Fibroblast activation protein-targeted radioligands with pharmacokinetic modulators

Abstract

A conjugate of the formula (I) wherein F is a ligand (radical thereof) that binds fibroblast activation protein alpha (FAPα); L is a functionalized linker that binds with F, A, and X; A is a pharmacokinetic extender that binds or associates or binds with a protein in the blood of an animal; and X is chelator; a method of imaging cancer-associated fibroblasts (CAFs) and activated myofibroblasts in a subject; and a method of treating a disease characterized by upregulation of FAPα, such as cancer, in a subject.

Description

[0001] FIBROBLAST ACTIVATION PROTEIN-TARGETED RADIOLIGANDS WITH PHARMACOKINETIC MODULATORS

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 622,988, filed lanuary 19, 2024, the entire contents of which are hereby incorporated by reference as if fully set forth.

[0004] TECHNICAL FIELD

[0005] The application relates to conjugates and methods for improving the tumor retention of theragnostic radioligands targeted to cancer-associated fibroblasts (CAFs) through structural modification of the linker. The present application also relates to conjugates and methods comprising secondary effector molecules that modulate the pharmacokinetics of the theragnostic radioligands through noncovalent association to serum proteins.

[0006] BACKGROUND

[0007] Many solid tumors comprise infiltrating immune and inflammatory cells such as cancer- associated fibroblasts (CAFs), T cells, tumor-associated macrophages (TAMs), myeloid- suppressor cells, blood and lymphatic vasculature networks, etc. They aid in the growth and development of the tumor by secreting growth factors, suppressing the immune system, promoting metastasis, contributing to drug resistance, etc.

[0008] CAFs are one of the major types of cells present in the tumor stroma and perform several critical roles to promote tumor growth. These functions include extracellular matrix (ECM) production and remodeling, cytokine secretion, and angiogenesis. These functions lead to accelerated tumor growth, signaling factor secretion to increase chemoresistance, dense tumor stroma to impede physically antibodies and immune cells, and enhanced cell motility, directly resulting in metastasis.

[0009] A common biomarker for CAFs is fibroblast activation protein alpha (FAPα). FAPα is a serine protease primarily expressed on the plasma membrane of activated fibroblasts in diseases such as fibrosis, rheumatoid arthritis, wound healing, and cancer. More than 90% of epithelial carcinomas show FAPoc expression in immunohistochemical (IHC) stainings. Additional FAPα expression has been found in a subset of primary glioma cell cultures and TAMs. At least 28 different types of human cancers have been detected in positron emission tomography (PET) scans with a FAP -targeted radiotracer. Furthermore, FAPα expression is very low or nonexistent in most adult tissues. Hence, because the expression is restricted to the surfaces of diseased cells, such as carcinomas, FAPα is uniquely qualified as a receptor for selectively delivering pharmaceuticals to tumors via ligand-targeting. FAPα is already being exploited as a ligand-target for imaging with near infrared (NIR) dyes or PET agents. Similarly, therapies to kill tumor cells, such as antimitotic or radiotherapeutic agents, are being delivered to the tumor microenvironment by targeting FAPα. However, the maximum radioactivity that can be safely delivered by current FAPα-targeted radioligands is too low to provide a consistently effective therapeutic dose. Additionally, the dose retained by the tumor, once delivered, is similarly too short to provide a robust and effective tumor-killing response.

[0010] Albumin binders have already been exploited to increase the circulation time, tumor uptake, and tumor retention of other radioligands such as folate, PSMA, DOTATATE, integrin- targeting peptide, etc. Linker structures, however, have not been thoroughly investigated for FAP radioligands conjugated to albumin-binders. Furthermore, relatively few albumin-binders have been evaluated when conjugated to FAP radioligands.

[0011] There is, therefore, an unmet need for novel FAPα-targeted radioligands that can maximize tumor-to-healthy organ ratios. It is an object of the present disclosure to provide targeted delivery of radiolabeled functional groups using an improved linker structure for FAP radioligands conjugated to a pharmacokinetic extender, such as an albumin binder. This and other objects and advantages, as well as inventive features, will be apparent from the detailed description provided herein.

[0012] SUMMARY

[0013] Provided is a conjugate of the formula:

[0014] X

[0015] A wherein F is a ligand (i.e., a radical thereof) that binds to fibroblast activation protein alpha (FAPα); L is a functionalized linker that forms chemical bonds with F, A, and X; A is a pharmacokinetic extender that associates or binds with a protein in the blood of an animal; and X is a radiolabeled functional group (i.e., a radical thereof). The conjugate can target cells expressing fibroblast activation protein alpha (FAPα), such as FAPαexpressing cancer- associated fibroblasts (CAFs).

[0016] In some embodiments, L comprises a primary spacer, a branching linker extending from the primary spacer, and a secondary spacer extending from the branching linker, wherein F is linked via the primary spacer and comprises a ligand (i.e., a radical thereof) that binds to fibroblast activation protein alpha (FAPα), X is linked via the branching linker and comprises a chelating agent optionally bound to a metal ion, and A is linked via the secondary spacer and comprises an albumin binder a chelating agent bound to a metal ion. In some embodiments, L is selected from the group consisting of:

[0017] The chelating agent is l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid (DOTA) in some embodiments. In some embodiments, F is FAP6.

[0018] In certain embodiments, the metal ion is selected from 18F, 44Sc, 47Sc, 52Mn, 55Co, 64Cu, 67Cu, 66Ga, 67Ga, 68Ga, 86Y, 89Zr, 90 Y, 99mTc, U lin, 114mln, 117mSn, 1241, 1251, 1311, 149Tb, 153Sm, 152Tb, 155Tb, 161Tb, 177Lu, 186Re, 188Re, 212Pb, 212Bi, 213Bi, 223Ra, 224Ra, 210At, 211At, 225Ab, 225 Ac, and 227Th.

[0019] In some embodiments, the metal ion is a radiolabel. In some embodiments, the metal ion is a radi opharmaceutical .

[0020] In some embodiments, the ligand has a molecular weight below 10,000. In certain embodiments, the protein is albumin or a pre-albumin.

[0021] In some embodiments, the radiolabeled functional group is a PET imaging agent, a single photon emission computed tomography (SPECT) imaging agent, a radio-imaging agent, or a radiotherapeutic agent.

[0022] In some preferred embodiments, the animal is a human.

[0023] In certain embodiments, F can have the structure: wherein T is CH2 (wherein either or both H can be independently substituted), NH (wherein H can be substituted), O, or S;

[0024] Ri andR2 are independently selected from the group consisting of -H, -CN, -CHO, -B(0H)2, -C(O)alkyl, -C(O)aryl-, -C=C-C(O)aryl, -C=C-S(O)2aryl, -CO2H, -SO3H, -SO2NH2, -PO3H2, -SO2F, 5-tetrazolyl, C1-C4 alkyl, isonitrile, and boronic acid;

[0025] R3 andR4 are independently selected from the group consisting of -H, -OH, F, Cl, Br, I, -C1-6 alkyl, -O-C1-6 alkyl, -S-C1-6 alkyl, isonitrile, and boronic acid;

[0026] Rs-Rs are independently selected from the group consisting of H, alkyl, and halo; and R9-R11 are independently selected from the group consisting of H, -C1-6 alkyl, -O-C1-6 alkyl, -S-C1-6 alkyl, F, Cl, Br, I, -CN, -CHO, -B(OH)2, -C(O)alkyl, -C(O)aiyl-, -C=C-C(O)aryl, -C=C-S(O)2aryl, -CO2H, -SO3H, -SO2NH2, -PO3H2, -SO2F, 5-tetrazolyl, C1-C4 alkyl, isonitrile, and boronic acid.

[0027] In some conjugates, L can comprise at least one of an amino acid, a polyethylene glycol (PEG) monomer, a PEG oligomer, a PEG polymer, a heterocycle, or a combination of two or more of the foregoing. In certain conjugates, L can comprise, or can further comprise, at least one of an ether, a thioether, a tertiary amine, a C1-6 alkyl, piperazine, piperidine, a bicycloheptane, a substituted benzene, or a combination of two or more of the foregoing. L can comprise, or can further comprise, an oligomer of peptidoglycans, glycans, anions, heterocycles, or a combination of two or more of the foregoing. L can comprise, or can further comprise, at least one of a diaminobutyric acid group, a substituted benzene group, a lysine group, a 2,3- diaminopropionic acid group, a tyrosine group, a glutamic acid group, a cysteine group, or a combination of two or more of the foregoing.

[0028] In some embodiments, L can comprise, or can further comprise, at least one of a 2,3- diaminopropionic acid group, a glutamic acid group, a lysine group, a cysteine group, and / or at least one heterocycle comprising one or more of the following structures:

[0029]

[0030]

[0031] In certain embodiments, A can comprise one of the following structures:

[0032]

[0033]

[0034] Foscan

[0035] Tookad

[0036] or an albumin-binding small protein scaffold such as, but not limited to, ABD035, ABDCon, DARPins, dsFv CA645, Nanobody, and VNAR (E06).

[0037] In certain embodiments, X can be, or can comprise, a chelating agent and a radionucleotide, where the chelating agent is selected from: DOTA (1,4,7,10- tetraazacyclododecane-l,4,7,10-tetraacetic acid) or a derivative thereof; TETA (1,4,8,11- tetraazacyclotetradecane-1,4,8,11 -tetraacetic acid) or a derivative thereof; SarAr (l-N-(4- aminobenzyl)-3,6,10,13, 16,19-hexaazabicyclo[6.6.6]-eicosane-l,8-diamine or a derivative thereof; NOTA (l,4,7-triazacyclononane-l,4,7-triacetic acid) or a derivative thereof; NETA (4- [2-(bis-carboxymethylamino)-ethyl]-7-carboxymethyl-[l,4,7]triazonan-l -yl) acetyl acid or a derivative thereof; TRAP (l,4,7-triazacyclononane-l,4,7-tris[methyl(2-carboxyethyl)phosphinic acid) or a derivative thereof; HBED (N,N0-bis(2-hydroxybenzyl)-ethylenediamine-N,N0- diacetic acid) or a derivative thereof; 2,3-HOPO (3-hydroxypyridin-2-one) or a derivative thereof; PCTA (3,6,9,15-tetraazabicyclo[9.3.1]-pentadeca-l(15),l l,13-triene-3, 6, 9, -triacetic acid) or a derivative thereof; DFO (desferri oxamine) or a derivative thereof; DTPA (diethylenetriaminepentaacetic acid) or a derivative thereof; OCTAPA (N,N0-bis(6-carboxy-2- pyridylmethyl)-ethylenediamine-N, NO-diacetic acid) or a derivative thereof; or H2-MACR0PA (N,N'-bis[(6-carboxy-2-pyridipmethyl]-4,13-diaza-18-crown-6) or a derivative thereof; FEdedpa (l,2-[[carboxy)-pyridin-2-yl]-methylamino]ethane or a derivative thereof; EC20-head comprising P-l-diaminopropionic acid, Asp, and Cys; Sarcophagine or a derivative thereof; TCMC (DOTAM) or a derivative thereof; Cyclen (S-2-(4-nitrobenzyl)- 1,4, 7,10- tetraazacyclododecane) or another derivative thereof; DO3A (1,4,7, 10-tetraazacy clododecane- l,4,7-tris(acetic acid)- 10-(2 -thioethyl )acetami de) or a derivative thereof; N02A (1,4,7, 10- tetraazacy clododecane-1, 4, 7-tris(acetic acid)-10-(2-thioethyl)acetamide) or a derivative thereof; PCTA (3,6,9,15-tetraazabicyclo[9.3.1] pentadeca- 1(15), 1 l,13-triene-4-S-(4-aminobenzyl)-3,6,9- triacetic acid) or a derivative thereof; Deferoxamine (l-(4-isothiocyanatophenyl)-3-[6, 17- dihydroxy-7,10,18,21-tetraoxo-27-(N-acetylhydroxylamino)- 6,11,17, 22- tetraazaheptaei cosine] thiourea) or a derivative thereof; HEHA (2-(4-isothiocyanatobenzyl)-l,2,7,10,13-

[0038] hexaazacyclooctadecane-l,4,7,10,13,16-hexaacetic acid) or a derivative thereof;

[0039]

[0040] In some conjugates, X can be, or can comprise, a radionuclide, such as18F,44Sc,47Sc,52Mn,55Co,64Cu,67Cu,66Ga,67Ga,68Ga,86Y,89Zr,90Y, "mTc,mIn,114mIn,117raSn,124I,125I,131I,149Tb,153Sm,152Tb,155Tb,161Tb,177Lu,186Re,188Re,212Pb,212Bi,213Bi,223Ra,224Ra,210At,211At,223Ab,225Ac, or227Th, wherein the radionuclide is covalently bonded to the conjugate alone or in a derivatized, chelated combination with an above-described bifunctional chelator.

[0041] Some conjugates may be of the formula:

[0042]

[0043] R28is F, Cl, Br, I, -NO2, -CH3- ibuprofen, or Evans Blue.

[0044] In other embodiments, the conjugate may have the formula:

[0045] R28is F, Cl, Br, I, -NO2, -CH3- ibuprofen, or Evans Blue.

[0046] In yet other embodiments, the conjugate may have the formula; wherein

[0047] R28is F, Cl, Br, I, -NO2, -CEE- ibuprofen, or Evans Blue; and n is 1-20, 1-10, or 1-5.

[0048] Further embodiments of the conjugate may have the formula: n is 1-20, 1-10, or 1-5.

[0049] Still other embodiments of the conjugate may have the formula:

[0050] n is 1-20, 1-10, or 1-5.

[0051] Also provided is a conjugate having the structure:

[0052] (FAP6-19).

[0053] In some embodiments, provided is a chelated complex represented by Formula (I): or a pharmaceutically acceptable salt thereof, wherein:

[0054] R1is an albumin binding ligand;

[0055] R2is a ligand chelated to a therapeutic radionuclide or a diagnostic radionuclide, and n is an integer from 1 to 6. R1can be:

[0056] wherein R27is selected from the group consisting of halogen and C1-6 alkyl. R27can be iodo. R27can be fluoro. R27can be methyl. R2can be a ligand chelated to a therapeutic radionuclide. R2can be a ligand chelated to a diagnostic radionuclide. The ligand can be:

[0057] In some embodiments, the ligand can be:

[0058] In some embodiments, provided is a chelated complex of a compound represented by a formula selected from the group consisting of: FAP6-17

[0059] FAP6-19 or a pharmaceutically acceptable salt thereof, and a radionuclide selected from a therapeutic radionuclide and a diagnostic radionuclide. In some embodiments, the therapeutic radionuclide can be selected from the group consisting of177Lu,90Y, and211At. In certain embodiments, the therapeutic radionuclide can be177Lu. The diagnostic radionuclide can be selected from the group consisting of "mTc,niIn,18F,68Ga,1241,125I, and131I. The diagnostic radionuclide can be11’In.

