ALK5 inhibitors complexes and uses thereof

Abstract

To provide compositions for the treatment of fibrosis and cancer.SOLUTION: The present disclosure relates to targeted drug conjugates comprising an ALK5 inhibitor and a targeting moiety that directs the ALK5 inhibitor to cells involved in fibrosis and cancers, e.g., myofibroblasts, activated fibroblasts, and migrating fibroblasts, and uses thereof, particularly wherein the ALK5 inhibitor is N-methyl-2 - (4 - (4 - (3 - (6 - methylpyridin-2-yl) - 1H-pyrazol-4-yl) pyridin-2-yl) phenoxy) ethan-1-amine.SELECTED DRAWING: None

Description

[Technical Field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Application No. 62 / 958,461, filed January 8, 2020. The benefit of prior art is claimed, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] 2.Background Fibrosis Idiopathic pulmonary fibrosis (IPF) is a disease of the lungs characterized by progressive hardening and scarring of lung tissue. It is a devastating chronic disease that affects the elderly (Lederer et al., 2018, NEJM, 378:1811-23, Barratt et al., al., 2018, J Clin Med 7(8):201). Approximately 130,000 patients die each year in the United States. It has a 5-year mortality rate of 80%. There is no cure for this disease, only treatment options (Somogyi et al., 2019, Eur Respir Rev, 28(153):190021). IPF is a condition that occurs when an exogenous irritant (e.g., smoking) causes persistent inflammatory bowel movements. It begins with repeated alveolar epithelial damage due to fibroblast activation, which is one of the drivers of fibrosis. Fibrosis is essentially a wound that cannot heal due to certain lung injuries.

[0003] The differentiation of lung fibroblasts into myofibroblasts is a primary step in the development of tissue fibrosis. (Yazdani et al., 2017, Adv Drug Deliv Rev 121:101-116, Huang et al., 2014, Au stin J Pulm Resp, 1(1):3-9). Myofibroblasts are involved in fibrogenesis and are present in fibrotic areas. Three possible causes of myofibroblasts in IPF 1) Myofibroblasts form in a process known as epithelial-mesenchymal transition (EMT). 1) resident lung epithelial cells that convert to alveoli, and 2) resident lung fibroblasts that convert to myofibroblasts (FMT). fibroblasts, and / or 3) myocytes that are recruited into the lung and cause fibrosis and scarring Fibroblasts (Pardali et al., 2017, Int J Mol Sci, 18(10)). The combination results in an increase in lung-resident myofibroblasts, causing fibrotic disease. Inhibiting fibroblasts is a key step in reversing fibrotic lung disease.

[0004] The pleiotropic cytokine transforming growth factor-beta (TGF-β) TGF-β is involved in the development, maintenance, and homeostasis of most tissues in the body. , through binding to TGF-β receptor II and TGF-β receptor I / ALK5 ALK5 is a serine-threonine kinase receptor that initiates signal transduction. , which phosphorylates the downstream signaling mediators, Smad2 and Smad3. Activated Smad2 / 3 form a complex with Smad4, translocate into the nucleus, and regulate gene expression. This is determined by the cellular context (Derynck et al., 2003, Nature, 425( 6958):577-84). In the lung, TGF-β stimulates alveolar macrophages, neutrophils, and activated A wide variety of cell types, including alveolar epithelial cells, endothelial cells, fibroblasts, and myofibroblasts TGF-β is produced by alpha- Extracellular proteins, including α-smooth muscle actin (αSMA), collagen, and fibronectin It is one of the most potent inducers of extracellular matrix (ECM) production (Pohlers et al., 2009 , Biochim Biophys Acta, 1792(8):746-56, Kim et al., 2018, Cold Spring Harb Persp During the disease progression of IPF, TGF-β upregulates collagen expression and EC M deposition, myofibroblast expansion, fibroblast-to-myofibroblast transition, and epithelial-mesenchymal Enhances transformation (EMT) (Pardali et al., 2017, Int J Mol Sci, 18(10), Yue et al., 2010, Curr Enzym Inhib, 6(2)). Furthermore, TGF-β expression is associated with pulmonary fibrosis. It is increased in both animal models and fibrotic human lungs (Tashiro et al., 2017, Fr Ont Med (Lausanne), 4:118). In animal models of pulmonary fibrosis, elevated TGF-β levels The increase in IL-12 precedes collagen synthesis and deposition. It is a driver of pulmonary fibrosis in vivo. As further evidence for the role of TGF-β in the pathogenesis of pulmonary edema, adenovirus-expressed T cells were used in the lung. Animal models of transgenic lung-specific expression of TGF-β1 or TGF-β1 have been reported to develop pulmonary fibroblasts. It was sufficient to induce fibrosis (Lee et al., Korean J Intern Med, 29: 281). In a classical mouse model of rheomycin-induced IPF, TGF-β levels were significantly elevated in the lungs. and TGF-β1 expression in Smad3 knockout mice or specifically in fibroblasts. by blocking TGF-β signaling either by dominant-negative expression of -βRII , and the severity of the disease was reduced (Fernandez et al., 2012, Proc Am Thorac Soc, 9(3):111-1 16, Degryse et al., 2011, Am J Physiol Lung Cell Mol Physiol, 300(6):887-897, Li et al., 2011, J Clin Invest, 121(1):277-87). Therapeutically, the small molecule TGF-β receptor Treatment with inhibitors or anti-TGF-β antibodies also improved disease in bleomycin and radiation inhibited adenoma-induced fibrosis (Giri et al., 1993, Thorax, 48:959-66; Flechsig et al., 2012, Clin Cancer Res, 18(13):3616-27).

[0005] Due to the important role of TGF-β in causing IPF, Targeted therapies are being investigated for the treatment of IPF. Due to the widespread expression of IL-1 and its receptors, the risk of host tissue toxicity is a significant limitation of safe and effective therapy. making development difficult (Anderton et al., 2011, Toxicologic Path, 39:916-24; Stauber et al., 2014, Clinical Tox, 4(3):1-10, Lonning et al, 2011, Curr Pharma Biotech, 1 2:2176-89). For example, αvβ6 integrin antibodies block TGF-β activation systemically. A phase 2 trial of (BG00011) was recently terminated due to safety issues (Arefayene, e t al., 2018, European Respiratory Journal, 52 (suppl 62) PA596). Most drugs and Similarly, toxicity and therapeutic window must be balanced, and the scope of action of TGF-β inhibitors Because the range of potential side effects is so wide, safety and risk of toxicity are paramount. Selective and potent TGF-β inhibitors have the potential to halt disease progression but have limited patient survival. can also be improved.

[0006] In 2014, two IPF drugs, the antifibrotic molecule pirfenidone and tyrosine Nintedanib, a kinase inhibitor, has been approved, both of which inhibit TGF-β, along with other pathways. -β signaling (Gan et al., 2011, Ther Clin Risk Manag, 7: 39-47, Margaritopoulos et al., 2016, Core Evid, 11:11-22, Lunardi et al., 2018, Arch Pathol Lab Med, 142:1090-1097). In general, pirfenidone and nintedanib Treatment reduces the risk of IPF disease progression in 50% of patients with mild to moderate disease (Ren et al., 2017, Saudi Med J, 38(9):889-894, Case et al., 2017, BMJ Open Re sp Res, 4:e000192). However, lung function (the ability of the patient to exhale air) has decreased to less than 50%. IPF patients with severely reduced forced vital capacity (FVC), or comorbidities Elderly patients, or those whose disease has not been officially diagnosed as IPF, were excluded from these trials. While both drugs can slow the disease, neither completely halts disease progression. Studies in IPF have not shown that fibrosis can be prevented or reversed. Keen attention is being given to the significant unmet needs that remain.

[0007] Other therapies such as IFN-γ inhibitors, angiogenesis inhibitors, and TNF-α blockers , have been found to be unsuccessful in treating IPF (Yazdani et al., 2017, Adv Drug D eliv Rev, 121:101-116, Somogyi et al., 2019, Eur Respir Rev, 28(153):190021). Ongoing trials in IPF are evaluating plasma amyloid protein P (pentraxin 2, PTX-2) It contains a circulating protein that binds to monocytes and inhibits their differentiation into prefibrotic fibrocytes. Pentraxin levels are increased by I Low in PF patients, ongoing phase 2 trial demonstrates improvement in lung function during the 6-minute walk test In a phase 2 trial, pamrevlumab inhibited connective tissue growth factor (CTG), which reduced fibrosis. F) but has been shown to improve pulmonary function (FVC) in patients with IPF. ) (Somogyi et al., 2019, Eur Respir Rev, 28(153):190021) In contrast, tralokinumab suppresses IL-1 expression, which reduces the expression of TGF-β and CCl2. Simtuzumab is an anti-LOXL2 antibody that reduces ECM cross-linking. Both failed in phase 2 trials due to lack of improvement in respiratory function (Raghu, 2017, E European Respiratory Review, 26:170071). Many of the therapies target TGF-β function directly or However, despite these efforts, no improvements have been observed for patients with IPF. There is an unmet need for improved treatments, particularly disease-selective and safe modifying therapies. still remains.

[0008] Fibrosis is driven by TGF-β in several diseases other than IPF, and other types pulmonary fibrosis (e.g., associated with systemic sclerosis), liver fibrosis (e.g., nonalcoholic fatty liver disease) These include nonalcoholic steatohepatitis (NASH), renal fibrosis, and cardiac fibrosis (Meng et al., 2016, Nat Rev Nephrol. 12(6):325-38, Biernacka et al., 2011, Growth Factors, 29(5):196-20 2, Gyorfi et al., 2017, Matrix Biology, 68-69:8-27). Elephants, in particular pulmonary fibrosis, e.g., IPF, liver fibrosis, e.g., liver fibrosis associated with NASH, renal TGF-β-induced fibrosis in patients suffering from fibrosis, cardiac fibrosis, and systemic sclerosis Therapies that can reverse the myofibroblast activation and reduce fibrosis are currently being investigated. There is an unaddressed need to

[0009] Cancer TGF-β signaling is also implicated in tumor progression, and the potential of TGF-β as a cancer therapy is unclear. Inhibition of the β pathway has long been of interest (Syed, 2016, J Cell Biochem. 117(6):1279- 87) However, because TGF-β receptors are ubiquitously expressed, problems of host toxicity and tumor Many TGF-β inhibitors are still in the preclinical discovery stage due to concerns that they may inadvertently promote tumor growth. It remains as it is.

[0010] TGF-β binds to tumor cells, cancer-associated fibroblasts (CAFs), and / or surrounding tumor cells. CAFs are secreted by tumor microenvironment (TME) cells. Among the stromal cells of the TME, It is the most abundant and is critically involved in cancer progression (Pure and Blomberg, 2018, Oncog ene, 37(32):4343-4357, Calon et al., 2014, Seminars in Cancer Bio, 25:15-22, Che n and Song, 2019, Nat Rev Drug Disc. 18:90). TGF-β mediates epithelial-mesenchymal transition (EM T) induces differentiation of tissue-resident fibroblasts and epithelial cells, and promotes their survival. CAFs are important drivers of activation, recruitment, and viability, supporting the AF is involved in tumor growth, angiogenesis, cancer stemness, ECM remodeling, tissue invasion, and metastasis. , and has an effect on chemoresistance (Harryvan and van der Burg, 2019, J Clin Med, 8:1989). CAFs are complex and often comprise intracellular alpha-smooth muscle actin. These include increased expression of fibroblast activation protein (FAP) and sarcolemmal activation protein (SMA) on the cell surface. Cellular heterogeneity identified using a combination of various intracellular and cell surface markers, including This is a large group (Pure and Blomberg, 2018, Oncogene, 37(32):4343-4357). In patients with thyroid and colorectal cancer, TGF-β signaling is associated with a compromised immune or This promotes "cold" tumors, and T cells remain captured outside the tumor by CAFs. This physically blocks them from infiltrating the tumor (Hegde, 2020, Immunity, 52: 17-35, Gajewski, 2015, Semin Oncol, 42: 663-671, Mariathasan and Powles, 2018, Nature, 554: 544-48).

[0011] TGF-β therapy has received attention for the treatment of cancer, but historically, widespread use of TGF-β has been limited. expression and its receptors, and the development, maintenance, and homeostasis of tissues, including the heart and bone. Due to its role in stasis, its full therapeutic potential has not been reached. GF-β is an early tumor suppressor involved in controlling early tumor growth and is a systemic TGF-β therapy has been shown to cause tissue toxicity and increased early tumor growth. (Anderton and Heier, 2011, Toxicologic Path, 39: 916, Stauber et al., 2014, Clinical Tox, 4(3):1-10, Lonning and McPherson, 2011, Curr Pharma Biotech, 12:2 176-89).

[0012] Therefore, inhibition of TGF-β signaling may be therapeutic while minimizing toxicity to host tissues. TGF-α to therapeutically useful cell types, e.g., cancer-associated fibroblasts ("CAFs"). There is a need for targeted -β inhibitors. Summary of the Invention

[0013] 3. Overview The present disclosure relates to compositions and methods for the treatment of fibrosis and cancer. The method advantageously targets primarily and preferably only those cells that provide therapeutic benefit. and thereby avoiding pleiotropic off-target effects, thereby enhancing the systemic efficacy of TGF-β inhibitors. Avoid administration-related targeted host toxicity.

[0014] In particular, the compositions and methods provide a method for targeting myofibroblasts, active fibroblasts (e.g., cancer-associated fibroblasts (“CAFs”)), and a targeting moiety. For example, antibody or antibody fragment-mediated fibroblast-to-myofibroblast transition (each cell type " Without being bound by theory, the ALK5 inhibitor is directed to fibroblasts (target cells). The use of a targeting moiety allows for the localization and internalization of the ALK5 inhibitor to target cells. thereby inhibiting the TGFβ pathway in target cells while limiting systemic toxicity. For example, myofibroblasts or fibroblasts that transition to myofibroblasts can be produced. Inhibition of the TGFβ pathway in cells can result in the inhibition of fibrogenesis (fibrosis or fibrosis). Inhibition of the TGFβ pathway in CAFs may improve tumor progression. Without being bound by theory, CA Selective blockade of TGF-β signaling in F1 may contribute to: 1) CAF-mediated immune cell infiltration; 2) can remove the blockage and / or cause tumor clearance and / or 3) may reduce the viability of CAFs and / or 4) may avoid the toxicity issues associated with systemic TGF-β inhibitors. can be.

[0015] Thus, the present disclosure provides targeted drug conjugates (TDCs) in which the drug is an ALK5 inhibitor. The TDCs of the present disclosure provide a targeting moiety, e.g., a cell surface molecule (e.g., human) on a target cell. or a target molecule (a cell surface molecule of human myofibroblasts). The binding moiety is a non-immunoglobulin peptide or polypeptide that binds to the cell surface of the target cell surface molecule. Without being bound by theory, the TDCs of the present disclosure may be capable of targeting target cells. promotes dedifferentiation of cells into resting fibroblasts and / or induces apoptosis of target cells It is believed that promoting this system can provide therapeutic benefits. , describes exemplary targeting moieties that can be used in the TDCs of the present disclosure. In embodiments, the ALK5 inhibitor is an imidazole-benzodioxole compound, ... dazole-quinoxaline compounds, pyrazole-pyrrolo compounds, or thiazole-based compounds Exemplary ALK5 inhibitors are described in Section 5.3 and Tables 1-3. In some embodiments, the ALK5 inhibitor is N-methyl-2-(4-(4-(3-(6- Methylpyridin-2-yl)-1H-pyrazol-4-yl)pyridin-2-yl)phenyl The compound is (compound C)(hydroxy)ethan-1-amine (referred to herein as "Compound C").

[0016] The ALK5 inhibitor can be directly conjugated to the targeting moiety or can be conjugated via a linker. The linker can be a non-cleavable linker or Or preferably, it may be a cleavable linker. Exemplary non-cleavable and cleavable linkers are: Anchoring is described in Section 5.4. ALK5 inhibitor molecules linked to targeting moieties The average number of ALK5 inhibitor molecules per targeting moiety can vary, generally between 2 and 8. Drug loading is detailed in Section 5.5.

[0017] The present disclosure further provides pharmaceutical compositions comprising the TDCs of the present disclosure. Exemplary pharmaceutical excipients that can be used to formulate pharmaceutical compositions include those listed in Section 5. It is described in section 6.

[0018] The present disclosure relates to a method for administering a TDC of the present disclosure or a pharmaceutical composition of the present disclosure to a subject in need thereof. The present invention further provides a method for treating fibrosis and a method for treating cancer by administering the compound of formula (I) to a subject. The disclosed TDCs and pharmaceutical compositions can be used as monotherapy or as part of a combination therapy, e.g., For example, other treatments such as pirfenidone or nintedanib (which may be associated with fibrosis or or chemotherapeutic agents (when treating subjects with cancer) In another example, TDC and a pharmaceutical composition can be administered in combination. The composition is administered in combination with a checkpoint inhibitor when treating a subject with cancer. Examples of conditions that can be treated with the TDCs and pharmaceutical compositions of the present disclosure include: Typical classes, as well as exemplary combination therapies, are described in Section 5.7. [Brief explanation of the drawings]

[0019] 4. Brief description of the drawings [Figure 1-1] Figure 1 shows the inhibition of TGF-β-induced luciferase activity in HEK293T cells by compounds A to D. Figure 1A: Compound A, Figure 1B: Compound B, Figure 1C: Compound C, and Figure 1D: Compound D. [Figure 1-2] This is a continuation of Figure 1-1. [Figure 2-1] We demonstrate that anti-FAP antibodies bind to HEK cells only when human FAP cDNA is transfected and expressed on the cell surface. Figure 2A: Unstained HEK cells. Figure 2B: HEK cells stained with anti-FAP antibodies. Figure 2C: HEK cells transfected with FAP cDNA and stained with anti-FAP antibodies. [Figure 2-2] This is a continuation of Figure 2-1. [Figure 3-1] The linkers and payloads used in targeted drug conjugates SYN-301 (Figure 3A) and SYN-302 (Figure 3B) are shown. [Figure 3-2]This is a continuation of Figure 3-1. [Figure 4-1] Figure 4 shows that SYN-301 suppresses TGF-β signaling in HEK cells expressing human FAP protein. Figure 4A: Relative luciferase reporter expression in HEK cells expressing human FAP protein. Figure 4B: Relative luciferase reporter expression in untransfected HEK cells. [Figure 4-2] This is a continuation of Figure 4-1. [Figure 5-1] Figure 5A shows that 50-60% of WI-38 cells express FAP. Figure 5A: Unstained WI-38 cells. Figure 5B: WI-38 cells stained with anti-FAP antibody. Figure 5C: WI-38 cells stained with SYN-301. Figure 5D: WI-38 cells stained with SYN-302. Figure 5E: WI-38 cells stained with isotype control ADC. [Figure 5-2] This is a continuation of Figure 5-1. [Figure 5-3] This is a continuation of Figure 5-1. [Figure 6] Shown is the % FAP internalization induced by anti-FAP antibody (63%), SYN-301 (63%), and SYN-302 (52%). [Figure 7-1] The effects of SYN-301 and SYN-302 on the RNA expression of collagen and fibronectin (FIG. 7A), and LRRC15 (FIG. 7B) in WI-38 cells are shown. [Figure 7-2] This is a continuation of Figure 7-1. DETAILED DESCRIPTION OF THE INVENTION

[0020] 5. Detailed Description The present disclosure relates to targeted drug conjugates (TDCs) useful for treating fibrosis and cancer. and comprising a targeting moiety covalently attached to an ALK5 inhibitor, either directly or through a linker. Targeted drug conjugates (TDCs) are provided. A general description of the TDCs of the present disclosure is presented in Section 5.1. The targeting moiety of the TDC may comprise, for example, an entire antibody or a fragment thereof. Targeting moieties that can be used in C are detailed in Section 5.2. ALK5 inhibitors that can be used in the present disclosure are detailed in Section 5.3. C typically contains a linker between the targeting moiety and the ALK5 inhibitor. Exemplary linkers that can be used in DCs are detailed in Section 5.4. The TDCs can contain a varying number of ALK5 inhibitor moieties per targeting moiety. Drug loading is described in detail in Section 5.5. The present disclosure provides pharmaceutical formulations comprising the TDCs of the present disclosure. Pharmaceutical formulations comprising TDC are described in Section 5.6. Further provided are methods of treating fibrosis and methods of treating cancer using the TDCs of the present disclosure. This product is used as a monotherapy or as part of a combination therapy for the treatment of fibrosis or cancer. Methods for using the disclosed TDCs are described in Section 5.7.

[0021] 5.1.Drug Conjugates The TDCs of the present disclosure generally are designed so that the covalent bond does not interfere with binding of the targeting moiety to the target. covalently attached to a targeting moiety such as an antibody or antibody fragment, typically via a linker. It consists of an ALK5 inhibitor.

[0022] Techniques for conjugating drugs to targeting moieties such as antibodies and antibody fragments are known in the art. are well known in the art (see, e.g., Hellstrom et al., Controlled Drug Delivery, 2nd Ed., a t pp. 623-53 (Robinson et al., eds., 1987)), Thorpe et al., 1982, Immunol. Rev. 62:119-58, Dubowchik et al., 1999, Pharmacology and Therapeutics 83:67-123, and and Zhou, 2017, Biomedicines 5(4):E64). ALK5 inhibitors are preferably administered at the The targeting moiety in the TDC of the present disclosure is bound via specific conjugation to AL. K5 inhibitors may be modified by one or more naturally occurring or engineered cysteines, lysines, or is a glutamine residue, one or more unnatural amino acids (e.g., p-acetylphenyl alanine (pAcF), p-azidomethyl-L-phenylalanine (pAMF), or is selenocysteine ​​(Sec)), one or more glycans (e.g., fucose, 6 -Thiofucose, galactose, N-acetylgalactosamine (GalNAc), N-acetyl cetylglucosamine (GlcNAc) or sialic acid (SA)), or 4 to 6 conjugated to a targeting moiety via one or more short peptide tags of amino acids See, for example, Zhou, 2017, Biomedicines 5(4):E64, the contents of which are incorporated herein by reference. No. 6,239,999, which is incorporated herein by reference in its entirety.

[0023] In one example, the targeting moiety is another protein (or a portion thereof, e.g., protein for an amino acid sequence of at least 10, 20, or 50 amino acids of via a covalent bond (e.g., a peptide bond) through the N-terminus or C-terminus of the targeting moiety The targeting moiety can be N-terminally linked to another protein. For example, an antibody or antibody fragment may be linked to another protein by the N-terminus of the antibody constant domain. The ends can be ligated. Recombinant DNA procedures can be used to create such fusions. For example, WO 86 / 01533 and European Patent Application No. In another example, the effector molecule The compound may increase the in vivo half-life and / or enhance the ability of the compound to target cells. Examples of suitable effector molecules of this type include poly(A)- and poly(B)-transferases (PTDs) that can enhance the delivery of TDCs. albumin, albumin-binding protein, or albumin-binding compound. For example, the PCT publication WO 2005 / 117984 pamphlet This is what is being done.

[0024] A metabolic process or reaction is an enzymatic process, such as the transcription of the peptide linker of a TDC. Enzyme-degrading cleavage or addition of functional groups such as hydrazones, esters, or amides Intracellular metabolites include, but are not limited to, those that enter cells, diffuse into cells, and are taken up by cells. These include peptides and free drugs that are cleaved intracellularly after transport or after transport.

[0025] The terms "cleaved intracellularly" and "intracellular cleavage" refer to the metabolism of a drug conjugate within a cell. refers to a process or metabolic reaction that results in a shared bond between the drug moiety (D) and the targeting moiety. The bond, e.g., the linker, is broken, resulting in free drug dissociated from the targeting moiety within the cell. Thus, the cleaved portion of TDC is an intracellular metabolite.

[0026] 5.2. Targeting part The present disclosure provides drug conjugates in which the targeting moiety binds to a cell surface molecule of a target cell. The targeting moiety typically comprises an antibody or antibody fragment (such conjugates are sometimes Alternatively, the targeting moiety may be a non- Immunoglobulin-based, e.g., non-immunoglobulin-based peptides or polypeptides (e.g., A targeting moiety may be a ligand for a receptor expressed on the surface of a target cell. Hence the term "targeting moiety." However, peptides (e.g., peptides having a length of 10 to 40 amino acids), single-chain polypeptides ( For example, polypeptides greater than 40 amino acids in length, such as single chain variable regions or scFvs ), and molecules containing multiple polypeptide chains (e.g., polymeric immunoglobulin molecules). It should be understood that this includes

[0027] Unless otherwise indicated, the term "antibody" refers to an antibody that specifically binds to a particular antigen or Refers to immunoglobulin molecules that react immunologically, and includes polyclonal and monoclonal antibodies. Antibodies include, but are not limited to, antibodies, genetically engineered antibodies, and otherwise modified forms of antibodies. , chimeric antibodies, humanized antibodies, heteroconjugate antibodies (e.g., bispecific antibodies) antibody, bispecific antibody (diabody), trispecific antibody, and tetraspecific antibody), and fragments such as Fab', F(ab')2, Fab, Fv, rIgG, and scFv fragments. Furthermore, unless otherwise indicated, the term "monoclonal antibody" includes antibody fragments of antibodies containing the antibody fragment. A "monoclonal antibody" (mAb) is a complete antibody capable of specifically binding to a protein. molecules, as well as antibody fragments (e.g., Fab and F(ab')2 fragments) Fab and F(ab')2 fragments lack the Fc fragment of an intact antibody. , are cleared more rapidly from the circulation of animals or plants and cause less non-specific tissue damage than intact antibodies. The nucleotide sequence may have a binding site (Wahl et al., 1983, J. Nucl. Med. 24:316).

[0028] References to "VH" include the heavy chain of an antibody, including the heavy chain of an Fv, scFv, or Fab. References to "VL" refer to the variable region of a globulin heavy chain. or Fab light chain. Ig is a glycoprotein with the same structural characteristics as an antibody. While antibodies exhibit binding specificity for a target molecule, immunoglobulins lack target specificity. Natural antibodies and immunoglobulins include both antibodies and other antibody-like molecules. and two identical heavy chains (H), usually about 150,000 daltons Each heavy chain contains at its amino terminus a variable domain (VH) Each light chain has a variable domain at the amino terminus followed by several constant domains. It has a constant domain at the carboxy terminus and a variable length domain (VL).

[0029] For optimal delivery of the ALK5 inhibitor within the cell, the targeting moiety preferably is an internalized Internalizing targeting moieties bind to their targets on the cell surface, e.g., internalizing antibodies. After binding to the molecule, it is internalized by the cell as a result of the binding. This effect is due to the The internalization of an antibody after binding to an antigen is determined by the Enabling processes are known to those skilled in the art and are described, for example, in PCT Publication WO 2004 / 020944. This is described on page 80 of the pamphlet No. 7 / 070538. As described in Section 5.4, ALK5 inhibitors are linked to the targeting moiety using a cleavable linker. Upon binding, ALK5 inhibitors may be inhibited by lysosomal cleavage or by other cellular mechanisms. and can be released from the targeting moiety.

[0030] The term "antibody fragment" refers to a portion of a full-length antibody, generally the target binding or variable region. Examples of fragments include Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, and scFv fragments. These include antibody fragments, dsFv fragments, and single domain antibodies.

