CN110746416A

CN110746416A - Glutaminase GLS1 inhibitor containing triazole structure or pharmaceutically acceptable salt thereof, and preparation method and application thereof

Info

Publication number: CN110746416A
Application number: CN201910839919.2A
Authority: CN
Inventors: 李志裕; 卞金磊; 徐熙; 孟颖; 王举波
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2019-09-05
Filing date: 2019-09-05
Publication date: 2020-02-04
Also published as: WO2021042723A1

Abstract

The invention discloses a glutaminase GLS1 inhibitor containing a triazole structure or pharmaceutically acceptable salts thereof, a preparation method and application thereofRelated diseases and disorders. The compounds can be effectively combined with an allosteric site of glutaminase, so that the conformation of the glutaminase is changed, and the glutaminase is blocked from performing biological functions. In vitro experiments show that: the compound has good inhibitory activity on various glutamine-dependent cancer cells, such as colon cancer, triple negative breast cancer, lung cancer and the like

Description

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

Technical Field

The invention relates to a glutaminase GLS1 inhibitor or a pharmaceutically acceptable salt thereof, a preparation method and application thereof, in particular to a glutaminase GLS1 inhibitor containing a triazole structure or a pharmaceutically acceptable salt thereof, a preparation method and application thereof.

Background

Metabolic reprogramming is an important marker for tumors that reprogram metabolically to meet the energy and substance synthesis requirements for rapid proliferation. The classical tumor metabolism reprogramming has two characteristics, one is aerobic glycolysis of glucose; the second is the reliance on glutamine to complement the tricarboxylic acid cycle. Glutamine can be used as a carbon source to supplement tricarboxylic acid cycle, can also provide a nitrogen source for the synthesis of biomacromolecules such as protein, amino hexose and nucleotide, and is also one of precursors of glutathione, so that glutamine is an important way for an organism to resist oxidative stress and maintain redox homeostasis. Glutaminase (GLS) catalyzes the deamination of glutamine to glutamic acid, which is the rate-limiting enzyme for glutamine glycolysis, and controls the glutamine metabolism mainly through GLS, holding the entrance of glutamine catabolism.

Glutamine is highly dependent on an important metabolic feature of tumor cells, also known as "glutamine addiction", Glutaminase (GLS) catalyzes the reaction of glutamine to produce glutamic acid, the first metabolic enzyme of glutamine glycolysis. GLS can be classified into kidney-type glutaminase (GLS1) and liver-type glutaminase (GLS 2). GLS1 was highly expressed and GLS2 was low expressed in most tumors. GLS1 has a "pro-cancer effect", whereas GLS2 has an "anti-cancer effect". GLS plays an important role in the development of a variety of tumors. GLS is of great importance in the diagnosis, progression and prognostic assessment of tumors. GLS1 is a potential target for tumor metabolism treatment, and a specific inhibitor thereof is expected to become a novel anti-tumor metabolism medicament.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to provide a glutaminase GLS1 inhibitor containing a triazole structure or a pharmaceutically acceptable salt thereof.

The invention also aims to provide a pharmaceutical composition which contains one or more glutaminase GLS1 inhibitors containing triazole structures in a general formula (I) or pharmaceutically acceptable salts thereof in a therapeutically effective amount, and a pharmaceutically acceptable carrier.

The invention also aims to provide a pharmaceutical composition which contains a therapeutically effective amount of one or more of the glutaminase GLS1 inhibitor containing the triazole structure with the general formula (I) or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable auxiliary materials.

The invention also aims to provide a preparation method of the glutaminase GLS1 inhibitor containing the triazole structure or the pharmaceutically acceptable salt thereof.

The last purpose of the invention is to provide the application of the glutaminase GLS1 inhibitor containing the triazole structure or the pharmaceutically acceptable salt thereof in preparing medicines for treating GLS1 mediated diseases.

The technical scheme is as follows: the invention provides a glutaminase GLS1 inhibitor containing a triazole structure or pharmaceutically acceptable salts thereof,

wherein n is an integer of 1 to 4;

l is: CH (CH)₂SCH₂、CH₂CH₂、CH₂CH₂CH₂、CH₂、CH₂S、SCH₂、CH₂NHCH₂CH ═ CH or

The hydrogen in the radical may be substituted by alkyl; -CH₂CH₂、CH₂CH₂CH₂Single CH in the radical₂May be substituted by hydroxy; r₁And R₂The two groups together with the atoms to which they are attached may optionally form a cycloalkane;

X₁、X₂respectively as follows: s, O and CH ═ CH, where any hydrogen in CH may be substituted by alkyl;

y is: h or CH₂O(CO)R₅，R₅Comprises the following steps: H. substituted or unsubstituted alkyl, alkoxy, amino, heterocycloalkyl, arylcycloalkyl or heterocycloalkoxy;

R₁、R₂respectively as follows: H. alkyl, alkoxy or hydroxy;

R₃comprises the following steps: alkanes, substituted alkanes, aromatics, aromatic alkanes, cyano groups, cycloalkanes, cycloparaffins, hydrogen, halogens, halogen-substituted alkanes, heteroatomic aromatics, heteroatomic aromatic alkanes, heteroatomic cycloalkanes, C (R)₆)(R₇)(R₈)、 N(R₉)(R₁₀)、OR₁₁Any hydroxy group may be acetylated to C (O) R₇；

R₄Comprises the following steps: alkanes, substituted alkanes, cycloalkanes, aromatics, substituted aromatics, or substituted aromatics;

R₆、R₇、R₈respectively as follows: hydrogen, substituted or unsubstituted alkyl, hydroxy, hydroxyalkyl, amino, acetamido, alkene, alkyne, alkoxy, aryl, arylalkyl, cycloalkane, heterocycle, or heteroatom aromatic;

R₉、R₁₀respectively as follows: hydrogen, substituted or unsubstituted alkyl, hydroxy, hydroxyalkyl, amino, acetamido, alkene, alkyne, alkoxy, aryl, arylalkyl, cycloalkane, heterocycle, heteroatomic aromatic hydrocarbon, any hydroxy group can be acetylated to C (O) R₇；

R₁₁Comprises the following steps: hydrogen, substituted or unsubstituted alkyl, hydroxy, hydroxyalkyl, amino, acetamido, alkene, alkyneHydrocarbons, alkoxy, aryl, arylalkyl, cycloalkanes, heterocycles, heteroaromatics, any hydroxy group being acetylated to C (O) R₇。

Further, L is CH₂CH₂。

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

wherein R is-CH₂CH₂OH、-C(CH₃)₂OH、-C(CH₃)(OH)(C₂H₅)、-CH₂CH₂COOH、-COOC₂H₅、

-Ph、4’-CH₃-Ph、4’-CF₃-Ph、4’-F-Ph、4’-Cl-Ph、4’-Br-Ph、4’-OCH₃-Ph、3’-OCH₃-Ph、3’-OH-Ph、 3’-NH₂-Ph、4’-NH₂-Ph、2’-Pyridine；

Wherein R is-Ph, 4 '-CN-Ph, 4' -NO₂-Ph、4’-F-Ph、4’-Cl-Ph、4’-Br-Ph、4’-CH₃-Ph、4’-OCH₃-Ph、 3’-OCH₃-4’-OCH₃-Ph、2’-CH₃-4’-CH₃-Ph、2’-CH₃-6’-CH₃-Ph；

Wherein R is 4' -CH₃-Ph、4’-CN-Ph、4’-NO₂-Ph、4’-F-Ph、4’-Cl-Ph、4’-Br-Ph、4’-OCH₃-Ph、 3’-OCH₃-4’-OCH₃-Ph、2’-CH₃-4’-CH₃-Ph、2’-CH₃-6’-CH₃-Ph、4’-CF₃-Ph、4’-OCF₃-Ph；

Wherein R is-Ph, 4 '-CN-Ph, 4' -NO₂-Ph、4’-F-Ph、4’-Cl-Ph、4’-Br-Ph、4’-CH₃-Ph、4’-OCH₃-Ph、 3’-OCH₃-4’-OCH₃-Ph、4’-OCF₃-Ph、2’-CH₃-6’-CH₃-Ph、4’-CF₃-Ph；

Wherein R is-Ph, 4 '-CN-Ph, 4' -NO₂-Ph、4’-F-Ph、4’-Cl-Ph、4’-Br-Ph、4’-OCH₃-Ph、3’-OCH₃-4’-OCH₃-Ph、4’-CH₃-Ph、2’-CH₃-6’-CH₃-Ph、4’-CF₃-Ph、4’-OCF₃-Ph；

Wherein R is-Ph, 4 '-CN-Ph, 4' -NO₂-Ph、4’-F-Ph、4’-Cl-Ph、4’-Br-Ph、4’-CH₃-Ph、4’-OCH₃-Ph、 3’-OCH₃-4’-OCH₃-Ph、4’-OCF₃-Ph、2’-CH₃-6’-CH₃-Ph、4’-CF₃-Ph。

A pharmaceutical composition comprises one or more of the glutaminase GLS1 inhibitor containing triazole structure with the general formula (I) or pharmaceutically acceptable salt thereof with effective treatment amount, and a pharmaceutically acceptable carrier.

A pharmaceutical composition comprises a therapeutically effective amount of one or more of the glutaminase GLS1 inhibitors containing triazole structures with the general formula (I) or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable auxiliary materials.

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

the compound II reacts with different alkynes or azides respectively to obtain corresponding compounds III-1 or III-2, and the compounds III-1 and III-2 react with different azides or alkynes respectively under the catalysis of CuI to obtain a final product of the glutaminase GLS1 inhibitor containing a triazole structure, which has a general formula (I).

The glutaminase GLS1 inhibitor containing a triazole structure with the general formula (I) or the medicinal salt thereof can be used for preparing medicines for treating GLS1 mediated diseases.

