BR122025008872A2 - USE OF TRISEUTRITIONAL BENZOTRIAZOLE DERIVATIVES AS DIHYDROOROTATE OXYGENASE INHIBITORS - Google Patents

Abstract

The present invention provides methods for treating cancer in a subject and inhibiting tumor growth, metastasis, or the enzymatic activity of dihydroorotate oxygenase in a tumor or cancer cell. At least one trisubstituted benzotriazole derivative of formula (I) is administered to the subject or is contacted with the cancer cell. The compounds of formula (I) have substituents R1, R2, and R3 that have the meanings given in the specification, and pharmaceutically acceptable salts thereof.

Description

BACKGROUND OF THE INVENTION Cross-reference to related orders

[001] 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, and now U.S. Patent No. 9,937,155, both of which are incorporated by this reference in their entirety.

Field of Invention

[002] The present invention relates to novel trisubstituted benzotriazole derivatives of formula (I) which are inhibitors of dihydroorotate dehydrogenase. In particular, the invention relates to novel compounds which inhibit the activity of the enzyme DHODH, to a process for their manufacture and pharmaceutical compositions containing them, and to their use in the treatment and prevention of diseases or disorders, in particular their use in diseases or disorders where there is an advantage in inhibiting DHODH.

Description of Related Art

[003] DHODH is a protein that catalyzes one of the steps in the denovo pyrimidine nucleotide biosynthetic pathway. (Greene et al. Biochem Pharmacol 1995, 50:861-7; Davis J.P et al. FASEB J 1996, 10(6): Abst C23). It catalyzes the only oxidation/reduction reaction in this pathway, which is the conversion step of DHO (dihydroorotate) to orotate with the help of the flavin cofactor and an electron acceptor. Dihydroorotate dehydrogenase inhibitors have been found to have broader applications as chemotherapeutic agents. (Kensler et al. 1989 in: 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, 526-528 (1993)).

[004] As an example for DHODH inhibitors, the quinoline derivative Brequinar (6-fluoro-2-(2’-fluoro[1,1’-biphenyl]-4-yl)-3-methyl-4-quinolinecarboxylic acid) exhibits anticancer activity against murine leukemia L1210 (Andreson LW. Et al. Cancer Commun. 1989; 1(6), 381-7; Chen SF. et al. Cancer Res. 1986 Oct; 46(10): 5014-9). Brequinar has also been shown to potentiate the antitumor activity of 5-fluorouracil in a murine colon tumor model by tissue-specific modulation of uridine nucleotide pools. (G Pizzorno et al., Cancer Res. 1992 Apr 1; 52:1660-5).

[005] DHODH inhibitors may also be useful in the treatment of viral-mediated diseases (see US 6,841,561). Furthermore, DHODH inhibition is known to be a promising target for the treatment of transplant rejection, rheumatoid arthritis, psoriasis, as well as autoimmune diseases (Kovarik, J. M. et al. Expert Opin. Emerg. Drugs 2003, 8, 47; Allison, A. C. Transplantation Proc (1993) 25(3) Suppl. 2, 8-18); Makowka, L., Immunolog Rev. (1993) 136, 51-70; Davis J. P et al. Biochemistry 1996, 35: 1270-3).

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

[007] In vivo, leflunomide is rapidly transformed to its active metabolite teriflunomide, which exerts its anti-inflammatory, antiproliferative, and immunosuppressive effects through mechanisms that are not completely understood. Teriflunomide is not only a potential inhibitor of protein tyrosine kinase in vivo, but a 100-1,000-fold greater inhibitor of DHODH (Davis JP et al. FASEB J 1996, 10(6): Abst C23; Davis JP et al. Biochemistry 1996, 35:1270-3).

[008] With the increasing number of patients affected by autoimmune and related diseases, there is an unmet need for new drugs that can treat these diseases more effectively. There is still a crucial need for immunosuppressive agents, which are additionally useful in a wide variety of autoimmune and chronic inflammatory diseases, including systemic lupus erythematosus, chronic rheumatoid arthritis, multiple sclerosis, type I diabetes mellitus, inflammatory bowel diseases, 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. They may also be useful as part of chemotherapeutic regimens for the treatment of cancers, lymphomas and leukemias, alone or in combination with antitumor compounds well known to those skilled in the art.

SUMMARY OF THE INVENTION

[009] The present invention is directed to a method for treating cancer in an individual in need of such treatment. In one embodiment, the cancer is selected from 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 or neck cancer, skin cancer, cutaneous or intraocular malignant endometrial cancer, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, soft tissue sarcoma, urethral cancer, penile cancer, solid tumors of the prostate, and other malignant tumors of the esophagus. children, lymphocytic lymphoma of the bladder, kidney or ureter cancer, renal pelvis carcinoma, central nervous system (CNS) neoplasm, primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, T-cell lymphoma, environmentally induced cancer, and a PTEN mutant 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 the step of administering to the individual one or more times a therapeutically effective amount of at least one compound according to formula (I): or a pharmaceutically acceptable salt thereof. In the structure, the dotted lines [...] in the ring may represent an optional bond that may be present in any stable combination. R1 may be hydrogen and alkyl. R2 may be -A-R4. A may be arylene or tetrasubstituted arylene where the substituent is halogen. R3 may be hydroxy and amino. R4 may be an optionally substituted aryl and an optionally substituted heteroaryl. The optional substituents may be one or more R5. R5 may be alkyl and -(CH2)nN(Ra)Rb. Ra and Rb may independently be hydrogen, alkyl and -C(O)alkyl or alternatively Ra and Rb may 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, wherein the optional substituent is alkyl and 'n' may be an integer 0 and 1.

[0010] The present invention is also directed to a method for inhibiting the growth and/or metastasis of tumor cells in an individual. The method comprises the step of administering to the individual one or more times a therapeutically effective amount of at least one compound according to formula (I): or a pharmaceutically acceptable salt thereof. In the structure, the dotted lines [...] in the ring may represent an optional bond that may be present in any stable combination. R1 may be hydrogen and alkyl. R2 may be -A-R4. A may be arylene or tetrasubstituted arylene where the substituent is halogen. R3 may be hydroxy and amino. R4 may be an optionally substituted aryl and an optionally substituted heteroaryl. The optional substituents may be one or more R5. R5 may be alkyl and -(CH2)nN(Ra)Rb. Ra and Rb may independently be hydrogen, alkyl and -C(O)alkyl or alternatively Ra and Rb may 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, wherein the optional substituent is alkyl and 'n' may be an integer 0 and 1.

[0011] The present invention is further directed to a method for inhibiting the activity of the enzyme dihydro-orotate oxygenase in a tumor cell. The method comprises the step of contacting the tumor cell one or more times with a therapeutically effective amount of at least one compound according to formula (I): or a pharmaceutically acceptable salt thereof. In the structure, the dotted lines [...] in the ring may represent an optional bond that may be present in any stable combination. R1 may be hydrogen and alkyl. R2 may be -A-R4. A may be arylene or tetrasubstituted arylene, wherein the substituent is halogen. R3 may be hydroxy and amino. R4 may be an optionally substituted aryl and an optionally substituted heteroaryl. The optional substituents may be one or more R5. R5 may be alkyl and -(CH2)nN(Ra)Rb. Ra and Rb may independently be hydrogen, alkyl and -C(O)alkyl or alternatively Ra and Rb may 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, wherein the optional substituent is alkyl and 'n' may be an integer 0 and 1.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1 shows the sensitivity of a panel of ~400 human cancer lines of hematopoietic and non-hemapoietic origin to growth inhibition by Compound 1 of the invention. Gray circles represent cell lines classified as sensitive (exhibiting >75% maximal growth inhibition and a GI50 value <1.5 µM.

[0013] FIG. 2 shows the sensitivity of an additional panel of hematologic lineage human cancer lines to growth inhibition by Compound 1 of the invention.

[0014] 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 compound in the indicated cancer lines.

[0015] FIG. 4A shows the relative growth rate vs. concentration sensitivity profiles of the MV411, Kasumi-1, THP-1, DB, Toledo, and WSU-DLCL2 cell lines toward varying concentrations of compound 1.

[0016] FIG. 4B shows the relative growth rate vs. concentration sensitivity profiles of the MV411, Kasumi-1, THP-1, DB, Toledo, and WSU-DLCL2 cell lines toward varying concentrations of cytarabine.

