2, 4-disubstituted thiadiazole derivatives and their use as GLS1 inhibitors

Abstract

The invention provides a 2, 4-disubstituted thiadiazole derivative and application of the 2, 4-disubstituted thiadiazole derivative as a GLS1 inhibitor, and belongs to the field of chemical medicines. The 2, 4-disubstituted thiadiazole derivative is a compound as shown in a formula I, or a salt thereof, or a stereoisomer thereof. The 2, 4-disubstituted thiadiazole derivative disclosed by the invention can be used as a GLS1 inhibitor and is used for preventing and / or treating diseases related to GLS1, such as cancers. Meanwhile, the 2, 4-disubstituted thiadiazole derivative disclosed by the invention is good in metabolism and has a good clinical application prospect. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of chemical medicine, and specifically relates to 2,4-disubstituted thiadiazole derivatives and their use as GLS1 inhibitors. Background Art

[0002] Tumor cells are different from normal cells due to "metabolic reprogramming". Their characteristic is that their unique energy metabolism can maintain their rapid growth and reproduction. The Warburg effect points out that compared with normal cells, tumor cells tend to aerobic glycolysis of glucose, that is, glucose is metabolized into lactic acid and excreted outside the cell, resulting in a significant reduction in metabolites produced by the tricarboxylic acid cycle (TCA). Therefore, the simple glycolysis process can no longer meet the energy supply required for the growth and proliferation of tumor cells. In order to maintain the operation of TCA, it must be effectively supplemented through other pathways, namely the "anaplastic effect". Studies have found that glutamate produced by glutamine hydrolysis can be converted into α-ketoglutaric acid (α-Ketoglutaric Acid, α-KG) and enter TCA, which is the most important pathway in the anaplastic effect. A large number of studies have shown that glutamine metabolism is abnormally active in tumor cells and plays a vital role in maintaining tumor cell growth, proliferation, and metastasis.

[0003] Glutamine is the most abundant conditionally essential amino acid in blood and muscle, accounting for about 20% and 40% respectively, which indicates its extensive participation in intracellular metabolic reactions. Stable and high concentrations of glutamine in the blood provide abundant carbon and nitrogen atoms to support biosynthesis, energy and cell homeostasis. Tumor cells use this energy to drive their own growth and proliferation. Under the action of many transport proteins, glutamine is transported into cells, and under the action of mitochondrial glutaminase (Glutaminase, GLS), it is converted into glutamate and free ammonium ions, and then further metabolized through two different pathways. Glutamate, a product of GLS catalysis, is then converted into α-KG, which enters the TCA cycle and produces ATP by producing NADH and FADH2. Many rapidly growing tumor cells consume and utilize glutamine in large quantities, showing the phenomenon of "glutamine dependence". More and more studies have shown that glutamine is not only an important carbon source for the synthesis of amino acids and fatty acids in tumor cells, but also an important nitrogen source for the biosynthesis of purine and pyrimidine nucleotides. Glutamine replenishment can provide TCA with intermediate metabolites that are lacking due to the enhanced anabolism of tumor cells, providing them with the energy required for growth and reproduction. At the same time, it can also generate metabolic intermediates for the synthesis of endogenous proteins, lipids, nucleic acids and other biological substances that are essential for cell growth and proliferation, and maintain the homeostasis of biosynthesis and energy metabolism in tumor cells. This is why glutamine metabolism has received so much attention.

[0004] GLS in mitochondria is currently recognized as the most critical target protein in the glutamine metabolic pathway and the most intensively studied drug target in the glutamine metabolic pathway. GLS is mainly responsible for catalyzing the hydrolysis of glutamine to glutamate, which is further converted into α-KG and enters TCA, participating in ATP generation and providing carbon and nitrogen sources for biosynthesis. In mammals, glutaminase is mainly divided into two subtypes: kidney-type glutaminase (GLS1) and liver-type glutaminase (GLS2). A large number of studies have shown that GLS1 plays a decisive role in tumors with active glutamine metabolism. For example, SIRT5 (Sirtuin5) overexpressed in lung cancer can desuccinylate GLS1, thereby inhibiting the activity of GLS1. In addition, inhibiting the glutaminase metabolic axis regulated by MYC is an effective synthetic lethal method that can be used to treat chemoresistant ovarian cancer.

[0005] The first non-competitive glutaminase inhibitors discovered for the key target GLS1 were glutamine analogs such as 6-diazo-5-oxo-L-norleucine (DON) and azaserine. These compounds inhibit the activity of glutaminase by irreversibly binding to the active site of glutaminase. However, the over-activated diazo group leads to poor selectivity. In addition to targeting GLS1, they also have significant inhibitory effects on NAD synthetase, CTP synthetase, and FGAR aminotransferase. Therefore, the clinical development of DON and its diazo analogs has been greatly limited due to systemic side effects caused by off-target and a narrow therapeutic window. The two subsequently developed allosteric small molecule glutaminase inhibitors 968 and BPTES have significantly improved selectivity and reduced side effects, but due to their poor in vitro kinase activity, they are only used in preclinical studies. CB-839 and IPN60090, modified with BPTES as the lead compound, are new reversible non-competitive glutaminase inhibitors. Their mechanism of action is similar to that of BPTES, but the anti-tumor activity of the two small molecules in vivo has been greatly improved, so they are also undergoing phase II clinical studies. Tumor immune checkpoint inhibitors PD1 and PDL1 have brought new breakthroughs in tumor treatment, but the efficacy of tumor treatment is only about 20%, and among these patients who benefit, 15%-35% of patients have recurrence of the disease after use. New studies have shown that this drug resistance failure is closely related to tumor metabolic reprogramming, directly inducing tumor cell apoptosis by targeting the metabolic glutamine pathway, reshaping the tumor microenvironment, enhancing cellular immune function, and activating T effector cells, NK cells, and NKT cells. The DON prodrug small molecule JHU083 reported in Science in 2019 confirms this well. JHU083 has the dual functions of destroying the tumor microenvironment and activating the immune system. It can not only break the resistance of tumor cells to immune checkpoint inhibitors, but also directly activate T cells to kill tumors. The researchers named this treatment "Metabolic Checkpoint" inhibitors. These studies have confirmed that there are huge differences in the metabolic plasticity of tumor cells and T cells, providing new ideas and directions for targeting tumor glutamine metabolism and tumor immunity. However, there are currently only two GLS1 inhibitors undergoing clinical research, of which CB-839 is progressing slowly and IPN60090 has just entered Phase I clinical trials. Therefore, the development of new GLS1 inhibitors has strong clinical research significance. Summary of the invention

[0006] The object of the present invention is to provide 2,4-disubstituted thiadiazole derivatives and their use as GLS1 inhibitors.

