Synthetic bifunctional decomposition agent for integrins

Abstract

A composition and method for the selective degradation of integrins, for example, used in the treatment of fibrosis, is provided. The compound of interest is a bifunctional integrin degradation molecule comprising a TG2 binding moiety linked to an integrin binding moiety.

Description

[Background technology]

[0001] Integrins, major extracellular matrix receptors, are involved in a variety of fibrotic diseases, including renal fibrosis, cardiac fibrosis, hepatic fibrosis, pulmonary fibrosis, cystic fibrosis, and scleroderma fibrosis. In preclinical studies and active clinical trials, 20+ αV integrin inhibitors targeting pulmonary, hepatic, and renal fibrotic diseases, as well as ocular diseases, exist. While αV integrin inhibitors are not yet approved by the FDA, several drugs have progressed to Phase II clinical trials.

[0002] Transglutaminase 2 (TG2) has been shown to interact with αVβ1, αVβ3, and αVβ5 integrins. Furthermore, its enzymatic activity is upregulated in pathological conditions such as renal fibrosis and pulmonary fibrosis. Three of the five αV integrins (αVβ1, αVβ3, and αVβ5) are expressed in renal and pulmonary fibroblasts, which are important cell types that produce extracellular matrix fibers in renal and pulmonary fibrosis. αVβ6 was initially thought to be important in renal and pulmonary fibrosis, but it was later found that αVβ6 is expressed in epithelial cells but not in fibroblasts. Independent studies using human primary pulmonary and primary renal fibroblasts, as well as rat primary renal fibroblasts, have shown that αVβ5 and αVβ3 are highly expressed in fibroblasts and are upregulated during fibrosis induction. Overexpression of αV integrins, including αVβ3 and αVβ5, in human fibroblasts promotes potential TGFβ1 activation, which plays a crucial role in fibrosis progression. Importantly, TG2 activation also promotes potential TGFβ1 activation and has been identified as a promising drug target for diseases such as renal fibrosis and pulmonary fibrosis.

[0003] Fibrosis and other conditions associated with integrin dysregulation remain significant medical challenges. The development of treatments is of interest and will be addressed herein. [Overview of the project]

[0004] A bifunctional integrin degradation molecule is disclosed, comprising a TG2-binding moiety linked to an integrin-binding moiety. The integrin-binding moiety binds to the integrin of interest, while the TG2-binding moiety is a substrate or inhibitor of TG2. Selective TG2 binding induces receptor-mediated endocytosis of the bifunctional molecule, along with both the bound TG2 and integrin (Figure 1). This endocytosis occurs via the cell surface receptor LRP-1. Upon entering the endolysosome compartment, the bound TG2 and integrin are released and degraded. These bifunctional molecules may be called LYTACs (lysosome-targeted chimeras).

[0005] In some embodiments, the integrin-binding moiety selectively binds to the integrin of interest. In some embodiments, the integrin-binding moiety is selective for a class of integrin. In some embodiments, the integrin-binding moiety is selective for one or more αV-containing integrins, e.g., αVβ1, αVβ3, αVβ5, αVβ6, αVβ8. The integrin of interest includes human integrin proteins. The integrin-binding moiety may include an RGD motif or an RGD mimetic. The integrin-binding moiety may include antibodies, nanobodies, aptamers, etc., or binding domains derived therefrom; peptides; cyclic peptides; synthetic molecules, etc., as known in the art.

[0006] In one embodiment, the TG2 binding portion comprises or consists of a polypeptide comprising a pentapeptide sequence: Pro-XZR I, During the ceremony: X is selected from amino acids that engage with TG2 via the formation of a covalent enzyme-compound intermediate, including but not limited to glutamine, α-diazoketones, α-halo-ketones, αβ-unsaturated carbonyl compounds, and αβ-unsaturated sulfones; Z is Selected from TIFF2026525300000001.tif23161 and dipeptide YP, where P is proline and Y is any amino acid, including but not limited to primary / secondary amines, alcohols, and carboxylic acids, including unnatural amino acids having readily addable functional groups; R is a natural or unnatural aromatic amino acid, such as tyrosine (Y), phenylalanine (F), and tryptophan (W), naphthylalanine, etc.; the C-terminus of the pentapeptide may be a carboxylic acid, ester, or amide.

[0007] In some embodiments, the TG2 binding moiety comprises or consists of the peptide sequence Ac-PQLPF-NH2; or a modified peptide in which a reactive glutamine residue is replaced with an electrophilic attack group. In situations where the TG2 binding moiety is an enzyme inhibitor, TG2 inhibition itself may be clinically useful, as TG2 is a drug target for renal fibrosis and pulmonary fibrosis.

[0008] The TG2 binding portion of formula I is linked to the integrin binding portion at the C-terminus of the pentapeptide, and / or to Z via a linker, via covalent direct conjugation, via non-covalent high-affinity pairing, etc.

[0009] In one embodiment, the bifunctional molecule has a structure selected from formulas II, III, and IV, as shown below.

[0010] [ka]

[0011] [ka]

[0012] R in the formula 1 These include Cl, Br, I, OSO2CF3, etc. R in the formula 2These include N(CH3)2, OCH3;NH2, CH3, etc.

[0013] [ka]

[0014] Y is any amino acid, or The filename is TIFF2026525300000005.tif27161; R is an aromatic amino acid, such as phenylalanine, tyrosine, or tryptophan; Link is a linker, for example, PEG n (n=1~10), PEG n Small organic linkers such as diamines, piperazines, 4-aminopiperidines, 3,9-diazaspiro[5.5]undecane, linkers suitable for click chemistry, hydrocarbons, or GGGGS, G n (GGS) n These are peptide linkers; and The integrin ligand is the integrin-binding moiety disclosed above.

[0015] In certain specific embodiments, the bifunctional integrin degradation molecule has the following structure.

[0016] [ka]

[0017] In certain specific embodiments, the bifunctional integrin degradation molecule has the following structure.

[0018] [ka]

[0019] In the formulas, n = 0 to 3; T is a TG2 bond portion identified by any of the formulas I to IV disclosed above, which is coupled to the integrin bond portion via an amide bond.

[0020] In certain specific embodiments, the bifunctional integrin degradation molecule has the following structure.

[0021] [ka]

[0022] In the formulas, T is a TG2 bond portion identified by any of the formulas I to IV disclosed above, which is coupled to the integrin bond portion via an amide bond.

[0023] In certain specific embodiments, the bifunctional integrin degradation molecule has the following structure.

[0024] [ka]

[0025] In the formulas, X = O, NH, or CH2; T is a TG2 bond moiety identified by any of the formulas I to IV disclosed above, which is coupled to the integrin bond moiety via an amide bond.

[0026] In certain specific embodiments, the bifunctional degrading agent consists of an αVβ5-specific integrin-binding moiety having the following structure.

[0027] [ka]

[0028] In the formula, T1 is H or a TG2 bond, and T2 is H or a TG2 bond identified by any of the formulas I to IV disclosed above, wherein one of T1 and T2 is a TG2 bond, and usually only one of T1 and T2 is a TG2 bond.

[0029] In some embodiments, therapeutic formulations are provided comprising the difunctional integrin degradation molecule of the present disclosure and pharmaceutically acceptable excipients. The difunctional integrin degradation molecule can selectively bind to and degrade αV integrin. αV integrin may be αVβ5 integrin. The formulation may be a unit-dose formulation. Alternatively, the unit-dose formulation may comprise a lyophilized difunctional integrin degradation molecule.

[0030] In some embodiments, a method for treating fibrosis is provided, which comprises administering an effective dose of the bifunctional integrin-degrading molecule of the present disclosure to an individual in need. The individual may be monitored during treatment to determine whether the treatment is effective in reducing or preventing fibrosis compared to an untreated individual.

[0031] In some embodiments, fibrosis is renal fibrosis. When fibrosis is renal fibrosis, it may be caused by hypertension, diabetes, obstruction, or infection. In some embodiments, fibrosis is pulmonary fibrosis, such as idiopathic pulmonary fibrosis. In some embodiments, fibrosis is intestinal fibrosis, a common complication of inflammatory bowel disease. In some embodiments, fibrosis is selected from cutaneous fibrosis, primary sclerosing cholangitis, cirrhosis, non-alcoholic steatohepatitis (NASH), and non-alcoholic fatty liver disease (NAFLD). In some embodiments, fibrosis is associated with cancer and tumor growth, i.e., tumor-associated histofibrosis, including, but not limited to, lung cancer, liver cancer, skin cancer, sarcoma, osteosarcoma, etc. In other embodiments, fibrosis is associated with chronic inflammation or injury.

[0032] Compositions and kits for carrying out the methods disclosed herein are also provided.

[0033] This invention will be best understood from the following detailed description in conjunction with the accompanying drawings. It should be emphasized that, in general implementation, various features in the drawings are not to scale. Conversely, the dimensions of various features are arbitrarily enlarged or reduced for clarity. The drawings include the following figures. [Brief explanation of the drawing]

[0034] [Figure 1] A schematic diagram of integrin degradation. [Figure 2] Compound 100 degrades cell surface integrin αvβ5. (A) Dose response to 100. Cell surface integrins are reduced with treatment up to approximately 1 μM; as is common with bifunctional molecules, higher concentrations result in less efficient endocytosis. (B) Quantification of (A). (C) Time dependence of treatment with 100. (D) Treatment with integrin ligand RGDVF alone does not result in a corresponding reduction in surface integrin levels (10 μM RGDVF, 6-hour treatment). (E) RAP treatment rescues some of the membrane integrin levels (10 μM 100, 6-hour treatment). [Figure 3] Compound 101 degrades cell surface integrin αvβ5. (A) Dose response to 101. Cell surface integrins are reduced with treatment up to approximately 1 μM; as is common with bifunctional molecules, higher concentrations result in less efficient endocytosis. (B) Quantification of (A). Note: 0 μM conditions are the same for the data shown for 100 and 101. [Figure 4] Antifibrotic effects of compound 101. (A) Treatment of human lung fibroblasts (IMR90 cells) with 0, 1, 5, and 20 ng / mL TGFβ1 for 2 days induced αSMA expression, which is used as a molecular marker of fibroblast-to-myofibroblast transition. (B) Treatment of IMR90 cells with 20 ng / mL TGFβ1 in the presence of 0, 1, 3, 10, or 30 μM 101 showed a significant decrease in αSMA expression, with the greatest effect observed at 3 μM 101. The multiplicative change represents the normalized αSMA intensity relative to the mean in the absence of 101. (C) Integrin ligand alone, RGDVF, did not affect αSMA expression at any of the test concentrations. [Figure 5]Effects of compound 101 on macrophages derived from the RAW264.7 cell line. (A) Integrin β5 expression increased when M0 macrophages were differentiated into M2 macrophages in the presence of 20 ng / mL of IL-4. Treatment with 1, 3, and 10 μM of 101 resulted in the degradation of integrin β5, as assessed by Western blotting. RGDVF alone did not have a comparable effect. Treatment with 101 also reduced both (B) total released TGFβ and (C) the active form of TGFβ produced by these macrophages, whereas RGDVF did not show a corresponding effect. [Figure 6] Effects of 103 on primary human lung fibroblasts. Integrin β5 expression in these cells increased for 2 days in the presence of 20 ng / mL of TGFβ1. Treatment with 103 for 24 hours induced dose-dependent changes in integrin β5. Integrin β5 degradation reached a maximum at a 1 μM 103 concentration, approximately to pre-TGFβ levels. At higher concentrations, integrin β5 increased, likely due to a well-established hook effect related to the bifunctional molecule. [Figure 7] The long-term sustained effect of 103 on integrin degradation was investigated. The time-dependent degradation was assessed by adding 103 to cultures of primary human lung fibroblasts for 1, 2, 4, 8, and 16 hours, followed by rinsing and cell analysis 16 hours later. Treatment with 103 for only 1–2 hours was sufficient to maintain reduced integrin levels for at least 16 hours. [Figure 8] Effect of compound 105 on αvβ5 integrin degradation in primary human lung fibroblasts. Cells were treated with 20 ng / mL TGFβ1 for 2 days to induce myofibroblast migration from fibroblasts. Subsequently, treatment with compound 5 for 24 hours induced integrin β5 degradation. Integrin β5 degradation was maximized in the presence of 3 μM of the compound. At higher degradation agent concentrations of 10 and 100 μM, the β5 degradation effect decreased. This is due to the well-established hook effect associated with the bifunctional molecule. Experimental details are similar to those shown in Figure 6. [Modes for carrying out the invention]

[0035] definition The present invention is not limited to the specific methodologies, products, apparatus, and factors described, and it should be understood that such methods, apparatus, and formulations may naturally vary. It should also be understood that the terms used herein are for the sole purpose of describing specific embodiments and are not intended to limit the scope of the present invention, which is limited only by the appended claims.

[0036] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” refer to multiple subjects unless otherwise explicitly stated in the context. Therefore, for example, a reference to “candidate drug” refers to one or a mixture of such candidates, and a reference to “method” includes references to equivalent processes and methods known to those skilled in the art.