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

The invention discloses a novel targeted drug with high efficiency and low toxicity for treating cancers, wherein a triazolyl group is introduced into the structure according to the crystal structures of glutaminase and a representative GLS1 inhibitor BPTES and CB839 compound, the influence of different substituents on the triazolyl group on the activity is examined, the optimal configuration is optimized, and the structure-activity relationship is summarized. The designed and synthesized compound has targeting property, can obviously inhibit the activity of glutaminase and block glutamine from being hydrolyzed into glutamic acid, thereby cutting off the energy supply of tumor cells, has very strong tumor inhibition capability on glutamine dependence, and can obtain good treatment effect by drug combination.

Has the advantages that: according to the invention, a series of novel glutaminase inhibitor compounds containing triazole structures are designed and synthesized based on the crystal structure of the allosteric site of glutaminase, and the bioactivity of glutaminase can be obviously inhibited. Experimental results show that the compounds can obviously inhibit the activity of glutaminase at the molecular level, block the hydrolysis of glutamine into glutamic acid, show good anti-tumor effect at the cellular level and animal level, and can be used for preparing anti-tumor drugs.

Drawings

FIG. 1 shows the thermo-stable migration of GLS1 protein by GLS1 inhibitor;

FIG. 2 shows (A) the determination of the affinity of the compound CPU-210 to the GLS1 protein by surface plasmon resonance assay; (B) the affinity of the compound CPU-301 and the GLS1 protein is measured by a surface plasmon resonance experiment;

FIG. 3 shows the intracellular glutamate assay performed by the compounds CPU-301 and CB 839;

FIG. 4 is an experiment showing that compounds CPU-301 and CB839 induce an increase in intracellular ROS levels.

Detailed Description

Example 1

Preparation of Compound I of formula (CPU101-CPU118)

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

Intermediate 1 (1)0g, 57.4mmol) was dissolved in 100ml of OCL₃Then, thiosemicarbazide (5.23g, 57.4mmol) was added and reacted at 85 ℃ for 4 hours. After the TLC detection reaction is completed, the reaction liquid is cooled to room temperature, water is added for dilution, then the pH value is adjusted to 7 by 6M NaOH, solid is precipitated, the filtration is carried out, and a filter cake is dried to obtain white solid with the yield of 41.85%. Hrms (esi): m/z, calcd for C₉H₁₅N₃O₂S[M+H]⁺，230.0963；found：230.0962.

Preparation of ethyl 5- (5- (2-phenylacetylamino) -1, 3, 4-thiadiazole) 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 24h, after completion of the TLC detection reaction the solvent was removed under reduced pressure and slurried with water to give a white solid in 92.5% yield. Hrms (esi): m/z, calcd for C₁₇H₂₁N₃O₃S₂[M+H]⁺，348.1382；found：348.1371.

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

Compound 3(2.0g, 5.76mmol) was dissolved in 4N NaOH (15mL) and MeOH (10mL) and stirred at room temperature for 3 h. After the reaction was completed, the solvent was removed under reduced pressure, pH was adjusted to 7 with 4N HCl, a white solid was precipitated, filtered and dried, and yield was 90.4%. Hrms (esi): m/z, calcd for C₁₅H₁₇N₃O₃S₂[M+H]⁺，320.1069；found：320.1064.

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

Intermediate 4(2g, 6.2mmol) was dissolved in 10mL POCl₃Then, thiosemicarbazide (0.629g, 6.8mmol) was added and reacted at 85 ℃ for 4 hours. After the TLC detection reaction is completed, the reaction liquid is cooled to room temperature, water is added for dilution, then the pH value is adjusted to 7 by 6M NaOH, solid is separated out, the filtration is carried out, a filter cake is dried to obtain black solid, and the yield is 43.5%. Hrms (esi): m/z, calcd for C₁₆H₁₈N₆OS₂[M+H]⁺，375.1065；found：375.1056.

Preparation of 2-chloro-N- (5- (4- (5- (2-phenylacetylamino) -1, 3, 4-thiadiazolyl) butyl) -1, 3, 4-thiadiazole) acetamide (6)

Intermediate 5(3g, 8.02mmol) was dissolved in 10mL DMF solution, TEA (2.43g, 24.06mmol) was added, and chloroacetyl chloride (1.8g, 16.04mmol) was added dropwise. The reaction mixture was stirred at room temperature for 12 h. Cooled to room temperature and the reaction mixture was poured into water. The solid precipitated and was filtered off to give a white solid in 84.2% yield. Hrms (esi): m/z, calcd for C₁₈H₁₉ClN₆O₂S₂[M+H]⁺，451.0772；found：451.0785.

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

To a solution of Compound 6(3.5g, 7.78mmol) in DMF was added NaN₃(1.52g, 23.3 mmol). The reaction was stirred at room temperature for 12 h. Then, the reaction solution was poured into water, extracted with DCM (30mL × 3), the organic layers were combined, washed with water three times, washed with saturated brine three times, dried over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure, and column chromatography was performed to obtain a white solid with a yield of 90%. Hrms (esi): m/z, calcd for C₁₈H₂₀N₉O₂S₂[M+H]⁺，458.1176；found：458.1148

Preparation of Compound CPU-101

To a solution of compound 7(0.2g, 0.44mmol) in DMF were added CuI (16.7mg, 0.088mmol) in water and 3-butyn-1-ol (8a) (77.05mg, 1.1 mmol). The reaction mixture was stirred at 100 ℃ for 0.5h under microwave 300W. The reaction solution was poured into water to precipitate a white solid. And (4) carrying out suction filtration to obtain a crude product, and carrying out column chromatography to obtain a white solid with the yield of 45.4%. m.p.208-210 ℃;¹H NMR(300MHz，DMSO-d₆)：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 C₂₅N₉O₃S₂[M+H]⁺，528.1595；found：528.1577.

preparation of Compound CPU-102

The compound CPU-102 was prepared in 39.4% yield using 2-methyl-3-butyn-2-ol (8b) instead of 8a as described for the preparation of CPU-101. m.p.210-212 ℃;¹H NMR(300MHz，DMSO-d₆)：δ 12.93(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 C₂₃H₂₇N₉O₃S₂[M+H]⁺，542.1751； found：542.1740.

preparation of the Compound CPU-103

The compound CPU-103 was prepared in 40.8% yield using 3-methyl-1-pentyn-3-ol (8c) instead of 8a as described for the preparation of CPU-101. m.p.220-223 ℃;¹H NMR(300MHz，DMSO-d₆)：δ 12.93(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 C₂₄H₂₉N₉O₃S₂[M+H]⁺，556.1908；found：556.1891.

preparation of the Compound CPU-104

CPU-104 was prepared as described for CPU-101 using 3-butyn-1-oic acid (8d) instead of 8a, in 35.7% yield. m.p.220-224 ℃;¹H NMR(300MHz，DMSO-d₆)：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 C₂₃H₂₅N₉O₄S₂[M+H]⁺，556.1544；found：556.1521.

preparation of Compound CPU-105

CPU-105 was prepared in 37.8% yield as described for CPU-101, substituting 8a for ethyl propiolate (8 e). m.p. 204-208 ℃;¹H NMR(300MHz，DMSO-d₆)：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 C₂₃H₂₅N₉O₄S₂[M+H]⁺，556.1544；found：556.1504.

preparation of Compound CPU-106

Compound CPU-106 was prepared in 42.3% yield as described for preparation of CPU-101, except that ethynylcyclopropane (8f) was used in place of 8 a. m.p.215-218 ℃;¹H NMR(300MHz，DMSO-d₆)：δ 12.91(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 C₂₃H₂₅N₉O₂S₂[M+ H]⁺，524.1645；found：524.1633.

preparation of Compound CPU-107

Compound CPU-107 was prepared in 45.1% yield using phenylacetylene (8g) instead of 8a as described for preparation of CPU-101. m.p.218-220 ℃;¹H NMR(300MHz，DMSO-d₆)：δ 12.64(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 C₂₆H₂₅N₉O₂S₂[M+H]⁺，560.1645；found：560.1617

preparation of Compound CPU-108

The compound CPU-108 was prepared in 33.3% yield using 4-benzylacetylene (8h) instead of 8a as described for the preparation of CPU-101. m.p.237-242 ℃;¹H NMR(300MHz，DMSO-d₆)：δ 12.99(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 C₂₇H₂₇N₉O₂S₂[M+H]⁺，574.1802；found：574.1806.

preparation of Compound CPU-109

As described for the preparation of CPU-101, use of Pair threeThe compound CPU-109 was prepared in 40.9% yield from fluoromethylphenylacetylene (8i) instead of 8 a. m.p.109-111 ℃;¹H NMR(300MHz，DMSO-d₆)：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 C₂₇H₂₄F₃N₉O₂S₂[M+H]⁺， 628.1519；found：628.1528.

preparation of Compound CPU-110

Compound CPU-110 was prepared in 41.5% yield using 4-fluorophenylacetylene (8j) instead of 8a as described for preparation of CPU-101. m.p.249-250 ℃;¹H NMR(300MHz，DMSO-d₆)：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 C₂₆H₂₄FN₉O₂S₂[M+H]⁺，578.1551；found：578.1533.

preparation of Compound CPU-111

The compound CPU-111 was prepared in 43.9% yield using 4-chlorophenylacetylene (8k) instead of 8a as described for the preparation of CPU-101. m.p.232-240 ℃;¹HNMR(300MHz，DMSO-d₆)：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 C₂₆H₂₄ClN₉O₂S₂[M+H]⁺， 594.1256；found：594.1243.

preparation of the Compound CPU-112

The compound CPU-112 was prepared in 30.6% yield as described for preparation CPU-101, using 4-bromobenzeneacetylene (81) instead of 8 a. m.p.235-240 ℃;¹HNMR(300MHz，DMSO-d₆)：δ 12.96(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 C₂₆H₂₄BrN₉O₂S₂[M +H]⁺，638.0751；found：638.0739.

