CN117355304A - ALK-5 inhibitors and uses thereof - Google Patents

Abstract

Provided herein are compounds (e.g., compounds having formulas (I), (II), (III), and (IV), or compounds listed in table 1), and pharmaceutically acceptable salts thereof, pharmaceutical compositions of one of the foregoing, and kits comprising the foregoing. The compounds provided herein are inhibitors of an activin receptor-like kinase (e.g., ALK-5) and are useful for treating and/or preventing a disease (e.g., a proliferative disease, such as cancer) in a subject, for inhibiting tumor growth in a subject, and/or for inhibiting the activity of an activin receptor-like kinase (e.g., ALK-5) in vitro or in vivo.

Description

ALK-5 inhibitors and uses thereof

Cross Reference to Related Applications

The present application claims the benefit of U.S. provisional application No. 63/166,610, filed on day 2021, 3, 26, and U.S. provisional application No. 63/215,122, filed on day 2021, 6, 25. The entire teachings of the above application are incorporated herein by reference.

Background

Activin receptor-like kinase 5 (ALK-5), also known as the TGF-beta receptor type 1 (tgfp R1), is a therapeutic target for example for proliferative diseases such as cancer, as it is believed to have an effect of promoting tumor growth, survival and metastasis. ALK-5 is a member of the TGF- β receptor superfamily, which is thought to regulate a broad range of cellular processes. Modulation of TGF- β signaling is important for controlling cellular processes associated with cell proliferation. See, e.g., akhurst, r.j. And Hata, a., "Targeting the TGF-beta Signalling Pathway in Disease [ TGF-beta signaling pathway targeting disease ]", nat. Rev. Drug Disc. [ natural review drug discovery ],11 pages 790-811 (2012) and Hallberg and Palmer, "The role of the ALK receptor in cancer biology [ role of ALK receptor in cancer biology ]", annals of Oncology [ annual oncology ],2016,27, iii4.

Typically, during TGF-beta signaling, type I and type II receptors are brought together, both of which are serine/threonine kinases. To date, seven type I receptors are known: activin receptor-like kinases 1 to 7 (ALK-1 to ALK-7). In some cases, TGF-beta signals through a combination of T beta R-II (type II receptor) and ALK-5. Upon activation, type I receptors transduce signals through various proteins, e.g., activated type I receptors phosphorylate receptor-mediated SMAD subfamily members, which enable their complexation with mediator SMAD. The resulting activated SMAD complexes accumulate in the nucleus where they play a role in the transcription of the target gene. Blocking this TGF-beta signaling pathway by ALK inhibition (especially ALK-5 inhibition) is an attractive target for therapy because this pathway plays a complex role in cell proliferation, differentiation, adhesion, migration, and apoptosis. Cell mutations have been observed in proliferative and fibrotic diseases, in which normal proliferative repression of TGF- β signaling is conferred, allowing uncontrolled proliferation of cells, see e.g. Blobe, g.c. et al, "Role of Transforming Growth Factor β in Human Disease [ transforming growth factor β role in Human Disease ]", N Engl J Med [ new england journal of medicine ] (342), pages 1350-1358 (2000); ballester, B.et al, "Idiopathic Pulmonary Fibrosis and Lung cancer: mechanisms and Molecular Targets [ idiopathic pulmonary fibrosis with lung cancer: mechanism and molecular targets ] ", int.j.of Molecular Sciences [ journal of international molecular science ]20 (593), doi:10.3390/ijms20030593 (2019), and Huang, j.j. And Blobe, g.c." Dichotomous Roles of TGF- βin Human Cancer [ dual role of TGF- β ] in Human Cancer, "Biochem soc. Trans [ journal of biochemistry ]342 (2016); 1441-1454 (https:// doi.org/10.1042/BST 20160065).

TGF- β is an important pathway in cancer that promotes tumor growth and immune escape, and plays a role in other cancer processes such as metastasis and angiogenesis. Upregulation of components of the TGF-beta pathway, including ligands and receptors, is observed in many types of cancer and is often associated with adverse consequences (de Reyni, A., javelaud, D., elarouci, N.et al, sci Rep [ science report ]10,14491 (2020), https:// doi.org/10.1038/s 41598-020-71559-w). Abnormal TGF-beta signaling has been shown to be involved in the pathogenesis of a variety of cancer types including triple negative breast cancers (Bhola, neil E. Et al, "TGF-. Beta. inhibition enhances chemotherapy action against triple-negative breast cancer [ TGF-. Beta.inhibition enhances chemotherapy against triple negative breast cancers ]" The Journal of clinical investigation [ journal of clinical research ]123.3 (2013) https:// doi/10.1172/JCI 65416; vishnubalaji, rad-rishanan and Nehad M. "Epigenetic regulation of Triple Negative Breast Cancer (TNBC) [ TGF-. Beta.signaling ]" epigenetic regulation of Triple Negative Breast Cancers (TNBC) [ scientific report ] "Scientific Reports.1 (https.)," support/doi. 41598-021-94514-9), pancreatic cancers (Gokeel. Progress in cancer genetics: lessons from pancreatic cancer [ cancer genetic progress: pancreatic cancer ], [ 21 ], [ 62 ]/6. Support ], "support (6. Support) and support [ support 6. Support ]," support (support) and support [ 10. Support ], "support 6. Support (support) and support 6. Support (support) 6. Support) of the foundation [ support (support) of the three negative breast cancer ] (TNBC) [ support ]," support ] (support) 11.1. Support (support) of three negative breast cancer (TNBC.) [ support ] (support) and (support) 4.1). Diana et al "active-like kinase 5 (ALK 5) inactivation in the mouse uterus results in metastatic endometrial carcinoma [ mouse endometrium Activin-like kinase 5 (ALK 5) inactivation resulted in metastatic endometrial cancer ]" Proceedings of the National Academy of Sciences [ national academy of sciences of the United states of America ]116.9 (2019) https:// doi.org/10.1073/pnas.1806838116, newstep, daniel et al "block of TGF-beta signaling with novel synthetic antibodies limits immune exclusion and improves chemotherapy response in metastatic ovarian cancer models [ blocking TGF-beta signaling with novel synthetic antibodies in metastatic ovarian cancer models to limit immune rejection and improve chemotherapeutic response ]," Oncoimmunogy [ tumor immunology ]8.2 (2019) https:// doi.org/10.1080/2162402X.2018.1539613.

Signaling of this pathway begins with release of the potential ligand (TGF-beta) and binding to a specific serine/threonine residue on a specific receptor (TGF-beta R2) which then binds to and phosphorylates a second receptor (TGF-beta R1, also known as ALK 5). This complex in turn phosphorylates and activates members of the SMAD protein family that translocate to the nucleus and regulate expression of target genes of the TGF- β pathway (Weiss, alexander and Liliana attisano. "The TGFbeta superfamily signaling pathway [ tgfβ superfamily signaling pathway ]," Wiley Interdisciplinary Reviews: developmental Biology [ wili trans-discipline comment: developmental biology ]2.1 (2013) https:// doi.org/10.1002/wtev.86).

Activation of The TGF-beta pathway can lead to immune escape of tumor cells by epithelial to mesenchymal transition (EMT) (Wang, G., xu, D., zhang, Z. Et al The pan-cancer landscape of crosstalk between epithelial-mesenchymal transition and immune evasion relevant to prognosis and immunotherapy response [ Pan cancer Condition of crosstalk between epithelial to mesenchymal transition and immune escape associated with prognostic and immunotherapeutic responses ]. Npj Precis.Onc. [ npj precision oncology ]5,56 (2021). Https:// doi.org/10.1038/s 41698-021-00200-4). It can also lead to immunosuppression by direct suppression of congenital and adaptive immune cells and stimulation of suppressive tregs and MDSCs (de Streel, gregoire and Sophie lucas. "Targeting immunosuppression by TGF- β1for cancer immunotherapy [ targeting immunosuppression caused by TGF- β1for cancer immunotherapy ]" Biochemical Pharmacology [ biochemistry ] (2021) https:// doi.org/10.1016/j.bcp.2021.114697). In addition, TGF-beta strongly modulates tumor microenvironment by altering the levels of ECM proteins and signaling molecules, resulting in immune cell depletion (Ghahremannifard, P.; chanda, A.; bonni, S.; bose, P.TGF-. Beta. Mediated Immune Evasion in Cancer-Spotlight on Cancer-Associated Fibroblasts [ TGF-. Beta.mediates immune escape in cancer-focusing cancer-associated fibroblasts ]. Cancer [ cancer ]2020,12,3650.Https:// doi.org/10.3390/cancer 12123650).

Ovarian Granuloma (GCT) accounts for about 5% of malignant ovarian cancers, and according to recent reports, 95% -97% of adult granulomas carry a unique somatic mutation 402C > C in The FOXL2 gene (Jamieson, s., butzow, r., andersson, n. et al The FOXL 2C 134W mutation is characteristic of adult granulosa cell tumors of The ovary [ FOXL 2C 134W mutation is characteristic of adult ovarian granuloma ]. Mod Pathol [ modern pathology ]23,1477-1485 (2010). Https:// doi.org/10.1038/modpathol.2010.145). The 402C > G Mutation results in an amino acid substitution, i.e., substitution of a cysteine to tryptophan (C134W) (Shah SP, kobel M, senz J, morin RD, clarke BA et al (2009) Mutation of FOXL2 in granulosa-cell tumors of the ovary [ FOXL2Mutation in ovarian granulocytoma ]. NEngl J Med [ J.New England medical journal ] 360:2719-2729), which is located in the second wing on the surface of the fork domain. Computer modeling shows that this change does not disrupt the folding of the FOXL2 fork domain or its interaction with DNA. Furthermore, mutations have been shown not to affect the localization of the FOXL2 protein (Benayoun BA, caburet S, dipieromaria A, georges A, D' Haene B et al (2010) Functional exploration of the adult ovarian granulosa cell tumor-associated somatic FOXL2mutation p.Cys134Trp (c.402C > G) [ function discovery of somatic FOXL2 mutations p.Cys134Trp (c.402C > G) associated with adult ovarian granulomas ]. PloS one [ journal of public science 5:e8789). Thus, it is believed that the pathogenicity of mutant FOXL2 occurs by altering its interaction with other proteins. Such candidate proteins include SMAD transcription factors and effectors of TGF- β and BMP family signaling (Kobel M, gilks CB, huntsman DG (2009) add-type granulosa cell tumors and FOXL2mutation [ Adult granulocytoma and FOXL2mutation ]. Cancer Res [ research ] 69:9160-9162). Furthermore, many transcriptional targets of mutant FOXL2 are known TGF- β signaling genes. Thus, the deregulation of this critical antiproliferative pathway is that unidirectional mutant FOXL2 promotes the onset of adult GCT (Rosario R, araki H, print CG, shelling AN (2012) The transcriptional targets of mutant FOXL2 in granulosa cell tumors [ transcription target of mutant FOXL2 in granulocytoma ]. PloS one [ journal of public science library ]; https:// doi. Org/10.1371/journ. Fine.0046270).

Activin receptor-like kinases are considered important therapeutic targets for proliferative diseases (such as cancer) because they play a role in promoting tumor growth, survival and metastasis. For example, many small molecule ALK-5 inhibitors have been demonstrated to have antiproliferative activity in a variety of cancers and tumor types. Small molecule SB-431542 was developed as an ALK-5 inhibitor and was found to inhibit other activin receptor-like kinases (ALK-4 and ALK-7). See, e.g., inman et al, "SB-431542 is a Potent and Specific Inhibitor of Transforming Growth Factor-beta Superfamily Type I Activin Receptor-Like Kinase (ALK) Receptors ALK4, ALK5, and ALK7[ SB-431542 is a potent and specific inhibitor of transforming growth factor-beta superfamily type I activin receptor-Like Kinase (ALK) Receptors ALK4, ALK5, and ALK7 ]", molecular Pharmacology [ molecular pharmacology ],2002,62,65. In addition, small molecule ALK-4, ALK-5 and ALK-7 inhibitors a-83-01 were developed and found to inhibit SMAD signaling and epithelial to mesenchymal transition (EMT), indicating that such inhibitors are useful in the treatment of a variety of advanced cancers. See, e.g., tojo et al, "The ALK-5 inhibitor A-83-01 inhibits SMAD signaling and epithelial-to-mesenchymal transition by transforming growth factor-beta [ ALK-5 inhibitor A-83-01 inhibits SMAD signaling and epithelial to mesenchymal transition by transforming growth factor-beta ]", cancer Sci. [ Cancer science ],2005,96,791. In the same manner, the role of ALK-5 in TGF-beta signaling may play a role in the generation of cancer-related fibroblasts and other fibrotic conditions. See, e.g., blobe, G.C. et al, "Role of Transforming Growth Factor βin Human Disease [ transforming growth factor β role in Human Disease ]", N Engl J Med [ New England medical journal ] (342), pages 1350-1358 (2000); ballester, B.et al, "Idiopathic Pulmonary Fibrosis and Lung cancer: mechanisms and Molecular Targets [ idiopathic pulmonary fibrosis with lung cancer: mechanism and molecular targets ] ", int.j.of Molecular Sciences [ journal of international molecular science ]20 (593), DOI 10.3390/ijms20030593 (2019), liu, L et al," Smad2 and Smad3 Have Differential Sensitivity in Relyaing TGFb Signaling and Inversely Regulate Early Linage Specification [ Smad2 and Smad3 have different sensitivities in dependence on TGFb signaling and reverse modulate early lineage specification ] ", scientific Reports [ science report ] [6:21602/DOI 10.1038/srep21602], pages 2 months-14 of 2015, huang, j.j. And Blobe, g.c." Dichotomous Roles of TGF- βin Human Cancer [ TGF- β dual role in Human Cancer ] ", biochem soc. Trans [ biochemistry society ]342 (2016); 1441-1454 (https: the term "related Art"// doi.org/10.1042/BST 20160065), akhurst, R.J., and Hata, A., "Targeting the TGF-. Beta. Signalling Pathway in Disease [ TGF-. Beta.signaling pathway targeting disease ]", nat.Rev.drug Disc. [ Nature comment drug discovery ],11 pages 790-811 (2012), leslie, K.O., "Idiopathic Pulmonary Fibrosis May Be a Disease of Recurrent, tractional Injury to the Periphery of the Aging Lung-A Unifying Hypothesis Regarding Etiology and Pathogenesis [ idiopathic pulmonary fibrosis may be recurrent traction injury disease to the aging lung week-unified assumption regarding etiology and pathogenesis ]" Arch Pathol Lab Med [ pathology & laboratory medical record ] (136) [ [591-600 (2012), knupppel, L. ] et al, "A Novel Antifibrotic Mechanism of Nintedanib and Pirfenidone-Inhibition of Collagen Fibril Assembly [ novel mechanism of anti-fibrosis of Nidani-b and pirfenidone-inhibition of collagen fiber assembly ]", am.J. of Resp.cell and Mobile.Bio. [ journal of American respiratory and molecular biology ]1 (57), pages 77-90 (2017), laping, N.J. et al, "Inhibition of TGF-b1-Induced Extracellular Matrix [ inhibition of extracellular matrix induced by TGF-b1 ]", mol.Pharmacol. [ volume 62, stage 1, pages 580-64 (2002), moore, B.B. and Moore, T.A., viruses in Idiopathic Pulmonary Fibrosis-Etiology and Exacerbation [ virus cause and exacerbation in idiopathic pulmonary fibrosis ], ann Am Thorac.Soc. [ annual book of American society of thoracic sciences ], volume 12 (journal 2) pages S186-S192 (2015) - [ DOI:10.1513/Annalsats.201502-088AW ], cho, M.E. and Kopp, J.B. "Pirfenidone: an Anti-Fibrotic and Cytoprotective Agent as Thereapy for Progressive Kidney Disease [ Pirfenidone: anti-fibrotic and cytoprotective agents as therapies for progressive renal disease ] ", expert opin. Invest. Drugs [ experimental drug specialist opinion ],19 (2), pages 275-283 (2010) [ DOI:10.1517/13543780903501539], and B.Rybinski et al," The Wound Healing, chronic Fibrosis, and Cancer Progresion Triad [ triple signs of wound healing, chronic Fibrosis and cancer progression ], physiol Genomics [ physiogenomics ].46 (7); 2014,223-244 PMID:24520152.

In a breast cancer model, the small molecule ALK-5 inhibitor, golomb Lu Nise, was found to inhibit tumor growth. In the colon cancer model, the combination of the golgi Lu Nise replacement with the PD-L1 inhibitor showed tumor growth inhibition and regression, indicating a synergistic effect between ALK-5 inhibition and PD-1/PD-L1 inhibition. See, e.g., holmegaard et al, "Targeting the TGF beta pathway with galunisertib, a tgfβ RI small molecule inhibitor, proteins anti-tumor immunity leading to durable, complete responses, as monotherapy and in combination with checkpoint blockade [ targeting tgfβ pathway with the tgfβ RI small molecule inhibitor gos Lu Nise as monotherapy and in combination with checkpoint blocking promotes anti-tumor immunity, resulting in a durable, complete response ]", journal for ImmunoTherapy of Cancer [ journal of cancer immunotherapy ],2018,6,47. Furthermore, the development of golgi Lu Nise has been pursued for the treatment of various other cancers, including glioblastoma, pancreatic cancer, hepatocellular carcinoma (HCC) and myelodysplastic syndrome, sometimes in combination with PD-1/PD-L1 inhibitors. See, e.g., herbertz et al, "Clinical Development of galunisertib (LY 2IS7299 monohydrate), a small molecule inhibitor of transforming growth factor-beta signaling pathway [ transforming growth factor-. Beta.small molecule inhibitor of the signaling pathway, gossyp Lu Nise alternative (LY 2IS7299 monohydrate), clinical Development ]", drug Design, development, and Therapy [ Drug Design, development and Therapy ],2015,9,4479.

Another small molecule ALK-5 inhibitor, TEW-7197 (also known as valvulrib) has also been investigated for the treatment of cancers such as melanoma, prostate cancer, breast cancer, HCC and glioblastoma. See, e.g., herbertz et al, "Clinical Development of galunisertib (LY 2 IS7299 monohydrate), a small molecule inhibitor of transforming growth factor-beta signaling pathway [ transforming growth factor-. Beta.small molecule inhibitor of the signaling pathway, gossyp Lu Nise alternative (LY 2 IS7299 monohydrate), clinical Development ]", drug Design, development, and Therapy [ Drug Design, development and Therapy ],2015,9,4479.

ALK inhibitors (especially ALK-5 inhibitors) are promising therapeutic agents for a variety of indications, and are currently under investigation. For example, studies have shown that TGF-beta R1/ALK-5 mutants can induce expression of Foxp3, which Foxp3 expression has been found to play a key role in the immunological resistance of different tumor types, including pancreatic cancer. See, e.g., hinz et al, "Foxp3 Expression in Pancreatic Carcinoma Cells as Novel Mechanism of Immune Evasion in Cancer [ Foxp3 expression in pancreatic Cancer cells as a new mechanism of immune escape in Cancer ]", cancer Res [ Cancer research ]2007,67,8344. Thus, cancers that are traditionally resistant to apoptosis via chemical and/or radiation-based therapies may respond when combined with ALK-5 inhibition.

Studies have also shown that ALK-5 inhibitors may also be useful in the treatment of proliferative disorders other than cancer, including systemic sclerosis and other fibrotic disorders (including those associated with cancer), see, for example, those described in the following documents: mori et al, "active Receptor-Like Kinase 5Signaling Blocks Profibrotic Transforming Growth Factor beta Responses in Skin Fibroblasts [ Activin Receptor-Like Kinase 5signaling blocks the pro-fibrotic transforming growth factor beta response in skin fibroblasts ]", arthritis & Rheumatism [ Arthritis and Rheumatism ],2004,8,4008, akhurst, R.J. and Hata, A., "Targeting the TGF-beta Signalling Pathway in Disease [ TGF-beta signaling pathway targeting disease ]", nat.Rev. Drug Disc. [ Nature review drug discovery ],11 pages 790-811 (2012), and Cox, T.R and Erler, J.T., "Molecular Pathways Connecting Fibrosis and Solid Tumor Methastasis [ molecular pathway connecting fibrosis and solid tumor metastasis ]", clin Cancer Res. [ clinical Cancer research ],2014,20 (14), pages 3637-3643.

Epithelial-to-mesenchymal transition (EMT) is a term describing the obtaining of migration and invasion properties of epithelial cells while losing their intercellular adhesiveness and polarity. These cells exhibit a more undifferentiated mesenchymal phenotype, which can then differentiate into a variety of other cell types. The process of EMT is essential for normal embryonic development and wound healing, but is also associated with diseases such as cancer and fibrosis. In cancer, EMT is thought to be particularly conducive to metastasis and resistance to chemotherapy. Hao, y et al, int.j.mol.sci. [ journal of international molecular science ] month 6 2019; 20 (11); 2767. pleiotropic cytokines TGF- β are considered to be the primary driver of EMT and inhibiting this pathway may be beneficial in a variety of diseases (such as cancer and fibrosis). Katsuno, y. And Derynck, r.dev.cell [ developmental cells ]2021, 3 months 22 days; 56 (6):726-746.

Elevated ALK-5 levels are also associated with cardiac pathologies and cardiovascular diseases, including not only cardiac remodeling and fibrosis (e.g., post myocardial infarction) and cardiac hypertrophy, but also dilated, ischemic and hypertrophic cardiomyopathy, valvular diseaseAnd cardiac arrhythmias (such as atrial fibrillation). Khan, r. and Sheppard, r. "Fibrosis in heart disease: understanding the role of transforming growth factor-beta ₁ in cardiomyopathy, valvular disease and arrhythmia [ fibrosis in heart disease: understanding transforming growth factor-beta ₁ Effects in cardiomyopathy, valvular disease and arrhythmia]", immunology [ Immunology ]]2006,118:10-24; bujak, M. and Frangagiannis, N.G. "The role of TGF-beta in myocardial infarction and cardiac remodeling [ effect of TGF-beta on myocardial infarction and cardiac remodeling ]]"Cardiovascular Research [ cardiovascular research]74 (2007), 184-195; dobaczewski, M.et al, "Transforming Growth Factor (TGF) -beta signaling in cardiac remodeling [ Transforming Growth Factor (TGF) -beta signaling in cardiac remodeling ]]"J.mol.cell Cardiol. [ journal of molecular and cell cardiology ]]2011,51 (4) 600-606; and Acronero, F.et al, "Genetic Analysis of Connective Tissue Growth Factor as an Effector of Transforming Growth Factor beta Signaling and Cardiac Remodeling [ Gene analysis of connective tissue growth factor as an effector of transforming growth factor beta signaling and cardiac remodeling ] ]", molecular and Cellular Biology [ molecular and cell biology ]]2015,35(12):2154-2164。

Despite advances, there remains a need for additional compounds to advance research and medical care for patients suffering from proliferative diseases (such as tumors and cancers) and fibrotic diseases (those associated with proliferative diseases and those not associated with proliferative disorders).

Disclosure of Invention

Provided herein are inhibitors of activin receptor-like kinases (e.g., ALK-5), including compounds having any formula herein and pharmaceutically acceptable salts thereof, pharmaceutical compositions and kits comprising the same, and methods of using any of the foregoing compounds, salts, compositions and kits (e.g., for treating and/or preventing a disease in a subject). Also provided herein are methods of preparing the compounds, pharmaceutically acceptable salts, and pharmaceutical compositions described herein.

In some embodiments, compounds having formula (I) are provided:

or a pharmaceutically acceptable salt thereof, wherein R ¹ 、R ² 、R ³ And ring G is as defined herein.

The compounds provided herein are inhibitors of activin receptor-like kinases (e.g., ALK-5) useful in the treatment and/or prevention of diseases (e.g., inhibitors related to modulating or targeting tgfp signaling pathway, e.g., because they are related to the treatment, alleviation or prevention of fibrotic, inflammatory and/or proliferative diseases (e.g., cancers associated with tgfp 1 signaling, pulmonary fibrosis, and heart disease)). See, for example, the relationship of these diseases and conditions to: the role of various signaling pathways that may be involved in the treatment of these diseases and disorders are described, for example, in Akhurst, r.j. And Hata, a., "Targeting the TGF- β Signalling Pathway in Disease [ TGF- β signaling pathway targeting disease ]", nat. Rev. Drug Disc. [ natural review drug discovery ],11 pages 790-811 (2012); cox, t.r and Erler, j.t. "Molecular Pathways Connecting Fibrosis and Solid Tumor Metastasis [ molecular pathways linking fibrosis and solid tumor metastasis ]", clin Cancer Res [ clinical Cancer research ],2014,20 (14), pages 3637-3643; radisky, d.c. et al, "Fibrosis and Cancer: do Myofibroblasts Come Also From Epithelial Cells via EMT? [ fibrosis and cancer: is myofibroblasts also derived from epithelial cells via EMT? J.Cell Biochem [ journal of cell biochemistry ],2101 (4), pages 830-839 [ DOI:10.1002/jcb.21186], and the effects of viral complications in IPF, such as described in Moore, B.B. and Moore, T.A., viruses in Idiopathic Pulmonary Fibrosis-Etiology and Exacerbation [ viruses in idiopathic pulmonary fibrosis-etiology and exacerbation ], ann Am Thorac.Soc. [ annual. Journal of the American society of pectoral science ], volume 12 (journal of Proc 2) pages S186-S192 (2015) - [ DOI: 10.1513/Annalsats.02-088 AW ], and effects of TGF signaling in cardiac remodeling, such as described in Dobacwski, M.et al, "Transforming Growth Factor (TGF) -beta signaling in cardiac remodeling [ Transforming Growth Factor (TGF) -beta signaling in cardiac remodeling ], J.Cell Cardiol [ molecule and cell cardiology 2011,51 (4-600): journal of cell biology.

In certain embodiments, the compounds provided herein are selective ALK-5 inhibitors, e.g., selective for ALK-5 over other kinases (e.g., over other activin receptor-like kinases such as ALK-2 and/or JAK 2). In certain embodiments, for example, the compound having formula (I) is selected from the compounds cited in table 1 (below) and pharmaceutically acceptable salts thereof.

In various aspects and embodiments disclosed herein, explicit reference to a compound having formula (I) is alternatively understood to refer to any of its disclosed subgenera, e.g., any of the compound having formula (II) (hereinafter), the compound having formula (III) (hereinafter), the compound having formula (IV) (hereinafter), the compound of table 1 (hereinafter), or the specific compounds disclosed herein.

In another aspect, provided herein are pharmaceutical compositions comprising a compound having formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers or excipients. In certain embodiments, the pharmaceutical compositions provided herein comprise a therapeutically and/or prophylactically effective amount of a compound having formula (I) or a pharmaceutically acceptable salt thereof. The pharmaceutical compositions described herein can be used to treat and/or prevent a disease (e.g., an inflammatory, fibrotic, or proliferative disease, such as cancer, or a combination of two or more cancers, as further described herein) in a subject. The pharmaceutical compositions provided herein may further comprise one or more additional therapeutic agents (e.g., antiproliferative agents, such as anticancer agents).

In another aspect, provided herein are methods of treating and/or preventing a disease in a subject, comprising administering to the subject a therapeutically and/or prophylactically effective amount of a compound having formula (I), (II), (III), or (IV), or a compound of table 1, or any of the compounds in the form of a pharmaceutically acceptable salt, or a pharmaceutical composition of any of the foregoing. For example, provided herein are methods for treating a disease (e.g., an inflammatory, fibrotic, or proliferative disease (e.g., cancer)) in a subject, comprising administering to the subject a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising any of the foregoing, e.g., a compound of formula (I), formula (II), formula (III), formula (IV), or table 1, or any of the specific compounds disclosed herein, or any of the foregoing, or a pharmaceutical composition of any of the foregoing, in the form of a pharmaceutically acceptable salt.

In certain embodiments, the proliferative disease is cancer. In certain embodiments, the proliferative disease is a solid tumor cancer. In some embodiments, the proliferative disease is hematological cancer. In some embodiments, the cancer is associated with an activity (e.g., aberrant or increased activity) of an activin receptor-like kinase (e.g., ALK-5) in the subject or cell. In some embodiments, cancer is associated with a tgfp signaling pathway that is critical for disease progression and can be alleviated by ALK-5 inhibition. In some embodiments, the cancer is associated with a FOXL2 mutation (e.g., a tumor-associated somatic FOXL2 mutation p.cys134trp (c.402 c > G)). In some embodiments, FOXL2 mutations affect one or more transcriptional targets, which are TGF- β signaling genes.

In certain embodiments, the cancer is lung cancer (e.g., non-small cell lung cancer (NSCLC)), brain cancer (e.g., neuroblastoma, glioblastoma), thyroid cancer (e.g., thyroid undifferentiated carcinoma (ATC)), breast cancer, colorectal cancer (e.g., colon cancer), liver cancer (e.g., hepatocellular carcinoma (HCC)), pancreatic cancer (pancreatic cancer) (e.g., pancreatic cancer (pancreatic carcinoma)), skin cancer (e.g., melanoma), prostate cancer, or hematological cancer (e.g., anaplastic Large Cell Lymphoma (ALCL), myelodysplastic syndrome (MDS), myelofibrosis (MF)). In certain embodiments, the cancer is Myelofibrosis (MF).

In some embodiments, the proliferative disease is a cancer, e.g., anaplastic astrocytoma, pancreatic cancer, e.g., pancreatic ductal adenocarcinoma and associated CAF, metastatic melanoma, colorectal cancer, breast cancer, prostate cancer, renal cancer, hepatocellular carcinoma, ovarian cancer, HPV-related cancer (e.g., cervical cancer, oropharyngeal cancer, anal cancer, vulvar/vaginal cancer, penile cancer), multiple myeloma, myelodysplastic syndrome, or myelofibrosis. In some embodiments, the cancer is treated by: targeting tumor stromal cells (e.g., in a tumor microenvironment), such as cancer-associated fibroblasts (CAF), astrocytes, or myofibroblasts, and/or tumor-associated immune cells (e.g., in a tumor-immune microenvironment), e.g., to modulate the tumor-stromal microenvironment and/or tumor-immune microenvironment.

In some embodiments, the disease is a fibrotic disorder, e.g., idiopathic pulmonary fibrosis, cardiac fibrosis or a disorder associated with cardiac fibrosis, liver fibrosis, cirrhosis, nonalcoholic steatohepatitis, peyronie's, dupuytren's contracture, cystic fibrosis, beta thalassemia, light keratosis, hypertension, systemic inflammatory disorders, dry eye, ulcers, corneal fibrosis, wet age-related macular degeneration, psoriasis, wound closure, chronic kidney disease, renal fibrosis, systemic sclerosis, or chronic Chagas heart disease (chronic Chagas ' heart disease). In some embodiments, the fibrotic condition is cardiac fibrosis or a condition associated with cardiac fibrosis, such as valvular disease, cardiac arrhythmia (e.g., atrial fibrillation), myocardial remodeling (e.g., post-infarction), cardiomyopathy (e.g., dilated, ischemic, or hypertrophic cardiomyopathy), restenosis (e.g., in-stent restenosis, restenosis after angioplasty). In some embodiments, the fibrotic disorder is diprote contracture. In some embodiments, the fibrotic disorder is acute exacerbation of, for example, idiopathic pulmonary fibrosis or familial pulmonary fibrosis, vascular fibrosis, kidney fibrosis (kidney fibrosis), skin fibrosis (skin fibrosis) (cutaneous fibrosis) or endometrial fibrosis, e.g., keloid, scleroderma or nephrogenic systemic fibrosis), gastrointestinal fibrosis (e.g., crohn's disease), bone marrow fibrosis (bone marrow fibrosis) (bone marrow fibrosis (myelofilrosis)), atherosclerosis fibrosis (e.g., atherosclerosis fibrosis of the knee, shoulder or another joint), diprote contracture, mediastinal fibrosis, retroperitoneal fibrosis, systemic sclerosis or autoimmune hepatitis. In some embodiments, the fibrotic disorder is a cancer-related fibrotic disorder; pulmonary fibrosis, which is commonly referred to as "pulmonary scarring" (e.g., pulmonary fibrosis, such as idiopathic pulmonary fibrosis or acute exacerbation of familial pulmonary fibrosis). In some embodiments, the fibrotic disorder is pulmonary fibrosis, e.g., pulmonary fibrosis, such as idiopathic pulmonary fibrosis, acute exacerbation of idiopathic pulmonary fibrosis, or familial pulmonary fibrosis. In embodiments, liver fibrosis is liver fibrosis (e.g., keloids, scleroderma, kidney-derived systemic fibrosis, biliary fibrosis (biliary fibrosis)) or cirrhosis (e.g., primary biliary cholangitis (biliary cirrhosis) or primary sclerosing cholangitis).

Also provided herein are methods of inhibiting or preventing tumor growth in a subject, comprising administering to the subject a therapeutically effective amount of a compound having formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the foregoing.

Also provided herein are methods of treating cachexia in a subject (e.g., a subject in need thereof), comprising administering to the subject a therapeutically effective amount of a compound having formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising any of the foregoing, e.g., a compound having formula (I), formula (II), formula (III), formula (IV), or table 1, or any of the specific compounds disclosed herein, or any of the foregoing compounds in the form of a pharmaceutically acceptable salt, or a pharmaceutical composition of any of the foregoing.

Also provided herein are methods for promoting immune infiltration in a tumor-immune microenvironment in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound having formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising any of the foregoing, e.g., a compound having formula (I), formula (II), formula (III), formula (IV), or table 1, or any of the specific compounds disclosed herein, or any of the foregoing compounds in the form of a pharmaceutically acceptable salt, or a pharmaceutical composition of any of the foregoing.

Also provided herein are methods for inhibiting epithelial-to-mesenchymal transition in a tumor (e.g., in a subject in need thereof), comprising contacting the tumor with: (e.g., an effective amount) of a compound having formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising any of the foregoing, e.g., a compound having formula (I), formula (II), formula (III), formula (IV), or table 1, or any of the specific compounds disclosed herein, or any of the foregoing compounds in the form of a pharmaceutically acceptable salt, or a pharmaceutical composition of any of the foregoing. In some embodiments, the tumor is present in a subject in need thereof, and the method comprises administering to the subject a therapeutically effective amount of a compound having formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

Also provided herein are methods for modulating (e.g., promoting, upregulating) an antigen-presenting pathway in a tumor (e.g., in a subject in need thereof), comprising contacting the tumor with: (e.g., an effective amount) of a compound having formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising any of the foregoing, e.g., a compound having formula (I), formula (II), formula (III), formula (IV), or table 1, or any of the specific compounds disclosed herein, or any of the foregoing compounds in the form of a pharmaceutically acceptable salt, or a pharmaceutical composition of any of the foregoing. In some embodiments, the tumor is present in a subject in need thereof, and the method comprises administering to the subject a therapeutically effective amount of a compound having formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

Also provided herein are methods of modulating a tumor-immune microenvironment in a subject, comprising administering to the subject a therapeutically effective amount of a compound having formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising any of the foregoing, e.g., a compound having formula (I), formula (II), formula (III), formula (IV), or table 1, or any of the specific compounds disclosed herein, or any of the foregoing compounds in the form of a pharmaceutically acceptable salt, or a pharmaceutical composition of any of the foregoing.

Also provided herein are methods of increasing tumor blood vessel or blood flow to a tumor, or both, in a subject, comprising administering to the subject a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising any of the foregoing, e.g., a compound of formula (I), formula (II), formula (III), formula (IV), or table 1, or any of the specific compounds disclosed herein, or any of the foregoing, or a pharmaceutical composition of any of the foregoing, in the form of a pharmaceutically acceptable salt.

Also provided herein are methods of inhibiting metastasis of cancer in a subject, comprising administering to the subject a therapeutically effective amount of a compound having formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising any of the foregoing, e.g., a compound having formula (I), formula (II), formula (III), formula (IV), or table 1, or any of the specific compounds disclosed herein, or any of the foregoing compounds in the form of a pharmaceutically acceptable salt, or a pharmaceutical composition of any of the foregoing.

Also provided herein are methods for inhibiting the activity of an activin receptor-like kinase (e.g., ALK-5) in vivo or in vitro, comprising contacting the activin receptor-like kinase (e.g., ALK-5) with: a compound having the formula (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the foregoing. In certain embodiments, inhibition occurs in a subject. In certain embodiments, inhibition occurs in vitro (e.g., in a cell line or biological sample). In certain embodiments, the inhibition is selective ALK-5 inhibition.

In another aspect, provided herein are compounds having formula (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the foregoing, for use as described herein, including but not limited to treating and/or preventing a disease (e.g., a proliferative disease (e.g., cancer), a fibrotic disease (e.g., cardiac fibrosis or hypertrophic disorder), or an inflammatory disorder) in a subject, for inhibiting tumor growth in a subject, or for inhibiting the activity of an activin-receptor-like kinase (e.g., ALK-5) in vitro or in vivo. In yet another aspect, provided herein is the use of a compound having formula (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition of the foregoing, for the manufacture of a medicament for treating and/or preventing a disease (e.g., an inflammatory disorder, a fibrotic disease (e.g., cardiac fibrosis or hypertrophic disorder), or a proliferative disease (e.g., cancer or a combination of two or more cancers)) in a subject, for inhibiting tumor growth in a subject, or for inhibiting ALK-5 activity in a subject.

The methods and uses provided herein may further comprise administering one or more additional therapeutic agents (e.g., anti-cancer agents, or immunotherapy or other agents described herein) to the subject. In certain embodiments, the PD-1 or PD-L1 inhibitor is administered in combination with a compound, pharmaceutically acceptable salt, or pharmaceutical composition described herein. The methods provided herein may further comprise treating the subject with radiation therapy or surgery.

Also provided herein are methods for increasing the activity of one or more therapeutic agents (e.g., anticancer agents and/or immunotherapies) for treating cancer in a subject (e.g., a subject in need thereof, such as a subject having cancer and/or receiving one or more therapeutic agents), comprising administering to the subject a therapeutically effective amount of a compound having formula (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

In another aspect, provided herein are kits comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the foregoing. Kits described herein can include a single dose or multiple doses of a compound, pharmaceutically acceptable salt, or pharmaceutical composition. The provided kits can be used in methods of the disclosure (e.g., methods of treating and/or preventing a disease in a subject). The kits of the present disclosure may further include instructions for using the kits (e.g., instructions for using the compounds, pharmaceutically acceptable salts, or compositions included in the kits).

Also provided herein are methods of preparing the compounds of the present disclosure, such as compounds having formula (I), (II), (III), (IV) or table 1, and pharmaceutically acceptable salts thereof.

Drawings

The patent or application contains at least one drawing in color. Copies of this patent or patent application publication and color drawings will be provided by the office upon request and payment of the necessary fee.

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the disclosure and, together with the description, serve to provide non-limiting examples of the disclosure.

FIG. 1A is a LanthaScreen ^TM Illustration of Eu kinase binding assay.

FIG. 1B shows ALK5 inhibition of selected compounds as a function of compound concentration using the RDSR cell-based assay technique described in example 3.

Figure 2A shows that compound number 04 shows complete concentration-dependent inhibition of TGF- β1 mediated αsma expression in all three IPF donors. The data are presented as normalized data for αsma staining (percent inhibition, PIN) and normalized data for cell nucleus count (percent remaining cells). At the highest test concentration (10 μm), marginal loss of nuclei (< 25%) was observed in donor IPF 03. No modulation of the number of nuclei was observed in the donors IPF06 and IPF08, indicating the absence of potential cytotoxic or antiproliferative effects.

Figure 2B shows that compound number 01 shows complete concentration-dependent inhibition of TGF- β1 mediated αsma expression in all three IPF donors. The data are presented as normalized data for αsma staining (percent inhibition, PIN) and normalized data for cell nucleus count (percent remaining cells). In donor IPF03, nuclear loss (> 25%) was observed at the highest test concentration (10 μm), indicating a potentially cytotoxic or antiproliferative effect at this concentration. No modulation of the number of nuclei was observed in the donors IPF06 and IPF08, indicating the absence of potential cytotoxic or antiproliferative effects.

Figure 3A shows p-SMAD-2 levels normalized to β -tubulin in tumors harvested from mice treated with the indicated compounds in an a549 xenograft assay.

Fig. 3B shows p-STAT3 levels normalized to β -tubulin in tumors harvested from mice treated with the indicated compounds in an a549 xenograft assay.

Fig. 4A shows p-SMAD-2 levels normalized to GAPDH in tumors harvested from mice treated with the indicated compounds of 50mpk (PO) in a longitudinal a549 xenograft study described in example 8.

Fig. 4B shows PK/PD characteristics of valatoxin in the longitudinal a549 xenograft study described in example 8.

Fig. 4C shows PK/PD characteristics of compound 04 in the longitudinal a549 xenograft study described in example 8.

Figure 5 shows the inhibition of SMAD signaling triggered by myostatin in the presence of compound 01, compound 04, or valtoletin.

Fig. 6A shows relative p-SMAD-2 levels normalized to GAPDH (as a percentage of vehicle) in tumors harvested from mice treated with the indicated compounds of 50mpk (PO) in the longitudinal a549 xenograft study described in example 10.

Fig. 6B shows PK/PD characteristics of valatoxin in the longitudinal a549 xenograft study described in example 10.

Fig. 6C shows PK/PD characteristics of compound 04 in the longitudinal a549 xenograft study described in example 10.

Fig. 6D shows PK/PD characteristics of compound 01 in the longitudinal a549 xenograft study described in example 10.

Fig. 7A shows the overall study design of the study described in example 11.

Fig. 7B shows the amount of hydroxyproline measured from a portion of lung tissue harvested from the remaining mice on the twenty-first day of the study described in example 11.

Fig. 7C shows histological analysis of five animals randomly selected from each treatment group using the modified Ashcroft scale in the study described in example 11.

Fig. 7D shows representative images of lung tissue stained with hematoxylin and eosin (H & E) (upper row) or Masson trichrome stain (lower row) obtained from the indicated treatment group in the study described in example 11.

Fig. 7E shows representative images of H & E (upper row) or Masson trichromatic stain (lower row) stained lung tissue obtained from the indicated treatment group in the study described in example 11.

FIG. 8A shows fold change in CDH1 (E-cadherin) gene expression levels in A549 lung fibroblasts after treatment with indicated compounds in the assay described in example 12.

FIG. 8B shows fold change in CDH2 (N-cadherin) gene expression levels in A549 lung fibroblasts after treatment with indicated compounds in the assay described in example 12.

Fig. 8C shows the fold change in SNAI1 (snail) gene expression levels in a549 lung fibroblasts after treatment with the indicated compounds in the assay described in example 12.

Fig. 8D shows the fold change in SNAI2 (slug) gene expression levels in a549 lung fibroblasts after treatment with the indicated compounds in the assay described in example 12.

Fig. 8E shows fold change in VIM (vimentin) gene expression levels in a549 lung fibroblasts after treatment with the indicated compounds in the assay described in example 12.

FIG. 8F shows fold change in SPARC gene expression levels in A549 lung fibroblasts after treatment with the indicated compounds in the assay described in example 12.

Fig. 8G shows fold change in GALNT6 gene expression levels in a549 lung fibroblasts after treatment with the indicated compounds in the assay described in example 12.

FIG. 8H shows the fold change in CTNNB1 (β -catenin) gene expression levels in A549 lung fibroblasts after treatment with the indicated compounds in the assay described in example 12.

FIG. 8I shows the fold change in TGFB1 gene expression levels in A549 lung fibroblasts after treatment with the indicated compounds in the assay described in example 12.

FIG. 8J shows fold changes in MAML3 gene expression levels in A549 lung fibroblasts after treatment with the indicated compounds in the assay described in example 12.

Fig. 9A shows the percentage change in body weight and the results of the subjects treated with compound 01 or compound 04 in the acute MTD study described in example 13.

Figure 9B shows the percent change in body weight and the results tested in the chronic MTD study described in example 13 for subjects treated with compound 04.

Fig. 9C shows the percentage change in body weight and the results tested in the chronic MTD study described in example 13 for subjects treated with compound 01.

Fig. 9D shows survival results for subjects treated with compound 04 in the chronic MTD study described in example 13.

Fig. 9E shows survival results of subjects treated with compound 01 in the chronic MTD study described in example 13.

FIG. 10 shows Median Fluorescence Intensity (MFI) of phosphoric acid-SMAD 2 as a function of concentration of indicated compounds from the phosphoric acid-SMAD 2 assay described in example 14.

FIG. 11 shows the results of the JAK selectivity assay described in example 15.

FIG. 12 shows the fibrillated HepaRG described in example 16 ^{T M} Results of NP 3D model.

Detailed Description

The description of the example embodiments is as follows.

Provided herein are compounds (e.g., compounds having formula (I), (II), (III), (IV) or table 1, or any of the compounds specifically exemplified herein (also referred to herein as "exemplified compounds"), and pharmaceutically acceptable salts thereof, pharmaceutical compositions of the foregoing, and kits comprising one or more of the foregoing). The compounds provided herein are inhibitors of an activin receptor-like kinase (e.g., ALK-5) and are therefore useful for treating and/or preventing a disease (e.g., a proliferative disease (e.g., cancer), a fibrotic disease, an inflammatory disease), for inhibiting tumor growth in a subject, and/or for inhibiting the activity of an activin receptor-like kinase (e.g., ALK-5) in vitro or in vivo. In certain embodiments, the compounds provided herein are ALK-5 inhibitors (e.g., selective ALK-5 inhibitors). Also provided herein are methods and synthetic intermediates useful for preparing the compounds described herein.

Definition of the definition

Chemical elements are identified according to Handbook of Chemistry and Physics [ handbook of chemistry and physics ], CAS version periodic table of elements within the 75 th edition cover, and specific functional groups are generally defined as described herein. In addition, the general principles of organic chemistry and specific functional moieties and reactivities are described in Organic Chemistry [ organic chemistry ], thomas Sorrell, university Science Books [ university science book press ], soxalato (sausalato), 1999; smith and March, march's Advanced Organic Chemistry [ March higher organic chemistry ], 5 th edition, john Wiley & Sons, inc. [ John Wili father-son publishing company ], new York, 2001; larock, comprehensive Organic Transformations [ comprehensive organic transformation ], VCH Publishers, inc. [ VCH publishing company ], new York, 1989; and Carruther, some Modern Methods of Organic Synthesis [ some modern methods of organic synthesis ], 3 rd edition, cambridge University Press [ Cambridge university Press ], cambridge City, 1987.

Unless otherwise indicated, references herein to "compounds of the present disclosure" and the like refer to compounds having any of the structural formulae described herein (e.g., compounds having formula (I), sub-formulae of compounds having formula (I)), including the described compounds as well as isomers, such as stereoisomers (including diastereomers, enantiomers, and racemates), geometric isomers, conformational isomers (including rotamers and atropisomers), tautomers, isotopically labeled compounds (including deuterium substitutions), and inherently formed moieties thereof (e.g., polymorphs and/or solvates, such as hydrates). Salts are also included when there is a moiety capable of forming a salt, particularly a pharmaceutically acceptable salt.

The compounds of the present disclosure may have asymmetric centers, chiral axes, and chiral planes (e.g., as described in e.l.eliel and s.h.wilen, stereo-chemistry of Carbon Compounds [ stereochemistry of carbon compounds ], john Wiley & Sons [ John wili parent publication company ], new york, 1994, pages 1119-1190), and these compounds exist as racemic mixtures, individual isomers (e.g., diastereomers, enantiomers, geometric isomers (including rotamers and atropisomers), tautomers), and intermediate mixtures, all of which are included in the disclosure.

As used herein, the term "isomer" refers to different compounds having the same molecular formula but different arrangements and configurations of atoms.

"enantiomers" are a pair of stereoisomers that are non-superimposable mirror images of each other. A1:1 mixture of a pair of enantiomers is a "racemic" mixture. "racemate" or "racemic" is used to refer to a racemic mixture, where appropriate. When expressing the stereochemistry of compounds of the present disclosure, a conventional RS system (e.g., (1 s,2 s)) is used to express a single stereoisomer with two chiral centers of known relative and absolute configurations; individual stereoisomers with known relative configurations but unknown absolute configurations are indicated by asterisks (e.g., (1R, 2R)); and the racemate is represented by two letters (e.g., (1 rs,2 rs) as a racemic mixture of (1 r,2 r) and (1 s,2 s), and (1 rs,2 sr) as a racemic mixture of (1 r,2 s) and (1 s,2 r). "diastereomers" are stereoisomers that have at least two asymmetric atoms but are not mirror images of each other. Absolute stereochemistry was specified according to the Cahn-Ingold-Prelog R-S system. When the compound is a pure enantiomer, the stereochemistry of each chiral carbon may be specified by R or S. Resolved compounds of unknown absolute configuration can be designated (+) or (-) depending on their direction of rotation (right-hand or left-hand) of plane polarized light at the wavelength of the sodium D-line. Alternatively, resolution of a compound may be defined by the respective retention times of the corresponding enantiomer/diastereomer via chiral HPLC.