[0037] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as those generally understood by those skilled in the art in which the invention pertains. All publications referenced herein are incorporated herein by reference for the purpose of describing and disclosing devices, formulations and methodologies described in those publications and that may be used in connection with the inventions described herein.

[0038] Where a range of values ​​is provided, unless otherwise explicitly stated in the context, each value between the upper and lower limits of that range, up to one-tenth of the lower limit, and any other values ​​or values ​​in between within that stated range are understood to be included in the invention. The upper and lower limits of these smaller ranges may independently be included within smaller ranges and are also included in the invention, subject to any specifically excluded limits within the stated range. Where a stated range includes one or both limits, the range excluding one or both of those limits is also included in the invention.

[0039] The following description includes numerous specific details to provide a more complete understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be carried out without one or more of these specific details. In other examples, well-known features and procedures familiar to those skilled in the art are omitted to avoid obscuring the invention.

[0040] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple references unless otherwise explicitly stated in the context. Therefore, for example, a reference to “a cell” includes multiple such cells, and a reference to “the peptide” includes one or more peptides and their equivalents, such as polypeptides known to those skilled in the art.

[0041] The publications discussed herein are provided solely for their disclosure prior to the filing date of this application. Nothing herein should be construed as an acknowledgment that the present invention has no prior rights to such publications by prior art. Furthermore, the publication dates presented may differ from the actual publication dates and may need to be independently verified.

[0042] The terms “individual,” “host,” “subject,” and “patient” are used interchangeably herein and are not limited to any one term, but refer to animals including humans and non-human primates, e.g., monkeys and humans; rodents, e.g., rats and mice; Bovidae; Equidae; Sheep; Felidae; Canidae; etc. “Mammal” means one or more members of any mammalian species, including, for example, Canidae; Felidae; Equidae; Bovidae; Sheep; Rodents, etc., and primates, e.g., non-human primates and humans. Non-human animal models, e.g., mammals, e.g., non-human primates, mice, Lagomorpha, etc., may be used in experimental studies.

[0043] As used herein, the terms “determine,” “measure,” “evaluate,” and “assay” are used interchangeably and include both quantitative and qualitative determinations.

[0044] The term “diagnosis” is used herein to mean the identification of a molecular or pathological condition, disease or symptom in a subject, individual or patient.

[0045] The term “prognosis” is used herein to refer to the prediction of the likelihood of death or disease progression, including recurrence, transmission, and drug resistance, in a subject, individual, or patient. The term “prediction” is used herein to refer to the act of predicting or estimating, based on observation, experience, or scientific reasoning, the likelihood that a subject, individual, or patient will experience a particular event or clinical outcome. For example, a physician may attempt to predict the likelihood of a patient surviving.

[0046] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to mammals being evaluated for and / or treated for treatment. In one embodiment, the mammal is human. Thus, the terms “subject,” “individual,” and “patient” encompass individuals having fibrosis, including but not limited to tumor fibrosis, cardiac fibrosis, hepatic fibrosis, renal fibrosis, pulmonary fibrosis, skin scars and keloids, and Alzheimer’s disease. While the subject may be human, it may also include other mammals, particularly those useful as laboratory models for human diseases, such as mice and rats.

[0047] Integrins are a family of cell surface receptors that play a crucial role in mediating cell adhesion to the extracellular matrix. In mammals, integrins have 18 distinct α-subunits and 8 distinct β-subunits, forming 24 distinct αβ heterodimers. Five members of the integrin family, the αV family (αVβ1, αVβ3, αVβ5, αVβ6, and αVβ8), play important roles in the progression of fibrosis. For example, gene deletion of αV integrins or pan-αV blockade using small molecule drugs dramatically reduces fibrosis in multiple organs, including the liver, lungs, and kidneys.

[0048] One key mechanism by which integrins interact with their ligands involves the recognition of a specific amino acid sequence known as the Arg-Gly-Asp (RGD) motif. The RGD motif is commonly found on various extracellular matrix proteins, such as fibronectin and vivonectin, and acts as a binding site for integrins, particularly the αvβ1, αvβ3, αvβ5, and α5β1 integrin subtypes. When an integrin receptor encounters the RGD motif, it undergoes a conformational change that allows it to bind firmly to the motif, leading to the initiation of a signaling cascade that regulates cell adhesion and various cellular processes, including migration, proliferation, and survival.

[0049] Adhesion proteins containing the RGD sequence include fibronectin, vitronectin, osteopontin, fibrinogen, von Willebrand factor, thrombospondin, laminin, enterin, tenascin, milk fat globule-epidermal growth factor 8 (MFEG8), TGFβ1 / 3 latency-related peptide (LAP-TGFβ1 / 3), and bone sialoproteins. The RGD sequence exhibits specificity for approximately half of 20 known integrins, including α5β1, α8β1, αvβ1, αvβ3, αvβ5, αvβ6, αvβ8, and αiiibβ3 integrins, and to a lesser extent, for α2β1, α3β1, α4β1, and α7β1 integrins.

[0050] The compounds of this disclosure include an integrin-binding moiety. The moiety may include, for example, a peptide ligated to either or both ends of an RGD sequence, such as the structure RGDXX, XXRGDXX, etc., where X is any amino acid. The identity of residue "X" can be varied in that X residues adjacent to the binding motif (RGD, RYD, etc.) combine to provide a specific structure that selectively recognizes a ligand. Alternatively, the integrin-binding moiety may include antibodies, nanobodies, aptamers, etc., or binding domains derived therefrom; cyclic peptides; synthetic molecules, etc., as known in the art.

[0051] In some embodiments, the integrin-binding moiety selectively binds to the integrin of interest. In some embodiments, the integrin-binding moiety is selective for a class of integrins. In some embodiments, the integrin-binding moiety is selective for one or more αV-containing integrins, e.g., αVβ1, αVβ3, αVβ5, αVβ6, αVβ8. The integrin of interest includes human integrin proteins. The integrin-binding moiety may contain an RGD motif. The integrin-binding moiety may contain antibodies, nanobodies, aptamers, etc., or binding domains derived therefrom; peptides; cyclic peptides; synthetic molecules, etc., as known in the art.

[0052] In some embodiments, the integrin binding site is selective for binding to αVβ5. The binding site of interest includes RGD-containing peptides, e.g., peptides of formula RGDX1X2 (wherein X is any amino acid, e.g., RGDVF, RGDNF, RGDNY, etc.); small molecules, e.g., compounds reported by Lippa et al.; cyclic peptides, e.g., c(RGDfV), c(Arg-Gly-Asp-d-Phe-[NMe]Val); antibodies, nanobodies, aptamers, etc. For example, see Yamada et al. (2022) FASEB J.36(7):e22389; Lippa et al. (2020) Eur.J.Med.Chem.208,112719; Kapp et al. (2017) Sci Rep.11;7:39805; Belvisi et al. (2005) Mol Cancer Ther.4(11):1670-80, Stuiver and Smith (1995) Hybridoma 14(6):545-50, etc. Each of these is specifically incorporated herein by reference.

[0053] Examples of target pan-αv integrin-binding monoclonal antibodies include intetumumab (O'Day et al, 2011), abituzumab (Hussain et al, 2016), Ab-31 (Zhang et al, 2021), and 17-E6 (Mitjans et al, 1998). Examples of target αVβ5 integrin-selective antibodies include the monoclonal antibodies P1F6 (Abcam #ab177004), P5H9 (BioTechne #MAB2528), and ALULA (BD Biosciences #AB2739376).

[0054] The target peptidomethyl integrin binding moieties include peptides of formula RGDX1X2, such as RGDVF, RGDNF, and RGDNY. Other target linear peptides include, for example, RWrNK (Zhang et al, 2019) and members of the disintegrin family (Oliveira et al, 2022).

[0055] Examples of target cyclic integrin-conjugated peptides include c(RGDfV) and c(Arg-Gly-Asp-d-Phe-[NMe]Val).

[0056] Examples of small molecules that bind to multiple target αV-integrins include MK-0429 (Coleman et al, 2004), bexotegrast, CWHM-12, and ST1646.

[0057] Small molecules that selectively bind to αVβ5 integrins include compounds described by Lippa, et al (2020).

[0058] Transglutaminase 2 (TG2) is a member of the human transglutaminase family of enzymes, abundantly expressed in various tissues and found both intracellularly and extracellularly (Lorand and Graham, 2003). It possesses catalytic activity to deamidate glutamine side chains on substrate peptides or proteins, or to crosslink them with biomolecules or protein-bound amines. An example of a high-affinity TG2 substrate is Sequence ID No. 1, LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF, a 33-mer gluten peptide that reveals an HLA-DQ2 epitope upon TG2-catalyzed deamidation at the underlined glutamine residue. Upon covalent bonding with the Cys277 residue of the active site of human TG2, several TG2 ligands promote efficient receptor-mediated endocytosis in a manner dependent on the activity of low-density lipoprotein receptor-associated protein 1 (LRP1) (Loppinet et al., 2023 Cell Chemical Biology 30, 55-68, specifically incorporated herein by reference). Receptor-mediated endocytosis results in the transport of TG2 and its binding ligand partners to lysosomes, where these substances are enzymatically degraded (Figure 1).

[0059] In one embodiment, the TG2 binding portion comprises or consists of a polypeptide comprising a pentapeptide sequence: Pro-XZR I, During the ceremony X is selected from amino acids that engage with TG2 via the formation of a covalent enzyme-compound intermediate, including but not limited to glutamine, α-diazoketones, α-halo-ketones, αβ-unsaturated carbonyl compounds, and αβ-unsaturated sulfones; Z is Selected from TIFF2026525300000011.tif23161 and dipeptide YP, where P is proline and Y is any amino acid, including but not limited to primary / secondary amines, alcohols, and carboxylic acids, including non-natural amino acids having readily addable functional groups; R is a natural or unnatural aromatic amino acid, such as tyrosine (Y), phenylalanine (F), and tryptophan (W), naphthylalanine, etc.; the C-terminus of the pentapeptide may be a carboxylic acid, ester, or amide. In some embodiments, the TG2 binding moiety comprises or consists of the peptide sequence Ac-PQLPF-NH2; or a modified peptide in which a reactive glutamine residue is replaced with an electrophilic attack group.

[0060] The TG2 binding portion of formula I is linked to Z via the integrin binding portion at the N-terminus or C-terminus of the pentapeptide, and / or via a linker, via covalent direct conjugation, or via non-covalent high-affinity pairing. In one embodiment, the bifunctional molecule has a structure selected from the following:

[0061] [ka]

[0062] [ka]

[0063] R 1include Cl, Br, I, OSO2CF3, etc.; R 2 include N(CH3)2, OCH3, NH2, CH3, etc.

[0064]

Chem.

[0065] Y is any amino acid, or TIFF2026525300000015.tif27161; R is an aromatic amino acid, such as phenylalanine, tyrosine, tryptophan, etc.; Link is a linker, such as PEG n PEG n small organic linkers such as diamines, piperazine, 4-aminopiperidine, 3,9-diazaspiro[5.5]undecane, linkers suitable for click chemistry, hydrocarbons, etc., or peptide linkers such as GGGGS, G n , (GGS) n etc.

[0066] The integrin ligand is the integrin-binding moiety disclosed above.

[0067] In certain specific embodiments, the bifunctional integrin-degrading molecule has the following structure.

[0068]

Chem.

[0069] In certain specific embodiments, the bifunctional integrin-degrading molecule has the following structure.

[0070]

Chem.

[0071] In the formulas, n = 0 to 3; T is a TG2 bond portion identified by any of the formulas I to IV disclosed above, which is coupled to the integrin bond portion via an amide bond.

[0072] In certain specific embodiments, the bifunctional integrin degradation molecule has the following structure.

[0073] [ka]

[0074] In the formulas, T is a TG2 bond portion identified by any of the formulas I to IV disclosed above, which is coupled to the integrin bond portion via an amide bond.

[0075] In certain specific embodiments, the bifunctional integrin degradation molecule has the following structure.

[0076] [ka]

[0077] In the formulas, X = O, NH, or CH2; T is a TG2 bond moiety identified by any of the formulas I to IV disclosed above, which is coupled to the integrin bond moiety via an amide bond.

[0078] In certain specific embodiments, the bifunctional degrading agent consists of an αVβ5-specific integrin-binding moiety having the following structure.

[0079] [ka]

[0080] In the formula, T1 is H or a TG2 bond, and T2 is H or a TG2 bond identified by any of the formulas I to IV disclosed above, wherein one of T1 and T2 is a TG2 bond, and usually only one of T1 and T2 is a TG2 bond.

