Bithiazole derivatives as inhibitors of lysyl oxidase.

Abstract

Described herein are novel bithiazole compounds that block the activity of LOX family members, have favorable IC50 values, non-cytotoxicity below 10 nM, induce cellular sensitization to the potent activity of doxorubicin in recombinant LOX / LOXL2 activity, and have a drug-like chemical structure that does not have the PAINS flag, and methods of treatment using the compounds for cancer, organ fibrosis, neurodegeneration, and cardiovascular diseases.

Description

[Technical field]

[0001] Statement regarding federally sponsored research

[0001] This disclosure was made with government support under National Institutes of Health Grant No. P20GM109091. The Government has certain rights in this disclosure.

[0002] Technical Field

[0002] The subject matter of the invention disclosed herein is generally directed to pharma- ceutically acceptable compositions and pharma- ceutically acceptable salts thereof, as well as treatment regimens for inhibiting the activity of LOX or the LOX family; the disclosed compounds are useful for treating a variety of diseases, disorders, or conditions associated with LOX activity; and the compounds are also useful in the study of the LOX enzyme in biological and pathological phenomena. [Background technology]

[0003]

[0003] The lysyl oxidase (LOX) family is a copper-dependent amine oxidase. There are five distinct members of the LOX family proteins (LOX, LOXL1, LOXL2, LOXL3 and LOXL4) that share a high degree of homology in the catalytically active carboxy terminus. See Barker, HE, Cox, TR & Erler, JT The rationale for targeting the LOX family in cancer. Nat Rev Cancer 12, 540-552, doi:10.1038 / nrc3319 (2012). Importantly, LOX and LOXL1 are synthesized as propeptides and are cleaved by bone morphogenetic protein 1 (BMP1) metalloprotease to yield functional enzymes. See Trackman, PC, Bedell-Hogan, D., Tang, J. & Kagan, H. Post-translational glycosylation and proteolytic processing of a lysyl oxidase precursor. Journal Of Biological Chemistry 267, 8666-8671 (1992). The LOX family of proteins mediates the conversion of lysine residues in collagen I and elastin precursors to highly reactive aldehydes; thus resulting in cross-linking and stabilization of ECM proteins, which regulate cell adhesion, motility, and invasion. See Kagan, HM & Li, W. Lysyl oxidase: properties, specificity, and biological roles inside and outside of the cell. Journal of Cellular Biochemistry 88, 660-672, doi:10.1002 / jcb.10413 (2003). LOX family members play important roles during embryogenesis and organ development. See Maki, JM et al. Lysyl oxidase is essential for normal development and function of the respiratory system and for the integrity of elastic and collagen fibers in various tissues.The American Journal of Pathology 167, 927-936(2005); Brody, JS, Kagan, H. & Manalo, A. Lung lysyl oxidase activity: relation to lung growth. American Review of Respiratory Disease 120, 1289-1295(1979); and Wei, S., Gao, L., Wu, C., Qin, F. & Yuan, J. Role of the lysyl oxidase family in organ development. Experimental and Therapeutic Medicine 20, 163-172(2020). Although all LOX family members are widely distributed in organs, all of them are expressed in the heart and colon. Yang, N., Cao, D.-F., Yin, X.-X., Zhou, H.-H. & Mao, X.-Y. Lysyl oxidases: emerging biomarkers and therapeutic targets for various diseases. Biomedicine & Pharmacotherapy 131, 110791 (2020), Martins, RP, Leach, RE & Krawetz, SA Whole-body gene expression by data mining.Genomics 72,34-42(2001), Jourdan-Le Saux, C., Tomsche, A., Ujfalusi, A., Jia, L. & Csiszar, K. Central nervous system, uterus, heart, and leukocyte expression of the LOXL3 gene, encoding a novel lysyl oxidase-like protein. Genomics 74, 211-218 (2001), Asuncion, L. et al. A novel human lysyl oxidase-like gene (LOXL4) on chromosome 10q24 has an altered scavenger receptor cysteine-rich domain.Matrix Biology 20, 487-491(2001), Kim,Y., Boyd,CD&Csiszar,KA New Gene with Sequence and Structural Similarity to the Gene Encoding Human Lysyl Oxidase(*).Journal of Biological Chemistry 270,7176-7182(1995), and Maki,JM, Tikkanen,H.&Kivirikko,KI Cloning and characterization of a fifth human lysyl oxidase isoenzyme:the third member of the lysyl oxidase-related subfamily with four scavenger receptor cysteine-rich domains, Matrix biology 20,493-496(2001). Importantly, all members have been linked to cancer so far, especially those with metastasis and, more recently, chemotherapy resistance; LOX (the first identified member) and LOXL2 are the most intensively studied members of the family. See Xiao, Q. & Ge, G. Lysyl oxidase, extracellular matrix remodeling and cancer metastasis. Cancer Microenviron 5, 261-273, doi:10.1007 / s12307-012-0105-z (2012).

[0004]

[0004] The inventors report that hypoxia-induced LOX expression is a driver of TNBC chemoresistance. See Saatci, O. et al., Targeting lysyl oxidase (LOX) overcomes chemotherapy resistance in triple negative breast cancer. Nat Commun 11, 2416, doi:10.1038 / s41467-020-16199-4 (2020). The inventors demonstrate that LOX not only strengthens ECM cross-linking and generates a barrier to chemotherapy, but also stimulates cell proliferation and therapy resistance via activation of pro-survival integrin signaling in TNBC. See ibid. Inhibition of LOX activity via β-aminopropionitrile (BAPN, a LOX family inhibitor) or shRNA-mediated knockdown of LOX reduces collagen cross-linking, causes doxorubicin accumulation in TNBC cells, and reduces FAK / Src signaling, resulting in chemotherapy sensitization both in vitro and in vivo. In gastric cancer, LOX expression has been shown to be increased and correlated with the depth of invasion and lymph node metastasis, while siRNA-mediated inhibition of LOX suppressed hypoxia-induced EMT in gastric cancer cells. See Kasashima, H. et al., Lysyl oxidase-like 2 (LOXL2) from stromal fibroblasts stimulates the progression of gastric cancer. Cancer Letters 354, 438-446 (2014). High LOX expression is also associated with EMT markers and predicts early recurrence and poor survival in HCC patients. See Umezaki, N. et al., Lysyl oxidase induces epithelial-mesenchymal transition and predicts intrahepatic metastasis of hepatocellular carcinoma. Cancer science 110, 2033-2043 (2019). Another study showed that LOX expression is required for mutant p53-driven invasion in pancreatic cancer, and the combination of gemcitabine with LOX inhibition resulted in tumor necrosis. See Miller, BW et al.Targeting the LOX / hypoxia axis reverses many of the hallmarks of lethal pancreatic cancer: inhibition of LOX blocks metastasis and promotes drug efficacy. EMBO Molecular Medicine 7, 1063-1076 (2015). Furthermore, LOX mRNA is increased in human clear cell renal cell carcinoma (ccRC), and ectopic expression of LOX leads to migration and collagen stiffness. See Di Stefano, V. et al., Major action of endogenous lysyl oxidase in clear cell renal cell carcinoma progression and collagen stiffness revealed by primary cell cultures. The American Journal of Pathology 186, 2473-2485 (2016) and Takahashi, M. et al., Gene expression profiling of clear cell renal cell carcinoma: gene identification and prognostic classification. Proceedings of the National Academy of Sciences 98, 9754-9759 (2001).

[0005]

[0005] Of the other members of the LOX family, LOXL1 is the least studied member in the context of cancer. Several studies have demonstrated its tumorigenic potency in different cancers. Increased expression of LOXL1 was observed in primary lung cancer tissues, and overexpression of LOXL1 in mice results in a greater number of lung metastatic nodules. See Lee, G.-H. et al. Lysyl oxidase-like-1 promotes lung metastasis when accompanied by lactate accumulation and monocarboxylate transporter expression. Oncology Letters 2, 831-838 (2011). LOXL1 mRNA was shown to be increased in patients with pancreatic cancer compared to normal pancreatic tissue. See LeCalve, B. et al. Lysyl oxidase family activity promotes chemotherapy resistance in pancreatic ductal adenocarcinoma by limiting anticancer drug distribution within the tumor. Oncotarget 7, 32100 (2016).

[0006]

[0006] The role of LOXL2 has been widely studied in cancer and fibrosis. In breast cancer, high expression of both LOX and LOXL2 strongly correlates with the invasive potential of metastatic breast tumors. See Kirschmann, DA et al., A molecular role for lysyl oxidase in breast cancer invasion. Cancer Research 62, 4478-4483 (2002). High levels of LOXL2 expression have also been detected in HCC cells and tissues, which are associated with poor disease-free and overall survival in HCC patients. See Ninomiya, G. et al., Significance of Lysyl oxidase-like 2 gene expression on the epithelial-mesenchymal status of hepatocellular carcinoma. Oncology Reports 39, 2664-2672 (2018). Higher expression of LOXL2 promotes the invasive potential of pancreatic cancer cells and higher distant recurrence rates have been observed in LOXL2-positive tumors in pancreatic cancer patients. See Park, JS et al., Emerging role of LOXL2 in the promotion of pancreas cancer metastasis. Oncotarget 7, 42539 (2016). In primary gastric tumors, high LOXL2 expression correlates with high tumor invasion, high lymph node metastasis, and poor overall patient survival. See Peng, L. et al., Secreted LOXL2 is a novel therapeutic target that promotes gastric cancer metastasis via the Src / FAK pathway. Carcinogenesis 30, 1660-1669 (2009).

[0007]

[0007] LOXL3 overexpression has been observed in gastric cancer, and a tumorigenic role for LOXL3 associated with increased invasion has been shown in gastric cancer. See Kasashima, supra. In melanoma, LOXL3 controls genome integrity by binding to several key members of the DNA damage response and promotes tumor growth in vivo. See Santamaria, PG, et al., Lysyl oxidase-like 3 is required for melanoma cell survival by maintaining genomic stability. Cell Death & Differentiation 25, 935-950 (2018). In addition to gastric cancer and melanoma, LOXL3 expression was also upregulated in breast cancer (see Jeong, YJ, et al., Association between lysyl oxidase and fibrotic focus in relation with inflammation in breast cancer. Oncology Letters 15, 2431-2440 (2018)), myeloproliferative neoplasms (see Tadmor, T., et al., The expression of lysyl-oxidase gene family members in myeloproliferative neoplasms. American Journal of Hematology 88, 355-358 (2013)), and ovarian cancer. See Dufresne, J., et al., The plasma peptides of ovarian cancer. Clinical Proteomics 15, 1-19 (2018). High LOXL4 expression is associated with poor prognosis in HCC patients and induces HCC intrahepatic and pulmonary metastasis in vivo. See Li, R. et al., Exosome-mediated secretion of LOXL4 promotes hepatocellular carcinoma cell invasion and metastasis. Molecular cancer 18, 1-19 (2019).LOXL4 overexpression is associated with poor overall survival in breast cancer patients (See Choi, SK, Kim, HS, Jin, T. & Moon, WKLOXL4 knockdown enhances tumor growth and lung metastasis through collagen-dependent extracellular matrix changes in triple-negative breast cancer. Oncotarget 8, 11977 (2017)), and LOXL4 inhibition inhibited lung adenocarcinoma cell migration and invasion. See Xie, S., Liu, G., Huang, J., Hu, HB & Jiang, W. miR-210 promotes lung adenocarcinoma proliferation, migration, and invasion by targeting lysyl oxidase-like 4. Journal of Cellular Physiology 234, 14050-14057 (2019). Another study in head and neck squamous cell carcinoma (HNSCC) showed that high LOXL4 expression correlated with tumor stage and lymph node metastasis. See Weise, JB et al., LOXL4 is a selectively expressed candidate diagnostic antigen in head and neck cancer. European journal of cancer 44, 1323-1331 (2008).

[0008]

[0008] Human fibrotic diseases, consisting of idiopathic pulmonary fibrosis, hepatic fibrosis, cardiovascular fibrosis and renal fibrosis, share the common feature of excessive deposition of ECM fibrous proteins, such as collagen, in damaged tissues, leading to disruption of ECM homeostasis and organ failure. Kim, Y.-M., Kim, E.-C. & Kim, Y. The human lysyl oxidase-like 2 protein functions as an amine oxidase toward collagen and elastin. Molecular Biology Reports 38, 145-149 (2011), Chen, L., Li, S. & Li, W. LOX / LOXL in pulmonary fibrosis: potential therapeutic targets. Journal of Drug Targeting 27, 790-796(2019), Yang, J. et al., Targeting LOXL2 for cardiac interstitial fibrosis and heart failure treatment.Nature Communications 7, 1-15(2016), Chien, JW et al., Serum lysyl oxidase-like 2 levels and idiopathic pulmonary fibrosis disease progression.European Respiratory Journal 43, 1430-1438(2014), Steppan, J. et al., Lysyl oxidase-like 2 depletion is protective in age-associated vascular stiffening.American Journal of Physiology-Heart and Circulatory Physiology 317, H49-H59(2019), Nave, AH et al., Lysyl oxidases play a causal role in vascular remodeling in clinical and experimental pulmonary arterial hypertension.See Arteriosclerosis, Thrombosis, and Vascular Biology 34, 1446-1458 (2014) and Martinez-Revelles, S. et al., Lysyl oxidase induces vascular oxidative stress and contributes to arterial stiffness and abnormal elastin structure in hypertension: role of p38MAPK. Antioxidants & Redox Signaling 27, 379-397 (2017). In addition to the role of LOX family members in cancer, their upregulation and increased activity have a fundamental role in fibrosis. For example, the expression of both LOX and LOXL1 is increased upon TGF-β stimulation, which promotes fibrogenesis in hepatic stellate cells and cardiac fibroblasts. See Lu, M., Qin, Q., Yao, J., Sun, L. & Qin, X. Induction of LOX by TGF-β1 / Smad / AP-1 signaling aggravates rat myocardial fibrosis and heart failure. IUBMB Life 71, 1729-1739 (2019) and Ma, L. et al., Knockdown of LOXL1 inhibits TGF-β1-induced proliferation and fibrogenesis of hepatic stellate cells by inhibition of Smad2 / 3 phosphorylation. Biomedicine & Pharmacotherapy 107, 1728-1735 (2018). Furthermore, LOX expression is increased in myocardial fibrosis and directly contributes to adverse myocardial remodeling in heart disease. Spurney, CFSee Kumari, S., Panda, TK & Pradhan, T. Lysyl oxidase: its diversity in health and diseases. Indian Journal of Clinical Biochemistry 32, 134-141 (2017). However, early induction and overexpression of LOX led to arteriosclerosis in Hutchinson-Gilford Progeria Syndrome. Moreover, LOX is strongly upregulated in young mice, and inhibition of LOX leads to improved arterial structure and cardiac function. See von Kleeck, R. et al., Arterial stiffness in Hutchinson-Gilford Progeria Syndrome corrected by inhibition of Lysyl Oxidase. Furthermore, both cardiac hypertrophy and dysfunction are associated with LOX overexpression, which may be targeted in the clinical management of hypertrophy and heart failure. See Galan, M. et al., Lysyl oxidase overexpression accelerates cardiac remodeling and aggravates angiotensin II-induced hypertrophy. The FASEB Journal 31, 3787-3799 (2017). Overall, these studies indicate a key role for LOX and other LOX family members in fibrosis.

[0009] In addition to the role of LOX family members in cancer and fibrotic diseases, abnormal lysyl oxidase activity has also been observed in other pathological conditions, including diabetes, neurodegeneration and adipose tissue dysfunction. See Wilhelmus, MM, Bol, JG, van Duinen, SG & Drukarch, B. Extracellular matrix modulator lysyl oxidase colocalizes with amyloid-beta pathology in Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis-Dutch type. Experimental Gerontology 48, 109-114 (2013), and Pastel, E. et al., Lysyl oxidase and adipose tissue dysfunction. Metabolism 78, 118-127 (2018). For example, LOX overexpression is induced by hypoxia and inflammation in adipose tissue with obesity. See Pastel, supra. Furthermore, LOX expression was induced in diabetes and led to apoptosis in rat retinal endothelial cells, and simultaneously siRNA knockdown of LOX reduced diabetes-induced LOX expression and prevented vascular leakage associated with diabetic retinopathy.See Chronopoulos, A., Tang, A., Beglova, E., Trackman, PC & Roy, S. High glucose increases lysyl oxidase expression and activity in retinal endothelial cells: mechanism for compromised extracellular matrix barrier function. Diabetes 59, 3159-3166 (2010). High LOX and LOXL2 levels were observed in choroidal neovascularization, and inhibition of LOX and LOXL2 with neutralizing anti-LOX and LOXL2 antibodies demonstrated a functional contribution of LOX and LOXL2 in diabetic retinopathy.See Roy, S., Ha, J., Trudeau, K. & Beglova, E. Vascular basement membrane thickening in diabetic retinopathy. Current eye research 35, 1045-1056 (2010). Under physiological conditions, LOX expression is low in rat brain but accumulates in lesions after brain injury. See Gilad, GM, Kagan, HM & Gilad, VH Lysyl oxidase, the extracellular matrix-forming enzyme, in rat brain injury sites. Neuroscience letters 310, 45-48 (2001). Interestingly, elevated LOX activity is observed during plaque formation in Alzheimer's disease (AD) via modulation of the ECM. See Gilad, GM, Kagan, HM & Gilad, VEvidence for increased lysyl oxidase, the extracellular matrix-forming enzyme, in Alzheimer's disease brain. Neuroscience Letters 376, 210-214 (2005).

