of 90.4%. HRMS(ESI):m/z,calcd for C ₁₅ H ₁₇ N ₃ O ₃ S ₂ [M+H] ⁺ ,320.1069; found:320.1064.

N-(5-(4-(5-amino-1,3,4-thiadiazolyl)butyl)-1,3,4-thiadiazolyl)-2-phenylacetamide (5) preparation

Intermediate 4 (2g, 6.2mmol) was dissolved in 10mL POCl _3, followed by addition of thiosemicarbazide (0.629g, 6.8mmol), the reaction 4h 85 ℃. After the completion of the reaction detected by TLC, the reaction solution was cooled to room temperature, diluted with water, and then adjusted to pH=7 with 6M NaOH, a solid was precipitated, filtered off with suction, and the filter cake was dried to obtain a black solid with a yield of 43.5%. HRMS(ESI):m/z,calcd for C ₁₆ H ₁₈ N ₆ OS ₂ [M+H] ⁺ ,375.1065; found:375.1056.

2-Chloro-N-(5-(4-(5-(2-phenylacetylamino)-1,3,4-thiadiazolyl)butyl)-1,3,4-thiadiazole)ethyl Preparation of amide (6)

Intermediate 5 (3g, 8.02mmol) was dissolved in 10mL DMF solution, TEA (2.43g, 24.06mmol) was added, and then chloroacetyl chloride (1.8g, 16.04mmol) was added dropwise. The reaction mixture was stirred at room temperature for 12 h. After cooling to room temperature, the reaction mixture was poured into water. A solid precipitated, and a white solid was obtained by suction filtration with a yield of 84.2%. HRMS(ESI):m/z,calcd for C ₁₈ H ₁₉ ClN ₆ O ₂ S ₂ [M+H] ⁺ ,451.0772; found:451.0785.

2-azido-N-(5-(4-(5-(2-phenylacetylamino)-1,3,4-thiadiazolyl)butyl)-1,3,4-thiadiazole ) Preparation of acetamide (7)

To the DMF solution of compound 6 (3.5 g, 7.78 mmol) was added NaN ₃ (1.52 g, 23.3 mmol). The reaction solution was stirred at room temperature for 12 hours. Then the reaction solution was poured into water, extracted with DCM (30mL×3), the organic layers were combined, washed three times with water, washed three times with saturated brine, and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and a white solid was obtained by column chromatography. The yield was 90%. HRMS(ESI):m/z,calcd for C ₁₈ H ₂₀ N ₉ O ₂ S ₂ [M+H] ⁺ ,458.1176; found:458.1148

Preparation of compound CPU-101

To the DMF solution of compound 7 (0.2 g, 0.44 mmol) was added an aqueous solution of CuI (16.7 mg, 0.088 mmol) and 3-butyn-1-ol (8a) (77.05 mg, 1.1 mmol). The reaction mixture was stirred in a microwave at 300W, 100°C for 0.5h. The reaction liquid was poured into water, and a white solid was precipitated. The crude product was obtained by suction filtration and column chromatography to obtain a white solid with a yield of 45.4%. mp208～210℃; ¹ H NMR(300MHz,DMSO-d ₆ ): δ12.66(s,2H),7.90(s,1H),7.31(s,5H),5.43(s,2H),4.69(s ,1H),3.79(s,2H),3.64(s,2H),3.00(s,4H),2.79(s,2H),1.74(s,4H)ppm.HRMS(ESI):m/z,calcd for C ₂₅ N ₉ O ₃ S ₂ [M+H] ⁺ ,528.1595; found:528.1577.

Preparation of compound CPU-102

As described in the preparation of CPU-101, the compound CPU-102 was prepared using 2-methyl-3-butyn-2-ol (8b) instead of 8a, and the yield was 39.4%. mp210～212℃; ¹ H NMR(300MHz,DMSO-d ₆ ): δ12.93(s,1H),12.67(s,1H),7.92(s,1H),7.32(s,5H),5.44(s ,2H),5.15(s,1H),3.80(s,2H),3.01(s,4H),1.75(s,4H),1.48(s,6H)ppm.HRMS(ESI):m/z,calcd for C ₂₃ H ₂₇ N ₉ O ₃ S ₂ [M+H] ⁺ ,542.1751; found:542.1740.

Preparation of compound CPU-103

As described in the preparation of CPU-101, 3-methyl-1-pentyn-3-ol (8c) was used instead of 8a to prepare compound CPU-103 with a yield of 40.8%. mp220～223℃; ¹ H NMR(300MHz,DMSO-d ₆ ): δ12.93(s,1H),12.65(s,1H),7.90(s,1H),7.32(s, 5H), 5.45(s ,2H),5.00(s,1H),3.80(s,2H),3.02(s,4H),1.75(s,6H),1.44(s,3H),0.76(s,3H)ppm.HRMS(ESI) ):m/z,calcd for C ₂₄ H ₂₉ N ₉ O ₃ S ₂ [M+H] ⁺ ,556.1908; found:556.1891.

Preparation of compound CPU-104

As described in the preparation of CPU-101, using 3-butyn-1-acid (8d) instead of 8a to prepare CPU-104, the yield was 35.7%. mp220～224℃; ¹ H NMR(300MHz,DMSO-d ₆ ): δ12.96-12.86(m,1H),12.64(s,2H),7.89(s,1H),7.32(s,5H),5.43 (s,2H),3.79(s,2H),3.00(s,6H),2.58(d,J=7.3Hz,2H),1.75(s,4H)ppm.HRMS(ESI):m/z,calcd for C ₂₃ H ₂₅ N ₉ O ₄ S ₂ [M+H] ⁺ ,556.1544; found:556.1521.

Preparation of compound CPU-105

As described in the preparation of CPU-101, ethyl propiolate (8e) was used instead of 8a to prepare CPU-105, with a yield of 37.8%. mp204～208℃; ¹ H NMR(300MHz,DMSO-d ₆ ): δ12.61(s,1H),8.76(s,1H),7.31(s,5H),5.57(s,2H),4.32(d ,J=7.1Hz,2H),3.79(s,2H),2.99(s,4H),1.75(s,4H),1.31(t,J=6.6Hz,3H)ppm.HRMS(ESI):m/ z,calcd for C ₂₃ H ₂₅ N ₉ O ₄ S ₂ [M+H] ⁺ ,556.1544; found:556.1504.

Preparation of compound CPU-106

As described in the preparation of CPU-101, the difference is that ethynylcyclopropane (8f) was used instead of 8a to prepare compound CPU-106, and the yield was 42.3%. mp215～218℃; ¹ H NMR(300MHz,DMSO-d ₆ ): δ12.91(s,1H),12.64(s,1H),7.85(s,1H),7.32(s,5H),5.40(s ,2H),3.79(s,2H),3.01(s,4H),1.96(s,1H),1.75(s,4H),0.90(s,2H),0.72(s,2H)ppm.HRMS(ESI) ):m/z,calcd for C ₂₃ H ₂₅ N ₉ O ₂ S ₂ [M+H] ⁺ ,524.1645; found:524.1633.

Preparation of compound CPU-107

As described in the preparation of CPU-101, phenylacetylene (8g) was used instead of 8a to prepare compound CPU-107 with a yield of 45.1%. mp218～220℃; ¹ H NMR(300MHz,DMSO-d ₆ ):δ12.64(s,1H),8.60(s,1H),7.85(s,1H),7.38(d,J=45.5Hz,9H ),5.55(s,2H),3.78(s,2H),3.00(s,4H),1.74(s,4H)ppm.HRMS(ESI):m/z,calcd for C ₂₆ H ₂₅ N ₉ O ₂ S ₂ [M+H] ⁺ ,560.1645; found:560.1617

Preparation of compound CPU-108

As described in the preparation of CPU-101, the compound CPU-108 was prepared by using 4-benzylacetylene (8h) instead of 8a, and the yield was 33.3%. mp237～242℃; ¹ H NMR(300MHz,DMSO-d ₆ ): δ12.99(s,1H),12.66(s,1H),8.55(s,1H),7.75(s,2H),7.33(s ,2H),7.29(s,5H),5.54(s,2H),3.79(d,J=12.5Hz,2H),3.01(s,4H),2.34(d,J=13.4Hz,3H),1.74 (s,4H)ppm.HRMS(ESI):m/z,calcd for C ₂₇ H ₂₇ N ₉ O ₂ S ₂ [M+H] ⁺ ,574.1802; found:574.1806.

Preparation of compound CPU-109

As described in the preparation of CPU-101, the compound CPU-109 was prepared by using p-trifluoromethylphenylacetylene (8i) instead of 8a, and the yield was 40.9%. mp109～111℃; ¹ H NMR(300MHz,DMSO-d ₆ ):δ12.68(s,1H),8.79(s,1H), 8.10(d,J=7.7Hz,2H),7.83(d,J ＝7.9Hz,2H),7.34-7.24(m,5H),5.60(s,2H),3.79(s,2H),3.00(s,4H),1.75(s,4H)ppm.HRMS(ESI): m/z,calcd for C ₂₇ H ₂₄ F ₃ N ₉ O ₂ S ₂ [M+H] ⁺ ,628.1519; found:628.1528.

Preparation of compound CPU-110

As described in the preparation of CPU-101, 4-fluorophenylacetylene (8j) was used instead of 8a to prepare compound CPU-110 with a yield of 41.5%. mp249～250℃; ¹ H NMR(300MHz,DMSO-d ₆ ): δ12.59(s,1H),8.51(s,1H),7.91-7.76(m,2H),7.23(s,5H),7.18 (d,J=8.3Hz,2H),5.46(s,2H),3.71(s,2H),2.92(s,4H),1.67(s,4H)ppm.HRMS(ESI):m/z,calcd for C ₂₆ H ₂₄ FN ₉ O ₂ S ₂ [M+H] ⁺ ,578.1551; found:578.1533.

Preparation of compound CPU-111

As described in the preparation of CPU-101, the compound CPU-111 was prepared by using 4-chlorophenylacetylene (8k) instead of 8a, and the yield was 43.9%. mp232～240℃; ¹ HNMR(300MHz,DMSO-d ₆ ):δ12.64(s,1H),8.63(s,1H),7.88(d,J=8.1Hz,2H),7.51(d,J= 8.2Hz, 2H), 7.27 (d, J = 14.2Hz, 5H), 5.55 (s, 2H), 3.78 (s, 2H), 2.99 (s, 4H), 1.74 (s, 4H) ppm.HRMS (ESI) ):m/z,calcd for C ₂₆ H ₂₄ ClN ₉ O ₂ S ₂ [M+H] ⁺ ,594.1256; found:594.1243.

Preparation of compound CPU-112

As described in the preparation of CPU-101, the compound CPU-112 was prepared by using 4-bromophenylacetylene (8l) instead of 8a, and the yield was 30.6%. mp 235～240℃; ¹ HNMR(300MHz,DMSO-d ₆ ): δ12.96(s,1H),12.62(s,1H),8.63(s,1H),7.82(d,J=8.0Hz,2H ), 7.65(d,J=8.2Hz,2H),7.38-7.24(m,5H),5.55(s,2H),3.78(s,2H),2.99(s,4H),1.74(s,4H) ppm.HRMS(ESI):m/z,calcd for C ₂₆ H ₂₄ BrN ₉ O ₂ S ₂ [M+H] ⁺ ,638.0751; found:638.0739.

Preparation of compound CPU-113

As described in the preparation of CPU-101, the compound CPU-113 was prepared by using 4-ethynyl anisole (8m) instead of 8a, and the yield was 42.2%. mp215～216℃; ¹ HNMR(300MHz,DMSO-d ₆ ): δ13.02(s,1H), 12.66(s,1H), 8.48(s,1H), 7.79(d,J=8.1Hz,2H) ,7.29(dd,J=14.4,3.7Hz,5H),7.03(d,J=8.3Hz,2H),5.54(s,2H),3.79(s,5H),3.00(s,4H),1.75( s,4H)ppm.HRMS(ESI):m/z,calcd for C ₂₇ H ₂₇ N ₉ O ₃ S ₂ [M+H] ⁺ ,590.1751; found:590.1754.

Preparation of compound CPU-114

As described in the preparation of CPU-101, 3-ethynyl anisole (8n) was used instead of 8a to prepare compound CPU-114 with a yield of 33.3%. mp227～229℃; ¹ HNMR(300MHz,DMSO-d ₆ ): δ13.00(s,1H), 12.65(s,1H), 8.62(s,1H), 7.47-7.36(m,3H), 7.29( d, J = 14.5 Hz, 5H), 6.92 (d, J = 7.2 Hz, 1H), 5.55 (s, 2H), 3.80 (d, J = 8.9 Hz, 5H), 3.00 (s, 4H), 1.75 ( s,4H)ppm.HRMS(ESI):m/z,calcd for C ₂₇ H ₂₇ N ₉ O ₃ S ₂ [M+H] ⁺ ,590.1751; found:590.1741.