[0081] The term "amino acid" includes both naturally occurring and synthetic amino acids, and both D and L forms of acids as well as racemic forms. More specifically, amino acids contain up to 10 carbon atoms. They may contain additional carboxyl groups, as well as heteroatoms such as nitrogen and sulfur. Preferably, amino acids are α and β-amino acids. The term α-amino acid refers to an amino acid in which the amino group is bonded to the carbon directly attached to the carboxyl group, i.e., the α-carbon. The term β-amino acid refers to an amino acid in which the amino group is bonded to the carbon one carbon away from the carboxyl group, i.e., the β-carbon. Amino acids as described herein are referred to by their standard IUPAC single-letter notation, where "X" represents any amino acid.

[0082] In the context of peptides, the term “substantial identity” indicates that a peptide contains a sequence that has at least 70% sequence identity with respect to a reference sequence, preferably 80%, more preferably 85%, and most preferably at least 90% or at least 95%, across a specified comparison window. As used herein, “percentage of sequence identity” means a value determined by comparing two optimally aligned sequences across a comparison window, where portions of the polynucleotide sequence within the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions in which identical nucleic acid bases or amino acid residues are present in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity.

[0083] The terms "specific binding," "selective binding," and "specifically binding" refer to the preferential non-covalent or covalent binding to a molecule compared to other molecules or parts in a solution or reaction mixture (for example, an antibody specifically binds to a particular polypeptide or epitope compared to other available polypeptides). In some embodiments, the affinity of one molecule to another molecule to which it specifically binds is 10 -5 M or less (for example, 10 -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, 10 -12 M or less, 10 -13 M or less, 10 -14 M or less, 10 -15 M or less, or 10 -16 K (M or below) D It is characterized by a (dissociation constant). "Affinity" refers to the strength of the binding, and increased binding affinity correlates with a lower Kd.

[0084] Linker. The TG2 binding portion and the integrin binding portion can be separated by a linker, such as a polypeptide linker or a non-peptide linker. In some embodiments, the linker is a rigid linker, and in other embodiments, the linker is a flexible linker. In some embodiments, the linker portion is a peptide linker. In some embodiments, the peptide linker contains 1 to 10 amino acids. In some embodiments, the peptide linker contains 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. Exemplary linkers include linear peptides having at least two amino acid residues, such as Gly-Gly, Gly-Ala-Gly, Gly-Pro-Ala, and Gly-Gly-Gly-Gly-Ser. Suitable linear peptides include polyglycine, polyserine, polyproline, polyalanine, and oligopeptides consisting of alanyl and / or selinyl and / or prolinyl and / or glycyl amino acid residues. In one embodiment, the linker comprises the amino acid sequence GTSTGSGKSSEGKG or (GGGGS)n, where n is 1, 2, 3, 4, 5, etc. However, many such linkers are known and used in the art and can serve this purpose. Examples of linkers used in bifunctional small molecules are provided in Cao et al. (2022). For example, PEG n PEG n Small organic linkers such as diamines, piperazines, 4-aminopiperidines, 3,9-diazaspiro[5.5]undecanes, and linkers suitable for click chemistry can be used.

[0085] In some embodiments, the linker is a small aliphatic or aromatic group having at least one terminal nitrogen that forms an amide bond with the carboxyl terminus of the TG2 bond moiety, such as a linker known and used in click chemistry reactions. See Fantoni et al. (2021) Chem. Rev. 2021, 121, 12, 7122-7154, incorporated herein by reference.

[0086] A “cleavable linker” is a linker having one or more cleavable groups that can be cleaved as a result of a reaction or condition. The term “cleavable group” refers to a portion that enables the release of a component of the solid support or oligomer of the present invention by cleaving a bond that links the released portion to the rest of the conjugate. The exemplary cleavage mechanisms used in both the preparation and use of the oligomers and solid supports of the present invention are mediated enzymatically or otherwise by chemical means.

[0087] The present invention includes, but is not limited to, a group that can be cleaved by one or more sites in addition to the enzymatically cleavable group, by the action of a non-enzymatic agent. Examples of non-enzymatic cleavage agents include, but are not limited to, acids, bases, light (e.g., nitrobenzyl derivatives, phenacyl groups, orthohydroxynamate esters, benzoin esters, piperidine, piperazine, 3-azaspiro[5.5]undecane) and heat. Many cleavable groups are known in the art. For example, see Jung et al., Biochem. Biophys. Acta, 761:152-162 (1983); Joshi et al., J. Biol. Chem., 265:14518-14525 (1990); Zarling et al., J. Immunol., 124:913-920 (1980); Bouizar et al., Eur. J. Biochem., 155:141-147 (1986); Park et al., J. Biol. Chem., 261:205-210 (1986); Browning et al., J. Immunol., 143:1859-1867 (1989). Furthermore, a wide range of bifunctional (both homobifunctional and heterobifunctional) spacer arms capable of cleaving are commercially available.

[0088] Exemplary cleavable groups can be cleaved by reagents, such as sodium hydroxide, ammonia, or other amines. In various embodiments, the cleavable linker is readily cleaved at room temperature or under heating. An example of a linker used is a valine-citrulline dipeptide linker cleaved by cathepsin B in lysosomes.

[0089] Chemical groups used for linking binding domains include, as is known in the art, carbamates, amides (amine + carboxylic acid), esters (alcohol + carboxylic acid), thioethers (haloalkane + sulfhydryl; maleimide + sulfhydryl), Schiff bases (amine + aldehyde), urea (amine + isocyanate), thiourea (amine + isothiocyanate), sulfonamides (amine + sulfonyl chloride), disulfides, hydrazones, and lipids.

[0090] The linkage between the bonding domains can be linear or branched, usually linear, and may contain one or more unsaturated bonds; typically having 1 to about 300 carbon atoms; more commonly having about 1 to 25 carbon atoms; and may also contain spacers, such as alkyl spacers, which may have about 3 to 12 carbon atoms. This type of spacer may also contain heteroatoms or functional groups, including amines, ethers, phosphodiesters, etc. Specific structures of interest include: (CH2CH2O)n (wherein n is 1 to about 12); (CH2CH2NH)n (wherein n is 1 to about 12); [(CH2)n(C=O)NH(CH2)] m ] z (In the formula, n and m are between 1 and approximately 6, and z is between 1 and approximately 10); [(CH2)nOPO3(CH2) m ] z (wherein n and m are 1 to about 6, and z is 1 to about 10). Such linkers may include polyethylene glycol, which may be linear or branched. Examples of more rigid linkers include piperidine, piperazine, and 3-azaspiro[5.5]undecane.

[0091] The compounds may include homo- or heterobifunctional linkers having a group at one end that can form a stable link to the cargo. Exemplary entities include: azidobenzoyl hydrazide, N-[4-(p-azidosalicylamino)butyl]-3'-[2'-pyridyldithio]propionamide), bis-sulfosuccinimidyl sberate, dimethyl adipimidate, disuccinimidyl tartrate, N-γ-maleimidobutyryloxysuccinimidate, N-hydroxysulfosuccinimidyl-4-azidobenzoate, N-succinimidyl[4-azidophenyl]-1,3'-dithiopropionate, N-succinimidyl[4-iodoacetyl]aminobenzoate, glutaraldehyde, NHS-PEG-MA L; succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate; 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide (SPDP); N,N'-(1,3-phenylene)bismaleimide; N,N'-ethylene-bis-(iodoacetamide); or 4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid N-hydroxysuccinimide (SMCC); m-maleimidobenzoyl-N-hydroxysuccinimide (MBS); and succinimide 4-(p-maleimidophenyl)butyrate (SMPB), a chain-extended analog of MBS. The succinimidyl group of these crosslinking agents reacts with primary amines, and thiol-reactive maleimides form covalent bonds with thiols of cysteine ​​residues.

[0092] Other reagents useful for this purpose include p,p'-difluoro-m,m'-dinitrodiphenyl sulfone (which forms irreversible crosslinks with amino and phenol groups); dimethyl adipimidate (specific to amino groups); phenol-1,4-disulfonyl chloride (which mainly reacts with amino groups); hexamethylene diisocyanate or diisothiocyanate, or azophenyl-p-diisocyanate (which mainly reacts with amino groups); disdiazobenzidine (which mainly reacts with tyrosine and histidine); O-benzotriazolyloxytetramethyluronium hexafluorophosphate (HATU), dicyclohexylcarbodiimide, bromotris(pyrrolidino)phosphonium bromide (PyBroP); N,N-dimethylaminopyridine (DMAP); 4-pyrrolidinopyridine; and N-hydroxybenzotriazole. A homobifunctional crosslinking reagent is bismaleimide hexane ("BMH").

[0093] The term "alkyl" refers to a C1-C chain that can be linear, branched, or cyclic. 20 This refers to alkyl groups. "Lower alkyl" as in "lower alkyl" or "substituted lower alkyl" refers to C1-C 10 It means alkyl. The terms "alkyl," "lower alkyl," or "cycloalkyl" refer to methyl, ethyl, isopropyl, propyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, neopentyl, hexyl, cyclopropyl, cyclopropylmethyl, cyclobutyl, cyclobutylmethyl, cyclopentyl, cyclopentylmethyl, cyclohexyl, cyclohexylmethyl, C6-C 12 The set includes a spiro ring, cyclopropyl ethyl, cyclobutyl ethyl, dekalinyl, bicyclo-[1.1.1]-pentyl, norboranyl, bisilo-[2.2.2]-octyl, cubyl, adamantanyl, and related cage hydrocarbon moieties. In certain embodiments, the alkyl is C1-C 20 It is an alkyl group. In certain embodiments, the alkyl group is polydeuterated.

[0094] "Substituting alkyl" typically refers to heterocycloalkyl, aryl, substituted aryl, heteroaryl, nitro, cyano (also called nitrile in this specification), azide, halo, -OR, -SR, -SF5, -CHO, -COR, -C(O)OR, -C(O)-NR2, -OC(O)R, -OC(O)NR2, -OC(O)OR, -P(O)(OR)2, -OP(O)(OR)2, -NR2, -N + R3 (counterions may be present), -CONR2, -NRCOR, -NHC(O)OR, -NHC(O)NR2, -NHC(NH)NR2, SO3 - These are alkyl groups mono, di, or tri-substituted with -SO2OR, -OSO2R, -SO2NR2, or -NRSO2R, where each R is independently hydrogen, lower alkyl, R'-substituted lower alkyl, aryl, R'-substituted aryl, heteroaryl, heteroaryl(alkyl), R'-substituted aryl(alkyl), or aryl(alkyl), where each R' is independently hydroxy, halo, alkyloxy, cyano, thio, SF5, nitro, alkyl, halo-alkyl, or amino. Substituted alkyl groups substituted with 1 to 3 substituents selected from the group consisting of alkynyl, cyano, halo, alkyloxy, thio, nitro, amino, or hydroxy are of particular interest.

[0095] The term "aryl" refers to an aromatic ring having (4n+2) π electrons, which may contain 6 to 20 ring carbon atoms and may consist of a monocycle (e.g., phenyl), or two or more fused rings, e.g., two or three fused rings (e.g., naphthyl), or two or more aromatic rings linked by single bonds, e.g., two or three aromatic rings (e.g., biphenylyl). In certain cases, aryl is C6-C 16 Or C6~C 14 In certain embodiments, the alkyl group has one or more hydrogen atoms replaced by deuterium.

[0096] A heteroaryl is an aromatic ring system containing (4n+2) π electrons, composed of 1 to 10 ring carbon atoms and 1 to 5 heteroatoms selected from O, N, S, and Se, and having a monoring (e.g., thiophene, pyridine, pyrazine, imidazole, oxazole, tetrazole, etc.), or two or more fused rings, for example, 2 to 3 fused rings (e.g., indole, benzimidazole, quinolone, quinoxaline, phenothiazine, etc.), or two or more aromatic rings linked by a single bond, for example, 2 to 3 aromatic rings (e.g., bipyridyl). In some cases, a heteroaryl is C1-C 16 , as well as the selection of 1 to 5 heteroatoms consisting of S, Se, N, and O.

[0097] The terms "heterocycloalkyl," "heterocyclic," "heterocyclic group," or "heterocyclyl" refer to saturated or unsaturated non-aromatic ring systems containing 1 to 10 ring carbon atoms and 1 to 5 heteroatoms selected from O, N, S, and Se, and having monocyclic rings (e.g., tetrahydrofuran, aziridine, azetidine, pyrrolidine, piperidine, tetrathiopyran, hexamethylene oxide, oxazepane, etc.) or two or more fused rings, such as 2 to 3 fused rings (e.g., indoline, tetrahydrobenzodiazapine, etc., e.g., condensed, bridged, and spirocyclic systems having 3 to 15 ring atoms containing 1 to 4 heteroatoms). In specific cases, heterocycloalkyl refers to a C1-C 16 , as well as the selection of 1 to 5 heteroatoms consisting of S, Se, N, and O. In fused ring systems, one or more of the rings may be cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, provided that the bonding site is via a non-aromatic ring. In certain embodiments, the nitrogen and / or sulfur atoms of the heterocyclic group are optionally oxidized to provide an N-oxide, -S(O)-, or -SO2- moiety.