[0007] The present invention provides a compound represented by formula I, or a salt thereof, or a stereoisomer thereof:

[0008]

[0009] in,

[0010] L is selected from

[0011] R 1 is a substituent at any position on the benzene ring, selected from

[0012] n is 0, 1, 2 or 3;

[0013] R 2 is selected from substituted or unsubstituted phenyl, substituted or unsubstituted 5-6 membered heterocycloalkyl;

[0014] The substituent of the phenyl group is selected from substituted or unsubstituted 5-6 membered heterocycloalkyl and C1-C6 alkyl;

[0015] The substituent of the heterocycloalkyl group is selected from substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkenyl;

[0016] The substituent of the alkyl group is selected from substituted or unsubstituted phenyl;

[0017] The two substituents on the same carbon atom of the alkenyl group are linked to form =0.

[0018] Furthermore, the compound is represented by Formula II:

[0019]

[0020] in,

[0021] R 1 is a substituent at any position on the benzene ring, selected from

[0022] n is 0, 1, 2 or 3;

[0023] R 2 Selected from

[0024] R 3 Selected from

[0025] R 5 Selected from C1-C6 alkyl;

[0026] R 4 Selected from C1-C6 alkyl,

[0027] Furthermore, the compound is represented by formula III:

[0028]

[0029] in,

[0030] R 1 is a substituent at any position on the benzene ring, selected from

[0031] n is 0, 1, 2 or 3;

[0032] R 2 Selected from

[0033] R 3 is a substituent at any position on the benzene ring, selected from

[0034] R 5 Selected from C1~C6 alkyl.

[0035] Furthermore, the compound is represented by formula IV:

[0036]

[0037] in,

[0038] L' connects any position of two benzene rings, selected from

[0039] R 5 Selected from C1~C6 alkyl.

[0040] Furthermore, the compound is one of the following compounds:

[0041]

[0042] The present invention also provides use of the aforementioned compound, or a salt thereof, or a stereoisomer thereof in the preparation of a GLS1 inhibitor.

[0043] The present invention also provides use of the aforementioned compound, or a salt thereof, or a stereoisomer thereof in the preparation of a drug for preventing and / or treating a disease associated with GLS1.

[0044] Furthermore, the drug is a drug for preventing and / or treating cancer.

[0045] Furthermore, the cancer is lung cancer, liver cancer, colon cancer, rectal cancer, breast cancer, ovarian cancer, or cervical cancer.

[0046] The present invention also provides a medicine, which is a preparation prepared by using the aforementioned compound, or its salt, or its stereoisomer as an active ingredient, and adding pharmaceutically acceptable excipients or auxiliary ingredients.

[0047] The compounds and derivatives provided in the present invention can be named according to the IUPAC (International Union of Pure and Applied Chemistry) or CAS (Chemical Abstracts Service, Columbus, OH) nomenclature system.

[0048] Definitions of terms used in the present invention: Unless otherwise stated, the initial definitions provided for groups or terms in this document apply to the groups or terms throughout the specification; for terms that are not specifically defined in this document, the meaning that a person skilled in the art can give them should be given based on the disclosure and context.

[0049] "Substitution" refers to the replacement of a hydrogen atom in a molecule by another different atom or molecule.

[0050] The minimum and maximum carbon atom content in a hydrocarbon group is indicated by a prefix, for example, the prefix C a ~C b Alkyl refers to any alkyl group containing from "a" to "b" carbon atoms. Thus, for example, "C1-C6 alkyl" refers to an alkyl group containing from 1 to 6 carbon atoms.

[0051] "Alkyl" refers to a saturated hydrocarbon chain having a specified number of carbon atoms. For example, C1-C6 alkyl refers to an alkyl group having 1 to 6 carbon atoms, i.e., 1, 2, 3, 4, 5 or 6 carbon atoms. Alkyl groups can be straight or branched. Representative branched alkyl groups have one, two or three branches. Alkyl groups include methyl, ethyl, propyl (n-propyl and isopropyl), butyl (n-butyl, isobutyl and tert-butyl), pentyl (n-pentyl, isopentyl and neopentyl) and hexyl, etc.

[0052] "Halogen" is fluorine, chlorine, bromine or iodine.

[0053] "Cycloalkyl" refers to a saturated or unsaturated all-carbon monocyclic or polycyclic ring (including fused rings, spiro rings or bridged rings) without a conjugated π electron system, such as but not limited to: wait.

[0054] "Heterocycloalkyl" refers to a cycloalkyl group in which at least one carbon atom is replaced by a heteroatom, wherein the heteroatom is O, N or S, and is a saturated or unsaturated monocyclic or polycyclic ring (including fused rings, spirocyclic rings or bridged rings) without a conjugated π electron system, such as but not limited to: wait.

[0055] "Aryl" refers to an all-carbon monocyclic or polycyclic ring (including fused rings, spirocyclic rings or bridged rings) with a conjugated π electron system, including but not limited to phenyl, naphthyl, phenanthrenyl, anthracenyl, fluorenyl and indenyl. The aromatic ring may be fused to other cyclic groups (including saturated and unsaturated rings), but cannot contain heteroatoms such as O, N or S, and the point of attachment to the parent must be on a carbon atom on the ring with a conjugated π electron system, including but not limited to wait.

[0056] "Heteroaryl" refers to an aromatic group in which at least one carbon atom on the ring of the conjugated π-electron system is replaced by a heteroatom, wherein the heteroatom is O, N or S, such as but not limited to thienyl, furyl, isothiazolyl and the like.

[0057] The salts in the present invention are pharmaceutically acceptable salts, and the pharmaceutically acceptable salts include acetate, adipate, aspartate, benzoate, benzenesulfonate, bicarbonate, carbonate, bisulfate, sulfate, borate, camphorsulfonate, citrate, cyclamate, edisylate, ethanesulfonate, formate, fumarate, glucoheptonate, gluconate, glucuronate, hexafluorophosphate, hydrochloride, hydrobromide, hydroiodide, isethionate, lactate, malate, maleate, malonate, methanesulfonate, methylsulfate, naphthoate, teasulfonate, nicotinate, nitrate, orotate, oxalate, palmitate, dihydroxynaphthoate, phosphate, hydrogen phosphate, dihydrogen phosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, toluenesulfonate, trifluoroacetate, xinafoate, methanesulfonate, p-toluenesulfonate, quaternary ammonium salt or succinate, etc.

[0058] Compared with the prior art, the present invention has the following beneficial effects:

[0059] The present invention provides a 2,4-disubstituted thiadiazole derivative, which can be used as a GLS1 inhibitor for preventing and / or treating diseases related to GLS1, such as cancer. At the same time, the 2,4-disubstituted thiadiazole derivative of the present invention has good metabolism and has good clinical application prospects.

[0060] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0061] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 The results of different concentrations of compound 11 and BPTES inhibiting the GLS1-mediated ROS pathway: A is an image of ROS changes after A549 cells were treated with different concentrations of compounds; B is the change in ROS amount in A549 cells after statistical analysis; C is an image of ROS changes after HCT116 cells were treated with different concentrations of compounds; D is the change in ROS amount in HCT116 cells after statistical analysis.