[0031] An "Fv" fragment is the minimum antibody fragment which contains a complete target recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight, non-covalent association. In this structure, the three CDRs of each variable domain interact with each other. These CDRs define an antigen-binding site on the surface of the VH-VL dimer. , which confers target binding specificity to the antibody. However, in some instances, a single variable domain The main (or half of the Fv containing only the three target-specific CDRs) recognizes the target. , may have the ability to bind.

[0032] "Single-chain Fv" or "scFv" antibody fragments are fragments of an antibody comprising the fragments of an antibody molecule in a single polypeptide chain. Generally, the scFv polypeptide comprises a VH and VL domain of the scFv. The polypeptide between the VH and VL domains enables it to form the desired structure for binding. Various scFv linkers have been described in the art. Ru. For example, Shen et al., 2008, Anal Chem. 80(6): 1910-1917, Yusakul, et al., 201 6, Biosci Biotechnol Biochem. 80(7):1306-12. Exemplary scFv linkers include: Sequence (GGGGS) n and n is 1 to 10.

[0033] "Disulfide-stabilized Fv" or "dsFv" antibody fragments have interdomain disulfide bonds. Contains antibody VH and VL domains stabilized by sulfide bonds. Brinkmann U. , 2010, Disulfide-Stabilized Fv Fragments. In: Kontermann R., Dubel S. (eds) Ant ibody Engineering. Springer, Berlin, Heidelberg.

[0034] A "single domain antibody" is an antibody that exhibits sufficient affinity for a target (e.g., a FAP), e.g., For example, a single VH or VL domain (e.g., of a human or mouse antibody). In embodiments, the single domain antibody is a camel V H H antibody fragments (e.g., Riechmann (See, e.g., J. Immunological Methods, 1999, 231:25-38). The use of single domain antibodies in this study has advantages over full-length antibodies, including their small size, high solubility, and high This may be advantageous due to its excellent stability and excellent tissue penetration in vivo. Various methods for generating main antibodies have been described, e.g., U.S. Pat. No. 10,030, 068, U.S. Patent Application Publication No. 2006 / 0246058, U.S. Patent No. 7 , 371,849, Vincke et al, 2008, JBC, 284(5):3273-3284.

[0035] The Fab fragment contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments contain heavy chains containing one or more cysteines from the antibody hinge region. It differs from Fab fragments by the addition of several residues at the carboxyl terminus of the CH1 domain. F(ab') fragments are formed by dimerization at the hinge cysteines of the F(ab')2 pepsin digestion product. Further chemical coupling of antibody fragments is known to those skilled in the art. It is being done.

[0036] In certain embodiments, the antibodies of the present disclosure are monoclonal antibodies. The term "monoclonal antibody" as used herein refers to an antibody produced through hybridoma technology. The term "monoclonal antibody" refers to an antibody derived from a single clone. refers to any eukaryotic, prokaryotic, or phage clone, but does not include the method of producing it. Monoclonal antibodies useful in connection with the present disclosure may be hybridoma, recombinant, or and phage display technology, or a combination thereof. Antibodies of the present disclosure can be prepared using a wide variety of techniques known in the art. These include primatized, humanized, or human antibodies.

[0037] The antibodies of the present disclosure may be chimeric antibodies. As used herein, the term "chimeric" antibody The antibody can be used with non-human immunoglobulins, such as rat or mouse antibodies, and with human immunoglobulins. Variable sequences derived from human immunoglobulin constant regions typically selected from Methods for producing chimeric antibodies are known in the art. See, for example, Morrison, 1985, Science 229(4719):1202-7; Oi et al., 1986, BioTechni ques 4:214-221, Gillies et al., 1985, J. Immunol. Methods 125:191-202, U.S. Pat. Nos. 5,807,715, 4,816,567, and U.S. Pat. See U.S. Pat. No. 4,816,397, the contents of which are incorporated herein by reference in their entirety. will be incorporated into

[0038] The antibodies of the present disclosure may be humanized. "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulins containing minimal sequences derived from non-human immunoglobulins; a globulin chain, or a fragment thereof (e.g., Fv, Fab, Fab', F(ab')2 of an antibody or other target-binding subdomain). Generally, humanized antibodies contain at least one , typically comprising substantially all of the two variable domains, and all or substantially all of the CDR regions. essentially all correspond to those of non-human immunoglobulins, and all or substantially all of the FR regions Humanized antibodies also contain immunoglobulin constant regions. at least a portion of the Fc region, typically of the human immunoglobulin consensus sequence. Methods for humanizing antibodies are known in the art. See, for example, Riechmann et al. al., 1988, Nature 332:323-7; U.S. Patent No. 5,530,101 to Queen et al. Specification, U.S. Patent No. 5,585,089 Specification, U.S. Patent No. 5,693,761 Specification No. 5,693,762, U.S. Pat. No. 6,180,370, European Patent Application Publication No. 239400, PCT Publication No. WO 91 / 09967 Brochure, U.S. Patent No. 5,225,539, European Patent Application Publication No. 59210 6, European Patent Application Publication No. 519596, Padlan, 1991, Mol. Immunol., 28:489-498, Studnicka et al., 1994, Prot. Eng. 7:805-814, Roguska et al., 1994, See Proc. Natl. Acad. Sci. 91:969-973, U.S. Pat. No. 5,565,332. , all of which are incorporated herein by reference in their entireties.

[0039] The antibodies of the present disclosure can be human antibodies. Fully "human" antibodies are suitable for the therapeutic treatment of human patients. As used herein, a "human antibody" refers to an antibody that is produced by a human immunoglobulin G (HIG) or human immunoglobulin G (HIN) gene. a human immunoglobulin library containing antibodies having the amino acid sequence of an immunoglobulin; or Isolated from animals transgenic for one or more human immunoglobulins Human antibodies include antibodies that are produced by human immunoglobulins and do not express endogenous immunoglobulins. The art includes phage display methods using sequence-derived antibody libraries. These can be prepared by a variety of methods known in the art, for example, U.S. Pat. No. 7 and U.S. Pat. No. 4,716,111, and the International Publication of the PCT Publication International Publication No. 98 / 46645, International Publication No. 98 / 50433, International Publication No. 98 / 24893, International Publication No. 98 / 16654 , International Publication No. 96 / 34096 Pamphlet, International Publication No. 96 / 33735 Pamphlet See WO 91 / 10741, each of which is incorporated herein by reference. Human antibodies are also derived from cells expressing functional endogenous immunoglobulins. It is not possible to produce transgenic mice that express human immunoglobulin genes. For example, PCT Publication No. WO 99 / 044444 can be produced using a transgenic mouse. Pamphlet No. 8 / 24893, Pamphlet No. 92 / 01047 ... No. 96 / 34096, International Publication No. 96 / 33735, U.S. Patent No. 5,413,923, U.S. Patent No. 5,625,126, U.S. Patent No. 5,633,425, U.S. Pat. No. 5,569,825, U.S. Pat. ,661,016, U.S. Pat. No. 5,545,806, U.S. Pat. No. 5,8 No. 14,318, U.S. Pat. No. 5,885,793, U.S. Pat. No. 5,916 ,771 and U.S. Pat. No. 5,939,598, the contents of which are incorporated herein by reference. , which is incorporated herein by reference in its entirety. In addition, Medarex (Princ eton, NJ), Astellas Pharma (Deerfield, Ill. .), Amgen (Thousand Oaks, Calif.), and Regene ron (Tarrytown, NY) and other companies using similar technology. Contracts can be made to provide human antibodies directed against selected antigens. Fully human antibodies that recognize epitopes can be generated using a technique called "guided selection." In this approach, selected non-human monoclonal antibodies can be generated using the A clonal antibody, e.g., a mouse antibody, can be used to select a fully human antibody that recognizes the same epitope. induces selection (Jespers et al., 1988, Biotechnology 12:899-903).

[0040] The antibodies of the present disclosure may be primatized. The term "primatized antibody" refers to an antibody that contains monkey variable regions and The term "primatized antibody" refers to an antibody that contains a human constant region and a human constant region. Methods for producing primatized antibodies are known in the art. For example, U.S. Pat. No. 5,658,570 and U.S. Pat. See U.S. Pat. Nos. 81,722 and 5,693,780, of which: The contents of which are incorporated herein by reference in their entirety.

[0041] Antibodies of the present disclosure include derivatized antibodies. For example, but not limited to, derivatized antibodies are typically Typically, glycosylation, acetylation, pegylation, phosphorylation, amidation, known protecting groups / blocking groups Derivatization with locking groups, proteolytic cleavage, cellular ligands or other proteins (See Section 5.1 for a discussion of antibody complexes.) Any of the chemical modifications may be, but are not limited to, specific chemical cleavage, acetylation, formylation, etc. This can be done by known techniques, including cleavage, metabolic synthesis of tunicamycin, etc. , derivatives containing one or more unnatural amino acids, e.g., using ambrx technology (See, e.g., Wolfson, 2006, Chem. Biol. 13(10):1011-2).

[0042] In yet another embodiment of the present disclosure, the antibody or fragment thereof is modified in its sequence to include: of at least one constant region-mediated biological effector relative to the corresponding wild-type sequence. For example, in some embodiments, The antibodies of the present disclosure may be modified to have at least one constant region-mediated binding affinity relative to the unmodified antibody. These can reduce the function of immune effectors, such as Fc receptors (FcγR) or FcγR and C1q binding can be reduced by FcγR R or specific regions required for C1q interaction, immunoglobulin constant region segments of antibodies This can be reduced by mutating the nucleotide sequence (e.g., Canfield and Morrison, 1991, J. Exp. Med. 173:1483-1491, Lund et al., 1991, J. Immunol. 147:2657-2662, Lo. et al., 2 017, J Biol Chem 292: 3900-08; Wang et al., 2018, Protein Cell 9:63-73).

[0043] The reduction in FcγR binding ability of antibodies also appears to be due to the presence of other effectors that are dependent on FcγR interactions. - Functions such as opsonization, phagocytosis, and antibody-dependent cellular cytotoxicity ("ADCC") are also poor. Although C1q binding may be reduced, reduced C1q binding reduces complement dependent cytotoxicity (CDCC) Thus, by reducing or eliminating effector function, the drug conjugates of the present disclosure may The targeted target cells may be prevented from being destroyed via ADCC or CDCC. Thus, in some embodiments, the effector functions of an antibody are determined by the Fc portion of the antibody. It is modified by selective mutations of the nucleotides, so that it maintains antigen specificity and internalization ability. In another embodiment, the effector functions of the antibody are eliminated. are not modified to reduce or eliminate ADCC / CDCC function. TDs of the present disclosure include antibodies or antibody fragments with ADCC / CDCC function without C promotes apoptosis of target cells, thereby further inhibiting fibrogenesis. This is thought to improve the therapeutic effect of inhibiting TGFβ signaling.

[0044] Numerous mutations have been described in the art for reducing FcγR and C1q binding. and such mutations may be included in the drug conjugates of the present disclosure. In U.S. Pat. No. 6,737,056, positions 238, 265, 269, 270, 2 92, 294, 295, 298, 303, 324, 327, 329, 333, 335, 3 38, 373, 376, 414, 416, 419, 435, 438, or 439 Single-position Fc region amino acid modification reduces binding to and FcγRII It is disclosed that this results in an asparagine residue at amino acid position 298, and a serine or thiamin residue at amino acid position 300; It has been disclosed that leonine residues reduce FcγR binding. In the brochure No. 2014 / 190441, L234D / L235E:L234R / L235R / E233K, L234D / L235E / D265S:E233K / L23 4R / L235R / D265S, L234D / L235E / E269K:E233K / L 234R / L235R / E269K, L234D / L235E / K322A:E233K / L234R / L235R / K322A, L234D / L235E / P329W:E23 3K / L234R / L235R / P329W, L234D / L235E / E269K / D 265S / K322A:E233K / L234R / L235R / E269K / D265S / K322A, L234D / L235E / E269K / D265S / K322E / E33 3K:E233K / L234R / L235R / E269K / D265S / K322E / E A modified Fc domain with a 333K mutation that reduces FcγR binding has been described. wherein the set of mutations preceding the semicolon is in the first Fc polypeptide. and the mutation following the semicolon is in the second Fc polypeptide of the Fc dimer. N297A is a mutation that can reduce γR receptor binding as well as C1q binding. , N297Q, N297G, D265A / N297A, D265A / N297G, L23 5E, L234A / L235A, and L234A / L235A / P329A. (Lo. et al., 2017, J Biol Chem 292: 3900-08, Wang et al., 2018, Protein Cell 9:63-73).

[0045] Mutations in the constant region to reduce effector function, such as the Fc domain described above, Instead of mutating the antibody fragment, effector functions can be mutated by modifying the antibody fragment (e.g., Fab, Fab'). or F(ab')2 fragments).

[0046] In other embodiments of the present disclosure, the antibody or fragment thereof is modified to have a specific activity relative to the unmodified antibody. Gaining or improving the function of at least one constant region-mediated biological effector For example, FcγR interaction can be improved (see, e.g., U.S. Pat. No. 6,223,999). (See Publication No. 2006 / 0134709). For example, the antibodies of the present disclosure may bind to FcγR IIA, FcγRIIB, and / or FcγRIIIA, respectively, with the corresponding wild-type constant regions It may have a constant region that binds with higher affinity.

[0047] Thus, the antibodies of the present disclosure may have biological activities that reduce opsonization, phagocytosis, or ADCC. Such alterations are known in the art. For example, ADCC Modifications in antibodies that reduce activity are described in U.S. Pat. No. 5,834,597. are.

[0048] In yet another embodiment, the antibody or fragment thereof is selected from the group consisting of antibodies, e.g., antibodies involved in FcRn interactions. by mutating immunoglobulin constant region segments in specific regions, Antibodies or antibodies that increase or decrease its binding affinity to the fetal Fc receptor, FcRn or fragments thereof (see, for example, WO 2005 / 123780). Such mutations can enhance antibody binding to FcRn, leading to increased antibody differentiation. protects it from degradation and extends its half-life.

[0049] In still other embodiments, the antibody is prepared using the methods described, for example, in Jung and Pluckthun, 1997, Protein Engineering neering 10(9):959-966, Yazaki et al., 2004, Protein Eng. Des Sel. 17(5):481-9, and its ultra- It has one or more amino acids inserted into one or more of the variable regions.

[0050] The target of the targeting moiety depends on the desired therapeutic use of the TDC. Typically, the target is the AL Desirable cells for delivery of K5 inhibitors, such as myofibroblasts or cancer-associated fibroblasts. The targeting moiety is preferably a molecule present on the surface of a cell, and is internalized upon binding to the target. Internalizing targeting moieties, e.g., antibodies, are described, for example, in Franke et al., 2000, Cancer Biology. other. Radiopharm. 15:459 76, Murray, 2000, Semin. Oncol. 27:64 70, Breitling et al., Recombinant Antibodies, John Wiley and Sons, New York, 1998. In certain embodiments, the targeting moiety significantly blocks the activity of the target cell surface molecule. For example, agonist antibodies or fragments thereof, or non-agonist antibodies or fragments thereof Fragments of the αvβ6 polypeptide may be used as targeting moieties, for example, when the target molecule is FAP or αvβ6. It is possible.

[0051] Preferably, the targeting moiety is capable of targeting other cell types, e.g., quiescent fibroblasts, lung epithelial cells, , hepatocytes, T cells, cells that do not express collagen, and / or α-smooth muscle actin ( Myofibroblasts, activated fibroblasts, and myofibroblasts were more likely to express αSMA than cells that do not. selectively binds to quiescent, migrating, or transitioning fibroblasts, or a combination thereof. Quiescent fibroblasts can be identified as single spindle-shaped cells, whereas activated fibroblasts The fibroblasts express αSMA and vimentin and assume a stellate shape. The binding site is expressed on the surface of one or more target cells, but is rarely expressed on other cell types. This can be achieved by targeting cell surface molecules that are absent or not expressed. The activity can be determined by various assays known in the art, such as flow cytometry. In some embodiments, the targeting moiety of a TDC of the present disclosure can be measured. For example, there is less activity in myofibroblasts than in resting fibroblasts as measured by FACS. At least 2-fold or at least 3-fold (e.g., 2-1000-fold, 2-100-fold, 2-50-fold) , 2~10x, 3~1000x, 3~100x, 3~50x, 3~10x, 5~1000 times, 5~100 times, 5~50 times, 5~10 times, 20~1000 times, 20~100 times, 20 ~50x, 50-1000x, 50-100x, 100-1000x, or 1000x In some embodiments, the targeting moiety of a TDC of the present disclosure is For example, activated fibroblasts (e.g., quiescent fibroblasts) were more likely to be present than resting fibroblasts, as measured by FACS. At least 2-fold or at least 3-fold (e.g., 2-1000-fold) , 2~100x, 2~50x, 2~10x, 3~1000x, 3~100x, 3~50x , 3~10x, 5~1000x, 5~100x, 5~50x, 5~10x, 20~100 0x, 20~100x, 20~50x, 50~1000x, 50~100x, 100~1 In some embodiments, the present invention provides a method for detecting a target gene that has a selectivity of 1000-fold or more than 1000-fold. The targeting moieties of the TDCs shown are more potent than quiescent fibroblasts, as measured, for example, by FACS. At least two-fold or at least three-fold (e.g., For example, 2 to 1000 times, 2 to 100 times, 2 to 50 times, 2 to 10 times, 3 to 1000 times, 3 to 100x, 3~50x, 3~10x, 5~1000x, 5~100x, 5~50x, 5~ 10x, 20~1000x, 20~100x, 20~50x, 50~1000x, 50~ 100-fold, 100-1000-fold, or greater than 1000-fold selectivity. Examples of cell surface molecules suitable for further targeting include, but are not limited to, fibroblast activation Protein (FAP), platelet-derived growth factor receptor beta (PDGFR-β), fibroblast Follicular growth factor receptor 1 (FGFR1), peroxisome proliferator-activated receptor gamma (P PAR-γ), fibroblast-specific protein 1 (FSP1), glial fibrillary acidic protein GFAP, fascin, CD147, CXC chemokine receptor type 4 (CXCR4 ), alpha V beta 6 (αvβ6), AXL, and MERTK. and MERTK are members of the TAM receptor kinase family. Further examples of cell surface molecules suitable for targeting include 15-containing leucine-rich Chile repeat (LRRC15).

[0052] In some embodiments, the targeting moiety of a TDC of the present disclosure binds to a FAP. In some embodiments, the targeting moiety of the TDC of the present disclosure binds to PDGFR-β. In embodiments, the targeting moiety of the TDC of the present disclosure binds to FGFR1. In other embodiments, the targeting moiety of the TDC of the present disclosure binds to PPAR-γ. In another embodiment, the targeting moiety of the TDC of the present disclosure binds to FSP1. The targeting moiety of the TDC of the present disclosure binds to GFAP. The targeting moiety of the TDC binds to fascin. In other embodiments, the targeting moiety of the TDC of the present disclosure The targeting moiety binds to CD147. In other embodiments, the targeting moiety of the TDCs of the present disclosure In other embodiments, the targeting moiety of the TDC of the present disclosure binds to CXCR4. In other embodiments, the targeting moiety of a TDC of the present disclosure binds to AXL. In other embodiments, the targeting moiety of the TDC of the present disclosure binds to MERTK. In other embodiments, the targeting moiety of a TDC of the present disclosure binds to LRRC15. .

[0053] Fibroblast activation protein (FAP) is a dipeptidyl peptidase (DPP) pharmacophore. It is a member of the membrane complex and is expressed as a type II integral membrane protein. P contains a short cytoplasmic tail (residues 1–4), a transmembrane region (residues 5–25), and an extracellular domain. FAP contains the main (residues 26 to 760) (www.uniprot.org / uniprot / Q12884). , which is active at the cell surface as a 170 kD dimer, but the extracellular domain also binds to the membrane. FAP is a dipeptidase and endopeptidase. Like other members of the DPP family of enzymes, FAP has both protease and protease activity. It is a loryl-specific serine protease, but also a FAP with gelatinase activity. It contains denatured collagen I and III, human fibroblast growth factor 21 (FGF-21), FAP allows the degradation of human alpha-2 antiplasmin, as well as human alpha-2 antiplasmin. It is expressed during development but is very rare in healthy adult tissues. Increased FAP expression in blastocytes is involved in active tissue remodeling, including inflammation and wound healing. FAP is expressed at sites of inflammation, fibrosis, and cancer. It is a marker for activated fibroblasts and myofibroblasts, making it a promising candidate for FAP-targeted therapy. The method is not limited to cancer patients, but is also applicable to IPF, as well as NASH (liver), cardiac, and / or or can be widely applied to the treatment of other fibrotic diseases such as renal fibrosis. AP expression can be increased by TGF-β, and the promoter of FAP is Smad This limited tissue expression and localized expression in fibrous tissues is Therefore, FAPs are being used for imaging and therapeutic targeting of diseased tissue. FAP is highly expressed in cancer-associated fibroblasts (CAFs), which support cancer growth and metastasis. FAP is readily internalized within cells, providing excellent cell targeting and delivery capabilities. Anti-FAP antibodies conjugated to a cytotoxic payload are used in combination with chemotherapy. demonstrated tumor clearance, while anti-FAP conjugated to radionucleotides It resulted in high survival in a mouse tumor model in vivo (Ostermann et al., 20 08, Clin Cancer Res, 2008. 14(14):4584-92, Fang et al., 2016, Int J Cancer, 138( 4):1013-23, Fischer et al., Clin Cancer Res 18(22):6208-18). Uncomplexed The human anti-FAP antibody, sibroluzumab, has been shown to be effective in patients with metastatic FAP+ cancer. While the drug did not show single-agent efficacy in tumors, it specifically accumulated in tumors and not in normal tissues. The soluble form of FAP was well tolerated in patients with limited adverse events. Antiplasmin cleaving enzyme (APCE), also known as antiplasmin cleaving enzyme (APCE), is a cytoplasmic tetanus of membrane-bound FAP. Soluble FAPs are expressed at levels that increase with disease severity. bell, and has been shown to be elevated in patients with cirrhosis (de Willige et al., 2013, JT 11(11):2029-36). Preferably, the targeting moiety that targets the FAP is Without being bound by theory, soluble FAP preferentially binds to membrane-bound FAP over soluble FAP. AP binds to soluble forms of FAP compared to TDC, which preferentially binds to membrane-bound forms of FAP. This suggests that the tDCS may act as a sink to reduce its in vivo activity. can be done.

[0054] Examples of antibodies that bind to FAP are described in WO 2012 / 020006, International Publication No. 2016 / 116399 (e.g., antibody F5), and International Publication No. No. 2016 / 110598, the contents of which are hereby incorporated by reference. In some embodiments, the targets of the TDCs of the present disclosure are The oxidized portion is described in International Publication No. 2012 / 020006 and International Publication No. 2016 / 1 16399 pamphlet or International Publication No. 2016 / 110598 pamphlet or a fragment thereof (e.g., a Fab fragment, a Fab' fragment, F(ab')2 fragment, Fv fragment, scFv fragment, dsFv fragment, or single domain antibody In some embodiments, the targeting moiety of a TDC of the present disclosure comprises a sibrotuzumab. Mab (Boehringer Ingelheim), or a fragment thereof (e.g., Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, scFv fragments, dsFv fragments, or single domain antibodies).

[0055] Examples of antibodies that bind to PDGFR-β are described in WO 2017 / 106609. and WO 2014 / 109999, among which The contents of which are incorporated herein by reference in their entirety. The targeting moieties of the disclosed TDCs may be those disclosed in WO 2017 / 106609 or WO 2017 / 106609. an anti-PDGFR-β antibody described in brochure no. 2014 / 109999; or Its fragments (e.g., Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, scF In some embodiments, the antibody comprises a fusion protein (e.g., a fusion protein fragment, a dsFv ... or a single domain antibody). Thus, the targeting moiety of the TDC of the present disclosure is IMC-2C5 (ImClone), or a fragment thereof. fragments (e.g., Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, scFv fragments) , dsFv fragments, or single domain antibodies).

[0056] Examples of antibodies that bind to FGFR1 include those disclosed in WO 2018 / 095932. and International Publication No. 2012 / 125124, the contents of which are as follows: , which is incorporated herein by reference in its entirety. In some embodiments, the present disclosure The targeting moiety of the TDC is described in WO 2018 / 095932 or WO 20 Anti-FGFR1 antibodies or fragments thereof described in the pamphlet No. 12 / 125124 (e.g., Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, scFv fragments, In some embodiments, the present disclosure provides a method for the production of antibodies against Fv fragments, dsFv fragments, or single domain antibodies. The targeting moiety of the TDC shown is identified by sequence number 1001 of WO 2012 / 125124. 45, and a heavy chain having the amino acid sequence of WO 2012 / 125124. An antibody comprising a light chain having the amino acid sequence of SEQ ID NO: 50 of the present invention, or a fragment thereof (e.g., , Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, scFv fragment, dsFv fragments, or single domain antibodies).

[0057] Examples of antibodies that bind to PPAR-γ are described in WO 2005 / 026336. No. 6,299,499, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the targeting moiety of the TDCs of the present disclosure is a targeting moiety of the TDCs described in WO 2005 / 026 The anti-PPAR-γ antibodies described in the pamphlet of No. 336, or fragments thereof (e.g., Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, scFv fragment, dsFv fragment In some embodiments, the TDCs of the present disclosure comprise a fragment, or a single domain antibody. The targeting moiety of Pγ48.34A is disclosed in WO 2005 / 026336. or a fragment thereof (e.g., Fab fragment, Fab' fragment, F(ab') 2 fragments, Fv fragments, scFv fragments, dsFv fragments, or single domain antibodies).

[0058] Examples of antibodies that bind to FSP1 are described in WO 2011 / 157724. No. 6,239,999, the contents of which are incorporated herein by reference in their entirety. In embodiments of the present disclosure, the targeting moiety of the TDC is The anti-FSP1 antibody or its fragment (e.g., Fab fragment) described in Pamphlet No. 4 fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, scFv fragment, dsFv fragment, if In some embodiments, the targeting of the TDCs of the present disclosure includes The moiety may be the antibody MAB4137 (R&D Systems), or a fragment thereof (e.g., F ab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, scFv fragment, dsFv fragment , or single domain antibodies).

[0059] Examples of antibodies that bind to GFAP are described in WO 2018 / 081649. No. 6,239,999, the contents of which are incorporated herein by reference in their entirety. In embodiments of the present disclosure, the targeting moiety of the TDC is Anti-GFAP antibodies described in Pamphlet No. 9, or fragments thereof (e.g., Fab fragments) fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, scFv fragment, dsFv fragment, if In some embodiments, the targeting of the TDCs of the present disclosure includes The portion is from GFAP-1 to GFAP in the pamphlet of WO 2018 / 081649. one of the antibodies designated as -19, or a fragment thereof (e.g., a Fab fragment, a Fab' fragment, F(ab')2 fragment, Fv fragment, scFv fragment, dsFv fragment, or single domain antibody body).

[0060] Examples of antibodies that bind to fascin include FCN01 (ThermoFisher), ab 126772 (Abcam), and ab183891 (Abcam). In some embodiments, the targeting moiety of the TDC of the present disclosure is FCN01, ab1267 72, or ab183891, or a fragment thereof (e.g., Fab fragment, Fab F(ab')2 fragment, Fv fragment, scFv fragment, dsFv fragment, or single domain fragment Main antibody).