[0010] Considering the multifaceted functions of LOX family members in fibrosis, cancer, and other diseases, there is great interest in identifying and developing potent and tolerable LOX or LOX family inhibitors.BAPN is the oldest and most commonly used research tool for inhibiting LOX, inhibits all LOX family members, and shows toxicity in clinical settings.See Keiser, HR & Sjoerdsma, A. Studies on beta-aminopropionitrile in patients with scleroderma. Clin Pharmacol Ther 8, 593-602, doi:10.1002 / cpt196784593 (1967) and Spencer, P. & Schaumburg, H. Lathyrism: a neurotoxic disease. Neurobehavioral toxicology and teratology 5, 625-629 (1983). Simtuzumab, a humanized monoclonal antibody that binds to LOXL2, has been tested in three different clinical trials in patients with fibrosis, but all of these studies were terminated due to lack of efficacy (NCT01769196) or did not provide benefit (NCT01672866 and NCT01672879). Furthermore, simtuzumab in combination with chemotherapy has not reported clinical benefit in the first and second line settings in patients with pancreatic and colorectal adenocarcinoma, respectively.See Benson, AB, 3rd et al., A Phase II Randomized, Double-Blind, Placebo-Controlled Study of Simtuzumab or Placebo in Combination with Gemcitabine for the First-Line Treatment of Pancreatic Adenocarcinoma. Oncologist 22, 241-e215, doi:10.1634 / theoncologist.2017-0024(2017); and Hecht, JR et al., A Phase II, Randomized, Double-Blind, Placebo-Controlled Study of Simtuzumab in Combination with FOLFIRI for the Second-Line Treatment of Metastatic KRAS Mutant Colorectal Adenocarcinoma. Oncologist 22, 243-e223, doi:10.1634 / theoncologist.2016-0479(2017). Recently, the LOXL2 / LOXL3 inhibitor, the phenoxypyridine-containing small molecule PAT-1251, has shown promising results as an anti-fibrotic agent in preclinical models (see Rowbottom, MW et al., Identification of 4-(Aminomethyl)-6-(trifluoromethyl)-2-(phenoxy)pyridine Derivatives as Potent, Selective, and Orally Efficacious Inhibitors of the Copper-Dependent Amine Oxidase, Lysyl Oxidase-Like 2 (LOXL2). J Med Chem 60, 4403-4423, doi:10.1021 / acs.jmedchem.7b00345 (2017)) and is currently in a Phase IIa clinical trial for myelofibrosis (NCT04679870).The haloarylamine-based small molecule inhibitors PXS-S1A and PXS-S2A were potent against LOX / LOXL2 and LOXL2, respectively, with the former inhibiting lung and liver metastasis. See Chang, J. et al., Pre-clinical evaluation of small molecule LOXL2 inhibitors in breast cancer. Oncotarget 8, 26066-26078, doi:10.18632 / oncotarget.15257 (2017). CCT365623, an orally bioavailable aminomethylenethiophene (AMT)-based inhibitor, was active against LOX / LOXL2 and led to a marked reduction in metastasis in breast cancer animal models. See Leung, L. et al., Anti-metastatic Inhibitors of Lysyl Oxidase (LOX): Design and Structure-Activity Relationships. J Med Chem 62, 5863-5884, doi:10.1021 / acs.jmedchem.9b00335 (2019). The pan-lysyl oxidase inhibitor PXS-5505 has been well tolerated in healthy volunteers and is currently being tested in patients with primary post-polycythemia vera (myeloproliferative neoplasm) or post-essential thrombocythemia myelofibrosis. See How, J. et al., Evaluation of a Pan-Lysyl Oxidase Inhibitor, Pxs-5505, in Myelofibrosis: A Phase I, Randomized, Placebo Controlled Double Blind Study in Healthy Adults. Blood 136, doi:10.1182 / blood-2020-139223 (2020). Recently, the FDA approved a study of PXS-5505 in combination with a PD-L1 inhibitor and an anti-VEGF drug as a first-line treatment in newly diagnosed patients with unresectable HCC carcinoma (NCT05109052). Furthermore, cyclosporine (CsA) is one of the most commonly used drugs after solid organ transplantation and is also a major cause of uremia.Importantly, combinatorial treatment with CsA and the pan-LOX inhibitor PXS-5505 attenuated uremia and progressive nephropathy. See Nguyen, LT et al., Lysyl oxidase inhibitors attenuate cyclosporin A-induced nephropathy in mouse.Scientific reports 11,1-12(2021). Despite the existence of several ongoing clinical trials using LOX family inhibitors in several diseases, there are still no FDA-approved LOX inhibitors, indicating the need to develop novel, effective, and tolerable inhibitors against LOX or LOX family members.

[0011] According to the American Cancer Society, it is estimated that 1.8 million new cancer cases will be diagnosed in the United States and 606,520 will die from cancer. This figure can be multiplied by at least eight to estimate the global cancer incidence / mortality. Cardiac disease is the leading cause of death, followed by cancer. Neurodegenerative diseases and fibrosis are also very common. Cancer is a global challenge and various treatment options are available for patients, e.g. chemotherapy, immunotherapy and antibody-drug conjugates. See www.cancer.org. Summary of the Invention [Problem to be solved by the invention]

[0012]

[0012] Accordingly, it is an object of the present disclosure to provide novel compounds that block the activity of lysyl oxidase family members and have the potential to be further developed as cancer drugs to inhibit resistance to conventional therapies and metastasis, or as drugs to treat fibrotic diseases. [Means for solving the problem]

[0013]

[0013] Citation or identification of any reference in this application is not an admission that such reference is available as prior art to the present disclosure.

[0014] summary The above objects are achieved in accordance with the present disclosure by providing a method for inhibiting lysyl oxidase, comprising administering to said patient a therapeutically effective amount of a compound of formula I:

[0015]

[0014] [ka]

[0015]

[0016] wherein R1 is CH3, R2 is H, NH2 and NHR, R3 is H or CH3, R4 is H, R5 is H, R6 is NO2, C2OH, NHCOCH3 or OH, R7 is H, Q1 is N, Q2 is S, Q3 is N, Q4 is CH, Q5 is S, Q6 is CH, Q7 is CH, Q8 is C; and Q9 is CH, or a pharma- ceutically acceptable salt thereof, which inhibits cell-based lysyl oxidase activity and / or a recombinant protein-based lysyl oxidase activity assay.

[0016]

[0017] In addition, a therapeutically effective amount of the compound or a pharma- ceutically acceptable salt thereof may be administered to treat neurodegenerative diseases, angiogenesis-related diseases, Alzheimer's disease; fibrosis including hepatic fibrosis, pulmonary fibrosis, renal fibrosis, myocardial fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis, radiation-induced fibrosis, ocular fibrosis, Peyronie's disease, and scleroderma; or respiratory disorders, abnormal wound healing and repair, postoperative surgery, cardiac arrest-related fibrosis; disorders such as Crohn's disease and inflammatory bowel disease, all associated with excessive or abnormal deposition of fibrotic material; hepatic, renal, and pancreatic fibrosis, diabetes, cerebral hemorrhage with amyloidosis, cardiac hypertrophy, Hutchinson-Gilford Progeria syndrome, retinopathy, chemotherapy resistance; and / or renal disorders including renal fibrosis, renal fibrosis, acute kidney injury, chronic kidney disease, diabetic nephropathy, glomerulosclerosis, vesicoureteral reflux, tubulointerstitial renal fibrosis, and / or glomerulonephritis. In addition, the cancers include lung cancer; breast cancer; colorectal cancer; anal cancer; pancreatic cancer; prostate cancer; ovarian cancer; liver and bile duct cancer; esophageal cancer; non-Hodgkin's lymphoma; bladder cancer; uterine cancer; glioma, glioblastoma, medulloblastoma, and other brain tumors; myelofibrosis; kidney cancer; head and neck cancer; gastric cancer; multiple myeloma; testicular cancer; germ cell tumors; neuroendocrine tumors; cervical cancer; oral cancer, carcinoids of the digestive tract, breast, and other organs; signet ring cell carcinoma; sarcoma, fibrosarcoma, hemangioma, hemangiomatosis, hemangiopericytoma, pseudoangiomatous stromal hyperplasia, and sarcoma. hyperplasia, myofibroblastoma, fibromatosis, inflammatory myofibroblastic tumor, lipoma, angiolipoma, granular cell tumor, neurofibroma, schwannoma, hemangiosarcoma, liposarcoma, rhabdomyosarcoma, osteosarcoma, mesenchymal tumors including leiomyoma and / or leiomyosarcoma. Again, the compound or a pharmaceutically acceptable salt thereof may be selected from the group comprising:

[0018]

[0017] [ka]

[0018]

[0043]

[0019] Additionally, the compound or a pharma- ceutically acceptable salt thereof may have the formula:

[0020]

[0019] [ka]

[0020]

[0043]

[0021] In fact, the compound or a pharma- ceutically acceptable salt thereof may have the formula:

[0022]

[0021] [ka]

[0022]

[0043]

[0023] Nevertheless, the compound or a pharma- ceutically acceptable salt thereof may have the formula:

[0024]

[0023] [ka]

[0024]

[0043]

[0025] Additionally, a therapeutically effective amount of the compound or a pharma- ceutically acceptable salt thereof may be used as an anti-cancer agent, including, but not limited to, lung cancer; breast cancer; colorectal cancer; anal cancer; pancreatic cancer; prostate cancer; ovarian cancer; liver and bile duct cancer; esophageal cancer; non-Hodgkin's lymphoma; bladder cancer; uterine cancer; glioma, glioblastoma, medulloblastoma, and other brain tumors; myelofibrosis, kidney cancer; head and neck cancer; gastric cancer; multiple myeloma; testicular cancer; germ cell tumors; neuroendocrine tumors; The compound is selected from the group including cervical cancer; oral cancer, gastrointestinal, breast, and other organ carcinoids; signet ring cell carcinoma; mesenchymal tumors including sarcoma, fibrosarcoma, hemangiomas, hemangiomatoses, hemangiopericytomas, pseudohemangiomatoid stromal hyperplasia, myofibroblastomas, fibromatoses, inflammatory myofibroblastic tumors, lipomas, hemangiolipomas, granular cell tumors, neurofibromas, schwannomas, hemangiosarcomas, liposarcomas, rhabdomyosarcomas, osteosarcomas, leiomyomas, and / or leiomyosarcomas. Again, a therapeutically effective amount of the compound or a pharmacologic acceptable salt thereof may be administered to reduce the cardiotoxic side effects of chemotherapy and / or immunotherapy. Again, a therapeutically effective amount of the compound or a pharmacologic acceptable salt thereof may be administered as a single agent or in combination with chemotherapy, immunotherapy, and / or radiation therapy in both adjuvant and neoadjuvant settings. Again, a therapeutically effective amount of the compound or a pharmacologic acceptable salt thereof may be administered to induce inhibition of metastasis. Additionally, a therapeutically effective amount of the compound or a pharma- ceutically acceptable salt thereof may be administered with a second therapeutic agent selected from an anti-cancer agent, an anti-inflammatory agent, an antihypertensive agent, an anti-fibrotic agent, an anti-angiogenic agent, and / or an immunosuppressant.

[0025]

[0026] The present disclosure also provides a compound of formula:

[0027]

[0026] [ka]

[0027] The present invention provides novel compounds for inhibiting lysyl oxidase having the formula:

[0028] Further, the compound or a pharma- ceutically acceptable salt thereof may be administered in a therapeutically effective amount to treat neurodegenerative diseases, angiogenesis-related diseases, Alzheimer's disease; fibrosis including liver fibrosis, pulmonary fibrosis, renal fibrosis, myocardial fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis, radiation-induced fibrosis, ocular fibrosis, Peyronie's disease and scleroderma; or respiratory disorders, abnormal wound healing and repair, post-operative surgery, cardiac arrest-associated fibrosis; disorders such as Crohn's disease and inflammatory bowel disease, all associated with excessive or abnormal deposition of fibrotic material; fibrosis of the liver, kidney and pancreas, diabetes, cerebral hemorrhage with amyloidosis, cardiac hypertrophy, Hutchinson-Gilford Progeria syndrome, retinopathy, chemotherapy resistance; and / or renal disorders including renal fibrosis, renal fibrosis, acute kidney injury, chronic kidney disease, diabetic nephropathy, glomerulosclerosis, vesicoureteral reflux, tubulointerstitial renal fibrosis and / or glomerulonephritis. Furthermore, in this case, the novel compound or its pharmaceutically acceptable salt may be administered as a single agent or in combination with chemotherapy, immunotherapy and / or radiation therapy in both adjuvant and neoadjuvant settings.Nevertheless, the compound or its pharmaceutically acceptable salt may be administered with a second therapeutic agent selected from an anti-cancer agent, an anti-inflammatory agent, an antihypertensive agent, an antifibrotic agent, an antiangiogenic agent, and / or an immunosuppressant.

[0028]

[0029] The present disclosure further provides a compound of formula:

[0030]

[0029] [ka]

[0030] The present invention provides novel compounds for inhibiting lysyl oxidase having the formula:

[0031] In addition, the novel compounds or pharma- ceutically acceptable salts thereof may be administered in therapeutically effective amounts to treat neurodegenerative diseases, angiogenesis-related diseases, Alzheimer's disease; fibrosis including liver fibrosis, pulmonary fibrosis, renal fibrosis, myocardial fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis, radiation-induced fibrosis, ocular fibrosis, Peyronie's disease and scleroderma; or respiratory disorders, abnormal wound healing and repair, post-operative surgery, cardiac arrest-associated fibrosis; disorders such as Crohn's disease and inflammatory bowel disease, all associated with excessive or abnormal deposition of fibrotic material; fibrosis of the liver, kidney and pancreas, diabetes, cerebral hemorrhage with amyloidosis, cardiac hypertrophy, Hutchinson-Gilford Progeria syndrome, retinopathy, chemotherapy resistance; and / or renal disorders including renal fibrosis, renal fibrosis, acute kidney injury, chronic kidney disease, diabetic nephropathy, glomerulosclerosis, vesicoureteral reflux, tubulointerstitial renal fibrosis and / or glomerulonephritis. Furthermore, the compound or its pharmaceutically acceptable salt may be administered as a single agent or in combination with chemotherapy, immunotherapy and / or radiation therapy in both adjuvant and neoadjuvant settings.Nevertheless, the compound or its pharmaceutically acceptable salt may be administered with a second therapeutic agent selected from an anti-cancer agent, an anti-inflammatory agent, an antihypertensive agent, an antifibrotic agent, an antiangiogenic agent, and / or an immunosuppressant.

[0031]

[0032] These and other aspects, objects, features, and advantages of the exemplary embodiments will become apparent to those skilled in the art upon consideration of the following detailed description of the exemplary embodiments.

[0033] An understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments in which the principles of the disclosure may be utilized, and the accompanying drawings, in which: [Brief description of the drawings]

[0032] [Figure 1]

[0034] FIG. 1 shows a diagram of extracellular matrix remodeling. [Diagram 2]

[0035] FIG. 2 shows a graphical and visual analysis of postnatal tumor cell proliferation and metastasis data obtained from treatment with CCT365623. [Diagram 3]

[0036] FIG. 3 shows an illustration of the interactions between LOXL2, tumor cells, and collagen fibers. [Figure 4]

[0037] FIG. 4 shows the molecular structure of PXS-5153A. [Diagram 5]

[0038] FIG. 5 illustrates one embodiment of a process for identifying and characterizing novel LOX / LOXL inhibitors. [Figure 6]

[0039] FIG. 6 is a graph showing relative lysyl oxidase activity and signal background for potential inhibitors. [Figure 7]

[0040] FIG. 7 is a graph showing a comparison of cell viability and lysyl oxidase activity for target compounds. [Figure 8-1]

[0041] FIG. 8 is a graph showing cell viability and lysyl oxidase activity for 20 specific target compounds. [Figure 8-2] FIG. 8 is a graph showing cell viability and lysyl oxidase activity for 20 specific target compounds. [Figure 9]

[0042] FIG. 9 is a graph showing a doxorubicin sensitization screen that revealed five target compounds as doxorubicin sensitizers. [Figure 10]

[0043] FIG. 10 is a graph showing LOX and LOX2 activity for compounds 6232 and 6229. [Figure 11]

[0044] FIG. 11 is a graph showing SAR analysis of 6232 indicating additional analog compounds. [Figure 12]

[0045] FIG. 12 is a graph showing testing of analogs for lysyl oxidase activity and cell proliferation rate versus drug dose. [Figure 13]

[0046]

[0047] FIG. 13 is a table showing the structure-activity relationship of Table 1, 4'-methyl-N2-phenyl-[4,5'-bithiazole]-2,2'-diamine Lox inhibitors. [Figure 14]

[0048] FIG. 14 is a graph showing testing of analogs 6403, 6405, and 6425 for toxicity in normal cells. [Figure 15]

[0049] FIG. 15 is a graph showing in vivo toxicity testing for compound 6403. [Figure 16]

[0050] FIG. 16 is a graph showing a graphical representation of growth inhibition for compounds 6403 and 6415. [Figure 17]

[0051] FIG. 17 shows the reduction of ECM cross-linking via the use of compounds 6403 and 5415. [Figure 18]

[0052] FIG. 18 is a graph showing LOX and LOXL2 activity graphs for compound 6415. [Figure 19]

[0053] FIG. 19 is a graph showing a graphic analysis of cell-based lysyl oxidase activity and cell viability assays in Caki-1 and ACHN cell lines treated with increasing doses of BAPN and 6232. [Figure 20]

