WO2025080100A1 - Novel di-amide compound, preparation method therefor, and pharmaceutical composition for prevention or treatment of cancer or autoimmune diseases, comprising same as active ingredient - Google Patents

Abstract

The present invention relates to a novel glutaminase inhibitor compound and a pharmaceutical composition for the prevention or treatment of cancer or autoimmune diseases, comprising same as an active ingredient. A compound represented by chemical formula 1 disclosed in the present specification exhibits excellent inhibitory activity against glutaminase and has excellent cytotoxicity against cancer cells, and thus is expected to be effective in cancer or autoimmune diseases.

Description

Novel di-amide compound, method for preparing same, and pharmaceutical composition for preventing or treating cancer or autoimmune disease containing same as active ingredient

The present invention relates to a compound that inhibits glutaminase and is useful for cancer or autoimmune diseases.

Recently, metabolic anticancer drugs that selectively suppress cancer by targeting tumor-specific metabolic pathways have emerged rapidly. Since cancer uses selective metabolic pathways for proliferation and survival, metabolic reorganization is a key characteristic of the cancer phenotype. Therefore, many studies are being conducted to determine which specific metabolic circuits cancer cells with specific genetic mutations reorganize to proliferate.

Glucose and glutamine are the primary metabolic fuels used for cancer growth. Most cancers utilize high levels of glucose metabolism to meet the protein anabolic and catabolic demands required to sustain rapid tumor growth. When metabolic stress is applied, such as by anticancer drugs, alternative nutrients such as amino acids are used to overcome this.

Glutamine is the most abundant amino acid found in blood and is a major source of carbon and nitrogen atoms required for the biosynthesis of nucleotides, nonessential amino acids, and fatty acids, which are important precursors for macromolecular synthesis. Glutamine and glutamine-derived metabolites are also known to participate in energy production, redox homeostasis, gene transcription, and intracellular signaling in cancer cells.

Thus, changes in glutamine metabolism in cancer cells can affect the progression and deterioration of cancer by activating various signaling systems related to energy metabolism and redox reactions as well as the continuous proliferation of cancer cells, and by acting toward suppressing cell aging processes and apoptosis. Therefore, targeting glutamine metabolism is an attractive therapeutic option in many cancer subtypes.

In particular, glutaminase (GLS1), an enzyme related to glutamine metabolism, is attracting attention as an important target of tumor metabolism, along with research reports that the readjustment of glutaminolysis, a series of processes in which glutamine is converted to glutamate and then to alpha-ketoglutarate (AKG), is closely related to metastasis and anticancer drug resistance (Non-patent literature 1, Medina, et al, J. Nutr ., September 1, 2001, Vol. 131, No. 9, 2539S-2542S).

In particular, KEAP1, known as the third most frequently mutated gene in lung adenocarcinomas, frequently co-occurs with KRAS oncogenic mutations. It has been reported that cancers with KEAP1- or NRF2-mutations are significantly dependent on upregulated glutaminolysis, and this property can be therapeutically exploited through pharmacological inhibition of glutaminase (Non-patent literature 2, R. Romero, et al, Nature Medicine, November 2017, Vol. 23, No. 11).

Additionally, glutaminase is known to be related to autoimmune diseases (Non-patent literature 3, Michihito Kono, et al, Arthritis & Rheumatology, 2019 Nov;71(11):1869-1878).

Korean Patent Publication No. 10-2015-0091389 discloses glutaminase inhibitor compounds, among which the CB-839 compound is attracting attention as an anticancer agent.

The present inventors synthesized novel compounds to develop a new inhibitor of glutaminase, confirmed that they exhibited inhibitory activity against glutaminase, and completed the present invention.

One object of the present invention is to provide a novel di-amide compound that inhibits glutaminase.

Another object of the present invention is to provide a novel pharmaceutical composition for preventing or treating cancer or autoimmune diseases.

To achieve the above purpose,

A compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof is provided.

L is C2-6 alkylene,

R ¹ and R ² are each independently C1-10 alkyl, C6-10 aryl substituted C1-10 alkyl or C1-10 alkyl substituted with 5 to 10 membered heteroaryl containing 1 to 3 heteroatoms selected from N, O and S,

wherein the aryl and heteroaryl are each independently substituted with one or more selected from the group consisting of unsubstituted or halogen, C1-10 alkyl unsubstituted or substituted with one or more halogens, C1-10 alkoxy unsubstituted or substituted with one or more halogens, and 3 to 6 membered cycloalkyloxy substituted with one or more halogens.

In another aspect, the present invention provides a pharmaceutical composition for preventing or treating cancer or autoimmune disease, containing a compound represented by the chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

In another aspect, the present invention provides a health functional food composition for preventing or improving cancer or autoimmune disease, containing a compound represented by the chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

In another aspect, the present invention provides a method for treating cancer or an autoimmune disease by administering an effective amount of a compound represented by the chemical formula 1 or a pharmaceutically acceptable salt thereof to a subject in need thereof.

In another aspect, the present invention provides a compound represented by the chemical formula 1 or a pharmaceutically acceptable salt thereof for use in the prevention or treatment of cancer or autoimmune disease.

In another aspect, the present invention provides the use of a compound represented by the above formula 1 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the prevention or treatment of cancer or an autoimmune disease.

The compound represented by chemical formula 1 described herein exhibits excellent inhibitory activity against glutaminase and excellent cytotoxicity against cancer cells, and is therefore expected to be useful for cancer or autoimmune diseases.

Figure 1a shows the results of measuring glutamine levels after treating ALK-TKI resistant cells with a compound according to the present invention.

Figure 1b shows the results of measuring glutamate levels after treating ALK-TKI resistant cells with a compound according to the present invention.

Figure 2a shows the change in tumor volume after administration of compound Example 22 to mice administered with H3122-LR pool cell line for a certain period of time, with CB-839 as a control.

Figure 2b shows the change in body weight after administration of compound Example 22 to mice administered with H3122-LR pool cell line for a certain period of time, with CB-839 as a control.

Hereinafter, the present invention will be described in detail.

Meanwhile, the embodiments of the present invention may be modified into various other forms, and the scope of the present invention is not limited to the embodiments described below. In addition, the embodiments of the present invention are provided to more completely explain the present invention to a person having average knowledge in the relevant technical field.

Furthermore, reference throughout the specification to an element “including” means that, unless otherwise specifically stated, it may include other elements, rather than excluding other elements.

The term "alkylene" or "alkyl", unless otherwise specified, includes straight-chain or branched-chain saturated hydrocarbon residues. For example, "C1-6 alkyl" means an alkyl having a skeleton of 1 to 6 carbons. Specifically, C1-6 alkyl includes methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, n-pentyl, i-pentyl, t-pentyl, sec-pentyl, neopentyl, hexyl, and the like, and C6 alkyl is a fully saturated hydrocarbon having 6 carbons, including straight-chain or branched-chain structural isomers. Alkyl means a monovalent substituent, and alkylene means a divalent substituent.

The term “alkenyl” means an unsaturated hydrocarbon residue containing one or more double bonds between carbons in the chain, for example, 1-3, 1-2 double bonds.

The term “cycloalkyl”, unless otherwise specified, means a cyclic hydrocarbon residue which is a saturated hydrocarbon residue forming a ring, and “3-6cycloalkyl” means a cyclic hydrocarbon residue containing from 3 to 6 carbon atoms as ring atoms.

The term “cycloalkenyl”, unless otherwise specified, means a cyclic hydrocarbon residue containing one or more, for example one or two, double bonds in the ring, which is non-aromatic.

The term “heterocycloalkyl”, unless otherwise specified, means a monovalent saturated moiety consisting of 1 to 3 rings containing one or more (e.g., 1-5, 1-4, 1-3, 1-2, 1) heteroatoms selected from N, O or S. It may be bicyclic or tricyclic containing 2 or 3 rings, wherein these 2 or 3 rings may be bridged, fused or spiro-shaped heterocycloalkyl. When composed of multiple rings, the heteroatoms may be present in all or only some of the rings. “Atomic heterocycloalkyl” means a heterocycloalkyl having 3 to 10 ring atoms. Meanwhile, when referring to a 3-10 membered heterocycloalkyl group containing at least one N, it refers to a group that necessarily contains at least one nitrogen atom (N) as a heteroatom forming the ring.

The term “aryl” refers to an aromatic radical having a single ring or two or three fused hydrocarbon aromatic rings, C6-10 aryl refers to an aromatic ring compound containing 6 to 10 ring-forming carbons, including phenyl and naphthyl.

The term “heteroaryl” means, unless otherwise specified, a single ring or an aromatic radical having two or three fused rings, which has one or more aromatic rings (the remaining ring atoms are C) containing one or more (e.g., 1-5, 1-4, 1-3, 1-2, 1) heteroatoms selected from N, O or S as ring atoms. When composed of multiple rings, the heteroatoms may be present in all or only some of the rings. “5-10 membered heteroaryl” means a heteroaryl having 5-10 ring atoms. On the other hand, when referring to a 5-10 membered heteroaryl containing one or more Ns, it means that it necessarily contains one or more nitrogen atoms (N) as a ring heteroatom. And,

“Alkoxy” refers to a substituent in which alkyl is connected through an oxygen atom, and C1-10 alkoxy refers to a substituent in which C1-10 alkyl is connected to an oxygen atom and substituted through oxygen.

“Cycloalkyloxy” refers to a substituent in which cycloalkyl is linked through an oxygen atom.

“Halogen” means a halogen atom such as F, Cl, Br, I, and when it is said to be “halo” substituted, it means substituted with one or more halogen atoms. In this case, the halogen atoms to be substituted can be selected within the range of 1 to 5.

In this specification,

In one aspect of the present invention,

A compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof is provided.

In one embodiment of the present invention,

L is C2-6 alkylene,

R ¹ and R ² are each independently C1-10 alkyl, C6-10 aryl substituted C1-10 alkyl or C1-10 alkyl substituted with 5 to 10 membered heteroaryl containing 1 to 3 heteroatoms selected from N, O and S,

wherein the aryl and heteroaryl are each independently substituted with one or more selected from the group consisting of unsubstituted or halogen, C1-10 alkyl unsubstituted or substituted with one or more halogens, C1-10 alkoxy unsubstituted or substituted with one or more halogens, and 3 to 6 membered cycloalkyloxy substituted with one or more halogens.

In another embodiment of the present invention,

L is (CH ₂ ) _n , where n is an integer from 2 to 6, or n is an integer from 3 to 5, or n is 4.

In another embodiment of the present invention,

R ¹ and R ² are each independently C1-6 alkyl, C1-6 alkyl substituted with C6 aryl, or C1-6 alkyl substituted with 5-6 membered heteroaryl containing 1-3 N,

wherein said aryl and heteroaryl are each independently unsubstituted or substituted with halogen, C1-6 alkyl unsubstituted or substituted with one or more halogens, C1-6 alkoxy unsubstituted or substituted with one or more halogens, and 3 to 5 membered cycloalkyloxy substituted with one or more halogens, and are substituted with 1 to 3 members.