[0060] In some embodiments, provided is a chelated complex selected from the group consisting of [177LU]LU-FAP6-16, [177LU]LU-FAP6-17, and [177Lu]Lu-FAP6-19. In other embodiments, provided is a chelated complex selected from the group consisting of [inIn]In-FAP6-16, [niIn]In-FAP6-17, and [i nIn]In-FAP6-19.

[0061] In some embodiments, provided is a compound represented by Formula (II): or a pharmaceutically acceptable salt thereof, wherein:

[0062] R3is an albumin binding ligand;

[0063] R4is selected from the group consisting of:

[0064] n is an integer from 1 to 6. R3can be: wherein R27is selected from the group consisting of halogen and C1-6 alkyl. R27can be iodo.

[0065] R27can be fluoro. R27can be methyl. R4can be:

[0066]

[0067] In some embodiments, provided is a compound selected from the group consisting of:

[0068]

[0069] FAP6-19 or a pharmaceutically acceptable salt thereof.

[0070] In view of the above, also provided is a pharmaceutical composition comprising a conjugate, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0071] Additionally, also provided is an imaging agent comprising a conjugate, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0072] A method of imaging cancer-associated fibroblasts (CAFs) in a subject is also provided. The method comprises (i) administering to the subject an effective amount of a composition comprising a conjugate (e.g., a chelated conjugate) and a pharmaceutically acceptable excipient and (ii) imaging the subject to identify the location of the CAFs in the patient.

[0073] Some embodiments concern any of the compositions herein for use in a method of treating a disease mediated by CAFs in a subject. In certain embodiments, the disease is a cancer, such as those discussed herein.

[0074] Further provided is a method of treating fibroblast activation protein alpha (FAPα)- expressing cancer in a subject. The method comprising administering to the subject an effective amount of a composition comprising a conjugate and a pharmaceutically acceptable excipient, whereupon FAPα-expressing cancer in the subject is treated. The cancer can be prostate cancer, renal cancer, breast cancer, thyroid cancer, gastric cancer, colorectal cancer, bladder cancer, pancreatic cancer, lung cancer, liver cancer, a brain tumor, melanoma, a neuroendocrine tumor, ovarian cancer, esophageal cancer, cholangiocellular carcinoma, head-neck cancer, carcinoma of unknown primary, medullary thyroid cancer, thymus cancer, small-intestine cancer, or a sarcoma, for example.

[0075] Yet other aspects include method of treating a disease characterized by upregulation of FAPα. In some embodiments, the disease is a cancer.

[0076] FIGURES

[0077] FIG. 1 depicts a structure having a targeting ligand bound to a primary spacer , which is connected to a branched linker. The branched linker is further attached to a radioactive payload and an albumin-binder. A secondary spacer may be utilized between the branched linker and the albumin-binder.

[0078] FIG. 2A depicts the synthetic scheme of intermediates required for the synthesis of the FAP6 conjugates.

[0079] FIG. 2B depicts a synthetic scheme for conjugates and provides the structure of FAP6- 01.

[0080] FIG. 3A depicts the LC / MS characterization of the structure FAP6-19.

[0081] FIG. 3B depicts the LC / MS characterization of the structure FAP6-17.

[0082] FIG. 3C depicts the LC / MS characterization of the structure FAP6-16.

[0083] FIG. 4A depicts a radio-chromatogram of [177Lu]Lu-FAP6-19.

[0084] FIG. 4B depicts a radio-chromatogram of [177Lu]Lu-FAP6-17.

[0085] FIG. 4C depicts a radio-chromatogram of [177Lu]Lu-FAP6-16. Radiolabeling conditions for FIGS. 4A-4C include 500 pM of FAP6 conjugate in 0.5 M NaOAc (pH = 6), 45°C for 10 minutes.

[0086] FIG. 5A depicts a radio-chromatogram of [111In]In-FAP6-19.

[0087] FIG. 5B depicts a radio-chromatogram of [i nIn]In-FAP6-17.

[0088] FIG. 5C depicts a radio-chromatogram of [111In]In-FAP6-16. Radiolabeling conditions for FIGS. 5A-5C include 500 pM of FAP6 conjugate in 0.5 M NaOAc (pH=6), 45°C for 10 minutes.

[0089] FIG. 6 depicts binding curves of FAP6 radioligands chelated with Lu- 177 binding to HEK-huFAP cells. Assay was executed as previously described. Data are presented as means with SEM bars. FIG. 7 depicts a cellular uptake assay of FAP6 radioligands chelated with In-11 1 using HT1080-huFAP. Assay was executed as reported in the literature. Data are presented as means normalized to FAP6-01 with SEM bars. Statistical analyses were performed by one-way, nonpaired ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

[0090] FIG. 8 depicts a receptor internalization assay of FAP6 radioligands chelated with In-111 using HT1080-huFAP. Assay was executed as reported in the literature. Data are presented as means normalized to initial FAP6-01 measurements at 1 hour with SEM bars. Statistical analyses were performed by one-way, non-paired ANOVA (*P < 0.05, **P < 0.01, ***p < 0.001, ****p < 0.0001).

[0091] FIG. 9 depicts a retention study using FAP6 radioligands chelated with In-111 using HT1080-huFAP. Assay was executed as reported in the literature (Wang, Z.; Tian, R.; Niu, G.; Ma, Y.; Lang, L.; Szajek, L. P.; Kiesewetter, D O.; Jacobson, O.; Chen, X., Single Low-Dose Injection of Evans Blue Modified PSMA-617 Radioligand Therapy Eliminates Prostate-Specific Membrane Antigen Positive Tumors. Bioconjugate Chemistry 2018, 29 (9), 3213-3221). Data are presented as means normalized to initial FAP6-01 measurements at 0 hours after replacing with nonradioactive material (i.e., after incubation with FAP6 radioligand for one hour) with SEM bars. Statistical analyses were performed by one-way, non-paired ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001, ****p < 0.0001).

[0092] FIG. 10 SPECT / CT scans at (A) 3 hours, (B) 24 hours, (C) 72 hours, and (D) 120 hours post-injection of FAP6 radioligands chelated with In-111 using Balb / c mice bearing 4T1 tumors. I Decay-corrected quantification of tracer uptake in the tumors. Radiolabeling, radiotracer administration, SPECT / CT scans, reconstruction, and analysis were performed as described previously. Tumor locations are indicated by hashed circle markings.

[0093] FIG. 11 depicts a biodistribution study of FAP6 radioligands chelated with Lu-177 at (A) 3 hours, (B) 24 hours, and (C) 120 hours post-injection using Balb / c mice bearing 4T1 tumors. (D) Graphs providing tumor-to-blood, -liver, and -kidney ratios. Study was performed as previously described. Data are presented as means with SEM bars. Statistical analyses were performed by one-way, non-paired ANOVA (*P < 0.05, **P < 0.01, ***p < 0.001, ****p < 0.0001).

[0094] FIG. 12A depicts dosimetry estimates based on the biodistribution data presented in

[0095] FIG10 FIG. 12B depicts tumor-to-healthy organ ratios based on the dosimetry calculations from part A. Calculations were performed as previously described (Lindeman, S. D.; Mukkamala, R.; Horner, A.; Tudi, P.; Booth, O. C.; Huff, R. R.; Hinsey, J.; Hovstadius, A.; Martone, P.; Zhang, F.; Srinivasarao, M.; Cox, A.; Low, P. S., Fibroblast activation protein targeted radioligand therapy for treatment of solid tumors. J. Nucl. Med. 2022, jnumed.122.264494).

[0096] FIG. 13 depicts a radiotherapy study with FAP radioligands chelated with Lu-177, using 4T1 tumor growth chart in Balb / c mice. 5 nmol of each FAP conjugate radiolabeled with ~1.0 mCi of Lu- 177 was administered per mouse by intravenous lateral tail-vein injections. Blind measurements were subsequently taken with a caliper by 2 researchers 3x per week. Other experimental parameters were followed as previously described.

[0097] FIG. 14 presents SPECT / CT scans at (A) 24 hours, (B) 72 hours, post-injection of [177LU]LUFAP6 -19 radioligands in Balb / c mice bearing 4T1 tumors. Arrows indicate tumor locations.

[0098] FIG. 15 illustrates numerous prophetic structures of additional FAP6 radioligands with various linker modifications, PK modulators, FAP ligands, and / or chelators that is in no way meant to be comprehensive. All possible combinations and permutations of the presented modifications are also anticipated.

[0099] DETAILED DESCRIPTION

[0100] In some aspects, the present disclosure concerns conjugate structures having a targeting ligand bound to a primary spacer which is connected to a branched linker as depicted in FIG. 1. The branched linker may be further attached to a radioactive payload and an albumin-binder. A secondary spacer may be utilized between the branched linker and the albumin-binder. Such structures may involve novel FAPα-targeted radioligands that can provide targeted delivery of radiolabeled functional groups to tumors and minimize exposure of the radioligands to healthy tissue.

[0101] In certain aspects, the present disclosure provides a conjugate of the formula: wherein F is a ligand (such as a ligand with a molecular weight below 10,000) that binds fibroblast activation protein alpha (FAPα); L is a functionalized linker that forms chemical bonds with F, A, and X; A is a pharmacokinetic extender (e.g., a fluorescent dye, an albumin binder, a pre-albumin ligand, etc.) that can associate or bind with a protein (e.g., albumin, a pre-albumin, etc.) in the blood of an animal (e.g., a human); and X is a radiolabeled functional group (e.g., a positron emission tomography (PET) imaging agent, a single photon emission computed tomography (SPECT) imaging agent, a radio-imaging agent, a radiotherapeutic agent, etc.). The conjugate can target cells expressing fibroblast activation protein alpha (FAPD), such as FAPL- expressing cancer-associated fibroblasts (CAFs).

[0102] By combining a ligand that binds fibroblast activation protein alpha (FAPα), the F function, with the A function (blood protein-binding function) and the radiolabeled functional group (X) with the linker (L), the instant disclosed compositions and treatments enable improved targeted delivery of radiolabeled functional groups for use in cancer treatment. By optimizing the combination of the linkers and associated F, A, and X functions, the instant compositions are designed to give improved retention and uptake of the conjugates with tumors while minimizing association with healthy tissue of a subject.

[0103] In some embodiments, L comprises a primary spacer, a branching linker extending from the primary spacer, and a secondary spacer extending from the branching linker, wherein F is linked via the primary spacer and comprises a ligand (i.e., a radical thereof) that binds to fibroblast activation protein alpha (FAPα), X is linked via the branching linker and comprises a chelating agent optionally bound to a metal ion, and A is linked via the secondary spacer and comprises an albumin binder a chelating agent bound to a metal ion. In some embodiments, the primary spacer and the branching linker make up the linkers (L) disclosed herein.

[0104] In some embodiments, L is selected from the group consisting of:

[0105] The chelating agent is l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid (DOTA) in some embodiments. In some embodiments, F is FAP6.

[0106] In certain embodiments, the metal ion is selected froml8F, 44Sc, 47Sc, 52Mn, 55Co, 64Cu, 67Cu, 66Ga, 67Ga, 68Ga, 86Y, 89Zr, 90Y, 99mTc, U lin, 114mln, 117mSn, 1241, 1251, 1311, 149Tb, 153Sm, 152Tb, 155Tb, 161Tb, 177Lu, 186Re, 188Re, 212Pb, 212Bi, 213Bi, 223Ra, 224Ra, 210At, 211At, 225Ab, 225 Ac, and 227Th.

[0107] In some embodiments, the metal ion is a radiolabel. In other embodiments, In some embodiments, the metal ion is a radiopharmaceutical.

[0108] In certain embodiments, F can have the structure: wherein T is CFb (wherein either or both H can be independently substituted), NH (wherein H can be substituted), O, or S;

[0109] Ri andR? are independently selected from the group consisting of -H, -CN, -CHO, -B(0H)2, -C(O)alkyl, -C(O)aryl-, -C=C-C(O)aryl, -C=C-S(O)2aryl, -CO2H, -SO3H, -SO2NH2, -PO3H2, -SO2F, 5-tetrazolyl, C1-C4 alkyl, isonitrile, and boronic acid;

[0110] R3 andR4 are independently selected from the group consisting of -H, -OH, F, Cl, Br, I, -C1-6 alkyl, -O-C1-6 alkyl, -S-C1-6 alkyl, isonitrile, and boronic acid;

[0111] Rs-Rs are independently selected from the group consisting of H, alkyl, and halo; and R9-R11 are independently selected from the group consisting of H, -C1-6 alkyl, -O-C1-6 alkyl, -S-C1-6 alkyl, F, Cl, Br, I, -CN, -CHO, -B(OH)2, -C(O)alkyl, -C(O)aryl-, -C=C-C(O)aryl, -C=C-S(O)2aryl, -CO2H, -SO3H, -SO2NH2, -PO3H2, -SO2F, 5-tetrazolyl, C1-C4 alkyl, isonitrile, and boronic acid. L can comprise at least one of an amino acid, a polyethylene glycol (PEG) monomer, a PEG oligomer, a PEG polymer, a heterocycle, or a combination of two or more of the foregoing. L can comprise, or can further comprise, at least one of an ether, a thioether, a tertiary amine, a C1-6 alkyl, piperazine, piperidine, a bicyclo-heptane, a substituted benzene, or a combination of two or more of the foregoing. L can comprise, or can further comprise, an oligomer of peptidoglycans, glycans, anions, heterocycles, or a combination of two or more of the foregoing. L can comprise, or can further comprise, at least one of a diaminobutyric acid group, a substituted benzene group, a lysine group, a 2,3 -diaminopropionic acid group, a tyrosine group, a glutamic acid group, a cysteine group, or a combination of two or more of the foregoing. L can comprise, or can further comprise, at least one of a 2,3 -diaminopropionic acid group, a glutamic acid group, a lysine group, a cysteine group, and / or at least one heterocycle comprising one or more of the following structures:

[0112]

[0113]

[0114]

[0115]

[0116]

[0117] Foscan

[0118] Tookad

[0119] or an albumin-binding small protein scaffold such as, but not limited to, ABD035, ABDCon, DARPins, dsFv CA645, Nanobody, and VNAR (E06). X can be, or can comprise, a chelating agent and radionucleotide, where the chelating agent is selected from DOTA (1,4,7,10- tetraazacyclododecane-l,4,7,10-tetraacetic acid) or a derivative thereof; TETA (1,4,8,11- tetraazacyclotetradecane-1,4,8,11 -tetraacetic acid) or a derivative thereof; SarAr (l-N-(4- aminobenzyl)-3,6,10,13,16,19-hexaazabicyclo[6.6.6]-eicosane-l,8-diamine or a derivative thereof; NOTA (l,4,7-triazacyclononane-l,4,7-triacetic acid) or a derivative thereof; NETA (4- [2-(bis-carboxymethylamino)-ethyl]-7-carboxymethyl-[l,4,7]triazonan-l-yl) acetyl acid or a derivative thereof; TRAP (l,4,7-triazacyclononane-l ,4,7-tris[methyl(2-carboxyethyl)phosphinic acid) or a derivative thereof; HBED (N,N0-bis(2-hydroxybenzyl)-ethylenediamine-N,N0- diacetic acid) or a derivative thereof; 2,3-HOPO (3-hydroxypyridin-2-one) or a derivative thereof; PCTA (3,6,9,15-tetraazabicyclo[9.3.1]-pentadeca-l(15),l l,13-triene-3, 6, 9, -triacetic acid) or a derivative thereof; DFO (desferri oxamine) or a derivative thereof; DTPA (diethylenetriaminepentaacetic acid) or a derivative thereof; OCTAPA (N,N0-bis(6-carboxy-2- pyridylmethyl)-ethylenediamine-N, NO-diacetic acid) or a derivative thereof; or H2-MACR0PA (’,N'-bis[(6-carboxy-2-pyridipmethyl]-4,13-diaza-18-crown-6) or a derivative thereof; Fkdedpa (l,2-[[carboxy)-pyridin-2-yl]-methylamino]ethane or a derivative thereof; EC20-head comprising P-l-diaminopropionic acid, Asp, and Cys; Sarcophagine or a derivative thereof; TCMC (DOTAM) or a derivative thereof; Cyclen (S-2-(4-Nitrobenzyl)-l,4,7,10- tetraazacyclododecane) or another derivative thereof; D03A (1,4,7, 10-Tetraazacyclododecane- l,4,7-tris(acetic acid)-10-(2-thioethyl)acetamide) or a derivative thereof; N02A (1,4,7,10- tetraazacyclododecane-l,4,7-tris(acetic acid)-10-(2-thioethyl)acetamide) or a derivative thereof; PCTA (3,6,9,15-tetraazabicyclo[9.3.1] pentadeca- 1(15), 1 l,13-triene-4-S-(4-aminobenzyl)-3,6,9- triacetic acid) or a derivative thereof; Deferoxamine (l-(4-isothiocyanatophenyl)-3-[6,17- dihydroxy-7,10,18,21-tetraoxo-27-(N-acetylhydroxylamino)- 6,11,17, 22- tetraazaheptaei cosine] thiourea) or a derivative thereof; HEHA (2-(4-isothicyanatobenzyl)-l,2,7,10,13-

[0120] hexaazacyclooctadecane-l,4,7,10,13,16-hexaacetic acid) or a derivative thereof;

[0121] 227Th, wherein the radionuclide is covalently bonded to the conjugate alone or in a derivatized, chelated combination with an above-described bifunctional chelator.

[0122] Also provided is a conjugate having the structure:

[0123] (FAP6-19).

[0124] The conjugate wherein F has the structure27’29: represents a functionalized 5- to 10-membered N-containing aromatic or non-aromatic mono- or bicyclic heterocycle, said heterocycle optionally further comprising 1 to 3 heteroatoms selected from O, N, and S.

[0125] Z is a bond, (un)substituted CH2, NH, O, or S.

[0126] T is (un) substituted CH2, NH, O, or S.

[0127] Ri, R2 are the same or different and independently selected from the group consisting of -H, -CN, -CHO, -B(OH)2, -C(O)alkyl, -C(O)aryl-, -C=C-C(O)aryl, -C=C-S(O)2aryl, -CO2H, -SO3H, - SO2NH2, -PO3H2, -SO2F, 5-tetrazolyl, C1-C4 alkyl, isonitrile, and boronic acid.

[0128] R3, R4 are the same or different and are independently selected from the group consisting of -H, -OH, F, Cl, Br, I, -C1-6alkyl, -O- C1-6alkyl, -S-C1-6alkyl, isonitrile, and boronic acid. R5-R.8 are the same or different and are independently selected from group consisting of H, alkyl and halo.

[0129] The structure indicates a point of attachment of F to L, A, or X, wherein the point of attachment can be through any of the carbon atoms of the 5- to 10- membered N-containing aromatic or non-aromatic mono- or bicyclic heterocycle or 1°, 2° amines or with functionalized alkyl or cycloalkyl motif, as well as stereoisomers and pharmaceutically acceptable salts thereof.

[0130] In some preferred embodiment the aforementioned FAPot targeted ligand (F) may be

[0131] T = (un)substituted CH2, NH, O, or S;

[0132] Ri, R2 are the same or different and are independently selected from the group consisting of -H, -CN, -CHO, -B(OH)2, -C(O)alkyl, -C(O)aryl-, -C=C-C(O)aryl, -C=C-S(O)2aryl, -CO2H, - SO3H, -SO2NH2, -PO3H2, -SO2F, 5-tetrazolyl, C1-C4 alkyl, isonitrile, and boronic acid.

[0133] R3, R4 are the same or different, and are independently selected from the group consisting of -H, -OH, F, Cl, Br, I, -C1-6 alkyl, -O-C1-6 alkyl, -S-C1-6 alkyl, isonitrile, and boronic acid.

[0134] Rs-Rs are the same or different and are independently selected from group consisting of H, alkyl and halo.

[0135] Rs-Rn are the same or different and are independently selected from group consisting of H, -C1-6alkyl, -O-C1-6alkyl, -S-C1-6 alkyl, F, Cl, Br and I.

[0136] In yet more particular aspects, F is selected from the group consisting of:

[0137]

[0138] In some embodiments, the conjugate comprises L as a linker selected from the group consisting of pegylated-, alkyl-, sugar-, peptide-, heterocyclic- based dual linker. In certain embodiments, L is either a non-releasable linker or a releasable (induced by pH changes, enzyme cleavage, etc.) linker bivalently and covalently attached to the PK-modulator A or the radiolabeled functional group X and the FAPα binding ligand F.

[0139] The conjugate, or a pharmaceutically acceptable salt thereof, the linker comprises a moiety of the formula:

[0140] wherein x is an integer from 0 to 10 and y is an integer from 3 to 100; and a moiety of the structure:

[0141]

[0142] derivatized from any carbon, oxygen, or amine.

[0143] In some embodiments, the conjugate of or a pharmaceutically acceptable salt thereof, the PK-modulator A comprises a small organic compound, peptide, or albumin-binding small protein scaffold domain selected from the group consisting of

[0144] (PK-modulator small organic compounds) wherein each of Ri2-i9is independently selected from -H, -Ci-Ce alkyl, -F, -Cl, -Br, -I, -CN, - CHO, -B(OH)2, -C(O)alkyl, -C(O)aryl-, -C=C-C(O)aryl, -C=C-S(O)2aryl, -CO2H, -SO3H, - SO2NH2, -PO3H2, and -SO2F; each of R20 and R21 is independently selected from -H, -Ci-Ce alkyl, -F, -Cl, -Br, -I, -O-C1-6 alkyl, -CN, -CHO, -B(OH)2, -C=C-C(O)aryl, -C=C-S(O)2aryl, - CO2H, -SO3H, -SO2NLJ- -PO3H2, -SO2F, and CF3;

[0145] (albumin-binding peptides) and wherein the PK-modulator albumin-binding small protein scaffold may include, but is not limited to, ABD035, ABDCon, DARPins, dsFv CA645, Nanobody, and VNAR (E06);

[0146]

[0147] Foscan

[0148] Tookad

[0149] and wherein the PK-modulator is a fluorescent dye, which may include, but is not limited to, S0456, rhodamine, IR700, fluorescein, or IR800. In yet more particular aspects, A is selected from the group consisting of

[0150]

[0151] Provided is a multivalent conjugate having a structure (F-S)m-Y-L-X, wherein F is or comprises a fibroblast activation protein (FAP) ligand; S is or comprises a spacer that provides optimal length for the arms of the multivalent drug to reach to multiple adjacent FAPs on the target cell or subpockets of the same FAP receptor, Y is or comprises a template that connects multiple arms of the conjugate, L is or comprises a linker, and X a radio-imaging agent or radiotherapeutic agent, such as a radio-imaging agent or a radiotherapeutic agent that is effective against cancer cells, cancer-associated fibroblasts, and / or a tumor microenvironment factor, where m = 2-6. S can comprise an oligoethylene or a polyethyleneglycol, an alkyl chain, an oligopeptide, a polypeptide, a rigid linker, a squaramide, a heterocycle. S can comprise an oligoethylene glycol or a polyethylene glycol. S can comprise an oligopeptide or polypeptide. S can comprise a peptidoglycan. S can comprise a rigid linker such as oligoproline and oligopiperidine, or a different heterocycle. S can comprise a linker of length between 15-200 angstroms. Y can be a template that connects multiple arms of the conjugate that has the structure selected from the following templates: lysine, diaminobutyric acid, ascorbic acid, glutamic acid, cysteine, serine, tyrosine, tryptophan, histidine, threonine, or any other natural or unnatural amino acid.

[0152] Y can be a template that connects multiple arms of the conjugate that has the repeating structures

[0153] Y can be a template that connects multiple arms of the conjugate that has the citric acidbased template

[0154] Y can be a template that connects multiple arms of the conjugate that has the citric acidbased template.

[0155]

[0156] Y can be a template that connects multiple arms of the conjugate that has the citric acidbased template.

[0157] In embodiments, L can be a linker selected from the group consisting of pegylated-, alkyl-, sugar-, peptide- , heterocyclic-, and / or aromatic- based dual linker. In embodiments, L can be either a non-releasable linker or a releasable linker bivalently covalently attached to X and F. In embodiments, the linker can comprise a moiety of the formula: The linker can comprise a portion selected from the group consisting of wherein each of R22 and R23 is independently H or Ci-Ce alkyl; and z is an integer from 1 to 8. L can comprise a structure:

[0158] The linker can comprise a portion selected from the group consisting of: wherein R26 is H or Ci-Ce alkyl; R24a, R24b, and R25a. R25b each is independently H or Ci-Ce alkyl.

[0159] X can comprise a radioactive theragnostic agent selected from a group consisting of18F,44Sc,47Sc,52Mn,55Co,64Cu,67Cu,66Ga,67Ga,68Ga,86Y,89Zr,90Y, "mTc,11’in,114mIn, ”7mSn,124I,125I,1311,149Tb,153Sm,152Tb,155Tb,161Tb,177Lu,186Re,188Re,203Pb,2’2Pb,212Bi,2’3Bi,223Ra:224Ra,210At,21’At,225Ab,225Ac, and227111.

[0160] X can be a radiolabelled prosthetic group comprising a radioisotope selected from the group consisting of18P,124I,125I,131I, and211At.

[0161] The radiolabeled prosthetic group X can be selected from the group consisting of:

[0162]

[0163] X can comprise a chelating agent, which includes, but is not limited to, DOTA (l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid) or a derivative thereof; TETA (1,4, 8,11-tetraazacy clotetradecane- 1,4, 8,11 -tetraacetic acid) or a derivative thereof; SarAr (l-N-(4-aminobenzyl)-3,6,10,13,16,19-hexaazabicyclo[6.6.6]-eicosane-l,8-diamine or a derivative thereof;

[0164] NOTA (l,4,7-triazacyclononane-l,4,7-triacetic acid) or a derivative thereof;

[0165] NETA (4-[2-(bis-carboxymethylamino)-ethyl]-7-carboxymethyl-[l,4,7]triazonan-l-yl) acetyc acid or a derivative thereof

[0166] TRAP (l,4,7-triazacyclononane-l,4,7-tris[methyl(2-carboxyethyl)phosphinic acid) or a derivative thereof;

[0167] HBED (N,N0-bis(2-hydroxybenzyl)-ethylenediamine-N, NO-diacetic acid) or a derivative thereof;

[0168] 2,3-HOPO (3 -hydroxy pyridin-2-one) or a derivative thereof;

[0169] PCTA (3,6,9,15-tetraazabicyclo[9.3.1]-pentadeca-l(15),l l,13-triene-3, 6, 9, -triacetic acid) or a derivative thereof;

[0170] DFO (desferri oxamine) or a derivative thereof;

[0171] DTPA (diethylenetriaminepentaacetic acid) or a derivative thereof;

[0172] OCTAPA (N,N0-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N, NO-diacetic acid) or a derivative thereof; or H2-MACR0PA (N,N’-bis[(6-carboxy-2-pyridipmethyl]-4,13-diaza-l 8- crown-6) or a derivative thereof.

[0173] Phdedpa (l,2-[[carboxy)-pyridin-2-yl]-methylamino]ethane or a derivative thereof;

[0174] EC20-head comprising P-l-diaminopropionic acid, Asp, and Cys.

[0175] Sarcophagine or a derivative thereof.

[0176] TCMC (DOT AM) or a derivative thereof.

[0177] Cyclen (S-2-(4-Nitrobenzyl)-l,4,7,10-tetraazacyclododecane) or another derivative thereof.

[0178] D03A (l,4,7,10-Tetraazacyclododecane-l,4,7-tris(acetic acid)-10-(2-thioethyl)acetamide) or another derivative thereof.

[0179] N02A (l,4,7,10-Tetraazacyclododecane-l,4,7-tris(acetic acid)-10-(2-thioethyl)acetamide) or another derivative thereof.

[0180] PCTA (3,6,9,15-Tetraazabicyclo[9.3.1] pentadeca- 1(15), 11,13-tri ene-4-S-(4-aminobenzyl)- 3,6,9-triacetic acid) or another derivative thereof.

