Methods of use for trisubstituted benzotriazole derivatives as dihydroorotate oxygenase inhibitors

Abstract

To provide methods of use for trisubstituted benzotriazole derivatives as dihydroorotate oxygenase inhibitors.SOLUTION: The present invention provides methods for treating a cancer in a subject and methods for inhibiting tumor growth, metastasis, or a dihydroorotate oxygenase enzyme activity of a tumor or cancer cell. At least one trisubstituted benzotriazole derivative with the formula (I) is administered to the subject or is contacted with the cancer cell. Compounds of formula (I) and pharmaceutically acceptable salts thereof have substituents R1, R2, and R3 which have the meanings given in the specification.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Patent Application No. 15 / 899,707, filed February 20, 2018, and U.S. Patent Application No. 15 / 494,820, filed April 24, 2017, now U.S. Patent No. 9,937,155, both of which are incorporated by reference in their entireties.

[0002] The present invention relates to novel trisubstituted benzotriazole derivatives of formula (I) that are inhibitors of dihydroorotate dehydrogenase. In particular, the present invention refers to novel compounds that inhibit DHODH enzyme activity, methods for their preparation and pharmaceutical compositions containing them, and their use for the treatment and prevention of diseases or disorders, particularly diseases or disorders in which inhibition of DHODH is beneficial. [Background technology]

[0003] DHODH is a protein that catalyzes one of the steps in the de novo pyrimidine nucleotide biosynthetic pathway. (Greene et al., Biochem Pharmacol 1995, 50:861-7; Davis JP et al., FASEB J 1996, 10(6):Abst C23). DHODH catalyzes the only oxidation / reduction reaction in the pathway, which converts DHO (dihydroorotate) to orotic acid with the help of a flavin cofactor and an electron acceptor. Inhibitors of dihydroorotate dehydrogenase have been found to have broader applications as chemotherapeutic agents. (Kensler et al., 1989, Design of Enzyme Inhibitors as Drugs; Sandler, M., and Smith, HJ, eds., pp. 379-401, Oxford Univ. Press, Oxford England; Cody et al., Am. J. Clin. Oncol. 16, pp. 526-528 (1993)).

[0004] One example of a DHODH inhibitor is the quinoline derivative brequinar (6-fluoro-2-(2'-fluoro[1,1'-biphenyl]-4-yl)-3-methyl-4-quinolinecarboxylic acid), which exhibits anticancer activity against L1210 mouse leukemia (Andreson LW et al., Cancer Commun. 1989;1(6):381-7; Chen SF et al., Cancer Res. 1986, October;46(10):5014-9). Brequinar has also been shown to enhance the antitumor activity of 5-fluorouracil in a mouse colon 38 tumor model through tissue-specific modulation of uridine nucleotide pools (G Pizzorno et al., Cancer Res. 1992, April 1;52:1660-5).

[0005] DHODH inhibitors can also be useful in treating virus-mediated diseases (referring to US6,841,561).In addition, it is known that DHODH inhibition is a promising target for treating transplant rejection, rheumatoid arthritis, psoriasis and autoimmune diseases (Kovarik, JM et al., Expert Opin. Emerg. Drugs 2003, vol. 8, p. 47; Allison, AC Transplantation Proc. (1993) vol. 25 (no. 3) supplement 2, pp. 8-18; Makowka, L., Immunol Rev. (1993) vol. 136, p. 51-70; Davis JP et al., Biochemistry 1996, vol. 35: pp. 1270-3).

[0006] Leflunomide, a well-known DHODH inhibitor, is a currently commercially available low molecular weight drug of the isoxazole class (see EP0527736, JP1993506425, JP1999322700, JP1999343285, US5494911, US5532259, WO19991017748), a synthetic drug used in the treatment of rheumatoid arthritis and also currently under evaluation for use in the treatment of inflammatory bowel disease and chronic allograft rejection.

[0007] Leflunomide is rapidly converted in vivo to its active metabolite, teriflunomide, which exerts its anti-inflammatory, antiproliferative, and immunosuppressive effects through mechanisms that are not fully understood. Teriflunomide is a potent inhibitor of protein tyrosine kinases in vivo, as well as a 100- to 1,000-fold more potent inhibitor of DHODH (Davis JP et al., FASEB J 1996, 10(6): Abst C23; Davis JP et al., Biochemistry 1996, 35:1270-3). The number of patients affected by autoimmune disease and related diseases is increasing, but there is an unmet need for new drugs that can more effectively treat these diseases.Still, there is a critical need for more effective immunosuppressants in a wide variety of autoimmune and chronic inflammatory diseases, including systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, type I diabetes, inflammatory bowel disease, biliary cirrhosis, uveitis, and other disorders such as Crohn's disease, ulcerative colitis, bullous pemphigoid, sarcoidosis, psoriasis, autoimmune myositis, Wegener's granulomatosis, ichthyosis, Graves' ophthalmopathy, atopic dermatitis and asthma.In addition, these immunosuppressants can be used alone or in combination with antitumor compounds known to those skilled in the art as part of chemotherapy regimens for the treatment of cancer, lymphoma and leukemia. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Greene et al., Biochem Pharmacol 1995, 50:861-7 Summary of the Invention [Means for solving the problem]

[0009] The present invention is directed to a method for treating cancer in a subject in need of such treatment. In one embodiment, the cancer is acute myeloid leukemia, multiple myeloma, B-prolymphocytic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, anaplastic large cell lymphoma, mantle cell lymphoma, lung cancer, breast cancer, triple-negative breast cancer, melanoma, glioblastoma, prostate cancer, colon cancer, pancreatic cancer, bone cancer, head and neck cancer, skin cancer, cutaneous or intraocular malignant endometrium, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, sarcoma of soft tissue, urethral cancer, penile cancer, solid tumors of childhood, lymphocytic lymphoma. The cancer is selected from bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) neoplasm, primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancer and PTEN mutation cancer.In another embodiment, the cancer is selected from acute myeloid leukemia, multiple myeloma, B-prolymphocytic leukemia, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, anaplastic large cell lymphoma, mantle cell lymphoma, triple-negative breast cancer, melanoma, prostate cancer and esophageal cancer.The method comprises administering to the subject a therapeutically effective amount of a compound of formula (I) [ka] or a pharmaceutically acceptable salt thereof. In this structure, the dotted lines [....] in the rings can represent optional bonds, which can exist in any stable combination. R1 can be hydrogen and alkyl. R2 can be -A-R4. A can be arylene or tetrasubstituted arylene, and the substituents are halogen. R3 can be hydroxy and amino. R4 can be optionally substituted aryl and optionally substituted heteroaryl. The optional substituents can be one or more R5. R5 can be alkyl and -(CH2) n N(R a )R b It can be. R a and R b may independently be hydrogen, alkyl, and —C(O)alkyl, or alternatively R a and R b can be taken together with the nitrogen atom to which they are attached to form an optionally substituted 4-6 membered heterocyclyl containing 0-2 additional heteroatoms independently selected from O and N, the optional substituents being alkyl and "n" being an integer between 0 and 1.

[0010] The present invention is also directed to a method for inhibiting tumor cell growth and / or metastasis in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound of formula (I) [ka] or a pharmaceutically acceptable salt thereof. In this structure, the dotted lines [....] in the rings can represent optional bonds that can exist in any stable combination. R1 can be hydrogen and alkyl. R2 can be -A-R4. A can be arylene or tetrasubstituted arylene, and the substituents are halogen. R3 can be hydroxy and amino. R4 can be optionally substituted aryl and optionally substituted heteroaryl. The optional substituents can be one or more R5. R5 can be alkyl and -(CH2) n N(R a )R b It can be. R a and R b may independently be hydrogen, alkyl, and —C(O)alkyl, or alternatively R a and R b can be taken together with the nitrogen atom to which they are attached to form an optionally substituted 4-6 membered heterocyclyl containing 0-2 additional heteroatoms independently selected from O and N, the optional substituents being alkyl and "n" being an integer between 0 and 1.

[0011] The present invention is further directed to a method for inhibiting dihydrorotate oxygenase enzyme activity in tumor cells, the method comprising treating the tumor cells with a therapeutically effective amount of a compound of formula (I): [ka] or a pharmaceutically acceptable salt thereof. In this structure, the dotted lines [....] in the ring can represent optional bonds, which can be present in any stable combination. R1 can be hydrogen and alkyl. R2 can be -A-R4. A can be arylene or tetrasubstituted arylene, and the substituents are halogen. R3 can be hydroxy and amino. R4 can be optionally substituted aryl and optionally substituted heteroaryl. The optional substituents can be one or more R5. R5 can be alkyl and -(CH2) n N(R a )R b It can be. R a and R b may independently be hydrogen, alkyl, and —C(O)alkyl, or alternatively R a and R b can be taken together with the nitrogen atom to which they are attached to form an optionally substituted 4-6 membered heterocyclyl containing 0-2 additional heteroatoms independently selected from O and N, the optional substituents being alkyl and "n" being an integer between 0 and 1. [Brief explanation of the drawings]