Geometrical isomers may occur when a compound contains a double bond or some other feature that imparts some amount of structural rigidity to the molecule. If the compound contains a double bond, the double bond may be in the E-or Z-configuration. If the compound contains a disubstituted cycloalkyl group, the cycloalkyl substituent may have a cis or trans configuration.

Conformational isomer (conformational isomer) (or conformational isomer) is an isomer that can be distinguished by rotation about one or more bonds. Rotamers are conformational isomers that differ by rotation around only one bond.

As used herein, the term "atropisomer" refers to structural isomers based on axial or planar chirality resulting from limited rotation in the molecule.

Optically active (R) -and (S) -isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques (e.g., separation on chiral SFC or HPLC chromatography columns such as those available from large xylonite company (DAICEL corp.))And->Columns or other equivalent columns, using a suitable solvent or mixture of solvents to effect a suitable separation).

The compounds of the present disclosure may be isolated in optically active or racemic forms. The optically active forms can be prepared by resolution of the racemic forms or by synthesis from optically active starting materials. All methods for preparing the compounds of the present disclosure, and intermediates produced therein, are considered to be part of the present disclosure. When enantiomeric or diastereomeric products are prepared, they can be separated by conventional methods, for example by chromatography or fractional crystallization.

Depending on the process conditions, the end products of the present disclosure are obtained in free (neutral) or salt form. Both the free form and the salt of these end products are within the scope of the present disclosure. One form of the compound may be converted to another form if desired. The free base or acid may be converted to a salt; a salt may be converted to the free compound or another salt; mixtures of isomeric compounds of the present disclosure may be separated into the individual isomers.

Any formula given herein is also intended to represent unlabeled as well as isotopically-labeled forms of the compounds, unless otherwise indicated. Isotopically-labeled compounds have structures described by the formulae given herein, except that one or more atoms are replaced by an atom having a selected atomic mass or mass number. Examples of isotopes that can be incorporated into compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, such as, respectively ² H、 ³ H、 ¹¹ C、 ¹³ C、 ¹⁴ C、 ¹⁵ N、 ¹⁸ F、 ³¹ P、 ³² P、 ³⁵ S、 ³⁶ Cl、 ¹²³ I、 ¹²⁴ I and ¹²⁵ I. the present disclosure includes various isotopically-labeled compounds as defined herein, for example, wherein a radioisotope such as ³ H and ¹⁴ those of C, or in which non-radioactive isotopes such as ² H and ¹³ those of C. Such isotopically-labeled compounds are useful in metabolic studies (with ¹⁴ C) Reaction kinetics studies (using, for example ² H or ³ H) Detection or imaging techniques, such as Positron Emission Tomography (PET) or Single Photon Emission Computed Tomography (SPECT), including drug or substrate tissue distribution assays, or for radiation therapy of patients. In particular the number of the elements to be processed, ¹⁸ f or labeled compounds may be particularly suitable for PET or SPECT studies.

In addition, the use of heavier isotopes, particularly deuterium (i.e., ² h or D) substitution may provide for substitution by higherFor example, increasing the half-life in vivo or reducing the dosage requirement or improving the therapeutic index. It should be understood that deuterium is considered a substituent of the compounds of the present disclosure in this context. The concentration of such heavier isotopes, in particular deuterium, may be defined by an isotopic enrichment factor. As used herein, the term "isotopically enriched factor" refers to the ratio between the isotopic abundance and the natural abundance of a given isotope. If substituents in compounds of the present disclosure are denoted as deuterium, such compounds have an isotopic enrichment factor for each named deuterium atom of at least 3500 (52.5% deuterium incorporated at each named deuterium atom), at least 4000 (60% deuterium incorporated), at least 4500 (67.5% deuterium incorporated), at least 5000 (75% deuterium incorporated), at least 5500 (82.5% deuterium incorporated), at least 6000 (90% deuterium incorporated), at least 6333.3 (95% deuterium incorporated), at least 6466.7 (97% deuterium incorporated), at least 6600 (99% deuterium incorporated) or at least 6633.3 (99.5% deuterium incorporated).

Isotopically-labeled compounds of the present disclosure can generally be prepared by conventional techniques known to those skilled in the art or by methods disclosed in the schemes or examples and preparations described below (or methods analogous to those described below) by substituting an appropriate or readily available isotopically-labeled reagent for an otherwise used non-isotopically-labeled reagent. Such compounds have a variety of potential uses, for example as standards and reagents for determining the ability of potential pharmaceutical compounds to bind to a target protein or receptor, or for imaging compounds of the present disclosure that bind to biological receptors in vivo or in vitro.

When a range of values is recited, each value and subrange within the range is intended to be covered. For example, "C _1-6 Alkyl "is intended to cover C ₁ 、C ₂ 、C ₃ 、C ₄ 、C ₅ 、C ₆ 、C _1-6 、C _1-5 、C _1-4 、C _1-3 、C _1-2 、C _2-6 、C _2-5 、C _2-4 、C _2-3 、C _3-6 、C _3-5 、C _3-4 、C _4-6 、C _4-5 And C _5-6 An alkyl group.

The term "alkyl" refers to a group that is a straight or branched saturated hydrocarbon group. In some embodiments, the alkyl group has 1 to 6 carbon atoms ("C _1-6 Alkyl "). In some embodiments, the alkyl group has 1 to 5 carbon atoms ("C _1-5 Alkyl "). In some embodiments, the alkyl group has 1 to 4 carbon atoms ("C _1-4 Alkyl "). In some embodiments, the alkyl group has 1 to 3 carbon atoms ("C _1-3 Alkyl "). In some embodiments, the alkyl group has 1 to 2 carbon atoms ("C _1-2 Alkyl "). In some embodiments, the alkyl group has 1 carbon atom ("C ₁ Alkyl "). In some embodiments, the alkyl group has 2 to 6 carbon atoms ("C _2-6 Alkyl "). C (C) _1-6 Examples of alkyl groups include methyl (C ₁ ) Ethyl (C) ₂ ) Propyl (C) ₃ ) (e.g., n-propyl, isopropyl), butyl (C) ₄ ) (e.g., n-butyl, t-butyl, sec-butyl, isobutyl), pentyl (C) ₅ ) (e.g., n-pentyl, 3-pentyl, neopentyl, 3-methyl-2-butyl, t-pentyl) and hexyl (C) ₆ ) (e.g., n-hexyl and all branched alkyl groups containing 6 carbon atoms), and the like. When an alkyl group is defined herein as "substituted", any limitation presented herein is incorporated, and unless otherwise indicated, "substituted alkyl" means that one or more positions on the carbon skeleton of the alkyl group that would normally be occupied by a proton is replaced by another substituent (e.g., a methyl group substituted with one or more halogens (including-F, -Cl and/or-Br), and including-CH, e.g., when substituted with F ₂ F、-CHF ₂ and-CF ₃ )。

The term "carbocyclyl", "carbocycle" or "carbocyclic" refers to a non-aromatic cyclic hydrocarbon substituent (meaning that the defined ring does not contain heteroatoms), wherein the defined ring has the indicated number of ring carbon atoms in a monocyclic, bicyclic, bridged or spiro configuration. Although carbocycles are non-aromatic, they may contain one or more double bonds located within the ring such that they are not conjugated. In some embodiments, one or more of the ring carbon atoms may be oxidized (e.g., cyclic ketone). At the position of In some embodiments, the carbocyclic group (moiety) has 3 to 10 carbon atoms ("C _3-10 Carbocycles "). In some embodiments, the carbocyclic group has 3 to 8 ring carbon atoms ("C _3-8 Carbocycles "). In some embodiments, the carbocyclic group has 3 to 7 ring carbon atoms ("C _3-7 Carbocycles "). In some embodiments, the carbocyclic group has 3 to 6 ring carbon atoms ("C _3-6 Carbocycles "). In some embodiments, the carbocyclic group has 4 to 6 ring carbon atoms ("C _4-6 Carbocycles "). Exemplary C _3-6 Carbocyclic groups include, but are not limited to, cyclopropyl (C ₃ ) Cyclopropenyl (C) ₃ ) Cyclobutyl (C) ₄ ) Cyclobutenyl (C) ₄ ) Cyclopentyl (C) ₅ ) Cyclopentenyl (C) ₅ ) Cyclohexyl (C) ₆ ) Cyclohexenyl (C) ₆ ) Cyclohexadienyl (C) ₆ ) Etc. In some embodiments, the carbocyclic group is cyclopropyl (C ₃ ). As illustrated by the foregoing examples, in certain embodiments, the carbocyclic group is a single ring ("monocyclic carbocycle") or multiple rings (e.g., containing a fused, bridged, or spiro ring system, such as a bicyclic system ("bicyclic carbocycle") or a tricyclic system ("tricyclic carbocycle")), and may be saturated or may contain one or more carbon-carbon double or triple bonds. In some embodiments, the carbocyclic group is a bicyclic carbocycle, such as a spiro ring preferably containing 6-9 carbon atoms. It is understood that the minimum number of carbon atoms in a bicyclic carbocycle is four and the minimum number of carbon atoms in a spiro carbocycle is five. Thus, it is to be understood that for monocyclic, bicyclic or spiro C ₃ -C ₁₀ The description of carbocycles refers to monocyclic C ₃ -C ₁₀ Carbocyclyl, bicyclo C ₄ -C ₁₀ Carbocyclyl or spirocyclic C ₅ -C ₁₀ Carbocyclyl. In some embodiments of spiro carbocyclyl, the carbocycle is preferably C _5-10 Spiro carbocyclyl, e.g. C _6-9 Spiro carbocyclyl.

The term "hydroxyl" refers to-OH.

The term "heterocyclyl", "heterocycle" or "heterocyclic" refers to a compound prepared from a compound having the indicated number of atoms selected from carbon atoms and at least 1, up to 3 heteroatoms selected from N, S and O, either identical or independentlyThe ring of the child defines a non-aromatic substituent, and the heteroatoms are selected to bond such that they form a stable chemical entity. The term "C ₅ -C ₁₀ Heterocyclyl "refers to a heterocyclyl group having 5 to 10 ring atoms selected from carbon atoms and at least 1, up to 3 heteroatoms, the same or independently selected from N, S and O, selected to bond such that they form a stable chemical entity. The heterocyclic ring may be saturated or may contain one or more sites of unsaturation, provided that the bonding scheme does not provide aromatic delocalization. The heterocyclic nucleus may be a single ring ("monocyclic heterocycle") or multiple rings (e.g., a fused, bridged or spiro ring system, such as a bicyclic ring system ("bicyclic heterocycle") or a tricyclic ring system ("tricyclic heterocycle")) so long as at least one of the ring moieties defined by the ring members contains heteroatoms, and the polycyclic heterocyclic substituent may, but need not, include one or more heteroatoms in the multiple rings. If a heterocycle is indicated as substituted, one or more substituents bonded to the "substituted heterocycle" core may be bonded via any of the ring member atoms that provide a stable bonding arrangement. In certain embodiments, the heterocyclic group is an unsubstituted 3-10 membered heterocyclic ring. In certain embodiments, the heterocyclic group is a substituted 3-10 membered heterocyclic ring. In some embodiments, the heterocyclyl group has 5 to 10 ring atoms. In some embodiments, it is preferred to select heterocyclic substituents that are 6 membered ring systems. In some embodiments, it is preferred to select heterocyclic substituents that are 10 membered spiro substituents. It is understood that the minimum number of ring atoms in the bicyclic heterocycle is four and the minimum number of ring atoms in the spiro carbocycle is five. Thus, it is to be understood that for monocyclic, bicyclic or spiro C ₃ -C ₁₀ The heterocyclic ring description refers to a single ring C ₃ -C ₁₀ Heterocyclyl, bicyclo C ₄ -C ₁₀ Heterocyclyl or spirocyclic C ₅ -C ₁₀ A heterocyclic group. In some embodiments of the spirocyclic heterocyclyl, the heterocycle is preferably C _5-10 Spirocyclic heterocyclic groups, e.g. C _6-9 A spiro heterocyclyl group.

The term "aryl" refers to an aromatic moiety of up to 10 carbon atoms defining an aromatic ring system. Such takingThe substituents are bonded to the substrate via any ring carbon atom that provides a stable structure. These moieties may comprise a monocyclic or bicyclic structure (e.g., fused rings) as defined or limited when used. In some embodiments, the aryl group has 6 ring carbon atoms ("C ₆ Aryl "; for example, phenyl). In some embodiments, the aryl group has 10 ring carbon atoms ("C ₁₀ Aryl "; for example naphthyl, such as 1-naphthyl and 2-naphthyl). In some embodiments, as defined or illustrated herein, an aryl moiety comprises a substituent on an aryl ring as defined above that is bonded to form a fused carbocyclic ring structure with the aryl moiety, the size of the carbocycle in the fused structure being defined in use. If an aryl moiety is defined herein as substituted, this means that a particular substituent may replace one or more protons bonded to a carbon atom defining an aryl ring in a manner that provides a stable species. In some embodiments, the aryl moiety is a 6-membered aryl ring (e.g., optionally substituted phenyl).

The term "heteroaryl" refers to an aromatic moiety defining up to 10 carbon atoms of an aromatic ring system, wherein one or more of the atoms defining the aromatic ring system are independently selected from O, N or S. Heteroaryl substituents may be bonded to the substrate via any atom in the heteroaryl ring that provides a stable bond. Heteroaryl substituents may be optionally substituted as defined herein for use. In some embodiments, the heteroaryl group has 5 to 10 ring atoms ("C ₅ -C ₁₀ Heteroaryl "). In some embodiments, the heteroaryl group has 6 to 10 ring atoms ("C ₆ -C ₁₀ Heteroaryl "), e.g., 6 ring atoms (" C ₆ Heteroaryl "; for example pyridyl) or 10 ring atoms ("C) ₁₀ Heteroaryl ").

The term "optionally substituted" as used in the definition of substituents herein means that the defined moiety may be present without any substituent (i.e., unsubstituted), or may be present in a form wherein one or more of the optional substituents, normally occupied by a proton, are designated as being substituted (i.e., substituted). In all embodiments, when optional substituents are present, they are present in an amount and in a bonding configuration that provides stable compounds, e.g., compounds that do not spontaneously undergo conversion as by rearrangement, cyclization, elimination, or other reaction; however, it does contemplate arrangements that provide tautomers or other similar bonding arrangements. Unless otherwise indicated, a "substituted" moiety has substituents at one or more substitutable positions of the moiety, and when more than one position in any given structure is substituted, the substituents are independently selected from the allowed substituents recited. Unless otherwise defined when used, it is contemplated that the term "substituted" includes substitution with all permissible substituents of organic compounds and includes any one of the substituents described herein which result in the formation of stable compounds. The present disclosure contemplates any and all such combinations to obtain stable compounds. For purposes of this disclosure, a heteroatom (e.g., nitrogen) may have a hydrogen substituent and/or any suitable substituent as described herein that satisfies the valency of the heteroatom and results in the formation of a stabilizing moiety. In some embodiments, where the trivalent nitrogen may be quaternized or where the quaternary nitrogen may be deprotonated to the trivalent form, the representation of either form contemplates conversion between the two forms, and such representation is not intended to be limited in any way by the exemplary substituents described herein.

As used herein, unless defined differently, the term "halo or halogen" refers to fluorine (fluoro), -F), chlorine (chloro), -Cl), bromine (bromobr) or iodine (iodine), -I), unless the term is more limited as used herein. In some embodiments, the halogen is fluorine, chlorine or bromine. In some embodiments, the halogen is fluorine or chlorine.

The term "sulfonamide" refers to-SO ₂ R 'R ", wherein R' and R" are the same or different and are each independently selected from hydrogen, alkyl or carbocyclyl. In some embodiments, R 'and R' are each independently selected from hydrogen, C ₁ -C ₅ Alkyl or C ₃ -C ₅ Cycloalkyl groups. In some embodiments, the sulfonamide is-SO ₂ NH ₂ 。

In certain embodiments, certain features of the compound substituents may be protected with protecting groups known to those of ordinary skill, such as those described in detail in Protecting Groups in Organic Synthesis [ protecting groups in organic synthesis ], t.w.greene and p.g.m.wuts, 3 rd edition, john Wiley & Sons [ John wili parent publishing company ],1999, which is incorporated herein by reference. All such transformations are contemplated by the representation of the unprotected form of the compound.

As used herein, the term "salt" refers to any and all salt forms that the compounds disclosed herein can be prepared from, and encompasses pharmaceutically acceptable salts. Pharmaceutically acceptable salts are preferred. However, other salts may be useful, for example, in isolation or purification steps that may be employed during preparation, and thus, are contemplated as being within the scope of the present disclosure. Generally, salts of the compounds described herein will be those that provide compositions suitable for administration to a human or animal subject via any suitable route of administration of the pharmaceutical composition.

The phrase "pharmaceutically acceptable" means that the substance or composition modified by the phrase must be suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and within the scope of sound medical judgment. If the substance is part of a composition or formulation, the substance must also be chemically and/or toxicologically compatible with the other ingredients in the composition or formulation.

The term "pharmaceutically acceptable salts" refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, bere et al, J.pharmaceutical Sciences [ journal of pharmaceutical Sciences ],1977,66,1-19 (which is incorporated herein by reference) describe in detail pharmaceutically acceptable salts, and for example, a list of suitable salts is found in Allen, L.V., jr., editions, remington: the Science and Practice of Pharmacy [ Lemington: pharmaceutical science and practice ], 22 nd edition, pharmaceutical Press [ medical press ], london, uk (2012).

Pharmaceutically acceptable salts include those derived from suitable inorganic and organic acids and inorganic and organic bases. Examples of pharmaceutically acceptable, non-toxic acid addition salts are salts of amino groups formed with inorganic acids (such as hydrochloric, hydrobromic, phosphoric, sulfuric and perchloric acids) or organic acids (such as acetic, oxalic, maleic, tartaric, citric, succinic or malonic acid) or by using other methods known in the art (such as ion exchange). Other pharmaceutically acceptable salts include adipic acid salts, alginates, ascorbates, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphoric acid salts, camphorsulfonate, citrate, cyclopentapropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, caproate, hydroiodic acid salts, 2-hydroxy-ethanesulfonate, lactobionic aldehyde, lactate, laurate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like.

Salts derived from suitable bases include alkali metals, alkaline earth metals, ammonium and N ⁺ (C _1-4 Alkyl group ₄ ^- And (3) salt. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Additional pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates. As described hereinThe compounds are also provided as the free base and may be administered as the free base. Generally, salts of the compounds described herein will be those that provide compositions suitable for administration to a human or animal subject via any suitable route of administration of a pharmaceutical composition comprising the salt.

Salts (e.g., pharmaceutically acceptable salts) of the compounds described herein can be synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods. Typically, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent or in a mixture of both; in general, nonaqueous media like diethyl ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred.

It is to be understood that when a compound described herein contains more than one basic moiety or more than one acidic moiety, each such moiety may independently participate in forming an acid addition salt form or a base addition salt form, all possible salt forms being encompassed by the present disclosure. Furthermore, when two or more portions of a compound are in salt form, the anions or cations forming the two or more salt forms may be the same or different. Typically, the anions or cations forming two or more salt forms are the same. Typical molar ratios of anions or cations in salts of compounds of the present disclosure to compounds described herein are 3:1, 2:1, 1:1, 2:1, 3:1, 4:1, and 5:1. In some embodiments, the molar ratio of anions or cations (e.g., anions) to the compound in the salts of the compounds described herein is 1:1.

The compounds described herein are also provided as free bases and may be administered as free bases.

"pharmaceutically acceptable carrier" refers to a medium commonly accepted in the art for delivery of bioactive agents to animals, particularly mammals, and includes solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antimicrobial agents, antifungal agents), isotonicity agents, absorption delaying agents, salts, preservatives, pharmaceutical stabilizers, binders, buffers (e.g., maleic acid, tartaric acid, lactic acid, citric acid, acetic acid, sodium bicarbonate, sodium phosphate, etc.), disintegrants, lubricants, sweeteners, flavoring agents, dyes, etc., and combinations thereof, as known to those of skill in the art (see, e.g., allen, l.v., jr. Et al, remington: the Science and Practice of Pharmacy [ lein: pharmaceutical science and practice ] (volume 2), 22 nd edition, pharmaceutical Press [ medical publishing ] (2012)).

The terms "composition" and "formulation" are used interchangeably.

By "subject" considered for administration is meant a human (i.e., male or female of any age group, such as a pediatric subject (e.g., infant, child, or adolescent) or an adult subject (e.g., young, middle-aged, or elderly)) or non-human animal. In certain embodiments, the non-human animal is a mammal (e.g., a primate (e.g., a cynomolgus monkey or rhesus monkey), a commercially relevant mammal (e.g., a cow, pig, horse, sheep, goat, cat, or dog)), or a bird (e.g., a commercially relevant bird such as a chicken, duck, goose, or turkey). In certain embodiments, the non-human animal is a fish, reptile, or amphibian. The non-human animal may be male or female at any stage of development. The non-human animal may be a transgenic animal or a genetically engineered animal. The term "patient" refers to a human subject in need of treatment for a disease.

The term "administering (administer, administering or administeration)" refers to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing a compound or a pharmaceutically acceptable salt thereof, or a combination of the foregoing, in or to a subject.

The terms "treatment (treatment, treat and treating)" refer to the administration of a drug or medical care to a subject (e.g., a human) suffering from a disease or disorder of interest (e.g., cancer), and include: (i) Preventing the occurrence of a disease or disorder in a subject, particularly when such a subject is susceptible to the disorder but has not been diagnosed as having the disorder; (ii) inhibiting a disease or disorder, such as arresting its development; (iii) Moderating the disease or disorder, e.g., causing regression of the disease or disorder; and/or (iv) alleviating symptoms caused by the disease or condition (e.g., pain, weight loss, cough, fatigue, weakness, etc.). Thus, treatment includes reversing, alleviating, delaying the onset of, and/or inhibiting the progression of a disease (e.g., a disease described herein). In some embodiments, the treatment may be administered after one or more signs or symptoms of the disease have occurred or have been observed. In other embodiments, the treatment may be administered in the absence of signs or symptoms of the disease. For example, the treatment may be administered to a susceptible subject prior to onset of symptoms. Treatment may also be continued after the symptoms subside, for example, to delay or prevent recurrence.

An "effective amount" of a compound refers to an amount sufficient to elicit the desired biological response. The effective amount of the compound may vary depending upon such factors as the desired biological endpoint, the pharmacokinetics of the compound, the condition being treated, the mode of administration, and the age and health of the subject. In certain embodiments, the effective amount is a therapeutically effective amount. In certain embodiments, the effective amount is a prophylactically effective amount. In certain embodiments, the effective amount is the amount of the compound in a single dose. In certain embodiments, the effective amount is the combined amount of the compounds in multiple doses.

A "therapeutically effective amount" of a compound refers to an amount sufficient to provide a therapeutic benefit in the treatment of a disorder or to delay or minimize one or more symptoms associated with the disorder. A therapeutically effective amount of a compound means an amount of a therapeutic agent alone or in combination with other therapies that provides a therapeutic benefit in the treatment of a disorder. The term "therapeutically effective amount" may encompass an amount that improves overall therapy, reduces or avoids symptoms, signs, or etiologies of a disorder, and/or enhances the therapeutic efficacy of another therapeutic agent. In certain embodiments, a therapeutically effective amount is an amount sufficient to treat any of the diseases or conditions described herein.

A "prophylactically effective amount" of a compound refers to an amount sufficient to prevent a disorder or one or more symptoms associated with the disorder or prevent recurrence thereof. A prophylactically effective amount of a compound means an amount of a therapeutic agent alone or in combination with other agents that provides a prophylactic benefit in the prevention of a disorder. The term "prophylactically effective amount" may encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent.

As used herein, "inhibit (inhibition, inhibiting, inhibit)" and "inhibitor" and the like refer to the ability of a compound to reduce, slow, stop or prevent the activity of a biological process (e.g., the activity of an activin receptor-like kinase (e.g., ALK-5) in a subject or cell) or thereby alter disease progression, by, for example, altering a signaling pathway (e.g., altering TGF- β1 signaling).

In certain embodiments, the compounds described herein are "selective inhibitors" of one protein kinase relative to other kinases. In certain embodiments, the compounds provided herein are selective ALK-5 inhibitors, i.e., selective for ALK-5 relative to other kinases (e.g., relative to other activin receptor-like kinases such as ALK-2; janus kinases (JAKs), such as JAK1, JAK2, and/or JAK 3). The selectivity of a compound described herein in inhibiting the activity of ALK-5 relative to a different kinase (e.g., a different activin receptor-like kinase) may be through EC of the compound to inhibit the activity of the different kinase ₅₀ Or IC (integrated circuit) ₅₀ EC value against ALK-5 inhibiting Activity of the Compound ₅₀ Or IC (integrated circuit) ₅₀ The quotient of the values is measured. The selectivity of a compound described herein for ALK-5 over a different kinase (e.g., a different activin receptor-like kinase) may also be achieved by the K of the adduct of the compound with the different kinase _d Value of K relative to adduct of the compound with ALK-5 _d The quotient of the values is measured, for example, for IC of ALK-5 under comparable test conditions ₅₀ IC for ALK-2 inhibition as observed ₅₀ At least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 30-fold, at least 50-fold, at least 100-fold, or greater than 100-fold.

Compounds of formula (I)

In some embodiments, compounds having formula (I) are provided:

or a pharmaceutically acceptable salt thereof, wherein:

R ¹ is C ₁ -C ₅ Alkyl, C ₃ -C ₅ Carbocycles or halogens;

R ² is-H, halogen, C optionally substituted by one or more-F ₁ -C ₃ Alkyl, or cyclopropyl optionally substituted with one or more-F;

R ³ is-H, halogen, C optionally substituted by one or more-F ₁ -C ₃ Alkyl, or cyclopropyl optionally substituted with one or more-F; and is also provided with

Ring G isWherein R is ¹⁰ 、R ¹¹ And R is ¹² One of them is a bond with-N (H) -to which ring G of formula (I) is attached, R ¹⁰ 、R ¹¹ And R is ¹² One of them is-H, and R ¹⁰ 、R ¹¹ And R is ¹² One of them is C ₁ -C ₄ An alkyl group; or (b)

Ring G is C optionally substituted with ₆ -C ₁₀ Aryl:

(i) One or more halogens;

(ii) Sulfonamide;

(iii) Optionally by one or more C ₁ -C ₆ Alkyl-substituted monocyclic, bicyclic or spiro C ₃ -C ₁₀ Carbocycles, or C optionally substituted by hydroxy or one or more halogens ₃ -C ₆ A carbocycle, wherein the carbocycle is attached to ring G by a single bond or a methylene or ethylene linker at a position on ring G that is meta or para with respect to the-N (H) -attached to ring G; or (b)

(iv) May contain up to 3 heteroatoms independently selected from N and O and optionally and independently substituted with one or more C' s ₁ -C ₆ Alkyl-substituted monocyclic, bicyclic, bridged or spiro C ₃ -C ₁₀ Heterocycles, or C optionally substituted by hydroxy or one or more halogens ₃ -C ₆ Carbocycles in which the heterocycle is attached to ring G at a position meta or para to the-N (H) -ring G by a single bond or a methylene or ethylene groupThe joint is attached to the ring G.

In some embodiments, ring G isWherein R is ¹⁰ 、R ¹¹ And R is ¹² One of them is a bond with-N (H) -to which ring G of formula (I) is attached, R ¹⁰ 、R ¹¹ And R is ¹² One of them is-H, and R ¹⁰ 、R ¹¹ And R is ¹² One of them is C ₁ -C ₄ An alkyl group. In further embodiments, R ¹⁰ Is C ₁ -C ₄ Alkyl, R ¹¹ Is a bond to-N (H) -attached to ring G of formula (I), and R ¹² is-H.

In some embodiments, compounds having formula (I) are provided:

or a pharmaceutically acceptable salt thereof, wherein:

R ¹ is C ₁ -C ₅ Alkyl, C ₃ -C ₅ Carbocycles or halogens;

R ² is-H, halogen, C optionally substituted by one or more-F ₁ -C ₃ Alkyl, or cyclopropyl optionally substituted with one or more-F;

R ³ is-H, halogen, C optionally substituted by one or more-F ₁ -C ₃ Alkyl, or cyclopropyl optionally substituted with one or more-F; and is also provided with

Ring G isWherein->Indicating the point at which ring G is attached to-N (H) -; or (b)

Ring G is C optionally substituted with ₆ -C ₁₀ Aryl:

(v) One or more halogens;

(vi) Sulfonamide;

(vii) Optionally by one or more C ₁ -C ₆ Alkyl-substituted monocyclic, bicyclic or spiro C ₃ -C ₁₀ Carbocycles, or C optionally substituted by hydroxy or one or more halogens ₃ -C ₆ A carbocycle, wherein the carbocycle is attached to ring G by a single bond or a methylene or ethylene linker at a position on ring G that is meta or para with respect to the-N (H) -attached to ring G; or (b)

(viii) May contain up to 3 heteroatoms independently selected from N and O and optionally and independently substituted with one or more C' s ₁ -C ₆ Alkyl-substituted monocyclic, bicyclic, bridged or spiro C ₃ -C ₁₀ Heterocycles, or C optionally substituted by hydroxy or one or more halogens ₃ -C ₆ Carbocycles wherein the heterocycle is attached to ring G by a single bond or a methylene or ethylene linker at a position on ring G that is meta or para to the-N (H) -attached to ring G.

In some embodiments, R ¹ Is C ₁ -C ₅ Alkyl or C ₃ -C ₅ Carbocycles.

In some embodiments, R ¹ Is methyl or cyclopropyl.

In some embodiments, R ¹ Is halogen.

In some embodiments, R ¹ Is methyl or halogen.

In some embodiments, R ¹ Is methyl or chlorine.

In some embodiments, R ² is-H, halogen, -CH ₃ 、-CF ₃ Or cyclopropyl.

In some embodiments, R ² is-H.

In some embodiments, R ³ is-H, halogen, -CH ₃ 、-CF ₃ Or cyclopropyl.

In some embodiments, R ³ is-H or halogen.

In some embodiments, R ³ is-H or fluorine.

In some embodiments, R ³ is-H.

In some embodiments, ring G is

In some embodiments, ring G is phenyl optionally substituted with:

(i) One or more halogens;

(ii) Sulfonamide;

(iii) Optionally by one or more C ₁ -C ₆ Alkyl-substituted monocyclic, bicyclic or spiro C ₃ -C ₁₀ Carbocycles, or C optionally substituted by hydroxy or one or more halogens ₃ -C ₆ A carbocycle, wherein the carbocycle is attached to ring G by a single bond or a methylene or ethylene linker at a position on ring G that is meta or para with respect to the-N (H) -attached to ring G; or (b)

(iv) May contain up to 3 heteroatoms independently selected from N and O and optionally and independently substituted with one or more C' s ₁ -C ₆ Alkyl-substituted monocyclic, bicyclic, bridged or spiro C ₃ -C ₁₀ Heterocycles, or C optionally substituted by hydroxy or one or more halogens ₃ -C ₆ Carbocycles wherein the heterocycle is attached to ring G by a single bond or a methylene or ethylene linker at a position on ring G that is meta or para to the-N (H) -attached to ring G.

In some embodiments, ring G is C substituted with ₆ -C ₁₀ Aryl (in some embodiments, phenyl):

(i) One or more halogens;

(ii) Sulfonamide;

(iii) Optionally by one or more C ₁ -C ₆ Alkyl-substituted monocyclic, bicyclic or spiro C ₃ -C ₁₀ Carbocycles, or C optionally substituted by hydroxy or one or more halogens ₃ -C ₆ A carbocycle, wherein the carbocycle is attached to ring G by a single bond or a methylene or ethylene linker at a position on ring G that is meta or para with respect to the-N (H) -attached to ring G; or (b)

(iv) May contain up to 3 groups independently selected from N and OHeteroatom and optionally and independently substituted with one or more C ₁ -C ₆ Alkyl-substituted monocyclic, bicyclic, bridged or spiro C ₃ -C ₁₀ Heterocycles, or C optionally substituted by hydroxy or one or more halogens ₃ -C ₆ Carbocycles wherein the heterocycle is attached to ring G by a single bond or a methylene or ethylene linker at a position on ring G that is meta or para to the-N (H) -attached to ring G.

In some embodiments, ring G is substituted with one or more halogens.

In some embodiments, ring G is substituted with a sulfonamide.

In some embodiments, ring G is substituted with: optionally by one or more C ₁ -C ₆ Alkyl-substituted monocyclic, bicyclic or spiro C ₃ -C ₁₀ Carbocycles, or C optionally substituted by hydroxy or one or more halogens ₃ -C ₆ A carbocycle, wherein the carbocycle is attached to ring G by a single bond or a methylene or ethylene linker at a position on ring G that is meta or para relative to the-N (H) -attached to ring G.

In some embodiments, ring G is substituted with: optionally by one or more C ₁ -C ₆ Alkyl-substituted monocyclic C ₃ -C ₇ Carbocycles, or C optionally substituted by hydroxy or one or more halogens ₃ -C ₆ Carbocycles.

In some embodiments, ring G is substituted with: optionally by one or more C ₁ -C ₆ An alkyl-substituted cyclohexyl group, or C optionally substituted with hydroxy or one or more halogens ₃ -C ₆ Carbocycles.

In some embodiments, the carbocycle attached to ring G is unsubstituted.

In some embodiments, the carbocycle attached to ring G is attached to ring G by a single bond.

In some embodiments, ring G is substituted with: may contain up to 3 heteroatoms independently selected from N and O and optionally and independently substituted with one or more C' s ₁ -C ₆ Alkyl-substituted monocyclic, bicyclic, bridged or spiro C ₃ -C ₁₀ Heterocycles, or C optionally substituted by hydroxy or one or more halogens ₃ -C ₆ Carbocycles wherein the heterocycle is attached to ring G by a single bond or a methylene or ethylene linker at a position on ring G that is meta or para to the-N (H) -attached to ring G.

In some embodiments, ring G is substituted with: may contain up to 3 heteroatoms independently selected from N and O and optionally and independently substituted with one or more C' s ₁ -C ₆ Alkyl-substituted monocyclic C ₅ -C ₆ Heterocycles, or C optionally substituted by hydroxy or one or more halogens ₃ -C ₆ Carbocycles.

In some embodiments, ring G is substituted with: may contain up to 2 heteroatoms independently selected from N and O and optionally and independently substituted with one or more C' s ₁ -C ₆ Alkyl-substituted monocyclic C ₆ Heterocycles, or C optionally substituted by hydroxy or one or more halogens ₃ -C ₆ Carbocycles.

In some embodiments, ring G is substituted with: optionally and independently is one or more C ₁ -C ₆ Alkyl-substituted piperazinyl, morpholinyl, piperidinyl or oxacyclohexyl, or C optionally substituted with hydroxy or one or more halogens ₃ -C ₆ Carbocycles.

In some embodiments, the heterocycle attached to ring G is unsubstituted or monosubstituted.

In some embodiments, the heterocycle attached to ring G is unsubstituted.

In some embodiments, the heterocycle attached to ring G is attached to ring G by a single bond.

In some embodiments, the carbocycle or heterocycle attached to ring G is optionally and independently methyl, CF ₃ CH ₂ -or HOCH ₂ CH ₂ -substitution.

In some embodiments, the carbocycle or heterocycle attached to ring G is attached to ring G at a position on ring G that is meta relative to the-N (H) -attached to ring G.

In some embodiments, the carbocycle or heterocycle attached to ring G is attached to ring G at a position on ring G that is para relative to the-N (H) -attached to ring G.

In some embodiments, compounds having formula (II) are provided:

or a pharmaceutically acceptable salt thereof. Variable R ¹ The values and alternative values of (a) are as described for the compounds having formula (I).

In some embodiments, compounds having formula (III) are provided:

or a pharmaceutically acceptable salt thereof, wherein:

ring J is attached to the phenylene at a position meta or para to the-N (H) -attached to the phenylene;

A ¹ is-N (R) ⁴ ) -, -O-or>C(H)(R ⁴ )；

R ⁴ is-H, or C ₁ -C ₆ Alkyl or C ₃ -C ₆ Carbocycles, each of which is optionally substituted with hydroxy or one or more halogens;

A ² is that>N-or>C(H)-；

Z is>CH ₂ The method comprises the steps of carrying out a first treatment on the surface of the And X and Y are independently>CH ₂ Or (b)>C(CH ₃ ) ₂ Or X and Y are both>CH-and are bonded together by methylene or ethylene bridges; or (b)

Y is>CH ₂ Or (b)>C(CH ₃ ) ₂ And X and Z are both>CH-and are bonded together by methylene or ethylene bridges; and is also provided with

n is 0, 1 or 2. Variable R ¹ The values and alternative values of (a) are as described for the compounds having formula (I).

In some embodiments, A ¹ Is that>C(H)(R ⁴ )。

In some embodiments, A ¹ is-N (R) ⁴ ) -or-O-。

In some embodiments, A ¹ is-N (R) ⁴ )-。

In some embodiments, A ¹ is-O-.

In some embodiments, R ⁴ is-H, or C ₁ -C ₆ Alkyl optionally substituted with hydroxy or one or more halogens.

In some embodiments, R ⁴ is-H, methyl, CF ₃ CH ₂ -or HOCH ₂ CH ₂ -。

In some embodiments, R ⁴ is-H or methyl.

In some embodiments, A ² Is that>C(H)-。

In some embodiments, A ² Is that>N-。

In some embodiments, ring J is:

in some embodiments, ring J is:

in some embodiments, ring J is attached to the phenylene at a position meta to the-N (H) -attached to the phenylene.

In some embodiments, ring J is attached to the phenylene at a position ortho to the-N (H) -attached to the phenylene.

In some embodiments, n is 0 or 1.

In some embodiments, n is 0.

In some embodiments, compounds having formula (IV) are provided:

or a medicine thereofA pharmaceutically acceptable salt, wherein the variables (e.g., R ¹ Ring J, A ¹ 、A ² ) The values and alternative values of (a) are as described for compounds having formula (I) and/or formula (III).

In some embodiments, a compound or pharmaceutically acceptable salt thereof is provided having one of the following structures:

statement of list of chemical groups in any definition of a variable herein includes definition of the variable as any single group or combination of listed groups. The recitation herein of an embodiment for a variable includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

Pharmaceutical compositions, kits and administration

Provided herein are pharmaceutical compositions comprising a compound having formula (I), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient. In certain embodiments, the pharmaceutical compositions provided herein comprise a therapeutically and/or prophylactically effective amount of a compound having formula (I) or a pharmaceutically acceptable salt thereof. In certain embodiments, the pharmaceutical composition comprises a therapeutically effective amount. The pharmaceutical compositions provided herein may further comprise one or more additional therapeutic agents (e.g., antiproliferative agents, such as anticancer agents), including any of the additional therapeutic agents described herein in combination with the combination therapy.

The pharmaceutical compositions described herein may be prepared by any method known in the pharmacological arts. Typically, such methods of preparation include associating a compound described herein (e.g., "active ingredient") with a carrier or excipient, and/or one or more other auxiliary ingredients, and then shaping and/or packaging the product into the desired single or multi-dose unit, if needed and/or desired. In some embodiments, the pharmaceutical composition is suitable for oral administration.

The pharmaceutical compositions may be prepared, packaged and/or sold in bulk as single unit doses and/or as a plurality of single unit doses. A "unit dose" is a discrete amount of a pharmaceutical composition comprising a predetermined amount of an active ingredient. The amount of active ingredient is typically equal to the dose of active ingredient to be administered to the subject and/or a convenient fraction of such dose, such as half or one third of such dose.

The relative amounts of the active ingredients (e.g., a compound having formula (I) or a pharmaceutically acceptable salt thereof), pharmaceutically acceptable excipients, and/or any additional ingredients in the pharmaceutical compositions described herein will vary depending, for example, on the nature, size, and/or condition of the subject receiving the treatment, and on the route of administration of the composition. The composition may comprise from 0.1% to 100% (w/w) of the active ingredient.

Pharmaceutically acceptable excipients used to manufacture the provided pharmaceutical compositions include inert diluents, dispersants and/or granulating agents, surfactants and/or emulsifying agents, disintegrants, binders, preservatives, buffers, lubricants and/or oils. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring and perfuming agents may also be present in the composition.

Examples of diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, sugar powder, and mixtures thereof.

Examples of granulating and/or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clay, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponges, cation exchange resins, calcium carbonate, silicates, sodium carbonate, crosslinked poly (vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, crosslinked sodium carboxymethyl cellulose (crosslinked carboxymethyl cellulose), methyl cellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and mixtures thereof.

Examples of surfactants and/or emulsifiers include natural emulsifiers (e.g., gum arabic, agar, alginic acid, sodium alginate, tragacanth, carrageenan (chondrux), cholesterol, xanthan gum, pectin, gelatin, egg yolk, casein, lanolin, cholesterol, waxes and lecithin), colloidal clays (e.g., bentonite (aluminum silicate) and Veegum (magnesium aluminum silicate)), long chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, glyceryl triacetate, ethylene glycol distearate, glyceryl monostearate and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxypolymethylene, polyacrylic acid, acrylic acid polymers and carboxyvinyl polymers), carrageenan, cellulose derivatives (e.g., carboxymethyl cellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate)20 Polyoxyethylene sorbitan (>60 Polyoxyethylene sorbitan monooleate (+)>80 Sorbitan monopalmitate ()>40 Sorbitan monostearate () >60 Sorbitan tristearate ()>65 Glycerol monooleate, sorbitan monooleate ()>80 Polyoxyethylene esters (e.g., polyoxyethylene monostearate ()>45 Polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylenestearate and +.>) Sucrose fatty acid ester, polyethylene glycol fatty acid ester (e.g.)>) Polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether ()>30 Poly (vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl laurate, sodium lauryl sulfate, < >>F-68, poloxamer P-188, cetyltrimethylamine bromide, cetylpyridinium chloride, benzalkonium chloride, sodium docusate, and/or mixtures thereof.

Examples of binders include starches (e.g., corn starch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, and the like), natural and synthetic gums (e.g., gum arabic, sodium alginate, irish moss extract, pan Waer gum, gum ghatti, mucilage of the psyllium husk, carboxymethylBase cellulose, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, microcrystalline cellulose, cellulose acetate, poly (vinyl-pyrrolidone), magnesium aluminum silicate And larch arabinogalactan), alginate, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylate, wax, water, alcohol, and/or mixtures thereof.

Examples of preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, antiprotozoal preservatives, alcohol preservatives, acid preservatives, and other preservatives. In certain embodiments, the preservative is an antioxidant. In other embodiments, the preservative is a chelating agent.

Examples of antioxidants include alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulphite, sodium metabisulfite and sodium sulfite.

Examples of chelating agents include ethylenediamine tetraacetic acid (EDTA) and salts and hydrates thereof (e.g., sodium ethylenediamine tetraacetate, disodium ethylenediamine tetraacetate, trisodium ethylenediamine tetraacetate, disodium calcium ethylenediamine tetraacetate, dipotassium ethylenediamine tetraacetate, etc.), citric acid and salts and hydrates thereof (e.g., citric acid monohydrate), fumaric acid and salts and hydrates thereof, malic acid and salts and hydrates thereof, phosphoric acid and salts and hydrates thereof, and tartaric acid and salts and hydrates thereof. Exemplary antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethanol, glycerol, hexetidine, imidazolidinyl urea, phenol, phenoxyethanol, phenethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.

Examples of antifungal preservatives include butyl parahydroxybenzoate, methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.

Examples of alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate esters and phenylethanol.

Examples of acidic preservatives include vitamin a, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.

Other preservatives include tocopherol, tocopheryl acetate, detoxime mesylate (deteroxime mesylate), trimethoprim bromide, butylated Hydroxyanisole (BHA), butylated Hydroxytoluene (BHT), and combinations thereof ethylenediamine, sodium Lauryl Sulfate (SLS), sodium Lauryl Ether Sulfate (SLES), sodium bisulphite, sodium metabisulfite, potassium sulfite, potassium metabisulfite,Plus、Methyl parahydroxybenzoate,/-hydroxybenzoate>115、/>II、/>And->

Examples of buffers include citrate buffer, acetate buffer, phosphate buffer, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glucuronate, calcium glucoheptonate, calcium gluconate, D-gluconate, calcium glycerophosphate, calcium lactate, propionic acid, calcium fructonate, valerate, dicalcium phosphate, phosphoric acid, tricalcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dipotassium phosphate, monopotassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, disodium phosphate, monosodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, ringer's solution, ethanol, and mixtures thereof.

Examples of lubricants include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behenate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and mixtures thereof.

Examples of natural oils include almond (almonds) oil, apricot kernel (apricots kernel) oil, avocado oil, babassu oil, bergamot oil, blackcurrant seed oil, borage oil, juniper oil, chamomile oil, canola oil, caraway oil, carnauba oil, castor oil, cassia oil, cocoa butter oil, coconut oil, cod liver oil, coffee oil, corn oil, cotton seed oil, emu oil, eucalyptus oil, evening primrose oil, fish oil, linseed oil, geraniol oil, fenugreek oil, grape seed oil, hazelnut oil, sea cable oil, isopropyl myristate oil, jojoba oil, chestnut oil, lavender oil, corn oil, and mixtures thereof Lavender oil, lemon oil, litsea cubeba oil, macadamia nut oil, mallow oil, mango seed oil, white pool seed oil, mink oil, nutmeg oil, olive oil, orange oil, thorn sea bream oil, palm kernel oil, peach kernel oil, peanut oil, poppy seed oil, pumpkin seed oil, rapeseed oil, rice bran oil, rosemary oil, safflower oil, sandalwood oil, crane (sasquana) oil, savoury oil, sea buckthorn oil, sesame oil, shea butter, silicone oil, soybean oil, sunflower oil, tea tree oil, thistle oil, chinese toosendan oil, vetiver oil, walnut oil and wheat germ oil. Exemplary synthetic oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and mixtures thereof.

For oral administration orLiquid dosage forms for parenteral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage form may contain inert diluents commonly used in the art (such as, for example, water or other solvents), stabilizers and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, dimethylformamide, oils (e.g., cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols, and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. In certain embodiments for parenteral administration, the active ingredient is mixed with a solubilizing agent, such asAlcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and mixtures thereof.

Injectable formulations (e.g., sterile injectable aqueous or oleaginous suspensions) may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may be a sterile injectable solution, suspension or emulsion in a non-toxic, parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids (such as oleic acid) are useful in the preparation of injectables.

The injectable formulation may be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which may be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

In order to prolong the effect of the drug, it is often desirable to slow down the absorption of the subcutaneously or intramuscularly injected drug. This can be achieved by using a liquid suspension of a crystalline material or an amorphous material which is poorly water soluble. The rate of absorption of the drug then depends on its rate of dissolution, which in turn may depend on the crystal size as well as the crystal form. Alternatively, delayed absorption of a parenterally administered drug may be achieved by dissolving or suspending the drug in an oil vehicle.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is mixed with the following substances: at least one inert, pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate and/or (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, (c) humectants such as glycerin, (d) disintegrants such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, (e) solution retarding agents such as waxes, (f) absorption accelerators such as quaternary ammonium compounds, (g) wetting agents such as, for example, cetyl alcohol and glyceryl monostearate, (h) absorbents such as kaolin and bentonite, and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may comprise buffering agents.

Solid compositions of a similar type may be used as fillers in soft and hard filled gelatin capsules using such excipients as lactose or milk sugar, high molecular weight polyethylene glycols and the like. Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmacological arts. They may optionally contain opacifying agents and may be compositions such that they release only or preferentially one or more active ingredients, optionally in a delayed manner, in a certain part of the intestinal tract. Examples of encapsulation compositions that may be used include polymeric materials and waxes. Solid compositions of a similar type may be used as fillers in soft and hard filled gelatin capsules using such excipients as lactose or milk sugar, high molecular weight polyethylene glycols and the like.