[0181] Deferoxamine (1 -(4-isothiocyanatophenyl)-3-[6,17-dihydroxy-7, 10, 18,21 -tetraoxo-27-(N- acetylhydroxylamino)- 6,11,17, 22- tetraazaheptaei cosine] thiourea) or another derivative thereof.

[0182] HYNIC (63ydrazine nicotinamide) or another derivative thereof.

[0183] Representative chelating agents include, but are not limited to: A conjugate can have the structure of wherein S comprises any spacer, S’ comprises any heterocycle, Y comprises any trifunctional linker, L comprises any combination of spacing moieties, and X comprises a radiolabeled functional group.

[0184] The conjugate can have the structure of the FAPoc binding ligand. F can have a binding affinity to FAPoc in the range between about 1 pM and 100 nM.

[0185] In certain embodiments, provided herein is a chelated complex of a compound represented by Formula (I): or a pharmaceutically acceptable salt thereof, wherein: R1is an albumin binding ligand; R2is a ligand chelated to a therapeutic radionuclide or a diagnostic radionuclide, and n is an integer from 1 to 6.

[0186] In certain embodiments, R1is:

[0187] In certain embodiments, R1is: wherein R27is selected from the group consisting of halogen and C1-6 alkyl.

[0188] In certain embodiments, R27is iodo. In certain embodiments, R27is fluoro. In certain embodiments, R27is methyl. In certain embodiments, R2is a ligand chelated to a therapeutic radionuclide. In certain embodiments, R2is a ligand chelated to a diagnostic radionuclide. In certain embodiments, the ligand is:

[0189] In certain embodiments, the ligand is:

[0190] In some embodiments, the conjugate may be of the formula:

[0191] NH(CH3);

[0192] R28is F, Cl, Br, I, -NO2, -CH3- ibuprofen, or Evans Blue;

[0193] Z is -(CH2)-, -CH2S(CH2)3-, -CH(CH3)S(CH2)2-, -(CH2)4-, -CH2-S-, -NHCH2-,

[0194] -CH2N(CH3)CH2CH2CH2-, -CH2SCH2CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2NH(CH2)3-, a is 0 or 1; and n is 1-20.

[0195] In some embodiments, the conjugate may have the formula:

[0196]

[0197] R28is F, Cl, Br, I, -NCh, -CH3- ibuprofen, or Evans Blue.

[0198] In other embodiments, the conjugate may have the formula:

[0199]

[0200] R28is F, Cl, Br, I, -NO2, -CH3- ibuprofen, or Evans Blue.

[0201] In yet other embodiments, the conjugate may have the formula;

[0202] R28is F, Cl, Br, I, -NO2, -CH3- ibuprofen, or Evans Blue; and n is 1 -20, 1 -10, or 1-5.

[0203] Further embodiments of the conjugate may have the formula: n is 1-20, 1-10, or 1-5.

[0204] Still other embodiments of the conjugate may have the formula: n is 1-20, 1-10, or 1-5.

[0205] In certain embodiments, provided herein is a chelated complex of a compound represented by a formula selected from the group consisting of:

[0206]

[0207] FAP6-19 or a pharmaceutically acceptable salt thereof, and a radionuclide selected from a therapeutic radionuclide and / or a diagnostic radionuclide.

[0208] In certain embodiments, the therapeutic radionuclide is selected from the group consisting of177Lu,90Y, and211At . In certain embodiments, the therapeutic radionuclide is177LU. In certain embodiments, the diagnostic radionuclide is selected from the group consisting of "mTc,1 HIn,18F,68Ga,124I,125I, and131I. In certain embodiments, the diagnostic radionuclide isU1ln.

[0209] In certain embodiments, provided herein is a chelated complex selected from the group consisting of [177Lu]Lu-FAP6-16, [177Lu]Lu-FAP6-17, and [177Lu]Lu-FAP6-19.

[0210] In certain embodiments, provided herein is a chelated complex selected from the group consisting of [i nIn]In-FAP6-16, [i nIn]In-FAP6-17, and [ulIn]In-FAP6-19.

[0211] In certain embodiments, provided herein is a chelated complex represented by Formula (LA): or a pharmaceutically acceptable salt thereof, wherein: R1is an albumin binding ligand; and R2is a ligand chelated to a therapeutic radionuclide or a diagnostic radionuclide.

[0212] In certain embodiments, the present disclosure also provides a chelated complex of a compound represented by a formula selected from the group consisting of:

[0213]

[0214] FAP6-19 or a pharmaceutically acceptable salt thereof, and a radionuclide selected from a therapeutic radionuclide and a diagnostic radionuclide.

[0215] In certain embodiments, the present disclosure also provides a chelated complex selected from the group consisting of: [177Lu]Lu-FAP6-16

[0216] [177Lu]Lu-FAP6-19 or a pharmaceutically acceptable salt thereof.

[0217] In certain embodiments, the present disclosure also provides a chelated complex selected from the group consisting of:

[0218]

[0219] [mIn]In-FAP6-19 or a pharmaceutically acceptable salt thereof. In certain embodiments, provided herein is a compound represented by Formula (II):

[0220] Formula (II) or a pharmaceutically acceptable salt thereof, wherein: R3is an albumin binding ligand; R4is selected from the group consisting of:

[0221] n is an integer from 1 to 6.

[0222] In certain embodiments, R3is:

[0223] In certain embodiments, R3is: wherein R27is selected from the group consisting of halogen and C1-6 alkyl.

[0224] In certain embodiments, R27is iodo. In certain embodiments, R27is fluoro. In certain embodiments, R27is methyl. In certain embodiments, R4is:

[0225]

[0226] In some embodiments, provided is a compound selected from the group consisting of:

[0227]

[0228] FAP6-19 or a pharmaceutically acceptable salt thereof.

[0229] Also described herein, in certain embodiments, is a compound represented by Formula

[0230] (II-A):

[0231] Formula (II- A) or a pharmaceutically acceptable salt thereof, wherein: R3is an albumin binding ligand; and R4is selected from the group consisting of:

[0232] In some embodiments, provided is a compound represented by Formula (I): or a pharmaceutically acceptable salt thereof, wherein:

[0233] R1is an albumin binding ligand;

[0234] R2is a ligand chelated to a therapeutic radionuclide or a diagnostic radionuclide, and n is an integer from 1 to 6. R1can be: wherein R27is selected from the group consisting of halogen and C1-6 alkyl. R27can be iodo.

[0235] R27can be fluoro. R27can be methyl. R2can be a ligand chelated to a therapeutic radionuclide.

[0236] R2can be a ligand chelated to a diagnostic radionuclide. The ligand can be:

[0237] In certain embodiments, the ligand can be:

[0238] In some embodiments, provided is a chelated complex of a compound represented by a formula selected from the group consisting of:

[0239] FAP6-19 or a pharmaceutically acceptable salt thereof, and a radionuclide selected from a therapeutic radionuclide and a diagnostic radionuclide. The therapeutic radionuclide can be selected from the group consisting of177Lu,90Y, and211At. The therapeutic radionuclide can be177Lu. The diagnostic radionuclide can be selected from the group consisting of "mTc,i nIn,18F,68Ga,124I,125I, and131I. The diagnostic radionuclide can beniIn.

[0240] In some embodiments, provided is a chelated complex selected from the group consisting of177LU-FAP6-16,177LU-FAP6-17, and177Lu-FAP6-19. In other embodiments, provided is a chelated complex selected from the group consisting ofi nIn-FAP6-16,111In-FAP6-17, andi nIn- FAP6-19.

[0241] In some embodiments, the chelated complex comprises a compound represented by Formula (II):

[0242] Formula (II) or a pharmaceutically acceptable salt thereof, wherein:

[0243] R3is an albumin binding ligand; R4is selected from the group consisting of:

[0244] n is an integer from 1 to 6. R3can be: wherein R27is selected from the group consisting of halogen and C1-6 alkyl. R27can be iodo.

[0245] R27can be fluoro. R27can be methyl. R4can be:

[0246] In some embodiments, the chelated complex comprises the formula: a is 0 or 1; and n is 1-20.

[0247] In some embodiments, the chelated complex comprises a compound of the formula:

[0248]

[0249] R28is F, Cl, Br, I, -NO2, -CH3- ibuprofen, or Evans Blue.

[0250] In other embodiments, the chelated complex comprises a compound may have the formula:

[0251]

[0252] R28is F, Cl, Br, I, -NO2, -CH3- ibuprofen, or Evans Blue.

[0253] In yet other embodiments, the chelated complex comprises a compound of the formula;

[0254] R28is F, Cl, Br, I, -NO2, -CH3- ibuprofen, or Evans Blue; and n is 1 -20, 1 -10, or 1-5.

[0255] In further embodiments, the chelated complex comprises a compound of the formula:

[0256] n is 1-20, 1-10, or 1-5.

[0257] Still other embodiments of the chelated complex comprise a compound may have the formula: n is 1-20, 1-10, or 1-5.

[0258] Conjugates can be synthesized in accordance with methods known in the art and exemplified herein. See, e.g., Examples 1-3 and Figures 2A and 2B.

[0259] Conjugates, in certain embodiments, can be administered in the form of pharmaceutical compositions. Provided, therefore, in certain embodiments, are pharmaceutical compositions that comprise, as the active ingredient, one or more of the conjugates, or pharmaceutically acceptable salts thereof, and one or more pharmaceutically acceptable excipients, including inert solid diluents and fillers, diluents, including sterile aqueous solution and various organic solvents, permeation enhancers, solubilizers and adjuvants. In certain embodiments, the pharmaceutical compositions can be administered in combination with other therapeutic agents.

[0260] Provided herein, in certain embodiments, is a pharmaceutical composition comprising a) a conjugate, or a pharmaceutically acceptable salt thereof; and b) a pharmaceutically acceptable excipient.

[0261] In certain embodiments, the pharmaceutical compositions are administered in a single dose by any of the accepted modes of administration of agents having similar utilities, including rectal, buccal, intranasal and transdermal routes, by intra-arterial injection, intravenously, intraperitoneally, parenterally, intramuscularly, subcutaneously, orally, topically, as an inhalant, or via an impregnated or coated device such as a stent, for example, or an artery-inserted cylindrical polymer. In certain embodiments, the pharmaceutical compositions are administered in multiple doses.

[0262] One mode for administration is parenteral, for example by injection. Administration by injection comprises, in some embodiments, aqueous or oil suspensions, or emulsions, with sesame oil, corn oil, cottonseed oil, or peanut oil, as well as elixirs, mannitol, dextrose, or a sterile aqueous solution, and similar pharmaceutical vehicles. Aqueous solutions in saline are also conventionally used for injection. In some embodiments, compositions suitable for injection comprise ethanol, glycerol, propylene glycol, liquid polyethylene glycol, or the like (and suitable mixtures thereof), cyclodextrin derivatives, or vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, or the like.

[0263] Sterile injectable solutions are prepared by incorporating a compound according to the present disclosure in the required amount in the appropriate solvent with various other ingredients as enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0264] Oral administration is another route for administration of compounds in accordance with the disclosure. Oral administration includes, for example, capsule or enteric coated tablets, or the like. In making the pharmaceutical compositions that include at least one compound described herein, the active ingredient is, in some embodiments, diluted by an excipient and / or enclosed within such a carrier that can be in the form of a capsule, sachet, paper or other container. When the excipient serves as a diluent, it can be in the form of a solid, semi-solid, or liquid material (as above), which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments comprising the active compound, soft and hard gelatin capsules, sterile injectable solutions, or sterile packaged powders.

[0265] The compositions can be formulated to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art. Controlled release drug delivery systems for oral administration include osmotic pump systems and dissolutional systems containing polymer-coated reservoirs or drug-polymer matrix formulations. Another formulation for use in the methods of the present disclosure employs transdermal delivery device" ("patches"). Such transdermal patches may be used to provide continuous or discontinuous infusion of the compounds of the present disclosure in controlled amounts. Such patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents.

[0266] The compositions are preferably formulated in a unit dosage form. The term "unit dosage forms" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient (e.g., a tablet, capsule, or ampoule). The compounds are generally administered in a pharmaceutically effective amount. It will be understood, however, that the amount of the compound actually administered usually will be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered and its relative activity, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.

[0267] For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical excipient to form a solid pre-formulation composition containing a homogeneous mixture of a compound of the present disclosure. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules.

[0268] In certain embodiments, the tablets or pills can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action, or to protect from the acid conditions of the stomach. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer that serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

[0269] Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect.

[0270] Compositions in preferably pharmaceutically acceptable solvents may be nebulized by use of inert gases. Nebulized solutions may be inhaled directly from the nebulizing device or the nebulizing device may be attached to a facemask tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may be administered, preferably orally or nasally, from devices that deliver the formulation in an appropriate manner.

[0271] A method of imaging cancer-associated fibroblasts (CAFs) in a subject is also provided. The method comprising (i) administering to the subject an effective amount of a composition comprising a conjugate (e.g., a chelated conjugate) and a pharmaceutically acceptable excipient and (ii) imaging the subject to identify the location of the CAFs in the patient. Some embodiments concern any of the compositions herein for use in a method of treating a disease mediated by CAFs in a subject. In certain embodiments, the disease is a cancer discussed herein.

[0272] Further provided is a method of treating fibroblast activation protein alpha (FAPα)- expressing cancer in a subject. The method comprises administering to the subject an effective amount (i.e., a therapeutically effective amount) of a composition comprising a conjugate and a pharmaceutically acceptable excipient, whereupon FAPα-expressing cancer in the subject is treated. The cancer can be prostate cancer, renal cancer, breast cancer (e.g., inflammatory breast cancer or triple-negative breast cancer), thyroid cancer, gastric cancer, colorectal cancer, bladder cancer, pancreatic cancer, mesothelioma, lung cancer, liver cancer, a brain tumor, melanoma, a neuroendocrine tumor, ovarian cancer, esophageal cancer, cholangiocellular carcinoma, headneck cancer, carcinoma of unknown primary, medullary thyroid cancer, thymus cancer, smallintestine cancer, a sarcoma, and metastases of any of the foregoing, such as brain metastases.