preparation of Compound CPU-113

Compound CPU-113 was prepared using 4-ethynylanisole (8m) instead of 8a with a 42.2% yield as described for preparation of CPU-101. m.p.215-216 ℃;¹HNMR(300MHz，DMSO-d₆)：δ 13.02(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＝8.3 Hz，2H)，5.54(s，2H)，3.79(s，5H)，3.00(s，4H)，1.75(s，4H)ppm.HRMS(ESI)：m/z，calcd for C₂₇H₂₇N₉O₃S₂[M+H]⁺，590.1751；found：590.1754.

preparation of the Compound CPU-114

Compound CPU-114 was prepared in 33.3% yield using 3-ethynylanisole (8n) instead of 8a as described for the preparation of CPU-101. m.p.227-229 ℃;¹HNMR(300MHz，DMSO-d₆)：δ 13.00(s，1H)，12.65(s，1H)，8.62(s，1H)，7.47-7.36(m，3H)，7.29(d，J＝14.5Hz，5H)，6.92(d，J＝7.2Hz，1H)， 5.55(s，2H)，3.80(d，J＝8.9Hz，5H)，3.00(s，4H)，1.75(s，4H)ppm.HRMS(ESI)：m/z， calcd for C₂₇H₂₇N₉O₃S₂[M+H]⁺，590.1751；found：590.1741.

preparation of Compound CPU-115

The compound CPU-115 was prepared in 38.7% yield using 3-hydroxyacetylene (8o) instead of 8a as described for the preparation of CPU-101. m.p.235-239 ℃;¹HNMR(300MHz，DMSO-d₆)：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 C₂₆H₂₅N₉O₃S₂[M+H]⁺， 576.1595；found：576.1576.

preparation of the Compound CPU-116

Compound CPU-116 was prepared in 42.5% yield using m-aminophenylacetylene (8p) instead of 8a as described for the preparation of CPU-101. m.p.232-235 ℃;¹HNMR(300MHz，DMSO-d₆)：δ 12.65(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 C₂₆H₂₆N₁₀O₂S₂[M+H]⁺，575.1754；found：575.1741.

preparation of Compound CPU-117

Compound CPU-117 was prepared in 40.0% yield using p-aminophenylacetylene (8q) instead of 8a as described for the preparation of CPU-101. m.p.201-204 ℃;¹HNMR(300MHz，DMSO-d₆)：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 C₂₆H₂₆N₁₀O₂S₂[M+H]⁺， 575.1754；found：575.1759.

preparation of compound CPU-118

Compound CPU-118 was prepared in 38.6% yield using 2-ethynylpyridine (8r) instead of 8a as described for the preparation of CPU-101. m.p.223-227 ℃;¹HNMR(300MHz，DMSO-d₆)：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 C₂₄N₁₀O₂S₂[M+H]⁺，561.1598；found：561.1597.

example 2

Preparation of Compound II of general formula (CPU201-CPU 211).

Synthesis of intermediate 10

Intermediate 5(0.255g, 0.68mmol), pentynoic acid (0.073g, 0.75mmol), HATU (0.3 mmol)8g, 1.02mmol) was dissolved in DMF (2mL) and stirred at room temperature for 10 min. DIPEA (0.26g, 2.04mmol) was added and stirring continued. The reaction was substantially complete after 20 min. Pouring the reaction solution into water, separating out white solid, filtering, and drying the filter cake to obtain the white solid. The yield was 61.5%. Hrms (esi): m/z, calcd for C₂₁H₂₂N₆O₂S₂[M+H]⁺，455.1324； found：455.1310.

Preparation of Compound CPU-201

To a solution of intermediate 10(0.1g, 0.202mmol) in DMF was added a solution of CuI (7.78mg, 0.041mmol) in water and benzyl azide (11a) (63.3mg, 0.476 mmol). The reaction mixture was stirred at 100 ℃ for 0.5 hour under microwave 300W. The reaction solution was poured into water to precipitate a white solid. And (4) carrying out suction filtration to obtain a crude product, and carrying out column chromatography to obtain a white final product with the yield of 38.7%.¹HNMR(300MHz，DMSO-d₆)：δ 7.90(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，calcdfor C₂₈H₂₉N₉O₂S₂[M+H]⁺，588.1958；found：588.1960.

Preparation of Compound CPU-202

Compound CPU-202 was prepared in 43.6% yield using 4-cyanobenzylazide (11b) instead of 11a as described for preparation of CPU-201.¹HNMR(300MHz，DMSO-d₆)：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 C₂₉H₂₈N₁₀O₂S₂[M+H]⁺，613.1911；found：613.1903.

Preparation of Compound CPU-203

The compound CPU-203 was prepared in 40.2% yield using 4-nitrobenzyl azide (11c) instead of 11a as described for the preparation of CPU-201.¹HNMR(300MHz，DMSO-d₆)：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＝6.9Hz，2H)，1.75(s，4H)ppm.HRMS(ESI)：m/z，calcd forC₂₈H₂₈N₁₀O₄S₂[M+H]⁺，633.1809；found：633.1808.

Preparation of Compound CPU-204

Compound CPU-204 was prepared using 4-fluorobenzylazide (11d) instead of 11a with a yield of 34.8% as described for preparation of CPU-201. m.p.203-208 ℃;¹HNMR(300MHz，DMSO-d₆)：δ 12.65(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 C₂₈H₂₈FN₉O₂S₂[M+H]⁺， 606.1864；found：606.1878.

preparation of Compound CPU-205

Compound CPU-205 was prepared as described for preparation of CPU-201 using 4-chlorobenzyl azide (11e) instead of 11a, at a yield of 39.4%. m.p.203-204 ℃;¹HNMR(300MHz，DMSO-d₆)：δ 12.70(s，1H)，12.45(s，1H)， 7.94(s，1H)，7.39(dd，J＝18.3，6.0Hz，5H)，7.29(d，J＝7.9Hz，4H)，5.58(s，2H)，3.84(s， 2H)，3.02(d，J＝13.0Hz，6H)，2.87(d，J＝6.5Hz，2H)，1.79(s，4H)ppm.HRMS(ESI)：m/z， calcd forC₂₈H₂₈ClN₉O₂S₂[M+H]⁺，622.1569；found：622.1528.

preparation of Compound CPU-206

Compound CPU-206 was prepared as described for preparation of CPU-201 using 4-bromobenzylazide (11f) instead of 11a, with a yield of 36.5%. m.p.217-219 ℃;¹HNMR(300MHz，DMSO-d₆)：δ 12.69(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 forC₂₈H₂₈BrN₉O₂S₂[M+H]⁺，666.1064；found：666.1059.

preparation of Compound CPU-207

Compound CPU-207 was prepared as described for preparation of CPU-201 using 4-methylbenzylazide (11g) instead of 11a in 44.3% yield. m.p.216-217 ℃;¹HNMR(300MHz，DMSO-d₆)：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 forC₂₉H₃₁N₉O₂S₂[M+H]⁺，602.2115；found：602.2143.

preparation of Compound CPU-208

The compound CPU-208 was prepared in 46.4% yield using 4-methoxybenzylazide (11h) instead of 11a as described for preparation of CPU-201. m.p.237-240 ℃;¹HNMR(300MHz，DMSO-d₆)：δ 12.67(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 C₂₉H₃₁N₉O₃S₂[M+H]⁺，618.2064；found：618.2070.

preparation of Compound CPU-209

Compound CPU-209 was prepared in 48.1% yield using 3, 4-dimethoxybenzyl azide (11i) instead of 11a as described for preparation of CPU-201. m.p.232-236 ℃;¹HNMR(300MHz，DMSO-d₆)：δ 12.66(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＝8.1Hz，1H)， 6.79(d，J＝8.3Hz，1H)，5.43(s，2H)，3.80(s，2H)，3.74-3.67(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 C₃₀H₃₃N₉O₄S₂[M+ H]⁺，648.217；found：648.2167.

preparation of Compound CPU-210

Compound CPU-210 was prepared in 30.9% yield using 2, 4-dimethylbenzyl azide (11j) instead of 11a as described for preparation of CPU-201. m.p.228-231 ℃;¹HNMR(300MHz，DMSO-d₆)：δ 12.67(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＝8.2Hz，6H)，1.75(s，4H)ppm.HRMS(ESI)：m/z， calcd forC₃₀H₃₃N₉O₂S₂[M+H]⁺，616.2271；found：616.2278.

preparation of Compound CPU-211

Compound CPU-211 was prepared in 38.6% yield using 2, 6-dimethylbenzyl azide (11k) instead of 11a as described for CPU-201. m.p.205-210 ℃;¹HNMR(300MHz，DMSO-d₆)：δ 12.66(s，1H)， 12.37(s，1H)，7.57(s，1H)，7.30(s，5H)，7.15-7.08(m，1H)，7.02(d，J＝7.2Hz，2H)，5.50(s， 2H)，3.78(s，2H)，2.95(d，J＝22.9Hz，6H)，2.77(s，2H)，2.26(s，6H)ppm.HRMS(ESI)：m/z， calcdfor C₃₀H₃₃N₉O₂S₂[M+H]⁺，616.2271；found：616.2288.

example 3

Preparation of Compound III of formula (CPU301-CPU310)