Preparation of compound CPU-115

As described in the preparation of CPU-101, the compound CPU-115 was prepared by using 3-hydroxyacetylene (8o) instead of 8a, and the yield was 38.7%. mp235～239℃; ¹ HNMR(300MHz,DMSO-d ₆ ): δ13.02-12.90(m,1H), 12.63(s,1H), 9.55(s,1H), 8.50(s,1H), 7.27( d,J=16.6Hz,8H),6.73(s,1H),5.52(s,2H),3.78(s,2H),2.99(s,4H),1.74(s,4H)ppm.HRMS(ESI) :m/z,calcd for C ₂₆ H ₂₅ N ₉ O ₃ S ₂ [M+H] ⁺ ,576.1595; found:576.1576.

Preparation of compound CPU-116

As described in the preparation of CPU-101, the compound CPU-116 was prepared using m-aminophenylacetylene (8p) instead of 8a, with a yield of 42.5%. mp232～235℃; ¹ HNMR(300MHz,DMSO-d ₆ ):δ12.65(s,2H),8.42(s,1H),7.31(s,9H),5.51(s,2H),4.15(s, 1H),3.77(s,3H),2.99(s,4H),1.73(s,4H)ppm.HRMS(ESI):m/z,calcd for C ₂₆ H ₂₆ N ₁₀ O ₂ S ₂ (M+H ] ⁺ ,575.1754; found:575.1741.

Preparation of compound CPU-117

As described in the preparation of CPU-101, the compound CPU-117 was prepared by using p-aminophenylacetylene (8q) instead of 8a, and the yield was 40.0%. mp201～204℃; ¹ HNMR(300MHz,DMSO-d ₆ ): δ12.66(s,1H), 8.28(s,1H), 7.52(d,J=6.5Hz,2H), 7.31(s,5H) ,7.27(d,J=4.0Hz,2H),6.61(s,2H),5.49(s,2H),3.79(s,2H),3.00(s,4H),1.74(s,4H)ppm.HRMS (ESI):m/z,calcd for C ₂₆ H ₂₆ N ₁₀ O ₂ S ₂ [M+H] ⁺ ,575.1754; found:575.1759.

Preparation of compound CPU-118

As described in the preparation of CPU-101, the compound CPU-118 was prepared using 2-ethynylpyridine (8r) instead of 8a, and the yield was 38.6%. mp223～227℃; ¹ HNMR(300MHz,DMSO-d ₆ ):δ12.62(s,2H),8.67(s,2H),7.95(s,2H),7.29(s,5H),7.25(s, 1H),5.59(s,2H),3.77(s,2H),2.98(s,4H),1.73(s,4H)ppm.HRMS(ESI):m/z,calcd for C ₂₄ N ₁₀ O ₂ S ₂ [M+H] ⁺ ,561.1598; found:561.1597.

Example 2

Preparation of compound II (CPU201-CPU211) of general formula.

Synthesis of Intermediate 10

Intermediate 5 (0.255g, 0.68mmol), pentynoic acid (0.073g, 0.75mmol), HATU (0.38g, 1.02mmol) were dissolved in DMF (2mL) and stirred at room temperature for 10min. DIPEA (0.26 g, 2.04 mmol) was added and stirring was continued. The reaction was almost complete after 20 minutes. The reaction liquid was poured into water, a white solid was separated out, filtered with suction, and the filter cake was dried to obtain a white solid. The yield was 61.5%. HRMS(ESI):m/z,calcd for C ₂₁ H ₂₂ N ₆ O ₂ S ₂ [M+H] ⁺ ,455.1324; found:455.1310.

Preparation of compound CPU-201

To the DMF solution of Intermediate 10 (0.1 g, 0.202 mmol) was added an aqueous solution of CuI (7.78 mg, 0.041 mmol) and benzyl azide (11a) (63.3 mg, 0.476 mmol). The reaction mixture was stirred in a microwave at 300W and 100°C for 0.5 hours. The reaction liquid was poured into water, and a white solid was precipitated. The crude product was obtained by suction filtration and column chromatography to obtain the white final product with a yield of 38.7%. ¹ HNMR (300MHz, DMSO-d ₆ ): δ7.90 (s, 1H), 7.30 (t, J = 11.1Hz, 10H), 5.54 (s, 2H), 3.77 (s, 2H), 3.04-2.92 ( m,6H),2.82(d,J=6.9Hz,2H),1.75(s,4H)ppm.HRMS(ESI):m/z,calcd for C ₂₈ H ₂₉ N ₉ O ₂ S ₂ [M+H ] ⁺ ,588.1958; found:588.1960.

Preparation of compound CPU-202

As described in the preparation of CPU-201, the compound CPU-202 was prepared by using 4-cyanobenzyl azide (11b) instead of 11a, and the yield was 43.6%. ¹ HNMR (300MHz, DMSO-d ₆ ): δ 12.67 (s, 1H), 12.44 (s, 1H), 7.95 (s, 1H), 7.80 (s, 2H), 7.32 (s, 7H), 5.67 ( s,2H),3.80(s,2H),3.00(s,6H),2.84(s,2H),1.75(s,4H)ppm.HRMS(ESI):m/z,calcd for C ₂₉ H ₂₈ N ₁₀ O ₂ S ₂ [M+H] ⁺ ,613.1911; found:613.1903.

Preparation of compound CPU-203

As described in the preparation of CPU-201, the compound CPU-203 was prepared by using 4-nitrobenzyl azide (11c) instead of 11a, and the yield was 40.2%. ¹ HNMR (300MHz, DMSO-d ₆ ): δ 12.65 (s, 1H), 12.45 (s, 1H), 8.19 (d, J = 8.6 Hz, 2H), 7.97 (s, 1H), 7.45 (d, J = 8.6Hz, 2H), 7.31 (t, J = 6.6Hz, 5H), 5.73 (s, 2H), 3.80 (s, 2H), 3.03-2.95 (m, 6H), 2.84 (t, J = 6.9 Hz,2H),1.75(s,4H)ppm.HRMS(ESI):m/z,calcd for C ₂₈ H ₂₈ N ₁₀ O ₄ S ₂ [M+H] ⁺ ,633.1809; found: 633.1808.

Preparation of compound CPU-204

As described in the preparation of CPU-201, the compound CPU-204 was prepared by using 4-fluorobenzyl azide (11d) instead of 11a, and the yield was 34.8%. mp203～208℃; ¹ HNMR(300MHz,DMSO-d ₆ ): δ12.65(s,1H),12.40(s,1H),7.87(s,1H),7.30(s,5H),7.14(t, J = 7.8Hz, 3H), 5.51 (s, 2H), 3.78 (s, 2H), 2.98 (s, 6H), 2.80 (s, 2H), 1.73 (s, 4H) ppm. HRMS (ESI): m /z,calcd for C ₂₈ H ₂₈ FN ₉ O ₂ S ₂ [M+H] ⁺ ,606.1864; found:606.1878.

Preparation of compound CPU-205

As described in the preparation of CPU-201, the compound CPU-205 was prepared by using 4-chlorobenzyl azide (11e) instead of 11a, and the yield was 39.4%. mp203～204℃; ¹ HNMR (300MHz, DMSO-d ₆ ): δ 12.70 (s, 1H), 12.45 (s, 1H), 7.94 (s, 1H), 7.39 (dd, J = 18.3, 6.0 Hz, 5H), 7.29 (d, J = 7.9 Hz, 4H), 5.58 (s, 2H), 3.84 (s, 2H), 3.02 (d, J = 13.0 Hz, 6H), 2.87 (d, J = 6.5 Hz, 2H),1.79(s,4H)ppm.HRMS(ESI):m/z,calcd for C ₂₈ H ₂₈ ClN ₉ O ₂ S ₂ [M+H] ⁺ ,622.1569; found: 622.1528.

Preparation of compound CPU-206

As described in the preparation of CPU-201, the compound CPU-206 was prepared by using 4-bromobenzyl azide (11f) instead of 11a, and the yield was 36.5%. mp217～219℃; ¹ HNMR(300MHz,DMSO-d ₆ ): δ12.69(s,1H), 12.44(s,1H), 7.93(s,1H), 7.56(d,J=8.2Hz,2H) ,7.42-7.29(m,5H),7.23(d,J=8.2Hz,2H),5.57(s,2H),3.84(s,2H),3.02(d,J=14.2Hz,6H),2.86( s,2H),1.80(s,4H)ppm.HRMS(ESI):m/z,calcd for C ₂₈ H ₂₈ BrN ₉ O ₂ S ₂ [M+H] ⁺ ,666.1064; found: 666.1059.

Preparation of compound CPU-207

As described in the preparation of CPU-201, the compound CPU-207 was prepared by using 4-methylbenzyl azide (11 g) instead of 11a, and the yield was 44.3%. mp216～217℃; ¹ HNMR(300MHz,DMSO-d ₆ ):δ12.67(s,1H),12.41(s,1H),7.85(s,1H),7.30(dd,J=14.6,3.9Hz, 5H), 7.13(s, 4H), 5.47(s, 2H), 3.80(s, 2H), 3.06-2.91(m, 6H), 2.83(d, J=6.2Hz, 2H), 2.25(s, 3H ),1.75(s,4H)ppm.HRMS(ESI):m/z,calcd for C ₂₉ H ₃₁ N ₉ O ₂ S ₂ [M+H] ⁺ ,602.2115; found: 602.2143.

Preparation of compound CPU-208

As described in the preparation of CPU-201, the compound CPU-208 was prepared by using 4-methoxybenzyl azide (11h) instead of 11a, and the yield was 46.4%. mp237～240℃; ¹ HNMR(300MHz,DMSO-d ₆ ): δ12.67(s,1H),12.42(s,1H),7.84(s,1H),7.32(s,5H),7.22(d, J = 8.3Hz, 3H), 6.88 (d, J = 8.2Hz, 2H), 5.45 (s, 2H), 3.80 (s, 2H), 3.72 (s, 3H), 2.97 (d, J = 18.2Hz, 6H),2.82(s,2H),1.75(s,4H)ppm.HRMS(ESI):m/z,calcd for C ₂₉ H ₃₁ N ₉ O ₃ S ₂ [M+H] ⁺ ,618.2064; found: 618.2070.

Preparation of compound CPU-209

As described in the preparation of CPU-201, 3,4-dimethoxybenzyl azide (11i) was used instead of 11a to prepare compound CPU-209 with a yield of 48.1%. mp232～236℃; ¹ HNMR(300MHz,DMSO-d ₆ ): δ12.66(s,1H), 12.41(s,1H), 7.84(s,1H), 7.30(t,J=7.8Hz,5H) , 6.95 (s, 1H), 6.88 (d, J = 8.1 Hz, 1H), 6.79 (d, J = 8.3 Hz, 1H), 5.43 (s, 2H), 3.80 (s, 2H), 3.74 to 3.67 ( m,6H),3.06-2.90(m,6H),2.83(d,J=6.8Hz,2H),1.75(s,4H)ppm.HRMS(ESI):m/z,calcd for C ₃₀ H ₃₃ N ₉ O ₄ S ₂ [M+H] ⁺ ,648.217; found:648.2167.

Preparation of compound CPU-210

As described in the preparation of CPU-201, 2,4-dimethylbenzyl azide (11j) was used instead of 11a to prepare compound CPU-210, and the yield was 30.9%. mp228～231℃; ¹ HNMR(300MHz,DMSO-d ₆ ): δ12.67(s,1H), 12.41(s,1H), 7.72(s,1H), 7.32(s,5H), 6.96(d, J = 17.0Hz, 3H), 5.48 (s, 2H), 3.80 (s, 2H), 3.01 (s, 6H), 2.81 (s, 2H), 2.21 (d, J = 8.2 Hz, 6H), 1.75 ( s,4H)ppm.HRMS(ESI):m/z,calcd for C ₃₀ H ₃₃ N ₉ O ₂ S ₂ [M+H] ⁺ ,616.2271; found:616.2278.

Preparation of compound CPU-211

As described in the preparation of CPU-201, 2,6-dimethylbenzyl azide (11k) was used instead of 11a to prepare compound CPU-211 with a yield of 38.6%. mp205～210℃; ¹ HNMR(300MHz,DMSO-d ₆ ): δ12.66(s,1H), 12.37(s,1H), 7.57(s,1H), 7.30(s,5H), 7.15-7.08( m, 1H), 7.02 (d, J = 7.2 Hz, 2H), 5.50 (s, 2H), 3.78 (s, 2H), 2.95 (d, J = 22.9 Hz, 6H), 2.77 (s, 2H), 2.26(s,6H)ppm.HRMS(ESI):m/z,calcd for C ₃₀ H ₃₃ N ₉ O ₂ S ₂ [M+H] ⁺ ,616.2271; found: 616.2288.

Example 3

Preparation of general formula compound III (CPU301-CPU310)

Preparation of 5-(3-butynyl)-1,3,4-thiadiazol-2-amine (14)

Thiosemicarbazide (1.39 g, 0.015 mol) was added to 13 (1.5 g, 0.015 mol) of POCl ₃ solution, and the reaction mixture was stirred at 80° C. for 4 hours. After the reaction was completed, it was cooled to room temperature, the mixture was adjusted to pH=9 with 6M NaOH, a solid precipitated out, filtered with suction, and the filter cake was dried; the filtrate was extracted 3 times with ethyl acetate, the organic layers were combined, dried with anhydrous sodium sulfate, and rotated The yellow-brown solid obtained by drying was combined with the above filter cake, and the yield was 88.3%. ¹ HNMR (300MHz, DMSO-d ₆ ): δ 6.99 (s, 2H), 2.95 (t, J = 7.0 Hz, 2H), 2.83 (d, J = 2.4 Hz, 1H), 2.50 (dd, J = 7.1,4.6Hz,2H)ppm.HRMS(ESI):m/z,calcd for C6H7N3S[M+H]+,154.0433; found:154.0434.