[0273] In certain embodiments, provided herein is a method of treating a disease mediated by CAFs in a subject. The method comprises administering to the subject an effective amount (i.e., a therapeutically effective amount) of a composition comprising a conjugate and a pharmaceutically acceptable excipient. In certain embodiments, the disease is cancer. In certain embodiments, the cancer is selected from the group consisting of breast cancer (e.g., inflammatory breast cancer or triple-negative breast cancer), pancreatic cancer, mesothelioma, esophageal cancer, cholangiocellular carcinoma, head and neck cancer, and carcinoma of unknown primary origin, and metastases thereof, such as brain metastases.

[0274] Dosage regimens may be adjusted to provide the optimum desired response. The skilled artisan would appreciate, based upon the disclosure provided herein, that the dose and dosing regimen is adjusted in accordance with methods well-known in the therapeutic arts. That is, the maximum tolerable dose can be readily established, and the effective amount providing a detectable therapeutic benefit to a patient may also be determined, as can the temporal requirements for administering each agent to provide a detectable therapeutic benefit to the patient. Accordingly, while certain dose and administration regimens are exemplified herein, these examples in no way limit the dose and administration regimen that may be provided to a patient in practicing the present disclosure. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated and may include single or multiple doses. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. For example, doses may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects such as toxic effects and / or laboratory values. Thus, the present disclosure encompasses intra-patient dose-escalation as determined by the skilled artisan. Determining appropriate dosages and regimens for administration of the chemotherapeutic agent are well-known in the relevant art and would be understood to be encompassed by the skilled artisan once provided the teachings disclosed herein.

[0275] The amount of the conjugate administered will be dependent on the subject being treated, the severity of the disorder or condition, the rate of administration, the disposition of the compound and the discretion of the prescribing physician. However, an effective dosage is in the range of about 0.001 to about 100 mg per kg body weight per day. In some instances, dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed without causing any harmful side effect, provided that such larger doses are first divided into several small doses for administration throughout the day.

[0276] In another aspect provided herein is a kit, comprising materials useful for the treatment or prevention of the diseases and disorders described above. In some embodiments, the kit comprises a container comprising a conjugate. In some embodiments, the kit further comprises a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, blister pack, etc. The container can be formed from a variety of materials such as glass or plastic. The container can hold a conjugate, or a pharmaceutically acceptable salt thereof) or a composition thereof which is effective for treating or preventing the condition and can have a sterile access port (for example, the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a conjugate, or a pharmaceutically acceptable salt thereof) or a pharmaceutically acceptable salt thereof. The label or package insert indicates that the composition is used for treating the condition of choice. The label or package insert can also indicate that the composition can be used to treat other disorders. Alternatively, or additionally, the article of manufacture can further comprise a second container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate- buffered saline, Ringer’s solution and dextrose solution. It can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, fdters, needles, and syringes.

[0277] The kit can further comprise directions for the administration of a conjugate, or a pharmaceutically acceptable salt thereof) or a pharmaceutically acceptable salt thereof and, if present, the second pharmaceutical formulation. For example, if the kit comprises a first composition comprising a conjugate, or a pharmaceutically acceptable salt thereof) or a pharmaceutically acceptable salt thereof and a second pharmaceutical formulation, the kit can further comprise directions for the simultaneous, sequential or separate administration of the first and second pharmaceutical compositions to a patient in need thereof.

[0278] In another embodiment, the kits are suitable for the delivery of solid oral forms of a conjugate, or a pharmaceutically acceptable salt thereof) or a pharmaceutically acceptable salt thereof, such as tablets or capsules. Such a kit preferably includes a number of unit dosages. Such kits can include a card having the dosages oriented in the order of their intended use. An example of such a kit is a blister pack. Blister packs are well-known in the packaging industry and are widely used for packaging pharmaceutical unit dosage forms. If desired, a memory aid can be provided, for example in the form of numbers, letters, or other markings or with a calendar insert, designating the days in the treatment schedule in which the dosages can be administered.

[0279] In some embodiments, a kit comprises (a) a first container with a conjugate, or a pharmaceutically acceptable salt thereof, contained therein; and optionally (b) a second container with a second pharmaceutical formulation contained therein. Alternatively, or additionally, the kit can further comprise a third container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer’s solution and dextrose solution. It can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0280] In some embodiments wherein the kit comprises a conjugate, or a pharmaceutically acceptable salt thereof, and a second therapeutic agent, the kit can comprise a container for containing the separate compositions such as a divided bottle or a divided foil packet, however, the separate compositions can also be contained within a single, undivided container. Typically, the kit comprises directions for the administration of the separate components. The kit form is particularly advantageous when the separate components are preferably administered in different dosage forms (e.g., oral and parenteral), are administered at different dosage intervals, or when titration of the individual components of the combination is desired by the prescribing physician.

[0281] EXAMPLES

[0282] Selected abbreviations: Boc: tert-butyloxycarbonyl; DIPEA: N,N-diisopropylethyl amine; DCM: dichloromethane; DIPEA: diisopropylethylamine; DMF: N,N-dimethylformamide;

[0283] DOTA: dodecane tetraacetic acid; ESI: electrospray ionization; EtOAc: ethyl acetate; Fmoc: 9- fluorenylmethyloxycarbonyl; HATU: hexafluorophosphate azabenzotriazole tetramethyl uronium; MS: mass spectrometry; NMR: nuclear magnetic resonance; OMs: methanesulfonate; RT: room temperature; TEA: triethylamine; TFA: trifluoroacetic acid.

[0284] Example 1. Synthesis of tert-butyl 4-(piperazin-l-ylmethyl)isoindoline-2-carboxylate

[0285] (Compound 4a).

[0286] Diisopropylethylamine (84 mL, 0.488 mmol) was added to a stirred solution tert-butyl 4- (((methylsulfonyl)oxy)methyl)isoindoline-2-carboxylate 1A (32 g, 0.0977 mol) in dichloromethane (43 mL) at ambient temperature. A solution of piperazine (33 g, 0.39 mmol) in di chloromethane (165 mL)) was added and resultant reaction mixture stirred at ambient temperature for 4 h. Upon completion the reaction mixture was diluted with ice-cold water (30 mL) and stirred for 15 min. Layers were separated and the aqueous layer was extracted with di chloromethane (2 * 100 mL). The combined organic layer was washed with ice cold water (80 mL), dried over anhydrous sodium sulphate and concentrated under reduced pressure to afford crude material. The crude material was purified by silica gel column chromatography using 5% MeOH in dichloromethane eluant. Desired fractions were concentrated under reduced pressure to afford Zc / 7-butyl 4-(piperazin-l-ylmethyl)isoindoline-2-carboxylate (Compound 4a) (15 g, 48.3%) as a pale yellow gummy liquid. MS (ESI) m / z: 318.2 [M+H]+.

[0287] Example 2. Synthesis of 2,2',2"-(10-(2-(((S)-6-(4-((2-(2-((3S,5S)-5-((S)-2-cyano-4,4- difluoropyrrolidine-l-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4- yl)methyl)piperazin-l-yl)-5-(4-((4-(4-iodophenyl)butanamido)methyl)benzamido)-6- oxohexyl)amino)-2-oxoethyl)-l,4,7,10-tetraazacyclododecane-l,4,7-triyl)triacetic acid (FAP6-19).

[0288]

[0289] Step-1: Synthesis of 4-((4-(4-iodophenyl)butanamido)methyl)benzoic acid: Trifluoroacetic acid (160 mL, 4 Vol) was added to a solution of tert-butyl 4-((4-(4- iodophenyl)butanamido)methyl)benzoate 1 (40 g, 0.083 mol) in dichloromethane (200 mL) at 0 °C. The resultant reaction mixture was stirred at room temperature for 5 hours then concentrated under reduced pressure. The material was triturated with diethyl ether (200 mL) and filtered to afford 4-((4-(4-iodophenyl)butanamido)methyl)benzoic acid 2 (31 g, yield: 88%) as a brown solid. MS (ESI) m / z: 424.42 [M+H]+.

[0290] Step-2: Synthesis of tert-butyl N6-(((9H-fluoren-9-yl)methoxy)carbonyl)-N2-(4-((4-(4- iodophenyl)butanamido)methyl)benzoyl)-L-lysinate: To a solution of 4-((4-(4- iodophenyl)butanamido)methyl)benzoic acid 2 (50 g, 0.118 mol) in DMF (350 mL) at 0 °C, was added diisoproylethylamine (61.7 mL, 0.354) followed by HATU (67.2 g, 0.177 mol). The mixture was stirred for 30 minutes, and then tert-butyl N6-(((9H-fluoren-9- yl)methoxy)carbonyl)-L-lysinate (50 g, 0.118 mol) was added and the mixture was stirred at room temperature for 6 hours. The reaction mixture was cooled to room temperature, diluted with EtOAc (30 mL) and ice-cold water (30 mL) and stirred for 15 minutes. The organic layer was separated, and the aqueous layer was extracted with EtOAc (2 * 30 mL). The combined organic layer was washed with ice cold water (50 mL), dried over anhydrous sodium sulphate, then filtered. The organic layer was concentrated under vacuum and resultant crude was purified by silica gel column chromatography using 2% MeOH in dichloromethane as an eluant.

[0291] Fractions were concentrated under reduced pressure to afford tert-butylN6-(((9H-fluoren-9- yl)methoxy)carbonyl)-N2-(4-((4-(4-iodophenyl)butanamido)methyl)benzoyl)-L-lysinate 3 (55 g, yield: 56%) as a brown solid. MS (ESI) m / z: 830.6 [M+H]+.

[0292] Step-3: Synthesis of N6-(((9H-fluoren-9-yl)methoxy)carbonyl)-N2-(4-((4-(4- iodophenyl)butanamido)methyl)benzoyl)-L-lysine: Trifluoracetic acid (220 mL, 4 vol) was added to a solution of tert-butyl N6-(((9H-fluoren-9-yl)methoxy)carbonyl)-N2-(4-((4-(4- iodophenyl)butanamido)methyl)benzoyl)-L-lysinate 3 (55 g, 0.065 mol) in DCM (275 mL) at 0 °C then warmed to room temperature and stirred for 2 hours. The reaction mixture was concentrated under vacuum to afforded crude material that was triturated with diethyl ether (200 mL), filtered and dried under vacuum to afford N6-(((9H-fluoren-9-yl)methoxy)carbonyl)-N2- (4-((4-(4-iodophenyl)butanamido)methyl)benzoyl)-L -lysine 4 (39 g, yield: 78 %) as a brown solid. MS (ESI) m / z: 774.4 [M+H]1.

[0293] Step-4: Synthesis of tert-butyl 4-((4-(N6-(((9H-fluoren-9-yl)methoxy)carbonyl)-N2-(4- ((4-(4-iodophenyl)butanamido)methyl)benzoyl)-L-lysyl)piperazin-l-yl)methyl)isoindoline-2- carboxylate: Diisopropylethylamine (3.38 mL, 19.37 mmol) and HATU (3.24 g, 8.53 mmol) were added to a solution of N6-(((9H-fluoren-9-yl)methoxy)carbonyl)-N2-(4-((4-(4- iodophenyl)butanamido)methyl)benzoyl)-L-lysine 4 (6 g, 7.75 mmol) in DMF (18 mL, 3 Vol) at 0 °C then warmed to room temperature and stirred for 30 minutes. tert-Butyl 4-(piperazin-l- ylmethyl)isoindoline-2-carboxylate (Compound 4A) (2.45 g, 7.75 mmol) was added and the reaction mixture was stirred at room temperature for 2 hours. After completion of reaction, reaction mixture was cooled to room temperature and diluted with EtOAc (100 mL), ice cold water (100 mL), and stirred for 15 minutes. The organic layer was separated, and aqueous layer was further extracted with EtOAc (2 * 100 mL). The combined organic layer was washed with ice cold water (50 mL), dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure to afford tert-butyl4-((4-(N6-(((9H-fluoren-9-yl)methoxy)carbonyl)-N2-(4-((4- (4-iodophenyl)butanamido)methyl)benzoyl)-L-lysyl)piperazin-l-yl)methyl)isoindoline-2- carboxylate 5 (7 g, yield: 84%) as a brown solid. MS (ESI) m / z: 1073.7 [M+H]+,1H-NMR (400 MHz, DMSO-d6): δ 8.47 (d, J= 7.6 Hz, 1H), 8.36 (t, J= 6Hz, 1H), 7.88-7.82 (m, 4H), 7.66-7.60 (m, 4H), 7.39 (t, J= 6 Hz, 2H), 7.28-7.31 (m, 4H), 7.7-7.2 (m, 2H), 7.2-7.1 (m, 1H), 7.01 (d, J = 8.4 Hz, 2H), 4.9-4.8 (m, 1H), 4 7-4.5 (m, 4H), 4.3-4.1 (m, 5H), 3.6-3.4 (m, 5H), 3.4-3.4 (m, 3H), 2.9-3.0 (m, 2H), 2.5-2.4 (m, 4 H), 2.4-2.2 (m, 2H), 2.16 (t, J= 8.4 Hz, 2H), 1.8-1.7 (m, 2H), 1.7- 1.6 (m, 2H), 1.4-1.3 (m, 11 H), 1.3-1.2 (m, 2H).

[0294] Step-5: Synthesis of (9H-fluoren-9-yl)methyl (S)-(5-(4-((4-(4-iodophenyl)butanamido) methyl)benzamido)-6-(4-(isoindolin-4-ylmethyl)piperazin-l-yl)-6-oxohexyl)carbamate: Trifluoracetic acid (3 mL, 32.61 mmol) was added to a solution of tert-butyl 4-((4-(N6-(((9H- fluoren-9-yl)methoxy)carbonyl)-N2-(4-((4-(4-iodophenyl)butanamido)methyl)benzoyl)-L- lysyl)piperazin-l-yl)methyl)isoindoline-2-carboxylate 9 (7 g, 6.52 mmol) in dichloromethane (21 mL, 3 Vol) at 0 °C then warmed to room temperature and stirred for 4 hours. The reaction mixture was concentrated under reduced pressure to afford crude material. The crude material was triturated with diethyl ether (2*50 mL) to afford (9H-fluoren-9-yl)methyl (S)-(5-(4-((4-(4- iodophenyl)butanamido)methyl)benzamido)-6-(4-(isoindolin-4-ylmethyl)piperazin-l-yl)-6- oxohexyl)carbamate 10 (4 g, yield: 63 %) as a brown solid. MS (ESI) m / z: 974.5 [M+H]+.