[0063] Figure 2 The in vivo antitumor activity results of compound 11 and BPTES of the present invention: A is the antitumor effect of compound 11 in the A549 model; B is the tumor weight of mice in the A549 model; C is the weight change of mice in the A549 model; D is the antitumor effect of compound 11 in the HCT116 model; E is the tumor weight of mice in the HCT116 model; F is the weight change of mice in the HCT116 model. DETAILED DESCRIPTION

[0064] The raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

[0065] The synthetic route one of the present invention is as follows:

[0066]

[0067] (a) K2CO3, piperidine derivative, DMF, 80°C, 3 hours;

[0068] (b) DMF, N,N-diisopropylethylamine, phenylacetyl chloride, room temperature, 30 minutes;

[0069] (c) NaOH, MeOH / H2O, 50°C, 1 hour;

[0070] (d) NaH, methyl 4(or 3)-(bromomethyl)benzoate, DMF, 0°C, 2 hours;

[0071] (e) NaOH, MeOH / H2O, 50°C, 1 hour;

[0072] (f)R 2 NH2, HATU, N,N-diisopropylethylamine, DMF, room temperature, 2 hours.

[0073] The second synthetic route of the present invention is as follows:

[0074]

[0075] (a) K2CO3, BOC-piperazine, DMF, 80°C, 3 hours;

[0076] (b) DMF, N,N-diisopropylethylamine, phenylacetyl chloride, room temperature, 30 minutes;

[0077] (c) CF3COOH, DCM, room temperature, 2 h;

[0078] (d) K2CO3, methyl 4(or 3)-(bromomethyl)benzoate, acetonitrile, 60°C, 2 hours;

[0079] (e) NaOH, MeOH / H2O, 50°C, 1 hour;

[0080] (f)R 2 NH2, HATU, N,N-diisopropylethylamine, DMF, room temperature, 2 hours.

[0081] The synthetic route three of the present invention is as follows:

[0082]

[0083] (a) K2CO3, 4 (or 3)-nitrobenzyl bromide, acetonitrile, 60°C, 2 hours;

[0084] (b) Pd / C, H2, MeOH, 40°C, 24 h;

[0085] (c)R 2 COOH, HATU, N,N-diisopropylethylamine, DMF, room temperature, 2 hours.

[0086] Example 1. Synthesis of 2-(1-(5-amino-1,3,4-thiadiazol-2-yl)piperidin-4-yl)-1-ethanol (2)

[0087] 2-Amino-5-bromo-1,3,4-thiadiazole (5.0 g, 27.7 mmol) was added to DMF (50 mL), followed by potassium carbonate (9.6 g, 69.3 mmol) and 4-piperidineethanol (3.6 g, 27.7 mmol). The mixture was reacted at 80 °C for 3 h. After the reaction was completed, the reaction solution was added to a large amount of water, and a large amount of solid was precipitated. The solid was filtered and the filter cake was dried to obtain the target product with a yield of 88%. 1 H NMR (400MHz, DMSO-d6) δ: 6.58 (s, 2H), 4.41 (s, 1H), 3.52 (m, 2H), 3.44 (d, J = 5.2Hz, 2H) ,2.78(t,J=11.9Hz,2H),1.71(m,2H),1.45(m,1H),1.37(d,J=6.3Hz,2H),1.14(m,2H).

[0088] Example 2. Synthesis of methyl 4-(((1-(5-amino-1,3,4-thiadiazol-2-yl)piperidin-4-yl)oxy)methyl)benzoate (4)

[0089] At 0°C, N,N-diisopropylethylamine (6.8 g, 67.5 mmol) was added to a DMF (50 mL) solution of compound 2 (5.4 g, 27 mmol), followed by the slow addition of phenylacetyl chloride (8.8 g, 56.7 mmol). After the reaction was completed (30 minutes at room temperature), the solvent was concentrated under reduced pressure, methanol (50 mL) and NaOH (aqueous solution, 27 mL, 2.5 mol / L) were added to the mixture, and stirred at 50°C for 1 hour. The mixture was then adjusted to pH 6-7 and extracted with DCM. The organic phase was concentrated, mixed, and separated by silica gel chromatography (1% DCM / MeOH to 7% MeOH / DCM) to obtain compound 4 as a white solid with a yield of 45%. 1 HNMR(400MHz,DMSO-d6)δ:12.25(s,1H),7.40-7.20(m,5H),4.39(s,1H),3.75(d,J=19.2Hz,4H),3.46(d,J=5.2Hz,2H ),2.99(t,J=11.9Hz,2H),1.71(d,J=12.5Hz,2H),1.62(m,1H),1.37(d,J=6.3Hz,2H),1.19(dd,J=21.1,11.1Hz,2H).

[0090] Example 3. Synthesis of methyl 4-((2-(1-(5-(2-phenylacetylamino)-1,3,4-thiadiazol-2-yl)piperidin-4-yl)ethoxy)methyl)benzoate (5)

[0091] At 0°C, compound 4 (3.8 g, 12.2 mmol) was dissolved in DMF (20 mL), followed by the slow addition of NaH (540 mg, 13.4 mmol, 60%), and then methyl 4-(bromomethyl)benzoate (2.8 g, 12.2 mmol). The mixture was stirred at 0°C for 2 hours, then quenched with 240 mL H2O. The solution was extracted with DCM, the organic layer was concentrated and chromatographed in 20% EtOAc / hexane to give the desired product in 56% yield. 1H NMR(400MHz, DMSO-d6)δ:12.25(s,1H),7.95(d,J=8.2Hz,2H),7.46(d,J=8.1Hz,2H),7.35-7.19(m,5H),4.57-4.47(m,2H),4.34(dd,J=9.7,4.8Hz ,2H),3.85(s,3H),3.81-3.66(m,4H),3.01(q,J=10.1Hz,2H),1.81-1.61 (m,4H),1.52(dd,J=12.8,6.3Hz,1H),1.25(ddd,J=15.3,9.3,5.5Hz,2H).

[0092] Example 4. Synthesis of methyl 3-((2-(1-(5-(2-phenylacetylamino)-1,3,4-thiadiazol-2-yl)piperidin-4-yl)ethoxy)methyl)benzoate (6)

[0093] The preparation method of compound 6 is the same as that of compound 5, except that methyl 3-(bromomethyl)benzoate is used instead of methyl 4-(bromomethyl)benzoate, and the yield is 43%. 1 H NMR(400MHz, DMSO-d6)δ:12.24(s,1H),7.42(m,2H),7.35-7.19(m,5H),7.21(m,2H),4.57-4.43(m,2H),4.34(dd,J=9.7,4.8Hz,2H),3.8 8(s,3H),3.83-3.64(m,4H),3.01(q,J=10.1Hz,2H),1.82-1.64(m,4H),1.54(dd,J=12.8,6.3Hz,1H),1.26(ddd,J=15.3,9.3,5.5Hz,2H).