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

To 13(1.5g, 0.015mol) of POCl₃Thiosemicarbazide (1.39g, 0.015mol) was added to the solution and the reaction mixture was stirred at 80 ℃ for 4 hours. After the reaction is finished, cooling to room temperature, adjusting the pH of the mixture to 9 with 6M NaOH, separating out a solid, performing suction filtration, and drying a filter cake; the filtrate was extracted 3 times with ethyl acetate, the organic layers were combined, dried over anhydrous sodium sulfate, and spin-dried to give a tan solid which was combined with the above filter cake in 88.3% yield.¹HNMR(300MHz，DMSO-d₆)：δ 6.99(s，2H)，2.95(t，J＝7.0Hz，2H)，2.83(d，J＝2.4Hz，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 and then 2-pyridineacetic acid hydrochloride (3.74g, 0.03mol), HATU (8.8g, 0.039mmol) were added, followed by stirring at room temperature for 15min and the addition of DIPEA (7.5g, 0.059 mmol). The reaction was stirred at room temperature for 2 hours to complete the reaction and the reaction mixture was poured into water. The aqueous layer was extracted with ethyl acetate (30 mL. times.3). The organic layer was dried over anhydrous sodium sulfate and spin dried to give a pale yellow solid in 92.6% yield.¹HNMR(300MHz，DMSO-d₆)：δ12.71(s，1H)，8.50(d，J＝4.8Hz，1H)，7.78(td， J＝7.7，1.7Hz，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 C₁₃H₁₂N₄OS[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 a solution of compound 15(2g, 7.4mmol) in DMA (10mL) were added 16(1.35g, 6.11mmol), CuI (0.117g, 0.613mmol), TEA (3.1g, 30.65mmol) and tetrakis (triphenylphosphine) palladium (0.708g, 0.613 mmol). The mixture was stirred at 60 ℃ for 3.5h under nitrogen. After cooling to room temperature, the reaction mixture was diluted in 30mL of isopropyl ether. The lower reddish brown oil was collected by separation, and a gray solid precipitated after addition of water. Column chromatography gave the title compound in 68.5% yield.¹HNMR(300MHz，DMSO-d₆)：δ12.69(s，1H)，8.49(d，J＝4.0Hz，1H)，7.76(t， J＝7.1Hz，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 forC₁₇H₁₅N₇OS[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(1g, 2.74mmol) was dissolved in 200mL of methanol, Raney nickel (2mL) was added, and H was added₂And stirred at room temperature for 48 hours. After completion of the reaction as indicated by TLC detection, the catalyst was filtered off. The filtrate was rotary dried to afford intermediate 18 as a yellow solid in 97.2% yield.¹HNMR(300MHz，DMSO-d₆)：δ12.67(s，1H)，8.50(d，J＝ 4.9Hz，1H)，7.77(td，J＝7.7，1.8Hz，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＝ 6.9Hz，2H)，2.70(t，J＝6.9Hz，2H)，1.70(s，4H)ppm.HRMS(ESI)：m/z，calcd for C₁₇H₁₉N₇OS[M+H]⁺，370.1445；found：370.1451.

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

Intermediate 18(0.25g, 0.68mmol), pentynoic acid (0.073g, 0.75mmol), HATU (0.38g, 1.02mmol) were dissolved in DMF and stirred at room temperature for 10 min. DIPEA (0.26g, 2.04mmol) was added and the reaction was continued, with TLC every five minutes to confirm the progress of the reaction. The reaction was substantially complete after 20 min. Pouring the reaction solution into water, separating out solid, filtering, and drying the filter cake to obtain white solid with the yield of 60.4%.¹H NMR(300MHz，DMSO-d₆)：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 forC₂₂H₂₃N₇O₂S[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 heating at 120 deg.c and 300W,the reaction was carried out for 5min, and the reaction solution was blue and turbid. Pouring the reaction liquid into water, separating out solid, filtering, drying the filter cake to obtain a crude product, and carrying out column chromatography to obtain a blue solid with the yield of 30.1%. m.p.160-164 ℃;¹H NMR(300MHz，DMSO-d6)：δ 12.69(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 C₃₀H₃₂N₁₀O₂S[M+H]⁺，597.2509；found： 597.2487.

preparation of Compound CPU-307

Compound CPU-307 was prepared as described for preparation CPU-301 using 4-methoxybenzylazidobenzene (11h) instead of 11g to give a blue solid. The yield was 32.7%. m.p.115-120 ℃;¹H NMR(300MHz，DMSO-d6)：δ 12.68(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 C₃₀H₃₂N₁₀O₃S[M+H]⁺， 613.2458；found：613.2439.

preparation of Compound CPU-308

The compound CPU-308 was prepared as described for preparation CPU-301 using 3, 4-dimethoxybenzyl azide (11i) instead of 11g to give a blue solid. The yield was 32.2%. m.p.110-115 ℃;¹H NMR(300MHz，DMSO-d₆+D₂O)： δ 8.51(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 C₃₁H₃₄N₁₀O₄S[M+H]⁺，643.2563；found：643.2574.

preparation of Compound CPU-309

Use of 3, 4-Dimethyl benzyl azide (11j) substituted for 11g of the preparative compound, CPU-309, gave a blue solid. The yield was 33.4%. m.p.110-111 ℃;¹H NMR(300MHz，DMSO-d₆+D₂O)：δ 8.49(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 C₃₁H₃₄N₁₀O₂S[M+H]⁺，611.2665；found：611.2672.

preparation of Compound CPU-310

Compound CPU-310 was prepared as described for preparation CPU-301 using 2, 6-dimethylbenzylazide (11k) instead of 11g, to afford a blue solid. The yield was 35.5%. m.p.210-215 ℃;¹H NMR(300MHz，DMSO-d₆)：δ 7.99(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 C₃₁H₃₄N₁₀O₂S[M+H]⁺，611.2665；found：611.2655.

preparation of Compound CPU-311

The compound CPU-312 was prepared as described for preparation CPU-301 using 4-trifluoromethylbenzyl azide (111) instead of 11g to give a blue solid. The yield was 25.5%. m.p.210-215 ℃;¹H NMR(300MHz，DMSO-d₆)：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 C₃₀H₂₉F₃N₁₀O₂S[M+H]⁺，651.2226；found：651.2188.

preparation of Compound CPU-312

Compound CPU-312 was prepared as described for preparation of CPU-301, using 4-trifluoromethoxybenzyl azide (11m) instead of 4-methylbenzyl azide, to afford a blue solid. The yield was 37.2%. m.p.210～215℃；¹H NMR(300MHz， DMSO-d₆)：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 C₃₀H₂₉F₃N₁₀O₃S[M+H]⁺，667.2175； found：667.2208.

Example 4

Preparation of Compound IV of formula (CPU401-CPU412)

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

Compound 21(0.5g, 2.8mmol), compound 22(0.556g, 2.8mmol) and NaHCO₃(0.448g, 11.2mmol) was dissolved in 10mL of ethanol, heated to reflux, stirred, and oil-bathed to 80 ℃. After 4.5h of reaction, the reaction was completed by TLC detection and stopped. The solvent was spin dried under reduced pressure to give a white brown solid. Adding water, pulping, and vacuum filtering to obtain off-white solid. The yield was 88%. Hrms (esi): m/z, calcd for C₁₂H₂₁N₅O₂S[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)

Compound 23(0.1g, 0.33mmol), phenylacetic acid (0.05g, 0.363mmol), HATU (0.25g, 0.66mmol) was placed in 5mL of N, N-Dimethylformamide (DMF) and stirred at room temperature as a pale yellow clear solution. After 15min of reaction, DIPEA (0.128g, 0.99mmol) was added. After 1h of reaction, the reaction was complete by TLC detection. The reaction solution was poured into water and filtered under suction to give a white solid with a yield of 87.6%. Hrms (esi): m/z, calcd for C₂₀H₂₇N₅O₃S[M +H]⁺，418.1913；found：418.1908.

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

Compound 24(0.1g, 0.24mmol) was dissolved in 1mL of dichloromethane and turbidity appeared. 1mL of trifluoroacetic acid was added thereto, and the reaction mixture was clarified. And (5) carrying out water bath at normal temperature and stirring. After 2h reaction, the completion of the reaction was checked by TLC. The pH was adjusted to 7 with saturated aqueous sodium bicarbonate solution, and a solid precipitated. And filtering to obtain yellow solid. The yield was 73%.¹H NMR (300 MHz，DMSO-d₆)：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，calcdfor C₁₅H₁₉N₅OS[M+H]⁺，318.1389；found：318.1375.

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

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 of ethanol and heated to reflux. After 3h reaction, the reaction was completed by TLC detection and stopped. The solvent was spin-dried under reduced pressure and water was added to precipitate a solid. Filtering, and carrying out column chromatography to obtain pink solid. The yield was 40%.¹HNMR(300MHz，DMSO-d₆)：δ 12.19(s，1H)，7.38-7.26(m，6H)，6.47(s，2H)，3.71 (singletoverlapping 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 C₁₇H₂₀N₈OS₂[M+H]⁺，417.1280； found：417.1266.

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

Compound 26(0.05g, 0.12mmol), pentynoic acid (0.01g, 0.12mmol), and HATU (0.07g, 0.18mmol) were placed in a single-necked reaction flask, and the reaction solution was an orange-colored clear solution. Stirred at room temperature for 20 min. DIPEA (0.06g, 0.48 mmol) was added and stirring continued at room temperature. After the reaction for 1h, the TLC detection shows that the reaction is complete and the reaction is stopped. Pouring the reaction solution into distilled water, and separating outAnd (3) a solid. And (5) carrying out suction filtration and drying. The yield was 60%.¹H NMR(300MHz，DMSO-d₆)：δ 12.14 (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，calcdfor C₂₂H₂₄N₈O₂S₂[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 appeared black first and yellow after stirring for a while. Then compound 27(0.1g, 0.2016mmol) and 4-nitrobenzyl azide (11c) (0.07g, 0.476mmol) were added. Reacting at room temperature under the protection of nitrogen, and detecting by TLC after reacting for 3h to complete the reaction. Pouring the reaction solution into water, filtering to obtain a crude product, and carrying out column chromatography to obtain a white final product with the yield of 37.1%. m.p.250-255 ℃; hrms (esi): m/z, calcd for C₂₉H₃₀N₁₂O₄S₂[M+H]⁺，675.2033； found：675.2029.

Preparation of Compound CPU-404 was prepared as described for preparation of CPU-403 using 4-fluorobenzyl azide (11d) instead of 11c to give a white solid. The yield was 33.6%. m.p.218-222 ℃;¹H NMR(300MHz，DMSO-d6)：δ 12.19(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 C₂₉H₃₀FN₁₁O₂S₂[M+H]⁺，648.2088； found：648.2079.

preparation of Compound CPU-405

Compound CPU-405 was prepared as described for preparation of CPU-403 using 4-chlorobenzyl azide (11e) instead of 11c to give a white solid. The yield was 37.5%. m.p.232-237 ℃; hrms (esi): m/z, calcd for C₂₉H₃₀ClN₁₁O₂S₂[M +H]⁺，664.1792；found：664.1796.