[0017] FIG. 4C shows the relative growth rate vs. concentration sensitivity profiles of the MV411, Kasumi-1, THP-1, DB, Toledo, and WSU-DLCL2 cell lines toward varying concentrations of doxorubicin.

[0018] FIG. 5A shows MOLM-13 tumor growth curves in CB 17 SCID mice when left untreated (vehicle) or treated with 100 mg/kg of Compound 1 twice daily, measured over 14 days.

[0019] FIG. 5B shows the pharmacokinetic profiles of Compound 1 (dosage = 100 mg/kg twice daily) in the plasma of CB 17 SCID mice and in implanted MOLM-13 tumors at the indicated time points after the last dose at the end of the study.

[0020] FIG. 5C shows DHO levels in untreated (vehicle) MOLM-13 tumors and Compound 1-treated tumors measured over 12 hours after the last dose at the end of the study.

[0021] FIG. 5D shows uridine levels in untreated (vehicle) MOLM-13 tumors and Compound 1-treated tumors measured over 12 hours after the last dose at the end of the study.

[0022] FIG. 6A shows patient-derived AML_1 tumor growth curves in CB 17 SCID mice when left untreated (vehicle) or treated with 100 mg/kg of Compound 1 twice daily.

[0023] FIG. 6B shows patient-derived AML_2 tumor growth curves in CB 17 SCID mice when left untreated (vehicle) or treated with 100 mg/kg of Compound 1 twice daily.

[0024] FIG. 6C shows patient-derived AML_3 tumor growth curves in CB 17 SCID mice when left untreated (vehicle) or treated with 100 mg/kg Compound 1 twice daily.

[0025] FIG. 6D shows patient-derived AML_4 tumor growth curves in CB 17 SCID mice when left untreated (vehicle) or treated with 100 mg/kg of Compound 1 twice daily.

[0026] FIG. 6E shows patient-derived AML_5 tumor growth curves in CB 17 SCID mice when left untreated (vehicle) or treated with 100 mg/kg of Compound 1 twice daily.

[0027] FIG. 7A shows patient-derived DLBCL_1 (triple translocation model) tumor growth curves in CB 17 SCID mice when left untreated (vehicle) or treated with 100 mg/kg of Compound 1 twice daily.

[0028] FIG. 7B shows patient-derived DLBCL_2 tumor growth curves in CB 17 SCID mice when left untreated (vehicle) or treated with 100 mg/kg of Compound 1 twice daily.

[0029] FIG. 8 is a curve showing the relative growth rate of the dual translocation diffuse large B-cell lymphoma cell lines OCILY18, SC-1, and CARNAVAL treated with various concentrations of Compound 1 for 96 hours.

[0030] FIG. 9A shows OCILY-19 double translocation diffuse large B-cell lymphoma (DLBCL) tumor growth curves in CB 17 SCID mice when left untreated (vehicle) or treated with 10 mg/kg Compound 1 twice daily; 30 mg/kg Compound 1 twice daily; 100 mg/kg Compound 1 twice daily; and 200 mg/kg Compound 1 QD, all measured over 14 days.

[0031] FIG. 9B shows the pharmacokinetic profiles of Compound 1, administered at the dosages described for FIG. 9A, in the plasma of CB 17 SCID mice, at the indicated time points after the last dose at the end of the study.

[0032] FIG. 9C shows DHO levels in untreated OCILY-19 tumors (vehicle) and in tumors treated with Compound 1 at the indicated doses, measured over 12 hours after the last dose at the end of the study.

[0033] FIG. 9D shows uridine levels in untreated OCILY-19 tumors (vehicle) and in tumors treated with Compound 1 at the indicated doses, measured over 12 hours after the last dose at the end of the study.

[0034] FIG. 10 is a curve showing the relative growth rate of the triple negative breast cancer line DU4475 treated with various concentrations of Compound 1 for 96 hours.

DETAILED DESCRIPTION OF THE INVENTION

[0035] In one embodiment, the present invention provides trisubstituted benzotriazole derivatives as dihydroorotate oxygenase inhibitors.

[0036] These derivatives are useful as a medicine in the treatment of autoimmune and inflammatory disorders, such as multiple sclerosis, rheumatoid arthritis and cancer-like diseases.

[0037] In a specific embodiment, the present invention provides compounds of formula (I), or a pharmaceutically acceptable salt or a pharmaceutically acceptable regioisomer thereof, wherein; the dotted lines [...] in the ring represent an optional bond which may be present in any stable combination; R1 is selected from hydrogen and alkyl; R2 is -A-R4; A is arylene or tetrasubstituted arylene; wherein the substituent is halogen; R3 is selected from hydroxy and amino; R4 is selected from optionally substituted aryl and optionally substituted heteroaryl; wherein the optional substituents are selected from one or more R5; R5 is selected from alkyl and -(CH2)nN(Ra)Rb; Ra and Rb are independently selected from hydrogen, alkyl, and -C(O)alkyl; alternatively, Ra and Rb may 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; where the optional substituent is alkyl; and 'n' is an integer selected from 0 and 1.

[0038] The embodiments below are illustrative of the present invention and are not intended to limit the claims to the specific embodiments exemplified.

[0039] According to one embodiment, specifically provided are compounds of formula (I), wherein R1 is alkyl; in particular alkyl is methyl.

[0040] According to another embodiment, specifically provided are compounds of formula (I), in which R2 is -A-R4; wherein -A- is selected from arylene and tetrasubstituted arylene.

[0041] In accordance with the preceding embodiment, specifically provided are compounds of formula (I), wherein R2 is selected from

[0042] According to one of the previous embodiments, specifically provided are compounds of formula (I), in which R4 is selected from optionally substituted phenyl; in which optional substituents are selected from methyl, acetylamino, isopropylaminomethyl, methylaminomethyl, dimethylaminomethyl,

[0043] According to one of the previous embodiments, specifically provided are compounds of formula (I), in which R4 is selected from 2,5-dimethyl-1H-pyrrole;

[0044] According to yet another embodiment, specifically provided are compounds of formula (I) in which R3 is -OH and -NH2.

[0045] According to yet another specific embodiment, compound of formula (I) is a compound of formula (1a) in which the dotted lines [---], R1, R3 and R4 are the same as those described in formula (I).

[0046] According to yet another specific embodiment, the is a compound of formula (Ib) where the dotted lines [-], R1, R3 and R4 are the same as those described in formula (I).

[0047] In another embodiment of the present invention, a process for the preparation of trisubstituted benzotriazole derivatives of formula (I).

[0048] The procedure for the compounds of formula (I) is detailed herein in the present invention below in the descriptive report, including the general synthesis of various intermediates involved in the process of manufacturing the compounds according to the present invention.

[0049] More particularly, the invention provides use of compounds of formula (I) or a pharmaceutically acceptable salt or a regioisomer thereof, including mixtures thereof in all ratios, as a drug, inhibiting the activity of the enzyme dihydro-orotate-oxygenase in the treatment of disorders such as multiple sclerosis and other diseases, such as, inflammatory disorders, rheumatoid arthritis and cancer.

[0050] The trisubstituted benzotriazole derivatives of formula (I) of the present invention have a therapeutic role in inhibiting the enzyme dihydro-orotate dehydrogenase (DHODH or DHOD). The compounds of formula (I) may be useful in the treatment and/or prevention of, but not limited to, autoimmune and chronic inflammatory diseases, including systemic lupus erythematosus, chronic rheumatoid arthritis, multiple sclerosis, type I diabetes mellitus, inflammatory bowel diseases, 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. Compounds of formula (I) and related formulae may also be useful as part of chemotherapeutic regimens for the treatment of cancers, lymphomas and leukemias alone or in combination with classical antitumor compounds well known to those skilled in the art.

[0051] Without limiting the scope of the present invention, the following definitions are provided in order to help those skilled in the art to understand the detailed description of the present invention.

[0052] “Alkyl” refers to a hydrocarbon chain which may be a straight or branched chain, containing the indicated number of carbon atoms, for example, a C1C6 alkyl group may have from 1 to 6 carbon atoms (inclusive) therein. 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 may be unsubstituted or substituted with one or more suitable groups.

[0053] “Amino” refers to an -N- group, the nitrogen atom of said group being bonded to a hydrogen, alkyl, cycloalkyl, aryl, heterocyclyl, or any suitable groups. Representative examples of an amino group include, but are not limited to, -NH2, -NHCH3, and -NH-cyclopropyl. An amino group may be unsubstituted or substituted with one or more of the suitable groups.