In another embodiment of the present invention,

The above aryl is phenyl and the above heteroaryl is pyridyl.

In another embodiment of the present invention,

R ¹ and R ² are each independently methyl, phenylmethyl or pyridylmethyl,

The phenyl of the above phenylmethyl and the pyridyl of the pyridylmethyl are each independently unsubstituted or substituted with 1 to 3 groups selected from the group consisting of F, Cl, methyl, methoxy, CF ₃ , OCF ₃ and 3,3-difluorocyclobutyloxy.

In another embodiment of the present invention,

The compound represented by the above chemical formula 1 is one selected from the group of compounds below.

1) 5,5'-(butane-1,4-diyl)bis(N-(2-fluoro-5-methoxybenzyl)-1,3,4-thiadiazole-2-carboxamide);

2) 5,5'-(butane-1,4-diyl)bis(N-((5-methoxypyridin-2-yl)methyl)-1,3,4-thiadiazole-2-carboxamide);

3) 5,5'-(Butane-1,4-diyl)bis(N-((6-methoxypyridin-2-yl)methyl)-1,3,4-thiadiazole-2-carboxamide);

4) 5,5'-(butane-1,4-diyl)bis(N-benzyl-1,3,4-thiadiazole-2-carboxamide);

5) 5,5'-(Butane-1,4-diyl)bis(N-(2-fluoro-5-(trifluoromethoxy)benzyl)-1,3,4-thiadiazole-2-carboxamide);

6) N-(2-Fluoro-5-(trifluoromethoxy)benzyl)-5-(4-(5-(methylcarbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide;

7) 5,5'-(Butane-1,4-diyl)bis(N-(2-chloro-5-methoxybenzyl)-1,3,4-thiadiazole-2-carboxamide);

8) N-(2-chloro-5-methoxybenzyl)-5-(4-(5-(methylcarbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide;

9) N-Methyl-5-(4-(5-((2-methyl-5-(trifluoromethoxy)benzyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide;

10) 5,5'-(Butane-1,4-diyl)bis(N-(2-methyl-5-(trifluoromethoxy)benzyl)-1,3,4-thiadiazole-2-carboxamide);

11) N-(2-Fluoro-5-(trifluoromethoxy)benzyl)-5-(4-(5-((pyridin-2-ylmethyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide;

12) N-(2-Methyl-5-(trifluoromethoxy)benzyl)-5-(4-(5-((pyridin-2-ylmethyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide;

13) N-(2-chloro-5-methoxybenzyl)-5-(4-(5-((pyridin-2-ylmethyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide;

14) 5,5'-(Butane-1,4-diyl)bis(N-((4-methoxypyridin-2-yl)methyl)-1,3,4-thiadiazole-2-carboxamide);

15) N-((4-methoxypyridin-2-yl)methyl)-5-(4-(5-(methylcarbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide;

16) N-((4-methoxypyridin-2-yl)methyl)-5-(4-(5-((pyridin-2-ylmethyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide;

17) 5,5'-(Butane-1,4-diyl)bis(N-((4-chloropyridin-2-yl)methyl)-1,3,4-thiadiazole-2-carboxamide);

18) N-((4-chloropyridin-2-yl)methyl)-5-(4-(5-(methylcarbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide;

19) N-((4-chloropyridin-2-yl)methyl)-5-(4-(5-((pyridin-2-ylmethyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide;

20) N-Methyl-5-(4-(5-(((4-(trifluoromethyl)pyridin-2-yl)methyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide;

21) N-(pyridin-2-ylmethyl)-5-(4-(5-(((4-(trifluoromethyl)pyridin-2-yl)methyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide;

22) 5,5'-(Butane-1,4-diyl)bis(N-((4-(trifluoromethyl)pyridin-2-yl)methyl)-1,3,4-thiadiazole-2-carboxamide);

23) N-((4-(trifluoromethyl)pyridin-2-yl)methyl)-5-(4-(5-((2,3,4-trimethoxybenzyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide;

24) N-Benzyl-5-(4-(5-(((4-(trifluoromethyl)pyridin-2-yl)methyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide;

25) 5,5'-(Butane-1,4-diyl)bis(N-((3-fluoropyridin-2-yl)methyl)-1,3,4-thiadiazole-2-carboxamide);

26) 5,5'-(Butane-1,4-diyl)bis(N-((6-fluoropyridin-2-yl)methyl)-1,3,4-thiadiazole-2-carboxamide);

27) N-((6-fluoropyridin-2-yl)methyl)-5-(4-(5-(((4-(trifluoromethyl)pyridin-2-yl)methyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide;

28) 5,5'-(Butane-1,4-diyl)bis(N-((4-(trifluoromethyl)pyridin-3-yl)methyl)-1,3,4-thiadiazole-2-carboxamide);

29) N-((3-fluoropyridin-2-yl)methyl)-5-(4-(5-(((4-(trifluoromethyl)pyridin-2-yl)methyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide;

30) N-(2,3-dimethoxybenzyl)-5-(4-(5-(((4-(trifluoromethyl)pyridin-2-yl)methyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide;

31) N-((3,4-dimethoxypyridin-2-yl)methyl)-5-(4-(5-(((4-(trifluoromethyl)pyridin-2-yl)methyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide;

32) N-((4-methoxypyridin-2-yl)methyl)-5-(4-(5-(((4-(trifluoromethyl)pyridin-2-yl)methyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide;

33) N-((4-(trifluoromethyl)pyridin-2-yl)methyl)-5-(4-(5-(((4-(trifluoromethyl)pyridin-3-yl)methyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide;

34) N-(2-Fluoro-5-methoxybenzyl)-5-(4-(5-(((4-(trifluoromethyl)pyridin-2-yl)methyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide;

35) N-(2-Fluoro-5-(trifluoromethoxy)benzyl)-5-(4-(5-(((4-(trifluoromethyl)pyridin-2-yl)methyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide;

36) 5,5'-(Butane-1,4-diyl)bis(N-((4-(3,3-difluorocyclobutyloxy)pyridin-2-yl)methyl)-1,3,4-thiadiazole-2-carboxamide);

37) N-((4-(3,3-difluorocyclobutyloxy)pyridin-2-yl)methyl)-5-(4-(5-(((4-(trifluoromethyl)pyridin-2-yl)methyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide;

38) N-((4-(3,3-difluorocyclobutyloxy)pyridin-2-yl)methyl)-5-(4-(5-(methylcarbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide;

39) N-((4-(3,3-difluorocyclobutyloxy)pyridin-2-yl)methyl)-5-(4-(5-((pyridin-2-ylmethyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide.

The compound represented by the above chemical formula 1 of the present invention can be provided in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. The acid addition salt is obtained from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, phosphorous acid, and the like, aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates and alkanedioates, aromatic acids, aliphatic and aromatic sulfonic acids, trifluoroacetic acid, acetate, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, fumaric acid, and the like. The types of these pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, iodide, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, Includes phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, and the like.

The acid addition salt according to the present invention can be prepared by a conventional method, for example, by dissolving the derivative of chemical formula 1 in an organic solvent such as methanol, ethanol, acetone, methylene chloride, acetonitrile, etc., adding an organic acid or inorganic acid, filtering and drying the resulting precipitate, or by distilling the solvent and an excess acid under reduced pressure, drying, and crystallizing in the presence of an organic solvent.

In another aspect of the present invention,

The present invention provides a method for producing a compound represented by the chemical formula 1.

One embodiment of the manufacturing method comprises:

As shown in reaction scheme 1 below,

A step of forming an amide bond between a compound of chemical formula 2 and H ₂ NR ¹ ; and

A step of forming an amide bond between a compound of chemical formula 2' and H ₂ NR ² .

[Reaction Formula 1]

Here, L, R ¹ and R ² are the same as defined in the chemical formula 1 above,

Y is C1-C10 alkyl.

The step of forming an amide bond between the above H ₂ NR ¹ and H ₂ NR ² can be performed sequentially. The step of forming an amide bond between H ₂ NR ² can be performed after isolating the compound of chemical formula 2', or, in some cases, can be performed by introducing H ₂ NR ² without isolating the compound of chemical formula 2'.

R ¹ and R ² may be different or the same, but if they are the same, they can be performed at once without having to proceed sequentially.

In another embodiment of the manufacturing method,

As shown in reaction formula 2 below,

A step of forming an amide bond of a compound of chemical formula 2 and a mixture of H ₂ NR ¹ and H ₂ NR ² amines;

[Reaction Formula 2]

Here, L, R ¹ and R ² are the same as defined in the chemical formula 1 above,

Y is C1-C10 alkyl.

R ¹ and R ² may be different or the same, and in different cases, the amine reactants may be introduced at once in the form of a mixture. In the same case, the amine may be introduced as a single amine compound rather than a mixture.

The above manufacturing method is not limited to one embodiment of the present invention presented as an example, and can be performed by modifying the solvent, reactant, temperature conditions, etc. under general organic chemical knowledge.

In another aspect of the present invention,

A pharmaceutical composition for preventing or treating cancer or autoimmune disease is provided, containing a compound represented by the above chemical formula 1, a solvate thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

The above compound is characterized by inhibiting glutaminase.

The above compound is cytotoxic to cancer cells, and the cancer cells may be cancer cells resistant to existing anticancer drugs, and for example, cancer cells resistant to Ceritinib.

In the present invention, “cancer” includes pseudomyxoma, intrahepatic cholangiocarcinoma, hepatoblastoma, liver cancer, thyroid cancer, testicular cancer, myelodysplastic syndrome, glioblastoma, oral cancer, lip cancer, mycosis fungoides, acute myeloid leukemia, acute lymphoblastic leukemia, basal cell carcinoma, ovarian epithelial cancer, ovarian germ cell cancer, male breast cancer, brain cancer, pituitary adenoma, multiple myeloma, gallbladder cancer, bile duct cancer, colon cancer, chronic myeloid leukemia, chronic lymphocytic leukemia, retinoblastoma, choroidal melanoma, ampulla of Vater cancer, bladder cancer, peritoneal cancer, parathyroid cancer, adrenal cancer, paranasal sinus cancer, tongue cancer, astrocytoma, small intestine cancer, meningioma, esophageal cancer, glioma, renal pelvis cancer, kidney cancer, heart cancer, duodenal cancer, malignant soft tissue cancer, malignant bone cancer, malignant lymphoma, malignant mesothelioma, It may be selected from the group consisting of malignant melanoma, ocular cancer, vulvar cancer, ureteral cancer, urethral cancer, cancer of unknown primary site, gastric lymphoma, gastric cancer, gastric carcinoid, gastrointestinal stromal cancer, Wilms' cancer, breast cancer, penile cancer, oropharyngeal cancer, gestational trophoblastic disease, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, metastatic bone cancer, metastatic brain cancer, mediastinal cancer, rectal cancer, vaginal cancer, spinal cancer, acoustic neuroma, pancreatic cancer, salivary gland cancer, Kaposi sarcoma, Paget's disease, tonsil cancer, squamous cell carcinoma, lung cancer, skin cancer, anal cancer, rhabdomyosarcoma, laryngeal cancer, pleural cancer, and thymic cancer.