The active ingredient may be in microencapsulated form together with one or more excipients as indicated above. Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, controlled release coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms, the active ingredient may be admixed with at least one inert diluent (such as sucrose, lactose or starch). As is common practice, such dosage forms may contain additional substances other than inert diluents, for example, tableting lubricants and other tableting aids such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may comprise buffering agents. They may optionally contain opacifying agents and may be compositions such that they release only or preferentially one or more active ingredients, optionally in a delayed manner, in a certain part of the intestinal tract. Examples of encapsulants that may be used include polymeric materials and waxes.

Dosage forms for topical and/or transdermal administration of the compounds described herein may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants and/or patches. Typically, the active ingredient is admixed under sterile conditions with a pharmaceutically acceptable carrier or excipient and/or any required preservatives and/or buffers which may be required. In addition, the present disclosure contemplates the use of transdermal patches, which generally have the additional advantage of providing controlled delivery of the active ingredient to the body. Such dosage forms may be prepared, for example, by dissolving and/or partitioning the active ingredient in an appropriate medium. Alternatively or additionally, the rate may be controlled by providing a rate controlling membrane and/or by dispersing the active ingredient in the polymer matrix and/or gel.

Suitable devices for delivering the intradermal pharmaceutical compositions described herein include short needle devices. The intradermal composition can be administered through a device that limits the effective penetration length of the needle into the skin. Alternatively or additionally, conventional syringes may be used for classical mango diagram (mantoux) methods of intradermal administration. Jet injection devices that deliver a liquid formulation to the dermis via a liquid jet injector and/or needle that pierces the stratum corneum and produces a jet that reaches the dermis are suitable. A flicking powder/particle delivery device using compressed gas to accelerate the compound in powder form through the outer layer of skin to the dermis is suitable.

Formulations suitable for topical application include, but are not limited to, liquid and/or semi-liquid formulations, such as liniments, lotions, oil-in-water and/or water-in-oil emulsions, such as creams, ointments and/or pastes, and/or solutions and/or suspensions. Topically administrable formulations may, for example, comprise from about 1% to about 10% (w/w) of the active ingredient, although the concentration of the active ingredient may be up to the solubility limit of the active ingredient in the solvent. Formulations for topical application may further comprise one or more of the additional ingredients described herein.

Compositions for rectal or vaginal administration are typically suppositories which can be prepared by combining the conjugates described herein with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax, which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.

The pharmaceutical compositions described herein may be prepared, packaged and/or sold as a formulation suitable for pulmonary administration via the oral cavity. Such formulations may comprise dry particles containing the active ingredient and having a diameter in the range of about 0.5 to about 7 nanometers, or about 1 to about 6 nanometers. Such compositions are conveniently in the form of dry powders for administration using devices comprising a dry powder reservoir into which a flow of propellant may be directed to disperse the powder and/or using self-propelled solvent/powder dispensing containers such as devices comprising an active ingredient dissolved and/or suspended in a low boiling point propellant in a sealed container. Such powders comprise particles, wherein at least 98% by weight of the particles have a diameter greater than 0.5 nm and at least 95% by number of the particles have a diameter less than 7 nm. Alternatively, at least 95% by weight of the particles have a diameter greater than 1 nanometer and at least 90% by number of the particles have a diameter less than 6 nanometers. The dry powder composition may include a solid fine powder diluent (such as sugar) and is conveniently provided in unit dosage form.

Low boiling point propellants typically include liquid propellants having a boiling point below 65°f at atmospheric pressure. Typically, the propellant may comprise 50% to 99.9% (w/w) of the composition and the active ingredient may comprise 0.1% to 20% (w/w) of the composition. The propellant may further comprise additional ingredients such as liquid nonionic surfactants and/or solid anionic surfactants and/or solid diluents (which may have particle sizes of the same order as the particles comprising the active ingredient).

Pharmaceutical compositions described herein formulated for pulmonary delivery may provide the active ingredient in the form of droplets of a solution and/or suspension. Such formulations may be prepared, packaged as or sold as aqueous and/or diluted alcoholic solutions and/or suspensions containing the active ingredient (optionally sterile) and may be conveniently applied using any of the nebulisation and/or evaporation devices. Such formulations may further comprise one or more additional ingredients including, but not limited to, flavoring agents (e.g., sodium saccharin), volatile oils, buffers, surfactants, and/or preservatives (e.g., methyl hydroxybenzoate). The average diameter of the droplets provided by this route of administration may be in the range of about 0.1 to about 200 nanometers.

Formulations described herein that can be used for pulmonary delivery can be used for intranasal delivery of the pharmaceutical compositions described herein. Another formulation suitable for intranasal administration is a meal comprising the active ingredient and having an average particle of about 0.2 to 500 microns. Such formulations are administered by rapid inhalation through the nasal passages from a powder container close to the nostrils.

Formulations for nasal administration may, for example, comprise from about as little as 0.1% (w/w) to as much as 100% (w/w) of the active ingredient, and may comprise one or more of the additional ingredients described herein.

The pharmaceutical compositions described herein may be prepared, packaged into formulations and/or sold as formulations for oral administration. Such formulations may, for example, be in the form of tablets and/or lozenges prepared using conventional methods and may contain, for example, from 0.1% to 20% (w/w) of the active ingredient, the balance comprising the orally dissolvable and/or degradable composition and optionally one or more of the additional ingredients described herein. Alternatively, formulations for oral administration may comprise powders and/or aerosolized and/or atomized solutions and/or suspensions containing the active ingredient. Such powdered, aerosolized, and/or aerosolized formulations, when dispersed, may have an average particle size and/or droplet size in the range of about 0.1 to about 200 nanometers, and may further comprise one or more of the additional ingredients described herein.

The pharmaceutical compositions described herein may be prepared, packaged into formulations and/or sold as formulations for ophthalmic administration. Such formulations may, for example, be in the form of eye drops comprising, for example, 0.1% to 1.0% (w/w) solutions and/or suspensions of the active ingredient in aqueous or oily liquid vehicles or excipients. Such drops may further comprise buffers, salts, and/or one or more other additional ingredients described herein. Other useful ophthalmically administrable formulations include those containing the active ingredient in microcrystalline form and/or in liposomal formulations. Ear drops and/or eye drops are also contemplated as falling within the scope of the present disclosure.

Although the description of pharmaceutical compositions provided herein is primarily directed to pharmaceutical compositions suitable for administration to humans, those skilled in the art will appreciate that such compositions are generally suitable for administration to all kinds of animals. Modifications to pharmaceutical compositions suitable for administration to humans in order to adapt the compositions to a variety of animals are well known and common skilled veterinary pharmacologists can design and/or perform such modifications using common experimentation.

The compounds, salts, and compositions provided herein can be administered by any route including enteral (e.g., oral), parenteral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, intradermal, rectal, intravaginal, intraperitoneal, topical (e.g., by powder, ointment, cream, and/or drops), ophthalmic, mucosal, nasal, oral, sublingual routes; by intratracheal instillation, bronchial instillation and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol. Routes of particular concern are oral administration, intravenous administration (e.g., systemic intravenous injection), regional administration via blood and/or lymph supply, and/or direct administration to the affected site. Generally, the most suitable route of administration will depend on a variety of factors, such as the nature of the agent (e.g., its stability in the gastrointestinal environment) and/or the condition of the subject (e.g., whether the subject is able to tolerate oral administration). In some embodiments, the pharmaceutical composition is formulated for oral administration.

The compounds, salts, and compositions provided herein are typically formulated in dosage unit form for ease of administration and uniformity of dosage. However, it should be understood that the total daily use of the compositions described herein will be determined by the physician within the scope of sound medical judgment. The particular therapeutically effective dose level of any particular subject or organism will depend on a variety of factors, including the severity of the disease or disorder being treated; the activity of the particular active ingredient employed; the specific composition employed; age, body weight, general health, sex, and diet of the subject; the time of administration, route of administration and rate of excretion of the particular active ingredient employed; duration of treatment; a medicament for use in combination or simultaneously with the particular active ingredient employed; and similar factors well known in the medical arts.

The exact amount of compound, salt or composition required to achieve an effective amount will vary from subject to subject, depending, for example, on the species, age and general condition of the subject, the severity of the side effects or disorders, the nature of the particular compound, the mode of administration, and the like. An effective amount may be included in a single dose (e.g., a single oral dose) or multiple doses (e.g., multiple oral doses). In certain embodiments, when multiple doses are administered to a subject or applied to tissue or cells, any two of the multiple doses comprise different or substantially the same amounts of a compound described herein. In certain embodiments, when multiple doses are administered to a subject or to tissue or cells, the multiple doses are administered to the subject or the multiple doses are administered to the tissue or cells at a frequency of three doses per day, two doses per day, one dose every other day, one dose every three days, one dose per week, two weeks, three weeks, or four weeks. In certain embodiments, the multiple doses are administered to the subject or the multiple doses are applied to the tissue or cells at a frequency of one dose per day. In certain embodiments, the multiple doses are administered to the subject or the multiple doses are applied to the tissue or cells at a frequency of two doses per day. In certain embodiments, the multiple doses are administered to the subject or the multiple doses are applied to the tissue or cells at a frequency of three doses per day. In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, the duration between the first and last dose of the multiple doses is one day, two days, four days, one week, two weeks, three weeks, one month, two months, three months, four months, six months, nine months, one year, two years, three years, four years, five years, seven years, ten years, fifteen years, twenty years, or the lifetime of the subject, tissue or cell. In certain embodiments, the duration between the first and last dose of the plurality of doses is three months, six months, or one year. In certain embodiments, the duration between the first and last dose of the plurality of doses is the lifetime of the subject, tissue, or cell. In certain embodiments, a dose described herein (e.g., a single dose, or any of a plurality of doses) independently comprises 0.1 μg to 1 μg, 0.001mg to 0.01mg, 0.01mg to 0.1mg, 0.1mg to 1mg, 1mg to 3mg, 3mg to 10mg, 10mg to 30mg, 30mg to 100mg, 100mg to 300mg, 300mg to 1,000mg, or 1g to 10g (inclusive) of a compound described herein. In certain embodiments, the dosages described herein independently comprise 1mg to 3mg (inclusive) of the compounds described herein. In certain embodiments, the dosages described herein independently comprise 3mg to 10mg (inclusive) of the compounds described herein. In certain embodiments, the dosages described herein independently comprise 10mg to 30mg (inclusive) of the compounds described herein. In certain embodiments, the dosages described herein independently comprise 30mg to 100mg (inclusive) of the compounds described herein. In some embodiments, the expected dose is in the range of 1mg/Kg subject mass and 150mg/Kg subject weight, e.g., at least about 1mg/Kg, at least about 10mg/Kg, at least about 20mg/Kg, at least about 30mg/Kg, at least about 40mg/Kg, at least about 50mg/Kg, at least about 60mg/Kg, at least about 70mg/Kg, at least about 80mg/Kg, at least about 90mg/Kg, at least about 100mg/Kg, at least about 110mg/Kg, at least about 120mg/Kg, at least about 130mg/Kg, at least about 140mg/Kg, or about 150mg/Kg.

The dosage ranges as described herein provide guidance for administration of the provided pharmaceutical compositions to adults. The amount administered to, for example, a child or adolescent may be determined by a physician or skilled artisan and may be lower than or the same as the amount administered to an adult.

The disclosure also encompasses kits (e.g., pharmaceutical packs). Kits may be provided that include a compound, salt, or pharmaceutical composition described herein, and a container (e.g., a vial, ampoule, bottle, syringe and/or dispenser package, or other suitable container). In some embodiments, the kit provided may optionally further comprise a second container comprising a pharmaceutical excipient for diluting or suspending the pharmaceutical composition or compound or salt described herein. In some embodiments, the pharmaceutical compositions or compounds or salts described herein provided in the first container and the second container are combined to form one unit dosage form.

Thus, in one aspect, a kit is provided comprising a first container comprising a compound, salt or pharmaceutical composition described herein. In certain embodiments, the kit can be used to treat a disease (e.g., a proliferative disease, such as cancer) in a subject in need thereof. In certain embodiments, the kit may be used to prevent a disease in a subject in need thereof. In certain embodiments, the kit may be used to reduce the risk of developing a disease in a subject in need thereof.

In certain embodiments, the kits described herein further comprise instructions for using the kits. Kits described herein may also include information required by regulatory authorities, such as the U.S. Food and Drug Administration (FDA). In certain embodiments, the information included in the kit is prescription information. The kits described herein may include one or more additional therapeutic agents described herein as separate compositions or in a combination comprising a compound or pharmaceutical composition described herein.

In the combinations and/or kits described herein, the compounds of the present disclosure and the other therapeutic agents may be manufactured and/or formulated by the same or different manufacturers. Furthermore, the compounds of the present disclosure and other therapeutic agents may be used together in combination therapies: (i) Prior to publishing the combination product to a physician (e.g., in the case of a kit comprising a compound of the disclosure and other therapeutic agent); (ii) By (or at the direction of) a physician shortly before administration; (iii) In the patient himself, for example during sequential administration of the compounds of the present disclosure and other therapeutic agents.

The pharmaceutical composition (or formulation) for application may be packaged in a variety of ways depending on the method used to administer the drug. Generally, the articles for dispensing include containers in which the pharmaceutical formulation is stored in a suitable form. Suitable containers are well known to those skilled in the art and include materials such as bottles (plastic and glass), sachets, ampoules, plastic bags, metal cylinders, and the like. The container may also include a tamper-proof fit to prevent light access to the contents of the package. In addition, the container has a label placed thereon that describes the contents of the container. The tag may also include an appropriate warning.

In some embodiments, the concentration of the one or more therapeutic agents provided in the pharmaceutical composition is less than 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%,14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/v.

In some embodiments of the present invention, in some embodiments, the concentration of the one or more therapeutic agents provided in the pharmaceutical composition is greater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25%, 19%, 18.75%, 18.50%, 18.25%, 18%, 17.75%, 17.50%, 17.25%, 17%, 16.75%, 16.50%, 16.25%, 16%, 15.75%, 15.50%, 15.25%, 15%, 14.75%, 14.50%, 14.25%, 14%, 13.75%, 13.50%, 13.25%, 13%, 12.75%, 12.50%, 12.25%, 12.75%, 11.50%, 11.25%, 11%, 10.75%, 10.50%, 10.25%, 10%, 9.75%, 9.50%, 9.25%, 9%, 8.75%, 8.50%, 8.25%, 8% >. 7.75%, 7.50%, 7.25%, 7%, 6.75%, 6.50%, 6.25%, 6%, 5.75%, 5.50%, 5.25%, 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0002% w/v/w/v.

In some embodiments, the concentration of the one or more therapeutic agents provided in the pharmaceutical composition is in a range of about 0.0001% to about 50%, about 0.001% to about 40%, about 0.01% to about 30%, about 0.02% to about 29%, about 0.03% to about 28%, about 0.04% to about 27%, about 0.05% to about 26%, about 0.06% to about 25%, about 0.07% to about 24%, about 0.08% to about 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12%, about 1% to about 10% w/w, w/v or v/v.

In some embodiments, the concentration of the one or more therapeutic agents provided in the pharmaceutical composition is in the range of about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to about 1%, about 0.1% to about 0.9% w/w, w/v, or v/v.

Therapeutic methods and uses

As shown herein, the compounds provided herein are inhibitors of activin receptor-like kinases (e.g., ALK-5). In some embodiments, the compounds provided herein are useful for treating and/or preventing a disease (e.g., a fibrotic disease (e.g., IPF or cardiac fibrosis or a cardiac disease associated with tgfp signaling), and a proliferative disease (e.g., cancer)) in a subject, such as inhibiting tumor growth in a subject, or inhibiting the activity of an activin receptor-like kinase (e.g., ALK-5) in vitro or in vivo. In some embodiments, the compounds provided herein may be used to modulate, prevent, or provide treatment for conditions and/or diseases whose progression is driven by or utilizes a tgfβ -signaling pathway for disease progression, as described herein.

As also shown herein, the compounds provided herein can inhibit epithelial to mesenchymal transition (EMT). In some embodiments, the compounds provided herein may be used to inhibit EMT in vitro or in vivo. In some embodiments, the compounds provided herein may be used to modulate, prevent, or provide treatment for conditions and/or diseases whose progression is driven by EMT, as described herein. Examples of such conditions and/or diseases include cancer (e.g., metastatic cancer, chemotherapy-resistant cancer) and fibrosis (e.g., cancer-associated fibrosis, idiopathic pulmonary fibrosis).

Provided herein are methods of treating and/or preventing (e.g., treating) a disease or disorder (e.g., a fibrotic disease or disorder, alone or in combination with an infectious, inflammatory, or proliferative disease or disorder (benign or malignant), an inflammatory disease or disorder, or a proliferative disease or disorder, such as cancer) in a subject (e.g., a subject in need thereof), comprising administering to the subject a therapeutically and/or prophylactically effective amount of a compound having formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. Also provided herein are compounds having formula (I) and pharmaceutically acceptable salts thereof, as well as pharmaceutical compositions of the foregoing, for use in the treatment and/or prevention of the diseases or disorders described herein. Also provided herein is the use of a compound having formula (I) and pharmaceutically acceptable salts thereof, as well as pharmaceutical compositions of the foregoing, for the manufacture of a medicament for the treatment and/or prevention of a disease or condition described herein. In certain embodiments, the disease is a disease associated with the activity of an activin receptor-like kinase (e.g., ALK-5) in a subject or cell. In certain embodiments, the activity is aberrant (e.g., increased) activity.

In certain embodiments, the disease or disorder is a proliferative disease. Provided herein are methods for treating a proliferative disease (e.g., cancer) in a subject (e.g., a subject in need thereof) comprising administering to the subject a therapeutically effective amount of a compound having formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. Also provided herein are compounds having formula (I) and pharmaceutically acceptable salts thereof, as well as pharmaceutical compositions of the foregoing, for use in the treatment of proliferative diseases (e.g., cancer). Also provided herein is the use of a compound having formula (I) and pharmaceutically acceptable salts thereof, as well as pharmaceutical compositions of the foregoing, for the manufacture of a medicament for the treatment of a proliferative disease (e.g., cancer). In certain embodiments, the proliferative disease is associated with the activity of an activin receptor-like kinase (e.g., ALK-5) in a subject or cell. In certain embodiments, the activity is an aberrant or increased activity.

"proliferative disease" refers to a disease that occurs due to abnormal growth or elongation caused by cell proliferation (Walker, cambridge Dictionary of Biology [ Cambridge Biodictionary ]; cambridge University Press [ Cambridge university Press ]: cambridge, UK, 1990). Proliferative diseases may be associated with the following: 1) Pathological proliferation of normal resting cells; 2) Pathologic migration of cells from their normal location (e.g., metastasis of tumor cells); 3) Pathological expression of proteolytic enzymes such as matrix metalloproteinases (e.g., collagenases, gelatinases, and elastases); and/or 4) pathologic angiogenesis, as in proliferative retinopathies and tumor metastasis. Exemplary proliferative diseases include cancer (i.e., "malignant neoplasms"), benign neoplasms, angiogenesis, inflammatory diseases, and autoimmune diseases.

The terms "neoplasm" and "tumor" are used interchangeably herein and refer to an abnormal mass of tissue, wherein the mass of tissue grows beyond and out of coordination with the growth of normal tissue. A neoplasm may be "benign" or "malignant" depending on the following characteristics: the extent of cell differentiation (including morphology and functionality), growth rate, local invasion and metastasis.

"benign neoplasms" are generally well differentiated, have characteristically slower growth than malignant neoplasms, and remain localized to the site of origin. In addition, benign neoplasms do not have the ability to infiltrate, invade or metastasize to distant sites. Exemplary benign neoplasms include, but are not limited to, lipomas, chondriomas, adenomas, acrochordons, senile hemangiomas, seborrheic keratosis, nevus sparks, and sebaceous hyperplasia. In some cases, certain "benign" tumors may later produce malignant neoplasms, which may be caused by additional genetic changes in the tumor cell subpopulation of the tumor, and these tumors are referred to as "pre-malignant neoplasms. An exemplary premalignant neoplasm is teratoma.

In contrast, "malignant neoplasms" are often poorly differentiated (anaplastic) and have characteristic rapid growth with progressive infiltration, invasion, and destruction of surrounding tissues. In addition, malignant neoplasms often have the ability to metastasize to distant sites. The term "metastasis" refers to the spread or migration of cancerous cells from a primary or original tumor to another organ or tissue, and is typically identified by the presence of a "secondary tumor" or "secondary cell mass" of the tissue type of the primary or original tumor (but not of the organ or tissue in which the secondary (metastatic) tumor is located).

In certain embodiments, the disease or disorder to be treated is cancer. Provided herein are methods for treating cancer in a subject (e.g., a subject in need thereof) comprising administering to the subject a therapeutically effective amount of one or more of the example compounds, or one or more of the example compounds in pharmaceutically acceptable salt form, or a pharmaceutical composition of the foregoing. In some embodiments, example compounds are those having formula (I), (II), (III), (IV) or compounds of table 1. Also provided herein are compounds having formula (I), (II), (III), (IV) or table 1, and pharmaceutically acceptable salts thereof, as well as pharmaceutical compositions of the foregoing, for use in the treatment of cancer. Also provided herein is the use of a compound having formula (I), (II), (III), (IV) or table 1, and pharmaceutically acceptable salts thereof, and pharmaceutical compositions of the foregoing, for the manufacture of a medicament for the treatment of cancer. In certain embodiments, the cancer is associated with the activity of an activin receptor-like kinase (e.g., ALK-5) in a subject or cell. In certain embodiments, the cancer is associated with ALK-5 activity in the subject or cell. In certain embodiments, the activity is aberrant (e.g., increased) activity.

The term "cancer" refers to a class of diseases characterized by the production of abnormal cells that proliferate uncontrolled and have the ability to infiltrate and destroy normal body tissues. In certain embodiments, the cancer is a solid tumor. In certain embodiments, the cancer is a hematologic cancer.

In certain embodiments, the cancer is associated with the activity of an activin receptor-like kinase (e.g., ALK-5) in a subject or cell. In certain embodiments, the cancer is associated with ALK-5 activity in the subject or cell. In certain embodiments, the activity is increased (e.g., aberrant) activin receptor-like kinase (e.g., ALK-5) activity.

In certain embodiments, the cancer expresses or has a mutant fork cassette L2 (FOXL 2) and/or FOXL2 (exampleSuch as FOXL2 ^C134W )。FOXL2 ^C134W Is about 97% AGCT (rare ovarian cancer subtype [ ]>5%) of the characteristic. An example of a cancer that expresses or has mutant FOXL2 is ovarian cancer (e.g., AGCT). Other sex cord stromal tumors (such as JGCT, follicular cytoma, SLCT, male AGCT, and gonadoblastoma) are other examples of cancers that express or have mutant FOXL2 and/or FOXL 2.

In some embodiments, provided herein is a method for treating cancer (e.g., ovarian cancer, such as adult granuloma), comprising determining whether a subject carries a FOXL2 mutation (e.g., FOXL2 ^C134W ) The method comprises the steps of carrying out a first treatment on the surface of the And if the subject is identified as having a FOXL2 mutation, treating the subject with a therapeutically effective amount of a compound of the present disclosure, e.g., a compound having formula (I), (II), (III) or (IV) or table 1 or table 4, such as one or more of Ex-10, ex-11, ex-12, ex-13, ex-33, ex-34, ex-57, or Ex-58, or a pharmaceutically acceptable salt of the foregoing, or a composition thereof.

In some embodiments, the cancer has FOXL 2-driven tumor growth.

In some embodiments, the cancer is associated with an elevated level of pSmad2 and/or αvβ6 and/or αsmooth muscle actin (α -SMA). In some embodiments, the cancer is associated with elevated levels of phosphorylated SMAD2 (pSMAD 2) or alpha smooth muscle actin (alpha-SMA).

Except for FOXL2 mutants (e.g. FOXL2 ^C134W ) In addition to pSMAD2, αvβ6, and α -SMA, other biomarkers that can predict (e.g., and serve as patient selection criteria) and/or indicate (e.g., and during and/or after treatment for assessing a certain aspect of treatment) the efficacy of the treatment disclosed herein also include CD31 (e.g., elevated levels of CD 31), CD45 (e.g., elevated levels of CD 45), and/or HLA (e.g., low levels of HLA).

In some embodiments, the cancer (e.g., solid tumor cancer) exhibits an exclusive phenotype or a desert type phenotype. In some embodiments, the cancer (e.g., solid tumor cancer) exhibits an exclusive phenotype. In some embodiments, the cancer (e.g., solid tumor cancer) exhibits a desert-type phenotype.

A variety of cancers (including solid tumors, leukemias, lymphomas, and myelomas) are suitable for use in the methods disclosed herein. In some embodiments, the cancer is a solid tumor cancer. In some embodiments, the cancer comprises a solid tumor (e.g., colorectal tumor, breast tumor, prostate tumor, lung tumor, pancreatic tumor, kidney tumor, or ovarian tumor). Accordingly, in some embodiments, the cancer is a solid tumor cancer. In some embodiments, the cancer is selected from one or more of lung system cancer, brain cancer, gastrointestinal cancer, skin cancer, genitourinary system cancer, head and neck cancer, sarcoma, carcinoma, and neuroendocrine cancer. In various embodiments, the solid tumor cancer is breast cancer, bladder cancer, endometrial cancer, esophageal cancer, liver cancer, pancreatic cancer, lung cancer, cervical cancer, colon cancer, colorectal cancer, gastric cancer, kidney cancer, ovarian cancer, prostate cancer, testicular cancer, uterine cancer, virus-induced cancer, melanoma, or sarcoma. In some embodiments, the cancer is bladder cancer. In some embodiments, the cancer is lung cancer (e.g., non-small cell lung cancer). In other embodiments, the cancer is liver cancer. In some embodiments, the cancer is a sarcoma, bladder cancer, or renal cancer. In some embodiments, the cancer is a prostate cancer (e.g., castration-resistant prostate cancer, castration-sensitive prostate cancer). In other embodiments, the cancer is bladder cancer, pancreatic cancer, colorectal cancer, glioblastoma, renal cancer, non-small cell lung cancer, prostate cancer, sarcoma, skin cancer, thyroid cancer, testicular cancer, or vulvar cancer. In some embodiments, the cancer is endometrial, pancreatic, testicular, renal, melanoma, colorectal, thyroid, bladder, pancreatic, vulvar, sarcoma, prostate, lung, or anal cancer. In some embodiments, the cancer is a sarcoma. In some embodiments, the cancer is renal cell carcinoma.

In some embodiments, the cancer is a non-solid tumor cancer. In some embodiments, the cancer is a hematologic cancer. Hematological cancers that can be treated according to the methods described herein include leukemia (e.g., acute leukemia, chronic leukemia), lymphoma (e.g., B-cell lymphoma, T-cell lymphoma), and multiple myeloma. In some embodiments, the cancer is leukemia. In some embodiments, the cancer is acute leukemia. In some embodiments, the cancer is acute myeloid leukemia or acute lymphoblastic leukemia. In some embodiments, the cancer is chronic leukemia. In some embodiments, the cancer is chronic myelogenous leukemia or chronic lymphocytic leukemia. In some embodiments, the cancer is lymphoma. In some embodiments, the cancer is multiple myeloma. In some embodiments, the hematological cancer is selected from multiple myeloma, myelodysplastic syndrome (MDS), acute Myeloid Leukemia (AML), acute Lymphoblastic Leukemia (ALL), acute lymphoblastic leukemia, lymphocytic lymphoma, mycosis fungoides, chronic Lymphocytic Leukemia (CLL), mantle cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hodgkin's lymphoma, non-hodgkin's lymphoma, or myelofibrosis.

In some embodiments, the cancer is a pre-metastatic cancer. In some embodiments, the cancer is a metastatic cancer.

Examples of cancers treatable according to the methods described herein include, but are not limited to, breast cancer, prostate cancer, and colon cancer; all forms of bronchogenic lung cancer; bone marrow cancer; melanoma; hepatoma; neuroblastoma; papillomas; APUD tumor; a vaginosis tumor; gill tumor; malignant carcinoid syndrome; carcinoid heart disease; and cancers (e.g., wok's cancer, basal cell carcinoma, basal squamous carcinoma, bron-pierce cancer, ductal carcinoma, ehrlichia tumor, krebs 2 cancer, merkel cell carcinoma, mucinous cancer, lung cancer (e.g., large cell lung cancer such as squamous cell carcinoma, non-small cell lung cancer), oat cell carcinoma, papillary cell carcinoma, hard cell carcinoma, bronchiolar cell carcinoma, bronchogenic carcinoma, squamous cell carcinoma, and transitional cell carcinoma). Additional examples of cancers that may be treated according to the methods described herein include, but are not limited to, tissue cell disorders; leukemia; malignant histiocytosis; hodgkin's disease; hypereosinophilia (small immunoproliferation); non-hodgkin's lymphoma; plasmacytoma; reticuloendotheliosis; melanoma; chondroblastoma; cartilage tumor; chondrosarcoma; fibrotic sarcoma of the skin, fibrotic cancer (myelofibrosis, pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), renal cancer, liver cancer, lung cancer (e.g., large cell lung cancer, such as squamous cell carcinoma), breast cancer (e.g., inflammatory breast cancer), ovarian cancer (e.g., highly severe ovarian cancer), endometrial cancer, uterine sarcoma (e.g., uterine leiomyosarcoma), renal cell carcinoma, sarcoma (e.g., soft tissue sarcoma), malignant fibrohistiocytoma, fibrosarcoma (e.g., fibrosarcoma of the skin of the carina), and hepatocellular carcinoma); fibroids; fibrosarcoma; giant cell tumor; histiocytoma; a fatty tumor; liposarcoma; mesothelioma; a myxoma; myxosarcoma; osteoma; osteosarcoma; pediatric malignancy, chordoma; craniopharyngeal pipe tumor; a vegetative cell tumor; hamartoma; a mesenchymal neoplasm; mesonephroma; myosarcoma; enameloblastoma; cementoma; dental tumor; teratoma; thymoma; nourishing cell tumor. In addition, the following types of cancers suitable for treatment are also contemplated: adenoma; gall bladder tumor; cholesteatoma; cylindrical tumors; cystic adenocarcinoma; cystic adenoma; granulocytoma; ampholytic blastoma; hepatocellular carcinoma, hepatoma; sweat gland tumor; islet cell tumor; testicular stromal cell tumor; papillomas; testicular support cell neoplasm; membranous cell neoplasms; smooth myoma; leiomyosarcoma; myoblasts; myomas; myosarcoma; rhabdomyomas; rhabdomyosarcoma; ventricular tube membranoma; ganglioma; glioma; medulloblastoma; meningioma; a schwannoma; neuroblastoma; neuroepithelial tumors; neurofibromatosis; neuroma; paraganglioma; non-chromaphilic paragangliomas. Further examples of cancers treatable according to the methods described herein include, but are not limited to, angiokeratomas; vascular lymphoid hyperplasia is accompanied by eosinophilia; sclerosing hemangioma; hemangiomatosis; glomeroclavicular tumor; vascular endothelial tumors; hemangioma; vascular endothelial cell tumor; hemangiosarcoma; lymphangioma; lymphangiomyomas; lymphangiosarcoma; pineal tumor; carcinoma sarcoma; chondrosarcoma; she Zhuangnang sarcoma; fibrosarcoma; hemangiosarcoma; leiomyosarcoma; leukemia sarcoma; liposarcoma; lymphangiosarcoma; myosarcoma; myxosarcoma; ovarian cancer; rhabdomyosarcoma; sarcoma; neoplasms; neurofibromatosis; atypical hyperplasia of cervix.

Additional examples of cancers that may be treated according to the methods described herein include, but are not limited to, acute Lymphoblastic Leukemia (ALL); acute Myeloid Leukemia (AML); adrenal cortex cancer; adrenal cortex cancer, childhood; AIDS-related cancers (e.g., kaposi's sarcoma, AIDS-related lymphoma, primary CNS lymphoma); anal region cancer; anal cancer; appendiceal cancer; astrocytoma, childhood; atypical teratoid/rhabdoid tumors, childhood, central Nervous System (CNS); CNS neoplasms (e.g., primary CNS lymphoma, spinal axis tumor, medulloblastoma, brain stem glioma or pituitary adenoma), barrett's esophagus (e.g., pre-malignant syndrome) and mycosis fungoides, basal cell carcinoma of the skin; bile duct cancer; bladder cancer; bladder cancer, childhood; bone cancer (including ewing's sarcoma, osteosarcoma, and malignant fibrous histiocytoma); brain tumor/cancer; breast cancer; burkitt's lymphoma; carcinoid tumor (gastrointestinal); carcinoid tumor, children; heart Tumors (cardioc or Heart tunes), children; embryo tumor, childhood; germ cell tumor, childhood; primary CNS lymphoma; cervical cancer; cervical cancer in children; cholangiocellular carcinoma; chordoma, childhood; chronic Lymphocytic Leukemia (CLL); chronic Myelogenous Leukemia (CML); chronic myeloproliferative neoplasms; colorectal cancer; colorectal cancer in children; craniopharyngeal pipe tumor, children; cutaneous T cell lymphomas (e.g., mycosis fungoides and sezary syndrome); in situ catheter carcinoma (DCIS); embryonic tumors, central nervous system, childhood; endocrine system cancer (e.g., thyroid cancer, pancreatic cancer, parathyroid cancer, or adrenal cancer), endometrial cancer (uterine cancer); ventricular tube tumor, children; esophageal cancer; esophageal cancer in children; glioma of nasal cavity; ewing's sarcoma; extracranial germ cell tumor, childhood; extragonadal germ cell tumors; eye cancer; intraocular melanoma in children; intraocular melanoma; retinoblastoma; fallopian tube cancer; malignant osteofibrohistiocytoma and osteosarcoma; gallbladder cancer; gastric Cancer (Gastric or stomachcancer); gastric Cancer (Gastric or stomachcancer) in children; gastrointestinal cancer tumor; gastrointestinal stromal tumor (GIST); gastrointestinal stromal tumor in children; germ cell tumor; childhood central nervous system germ cell tumors (e.g., childhood extracranial germ cell tumors, extragonadal germ cell tumors, ovarian germ cell tumors, testicular cancer); gestational trophoblastic disease; gynecological tumors (e.g., uterine sarcoma, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, or vulvar cancer), hairy cell leukemia; cancer of the head and neck; heart tumor, childhood; hepatocellular (liver) carcinoma; histiocytosis, langerhans cells; hodgkin lymphoma; a cancer of the pharynx; cutaneous or intraocular melanoma; intraocular melanoma in children; islet cell tumors, pancreatic neuroendocrine tumors; kaposi's sarcoma; kidney (renal cell) carcinoma; langerhans cell histiocytosis; laryngeal carcinoma; leukemia; lip and oral cavity cancer; liver cancer; lung cancer (non-small cells and small cells); lung cancer in children; lymphomas; male breast cancer; osteomalignant fibrous histiocytoma and osteosarcoma; melanoma; melanoma in children; melanoma, intraocular (eye); intraocular melanoma in children; merkel cell carcinoma; mesothelioma, malignant; mesothelioma in children; metastatic cancer; metastatic squamous neck cancer with latent primary; central tract cancer with NUT gene alterations; oral cancer; multiple endocrine tumor syndrome; multiple myeloma/plasma cell tumor; mycosis fungoides; myelodysplastic syndrome, myelodysplastic/myeloproliferative neoplasm; myeloid leukemia, chronic (CML); myeloid leukemia, acute (AML); myeloproliferative neoplasms, chronic; nasal and sinus cancer; nasopharyngeal carcinoma; neuroblastoma; non-hodgkin's lymphoma; non-small cell lung cancer; oral, lip and mouth cancers and oropharyngeal cancers; osteosarcoma and osteomalignant fibrous histiocytoma; ovarian cancer; ovarian cancer in children; pancreatic cancer; pancreatic cancer in children; pancreatic neuroendocrine tumors; papillomatosis (childhood larynx); paraganglioma; children paraganglioma; paranasal sinus and nasal cavity cancer; parathyroid cancer; penile cancer; pharyngeal cancer; pheochromocytoma; pheochromocytoma in children; pituitary tumor; plasma cell tumor/multiple myeloma; pleural pneumoblastoma; gestational breast cancer; primary Central Nervous System (CNS) lymphomas; primary peritoneal cancer; prostate cancer; rectal cancer; recurrent cancer; renal cell (kidney) carcinoma; retinoblastoma; rhabdomyosarcoma, childhood; salivary gland cancer; sarcomas (e.g., pediatric rhabdomyosarcoma, pediatric hemangioma, ewing's sarcoma, kaposi's sarcoma, osteosarcoma (bone carcinoma), soft tissue sarcoma, uterine sarcoma); cerclari syndrome; skin cancer; skin cancer in children; small cell lung cancer; small intestine cancer; soft tissue sarcoma; squamous cell carcinoma of the skin; squamous neck cancer with latent primary, metastatic; gastric Cancer (stomachs or Gastric Cancer); gastric Cancer (stomachs or Gastric Cancer) in children; t cell lymphoma, skin (e.g., granulomatosis mycosis fungoides and sezary syndrome); testicular cancer; testicular cancer in children; laryngeal cancer (e.g., nasopharyngeal cancer, oropharyngeal cancer, and hypopharyngeal cancer); thymoma and thymus cancer; thyroid cancer; transitional cell carcinoma of the renal pelvis and ureter; ureters and renal pelvis (e.g., renal cell carcinoma, renal pelvis carcinoma), benign prostatic hypertrophy, parathyroid carcinoma, transitional cell carcinoma; urethral cancer; uterine cancer, endometrial cancer; uterine sarcoma; vaginal cancer; vaginal cancer in children; vascular tumors; vulvar cancer; and wilms' tumor and other childhood kidney tumors.

Metastasis of the above cancers can also be treated according to the methods described herein.

In certain embodiments, the cancer is a hematological cancer (e.g., leukemia (e.g., acute Lymphoblastic Leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute Myelogenous Leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic Myelogenous Leukemia (CML) (e.g., B-cell CML, T-cell CML), chronic Lymphoblastic Leukemia (CLL) (e.g., B-cell CLL, T-lymphocyte CLL)); lymphomas (e.g., hodgkin's Lymphoma (HL) (e.g., B-cell HL, T-cell HL)), non-hodgkin's lymphomas (NHL) (e.g., B-cell NHL, such as Diffuse Large Cell Lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma)), follicular lymphomas, chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), mantle Cell Lymphoma (MCL), marginal zone B-cell lymphomas (e.g., mucosa-associated lymphoid tissue (MALT) lymphomas, lymph node marginal zone B-cell lymphomas, spleen marginal zone B-cell lymphomas), primary mediastinal B-cell lymphomas, burkitt's lymphomas, lymphoplasmacytic lymphomas (i.e., waldenstrom's macroglobulinemia), hairy Cell Leukemia (HCL), immunocytoblast lymphomas, precursor B lymphoblastoid lymphomas and primary Central Nervous System (CNS) lymphomas, T-cell lymphomas such as precursor T-lymphoblastomas/leukemias, peripheral T Cell Lymphoma (PTCL) (e.g., cutaneous T Cell Lymphoma (CTCL) (e.g., granulomatosis mycotica, sezary syndrome)), angioimmunoblastic T cell lymphoma, extranodal natural killer T cell lymphoma, enteropathic T cell lymphoma, subcutaneous panniculitis-like T cell lymphoma, anaplastic large cell lymphoma); heavy chain diseases (e.g., alpha chain diseases, gamma chain diseases, mu chain diseases); myeloproliferative disorders (MPD) (e.g., polycythemia Vera (PV), essential Thrombocythemia (ET), agnostic myeloplasia (AMM) (also known as Myelofibrosis (MF)), chronic idiopathic myelofibrosis, chronic Myelogenous Leukemia (CML), chronic Neutrophilic Leukemia (CNL), hypereosinophilic syndrome (HES)); multiple Myeloma (MM); plasmacytoma formation; familial hypereosinophilia; inflammatory myofibroblastic tumor; immune cell amyloidosis). In certain embodiments, the cancer is leukemia. In certain embodiments, the cancer is Acute Lymphoblastic Leukemia (ALL). In certain embodiments, the cancer is early T cell precursor (ETP) -Acute Lymphoblastic Leukemia (ALL).

In certain embodiments, the cancer is anaplastic astrocytoma, pancreatic cancer, skin cancer, melanoma, metastatic melanoma, colorectal cancer, breast cancer, prostate cancer, renal cancer, hepatocellular cancer, ovarian cancer, HPV-related cancer (e.g., cervical cancer, oropharyngeal cancer, anal cancer, vulval/vaginal cancer, and penile cancer), multiple myeloma, myelodysplastic syndrome, myelofibrosis.

In certain embodiments, the cancer is a liver cancer (e.g., hepatocellular carcinoma (hepatocellular cancer, HCC) (e.g., hepatocellular carcinoma (hepatocellular carcinoma), hepatoblastoma, hepatocellular adenoma), malignant liver cancer, hemangioma, bile cancer (e.g., cholangiocarcinoma)). In some embodiments, the cancer is liver cancer, the cancer is hepatocellular carcinoma (HCC). In some embodiments, the cancer is lung cancer (e.g., non-small cell lung cancer (NSCLC)). In some embodiments, the cancer is brain cancer (e.g., neuroblastoma, glioblastoma). In some embodiments, the cancer is brain cancer, and the cancer is anaplastic astrocytoma. In some embodiments, the cancer is thyroid cancer (e.g., thyroid undifferentiated carcinoma (ATC)). In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is renal cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is an HPV-associated cancer, e.g., HPV-associated cervical cancer, HPV-associated oropharyngeal cancer, HPV-associated anal cancer, HPV-associated vulva/vaginal cancer, and HPV-associated penile cancer. In some embodiments, the cancer is colorectal cancer (e.g., colon cancer). In some embodiments, the cancer is pancreatic cancer (pancreatic cancer) (e.g., pancreatic cancer (pancreatic carcinoma)). In some embodiments where the cancer is pancreatic cancer, the cancer is pancreatic ductal adenocarcinoma and associated fibrotic CAF. In some embodiments, the cancer is skin cancer. In some embodiments, the cancer is a skin cancer, and the cancer is metastatic melanoma. In some embodiments, the cancer is prostate cancer.

In some embodiments, the proliferative disease is a hematological cancer (e.g., anaplastic Large Cell Lymphoma (ALCL), myelodysplastic syndrome, multiple myeloma, and myelofibrosis).

In certain embodiments, the cancer is a musculoskeletal cancer (e.g., bone cancer (e.g., osteosarcoma, osteoid tumor, malignant fibrous histiocytoma, ewing's sarcoma, chordoma, malignant giant cell tumor chordoma, chondrosarcoma, osteochondral tumor, benign chondrioma, chondroblastoma, chondromucoid fibroma, myelodysplastic syndrome (MDS)), muscle cancer (e.g., rhabdomyosarcoma, rhabdomyoma), connective tissue cancer, synovial tumor).

In certain embodiments, the cancer is a nervous system cancer (e.g., brain cancer (e.g., astrocytoma, medulloblastoma, glioma (e.g., astrocytoma, oligodendroglioma), glioblastoma multiforme, medulloblastoma, ependymoma, germ cell tumor (i.e., pineal tumor), oligodendroglioma, schwannoma, retinoblastoma, congenital tumor, craniopharyngeal tube tumor), spinal cord cancer, neurofibromatosis (e.g., neurofibromatosis (NF) type 1 or type 2, schwannoma), neuroblastoma, primary extraneuroblastoma (PNT), meningioma (e.g., meningioma, glioma), craniocerebral cancer, auditory neuroma, ependymoma, angioblastoma, and ocular cancer (e.g., intraocular melanoma, retinoblastoma)), in certain embodiments, the disease to be treated is brain tumor.

In certain embodiments, the cancer is selected from endocrine/exocrine cancers (e.g., thyroid cancer (e.g., papillary thyroid cancer, follicular thyroid cancer; medullary thyroid cancer, multiple endocrine tumor type 2A, multiple endocrine tumor type 2B, familial medullary thyroid cancer, pheochromocytoma, paraganglioma), pancreatic cancers (e.g., pancreatic adenocarcinoma, intraductal papillary mucinous tumor (IPMN), islet cell tumor, ductal adenocarcinoma, insulinoma, glucagon tumor, schwann intestinal peptide tumor), adrenal gland cancers, neuroendocrine cancers (e.g., gastrointestinal pancreatic neuroendocrine tumor (GEP-NET), carcinoid tumor), sebaceous gland cancers, sweat gland cancers). In certain embodiments, the cancer is sweat gland cancer (sweat gland cancer) (e.g., sweat gland cancer (sweat gland carcinoma)).

In certain embodiments, the cancer is a head and neck cancer (e.g., head and neck Squamous Cell Carcinoma (SCCHN), adenoid cystic carcinoma).

In certain embodiments, the cancer is an oral cancer (e.g., oral cancer (buccal cavity cancer), lip cancer, tongue cancer, oral cancer (mouth cancer), pharynx cancer, hypopharynx cancer (e.g., hypopharynx cancer), larynx cancer (e.g., larynx cancer (laryngeal cancer), pharynx cancer, nasopharynx cancer, oropharynx cancer), salivary gland cancer).

In certain embodiments, the cancer is esophageal cancer (e.g., esophageal squamous cell carcinoma, esophageal adenocarcinoma, barrett's adenocarcinoma, esophageal leiomyosarcoma).

In certain embodiments, the cancer is gastrointestinal cancer (e.g., anal cancer, colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma), gallbladder cancer, gastric cancer (gastric cancer) (e.g., gastric adenocarcinoma)), gastrointestinal stromal tumor (GIST), small intestine cancer (small bowel cancer) (e.g., appendiceal cancer, small intestine cancer (small bowel carcinoma), e.g., small intestine adenocarcinoma), small intestine cancer (small intestine cancer), large intestine cancer (large bowel cancer), large intestine cancer (large intestine cancer)).

In certain embodiments, the cancer is a cardiovascular cancer (e.g., primary heart tumor, vascular sarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma), endothelial sarcoma (e.g., kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma), cardiac myxoma, cardiac rhabdomyoma).

In certain embodiments, the cancer is lung cancer (e.g., bronchogenic cancer, bronchogenic carcinoma), alveolar cancer, mesothelioma, small Cell Lung Cancer (SCLC), non-small cell lung cancer (NSCLC), lung adenocarcinoma, chondromatous hamartoma, papillary adenocarcinoma).

In certain embodiments, the cancer is a genitourinary system cancer (e.g., bladder cancer (e.g., urothelial cancer), urinary tract cancer, kidney cancer (e.g., nephroblastoma (also known as wilms tumor), renal cell carcinoma), testicular cancer (e.g., seminoma, testicular embryo cancer), germ cell cancer, prostate cancer (e.g., prostate adenocarcinoma), penile cancer (e.g., paget's disease of the penis and scrotum).

In certain embodiments, the cancer is a gynaecological cancer (e.g., breast adenocarcinoma, breast papillary carcinoma, breast cancer (mammary cancer), breast medullary carcinoma, triple negative breast cancer, HER-2 positive breast cancer, HER2 negative breast cancer), endometrial cancer (endometrial cancer) (e.g., uterine cancer (e.g., uterine sarcoma, choriocarcinoma), endometrial cancer (endometrial carcinoma)), cervical cancer (e.g., cervical adenocarcinoma), ovarian cancer (e.g., cystic adenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma), germ cell carcinoma, vulvar cancer (e.g., paget's disease of the vulva), vaginal cancer, fallopian tube cancer).

In certain embodiments, the cancer is a skin cancer (e.g., squamous Cell Carcinoma (SCC), keratoacanthoma (KA), melanoma, basal Cell Carcinoma (BCC), skin fibroma).

In certain embodiments, the cancer is a soft tissue cancer (e.g., intraepithelial tumor, epithelial cancer, epithelial sarcoma, adenocarcinoma, adenoma, fibrosarcoma, liposarcoma, lipoma, mucinous tumor, teratoma).

Myeloproliferative neoplasms are also treatable according to the methods described herein. Non-limiting examples of myeloproliferative neoplasms include myelofibrosis, polycythemia vera, and essential thrombocythemia.

In certain embodiments, the cancer is a rare cancer. The term "rare cancer" refers to cancers that occur in a relatively small number of patients.