[0054] “Aryl” refers to an optionally substituted monocyclic, bicyclic, or polycyclic aromatic carbocyclic ring system of about 6 to 14 carbon atoms. Examples of a C6-C14 aryl group include, but are not limited to, phenyl, naphthyl, biphenyl, anthryl, tetrahydronaphthyl, fluorenyl, indanyl, biphenylenyl, and acenaphthyl. An aryl group may be unsubstituted or substituted with one or more suitable groups.

[0055] “Arylene” means a monocyclic or bicyclic, saturated, unsaturated or divalent aromatic carbocyclic ring having 6 to 14 carbon atoms which may be unsubstituted or substituted with one or more suitable groups.

[0056] “Halogen” or “halo” includes fluorine, chlorine, bromine or iodine.

[0057] “Hydroxy” refers to the -OH group.

[0058] The term “heterocyclyl” includes the definitions of “heterocycloalkyl” and “heteroaryl”. The term “heterocycloalkyl” refers to a 3- to 10-membered, saturated or partially saturated, non-aromatic monocyclic or polycyclic ring system having at least one heteroatom or heterogroup selected from O, N, S, S(O), S(O)2, NH, and C(O). Exemplary heterocycloalkyl groups include piperdinyl, piperazinyl, morpholinyl, thiomorpholyl, 1,3-dioxolanyl, 1,4-dioxanyl, and the like. A heterocycloalkyl group may be unsubstituted or substituted with one or more suitable groups.

[0059] “Heteroaryl” refers to an unsaturated, monocyclic, bicyclic, or polycyclic aromatic ring system containing at least one heteroatom selected from oxygen, sulfur, and nitrogen. Examples of C5-C10 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, lH-tetrazole, 1-methyltetrazole, benzoxazole, benzothiazole, benzofuran, benzisoxazole, benzimidazole, N-methylbenzimidazole, azabenzimidazole, indazole, quinazoline, quinoline and isoquinoline. Bicyclic heteroaryl groups include those 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, a nitrogen atom together with an oxygen or a sulfur atom in the ring, or a ring O or S atom. A heteroaryl group may be unsubstituted or substituted with one or more suitable groups.

[0060] “Heteroatom” refers to a sulfur, nitrogen, or oxygen atom.

[0061] “Optionally substituted or substituted”, as used herein, means that at least one hydrogen atom of the optionally substituted group has been replaced with suitable substitutions, as exemplified, 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)NH2, -C(O)NH(C1-C6 alkyl), -C(O)NH(cycloalkyl), -C(O)NH(heterocyclyl), - C(O)NH(heteroaryl), -C(O)N(C1-C6 alkyl)(alkyl C1-C6), - S(O)NH(C1-C6 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, or unsubstituted arylalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenylalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted amino, substituted or unsubstituted amino, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted heterocyclic ring.

[0062] The particular compounds of the present invention without departing from the scope of the definitions given in the compounds of formula (I) and specific compounds emanating from the formula (I) are summarized in the table of the present invention below which covers the entire scope of the compounds in the compound of formula (I). or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable regioisomer thereof.

[0063] In yet another embodiment, the present invention relates to compounds of formula (I) for use in the treatment of inflammatory disorders and autoimmune diseases or hyperactive immune response. More preferably, the present invention relates to the use of compounds of formula (I) for the treatment of multiple sclerosis, rheumatoid arthritis and transplant rejection.

[0064] Other embodiments of the invention include the use of compounds of formula (I) or pharmaceutically acceptable derivatives, salts and regioisomers thereof, including mixtures thereof in all ratios as a medicament.

[0065] Use of compounds as above and pharmaceutically usable derivatives, salts and regioisomers thereof, including mixtures thereof in all ratios, for the preparation of a medicament for the treatment and/or prophylaxis of a disorder associated with dihydroorotate dehydrogenase.

[0066] Use of compounds as above, wherein the dihydro-orotate dehydrogenase associated disorder is an autoimmune disorder or condition associated with an overactive immune response.

[0067] Use of compounds as above and pharmaceutically usable derivatives, salts and regioisomers thereof, including mixtures thereof in all ratios, for the preparation of a medicament for the treatment and/or prophylaxis of an immunoregulatory abnormality.

[0068] Use of compounds as above, wherein the immunoregulatory abnormality is multiple sclerosis or rheumatoid arthritis.

[0069] Use of the compounds as above for the preparation of a medicament for the treatment and prophylaxis of cancerous diseases, inflammatory bowel diseases or rheumatoid arthritis.

[0070] 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 regioisomers thereof, including mixtures thereof in all ratios, and at least one additional active ingredient.

[0071] The present invention additionally provides a pharmaceutical composition comprising at least one compound according to formula (I) and/or pharmaceutically usable derivatives, salts and regioisomers thereof, including mixtures thereof in all ratios, eventually an additional active ingredient, and excipients

[0072] The term “pharmaceutically acceptable salt” or “pharmaceutically acceptable derivatives” is considered to be an active ingredient comprising a compound of formula (I) in the form of one of its salts, in particular if this salt form confers improved pharmacokinetic properties on the active ingredient compared to the free form of the active ingredient or any other salt form of the active ingredient previously used. The pharmaceutically acceptable salt form of the active ingredient may also provide this active ingredient for the first time with a desired pharmacokinetic property that it did not previously possess and may even have a positive influence on the pharmacodynamics of this active ingredient with regard to its therapeutic efficacy in the body.

[0073] The term “regioisomer” or “regioisomers” refers to positional isomers, which is a category of structural isomers in which the position or substituent changes position in the parent structure. Here, the term regioisomer, without departing from the scope of the compound of formula (I), inherently includes all regioisomers as a pure regioisomer or mixture of two or more regioisomers. Since the pharmaceutical activity of the regioisomers of the compounds of the present invention may differ, it may be desirable to use the regioisomers. In such cases, the regioisomers may be separated at any of the possible stages, as an intermediate product or as a final product, by the process well known to the person skilled in the art or even used as such in the synthesis. The regioisomers of the compounds of formula (I) refer to the following structures:

[0074] Pharmaceutical formulations may be adapted for administration via any suitable method desired, for example, by 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 may be prepared using all processes known in the art of pharmacy, for example, by combining the active ingredient with excipient(s) or adjuvant(s).

[0075] Pharmaceutical formulations adapted for oral administration may be administered as separate units, such as, for example, capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or foaming foods; or oil-in-water liquid emulsions or water-in-oil liquid emulsions.

[0076] For example, in the case of oral administration as a tablet or capsule, the active ingredient component may be combined with an oral, non-toxic, pharmaceutically acceptable inert excipient, such as, for example, ethanol, glycerol, water, and the like.

[0077] Powders are prepared by grinding the compound to a suitable fine size and mixing it with a similarly ground pharmaceutical excipient, such as, for example, an edible carbohydrate such as, for example, starch or mannitol. Likewise, a flavoring, preservative, dispersant, and colorant may be present.

[0078] Capsules are produced by preparing a powder mixture as described above and filling them with shaped gelatin shells. Glidants and lubricants, such as, for example, highly dispersed silicic acid, talc, magnesium stearate, calcium stearate, or polyethylene glycol in solid form, may be added to the powder mixture prior to the filling operation. A disintegrant or solubilizer, such as, for example, agar-agar, calcium carbonate, or sodium carbonate, may also be added to improve the availability of the drug after ingestion of the capsule.

[0079] Additionally, if desired or necessary, suitable binders, lubricants, and disintegrants, as well as colorants, may also be incorporated into the mixture. Suitable binders include starch, gelatin, natural sugars such as, for example, glucose or beta-lactose, sweeteners made from corn, natural and synthetic rubber such as, for example, acacia, tragacanth, or sodium alginate, carboxymethyl cellulose, polyethylene glycol, waxes, and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like. Tablets are formulated, for example, by preparing a powder mixture, granulating or dry-pressing the mixture, adding a lubricant and a disintegrant, and pressing the entire mixture to form tablets. A powder mix is prepared by mixing the ground compound appropriately with a diluent or base, as described above, and optionally with a binder such as carboxymethyl cellulose, alginate, gelatin, or polyvinylpyrrolidone, a dissolution retardant such as paraffin, an absorption accelerator such as a quaternary salt, and/or an absorbent such as bentonite, kaolin, or dicalcium phosphate. The powder mix can be granulated by moistening it with a binder such as syrup, starch paste, acadia mucilage, or solutions of cellulose or polymeric materials and pressing it through a sieve. As an alternative to granulation, the powder mix can be passed through a tabletting machine, resulting in unevenly shaped pieces that are broken down to form granules. Granules can be lubricated by adding stearic acid, a stearate salt, talc, or mineral oil to prevent sticking to tablet molds. The lubricated mixture is then pressed into tablets. The active ingredients can also be combined with a free-flowing inert excipient and then pressed directly into tablets without granulation or dry pressing. A transparent or opaque protective layer consisting of a shellac sealing layer, a sugar or polymeric layer, and a glossy wax layer may be present. Colorants may be added to these coatings to differentiate between different dosage units.