Preparation of N-(5-(3-butynyl)-1,3,4-thiadiazole)-2-(2-pyridine)acetamide (15)

Intermediate 14 (3g, 0.02mol) was dissolved in 30mL DMF, then added 2-pyridineacetic acid hydrochloride (3.74g, 0.03mol), HATU (8.8g, 0.039mmol), stirred at room temperature for 15min and then added DIPEA (7.5 g, 0.059mmol). After stirring at room temperature for 2 hours, the reaction was almost complete, and the reaction mixture was poured into water. The aqueous layer was extracted with ethyl acetate (30 mL×3). The organic layer was dried with anhydrous sodium sulfate and spin-dried to obtain a pale yellow solid with a yield of 92.6%. ¹ HNMR (300MHz, DMSO-d ₆ ): δ12.71 (s, 1H), 8.50 (d, J = 4.8 Hz, 1H), 7.78 (td, J = 7.7, 1.7 Hz, 1H), 7.40 (d, J = 7.7Hz, 1H), 7.29 (dd, J = 6.7, 5.0Hz, 1H), 4.02 (s, 2H), 3.16 (t, J = 7.0Hz, 2H), 2.88 (t, J = 2.6Hz, 1H),2.62(td,J＝7.0,2.6Hz,2H)ppm.HRMS(ESI):m/z,calcd for C ₁₃ H ₁₂ N ₄ OS[M+H] ⁺ ,273.0805; found:273.0807.

Preparation of N-(5-(4-(3-aminopyridazine)-3-butynyl)-1,3,4-thiadiazole)-2-(2-pyridine)acetamide (17)

To the DMA (10mL) solution of compound 15 (2g, 7.4mmol) was added 16 (1.35g, 6.11mmol), CuI (0.117g, 0.613mmol), TEA (3.1g, 30.65mmol) and tetrakis (triphenylphosphine) ) Palladium (0.708 g, 0.613 mmol). The mixture was stirred at 60°C for 3.5h under the protection of nitrogen. After cooling to room temperature, the reaction mixture was diluted in 30 mL of isopropyl ether. The lower layer of reddish brown oil was collected by liquid separation, and after adding water, a gray solid was precipitated. The target compound was obtained by column chromatography with a yield of 68.5%. ¹ HNMR (300MHz, DMSO-d ₆ ): δ12.69 (s, 1H), 8.49 (d, J = 4.0 Hz, 1H), 7.76 (t, J = 7.1 Hz, 1H), 7.39 (d, J = 7.8Hz,1H),7.33-7.17(m,2H),6.69(d,J=9.1Hz,1H),6.63(s,2H),4.01(s,2H),3.31-3.23(m,2H), 2.90(t,J＝6.8Hz,2H)ppm.HRMS(ESI):m/z,calcd for C ₁₇ H ₁₅ N ₇ OS[M+H] ⁺ ,366.1132; found: 366.1135.

Preparation of N-(5-(4-(3-aminopyridazine)butyl)-1,3,4-thiadiazole)-2-(2-pyridine)acetamide (18)

Intermediate 17 (1 g, 2.74 mmol) was dissolved in 200 mL of methanol solution, Raney nickel (2 mL) was added, and H _{2 was} passed through, and the mixture was stirred at room temperature for 48 hours. After TLC detection showed that the reaction was completed, the catalyst was removed by filtration. The filtrate was spin-dried to obtain Intermediate 18 as a yellow solid with a yield of 97.2%. ¹ HNMR (300MHz, DMSO-d ₆ ): δ12.67 (s, 1H), 8.50 (d, J = 4.9 Hz, 1H), 7.77 (td, J = 7.7, 1.8 Hz, 1H), 7.40 (d, J=7.8Hz,1H), 7.29(dd,J=6.9,5.3Hz,1H), 7.19(d,J=9.0Hz,1H), 6.76(d,J=9.1Hz,1H), 6.28(s, 2H), 4.01 (s, 2H), 3.00 (t, J = 6.9 Hz, 2H), 2.70 (t, J = 6.9 Hz, 2H), 1.70 (s, 4H) ppm. HRMS (ESI): m/z ,calcd for C ₁₇ H ₁₉ N ₇ OS[M+H] ⁺ ,370.1445; found:370.1451.

Preparation of N-(3-(4-(5-(2-(2-pyridine)acetamido)-1,3,4-thiadiazole)butyl)pyridazine)-4-pentynamide (19)

Intermediate 18 (0.25 g, 0.68 mmol), pentynoic acid (0.073 g, 0.75 mmol), HATU (0.38 g, 1.02 mmol) were dissolved in DMF, and stirred at room temperature for 10 min. DIPEA (0.26g, 2.04mmol) was added to continue the reaction, and the progress of the reaction was confirmed by TLC every five minutes. The reaction was almost complete after 20 minutes. The reaction liquid was poured into water, and solids were separated out, filtered with suction, and the filter cake was dried to obtain a white solid with a yield of 60.4%. ¹ H NMR (300MHz, DMSO-d ₆ ): δ 12.65 (s, 1H), 11.05 (s, 1H), 8.47 (s, 1H), 8.22 (d, 1H), 7.75 (s, 1H), 7.56 (d, 1H), 7.38(d, 2H), 3.98(s, 2H), 2.99(s, 3H), 2.87(s, 3H), 2.77(t, 1H), 2.63(s, 2H), 1.72( s,4H)ppm.HRMS(ESI):m/z,calcd for C ₂₂ H ₂₃ N ₇ O ₂ S[M+H] ⁺ ,450.1712; found: 450.1721.

Preparation of compound CPU-301

Cuprous iodide (0.38mg, 0.002mmol) was dissolved in water, and Intermediate 19 (5mg, 0.01mmol) and 11g (4.89mg, 0.03mmol) were dissolved in DMF and added to the above reaction solution respectively. Microwave, 120°C, 300W, react for 5min, the reaction liquid is blue and turbid. The reaction solution was poured into water, and solids were separated out, filtered with suction, and the filter cake was dried to obtain a crude product. A blue solid was obtained by column chromatography with a yield of 30.1%. mp160～164℃; ¹ H NMR(300MHz,DMSO-d6):δ12.69(s,1H),11.04(s,1H),8.50(s,1H),8.22(d,1H),7.86(s, 1H), 7.78(s, 1H), 7.57(s, 1H), 7.41(d, 1H), 7.29(s, 1H), 7.14(d, 4H), 5.48(s, 2H), 4.01(s, 2H) ),3.02-2.74(m,8H),2.26(s,3H),1.75(s,2H),1.24(s,2H)ppm.HRMS(ESI):m/z,calcd for C ₃₀ H ₃₂ N ₁₀ O ₂ S[M+H] ⁺ ,597.2509; found:597.2487.

Preparation of compound CPU-307

As described in the preparation of CPU-301, the compound CPU-307 was prepared by using 4-methoxybenzyl azide (11h) instead of 11 g to obtain a blue solid. The yield was 32.7%. mp115～120℃; ¹ H NMR(300MHz,DMSO-d6): δ12.68(s,1H), 11.03(s,1H), 8.50(s,1H), 8.21(d,1H), 7.96-7.77( m, 3H), 7.56(d, 1H), 7.41(s, 1H), 7.24(d, 2H), 6.88(d, 2H), 5.45(s, 2H), 4.01(s, 2H), 3.72(s ,3H),3.01-2.75(m,8H),1.74(s,2H),1.23(s,2H)ppm.HRMS(ESI):m/z,calcd for C ₃₀ H ₃₂ N ₁₀ O ₃ S(M +H] ⁺ ,613.2458; found:613.2439.

Preparation of compound CPU-308

As described in the preparation of CPU-301, 3,4-dimethoxybenzyl azide (11i) was used instead of 11g to prepare compound CPU-308 to obtain a blue solid. The yield was 32.2%. mp110～115℃; ¹ H NMR(300MHz,DMSO-d ₆ +D ₂ O):δ8.51(s,1H),8.19(t,1H),7.95-7.77(m,2H),7.56(d, 1H), 7.43 (d, 1H), 7.30 (s, 1H), 6.98-6.81 (m, 3H), 5.43 (s, 2H), 3.70 (s, 6H), 3.00-2.73 (m, 8H), 1.73 (s,2H),1.23(s,2H)ppm.HRMS(ESI):m/z,calcd for C ₃₁ H ₃₄ N ₁₀ O ₄ S[M+H] ⁺ ,643.2563; found:643.2574.

Preparation of compound CPU-309

As described in the preparation of CPU-301, 3,4-dimethylbenzyl azide (11j) was used instead of 11g to prepare compound CPU-309 to obtain a blue solid. The yield was 33.4%. mp110～111℃; ¹ H NMR(300MHz,DMSO-d ₆ +D ₂ O): δ8.49(s,1H),8.16(s,1H),7.71(s,2H),7.56(d,1H) ,7.40(s,1H),7.29(s,1H),7.02-6.91(m,3H),5.47(s,2H),3.00-2.89(m,6H),2.79(s,2H),2.20(t ,6H),1.72(s,2H),1.22(s,2H)ppm.HRMS(ESI):m/z,calcd for C ₃₁ H ₃₄ N ₁₀ O ₂ S[M+H] ⁺ ,611.2665; found: 611.2672.

Preparation of compound CPU-310

As described in the preparation of CPU-301, 2,6-dimethylbenzyl azide (11k) was used instead of 11g to prepare compound CPU-310, and a blue solid was obtained. The yield was 35.5%. mp210～215℃; ¹ H NMR(300MHz,DMSO-d ₆ ): δ7.99(s,2H),7.71(s,2H),7.64(s,2H),7.43(d,2H),7.14(s ,2H),5.53(s,2H),4.00(s,1H),3.02(s,1H),2.89(s,3H),2.77-2.73(m,3H),2.33(s,6H),1.74( s,2H),1.23(s,2H)ppm.HRMS(ESI):m/z,calcd for C ₃₁ H ₃₄ N ₁₀ O ₂ S[M+H] ⁺ ,611.2665; found: 611.2655.

Preparation of compound CPU-311

As described in the preparation of CPU-301, the compound CPU-312 was prepared by using 4-trifluoromethylbenzyl azide (11l) instead of 11g to obtain a blue solid. The yield was 25.5%. mp210～215℃; ¹ H NMR(300MHz,DMSO-d ₆ ):δ12.67(s,1H),11.03(s,1H),8.50(s,1H),8.20(s,1H),7.96(s ,1H), 7.68(s, 3H), 7.42(s, 4H), 7.28(d, 1H), 5.68(s, 2H), 4.02(s, 2H), 2.97(s, 4H), 2.88(d, 4H),1.74(s,4H)ppm.HRMS(ESI):m/z,calcd for C ₃₀ H ₂₉ F ₃ N ₁₀ O ₂ S[M+H] ⁺ ,651.2226; found:651.2188.

Preparation of compound CPU-312

As described in the preparation of CPU-301, the compound CPU-312 was prepared by using 4-trifluoromethoxybenzyl azide (11m) instead of 4-methylbenzyl azide, and a blue solid was obtained. The yield was 37.2%. mp210～215℃; ¹ H NMR(300MHz,DMSO-d ₆ ): δ12.66(s,1H),11.02(s,1H),8.50(s,1H),8.22(d,1H),7.93(s ,1H),7.77(t,1H),7.57(d,1H),7.37-7.31(m,6H),5.60(s,2H),4.01(s,2H),3.02(s,4H),2.93( t,2H),2.82(d,2H),1.75(s,4H)ppm.HRMS(ESI):m/z,calcd for C ₃₀ H ₂₉ F ₃ N ₁₀ O ₃ S[M+H] ⁺ ,667.2175 ;Found:667.2208.

Example 4

Preparation of general formula compound IV (CPU401-CPU412)

Preparation of 1-(4-(5-amino-1,3,4-thiadiazole)amino)piperidine-carboxylic acid tert-butyl ester (23)

Compound 21 (0.5 g, 2.8 mmol), compound 22 (0.556 g, 2.8 mmol) and NaHCO ₃ (0.448 g, 11.2 mmol) were dissolved in 10 mL of ethanol, heated to reflux, stirred, and oil bathed to 80°C. After 4.5 hours of reaction, the reaction was completed as detected by TLC, and the reaction was stopped. The solvent was spin-dried under reduced pressure to obtain a white brown solid. Add water to make a slurry and filter with suction to obtain an off-white solid. The yield was 88%. HRMS(ESI):m/z,calcd for C ₁₂ H ₂₁ N ₅ O ₂ S[M+H] ⁺ ,300.1494; found:300.1492.