[0012] [Figure 1] Figure 1 shows the sensitivity of a panel of approximately 400 human cancer lines of hemapoietic and non-hematopoietic origin to growth inhibition by Compound 1 of the present invention. Grey circles represent cell lines scored as highly sensitive (exhibiting ≥ 75% maximal growth inhibition and GI50 values ​​< 1.5 μM). [Figure 2] FIG. 2 shows the sensitivity of a further panel of heme-lineage human cancer lines to growth inhibition by compound 1 of the present invention. [Figure 3] FIG. 3 shows the ability of physiological (5 μM) and supraphysiological (25 μM, 100 μM) concentrations of exogenous uridine to rescue the cytotoxic effects of 10 μM of the compounds against the indicated cancer lines. [Figure 4A]FIG. 4A shows the relative growth rates versus concentration sensitivity profiles of MV411, Kasumi-1, THP-1, DB, Toledo, and WSU-DLCL2 cell lines to various concentrations of Compound 1. [Figure 4B] FIG. 4B shows the relative growth rates versus concentration sensitivity profiles of MV411, Kasumi-1, THP-1, DB, Toledo, and WSU-DLCL2 cell lines to various concentrations of cytarabine. [Figure 4C] FIG. 4C shows the relative growth rates versus concentration sensitivity profiles of MV411, Kasumi-1, THP-1, DB, Toledo, and WSU-DLCL2 cell lines to various concentrations of doxorubicin. [Figure 5A] FIG. 5A shows MOLM-13 tumor growth curves in CB17 SCID mice left untreated (vehicle) or treated BID with 100 mg / kg Compound 1 measured over the course of 14 days. [Figure 5B] FIG. 5B is a graph showing the pharmacokinetic profile of Compound 1 (dose=100 mg / kg, BID) in plasma and implanted MOLM-13 tumors of CB17 SCID mice at the indicated time points after the final dose at the end of the study. [Figure 5C] FIG. 5C shows DHO levels in untreated (vehicle) MOLM-13 tumors and tumors treated with Compound 1, measured over the course of 12 hours after the final dose at the end of the study. [Figure 5D] FIG. 5D shows uridine levels in untreated (vehicle) MOLM-13 tumors and tumors treated with Compound 1, measured over the course of 12 hours after the final dose at the end of the study. [Figure 6A] FIG. 6A shows patient-derived AML_1 tumor growth curves in CB17 SCID mice when left untreated (vehicle) or treated BID with 100 mg / kg Compound 1. [Figure 6B]FIG. 6B shows patient-derived AML_2 tumor growth curves in CB17 SCID mice when left untreated (vehicle) or treated BID with 100 mg / kg Compound 1. [Figure 6C] FIG. 6C shows patient-derived AML_3 tumor growth curves in CB17 SCID mice when left untreated (vehicle) or treated BID with 100 mg / kg Compound 1. [Figure 6D] FIG. 6D shows patient-derived AML_4 tumor growth curves in CB17 SCID mice when left untreated (vehicle) or treated BID with 100 mg / kg Compound 1. [Figure 6E] FIG. 6E shows patient-derived AML_5 tumor growth curves in CB17 SCID mice when left untreated (vehicle) or treated BID with 100 mg / kg Compound 1. [Figure 7A] FIG. 7A shows patient-derived DLBCL_1 (triple-hit model) tumor growth curves in CB17 SCID mice when left untreated (vehicle) or treated BID with 100 mg / kg Compound 1. [Figure 7B] FIG. 7B shows patient-derived DLBCL_2 tumor growth curves in CB17 SCID mice when left untreated (vehicle) or treated BID with 100 mg / kg Compound 1. [Figure 8] FIG. 8 is a curve showing the relative growth rates of OCILY18, SC-1, and CARNAVAL double-hit diffuse large B-cell lymphoma (DLBCL) cell lines treated with various concentrations of Compound 1 for 96 hours. [Figure 9A] Figure 9A shows OCILY-19 double-hit diffuse large B-cell lymphoma (DLBCL) tumor growth curves in CB17 SCID mice when left untreated (vehicle) or treated with 10 mg / kg Compound 1 BID, 30 mg / kg Compound 1 BID, 100 mg / kg Compound 1 BID, and 200 mg / kg Compound 1 QD, all measured over the course of 14 days. [Figure 9B] FIG. 9B shows the pharmacokinetic profile of Compound 1 administered at the doses indicated in FIG. 9A in the plasma of CB17 SCID mice at the indicated time points after the final dose at the end of the study. [Figure 9C] FIG. 9C shows DHO levels in untreated (vehicle) OCILY-19 tumors and tumors treated with the indicated doses of Compound 1, measured over the course of 12 hours after the final dose at the end of the study. [Figure 9D] FIG. 9D shows uridine levels in untreated (vehicle) OCILY-19 tumors and tumors treated with the indicated doses of Compound 1, measured over the course of 12 hours after the final dose at the end of the study. [Figure 10] FIG. 10 is a curve showing the relative growth rate of the DU4475 triple-negative breast cancer line treated with various concentrations of Compound 1 for 96 hours. DETAILED DESCRIPTION OF THE INVENTION

[0013] In one embodiment, the present invention provides trisubstituted benzotriazole derivatives as dihydroorotate oxygenase inhibitors.

[0014] These derivatives are useful as pharmaceuticals in the treatment of diseases such as autoimmune and inflammatory disorders, eg multiple sclerosis, rheumatoid arthritis and cancer.

[0015] In one particular embodiment, the present invention provides a compound of formula (I) [ka] [In the formula, The dotted lines [....] in the ring represent optional bonds which may exist in any stable combination, and R1 is selected from hydrogen and alkyl; R2 is -A-R4, A is arylene or tetrasubstituted arylene, the substituents being halogen; R3 is selected from hydroxy and amino; R4 is selected from optionally substituted aryl and optionally substituted heteroaryl, and the optional substituents are selected from one or more R5; R5 is alkyl and -(CH2) n N(R a )R b is selected from R a and R b are independently selected from hydrogen, alkyl, and —C(O)alkyl; Or R a and R b can be taken together with the nitrogen atom to which they are attached to form an optionally substituted 4-6 membered heterocyclyl containing 0-2 additional heteroatoms independently selected from O and N, the optional substituents being alkyl and "n" being an integer selected from 0 and 1. or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable positional isomer thereof.

[0016] The following embodiments are illustrative of the present invention and are not intended to limit the scope of the claims to the specific embodiments illustrated.

[0017] According to one embodiment, in particular there are provided compounds of formula (I) in which R1 is alkyl, in particular alkyl is methyl.

[0018] According to another embodiment, there are specifically provided compounds of formula (I) wherein R2 is -A-R4, where -A- is selected from arylene and tetrasubstituted arylene.

[0019] According to the above embodiment, specifically, R2 is [ka] Compounds of formula (I) are provided, wherein the compound is selected from:

[0020] According to one of the aforementioned embodiments, in particular, R4 is selected from optionally substituted phenyl, the optional substituents being methyl, acetylamino, isopropylaminomethyl, methylaminomethyl, dimethylaminomethyl, [ka] Compounds of formula (I) are provided, wherein the compound is selected from:

[0021] According to one of the aforementioned embodiments, there is specifically provided a compound of formula (I) in which R4 is selected from 2,5-dimethyl-1H-pyrrole.

[0022] According to yet another embodiment, there are specifically provided compounds of formula (I) wherein R3 is -OH and -NH2.

[0023] According to yet another particular embodiment, the compound of formula (I) has the formula (Ia) [ka] wherein the dotted line [---], R1, R3, and R4 are the same as those described in formula (I). is a compound of

[0024] According to yet another particular embodiment, the compound of formula (I) has the formula (Ib) [ka] wherein the dotted line [---], R1, R3, and R4 are the same as those described in formula (I). is a compound of

[0025] In another embodiment of the present invention, the present invention provides a process for preparing the trisubstituted benzotriazole derivatives of formula (I).

[0026] The procedures for the compounds of formula (I) are described in detail herein below step by step, including the general synthesis of the various intermediates involved in the preparation of compounds according to the invention.

[0027] More specifically, the present invention provides the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof or positional isomer thereof, including mixtures thereof in any ratio, as a pharmaceutical agent by inhibiting dihydroorotate oxygenase enzyme activity in the treatment of disorders such as multiple sclerosis, and other diseases such as inflammatory disorders, rheumatoid arthritis and cancer.

[0028] The trisubstituted benzotriazole derivatives of formula (I) of the present invention have the therapeutic role of inhibiting dihydroorotate dehydrogenase (DHODH or DHOD) enzyme.The compounds of formula (I) can be useful for treating and / or preventing autoimmune and chronic inflammatory diseases, including, but not limited to, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, type I diabetes, inflammatory bowel disease, biliary cirrhosis, uveitis, and other disorders such as Crohn's disease, ulcerative colitis, bullous pemphigoid, sarcoidosis, psoriasis, autoimmune myositis, Wegener's granulomatosis, ichthyosis, Graves' ophthalmopathy, atopic dermatitis, and asthma.The compounds of formula (I) and related formulas can also be used alone or in combination with typical antitumor compounds well known to those skilled in the art as part of chemotherapy regimens for the treatment of cancer, lymphoma, and leukemia.

[0029] To aid those skilled in the art in understanding the detailed description of the present invention, without limiting the scope of the present invention, the following definitions are provided.

[0030] "Alkyl" refers to a hydrocarbon chain, which may be straight or branched, containing the specified number of carbon atoms; for example, a C1-C6 alkyl group can have 1 to 6 (inclusive) carbon atoms in it. Examples of C1-C4 and C1-C6 alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, neopentyl, and isohexyl. An alkyl group can be unsubstituted or substituted with one or more suitable groups.

[0031] "Amino" refers to an -N- group, wherein the nitrogen atom of said group is bonded to a hydrogen, alkyl, cycloalkyl, aryl, heterocyclyl, or any suitable group. Representative examples of amino groups include, but are not limited to, -NH, -NHCH, and -NH-cyclopropyl. An amino group can be unsubstituted or substituted with one or more suitable groups.

[0032] "Aryl" refers to an optionally substituted monocyclic, bicyclic or polycyclic aromatic carbocyclic ring system of about 6 to 14 carbon atoms. 14 Aryl group Examples include, but are not limited to, phenyl, naphthyl, biphenyl, anthryl, tetrahydronaphthyl, fluorenyl, indanyl, biphenylenyl, and acenaphthyl. Aryl groups can be unsubstituted or substituted with one or more suitable groups.

[0033] "Arylene" refers to a divalent monocyclic or bicyclic saturated, unsaturated, or aromatic carbocyclic ring having 6 to 14 carbon atoms, which may be unsubstituted or substituted with one or more suitable groups.

[0034] "Halogen" or "halo" includes fluorine, chlorine, bromine or iodine.

[0035] "Hydroxy" refers to the group --OH.

[0036] The term "heterocyclyl" encompasses the definitions of "heterocycloalkyl" and "heteroaryl." The term "heterocycloalkyl" refers to a 3- to 10-membered non-aromatic, saturated or partially saturated, monocyclic or polycyclic ring system having at least one heteroatom or heterogroup selected from O, N, S, S(O), S(O), NH, and C(O). Exemplary heterocycloalkyl groups include piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, 1,3-dioxolanyl, 1,4-dioxanyl, and the like. Heterocycloalkyl groups can be unsubstituted or substituted with one or more suitable groups.