[0080] Oral liquids, such as, for example, solution, syrups and elixirs, can be prepared in dosage unit form, such that a given quantity comprises a pre-specified amount of the compounds. Syrups can be prepared by dissolving the compounds in an aqueous solution with a suitable flavoring, while elixirs are prepared using a non-toxic alcoholic vehicle. Suspensions can be formulated by dispersing the compounds in a non-toxic vehicle. Solubilizers and emulsifiers, such as, for example, ethoxylated isostearyl alcohols and polyoxyethylene sorbitol ethers, preservatives, flavoring additives, such as, for example, peppermint oil or natural sweeteners or saccharin, or other artificial sweeteners and the like, can also be added.

[0081] Dosage unit formulations for oral administration may, if desired, be encapsulated in microcapsules. The formulation may also be prepared in such a manner that the release is prolonged or delayed, such as, for example, by coating or incorporating particulate matter into polymers, wax, and the like.

[0082] Novel trisubstituted benzotriazole derivatives of formula (I) and pharmaceutically acceptable salts thereof and physiologically functional derivatives thereof and the other active ingredients may also be administered in the form of liposome delivery systems, such as, for example, small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. The liposomes may be formed from suitable lipids or phospholipids or both, such as, for example, cholesterol, stearylamine or phosphatidylcholines or the like.

[0083] Pharmaceutical formulations adapted for transdermal administration may be administered as independent patches for close and prolonged contact with the epidermis of the recipient. Thus, for example, the active ingredient may be dispensed from the patch by iontophoresis, as described in general terms in Pharmaceutical Research, 3(6), 318 (1986).

[0084] Pharmaceutical compounds adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils.

[0085] For the treatment of the eye or other external tissue, e.g., the mouth and skin, the formulations are preferably applied as a topical ointment or cream. When formulated to result in an ointment, the active ingredient may be employed with a paraffinic or water-miscible cream base. Alternatively, the active ingredient may be formulated to result in a cream with an oil-in-water cream base or a water-in-oil base.

[0086] 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.

[0087] Pharmaceutical formulations adapted for topical application in the mouth include pills, pastilles and mouthwashes.

[0088] Pharmaceutical formulations adapted for rectal administration may be administered in the form of suppositories or enemas.

[0089] Pharmaceutical formulations adapted for nasal administration in which the carrier substance is a solid comprise a coarse powder having a particle size, for example, in the range of 20 to 500 microns, which is administered in the manner in which snuff is absorbed, i.e., by rapid inhalation through the nasal passages from a container containing the powder held close to the nose. Formulations suitable for administration as a nasal spray or nasal drops with a liquid as the carrier substance encompass solutions of active ingredients in water or oil.

[0090] Pharmaceutical formulations adapted for administration by inhalation include finely particulate powders or mists, which may be generated by various types of pressurized aerosol dispensers, nebulizers, or insufflators.

[0091] Pharmaceutical formulations adapted for vaginal administration may be administered as pessaries, tampons, creams, gels, pastes, foams, or spray formulations. Pharmaceutical formulations adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions comprising antioxidants, buffers, bacteriostats, and solutes, by which the formulation is made isotonic with the blood of the recipient to be treated; and aqueous and non-aqueous sterile suspensions, which may comprise suspending media and thickeners. The formulations may be administered in single-dose or multi-dose containers, e.g., sealed ampoules and vials, and stored in a freeze-dried (lyophilized) state, so that only the addition of the sterile carrier liquid, e.g., water for injection, immediately before use is necessary.

[0092] Solutions and suspensions for injection prepared according to the recipe can be prepared from sterile powders, granules and tablets.

[0093] It goes without saying that, in addition to the constituents particularly mentioned above, the formulations may also comprise other agents usual in the art with regard to the particular type of formulation; thus, for example, formulations that are suitable for oral administration may comprise flavorings.

[0094] A therapeutically effective amount of a compound of formula (I) and the other active ingredient depends on a number of factors, including, for example, the age and weight of the animal, the precise disease state requiring treatment and its severity, 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 is generally in the range of 0.1 to 100 mg/kg of body weight of the recipient (mammal) per day, and particularly typically in the range of 1 to 10 mg/kg of body weight per day. Thus, the actual amount per day for an adult mammal weighing 70 kg is usually between 70 and 700 mg, where this amount may be administered as a single dose per day or usually in a series of partial doses (such as, for example, two, three, four, five, or six) per day, so that the total daily dose is the same. An effective amount of a salt or solvate or a physiologically functional derivative thereof can be determined as a fraction of the effective amount of the compound per se.

[0095] In yet another embodiment, the present invention relates to a method for treating a cancer in an individual in need of such treatment, comprising the step of administering to the individual one or more times a therapeutically effective amount of at least one compound or pharmaceutically acceptable salt thereof disclosed in the present invention.

[0096] In yet another embodiment, the present invention relates to a method for inhibiting the growth and/or metastasis of tumor cells in a subject, comprising the step of administering to the subject one or more times a therapeutically effective amount of at least one compound or pharmaceutically acceptable salt thereof as disclosed in the present invention.

[0097] In yet another embodiment, the present invention relates to a method for inhibiting an activity of the enzyme dihydroorotate oxygenase in a tumor cell, comprising the step of contacting the tumor cell one or more times with a therapeutically effective amount of at least one compound or pharmaceutically acceptable salt as disclosed in the present invention. In this embodiment, the tumor cells are contacted in vivo, ex vivo or in vitro.

[0098] The compounds, pharmaceutically acceptable salts thereof, and pharmaceutical formulations and compositions disclosed in the present invention are useful for treating cancer in an individual in need of such treatment. Concomitantly, tumor cell growth and/or metastasis or dihydroorotate oxygenase enzymatic activity may be inhibited. The compounds and the pharmaceutical composition may be administered one or more times to achieve a therapeutic effect. As is known in the art, the skilled person is able to determine the dose, dosage regimens, and routes of administration depending on the condition to be treated and the individual requiring treatment. Representative examples of a cancer include hematologic 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 a 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 a cancer include a solid cancer such as, but not limited to, lung cancer, breast cancer, triple-negative breast cancer, melanoma, glioblastoma, prostate cancer, colon cancer, pancreatic cancer, bone cancer, head or neck cancer, skin cancer, cutaneous or intraocular malignant endometrial cancer, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, soft tissue sarcoma, cancer of the urethra, cancer of the penis, solid tumors of children, lymphocytic lymphoma of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, central nervous system (CNS) neoplasm, primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, squamous cell cancer, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, including those induced by asbestos, and PTEN-mutant cancers.

[0099] In a further aspect, the present invention relates to a process for the preparation of trisubstituted benzotriazole derivatives of formula (I).

[00100] Dihydroorotate dehydrogenase inhibitors according to formula (I) can be prepared from readily available starting materials using the following general methods and procedures. It will be appreciated that where typical or preferred experimental conditions are given (i.e., reaction temperatures, time, moles of reactants, solvents, etc.), other experimental conditions can also be used unless otherwise indicated. Optimum reaction conditions may vary with the specific reactants or solvents used, but such conditions can be determined by one skilled in the art using routine optimization procedures. Furthermore, using the procedures described in detail, one skilled in the art can prepare additional compounds of the present invention claimed herein. All temperatures are in degrees Celsius (°C) unless otherwise indicated.