The above autoimmune disease may be any one of psoriasis, rheumatoid arthritis, vasculitis, inflammatory bowel disease, dermatitis, osteoarthritis, asthma, inflammatory muscle disease, allergic disease (e.g., allergic rhinitis), vaginitis, interstitial cystitis, scleroderma, osteoporosis, eczema, rejection of allogeneic or xenogeneic transplantation (organ, bone marrow, stem cell and other cells and tissues), graft-versus-host disease, lupus erythematosus, inflammatory disease, type I diabetes, pulmonary fibrosis, dermatomyositis, Sjogren's syndrome, thyroiditis (e.g., Hashimoto's and autoimmune thyroiditis), myasthenia gravis, autoimmune hemolytic anemia, multiple sclerosis, cystic fibrosis, chronic relapsing hepatitis, primary biliary cirrhosis, allergic conjunctivitis and atopic dermatitis.

In the present invention, the term “containing as an active ingredient” means containing in a dosage range that brings about the effect of preventing, improving, or treating cancer or autoimmune disease, and the dosage range may vary depending on the severity and formulation, and the number of applications may also vary depending on the age, weight, and constitution of the subject. In one specific example of the present invention, the compound represented by Chemical Formula 1 in the pharmaceutical composition of the present invention is contained in an amount of, for example, 0.001 mg/kg or more, preferably 0.1 mg/kg or more, more preferably 10 mg/kg or more, still more preferably 100 mg/kg or more, still more preferably 250 mg/kg or more, and most preferably 0.1 g/kg or more. The quantitative upper limit of the compound represented by Chemical Formula 1 contained in the pharmaceutical composition of the present invention can be selected and implemented within an appropriate range by a person skilled in the art.

The pharmaceutical composition according to the present invention may contain an effective amount of a compound represented by chemical formula 1 alone or may contain one or more pharmaceutically acceptable carriers, excipients or diluents.

The above pharmaceutically acceptable carrier, excipient or diluent refers to a substance that is physiologically acceptable and does not typically cause allergic reactions such as gastrointestinal upset, dizziness or similar reactions when administered to humans. Examples of the carrier, excipient and diluent include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be additionally included.

The compound represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof can be administered in various oral and parenteral dosage forms during clinical administration. When formulating, it is prepared using diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing one or more compounds with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, and emulsions. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

A pharmaceutical composition containing the compound represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient can be administered parenterally, and parenteral administration is by subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection.

At this time, in order to formulate a formulation for parenteral administration, the compound represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof is mixed with a stabilizer or buffer in water to prepare a solution or suspension, which can be prepared in an ampoule or vial unit dosage form. The composition may be sterilized and/or contain auxiliary agents such as preservatives, stabilizers, wetting agents or emulsifying promoters, salts for regulating osmotic pressure and/or buffers, and other therapeutically useful substances, and may be formulated according to conventional methods such as mixing, granulating, or coating.

Oral dosage forms include, for example, tablets, pills, hard/soft capsules, solutions, suspensions, emulsifiers, syrups, granules, elixirs, and troches, and these dosage forms contain, in addition to the active ingredient, diluents (for example, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and/or glycine), lubricants (for example, silica, talc, stearic acid and its magnesium or calcium salts, and/or polyethylene glycol). Tablets may contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidine, and, if desired, disintegrating agents or effervescent mixtures such as starch, agar, alginic acid or its sodium salt, and/or absorbents, coloring agents, flavoring agents, and sweetening agents.

In the present invention, the term “prevention” means preventing the occurrence of symptoms of cancer or autoimmune disease in advance by administering, ingesting or applying the pharmaceutical composition or health functional food composition of the present invention to a subject not suffering from cancer or an autoimmune disease, thereby suppressing or blocking the symptoms of cancer or an autoimmune disease.

In the present invention, the term “treatment” includes complete cure of symptoms of cancer or autoimmune disease as well as partial cure, improvement and alleviation of symptoms of cancer or autoimmune disease as a result of administering the pharmaceutical composition of the present invention to a subject suffering from cancer or an autoimmune disease.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount.

In the present invention, the term “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment or improvement, and the effective dosage level can be determined according to factors including the type and severity of the individual, age, sex, activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, treatment period, concurrently used drugs, and other factors well known in the medical field.

Furthermore, the compound represented by the above chemical formula 1 can be used in the form of not only its pharmaceutically acceptable salt but also a solvate, hydrate, etc. that can be prepared therefrom.

In addition, a pharmaceutical composition for preventing or treating cancer containing the compound represented by the above chemical formula 1, or a pharmaceutically acceptable salt thereof, as an active ingredient, may be administered as an individual therapeutic agent, or may be used in combination with other anticancer agents currently in use.

In addition, a pharmaceutical composition for preventing or treating cancer containing the compound represented by the chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient can enhance the anticancer effect by co-administration with an anticancer agent.

In another aspect of the present invention,

A health functional food composition for preventing or improving cancer or autoimmune disease is provided, containing a compound represented by the above chemical formula 1, a solvate thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

In the present invention, the term “improvement” means alleviating or alleviating the symptoms of cancer or an autoimmune disease by administering, ingesting, or applying the pharmaceutical composition or food composition of the present invention to a subject suffering from cancer or an autoimmune disease.

The matters mentioned in the pharmaceutical composition and health functional food composition of the present invention are equally applicable unless they are contradictory to each other.

Hereinafter, the present invention will be described in detail through examples and experimental examples.

However, the examples and experimental examples described below are only intended to specifically illustrate one aspect of the present invention, and the present invention is not limited thereto.

The reaction scheme below illustrates a general method for preparing compounds according to the present invention.

The compound of chemical formula 1, which is a glutaminase inhibitor according to the present invention, can be prepared from the compound of chemical formula 2 as an intermediate according to two methods.

When conducting an amidation reaction with an amine, the two types of amines can be reacted sequentially, or they can be introduced in a mixed form at once and reacted.

The introduction of a mixed form at one time is desirable for producing a compound with a symmetrical structure when two types of amines are the same, i.e., R ¹ and R ² are the same.

[Reaction Formula 3]

In the above reaction scheme 3, Y, L, R ¹ and R ² follow the definitions described above.

A brief description is given of the general manufacturing method of the above reaction scheme 3.

Step 1 is the step of forming an amide bond by reacting the compound of chemical formula 4 and the compound of chemical formula 5.

The compound of chemical formula 5 is activated and can be carried out by employing known amidation reaction conditions. The compound of chemical formula 4 is added at a low temperature, for example, 20° C. or lower, 10° C. or lower, and then the temperature is raised to room temperature. The reaction is performed until completion, for example, 1 hour or longer, 6 hours or longer, or 12 hours or longer. The reaction can be performed in an organic solvent, and it is preferable to use an aprotic organic solvent. Dichloromethane, acetone, ethyl acetate, THF, DMF, DMSO, benzene, toluene, diethyl ether, etc. can be used. A base can be added due to the generated hydrogen chloride, etc., and an amine base or an inorganic base can be used. As the amine base, it is preferable to use a tertiary amine such as triethylamine or pyridine. Afterwards, a further purification process can be performed.

Step 2 is a ring formation reaction with thiadiazole. After adding P ₂ S ₅ at room temperature, it can be performed at 50 to 120 ℃, 60 to 110 ℃, or 70 to 100 ℃, but is not limited thereto. The reaction time is performed until completion, and can be performed for, for example, 1 hour or more, 6 hours or more, or 12 hours or more. The solvent is the organic solvent of step 1, but is selected considering the boiling point.

The subsequent step is a step of forming an amide bond by reacting with an amine. The amide bond forming step is performed in an organic solvent, and the organic solvents mentioned above can be used, for example, an alcohol solvent such as ethanol, propanol, or isopropanol. After adding the amine reactant at room temperature, the reaction can be performed at 50° C. to 120° C., 60° C. to 110° C., or 70° C. to 100° C., but is not limited thereto. The reaction time is performed until completion, and for example, can be performed for 1 hour or more, 6 hours or more, or 12 hours or more.

Below, specific examples of methods for producing intermediate compounds are presented.

Manufacturing Example. Manufacturing of Intermediate Compounds

Manufacturing example 1

Diethyl 2,2'-(adipoylbis(hydrazine-2,1-diyl))bis(2-oxoacetate)

Triethylamine (2.80 mL, 20.1 mmol) was added to a solution of adipohydrazide (1.00 g, 5.74 mmol) in dichloromethane (60 mL) under stirring at room temperature (R.T). Ethyl 2-chloro-2-oxoacetate (2.35 g, 17.2 mmol) was added dropwise to the reaction mixture at 0 °C. The reaction mixture was slowly warmed to room temperature and maintained for 24 h. The reaction mixture was diluted with dichloromethane, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using 1-10% methanol in dichloromethane as an eluent to obtain the target product as a yellow solid (1.03 g, 48%).

¹ H NMR (500 MHz, DMSO) δ 10.76 - 10.68 (m, 2H), 10.01 - 9.92 (m, 2H), 4.27 (q, J = 7.1 Hz, 4H), 2.21 - 2.12 (m, 4H), 1.55 (p, J = 3.5 Hz, 4H), 1.29 (t, J = 7.1 Hz, 6H).

Manufacturing example 2

Diethyl 5,5'-(butane-1,4-diyl)bis(1,3,4-thiadiazole-2-carboxylate)

Phosphorus pentasulfide (2.23 g, 10.0 mmol) was added to a solution of Preparation 1 (1.50 g, 4.01 mmol) in toluene (26.7 mL) at room temperature under stirring. The reaction mixture was heated at 70 °C for 24 h. The reaction mixture was cooled to room temperature, diluted with dichloromethane and methanol, filtered and concentrated under reduced pressure. The crude was purified by silica gel column chromatography using 1-10% methanol in dichloromethane as an eluent to obtain the target compound as a yellow solid (862 mg, 58%).

¹ H NMR (400 MHz, DMSO) δ 4.42 (q, J = 7.1 Hz, 4H), 3.25 (t, J = 6.9 Hz, 4H), 1.86 (p, J = 3.5 Hz, 4H), 1.34 (t, J = 7.1 Hz, 6H).