Preparation of compound CPU-406 Compound CPU-406 was prepared as described for preparation of CPU-403, using 4-bromobenzylazide (11f) in place of 11c to give a white solid. The yield was 35.4%. m.p.258-261 ℃;¹H NMR(300MHz，DMSO-d₆)：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 forC₂₉H₃₀BrN₁₁O₂S₂[M+H]⁺， 710.1267；found：710.1272.

preparation of Compound CPU-407

Compound CPU-407 was prepared as described for preparation of CPU-403, using 4-methylbenzyl azide (11g) instead of 11c, to give a white solid. The yield was 32.3%. m.p. 250-254 ℃; 1HNMR (300MHz, DMSO-d 6): δ 12.19(s, 1H), 12.04(s, 1H), 7.85(s, 1H), 7.35-7.25(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 C₃₀H₃₃N₁₁O₂S₂[M+H]⁺，644.2338；found：644.2322.

Preparation of Compound CPU-408 was prepared as described for preparation of CPU-403, using 4-methoxybenzylazide (11h) instead of 11c, to give a white solid. The yield was 39.2%. m.p.237-241 ℃;¹H NMR(300MHz，DMSO-d₆)：δ 12.19(s，1H)， 12.04(s，1H)，7.83(s，1H)，7.35-7.28(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 forC₃₀H₃₃N₁₁O₃S₂[M+H]⁺，660.2288； found：660.2268.

preparation of the compound CPU-409 asPreparation of CPU-403 the compound CPU-409 was prepared as described for CPU-403 using 3, 4-dimethoxybenzyl azide (11i) instead of 11c to give a white solid. The yield was 33.3%. m.p.150-155 ℃;¹H NMR(300MHz，DMSO-d₆)：δ 12.19(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，calcdfor C₃₁H₃₅N₁₁O₄S₂[M+H]⁺， 690.2393；found：690.2370.

preparation of compound CPU-410 Compound CPU-410 was prepared as described for preparation of CPU-403, using 4-trifluoromethoxybenzyl azide (11m) instead of 11c, to give a white solid. The yield was 32.5%. m.p.275-280 ℃;¹H NMR(300MHz，DMSO-d₆)：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 C₃₀H₃₀F₃N₁₁O₂S₂[M+H]⁺，698.2056；found：698.2042.

preparation of Compound CPU-411 was prepared as described for preparation of CPU-403 using 2, 6-dimethylbenzylazide (11k) instead of 11c to give a white solid. The yield was 38.7%. m.p.258-263 ℃;¹H NMR(300MHz，DMSO-d₆)：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＝9Hz，2H)，6.80(d，J＝ 9Hz，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，calcdfor C₃₁H₃₅N₁₁O₃S₂[M+ H]⁺，658.2495；found：658.2475.

preparation of compound CPU-412 Compound CPU-412 was prepared as described for preparation CPU-403 using 4-trifluoromethylbenzyl azide (111) instead of 11c to give a white solid. The yield was 32.6%.m.p.273～278℃；¹H NMR(300MHz，DMSO-d₆)：δ 12.17(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 C₃₀H₃₀F₃N₁₁O₃S₂[M+H]⁺，714.2005；found：714.1987.

Example 5

Preparation of Compound VI (CPU601-CPU 612).

Preparation of N- (1- (3-aminopyridazine) piperidine) carbamic acid tert-butyl ester (31)

29(1g, 4.5mmol), 30(2.72g, 13.6mmol) and triethylamine (1.5mL) were dissolved in n-butanol. The reaction mixture was stirred at 180 ℃ under microwave 400W for 1.5h, and the solvent was spin-dried to give an oily product. After dissolving in dichloromethane, the mixture was washed twice with water and twice with saturated salt water. The organic layer was dried over anhydrous sodium sulfate, and the product was obtained as a yellow oily product, which was subjected to column chromatography to obtain a yellow-white solid. The yield was 25.7%.¹H NMR(300MHz，DMSO-d₆)：δ7.13(d，J＝9.63Hz，1H)，6.80(s， 1H)，6.74(d，J＝9.63Hz，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 forC₁₄H₂₃N₅O₂[M+H]⁺，294.1925；found：294.1925.

Preparation of N- (1- (3- (pent-4-ynylamido) pyridazine) piperidine) carbamic acid tert-butyl ester (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 10-15 min. DIPEA (0.26g, 2.011mmol) was added and the reaction was continued, the progress of the reaction being determined by thin layer chromatography every five minutes. The reaction was substantially complete after 20 min. Pouring the reaction liquid into water, separating out solid, and filteringAnd drying the filter cake to obtain a white solid. The yield was 60.4%.¹H NMR(300MHz，DMSO-d₆)：δ 10.67(s，1H)， 8.02(d，J＝9.75Hz，1H)，7.35(d，J＝9.75Hz，1H)，6.82(s，1H)，4.19(d，2H)，3.50(s，1H)， 2.99(t，J＝13.95Hz，2H)，2.78(s，1H)，2.62(t，J＝13.95Hz，2H)，2.44-2.42(m，2H)， 1.81-1.78(m，2H)，1.39(s，11H)ppm.HRMS(ESI)：m/z，calcd for C₁₉H₂₇N₅O₃[M+H]⁺， 374.2187；found：374.2165.

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

Intermediate 32(0.2g, 0.54mmol), trifluoroacetic acid (0.2mL, 2.6mmol) and dichloromethane were stirred and reacted at room temperature for 1 h. The reaction was stopped and dichloromethane was removed under reduced pressure. The pH was adjusted to 7 with saturated sodium bicarbonate, a white solid precipitated and was filtered off with suction. The yield was 100% by thin layer chromatography.¹H NMR(300MHz，DMSO-d₆)：δ10.69(s，1H)，8.05 (d，J＝9.81Hz，1H)，7.39(d，J＝9.81Hz，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＝5.19Hz，2H)，2.47-2.44(m，2H)，1.97-1.93(m，2H)，1.56(m， 2H)ppm.HRMS(ESI)：m/z，calcd for C₁₄H₁₉N₅O[M+H]⁺，274.1662；found：274.1655.

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

Intermediate 33(200mg, 7.4mmol), 2-amino-5-bromo-1, 3, 4-thiadiazole (1320mg, 7.4mmol), NaHCO₃(1184mg, 17.6mmol) was dissolved in 0.5mL ethanol and refluxed at 80 ℃ giving a brown turbid solution. After three hours of reaction, the reaction was complete. Spin-drying ethanol to obtain white solid, adding water, pulping, separating out solid, and vacuum filtering. The filter cake was the product as a tan solid. The yield was 69.7%. Hrms (esi): m/z, calcd for C₁₆H₂₀N₈O₂S[M+H]⁺， 373.1554；found：373.1546.

Preparation of N- (6- (4- (N- (5- (2-pyridyl) acetamido) -1, 3, 4-thiadiazole) amino-piperidine) -pyridazine) pent-4-ynylamide (35)

Intermediate 34(5mg, 0.013mmol), 2-pyridineacetic acid hydrochloride (2.6mg, 0.015mmol), and HATU (7.6mg, 0.02mmol) were dissolved in 1mL DMF and stirred for 10-15min, the solution was a pale yellow clear liquid. DIPEA (5.2mg, 0.04mmol) was added and the reaction was continued for 20 minutes to complete the reaction. The reaction solution was poured into water to precipitate a solid, which was filtered with suction to give a white solid as a filter cake in 75.8% yield.¹H NMR(300MHz，DMSO-d₆)：δ 12.14(s，1H)，10.68(s， 1H)，8.50(d，J＝4.11Hz，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，Je7.05Hz，2H)，2.78(s，1H)，2.63(t， J＝13.86Hz，2H)，2.47-2.44(m，2H)，1.97-1.93(m，2H)，1.56(m，2H)ppm.HRMS(ESI)： m/z，calcd for C₂₃H₂₅N₉O₂S[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 a mixture of intermediate 35(5mg, 0.01mmol), 4-nitrobenzyl azide (11c) (3.99mg, 0.03mmol) in DMF. Microwave reaction at 120 deg.c and 300W for 5 min. The reaction solution was blue turbid and poured into water to precipitate a grey solid. Purifying by column chromatography to obtain blue solid. The yield was 33.7%. m.p.243-247 ℃; hrms (esi): m/z, calcd for C₃₀H₃₁N₁₃O₄S[M+H]⁺，670.2421； found：670.2433.

Preparation of Compound CPU-604

Compound CPU-604 was prepared as described for preparation of CPU-603, using 4-fluorobenzyl azide (11d) instead of 11c, to give a blue solid. The yield was 32.7%. m.p.232-235 ℃;¹H NMR(300MHz，DMSO-d₆)：δ 12.21(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＝8.19Hz，2H)，2.92(t，J＝5.25Hz，2H)，2.74(t，J＝5.25Hz，2H)，2.07(d，J＝7.98Hz，2H)，1.49(d，J＝7.98Hz，2H)ppm.HRMS(ESI)：m/z，calcd for C₃₀H₃₁FN₁₂O₂S[M+ H]⁺，643.2470；found：643.2466.

preparation of compound CPU-605 Compound CPU-605, a blue solid, was prepared as described for preparation of CPU-603, using 4-chlorobenzyl azide (11e) instead of 11 c. The yield was 33.8%. m.p.240-241 ℃;¹H NMR(300MHz，DMSO-d₆)：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＝7.68Hz，2H)，1.49(d，J＝7.68Hz，2H)ppm. HRMS(ESI)：m/z，calcd for C₃₀H₃₁ClN₁₂O₂S[M+H]⁺，659.2175；found：659.2221.

preparation of Compound CPU-606

The compound CPU-606, a blue solid, was prepared as described for CPU-603, using 4-bromobenzylazide (11f) instead of 11 c. The yield was 34.8%. m.p.240-245 ℃; hrms (esi): m/z, calcd for C₃₀H₃₁BrN₁₂O₂S[M+H]⁺，703.1670；found：703.1669.