[0295] Step-6: Synthesis of ((S)-6-(4-((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-l- carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)piperazin-l-yl)-5-(4-((4-(4- iodophenyl)butanamido)methyl)benzamido)-6-oxohexyl)carbamate: To a solution of 2-((3S,5S)- 5-((S)-2-cyano-4,4-difluoropyrrolidine-l-carbonyl)-2-oxopyrrolidin-3-yl)acetic acid (CDA-5) (1.2 g, 4.11 mmol) in DMF (20 mL) at 0 °C was added diisopropylethylamine (1.76 mL, 10.27 mmol) followed by HATU (1.56 g, 4.11 mmol). Then warmed to room temperature and stirred for 30 minutes. (9H-fluoren-9-yl)methyl (S)-(5-(4-((4-(4- iodophenyl)butanamido)methyl)benzamido)-6-(4-(isoindolin-4-ylmethyl)piperazin-l-yl)-6- ohexyl)carbamate 6 (4 g, 4.11 mmol) as added to the mixture. Upon completion the reaction was diluted with EtOAc (30 mL), ice cold water (30 mL), and stirred for 15 minutes. The organic layer was separated, and the aqueous layer was further extracted with EtOAc (2 * 30 mL). Combined organic layer was washed with ice cold water (50 mL) and dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure to afford (9H-fluoren-9- yl)methyl ((S)-6-(4-((2-(2-((3 S, 5 S)-5-((S)-2-cyano-4,4-difluoropyrrolidine- 1 -carbonyl)-2- oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)piperazin-l-yl)-5-(4-((4-(4- iodophenyl)butanamido)methyl)benzamido)-6-oxohexyl)carbamate 7 (4.3 g, yield: 84%) as a brown solid. MS (ESI) m / z: 1258.3 [M+H]+. Step-7: Synthesis of V-((S)-6-amino-l -(4-((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrr olidine- 1 -carbonyl)-2-oxopyrrolidin-3 -yl)acetyl)isoindolin-4-yl)methyl)piperazin- 1 -yl)- 1 - oxohexan-2-yl)-4-((4-(4-iodophenyl)butanamido)methyl)benzamide: Diethyl amine (48 mL, 4Vol) was added to a solution of (9H-fluoren-9-yl)methyl ((S)-6-(4-((2-(2-((3S,5S)-5-((S)-2- cyano-4,4-difluoropyrrolidine-l-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4- yl)methyl)piperazin-l-yl)-5-(4-((4-(4-iodophenyl)butanamido)methyl)benzamido)-6- oxohexyl)carbamate 7 (12 g, 9.5 mmol) in acetonitrile (120 mL, 10 volumes) at room temperature and stirred for 4 hours. The reaction mixture was concentrated under vacuum, and resultant crude compound (13 g) was triturated with cold EtOAc (30 mL) and filtered. The filtered solid was dissolved in 10% MeOH in DCM (20 mL), added diethyl ether (30 mL) to precipitate a solid. The regenerated solid was filtered to affordA-((S)-6-amino-l-(4-((2-(2- ((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-l-carbonyl)-2-oxopyrrolidin-3- yl)acetyl)isoindolin-4-yl)methyl)piperazin- 1 -yl)- 1 -oxohexan-2-yl)-4-((4-(4- iodophenyl)butanamido)methyl)benzamide 13 (9.5 g, crude) as a pale brown solid. MS (ESI) m / z: 1035.9 [M+H]+.

[0296] Step-8: Synthesis of 2,2',2"-(10-(2-(((S)-6-(4-((2-(2-((3S,5S)-5-((S)-2-cyano-4,4- difluoropyrrolidine-l-carbonyl)-2-oxopyrrolidin-3-yl)acetyl)isoindolin-4-yl)methyl)piperazin-l- yl)-5-(4-((4-(4-iodophenyl)butanamido)methyl)benzamido)-6-oxohexyl)amino)-2-oxoethyl)- l,4,7,10-tetraazacyclododecane-l,4,7-triyl)triacetic acid: To a solution ofN-((S)-6-amino-l-(4- ((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-l-carbonyl)-2-oxopyrrolidin-3- yl)acetyl)isoindolin-4-yl)methyl)piperazin- 1 -yl)- 1 -oxohexan-2-yl)-4-((4-(4- iodophenyl)butanamido)methyl)benzamide 8 (9.5 g, 9.18 mmol) in acetonitrile (190 mL, 20 Vol) at room temperature was added diisopropylethylamine (14.4 mL, 82.62 mmol, 9 equivalents) followed by 2,2',2"-( 10-(2-((2,5-dioxopyrrolidin- 1 -yl)oxy)-2-oxoethyl)- 1,4,7,10- tetraazacyclododecane-l,4,7-triyl)triacetic acid (6.9 g, 11.02 mmol). The reaction mixture was stirred at room temperature for 16 hours. Upon completion the reaction mixture was concentrated under reduced pressure to afford crude material (15.6 g). The material was triturated with EtOAc (150 mL) and filtered to afford crude material (13.1 g). The crude material was further purified by DAC (Dynamic axial compression column chromatography) using the below condition.

[0297] The required fractions were collected and concentrated under vacuum at 35 °C to afford solid compound, which was the co-distilled with acetonitrile (2*50 mL). Compound obtained was dissolved in 10% MeOH in DCM and precipitated with diethyl ether to afford 2,2',2"-(10-(2- (((S)-6-(4-((2-(2-((3S,5S)-5-((S)-2-cyano-4,4-difluoropyrrolidine-l-carbonyl)-2-oxopyrrolidin-3- yl)acetyl)isoindolin-4-yl)methyl)piperazin-l-yl)-5-(4-((4-(4- iodophenyl)butanamido)methyl)benzamido)-6-oxohexyl)amino)-2-oxoethyl)-l,4,7,10- tetraazacyclododecane-l,4,7-triyl)triacetic acid (FAP6-19) (1.38 g, yield: 10.6%) as a white solid. MS (ESI) m / z: 1418.53 [M-H] ; 'H-NMR (400 MHz, DMSO-d6): 8 8.6-8.5 (m, 1H), 8.5- 8.45 (m, 2H), 8.09-8.06 (m, 1H), 7.83 (d, J= 8 Hz, 2H), 7.63 (d, J= 8 Hz, 2H), 7.46-7.40 (m, 3H), 7.31 (d, .7 = 8 Hz, 2H), 7.01 (d, J= 8 Hz, 2H), 5.11-5.09 (m, 1H), 5.0-4.6 (m, 5H), 4.4-4.2 (m, 3H), 4.2-4.1 (m, 4H), 4.0-3.9 (m, 2H), 3.6-3.5 (m, 9H), 3.4-3.3 (m, 12H), 3.3-3.0 (m, 13H), 2 9-2.7 (m, 6H), 2.4-2.0 (m, 4H), 1.8-1.6 (m, 4H), 1.4-1.2 (m, 4H).

[0298] Example 3. Syntheses FAP6-16 and FAP6-17.

[0299] FAP6-16 and FAP6-17 were prepared using procedures similar to those described in Examples 1 and 2. FIGS. 2A-2B depict LC / MS characterizations of compounds prepared by the above procedures. Example 4. Preparation ofinIn- or177Lu-chelated compounds

[0300] Exemplary non-chelated compounds of the disclosure (FAP6-16, FAP6-17, or FAP6-19) were diluted in ammonium acetate (0.5 M, pH 8.0).1 1 Jln[ InCh] was added to obtain a specific activity of no more than 4.0 MBq / nmol, orj7 / Lu[LuCh] was added to obtain a specific activity of no more than 11.0 MBq / nmol. The resulting solutions were heated to 90 °C for 10-20 minutes, and the radiopurities of the products were analyzed by radio-high-performance li quid chromatography. Radiopurity exceeded 95% in all studies.

[0301] FIGS. 3A-3C depict radio-chromatograms of exemplary radio-chromatogram of exemplary177Lu-chelated compounds of the disclosure. FIGS. 4A-4C depict radiochromatograms of exemplary radio-chromatogram of exemplaryluIn-chelated compounds of the disclosure.

[0302] Example 5. Binding studies of177Lu-chelated radioligands

[0303] Cell binding assay: HEK-huFAP cancer-associated fibroblasts (CAFs) grown to confluency in 24-we!l amine-coated plates were incubated with increasing concentrations of1' 'Lu-radioligands (e.g., [!' 'Lu]Lu-FAP6-16, [s' 'Lu]Lu-FAP6-17, or f1 / / Lu]Lu-FAP6-19) in 0.25 mL media containing 1% fetal bovine serum (FBS). The cells were then washed 3x with phosphate-buffered saline (PBS), dissolved in 1.0 M NaOH, and analyzed by a gamma counter. Results are depicted in FIG. 5.

[0304] Example 6. Cellular uptake, receptor internalization, and retention studies ofinIn- chelated radioligands

[0305] 2 x 105HT1080-huFAP cells were plated in a 24-well plate in supplemented media 24 hours before the studies. The medium was removed, and then In-11 1 -chelated compounds were added at 50 nM in 0.5 mL of medium containing 1% fetal bovine serum (FBS). The plates were then incubated for 1, 4, and 24 hours at 37 °C. Samples were prepared for each experiment in triplicate as follows: Cellular Uptake: At each time point (1 hour, 4 hours, and 24 hours), the medium was removed, and the wells were washed thrice with 1 mL of phosphate buffered saline, followed by adding 0.25 mL of 1 M NaOH to facilitate cell lysis.

[0306] Receptor Internalization: At the indicated times, cells were washed once with PBS, then a 0.5 mL acid medium containing 50 mM glycine and 100 mM NaCl at a pH of 2.8 was added to the sample for 1 minute. After removing the supernatant, the cells were washed one more time with phosph ale-buffered saline (PBS) before adding 0.25 mL of 1 M NaOH to facilitate cell lysis.

[0307] Cell retention / efflux: 50 nM of ’’’In-chelated compounds were added to plates in a 1% FBS medium and were incubated for 1 hours at 37 °C. Cells were washed once with phosphate- buffered saline (PBS), then incubated with radioactive-free medium for 1 hour, 4 hours, and 24 hours at 37 °C. At the indicated times, wells were washed twice with PBS, and dissolved in 1.0 M NaOH.

[0308] 0.2 mL of cell lysates from each sample were transferred to individual tubes and counted in a gamma counter. For cellular update studies, exemplary results are depicted in FIG. 6. For receptor internalization studies, exemplary results are depicted in FIG. 7, For cell retention studies, exemplary results are depicted in FIG. 8.

[0309] Example 7. Imaging studies ofmIn-chelated radioligands

[0310] 4T1 tumor-bearing mice were intravenously injected with 5 nmol of compound radiolabeled with about 13 MBq ofnTn. At indicated times (3 hours, 24, hours, 72 hours, and 120 hours), the mice were anesthetized and then scanned using a single-photon emission computed tomography / computed tomography (SPECT / CT) dual-modality instrument. SPECT / CT scans were reconstructed using software. The datasets were fused, filtered, and processed using software. Exemplary results are depicted in FIGS. 9.

[0311] Example 8. Biodistribution studies of177Lu-chelated radioligands

[0312] 4T1 tumor-bearing mice (w = 3 -4) were intravenously injected with exemplary ’•'•'Lu- chelated compounds of the disclosure. At indicated times, the mice were euthanized, and organs of interest were harvested, washed, dried, weighed, and analyzed by a gamma counter.

[0313] Exemplary biodistribution results are depicted in FIGS. 10A-10E.

[0314] Example 9. Dosimetry of177Lu-chelated radioligands

[0315] From the studies of Example 9 above, dosimetry calculations of177Lu-radiolabeled compounds were determined using software. Exemplary estimates are provided in FIGS. 11A- 11C

[0316] Example 10. Tumor radiotherapy studies of177Lu-chelated radioligands

[0317] Mice bearing 4T1 tumors were randomized into control and treatment groups to ensure equal starting tumor volumes. Each cohort received a single intravenous injection of either vehicle alone or vehicle with 1 mCi of177Lu-radiolabeled compounds on Day 0 at a specific activity of 0.2 mCi / nmol. Tumors were measured with a caliper in 2 perpendicular directions 3x / week by 2 independent researchers in blinded fashion, then averaged together. The mice were euthanized on reaching one of the predefined endpoint criteria according to the regulations of the Institutional Animal Care and Use Committee. Exemplary results are depicted in FIG. 12A-12B.

[0318] Example 11. Tumor Size over Time for Treatment with177Lu-chelated radioligands

[0319] FIG. 13 depicts a radiotherapy study with FAP radioligands chelated with Lu-177, using 4T1 tumor growth chart in Balb / c mice. 5 nmol of each FAP conjugate radiolabeled with ~1.0 mCi of Lu-177 was administered per mouse by intravenous lateral tail-vein injections. Blind measurements were subsequently taken with a caliper by 2 researchers 3x per week. Other experimental parameters were followed as previously described (Lindeman, S. D.; Mukkamala, R.; Horner, A.; Tudi, P.; Booth, O. C.; Huff, R. R.; Hinsey, J.; Hovstadius, A.; Martone, P.; Zhang, F.; Srinivasarao, M.; Cox, A.; Low, P. S., Fibroblast activation protein targeted radioligand therapy for treatment of solid tumors. J. Nucl. Med. 2022, jnumed.122.264494).

[0320] FIG. 14 presents SPECT / CT scans at (A) 24 hours, (B) 72 hours, post-injection of [177LU]LUFAP6 -19 radioligands in Balb / c mice bearing 4T1 tumors. Example 12. Synthesis and Characterization of FAP6 Conjugates

[0321] FIG. 2A depicts the synthetic scheme of intermediates required for the synthesis of the FAP6 conjugates. In FIG. 2B, a synthetic scheme for conjugates is presented and provides the structure ofFAP6-01. LC / MS characterizations of the structures FAP6- 19, FAP6-17d, and FAP- 16 are presented in FIGS. 3A-3C, respectively. Radio-chromatograms of [177Lu]Lu-FAP6-19, [177Lu]Lu-FAP6-17, and [177Lu]Lu-FAP6-16 are presented in FIGS. 4A-C, respectively. Radiolabeling conditions for FIGS. 4A-4C include 500 pM of FAP6 conjugate in 0.5 M NaOAc (pH = 6), 45°C for 10 minutes.