[0054] FIG. 20 shows the effect of ECM cross-linking in ACHN and Caki-1 cell lines when treated with compound 6232. [Figure 21]

[0055] FIG. 21 shows the reduction in ECM cross-linking upon treatment with compound 6232. [Figure 22]

[0056] FIG. 22 shows the expression of LOX family members in cancer versus normal tissues by using the TCGA dataset. [Diagram 23]

[0057] FIG. 23 is a graph showing the association between LOX, LOXL1 and LOXL2 expression and survival in breast, pancreatic and renal cancer patients. [Figure 24]

[0058] FIG. 24 shows that LOX expression is increased in doxorubicin resistance and its high expression is associated with worse survival in TNBC patients treated with chemotherapy. [Diagram 25]

[0059] FIG. 25 shows that LOX inhibition overcomes doxorubicin resistance in 3D cultures. [Figure 26]

[0060] FIG. 26 shows that inhibition of LOX overcomes resistance in acquired doxorubicin-resistant MDA-MB-231 xenografts. [Figure 27]

[0061] FIG. 27 shows that targeting LOX in TNBC PDX organoids or tumors overcomes resistance. [Figure 28]

[0062] FIG. 28 shows an overview of our LOX or LOX family inhibitor discovery pipeline. [Figure 29]

[0063] FIG. 29 is a graph depicting characterization and selection of top hits from high-throughput screening (HTS) of a diversified small molecule library to identify novel LOX inhibitors. [Figure 30-1]

[0064] FIG. 30 is a table showing LOX inhibitors generated based on SAR analysis. [Figure 30-2] FIG. 30 is a table showing LOX inhibitors generated based on SAR analysis. [Figure 30-3] FIG. 30 is a table showing LOX inhibitors generated based on SAR analysis. [Diagram 31]

[0065] FIG. 31 shows that novel 6232 analogs 6403 and 6415 effectively inhibit lysyl oxidase activity, overcome doxorubicin resistance, and reduce collagen cross-linking and fibronectin assembly. [Diagram 32]

[0066] FIG. 32 is a graph showing that 6232 derivatives 6403 and 6415 do not exhibit cytotoxicity in normal cells. [Diagram 33]

[0067] FIG. 33 is a graph showing that 6403 inhibits LOX recombinant protein, while 6415 inhibits both LOX and LOXL2 recombinant proteins. [Diagram 34]

[0068] FIG. 34 is a graph showing that 6232, 6403 and 6415 are more potent than the LOX family inhibitor BAPN. [Diagram 35]

[0069] FIG. 35 is a graph showing that 6403 and 6415 resulted in doxorubicin chemotherapy sensitization in 3D cultures of TNBC cell lines. [Diagram 36]

[0070] FIG. 36 shows that the combination of 6403 and 6415 with doxorubicin reduces proliferation of TNBC organoids. [Figure 37]

[0071] FIG. 37 is a graph showing that LOX inhibitors, 6232, 6403 and 6415, reduced the migration ability of TNBC cell lines. [Figure 38]

[0072] FIG. 38 is a graph showing dose-dependent inhibition of cellular LOX activity and proliferation inhibition in 3D upon treatment with 6232 and its analogs 6403 and 6415 in the ACHN cancer cell line. [Figure 39]

[0073] FIG. 39 is a graph showing inhibition of cellular LOX activity in pancreatic and renal cancer cell lines. [Diagram 40]

[0074] FIG. 40 shows that treatment with 6232, and its analogs 6403 and 6415, inhibited TGF-β-induced fibrosis in HFF cells. [Diagram 41]

[0075] FIG. 41 shows in vivo studies of 6403 for PK and toxicity. [Diagram 42]

[0076] FIG. 42 is a graph showing the efficacy of 6403 as a single agent in ccRC xenografts. [Diagram 43]

[0077] FIG. 43 shows that 6403 overcomes doxorubicin resistance without significant toxicity in the syngeneic mouse model, 4T1. [Diagram 44]

[0078] FIG. 44 is a table showing the structure-activity relationship of Table 2, 4'-methyl-N2-phenyl-[4,5'-bithiazole]-2,2'-diamine Lox inhibitors. [Diagram 45]

[0079] FIG. 45 shows a synthetic scheme for Formula I. [Figure 46]

[0080] FIG. 46 shows the synthetic routes for analogs 6403 and 6415.

[0033]

[0081] The figures herein are for illustration purposes only and are not necessarily drawn to scale. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0082] Before describing the present disclosure in more detail, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0035]

[0083] Unless otherwise specified, terms and phrases used in this document, and variations thereof, unless expressly specified otherwise, should be construed as open ended as opposed to exclusive. Similarly, a group of items joined by the conjunction "and" should not be read as requiring every single one of the items to be present in the group, but rather should be read as "and / or" unless otherwise expressly specified. Similarly, a group of items joined by the conjunction "or" should not be read as requiring mutual exclusivity between the groups, but rather should also be read as "and / or" unless otherwise expressly specified.

[0036]

[0084] Additionally, although items, elements, or components of the disclosure may be described or claimed in the singular, the plural is contemplated within its scope unless a limitation to the singular is expressly stated. In some instances, the presence of broad words and phrases, such as "one or more," "at least," "including but not limited to," or other similar phrases, should not be read to imply that a narrower case is intended or required in the absence of such broad phrase.

[0037]

[0085] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure, the preferred methods and materials are described.

[0038]

[0086] All publications and patents cited in this specification are cited to disclose and describe the methods and / or materials associated with the cited publications. All such publications and patents are incorporated herein by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. Such incorporation by reference is expressly limited to the methods and / or materials described in the cited publications and patents, and does not extend to any lexical definitions from the cited publications and patents. Any lexical definitions in the cited publications and patents that are not expressly repeated in this application should not be treated as such and should not be read as defining any terms that appear in the appended claims. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publications provided may differ from the actual publication dates, which may need to be independently confirmed.

[0039]

[0087] As will be apparent to those skilled in the art upon reading this disclosure, each individual embodiment described and illustrated herein has separate components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0040]

[0088] When a range is stated, further embodiments include from one particular value and / or to the other particular value. The description of a numerical range by endpoints includes all numbers and fractions contained within the respective ranges as well as the endpoints recited. When a range of values ​​is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit between the upper and lower limits of that range, and any other stated or intervening values ​​in that stated range, are encompassed within the scope of the disclosure, unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges, and are also encompassed within the scope of the disclosure, subject to any specific excluded limitations in the stated range. When a stated range includes one or both of the limits, ranges excluding either or both of the included limits are also included in the disclosure. For example, when a stated range includes one or both of the limits, ranges excluding either or both of the included limits are also included in the disclosure. For example, the phrase "from x to y" includes the range from "x" to "y" as well as the range from greater than "x" to less than "y". Ranges may also be expressed as upper limits, e.g., "less than about x, y, z," and should be considered to include the specific ranges of "about x," "about y," and "about z," as well as the ranges "less than x," "less than y," and "less than z." Similarly, the phrase "about x, y, z, or more" should be considered to include the specific ranges of "about x," "about y," and "about z," as well as the ranges "greater than x," "greater than y," and "greater than z." Additionally, the phrase "about 'x' to 'y'" includes "about 'x' to about 'y'," where 'x' and 'y' are numerical values.

[0041]

[0089] It should be noted that ratios, concentrations, amounts, and other numerical data may be presented herein in a range format. It will be further understood that the endpoints of each range are significant both in relation to the other endpoint and independently of the other endpoint. It is also understood that there are numerous values ​​disclosed herein, and each value is also disclosed herein as "about" a particular value in addition to the value itself. For example, if a value of "10" is disclosed, "about 10" is also disclosed. Ranges may be presented herein as "about" one particular value and / or "about" another particular value. Similarly, when values ​​are presented as approximations using the antecedent "about", it will be understood that the particular value forms a further aspect. For example, if a value of "about 10" is disclosed, "10" is also disclosed.

[0042]

[0090] Such range formats are used for convenience and brevity, and thus should be understood to be interpreted in a flexible manner not only to include the numerical values ​​explicitly recited as the limits of the range, but also to include all individual numerical values ​​or subranges encompassed within the ranges as if each numerical value and subrange were specifically recited. By way of example, a numerical range of "about 0.1% to 5%" should be considered to include not only about 0.1% to about 5% of the explicitly recited values, but also the individual values ​​(e.g., about 1%, about 2%, about 3%, and about 4%) and subranges within the recited ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible subranges).

[0043]

[0091] As used herein, the singular forms "a," "an," and "the" include both singular and plural referents unless the context clearly dictates otherwise.

[0044]

[0092] As used herein, "about," "approximately," "substantially," and the like, when used in connection with a measurable variable, such as a parameter, amount, temporal duration, and the like, are meant to encompass variations of and from the specified value, including those within the range of experimental error, as may be determined, for example, by a given data set, art-accepted standards, and / or, for example, by a given confidence interval (e.g., a confidence interval from the mean of 90%, 95%, or more), so long as such variations are appropriate to make in this disclosure, such as variations of + / -10% or less, + / -5% or less, + / -1% or less, and + / -0.1% or less of and from and to the specified value. As used herein, the terms "about," "approximate," "at or about," and "substantially" may mean that the amount or value in question can be an exact value or a value that provides an equivalent result or effect as recited in the claims or teachings herein. That is, it is understood that the amounts, sizes, formulations, parameters, and other quantities and characteristics are not and do not need to be exact, but may be approximate and / or larger or smaller as necessary, reflecting tolerances, conversion factors, rounding, measurement errors, and other factors known to those skilled in the art that would produce equivalent results or effects. In some circumstances, values ​​that provide equivalent results or effects cannot be reasonably determined. In general, amounts, sizes, formulations, parameters, or other quantities or characteristics are "about", "approximate", or "at or about", whether or not expressly specified as such. When "about", "approximate", or "at or about" is used before a quantitative value, it is understood that the parameter also includes the specific quantitative value itself, unless otherwise specified.

[0045]

[0093] As used herein, a "biological sample" may include whole cells and / or viable cells and / or cell debris. A biological sample may include (or be derived from) a "body fluid." The present disclosure encompasses embodiments in which the body fluid is selected from amniotic fluid, aqueous humor, vitreous humor, bile, serum, breast milk, cerebrospinal fluid, earwax, chyle, chyme, endolymph, perilymph, exudate, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal mucus and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, mucous secretions, saliva, sebum (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vomit, and mixtures of one or more of these. Biological samples include cell cultures, body fluids, and cell cultures from body fluids. Bodily fluids may be obtained from a mammalian organism, for example, by puncture or other withdrawal or sampling procedures.

[0046]

[0094] As used herein, "agent" refers to any substance, compound, molecule, etc. that can be administered to a subject to which it is administered. An agent may be inactive. An agent may be an active agent. An agent may be a primary active agent, in other words, a component of a composition that is the result in whole or in part of the composition's effect. An agent may be a secondary agent, in other words, a component of a composition that is the result of additional, partial and / or other effects of the composition.

[0047]

[0095] As used herein, an "active agent" or "active ingredient" refers to a substance, compound, or molecule that exhibits biological activity or induces a biological or physiological effect in a subject to which it is administered. In other words, an "active agent" or "active ingredient" refers to a component or components of a composition that is the result, in whole or in part, of the effect of the composition.

[0048]

[0096] As used herein, "administering" refers to any suitable administration to the agent to be delivered and / or to the subject to whom said agent is given, and may be oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intraarticular, parenteral, intraarteriolar, intradermal, intraventricular, intraosseous, intraocular, intracranial, intraperitoneal, intralesional, intranasal, intracardiac, intraarticular, intracavernous, intraarachnoid, intravitreal, intracerebral, and intraventricular, intratympanic, intracochlear, rectal, intravaginal, by inhalation, by catheter, stent, or via implanted reservoir or other device that actively or passively (e.g., by diffusion) administers the composition to the perivascular space and adventitia. For example, a medical device, e.g., a stent, may contain a composition or formulation that can be placed on its surface and then dissolved or distributed to the surrounding tissues and cells. The term "parenteral" may include subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.The routes of administration include, for example, auricular (ear), buccal, conjunctival, cutaneous, dental, electroosmotic, intracervical, intranasal, tracheal, intestinal, epidural, extra-amniotic, extracorporeal, hemodialysis, infiltration, interstitial, intraperitoneal, intra-amniotic, intra-arterial, intra-articular, intrabiliary, intra-bronchial, intra-vesical, intracardiac, intracartilage, intrasacral, intracavernous, intracavity, intracerebral, intracisternal, intracorneal, intracoronary (tooth), intracoronary, and pubic. Within the pedicle cavernosa, within the skin, within the intervertebral disc, within the tube, within the duodenum, within the dura mater, within the epidermis, within the esophagus, within the stomach, within the gums, within the ileum, within the lesion, within the cavity, within the lymph, within the marrow, within the meninges, within the muscle, within the eye, within the ovary, within the pericardium, within the abdominal cavity, within the pleura, within the prostate, within the lung, within the paranasal sinuses, within the medullary cavity, within the synovial bursa, within the tendon, within the testis, within the subarachnoid space, within the thoracic cavity, within the tube, within the tumor, within the tympanic membrane, within the uterus The administration route may be intravenous, intravascular, intravenous, intravenous bolus, intravenous drip, intraventricular, intravesical, intravitreal, iontophoretic, irrigation, translaryngeal, nasal, nasogastric, occlusive dressing technique, ocular, oral, or oropharyngeal, other, parenteral, transdermal, periarticular, epidural, perineural, periodontal, rectal, respiratory (inhalation), retrobulbar, soft tissue, subarachnoid, subconjunctival, subcutaneous, sublingual, submucosal, topical, transdermal, transmucosal, transplacental, transtracheal, transtympanic, ureteral, urethral, ​​and / or vaginal, and / or any combination of the above routes of administration, which typically depend on the disease to be treated, the subject to be treated, and / or the agent to be administered.

[0049]

[0097] As used herein, "cancer" includes, but is not limited to, acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, Kaposi's sarcoma, AIDS-related lymphoma, primary central nervous system (CNS) lymphoma, anal cancer, appendix cancer, astrocytoma, atypical teratoma / rhabdoid tumor, basal cell carcinoma of the skin, bile duct cancer, bladder cancer, bone cancer (including but not limited to Ewing's sarcoma, osteosarcoma, and malignant fibrous histiocytoma), brain tumor, breast cancer, bronchial tumor, Burkitt's lymphoma, carcinoid tumor, cardiac tumor, germ cell tumor, embryonal tumor, cervical cancer, gallbladder ... ductal carcinoma, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative neoplasms, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, localized ductal carcinoma, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, extracranial germ cell tumors, extragonadal germ cell tumors, eye cancer (including but not limited to intraocular melanoma and retinoblastoma), fallopian tube cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, central nervous system germ cell tumors, extracranial germ cell tumors, extragonadal germ cell tumors, ovarian germ cell tumors, testicular cancer, gestational trophoblastic disease, hairy cell leukemia, head and neck cancer, hepatocellular ( liver) cancer, Langerhans cell histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, islet cell tumors, pancreatic neuroendocrine tumors, kidney (renal cell) cancer, laryngeal cancer, leukemia, lip cancer, oral cavity cancer, lung cancer (non-small cell and small cell), lymphoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell cervical cancer, midline pathway carcinoma with and without NUT gene alterations, multiple endocrine neoplasia syndrome, multiple myeloma, plasma cell neoplasms, mycosis fungoides, myelodysplastic syndromes, myelodysplastic / myeloproliferative neoplasms, chronic myeloid leukemia, nasal cancer, nasal cavity cancer, non-Hodgkin's lymphoma, pancreatic It may refer to one or more types of cancer, including pancreatic cancer, paraganglioma, cancer of the paranasal sinuses, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary cancer, peritoneal cancer, prostate cancer, rectal cancer, rhabdomyosarcoma, salivary gland cancer, uterine sarcoma, Sézary syndrome, skin cancer, small intestine cancer, large intestine cancer (colon cancer), soft tissue sarcoma, T-cell lymphoma, throat cancer, oropharyngeal cancer, nasopharyngeal cancer, hypopharyngeal cancer, thymoma, thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, urethral cancer, uterine cancer, vaginal cancer, cervical cancer, vascular tumors and cancer, vulvar cancer, and Wilms' tumor.

[0050]

[0098] As used herein, "chemotherapeutic agent" or "chemotherapy" refers to therapeutic agents utilized to prevent or treat cancer.

[0099] As used herein, a "control" may refer to a substitute subject or sample used in an experiment for purposes of comparison and included to minimize or distinguish the effects of variables other than the independent variable.

[0051]

[0100] The term "optionally" or "optionally" means that the subsequently described event, circumstance, or substituent may or may not occur, and that the description includes cases where the event or circumstance occurs and cases where it does not occur.

[0052]

[0101] As used herein, "dose," "unit dose," or "dosage" may refer to physically discrete units suitable for use in a subject, each unit containing a predetermined amount of these pharmaceutical preparations calculated to produce a response or responses associated with its administration desired.

[0053]

[0102] The term "molecular weight" as used herein may generally refer to the mass or average mass of a material. In the case of a polymer or oligomer, molecular weight may refer to the relative average chain length or relative chain mass of the bulk polymer. In practice, the molecular weight of polymers and oligomers can be estimated or characterized in a variety of ways, including gel permeation chromatography (GPC) or capillary viscometry. GPC molecular weight is the number average molecular weight (M n ) as opposed to the weight average molecular weight (M w Capillary viscometry provides an estimate of molecular weight as the intrinsic viscosity determined from a dilute polymer solution using a particular set of concentration, temperature, and solvent conditions.

