Methods for fibroblast activation protein (FAP)-targeted imaging and therapy of cancers and other fibrotic and inflammatory diseases

Abstract

To provide: fibroblast activation protein (FAP)-targeting compounds (e.g., conjugates); methods for imaging cancer or fibrosis; and methods for treating an inflammatory disease / disorder and cancer.SOLUTION: The invention provides a compound represented by a structure of formula (X), Am-L-B, where: A is a radical of a fibroblast activation protein α (FAPα) ligand; L is a linker connecting one or more A groups to B; B is a radical of an optical dye, a photodynamic therapeutic agent, a radio-imaging agent, a radiotherapeutic agent, a chemotherapeutic agent, an antifibrotic agent, or an anticancer agent; and m is 1-6. The invention also provides a method for imaging cancer or fibrosis in a subject with the cancer or the fibrosis, the method comprising administering an effective amount of the compound to a subject in need thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 901,792, filed September 17, 2019, which is incorporated herein by reference in its entirety.

[0002] Technical Field The present disclosure relates to the development of certain chemical compounds and radicals, such as ligands that target fibroblast activation protein (FAP-α). In some cases, the radicals are conjugated to drugs or imaging agents. In certain cases, such conjugated compounds (also referred to herein as "conjugates") are used in various methods, such as treating and / or imaging (e.g., FAP-positive) cancer, fibrotic diseases, and / or inflammatory disorders. In some cases, cancer-associated fibroblasts (CAFs) and / or activated myofibroblasts (e.g., in cancer and / or other fibrotic and inflammatory diseases) are specifically targeted (e.g., in therapeutic and / or imaging methods). In some cases, the chemical compounds and / or ligands provided herein have good or improved internalization and residence time in tumors and other disease sites. [Background technology]

[0003] background Radiation therapy and chemotherapy are considered for a variety of cancers, fibrotic diseases, and inflammatory disorders. Such therapies are often not considered as first-line treatments because of their potential adverse (e.g., systemic) effects.

[0004] As a result, there is a need for targeted therapies that treat diseases (e.g., cancer, fibrotic diseases, and / or inflammatory diseases) with radiation and / or chemotherapeutic agents that are suitable for targeting diseased cells and tissues with minimal or reduced off-target or systemic effects.

[0005] In some cases, the survival and growth of certain tumors depend on the percentage of tumor stroma (TSP). High TSP may be associated with poorer long-term patient survival compared with low TSP (>50% vs. ≦50%, respectively). TSP may also be an important prognostic factor for tumor recurrence, growth, and metastasis.

[0006] In certain cases, cancer-associated fibroblasts (CAFs) are abundant in the tumor stroma and perform several important functions to promote tumorigenesis. These functions include, by way of non-limiting example, cytokine secretion and / or extracellular matrix (ECM) production and remodeling. In some cases, such effects result in angiogenesis that promotes tumor growth, signaling factors that increase chemotherapy resistance, a denser ECM that creates an immunosuppressive environment, and enhanced cell motility that promotes metastasis. In some cases, such processes correspond to the behavior of pathogenic fibroblasts in fibrotic diseases.

[0007] In some cases, a common marker for CAFs is fibroblast activation protein alpha (FAPα). Furthermore, FAPα is a serine protease found (primarily) on the cell surface of activated fibroblasts in diseased cells and tissues, for example, in fibrotic diseases, inflammatory diseases, and / or cancers (e.g., fibrosis, rheumatoid arthritis, wound healing, and cancer). More than 90% of epithelial cancers show FAPα expression in immunohistochemical (IHC) staining. Further FAPα expression has been found in primary glioma cell cultures and a subset of tumor-associated macrophages (TAMs). However, FAPα expression is very low or absent in most adult tissues. Therefore, because its expression is restricted to the surface of diseased cells, such as carcinomas, FAPα is less suitable as a receptor for selectively delivering therapeutic agents to tumors via ligand targeting. Summary of the Invention

[0008] overview A compound (e.g., a conjugate) of formula X is provided: A m -LB(X) During the ceremony, A is a radical of a fibroblast activation protein alpha (FAPα) ligand (targeting moiety) (e.g., molecular weight less than 10,000); L is a (e.g., bifunctional) linker that connects one or more A groups to B (e.g., via a first covalent bond connecting L to A and a second covalent bond bonding L to B); B is (e.g., a radical of) an imaging agent, a photodynamic therapy agent, a radioactive imaging agent, a radioactive therapeutic agent, a chemotherapeutic agent, an antifibrotic agent, or an anticancer agent (e.g., an anticancer agent that is effective against cancer cells or cancer-associated fibroblasts, myofibroblasts, or other tumor microenvironment factors); and m is 1 to 6.

[0009] Also provided are compounds (e.g., conjugates) of Formula I, ALB (I) During the ceremony, A comprises (e.g., a radical of) a FAPα ligand (e.g., a targeting moiety); L comprises a (e.g., bifunctional) linker connecting one or more A groups to B; and B includes (e.g., a radical of) an optical imaging agent, a photodynamic therapy agent, a radioactive imaging agent, a radioactive therapeutic agent, a chemotherapeutic agent, an antifibrotic agent, or an anticancer agent (e.g., an anticancer agent effective against cancer cells or cancer-associated fibroblasts, myofibroblasts, or other tumor microenvironment factors).

[0010] There is also provided a method for imaging cancer or fibrosis in a subject having cancer or fibrosis, the method comprising administering to a subject in need thereof an effective amount of a compound.

[0011] Also provided is a method for treating an inflammatory disease or disorder, the method comprising administering to a subject suffering therefrom a therapeutically effective amount of a compound.

[0012] Still yet further provided is a method for treating cancer, the method comprising administering to a subject suffering therefrom a therapeutically effective amount of a compound. [The present invention 1001] A compound represented by the structure of formula (X): A m -LB(X) During the ceremony, A is the radical of fibroblast activation protein α (FAPα) ligand; L is a linker connecting one or more A groups to B; B is a radical of an optical dye, a photodynamic therapy agent, a radioactive contrast agent, a radioactive therapeutic agent, a chemotherapeutic agent, an antifibrotic agent, or an anticancer agent; and m is 1 to 6. [The present invention 1002] A has the structure of formula (XA), TIFF2025131780000001.tif22128 formula, Q is aryl, heteroaryl, or heterocyclyl; Z is a bond, substituted or unsubstituted C1-C3 alkylene, substituted or unsubstituted heteroalkylene, amino, -O-, or -S-; T is substituted or unsubstituted methylene, substituted or unsubstituted amino, -O-, or -S-; R 1 and R 2 are each independently selected from the group consisting of -H, -CN, -CHO, -B(OH)2, -C(O)alkyl, -C(O)aryl-, -C=CC(O)aryl, -C=CS(O)2aryl, -CO2H, -SO3H, -SON2NH2, -PO3H2, -SO2F, -CONH2, and 5-tetrazolyl; R 3 and R 4 are each independently -H, -OH, F, Cl, Br, I, -C 1~6 Alkyl, -OC 1~6 Alkyl, and -SC 1~6 alkyl; and R 5 , R 6 , R7 , and R 8 are each independently selected from the group consisting of H, alkyl, and halo; 1001 compounds of the present invention. [The present invention 1003] A has the structure of formula (XB), TIFF2025131780000002.tif43128In formula, Q is aryl, heteroaryl, or heterocyclyl; T is substituted or unsubstituted methylene, substituted or unsubstituted amino, -O-, or -S-; J is C(R J )2, where each R J are independently H or alkyl, or both R J together to form oxo; R 1 and R 2 are each independently selected from the group consisting of -H, -CN, -CHO, -B(OH)2, -C(O)alkyl, -C(O)aryl-, -C=CC(O)aryl, -C=CS(O)2aryl, -CO2H, -SO3H, -SON2NH2, -PO3H2, -SO2F, -CONH2, and 5-tetrazolyl; R 3 and R 4 are each independently -H, -OH, F, Cl, Br, I, -C 1~6 Alkyl, -OC 1~6 Alkyl, and -SC 1~6 selected from the group consisting of alkyl; R 5 , R 6 , R 7 , and R 8 are each independently selected from the group consisting of H, alkyl, and halo; and R 9 , R 10 , and R 11 are independently H, -C 1~6 Alkyl, -C 1~6 Haloalkyl, -OC 1~6 Alkyl, -SC 1~6selected from the group consisting of alkyl, F, Cl, Br, and I; The compound of the present invention 1001 or 1002. [The present invention 1004] A, Any of compounds 1001 to 1003 of the present invention selected from the group consisting of TIFF2025131780000003.tif219147. [The present invention 1005] A, Any of compounds 1001 to 1003 of the present invention selected from the group consisting of TIFF2025131780000004.tif89129. [The present invention 1006] Any of compounds 1001 to 1005 of the present invention, wherein A has a binding affinity to FAPα of about 1 nM to about 25 nM. [The present invention 1007] 1006. The compound of any of claims 1001 to 1006, wherein L comprises one or more linker groups, each linker group independently selected from the group consisting of alkyl(ene), heteroalkyl(heteroalkylene), heterocycloalkyl(heterocycloalkylene), heteroaryl, aryl, alkoxy, thioether, disulfide, carboxylic acid, anhydride, carbonate, carbamate, thioether, sugar, and peptide. [The present invention 1008] 8. The compound of any of claims 1001 to 1007, wherein L comprises one or more linker groups, each linker group independently selected from the group consisting of polyethylene glycol (PEG), alkyl(ene), disulfide, amide, carboxylic acid, anhydride, carbonate, ester, carbamate, thioether, phenyl, and triazole. [The present invention 1009] 9. The compound of any of claims 1001-1008, wherein L comprises one or more linker groups, each linker group independently selected from the group consisting of polyethylene glycol (PEG), alkyl(ene), disulfide, amide, carboxylic acid, carbonate, and ester. [The present invention 1010] The compound of any one of claims 1001 to 1008, wherein L is a releasable linker. [The present invention 1011] 9. The compound of any of claims 1001-1008, wherein L comprises one or more linker groups, each linker group independently selected from the group consisting of polyethylene glycol (PEG), alkyl(ene), amide, phenyl, and triazole. [The present invention 1012] The compound of any one of claims 1001 to 1008 and 1011, wherein L is a non-releasable linker. [The present invention 1013] L is (L 1 ) o -Y-(L2) p and where: Each L 1 is the first linker; Each L 2 is the second linker; Y is a third linker; o is an integer from 1 to 5; and p is an integer from 1 to 5; Any of compounds 1001 to 1012 of the present invention. [The present invention 1014] Each L 1 and L 2 independently comprise one or more linker groups, each linker group independently selected from the group consisting of alkyl(ene), heteroalkyl(heteroalkylene), heterocycloalkyl(heterocycloalkylene), heteroaryl, aryl, alkoxy, thioether, disulfide, carboxylic acid, anhydride, carbonate, carbamate, thioether, sugar, and peptide. [The present invention 1015] Each L 1 and L 2independently comprise one or more linker groups, each linker group independently selected from the group consisting of polyethylene glycol (PEG), alkyl(ene), disulfide, amide, carboxylic acid, anhydride, carbonate, ester, carbamate, thioether, phenyl, and triazole. [The present invention 1016] Each L 1 and L 2 independently comprise one or more linker groups, each linker group independently selected from the group consisting of polyethylene glycol (PEG), alkyl(ene), disulfide, amide, carboxylic acid, carbonate, and ester. [The present invention 1017] Each L 1 and L 2 independently comprise one or more linker groups, each linker group independently selected from the group consisting of polyethylene glycol (PEG), alkyl (alkylene), amide, phenyl, and triazole. [The present invention 1018] The compound of any one of claims 1013 to 1017, wherein Y has an amine core or an aromatic core. [The present invention 1019] The compound of any one of claims 1013 to 1017, wherein Y has an amine core, an aromatic core, or an alkylene core. [The present invention 1020] L, L 1 , L 2 or any combination thereof independently have a length of 15 to 200 angstroms (Å). [The present invention 1021] L, L 1 , L 2 or any combination thereof independently having the following structure: 10. The compound of any one of claims 1001 to 1020, comprising at least one linker group having TIFF2025131780000005.tif22143, wherein n is 0 to 10. [The present invention 1022] L, L 1 , L 2 or any combination thereof independently having the following structure: Any of compounds of inventions 1001 to 1021, comprising at least one linker group having TIFF2025131780000006.tif18128. [The present invention 1023] L, L 1 , L 2 or any combination thereof, having the following structure: Any of compounds of inventions 1001 to 1022, comprising at least one linker group having TIFF2025131780000007.tif47135, wherein n is 1 to 32. [The present invention 1024] L has the following structure: Any of compounds 1001 to 1023 of the present invention having TIFF2025131780000008.tif20128. [The present invention 1025] The compound of any one of claims 1001 to 1024, wherein B is a radical of a (eg, fluorescent) dye. [The present invention 1026] The compound of any one of claims 1001 to 1025, wherein the compound is a contrast agent and B is a radical of a fluorescent dye. [The present invention 1027] The compound of any one of claims 1001 to 1024, wherein B is a radical of an anticancer agent or an antifibrotic agent. [The present invention 1028] The compound of any one of claims 1001 to 1024 or 1027, wherein said compound is a chemotherapeutic agent and B is a radical of an antifibrotic agent or an anticancer agent. [The present invention 1029] The compound of any one of claims 1001 to 1024, 1027, or 1028, wherein B is a radical of a phosphoinositide 3-kinase (PI3K) inhibitor. [The present invention 1030] B has the following formula: TIFF2025131780000009.tif23128, wherein X is a radical represented by Any of compounds 1001 to 1024 or 1027 to 1029 of the present invention selected from the group consisting of TIFF2025131780000010.tif30153. [The present invention 1031] B has the following structure: Any of compounds 1001 to 1024 or 1027 to 1030 of the present invention, which is a radical of TIFF2025131780000011.tif34128. [The present invention 1032] The following structure: Compound TIFF2025131780000012.tif64132. [The present invention 1033] The following structure: Compound TIFF2025131780000013.tif59128. [The present invention 1034] The following structure: Compound TIFF2025131780000014.tif71154. [This invention 1035] The following structure: Compound TIFF2025131780000015.tif212118. [The present invention 1036] A pharmaceutical composition comprising any one of the compounds of the present invention 1001 to 1035 and a pharmaceutically acceptable carrier. [This invention 1037] A method for imaging cancer or fibrosis in a subject having cancer or fibrosis, comprising administering an effective amount of any of the compounds of the present inventions 1001 to 1026 and 1035 to a subject in need thereof. [The present invention 1038] A method for treating an inflammatory disease or disorder, comprising the step of administering a therapeutically effective amount of any of the compounds of the present inventions 1001 to 1035 to a subject in need thereof. [This invention 1039] A method for treating cancer, comprising the step of administering a therapeutically effective amount of any of the compounds of the present inventions 1001 to 1035 to a subject in need thereof. [Brief explanation of the drawings]

[0013] The drawings illustrate generally, by way of example, but not by way of limitation, various aspects discussed in the present document. [Figure 1]

[0023] Figure 1 shows the retrosynthesis of a ligand that targets fibroblast activation protein (FAP). [Figure 2] 1 shows the structure of a targeting compound with a targeting ligand. [Figure 3] Shows increased binding with increasing concentrations of targeting ligand (e.g., (A) at 50 nM, (B) at 25 nM, (C) at 12.5 nM, (D) at 6.25 nM) to fibroblasts with high concentrations of fibroblast activation protein (FAP). [Figure 4] Binding of targeting ligand at a single concentration to fibroblasts with high surface concentrations of FAP after (A) 1 hour, (B) 8 hours, (C) 24 hours, and (D) 48 hours of culture is shown. [Figure 5] Binding of targeting ligand to fibroblasts with high surface concentrations of FAP using at least a 100-fold excess of competing ligand for 1 hour (e.g., A: 25 nM targeting ligand, 2.5 μM competitor; B: 25 nM targeting ligand, 5 μM competitor). [Figure 6] Binding of targeting ligands to similar fibroblasts without high surface concentrations of FAP (e.g., (A) at 100 nM and (B) at 200 nM) is shown. [Figure 7] Binding curves of targeting ligands to fibroblasts with high surface concentrations of FAP (or FAP fibroblasts) are shown. [Figure 8] Binding curves of targeting ligand to FAP fibroblasts (targeting ligand only (circles) and targeting ligand and competitor (squares)) and FAP fibroblasts (triangles) are shown. [Figure 9A] 1 shows imaging results demonstrating in vivo tumor-specific targeting of the targeting ligand 2 to 32 hours after administration to mammals bearing tumors with a high FAP environment. [Figure 9B] 1 shows imaging results demonstrating in vivo tumor-specific targeting of the targeting ligand 48 to 122 hours after administration to mammals bearing tumors with a high FAP environment. [Figure 9C] This figure shows the biodistribution of a targeting ligand in the tumor, heart, liver, lungs, spleen, kidneys, intestine, and stomach 122 hours after administration to tumor-bearing mammals (MDA-MB-231 xenograft mice) with a high FAP environment. Black or white ovals or circles in the image highlight the locations where the targeting ligand is present. Darker shading within the oval or circle represents a higher concentration of targeting ligand than lighter shading within the oval or circle. [Figure 10] In vivo imaging of a targeting ligand is shown 2 and 6 hours after administration to a tumor-bearing mammal (MDA-MB-231 xenograft mouse) with a high FAP environment, both in the presence (right) and absence (left) of an unlabeled competitor. Black ovals or circles in the images highlight the location of the targeting ligand. Darker shading within the oval or circle represents a higher concentration of targeting ligand than lighter shading within the oval or circle. [Figure 11A] Shown is in vivo imaging of the targeting ligand 2 to 32 hours after administration to another tumor-bearing mammal (KB xenograft mouse) with a high FAP environment. [Figure 11B] Shown is in vivo imaging of the targeting ligand 48 to 122 hours after administration to another tumor-bearing mammal (KB xenograft mouse) with a high FAP environment. [Figure 11C]Figure 1 shows the biodistribution of a targeting ligand in the tumor, heart, liver, lungs, spleen, kidneys, intestine, and stomach 122 hours after administration to a mammal bearing a tumor with a high FAP environment. Black ovals or circles in the image highlight locations where the targeting ligand is present. Darker shading within the oval or circle represents a higher concentration of targeting ligand than lighter shading within the oval or circle. [Figure 12] In vivo imaging of the targeting ligand is shown 2 and 6 hours after administration to another tumor-bearing mammal (KB xenograft mouse) with a high FAP environment, both in the presence (right) and absence (left) of unlabeled competitor. Black ovals or circles in the images highlight the location of the targeting ligand. Darker shading within the oval or circle represents a higher concentration of targeting ligand than lighter shading within the oval or circle. [Figure 13] Biodistribution of targeting ligand in tumor, heart, liver, lung, spleen, kidney, intestine, and stomach at 2, 4, 6, 6 (kidney coverage (KC)), 15, 24, and 122 hours after administration to mammals (KB xenograft mice) bearing another tumor with a high FAP environment. Black or white arrows, ellipses, or circles in the images highlight the location of the targeting ligand. Darker shading near the arrowhead or within the ellipse or circle represents a higher concentration of targeting ligand than lighter shading. [Figure 14A] In vivo imaging of the targeting ligand 6 hours after injection into mammals bearing different tumors with a high FAP environment (FADu xenograft mice M1, M2, M3). [Figure 14B] 1 shows the biodistribution of FAP targeting compounds in tumor, heart, liver, lung, spleen, kidney, intestine, muscle, and stomach after 6 hours. [Figure 14C] 1 shows in vivo imaging of a competition experiment between an exemplary FAP-targeting compound and an unlabeled competitor 6 hours after injection into mammals bearing different tumors with a high FAP environment (FADu xenograft mice M1, M2, and M3). [Figure 14D] Figure 1 shows the biodistribution of FAP-targeting compounds in tumor, heart, liver, lung, spleen, kidney, intestine, muscle, and stomach after 6 hours. Black or white arrows, ellipses, or circles in the images highlight the locations of targeting ligand. Darker shading near the arrowhead or within the ellipse or circle represents higher concentrations of targeting ligand than lighter shading. [Figure 15A] 1 shows in vivo imaging of the targeting ligand after administration to mammals bearing different tumors with a high FAP environment (HT29 xenograft mice M1, M2, and M3). [Figure 15B] 1 shows the biodistribution of FAP targeting compounds in tumor, heart, liver, lung, spleen, kidney, intestine, muscle, and stomach after 6 hours. [Figure 15C] Figure 15C shows the in vivo imaging of the competition experiment between targeting ligand and unlabeled competitor after administration. Figure 15C shows the biodistribution of FAP targeting compound in tumor, heart, liver, lung, spleen, kidney, intestine, muscle and stomach after 6 hours. Black or white arrows, ellipses or circles in the image highlight the location where targeting ligand is present. Darker shades near the arrowhead or in the ellipses or circles represent higher concentrations of targeting ligand than lighter shades. [Figure 15D] Figure 1 shows the biodistribution of FAP-targeting compounds in tumors, heart, liver, lungs, spleen, kidneys, intestines, muscle, and stomach after 6 hours. Black or white arrows, ellipses, or circles in the images highlight the locations of targeting ligands. Darker shading near the arrowheads or within the ellipses or circles represents higher concentrations of targeting ligand than lighter shading in HT29 xenograft mice. [Figure 16A] 1 shows in vivo imaging of targeting ligands after administration to mammals bearing tumors with a high FAP environment (KB tumor xenografted mice (e.g., M1, M2, and M3)). [Figure 16B] Biodistribution in tumor, heart, liver, lung, spleen, kidney, intestine, muscle, and stomach is shown. [Figure 16C] 1 shows in vivo imaging of a competition experiment between a targeting ligand and an unlabeled competitor after administration to mammals bearing tumors with a high FAP environment (KB tumor xenografted mice (e.g., M1, M2, and M3)). [Figure 16D] Figure 1 shows the biodistribution in tumor, heart, liver, lung, spleen, kidney, intestine, muscle, and stomach for a competition study. Black or white arrows, ellipses, or circles in the images highlight the location of the targeting ligand. Darker shading near the arrowhead or within the ellipse or circle represents a higher concentration of targeting ligand than lighter shading. [Figure 17A] 1 shows in vivo imaging of targeting ligands after administration to tumor-bearing mammals (MDA-MB-231 tumor xenografted mice (e.g., M1, M2, and M3)) with a high FAP environment. [Figure 17B] 1 shows the biodistribution of FAP targeting compounds in tumor, heart, liver, lung, spleen, kidney, intestine, muscle, and stomach. [Figure 17C] 1 shows in vivo imaging of a competition experiment between a targeting ligand and 500 nmol of unlabeled competitor. [Figure 17D] Figure 1 shows the biodistribution of FAP-targeting compounds in tumors, heart, liver, lungs, spleen, kidneys, intestines, muscle, and stomach in a competition study. Black or white arrows, ellipses, or circles in the images highlight the locations where the targeting ligand is present. Darker shading near the arrowhead or within the ellipse or circle represents a higher concentration of targeting ligand than lighter shading. [Figure 18A] 1 shows in vivo imaging of targeting ligands after administration to mammals bearing different tumors with a high FAP environment (U87MG tumor xenografted mice (e.g., M1, M2, and M3)). [Figure 18B] 1 shows the biodistribution of FAP targeting compounds in tumor, heart, liver, lung, spleen, kidney, intestine, muscle, and stomach. [Figure 18C]1 shows in vivo imaging of a competition experiment between a targeting ligand and an unlabeled competitor. [Figure 18D] Biodistribution in tumor, heart, liver, lung, spleen, kidney, intestine, muscle, and stomach in a competition study is shown. Black or white arrows, ellipses, or circles in the images highlight the location of the targeting ligand. Darker shading near the arrowhead or within the ellipse or circle represents a higher concentration of targeting ligand than lighter shading. [Figure 19A] 1 shows in vivo imaging of the targeting ligand after administration to mammals bearing different tumors with a high FAP environment (PANC1 tumor xenografted mice (e.g., M1, M2, and M3)). [Figure 19B] 1 shows the biodistribution of FAP targeting compounds in tumor, heart, liver, lung, spleen, kidney, intestine, muscle, and stomach. [Figure 19C] 1 shows in vivo imaging of a competition experiment between a targeting ligand provided herein and an unlabeled competitor. [Figure 19D] Biodistribution in tumor, heart, liver, lung, spleen, kidney, intestine, muscle, and stomach in a competition study is shown. Black or white arrows, ellipses, or circles in the images highlight the location of the targeting ligand. Darker shading near the arrowhead or within the ellipse or circle represents a higher concentration of targeting ligand than lighter shading. [Figure 20A] Shown is in vivo imaging of the targeting ligand 2 hours after injection upon administration to a mammal bearing another tumor (4T1 tumor xenografted mouse) with a high FAP environment. [Figure 20B] Figure 1 shows the biodistribution of FAP-targeting compounds in tumor, heart, liver, lung, spleen, kidney, intestine, muscle, and stomach after 6 hours. Black or white arrows, ellipses, or circles in the images highlight the locations of targeting ligand. Darker shading near the arrowhead or within the ellipse or circle represents higher concentrations of targeting ligand than lighter shading. [Figure 20C]Figure 1 shows the biodistribution of FAP-targeting compounds in tumors, heart, liver, lungs, spleen, kidneys, intestines, muscle, and stomach after 6 hours. Black or white arrows, ellipses, or circles in the images highlight the locations of targeting ligands. Darker shading near the arrowhead or within the ellipses or circles represents higher concentrations of targeting ligand in 4T1 xenografted mice than lighter shading. [Figure 21] 1 shows displacement binding curves of targeting ligands in HEK-FAP cells. [Figure 22] 1 shows displacement binding curves of targeting ligands in HEK-FAP cells. [Figure 23] 1 shows a Western blot of Akt (protein kinase B) phosphorylation levels in transforming growth factor (TGF)-β-stimulated human lung fibroblasts after treatment with a phosphoinositide 3-kinase inhibitor (PI3Ki) or a targeting compound provided herein (e.g., at a concentration of 1 nM, 10 nM, or 100 nM). [Figure 24] 1 shows the relative change in collagen 1A1 mRNA expression in transforming growth factor (TGF)-β-stimulated human lung fibroblasts after treatment with a phosphoinositide 3-kinase inhibitor (PI3Ki) or a targeting compound provided herein (e.g., at a concentration of 1 nM, 10 nM, or 100 nM). DETAILED DESCRIPTION OF THE INVENTION