[0037] "Heteroaryl" refers to an unsaturated, monocyclic, bicyclic, or polycyclic aromatic ring system containing at least one heteroatom selected from oxygen, sulfur, and nitrogen. 10Examples of heteroaryl groups include furan, thiophene, indole, azaindole, oxazole, thiazole, thiadiazole, isoxazole, isothiazole, imidazole, N-methylimidazole, pyridine, pyrimidine, pyrazine, pyrrole, N-methylpyrrole, pyrazole, N-methylpyrazole, 1,3,4-oxadiazole, 1,2,4-triazole, 1-methyl-1,2,4-triazole, 1H-tetrazole, 1-methyltetrazole, benzoxazole, benzothiazole, benzofuran, benzisoxazole, benzimidazole, N-methylbenzimidazole, azabenzimidazole, indazole, quinazoline, quinoline, and isoquinoline. Bicyclic heteroaryl groups include groups in which a phenyl, pyridine, pyrimidine, or pyridazine ring is fused to a 5- or 6-membered monocyclic heterocyclyl ring having one or two nitrogen atoms in the ring, one nitrogen atom together with either one oxygen or one sulfur atom in the ring, or one O or S ring atom. Heteroaryl groups can be unsubstituted or substituted with one or more suitable groups.

[0038] "Heteroatom" refers to a sulfur, nitrogen, or oxygen atom.

[0039] "Optionally substituted or substituted," as used herein, means that at least one hydrogen atom of an optionally substituted group has been replaced with a suitable substituent, such as, but not limited to, halogen, nitro, cyano, hydroxy, oxo (=O), thio (=S), -N(C1-C3 alkyl)C(O)(C1-C6 alkyl), -NHC(O)(C1-C6 alkyl), -NHC(O)(cycloalkyl), -NHC(O)(aryl), -NHC(O)(heterocyclyl), -NHC(O)(heteroaryl), -NHC(O)H, -C(O)NH, -C(O)NH(C1-C6 alkyl), -C(O)NH(cycloalkyl), -C(O)NH(heterocyclyl), -C(O)NH(heteroaryl), -C(O)N(C1-C6 alkyl)(C1-C6 alkyl), -S(O)NH(C1-C 6 alkyl), -S(O)2NH(C1-C6 alkyl), -S(O)NH(cycloalkyl), -S(O)2NH(cycloalkyl), carboxy, -C(O)O(C1-C6 alkyl), -C(O)(C1-C6 alkyl), =N-OH, substituted or unsubstituted alkyl, substituted or unsubstituted haloalkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted haloalkoxy, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenylalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted amino, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted heterocyclic ring.

[0040] Specific compounds of the present invention and specific compounds arising from formula (I) that do not deviate from the scope of the definition given under compounds of formula (I) are summarized in the tables herein below, encompassing the entire range of compounds included in compounds of formula (I). [ka] [ka] [ka] or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable positional isomer thereof.

[0041] In yet another embodiment, the present invention relates to a compound of formula (I) for use in the treatment of inflammatory disorders and autoimmune diseases or overactive immune responses. More preferably, the present invention relates to the use of a compound of formula (I) for the treatment of multiple sclerosis, rheumatoid arthritis and transplant rejection.

[0042] A further embodiment of the present invention includes the use of a compound of formula (I) or a pharmaceutically acceptable derivative, salt and positional isomer thereof, including mixtures thereof in any ratio, as a pharmaceutical.

[0043] Use of the aforementioned compounds and pharmaceutically usable derivatives, salts and positional isomers thereof, including mixtures thereof in any ratio, for preparing a medicament for the treatment and / or prevention of dihydroorotate dehydrogenase-associated disorders.

[0044] The use of the aforementioned compounds, wherein the dihydroorotate dehydrogenase-associated disorder is an autoimmune disorder or a condition associated with an overactive immune response.

[0045] Use of the aforementioned compounds and pharmaceutically usable derivatives, salts and positional isomers thereof, including mixtures thereof in any ratio, for preparing a medicament for the treatment and / or prevention of immune dysregulation.

[0046] The use of the aforementioned compounds, wherein the immunoregulatory dysregulation is multiple sclerosis or rheumatoid arthritis.

[0047] Use of the aforementioned compounds for preparing a medicament for the treatment and prevention of cancer diseases, inflammatory bowel diseases or rheumatoid arthritis.

[0048] In a further embodiment, the present invention relates to a pharmaceutical formulation comprising at least one compound according to formula (I) and / or pharmaceutically usable derivatives, salts and positional isomers thereof, including mixtures thereof in any ratio, and at least one further active ingredient.

[0049] The present invention further provides pharmaceutical compositions comprising at least one compound according to formula (I) and / or pharmaceutically usable derivatives, salts and positional isomers thereof, including mixtures thereof in any ratio, finally one further active ingredient, and an excipient.

[0050] The term "pharmaceutically acceptable salt" or "pharmaceutically acceptable derivative" is understood to mean an active ingredient, including a compound of formula (I), in the form of one of its salts, especially if the salt form confers improved pharmacokinetic properties to the active ingredient compared to the free form of the active ingredient or any other salt form of the active ingredient previously used. Also, a pharmaceutically acceptable salt form of an active ingredient can provide the active ingredient for the first time with desirable pharmacokinetic properties that it did not previously possess, and can even have a positive effect on the pharmacodynamics of the active ingredient in the body with regard to its therapeutic efficacy.

[0051] The term "regioisomer" or "regioisomers" refers to isomers of the same structure as Or, it refers to positional isomers, which is a category of structural isomers in which the position of a substituent is changed from that on the parent structure. The term positional isomer in this specification inherently includes any positional isomer, either as a pure positional isomer or a mixture of two or more of its positional isomers, without departing from the scope of the compound of formula (I). Since the pharmaceutical activity of the positional isomers of the compounds of the present invention may be different, it may be desirable to use positional isomers. In these cases, positional isomers can be separated at any possible stage, either as intermediates or final products, by methods well known to those skilled in the art, or can be used directly in synthesis. Positional isomers of compounds of formula (I) refer to the following structures: [ka]

[0052] Pharmaceutical formulations can be adapted for administration via any desired and appropriate method, for example, oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual, or transdermal), vaginal, or parenteral (including subcutaneous, intramuscular, intravenous, or intradermal) methods. Such formulations can be prepared using any method known in the pharmaceutical art, for example, by combining the active ingredient with the excipient(s) or adjuvant(s).

[0053] Pharmaceutical formulations adapted for oral administration can be administered as discrete units, such as, for example, capsules or tablets, powders or granules, solutions or suspensions in aqueous or non-aqueous liquids, edible foams or frothy food products, or oil-in-water or water-in-oil liquid emulsions.

[0054] For example, when administered orally as a tablet or capsule, the active ingredient component can be combined with an orally non-toxic, pharmaceutically acceptable inert excipient, such as, for example, ethanol, glycerol, water, etc. Powders are prepared by grinding the compound to a suitable fine size and mixing it with a similarly ground pharmaceutical excipient, such as an edible carbohydrate, for example, starch or mannitol, etc. Flavorings, preservatives, dispersing agents, and dyes can also be present.

[0055] Capsules are produced by preparing a powder mixture as described above and filling formed gelatin shells with it. Glidants and lubricants, such as solid forms of highly dispersed silicic acid, talc, magnesium stearate, calcium stearate, or polyethylene glycol, can be added to the powder mixture before the filling operation. Disintegrants or solubilizers, such as agar-agar, calcium carbonate, or sodium carbonate, can also be added to improve the availability of the medicament after the capsule is ingested.

[0056] Furthermore, if desired or necessary, suitable binders, lubricants, disintegrants, and dyes can also be incorporated into the mixture.Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, sweeteners made from corn, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, wax, etc.Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, etc.Disintegrants include, but are not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, etc.Tablets can be formulated, for example, by preparing a powder mixture, granulating or dry-pressing the mixture, adding a lubricant and a disintegrant, and compressing the entire mixture to produce tablets. Powder mixtures are prepared by mixing the com- pounds comminuted in a suitable manner as described above with a diluent or base, optionally containing binders such as carboxymethylcellulose, alginate, gelatin or polyvinylpyrrolidone, solution retarders such as paraffin, absorption accelerators such as quaternary salts, and / or absorbents such as bentonite, kaolin or the like. or dicalcium phosphate. The powder mixture can be granulated by wetting it with a binder, such as syrup, starch paste, acadia mucilage or cellulose solution, or polymeric materials, and then pressing it through a screen. As an alternative to granulation, the powder mixture can be passed through a tablet press, resulting in irregularly shaped lumps that are broken down until granules are formed. The granules can be lubricated by adding stearic acid, a stearate salt, talc, or mineral oil to prevent sticking to the tablet die. The lubricated mixture is then compressed to produce tablets. The active ingredient can also be combined with a free-flowing inert excipient and then directly compressed to produce tablets without the need for granulation or dry-pressing. A clear or opaque protective layer consisting of a shellac sealing layer, a layer of sugar or polymeric material, and a gloss layer of wax can be present. Dyes can be added to these coatings to distinguish between different dosage units.

[0057] Oral liquid, such as solution, syrup and elixir, can be prepared in the form of dosage unit, so that a given amount contains a predetermined amount of compound.Syrup can be prepared by dissolving compound in aqueous solution with suitable flavoring, while elixir is prepared using non-toxic alcoholic vehicle.Suspension can be prepared by dispersing compound in non-toxic vehicle.Solubilizer and emulsifier, such as ethoxylated isostearyl alcohol and polyoxyethylene sorbitol ether, preservative, flavor additive, such as peppermint oil or natural sweetener or saccharin, or other artificial sweetener, etc. can also be added.

[0058] Dosage unit formulations for oral administration may, if desired, be encapsulated in microcapsules. The formulations may also be prepared in such a way as to provide extended or delayed release, for example, by coating or embedding particulate material in polymers, waxes, etc.

[0059] The new trisubstituted benzotriazole derivatives of formula (I) and their pharmaceutically acceptable salts, physiologically functional derivatives, and other active ingredients can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. Liposomes can be formed from suitable lipids or phospholipids, or both, such as, for example, cholesterol, stearylamine, or phosphatidylcholine.