[00101] In a further aspect, the compounds of the present invention may also contain unnatural proportions of atomic isotopes in one or more of the atoms constituting such compounds. For example, the present invention also encompasses isotopically labeled variants of the present invention that are identical to those recited herein, but for the fact that one or more atoms of the compound are replaced by an atom having an atomic mass or mass number different from the predominant atomic mass or mass number usually found in nature for the atom. All isotopes of any specific atom or element, as specified, are contemplated within the scope of the compounds of the invention and their uses. Exemplary isotopes that may be incorporated into the compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, and iodine, such as, 2H (“D”), 3H, 11C, 13C, 14C, 13N, 15N, 15O, 17O, 18O, 32P, 33P, 35S, 18F, 36Cl, 123I, and 125I. The isotopically labeled compounds of the present invention can generally be prepared following procedures analogous to those disclosed in the Schemes and/or Examples below in the present invention, substituting an isotopically labeled reagent for a non-isotopically labeled reagent.

[00102] The following abbreviations refer respectively to the definitions below: AcOH (acetic acid), ACN (acetonitrile), ATP (adenoside triphosphate), BSA (bovine serum albumin), CHCl3 (chloroform), CS2CO3 (cesium carbonate), DCM (dichloromethane), DIPEA (diisopropyl ethylamine), DMSO (dimethyl sulfoxide), DMF (N,N-Dimethylformamide), EDCI. HCl (l-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 (minute), MeOH (methanol), MeI (iodide methyl), MgSO4 (magnesium sulfate), NH4Cl (ammonium chloride), NH4(CO3)2 (ammonium carbonate), 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), room temperature at room temperature (25°C-35°C), TEA (triethylamine), TFA (trifluoroacetic acid), THF (tetrahydrofuran), t-BuOK (tert-butoxide of potassium), TMSI (Trimethylsilyl iodide), TLC (thin layer chromatography), H2O- Water; mL-Milli Liter; h - hour; N - Normality; M - Molarity; s - singlet; d - doublet; t - tri singlet; m - multiplet; 1H NMR - proton nuclear magnetic resonance; MS - mass spectroscopy; LC - Liquid chromatography; HPLC - High performance liquid chromatography, - Coupling constant; 1H - proton; MHz - Mega Hertz (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 - degree Celsius, Molecular ion M+ -, % - Percentage; µ - Micron; and d - Delta; anh. - anhydrous; pH - potential of Hydrogen;

[00103] Another embodiment of the present invention provides useful methods for preparing compounds of formula (I) are set forth in the Examples below and generalized in Scheme-I. One 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. Wherein all symbols/variables are as defined above unless otherwise indicated. The process is represented by Scheme-I:

[00104] Compounds of the invention can be prepared using the synthetic transformations illustrated in Scheme I. Starting materials are commercially available, can be prepared by the procedures described herein, by literature procedures, or by procedures that would be well known to one skilled in the art of organic chemistry. The starting material 5-substituted methyl 2,3-diaminobenzoate is prepared by the procedures described in WO 2010115736A2.

[00105] Step-a: Compound-i is reacted with sodium nitrite in acidic medium using General Procedure-A to obtain compound-ii.

[00106] Step-b: Compound-ii is further subjected to N-alkylation using methyl iodide under basic conditions as described in General Procedure-B to obtain compounds of formula-iii.

[00107] Step c: The compounds of formula-iii are reacted with diborane bispinacolate in basic medium in the presence of suitable palladium catalyst using General Procedure-C to obtain the compounds of formula-iv.

[00108] Step d: The compounds of formula-iv treated with substituted aryl halide in the presence of suitable palladium catalyst using conditions such as described in General Procedure-D to provide the compounds of formula-v.

[00109] Step-e: Alternatively, compounds of formula-v may be prepared from compounds of formula-iii using appropriate boronic acids under suitable conditions such as described in General Procedure-D.

[00110] Step-f: The resultant compounds of formula-v undergo ester hydrolysis under basic conditions as described in General Procedure-F to provide compounds of formula (I) (wherein R3 = OH).

[00111] Step g: The carboxylic acids of formula (I) were treated with ammonium chloride using the conditions described in general procedure-G to obtain the respective compounds of formula (I) (where R3 = NH2).

[00112] If the above 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), suitable methods of preparation known to a person skilled in the art should be used. In general, the synthetic routes for any individual compound of formula (I) will depend on the specific substituents of each molecule and the ready availability of the required intermediates; again these factors being appreciated by those of ordinary skill in the art.

[00113] The compounds of this invention may be isolated in association with solvent molecules by crystallization from the evaporation of a suitable solvent. Pharmaceutically acceptable acid addition salts of compounds of formula (I) that contain a basic center may be prepared in a conventional manner. For example, a solution of the free base may be treated with a suitable acid, either neat or in a suitable solution, and the resulting salt isolated by filtration or by vacuum evaporation of the reaction solvent. Pharmaceutically acceptable base addition salts may be obtained analogously by treating a solution of the compound of formula (I) with a suitable base. Both types of salts may be formed or interconverted using ion exchange resin techniques.

[00114] Although the invention is illustrated by some of the following examples, it should not be construed as being so limited; rather, the invention covers the generic field as previously disclosed. Various modifications and embodiments may be made without departing from the spirit and scope.

EXAMPLES General:

[00115] The MS data provided in the examples described below were obtained as follows:

[00116] Mass spectrum: LC/MS Waters ZMD (ESI) or Waters Acquity SQD (ESI).

[00117] The NMR data selected in the examples below are as follows: 1H-NMR: Bruker DPX-300MHz or Bruker DPX 400 MHz.

[00118] The HPLC data used in the examples below are used as follows.

[00119] Condition A: Xbridge™ Cg 50 mm x 4.6 mm column waters at a flow rate of 2 mL/min; 8 min gradient from 0.1% TFA in H2O to 0.07% TFA in CH3CN.

[00120] Condition B: C18 BDS (4.6X250) mm, SC\244 at a flow rate of 0.7 mL/min; 10 min gradient from 0.1% TFA in H2O to CH3CN.

[00121] Preparative conditions for HPLC: Column - Zorbax Eclipse XDB C18 PrepHT (150 x 21.2 mm, 5 µ); Mobile phase: (A) 0.01% TFA or 0.1% TFA; (B) ACN or ACN:MeOH (1:1); Flow: 20 ml/min.

[00122] HPLC preparation purifications were performed with a Waters Fractionlynx mass-directed autopurification equipped with a 5 µm OBD Sunfire Prep C18 19x100 mm column, unless otherwise noted. All HPLC purifications were performed with an ACN/H2O or ACN/H2O/HCOOH (0.1%) gradient.

[00123] The invention compounds were named according to the standards used in the ACD/Batch Name program of “Advanced Chemistry Development Inc., ACD/Labs (version 7.00)”. Product version: 7.10 build: September 15, 2003.

[00124] The procedure for the compounds of formula (I) is detailed in the present invention below the general procedures, including a general description of various intermediates involved in the process of manufacturing compounds according to the present invention. General Procedure - A: Preparation of substituted [1,2,3]benzotriazoles

[00125] A flask containing 6-substituted or substituted diaminoester (1-3 equiv) in acetic acid is stirred for 10-20 min, preferably 10 min, followed by the addition of (sodium nitrogen, potassium nitrite, preferably acid nitrite) (2.5-3.5, preferably 2.5 equiv) in water. The reaction mixture was stirred for 1-2 h, preferably 1 h, at room temperature. The separated solid is collected by filtration and dried under vacuum to obtain the target products. Illustrative Example of General Procedure-A: Preparation # Al: Synthesis of methyl 6-bromo-1H-benzo[d][1,2,3]triazole-4-carboxylate:

[00126] A solution of methyl 2,3-diamino-5-bromobenzoate (1.0 g, 4.08 mmol) (Ref: WO2010/115736 A2) in acetic acid (15 mL) was stirred for 10 min at RT. Sodium nitrite was added (0.309 g, 4.48 mmol) in water (2 mL) and the reaction mixture stirred for about 30 min at room temperature. The precipitated solid was filtered, washed with water and dried under vacuum to obtain the desired product (0.8 g, 77%); 1H NMR (400 MHz, DMSO-d6): d 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

[00127] To a stirred solution derived from substituted benzotriazole carboxylate (1 equiv) in an organic solvent (such as DMF, THF, dioxane, preferably DMF) is added a suitable base (such as K2CO3, CS2CO3, NaH etc., preferably K2CO3 2 to 5 equivalents, preferably 2 equiv) followed by alkyl halogen (2 to 5 equiv, preferably 3 equiv). A reaction mixture stirred at RT for about 1 to 10 h (preferably 3 h). A reaction mixture is poured into ice-cold water and the separated solid is collected by filtration and dried under vacuum. The regioisomers were separated by column chromatography to obtain the desired products. Illustrative Example of General Procedure-B: Preparation # Bl: 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