Preparation of Compound CPU-607

Compound CPU-607 was prepared as described for preparation of CPU-603, using 4-methoxybenzylazide (11h) instead of 11c, to give a blue solid. The yield was 31.6%. m.p.148-152 ℃;¹H NMR(300MHz，DMSO-d₆)：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＝5.25Hz，2H)，5.45(s，2H)，4.16(d，J＝6.96Hz，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 C₃₁H₃₄N₁₂O₃S[M+H]⁺，655.2670； found：655.2672.

preparation of Compound CPU-608

Compound CPU-608 was prepared as described for preparation CPU-603, using 3, 4-dimethoxybenzyl azide (11i) instead of 11c, to give a blue solid. The yield was 33.9%. m.p.108-111 ℃;¹H NMR(300MHz，DMSO-d₆)： 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＝8.16Hz，1H)，6.83(d，J＝8.16Hz，1H)，5.46(s，2H)， 4.20(d，J＝6.96Hz，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 C₃₂H₃₆N₁₂O₄S[M+H]⁺，685.2776；found：685.2775.

preparation of Compound CPU-609

Compound CPU-609 was prepared as described for preparation of CPU-603, using 4-methylbenzyl azide (11g) instead of 11c, to give a blue solid. The yield was 38.5%. m.p.219-222 ℃;¹H NMR(300MHz，DMSO-d₆)：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＝9.27Hz，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 C₃₁H₃₄N₁₂O₂S[M+H]⁺，639.2721；found：639.2716.

preparation of Compound CPU-610

Compound CPU-610 was prepared as described for preparation of CPU-603, using 2, 6-dimethylbenzyl azide (11k) instead of 11c, to give a blue solid. The yield was 39.1%. m.p.161-165 ℃;¹H NMR(300MHz，DMSO-d₆)：δ 12.18(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 C₃₂H₃₆N₁₂O₂S[M+H]⁺，653.2878；found：653.2807.

preparation of Compound CPU-611

Compound CPU-611 was prepared as described for preparation of CPU-603, using 4-trifluoromethylbenzyl azide (111) instead of 11c, to give a blue solid. The yield was 35.4%. m.p.230-233 ℃; hrms (esi): m/z, calcd for C₃₁H₃₁F₃N₁₂O₂S[M +H]⁺，693.2439；found：693.2434.

Preparation of Compound CPU-612

Compound CPU-612 was prepared as described for preparation of CPU-603, using 4-trifluoromethoxybenzyl azide (11m) instead of 11c, to give a blue solid. The yield was 36.9%. m.p.245-250 ℃;¹H NMR(300MHz，DMSO-d₆)： 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＝7.56Hz，2H)，1.47(d，J＝7.56Hz，2H)ppm.HRMS (ESI)：m/z，calcd forC₃₁H₃₁F₃N₁₂O₃S[M+H]⁺，709.2388；found：709.2410.

example 6

Preparation of Compound VIII of general formula (CPU801-CPU 812).

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

Compound 38(0.1g, 0.36mmol) and pentynoic acid (0.038g, 0.39mmol), HATU (0.2g, 0.54mmol) were dissolved in DMF and stirred. DIPEA (0.14g, 1.08mmol) was added thereto and stirred. And (7) detecting by TLC. Pouring the reaction liquid into water, separating out solid, filtering to obtain white solid, and stoving. The yield thereof was found to be 83.4%. Hrms (esi): m/z, calcd for C₁₈H₂₆N₅O₃[M+H]⁺，360.2036；found：360.2034.

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

To compound 39(0.5g, 1.39mmol) was dissolved in 5mL of dichloromethane, 5mL of CF was added₃COOH, reacting for 4 hours at room temperature, and detecting the reaction progress by TLC. DCM, saturated NaHCO, was spin dried under reduced pressure₃Adjusting the pH value to 7-8, separating out off-white solid, and performing suction filtration to obtain the off-white solid with the yield of 75%. Hrms (esi): m/z, calcd for C₁₃H₁₈N₅O[M+ H]⁺，260.1511；found：260.1513.

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

Compound 40(10mg, 0.039mmol), thiadiazole (6.95mg, 0.039mmol) and NaHCO₃(6.24mg, 0.156mmol) was dissolved in 2mL of ethanol, heated to 80 ℃ and refluxed for 4h, and the progress of the reaction was checked by TLC. After the reaction is finished, the solvent is dried in a spinning mode under reduced pressure to obtain a yellow brown solid, 5mL of water is used for pulping, the light yellow solid is obtained through pumping filtration, and the light yellow solid is dried, wherein the yield is 92%. Hrms (esi): m/z, calcd for C₁₅H₁₈N₈OS[M+H]⁺，359.1403；found：359.1413.

Preparation of N- (6- (3- (N- (5- (2-piperidine) acetamido) 1, 3, 4-thiadiazole) amino) -pyrrolidine) pyridazin-pent-4-yneamide (42)

Compound 41(0.1g, 0.28mmol) was dissolved in 2mL DMF with HATU (0.16g, 0.42mmol), 2-pyridineacetic acid hydrochloride (0.054g, 0.31mmol), stirred for 2min, reacted with DIPEA (0.11g, 0.85mmol) for 10min, and detected by TLC. Pouring the reaction liquid into water, separating out solid, and performing suction filtration to obtain a light yellow solid, wherein the yield is 54.5%. Hrms (esi): m/z, calcd for C₂₂H₂₃N₉O₂S[M+H]⁺，478.1774；found：478.1768.

Preparation of Compound CPU-804

To a solution of compound 42(0.1g, 0.21mmol) in DMF was added CuI (7.78mg, 0.041mmol) in water and 4-fluorobenzyl azide (11f) (55.85mg, 0.42 mmol). The reaction mixture was stirred at 120 ℃ for 2min under microwave 300W and checked by TLC. The reaction solution was poured into water to precipitate a solid. DrawerThe crude product was filtered and chromatographed on column (DCM/MeOH 20: 1) to give a blue solid. The yield was 35.5%. m.p.210-212 ℃; hrms (esi): m/z, calcd for C₂₉H₂₉FN₁₂O₂S[M+H]⁺，629.2319；found：629.2317.

Preparation of Compound CPU-805

Compound CPU-805 was prepared as described for preparation of CPU-804 using 4-chlorobenzyl azide (11e) instead of 11f to give a blue solid. The yield was 31.1%. m.p.233-235 ℃; hrms (esi): m/z, calcd for C₂₉H₂₉ClN₁₂O₂S[M +H]⁺，645.2024；found：645.1999.

Preparation of Compound CPU-807

Compound CPU-807 was prepared as described for preparation of CPU-804 using 4-methylbenzylazide (11g) instead of 11f to afford a blue solid. The yield was 34.7%. m.p.242-247 ℃; hrms (esi): m/z, calcd for C₃₀H₃₂N₁₂O₂S[M+ H]⁺，625.2570；found：625.2562.

Preparation of Compound CPU-808

Compound CPU-808 was prepared as described for preparation of CPU-804 using 4-methoxybenzylazide (11h) instead of 11f to give a blue solid. The yield was 32.5%. m.p.221-222 ℃; hrms (esi): m/z, calcd for C₃₀H₃₂N₁₂O₃S[M +H]⁺，641.2519；found：641.2512.

Preparation of Compound CPU-809

Compound CPU-809 was prepared as described for preparation CPU-804 using 3, 4-dimethoxybenzyl azide (11i) instead of 11f to give a blue solid. The yield was 30.8%. m.p.219-220 ℃; hrms (esi): m/z, calcd for C₃₁H₃₄N₁₂O₄S[M+H]⁺，671.2625；found：671.2612.

Preparation of Compound CPU-810

Compound CPU-810 was prepared as described for preparation of CPU-804 using 4-trifluoromethoxybenzyl azide (11m) instead of 11f to give a blue solid. The yield was 36.7%. m.p.243-247 ℃; hrms (esi): m/z, calcd for C₃₀H₂₉F₃N₁₂O₃S[M+H]⁺，695.2237；found：695.2239.

Preparation of Compound CPU-811

Compound CPU-811 was prepared as described for preparation of CPU-804 using 2, 6-dimethylbenzyl azide (11k) instead of 11f to give a blue solid. The yield was 37.2%. m.p.256-258 ℃; hrms (esi): m/z, calcd for C₃₁H₃₄N₁₂O₂S[M +H]⁺，639.2727；found：639.2724.

Preparation of Compound CPU-812

Compound CPU-812 was prepared as described for preparation of CPU-804 using 4-trifluoromethylbenzyl azide (111) instead of 11f to give a blue solid. The yield was 33.3%. m.p.219-221 ℃; hrms (esi): m/z, calcd for C₃₀H₂₉F₃N₁₂O₂S [M+H]⁺，679.2287；found：679.2276.

Example 7

Preparation of Compound X of general formula (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 triethylamine was added. The reaction mixture was stirred at microwave 400W, 180 ℃ for 1.5 h. Spin-drying solvent, adding dichloromethane for dissolution, washing with water for three times, washing with saturated ammonium chloride for three times, drying with anhydrous sodium sulfate, and performing column chromatography to obtain white solid with yield of 37%. Hrms (esi): m/z, calcd for C₁₃H₂₂N₅O₂[M+H]⁺，280.1773；found：280.1777.

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

Compound 45(0.1g, 0.36mmol) and 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 to the chamberThe reaction is carried out for 1 h. And (7) detecting by TLC. Pouring the reaction liquid into water, separating out solid, filtering to obtain white solid, and stoving. The yield thereof was found to be 83.4%. Hrms (esi): m/z, calcdfor C₁₈H₂₆N₅O₃[M+H]⁺，360.2036；found：360.2034.