[0094] Example 5. Synthesis of 4-((2-(1-(5-(2-phenylacetylamino)-1,3,4-thiadiazol-2-yl)piperidin-4-yl)ethoxy)methyl)benzoic acid (7)

[0095] Compound 5 (3.2 g, 6.8 mmol) was dissolved in MeOH (40 mL), and then NaOH (aqueous solution, 6.8 mL, 2.5 mol / L) was added to the solution and heated to 50°C for 1 hour. The pH of the solution was adjusted to 6-7 using 13% HCl, and a solid was precipitated, filtered, and the filter cake was dried to obtain a white solid with a yield of 92%. 1H NMR(400MHz, DMSO-d6)δ:12.54(s,1H),7.93(d,J=8.1Hz,2H),7.43(d,J=8.0Hz,2H),7.39-7.19(m,6H),4.51(d,J=16.5Hz,2H),3.85-3.74(m,4H),3 .48(dt,J=17.6,5.3Hz,2H),3.10(t,J=12.5Hz,2H),1.71(t,J=14.1Hz,2H ),1.52(dd,J=12.4,6.1Hz,2H),1.24(s,2H),1.08(dt,J=11.8,7.0Hz,1H).

[0096] Example 6. Synthesis of 3-((2-(1-(5-(2-phenylacetylamino)-1,3,4-thiadiazol-2-yl)piperidin-4-yl)ethoxy)methyl)benzoic acid (8)

[0097] Compound 6 was used as the raw material and prepared according to the method described for compound 7 with a yield of 94%. 1 HNMR(400MHz,DMSO-d6)δ:12.45(s,1H),7.52(m,2H),7.45-7.39(m,5H),7.31(m,2H),4.65-4.53(m,2H),4.44(dd,J=9.7,4.8Hz,2 H),3.93-3.74(m,4H),3.21(q,J=10.1Hz,2H),1.92-1.74(m,4H),1.64(dd,J=12.8,6.3Hz,1H),1.36(ddd,J=15.3,9.3,5.5Hz,2H).

[0098] Example 7. Synthesis of N-(3-(4-methylpiperazin-1-yl)phenyl)-4-((2-(1-(5-(2-phenylacetylamino)-1,3,4-thiadiazol-2-yl)piperidin-4-yl)ethoxy)methyl)benzamide (9)

[0099]

[0100] Compound 7 (100 mg, 0.21 mmol) was dissolved in DMF (10 mL), followed by the addition of HATU (80 mg), N,N-diisopropylethylamine (56.7 mg, 0.53 mmol), and 3-(4-methylpiperazin-1-yl)aniline (40 mg, 0.21 mmol). After the reaction was completed (reacted at room temperature for 2 hours), 20 mL of water was added to precipitate the solid, which was filtered off with suction. After the filter cake was dried, it was slurried with petroleum ether / ethyl acetate (10:1, 10 mL), filtered off with suction, and dried to obtain the target product with a yield of 79%.1 H NMR (400MHz, DMSO-d6) δ: 12.24 (s, 1H), 10.03 (s, 1H), 7.93 (d, J=8.2Hz, 2H), 7.45 (d, J= 8.2Hz,2H),7.41(s,1H),7.37-7.21(m,6H),7.16(t,J=8.1Hz,1H),6.69(d,J=8.2Hz,1H ),4.54(s,2H),3.81-3.68(m,4H),3.52(t,J=6.2Hz,2H),3.13(s,3H),3.00(t,J=11.3H z,2H),2.24(s,3H),1.66(dd,J=42.8,23.9Hz,4H),1.53(d,J=6.3Hz,2H),1.24(s,3H). 13 C NMR(101MHz,DMSO-d6)δ:169.06,167.93,165.65,151.23,150.24,142.84,140.47,135.39,134.40,129.64,128.87,128.13 ,127.51,127.29,112.07,111.84,108.21,71.79,67.88,54.02,49.82,47.62,44.68,41.93,36.04,32.58,31.20.HRMS (ESI + )calcd for C 36 H 43 N7O3S[M+H] + 654.3226, found 654.3214.

[0101] Example 8. Synthesis of N-((1-butylpiperidin-4-yl)methyl)-4-((2-(1-(5-(2-phenylacetylamino)-1,3,4-thiadiazol-2-yl)piperidin-4-yl))ethoxy)methyl)benzamide (10)

[0102]

[0103] Compound 10 was prepared in a similar manner to compound 9 with a yield of 66%. 1H NMR(400MHz, CDCl3-d)δ:8.42(t,J=5.7Hz,1H),7.89-7.74(m,2H),7.51-7.10(m ,7H),4.50(s,2H),3.98-3.58(m,4H),3.49(t,J=6.4Hz,2H),3.14(t,J=6.3Hz,2H ),2.99(td,J=12.6,2.7Hz,2H),2.89(d,J=15.1Hz,2H),2.29(s,2H),1.89(s,2H) ,1.74-1.36(m,10H),1.23(ddd,J=28.9,15.0,9.4Hz,6H),0.87(t,J=7.3Hz,3H). 13 C NMR(101MHz,DMSO-d6)δ:169.03,167.91,166.52,150.24,142.22,135.38,134.14,129.63,128.86,128.61,127.67,127.45, 127.28,71.82,67.77,58.10,55.38,53.40,49.81,45.10,41.93,36.04,32.55,31.20,29.97,28.70,20.57,14.36.HRMS(ESI + )calcd for C 35 H 48 N6O3S[M+H] + 633.3587, found 633.3590.

[0104] Example 9. Synthesis of N-((1-(pent-4-enoyl)piperidin-4-yl)methyl)-4-((2-(1-(5-(2-phenylacetylamino))-1,3,4-thiadiazol-2-yl)piperidin-4-yl)ethoxy)methyl)benzamide (11)

[0105]

[0106] Compound 11 was prepared in a similar manner to compound 9 with a yield of 63%. 1H NMR (400MHz, DMSO-d6) δ: 12.23 (s, 1H), 8.46 (t, J = 5.9Hz, 1H), 7.82 (d, J = 8.0Hz, 2H), 7.48-7.15 (m, 7H), 5.83 (ddt, J = 16. 8,10.3,6.5Hz,1H),5.03(dq,J=17.2,1.8Hz,1H),4.94(dd,J=10.2,1.9Hz,1H),4.50(s,2H),4.36(d,J=13.1Hz,1H),3.86 (d,J=13.9Hz,1H),3.76(d,J=13.2Hz,4H),3.49(t,J=6.4Hz,2H),3.19-3.12(m,2H),2.99(td,J=13.0,3.1Hz,3H),2.38( td,J=7.2,6.7,2.4Hz,2H),2.23(q,J=7.2Hz,2H),1.86-1.59(m,6H),1.51(q,J=6.5Hz,2H),1.22(tt,J=11.9,6.1Hz,2H). 13 C NMR(101MHz,DMSO-d6)δ:170.03,169.03,167.92,166.59,150.23,142.27,138.51,135.37,134.10,129.62,128.86,127.67,127.46, 127.28,115.42,71.81,67.77,49.81,45.27,44.95,41.94,41.44,36.40,36.03,32.55,32.10,31.19,30.78,30.01,29.47.HRMS(ESI + )calcd for C 36 H 46 N6O4S[M+H] + 659.3379, found 659.3369.