[0061] Examples of antibodies that bind to CD147 include those disclosed in WO 2015 / 160853. , International Publication No. 2018 / 121578, and International Publication No. 2018 / 16 No. 5619, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the targeting moiety of the TDC of the present disclosure is incorporated into the International International Publication No. 2015 / 160853, International Publication No. 2018 / 121578 FRET or as described in the pamphlet of International Publication No. 2018 / 165619 Anti-CD147 antibodies, or fragments thereof (e.g., Fab fragments, Fab' fragments, F(ab') 2 fragments, Fv fragments, scFv fragments, dsFv fragments, or single domain antibodies). In some embodiments, the targeting moiety of the TDC of the present disclosure is The antibody designated as 3A11 in International Publication No. 65619 or International Publication No. 2018 / 165619, or fragments of such antibodies. fragments (e.g., Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, scFv fragments) , dsFv fragments, or single domain antibodies).

[0062] Examples of antibodies that bind to CXCR4 are described in WO 2011 / 098762. , International Publication No. 2008 / 060367, and International Publication No. 2006 / 08 No. 9141, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the targeting moiety of the TDC of the present disclosure is incorporated into the International International Publication No. 2011 / 098762, International Publication No. 2008 / 060367 FRET or as described in the pamphlet of International Publication No. 2006 / 089141 Anti-CXCR4 antibodies, or fragments thereof (e.g., Fab fragments, Fab' fragments, F(ab') 2 fragments, Fv fragments, scFv fragments, dsFv fragments, or single domain antibodies). In some embodiments, the targeting moiety of the TDC of the present disclosure is Antibodies C-9P21, B-1M22, and C-1I described in brochure No. 98762 24, D-1K21, or 9N10, or a fragment thereof (e.g., a Fab fragment, Fab F(ab')2 fragment, Fv fragment, scFv fragment, dsFv fragment, or single domain fragment Main antibody).

[0063] Examples of antibodies that bind to αvβ6 are described in WO 2008 / 112004 and and International Publication No. 2013 / 123152, the contents of which are as follows: In some embodiments, the present disclosure provides a method for the preparation of a medicament for the treatment of a pulmonary arthritis. The targeting moiety of the TDC may be any of the compounds described in WO 2008 / 112004 or WO 201 Anti-αvβ6 antibodies or fragments thereof (e.g., For example, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, scFv fragments, ds In some embodiments, the antibodies of the present disclosure include antibodies having a nucleotide sequence similar to that of the antibody of the present disclosure. The targeting moiety of the TDC may be the antibody STX-100 (Biogen), or a fragment thereof (e.g. , Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, scFv fragment, dsFv fragments, or single domain antibodies).

[0064] Examples of antibodies that bind to AXL are described in WO 2009 / 062690; International Publication No. 2010 / 130751, International Publication No. 2015 / 193430 brochure and WO 2016 / 005593 brochure The contents of which are incorporated herein by reference in their entirety. In this disclosure, the targeting moiety of the TDC is , International Publication No. 2010 / 130751, International Publication No. 2015 / 193 430 or International Publication No. 2016 / 005593 The anti-AXL antibodies described herein, or fragments thereof (e.g., Fab fragments, Fab' fragments, F( ab') a double-stranded antibody fragment, Fv fragment, scFv fragment, dsFv fragment, or single domain antibody) In some embodiments, the targeting moiety of a TDC of the present disclosure comprises ADCT-60 1 (ADC Therapeutics), or a fragment thereof (e.g., a Fab fragment) , Fab' fragment, F(ab')2 fragment, Fv fragment, scFv fragment, dsFv fragment, or includes single domain antibodies).

[0065] Examples of antibodies that bind to MERTK include those described in WO 2016 / 106221. , International Publication No. 2019 / 005756, and International Publication No. 2019 / 08 No. 4307, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the targeting moiety of the TDC of the present disclosure is incorporated into the International International Publication No. 2016 / 106221, International Publication No. 2019 / 005756 FRET or as described in the pamphlet of International Publication No. 2019 / 084307 Anti-MERTK antibody, or a fragment thereof (e.g., Fab fragment, Fab' fragment, F(ab') 2 fragments, Fv fragments, scFv fragments, dsFv fragments, or single domain antibodies). In some embodiments, the targeting moiety of a TDC of the present disclosure is the antibody RGX-019 (R genix), or a fragment thereof (e.g., Fab fragment, Fab' fragment, F(ab')2 fragment) fragments, Fv fragments, scFv fragments, dsFv fragments, or single domain antibodies).

[0066] LRRC15 is involved in many cancers, including breast cancer, head and neck cancer, lung cancer, pancreatic cancer, and ovarian cancer. Cancer-associated fibroblasts in colon cancer, renal cancer, esophageal cancer, gastric adenocarcinoma, and bladder cancer It is expressed in the cytoplasm of the thyroid gland (Purcell et al., 2018, Cancer Res. 78(14):4059-4072, Dominguez et al. l., 2019, Cancer Discovery 10(2):232-253). Thus, in some embodiments, The TDCs of the present disclosure target LRRC15. Examples of antibodies that bind to LRRC15 are available from International Publication No. 2017 / 095805, the contents of which are incorporated herein by reference. The entire disclosure of which is incorporated herein. Antibodies that bind to LRRC15 are also commercially available. For example, Abcam catalog number ab150376 and Creative B The product is from Iolabs and has the catalog number TAB-0709CL. i.e.) is an MMAE containing an ADC directed against LRRC15 (Purcell et al., 2018, Cancer Res. 78(14):4059-4072). In some embodiments, the present disclosure The targeting moiety of the TDC is described in WO 2017 / 095805. the antibody of ABBV-085, one of the commercially available antibodies described in this paragraph or a fragment thereof (e.g., Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, fragments, scFv fragments, dsFv fragments, or single domain antibodies).

[0067] ALK5 inhibitors The ALK5 inhibitors of the present disclosure preferably competitively and reversibly inhibit the cytoplasmic activity of the ALK5 receptor. It is a small molecule that binds to the ATP binding site in the kinase domain.

[0068] ALK5 inhibitors inhibit the activity of ALK5 against other TGF-β family receptors, such as ALK4 and and / or ALK7 and / or TGF-β receptor II, or It can be, but need not be, selective. The inhibitors have activity against both ALK5 and TGF-β receptor II. In one embodiment, the ALK5 inhibitor has limited inhibitory activity against the BMP II receptor. It has.

[0069] As measured in an in vitro cell assay using HEK293T cells, IC of the indicated ALK5 inhibitors 50 is preferably 100 nM or less, more preferably 50 nM and most preferably 20 nM or less. An exemplary cellular assay is described in Section 6.6 below. will be explained.

[0070] Specific examples of ALK5 inhibitors suitable for use in the TDC of the present disclosure include imidazole-benzaldehyde Zodioxole compounds, imidazole-quinoxaline compounds, pyrazole-pyrrolo compounds and thiazole-based compounds.

[0071] According to one embodiment of the present disclosure, the imidazole-benzodioxole ALK5 inhibitor is , which has the following formula:

[0072] [ka] In the formula, R 1 is hydrogen or lower alkyl having 1 to about 5 carbon atoms, and R 2 teeth , hydrogen, or lower alkyl having 1 to about 5 carbon atoms; R 3 is amide, tolyl, alkynyl having 1 to about 3 carbon atoms, carboxyl, or alkynyl having 1 to about 5 carbon atoms A is a direct bond or an alkanol having 1 to about 5 carbon atoms. and B is a direct bond or an alkyl having 1 to about 5 carbon atoms. In another preferred embodiment of the present disclosure, R 2 is hydrogen or methyl, A has one carbon atom, B is a direct bond to the benzyl group, and R 3 is an amide In a combined preferred embodiment of the present disclosure, R 2 is hydrogen or methyl where A has 1 carbon atom and B is a direct bond to the benzyl group.

[0073] In accordance with another aspect of the present disclosure, the imidazole-quinoxaline ALK5 inhibitor is It has the following formula:

[0074] [ka] In the formula, R 1 is hydrogen or lower alkyl having 1 to about 5 carbon atoms, and R 2 teeth , hydrogen, halogen, or lower alkyl having 1 to about 5 carbon atoms; R 3 teeth, amide, nitrile, alkynyl having 1 to about 3 carbon atoms, carboxyl, or and A is an alkanol having from 1 to about 5 carbon atoms, and B is a direct bond or an alkyl having 1 to about 5 carbon atoms. In another preferred embodiment of the present disclosure, R 2 is hydrogen or methyl wherein halogen includes fluorine or chlorine, A has 1 carbon atom, and B is Direct bond to the benzyl group, R 3 is an amide. In embodiments, R 2 is hydrogen or methyl, A has 1 carbon atom, B is a direct bond to the benzyl group.

[0075] According to another embodiment of the present disclosure, the pyrazole ALK5 inhibitor has the formula:

[0076] [ka] In the formula, R 2 is hydrogen, halogen, or lower alkyl having 1 to about 5 carbon atoms. R 4 is hydrogen, halogen, lower alkyl having 1 to about 5 carbon atoms, Alkoxy, haloalkyl, carboxyl, carboxyalkyl esters having carbon atoms of The alkyl group may be a terephthalate, a nitrile, an alkylamine, or a group having the formula:

[0077] [ka] In the formula, R 5 is a lower alkyl having 1 to about 5 carbon atoms, halogen, or morpholino No, R 6 pyrrole, cyclohexyl, morpholino, pyrazole, pyran, imino A is a direct bond or or alkyl having 1 to about 5 carbon atoms.

[0078] In accordance with another embodiment of the present disclosure, the pyrazole-pyrrolo ALK5 inhibitor has the formula: Has.

[0079] [ka] In the formula, R 7 is hydrogen, halogen, lower alkyl having 1 to about 5 carbon atoms, alkano alkylamine, morpholino, or alkylamine; R 2 is hydrogen, halogen, or 1~ is a lower alkyl having about 5 carbon atoms, and R 8 is hydrogen, hydroxyl, amino, a halogen, or a group having the formula:

[0080] [ka] In the formula, R 5 is piperazinyl and R 6 are morpholino, piperidinyl, and piperazinyl , alkoxy, hydroxyl, oxane, halogen, thioalkyl, or alkylamino and A is lower alkyl having 1 to about 5 carbon atoms.

[0081] According to another embodiment of the present disclosure, the thiazole ALK5 inhibitor has the formula:

[0082] [ka]

[0083] In the formula, R 9 is hydrogen, halogen, or lower alkyl having 1 to about 5 carbon atoms Yes, R 10 is hydrogen or lower alkyl having 1 to about 5 carbon atoms.

[0084] In certain embodiments, the ALK5 inhibitor is a compound designated A-N in Table 1 below. The compound is selected from any one of the following:

[0085] [Table 1-1]

[0086] [Table 1-2]

[0087] [Table 1-3]

[0088] In more specific embodiments, the ALK5 inhibitor is designated 1-283 in Table 2 below. The compound is selected from any of the compounds listed above.

[0089] [Table 2-1]

[0090] [Table 2-2]

[0091] [Table 2-3]

[0092] Table 2-4

[0093] Table 2-5

[0094] Table 2-6

[0095] Table 2-7

[0096] Table 2-8

[0097] Table 2-9

[0098] Table 2-10

[0099] Table 2-11

[0100] Table 2-12

[0101] Table 2-13

[0102] [Table 2-14]

[0103] [Table 2-15]

[0104] [Table 2-16]

[0105] [Table 2-17]

[0106] [Table 2-18]

[0107] The preparation and use of ALK5 inhibitors is well known in the scientific and patent literature, It is well documented. PCT Publication WO 2000 / 61576, and U.S. Patent Application Publication No. 2003 / 0149277, which discloses triaryl imidazoline. The present application discloses benzodiazepine derivatives and their use as ALK5 inhibitors. In WO 2001 / 62756, pyridinyl imidazole derivatives and and its use as an ALK5 inhibitor. PCT Publication WO 2009 / 024444 is hereby incorporated by reference. The 2 / 055077 pamphlet identifies imidazolyl cyclic acetamides as ALK5 inhibitors. The use of benzophenone derivatives is disclosed in PCT Publication No. WO 2003 / 087304. In FRET, trisubstituted heteroaryls and ALK5 and / or ALK4 inhibition Its use as an anti-inflammatory agent is disclosed in WO 2005 / 103028. U.S. Patent Application Publication No. 2008 / 0319012, and U.S. Patent No. 7,400,400. No. 7,958 discloses 2-pyridyl compounds as ALK5 and / or ALK4 inhibitors. Substituted imidazoles are disclosed. One representative compound, IN-1130, has several It exhibits ALK5 and / or ALK4 inhibitor activity in several animal models. The patent and patent publications provide additional examples of ALK5 inhibitors, exemplary synthetic schemes, and Methods of using ALK5 inhibitors are provided: U.S. Pat. No. 6,465,493; U.S. Pat. No. 6,906,089, U.S. Pat. No. 7,365,066, U.S. Pat. No. 7,087,626, U.S. Pat. No. 7,368,445, U.S. Pat. No. 7,265,225, U.S. Pat. No. 7,405,299, U.S. Pat. 407,958, U.S. Pat. No. 7,511,056, U.S. Pat. No. 7,61 2,094, U.S. Pat. No. 7,691,865, U.S. Pat. No. 7,863, 288, U.S. Pat. No. 8,410,146, U.S. Pat. No. 8,410,14 No. 6, U.S. Patent No. 8,420,685, U.S. Patent No. 8,513,222 Specification, U.S. Patent No. 8,614,226 Specification, U.S. Patent No. 8,791,113 Specification No. 8,815,893; No. 8,846,931; U.S. Patent No. 8,912,216, U.S. Patent No. 8,987,301, U.S. Patent No. 9,051,307, U.S. Patent No. 9,051,318, U.S. Patent No. 9,073,918 and PCT Publication No. WO 2004 / 06539 Pamphlet No. 2, International Publication No. 2009 / 050183 Pamphlet, International Publication No. 20 Pamphlet No. 09 / 133070, Pamphlet No. WO 2011 / 146287 and WO 2013 / 009140. The aforementioned patents and patent publications is incorporated by reference in its entirety.

[0108] Several ALK5 inhibitors are commercially available, including SB-525334 (CAS 356 559-20-1), SB-505124(CAS 694433-59-5), SB- 431542(CAS 301836-41-9), SB-202474(EMD4 B iosciences Merck KGaA, Darmstadt, Germany) , LY-364947(CAS 396129-53-6), IN-1130, GW-7 88388, and D4476 (EMD4 Biosciences Merck KG aA, Darmstadt, Germany).

[0109] The structures and names of the ALK5 inhibitors described herein may be used in conjunction with antibodies and / or linkers. This refers to a molecule before binding to a molecule.

[0110] Preferred ALK5 inhibitors are those which have a free NH or NH2 group, preferably alkyl, heteroaryl, NH or NH2 groups attached to alkyl, heteroaryl, or aryl groups Attached to the linker via the aryl or NH or NH2 group moiety of the aryl group (For example, compounds 1 to 23, 26 to 29, and 31 shown in Table 2) , 35, 37, 39, 40, 42, 43, 45-48, 50-85, 87-90, 93, 96, 98-104, 106, 108, 109, 111, 112, 114, 116-12 0, 132, 146, 149, 156, 184, 187, 193, 218, 260-27 7, 282, and 283). ALK5 inhibitors can be derivatized to free NH or NH groups. The derivatized ALK5 inhibitors are preferably designed so that their activity is mediated by Although this can be determined experimentally, the inhibitory activity of adding NH or NH2 groups is nullified. The structure-activity relationship (SAR) of the inhibitor should be taken into account to reduce the possibility of Exemplary derivatized counterparts of some of the compounds shown in Table 1 are listed below in Table 3. is shown.

[0111] [Table 3]

[0112] Linker Typically, the TDC includes a linker between the ALK5 inhibitor and the targeting moiety. - is a moiety containing a covalent bond or chain of atoms that covalently attaches a targeting moiety to a drug moiety In various embodiments, the linker may be an alkyldiyl, an aryldiyl, a heterodiyl, or Divalent groups such as aryldiyl, -(CR2) n O(CR2) n -, alkyloxy (e.g. e.g., polyethyleneoxy, PEG, polymethyleneoxy) and alkylamino (e.g., Moieties such as repeating units of polyethyleneamino, Jeffamine™, and succinate, succinamide, diglycolate, malonate, and caproamide Examples include diacid esters and amides containing various PEG-containing linkers. are known in the art and are available from companies such as BroadPharm Exemplary PEG-containing linkers include Mal -PEG2-Val-Cit-PAB-OH (BroadPharm catalog number BP- 23203), Mal-PEG4-Val-Cit-PAB-OH(BroadPhar (mCatalog No. BP-23204), Mal-PEG4-Val-Cit-PAB-PN P (BroadPharm catalog number BP-23668), Mal-amido-PE G2-Val-Cit-PAB-PNP (BroadPharm catalog number BP-23 675), Azido-PEG3-Val-Cit-PAB-OH (BroadPharm Catalog No. BP-23206), Azido-PEG4-Val-Cit-PAB-OH (B roadPharm Catalog No. BP-23207), Azide-PEG3-Val-Ci t-PAB-PNP (BroadPharm catalog number BP-23368), Fmoc -PEG4-Ala-Ala-Asn-PAB (BP-23328), azide-PEG5 -Ala-Ala-Asn-PAB (BroadPharm catalog number BP-2332 9), Fmoc-PEG3-Ala-Ala-Asn(Trt)-PAB(BroadP Harm Catalog No. BP-23285), Azido-PEG4-Ala-Ala-Asn (Trt)-PAB (BroadPharm catalog number BP-23284), and F moc-PEG3-Ala-Ala-Asn(Trt)-PAB-PNP(BroadP In some embodiments, the compound may be selected from the group consisting of benzodiazepines (Bordeaux Pharmaceuticacid, Catalog No. BP-23297). The TDC linker may be a PEG or a peptide, such as those described in this section, e.g., Val-Cit. It contains one of the dipeptides known to be

[0113] The linker may comprise one or more linker moieties, such as a stretcher and a spacer moiety. For example, a peptidyl linker may comprise two or more amino acids, and optionally and containing one or more stretcher and / or spacer peptidyl moieties. A variety of linker moieties are known in the art, some of which are , as described below.

[0114] The linker may be a "cleavable linker" that facilitates release of the drug inside the cell. For example, Acid-labile linkers (e.g., hydrazones), protease-sensitive linkers (e.g., peptidases) (sensitive) linker, photolabile linker, dimethyl linker, or disulfide-containing linker (Chari et al., 1992, Cancer Research 52:127-131, U.S. Patent No. 5,208,020 No. 10 / 109,493, can be used.

[0115] Examples of linkers and linker moieties known in the art include maleimidocaproyl (mc); Maleimidocaproyl-p-aminobenzylcarbamate; Maleimidocapro yl-peptide-aminobenzylcarbamate linkers, e.g., maleimidocaproyl -L-phenylalanine-L-lysine-p-aminobenzylcarbamate, and maleic Midocaproyl-L-valine-L-citrulline-p-aminobenzylcarbamate (vc );N-Succinimidyl 3-(2-pyridyldithio)proprionate (N-succinimidyl also known as imidinyl 4-(2-pyridyldithio)pentanoate or SPP); 4-Succinimidyl-oxycarbonyl-2-methyl-2-(2-pyridyldithio)- Toluene (SMPT); N-Succinimidyl 3-(2-pyridyldithio)propionate N-Succinimidyl 4-(2-pyridyldithio)butyrate (SPDP); N-Succinimidyl 4-(2-pyridyldithio)butyrate (SPD B); 2-Iminothiolane; S-Acetyl succinic anhydride; Disulfide benzylcarbamate Carboxylate;Hydrazone linker;N-(α-maleimidoacetoxy)succinic acid Imidoester; N-[4-(p-azidosalicylamido)butyl]-3'-(2'-pi Lysyldithio)propionamide (AMAS); N[β-maleimidopropyloxy]s Succinimide ester (BMPS); [N-ε-Maleimidocaproyloxy]succinimide Imidoester (EMCS); N-[γ-Maleimidobutyryloxy]succinimide ester Succinimidyl-4-[N-maleimidomethyl]cyclohexane (GMBS); -1-carboxy-[6-amidocaproate] (LC-SMCC); succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate (LC-SPD P); m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS); N-Succinimidyl [4-iodoacetyl]aminobenzoate (SIAB); Succinimidyl Maleimidomethyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SM CC); N-Succinimidyl 3-[2-pyridyldithio]-propionamide (SPD P); [N-ε-maleimidocaproyloxy] sulfosuccinimide ester (sulfo -EMCS); N-[γ-maleimidobutyryloxy]sulfosuccinimide ester ( Sulfo-GMBS; 4-sulfosuccinimidyl-6-methyl-α-(2-pyridyldimethyl) Thio)toluamido]hexanoate-)(sulfo-LC-SMPT); sulfosuccinimide 6-(3'-[2-pyridyldithio]-propionamido)hexanoate(sulfonyl) m-Maleimidobenzoyl-N-hydroxysulfosuccinimide N-Sulfosuccinimidyl [4-iodoacetyl] ester (Sulfo-MBS); Sulfosuccinimidyl 4-[N-maleimide] Methyl]cyclohexane-1-carboxylate (sulfo-SMCC); sulfosuccin Imidyl 4-[p-maleimidophenyl]butyrate (sulfo-SMPB); Licor-bis(succinic acid N-hydroxysuccinimide ester) (EGS); Disc DST (Dysprosyl tartrate); 1,4,7,10-tetraazacyclododecane 1,4,7,10-tetraacetic acid (DOTA); diethylenetriamine-pentaacetic acid (DTPA); thiourea linkers; and oxime-containing linkers.

[0116] In some embodiments, the linker is cleavable under intracellular or extracellular conditions. Thus, cleavage of the linker releases the ALK5 inhibitor from the targeting moiety in the appropriate environment. In yet other embodiments, the linker is not cleavable and the drug is released, e.g. For example, the targeting moiety is released by degradation in the lysosome (U.S. Patent Application Publication No. 2005 / 010999). See US Pat. No. 6,023,649, the entire contents of which are incorporated herein by reference for all purposes. incorporated).

[0117] An example of a non-cleavable linker that can be used in the TDCs of the present disclosure is N-maleimide. Methylcyclohexane 1-carboxylate, maleimidocaproyl, or mercapto An example is an acetamidocaproyl linker.

[0118] In some embodiments, the linker is a protein that is expressed in an intracellular environment (e.g., a lysosome or The linker can be cleaved by a cleavage agent present in the endosome or caveolae. intracellular, including, but not limited to, lysosomal or endosomal proteases It can be a peptidyl linker that is cleaved by a peptidase or protease enzyme. In some embodiments, the peptidyl linker is at least 2 amino acids in length, or It contains a peptidyl moiety that is at least three amino acids in length, or longer.

[0119] Cleavage agents may include, but are not limited to, cathepsins B and D, and plasmin. All of these hydrolyze dipeptide drug derivatives that release the active drug within the target cell. It is known that (e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:6 For example, peptidyl linkers can be used to bind thiol-dependent proteases such as cathepsins. linker) Other examples of such linkers are described, for example, in U.S. Pat. 45, which is incorporated herein by reference in its entirety for all purposes. be absorbed.

[0120] In some embodiments, the peptidyl group is cleavable by an intracellular protease. The linker is a Val-Cit linker or a Phe-Lys linker (e.g., va U.S. Patent No. 6,214,344 describes the synthesis of doxorubicin with an l-cit linker. See specification No. 5).

[0121] In other embodiments, the cleavable linker is pH sensitive, i.e., sensitive to certain pH values. Typically, pH-sensitive linkers are hydrolyzable under acidic conditions. For example, an acid-labile linker (e.g., Hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic acid amides, ol Other suitable esters, acetals, ketals, etc., can be used (see, e.g., U.S. Pat. No. 6,139,523). No. 5,122,368, U.S. Pat. No. 5,824,805, U.S. Pat. , 622,929, Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67- 123, see Neville et al., 1989, Biol. Chem. 264:14653-14661). - is relatively stable under neutral pH conditions such as those in blood, but is stable under pH conditions close to that of the lysosome. In certain embodiments, the hydrolyzable linker is unstable at H 5.5 or below 5.0. The linker is attached to the therapeutic agent via a thioether linker (e.g., an acylhydrazone bond). thioethers) (see, for example, U.S. Pat. No. 5,622,929).

[0122] In yet other embodiments, the linker is cleavable under reducing conditions (e.g., di Disulfide linkers). A variety of disulfide linkers are known in the art, For example, SATA (N-succinimidyl-5-acetylthioacetate), SPDP ( N-Succinimidyl-3-(2-pyridyldithio)propionate), SPDB(N- succinimidyl-3-(2-pyridyldithio) butyrate), and SMPT (N- Succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl) dithio)-toluene)-, SPDB, and SMPT can be used to form (See, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C.W. Vogel ed., Oxford U. Press, 1987; see also U.S. Pat. No. 4,880 (See also ,935)

[0123] In other embodiments, the linker is a malonic acid linker (Johnson et al., 1995, Anti Cancer Res. 15:1387-93), maleimidobenzoyl linker (Lau et al., 1995, Bioorg -Med-Chem. 3(10):1299-1304), or the 3'-N-amide analogue (Lau et al., 1995, Bi oorg-Med-Chem. 3(10):1305-12).

[0124] In some embodiments, the linker allows multiple drug molecules to be linked to a single targeting moiety molecule. It is a multivalent linker that can be used to link multiple antibodies (e.g., single antibody molecules). For example, the Fleximer linker technology developed by Mersana uses ester bonds. The drug molecules are incorporated into the solubilized polyacetal backbone via the The method is based on the ability to produce highly loaded TDCs (e.g., ) with a drug-antibody ratio (DAR) of up to 20. Exemplary multivalent linkers include, for example, For example, International Publication No. 2009 / 073445 and International Publication No. 2010 / 068 Pamphlet No. 795, Pamphlet No. 2010 / 138719, Pamphlet No. Pamphlet No. 2011 / 120053, Pamphlet No. 2011 / 171020 , International Publication No. 2013 / 096901, International Publication No. 2014 / 008 Pamphlet No. 375, Pamphlet No. 2014 / 093379, Pamphlet No. 2014 / 093394 pamphlet and International Publication No. 2014 / 093640 pamphlet and a brochure, the contents of which are incorporated herein by reference in their entirety. do.

[0125] In many cases, the linker is substantially insensitive to the extracellular environment. In this case, "substantially insensitive to the extracellular environment" in the context of a linker means that the TDC Less than about 20%, 15%, 10%, 5%, 3%, or less than about 1% risk in the sample This means that the anchor is cleaved when the TDC is present in an extracellular environment (e.g., plasma). do.

[0126] Whether the linker is substantially insensitive to the extracellular environment can be determined, for example, by administering TDCs in plasma. Incubate for a predetermined time (e.g., 2, 4, 8, 16, or 24 hours), then plasma The amount of free drug present in the solution can be determined by quantifying the amount of free drug present in the solution.

[0127] In other, non-mutually exclusive, embodiments, the linker may be capable of promoting cellular internalization. In certain embodiments, the linker can be used when conjugated to a therapeutic agent (i.e., i.e., in the context of the linker-therapeutic agent moiety of the TDCs described herein), intracellular In yet another embodiment, the linker facilitates the synthesis of the ALK5 inhibitor and the antibody. When complexed with both, it promotes cellular internalization.