In certain embodiments, the cancer is lung cancer (e.g., non-small cell lung cancer (NSCLC)), brain cancer (e.g., neuroblastoma, glioblastoma), thyroid cancer (e.g., thyroid undifferentiated carcinoma (ATC)), breast cancer, colorectal cancer (e.g., colon cancer), liver cancer (e.g., hepatocellular carcinoma (HCC)), pancreatic cancer (pancreatic cancer) (e.g., pancreatic cancer (pancreatic carcinoma)), skin cancer (e.g., melanoma), prostate cancer, or hematological cancer (e.g., anaplastic Large Cell Lymphoma (ALCL), myelodysplastic syndrome).

In some embodiments, it is preferred to treat cancers driven by TGF-b signaling with one or more ALK-5 inhibitors described herein, e.g., compounds having formula (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt thereof.

In some embodiments, a proliferative disease (e.g., cancer) is treated by: targeting tumor stromal cells (e.g., in a tumor microenvironment) (e.g., cancer-associated fibroblasts (CAF), astrocytes, or myofibroblasts), and/or immune cells (e.g., tumor-associated immune cells) (e.g., in a tumor-immune microenvironment), e.g., to modulate the tumor-stromal microenvironment and/or tumor-immune microenvironment.

Cachexia is associated with chronic disease and manifests as involuntary weight loss (e.g., greater than 5% of the pre-illness weight) caused by skeletal muscle and adipose tissue atrophy. This condition is different from other conditions (like anorexia) in that the fat reserves are depleted, but the muscle mass remains substantially unchanged. Cachexia affects more than half of cancer patients, resulting in poor quality of life (fatigue and weakness), and sometimes may even affect the therapeutic strategy of some individuals. Myostatin is a superfamily member of transforming growth factor-beta (TGF-beta) that is widely characterized as a negative regulator of muscle growth and development. Without wishing to be bound by any particular theory, it is believed that blocking this pathway would be potentially beneficial to cancer patients, particularly advanced disease and metastatic patients with prominent cachexia. Thus, in some embodiments, the disease or disorder is cachexia (e.g., cancer cachexia).

In some embodiments, the disease or disorder is a fibrotic disease or disorder (e.g., a fibrotic disorder). In some embodiments, the fibrotic disorder is associated with a proliferative disease. In some embodiments, the fibrotic disorder is present without co-morbidity. In some embodiments, the fibrotic condition is idiopathic pulmonary fibrosis, cardiac fibrosis, a condition associated with cardiac fibrosis (e.g., valvular disease, cardiac arrhythmia (e.g., atrial fibrillation), myocardial remodeling (e.g., post-infarction), cardiomyopathy (e.g., dilated, ischemic, or hypertrophic cardiomyopathy), restenosis (e.g., in-stent restenosis, restenosis after angioplasty), liver fibrosis, cirrhosis, nonalcoholic steatohepatitis, peclet's disease, dipteret's contracture, cystic fibrosis, beta thalassemia, light keratosis, hypertension, systemic inflammatory disorders, dry eye, ulcers, corneal fibrosis, wet age-related macular degeneration, psoriasis, wound closure, chronic kidney disease, kidney fibrosis, systemic sclerosis, or chronic Chagas's heart disease, the fibrotic condition is idiopathic pulmonary fibrosis, liver cirrhosis, nonalcoholic steatohepatitis, pecimen's disease, cystic fibrosis, beta thalassemia, light keratosis, hypertension, systemic inflammatory disorders, dry eye, ulcers, corneal fibrosis, wet age-related macular degeneration, psoriasis, wound closure, chronic kidney disease, kidney fibrosis, systemic sclerosis, or chronic Chagas heart disease in some embodiments, the condition is idiopathic pulmonary fibrosis in some embodiments, the fibrotic condition is cardiac fibrosis or a condition associated with cardiac fibrosis (e.g., valvular disease, cardiac arrhythmia (e.g., atrial fibrillation), myocardial remodeling (e.g., post-infarction), cardiomyopathy (e.g., dilated, ischemic, or hypertrophic cardiomyopathy), restenosis (e.g., restenosis within a stent, restenosis after angioplasty)). In some embodiments, the fibrotic disorder is diprote contracture. In some embodiments, the fibrotic disorder is a desmoid tumor (fibromatosis).

As used herein, the terms "fibrosis", "fibrotic disease", "fibrotic disorder", "fibrotic lesion" and "fibrotic disease and/or disorder" (collectively referred to herein as fibrosis) refer to a disease or disorder in a subject that involves the formation of excess fibrous connective tissue in an organ or tissue. Fibrosis may occur with another disease state or condition, such as inflammation, cancer, viral or bacterial infection, and the like.

Fibrosis may be associated with another disease, disorder or condition (e.g., inflammation, inflammatory disease, disorder or condition (e.g., psoriasis), proliferative disease (e.g., cancer), viral or bacterial infection, etc.), or may occur independently. For example, fibrosis may be before (e.g., is the cause of) or after (e.g., is caused by) another disease, disorder, or condition. Fibrosis is also or alternatively present with another disease, disorder or condition (e.g., inflammation, inflammatory disease, disorder or condition (e.g., psoriasis), proliferative disease (e.g., cancer), viral or bacterial infection, etc.), whether or not associated; or may be present without an accompanying disease, disorder or condition (e.g., a related disease, disorder or condition). In some embodiments, fibrosis is present in the absence of a related disease, disorder, or condition. In some embodiments, fibrosis is present with an associated disease, disorder, or condition.

Although the occurrence of fibrosis associated with another disease, disorder or condition is not uncommon (e.g., the presence of fibrosis associated with cancer), the etiology of fibrosis is not well understood and fibrosis also occurs independently and/or in the absence of other diseases, disorders or conditions. However, it is believed that similar mechanisms and signaling pathways are present in many related diseases, disorders or conditions that affect organs or tissues in which fibrosis is also present (e.g., IPF is present with lung cancer). For example, it is believed that fibrosis, along with many diseases that often exist with it, progresses via the signaling cascade involved in the overexpression of TGF-beta proteins, see, e.g., ballester, B et al, idiopathic Pulmonary Fibrosis and lung Cancer: mechanisms and Molecular targets [ idiopathic pulmonary fibrosis with lung cancer: mechanism and molecular targets ], int.j.mol.sci. [ journal of international molecular science ]2019,20,593; doi 10.3390/ijms20030593.

Fibrosis may be co-morbid with (e.g., infection, as described herein, such as a viral or bacterial infection; an inflammatory disease, disorder or condition, as described herein, such as psoriasis; or a proliferative disease, as described herein, such as cancer, particularly fibrotic cancer), caused by and/or exacerbated by such. Thus, in some embodiments, the disease, disorder, or condition associated with fibrosis is a co-disease, causal, and/or exacerbated disease, disorder, or condition. In some embodiments, fibrosis is co-morbid with an associated disease, disorder, or condition. For example, fibrosis may co-morbid with: infections, such as viral or bacterial infections; an inflammatory disease, disorder or condition, such as psoriasis, as described herein; or a proliferative disease, as described herein, such as cancer, in particular fibrotic cancer. In some embodiments, fibrosis is caused by a related disease, disorder, or condition (e.g., fibrosis is caused by an infection, e.g., a viral or bacterial infection, an inflammatory disease, disorder, or condition, such as an inflammatory disease, disorder, or condition described herein, e.g., psoriasis, or a proliferative disease, such as cancer, as described herein). In some embodiments, fibrosis is co-morbid with and/or caused by a related disease, disorder or condition (e.g., an infection, such as a viral or bacterial infection, an inflammatory disease, disorder or condition, such as an inflammatory disease, disorder or condition described herein, such as psoriasis, or a proliferative disease, such as a cancer, particularly a fibrotic cancer, as described herein). In some embodiments, fibrosis is exacerbated by a related disease, disorder, or condition. For example, fibrosis may be exacerbated by: infections, such as viral or bacterial infections; an inflammatory disease, disorder or condition, such as psoriasis, as described herein; or a proliferative disease, as described herein, such as cancer, in particular fibrotic cancer.

The formation of excess fibrous connective tissue leading to fibrosis is believed to occur during repair or reaction of an organ or tissue. This may be a reactive, benign or pathological state. Physiologically, the role of fibrosis is to deposit connective tissue, which may interfere with or completely inhibit the normal structure and function of a potential organ or tissue. For example, pulmonary fibrosis is a respiratory disease in which scarring in the lung tissue occurs, leading to serious respiratory problems. Scar formation typically involves the accumulation of excess fibrous connective tissue and generally results in thickening of the wall and a decrease in the supply of oxygen in the blood. Reduced oxygen supply in the blood can in turn lead to heart failure and even death. Replacement of normal lungs with scar tissue results in an irreversible decrease in oxygen diffusion capacity. Some types of pulmonary fibrosis are thought to be caused by abnormal wound healing rather than chronic inflammation. Once scarring occurs, it is often permanent. Idiopathic Pulmonary Fibrosis (IPF) is a type of pulmonary fibrosis, which is a fatal pulmonary disease of unknown etiology but may exist with inflammation, cancer, and/or viral infection.

In general, progression of fibrosis is divided into three stages (illustrated for pulmonary fibrosis, but common in many fibrotic conditions): stage of injury ("stage 1"), stage of epithelial-fibroblast interaction ("stage 2"), and stage of abnormal repair and fibrosis ("stage 3"). In stage 1, epithelial cells are typically damaged, and one or more of the following events may occur: epithelial lesions, endothelial lesions, such as in pulmonary fibrosis, destruction of alveolar capillary basement membrane, vascular leakage, platelet activation, and fibrin clot activation. In stage 2, fibroblasts typically begin to interact with damaged epithelium, and one or more of the following events may occur: release of pro-fibrotic cytokines, recruitment, proliferation and differentiation of (myofibroblasts), formation of temporary matrix, angiogenesis and defective re-epithelialization. At stage 3, epithelial lesions are typically repaired abnormally, resulting in fibrosis, and one or more of the following events may occur: extracellular matrix (ECM) accumulates too much, lacking matrix degradation, such as progressive lung remodeling and cellular changes in pulmonary fibrosis (in which lung tissue becomes similar to honeycomb).

Although it is not uncommon for fibrosis to occur with other disease conditions, for example, the presence of cancer with fibrosis, viral infection with fibrosis, or chronic inflammation with fibrosis, the etiology of fibrotic disease is not well understood and can occur in the absence of other disease states. However, it is believed that similar mechanisms and signaling pathways are present in both fibrotic conditions and in a variety of concomitant diseases (including cancer, infection and systemic inflammation) that act on organs or tissues where fibrosis is also present (e.g., IPF is present with lung cancer). Accordingly, it is believed that fibrosis, along with many diseases that often exist with it, progresses via the signaling cascade involved in tgfβ proteins and overexpression of the proteins, see, e.g., ballaster, B; et al, idiopathic Pulmonary Fibrosis and lung Cancer: mechanisms and Molecular targets [ idiopathic pulmonary fibrosis with lung cancer: mechanism and molecular targets ], int.j.mol.sci. [ journal of international molecular science ]2019,20,593; doi 10.3390/ijms20030593.

Accordingly, in some embodiments, the compounds described herein may be used to treat (e.g., provide therapy, reverse course of disease), ameliorate (e.g., reduce associated symptoms), prevent (e.g., prophylactic treatment), or manage (e.g., slow or stop progression of) a fibrotic disease (collectively referred to herein as "treating a fibrotic disease"), such as one or more of the fibrotic diseases described herein. In some embodiments, the fibrosis to be treated is present without any concomitant disease. In some embodiments, the fibrosis to be treated is present with an infection (e.g., a viral or bacterial infection). In some embodiments, the fibrosis to be treated is present with an inflammatory disorder. In some embodiments, the inflammatory disorder present is each and several of those described herein. In some embodiments, treating comprises identifying a patient having fibrosis (with or without co-morbid, causal, or exacerbated condition), or a patient at risk of developing fibrosis (with or without co-morbid, causal, or exacerbated condition), and administering thereto a therapeutically effective amount of one or more of the compounds described herein, e.g., one or more compounds having formula (I), (II), (III), (IV), or table 1, or a pharmaceutically acceptable salt of any of the foregoing.

In some embodiments, the fibrosis to be treated is present with cancer. In some embodiments, fibrosis is co-morbid with a cancerous condition. In some embodiments, the cancer is the cause of a fibrotic disorder. In some embodiments, the fibrotic disorder is exacerbated by cancer. In some embodiments, the cancer present is each and several of those described in detail herein, whether as a co-morbid, causal, or exacerbated condition.

In some embodiments, the fibrosis to be treated is present with a viral infection. In some embodiments, the viral infection is co-morbid with a fibrotic condition. In some embodiments, the viral infection is the cause of a fibrotic disorder. In some embodiments, the fibrotic disorder is exacerbated by a viral infection. In some embodiments, the viral infection present is each and several of the viral infections mentioned herein.

In some embodiments, the treatment of a fibrotic disease is performed by administering one or more of the compounds described herein (e.g., one or more compounds having formula (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt of any of the foregoing), which may be alone or in combination with another disorder (which may be a co-morbid, exacerbated, or causative disorder of fibrosis) selected from each and several of a viral infection, cancer, or inflammatory disorder, such as each and several of those described herein. In some embodiments, the treatment of a fibrotic disease (with or without an accompanying disorder) (e.g., one or more of those described herein) is performed by administering two or more compounds as described herein (e.g., two or more compounds having formula (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt of any of the foregoing). In some embodiments, the treatment of a fibrotic disease (with or without an accompanying disorder) (e.g., one or more of those described herein) is performed by administering a therapeutic agent comprising one or more compounds described herein (e.g., one or more of the example compounds or pharmaceutically acceptable salts thereof) in combination with one or more additional therapeutic agents (e.g., at least one example compound or pharmaceutically acceptable salt thereof; at least one additional therapeutic agent). In some embodiments, the combination therapy is provided by administering a compound having formula (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt of any one thereof, and one or more additional therapeutic agents. In some embodiments, the combination of therapeutic agents comprises one exemplary compound or pharmaceutically acceptable salt thereof, and more than one additional therapeutic agent.

In some embodiments, administration of the exemplary compounds or pharmaceutically acceptable salts thereof (alone or in combination with one or more additional therapeutic agents) occurs during a single stage of the disease (e.g., stage 1, stage 2, stage 3). In some embodiments, the fibrosis treatment includes administration of a combination therapy distributed across multiple stages of the disease. As a non-limiting example, an exemplary compound or pharmaceutically acceptable salt thereof (e.g., one or more of the exemplary compounds) may be administered during stage 1, stage 2, or stage 3 of the disease, while one or more additional therapeutic agents may be administered during different stages of the disease. For example, in some embodiments, the treatment of a fibrotic disease (as described herein) is accomplished by administering an exemplary compound (e.g., one or more of: a compound having formula (I), (II), (III), (IV), or Table 1, or a pharmaceutically acceptable salt of any of the foregoing). In some embodiments, when a combination is used to treat a proliferative disease, the combination is one or more of the compounds having formula (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt of any one thereof, and an immunooncology agent. In some embodiments, the exemplary compound and one or more additional therapeutic agents in combination therapy are administered during all phases of fibrosis. In some embodiments, the exemplary compounds or pharmaceutically acceptable salts thereof are provided during some phases but not others. In some embodiments in which combination therapies are employed, the exemplary compounds or pharmaceutically acceptable salts thereof are administered during all phases of the disease, and the additional therapeutic agents in combination therewith are administered during some phases of the disease but not others.

In some embodiments, an exemplary compound or pharmaceutically acceptable salt thereof is administered to a subject in need thereof in an amount effective to treat a fibrotic disease, e.g., in an amount effective to slow or stop progression of a disease or disorder (e.g., idiopathic pulmonary fibrosis, acute exacerbation of IPF, heart disease, liver fibrosis, cirrhosis, non-alcoholic steatohepatitis, pecan's disease, dipivefrong contracture, cystic fibrosis, beta thalassemia, light keratosis, hypertension, systemic inflammatory disorders, dry eye, ulcers, corneal fibrosis, wet age-related macular degeneration, psoriasis, wound closure, chronic kidney disease, kidney fibrosis, systemic sclerosis, and chronic chagas's heart disease); increasing survival time of a subject having a disease or disorder (e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% when compared to a subject not administered the example compound or pharmaceutically acceptable salt thereof); increasing survival of a population of subjects (e.g., increased survival of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% after entry into an intensive care unit when compared to a population of subjects not administered the example compound or pharmaceutically acceptable salt thereof); reducing the risk of a subject developing a fibrotic disorder (e.g., pulmonary fibrosis or IPF) when compared to a subject not administered the example compound or a pharmaceutically acceptable salt thereof; protecting organ function (e.g., lung function or liver function) when compared to a subject not administered the example compound or pharmaceutically acceptable salt thereof; and/or preventing or reducing the risk of acute exacerbation of the disorder when compared to a subject not administered the example compound or pharmaceutically acceptable salt thereof.

In some embodiments, methods of inhibiting fibrosis in a tissue are provided, the methods comprising contacting the tissue (e.g., in vitro, ex vivo, in vivo) with a compound of the present disclosure (e.g., an effective amount of a compound of the present disclosure). In various embodiments, the effective amount is an amount effective to inhibit the formation or deposition of tissue fibrosis, and/or reduce the size, cellularity, composition, cell content, or collagen content of the fibrotic lesion. In some embodiments of the methods described herein, the method involves contacting the tissue with an amount of a compound having formula (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt of any one thereof, sufficient to reduce or inhibit fibrosis. In some embodiments of the methods described herein, the methods may include inhibiting the formation or deposition of tissue fibrosis, and/or reducing the size, cellularity, composition, cell content, or collagen content of a fibrotic lesion. In some embodiments, the fibrotic lesions are in a subject (e.g., a human subject). In some embodiments, the method of inhibiting is applied to a subject who has developed a concomitant disorder with, resulting in, or exacerbating the fibrosis, e.g., cancer, inflammation, or viral infection.

In some embodiments, methods of treating fibrosis in a tissue are provided, the methods comprising administering a compound described herein, e.g., one or more of the following: a compound having the formula (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt of any of the foregoing. In some embodiments of the methods described herein, the methods involve contacting the tissue with an amount of a compound described herein, or a pharmaceutically acceptable salt thereof, sufficient to reverse progression or eliminate fibrosis. In some embodiments of the methods described herein, the methods may include reversing or eliminating the formation or deposition of tissue fibrosis, and/or reducing the size, cellularity, composition, cell content, or collagen content of the fibrotic lesions. In some embodiments, the fibrotic lesions are in a subject (e.g., a human subject). In some embodiments, the method of treating is applied to a subject who has developed a concomitant disorder with, resulting in, or exacerbating the fibrosis, e.g., cancer, inflammation, or viral infection.

In some embodiments, the treatment, alleviation or prevention (e.g., prophylactic treatment) of a fibrotic disorder (e.g., pulmonary fibrosis) present with (co-morbid with, caused by, and/or exacerbated by) a cancer is provided by administration of one or more compounds as described herein (e.g., one or more compounds having the formula (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt of any of the foregoing).

In some embodiments, the treatment, alleviation or prevention of a fibrotic disorder present with a cancerous disorder (e.g., acute exacerbation of idiopathic pulmonary fibrosis) is carried out by administering one or more compounds (e.g., a compound having formula (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt of any of the foregoing).

In some embodiments, the treatment of a fibrotic disease (e.g., one or more of those described herein) present with cancer is performed by administering a therapeutic agent comprising one or more compounds described herein (e.g., a compound having formula (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt of any of the foregoing) in combination with one or more additional therapeutic agents. In some embodiments, the combination treatment of fibrosis present with cancer is provided by administering two or more ALK-5 inhibitors (e.g., two or more compounds having formula (I), (II), (III), (IV) or table 1 or a pharmaceutically acceptable salt of either), and one or more additional therapeutic agents.

In some embodiments, the treatment, alleviation or prevention of fibrosis co-morbid with a viral infection is performed by administering one or more ALK-5 inhibitor compounds (e.g., a compound having formula (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt of any of the foregoing). In some embodiments, the treatment of a fibrotic disease (e.g., one or more of those described herein) present with a viral infection is performed by administering two or more ALK-5 inhibitor compounds (e.g., two or more compounds having formula (I), (II), (III), (IV), or table 1, or a pharmaceutically acceptable salt of any one thereof).

In some embodiments, the treatment of a fibrotic disease (e.g., one or more of those described herein) present with a viral infection is performed by administering a therapeutic agent comprising one or more compounds described herein (e.g., a compound having formula (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt of any of the foregoing) in combination with one or more additional therapeutic agents.

In some embodiments, the treatment, alleviation or prevention of a fibrotic disorder present with a viral infection (e.g., acute exacerbation of idiopathic pulmonary fibrosis) is carried out by administration of one or more compounds (e.g., a compound having formula (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt of any of the foregoing).

In some embodiments, treatment, alleviation or prevention of a fibrotic disorder (e.g., pulmonary fibrosis) co-morbid with, caused by, or exacerbated by an inflammatory disorder is provided by administration of one or more compounds described herein (e.g., a compound having formula (I), (II), (III), (IV), or table 1, or a pharmaceutically acceptable salt of any of the foregoing). In some embodiments, the treatment, alleviation or prevention of a fibrotic disorder (e.g., pulmonary fibrosis) present with an inflammatory disorder (e.g., each and several of those described herein) is carried out by administering two or more compounds described herein (e.g., a compound having formula (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt of any of the foregoing).

In some embodiments, treatment, alleviation or prevention of a fibrotic disorder (e.g., pulmonary fibrosis) that is co-morbid with, caused by, or exacerbated by an inflammatory disorder (e.g., each and several of those described herein) is carried out by administration of a therapeutic agent comprising one or more compounds described herein (e.g., a compound having formula (I), (II), (III), (IV), or table 1, or a pharmaceutically acceptable salt of any of the foregoing) in combination with one or more additional therapeutic agents. In some embodiments, combination therapy is provided by administering two or more ALK-5 inhibitor compounds (e.g., two or more compounds having formula (I), (II), (III), (IV) or table 1 or a pharmaceutically acceptable salt of any of the foregoing), and one or more additional therapeutic agents. In some embodiments, the treatment, alleviation or prevention of a fibrotic disorder present with an inflammatory disorder (e.g., acute exacerbation of idiopathic pulmonary fibrosis) is carried out by administration of one or more compounds (e.g., a compound having formula (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt of any of the foregoing).

In some embodiments, a fibrotic disorder (e.g., pulmonary fibrosis) is present with one or more additional disorders (concomitant disorders) (e.g., inflammatory disorders, cancer, and/or viral infection). The concomitant disorder may be the cause of the fibrotic disorder or exacerbation of the fibrotic disorder, or may be a co-disease of the fibrotic disorder. In some embodiments, the concomitant disorder is a viral infection. In some embodiments, the concomitant disorder is cancer. In some embodiments, the concomitant disorder is an inflammatory disorder. In some embodiments, where treatment, alleviation or prevention of a fibrotic disorder (e.g., pulmonary fibrosis) present with, caused by, and/or exacerbated by cancer, a viral infection, or an inflammatory disorder is provided, the fibrotic disorder is pulmonary fibrosis. In some embodiments, the fibrotic disorder is idiopathic pulmonary fibrosis. In some embodiments, the fibrotic disorder is an acute exacerbation of idiopathic pulmonary fibrosis.

In some embodiments, the fibrotic disorder (e.g., pulmonary fibrosis) to which the treatment is administered is present without concomitant disease states. In some embodiments, the treatment of a fibrotic disorder present without concomitant disease states is provided by administering a compound described herein, or a pharmaceutically acceptable salt thereof (e.g., a compound having formula (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt of any of the foregoing). In some embodiments, the treatment of a fibrotic disorder present without concomitant disease states is provided by administering a therapeutically effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof (e.g., a compound having formula (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt of any of the foregoing). In some embodiments, treatment, alleviation or prevention of fibrotic disorders (e.g., pulmonary fibrosis) that do not exist with concomitant cancer, viral infection, or inflammatory disorders is provided. In some embodiments, the fibrotic disorder is pulmonary fibrosis. In some embodiments, the fibrotic disorder is idiopathic pulmonary fibrosis. In some embodiments, the fibrotic disorder is an acute exacerbation of idiopathic pulmonary fibrosis.

In some embodiments, the methods according to the present disclosure are performed by administering a compound described herein (alone or as part of a combination therapy) (e.g., alone or in a combination of two or more of the following: a compound of formula (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt of any of the foregoing), a fibrotic disorder such as, but not limited to, pulmonary fibrosis (commonly referred to as "pulmonary scarring") (e.g., pulmonary fibrosis, e.g., idiopathic pulmonary fibrosis, or acute exacerbation of familial pulmonary fibrosis), liver fibrosis (liver fibrosis, e.g., keloid, scleroderma, or systemic fibrosis of renal origin), biliary fibrosis (bile fibrosis), liver cirrhosis, e.g., primary biliary cholangitis (biliary cirrhosis), primary sclerosing cholangitis), fibrosis of cardiac tissue (cardiac fibrosis or restenosis (e.g., intractable restenosis, post-angioplasty restenosis)), vascular fibrosis, renal fibrosis (renal fibrosis), skin fibrosis (epidermal fibrosis or endometrial fibrosis, e.g., keloid, scleroderma, or systemic fibrosis of renal origin), gastrointestinal fibrosis (e.g., keloid, scleroderma, or systemic fibrosis of renal origin), bone marrow fibrosis (e.g., keloid, atherosclerosis, or other fibrotic joint fibrosis (e.g., atherosclerosis), or atherosclerosis, etc. Pecimen's disease, retroperitoneal fibrosis, systemic sclerosis, autoimmune hepatitis, or two or more thereof.

In some embodiments, the fibrotic condition to be treated is pulmonary fibrosis. In some embodiments, the fibrotic condition to be treated is liver fibrosis. In some embodiments, the fibrotic condition to be treated is cirrhosis. In some embodiments, the fibrotic condition to be treated is non-alcoholic steatohepatitis. In some embodiments, the fibrotic condition to be treated is pecan disease. In some embodiments, the fibrotic condition to be treated is cystic fibrosis. In some embodiments, the fibrotic condition to be treated is β -thalassemia. In some embodiments, the fibrotic condition to be treated is a keratosis. In some embodiments, the fibrotic condition to be treated is hypertension. In some embodiments, the fibrotic condition to be treated is a chronic kidney disease, such as kidney fibrosis. In some embodiments, the fibrotic condition to be treated is chronic Chagas heart disease.

In some embodiments, the fibrotic condition to be treated is dry eye, an ulcer, corneal fibrosis, wet age-related macular degeneration, chronic wounds (incapable of healing), or systemic sclerosis. In some embodiments, the fibrotic condition to be treated is psoriasis. In some embodiments, the fibrotic disorder is idiopathic pulmonary fibrosis, liver cirrhosis, non-alcoholic steatohepatitis, pecan's disease, dipteret's contracture, cystic fibrosis, beta thalassemia, light keratosis, hypertension, systemic inflammatory disorders, dry eye, ulcers, corneal fibrosis, wet age-related macular degeneration, psoriasis, wound closure, chronic kidney disease, kidney fibrosis, systemic sclerosis, or chronic chagas heart disease. In some embodiments, the fibrotic condition is cardiac fibrosis or a condition associated with cardiac fibrosis, such as valvular disease, cardiac arrhythmia (e.g., atrial fibrillation), myocardial remodeling (e.g., post-infarction), cardiomyopathy (e.g., dilated, ischemic, or hypertrophic cardiomyopathy), restenosis (e.g., in-stent restenosis, restenosis after angioplasty). In some embodiments, the fibrotic disorder is diprote contracture.

In some embodiments, a fibrotic disorder (e.g., pulmonary fibrosis) may be present with, may be caused by, and/or may be exacerbated by (concomitant with) a viral infection. In some embodiments, the viral infection present may be an Orthomyxoviridae (Orthomyxoviridae) viral infection (e.g., influenza a virus infection or influenza B virus infection), a Pneumoviridae (Pneumoviridae) viral infection (e.g., metapneumoviridae (HMPV) infection) or an orthopneumoviridae (orthopneumoviridae) infection (e.g., respiratory Syncytial Virus (RSV) (e.g., human Respiratory Syncytial Virus (HRSV) infection (e.g., human respiratory syncytial virus A2 (HRSV-A2) infection) or respiratory syncytial virus B1 (HRSV-B1) infection)), an Orthohepadnavirus (Orthohepadnavirus) viral infection (e.g., hepatitis B virus infection), a Hepatitis (Hepacivirus) infection (e.g., hepatitis c virus (Hepatitis C virus) infection), a paramyxovirus (e.g., human respiratory syncytial virus (iv) infection) or a virus (e.g., human respiratory parainfluenza iv) infection (iv-3) infection or a virus (p-iv) infection (e.g., human respiratory syncytial virus 2-iv) infection (iv-3) Adenoviridae (Adenoviridae) viral infections (e.g., mammalian adenovirus (Mastadenorus) infections (e.g., human adenovirus B (HAdV-B) infections or human adenovirus C (HAdV-C) infections)), enterovirus (Enterovirus) viral infections (e.g., rhinovirus A (Rhinovirus A) infections, rhinovirus B (Rhinovirus B) infections or Rhinovirus C (Rhinovirus C) infections).

In some embodiments, a treatment is provided for each and several of the fibrotic disorders described herein, wherein each and several of the foregoing viral infections are present as co-morbid disorders, comprising administering one or more compounds described herein, e.g., a compound having formula (I), (II), (III), (IV), or table 1, or a pharmaceutically acceptable salt of any of the foregoing. In some embodiments, the treatment of a fibrotic disease (e.g., each and several of those described herein) is performed by administering two or more compounds as described herein (e.g., two or more compounds having formula (I), (II), (III), (IV) or table 1 or a pharmaceutically acceptable salt of any of the foregoing). In some embodiments, the treatment of a fibrotic disease co-morbid with a viral infection (e.g., each and several of those described herein) is performed by administering a combination comprising one or more compounds described herein (e.g., one or more compounds having formula (I), (II), (III), (IV) or table 1 or a pharmaceutically acceptable salt of any of the foregoing) in combination with one or more additional therapeutic agents. In some embodiments, the combination therapy is provided by administering a compound having formula (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt thereof, and one or more additional therapeutic agents.

In some embodiments, a treatment is provided for each and several of the fibrotic disorders described herein, wherein each and several of the viral infections are present as exacerbated disorders, comprising administering one or more compounds described herein, e.g., a compound having formula (I), (II), (III), (IV), or table 1, or a pharmaceutically acceptable salt thereof. In some embodiments, the treatment of fibrotic diseases (e.g., each and several of those described herein) present with an exacerbated viral infection is performed by administering two or more compounds as described herein (e.g., two or more compounds having formula (I), (II), (III), (IV) or table 1 or a pharmaceutically acceptable salt of any of the foregoing). In some embodiments, the treatment of a fibrotic disease (e.g., one or more of those described herein) present with an exacerbated viral infection is performed by administering a combination of a therapeutic agent comprising one or more compounds described herein (e.g., one or more compounds having formula (I), (II), (III), (IV) or table 1 or a pharmaceutically acceptable salt of any of the foregoing) with one or more additional therapeutic agents. In some embodiments, the combination therapy is provided by administering a compound having formula (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt thereof, and one or more additional therapeutic agents.

In some embodiments, a treatment is provided for fibrosis present with each and several of these viral infections as the cause of fibrosis, the treatment comprising administration of one or more ALK-5 inhibitor compounds as described herein, e.g., an ALK-5 inhibitor compound having formula (I), (II), (III), (IV), or table 1, or a pharmaceutically acceptable salt thereof. In some embodiments, the treatment of a fibrotic disease (e.g., each and several of those described herein) present with a causative viral infection is performed by administering two or more compounds as described herein (e.g., two or more compounds having formula (I), (II), (III), (IV) or table 1 or a pharmaceutically acceptable salt of any of the foregoing). In some embodiments, the treatment of a fibrotic disease (e.g., one or more of those described herein) present with a causative viral infection is performed by administering a combination of a therapeutic agent comprising one or more compounds described herein (e.g., one or more compounds having formula (I), (II), (III), (IV) or table 1 or a pharmaceutically acceptable salt of any of the foregoing) with one or more additional therapeutic agents. In some embodiments, the combination therapy is provided by administering an ALK-5 inhibitor compound having formula (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt thereof, and one or more additional therapeutic agents.

In some embodiments, a fibrotic disorder (e.g., pulmonary fibrosis) may be present with, may be caused by, and/or may be exacerbated by an inflammatory disorder. As used herein, the terms "inflammatory disease," "inflammatory disorder," and "inflammatory disease and/or disorder" refer to a disease or disorder in a subject that involves the response of one or more body tissues to a stimulus recognized by the body as being harmful. In some embodiments, the inflammatory disorder is an autoimmune disorder. Exemplary inflammatory conditions include non-alcoholic fatty liver disease (NAFLD), alcoholic Steatohepatitis (ASH), non-alcoholic steatohepatitis (NASH), primary Biliary Cholangitis (PBC), primary sclerosing cholangitis, and autoimmune hepatitis. NAFLD is a condition in which fat is deposited in the liver for reasons other than excessive alcohol consumption, whereas NASH is a late form of NAFLD in which the liver is both inflamed and damaged. Abnormal injury repair of NASH can lead to cirrhosis. ASH is a condition in which the liver is inflamed and damaged in connection with drinking, and it may include liver fibrosis and/or cirrhosis. PBC is an autoimmune disease of the liver, and abnormal repair of liver damage can lead to scarring, fibrosis, and/or cirrhosis. Primary sclerosing cholangitis may be characterized by cholangitis and scarring, which may lead to fibrosis and/or cirrhosis. Autoimmune hepatitis can cause liver inflammation, and abnormal repair of liver inflammation can lead to fibrosis and/or cirrhosis.

In some embodiments, a treatment is provided for fibrosis present with each and several of these inflammatory conditions present as a fibrotic co-morbid condition, comprising administering one or more compounds as described herein, e.g., a compound having formula (I), (II), (III), (IV), or table 1, or a pharmaceutically acceptable salt thereof. In some embodiments, the treatment of a fibrotic disease (e.g., each and several of those described herein) co-morbid with an inflammatory disorder is performed by administering two or more compounds as described herein (e.g., two or more compounds having formula (I), (II), (III), (IV) or table 1 or a pharmaceutically acceptable salt of any of the foregoing). In some embodiments, the treatment of a fibrotic disease (e.g., one or more of those described herein) co-morbid with an inflammatory disorder is performed by administering a combination of a therapeutic agent comprising one or more compounds described herein (e.g., one or more compounds having formula (I), (II), (III), (IV) or table 1 or a pharmaceutically acceptable salt of any of the foregoing) with one or more additional therapeutic agents. In some embodiments, the combination therapy is provided by administering a compound having formula (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt thereof, and one or more additional therapeutic agents.

In some embodiments, a treatment is provided for each of these inflammatory disorders that are present as exacerbating disorders of fibrosis, comprising administering one or more compounds as described herein, e.g., a compound having formula (I), (II), (III), (IV), or table 1, or a pharmaceutically acceptable salt thereof. In some embodiments, the treatment of fibrotic diseases (e.g., each and several of those described herein) present with an exacerbated inflammatory disorder is performed by administering two or more compounds having formula (I), (II), (III), (IV), or table 1, or a pharmaceutically acceptable salt of any of the foregoing. In some embodiments, the treatment of a fibrotic disease (e.g., one or more of those described herein) present with an exacerbated inflammatory disorder is performed by administering a combination of a therapeutic agent comprising one or more compounds described herein (e.g., one or more compounds having formula (I), (II), (III), (IV) or table 1 or a pharmaceutically acceptable salt of any of the foregoing) with one or more additional therapeutic agents. In some embodiments, the combination therapy is provided by administering a compound having formula (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt thereof, and one or more additional therapeutic agents.

In some embodiments, a treatment is provided for each of the inflammatory disorders present as a cause of fibrosis, comprising administering one or more compounds as described herein, e.g., a compound having formula (I), (II), (III), (IV), or table 1, or a pharmaceutically acceptable salt thereof. In some embodiments, the treatment of fibrotic diseases (e.g., each and several of those described herein) present with a causative inflammatory disorder is performed by administering two or more compounds having formula (I), (II), (III), (IV), or table 1, or a pharmaceutically acceptable salt of any of the foregoing. In some embodiments, the treatment of a fibrotic disease (e.g., one or more of those described herein) present with a causative inflammatory disorder is performed by administering a combination of a therapeutic agent comprising one or more compounds described herein (e.g., one or more compounds having formula (I), (II), (III), (IV) or table 1 or a pharmaceutically acceptable salt of any of the foregoing) with one or more additional therapeutic agents. In some embodiments, the combination therapy is provided by administering a compound having formula (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt thereof, and one or more additional therapeutic agents.

Provided herein is a method of treating a fibrotic, inflammatory or proliferative disease or disorder susceptible to inhibition of a tgfβ signaling pathway, the method comprising administering to a subject suffering from the fibrotic, inflammatory or proliferative disease or disorder an amount of a compound as described herein, or a pharmaceutically acceptable salt form thereof, or a pharmaceutical composition of the foregoing, effective to inhibit tgfβ signaling.

Also provided herein is a method of inhibiting tgfβ signaling in a subject suffering from a disease or condition facilitated by tgfβ signaling (particularly TGF- β1 signaling), the method comprising administering an amount of at least one compound as described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the foregoing, effective to substantially suppress tgfβ signaling to alter the course of the disease or condition.

Also provided herein is a method of inhibiting an epithelial to mesenchymal transition (EMT) in a subject having a disease or disorder facilitated by EMT, the method comprising administering an amount of at least one compound as described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the foregoing, effective to sufficiently inhibit EMT to alter the course of the disease or disorder (e.g., a therapeutically effective amount).

Additionally, provided herein are methods of inhibiting tumor growth in a subject (e.g., a subject in need thereof) comprising administering to the subject a therapeutically effective amount of a compound having formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the foregoing. Also provided herein are compounds having formula (I) and pharmaceutically acceptable salts thereof, as well as pharmaceutical compositions of the foregoing, for inhibiting tumor growth. Also provided herein is the use of a compound having formula (I) and pharmaceutically acceptable salts thereof, as well as pharmaceutical compositions of the foregoing, for the manufacture of a medicament for inhibiting tumor growth.

Also provided herein are methods for inhibiting the activity of an activin receptor-like kinase (e.g., ALK-5) in vivo or in vitro, comprising contacting the activin receptor-like kinase (e.g., ALK-5) with one or more of the exemplified compounds (e.g., one or more compounds having formula (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt thereof) or a pharmaceutical composition of the foregoing. Also provided herein are compounds having formula (I), (II), (III), (IV) or table 1, and pharmaceutically acceptable salts thereof, as well as pharmaceutical compositions of the foregoing, for inhibiting the activity of activin receptor-like kinase (e.g., ALK-5) in vivo or in vitro. Also provided herein is the use of a compound having formula (I), (II), (III), (IV) or table 1, and pharmaceutically acceptable salts thereof, and pharmaceutical compositions of the foregoing, for the manufacture of a medicament for inhibiting the activity of an activin receptor-like kinase (e.g., ALK-5) in vivo or in vitro. In certain embodiments, inhibition occurs in a subject. In certain embodiments, inhibition occurs in vitro (e.g., in a cell line or biological sample). In certain embodiments, these methods and uses are for inhibiting ALK-5. In certain embodiments, inhibition is selective for ALK-5, i.e., selective for ALK-5 relative to other kinases (e.g., selective for ALK-5 relative to other activin receptor-like kinases (e.g., ALK-2; JAK 2)). In certain embodiments, inhibition is selective for ALK-5 over ALK-2.

Also provided herein are methods for targeting tumor stromal cells or immune cells (e.g., tumor-associated immune cells) and/or (e.g., and thereby) modulating (e.g., normalizing) a tumor microenvironment (e.g., tumor-stromal microenvironment and/or tumor-immune microenvironment) in vivo or in vitro, comprising contacting tumor stromal cells or immune cells (e.g., tumor-associated immune cells) with one or more of the exemplified compounds (e.g., one or more compounds having formula (I), (II), (III), (IV) or table 1, or pharmaceutically acceptable salts thereof) or pharmaceutical compositions of the foregoing. Also provided herein are compounds of formula (I), (II), (III), (IV) or table 1, and pharmaceutically acceptable salts thereof, as well as pharmaceutical compositions of the foregoing, for targeting tumor stromal cells or immune cells (e.g., tumor-associated immune cells) and/or (e.g., and thereby) modulating (e.g., normalizing) a tumor microenvironment (e.g., tumor-stromal microenvironment and/or tumor-immune microenvironment) in vivo or in vitro. Also provided herein is the use of a compound having formula (I), (II), (III), (IV) or table 1, and pharmaceutically acceptable salts thereof, as well as pharmaceutical compositions of the foregoing, for the manufacture of a medicament for targeting tumor stromal cells or immune cells (e.g., tumor-associated immune cells) and/or (e.g., and thereby) modulating (e.g., normalizing) a tumor microenvironment (e.g., tumor-stromal microenvironment and/or tumor-immune microenvironment) in vivo or in vitro. In certain embodiments, inhibition occurs in a subject. In certain embodiments, inhibition occurs in vitro (e.g., in a cell line or biological sample). In certain embodiments, the tumor stromal cells are cancer-associated fibroblasts (CAF), astrocytes, or myofibroblasts.

Tumor microenvironments generally favor tumor growth and survival by favoring cancer biology over healthy cellular function. In particular, the "exclusionary" or "desert" phenotype creates an optimal microenvironment for cancer cells to avoid immune surveillance, to give the microenvironment high acidity and hypoxia, and to create high interstitial pressure. The tumor microenvironment prevents beneficial effects such as immune oncology agents, while poor perfusion and interstitial pressure prevent drug delivery.

"desert phenotype" as used herein to describe cancer refers to the immunophenotype of a tumor characterized by the absence or substantial absence of T cells within the tumor and at one or more edges thereof. The phenotype may be caused by factors including, but not limited to, inadequate priming, defective antigen presentation, and/or antigen deficiency.

As used herein to describe the "exclusion phenotype" of a cancer refers to the immunophenotype of a tumor, characterized by T cells located only or substantially only at one or more edges of the tumor. In the "exclusion phenotype", no or substantially no T cells are present in the tumor bed. The phenotype may be caused by factors including, but not limited to, matrix barriers, abnormal vascular systems, chemokine deficiency, oncogenic pathways, and/or hypoxia.

The tumor microenvironment may be beneficially modulated by promoting an invasive phenotype. As used herein, reference to "invasive phenotype" and "immunoinflammatory phenotype" of a cancer refers to the immunophenotype of a tumor, characterized by T cells located throughout or substantially throughout the tumor bed. Promotion of this desired phenotype may be affected by, for example: inhibition of tgfβ, increasing angiogenesis (e.g., angiogenesis), reducing tumor induration, increasing antigen presentation, deactivating cancer-associated fibroblasts, increasing infiltration of T cells into the tumor bed, or any combination thereof.

Without wishing to be bound by any particular theory, it is believed that the compounds of the present disclosure may modulate the tumor microenvironment (e.g., tumor-stroma microenvironment and/or tumor-immune microenvironment), for example, by promoting an invasive phenotype. Accordingly, in some embodiments, provided herein is a method for modulating (e.g., normalizing) a tumor microenvironment (e.g., tumor-stromal microenvironment and/or tumor-immune microenvironment) in vitro or in vivo (e.g., in a subject such as a subject having a tumor), the method comprising contacting the tumor and/or the tumor microenvironment with an effective amount of a compound of the disclosure (e.g., one or more compounds having formulae (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt thereof) or a pharmaceutical composition thereof. In some embodiments wherein modulation occurs in a subject in need thereof, the method comprises administering to the subject a therapeutically effective amount of a compound of the present disclosure or a pharmaceutical composition thereof.

Without wishing to be bound by any particular theory, it is believed that the exemplary compounds may normalize the tumor microenvironment and thereby improve vascular perfusion and drug delivery. Enhanced drug delivery is expected to in turn enhance the efficacy of drugs (such as immunomodulators (e.g., immunooncology agents), including any of the immunomodulators described herein). Accordingly, provided herein are also methods for modulating (e.g., normalizing) a tumor microenvironment (e.g., tumor-stroma microenvironment and/or tumor-immune microenvironment) in vivo or in vitro, comprising contacting the tumor with one or more of the example compounds (e.g., one or more compounds having formula (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt thereof) or a pharmaceutical composition of the foregoing. Also provided herein are compounds having formula (I), (II), (III), (IV) or table 1, and pharmaceutically acceptable salts thereof, as well as pharmaceutical compositions of the foregoing, for modulating (e.g., normalizing) a tumor microenvironment (e.g., tumor-matrix microenvironment and/or tumor-immune microenvironment) in vivo or in vitro. Also provided herein is the use of a compound having formula (I), (II), (III), (IV) or table 1, and pharmaceutically acceptable salts thereof, and pharmaceutical compositions of the foregoing, for the manufacture of a medicament for modulating (e.g., normalizing) a tumor microenvironment (e.g., tumor-matrix microenvironment and/or tumor-immune microenvironment) in vivo or in vitro. In certain embodiments, inhibition occurs in a subject. In certain embodiments, inhibition occurs in vitro (e.g., in a cell line or biological sample).

Also provided is a method for promoting immune infiltration (e.g., infiltration of immune cells (e.g., T cells)) in vitro or in vivo (e.g., in a subject (e.g., a subject having a tumor), the method comprising contacting the tumor with an effective amount of a compound of the disclosure (e.g., one or more compounds having formula (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt thereof), or a pharmaceutical composition thereof. In some embodiments, wherein the method occurs in a subject in need thereof, the method comprises administering to the subject a therapeutically effective amount of a compound of the present disclosure or a pharmaceutical composition thereof.

Also provided is a method for promoting tumor vascularization (e.g., angiogenesis) in vitro or in vivo (e.g., in a subject (e.g., a subject having a tumor), the method comprising contacting the tumor with an effective amount of a compound of the disclosure (e.g., one or more compounds having formula (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt thereof) or a pharmaceutical composition thereof. In some embodiments, wherein the method occurs in a subject in need thereof, the method comprises administering to the subject a therapeutically effective amount of a compound of the present disclosure or a pharmaceutical composition thereof.

In some embodiments, provided herein is a method for inhibiting metastasis of cancer, the method comprising administering to the subject (e.g., a therapeutically effective amount of a compound of the disclosure or a pharmaceutical composition thereof).

In one embodiment, provided herein is a method of treating cachexia in a subject in need thereof, the method comprising administering to the subject an effective amount (e.g., a therapeutically effective amount) of a compound of any one of formulas (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising one or more compounds of formulas (I), (II), (III), (IV), or table 1, or a pharmaceutically acceptable salt thereof.

In one embodiment, provided herein is a method of promoting immune infiltration in a tumor-immune microenvironment in a subject in need thereof, the method comprising administering to the subject an effective amount (e.g., a therapeutically effective amount) of a compound having any one of formulas (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising one or more compounds having formulas (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt thereof.

In one embodiment, provided herein is a method of inhibiting epithelial to mesenchymal transition in a tumor in a subject in need thereof, the method comprising administering to the subject an effective amount (e.g., a therapeutically effective amount) of a compound having any one of formulas (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising one or more compounds having formulas (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt thereof.

In one embodiment, provided herein is a method for modulating a tumor-immune microenvironment in a subject in need thereof, the method comprising administering to the subject an effective amount (e.g., a therapeutically effective amount) of a compound having any one of formulas (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising one or more compounds having formulas (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt thereof.

In one embodiment, provided herein is a method for increasing tumor blood vessel or blood flow to a tumor, or both, in a subject in need thereof, the method comprising administering to the subject an effective amount (e.g., a therapeutically effective amount) of a compound of any one of formulas (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising one or more compounds of formulas (I), (II), (III), (IV), or table 1, or a pharmaceutically acceptable salt thereof.

In one embodiment, provided herein is a method for inhibiting metastasis of cancer in a subject in need thereof, the method comprising administering to the subject an effective amount (e.g., a therapeutically effective amount) of a compound having any one of formulas (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising one or more compounds having formulas (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt thereof.

Exemplary compounds and pharmaceutically acceptable salts thereof, as well as pharmaceutical compositions of the foregoing, may be administered via a variety of routes of administration including, for example, oral, dietary, topical, transdermal, rectal, parenteral (e.g., intra-arterial, intravenous, intramuscular, subcutaneous, intradermal injection), intravenous infusion and inhalation (e.g., intrabronchial, intranasal or oral inhalation, intranasal drops) routes of administration, depending on the compound and the particular disease to be treated. Administration may be local or systemic, as indicated. The preferred mode of administration may vary depending on the particular compound selected. In some embodiments, the compounds of the present disclosure are administered orally. In some embodiments, the compounds of the present disclosure are administered intravenously.