[0322] Example 13. Biodistribution Study of FAP6 Radioligands

[0323] FIG. 11 depicts a biodistribution study of FAP6 radioligands chelated with Lu-177 at (A) 3 hours, (B) 24 hours, and (C) 120 hours post-injection using Balb / c mice bearing 4T1 tumors. (D) Graphs providing tumor-to-blood, -liver, and -kidney ratios. Study was performed as previously described. Data are presented as means with SEM bars. Statistical analyses were performed by one-way, non-paired ANOVA (*P < 0.05, **P < 0.01, ***p < 0.001, ****p < 0.0001). In FIG. 12A, dosimetry estimates based on the biodistribution data presented in FIG. 11 are presented. FIG. 12B depicts tumor-to-healthy organ ratios based on the dosimetry calculations from part A. Calculations were performed as previously described (Lindeman, S. D ; Mukkamala, R.; Homer, A.; Tudi, P.; Booth, O. C.; Huff, R. R.; Hinsey, J.; Hovstadius, A.; Martone, P.; Zhang, F.; Srinivasarao, M.; Cox, A.; Low, P. S., Fibroblast activation protein targeted radioligand therapy for treatment of solid tumors. J. Nucl. Med. 2022, jnumed.122.264494).

[0324] Example 14. Synthesis of Conjugates

[0325] FIGS. 15A-15T illustrate numerous, non-limiting, prophetic structures of additional FAP6 radioligands with various linker modifications, PK modulators, FAP ligands, and / or chelators.

[0326] Group A structures, illustrated in FIG. 15A-15I, are prosthetic structure that may be synthesized using various key intermediates, starting materials, and general experimental conditions as described previously (Mukkamala R, Lindeman SD, Kragness KA, Shahriar I, Srinivasarao M, Low PS. Design and characterization of fibroblast activation protein targeted pan-cancer imaging agent for fluorescence-guided surgery of solid tumors. Journal of Materials Chemistry B. 2022;10:2038-2046; Spencer DL, Ramesh M, Autumn H, et al. Fibroblast Activation Protein-Targeted Radioligand Therapy for Treatment of Solid Tumors. Journal of Nuclear Medicine. 2023;64:759; Lindeman SD, Booth OC, Tudi P, et al. FAP Radioligand Linker Optimization Improves Tumor Dose and Tumor-to-Healthy Organ Ratios in 4T1 Syngeneic Model. Journal of Medicinal Chemistry. 2024;67: 11827-11840 and Mukkamala R, Carlson DJ, Miller NK, et al. Design of a Fibroblast Activation Protein-Targeted Radiopharmaceutical Therapy with High Tumor-to-Healthy-Tissue Ratios. Journal of Nuclear Medicine. 2024:jnumed.124.267756).

[0327] Group B structures, illustrated in FIG. 15J, are prophetic structures that may be synthesized using various chemical modifications from commercially available starting materials. Initially, the coupling of amine A with acid B(7) yields the intermediate compound C. Subsequently, a Suzuki-Miyaura cross-coupling reaction ( Ahmed A, Mushtaq I, Chinnam S. Suzuki-Miyaura cross-couplings for alkyl boron reagent: recent developments — a review. Future Journal of Pharmaceutical Sciences. 2023;9:67) between C and various substituted benzyl boronate derivatives produces the intermediates D. Further chemical modifications of these intermediates ultimately result in the final conjugates in group B series (Scheme-1 of FIG. 15J).

[0328] Group C structures, illustrated in FIG. 15K, are prophetic structures that may be synthesized from a key fragment F, which is readily synthesized using literature condition (f Chinchilla R, Najera C. The Sonogashira Reaction: A Booming Methodology in Synthetic Organic Chemistry. Chemical Reviews. 2007;107:874-922), and then coupled with another fragment E followed by few chemical transformations provide a group C conjugates (Scheme-2 of FIG. 15K)

[0329] Group D structures, illustrated in FIG. 15L, are prophetic structures that may be readily synthesized from the key intermediate compounds K and Na. Intermediate K is synthesized in a single step from the reported molecule J by reacting with PPha. While intermediate Na is obtained by coupling of commercially available starting materials La or Lb with the known intermediate E. The resulting coupled products (Ma and Mb) undergo a Wittig reaction with Wittig salt K, followed by hydrogenation to yield intermediate Na. Then compounds Na is converted to group D conjugates using routinely used synthetic steps provide the group-D conjugate (Scheme-3 of FIG. 15L).

[0330] Group E structures, illustrated in FIG. 15M, are prophetic structures that may be synthesized starting from intermediate compounds (Mb), which are reduced to their corresponding alcohols (Pa). Subsequently, base-mediated alkylation of Pa with isoindolebromide yields the intermediate compounds Pb. Finally, multiple functional group transformations on Pb lead to the Group D conjugates, as depicted in Scheme 4 of FIG. 15M.

[0331] Similarly, the Group F structures, illustrated in FIG. 15N, may be synthesized from the key intermediate Mb. Reductive amination with methylamine yields compound Qa, which undergoes subsequent alkylation and several transformations with appropriate starting material to produce the Group F conjugates, as shown in Scheme 5 of FIG. 15N.

[0332] Conjugate structures in Group E (Fig. 15M) and Group F (Fig. 15N) may be synthesized by reacting the appropriate intermediates from Scheme 3 and Scheme 4 with the key intermediate R as illustrated in FIG. 150. The synthesis of intermediate R was carried out following established literature protocols. ( Cui X-Y, Li Z, Kong Z, et al. Covalent targeted radioligands potentiate radionuclide therapy. Nature. 2024;630:206-213).

[0333] Additional conjugate structures of Groups E and F are depicted in FIG. 15P and FIG. 15Q.

[0334] Groups G, H and I, depicted in FIGs. 15R-15T, present additional structures that represent embodiments of the instant disclosure.

[0335] DEFINITIONS

[0336] As used herein the specification, “a” or “an” may mean one or more. As used herein, when used in conjunction with the word "comprising", the words “a” or “an” may mean one or more than one. As used herein “another” may mean at least a second or more. Still further, the terms “having”, “including”, “containing” and “comprising” are interchangeable and one of skill in the art is cognizant that these terms are open ended terms. Some embodiments of the disclosure may consist of or consist essentially of one or more elements, method steps, and / or methods of the disclosure. It is contemplated that any method, compound, or composition described herein can be implemented with respect to any other method, compound, or composition described herein. "About" and "approximately" shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, and more typically, within 5% of a given value or range of values.

[0337] As used herein, “alkyl” refers to a radical of a straight-chain or branched saturated hydrocarbon group, e.g., having 1 to 20 carbon atoms (“C1-20 alkyl”). In some embodiments, an alkyl group has 1 to 10 carbon atoms (“C1-10 alkyl”). In some embodiments, an alkyl group has 1 to 9 carbon atoms (“C1-9 alkyl”). In some embodiments, an alkyl group has 1 to 8 carbon atoms (“C1-8 alkyl”). In some embodiments, an alkyl group has 1 to 7 carbon atoms (“C1-7 alkyl”). In some embodiments, an alkyl group has 1 to 6 carbon atoms (“C1-6 alkyl”). In some embodiments, an alkyl group has 1 to 5 carbon atoms (“C1-5 alkyl”). In some embodiments, an alkyl group has 1 to 4 carbon atoms (“Ci^i alkyl”). In some embodiments, an alkyl group has 1 to 3 carbon atoms (“C1-3 alkyl”). In some embodiments, an alkyl group has 1 to 2 carbon atoms (“C1-2 alkyl”). In some embodiments, an alkyl group has 1 carbon atom (“Ci alkyl”). Examples of Ci 6 alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, hexyl, and the like. In some embodiments, “alkyl” is unsubstituted or substituted with one or more substituents as described herein.

[0338] As used herein, “halogen” or “halo” refers to -F, -Cl, -Br, or -I.

[0339] It will be appreciated that the compounds, as described herein, may be substituted with any number of substituents or functional moieties. In general, the term “substituted” whether preceded by the term “optionally” or not, and substituents contained in formulas of this disclosure, refer to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent. When more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position.

[0340] Unless otherwise indicated, any heteroatom with unsatisfied valences is assumed to have hydrogen atoms sufficient to satisfy the valences.

[0341] Certain compounds of the present disclosure may exist in particular geometric or stereoisomeric forms. The present disclosure contemplates all such compounds, including cisand trans-i somers, R- and S-enantiomers, diastereomers, (d)-isomers, (l)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the disclosure. Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this disclosure.

[0342] All stereoisomers of the present compounds (for example, those which may exist due to asymmetric carbons on various substituents), including enantiomeric forms and diastereomeric forms, are contemplated within the scope of this disclosure. Individual stereoisomers of the compounds of the disclosure may, for example, be substantially free of other isomers (e.g., as a pure or substantially pure optical isomer having a specified activity), or may be admixed, for example, as racemates or with all other, or other selected, stereoisomers. The chiral centers of the present disclosure may have the S or R configuration as defined by the International Union of Pure and Applied Chemistry (IUPAC) 1974 Recommendations. The racemic forms can be resolved by physical methods, such as, for example, fractional crystallization, separation or crystallization of diastereomeric derivatives, or separation by chiral column chromatography. The individual optical isomers can be obtained from the racemates by any suitable method, including without limitation, conventional methods, such as, for example, salt formation with an optically active acid followed by crystallization.

[0343] All configurational isomers of the compounds of the present disclosure are contemplated, either in admixture or in pure or substantially pure form. The definition of compounds of the present disclosure embraces both cis (Z) and trans (E) alkene isomers, as well as cis and trans isomers of cyclic hydrocarbon or heterocyclic rings.

[0344] Isomeric mixtures containing any of a variety of isomer ratios may be utilized in accordance with the present disclosure. For example, where only two isomers are combined, mixtures containing 50:50, 60:40, 70:30, 80:20, 90: 10, 95:5, 96:4, 97:3, 98:2, 99: 1, or 100:0 isomer ratios are all contemplated by the present disclosure.

[0345] The present disclosure also includes isotopically labeled compounds, which are identical to the compounds disclosed herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, sulfur, fluorine, and chlorine, such as2H,3H,13C,nC,14C,15N,18O,170,31P,32P,35S,18F, and36C1, respectively. Compounds of the present disclosure, or a stereoisomer, tautomer, or pharmaceutically acceptable salt or solvate thereof, which contain the aforementioned isotopes and / or other isotopes of other atoms are within the scope of this disclosure.

[0346] As used herein, “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge etal., describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66: 1-19. Pharmaceutically acceptable salts of the compounds of this disclosure include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Pharmaceutically acceptable salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(Ci-4alkyl)4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.

[0347] As used herein, “pharmaceutically acceptable excipient” refers to any substance in a pharmaceutical formulation other than an active pharmaceutical ingredient(s). Exemplary pharmaceutical excipients include those that aid the manufacturing process; protect, support or enhance stability; increase bioavailability; or increase patient acceptability. They may also assist in product identification or enhance the overall safety or function of the product during storage or use.

[0348] As used herein, a “subject” to which administration is contemplated includes, but is not limited to, humans (i.e., a male or female of any age group, e.g., a pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult or senior adult)) and / or a non-human animal, e.g., a mammal such as primates (e.g., cynomolgus monkeys, rhesus monkeys), cattle, pigs, horses, sheep, goats, rodents, cats, and / or dogs. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. The terms “human,” “patient,” “subject,” and “individual” are used interchangeably herein. None of these terms require the active supervision of medical personnel.

[0349] Disease, disorder, and condition are used interchangeably herein.

[0350] As used herein, and unless otherwise specified, the terms “treat,” “treating” and “treatment” contemplate an action that occurs while a subject is suffering from the specified disease, disorder or condition, which reduces the severity of the disease, disorder or condition, or reverses or slows the progression of the disease, disorder or condition (also “therapeutic treatment”).

[0351] In general, the “effective amount” of a compound refers to an amount sufficient to elicit the desired biological response. As will be appreciated by those of ordinary skill in this art, the effective amount of a compound of the disclosure may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the age, weight, health, and condition of the subject. A “therapeutically effective amount” of a compound is an amount sufficient to provide a therapeutic benefit in the treatment of a disease, disorder or condition, or to delay or minimize one or more symptoms associated with the disease, disorder or condition. A therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the disease, disorder or condition. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of disease or condition, or enhances the therapeutic efficacy of another therapeutic agent. A “prophylactically effective amount” of a compound is an amount sufficient to prevent a disease, disorder or condition, or one or more symptoms associated with the disease, disorder or condition, or prevent its recurrence. A prophylactically effective amount of a compound means an amount of a therapeutic agent, alone or in combination with other agents, which provides a prophylactic benefit in the prevention of the disease, disorder or condition. The term “prophylactically effective amount” can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent. A “prophylactic treatment” contemplates an action that occurs before a subject begins to suffer from the specified disease, disorder or condition.

[0352] When the term “ligand” is used herein, it is understood that the ligand is a radical that is bound to a further chemical moiety.

[0353] As used herein, the phrase “albumin-binder” indicates that the moiety can bind or associate with albumin.

[0354] The term “chelator” refers to a structure that can chelate with a radioisotope.

[0355] As used herein, a “PET” imaging agent refers to a radioactive substance used in positron emission tomography (PET).

[0356] SPECT refers to single-photon emission computed tomography which is a nuclear medicine tomographic imaging technique.

[0357] As used herein, “[177Lu]Lu-FAP6-01” refers to a chelated complex of a compound represented by the formula:

[0358] As used herein, “[111In]In-FAP6-01” refers to a chelated complex represented by the formula:

[0359] As used herein, “[177Lu]Lu-FAP2286” refers to a chelated complex represented by the formula:

[0360] As used herein, “[177Lu]Lu-F API-46” refers to a chelated complex represented by the formula:

Claims

WHAT IS CLAIMED IS:1 . A conjugate of the formula:wherein F is a ligand that binds fibroblast activation protein alpha (FAPα); L is a functionalized linker that binds with F, A, and X; A is a pharmacokinetic extender that binds or associates or binds with a protein in the blood of an animal; and X is a chelator, wherein the conjugate can target fibroblast activation protein alpha- (FAPα-) expressing cancer-associated fibroblasts (CAFs).