Manufacturing example 3

Ethyl 5-(4-(5-(methylcarbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxylate

Methaneamine (33.5 mg, 1.08 mmol) was added to a solution of the compound of Preparation Example 2 (400 mg, 1.08 mmol) in ethanol (4.0 mL) at room temperature under stirring. The reaction mixture was heated at 80 °C for 2 h. The reaction mixture was cooled to room temperature, diluted with dichloromethane, and concentrated under reduced pressure. The crude was purified by silica gel column chromatography using 1-10% methanol in dichloromethane as an eluent to obtain the target compound as a white solid (75.0 mg, 21%).

^1H NMR (500 MHz, DMSO) δ 9.17 - 9.09 (m, 1H), 4.42 (q, J = 7.1 Hz, 2H), 3.28 - 3.17 (m, 4H), 2.81 (d, J = 4.8 Hz, 3H), 1.85 (p, J = 3.1 Hz, 4H), 1.34 (t, J = 7.1 Hz, 3H).

Manufacturing example 4

Ethyl 5-(4-(5-((pyridin-2-ylmethyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxylate

Pyridin-2-ylmethaneamine (32.1 mg, 0.297 mmol) was added to a stirred solution of the compound of Preparation Example 2 (100 mg, 0.270 mmol) in isopropanol (3.0 mL) at room temperature.

Similar to Manufacturing Example 3, the target compound was obtained as a yellow solid (23.3 mg, 20%).

^1H NMR (500 MHz, DMSO) δ 9.72 (t, J = 6.1 Hz, 1H), 8.52 (ddd, J = 4.9, 1.9, 1.0 Hz, 1H), 7.77 (td, J = 7.7, 1.8 Hz, 1H), 7.38 - 7.32 (m, 1H), 7.32 - 7.26 (m, 1H), 4.58 (d, J = 6.1 Hz, 2H), 4.42 (q, J = 7.1 Hz, 2H), 3.27 - 3.19 (m, 4H), 1.91 - 1.82 (m, 4H), 1.35 - 1.32 (m, 3H).

Manufacturing example 5

Ethyl 5-(4-(5-(((4-(trifluoromethyl)pyridin-2-yl)methyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxylate

Similar to Manufacturing Example 4, the target compound was obtained as a yellow solid (80.2 mg, 20%).

¹ H NMR (500 MHz, DMSO) δ 9.81 (t, 1H), 8.82 (d, 1H), 7.73 - 7.65 (m, 3H), 4.70 (d, 2H), 4.42 (q, J = 7.1 Hz, 2H), 3.25 - 3.23 (m, 4H), 1.87 - 1.86 (m, 4H), 1.35 - 1.34 (m, 3H).

Example. Preparation of di-amide compound

The chemical structures of the example compounds are shown in Table 1 below.

Example 1

5,5'-(butane-1,4-diyl)bis(N-(2-fluoro-5-methoxybenzyl)-1,3,4-thiadiazole-2-carboxamide)

To a solution of the compound of Preparation Example 2 (20.0 mg, 0.0540 mmol) in isopropanol (1.0 mL) was stirred (2-fluoro-5-methoxyphenyl)methaneamine (83.8 mg, 0.540 mmol) at room temperature. The reaction mixture was heated at 80 °C for 18 h. It was then cooled and filtered to obtain the target compound.

white solid (31.0 mg, 98%): ¹ H NMR (500 MHz, DMSO) δ 9.73 (s, 2H), 7.12 (t, J = 9.4 Hz, 2H), 6.96 - 6.90 (m, 2H), 6.89 - 6.83 (m, 2H), 4.53 - 4.44 (m, 4H), 3.71 (s, 6H), 3.22 (s, 4H), 1.86 (s, 4H); MS: 589.7 [M+H ⁺ ].

Example 2

5,5'-(butane-1,4-diyl)bis(N-((5-methoxypyridin-2-yl)methyl)-1,3,4-thiadiazole-2-carboxamide)

The target compound was obtained as in Example 1.

white solid (21.0 mg, 70%): ¹ H NMR (500 MHz, DMSO) δ 9.66 (s, 2H), 8.23 (s, 2H), 7.37 (d, J = 9.0 Hz, 2H), 7.30 (d, J = 8.8 Hz, 2H), 4.52 (t, J = 4.5) Hz, 4H), 3.81 (s, 6H), 3.23 (s, 4H), 1.87 (s, 4H); MS: 555.7 [M+H ⁺ ].

Example 3

5,5'-(butane-1,4-diyl)bis(N-((6-methoxypyridin-2-yl)methyl)-1,3,4-thiadiazole-2-carboxamide)

The target compound was obtained as in Example 1.

white solid (25.1 mg, 84%): ¹ H NMR (500 MHz, DMSO) δ 9.70 (s, 2H), 7.67 (t, J = 8.1 Hz, 2H), 6.90 (d, J = 7.3 Hz, 2H), 6.70 (d, J = 8.4 Hz, 2H), 4.50 (t, J = 4.6 Hz, 4H), 3.85 - 3.82 (m, 6H), 3.24 (s, 4H), 1.88 (s, 4H); MS: 555.7 [M+H ⁺ ].

Example 4

5,5'-(butane-1,4-diyl)bis(N-benzyl-1,3,4-thiadiazole-2-carboxamide)

The target compound was obtained as in Example 1.

white solid (18.8mg, 28%): ¹ H NMR (500 MHz, DMSO) δ 9.78 (s, 2H), 7.37 - 7.30 (m, 8H), 7.26 (q, J = 4.4 Hz, 2H), 4.51 - 4.42 (m, 4H), 3.21 (s, 4H), 1.85 (s, 4H); MS: 493.7 [M+H ⁺ ].

Example 5

5,5'-(butane-1,4-diyl)bis(N-(2-fluoro-5-(trifluoromethoxy)benzyl)-1,3,4-thiadiazole-2-carboxamide)

The target compound was obtained as in Example 1.

white solid (15.9 mg, 56%): ¹ H NMR (500 MHz, DMSO) δ 9.83 (t, J = 6.1 Hz, 1H), 7.40 (d, J = 6.3 Hz, 2H), 7.38 - 7.35 (m, 4H), 4.56 - 4.52 (m, 4H), 3.26 - 3.20 (m, 4H), 1.86 (p, J = 3.7 Hz, 4H); MS: 697.5 [M+H ⁺ ].

Example 6

N-(2-fluoro-5-(trifluoromethoxy)benzyl)-5-(4-(5-(methylcarbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide

The target compound was obtained in the same manner as in Example 1 using the starting material of Manufacturing Example 3.

white solid (4.8 mg, 33%): ¹ H NMR (500 MHz, DMSO) δ 9.83 (t, J = 6.1 Hz, 1H), 9.14 (t, J = 4.8 Hz, 1H), 7.41 (d, J = 6.3 Hz, 1H), 7.39 - 7.35 (m, 2H), 4.54 (d, J = 5.6 Hz, 2H), 3.24 - 3.17 (m, 4H), 2.84 - 2.79 (m, 3H), 1.85 (p, 4H); MS: 519.6 [M+H ⁺ ].

Example 7

5,5'-(butane-1,4-diyl)bis(N-(2-chloro-5-methoxybenzyl)-1,3,4-thiadiazole-2-carboxamide)

The target compound was obtained as in Example 1.

white solid (16.0 mg, 64%): ¹ H NMR (500 MHz, DMSO) δ 9.76 (t, J = 6.1 Hz, 2H), 7.38 (d, J = 8.5 Hz, 2H), 6.93 - 6.88 (m, 4H), 4.51 (d, J = 6.1 Hz, 4H), 3.73 (s, 6H), 3.27 - 3.21 (m, 4H), 1.88 (p, J = 3.6 Hz, 4H); MS: 621.7 [M+H ⁺ ].

Example 8

N-(2-chloro-5-methoxybenzyl)-5-(4-(5-(methylcarbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide

The target compound was obtained in the same manner as in Example 1 using the starting material of Manufacturing Example 3.

white solid (7.2 mg, 53%): ¹ H NMR (500 MHz, DMSO) δ 9.76 (t, J = 6.1 Hz, 1H), 9.13 (q, J = 4.7 Hz, 1H), 7.38 (d, J = 8.4 Hz, 1H), 6.95 - 6.87 (m, 2H), 4.55 - 4.47 (m, 2H), 3.26 - 3.18 (m, 4H), 2.81 (d, J = 4.8 Hz, 3H), 1.86 (p, J = 3.8 Hz, 4H); MS: 481.6 [M+H ⁺ ].

Example 9

N-Methyl-5-(4-(5-((2-methyl-5-(trifluoromethoxy)benzyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide

The target compound was obtained in the same manner as in Example 1 using the starting material of Manufacturing Example 3.

white solid (7.1 mg, 49%): ¹ H NMR (500 MHz, DMSO) δ 9.80 (t, J = 6.2 Hz, 1H), 9.13 (t, J = 4.8 Hz, 1H), 7.31 (d, J = 8.3 Hz, 1H), 7.21 (s, 1H), 7.17 (d, J = 8.1 Hz, 1H), 4.47 (d, J = 6.1 Hz, 2H), 3.25 - 3.18 (m, 4H), 2.81 (d, J = 4.8 Hz, 3H), 2.35 (s, 3H), 1.86 (p, 4H); MS: 515.7 [M+H ⁺ ].

Example 10

5,5'-(butane-1,4-diyl)bis(N-(2-methyl-5-(trifluoromethoxy)benzyl)-1,3,4-thiadiazole-2-carboxamide)

The target compound was obtained as in Example 1.

white solid (15.1 mg, 81%): ¹ H NMR (500 MHz, DMSO) δ 9.80 (t, J = 6.1 Hz, 2H), 7.31 (d, J = 8.3 Hz, 2H), 7.21 (d, J = 2.6 Hz, 2H), 7.17 (d, J = 8.4 Hz, 2H), 4.47 (d, J = 6.1 Hz, 4H), 3.25 - 3.20 (m, 4H), 2.35 (s, 6H), 1.87 (p, J = 3.9 Hz, 4H); MS: 689.8 [M+H ⁺ ].

Example 11

N-(2-fluoro-5-(trifluoromethoxy)benzyl)-5-(4-(5-((pyridin-2-ylmethyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide

The target compound was obtained in the same manner as in Example 1 using the starting material of Manufacturing Example 4.

white solid (9.2 mg, 33%): ¹ H NMR (500 MHz, DMSO) δ 9.82 (t, J = 6.1 Hz, 1H), 9.72 (t, J = 6.1 Hz, 1H), 8.53 - 8.51 (m, 1H), 7.77 (td, J = 7.7, 1.8 Hz, 1H), 7.43 - 7.32 (m, 4H), 7.32 - 7.26 (m, 1H), 4.58 (d, J = 6.1 Hz, 2H), 4.54 (d, J = 6.1 Hz, 2H), 3.24 (t, J = 6.7 Hz, 4H), 1.87 (p, J = 3.6 Hz, 4H); MS: 596.5 [M+H ⁺ ].