[0014] Detailed Description definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art of chemistry and biology. Additionally, as used in this specification and the appended claims, the singular forms "a," "an," and "the" also include the plural of the referent unless the context clearly dictates otherwise. Thus, for example, if a compound / composition is substituted with "an" alkyl or aryl, the compound / composition may also be substituted with at least one alkyl and / or at least one aryl. Furthermore, unless otherwise specified, the term "about" refers to a range of plus or minus 10% for percentages and plus or minus 1.0 units for unit values; for example, about 1.0 refers to a range of 0.9 to 1.1.

[0015] A "therapeutically effective amount" (or "effective amount") of a compound, with respect to use in therapy, refers to the amount of compound in a formulation that, when administered (to a mammal such as a human) as part of a desired dosing regimen, alleviates the symptoms, improves the condition, or delays the onset of a disease state in accordance with clinically acceptable criteria for the disorder or condition being treated or for cosmetic purposes, e.g., at a reasonable benefit / risk ratio applicable to any medical treatment.

[0016] The term "prophylactic or therapeutic" treatment is art-recognized and includes administration to a patient of one or more compounds of the present disclosure. When administered prior to the clinical appearance of an undesirable condition (e.g., a disease or other undesirable condition in a host animal), the treatment is prophylactic (i.e., protects the host from the development of the undesirable condition), whereas when administered after the appearance of the undesirable condition, the treatment is therapeutic (i.e., intended to reduce, ameliorate, or stabilize an existing undesirable condition or its side effects).

[0017] The terms "patient," "individual," or "subject" refer to a mammal in need of a particular treatment. The patient or subject may be a primate, dog, cat, or horse. The patient or subject may be a bird. The bird may be a domesticated bird, such as a chicken. The bird may be poultry. The patient or subject may be a human.

[0018] "Oxo" refers to the =O radical.

[0019] "Alkyl" refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, such as those having 1 to 15 carbon atoms (e.g., C1-C 15 The disclosure provided herein of "alkyl" is intended to include the independent description of saturated "alkyl" unless otherwise specified. Alkyl can contain 1 to 13 carbon atoms (e.g., C1-C 13 alkyl). An alkyl can contain 1 to 8 carbon atoms (e.g., C1-C8 alkyl). An alkyl can contain 1 to 5 carbon atoms (e.g., C1-C5 alkyl). An alkyl can contain 1 to 4 carbon atoms (e.g., C1-C4 alkyl). An alkyl can contain 1 to 3 carbon atoms (e.g., C1-C3 alkyl). An alkyl can contain 1 to 2 carbon atoms (e.g., C1-C2 alkyl). An alkyl can contain 1 carbon atom (e.g., C1 alkyl). An alkyl can contain 5 to 15 carbon atoms (e.g., C5-C 15 alkyl). The alkyl can contain 5 to 8 carbon atoms (e.g., C5-C8 alkyl). The alkyl can contain 2 to 5 carbon atoms (e.g., C2-C5 alkyl). The alkyl can contain 3 to 5 carbon atoms (e.g., C3-C5 alkyl). In another embodiment, the alkyl group is selected from the group consisting of methyl, ethyl, 1-propyl (n-propyl), 1-methylethyl (iso-propyl), 1-butyl (n-butyl), 1-methylpropyl (sec-butyl), 2-methylpropyl (isobutyl), 1,1-dimethylethyl (tert-butyl), and 1-pentyl (n-pentyl). The alkyl is attached to the remainder of the molecule by a single bond.

[0020] "Alkoxy" refers to a radical attached through an oxygen atom of the formula --O-alkyl, where alkyl is an alkyl chain as defined above.

[0021] "Alkylene" or "alkylene chain" generally refers to a straight- or branched-chain divalent alkyl group, such as those having 1 to 12 carbon atoms, that attaches the remainder of the molecule to a radical group, e.g., methylene, ethylene, propylene, i-propylene, n-butylene, etc.

[0022] "Aryl" refers to a radical derived from an aromatic monocyclic or polycyclic hydrocarbon ring system by removing a hydrogen atom from a ring carbon atom. The aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and carbon atoms from 5 to 18 carbon atoms, where at least one of the rings within the ring system is fully unsaturated, i.e., contains a cyclic delocalized (4n+2) π-electron system according to Hückel theory. Ring systems from which aryl groups are derived include, but are not limited to, groups such as benzene, fluorene, indane, indene, tetralin, and naphthalene.

[0023] An "aralkyl" or "aryl-alkyl" is a group of the formula -R c -aryl radical, where R c is an alkylene chain as defined above, for example, methylene, ethylene, etc. The alkylene chain part of the aralkyl radical may be optionally substituted as described above for an alkylene chain.

[0024] "Carbocyclyl" or "cycloalkyl" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical, consisting solely of carbon and hydrogen atoms, including fused or bridged ring systems having 3 to 15 carbon atoms. A carbocyclyl can contain 3 to 10 carbon atoms. A carbocyclyl can contain 5 to 7 carbon atoms. A carbocyclyl is attached to the rest of the molecule by a single bond. A carbocyclyl or cycloalkyl can be saturated (i.e., containing only single C-C bonds) or unsaturated (i.e., containing one or more double or triple bonds). Examples of saturated cycloalkyls include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Unsaturated carbocyclyls are also referred to as "cycloalkenyls." Examples of monocyclic cycloalkenyls include, for example, cyclopentenyl, cyclohexenyl, cycloheptenyl, and cyclooctenyl. Polycyclic carbocyclyl radicals include, for example, adamantyl, norbornyl (ie, bicyclo[2.2.1]heptanyl), norbornenyl, decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptanyl, and the like.

[0025] "Carbocyclylalkyl" has the formula -R c -carbocyclyl radical, where R c is an alkylene chain as defined above.

[0026] "Halo" or "halogen" refers to a bromo, chloro, fluoro, or iodo substituent.

[0027] "Haloalkyl" refers to an alkyl radical, as defined above, that is substituted by one or more halogen radicals, as defined above, e.g., trifluoromethyl, difluoromethyl, fluoromethyl, 2,2,2-trifluoroethyl, 1-fluoromethyl-2-fluoroethyl, and the like.

[0028] The term "heteroalkyl" refers to an alkyl group, as defined above, in which one or more skeletal carbon atoms of the alkyl have been replaced with a heteroatom (with the appropriate number of substituents or valences, e.g., -CH2- may be replaced with -NH- or -O-). For example, each substituted carbon atom is independently replaced with a heteroatom, e.g., carbon replaced with nitrogen, oxygen, selenium, or other suitable heteroatom. In some cases, each substituted carbon atom is independently replaced with oxygen, nitrogen (e.g., with -NH-, -N(alkyl)-, or -N(aryl)- or other substituents contemplated herein), or sulfur (e.g., -S-, -S(=O)-, or -S(=O)2-). A heteroalkyl is attached to the remainder of the molecule at a carbon atom of the heteroalkyl. A heteroalkyl is attached to the remainder of the molecule at a heteroatom of the heteroalkyl. A heteroalkyl is a C1-C 18 Heteroalkyl is a C1-C 12 Heteroalkyl is a C1-C6 heteroalkyl. Heteroalkyl is a C1-C4 heteroalkyl. Heteroalkyl can include alkoxy, alkoxyalkyl, alkylamino, alkylaminoalkyl, aminoalkyl, heterocycloalkyl, heterocycloalkyl, and heterocycloalkylalkyl, as defined herein.

[0029] "Heteroalkylene" refers to a divalent heteroalkyl group, as defined above, linking one portion of the molecule to another portion of the molecule.

[0030] "Heterocyclyl" refers to a stable 3- to 18-membered non-aromatic ring radical that may contain 2 to 12 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen, and sulfur. Unless otherwise specified in the specification, a heterocyclyl radical is a monocyclic, bicyclic, tricyclic, or tetracyclic ring system that may include aromatic, fused, and / or bridged ring systems. The heteroatoms in a heterocyclyl radical may be oxidized. The heterocyclyl radical may be partially or fully saturated. The disclosure provided herein for "heterocyclyl" is intended to include independent descriptions of heterocyclyl, including aromatic and non-aromatic ring structures, unless otherwise specified. A heterocyclyl is attached to the remainder of the molecule through any atom of the ring. Examples of such heterocyclyl radicals are dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl, 1,3-benzodioxolyl, 1,4-benzodioxanyl, tetrahydroquinolinyl, 5,6,7,8-tetrahydroquinazolinyl, 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl, 6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,5-c]pyridazinyl, indolinyl, isoindolinyl In some embodiments, the aryl group may be substituted or unsubstituted, including, but not limited to, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl.

[0031] "N-heterocyclyl" or "N-linked heterocyclyl" refers to a heterocyclyl radical, as defined above, containing at least one nitrogen, where the point of attachment of the heterocyclyl radical to the rest of the molecule is through a nitrogen atom in the heterocyclyl radical. Examples of such N-heterocyclyl radicals include, but are not limited to, 1-morpholinyl, 1-piperidinyl, 1-piperazinyl, 1-pyrrolidinyl, pyrazolidinyl, imidazolinyl, and imidazolidinyl.

[0032] "Heteroaryl" refers to a radical derived from a 3- to 18-membered aromatic ring radical, which may contain 2 to 17 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen, and sulfur. As used herein, a heteroaryl radical is a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, in which at least one of the rings within the ring system is fully unsaturated, i.e., contains a cyclic delocalized (4n+2) π-electron system according to Hückel theory. Heteroaryl includes fused or bridged ring systems. Heteroatoms in a heteroaryl radical may be oxidized. If present, one or more nitrogen atoms may be quaternized. A heteroaryl is attached to the remainder of the molecule through any atom of the ring. Examples of heteroaryl include azepinyl, acridinyl, benzimidazolyl, benzoindolyl, benzofuranyl, benzoxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, benzo[b][1,4]oxazinyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzothieno[3,2-d]pyrimidinyl, benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, cyclopenta[d]pyrimidinyl, and 6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidinyl. , 5,6-dihydrobenzo[h]quinazolinyl, 5,6-dihydrobenzo[h]cinnolinyl, 6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, furo[3,2-c]pyridinyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyrimidinyl, 5,6,7 ,8,9,10-Hexahydrocycloocta[d]pyridazinyl, 5,6,7,8,9,10-Hexahydrocycloocta[d]pyridinyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, isoquinolyl, indolizinyl, isoxazolyl, 5,8-methano-5,6,7,8-tetrahydroquinazolinyl, naphthyridinyl, 1,6-naphthyridinonyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 5,6,6a,7,8,9,10,10a-octahydrobenzo[h]quinazolinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, Including, but not limited to, pyrazolo[3,4-d]pyrimidinyl, pyridinyl, pyrido[3,2-d]pyrimidinyl, pyrido[3,4-d]pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl, quinoxalinyl, quinolinyl, isoquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, thieno[2,3-d]pyrimidinyl, thieno[3,2-d]pyrimidinyl, thieno[2,3-c]pridinyl, and thiophenyl (i.e., thienyl).

[0033] The compounds disclosed herein may contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomers defined in terms of absolute stereochemistry as (R)- or (S)-. Unless otherwise specified, all stereoisomeric forms of the compounds disclosed herein are intended to be contemplated by the present disclosure. When a compound described herein contains an alkene double bond, unless otherwise specified, the present disclosure is intended to include both E and Z geometric isomers (e.g., cis or trans). Similarly, all possible isomers, as well as their racemic and optically pure forms, and all tautomeric forms, are also intended to be included. The term "geometric isomer" refers to the E or Z geometric isomer (e.g., cis or trans) of the alkene double bond. The term "positional isomer" refers to structural isomers around a central ring, such as ortho, meta, and para isomers around a benzene ring.

[0034] As used herein, the term "linker" generally refers to a portion of a compound that forms a chemical bond with A (e.g., a binding ligand) and / or B (e.g., a therapeutic or imaging agent). In particular, a "linker" can connect two or more functional portions of a molecule to form a compound provided herein. Illustratively, a linker can include atoms selected from C, N, O, S, Si, and P; C, N, O, S, and P; or C, N, O, and S. A linker can connect different functionally capable portions of a compound, such as a FAP ligand and a PI3K inhibitor. A linker can include several linker groups in a contiguous backbone, for example, ranging from about 2 to about 100 atoms. A linker can be a releasable linker. A linker can be a non-releasable linker.

[0035] The compound can be a monovalent conjugate (e.g., a compound comprising one binding ligand (e.g., one FAP-binding ligand, as described elsewhere herein)). The compound can be a bivalent conjugate (a compound comprising one or more binding ligands (e.g., one or more FAP-binding ligands, as described elsewhere herein) conjugated (e.g., via a linker) to a therapeutic or imaging agent (as described elsewhere herein). The compound can be a multivalent conjugate (e.g., a compound comprising two or more binding ligands (e.g., two or more FAP-binding ligands, as described elsewhere herein) conjugated to a multipoint linker).

[0036] A binding ligand (also referred to herein as a targeting ligand or targeting moiety) can be a compound (or a radical thereof) that binds to a biomolecule (e.g., a polypeptide (e.g., an enzyme)) that is localized to a particular cell, tissue, organ, etc. The binding ligand can be a fibroblast activation protein (FAP) ligand (or a radical thereof). The binding ligand can be a fibroblast activation protein alpha (FAPα) ligand (or a radical thereof).

[0037] The therapeutic agent (or radical thereof) can be any entity capable of eliciting a desired physiological response. The therapeutic agent (or radical thereof) can be an anti-fibrotic agent, an anti-cancer agent, a chemotherapeutic agent, a radiotherapeutic agent, etc. The therapeutic agent can be a compound (or, e.g., a radical thereof) that is effective against (e.g., effective in eliminating, destroying, reducing (e.g., reducing the amount of), or lessening the influence of) cancer cells or pro-fibrotic cells (e.g., cancer-associated fibroblasts, myofibroblasts, etc. (e.g., other tumor microenvironment factors)). Examples of therapeutic agents (or radicals thereof) include, but are not limited to, photodynamic therapeutic agents, radiotherapeutic agents, chemotherapeutic agents, anti-fibrotic agents, and anti-cancer agents. The therapeutic agent provided herein can be a phosphoinositide-3-kinase (PI3K) inhibitor (or radical thereof). The therapeutic agent can be an anti-cancer agent (or radical thereof). The therapeutic agent can be an anti-fibrotic agent (or radical thereof). The therapeutic agent can be a compound (or a radical thereof) selected from a transforming growth factor (TGF) beta / Smad inhibitor, a Wnt / beta-catenin inhibitor, a kinase inhibitor (e.g., a kinase inhibitor for vascular endothelial growth factor receptor (VEGFR), a kinase inhibitor for fibroblast growth factor receptor (FGFR), a kinase inhibitor for platelet-derived growth factor receptor (PDGFR), a kinase inhibitor for focal adhesion kinase (FAK), or a kinase inhibitor for Rho-associated protein kinase (ROCK)), a toll-like receptor agonist (TLR), a nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) inhibitor, an inhibitor of collagen synthesis, and a phosphoinositide-3-kinase (PI3K) inhibitor. The therapeutic agent can be a phosphoinositide-3-kinase (PI3K) inhibitor (or a radical thereof).

[0038] An imaging agent can be a compound (or radical thereof) that emits a detectable signal (e.g., an electromagnetic signal (e.g., a radio signal, a fluorescent signal, a gamma ray) or a mass). Examples of imaging agents include, but are not limited to, radiological imaging agents (e.g., a PET imaging agent or a SPECT imaging agent), fluorescent imaging agents (e.g., a fluorescent dye), and the like.

[0039] compound A compound (e.g., a conjugate) of formula X is provided: A m -LB(X) During the ceremony, A is a radical of a fibroblast activation protein alpha (FAPα) ligand (targeting moiety) (e.g., molecular weight less than 10,000); L is a (e.g., bifunctional) linker that connects one or more A groups to B (e.g., via a first covalent bond connecting L to A and a second covalent bond bonding L to B); B is (e.g., a radical of) an optical imaging agent, a photodynamic therapy agent, a radioactive imaging agent, a radioactive therapeutic agent, a chemotherapeutic agent, an antifibrotic agent, or an anticancer agent (e.g., an anticancer agent that is effective against cancer cells or cancer-associated fibroblasts, myofibroblasts, or other tumor microenvironment factors); and m is 1 to 6.

[0040] A can be the radical of a FAPα ligand having a molecular weight of less than 10,000, 7,500, 5,000, 2,500, 1,000, 760, or 500; about 500 to about 10,000 g / mol; about 1,000 to about 7,500 g / mol; about 750 g / mol to about 1,500 g / mol; about 1,000 to about 5,000 g / mol; or about 500 to about 2,500 g / mol.

[0041] m can be 1. m can be 2. m can be 3. m can be 4. m can be 5. m can be 6. m can be 1 to 3, 2 to 4, or 1 to 5.

[0042] The present disclosure also relates to compounds (e.g., conjugates) of Formula I: ALB (I) During the ceremony, A comprises (e.g., a radical of) a FAPα ligand (e.g., a targeting moiety); L comprises a (e.g., bifunctional) linker connecting one or more A groups to B; and B includes (e.g., a radical of) an optical imaging agent, a photodynamic therapy agent, a radioactive imaging agent, a radioactive therapeutic agent, a chemotherapeutic agent, an antifibrotic agent, or an anticancer agent (e.g., an anticancer agent effective against cancer cells or cancer-associated fibroblasts, myofibroblasts, or other tumor microenvironment factors).

[0043] The targeting moiety can bind to activated fibroblasts expressing FAPα, and such activated fibroblasts are involved in cancer or inflammatory diseases. The targeting moiety can have a molecular weight of less than 10,000. L can include a bifunctional linker. The (e.g., bifunctional) linker can form a chemical bond with A and B. L can be a (e.g., bifunctional) linker that connects one or more A groups to B (e.g., via a first covalent bond connecting L to A and a second covalent bond connecting L to B). B can include (e.g., a radical of) an imaging agent, radiocontrast agent, photodynamic therapy agent, chemotherapeutic agent, antifibrotic agent, and / or radiotherapy agent, where B is an anticancer agent effective against cancer cells or cancer-associated fibroblasts, myofibroblasts, or other tumor microenvironment factors.

[0044] A can have the structure of formula IA and stereoisomers and pharmaceutically acceptable salts thereof: TIFF2025131780000016.tif22128 formula, TIFF2025131780000017.tif11128 is a functionalized 5-10 membered N-containing aromatic or non-aromatic monocyclic or bicyclic heterocycle, which may further contain 1-3 heteroatoms selected from oxygen, nitrogen, and sulfur; Z is a bond, substituted or unsubstituted alkylene (e.g., —CH—), substituted or unsubstituted amino (e.g., —NH—), —O—, or —S—; T is substituted or unsubstituted methylene (-CH-), substituted or unsubstituted amino (-NH-), -O-, or -S-; R 1 and R 2 are each independently selected from the group consisting of -H, -CN, -CHO, -B(OH)2, -C(O)alkyl, -C(O)aryl-, -C=CC(O)aryl, -C=CS(O)2aryl, -CO2H, -SO3H, -SON2NH2, -PO3H2, -SO2F, and 5-tetrazolyl; R 3 and R 4 are each independently -H, -OH, F, Cl, Br, I, -C 1~6 Alkyl, -OC 1~6 Alkyl, and -SC 1~6 selected from the group consisting of alkyl; R 5 , R 6 , R 7 , and R 8 are each independently selected from the group consisting of H, alkyl, and halo; and TIFF2025131780000018.tif9128 is a point of attachment of the FAPα binding ligand (e.g., via a linker L or imaging agent / therapeutic moiety B), where the point of attachment is at a carbon atom or one of a 5-10 membered N-containing aromatic or non-aromatic monocyclic or bicyclic heterocycle. o , 2 o This can be either through an amine or through a functionalized alkyl or cycloalkyl motif.