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

To compound 46(0.5g, 1.39mmol) was dissolved in 5mL of dichloromethane, 5mL of CF was added₃COOH, reacting for 4 hours at room temperature, and detecting the reaction progress by TLC. DCM, saturated NaHCO, was spin dried under reduced pressure₃Adjusting the pH value to 7-8, separating out off-white solid, and performing suction filtration to obtain the off-white solid with the yield of 75%. Hrms (esi): m/z, calcd for C₁₃H₁₈N₅O[M+ H]⁺，260.1511；found：260.1513.

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

Compound 47(10mg, 0.039mmol), thiadiazole (6.95mg, 0.039mmol) and NaHCO₃(6.24mg, 0.156mmol) was dissolved in 2mL of ethanol, heated to 80 ℃ and refluxed for 4h, and the progress of the reaction was checked by TLC. After the reaction is finished, the solvent is dried in a spinning mode under reduced pressure to obtain a yellow brown solid, 5mL of water is used for pulping, the light yellow solid is obtained through pumping filtration, and the light yellow solid is dried, wherein the yield is 92%. Hrms (esi): m/z, calcd for C₁₅H₁₈N₈OS[M+H]⁺，359.1403；found：359.1413.

Preparation of N- (6- (3- (N- (5- (2-piperidine) acetamido) 1, 3, 4-thiadiazole) amino) -pyrrolidine) pyridazin-pent-4-yneamide (49)

Compound 48(0.1g, 0.28mmol) was dissolved in 2mL DMF with HATU (0.16g, 0.42mmol), 2-pyridineacetic acid hydrochloride (0.054g, 0.31mmol), stirred for 20min, reacted with DIPEA (0.11g, 0.85mmol) for 10min, and detected by TLC. Pouring the reaction liquid into water, separating out solid, and performing suction filtration to obtain a light yellow solid, wherein the yield is 54.5%. Hrms (esi): m/z, calcdfor C₂₂H₂₃N₉O₂5[M+H]⁺，478.1774；found：478.1768.

Preparation of Compound CPU-1001

To compound 49(0.1g, 0)21mmol) was added to a solution of CuI (7.78mg, 0.041mmol) in water and 4-methylbenzyl azide (11g) (55.85mg, 0.42 mmol). The reaction mixture was stirred at 120 ℃ for 2min under microwave 300W and checked by TLC. The reaction solution was poured into water to precipitate a solid. The crude product was filtered off with suction and chromatographed on a column (DCM/MeOH 20: 1) to give a blue solid. The yield was 33.5%. m.p.208-212 ℃; hrms (esi): m/z, calcd for C₃₀H₃₂N₁₂O₂5[M+H]⁺，625.2570；found：625.2562.

Preparation of Compound CPU-1002

The compound, CPU-1002, was prepared as described for preparation of CPU-1001, using 4-methoxybenzylazide (11h) instead of 11g, to afford a white solid. The yield was 41.3%. m.p.219-222 ℃; hrms (esi): m/z, calcd for C₃₀H₃₂N₁₂O₃S[M +H]⁺，641.2519；found：641.2512.

Preparation of Compound CPU-1003

The compound, CPU-1003, was prepared as described for preparation of CPU-1001, using 4-trifluoromethylbenzyl azide (111) instead of 11g, to afford a white solid. The yield was 37.6%. m.p.194-198 ℃; hrms (esi): m/z, calcd for C₃₀H₂₉F₃N₁₂O₂S[M+H]⁺，679.2287；found：679.2276.

Preparation of Compound CPU-1004

The compound CPU-1004 was prepared as described for preparation of CPU-1001 using 4-trifluoromethoxybenzyl azide (11m) instead of 11g to give a white solid. The yield was 39.0%. m.p.199-204 ℃; hrms (esi): m/z, calcd for C₃₀H₂₉F₃N₁₂O₃S[M+H]⁺，695.2237；found：695.2239.

Example 8

Preparation of Compound of formula XII (CPU1201-CPU 1212).

Preparation of Compound CPU-1204

To a mixture of compound 34(0.1g,0.26mmol) was added to a solution of CuI (7.78mg, 0.041mmol) in water and 4-fluorobenzyl azide (11d) (55.85mg, 0.42 mmol). The reaction mixture was stirred at 120 ℃ for 5min under microwave 300W and checked by TLC. The reaction solution was poured into water to precipitate a solid. The crude product was filtered off with suction and chromatographed on a column (DCM/MeOH 20: 1) to give a white solid. The yield was 35.5%. m.p.178-180 ℃; hrms (esi): m/z, calcd for C₂₃H₂₆FN₁₁OS[M +H]⁺，524.2105；found：524.2098.

Preparation of compound CPU-1205

Compound CPU-1205 was prepared as described for preparation of CPU-1204, using 4-chlorobenzyl azide (11e) instead of 11d, to give a white solid. The yield was 37.3%. m.p.200-203 ℃; hrms (esi): m/z, calcd for C₂₃H₂₆ClN₁₁OS[M+ H]⁺，540.1809；found：540.1830.

Preparation of Compound CPU-1206

Compound CPU-1206 was prepared as described for preparation of CPU-1204, using 4-bromobenzylazide (11f) instead of 11d, to give a white solid. The yield was 32.1%. m.p.175-177 ℃; hrms (esi): m/z, calcd for C₂₃H₂₆BrN₁₁OS[M+ H]⁺，584.1304；found：584.1297.

Preparation of Compound CPU-1207

Compound CPU-1207 was prepared as described for preparation of CPU-1204, using 4-methylbenzylazide (11g) instead of 11d, to give a white solid. The yield was 37.2%. m.p.200-205 ℃; hrms (esi): m/z, calcd for C₂₄H₂₉N₁₁OS[M+ H]⁺，520.2356；found：520.2376.

Preparation of Compound CPU-1208

Compound CPU-1208 was prepared as described for preparation of CPU-1204 using 4-methoxybenzylazide (11h) instead of 11d to give a white solid. The yield was 34.6%. m.p.210-212 ℃; hrms (esi): m/z, calcd for C₂₄H₂₉N₁₁O₂S[M +H]⁺，536.2305；found：536.2299.

Preparation of Compound CPU-1209

As described in preparation of CPU-1204, makeCompound CPU-1209 was prepared using 3, 4-dimethoxybenzyl azide (11i) instead of 11d, to give a white solid. The yield was 39.5%. m.p.200-202 ℃; hrms (esi): m/z, calcd for C₂₅H₃₁N₁₁O₃5[M+H]⁺，566.2410；found：566.2403.

Preparation of Compound CPU-1210

Compound CPU-1210 was prepared as described for preparation of CPU-1204, using 4-trifluoromethoxybenzyl azide (11m) instead of 11d, to give a white solid. The yield was 34.4%. m.p.210-213 ℃; hrms (esi): m/z, calcd for C₂₄H₂₇F₃N₁₁O₂S[M+H]⁺，590.2022；found：590.2010.

Preparation of Compound CPU-1212

Compound CPU-1212 was prepared as described for preparation of CPU-1204 using 4-trifluoromethylbenzyl azide (111) instead of 11g to give a white solid. The yield was 37.1%. m.p.175-180 ℃; hrms (esi): m/z, calcd for C₂₄H₂₇F₃N₁₁OS[M+H]⁺，574.2073；found：574.2079.：

Example 9: is the pharmacological test and result of the part compound of the invention:

(1) in vitro tumor cell proliferation inhibition assay

The purpose of the test is as follows: the proliferation inhibitory effect of the test compound on tumor cells was observed. The mechanism research shows that: triple negative breast cancer MDA-MB-436 cells and colon cancer HCT116 cells are highly sensitive to glutamine, depend excessively on glutamine to maintain the growth and reproduction of cells, and present 'glutamine addiction'. Meanwhile, the tumor gene map shows that: the glutaminase GLS1 gene level and protein level of MDA-MB-436 and HCT116 cells were highly expressed, and GLS1 showed strong enzyme activity. Therefore, the effect of the compound on the proliferation inhibition of the two cell lines can be reflected in that the designed compound achieves the effect of resisting the proliferation of tumor cells by inhibiting GLS 1.

The test principle is as follows: the MTT assay is based on the living cell metabolite reducing agent MTT (known as 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyltetrazolium bromide salt, trade name: thiazole blue). MTT is a yellow compound, a dye that accepts hydrogen ions, and acts on the respiratory chain in mitochondria of living cells, and the tetrazolium ring is cleaved by succinate dehydrogenase and cytochrome C to produce blue formazan crystals, the amount of which is only proportional to the number of living cells (i.e., when cells die, succinate dehydrogenase disappears, and MTT cannot be reduced). The formazan crystals formed by reduction can be dissolved in DMSO, and the OD value of the optical density at 492nm is measured by a microplate reader to reflect the number of living cells.

The test method comprises the following steps: 1) inoculating cells: after the cells grow to a logarithmic growth phase in a culture solution containing 10% FBS, digesting the cells by using pancreatin to prepare a single cell suspension, and respectively inoculating 6000 HCT116 cells or 4000 MDA-MB-436 cells per well into a 96-well plate; 2) administration: 37 ℃ and 5% CO₂After 24h of culture, dissolving the compound to be detected by DMSO, diluting the dissolved compound to 0.1M/L by culture solution respectively, administering with concentration gradient of 10nM, 100nM, 1 μ M, 10 μ M and 100 μ M respectively, and setting blank group and solvent control group; 3)37 ℃ and 5% CO₂Continuously culturing for 72 h; 4) color appearance: mu.l of MTT solution (5mg/ml) was added to each well, incubation was continued for 4 hours, and the culture supernatant in the wells was carefully aspirated. Adding 150 mul DMSO into each hole, and oscillating for 10 minutes to fully melt the crystal; 5) color comparison: selecting 492nm wavelength, measuring the light absorption value of each pore on an enzyme-linked immunosorbent instrument, recording the result, calculating the growth inhibition rate, and drawing a growth inhibition curve.