Preparation of 1-(4-((5-(2-phenylacetamido)-1,3,4-thiadiazole)amino)piperidine-carboxylic acid tert-butyl ester (24)

Put compound 23 (0.1g, 0.33mmol), phenylacetic acid (0.05g, 0.363mmol), HATU (0.25g, 0.66mmol) in 5mL N,N-dimethylformamide (DMF), stir at room temperature, Light yellow clear solution. After reacting for 15 min, DIPEA (0.128 g, 0.99 mmol) was added. After reacting for 1h, the reaction was complete as detected by TLC. The reaction solution was poured into water and filtered with suction to obtain a white solid with a yield of 87.6%. HRMS(ESI):m/z,calcd for C ₂₀ H ₂₇ N ₅ O ₃ S[M+H] ⁺ ,418.1913; found: 418.1908.

Preparation of 2-phenyl-N-(5-(4-aminopiperidine)-1,3,4-thiadiazole)acetamide (25)

Compound 24 (0.1 g, 0.24 mmol) was dissolved in 1 mL of dichloromethane, and turbidity appeared. Then 1 mL of trifluoroacetic acid was added, and the reaction solution was clear. Stir in a water bath at room temperature. After reacting for 2h, the reaction was completed as detected by TLC. Adjust the pH to 7 with saturated aqueous sodium bicarbonate solution, and a solid precipitated out. A yellow solid was obtained by suction filtration. The yield was 73%. ¹ H NMR (300MHz, DMSO-d ₆ ): δ7.51-7.48 (m, 1H), 7.31 (s, 6H), 6.47 (s, 2H), 3.71 (singlet overlapping with m, 3H), 3.16 (s ,2H),2.89-2.86(m,2H),2.04(s,2H),1.59-1.57(m,2H)ppm.HRMS(ESI):m/z,calcd for C ₁₅ H ₁₉ N ₅ OS(M +H] ⁺ ,318.1389; found:318.1375.

2-Phenyl-N-(N-((1-(5-amino-1,3,4-thiadiazole)piperidine)amino)-1,3,4-thiadiazole)acetamide (26) Preparation

Compound 25 (0.1g, 0.315mmol), 2-amino-5-bromo-1,3,4-thiadiazole (0.06g, 0.315mmol) and sodium bicarbonate (0.05g, 1.26mmol) were dissolved in 5mL ethanol In, heat to reflux. After reacting for 3 hours, the reaction was completed as detected by TLC, and the reaction was stopped. The solvent was spin-dried under reduced pressure, and a solid was precipitated after adding water. Suction filtration, column chromatography to obtain a pink solid. The yield is 40%. ¹ H NMR (300MHz, DMSO-d ₆ ): δ12.19 (s, 1H), 7.38-7.26 (m, 6H), 6.47 (s, 2H), 3.71 (singlet overlapping with m, 3H), 3.61-3.56 (m,2H),3.10-3.03(m,2H),2.03-2.00(m,2H),1.55-1.44(m,2H)ppm.HRMS(ESI):m/z,calcd for C ₁₇ H ₂₀ N ₈ OS ₂ [M+H] ⁺ ,417.1280; found:417.1266.

N-(5-(4-(5-(2-phenylacetylamino)-1,3,4-thiadiazole)amino)piperidine)-1,3,4-thiadiazole)pentan-4- Preparation of alkynamide (27)

Compound 26 (0.05 g, 0.12 mmol), pentynoic acid (0.01 g, 0.12 mmol), and HATU (0.07 g, 0.18 mmol) were placed in a single-necked reaction flask. The reaction solution was orange-pink and clear. Stir at room temperature for 20 min. DIPEA (0.06 g, 0.48 mmol) was added, and stirring was continued at room temperature. After reacting for 1 hour, the reaction was completed by TLC detection, and the reaction was stopped. The reaction liquid was poured into distilled water, and a solid was precipitated. Filter by suction and dry. The yield is 60%. ¹ H NMR (300MHz, DMSO-d ₆ ): δ12.14 (s, 2H), 7.38-7.31 (m, 6H), 3.78-3.71 (m, 5H), 3.24-3.16 (m, 2H), 2.89 ( s,1H),2.81-2.69(m,2H),2.61-2.59(m,2H),2.07-2.04(m,2H),1.54-1.51(m,2H)ppm.HRMS(ESI):m/z ,calcd for C ₂₂ H ₂₄ N ₈ O ₂ S ₂ [M+H] ⁺ ,497.1542; found:497.1548.

Preparation of compound CPU-403

Copper sulfate pentahydrate (0.05g, 0.2016mmol) and sodium ascorbate (0.04g, 0.2016mmol) were dissolved in 2mL of water. The solution first appeared black, and then turned yellow after stirring for a while. Then compound 27 (0.1 g, 0.2016 mmol) and 4-nitrobenzyl azide (11c) (0.07 g, 0.476 mmol) were added. Under nitrogen protection, the reaction was carried out at room temperature. After 3 hours of reaction, TLC detection showed that the reaction was complete. The reaction solution was poured into water, filtered with suction to obtain a crude product, and column chromatography was used to obtain a white final product with a yield of 37.1%. mp250～255℃; HRMS(ESI): m/z, calcd for C ₂₉ H ₃₀ N ₁₂ O ₄ S ₂ [M+H] ⁺ ,675.2033; found: 675.2029.

The preparation of compound CPU-404 was as described in the preparation of CPU-403, using 4-fluorobenzyl azide (11d) instead of 11c to prepare compound CPU-404 to obtain a white solid. The yield was 33.6%. mp218～222℃; ¹ H NMR(300MHz,DMSO-d ₆ ): δ12.19(s,1H),12.04(s,1H),7.88(s,1H),7.41-7.28(m,8H),7.20 -7.14(m,2H),5.54(s,2H),3.79-3.72(m,5H),3.24-3.16(m,2H), 2.93(t,J=12Hz,2H),2.76(t,J= 12Hz,2H),2.11-2.04(m,2H),1.57-1.48(m,2H)ppm.HRMS(ESI):m/z,calcd for C ₂₉ H ₃₀ FN ₁₁ O ₂ S ₂ [M+H] ⁺ ,648.2088; found:648.2079.

Preparation of compound CPU-405

As described in the preparation of CPU-403, the compound CPU-405 was prepared by using 4-chlorobenzyl azide (11e) instead of 11c to obtain a white solid. The yield was 37.5%. mp232～237℃; HRMS(ESI): m/z, calcd for C ₂₉ H ₃₀ ClN ₁₁ O ₂ S ₂ [M+H] ⁺ ,664.1792; found: 664.1796.

The preparation of compound CPU-406 was as described in the preparation of CPU-403, using 4-bromobenzyl azide (11f) instead of 11c to prepare compound CPU-406 to obtain a white solid. The yield was 35.4%. mp258～261℃; ¹ H NMR(300MHz,DMSO-d ₆ ): δ12.18(s,1H), 12.04(s,1H), 7.89(s,1H), 7.54(d,J=9Hz,2H) ,7.35-7.25(m,6H),7.20(d,J=9Hz,2H),5.53(s,2H),3.78-3.71(m,5H),3.24-3.16(m,2H),2.95-2.93( m,2H),2.78-2.74(m,2H),2.11-2.04(m,2H),1.54-1.48(m,2H)ppm.HRMS(ESI):m/z,calcd for C ₂₉ H ₃₀ BrN ₁₁ O ₂ S ₂ [M+H] ⁺ ,710.1267; found:710.1272.

Preparation of compound CPU-407

As described in the preparation of CPU-403, the compound CPU-407 was prepared by using 4-methylbenzyl azide (11 g) instead of 11c to obtain a white solid. The yield was 32.3%. mp250～254℃; 1H NMR (300MHz, DMSO-d6): δ 12.19 (s, 1H), 12.04 (s, 1H), 7.85 (s, 1H), 7.35 to 7.25 (m, 6H), 7.13 (s) ,2H), 5.48(s, 2H), 3.78-3.71(m, 5H), 3.23-3.16(m, 2H), 2.92(s, 2H), 2.75(s, 2H), 2.26(s, 3H), 2.07-2.04(m,2H),1.54-1.50(m,2H)ppm.HRMS(ESI):m/z,calcd for C ₃₀ H ₃₃ N ₁₁ O ₂ S ₂ [M+H] ⁺ ,644.2338; found :644.2322.

Preparation of compound CPU-408

As described in the preparation of CPU-403, the compound CPU-408 was prepared by using 4-methoxybenzyl azide (11h) instead of 11c to obtain a white solid. The yield was 39.2%. mp237～241℃; ¹ H NMR (300MHz, DMSO-d ₆ ): δ 12.19 (s, 1H), 12.04 (s, 1H), 7.83 (s, 1H), 7.35 to 7.28 (m, 6H), 7.26 -7.21 (m, 2H), 6.90-6.87 (m, 2H), 5.45 (s, 2H), 3.78-3.69 (m, 8H), 3.25-3.16 (m, 2H), 2.91 (s, 2H), 2.75 (s,2H),2.07-2.04(m,2H),1.54-1.48(m,2H)ppm.HRMS(ESI):m/z,calcd for C ₃₀ H ₃₃ N ₁₁ O ₃ S ₂ (M+H ] ⁺ ,660.2288; found:660.2268.

Preparation of compound CPU-409

As described in the preparation of CPU-403, using 3,4-dimethoxybenzyl azide (11i) instead of 11c to prepare compound CPU-409, a white solid was obtained. The yield was 33.3%. mp150～155℃; ¹ H NMR(300MHz,DMSO-d ₆ ): δ12.19(s,1H),12.01(s,1H),7.58(s,1H),7.32-7.25(m,6H),7.17 -7.12 (m, 1H), 7.06-7.04 (m, 1H), 5.52 (s, 2H), 3.78-3.71 (m, 5H), 3.24-3.16 (m, 2H), 2.89 (s, 2H), 2.73 (s,2H),2.29(s,6H),2.10-2.04(m,2H),1.54-1.51(m,2H)ppm.HRMS(ESI):m/z,calcd for C ₃₁ H ₃₅ N ₁₁ O ₄ S ₂ [M+H] ⁺ ,690.2393; found:690.2370.

Preparation of compound CPU-410

As described in the preparation of CPU-403, the compound CPU-410 was prepared by using 4-trifluoromethoxybenzyl azide (11m) instead of 11c to obtain a white solid. The yield was 32.5%. mp275～280℃; ¹ H NMR(300MHz,DMSO-d ₆ ):δ7.92(s,1H),7.35-7.25(m,10H),5.60(s,2H),3.78-3.71(m,5H) ,3.24-3.16(m,2H),2.94(s,2H),2.77(s,2H),2.09-1.99(m,2H),1.59-1.51(m,2H)ppm.HRMS(ESI):m/ z,calcd for C ₃₀ H ₃₀ F ₃ N ₁₁ O ₂ S ₂ [M+H] ⁺ ,698.2056; found:698.2042.

Preparation of compound CPU-411

As described in the preparation of CPU-403, 2,6-dimethylbenzyl azide (11k) was used instead of 11c to prepare compound CPU-411 to obtain a white solid. The yield was 38.7%. mp258～263℃; ¹ H NMR(300MHz,DMSO-d ₆ ): δ12.18(s,1H),12.05(s,1H),7.88(s,1H),7.35-7.31(m,6H),6.95 (s, 2H), 6.89 (d, J = 9 Hz, 2H), 6.80 (d, J = 9 Hz, 2H), 5.44 (s, 2H), 3.72-3.71 (m, 11H), 3.23-3.16 (m, 2H),2.91(s,2H),2.76(s,2H),2.07-2.04(m,2H),1.53-1.51(m,2H)ppm.HRMS(ESI):m/z,calcd for C ₃₁ H ₃₅ N ₁₁ O ₃ S ₂ [M+H] ⁺ ,658.2495; found:658.2475.

Preparation of compound CPU-412

As described in the preparation of CPU-403, the compound CPU-412 was prepared by using 4-trifluoromethylbenzyl azide (11l) instead of 11c to obtain a white solid. The yield was 32.6%. mp273～278℃; ¹ H NMR(300MHz,DMSO-d ₆ ): δ12.17(s,1H),12.04(s,1H),7.95(s,1H),7.71-7.69(m,2H),7.43 -7.31(m,8H), 5.68(s, 2H), 3.78-3.71(m, 5H), 3.23-3.19(m, 2H), 2.95(s, 2H), 2.78(s, 2H), 2.08-2.04 (m,2H),1.53-1.51(m,2H)ppm.HRMS(ESI):m/z,calcd for C ₃₀ H ₃₀ F ₃ N ₁₁ O ₃ S ₂ [M+H] ⁺ ,714.2005; found: 714.1987.

Example 5

Preparation of compound VI (CPU601-CPU612) of general formula.

Preparation of tert-butyl N-(1-(3-aminopyridazine)piperidine)carbamate (31)

29 (1 g, 4.5 mmol), 30 (2.72 g, 13.6 mmol) and triethylamine (1.5 mL) were dissolved in n-butanol. The reaction mixture was stirred in a microwave at 400W and 180°C for 1.5h, and the solvent was spin-dried to obtain an oily product. After adding dichloromethane to dissolve, wash twice with water and twice with saturated saline. The organic layer was dried with anhydrous sodium sulfate to obtain a yellow oily product and column chromatography to obtain a yellow-white solid. The yield was 25.7%. ¹ H NMR (300MHz, DMSO-d ₆ ): δ7.13 (d, J = 9.63Hz, 1H), 6.80 (s, 1H), 6.74 (d, J = 9.63 Hz, 1H), 5.60 (s, 2H) ),3.94(d,2H),3.43(singlet overlapping with m,3H),2.80-2.73(m,2H),1.78(d,J=10.74Hz,2H),1.39(s,9H)ppm.HRMS( ESI):m/z,calcd for C ₁₄ H ₂₃ N ₅ O ₂ [M+H] ⁺ ,294.1925; found:294.1925.