[0045] A can have a structure represented by formula IB: TIFF2025131780000019.tif44128T is a substituted or unsubstituted methylene (-CH2-), a substituted or unsubstituted amino (-NH-), -O-, or -S-; R 1 and R 2are each independently selected from the group consisting of -H, -CN, -CHO, -B(OH)2, -C(O)alkyl, -C(O)aryl-, -C=CC(O)aryl, -C=CS(O)2aryl, -CO2H, -SO3H, -SON2NH2, -PO3H2, -SO2F, and 5-tetrazolyl; R 3 and R 4 are each independently -H, -OH, F, Cl, Br, I, -C 1~6 Alkyl, -OC 1~6 Alkyl, and -SC 1~6 selected from the group consisting of alkyl; R 5 , R 6 , R 7 , and R 8 are each independently selected from the group consisting of H, alkyl, and halo; and R 9 , R 10 , and R 11 are independently H, -C 1~6 Alkyl, -OC 1~6 Alkyl, -SC 1~6 is selected from the group consisting of alkyl, F, Cl, Br, and I.

[0046] A can have a structure represented by formula IC: TIFF2025131780000020.tif43128T is a substituted or unsubstituted methylene (-CH2-), a substituted or unsubstituted amino (-NH-), -O-, or -S-; R 1 and R 2 are each independently selected from the group consisting of -H, -CN, -CHO, -B(OH)2, -C(O)alkyl, -C(O)aryl-, -C=CC(O)aryl, -C=CS(O)2aryl, -CO2H, -SO3H, -SON2NH2, -PO3H2, -SO2F, and 5-tetrazolyl; R 3 and R 4 are each independently -H, -OH, F, Cl, Br, I, -C 1~6 Alkyl, -OC 1~6Alkyl, and -SC 1~6 selected from the group consisting of alkyl; R 5 , R 6 , R 7 , and R 8 are each independently selected from the group consisting of H, alkyl, and halo; and R 9 , R 10 , and R 11 are independently H, -C 1~6 Alkyl, -OC 1~6 Alkyl, -SC 1~6 is selected from the group consisting of alkyl, F, Cl, Br, and I.

[0047] A has the following formula: TIFF2025131780000021.tif90128, During the ceremony, T is substituted or unsubstituted methylene (-CH-), substituted or unsubstituted amino (-NH-), -O-, or -S-; R 1 and R 2 are each independently selected from the group consisting of -H, -CN, -CHO, -B(OH)2, -C(O)alkyl, -C(O)aryl-, -C=CC(O)aryl, -C=CS(O)2aryl, -CO2H, -SO3H, -SON2NH2, -PO3H2, -SO2F, and 5-tetrazolyl; R 3 and R 4 are each independently -H, -OH, F, Cl, Br, I, -C 1~6 Alkyl, -OC 1~6 Alkyl, and -SC 1~6 selected from the group consisting of alkyl; R 5 , R 6 , R 7 , and R 8 are each independently selected from the group consisting of H, alkyl, and halo; and R 9 , R 10 , and R 11are independently H, -C 1~6 Alkyl, -OC 1~6 Alkyl, -SC 1~6 is selected from the group consisting of alkyl, F, Cl, Br, and I.

[0048] A can have a structure represented by formula XA, TIFF2025131780000022.tif22128 formula, Q is aryl, heteroaryl, or heterocyclyl (e.g., including aryl and non-aryl ring structures) (e.g., a 5-10 membered N-containing aromatic or non-aromatic monocyclic or bicyclic heterocycle, which may further contain 1-3 heteroatoms selected from O, N, and S); Z is a bond, substituted or unsubstituted C1-C3 alkylene (e.g., -CH2-), substituted or unsubstituted heteroalkyl (e.g., 1-3 atoms in length), amino (e.g., NH), -O-, or -S-; T is substituted or unsubstituted methylene (-CH2-), substituted or unsubstituted amino (-NH-), -O-, or -S- (e.g., where the substituents on T are C1-C3 alkyl, haloalkyl, or halo); R 1 and R 2 are each independently selected from the group consisting of -H, -CN, -CHO, -B(OH)2, -C(O)alkyl, -C(O)aryl-, -C=CC(O)aryl, -C=CS(O)2aryl, -CO2H, -SO3H, -SON2NH2, -PO3H2, -SO2F, -CONH2, and 5-tetrazolyl; R 3 and R 4 are each independently -H, -OH, F, Cl, Br, I, -C 1~6 Alkyl, -OC 1~6 Alkyl, and -SC 1~6 alkyl; and R 5 , R 6 , R 7 , and R 8are each independently selected from the group consisting of H, alkyl, and halo.

[0049] Q is L (e.g., L or L 1 ). Q can be aryl, heteroaryl, or heterocyclyl. Heterocyclyl can include aryl and non-aryl ring structures. Q can be bonded to L at the heteroalkyl, alkyl, or aryl position of Q. Q can be bonded to L at the aryl position of Q. Q can be bonded to L through a nitrogen atom (e.g., of L). Q can be bonded to L through a triazolyl or amido (e.g., of L). Heteroaryl can include aryl and non-aryl ring structures. Heteroaryl or heterocyclyl can contain 1 to 3 heteroatoms selected from O, N, and S. Heterocyclyl can contain 1 to 3 heteroatoms selected from O, N, and S. Q can be a 5- to 10-membered N-containing aromatic or non-aromatic monocyclic or bicyclic heterocycle (e.g., which may contain aryl and non-aryl ring structures). Q can be an N-linked heterocyclyl (e.g., which may contain aryl and non-aryl ring structures). Q can be a C6-C9-N-linked heterocyclyl (e.g., which may include aryl and non-aryl ring structures). The N-linked heterocyclyl is linked to Z via an N-heterocycloalkyl. Q can be (e.g., N-linked) isoindolinyl (e.g., where N is linked to Z).

[0050] Z can be a bond, substituted or unsubstituted C1-C3 alkylene, substituted or unsubstituted heteroalkylene (e.g., 1-3 atoms in length), amino (e.g., NH), -O-, or -S-. Z can be a bond. Z can be substituted methylene. Z can be -CH2-. Z can be substituted ethylene. Z can be ethylene substituted with oxo. Z can be -C(CO)CH2-. Z can be -CH2CH2-. Z can be C1-C3 heteroalkylene.

[0051] A is a function of the formula XB: TIFF2025131780000023.tif42128, During the ceremony, Q is aryl, heteroaryl, or heterocyclyl (e.g., including aryl and non-aryl ring structures); (e.g., a 5-10 membered N-containing aromatic or non-aromatic monocyclic or bicyclic heterocycle, which may further contain 1-3 heteroatoms selected from O, N, and S); T is substituted or unsubstituted methylene (-CH2-), substituted or unsubstituted amino (-NH-), -O-, or -S- (e.g., where the substituents on T are C1-C3 alkyl, haloalkyl, or halo); J is C(R J )2, where each R J are independently H or alkyl, or both R J together to form oxo; R 1 and R 2 are each independently selected from the group consisting of -H, -CN, -CHO, -B(OH)2, -C(O)alkyl, -C(O)aryl-, -C=CC(O)aryl, -C=CS(O)2aryl, -CO2H, -SO3H, -SON2NH2, -PO3H2, -SO2F, -CONH2, and 5-tetrazolyl; R 3 and R 4 are each independently -H, -OH, F, Cl, Br, I, -C 1~6 Alkyl, -OC 1~6 Alkyl, and -SC 1~6 selected from the group consisting of alkyl; R 5 , R 6 , R 7 , and R 8 are each independently selected from the group consisting of H, alkyl, and halo; and R 9 , R 10 , and R 11 are independently H, -C 1~6 Alkyl, -C 1~6 Haloalkyl, -OC1~6 Alkyl, -SC 1~6 is selected from the group consisting of alkyl, F, Cl, Br, and I.

[0052] J is L (e.g., L or L 1 J can be bonded to L through a nitrogen atom. J can be bonded to L through a triazolyl or amide (e.g., of L). J can be bonded to C(R J )2, where each R J are independently H or alkyl, or both R J together form oxo. J can be a C1-C3 alkyl. J can be -CH2-. J can be -CH2CH2-. J can be C=O.

[0053] T can be substituted or unsubstituted methylene (e.g., -CH2-), substituted or unsubstituted amino (e.g., -NH-), -O-, or -S-. The substituents on T can be C1-C3 alkyl, C1-C3 haloalkyl, or (for methylene) halo. T can be (-CH2-). The substituents on T can be C1-C3 alkyl, haloalkyl, or halo. T can be unsubstituted.

[0054] R 1 and R 2 are each independently selected from the group consisting of -H, -CN, -CHO, -B(OH), -C(O)alkyl, -C(O)aryl-, -C=CC(O)aryl, -C=CS(O)aryl, -COH, -SOH, -SONH, -POH, -SOF, -CONH, and 5-tetrazolyl. 1 and R 2 may each independently be selected from the group consisting of H, —CN, —CHO, and —B(OH). 1 and R 2 may each independently be selected from the group consisting of H, —CN, —CHO, and —CONH2. 1 can be H. R 2 R can be -CN, -CHO, -B(OH)2, or -CONH2.1 can be H, and R 2 R can be -CN, -CHO, -B(OH)2, or -CONH2. 1 can be H, and R 2 R can be -CN. 1 can be H, and R 2 R can be -CHO. 1 can be H, and R 2 can be -B(OH)2. 1 can be H, and R 2 can be -CONH2.

[0055] R 3 and R 4 are each independently -H, -OH, F, Cl, Br, I, -C 1~6 Alkyl, -OC 1~6 Alkyl, and -SC 1~6 R may be selected from the group consisting of alkyl. 3 and R 4 Each R can independently be -H or -F. 3 can be H, and R 4 can be -F. R 3 can be F and R 4 can be -F.

[0056] R 1 can be H, and R 2 can be -CN, R 3 can be H, and R 4 can be -F. R 1 can be H, and R 2 can be -CN, R 3 can be F and R 4 can be -F. R 1 can be H, and R 2 can be -CHO, and R 3 can be H, and R 4 can be -F. R 1 can be H, and R 2 can be -CHO, and R 3 can be F and R 4 can be -F. R 1 can be H, and R 2can be -B(OH)2, and R 3 can be H, and R 4 can be -F. R 1 can be H, and R 2 can be -B(OH)2, and R 3 can be F and R 4 can be -F. R 1 can be H, and R 2 can be -CONH2, R 3 can be H, and R 4 can be -F. R 1 can be H, and R 2 can be -CONH2, R 3 can be F and R 4 can be -F.

[0057] R 5 , R 6 , R 7 , and R 8 Each R may be independently selected from the group consisting of H, alkyl, and halo. 5 , R 6 , R 7 , and R 8 can each be H.

[0058] R 9 , R 10 , and R 11 are independently H, -C 1~6 Alkyl, -C 1~6 Haloalkyl, -OC 1~6 Alkyl, -SC 1~6 R may be selected from the group consisting of alkyl, F, Cl, Br, and I. 9 , R 10 , and R 11 are independently H, -C 1~6 R may be selected from the group consisting of haloalkyl, F, and Cl. 9 and R 11 can be H, and R 10 is H, -C 1~6 R can be haloalkyl, F, or Cl. 9 and R 11 can be H, and R 10R can be H, —CF3, F, or Cl. 9 and R 11 can be H, and R 10 R can be -CF3. 9 and R 11 can be H, and R 10 can be F. R 9 and R 11 can be H, and R 10 R can be Cl. 9 , R 10 , R 11 can be H.

[0059] A can be attached to L through a nitrogen atom (e.g., in L). A can be attached to L through a triazolyl or amide (e.g., in L).

[0060] A is, TIFF2025131780000024.tif164128TIFF2025131780000025.tif90128.

[0061] A is, TIFF2025131780000026.tif89129.

[0062] A (eg, a FAPα-binding ligand) can have a binding affinity for a FAP (eg, FAPα) in the range of about 1 nM to about 25 nM, for example, 1 nM to about 25 nM or about 1 nM to 25 nM.

[0063] L can be a linker, e.g., any suitable linker. L can be a non-releasable linker. L can be a releasable linker.

[0064] L may comprise one or more linker groups, each independently selected from the group consisting of alkyl(alkylene), heteroalkyl(heteroalkylene), heterocycloalkyl(heterocycloalkylene), heteroaryl, aryl, alkoxy, thioether, disulfide, carboxylic acid, anhydride, carbonate, carbamate, thioether, sugar, and peptide. L may comprise one or more linker groups, each independently selected from the group consisting of polyethylene glycol (PEG), alkyl(alkylene), disulfide, amide, carboxylic acid, anhydride, carbonate, ester, carbamate, thioether, triazole, sugar, and peptide. L may comprise one or more linker groups, each independently selected from the group consisting of polyethylene glycol (PEG), alkyl(alkylene), disulfide, amide, carboxylic acid, carbonate, ester, phenyl, triazole, and carbamate. L may comprise one or more linker groups, each independently selected from the group consisting of polyethylene glycol (PEG), alkyl(alkylene), disulfide, amide, carboxylic acid, phenyl, triazole, ester, and carbonate. L may comprise one or more linker groups, each independently selected from the group consisting of polyethylene glycol (PEG), alkyl(alkylene), disulfide, amide, carboxylic acid, ester, and carbonate. L may comprise one or more linker groups, each independently selected from the group consisting of polyethylene glycol (PEG), alkyl(alkylene), disulfide, and amide. L may comprise one or more linker groups, each independently selected from the group consisting of alkyl(alkylene), disulfide, and amide. L may comprise one or more linker groups, each independently selected from the group consisting of amide, alkyl(alkylene), PEG, phenyl, and triazole. L can include one or more linker groups, each independently selected from the group consisting of PEG, alkyl(ene), and amide.L may include one or more linker groups, each independently selected from the group consisting of alkyl(ene) and amide. L may include one or more linker groups, each independently selected from the group consisting of PEG and amide. The linker may include one or more triazole linker groups. The linker may include one or more disulfide linker groups. The linker may include one or more amide linker groups. The linker may include one or more PEG linker groups.

[0065] L may contain one or more releasable groups.

[0066] L can be (covalently) bonded to A through an amide linker group. L can be (covalently) bonded to B through an amide linker group. L can be (covalently) bonded independently to A and B through amide linker groups.

[0067] L can be (covalently) bonded to A through a triazole linker group. L can be (covalently) bonded to B through a triazole linker group. L can be (covalently) bonded independently to A and B through triazole linker groups.

[0068] L may be (covalently) attached to A through a triazole linker group. L may be (covalently) attached to B through an amide linker group. L may be attached to A through a triazole linker group and to B through an amide linker group.

[0069] L may be (covalently) attached to A through an amide linker group. L may be (covalently) attached to B through a carbamate linker group. L may be attached to A through an amide linker group and to B through a carbamate linker group.

[0070] The linker can be a bivalent linker (e.g., connecting one A to one B). The linker can be a polyvalent linker (e.g., connecting two or more As to one B). The linker can be a releasable linker. The linker can be a non-releasable linker.

[0071] L is (L 1 ) o -Y-(L2) p It can be, where: Each L 1 is the first linker; Each L 2 is a second linker; Y is a third linker; o is an integer from 1 to 5; and p is an integer of 1 to 5.

[0072] L 1 and L 2 can be identical. 1 and L 2 can be different. 1 can be attached to the A group (and the Y group). 2 may be connected to the B group (and the Y group). o and m may be the same, for example, 1 to 6, 1 to 3, or 1. p may be 1. o may be 1. p and o may each be 1.

[0073] Each L 1 and L 2 Each L independently comprises an oligoethylene glycol (chain), a polyethylene glycol (chain), an alkyl (chain), an oligopeptide (chain), or a polypeptide (chain). 1 and L 2 independently comprise an oligoethylene glycol (chain) or a polyethylene glycol (chain).

[0074] Each L 1 and L 2 independently comprises a triazole or an amide.

[0075] Each L 1and L 2 Each L independently comprises an oligopeptide (chain) or a polypeptide (chain). 1 and L 2 Each independently contains a peptidoglycan (chain).

[0076] Each L 1 and L 2 independently comprise an oligoproline or an oligopiperidine.

[0077] Each L 1 and L 2 can independently be 15 to 200 angstroms (Å) in length.

[0078] o can be an integer from 1 to 5. o can be an integer from 1 to 3. o can be 1.

[0079] p can be an integer of 1 to 5. p can be an integer of 1 to 3. p can be 1.

[0080] Formula II: (AS) m YLB Also provided is a multivalent conjugate having During the ceremony, A is a radical of a FAPα ligand (targeting moiety) (e.g., molecular weight less than 10,000); S is a spacer (e.g., having a length that allows the arms of a multivalent targeting ligand (e.g., a drug) to reach multiple adjacent FAPs on a target cell); Y is a linker; L is a (e.g., bifunctional) linker that connects one or more A groups to B (e.g., via a first covalent bond connecting L to A and a second covalent bond linking L to B); and B is a radical of a fluorescent dye, a photodynamic therapy agent, a radioactive contrast agent, a radioactive therapeutic agent, a chemotherapeutic agent, an antifibrotic agent, or an anticancer agent (e.g., an anticancer agent that is effective against cancer cells or cancer-associated fibroblasts, myofibroblasts, or other tumor microenvironment factors); and m is 2 to 6.

[0081] The spacer can be of an optimal length for the arms of the multivalent drug to reach multiple adjacent FAPs on the target (eg, cancer or profibrotic) cells.

[0082] S may comprise an oligoethylene, polyethylene glycol, an alkyl chain, an oligopeptide, or a polypeptide. S may be an oligoethylene glycol or a polyethylene glycol.

[0083] S can be an oligopeptide or a polypeptide.

[0084] S can be peptidoglycan.

[0085] The spacer can be a rigid linker. S can be, for example, a rigid linker such as an oligoproline or an oligopiperidine.

[0086] S may have a length of at least 15 angstroms (Å). S may have a length of up to 200 angstroms (Å). S may have a length of 15 to 200 angstroms (Å).

[0087] Y can be a linker connecting multiple arms of a compound (e.g., a conjugate). Y can have a repeating structure. Y can include a releasable bond. L can include a disulfide bond. Y can include at least one citrate group (or radical thereof). Y can include one or more triazoles. Y can include one or more amines. Y can include one or more amides. Y can have an aromatic core (e.g., an aryl core or a heteroaryl core). Y can have an alkyl(ene) core. Y can have an amine core. Y can be an N(L 1 ) 3 (e.g., where L 1 (where Y is a group consisting of three L 1 (e.g., where L 1are described elsewhere in this specification). Y is C(L 1 ) 4 (e.g., where L 1 may be as described elsewhere herein).

[0088] Y is one L 1 Y can be bonded to one L 2 Y can be bonded to one L 1 and one L 2 Y can be independently bonded to L 1 and L 2 Y can be attached to L by an amide bond.

[0089] Y can be a linker (e.g., a polyvalent linker) that connects multiple arms of a compound (e.g., a conjugate). Y can have a repeating structure. Y can include at least one citrate group (or radical thereof). The linker can have the following structure: TIFF2025131780000027.tif30128.

[0090] Y can be a linker (e.g., a polyvalent linker) that connects multiple arms of a compound (e.g., a conjugate) and has the following structure: It may contain a linker (e.g., repeat unit) of TIFF2025131780000028.tif65128.

[0091] Y can be a linker connecting multiple arms of a compound (e.g., a conjugate) that can have a citrate-based linker. Y can be a linker (e.g., a multivalent template) connecting multiple arms of a compound (e.g., a conjugate) and has the following structure: TIFF2025131780000029.tif51128 (e.g., citrate-based) linker.

[0092] Y can be a linker (e.g., a polyvalent linker) that connects multiple arms of a compound (e.g., a conjugate) and has the following structure: TIFF2025131780000030.tif62128 (e.g., citrate-based) linker.

[0093] Y can be a linker (e.g., a polyvalent linker) that connects multiple arms of a compound (e.g., a conjugate) and has the following structure: TIFF2025131780000031.tif38128 (e.g., citrate-based) linker.

[0094] L can include at least one linker group, each linker group selected from the group consisting of polyethylene glycol (PEG), alkyl, sugar, and peptide. The linker can be a polyethylene glycol-based (PEG-based) (e.g., PEG-based), alkyl-based, sugar-based, and peptide-based dual linker.

[0095] L can be a non-releasable linker (e.g., bivalently (e.g., covalently) linked to B and A). L can be a releasable linker (e.g., bivalently (e.g., covalently) linked to B and A).

[0096] L, L1, L2, or any combination thereof, may have the following structure: TIFF2025131780000032.tif22166, where n is 0-10.

[0097] L has the following structure: TIFF2025131780000033.tif22166, where n is 0-10.

[0098] L has the following structure: TIFF2025131780000034.tif20128, where n is 0-10.

[0099] L has the following structure: TIFF2025131780000035.tif46136, where n is 1 to 32.

[0100] L has the following structure: TIFF2025131780000036.tif22137, where n is 1 to 32.

[0101] L has the following structure: TIFF2025131780000037.tif32128, where: R 12 and R 13 may each independently be H or C1-C6 alkyl; and z is an integer of 1 to 8.

[0102] L has the following structure: TIFF2025131780000038.tif32128, where: R 12 and R 13 may each independently be H or C1-C6 alkyl; and z is an integer of 1 to 8.

[0103] L, L 1 , L 2 or any combination thereof, having the following structure: It may include one or more linker groups having TIFF2025131780000039.tif18128.

[0104] L has the following structure: It may include one or more linker groups having TIFF2025131780000040.tif16128.

[0105] L has the following structure: TIFF2025131780000041.tif19128, where: R 16 is H or C1-C6 alkyl; and R 14a , R 14b , and R 15a , R 15b may each independently be H or C1-C6 alkyl.

[0106] L has the following structure: TIFF2025131780000042.tif20128.

[0107] L has the following structure: TIFF2025131780000043.tif95144, where n is 0-15.

[0108] B can be bonded to L through a carbon atom or nitrogen atom (e.g., of L). B can be bonded to L through a triazolyl. B can be bonded to L through an oxo (e.g., an ester). B can be bonded to L through an amide (e.g., of L).

[0109] B can be an optical dye (or a radical thereof). B can be a fluorescent dye (or a radical thereof) (e.g., useful for fluorescence-guided surgery (FGS)). The fluorescent dye can include a fluorochrome group, each fluorochrome group selected from the group consisting of carbocyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, polymethine, acoumarine, rhodamine, xanthene, fluorescein, and the like. The fluorescent dye (or radical thereof) can be boron dipyrromethane (BODIPY), CyS, CyS.S, Cy7, VivoTag-680, VivoTag-S680, VivoTag-S7S0, AlexaFluor660, AlexaFluor680, AlexaFluor700, AlexaFluor7S0, AlexaFluor790, Dy677, Dy676, Dy682, Dy7S2, Dy780, DyLightS47, Dylight647, HiLyte Fluor 647, HiLyte Fluor 680, HiLyte Fluor 7S0, IRDye 800CW, IRDye 800RS, IRDye 700DX, ADS780WS, ADS830WS, ADS832WS, S0456, and the like.

[0110] The payload can have an excitation wavelength of 600 nanometers (nm) to 1000 nm. The payload can have an emission wavelength of 700 nm to 1800 nm.

[0111] B has the following structure: TIFF2025131780000044.tif167139TIFF2025131780000045.tif194126 may be a fluorescent dye group (or radical thereof).

[0112] The compound may be a FAP-targeting ligand (or a radical thereof) attached to a linker comprising one or more linker groups, each linker group being selected from alkyl, pegylated, and peptidoglycan, wherein the linker may further be attached to a fluorescent dye as described herein.

[0113] ALB has the following structure: TIFF2025131780000046.tif141135, where: n is an integer from 1 to 5; and B is The file is TIFF2025131780000047.tif116128.