Example 12

N-(2-Methyl-5-(trifluoromethoxy)benzyl)-5-(4-(5-((pyridin-2-ylmethyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide

The target compound was obtained in the same manner as in Example 1 using the starting material of Manufacturing Example 4.

white solid (15.4 mg, 75%): ¹ H NMR (500 MHz, DMSO) δ 9.80 (t, J = 6.1 Hz, 1H), 9.72 (t, J = 6.1 Hz, 1H), 8.54 - 8.51 (m, 1H), 7.77 (td, J = 7.7, 1.8 Hz, 1H), 7.37 - 7.26 (m, 3H), 7.21 (s, 1H), 7.20 - 7.15 (m, 1H), 4.59 (d, J = 6.1 Hz, 2H), 4.47 (d, J = 6.0 Hz, 2H), 3.23 (t, 4H), 2.35 (s, 3H), 1.87 (p, 4H); MS: 592.5 [M+H ⁺ ].

Example 13

N-(2-chloro-5-methoxybenzyl)-5-(4-(5-((pyridin-2-ylmethyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide

The target compound was obtained in the same manner as in Example 1 using the starting material of Manufacturing Example 4.

white solid (14.5 mg, 56%): ¹ H NMR (500 MHz, DMSO) δ 9.76 (t, J = 6.1 Hz, 1H), 9.72 (t, J = 6.2 Hz, 1H), 8.54 - 8.50 (m, 1H), 7.77 (td, J = 7.7, 1.8 Hz, 1H), 7.41 - 7.33 (m, 2H), 7.31 - 7.26 (m, 1H), 6.94 - 6.88 (m, 2H), 4.59 (d, J = 6.1 Hz, 2H), 4.51 (d, J = 6.1 Hz, 2H), 3.73 (s, 3H), 3.28 - 3.20 (m, 4H), 1.92 - 1.83 (m, 4H); MS: 558.4 [M+H ⁺ ].

Example 14

5,5'-(butane-1,4-diyl)bis(N-((4-methoxypyridin-2-yl)methyl)-1,3,4-thiadiazole-2-carboxamide)

The target compound was obtained as in Example 1.

white solid (21.0 mg, 94%): ¹ H NMR (500 MHz, DMSO) δ 9.64 (s, 2H), 8.34 (d, J = 5.7 Hz, 2H), 6.94 - 6.86 (m, 4H), 4.54 (d, J = 6.1 Hz, 4H), 3.81 (s, 6H), 3.28 - 3.20 (m, 4H), 1.95 - 1.81 (m, 4H); MS: 555.5 [M+H ⁺ ].

Example 15

N-((4-methoxypyridin-2-yl)methyl)-5-(4-(5-(methylcarbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide

The target compound was obtained in the same manner as in Example 1 using the starting material of Manufacturing Example 3.

white solid (10.6 mg, 56%): ¹ H NMR (500 MHz, DMSO) δ 9.65 (t, J = 6.1 Hz, 1H), 9.17 - 9.09 (m, 1H), 8.34 (d, J = 5.6 Hz, 1H), 6.95 - 6.85 (m, 2H), 4.53 (d, J = 6.1 Hz, 2H), 3.81 (s, 3H), 3.28 - 3.15 (m, 4H), 2.81 (d, J = 4.8 Hz, 3H), 1.86 (p, 4H); MS: 448.4 [M+H ⁺ ].

Example 16

N-((4-methoxypyridin-2-yl)methyl)-5-(4-(5-((pyridin-2-ylmethyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide

The target compound was obtained in the same manner as in Example 1 using the starting material of Manufacturing Example 4.

white solid (10.3 mg, 57%): ¹ H NMR (500 MHz, DMSO) δ 9.72 (t, J = 6.2 Hz, 1H), 9.65 (t, J = 6.0 Hz, 1H), 8.55 - 8.50 (m, 1H), 8.34 (d, J = 5.7 Hz, 1H), 7.77 (td, J = 7.7, 1.8 Hz, 1H), 7.35 (d, J = 7.9 Hz, 1H), 7.32 - 7.26 (m, 1H), 6.93 - 6.87 (m, 2H), 4.59 (d, J = 6.1 Hz, 2H), 4.53 (d, J = 6.0 Hz, 2H), 3.81 (s, 3H), 3.29 - 3.20 (m, 4H), 1.88 (p, J = 3.5 Hz, 4H); MS: 525.5 [M+H ⁺ ].

Example 17

5,5'-(butane-1,4-diyl)bis(N-((4-chloropyridin-2-yl)methyl)-1,3,4-thiadiazole-2-carboxamide)

The target compound was obtained as in Example 1.

white solid (15.4 mg, 68%): ¹ H NMR (500 MHz, DMSO) δ 9.75 (t, J = 6.1 Hz, 2H), 8.51 (d, J = 5.3 Hz, 2H), 7.53 - 7.41 (m, 4H), 4.60 (d, J = 6.1 Hz, 4H), 3.28 - 3.20 (m, 4H), 1.88 (p, J = 3.8 Hz, 4H); MS: 563.4 [M+H ⁺ ].

Example 18

N-((4-chloropyridin-2-yl)methyl)-5-(4-(5-(methylcarbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide

The target compound was obtained in the same manner as in Example 1 using the starting material of Manufacturing Example 3.

white solid (8.0 mg, 42%): ¹ H NMR (500 MHz, DMSO) δ 9.75 (t, J = 6.2 Hz, 1H), 9.16 - 9.10 (m, 1H), 8.51 (d, J = 5.3 Hz, 1H), 7.51 - 7.43 (m, 2H), 4.60 (d, J = 6.1 Hz, 2H), 3.27 - 3.19 (m, 4H), 2.81 (d, J = 4.8 Hz, 3H), 1.86 (p, J = 5.5 Hz, 4H); MS: 452.3 [M+H ⁺ ].

Example 19

N-((4-chloropyridin-2-yl)methyl)-5-(4-(5-((pyridin-2-ylmethyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide

The target compound was obtained in the same manner as in Example 1 using the starting material of Manufacturing Example 4.

white solid (9.1 mg, 50%): ¹ H NMR (500 MHz, DMSO) δ 9.79 - 9.69 (m, 2H), 8.56 - 8.48 (m, 2H), 7.77 (td, J = 7.7, 1.8 Hz, 1H), 7.52 - 7.43 (m, 2H), 7.35 (d, J = 7.9 Hz, 1H), 7.31 - 7.26 (m, 1H), 4.63 - 4.56 (m, 4H), 3.27 - 3.21 (m, 4H), 1.88 (p, J = 3.7 Hz, 4H); MS: 529.4 [M+H ⁺ ].

Example 20

N-Methyl-5-(4-(5-(((4-(trifluoromethyl)pyridin-2-yl)methyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide

The target compound was obtained in the same manner as in Example 1 using the starting material of Manufacturing Example 3.

white solid (17.5 mg, 64%): ¹ H NMR (500 MHz, DMSO) δ 9.80 (t, J = 6.1 Hz, 1H), 9.16 - 9.10 (m, 1H), 8.82 (d, J = 5.1 Hz, 1H), 7.74 (s, 1H), 7.70 (d, J) = 5.2 Hz, 1H), 4.70 (d, J = 6.1 Hz, 2H), 3.26 - 3.18 (m, 4H), 2.81 (d, J = 4.7 Hz, 3H), 1.86 (p, J = 3.8 Hz, 4H); MS: 486.4 [M+H ⁺ ].

Example 21

N-(pyridin-2-ylmethyl)-5-(4-(5-(((4-(trifluoromethyl)pyridin-2-yl)methyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide

The target compound was obtained in the same manner as in Example 1 using the starting material of Manufacturing Example 4.

white solid (16.2 mg, 83%): ¹ H NMR (500 MHz, DMSO) δ 9.81 (t, J = 6.1 Hz, 1H), 9.72 (t, J = 6.1 Hz, 1H), 8.82 (d, J = 5.1 Hz, 1H), 8.56 - 8.50 (m, 1H), 7.77 (td, J = 7.7, 1.8 Hz, 1H), 7.74 (s, 1H), 7.69 (d, J = 5.1 Hz, 1H), 7.35 (d, J = 7.9 Hz, 1H), 7.32 - 7.26 (m, 1H), 4.71 (d, J = 6.1 Hz, 2H), 4.59 (d, J = 6.1 Hz, 2H), 3.24 (t, J = 6.5 Hz, 4H), 1.88 (p, J = 3.6 Hz, 4H); MS: 563.4 [M+H ⁺ ].

Example 22

5,5'-(butane-1,4-diyl)bis(N-((4-(trifluoromethyl)pyridin-2-yl)methyl)-1,3,4-thiadiazole-2-carboxamide)

The target compound was obtained as in Example 1.

white solid (15.8 mg, 62%): ¹ H NMR (500 MHz, DMSO) δ 9.83 (s, 2H), 7.42 (d, J = 6.0 Hz, 2H), 7.39 - 7.35 (m, 4H), 4.52 (s, 4H), 3.01 (t, 4H), 1.86 (p, J = 3.6 Hz, 4H); MS: 631.5 [M+H ⁺ ].

Example 23

N-((4-(trifluoromethyl)pyridin-2-yl)methyl)-5-(4-(5-((2,3,4-trimethoxybenzyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide

The target compound was obtained in the same manner as in Example 1 using the starting material of Manufacturing Example 5.

white solid (7.9 mg, 36%): ¹ H NMR (500 MHz, DMSO) δ 9.79 (t, J = 6.0 Hz, 1H), 9.51 (t, J = 6.1 Hz, 1H), 8.81 (d, J = 5.1 Hz, 1H), 7.72 (s, 1H), 7.68 (d, J = 5.2 Hz, 1H), 6.95 (d, J = 8.6 Hz, 1H), 6.76 (d, J = 8.7 Hz, 1H), 4.69 (d, J = 6.0 Hz, 2H), 4.40 (d, J = 6.1 Hz, 2H), 3.81 (s, 3H), 3.76 (s, 3H), 3.74 (s, 3H), 3.25 - 3.18 (m, 4H), 1.85 (p, J = 4.2 Hz, 4H); MS: 652.6 [M+H ⁺ ].