[0098] Examples of heterocyclic and heteroaryl compounds include, but are not limited to, azetidine, pyrrole, imidazole, benzimidazole, pyrazole, benzopyrazole, tetrazole, 1,2,3-triazole, benzotriazole, 1,2,4-triazole, pyridine, pyrazine, pyrimidine, pyridazine, indidine, isoindole, indole, dihydroindole, indazole, purine, quinoridine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carborin, phenanthidine, acridine, phenanthroline, isothiazole, benzoisothia Examples include zole, phenazine, isoxazole, benzoisoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, benzothiazole, thiazolidine, furan, benzofuran, thiophene, benzothiophene, benzo[b]thiophene, morpholinil, thiomorpholinil (also called thiamorpholinil), 1,1-dioxothiomorpholinil, piperidinil, pyrrolidine, tetrahydrofuranil, and benzotetrahydrofuranil.

[0099] Substituted heterocycloalkyl, aryl, heteroaryl are hydrogen, 1-3 alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkyl(alkyl), aryl, substituted aryl, aryl(alkyl), -SO2NR 5 R 5 -PO3H2, -NR 5 SO2R 6 or -NR 5 C(=O)R 6 It is arbitrarily substituted with, and in the formula, R 5 and R 6 These are independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkyl(alkyl), aryl, optionally substituted heterocycloalkyl, aryloxy, heteroaryl, heteroaryl(alkyl), or R 5 and R6 Together, -(CH2) 3-6 -or-(CH2) 0-3 X(CH2) 0-3 - and in the formula, X = NR, O, S, SO2, substituted aryl(alkyl), halo(alkyl), SF5, NR 5 3 + , azide, cyano (also called nitrile in this specification), -OR 5 , -SR 5 , -NR 5 R 6 Halogen, Nitro, SCH3, OCF3, SO2CH3, SCF3, SO2CF3, CF3, -SO2OR 5 , -OSO2R 5 ,CCl3,-C(=O)R 5 , -C(=O)OR 5 -C(=O)NR 5 R 6 -OC(=O)R 5 That is the case.

[0100] The term "substituted," as in "substituted alkyl" and "substituted aryl," means, as implied in some of the definitions above, that in the hydrocarbyl, alkyl, aryl, or other part, at least one hydrogen atom bonded to a carbon (or other) atom is replaced by one or more non-hydrogen substituents. Examples of such substituents include, but are not limited to, functional groups and hydrocarbyl moieties C1-C24 alkyl (containing C1-C18 alkyl, further containing C1-C12 alkyl, further containing C1-C6 alkyl), C2-C24 alkenyl (containing C2-C18 alkenyl, further containing C2-C12 alkenyl, further containing C2-C6 alkenyl), C2-C24 alkynyl (containing C2-C18 alkynyl, further containing C2-C12 alkynyl, further containing C2-C6 alkynyl), C5-C30 aryl (containing C5-C20 aryl, further containing C5-C12 aryl), and C6-C30 aralkyl (containing C6-C20 aralkyl, further containing C6-C12 aralkyl). The above hydrocarbyl moieties may be further substituted with one or more functional groups or additional hydrocarbyl moieties, for example, those specifically listed. Unless otherwise indicated, any group described herein should be interpreted as including substituted and / or heteroatom-containing moieties in addition to non-substituted moieties.

[0101] "Sulfonyl" refers to the group SO2-alkyl, SO2-substituted alkyl, SO2-alkenyl, SO2-substituted alkenyl, SO2-alkynyl, SO2-substituted alkynyl, SO2-cycloalkyl, SO2-substituted cycloalkyl, SO2-cycloalkenyl, SO2-substituted cycloalkenyl, SO2-aryl, SO2-substituted aryl, SO2-heteroaryl, SO2-substituted heteroaryl, SO2-heterocyclic, and SO2-substituted heterocyclic, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. Examples of sulfonyls include methyl-SO2-, phenyl-SO2-, and 4-methylphenyl-SO2-. Sulfonimidoyl refers to S(O)(NH) bonded in the same way as sulfonyl as defined above.

[0102] The term "water-soluble group" refers to a functional group that is well solvable in an aqueous environment and imparts improved water solubility to the compound to which it is bound. Examples of water-soluble groups of interest include, but are not limited to, polyalcohols, linear or cyclic sugars, primary, secondary, tertiary or quaternary amines and polyamines, sulfate groups, sulfonate groups, sulfinate groups, carboxylate groups, phosphate groups, phosphonate groups, phosphinate groups, ascorbate groups, glycols including polyethylene glycol (PEG) and modified PEG, and polyethers. In some examples, water-soluble groups include primary, secondary, tertiary and quaternary amines, carboxylates, phosphonates, phosphates, sulfonates, sulfates, -N(H) 0-1 (CH2CH2OH) 1-2 ,-NHCH2CH2N(CH3) 2-3 -NHCH2CH2SO3H, -NHCH2CH2PO3H2, and -NHCH2CH2CO2H, -(CH2CH2O) yy CH2CH2XR yy ,-(CH2CH2O) yy CH2CH2X-, -X(CH2CH2O) yyCH2CH2-, glycol, oligoethylene glycol, and polyethylene glycol, where yy is selected from 1 to 1000, and X is O, S, and NR. ZZ Selected from, R ZZ and R YY The element is independently selected from H and C1-3 alkyl groups.

[0103] The term "carboxyl equivalent" refers to standard pharmaceutically acceptable bioequivalent substituents of carboxylic acids, amides, and esters. These include, but are not limited to, acylcyanamides, tetrazoles, hydroxychromium, 3-hydroxy-1,2,4-triazole, 1-hydroxypyrazole, 2,4-dihydroxyimidazole, 1-hydroxyimidazole, 1-hydroxy-1,2,3-triazole, alkylsulfonylcarboxamides, hydroxyisoxazole, 5-hydroxy-1,2,4-oxadiazole, thiazole, 1,2,4-oxadiazole, 1,2,4-oxadiazolone, oxazole, triazole, thiazole, other hydroxamic acids, sulfonimides, acylsulfonamides, sulfonylureas, oxadiazolone, thiazolidinediones, oxadiazole, thiadiazole, isothiazole, difluorophenols, tetramic acid, tetronic acid, squalic acid, hydroxyquinoline, hydroxyquinoline-2-one, boronic acid, and phosphoric acid.

[0104] The term "functional group" includes halo, hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C20 aryloxy, acyl (including C2-C24 alkylcarbonyl (-CO-alkyl) and C6-C20 arylcarbonyl (-CO-aryl)), acyloxy (-O-acyl), C2-C24 alkoxycarbonyl (-(CO)-O-alkyl), C6-C20 aryloxycarbonyl (-(CO)-O-aryl), halocarbonyl (-CO)-X (wherein in the formula, X is a halo), C2-C24 alkylcarbonate (-O-(CO)-O-alkyl), C6-C20 arylcarbonate (-O-(CO)-O-aryl), carboxy(-COOH), carboxylat(-COO-), carbamoyl(-(CO)-NH2), monosubstituted C1-C24 alkylcarbamoyl(-(CO)-NH(C1-C24 alkyl)), disubstituted alkylcarbamoyl(-(CO)-N(C1-C24 alkyl)2), monosubstituted arylcarbamoyl(-(CO)-NH-aryl), thiocarbamoyl(-(CS)-NH 2) Carbamide (-NH-(CO)-NH2), cyano (-C≡N), isocyano (-N+≡C-), cyanato (-OC≡N), isocyanato (-O-N+≡C-), isothiocyanato (-SC≡N), azide (-N=N+=N-), formyl (-(CO)-H), thioformyl (-(CS)-H), amino (-NH2), mono and di(C1-C24 alkyl) substituted aminos, mono and di(C5-C20 aryl) substituted aminos, C2-C24 alkylamide (-NH-(CO)-alkyl), C5-C20 arylamide (-NH-(CO) -aryl), imino (-CR=NH, where R=hydrogen, C1-C24 alkyl, C5-C20 aryl, C6-C20 alkaryl, C6-C20 aralkyl, etc.), alkylimino (-CR=N(alkyl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), arylimino (-CR=N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (-NO2), nitroso (-NO), sulfo (-SO2-OH), sulfonate (-SO2-O-), C1-C24 alkylsulfanyl (-S-alkyl;This refers to chemical groups such as alkylthio (also called alkylthio), arylsulfanyl (-S-aryl; also called arylthio), C1-C24 alkylsulfinyl (-(SO)-alkyl), C5-C20 arylsulfinyl (-(SO)-aryl), C1-C24 alkylsulfonyl (-SO2-alkyl), C5-C20 arylsulfonyl (-SO2-aryl), phosphono (-P(O)(OH)2), phosphonato (-P(O)(O-)2), phosphinato (-P(O)(O-)), phospho (-PO2) and phosphino (-PH2), mono- and di-(C1-C24 alkyl) substituted phosphino, and mono- and di-(C5-C20 aryl) substituted phosphines. Furthermore, the above functional groups may be further substituted with one or more additional functional groups or one or more hydrocarbyl moieties, for example, those specifically listed above, where permitted by the particular group.

[0105] When the term "substituted" appears before a list of possible substituents, the term is intended to apply to all members of that base. For example, the phrase "substituted alkyl and aryl" should be interpreted as "substituted alkyl and substituted aryl."

[0106] In addition to the disclosures herein, the term “substituted” may also mean, when used to modify a given group or radical, that one or more hydrogen atoms of the given group or radical are each independently replaced by the same or different substituents as defined below.

[0107] In addition to the groups disclosed with respect to individual terms herein, one or more hydrogen atoms on a saturated carbon atom in a specified group or radical (any two hydrogen atoms on a single carbon atom = O, = NR) 70 、=N-OR 70 The substituents to replace (which can be replaced by =N2 or =S) are -R unless otherwise specified. 60 , halo, =O, -OR 70 , -SR 70 , -NR 80 R80 、トリハロメチル、-CN、-OCN、-SCN、-NO、-NO2、=N2、-N3、-SO2R 70 、-SO2O - M + 、-SO2OR 70 、-OSO2R 70 、-OSO2O - M + 、-OSO2OR 70 、-P(O)(O - )2(M + )2、-P(O)(OR 70 )O - M + 、-P(O)(OR 70 )2、-C(O)R 70 、-C(S)R 70 、-C(NR 70 )R 70 、-C(O)O - M + 、-C(O)OR 70 、-C(S)OR 70 、-C(O)NR 80 R 80 、-C(NR 70 )NR 80 R 80 、-OC(O)R 70 、-OC(S)R 70 、-OC(O)O - M + 、-OC(O)OR 70 、-OC(S)OR 70 、-NR 70 C(O)R 70 、-NR 70 C(S)R 70 、-NR 70 CO2 - M + 、-NR 70 CO2R 70 、-NR 70 C(S)OR 70 、-NR 70 C(O)NR 80 R 80 、-NR 70 C(NR 70 )R 70 および-NR 70 C(NR 70 )NR80 R 80 And in the formula, R 60 The group consists of optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl, and each R 70 These are independently hydrogen or R 60 And each R 80 R is independent of R 70 Either or two R's 80 However, together with the nitrogen atom to which they are bonded, they form a 5, 6, or 7-membered heterocycloalkyl group which may optionally contain 1 to 4 additional identical or different heteroatoms selected from the group consisting of O, N, and S, and the N may have a -H or C1-C3 alkyl substitution; each M + M+ is a counterion with a net single positive charge. Each M+ independently has, for example, K + kaNa + Li + Alkaline ions such as; + N(R 60 ) Ammonium ions such as 4; or alkaline earth ions, for example [Ca 2+ ] 0.5 [Mg 2+ ] 0.5 or [Ba 2+ ] 0.5 It may also be the case that one of the counterions of such a divalent alkaline earth ion may be the ionized form of the compound of the present invention and the other may be a typical counterion such as a chloride, or that the two ionized compounds disclosed herein may function as counterions of such a divalent alkaline earth ion, or that the biionized compound of the present invention may function as a counterion of such a divalent alkaline earth ion). For example, -NR 80 R 80 -NH2, -NH-alkyl, N-pyrrolidinyl, N-piperazinyl, 4N-methyl-piperazin-1-yl, N-morpholinyl, -N(H) 0-1 (CH2CH2OH) 1-2 ,-NHCH2CH2N(CH3)2-3 This means that it includes -NHCH2CH2SO3H, -NHCH2CH2PO3H2, and -NHCH2CH2CO2H.