[0114] ALB has the following structure: TIFF2025131780000048.tif183134TIFF2025131780000049.tif65128, where: n is an integer from 1 to 5; and B is TIFF2025131780000050.tif116128.

[0115] The compound (eg, conjugate) may have the following structure: [Table 1] TIFF2025131780000052.tif169142TIFF2025131780000053.tif214140TIFF2025131780000054.tif187145

[0116] Below is a scheme showing a method for producing an unlabeled (competing) FAP ligand, as used in the Examples and Figures herein. TIFF2025131780000055.tif59149

[0117] A drawback of certain therapeutic agents (e.g., chemotherapeutic or radiotherapeutic agents) is the inability of such agents to achieve and / or maintain therapeutically effective concentrations at the target location (e.g., at a cancer, tumor, or fibrotic tissue) without causing undesirable, toxic, and / or fatal (e.g., systemic) effects. General systemic administration of such agents, or even local administration (e.g., which may be route-specific but not target tissue-specific), can, in some cases, result in off-target therapeutic agents, thereby increasing side effects. In some cases, compounds (e.g., compounds containing targeting ligands) that localize the payload (e.g., therapeutic agent) to the target site (e.g., tumor or fibrotic tissue) can improve the residence time of the payload at the target site. In some cases, increasing the residence time of the therapeutic agent at the target site promotes increased and / or effective concentrations of the therapeutic agent even at low or tolerable doses. In some cases, an effective concentration of the active payload is maintained at the target location for a sufficient time to achieve a therapeutic concentration (e.g., at an acceptable dose and / or at a dose that would not be sufficient to achieve a therapeutic concentration if the free payload were similarly administered) and / or for a sufficient time to reduce the frequency of dosing (e.g., compared to that required for administration of the free payload). Furthermore, increasing the residence time of the therapeutic payload at the target location may, in some cases, mean that off-target effects may be reduced (e.g., because the active agent is maintained at the target location). In some cases, the compound facilitates, for example, administration of the active agent or payload with reduced administration frequency, reduced side effects, or a combination thereof (e.g., compared to administration of an otherwise similar free active agent or payload).

[0118] The targeting ligands provided herein are synthesized according to the retrosynthetic scheme shown in Figure 1. The retrosynthetic scheme of Figure 1 is used to synthesize the compounds shown in Figure 2. However, any suitable synthetic scheme or process may be used to produce the compounds. For example, specific synthetic schemes are shown in the Examples. All such synthetic schemes are incorporated into the detailed description herein in connection with any process, step, or compound (e.g., scheme end products, scheme intermediates, and / or scheme reagents).

[0119] The compound targets (e.g., localizes to) cells expressing a FAP (e.g., FAP5). The compound binds to a FAP (e.g., FAP5) expressed by (e.g., cancer cells or profibrotic cells) (e.g., incorporated into the cell membrane of) the cell. For example, as shown by the gray shading in Figures 3, 4, 5, and 6, Compound 1 (at various concentrations, e.g., 50 nM, 25 nM, 12.5 nM, and 6.5 nM (Figure 3)) localizes to cells expressing FAP5 (e.g., HT1080-FAP cells). Furthermore, Compound 1 localizes and internalizes the ligand to the membrane of FAP5-expressing cells (e.g., where FAP5 is located) and remains localized and internalized in the cells (e.g., when administered at a concentration of 12.5 nM) for at least 1 hour (e.g., 1 hour, 8 hours, 24 hours, or more) (e.g., Figure 4).

[0120] Figure 3 shows the binding of targeting ligand to (FAP HT1080) cells for 1 hour (e.g., at (A) 50 nM, (B) 25 nM, (C) 12.5 nM, and (D) 6.25 nM). Higher concentrations of targeting ligand demonstrate more surface binding. Figure 4 shows the binding of targeting ligand to FAP HT1080 cells for (A) 1 hour, (B) 8 hours, (C) 24 hours, and (D) 48 hours (e.g., at 12.5 nM). At early time points, the compound (targeting ligand) is observed on the surface of the cells, and over time, the compound is internalized within the cells. Figure 5 shows the binding of targeting ligands to FAP HT1080 cells over 1 hour using at least a 100-fold excess of competing ligand (e.g., A: 25 nM targeting ligand, 2.5 µM competitor; B: 25 nM targeting ligand, 5 µM competitor). Figure 6 shows the binding of targeting ligands to non-FAP HT1080 cells (e.g., (A) at 100 nM and (B) at 200 nM). At comparable time points, very little compound (targeting ligand) is observed on the surface of such cells after 1 hour (compared to Figures 3-5, which show good surface binding of compound after a similar time period to cells with a high FAP surface concentration, even at much lower concentrations).

[0121] The compound may have a strong (e.g., binding) affinity for FAP (e.g., FAP5)-expressing cells. In some cases, this strong binding affinity allows the compound to localize to FAP (e.g., FAP5)-expressing cells for an extended period of time (e.g., a period sufficient to measure a signal from the compound localized to a tissue of interest (e.g., tumor or fibrotic tissue) or to deliver a payload (e.g., therapeutic agent)). The compound may have a binding affinity of 0.01 nM to 1 μM for FAP (e.g., FAP5)-expressing cells. For example, Compound 1 has a K of 5 nM to 15 nM in cells expressing FAP5. d (Figure 7). Furthermore, as shown in Figure 8, Compound 1 does not bind to HT1080 cells that do not express FAP5, and Compound 1 does not bind to HT1080 cells that express FAP5 in the presence of unlabeled FAP5 ligand (e.g., Compound 8).

[0122] Figure 7 shows the binding curves of the targeting ligand to HT1080-FAP cells. Figure 8 shows the binding curves of the targeting ligand to HT1080-FAP cells (targeting ligand only (circles) and targeting ligand and competitor (squares)) and HT1080 cells (triangles).

[0123] The compound can target cancer cells (or tumors, etc.). The compounds provided herein can target tumors with minimal off-target effects. The compound can rapidly accumulate and maintain its concentration in tumors for a long period of time. As shown, the ligand demonstrates good accumulation at the target location (tumor), while demonstrating limited accumulation at off-target locations. Furthermore, as shown, there is little or no accumulation of the compound (FAP ligand conjugate) in non-tumor organs. Specifically, as shown, in some cases, high concentrations of the compound can be achieved at the target location (e.g., tumor) for a long period of time (e.g., up to 5 days or more), while there is little or no accumulation at any point in the heart, liver, lungs, spleen, stomach, or intestines. There is some short-term accumulation in the kidney, but it is mostly resolved by 15 hours (e.g., compared to high concentrations at the target location for many days). In certain cases, this means that certain therapeutic agents that would otherwise need to be administered once or twice a day to maintain therapeutic concentrations and / or provide therapeutic effects (even if such results are achieved without unacceptable side effects and / or death) can be administered much less frequently (e.g., once or twice a week) in the forms provided herein. Furthermore, as described, there is little off-target accumulation of the compounds provided herein, indicating the tolerability of administering such compounds in effective amounts. In some cases, the compound accumulates in the subject's kidney. In some cases, the compound accumulates (e.g., in the kidney) for up to 6 hours. In some cases, the compound is significantly cleared from organs (e.g., kidneys) by 24 hours (e.g., by 24 hours, by 15 hours, etc.). In some cases, the compound remains localized in the tumor for at least one day (e.g., 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, etc.).

[0124] For example, Figures 9A-20C show that a compound (e.g., Compound 8) localizes to tumors (e.g., expressing FAP5 in MDA-MB-231 xenografted mice (e.g., Figures 9A-10 and Figures 17A-17D), KB xenografted mice (e.g., Figures 11A-13D and Figures 16A-16D), FADu xenografted mice (e.g., Figures 14A-14D), HT29 xenografted mice (e.g., Figures 15A-15D), U87MG tumor xenografted mice (e.g., Figures 18A-18D), PANC1 xenografted mice (e.g., Figures 19A-19D), and 4T1 tumor xenografted mice (e.g., Figures 20A-20C)). This data shows that the compounds provided herein localize to tumors (only) (e.g., in some tumor types) for at least 1 day, 2 days, 3 days, 4 days, 5 days, or longer. Furthermore, although the compound (e.g., compound 8) shows some accumulation in the kidney (e.g., generally starting after 2 hours), peak kidney accumulation occurs after 6 hours, after which it rapidly declines and disappears. For example, a significant decrease in kidney exposure is achieved after only 15 hours, and by 24 hours it is almost completely gone. In contrast, the compound is rapidly taken up (e.g., by 2 hours) and remains in the tumor much longer than its presence in the kidney (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, or longer). Taken together, these in vivo data demonstrate that the conjugates provided herein target the kidney and enable delivery of a payload (e.g., an imaging agent or a chemotherapeutic agent) for an extended period of time (e.g., over several days).

[0125] Figures 9A and 9B show the results of MDA-MB-231 xenograft mice (400 mm) from 2 to 122 hours after transplantation. 39A and 9B show in vivo imaging of a targeting ligand provided herein (e.g., at a dose of 10 nmol) against a mammal with a tumor of 10 nmol (having a tumor size of 10 nmol). Figure 9C shows the biodistribution in the tumor, heart, liver, lungs, spleen, kidneys, intestine, and stomach after 122 hours. Black or white ovals or circles in the images highlight the locations where the targeting ligand is present. Darker shading within the oval or circle represents a higher concentration of targeting ligand than lighter shading within the oval or circle. More broadly, Figures 9A and 9B show imaging results demonstrating in vivo tumor-specific targeting of the targeting ligand between 2 hours and 122 hours after administration to a mammal with a tumor with a high FAP environment. Figure 9C shows the biodistribution in the tumor, heart, liver, lungs, spleen, kidneys, intestine, and stomach after 122 hours after administration to a mammal with a tumor with a high FAP environment. As can be seen, there is (i) good targeting of the tumor for several days, and (ii) little to no compound is seen elsewhere when looking at the mammal systemically or at mammalian organs specifically.

[0126] Figure 10 shows in vivo imaging of the targeting ligand provided herein in MDA-MB-231 xenograft mice from 2 to 6 hours, both in the presence and absence of unlabeled competitor. In studies without competitor, mice were administered 10 nmol of labeled ligand. In studies with competitor, mice were administered 10 nmol of labeled ligand and 1,000 nmol of unlabeled ligand. At each time point, the leftmost mouse represents a mouse treated with targeting ligand only, and the rightmost mouse represents a mouse treated with targeting ligand and unlabeled competitor. Black ovals or circles in the images highlight the location of the targeting ligand. Darker shading within the oval or circle represents a higher concentration of targeting ligand than lighter shading within the oval or circle. As can be seen, without the use of FAP competitor, successful targeting of the tumor location was achieved at 2 and 6 hours. In contrast, the presence of a FAP competitor limits tumor targeting of the compound, demonstrating in some cases that the ability of the compound to target the FAP is important for the compound to be able to target the desired location. Furthermore, in some cases, such information demonstrates that in locations where the FAP concentration is not high, uneven or undesirable accumulation of the targeting compound is unlikely to occur.

[0127] Figures 11A and 11B show KB xenograft mice (600 mm 3Figure 11C shows in vivo imaging of a targeting ligand provided herein (e.g., at a dose of 5 nmol) against a tumor (having a tumor size of 1000 nmol) from 2 hours to 122 hours. Biodistribution in the tumor, heart, liver, lungs, spleen, kidneys, intestine, and stomach after 122 hours is shown in Figure 11C. Black ovals or circles in the images highlight the location of the targeting ligand. Darker shading within the oval or circle represents a higher concentration of targeting ligand than lighter shading within the oval or circle. High concentrations of the active agent are seen at the tumor site for 5 days or more. In contrast, the absence of the FAP targeting compound is evident in other organs. Furthermore, these data demonstrate similar effects in different systems, including MDA-MB-231 xenografted mice in Figure 9 and KB xenografted mice in Figure 11.

[0128] Figure 12 shows in vivo imaging of KB xenograft mice treated with the targeting ligand provided herein from 2 to 6 hours, both in the presence and absence of unlabeled competitor. In studies without competitor, mice were administered 10 nmol of labeled ligand. In studies with competitor, mice were administered 10 nmol of labeled ligand and 1,000 nmol of unlabeled ligand. At each time point, the leftmost mouse represents a mouse treated with targeting ligand alone, and the rightmost mouse represents a mouse treated with targeting ligand and unlabeled competitor. Black ovals or circles in the images highlight the location of the targeting ligand. Darker shading within the oval or circle represents a higher concentration of targeting ligand than lighter shading within the oval or circle. As can be seen, without the use of FAP competitor, successful targeting of the tumor location was achieved at 2 and 6 hours. In contrast, the presence of FAP competitors limits the tumor targeting of compounds, and in some cases, it is demonstrated that the ability of compounds to target FAP is important for compounds provided herein to be able to target desired locations.Furthermore, in some cases, this information demonstrates that in places where FAP concentration is not high, uneven or undesirable accumulation of targeting compounds may not occur.Furthermore, these data demonstrate similar effects in different systems, including MDA-MB-231 xenograft mice in Figure 10 and KB xenograft mice in Figure 12.

[0129] Figure 13 shows the biodistribution (in KB tumor-bearing mice) of targeting ligand (injected into the tail vein at a dose of 10 nmol) in the tumor, heart, liver, lung, spleen, kidney, intestine, and stomach after 2 hours, 4 hours, 6 hours, 6 hours (kidney coverage (KC)), 15 hours, 24 hours, and 122 hours. Black or white arrows, ellipses, or circles in the images highlight the location of the targeting ligand. Darker shading near the arrowhead or within the ellipse or circle represents a higher concentration of targeting ligand than lighter shading.

[0130] Figure 14A shows in vivo imaging (whole body distribution) of a targeting ligand (e.g., at a dose of 5 nmol) for FADu xenografted mice (e.g., M1, M2, M3) 6 hours after injection. The biodistribution in the tumor, heart, liver, lung, spleen, kidney, intestine, muscle, and stomach after 6 hours is shown in Figure 14B. Figure 14C shows in vivo imaging (whole body distribution) of a competition experiment between a targeting ligand (e.g., at a dose of 5 nmol) for FADu xenografted mice (e.g., M1, M2, M3) 6 hours after injection and 500 nmol of unlabeled competitor. The biodistribution in the tumor, heart, liver, lung, spleen, kidney, intestine, muscle, and stomach after 6 hours is shown in Figure 14D. Black or white arrows, ovals, and The circles or circles highlight the locations where the targeting ligand is present. Darker shading near the arrowhead or within the oval or circle represents a higher concentration of targeting ligand than lighter shading. As can be seen in Figures 14A and 14B, the FAP targeting compound targets tumors with little off-target accumulation. Figures 14C and 14D demonstrate that in the presence of a FAP targeting competitor, there is less accumulation of the FAP targeting compound in tumors (e.g., due to a competitor to the FAP), and Figure 14D demonstrates that there are more off-target effects (e.g., in the stomach and kidney) when there is a competitor to the FAP.

[0131] Furthermore, as can be seen in Figures 13 and 14B, high concentrations of the active agent are observed at the tumor site for 5 days or more. In contrast, other organs are apparently free of the FAP targeting compound. While there appears to be some accumulation of the active agent in the kidney, such accumulation appears to peak around 6 hours, with a significant decrease in kidney concentrations by 15 hours and near complete disappearance by 24 hours. In some cases, the use of targeting ligands to deliver active payloads facilitates successful delivery of the payload to the target site with favorable (e.g., minimal or no) off-target effects or side effects. Furthermore, in some cases, the ability to maintain delivery to the target site for several days with a single administration facilitates reduced frequency of treatment administration, improved patient compliance (e.g., through reduced required dosing frequency), reduced side effects (e.g., less frequent dosing further reduces off-target effects / side effects), and / or other benefits.

[0132] Figure 15A shows in vivo imaging (whole-body distribution) of a targeting ligand (5 nmol) in HT29 xenografted mice (e.g., M1, M2, M3) 6 hours after injection. Figure 15B shows the biodistribution in the tumor, heart, liver, lungs, spleen, kidneys, intestines, muscle, and stomach after 6 hours. Figure 15C shows in vivo imaging (whole-body distribution) of a competition experiment between a targeting ligand (5 nmol) and an unlabeled competitor (500 nmol) in HT29 xenografted mice (e.g., M1, M2, M3) 6 hours after injection. Figure 15D shows the biodistribution in the tumor, heart, liver, lungs, spleen, kidneys, intestines, muscle, and stomach after 6 hours. Black or white arrows, ellipses, or circles in the images highlight the location of the targeting ligand. Darker shading near the arrowhead or within the ellipse or circle represents a higher concentration of targeting ligand than lighter shading. Figure 16A shows in vivo imaging (whole-body distribution) of a targeting ligand (5 nmol) in KB tumor xenografted mice (e.g., M1, M2, M3) 6 hours after injection. Figure 16B shows the biodistribution in the tumor, heart, liver, lungs, spleen, kidneys, intestine, muscle, and stomach after 6 hours. Figure 16C shows in vivo imaging (whole-body distribution) of a competition experiment between a targeting ligand (5 nmol) and an unlabeled competitor (500 nmol) in KB tumor xenografted mice (e.g., M1, M2, M3) 6 hours after injection. Figure 16D shows the biodistribution in the tumor, heart, liver, lungs, spleen, kidneys, intestine, muscle, and stomach after 6 hours. Black or white arrows, ellipses, or circles in the images highlight the location of the targeting ligand. Darker shading near the arrowhead or within the ellipse or circle represents a higher concentration of targeting ligand than lighter shading. Figure 17A shows in vivo imaging (whole body distribution) of a targeting ligand provided herein (e.g., at a concentration of 5 nmol) in MDA-MB-231 tumor xenograft mice (e.g., M1, M2, M3) 6 hours after injection. Figure 17B shows the biodistribution in the tumor, heart, liver, lung, spleen, kidney, intestine, muscle, and stomach (e.g., M1, M1 kidney coating (KC), M2, and M3) after 6 hours.Figure 17C shows in vivo imaging (whole-body distribution) of a competition experiment between a targeting ligand (e.g., at a concentration of 5 nmol) and an unlabeled competitor (500 nmol) in MDA-MB-231 tumor-bearing mice (e.g., M1, M2, M3) 6 hours after injection. Figure 17D shows the biodistribution in the tumor, heart, liver, lungs, spleen, kidneys, intestine, muscle, and stomach after 6 hours. Black or white arrows, ellipses, or circles in the images highlight the location of the targeting ligand. Darker shading near the arrowhead or within the ellipse or circle represents a higher concentration of targeting ligand than lighter shading. Figure 18A shows in vivo imaging (whole-body distribution) of a targeting ligand (5 nmol) in U87MG tumor xenografted mice (e.g., M1, M2, M3) 6 hours after injection. Biodistribution in the tumor, heart, liver, lungs, spleen, kidney, intestine, muscle, and stomach (e.g., M1, M1 kidney coating (KC), M2, M3 KC, and M3) after 6 hours is shown in Figure 18B. Figure 18C shows in vivo imaging (whole-body distribution) of a competition experiment between a targeting ligand (5 nmol) and an unlabeled competitor (500 nmol) in U87MG tumor-bearing mice (e.g., M1, M2, M3) 6 hours after injection. Biodistribution in the tumor, heart, liver, lungs, spleen, kidney, intestine, muscle, and stomach after 6 hours is shown in Figure 18D. Black or white arrows, ellipses, or circles in the images highlight the location of the targeting ligand. Darker shading near the arrowhead or within the ellipse or circle represents a higher concentration of targeting ligand than lighter shading. Figure 19A shows in vivo imaging (whole body distribution) of a targeting ligand provided herein (e.g., at a dose of 5 nmol) in PANC1 tumor xenograft mice (e.g., M1, M2, M3) 6 hours after injection. Figure 18B shows the biodistribution in the tumor, heart, liver, lung, spleen, kidney, intestine, muscle, and stomach (e.g., M1, M1 kidney coating (KC), M2 KC, M2, M3 KC, and M3) 6 hours after injection.Figure 18C shows in vivo imaging (whole-body distribution) of a competition experiment between a targeting ligand (e.g., at a dose of 5 nmol) and an unlabeled competitor (500 nmol) in PANC1 tumor-bearing mice (e.g., M1, M2, M3) 6 hours after injection. Figure 18D shows the biodistribution in the tumor, heart, liver, lungs, spleen, kidneys, intestine, muscle, and stomach after 6 hours. Black or white arrows, ellipses, or circles in the images highlight the location of the targeting ligand. Darker shading near the arrowhead or within the ellipse or circle represents a higher concentration of targeting ligand than lighter shading. Figure 20A shows in vivo imaging (whole-body distribution) of a targeting ligand and an unlabeled competitor compared to the targeting ligand alone (e.g., at a dose of 5 nmol) in 4T1 tumor xenografted mice 2 hours after injection. Figure 20B shows in vivo imaging (whole-body distribution) of targeting ligand and unlabeled competitor compared to targeting ligand alone (e.g., at a dose of 5 nmol) in 4T1 tumor xenografted mice 6 hours after injection. Figure 20C shows the biodistribution in the tumor, heart, liver, lung, spleen, kidney, intestine, muscle, and stomach (e.g., targeting, targeted kidney coverage (KC), and competition) after 6 hours. Black or white arrows, ellipses, or circles in the images highlight the location of the targeting ligand. Darker shading near the arrowhead or within the ellipse or circle represents a higher concentration of targeting ligand than lighter shading. These results are consistent with those shown in other figures, further demonstrating the consistency of the results across various tumor types and / or models.

[0133] In addition, Figures 21-22 show that several compounds (e.g., Compound 21, Compound 1, Compound 5, and Compound 6) target cells expressing FAP (e.g., FAP5) with very high affinity (e.g., 1 nM-10 nM).

[0134] Furthermore, in conjunction with Figures 9A-22, which show that the compound localizes to the target site, Figures 23 and 24 demonstrate that a compound (e.g., compound 11) is effective against FAP (e.g., FAP5)-expressing cells (e.g., reducing fibrotic responses). For example, a compound (e.g., compound 11) is as effective as a PI3Ki alone in reducing pathological biological responses (e.g., reducing Akt phosphorylation in TGF-β-stimulated human lung fibroblasts (Figure 23) and reducing the relative expression of collagen 1A1 mRNA in TGF-β-stimulated human lung fibroblasts (Figure 24)). Thus, while a payload (e.g., a PI3Ki) alone effectively reduces pathological biological responses, the compounds provided herein effectively reduce pathological biological responses and increase residence time at the target site (e.g., tumor or fibrotic tissue), ultimately increasing the effective concentration of the payload at the target site (e.g., reducing side effects, reducing dosage, etc.).

[0135] B can be an imaging agent. B can be a radiological imaging agent. B can be a photodynamic therapy agent. B can be a chemotherapeutic agent. B can be an antifibrotic agent. B can be a radiotherapeutic agent. B can be an anticancer agent. B can be an anticancer agent that is effective against cancer cells or cancer-associated fibroblasts, myofibroblasts, or other tumor microenvironment factors.

[0136] B may comprise a radioactive isotope. The radioactive isotope may be any suitable radioactive isotope. The radioactive isotope may be 99m Tc, 111 In, 18 F, 68 Ga, 124 I, 125 I, and 131 It may be selected from the group consisting of I.

[0137] B may comprise a radiotherapeutic nuclide. 177 Lu, 90 Y, and 211 At may be selected from the group consisting of

[0138] B may be a chelating agent, and in the case of radiotherapeutic nuclides, B may chelate the nuclide.