[0128] In many embodiments, the linker is self-immolative. The term "self-immolative" refers to the covalent bonding of two spaced apart chemical moieties to form a stable triad. refers to a bifunctional chemical moiety that can be broken down into two groups when its bond to the first moiety is cleaved. When the second chemical moiety is present, it spontaneously separates from the second chemical moiety. 7 / 059404 pamphlet, International Publication No. 2006 / 110476 pamphlet, International Publication No. 2005 / 112919, International Publication No. 2010 / 062171 No. 2009 / 017394, International Publication No. 200 7 / 089149 pamphlet, International Publication No. 2007 / 018431 pamphlet, International Publication No. 2004 / 043493 and International Publication No. 2002 / 083 See Brochure No. 180, which describes drugs and cutting agents that are optionally self-immolating phosphorus. and drug-cleavable agent conjugates linked through a carrier, all of which are expressly incorporated by reference. Self-immolative spaces that can be used to generate self-immolative linkers are incorporated. Examples of Sar units are set forth in the following formula:

[0129] Various exemplary linkers that can be used in the present compositions and methods are described in PCT Publication No. International Publication No. 2004 / 010957, U.S. Patent Application Publication No. 2006 / 00 74008, U.S. Patent Application Publication No. 2005 / 0238649, and U.S. and US Patent Application Publication No. 2006 / 0024317 (each of which is a part of the entire (which is incorporated herein by reference in its entirety for this purpose).

[0130] The TDCs of the present disclosure may be of Formula I below, where the antibody or other targeting moiety (" in Formula I") Ab) is a compound that binds one or more ALK5 inhibitors via an optional linker (L). complexed with a biological moiety (D). Ab-(LD)p I

[0131] Thus, the targeting moiety can be linked to the drug either directly or via a linker. In Formula I, p represents the number of drugs per targeting moiety (i.e., A The average number of targeting moieties is, for example, from about 1 to about 20 per targeting moiety. The number of drug moieties per targeting moiety can range from 2 to 10, and in certain embodiments, There may be about 8 drug moieties. Further details on drug loading are provided in Section 5.5 below. do.

[0132] In some embodiments, the linker moiety is linked to another A linker component or "stretcher" may be included that connects the targeting moiety to the drug moiety. An exemplary Stretcher is shown below (where the wavy line on the left indicates the covalent bond to the targeting moiety): The wavy line on the right indicates the site of covalent attachment to another linker component or drug moiety. vinegar).

[0133] [ka] U.S. Patent No. 9,109,035; Ducry et al., 2010, Bioconjugate Chem. 21: See 5-13.

[0134] In some embodiments, the linker component may comprise an amino acid unit. In one embodiment, the amino acid unit allows the linker to be cleaved by a protease. This allows the release of TDCs from the TDCs upon exposure to intracellular proteases such as lysosomal enzymes. For example, Doronina et al., 2003, Nat. Biotechnol. 21:778-7 84. Exemplary amino acid units include, but are not limited to, dipeptides, tripeptides, Exemplary dipeptides include valinyl peptides, tetrapeptides, and pentapeptides. citrulline (VC or val-cit), alanine-phenylalanine (AF) or ala-phe), phenylalanine-lysine (FK or phe-lys), or or N-methyl-valine-citrulline (Me-val-cit). Examples of tides are glycine-valine-citrulline (gly-val-cit), and An exemplary tetracarboxylic acid is lysine-glycine-glycine (gly-gly-gly). As a peptide, glycine-glycine-phenylalanine-glycine (gly-gly- The amino acid units include naturally occurring amino acid residues, as well as It may contain minor amino acids and unnatural amino acid analogs, for example, the amino acid citrulline. The site binds to specific enzymes, such as cathepsins B, C, and D, or plasmin proteases. can be designed and optimized in selection for enzymatic cleavage.

[0135] In some embodiments, the linker component is either directly or in combination with a stretcher and "Spectroscopy" links the targeting moiety to the drug moiety either by a targeting unit and / or an amino acid unit. Spacer units may be "self-immolative" or "non-self-immolative." A "non-self-immolative" spacer unit is one in which some or all of the spacer units are part of the enzyme TDC. These remain attached to the drug moiety upon cleavage by an enzyme (e.g., a protease). Examples of spacer units include, but are not limited to, glycine spacer units and glycine-glycine spacer units. A "self-immolative" spacer unit is a unit that is attached to a separate hydrolysis site. In certain embodiments, the linker In one such embodiment, the spacer unit comprises a p-aminobenzyl unit. p-Aminobenzyl alcohol is linked to the amino acid unit via an amide bond, and carbaminium The phosphate, methylcarbamate, or carbonate salts are the cytotoxic agents of benzyl alcohol. See, e.g., Hamann et al., 2005, Expert Opin. Ther. Patents 15:1087- See 1103. In one embodiment, the spacer unit is p-aminobenzyloxycarbo In certain embodiments, the phenyl group of the p-aminobenzyl unit is The Len part is Q m where Q is -C1-C8 alkyl, -O-(C1-C8 alkyl), -halogen, -nitro, or -cyano, and m is an integer ranging from 0 to 4. Examples of self-immolative spacer units include, but are not limited to, p-aminobenzyl alkyl. Aromatic compounds that are electronically similar to coal (e.g., U.S. Patent Application Publication No. 2005 / 025 6030), such as 2-aminoimidazole-5-methanol derivatives (Ha y et al., 1999, Bioorg. Med. Chem. Lett. 9:2237), and ortho- or para- Further examples include substituted and unsubstituted 4-aminobutyric acid amides ( Rodrigues et al., 1995, Chemistry Biology 2:223), appropriately substituted bicyclo[2. 2.1] and bicyclo[2.2.2] ring systems (Storm et al., 1972, Amer. Chem. Soc. 9 4:5815), and 2-aminophenylpropionic acid amide (Amsberry et al., 1990, J Org. Chem. 55:5867), which undergoes ring formation upon hydrolysis of the amide bond. can be used. Exclusion of amine-containing drugs substituted at the a-position of glycine (Kingsbury (Y et al., 1984, J. Med. Chem. 27:1447) is also a useful self-immolative spacer for TDC. This is an example.

[0136] In one embodiment, the spacer unit is a branched bis(hydroxymethyl) (BHMS) units, which are used to incorporate and release multiple drugs. It is possible.

[0137] [ka] wherein A and D are defined above for Formula I, and A is a stretcher where a is an integer ranging from 0 to 1, W is an amino acid unit, and w is a number ranging from 0 to 1. 2, and Q is -C1-C8 alkyl, -O-(C1-C8 alkyl), -halogen, -nitro, or -cyano; m is an integer ranging from 0 to 4; and n is , 0 or 1, and p ranges from 1 to about 20.

[0138] The linker may include any one or more of the linker components described above. In the form, the linker is shown in parentheses in the formula of TDC below: Ab-(-[Aa-Ww-Yy]-D) p II where Ab, A, a, W, w, D, and p are defined in the previous paragraph, and Y is a (where y is a pacer unit and y is 0, 1, or 2). Exemplary of such linkers are: Embodiments are described in U.S. Patent Application Publication No. 2005 / 0238649, which , which is incorporated herein by reference.

[0139] Exemplary linker moieties and combinations thereof are shown below in the context of a TDC of Formula II:

[0140] [ka]

[0141] Linker components, including stretcher, spacer, and amino acid units, can be prepared using methods known in the art. Methods known in the art, such as those described in U.S. Patent Application Publication No. 2005 / 0238649 It can be synthesized by

[0142] Drug Load Drug loading is expressed as p and is the number of ALK5 receptors per targeting moiety (e.g., per antibody) in the molecule. is the average number of inhibitor moieties. The average number is often a fraction or decimal, but is based on the drug loading ("p") is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 1 per targeting moiety 2, 13, 14, 15, 16, 17, 18, 19, 20 or more parts (D) Generally, ALK5 inhibitor loading averages between 2 and 8 drugs per targeting moiety. moieties, more preferably 2 to 4 drug moieties per antibody, or 5 to 6 drug moieties per targeting moiety. This results in seven drug moieties.

[0143] As will be appreciated by those skilled in the art, in many instances reference to TDCs (sometimes referred to as pharmaceutical compositions) (in the context of a product) is shorthand for a group or collection of TDC molecules, each molecule or multiple ALK5 inhibitor moieties covalently linked to a targeting moiety at a distinct molecular base. Although the ratio may vary from TDC molecule to TDC molecule in a population, the drug loading ratio is the average for a population or population. In some embodiments, the population or collection comprises 1 to 30 drug moieties. In some embodiments, 1 to 20, 1 to 15, 2 to 12 1, 2-8, 4-15, or 6-12 drug moieties covalently attached to Preferably, the population average is greater than or equal to the average of the TDC molecules described in the preceding paragraph. and, for example, 2 to 8 drug moieties per targeting moiety, more preferably 1 to 8 drug moieties per targeting moiety. 4-8 drug moieties per minute, or 5-7 drug moieties per targeting moiety.

[0144] Some TDC populations are compositions comprising the TDCs described herein, and a drug moiety. targeting moieties that lack the ability to bind to ALK5 inhibitors, e.g., in the form of antibodies that fail to bind to ALK5 inhibitors. It is possible.

[0145] of ALK5 inhibitor moieties per targeting moiety in the preparation of TDC from the conjugation reaction Average counts were obtained using mass spectroscopy, hydrophobic interaction chromatography (HIC), and ELISA. It can be characterized by conventional means such as the A assay.

[0146] The quantitative distribution of TDCs with respect to p can also be determined. The isolation, purification, and characterization of a single tDC demonstrates that p is a tDC with other ALK5 inhibitor loading. A certain value from can be achieved by means such as electrophoresis.

[0147] For some drug conjugates, p may be limited by the number of binding sites on the targeting moiety. For example, if the bond is a cysteine ​​thiol, then in the above exemplary embodiment: The targeting moiety (e.g., an antibody) has only one or more cysteine ​​thiol groups. or one or more sufficiently reactive groups to which a linker can be attached. In certain embodiments, higher drug loading, e.g., At p>5, certain drug conjugates may aggregate, become insoluble, become toxic, or have reduced cell permeability. In certain embodiments, the drug loading on the TDCs of the present disclosure is from 1 to about 8 , about 2 to about 6, about 3 to about 5, about 3 to about 4, about 3.1 to about 3.9, about 3.2 to about 3.8, about 3.2 to about 3.7, about 3.2 to about 3.6, about 3.3 to about 3.8, or about 3.3 to about 3. In fact, for a given TDC, the optimal ratio of drug moieties per antibody is It has been shown that the β-glucan content may be less than 8, and may be about 2 to about 5. See US Pat. No. 05 / 0238649, which is incorporated herein by reference in its entirety. .

[0148] In certain embodiments, less than the theoretical maximum amount of drug moiety is added during the conjugation reaction. The targeting moiety is conjugated to a drug-linker, for example, as discussed below. It may contain lysine residues that do not react with the intermediate or linker reagent. does not contain many free reactive cysteine ​​thiol groups that can be linked to a biological moiety, In this case, many cysteine ​​thiol residues in antibodies exist as disulfide bridges. In certain embodiments, the antibody or other targeting moiety is subjected to partial or total reducing conditions. Under these conditions, dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP ) to generate a reactive cysteine ​​thiol group. In certain embodiments, the antibody or other targeting moiety is subjected to denaturing conditions to denature the lysine or presenting a reactive nucleophilic group such as cysteine.

[0149] The loading (drug / antibody ratio) of a TDC can be determined, for example, by (i) the drug-linker ratio to the targeting moiety. (ii) limiting the molar excess of intermediates or linker reagents; (iii) partial reducing conditions for cysteine ​​thiol modification; (iv) limiting the number and location of cysteine ​​residues in the linker-drug Product combinations (see, for example, PCT Publication WO 2006 / 034488 (see (which is incorporated herein in its entirety by reference) a) and / or b) by targeted modification to control the number and / or position of the target molecule. This can be controlled in a variety of ways by manipulating the amino acid sequence of the functionalized moiety.

[0150] Two or more nucleophilic groups react with a drug-linker intermediate or linker reagent, followed by the formation of a drug-linker intermediate or linker reagent. When reacted with a targeting moiety reagent, the resulting product then contains one or more targeting moieties attached to the targeting moiety. It should be understood that is a mixture of TDC compounds having a distribution of multiple drug moieties. The average number of drugs per targeting moiety was determined from the mixture by dual ELISA antibody assay. can be calculated, which is targeting moiety specific and drug specific. TDC molecules are identified in the mixture by mass spectroscopy and HPLC, e.g., hydrophobic interaction. They can be separated by chromatography.

[0151] In some embodiments, a uniform TDC with a single loading value is obtained by electrophoresis or It can be isolated from complex mixtures by chromatography.

[0152] 5.6. Formulation and Administration Suitable routes of administration of TDC include, but are not limited to, oral, parenteral, rectal, transmucosal, and intestinal. Intrathecal, intramedullary, intrathecal, direct intraventricular, intravenous, intravitreal, intracavity, intraperitoneal, or intratumoral injection The preferred route of administration is parenteral, more preferably intravenous. Alternatively, one could try to affect fibrosis in a local rather than systemic manner, e.g. via direct injection of the compound into the affected area or via direct injection of the compound into a solid tumor The compounds may be administered via injection.

[0153] The immunoconjugates can be formulated according to known methods for preparing pharmaceutically useful compositions. whereby the TDC combines the mixture with pharmaceutically useful excipients. Phosphate buffered saline is one example of a pharmaceutically useful excipient. Other useful excipients are and are well known to those skilled in the art. See, e.g., Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 5th Edition (Lea & Febiger 1990), and Gennaro (ed.), Re minton's Pharmaceutical Sciences, 18th Edition (Mack Publishing Company 1990), and its revised editions.

[0154] In a preferred embodiment, TDC is N-(2-acetamido)-2-aminoethane Sulfonic acid (ACES); N-(2-acetamido)iminodiacetic acid (ADA); N,N- Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES); 4-(2-hydroxyethyl)-2-aminoethanesulfonic acid hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES); 2-(N-morpho 3-(N-morpholino)ethanesulfonic acid (MES); 3-(N-morpholino)propanesulfonic acid (MO PS; 3-(N-morpholinyl)-2-hydroxypropanesulfonic acid (MOPSO) and piperazine-N,N'-bis(2-ethanesulfonic acid) [Pipes] Formulated in Good's biological buffer (pH 6-7) using a buffer selected from the group A more preferred buffer solution is one having a concentration in the range of 20 to 100 mM, more preferably about MES or MOPS at a concentration of 25 mM. Most preferred is 2 mM MES or MOPS at pH 6.5. The formulation contained 25 mM trehalose as an additive, and 0.01 % v / v Polysorbate 80 and as a result of the added additives, the final buffer The concentration is adjusted to 22.25 mM. The preferred method of storage is at 2°C to 8°C. as a lyophilized formulation of the complex, stored at temperatures ranging from -20°C to 2°C. do.

[0155] TDCs can be administered intravenously, e.g., via bolus injection, slow infusion, or continuous infusion. Preferably, the TDC is less than about 4 hours, more preferably about Infused over less than 3 hours. For example, the first 25-50 mg should be administered within 30 minutes, preferably Inject one part in exactly 15 minutes and the rest over the next 2-3 hours. The formulations may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles. and may contain formulatory agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the active ingredient can be dissolved in a suitable vehicle, such as a sterile pipette, before use. It may be in powder form for constitution with halogen-free water.

[0156] Additional pharmaceutical methods can be utilized to control the duration of action of TDC. Release preparations are prepared through the use of polymers that complex or adsorb TDCs. For example, poly(ethylene-co-vinyl acetate) can be used as a biocompatible polymer. ) matrix, and a polyanhydride copolymer of stearic acid dimer and sebacic acid. Sherwood et al., 1992, Bio / Technology 10:1446. The release rate of TDC from such a matrix depends on the molecular weight of the TDC, the amount of TDC in the matrix, It depends on the amount of C and the size of the dispersed particles. Saltzman et al., 1989, Biophys. J. 55: 163, Sherwood et al., supra. Other solid dosage forms are described in Ansel et al., Pharmaceutical Dosage Forms Age Forms And Drug Delivery Systems, 5th Edition (Lea & Febiger 1990), and Gen naro (ed.), Remington's Pharmaceutical Sciences, 18th Edition (Mack Publishing C Company 1990) and its revised edition.

[0157] In general, the dosage of TDC administered to humans depends on the patient's age, weight, height, sex, and total body mass. Approximately 0.3mg / kg to 5mg, depending on factors such as health status and past medical history Doses of TDC in the range of 1 / kg can be given to recipients as a single intravenous infusion. Although it may be desirable, lower or higher doses may be administered depending on the circumstances. For example, a dose of 0.3 to 5 mg / kg for a 70 kg patient is ~350 mg, which is 12-20 mg for a 1.7 m patient. 6 mg / m 2 At a dosage of Dosage may be adjusted as needed, e.g., once weekly for 2-10 weeks, once weekly for 8 weeks, or once weekly for 10 weeks. This can be repeated once a week for 1 week or once a week for 4 weeks. Depending on the situation, it may be done less frequently, for example, every other week for several months, or It can also be given monthly or quarterly for a period of 12 months. Not specified, but 0.3mg / kg, 0.5mg / kg, 0.7mg / kg, 1.0mg / kg, 1.2mg / kg, 1.5mg / kg, 2.0mg / kg, 2.5mg / kg, 3 .0mg / kg, 3.5mg / kg, 4.0mg / kg, 4.5mg / kg, and 5. A more preferred dosage is 0.6 mg / kg for one week of administration. / kg, and 1.2 mg / kg for less frequent administration. Any amount ranging from 1000 mg / kg to 1000 mg / kg can be used. The dosage is preferably 100 mg / kg per week. It is administered once or multiple times over a 4-week period, more preferably over a 8-week period, more preferably over a 16-week period, or can use a longer minimum dose schedule, and the frequency of administration is The schedule of administration depends on the adverse side effects and recovery from them, most commonly associated with hematologic toxicity. The regimen may be, for example, (i) once a week; (ii) every other week; or (iii) a weekly regimen. (iv) 2 weeks of therapy followed by 1 week, 2 weeks, or 4 weeks of rest; (v) 3 weeks of therapy followed by 1 week, 2 weeks, or 3 weeks of rest; , 4 weeks, or 5 weeks off; (vi) 4 weeks of therapy followed by 1 week, 2 weeks, or 3 weeks (vii) 5 weeks of therapy followed by 1, 2, or 3 weeks (viii) once a month; The cycle may include administration once or twice weekly in a cycle of 2, 4, 6, or 7 days. , which can be repeated 8, 10, or 12 times or more.

[0158] Alternatively, TDC should be administered in one dose every two or three weeks for a total of at least three doses. It can be administered repeatedly, or twice a week for 4 to 6 weeks. Doses can be administered once every other week, or even less frequently, allowing patients can reverse any drug-related toxicity. Alternatively, the dosage schedule can be shortened, i.e., every 2 or 3 weeks for 2 to 3 months The administration schedule may optionally be repeated at other intervals, and the dosage may be adjusted according to the dose and Can be administered via various parenteral routes with appropriate scheduling .

[0159] 5.7. Treatment Method Fibrosis The TDCs of the present disclosure are useful in treating various fibrotic conditions, such as fibrosis associated with systemic sclerosis (SC). It can be used to treat nonalcoholic steatohepatitis (also known as steatohepatitis) or nonalcoholic steatohepatitis (NASH). Patients often suffer from pulmonary fibrosis, skin fibrosis, and esophageal fibrosis, but fibrosis is Qualitatively, it can occur in any organ. Patients with NASH often suffer from liver fibrosis. TDCs may be used, for example, as monotherapy or in combination with standard-of-care agents or regimens. In some embodiments, the compound may be used as part of a combination therapy. The methods include pirfenidone, nintedanib, pentraxin-2, pamrevlumab, and prednisolone. in combination with steroids, cortisone, cyclophosphamide, azathioprine, or a combination thereof. In some embodiments, the combination therapy includes administering a pharmacokinetic (TDC) to a patient in need thereof. including administering TDC in combination with rufenidone and / or nintedanib .

[0160] Examples of conditions that can be treated using the TDCs of the present disclosure include, but are not limited to, pulmonary Fibrosis, e.g., IPF, liver fibrosis, e.g., liver fibrosis associated with NASH, kidney fibrosis, cardiac The TDCs of the present disclosure are useful in treating various diseases, including fibrosis, skin fibrosis, esophageal fibrosis, and systemic sclerosis. , before the onset of signs and / or symptoms of fibrosis, a disease associated with fibrosis, e.g., systemic The present invention can be administered to a subject having, for example diagnosed with, non-steroidal anti-inflammatory drug (NSAID) or non-steroidal anti-inflammatory drug (NASH). Alternatively, or in addition, TDCs may be used to treat fibrotic disease where signs and / or symptoms of fibrosis are observed. and then administering the compound to a subject having, e.g., diagnosed with, a disease associated with fibrosis. can.

[0161] The use of the TDCs of the present disclosure in combination with one or more therapies may involve changing the order in which the therapies are administered. For example, but not by way of limitation, the TDCs of the present disclosure can be administered to a subject before the subject is treated with one or more therapies: It can be administered during or after treatment. The TDCs of the present disclosure may be administered prior to (e.g., after) treatment of a patient with another therapy (e.g., a second therapeutic agent as described above). For example, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours Hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks 1, 8 weeks, or 12 weeks before treatment), concurrently with treatment, or subsequent to treatment (e.g., 5 minutes , 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 4 8 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks In some embodiments, the TDC of the present disclosure is administered within 12 weeks of the initial administration of the TDC. is incorporated into the same regimen as a second therapeutic agent.

[0162] Cancer The TDCs of the present disclosure (e.g., TDCs targeted to FAP) can be used to treat various cancers. TDCs can be used, for example, as standard-of-care agents or regimens, as monotherapy. In some embodiments, the compound may be used alone or as part of a combination therapy regimen. In this context, combination therapy may involve immunotherapy, such as checkpoint inhibitor therapy, chimeric antigen receptor (CAR) therapy, or CAR) therapy, adoptive T cell therapy (e.g., autologous T cell therapy), oncolytic virus therapy, Dendritic cell vaccine therapy, stimulator of interferon genes (STING) agonist therapy therapy, toll-like receptor (TLR) agonist therapy, intratumoral CpG therapy, cytokine therapy (e.g., IL2, IL12, IFN-α, or INF-γ therapy), or In some embodiments, the combination comprises administering TDC in combination with the combination. Therapies include ADCs with cytotoxic payloads, such as OMTX705 (Oncomat This includes administering a TDC in combination with a FAP-targeting ADC, such as ryx. In some embodiments, the combination therapy includes immunoconservative chemotherapy (e.g., 5-fluorouracil). antimetabolites such as cyclophosphamide, gemcitabine, or methotrexate; antihistamines such as benzodiazepine, dacarbazine, mechlorethamine, diaziquone, or temozolomide alkylating agents, anthracyclines such as doxorubicin or epirubicin, vincristine, antimicrotubule agents such as cisplatin, platinum compounds such as cisplatin or oxaliplatin a taxane such as paclitaxel or docetaxel, or etoposide or are topoisomerase inhibitors such as mitoxantrone or vincristine This involves administering TDC in combination with a vinca alkaloid.

[0163] Examples of cancers that can be treated using the TDCs of the present disclosure include, but are not limited to, urinary tract cancers. urothelial cancer (e.g., bladder cancer, urethral cancer, and ureteral cancer), lung cancer (e.g., adenocarcinoma, squamous cell carcinoma) epithelial cell carcinoma, non-small cell lung cancer (NSCLC) such as large cell carcinoma, and small cell lung cancer), Breast cancer, colorectal cancer (e.g., adenocarcinoma, carcinoid tumor, gastrointestinal stromal tumor, and colon These include rectal lymphoma, pancreatic cancer, prostate cancer, and esophageal cancer. Other examples of cancers that can be treated with methicillin include head and neck cancer, ovarian cancer, renal cancer, and gastric cancer. Adenocarcinoma is one example.

[0164] The TDCs of the present disclosure can be used in combination with checkpoint inhibitors, such as PD1, PDL1, and CTLA4. Checks targeting TIGIT, LAG3, OX40, CD40, or VISTA Checkpoint inhibitors can be used in combination with antibodies. Exemplary checkpoint inhibitors that target PD1 include pemafibrate, pembrolizumab, pembrolizumab globulin, and small molecules. These include brolizumab, nivolumab, cemiplimab, and dostallimab. Exemplary checkpoint inhibitors targeting 1 include atezolizumab, avelumab, These include durvalumab, BMS-1001, and BMS-1166. An exemplary checkpoint inhibitor that targets TIGIT is ipilimumab. Exemplary checkpoint inhibitors that target gliomas include etigilimab, thiramimab, and thiramimab. Exemplary agents targeting LAG3 include tiragolumab, and AB154. Effective checkpoint inhibitors include LAG525, Sym022, and relatlimab. Exemplary checkpoint inhibitors targeting OX40 include OX40-specific steroids (e.g., OX40-specific steroids), ... and OX40-specific steroids (e.g., OX40-specific steroids). As inhibitors of vasodilator activity, MEDI6469, PF-04518600, and BMS 986 178. Exemplary checkpoint inhibitors that target CD40 include Sec1 These include licrelumab, CP-870,893, and APX005M. An exemplary checkpoint inhibitor that targets VISTA is HMBD-002. For the treatment of urothelial cancer, the TDCs of the present disclosure are used in combination with cisplatin, mitomycin, C, carboplatin, docetaxel, paclitaxel, doxorubicin, 5-FU, methotrexate including, but not limited to, trexate, vinblastine, ifosfamide, and pemetrexed It can be used in combination with standard of care treatments that do not involve ipsilateral It can be used in combination with checkpoint inhibitors such as limumab.

[0165] For the treatment of non-small cell lung cancer (NSCLC), the TDCs of the present disclosure may be administered in combination with cisplatin, which may be administered intravenously. tin, carboplatin, paclitaxel, gemcitabine, vinorelbine, irinotecan, Used in combination with standard-of-care chemotherapy treatments such as etoposide or vinblastine In addition, TDC can be used in combination with targeted drugs such as bevacizumab or Erbitux. In addition, TDC can be used in combination with pembrolizumab, Volumab, cemiplimab, dostallimab, atezolizumab, avelumab, durvalumab It can be used in combination with other anti-cancer drugs, such as ipilimumab, or checkpoint inhibitors. Cut.

[0166] For the treatment of breast cancer, the TDCs of the present disclosure may be used in combination with anthracyclines (doxorubicin or epirubicin), and taxanes (paclitaxel or docetaxel), as well as Standard-of-care chemotherapy such as fluorouracil, cyclophosphamide, and carboplatin In addition, the TDCs of the present disclosure can be used in combination with targeted therapies. It can be used in combination with other drugs as a targeted therapy for HER2 / neu-positive tumors. These include rastuzumab and pertuzumab, which are used to treat estrogen receptor (ER)-positive tumors. Targeted therapies include tamoxifen, toremifene, and fulvestrant. In addition, TDC can be combined with checkpoint inhibitors such as atezolizumab. It can be used as such.