[0054]

[0103] As used herein, a "pharmaceutical formulation" refers to a combination of an active agent, compound, or ingredient with a pharma- ceutically acceptable carrier or excipient, rendering the composition suitable for diagnostic, therapeutic, or prophylactic use in vitro, in vivo, or ex vivo.

[0055]

[0104] As used herein, a "pharmaceutically acceptable carrier or excipient" refers to a carrier or excipient that is useful in preparing pharmaceutical formulations and is generally safe, non-toxic, and not biologically or otherwise undesirable, and includes carriers or excipients that are acceptable for veterinary use as well as for human pharmaceutical use. As used in the specification and claims, a "pharmaceutically acceptable carrier or excipient" includes both one such carrier or excipient and more than one such carrier or excipient.

[0056]

[0105] As used herein, "polymer" refers to a molecule composed of repeating units of monomers linked together. "Polymer" is understood to include, but is not limited to, homopolymers, copolymers, such as block, graft, random and alternating copolymers, terpolymers, etc., as well as blends and modifications thereof. A "polymer" may be a three-dimensional network (e.g., repeating units are linked together side-to-side, front-to-back, top-to-bottom), a two-dimensional network (e.g., repeating units are linked together side-to-side, top-to-bottom, and in sheet form), or a one-dimensional network (e.g., repeating units are linked side-to-side to form chains). A "polymer" may be composed of natural or synthetic monomers and combinations thereof. A polymer may be biological (e.g., monomers are biologically significant (e.g., amino acids)), natural, or synthetic.

[0057]

[0106] As used herein, the term "radiosensitizer" refers to an agent that can selectively enhance cell killing by radiation in a desired cell population, e.g., tumor cells, while at the same time not exhibiting single-agent toxicity to tumor or normal cells.

[0058]

[0107] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to vertebrates, preferably mammals, more preferably humans. Mammals include, but are not limited to, murines, simians, humans, farm animals, sports animals, and pets. Tissues, cells, and their progeny of biological entities obtained in vivo or cultured in vitro are also encompassed by the term "subject."

[0059]

[0108] As used herein, "substantially pure" may mean that the species of interest is the predominant species present (i.e., on a molar basis, it outweighs other individual species in the composition), and preferably a substantially purified fraction is one in which the species of interest accounts for about 50 percent of all species present. Generally, a substantially pure composition will account for greater than about 80 percent of all species present in the composition, more preferably greater than about 85%, 90%, 95%, and 99%. Most preferably, the species of interest is purified to substantial homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) and the composition consists essentially of a single species.

[0060]

[0109] When used interchangeably herein, the terms "sufficient" and "effective" may refer to the amount (e.g., mass, volume, dosage, concentration, and / or time) required to achieve one or more desired and / or specified results. For example, a therapeutically effective amount refers to the amount required to achieve one or more therapeutic effects.

[0061]

[0110] As used herein, "tangible expression media" refers to media that are physically concrete or accessible and are not merely abstract thoughts or unrecorded spoken words. "Tangible expression media" includes, but is not limited to, words in cellulose or plastic materials, or data stored in a suitable computer-readable memory form. Data can be stored in a unit device, such as flash memory or CD-ROM, or in a server that can be accessed by users, for example, via a web interface.

[0062]

[0111] As used herein, "therapeutic" may refer to treating, curing, and / or ameliorating a disease, disorder, condition, or side effect, or reducing the rate of progression of a disease, disorder, condition, or side effect. Thus, a "therapeutically effective amount" may refer to the amount of a compound capable of producing a therapeutic effect.

[0063]

[0112] As used herein, the terms "treating" and "treatment" may generally refer to obtaining a desired pharmacological and / or physiological effect. The effect may be, but need not necessarily be, preventative in terms of preventing or partially preventing these diseases, symptoms or conditions, such as cancer and / or indirect radiation damage. The effect may also be therapeutic in terms of partially or completely curing a disease, condition, symptom, or adverse effects caused by a disease, disorder, or condition. The term "treatment" as used herein encompasses any treatment of cancer and / or indirect radiation damage in a subject, particularly a human and / or a companion animal, and may include any one or more of: (a) preventing the disease or damage from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., alleviating or ameliorating the disease and / or its symptoms or conditions. The term "treatment", as used herein, may refer to therapeutic treatment alone, prophylactic treatment alone, or both therapeutic and prophylactic treatment. Those in need of treatment (subjects in need of treatment) may include those who already have a disorder and / or those in whom a disorder should be prevented. As used herein, the term "treating" may include inhibiting a disease, disorder, or condition, e.g., preventing its progression; alleviating a disease, disorder, or condition, e.g., causing regression of a disease, disorder, and / or condition. Treating a disease, disorder, or condition may include improving at least one symptom of a particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, e.g., treating a subject's pain by administering a painkiller, even though such an agent does not treat the cause of the pain.

[0064]

[0113] As used herein, the terms "weight percent", "wt%" and "wt.%" can be used interchangeably and refer to the weight percent of a given component based on the total weight of the composition of which it is a component, unless otherwise specified. That is, all wt% values ​​are based on the total weight of the composition, unless otherwise specified. It should be understood that the sum of the wt% values ​​for all components in a disclosed composition or formulation equals 100. Alternatively, if the wt% values ​​are based on the total weight of a subset of components in a composition, it should be understood that the sum of the wt% values ​​of the specified components in a disclosed composition or formulation equals 100.

[0065]

[0114] As used herein, "water soluble" generally means that at least about 10 g of a substance is soluble in 1 L of water, ie, at neutral pH and 25°C.

[0066]

[0115] Various embodiments are described hereinbelow. It should be noted that specific embodiments are not intended as exhaustive descriptions or limitations to the broader aspects discussed herein. An aspect described with a particular embodiment is not necessarily limited to that embodiment, but may occur in other embodiments. Throughout this specification, references to "one embodiment," "embodiment," "exemplary embodiment" mean that the particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. Thus, the appearance of the phrases "in one embodiment," "in an embodiment," or "exemplary embodiment" in various places throughout this specification may, but do not necessarily, all refer to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments, as would be apparent to one of ordinary skill in the art from this disclosure. Furthermore, some embodiments described herein include some features but not other features included in other embodiments, and combinations of features of different embodiments are intended to be within the scope of this disclosure. For example, in the appended claims, any of the claimed embodiments may be used in any combination.

[0067]

[0116] All patents, patent applications, published applications, and publications, databases, websites and other published samples mentioned herein are hereby incorporated by reference to the same extent as if each individual publication, published patent document, or patent application was specifically and individually indicated to be incorporated by reference.

[0068] kit

[0117] Any of the compounds and / or formulations described herein can be presented as a combination kit. As used herein, the term "combination kit" or "kit of parts" refers to a combination of elements contained therein or a single element, such as a compound, composition, formulation, particle, cell, and any additional components used to package, sell, trade, deliver, and / or administer the active ingredient. Such additional components include, but are not limited to, packages, syringes, blister packages, bottles, and the like. When one or more of the compounds, compositions, formulations, particles, cells described herein, or combinations thereof (e.g., drugs) contained in the kit are administered simultaneously, the combination kit may contain the active agent in a single formulation, such as a pharmaceutical formulation (e.g., tablet, liquid preparation, dehydrated preparation, etc.) or in separate formulations. When the compounds, compositions, formulations, particles, and cells described herein, or combinations thereof and / or kit components are administered simultaneously, the combination kit may contain each drug or other component in separate pharmaceutical formulations. Separate kit components may be included in a single package or in separate packages within the kit.

[0069]

[0118] In some embodiments, the combination kit also includes an instruction manual printed on or contained in a tangible medium. The instruction manual can provide information on the content of the compound and / or formulation, safety information on the content of the compound and formulation (e.g., pharmaceutical formulation), dosage, indications for use, and / or information on the recommended treatment regimen for the compound and / or pharmaceutical formulation contained therein. In some embodiments, the instruction manual can provide instructions and protocols for administering the compounds and / or formulations described herein to a subject in need thereof. In some embodiments, the instruction manual can provide one or more embodiments of the method for administering these pharmaceutical formulations, such as any of the methods described in more detail elsewhere herein.

[0070]

[0119] Although dramatic advances have been made in cancer treatment and cure, disease progression after primary treatment with spread of cancer to distant organs is often unavoidable and associated with cancer death. Proteins that remodel the extracellular matrix (ECM) that surrounds tumors and aids their growth and invasion into distant organs are attractive therapeutic targets not only for cancer but also for other diseases that involve excessive ECM deposition, such as fibrotic diseases, neurodegenerative and cardiovascular diseases.

[0071]

[0120] The lysyl oxidase family of proteins consists of five members (LOX and four related enzymes, LOXL1-4). The main function of lysyl oxidase is to catalyze the cross-linking of extracellular matrix (ECM) components, mainly collagen and elastin, and remodel the ECM. Given that lysyl oxidase plays a key role in maintaining tissue homeostasis, its deregulation may lead to various diseases, including cancer, organ fibrosis, neurodegeneration, and cardiovascular diseases. Each LOX enzyme has some overlapping as well as unique functions. LOX and LOXL2 proteins are involved in the progression and metastasis of several types of cancer, e.g., adenocarcinoma of the breast, pancreas, and lung. Its role in promoting breast cancer metastasis is associated with its ability to increase tumor stiffness and reinforce collagen cross-links at metastatic sites, thus preparing the microenvironment necessary for the growth of metastases. They may also stimulate endothelial cells and promote angiogenesis. Furthermore, published data from us and others suggest a role for LOX proteins in cancer therapy resistance. LOX / LOXL can regulate cancer cell invasion by promoting epithelial-mesenchymal transition (EMT), stabilizing EMT transcription factors or suppressing epithelial gene transcription. They also have non-canonical functions, e.g., regulating gene transcription. Most importantly, high expression of LOX / LOXL is associated with poor prognosis in various cancers. In addition to cancer, LOX family proteins are also associated with fibrotic diseases, broadening the applicability of LOX / LOXL targeting strategies. In an effort to identify novel compounds targeting LOX proteins, we performed a high-throughput screening program and identified a number of initial hit compounds that reduced lysyl oxidase activity and minimized cytotoxicity in cellular assays. We then purchased new batches of shortlisted compounds and verified their dose-dependent effects on cell viability and lysyl oxidase activity. We then tested the effects of the compounds on chemotherapy sensitization and also tested their direct inhibitory effects on LOX and LOXL2 activity using recombinant proteins.The final filter was an assessment of the compound's drug potential and the ease of generating novel analogues by synthetic chemistry. One family based on the core structure of 4'-methyl-N2-phenyl-[4,5'-bithiazole]-2,2'-diamine met all of these criteria, and our current lead inhibitor from this family is 4-((2'-amino-4'-methyl-[4,5'-bithiazole]-2-yl)(methyl)amino)phenol. This compound is a non-selective inhibitor of the LOX family of enzymes.

[0072]

[0121] In cancer, the ECM is frequently remodeled by the cancer cells themselves or by tumor-associated stromal cells, leading to the promotion of tumor stiffening, thus enhancing proliferation, migration and invasion. ECM remodeling at metastatic sites may also induce colonization and metastatic growth. In addition to cancer, disruption of ECM homeostasis may also lead to organ fibrosis, neurodegeneration and cardiovascular diseases. Figure 1 shows a diagram of extracellular matrix remodeling. See Hung-Yu Lin et al., 2020, International Journal of Molecular Sciences.

[0073]

[0122] Considering the crucial functions that LOX / LOXL have in ECM remodeling, targeting them is an attractive therapeutic strategy that would be of benefit in the treatment of ECM-driven diseases. BAPN is the most widely used LOX family inhibitor. However, due to its simple chemical structure, it has very low specificity and causes severe toxicity. Also, it lacks a suitable position for chemical modification, thus making preclinical optimization impossible. Several studies have reported LOXL2-specific inhibitors and LOX / LOXL inhibitors. See Tang et al., Lysyl oxidase drives tumor progression by trapping EGF receptors at the cell surface (2017); Tang et al., developed an orally bioavailable LOX / LOXL2 inhibitor named CCT365623. This inhibitor is an aminomethylenethiophene (AMT)-based inhibitor. In a breast cancer model, daily treatment with CCT365623 by oral gavage at a dose of 70 mg / kg reduced tumor growth and reduced metastasis. Referring to Figure 2, which shows a graphical and visual analysis of postnatal tumor cell proliferation and metastasis data obtained from treatment with CCT365623. See Tang et al., Lysyl Oxidase Drives Tumor Progression by Trapping EFT Receptors at the Cell Surface, DOI:10.1038 / ncomms14909.

[0074]

[0123] Furthermore, simtuzumab, a humanized monoclonal antibody that targets non-competitive LOXL2, has shown beneficial effects in various preclinical models of fibrosis and cancer. It has also been tested in several clinical trials in the context of fibrotic diseases as well as cancer. With regard to cancer, a phase II clinical trial was conducted with simtuzumab in combination with gemcitabine to treat patients with pancreatic cancer (NCT01472198). Although the antibody was well tolerated, the clinical benefit was minimal due to the lack of efficacy. Referring to Figure 3, this shows an illustration of the interaction between LOXL2 and tumor cells and collagen fibers. See Sandra Ferreira S. et al., Antioxidants, 2021. The lack of efficacy of antibodies targeting LOXL2 may be due to the fact that simtuzumab targets only extracellular LOXL2 and the effect is apparently insufficient due to some intracellular functions of LOX family proteins (Sandra Ferreira S. et al., Antioxidants, 2021).

[0075]

[0124] Therefore, it would be even more effective to target the LOX enzyme with a small molecule inhibitor that inhibits both intracellular and extracellular LOX functions. Figure 4 shows the molecular structure of PXS-5153A. See (Schilter H et al., 2018, J. Cell. Mol. Med). The small molecule PXS-5153A demonstrated complete and irreversible enzyme inhibition for LOXL2 and LOXL3. PXS-5153A has been shown to reduce LOXL2-mediated collagen oxidation and cross-linking in a dose-dependent manner in vitro. This dual LOXL2 / LOXL3 inhibitor has shown beneficial effects in models of liver fibrosis and myocardial infarction (Schilter H et al., 2018, J. Cell. Mol. Med.). Pharmaxis has developed an orally bioavailable pan-lysyl oxidase inhibitor, PXS-5505, that inhibits all lysyl oxidase family members. The compound showed a significant reduction in fibrosis in vivo in models of kidney fibrosis, lung fibrosis, myelofibrosis (a rare type of blood cancer) and pancreatic cancer. A favorable safety profile was also demonstrated and was well tolerated in healthy male volunteers (How, J. et al., 2020, Blood). A phase 1c / 2a clinical trial in myelofibrosis patients is currently planned, with results expected by 2023.

[0076]

[0125] The present disclosure provides, among other things, a method for identifying and characterizing novel LOX / LOXL inhibitors. Figure 5 illustrates one embodiment of a process for identifying and characterizing novel LOX / LOXL inhibitors. 20 compounds from a library of drug-like molecules and bioactive compounds were identified as potential lysyl oxidase inhibitors without major cytotoxicity. 4,480 small molecules (1,280 compounds) from the EXPRESS-Pick diversification library by ChemBridge and the Tocriscreen library of bioactive compounds by Tocris. Figure 6 shows the relative lysyl oxidase activity and signal background for potential inhibitors. Figure 7 shows a comparison of cell viability and lysyl oxidase activity for target compounds. Figure 8 shows cell viability and lysyl oxidase activity for 20 specific target compounds. Additionally, 19 shortlisted compounds were newly obtained and their effects on cell viability and lysyl oxidase activity were verified. Doxorubicin sensitization screening was performed on the 19 shortlisted candidates, and five compounds were identified as doxorubicin sensitizers. Figure 9 shows the doxorubicin sensitization screen that revealed five target compounds as doxorubicin sensitizers. Of the five candidates, two compounds (6232 and 6229) reduced the activity of both recombinant LOX and LOXL2 proteins, but LOXL2 inhibition was weaker compared to BAPN. Figure 10 shows the LOX and LOX2 activity for compounds 6232 and 6229. SAR analysis with 6232 resulted in inhibition of several analogs: lysyl oxidase activity in cell-based assays. Figure 11 shows the SAR analysis of 6232 showing additional analog compounds. Six of the promising analogs were further tested at multiple doses. Figure 12 shows the analogs tested for lysyl oxidase activity and cell proliferation rate versus drug dose. Figure 13 shows the structure-activity relationship of Table 1, 4'-methyl-N2-phenyl-[4,5'-bithiazole]-2,2'-diamine Lox inhibitors. The shortlisted analogs (6403, 6405 and 6425) were tested for toxicity in normal cells.Figure 14 shows the testing of analogs 6403, 6405, and 6425 for toxicity in normal cells. No significant effect was observed on the viability of three different normal cell lines. Figure 15 shows the in vivo toxicity testing for compound 6403. Figure 16 shows a graphic representation of the growth inhibition for compounds 6403 and 6415. No significant changes were observed in body weight or blood cell count even at higher doses. Figure 17 shows the reduction in ECM cross-linking through the use of compounds 6403 and 5415. Figure 18 shows a graph of LOX and LOXL2 activity for compound 6415.