[00128] To a stirred solution of methyl 6-bromo-1H-benzo[d][1,2,3]triazole-4-carboxylate (4.5 g, 17.5 mmol, preparation # 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, dried under vacuum. The obtained crude compound was purified by column chromatography on silica gel (100-200 mesh) using ethyl acetate in hexane 10% to obtain Isomer I (B.1.a) (1.9 g); 1H NMR (400 MHz, CDCl3) d 8.40 (s, 1H), 8.22 (s, 1H), 4.57 (s, 3H), 4.01 (s, 3H) and LC-MS m/z: 272 (M+2)+; 15-20% ethyl acetate in hexane to give isomer-II (B.1.b) (1.4 g); 1H NMR (400 MHz, CDCl3) d 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)+; 20-25% ethyl acetate in hexane to give isomer-III (B.1.c) (1.0 g); 1H NMR (400 MHz, DMSO-d6): d 8.67 (s, 1H), 8.13 (s, 1H), 4.45 (s, 3H), 3.96 (s, 3H) and LC-MS m/z: 272.0 (M+2)+. General Procedure - C: Preparation of boronic ester

[00129] A mixture of aryl-halo derivative (1.0 to 3.0 equiv, preferably 1.0 equiv), suitable inorganic base (such as KOAC or Na2CO3 or K2CO3 or Cs2CO3, preferably KOAC), diborane bispinacolate (1.0 to 3.0 equiv, preferably 1.1 equiv) in dioxane is degassed with nitrogen for about 10 to 15 min and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (0.001 to 0.010 equiv, preferably 0.05 equiv) is added. The reaction mixture is stirred at reflux temperature under nitrogen for about 3 to 12 h (preferably about 6 h). The reaction mixture is cooled to RT and evaporated to dryness under reduced pressure. The residue obtained is redissolved in EtOAc, washed successively with water and brine. The organic solution is dried over Na2SO4, filtered and concentrated under reduced pressure. The product is purified by crystallization or trituration from an appropriate solvent(s) or by preparative HPLC or flash chromatography. Illustrative Example of General Procedure-C: Preparation # C.1: Synthesis of 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[d][1,2,3]triazole-7-methylcarboxylate

[00130] A mixture of methyl 5-bromo-1-methyl-1H-benzo[d][1,2,3]triazole-7-carboxylate (1.0 g, 3.7 mmol, preparation # B.1.a), potassium acetate (0.627 g, 5.92 mmol), diboron bispinacolate (0.93 g, 3.7 mmol) in dioxane (60 mL) was degassed with nitrogen for about 15 min and [1,1-bis(diphenylphosphino)-ferrocene]dichloropalladium(II) (0.151 g, 0.018 mmol) added. The reaction mixture was stirred at reflux temperature for 6 h under nitrogen. The reaction mixture was cooled to RT and evaporated to room temperature under reduced pressure. The removed residue was redissolved in EtOAc, washed successively with water and brine and concentrated. The obtained crude compound was purified by column chromatography on silica gel (60-120 mesh) using 30% ethyl acetate in hexane to obtain the desired product (0.9 g, 77%); 1H NMR (400 MHz, DMSO-d6): d 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)+.

[00131] Other compounds synthesized using general procedure C are monitored in Table C.1. General Procedure-D: Suzuki Reaction

[00132] A mixture of acetonitrile and water (8:2) is degassed with nitrogen for about 10 to 15 minutes and then a suitable base (such as Na2CO3 or K2CO3 or Cs2CO3, preferably Na2CO3) is added, followed by arylbromo derivative (1.0 to 3.0 equiv, preferably 1.0 equiv) and appropriate boronic acid (1.0 to 3.0 equiv, preferably 1.5 equiv). The reaction mixture is again degassed for 15 min and finally [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (0.001 to 0.010 equiv, preferably 0.05 equiv) is added. A reaction mixture is stirred at reflux temperature under nitrogen for about 3 to 12 h (preferably about 4 h). The reaction mixture is cooled to RT and evaporated to dryness under reduced pressure. The residue is redissolved in EtOAc, washed successively with water and brine. The organic solution is dried over Na2SO4, filtered, and concentrated under reduced pressure. The product is purified by crystallization or trituration from an appropriate solvent(s), or by preparative HPLC or flash chromatography. Illustrative Example of General Procedure-D: Preparation # D1: Synthesis of methyl 1-methyl-5-(2'-methyl-[1,1'-biphenyl]-4-yl)-1H-benzo[d][1,2,3]triazole-7-carboxylate

[00133] A mixture of acetonitrile (80 mL) and water (15 mL) was degassed with nitrogen for 10 min. 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,r-biphenyl]-4-yl)-1,3,2-dioxaborolane (3.81 g, 12.0 mmol) (C.1.5). The reaction mixture was again degassed for 15 min. Finally [1,1-bis(diphenylphosphino)-ferrocene]dichloropalladium(II) (0.526 g, 0.64 mmol) was added. The reaction mixture was stirred at reflux temperature for 5 h under nitrogen. The reaction mixture was cooled to RT and evaporated to dryness under reduced pressure. The obtained residue was redissolved in EtOAc, washed successively with water and brine, and concentrated. The obtained crude compound was purified by column chromatography on silica gel (60-120 mesh) using 30% ethyl acetate in hexane to obtain the desired product (3.6 g, 77%); 1H NMR (400 MHz, CDC13): d 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)+.

[00134] Other compounds synthesized using General Procedure D are described in Table D.1. General Procedure-E: Reductive Animation

[00135] A mixture of aldehyde and appropriate amine in organic solvent (such as 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 in small portions, followed by a catalytic amount of acetic acid. The resulting reaction mixture is stirred at room temperature for 2-4 hours. The progress of the reaction is monitored by TLC, and the reaction mixture is quenched with an aq. sodium bicarbonate solution. It is further extracted with ethyl acetate, the combined organic layers are dried over sodium sulfate, and concentrated under vacuum to obtain the target compound. Optionally, the target compound can be purified by crystallization or trituration from an appropriate solvent(s), or by preparative HPLC or flash chromatography. Illustrative Example of General Procedure-E: Preparation #E.1: Synthesis of methyl 1-methyl-5-(2'-(morpholinomethyl)-[1,1'-biphenyl]-4-yl-1H-benzo[d][1,2,3]triazole-7-carboxylate

[00136] 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 for 30 min at room temperature. The reaction mixture was cooled to 0 °C, sodium triacetoxyborohydride (0.342 g, 1.6 mmol) followed by acetic acid (0.2 mL) was added. The reaction mixture was stirred for 2 h at room temperature. The reaction mixture was quenched with aq. sodium bicarbonate solution (50 mL). It was extracted with ethyl acetate (3 x 50 mL), the combined organic layers were dried over sodium sulfate and concentrated under reduced pressure. The crude product obtained was taken to the following without purification (0.200 g); 1H NMR (400 MHz, DMSO-d6): d 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

[00137] To a flask containing an appropriate alkyl ester in an aqueous organic solvent (such as THF or methanol, 1,4-dioxane, preferably 1,4-dioxane), 1.5 equiv. of aqueous sodium hydroxide solution is added and the reaction mixture is refluxed for 1-8 h. (preferably 4 h). The 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 obtain the target carboxylic acid derivative. Optionally, the target compound can be purified by crystallization or trituration from an appropriate solvent(s), 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)

[00138] 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 aq. NaOH (15 mL). The reaction mixture was refluxed for 4 h. 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~2). The separated solid is collected by filtration and dried under vacuum to obtain the title compound as an off-white solid (1.1 g, 95%); 1H NMR (400 MHz, DMSO-6): d 13.35 (bs, 1H), 8.52 (s, 1H), 8.38 (s, 1H), 7.89-7.87 (d, 7=8.0 Hz, 2H), 7.51-7.49 (d, 7=8.4 Hz, 2H), 7,327.25 (m, 4H), 4.58 (s, 3H) 2.30 (s, 3H) and LC-MS m/z = 344.1 (M+H)+.