Example 24

N-Benzyl-5-(4-(5-(((4-(trifluoromethyl)pyridin-2-yl)methyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide

The target compound was obtained in the same manner as in Example 1 using the compound of Manufacturing Example 5 as a starting material.

white solid (13.8 mg, 82%): ¹ H NMR (500 MHz, DMSO) δ 9.83 - 9.76 (m, 2H), 8.82 (d, J = 5.1 Hz, 1H), 7.73 (s, 1H), 7.70 (d, J = 4.7 Hz, 1H), 7.33 (d, J) = 4.4 Hz, 4H), 7.29 - 7.23 (m, 1H), 4.70 (d, J = 6.0 Hz, 2H), 4.47 (d, J = 6.3 Hz, 2H), 3.25 - 3.20 (m, 4H), 1.86 (p, J = 3.8 Hz, 4H); MS: 562.5 [M+H ⁺ ].

Example 25

5,5'-(butane-1,4-diyl)bis(N-((3-fluoropyridin-2-yl)methyl)-1,3,4-thiadiazole-2-carboxamide)

The target compound was obtained as in Example 1.

white solid (15.9 mg, 74%): ¹ H NMR (500 MHz, DMSO) δ 9.53 (s, 2H), 8.38 (dt, J = 4.7, 1.5 Hz, 2H), 7.75 - 7.68 (m, 2H), 7.44 - 7.38 (m, 2H), 4.66 (d, J = 4.5 Hz, 4H), 3.24 - 3.18 (m, 4H), 1.85 (p, J = 4.0 Hz, 4H); MS: 531.4 [M+H ⁺ ].

Example 26

5,5'-(butane-1,4-diyl)bis(N-((6-fluoropyridin-2-yl)methyl)-1,3,4-thiadiazole-2-carboxamide)

The target compound was obtained as in Example 1.

white solid (15.0 mg, 70%): ¹ H NMR (500 MHz, DMSO) δ 9.78 (s, 2H), 7.97 (q, J = 8.1 Hz, 2H), 7.31 (dd, J = 7.4, 2.5 Hz, 2H), 7.07 (dd, J = 8.2, 2.5 Hz, 2H), 4.53 (d, J = 5.2 Hz, 4H), 3.24 (t, 4H), 1.88 (p, J = 3.6 Hz, 4H); MS: 531.4 [M+H ⁺ ].

Example 27

N-((6-fluoropyridin-2-yl)methyl)-5-(4-(5-(((4-(trifluoromethyl)pyridin-2-yl)methyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide

The target compound was obtained in the same manner as in Example 1 using the compound of Manufacturing Example 5 as a starting material.

white solid (7.5 mg, 43%): ¹ H NMR (500 MHz, DMSO) δ 9.80 (q, J = 6.4 Hz, 2H), 8.82 (d, J = 5.1 Hz, 1H), 7.97 (q, J = 8.1 Hz, 1H), 7.73 (s, 1H), 7.69 (d, J = 5.2 Hz, 1H), 7.30 (dd, J = 7.5, 2.5 Hz, 1H), 7.07 (dd, J = 8.2, 2.5 Hz, 1H), 4.70 (d, J = 5.9 Hz, 2H), 4.53 (d, J = 6.0 Hz, 2H), 3.27 - 3.21 (m, 4H), 1.88 (p, J = 3.7 Hz, 4H); MS: 581.5 [M+H ⁺ ].

Example 28

5,5'-(butane-1,4-diyl)bis(N-((4-(trifluoromethyl)pyridin-3-yl)methyl)-1,3,4-thiadiazole-2-carboxamide)

The target compound was obtained as in Example 1.

white solid (21.1 mg, 83%): ¹ H NMR (500 MHz, DMSO) δ 9.90 (t, J = 5.9 Hz, 2H), 8.82 (s, 2H), 8.78 (d, J = 5.1 Hz, 2H), 7.75 (d, J = 5.1 Hz, 2H), 4.71 (d, J = 5.6 Hz, 4H), 3.23 (t, J = 6.7 Hz, 4H), 1.87 (p, 4H); MS: 631.5 [M+H ⁺ ].

Example 29

N-((3-fluoropyridin-2-yl)methyl)-5-(4-(5-(((4-(trifluoromethyl)pyridin-2-yl)methyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide

The target compound was obtained in the same manner as in Example 1 using the compound of Manufacturing Example 5 as a starting material.

white solid (6.5 mg, 37%): ¹ H NMR (500 MHz, DMSO) δ 9.80 (t, J = 6.1 Hz, 1H), 9.54 (t, J = 5.8 Hz, 1H), 8.82 (d, J = 5.1 Hz, 1H), 8.39 (dt, J = 4.7, 1.5) Hz, 1H), 7.76 - 7.67 (m, 3H), 7.46 - 7.39 (m, 1H), 4.70 (d, J = 6.0 Hz, 2H), 4.69 - 4.65 (m, 2H), 3.28 - 3.20 (m, 4H), 1.92 - 1.83 (m, 4H); MS: 581.5 [M+H ⁺ ].

Example 30

N-(2,3-dimethoxybenzyl)-5-(4-(5-(((4-(trifluoromethyl)pyridin-2-yl)methyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide

The target compound was obtained in the same manner as in Example 1 using the compound of Manufacturing Example 5 as a starting material.

white solid (6.1 mg, 33%): ¹ H NMR (500 MHz, DMSO) δ 9.80 (t, J = 6.1 Hz, 1H), 9.60 (t, J = 6.2 Hz, 1H), 8.82 (d, J = 5.1 Hz, 1H), 7.73 (d, J = 1.6 Hz, 1H), 7.69 (d, J = 5.1 Hz, 1H), 7.02 (t, J = 7.9 Hz, 1H), 6.96 (dd, J = 8.2, 1.6 Hz, 1H), 6.86 (dd, J = 7.6, 1.6 Hz, 1H), 4.70 (d, J = 6.0 Hz, 2H), 4.49 (d, J = 6.2 Hz, 2H), 3.81 (s, 3H), 3.77 (s, 3H), 3.26 - 3.20 (m, 4H), 1.87 (p, J = 3.7 Hz, 4H); MS: 622.5 [M+H ⁺ ].

Example 31

N-((3,4-dimethoxypyridin-2-yl)methyl)-5-(4-(5-(((4-(trifluoromethyl)pyridin-2-yl)methyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide

The target compound was obtained in the same manner as in Example 1 using the compound of Manufacturing Example 5 as a starting material.

white solid (5.2 mg, 28%): ¹ H NMR (500 MHz, DMSO) δ 9.80 (t, J = 6.1 Hz, 1H), 9.26 (t, J = 5.6 Hz, 1H), 8.82 (d, J = 5.1 Hz, 1H), 8.19 (d, J = 5.5 Hz, 1H), 7.73 (s, 1H), 7.69 (d, J = 5.1 Hz, 1H), 7.09 (d, J = 5.6 Hz, 1H), 4.70 (d, J = 6.0 Hz, 2H), 4.60 (d, J = 5.5 Hz, 2H), 3.90 (s, 3H), 3.81 (s, 3H), 3.27 - 3.20 (m, 4H), 1.92 - 1.83 (m, 4H); MS: 623.5 [M+H ⁺ ].

Example 32

N-((4-methoxypyridin-2-yl)methyl)-5-(4-(5-(((4-(trifluoromethyl)pyridin-2-yl)methyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide

The target compound was obtained in the same manner as in Example 1 using the compound of Manufacturing Example 5 as a starting material.

white solid (10.1 mg, 57%): ¹ H NMR (500 MHz, DMSO) δ 9.80 (t, J = 5.9 Hz, 1H), 9.64 (t, J = 6.1 Hz, 1H), 8.82 (d, J = 5.1 Hz, 1H), 8.34 (d, J = 5.4 Hz, 1H), 7.74 (d, J = 1.8 Hz, 1H), 7.72 - 7.67 (m, 1H), 6.93 - 6.86 (m, 2H), 4.70 (d, J = 5.6 Hz, 2H), 4.53 (d, J = 5.9 Hz, 2H), 3.81 (s, 3H), 3.27 - 3.21 (m, 4H), 1.88 (p, 4H); MS: 593.5 [M+H ⁺ ].

Example 33

N-((4-(trifluoromethyl)pyridin-2-yl)methyl)-5-(4-(5-(((4-(trifluoromethyl)pyridin-3-yl)methyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide

The target compound was obtained in the same manner as in Example 1 using the compound of Manufacturing Example 5 as a starting material.

white solid (7.5 mg, 40%): ¹ H NMR (500 MHz, DMSO) δ 9.90 (s, 1H), 9.80 (t, J = 6.0 Hz, 1H), 8.82 (t, J = 2.6 Hz, 2H), 8.78 (d, J = 5.1 Hz, 1H), 7.75 (d, J = 5.1 Hz, 1H), 7.73 (s, 1H), 7.69 (d, J = 5.2 Hz, 1H), 4.70 (d, J = 5.5 Hz, 4H), 3.27 - 3.21 (m, 4H), 1.92 - 1.84 (m, 4H); MS: 631.5 [M+H ⁺ ].

Example 34

N-(2-Fluoro-5-methoxybenzyl)-5-(4-(5-(((4-(trifluoromethyl)pyridin-2-yl)methyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide

The target compound was obtained in the same manner as in Example 1 using the compound of Manufacturing Example 5 as a starting material.

white solid (9.1 mg, 50%): ¹ H NMR (500 MHz, DMSO) δ 9.80 (t, J = 6.1 Hz, 1H), 9.74 (t, J = 6.1 Hz, 1H), 8.82 (d, J = 5.1 Hz, 1H), 7.73 (s, 1H), 7.69 (d, J = 5.2 Hz, 1H), 7.12 (t, J = 9.3 Hz, 1H), 6.92 (dd, J = 6.1, 3.2 Hz, 1H), 6.86 (dt, J = 8.9, 3.6 Hz, 1H), 4.70 (d, J = 5.9 Hz, 2H), 4.48 (d, J = 6.0 Hz, 2H), 3.26 - 3.19 (m, 4H), 1.91 - 1.83 (m, 4H); MS: 610.5 [M+H ⁺ ].

Example 35

N-(2-Fluoro-5-(trifluoromethoxy)benzyl)-5-(4-(5-(((4-(trifluoromethyl)pyridin-2-yl)methyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide

To a solution of the compound of Preparation Example 5 (12.0 mg, 0.0242 mmol) in isopropanol (0.24 mL) was stirred (2-fluoro-5-(trifluoromethoxy)phenyl)methanamine (20.0 mg, 0.0967 mmol) at room temperature. The reaction mixture was heated at 80 °C for 18 h. It was then cooled and filtered to obtain the target compound.

white solid (11.1 mg, 69%): ¹ H NMR (300 MHz, MeOD) δ 8.79 (d, J = 5.2 Hz, 1H), 7.73 (s, 1H), 7.62 (d, J = 5.2 Hz, 1H), 7.36 (d, J = 6.0 Hz, 1H), 7.26 - 7.10 (m, 3H), 4.82 (s, 2H), 4.67 (s, 2H), 3.31 - 3.25 (m, 1H), 2.04 - 1.94 (m, 4H); MS: 664.7 [M+H ⁺ ].