TABLE 1 inhibition of tumor cell proliferation (MDA-MB-436, HCT116) by partial compounds of the invention

(2) Test for the inhibitory Effect of Compounds on Glutamine hydrolase GLS1 in vitro

The purpose of the test is as follows: and confirming whether the test compound affects the growth and reproduction of tumor cells by acting on GLS1 so as to block glutamine metabolism.

The test steps are as follows: human GLS1 protein (0.1mM hKGA) was incubated with a concentration of compound at 25 ℃ for 10min at 50mM Tris-AcetatepH ═ 8.6, 0.2mM EDTA. Then, 200mM glutamine was added to start the first reaction, and the reaction was carried out at 37 ℃ for 60 minutes. The reaction was quenched by addition of 0.6M HCl. 3.7units GDH, 160 mM Tris-Acetate pH 9.4, 400mM hydrazine, 5mM ADP, 2mM NAD were added⁺Incubate at 25 ℃ for 30 min. Finally, the absorbance value of the sample at 340nm was measured.

TABLE 2 test results of the inhibition of Glutamine hydrolase GLS1 by some of the compounds of the invention

(3) Protein heat stability migration experiment

Purpose of the experiment: the interaction between the small molecule compound and the glutaminase GLS1 protein was examined. It was confirmed whether the inhibitor acts directly on the GLS1 protein.

The experimental process comprises the following steps: the change in fluorescence value of the system was monitored using SYPRO Orange (Invitrogen) as a fluorescent dye, and the excitation and emission wavelengths were set to 492nm (FAM) and 610nm (ROX), respectively. mu.M GLS1 protein, 5 Xfluorescent dye and different concentrations of compounds were added to 20. mu.L of reaction buffer (25mM HEPES pH 8.0, 150mM NaCl). The temperature of the System was gradually raised from 25 ℃ to 95 ℃ at a 1% temperature rise rate on a 7500Fast RT-PCR System (ABI) instrument, while the change in fluorescence intensity with temperature was recorded at 20-second intervals. Further, the dissolution temperature (Tm) of EED under different compound concentration conditions was calculated using Boltzmann fitting method in Protein thermal Shift Software Version 1.1(ABI) program. The experimental results are shown in fig. 1:

(4) indicating plasma resonance experiments

Surface plasmon resonance test samplingThis was done with BIACORE T200 instrument (GE Healthcare). Freshly purified EED protein (concentration 10mg/ml) was used with 10mM CH₃COONa (pH4.2) was diluted to 0.1mg/ml, and GLS1 protein was coupled to CM5 chip by standard amino coupling method. After stepwise dilution of the GLS1 inhibitor with HBS-EP buffer (10mM HEPES (pH7.4), 150mM NaCl, 3mM EDTA, 0.005% (v/v) surfactant P20), the sample was continuously injected at a flow rate of 20. mu.l/s for 60 seconds and dissociated for 120 seconds, and the change in response signal with time during the process was recorded. The binding rate constant (K) of the GLS1 inhibitor to GLS1 protein was calculated using the kinetic analysis module of the BIAEevaluation Software (GE Healthcare) program_on) Dissociation rate constant (K)_off) And dissociation constant (K)_d). The experimental results are shown in fig. 2:

(5) assay for cellular glutamic acid level

The mechanism research shows that: GLS1 inhibitors block glutamine hydrolysis by inhibiting the activity of GLS1, resulting in a decrease in the glutamate, the hydrolysis product of intracellular glutamine. Therefore, the inhibition of GLS1 by the compound can be indirectly reflected by detecting the content of glutamic acid in cells. The glutamic acid content of CPU-301 and CB 839-treated HCT116 cells was measured, and the results are shown in FIG. 3. The experimental results show that: the compounds CPU-301 and CB839 can obviously reduce the content of intracellular glutamic acid in a concentration-dependent manner, and indirectly prove that the compound CPU-301 plays a role by inhibiting GLS 1.

(6) Assay of ROS content in cells

ROS are key regulators of cancer cell growth. Induction of oxidative stress can lead to preferential killing of cancer cells. Various drugs having direct or indirect effects on ROS have been used for effective cancer treatment. The mechanism research shows that: the GLS1 inhibitor can promote the increase of ROS level in tumor cells by blocking glutamine metabolism, thereby playing a certain role in killing tumors. Active oxygen levels in CPU-301 and CB839 treated HCT116 cells were determined and are shown in FIG. 4. The experimental results show that: the compounds CPU-301 and CB839 can obviously induce the increase of the intracellular ROS level in a concentration-dependent manner, and cause a certain killing effect on tumor cells.

Claims

1. A glutaminase GLS1 inhibitor containing triazole structure with a general formula (I) or a medicinal salt thereof,

wherein n is an integer of 1 to 4;

Wherein CH or CH₂Any of the hydrogens in (a) may be substituted with an alkyl or alkoxy group; the hydrogen in the-NH group may be substituted by an alkyl group; -CH₂CH₂、CH₂CH₂CH₂Single CH in the radical₂May be substituted by hydroxy; r₁And R₂The two groups together with the atoms to which they are attached may optionally form a cycloalkane;

R₁、R₂respectively as follows: H. alkyl, alkoxy or hydroxy;

R₃comprises the following steps: alkanes, substituted alkanes, aromatics, aromatic alkanes, cyano groups, cycloalkanes, cycloparaffins, hydrogen, halogens, halogen-substituted alkanes, heteroatomic aromatics, heteroatomic aromatic alkanes, heteroatomic cycloalkanes, C (R)₆)(R₇)(R₈)、N(R₉)(R₁₀)、OR₁₁The hydroxyl group in the above substituent group may be acetylated to C (O) R₇；

R₆、R₇、R₈respectively as follows: hydrogen, substituted or unsubstituted alkyl, hydroxy, hydroxyalkyl, amino, acetamido, alkene, alkyne, alkoxy, aryl, arylalkyl, cycloalkane, heterocycle, or heteroatom aromatics;

R₉、R₁₀respectively as follows: hydrogen, substituted or unsubstituted alkyl, hydroxy, hydroxyalkyl, amino, acetamido, alkene, alkyne, alkoxy, aryl, arylalkyl, cycloalkane, heterocycle, heteroatom aromatics, wherein the hydroxy group in the above-mentioned substituent groups can be acetylated to C (O) R₇；

R₁₁Comprises the following steps: hydrogen, substituted or unsubstituted alkyl, hydroxy, hydroxyalkyl, amino, acetamido, alkene, alkyne, alkoxy, aryl, arylalkyl, cycloalkane, heterocycle, heteroatom aromatics, wherein the hydroxy group in the above-mentioned substituent groups can be acetylated to C (O) R₇。

2. The glutaminase GLS1 inhibitor containing triazole structure shown in the general formula (I) or the pharmaceutically acceptable salt thereof according to claim 1, wherein the inhibitor is characterized in that: l is CH₂CH₂。

3. The glutaminase GLS1 inhibitor containing triazole structure in the general formula (I) according to claim 1 or a pharmaceutically acceptable salt thereof is any one of the following:

-Ph、4’-CH₃-Ph、4’-CF₃-Ph、4’-F-Ph、4’-Cl-Ph、4’-Br-Ph、4’-OCH₃-Ph、3’-OCH₃-Ph、3’-OH-Ph、3’-NH₂-Ph、4’-NH₂-Ph、2’-Pyridine；

Wherein R is-Ph, 4 '-CN-Ph, 4' -NO₂-Ph、4’-F-Ph、4’-Cl-Ph、4’-Br-Ph、4’-CH₃-Ph、4’-OCH₃-Ph、3’-OCH₃-4’-OCH₃-Ph、2’-CH₃-4’-CH₃-Ph、2’-CH₃-6’-CH₃-Ph；

Wherein R is 4' -CH₃-Ph、4’-CN-Ph、4’-NO₂-Ph、4’-F-Ph、4’-Cl-Ph、4’-Br-Ph、4’-OCH₃-Ph、3’-OCH₃-4’-OCH₃-Ph、2’-CH₃-4’-CH₃-Ph、2’-CH₃-6’-CH₃-Ph、4’-CF₃-Ph、4’-OCF₃-Ph；

Wherein R is-Ph, 4 '-CN-Ph, 4' -NO₂-Ph、4’-F-Ph、4’-Cl-Ph、4’-Br-Ph、4’-CH₃-Ph、4’-OCH₃-Ph、3’-OCH₃-4’-OCH₃-Ph、4’-OCF₃-Ph、2’-CH₃-6’-CH₃-Ph、4’-CF₃-Ph；

Wherein R is-Ph, 4 '-CN-Ph, 4' -NO₂-Ph、4’-F-Ph、4’-Cl-Ph、4’-Br-Ph、4’-CH₃-Ph、4’-OCH₃-Ph、3’-OCH₃-4’-OCH₃-Ph、4’-OCF₃-Ph、2’-CH₃-6’-CH₃-Ph、4’-CF₃-Ph。

4. A pharmaceutical composition, which comprises a therapeutically effective amount of one or more glutaminase GLS1 inhibitors containing triazole structure according to any of claims 1 to 3, or pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier.

5. A pharmaceutical composition, which comprises a therapeutically effective amount of one or more glutaminase GLS1 inhibitors containing triazole structure according to any one of claims 1 to 3 or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable excipients.

6. The preparation method of the glutaminase GLS1 inhibitor containing triazole structure with the general formula (I) or the pharmaceutically acceptable salt thereof according to claim 1, comprising the following steps:

and (3) respectively reacting the compound II with different alkynes or azides to obtain corresponding compounds III-1 or III-2, and respectively reacting the compounds III-1 and III-2 with different azides or alkynes under the catalysis of CuI to obtain a final product of the glutaminase GLS1 inhibitor containing a triazole structure, which has the general formula (I).

7. Use of the glutaminase GLS1 inhibitor containing a triazole structure, which has the general formula (I) according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof in preparing a medicament for treating GLS 1-mediated diseases.

8. The use according to claim 7, wherein the disease is colon cancer, triple negative breast cancer or lung cancer.