[0167] For the treatment of colorectal cancer, the TDCs of the present disclosure may be used in combination with 5-FU, capecitabine, irinotecan, Targeted drugs include, but are not limited to, rifacil, oxaliplatin, trifluridine, and tipiracil. In addition, the TDCs of the present disclosure can be used in combination with targeted therapy. It can be used in combination with targeted therapy such as bevacizumab and ramucirumab. In addition, TDCs include pembrolizumab, in combination with checkpoint inhibitors such as nivolumab, ipilimumab, or ipilimumab It is possible.

[0168] For pancreatic cancer, the TDCs of the present disclosure include gemcitabine, 5-fluorouracil (FLU), and il), irinotecan, oxaliplatin, paclitaxel, capecitabine, cisplatin or in combination with standard-of-care chemotherapy agents such as docetaxel In addition, TDC can be combined with targeted therapies such as erlotinib, which inhibits EGFR. It can be used as such.

[0169] For prostate cancer, the TDC of the present disclosure is optionally administered with the steroid prednisone. In combination with standard-of-care chemotherapy agents, including docetaxel or cabazitaxel In addition, TDC can be combined with checkpoint inhibitors such as ipilimumab. They can be used in combination.

[0170] For espohageal cancer, the TDCs of the present disclosure are used in combination with carboplatin and paclitaxel. cisplatin and 5-FU, epirubicin, cisplatin, and 5-FU, Docetaxel, cisplatin, and 5-FU, cisplatin with capecitabine, oxaliplatin Saliplatin and either 5-FU or capecitabine, irinotecan or trimethoprim-4 Use in combination with standard-of-care chemotherapy agents such as fluridin and tipiracil In addition, TDC can be combined with targeted therapies such as trastuzumab or ramucirumab. In addition, TDC can be used in combination with chemotherapy, such as pembrolizumab. It can be used in combination with a cross-linking inhibitor.

[0171] The use of the TDCs of the present disclosure in combination with one or more therapies may involve changing the order in which the therapies are administered. For example, but not by way of limitation, the TDCs of the present disclosure can be administered to a subject before the subject is treated with one or more therapies: It can be administered during or after treatment. The TDCs of the present disclosure may be administered prior to (e.g., after) treatment of a patient with another therapy (e.g., a second therapeutic agent as described above). For example, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours Hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks 1, 8 weeks, or 12 weeks before treatment), concurrently with treatment, or subsequent to treatment (e.g., 5 minutes , 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 4 8 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks In some embodiments, the TDC of the present disclosure is administered within 12 weeks of the initial administration of the TDC. is incorporated into the same regimen as a second therapeutic agent. [Example]

[0172] The following abbreviations appear throughout the examples: Boc - tert-butyloxycarbonyl DCM - dichloromethane DMA - Dimethylamine DMF - dimethylformamide DIPEA - N,N-Diisopropylethylamine EtOAc - ethyl acetate EtOH - ethanol Fmoc - Fluorenylmethyloxycarbonyl HOBt - Hydroxybenzotriazole MeOH - methanol NaHMDS - sodium hexamethyldisilazide RT - Room temperature, approximately 21°C TBTU - O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyl Thiuronium tetrafluoroborate TEA - Triethylamine THF - tetrahydrofuran TFA - Trifluoroacetic acid TMS-Imidazole - 1-(Trimethylsilyl)imidazole

[0173] 6.1. [Example 1] 4-(6-methylpyridin-2-yl)-5-(1,5-naphthyridin-2-yl)-1 Synthesis of ,3-thiazol-2-amine (Compound A) Compound A was prepared according to the general method in Scheme 1 below.

[0174] [ka]

[0175] 6.1.1. 2-Methyl-1,5-naphthyridine (A1) Concentrated sulfuric acid (2.5 ml), sodium m-nitrobenzenesulfonate (2.08 g, 9. 24 mmol), boronic acid (445 mg, 7.21 mmol), and ferrous sulfate heptahydrate A mixture of glycerol (1. 5 ml), followed by 5-amino-2-methylpyridine (A-SM) (500 mg, 4.6 2 mmol) and water (2.5 ml) were added to the reaction mixture and heated at 135° C. for 18 hours. After the reaction was complete as determined by TLC, the reaction mixture was cooled to approximately 21°C and diluted with 4N N The solution was basified using NaOH and extracted with EtOAc (2 x 100 ml). The combined extracts were washed with water (200 ml), dried over Na2SO4, and evaporated under reduced pressure to give crude Compound A1 was obtained. The crude product was purified by silica gel column chromatography using (2% MeOH / CH2Cl2). Compound A1 (200 ml) was purified by column chromatography to give a light brown crystalline solid. g, 30%).

[0176] 1H NMR (500 MHz, CDCl3): δ 8.92 (d, J = 3.0 Hz, 1H), 8.35 (d, J = 9.0 Hz, 1H) , 8.31 (d, J = 5.9 Hz, 1H), 7.62 (dd, J = 8.5, 4.5 Hz, 1H), 7.54 (d, J = 5.9 Hz, 1H), 2.8 (s, 3H)

[0177] LC-MS(ESI):m / z 145[M+H] +

[0178] 6.1.2. 1-(6-methylpyridin-2-yl)-2-(1,5-naphthyridine- 2-yl)ethan-1-one (A2) A1 (200 mg, 1.38 mmol) and methyl 6-methylpicolinate (209 A solution of 1.38 mg (1.38 mmol) in anhydrous THF (10 ml) was placed under a N2 atmosphere. Cooled to -78°C. Potassium bis(trimethylsilyl)amide (0.5M in toluene) , 6.9 ml, 3.47 mmol) was added dropwise over a period of 5 minutes. The mixture was stirred at -78°C for 1 hour, then warmed to approximately 21°C and maintained for 20 hours. (determined by TLC), the reaction mixture was quenched with saturated ammonium chloride solution (20 ml). The aqueous layer was extracted with EtOAc (2 x 20 ml). The combined organic extracts were washed with water ( 100 ml), dried over Na2SO4 and evaporated to give crude compound A2. The material was purified by column chromatography (1% MeOH / CH2Cl2) to give Compound A2 (110 mg, 30.5%) was obtained as an orange-yellow solid.

[0179] 1H NMR (400 MHz, CDCl: enol form): δ 15.74 (brs, -OH), 8.69 (t, J = 3.6, 1H), 8.12 (d, J = 9.2 Hz, 1H), 8.06 (dd, J = 8.4, 4.4 Hz, 2H), 7.82 (t, J = 7.6 Hz, 1H), 7.55 (dd, J = 8.4, 4.8 Hz, 1H) 7.45 (d, J= 9.6 Hz,1H), 7.3 (dd, J = 7.6, 4.0 Hz, 1H), 7.16 (s, 1H), 2.75(s, 3H)

[0180] LC-MS(ESI):m / z 264[M+H] +

[0181] 6.1.3. 4-(6-methylpyridin-2-yl)-5-(1,5-naphthyridine- 2-yl)-1,3-thiazol-2-amine (Compound A) A solution of A2 (110 mg, 0.418 mmol) in 1,4-dioxane (10 ml) was treated with bromine (0.025 ml, 0.501 mmol). Stirring at approximately 21° C. for 1 h followed by concentration under reduced pressure gave crude A3 (120 mg). This was used in the next step without further purification. Crude A3 (120 mg) was The solution was dissolved in 15 ml of ethanol. Then, thiourea (3.5 mg, 0.046 mmol) was added and the reaction mixture was stirred for 78 hours (until complete consumption of the starting material was observed by TLC). The reaction mixture was cooled to approximately 21° C. and ammonia solution (25%, 1.5 ml) was added with gentle stirring. The solvent was evaporated and the residue was dissolved in CH It was dissolved in Cl2 (2 x 20 ml) and washed with water (50.0 ml). The organic layer was washed with 1N HCl (30 ml x 2). The combined aqueous layers were washed with 35% hydroxide It was basified with aqueous sodium (20 ml) and extracted with CH2Cl2 (2 x 20 ml). The organic layer was dried over sodium sulfate and evaporated to give crude compound A. Crude compound A was purified by acetone distillation. Compound A (35%) was purified by recrystallization from 2 ml of toluene as a yellow crystalline solid. mg, 49% yield over two steps).

[0182] 1 H NMR (400 MHz, CDCl3): δ 8.86 (dd, J = 4.4, 1.6 Hz, 1H), 8.29 (t, J = 8.4 H z, 1H), 8.06 (d, J = 9.2 Hz,1H), 7.64 (t, J = 7.6 Hz, 1H), 7.60-7.55 (m, 2H), 7 .46 (d, J = 8 Hz, 1H), 7.20 (d, J = 7.6, 1H), 5.32 (brs, 2H), 2.57 (s, 3H)

[0183] LC-MS(ESI):m / z 320[M+H] +

[0184] UPLC purity: 97.6%

[0185] 6.2. [Example 2] N-methyl-2-(4-{4-[3-(pyridin-2-yl)-1H-pyrazole-4- Synthesis of (phenyl)pyridin-2-yl}phenoxy)ethan-1-amine (Compound B) Compound B was prepared according to the general method in Scheme 2 below.

[0186] [ka]

[0187] 6.2.1. tert-Butyl (2-chloroethyl) (methyl)carbamate (B7) To a stirred solution of Boc-anhydride (1.7 ml, 7.30 mmol) in THF (4 ml) At the same time, a solution of B6 (1 g, 7.69 mmol) in water (4 ml) and TEA (1 ml, 7.69 mmol) in THF (4 ml) was added over 1 hour. The resulting mixture was stirred for 16 hours at approximately 21° C. The reaction mixture was diluted with saturated NaCl solution (20 ml) and extracted with DCM (3 x 50 ml). The combined organic extracts were diluted with Na 2SO4 and concentrated in vacuo to give the crude compound, which was extracted with 10% EtOAc / Hexane. The product was purified by silica gel column chromatography using hexane to give a pale yellow liquid. Compound B7 (1 g, 5.18 mmol, 71%) was obtained.

[0188] 1 H NMR (400 MHz, CDCl3): δ 3.58-3.52 (m, 4H), 2.93 (s, 3H), 1.46 (s, 9H)

[0189] 6.2.2. tert-Butylmethyl (2-(4-(4,4,5,5-tetramethyl- 1,3,2-Dioxaborolan-2-yl)phenoxy)ethyl)carbamate (Int -B) 4-Hydroxyphenylboronic acid pinacol ester (789 mg, 3.58 mmol) To a stirred solution of B7 (900 mg, 4.66 mmol), KI (18 mg, 0.10 mmol), and Cs2CO3 (2.57 g, 7.88 mmol ) was added under an argon atmosphere. The reaction mixture was heated to 65° C. and stirred for 16 hours. The reaction mixture was poured into water (20 ml) and extracted with EtOAc (3 x 20 ml). The combined organic layers were concentrated under reduced pressure to give the crude product, which was extracted with 7% EtOAc / hexanes. Int-B (58) was purified by column chromatography using HCl to give Int-B (58) as a pale yellow solid. 0 mg, 1.53 mmol, 43%) was obtained.

[0190] 1 H NMR (400 MHz, CDCl3):δ 7.74 (d, J = 8.4 Hz, 2H), 6.87 (d, J = 8.8 Hz, 2 H) , 4.16-4.06 (m, 2H), 3.65-3.59 (m, 2H), 2.97 (s, 3H), 1.45 (s, 9H), 1.33 (s, 12H) )

[0191] 6.2.3. 2-(2-Bromopyridin-4-yl)-1-(pyridin-2-yl) ester Tan-1-one (B2) 2-Bromo-4-methylpyridine (B1) (2 g, 11.62 mmol) in THF (3 To a stirred solution of 100 ml of NaHMDS (2M in THF, 1 A solution of 2.7 ml of 25.58 mmol of HCl was added dropwise. The yellow solution was heated at -78°C for 30 minutes. The mixture was stirred for 1 minute. Then, ethyl picolinate (1.72 ml, 12.79 mmol) was added to the TH A solution of F (10 ml) was added and the reaction mixture was warmed to approximately 21° C. and stirred for 16 h. The solvent was evaporated under reduced pressure and the solid residue was triturated with diethyl ether, filtered and The solid was then diluted with saturated NH4Cl solution (30 ml) and The organic layer was extracted with EtOAc (2 x 200 ml). The organic layer was dried over Na2SO4 and concentrated. The crude product was purified by silica gel column chromatography using 10% EtOAc / hexane. The compound was purified by HPLC to give compound B2 (2.06 g, 7.46 mmol) as a yellow solid. l, 64.3%).

[0192] 1 H NMR (400 MHz, CDCl3):δ 8.75 (d, J = 5.2 Hz, 1H), 8.32 (d, J= 5.2 Hz, 1H), 8.08 (d, J = 8.0 Hz, 1H), 7.89 (t, J =7.6 Hz 1H), 7.56-7.51(m, 2H), 7.28-7.25 (m , 1H), 4.55 (s, 2H)

[0193] LC-MS(ESI):m / z 277[M] +

[0194] 6.2.4. 2-Bromo-4-[3-(pyridin-2-yl)-1H-pyrazole-4 -yl]pyridine (B3) A solution of B2 (850 mg, 3.07 mmol) in dry DMF (3.4 ml) was added to The mixture was treated with glacial acetic acid (0.45 ml, 7.39 mmol) in DMF under atmospheric pressure. 0.6 ml, 4.61 mmol) was added dropwise and the mixture was heated at approximately 21°C for 2 hours under argon. The mixture was stirred under an atmosphere of 1000 kJ / cm². Hydrazine monohydrate (1.15 ml, 23.09 mmol) was added dropwise. The resulting mixture was heated at 50°C for 3 hours and then at approximately 21°C for 16 hours. The reaction mixture was poured into water (30 ml) and extracted with CH2Cl2 (3 x 30 ml). The organic layer was dried over Na2SO4 and filtered. The solvent was evaporated under reduced pressure to give the crude compound. The crude product was purified by silica gel column chromatography using 30% EtOAc / hexane. The compound was purified by chromatography to give compound B3 (560 mg, 1.86 mmol) as a yellow solid. , 60.6%).

[0195] 1 H NMR (500 MHz, CDCl3):δ 8.74 (brs, 1H), 8.34 (d, J = 5.0 Hz, 1H), 7.83 (brs , 1H), 7.81 (t, J = 6.0 Hz, 1H), 7.56 (s, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.39-7. 84 (m, 1H), 7.31-7.26 (m, 1H)

[0196] LC-MS(ESI):m / z 301[M] +

[0197] 6.2.5. 2-Bromo-4-(3-(pyridin-2-yl)-1-trityl-1H- Pyrazol-4-yl)pyridine (B4) To a stirred solution of B3 (500 mg, 1.66 mmol) in acetone (10 ml), CO3 (1.37 g, 9.99 mmol) and trityl chloride (464 mg, 2.4 9 mmol) was added. The reaction mixture was then heated to reflux and stirred for 24 hours. The mixture was filtered, and the filtrate was concentrated and then diluted with CH2Cl2 (20 mL) and water (10 mL). The organic phase was dried over Na2SO4 and concentrated. The crude solid was partitioned between 2% MeO Purification by silica gel column chromatography using HCl / CH2Cl2 gave a pale yellow Compound B4 (402 mg, 0.74 mmol, 44%) was obtained as a white solid.

[0198] 1 H NMR (500 MHz, CDCl): δ 8.53 (d, J = 4.5 Hz, 1H), 8.20 (d, J = 5.5 Hz, 1H) , 7.75-7.05 (m, 2H), 7.56 (s, 1H), 7.51 (s, 1H), 7.35-7.32 (m, 9H), 7.25-7.22 (m , 8H)

[0199] 6.2.6. tert-Butylmethyl (2-(4-(4-(3-(pyridin-2-yl)methyl) )-1-trityl-1H-pyrazol-4-yl)pyridin-2-yl)phenoxy)e Chill) Carbamate (B5) To a stirred solution of B4 (100 mg, 0.18 mmol) in toluene (2 ml), EtO Int-B (185 mg, 0.49 mmol) in H (0.75 ml) followed by 2 M A solution of Na2CO3 (0.45 ml) was added under an argon atmosphere. The mixture was degassed with argon for 20 minutes, and then Pd(PPh3)4 (16 mg, 0.01 mmol) was added. After complete consumption of the starting material (monitored by TLC), the reaction The mixture was poured into water and extracted with toluene (3 x 15 ml). and concentrated under reduced pressure to give the crude product, which was purified by 30% EtOAc / hexanes Compound (II) was purified by silica gel column chromatography using B5 (70 mg, 0.09 mmol, 53%) was obtained.

[0200] 1 H NMR (400 MHz, CDCl3): δ 8.53 (s, 1H), 8.49 (d, J= 4.8 Hz, 1H),7.82 (d, J = 8.8 Hz, 2H) 7.74-7.76 (m, 3H), 7.60 (s, 1H), 7.40-7.34 (s, 8H), 7.31-7.30 (m, 2 H), 7.24-7.19 (m, 4H), 7.12- 7.10 (m, 1H), 6.93(d, J = 8.8 Hz, 2H), 4.19-4.12 (m , 2H), 3.66-3.58 (m, 2H), 2.98 (s, 3H), 1.46 (s, 9H).

[0201] 6.2.7. N-methyl-2-(4-(3-(pyridin-2-yl)-1H-pyridin-2-yl)-1H-pyridin-2-yl)-1H-pyridin-2-yl (4-pyridin-2-yl)phenoxy)ethan-1-amine hydrochloride (chemical Compound B) To a stirred solution of B5 (70 mg, 0.09 mmol) in CH2Cl2 (6 ml) was added 1, 4N HCl in 4-dioxane (0.5 ml) was added at 0°C. The reaction mixture was The mixture was stirred under an atmosphere of ammonium hydroxide for 1 hour. After complete consumption of the starting material (monitored by TLC), The solvent was evaporated under reduced pressure to give the crude compound, which was triturated with n-pentane (2 x 1 ml). Crushed and dried to give the HCl salt of Compound B as a colorless solid (25 mg, 0.06 mm ol, 69%).

[0202] 1 H NMR (400 MHz, DMSO-d6):δ 8.94 (brs, 2H), 8.62-8.56 (m, 3H), 8.30 (brs, 1H) , 8.03-7.96 (m, 3H), 7.86 (d, J = 7.6 Hz, 1H),7.69 (brs, 1H), 7.49 (dd, J =7.2, 5.6 Hz, 1H), 7.29 (d, J=7.6 Hz, 1H), 7.20 (d, J = 8.4 Hz, 1H), 4.36 (t, J = 4.8 Hz, 2H), 3.39-3.35 (m, 2H), 2.67-2.63 (m, 3H)

[0203] LC-MS(ESI):m / z 372[M+H] +

[0204] 6.3. [Example 3] N-methyl-2-(4-{4-[3-(6-methylpyridin-2-yl)-1H-pyrazoline (4-yl)pyridin-2-yl}phenoxy)ethan-1-amine (Compound C) synthesis Compound C was prepared according to the general method in Scheme 3 below.

[0205] [ka]

[0206] 6.3.1. 2-(2-Bromopyridin-4-yl)-1-(6-methylpyridine-2 -yl)ethan-1-one (C2) 2-Bromo-4-methylpyridine (B1) (1 g, 5.81 mmol) in THF (15 To a stirred solution of NaHMDS (2M in THF, 6. A solution of 39 ml of 12.8 mmol of HCl was added dropwise. The yellow solution was heated at -78°C for 30 minutes. Then, 6-methylpicolinic acid methyl ester (1.19 ml, 8.72 mm A solution of 100 ml of HCl in THF (7 ml) was added and the reaction mixture was allowed to warm to a maximum of approximately 21°C. The mixture was stirred for 16 hours, the solvent was evaporated under reduced pressure, and the solid residue was triturated with diethyl ether. The solid was then dissolved in saturated NH4Cl solution (20 ml) and washed with diethyl ether. The organic layer was diluted with 1 ml of NaSO and the aqueous phase was extracted with EtOAc (2 x 150 ml). The crude product was purified by silica gel chromatography using 10% EtOAc / hexanes. Purification by gel column chromatography gave compound C2 (1.1 g, 3.79 mmol, 65.4%) was obtained.

[0207] 1 H NMR (500 MHz, CDCl3): δ 8.30 (d, J = 5.0 Hz, 1H), 7.86 (d, J = 8 Hz, 1H), 7.73 (t, J = 7.5 Hz, 1H), 7.51 (s, 1H), 7.36 (d, J = 8 Hz, 1H), 7.24 (d, J = 5 Hz, 1H), 4.52 (s, 2H), 2.64 (s, 3H)

[0208] LC-MS(ESI):m / z 291[M] +

[0209] 6.3.2. 2-Bromo-4-[3-(6-methylpyridin-2-yl)-1H-pyrazol-2-yl] [4-[ ... A solution of C2 (300 mg, 1.03 mmol) in dry DMF (1 ml) was heated under argon The mixture was treated with glacial acetic acid (0.14 ml, 2.48 mmol) in DMF under reduced pressure. 2 ml, 1.55 mmol) was added dropwise and the mixture was stirred at approximately 21°C for 1 hour under argon. The mixture was stirred under atmospheric pressure. Hydrazine monohydrate (0.37 ml, 7.75 mmol) was added dropwise. The resulting mixture was heated at 50°C for 3 hours and then at approximately 21°C for 16 hours. The mixture was poured into water (20 ml) and extracted with CH2Cl2 (3 x 20 ml). The layer was dried over Na2SO4 and filtered. The solvent was evaporated under reduced pressure to give crude C3. C3 was purified by silica gel column chromatography using 2% MeOH / DCM Purification gave purified C3 (172 mg, 0.54 mmol, 53%) as a yellow solid. Ta.

[0210] 1 H NMR (500 MHz, CDCl3):δ 11.40 (brs, 1H), 8.37 (d, J = 5.0 Hz, 1H), 7.74 (s, 1H), 7.64 (s, 1H), 7.58 (t, J = 8.0 Hz, 1H), 7.34 (d, J= 6.0 Hz, 1H), 7.26 (d, J = 8.0 Hz, 1H), 7.17 (d, J = 8.0 Hz, 1H), 2.60 (s, 3H)

[0211] LC-MS(ESI):m / z 315[M+H] +

[0212] 6.3.3. 2-Bromo-4-(3-(6-methylpyridin-2-yl)-1-trimethylpyridinyl)- (1H-pyrazol-4-yl)pyridine (C4) To a stirred solution of C3 (40 mg, 0.12 mmol) in acetone (2 ml) was added KCO 3 (53 mg, 0.38 mmol) and trityl chloride (53 mg, 0.19 mmol) l) was added. The reaction mixture was then heated to reflux and stirred for 24 hours. Filter, concentrate the filtrate, then partition between CH2Cl2 (5 mL) and water (5 mL). The organic phase was dried over Na2SO4 and concentrated. The crude solid was extracted with 2% MeOH / CH2Cl Purification by silica gel column chromatography using l2 gave a pale yellow solid Compound C4 (30 mg, 0.05 mmol, 41%) was obtained.

[0213] 1H NMR (400 MHz, CDCl3):δ 8.22 (d, J = 4.8 Hz, 1H), 7.73 (s, 1H), 7.59 (s, 3H ), 7.39-7.35 (m, 9H), 7.31 (s, 1H), 7.28-7.25 (m, 6H), 7.24 (d, J = 12 Hz, 1H), 2.53 (s, 3H)

[0214] LC-MS(ESI):m / z 558[M+H] +

[0215] 6.3.4. tert-Butylmethyl (2-(4-(4-(3-(6-methylpyridine) -2-yl)-1-trityl-1H-pyrazol-4-yl)pyridin-2-yl)phenyl (C5)(oxy)ethyl)carbamate To a stirred solution of C4 (150 mg, 0.26 mmol) in toluene (5 ml), EtO Int-B (152 mg, 0.40 mmol) in 1 ml of H, followed by 2 M Na A solution of CO3 (0.7 ml) was added under an argon atmosphere. The reaction mixture was then purged with argon. Degass for 20 minutes, then add Pd(PPh3)4 (25 mg, 0.02 mmol) After complete consumption of the starting material (monitored by TLC), the reaction mixture was The mixture was poured into water and extracted with toluene (3 x 10 ml). The organic layer was dried over Na2SO4. The mixture was concentrated under reduced pressure to give crude C5, which was purified by 30% EtOAc / hexanes. Purification by silica gel column chromatography gave purified C5 (5 1 mg, 0.07 mmol, 26%) was obtained.

[0216] 1H NMR (400 MHz, CDCl3): δ 8.48 (d, J = 5.2 Hz, 1H), 7.82 (d, J = 8.8 Hz, 3H) , 7.74 (s, 1H), 7.60 (s, 1H), 7.56 (d, J = 15.2Hz, J = 7.6Hz, 2H), 7.35-7.33 (m , 8H), 7.28-7.27 (m, 6H), 7.08 (d, J = 6.8 Hz, 2H), 6.93 (d, J = 8.8 Hz, 2H), 4. 16-4.08 (m, 2H), 3.63-3.58 (m, 2H), 2.98 (s, 3H), 2.41 (s, 3H), 1.46 (s, 9H)

[0217] 6.3.5. N-methyl-2-(4-{4-[3-(6-methylpyridin-2-yl) -1H-pyrazol-4-yl]pyridin-2-yl}phenoxy)ethan-1-amine (Compound C) To a stirred solution of C5 (51 mg, 0.07 mmol) in CH2Cl2 (5 ml), 1, 4N HCl in 4-dioxane (0.3 ml) was added at 0° C. Then the reaction mixture The mixture was stirred under argon atmosphere for 1 hour. After complete consumption of the starting material (monitored by TLC), The solvent was evaporated under reduced pressure to give crude compound C. The crude compound C was then purified by Triturate with ethanol (2 × 1 ml) and dry to give compound C as the HCl salt as a brown solid. Obtained (20 mg, 0.05 mmol, 74%).

[0218] 1 H NMR (400 MHz, DMSO-d6):δ 8.93 (brs, 2H), 8.61 (d, J = 5.6 Hz, 1H),8.56 (br s, 1H), 8.33 (brs, 1H), 8.03 (d, J = 8.8 Hz, 2H), 7.88 (t, J = 7.6 Hz, 1H), 7.78 -7.74 (m,1H), 7.65 (d, J = 7.2 Hz, 1H), 7.38 (d, J = 7.6 Hz, 1H), 7.20 (d, J = 8 .4 Hz, 2H), 4.36 (t, J = 5.2 Hz, 2H), 3.36 (t, J = 5.2 Hz, 2H), 2.66-2.63 (m, 3H ), 2.50-2.46 (m, 3H)

[0219] LC-MS(ESI):m / z 386[M+H] +

[0220] 6.4. [Example 4] (Z)-N-ethyl-3-(((4-(N-(2-(methylamino)ethyl)methylsulfonyl) (phenyl)amino)(phenyl)methylene)-2-oxoindoline-6- Synthesis of carboxamide (compound D) Compound D was prepared according to the general method in Scheme 4 below.

[0221] [ka]

[0222] 6.4.1. Methyl 1-acetyl-2-oxoindoline-6-carboxylate (D 2) Methyl 2-oxoindoline-6-carboxylate (D1) (2.0 g, 10.47 A stirred solution of 100 mmol of acetic acid in acetic anhydride (16 ml) was heated to 130°C under an inert atmosphere for 6 hours. After complete consumption of the starting material (monitored by TLC), the reaction mixture was The mixture was cooled to about 21° C. The precipitate was filtered, washed with n-hexane (2×50 ml) and evaporated under vacuum. The mixture was dried at rt to give compound D2 (1.5 g, 61.5%) as a yellow solid.