[0107] Example 10. Synthesis of N-((1-(2-methylbenzyl)piperidin-4-yl)methyl)-4-((2-(1-(5-(2-phenylacetylamino)-1,3,4-thiadiazol-2-yl))piperidin-4-yl)ethoxy)methyl)benzamide (12)

[0108]

[0109] Compound 12 was prepared in a similar manner to compound 9 with a yield of 63%. 1H NMR(400MHz, DMSO-d6)δ:12.24(s,1H),8.47(s,1H),7.85-7.69(m,2H),7.51-7. 41(m,2H),7.41-6.99(m,9H),4.49(s,2H),3.74(d,J=18.1Hz,4H),3.48(q,J=5.9 ,5.4Hz,2H),3.15(s,2H),2.97(td,J=12.6,2.6Hz,2H),2.91-2.60(m,2H),2.31( s,3H),1.90(s,2H),1.75-1.56(m,5H),1.50(q,J=6.4Hz,2H),1.27-1.14(m,3H). 13 C NMR(101MHz,DMSO-d6)δ:169.03,167.92,166.54,164.09,163.96,161.65,161.51,150.24,142.23,135.36,134.14,129.62,128.86,1 27.66,127.46,127.28,111.99,111.69,71.81,67.76,61.52,53.28,49.80,45.15,41.93,36.03,35.94,32.54,31.19,30.16.HRMS(ESI + )calcd for C 39 H 48 N6O3S[M+H] + 681.3587, found 681.3574.

[0110] Example 11. Synthesis of N-((1-hexylpiperidin-4-yl)methyl)-3-((2-(1-(5-(2-phenylacetylamino)-1,3,4-thiadiazol-2-yl)piperidin-4-yl)ethoxy)methyl)benzamide (13)

[0111]

[0112] Compound 13 was prepared in a similar manner to compound 9 with a yield of 70%. 1H NMR (400MHz, DMSO-d6) δ: 12.22 (s, 1H), 8.47 (t, J = 5.8Hz, 1H), 7.85-7.69 (m, 2H), 7 .55-7.40(m,2H),7.40-7.12(m,5H),4.50(s,2H),3.83-3.63(m,4H),3.49(t,J=6.3 Hz,2H),3.14(t,J=6.3Hz,2H),2.97(tt,J=16.5,8.1Hz,4H),2.32(s,2H),1.95(s,2 H),1.73-1.36(m,10H),1.23(qd,J=12.3,11.6,4.6Hz,10H),0.85(t,J=6.6Hz,3H). 13 C NMR(101MHz,DMSO-d6)δ:179.20,166.64,161.08,160.58,137.19,136.39,135.26,131.24,129.82,129.01,128.53,127.65,126.89,12 6.65,58.52,56.42,55.37,53.15,51.99,48.33,46.42,44.18,34.16,31.90,31.21,27.52,26.28,24.19,22.36,21.55,14.31.HRMS(ESI + )calcd for C 37 H 52 N6O3S[M+H] + 661.3900, found 661.3903.

[0113] Example 12: Synthesis of 1,3,4-thiadiazole-2-yl)piperidin-4-yl)ethoxy)methyl)benzamide (14)

[0114]

[0115] Compound 14 was prepared in a similar manner to compound 9 with a yield of 77%. 1H NMR(400MHz, DMSO-d6)δ:12.24(s,1H),8.47(s,1H),7.96-7.73(m,2H),7.56-6.86(m,10H),4.49(s,2H),3.74(d,J=16.1Hz,4H),3 .40(dd,J=29.4,22.3Hz,4H),3.15(s,2H),2.97(t,J=11.8Hz,2H),2.81(s,2H),2.30(s,3H),2.03-1.32(m,9H),1.30-1.04(m,4H). 13 C NMR(101MHz,Chloroform-d)δ:170.84,167.53,160.26,155.69,136.79,13 6.30,136.24,135.43,132.92,129.18,129.15,128.84,128.80,128.30,12 7.61,127.43,127.13,126.28,126.19,125.86,72.61,69.80,56.61,52.31 ,46.30,44.51,43.30,35.91,35.78,34.23,30.54,29.22,19.47.HRMS(ESI + )calcd for C 39 H 48 N6O3S[M+H] + 681.3587, found 681.3570.

[0116] Example 13. Synthesis of tert-butyl 4-(5-amino-1,3,4-thiadiazol-2-yl)piperazine-1-carboxylate (15)

[0117] Potassium carbonate (10.4 g, 75 mmol) and BOC-piperazine (3.6 g, 36 mmol) were added to a DMF (80 mL) solution of compound 1 (5.4 g, 30 mmol) and reacted at 80 °C for 3 hours. After the reaction was completed, the mixture was poured into water (240 mL) and extracted with dichloromethane (150 mL). The organic phase was concentrated under reduced pressure to obtain a solid, which was then slurried with a mixture of petroleum ether / ethyl acetate (10:1, 50 ml), filtered and dried to obtain the target compound with a yield of 93%. 1 H NMR (400MHz, CDCl3-d) δ: 6.68 (s, 2H), 3.57 (m, 4H), 3.32 (m, 4H), 1.42 (s, 9H).

[0118] Example 14. Synthesis of tert-butyl 4-(5-(2-phenylacetylamino)-1,3,4-thiadiazol-2-yl)piperazine-1-carboxylate (16)

[0119] Intermediate 16 was prepared as described in the preparation of Intermediate 3 in 88% yield. 1 H NMR(400MHz, DMSO-d6)δ:12.25(s,1H),7.49-7.39(m,5H),5.68(s,2H),3.57(m,4H),3.45(m,4H),1.45(s,9H).

[0120] Example 15. Synthesis of 2-phenyl-N-(5-(piperazine-1-yl)-1,3,4-thiadiazol-2-yl)acetamide (17)

[0121] To a solution of compound 16 (4.0 g, 10 mmol) in DCM (50 mL) was added trifluoroacetic acid (2 mL) and stirred at room temperature for 2 h. The mixture was then concentrated in vacuo to afford the target compound without further purification in a yield of 95%.