[0139] B may comprise a radiolabeled prosthetic group (or radical thereof). 18 F, 124 I, 125 I, 131 I, and 211 The radioisotope may be selected from the group consisting of At.

[0140] AL- has the following structure: TIFF2025131780000056.tif166128TIFF2025131780000057.tif64128, where n is an integer from 1 to 5.

[0141] B (e.g., a radiolabeled prosthetic group (or radical thereof)) has the following structure: TIFF2025131780000058.tif48128, where: Each X is independently 18 F, 124 I, 125 I, 131 I, and 2ll a radioisotope selected from the group consisting of At; Each R or R 1 is independently H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl; and Each n is independently an integer selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.

[0142] Representative radiolabeled prosthetic groups (e.g., B) are: This includes, but is not limited to, TIFF2025131780000059.tif60128.

[0143] B can be a chelating group (e.g., a chelating agent (or radical thereof)). Representative chelating groups (including the free base, e.g., COH (COOH) with one or more protons (H+) removed to form COO-) are: Includes, but is not limited to, TIFF2025131780000060.tif153138TIFF2025131780000061.tif167128.

[0144] B can be an antifibrotic agent or a radical thereof.

[0145] B can be a PI-3 kinase inhibitor or a radical thereof.

[0146] B can be a transforming growth factor β (TGFβ) / Smad inhibitor or a radical thereof.

[0147] B can be a Wingless-associated integration site (Wnt) / β-catenin inhibitor or a radical thereof.

[0148] B can be a kinase inhibitor or radical thereof for vascular endothelial growth factor receptor (VEGFR1, VEGFR2, VEGFR3), fibroblast growth factor receptor (FGFR1 or FGFR2), or platelet-derived growth factor receptor (PDGFR).

[0149] B can be a kinase inhibitor or radical thereof for focal adhesion kinase (FAK) or Rho-associated protein kinase (ROCK).

[0150] B can be an agonist of a Toll-like receptor (TLR) or a radical thereof.

[0151] B can be an inhibitor of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) or a radical thereof.

[0152] B can be an inhibitor of collagen synthesis or a radical thereof.

[0153] B binds to L via a hydroxyl radical.

[0154] A PI-3 kinase inhibitor (or radical thereof) (e.g., a compound or conjugate comprising a PI-3 kinase inhibitor (or radical thereof)) can have the structure of Formula III: TIFF2025131780000062.tif25128In the formula, X is TIFF2025131780000063.tif28142.

[0155] X can be a radical of B (e.g., the radical is on a heteroatom (e.g., S, N, or O of X)). B can be bonded to L through X (e.g., a hydroxyl radical of X).

[0156] The compound (eg, conjugate) may have the following structure: [Table 2] X is TIFF2025131780000065.tif29128.

[0157] The PI-3 kinase inhibitor (or radical thereof) (e.g., a compound or conjugate comprising a PI-3 kinase inhibitor (or radical thereof)) may be: It may have the structure TIFF2025131780000066.tif34128.

[0158] The compound (eg, conjugate) may have the following structure: [Table 3] TIFF2025131780000068.tif99146

[0159] The compound (eg, conjugate) may have the following structure: [Table 4]

[0160] The compound (e.g., conjugate) may have the following structure: TIFF2025131780000070.tif64132.

[0161] The compound (e.g., conjugate) may have the following structure: TIFF2025131780000071.tif63128.

[0162] Treatment methods Also provided are methods for treating inflammatory diseases or disorders. The method for treating inflammatory diseases or disorders by regulating the activity of activated fibroblasts. The method can include administering a compound (e.g., a conjugate) of any formula provided herein (e.g., Formula (I), Formula (IA), Formula (IB), Formula (IC), Formula (II), Formula (III), Formula (X), Formula (XA), Formula (XB), Table 2, Table 3, or Table 4).

[0163] Methods for treating cancer are provided. The method of treating cancer can be by modulating the activity of activated fibroblasts. The method can include a compound (e.g., a conjugate) of any formula provided herein (e.g., Formula (I), Formula (IA), Formula (IB), Formula (IC), Formula (II), Formula (III), Formula (X), Formula (XA), Formula (XB), Table 2, Table 3, or Table 4). The method can include contacting cancer-activated fibroblasts (CAFs) (e.g., CAFs of a cancer patient) with a compound (e.g., a conjugate) of any formula provided herein (e.g., Formula (I), Formula (IA), Formula (IB), Formula (IC), Formula (II), Formula (III), Formula (X), Formula (XA), Formula (XB), Table 2, Table 3, or Table 4).

[0164] Also provided is a method for treating fibrosis. The method for treating fibrosis can be by regulating the activity of activated fibroblasts. The method can include administering a compound (e.g., a conjugate) of any formula provided herein (e.g., Formula (I), Formula (IA), Formula (IB), Formula (IC), Formula (II), Formula (III), Formula (X), Formula (XA), Formula (XB), Table 2, Table 3, or Table 4).

[0165] The method may be chemotherapy or radiation therapy.

[0166] Methods are provided for imaging cancer or fibrosis in a subject with cancer or fibrosis.

[0167] Also provided are compositions and methods for optical imaging. The compositions and methods may be for fluorescence-guided surgery. The compositions and methods may be for radiological imaging.

[0168] The method includes providing to a patient in need thereof a pharmaceutically effective amount of a conjugated ALB, wherein A is a fibroblast activation protein alpha (FAPα) targeting moiety having a molecular weight of less than 10,000; L comprises a bifunctional linker capable of forming a chemical bond with A and B; and B comprises an optical dye (e.g., a fluorescent dye), a photodynamic therapy agent, a radioactive contrast agent, a radioactive therapeutic agent, a chemotherapeutic agent, an antifibrotic agent, or an anti-cancer agent effective against cancer cells or cancer-associated fibroblasts, myofibroblasts, or other tumor microenvironment factors.

[0169] Pharmaceutical Compositions, Routes of Administration, and Dosages In certain aspects, the present disclosure is directed to a pharmaceutical composition comprising a compound and a pharmaceutically acceptable carrier. In certain aspects, a pharmaceutical composition comprises a plurality of compounds and a pharmaceutically acceptable carrier.

[0170] In certain embodiments, the pharmaceutical composition further comprises at least one additional pharmaceutically active agent, which can be an agent useful in the treatment of ischemia-reperfusion injury.

[0171] Pharmaceutical compositions may be prepared by combining one or more compounds with a pharmaceutically acceptable carrier and, optionally, one or more additional pharmaceutically active agents.

[0172] As stated above, "effective amount" refers to any amount sufficient to achieve a desired biological effect. By selecting from among various active compounds and important factors such as potency, relative bioavailability, patient weight, severity of adverse side effects, and mode of administration, combined with the teachings provided herein, one can design an effective prophylactic or therapeutic treatment regimen that is effective for treating a particular subject without causing substantial undesirable toxicity. The effective amount for any particular application may vary depending on factors such as the disease or condition being treated, the specific compound being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular compound and / or other therapeutic agent without necessitating undue experimentation. A maximum dose, i.e., the highest safe dose according to any medical judgment, may be used. Multiple doses per day may be contemplated to achieve an appropriate systemic level of the compound. The appropriate systemic level may be determined, for example, by measuring the patient's peak or sustained plasma levels of the drug. "Dose" and "administration" are used interchangeably herein.

[0173] Generally, the daily oral dose of the compound for a human subject is about 0.01 milligrams / kg / day to 1,000 milligrams / kg / day. Oral doses ranging from 0.5 to 50 milligrams / kg can produce therapeutic results, with single or multiple daily administrations. The dosage may be adjusted appropriately to achieve the desired local or systemic drug level, depending on the mode of administration. For example, intravenous administration may vary from one to several orders of magnitude lower daily doses. If the subject's response is inadequate at such doses, even higher doses (or an effective higher dose via another, more localized delivery route) may be employed, as tolerated by the patient. Multiple daily doses are contemplated to achieve adequate systemic levels of the compound.

[0174] For any compound, the therapeutically effective amount can be first determined from animal models.The therapeutically effective dose can also be determined from human data for compounds that have been tested in humans and for compounds known to exhibit similar pharmacological activity, such as other related active agents.Higher doses may be required for parenteral administration.The applied dose can be adjusted based on the relative bioavailability and efficacy of the administered compound.Adjusting doses to achieve maximum efficacy based on the methods described above and other methods is well known in the art and is well within the capabilities of those skilled in the art.

[0175] For clinical use, any compound may be administered in an amount equal to or equivalent to 0.2 to 2,000 milligrams (mg) per kilogram (kg) of subject body weight per day. The compound may be administered in an amount equal to or equivalent to 2 to 2,000 mg of compound per kg of subject body weight per day. The compound may be administered in an amount equal to or equivalent to 20 to 2,000 mg of compound per kg of subject body weight per day. The compound may be administered in an amount equal to or equivalent to 50 to 2,000 mg of compound per kg of subject body weight per day. The compound may be administered in an amount equal to or equivalent to 100 to 2,000 mg of compound per kg of subject body weight per day. The compound may be administered in an amount equal to or equivalent to 200 to 2,000 mg of compound per kg of subject body weight per day. When a precursor or prodrug of the compound is administered, it is administered in an amount equivalent to, i.e., sufficient to deliver, the above-mentioned amounts of compound.

[0176] The compound formulations can be administered to human subjects in therapeutically effective amounts. Typical dosage ranges are about 0.01 micrograms / kg to about 2 mg / kg of body weight per day. The administered drug dosage can vary depending on variables such as the type and severity of the disorder, the general health of the particular subject, the specific compound administered, the excipients used to formulate the compound, and its route of administration. Routine experimentation can be used to optimize the dosage and frequency of administration of any particular compound.

[0177] The compounds can be administered at concentrations ranging from about 0.001 micrograms / kg to greater than about 500 mg / kg, for example, concentrations of 0.001 micrograms / kg, 0.01 micrograms / kg, 0.05 micrograms / kg, 0.1 micrograms / kg, 0.5 micrograms / kg, 1.0 micrograms / kg, 10.0 micrograms / kg, 50.0 micrograms / kg, 100.0 micrograms / kg, 500 micrograms / kg, 1.0 mg / kg, 5.0 mg / kg, 10.0 mg / kg, 15.0 mg / kg, 20.0 mg / kg, 25.0 mg / kg , 30.0 mg / kg, 35.0 mg / kg, 40.0 mg / kg, 45.0 mg / kg, 50.0 mg / kg, 60.0 mg / kg, 70.0 mg / kg, 80.0 mg / kg, 90.0 mg / kg, 100.0 mg / kg, 150.0 mg / kg, 200.0 mg / kg, 250.0 mg / kg, 300.0 mg / kg, 350.0 mg / kg, 400.0 mg / kg, 450.0 mg / kg to greater than about 500.0 mg / kg or any increment thereof, it being understood that all values ​​and ranges between these values ​​and ranges are meant to be encompassed.

[0178] The compound may be administered at a dosage ranging from about 0.2 milligrams / kg / day to more than about 100 mg / kg / day. For example, dosages may range from 0.2 mg / kg / day to 100 mg / kg / day, 0.2 mg / kg / day to 50 mg / kg / day, 0.2 mg / kg / day to 25 mg / kg / day, 0.2 mg / kg / day to 10 mg / kg / day, 0.2 mg / kg / day to 7.5 mg / kg / day, 0.2 mg / kg / day to 5 mg / kg / day, 0.25 mg / kg / day to 100 mg / kg / day, 0.25 mg / kg / day to 50 mg / kg / day, 0.25 mg / kg / day to 25 mg / kg / day, 0.25 mg / kg / day to 10mg / kg / day, 0.25mg / kg / day~7.5mg / kg / day, 0.25mg / kg / day~5mg / kg / day, 0.5mg / kg / day~50mg / kg / day, 0.5mg / kg / day~25mg / kg / day, 0.5mg / kg / day~ 20mg / kg / day, 0.5mg / kg / day ~ 15mg / kg / day, 0.5mg / kg / day ~ 10mg / kg / day, 0.5mg / kg / day ~ 7.5mg / kg / day, 0.5mg / kg / day ~ 5mg / kg / day, 0.75mg / kg / day ~ 50 mg / kg / day, 0.75mg / kg / day ~ 25mg / kg / day, 0.75mg / kg / day ~ 20mg / kg / day, 0.75mg / kg / day ~ 15mg / kg / day, 0.75mg / kg / day ~ 10mg / kg / day, 0.75mg / kg / day ~7.5mg / kg / day, 0.75mg / kg / day ~5mg / kg / day, 1.0mg / kg / day ~50mg / kg / day, 1.0mg / kg / day ~25mg / kg / day, 1.0mg / kg / day ~20mg / kg / day, 1.0mg / kg / day ~1 The dose may be 5 mg / kg / day, 1.0 mg / kg / day to 10 mg / kg / day, 1.0 mg / kg / day to 7.5 mg / kg / day, 1.0 mg / kg / day to 5 mg / kg / day, 2 mg / kg / day to 50 mg / kg / day, 2 mg / kg / day to 25 mg / kg / day, 2 mg / kg / day to 20 mg / kg / day, 2 mg / kg / day to 15 mg / kg / day, 2 mg / kg / day to 10 mg / kg / day, 2 mg / kg / day to 7.5 mg / kg / day, or 2 mg / kg / day to 5 mg / kg / day.

[0179] The compounds can be administered at dosages ranging from about 0.25 milligrams / kg / day to about 25 mg / kg / day. For example, dosages can be 0.25 mg / kg / day, 0.5 mg / kg / day, 0.75 mg / kg / day, 1.0 mg / kg / day, 1.25 mg / kg / day, 1.5 mg / kg / day, 1.75 mg / kg / day, 2.0 mg / kg / day, 2.25 mg / kg / day, 2.5 mg / kg / day, 2.75 mg / kg / day, 3.0 mg / kg / day, 3.25 mg / kg / day, 3.5 mg / kg / day, 3.75 mg / kg / day, 4.0 mg / kg / day, 5.0 mg / kg / day, 6.0 mg / kg / day, 7.0 mg / kg / day, 8.0 mg / kg / day, 9.0 mg / kg / day, 10.0 mg / kg / day, 11.0 mg / kg / day, 12.0 mg / kg / day, 13.0 mg / kg / day, 14.0 mg / kg / day, 15.0 mg / kg / day, 16.0 mg / kg / day, 17.0 mg / kg / day, 18.0 mg / kg / day, 19.0 mg / kg / day, 20.0 mg / kg / day, 21.0 mg / kg / day, 22.0 mg / kg / day, 23.0 mg / kg / day, 24.0 mg / kg / day, 25.0 mg / kg / day, 26.0 mg / kg / day, 27.0 mg / kg / day, 28.0 mg / kg / day, 29.0 mg / kg / day, 30.0 mg / kg / day, 31.0 mg / kg / day, 32.0 mg / kg / day g / day, 4.25mg / kg / day, 4.5mg / kg / day, 4.75mg / kg / day, 5mg / kg / day, 5.5mg / kg / day, 6.0mg / kg / day, 6.5mg / kg / day, 7.0mg / kg / day, 7. 5mg / kg / day, 8.0mg / kg / day, 8.5mg / kg / day, 9.0mg / kg / day, 9.5mg / kg / day, 10mg / kg / day, 11mg / kg / day, 12mg / kg / day, 13mg / kg / day, 14 mg / kg / day, 15mg / kg / day, 16mg / kg / day, 17mg / kg / day, 18mg / kg / day, 19mg / kg / day, 20mg / kg / day, 21mg / kg / day, 22mg / kg / day, 23mg / k g / day, 24mg / kg / day, 25mg / kg / day, 26mg / kg / day, 27mg / kg / day, 28mg / kg / day, 29mg / kg / day, 30mg / kg / day, 31mg / kg / day, 32mg / kg / day, 3 It may be 3 mg / kg / day, 34 mg / kg / day, 35 mg / kg / day, 36 mg / kg / day, 37 mg / kg / day, 38 mg / kg / day, 39 mg / kg / day, 40 mg / kg / day, 41 mg / kg / day, 42 mg / kg / day, 43 mg / kg / day, 44 mg / kg / day, 45 mg / kg / day, 46 mg / kg / day, 47 mg / kg / day, 48 mg / kg / day, 49 mg / kg / day, or 50 mg / kg / day.

[0180] The compound or precursor thereof can be administered at a concentration ranging from 0.01 micromolar to greater than or equal to 500 micromolar. For example, doses can be 0.01 micromolar, 0.02 micromolar, 0.05 micromolar, 0.1 micromolar, 0.15 micromolar, 0.2 micromolar, 0.5 micromolar, 0.7 micromolar, 1.0 micromolar, 3.0 micromolar, 5.0 micromolar, 7.0 micromolar, 10.0 micromolar, 15.0 micromolar, 20.0 micromolar, 25.0 micromolar, 30.0 micromolar, 35.0 micromolar, 40.0 micromolar, 50.0 micromolar, 60.0 micromolar, 70.0 micromolar, 80.0 micromolar, 90.0 micromolar, 100.0 micromolar, 110.0 micromolar, 120.0 micromolar, 130.0 micromolar, 140.0 micromolar, 150.0 micromolar, 160.0 micromolar, 170.0 micromolar, 180.0 micromolar, 190.0 micromolar, 210.0 micromolar, 220.0 micromolar, 230.0 micromolar, 240.0 micromolar, 250.0 micromolar, 260.0 micromolar, 270.0 micromolar, 280.0 micromolar, 290.0 micromolar, 300.0 micromolar, 310.0 micromolar, 320.0 micromolar, 330.0 micromolar, 340.0 micromolar, 350.0 micromolar, 360.0 micromolar, The range may be from 400.0 micromolar, 45.0 micromolar, 50.0 micromolar, 60.0 micromolar, 70.0 micromolar, 80.0 micromolar, 90.0 micromolar, 100.0 micromolar, 150.0 micromolar, 200.0 micromolar, 250.0 micromolar, 300.0 micromolar, 350.0 micromolar, 400.0 micromolar, 450.0 micromolar to greater than about 500.0 micromolar or any increment therein, and it is understood that all values ​​and ranges therebetween are meant to be encompassed.

[0181] The compound or its precursor may be administered at a concentration ranging from 0.10 micrograms / mL to 500.0 micrograms / mL. For example, the concentration may be 0.10 micrograms / mL, 0.50 micrograms / mL, 1 microgram / mL, 2.0 micrograms / mL, 5.0 micrograms / mL, 10.0 micrograms / mL, 20 micrograms / mL, 25 micrograms / mL, 30 micrograms / mL, 35 micrograms / mL, 40 micrograms / mL, 45 micrograms / mL, 50 micrograms / mL, 60.0 micrograms / mL, 70.0 micrograms / mL, 80.0 micrograms / mL, 90.0 micrograms / mL, 100.0 micrograms / mL, 150.0 micrograms / mL, 200.0 micrograms / mL, 250.0 micrograms / mL, 250.0 micrograms / mL, 300.0 micrograms / mL, 350.0 micrograms / mL, 400.0 micrograms / mL, 450.0 micrograms / mL to greater than about 500.0 micrograms / mL, or any increment thereof. It is understood that all values ​​and ranges between these values ​​and ranges are meant to be encompassed.

[0182] The formulation can be administered in a pharmaceutically acceptable solution, which may typically contain pharmaceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients. For use in therapy, an effective amount of the compound can be administered to a subject by any method that delivers the compound to the desired surface. Administering a pharmaceutical composition can be achieved by any means known to those skilled in the art. Routes of administration include, but are not limited to, intravenous, intramuscular, intraperitoneal, intravesical (bladder), oral, subcutaneous, direct injection (e.g., into tumors or abscesses), mucosal (e.g., topically to the eye), inhalation, and topical.

[0183] For intravenous and other parenteral administration routes, the compounds can be formulated as lyophilized preparations, as lyophilized preparations of liposome-intercalated or liposome-encapsulated active compounds, as lipid complexes in aqueous suspension, or as salt complexes. Lyophilized preparations are generally reconstituted in a suitable aqueous solution, such as sterile water or physiological saline, immediately prior to administration.

[0184] For oral administration, compounds can be easily formulated by combining active compounds with pharmaceutically acceptable carriers known in the art.Such carriers allow compounds to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral ingestion by the subject to be treated.Pharmaceutical preparations for oral use can be obtained as solid excipients, and optionally, the resulting mixture is ground, and after adding suitable excipients as needed, the granular mixture is processed to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP).If necessary, disintegrants such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or its salts, such as sodium alginate, can be added. Optionally, oral formulations may also be formulated in saline or buffers such as EDTA for neutralizing internal acidic conditions, or may be administered without any carrier.

[0185] Oral dosage forms of the compounds are also contemplated. The compounds can be chemically modified to enable effective oral delivery of the derivatives. Generally, the contemplated chemical modifications are the attachment of at least one moiety to the compound itself, where the moiety (a) inhibits acid hydrolysis and (b) allows uptake into the bloodstream from the stomach or intestine. Increasing the overall stability of the compound and increasing its circulation time in the body are also desirable. Examples of such moieties include polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, and polyproline. Abuchowski and Davis, "Soluble Polymer-Enzyme Adducts," In: Enzymes as Drugs, Hocenberg and Roberts, eds., Wiley-Interscience, New York, NY, pp. 367-383 (1981); Newmark et al., J Appl Biochem 4:185-189 (1982). Other polymers that can be used are poly-1,3-dioxolane and poly-1,3,6-tioxocane.As indicated above, polyethylene glycol moieties are preferred for pharmaceutical applications.

[0186] The location of release of the compound may be the stomach, the small intestine (duodenum, jejunum, or ileum), or the large intestine. Those skilled in the art have available formulations that will not dissolve in the stomach but will release the substance in the duodenum or elsewhere in the intestine. Release may avoid harmful effects on the stomach environment either by protecting the compound or by releasing the compound beyond the stomach environment, for example, in the intestine.

[0187] To ensure adequate gastric resistance, a coating that is impermeable down to at least pH 5.0 is essential. Examples of more common inactive ingredients used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP50, HPMCP55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and shellac. These coatings may also be used as mixed films.

[0188] Coatings or mixtures of coatings may also be used on tablets not intended for gastric protection. This may include sugar coatings or coatings that make the tablet easier to swallow. Capsules may consist of a hard shell (such as gelatin) for delivery of dry therapeutics (e.g., powder); a soft gelatin shell may be used for liquid forms. The shell material for cachets may be thick starch or other edible paper. Moist massing techniques may be used for pills, troches, molded tablets, or crushed tablets.

[0189] The therapeutic agent may be included in the formulation as fine multiparticulates in the form of granules or pellets with a particle size of about 1 mm. Formulation of material for capsule administration may also be as a powder, lightly compressed plugs, or even tablets. The therapeutic agent may be prepared by compression.

[0190] Colorants and flavoring agents may all be included. For example, the compound may be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage, that contains colorants and flavoring agents.

[0191] Therapeutic substances may be diluted or augmented with inert materials. These diluents may include carbohydrates, particularly mannitol, α-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans, and starch. Certain inorganic salts, including calcium triphosphate, magnesium carbonate, and sodium chloride, may also be used as fillers. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress, and Avicell.

[0192] Disintegrants may be included in the formulation of the therapeutic agent into a solid dosage form. Materials used as disintegrants include, but are not limited to, starch, including the commercially available starch-based disintegrant Explotab. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethylcellulose, natural sponge, and bentonite may also be used. Another form of disintegrant is an insoluble cation exchange resin. Powdered gums may be used as disintegrants and binders, including powdered gums such as agar, Karaya, or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants.