[0077]

[0126] The inhibitory effect of our parent compound 6232 has also been tested in ccRCC with pseudohypoxic conditions (i.e., constitutive activation of HIF1α and HIF2α independent of oxygen levels). Cell-based lysyl oxidase activity and cell viability assays in Caki-1 and ACHN cell lines treated with increasing doses of BAPN and 6232. Figure 19 shows a graphic analysis of cell-based lysyl oxidase activity and cell viability assays in Caki-1 and ACHN cell lines treated with increasing doses of BAPN and 6232. Figure 20 shows the effect of ECM cross-linking in ACHN and Caki-1 cell lines when treated with compound 6232. Figure 21 shows the decrease in ECM cross-linking when treated with compound 6232.

[0078]

[0127] Since all available LOX inhibitors are not very potent or have toxicity issues, we developed a robust screening pipeline to identify potent and safe LOX-specific and pan-LOX inhibitors. A high-throughput cell-based LOX activity (Abcam) screen was performed on over 5,000 drug-like compounds. Toxic molecules were counter-screened and selected using cytotoxicity assays. Of the most potent hits (20 in total), five were shortlisted based on their ability to sensitize TNBC cells to doxorubicin and cross-link collagen-I, and tested in recombinant LOX and LOXL2 protein-based activity assays to determine their on-target activity and selectivity. One of the compounds (6232 (phenylbisthiazolediamine core structure)) was selected for SAR and lead optimization studies based on favorable chemical properties for drug-likeness, synthetic feasibility, and the potential to generate novel and chemically distinct LOX inhibitors. After SAR analysis, several analogs showed similar or increased activity against both 6232 and 6232 derivatives 6403 and 6415, resulting in doxorubicin sensitization. Lysyl oxidase activity assays using recombinant proteins showed that 6232 derivative 6403 (in which NO2 is replaced by acetamide) is a more potent and selective inhibitor of LOX compared to LOXL2, while 6415 is closer to a LOX / LOXL2 dual inhibitor. Importantly, 6403 showed potent cellular lysyl oxidase inhibition (IC50=1.3 μM for LOX inhibition) when compared to BAPN (IC50>100 μM). 6403 showed favorable pharmacokinetic (PK) properties in mice and did not result in any observable organ damage. As single agents, we tested our LOX inhibitors against kidney cancer in an in vivo xenograft model. 6403 successfully reduced tumor growth in LOX-expressing ACHN xenografts without affecting body weight.Finally, we tested the chemotherapy-sensitizing effect of 6403 in a syngeneic TNBC murine tumor model, 4T1, which has an intact immune system.The combination of 6403 and doxorubicin significantly reduced tumor growth in this LOX-expressing model compared to 6403, and the doxorubicin alone group was accompanied by no significant changes in body weight and blood counts after 21 days of treatment. Overall, we have obtained both a LOX-specific inhibitor and a broader LOX family inhibitor that are novel and have great potential for drug development.

[0079]

[0128] Among the LOX family members, the expression of LOX, LOXL1 and LOXL2 was higher in a broad spectrum of cancers compared to normal tissues in the Cancer Genome Atlas (TCGA) dataset (see Figure 22A). LOX, LOXL1 and LOXL2 are highly expressed in TNBC compared to normal breast (see Figure 22B), highly expressed in pancreatic tumors compared to normal tissues, and highly expressed in renal cancer compared to normal tissues (see Figure 22C). These are tumors known to be stroma-rich and fibrotic.

[0080]

[0129] In the same TCGA dataset, higher expression of LOX, LOXL1 and LOXL2 is associated with worse disease-free survival (DFS) in kidney renal cell carcinoma (KIRC). Furthermore, higher expression of LOX and LOXL2 is associated with worse recurrence-free survival (RFS) in pancreatic adenocarcinoma (PAAD) (in TCGA dataset) and chemotherapy-treated breast cancer (KM Plotter database) (see Figure 23A, B and C). These results suggest that higher expression of LOX family members, especially LOX and LOXL2, is associated with worse disease progression in cancer, especially in hard tumors.

[0081]

[0130] We identified hypoxia-inducible factor alpha (HIF1α)-induced LOX as significantly overexpressed in conjunction with increased fibrillar collagen in tumors of our clinically relevant in vivo model of doxorubicin resistance (see Figures 24A and B). We demonstrated that higher LOX mRNA / protein levels were significantly associated with worse disease-free survival (DFS) in chemotherapy-treated TNBC patients using multiple published datasets and our own patient cohorts (see Figures 24C and D).

[0082]

[0131] The non-specific LOX family inhibitor BAPN was used to resensitize three human TNBC cell lines (MDA-MB-231, FIG. 25A, MDA-MB-15714, and MDA-MB-43614) embedded in different collagen I (LOX substrates) to doxorubicin. These results were also reproduced by specific LOX inhibition upon siRNA-mediated knockdown (see FIG. 25B). Immunofluorescence (IF) staining of collagen and fibronectin upon incubation of BAPN-treated MDA-MB-231 cells with rat tail collagen I or ECM produced by human foreskin fibroblast (HFF) cells demonstrated reduced ECM deposition and assembly and enhanced drug penetration upon LOX inhibition (see FIG. 25C and D). This resulted in the deactivation of downstream FAK / Src signaling (see FIG. 25E) and increased apoptosis (see FIG. 25F). Importantly, inhibition of FAK or Src kinases recapitulated LOX inhibition-mediated chemotherapeutic sensitization (see FIG. 25G).

[0083]

[0132] To test the ability of LOX inhibition to overcome doxorubicin resistance in vivo in chemotherapy-refractory TNBC, we first treated MDA-MB-231 xenografts with doxorubicin until tumor growth promotion was achieved. At this point, BAPN (100 mg / kg) was added to half of the doxorubicin-resistant tumors, while the remaining tumors continued to receive doxorubicin alone (2.5 mg / kg). Surprisingly, the addition of LOX inhibitors led to a significant decrease in tumor growth (see FIG. 26A) and improved survival. We confirmed a decrease in LOX activity (see FIG. 26B) and a decrease in fibrillar collagen (see FIG. 26C).

[0084]

[0133] To evaluate the clinical relevance of targeting LOX, we obtained RNA-seq data from 15 patient-derived xenograft (PDX) models of TNBC from Jackson Lab along with their drug response data (see FIG. 27A), performed gene expression-based assessment of hypoxia and focal adhesion (FA) scores for each model, and correlated these scores with LOX expression (see FIG. 27B). Importantly, LOX expression had good correlation with both scores (r=0.649 and r=0.625). Based on RNA-seq analysis and drug response data, we selected one resistant model, TM01278, which showed resistance to doxorubicin, expressed high levels of LOX, and was accompanied by strong hypoxia and focal adhesion scores. The combination of LOX inhibitors with doxorubicin significantly reduced tumor organoid size in 3D organoid cultures containing collagen I (see FIG. 27C) and reduced tumor growth (see FIG. 27D). Importantly, LOX activity and fibrillar collagen content were decreased upon LOX inhibition with BAPN (see FIG. 27E), which was accompanied by enhanced doxorubicin penetration (see FIG. 27F) and reduced FAK / Src signaling (see FIG. 27G).

[0085]

[0134] Since all available LOX inhibitors are either not very potent or toxic, we developed a robust screening pipeline shown in Figure 28 to identify potent and safe LOX-specific and pan-LOX inhibitors. Briefly, a high-throughput cell-based LOX activity (Abcam) screen (HTS) was performed on over 5,000 drug-like compounds from the EXPRESS-Pick diversification library (4,480 molecules, ChemBridge) and the Tocriscreen library of bioactive compounds (1,280 compounds, Tocris) at a single concentration of 10 μM each. The screen identified 25 compounds from the ChemBridge library and 56 hits from the Tocris library that successfully inhibited more than 90% of LOX activity. The toxic molecules were counter-screened and selected using a cytotoxicity assay.

[0086]

[0135] The most potent hits (20 in total) were then validated by re-sorting new compounds and testing them in LOX activity and cell viability assays (see Figure 29A). Of these 20 compounds, five were shortlisted based on their ability to sensitize TNBC cells to doxorubicin (see Figure 29B). These five compounds were further tested in recombinant LOX and LOXL2 protein-based activity assays to determine their target activity and selectivity. Two compounds (6229 and 6232) showed strong inhibition of recombinant LOX and moderate inhibition of LOXL2 at 10 μM, a concentration 1000-fold lower than that required for the LOX family inhibitor BAPN (see Figures 29C and D).

[0087]

[0136] A diverse set of 6232 analogs (phenylbisthiazolediamine core structure, see Figure 30) was commercially available from ChemBridge, and a total of 19 compounds were obtained. We found that several analogs had similar or higher activity to 6232, providing initial insight into the SAR (see Tables 2-44, Figures 30 and 31A). These data indicated the importance of the free amino group on the terminal thiazole ring and suggested that a variety of substituents could be tolerated on the aniline ring without loss of activity. Of the 19 analogs tested, 6403 and 6415 were the most potent, with cellular LOX activity of 1.3 μM and 3.5 μM, respectively, and therefore these analogs are considered preliminary lead compounds. We also synthesized two novel derivatives based on the phenylbisthiazolediamine core structure. Compounds 6425 and 6426 contain additional variations in the R4 group (6425 is a methoxypropanamide and 6426 is a pentamide) and show high potency, with cellular LOX activities of 7.5 μM and 2 μM, respectively. Both 6425 and 6426 are novel compounds and have not been reported in Chemical Abstracts.

[0088]

[0137] Treatment with both 6403 and 6415 resulted in doxorubicin sensitization (Figure 31B) and reduced collagen crosslinking / fibronectin assembly (see Figure 31C). Furthermore, the cytotoxicity of 6403 and 6415 was tested against normal cell lines; MCF12A, HUVEC and HFF (see Figure 32) and no significant changes in cell viability were detected.

[0089]

[0138] Lysyl oxidase activity assays using recombinant proteins showed that 6403 is a more potent and selective inhibitor of LOX compared to LOXL2, fulfilling our essential requirements for LOX specificity (see Figure 33A), while 6415 is closer to being a LOX / LOXL2 inhibitor (see Figure 33B).

[0090]

[0139] 6232, 6403 and 6415 demonstrated more potent inhibition of cellular lysyl oxidase (eg, IC50=1.3 μM for 6403) compared to BAPN (IC50>100 μM) (see FIG. 34).

[0091]

[0140] Treatment with both 6403 and 6415 resulted in doxorubicin sensitization in collagen I-embedded MDA-MB-231 and HCC1143TNBC cell lines (see FIG. 35).

[0092]

[0141] We then generated organoid cultures of this doxorubicin-resistant PDX tumor (TM01278) and demonstrated that the combination of LOX inhibitors, 6403 and 6415, and doxorubicin significantly reduced organoid size (see FIG. 36A) and organoid viability (see FIG. 36B) after 9 days of treatment compared to single agent treatments.

[0093]

[0142] The effect of LOX inhibitors, 6232, 6403 and 6415, was tested on cell migration by using RTCA-CIM plates (Roche, USA). All three compounds inhibited the migration ability of MDA-MB-231 cells, with 6403 being the most effective in this cell line (see Figure 37).

[0094]

[0143] Previous studies have shown that LOX is overexpressed in different cancers and strongly associated with poor outcomes in patients with pancreatic (see Miller, BW et al., Targeting the LOX / hypoxia axis reverses many of the features that make pancreatic cancer deadly: inhibition of LOX abrogates metastasis and enhances drug efficacy. EMBO Mol Med 7, 1063-1076, doi:10.15252 / emmm.201404827(2015)), and renal (see Li, T. et al., Lysyl oxidase family members in urological tumorigenesis and fibrosis. Oncotarget 9, 20156-20164, doi:10.18632 / oncotarget.24948(2018)) tumors, making it an attractive therapeutic target for these clinically challenging diseases. We observed dose-dependent inhibition of cellular lysyl oxidase activity (see FIG. 38A) and decreased cell viability in 3D culture (see FIG. 38B) upon treatment with 6232, 6403 and 6415 in ACHN kidney cancer cell line (see FIG. 38). In addition to ACHN, treatment with 6232, 6403 and 6415 inhibited cellular LOX activity in another kidney cancer cell line, Caki-1, and pancreatic cancer cell lines, Mia-Paca-2 and PANC1 (see FIG. 39). Inhibition of LOX activity via our novel lead inhibitors will be beneficial not only for TNBC, but also for other tumor types characterized by high curability and chemotherapy resistance, such as pancreatic cancer, or LOX-driven cancers, such as kidney cancer in the future.

[0095]

[0144] We tested the effect of our LOX inhibitor on TGF-beta-induced fibrosis. The immunofluorescence staining of fibrosis markers is substantially reduced upon treatment with LOX inhibitor (see Figure 40). ALK5 inhibitor was used as a positive control.

[0096]

[0145] Our lead LOX inhibitor 6403 showed favorable pharmacokinetic (PK) properties in mice with large area under the curve (AUC), long half-life (T1 / 2), high maximum concentration (Cmax) and volume of distribution (Vd) with low clearance (Cl) (see Figure 41A) without causing any observable organ damage (see Figure 41B) or body weight change (see Figure 41C). Importantly, our parent molecule 6232, and its derivatives 6403 and 6415, did not inhibit monoamine oxidase A or B (MAO-A / -B) activity in cell-free enzyme activity assays (see Figure 41D), demonstrating its selectivity for the LOX enzyme.

[0097]

[0146] 6403 successfully reduced tumor growth in LOX-expressing ACHN ccRCC xenografts without affecting body weight and major blood cell counts (see FIG. 42).

[0098]

[0147] Finally, we tested the chemotherapy sensitizing effect of 6403 in the syngeneic TNBC tumor model 4T1 with an intact immune system. The combination of 6403 with doxorubicin significantly reduced tumor growth in this LOX-expressing model compared to the 6403 and doxorubicin alone group (see Figures 43A and B), without significant changes in body weight and blood counts 21 days after treatment (see Figures 43C and D).

[0099]

[0148] It has now been found that compounds of the present invention, and pharma- ceutically acceptable compositions thereof, are effective as inhibitors of LOX or LOX family activity. Such compounds have the general formula I:

[0149]

[0150]

[0100] [ka]

[0101]

[0151] or a pharma- ceutically acceptable salt thereof, wherein each variable is as defined and described herein. The compounds of the present invention and pharma- ceutically acceptable compositions thereof are useful for treating a variety of diseases, disorders, or conditions associated with LOX activity. Such diseases, disorders, or conditions include those described herein. The compounds provided by the present invention are also useful for studying LOX enzymes in biological and pathological phenomena.

[0102]

[0152] I or a pharma- ceutically acceptable salt thereof,

[0153] Q1 is =N- or =CH-

[0154] Q2 is NH, O or S

[0155] Q3 is =N- or =CH-

[0156] Q4 is NH, O or S, and Q5 is =N- or =CH-.

[0157] Q4=N- or =CH-; Q5 is NH, O or S.

[0158] Q6 is =N- or =CH-R4

[0159] Q7 is =N- or =CH-R5

[0160] Q8 is =N- or =CH-R6

[0161] Q9 is =N- or =CH-R7

[0162] R1-R7 are independently halogen, -CN, -NO2, -NH2, -NHR, -CH2R, -OR, -SR, -NR2, -S(0)2R, -S(O)2NR2, -S(O)R, -C(O)R, -C(O)OR, -C(O)NR2, -C(O)N(R)OR, -N(R)C(O)OR, -N(R)C(O)NR2, or -N(R)S(O)2R;

[0163] Each R is independently hydrogen or an optionally substituted group selected from a C1-6 aliphatic, phenyl, 4-7 membered saturated or partially unsaturated heterocyclic having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or: two R groups on the same nitrogen together with the intervening atoms form a 4-7 membered saturated, partially unsaturated, or heteroaryl ring having, in addition to the nitrogen, 0-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur (see Table 2, Figure 44).

[0103]

[0164] The chemical structure for 6232 is:

[0165]

[0104] [ka]

[0105] It is.

[0166] The chemical structure for 6403 is:

[0167]

[0106] [ka]

[0107] It is.

[0168] The chemical structure for 6405 is:

[0169]

[0108] [ka]

[0109] It is.

[0170] The chemical structure for 6415 is:

[0171]

[0110] [ka]

[0111] It is.

[0172]

[0173] Figure 45 shows a synthetic scheme for Formula I. Figure 46 shows the synthetic routes for analogs 6403 and 6415.

[0112]

[0174] Text description of the illustration image024.gif.

[0175] Figure 1 shows a diagram of extracellular matrix remodeling (Hung-Yu Lin et al., 2020, International Journal of Molecular Sciences).

[0113]

[0176] Figure 2 shows a graphical and visual analysis of postnatal tumor cell proliferation and metastasis data obtained from treatment with CCT365623 (Tang, HaoRan et al., 2017, Nature Communications). In a breast cancer model, daily treatment with CCT365623 by oral gavage at a dose of 70 mg / kg reduced tumor proliferation and reduced metastasis.

[0114]

[0177] Figure 3 shows an illustration of the interaction between LOXL2, tumor cells and collagen fibers (Ferreira, Sandra et al., 2021, Antioxidants).

[0178] Figure 4 shows the molecular structure of PXS-5153A (Schilter H et al., 2018, J. Cell. Mol. Med).

[0115]

[0179] FIG. 5 shows one embodiment of a process for identifying and characterizing novel LOX / LOXL inhibitors.

[0180] 6 shows a preliminary study of the LOX activity assay for compound screening. The left panel shows the relative lysyl oxidase activity, and the right panel shows the signal to background ratio for the common LOX inhibitor, BAPN.

[0116]

[0181] Figure 7 shows a comparison of cell viability percentage and lysyl oxidase activity for target compounds using MDA-MB-231 cells. Twenty compounds from a library of drug-like molecules and bioactive compounds were identified as potential lysyl oxidase inhibitors without causing major cytotoxicity.