[0122] Example 16. Synthesis of methyl 4-((4-(5-(2-phenylacetamido)-1,3,4-thiadiazol-2-yl)piperazin-1-yl)methyl)benzoate (18)

[0123] Compound 17 (2.8 g, 9.2 mmol) was dissolved in acetonitrile (60 mL), and potassium carbonate (3.2 g, 23.1 mmol) and methyl 4-(bromomethyl)benzoate (2.7 g, 12 mmol) were added to the mixture and stirred at 60° C. for 2 h. After cooling to room temperature, the mixture was filtered off with suction, the filtrate was concentrated, and the mixture was stirred with silica gel, followed by chromatography (10% petroleum ether / EtOAc to 50% petroleum ether / EtOAc) to give compound 18 in a yield of 76%. 1 H NMR(400MHz, DMSO-d6)δ:12.28(s,1H),7.93(d,J=8.1Hz,2H),7.47(d,J=8.0Hz,2H),7.38-7.18 (m,5H),5.75(s,2H),3.85(s,3H),3.74(s,2H),3.59(s,2H),3.38(s,4H),2.53(d,J=2.6Hz,2H).

[0124] Example 17. Synthesis of methyl 3-((4-(5-(2-phenylacetamido)-1,3,4-thiadiazol-2-yl)piperazin-1-yl)methyl)benzoate (19)

[0125] Compound 19 was synthesized according to the preparation method of compound 18, except that 4-(bromomethyl)benzoic acid methyl ester was replaced by 3-(bromomethyl)benzoic acid methyl ester, with a yield of 71%. 1 H NMR(400MHz, DMSO-d6)δ:12.27(s,1H),7.80(d,J=1.8Hz,1H),7.75(dt,J=7.5,1.6Hz,1H),7.51-7.39(m,2H) ,7.38-7.18(m,5H),5.78(s,2H),3.90(s,3H),3.77(s,2H),3.63(s,2H),3.41(s,4H),2.56(d,J=2.6Hz,2H).

[0126] Example 18. Synthesis of 4-((4-(5-(2-phenylacetamido)-1,3,4-thiadiazol-2-yl)piperazin-1-yl)methyl)benzoic acid (20)

[0127] Compound 20 was synthesized according to the preparation method of compound 7 with a yield of 70%. 1 HNMR(400MHz, DMSO-d6)δ:12.49(s,1H),11.74(s,1H),8.00(d,J=8.2Hz,2H),7.78(d,J=8.2Hz,2H),7.38-7.20( m,5H),4.45(s,2H),4.04-3.82(m,2H),3.75(d,J=12.2Hz,2H),3.51(ddd,J=21.0,10.1,4.6Hz,4H),3.32(s,2H).

[0128] Example 19. Synthesis of 3-((4-(5-(2-phenylacetamido)-1,3,4-thiadiazol-2-yl)piperazin-1-yl)methyl)benzoic acid (21)

[0129] Compound 21 was synthesized according to the preparation method of compound 7 with a yield of 92%. 1 HNMR(400MHz,DMSO-d6)δ:12.47(s,1H),11.65(s,1H),7.82(d,J=1.8Hz,1H),7.76(dt,J=7.5,1.6Hz,1H),7.5 3-7.41(m,2H),7.39-7.20(m,5H),5.80(s,2H),3.80(s,2H),3.65(s,2H),3.43(s,4H),2.60(d,J=2.6Hz,2H).

[0130] Example 20. Synthesis of N-(3-(4-methylpiperazin-1-yl)phenyl)-4-((4-(5-(2-phenylacetylamino)-1,3,4-thiadiazol-2-yl)piperazin-1-yl)methyl)benzamide (22)

[0131]

[0132] Compound 22 was synthesized according to the preparation method of compound 9 with a yield of 62%. 1 HNMR(400MHz,DMSO-d6)δ:12.31(s,1H),10.03(s,1H),7.96-7.88(m,2H), 7.47(d,J=8.0Hz,2H),7.41(t,J=2.2Hz,1H),7.38-7.21(m,6H),7.17(t,J= 8.1Hz,1H),6.69(dd,J=8.3,2.4Hz,1H),3.74(s,2H),3.61(s,2H),3.43-3. 37(m,4H),3.13(dd,J=6.3,3.7Hz,4H),2.47(t,J=5.1Hz,4H),2.23(s,3H). 13 C NMR(101MHz,DMSO-d6)δ:169.18,167.92,165.81,150.76,150.69,141.98,140.55,135.33,134.31,129.64,129. 26,128.88,128.13,127.31,112.51,112.14,108.41,61.82,53.25,52.01,49.33,46.83,43.44,41.94.HRMS(ESI + )calcd for C 33 H 38 N8O2S[M+H] + 611.2917, found 611.2906.

[0133] Example 21. Synthesis of N-(3-(4-methylpiperazin-1-yl)phenyl)-3-((4-(5-(2-phenylacetylamino)-1,3,4-thiadiazol-2-yl)piperazin-1-yl)methyl)benzamide (23)

[0134]

[0135] Compound 23 was synthesized according to the preparation method of compound 9 with a yield of 67%. 1HNMR(400MHz, DMSO-d6)δ:12.31(s,1H),10.07(s,1H),7.87(d,J=14.6Hz,2H),7.74-6.81(m,10H),6.69(d, J=8.3Hz,1H),3.67(d,J=50.5Hz,4H),3.40(s,4H),3.25-2.98(m,4H),2.49(d,J=17.3Hz,8H),2.23(s,3H). 13 C NMR(101MHz,DMSO-d6)δ:169.13,167.92,165.89,151.66,150.67,140.37,138.64,135.58,135.33,132.49,129.64,129.45, 128.88,128.76,128.61,127.30,126.85,111.76,111.64,108.04,62.13,54.84,52.06,49.37,48.41,45.88,41.95.HRMS(ESI + )calcd for C 33 H 38 N8O2S[M+H] + 611.2917, found 611.2905.

[0136] Example 22. Synthesis of N-(5-(4-(4-nitrobenzyl)piperazine-1-yl)-1,3,4-thiadiazole-2-yl)-2-phenylacetamide (24)

[0137] Compound 24 was synthesized in a similar manner to compound 18 with a yield of 76%. 1 HNMR(400MHz,DMSO-d6)δ:12.30(s,1H),8.10(d,J=8.1Hz,2H),7.85(d,J=8.0Hz,2H),7.4 8-7.20(m,5H),5.80(s,2H),3.84(s,2H),3.66(s,2H),3.48(s,4H),2.62(d,J=2.6Hz,2H).

[0138] Example 23. Synthesis of N-(5-(4-(3-nitrobenzyl)piperazine-1-yl)-1,3,4-thiadiazole-2-yl)-2-phenylacetamide (25)

[0139] Compound 25 was synthesized in a similar manner to compound 18 with a yield of 66%. 1HNMR(400MHz,DMSO-d6)δ:12.32(s,1H),8.13(d,J=1.8Hz,1H),7.90(dt,J=7.5,1.6Hz,1H),7.55-7.41 (m,2H),7.40-7.19(m,5H),5.88(s,2H),3.91(s,2H),3.72(s,2H),3.53(s,4H),2.70(d,J=2.6Hz,2H).