[0108] In addition to the disclosures herein, the hydrogen substituents on unsaturated carbon atoms in “substituted” alkenes, alkynes, aryl and heteroaryl groups are, unless otherwise specified, -R 60 Hello, -O - M + , -OR 70 , -SR 70 , -S - M + , -NR 80 R 80 , trihalomethyl, -CF3, -CN, -OCN, -SCN, -NO, -NO2, -N3, -SO2R 70 , -SO3 - M + , -SO3R 70 , -OSO2R 70 , -OSO3 - M + -OSO3R 70 , -PO3 -2 (M + )2, -P(O)(OR 70 )O - M + , -P(O)(OR 70 )2, -C(O)R 70 ,-C(S)R 70 -C(NR 70 )R 70 , -CO2 - M + , -CO2R 70 , -C(S)OR 70 -C(O)NR 80 R 80 -C(NR 70 )NR 80 R 80 -OC(O)R 70 ,-OC(S)R 70 , -OCO2 - M + , -OCO2R 70 , -OC(S)OR 70 , -NR 70 C(O)R 70, -NR 70 C(S)R 70 , -NR 70 CO2 - M + , -NR 70 CO2R 70 , -NR 70 C(S)OR 70 , -NR 70 C(O)NR 80 R 80 , -NR 70 C(NR 70 )R 70 and -NR 70 C(NR 70 )NR 80 R 80 And in the formula, R 60 , R 70 , R 80 and M + This is as previously defined, except that in the case of substituted alkenes or alkynes, the substituent is -O - M + , -OR 70 , -SR 70 or -S - M + isn't it.

[0109] In addition to the groups disclosed with respect to the individual terms herein, substituents on the nitrogen atom of a "substituted" heteroalkyl and cycloheteralkyl group are -R unless otherwise specified. 60 , -O - M + , -OR 70 , -SR 70 , -S - M + , -NR 80 R 80 , trihalomethyl, -CF3, -CN, -NO, -NO2, -S(O)2R 70 -S(O)2O - M + -S(O)2OR 70 -OS(O)2R 70 -OS(O)2O - M + -OS(O)2OR 70 ,-P(O)(O - )2(M+ )2, -P(O)(OR 70 )O - M + , -P(O)(OR 70 )(OR 70 ), -C(O)R 70 ,-C(S)R 70 -C(NR 70 )R 70 , -C(O)OR 70 , -C(S)OR 70 -C(O)NR 80 R 80 -C(NR 70 )NR 80 R 80 -OC(O)R 70 ,-OC(S)R 70 , -OC(O)OR 70 , -OC(S)OR 70 , -NR 70 C(O)R 70 , -NR 70 C(S)R 70 , -NR 70 C(O)OR 70 , -NR 70 C(S)OR 70 , -NR 70 C(O)NR 80 R 80 , -NR 70 C(NR 70 )R 70 and -NR 70 C(NR 70 )NR 80 R 80 And in the formula, R 60 , R 70 , R 80 and M + This is as defined earlier.

[0110] In some embodiments, the formulas and compounds disclosed herein are peptide mimetic compounds that typically replace the Y-Pro dipeptide moiety. This includes TIFF2026525300000021.tif24161. Such peptide mimetic compounds are developed to enhance pharmacokinetic characteristics such as oral bioavailability and half-life. The synthesis of this peptide mimetic compound was described by Dragovich, et al (2002). The peptide mimetic compound may be referred to as "HWE" in relation to Horner-Wadsworth-Emmons, which is the named chemical reaction used to produce the peptide mimetic compound.

[0111] As used herein, the term “electrophilic attack group” may, but is not limited to, the following:

[0112] [ka]

[0113] In some embodiments, X is glutamine. In some embodiments, X is The filename is TIFF2026525300000023.tif41161.

[0114] Salts include, but are not limited to, Na, K, Ca, Mg, ammonium, tetraalkylammonium, arylsulfonates and alkylsulfonates, phosphates, carboxylates, sulfates, Cl, Br, and guanidinium.

[0115] Unless otherwise specified, a reference to an atom implies the inclusion of its isotopes. For example, a reference to H means, 1 H, 2 H (i.e., D) and 3 This means that H (i.e., T) is included, and the reference to C is, 12 C and all isotopes of carbon ( 13 This means including C, etc.

[0116] In addition to the disclosures herein, in certain embodiments, the substituted group has one, two, three or four substituents, one, two or three substituents, one or two substituents, or one substituent.

[0117] Unless otherwise indicated, the nomenclature of substituents not explicitly defined herein is obtained by naming the terminal portion of the functional group, followed by the functional groups adjacent toward the bond site. For example, the substituent "heterocycloalkyl(alkyl)" refers to the group (heterocycloalkyl)-(alkyl)-.

[0118] With respect to any of the groups disclosed herein that contain one or more substituents, it is understood that such groups do not contain any substitutions or substitution patterns that are sterically impractical and / or synthetically unfeasible. Furthermore, the compounds of interest include all stereochemical isomers resulting from the substitutions of these compounds.

[0119] In certain embodiments, substituents may contribute to the optical isomerism and / or stereoisomerism of the compound. Salt, solvate, hydrate, and prodrug forms of the compound are also of interest. Polymorphic, pseudopolymorphic, amorphous, and cocrystalline forms of the compound are also of interest. All such forms are encompassed in this disclosure. Therefore, the compounds described herein include their pharmaceutically acceptable salt, solvate, hydrate, prodrug, and isomeric forms, including their pharmaceutically acceptable salt, solvate, hydrate, prodrug, and isomeric forms. In certain embodiments, the compound may be metabolized to pharmaceutically active derivatives.

[0120] A “prodrug” is a derivative of a compound described herein, the pharmacological effect of which derives from conversion to an active compound by an in vivo chemical or metabolic process, including release by linker cleavage. Prodrugs include compounds in which an amino acid residue, or a polypeptide chain of two or more (e.g., two, three, or four) amino acid residues, is covalently bonded to a free amino, hydroxyl, or carboxylic acid group of the compound via an amide or ester bond. Additional types of prodrugs are also included. For example, a free carboxyl group can be derivatized as an amide or alkyl ester. Prodrug esters as used herein include esters and carbonates formed by reacting one or more hydroxyls of a compound of the method of the present invention with an alkyl, alkoxy, or aryl-substituted acylating agent using procedures known to those skilled in the art to produce acetates, pivalates, methyl carbonates, benzoates, etc.

[0121] As a further example, free hydroxyl groups can be derivatized using groups including, but not limited to, hemisuccinates, phosphate esters, dimethylaminoacetates, and phosphoryloxymethyloxycarbonyls, as outlined in Advanced Drug Delivery Reviews, 1996, 19, 115. Carbamate prodrugs of hydroxyl and amino groups are also included, as are carbonate prodrugs, sulfonate prodrugs, sulfonate esters, and sulfate esters of hydroxyl groups. Free amines can also be derivatized to amides, sulfonamides, or phosphoamides. All prodrug moieties described may incorporate groups including, but not limited to, ethers, amines, and carboxylic acid functional groups. Furthermore, any compound that can be converted in vivo to provide a bioactive agent (e.g., a compound of formula I) is a prodrug within the scope of the present invention. Various forms of prodrugs are well known in the art. A comprehensive description of prodrugs and prodrug derivatives can be found in (a) The Practice of Medicinal Chemistry, Camille G. Wermuth et al., (Academic Press, 1996); (b) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985); and (c) A Textbook of Drug Design and Development, P. Krogsgaard-Larson and H. Bundgaard, eds., (Harwood Academic Publishers, 1991).

[0122] As used herein, “therapeutic dose” refers to an amount of therapeutic agent sufficient to treat or manage a disease or disorder. A therapeutic dose may also refer to an amount of therapeutic agent sufficient to delay or minimize the onset of a disease. A therapeutic dose may also refer to an amount of therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease. Furthermore, with respect to the therapeutic agents of the present invention, a therapeutic dose means an amount of therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease, either alone or in combination with other therapies.

[0123] As used herein, the term “medication regimen” refers to a set of unit doses (typically two or more) administered individually to a subject, typically divided into periods. In some embodiments, a given therapeutic agent has a recommended medication regimen which may comprise one or more doses. In some embodiments, a medication regimen comprises multiple doses, each separated from the others over periods of equal length. In some embodiments, a medication regimen comprises multiple doses and at least two distinct periods separating the individual doses. In some embodiments, all doses within a medication regimen are of the same unit dose amount. In some embodiments, the different doses within a medication regimen are of different amounts. In some embodiments, a medication regimen comprises a first dose in the amount of a first dose, followed by one or more additional doses in the amount of a second dose different from the first dose. In some embodiments, a medication regimen comprises a first dose in the amount of a first dose, followed by one or more additional doses in the amount of a second dose, the same as the first dose. In some embodiments, a medication regimen correlates with a desired or beneficial outcome when administered across a relevant population (i.e., it is a therapeutic medication regimen).

[0124] The term “diagnosis” is used herein to mean the identification of a molecular or pathological condition, disease or symptom, such as the identification of fibrosis. The methods of the present invention may further include the analysis of fibrosis or fibrotic activity after treatment by the claimed methods. The analysis of fibrosis may be performed by obtaining a biological sample and examining the molecular or pathological condition, disease or symptom, etc.

[0125] As used herein, terms such as “treatment” and “to treat” refer to administering a drug or performing a procedure for the purpose of obtaining an effect. The effect may be prophylactic in that it completely or partially prevents the disease or its symptoms, and / or therapeutic in that it results in a partial or complete cure of the disease and / or its symptoms. As used herein, “treatment” encompasses any treatment of fibrosis in mammals, in particular humans, and includes (a) preventing the onset of fibrosis; (b) inhibiting fibrosis in progress, i.e., halting its development; and (c) reducing fibrosis, i.e., causing regression of fibrosis.

[0126] Treatment can refer to any sign of success in treating, improving, or preventing fibrosis, and may include any objective or subjective parameters such as, for example, relief; remission; reduction of symptoms or making disease symptoms more tolerable to the patient; slowing the rate of degeneration or deterioration; or mitigating debilitation at the end of degeneration. Treatment or improvement of symptoms may be based on objective or subjective parameters, including the results of examinations by a physician. Accordingly, the term “treatment” includes the administration of the compounds or agents of the present invention to prevent or delay, alleviate, or halt or inhibit the onset of symptoms or symptoms associated with fibrosis. The term “therapeutic effect” refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in a subject.

[0127] "In combination," "combination therapy," and "combination product" refer, in certain embodiments, to the simultaneous administration to a patient of the first therapeutic agent (i.e., the first therapeutic agent) and the compounds used herein. When administered in combination, each component may be administered simultaneously or sequentially in any order at different time points. Thus, each component may be administered separately but may be administered in sufficiently close time proximity to provide the desired therapeutic effect. The first therapeutic agent intended for use in conjunction with the method of the present invention includes any other agents for use in the treatment of fibrosis. Examples of such therapeutic agents, but not limited to, include antifibrotic agents.

[0128] The “simultaneous administration” of known therapeutic agents and the pharmaceutical compositions of the present invention means the administration of the therapeutic agent and inhibitor at a time when both the known therapeutic agent and the compositions of the present invention are therapeutic. Such simultaneous administration may include simultaneous (i.e., at the same time), pre-administration, or post-administration of the drugs with respect to the administration of the compounds of the present invention. Those skilled in the art will not have difficulty determining the appropriate timing, order, and dosage of administration of the particular drugs and compositions of the present invention. Therapeutic agents to which simultaneous administration by the method of the present invention is intended include any other agents for use in the treatment of fibrosis.

[0129] As used herein, terms such as “correlated” or “correlated with” refer to a statistical association between two instances of events, where events include numbers, datasets, etc. For example, if the events include numbers, a positive correlation (also known herein as a “direct correlation”) means that as one increases, the other also increases. A negative correlation (also known herein as an “inverse correlation”) means that as one increases, the other decreases.

[0130] A “dosage unit” refers to a physically distinct unit suitable as a unit dose for a particular individual being treated. Each unit may contain a predetermined amount of the active compound calculated to produce the desired therapeutic effect in combination with the necessary pharmaceutical carrier. The specifications of the dosage unit form may be determined by (a) the inherent characteristics of the active compound and the specific therapeutic effect to be achieved, as well as (b) the limitations inherent in the technology for formulating such an active compound.

[0131] "Pharmacologically acceptable excipients" generally means excipients that are safe, non-toxic, and useful for preparing a desirable pharmaceutical composition, and include excipients acceptable for veterinary and human pharmaceutical use. Such excipients may be solid, liquid, semi-solid, or, in the case of aerosol compositions, gas. The terms "pharmaceutically acceptable" and "physiologically acceptable," and their grammatical variations, when they refer to compositions, carriers, diluents, and reagents, are interchangeable and indicate that the material can be administered to or to humans without producing undesirable physiological effects to the extent that they interfere with the administration of the composition.

[0132] "Therapeutic dose" refers to the amount of a disease that, when administered to a subject, is sufficient to treat that disease.

[0133] The phrase “determination of treatment effectiveness” and its variations may include any method for determining whether a treatment is beneficial to a subject. The term “treatment effectiveness” and its variations are generally indicated by the alleviation of one or more signs or symptoms associated with the disease and can be readily determined by those skilled in the art. “Treatment effectiveness” may also refer to the prevention or improvement of toxic signs and symptoms typically associated with standard or non-standard treatment of a disease. Determination of treatment effectiveness is usually indication and disease-specific and may include any method known or available in the art for determining whether a treatment is providing a beneficial effect to a patient. For example, evidence of treatment effectiveness may include, but is not limited to, remission of the disease or indication. Furthermore, treatment effectiveness may also include, but is not limited to, general improvements in the subject’s overall health, such as improved quality of life, increased predicted subject survival, reduced depression, or reduced relapse rates for the indication (increased remission time). (See, for example, Physicians' Desk Reference (2010).)