[0117]

[0182] FIG. 8 shows validation of the effects of the 19 newly derived shortlisted compounds on cell viability and lysyl oxidase activity.

[0183] Figure 9 shows a doxorubicin sensitization screen that revealed five target compounds as doxorubicin sensitizers, with BAPN used as a positive control.

[0118]

[0184] Figure 10 shows the effect of five compounds on LOX and LOL2 activity. Of the five candidates, two compounds (6232 and 6229) reduced the activity of both recombinant LOX protein (A) and recombinant LOXL2 protein (B), but the LOXL2 inhibition was weaker than that of BAPN.

[0119]

[0185] Figure 11 shows the effect of 18 different 6232 analogs identified by SAR analysis on lysyl oxidase activity. Analogs were used at a concentration of 5 μM, while BAPN was used at 10 mM.

[0120]

[0186] Figure 12 shows the effect of the shortlisted analogs at multiple doses (2.5, 5, 10 and 20 μM) on lysyl oxidase activity and cell proliferation rate. BAPN was used at a concentration of 10 mM.

[0121]

[0187]

[0188] FIG. 13 shows the structure-activity relationship of Table 1, 4'-methyl-N2-phenyl-[4,5'-bithiazole]-2,2'-diamine Lox inhibitors.

[0189] Figure 14 shows testing of shortlisted analogs 6403, 6405, and 6415 for toxicity in normal HUVEC, MCF12A, and HFF cells. No significant effect on the viability of normal cell lines was detected.

[0122]

[0190] Figure 15 shows in vivo toxicity studies for compound 6403. The top panel shows the body weight of mice treated with different doses of 6403 and administered daily. The bottom panel shows blood cell counts from mice treated with vehicle vs. 200 mg / kg of 6403. No significant effects on body weight and blood cell counts were detected.

[0123]

[0191] FIG. 16 shows the percentage of growth inhibition in the TNBC cell line, MDA-MB-231, embedded in collagen and treated with a combination of doxorubicin and compounds 6232, 6403, 6405 and 6415.

[0124]

[0192] FIG. 17 shows the reduction in ECM cross-linking upon BAPN, 6232, 6403, 6405, or 6415 treatment, as assessed by collagen (green) and fibronectin (red) staining.

[0125]

[0193] Figure 18 shows the effect of compound 6415 on LOX and LOXL2 activity. 6415 was used at concentrations of 5 and 10 μM in recombinant protein-based lysyl oxidase activity assays to inhibit the activity of LOX and LOXL2 recombinant proteins. BAPN was used as a control at a dose of 10 mM.

[0126]

[0194] FIG. 19 shows lysyl oxidase activity and cell viability in Caki-1 and ACHN cell lines treated with increasing doses of BAPN and 6232.

[0195] FIG. 20 shows the cell morphology of ACHN and Caki-1 cell lines when treated with 10 μM of compound 6232.

[0127]

[0196] FIG. 21 shows the decrease in ECM cross-linking upon treatment of Caki-1 cells with 10 μM compound 6232 and 10 mM BAPN.

[0197] Figure 22. Expression of LOX family members in cancer vs. normal tissues using TCGA dataset. A. Pan-cancer expression of LOX family members in TCGA dataset. B. Expression of LOX, LOXL1 and LOXL2 in TNBC vs. normal breast. C. Expression of LOX, LOXL1 and LOXL2 in pancreatic tumor vs. normal tissues and kidney cancer vs. normal tissues. ACC-adenoid cystic carcinoma; BLCA-bladder urothelial carcinoma; BRCA-breast cancer; CESC-cervical squamous cell carcinoma; CHOL-cholangiocarcinoma; COAD-colon adenocarcinoma; DLBC-diffuse large B-cell lymphoma; ESCA-esophageal cancer; GBM-glioblastoma; HNSC-head and neck squamous cell carcinoma; KICH-kidney chromophobe; KIRC-kidney renal clear cell carcinoma; KIRP-kidney renal papillary cell carcinoma; LAML-acute myeloid leukemia; LGG-low grade glioma; LI HC-liver hepatocellular carcinoma;LUAD-lung adenocarcinoma;LUSC-lung squamous cell carcinoma;MESO-mesothelioma;OV-ovarian cancer;PAAD-pancreatic adenocarcinoma;PCPG-pheochromocytoma and paraganglioma;PRAD-prostate adenocarcinoma;READ-rectal adenocarcinoma;SARC-sarcoma;SKCM-skin cutaneous melanoma;STAD-gastric adenocarcinoma;TGTC-tendon sheath giant cell tumor;THCA-thyroid cancer;THYM-thymoma;UCEC-uterine endometrial carcinoma;UCS-uterine carcinosarcoma;UVM-uveal melanoma.

[0128]

[0198] Figure 23. Association of LOX, LOXL1 and LOXL2 expression and survival in breast, pancreatic and renal cancer patients. In the TCGA dataset, LOX, LOXL1 and LOXL2 expression is associated with worse DFS in KIRC, while high expression of LOX and LOXL2 is associated with worse recurrence-free survival (RFS) in PAAD (in the TCGA dataset) and chemotherapy-treated breast cancer (KM Plotter database).

[0129]

[0199] Figure 24. LOX expression is increased in doxorubicin resistance and its high expression is associated with worse survival in chemotherapy-treated TNBC patients. A. LOX mRNA expression in sensitive vs. resistant tumor xenografts. Picrosirius staining of tumors from BA. C. Kaplan-Meier survival curves depicting DFS in chemotherapy-treated TNBC patients (n=77) separated from median LOX protein expression. D. IHC images of TNBC patient tissues with low and high LOX protein expression. In all figures, *, P<0.05; **, P<0.01.

[0130]

[0200] Figure 25. LOX inhibition overcomes doxorubicin resistance in 3D cultures. A, B. Percentage growth inhibition of 3D collagen I-embedded MDA-MB-231 cells treated with BAPN in combination with doxorubicin (A) or transfected with siLOX in combination with doxorubicin (B). C. IF staining of collagen I (green) and fibronectin (red) in HFF-derived ECM incubated with vehicle or BAPN-treated MDA-MB-231 cells. D. Relative doxorubicin fluorescence intensity upon BAPN-treatment in collagen I-embedded MDA-MB-231 cells. E. Western blot of LOX and FAK / Src signaling in collagen I-embedded MDA-MB-231 cells treated with doxorubicin (1 μM) and BAPN (5 mM) for 24 h. Annexin V staining in 3D collagen I embedded MDA-MB-231 cells treated with Doxo+BAPN combination from FE. G. % proliferation inhibition of 3D collagen I embedded MDA-MB-231 cells treated with doxorubicin in combination with FAK (500 nM) or Src (500 nM) inhibitors.

[0131]

[0201] Figure 26. Inhibition of LOX overcomes resistance in acquired doxorubicin-resistant MDA-MB-231 xenografts. A. Tumor growth in xenografts treated with low-dose doxorubicin until resistance developed, followed by treatment with a combination of doxorubicin (2.5 mg / kg) and the LOX inhibitor, BAPN (100 mg / kg). Relative LOX activity in tumors from BA. Representative images of picrosirius red in tumors from CA.

[0132]

[0202] Figure 27. Targeting LOX in TNBC PDX organoids or tumors overcomes resistance. A. Scheme showing the selection pipeline of PDXs for in vivo testing. B. Correlation analysis of LOX mRNA expression with hypoxia and focal adhesion scores in 15 different TNBC PDX models. Red dots indicate selected TM01278 locations. C. Representative images of TM01278 PDX organoids 0 and 9 days after treatment with doxorubicin (40 nM) and BAPN (25 mM) treatment alone or in combination (left), and quantification of organoid diameters during 9 days of combination therapy (right) (n=12 (vehicle, Doxo, BAPN), n=11 (Doxo+BAPN)). D. Tumor growth of TM01278 PDXs upon treatment with doxorubicin (2 mg / kg) and BAPN (100 mg / kg) alone or in combination (n=5). Inset shows LOX expression in PDX tumors. Quantification of picrosirius red staining in PDX tumors from ED (n=4). Doxorubicin fluorescence quantification in tumors from FD (n=6). G. Western blot of FAK / Src signaling in PDX treated with doxorubicin and BAPN alone or in combination.

[0133]

[0203] FIG. 28. Overview of our LOX or LOX family inhibitor discovery pipeline.

[0204] Figure 29. Characterization and selection of top hits from high-throughput screening (HTS) of a diversified small molecule library to identify novel LOX inhibitors. A. Inhibition of cell-based LOX activity and cell viability upon treatment with 10 μM of the top 20 candidate LOX inhibitors. B. Doxorubicin sensitization upon combination of five selected compounds (5 μM) with doxorubicin (1 μM). C, D. LOX activity assays using recombinant LOX (C) LOXL2 (D) protein incubated with 10 μM of 6229 and 6232 and 10 mM BAPN. μM = microM.

[0134]

[0205] Figure 30. LOX inhibitors generated based on SAR analysis.

[0206] Figure 31. Novel 6232 analogs 6403 and 6415 effectively inhibit lysyl oxidase activity, overcome doxorubicin resistance, and reduce collagen cross-linking and fibronectin assembly. A. Inhibition of LOX activity in MDA-MB-231 cells treated with 5 μM LOX inhibitor. B. Doxorubicin sensitization with 6232 and its analogs 6403 and 6415 in 3D collagen-I cultures. C. Collagen and fibronectin staining of vehicle or 6232 or 6403 or 6415 treated MDA-MB-231 cells embedded in collagen-I. BAPN is used as a positive control.

[0135]

[0207] Figure 32. 6232 derivatives 6403 and 6415 show no cytotoxicity in normal cells. Viability assays in normal breast cell line, MCF12A, human foreskin fibroblast cell line, HFF, and human endothelial cell line, HUVEC, treated with 6403 or 6415.

[0136]

[0208] Figure 33. 6403 inhibits LOX recombinant protein, while 6415 inhibits both LOX and LOXL2 recombinant proteins. A. Lysyl oxidase activity assay with recombinant LOX and LOXL2 proteins in the presence or absence of 10 μM 6403. B. Activity assay with recombinant LOX and LOXL2 proteins in the presence or absence of 10 μM 6415.

[0137]

[0209] Figure 34. 6232, 6403 and 6415 are more potent than the LOX family inhibitor BAPN. IC50 on cellular lysyl oxidase activity upon treatment of MDA-MB-231 cells with different LOX inhibitors. μM = microM.

[0138]

[0210] Figure 35. 6403 and 6415 resulted in doxorubicin chemotherapy sensitization in 3D cultures of TNBC cell lines. Doxorubicin sensitization with 6232 analogs 6403 and 6415 in 3D collagen-I cultures of MDA-MB-231 (Doxo1: 0.75 μM, Doxo2: 1 μM, 6403: 5 μM, 6415: 5 μM) (A) and HCC1143 (Doxo1: 1 μM, Doxo2: 2 μM, 6403: 5 μM, 6415: 5 μM) (B).

[0139]

[0211] Figure 36. Combination of 6403 and 6415 with doxorubicin reduces proliferation of TNBC organoids. A. Representative images of TM01278 PDX organoids 0 and 9 days after treatment with doxorubicin (50 nM) and 6403 and 6415 treatments alone or in combination. B. Quantification of organoid diameter and viability over 9 days of combination therapy.

[0140]

[0212] Figure 37. LOX inhibitors, 6232, 6403 and 6415, reduced the migration ability of TNBC cell lines. RTCA-CIM plates were used to measure real-time migration of MDA-MB-231 cells upon treatment with 20 μM 6232, 6403 and 6415 for 48 hours. (*p<0.05; **p<0.01)

[0213] Figure 38. Dose-dependent inhibition of cellular LOX activity and proliferation inhibition in 3D upon treatment with 6232 and its analogs 6403 and 6415 in ACHN cancer cell lines. Inhibition of cell-based LOX activity in ACHN (A) and proliferation inhibition in collagen:matrigel (1:1) culture cancer cell lines upon treatment with LOX inhibitors 6232, 6403 and 6415 (μM range) (B). BAPN is used as a positive control (mM range) in the 3D cell viability assay. Proliferation inhibition was measured by using the 3D Cell Titer Glo kit.

[0141]

[0214] Figure 39. Inhibition of cellular LOX activity in pancreatic and renal cancer cell lines. Inhibition of cellular LOX activity upon treatment with BAPN, 6232, 6403 and 6415 in renal cell line Caki-1 (A) and pancreatic cancer cell lines Mia-Paca-2 (B) and PANC1 (C).

[0142]

[0215] Figure 40. Treatment with 6232, and its analogs 6403 and 6415, inhibited TGF-β-induced fibrosis in HFF cells. HFF cells serum-starved for 24 h were pretreated with inhibitors for 1 h. Fibrosis was then induced in HFF cells by 10 μM TGF-β for 48 h. ALK5 (10 μM) inhibitor is used as a negative control. LOX inhibitors 6232, 6403 and 6415 were used at a concentration of 10 μM. HFF cells were stained with alpha-SMA and fibronectin primary antibodies. Cell nuclei were labeled with DAPI.

[0143]

[0216] Figure 41. In vivo study of 6403 for PK and toxicity. A. Summary of PK parameters of 6403 at 50 mg / kg given orally, n=3. B. H&E staining of organs of BALB / c mice treated daily with 50 or 200 mg / kg 6403 for 1 week. C. Body weight of mice treated with vehicle or 25, 50, 75, 100 or 200 mg / kg 6403 for 5 days. D. Cell-free enzyme MAO-A and MAO-B assays.

[0144]

[0217] Figure 42. Efficacy of 6403 as a single agent in ccRC xenografts. Tumor volume (A), % body weight change (B), and blood counts (C) of ACHN xenografts treated with 6403 (50 mg / kg, PO, daily). μg = micrograms. LOX expression in ACHN cells is provided by inset next to the graph in A.

[0145]

[0218] Figure 43. 6403 overcomes doxorubicin resistance without significant toxicity in a syngeneic mouse model, 4T1. A. Tumor growth in 4T1 models treated with doxorubicin (2 mg / kg, IV, once a week), 6403 (50 mg / kg, PO, daily) or their combination. Representative photos of tumors are shown in the right panel. Western blot analysis of LOX in 4T1 tumors from BA. % body weight change in mice from CA. D. Blood cell counts in mice from A at the end of the experiment.

[0146]

[0219] method

[0220] Cell culture and reagents

[0221] Human TNBC cell lines, MDA-MB-231, and HCC1143; pancreatic cancer cell lines, PANC1, and Mia-PACA-2; and renal cancer cell lines, ACHN, and Caki-1. All cells were cultured in Dulbecco's modified Eagle's medium (Gibco, USA) supplemented with 50 U / ml penicillin / streptomycin, 1% non-essential amino acids, and 10% fetal bovine serum (Gibco, USA). Cell lines were regularly authenticated and tested for mycoplasma contamination using the MycoAlert mycoplasma detection kit (Lonza, NJ, USA). The cumulative culture time of cells between thawing and use in this study was less than 20 passages.

[0147]

[0222] Western blotting

[0223] Protein isolation and Western blotting were performed as previously described (see Mishra, R. R. et al., Reactivation of cAMP Pathway by PDE4D Inhibition Represents a Novel Druggable Axis for Overcoming Tamoxifen Resistance in ER-positive Breast Cancer. Clinical Cancer Research: An Official Journal of The American Association for Cancer Research 24, 1987-2001, doi:10.1158 / 1078-0432. CCR-17-2776 (2018) and Saatci, O. et al., Targeting PLK1 overcomes T-DM1 resistance via CDK1-dependent phosphorylation and inactivation of Bcl-2 / xL in HER2-positive breast cancer. Oncogene 37, 2251-2269, doi:10.1038 / s41388-017-0108-9 (2018)). Briefly, total protein lysates were isolated using RIPA buffer in the presence of protease and phosphatase inhibitor cocktails, and protein concentrations were measured using a BCA protein assay reagent kit (Thermo Fisher Scientific, MA, USA). Equal amounts of protein were separated using 8–10% SDS-PAGE gels. Separated proteins were transferred to PVDF membranes (Bio-Rad, CA, USA) using a Trans-Blot turbo transfer system (Bio-Rad, CA, USA) and incubated with primary antibodies (LOX (Novus), LOXL1 (Santa-Cruz), and LOXL2 (Abcam)). Horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies (Cell signaling Technology, MA, USA) were used as secondary antibodies, and signals were detected by enhanced chemiluminescence (Thermo Fisher Scientific, MA, USA).Images were acquired using Image Lab software (Biorad, CA, USA).

[0148]

[0224] Cell embedding in type I collagen:matrigel (1:1) mix

[0225] Collagen solutions were prepared from rat tail collagen I (Corning, USA) at concentrations of 0.5–1 mg / ml using a neutralization step involving the addition of 1N NaOH solution. Cells were trypsinized and resuspended in collagen:matrigel mix (1:1). 8 × 10 3 Cells were seeded into 96-well or μ-slide 8-well glass bottom chambers. After 1 hour incubation at room temperature, media was added on top of the solidified collagen:matrigel mix. Drug treatments were performed 12 hours after cells were seeded.

[0149] [0226 ] Immunofluorescence staining

[0227] Immunofluorescence staining of cells was performed in μ-slide 8-well glass-bottom chambers (Ibidi, Germany). Cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.5% TritonX-100 for 10 min, and blocked with 5% BSA-PBS. Samples were incubated with primary antibodies (collagen I, fibronectin, F-actin, a-SMA) and secondary Alexa Fluor 647- or 488-labeled antibodies for 1.5 h at room temperature. Cells were also counterstained with DAPI for 5 min. Images were acquired using a ZEN 2012 SP5 (Black) LSM 700 (Carl Zeiss, DE).