[0193] Binders may be used to hold the therapeutic substance together to form a hard tablet and include materials from natural sources such as acacia, tragacanth, starch, and gelatin. Others include methylcellulose (MC), ethylcellulose (EC), and carboxymethylcellulose (CMC). Polyvinylpyrrolidone (PVP) and hydroxypropylmethylcellulose (HPMC) can both be used in alcoholic solutions to granulate the therapeutic substance.

[0194] Antifriction agents may be included in the formulation of the therapeutic agent to prevent sticking during the formulation process. Lubricants may be used as a layer between the therapeutic agent and the mold wall, and may include, but are not limited to, stearic acid, including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils, and waxes. Soluble lubricants such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycols of various molecular weights, and Carbowax 4000 and 6000 may also be used.

[0195] Glidants may be added which may improve the flow properties of the drug during formulation and aid in rearrangement during compression. Glidants may include starch, talc, pyrogenic silica, and hydrated silicoaluminate.

[0196] Surfactants may be added as wetting agents to aid in dissolving the therapeutic substance in the aqueous environment. Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate, and dioctyl sodium sulfonate. Cationic detergents that can be used include benzalkonium chloride and benzethonium chloride. Possible nonionic detergents that can be included in the formulation as surfactants include lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50, and 60, glycerol monostearate, polysorbate 40, 60, 65, and 80, sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose. These surfactants may be present in the formulation of the compound or its derivatives either alone or as a mixture in different ratios.

[0197] Orally usable pharmaceutical preparations include push-fit capsules made of gelatin and soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. Push-fit capsules may contain the active ingredient mixed with a filler such as lactose, a binder such as starch, and / or a lubricant such as talc or magnesium stearate, and optionally, stabilizers. In soft capsules, the active compound may be dissolved or suspended in a suitable liquid, such as fatty oils, liquid paraffin, or liquid polyethylene glycol. Additionally, stabilizers may be added. Microspheres formulated for oral administration may also be used. Such microspheres are well-defined in the art. All formulations for oral administration should be in a dosage suitable for such administration.

[0198] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0199] For topical administration, the compounds may be formulated as solutions, gels, ointments, creams, suspensions, etc., as is well known in the art. Systemic formulations include those designed for administration by injection, e.g., subcutaneous, intravenous, intramuscular, intrathecal, or intraperitoneal injection, as well as those designed for transdermal, transmucosal, oral, or pulmonary administration.

[0200] For administration by inhalation, compound can be conveniently delivered in the form of aerosol spray from pressurized pack or nebulizer, using suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.In the case of pressurized aerosol, dosage unit can be determined by providing a valve for delivering a metered amount.Capsules and cartridges such as gelatin for use in inhaler or insufflator can be formulated to contain the powder mixture of compound and suitable powder base, for example, lactose or starch.

[0201] Pulmonary delivery of the compound (or salt thereof) is also contemplated. During inhalation, the compound is delivered to the lungs of a mammal and passes through the lung epithelial lining into the bloodstream. Other reports of inhaled molecules include Adjei et al., Pharm Res 7:565-569 (1990); Adjei et al., Int J Pharmaceutics 63:135-144 (1990) (leuprolide acetate); Braquet et al., J Cardiovasc Pharmacol 13(suppl. 5):143-146 (1989) (endothelin-1); Hubbard et al., Annal Int Med 3:206-212 (1989) (α1-antitrypsin); Smith et al., 1989, J Clin Invest 84:1145-1146 (α1-proteinase); Oswein et al., 1990, "Aerosolization of Proteins," Proceedings of Symposium on Respiratory Drug Delivery II, Keystone, Colorado, March, (recombinant human growth hormone); Debs et al., 1988, J Immunol 140:3482-3488 (interferon-γ and tumor necrosis factor α) and Platz et al., U.S. Pat. No. 5,284,656 (granulocyte colony-stimulating factor; incorporated by reference). Methods and compositions for pulmonary delivery of drugs for systemic effect are described in U.S. Pat. No. 5,451,569, issued September 19, 1995 to Wong et al. (specifically incorporated by reference for its disclosure in this regard).

[0202] Contemplated for use are a wide variety of mechanical devices designed for pulmonary delivery of therapeutic products, including, but not limited to, nebulizers, metered dose inhalers, and dry powder inhalers, all of which are well known to those skilled in the art.

[0203] Nasal delivery of pharmaceutical compositions is also contemplated. Nasal delivery allows the pharmaceutical composition to enter the bloodstream immediately after administration of the therapeutic product to the nose, without the need for deposition of the product in the lungs. Formulations for nasal delivery include formulations using dextran or cyclodextrin.

[0204] The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion, when it is desirable to deliver them systemically. Injectable preparations may be provided in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulating agents such as suspending, stabilizing, and / or dispersing agents.

[0205] Pharmaceutical preparations for parenteral administration include aqueous solutions of water-soluble active compounds.In addition, suspensions of active compounds can be prepared as suitable oily injection suspensions.Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes.Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran.Optionally, suspensions can also contain suitable stabilizers or agents that increase the solubility of compounds, allowing the preparation of highly concentrated solutions.

[0206] Alternatively, the active compound may be in powder form for constitution with a suitable vehicle, eg, sterile pyrogen-free water, before use.

[0207] The compounds may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, eg, containing conventional suppository bases such as cocoa butter or other glycerides.

[0208] In addition to the formulations described above, the compounds can also be formulated as sustained-release preparations. Such long-acting preparations can be formulated using suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0209] The pharmaceutical compositions may also comprise suitable solid- or gel-phase carriers or excipients. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

[0210] Suitable liquid or solid pharmaceutical preparation forms include, for example, aqueous solutions or saline solutions for inhalation, microencapsulated, encapsulated, coated with fine gold particles, contained in liposomes, atomized, aerosolized, pellets for skin implantation, or dried on a sharp object and rubbed onto the skin. Pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, droplets, or preparations for extended release of active compounds, in which excipients and additives and / or auxiliaries, such as disintegrants, binders, coating agents, swelling agents, lubricants, flavoring agents, sweeteners, or solubilizers, are conventionally used as described above. Pharmaceutical compositions are suitable for use in various drug delivery systems. For a brief review of methods for drug delivery, see Langer R, Science 249:1527-1533 (1990).

[0211] The compound and, optionally, one or more other therapeutic agents may be administered as such (neat) or in the form of a pharmaceutically acceptable salt. When used in medicine, the salt should be pharmaceutically acceptable, although pharmaceutically unacceptable salts may be conveniently used to prepare pharmaceutically acceptable salts. Such salts include, but are not limited to, those prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, p-toluenesulfonic acid, tartaric acid, citric acid, methanesulfonic acid, formic acid, malonic acid, succinic acid, naphthalene-2-sulfonic acid, and benzenesulfonic acid. Such salts may also be prepared as alkali metal or alkaline earth salts, such as sodium, potassium, or calcium salts of the carboxylic acid group.

[0212] Suitable buffering agents include acetic acid and salts (1-2% w / v), citric acid and salts (1-3% w / v), boric acid and salts (0.5-2.5% w / v), and phosphoric acid and salts (0.8-2% w / v). Suitable preservatives include benzalkonium chloride (0.003-0.03% w / v), chlorobutanol (0.3-0.9% w / v), parabens (0.01-0.25% w / v), and thimerosal (0.004-0.02% w / v).

[0213] Pharmaceutical compositions contain an effective amount of the compounds described herein contained in a pharmaceutically acceptable carrier, and optionally one or more therapeutic substances. The term "pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid fillers, diluents or encapsulating materials suitable for administration to humans or other vertebrates. The term "carrier" refers to a natural or synthetic organic or inorganic component with which active ingredients are combined to facilitate application. The components of pharmaceutical compositions can also be mixed with the compound and with each other in a manner that does not cause any interaction that would substantially impair the desired pharmaceutical effect.

[0214] Therapeutic agents, particularly including, but not limited to, compounds, may be provided in particles. As used herein, particle refers to nanoparticles or microparticles (or, in some cases, larger particles) that may be composed entirely or partially of the compounds described herein or other therapeutic agents. Particles may contain a therapeutic agent within a core surrounded by a coating, including, but not limited to, an enteric coating. The therapeutic agent may also be dispersed throughout the particle. The therapeutic agent may also be adsorbed onto the particle. Particles may have any order of release kinetics, including zero-order release, first-order release, second-order release, delayed release, sustained release, immediate release, and any combination thereof. In addition to the therapeutic agent, particles may contain any of the materials routinely used in the pharmaceutical and medical arts, including, but not limited to, erodible, nonerodible, biodegradable, or nonbiodegradable materials, or combinations thereof. Particles may be microcapsules containing the compound in solution or in a semi-solid state. Particles may be virtually any shape.

[0215] Both non-biodegradable and biodegradable polymeric materials can be used to manufacture particles for delivering therapeutic agents. Such polymers can be natural or synthetic. The polymer is selected based on the desired period of release. Bioadhesive polymers of particular interest include the bioerodible hydrogels described in Sawhney et al., Macromolecules 26:581-587 (1993), the teachings of which are specifically incorporated herein by reference. These include polyhyaluronic acid, casein, gelatin, gluten, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate).

[0216] The therapeutic substance may be contained in a controlled release system. The term "controlled release" is intended to refer to any drug-containing formulation in which the manner and profile of drug release from the formulation is controlled. This refers to immediate and non-immediate release formulations, which include, but are not limited to, sustained and delayed release formulations. The term "sustained release" (also called "extended release") is used in its conventional sense to refer to a drug formulation that provides gradual release of drug over an extended period of time and can result in a substantially constant blood concentration of drug over an extended period of time. The term "delayed release" is used in its conventional sense to refer to a drug formulation in which there is a time delay between administration of the formulation and the release of drug therefrom. "Delayed release" may or may not involve gradual release of drug over an extended period of time, and thus may or may not be a "sustained release."

[0217] The use of long-term sustained-release implants may be particularly suitable for treating chronic conditions. As used herein, "long-term" release means that the implant is constructed and adapted to deliver therapeutic levels of the active ingredient for at least 7 days, and up to 30-60 days. Long-term sustained-release implants are well known to those skilled in the art and include some of the release systems described above.

[0218] It will be understood by those skilled in the relevant art that other suitable modifications and adaptations to the compositions and methods described herein will be readily apparent from the description contained herein in light of information known to those skilled in the art, and may be made without departing from the scope of the disclosure or any of its aspects. Having thus described the present disclosure in detail, it will be more clearly understood by reference to the following examples, which are intended to be merely illustrative and are not intended to be limiting of the disclosure. [Example]

[0219] Chemical Examples : Example 1: Synthesis of trans-2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetic acid: Trans-2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetic acid was synthesized according to Scheme 1. TIFF2025131780000072.tif83149

[0220] Process-I: Under nitrogen, benzyl (2S)-N-tert-butoxylcarbonylpyroglutamate (compound 1, 5 grams (g), 15.67 millimoles (mmol)) was dissolved in tetrahydrofuran (THF, 75 milliliters (mL)) and cooled to -78°C. Lithium bis(trimethylsilyl)amide (LiHMDS) (1.0 M in THF, 34.5 mL, 34.5 mmol) was added dropwise over 5 minutes (min), and stirring was continued for 1 hour (h). tert-Butyl bromoacetate (4.36 mL, 31.34 mmol) was added dropwise over 5 minutes, and stirring was continued for an additional 2 hours at -78°C. The reaction mixture was quenched with saturated aqueous ammonium chloride solution (100 mL) and extracted into ethyl acetate (2 x 100 mL). The organic layer was washed with water and saturated aqueous sodium chloride solution and then dried over anhydrous magnesium sulfate (MgSO). The solvent was removed in vacuo to give a brown oil, which was purified by Combi-Flash silica gel chromatography (eluting with hexane / EtOAc) to give a mixture of compound 2a (2-benzyl 1-(tert-butyl) trans-(2S,4S)-4-(2-(tert-butoxy)-2-oxoethyl)-5-oxopyrrolidine-1,2-dicarboxylate) and compound 2b (2-benzyl 1-(tert-butyl) cis-(2S,4S)-4-(2-(tert-butoxy)-2-oxoethyl)-5-oxopyrrolidine-1,2-dicarboxylate) (6.2 gm, 89%) as a viscous liquid.

[0221] Process-II: A mixture of compounds 2a and 2b (6 g, 13.85 mmol) was dissolved in CHCl (90 mL) and cooled to 0 °C. DBU (6.2 mL, 41.55 mmol) was added dropwise, and the mixture was stirred at 0 °C for 30 minutes and then at room temperature for 24 hours. The reaction mixture was diluted with CHCl (20 mL) and washed with water (100 mL). The organic layer was dried over MgSO, filtered, and concentrated in vacuo to give the crude product, which was purified by column chromatography as described in Step I to give 2-benzyl 1-(tert-butyl) trans-(2S,4S)-4-(2-(tert-butoxy)-2-oxoethyl)-5-oxopyrrolidine-1,2-dicarboxylate (compound 3, 4.5 g, 75%) as a pale yellow liquid.

[0222] Process-III: To a solution of compound 3 (4 g, 9.23 mmol) in MeOH (40 mL) was added 5% Pd / C (400 mg) under a nitrogen atmosphere. The mixture was vigorously stirred under a hydrogen atmosphere at room temperature for 12 hours. The mixture was filtered through a Celite pad and concentrated in vacuo to give trans-(2S,4S)-4-(2-(tert-butoxy)-2-oxoethyl)-1-(tert-butoxycarbonyl)-5-oxopyrrolidine-2-carboxylic acid (compound 4, 2.7 g, 87%) as a white solid.

[0223] Process-IV: To a stirred solution of compound 4 (2.5 g, 7.28 mmol) in anhydrous DMF (20 mL), HATU (3.3 g, 8.7 mmol) and DIPEA (3.6 mL, 21.84 mmol) were added, and stirring was continued for 10 min for activation of the acid functional group. (S)-4,4-Difluoropyrrolidine-2-carboxamide hydrochloride (1.3 g, 8.736 mmol) followed by DIPEA (1.46 mL, 8.73 mmol) was added to the above reaction mixture, and stirring was continued for 5 h at room temperature under a nitrogen atmosphere. The reaction mixture was diluted with water (30 mL), brine (30 mL), and extracted with ethyl acetate (2 × 50 mL). The combined organic extracts were dried over anhydrous sodium sulfate, filtered, the filtrate evaporated under reduced pressure and the crude residue obtained was purified by Combiflash using DCM / MeOH as the mobile phase to give tert-butyl trans-(3S,5S)-3-(2-(tert-butoxy)-2-oxoethyl)-5-((S)-2-carbamoyl-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidine-1-carboxylate (5, 2.4 g 88%) as a sticky solid.

[0224] Process-V: To a mixture of compound 5 (2 g, 4.21 mmol) and imidazole (333 milligrams (mg), 4.73 mmol) in pyridine (13 mL) cooled to −20 °C under nitrogen was added phosphoryl chloride (POCl) (1.02 mL, 10.94 mmol). After stirring at −20 °C for 30 min to 1 h, the mixture was evaporated to dryness in vacuo. The resulting brown solid was dissolved in CHCl (40 mL) and washed with 1.0 N aqueous citric acid (40 mL). The organic phase was dried over magnesium sulfate, filtered, and concentrated under reduced pressure to give the crude material as a viscous oil. The crude material was purified by Combiflash (eluting with hexane / EtOAc) to give tert-butyl trans-(3S,5S)-3-(2-(tert-butoxy)-2-oxoethyl)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidine-1-carboxylate (compound 6, 1.3 g, 68%) as a white solid.

[0225] Process-VI: To a solution of compound 6 (1.0 g, 2.1 mmol) in acetonitrile (CHCN) (5 mL) at 0° C., TFA (5 mL) was added dropwise over 5 minutes. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated in vacuo and crystallized from ethyl acetate (EA) / ether to give trans-2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetic acid (compound 7, 500 mg, 76%) as a white powder.

[0226] Example 2: Synthesis of tert-butyl 4-(azidomethyl)isoindoline-2-carboxylate and tert-butyl 4-(aminomethyl)isoindoline-2-carboxylate: tert-Butyl 4-(azidomethyl)isoindoline-2-carboxylate and tert-butyl 4-(aminomethyl)isoindoline-2-carboxylate were synthesized according to Scheme 2. TIFF2025131780000073.tif29149

[0227] Process-I To a stirred solution of isoindoline methyl ester hydrochloride (compound 8, 1.00 g, 5.64 mmol) in DCM (20 mL) at room temperature, BocO (4.9 mL, 22.59 mmol) was added in one portion, followed by the dropwise addition of triethylamine (2.9 mL, 22.59 mmol). Stirring was continued for 12 h, and the reaction mixture was diluted with water (30 mL) and extracted into DCM (2 × 25 mL). The organic layer was dried over anhydrous MgSO, filtered, and the filtrate was evaporated under reduced pressure. The crude residue was purified by CombiFlash using hexane and ethyl acetate as the mobile phase to give 2-(tert-butyl)4-methyl isoindoline-2,4-dicarboxylate (compound 9, 1.2 g, 92%) as a white, colorless, viscous liquid. LC-MS (m / z); [M+H] C 15 H 20 Calculated and measured value of NO4: 278.13 g / mol.

[0228] Process-II: To a stirred solution of 2-(tert-butyl)4-methyl isoindoline-2,4-dicarboxylate (compound 9, 1.0 g, 3.61 mmol) in THF (10.0 mL) at room temperature under a nitrogen atmosphere was added sodium borohydride (1.37 g, 36.101 mmol). Methanol (MeOH, 10 mL) was added dropwise over 5 minutes to the stirred mixture. The reaction was warmed to 55°C and stirred for 5 hours. The reaction mixture was cooled to 0°C, quenched slowly with saturated aqueous ammonium chloride, and extracted into EtOAc (60 mL). The organic phase was collected, dried over sodium sulfate, and the solvent was evaporated to give a crude residue that was purified by Combiflash to give tert-butyl 4-(hydroxymethyl)isoindoline-2-carboxylate (compound 10, 700 mg, 70%) as a white sticky solid. LC-MS (m / z): [M+H]: C 14 H 20 Calculated NO3: Measured: 250.14 g / mol.

[0229] Process-III: tert-Butyl 4-(hydroxymethyl)isoindoline-2-carboxylate (compound 10, 500 mg, 2.00 mmol) followed by PPh3 (790 mg, 3.01 mmol) was dissolved in DMF (10 mL) and freshly recrystallized NBS (532 mg, 3.01 mmol) was added. The reaction mixture was stirred at room temperature under a nitrogen atmosphere for 4–5 h. The reaction mixture was diluted with water (40 mL) and extracted with ethyl acetate (2 × 25 mL). The organic layer was washed with water and brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was evaporated under reduced pressure and purified in a combi-flask to give tert-butyl 4-(bromomethyl)isoindoline-2-carboxylate (compound 11, 450 mg, 72%) as a white solid. LC-MS (m / z): [M+H] C 14 H 19 Calculated value of BrNO2: Found: 312.05 g / mol.

[0230] Process-IV: To a stirred solution of tert-butyl 4-(bromomethyl)isoindoline-2-carboxylate (compound 11, 400 mg, 1.286 mmol) in DMF was added NaN (420 mg, 6.430 mmol), and stirring was continued at 65 °C for 6 h. The reaction mixture was diluted with water and extracted into ethyl acetate. The organic layer was washed with water and brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was evaporated under reduced pressure and purified by combi-flask to give tert-butyl 4-(azidomethyl)isoindoline-2-carboxylate (compound 12a, 300 mg, 85%) as a colorless viscous liquid. LC-MS (m / z): [M+H] C 14 H 19 Calculated and measured N4O2: 274.14 g / mol.

[0231] Process-V: To a stirred solution of tert-butyl 4-(azidomethyl)isoindoline-2-carboxylate (compound 12a, 1.0 equivalent (eq)), THF was added followed by PPh3 (1.5 eq) and water (3.0 eq), and stirring was continued at room temperature for 12 hours. The reaction mixture was evaporated under reduced pressure, and the resulting crude residue was purified by using a combi-flask with methanol and dichloromethane as the mobile phase to give tert-butyl 4-(aminomethyl)isoindoline-2-carboxylate (compound 12b, 85%) as a white solid. LC-MS (m / z): [M+H] C 14 H 21 Calculated and measured N2O2: 249.15 g / mol.

[0232] Example 3: 5-((2-(4-(((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)amino)-4-oxobutanamido)ethyl)carbamoyl)-2-(6-(dimethylamino)-3-(dimethyliminio)-9,9a-dihydro-3H-xanthen-9-yl)benzoate was synthesized according to Scheme 3.

[0233] Synthesis of 5-((2-(4-(((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)amino)-4-oxobutanamido)ethyl)carbamoyl)-2-(6-(dimethylamino)-3-(dimethyliminio)-9,9a-dihydro-3H-xanthen-9-yl)benzoate TIFF2025131780000074.tif138128

[0234] Process-I Trifluoroacetic acid (TFA) was added to a stirred solution of tert-butyl 4-(azidomethyl)isoindoline-2-carboxylate (compound 12a) in DCM, and stirring was continued for 1 h. In a separate flask, trans-2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetic acid (compound 7) was dissolved in DMF, followed by the addition of HATU (1.3 eq) and DIPEA (3.0 eq) for preactivation of the acid function. The resulting amine was added to the mixture containing the preactivated acid, and the mixture was stirred at room temperature for 3 h. The reaction mixture was diluted with water and extracted with ethyl acetate. The organic layer was evaporated and the resulting crude residue was purified by Combiflash using methanol and DCM as the mobile phase to give (S)-1-((2S,4S)-4-(2-(4-(azidomethyl)isoindolin-2-yl)-2-oxoethyl)-5-oxopyrrolidine-2-carbonyl)-4,4-difluoropyrrolidine-2-carbonitrile (compound 13). LC-MS (m / z): C 21 H 22 Calculated and measured value of F2N7O3: 458.17.

[0235] Process-II: Using the same procedure as in Step V provided in Example 2, (S)-1-((2S,4S)-4-(2-(4-(azidomethyl)isoindolin-2-yl)-2-oxoethyl)-5-oxopyrrolidine-2-carbonyl)-4,4-difluoropyrrolidine-2-carbonitrile (Compound 13) was reacted with triphenylphosphine (PPh), water, and tetrahydrofuran to give (S)-1-((2S,4S)-4-(2-(4-(aminomethyl)isoindolin-2-yl)-2-oxoethyl)-5-oxopyrrolidine-2-carbonyl)-4,4-difluoropyrrolidine-2-carbonitrile (Compound 14), which was carried on to the next step without purification. (S)-1-((2S,4S)-4-(2-(4-(aminomethyl)isoindolin-2-yl)-2-oxoethyl)-5-oxopyrrolidine-2-carbonyl)-4,4-difluoropyrrolidine-2-carbonitrile (compound 14) was dissolved in 4-((2-((tert-butoxycarbonyl)amino)ethyl)amino)-4-oxobutanoic acid, HATU (1.3 eq), DI Reaction with PEA (3.0 eq) and DCM gave tert-butyl (2-(4-(((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)amino)-4-oxobutanamido)ethyl)carbamate (compound 15).