Combination therapy

In addition to administration as monotherapy, the exemplary compounds and pharmaceutically acceptable salts thereof, as well as pharmaceutical compositions of the foregoing, may be administered in combination with other therapeutic agents and/or modes of treatment. Accordingly, in some embodiments, the methods further comprise administering one or more additional therapies (e.g., therapeutic agents) to the subject. Suitable additional therapies (e.g., therapeutic agents) for the methods, compositions, and combinations disclosed herein include those discussed herein.

The term "combination therapy" refers to the administration of two or more therapeutic agents to treat a disease, disorder, or condition described herein. Such administration encompasses co-administration of the therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of active ingredients. Alternatively, such administration encompasses co-administration in multiple or separate containers (e.g., capsules, powders, and liquids) for each active ingredient. Such administration also encompasses the use of each type of therapeutic agent at about the same time or at different times in a sequential manner. The exemplary compound or pharmaceutically acceptable salt thereof, or a combination of the foregoing, and one or more additional therapeutic agents may be administered via the same route of administration or via different routes of administration. The powder and/or liquid may be reconstituted or diluted to the desired dosage prior to administration. Typically, a treatment regimen will provide a beneficial effect of the pharmaceutical combination in treating a disease, condition, or disorder described herein.

In some embodiments, a compound of the disclosure and one or more additional therapies are co-administered, e.g., in a simultaneous or substantially simultaneous manner. In some embodiments, the compound of the present disclosure and one or more additional therapies are administered at about the same time or sequentially at different times. For example, a compound of the present disclosure may be administered prior to one or more additional therapies. Alternatively, the compounds of the present disclosure may be administered after one or more additional therapies.

In some embodiments, therapies for use in combination with the compounds of the present disclosure provide a known agent that, when used in combination with a compound as described herein, modulates other pathways, or other components of the same pathway, or even overlapping groups of target enzymes. The compound having formula (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt thereof, or a combination of the foregoing may be administered in combination with one or more additional therapies (e.g., therapeutic agents), such as improving activity, potency and/or efficacy in: treating a disease in a subject in need thereof, preventing a disease in a subject in need thereof, reducing the risk of developing a disease in a subject in need thereof, and/or inhibiting the activity of a protein kinase in a subject or cell; improving bioavailability; the safety is improved; reduce drug resistance; reducing and/or modifying metabolism; inhibit excretion; and/or modifying the distribution in a subject or cell of a compound having formula (I), (II), (III), (IV) or table 1, or a pharmaceutically acceptable salt thereof, or a combination of the foregoing. It will also be appreciated that the one or more additional therapies employed may achieve a desired effect for the same disorder, and/or that they may achieve different effects. In one aspect, such therapies include, but are not limited to, combinations of a compound as described herein with a chemotherapeutic agent, a therapeutic antibody, and radiation therapy to provide synergistic or additive therapeutic effects.

The compositions for use in combination therapies will be formulated together as a pharmaceutical combination, or provided for separate administration (e.g., incorporated in a kit). Accordingly, further embodiments are pharmaceutical combinations comprising an example compound or a pharmaceutically acceptable salt thereof, or a combination of the foregoing (e.g., a therapeutically effective amount of an example compound or a pharmaceutically acceptable salt thereof, or a combination of the foregoing), and one or more other therapeutic agents (e.g., a therapeutically effective amount of one or more other therapeutic agents). The pharmaceutical combination may also comprise one or more pharmaceutically acceptable carriers, such as one or more of the pharmaceutically acceptable carriers described herein.

When administered in combination with another therapy, the example compounds or pharmaceutically acceptable salts thereof, or combinations of the foregoing, can be administered before, after, or concurrently with the other therapy (e.g., one or more additional therapeutic agents). When two or more therapeutic agents are co-administered simultaneously (e.g., in parallel), the example compounds or pharmaceutically acceptable salts thereof, and one or more other therapeutic agents may be in separate formulations or the same formulation. Alternatively, the example compound or a pharmaceutically acceptable salt thereof, or a combination of the foregoing, and the other therapies may be administered sequentially (e.g., as separate compositions) within an appropriate time frame (e.g., sufficient to allow overlapping pharmaceutical effects of the example compound or a pharmaceutically acceptable salt thereof, or a combination of the foregoing, and the other therapies) as determined by a skilled clinician.

Additional therapeutic agents include therapeutically active agents. Therapeutic agents also include prophylactically active agents. Therapeutic agents include small organic molecules such as pharmaceutical compounds (e.g., compounds approved by the U.S. food and drug administration for human or veterinary use, as provided in federal regulations (Code of Federal Regulations, CFR)), peptides, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucins, lipoproteins, synthetic polypeptides or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNA, RNA, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins, and cells. Each additional therapeutic agent may be administered at a dosage and/or schedule determined for that therapeutic agent. The additional therapeutic agents may also be administered together with each other and/or with the compounds or compositions described herein in a single dose, or separately in different doses. The particular combination employed in the regimen will take into account, for example, the compatibility of the compounds described herein with one or more additional therapeutic agents and/or the desired therapeutic and/or prophylactic effects to be achieved. Typically, the level of use of one or more additional therapeutic agents in a combination is not expected to exceed the level at which they are used alone. In some embodiments, the level used in combination will be lower than the level used alone.

In certain embodiments, the additional therapeutic agent is selected from the group consisting of: antimetabolites, DNA fragmentation agents, DNA cross-linking agents, intercalating agents, protein synthesis inhibitors, topoisomerase I poisons (e.g., camptothecins or topotecan), topoisomerase II poisons, microtubule-directing agents, kinase inhibitors, hormones, and hormone antagonists.

Examples of therapies for use in combination with a compound of the disclosure (e.g., in combination therapy, in a pharmaceutical combination) include standard of care therapies and/or regimens (e.g., standard of care agents), such as first-line standard of care therapies (e.g., chemotherapy) or last-line standard of care therapies (e.g., chemotherapy). Standard of care therapy is therapy that a clinician should use for a certain type of patient, disorder, and/or clinical condition.

In some embodiments, the compounds of the present disclosure are administered in combination with standard of care therapies for ovarian cancer. For example, non-limiting examples of standard of care therapies for ovarian cancer include platinum analogs (e.g., cisplatin, paclitaxel, carboplatin) or combinations including platinum analogs (e.g., docetaxel and carboplatin; paclitaxel and carboplatin; carboplatin and liposomal doxorubicin (dox); paclitaxel, carboplatin and bevacizumab (bev); carboplatin and gemcitabine (gem)/(bev), carboplatin, liposomal dox and bev; carboplatin, paclitaxel and bev; cisplatin and gemcitabine; oxaliplatin); altretamine; capecitabine (capecitabine); ifosfamide; irinotecan; melphalan; paclitaxel (e.g., albumin-bound paclitaxel); pemetrexed; or vinorelbine. Non-limiting examples of standard of care therapies for ovarian cancer also include targeted therapies, such as antibody therapies (e.g., bevacizumab); PARP inhibitors (e.g., olaparib, lu Kapa, nilaparib, veliparib, talazapanib); tyrosine Kinase Inhibitors (TKIs) (e.g., pazopanib); immunotherapy; immune checkpoint inhibitors (e.g., PD-1 or PD-L1 inhibitors); pembrolizumab; or hormone therapy (e.g., tamoxifen, anastrozole, exemestane, letrozole, LHRH agonists such as leuprolide acetate, megestrol acetate). Non-limiting examples of standard of care therapies for ovarian cancer further include hormonal therapies (e.g., anastrozole, exemestane, letrozole, leuprolide acetate, megestrol acetate, tamoxifen). Non-limiting examples of standard of care therapies for ovarian cancer additionally include cyclophosphamide; etoposide; sorafenib; or vinorelbine.

In some embodiments, the compounds of the present disclosure are administered in combination with standard of care therapies for pancreatic cancer. Non-limiting examples of standard of care therapies for pancreatic cancer include FOLFIRINOX (a chemotherapeutic regimen consisting of folinic acid, bolus fluorouracil, irinotecan, and oxaliplatin); improved FOLFIRINOX regimen (a chemotherapy regimen consisting of folinic acid, continuous infusion of fluorouracil, irinotecan, and oxaliplatin); gemcitabine and albumin paclitaxel; gemcitabine and capecitabine; olaparib; gemcitabine and erlotinib; gemcitabine, docetaxel, and capecitabine; larotinib; pembrolizumab; gemcitabine; and a triple combination of albumin paclitaxel, gemcitabine, and cisplatin.

In some embodiments, the compounds of the present disclosure are administered in combination with standard of care therapies for prostate cancer, including castration-resistant prostate cancer. Non-limiting examples of standard of care therapies for prostate cancer include PARP inhibitors (e.g., olaparib, lu Kapa ni, nilaparib, veliparib, talazapanib), LHRH agonists (e.g., goserelin acetate, histrelin acetate, leuprolide acetate, and tranexamine pamoate); LHRH antagonists (e.g., degarelix); anti-androgen receptors (e.g., bicalutamide, flutamide, nilutamide, enzalutamide, apamide, darulomine); corticosteroids (e.g., prednisone, methylprednisolone, hydrocortisone, dexamethasone); estrogens (e.g., diethylstilbestrol); androgen synthesis inhibitors (e.g., ketoconazole, abiraterone acetate); androgen deprivation therapy.

In some embodiments, the compounds of the present disclosure are administered in combination with standard of care therapies for multiple myeloma. Non-limiting examples of standard of care therapies for multiple myeloma include proteasome inhibitors such as bortezomib, carfilzomib and malizomib.

Generally, organizations such as the national integrated cancer network (NCCN) have published guidelines and/or therapeutic algorithms for best practices for treating certain patients, conditions, and/or clinical situations. See ncn. These guidelines generally establish, describe, and/or summarize standard of care therapies.

In some embodiments, radiation therapy may be administered in combination with a compound as described herein. Exemplary radiation therapies include external beam therapy, internal radiation therapy, implant radiation, stereotactic radiosurgery, whole body radiation therapy, and permanent or temporary interstitial brachytherapy. As used herein, the term "brachytherapy" refers to radiation therapy delivered by spatially limited radioactive material inserted into the body at or near a tumor or other proliferative tissue disease site. The term is intended to include, but is not limited to, exposure to radioisotopes (e.g., at211, I131, I125, Y90, re186, re188, sm153, bi212, P32, and radioisotopes of Lu). Suitable radiation sources for use as cell conditioning agents of the present disclosure include solids and liquids. As non-limiting examples, the radiation source may be a radionuclide, such as I125, I131, yb169, ir192 as a solid source, I125 as a solid source, or other radionuclide that emits photons, beta particles, gamma radiation, or other therapeutic rays. The radioactive material may also be a fluid made from any solution of one or more radionuclides (e.g., a solution of I125 or I131), or the radioactive fluid may be produced using a slurry of a suitable fluid containing small particles of a solid radionuclide (e.g., au198, Y90). Furthermore, one or more radionuclides may be embodied in a gel or a radioactive microsphere.

Without being limited by any theory, the compounds of the present disclosure may make abnormal cells more susceptible to radiation therapy for the purpose of killing such cells and/or inhibiting the growth of such cells. Accordingly, some embodiments include a method for sensitizing abnormal cells in a mammal to radiation therapy comprising administering to the mammal an amount of a compound as described herein effective to sensitize the abnormal cells to radiation therapy. The amount of a compound of the present disclosure in this method can be determined according to the means used to confirm effective amounts of such compounds and salts described herein. In some embodiments, the standard of care therapy comprises radiation therapy.

DNA damaging agents may also be used in combination with the compounds of the present disclosure. Non-limiting examples of DNA damaging agents include radiation, topoisomerase inhibitors, PARP inhibitors, DNA cross-linking agents, and standard of care agents that induce DNA damage, such as DNA cross-linking agents. Specific non-limiting examples of DNA damaging agents include albumin-bound paclitaxel (abaxane), gemcitabine, paclitaxel, and temozolomide.

Agents that induce Endoplasmic Reticulum (ER) stress may also be used in combination with the compounds of the present disclosure. Non-limiting examples of agents that induce ER stress include agents that increase Reactive Oxygen Species (ROS) (e.g., nalbuphine), chaperonin inhibitors, HSP90 inhibitors, HSP70 inhibitors, PDI inhibitors, and proteasome inhibitors. Further non-limiting examples of agents that induce ER stress include GSK2606414, GSK2656157, STF-083010, tyrosine kinase inhibitors (e.g., sorafenib), phospho-eukaryotic initiation factor 2α phosphatase (e.g., sal 003), diindolmethane derivatives, proteasome inhibitors (e.g., bortezomib), angelolide a, andrographolide, tolfenamic acid, cantharidin, carnosic acid, castin, cryptotanshinone, curcumin, huang Kawa piperin B, fucoidan sulfate, 2-3, 4-dihydroxyphenylethanol, 7-dimethoxyflavone, SMIP004 (N- (4-butyl-2-methyl-phenylacetamide), licochalcone a, methyl liensine, paeonol, leopamine, kavalin, polyphenylene E, paris saponin D, resveratrol, gamma-tocotrienol, omega-hydroxyundecanoic-9-enoic acid, ampelopsin, brassinosteroid, brassinosteroids, succinin a, D-jingnine, D-5, D-methylenoid, D-35 a, D-methylenoid, D-35, D-75, D-methylenoid, D-35, D-75, and the like.

Non-limiting examples of chemotherapeutic agents for use in combination with the compounds of the present disclosure (e.g., in combination therapy, in pharmaceutical combination) include capecitabineN4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatinCisplatin->Cladribine>Cyclophosphamide (/ -s)>Or (b)) Cytarabine, cytosine cytarabine +.>Cytarabine liposome injectionDacarbazine->Doxorubicin hydrochlorideFludarabine phosphate->5-fluorouracilGemcitabine (difluoro deoxycytidine), irinotecan +.>L-asparaginase->6-mercaptopurine->Methotrexate>Pennisetum, 6-thioguanine, thiotepa and topotecan hydrochloride for injection>Another example is bortezomib. Further examples include gemcitabine, albumin paclitaxel +>Erlotinib, fluorouracil and FOLFIRINOX (a chemotherapeutic regimen consisting of folinic acid, fluorouracil, irinotecan and oxaliplatin), or any combination of two or more of the foregoing, e.g., for the treatment of pancreatic cancer (e.g., advanced pancreatic cancer, pancreatic ductal adenocarcinoma).

Particularly interesting anticancer agents for use in combination with the compounds of the present disclosure include:

topoisomerase inhibitors, including type I topoisomerase inhibitors (e.g., irinotecan, topotecan, and camptothecin) and type 2 topoisomerase inhibitors (e.g., etoposide, doxorubicin, and epirubicin).

Poly (ADP-ribose) polymerase (PARP) inhibitors such as Olaparib, lu Kapa, nilapatinib, taraxazopanib, velippanib, pa Mi Pani and Enipab.

DNA cross-linking agents such as cisplatin, carboplatin, and oxaliplatin.

Agents that increase Reactive Oxygen Species (ROS) levels, such as nalbupcine.

PARP inhibitors such as olaparib, lu Kapa, nilaparib, veliparib and talazapanib.

Purine antimetabolites and/or inhibitors of purine de novo synthesis: pemetrexedGemcitabine5-fluorouracil (/ -)>And->) Methotrexate->Capecitabine->Fluorouridine->Decitabine>Azacytidine (+)>And->) 6-mercaptopurine->Cladribine (>And) Fludarabine->Pennisetum>Nelarabine->Clofarabine ()>And->) And cytarabine->

Anti-angiogenic agents include, for example, MMP-2 (matrix-metalloproteinase 2) inhibitors, rapamycin, temsirolimus (CCI-779), everolimus (RAD 001), sorafenib, sunitinib, and bevacizumab. Examples of useful COX-II inhibitors include CELEBREX ^TM (alecoxib), valdecoxib and rofecoxib. Examples of useful matrix metalloproteinase inhibitors are described in WO 96/33172 (publication of 1996, 10 and 24), WO 96/27583 (publication of 1996, 7), european patent application No. 97304971.1 (publication of 1997, 7 and 8), european patent application No. 99308617.2 (publication of 1999, 10 and 29), WO 98/07697 (publication of 1998, 2 and 26), WO 98/03516 (publication of 1991, 1 and 29), WO 98/34918 (publication of 1998 and 13), WO 98/34915 (publication of 1998 and 8), WO 98/33768 (publication of 1998, 8 and 6), WO 98/30556 (publication of 1998, 7 and 16), european patent publication 606,046 (publication of 1994, 7 and 13), european patent publication 931,788 (publication of 1999, 7 and 28), WO 90/05719 (publication of 1990, 5 and 31), WO 99/52910 (21 of 1999, 10 and 10) Publication), WO 99/52889 (published 10.21 1999), WO 99/29667 (published 6.17 1999), PCT International application No. PCT/IB98/01113 (published 7.21 1998), european patent application No. 99302232.1 (published 3.25 1999), british patent application No. 9912961.1 (published 6.3 1999), U.S. provisional application No. 60/148,464 (published 8.12 1999), U.S. patent 5,863,949 (published 1.26 1999), U.S. patent 5,861,510 (published 1.19 1999) and European patent publication 780,386 (published 6.25 1997), all of which are incorporated herein by reference in their entirety. Examples of MMP-2 and MMP-9 inhibitors include those with little or no inhibition of MMP-1 activity. Other embodiments include those that selectively inhibit MMP-2 and/or AMP-9 relative to other matrix-metalloproteinases (i.e., MAP-1, MMP-3, MMP-4, MMP-5, MMP-6, MMP-7, MMP-8, MMP-10, MMP-ll, MMP-12, and MMP-13). In some embodiments, some specific examples of useful MMP inhibitors are AG-3340, RO 323555, and RS13-0830.

Autophagy inhibitors include, but are not limited to, chloroquine, 3-methyladenine, hydroxychloroquine (Plaquenil) ^TM ) Bafilomycin A1, 5-amino-4-imidazolecarboxamide nucleoside (AICAR), okadaic acid, autophagy-inhibiting algal toxins of the type 2A or type 1 that inhibit protein phosphatases, analogs of cAMP, and agents that increase cAMP levels, such as adenosine, LY204002, N6-mercaptopurine nucleoside, and vinca alkaloid. In addition, antisense or siRNA that inhibits the expression of proteins including, but not limited to, ATG5 (which is involved in autophagy) may also be used.

In other embodiments, agents that may be used in methods of combination therapy with a compound as described herein include, but are not limited to: erlotinib, afatinib, iressa (gefitinib), GDC0941, MLN1117, BYL719 (apitrest), BKM120 (bupirise), CYT387, GLPG0634, baratinib, letatinib, molatinib, panatinib, lu Suoti, TG101348, crizotinib, tivantinib, AMG337, cabatinib, furatinib, onanital, NVP-AEW541, dasatinib, ponatinib, secatinib, bosutinib, trimatinib, sematinib, tematinib cobratinib, PD0325901, RO5126766, axitinib, bevacizumab, bosutinib, cetuximab, fotamtinib, imatinib, lapatinib, lenvatinib, ibrutinib, nilotinib, panitumumab, pazopanib, piplitanib, ranibizumab, lu Suoti, sorafenib, sunitinib, SU6656, trastuzumab, tofacitinib, vandetanib, vitamin Mo Feini, irinotecan, paclitaxel, docetaxel, rapamycin, or MLN0128.

B cell receptor signaling antagonists (e.g., bruton's Tyrosine Kinase (BTK) inhibitors): ibrutinib.

Bromodomain inhibitors. Bromodomain inhibitors inhibit at least one bromodomain protein, such as Brd2, brd3, brd4, and/or BrdT, e.g., brd4. In some of these embodiments, the bromodomain inhibitor is JQ-1 (Nature, 2010, 12, 23; 468 (7327): 1067-73), BI2536 (ACS Chem. Biol [ ACS chemical biology ]2014, 5 months 16 days; 1160-71; boringer Ghan corporation (Boehringer Ingelheim)), TG101209 (ACS Chem. Biol. [ ACS Chem. Biol. ]2014, month 16, 9 (5): 1160-71), OTX015 (mol. Cancer therapeutic agent) 2013, month 11, 12, C244; oncoethix corporation), IBET762 (J Med Chem. [ J. Pharmaceutical Chem. ]2013Oct 10;56 (19): 7498-500), gaussin Shike corporation (GlaxoSmithKline)), IBET151 (Bioorg. Med. Chem. Lett. [ biological organic and pharmaceutical Chem communications ]2012, month 15, 22 (8): 2968-72, gauss Ghan corporation), I-1 (J. Chem.) [ pharmaceutical chemical journal ] 11, month 26, 55 (22 9831-7): cand 2013Oct 10;56 (19): 7498-500), and BI 35 (35, and 37) are in the pharmaceutical world 35, or the group of the pharmaceutical structures of the CPI, 35, the group of which are carried out by the company, such as the PYREGINSHINQI, PY 1 (GlaxoSmith Kline) or the PY 1, PYREG 35, PYREG.CHINGY. CHINGY. 5, PY. Kline, [ 13, PY. 5, 35, and YX. PY. GY. 5, PY. 5, and XYX. GY. 5, and, GY. Stands, and, GY. 5, and, GY. Stands, and, GY. 11, and, C.Y. Stands, and, C, and, 35, and, 35, among, and, among, and, among, among,.

Histone Deacetylase (HDAC) inhibitors. HDAC inhibitors inhibit at least one HDAC protein. HDAC proteins can be divided into classes based on homology to yeast HDAC proteins, wherein class I consists of HDAC1, HDAC2, HDAC3 and HDAC 8; class IIa consists of HDAC4, HDAC5, HDAC7 and HDAC 9; class IIb consists of HDAC6 and HDAC 10; and class IV consists of HDAC 11. In some of these embodiments, the HDAC inhibitor is trichostatin a, vorinostat (proc.Natl. Acad. Sci.U.S. A. [ Proc. Natl. Acad. Sci. U.S. A. ]1998, 3 months, 17 days; 95 (6) 3003-7), ji Weisi he, ibesstat (mol. Cancer therapist, 5 months 2006; 5 (5): 1309-17), belinostat (mol. Cancer Ther. [ molecular cancer therapeutic agent ] 8 months 2003; 2 (8): 721-8), panobinostat (Clin. Cancer Res. [ clinical cancer research ]1 month 8, 12 (15): 4628-35), reminostat (Clin. Cancer Res. [ clinical cancer research ]1 month 10, 19 (19): 5494-504), quiniuostat (Clin. Cancer Res. [ clinical cancer research ]1 month 8, 19 (15): 4262-72), depsipeptide (Blood. [ Blood ]2001 month 11 months 1, 98 (9): 2865-8), enronostat (Proc. Natl. Acad. Sci. U.S.A. [ national institute ] 13 month 13, 96 (8) 1999), biotechnology (Mo Xisi.35:35, and Biol. 35:35:35:20 (35:35.69). For example, in some embodiments, the HDAC inhibitor is panobinostat, vorinostat, MS275, belinostat, or LBH589. In some embodiments, the HDAC inhibitor is panobinostat or SAHA.

Epidermal Growth Factor Receptor (EGFR) inhibitors: erlotinib hydrochlorideOritinib, lapatinib, lenatinib, vandanib and gefitinib +.>The combination of a compound as described herein with an EGFR inhibitor and/or an EGFR antibody may be useful, for example, in the treatment of cancers associated with EGFR dysregulation, such as non-small cell lung cancer (NSCLC), pancreatic cancer, breast cancer, and colon cancer. EGFR may be deregulated, for example, due to activating mutations in exons 18, 19, 20 or 21. In particular embodiments, the EGFR inhibitor is erlotinib or octtinib. In particular embodiments, a combination of a compound as described herein and an EGFR inhibitor is used for treatmentEGFR mutated NSCLC. In particular embodiments, the combination of a compound as described herein with an EGFR inhibitor is used to treat EGFR inhibitor resistant cancers, and the compound as described herein sensitizes the cancer to EGFR inhibitors.

EGFR antibody: cetuximabXitumumab and panitumumab resistant.

MTAP inhibitors: (3R, 4S) -1- ((4-amino-5H-pyrrolo [3,2-d ] pyrimidin-7-yl) methyl) -4- ((methylsulfanyl) methyl) pyrrolidin-3-ol (MT-DADMe-immunomycin-A, CAS 653592-04-2).

Methylthioadenosine: ((2R, 3R,4S, 5S) -2- (6-amino-9H-purin-9-yl) -5- ((methylthio) methyl) tetrahydrofuran-3, 4-diol, CAS 2457-80-9).

MET inhibitors: carmattinib (INC 280, CAS 1029712-80-8).

Platelet Derived Growth Factor (PDGF) receptor inhibitors: imatinibLi Nifa Ni (N- [4- (3-amino-1H-indazol-4-yl) phenyl)]-N' - (2-fluoro-5-methylphenyl) urea, also known as ABT 869, available from genetec company (Genentech); sunitinib malate->Quinizarinib (AC 220, CAS 950769-58-1); pazopanib->Axitinib->Sorafenib->Nidaminib (BIBF 1120, CAS 928326-83-4); tiratinib (BAY 57-9352, CAS 332012-40-5); varanib dihydrochloride (PTK 787, CAS 212141-51-0);motif Sha Ni diphosphate (AMG 706, CAS 857876-30-3, N- (2, 3-dihydro-3, 3-dimethyl-1H-indol-6-yl) -2- [ (4-pyridylmethyl) amino)]3-Pyridinecarboxamide, described in PCT publication number WO 02/066470).

Phosphoinositide 3-kinase (PI 3K) inhibitors: 4- [2- (1H-indazol-4-yl) -6- [ [4- (methylsulfonyl) piperazin-1-yl ]]Methyl group]Thieno [3,2-d]Pyrimidin-4-yl]Morpholine (also known as GDC 0941 and described in PCT publication Nos. WO 09/036082 and WO 09/055730); 4- (trifluoromethyl) -5- (2, 6-dimorpholinopyrimidin-4-yl) pyridin-2-amine (also known as BKM120 or NVP-BKM120 and described in PCT publication No. WO 2007/084786); aprilsedge (BYL 719): (5Z) -5- [ [4- (4-pyridinyl) -6-quinolinyl ] ]Methylene group]-2, 4-thiazolidinedione (GSK 1059615, CAS 958852-01-2); 5- [ 8-methyl-9- (1-methylethyl) -2- (4-morpholinyl) -9H-purin-6-yl]-2-pyrimidinamine (VS-5584, CAS 1246560-33-7) and everolimus

Cyclin Dependent Kinase (CDK) inhibitors: rabociclib (LEE 011, CAS 1211441-98-3); aloesin a (aloisine a); alvocidb (also known as fravidipine or HMR-1275,2- (2-chlorophenyl) -5, 7-dihydroxy-8- [ (3 s,4 r) -3-hydroxy-1-methyl-4-piperidinyl ] -4-chromene and is described in U.S. patent No. 5,621,002); crizotinib (PF-02341066,CAS 877399-52-5); 2- (2-chlorophenyl) -5, 7-dihydroxy-8- [ (2R, 3S) -2- (hydroxymethyl) -1-methyl-3-pyrrolidinyl ] -4H-1-benzopyran-4-one, hydrochloride (P276-00, CAS 920113-03-7); 1-methyl-5- [ [2- [5- (trifluoromethyl) -1H-imidazol-2-yl ] -4-pyridinyl ] oxy ] -N- [4- (trifluoromethyl) phenyl ] -1H-benzimidazol-2-amine (RAF 265, CAS 927880-90-8); indianside Su Lan (E7070); luo Kewei (CYC 202); 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2,3-d ] pyrimidin-7-one hydrochloride (PD 0332991); dinaciclib (SCH 727965); n- [5- [ [ (5-tert-butyloxazol-2-yl) methyl ] thio ] thiazol-2-yl ] piperidine-4-carboxamide (BMS 387032,CAS 345627-80-7); 4- [ [ 9-chloro-7- (2, 6-difluorophenyl) -5H-pyrimido [5,4-d ] [2] benzazepin-2-yl ] amino ] -benzoic acid (MLN 8054, CAS 869363-13-3); 5- [3- (4, 6-difluoro-1H-benzoimidazol-2-yl) -1H-indazol-5-yl ] -N-ethyl-4-methyl-3-pyridinemethylamine (AG-024322,CAS 837364-57-5); 4- (2, 6-dichlorobenzoylamino) -1H-pyrazole-3-carboxylic acid N- (piperidin-4-yl) amide (AT 7519, CAS 844442-38-2); 4- [ 2-methyl-1- (1-methylethyl) -1H-imidazol-5-yl ] -N- [4- (methylsulfonyl) phenyl ] -2-pyrimidinamine (AZD 5438, CAS 602306-29-6); palbociclib (PD-0332991); (2R, 3R) -3- [ [2- [ [3- [ [ S (R) ] -S-cyclopropylsulfonyl imino ] -phenyl ] amino ] -5- (trifluoromethyl) -4-pyrimidinyl ] oxy ] -2-butanol (BAY 10000394).

p53-MDM2 inhibitors: (S) -1- (4-chloro-phenyl) -7-isopropoxy-6-methoxy-2- (4- { methyl- [4- (4-methyl-3-oxo-piperazin-1-yl) -trans-cyclohexylmethyl ] -amino } -phenyl) -1, 4-dihydro-2H-isoquinolin-3-one, (S) -5- (5-chloro-1-methyl-2-oxo-1, 2-dihydro-pyridin-3-yl) -6- (4-chloro-phenyl) -2- (2, 4-dimethoxy-pyrimidin-5-yl) -1-isopropyl-5, 6-dihydro-1H-pyrrolo [3,4-d ] imidazol-4-one, [ (4S, 5R) -2- (4-tert-butyl-2-ethoxyphenyl) -4, 5-bis (4-chlorophenyl) -4, 5-dimethylimidazol-1-yl ] - [4- (3-methanesulfonylpropyl) piperazin-1-yl ] methanone (RG 7112), 4- [ [ (2 r,3S,4r, 5S) -3- (3-chloro-2-fluorophenyl) -4- (4-chloro-2-fluorophenyl) -4-cyano-5- (2, 2-dimethylpropyl) pyrrolidine-2-carbonyl ] amino ] -3-methoxybenzoic acid (RG 7388), SAR299155, 2- ((3 r,5r, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1- ((S) -1- (isopropylsulfonyl) -3-methylbutan-2-yl) -3-methyl-2-oxopiperidin-3-yl) acetic acid (AMG 232), { (3 r,5r, 6S) -5- (3-chlorophenyl) -6- (4-chlorophenyl) -1- [ (2S, 3S) -2-hydroxy-3-pentyl ] -3-methyl-2-oxo-3-piperidinyl } acetic acid (AM-8553), (±) -4- [4, 5-bis (4-chlorophenyl) -2- (2-isopropoxy-4-methoxy-phenyl) -4, 5-dihydro-imidazol-3-yl) acetic acid (AMG 232), { (3 r,5r, 6S) -5- (3-chlorophenyl) -1- [ (2S, 3-hydroxy-3-pentyl ] -3-methyl-3-piperidinyl } acetic acid (AM-8553), (±4-4-bis (4-chlorophenyl) -4-methoxy-phenyl) -4-methoxy-4-2-carbonyl ] -1-carbonyl } -1-piperazin 2-methyl-7- [ phenyl (phenylamino) methyl ] -8-hydroxyquinoline (NSC 66811), 1-N- [2- (1H-indol-3-yl) ethyl ] -4-N-pyridin-4-ylbenzene-1, 4-diamine (JNJ-26854165), 4- [4, 5-bis (3, 4-chlorophenyl) -2- (2-isopropoxy-4-methoxy-phenyl) -4, 5-dihydro-imidazole-1-carboxy ] -piperazin-2-one (Caylin-1), 4- [4, 5-bis (4-trifluoromethyl-phenyl) -2- (2-isopropoxy-4-methoxy-phenyl) -4, 5-dihydro-imidazol-1-carboxy ] -piperazin-2-one (Caylin-2), 5- [ [ 3-dimethylamino) propyl ] amino ] -3, 10-dimethylpyrimidinyl [4,5-b ] quinoline-2, 4 (3H, 10H) -dione dihydrochloride (HLI 373), and trans-4-iodo-4' -boron-c-insulator-072 (SC 204072).

Mitogen activated protein kinase (MEK) inhibitors: XL-518 (also known as GDC-0973, CAS number 1029872-29-4, available from ACC corporation (ACC Corp.)); sematinib (5- [ (4-bromo-2-chlorophenyl) amino ] -4-fluoro-N- (2-hydroxyethoxy) -1-methyl-1H-benzimidazole-6-carboxamide, also known as AZD6244 or ARRY 142886, described in PCT publication No. WO 2003/077914); 2- [ (2-chloro-4-iodophenyl) amino ] -N- (cyclopropylmethoxy) -3, 4-difluoro-benzamide (also known as CI-1040 or PD184352 and described in PCT publication No. WO 2000/035436); n- [ (2R) -2, 3-dihydroxypropoxy ] -3, 4-difluoro-2- [ (2-fluoro-4-iodophenyl) amino ] -benzamide (also known as PD0325901 and described in PCT publication No. WO 2002/006213); 2, 3-bis [ amino [ (2-aminophenyl) thio ] methylene ] -succinonitrile (also known as U0126 and described in U.S. patent No. 2,779,780); n- [3, 4-difluoro-2- [ (2-fluoro-4-iodophenyl) amino ] -6-methoxyphenyl ] -1- [ (2R) -2, 3-dihydroxypropyl ] -cyclopropanesulfonamide (also known as RDEA119 or BAY869766 and described in PCT publication No. WO 2007/014011); (3 s,4r,5z,8s,9s,11 e) -14- (ethylamino) -8,9,16-trihydroxy-3, 4-dimethyl-3, 4,9; 19-tetrahydro-1H-2-benzoxazolecane-1, 7 (8H) -dione (also known as E6201 and described in PCT publication No. WO 2003/076424); 2 '-amino-3' -methoxyflavone (also known as PD98059, available from Biaffin GmbH & co., KG, germany); (R) -3- (2, 3-dihydroxypropyl) -6-fluoro-5- (2-fluoro-4-iodophenylamino) -8-methylpyrido [2,3-d ] pyrimidine-4, 7 (3 h,8 h) -dione (TAK-733, cas 1035555-63-5); piomaseti (AS-703026, CAS 1204531-26-9); tramatinib dimethyl sulfoxide (GSK-1120212, CAS 1204531-25-80); 2- (2-fluoro-4-iodophenylamino) -N- (2-hydroxyethoxy) -1, 5-dimethyl-6-oxo-1, 6-dihydropyridine-3-carboxamide (AZD 8330); 3, 4-difluoro-2- [ (2-fluoro-4-iodophenyl) amino ] -N- (2-hydroxyethoxy) -5- [ (3-oxo- [1,2] oxazinyl-2-yl) methyl ] benzamide (CH 4987555 or Ro 4987555); 5- [ (4-bromo-2-fluorophenyl) amino ] -4-fluoro-N- (2-hydroxyethoxy) -1-methyl-1H-benzimidazole-6-carboxamide (MEK 162).

B-RAF inhibitors: rigefenib(BAY 73-4506, cas 755037-03-7); FIG. Vizani (AV 951, CAS 475108-18-0); vitamin Mo Feini%PLX-4032, CAS 918504-65-1); kang Naifei Ni (also known as LGX 818); 1-methyl-5- [ [2- [5- (trifluoromethyl) -1H-imidazol-2-yl]-4-pyridinyl]Oxy group]-N- [4- (trifluoromethyl) phenyl-1H-benzoimidazol-2-amine (RAF 265, CAS 927880-90-8); 5- [1- (2-hydroxyethyl) -3- (pyridin-4-yl) -1H-pyrazol-4-yl]-2, 3-indan-1-one oxime (GDC-0879, cas 905281-76-7); 5- [2- [4- [2- (dimethylamino) ethoxy ]]Phenyl group]-5- (4-pyridinyl) -1H-imidazol-4-yl]-2, 3-dihydro-1H-inden-1-one oxime (GSK 2118436 or SB 590885); (+/-) - (5- (2- (5-chloro-2-methylphenyl) -1-hydroxy-3-oxo-2, 3-dihydro-1H-isoindol-1-yl) -1H-benzimidazol-2-yl) carbamic acid methyl ester (also known as XL-281 and BMS 908662), dabrafenib>N- (3- (5-chloro-1H-pyrrolo [2, 3-b)]Pyridine-3-carbonyl) -2, 4-difluorophenyl-propane-1-sulfonamide (also known as PLX 4720).

ALK inhibitors: crizotinib

PIM kinase inhibitors:or a pharmaceutically acceptable salt thereof.

Proteasome inhibitors: bortezomibN-5-benzyloxycarbonyl-Ile-Glu (O-tert-butyl) -Ala-leucine (PSI), carfilzomib and I Sha Zuomi, maruzomib (NPI-0052), delanzomib (CEP-18770) and O-methyl-N- [ (2-methyl-5-thiazolyl) carbonyl ]-L-seryl-O-methyl-N- [ (1S) -2- [ (2R) -2-methyl-2-oxiranyl]-2-oxo-1- (phenylmethyl) ethyl]L-serinamide (ospemide, ONX-0912, PR-047) (e.g. boron)Tizomib). RNAi screening identified TNK1 as a potential modulator of proteasome inhibitor sensitivity in myeloma. Zhu et al Blood](2011) 117 (14):3847-3857. In some embodiments, a compound described herein (e.g., a compound having formula I or a sub-formula thereof, or a pharmaceutically acceptable salt of the foregoing) is administered in combination with a proteasome inhibitor described herein (e.g., bortezomib), e.g., for treating multiple myeloma.

Further non-limiting examples of therapeutic agents that may be used in combination with a compound as described herein are chemotherapeutic agents, cytotoxic agents and non-peptide small molecules, such as(imatinib mesylate), ->(bortezomib), casodex (bicalutamide), ->(gefitinib) and doxorubicin, and many chemotherapeutic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclophosphamide +.>Alkyl sulfonates such as busulfan (busulfan), imperoshu (imposulfan) and piposulfan (piposulfan); aziridines, such as benzodopa (benzodopa), carboquinone (carboquone), mideperide (metadopa), and udelpirane (uredopa); ethyleneimine and methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphamide, and trimethylol melamine; nitrogen mustards such as chlorambucil, napthalen mustards, cholesteryl phosphoramide (cholosphamide), estramustine, ifosfamide, mechlorethamine hydrochloride, melphalan, neonitrogen mustard, mechlorethamine, prednisone, trofosamine, uracil mustard; nitrosoureas such as carmustine, chlorourea, fotemustine, lomustine, nimustine, and ramustine; antibiotics Such as aclacinomycin (aclacinomycin), actinomycin, amphotericin (authamycin), diazoserine, bleomycin, actinomycin C (cactinomycin), calicheamicin (calicheamicin), cartubicin (carbicin), carminomycin, carcinophilin, and the like>Chromomycin, dactinomycin, daunorubicin, ditobacin (detorubicin), 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, doxycycline, mitomycin, mycophenolic acid, norgamycin, olivomycin, pelomycin, pofeomycin (potfiromycin), puromycin, tri-iron doxorubicin, rodubicin, streptozocin, streptozotocin, tuberculin, ubenimex, jiostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as, for example, dimethyl folic acid (denopterin), methotrexate, ptertrexate (pteroprerin), trimeoxate (trimetrexate); purine analogs such as fludarabine, 6-mercaptopurine, thiopurine (thiamiprine), thioguanine; pyrimidine analogs such as ambcitabine, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, fluorouridine; androgens, such as carbosterone, drotasone propionate, cyclothioandrostane, emasculan, and testosterone; anti-epinephrine such as aminoglutethimide (amitothecide), mitotane (mitotane), trilostane (trilostane); folic acid supplements, such as folinic acid; acetylglucuronolactone (aceglatone); aldehyde phosphoramidate glycoside (aldophosphamide glycoside); aminolevulinic acid (aminolevulinic acid); amsacrine; bai-vertical Buxi; bisantrene (bisantrene); edatraxate (edatraxate); ground phosphoramide (defofame); colchicine (demecolcine); deaquinone; ornithine difluoride; ammonium (elliptinium acetate) according to Li Yi; etodolac (etoglucid); gallium nitrate; hydroxyurea; mushroom polysaccharide (lentinan); lonidamine; mitoguazone (mitoguazone); mitoxantrone; mo Pai dar alcohol; diamine nitroacridine; prastatin; chlorambucil (phenamet); pirarubicin (pirarubicin); podophylloic acid (podophyllinic acid); 2-Ethyl acyl Hydrazine; procarbazine (procarbazine); psk.rtm.; raschig (razoxane); a sirzopyran; germanium spiroamine (spirogmanium); tenuazonic acid (tenuazonic acid); triiminoquinone (triaziquone); 2,2',2 "-trichlorotriethylamine; uratam (urethan); vindesine; dacarbazine; mannitol; dibromomannitol (mitobronitol); dibromodulcitol (mitolactol); pipobromine; lid tosine (tetracooxin); arabinoside (arabinoside) ("Ara-C"); cyclophosphamide; thiotepa; taxanes, such as paclitaxel (taxol (tm), bai-time metastine nobiletin oncology company (Bristol-Myers Squibb Oncology, princeton, n.j.)), docetaxel (taxotere (tm), roner-planck company (Rhone-Poulenc Rorer, antonny, france), and cabazitaxel (JEVTANA, sanofi Genzyme); retinoic acid; espimira (esperamicins); capecitabine (capecitabine); and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

Many chemotherapeutic agents are currently known in the art and may be used in combination with the compounds as described herein. In some embodiments, the chemotherapeutic agent is selected from the group consisting of mitotic inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifiers, anti-hormones, angiogenesis inhibitors, and anti-androgens.

Further non-limiting examples of chemotherapeutic agents for use in combination with a compound as described herein (e.g., in combination therapy, in pharmaceutical combination) include capecitabineN4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin +.>Cisplatin->Cladribine>Cyclophosphamide (/ -s)>Or->) Cytarabine, cytosine cytarabine +.>Cytarabine liposome injectionDacarbazine->Doxorubicin hydrochlorideFludarabine phosphate->5-fluorouracilGemcitabine (difluoro deoxycytidine), irinotecan +.>L-asparaginase->6-mercaptopurine->Methotrexate>Pennisetum, 6-thioguanine, thiotepa and topotecan hydrochloride for injection>

Further non-limiting examples of common anticancer drugs include ABVD, AVICINE, aba Fu Shan, acridine carboxamide, adalimumab, 17-N-allylamino-17-desmethoxygeldanamycin, alpha latin (Alpharadin), avoxidine, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone, amantadine, anthraquinone, anti-CD 22 immunotoxin, antitumor drug, antitumor herbal, apazinone, altimod, azathioprine, belotecan, bendamustine, BIBW 2992, biridar (biricarb), cloth Luo Sida mycin (brostabillin), bryostatin, butylsulfanilide sulfonylimine, CBV (chemotherapy), calyxin (calyclin), cell cycle non-specific antitumor agent, dichloroacetic acid, discodermolide (Discodermolide) elsamigrin, epothilone, eribulin (Eribulin), everolimus, everbutin (Exatecan), everolimus, evergreen (exoecan), everolimus (exisulin), ferrored (Ferruginol), furazolidone (Forodesine), fosfestokes (fosfesfestoll), ICE chemotherapy regimen, IT-101, imacortison (imaxon), imiquimod, indolocarbazole, ilofofene, lanigidad (lanquidar), ralostazol (Larotaxel), lenalidomide, methione, lurtolterone, maphosphamide (mafosfomide), mitozolamide, naftid, nedaplatin, oxapanil, ortataxel (ortalaxel), PAC-1, pawtawave), picoline (pirone), proteasome inhibitors, butterfly mycin (Rebeccamycin), requimod, lubitecan (Rubitecan), SN-38, salidroamide A (Salinosporamide A), sapatabine (sapatabine), stanford V (Stanford V), swainsonine (Swainsonine), talaporfin (Talaporfin), tarquada (tarquidar), tegafur-uracil, temoda (Temodar), tesetaxel (Tese) taxel), triplatinum tetranitrate, tris (2-chloroethyl) amine, qu Shaxi tabine, uramestin, vadimezan (Vadimezan), vinflunine, ZD6126 or zol Su Kui dar (Zosuquidar).

Also included as suitable chemotherapeutics are anti-hormonal agents, such as antiestrogens, which act to modulate or inhibit hormonal effects on tumors, including, for example, tamoxifen (nolvadex), raloxifene (raloxifene), aromatase-inhibiting 4 (5) -imidazole, 4-hydroxy tamoxifen, trawoxifene, raloxifene (keoxifene), LY 117018, onapristone, and toremifene (farston); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; vinblastine; noon An Tuo (novantrone); teniposide; daunomycin; aminopterin (aminopterin); hilded (xeloda); ibandronate (ibandronate); camptothecin-11 (CPT-11); topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO).

Non-limiting examples of therapeutic agents that can be used in combination with a compound as described herein are mTOR inhibitors. Exemplary mTOR inhibitors include, for example, temsirolimus; li Dafu (formal name of Delrolimus, (1R, 2R, 4S) -4- [ (2R) -2[ (1R, 9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S, 35R) -1, 18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentoxy-11, 36-dioxa-4-azatricyclo [ 30.3.1.0) ^4,9 ]Trihexadecan-16,24,26,28-tetraen-12-yl]Propyl group]-2-methoxycyclohexyldimethylphosphinate, also known as AP23573 and MK8669, and described in PCT publication No. WO 03/064383; everolimus @Or RAD 001); rapamycin (AY 22989,)>) The method comprises the steps of carrying out a first treatment on the surface of the Western Ma Mode (CAS 164301-51-3); temsirolimus, (5- {2, 4-bis [ (3S) -3-methylmorpholin-4-yl)]Pyrido [2,3-d ]]Pyrimidin-7-yl } -2-methoxyphenyl) methanol (AZD 8055); 2-amino-8- [ trans-4- (2-hydroxyethoxy) cyclohexyl]-6- (6-methoxy-3-pyridinyl) -4-methyl-pyrido [2,3-d]Pyrimidin-7 (8H) -one (PF 04691502, CAS 1013101-36-4); n ² - [1, 4-dioxo-4- [ [4- (4-oxo-8-phenyl-4H-1-benzopyran-2-yl) morpholinium-4-yl ] ]Methoxy group]Butyl group]L-arginyl glycyl-L-alpha-aspartyl L-serine-inner salt (SEQ ID NO: 1482) (SF 1126, CAS 936487-67-1) and XL765.

In certain other embodiments, there is provided a method for treating cancer, the method comprising administering to a subject in need thereof an effective amount of a compound as described herein and a CDK inhibitor.

In embodiments, the CDK inhibitor is a CDK2, CDK4, CDK6, CDK7, CDK8, CDK9, CDK10, and/or CDK11 inhibitor. In some embodiments, the CDK inhibitor is a CDK7, CDK9 inhibitor, or both. In some embodiments, the CDK inhibitor is denafil (ACS Med. Chem. Lett. [ ACS pharmaceutical chemistry communication ] month 5, 17, 1 (5): 204-8;Mol.Cancer Ther. [ molecular cancer therapeutic agent ] month 8, 9 (8): 2344-53; mercury, sharp and Dohme)), AT7519 (J. Med. Chem. ]2008, month 8, 28, 51 (16): 4986-99; altaix pharmaceutical Co (Astex Pharmaceutical)) or Pabosinib (J. Med. Chem. ]2005, month 4, 7; 48 (7): 2388-406; condui (Pfizer)). In certain embodiments, the CDK inhibitor is a CDK9 inhibitor, such as avoxidine. The avoxidine may be administered as the free base, as a pharmaceutically acceptable salt, or as a prodrug. In certain embodiments, the CDK9 inhibitor is avoxidine. In other embodiments, the CDK9 inhibitor is a pharmaceutically acceptable salt of avocado. In other embodiments, the CDK9 inhibitor is a prodrug of avoxidine. Prodrugs of avoxidine include those disclosed in WO 2016/187316, the entire disclosure of which is hereby incorporated by reference in its entirety.