Preparation of tert-butyl N-(1-(3-(pent-4-ynamido)pyridazine)piperidine)carbamate (32)

Intermediate 31 (0.2g, 0.68mmol), pentynoic acid (0.073g, 0.75mmol), HATU (0.38g, 1.02mmol) were dissolved in DMF, and stirred at room temperature for 10-15min. DIPEA (0.26g, 2.011mmol) was added, the reaction was continued, and the progress of the reaction was confirmed by thin-layer chromatography every five minutes. The reaction was almost complete after 20 minutes. The reaction liquid was poured into water, and solids were separated out, filtered with suction, and the filter cake was dried to obtain a white solid. The yield was 60.4%. ¹ H NMR (300MHz, DMSO-d ₆ ): δ10.67 (s, 1H), 8.02 (d, J = 9.75 Hz, 1H), 7.35 (d, J = 9.75 Hz, 1H), 6.82 (s, 1H) ), 4.19 (d, 2H), 3.50 (s, 1H), 2.99 (t, J = 13.95 Hz, 2H), 2.78 (s, 1H), 2.62 (t, J = 13.95 Hz, 2H), 2.44-2.42 (m,2H),1.81-1.78(m,2H),1.39(s,11H)ppm.HRMS(ESI):m/z,calcd for C ₁₉ H ₂₇ N ₅ O ₃ [M+H] ⁺ ,374.2187 ;Found:374.2165.

Preparation of N-(6-(4-aminopiperidine)pyridazine)pent-4-ynamide (33)

Intermediate 32 (0.2 g, 0.54 mmol), trifluoroacetic acid (0.2 mL, 2.6 mmol) and dichloromethane were stirred and reacted at room temperature for 1 h. The reaction was stopped and the dichloromethane was removed under reduced pressure. Adjust pH=7 with saturated sodium bicarbonate, a white solid precipitated out, and filtered with suction. Analyzed by thin layer chromatography, the yield was 100%. ¹ H NMR (300MHz, DMSO-d ₆ ): δ10.69 (s, 1H), 8.05 (d, J = 9.81 Hz, 1H), 7.39 (d, J = 9.81 Hz, 1H), 4.30 (d, 2H ), 3.51 (s, 1H), 3.00 (t, J = 5.19 Hz, 2H), 2.92 (s, 1H), 2.77 (t, J = 5.19 Hz, 2H), 2.47-2.44 (m, 2H), 1.97 -1.93(m,2H),1.56(m,2H)ppm.HRMS(ESI):m/z,calcd for C ₁₄ H ₁₉ N ₅ O[M+H] ⁺ ,274.1662; found:274.1655.

Preparation of N-(6-(4-(N-(5-amino)-1,3,4-thiadiazole)amino-piperidine)-pyridazine)pent-4-ynamide (34)

Intermediate 33 (200mg, 7.4mmol), 2-amino-5-bromo-1,3,4-thiadiazole (1320mg, 7.4mmol), NaHCO ₃ (1184mg, 17.6mmol) were dissolved in 0.5mL ethanol, Reflux at 80°C, and the reaction liquid was brown and turbid. After three hours of reaction, the reaction was complete. The ethanol was spin-dried to obtain a white solid, and water was added to make a slurry, and a solid precipitated, and filtered with suction. The filter cake is the product, which is a yellow-brown solid. The yield was 69.7%. HRMS(ESI):m/z,calcd for C ₁₆ H ₂₀ N ₈ O ₂ S[M+H] ⁺ ,373.1554; found:373.1546.

N-(6-(4-(N-(5-(2-pyridine)acetamido)-1,3,4-thiadiazole)amino-piperidine)-pyridazine)pent-4-ynamide (35) Preparation

Intermediate 34 (5mg, 0.013mmol), 2-pyridineacetic acid hydrochloride (2.6mg, 0.015mmol), HATU (7.6mg, 0.02mmol) were dissolved in 1mL DMF, stirred for 10-15min, the solution was pale yellow Clear liquid. DIPEA (5.2mg, 0.04mmol) was added, the reaction was continued for 20 minutes, and the reaction was complete. The reaction liquid was poured into water, and solids were separated out and filtered with suction. The filter cake was a white solid with a yield of 75.8%. ¹ H NMR (300MHz, DMSO-d ₆ ): δ12.14 (s, 1H), 10.68 (s, 1H), 8.50 (d, J = 4.11 Hz, 1H), 8.04-7.79 (m, 1H), 7.78 -7.73(m,1H),7.38-7.26(m,4H),4.17(d,J=13.62Hz,2H),3.92(s,2H),3.79(s,1H),3.08(t,J=7.05 Hz, 2H), 2.78 (s, 1H), 2.63 (t, J = 13.86 Hz, 2H), 2.47-2.44 (m, 2H), 1.97-1.93 (m, 2H), 1.56 (m, 2H) ppm. HRMS(ESI):m/z,calcd for C ₂₃ H ₂₅ N ₉ O ₂ S[M+H] ⁺ ,492.1925; found:492.1919.

Preparation of compound CPU-603

Cuprous iodide (0.38mg, 0.002mmol) was dissolved in water and added to the DMF mixture of Intermediate 35 (5mg, 0.01mmol) and 4-nitrobenzylazide (11c) (3.99mg, 0.03mmol). Microwave, 120°C, 300W, reaction for 5min. The reaction liquid was blue and turbid, and a gray solid precipitated out when poured into water. Purified by column chromatography to obtain a blue solid. The yield was 33.7%. mp243～247℃; HRMS(ESI): m/z,calcd for C ₃₀ H ₃₁ N ₁₃ O ₄ S[M+H] ⁺ ,670.2421; found: 670.2433.

Preparation of compound CPU-604

As described in the preparation of CPU-603, the compound CPU-604 was prepared by using 4-fluorobenzyl azide (11d) instead of 11c to obtain a blue solid. The yield was 32.7%. mp232～235℃; ¹ H NMR(300MHz,DMSO-d ₆ ): δ12.21(s,1H), 10.71(s,1H), 8.49(s,1H), 8.09(s,1H), 8.02-7.90 (m,1H),7.78-7.75(m,1H),7.39-7.37(m,1H),7.35-7.27(m,5H),7.19-7.14(m,2H),5.54(s,2H),4.17 (d, 2H), 3.93 (s, 2H), 3.79 (s1H), 3.09 (t, J = 8.19 Hz, 2H), 2.92 (t, J = 5.25 Hz, 2H), 2.74 (t, J = 5.25 Hz ,2H),2.07(d,J＝7.98Hz,2H),1.49(d,J＝7.98Hz,2H)ppm.HRMS(ESI):m/z,calcd for C ₃₀ H ₃₁ FN ₁₂ O ₂ S[ M+H] ⁺ ,643.2470; found:643.2466.

Preparation of compound CPU-605

As described in the preparation of CPU-603, the compound CPU-605 was prepared using 4-chlorobenzyl azide (11e) instead of 11c as a blue solid. The yield was 33.8%. mp240～241℃; ¹ H NMR(300MHz,DMSO-d ₆ ): δ12.21(s,1H),10.69(s,1H),8.50(s,1H),8.01(s,1H),7.91(s ,1H),7.79(t,J=11.25Hz,1H),7.40-7.38(m,5H),7.28-7.26(m,3H),5.55(s,2H),4.17(d,2H),3.93( s, 2H), 3.80 (s1H), 3.10 (t, J = 8.61 Hz, 2H), 2.74 (s, 2H), 2.59 (s, 2H), 2.07 (d, J = 7.68 Hz, 2H), 1.49 ( d,J=7.68Hz,2H)ppm.HRMS(ESI):m/z,calcd for C ₃₀ H ₃₁ ClN ₁₂ O ₂ S[M+H] ⁺ ,659.2175; found: 659.2221.

Preparation of compound CPU-606

As described in the preparation of CPU-603, the compound CPU-606 was prepared using 4-bromobenzyl azide (11f) instead of 11c as a blue solid. The yield was 34.8%. mp240～245℃; HRMS(ESI): m/z, calcd for C ₃₀ H ₃₁ BrN ₁₂ O ₂ S[M+H] ⁺ ,703.1670; found: 703.1669.

Preparation of compound CPU-607

As described in the preparation of CPU-603, the compound CPU-607 was prepared by using 4-methoxybenzyl azide (11h) instead of 11c to obtain a blue solid. The yield was 31.6%. mp148～152℃; ¹ H NMR(300MHz,DMSO-d ₆ ): δ12.23(s,1H), 10.70(s,1H), 8.54(d,J=8.43Hz,1H), 8.03(m,1H) ), 7.85-7.78 (m, 2H), 7.40-7.22 (m, 6H), 6.89 (d, J = 5.25 Hz, 2H), 5.45 (s, 2H), 4.16 (d, J = 6.96 Hz, 2H) ,3.93(s,2H),3.79-3.72(singlet overlapping with m,4H),3.10(t,2H),2.91(s,1H),2.73(t,2H),2.07(d,2H),1.47( m,2H)ppm.HRMS(ESI):m/z,calcd for C ₃₁ H ₃₄ N ₁₂ O ₃ S[M+H] ⁺ ,655.2670; found:655.2672.

Preparation of compound CPU-608

As described in the preparation of CPU-603, using 3,4-dimethoxybenzyl azide (11i) instead of 11c to prepare compound CPU-608, a blue solid was obtained. The yield was 33.9%. mp108～111℃; ¹ H NMR(300MHz,DMSO-d ₆ ): δ12.25(s,1H), 10.73(s,1H), 8.53(s,1H), 8.10-8.03(m,1H), 7.88 (s, 1H), 7.82 (s, 1H), 7.42 (m, 3H), 7.32 (s, 1H), 6.99 (s, 1H), 6.91 (d, J = 8.16 Hz, 1H), 6.83 (d, J = 8.16 Hz, 1H), 5.46 (s, 2H), 4.20 (d, J = 6.96 Hz, 2H), 3.95 (s, 2H), 3.74 (singlet overlapping with m, 7H), 3.15 (t, J = 12.03Hz,2H),2.94(s,2H),2.76(t,2H),2.11(d,2H),1.49(m,2H)ppm.HRMS(ESI):m/z,calcd for C ₃₂ H ₃₆ N ₁₂ O ₄ S[M+H] ⁺ ,685.2776; found:685.2775.

Preparation of compound CPU-609

As described in the preparation of CPU-603, the compound CPU-609 was prepared by using 4-methylbenzyl azide (11 g) instead of 11c to obtain a blue solid. The yield was 38.5%. mp219～222℃; ¹ H NMR(300MHz,DMSO-d ₆ ): δ12.21(s,1H), 10.69(s,1H), 8.50(s,1H), 8.01(m,1H),7.85-7.75 (m, 2H), 7.38-7.37 (m, 4H), 7.14 (s, 4H), 5.48 (s, 2H), 4.15 (d, J = 9.27 Hz, 2H), 3.93 (s, 2H), 3.82 ( s1H), 3.13(s, 2H), 2.92(s, 2H), 2.73(s, 2H), 2.26(s, 3H), 2.06(d, 2H), 1.46(m, 2H) ppm.HRMS(ESI) :m/z,calcd for C ₃₁ H ₃₄ N ₁₂ O ₂ S[M+H] ⁺ ,639.2721; found:639.2716.

Preparation of compound CPU-610

As described in the preparation of CPU-603, 2,6-dimethylbenzyl azide (11k) was used instead of 11c to prepare compound CPU-610 to obtain a blue solid. The yield was 39.1%. mp161～165℃; ¹ H NMR(300MHz,DMSO-d ₆ ): δ12.18(s,1H), 10.63(s,1H), 8.50(s,1H), 7.98(s,1H), 7.77(s ,1H),7.59(s,1H),7.37-7.30(m,3H),7.14-7.06(m,4H),5.53(s,2H),4.17(d,J＝8.70Hz,2H),3.93( s, 2H), 3.81 (s1H), 3.17 (s, 2H), 2.89 (s, 2H), 2.71 (s, 2H), 2.30 (s, 6H), 2.09 (d, 2H), 1.48 (m, 2H) )ppm.HRMS(ESI):m/z,calcd for C ₃₂ H ₃₆ N ₁₂ O ₂ S[M+H] ⁺ ,653.2878; found:653.2807.

Preparation of compound CPU-611

As described in the preparation of CPU-603, the compound CPU-611 was prepared by using 4-trifluoromethylbenzyl azide (11l) instead of 11c to obtain a blue solid. The yield was 35.4%. mp230～233℃; HRMS(ESI): m/z, calcd for C ₃₁ H ₃₁ F ₃ N ₁₂ O ₂ S[M+H] ⁺ ,693.2439; found: 693.2434.