[0060] Pharmaceutical formulations adapted for transdermal administration can be administered as independent plasters for prolonged contact with the recipient's epidermis. Thus, for example, the active ingredient can be delivered from the plaster by iontophoresis, as generally described in Pharmaceutical Research, Vol. 3 (No. 6), p. 318 (1986).

[0061] Pharmaceutical compounds adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils.

[0062] For treating eyes or other external tissues, such as mouth and skin, the preparation is preferably applied as a topical ointment or cream.When preparing to obtain an ointment, active ingredient can be used with either a paraffinic or water-miscible cream base.Alternatively, active ingredient can be formulated with an oil-in-water cream base or a water-in-oil base to obtain a cream.

[0063] Pharmaceutical formulations adapted for topical application to the eye include eye drops, in which the active ingredient is dissolved or suspended in a suitable carrier, in particular an aqueous solvent.

[0064] Pharmaceutical formulations adapted for topical application in the mouth encompass lozenges, pastilles and mouthwashes.

[0065] Pharmaceutical formulations adapted for rectal administration can be administered in the form of suppositories or enemas.

[0066] Pharmaceutical formulations adapted for nasal administration wherein the carrier substance is a solid include coarse powder having a particle size in the range 20 to 500 microns, which is administered in the manner of sniffing, i.e., by rapid inhalation through the nostrils from a container containing the powder held close to the nose. Formulations suitable for administration as a nasal spray or nasal drops, which use a liquid as the carrier substance, include aqueous or oil solutions of the active ingredient.

[0067] Pharmaceutical formulations adapted for administration by inhalation encompass fine particle dusts or mists, which can be generated by means of various types of pressurized dispensers, aerosols, nebulizers or inhalers.

[0068] Pharmaceutical preparations adapted for vaginal administration can be administered as pessaries, tampons, creams, gels, pastes, foams or spray preparations. Pharmaceutical preparations adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions containing antioxidants, buffers, bacteriostats and solutes to make the preparation isotonic with the blood of the treated recipient, as well as aqueous and non-aqueous sterile suspensions that may contain suspending media and thickeners. Preparations can be administered in single-dose or multi-dose containers, such as sealed ampoules and vials, and can be stored in a freeze-dried (lyophilized) state so that only a sterile carrier liquid for injection, such as water, is added immediately before use.

[0069] Injection solutions and suspensions prepared according to the formula may be prepared from sterile powders, granules, and tablets.

[0070] It will be appreciated that the formulations may include, in addition to the components particularly listed above, other agents conventional in the art having regard to the particular type of formulation; thus, for example, formulations suitable for oral administration may include flavorings.

[0071] The therapeutically effective amount of a compound of Formula (I) and other active ingredients will depend on several factors, including, for example, the age and weight of the animal, the precise disease state and its severity requiring treatment, the nature of the formulation, and the method of administration, and is ultimately determined by the treating physician or veterinarian. However, an effective amount of a compound generally ranges from 0.1 to 100 mg per kg of recipient (mammal) body weight per day, with a more typical range being 1 to 10 mg per kg of body weight per day. Thus, the actual daily amount for an adult mammal weighing 70 kg will usually be between 70 and 700 mg, which can be administered as individual doses per day, or in a series of subdoses (e.g., two, three, four, five, or six) per day, usually resulting in the same total daily dose. An effective amount of a salt or solvate or physiologically functional derivative thereof can be determined as a fraction of the effective amount of the compound itself.

[0072] In yet another embodiment, the present invention relates to a method for treating cancer in a subject in need thereof, comprising administering to the subject one or more times a therapeutically effective amount of at least one compound disclosed herein, or a pharmaceutically acceptable salt thereof.

[0073] In yet another embodiment, the present invention relates to a method for inhibiting tumor cell growth and / or metastasis in a subject, comprising administering to the subject one or more times a therapeutically effective amount of at least one compound disclosed herein, or a pharmaceutically acceptable salt thereof.

[0074] In yet another embodiment, the present invention relates to a method for inhibiting dihydroorotate oxygenase enzyme activity in tumor cells, comprising contacting the tumor cells one or more times with a therapeutically effective amount of at least one compound disclosed herein or a pharmaceutically acceptable salt thereof. In this embodiment, the tumor cells are contacted in vivo, ex vivo, or in vitro.

[0075] The compounds, pharmaceutically acceptable salts thereof, and pharmaceutical formulations and compositions disclosed herein are useful for treating cancer in subjects in need thereof. Accordingly, tumor cell growth and / or metastasis or dihydroorotate oxygenase enzyme activity in the cells thereof can be inhibited. The compounds and pharmaceutical compositions can be administered once or multiple times to achieve a therapeutic effect. As is known in the art, those skilled in the art are fully capable of determining the dosage, administration regimen, and administration route depending on the condition being treated and the subject in need of treatment. Representative examples of cancer include hematological malignancies, such as, but not limited to, acute myeloid leukemia, multiple myeloma, B-prolymphocytic leukemia, acute lymphoblastic leukemia, and chronic lymphocytic leukemia. Representative examples of cancer include lymphomas, such as, but not limited to, Hodgkin's disease, non-Hodgkin's lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, anaplastic large cell lymphoma, and mantle cell lymphoma. Representative examples of cancers include solid cancers, including, but not limited to, lung cancer, breast cancer, triple-negative breast cancer, melanoma, glioblastoma, prostate cancer, colon cancer, pancreatic cancer, bone cancer, head and neck cancer, skin cancer, cutaneous or intraocular malignant endometrium, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, solid tumors of childhood, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, including those induced by asbestos, and PTEN-mutated cancers.

[0076] In a further aspect, the present invention relates to a process for preparing the trisubstituted benzotriazole derivatives of formula (I).

[0077] Dihydroorotate dehydrogenase inhibitors according to formula (I) can be prepared from readily available starting materials using the following general methods and procedures. Where typical or preferred experimental conditions (i.e., reaction temperature, time, moles of reagents, solvents, etc.) are given, it will be understood that other experimental conditions can also be used unless otherwise specified. Optimal reaction conditions may vary with the specific reactants or solvents used, but such conditions can be determined by those skilled in the art using routine optimization procedures. Furthermore, those skilled in the art can prepare additional compounds of the present invention claimed herein by utilizing the procedures described in detail. All temperatures are in degrees Celsius (°C) unless otherwise noted.

[0078] In a further aspect, the compounds of the present invention can also contain unnatural proportions of atomic isotopes in one or more of the atoms that constitute such compounds.For example, the present invention also encompasses the isotopically labeled variants of the present invention that are identical to those listed herein, except that one or more atoms of the compound are replaced by atoms that have atomic masses or mass numbers that are different from the main atomic masses or mass numbers that are normally found in nature for the atoms.All isotopes of any specific atom or element as specified are intended to be included in the scope of the compounds of the present invention and their use.Exemplary isotopes that can be incorporated into the compounds of the present invention include hydrogen, carbon, nitrogen, oxygen, phosphorous, sulfur, fluorine, and the like. Isotopes of boron, chlorine and iodine, e.g. 2 H("D"), 3 H, 11 C. 13 C. 14 C. 13 N, 15 N, 15 O. 17 O. 18 O. 32 P, 33 P, 35 S, 18 F, 36 Cl, 123 I and 125Isotopically labeled compounds of the invention can generally be prepared by following procedures similar to those disclosed in the following schemes and / or examples herein, substituting an isotopically labeled reagent for a non-isotopically labeled reagent.

[0079] The following abbreviations refer to the following definitions: AcOH (acetic acid), ACN (acetonitrile), ATP (adenosine triphosphate), BSA (bovine serum albumin), CHCl3 (chloroform), Cs2CO3 (cesium carbonate), DCM (dichloromethane), DIPEA (diisopropylethylamine), DMSO (dimethyl sulfoxide), DMF (N,N-dimethylformamide), EDCI.HCl (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), Et3N (triethylamine), EtOAc (ethyl acetate), EtOH (ethanol), HOBT (hydroxybenzotriazole), HCl (hydrogen chloride), K2CO3 (potassium carbonate), KOAc (potassium acetate), min (minutes), MeOH (methanol), MeI (methyl iodide), MgSO4 (magnesium sulfate), NH4Cl (ammonium chloride), NH4(CO3)2 (ammonium carbonate) palladium(II), Pd(dppf)2Cl2 ([1,1-bis(diphenylphosphino)-ferrocene]dichloropalladium(II)), NaH (sodium hydride), NaNO2 (sodium nitrite), NaHCO3 (sodium bicarbonate), PetEther (petroleum ether), PBS (phosphate buffered saline), RT - room temperature (25°C to 35°C), TEA (triethylamine), TFA (trifluoroacetic acid), THF (tetrahydrofuran), t-BuOK (potassium tert-butoxide), TMSI (trimethylsilyl iodide), TLC (thin layer chromatography), H2O - water, mL - milliliter, hr / h - hour, N - linear (normality), M - molarity, s - singlet, d - doublet, t - triplet, m - multiplet , 1 HNMR - proton nuclear magnetic resonance, MS - mass spectrometry, LC - liquid chromatography, HPLC - high performance liquid chromatography, J-coupling constant, 1H - proton, MHz - megahertz (frequency), Hz - hertz, ppm - parts per million, bs - broad singlet, ES - electrospray, Conc. - concentrated, g - gram, mmol or mM - millimolar, μM - micromolar, nM - nanomolar, UV - ultraviolet, °C - degrees Celsius, M + - molecular ion, % - percentage, μ - micron, and δ - delta, anh. - anhydrous, pH - hydrogen potential.

[0080] Another embodiment of the present invention provides a method useful for synthesizing compounds of formula (I), which is described in the Examples below and generalized in Scheme I. Those skilled in the art will recognize that Scheme I can be adapted to produce compounds of formula (I) and pharmaceutically acceptable salts of compounds of formula (I) according to the present invention, where all symbols / variables are as previously defined unless otherwise specified. This method is represented by Scheme I. [ka]

[0081] The compounds of the present invention can be prepared using the synthetic transformations illustrated in Scheme I. The starting materials are commercially available, can be prepared by the procedures described herein, by literature procedures, or by procedures well known to those skilled in the art of organic chemistry. The starting material, 5-substituted methyl 2,3-diaminobenzoate, is prepared by the procedures described in WO2010115736A2.