[0150]

[0228] Lysyl oxidase activity assay

[0229] For cell-based lysyl oxidase activity, a fluorometric lysyl oxidase activity assay kit (Abcam, USA) was used according to the manufacturer's instructions. Briefly, after treatment, 50 μl of supernatant from each well was incubated with the reagent mix, and the fluorescence at Ex / Em=540 / 590 nm wavelength was measured with a multimode reader.

[0151]

[0230] For in vitro lysyl oxidase activity (for recombinant proteins), a lysyl oxidase activity kit (Biovision, USA) was used according to the manufacturer's instructions. Briefly, 1 μg LOX (Novus, USA), 0.2 μg LOXL1 (Abcam, USA) and 0.1 μg LOXL2 (Abcam, USA) were incubated with the reagent mix in the presence of inhibitors or vehicle. The lysyl oxidase activity of the wells was measured in luminescence mode on a multimode reader (every 30 s, for a total of 2.5 h).

[0152]

[0231] Chemotherapy sensitization in 3D cultures

[0232] Cells were embedded in type I collagen: Matrigel mix as described above. Cells were grown for 48 hours in the presence of doxorubicin or LOX inhibitor alone, or LOX inhibitor and doxorubicin in combination. After treatment, cell viability was measured by using 3D Cell Titer Glo (Promega, USA) kit.

[0153]

[0233] PDX-derived TNBC organoids

[0234] TNBC organoids were established from fresh surgical tissue by cutting the tumor into small pieces and incubating in collagenase A solution containing ROCK inhibitor for 30 minutes at 37°C on a shaker. Collagenase activity was inhibited by adding FBS and pipetting was performed to ensure the formation of a solution with mostly single cells. After several washes with PBS, the cell pellet was dissolved in matrigel. Breast organoid medium containing ROCK and GSK inhibitors was added after the matrigel solidified. See Sachs, N. et al., A Living Biobank of Breast Cancer Organoids Captures Disease Heterogeneity. Cell 172, 373-386 e310, doi:10.1016 / j.cell.2017.11.010 (2018). For drug testing studies, organoids were disrupted into single cells by digestion with TrypLE (A1217701, Gibco, NY, USA) in the presence of 10 μM Rock inhibitor (s1049, Selleckchem, TX, USA) for 30 min at 37°C. Organoids were plated in wells of a 96-well plate (20.000 cells / well) with a collagen-coated surface and medium containing 2% Matrigel (356252, Corning, NY, USA). Drugs were added 72 hours after plating. Cells were grown for 9 days in the presence of drug or vehicle, and pictures from each well were taken to show organoid size. Organoid viability was measured by using a 3D Cell Titer Glo (Promega, USA) kit.

[0154]

[0235] Migration assay

[0236] RTCA CIM plates were used to evaluate the migration ability of cells upon treatment with LOX inhibitors according to the manufacturer's instructions. Briefly, 160 μl of 10% FBS containing medium was added to each bottom chamber / 50 μl of FBS-free medium to the top chamber and incubated at 37°C for 1 h. After background measurement, 5 × 10 4Cells were seeded in the upper chamber in FBS-free medium in the presence of LOX inhibitor or vehicle. After 30 min incubation at room temperature, real-time cell migration was recorded for 48 h.

[0155]

[0237] Fibrosis assay

[0238] Human foreskin fibroblast (HFF) cells (see Li, M. et al., Icaritin inhibits skin fibrosis through regulating AMPK and Wnt / β-catenin signaling. Cell Biochemistry and Biophysics 79, 231-238 (2021)) were seeded in μ-slide 8-well glass-bottom chambers (Ibidi, Germany). The next day, the cell medium was replaced with 0.5% FBS-containing medium and incubated for 24 hours. The cells were then pretreated with either an ALK5 inhibitor (Sellekchem, USA) or a LOX inhibitor for 1 hour, and the cells were incubated with 10 ng / ml TGF-beta (R&D Biosystems, USA) for 48 hours. Marker staining followed the same procedure as described in the immunofluorescence section above.

[0156]

[0239] PK analysis

[0240] The PK profile of the lead LOX inhibitor, 6403, was studied in BALB / c mice. Three mice per group were dosed at 50 mg / kg by oral gavage. Serum samples were collected at 0.25, 0.5, 2, 7 and 24 hours. 6403 was diluted with serum from healthy mice as a standard and serum levels of the LOX inhibitor were quantified by LC-MS / MS method and compared to standard readings followed by C max ,A.U.C.,T. 1 / 2 PK parameters including C value, clearance (Cl) and oral bioavailability (F%) were calculated. max represents the maximum concentration of drug observed in serum. AUC is a measure of the total exposure of the drug in serum. 1 / 2, half-life is the time required to eliminate half of the drug. Cl is defined as the volume of blood from which all the drug is removed per minute.

[0157]

[0241] MAO-GLO assay

[0242] MAO-GLO assay (Promega, USA) was used to measure MAO-A and MAO-B activity upon treatment with LOX inhibitors according to the manufacturer's instructions. For enzyme activity, substrates were used at concentrations of 160 μM and 16 μM for MAO-A and MAO-B, respectively. MAO-A and MAO-B enzymes (Active Motif, USA) were used at 0.2 μg per well. Reaction mix, enzyme, and drug (30, 10, 3, 1, 0.3, and 0.1 nM) were mixed and incubated at room temperature for 3 hours. Then, 50 μl of luciferin was added into the wells and incubated at room temperature for 20 minutes. Luciferase signal was measured by using a multimode reader.

[0158]

[0243] Syngeneic and xenograft mouse tumor models

[0244] For kidney xenografts, 4 × 10 6 ACHN cells were mixed with 1:1 DMEM and Matrigel and injected into the flank of BALB / c nude mice. The weight of the mice and the tumor volume were measured every 2 days using a caliper. When the tumor size was 100 mm 3 Upon reaching 10 days, mice were randomly assigned to vehicle and 6403 treatment groups. Mice were treated daily by oral gavage with vehicle (50% PEG400, 1% Tween80 in acetate buffer, pH=4) or 50 mg / kg of 6403.

[0159]

[0245] 500,000 4T1 cells were injected into the MFP of 6-8 week old female BALB / c mice. When tumors reached 100 mm3, mice were randomized into 4 groups and treated with vehicle, doxorubicin (2 mg / kg, once a week, IV), 6403 (50 mg / kg, daily, oral gavage), or a combination of doxorubicin and 6403. Tumor volumes were measured using calipers every 2 days, and body weights were also recorded. After 3 weeks of treatment, blood was collected and blood cells were quantified using a VetScan HM5 Hematology analyzer.

[0160]

[0246] Bioinformatic analysis

[0247] Patient data for normal vs. tumor comparisons were retrieved from the NCBI GEO database (Accession ID: GSE76250). Patient data for survival analysis were retrieved from the TCGA database using cBio Portal (see Cerami, E. et al., Vol. 2 401-404 (AACR, 2012) and Lopes, CT et al., Cytoscape Web: an interactive web-based network browser. Bioinformatics 26, 2347-2348 (2010)) and the online survival analysis tool, KM-plotter. See Gyorffy, B. et al., An online survival analysis tool to rapidly assess the effect of 22,277 genes on breast cancer prognosis using microarray data of 1,809 patients. Breast Cancer Research and Treatment 123, 725-731 (2010). Survival curves were generated based on the 25th percentile separation using the Kaplan-Meier method, and the significance between groups was calculated by the log-rank test. The significance between normal vs. tumor expression was calculated using the unpaired Student's t-test. Box plots show the median values ​​and the 25th to 75th quartiles. The upper and lower whiskers show the minimum and maximum values ​​in the corresponding groups. Graphs and curves were drawn using GraphPad software (GraphPad software Inc., La Jolla, CA, USA). Significance thresholds were described as *P<0.05; **P<0.01; ***P<0.001; ns, not significant.

[0161]

[0248] The compounds disclosed herein have a wide range of impacts on disease treatment.Indeed, the compounds disclosed herein or their pharma- ceutically acceptable salts may be administered in a therapeutically effective amount to inhibit LOX and LOX family members in both LOX-specific and pan-LOX manners.Indeed, the compounds disclosed herein may be administered as anti-cancer drugs, including cancers such as lung cancer; breast cancer; colorectal cancer; anal cancer; pancreatic cancer; prostate cancer; ovarian cancer; liver and bile duct cancer; esophageal cancer; non-Hodgkin's lymphoma; bladder cancer; uterine cancer; glioma, glioblastoma, medulloblastoma, and other brain tumors; myelofibrosis, kidney cancer; head and neck cancer; gastric cancer; multiple myeloma; testicular cancer; germ cell tumors; neuroendocrine tumors; uterine cancer; cervical cancer; carcinoids of the oral cavity, gastrointestinal tract, breast, and other organs; signet ring cell carcinoma; mesenchymal tumors, including sarcoma, fibrosarcoma, hemangioma, hemangiomatosis, hemangiopericytoma, pseudohemangiomatoid stromal hyperplasia, myofibroblastoma, fibromatosis, inflammatory myofibroblastic tumor, lipoma, angiolipoma, granular cell tumor, neurofibroma, neurilemmoma, angiosarcoma, liposarcoma, rhabdomyosarcoma, osteosarcoma, leiomyoma, or leiomyosarcoma.

[0162]

[0249] Indeed, compounds 6232, 6403, and 6415 can be used to target cancers and diseases caused by LOX family members and to induce inhibition of metastasis, although the present disclosure is not limited to only these compounds and should not be read restrictively or exclusively in this sense. The compounds disclosed herein inhibit both cell-based and recombinant protein-based lysyl oxidase activity. The compounds may be pharma- ceutically acceptable salts thereof, or pharmaceutical compositions used in the treatment of fibrosis, neurodegeneration, angiogenesis-related diseases and cancers that exhibit abnormal LOX family expression and / or lysyl oxidase activity. Indeed, the compounds disclosed herein may be used as single agents and / or in combination with standard care therapy, as well as chemotherapy, immunotherapy, or radiation therapy in both adjuvant and neoadjuvant settings. Furthermore, the compounds disclosed herein may be used with small molecule treatments to address the diseases / disorders herein. The compounds disclosed herein may be used to treat diabetes-induced fibrosis and retinopathy. Fibrosis may include liver fibrosis, pulmonary fibrosis, renal fibrosis, myocardial fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis, radiation-induced fibrosis, ocular fibrosis, Peyronie's disease and scleroderma, or respiratory disorders, abnormal wound healing and repair, post-operative surgery, cardiac arrest-related fibrosis; excessive or abnormal deposition of fibrotic material associated with disorders such as Crohn's disease and inflammatory bowel disease; liver, kidney and pancreatic fibrosis. The compound may also be used to treat angiogenesis-related diseases. In fact, the compounds disclosed herein may be administered with or in combination with a second therapeutic agent selected from anti-cancer agents, anti-inflammatory agents, antihypertensive agents, anti-fibrotic agents, anti-angiogenic agents, and immunosuppressants. The compound may also be used to treat kidney disorders such as renal fibrosis, renal fibrosis, acute kidney injury, chronic kidney disease, diabetic nephropathy, glomerulosclerosis, vesicoureteral reflux, tubulointerstitial kidney fibrosis and glomerulonephritis. Additionally, the compounds of the present disclosure may be targeted to healing and treating hypertrophic scars, keloids, and diabetic skin, and may be used for cosmetic applications.See Pathol Biol (Paris) 2005 Sep, 53(7):448-56. doi:10.1016 / j.patbio.2004.12.033.

[0163]

[0250] Exemplary Synthetic Routes

[0251] Compounds 6232, 6398, 6399, 6400, 6401, 6402, 6403, 6404, 6405, 6406, 6407, 6408, 6409, 6410, 6411, 6412, 6413, 6414, and 6415 were purchased from Chembridge Corporation (San Diego, CA, USA, 92121) and tested as is. The synthesis of compounds 6424 (synthesized as the formate salt of 6403), 6432 (synthesized as the formate salt of 6405), 6433 (synthesized as the formate salt of 6232), and 6439 (synthesized as the formate salt of 6415) is described below. Compounds 6425 and 6426 have not been synthesized before and are novel. Compounds purchased from Chembridge may be resynthesized using the same general method. A general scheme encompassing the synthesis of 4-methylthiazol-2-amine and future derivatives containing substitution at the aniline ring is shown below:

[0252]

[0164] [ka]

[0165]

[0253] Synthesis of 1-(2-amino-4-methyl-thiazol-5-yl)-2-bromo-ethanone (1)

[0254]

[0166] [ka]

[0167]

[0255] 1-(2-amino-4-methyl-thiazol-5-yl)ethanone (5 g) was dissolved in HBr (20 ml) and warmed to 60° C. Then Br2 (4.6 g) was dissolved in 1,4-dioxane and added in a dropwise fashion. The reaction was stirred at 60° C. for 3 hours. After cooling to room temperature, the mixture was basified with Na2CO3 after which a solid product came out of solution. The solid was filtered off. The remaining solution was extracted with ethyl acetate, then the organic layer was washed with brine, dried over sodium sulfate and rotary evaporated to dryness. The remaining crude material was combined with the filtered solid and dried in a vacuum desiccator (7.5 g, 50% purity), ESI-MS (pos): m / z 235 (M+H). + (Expected value 235.10).

[0168]

[0256] Synthesis of N-[4-[[4-(2-amino-4-methyl-thiazol-5-yl)thiazol-2-yl]amino]phenyl]acetamide (6403, 6424)

[0257] Synthesis of N-[4-(carbamothioylamino)phenyl]acetamide

[0258]

[0169] [ka]

[0170]

[0259] N-(4-aminophenyl)acetamide (6 g) was dissolved in anhydrous DCM and benzoyl isothiocyanate (8.15 g) was added in a dropwise fashion at room temperature. The reaction was then stirred overnight. The solvent was removed and the product was redissolved in 1:1 THF / 1M NaOH and then refluxed at 75° C. for 3 h. The sample was cooled to room temperature, diluted with water (approximately 150 mL) and extracted with ethyl acetate (4 times) to give N-[4-(carbamothioylamino)phenyl]acetamide (2.2 g, 23% yield, 86% purity). ESI-MS (pos): m / z 210.0309 (M+H). + (Expected value 210.06).

[0171]

[0260] Synthesis of N-[4-[[4-(2-amino-4-methyl-thiazol-5-yl)thiazol-2-yl]amino]phenyl]acetamide (6403, 6424)

[0261]

[0172] [ka]

[0173]

[0262] N-[4-(carbamothioylamino)phenyl]acetamide (1.1 g) and 1-(2-amino-4-methyl-thiazol-5-yl)-2-bromo-ethanone (1.85 g) were dissolved in 30 mL of ethanol and the solution was heated to 90 °C for 2-3 h. After completion of the starting material, the solvent was removed under reduced pressure at a water bath temperature of 45 °C. The crude material was redissolved in 3 mL of DMSO and purified by medium pressure liquid chromatography using a C18 reverse phase column and acetonitrile-water as eluent with 1% aqueous formic acid additive to give the desired product (1.3 g, 99% pure) in good yield (71.6%). 1 H-NMR (400 MHz, D6DMSO) δ ppm 10.20 (s, 1H), 9.849 (s, 1H), 8.141 (s, 1H), 7.500-7.731(m, 6H), 2.353 (s, 3H), 2.025 (s, 3H).ESI-MS(pos):m / z346.1247(M+H) + (Expected value 346.08).

[0174]

[0263] Synthesis of 4-[[4-(2-amino-4-methyl-thiazol-5-yl)thiazol-2-yl]-methyl-amino]phenol (6415, 6439)

[0264] 1) Synthesis of 4-(methylamino)phenol

[0265]

[0175] [ka]

[0176]

[0266] 4-Aminophenol (5 g) was placed in an oven-dried 250 mL round bottom flask and 20 mL of dimethyl carbonate (20 mL) was added in a sequential manner, followed by dimethoxyethane (20 mL) and NaY catalyst (5 g). The mixture was heated to 86 °C for 16 h. The solvent and excess reagents were then evaporated, the crude material was redissolved in 70-80 mL of methanol, the solid catalyst was filtered through a celite pad, and the methanol was concentrated under reduced pressure at a water bath temperature of 45 °C to give the product (5.6 g, 14% yield). ESI-MS (pos): m / z 124 (M+H) + .Expected value 124.

[0177]

[0267] 2) Synthesis of 1-(4-aminophenyl)-1-methylthiourea

[0268]

[0178] [ka]

[0179]

[0269] Crude 4-(methylamino)phenol (2.5 g) was dissolved in anhydrous DCM and then benzoyl isothiocyanate (5.0 g) was added in a dropwise fashion at room temperature (25° C.). The reaction mixture was allowed to warm slightly during the process of addition. The reaction was then stirred at room temperature overnight. After 16 hours, the DCM was evaporated to dryness and then a 1:1 THF / 1M NaOH solution was added until the solids were completely dissolved. The reaction was then refluxed at 75° C. for 8 hours. The organic and aqueous layers were separated and the aqueous layer was acidified with concentrated HCl (pH=3-4) and then extracted with ethyl acetate, the solids were filtered and the organic layer was concentrated under reduced pressure. LCMS showed the desired mass peak at 183.