Preparation of compound CPU-612

As described in the preparation of CPU-603, the compound CPU-612 was prepared by using 4-trifluoromethoxybenzyl azide (11m) instead of 11c to obtain a blue solid. The yield was 36.9%. mp245～250℃; ¹ H NMR(300MHz,DMSO-d ₆ ): δ12.21(s,1H), 10.69(s,1H), 8.50(s,1H), 8.01(m,2H), 7.77-7.75 (m,J=5.25Hz,1H), 7.37-7.35(m,8H), 5.60(s, 2H), 4.17(d, 2H), 3.92(s, 2H), 3.79(s1H), 3.12-3.06( m, 2H), 2.93 (s, 2H), 2.74 (s, 2H), 2.07 (d, J = 7.56 Hz, 2H), 1.47 (d, J = 7.56 Hz, 2H) ppm. HRMS (ESI): m /z,calcd for C ₃₁ H ₃₁ F ₃ N ₁₂ O ₃ S[M+H] ⁺ ,709.2388; found:709.2410.

Example 6

Preparation of general formula compound VIII (CPU801-CPU812).

Preparation of tert-butyl N-((3-amino-pyridazine)pyrrolidine)carbamate (38)

Compound 38 (0.1g, 0.36mmol), pentynoic acid (0.038g, 0.39mmol), HATU (0.2g, 0.54mmol) were dissolved in DMF and stirred. Then DIPEA (0.14g, 1.08mmol) was added and stirred. TLC detection. The reaction liquid was poured into water to separate out a solid, which was filtered with suction to obtain a white solid, which was dried. The yield was 83.4%. HRMS(ESI):m/z,calcd for C ₁₈ H ₂₆ N ₅ O ₃ [M+H] ⁺ ,360.2036; found: 360.2034.

Preparation of N-(6-(3-amino)-pyrrolidine)pyridazine-pent-4-ynamide (40)

Compound 39 (0.5 g, 1.39 mmol) was dissolved in 5 mL of dichloromethane, 5 mL of CF ₃ COOH was added, and the reaction was carried out at room temperature for 4 hours. The progress of the reaction was detected by TLC. The DCM was spin-dried under reduced pressure, the _{pH was adjusted to 7-8 with saturated NaHCO 3} , an off-white solid was precipitated, and the off-white solid was obtained by suction filtration, and the yield was 75%. HRMS(ESI):m/z,calcd for C ₁₃ H ₁₈ N ₅ O[M+H] ⁺ ,260.1511; found:260.1513.

Preparation of N-(6-(3-(N-(5-amino)1,3,4thiadiazole)amino)-pyrrolidine)pyridazine-pent-4-ynamide (41)

Compound 40 (10 mg, 0.039 mmol), thiadiazole (6.95 mg, 0.039 mmol) and NaHCO ₃ (6.24 mg, 0.156 mmol) were dissolved in 2 mL ethanol, heated at 80°C and refluxed for 4 hours, and the reaction progress was detected by TLC. After the completion of the reaction, the solvent was spin-dried under reduced pressure to obtain a yellow-brown solid, which was slurried with 5 mL of water, filtered with suction to obtain a pale yellow solid, and dried. The yield was 92%. HRMS(ESI):m/z,calcd for C ₁₅ H ₁₈ N ₈ OS[M+H] ⁺ ,359.1403; found:359.1413.

N-(6-(3-(N-(5-(2-piperidine)acetamido)1,3,4thiadiazole)amino)-pyrrolidine)pyridazine-pent-4-ynamide (42 ) Preparation

Compound 41 (0.1g, 0.28mmol) and HATU (0.16g, 0.42mmol), 2-pyridineacetic acid hydrochloride (0.054g, 0.31mmol) were dissolved in 2mL DMF, stirred for 2min, added DIPEA (0.11g, 0.85 mmol) reaction for 10 min, TLC detection. The reaction solution was poured into water, a solid was precipitated, and a light yellow solid was obtained by suction filtration, and the yield was 54.5%. HRMS(ESI):m/z,calcd for C ₂₂ H ₂₃ N ₉ O ₂ S[M+H] ⁺ ,478.1774; found:478.1768.

Preparation of compound CPU-804

To the DMF solution of compound 42 (0.1 g, 0.21 mmol) was added an aqueous solution of CuI (7.78 mg, 0.041 mmol) and 4-fluorobenzyl azide (11f) (55.85 mg, 0.42 mmol). The reaction mixture was stirred in a microwave at 300W, 120°C for 2 min, and detected by TLC. The reaction liquid was poured into water, and a solid was precipitated. The crude product was obtained by suction filtration, and then chromatographed by column chromatography (DCM/MeOH=20:1) to obtain a blue solid. The yield was 35.5%. mp210～212℃; HRMS(ESI): m/z, calcd for C ₂₉ H ₂₉ FN ₁₂ O ₂ S[M+H] ⁺ ,629.2319; found: 629.2317.

Preparation of compound CPU-805

As described in the preparation of CPU-804, the compound CPU-805 was prepared by using 4-chlorobenzyl azide (11e) instead of 11f to obtain a blue solid. The yield was 31.1%. mp233～235℃; HRMS(ESI): m/z, calcd for C ₂₉ H ₂₉ ClN ₁₂ O ₂ S[M+H] ⁺ ,645.2024; found:645.1999.

Preparation of compound CPU-807

As described in the preparation of CPU-804, the compound CPU-807 was prepared by using 4-methylbenzyl azide (11 g) instead of 11f to obtain a blue solid. The yield was 34.7%. mp242～247℃; HRMS(ESI):m/z,calcd for C ₃₀ H ₃₂ N ₁₂ O ₂ S[M+H] ⁺ ,625.2570; found:625.2562.

Preparation of compound CPU-808

As described in the preparation of CPU-804, the compound CPU-808 was prepared by using 4-methoxybenzyl azide (11h) instead of 11f to obtain a blue solid. The yield was 32.5%. mp221～222℃; HRMS(ESI): m/z,calcd for C ₃₀ H ₃₂ N ₁₂ O ₃ S[M+H] ⁺ ,641.2519; found: 641.2512.

Preparation of compound CPU-809

As described in the preparation of CPU-804, using 3,4-dimethoxybenzyl azide (11i) instead of 11f to prepare compound CPU-809, a blue solid was obtained. The yield was 30.8%. mp219～220℃; HRMS(ESI): m/z, calcd for C ₃₁ H ₃₄ N ₁₂ O ₄ S[M+H] ⁺ ,671.2625; found: 671.2612.

Preparation of compound CPU-810

As described in the preparation of CPU-804, the compound CPU-810 was prepared by using 4-trifluoromethoxybenzyl azide (11m) instead of 11f to obtain a blue solid. The yield was 36.7%. mp243～247℃; HRMS(ESI): m/z, calcd for C ₃₀ H ₂₉ F ₃ N ₁₂ O ₃ S[M+H] ⁺ ,695.2237; found: 695.2239.

Preparation of compound CPU-811

As described in the preparation of CPU-804, 2,6-dimethylbenzyl azide (11k) was used instead of 11f to prepare compound CPU-811 to obtain a blue solid. The yield was 37.2%. mp256～258℃; HRMS(ESI): m/z, calcd for C ₃₁ H ₃₄ N ₁₂ O ₂ S[M+H] ⁺ ,639.2727; found: 639.2724.

Preparation of compound CPU-812

As described in the preparation of CPU-804, the compound CPU-812 was prepared by using 4-trifluoromethylbenzyl azide (11l) instead of 11f to obtain a blue solid. The yield was 33.3%. mp219～221℃; HRMS(ESI): m/z, calcd for C ₃₀ H ₂₉ F ₃ N ₁₂ O ₂ S[M+H] ⁺ ,679.2287; found: 679.2276.

Example 7

Preparation of general formula compound X (CPU1001-CPU1012)

Preparation of tert-butyl N-((3-amino-pyridazine)pyrrolidine)carbamate (45)

29 (0.1g, 0.45mmol) and 44 (0.17g, 0.9mmol) were dissolved in n-butanol, and 0.2mL of triethylamine was added. The reaction mixture was stirred in a microwave at 400W, 180°C for 1.5h. Rotate the solvent to dryness, add dichloromethane to dissolve, wash three times with water, wash three times with saturated ammonium chloride, and dry with anhydrous sodium sulfate. The crude oil is obtained by column chromatography to obtain a white solid with a yield of 37%. HRMS(ESI):m/z,calcd for C ₁₃ H ₂₂ N ₅ O ₂ [M+H] ⁺ ,280.1773; found:280.1777.

Preparation of tert-butyl N-((3-(pent-4-ynamido)-pyridazine)pyrrolidine)carbamate (46)

Compound 45 (0.1g, 0.36mmol), pentynoic acid (0.038g, 0.39mmol), HATU (0.2g, 0.54mmol) were dissolved in DMF and stirred. After 20min, DIPEA (0.14g, 1.08mmol) was added and reacted at room temperature for 1h. TLC detection. The reaction liquid was poured into water to separate out a solid, which was filtered with suction to obtain a white solid, which was dried. The yield was 83.4%. HRMS(ESI):m/z,calcd for C ₁₈ H ₂₆ N ₅ O ₃ [M+H] ⁺ ,360.2036; found: 360.2034.

Preparation of N-(6-(3-amino)-pyrrolidine)pyridazine-pent-4-ynamide (47)

Compound 46 (0.5 g, 1.39 mmol) was dissolved in 5 mL of dichloromethane, 5 mL of CF ₃ COOH was added, and the reaction was carried out at room temperature for 4 hours, and the reaction progress was detected by TLC. The DCM was spin-dried under reduced pressure, the _{pH was adjusted to 7-8 with saturated NaHCO 3} , an off-white solid was precipitated, and the off-white solid was obtained by suction filtration, and the yield was 75%. HRMS(ESI):m/z,calcd for C ₁₃ H ₁₈ N ₅ O[M+H] ⁺ ,260.1511; found:260.1513.

Preparation of N-(6-(3-(N-(5-amino)1,3,4thiadiazole)amino)-pyrrolidine)pyridazine-pent-4-ynamide (48)

Compound 47 (10 mg, 0.039 mmol), thiadiazole (6.95 mg, 0.039 mmol) and NaHCO ₃ (6.24 mg, 0.156 mmol) were dissolved in 2 mL of ethanol, heated at 80°C and refluxed for 4 hours, and the reaction progress was detected by TLC. After the completion of the reaction, the solvent was spin-dried under reduced pressure to obtain a yellow-brown solid, which was slurried with 5 mL of water, filtered with suction to obtain a pale yellow solid, and dried. The yield was 92%. HRMS(ESI):m/z,calcd for C ₁₅ H ₁₈ N ₈ OS[M+H] ⁺ ,359.1403; found:359.1413.

N-(6-(3-(N-(5-(2-piperidine)acetamido)1,3,4thiadiazole)amino)-pyrrolidine)pyridazine-pent-4-ynamide (49 ) Preparation

Compound 48 (0.1g, 0.28mmol) and HATU (0.16g, 0.42mmol), 2-pyridineacetic acid hydrochloride (0.054g, 0.31mmol) were dissolved in 2mL DMF, stirred for 20min, added DIPEA (0.11g, 0.85 mmol) reaction for 10 min, TLC detection. The reaction solution was poured into water, a solid was precipitated, and a light yellow solid was obtained by suction filtration, and the yield was 54.5%. HRMS(ESI):m/z,calcd for C ₂₂ H ₂₃ N ₉ O ₂ S[M+H] ⁺ ,478.1774; found:478.1768.

Preparation of compound CPU-1001

To the DMF solution of compound 49 (0.1 g, 0.21 mmol) was added an aqueous solution of CuI (7.78 mg, 0.041 mmol) and 4-methylbenzyl azide (11 g) (55.85 mg, 0.42 mmol). The reaction mixture was stirred in a microwave at 300W, 120°C for 2 min, and detected by TLC. The reaction liquid was poured into water, and a solid was precipitated. The crude product was obtained by suction filtration, and then chromatographed by column chromatography (DCM/MeOH=20:1) to obtain a blue solid. The yield was 33.5%. mp208～212℃; HRMS(ESI):m/z,calcd for C ₃₀ H ₃₂ N ₁₂ O ₂ S[M+H] ⁺ ,625.2570; found:625.2562.

Preparation of compound CPU-1002

As described in the preparation of CPU-1001, the compound CPU-1002 was prepared by using 4-methoxybenzyl azide (11h) instead of 11 g to obtain a white solid. The yield was 41.3%. mp219～222℃; HRMS(ESI): m/z,calcd for C ₃₀ H ₃₂ N ₁₂ O ₃ S[M+H] ⁺ ,641.2519; found: 641.2512.

Preparation of compound CPU-1003

As described in the preparation of CPU-1001, the compound CPU-1003 was prepared by using 4-trifluoromethylbenzyl azide (11 l) instead of 11 g to obtain a white solid. The yield was 37.6%. mp194～198℃; HRMS(ESI): m/z, calcd for C ₃₀ H ₂₉ F ₃ N ₁₂ O ₂ S[M+H] ⁺ ,679.2287; found: 679.2276.

Preparation of compound CPU-1004

As described in the preparation of CPU-1001, the compound CPU-1004 was prepared by using 4-trifluoromethoxybenzyl azide (11m) instead of 11 g to obtain a white solid. The yield was 39.0%. mp199～204℃; HRMS(ESI): m/z, calcd for C ₃₀ H ₂₉ F ₃ N ₁₂ O ₃ S[M+H] ⁺ ,695.2237; found: 695.2239.