[0134] Fibrosis is the formation of excess connective tissue that leads to interstitial sclerosis and scar formation. Fibroblasts are connective tissue cells of mesenchymal origin. They are stromal cells that control tissue integrity. Fibroblasts maintain ECM homeostasis through both ECM deposition and the secretion of matrix metalloproteinases (MMPs) in order to remodel the ECM. Fibroblasts also regulate adjacent epithelial cells that direct epithelial proliferation and differentiation.

[0135] Fibroblasts are considered the major effectors of fibrosis in both normal and pathological contexts. During inflammation, fibroblasts are "activated" and called myofibroblasts, the body's primary collagen-producing cells. Fibroblasts associated with normal wound healing differ phenotype from cancer-associated fibroblasts. Fibroblasts within TMEs are called cancer-associated fibroblasts (CAFs) and have a unique expression profile and function that significantly contributes to cancer-associated fibrosis. In contrast to normal fibroblasts, CAFs have increased autocrine signaling capacity and proliferative tendencies.

[0136] Exemplary forms of fibrosis include, but are not limited to, tumor fibrosis, cardiac fibrosis, hepatic fibrosis such as cirrhosis, renal fibrosis and bladder fibrosis, pulmonary fibrosis, intestinal fibrosis associated with inflammatory bowel disease, skin scarring and keloids, wound healing and adhesions, post-irradiation fibrosis, fibrosis associated with chronic graft-versus-host disease (GvHD), and Alzheimer's disease. In further embodiments, cardiac fibrosis is associated with hypertension, hypertensive heart disease (HHD), myocardial infarction (M1), cardiac scarring associated with ischemic congestive heart failure, cardiomyopathy, post-myocardial infarction defects in cardiac function, atherosclerosis, and restenosis. Renal fibrosis may include, but is not limited to, diabetic nephropathy, vesicoureteral reflux, tubulointerstitial renal fibrosis, glomerulonephritis or glomerulonephritis (GN), focal segmental glomerulosclerosis, membranous glomerulonephritis, or mesocapillary GN. Hepatic fibrosis may include, but is not limited to, cirrhosis and associated conditions such as chronic viral hepatitis, non-alcoholic fatty liver disease (NAFLD), alcoholic steatohepatitis (ASH), non-alcoholic steatohepatitis (NASH), primary biliary cirrhosis (PBC), biliary cirrhosis, and autoimmune hepatitis. Pulmonary fibrosis may include, but is not limited to, idiopathic pulmonary fibrosis (IPF) or idiopathic fibrotic alveolitis, chronic fibrotic interstitial pneumonia, interstitial lung disease (ILD), and diffuse parenchymal lung disease (DPLD), pulmonary scarring including, but not limited to, damage due to bacterial, viral, or fungal infections, emphysema, and chronic obstructive pulmonary disease (COPD). Chronic asthma may also be prevented, treated, or ameliorated by the compositions described herein. This includes fibrosis of the eye and lens, such as glaucoma; age-related macular degeneration (exudative and dry AMD); fibrosis of the lens; periorbital fibrosis such as IgG4-related disease; and hyperthyroidism. Uterine fibroids are also treated.

[0137] One example of pulmonary fibrosis is idiopathic pulmonary fibrosis (IPF), the most common form of idiopathic interstitial pneumonia, which leads to progressive pulmonary fibrosis. Symptoms and signs develop over several months to several years and include exertional dyspnea, cough, and fine (Velcro) rales. Diagnosis is based on medical history, physical examination, high-resolution CT, and / or lung biopsy, if necessary. Treatment may include antifibrotic drugs and oxygen therapy. Most patients worsen. The median survival time is about 3 years from diagnosis.

[0138] Various drugs have been attempted in various types of fibrosis, particularly pulmonary fibrosis, but with little success. Anti-inflammatory drugs, including prednisolone and azathioprine, have little effect on fibrosis, suggesting that inflammation is merely an initiating factor rather than a driving factor of the disease. The use of nonspecific antiproliferative agents such as colchicine and cyclophosphamide also prevents the repair of fibrous tissue, for example, by impairing epithelial growth. Treatment with IFN-γ has shown some usefulness, but is limited by serious side effects.

[0139] By the time a typical patient presents with fibrosis-related symptoms (e.g., dyspnea for pulmonary fibrosis, cirrhosis for hepatic fibrosis), fibrosis in the target organ is often very severe, with much of the target organ structure replaced by extracellular matrix. Stopping this ongoing fibrosis can extend lifespan and improve quality of life. Areas of the target organ where fibrosis is not widespread can be restored to normal structure with appropriate treatment.

[0140] In some embodiments, tumor fibrosis is associated with pancreatic cancer. Pancreatic cancer is characterized by a pronounced fibrotic / stromal response. Pancreatic stellate cells (PSCs) are the primary cause of fibrosis in the stroma, closely interacting with cancer cells to create a tumor-promoting environment that stimulates local tumor growth and distant metastasis. Pancreatic fibrosis begins when PSCs are activated and undergo morphological and functional changes, resulting in an extracellular matrix (ECM) deposition rate exceeding the rate of ECM degradation in the gland. It is now well established that pancreatic cancer cells activate PSCs and increase fibrosis. There is significant evidence that a strong stromal / fibrotic response around tumor elements (a feature of most pancreatic cancers) plays a crucial role in tumor progression. A key histopathological feature of pancreatic cancer, related to its innate clinical and biological aggression, is its fibrotic (stromal) response. Stromal production is stimulated by cancer cell-derived growth factors, including transforming growth factor-β (TGFβ), hepatocyte growth factor (HGF), fibroblast growth factor (FGF), insulin-like growth factor 1 (IGF-1), and epidermal growth factor (EGF). The fibrous reaction consists of extracellular matrix (ECM) proteins, mainly type I and type III collagen, fibronectin, and proteoglycans; small endothelial lining blood vessels; and a diverse population of cells, including inflammatory cells, fibroblasts, and astrocytes. Stromal formation can constitute up to 90% of the tumor volume, a characteristic unique to pancreatic cancer. The tumor microenvironment in pancreatic cancer plays a role in its resistance to chemotherapy.

[0141] Chronic inflammation leads to fibrosis, and chronic fibrosis is also a predisposing factor for cancer development. In addition to cancer-induced chronic inflammation as a driving factor for fibrosis, cancer treatment also plays a crucial role in creating a fibrous tumor microenvironment (TME). Organ fibrosis, most notably pulmonary fibrosis, is a known toxicity of several chemotherapy agents, including bleomycin, gemcitabine, and methotrexate. In vitro and in vivo studies have demonstrated that chemotherapy can promote an inflammatory and fibrous microenvironment through tissue damage associated with oxidative stress. Tissues exposed to chemotherapy undergo remodeling, involving fibroblast activation and proliferation, as well as ECM accumulation and crosslinking, after going through a similar stage of wound healing, including inflammation due to the influx of immune cells.

[0142] Cancer develops within a complex microenvironment crucial for tumor survival, growth, and metastasis. This tumor microenvironment (TME) consists of vascular structures, the extracellular matrix (ECM), stromal cells, immune cells, and a web of soluble signaling molecules that form a dynamic "organ" important to the pathophysiology of cancer. Within the TME, cancer-associated fibrosis emerges as a key regulator of cancer behavior. In fact, fibrosis is a prominent feature of cancer. Up to 20% of cancers, including hepatocellular carcinoma, gastric cancer, esophageal cancer, head and neck cancer, colon cancer, pancreatic cancer, cervical cancer, and vulvar cancer, are associated with chronic inflammation-associated fibrosis (due to either infectious or autoimmune etiologies).

[0143] Fibrosis has been reported to support cancer growth through various mechanisms, including direct cell interactions, immunomodulation, and ECM remodeling. As stromal progenitor cells, fibrosis significantly influences TME formation and is a key mediator. Fibrosis is important not only in the formation of established tumor sites but also in the formation of pre-metastatic niches. In vivo models demonstrate increased fibronectin expression in the stroma of future metastatic sites.

[0144] Fibrosis can be monitored during and after treatment, for example in diagnosis, to evaluate the effectiveness of treatment. The presence of fibrosis can be detected by means known in the art, for example, by examining tissue for excessive scarring. Prior to fibrosis, an individual may be determined to be susceptible based on an undesirable increase in inflammatory mediators that can exacerbate tissue damage, such as IL-1, TNF-α, and reactive oxygen species and reactive nitrogen species. Pro-fibrosis mediators such as TGF-β1 may be present. Activated myofibroblasts that may be resistant to the induction of apoptosis may also be present. Methods for monitoring fibrosis may include, for example, chest X-rays imaging scar tissue typical of pulmonary fibrosis, which can be useful for monitoring the course of the disease and treatment. Computed tomography (CT) scans combine X-ray images taken from many different angles to produce cross-sectional images of internal structures within the body. High-resolution CT scans can be particularly useful in determining the extent of lung damage caused by pulmonary fibrosis. Echocardiography visualizes the heart using sound waves. Functional tests, including lung function tests such as vital capacity measurement, pulse oximetry, exercise stress tests, and arterial blood gas analysis, may be useful, for example, in the treatment of pulmonary fibrosis.

[0145] Treatment method Individuals diagnosed with or at risk of developing a fibrotic disease are treated by administering the bifunctional molecule of this disclosure in a dose effective in reducing or preventing fibrosis by breaking down integrins. In some embodiments, fibrosis is renal fibrosis. When fibrosis is renal fibrosis, renal fibrosis may be caused by hypertension, diabetes, obstruction, or infection. In some embodiments, fibrosis is pulmonary fibrosis, e.g., idiopathic pulmonary fibrosis. In some embodiments, fibrosis is cutaneous fibrosis, renal fibrosis, intestinal fibrosis, hepatic fibrosis such as cirrhosis, non-alcoholic steatohepatitis (NASH), and non-alcoholic fatty liver disease (NAFLD). In some embodiments, fibrosis is associated with cancer and tumor growth, i.e., tumor-associated histofibrosis, including, but not limited to, lung cancer, skin cancer, and sarcoma. In other embodiments, fibrosis is associated with chronic inflammation or injury, including, but not limited to, irradiation of tissues, including, liver, lung, kidney, uterus, eye, and lens fibrosis, IgG4-related disease, and chronic GvHD.

[0146] The method involves administering a therapeutically effective amount or effective dose of the bifunctional molecule of this disclosure to a subject requiring treatment. In some embodiments, the effective dose of the therapeutic entity of the present invention described herein varies depending on many different factors, including the means of administration, the target site, the physiological state of the patient, whether the patient is human or animal, other pharmaceuticals administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is human, but non-human mammals, including transgenic mammals, can also be treated. The treatment dose needs to be adjusted to optimize safety and efficacy.

[0147] In some embodiments, fibrosis reduction is monitored during or after treatment. Monitoring may include, but is not limited to, detection of a decrease in fibrous cells, a decrease in adipose-infiltrating cells, etc.

[0148] The effective dose of the bifunctional molecules of this disclosure may vary depending on the drug, but generally ranges from about 0.01 mg / kg to about 0.05 mg / kg to about 0.1 mg / kg to about 50 mg / kg to about 40 mg / kg to about 30 mg / kg to about 20 mg / kg to about 10 mg / kg to about 5 mg / kg; up to about 1 mg / kg to about 0.5 mg / kg; the dose may vary depending on the specific drug and recipient.

[0149] The drug may be administered over one or more days, and in some embodiments, it may be administered daily, every other day, twice a week, or weekly for a period of about 1, 2, 3, 4, 5, 6, or 7 weeks or more, up to a chronic maintenance level.

[0150] In prophylactic applications, relatively low doses are administered over a long period at relatively infrequent intervals. Some patients continue treatment for life. In therapeutic applications, relatively high doses may be required at relatively short intervals until disease progression is reduced or terminated, preferably until the patient shows partial or complete improvement in the symptoms of the disease. Thereafter, the patient may be administered a prophylactic regimen.

[0151] In some other embodiments, for prophylactic application, a pharmaceutical composition or pharmacopoeia is administered to a patient who is susceptible to or otherwise at risk of the disease or symptoms, including the biochemical, histological and / or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes that appear during the onset of the disease, in an amount sufficient to eliminate or reduce the risk of the disease, reduce its severity, or delay its onset.

[0152] In other embodiments, for therapeutic application, the therapeutic entity of the present invention is administered to a patient suspected of or already suffering from such disease in an amount sufficient to cure or at least partially cessate the symptoms (biochemical, histological, and / or behavioral) of the disease, including its complications and intermediate pathological phenotypes in the onset of the disease. An amount sufficient to achieve therapeutic or prophylactic treatment is defined as a therapeutically or prophylactically effective dose. In both prophylactic and therapeutic regimens, the agent is usually administered in several doses until a satisfactory response is achieved.