[0236] Process-III To a stirred solution of tert-butyl (2-(4-(((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)amino)-4-oxobutanamido)ethyl)carbamate (compound 15) in DCM was added TFA and stirring was continued for 10 min. The reaction mixture was evaporated under reduced pressure and the resulting crude residue was treated with NHS rhodamine (1.0 eq) and DIPEA (2.0 eq) in DMF for 1 h. The reaction mixture was diluted with water and purified by UHPLC (A = 20 mM ammonium acetate buffer (pH = 7), B = acetonitrile, solvent gradient from 5% B to 95% in 60 min) to give 5-((2-(4-(((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)amino)-4-oxobutanamido)ethyl)carbamoyl)-2-(6-(dimethylamino)-3-(dimethyliminio)-9,9a-dihydro-3H-xanthen-9-yl)benzoate (compound 16). LC-MS (m / z): [M+H] C 52 H 56 Calculated and measured value of F2N9O9: 988.41 g / mol.

[0237] Example 4: N-(9-(2-carboxy-4-((1-(4-(1-((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)phenyl)-1,6-dioxo-9,12,15,18-tetraoxa-2,5-diazaicosan-20-yl)carbamoyl)phenyl)-6-(dimethylamino)-9,9a-dihydro-3H-xanthen-3-ylidene)-N-methylmethanaminium and 5-((1-(4-(1-((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)phenyl)-1,6-dioxo-9,12,15,18-tetraoxa-2,5-diazaicosan-20-yl)carbamoyl)-2-(6-hydroxy-3-oxo-9,9a-dihydro-3H-xanthen-9-yl)benzoic acid were synthesized according to Schemes 4a and 4b.

[0238] N-(9-(2-carboxy-4-((1-(4-(1-((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)phenyl)-1,6-dioxo-9,12,15,18-tetraoxa-2,5-diazaicosan-20-yl)carbamoyl)phenyl)-6-(dimethylamino)-9,9a-dihydro-3H-xanthen-3-ylidene)-N-methyl Synthesis of ethylmethanaminium and 5-((1-(4-(1-((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)phenyl)-1,6-dioxo-9,12,15,18-tetraoxa-2,5-diazaicosan-20-yl)carbamoyl)-2-(6-hydroxy-3-oxo-9,9a-dihydro-3H-xanthen-9-yl)benzoic acid: TIFF2025131780000075.tif82149

[0239] Process-I To a stirred solution of 4-ethynylbenzoic acid (compound 17, 500 mg, 3.424 mmol) in anhydrous DCM (10 mL) at room temperature under a nitrogen atmosphere, HATU (1.4 g, 3.76 mmol) was added followed by DIPEA (1.7 mL, 10.27 mmol), and stirring was continued for 10 minutes to activate the acid. N-Fmoc-ethylenediamine (1.0 g, 3.76 mmol) was added to the reaction mixture, and stirring was continued for an additional 3 hours. The reaction mixture was diluted with DCM (20 mL), and the resulting precipitate was filtered through a Buchner funnel. The resulting white solid was washed again with DCM (2 × 20 mL) and dried under vacuum for 1 hour to give (9H-fluoren-9-yl)methyl (2-(4-ethynylbenzamido)ethyl)carbamate (compound 18, 1.2 g, 85%). LRMS-LC / MS (m / z): [M+H] C 26 H 23 Calculated N2O3, measured value 411.16.

[0240] Process-II: To a mixture of tert-butyl 4-(azidomethyl)isoindoline-2-carboxylate (Compound 12a, 200 mg, 0.729 mmol) and (9H-fluoren-9-yl)methyl (2-(4-ethynylbenzamido)ethyl)carbamate (Compound 18, 360 mg, 0.874 mmol) in anhydrous DMF (5.0 mL), copper iodide (CuI, 70 mg, 0.364 mmol) was added, followed by DIPEA (0.3 mL, 1.458 mmol). The reaction mixture was stirred at 50 °C under a nitrogen atmosphere for 5 h. The mixture was cooled to room temperature, diluted with water (20 mL), and vigorously stirred for 15 min. The solid residue formed in the reaction mixture was filtered off, washed with 2×20 mL of water, and dried under vacuum for 1 hour to give tert-butyl 4-((4-(4-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)carbamoyl)phenyl)-1H-1,2,3-triazol-1-yl)methyl)isoindoline-2-carboxylate (compound 19, 480 mg, 96%) as a brown solid, which was carried on to the further step without purification. LRMS-LC / MS (m / z): [M+H] C 40 H 41 Calculated N6O5, measured value 684.31.

[0241] Process-III: To a stirred solution of compound tert-butyl 4-((4-(4-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)carbamoyl)phenyl)-1H-1,2,3-triazol-1-yl)methyl)isoindoline-2-carboxylate (compound 19, 400 mg, 0.584 mmol) in DCM and MeOH (1:0.5 mL) was added (Et)NH (1 mL) and stirring was continued for 2 hours. The reaction mixture was evaporated under reduced pressure and the crude residue obtained was treated with diethyl ether (3 x 10 mL). The residue was scratched to give a solid. The diethyl ether was decanted and the solid obtained was dried under vacuum for 1 hour before being carried on to the next step. The amine compound was followed by Fmoc-NH(PEG) nNHS esters (1.2 equiv., n = 3, 5, and 11) followed by DIPEA (2.0 equiv.) were dissolved in DCM (1 mL per 1 mmol), and the reaction mixture was stirred at room temperature under a nitrogen atmosphere for 1 h. The reaction mixture was evaporated under reduced pressure, and the resulting crude residue was purified by CombiFlash using DCM and MeOH as the mobile phase to give compounds 20a–c in 70–80% yield. i) LCMS of 20a: LC / MS (m / z): [M+H] C 51 H 62 N7O 10 Calculated value, measured value: 931.45. ii) LCMS of 20b: LC / MS (m / z): [M+H] C 55 H 69 N7O 12 Calculated value, measured value: 1020.50. iii) LCMS of 20c: LC / MS (m / z): [M+H]C 67 H 93 N7O 18 Calculated value, measured value: 1284.66.

[0242] Process-IV: To a stirred solution of compound 20a, 20b, or 20c (1.0 eq) in DCM (1.0 mL) at room temperature, TFA (10 eq) was added and stirring was continued for 30 min. The reaction mixture was evaporated and dried under vacuum. In a separate round-bottom flask, trans-2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetic acid (compound 7, 1.2 eq) was dissolved in DMF (0.5 mL), followed by HATU (1.3 eq) and DIPEA (5.0 eq). The reaction mixture was stirred at room temperature for 10 min under a nitrogen atmosphere for activation of the acid functional group in compound 7. The amine obtained from the Fmoc deprotection of compounds 20a–c was dissolved in DCM (1 mL) and added to the above reaction mixture. Stirring was continued for an additional 2 h. The reaction mixture was diluted with water (15 mL) and extracted with ethyl acetate (2 × 15 mL). The organic extract was dried over anhydrous sodium sulfate and concentrated.The resulting crude residue was purified by Combiflash using DCM and MeOH as the mobile phase to give the desired compound (1-(4-(1-((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)fluoro (9H-fluoren-9-yl)methyl (1-(4-(1-((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl) (9H-fluoren-9-yl)methyl (1-(4-(1-((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxo-1H-1,2,3-triazol-4-yl)phenyl)-1,6-dioxo-9,12,15,18,21,24-hexaoxa-2,5-diazahexacosan-26-yl)carbamate, (9H-fluoren-9-yl)methyl (isopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)phenyl)-1,6-dioxo-9,12,15,18,21,24,27,30,33,36,39,42-dodecaoxa-2,5-diazatetracontan-44-yl)carbamate was obtained in 40-60% yield. i) LCMS of compound 21a: LC / MS (m / z): [M+H] C 58 H 65 F2N 10 O 11 Calculated value, measured value: 1115.47. ii) LCMS of compound 21b: LC / MS (m / z): [M+H] C 62 H 72 F2N 10 O 13 Calculated value, measured value: 1203.52. iii) LCMS of compound 21c: LC / MS (m / z): [M+H] C74 H 97 F2N 10 O 19 Calculated value, measured value: 1467.68. TIFF2025131780000076.tif72149

[0243] Process-V: Deprotection of the Fmoc functional group of compounds 21a-c followed the same procedure as provided in Step III of Example 4. The free amine obtained from compound 21a was treated with NHS-rhodamine (1.1 eq) or 1.1 eq of NHS-FITC in DMF in the presence of DIPEA (1.1 eq) for 30 min, followed by purification by UHPLC (A = 20 mM ammonium acetate buffer (pH = 7), B = acetonitrile, solvent gradient from 5% B to 95% in 60 min) to give the desired rhodamine compound 22 or FITC (23) conjugate, respectively, in quantitative yield. LC / MS of compound 22: LC / MS (m / z): [M+H] C 68 H 78 F2N 12 O 13 Calculated value: 1308.57 g / mol. LCMS of compound 23: LC / MS (m / z): [M+H] C 64 H 67 F2N 10 O 15 Calculated and measured values: 1253.47 g / mol.

[0244] Example 5: 2-((E)-2-((E)-2-(4-(1-(4-(1-((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)phenyl)-1,6,21-trioxo-9,12,14,17-tetraoxa- Sodium 2,5,20-triazatricosan-23-yl)phenoxy)-3-(2-((E)-3,3-dimethyl-5-sulfonato-1-(4-sulfonatobutyl)indolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indol-1-ium-5-sulfonate was synthesized according to Scheme 5.

[0245] 2-((E)-2-((E)-2-(4-(1-(4-(1-((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)phenyl)-1,6,21-trioxo-9,12,14,17-tetramethyl Synthesis of sodium triaoxa-2,5,20-triazatricosan-23-yl)phenoxy)-3-(2-((E)-3,3-dimethyl-5-sulfonato-1-(4-sulfonatobutyl)indolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indol-1-ium-5-sulfonate: TIFF2025131780000077.tif108145

[0246] Process-I: The amine (1.0 eq) obtained by reduction of compound 21a with diethylamine, as provided in Step III of Example 4, was dissolved in DMF. 3-(4-hydroxyphenyl)propionic acid (1.2 eq), HATU (1.3 eq), and DIPEA (3.0 eq) were added, and the reaction mixture was stirred at room temperature for 2-3 hours, followed by evaporation under reduced pressure. The resulting crude residue was purified by UHPLC (A = 20 mM ammonium acetate buffer (pH = 7), B = acetonitrile, using a solvent gradient of 5% B to 95% B over 60 min) to give the desired compound 24a. LC / MS (m / z): [M+H] C 50 H 59 F2N 10 O 10 Calculated value, measured value: 997.43.

[0247] Process-II To a stirred solution of compound 24a in anhydrous DMSO at room temperature under an argon atmosphere, ClS0456 dye (1.0 eq) was added followed by Cs2CO3 (5.0 eq), and stirring was continued for an additional 3-4 h while monitoring the reaction progress by LCMS. The reaction mixture was diluted with water and purified by UHPLC (A = 20 mM ammonium acetate buffer (pH = 7), B = 0 acetonitrile, solvent gradient from 5% B to 35% over 60 min) to give 2-((E)-2-((E)-2-(4-(1-(4-(1-((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)-1H-1,2,3-triazolium stearate. Sodium (2-((E)-3,3-dimethyl-5-sulfonato-1-(4-sulfonatobutyl)indolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indol-1-ium-5-sulfonate (compound 25a) was obtained. LC-MS of 25a: LC / MS (m / z): [M+H] C 90 H105 F2N 12 NaO 23 Calculated value of S4: 1957.60.

[0248] Example 6: 4-((2-(4-(1-((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)benzamido)ethyl)amino)-4-oxobutanoic acid was synthesized according to Scheme 6.

[0249] Synthesis of 4-((2-(4-(1-((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)benzamido)ethyl)amino)-4-oxobutanoic acid TIFF2025131780000078.tif42149

[0250] Process-I The synthesis of (9H-fluoren-9-yl)methyl (2-(4-(1-((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)benzamido)ethyl)carbamate (Compound 26) followed a similar procedure as provided in Step-IV of Example 4 (LC / MS (m / z): [M+H] C 47 H 44Calculated value for F2N9O6, Found: 868.33). The free amine obtained by reducing compound 26 using a procedure similar to that provided in Step-III of Example 4 was dissolved in DCM, followed by the addition of succinic anhydride (1.5 eq) and DIPEA (2.0 eq). The solution was stirred at room temperature for 1 hour. The reaction mixture was evaporated and the resulting crude residue was purified by UHPLC (A = 20 mM ammonium acetate buffer (pH = 7), B = acetonitrile, solvent gradient from 5% B to 95% in 60 min) to give 4-((2-(4-(1-((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)benzamido)ethyl)amino)-4-oxobutanoic acid (compound 27). LC / MS (m / z): [M+H] C 36 H 38 Calculated and measured value of F2N9O7: 746.28.

[0251] Example 7: 2-((E)-2-((E)-2-(4-(1-(2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)-3,25-dioxo-6,9,12,15,18,21-hexaoxa-2,24-diazaheptacosane-27 Sodium (2-((E)-3,3-dimethyl-5-sulfonato-1-(4-sulfonatobutyl)indolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indol-1-ium-5-sulfonate was synthesized according to Scheme 7.

[0252] Synthesis of sodium 2-((E)-2-((E)-2-(4-(1-(2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)-3,25-dioxo-6,9,12,15,18,21-hexaoxa-2,24-diazaheptacosan-27-yl)phenoxy)-3-(2-((E)-3,3-dimethyl-5-sulfonato-1-(4-sulfonatobutyl)indolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indol-1-ium-5-sulfonate TIFF2025131780000079.tif109145

[0253] Process-1: To a stirred solution of 3-(4-(tert-butoxy)phenyl)propanoic acid (compound 28) in anhydrous DMF was added HATU (1.3 eq) and DIPEA (3.0 eq), followed by amino-PEG6-t-butyl ester (1.2 eq). The reaction mixture was diluted with water and then extracted into ethyl acetate (2 x 20 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated under reduced pressure. The crude residue was purified by Combiflash using methanol and dichloromethane as the mobile phase to give tert-butyl 3-(2-(2-(3-(4-(tert-butoxy)phenyl)propanamido)ethoxy)ethoxy)propanoate (compound 29) as a viscous liquid. LC / MS (m / z): [M+H]: C 32 H 56 NO 10 Calculated value, measured value: 614.38.

[0254] Process-II: tert-Butyl 3-(2-(2-(3-(4-(tert-butoxy)phenyl)propanamido)ethoxy)ethoxy)propanoate (compound 29) was dissolved in DCM. Trifluoroacetic anhydride was added and the reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was evaporated under reduced pressure and the crude residue obtained was dissolved in DMF. DIPEA (5.0 eq) and HATU (1.3 eq) were added, followed by tert-butyl 4-(aminomethyl)isoindoline-2-carboxylate (compound 12b, 1.1 eq). The mixture was stirred for 1 hour. The reaction mixture was diluted with water and extracted into ethyl acetate (2 x 20 mL), the organic layer was evaporated, and the crude residue was purified by Combiflash using methanol and dichloromethane as the mobile phase to give tert-butyl 4-(27-(4-hydroxyphenyl)-3,25-dioxo-6,9,12,15,18,21-hexaoxa-2,24-diazaheptacosyl)isoindoline-2-carboxylate (compound 30) as a viscous liquid. LC / MS (m / z): [M+H]: C 38 H 58 N3O 11 Calculated value, measured value: 732.40.

[0255] Step-III and Step-IV: Using the same procedures as provided in Step-IV of Example 4 and Step-II of Example 5, respectively, N-((2-(2-((3S,5R)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)-1-(3-(4-hydroxyphenyl)propanamido)-3,6,9,12,15,18-hexaoxahenicosan-21-amide (Compound 31) and 2-((E)-2-((E)-2-(4-(1-(2-(2-((3S,5S)-5-((S)-2-cyano-4,4 (Difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)-3,25-dioxo-6,9,12,15,18,21-hexaoxa-2,24-diazaheptacosan-27-yl)phenoxy)-3-(2-((E)-3,3-dimethyl-5-sulfonato-1-(4-sulfonatobutyl)indolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indol-1-ium-5-sulfonate sodium salt (compound 32) was synthesized. LCMS of compound 31: LC / MS (m / z): [M+H] C 45 H 61 F2N6O 12 Calculated value for , Found: 915.42. LCMS of compound 32: LC / MS (m / z): [M+H] C 83 H 104 F2N8Na3O 24 Calculated and measured values ​​of S4: 1831.56.

[0256] Example 8: 2-((E)-2-((E)-2-(4-(1-(1-((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)-3,19-dioxo-6,9,12,15-tetraoxa-2,18 Sodium (-diazahenicosan-21-yl)phenoxy)-3-(2-((E)-3,3-dimethyl-5-sulfonato-1-(4-sulfonatobutyl)indolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indol-1-ium-5-sulfonate was synthesized according to Scheme 8.

[0257] 2-((E)-2-((E)-2-(4-(1-(1-((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)-3,19-dioxo-6,9,12,15-tetraoxa Synthesis of sodium (-2,18-diazahenicosan-21-yl)phenoxy)-3-(2-((E)-3,3-dimethyl-5-sulfonato-1-(4-sulfonatobutyl)indolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indol-1-ium-5-sulfonate: TIFF2025131780000080.tif131146

[0258] Process-I To a mixture of tert-butyl 4-(azidomethyl)isoindoline-2-carboxylate (Compound 12a, 1.0 eq) and Fmoc propargylamine (1.2 eq) in anhydrous DMF (5.0 mL) was added CuI (0.5 eq) followed by DIPEA (2.0 eq). The reaction mixture was stirred at 50° C. under a nitrogen atmosphere for 5 hours. The reaction mixture was then cooled to room temperature, diluted with water (20 mL), and vigorously stirred for 15 minutes. The solid residue formed in the reaction mixture was filtered, washed with 2×20 mL of water, and dried under vacuum for 1 hour to give tert-butyl 4-((4-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-1H-1,2,3-triazol-1-yl)methyl)isoindoline-2-carboxylate (Compound 33, 96%) as a solid, which was used in the next step without further purification. To a stirred solution of compound 33 (1 mmole) in DCM (5 mL) was added (Et)NH (2 mL), and stirring was continued for 2 h while monitoring the progress of the reaction by LCMS. The reaction mixture was evaporated under reduced pressure, and the resulting crude residue was treated with diethyl ether. The ether layer was decanted, and the solid compound was dried under vacuum and subsequently dissolved in DCM. To this reaction mixture was added Fmoc-NH(PEG4)NHS ester (1.2 eq) and DIPEA (3.0 eq). The reaction mixture was stirred at room temperature for 1 h, evaporated under reduced pressure, and the crude residue was purified by Combiflash using methanol and DCM as the mobile phase to give compound 34.

[0259] Example 9: 2-((3S,5S)-5-((2S,4S)-2-cyano-4-fluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetic acid was synthesized according to Scheme 9. Synthesis of 2-((3S,5S)-5-((2S,4S)-2-cyano-4-fluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetic acid: TIFF2025131780000081.tif38149

[0260] Process-I: To a stirred solution of (2S,4S)-4-(2-(tert-butoxy)-2-oxoethyl)-1-(tert-butoxycarbonyl)-5-oxopyrrolidine-2-carboxylic acid (compound 4, 1.0 eq) in anhydrous DMF were added HATU (1.3), DIPEA (3.0 eq), and stirring was continued for 10 min for activation of the acid functional group in 4. 4-cis-fluoro-L-prolinamide hydrochloride (1.2) and DIPEA (2.0 eq) were added to the mixture, and stirring was continued for 5 h at room temperature under a nitrogen atmosphere. The reaction mixture was diluted with water and brine and extracted into ethyl acetate. The combined organic extracts were dried over anhydrous sodium sulfate and filtered. The filtrate was evaporated under reduced pressure and the resulting crude residue was purified by Combiflash using DCM / MeOH as the mobile phase to give tert-butyl (3S,5S)-3-(2-(tert-butoxy)-2-oxoethyl)-5-((2S,4S)-2-carbamoyl-4-fluoropyrrolidine-1-carbonyl)-2-oxopyrrolidine-1-carboxylate (compound 38) as a sticky solid.

[0261] Process-II: To a mixture of tert-butyl (3S,5S)-3-(2-(tert-butoxy)-2-oxoethyl)-5-((2S,4S)-2-carbamoyl-4-fluoropyrrolidine-1-carbonyl)-2-oxopyrrolidine-1-carboxylate (compound 38, 1.0 eq) and imidazole (1.2 eq) in pyridine cooled to −20 °C under nitrogen was added phosphoryl chloride (POCl) (1.0 eq). After stirring at −20 °C for 30 min to 1 h, the mixture was evaporated to dryness in vacuo. The resulting brown solid was dissolved in CHCl (40 mL) and washed with 1.0 N aqueous citric acid (40 mL). The organic phase was dried over magnesium sulfate, filtered, and concentrated under reduced pressure to give the crude material as a viscous oil. The crude material was purified by Combiflash (eluted with hexane / EtOAc) to give tert-butyl (3S,5S)-3-(2-(tert-butoxy)-2-oxoethyl)-5-((2S,4S)-2-cyano-4-fluoropyrrolidine-1-carbonyl)-2-oxopyrrolidine-1-carboxylate (compound 39) as a white solid.

[0262] Process-III: To a solution of tert-butyl (3S,5S)-3-(2-(tert-butoxy)-2-oxoethyl)-5-((2S,4S)-2-cyano-4-fluoropyrrolidine-1-carbonyl)-2-oxopyrrolidine-1-carboxylate (compound 39, 1 mmol) in CH3CN (5 mL) at 0 °C was added TFA (5 mL) dropwise over 5 min. The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated in vacuo and crystallized with EA / ether to give 2-((3S,5S)-5-((2S,4S)-2-cyano-4-fluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetic acid (compound 40) as a white powder. LC / MS (m / z): C 12 H 15 Calculated FN3O4, measured value 284.10.

[0263] Example 10: 4-((2-(4-(1-((2-(2-((3S,5S)-5-((2S,4S)-2-cyano-4-fluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)benzamido)ethyl)amino)-4-oxobutanoic acid was synthesized according to Scheme 10.

[0264] Synthesis of 4-((2-(4-(1-((2-(2-((3S,5S)-5-((2S,4S)-2-cyano-4-fluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)-1H-1,2,3-triazol-4-yl)benzamido)ethyl)amino)-4-oxobutanoic acid TIFF2025131780000082.tif47149

[0265] To a stirred solution of compound 19 (1.0 eq) in DMF (1.0 mL) at room temperature, TFA (10 eq) was added and stirring was continued for 30 min. The reaction mixture was evaporated and dried under vacuum. In a separate round-bottom flask, compound 40 (1.2 eq) was dissolved in DMF (0.5 mL), followed by HATU (1.3 eq) and DIPEA (5.0 eq). The reaction mixture was stirred at room temperature for 10 min under a nitrogen atmosphere for activation of the acid functional group in compound 40. The amine obtained from compound 19 was dissolved in DMF (1 mL) and added to the above reaction mixture, and stirring was continued for another 2 h. The reaction mixture was diluted with water (15 mL) and extracted into ethyl acetate (2 × 15 mL). The organic extract was dried over anhydrous sodium sulfate and concentrated. The crude residue obtained was purified by Combiflash using DCM and MeOH as the mobile phase to give the desired compound 41. LC / MS of compound 41: LC / MS (m / z): [M+H] C 47 H 45 Calculated for FN9O6, Found: 850.34. LCMS for compound 42: LC / MS (m / z): [M+H] C 36 H 40 Calculated value of FN9O7, measured value 728.29 g / mol.