[0082] Step-a: Compound-i is reacted with sodium nitrite in acidic medium using general procedure-A to give compound-ii.

[0083] Step-b: Compound-ii is further N-alkylated using methyl iodide under basic conditions such as those described in general procedure-B to give compounds of formula-iii.

[0084] Step-c: The compound of formula-iii is reacted with bispinacolatodiborane in the presence of a suitable palladium catalyst in basic medium using general procedure-C to give the compound of formula-iv.

[0085] Step-d: Compounds of formula-iv are treated with a substituted aryl halide in the presence of a suitable palladium catalyst using conditions such as those described in general procedure-D to give compounds of formula-v.

[0086] Step-e: Alternatively, compounds of formula-v can be prepared from compounds of formula-iii by using an appropriate boronic acid under appropriate conditions such as those described in general procedure-D.

[0087] Step-f: The obtained compound of formula-v undergoes ester hydrolysis under basic conditions such as those described in general procedure-F to give the compound of formula (I) (wherein R3 = OH).

[0088] Step-g: Carboxylic acids of formula (I) were treated with ammonium chloride using the conditions described in general procedure-G to give the respective compounds of formula (I) (wherein R3 = NH2).

[0089] Where the foregoing set of general synthetic methods is not applicable to obtain compounds according to formula (I) and / or intermediates required for the synthesis of compounds of formula (I), appropriate preparative methods known to those skilled in the art should be used. Generally, the synthetic route for any individual compound of formula (I) will vary depending on the specific substituents on each molecule and the availability of the necessary intermediates, and such factors will again be appreciated by those skilled in the art.

[0090] The compounds of the present invention can be isolated together with solvent molecules by crystallization by evaporation of an appropriate solvent. Pharmaceutically acceptable acid addition salts of compounds of formula (I) containing a basic center can be prepared in a conventional manner. For example, a solution of the free base can be treated with an appropriate acid, either neat or in an appropriate solution, and the resulting salt is isolated either by filtration or by evaporation of the reaction solvent under vacuum. Pharmaceutically acceptable base addition salts can be obtained in a similar manner by treating a solution of a compound of formula (I) with an appropriate base. Both types of salts can be formed or interconverted using ion exchange resin techniques.

[0091] The present invention is illustrated by some of the following examples, but should not be construed as being limited thereby, but rather encompasses the general scope disclosed hereinabove. Various modifications and embodiments can be made without departing from the spirit and scope of the invention. [Example]

[0092] overview The MS data provided in the examples below was obtained as follows: Mass Spectrum: LC / MS Waters ZMD (ESI) or Waters Acquity SQD (ESI).

[0093] The NMR data provided in the examples below was obtained as follows. 1 H-NMR: Bruker DPX-300MHz or Bruker DPX 400MHz.

[0094] The HPLC data provided in the examples below was obtained as follows.

[0095] Condition A: Column Waters Xbridge™ C8, 50 mm x 4.6 mm, flow rate 2 mL / min, 8 min gradient from 0.1% TFA in H2O to 0.07% TFA in CH3CN.

[0096] Condition B: C18 BDS (4.6 × 250) mm, SC\244, flow rate 0.7 mL / min, 10 min gradient from 0.1% TFA in H2O to CH3CN.

[0097] Preparative HPLC conditions: Column - Zorbax Eclipse XDB C18 PrepHT (150 x 21.2 mm, 5μ); Mobile phase: (A) 0.01% TFA or 0.1% TFA, (B) ACN or ACN:MeOH (1:1), Flow rate: 20 ml / min.

[0098] Preparative HPLC purifications were performed using a Waters mass-directed autopurification Fractionlynx equipped with a Sunfire Prep C18 OBD column 19 × 100 mm, 5 μm unless otherwise reported. All HPLC purifications were performed using a gradient of ACN / HO or ACN / HO / HCOOH (0.1%).

[0099] The compounds of the present invention are named according to the standards used in the program ACD / Name Batch of "Advanced Chemistry Development Inc., ACD / Labs (7.00 Release)" product version: 7.10 (created September 15, 2003).

[0100] The procedures for the compounds of formula (I) are described in detail herein under general procedures, including the general synthesis of the various intermediates involved in the preparation of compounds according to the invention. General Procedure-A: Preparation of Substituted [1,2,3]Benzotriazoles

[0101] A flask containing a hexa-substituted or substituted diaminoester (1-3 equivalents) in acetic acid is stirred for 10-20 minutes, preferably 10 minutes, followed by the addition of (sodium nitrite, potassium nitrite, preferably sodium nitrite) (2.5-3.5, preferably 2.5 equivalents) in water. The reaction mixture is stirred for 1-2 hours, preferably 1 hour, at RT. The separated solid is collected by filtration and dried under vacuum to give the desired product. Illustrative Example of General Procedure-A Preparation No. A.1: Synthesis of methyl 6-bromo-1H-benzo[d][1,2,3]triazole-4-carboxylate [ka]

[0102] A solution of methyl 2,3-diamino-5-bromobenzoate (1.0 g, 4.08 mmol) (see WO2010 / 115736A2) in acetic acid (15 mL) was stirred at RT for 10 minutes. Sodium nitrite (0.309 g, 4.48 mmol) in water (2 mL) was added, and the reaction mixture was stirred at RT for about 30 minutes. The precipitated solid was filtered, washed with water, and dried under vacuum to give the desired product (0.8 g, 77%). 1 H NMR (400 MHz, DMSO-d): δ 16.19 (s, 1H), 8.70 (s, 1H), 8.14 (s, 1H), 3.99 (s, 3H) and LC-MS m / z: 258 (M+H) + . General Procedure-B: N-Alkylation of Substituted Benzotrizoles

[0103] To a stirred solution of substituted benzotriazole-carboxylate derivative (1 equivalent) in an organic solvent (e.g., DMF, THF, dioxane, preferably DMF) is added an appropriate base (e.g., K2CO3, CS2CO3, NaH, etc., preferably 2-5 equivalents of K2CO3, preferably 2 equivalents), followed by the addition of alkyl halide (2-5 equivalents, preferably 3 equivalents). The reaction mixture is stirred at RT for about 1-10 hours (preferably 3 hours). The reaction mixture is poured into ice-cold water, and the separated solid is collected by filtration and dried under vacuum. Regioisomers are separated by column chromatography to give the desired products. Illustrative Example of General Procedure-B

[0104] Preparation No. B.1: Synthesis of methyl 5-bromo-1-methyl-1H-benzo[d][1,2,3]triazole-7-carboxylate, methyl 6-bromo-2-methyl-2H-benzo[d][1,2,3]triazole-4-carboxylate and methyl 6-bromo-1-methyl-1H-benzo[d][1,2,3]triazole-4-carboxylate [ka]

[0105] To a stirred solution of methyl 6-bromo-1H-benzo[d][1,2,3]triazole-4-carboxylate (4.5 g, 17.5 mmol, Preparation No. A.1) in DMF (25 mL) was added potassium carbonate (4.85 g, 35.15 mmol), followed by methyl iodide (7.48 g, 52.73 mmol). The reaction mixture was stirred at RT for 1 h. The reaction mixture was quenched with ice-cold water (100 mL), and the separated solid was collected by filtration and dried under vacuum. The resulting crude compound was purified by silica gel (100-200 mesh) column chromatography using 10% ethyl acetate in hexane to give Isomer-I (B.1.a) (1.9 g), 1 H NMR (400 MHz, CDCl3) δ 8.40 (s, 1H), 8.22 (s, 1H), 4.57 (s, 3H), 4.01 (s, 3H) and LC-MS m / z: 272 (M+2) + which was purified using 15-20% ethyl acetate in hexane to give Isomer-II (B.1.b) (1.4 g), 1 H NMR (400 MHz, CDCl3) δ 8.26 (s, 1H), 8.23 ​​(s, 1H), 4.58 (s, 3H), 4.04 (s, 3H) and LC-MS m / z: 272.0 (M+2) + which was purified using 20-25% ethyl acetate in hexane to give Isomer-III (B.1.c) (1.0 g), 1H NMR (400 MHz, DMSO-d): δ 8.67 (s, 1H), 8.13 (s, 1H), 4.45 (s, 3H), 3.96 (s, 3H) and LC-M S m / z: 272.0 (M+2) + obtained. General Procedure-C: Preparation of Boronic Esters

[0106] A mixture of an aryl halo derivative (1.0-3.0 equivalents, preferably 1.0 equivalent), an appropriate inorganic base (e.g., KOAC, Na2CO3, K2CO3, or Cs2CO3, preferably KOAC), and bis-pinacolatodiborane (1.0-3.0 equivalents, preferably 1.1 equivalents) in dioxane is degassed with nitrogen for about 10-15 minutes, and [1,1-bis(diphenylphosphino)-ferrocene]dichloropalladium(II) (0.001-0.010 equivalents, preferably 0.05 equivalents) is added. The reaction mixture is stirred at reflux under nitrogen for about 3-12 hours (preferably about 6 hours). The reaction mixture is cooled to RT and evaporated to dryness under reduced pressure. The resulting residue is redissolved in EtOAc and washed successively with water and brine solution. The organic solution is dried over Na2SO4, filtered, and concentrated under reduced pressure. The product is purified by crystallization or trituration from one or more appropriate solvents, or by preparative HPLC or flash chromatography. Illustrative Example of General Procedure-C Preparation No. C.1: Synthesis of methyl 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[d][1,2,3]triazole-7-carboxylate [ka]

[0107] A mixture of methyl 5-bromo-1-methyl-1H-benzo[d][1,2,3]triazole-7-carboxylate (1.0 g, 3.7 mmol, Preparation No. B.1.a), potassium acetate (0.627 g, 5.92 mmol), and bispinacolatodiborane (0.93 g, 3.7 mmol) in dioxane (60 mL) was degassed with nitrogen for approximately 15 minutes, and [1,1-bis(diphenylphosphino)-ferrocene]dichloropalladium(II) (0.151 g, 0.018 mmol) was added. The reaction mixture was stirred at reflux under nitrogen for 6 hours. The reaction mixture was cooled to RT and evaporated to dryness under reduced pressure. The resulting residue was redissolved in EtOAc, washed successively with water and brine solution, and concentrated. The crude compound obtained was purified by silica gel (60-120 mesh) column chromatography using 30% ethyl acetate in hexane to give the desired product (0.9 g, 77%). 1 H NMR (400 MHz, DMSO-d6): δ 8.46 (s, 1H), 8.31 (s, 1H), 4.59 (s, 3H), 3.94 (s, 3H), 1.35 (s, 12H) and LC-MS m / z=318. 2(M+H) + .