Example 8

Preparation of compound XII (CPU1201-CPU1212) of general formula.

Preparation of compound CPU-1204

To the DMF solution of compound 34 (0.1 g, 0.26 mmol) was added an aqueous solution of CuI (7.78 mg, 0.041 mmol) and 4-fluorobenzyl azide (11d) (55.85 mg, 0.42 mmol). The reaction mixture was stirred in a microwave at 300W, 120°C for 5 min, and detected by TLC. The reaction liquid was poured into water, and a solid was precipitated. The crude product was obtained by suction filtration, and then chromatographed by column chromatography (DCM/MeOH=20:1) to obtain a white solid. The yield was 35.5%. mp178～180℃; HRMS(ESI):m/z,calcd for C ₂₃ H ₂₆ FN ₁₁ OS[M+H] ⁺ ,524.2105; found:524.2098.

Preparation of compound CPU-1205

As described in the preparation of CPU-1204, the compound CPU-1205 was prepared by using 4-chlorobenzyl azide (11e) instead of 11d to obtain a white solid. The yield was 37.3%. mp200～203℃; HRMS(ESI): m/z,calcd for C ₂₃ H ₂₆ ClN ₁₁ OS[M+H] ⁺ ,540.1809; found: 540.1830.

Preparation of compound CPU-1206

As described in the preparation of CPU-1204, the compound CPU-1206 was prepared by using 4-bromobenzyl azide (11f) instead of 11d to obtain a white solid. The yield was 32.1%. mp175～177℃; HRMS(ESI):m/z,calcd for C ₂₃ H ₂₆ BrN ₁₁ OS[M+H] ⁺ ,584.1304; found:584.1297.

Preparation of compound CPU-1207

As described in the preparation of CPU-1204, the compound CPU-1207 was prepared by using 4-methylbenzyl azide (11 g) instead of 11d to obtain a white solid. The yield was 37.2%. mp200～205℃; HRMS(ESI):m/z,calcd for C ₂₄ H ₂₉ N ₁₁ OS[M+H] ⁺ ,520.2356; found:520.2376.

Preparation of compound CPU-1208

As described in the preparation of CPU-1204, the compound CPU-1208 was prepared by using 4-methoxybenzyl azide (11h) instead of 11d to obtain a white solid. The yield was 34.6%. mp210～212℃; HRMS(ESI): m/z, calcd for C ₂₄ H ₂₉ N ₁₁ O ₂ S[M+H] ⁺ ,536.2305; found: 536.2299.

Preparation of compound CPU-1209

As described in the preparation of CPU-1204, 3,4-dimethoxybenzyl azide (11i) was used instead of 11d to prepare compound CPU-1209, and a white solid was obtained. The yield was 39.5%. mp200～202℃; HRMS(ESI): m/z,calcd for C ₂₅ H ₃₁ N ₁₁ O ₃ S[M+H] ⁺ ,566.2410; found: 566.2403.

Preparation of compound CPU-1210

As described in the preparation of CPU-1204, the compound CPU-1210 was prepared by using 4-trifluoromethoxybenzyl azide (11m) instead of 11d to obtain a white solid. The yield was 34.4%. mp210～213℃; HRMS(ESI): m/z, calcd for C ₂₄ H ₂₇ F ₃ N ₁₁ O ₂ S[M+H] ⁺ ,590.2022; found: 590.2010.

Preparation of compound CPU-1212

As described in the preparation of CPU-1204, the compound CPU-1212 was prepared by using 4-trifluoromethylbenzyl azide (11 l) instead of 11 g to obtain a white solid. The yield was 37.1%. mp175～180℃; HRMS(ESI):m/z,calcd for C ₂₄ H ₂₇ F ₃ N ₁₁ OS[M+H] ⁺ ,574.2073; found:574.2079.:

Example 9: Pharmacological tests and results of some compounds of the present invention:

(1) In vitro inhibition test of the compound on tumor cell proliferation

Test purpose: to observe the inhibitory effect of test compounds on tumor cell proliferation. Mechanism studies have shown that triple-negative breast cancer MDA-MB-436 cells and colon cancer HCT116 cells are highly sensitive to glutamine, relying too much on glutamine to maintain cell growth and reproduction, showing "glutamine addiction." At the same time, the tumor gene map showed that the glutaminase GLS1 gene and protein levels of MDA-MB-436 and HCT116 cells were highly expressed, and GLS1 showed strong enzymatic activity. Therefore, the compound's inhibitory effect on the proliferation of these two cell lines can reflect that the designed compound achieves the anti-tumor cell proliferation effect by inhibiting GLS1.

Test principle: MTT analysis method uses living cell metabolite reducing agent MTT (full name 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide, trade name: Thiazole Blue ) As the basis. MTT is a yellow compound and a dye that accepts hydrogen ions. It can act on the respiratory chain in the mitochondria of living cells. Under the action of succinate dehydrogenase and cytochrome C, the tetrazolium ring is cracked to produce blue formazan crystals, formazan The amount of crystals produced is only proportional to the number of living cells (the succinate dehydrogenase disappears when the cells die, and MTT cannot be reduced). The reduced formazan crystals can be dissolved in DMSO, and the optical density OD value at 492nm is measured with a microplate reader to reflect the number of living cells.

Test method: 1) Inoculation of cells: After the cells grow to logarithmic growth phase in a culture medium containing 10% FBS, they are digested with trypsin and prepared into a single cell suspension, respectively, with 6000 HCT116 or 4000 MDA- per well. MB-436 cells were seeded into 96-well plates; 2) Dosing: _{After culturing at 37°C and 5% CO 2 for} 24 hours, dissolve the test compound with DMSO and dilute the dissolved compound to 0.1M/L with culture solution, respectively Concentration gradients of 10nM, 100nM, 1μM, 10μM, 100μM were administered, and a blank group and a solvent control group were set; 3) 37°C, 5% CO ₂ continued to incubate for 72 hours; 4) Color: add MTT solution (5mg/ ml) 20μl, continue to incubate for 4 hours, carefully aspirate and discard the culture supernatant in the well. Add 150μl DMSO to each well and shake for 10 minutes to fully dissolve the crystals; 5) Colorimetry: select a wavelength of 492nm, measure the light absorption value of each well on an enzyme-linked immunosorbent monitor, record the result, calculate the growth inhibition rate, and plot the growth inhibition curve.

Table 1. Some compounds of the present invention inhibit the proliferation of tumor cells (MDA-MB-436, HCT116) test results

化合物编号Compound number	IC ₅₀/μM(MDA-MB-436) IC ₅₀ /μM(MDA-MB-436)	IC ₅₀/μM(HCT116) IC ₅₀ /μM(HCT116)
CPU-101CPU-101	2.262.26	4.454.45
CPU-102CPU-102	16.416.4	13.613.6
CPU-103CPU-103	29.329.3	19.419.4
CPU-104CPU-104	1.061.06	5.705.70
CPU-105CPU-105	9.449.44	N.A.N.A.
CPU-106CPU-106	1.701.70	17.717.7
CPU-107CPU-107	3.463.46	3.513.51
CPU-201CPU-201	1.161.16	4.784.78
CPU-202CPU-202	2.952.95	5.145.14
CPU-203CPU-203	10.6710.67	1.051.05
CPU-204CPU-204	0.350.35	1.091.09
CPU-205CPU-205	1.11.1	3.413.41
CPU-206CPU-206	4.064.06	1.851.85
CPU-207CPU-207	1.791.79	1.571.57
CPU-301CPU-301	0.180.18	0.150.15
CPU-307CPU-307	0.630.63	0.230.23
CPU-308CPU-308	0.400.40	0.380.38
CPU-309CPU-309	0.200.20	0.720.72
CPU-310CPU-310	0.340.34	0.270.27
CPU-403CPU-403	2.02.0	0.810.81
CPU-404CPU-404	6.56.5	1.321.32
CPU-405CPU-405	8.68.6	2.402.40
CPU-406CPU-406	4.24.2	1.941.94
CPU-407CPU-407	2.22.2	1.021.02
CPU-408CPU-408	1.571.57	0.830.83
CPU-409CPU-409	1.581.58	0.960.96
CPU-410CPU-410	24.424.4	14.114.1
CPU-411CPU-411	0.210.21	0.920.92
CPU-412CPU-412	1.071.07	1.31.3
CPU-603CPU-603	2.832.83	7.257.25
CPU-604CPU-604	3.083.08	1.151.15
CPU-605CPU-605	1.421.42	1.021.02
CPU-606CPU-606	1.191.19	0.560.56
CPU-607CPU-607	2.042.04	1.451.45
CPU-608CPU-608	2.262.26	2.752.75
CPU-609CPU-609	0.470.47	0.570.57
CPU-610CPU-610	1.031.03	1.241.24
CPU-611CPU-611	1.021.02	1.241.24
CPU-612CPU-612	0.840.84	1.061.06
CPU-807CPU-807	1.141.14	0.680.68

CPU-808CPU-808	1.541.54	0.490.49
CPU-812CPU-812	1.731.73	0.790.79
CPU-1001CPU-1001	0.890.89	0.460.46
CPU-1002CPU-1002	0.380.38	0.650.65
CPU-1003CPU-1003	0.320.32	0.600.60
CPU-1004CPU-1004	2.502.50	1.391.39
CPU-1005CPU-1005	0.910.91	0.300.30
CPU-1006CPU-1006	0.420.42	0.510.51
CPU-1007CPU-1007	1.031.03	0.240.24

(2) In vitro inhibitory effect test of the compound on glutamine hydrolase GLS1

Test purpose: to confirm whether the test compound affects the growth and reproduction of tumor cells by acting on GLS1, thereby blocking glutamine metabolism.

Test procedure: human GLS1 protein (0.1mMhKGA) and a certain concentration of compound were incubated with 50mM Tris-Acetate pH=8.6 and 0.2mM EDTA at 25°C for 10 min. Then, 200mM glutamine was added to start the first step of the reaction, and the reaction was carried out at 37°C for 60 minutes. The reaction was quenched by adding 0.6M HCl. Then add 3.7units GDH, 160mM Tris-Acetate pH=9.4, 400mM hydrazine, 5mM ADP, 2mM NAD ⁺ , and incubate at 25℃ for 30min. Finally, detect the absorbance value of the sample at 340nm.

Table 2 Test results of the inhibitory effect of some compounds of the present invention on glutamine hydrolase GLS1

化合物编号Compound number	IC ₅₀/μM(GLS1) IC ₅₀ /μM(GLS1)
CPU-104CPU-104	2.462.46
CPU-105CPU-105	27.0427.04
CPU-107CPU-107	1.571.57
CPU-108CPU-108	4.574.57
CPU-109CPU-109	0.810.81
CPU-112CPU-112	1.761.76
CPU-117CPU-117	6.016.01
CPU-201CPU-201	0.710.71
CPU-202CPU-202	0.670.67
CPU-203CPU-203	1.971.97
CPU-204CPU-204	0.880.88
CPU-205CPU-205	0.990.99
CPU-206CPU-206	0.630.63
CPU-207CPU-207	0.330.33
CPU-301CPU-301	0.0540.054
CPU-307CPU-307	0.320.32
CPU-308CPU-308	0.660.66
CPU-309CPU-309	0.220.22
CPU-310CPU-310	0.650.65
CPU-403CPU-403	0.0400.040
CPU-404CPU-404	0.0160.016
CPU-405CPU-405	0.0320.032
CPU-406CPU-406	0.0160.016
CPU-407CPU-407	0.0120.012

CPU-408CPU-408	0.00810.0081
CPU-409CPU-409	0.0100.010
CPU-410CPU-410	0.0160.016
CPU-411CPU-411	0.00830.0083
CPU-412CPU-412	0.0140.014
CPU-603CPU-603	0.0390.039
CPU-604CPU-604	0.0120.012
CPU-605CPU-605	0.0090.009
CPU-606CPU-606	0.000770.00077
CPU-607CPU-607	0.0230.023
CPU-608CPU-608	0.0570.057
CPU-609CPU-609	0.0190.019
CPU-610CPU-610	0.0120.012
CPU-611CPU-611	0.0170.017
CPU612CPU612	0.0160.016
CPU-801CPU-801	0.0380.038
CPU-802CPU-802	0.0490.049
CPU-803CPU-803	0.0260.026
CPU-804CPU-804	0.0410.041
CPU-805CPU-805	0.0430.043
CPU-806CPU-806	0.0450.045
CPU-807CPU-807	0.00830.0083
CPU-808CPU-808	0.0110.011
CPU-809CPU-809	0.0240.024
CPU-810CPU-810	0.0130.013
CPU-811CPU-811	0.0110.011
CPU-812CPU-812	0.00990.0099
CPU-1204CPU-1204	11.5711.57
CPU-1205CPU-1205	5.545.54
CPU-1206CPU-1206	7.817.81
CPU-1207CPU-1207	3.583.58
CPU-1208CPU-1208	5.965.96
CPU-1209CPU-1209	12.3612.36
CPU-1210CPU-1210	3.333.33
CPU-1211CPU-1211	17.7917.79
CPU-1212CPU-1212	4.404.40

(3) Protein thermal stability migration experiment

Experimental purpose: to investigate the interaction between small molecule compounds and glutaminase GLS1 protein. Confirm whether the inhibitor acts directly on the GLS1 protein.