Example 36

5,5'-(butane-1,4-diyl)bis(N-((4-(3,3-difluorocyclobutoxy)pyridin-2-yl)methyl)-1,3,4-thiadiazole-2-carboxamide)

(4-(3,3-difluorocyclobutoxy)pyridin-2-yl)methaneamine trifluoroacetic acid salt (47.0 mg, 0.142 mmol) was added to a solution of the compound of Preparation Example 2 (15.0 mg, 0.0405 mmol) and triethylamine (0.07 mL, 0.486 mmol) in isopropanol (0.4 mL) under stirring at room temperature. The reaction mixture was heated at 80 °C for 24 h. It was then cooled and filtered to obtain the target compound.

white solid (17.3 mg, 60%): ¹ H NMR (400 MHz, DMSO) δ 9.63 (t, J = 6.1 Hz, 1H), 8.36 (d, J = 5.6 Hz, 1H), 6.86 (dt, J = 8.1, 2.5 Hz, 2H), 4.85 (dd, J = 8.2, 4.8 Hz, 1H), 4.53 (d, J = 6.1 Hz, 2H), 3.22 (pt, J = 11.7, 5.2 Hz, 6H), 2.72 (qd, J = 14.2, 4.8 Hz, 2H), 1.90 - 1.83 (m, 4H); MS: 707.5 [M+H ⁺ ].

Example 37

N-((4-(3,3-difluorocyclobutoxy)pyridin-2-yl)methyl)-5-(4-(5-(((4-(trifluoromethyl)pyridin-2-yl)methyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide

To a solution of compound of Preparation Example 5 (10.0 mg, 0.0200 mmol) and triethylamine (0.028 mL, 0.200 mmol) in isopropanol (0.2 mL) was stirred (4-(3,3-difluorocyclobutoxy)pyridin-2-yl)methaneamine trifluoroacetic acid salt (9.84 mg, 0.0300 mmol) at room temperature. The reaction mixture was heated at 80 °C for 18 h. It was then cooled and filtered to obtain the target compound.

white solid (9.1 mg, 68%): ¹ H NMR (400 MHz, DMSO) δ 9.78 (t, J = 6.1 Hz, 1H), 8.81 (d, J = 5.1 Hz, 1H), 8.35 (d, J = 5.6 Hz, 1H), 7.72 (s, 1H), 7.68 (d, J = 5.2 Hz, 1H), 6.90 - 6.82 (m, 2H), 4.93 - 4.79 (m, 1H), 4.70 (d, J = 6.1 Hz, 2H), 4.53 (d, J = 6.1 Hz, 2H), 3.29 - 3.12 (m, 6H), 2.71 (qd, J = 14.1, 4.7 Hz, 2H), 1.91 - 1.83 (m, 3H); MS: 669.9 [M+H ⁺ ].

Example 38

N-((4-(3,3-difluorocyclobutoxy)pyridin-2-yl)methyl)-5-(4-(5-(methylcarbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide

(4-(3,3-difluorocyclobutoxy)pyridin-2-yl)methaneamine trifluoroacetic acid salt (92.4 mg, 0.281 mmol) was added to a solution of the compound of Preparation Example 3 (40.0 mg, 0.113 mmol) and triethylamine (0.157 mL, 1.13 mmol) in isopropanol (1.1 mL) under stirring at room temperature. The reaction mixture was heated at 80 °C for 18 h. It was then cooled and filtered to obtain the target compound.

white solid (21.1 mg, 36%): ¹ H NMR (400 MHz, DMSO) δ 9.63 (t, J = 6.1 Hz, 1H), 9.11 (q, J = 5.0 Hz, 1H), 8.36 (d, J = 5.6 Hz, 1H), 6.91 - 6.82 (m, 2H), 4.85 (dd, J = 8.1, 4.6 Hz, 1H), 4.53 (d, J = 6.1 Hz, 2H), 3.30 - 3.14 (m, 6H), 2.81 (d, J = 4.8 Hz, 3H), 2.72 (qd, J = 14.3, 4.9 Hz, 2H), 1.86 (p, J = 3.4 Hz, 4H); MS: 524.3 [M+H ⁺ ].

Example 39

(4-(3,3-difluorocyclobutoxy)pyridin-2-yl)methaneamine trifluoroacetic acid salt (75.9 mg, 0.231 mmol) was added to a solution of the compound of Preparation Example 4 (40.0 mg, 0.093 mmol) and triethylamine (0.129 mL, 0.925 mmol) in isopropanol (0.9 mL) under stirring at room temperature. The reaction mixture was heated at 80 °C for 18 h. It was then cooled and filtered to obtain the target compound.

white solid (24.2 mg, 44%): ¹ H NMR (400 MHz, DMSO) δ 9.70 (t, J = 6.2 Hz, 1H), 9.64 (t, J = 6.1 Hz, 1H), 8.52 (d, J = 4.7 Hz, 1H), 7.77 (td, J = 7.7, 1.8 Hz, 1H), 7.35 (d, J = 7.9 Hz, 1H), 7.28 (dd, J = 7.5, 5.0 Hz, 1H), 6.91 - 6.82 (m, 2H), 4.92 - 4.78 (m, 1H), 4.58 (d, J = 6.1 Hz, 2H), 4.53 (d, J = 6.1 Hz, 2H), 3.29 - 3.15 (m, 6H), 2.72 (qd, J = 14.3, 4.8 Hz, 2H), 1.88 (p, J = 3.6 Hz, 4H); MS: 601.3 [M+H ⁺ ].

<Experimental Example 1> Analysis of glutaminase inhibitory activity outside the cell

To confirm the glutaminase inhibitory activity outside the cell, the inhibitory activity IC ₅₀ for glutaminase was measured when the example compound was treated. The experimental method is as follows.

To measure glutaminase activity, a GLS1 inhibitor screening assay kit (catalog number #79596) provided by BPS Bioscience was used. First, 8 μL of a solution containing 10 ng of GLS1 (elongated glutaminase) was dispensed into each well of a 384 plate (black, low volume, round bottom). 2 μL of a compound solution prepared at a concentration 5 times higher than the final concentration of the enzyme solution was added and reacted at room temperature for two hours. After the reaction was complete, L-glutamine, NAD+, and coupling reagent were mixed using GLS1 buffer solution, and 10 μL of this mixture was dispensed into each well. After an additional 30 minutes of reaction at room temperature, the enzyme activity was confirmed by measuring the fluorescence value. The IC ₅₀ values of glutaminase inhibitory activity were determined by measuring the activity of glutaminase at six different compound concentrations and analyzing the results using GraphPad Prism. The results are shown in Table 2 below.

Example GLS1 IC ₅₀ (μM) Example GLS1 IC ₅₀ (μM) 1 0.75 21 0.66 2 >10 22 0.032 3 >10 23 0.36 4 >10 24 0.05 5 0.02 25 3.3 6 >10 26 >10 7 >10 27 0.62 8 >10 28 >10 9 >10 29 2.1 10 >10 30 1.1 11 0.39 31 0.87 12 >10 32 7.3 13 6.9 33 3.1 14 5.5 34 0.53 15 >10 35 0.011 16 >10 36 0.03 17 0.044 37 0.046 18 0.037 38 1.7 19 5.6 39 1.3 20 >10

<Experimental Example 2> Cytotoxicity Experiment

In order to confirm the anticancer effect of the compound according to the present invention, IC ₅₀ was measured through cell viability according to concentration using CB-839 as a reference in the LR (Ceritinib (LDK378) resistant) pool cell line, which is an ALK-TKI resistant cell.

In the ALK-positive non-small cell lung cancer cell line H3122, which is sensitive to the ALK inhibitor Ceritinib, the concentration of Ceritinib was continuously increased from 0.01 μM to 1 μM. The surviving H3122 cell line was continuously cultured in a medium supplemented with 1 μM Ceritinib to establish an acquired-resistant non-small cell lung cancer cell line (hereinafter referred to as H3122-LR pool).

Sequencing results showed that this resistant cell line showed amplification of NRF2, a predictive biomarker for response to glutaminase inhibitors. Therefore, it was selected as a suitable model to test the anticancer effect of the compound. The experimental method is as follows.

After seeding cells in a 96-Well White/Clear Bottom Plate, the compounds and CB-839 reference were serially diluted and treated to the cells the next day. 72 hours after treatment, CellTiter-Glo reagent, which measures ATP present in living cells to determine cell viability, was added, and luminescence was measured using a GloMax® luminometer. The cell viability (% of control) at each drug concentration compared to the untreated control was calculated, and the drug concentration that exhibited 50% cell survival (IC ₅₀ : Half maximal inhibitory concentration) was calculated. The results are shown in Table 3 below.

Example LR pool IC ₅₀ (μM) Example LR pool IC ₅₀ (μM) 1 1.5 21 1 2 9.9 22 0.028 3 >10 23 0.72 4 >10 24 4.5 5 0.018 25 >10 6 2.2 26 >10 7 >10 27 1.6 8 2.3 28 >10 9 4.6 29 >10 10 0.59 30 0.41 11 0.38 31 1.3 12 1.4 32 0.3 13 2.7 33 0.67 14 >10 34 0.31 15 >10 35 0.026 16 4.5 36 - 17 >10 37 0.045 18 9 38 2.3 19 2.3 39 0.7 20 4.6

As shown in the results in Table 3, it was confirmed that the compounds of the present invention exhibit excellent cytotoxicity even against non-small cell lung cancer cells resistant to ALK inhibitors. Accordingly, the compounds of the present invention are expected to be useful as second-line treatments for patients with resistance when ALK inhibitors are used as first-line treatments, and also exhibit excellent therapeutic effects when used in combination with ALK inhibitors.

In addition to the H3122-LR pool cell line, cytotoxicity experiments were performed on other lung cancer cell lines, and the following results were obtained. The results are presented in Table 4, and the values in the table are IC ₅₀ (μM), which is the drug concentration that exhibits 50% cell survival.

Example A549 H460 H358 H2030 YU-1096 5 0.041 0.031 0.04 0.011 0.12 22 0.069 0.064 0.091 0.022 0.15 37 0.075 0.043 0.041

As shown in Table 4, it was confirmed that excellent cytotoxicity was exhibited against several different lung cancer patient-derived cell lines.

<Experimental Example 3> Analysis of glutaminase inhibitory activity in cells

To confirm the glutaminase inhibitory activity in H3122-LR pool cells, the levels of glutamine and glutamate were compared after treating the cells with the example compounds. The experimental method is as follows.

After seeding cells in 96-Well White/Clear Bottom Plate, compounds and CB-839 reference were treated with 0.3 μM each the next day. After culturing for 24 hours, intracellular glutamine/glutamate levels were measured using the Glutamine/Glutamate-Glo™ Assay kit. At this time, the culture medium was used by adding 5 mM glucose, 2 mM glutamine, and 10% dialyzed FBS to serum-free medium without glucose, glutamine, or pyruvate.