[0108] Other compounds synthesized using general procedure C are listed in Table C.1. General Procedure-D: Suzuki Reaction

[0109] A mixture of acetonitrile and water (8:2) is degassed with nitrogen for about 10-15 minutes, followed by the addition of an appropriate base (e.g., Na2CO3, K2CO3, or Cs2CO3, preferably Na2CO3), followed by the addition of an aryl bromo derivative (1.0-3.0 equivalents, preferably 1.0 equivalent) and an appropriate boronic acid (1.0-3.0 equivalents, preferably 1.5 equivalents). The reaction mixture is again degassed for 15 minutes, and finally [1,1-bis(diphenylphosphino)-ferrocene]dichloropalladium(II) (0.001-0.010 equivalents, preferably 0.05 equivalents) is added. The reaction mixture is stirred at reflux under nitrogen for about 3-12 hours (preferably about 4 hours). The reaction mixture is cooled to RT and evaporated to dryness under reduced pressure. The resulting residue is redissolved in EtOAc and washed successively with water and brine solution. The organic solution is dried over Na2SO4, filtered, and concentrated under reduced pressure. The product is purified by crystallization or trituration from one or more appropriate solvents, or by preparative HPLC or flash chromatography. Illustrative Example of General Procedure-D Preparation No. D.1: Synthesis of methyl 1-methyl-5-(2'-methyl-[1,1'-biphenyl]-4-yl)-1H-benzo[d][1,2,3]triazole-7-carboxylate [ka]

[0110] A mixture of acetonitrile (80 mL) and water (15 mL) was degassed with nitrogen for 10 minutes. Sodium carbonate (2.74 g, 25.9 mmol) was added, followed by methyl 5-bromo-1-methyl-1H-benzo[d][1,2,3]triazole-7-carboxylate (3.5 g, 12.9 mmol) and 4,4,5,5-tetramethyl-2-(2'-methyl-[1,1'-biphenyl]-4-yl)-1,3,2-dioxaborolane (3.81 g, 12.0 mmol) (C.1.5). The reaction mixture was degassed again for 15 minutes. Finally, [1,1-bis(diphenylphosphino)-ferrocene]dichloropalladium(II) (0.526 g, 0.64 mmol) was added. The reaction mixture was stirred at reflux for 5 hours under nitrogen. The reaction mixture was cooled to room temperature and evaporated to dryness under reduced pressure. The resulting residue was redissolved in EtOAc, washed successively with water and brine solution, and concentrated. The resulting crude compound was purified by silica gel (60-120 mesh) column chromatography using 30% ethyl acetate in hexane to give the desired product (3.6 g, 77%). 1 H NMR (400 MHz, CDCl3): δ 8.52 (s, 1H), 8.31 (s, 1H), 7.76-7.74 (d, J=8.0 Hz, 2H), 7.48-7.46 (d, J=7.6, 2H), 7.31-7.28 (m, 4H), 4.63 (s, 3H), 4.08 (s, 3H), 2.34 (s, 3H) and LC-MS m / z=358.2(M+H) + .

[0111] Other compounds synthesized using general procedure D are listed in Table D.1. General Procedure E: Reductive Amination

[0112] A mixture of an appropriate aldehyde and amine in an organic solvent (e.g., DCM, THF, ACN, DMF, DCE, or dioxane) is stirred at room temperature for 30 minutes to 4 hours. The resulting reaction mixture is cooled to 0°C, and a reducing agent such as sodium triacetoxyborohydride is added portionwise, followed by a catalytic amount of acetic acid. The resulting reaction mixture is stirred at room temperature for 2 to 4 hours. The progress of the reaction is monitored by TLC, and the reaction mixture is quenched with aqueous sodium bicarbonate. It is further extracted with ethyl acetate, and the combined organic layers are dried over sodium sulfate and concentrated in vacuo to give the desired compound. If necessary, the desired compound can be purified by crystallization or trituration from one or more appropriate solvents, or by preparative HPLC or flash chromatography. Illustrative Example of General Procedure-E Preparation No. E.1: Synthesis of methyl 1-methyl-5-(2'-(morpholinomethyl)-[1,1'-biphenyl]-4-yl)-1H-benzo[d][1,2,3]triazole-7-carboxylate [ka]

[0113] A solution of methyl 5-(2'-formyl-[1,1'-biphenyl]-4-yl)-1-methyl-1H-benzo[d][1,2,3]triazole-7-carboxylate (0.300 g, 0.8 mmol, D.1.8) and morpholine (0.070 g, 0.8 mmol) in DCE (15 mL) was stirred at room temperature for 30 minutes. The reaction mixture was cooled to 0 °C, and sodium triacetoxyborohydride (0.342 g, 1.6 mmol) was added, followed by acetic acid (0.2 mL). The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was quenched with aqueous sodium bicarbonate (50 mL). It was extracted with ethyl acetate (3 × 50 mL), and the combined organic layers were dried over sodium sulfate and concentrated under reduced pressure. The crude product obtained (0.200 g) was carried on to the next step without purification. 1H NMR (400 MHz, DMSO-d6): δ 8.69 (s, 1H), 8.696 (s, 1H), 8.424-8.422 (d, J=8 Hz, 2H), 7.912-7.891 (d, J=8 Hz, 2H), 7.607 (m, 1H), 7.531-7.324 (m, 3H), 4.50 (s, 3H), 4.0 (s, 3H), 3.560 (m, 4H), 3.55 (s, 2H), 3.308 (m, 4H) and LC-MS m / z=443.3(M+H) + . General Procedure-F: Ester Hydrolysis

[0114] To a flask containing the appropriate alkyl ester in an aqueous organic solvent (e.g., THF or methanol, 1,4 dioxane, preferably 1,4 dioxane), 1.5 equivalents of aqueous sodium hydroxide are added, and the reaction mixture is refluxed for 1 to 8 hours (preferably 4 hours). Completion of the reaction is monitored by TLC. Excess solvent is removed under vacuum, and the solution is acidified with 10% HCl solution. The separated solid is collected by filtration and dried under vacuum to give the desired carboxylic acid derivative. If necessary, the desired compound can be purified by crystallization or trituration from one or more appropriate solvents, or by preparative HPLC or flash chromatography. Illustrative Example of General Procedure-F Example #1: Synthesis of 1-methyl-5-(2'-methyl-[1,1'-biphenyl]-4-yl)-1H-benzo[d][1,2,3]triazole-7-carboxylic acid (Compound-1) [ka]

[0115] To a stirred solution of methyl 1-methyl-5-(2'-methyl-[1,1'-biphenyl]-4-yl)-1H-benzo[d][1,2,3]triazole-7-carboxylate (1.2 g, 3.361 mmol, D.1) in 1,4-dioxane (15 mL) was added 2N aqueous NaOH (15 mL). The reaction mixture was refluxed for 4 hours. After completion of the reaction, the reaction mixture was cooled to room temperature, excess solvent was removed under reduced pressure, and the solution was acidified with 10% HCl solution (pH approx. 2). The separated solid was collected by filtration and dried under vacuum to give the title compound as an off-white solid (1.1 g, 95%). 1 H NMR (400 MHz, DMSO-d6): δ 13.35 (bs, 1H), 8.52 (s, 1H), 8.38 (s, 1H), 7.89-7.87 (d, J=8.0 Hz, 2H), 7.51-7.49 (d, J=8.4 Hz, 2H), 7.32-7.25 (m, 4H), 4.58 (s, 3H) 2.30 (s, 3H) and LC-MS m / z=344.1 (M+H) + . General Procedure G: Amide Formation

[0116] To a flask containing the appropriate carboxylic acid derivative (1.0 equivalent) in an organic solvent (e.g., DMF, THF, or CHCl) is added EDCI.HCl (1.5 equivalents), HOBT (1.5 equivalents), and N-ethyl-N-isopropylpropan-2-amine (3 equivalents). After stirring for about 10 minutes at approximately 25°C, the appropriate amine (1.5 equivalents) is added, and the reaction is stirred for an additional 8-12 hours (preferably 12 hours). The solid that separates upon addition of water is collected by filtration and dried under vacuum to yield the amide derivative. If necessary, the resulting compound can be purified by crystallization or trituration from one or more appropriate solvents, or by preparative HPLC or flash chromatography. Illustrative Example of General Procedure-G Example #2: Synthesis of 1-methyl-5-(2'-methyl-[1,1'-biphenyl]-4-yl)-1H-benzo[d][1,2,3]triazole-7-carboxamide (Compound-2) [ka]

[0117] To a flask containing 1-methyl-5-(2'-methyl-[1,1'-biphenyl]-4-yl)-1H-benzo[d][1,2,3]triazole-7-carboxylic acid (0.150 g, 0.43 mmol, Compound-1) in DMF (3 mL) was added EDCI.HCl (0.100 g, 0.52 mmol), HOBT (0.070 g, 0.52 mmol), and N-ethyl-N-isopropylpropan-2-amine (0.168 g, 1.31 mmol). The mixture was stirred at about 25 °C for approximately 10 minutes, and ammonium chloride (0.070 g, 1.31 mmol) was added. The reaction was then stirred for an additional 12 hours and quenched with water (50 mL). The separated solid was collected by filtration and dried under vacuum to give the desired compound as an off-white solid (0.08 g, 53%). 1 H NMR (400 MHz, DMSO-d6): δ 8.47 (s, 1H), 8.37 (s, 1H), 8.05 (s, 1H), 8.00 (s, 1H), 7.90-7.88 (d, J=8.0 Hz, 2H), 7.51-7.49 (d, J=7.6 Hz, 2H), 7.35-7.27 (m, 4H), 4.61 (s, 3H), 2.30 (s, 3H) and LC-MS m / z =343.2(M+H) + .