[0153] According to the present invention, the composition can be administered parenterally, topically, intravenously, orally, subcutaneously, intra-arterially, intracranially, intraperitoneally, intranasally, or intramuscularly. The most typical route of administration is intravenous, but other routes may be equally effective.

[0154] For parenteral administration, the compositions of the present invention can be administered as an injectable dose of a solution or suspension of the substance in a physiologically acceptable diluent containing a pharmaceutical carrier, which may be a sterile liquid such as water, oil, saline, glycerol, or ethanol. Furthermore, auxiliary substances such as wetting agents or emulsifiers, surfactants, and pH buffers may be present in the composition. Other components of the pharmaceutical composition may be of petroleum, animal, plant, or synthetic origin, e.g., peanut oil, soybean oil, and mineral oil. Generally, glycols, such as propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions. Antibodies and / or polypeptides can be administered in the form of depot injections or implant preparations, which can be formulated to allow sustained release of the active ingredient. An exemplary composition contains 1 mg / mL of polypeptide formulated in an aqueous buffer consisting of 10 mM Tris, 210 mM sucrose, 51 mM L-arginine, and 0.01% polysorbate 20, adjusted to pH 7.4 with HCl or NaOH.

[0155] Typically, the composition is prepared as either a liquid solution or a suspension for injection. A solid form suitable for dissolving or suspending in a liquid vehicle before injection can also be prepared. As described above, to enhance the adjuvant effect, the preparation may also be emulsified or encapsulated in liposomes or microparticles such as polylactides, polyglycolides, or copolymers. (Langer, Science 249:1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28:97-119, 1997). The agents of the present invention can be administered in the form of depot injections or implant preparations that can be formulated to allow sustained or pulsatile release of the active ingredient.

[0156] Further formulations suitable for other modes of administration include oral, intranasal, and pulmonary formulations, suppositories, and subcutaneous and transdermal applications.

[0157] For suppositories, binders and carriers include, for example, polyalkylene glycols or triglycerides. Such suppositories can be formed from a mixture containing the active ingredient in an amount of 0.5% to 10%, preferably 1% to 2%. Oral formulations include excipients such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations, or powders and contain 10% to 95%, preferably 25% to 70%, of the active ingredient.

[0158] Topical application may result in transdermal or intradermal delivery. Topical administration can be facilitated by co-administration of the drug with cholera toxin or its detoxification derivatives or subunits or other similar bacterial toxins. Glenn et al., Nature 391:851, 1998. Co-administration can be achieved by using the components as a mixture or as linked molecules obtained by chemical cross-linking or expression as fusion proteins.

[0159] In some embodiments, the composition is delivered subcutaneously. Subcutaneous (SC) injection is currently the most common route for self-administered biopharmaceuticals such as proteins and peptides. In addition to simple injection, the formulation may contain additional agents to enhance SC delivery. Chemical penetration enhancers that can disrupt the skin barrier and provide an additional driving force for transporting the therapeutic agent. Chemical enhancers can be inserted into the highly ordered lipid bilayer of the stratum corneum to disrupt molecular packing or extract lipids to create lipid packing defects on the nanometer scale, thus resulting in higher transport efficiency. Nano / micro vesicles such as liposomes and nano / micro emulsions are useful as chemical penetration enhancers. They can not only improve skin permeability but also act as vehicles for drug solubilization and drug transport through the skin.

[0160] In addition to chemical penetration enhancers, an electrical device that promotes transport through the skin can be used to provide an additional driving force through electrical interactions or introduce a transient perturbation of the stratum corneum through high-voltage electrical pulses, thereby enhancing the delivery efficiency through the skin. Electroporation has evolved as another technique for electrically assisted transdermal drug delivery. The procedure of electroporation involves inducing a transient perturbation in the stratum corneum by using short high-voltage pulses to create microchannels across its lipid bilayer.

[0161] In addition to the electric field, mechanical forces are another alternative for creating transient channels on the surface of the skin for transdermal drug delivery. Ultrasound and jet injection are two representative mechanical force-triggered methods for drug delivery. Ultrasound can enhance the permeability of drugs across the skin by thermal or cavitation effects. Jet injection applies high-speed liquid to disrupt the surface of the skin and distribute the insulin solution into the skin tissue.

[0162] Microneedle (MN) technology offers an alternative method for transdermal protein delivery. Microscale needles can painlessly disrupt the stratum corneum and reach the epidermis and dermis for drug release. Solid MNs are designed to penetrate the skin to improve drug transport. Hollow MNs are used for injecting fluid drug formulations through openings in the skin caused by the needle, while soluble or degradable MNs are made from polymers containing encapsulated drugs. Examples of soluble polymers include hyaluronic acid (HA), carboxymethylcellulose (CMC), chitosan, alginates, polyvinylpyrrolidone (PVP), and polyvinyl alcohol (PVA).

[0163] Alternatively, transdermal delivery can be achieved using skin patches or transferosomes. (Paul et al., Eur. J. Immunol. 25:3521-24, 1995; Cevic et al., Biochem. Biophys. Acta 1368:201-15, 1998). Pharmaceutical compositions are generally sterile, substantially isotonic, and formulated in full compliance with U.S. Food and Drug Administration Good Manufacturing Practice (GMP) regulations. Preferably, the therapeutically effective dose provides therapeutic benefits without causing substantial toxicity.

[0164] In some embodiments, the compounds disclosed herein are formulated for sustained release, e.g., transdermal patches or implants. Subcutaneous sustained-release formulations include, for example, polymer-based micro / nanocarriers. Colloidal micro / nanoparticle systems with large surface area significantly enhance epithelial permeability and improve the bioavailability of protein and peptide drugs. A variety of FDA-approved biodegradable polymers are commercially available for drug delivery in micro / nanoparticle formulations. By modifying the polymer structure, these polymer carriers, such as poly(lactic acid-coglycolic acid) (PLGA), poly(lactic acid) (PLA), or chitosan (CS), can achieve structural stability and sustained release of protein and peptide drugs. Other polymer-based carriers include poly(hydroxybutyrate-co-hydroxyhexanoate (PHBHHx)). Another biodegradable biocompatible polymer carrier is microspheres of p(CPP:SA). After the formation of water-soluble degradation products, the anhydride bonds of this CPP:SA copolymer are hydrolyzed.

[0165] In situ gelation systems can be used as drug delivery systems. For example, temperature-sensitive hydrogels are polymer aqueous solutions at room temperature and convert to sol-gel form at physiological temperatures, which can be composed of PLGA and benzyl alcohol (BA) and benzyl benzoate (BB) in various ratios. Examples of thermogelling triblock copolymers include PLGA-PEG-PLGA triblock copolymers, poly(ε-caprolactone-coglycolic acid)-poly(ethylene glycol)-poly(ε-caprolactone-coglycolic acid) (PCGA-PEG-PCGA), and PLGA-PEG-PLGA. Poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO), also called Pluronic, is a nonionic amphiphilic triblock copolymer made by the sequential addition of propylene and ethylene oxide with a propylene glycol initiator. PF-127 gels can be used as a controlled peptide delivery system. Poly(ethylene glycol)-block-poly(alanine-cophenylalanine) (PEG-PAF) is another applicable material.

[0166] Liposomes, which are small lipid spheres composed of concentric lipid bilayers surrounding an aqueous compartment, are applicable as sustained delivery systems. Multivesicular liposomes (MLVs) consist of multiple non-concentric lipid layers. Since the diameter of MLVs is generally in the range of tens of microns, they can be loaded with large amounts of drug to maintain sustained drug release. Vesicular phospholipid gels (VPGs) are semi-solid liposome dispersions that contain aqueous compartments within or between the cores of vesicles. Therefore, they can be used to load and maintain the release of hydrophilic, amphiphilic, or lipophilic drugs. Another type of lipid gel is the phospholipid-based phase separation gel (PPSG), which converts from a sol state to a solid or semi-solid state after subcutaneous injection.

[0167] Inorganic micro / nanoparticles such as mesoporous silicon (PSi) and silica are applicable as long-term sustained drug delivery systems.

[0168] The toxicity of the drugs described herein can be determined by standard pharmaceutical procedures in cell culture or experimental animals, for example, by determining the LD50 (lethal dose for 50% of the population) or LD100 (lethal dose for 100% of the population). The dose ratio between the toxic effect and the therapeutic effect is the therapeutic index. Data obtained from these cell culture assays and animal studies can be used to establish a non-toxic dose range for human use. The doses of the proteins described herein are preferably within the range of circulating concentrations that include an effective dose with little or no toxicity. The dose may vary within this range depending on the dosage form used and the route of administration utilized. The exact formulation, route of administration, and dose can be selected by the individual physician in consideration of the patient's condition. (See, for example, Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Ch. 1).

[0169] A kit comprising the composition and instructions for use of the present invention is also within the scope of the present invention. The kit may further contain at least one additional reagent. The kit typically includes a label indicating the intended use of the contents of the kit. The term "label" includes any written or recorded material on or supplied with the kit, or otherwise accompanying the kit.

[0170] As will be obvious to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has separate components and features that can be readily separated or combined with features of any of several other embodiments without departing from the scope or spirit of the invention. Any method of description may be carried out in the order of the events described or in any other order that is logically possible. It will also be understood that the terms used herein are for the purpose of describing specific embodiments.

[0171] While the aforementioned inventions are described in some detail as examples and illustrations for the purpose of clarifying understanding, it will be readily apparent to those skilled in the art, in light of the teachings of the invention, that certain modifications and alterations can be made without departing from the spirit of the invention, and that there is no intention to limit the scope of the invention, which is limited only by the appended claims. Those skilled in the art will recognize, or can confirm by means of routine experimentation, many equivalents to the particular embodiments of the invention described herein. Such equivalents are intended to be encompassed within the appended claims. [Examples]

[0172] experiment Example 1 Targeted lysosomal degradation of αV integrin via the TG2 / LRP-1 pathway LYTACs (lysosome-targeted chimeras) are related bifunctional conjugates that crosslink endocytosis receptors (e.g., cation-independent mannose-6-phosphate receptors, asialoglycoprotein receptors) to target extracellular or cell surface proteins of interest, thereby enabling the internalization and lysosomal degradation of the target proteins. The discovery of LYTACs has sparked an intense search for novel receptor-mediated endocytosis mechanisms that can hijack for lysosomal delivery of pathogenic-related molecules in the extracellular matrix or on the plasma membrane of dysfunctional cells.

[0173] We recently characterized a novel receptor-mediated endocytosis pathway that enables gluten peptides, a major T cell antigen in the pathogenesis of celiac disease (CeD), to be simultaneously deamidated and enriched in the endolysosomal system of antigen-presenting cells (Loppinet et al., 2023; Figure 1). This pathway depends on both LRP-1, a cell surface receptor involved in receptor-mediated endocytosis, and transglutaminase 2 (TG2), an extracellular enzyme.

[0174] Given the exceptional ability of catalytically active extracellular TG2 to deliver low-abundance substrates to lysosomes in cells expressing the LRP-1 receptor, we have attempted to hijack the TG2 / LRP-1 pathway for targeted lysosomal delivery and subsequent degradation of molecules on the cell surface or in the extracellular matrix. This example describes the design, synthesis, and characterization of bifunctional molecules having ligands for integrins of the αV subfamily, which is the target protein family of interest, and high-affinity TG2 substrates or inhibitors.

[0175] Integrins are a family of more than 20 heterodimeric transmembrane proteins, each composed of α and β subunits. Dysregulation of integrin activity has been shown to be important in many cancers, inflammatory diseases, and fibrotic conditions. Therefore, integrin-targeted drugs have been approved for a variety of indications, including dry eye disease (rifitegrast), psoriasis (efalizumab), inflammatory bowel disease (vedolizumab), and cardiovascular thrombosis (abciximab, eptifibatide), with many others under development. Importantly, all approved integrin-targeted small molecules and antibody drugs are receptor antagonists that passively block integrin signaling. There remains a need to explore the therapeutic relevance of irreversibly eliminating targeted integrins from the cell surface via proteolysis.

[0176] The inventors sought to develop a prototype drug for targeted degradation of αvβ5 integrin, a representative member of the αV subfamily, which is inhibited by the pentapeptide sequence RGDVF. For this purpose, compound 100 was synthesized in which Ac-PQLPF-NH2 is linked to RGDVF via a GGGGS linker. TG2 exhibits similar specificity to 100 compared to Ac-PQLPF-NH2 (k cat / K M = 0.16 ± 0.04 μM -1 minutes -1Treatment of NRK cells with gradually increasing concentrations of 100 in the presence of 100 μg / mL α2-macroglobulin dose-dependently reduced the presence of αVβ5 on the cell surface. Interestingly, a hook effect was observed, with maximum degradation occurring at 1 μM HB320, and cell surface integrins increasing after this point (Figure 2A, Figure 2B). Degradation was also time-dependent, reaching maximum degradation after 6 hours of treatment (Figure 2C). Finally, the integrin ligand alone, RGDVF, did not result in a corresponding reduction of surface αVβ5 (Figure 2D). Further confirmation of the involvement of the LRP-1 pathway, pretreatment of cells with RAP, a known LRP-1 binder, rescued most of the surface αVβ5 (Figure 2E). The inventors also synthesized 101, which has HWE at the position of the reactive glutamine residue. Treatment with 101 yielded similar results to 100 (Figure 3).