[0223] 1 H NMR (400 MHz, DMSO-d6):δ 8.66 (s, 1H), 7.82 (d, J = 8.0 Hz, 1H), 7.48 (d, J = 8.0 Hz, 1H), 3.91 (s, 2H), 3.87 (s, 3H), 2.57 (s, 3H)

[0224] 6.4.2. Methyl(Z)-1-acetyl-3-(hydroxy(phenyl)methylene) -2-oxoindoline-6-carboxylate (D3) To a stirred solution of compound D2 (1.5 g, 6.43 mmol) in DMF (10 ml) was added T BTU (2.69g, 8.36mmol), benzoic acid (903mg, 7.40mmol) and triethylamine (2.2 ml) were added at 0° C. under an inert atmosphere. The mixture was warmed to approximately 21° C. and stirred for 16 h. After complete consumption of the starting material (TLC After 2 min (monitored by HCl), the reaction mixture was quenched with ice-cold water (30 ml) and × 40 ml). The combined organic extracts were dried over Na2SO4, filtered and vacuum Concentration at 25°C gave crude product D3, which was purified by silica gel chromatography using 80% EtOAc / hexanes. The compound was purified by gel column chromatography to give compound D3 (900 ml) as a yellow solid. g, 42%).

[0225] 1 H NMR (400 MHz, CDCl3): δ 14.01 (brs, 1H), 8.93 (s, 1H), 7.76-7.70 (m, 3H), 7.67-7.63 (m, 1H), 7.59-7.56 (m, 2H), 7.12 (d, J = 8.0 Hz, 1H), 3.90 (s, 3H), 2. 83 (s, 3H)

[0226] LC-MS(ESI):m / z 338.3[M+H] +

[0227] 6.4.3. (Z)-3-(hydroxy(phenyl)methylene)-2-oxoindoli Benzene-6-carboxylic acid (D4) To a stirred solution of compound D3 (900 mg, 2.67 mmol) in MeOH (15 ml) Then, 1N aqueous NaOH solution (15 ml) was added at approximately 21° C. The reaction mixture was stirred for 100 minutes. The mixture was heated to °C and stirred for 6 hours. After complete consumption of the starting material (monitored by TLC), The reaction mixture was cooled to approximately 21° C. and quenched with 1N aqueous HCl (13 ml), Stirred for 30 min. The precipitated solid was filtered and washed with 20% EtOAc / hexanes to give Compound D4 (580 mg, 77%) was obtained as an off-white solid, which was further purified This was used in the next step without further purification.

[0228] 1 H NMR (400 MHz, DMSO-d6):δ 12.76 (brs, 1H), 11.61 (brs, 1H), 7.77-7.50 (m, 8 H), 7.13 (brs, 1H)

[0229] 6.4.4. (Z)-N-ethyl-3-(hydroxy(phenyl)methylene)-2-ol Xoindoline-6-carboxamidelate (Fragment A) To a stirred solution of compound D4 (580 mg, 2.06 mmol) in DMF (10 ml) TBTU (729mg, 2.27mmol), HOBt (306mg, 2.27mmol) ), and N,N-diisopropylethylamine (1.9 ml, 10.32 mmol) , was added under an inert atmosphere at approximately 21° C. After 30 min, THF (2.1 ml, 4.12 2N ethylamine in 100 mmol) was added at 0°C and stirred for 1 hour. The mixture was warmed to approximately 21° C. and stirred for an additional 16 h. After complete consumption of the starting material (T (monitored by LC) and the volatiles were removed in vacuo. The residue was diluted with water (15 ml). The crude product was obtained by filtration and washing with 20% EtOAc / hexane (2 x 10 ml). This was purified by silica gel column chromatography using 10% MeOH / CH2Cl2. Purification by HCl gave Fragment A (410 mg, 64.5%) as an off-white solid. .

[0230] 1 H NMR (400 MHz, DMSO-d6):δ 13.62 (brs, 1H), 11.39 (brs, 1H), 8.35-8.33 (m, 1 H), 7.76-7.52 (m, 5H), 7.44-7.36 (m, 3H), 3.29-3.22 (m, 2H), 1.10 (t, J = 7.2 Hz , 3H)

[0231] LC-MS(ESI): m / z 307.1(MH + )

[0232] 6.4.5. N-(2-(dimethylamino)ethyl)-N-(4-nitrophenyl)methyl Tansulfonamide (D8) To a stirred solution of compound D7 (800 mg, 3.70 mmol) in acetone (15 ml) , potassium carbonate (1.32 g, 9.62 mmol), sodium iodide (110 mg, 0 Compound B6 (799 mg, 5.55 mmol) was added to the flask in an inert atmosphere. The mixture was added at 0°C under atmospheric pressure. The reaction mixture was heated to 50°C and stirred for 20 hours. After complete consumption (monitored by TLC), the volatiles were removed in vacuo. The residue was diluted with water. (20 ml) and extracted with EtOAc (2 x 40 ml). Drying over Na2SO4, filtering and concentrating in vacuo gave the crude product which was extracted with 5% Me Purification by silica gel column chromatography using OH / CH2Cl2 gave pale yellow chromatograms. Compound D8 (460 mg, 43%) was obtained as a yellow solid.

[0233] 1 H NMR (500 MHz, DMSO-d6): δ 8.27 (d, J = 9.5 Hz, 2H), 7.68 (d, J = 9.5 Hz, 2H ), 3.85 (t, J = 6.5 Hz, 2H), 3.13 (s, 3H), 2.31 (t, J = 6.5 Hz, 2H), 2.12 (s, 6H) )

[0234] LC-MS(ESI):m / z 288.3[M+H] +

[0235] 6.4.6. N-(4-aminophenyl)-N-(2-(dimethylamino)ethyl)methyl Tansulfonamide (Fragment B) To a stirred solution of compound D8 (460 mg, 1.60 mmol) in MeOH (10 ml) 10% Pd / C (40 mg) was added, and the mixture was heated at approximately 21°C under a hydrogen atmosphere (balloon pressure). After complete consumption of the starting material (monitored by TLC), the reaction mixture was filtered through a pad of Celite® and washed with MeOH (10 ml). The filtrate was concentrated in vacuo to give the crude product, which was extracted with 10% MeOH / CH2Cl2 Fragment B was purified by silica gel column chromatography using HCl as a pale yellow solid. (300 mg, 73%) was obtained.

[0236] 1 H NMR (400 MHz, DMSO-d6): δ 6.99 (d, J = 8.8 Hz, 2H), 6.54 (d, J = 8.8 Hz, 2H ), 5.25 (s, 2H), 3.55 (t, J = 7.2 Hz, 2H), 2.91 (s, 3H), 2.24 (t, J = 7.2 Hz, 2H ), 2.12 (s, 6H)

[0237] LC-MS(ESI):m / z 258.2[M+H] +

[0238] 6.4.7. (Z)-3-(((4-(N-(2-(dimethylamino)ethyl)methyl Sulfonamido)phenyl)amino)(phenyl)methylene)-N-ethyl-2-oxo Indoline-6-carboxamide (D5) Fragment A (200mg, 0.64mmol), Fragment B (500mg, 1.94mmol) and TMS-imidazole (455 mg, 3.24 mmol) in THF (5 ml) The solution was heated to 170° C. under microwave for 1 hour. Consumption of starting material (TLC and L After filtration (monitored by C-MS), the volatiles were removed in vacuo. The residue was diluted with water (10 ml). and extracted with EtOAc (3×25 ml) to give the crude product, which was purified by preparative HPLC. C to give compound D5 (150 mg, 42%) as a pale yellow solid.

[0239] 1 H NMR (400 MHz, DMSO-d6):δ 12.14 (s, 1H), 10.91 (s, 1H), 8.17 (t, J = 5.6 Hz , 1H), 7.64-7.57 (m, 3H), 7.53-7.51 (m, 2H), 7.34 (s, 1H), 7.17 (d, J = 8.8 Hz, 2H), 7.06 (d, J = 8.4 Hz, 1H), 6.84 (d, J = 8.8 Hz, 2H), 5.73 (d, J = 8.4 Hz, 1H) ), 3.58 (t, J = 6.8 Hz, 2H), 3.23-3.20 (m, 2H), 2.93 (s, 3H), 2.13 (t, J = 6.8 H z, 2H), 1.90 (s, 6H), 1.06 (t, J = 7.2 Hz, 3H)

[0240] LC-MS(ESI):m / z 548.6[M+H] +

[0241] 6.4.8. (Z)-N-ethyl-3-(((4-(N-(2-(methylamino)ethyl) (phenyl)methylsulfonamido)phenyl)amino)(phenyl)methylene)-2-oxoi Endrine-6-carboxamide (Compound D) To a stirred solution of compound D5 (70 mg, 0.12 mmol) in dry toluene (3 ml) , 2,2,2-trichloroethoxycarbonyl chloride (0.04 ml, 0.19 mmol) 1) was added at approximately 21°C under an inert atmosphere. The reaction mixture was heated to reflux (120°C). ) and maintained for 16 hours. After complete consumption of the starting material (monitored by TLC), The reaction mixture was cooled to approximately 21° C., diluted with EtOAc (30 mL) and washed with 1N HCl The organic layer was dried over Na2SO4, filtered and concentrated in vacuo. Condensation gave the monodemethylated di-troc protected compound (40 mg).

[0242] The crude product from the above reaction was dissolved in acetic acid (3 ml) and zinc powder (9 mg, 0.1 3 mmol) was added at approximately 21°C under an inert atmosphere. The reaction mixture was heated to 50°C. After complete consumption of the starting material (monitored by TLC), the reaction mixture was The mixture was cooled to approximately 21°C and the volatiles were removed in vacuo. The residue was diluted with water (20 ml). The combined organic extracts were diluted with saturated NaHC Washed with O3 solution (20 ml), dried over Na2SO4, filtered and concentrated under reduced pressure to give Crude compound D was obtained, which was then separated by silica gel column chromatography using 5-6% MeOH / CH2Cl2. The resulting solution was purified by column chromatography to give 12 mg of compound D with an HPLC purity of 83%. Ta.

[0243] The reaction was repeated on a 60 mg scale and the resulting crude product was combined with the previous batch. and purified by preparative HPLC to give compound D (8.0 mg, 6.3%) as a pale yellow solid. Got it.

[0244] 1 H NMR (400 MHz, CD3OD):δ 7.65-7.59 (m, 3H), 7.52.7.50 (m, 2H), 7.40 (s, 1H), 7.31 (d, J = 8.8 Hz, 2H), 7.07 (d, J = 8.4 Hz, 1H), 6.90 (d, J = 8.8 Hz, 2H), 5 .95 (d, J = 8.4 Hz, 1H), 3.95 (t, J = 5.6 Hz, 2H), 3.39-3.32 (m, 2H), 3.05 (t, J = 5.6 Hz, 2H), 2.93 (s, 3H), 2.71 (s, 3H), 1.19 (t, J = 7.2 Hz, 3H)

[0245] LC-MS(ESI):m / z 534.6[M+H] +

[0246] UPLC purity: 99.18%

[0247] 6.5. [Example 5] (Z)-N-ethyl-3-(((4-(N-(2-(methylamino)ethyl)methylsulfonyl) (phenyl)amino)(phenyl)methylene)-2-oxoindoline-6- Alternative synthesis of carboxamide (compound D) Compound D was also prepared according to the general method in Scheme 5 below.

[0248] [ka]

[0249] 6.5.1. N-(2-bromoethyl)-N-(4-nitrophenyl)methanesulfone Amide (D9) To a stirred solution of compound D7 (1.0 g, 4.65 mmol) in DMF (10 ml) was added water Sodium oxide (60% in mineral oil, 320 mg, 7.99 mmol) was added under an inert atmosphere. The mixture was added at 0° C. and stirred for 30 minutes at approximately 21° C. To this mixture, 1,2-dibromoethylene At approximately 21° C., ethanol (2.18 g, 11.60 mmol) was added. The mixture was stirred for 90 The mixture was heated to °C and stirred for 24 hours. The reaction was monitored by TLC. Cool to approximately 21°C, quench with ice-cold water (30 ml), and add EtOAc (2 x 40 ml). The combined organic extracts were dried over Na2SO4, filtered and concentrated in vacuo to give The crude product was purified by silica gel column chromatography using 5% MeOH / CH2Cl2. Chromatographic purification gave 1.2 as a mixture containing 40% unreacted starting material. g of D9 was obtained. The resulting mixture was used directly in the next reaction without further purification.

[0250] 1 H NMR (500 MHz, CDCl3):δ 8.29 (d, J = 8.5 Hz, 2H), 7.56 (d, J = 8.5 Hz, 2H), 4.12 (t, J = 7.0 Hz, 2H), 3.44 (t, J = 7.0 Hz, 2H), 3.01 (s, 3H)

[0251] 6.5.2. N-(2-(methylamino)ethyl)-N-(4-nitrophenyl)meth Sulfonamide (D10) To a stirred solution of compound D9 (1.2 g, impure) in THF (10 ml) was added triethylamine. Methylamine (2M in THF, 9.3 ml, 18.63 mmol) l) was added in a sealed tube under an inert atmosphere at approximately 21°C. The reaction mixture was heated to 80°C. After complete consumption of the starting material (monitored by TLC), the reaction mixture was heated and maintained for 16 hours. The reaction mixture was cooled to approximately 21° C. and concentrated under reduced pressure to give crude D10. by silica gel column chromatography using 15% MeOH / CH2Cl2 Purification gave compound D10 (500 mg, 39% overall over two steps) as a yellow solid. Yield) was obtained.

[0252] 1 H NMR (500 MHz, DMSO-d6): δ 8.94 (brs, 1H), 8.31 (d, J = 9.0 Hz, 2H), 7.80 (d , J = 8.5 Hz, 2H), 4.06 (t, J = 6.0 Hz, 2H), 3.15 (s, 3H), 3.00 (t, J = 6.0 Hz, 2H), 2.55 (s, 3H)

[0253] 6.5.3. tert-Butylmethyl (2-(N-(4-nitrophenyl)methylsulfonyl)methyl) (Homo)amino)ethyl)carbamate (D11) To a stirred solution of D10 (500 mg, 1.83 mmol) in CH2Cl2 (10 ml) , triethylamine (0.4 ml, 2.61 mmol) and Boc anhydride (659 mg, 3.02 mmol) was added at approximately 21°C under an inert atmosphere and maintained for 5 hours. After complete consumption of the material (monitored by TLC), the volatiles were removed in vacuo to give the crude product. The product was purified by silica gel column chromatography using 5% MeOH / CH2Cl2. D11 (320 mg, 47%) was obtained as a colorless thick syrup by roughing. Got it.

[0254] 1 H NMR (400 MHz, DMSO-d6): δ 8.27 (d, J = 8.4 Hz, 2H), 7.68 (d, J = 8.4 Hz, 2H ), 3.91 (t, J = 6.4 Hz, 2H), 3.28-3.25 (m, 2H), 3.07 (s, 3H), 2.72-2.70 (m, 3H), 1.33-1.27 (m, 9H)

[0255] LC-MS(ESI): m / z 274.2(M + -B℃)

[0256] 6.5.4. tert-Butyl (2-(N-(4-aminophenyl)methylsulfonyl) (mido)ethyl)(methyl)carbamate (Boc variant of fragment B) To a solution of compound D11 (250 mg, 0.67 mmol) in EtOH (10 ml), Raney-Ni (40 mg) was added and the mixture was stirred at approximately 21°C under a hydrogen atmosphere (balloon pressure) for 1 hour. After complete consumption of the starting material (monitored by TLC), the reaction mixture was The mixture was filtered through a pad of elite® and washed with EtOH (10 ml). The combined filtrate was concentrated in vacuo to give the crude product, which was extracted with 10% MeOH / CH2Cl2 The fraction was purified by silica gel column chromatography using The Boc variant of B (180 mg, 77%) was obtained.

[0257] H NMR (400 MHz, DMSO-d6): δ 7.01 (d, J = 8.4 Hz, 2H), 6.53 (d, J = 8.4 HZ, 2H) , 5.24 (s, 2H), 3.60 (t, J = 6.4 Hz, 2H), 3.18 (t, J = 6.4 HZ, 2H), 2.88 (s, 3H) , 2.75-2.71 (m, 3H), 1.36-1.33 (m, 9H)

[0258] LC-MS(ESI): m / z 244.2(M + -B℃)

[0259] 6.5.5. tert-Butyl (Z)-(2-(N-(4-(((6-(ethylcarbamoyl) (2-oxoindolin-3-ylidene)(phenyl)methyl)amino)phenyl (methylsulfonamido)ethyl)(methyl)carbamate (D10) Fragment A (70 mg, 0.22 mmol), Boc variant of fragment B (155 mg, 0.4 5 mmol) and TMS-imidazole (159 mg, 1.13 mmol) in THF( The solution in 3 ml) was heated in a microwave at 170° C. for 160 min. Consumption of starting material After (monitored by TLC and LC-MS), the volatiles were removed in vacuo to give the residue This was purified by preparative HPLC to give compound D10 (50 mg) as a pale yellow solid. , 36%).

[0260] 1 H NMR (400 MHz, CDCl3):δ 12.13 (brs, 1H), 8.01 (brs, 1H), 7.61-7.51 (m, 3H), 7.44-7.41 (m, 3H), 7.13-7.11 (m, 2H), 6.98 (d, J = 8.4 HZ, 1H), 6.75 (d, J = 8. 4 HZ, 2H), 5.96-5.91 (m, 2H), 3.74-3.71 (m, 2H), 3.49-3.41 (m, 2H), 3.30-3.27 (m , 2H), 2.80 (s, 6H), 1.40-1.36 (m, 9H), 1.19 (t, J = 7.2 HZ, 3H)

[0261] LC-MS(ESI):m / z 634.6[M+H] +

[0262] 6.5.6. (Z)-N-ethyl-3-(((4-(N-(2-(methylamino)ethyl) (phenyl)methylsulfonamido)phenyl)amino)(phenyl)methylene)-2-oxoi Endrine-6-carboxamide hydrochloride (Compound D as the HCl salt) Compound D10 (20 mg, 0.03 mmol) stirred in diethyl ether (3 ml) The solution was added with 4N HCl in 1,4-dioxane (0.3 ml) at 0° C. under an inert atmosphere. The reaction mixture was stirred at approximately 21° C. for 1 hour. Complete consumption of the starting material (T After HPLC monitoring, the volatiles were removed in vacuo to give the crude product, which was Trituration with pentane (2 × 4 ml) gave compound D (1) as the HCl salt as a pale yellow solid. 2 mg, 71%) was obtained.

[0263] 1 H NMR (400 MHz, CD3OD):δ 7.65-7.59 (m, 3H), 7.52.7.50 (m, 2H), 7.40 (s, 1H), 7.31 (d, J = 8.8 Hz, 2H), 7.07 (d, J = 8.4 Hz, 1H), 6.90 (d, J = 8.8 Hz, 2H), 5 .95 (d, J = 8.4 Hz, 1H), 3.95 (t, J = 5.6 Hz, 2H), 3.39-3.32 (m, 2H), 3.05 (t, J = 5.6 Hz, 2H), 2.93 (s, 3H), 2.71 (s, 3H), 1.19 (t, J = 7.2 Hz, 3H).

[0264] LC-MS(ESI):m / z 534.7[M+H] +

[0265] UPLC purity: 96.26%

[0266] 6.6. [Example 6] In vitro assays to test the activity of compounds AD Compounds A to D were tested to determine their potency in inhibiting TGF-β in HEK293T cells in vitro. We then determined whether β-induced luciferase activity could be inhibited by β-lactamase.

[0267] 30,000 HEK293T cells were seeded overnight in a 96-well white flat-bottom plate. The next day, 100 ng of SMAD luciferase reporter plasmid was added per well. The following day, the cells were transfected with Lipofectamine for 24 hours. , cells were treated with compounds A-D and 100 pM TGF-β for 24 hours. Luciferase activity was measured using the Dual-Glo® Luciferase Assay Kit (Promega). Assays were performed in duplicate for compounds A, B, and D. Three runs were performed for Compound C. The results are shown in Table 4.

[0268] [Table 4]

[0269] The activity data from Experiment 1 are shown in FIG.

[0270] Compounds A to C showed the greatest inhibitory activity.

[0271] 6.7. [Example 7] 4-((S)-2-((S)-2-(6-(2,5-dioxo-2H-pyrrole-1(5 H)-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido benzylmethyl(2-(4-(4-(3-(6-methylpyridin-2-yl)-1H -pyrazol-4-yl)pyridin-2-yl)phenoxy)ethyl)carbamate Growth Compound C can be prepared by the general method of Scheme 6 below, by the addition of valine-citrulline. Linked to a linker.

[0272] [ka]

[0273] L1 (122 mg, 0.165 mmol, 1.1 equiv.) and TEA (52 μl, 0. Compound C (58 mg, 0.150 mmol, 1.0 eq.) was added to A solution of 100 ml of ethanol (amount) in DMF (2 ml) was added at 0° C., and the reaction mixture was stirred at approximately 21° C. for 2 hours. The crude ADC-1 was purified by preparative HPLC to give a white solid. Purified ADC-1 (34 mg, 24% yield) was obtained as a pure product.

[0274] 6.8. [Example 8] Generation of antibody drug conjugate 1 (ADC1) Anti-human FAP antibody was incubated in complex buffer (25 mM sodium borate / 25 mM NaCl The antibody was dialyzed overnight into Tris ( 2-carboxyethyl)phosphine (TCEP) at a reduction ratio of 10-30 for 2 hours ADC-1 was dissolved in DMSO to a final concentration of 10 mM and then reduced to 15% The antibodies were conjugated to the IgG1A at conjugation ratios of 5 to 30 in the presence of DMSO. All reactions were performed at approximately 21 For several drug-antibody ratios (DAR), 50% propylene glycol was used. The final ADC was permeated overnight in PBS. The mixture was analyzed by HPLC-HIC, filtered using a 0.22 μm filter, and analyzed. Determine the DAR and analyze via HPLC-SEC to determine the level of aggregation. For HIC, samples were loaded onto TSKgel® Butyrate at a flow rate of 0.5 ml / min. Run on a LE-NPR column. Phase A is 25 mM sodium phosphate, pH 6. Phase B is 1.5M ammonium sulfate at pH 6.95, while Phase B is 75% 2M ammonium sulfate at pH 6.95. 5 mM sodium phosphate, and 25% isopropyl alcohol. For SEC analysis, a TSKgel® G3000SW column (Tosoh B A ioscience is used at a flow rate of 0.25 ml / min at 280 nM for 25 minutes.

[0275] 6.9. [Example 9] Synthesis of disulfide linker-linked compound C (ADC-2) Compound C can be converted to a disulfide according to the general method in Schemes 7A-B below. Linked to a linker.

[0276] [ka]

[0277] [ka]

[0278] 6.9.1. Synthesis of Intermediate A 2-Chlorotrityl chloride resin (L2) (4 g, 4 mmol) was dissolved in DCM (2 × 40 ml), swell in 50 ml of DCM for 10 minutes, then drain. -Cys(Trt)-OH(L3) (7.03 g, 12 mmol) in 40 ml of DCM and add to the vessel containing the 2-chlorotrityl chloride resin. IPEA (6.8 ml, 40 mmol) was added to the vessel and the mixture was incubated at approximately 21° C. for 2 hours. Stir. Then add 10 ml of methanol to the mixture and stir for 30 minutes. Then, the resulting resin (L4) is drained and washed five times with DMF. Upon protection, approximately 40 ml of 20% piperidine in DMF was added to resin L4, and the mixture was The resin is shaken and then drained to produce resin L5. Another 40 ml of 20% piperidine is added to the resin and shaken for 15 minutes. Resin L5 is drained and washed with DMF (6 x 40 ml).

[0279] The solution of Fmoc-amino acid was prepared by adding Fmoc-Asp(OtBu)-OH (4.93 g, 1 2mmol), Fmoc-Asp(OtBu)-OH (4.93g, 12mmol), F moc-Arg(Pbf)-OH(7.79g, 12mmol), Fmoc-Asp(O tBu)-OH (4.93 g, 12 mmol), and Fmoc-Glu-OtBu (5 0.1g, 12mmol) to HBTU / HOBT (4.55g, 12mmol / 1.62 g, 12 mmol) and DIPEA (2 ml, 12 mmol) were prepared separately. To manufacture.

[0280] The Fmoc-Asp(OtBu)-OH solution was added to resin L5 and shaken for 60 minutes. , yielding resin L6. Resin L6 was washed with DMF (6 x 40 ml) and then purified as described above. , deprotected with 20% piperidine in DMF. Resins L7, L8, L9, and L10 were then prepared. L10 was prepared by performing successive couplings using Fmoc-amino acid solutions. Resin L6 is prepared from resin L5 using the same procedure.

[0281] In an exemplary synthesis, dry resin L10 (8 g) was added to a flask and 80 ml of cleavage solution was added. The solution was added (TFA:TES:EDT:HO=90:5:3:2, v / v / v / v The reaction was allowed to proceed for 1.5 hours. The resin was then removed from the reaction mixture by filtration under pressure. The resin was then washed twice with TFA. The filtrates were combined and diluted with 10 times the volume of cold M TBE was added dropwise, and the precipitated peptide (Intermediate A) was then centrifuged and diluted with cold MTBF. Intermediate A was then dried under reduced pressure and purified by preparative HPLC to give: 1.1 g of intermediate A was obtained as a white solid (37% yield). LC-MS (ESI) m / z:752[M+H]+.

[0282] 6.9.2. 2-(Pyridin-2-yldisulfanyl)ethylmethyl (2-(4-( 4-(4-(6-methylpyridin-2-yl)-1H-pyrazol-3-yl)pyridine -2-yl)phenoxy)ethyl)carbamate (L12) Compound C (40 mg, 0.1038 mmol) and 4-nitrophenyl 2-(pyridinyl) (2-phenyl-2-yldisulfanyl)ethyl carbamate (L11) (80 mg, 0.2272 A solution of 10 mmol) in DMF (5 ml) was added with DIPEA (0.5 ml) and HOBt ( The mixture was heated under N2 at approximately 21 °C for 16 h. The mixture was stirred for 1 hour to produce L12. Crude L12 was purified by preparative HPLC to give a white solid. This gave 35 mg of purified L12 (56% yield).

[0283] 6.9.3. (2R,5S,8S,11S,14S,19S)-19-amino-5,8 ,14-tris(carboxymethyl)-11-(3-guanidinopropyl)-2-((( 2-(methyl(2-(4-(4-(4-(6-methylpyridin-2-yl)-1H-pyra) (3-yl)pyridin-2-yl)phenoxy)ethyl)carbamoyloxy) (ethyl)disulfanyl)methyl)-4,7,10,13,16-pentaoxo-3,6, 9,12,15-Pentaazaicosane-1,20-dioic acid (L13) Dissolve L12 (35 mg, 0.058 mmol) in THF / H2O (5 ml / 5 ml) To the solution, Intermediate A (80 mg, 0.106 mmol) was added under N2. The mixture was stirred at 21° C. for 16 hours to produce L13. Crude L13 was purified by preparative HPLC. This gave 23 mg of purified L13 (31% yield) as a white solid.