General Procedure - G: Amide Formation

[00139] To a flask containing appropriate carboxylic acid derivative (1.0 equiv) in an organic solvent (such as DMF, THF or CH2Cl2) is added EDCI.HCl (1.5 equiv), HOBT (1.5 equiv) and N-ethyl-N-isopropylpropane-2-amine (3 equiv). After stirring for about 10 minutes at approximately 25°C, the appropriate amine (1.5 equiv) is added and the reaction is stirred for an additional 8-12 h (preferably 12 h). The solid separated by addition of water is collected by filtration and dried under vacuum to obtain the amide derivative. Optionally, the obtained compound can be purified by crystallization or trituration from an appropriate solvent or 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)

[00140] To a flask containing a 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), 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) were added. The mixture was stirred at about 25°C for approximately 10 min, and ammonium chloride (0.070 g, 1.31 mmol) was added. The reaction was then stirred for about 12 additional hours and quenched with water (50 mL). The separated solid was collected by filtration and dried under vacuum to obtain the desired compound as an off-white solid (0.08 g, 53%); 1H NMR (400 MHz, DMSO-d6): d 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)+.

[00141] The intermediates below were prepared by a procedure similar to that described in General Procedure-C, with appropriate variations in reagents, amounts of reagents, and reaction conditions. The physicochemical characteristics of the compounds are summarized herein below in Table C.1. Table C.1.

[00142] The intermediates below were prepared by a procedure similar to a procedure described in General Procedure D with physicochemical variations of the compounds are summarized below in Table D.1. in the present invention. Table D1.

[00143] The compounds below were prepared by a procedure similar to a procedure described in General Procedure D with appropriate variations in reagents, reagent amounts, and reaction conditions. The physicochemical characteristics of the compounds are summarized in the present invention in the table below.

PHARMACOLOGICAL ACTIVITY Measurement of DHODH inhibitory enzyme activity (in vitro assays)

[00144] The DHODH activity assay is a coupled enzyme assay in which the oxidation of DHO and subsequent reduction of ubiquinone are stoichiometrically equivalent to the reduction of DCIP (2,6-dichlorophenol). The reduction of DCIP is accompanied by a loss of absorbance at 610nm.

[00145] Preparation of solutions/reagents:

[00146] Buffer preparation: 50 mM tris HCl, 150 mM KCl and pH 8.0, 0.8% triton.

[00147] Stock solution of 20 mM L-dihydroorotic acid in buffer.

[00148] Stock solution of 20 mM 2,6-dichloroindophenol sodium salt hydrate in buffer.

[00149] 20 mM decylubiquinone stock solution in buffer.

[00150] DMSO used as vehicle. Procedure

[00151] 5 µL of dimethyl sulfoxide or a compound of formula (I) in DMSO solution was added to the wells of a 96-well plate. Compounds of formula (I) were measured at 10 µM.

[00152] Protein was added along with buffer so that the total volume including DMSO was 87 µL. The compound and protein were incubated for half an hour at room temperature after mixing. 5 µL of 20 mM L-dihydroorotic acid solution, 5 µL of 2 mM decylubiquinone solution, and 3 µL of 2 mM 2,6-dichloroindophenol sodium salt hydrate solution were added to the above solution (total assay volume 100 µL). The mixture was shaken for 2 min, and absorbance was recorded every 10 min at 610 nanometers. The percent inhibition is calculated as follows:

[00153] The reaction containing the compound has compound, buffer, enzyme, and substrates.

[00154] Positive control contains DMSO, buffer, substrates.

[00155] No enzymatic reaction contains DMSO, buffer and substrates.

Determination of IC50

[00156] A 2 mM DMSO stock solution of the selected trisubstituted benzoimidazole and benzotriazole derivatives of formula (I) of the present invention to be examined was prepared. Subsequent 1/3 dilutions were made.

[00157] 5 µg of each compound of formula (I) loading was used for each 100 µg assay. Therefore, 5 µL of the mM loading provided 100 µL of a 100 µM solution of the compound of formula (I) when consisting of buffer, protein and substrate. See also: Ulrich et al. (2001) Eur. J. Biochem.268, 1861-1868.

[00158] The IC50 values of the selected compounds of the present invention have been provided in the table below. The compounds exhibiting IC50 values ≥0.1 µM have been grouped as 'a', the compounds exhibiting IC50 value in the range of 0.101 µM to 1.0 µM have been grouped as 'b' and the compounds exhibiting IC50 value >1.0 µM have been grouped as 'c'. Table: DHODH inhibition activity of the selected compounds.

CELL-BASED ACTIVITY Branch proliferation assay (IN VITRO TESTS)

[00159] The cell proliferation assay is a sensitive method for quantifying viable cells in a cytotoxicity or proliferation assay. 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 mitochondrial dehydrogenases in living cells. Mitochondrial dehydrogenases in viable cells cleave the tetrazolium ring of XTT, producing orange formazan crystals that are soluble in aqueous solutions. The XTT solution is potentiated by the addition of an 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, indicating the degree of cytotoxicity caused by the test material.

Preparation of solutions/reagents Medium preparation

[00160] Dissolve 17.7 g of IMDM powder (Iscove's Modified Dulbecco's Medium), 1.5 g of sodium bicarbonate pH 7.27.4 in 1 L of MiliQ water, add 1% Penicillin/Streptomycin and 10% FBS.

[00161] Dissolve 10.6 g of Ham's F12 powder, 1.5 g of sodium bicarbonate pH 7.2-7.4 in 1 L of MiliQ water, add 1% Penicillin/Streptomycin.

[00162] DMSO used as a vehicle.

[00163] IX PBS (phosphate buffered saline): dissolve 5 PBS tablets (Sigma: Cat#P4417) in 1L of MiliQ water.

Procedure (IC50 determination)

[00164] Ramos cells were resuspended at a density of 1 x 105 cells/ml in complete IMDM medium. 95 µL of this cell suspension was added to the 96-well plate to seed ~10,000 cells per well. The plates were incubated at 37°C under a humidified atmosphere of 5% CO2 for ~1 hour prior to compound addition.

[00165] Test compounds (Refer to Table 1) were dissolved in 100% DMSO to generate a 2/6/10/20 mM stock solution. A 200X concentration of the required final concentrations was prepared in DMSO. 10 µL of each concentration (200X) was then diluted in 90 µL of serum-free Ham's F12 medium to obtain a 20X intermediate concentration in the medium. The DMSO concentration at this step is 10% (intermediate dilution). 5 µL of each intermediate dilution was then added in triplicates to the previously seeded 96-well plate. 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 media blank for data analysis. 200 µL of 1X PBS was added to all corner wells of the assay plate. The plates were then incubated for 72 hours in a 5% CO2 incubator at 37°C.

[00166] On the day of completion, 100 µL of XTT solution (1 mg/ml XTT supplemented with 25 µM PMS in Ham's F12 medium) 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 plate using the VICTOR X5 multi-label plate reader at 450 nm wavelength. IC50 values were determined as concentrations that reduced cell viability by 50% and the curve was plotted with GraphPad Prism 6.0.

[00167] The inhibition percentage is calculated as follows:

[00168] Percent inhibition (%) was calculated by normalizing DMSO control values to 100% using the formula: % Inhibition = 100% - (Abs450test compound - Abs450positive control - blank*100

[00169] The test compound contains cells, test compound, IMDM medium, and 0.5% DMSO

[00170] The positive control contains cells, IMDM medium and 0.5% DMSO

[00171] Blank contains IMDM medium

Inhibition of in vitro growth of multiple human cancer cell lines by Compound 1

[00172] A tumor cell line panel screen was performed with the aim of identifying tumor cell subsets that were particularly sensitive to DHODH inhibition with Compound 1. Compound 1 is represented by the following structural formula:

[00173] These cell lines were treated with Compound 1 for a total of 72 hours.

[00174] Assessment of tumor growth rates following the 72-hour treatment, as shown in FIG. 1 and Table 1, revealed that a distinct subset of cell lines (represented by gray dots in FIG. 1) were sensitive to Compound 1. The majority of cell lines exhibiting high sensitivity to Compound 1 are of hematopoietic origin, although some solid tumors also exhibited high sensitivity (Table 1). For the purposes of generating FIG. 1, sensitive cell lines were defined as exhibiting >75% maximal growth inhibition and a GI50 value of <1.5 µM. Table 1 is a list of some of the cell lines sensitive to Compound 1, along with the GI50 values and maximal growth response. A maximal inhibition of 100 represents complete growth inhibition; a maximal inhibition value >100 represents cell death.