In one embodiment, a combination of a compound as described herein and an ATR inhibitor (e.g., AZD6738 or VX-970) is administered to a subject in need thereof. The administration may be prior to, concurrent with, or subsequent to administration of the ATR inhibitor. In a particular embodiment, a compound as described herein is administered to a subject in need thereof in combination with an ATR inhibitor (e.g., AZD6738 or VX-970) to treat non-small cell lung cancer. In related particular embodiments, a pharmaceutically acceptable salt of a compound as described herein is administered to a subject in need thereof in combination with an ATR inhibitor (e.g., AZD6738 or VX-970) to treat non-small cell lung cancer. In some of the foregoing embodiments, the salt is a tartrate salt. In some of the foregoing embodiments, the ATR inhibitor is AZD6738. In some of the foregoing embodiments, the ATR inhibitor is VX-970. In some embodiments, the salt is tartrate and the ATR inhibitor is AZD6738. In some embodiments, the salt is tartrate and the ATR inhibitor is VX-970. In some of the foregoing embodiments, the ATR inhibitor is a combination of AZD6738 and VX-970.

In some of the foregoing embodiments, the non-small cell lung cancer comprises TCGA lung adenocarcinoma, one or more LUAD tumors, TCGA lung squamous cell carcinoma, one or more luc tumors, one or more mdaccpro spect tumors, one or more mdaccbattle 1 tumors, one or more BATTLE2 tumors, or a combination thereof. In some embodiments, the non-small cell lung cancer comprises a tcgaluoad tumor, e.g., an ALK translocation-enriched tumor. In some embodiments, the non-small cell lung cancer comprises a tcgaluoad tumor, e.g., a tumor comprising one or more EGFR mutations.

In one embodiment, a compound as described herein is administered to a subject in need thereof, thereby sensitizing the subject to administration of an ATR inhibitor (e.g., AZD6738 or VX-970). In related embodiments, a pharmaceutically acceptable salt of a compound as described herein is administered to a subject in need thereof, thereby rendering the subject susceptible to administration of an ATR inhibitor (e.g., AZD6738 or VX-970). In a particular embodiment, a compound as described herein is administered to a subject in need thereof, thereby sensitizing the subject to administration of ATR inhibitors (e.g., AZD6738 or VX-970) to treat non-small cell lung cancer. In a related particular embodiment, a pharmaceutically acceptable salt of a compound as described herein is administered to a subject in need thereof, thereby sensitizing the subject to administration of ATR inhibitors (e.g., AZD6738 or VX-970) to treat non-small cell lung cancer. In some of the foregoing embodiments, the salt is a tartrate salt. In some of the foregoing embodiments, the ATR inhibitor is AZD6738. In some of the foregoing embodiments, the ATR inhibitor is VX-970. In some embodiments, the salt is tartrate and the ATR inhibitor is AZD6738. In some embodiments, the salt is tartrate and the ATR inhibitor is VX-970. In some of the foregoing embodiments, the ATR inhibitor is a combination of AZD6738 and VX-970.

Some patients may experience allergic reactions to a compound as described herein and/or one or more other therapeutic agents (e.g., one or more anticancer agents) during or after administration. Thus, an antiallergic agent may be administered in combination with a compound as described herein and/or one or more other therapeutic agents (e.g., one or more anticancer agents) to minimize the risk of an allergic reaction. Suitable antiallergic agents include corticosteroids (Knutson, S.et al, PLoS One [ public science library: complex]DOI 10.1371/journ.fine.011840 (2014)), such as dexamethasone (e.g.,) Beclomethasone (e.g.)>) Hydrocortisone (also known as cortisone, hydrocortisone sodium succinate, hydrocortisone sodium phosphate, trade name +.>Hydrocortisone phosphate,/>HYDROCORT/>And->Sold under the trade name prednisoloneAnd->Sold), prednisone (under the trade name +.>LIQUID/> And->Sold), methylprednisolone (also known as 6-methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, under the trade nameAndsales); antihistamines, such as diphenhydramine (e.g., +.>) Hydroxyzine and cyproheptadine; and bronchodilators, such as beta-adrenergic receptor agonists, albuterol (e.g., +. >) And terbutaline->

Some patients may be administered a compound described herein and/or one or more other treatmentsNausea is experienced during and after an agent (e.g., one or more anticancer agents). Thus, an antiemetic may be used in combination with a compound as described herein and/or one or more other therapeutic agents (e.g., one or more anticancer agents) to prevent nausea (upper stomach) and vomiting. Suitable antiemetics include aprepitantOndansetron->Granisetron hydrochlorideLorazepam->Dexamethasone->ProchlorlazineCasoprotein (>And->) And combinations thereof.

Drugs that alleviate the pain experienced are often prescribed during the treatment period to make the patient more comfortable. Common over-the-counter analgesics (e.g) May also be used in combination with a compound described herein and/or one or more other therapeutic agents (e.g., one or more anticancer agents). Opioid analgesics (e.g. hydrocodone/paracetamol or hydrocodone/acetaminophen (e.g.)>) Morphine (e.g.)>Or->) Oxycodone (e.g.)>Or->) Oxymorphone hydrochloride->And fentanyl (e.g.,) For moderate or severe pain, and may be used in combination with a compound described herein and/or one or more other therapeutic agents (e.g., one or more anticancer agents).

Particularly interesting immunomodulators (e.g., immunooncology agents) for use in combination with the compounds described herein include: alfupotato beads (available fromObtaining; polyethylene glycol feigiosteine->Lenalidomide (CC-5013, < >>) The method comprises the steps of carrying out a first treatment on the surface of the Thalidomide->Acipimide (actimid, CC 4047); and IRX-2 (a mixture of human cytokines including interleukin 1, interleukin 2 and interferon gamma, CAS 9)51209-71-5, available from IRX therapeutic company (IRX Therapeutics).

A chimeric antigen receptor T cell (CAR-T) therapy of particular interest for use in combination with a compound described herein includes: texarence (Novartis), alpranluki (axicabtagene ciloleucel) (Kite) and tozumab (Arizumab; roche).

Immune checkpoint inhibitors of interest for use in combination with the compounds described herein include: PD-1 inhibitors, e.g. pembrolizumab (also known as lamb Luo Lizhu mab, MK-3475, MK03475, SCH-900475 or) And other anti-PD-1 antibodies (e.g., hamid, O.et al (2013) New England Journal of Medicine, J. New England medical science]369 (2) 134-44, US 8,354,509 and WO 2009/114335, which are incorporated by reference in their entirety), nawuzumab (also known as MDX-1106, MDX-1106-04, ONO-4538, BMS-936558 or ) And other anti-PD-1 antibodies (as disclosed in U.S. Pat. No. 8,008,449 and WO 2006/121168, which are incorporated by reference in their entirety), cimipne Li Shan anti ∈ ->Stadalizumab (PDR 001), pidazumab (Curetech), MEDI0680 (Medimune), cimetidine Li Shan (REGN 2810), duotalizumab (TSR-042), PF-06801591 (Constipation), xindi Li Shan, terep Li Shan, tiarelizumab (BGB-A317), caririlizumab (INCSHR 1210, SHR-1210), AMP-224 (Amplimune), CBT-501 (CBT pharmaceutical (CBT Pharmaceuticals)), CBT-502 (CBT pharmaceutical), JS001 (Jun solid biosystems (Junshi Biosciences)), IBI308 (Xindabiosystems (Innovent Biologics)), INCSHR1210 (Saint Co (Incyte)) (also known as SHR-1210 (Hengrui Medicine)), and INCSHR1210 (ShengJib, BAR-state)Company (Beigene)), BGB-108 (BAT-I306 (Bio-therapeutic Solutions), GLS-010 (yu-heng pharmaceutical company (Gloria Pharmaceuticals); pharmaceutical biology, inc. (WuXi Biologics)), AK103, AK104, AK105 (Ming's Biopharma, akesio Biopharma); hangzhou Hansi Biol corporation (Hangzhou Hansi Biologics); hanzhong biosystems (Hanzhong Biologics)), LZM009 (Livzon), HLX-10 (Henlius Biotech), MEDI0680 (immunology Co., ltd.), PDF001 (Norhua Co., ltd.), PF-06801591 (pyro Co., ltd.), pittuzumab (CureTech Co., ltd.), also known as CT-011, and other anti-PD-1 antibodies (e.g., rosenblatt, J. Et al (2011) JImmunotherapy journal of immunotherapy) ]34 (5) 409-18, US 7,695,715, US 7,332,582 and US 8,686,119, which are incorporated by reference in their entirety), REGN2810 (Regenron), TSR-042 (Tesaro) (also known as ANB 011), or CS1003 (ken pharmaceutical Co (CStone Pharmaceuticals)). MEDI0680 (immunology corporation), also known as AMP-514.MEDI0680 and other anti-PD-1 antibodies are disclosed in US 9,205,148 and WO 2012/145493, which are incorporated by reference in their entirety. Additional known anti-PD-1 antibody molecules include, for example, those described in WO 2015/112800, WO 2016/092419, WO 2015/085847, WO 2014/179664, WO 2014/194302, WO 2014/209404, WO 2015/200119, US 8,735,553, US 7,488,802, US 8,927,697, US 8,993,731 and US 9,102,727, which are incorporated by reference in their entirety. In one embodiment, the PD-1 inhibitor is an anti-PD-1 antibody molecule, such as an antibody molecule titled "Antibody Molecules to PD-1and Uses Thereof[PD-1" published on 5,7, 30 and uses thereof]"U.S. Pat. No. 5/0210769, which is incorporated by reference in its entirety. In one embodiment, the anti-PD-1 antibody molecule comprises the CDRs, variable regions, heavy chains and/or light chains of BAP049-Clone-E or BAP049-Clone-B disclosed in US 2015/0210769. The antibody molecules described herein may be prepared by vectors, host cells and methods described in US2015/0210769, which is incorporated by reference in its entirety. In one embodiment, the PD-1 inhibitor is an inhibitor of PD-1 signaling Peptides of the way, for example as described in US 8,907,053, which is incorporated by reference in its entirety. In one embodiment, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In one embodiment, the PD-1 inhibitor is AMP-224 (B7-DCIg (Amplimume Corp.), such as disclosed in WO 2010/027827 and WO 2011/066342, which are incorporated by reference in their entirety.

Immune checkpoint inhibitors of interest for use in combination with the compounds described herein also include: PD-L1 inhibitors, e.g. alemtuzumab (also known as MPDL3280A, RG7446, RO5541267, YW243.55.S70 or) And other anti-PD-L1 antibodies, as disclosed in US 8,217,149, which is incorporated by reference in its entirety; avermectin (/ -herba)>Also known as MSB 0010718C) and other anti-PD-L1 antibodies, as disclosed in WO 2013/079174, which is incorporated by reference in its entirety; dewaruzumab (+)>Or MEDI 4736) and other anti-PD-L1 antibodies, as disclosed in US 8,779,108, which is incorporated by reference in its entirety; FAZ053 (nowa); BMS-936559 (Bristol-Myers Squibb), bai Shi Gui Bao Co. In certain embodiments, the PD-L1 inhibitor is KN035 (Kangan JieR Biopharmaceutical Co., ltd. (Alphamab); di Corp., 3D med.; gem Gift pharmaceutical Co., ascletis Pharma), en Wo Lishan anti (Terakang pharmaceutical Co., TRACON Pharmaceuticals), BMS 936559 (Bai Meissu Guibao Co., ltd.), CS1001 (Kelvin pharmaceutical Co., ligand pharmaceutical Co., ligand Pharmaceuticals)), CX-072 (CytomX therapeutic Co., cytomX Therapeutics), FAZ053 (Nohua Co., SHR-1316 (Henry pharmaceutical Co., ltd.), TQB2450 (Zhengtian pharmaceutical Co., ltd.), STI-A1014 (megafamily) Industry (Zhaoke Pharm); litsea big pharmaceutical factory (Lee's Pharm), lonza Group (Lonza), soronto treatment company (Sorrento Therapeutics), nantWorks), LYN00102 (Lynkcell), A167 (and platinum medicine company (Harbour BioMed), colon Group (Kelun Group)), BGB-A333 (BAIJI Shenzhou Co.), MSB2311 (Michaelis Bioscience (Mabspace Biosciences)) or HLX-20 (Shanghai Fuhongham Biotechnology Co., ltd.). In one embodiment, the anti-PD-L1 antibody molecule is BMS-936559 (BASEMERIUM Guibao), also known as MDX-1105 or 12A4.BMS-936559 and other anti-PD-L1 antibodies are disclosed in US 7,943,743 and WO 2015/081158, which are incorporated by reference in their entirety. In certain embodiments, the PD-L1 inhibitor is Ke Xili mab (Feng Ze Biotech), LY3300054 or lodalimab (Gillet Lilly), GS-4224 (Giled Sciences), STI-A1015 (Liu Han ocean, yuhan, soronto treatment Co.), BCD-135 (BIOCAD), ke Xili mab (Dana-Faber cancer institute (Dana-Farber Cancer Institute), TG therapeutic company (TG Therapeutics), APL-502 (Apollomics), AK106 (Ming Biotech), MSB2311 (Legeng group (Transcenta Holding)), TG-1501 (TG therapeutic), FAZ053 (Nohua). In certain embodiments, the PD-L1 inhibitor is MT-6035 (molecular template Co (Molecular Templates)), icaritin and ZKAB001 (Dragon's group, litsea big pharmaceutical Co., lee's Pharmaceutical Holdings), soronto treatment Co., shen Nuogen pharmaceutical Co., shenogen Pharma Group), TRIDENT antibody (macrogenetics, tripod pharmaceutical Co., zai Lab), YBL-007 (Anguo pharmaceutical Co., anh-Gook Pharmaceutical), Y-biologic Co., Y-biologic), HTI-1316 (Hengrui therapeutic Co., hengrui Therapeutics), PD-L1 oncology project (We, wittman scientific institute (Weizmann Institute of Sciences)), JS003 (Shanghai Jun solid Bio Co., shanghai Junshi Biosciences), ND021 (Nama therapeutic Co., numab Therapeutics), ke pharmaceutical Co., toca 521 (Tocagen)), STT01 (STCube). In certain embodiments, the PD-L1 inhibitor is D B004 (Duote Biol Co (DotBuo)), MT-5050 (molecular template Co), KD036 (Kadmon Co (Kadmon)). In one embodiment, the PD-L1 inhibitor is an anti-PD-L1 antibody molecule. In one embodiment, the PD-L1 inhibitor is an anti-PD-L1 antibody molecule, such as an antibody molecule titled "Antibody Molecules to PD-L1 and Uses therof [ PD-1 ] published in month 21 of 2016 and Uses Thereof]"U.S. Pat. No. 2016/0108123, incorporated herein by reference in its entirety. In one embodiment, the anti-PD-L1 antibody molecule comprises the CDRs, variable regions, heavy chains and/or light chains of BAP058-Clone O or BAP058-Clone N disclosed in US 2016/0108123.

Additional known anti-PD-L1 antibodies include, for example, those described in WO 2015/181342, WO 2014/100079, WO 2016/000619, WO 2014/022758, WO 2014/055897, WO 2015/061668, WO 2013/079174, WO 2012/145493, WO 2015/112805, WO 2015/109124, WO 2015/195163, US 8,168,179, US 8,552,154, US 8,460,927, and US 9,175,082, which are incorporated by reference in their entirety.

In some embodiments, the immune checkpoint inhibitor is a cytotoxic T lymphocyte-associated modulator. In some embodiments, the immune checkpoint inhibitor targets CTLA-4, such as ipilimumab Tremellizumab, ALPN-202 (alpine immunology Co (Alpine Immune Sciences)), RP2 (Repliline Co (replimene)), BMS-986249 (BAwhile Meishi Guibao Co.), BMS-986218 (BAwhile Meishi Guibao Co.), lycofulizumab (A Ji Nusi Co (Agenus), ledebe Vickers cancer institute (Ludwig Institute for Cancer Research), uroGen pharmaceutical Co (UroGen Pharma), lei Saida biopharmaceutical Co (Recepta Biopharma)), BCD-217 (Bokang Co.), onc-392 (trim Co., tumor immunology Co (Oncomene)), IBI310 (Xinda biosystems), KN046 (Kangning JieJie biosciences), MK-1308 (Merck pharmaceutical Co (Merck)&Co)), REGN4659 (regenerator pharmaceutical Co., ltd (Regeneron Pharmaceuticals)), xmAb20717 (Xencor), xmAb22841 (Senke Co., ltd.), anti-CTLA-4 NF (Bai-Shi-Guibao Co., ltd.),MEDI5752 (AstraZeneca), AGEN1181 (A Ji Nusi), MGD019 (macroscopical Co.), ATOR-1015 (crocodile bioscience (Alligator Bioscience)), BCD-145 (Bokang), PSB205 (Sound Biologics), CS1002 (Kirschner), ADU-1604 (Aduro Biotech)), PF-06753512 (PY), bioInvent-Transgene research (Chung Jie Co (Transgene)), AGEN2041 (A Ji Nusi, lei Saida biopharmaceutical Co.), ATOR-1144 (crocodile bioscience), CTLA-4 research (Soronto treatment Co.), and the like PD-L1/CTLA-4 research project (Soronto treatment Co.), HLX13 (Shanghai Fu Han Lini Biotechnology Co., ltd.), ISA203 (Isa pharmaceutical Co., ISA Pharmaceuticals), PRS-300 series A (Pi Aili S pharmaceutical Co., pieris Pharmaceuticals)), BA3071 (Boattla Co., bioatlas)), CTLA4 cancer research project (Piaosotida pharmaceutical Co., biosortia Pharmaceuticals)), RP3 (Leprin Co., ltd.), CG0161 (Cold Genesys), APL-509 (Guangkomeibo Co., JSR Co., ltd.), AGEN2041 (Ledeb Virgi cancer institute), APC 101 (advanced proteome Co., advanced Proteome)), CTLA-4 inhibitor (advanced proteome), BA3071 (ba3071, bai-002 (baichun pharmaceutical industry (BeyondSpring Pharmaceuticals)), CTLA-4 antibody (tikronet technologies (Tikcro Technologies)), immune-oncology research project II (olypass), PBP1701 (wisconsin biopharmaceutical company (Prestige BioPharma)), DB002 (multi-t biosystems), DB003 (multi-t biosystems), OR-2299 (OncoResponse), NK044 (corning jerry biopharmaceutical company). In certain embodiments, the CTLA-4 inhibitor is ipilimumab. In other embodiments, the CTLA4 inhibitor is tremelimumab.

Immune checkpoint inhibitors of interest for use in combination with the compounds described herein also include: LAG-3 inhibitors. In some embodiments, the LAG-3 inhibitor is selected from LAG525 (North China), BMS-986016 (Beziram Mitsui) or TSR-033 (Tesalo). In one embodiment, the LAG-3 inhibitor is an anti-LAG-3 antibody molecule. In one embodiment, the LAG-3 inhibitor is an anti-LAG-3 antibody molecule, as disclosed in US2015/0259420 entitled "antibody molecule of Antibody Molecules to LAG-3and Uses Thereof[LAG-3 and uses thereof," published on month 17 of 2015, which is incorporated by reference in its entirety. In one embodiment, the anti-LAG-3 antibody molecule comprises the CDRs, variable regions, heavy chains and/or light chains of BAP050-Clone I or BAP050-Clone J disclosed in US 2015/0259420. In one embodiment, the anti-LAG-3 antibody molecule is BMS-986016 (BASEMERIZA, inc.), also known as BMS986016.BMS-986016 and other anti-LAG-3 antibodies are disclosed in WO 2015/116539 and US 9,505,839, which are incorporated by reference in their entirety. In one embodiment, the anti-LAG-3 antibody molecule is TSR-033 (Texaro Corp.). In one embodiment, the anti-LAG-3 antibody molecule is IMP731 or GSK2831781 (glazin smith corporation (GSK) and sigma biomedical corporation (Prima BioMed)). IMP731 and other anti-LAG-3 antibodies are disclosed in WO 2008/132601 and US 9,244,059, which are incorporated by reference in their entirety. In one embodiment, the anti-LAG-3 antibody molecule is IMP761 (common biomedical company). Additional known anti-LAG-3 antibodies include, for example, those described in WO 2008/132601, WO 2010/019570, WO 2014/140180, WO 2015/116539, WO 2015/200119, WO 2016/028672, US 9,244,059, US 9,505,839, which are incorporated by reference in their entirety. In one embodiment, the anti-LAG-3 inhibitor is a soluble LAG-3 protein, such as IMP321 (common biomedical company), for example as disclosed in WO 2009/044273, which is incorporated by reference in its entirety.

Immune checkpoint inhibitors of interest for use in combination with the compounds described herein also include: tim-3 inhibitors. In some embodiments, the TIM-3 inhibitor is MGB453 (Nohua Corp.) or TSR-022 (Texaro Corp.). For one embodiment, the TIM-3 inhibitor is an anti-TIM-3 antibody molecule. In one embodiment, the TIM-3 inhibitor is an anti-TIM-3 antibody molecule, as disclosed in U.S. Pat. No. 2015/0218274 entitled "antibody molecule of Antibody Molecules to TIM-3and Uses Thereof[TIM-3 and its use" published on month 8 and 6 of 2015, which is incorporated by reference in its entirety. For one embodiment, the anti-TIM-3 antibody molecule comprises the CDRs, variable regions, heavy and/or light chains of ABTIM3-hum11 or ABTIM3-hum03 disclosed in US 2015/0218274. For one embodiment, the anti-TIM-3 antibody molecule is TSR-022 (An Napu TilsBio/Tesalo). For one embodiment, the anti-TIM-3 antibody molecule comprises one or more of the CDR sequences of APE5137 or APE5121 (or collectively all CDR sequences), the heavy or light chain variable region sequences, or the heavy or light chain sequences. APE5137, APE5121 and other anti-TIM-3 antibodies are disclosed in WO 2016/161270, which is incorporated by reference in its entirety. For one embodiment, the anti-TIM-3 antibody molecule is antibody clone F38-2E2. Additional known anti-TIM-3 antibodies include, for example, those described in WO 2016/111947, WO 2016/071448, WO 2016/144803, US 8,552,156, US 8,841,418 and US 9,163,087, which are incorporated by reference in their entirety.

In order to protect normal cells from therapeutic toxicity and limit organ toxicity, cytoprotective agents (e.g., neuroprotective agents, free radical scavengers, cardioprotective agents, anthracycline extravasation neutralizing agents, nutrients, etc.) may be used as adjuvant therapy in combination with the compounds of the present disclosure. Suitable cytoprotective agents include amifostineGlutamine, dimesnaMesna->Dexrazoxane (+)>Or->) Zali Luo den->And leucovorin (also known as calcium folinate, orange-philic)Bacterial factor (citrovorum factor) and folinic acid (folinic acid)).

The compounds described herein may also be advantageously used in combination with known methods of treatment, such as administration of hormones or, in particular, radiation. The compounds described herein may be particularly useful as radiosensitizers, especially for the treatment of tumors that exhibit poor sensitivity to radiotherapy.

In some embodiments, a compound described herein and a BTK inhibitor are co-administered. In other embodiments, the compounds described herein are administered after the BTK inhibitor. In still other embodiments, the compounds described herein are administered prior to the BTK inhibitor.

In various embodiments, the BTK inhibitor is ibrutinib. In some particular embodiments, the cancer is Chronic Lymphocytic Leukemia (CLL), small Lymphocytic Lymphoma (SLL), or both. In some embodiments, the subject has received a prior treatment regimen of CLL, SLL, or both. In some embodiments, the subject is refractory following a previous treatment regimen, the subject relapses CLL, SLL, or both after responding to a previous treatment regimen, or the subject has a detectable Minimal Residual Disease (MRD).

In another embodiment, a compound as described herein is administered to a subject in need thereof in combination with a Bcl-2 inhibitor (e.g., valnemulin). The administration may be prior to, concurrent with, or subsequent to administration of the Bcl-2 inhibitor. In certain embodiments, the subject is insensitive to treatment with a Bcl-2 inhibitor, unsuitable for treatment with a Bcl-2 inhibitor, or relapsed after treatment with a Bcl-2 inhibitor. In a particular embodiment, a compound as described herein is administered to a subject in need thereof in combination with a Bcl-2 inhibitor (e.g., valnemulin) to treat leukemia (e.g., CLL, SLL, or both).

In another embodiment, a compound as described herein is administered to a subject in need thereof in combination with an immunomodulatory agent (e.g., CAR-T therapy, which is an immune checkpoint inhibitor, such as a PD-1, PD-L1, or CTLA4 inhibitor). In some embodiments, the immunomodulatory agent is CAR-T therapy, including any of the CAR-T therapies described herein. In some embodiments, the immune modulator is an immune checkpoint inhibitor (e.g., a PD-1, PD-L1, or CTLA4 inhibitor), including any of the immune checkpoint inhibitors described herein. Without wishing to be bound by any particular theory, it is believed that the exemplary compounds may improve vascular perfusion to the tumor and thereby enhance drug delivery to the tumor. Enhanced drug delivery is expected to in turn enhance the efficacy of drugs, such as immunomodulators (e.g., immunooncology agents), including any of the immunomodulators described herein, for example, by making the tumor more susceptible to circulating drugs.

In another embodiment, a compound as described herein is administered to a subject in need thereof in combination with an immune checkpoint inhibitor (e.g., a PD-1 inhibitor (such as pembrolizumab or nivolumab), a PD-L1 inhibitor (such as alemtuzumab, avilamab, or Dewaruzumab), a CTLA-4 inhibitor, a LAG-3 inhibitor, or a Tim-3 inhibitor). Accordingly, the methods of the present disclosure include methods for treating cancer comprising administering to a subject in need thereof an effective amount of a compound as described herein and an immune checkpoint inhibitor. Administration of a compound described herein can be prior to, concurrent with, or subsequent to administration of an immune checkpoint inhibitor (e.g., a PD-1 inhibitor (such as pembrolizumab or nivolumab), a PD-L1 inhibitor (such as alemtuzumab, avilamab, or Devaluzumab), a CTLA-4 inhibitor, a LAG-3 inhibitor, or a Tim-3 inhibitor).

In some embodiments, a compound described herein and an immune checkpoint inhibitor are co-administered. In other embodiments, the compounds described herein are administered after the immune checkpoint inhibitor. In still other embodiments, the compounds described herein are administered prior to the immune checkpoint inhibitor.

In embodiments, a compound as described herein is administered to a subject in need thereof with a bromodomain inhibitor, a Histone Deacetylase (HDAC) inhibitor, or a combination of both.

In some embodiments, the methods of the present disclosure further comprise administering radiation therapy to the subject.

Embodiments further relate to a method of administering a compound as described herein in combination with a BTK inhibitor (e.g., ibrutinib) or a CDK9 inhibitor (e.g., avoxidine) provided herein, with radiation therapy for inhibiting abnormal cell growth or treating hyperproliferative disorders in a mammal to a subject in need thereof. Techniques for administering radiation therapy are known in the art, and these techniques may be used in combination therapies described herein. In this combination therapy, administration of a pharmaceutically acceptable salt of a compound described herein can be determined as described herein.

The compounds described herein may also be used in combination with an amount of one or more substances selected from the group consisting of anti-angiogenic agents, signal transduction inhibitors, antiproliferative agents, glycolytic inhibitors, or autophagy inhibitors.

In certain embodiments, the compounds described herein are administered in combination with erlotinib. In some embodiments, such a combination is used to treat pancreatic cancer. In other embodiments, such a combination is used to treat lung cancer. In further embodiments, the lung cancer is non-small cell lung cancer.

In certain embodiments, the compounds described herein are administered in combination with octreotide. In some embodiments, such a combination is used to treat lung cancer. In further embodiments, the lung cancer has EGFR mutations.

The structure of the active compound identified by code number, common or trade name may be taken from the actual version of the standard compilation "merck index" or from a database, such as an international patent (e.g., IMS world publication company (IMS World Publications)).

Numbered examples

1. A compound having the formula (I):

or a pharmaceutically acceptable salt thereof, wherein:

R ¹ is C ₁ -C ₅ Alkyl, or C ₃ -C ₅ Carbocycles or halogens;

R ² is-H, halogen, C optionally substituted by one or more-F ₁ -C ₃ Alkyl, or cyclopropyl optionally substituted with one or more-F;

R ³ is-H, halogen, C optionally substituted by one or more-F ₁ -C ₃ Alkyl, or cyclopropyl optionally substituted with one or more-F; and is also provided with

Ring G isWherein->Indicating the point at which ring G is attached to-N (H) -; or (b)

Ring G is C optionally substituted with ₆ -C ₁₀ Aryl:

(i) One or more halogens;

(ii) Sulfonamide;

(iii) Optionally by one or more C ₁ -C ₆ Alkyl-substituted monocyclic, bicyclic or spiro C ₃ -C ₁₀ Carbocycles, or C optionally substituted by hydroxy or one or more halogens ₃ -C ₆ A carbocycle, wherein the carbocycle is attached to ring G by a single bond or a methylene or ethylene linker at a position on ring G that is meta or para with respect to the-N (H) -attached to ring G; or (b)

(iv) May contain up to 3 heteroatoms independently selected from N and O and optionally and independently substituted with one or more C' s ₁ -C ₆ Alkyl-substituted monocyclic, bicyclic, bridged or spiro C ₃ -C ₁₀ Heterocycles, or C optionally substituted by hydroxy or one or more halogens ₃ -C ₆ Carbocycles wherein the heterocycle is attached to ring G by a single bond or a methylene or ethylene linker at a position on ring G that is meta or para to the-N (H) -attached to ring G.

2. The compound of numbered example 1 wherein R ¹ Is C ₁ -C ₅ Alkyl or C ₃ -C ₅ Carbocycles.

3. As described in numbered example 2Wherein R is a compound of formula (I) ¹ Is methyl or cyclopropyl.

4. The compound of numbered example 1 wherein R ¹ Is halogen.

5. The compound of numbered example 1 wherein R ¹ Is methyl or halogen.

6. The compound of numbered example 5 wherein R ¹ Is methyl or chlorine.

7. The compound of any one of numbered embodiments 1-6, wherein R ² is-H, halogen, -CH ₃ 、-CF ₃ Or cyclopropyl.

8. The compound of numbered example 7 wherein R ² is-H.

9. The compound of any one of numbered embodiments 1-8, wherein R ³ is-H, halogen, -CH ₃ 、-CF ₃ Or cyclopropyl.

10. The compound of numbered example 9 wherein R ³ is-H or halogen.

11. The compound of numbered example 9 wherein R ³ is-H or fluorine.

12. The compound of numbered example 9 wherein R ³ is-H.

13. The compound of any of numbered embodiments 1-12 wherein ring G is

14. The compound of any of numbered embodiments 1-12, wherein ring G is phenyl optionally substituted with:

(i) One or more halogens;

(ii) Sulfonamide;

(iii) Optionally by one or more C ₁ -C ₆ Alkyl-substituted monocyclic, bicyclic or spiro C ₃ -C ₁₀ Carbocycles, or C optionally substituted by hydroxy or one or more halogens ₃ -C ₆ A carbocycle, wherein the carbocycle is attached by a single bond or a methylene or ethylene group at a position on ring G that is meta or para with respect to the-N (H) -attached to ring G The head is attached to ring G; or (b)

(iv) May contain up to 3 heteroatoms independently selected from N and O and optionally and independently substituted with one or more C' s ₁ -C ₆ Alkyl-substituted monocyclic, bicyclic, bridged or spiro C ₃ -C ₁₀ Heterocycles, or C optionally substituted by hydroxy or one or more halogens ₃ -C ₆ Carbocycles wherein the heterocycle is attached to ring G by a single bond or a methylene or ethylene linker at a position on ring G that is meta or para to the-N (H) -attached to ring G.

15. The compound of numbered example 14 wherein ring G is phenyl substituted with:

(i) One or more halogens;

(ii) Sulfonamide;

(iii) Optionally by one or more C ₁ -C ₆ Alkyl-substituted monocyclic, bicyclic or spiro C ₃ -C ₁₀ Carbocycles, or C optionally substituted by hydroxy or one or more halogens ₃ -C ₆ A carbocycle, wherein the carbocycle is attached to ring G by a single bond or a methylene or ethylene linker at a position on ring G that is meta or para with respect to the-N (H) -attached to ring G; or (b)

(iv) May contain up to 3 heteroatoms independently selected from N and O and optionally and independently substituted with one or more C' s ₁ -C ₆ Alkyl-substituted monocyclic, bicyclic, bridged or spiro C ₃ -C ₁₀ Heterocycles, or C optionally substituted by hydroxy or one or more halogens ₃ -C ₆ Carbocycles wherein the heterocycle is attached to ring G by a single bond or a methylene or ethylene linker at a position on ring G that is meta or para to the-N (H) -attached to ring G.

16. The compound of numbered embodiment 14 or 15 wherein ring G is substituted with one or more halogens.

17. The compound of numbered example 14 or 15 wherein ring G is substituted with sulfonamide.

18. The compound of numbered embodiment 14 or 15 wherein ring G is substituted with: optionally by one or more C ₁ -C ₆ Alkyl substituted monocyclic and bicyclic ringsOr spiro C ₃ -C ₁₀ Carbocycles, or C optionally substituted by hydroxy or one or more halogens ₃ -C ₆ A carbocycle, wherein the carbocycle is attached to ring G by a single bond or a methylene or ethylene linker at a position on ring G that is meta or para relative to the-N (H) -attached to ring G.

19. The compound of numbered example 18, wherein ring G is substituted with: optionally by one or more C ₁ -C ₆ Alkyl-substituted monocyclic C ₃ -C ₇ Carbocycles, or C optionally substituted by hydroxy or one or more halogens ₃ -C ₆ Carbocycles.

20. The compound of numbered embodiment 18 or 19 wherein ring G is substituted with: optionally by one or more C ₁ -C ₆ An alkyl-substituted cyclohexyl group, or C optionally substituted with hydroxy or one or more halogens ₃ -C ₆ Carbocycles.

21. The compound of any of numbered embodiments 18-20, wherein the carbocycle attached to ring G is unsubstituted.

22. The compound of any of numbered embodiments 18-21, wherein the carbocycle attached to ring G is attached to ring G by a single bond.

23. The compound of numbered embodiment 14 or 15 wherein ring G is substituted with: may contain up to 3 heteroatoms independently selected from N and O and optionally and independently substituted with one or more C' s ₁ -C ₆ Alkyl-substituted monocyclic, bicyclic, bridged or spiro C ₃ -C ₁₀ Heterocycles, or C optionally substituted by hydroxy or one or more halogens ₃ -C ₆ Carbocycles wherein the heterocycle is attached to ring G by a single bond or a methylene or ethylene linker at a position on ring G that is meta or para to the-N (H) -attached to ring G.

24. The compound of numbered example 23 wherein ring G is substituted with: may contain up to 3 heteroatoms independently selected from N and O and optionally and independently substituted with one or more C' s ₁ -C ₆ Alkyl-substituted monocyclic C ₅ -C ₆ Heterocycles, or C optionally substituted by hydroxy or one or more halogens ₃ -C ₆ Carbocycles.

25. The compound of numbered embodiment 23 or 24 wherein ring G is substituted with: may contain up to 2 heteroatoms independently selected from N and O and optionally and independently substituted with one or more C' s ₁ -C ₆ Alkyl-substituted monocyclic C ₆ Heterocycles, or C optionally substituted by hydroxy or one or more halogens ₃ -C ₆ Carbocycles.

26. The compound of any of numbered embodiments 23-25, wherein ring G is substituted with: optionally and independently is one or more C ₁ -C ₆ Alkyl-substituted piperazinyl, morpholinyl, piperidinyl or oxacyclohexyl, or C optionally substituted with hydroxy or one or more halogens ₃ -C ₆ Carbocycles.

27. The compound of any of numbered embodiments 23-26, wherein the heterocycle attached to ring G is unsubstituted or monosubstituted.

28. The compound of numbered embodiment 27 wherein the heterocycle attached to ring G is unsubstituted.

29. The compound of any of numbered embodiments 23-28, wherein the heterocycle attached to ring G is attached to ring G by a single bond.

30. The compound of any of numbered embodiments 18-20, 22-27, and 29 wherein the carbocycle or heterocycle attached to ring G is optionally and independently methyl, CF ₃ CH ₂ -or HOCH ₂ CH ₂ -substitution.

31. The compound of any of numbered embodiments 1-12 and 14-30, wherein a carbocycle or heterocycle attached to ring G is attached to ring G at a position on ring G that is meta relative to-N (H) -attached to ring G.

32. The compound of any of numbered embodiments 1-12 and 14-30, wherein a carbocycle or heterocycle attached to ring G is attached to ring G at a position on ring G that is para relative to-N (H) -attached to ring G.

33. The compound of any one of numbered examples 1-6, having the structure:

or a pharmaceutically acceptable salt thereof.

34. The compound of any one of numbered examples 1-6, having the structure:

or a pharmaceutically acceptable salt thereof, wherein:

ring J is attached to the phenylene at a position meta or para to the-N (H) -attached to the phenylene;

A ¹ is-N (R) ⁴ ) -, -O-or>C(H)(R ⁴ )；

R ⁴ is-H, or C ₁ -C ₆ Alkyl or C ₃ -C ₆ Carbocycles, each of which is optionally substituted with hydroxy or one or more halogens;

A ² is that>N-or>C(H)-；

Z is>CH ₂ The method comprises the steps of carrying out a first treatment on the surface of the And X and Y are independently>CH ₂ Or (b)>C(CH ₃ ) ₂ Or X and Y are both>CH-and are bonded together by methylene or ethylene bridges; or (b)

Y is>CH ₂ Or (b)>C(CH ₃ ) ₂ And X and Z are both>CH-and are bonded together by methylene or ethylene bridges; and is also provided with

n is 0, 1 or 2.

35. The compound of numbered example 34 wherein a ¹ Is that>C(H)(R ⁴ )。

36. The compound of numbered example 34 wherein a ¹ is-N (R) ⁴ ) -or-O-.

37. The compound of numbered example 36 wherein a ¹ is-N (R) ⁴ )-。

38. The compound of numbered example 36 wherein a ¹ is-O-.

39. Example 34 as numberedThe compound of any one of claims-38, wherein R ⁴ is-H, or C ₁ -C ₆ Alkyl optionally substituted with hydroxy or one or more halogens.

40. The compound of example 39 numbered wherein R ⁴ is-H, methyl, hydroxyethyl or trifluoroethyl.

41. The compound of numbered embodiment 40 wherein R ⁴ is-H or methyl.

42. The compound of any of numbered embodiments 34-41 wherein a ² Is that>C(H)-。

43. The compound of any of numbered embodiments 34-41 wherein a ² Is that>N-。

44. The compound of numbered example 34, wherein ring J is:

45. the compound of any of numbered embodiments 34-44, wherein ring J is attached to the phenylene at a position meta to-N (H) -attached to the phenylene.

46. The compound of any of numbered embodiments 34-44, wherein ring J is attached to the phenylene at a position para relative to-N (H) -attached to the phenylene.

47. The compound of any of numbered embodiments 34-46, wherein n is 0 or 1.

48. The compound of numbered embodiment 47 wherein n is 0.

49. The compound of any one of numbered examples 1-6, 34-43, 45, and 46, having the structure:

or a pharmaceutically acceptable salt thereof.

50. The compound of numbered example 49, wherein ring J is:

51. a compound, or a pharmaceutically acceptable salt thereof, having the structure:

/>

52. the compound of numbered example 51, wherein the compound is of the formula:

or a pharmaceutically acceptable salt thereof.

53. The compound of numbered example 51, wherein the compound is of the formula:

or a pharmaceutically acceptable salt thereof.

54. The compound of numbered example 51, wherein the compound is of the formula:

or a pharmaceutically acceptable salt thereof.

55. The compound of numbered example 51, wherein the compound is of the formula:

or a pharmaceutically acceptable salt thereof.

56. The compound of numbered example 51, wherein the compound is of the formula:

or a pharmaceutically acceptable salt thereof.

57. The compound of numbered example 51, wherein the compound is of the formula:

Or a pharmaceutically acceptable salt thereof.

58. The compound of numbered example 51, wherein the compound is of the formula:

or a pharmaceutically acceptable salt thereof.

59. The compound of numbered example 51, wherein the compound is of the formula:

or a pharmaceutically acceptable salt thereof.

60. A pharmaceutical composition comprising a compound of any one of numbered examples 1-59, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.

61. A pharmaceutical combination comprising a compound of any one of numbered examples 1-59, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of numbered example 60, and one or more additional therapeutic agents.

62. A method of treating a proliferative disease in a subject, the method comprising administering to the subject a compound of any one of numbered embodiments 1-59, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of numbered embodiment 60.

63. The method of example 62, wherein the proliferative disease is cancer.

64. The method of example 63 of number, wherein the cancer is lung cancer, brain cancer, thyroid cancer, anaplastic astrocytoma, liver cancer, pancreatic cancer, skin cancer, melanoma, metastatic melanoma, colorectal cancer, breast cancer, prostate cancer, renal cancer, hepatocellular carcinoma, ovarian cancer, HPV-associated cancer, multiple myeloma, myelodysplastic syndrome, hematologic cancer, or myelofibrosis.

65. The method of example 64, wherein the cancer is non-small cell lung cancer (NSCLC).

66. The method of example 64, wherein the cancer is neuroblastoma or glioblastoma.

67. The method of example 64, wherein the cancer is thyroid undifferentiated carcinoma (ATC).

68. The method of numbered example 64, wherein the cancer is colon cancer.

69. The method of example 64, wherein the cancer is hepatocellular carcinoma (HCC).

70. The method of example 64, wherein the cancer is pancreatic cancer.

71. The method of example 64, wherein the cancer is Anaplastic Large Cell Lymphoma (ALCL) or myelodysplastic syndrome.

72. The method of example 64, wherein the cancer is anaplastic astrocytoma.

73. The method of example 64, wherein the cancer is pancreatic ductal adenocarcinoma.

74. The method of example 64, wherein the cancer is a related CAF cancer, metastatic melanoma, colorectal cancer, breast cancer, prostate cancer, renal cancer, hepatocellular carcinoma, ovarian cancer, HPV-related cancer, multiple myeloma, myelodysplastic syndrome, or myelofibrosis.

75. The method of numbered example 64, wherein the HPV-associated cancer is selected from the group consisting of: cervical cancer, oropharyngeal cancer, anal cancer, vulvar/vaginal cancer, or penile cancer.

76. The method of any one of embodiments 63-75 of the numbering, wherein the cancer is driven by TGF- β signaling.

77. The method of example 62, wherein the proliferative disease is a fibrotic disorder.

78. The method of example 77, wherein the fibrotic condition is idiopathic pulmonary fibrosis, cardiac fibrosis, a condition associated with cardiac fibrosis, valvular disease, cardiac arrhythmia, atrial fibrillation, myocardial remodeling, cardiomyopathy, dilated cardiomyopathy, ischemic cardiomyopathy, hypertrophic cardiomyopathy, restenosis, liver fibrosis, cirrhosis, non-alcoholic steatohepatitis, peclet's disease, dipteret's contracture, cystic fibrosis, beta thalassemia, light keratosis, hypertension, systemic inflammatory disorders, dry eye, ulcers, corneal fibrosis, wet age-related macular degeneration, psoriasis, wound closure, chronic kidney disease, renal fibrosis, systemic sclerosis, or chronic chagas heart disease.

79. A method of inhibiting tumor growth in a subject, the method comprising administering to the subject a compound of any one of numbered embodiments 1-59, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of numbered embodiment 60.

80. The method of any of numbered embodiments 62-79, further comprising administering to the subject one or more additional therapeutic agents.

81. The method of numbered embodiment 80, wherein at least one of the additional therapeutic agents is an anti-cancer agent.

82. The method of numbered embodiment 80 or 81, wherein at least one of the additional therapeutic agents is a PD-1 or PD-L1 inhibitor.

83. The method of any of numbered embodiments 62-82, further comprising treating the subject with radiation therapy or surgery.

84. A method of inhibiting ALK-5 activity in vivo or in vitro comprising contacting ALK-5 with a compound of any one of numbered examples 1-59, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of numbered example 60.

85. The method of numbered example 84, wherein the inhibiting occurs in the subject.

86. The method of example 84, wherein the inhibiting occurs in vitro.

87. The method of any one of numbered embodiments 62-85, wherein the subject is a human.

88. The method of any of numbered embodiments 84-87, wherein ALK-5 inhibition is at least 2-fold greater than ALK-2 inhibition under the same conditions.

89. A method of treating a fibrotic, inflammatory or proliferative disease or disorder susceptible to inhibition of a tgfβ signaling pathway, the method comprising administering to a subject suffering from the fibrotic, inflammatory or proliferative disease or disorder an amount of a compound of any one of examples 1-59, numbered, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of example 60, numbered, effective to inhibit tgfβ signaling.

90. The method of example 89, wherein the disease or disorder is a fibrotic disease or disorder.

91. The method of numbered embodiment 90, wherein the fibrotic disease or disorder is selected from idiopathic pulmonary fibrosis, cardiac fibrosis, a disorder associated with cardiac fibrosis, liver fibrosis, cirrhosis, non-alcoholic steatohepatitis, pecan's disease, dipterex's contracture, cystic fibrosis, beta thalassemia, light keratosis, hypertension, systemic inflammatory disorders, dry eye, ulcers, corneal fibrosis, wet age-related macular degeneration, psoriasis, wound closure, chronic kidney disease, kidney fibrosis, systemic sclerosis, or chronic chagas ' heart disease.

92. The method of example 91, wherein the fibrotic disease or disorder is idiopathic pulmonary fibrosis.

93. The method of example 89, wherein the disease or disorder is an inflammatory disease or disorder.

94. The method of example 89, wherein the disease or disorder is a proliferative disease or disorder.

95. The method of example 94, wherein the proliferative disease or disorder is selected from anaplastic astrocytoma, pancreatic cancer, metastatic melanoma, colorectal cancer, breast cancer, prostate cancer, renal cancer, hepatocellular carcinoma, ovarian cancer, HPV-related cancer, cervical cancer, oropharyngeal cancer, anal cancer, vulval/vaginal cancer, penile cancer, multiple myeloma, myelodysplastic syndrome, or myelofibrosis.

96. A method of inhibiting tgfβ signalling in a subject suffering from a disease or disorder promoted by tgfβ signalling, the method comprising administering to the subject an amount of at least one compound of any one of numbered examples 1-59, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of numbered example 60, effective to substantially inhibit tgfβ signalling to alter the course of the disease or disorder.

97. A method of treating cachexia in a subject, the method comprising administering to the subject a compound of any one of numbered embodiments 1-59, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of numbered embodiment 60.

98. A method of inhibiting an epithelial-to-mesenchymal transition (EMT) in a subject having a disease or disorder facilitated by the EMT, the method comprising administering to the subject an amount of at least one compound of any one of examples 1-59, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of example 60, in an amount effective to sufficiently inhibit the EMT to alter the course of the disease or disorder.