[0140] Example 24. Synthesis of N-(5-(4-(4-aminobenzyl)piperazine-1-yl)-1,3,4-thiadiazol-2-yl)-2-phenylacetamide (26)

[0141] 10% Pd / C (220 mg, 10%) was added to a solution of compound 24 (2.2 g, 5 mmol) in methanol (50 mL), and the mixture was reacted at 40°C for 24 hours under hydrogen. After the reaction was completed, the mixture was filtered through celite and the filtrate was concentrated under reduced pressure to obtain the target compound in a yield of 94%. 1 H NMR (400MHz, DMSO-d6) δ: 11.85 (s, 1H), 7.39-7.19 (m, 5H), 6.93 (d, J = 8.2Hz, 2H), 6.50 (t ,J=9.3Hz,2H),4.94(s,2H),3.72(s,2H),3.33(dd,J=9.8,4.7Hz,6H),2.47-2.34(m,4H).

[0142] Example 25. Synthesis of N-(5-(4-(3-aminobenzyl)piperazine-1-yl)-1,3,4-thiadiazol-2-yl)-2-phenylacetamide (27)

[0143] The preparation method of compound 27 is similar to that of compound 26, except that the starting material is replaced by compound 25, with a yield of 96%. 1 H NMR (400MHz, DMSO-d6) δ: 12.08 (s, 1H), 7.36-7.21 (m, 5H), 6.95 (t, J = 7.7Hz, 1H), 6.55 (s, 1H), 6.49 -6.39(m,2H),5.03(d,J=37.5Hz,2H),3.73(s,2H),3.37(dd,J=10.9,7.6Hz,6H),2.49-2.37(m,4H).

[0144] Example 26. Synthesis of 4-(4-methylpiperazin-1-yl)-N-(4-((4-(5-(2-phenylacetylamino)-1,3,4-thiadiazol-2-yl)piperazin-1-yl)methyl)phenyl)benzamide (28)

[0145]

[0146] Compound 28 was prepared by a method similar to compound 9 with a yield of 82%. 1 HNMR(400MHz, DMSO-d6)δ:12.29(s,1H),9.91(s,1H),7.87(d,J=8.5Hz,2H),7.72(d,J=8.1Hz,2H),7.38-7.18(m,7H),7.00(d, J=8.6Hz,2H),3.73(s,2H),3.47(s,2H),3.37(t,J=4.9Hz,4H),3.28(t,J=5.0Hz,4H),2.46(dd,J=9.9,5.2Hz,8H),2.23(s,3H). 13 C NMR(101MHz,DMSO-d6)δ:169.14,167.98,165.40,153.41,150.61,138.98,135.32,132.79,129.63,129.52, 128.88,127.31,123.98,120.54,119.08,113.99,62.00,54.69,51.97,49.39,47.27,46.01,41.93.HRMS(ESI + )calcd for C 33 H 38 N8O2S[M+H] + 611.2917, found 611.2905.

[0147] Example 27. Synthesis of 3-(4-methylpiperazin-1-yl)-N-(3-((4-(5-(2-phenylacetylamino)-1,3,4-thiadiazol-2-yl)piperazin-1-yl)methyl)phenyl)benzamide (29)

[0148]

[0149] Compound 29 was prepared by a method similar to compound 9 with a yield of 66%. 1HNMR(400MHz,DMSO-d6)δ:12.32(s,1H),10.17(s,1H),7.80-7.66(m,2H),7.48(s,1H),7.44-7.21(m,8H),7.15(d,J=7.8Hz,1H), 7.05(d,J=7.5Hz,1H),3.74(s,2H),3.52(s,2H),3.39(t,J=5.1Hz,8H),3.28(t,J=5.1Hz,4H),2.63(t,J=4.9Hz,4H),2.34(s,3H). 13 C NMR(101MHz,DMSO-d6)δ:169.15,167.93,165.90,151.67,150.68,140.38,138.66,135.58,135.34,132.50,129.63,129.44, 128.89,128.78,128.62,127.31,126.84,111.80,111.66,108.04,62.13,54.84,52.06,49.37,48.41,45.88,41.95.HRMS(ESI + )calcd for C 33 H 38 N8O2S[M+H] + 611.2917, found 611.2900.

[0150] The beneficial effects of the present invention are demonstrated below through specific test examples.

[0151] Test Example 1: Inhibitory activity of the compounds of the present invention on cell proliferation

[0152] Cell culture and antiproliferation assay: HCT116 and A549 cells were cultured in RPMI1640 (HyClone) medium supplemented with 10% fetal bovine serum (FBS) (Gibco, Milan, Italy), penicillin (100 units / ml, Gibco, Milan, Italy) and streptomycin (100 μg / mL). Cells were incubated at 37°C in a humidified atmosphere of 5% CO2. Cells in the logarithmic growth phase were seeded into 96-well culture plates at a density of (10,000-15,000) cells per well. After 24 hours, cells were treated with different concentrations of compounds for 72 hours in a final volume of 200 μL. After reaching the endpoint, 20 μL of MTT (5 mg / mL) was added to each well, and the cells were incubated for another 1-3 hours. After being treated with 20% SDS overnight, the absorbance value at a wavelength of 570 nM was obtained on a spectrophotometer (Molecular Devices, Sunnyvale, USA). IC was calculated using the percentage of growth compared to the untreated control 50 The CAS No. of the reference compound BPTES is 314045-39-1.

[0153] ROS assay: DCFH-DA was used to measure the intracellular ROS content (HCT116 and A549 cells) by flow cytometry. Briefly, 5 × 10 5 Cells were plated on 6-well plates, adhered overnight, and then treated with the specified concentrations of compound 11 (1, 5 and 10 μM) for 6 hours. Where indicated, antioxidant (NAC) pretreatment was performed for 1 hour. Cells were stained with 10 μM DCFH-DA at 37 ° C in the dark for 30 minutes. DCFH-DA fluorescence (generated in the presence of ROS) was analyzed using flow cytometry. Images were captured using a Nikon epifluorescence microscope (Nikon, Japan) equipped with a digital camera.

[0154] Table 1. Inhibitory activity of the compounds of the present invention on cell proliferation

[0155]

[0156]

[0157] a IC 50 = concentration of compound required to inhibit tumor cell proliferation by 50%; data are presented as mean±SEM of dose-response curves from at least three independent experiments.

[0158] The experimental results in Table 1 show that the compounds of the present invention have a good inhibitory effect on the proliferation of HCT116 and A549 cells.

[0159] Figure 1The experimental results show that the compounds of the present invention can inhibit GLS1 and thus inhibit the ROS pathway mediated by GLS1.