[0284] 6.9.4. (2R,5S,8S,11S,14S,19S)-19-(2-(ter t-Butoxycarbonylaminooxy)acetamido)-5,8,14-tris(carbo 2-((2-(methyl)-11-(3-guanidinopropyl)-2-(((2-(methyl)-2-(4 -(4-(4-(6-methylpyridin-2-yl)-1H-pyrazol-3-yl)pyridin Diazin-2-yl)phenoxy)ethyl)carbamoyloxy)ethyl)disulfanyl) Methyl)-4,7,10,13,16-pentaoxo-3,6,9,12,15-penta Azaicosane-1,20-dioic acid (L15) To a solution of L13 (32 mg, 0.025 mmol) in DMF (3 ml), 2,5-dichloro- Oxopyrrolidin-1-yl 2-(tert-butoxycarbonylaminooxy)acetate Add 28 mg (0.097 mmol) of L14 followed by 0.5 ml of TEA. The reaction mixture was stirred at approximately 21 °C under a N2 atmosphere for 16 h to produce L15. The crude L15 was purified by preparative HPLC to give 12 mg of purified L15 as a white solid. 15 (33% yield) was produced.

[0285] 6.9.5. (2R,5S,8S,11S,14S,19S)-19-(2-(amino) Oxy)acetamido)-5,8,14-tris(carboxymethyl)-11-(3-glucan anidinylpropyl)-2-(((2-(methyl(2-(4-(4(4-(6-methylpyridine (1H-pyrazol-3-yl)pyridin-2-yl)phenoxy)e (ethyl)carbamoyloxy)ethyl)disulfanyl)methyl)-4,7,10,13, 16-pentaoxo-3,6,9,12,15-pentaazaicosane-1,20-dioic acid ( ADC-2) To a mixture of L15 (12 mg, 0.0085 mmol) in DCM (5 ml), TFA (1 ml) was added. The mixture was stirred at approximately 21° C. for 30 minutes to produce ADC-2. The crude ADC-2 was concentrated and purified by preparative HPLC to give 3.5 mg of a white solid. of purified ADC-2 (31% yield).

[0286] 6.10. [Example 10] Generation of antibody drug conjugate 2 (ADC2) ADC-2 was prepared by converting antibody lysine residues to hydroxyl groups according to the general method in Scheme 8 below. The antibody bound to the anti-human FAP antibody via the IgG.

[0287] [ka]

[0288] The antibody was dialyzed in PBS at pH 7.4. S-4FB was added at different molar ratios to the antibody at pH 7. S-4FB was added to the antibody in PBS at 4°C and incubated at approximately 21°C for 3 hours. The coated antibody solution was diluted with 2-hydrazinopyridine solution (0.5 mL in 100 mM MES buffer). 100 mM, pH 5.0) at various conjugation ratios ranging from 5 to 50, and incubated at 37°C for 30 min. The S4FB / Ab molar substitution ratio was determined by UV-Vis at A354. The modified antibody was purified by Zeba™ spin desalting column, 50 mM phosphate buffer (PBS) Purification was performed using buffer exchanged into HCl (HCl 6.5, 150 mM NaCl) and then linker -SS-drug ADC-2 (10 mM in DMSO) and 2 at different molar ratios at 37 °C. Mix for 4 hours to generate ADC2. The next day, the ADC2 sample was diluted to PBS and Dialyze overnight. Filter the sample and then analyze it by HPLC-SEC, SDS-PAGE, and L. Test via C-MS.

[0289] If ADC2 aggregation at 5% is detected by HPLC-SEC, the aggregated components was measured using an SEC column (GE Healthcare Life Sciences, Sup AKTA with Erdex 200 increase 10 / 300GL) The mixture is separated and re-analyzed by HPLC-SEC.

[0290] 6.11. [Example 11] Synthesis and characterization of compound N Compound N was synthesized according to the general method in Scheme 9 below.

[0291] [ka]

[0292] [ka]

[0293] Compound N was compared to Compound C in several in vitro assays. Its IC50 activity and K in recombinant kinase assays i A summary of the values ​​is given in Table 5 Table 5 also shows the inhibitory activity of Compound C on TGF-β signaling in human HEK cells. Compound C was found to be 10 times more potent than compound N in the kinase assay. I understand.

[0294] [Table 5]

[0295] 6.12. [Example 12] Anti-FAP antibody binding to HEK cells Anti-FAP antibody (commercially available mouse IgG1, clone 427819) expressing human FAP The ability of the anti-FAP antibodies to bind to HEK293 cells was assessed by FACS. The FAP cDNA was transfected and expressed on the cell surface of HEK293 cells. (Fig. 2C) but not in parental HEK cells that were not transfected with human cDNA. There was no (Figure 2B).

[0296] 6.13. [Example 13] Production of targeted drug conjugates SYN-301 and SYN-302 Two targeted drug conjugates were synthesized. First, compound C was conjugated to MC-Val-Cit-P An ABC-cleavable linker was used to ligate anti-FAP antibody (commercially available mouse IgG1, clone 42 7819) (Figure 3A). This targeted drug conjugate is referred to herein as SYN-3 Second, the compound was linked to an anti-FAP antibody using a non-cleavable (MC) linker. This targeted drug conjugate is referred to herein as SYN-302 (Figure 3B). The drug-antibody ratio was determined using a PLRP-S column (1000 Å pore size, 5 μm particle size, 1 × 50 % aggregation was determined by reverse phase liquid chromatography using TSKgel ® G3000SWXL column by size exclusion chromatography. The SYN-301 preparations had measured drug-antibody ratios of 5.5 and 4% aggregation. (DAR), whereas the SYN-302 preparation measured 5 and 3.9% aggregation. The DAR was

[0297] 6.14. [Example 14] SYN-301 and SYN-30 in HEK cells expressing human FAP proteins Evaluation of TGF-β signaling inhibition by 2 Evaluating the ability of SYN-301 and SYN-302 to inhibit TGF-β signaling To demonstrate this, assays were performed using HEK293FT cells expressing human FAP. Ta.

[0298] HEK293FT cells were transfected with Mirus TransIT®-LT1 transfection. Constructs encoding human FAP, including transfection reagents, and luciferase The transporter gene luc2P (pGL4.48[luc2P / SBE / Hygro]; Pr TGF-β response containing three copies of the SMAD binding element triggers the expression of omega A control construct expressing Renilla luciferase was used. pGL4.74 encoding the enzyme; Promega) (1 μg per ml of cell culture: 1 μg The cells were transiently transfected with 0.125 μg of IgG. Cells were plated at 350,000 cells / ml (100 μl per well). After incubation, cells were incubated with SYN-301, SYN-302, or unconjugated anti-FAP for 4 hours. The cells were pretreated with either antibody or unconjugated Compound C. Then, 1 nM TGF-β was administered. The cells were incubated for 3 hours after addition of 1000 μg ... ual-Glo luciferase detection system, Promega).

[0299] The results are shown in Figures 4A-4B. SYN-301 with a cleavable linker inhibited the expression of FAP. We were able to inhibit TGF-β signaling in engineered HEK cells (Figure 4A). ) On the other hand, SYN-301 inhibits TGF-β signaling in parental HEK cells that do not express FAP. The non-cleavable linker was not observed to have a significant effect on IL-1 signal transduction (Figure 4B). SYN-302, which has a β-blocking activity, was less effective than SYN-301 in this assay. was observed (Figure 4A).

[0300] 6.15. [Example 15] FAP internalization induced by SYN-301 and SYN-302 SYN-301 and SYN-302 inhibit FAP in target cells that endogenously express FAP To assess the ability of WI-38 human lung fibroblasts to internalize IL-1, an internalization assay was performed. It was carried out using

[0301] WI-38 cells were incubated with anti-FAP antibodies, SYN-301, and SYN-302 for 30 minutes at 4°C. or isotype control ADC (cleavable ValCit-ALK5 inhibitor compound) Cell surface FAP expression was detected by incubation with a nonspecific antibody conjugated to substance C (control). The cells were then washed twice with cold PBS to remove any remaining antibody / antibody complexes in the supernatant. The solution was removed and then incubated at 37°C for 3 hours to induce receptor internalization. After a 2-h incubation, cells were washed and immunolabeled with a PE-conjugated rat anti-mouse secondary antibody. The remaining cell surface FAP expression was detected by incubating at 4°C. WI-38 cells incubated with anti-FAP antibody as a measure of specific FAP expression or compared with the composite.

[0302] The results are shown in Figures 5A to 5E and 6. 50 to 60% of WI-38 cells (Figures 5A to 5E ) express FAP, and anti-FAP antibodies, SYN-301 and SYN-302, were used to treat WI- 38 cells and were relatively able to bind and internalize FAP (63% and 63%, and 52%) (Figure 6).

[0303] 6.16. [Example 16] Functional characterization of SYN-301 and SYN-302 in WI-38 cells Type IV collagen (COL4A1), fibronectin (FN1), and 15 Increased expression of leucine-rich repeat-containing 15 (LRRC15) is a marker of increased fibrosis. SYN-301 and SYN-30 were tested using WI-38 human lung fibroblasts. The ability of 2 to reduce the expression of COL4A1, FN1, and LRRC15 was evaluated.

[0304] WI-38 human lung fibroblast cells were plated at 50,000 cells / ml in 24-well plates. Cells were plasma-starved for 18 hours and incubated overnight. Starvation reduces the plasma effect on TGFβ-regulated genes, followed by 1 At μg / ml, SYN-301, SYN-302, isotype control ADC, The cells were pretreated with FAP antibody or compound C. TGF-β was added and the cells were incubated for 19 hours. The cells were then scraped into RLT buffer (Qiagen) and the RNA was extracted with Qiagen. RNA was extracted using the Aggen RNaeasy Kit. RNA was reverse transcribed into cDNA. qPCR was then performed using TaqMan probes for COL4A1, FN1, and LRRC15. The assay was performed using a Reimer. GAPDH was used as a normalizer.

[0305] The results are shown in Figures 7A-7B. SYN-301 partially inhibited TGF-β-induced gene responses. It effectively blocked COL4A1 expression by approximately 25-30% (Fig. 7A) and FN1 expression. It reduced LRRC15 expression by approximately 20-25% (Fig. 7A) and 15-20%. SYN-302 was more modest in blocking TGF-β signaling (Fig. 7B). While there was a response, unconjugated anti-FAP antibody and isotype control A DCs did not inhibit TGF-β signaling.

[0306] 7. Specific Embodiments The present disclosure is illustrated by the following specific embodiments. 1. Myofibroblasts, activated fibroblasts, fibroblasts transitioning to myofibroblasts, or operably linked to a targeting moiety that binds to a cell surface molecule expressed on the surface of the combination. A targeted drug conjugate comprising an ALK5 inhibitor. 2. The targeting moiety of embodiment 1, wherein the targeting moiety binds to a cell surface molecule of a myofibroblast. Drug complexes. 3. The method of embodiment 1, or 3. The targeted drug conjugate of embodiment 2. 4. The targeting moiety binds to a cell surface molecule on fibroblasts that transition into myofibroblasts. 4. The targeted drug conjugate of any one of Forms 1 to 3. 5. The ALK5 inhibitor has an IC of at least 20 nM 50 Any of embodiments 1 to 4, having The targeted drug complex described in any one of claims 1 to 4. 6. The ALK5 inhibitor is an imidazole, pyrazole, or thiazole. 6. The targeted drug conjugate of any one of embodiments 1 to 5, which is a compound based on 7. The targeted drug complex of embodiment 6, wherein the ALK5 inhibitor is an imidazole-based compound. Merge. 8. The targeted drug combination of embodiment 6, wherein the ALK5 inhibitor is a pyrazole-based compound. body. 9. The targeted drug combination of embodiment 6, wherein the ALK5 inhibitor is a thiazole-based compound. body. 10. The ALK5 inhibitor is an imidazole-benzodioxole compound, or an imidazole 7. The target compound of embodiment 6, which is an imidazole-based compound, is a quinoxaline compound. Chemical drug conjugates. 11. Embodiment 1, wherein the ALK5 inhibitor is an imidazole-benzodioxole compound. 10. The targeted drug conjugate of claim 0. 12. The method of embodiment 10, wherein the ALK5 inhibitor is an imidazole-quinoxaline compound. The targeted drug conjugate described above. 13. The ALK5 inhibitor is a pyrazole-pyrrolo compound, a pyrazole-based compound. 7. The targeted drug conjugate of embodiment 6. 14. ALK5 inhibitors include imidazole-benzodioxole compounds, imidazole-ki a pyrazole-pyrrolo compound, a thiazole-based compound, 7. The targeted drug conjugate of claim 6. 15. The target of any one of embodiments 1 to 4, wherein the ALK5 inhibitor is compound C. Targeted drug complex. 16. The compound according to any one of embodiments 1 to 4, wherein the ALK5 inhibitor is compound N. Targeted drug complex. 17. Embodiment 1, in which the ALK5 inhibitor is linked to the targeting moiety via a linker. 17. The targeted drug complex of any one of claims 1 to 16. 18. The targeted drug of embodiment 17, wherein the linker is a linker containing PEG. Complex. 19. The target according to embodiment 17 or embodiment 18, wherein the linker is a polyvalent linker. Chemical drug conjugates. 20. The method of any one of embodiments 17 to 19, wherein the linker is a non-cleavable linker. The targeted drug conjugate described above. 21. The non-cleavable linker is N-maleimidomethylcyclohexane-1-carboxylate. a maleimidocaproyl, or mercaptoacetamidocaproyl linker; 21. The targeted drug conjugate of embodiment 20. 22. The non-cleavable linker is N-maleimidomethylcyclohexane 1-carboxylate 22. The targeted drug conjugate of embodiment 21, wherein: 23. As described in embodiment 21, wherein the non-cleavable linker is a maleimidocaproyl linker. Targeted drug conjugates. 24. An embodiment in which the non-cleavable linker is a mercaptoacetamidocaproyl linker. 22. The targeted drug conjugate according to embodiment 21. 25. Any one of embodiments 17 to 19, wherein the linker is a cleavable linker. Targeted drug conjugates. 26. The targeted drug complex of embodiment 25, wherein the cleavable linker is a peptide linker. Merge. 27. The cleavable linker is a dipeptide linker, a disulfide linker, or a hydrazoline linker. 26. The targeted drug conjugate of embodiment 25, wherein the linker is a carboxyl group. 28. The targeted drug of embodiment 27, wherein the cleavable linker is a dipeptide linker. Complex. 29. The targeting method of embodiment 26, wherein the peptide linker is a tripeptide linker. Drug complexes. 30. The target according to embodiment 26, wherein the peptide linker is a tetrapeptide linker. Chemical drug conjugates. 31. The peptide linker is glycine-glycine-phenylalanine-glycine (gly 31. The targeted drug conjugate of embodiment 30, wherein the linker is 32. The targeting agent of embodiment 27, wherein the cleavable linker is a disulfide linker. Material complex. 33. The targeted drug of embodiment 27, wherein the cleavable linker is a hydrazone linker. Complex. 34. The linker is a protease-sensitive valine-citrulline dipeptide linker. 28. The targeted drug conjugate of embodiment 27. 35. The linker is a protease-sensitive phenylalanine-lysine dipeptide linker. 28. The targeted drug conjugate of embodiment 27. 36. The method according to embodiment 27, wherein the linker is a glutathione-sensitive disulfide linker. The targeted drug conjugate described above. 37. The targeting method according to embodiment 27, wherein the linker is an acid-sensitive disulfide linker. Drug complexes. 38. A method for the preparation of a compound in which an ALK5 inhibitor is conjugated to a targeting moiety via site-specific conjugation. 38. The targeted drug conjugate of any one of embodiments 1 to 37. 39. The ALK5 inhibitor comprises one or more cysteines, lysines, or 39. The targeted drug conjugate of embodiment 38, wherein the conjugate is via a glutamine residue. 40. ALK5 inhibitors are conjugated via one or more cysteine ​​residues on the targeting moiety. 40. The targeted drug conjugate of embodiment 39, wherein the targeted drug conjugate incorporates 41. ALK5 inhibitors are conjugated via one or more lysine residues on the targeting moiety. 40. The targeted drug conjugate of embodiment 39, 42. ALK5 inhibitors are conjugated via one or more glutamine residues on the targeting moiety. 40. The targeted drug conjugate of embodiment 39, wherein the targeted drug conjugate incorporates 43. The ALK5 inhibitor is directed to a targeting moiety via one or more unnatural amino acid residues. 39. The targeted drug conjugate of embodiment 38, wherein the targeted drug conjugate is conjugated to 44. One or more unnatural amino acid residues are p-acetylphenylalanine (pAc F). 45. One or more unnatural amino acid residues are p-azidomethyl-L-phenylalanine 44. The targeted drug conjugate of embodiment 43, comprising pAMF. 46. ​​The one or more unnatural amino acid residues include selenocysteine ​​(Sec). 44. The targeted drug conjugate of embodiment 43. 47. ALK5 inhibitors are conjugated via one or more glycans on the targeting moiety. 39. The targeted drug conjugate of embodiment 38, wherein 48. The targeted drug of embodiment 47, wherein one or more glycans comprise fucose. Complex. 49. The target of embodiment 47, wherein one or more glycans contain 6-thiofucose. Targeted drug complex. 50. The targeting method of embodiment 47, wherein one or more glycans comprise galactose. Drug complexes. 51. One or more glycans contain N-acetylgalactosamine (GalNAc). 48. The targeted drug conjugate of embodiment 47. 52. One or more glycans contain N-acetylglucosamine (GlcNAc) 48. The targeted drug complex of embodiment 47. 53. The target of embodiment 47, wherein one or more glycans comprise sialic acid (SA). Targeted drug complex. 54. Any of embodiments 38 to 53, wherein the ALK5 inhibitor is conjugated via a linker. The targeted drug complex described in any one of claims 1 to 4. 55. The average number of ALK5 inhibitor molecules per targeting moiety molecule is in the range of 1 to 30. 55. The targeted drug conjugate of any one of embodiments 1 to 54. 56. The average number of ALK5 inhibitor molecules per targeting moiety molecule is in the range of 1 to 20; 55. The targeted drug conjugate of any one of embodiments 1 to 54. 57. The average number of ALK5 inhibitor molecules per targeting moiety molecule is in the range of 1 to 15. 55. The targeted drug conjugate of any one of embodiments 1 to 54. 58. The average number of ALK5 inhibitor molecules per targeting moiety molecule is in the range of 2 to 12. 55. The targeted drug conjugate of any one of embodiments 1 to 54. 59. The average number of ALK5 inhibitor molecules per targeting moiety molecule is in the range of 4 to 15. 55. The targeted drug conjugate of any one of embodiments 1 to 54. 60. The average number of ALK5 inhibitor molecules per targeting moiety molecule is in the range of 6 to 12; 55. The targeted drug conjugate of any one of embodiments 1 to 54. 61. A study in which the average number of ALK5 inhibitor molecules per targeting moiety molecule is in the range of 2 to 8. 55. The targeted drug conjugate of any one of embodiments 1 to 54. 62. The targeting agent of any one of embodiments 1 to 61, wherein the targeting moiety is internalized. Material complex. 63. Any one of embodiments 1 to 62, wherein the targeting moiety comprises an antibody or an antibody fragment. 1. A targeted drug conjugate as described herein. 64. The targeted drug conjugate of embodiment 63, wherein the targeting moiety comprises an antibody. 65. The targeted drug conjugate of embodiment 64, wherein the antibody is a monoclonal antibody. 66. The targeted drug conjugate of embodiment 65, wherein the antibody is human or humanized. 67. The targeted drug conjugate of embodiment 66, wherein the antibody is human. 68. The targeted drug conjugate of embodiment 66, wherein the antibody is humanized. 69. The targeted drug conjugate of embodiment 63, wherein the targeting moiety comprises an antibody fragment. 70. The targeted drug of embodiment 69, wherein the antibody fragment is a fragment of a monoclonal antibody. Complex. 71. The targeting agent of embodiment 70, wherein the antibody fragment is a fragment of a human or humanized antibody. Material complex. 72. The targeted drug conjugate of embodiment 71, wherein the antibody fragment is a fragment of a human antibody. 73. The targeted drug conjugate of embodiment 71, wherein the antibody fragment is a fragment of a humanized antibody. 74. The antibody fragment may be Fab, Fab', F(ab')2, Fv, scFv, dsFv, or or a single domain antibody. Complex. 75. The targeted drug conjugate of embodiment 74, wherein the antibody fragment is a Fab. 76. The targeted drug conjugate of embodiment 74, wherein the antibody fragment is Fab'. 77. The targeted drug conjugate of embodiment 74, wherein the antibody fragment is F(ab')2. 78. The targeted drug conjugate of embodiment 74, wherein the antibody fragment is an Fv. 79. The targeted drug conjugate of embodiment 74, wherein the antibody fragment is an scFv. 80. The scFv comprises a polypeptide linker between the VH and VL domains of the scFv. 79. The targeted drug complex of embodiment 79. 81. The targeted drug conjugate of embodiment 74, wherein the antibody fragment is a dsFv. 82. The targeted drug conjugate of embodiment 74, wherein the antibody fragment is a single domain antibody. 83. Single domain antibodies against camel V H H antibody fragment or humanized camel V H H antibody fragment 83. The targeted drug conjugate of embodiment 82. 84. Any one of embodiments 1 to 62, wherein the targeting moiety is non-immunoglobulin-based. 1. A targeted drug conjugate as described herein. 85. Any one of embodiments 1 to 84, wherein the cell surface molecule is a human cell surface molecule. 1. A targeted drug conjugate as described herein. 86. Cell surface molecules include FAP, PDGFR-β, FGFR1, PPAR-γ, FSP1 , GFAP, fascin, CD147, CXCR4, αvβ6, AXL, or MERTK 86. The targeted drug conjugate of any one of embodiments 1 to 85, wherein 87. The targeted drug conjugate of embodiment 86, wherein the cell surface molecule is FAP. 88. An embodiment in which the targeting moiety preferentially binds to membrane-bound FAPs over soluble FAPs. 88. The targeted drug conjugate of embodiment 87. 89. The targeted drug conjugate of embodiment 86, wherein the cell surface molecule is PDGFR-β. . 90. The targeted drug conjugate of embodiment 86, wherein the cell surface molecule is FGFR1. 91. The targeted drug conjugate of embodiment 86, wherein the cell surface molecule is PPAR-γ. 92. The targeted drug conjugate of embodiment 86, wherein the cell surface molecule is FSP1. 93. The targeted drug conjugate of embodiment 86, wherein the cell surface molecule is GFAP. 94. The targeted drug conjugate of embodiment 86, wherein the cell surface molecule is fascin. 95. The targeted drug conjugate of embodiment 86, wherein the cell surface molecule is CD147. 96. The targeted drug conjugate of embodiment 86, wherein the cell surface molecule is CXCR4. 97. The targeted drug conjugate of embodiment 86, wherein the cell surface molecule is αvβ6. 98. The targeted drug conjugate of embodiment 86, wherein the cell surface molecule is AXL. 99. The targeted drug conjugate of embodiment 86, wherein the cell surface molecule is MERTK. 100. The method of any one of embodiments 1 to 85, wherein the cell surface molecule is LRRC15. The targeted drug conjugate described above. 101. Apoptosis of myofibroblasts exposed to targeted drug conjugates 101. The targeted drug conjugate of any one of embodiments 1 to 100, 102. The method of claim 1, wherein the dedifferentiation of myofibroblasts contacted with the targeted drug complex is promoted. 100. A targeted drug complex described in any one of claims 100 to 100. 103. As described in embodiment 102, wherein dedifferentiation is measured by a decrease in smooth muscle actin expression. Targeted drug conjugates. 104. Fc domain with one or more amino acid substitutions that reduce effector function 104. The targeted drug conjugate of any one of embodiments 1 to 103, comprising a main. 105. One or more substitutions are N297A, N297Q, N297G, D265A / N297A, D265A / N297G, L235E, L234A / L235A, L234 A / L235A / P329A, L234D / L235E:L234R / L235R / E2 33K, L234D / L235E / D265S:E233K / L234R / L235R / D265S, L234D / L235E / E269K:E233K / L234R / L235 R / E269K, L234D / L235E / K322A:E233K / L234R / L2 35R / K322A, L234D / L235E / P329W:E233K / L234R / L235R / P329W, L234D / L235E / E269K / D265S / K322 A:E233K / L234R / L235R / E269K / D265S / K322A, and L234D / L235E / E269K / D265S / K322E / E333K:E23 Including 3K / L234R / L235R / E269K / D265S / K322E / E333K 105. The targeted drug conjugate of embodiment 104. 106. The targeting agent of embodiment 105, wherein the one or more substitutions include N297A. Material complex. 107. The targeting agent of embodiment 105, wherein the one or more substitutions include N297Q. Material complex. 108. The targeting agent of embodiment 105, wherein the one or more substitutions include N297G. Material complex. 109. The amino acid sequence of embodiment 105, wherein the one or more substitutions include D265A / N297A. The targeted drug conjugate described above. 110. The method of embodiment 105, wherein the one or more substitutions include D265A / N297G. The targeted drug conjugate described above. 111. The targeting agent of embodiment 105, wherein the one or more substitutions include L235E. Material complex. 112. The amino acid sequence according to embodiment 105, wherein one or more substitutions include L234A / L235A. The targeted drug conjugate described above. 113. One or more substitutions include L234A / L235A / P329A. 106. The targeted drug conjugate of embodiment 105. 114. One or more substitutions are: L234D / L235E:L234R / L235R / The targeted drug conjugate of embodiment 105, comprising E233K. 115. One or more substitutions are L234D / L235E / D265S:E233K / The targeted drug conjugate of embodiment 105, comprising L234R / L235R / D265S. . 116. One or more substitutions are L234D / L235E / E269K:E233K / The targeted drug conjugate of embodiment 105, comprising L234R / L235R / E269K. . 117. One or more substitutions are L234D / L235E / K322A:E233K / The targeted drug conjugate of embodiment 105, comprising L234R / L235R / K322A. . 118. One or more substitutions are L234D / L235E / P329W:E233K / The targeted drug conjugate of embodiment 105, comprising L234R / L235R / P329W. . 119. One or more substitutions are: L234D / L235E / E269K / D265S / K322A:E233K / L234R / L235R / E269K / D265S / K322 The targeted drug conjugate of embodiment 105, comprising A. 120. One or more substitutions are: L234D / L235E / E269K / D265S / K322E / E333K:E233K / L234R / L235R / E269K / D265 The targeted drug conjugate of embodiment 105, comprising S / K322E / E333K. 121. A targeted drug conjugate according to any one of embodiments 1 to 120, and a pharmaceutical A pharmaceutical composition comprising a carrier acceptable to 122. At least 30% of the targeted drug conjugate molecules in the pharmaceutical composition contain 1 to 30 ALK5 The pharmaceutical composition of embodiment 121, having a ratio of inhibitor:targeting moiety: 123. At least 30% of the targeted drug conjugate molecules in the pharmaceutical composition contain 1 to 20 ALK5 The pharmaceutical composition of embodiment 121, having a ratio of inhibitor:targeting moiety: 124. At least 30% of the targeted drug conjugate molecules in the pharmaceutical composition contain 1 to 15 ALK5 The pharmaceutical composition of embodiment 121, having a ratio of inhibitor:targeting moiety: 125. At least 30% of the targeted drug conjugate molecules in the pharmaceutical composition contain 2 to 12 ALK5 The pharmaceutical composition of embodiment 121, having a ratio of inhibitor:targeting moiety: 126. At least 30% of the targeted drug conjugate molecules in the pharmaceutical composition contain 4 to 15 ALK5 The pharmaceutical composition of embodiment 121, having a ratio of inhibitor:targeting moiety: 127. At least 30% of the targeted drug conjugate molecules in the pharmaceutical composition contain 6 to 12 ALK5 The pharmaceutical composition of embodiment 121, having a ratio of inhibitor:targeting moiety: 128. At least 30% of the targeted drug conjugate molecules in the pharmaceutical composition contain 2 to 8 ALK5 inhibitors. 122. The pharmaceutical composition of embodiment 121, having a ratio of cytotoxic agent:targeting moiety: 129. At least 40% of the targeted drug conjugate molecules in the pharmaceutical composition contain 1 to 30 ALK5 The pharmaceutical composition of embodiment 121, having a ratio of inhibitor:targeting moiety: 130. At least 40% of the targeted drug conjugate molecules in the pharmaceutical composition contain 1 to 20 ALK5 The pharmaceutical composition of embodiment 121, having a ratio of inhibitor:targeting moiety: 131. At least 40% of the targeted drug conjugate molecules in the pharmaceutical composition contain 1 to 15 ALK5 The pharmaceutical composition of embodiment 121, having a ratio of inhibitor:targeting moiety: 132. At least 40% of the targeted drug conjugate molecules in the pharmaceutical composition contain 2 to 12 ALK5 The pharmaceutical composition of embodiment 121, having a ratio of inhibitor:targeting moiety: 133. At least 40% of the targeted drug conjugate molecules in the pharmaceutical composition contain 4 to 15 ALK5 The pharmaceutical composition of embodiment 121, having a ratio of inhibitor:targeting moiety: 134. At least 40% of the targeted drug conjugate molecules in the pharmaceutical composition contain 6 to 12 ALK5 The pharmaceutical composition of embodiment 121, having a ratio of inhibitor:targeting moiety: 135. At least 40% of the targeted drug conjugate molecules in the pharmaceutical composition contain 2 to 8 ALK5 inhibitors. 122. The pharmaceutical composition of embodiment 121, having a ratio of cytotoxic agent:targeting moiety: 136. At least 50% of the targeted drug conjugate molecules of the pharmaceutical composition contain 1 to 30 ALK5 The pharmaceutical composition of embodiment 121, having a ratio of inhibitor:targeting moiety: 137. At least 50% of the targeted drug conjugate molecules in the pharmaceutical composition contain 1 to 20 ALK5 The pharmaceutical composition of embodiment 121, having a ratio of inhibitor:targeting moiety: 138. At least 50% of the targeted drug conjugate molecules in the pharmaceutical composition contain 1 to 15 ALK5 The pharmaceutical composition of embodiment 121, having a ratio of inhibitor:targeting moiety: 139. At least 50% of the targeted drug conjugate molecules in the pharmaceutical composition contain 2 to 12 ALK5 The pharmaceutical composition of embodiment 121, having a ratio of inhibitor:targeting moiety: 140. At least 50% of the targeted drug conjugate molecules in the pharmaceutical composition contain 4 to 15 ALK5 The pharmaceutical composition of embodiment 121, having a ratio of inhibitor:targeting moiety: 141. At least 50% of the targeted drug conjugate molecules in the pharmaceutical composition contain 6 to 12 ALK5 The pharmaceutical composition of embodiment 121, having a ratio of inhibitor:targeting moiety: 142. At least 50% of the targeted drug conjugate molecules in the pharmaceutical composition contain 2 to 8 ALK5 inhibitors. 122. The pharmaceutical composition of embodiment 121, having a ratio of cytotoxic agent:targeting moiety: 143. At least 60% of the targeted drug conjugate molecules of the pharmaceutical composition contain 1 to 30 ALK5 The pharmaceutical composition of embodiment 121, having a ratio of inhibitor:targeting moiety: 144. At least 60% of the targeted drug conjugate molecules of the pharmaceutical composition contain 1 to 20 ALK5 The pharmaceutical composition of embodiment 121, having a ratio of inhibitor:targeting moiety: 145. At least 60% of the targeted drug conjugate molecules of the pharmaceutical composition contain 1 to 15 ALK5 The pharmaceutical composition of embodiment 121, having a ratio of inhibitor:targeting moiety: 146. At least 60% of the targeted drug conjugate molecules in the pharmaceutical composition contain 2 to 12 ALK5 The pharmaceutical composition of embodiment 121, having a ratio of inhibitor:targeting moiety: 147. At least 60% of the targeted drug conjugate molecules in the pharmaceutical composition contain 4 to 15 ALK5 The pharmaceutical composition of embodiment 121, having a ratio of inhibitor:targeting moiety: 148. At least 60% of the targeted drug conjugate molecules in the pharmaceutical composition contain 6 to 12 ALK5 The pharmaceutical composition of embodiment 121, having a ratio of inhibitor:targeting moiety: 149. At least 60% of the targeted drug conjugate molecules in the pharmaceutical composition contain 2 to 8 ALK5 inhibitors. 122. The pharmaceutical composition of embodiment 121, having a ratio of cytotoxic agent:targeting moiety: 150. A method for treating fibrosis in a subject in need thereof, comprising administering to the subject a compound of any one of embodiments 1 to 150. 120, or the targeted drug conjugate of any one of embodiments 121 to 149. A method comprising administering the pharmaceutical composition of any one of claims 1 to 4. 151. The method of embodiment 150, wherein the fibrosis is pulmonary fibrosis. 152. The method of embodiment 151, wherein the fibrosis is idiopathic pulmonary fibrosis (IPF). 153. The method of embodiment 150, wherein the fibrosis is liver fibrosis. 154. The method of embodiment 150, wherein the fibrosis is renal fibrosis. 155. The method of embodiment 150, wherein the fibrosis is cardiac fibrosis. 156. The method of embodiment 150, wherein the fibrosis is dermal fibrosis. 157. The method of embodiment 150, wherein the fibrosis is esophageal fibrosis. 158. Embodiment 15, wherein the subject has NASH, e.g., has been diagnosed with NASH. The method described in 3. 159. The subject has systemic sclerosis, e.g., has been diagnosed with systemic sclerosis, 158. The method of any one of embodiments 150 to 157. 160. Treating a subject with systemic sclerosis, e.g., diagnosed with systemic sclerosis 121. A method comprising administering to a subject a targeted drug conjugate of any one of embodiments 1 to 120. or administering a pharmaceutical composition according to any one of embodiments 121 to 149. Including, a method. 161. A method for treating a subject with NASH, e.g., a subject who has been diagnosed with NASH, comprising: 120. The targeted drug conjugate of any one of embodiments 1 to 120, or 150. A method comprising administering a pharmaceutical composition according to any one of claims 121 to 149. 162. The method of embodiment 160 or embodiment 160, wherein the subject exhibits signs and / or symptoms of fibrosis. 162. The method of claim 161. 163. The method of embodiment 160 or embodiment 160, wherein the subject does not exhibit signs and / or symptoms of fibrosis. 162. The method of embodiment 161. 164. The targeted drug conjugate or pharmaceutical composition is administered with one or more second therapeutic agents. and optionally, one or more of the agents is administered as part of a combination therapy regimen that includes , and is not the targeted drug conjugate of any one of embodiments 1 to 120 (each referred to as a "second The method of any one of embodiments 150 to 163, wherein the therapeutic agent is a 165. The targeted drug conjugate or pharmaceutical composition is combined with a standard of care therapy or treatment regimen. 165. The method of embodiment 164, wherein the two or more of ... 166. A method of administering a combination therapy to a subject, the combination therapy comprising administering at least one second therapeutic agent to the subject. 166. The method of claim 164 or 165. 167. The second treatment is pirfenidone, nintedanib, pentraxin-2, or paclitaxel. including blumab, prednisone, cortisone, cyclophosphamide, or azathioprine 167. The method of any one of embodiments 164 to 166. 168. The method of embodiment 167, wherein the second therapeutic agent comprises pirfenidone. 169. The method of embodiment 167 or embodiment 168, wherein the second therapeutic agent comprises nintedanib. The method described. 170. Any of embodiments 167 to 169, wherein the second therapeutic agent comprises pentraxin-2. The method according to any one of claims 1 to 5. 171. Any of embodiments 167 to 170, wherein the second therapeutic agent comprises pamrevlumab. 10. The method according to claim 1. 172. Any one of embodiments 167 to 171, wherein the second therapeutic agent comprises prednisone. The method described in paragraph . 173. Any one of embodiments 167 to 172, wherein the second therapeutic agent comprises cortisone. The method described below. 174. Any of embodiments 167 to 173, wherein the second therapeutic agent comprises cyclophosphamide. The method according to any one of claims 1 to 5. 175. Any of embodiments 167 to 174, wherein the second therapeutic agent comprises azathioprine. 10. The method according to claim 1. 176. Any one of embodiments 164 to 175, comprising treating the subject with a combination therapy. The method described in paragraph . 177. Any of embodiments 164 to 176, comprising administering a second therapeutic agent to the subject. 1. The method according to claim 1. 178. A method of treating a subject having cancer, comprising administering to a subject in need thereof, The targeted drug conjugate of any one of claims 1 to 120, or embodiments 121 to 14. 10. A method comprising administering a pharmaceutical composition according to any one of claims 9 to 10. 179. The method of embodiment 178, wherein the cancer is urothelial cancer. 180. The method of embodiment 179, wherein the cancer is bladder cancer. 181. The method of embodiment 179, wherein the cancer is urethral cancer. 182. The method of embodiment 179, wherein the cancer is ureteral cancer. 183. The method of embodiment 178, wherein the cancer is lung cancer. 184. The method of embodiment 183, wherein the cancer is NSCLC. 185. The method of embodiment 184, wherein the NSCLC is adenocarcinoma. 186. The method of embodiment 184, wherein the NSCLC is squamous cell carcinoma. 187. The method of embodiment 184, wherein the NSCLC is large cell carcinoma. 188. The method of embodiment 183, wherein the cancer is small cell lung cancer. 189. The method of embodiment 178, wherein the cancer is breast cancer. 190. The method of embodiment 178, wherein the cancer is pancreatic cancer. 191. The method of embodiment 178, wherein the cancer is prostate cancer. 192. The method of embodiment 178, wherein the cancer is esophageal cancer. 193. The method of embodiment 178, wherein the cancer is colorectal cancer. 194. The method of embodiment 193, wherein the colorectal cancer is adenocarcinoma. 195. The method of embodiment 193, wherein the colorectal cancer is a carcinoid tumor. 196. The method of embodiment 193, wherein the colorectal cancer is a gastrointestinal stromal tumor. 197. The method of embodiment 193, wherein the colorectal cancer is colorectal lymphoma. 198. The method of embodiment 178, wherein the cancer is head and neck cancer. 199. The method of embodiment 178, wherein the cancer is ovarian cancer. 200. The method of embodiment 178, wherein the cancer is renal cancer. 201. The method of embodiment 178, wherein the cancer is gastric adenocarcinoma. 202. The targeted drug conjugate or pharmaceutical composition is administered with one or more second therapeutic agents. and optionally, one or more of the agents is administered as part of a combination therapy regimen that includes , and is not the targeted drug conjugate of any one of embodiments 1 to 120 (each referred to as a "second The method of any one of embodiments 178 to 201, wherein the therapeutic agent is a 203. The targeted drug conjugate or pharmaceutical composition is combined with a standard of care therapy or treatment regimen. 203. The method of embodiment 202, wherein the two or more of the following are administered in combination: 204. A method of administering a combination therapy to a subject, the method comprising administering at least one second therapeutic agent to the subject. 204. The method of claim 202 or 203. 205. The combination therapy includes immunotherapy, and optionally, the immunotherapy includes checkpoint inhibitor therapy. method, chimeric antigen receptor (CAR) therapy, adoptive T cell therapy, oncolytic virus therapy, dendritic Cellular vaccine therapy, STING agonist therapy, TLR agonist therapy, intratumoral CpG therapy 205. The method of any one of embodiments 202 to 204, wherein the method is a method of administering a medicament for the treatment of a rheumatoid arthritis, or a cytokine therapy. method. 206. The method of any one of embodiments 202 to 205, wherein the combination therapy includes checkpoint inhibitor therapy. 10. The method according to any one of claims 1 to 9. 207. Checkpoint inhibitor therapy, including T-cell checkpoint inhibitor therapy, is 207. The method of embodiment 206. 208. T-cell checkpoint inhibitor therapy includes an antibody or an antigen-binding fragment thereof; The method of embodiment 207. 209. Checkpoint inhibitor therapy inhibits PD1, PDL1, CTLA4, TIGIT,