Examples

Synthesis example

Scheme-1:

synthesis of 1- (2-amino-3-methylphenyl) ethan-1-one (1.2):

to a suspension of 2-amino-3-methylbenzoic acid (1) (150 g,993.3 mmol) in tetrahydrofuran (2.5L) was added MeLi (1.6M in diethyl ether) (2.48L, 3973.5 mmol) at 0℃and the resulting mixture was stirred at 25℃for 3h. The reaction mixture was quenched with saturated ammonium chloride solution (2000 mL) and extracted with EtOAc (2 x 10 l). The combined organic layers were washed with water (1.0L), brine (1.0L), dried over anhydrous sodium sulfate, and concentrated in vacuo to afford the crude compound which was triturated with n-pentane (2 x 500 ml) to afford the title compound (1.2), characterized in that ¹ H NMR(CDCl ₃ 400 MHz): delta 7.65 (d, j=8.4 hz, 1H), 7.21 (d, j=6.8 hz, 1H), 6.59 (t, j=8.0 hz, 1H), 6.41 (bs, 2H), 2.59 (s, 3H), 2.16 (s, 3H); LCMS (m+h): 150.1.

synthesis of 8-methylcinnolin-4-ol (1.3):

to a stirred solution of 1- (2-amino-3-methylphenyl) ethan-1-one (1.2) (126 g,845.6 mmol) in concentrated HCl (1.26L) at-5℃was added NaNO dropwise ₂ (70 g,1014.7 mmol) in water (95 mL) and stirred at 70℃for 3h. The reaction mixture was cooled to room temperature, filtered, and the residue was washed with diethyl ether (1.5L). The filtrate was neutralized to pH 7 with saturated sodium bicarbonate, and the precipitated solid was filtered and dried under vacuum to provide the title compound (3), characterized in that ¹ H NMR(CDCl ₃ 500 MHz): δ10.06 (bs, 1H), 8.14 (d, j=8.0 hz, 1H), 7.87 (s, 1H), 7.54 (d, j=7.0 hz, 1H), 7.32-7.29 (m, 1H), 2.56 (s, 3H); LCMS (m+h): 161.1.

synthesis of 4-chloro-8-methylcinnoline (1.4):

POCl at room temperature ₃ (380 mL) was added to compound (1.3) (38 g,187.0 mmol) and allowed to stir at 100deg.C for 8h. The reaction mixture was cooled to room temperature and excess POCl was distilled off ₃ The residue was poured into ice water (750 mL) and neutralized with saturated sodium bicarbonateTo pH 7, the precipitated solid was filtered off and dried under vacuum to give the title compound (4), characterized in that ¹ H NMR(CDCl ₃ 400 MHz): δ9.35 (s, 1H), 8.05 (d, j=7.6 hz, 1H), 7.77-7.71 (m, 2H), 3.05 (s, 3H); LCMS (m+h): 179.1.

synthesis of 4-azido-8-methylcinnoline (1.5):

to a stirred solution of compound (1.4) (30 g,168.5 mmol) in ethanol (400 mL), water (100 mL) was added NaN ₃ (54.77 g,842.69 mmol) and stirred at 75℃for 5h. The reaction mixture was cooled to room temperature and concentrated in vacuo. The residue was diluted with water (500 mL), the precipitated solid was filtered off and dried under vacuum to provide the title compound (5), characterized in that ¹ H NMR(CDCl ₃ 400 MHz): δ9.23 (s, 1H), 7.89 (d, j=8.4 hz, 1H), 7.69-7.61 (m, 2H), 3.02 (s, 3H); LCMS (m+h): 186.1.

synthesis of 8-methylcinnolin-4-amine (1.6):

to a stirred solution of 4-azido-8-methylcinnoline (1.5) (25 g,135.13 mmol) in ethanol, THF (750 mL, 500 mL) was added 10% Pd/C (50% moisture) (5.0 g) and the reaction was stirred under hydrogen for 1h. The reaction mixture was filtered through a celite pad and the residue was washed with methanol (2 x 1.0 l). The filtrate was concentrated under reduced pressure and co-distilled with toluene (2 x 500 ml) and triturated with ether (2 x 500 ml) to afford the title compound (6), characterized in that ¹ H NMR(DMSO-d ₆ 400 MHz): δ8.63 (s, 1H), 8.01 (d, j=8.4 hz, 1H), 7.56 (d, j=6.8 hz, 1H), 7.45 (t, j=8.0 hz, 1H), 7.08 (bs, 2H), 2.76 (s, 3H); LCMS (m+h): 160.1.

Synthesis of N- (2-bromopyridin-4-yl) -8-methylcinnolin-4-amine (1.8):

a solution of 8-methylcinnolin-4-amine (1.6) (10 g,62.8 mmol), 2-bromo-4-fluoropyridine (1.7) (13.2 g,75.00 mmol) in DMF (125 mL) and THF (125 mL) was added to a suspension of NaH (6.28 g,157.00 mmol) in THF (125 mL). The resulting reaction mixture was stirred at 25 ℃ for 3h. The reaction mixture was quenched with saturated ammonium chloride solution and concentrated in vacuo, the residue was diluted with water (500 mL), the precipitated solid was filtered, washed with diethyl ether (2 x 200 mL) and dried under vacuum to afford the title compoundThe title compound (8) is characterized in that ¹ H NMR(DMSO-d ₆ 400 MHz): δ10.40 (s, 1H), 9.11 (s, 1H), 8.19 (d, j=7.6 hz, 1H), 8.09 (d, j=11.2 hz, 1H), 7.73-7.63 (m, 2H), 7.38 (s, 1H), 7.27 (d, j=5.2 hz, 1H), 2.83 (s, 3H); LCMS (m+h): 315.04.

synthesis of N2- (3-methyl-1- (2, 2-trifluoroethyl) -1H-pyrazol-4-yl) -N4- (8-methylcinnolin-4-yl) pyridine-2, 4-diamine (Compound 01)

A mixture of N- (2-bromopyridin-4-yl) -8-methylcinnolin-4-amine (1.8) (35 g,111.11 mmol), 3-methyl-1- (2, 2-trifluoroethyl) -1H-pyrazol-4-amine (1.9) (19.88 g,111.11 mmol) and potassium carbonate (46.0 g,333.33 mmol) in DMF (250 mL) was degassed for 20min and Pd was added ₂ (dba) ₃ (10.176 g,11.11 mmol), xantphos (6.42 g,11.11 mmol) and the resulting reaction mixture was stirred at 160℃for 3h. The reaction mixture was cooled to room temperature and diluted with water (750 mL), the precipitated solid was filtered and dried under vacuum to provide the crude compound, which was purified by flash column chromatography (100-200 silica mesh) using 1% -5% methanol/dichloromethane as eluent, followed by trituration with DCM (100 mL) to give the title compound (10), characterized in that ¹ H NMR(DMSO-d ₆ 400 MHz): δ9.35 (s, 1H), 9.31 (s, 1H), 8.21-8.19 (m, 2H), 8.14 (s, 1H), 8.02 (d, j=5.6 hz, 1H), 7.74-7.67 (m, 2H), 6.80 (s, 1H), 6.66 (d, j=5.2 hz, 1H), 4.99 (q, j=9.2 hz, 2H), 2.88 (s, 3H), 2.17 (s, 3H); LCMS (m+h): 414.1; and (3) HPLC:98.06%.

N4- (8-methylcinnolin-4-yl) -N2- (3-morpholinophenyl) pyridine-2, 4-diamine (compound 03):

using a procedure similar to that described for the synthesis of compound 01 of scheme 1, N- (2-bromopyridin-4-yl) -8-methylcinnolin-4-amine (1.8) (0.55 g,1.74 mmol) was reacted with 3-morpholinoaniline (0.310 g,1.74 mmol) in DMSO and purified by flash column chromatography (100-200 silica mesh) using 60% -70% ethyl acetate/hexane as eluent to give the title compound (03), characterized in that ¹ H NMR(DMSO-d ₆ ,400MHz)：δ9.37(s,1H),9.36(s,1H),8.85(s,1H),8.20(d,J＝8.0Hz,1H),8.06(d,J＝5.6Hz,1H),7.74-7.68(m,2H),7.23(s,1H),7.15-7.07(m2H), 6.82 (s, 1H), 6.73 (d, j=5.2 hz, 1H), 6.50 (d, j=7.2 hz, 1H), 3.74 (t, j=4.0 hz, 4H), 3.07 (t, j=4.4 hz, 4H), 2.89 (s, 3H); LCMS (m+h): 413.22; and (3) HPLC:98.49%.

Synthesis of N4- (8-methylcinnolin-4-yl) -N2- (4- (tetrahydro-2H-pyran-4-yl) phenyl) pyridine-2, 4-diamine (Compound 08):

using a procedure similar to that described for the synthesis of compound 01 of scheme 1, N- (2-bromopyridin-4-yl) -8-methylcinnolin-4-amine (1.8) (0.5 g,1.58 mmol) was reacted with 4- (tetrahydro-2H-pyran-4-yl) aniline (0.28 g,1.58 mmol) in DMSO and purified by flash column chromatography (100-200 silica mesh) using 60% -70% ethyl acetate/hexane as eluent to give the title compound (08), characterized in that ¹ H NMR(DMSO-d ₆ 400 MHz): delta 9.36 (s, 2H), 8.89 (s, 1H), 8.20 (d, j=7.6 hz, 1H), 8.04 (d, j=5.6 hz, 1H), 7.74-7.68 (m, 2H), 7.55 (d, j=8.4 hz, 2H), 7.13 (d, j=8.4 hz, 2H), 6.80 (s, 1H), 6.74 (t, j=4.0 hz, 1H), 3.95-3.92 (m, 2H), 3.45-3.39 (m, 2H), 2.89 (s, 3H), 2.71-2.66 (m, 1H), 1.67-1.62 (m, 4H); LCMS (m+h): 412.2; and (3) HPLC:98.84%.

Synthesis of N4- (8-methylcinnolin-4-yl) -N2- (3- (tetrahydro-2H-pyran-4-yl) phenyl) pyridine-2, 4-diamine (Compound 07):

Using a procedure similar to that described for the synthesis of compound 01 of scheme 1, N- (2-bromopyridin-4-yl) -8-methylcinnolin-4-amine (1.8) (0.5 g,1.58 mmol) was reacted with 3- (tetrahydro-2H-pyran-4-yl) aniline (0.28 g,1.58 mmol) in DMSO and purified by flash column chromatography (100-200 silica mesh) using 60% -70% ethyl acetate/hexane as eluent to give the title compound (07), characterized in that ¹ H NMR(DMSO-d ₆ 400 MHz): δ9.37 (s, 2H), 8.84 (s, 1H), 8.20 (d, j=7.6 hz, 1H), 8.06 (d, j=5.6 hz, 1H), 7.75-7.68 (m, 2H), 7.54 (d, j=8.4 hz, 1H), 7.47 (s, 1H), 7.18 (t, j=8.0 hz, 1H), 6.82 (d, j=1.6 hz, 1H), 6.79-6.73 (m, 2H), 3.96-3.93 (m, 2H), 3.47-3.41 (m, 2H), 2.89 (s, 3H), 2.71-2.66 (m, 1H), 1.71-1.60 (m, 4H); LCMS (m+h): 412.2; and (3) HPLC:98.84%.

Synthesis of N4- (8-methylcinnolin-4-yl) -N2- (4- (4-methylpiperazin-1-yl) phenyl) pyridine-2, 4-diamine (Compound 06):

n- (2-bromopyridin-4-yl) -8-methylcinnolin-4-amine (1.8) (0.2 g,0.63 mmol) was treated with 4- (4-methylpiperazin-1-yl) aniline (0.12 g,0.63 mmol) in DMF using a procedure similar to that described for the synthesis of Compound 01 of scheme 1 and purified by preparative HPLC to give the title compound (06) characterized in that ¹ H NMR(DMSO-d ₆ 400 MHz): δ9.33 (s, 1H), 9.31 (s, 1H), 8.66 (s, 1H), 8.19 (d, j=8.0 hz, 1H), 8.00 (d, j=5.6 hz, 1H), 7.74-7.67 (m, 2H), 7.44 (d, j=8.0 hz, 2H), 6.87 (d, j=9.2 hz, 2H), 6.71-6.67 (m, 2H)), 3.04 (t, j=4.4 hz, 4H), 2.88 (s, 3H), 2.44 (t, j=4.8 hz, 4H), 2.21 (s, 3H); LCMS (m+h): 426.2; and (3) HPLC:95.52%.

Synthesis of N4- (8-methylcinnolin-4-yl) -N2- (4-morpholinophenyl) pyridine-2, 4-diamine (Compound 05):

using a procedure similar to that described for the synthesis of compound 01 of scheme 1, N- (2-bromopyridin-4-yl) -8-methylcinnolin-4-amine (1.8) (10.0 g,31.74 mmol) was treated with 4-morpholinoaniline (5.65 g,31.74 mmol) in DMSO and purified by flash column chromatography (100-200 silica mesh) using 1% -5% methanol/DCM as eluent to give the title compound (05) characterized in that ¹ H NMR(DMSO-d ₆ 400 MHz): δ9.33 (s, 2H), 8.69 (s, 1H), 8.20 (d, j=8.0 hz, 1H), 8.01 (d, j=5.6 hz, 1H), 7.74-7.67 (m, 2H), 7.47 (d, j=8.8 hz, 2H), 6.88 (d, j=8.8 hz, 2H), 6.71-6.72-6.68 (m, 2H)), 3.73 (t, j=4.4 hz, 4H), 3.01 (t, j=4.4 hz, 4H), 2.88 (s, 3H); LCMS (m+h): 413.2; and (3) HPLC:96.01%.

The synthesis scheme of the intermediate is as follows:

scheme-2:

synthesis of 3- (3, 6-dihydro-2H-pyran-4-yl) aniline (1.18):

3-Bromoaniline (1.16) (5.0 g,29.0 mmol), 2- (3, 6-dihydro-2H-pyran-4-yl) -4, 5-tetramethyl-1, 3, 2-dioxaborolan (1.17) (9.15 g,43.6 mmol) and K were reacted with argon ₂ CO ₃ (12.0 g,87.0 mmol) in 1, 4-dioxane (150 mL) and water (15 mL) was degassed for 20 min. Addition of Pd (dppf) Cl ₂ DCM (2.36 g,2.90 mmol) and stir the resulting reaction mixture at 100deg.C for 5h. The reaction mixture was cooled to room temperature and then extracted with ethyl acetate (2 x 200 ml). The combined organic layers were washed with water (100 mL) and brine (100 mL), dried over anhydrous sodium sulfate, and concentrated in vacuo to afford the crude compound which was purified by flash column chromatography on silica gel (100-200 mesh) using 30% ethyl acetate and hexane as eluent to afford the title compound 1.18, characterized in that ¹ H NMR(CDCl ₃ 400 MHz): delta 7.12 (t, j=8.0 hz, 1H), 6.81-6.79 (m, 1H), 6.70 (t, j=2.0 hz, 1H), 6.61-6.58 (m, 1H), 6.08-6.06 (m, 1H), 4.31-4.29 (m, 2H), 3.91 (t, j=5.2 hz, 2H) 3.66 (bs, 2H), 2.50-2.46 (m, 2H); LCMS (m+h): 176.04.

synthesis of 3- (tetrahydro-2H-pyran-4-yl) aniline (1.19):

to a stirred solution of 3- (3, 6-dihydro-2H-pyran-4-yl) aniline (1.18) (3.5 g,20.0 mmol) in methanol (200 mL) was added 10% Pd/C (50% moisture) (1.0 g). The resulting reaction mixture was stirred under a hydrogen pal apparatus (hydrogen par apparatus) at 20Psi for 16h. The reaction mixture was filtered through a pad of celite and washed with methanol (2 x 100 ml). The filtrate was concentrated under reduced pressure, co-distilled with toluene (2 x 25 ml), and washed with ether (2 x 25 ml) to afford the title compound (1.19), characterized in that ¹ H NMR(CDCl ₃ 400 MHz): delta 7.12-7.08 (m, 1H), 6.64-6.62 (m, 1H), 6.55-6.53 (m, 2H), 4.07-4.04 (m, 2H), 6.34 (bs, 2H), 3.54-3.47 (m, 2H), 2.68-2.62 (m, 1H), 1.84-1.72 (m, 4H); LCMS (m+h): 178.22.

scheme-3:

synthesis of 3-methyl-4-nitro-1- (2, 2-trifluoroethyl) -1H-pyrazole (1.22):

3-methyl-4-nitro-1H-pyrazole (1.20) (30.0 g,236.22 mmol), 1-trifluoro-2-iodoethane (1.21) (148 g,708.66 mmol) and K were reacted at 100deg.C ₂ CO ₃ (97.79 g,708.66 mmol) in DMF (100 mL) was stirred for 3h. The reaction mixture was cooled to room temperature and extracted with ethyl acetate (2 x 200 ml). The combined organic layers were washed with water (100 mL), brine (100 mL), dried over anhydrous sodium sulfate, and concentrated in vacuo to afford the crude compound which was purified by flash column chromatography using 10% ethyl acetate and hexane as eluent on silica gel (100-200 mesh) to afford the title compound 1.22, characterized in that ¹ H NMR(CDCl ₃ 300 MHz): delta 8.25 (s, 1H), 4.70-4.62 (m, 2H), 2.56 (s, 3H); LCMS (m+h): 210.01.

synthesis of 3-methyl-1- (2, 2-trifluoroethyl) -1H-pyrazol-4-amine (1.23):

to a stirred solution of 3-methyl-4-nitro-1- (2, 2-trifluoroethyl) -1H-pyrazole (1.22) (25 g,119.04 mmol) in ethanol (750 mL) was added 10% Pd/C (50% moisture) (5.0 g) and stirred under a hydrogen Parr apparatus at 40Psi for 6H. The reaction mixture was filtered through celite pad and washed with methanol (2 x 1000 ml), the filtrate was concentrated under reduced pressure to provide crude compound, which was purified by flash column chromatography using 50% -100% ethyl acetate and hexane as eluent on silica gel (100-200 mesh) to provide the title compound 1.23, characterized in that ¹ H NMR(CDCl ₃ 400 MHz): delta 7.02 (s, 1H), 4.53-4.46 (m, 2H), 2.80 (bs, 2H), 2.18 (s, 3H); LCMS (m+h): 180.01.

scheme-4:

1- (2-amino-3-chlorophenyl) ethan-1-one (1.25):

to a suspension of 2-amino-3-chlorobenzoic acid (1.24) (20.0 g,116.95 mmol) in tetrahydrofuran (300 mL) was added MeLi (1.6M in diethyl ether, 293mL,467.83 mmol) at 0deg.C and the resulting reaction mixture was stirred at 25deg.C for 2h. The reaction mixture was quenched with saturated ammonium chloride solution (50 mL) and extracted with ethyl acetate (2 x 200 mL). The combined organic layers were washed with water (100 mL), brine (100 mL), and driedDried over sodium sulfate and concentrated in vacuo to afford a crude product which was purified by flash column chromatography on silica gel (100-200 mesh) using 30% ethyl acetate and hexane as eluent to afford the title compound (1.25), characterized in that ¹ H NMR(DMSO-d ₆ 400 MHz): delta 7.66 (dd, j=1.6 hz,1.2hz, 1H), 7.41 (dd, j=1.6 hz,1.2hz, 1H), 7.82 (bs, 2H), 6.59 (t, j=8 hz, 1H), 2.59 (s, 3H); LCMS (m+h): 170.06.

synthesis of 8-chlorocinnolin-4-ol (1.26):

to a stirred solution of 1- (2-amino-3-chlorophenyl) ethan-1-one (1.25) (15.0 g,88.75 mmol) in concentrated HCl (100 mL) at-5deg.C was added NaNO dropwise ₂ (7.40 g,106.50 mmol) in water (25 mL) and the resulting reaction mixture was stirred at 70deg.C for 3h. The reaction mixture was cooled to room temperature and filtered, the residue was washed with diethyl ether (1.5L) and the filtrate was neutralized to pH 7 with saturated sodium bicarbonate, the precipitated solid was filtered and dried under vacuum to give the title compound (1.26), characterized in that ¹ H NMR CDCl ₃ 300 MHz): δ10.40 (bs, 1H), 8.18 (d, j=6.0 hz, 1H), 7.88 (s, 1H), 7.77-7.74 (m, 1H), 7.34 (t, j=8.1 hz, 1H); LCMS (M-H): 181.7.

synthesis of 4, 8-dichloro cinnoline (1.27):

POCl at room temperature ₃ (50 mL) was added to the compound 8-chlorocinnolin-4-ol (1.26) (4.5 g,25.0 mmol) and allowed to stir at 100deg.C for 8h. The reaction mixture was cooled to room temperature and excess POCl was taken up ₃ Distilling off. The residue was poured into ice water (50 mL) and basified to pH 7 with saturated sodium bicarbonate solution, the precipitated solid was filtered and dried under vacuum to provide the title compound (1.27), characterized in that ¹ H NMR CDCl ₃ 400 MHz): delta 9.46 (s, 1H), 8.17-8.13 (m, 1H), 8.02-8.00 (m, 1H), 7.81-7.34 (m, 1H); LCMS (m+h): 198.97.

synthesis of 4-azido-8-chlorocinnoline (1.28):

to a stirred solution of 4, 8-dichlorocinnoline (1.27) (4.3 g,21.82 mmol) in ethanol (50 mL), water (5 mL) was added NaN ₃ (7.10 g,109.13 mmol) and stirred at 75℃for 6h. Cooling the reaction mixture to a chamberWarm and concentrated under vacuum. The residue was diluted with water (50 mL) and the precipitated solid was filtered and dried under vacuum to afford the title compound (1.28), characterized in that ¹ H NMR(CDCl ₃ 400 MHz): delta 9.31 (s, 1H), 7.99-7.95 (m, 2H) 7.68-7.63 (m, 1H); LCMS (m+h): 205.95.

synthesis of 8-chlorocinnolin-4-amine (1.29):

to a stirred solution of 4-azido-8-chlorocinnoline (1.28) (4.0 g,19.51 mmol) in ethyl acetate (100 mL) was added 10% Pd/C (50% moisture) (0.5 g) and stirred under a hydrogen Parr apparatus at 20Psi for 16h. The reaction mixture was filtered through a pad of celite and washed with methanol (2 x 100 ml), the filtrate was concentrated under reduced pressure and co-distilled with toluene (2 x 25 ml) and washed with ether (2 x 25 ml) to provide the title compound (1.29), characterized in that ¹ H NMR(CDCl ₃ 300 MHz): delta 8.71 (s, 1H), 8.18 (dd j=7.8 hz,1.2hz, 1H) 7.91 (dd j=6.6 hz,6.0hz, 1H) 7.56-7.51 (m, 1H), 7.45 (bs, 2H); LCMS (m+h): 180.11.

synthesis of 8-chloro-N- (2-chloropyridin-4-yl) cinnolin-4-amine (1.31):

a solution of 8-chlorocinnolin-4-amine (1.29) (10 g,55.86 mmol), 2-chloro-4-fluoropyridine (1.30) (8.88 g,67.03 mmol) in DMF (200 mL), THF (50 mL) was added to a solution of NaH (5.6 g,139.65 mmol) in THF (50 mL) and the resulting reaction mixture was stirred at 25deg.C for 6h. The reaction mixture was quenched with cold water and concentrated in vacuo, the residue was diluted with water (500 mL), the precipitated solid was filtered, washed with diethyl ether (2 x 200 mL) and dried under vacuum to afford the title compound (31), characterized in that ¹ H NMR(DMSO-d ₆ 400 MHz): δ10.40 (s, 1H), 9.21 (s, 1H), 8.27-8.24 (m, 2H), 8.06 (d, j=6.6 hz, 1H), 7.73 (t, j=7.5 hz, 1H), 7.31.—7.29 (m, 2H); LCMS (m+h): 290.94.

synthesis of N4- (8-chlorocinnolin-4-yl) -N2- (3-morpholinophenyl) pyridine-2, 4-diamine (Compound 04):

8-chloro-N- (2-chloropyridin-4-yl) cinnolin-4-amine (60.0 g,206.89 mmol) was treated with 3-morpholinoaniline (36.82 g,206.89 mmol) in DMSO using a procedure similar to that described for the synthesis of Compound 01 of scheme 1 byFlash column chromatography (100-200 silica mesh) using 1% -5% methanol/DCM as eluent to give the title compound (04) characterised in that ¹ H NMR(DMSO-d ₆ 400 MHz): delta 9.58 (s, 1H), 9.43 (s, 1H), 8.89 (s, 1H), 8.38 (d, j=7.6 hz, 1H), 8.11-8.07 (m, 2H), 7.79-7.76 (m, 1H), 7.24 (s, 1H), 7.16-7.12 (m, 1H), 7.10-7.08 (m, 1H), 6.86 (s, 1H), 6.76 (s, 1H), 6.51 (d, j=5.8 hz, 1H), 3.75 (t, j=4.4 hz, 4H), 3.08 (t, j=4.8 hz, 4H); LCMS (m+h): 433.00; and (3) HPLC:98.71%.

Synthesis of N4- (8-chlorocinnolin-4-yl) -N2- (3-methyl-1- (2, 2-trifluoroethyl) -1H-pyrazol-4-yl) pyridine-2, 4-diamine (Compound 02):

using a procedure similar to that described for the synthesis of compound 01 of scheme 1, 8-chloro-N- (2-chloropyridin-4-yl) cinnolin-4-amine (2.5 g,8.59 mmol) was reacted with 4- ((4-methylpiperazin-1-yl) methyl) aniline 3-methyl-1- (2, 2-trifluoroethyl) -1H-pyrazol-4-amine (1.53 g,8.59 mmol) and purified by preparative HPLC to give the title compound (02) characterized in that ¹ H NMR(DMSO-d ₆ 500 MHz): δ9.53 (s, 1H), 9.41 (s, 1H), 8.38 (s, 1H), 8.27 (s, 1H), 8.14 (s, 1H), 8.07 (t, j=5.6 hz, 2H), 7.77 (d, j=8.0 hz, 1H), 6.83 (s, 1H), 6.70-6.69 (m, 1H), 5.00 (q, j=9.2 hz, 2H), 2.17 (s, 3H); LCMS (M-H): 434.02; and (3) HPLC:95.03%.

TABLE 1 exemplary Compounds

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Measurement examples

EXAMPLE 1 LanthaScreen for ALK5 and ALK2 ^TM Kinase assay

Determination of selected compounds of the present disclosure against ALK5 and ALK2Is a potent inhibitor of the activity of (a). Activity determination and selectivity SelectScreen by Semerle Feishmania technologies Co (Thermo Fisher Scientific) ^TM Biochemical kinase profiling services use their LanthaScreen ^TM Eu kinase binding assay screening.

LanthaScreen ^TM The principle of the Eu kinase binding assay is shown in fig. 1A. Alexa Fluor ^TM Binding of the conjugate or "tracer" to the kinase is detected by adding Eu-labeled anti-tag antibodies. Binding of the tracer and antibody to the kinase results in a high degree of FRET, whereas replacement of the tracer with a kinase inhibitor results in the loss of FRET. The assay is performed by mixing and reading the tested compounds with reagents; no color development step is required.

Kinase tracers from life technologies company (Life Technologies) are based on ATP-competitive kinase inhibitors, which makes them suitable for detecting any compound that binds to the ATP site. Inhibitors that bind to the ATP site include inhibitors of type I kinases that bind only to the ATP site, and inhibitors of type II that bind to the ATP site and a second site, commonly referred to as an allosteric site (e.g., Imatinib, sorafenib, BIRB-796).

Test compounds were screened in wells in 1% DMSO (final). For 10-point titration, 3-fold serial dilutions were made from the starting concentration (see table 2 below).

TABLE 2 details of kinase assay protocol

Buffers used in all assays were:

50mM HEPES ph7.5；0.01％ GRIJ-35；10mM MgCl ₂ ；1mM EGTA

* The tracer is derived from the company Siemens Feisher (ThermoFisher)

All kinase/antibody mixtures were diluted to 2-fold working concentrations in the indicated kinase buffers. 4X AlexaFluor labelled tracer was prepared in kinase buffer.

Assay protocol

Bar code, low volume, white, 384 well plate (Greiner) catalog number 784207

1.160 nL-100X test Compound in 100% DMSO

2.3.84 mu L of kinase buffer

3.8.0. Mu.L of-2X kinase/antibody mixture

4.4.0. Mu.L-4X tracer

5.30 seconds plate oscillation

6. Incubate at room temperature for 60 min

7. Reading and data analysis on a fluorescent plate reader

The following controls were prepared for each individual kinase and placed on the same plate as the kinase:

0% replacement control: maximum emissivity was established from 0% displacement control wells that did not contain known inhibitors of the reaction and therefore did not exhibit displacement of the tracer.

100% replacement control: minimum emissivity was established from 100% displacement control wells containing the highest concentration of known inhibitors used in the assay.

Inhibitors are known: a control standard curve (10 point titration) of known inhibitors was made for each individual kinase on the same plate as the kinase to ensure that the displacement of the inhibitor was within the expected IC50 range previously determined.

The following equation is used for each set of data points:

the generated data was plotted using plotting software XLfit from IDBS company. The dose response curve is curve fitted to model number 205. If the fit at the bottom of the curve is not between-20% and 20% inhibition, it is set to 0% inhibition. If the fit at the top of the curve is not between 70% and 130% inhibition, it is set to 100% inhibition.

Example 2 HotSpot ^TM JAK2 assay protocol

Basic reaction buffer: 20mM Hepes (pH 7.5), 10mM MgCl ₂ 、1mM EGTA、0.01％ Brij35、0.02mg/ml BSA、0.1mM Na ₃ VO ₄ 2mM DTT, 1% DMSO, to which was added a final concentration of 0.2mg/mL of a polyamino acid sodium salt (poly (Glu, tyr) sodium salt Glu: tyr (4:1), 5000-2000, from Sigma Aldrich, catalog number P7244).

The reaction procedure:

1. the substrate was prepared in freshly prepared reaction buffer.

2. The kinase was delivered to the substrate solution and gently mixed.

3. Compounds in 100% DMSO were delivered to the kinase reaction mixture by acoustic techniques (Echo 550; nanoliter range) and incubated for 20 minutes at room temperature.

4. Will be ³³ P-ATP is delivered to the reaction mixture to initiate the reaction.

5. Incubate for 2 hours at room temperature.

6. Kinase activity was detected by the P81 filter binding method.

The method is performed according to published protocols (see Anastassiadis T et al, "Comprehensive assay of kinase catalytic activity reveals features of kinase inhibitor selectivity [ comprehensive determination of kinase catalytic activity reveals a characteristic of kinase inhibitor selectivity ]", nat Biotechnol. [ Nature Biotechnology ]2011, 10 months 30; 29 (11): 1039-45.Doi:10.1038/nbt.2017.PMID:22037377; PMCID: PMC 3230241), and further details of the method are summarized therein.

The concentration of JAK2 enzyme used in this assay was 0.25nM. The JAK2 enzyme used is a recombinant human protein expressed in insect cells, catalytic domain (amino acids 808-1132), GST tagged. This material was obtained from Invitrogen, catalog number PV4210. Additional details are available from the product catalog number PV4210 of the sameidie company.

JAK2 kinase activity determinations were performed using the "HotSpot" assay platform at reaction biology company (Reaction Biology Corporation) (Malvern, PA). Briefly, specific kinase/substrate pairs are prepared in reaction buffer. The compound was delivered to the reaction, followed by addition of ATP (Sigma, st. Louis MO, mitsui) and ATP after about 20 minutes ³³ P ATP (Perkin Elmer, waltham MA, MA) to a final concentration of 10 μm. The reaction was allowed to proceed for 120 minutes at room temperature, then the reaction spot was placed on P81 ion exchange filter paper (Whatman inc., piscataway, NJ) of new jersey. Unbound phosphate was removed by extensive washing of the filter in 0.75% phosphoric acid. Kinase activity data, after subtraction of the background derived from the control reaction containing inactive enzyme, is expressed as a percentage of the remaining kinase activity in the test samples compared to the vehicle (dimethyl sulfoxide) reaction. IC (integrated circuit) ₅₀ Values and curve fits were obtained using Prism (GraphPad software).

Raw data were measured in duplicate as the percentage of compound activity of the tested kinase-inhibitor pairs. The Coefficient of Variation (CV) of the tested kinase inhibitor pairs and the differences from the duplicate observations (D) were calculated.

EXAMPLE 3 RDSR assay

TGF-beta (also known as TGF- β1) is a multifunctional, highly conserved cytokine, has many key functions in development, cell growth and apoptosis, and plays a key role in tissue repair reactions and acts as a potent immunomodulator. When activated TGF-beta homodimers bind to TGF-beta receptor 2, TGF-beta signaling is triggered, which in turn leads to recruitment and phosphorylation of TGF-beta receptor 1 (ALK 5). Activated TGF- β receptor 1 phosphorylates signal transduction molecules SMAD2 and SMAD 3. These molecules bind to the common mediator SMAD4 and translocate to the nucleus where they bind to short conserved DNA sequences called SMAD binding elements and induce transcription of multiple target genes.

Stable cellular reporters, RD SMAD reporters (RDSRs), were generated to test the ability of certain compounds disclosed herein to inhibit classical TGF- β1-induced SMAD signaling pathways in a cellular context. RD SMAD reporter (RDSR) cell lines were generated by stably integrating the SMAD cell reporter plasmid (Promega ), pGL4.48[ luc2P/SBE/Hygro ]) into the human rhabdomyosarcoma cell line RD (ATCC, CCL-136). Upon triggering SMAD signaling with, for example, the addition of TGF- β1, receptor-activated SMAD binds to SMAD Binding Elements (SBEs), resulting in expression of intracellular luciferase.

Rhabdomyosarcoma line RD (ATCC, CCL-136) was transfected with the SMAD reporter vector (Promega, E3671) and a polyclonal stable cell line was selected using hygromycin B. The transfected vector contained three copies of the Smad Binding Element (SBE) that driven transcription of the luciferase reporter luc2P (firefly north america). luc2P is a synthetically derived luciferase sequence with humanized codon optimization designed for high expression and reduced aberrant transcription. The luc2P gene contains the protein destabilizing sequence hPEST, which makes the luc2P protein level react faster to induction of transcription than those of luc 2. Intracellular luciferase was quantified by adding an equal volume (100 μl) of ONE-GLO substrate (Promega, E6120) and reading on an Envision plate reader within 10 minutes. Stable RDSR cell lines were tested by evaluating the response to human TGF-beta 1 (Andi Systems, 7754-BH-005) and myostatin (Andi Systems, 788-G8-010/CF) in a concentration-dependent manner after 24 hours of stimulation. IL-1 was used as a negative control and showed no response (data not shown). For compound evaluation, either the tool compounds (including valtoletin and PF-06952229 (TGF beta R1 inhibitor available from HY-136244 of medical chemistry Express company (MedChem Express)) or the test compounds were used as positive controls) were incubated with the cells for 1 hour at 37℃and then stimulated with 200pg/ml rhTGF-beta 1 for 24 hours. ONE-GLO (Promega) substrates were added to determine reporter activity and luminescence counts were collected on an Envision plate reader (Perkin Elmer). IC50 or EC50 were calculated using nonlinear regression curve fitting using prism software from graphic pad company.

The results of the biochemical assays described above are reported in table 3A.

TABLE 3A

Example 4 comparative RDSR assay

Comparative RDSR analysis was performed using IC50 values for selected compounds and standards obtained using the RDSR assay protocol described in example 3.

Tool compounds valtoletin (ALK 5 inhibitor, from medical chemistry express company, HY-19928) and PF-06952229 (TGF beta R1 inhibitor) were included in the study as comparator compounds. Selected example compounds or comparator compounds were incubated with cells for 1 hour at 37℃and then stimulated with 200pg/ml rhTGF-. Beta.1 for 24 hours. The activity of the reporter was determined by adding ONE-GLO (Promega) substrate and collecting luminescence counts on an Envision plate reader (Perkin Elmer).

Each of the example compounds and the comparator compounds were tested at drug concentrations of 20 micromolar to 1 nanomolar and IC50 values for each were calculated using Prism software from Graph Pad company. DMSO was used as negative control and all wells contained normalized DMSO concentrations. All drugs were incubated with RDSR cells for 1 hour at 37℃and then stimulated with 200pg/ml rhTGF-. Beta.1 for 24 hours. The results of the comparative RDSR assay showed that all compounds had an IC50 ALK-5 activity of less than 50nM, as shown in FIG. 1B.

These data demonstrate that certain example compounds inhibit ALK5 activity with efficacy similar to that exhibited by the comparator compounds valvulaze and PF-06952229.

Example 5 ALK2 off-target assay

Bone Morphogenic Proteins (BMPs) are a subfamily belonging to the TGF-beta ligand superfamily. BMP signaling pathways control many cellular processes during development and in adult tissues. At the cellular level, BMP homodimers bind to a heterotetrameric receptor complex consisting of two type I receptors (ALK 1, ALK2 or ALK 3) and two type II receptors (ACTRII, ACTRIIB or BMPRII). Upon complex formation, constitutively active type II receptors phosphorylate type I receptors. Activated type I receptors phosphorylate receptor-regulated SMAD proteins (SMAD 1/5/8) and activate these SMAD proteins, which in turn bind to the common mediator SMAD4 and translocate to the nucleus where they bind to the consensus DNA sequence of the promoter region (SMAD binding element, SBE) to induce transcription of the target gene.

Hepcidin is a small peptide synthesized primarily by the liver. It is the primary regulator of iron metabolism. BMP signaling positively regulates the transcription of hepcidin in liver cells.

To demonstrate that the selected compounds in the cellular context do not inhibit ALK2 (a close proximity to ALK 5), the effect of the selected compounds on BMP-6/ALK 2/hepcidin pathway activity was measured using hepcidin mRNA expression in human hepatocytes as a readout. In this study LDN214117 (an ALK2 inhibitor available from S7627 of the company celecoxib chemical (Selleck Chemicals)) was included as a positive control and valvulgare (an ALK5 inhibitor available from HY-19928 of the company medical chemistry express) was included as a negative control.

HepG2 human hepatocytes (ATCC HB-8065) were inoculated into EMEM (ATCC 30-2003) +10% FBS in 96-well plates and allowed to stand at 37℃with 5% CO ₂ Grow overnight. The following day, cells were pre-treated with one of the selected compounds (compound 01, 04 or 05) or control compounds at multiple concentrations, or with vehicle (DMSO) for one hour. Cells were then stimulated with recombinant human BMP-6 (Andi systems company 507-BP-020) at a final concentration of 40ng/ml for 3 hours in the presence of test compound or DMSO. BMP-6 free stimulation conditions were included for baseline hepcidin level measurements.

mRNA expression of hepcidin was measured by real-time RT-PCR using the Cells-to-CT 1 step TaqMan kit (Sieimer Feier A25603) according to the manufacturer's instructions. GAPDH levels were measured and used as a normalization control. Hepcidin primers: taqMan HAMP-FAM/MGB 20x (Semer Feishmania 4331182); GAPDH primer: taqMan GAPDH-VIC/MGB 20x (Semer Feishmania 4326317E). Relative hepcidin expression was obtained using the delta-delta Ct method.

The selected and control compounds LDN214117 and valatoxin were tested at drug concentrations of 20 μmol to 1.22nmol at 4-fold serial dilutions. All wells contained the same DMSO concentration. IC50 values were calculated using nonlinear regression model using Prism software from GraphPad. The results of the ALK-2 off-target assay are reported in Table 3B, where "++" represents 0.1. Mu.M <IC ₅₀ Is less than or equal to 1 mu M, and' ++ + "means IC ₅₀ >1μM。

TABLE 3B

Numbering of compounds	IC50
		05	++
04	++
		01	+++
LDN214117	++
		Vatochrotification	+++

Example 6 fibroblast-myofibroblast transformation (FMT) assay

Idiopathic Pulmonary Fibrosis (IPF) is a respiratory disease characterized by abnormal activation of fibroblasts of the lung and progressive fibrosis remodeling. Although the exact pathophysiological mechanisms of IPF remain unknown, TGF- β1 is thought to be the primary driver of the disease by mediating fibroblast to myofibroblast transformation (FMT). TGF-. Beta.1 induced myofibroblasts are thought to play a major role in fibrosis caused by excessive deposition of extracellular matrix.

To test the ability of selected compounds to inhibit TGF- β1 dependent fibroblast to myofibroblast transformation in an IPF-related disease model, FMT assays were performed using lung fibroblasts from IPF patients. In this assay, the conversion of fibroblasts to myofibroblasts is determined by the expression of the biomarker alpha smooth muscle actin (a-SMA).

Primary human bronchial fibroblasts derived from IPF patients were accessed on day zero and the medium was refreshed the next day. On the fifth day, the selected compound or control was added at an eight-point concentration response curve starting at 10 μm (semilog dilution). Each drug concentration condition was evaluated in biological duplicate. One hour after drug addition, cells were stimulated with 1.25ng/ml TGF- β1. Cells were fixed with formaldehyde 72 hours after TGF- β addition. High content imaging to assess cell number (using nuclear stained DAPI) and expression of a-SMA was performed. The following controls were run with selected compounds: valvulgare and approved IPF drug nildanib (starting concentration at 10 μm in an eight-point, semilogarithmic curve). As a negative control, 0.1% DMSO was also used, which matched the DMSO concentration in the treated wells. The following calculations were used to determine the cell number and percent inhibition of αsma expression (PIN):

Analysis of alpha SMA

Segmentation and quantification of the alpha SMA immunoreactivity by HCA algorithm, output Density x area (DxA)

Normalization of data of raw αsma (DxA) with respect to Percent Inhibition (PIN) values on a plate-by-plate basis

oμ _p Average alpha SMA value of positive control (TGF-. Beta.1+1. Mu.M SB 525334)

oμ _n Mean αsma value for vehicle control (TGF- β1+0.1% DMSO)

o X _i Is the alpha SMA value of the compound

IC of all Compounds ₅₀ Value (if computable)

NB：IC ₅₀ The value is based on inflection points

Analysis of% remaining cells

DAPI fluorescence was applied to quantification of imaged cell numbers based on HCA on a plate-by-plate basis

oμ _n Mean number of nuclei for vehicle control (TGF-. Beta.1+0.1% DMSO)

o X _i Number of compounds that are nuclei

By inducing complete inhibition of TGF- β1 mediated a-SMA expression (maximum (max) PIN greater than 75) in all donors, all compounds tested showed high efficiency. Tables 4A-4D summarize the results of all individual donors as well as controls. Figures 2A and 2B show individual experiments using three different IPF donor cells, and the inhibition ability of compound number 04 (figure 2A) and compound number 01 (figure 2B) to inhibit upregulation of αsma following TGF- β treatment.

Table 4A: summary for IPF03

¹ Potential cytotoxicity was defined as nuclear loss compared to the average nuclear count of vehicle controls>25%; indicating that observe>Minimum concentration at 25% nuclear loss

Table 4B: summary for IPF06

* Potential cytotoxicity was defined as >25% nuclear loss compared to the average nuclear count of vehicle controls, indicating the lowest concentration at which >25% nuclear loss was observed

* Incomplete sigmoid curves do not allow reliable potency determination

Table 4C: summary for IPF08

* Potential cytotoxicity was defined as >25% nuclear loss compared to the average nuclear count of vehicle controls, indicating the lowest concentration at which >25% nuclear loss was observed

Table 4D:

numbering of compounds	IPF03	IPF06	IPF08	Average IC50 (nM)
					05	60	50	40	50.0
04	40	50	30	40.0
					01	100	100	50	83.3
Vatochrotification	50	40	25	38.3
					Nidani cloth	600	1500	400	833.3

Example 7A 549 xenograft assay

To test the inhibition of target activity in vivo (ALK 5/TGF-bR1 inhibition) and inhibition of potential Janus kinase (JAK) signaling off-target activity by the compounds, an a549 murine xenograft model was used. ALK5 inhibitors (e.g., valtoletin) are expected to reduce the amount of the TGF-beta signaling molecule phospho-SMAD-2 (pSMAD 2) in a549 xenograft cells. In a549 cells, TGF- β mediated SMAD2 phosphorylation occurs at amino acid residues four hundred sixty-five and four hundred sixty-seven (both serine residues). JAK inhibitors (e.g., ruxotinib) are expected to reduce phosphorylation of Signal Transducers and Activator of Transcription (STAT) proteins (e.g., STAT 3) at amino acid residue 705 (tyrosine).

Female athymic nude mice (purchased from Charles River) were injected with approximately 350 ten thousand A549 cells (ATCC, CCL-185) at eight weeks of age. Specifically, cells were harvested and resuspended in normal RPMI medium (phenol red not added) and matrigel (Corning) 356237) at a one-to-one ratio, and 200 μl was injected into the right posterior flank of each mouse. Tumors were measured every three days by calipers and mice were randomly allocated to groups of three as tumors reached an average of 70-80 cubic millimeters. All compounds were resuspended in 1-methyl-2-pyrrolidone (sigma 494496) in water (90%) (10%) plus 20% Solutol (sigma 42966). Valatinib and ruxotinib as ALK5 signaling inhibitors and JAK signaling inhibitors, respectively, were included as controls. Both controls were given to three mice at 75 mg/kg. The drug suspension was sonicated for 15 minutes to produce a fine particle suspension, which was then administered to mice. Mice were dosed (oral gavage) with 100, 75, 50 or 10mg/kg of drug, three mice per group. A vehicle control group of three mice was used to establish baseline levels of phospho-SMAD-2 in tumor xenografts.

Four hours after drug administration, tumors were harvested, flash frozen and stored at minus eighty degrees celsius until further processing. Using Bio-Plex Pro ^TM The phospho-Smad 2 (Ser 465/Ser 467) set (burle corporation (BioRad) 171V 50019M) determines phospho-Smad-2 levels. Phosphostat 3 levels were determined using the bead/antibody set from the MILLIPLEXMAP STAT cell signaling bead 5-Plex kit (Millipore) 48-610 MAG. Both phosphosmad-2 and phosphostat 3 levels were compared to the beta-tubulin (millplex MAP beta-tubulin total magnetic bead MAPmate from each sample ^TM Miibo 46-713 MAG) or GAPDH (MILLIPLEX MAP GAPDH total magnetic beads MAPmate) ^TM Milbo 46-667 MAG) level. All analytes were analyzed in multiplex fashion using the Bio-Plex Pro cell signaling kit (Berle Corp. 171304006M). Briefly, frozen tumors (15 to 30 mg) were lysed in 100 μl lysis buffer in a bead mill tubeProcessed, and centrifuged. The resulting lysate was diluted 1:50 for the assay according to the manufacturer's instructions. The bead suspension was analyzed using the Luminex system (magix).

All compounds tested reduced the level of phospho-SMAD-2 in a dose-dependent manner (fig. 3A). Compound number 04 showed the strongest potency when administered at 75mg/kg, with 6% p-SMAD2 levels remaining compared to the average vehicle, which translates to 94% inhibition of biomarker p-SMAD 2. The percentage of p-SMAD-2 (Ser 465/467) compared to average vehicle treated mice is reported in Table 5A.

Table 5A: average p-SMAD2 (Ser 465/467)% of vehicle

Numbering of compounds	10mg/kg	50mg/kg	75mg/kg	100mg/kg
					Vatochrotification			32
Ruxotinib			120
					05	136	74	11	13
04	69	13	6	7
					01	97	33	13	8

The p-STAT3 level of the same tumor sample was determined. The JAK signaling inhibitor Lu Suoti ni showed an average 90% inhibition of STAT3 phosphorylation in tumor samples when administered at 75mg/kg (fig. 3B, table 5B). The clinical stage ALK5 inhibitor valtoletin showed a p-STAT3 level of 63% compared to vehicle control. The test compounds were comparable to, or showed lower p-STAT3 inhibition than, valatine (fig. 3B, table 5B), which demonstrates their specificity.

Table 5B: average p-STAT3 (Tyr 705)% of vehicle

Numbering of compounds	10mg/kg	50mg/kg	75mg/kg	100mg/kg
					Vatochrotification			63
Ruxotinib			10
					05	107	85	77	93
04	88	73	59	97
					01	92	70	66	76

EXAMPLE 8 longitudinal PK/PD analysis of p-SMAD2 in A549 xenograft mouse model

Xenograft studies performed in a similar manner to that described in example 7 were used to demonstrate the ability of compounds to inhibit TGF- β signaling over time. Accordingly, a longitudinal twenty-two hour, one dose study was performed in an a549 xenograft mouse model using compound 04 and the commercially available ALK5 inhibitor valtoletin. In this study, p-SMAD2 (Ser 465/Ser 467) and housekeeping Gene (GAPDH) were measured at 10 time points from zero (established with vehicle treated animals) up to 22 hours post-dose for a single drug dose of 50 mg/kg/subject (three subjects/dosing group).

In this study, xenografts were prepared and implanted into mice as follows. At five weeks of age, female athymic nude mice (purchased from Charles rituximab) were injected with approximately 210 ten thousand A549 cells (ATCC, CCL-185). Cells were harvested and resuspended in normal RPMI medium (no phenol red added) and matrigel (corning company 356237) at a one to one ratio, and two hundred microliters of cell suspension was injected into the right posterior flank of each mouse. Tumors were measured every three days by calipers and mice were randomly assigned to groups of three subjects as tumors reached an average of 70-80 cubic millimeters. Each of the test compounds (valatine, compound 04) was resuspended in 1-methyl-2-pyrrolidone (sigma, 494496) (10%) plus 20% Solutol (sigma 42966) in water (90%). The drug suspension was sonicated for 15 minutes to produce a fine particle suspension, and then administered to the test subject. The subject was dosed with the suspension (following oral gavage). A vehicle control group with three mice was used to establish baseline and time zero for phospho-SMAD-2 in tumor xenografts. The test compounds were administered to the corresponding subject group at 50 mg/kg.