[0160] Test Example 2: Inhibition of GLS1 enzyme activity by the compounds of the present invention

[0161] The activity of glutaminase (GLS1 enzyme) was determined according to relevant literature (Literature 1 is C. A. S. T. Altnecker, S. M. Ulrich, Y. Li, S. Ramachandran, M. K. McBrayer, R. J. De Berardinis, R. A. C. Erione, J. W. Erickson, Mechanism by which a recently discovered allosteric inhibitor blocks glutamine metabolism in transformed cells, Proc. Natl. Acad. Sci. USA 112 (2015) 394-399.; Literature 2 is Z. Chen, D. Li, N. Xu, J. Fang, Y. Yu, W. Hou, H. Ruan, P. Zhu, R. Ma, S. Lu, D. Cao, R. Wu, M. Ni, W. Zhang, W. Su, B. H. Ruan, Novel 1,3,4-Selenadiazole-containing kidney-type glutaminase inhibitors showed improved cellular uptake and antitumor activity,J.Med.Chem.62(2019)589-603.

[43] APFerreira,A.Cassago,A.Goncalves Kde,MMDias,D.Adamoski,CFAscencao,RVHonorato,JFde Oliveira,IMFerreira,C.Fornezari,J.Bettini,PSOliveira,AFPaes Leme, RVPortugal, ALAmbrosio, SMDias, Active glutaminase C self-assembles into a supratetrameric oligomer that can be disrupted by anallosteric inhibitor, J. Biol. Chem. 288 (2013) 28009-28020.) Obtained using a two-step assay. First, 10 μL of a solution of the compound of interest in DMSO was added to 80 μL of the initial assay mixture (0.1 μM GAC, 0.2 mM EDTA, and 50 mM Tris / acetate, pH 8.6).The samples were incubated at 25°C for 10 minutes, and then 10 μL 200mM glutamine was added to the reaction mixture. After adding glutamine, the reaction was incubated at 37°C for 60 minutes, and then quenched by adding 10 μL 0.6M HCl. Subsequently, 100 μL of the second reaction mixture (3.7 units of purified bovine liver glutamate dehydrogenase, 160mMTris / acetate [pH 9.4], 400mM hydrazine, 5mM ADP and 2mM NAD+) were added to the stopped first reaction mixture and incubated at 25°C for 30 minutes. The absorbance at 340nm was measured using a Multiskan Sky (Thermo Scientific) equipped with a 340nm excitation filter. The CAS No. of the control compound BPTES: 314045-39-1.

[0162] Human liver microsome stability: 10 μM compound was added to human microsomes (0.5 mg / mL) in phosphate buffer (100 mM, pH = 7.4), incubated at 37°C for 5 minutes, then 1 mM NADPH was added to start the reaction, and then the protein was precipitated with cold acetonitrile. Finally, the sample was centrifuged for further analysis by LC-MS / MS.

[0163] Table 2. GLS1 inhibitory activity, selectivity and metabolic activity of the compounds of the present invention

[0164]

[0165] a Data are presented as mean ± SEM of dose-response curves from at least three independent experiments. b Human liver microsomal metabolism.

[0166] From the results in Table 2, it can be seen that the compounds of the present invention have significant inhibitory activity against GLS1, but have no inhibitory activity against GLS2 of the same family; compared with the control compounds, the compounds of the present invention have significant advantages in in vitro metabolism.

[0167] Test Example 3: In vivo antitumor activity of the compounds of the present invention

[0168] In vivo antitumor efficacy in HCT116 and A549 xenograft models: HCT116 cells (0.1 mL, 1 × 10 7 HCT116 xenograft model was generated by injecting 100 cells / mL into the abdomen of 6-week-old BALB / c SPF nude mice. The average tumor volume reached 150 mm 3Nude mice were orally administered 0.9% saline solution (compound 11 at the specified concentration) and BPTES containing 5% DMSO (vehicle) once a day for 18 days. Tumor volume and body weight were measured every two days. After oral administration (18 days), mice were sacrificed and dissected. Tumors and major organs were collected. TGI was measured according to the following formula:

[0169] TGI=1-average tumor weight of treatment group / average tumor weight of control group.

[0170] The construction of A549 xenograft model was the same as that of HCT116 xenograft model, except that the administration time was 28 days. In the present invention, all animal experiments were approved by the Animal Care and Use Committee of Sichuan University (Chengdu, Sichuan, China).

[0171] Figure 2 The experimental results show that compared with the control compound, the compound of the present invention exhibits excellent anti-tumor activity in both tumor models.

[0172] In summary, the present invention provides a 2,4-disubstituted thiadiazole derivative, which can be used as a GLS1 inhibitor for preventing and / or treating diseases related to GLS1, such as cancer. At the same time, the 2,4-disubstituted thiadiazole derivative of the present invention has good metabolism and has good clinical application prospects.

Claims

1. A compound of formula I, or a salt thereof, or a stereoisomer thereof: in, L is selected from R 1 is a substituent at any position on the benzene ring, selected from n is 0, 1, 2 or 3; R 2 is selected from substituted or unsubstituted phenyl, substituted or unsubstituted 5-6 membered heterocycloalkyl; The substituent of the phenyl group is selected from substituted or unsubstituted 5-6 membered heterocycloalkyl and C1-C6 alkyl; The substituent of the heterocycloalkyl group is selected from substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkenyl; The substituent of the alkyl group is selected from substituted or unsubstituted phenyl; The two substituents on the same carbon atom of the alkenyl group are linked to form =0.

2. The compound according to claim 1, or a salt thereof, or a stereoisomer thereof, characterized in that: The compound is shown in formula II: in, R 1 is a substituent at any position on the benzene ring, selected from n is 0, 1, 2 or 3; R 2 Selected from R 3 Selected from R 5 Selected from C1-C6 alkyl; R 4 Selected from C1-C6 alkyl, 3. The compound according to claim 1, or a salt thereof, or a stereoisomer thereof, characterized in that: The compound is shown in formula III: in, R 1 is a substituent at any position on the benzene ring, selected from n is 0, 1, 2 or 3; R 2 Selected from R 3 is a substituent at any position on the benzene ring, selected from R 5 Selected from C1~C6 alkyl.

4. The compound according to claim 3, or a salt thereof, or a stereoisomer thereof, characterized in that: The compound is shown in formula IV: in, L' connects any position of two benzene rings, selected from R 5 Selected from C1~C6 alkyl.

5. The compound according to any one of claims 1 to 4, or a salt thereof, or a stereoisomer thereof, characterized in that: The compound is one of the following compounds:

6. Use of the compound according to any one of claims 1 to 5, or a salt thereof, or a stereoisomer thereof in the preparation of a GLS1 inhibitor.

7. Use of the compound according to any one of claims 1 to 5, or a salt thereof, or a stereoisomer thereof in the preparation of a medicament for preventing and / or treating a disease associated with GLS1.

8. The use according to claim 7, characterized in that: The medicine is a medicine for preventing and / or treating cancer.

9. The use according to claim 8, characterized in that: The cancer is lung cancer, liver cancer, colon cancer, rectal cancer, breast cancer, ovarian cancer, and cervical cancer.

10. A drug, characterized in that: The invention relates to a preparation prepared by taking the compound described in any one of claims 1 to 5, or its salt, or its stereoisomer as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.

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