As shown in Fig. 1a, in the case of Example Compounds 5 and 22, when 0.3 μM was treated, the glutamine concentration was high, whereas in Fig. 1b, the glutamate concentration was low. Since glutamine is converted to glutaminase by glutaminase, the concentration of glutamine is significantly higher than the concentration of glutamate, indicating that the compound according to the present invention is absorbed into cells and exhibits excellent cell activity through glutaminase inhibition even within cells.

<Experimental Example 4>

The in vivo activity was evaluated by administering CB-839 200 mg/kg and Example 22 100 mg/kg, respectively, to mice (n=8) administered with the H3122-LR pool cell line, which is most sensitive to CB-839, orally twice a day for 30 days, dissolved in [25% hydroxypropyl-b-cyclodextrin 10 mM citrate (pH 2.0)].

As shown in Fig. 2a, Example 22 at a lower dose than CB-839 (TGI 76.31%) showed similar cancer growth inhibition (TGI 75.67%), and as shown in Fig. 2b, no particular weight loss was observed for either substance, verifying the in vivo activity of Example 22.

Claims (14)

A compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof:

L is C2-6 alkylene, R ¹ and R ² are each independently C1-10 alkyl, C6-10 aryl substituted C1-10 alkyl or C1-10 alkyl substituted with 5 to 10 membered heteroaryl containing 1 to 3 heteroatoms selected from N, O and S, wherein the aryl and heteroaryl are each independently substituted with one or more selected from the group consisting of unsubstituted or halogen, C1-10 alkyl unsubstituted or substituted with one or more halogens, C1-10 alkoxy unsubstituted or substituted with one or more halogens, and 3 to 6 membered cycloalkyloxy substituted with one or more halogens. In claim 1, L is (CH ₂ ) _n , where n is an integer from 2 to 6, A compound or a pharmaceutically acceptable salt thereof. In claim 1, R ¹ and R ² are each independently C1-6 alkyl, C1-6 alkyl substituted with C6 aryl, or C1-6 alkyl substituted with 5-6 membered heteroaryl containing 1-3 heteroatoms, which is N, wherein the aryl and heteroaryl are each independently selected from the group consisting of unsubstituted or substituted with halogen, C1-6 alkyl unsubstituted or substituted with one or more halogens, C1-6 alkoxy unsubstituted or substituted with one or more halogens, and 3 to 5 membered cycloalkyloxy substituted with one or more halogens, a compound or a pharmaceutically acceptable salt thereof. In claim 1, A compound or a pharmaceutically acceptable salt thereof, wherein said aryl is phenyl and said heteroaryl is pyridyl. In claim 1, L is a compound represented by (CH ₂ ) _{n ,} wherein n is an integer from 3 to 5, or a pharmaceutically acceptable salt thereof. In claim 1, R ¹ and R ² are each independently methyl, phenylmethyl or pyridylmethyl, The phenyl of the above phenylmethyl and the pyridyl of the pyridylmethyl are each independently unsubstituted or substituted with 1 to 3 groups selected from the group consisting of F, Cl, methyl, methoxy, CF ₃ , OCF ₃ and 3,3-difluorocyclobutyloxy. A compound or a pharmaceutically acceptable salt thereof. In claim 1, The compound is a compound selected from the group consisting of: or a pharmaceutically acceptable salt thereof: 1) 5,5'-(butane-1,4-diyl)bis(N-(2-fluoro-5-methoxybenzyl)-1,3,4-thiadiazole-2-carboxamide); 2) 5,5'-(butane-1,4-diyl)bis(N-((5-methoxypyridin-2-yl)methyl)-1,3,4-thiadiazole-2-carboxamide); 3) 5,5'-(Butane-1,4-diyl)bis(N-((6-methoxypyridin-2-yl)methyl)-1,3,4-thiadiazole-2-carboxamide); 4) 5,5'-(butane-1,4-diyl)bis(N-benzyl-1,3,4-thiadiazole-2-carboxamide); 5) 5,5'-(Butane-1,4-diyl)bis(N-(2-fluoro-5-(trifluoromethoxy)benzyl)-1,3,4-thiadiazole-2-carboxamide); 6) N-(2-Fluoro-5-(trifluoromethoxy)benzyl)-5-(4-(5-(methylcarbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide; 7) 5,5'-(Butane-1,4-diyl)bis(N-(2-chloro-5-methoxybenzyl)-1,3,4-thiadiazole-2-carboxamide); 8) N-(2-chloro-5-methoxybenzyl)-5-(4-(5-(methylcarbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide; 9) N-Methyl-5-(4-(5-((2-methyl-5-(trifluoromethoxy)benzyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide; 10) 5,5'-(Butane-1,4-diyl)bis(N-(2-methyl-5-(trifluoromethoxy)benzyl)-1,3,4-thiadiazole-2-carboxamide); 11) N-(2-Fluoro-5-(trifluoromethoxy)benzyl)-5-(4-(5-((pyridin-2-ylmethyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide; 12) N-(2-Methyl-5-(trifluoromethoxy)benzyl)-5-(4-(5-((pyridin-2-ylmethyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide; 13) N-(2-chloro-5-methoxybenzyl)-5-(4-(5-((pyridin-2-ylmethyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide; 14) 5,5'-(Butane-1,4-diyl)bis(N-((4-methoxypyridin-2-yl)methyl)-1,3,4-thiadiazole-2-carboxamide); 15) N-((4-methoxypyridin-2-yl)methyl)-5-(4-(5-(methylcarbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide; 16) N-((4-methoxypyridin-2-yl)methyl)-5-(4-(5-((pyridin-2-ylmethyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide; 17) 5,5'-(Butane-1,4-diyl)bis(N-((4-chloropyridin-2-yl)methyl)-1,3,4-thiadiazole-2-carboxamide); 18) N-((4-chloropyridin-2-yl)methyl)-5-(4-(5-(methylcarbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide; 19) N-((4-chloropyridin-2-yl)methyl)-5-(4-(5-((pyridin-2-ylmethyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide; 20) N-Methyl-5-(4-(5-(((4-(trifluoromethyl)pyridin-2-yl)methyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide; 21) N-(pyridin-2-ylmethyl)-5-(4-(5-(((4-(trifluoromethyl)pyridin-2-yl)methyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide; 22) 5,5'-(Butane-1,4-diyl)bis(N-((4-(trifluoromethyl)pyridin-2-yl)methyl)-1,3,4-thiadiazole-2-carboxamide); 23) N-((4-(trifluoromethyl)pyridin-2-yl)methyl)-5-(4-(5-((2,3,4-trimethoxybenzyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide; 24) N-Benzyl-5-(4-(5-(((4-(trifluoromethyl)pyridin-2-yl)methyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide; 25) 5,5'-(Butane-1,4-diyl)bis(N-((3-fluoropyridin-2-yl)methyl)-1,3,4-thiadiazole-2-carboxamide); 26) 5,5'-(Butane-1,4-diyl)bis(N-((6-fluoropyridin-2-yl)methyl)-1,3,4-thiadiazole-2-carboxamide); 27) N-((6-fluoropyridin-2-yl)methyl)-5-(4-(5-(((4-(trifluoromethyl)pyridin-2-yl)methyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide; 28) 5,5'-(Butane-1,4-diyl)bis(N-((4-(trifluoromethyl)pyridin-3-yl)methyl)-1,3,4-thiadiazole-2-carboxamide); 29) N-((3-fluoropyridin-2-yl)methyl)-5-(4-(5-(((4-(trifluoromethyl)pyridin-2-yl)methyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide; 30) N-(2,3-dimethoxybenzyl)-5-(4-(5-(((4-(trifluoromethyl)pyridin-2-yl)methyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide; 31) N-((3,4-dimethoxypyridin-2-yl)methyl)-5-(4-(5-(((4-(trifluoromethyl)pyridin-2-yl)methyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide; 32) N-((4-methoxypyridin-2-yl)methyl)-5-(4-(5-(((4-(trifluoromethyl)pyridin-2-yl)methyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide; 33) N-((4-(trifluoromethyl)pyridin-2-yl)methyl)-5-(4-(5-(((4-(trifluoromethyl)pyridin-3-yl)methyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide; 34) N-(2-Fluoro-5-methoxybenzyl)-5-(4-(5-(((4-(trifluoromethyl)pyridin-2-yl)methyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide; 35) N-(2-Fluoro-5-(trifluoromethoxy)benzyl)-5-(4-(5-(((4-(trifluoromethyl)pyridin-2-yl)methyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide; 36) 5,5'-(Butane-1,4-diyl)bis(N-((4-(3,3-difluorocyclobutyloxy)pyridin-2-yl)methyl)-1,3,4-thiadiazole-2-carboxamide); 37) N-((4-(3,3-difluorocyclobutyloxy)pyridin-2-yl)methyl)-5-(4-(5-(((4-(trifluoromethyl)pyridin-2-yl)methyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide; 38) N-((4-(3,3-difluorocyclobutyloxy)pyridin-2-yl)methyl)-5-(4-(5-(methylcarbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide; and 39) N-((4-(3,3-difluorocyclobutyloxy)pyridin-2-yl)methyl)-5-(4-(5-((pyridin-2-ylmethyl)carbamoyl)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazole-2-carboxamide. As shown in reaction scheme 1 below, A step of forming an amide bond between a compound of chemical formula 2 and H ₂ NR ¹ ; and A step of forming an amide bond between a compound of chemical formula 2' and H ₂ NR ² , Method for producing a compound represented by chemical formula 1: [Reaction Formula 1]

Here, L, R ¹ and R ² are the same as defined in claim 1, Y is C1-C10 alkyl. As shown in reaction formula 2 below, A step of forming an amide bond of a compound of chemical formula 2 and a mixture of H ₂ NR ¹ and H ₂ NR ² amines; comprising; Method for producing a compound represented by chemical formula 1: [Reaction Formula 2]

Here, L, R ¹ and R ² are the same as defined in claim 1, Y is C1-C10 alkyl. A pharmaceutical composition for the prevention or treatment of cancer or autoimmune disease, containing the compound of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient. A pharmaceutical composition for the prevention or treatment of a disease requiring inhibition of glutaminase, containing the compound of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient. A health functional food containing the compound of Article 1 or a pharmaceutically acceptable salt thereof as an active ingredient for preventing or improving cancer or autoimmune disease. Use of a compound according to paragraph 1 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the prevention or treatment of cancer or autoimmune disease. A method for treating cancer or an autoimmune disease by administering an effective amount of a compound according to Article 1 or a pharmaceutically acceptable salt thereof to a subject in need thereof.

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