[0177] To demonstrate the anti-fibrotic activity of 101, human lung fibroblasts (IMR90 cells) were treated with 0, 1, 5, and 20 ng / mL of TGFβ1 for 2 days to induce myofibroblast transition. For this purpose, IMR-90 cells (ATCC, CCL-186) were cultured according to the ATCC protocol. As described by Nichols et al. (1977), IMR-90 cells are a human diploid fibroblast cell line. The cells were seeded in a 24-well glass-bottom plate coated with vitronectin, incubated at 37°C for 24 hours in 5% CO2, and then treated with 20 ng / mL of human transforming growth factor beta-1 (TGFβ1) (PeproTech#100-21) for 48 hours to induce alpha-smooth muscle actin (α-SMA) expression (Figure 4A). The cells were then treated with different concentrations of 101 or a control reagent for 24 hours. After treatment, cells were immunostained and α-SMA expression was detected using an established protocol. Primary antibody staining was performed using mouse anti-α-SMA antibody (Sigma-Aldrich, 1A4) at dilutions of 1:500 to 1:1000 in blocking buffer, incubated overnight at 4°C. After washing three times with PBS, cells were stained with anti-mouse secondary antibody in blocking buffer at room temperature for 30 to 60 minutes. Significant reductions in α-SMA expression were observed in the presence of 0, 1, 3, 10, or 30 μM 101 (Figure 4B), with the greatest effect observed at 3 μM 101. Integrin ligand alone and RGDVF did not affect α-SMA expression at any of the test concentrations (Figure 4C).

[0178] Macrophages are another important class of cells that contribute to the progression of fibrosis by secreting pro-fibrotic cytokines, including TGFβ, IL-1β, and PDGF, which promote the transition from fibroblasts to myofibroblasts. To demonstrate the anti-fibrotic activity of 101 against macrophages, the RAW 264.7 cell line (ATCC, TIB-71) was used as a model. Cells were cultured as recommended according to the method of Raschke et al. (1978) (https: / / www.atcc.org / products / tib-71). To induce differentiation into M2a macrophages, RAW 264.7 cells (referred to as M0 cells) were treated with 20 ng / mL interleukin-4 (IL-4) for 48 hours. After differentiation, cells were further treated with either 101 or RGDVF peptide in DMEM for 24 hours. To monitor TGFβ1 expression, a highly sensitive bioassay was utilized as described by Tesseur et al. (2006). This assay is for TGFβ1 deficiency (Tgfb1 - / - The study involved the use of MFB-F11 cells, which are mouse embryonic fibroblasts derived from mice. These cells were stably transfected with a reporter plasmid containing a TGF-β responsive Smad-binding element (SBE) linked to a secreted alkaline phosphatase (SEAP) reporter gene. The protocol involved culturing MFB-F11 cells and treating them with conditioned medium derived from RAW 264.7 cells. SEAP activity, indicating TGFβ1 expression, was measured using the Great EscAPe® SEAP Chemiluminescence Kit 2.0 (Takara#631738) according to the manufacturer's instructions.

[0179] The expression of integrin β5 increases upon differentiation of M0 macrophages into M2 macrophages by exposure to 20 ng / mL of IL-4 (Figure 5A). Treatment with 1, 3, and 10 μM of 101 resulted in degradation of integrin β5 as evaluated by Western blot (Figure 5A). RGDVF alone had no equivalent effect. 101 treatment also reduced both total released TGFβ and the active form of TGFβ produced by these macrophages, while RGDVF showed no corresponding effect (Figures 5B - 5C).

[0180] Compound 102 is another useful example of a peptide integrin degrader.

[0181] [Chemical formula]

[0182] In the formula, T is AcP(HWE)LPFGGGGS-. The synthesis of 102 follows the method used to synthesize the commercially available peptide syringotide, except that the Val residue of syringotide is replaced with a suitably protected Lys residue. Upon deprotection, the primary amine of the cyclic peptide binds to the carboxy terminus of the TG2 binding moiety via the GGGGS linker.

[0183] Example 2 Bifunctional Degraders of αV Integrin with Non-Peptidic Integrin Binding Moieties This example describes bifunctional molecules that induce degradation of cell surface integrins by engaging integrins via non-peptidic moieties. Such molecules are expected to have the advantage of improved DMPK properties over the peptidic compounds described in the previous examples. The synthesis of the compounds in this example follows standard methods described in the art, such as those found in the Reaxys database (www.reaxys.com).

[0184] The synthesis of compound 103 (based on the non-peptide pan-αV ligand, MK-0429) is outlined below. In this scheme, the Aza-Michael reaction between alli-imidazolidinone and methoxypyridinyl acrylate yields a racemate of the integrin engagement moiety; however, the enantioselective synthesis of the desired stereoisomer can be readily achieved by other established methods. The identity of 103 was confirmed by mass spectrometry and by verifying that human TG2 exhibits similar specificity to that of Ac-PQLPF-NH2 for this compound.

[0185] [ka]

[0186] The αvβ5-degrading activity of 103 was confirmed in primary human lung fibroblasts obtained from ATCC. Similar to IMR90 fibroblasts, integrin β5 expression in these cells increased for 2 days in the presence of 20 ng / mL TGFβ1. Treatment with 103 for 24 hours induced a dose-dependent change in integrin β5 (Figure 6). Integrin β5 degradation reached a maximum at a 1 μM concentration of 103, up to approximately pre-TGFβ levels. At higher concentrations of 103, integrin β5 increased, likely due to a well-established hook effect related to the bifunctional molecule. The time-dependence of degradation was evaluated by adding 103 to primary human lung fibroblast cultures for 1, 2, 4, 8, and 16 hours, followed by washing off the compound and cell analysis at 16 hours. Treatment with 103 for just 1-2 hours was sufficient to maintain reduced integrin levels for at least 16 hours (Figure 7), highlighting the sustained effect of the integrin degrader after rinsing. In contrast, integrin activity rapidly recovers after drug rinsing when this class of receptors is blocked using conventional antagonists.

[0187] Compound 104 is another example of an integrin degrader based on the non-peptide pan-αV ligand, MK-0429.

[0188] [ka]

[0189] Compound 105, an integrin degradation agent based on a non-peptide αVβ5-specific ligand (Lippa et al, 2019), was synthesized as outlined below. Its identity was confirmed by mass spectrometry and by verifying that human TG2 exhibits a similar degree of specificity to 105 as Ac-PQLPF-NH2.

[0190] [ka]

[0191] The αvβ5-degrading activity of 105 was also confirmed in primary human lung fibroblasts (Figure 8). Cells were treated with 20 ng / mL TGFβ1 for 2 days to induce the transition from fibroblasts to myofibroblasts. Subsequently, treatment with 105 for 24 hours induced the degradation of integrin β5. The degradation of integrin β5 was maximized in the presence of 3 μM of the compound.

[0192] The presence of bromophenyl substituents in parenteral nonpeptidic αVβ5-specific ligands, as reported by Lippa et al (2019), enables alternative strategies for binding the TG2 engager moiety, as demonstrated by the bifunctional integrin degrader 106.

[0193] [ka]

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[0232] Cross-reference of related applications In accordance with 35 U.S.C. § 119(e), this application claims priority to the filing date of U.S. Provisional Patent Application No. 63 / 525,911, filed on 10 July 2023, the disclosure of which is incorporated herein by reference.

Claims

1. Includes a TG2 binding site connected to the integrin binding site. A bifunctional integrin-degrading molecule.

2. The TG2 binding portion comprises or consists of a polypeptide containing the pentapeptide sequence Pro-X-Z-R (Formula I), In formula I, X is selected from amino acids that engage with TG2 via the formation of a covalent enzyme-compound intermediate selected from glutamine, α-diazoketone, α-halo-ketone, αβ-unsaturated carbonyl compounds, and αβ-unsaturated sulfones. Z is, and selected from dipeptide YP, where P is proline and Y is any amino acid or a non-natural amino acid. R is a natural or unnatural aromatic amino acid, and The C-terminus of the pentapeptide is a carboxylic acid, ester, or amide. The bifunctional molecule according to claim 1.

3. The TG2 binding portion is the peptide sequence Ac-PQLPF-NH ２ ; or comprising or derived from a modified peptide in which the reactive glutamine residue is replaced with an electrophilic attack group. A bifunctional molecule according to claim 1 or claim 2.

4. A bifunctional molecule according to claim 1 or claim 2, having a structure selected from the following. 【Chemistry 1】 【Chemistry 2】 R １ Cl, Br, I, OSO ２ CF ３ And; R ２ is N(CH ３ ) ２ , OCH ３ ; NH ２ , CH ３ ; and 【Transformation 3】 Y is any amino acid, or 【change】 And; R is an aromatic amino acid; Link is a linker.

5. The linker is PEG ｎ PEG ｎ Selected from diamines, piperazines, 4-aminopiperidines, 3,9-diazaspiro[5.5]undecanes, linkers suitable for click chemistry, hydrocarbons, and peptide linkers. The bifunctional molecule according to claim 4.

6. The linker is a small aliphatic or aromatic group having at least one terminal nitrogen that forms an amide bond with the carboxyl terminus of the TG2 bond portion. The bifunctional molecule according to claim 5.

7. A bifunctional molecule according to any one of claims 1 to 2 and 4 to 6, having any of the following structures. 【Chemistry 4】

8. The integrin binding portion selectively binds to the target integrin or integrin class. A bifunctional molecule according to any one of claims 1 to 7.

9. The integrin binding site is selective for one or more of αVβ1, αVβ3, αVβ5, αVβ6, and αVβ8. A bifunctional molecule according to any one of claims 1 to 7.

10. The integrin-binding portion is selective for binding to human αVβ5 integrin. A bifunctional molecule according to any one of claims 8 to 9.

11. The integrin-binding portion is a peptide containing an RGD sequence. A bifunctional molecule according to any one of claims 1 to 10.

12. The aforementioned peptide is of the formula RGDX １ X ２ It belongs to The bifunctional molecule according to claim 11.

13. The peptide has the sequence RGDVF, RGDNF, or RGDNY. The bifunctional molecule according to claim 12.

14. The integrin-binding portion is selected from peptides, cyclic peptides, small molecules, antibodies or antibody-binding fragments, nanobodies, and aptamers. A bifunctional molecule according to any one of claims 1 to 10.

15. The bifunctional molecule according to any one of claims 1 to 6, wherein the cyclic peptide has the following structure. 【Transformation 5】 In the formula, T is the TG2 bonded portion coupled to the cyclic peptide via an amide bond, and n = 0 to 3.

16. The compound is a bifunctional molecule according to any one of claims 1 to 6, wherein the compound has the following structure. 【Transformation 6】 In the formula, T is the TG2 bond portion coupled to the integrin bond portion via an amide bond.

17. The compound is a bifunctional molecule according to any one of claims 1 to 6, wherein the compound has the following structure. 【Transformation 7】 In the formula, T is the TG2 bond portion coupled to the integrin bond portion via an amide bond, and X is O, NH, or CH ２ That is the case.

18. The compound is a bifunctional molecule according to any one of claims 1 to 6, wherein the compound has the following structure. 【Transformation 8】 In the ceremony, T １ H is the TG2 bonding portion, and T ２ is the TG2 bond portion identified by H or any of the formulas I to IV disclosed above, wherein one of T1 and T2 is the TG2 bond portion, and typically T １ and T ２ Only one of them is the TG2 bond region.

19. A pharmaceutical composition comprising a bifunctional molecule according to any one of claims 1 to 18 and a pharmaceutically acceptable excipient.

20. A unit dose of the pharmaceutical composition according to claim 19.

21. A method for the prevention or treatment of fibrosis in mammalian patients, A method comprising administering a bifunctional molecule according to any one of claims 1 to 18 to a mammalian patient in a dose effective in preventing or reducing fibrosis.

22. The method according to claim 21, wherein the dose is effective in achieving pharmacological blockade of transglutaminase 2 and degradation of integrin.

23. The method according to claim 21 or 22, wherein the fibrosis is selected from pulmonary fibrosis, cutaneous fibrosis, cirrhosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), cancer-associated fibrosis, intestinal fibrosis, and renal fibrosis.

24. The method according to any one of claims 21 to 23, wherein the bifunctional molecule is administered locally to a site of cancer or fibrous tissue.

25. The method according to any one of claims 21 to 23, wherein the bifunctional molecule is administered systemically.

26. A method according to any one of claims 21 to 25, wherein the individual is A method, which involves analysis after prevention or treatment by a method including determining the degree of fibrosis in the patient or in a biological sample of the patient.

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