[0266] Example 11: 5-((2-(2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindoline-4-carboxamido)ethyl)carbamoyl)-2-(6-(dimethylamino)-3-(dimethyliminio)-3H-xanthen-9-yl)benzoate was synthesized according to Scheme 11.

[0267] Synthesis of 5-((2-(2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindoline-4-carboxamido)ethyl)carbamoyl)-2-(6-(dimethylamino)-3-(dimethyliminio)-3H-xanthen-9-yl)benzoate: TIFF2025131780000083.tif90146

[0268] Process-I To a stirred solution of 2-(tert-butyl)4-methyl isoindoline-2,4-dicarboxylate (compound 8, 200 mg, 0.72 mmol) in MeOH / THF / HO (0.2 mL / 0.6 mL / 0.2 mL) was added LiOH (172.8 mg, 7.2 mmol, 10 eq). The mixture was stirred for 5 h. The solvent was removed under reduced pressure, and the mixture was dissolved in water (1 mL), and the pH was adjusted to 7 with citric acid (1 M). The product was extracted with EA (3 mL × 3). The organic layers were combined, dried over sodium sulfate, and concentrated under reduced pressure to give 2-(tert-butoxycarbonyl)isoindoline-4-carboxylic acid (compound 51), which was used in the next step without further purification.

[0269] Process-II HATU (456 mg, 1.2 mmol, 1.2 eq) and DIPEA (258 mg, 2 mmol, 2.0 eq) were added to a solution of 2-(tert-butoxycarbonyl)isoindoline-4-carboxylic acid (compound 51, 263 mg, 1.0 mmol, 1.0 eq) in DMF (5 mL). The mixture was stirred for 10 minutes, and then (9H-fluoren-9-yl)methyl (2-aminoethyl)carbamate hydrochloride (350.9 mg, 1.1 mmol, 1.1 eq) was added. The mixture was diluted with ethyl acetate (2 mL) and washed with HO (2 mL × 3). The organic layers were combined, dried over sodium sulfate, and concentrated under reduced pressure. The residual oil was purified by Combiflash chromatography using hexane / ethyl acetate as eluent to give tert-butyl 4-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)carbamoyl)isoindoline-2-carboxylate (compound 52) in 352.5 mg yield as a white solid.

[0270] Process-III To a stirred solution of compound 52 (1.0 eq) in DMF (1.0 mL) at room temperature, TFA (10 eq) was added and stirring was continued for 30 min. The reaction mixture was evaporated and dried under vacuum. In a separate round-bottom flask, acid compound 7 (1.2 eq) was dissolved in DMF (0.5 mL), followed by HATU (1.3 eq) and DIPEA (5.0 eq). The reaction mixture was stirred at room temperature for 10 min under a nitrogen atmosphere for activation of the acid functional group in compound 7. The amine obtained from compound 52 was dissolved in DMF (1 mL) and added to the above reaction mixture, and stirring was continued for an additional 2 h. The reaction mixture was diluted with water (15 mL) and extracted with ethyl acetate (2 × 15 mL). The organic extract was dried over anhydrous sodium sulfate. The extract was concentrated, and the resulting crude residue was purified by Combiflash using DCM and MeOH as the mobile phase to give the desired compound 53. (Et)2NH was added to a solution of compound 53 in DCM and stirred at room temperature for 1 hour, evaporated under reduced pressure and the crude residue was treated with diethyl ether, dried and taken to the next step without purification.

[0271] N-(2-aminoethyl)-2-(2-((3S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindoline-4-carboxamide (10.0 mg, 0.02 mmol, 1.0 eq) was dissolved in DMF (1 mL), and then rhodamine-NHS (12.9 mg, 0.024 mmol, 1.2 eq) was added, followed by DIPEA (3.87 mg, 0.03 mmol, 1.5 eq). The mixture was stirred at room temperature for 2 h. The reaction mixture was diluted with water and purified by UHPLC (A = 20 mM ammonium acetate buffer (pH = 7), B = acetonitrile, solvent gradient from 5% B to 95% in 60 min) to give 5-((2-(2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindoline-4-carboxamido)ethyl)carbamoyl)-2-(6-(dimethylamino)-3-(dimethyliminio)-3H-xanthen-9-yl)benzoate (compound 54). LC / MS (m / z): [M+H] C 48 H46 Calculated value of F2N8O8, measured value 901.3 g / mol.

[0272] Example 12: 5-((2-(3-(2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)propanamido)ethyl)carbamoyl)-2-(6-(dimethylamino)-3-(dimethyliminio)-3H-xanthen-9-yl)benzoate was synthesized according to Scheme 12.

[0273] Synthesis of 5-((2-(3-(2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)propanamido)ethyl)carbamoyl)-2-(6-(dimethylamino)-3-(dimethyliminio)-3H-xanthen-9-yl)benzoate TIFF2025131780000084.tif81145

[0274] Process-I To a solution of tert-butyl 4-bromoisoindoline-2-carboxylate (compound 55) (297 mg, 1 mmol, 1.0 eq) in DMF (1 mL) under an argon atmosphere was added benzyl acrylate (486 mg, 3 mmol, 3.0 eq), Pd(OAc) (22.3 mg, 0.1 mmol, 0.1 eq), P(o-Tol) (60.8 mg, 0.2 mmol, 0.2 eq), and DIPEA (387 mg, 3.0 mmol, 3.0 eq). The resulting mixture was heated at 100 °C for 8 h. After completion, the reaction was quenched with water (3 mL). The aqueous layer was extracted with EA (10 mL × 3), and the combined organic phases were dried over sodium sulfate. The organic layer was concentrated under reduced pressure, and the residue was purified by Combiflash chromatography using hexane / ethyl acetate as the eluent to give 148 mg of tert-butyl (E)-4-(3-(benzyloxy)-3-oxoprop-1-en-1-yl)isoindoline-2-carboxylate (compound 56) as a yellowish oil without further purification. Compound 56 was hydrogenated using Pd / C in MeOH under a hydrogen atmosphere for 8 hours to give 3-(2-(tert-butoxycarbonyl)isoindolin-4-yl)propanoic acid (compound 57). LC / MS of 57: LC / MS (m / z) [M+H] C 16 H 22 Calculated NO4 value, measured value 292.15.

[0275] Compounds 58-61 were synthesized in a similar manner as provided in Example 11 to yield 5-((2-(3-(2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)propanamido)ethyl)carbamoyl)-2-(6-(dimethylamino)-3-(dimethyliminio)-3H-xanthen-9-yl)benzoate. LC / MS (m / z) of compound 61: [M+H] C 50 H 51 Calculated value of F2N8O8, measured value 929.3 g / mol.

[0276] Example 13 N-(4-((2-(2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)-2,3,7,7a-tetrahydro-1H-isoindole-4-carboxamido)ethyl)amino)-4-oxobutanoyl)-S-((2-((((5-(6-(5-((2,4-difluorophenyl)sulfonamido)-6-methoxypyridin-3-yl)quinolin-4-yl)pyridin-2-yl)methoxy)carbonyl)oxy)ethyl)thio)cysteine ​​was synthesized according to Scheme 13.

[0277] Synthesis of N-(4-((2-(2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)-2,3,7,7a-tetrahydro-1H-isoindole-4-carboxamido)ethyl)amino)-4-oxobutanoyl)-S-((2-((((5-(6-(5-((2,4-difluorophenyl)sulfonamido)-6-methoxypyridin-3-yl)quinolin-4-yl)pyridin-2-yl)methoxy)carbonyl)oxy)ethyl)thio)cysteine TIFF2025131780000085.tif120148

[0278] N-(2-aminoethyl)-2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindoline-4-carboxamide (compound 53, 97.6 mg, 0.2 mmol, 1.0 eq) was dissolved in DCM (2 mL), then succinic anhydride (24 mg, 0.24 mmol, 1.2 eq) was added followed by DIPEA (51.6 mg, 0.4 mmol, 2 eq). The resulting mixture was kept for 8 h. The solvent was removed under reduced pressure. The residue was purified by preparative RP-HPLC [A = 2 mM ammonium acetate buffer (pH 7.0), B = acetonitrile, solvent gradient from 5% B to 55% B in 60 min] to yield 4-((2-(2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindoline-4-carboxamido)ethyl)amino)-4-oxobutanoic acid (compound 71). LC / MS (m / z): [M+H] Exact mass: 588.21 g / mol. 4-((2-(2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindoline-4-carboxamido)ethyl)amino)-4-oxobutanoic acid (compound 71, 117.6 mg, 0.2 mmol, 1.0 eq) was introduced into solid-phase peptide coupling conditions using H-Cys(Trt)-2-Cl-Trt (1.2 eq) along with HATU (91.2 mg, 0.24 mmol, 1.2 eq) and DIPEA (51.6 mg, 0.4 mmol, 2.0 eq). The final product was cleaved from the resin using a cocktail solution of TFA:water:TIPS:ethanedithiol (95%:2.5%:2.5%:2.5%). The crude compound was precipitated in ether to give (4-((2-(2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindoline-4-carboxamido)ethyl)amino)-4-oxobutanoyl)cysteine ​​(Compound 72). LC / MS (m / z): [M+H] Exact mass: 691.22.(5-(6-(5-((2,4-difluorophenyl)sulfonamido)-6-methoxypyridin-3-yl)quinolin-4-yl)pyridin-2-yl)methyl(2-mercaptoethyl) carbonate (7.46 mg, 0.01 mmol) and (4-((2-(2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindoline-4-carboxamido)ethyl)amino)-4-oxobutanoyl)cysteine ​​(Compound 72, 6.91 mg, 0.01 mmol) were dissolved in DMF (1 mL) and stirred. Following completion of the reaction, the crude product was purified by preparative RP-HPLC [A = 2 mM ammonium acetate buffer (pH 7.0), B = acetonitrile, solvent gradient from 5% B to 75% B in 60 min] to give N-(4-((2-(2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-1-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)-2,3, 7,7a-Tetrahydro-1H-isoindole-4-carboxamido)ethyl)amino)-4-oxobutanoyl)-S-((2-((((5-(6-(5-((2,4-difluorophenyl)sulfonamido)-6-methoxypyridin-3-yl)quinolin-4-yl)pyridin-2-yl)methoxy)carbonyl)oxy)ethyl)thio)cysteine ​​(compound 73) was obtained. LRMS-LCMS (m / z): [M + H]+ C. 60 H 58 F4N 11 O 14 S3 calculated value 1328.3; measured value 1328.2).

[0279] cell culture FaDu, HT29, MDA-MB231, KB, 4T1 PANC1, U87MG, LaNCap, and human FAP-transfected HEK-FAP and HT1080-FAP cells were cultured at 37°C in a 5% CO2 and 95% humidified atmosphere in a medium consisting of RPMI1640, DMEM, and EMEM, 10% FBS, 1% penicillin-streptomycin, and 1% 2 mM glutamine. The cells used in this study were initiated by thawing frozen vials from master stocks stored from the original cell lines purchased from ATCC. All experiments were performed within 2–5 passages after thawing the cells. Mycoplasma testing was not performed on any of the cell lines.

[0280] Animal husbandry Five- to six-week-old female athymic nu / nu mice were purchased from Harlan Laboratories and fed regular rodent chow and water ad libitum. Animals were maintained on a standard 12-hour light-dark cycle. All animal procedures were approved by the Purdue Animal Care and Use Committee.

[0281] Confocal binding studies of FAP-targeting ligands Method 1: HT1080-FAP cells (1,000,000 cells / well) were seeded in 4-well confocal plates. Cells were grown as monolayers for 24 hours at 37°C and then incubated with various concentrations of conjugate ranging from 3.0 nM (lowest) to 25 nM (highest) in 1% FBS in PBS for 1 hour at 37°C. Cells were washed with 1% FBS (3 x 500 μL), and images were acquired using a confocal microscope while the cells remained in 500 μL of 1% FBS. Again, the PBS inside the cells was replaced with growth medium, and the cells were re-incubated at 37°C for 8 to 48 hours. Images acquired with various concentrations of compound at 37°C are shown in Figure 3, images obtained at different time points are shown in Figure 4, and images acquired with a 100-fold excess of competing ligand are shown in Figure 5.

[0282] Method 2: HT1080-FAP cells (100,000 cells) transfected with human FAP were plated in a 4-well confocal plate and incubated with different concentrations of compounds (50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, and 1.65 nM) at 37°C for 1 hour. Unbound fluorescence was removed by washing the cells three times with medium, and cell-bound fluorescence was imaged using an Olympus confocal microscope. Experiments were repeated three times.

[0283] Binding assay Method 1: HT1080-FAP cells (200,000 cells / well) were seeded in 24-well plates. Cells were grown as monolayers for 24 hours and then incubated with various concentrations of rhodamine conjugates targeting FAP in the presence or absence of excess competing ligand (a dye-free ligand). After incubation at 4°C for 1 hour, cells were washed three times with PBS to remove unbound fluorescence. Cells were then lysed in 1% SDS, and cell-bound fluorescence was measured using a Neo2 plate reader. The results are shown in Figure 6.

[0284] Method 2: 100,000 HT1080-hFAP and HT1080 cells were seeded onto amine-coated 24-well plates to allow for cell adhesion. Once a monolayer was formed, the cells were incubated with various concentrations of compounds in the presence or absence of excess unlabeled ligand. After 1 hour of incubation, the cells were washed three times with medium to remove unbound fluorescence and dissolved in 1% SDS. Cell-bound fluorescence was measured. TIFF2025131780000086.tif4128 and Cell-bound fluorescence was measured using a fluorescence spectrophotometer (NeO2 plate reader) set to TIFF2025131780000087.tif4128. Cell-bound fluorescence was plotted against various concentrations, and the apparent K was calculated using one-site binding (hyperbolic) curve fitting in GraphPad prism7. d The experiment was performed three times.

[0285] Ex vivo fluorescence imaging and biodistribution: Female nu / nu athymic (5-6 weeks old) mice were cultured in 0.1 mL sterile PBS at 5 × 10 6 KB, MDA-MB231, HT29, U87MG, FaDu, PANC1 (supplemented with 20% Matrigel), and BalbC mice were subcutaneously injected with 4T1 cells. Tumors were allowed to grow to approximately 250–600 mm before imaging studies began. 3 Tumor-bearing mice were allowed to grow until tumor size reached 10 ...

[0286] Western blot analysis: After 24 hours of co-culture, the supernatant was removed and the cells were washed with phosphate-buffered saline (PBS). Cells were harvested and lysed for Western blot analysis. Samples were analyzed by gel electrophoresis using a 10% sodium dodecyl sulfate polyacrylamide gel followed by blocking. The nitrocellulose membrane was then incubated with antibodies to detect phosphorylated Akt^Ser473 and α-actin, and the signals were visualized using the Odyssey CLx imaging system (Figure 23).

[0287] quantitative PCR RNA was extracted using a Quick-RNA Microprep Kit according to the manufacturer's specifications (Zymo Research, catalog number R1050). The extracted RNA was incubated with DNase I provided in the kit for 15 minutes at room temperature. RNA was reverse transcribed using a High-Capacity cDNA Reverse Transcription Kit according to the manufacturer's specifications (Thermo Fisher; catalog number 4368814). The cDNA was then mixed with Cyber-green supermix and primers for human collagen 1a1 and human alpha-smooth muscle actin, and quantitative PCR was performed to analyze the expression of these two profibrotic markers (Figure 24).

Claims

1. A compound represented by the structure of formula (X): A m -L-B (X) During the ceremony, A is a radical of fibroblast activation protein α (FAPα) ligand; L is a linker connecting one or more A groups to B; B is a radical of an optical dye, a photodynamic therapy agent, a radioimaging agent, a radiotherapeutic agent, a chemotherapeutic agent, an antifibrotic agent, or an anticancer agent; and m is 1 to 6.

2. A has the structure of formula (XA), During the ceremony, Q is aryl, heteroaryl, or heterocyclyl; Z is a bond, substituted or unsubstituted C 1 ~C 3 alkylene, substituted or unsubstituted heteroalkylene, amino, -O-, or -S-; T is substituted or unsubstituted methylene, substituted or unsubstituted amino, -O-, or -S-; R 1 and R 2 are each independently -H, -CN, -CHO, -B(OH) 2 , -C(O) alkyl, -C(O) aryl-, -C=CC(O) aryl, -C=CS(O) 2 Aryl, -CO 2 H, -SO 3 H, -SO 2 NH 2 , -PO 3 H 2 , -SO 2 F, -CONH 2 and 5-tetrazolyl; R 3 and R 4 are each independently -H, -OH, F, Cl, Br, I, -C 1～6 Alkyl, -OC 1～6 Alkyl, and -SC 1～6 alkyl; and R 5 , R 6 , R 7 , and R 8 are each independently selected from the group consisting of H, alkyl, and halo; The compound of claim 1.

3. A has the structure of formula (XB), During the ceremony, Q is aryl, heteroaryl, or heterocyclyl; T is substituted or unsubstituted methylene, substituted or unsubstituted amino, -O-, or -S-; J is C(R J ) 2 where each R J are independently H or alkyl, or both R J together to form oxo; R 1 and R 2 are each independently -H, -CN, -CHO, -B(OH) 2 , -C(O) alkyl, -C(O) aryl-, -C=CC(O) aryl, -C=CS(O) 2 Aryl, -CO 2 H, -SO 3 H, -SO 2 NH 2 , -PO 3 H 2 , -SO 2 F, -CONH 2 and 5-tetrazolyl; R 3 and R 4 are each independently -H, -OH, F, Cl, Br, I, -C 1～6 Alkyl, -OC 1～6 Alkyl, and -SC 1～6 selected from the group consisting of alkyl; R 5 , R 6 , R 7 , and R 8 are each independently selected from the group consisting of H, alkyl, and halo; and R 9 , R 10 , and R 11 are independently H, -C 1～6 Alkyl, -C 1～6 Haloalkyl, -OC 1～6 Alkyl, -SC 1～6 selected from the group consisting of alkyl, F, Cl, Br, and I; A compound according to claim 1 or claim 2.

4. A, 4. The compound of any one of claims 1 to 3, selected from the group consisting of:

5. A, 4. The compound of any one of claims 1 to 3, selected from the group consisting of:

6. 6. The compound of any one of claims 1 to 5, wherein A has a binding affinity for FAPα of about 1 nM to about 25 nM.

7. 7. The compound of any one of claims 1-6, wherein L comprises one or more linker groups, each linker group independently selected from the group consisting of alkyl(ene), heteroalkyl(ene), heterocycloalkyl(ene), heteroaryl, aryl, alkoxy, thioether, disulfide, carboxylic acid, anhydride, carbonate, carbamate, thioether, sugar, and peptide.

8. 8. The compound of any one of claims 1-7, wherein L comprises one or more linker groups, each linker group independently selected from the group consisting of polyethylene glycol (PEG), alkyl(ene), disulfide, amide, carboxylic acid, anhydride, carbonate, ester, carbamate, thioether, phenyl, and triazole.

9. 9. The compound of any one of claims 1-8, wherein L comprises one or more linker groups, each linker group independently selected from the group consisting of polyethylene glycol (PEG), alkyl(ene), disulfide, amide, carboxylic acid, carbonate, and ester.

10. 9. The compound of any one of claims 1 to 8, wherein L is a releasable linker.

11. 9. The compound of any one of claims 1-8, wherein L comprises one or more linker groups, each linker group independently selected from the group consisting of polyethylene glycol (PEG), alkyl(ene), amide, phenyl, and triazole.

12. 12. The compound of any one of claims 1 to 8 and 11, wherein L is a non-releasable linker.

13. L is (L 1 ) o -Y-(L 2 ) p and where: Each L 1 is the first linker; Each L 2 is the second linker; Y is a third linker; o is an integer from 1 to 5; and p is an integer from 1 to 5; 13. The compound of any one of claims 1 to 12.

14. Each L 1 and L 2 independently comprise one or more linker groups, each linker group independently selected from the group consisting of alkyl(ene), heteroalkyl(heteroalkylene), heterocycloalkyl(heterocycloalkylene), heteroaryl, aryl, alkoxy, thioether, disulfide, carboxylic acid, anhydride, carbonate, carbamate, thioether, sugar, and peptide.

15. Each L 1 and L 2 independently comprise one or more linker groups, each linker group independently selected from the group consisting of polyethylene glycol (PEG), alkyl(ene), disulfide, amide, carboxylic acid, anhydride, carbonate, ester, carbamate, thioether, phenyl, and triazole.

16. Each L 1 and L 2 independently comprise one or more linker groups, each linker group independently selected from the group consisting of polyethylene glycol (PEG), alkyl(ene), disulfide, amide, carboxylic acid, carbonate, and ester.

17. Each L 1 and L 2 independently comprise one or more linker groups, each linker group independently selected from the group consisting of polyethylene glycol (PEG), alkyl(ene), amide, phenyl, and triazole.

18. 18. The compound of any one of claims 13-17, wherein Y has an amine core, an aromatic core.

19. 18. The compound of any one of claims 13 to 17, wherein Y has an amine core, an aromatic core, or an alkylene core.

20. L, L 1 , L 2 20. The compound of any one of claims 1-19, wherein:

21. L, L 1 , L 2 or any combination thereof independently having the following structure:

21. The compound of any one of claims 1 to 20, comprising at least one linker group having the formula:

22. L, L 1 , L 2 or any combination thereof independently having the following structure:

22. The compound of any one of claims 1 to 21, comprising at least one linker group having the formula:

23. L, L 1 , L 2 or any combination thereof, having the following structure:

23. The compound of any one of claims 1-22, comprising at least one linker group having the formula:

24. L has the following structure:

24. The compound of any one of claims 1 to 23, having the formula:

25. 25. The compound of any one of claims 1 to 24, wherein B is a radical of a (e.g., fluorescent) dye.

26. 26. The compound of any one of claims 1 to 25, wherein the compound is an imaging agent and B is a radical of a fluorescent dye.

27. 25. The compound of any one of claims 1 to 24, wherein B is a radical of an anti-cancer or anti-fibrotic agent.

28. 30. The compound of any one of claims 1-24 or 27, wherein the compound is a chemotherapeutic agent and B is a radical of an antifibrotic or anticancer agent.

29. 30. The compound of any one of claims 1-24, 27, or 28, wherein B is a radical of a phosphoinositide 3-kinase (PI3K) inhibitor.

30. B has the following formula: wherein X is a radical represented by 30. The compound of any one of claims 1-24 or 27-29, selected from the group consisting of:

31. B has the following structure:

31. The compound of any one of claims 1 to 24 or 27 to 30, wherein the radical is

32. The following structure: Compound.

33. The following structure: Compound.

34. The following structure: Compound.

35. The following structure: Compound.

36. 36. A pharmaceutical composition comprising a compound of any one of claims 1 to 35 and a pharmaceutically acceptable carrier.

37. 36. A method for imaging cancer or fibrosis in a subject having cancer or fibrosis, comprising administering to a subject in need thereof an effective amount of a compound of any one of claims 1-26 and 35.

38. 36. A method for treating an inflammatory disease or disorder, comprising administering a therapeutically effective amount of a compound of any one of claims 1-35 to a subject in need thereof.

39. 36. A method for treating cancer, comprising administering a therapeutically effective amount of a compound of any one of claims 1-35 to a subject in need thereof.

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