[00175] A follow-up screen was performed on an expanded panel of hematologic lineage cell lines in a 4-day growth assay. Growth was assessed by Cell-Titer Glo measurements on day 0 and day 4. As shown in FIG. 2, 25% of the hematologic lines screened (20/80) exhibited sensitivity to Compound 1, of which diffuse large B-cell lymphoma (DLBCL) lines were particularly sensitive (8/11 or 73%). A subset of the hematologic lines that were intermediately 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 were subjected to extensive growth assays to assess whether increasing treatment time modulated their sensitivity profile. Specifically, these hematologic lines were pretreated with Compound 1 for three days at the indicated concentrations and then replated for a standard 4-day growth assay in fresh media/drug. The vast majority of the retested hematologic lines exhibited strong sensitivity to Compound 1 after 7 days of Compound 1 treatment (Table 2). Table 1. GI50 and maximal growth inhibition of various cell lines treated with Compound 1 Table 2. 4- and 7-day sensitivity of various cell lines - insensitive 2 * -intermediate 3 ** - sensitive Inhibition of cancer cell growth by Compound 1 is attributed to inhibition of DHODH

[00176] DHODH catalyzes the fourth step in de novo pyrimidine biosynthesis by oxidizing dihydroorotate to orotate in the inner mitochondrial membrane. Orotate then combines with phosphoribosyl pyrophosphate (PRPP) to form orotidine-5'-monophosphate (OMP). Uridine monophosphate (UMP) is finally produced from OMP in the cytosol, where it is used to prepare pyrimidines for RNA/DNA biosynthesis, as well as for other important biosynthetic functions such as protein/lipid glycosylation and production of phospholipids for membrane biosynthesis.

[00177] To confirm that the effects of Compound 1 on impaired cell growth/viability were due to specific inhibition of DHODH, cell growth assays were performed with varying amounts of uridine supplemented to the medium. Supplementing medium with near-physiological uridine concentrations (5 µM) partially rescued the effects of Compound 1, while supraphysiological concentrations (25 µM and 100 µM) completely rescued the growth effects of up to 10 µM Compound 1. These results indicate that the growth effects of Compound 1 are on target (FIG. 3). Comparison of the sensitivity profile of Compound 1 with the sensitivity profiles of cytarabine and doxorubicin

[00178] The sensitivity profile of Compound 1 against a subset of hematologic lines was compared to the sensitivity profiles of other agents that are used as standard of care (SOC) in hematologic malignancies. Consistent with its different mechanism of action, Compound 1 exhibited a sensitivity profile (FIG. 4A) distinct from the sensitivity profiles of cytarabine (FIG. 4B) and doxorubicin (FIG. 4C). Compound 1 effectively inhibits DHODH IN VIVO and blocks tumor growth in the AML xenograft model

[00179] In vivo efficacy studies were performed with Compound 1 to evaluate the translation of in vitro to in vivo effects on DHODH blockade and tumor cell growth inhibition. 1 x 106 MOLM-13 cells were implanted subcutaneously into CB 17 SCID mice. Mice (n = 15/group) were treated with vehicle or Compound 1 at 100 mg/kg twice daily PO once tumors reached an average of ~150 mm3. At the end of the study, tissues were collected at the indicated time points after the last dose for pharmacokinetic (PK) and pathway biomarker analyses.

[00180] Compound 1 administered at 100 mg/kg twice daily was well tolerated and resulted in near complete tumor growth inhibition (TGI) of the MOLM-13 acute myeloid leukemia (AML) xenograft model (FIG. 5A). The pharmacokinetic profile measured in plasma and tumor shows a decline in drug concentrations over 12 hours, supporting the twice-daily dosing regimen (FIG. 5B). Clear evidence of target engagement was observed by the dramatic increase in tumor levels of dihydroorotate (DHO), the DHODH substrate (FIG. 5C). Baseline DHO levels were below the quantifiable limit (BQL <120ng/g). Tumor uridine pools were concomitantly reduced by ~60%, depending on the time point evaluated (FIG. 5D). Compound 1 effectively inhibits DHODH IN VIVO and blocks tumor growth in patient-derived AML and DLBCL xenograft models

[00181] A screen was performed using a small number of mice/group to evaluate the efficacy of Compound 1 in patient-derived xenograft models of AML and DLBCL. Tumor-bearing mice (n = 3/group) were treated with vehicle or Compound 1 at 100 mg/kg twice daily PO.

[00182] As shown in FIGS. 6A-6E and 7A-7B, antitumor activity of Compound 1 was observed in all models tested, with >60% TGI in two of five AML models (i.e., AML_2 and AML_5) and one of two DLBCL models (DLBCL_1). The DLBCL_1 model is characterized as a triple translocation DLBCL model. IN VITRO Growth Inhibition of Dual Translocation Diffuse Large B-Cell Lymphoma Human Cancer Cell Lines by Compound 1

[00183] Three patient-derived DBLCL lymphoma cell lines classified as double-translocation DLBCL, namely OCILY18, SC-1, and CARNAVAL, were found to be highly sensitive to inhibition by Compound 1 in a 96-hour growth assay (FIG. 8). Compound 1 effectively blocks tumor growth in the patient-derived DLBCL xenograft model

[00184] A strong block in tumor growth in vivo was observed with Compound 1 in the OCILY-19 diffuse large B-cell lymphoma (DLBCL) xenograft model. 7 x 106 OCILY-19 cells were implanted subcutaneously into CB 17 SCID mice. Mice (n = 15-18/group) were treated with vehicle or Compound 1 at the indicated dose/frequency when tumors reached an average of ~150 mm3. Tissues were collected at the indicated time points after the last dose for PK and biomarker analyses.

[00185] The degree of pathway modulation and tumor growth inhibition was demonstrated to be dose- and schedule-dependent (FIG. 9A). The 100 mg/kg twice-daily dosing regimen resulted in superior efficacy compared to the 10 and 30 mg/kg twice-daily dose arms, and this correlated with a greater increase in tumor DHO (FIG. 9C) as well as a decrease in the total tumor uridine pool (FIG. 9D). The 200 mg/kg QD regimen was less effective than the 10 mg/kg twice-daily dosing regimen due to the short half-life of Compound 1 in mice, resulting in lower trough drug concentrations with QD dosing (see FIG. 9B). IN VITRO Growth Inhibition of Multiple Human Triple-Negative Breast Cancer Cell Lines by Compound 1

[00186] A panel of triple negative cancer (TNBC) cell lines was screened to evaluate whether a subset of TNBC could potentially benefit from treatment with Compound 1 as a single agent. Treatment with Compound 1 for 96 hours strongly impaired cell viability/growth in DU4475 to a comparable degree as observed in the cell lines of hematologic origin (see FIG. 10), while four other TNBC lines (HCC1143, HCC38, BTS49, and HCC1806) were insensitive under the conditions tested.

Claims (13)

1. Use of a compound represented by the following structural formula: or a pharmaceutically acceptable salt thereof, characterized in that it is for preparing a medicament for the treatment of a cancer selected from multiple myeloma, B prolymphocytic leukemia, non-Hodgkin's lymphoma, anaplastic large cell lymphoma, mantle cell lymphoma, triple negative breast cancer, melanoma, prostate cancer, esophageal cancer, chronic lymphocytic leukemia, Hodgkin's disease and follicular lymphoma. 2. Use according to claim 1, characterized in that the cancer is multiple myeloma. 3. Use according to claim 1, characterized in that the cancer is prolymphocytic leukemia B. 4. Use according to claim 1, characterized in that the cancer is non-Hodgkin's lymphoma. 5. Use according to claim 1, characterized in that the cancer is anaplastic large cell lymphoma. 6. Use according to claim 1, characterized in that the cancer is mantle cell lymphoma. 7. Use according to claim 1, characterized in that the cancer is triple negative breast cancer. 8. Use according to claim 1, characterized in that the cancer is melanoma. 9. Use according to claim 1, characterized in that the cancer is prostate cancer. 10. Use according to claim 1, characterized in that the cancer is esophageal cancer. 11. Use according to claim 1, characterized in that the cancer is chronic lymphocytic leukemia. 12. Use according to claim 1, characterized in that the cancer is Hodgkin's disease. 13. Use according to claim 1, characterized in that the cancer is follicular lymphoma.