[0118] The following intermediates were prepared by procedures similar to those described in General Procedure-C, with appropriate changes in reactants, amounts of reagents and reaction conditions. The physicochemical characteristics of the compounds are summarized in Table C.1 herein below.

[0119] [Table C1]

[0120] The following intermediates were prepared by procedures similar to those described in General Procedure-D, with appropriate changes in reactants, amounts of reagents and reaction conditions. The physicochemical characteristics of the compounds are summarized in Table D.1 herein below.

[0121] [Table D1-1] [Table D1-2] [Table D1-3] [Table D1-4]

[0122] The following compounds were prepared by procedures similar to those described in General Procedures-E, F and G, with appropriate changes in reactants, amounts of reagents and reaction conditions. The physicochemical characteristics of the compounds are summarized in the tables herein below. [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0123] Pharmacological activity Measurement of DHODH inhibitory enzyme activity (in vitro assay)

[0124] The DHODH activity assay is a coupled enzyme assay in which the oxidation of DHO and subsequent reduction of ubiquinone is stoichiometrically equivalent to the reduction of DCIP (2,6-dichlorophenol), which is accompanied by a decrease in absorbance at 610 nm.

[0125] Solution / reagent preparation:

[0126] Preparation of buffer: 50 mM Tris-HCl, 150 mM KCl, and pH 8.0, 0.8% Triton.

[0127] 20 mM L-dihydroorotic acid stock solution in buffer.

[0128] 20 mM 2,6-dichloroindophenol sodium salt hydrate stock solution in buffer.

[0129] 20 mM decylubiquinone stock solution in buffer.

[0130] DMSO was used as the vehicle. procedure

[0131] 5 μL of a dimethyl sulfoxide or DMSO solution of the compound of formula (I) was added to the wells of a 96-well plate. The compound of formula (I) was measured at 10 μM.

[0132] The protein was added with the buffer to a total volume of 87 μL, including DMSO. The compound and protein were mixed and then incubated at room temperature for 30 minutes. 5 μL of a 20 mM solution of L-dihydroorotic acid, 5 μL of a 2 mM solution of decylubiquinone, and 3 μL of a 2 mM solution of 2,6-dichloroindophenol sodium salt hydrate were added to the above solution (total assay volume 100 μL). The mixture was stirred for 2 minutes, and absorbance was recorded every 10 minutes at 610 nanometers. Percent inhibition was calculated as follows: 100*{(Abs of reactions containing compound 610 )-(Positive control Abs 610 ) (Abs without enzyme reaction 610 )-(Positive control Abs 610 ) A reaction containing a compound has a compound, a buffer, an enzyme, and a substrate. The positive control contains DMSO, buffer, enzyme and substrate. No enzyme reaction contains DMSO, buffer and substrate I C 50 Decision

[0133] A 2 mM DMSO stock solution of selected trisubstituted benzimidazole and benzotriazole derivatives of formula (I) of the present invention to be investigated was prepared and then diluted one-third.

[0134] Five microliters of each stock solution of the compound of formula (I) was used for each 100-microliter assay. Thus, when prepared using buffer, protein, and substrate, 5 microliters of a 2 mM stock solution yielded 100 microliters of a 100-micromolar solution of the compound of formula (I). Ulrich et al. (2001) Eur. J. See also Biochem. 268, 1861-1868.

[0135] IC of selected compounds of the present invention 50 The values ​​are provided in the table below and are 50 Compounds showing IC values ​​≤ 0.1 μM were grouped into "a" group, and compounds with IC values ​​ranging from 0.101 μM to 1.0 μM were grouped into "b" group. 50Compounds showing IC values ​​were divided into group "b" and 50 Compounds showing values ​​>1.0 μM were grouped into the "c" group.

[0136] [Table A]

[0137] Cell-based activity Ramos proliferation assay (in vitro assay)

[0138] The cell proliferation assay is a sensitive method for quantifying viable cells in cytotoxicity or proliferation assays. The XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide inner salt) system is a means of measuring the activity of viable cells via mitochondrial dehydrogenases. Mitochondrial dehydrogenases in viable cells cleave the tetrazolium ring of XTT to produce orange formazan crystals that are soluble in aqueous solution. The XTT solution is enhanced by adding the electron coupling agent phenazine methosulfate (PMS) to the reaction. The resulting orange color is measured spectrophotometrically at 450 nm. An increase or decrease in cell number results in a concomitant change in the amount of formazan formed, which indicates the degree of cytotoxicity caused by the test material. Preparation of solutions / reagents Preparation of culture medium

[0139] 17.7 g of IMDM (Iscove's Modified Dulbecco's Medium) powder, 1.5 g of sodium bicarbonate, pH 7.2-7.4 were dissolved in 1 L of MiliQ water, and 1% penicillin / streptomycin and 10% FBS were added.

[0140] 10.6 g of Ham's F12 powder and 1.5 g of sodium bicarbonate, pH 7.2-7.4, were dissolved in 1 L of MiliQ water, and 1% penicillin / streptomycin was added.

[0141] DMSO was used as the vehicle.

[0142] 1x PBS (Phosphate Buffered Saline): 5 PBS tablets (Sigma: Cat. No. P4417) were dissolved in 1 L of MiliQ water. Procedure (IC 50 (Decision of

[0143] Ramos cells were cultured at 1 × 10 in complete IMDM medium. 5 The cells were resuspended at a density of 10,000 cells / ml. 95 μL of this cell suspension was added to a 96-well plate, and approximately 10,000 cells were seeded per well. The plate was incubated at 37°C in a humidified atmosphere of 5% CO2 for approximately 1 hour before compound addition.

[0144] Test compounds (see Table 1) were dissolved in 100% DMSO to create stock solutions of 2, 6, 10, and 20 mM. 200x concentrations of the required final concentrations were prepared in DMSO. Next, 10 μL of each concentration (200x) was diluted with 90 μL of serum-free Ham's F12 medium to obtain 20x intermediate concentrations in the medium. The DMSO concentration at this step was 10% (intermediate dilutions). Next, 5 μL of each intermediate dilution was added in triplicate to pre-seeded 96-well plates. The final DMSO concentration was 0.5% in the experimental wells. Cells treated with 0.5% DMSO served as a positive control. 100 μL of complete IMDM medium served as a medium blank for data analysis. 200 μL of 1x PBS was added to all corner wells of the assay plate. The plates were then incubated at 37°C in a 5% CO2 incubator for 72 hours.

[0145] On the final day, 100 μl of XTT solution (1 mg / ml XTT in Ham's F12 medium supplemented with 25 μM PMS) was added to each well. The plates were incubated for 2 hours. The amount of formazan produced was determined by reading the absorbance of the plates at a wavelength of 450 nm using a VICTOR X5 multilabel plate reader. IC 50Values ​​were determined as the concentration that reduced cell viability by 50% and curves were plotted using GraphPad Prism 6.0.

[0146] Percent inhibition is calculated as follows: Percent inhibition (%) was calculated by normalizing the DMSO control value to 100% using the following formula: Inhibition % = 100% - (Abs450 試験化合物-ブランク ) / (Abs450 陽性対照-ブランク )*100 The test compound contains cells, test compound, IMDM medium and 0.5% DMSO. The positive control contains cells, IMDM medium, and 0.5% DMSO. The blank contains IMDM medium. [Table B]

[0147] In vitro growth inhibition of multiple human cancer cell lines by compound 1

[0148] A screen of a panel of tumor cell lines was conducted aimed at identifying tumor cell subsets that were particularly sensitive to inhibition of DHODH by compound 1. Compound 1 is represented by the following structural formula: [ka]

[0149] These cell lines were treated with Compound 1 for a total of 72 hours.

[0150] As shown in Figure 1 and Table 1, assessment of tumor growth rates after 72 hours of treatment revealed that a distinct subset of cell lines (illustrated by gray dots in Figure 1) were sensitive to Compound 1. The majority of cell lines highly sensitive to Compound 1 were of hematopoietic origin, although some solid tumors also showed high sensitivity (Table 1). For the purposes of generating Figure 1, sensitive cell lines were defined as those with ≥75% maximal growth inhibition and GI 50 Table 1 shows some of the cell lines sensitive to Compound 1, as defined by GI 50 The table shows the maximum growth response and the maximum inhibition value, where 100 represents complete growth inhibition and >100 represents cell killing.

[0151] A follow-up screen was performed on an expanded panel of heme-lineage cell lines in a 4-day growth assay. Growth was assessed by Cell-Titer Glo measurements on days 0 and 4. As shown in Figure 2, 25% (20 / 80) of the screened heme lines were sensitive to Compound 1, of which diffuse large B-cell lymphoma (DLBCL) lines were particularly sensitive (8 / 11 or 73%). A subset of heme lines that were moderately sensitive (defined as >50% and <75% growth rate inhibition) or insensitive (defined as <50% growth rate inhibition) to Compound 1 in this follow-up screen was subjected to an extended growth assay to assess whether extending the treatment time modulated their sensitivity profile. Specifically, these heme lines were pretreated with the indicated concentrations of Compound 1 for 3 days and then replated in fresh medium / drug for a standard 4-day growth assay. The majority of the retested heme strains showed strong sensitivity to Compound 1 after 7 days of treatment with Compound 1 (Table 2). [Table 1]

[0152] [Table 2]

[0153] Cancer cell growth inhibition by compound 1 is due to inhibition of DHODH

[0154] DHODH catalyzes the fourth step in de novo pyrimidine biosynthesis, oxidizing dihydroorotate to orotic acid in the inner mitochondrial membrane. Orotic acid then combines with phosphoribosyl pyrophosphate (PRPP) to form orotidine-5'-monophosphate (OMP). Uridine monophosphate (UMP) is ultimately generated from OMP in the cytosol, where it is utilized to generate pyrimidines for RNA / DNA biosynthesis and other important biosynthetic functions, such as protein / lipid glycosylation and phospholipid production for membrane biosynthesis.