Experimental procedure: SYPRO Orange (Invitrogen) was used as a fluorescent dye to monitor the changes in the fluorescence value of the system, and the excitation light and emission wavelengths were set to 492nm (FAM) and 610nm (ROX) respectively. Add 2μM GLS1 protein, 5X fluorescent dyes and different concentrations of compounds to 20μL reaction buffer solution (25mM HEPES pH 8.0, 150mM NaCl). On the 7500 Fast RT-PCR System (ABI) instrument, the temperature of the system was gradually increased from 25°C to 95°C at a heating rate of 1%, and the changes in fluorescence intensity with temperature were recorded at 20 second intervals. Furthermore, in the Protein Thermal Shift Software Version 1.1 (ABI) program, the Boltzmann fitting method is used to calculate the dissolution temperature (Tm) of EED under different compound concentration conditions. The experimental results are shown in Figure 1:

(4) Indicate the plasmon resonance experiment

The surface plasmon resonance test was done with BIACORE T200 instrument (GE Healthcare). The freshly purified EED protein (concentration 10 mg/ml) was diluted with 10 mM CH ₃ COONa (pH 4.2) to 0.1 mg/ml, and the GLS1 protein was coupled to the CM5 chip by standard amino coupling methods. Use HBS-EP buffer solution (10mM HEPES (pH 7.4), 150mM NaCl, 3mM EDTA, 0.005% (v/v)surfactant P20) to dilute the GLS1 inhibitor step by step, and then continuously inject the sample for 60s at a flow rate of 20μl/s. Dissociate for 120s, and record the response signal changes with time during the process. _{Calculate the binding rate constant (K on} ), dissociation rate constant (K _off ) and dissociation constant (K _d ) of the GLS1 inhibitor and GLS1 protein using the kinetic analysis module in the BIA Evaluation Software (GE Healthcare) program. The experimental results are shown in Figure 2:

(5) Cell-level glutamate content determination experiment

Mechanism studies have shown that: GLS1 inhibitors can block the hydrolysis of glutamine by inhibiting the activity of GLS1, which will result in the decrease of glutamate, the hydrolysate of glutamine in cells. Therefore, the inhibitory effect of the compound on GLS1 can be indirectly reflected by detecting the content of glutamate in the cell. We measured the glutamate content in HCT116 cells treated with CPU-301 and CB839, and the results are shown in Figure 3. The experimental results show that the compounds CPU-301 and CB839 can significantly reduce the intracellular glutamate content in a concentration-dependent manner, which indirectly proves that the compound CPU-301 works by inhibiting GLS1.

(6) ROS content determination experiment in cells

ROS is a key regulator of cancer cell growth. The induction of oxidative stress can lead to preferential killing of cancer cells. Various drugs that have a direct or indirect effect on ROS have been used for effective cancer treatment. Mechanism studies have shown that GLS1 inhibitors can promote the increase of ROS levels in tumor cells by blocking glutamine metabolism, thereby playing a certain role in killing tumors. We measured the content of reactive oxygen species in HCT116 cells treated with CPU-301 and CB839, and the results are shown in Figure 4. The experimental results show that the compounds CPU-301 and CB839 can obviously induce the increase of intracellular ROS level in a concentration-dependent manner, and cause a certain killing effect on tumor cells.

Claims

A glutaminase GLS1 inhibitor containing a triazole structure of the general formula (I) or a pharmaceutically acceptable salt thereof,

Wherein, n is an integer of 1-4;

L is: CH 2 SCH 2 , CH 2 CH 2 , CH 2 CH 2 CH 2 , CH 2 , CH 2 S, SCH 2 , CH 2 NHCH 2 , CH=CH or
Wherein any hydrogen in CH or CH 2 can be substituted by alkyl or alkoxy; hydrogen in -NH group can be substituted by alkyl; in -CH 2 CH 2 , CH 2 CH 2 CH 2 group A single CH 2 may be substituted by a hydroxyl group; the two groups R 1 and R 2 and the atoms to which they are attached may optionally form a cycloalkane together;

X 1 and X 2 are respectively: S, O and CH=CH, wherein any hydrogen in CH can be substituted by an alkyl group;

Y is: H or CH 2 O(CO) R 5 , R 5 is: H, substituted or unsubstituted alkyl, alkoxy, amino, heterocycloalkyl, aromatic cycloalkyl or heterocycloalkoxy ；

R 1 and R 2 are respectively: H, alkyl, alkoxy or hydroxyl;

R 3 is: alkanes, substituted alkanes, aromatic hydrocarbons, aromatic alkanes, cyano groups, cycloalkanes, cycloaromatic alkanes, hydrogen, halogen, halogen-substituted alkanes, heteroatom aromatic hydrocarbons, heteroatom aromatic alkanes, heteroatom cycloalkanes, C(R 6 )(R 7 )(R 8 ), N(R 9 )(R 10 ), OR 11 , the hydroxyl group in the above-mentioned substituent groups can be acetylated to C(O)R 7 ;

R 4 is: alkanes, substituted alkanes, cycloalkanes, aromatic hydrocarbons, aromatic alkanes, substituted aromatic hydrocarbons or substituted aromatic alkanes;

R 6 , R 7 , and R 8 are respectively: hydrogen, substituted or unsubstituted alkyl, hydroxyl, hydroxyalkyl, amino, acetamido, alkene, alkyne, alkoxy, aryl, arylalkyl, cycloalkane , Heterocyclic or heteroatom aromatic hydrocarbons;

R 9 and R 10 are respectively: hydrogen, substituted or unsubstituted alkyl, hydroxyl, hydroxyalkyl, amino, acetamido, alkene, alkyne, alkoxy, aryl, arylalkyl, cycloalkane, heterocycle , Heteroatomic aromatic hydrocarbons, the hydroxyl groups in the above substituent groups can be acetylated to C(O)R 7 ;

R 11 is: hydrogen, substituted or unsubstituted alkyl, hydroxy, hydroxyalkyl, amino, acetamido, alkene, alkyne, alkoxy, aryl, arylalkyl, cycloalkane, heterocycle, heteroatom aromatic Hydrocarbons, the hydroxyl groups in the above-mentioned substituent groups can be acetylated to C(O)R 7 .
The glutaminase GLS1 inhibitor with the general formula (I) containing a triazole structure or a pharmaceutically acceptable salt thereof according to claim 1, wherein the L is CH 2 CH 2 .
The glutaminase GLS1 inhibitor with the triazole structure containing the general formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, is any one of the following:

Where R is -CH 2 CH 2 OH, -C(CH 3 ) 2 OH, -C(CH 3 )(OH)(C 2 H 5 ), -CH 2 CH 2 COOH, -COOC 2 H 5,
-Ph, 4'-CH 3 -Ph, 4'-CF 3 -Ph, 4'-F-Ph, 4'-Cl-Ph, 4'-Br-Ph, 4'-OCH 3 -Ph, 3' -OCH 3 -Ph, 3'-OH-Ph, 3'-NH 2 -Ph, 4'-NH 2 -Ph, 2'-Pyridine;

Where R is -Ph, 4'-CN-Ph, 4'-NO 2 -Ph, 4'-F-Ph, 4'-Cl-Ph, 4'-Br-Ph, 4'-CH 3 -Ph , 4'-OCH 3 -Ph, 3'-OCH 3 -4'-OCH 3 -Ph, 2'-CH 3 -4'-CH 3 -Ph, 2'-CH 3 -6'-CH 3 -Ph ；

Among them, R is 4'-CH 3 -Ph, 4'-CN-Ph, 4'-NO 2 -Ph, 4'-F-Ph, 4'-Cl-Ph, 4'-Br-Ph, 4' -OCH 3 -Ph, 3'-OCH 3 -4'-OCH 3 -Ph, 2'-CH 3 -4'-CH 3 -Ph, 2'-CH 3 -6'-CH 3 -Ph, 4' -CF 3 -Ph, 4'-OCF 3 -Ph;

Where R is -Ph, 4'-CN-Ph, 4'-NO 2 -Ph, 4'-F-Ph, 4'-Cl-Ph, 4'-Br-Ph, 4'-CH 3 -Ph , 4'-OCH 3 -Ph, 3'-OCH 3 -4'-OCH 3 -Ph, 4'-OCF 3 -Ph, 2'-CH 3 -6'-CH 3 -Ph, 4'-CF 3 -Ph;

Where R is -Ph, 4'-CN-Ph, 4'-NO 2 -Ph, 4'-F-Ph, 4'-Cl-Ph, 4'-Br-Ph, 4'-CH 3 -Ph , 4'-OCH 3 -Ph, 3'-OCH 3 -4'-OCH 3 -Ph, 4'-OCF 3 -Ph, 2'-CH 3 -6'-CH 3 -Ph, 4'-CF 3 -Ph;

Where R is -Ph, 4'-CN-Ph, 4'-NO 2 -Ph, 4'-F-Ph, 4'-Cl-Ph, 4'-Br-Ph, 4'-OCH 3 -Ph , 3'-OCH 3 -4'-OCH 3 -Ph, 4'-CH 3 -Ph, 2'-CH 3 -6'-CH 3 -Ph, 4'-CF 3 -Ph, 4'-OCF 3 -Ph;

Where R is -Ph, 4'-CN-Ph, 4'-NO 2 -Ph, 4'-F-Ph, 4'-Cl-Ph, 4'-Br-Ph, 4'-CH 3 -Ph , 4'-OCH 3 -Ph, 3'-OCH 3 -4'-OCH 3 -Ph, 4'-OCF 3 -Ph, 2'-CH 3 -6'-CH 3 -Ph, 4'-CF 3 -Ph;

Where R is -Ph, 4'-CN-Ph, 4'-NO 2 -Ph, 4'-F-Ph, 4'-Cl-Ph, 4'-Br-Ph, 4'-CH 3 -Ph , 4'-OCH 3 -Ph, 3'-OCH 3 -4'-OCH 3 -Ph, 4'-OCF 3 -Ph, 2'-CH 3 -6'-CH 3 -Ph, 4'-CF 3 -Ph;

Where R is -Ph, 4'-CN-Ph, 4'-NO 2 -Ph, 4'-F-Ph, 4'-Cl-Ph, 4'-Br-Ph, 4'-CH 3 -Ph , 4'-OCH 3 -Ph, 3'-OCH 3 -4'-OCH 3 -Ph, 4'-OCF 3 -Ph, 2'-CH 3 -6'-CH 3 -Ph, 4'-CF 3 -Ph;

Where R is -Ph, 4'-CN-Ph, 4'-NO 2 -Ph, 4'-F-Ph, 4'-Cl-Ph, 4'-Br-Ph, 4'-CH 3 -Ph , 4'-OCH 3 -Ph, 3'-OCH 3 -4'-OCH 3 -Ph, 4'-OCF 3 -Ph, 2'-CH 3 -6'-CH 3 -Ph, 4'-CF 3 -Ph;

Where R is -Ph, 4'-CN-Ph, 4'-NO 2 -Ph, 4'-F-Ph, 4'-Cl-Ph, 4'-Br-Ph, 4'-CH 3 -Ph , 4'-OCH 3 -Ph, 3'-OCH 3 -4'-OCH 3 -Ph, 4'-OCF 3 -Ph, 2'-CH 3 -6'-CH 3 -Ph, 4'-CF 3 -Ph;

Where R is -Ph, 4'-CN-Ph, 4'-NO 2 -Ph, 4'-F-Ph, 4'-Cl-Ph, 4'-Br-Ph, 4'-CH 3 -Ph , 4'-OCH 3 -Ph, 3'-OCH 3 -4'-OCH 3 -Ph, 4'-OCF 3 -Ph, 2'-CH 3 -6'-CH 3 -Ph, 4'-CF 3 -Ph.
A pharmaceutical composition containing a therapeutically effective amount of one or more of the glutaminase GLS1 inhibitors having the general formula (I) containing the triazole structure according to any one of claims 1 to 3 Or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
A pharmaceutical composition containing a therapeutically effective amount of one or more of the glutaminase GLS1 inhibitors having the general formula (I) containing the triazole structure according to any one of claims 1 to 3 Or its pharmaceutically acceptable salts and pharmaceutically acceptable excipients.
The preparation method of the glutaminase GLS1 inhibitor with the triazole structure containing the triazole structure or the pharmaceutically acceptable salt thereof with the general formula (I) according to claim 1, comprising the following steps:

Compound II reacts with different alkynes or azides to obtain corresponding compounds III-1 or III-2. Compounds III-1 and III-2 undergo Click reactions with different azide compounds or alkynes, respectively, under the catalysis of CuI. The final product of the glutaminase GLS1 inhibitor containing the triazole structure with the general formula (I) is obtained.
The glutaminase GLS1 inhibitor with the triazole structure of the general formula (I) according to any one of claims 1-3 or a pharmaceutically acceptable salt thereof is used in the preparation of a medicament for the treatment of GLS1 mediated diseases use.
The use according to claim 7, wherein the disease is colon cancer, triple negative breast cancer or lung cancer.