2. The conjugate of claim 1, wherein the chelator is associated with a radioisotope.

3. The conjugate of claim 1 or claim 2, wherein L comprises a primary spacer, a branching linker extending from the primary spacer, and a secondary spacer extending from the branching linker, wherein F is linked via the primary spacer, X is linked via the branching linker and comprises a chelating agent., and A is linked via the secondary spacer and comprises an albumin binder.

4. The conjugate of claim 1, wherein the L is selected from the group consisting of:

5. The conjugate of any of claims 1-4, wherein the chelating agent is 1,4,7, 10- tetraazacyclododecane-l,4,7,10-tetraacetic acid (DOTA).

6. The conjugate of any one of claims 1-5, wherein F is FAP6.

7. The conjugate of any one of claims 1 -6, wherein the metal ion is selected from18F,44Sc,47Sc,52Mn,55Co,64Cu,67Cu,66Ga,67Ga,68Ga,86Y,89Zr,90Y, "mTc,mIn,114mIn,117mSn,124I,125I,131I,149Tb,153Sm,152Tb,155Tb,161Tb,177Lu,186Re,188Re,212Pb,212Bi,213Bi,223Ra,224Ra,210At,211At,225Ab,225Ac, and227Th.

8. The conjugate of any one of claims 1-7, wherein the metal ion is a radiolabel.

9. The conjugate of any one of claims 1-7, wherein the metal ion is a radiopharmaceutical.

10. The conjugate of any one of claims 1, wherein the ligand has a molecular weight below10,000.11 . The conjugate of any one of claims 1-10, wherein the protein is albumin or a prealbumin.

12. The conjugate of any one of claims 1, 9, and 10, wherein the radiolabeled functional group is a positron emission tomography (PET) imaging agent, a single photon emission computed tomography (SPECT) imaging agent, a radio-imaging agent, or a radiotherapeutic agent.

13. The conjugate of any one of claims 1 and 9-12, wherein F has the structure:wherein T is CH2 O, NH, or S;Ri andR? are independently selected from the group consisting of -H, -CN, -CHO, -B(OH)2, -C(O)alkyl, -C(O)aryl-, -C=C-C(O)aryl, -C=C-S(O)2aryl, -CO2H, -SO3H, -SO2NH2, -PO3H2, -SO2F, 5-tetrazolyl, C1-C4 alkyl, isonitrile, and boronic acid;R3 andR4 are independently selected from the group consisting of -H, -OH, F, Cl, Br, I, -C1-6 alkyl, -O-C1-6 alkyl, -S-C1-6 alkyl, isonitrile, and boronic acid;Rs-Rs are independently selected from the group consisting of H, alkyl, and halo; and R9-R11 are independently selected from the group consisting of H, -C1-6 alkyl, -O-C1-6 alkyl, -S-C1-6 alkyl, F, Cl, Br, I, -CN, -CHO, -B(OH)2, -C(O)alkyl, -C(O)aryl-, -C=C-C(O)aryl, -C=C-S(O)2aryl, -CO2H, -SO3H, -SO2NH2, -PO3H2, -SO2F, 5-tetrazolyl, C1-C4 alkyl, isonitrile, and boronic acid.

14. The conjugate of any one of claims 1 and 9-13, wherein L is or comprises at least one of an amino acid, a polyethylene glycol (PEG) monomer, a PEG oligomer, a PEG polymer, a heterocycle, or a combination of two or more of the foregoing.

15. The conjugate of any one of claims 1 and 9-14, wherein L is or comprises at least one of an ether, a thioether, a tertiary amine, a C1-6 alkyl, piperazine, piperidine, a bicyclo-heptane, a substituted benzene, or a combination of two or more of the foregoing.

16. The conjugate of any one of claims 1 and 9-15, wherein L is or comprises an oligomer of peptidoglycans, glycans, anions, heterocycles, or a combination of two or more of the foregoing.

17. The conjugate of any one of claims 1 and 9-16, wherein L is or comprises at least one of a diaminobutyric acid group, a substituted benzene group, a lysine group, a 2,3-diaminopropionic acid group, a tyrosine group, a glutamic acid group, a cysteine group, or a combination of two or more of the foregoing.

18. The conjugate of any one of claims 1 and 9-17, wherein L is or comprises at least one of a 2,3-diaminopropionic acid group, a glutamic acid group, a lysine group, a cysteine group, and / or at least one heterocycle of or comprising one or more of the following structures:

19. The conjugate of any one of claims 1 and 9-18, wherein A is or comprises one of the structures:FoscanTookador an albumin-binding small protein scaffold such as, but not limited to, ABD035, ABDCon, DARPins, dsFv CA645, Nanobody, and VNAR (E06).

20. The conjugate of any one of claims 1 and 9-19, wherein X is or comprises a chelating agent selected from:DOTA (l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid) or a derivative thereof;TETA (1,4,8, 11-tetraazacyclotetradecane-l, 4,8,11-tetraacetic acid) or a derivative thereof;SarAr (l-N-(4-Aminobenzyl)-3,6,10,13,16,19-hexaazabicyclo[6.6.6]-eicosane-l,8-diamine or a derivative thereof;NOTA (l,4,7-triazacyclononane-l,4,7-triacetic acid) or a derivative thereof;NETA (4-[2-(bis-carboxymethylamino)-ethyl]-7-carboxymethyl-[l,4,7]triazonan-l-yl) acetyl acid or a derivative thereof;TRAP (l,4,7-triazacyclononane-l,4,7-tris[methyl(2-carboxyethyl)phosphinic acid) or a derivative thereof;HBED (N,N0-bis(2-hydroxybenzyl)-ethylenediamine-N, NO-diacetic acid) or a derivative thereof;2,3-HOPO (3-hydroxypyridin-2-one) or a derivative thereof;PCTA (3,6,9,15-tetraazabicyclo[9.3.1]-pentadeca-l(15),l l,13-triene-3,6,9,-triacetic acid) or a derivative thereof;DFO (desferri oxamine) or a derivative thereof;DTPA (diethylenetriaminepentaacetic acid) or a derivative thereof;OCTAPA (N,N0-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N, NO-diacetic acid) or a derivative thereof; or H2-MACR0PA (N,N'-bis[(6-carboxy-2-pyridipmethyl]-4,13-diaza-18- crown-6) or a derivative thereof;Fbdedpa (l,2-[[carboxy)-pyridin-2-yl]-methylamino]ethane or a derivative thereof;EC20-head comprising P-l-diaminopropionic acid, Asp, and Cys;Sarcophagine or a derivative thereof;TCMC (DOTAM) or a derivative thereof;Cyclen (S-2-(4-nitrobenzyl)-l,4,7,10-tetraazacyclododecane) or another derivative thereof;D03A (l,4,7,10-tetraazacyclododecane-l,4,7-tris(acetic acid)-10-(2-thioethyl)acetamide) or a derivative thereof;N02A (l,4,7,10-tetraazacyclododecane-l,4,7-tris(acetic acid)-10-(2-thioethyl)acetamide) or a derivative thereof;PCTA (3,6,9,15-tetraazabicyclo[9.3.1] pentadeca- 1(15), 1 l,13-triene-4-S-(4-aminobenzyl)-3,6,9- triacetic acid) or a derivative thereof;Deferoxamine (l-(4-isothiocyanatophenyl)-3-[6, 17-dihydroxy-7, 10, 18,21 -tetraoxo-27-(N- acetylhydroxylamino)- 6,11,17, 22- tetraazaheptaei cosine] thiourea) or a derivative thereof; HEHA (2-(4-isothicyanatobenzyl)- 1 ,2,7, 10,13 -hexaazacyclooctadecane- 1 ,4,7, 10, 13 , 16- hexaacetic acid) or a derivative thereof;21. The conjugate of claim 20, wherein X comprises a radionuclide selected from18F,44Sc,47Sc,52Mn,55Co,64Cu,67Cu,66Ga,67Ga,68Ga,86Y,89Zr,90Y, "mTc,mIn,114mIn,117raSn,124I,125I,131I,149Tb,153Sm,152Tb,155Tb,161Tb,177Lu,186Re,188Re,212Pb,212Bi,213Bi,223Ra,224Ra,210At,211At,223Ab,225Ac, or227Th, wherein the radionuclide is chelated to the chelator.

22. The conjugate of claim 1 represented by Formula (II):Formula (II) or a pharmaceutically acceptable salt thereof, wherein:R3is an albumin binding ligand;R4is selected from the group consisting of:n is an integer from 1 to 6.

23. The conjugate of claim 22, wherein R3is:

24. The conjugate of claim 22 or 23, wherein R3is:wherein R27is selected from the group consisting of halogen and C1-6 alkyl.

25. The conjugate of claim 24, wherein R27is iodo.

26. The conjugate of claim 24, wherein R27is fluoro.

27. The conjugate of claim 24, wherein R27is methyl.

28. The conjugate of any one of claims 22-27, wherein R4comprises a chelating agent and a radionucleotide, wherein the chelating agent is:n is 1-20.

30. The conjugate of claim 1 having the formula:a is 0 or 1; and n is 1-20.

31. The conjugate of claim 1 having the formula:R28is F, Cl, Br, I, -N02, -CH3- ibuprofen, or Evans Blue.

32. The conjugate of claim 1 having the formula:R28is F, Cl, Br, I, -NO2, -CH3- ibuprofen, or Evans Blue.

33. The conjugate of claim 1 having the formula;R28is F, Cl, Br, I, -NO2, -CH3- ibuprofen, or Evans Blue; and n is 1-20.

34. The conjugate of claim 1 having the formula:

35. The conjugate of claim 1 having the formula:n is 1-10.

36. The conjugate of claim 1 selected from the group consisting of:FAP6-16FAP6-19 or a pharmaceutically acceptable salt thereof.

37. A conjugate of claim 36 having the structure:(FAP6-19).

38. A chelated complex represented by Formula (I):Formula (I) or a pharmaceutically acceptable salt thereof, wherein:R1is an albumin binding ligand;R2is a ligand chelated to a therapeutic radionuclide or a diagnostic radionuclide, and n is an integer from 1 to 6.

39. The chelated complex of claim 38, wherein R1is:

40. The chelated complex of claim 38 or 39, wherein R1is:wherein R27is selected from the group consisting of halogen and C1-6 alkyl.

41. The chelated complex of claim 40, wherein R27is iodo.

42. The chelated complex of claim 40, wherein R27is fluoro.

43. The chelated complex of claim 40, wherein R27is methyl.

44. The chelated complex of any one of claims 38-43, wherein R2is a ligand chelated to a therapeutic radionuclide.

45. The chelated complex of any one of claims 38-43, wherein R2is a ligand chelated to a diagnostic radionuclide.

46. The chelated complex of any one of claims 38-45, wherein the ligand is:

47. The chelated complex of any one of claims 38-46, wherein the ligand is:

48. A chelated complex of a compound represented by a formula selected from the group consisting of:FAP6-19 or a pharmaceutically acceptable salt thereof, and a radionuclide selected from a therapeutic radionuclide and a diagnostic radionuclide.

49. The chelated complex of any one of claims 38-48, wherein X comprises a therapeutic radionuclide selected from the group consisting of18F,44Sc,47Sc,52Mn,55Co,64Cu,67Cu,66Ga,67Ga,68Ga,86Y,89Zr,90Y, "mTc,U1ln,114mIn,117mSn,124I,125I,131I,149Tb,153Sm,152Tb,155Tb,161Tb,177LU,186Re,188Re,212Pb,212Bi,213Bi,223Ra,224Ra,210At,211At,225Ab,225Ac, and227Th.

50. The chelated complex of any one of claims 38-49, wherein the therapeutic radionuclide is177LU.51 . The chelated complex of any one of claims 38-48, wherein the diagnostic radionuclide is selected from the group consisting of "mTc,i nIn,18F,68Ga,124I,12?I, and131I.

52. The chelated complex of any one of claims 38-48, wherein the diagnostic radionuclide is11’In.

53. A chelated complex selected from the group consisting of[177Lu]Lu-FAP6-16, [177Lu]Lu- FAP6-17, and [177Lu]Lu-FAP6-19.

54. A chelated complex selected from the group consisting of [i nIn]In-FAP6-16, [niIn]- FAP6-17, and [ulIn]In-FAP6-19.

55. A pharmaceutical composition comprising a conjugate or compound of any one of claims 1-54, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

56. A method of imaging cancer-associated fibroblasts (CAFs) in a subject, the method comprising (i) administering to the subject an effective amount of a composition comprising a conjugate or chelated complex of any one of claims 1-54 and a pharmaceutically acceptableexcipient and (ii) imaging the subject to identify the location of the CAFs in the patient, whereupon CAFs in the subject are imaged.

57. A method of treating a disease characterized by upregulation of FAPα comprising administrating to a subject an effective amount of a composition comprising a conjugate or chelated complex of any one of claims 1-54.

58. The method of claim 57, wherein the disease is a cancer.

59. A method of treating fibroblast activation protein alpha (FAPα)-expressing cancer in a subject, the method comprising administering to the subject an effective amount of a composition comprising a conjugate or chelated complex of any of claims 1-54 and a pharmaceutically acceptable excipient, whereupon FAPα-expressing cancer in the subject is treated.

60. The method of claim 59, wherein the cancer is characterized by upregulation of FAPα.

61. The method of claim 56-60, wherein the cancer is prostate cancer, renal cancer, breast cancer, thyroid cancer, gastric cancer, colorectal cancer, bladder cancer, pancreatic cancer, lung cancer, liver cancer, a brain tumor, melanoma, a neuroendocrine tumor, ovarian cancer, esophageal cancer, cholangiocellular carcinoma, head-neck cancer, carcinoma of unknown primary, medullary thyroid cancer, thymus cancer, small -intestine cancer, or a sarcoma.

62. The conjugate or chelated complex of any of claims 1-54 for use in a method of treating a disease mediated by CAFs in a subject.

63. The conjugate or chelated complex of claim 62, wherein the disease is a cancer and the cancer is characterized by upregulation of FAPα.

64. The conjugate or chelated complex of claim 63, wherein the cancer is prostate cancer, renal cancer, breast cancer, thyroid cancer, gastric cancer, colorectal cancer, bladder cancer, pancreatic cancer, lung cancer, liver cancer, a brain tumor, melanoma, a neuroendocrine tumor, ovarian cancer, esophageal cancer, cholangiocellular carcinoma, head-neck cancer, carcinoma of unknown primary, medullary thyroid cancer, thymus cancer, small-intestine cancer, or a sarcoma.

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