[0180]

[0270] 3) Synthesis of 4-[[4-(2-amino-4-methyl-thiazol-5-yl)thiazol-2-yl]-methyl-amino]phenol (6415, 6435)

[0271]

[0181] [ka]

[0182]

[0272] 1-(4-aminophenyl)-1-methylthiourea (1.8 g) and 1-(2-amino-4-methyl-thiazol-5-yl)-2-bromo-ethanone (3.48 g) were dissolved in 30 mL of ethanol and the solution was heated to 90 °C for 2-3 h. After completion of the starting material, the solvent was removed under reduced pressure at a water bath temperature of 45 °C. The crude material was redissolved in 3 mL of DMSO and purified by medium pressure liquid chromatography using a C18 reverse phase column and acetonitrile-water as eluent with 1% aqueous formic acid additive to give the desired product (2.5 g, 99% pure) in good yield (79.5%). 1 H-NMR (400 MHz, D6DMSO) δ ppm 7.235-7.258 (m, 2H), 6.900 (s, 1H), 6.818-6.850 (m, 2H), 3.379 (s, 3H), 2.2848 (s, 3H).ESI-MS(pos):m / z319.1(M+H) + (Expected value 319.1).

[0183]

[0273] Synthesis of N-[4-[[4-(2-amino-4-methyl-thiazol-5-yl)thiazol-2-yl]amino]phenyl]-3-methoxy-propanamide (6425).

[0184]

[0274] 1) Synthesis of N-[4-(carbamothioylamino)phenyl]-3-methoxy-propanamide

[0275]

[0185] [ka]

[0186]

[0276] N-(4-aminophenyl)-3-methoxy-propanamide (0.24 g) was suspended in 2 mL of dry DCM and benzoyl isothiocyanate (0.26 g) was dissolved in 1 mL of dry DCM in portions. Benzoyl isothiocyanate was then added dropwise to the aniline over 1 min with stirring. The solution was then rotary evaporated to dryness and dissolved in a solution of THF / 1M NaOH (1:1). The solution was then refluxed at 75° C. for 3 h. The sample was then cooled to room temperature, diluted with water and extracted three times with ethyl acetate. The organic solution was then dried over sodium sulfate, filtered, rotary evaporated to dryness and purified by medium pressure liquid flash chromatography to give the product (0.053 g, 16.6% yield, 99% purity). ESI-MS (pos): m / z 254.1 (M+H) + (Expected value 254.1).

[0187]

[0277] 2) Synthesis of N-[4-[[4-(2-amino-4-methyl-thiazol-5-yl)thiazol-2-yl]amino]phenyl]-3-methoxy-propanamide (6425).

[0188]

[0278]

[0189] [ka]

[0190]

[0279] N-[4-(carbamothioylamino)phenyl]-3-methoxy-propanamide (0.019 g) and 1 (0.023 g) were dissolved in ethanol and refluxed for 90 min, then the sample was rotary evaporated to dryness, dissolved in DMSO, and purified by reversed-phase flash chromatography to give the product (0.029 g, 99% pure). ESI-MS (pos): m / z 390.1 (M+H). + (Expected value 390.1).

[0191]

[0280] Synthesis of N-[4-[[4-(2-amino-4-methyl-thiazol-5-yl)thiazol-2-yl]amino]phenyl]pentanamide (6526)

[0281] 1) Synthesis of N-[4-(carbamothioylamino)phenyl]pentanamide

[0282]

[0192] [ka]

[0193]

[0283] N-(4-aminophenyl)pentanamide (0.044 g) was suspended in 2 mL of dry DCM and benzoyl isothiocyanate (0.047 g) was dissolved in 1 mL of dry DCM in portions. Benzoyl isothiocyanate was then added dropwise to the aniline over 1 min with stirring. The solution was then rotary evaporated to dryness and dissolved in a solution of THF / 1M NaOH (1:1). The solution was then refluxed at 75° C. for 3 h. The sample was then cooled to room temperature, diluted with water and extracted three times with ethyl acetate. The organic solution was then dried over sodium sulfate, filtered, rotary evaporated to dryness and purified by medium pressure liquid flash chromatography to give the product (0.007 g). ESI-MS (pos): m / z 252.1 (M+H) + (Expected value 252.1).

[0194]

[0284] 2) Synthesis of N-[4-[[4-(2-amino-4-methyl-thiazol-5-yl)thiazol-2-yl]amino]phenyl]pentanamide (6526)

[0285]

[0195] [ka]

[0196]

[0286] N-[4-(carbamothioylamino)phenyl]pentanamide (0.0073 g) and 1 (0.0047 g) were dissolved in ethanol and refluxed for 90 min, then the sample was rotary evaporated to dryness, dissolved in DMSO, and purified by reversed-phase flash chromatography to give the product (0.038 g, 99% pure). ESI-MS (pos): m / z 388.1 (M+H). + (Expected value 388.1).

[0197]

[0287] Synthesis of 4-methyl-5-[2-(4-nitroanilino)thiazol-4-yl]thiazol-2-amine (6232, 6433).

[0288] 1) Synthesis of (4-nitrophenyl)thiourea

[0289]

[0198] [ka]

[0199]

[0290] 4-Nitroaniline (2.0 g) was suspended in 2 mL of dry DCM and benzoyl isothiocyanate (2.95 g) was dissolved in 1 mL of dry DCM in portions. Benzoyl isothiocyanate was then added dropwise to the aniline over 1 min with stirring. The solution was then rotary evaporated to dryness and dissolved in a solution of THF / 1M NaOH (1:1). The solution was then refluxed at 75° C. for 1 h. The sample was then cooled to room temperature, diluted with water and extracted three times with ethyl acetate. The organic solution was then dried over sodium sulfate, filtered, rotary evaporated to dryness and purified by medium pressure liquid flash chromatography to give the product (0.181 g). ESI-MS (pos): m / z 198.0 (M+H) + (Expected value 198.0).

[0200]

[0291] 2) Synthesis of 4-methyl-5-[2-(4-nitroanilino)thiazol-4-yl]thiazol-2-amine (6232, 6433)

[0292]

[0201] [ka]

[0202]

[0293] (4-Nitrophenyl)thiourea (0.18 g) and 1 (0.30 g) were dissolved in ethanol and refluxed for 90 min, then the sample was rotary evaporated to dryness, dissolved in DMSO, and purified by reverse phase flash chromatography to give the product (0.041 g, 99% pure). (Expected 334.0). 1 H-NMR (400 MHz, D3 methanol) δ ppm 8.027-8.124 (m, 3H), 7.742-7.766 (d, 2H), 6.667 (s, 1H), 2.326 (s, 3H).ESI-MS(pos):m / z334.0(M+H) + Estimated value: 334.0.

[0203]

[0294] Synthesis of 4-((2'-amino-4'-methyl-[4,5'-bithiazol]-2-yl)amino)benzenesulfonamide (6405, 6432).

[0295] 1) Synthesis of (4-sulfamoylphenyl)thiourea

[0296]

[0204] [ka]

[0205]

[0297] 4-Aminobenzenesulfonamide (0.10 g) was dissolved in HCl with warming and then cooled to room temperature. Potassium thiocyanate (0.056 g) was then added and the sample was microwaved at 90° C. for 2 hours. The sample was then transferred to a vial, cooled on ice, precipitated by scratching, and filtered to leave a white solid (0.006 g, 99% pure). ESI-MS (pos): m / z 232.0 (M+H) + (Expected value 232.3).

[0206]

[0298] 2) Synthesis of 4-((2'-amino-4'-methyl-[4,5'-bithiazol]-2-yl)amino)benzenesulfonamide (6405, 6432).

[0299]

[0207] [ka]

[0208]

[0300] (4-Sulfamoylphenyl)thiourea (0.13 g) and 1 (0.12 g) were dissolved in ethanol and refluxed for 90 min, then the sample was rotary evaporated to dryness, dissolved in DMSO, and purified by reverse phase flash chromatography to give the product (0.064 g, 99% pure). ESI-MS (pos): m / z 368.0 (M+H). + (Expected value 368.0).

[0209]

[0301] Various modifications and variations of the described methods, pharmaceutical compositions, and kits of the present disclosure will be apparent to those skilled in the art without departing from the scope and spirit of the present disclosure. Although the present disclosure has been described in connection with specific embodiments, it will be understood that further modifications are possible and the claimed disclosure should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the present disclosure that are obvious to those skilled in the art are intended to be within the scope of the present disclosure. This application is intended to cover any variation, use, or adaptation of the present disclosure, including such deviations from the present disclosure that generally follow the principles of the present disclosure and fall within the scope of known ordinary practice within the art to which the present disclosure pertains, and can be applied to the essential features hereinbefore described.

Claims

1. A pharmaceutical composition for inhibiting lysyl oxidase, comprising: Formula I: 【Chemistry 1】 (In the formula, R １ is CH ３ and R ２ , H, NH ２ or NHR, R ３ is H or CH ３ and R ４ is H, R ５ is H, R ６ NHCOCH ３、 NO ２ , C ２ OH, or OH, R ７ is H, Q1 is N; Q2 is S; Q3 is N; Q4 is CH; Q5 is S; Q6 is CH; Q7 is CH; Q8 is C; or a pharma- ceutically acceptable salt thereof, The composition, wherein the compound or a pharma- ceutically acceptable salt thereof inhibits cell-based lysyl oxidase activity and / or a recombinant protein-based lysyl oxidase activity assay.

2. 2. The composition of claim 1 for treating neurodegenerative diseases, angiogenesis-related diseases, Alzheimer's disease; fibrosis including liver fibrosis, pulmonary fibrosis, renal fibrosis, myocardial fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis, radiation-induced fibrosis, ocular fibrosis, Peyronie's disease and scleroderma; or respiratory disorders, abnormal wound healing and repair, post-operative surgery, cardiac arrest-related fibrosis; disorders such as Crohn's disease and inflammatory bowel disease, all associated with excessive or abnormal deposition of fibrotic material; liver, kidney and pancreas fibrosis, diabetes, cerebral hemorrhage with amyloidosis, cardiac hypertrophy, Hutchinson-Gilford Progeria syndrome, retinopathy, chemotherapy resistance; and / or renal disorders including renal fibrosis, renal fibrosis, acute kidney injury, chronic kidney disease, diabetic nephropathy, glomerulosclerosis, vesicoureteral reflux, hypertrophic scars, keloids, diabetic skin, tubulointerstitial renal fibrosis and / or glomerulonephritis.

3. The cancer is lung cancer; breast cancer; colorectal cancer; anal cancer; pancreatic cancer; prostate cancer; ovarian cancer.

3. The composition of claim 2, wherein the therapeutic agent is selected from the group comprising cancer; liver and bile duct cancer; esophageal cancer; non-Hodgkin's lymphoma; bladder cancer; uterine cancer; glioma, glioblastoma, medulloblastoma, and other brain tumors; myelofibrosis, kidney cancer; head and neck cancer; gastric cancer; multiple myeloma; testicular cancer; germ cell tumors; neuroendocrine tumors; cervical cancer; oral cancer, carcinoids of the digestive tract, breast, and other organs; signet ring cell carcinoma; sarcoma, fibrosarcoma, hemangiomas, hemangiomatoses, hemangiopericytomas, pseudohemangiomatous stromal hyperplasia, myofibroblastoma, fibromatosis, inflammatory myofibroblastic tumor, lipoma, angiolipoma, granular cell tumor, neurofibroma, neurilemmoma, angiosarcoma, liposarcoma, rhabdomyosarcoma, osteosarcoma, mesenchymal tumors including leiomyoma and / or leiomyosarcoma.

4. The compound or a pharma- ceutically acceptable salt thereof has the formula: 【Chemistry 2】 The composition of claim 1 having the formula:

5. The compound or a pharma- ceutically acceptable salt thereof has the formula: 【Chemistry 3】 The composition of claim 1 having the formula:

6. The compound or a pharma- ceutically acceptable salt thereof has the formula: 【Chemistry 4】 The composition of claim 1 having the formula:

7. The compound or a pharma- ceutically acceptable salt thereof has the formula: 【Chemistry 5】 The composition of claim 1 having the formula:

8. 2. The composition according to claim 1, wherein the composition is used as an anti-cancer agent, and the cancer is selected from the group consisting of lung cancer, breast cancer, colorectal cancer, anal cancer, pancreatic cancer, prostate cancer, ovarian cancer, liver and bile duct cancer, esophageal cancer, non-Hodgkin's lymphoma, bladder cancer, uterine cancer, glioma, glioblastoma, medulloblastoma, and other brain cancers, myelofibrosis, kidney cancer, head and neck cancer, gastric cancer, multiple myeloma, testicular cancer, germ cell tumors, neuroendocrine tumors, cervical cancer, and oral cancer. said composition being selected from the group comprising cavity carcinoma, carcinoid of the gastrointestinal tract, breast, and other organs; signet ring cell carcinoma; mesenchymal tumors including sarcoma, fibrosarcoma, hemangioma, hemangiomatosis, hemangiopericytoma, pseudohemangiomatous stromal hyperplasia, myofibroblastoma, fibromatosis, inflammatory myofibroblastic tumor, lipoma, angiolipoma, granular cell tumor, neurofibroma, neurilemmoma, angiosarcoma, liposarcoma, rhabdomyosarcoma, osteosarcoma, leiomyoma and / or leiomyosarcoma.

9. The composition of claim 1 for reducing the cardiotoxic side effects of chemotherapy and / or immunotherapy.

10. 10. The composition of claim 1, administered as a single agent or in combination with chemotherapy, immunotherapy and / or radiation therapy in both adjuvant and neoadjuvant settings.

11. The composition of claim 1 for inducing inhibition of metastasis.

12. 10. The composition of claim 1, administered with a second therapeutic agent selected from an anti-cancer agent, an anti-inflammatory agent, an anti-hypertensive agent, an anti-fibrotic agent, an anti-angiogenic agent, and / or an immunosuppressant.

13. formula: 【Chemistry 6】 A novel compound having the formula:

14. A pharmaceutical composition for inhibiting lysyl oxidase, comprising the novel compound of claim 13 or a pharma- ceutically acceptable salt thereof.

15. Neurodegenerative diseases, angiogenesis-related diseases, Alzheimer's disease; fibrosis including liver fibrosis, pulmonary fibrosis, renal fibrosis, myocardial fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis, radiation-induced fibrosis, ocular fibrosis, Peyronie's disease and scleroderma; or respiratory disorders, abnormal wound healing and repair, post-operative surgery, cardiac arrest-related fibrosis; excessive or abnormal deposition of fibrotic material associated with all of the disorders, e.g., Crohn's disease and inflammatory bowel disease; liver, kidney and pancreatic fibrosis, diabetes, Alzheimer's disease, and other conditions that may be associated with fibrosis.

14. A pharmaceutical composition comprising the novel compound of claim 13 or a pharma- ceutical acceptable salt thereof for treating cerebral hemorrhage with myloidosis, cardiac hypertrophy, Hutchinson-Gilford Progeria Syndrome, retinopathy, chemotherapy resistance; and / or renal disorders including renal fibrosis, renal fibrosis, acute kidney injury, chronic kidney disease, diabetic nephropathy, glomerulosclerosis, hypertrophic scars, keloids, diabetic skin, vesicoureteral reflux, tubulointerstitial renal fibrosis and / or glomerulonephritis.

16. 14. A pharmaceutical composition comprising the novel compound of claim 13 or a pharma- ceutically acceptable salt thereof, administered as a single agent or in combination with chemotherapy, immunotherapy and / or radiation therapy in both adjuvant and neoadjuvant settings.

17. 14. A pharmaceutical composition comprising the novel compound of claim 13 or a pharma- ceutical acceptable salt thereof, administered in combination with a second therapeutic agent selected from an anticancer agent, an anti-inflammatory agent, an antihypertensive agent, an antifibrotic agent, an antiangiogenic agent, and / or an immunosuppressant.

18. formula: 【Chemistry 7】 A novel compound having the formula:

19. A pharmaceutical composition comprising the novel compound of claim 18 for inhibiting lysyl oxidase.

20. 20. A pharmaceutical composition comprising the novel compound of claim 18 or a pharmacologic acceptable salt thereof for treating neurodegenerative diseases, angiogenesis-related diseases, Alzheimer's disease; fibrosis including liver fibrosis, pulmonary fibrosis, renal fibrosis, myocardial fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis, radiation-induced fibrosis, ocular fibrosis, Peyronie's disease and scleroderma; or respiratory disorders, abnormal wound healing and repair, post-operative surgery, cardiac arrest-related fibrosis; disorders such as Crohn's disease and inflammatory bowel disease, associated with excessive or abnormal deposition of fibrotic material; liver, kidney and pancreas fibrosis, diabetes, cerebral hemorrhage with amyloidosis, cardiac hypertrophy, Hutchinson-Gilford Progeria syndrome, retinopathy, chemotherapy resistance; and / or renal disorders including renal fibrosis, renal fibrosis, acute kidney injury, chronic kidney disease, diabetic nephropathy, glomerulosclerosis, vesicoureteral reflux, tubulointerstitial renal fibrosis and / or glomerulonephritis.

21. 20. A pharmaceutical composition comprising the novel compound of claim 18 or a pharma- ceutically acceptable salt thereof, administered as a single agent or in combination with chemotherapy, immunotherapy and / or radiation therapy in both adjuvant and neoadjuvant settings.

22. 20. A pharmaceutical composition comprising the novel compound of claim 18 or a pharma- ceutical acceptable salt thereof, administered in combination with a second therapeutic agent selected from an anticancer agent, an anti-inflammatory agent, an antihypertensive agent, an antifibrotic agent, an antiangiogenic agent, and / or an immunosuppressant.

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