[0033] Embodiments targeting LAG3, OX40, CD40, VISTA, or a combination thereof 209. The method of any one of 206 to 208. 210. The method of embodiment 209, wherein the checkpoint inhibitor therapy targets PD1. method. 211. The method of embodiment 210, wherein the second therapeutic agent is pembrolizumab. 212. The method of embodiment 210, wherein the second therapeutic agent is nivolumab. 213. The method of embodiment 210, wherein the second therapeutic agent is cemiplimab. 214. The method of embodiment 210, wherein the second therapeutic agent is dostarlimab. 215. The checkpoint inhibitor therapy targets PDL1, according to embodiment 209-209. 15. The method of any one of claims 14 to 14. 216. The method of embodiment 215, wherein the second therapeutic agent is atezolizumab. 217. The method of embodiment 215, wherein the second therapeutic agent is avelumab. 218. The method of embodiment 215, wherein the second therapeutic agent is durvalumab. 219. The checkpoint inhibitor therapy targets CTLA4, from embodiment 209. 218. The method of any one of claims 218 to 218. 220. The method of embodiment 219, wherein the second therapeutic agent is ipilimumab. 221. The checkpoint inhibitor therapy targets TIGIT, from embodiment 209. 220. The method of any one of claims 220 to 220. 222. The method of embodiment 221, wherein the second therapeutic agent is etigilimab. 223. The method of embodiment 221, wherein the second therapeutic agent is tiragolumab. 224. The method of embodiment 221, wherein the second therapeutic agent is AB154. 225. The checkpoint inhibitor therapy targets LAG3, according to embodiments 209-209. 24. The method of any one of claims 24 to 26. 226. The method of embodiment 225, wherein the second therapeutic agent is LAG525. 227. The method of embodiment 225, wherein the second therapeutic agent is Sym022. 228. The method of embodiment 225, wherein the second therapeutic agent is leratolimab. 229. The method of embodiment 225, wherein the second therapeutic agent is TSR-033. 230. The checkpoint inhibitor therapy of embodiment 209-209, wherein the checkpoint inhibitor therapy targets OX40. 29. The method of any one of 29. 231. The method of embodiment 230, wherein the second therapeutic agent is MEDI6469. 232. The method of embodiment 230, wherein the second therapeutic agent is PF-04518600. . 233. The method of embodiment 230, wherein the second therapeutic agent is BMS 986178. 234. The checkpoint inhibitor therapy of embodiment 209-209, wherein the checkpoint inhibitor therapy targets CD40. 33. The method of any one of claims 33 to 33. 235. The method of embodiment 234, wherein the second therapeutic agent is cericlerumab. 236. The method of embodiment 234, wherein the second therapeutic agent is CP-870,893. 237. The method of embodiment 234, wherein the second therapeutic agent is APX005M. 238. Checkpoint inhibitor therapy targets VISTA, from embodiment 209 237. The method of any one of claims 237 to 237. 239. The method of embodiment 238, wherein the second therapeutic agent is HMBD-002. 240. The method of any one of embodiments 202 to 23, wherein the second therapeutic agent is a chimeric antigen receptor (CAR). 10. The method of any one of 9. 241. Any one of embodiments 202 to 240, wherein the combination therapy includes adoptive T cell therapy. The method described below. 242. The method of embodiment 241, wherein the adoptive T cell therapy is autologous T cell therapy. 243. Any of embodiments 202 to 242, wherein the combination therapy includes oncolytic virus therapy. The method according to any one of claims 1 to 5. 244. Any of embodiments 202 to 243, wherein the combination therapy includes dendritic cell vaccine therapy. 1. The method according to claim 1. 245. Any of embodiments 202 to 244, wherein the combination therapy includes STING agonist therapy. The method according to any one of claims 1 to 4. 246. Any of embodiments 202 to 245, wherein the combination therapy includes TLR agonist therapy. 1. The method according to claim 1. 247. Any one of embodiments 202 to 246, wherein the combination therapy includes chemotherapy. How to do it. 248. The second therapeutic agent is an antimetabolite, alkylating agent, anthracycline, or antimicrotubule agent. agents, platinum compounds, taxanes, topoisomerase inhibitors, or vinca alkaloids , The method described in embodiment 247. 249. The method of embodiment 248, wherein the second therapeutic agent is an antimetabolite. 250. The method of embodiment 249, wherein the antimetabolite is 5-fluorouracil. 251. The method of embodiment 249, wherein the antimetabolite is gemcitabine. 252. The method of embodiment 249, wherein the antimetabolite is methotrexate. 253. The method of embodiment 248, wherein the second therapeutic agent is an alkylating agent. 254. The method of embodiment 253, wherein the alkylating agent is cyclophosphamide. 255. The method of embodiment 253, wherein the alkylating agent is dacarbazine. 256. The method of embodiment 253, wherein the alkylating agent is mechlorethamine. 257. The method of embodiment 253, wherein the alkylating agent is diaziconazole. 258. The method of embodiment 253, wherein the alkylating agent is temozolomide. 259. The method of embodiment 248, wherein the second therapeutic agent is an anthracycline. 260. The method of embodiment 259, wherein the anthracycline is doxorubicin. 261. The method of embodiment 259, wherein the anthracycline is epirubicin. 262. The method of embodiment 248, wherein the second therapeutic agent is an anti-microtubule agent. 263. The method of embodiment 262, wherein the anti-microtubule agent is vinblastine. 264. The method of embodiment 248, wherein the second therapeutic agent is a platinum compound. 265. The method of embodiment 264, wherein the platinum compound is cisplatin. 266. The method of embodiment 264, wherein the platinum compound is oxaliplatin. 267. The method of embodiment 248, wherein the second therapeutic agent is a taxane. 268. The method of embodiment 267, wherein the taxane is paclitaxel. 269. The method of embodiment 267, wherein the taxane is docetaxel. 270. The method of embodiment 248, wherein the second therapeutic agent is a topoisomerase inhibitor. . 271. The method of embodiment 270, wherein the topoisomerase inhibitor is etoposide. 272. The method according to embodiment 270, wherein the topoisomerase inhibitor is mitoxantrone. method. 273. The method of embodiment 248, wherein the second therapeutic agent is a vinca alkaloid. 274. The method of embodiment 273, wherein the vinca alkaloid is vincristine. 275. Any one of embodiments 202 to 274, wherein the combination therapy includes intratumoral CpG therapy. The method described in paragraph . 276. From embodiment 202, wherein the second therapeutic agent is an ADC with a cytotoxic payload. 275. The method of any one of claims 275. 277. The ADC according to embodiment 276, wherein the ADC with a cytotoxic payload targets FAP. How to post. 278. The method of embodiment 277, wherein the second therapeutic agent is OMTX705. 279. Any one of embodiments 202 to 278, wherein the second therapeutic agent is a cytokine. The method described in paragraph . 280. The method of embodiment 279, wherein the cytokine is IL2. 281. The method of embodiment 279, wherein the cytokine is IL12. 282. The method of embodiment 279, wherein the cytokine is IFN-α. 283. The method of embodiment 279, wherein the cytokine is IFN-γ. 284. Any one of embodiments 202 to 283, comprising treating the subject with a combination therapy. The method described in paragraph . 285. Any of embodiments 202 to 284, comprising administering a second therapeutic agent to the subject. 1. The method according to claim 1. 286. A method for promoting dedifferentiation of myofibroblasts into resting fibroblasts, comprising: Fibroblasts are treated with the targeted drug conjugate of any one of embodiments 1 to 120, or with a pharmaceutical composition according to any one of embodiments 121 to 149. method. 287. A method for promoting dedifferentiation of activated fibroblasts into resting fibroblasts. Thus, the activated fibroblasts are treated with the targeting agent of any one of embodiments 1 to 120. 149. The method includes: 288. Promotes dedifferentiation of myofibroblast-transforming fibroblasts into quiescent fibroblasts 121. A method for the treatment of fibroblasts that transition into myofibroblasts, comprising the steps of: 149. The targeted drug conjugate of any one of embodiments 121 to 149. with the pharmaceutical composition described. 289. Any of embodiments 286 to 288, wherein dedifferentiation comprises a decrease in smooth muscle actin expression. The method according to any one of claims 1 to 5. 290. A method for promoting apoptosis of myofibroblasts, comprising: The targeted drug conjugate of any one of embodiments 1 to 120, or embodiments 121 to 123. 50. A method comprising contacting a subject with a pharmaceutical composition according to any one of claims 49 to 50. 291. A method for promoting apoptosis of activated fibroblasts, comprising: The blast cells are treated with the targeted drug conjugate of any one of embodiments 1 to 120, or with the pharmaceutical composition of any one of Forms 121 to 149. . 292. A method for promoting apoptosis of fibroblasts that transition to myofibroblasts, comprising: 121. The target fibroblasts that transition to fibroblasts are selected from the group consisting of: 149. A method comprising: 293. Embodiment 286, in which the contacting is performed in vivo in a subject. 292. The method of claim 1, wherein 294. Embodiment 29, comprising administering a targeted drug complex or pharmaceutical composition to a subject. The method described in 3. 295. Compound C or a salt thereof. 296. Compound N or its salt. 297. A targeted drug conjugate comprising compound C operably linked to a targeting moiety. 298. A targeted drug conjugate comprising compound N operably linked to a targeting moiety. 299. Compound C, conjugated to a linker. 300. Compound N, conjugated to a linker.

[0307] While various specific embodiments are illustrated and described, it is to be understood that these embodiments do not depart from the spirit and scope of the present disclosure. It will be understood that various changes may be made without departing from the spirit and scope of the invention.

[0308] 8. Citation of References All publications, patents, patent applications, and other references cited in this application are hereby incorporated by reference in their entirety. Each patent, patent application, or other document is individually incorporated by reference for all purposes. and to the same extent as if set forth herein, are incorporated by reference in their entirety for all purposes. between the teachings of this specification and one or more of the references incorporated into this disclosure In the event of a conflict, the teachings of the present specification are intended.

Claims

1. Myofibroblasts, activated fibroblasts, fibroblasts transitioning to myofibroblasts, or any of these operably linked to a targeting moiety that binds to a cell surface molecule expressed on the surface of these combinations.

1. A targeted drug conjugate comprising an ALK5 inhibitor, wherein the ALK5 inhibitor is N-methyl. 2-(4-(4-(3-(6-methylpyridin-2-yl)-1H-pyrazole-4 -yl)pyridin-2-yl)phenoxy)ethan-1-amine, body.

2. 10. The method of claim 9, wherein the ALK5 inhibitor is linked to the targeting moiety via a linker.

2. The targeted drug complex described in 1.

3. 3. The targeted drug conjugate of claim 2, wherein the linker is a PEG-containing linker. body.

4. The ALK5 inhibitor is linked to the targeting molecule via a non-cleavable or cleavable linker.

4. The targeted drug conjugate of claim 1, wherein the targeted drug conjugate is linked to a moiety.

5. The ALK5 inhibitor is N-maleimidomethylcyclohexane-1-carboxylate , maleimidocaproyl, or mercaptoacetamidocaproyl linkers 5. The targeted drug of claim 4, which is linked to the targeting moiety via a cleavable linker. Complex.

6. The ALK5 inhibitor may be a dipeptide linker, a disulfide linker, or a hydrazoline linker.

4. The method of claim 3, wherein the targeting moiety is linked to the targeting moiety via a cleavable linker. The targeted drug complex described in

7. The linker may be a protease-sensitive valine-citrulline dipeptide linker, glutamate, or The linker is a thione-sensitive disulfide linker or an acid-sensitive disulfide linker. Item 7. The targeted drug complex described in item 6.

8. The target of claim 7, wherein the linker is a valine-citrulline dipeptide linker. Targeted drug complex.

9. The targeted drug complex of claim 6 , wherein the linker is a disulfide linker.

10. the ALK5 inhibitor comprises one or more cysteine ​​residues on the targeting moiety; and is conjugated via one or more lysine residues on the targeting moiety, and optionally The targeted drug conjugate of any one of claims 1 to 9, which is conjugated via a carrier.

11. the average number of ALK5 inhibitor molecules per targeting moiety molecule is in the range of 2 to 8. Item 11. The targeted drug complex according to any one of items 1 to 10.

12. 12. The method of claim 1, wherein the targeting moiety comprises an antibody or an antibody fragment. The targeted drug conjugate described above.

13. 13. The targeted drug conjugate of claim 12, wherein the targeting moiety comprises an antibody.

14. The targeted drug conjugate of claim 13 , wherein the antibody is a monoclonal antibody.

15. 15. The targeted drug conjugate of claim 14, wherein the antibody is human or humanized.

16. 13. The targeted drug conjugate of claim 12, wherein the targeting moiety comprises an antibody fragment.

17. The antibody fragment may be Fab, Fab', F(ab') ２ , Fv, scFv, dsFv, or or a single domain antibody.

18. 17. The targeted drug of claim 16, wherein the antibody fragment is a fragment of a human or humanized antibody. Complex.

19. The cell surface molecules include FAP, PDGFR-β, FGFR1, PPAR-γ, FSP1. , GFAP, fascin, αvβ6, CD147, CXCR4, αvβ6, AXL, or 19. The targeted drug conjugate of any one of claims 1 to 18, which is MERTK.

20. 20. The targeted drug conjugate of claim 19, wherein the cell surface molecule is FAP.

21. 21. The targeted drug conjugate of any one of claims 1 to 20, and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising a carrier comprising:

22. 21. A target according to any one of claims 1 to 20 for use in a method for treating fibrosis.

22. A targeted drug conjugate or pharmaceutical composition according to claim 21.

23. The fibrosis is pulmonary fibrosis, liver fibrosis, kidney fibrosis, cardiac fibrosis, skin fibrosis, or esophageal fibrosis.

23. The targeted drug conjugate or pharmaceutical composition for use according to claim 22, which is fibrosis.

24. 23. The target for use according to claim 22, wherein the fibrosis is idiopathic pulmonary fibrosis (IPF). Targeted drug conjugates or pharmaceutical compositions.

25. 25. Any of claims 22 to 24, wherein the targeted drug conjugate is administered as a monotherapy. A targeted drug conjugate or pharmaceutical composition for use according to claim 1.

26. 23. The method of claim 22, wherein the targeted drug conjugate is administered as part of a combination therapy regimen.

25. A targeted drug conjugate or pharmaceutical composition for use according to any one of claims 24.

27. 27. The method of claim 26, wherein the combination therapy regimen comprises pirfenidone or nintedanib. A targeted drug conjugate or pharmaceutical composition for the above-described use.

28. 21. A method for treating systemic sclerosis according to any one of claims 1 to 20. The targeted drug complex described above, or the pharmaceutical composition described in claim 21.

29. 21. A target according to any one of claims 1 to 20 for use in a method of treating cancer.

22. The compounded drug conjugate or pharmaceutical composition of claim 21.

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