Samples were obtained at 30 minutes, one hour, two hours, four hours, six hours, eight hours, twelve hours, sixteen hours and twenty-two hours after administration of the test compound. Tumors were harvested, flash frozen and stored at minus eighty degrees celsius until further processing. Plasma was collected from all animals by collection of whole blood via cardiac puncture followed by centrifugation in EDTA-containing tubes (BD company, microtainer tube, 365974). The group of three mice that received vehicle only served as the time zero for the two drug groups. Using Bio-Plex Pro ^TM The phospho-Smad 2 (Ser 465/Ser 467) set (burle 171V 50019M) determines phospho Smad-2 levels. The level of SMAD-2 phosphate was compared to GAPDH (MILLIPLEX MAP GAPDH total magnetic beads MAPmate) from each sample ^TM Milbo 46-667 MAG) level. All analytes were analyzed in multiplex fashion using the Bio-Plex Pro cell signaling kit (Berle Corp. 171304006M). Frozen tumor samples (15 to 30 mg) were lysed in 100 μl lysis buffer, processed in bead mill tubes, and centrifuged. The resulting lysate was diluted 1:50 for the assay according to the manufacturer's instructions. The bead suspension was analyzed using the Luminex system (magix).

As shown in fig. 4A, compound 04 and valtolterodine reduced p-SMAD2 levels (normalized to GAPDH) to similar levels 30 minutes after drug administration (53% of vehicle for valtolterodine and 55% of vehicle for compound 04). However, as shown in fig. 4A, valtolterodine cannot maintain containment of p-SMAD2 levels for an extended amount of time. As shown in fig. 4A, valtolterodine exhibited peak suppression (14% p-SMAD2 levels normalized to GAPDH) at one hour post-dosing compared to vehicle. The data in fig. 4A and 4B show that valtolterodine is cleared rapidly from the system, resulting in a rapid rise in p-SMAD2 levels after one hour. In contrast, compound 04 reached its peak suppression two hours after drug administration, achieving a p-SMAD2 level suppression to about 10% p-SMAD2 (normalized to GAPDH) compared to the mean value of the vehicle group.

Fig. 4B and 4C show PK/PD relationships of valtolterodine (fig. 4B) and compound 04 (fig. 4C). Cumulatively, the data in figures 4B and 4C show that compound 04 exhibits longer drug tumor and plasma exposure than valatine in vivo, resulting in prolonged suppression of tgfβ signaling molecule p-SMAD2 over time compared to equivalent administration levels of valatine.

Example 9 cachexia in cancer

Cachexia is associated with chronic disease and manifests as involuntary weight loss (e.g., greater than 5% of the pre-illness weight) caused by skeletal muscle and adipose tissue atrophy. This condition is different from other conditions (like anorexia) in that the fat reserves are depleted, but the muscle mass remains substantially unchanged. Cachexia affects more than half of cancer patients, resulting in poor quality of life (fatigue and weakness), and sometimes may even affect the therapeutic strategy of some individuals. Myostatin is a superfamily member of transforming growth factor-beta (TGF-beta) that is widely characterized as a negative regulator of muscle growth and development. Blocking this pathway would potentially be beneficial in cancer patients, particularly in advanced disease and metastatic patients with prominent cachexia.

To determine the ability of a compound to inhibit SMAD signaling triggered by myostatin, and thus likely to help reduce cachexia in a cancer patient, the following assay was performed. Human rhabdomyosarcoma cell line RD cell line was purchased from ATCC (CCL-136) and a stable SMAD reporter plasmid (Promega, pGL4.48[ luc2P/SBE/Hygro ]) was introduced to create a RD SMAD reporter (RDSR) cell line. Two tens of thousands of RDSR cells were plated in 96-well plates (Greiner bio-one company 655083) in DMEM medium supplemented with 10% fetal bovine serum. Cells were pre-incubated with selected compounds at various concentrations (8-point curve ranging from 10. Mu. Mol to 610 pmol) for 1 hour, followed by stimulation with 50ng/ml myostatin (Andi, 788-G8-010) for 24 hours. Luciferase signals were then read on an Envision plate reader with One-Glo substrate.

As seen in fig. 5, SMAD signaling triggered by myostatin was inhibited with compounds 01 and 04, with IC50 of 0.0571 μmol and 0.0191 μmol, respectively. Vatoxib inhibits myostatin induced SMAD signaling with an IC50 of 0.0164. Mu. Mol. This data demonstrates that the exemplary compounds can reduce involuntary weight loss, particularly muscle loss, in patients with cachexia.

EXAMPLE 10 longitudinal PK/PD analysis of p-SMAD2 in A549 xenograft mouse model

The study described in example 8 was extended to include compound 01. Accordingly, a longitudinal, twenty-two hour, one dose study was conducted using compound 01, using the same procedure used to prepare test subjects and the ability of the tested compounds to suppress TGF-beta signaling over time (as described in example 8), and the data was plotted along with the data obtained from compound 04 and the commercially available ALK5 inhibitor valtolitine in example 8 (re-plot according to example 10). For extension of this longitudinal study, p-SMAD2 (Ser 465/Ser 467) and housekeeping gene GAPDH were measured at 10 time points 22 hours after a single drug dose from zero up to 50 mg/kg/animal (three animals/group). A vehicle-only treatment group of three mice was used to establish baseline levels of p-SMAD2 in a549 xenograft tumor samples.

As in example 8, xenografts were prepared and implanted into mice as follows. At five weeks of age, female athymic nude mice (purchased from Charles rituximab) were injected with approximately 210 ten thousand A549 cells (ATCC, CCL-185). Specifically, cells were harvested and resuspended in normal RPMI medium (phenol red not added) and matrigel (Corning) 356237) at a one-to-one ratio, and 200 μl was injected into the right posterior flank of each mouse. Tumors were measured every three days by calipers and mice were randomly allocated to groups of three as tumors reached an average of 70-80 cubic millimeters. As in example 8, the tested compounds were resuspended in 1-methyl-2-pyrrolidone (sigma, 494496) (10%) plus 20% Solutol (sigma 42966) in water (90%) and the suspension sonicated for 15 minutes to produce a fine particle suspension, which was then administered to each of the subjects as oral gavage at 50 mg/kg/time point. A vehicle control group with three mice was used to establish baseline and time zero for phospho-SMAD-2 in tumor xenografts.

As in example 8Measurements were obtained, after a number of times (in particular, 30 minutes, one hour, two hours, four hours, six hours, eight hours, twelve hours, sixteen hours and twenty-two hours) following drug administration, tumors were harvested, flash frozen and stored at minus eighty degrees celsius until further processing. Plasma was collected from all animals by collection of whole blood via cardiac puncture followed by centrifugation in EDTA-containing tubes (BD company, microtainer tube, 365974). The group of three mice that received vehicle only served as the time zero for the two drug groups. Using Bio-Plex Pro ^TM The phospho-Smad 2 (Ser 465/Ser 467) set (burle corporation (BioRad) 171V 50019M) determines phospho-Smad-2 levels. The level of phospho-SMAD-2 was compared to GAPDH (MILLIPLEX MAP GAPDH total magnetic beads MAPmate) from each sample ^TM Milbo 46-667 MAG) level. All analytes were analyzed in multiplex fashion using the Bio-Plex Pro cell signaling kit (Berle Corp. 171304006M). Briefly, frozen tumors (15 to 30 mg) were lysed in 100 μl lysis buffer, processed in bead mill tubes, and centrifuged. The resulting lysate was diluted 1:50 for the assay according to the manufacturer's instructions. The bead suspension was analyzed using the Luminex system (magix).

Fig. 6A shows p-SMAD2 levels normalized to housekeeping protein (GAPDH), expressed as a percentage of mice treated in vehicle groups. Compound 01, compound 04 and valtolterodine reduced p-SMAD2 levels (normalized to GAPDH) to similar levels 30 minutes after drug administration (53% vehicle for valtolterodine, 55% vehicle for compound 04, and 57% vehicle for compound 01). However, valtolterodine was unable to maintain p-SMAD2 suppression for more than one hour and reached its peak suppression one hour after dosing, leaving a 14% level of p-SMAD2 compared to vehicle (normalized to GAPDH). The data in fig. 6A and 6B and table 6 show that valtoletin is rapidly cleared from mice, resulting in a rapid rise in p-SMAD2 levels after one hour. Compound 04 reached its peak suppression two hours after drug administration, one hour later than valatine, and reached lower p-SMAD2 levels (normalized to GAPDH) compared to 10% p-SMAD2 of valatine (compared to the mean of the vehicle group). Compound 01 also reached its peak p-SMAD2 inhibition 2 hours after dosing, reducing p-SMAD2 levels in tumor tissue to 24% of vehicle.

Table 6 average (average from 3 mice) percent of p-SMAD2 levels compared to vehicle group determined in A549 tumor samples after the indicated time following 50mg/kg dosing (following oral gavage). Each value represents the average of 3 mice/time point/group. Bold values represent peak biomarker inhibition.

Fig. 6B shows the PK/PD relationship of valtoletin, fig. 6C shows the PK/PD relationship of compound 04, and fig. 6D shows the PK/PD relationship of compound 01. Cumulatively, the data in figures 6B, 6C and 6D show that compound 04 and compound 01 exhibit longer drug tumor and plasma exposure in vivo, resulting in prolonged suppression of the tgfβ signaling molecule p-SMAD2 over time compared to valvulgare.

EXAMPLE 11 bleomycin-induced pulmonary fibrosis study

The most common animal model of pulmonary fibrosis is the bleomycin-induced rodent pulmonary fibrosis model. This model is commonly used to study biological and potential therapies for fibrotic diseases affecting the lung, such as Idiopathic Pulmonary Fibrosis (IPF). Bleomycin is a cytotoxic drug that is administered to mice or rats and causes direct cell damage triggered by DNA strand breaks, which leads to overproduction of reactive oxygen species, thereby triggering inflammation, pulmonary toxicity, activation of fibroblasts and subsequent fibrosis. Fibrosis is characterized by abnormal activation of lung epithelial cells and accumulation of fibroblasts and myofibroblasts that overproduce extracellular matrix (e.g., collagen). To test the ability of compound 01 and compound 04 to reduce fibrosis in vivo, bleomycin-induced mouse lung fibrosis models were used and endpoints such as lung hydroxyproline levels, surrogate markers of collagen and fibrosis, and histological scores of fibrosis were set.

Fig. 7A shows the overall study design. For this study, male C57BL/6J mice (purchased from charles) of about 6 to 7 weeks of age were weighed on the day prior to study day 0 to establish a baseline. All arms contained a total of 16 animals except the sham surgery/vehicle group (containing 10 animals). In the morning of study day 1, all animals except the sham surgery/vehicle group were given intranasal bleomycin at 4 international units/kg. Bleomycin was prepared in 0.9% sodium chloride solution. All drugs were prepared in 10% Tween 20. At night on study day 1, compound 04 or compound 01 was administered as 25 or 50mg/kg per oral gavage. For all subsequent days of the twenty-day study, animals were dosed twice daily with 25 or 50mg/kg of compound 04 or compound 01 as oral gavage. On the twenty-first day, lungs were harvested and either flash frozen or fixed in paraformaldehyde. The left lobe of the lung was subjected to hydroxyproline quantification, which was used as biomarker for fibrosis and total collagen. The remaining lung tissue was fixed in paraformaldehyde and processed for histological examination after hematoxylin-eosin (H & E) and Masson trichromatic staining. Histological sections were scored using the following modified Ashcroft scale (adapted from Hubner, R-H et al, standardized quantification of pulmonary fibrosis in histological samples [ normalized quantification of lung fibrosis in histological samples ], bioTechniques [ Biotechnology ] 2008:44:507-17, doi:10.2144/00011272):

Grade 0 = normal lung

Class 1 = minimum detectable alveolar wall thickening

Class 2 = slightly thickened alveolar wall

Grade 3 = moderately continuous thickened wall accompanying fibrous nodules

Grade 4 = thickened septal and confluent fibrotic mass, totally smaller than microscopy

10% of visual field

Grade 5 = increased fibrosis, in which lung structures are significantly impaired and microscopic formation occurs

10% -50% of the visual field of fibrous strips or small fibrous masses

Grade 6 = large continuous fibrotic masses, combined over 50% of the microscopic field of view

Grade 7 = severe deformation of structure and large fibrous area

Grade 8 = total pulmonary fiber occlusion in microscopic field of view

Mice were weighed on the seventh, fourteenth and twenty-first days to monitor weight changes and mortality. Clinical observations were recorded daily.

Fig. 7B shows the amount of hydroxyproline measured from a portion of lung tissue harvested from each mouse remaining on the twenty-first day of the study (μg hydroxyproline/mg lung tissue). Using the Dunnett multiple comparison test, the hydroxyproline amounts of all groups showed statistically significant differences compared to the bleomycin/vehicle group, except that the arms receiving 50mg/kg of compound 04 twice daily were not statistically significant (adjusted p-value = 0.0662). The most statistically significant reduction in hydroxyproline was seen in the arms of animals receiving 25mg/kg of compound 01 twice daily (adjusted p-value < 0.0001). The arms receiving 25mg/kg of compound 04 and 50mg/kg of compound 01 were calculated to be statistically significant compared to the bleomycin/vehicle group, with adjusted p-value=0.0031 and adjusted p-value=0.0024, respectively.

Fig. 7C shows histological analysis of five animals randomly selected from each treatment group using Ashcroft scores. The Ashcroft score was determined by a single pathologist using a modified Ashcroft scale applied to tissues stained with Masson trichromatic and H & E. Ashcroft score is used to indicate the severity of fibrosis and changes in lung tissue structure resulting from fibrosis. At least 2 animals in all treatment groups had significantly lower Ashcroft scores compared to the average Ashcroft score of the bleomycin/vehicle group. The group comparison between study arms receiving compound 04 and compound 01 (all doses) was not statistically significant compared to the bleomycin/vehicle group.

Figures 7D and 7E show images of lung tissue stained with Masson trichromatography obtained from one animal per treatment group. Each image is 5 times magnified and the entire tissue is shown in the lower right hand corner, with the rectangle indicating the magnified area relative to the entire tissue mass. In particular, fig. 7D and 7E show representative lung images from animals treated with sham surgery/vehicle (top left image in fig. 7D and 7E, same image), and representative lung images from animals treated with bleomycin/vehicle (top right image in tables 7D and 7E, same image). The lower left images in figures 7D and 7E were obtained from animals that responded to 25mg/kg of compound 04 or compound 01 administered twice daily (BID), respectively. The lower right images in figures 7D and 7E were obtained from animals that responded to 50mg/kg of compound 04 or compound 01, respectively, administered twice daily (BID). Masson trichromatography gives blue tissue staining when the tissue contains mature collagen (a marker useful for identifying fibrosis). As seen in the upper right images of fig. 7D and 7E, bleomycin/vehicle treatment resulted in dense lung tissue, with large blue spots indicating severely fibrotic tissue regions. Treatment with compound 04 or compound 01 reduced the formation of fibrosis, and lung tissue obtained from animals that responded to compound 04 or compound 01 was similar to healthier lungs (bottom images in fig. 7D and 7E). These data indicate that compounds 04 and 01 show efficacy in some animals induced to develop severe fibrosis, which may indicate the potential of these compounds to treat fibrotic disorders in humans.

Example 12 expression of epithelial to mesenchymal transition Gene in A549

To evaluate whether compounds 01 and 04 were able to inhibit epithelial to mesenchymal transition (EMT), an in vitro model of a549 lung fibroblasts was used. A549 cells were purchased from ATCC (CCL-185) and grown in RPMI medium supplemented with 10% Fetal Bovine Serum (FBS). On the day of the experiment, ten thousand a549 cells were plated in a flat, tissue culture-coated 96-well plate in a total volume of 80 μl. Drugs (including positive control compounds valtoletin, PF-06952229 and LY 3200882) were added in eight-point, one-to-three serial dilutions, with a maximum concentration of 5. Mu. Mol for each drug, making the total volume in each well 90. Mu.l. In all wells, DMSO was normalized to 10mmol stock solution used to solubilize each drug. After one hour of drug pretreatment, recombinant human TGF- β1 was added to a final concentration of 5ng/ml to a volume of 100 μl. Recombinant human TGF-. Beta.1 was purchased from Andi systems Co (7754-BH-005) and stock solutions thereof were prepared according to the manufacturer's instructions and diluted in RPMI medium supplemented with 10% FBS. At 37℃with 5% CO ₂ 96-well plates with a volume of 100 μl were incubated for 48 hours. At the end of the incubation period, the gene expression levels of the various EMT markers were assessed using custom designed QuantiGene Plex gene expression assays. The genes selected are CDH1 (E-cadherin), CDH2 (N-cadherin), SNAI1 (snail), SNAI2 (slug), VIM (vimentin), SPARC, GALNT6, CTNNB1 (β -catenin), TGFB1 and MAML3.

Briefly, cell lysates were prepared in 96-well plates using the QuantiGene sample processing kit-cultured cells (QS 0100). The cell lysate is then subjected to bead hybridization, multiple probe hybridization steps, and SAPE labeling according to the manufacturer's instructions. The plates were read on a Luminex magix instrument. With the exception of CDH1 (E-cadherin), all selected genes were expected to have increased expression following induction of EMT by TGF- β1 stimulation, CDH1 being known to be down-regulated when cell-to-mesenchymal phenotype transformation and cell adhesion are disrupted.

FIG. 8A shows inhibition of CDH1 (E-cadherin) down-regulation by positive control TGF-beta signaling inhibitors (vartoletin, PF-06952229, and LY 3200882), and compound 01 and compound 04. Figures 8B-8J show the positive control TGF-beta signaling inhibitors (valtoletin, PF-06952229 and LY 3200882), and the inhibition of the induction of EMT by compound 01 and compound 04 for nine genes (i.e., CDH2 (N-cadherin), SNAI1 (snail), SNAI2 (slug), VIM (VIM), SPARC, GALNT6, CTNNB1 (β -catenin), TGFB1 and MAML3 EMT). As indicated by these figures, the addition of compound 01 or compound 04 inhibited TGF- β triggered signaling and thus blocked EMT conversion in a549 cells in vitro similar to the TGFBR1/ALK5 inhibitors valtoletin, PF-06952229, and LY3200882 used as references in this example.

The information presented in the above figures indicates that compounds 01 and 04 may potentially treat the disease (EMT contributes to its progression), as well as complications such as cancer metastasis and cancer-associated tissue fibrosis, as well as other fibrotic diseases (such as idiopathic pulmonary fibrosis).

EXAMPLE 13 Maximum Tolerated Dose (MTD) study

Determining the MTD is critical to calculating a therapeutic index (also referred to as a therapeutic ratio), which is the ratio of the MTD to the dose required to remove a biomarker or display efficacy in the same species. Evaluation of acute and chronic Maximum Tolerated Doses (MTD) of compound 04 and compound 01 was performed by oral gavage administration of each compound to athymic nude mice according to the following procedure.

Female athymic nude mice (NCRNU) five weeks old were purchased from tacon biosciences (Taconic Biosciences) of renssella (Rensselaer NY) new york and acclimatized for one week. When engaged in the study, the mice were approximately four months of age. All compounds were resuspended in 1-methyl-2-pyrrolidone (sigma, 494496) (10% v/v) plus 20% solutol (sigma 42966) in water (90% v/v). At the beginning of the acute and chronic MTD study, mice were randomized according to baseline weight (day 1).

For the acute MTD study, mice were administered three ascending doses of each compound every other day (two animals/group). Mice were dosed with 100mg/kg on the first day, 500mg/kg on the third day, and 1000mg/kg on the fifth day by oral gavage. Body weight was measured daily and percent change in body weight was calculated.

For chronic MTD studies, three groups (three mice/group) were dosed daily with 100, 300, or 1000mg/kg of compound 04 or compound 01 and five days were observed continuously during recovery after dosing. Body weight was measured and clinical observations were recorded daily for ten consecutive days. Vehicle-only treatment groups comprising three mice were included to evaluate potential toxicity of vehicle. The percentage change in body weight per day was calculated based on the measurement on day 1.

Fig. 9A shows the percentage of body weight change observed in the acute MTD study. Each line represents an animal dosed with either compound 04 or compound 01. Animals of both drug treatment groups showed slight weight loss on day 2 after the first dose of 100 mg/kg. However, body weight was restored in all animals of both drug treatment groups throughout the study, even after administration of higher drug doses. These data indicate that both compounds are well tolerated in mice when administered at a single dose of 1000 mg/kg.

Figures 9B and 9C show the percentage of body weight change observed in chronic MTD studies using compound 04 (figure 9B) or compound 01 (figure 9C). Data are presented as the mean plus or minus standard deviation of three animals in each treatment group. As shown by these data, compound 04 was well tolerated at 300mg/kg. However, when treated with 100mg/kg of compound 04, two of the three animals in the 100mg/kg group lost more than 15% of their body weight. Mice treated with compound 04 showed signs of toxicity at 1000 mg/kg. At a dose of 1000mg/kg, all three mice died during the course of the study.

From these data, the MTD of Compound 04 was determined to be 300mg/kg. Based on an effective dose of 50mg/kg derived from studying p-SMAD2 inhibition in the a549 xenograft mouse model (as described above), the therapeutic index of compound 04 was estimated to be 6x (300 mg/kg MTD/50mg/kg PD efficacy).

Turning to the MTD study with compound 01, when compound 01 was administered to mice at 100mg/kg, the mice exhibited some clinical signs of distress, exhibited a bowed back posture, and one animal died on day seven, and one mouse exhibited weight loss of greater than 30%. However, at 300mg/kg, mice administered compound 01 exhibited only slight weight loss, with two of the three animals losing 10% to 15% of their body weight by day seven, and then regained more than 90% of their original body weight by day ten. At 1000mg/kg, compound 01 was intolerant: all three subjects died from toxicity during the course of the study.

Based on these data, the MTD of Compound 01 was determined to be less than 300mg/kg. Based on an effective dose of 75mg/kg derived from studying p-SMAD2 inhibition in the a549 xenograft mouse model (as described above), the therapeutic index of compound 01 was estimated to be less than 4x (300 mg/kg MTD/75mg/kg PD efficacy). Figures 9D and 9E show the overall mortality associated with the different treatment arms of the chronic MTD study involving compound 04 and compound 01, respectively, indicating that both compounds exceeded their 1000mg/kg MTD when administered for five consecutive days.

EXAMPLE 14 phosphate-SMAD 2 assay

TGF- β is a pleiotropic cytokine involved in extremely conserved pathways related to cell growth, differentiation and development. When activated TGF- β homodimers bind to TGF- β receptor 2 (TGFbR 2), TGF- β signaling is triggered, which in turn leads to recruitment and phosphorylation of TGF- β receptor 1 (TGFbR 1). Activated TGFbR1 phosphorylates signal transduction molecules SMAD2 and SMAD3, and then SMAD2 and SMAD3 bind to common mediator SMAD4 and translocate to the nucleus where they alter gene transcription. In this assay, phospho-SMAD 2 (S465/S467), which is a directly phosphorylated substrate of TGFbR1, is used as a readout of TGF- β pathway activity. Varrozen (a TGFbR1 inhibitor available from HY-19928 of medical chemistry express company) was included as a positive control.

RD SMAD reporter (RDSR) cell lines were generated by stably integrating the SMAD reporter plasmid (Promega, pGL4.48[ luc2P/SBE/Hygro ]) into the human rhabdomyosarcoma cell line RD (ATCC, CCL-136).

RDSR cells were inoculated into DMEM (ATCC 30-2002) +10% FBS in 96-well plates and allowed to stand at 5% CO ₂ And grown overnight at 37 ℃. The following day, cells were pretreated with multiple concentrations of compound 04, compound 01, or valatide, or with vehicle (DMSO) for one hour. Cells were then stimulated with recombinant human TGF-beta 1 (Andi systems Co 7754-BH-005) at a final concentration of 100ng/ml in the presence of Compound 04, compound 01, vatoxitinit or DMSO for 30 minutes. TGF- β1 free stimulation conditions were included for baseline phospho-SMAD 2 level measurements.

Using Bio-Plex Pro ^TM phospho-Smad 2 (Ser 465/Ser 467) beads/antibody set (burle 171V 50019M) determines phospho-Smad 2 levels. phosphate-SMAD 2 levels relative to beta-tubulin (millplex MAP beta-tubulin total magnetic bead MAPmate from each sample ^TM Miibo 46-713 MAG) or GAPDH (MILLIPLEX MAP GAPDH total magnetic beads MAPmate) ^TM Milbo 46-667 MAG) level.All samples were analyzed in multiplex fashion using the Bio-Plex Pro cell signaling kit (Berle Corp. 171304006M). Briefly, at the end of the treatment, RDSR cells were rinsed with ice-cold Phosphate Buffered Saline (PBS) and lysed in 55. Mu.l of lysis buffer for 30 min. The resulting lysate was used for the assay according to the manufacturer's instructions. The bead suspension was analyzed using the Luminex system (magix).

Compound 04, compound 01 and valatide were tested at drug concentrations of 20 μmol to 1.22nmol using 4-fold serial dilutions. All wells contained the same DMSO concentration. IC50 values were calculated using a nonlinear regression model of Prism software from GraphPad, where all compounds showed values less than 200 nM. A graphical representation of the longitudinal results from this phosphoric acid-SMAD 2 assay is shown in fig. 10.

EXAMPLE 15 JAK Selectivity assay

Janus kinase (JAK) signaling inhibitors, despite FDA approval, have been shown to cause anemia and lymphopenia, and inhibit the immune system in some people. These properties are undesirable in ALK5 inhibitors that are used in a repeat/chronic dosing regimen in non-oncological indications. To demonstrate the extent of JAK signaling inhibition of compound 01 and compound 04, in vitro cell assays were utilized and IC50 values were calculated and compared to various JAK inhibitor molecules.

To evaluate the efficacy of compound 04 and compound 01 in inhibiting JAK signaling, HEK blue IL-12 reporter assays were used. Cells were purchased from Invivogen (catalog number: hkb-il 12) and maintained according to the instructions of the supplier. On the day of the experiment, cells were harvested by repeatedly patting the flask to shake loose cells attached to the bottom of the flask. Cells were then harvested, washed and resuspended in DMEM medium supplemented with 10% fetal bovine serum. Mu.l of the cell suspension was inoculated into 384-well tissue culture coated plates containing a total of about 10,000 cells. Compound 01, compound 04 or tool compound was added at a maximum concentration of 20 μmol using an ECHO acoustic dispenser. Test compounds were added to duplicate wells in 3-fold dilution curves using ECHO "dose response" software. DMSO was backfilled to normalize DMSO in all wells. After one hour incubation in an incubator at 37 ℃, the plates were removed from the incubator and 7.5nl of human recombinant IL-12 (50 ug/ml) was delivered to all wells with ECHO (final concentration of IL-12 12.5 ng/ml). Recombinant human IL-12 was purchased from Andi systems Inc. (catalog number: 219-IL-005). After incubation in an incubator at 37 degrees celsius for 24 hours, the plates were removed, 5 μl of supernatant was removed and mixed with 45 μl of Quanti-blue solution (ex invitrogen company, rep-qbs) in a bottom transparent black 384 well plate. After incubation for 30 minutes at room temperature, the optical density was measured at 620nm using an Envision plate reader from perkin elmer. When secreted alkaline phosphatase triggered by IL-12 signaling is present in the supernatant, a color change can be detected at this wavelength. The lack of signal indicates that the test compound inhibited the reporter. Table 8 lists the tool compounds included in the assay as positive controls.

TABLE 8 tool Compounds

JAK tool compounds Lu Suoti ni, barytinib, phenanthrene Zhuo Tini and tofacitinib showed strong efficacy in this assay, and IC50 values for all JAK inhibitors tested were calculated to be below 300nmol. Lu Suoti Ni is described as the most potent JAK2 inhibitor, which is also the most potent in this assay, with an IC50 value of 31nmol. Compound 04 (3.7 μmol IC 50) and compound 01 (3.8 μmol IC 50) showed weak inhibition in this assay, indicating that these compounds have minimal JAK signaling potency. The ALK5 tool compound valtoletin also showed weak off-target activity of 18.5 μmol IC 50. The results of this measurement are shown in table 11. As shown in fig. 11, compounds 01 and 04 show similar properties to valcanite with regard to off-target JAK2 inhibition, which is distinguished from the various JAK2 inhibitors used for reference in the study.

Example 16 in vitro 3D liver fibrosis assay

Using Visikol OpenLiver for fibrosis ^TM HepaRG ^TM NP 3D modelAdditional fibrosis assays were performed on selected compounds. OpenLiver ^TM HepaRG ^TM The NP 3D model is characterized by the ability to generalize the pathology of a variety of liver diseases. Whether evaluating the likelihood of a compound inducing a disease or exploring a target for therapeutic remission of a disease state, hepavg ^TM NP 3D models can outline disease features such as hepatocyte lipid accumulation, insubstantial cellular phenotypic shift, and collagen matrix deposition.

The hepavg (longsha, NSHPRG, lot number HNS 1013) and non-parenchymal (NP) cells (astrocytes, kupfu cells and LSECS (longsha, lot number HUM 201221)) were thawed, assessed for viability, and were inoculated into ultra low adhesion round bottom plates (corning, 4515) at a ratio of hepatocytes to NP cells of 60:40. Cells were initially supplemented with hepavg thawing, plates and general purpose supplements (hepavg Thaw, plate&General Purpose Supplement) (Semerle, HPRG 770) and GlutaMax (Semerle, 35050061) overnight in William's E medium. The next day, the medium was half-exchanged with William's E medium supplemented with hepavg maintenance supplement (HPRG 720, zemoeimeric feier) and GlutaMax (35050061). Every 2-3 days, the medium was half-exchanged with fresh maintenance medium until spheroids were formed (about 7 days after access). The spheroids were then further cultured for 7 days, at which point the medium was exchanged for medium containing the treatment of interest. All cell cultures occurred at 37 ℃/5% CO ₂ In an incubator.

On the day of treatment, each well was pretreated with 1 μm valatine, compound 04 or compound 01 or DMSO alone (0.05%) for 1 hour. TGF-. Beta.1 (PeproTech, 100-21) was reconstituted to a stock concentration of 20 μg/mL in sterile 4mM HCl according to manufacturer's guidelines. The stock solution was further diluted in maintenance medium to a final concentration of 100 ng/mL. After pretreatment, each well was treated with 100ng/mL of TGF- β1 or vehicle control containing 0.5%4mM HCl. After the addition of the test compound, the spheroids were incubated for 72 hours.

At the time point of 72 hours, the spheroids marked for labeling were treated with a vitality dye (zemoeimer feier company, 65-0865-14) to determine vitality, fixed in neutral buffered formalin (Fisher), SF 100-20), washed three times in PBS, and then stored in PBS until labeling.

Spheroids were simultaneously permeabilized and blocked using goat serum diluted in Triton X100-containing PBS. Spheroids were then labeled with antibodies to pan collagen. Secondary antibodies were then used to fluorescent indicate the primary label and DAPI was added as a nuclear counterstain. All labelling was performed in the same solution used for permeabilization and blocking. Labeling and after light dehydration in methanol, with Visikol HISTO-M ^TM The spheroids were cleaned and then imaged. The materials used for this determination are listed in table 10.

Table 10.

The labeled spheroids were imaged at 10 x in z steps of 10 μm using an ImageExpress micro confocal high content imaging system of molecular instruments (Molecular Devices). The image is acquired and saved as a 16-bit 2048x 2048 TIF file for further processing.

Using Visikol HISTO-M ^TM The clean spheroid allows a clear image to be obtained throughout the volume of the spheroid, allowing analysis of the entire z-stack. The DAPI channel was used to determine the spheroid area for each z slice to calculate the spheroid volume. For pan collagen, a manual thresholding was performed to select bright areas of collagen + staining on z-stacks. The thresholded area or integrated density of each slice is summed and multiplied by the z step size (10 μm) to give the total volume in the spheroid (for ubiquity). Results for pan collagen are reported as normalized to the volume of spheroids.

TGF-beta induced overt collagen expression is as expected. Vatochrotinit, compound 04 and Compound 01 inhibited TGF- β induced overt collagen expression to different levels, with Compound 01 being the most potent (FIG. 12). The results of this assay indicate that the tested compounds will be useful in providing treatment of fibrotic diseases.

While example embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the embodiments encompassed by the appended claims.

Equivalents and scope

In the claims, articles such as "a" and "an" and "the" may mean one or more than one unless indicated to the contrary or otherwise apparent from the context. Unless indicated to the contrary or otherwise apparent from the context, if one, more than one, or all of the group members are present in, used in, or otherwise related to a given product or method, the claims or descriptions including an "or" between one or more members of the group are deemed satisfied. The present disclosure includes embodiments in which exactly one group member is present in, used in, or otherwise associated with a given product or method. The present disclosure includes embodiments in which more than one or all of the group members are present in, used in, or otherwise associated with a given product or method.

Furthermore, this disclosure covers all variations, combinations, and permutations that incorporate one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims into another claim. For example, any claim that depends from another claim may be modified to include one or more limitations found in any other claim that depends from the same base claim. Where elements are presented as a list (e.g., in the form of markush groups), each element subgroup is also disclosed, and any one or more elements may be removed from the group. It should be understood that, in general, where reference is made to the present disclosure or aspects of the disclosure containing particular elements and/or features, certain embodiments of the disclosure or aspects of the disclosure consist of or consist essentially of such elements and/or features. For the sake of simplicity, those embodiments are not specifically set forth herein in such utterances. It should also be noted that the terms "comprising" and "including" are intended to be open ended and allow for the inclusion of additional elements or steps. Endpoints are included for a given range. Furthermore, unless indicated otherwise or otherwise evident from the context and understanding of one of ordinary skill in the art, values expressed as ranges in the various embodiments of the disclosure may assume any particular value or subrange within one tenth of the unit of the range to the lower end of the range, unless the context clearly dictates otherwise.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. The scope of the embodiments described herein is not intended to be limited by the foregoing description, but rather is set forth in the following claims. Those skilled in the art will appreciate that various changes and modifications can be made to the invention without departing from the spirit or scope thereof, as defined by the appended claims.

Claims (71)

1. A compound of formula (I):

or a pharmaceutically acceptable salt thereof, wherein:

R ¹ is C ₁ -C ₅ Alkyl, C ₃ -C ₅ Carbocycles or halogens;

R ² is-H, halogen, C optionally substituted by one or more-F ₁ -C ₃ Alkyl, or cyclopropyl optionally substituted with one or more-F;

R ³ is-H, halogen, C optionally substituted by one or more-F ₁ -C ₃ Alkyl, or cyclopropyl optionally substituted with one or more-F; and is also provided with

Ring G isWherein->Indicating the point at which ring G is attached to-N (H) -; or (b)

Ring G is C optionally substituted with ₆ -C ₁₀ Aryl:

(i) One or more halogens;

(ii) Sulfonamide;

(iii) Optionally by one or more C ₁ -C ₆ Alkyl-substituted monocyclic, bicyclic or spiro C ₃ -C ₁₀ Carbocycles, or C optionally substituted by hydroxy or one or more halogens ₃ -C ₆ A carbocycle, wherein the carbocycle is attached to ring G by a single bond or a methylene or ethylene linker at a position on ring G that is meta or para with respect to the-N (H) -attached to ring G; or (b)

(iv) May contain up to 3 heteroatoms independently selected from N and O and optionally and independently substituted with one or more C' s ₁ -C ₆ Alkyl-substituted monocyclic, bicyclic, bridged or spiro C ₃ -C ₁₀ Heterocycles, or C optionally substituted by hydroxy or one or more halogens ₃ -C ₆ Carbocycles wherein the heterocycle is attached to ring G by a single bond or a methylene or ethylene linker at a position on ring G that is meta or para to the-N (H) -attached to ring G.

2. The compound of claim 1, wherein R ¹ Is C ₁ -C ₅ Alkyl, or C ₃ -C ₅ Carbocycles.

3. The compound of claim 1, wherein R ¹ Is halogen.

4. The compound of claim 1, wherein R ¹ Is methyl, cyclopropyl or chloro.

5. The compound of any one of claims 1-4, wherein R ² is-H, halogen, -CH ₃ 、-CF ₃ Or cyclopropyl.

6. The compound of claim 5, wherein R ² is-H.

7. The compound of any one of claims 1-6, wherein R ³ is-H, halogen, -CH ₃ 、-CF ₃ Or cyclopropyl.

8. The compound of claim 7, wherein R ³ is-H.

9. The compound of any one of claims 1-8, wherein ring G is

10. The compound of any one of claims 1-8, wherein ring G is C substituted with ₆ -C ₁₀ Aryl:

(i) One or more halogens;

(ii) Sulfonamide;

(iii) Optionally by one or more C ₁ -C ₆ Alkyl-substituted monocyclic, bicyclic or spiro C ₃ -C ₁₀ Carbocycles, or C optionally substituted by hydroxy or one or more halogens ₃ -C ₆ A carbocycle, wherein the carbocycle is attached to ring G by a single bond or a methylene or ethylene linker at a position on ring G that is meta or para with respect to the-N (H) -attached to ring G; or (b)

(iv) May contain up to 3 heteroatoms independently selected from N and O and optionally and independently substituted with one or more C' s ₁ -C ₆ Alkyl-substituted monocyclic, bicyclic, bridged or spiro C ₃ -C ₁₀ Heterocycles, or C optionally substituted by hydroxy or one or more halogens ₃ -C ₆ Carbocycles wherein the heterocycle is in a meta or para position on ring G relative to the-N (H) -attached to ring GIs attached to ring G by a single bond or a methylene or ethylene linker.

11. The compound of claim 10, wherein ring G is substituted with:

i) One or more halogens;

ii) a sulfonamide;

iii) Optionally by one or more C ₁ -C ₆ Alkyl-substituted monocyclic, bicyclic or spiro C ₃ -C ₁₀ Carbocycles, or C optionally substituted by hydroxy or one or more halogens ₃ -C ₆ A carbocycle, wherein the carbocycle is attached to ring G by a single bond or a methylene or ethylene linker at a position on ring G that is meta or para with respect to the-N (H) -attached to ring G;

iv) optionally substituted with one or more C ₁ -C ₆ Alkyl-substituted monocyclic C ₃ -C ₇ Carbocycles, or C optionally substituted by hydroxy or one or more halogens ₃ -C ₆ A carbocycle; or (b)

v) optionally with one or more C ₁ -C ₆ An alkyl-substituted cyclohexyl group, or C optionally substituted with hydroxy or one or more halogens ₃ -C ₆ Carbocycles.

12. The compound of claim 10 or 11, wherein the carbocycle attached to ring G is unsubstituted.

13. The compound of claim 10, wherein ring G is substituted with: may contain up to 3 heteroatoms independently selected from N and O and optionally and independently substituted with one or more C' s ₁ -C ₆ Alkyl-substituted monocyclic, bicyclic, bridged or spiro C ₃ -C ₁₀ Heterocycles, or C optionally substituted by hydroxy or one or more halogens ₃ -C ₆ Carbocycles wherein the heterocycle is attached to ring G by a single bond or a methylene or ethylene linker at a position on ring G that is meta or para to the-N (H) -attached to ring G.

14. As claimed in claim 13A compound as described wherein ring G is substituted with: may contain up to 3 heteroatoms independently selected from N and O and optionally and independently substituted with one or more C' s ₁ -C ₆ Alkyl-substituted monocyclic C ₅ -C ₆ Heterocycles, or C optionally substituted by hydroxy or one or more halogens ₃ -C ₆ Carbocycles.

15. The compound of claim 14, wherein ring G is substituted with: optionally and independently is one or more C ₁ -C ₆ Alkyl-substituted piperazinyl, morpholinyl, piperidinyl or oxacyclohexyl, or C optionally substituted with hydroxy or one or more halogens ₃ -C ₆ Carbocycles.

16. The compound of any one of claims 13-15, wherein the heterocycle attached to ring G is unsubstituted or monosubstituted.

17. The compound of claim 16, wherein the heterocycle attached to ring G is unsubstituted.

18. The compound of any one of claims 10-17, wherein the carbocycle or heterocycle attached to ring G is optionally and independently methyl, CF ₃ CH ₂ -or HOCH ₂ CH ₂ -substitution.

19. The compound of any one of claims 10-18, wherein a carbocycle or heterocycle attached to ring G is attached to ring G at a position on ring G that is meta relative to-N (H) -attached to ring G.

20. The compound of any one of claims 10-18, wherein a carbocycle or heterocycle attached to ring G is attached to ring G at a position on ring G that is para relative to-N (H) -attached to ring G.

21. The compound of any one of claims 1-20, wherein C of ring G ₆ -C ₁₀ Aryl groupIs phenyl.

22. The compound of any one of claims 1-4, having the structure:

or a pharmaceutically acceptable salt thereof.

23. The compound of any one of claims 1-4, having the structure:

or a pharmaceutically acceptable salt thereof, wherein:

ring J is attached to the phenylene at a position meta or para with respect to-N (H) -attached to the phenylene;

A ¹ is-N (R) ⁴ ) -, -O-or>C(H)(R ⁴ )；

R ⁴ is-H, or C ₁ -C ₆ Alkyl or C ₃ -C ₆ Carbocycles, each of which is optionally substituted with hydroxy or one or more halogens;

A ² is that>N-or>C(H)-；

Z is>CH ₂ The method comprises the steps of carrying out a first treatment on the surface of the And X and Y are independently>CH ₂ Or (b)>C(CH ₃ ) ₂ Or X and Y are both>CH-and are bonded together by methylene or ethylene bridges; or (b)

Y is>CH ₂ Or (b)>C(CH ₃ ) ₂ And X and Z are both>CH-and are bonded together by methylene or ethylene bridges; and is also provided with

n is 0, 1 or 2.

24. The compound of claim 23, wherein a ¹ Is that>C(H)(R ⁴ )。

25. The compound of claim 23, wherein a ¹ is-N (R) ⁴ ) -or-O-.

26. The compound of any one of claims 23-25, wherein R ⁴ is-H, or C ₁ -C ₆ Alkyl optionally substituted with hydroxy or one or more halogens.

27. The compound of claim 26, wherein R ⁴ is-H, methyl, hydroxyethyl or trifluoroethyl.

28. The compound of any one of claims 23-27, wherein a ² Is that>C(H)-。

29. The compound of any one of claims 23-28, wherein a ² Is that>N-。

30. The compound of claim 23, wherein ring J is:

31. the compound of any one of claims 23-30, wherein ring J is attached to the phenylene at a position meta to-N (H) -attached to the phenylene.

32. The compound of any one of claims 23-31, wherein n is 0 or 1.

33. The compound of claim 32, wherein n is 0.

34. The compound of any one of claims 1-4, 23-29, and 31, having the structure:

or a pharmaceutically acceptable salt thereof.

35. The compound of claim 34, wherein ring J is:

36. a compound, or a pharmaceutically acceptable salt thereof, having the structure:

37. The compound of claim 36, wherein the compound is of the formula:

or a pharmaceutically acceptable salt thereof.

38. The compound of claim 36, wherein the compound is of the formula:

or a pharmaceutically acceptable salt thereof.

39. The compound of claim 36, wherein the compound is of the formula:

or a pharmaceutically acceptable salt thereof.

40. The compound of claim 36, wherein the compound is of the formula:

or a pharmaceutically acceptable salt thereof.

41. The compound of claim 36, wherein the compound is of the formula:

or a pharmaceutically acceptable salt thereof.

42. The compound of claim 36, wherein the compound is of the formula:

or a pharmaceutically acceptable salt thereof.

43. The compound of claim 36, wherein the compound is of the formula:

or a pharmaceutically acceptable salt thereof.

44. The compound of claim 36, wherein the compound is of the formula:

or a pharmaceutically acceptable salt thereof.

45. A pharmaceutical composition comprising a compound of any one of claims 1-44, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.

46. A pharmaceutical combination comprising a compound of any one of claims 1-44, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 45, and one or more additional therapeutic agents.

47. A method of treating a proliferative disease in a subject, the method comprising administering to the subject a compound of any one of claims 1-44, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 45.

48. The method of claim 47, wherein the proliferative disease is cancer.

49. The method of claim 48, wherein the cancer is a hematologic cancer.

50. The method of claim 48, wherein the cancer comprises a solid tumor.

51. The method of claim 48, wherein the cancer is lung cancer, brain cancer, thyroid cancer, anaplastic astrocytoma, liver cancer, pancreatic cancer, skin cancer, melanoma, metastatic melanoma, colorectal cancer, breast cancer, prostate cancer, renal cancer, hepatocellular carcinoma, ovarian cancer, HPV-associated cancer, multiple myeloma, myelodysplastic syndrome, hematological cancer, or myelofibrosis.

52. The method of claim 48, wherein the cancer is non-small cell lung cancer (NSCLC), neuroblastoma, glioblastoma, thyroid undifferentiated carcinoma (ATC), colon cancer, hepatocellular carcinoma (HCC), pancreatic cancer, anaplastic Large Cell Lymphoma (ALCL), myelodysplastic syndrome, anaplastic astrocytoma, or pancreatic ductal adenocarcinoma.

53. The method of claim 48, wherein the cancer is adult granulocytoma.

54. The method of claim 48, wherein the cancer is an HPV-related cancer selected from cervical cancer, oropharyngeal cancer, anal cancer, vulvar/vaginal cancer, or penile cancer.

55. The method of claim 47, wherein the proliferative disease is a fibrotic disorder.

56. The method of claim 55, wherein the fibrotic condition is idiopathic pulmonary fibrosis, cardiac fibrosis, a condition associated with cardiac fibrosis, valvular disease, cardiac arrhythmia, atrial fibrillation, myocardial remodeling, cardiomyopathy, dilated cardiomyopathy, ischemic cardiomyopathy, hypertrophic cardiomyopathy, restenosis, liver fibrosis, cirrhosis, non-alcoholic steatohepatitis, peciniopathy, dipivefrong contracture, cystic fibrosis, beta thalassemia, light keratosis, hypertension, systemic inflammatory disorders, dry eye, ulcers, corneal fibrosis, wet age-related macular degeneration, psoriasis, wound closure, chronic kidney disease, renal fibrosis, systemic sclerosis, or chronic Chagas heart disease.

57. A method of inhibiting tumor growth in a subject, the method comprising administering to the subject the compound of any one of claims 1-44, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 45.

58. A method of inhibiting ALK-5 activity in vivo or in vitro, comprising contacting ALK-5 with a compound of any one of claims 1-44, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 45.

59. A method of treating an inflammatory disease, disorder, or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of any one of claims 1-44, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 45.

60. The method of claim 59, wherein the inflammatory disease, disorder, or condition is nonalcoholic fatty liver disease (NAFLD), alcoholic Steatohepatitis (ASH), nonalcoholic steatohepatitis (NASH), primary Biliary Cholangitis (PBC), primary sclerosing cholangitis, autoimmune hepatitis, skin inflammation, or psoriasis.

61. The method of claim 59 or 60, wherein the inflammatory disease, disorder, or condition is an autoimmune disease, disorder, or condition.

62. The method of claim 61, wherein the autoimmune disease, disorder or condition is osteoarthritis, rheumatoid arthritis, pain, inflammatory bowel disease, a respiratory disorder, or a skin disorder.

63. A method of treating a fibrotic, inflammatory or proliferative disease or disorder susceptible to inhibition of a tgfβ signaling pathway, the method comprising administering to a subject suffering from the fibrotic, inflammatory or proliferative disease or disorder an amount of a compound of any one of claims 1-44, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 45, effective to inhibit tgfβ signaling.

64. The method of any one of claims 47-57 and 59-63, wherein the subject is a human.

65. The method of any one of claims 47-57 and 59-64, wherein the proliferative disease, inflammatory disease, disorder or condition, tumor, cancer, or fibrosis, inflammatory or proliferative disease or condition expresses or has a mutant fork cassette L2 (FOXL 2) or FOXL2.

66. The method of any one of claims 47-57 and 59-65, further comprising administering to the subject one or more additional therapeutic agents selected from the group consisting of an anticancer agent and an immune checkpoint inhibitor.

67. The method of any one of claims 47-57 and 59-66, further comprising treating the subject with radiation therapy or surgery.

68. A method of inhibiting an epithelial-to-mesenchymal transition (EMT) in a subject suffering from a disease or disorder facilitated by EMT, the method comprising administering to the subject an amount of at least one compound of any one of claims 1-44, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 45, effective to sufficiently inhibit EMT to alter the course of the disease or disorder.

69. A method for enhancing the activity of one or more therapeutic agents for treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound of any one of claims 1-44, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 45.

70. The method of claim 69, further comprising administering to the subject one or more therapeutic agents selected from an anti-cancer agent or an immune checkpoint inhibitor.

71. The method of claim 66, 67, or 70, wherein the immune checkpoint inhibitor is a PD-1 or PD-L1 inhibitor.