[0155] To confirm that the effects of compound 1 on cell growth / survival were due to specific inhibition of DHODH, cell growth assays were performed by supplementing the medium with various amounts of uridine. Supplementing the medium with a near-physiological concentration of uridine (5 μM) partially rescued the effects of compound 1, whereas supraphysiological concentrations (25 μM and 100 μM) completely rescued the effects of compound 1 on growth up to 10 μM. These results demonstrate that the effects of compound 1 on growth are precise (Figure 3). Comparison of the sensitivity profile of Compound 1 with that of cytarabine and doxorubicin

[0156] The sensitivity profile of compound 1 against a subset of heme strains was compared with that of other drugs used as standard of care (SOC) for heme malignancies. Compound 1 exhibited a sensitivity profile (Figure 4A) that differed from that of cytarabine (Figure 4B) and doxorubicin (Figure 4C), consistent with its different mechanism of action. Compound 1 effectively inhibits DHODH in vivo and disrupts tumor growth in AML xenograft models

[0157] An in vivo efficacy study with Compound 1 was performed to evaluate the in vitro to in vivo translation of its effects on blocking DHODH and tumor cell growth inhibition. 6 MOLM-13 cells were implanted subcutaneously into CB17 SCID mice (n=15 / group). Mice (n=15 / group) were cultured until tumors averaged approximately 150 mm 3 Once the NIH score reached 0.05, mice were treated BID PO with vehicle or 100 mg / kg Compound 1. At the end of the study, tissues were collected at designated time points after the final dose for pharmacokinetic (PK) and pathway biomarker analysis.

[0158] Compound 1 administered BID at 100 mg / kg resulted in near-complete tumor growth inhibition (TGI) in a MOLM-13 acute myeloid leukemia (AML) xenograft model (Figure 5A). Pharmacokinetic profiles measured in plasma and tumor demonstrated a decline in drug concentrations by 12 hours, supporting the BID dosing regimen (Figure 5B). Clear evidence of target engagement was observed by a dramatic increase in tumor levels of the DHODH substrate, dihydroorotic acid (DHO) (Figure 5C). Baseline DHO levels were below the limit of quantitation (BQL < 120 ng / g). Tumor uridine pools were concomitantly reduced by approximately 60%, depending on the time point assessed (Figure 5D). Compound 1 effectively inhibits DHODH in patient-derived AML and DLBCL xenograft models in vivo and disrupts tumor growth

[0159] Next, screening was performed using a small number of mice per group to evaluate the efficacy of Compound 1 in AML and DLBCL patient-derived xenograft models. Tumor-bearing mice (n=3 / group) were treated with vehicle or 100 mg / kg Compound 1 PO BID.

[0160] As shown in Figures 6A-6E and 7A-7B, antitumor activity of Compound 1 was observed in all models tested, with TGI >60% in two of the five AML models (i.e., AML_2 and AML_5) and one of the two DLBCL models (DLBCL_1). The DLBCL_1 model is characterized as a triple-hit DLBCL model. Double-hit by compound 1 in human cancer cell lines of diffuse large B-cell lymphoma vitro growth inhibition

[0161] Three patient-derived DLBCL lymphoma cell lines classified as double-hit DLBCL, namely OCILY18, SC-1, and CARNAVAL, were found to be highly sensitive to inhibition by compound 1 in a 96-hour growth assay (Figure 8). Compound 1 effectively inhibits tumor growth in patient-derived DLBCL xenograft models

[0162] Potent inhibition of in vivo tumor growth by Compound 1 was observed in the OCILY-19 diffuse large B-cell lymphoma (DLBCL) xenograft model. 6 OCILY-19 cells were implanted subcutaneously into CB17 SCID mice (n = 15–18 / group). The mice were cultured until tumors averaged approximately 150 mm 3 Once the patient reached 100 mg / kg, they were treated with the designated dose / frequency of vehicle or Compound 1. Tissues were collected at the designated time points after the final dose for PK and biomarker analysis.

[0163] The degree of pathway modulation and tumor growth inhibition was demonstrated to be dose- and schedule-dependent (Figure 9A). The 100 mg / kg BID dosing regimen resulted in superior efficacy compared to the 10 and 30 mg / kg BID dose arms, which correlated with a higher increase in tumor DHO (Figure 9C) and a decrease in the total tumor uridine pool (Figure 9D). The 200 mg / kg QD regimen was less effective than the 100 mg / kg BID dosing regimen due to the short half-life of compound 1 in mice, resulting in lower trough drug concentrations with QD dosing (see Figure 9B). In vitro growth inhibition of multiple human triple-negative breast cancer cell lines by compound 1

[0164] A panel of triple-negative cancer cell lines (TNBC) was screened to assess whether a subset of TNBCs could potentially benefit from treatment with Compound 1 as a single agent. Treatment with Compound 1 for 96 hours strongly impaired cell viability / growth of DU4475 to the same extent as observed in heme-derived cell lines (see FIG. 10), whereas the other four TNBC lines (HCC1143, HCC38, BTS49, and HCC1806) were insensitive under the conditions tested. The present invention provides, for example, the following items. (Item 1) Subjects with acute myeloid leukemia, multiple myeloma, B-prolymphocytic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, anaplastic large cell lymphoma, mantle cell lymphoma, lung cancer, breast cancer, triple-negative breast cancer, melanoma, glioblastoma, prostate cancer, colon cancer, pancreatic cancer, bone cancer, head and neck cancer, skin cancer, or cutaneous or intraocular malignancies 1. A method of treating a cancer selected from endometrial, cervical, vaginal, vulvar, esophageal, small intestinal, endocrine system, thyroid, parathyroid, adrenal, soft tissue sarcoma, urethral, ​​penile, solid tumors of childhood, lymphocytic lymphoma, bladder, kidney or ureter, renal pelvis, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancer, and PTEN-mutated cancer, comprising administering to the subject a therapeutically effective amount of a compound of the following structural formula: [ka] [In the formula, The dotted lines [....] in the ring represent optional bonds that exist in any stable combination; R1 is selected from hydrogen and alkyl; R2 is -A-R4, A is arylene or tetrasubstituted arylene, the substituents being halogen; R3 is selected from hydroxy and amino; R4 is selected from aryl and heteroaryl optionally substituted with one or more R5; R5 is alkyl and -(CH2) n N(R a )R b is selected from R a and R b are independently selected from hydrogen, alkyl, and —C(O)alkyl; Or R a and R b can be taken together with the nitrogen atom to which they are attached to form a 4-6 membered heterocyclyl containing 0-2 additional heteroatoms independently selected from O and N, and optionally substituted with alkyl; n is an integer selected from 0 and 1. or a pharmaceutically acceptable salt thereof. 2. The method of item 1, wherein the cancer is selected from acute myeloid leukemia, multiple myeloma, B-prolymphocytic leukemia, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, anaplastic large cell lymphoma, mantle cell lymphoma, triple-negative breast cancer, melanoma, prostate cancer, and esophageal cancer. (Item 3) The method according to any one of items 1 and 2, wherein the cancer is acute myeloid leukemia. (Item 4) 3. The method of any one of items 1 and 2, wherein the cancer is multiple myeloma. (Item 5) 3. The method of any one of items 1 and 2, wherein the cancer is B-prolymphocytic leukemia. (Item 6) 3. The method of any one of items 1 and 2, wherein the cancer is non-Hodgkin's lymphoma. (Item 7) 3. The method of any one of items 1 and 2, wherein the cancer is diffuse large B-cell lymphoma. (Item 8) 3. The method of any one of items 1 and 2, wherein the cancer is anaplastic large cell lymphoma. (Item 9) 3. The method of any one of items 1 and 2, wherein the cancer is mantle cell lymphoma. (Item 10) 3. The method of any one of items 1 and 2, wherein the cancer is triple-negative breast cancer. (Item 11) 3. The method of any one of items 1 and 2, wherein the cancer is melanoma. (Item 12) 3. The method of any one of items 1 and 2, wherein the cancer is prostate cancer. (Item 13) 3. The method of any one of items 1 and 2, wherein the cancer is esophageal cancer. (Item 14) The compound is [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] 14. The method according to any one of items 1 to 13, wherein the compound is selected from the group consisting of: (Item 15) The compound is [ka] or a pharmaceutically acceptable salt thereof. (Item 16) The compound is [ka] or a pharmaceutically acceptable salt thereof. (Item 17) The compound is [ka] or a pharmaceutically acceptable salt thereof. (Item 18) The compound is [ka] or a pharmaceutically acceptable salt thereof. (Item 19) The compound is [ka] or a pharmaceutically acceptable salt thereof. (Item 20) The compound is [ka] or a pharmaceutically acceptable salt thereof.

Claims

1. For the treatment of acute myeloid leukemia, multiple myeloma, B-prolymphocytic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, anaplastic large cell lymphoma, mantle cell lymphoma, lung cancer, breast cancer, triple-negative breast cancer, melanoma, glioblastoma, prostate cancer, colon cancer, pancreatic cancer, bone cancer, head and neck cancer, skin cancer, cutaneous or intraocular malignant endometrium, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumors, lymphocytic lymphoma in a subject.

1. A method of treating a cancer selected from bladder cancer, kidney or ureter cancer, renal pelvis cancer, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancer, and PTEN-mutated cancer, comprising administering to the subject a therapeutically effective amount of a compound of the following structural formula: [In the formula, Dotted line in the ring [ . . . .] represents optional bonds present in any stable combination; R 1 is selected from hydrogen and alkyl; R 2 is -A-R 4 ; A is arylene or tetrasubstituted arylene, the substituents being halogen; R 3 is selected from hydroxy and amino; R 4 is selected from aryl and heteroaryl optionally substituted with one or more R 5 ; R 5 is selected from alkyl and —(CH 2 ) n N(R a )R b ; R a and R b are independently selected from hydrogen, alkyl, and —C(O)alkyl; or R a and R b together with the nitrogen atom to which they are attached can form a 4-6 membered heterocyclyl containing 0-2 additional heteroatoms independently selected from O and N, and optionally substituted with alkyl; n is an integer selected from 0 and 1. or a pharmaceutically acceptable salt thereof.

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