FAP-Targeted Radiopharmaceuticals and Imaging Agents and Related Uses

Abstract

The tumor stroma, which accounts for a large portion of the tumor mass, is an attractive target for the delivery of diagnostic and therapeutic compounds. Here, we focus specifically on a subpopulation of stromal cells known as cancer-associated fibroblasts, which are present in over 90% of epithelial cancers, including pancreatic, colon, and breast cancers. Cancer-associated fibroblasts are characterized by high expression of FAPs, which are undetectable in adult normal tissues but are associated with poor prognosis in cancer patients. The present invention provides small molecule radiopharmaceuticals and imaging agents based on FAP-specific inhibitors.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 993,874, filed March 24, 2020, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Tumor growth and spread are determined not only by cancer cells but also by non-malignant components of malignant lesions, which are encompassed by the term stroma. Stroma can represent over 90% of the mass in tumors with stromal desmoplastic reactions, such as breast, colon, and pancreatic cancers. In particular, a subpopulation of fibroblasts, called cancer-associated fibroblasts, is known to be involved in tumor growth, migration, and progression. Therefore, these cells are attractive targets for diagnostics and antitumor therapy.

[0003] A distinctive feature of cancer-associated fibroblasts is the expression of fibroblast activation protein (FAP), a type II membrane-bound glycoprotein belonging to the dipeptidyl peptidase 4 family. FAP possesses both dipeptidyl peptidase and endopeptidase activities. Endopeptidase activity distinguishes FAP from other members of the dipeptidyl peptidase 4 family. Substrates identified for endopeptidase activity to date include denatured type I collagen, α1-antitrypsin, and several neuropeptides. FAP plays a role in normal developmental processes and tissue modeling during embryogenesis. In normal adult tissues, it is expressed only sparingly or not at all. However, high expression occurs in wound healing, arthritis, atherosclerotic plaques, fibrosis, and in over 90% of epithelial cancers.

[0004] The presence of FAP in cancer-associated fibroblasts (CAFs) in many epithelial tumors, and the fact that overexpression is associated with poor prognosis in cancer patients, have led to the hypothesis that FAP activity is involved in cancer initiation, cancer cell migration, and cancer spread. Therefore, targeting this enzyme for imaging and endoradiotherapy may be considered a promising strategy for detecting and treating malignant tumors. Summary of the Invention [Means for solving the problem]

[0005] The tumor stroma, which accounts for a large portion of the tumor mass, is an attractive target for the delivery of diagnostic and therapeutic compounds. Here, we focus specifically on a subpopulation of stromal cells known as cancer-associated fibroblasts (CAFs), which are present in over 90% of epithelial cancers, including pancreatic, colon, and breast cancers. CAFs are characterized by high expression of FAPs, which are undetectable in adult normal tissues but are associated with poor prognosis in cancer patients.

[0006] The present invention provides small molecule radiopharmaceuticals and imaging agents based on FAP-specific inhibitors. In certain embodiments, the FAP-targeted agent has a structure represented by Formula I, or a pharmaceutically acceptable salt thereof: [ka] [In the formula, R represents a radioactive moiety, a chelator, a fluorescent moiety, a photoacoustic reporting molecule, a Raman-active reporting molecule, an imaging agent, a detectable nanoparticle, or an enzyme; R1 represents (C1-C6) alkyl; R2 is -B(-Y 1 )(-Y 2 ) or -CN; Y 1 and Y2 are independently -OH or together with the boron atom to which they are attached represent a group hydrolyzable to boronic acids or together with the boron atom to which they are attached form a 5- to 8-membered ring hydrolyzable to boronic acids; R3 represents H or (C1-C6) alkyl; R4 is absent or represents one, two, or three substituents each independently selected from the group consisting of (C1-C6) alkyl, -OH, -NH2, and halogen; X represents O or S; L represents a bond or a linker.

[0007] In certain preferred embodiments, the compounds of formula I contain one or more radioisotopes.

[0008] In certain preferred embodiments, the compounds of formula I contain one or more therapeutic radioisotopes.

[0009] In certain preferred embodiments, the compounds of formula I contain one or more diagnostic radioisotopes.

[0010] In certain preferred embodiments, R is a radioactive moiety.

[0011] In certain preferred embodiments, R is a chelator.

[0012] In further preferred embodiments, R is a chelator and the compound of Formula I comprises one or more radioisotopes. In certain aspects of such embodiments, the one or more radioisotopes can be therapeutic radioisotopes. In other certain aspects of such embodiments, the one or more radioisotopes can be diagnostic radioisotopes.

[0013] In further preferred embodiments, R is a chelator comprising one or more complexed radioactive isotopes. In certain aspects of such embodiments, the one or more radioactive isotopes can be therapeutic radioisotopes. In other specific aspects of such embodiments, the one or more radioactive isotopes can be diagnostic radioisotopes.

[0014] In certain preferred embodiments, R1 represents -CH3 or -CH2CH3, and even more preferably represents -CH3.

[0015] In certain preferred embodiments, R2 is -B(-Y 1 )(-Y 2 ), and even more preferably represents —B(OH) 2 .

[0016] In certain preferred embodiments, R3 represents H.

[0017] In certain preferred embodiments, R4 is absent.

[0018] In certain preferred embodiments, X represents O.

[0019] In certain preferred embodiments, the compound is represented by the following Formula II or Formula III, or a pharmaceutically acceptable salt thereof: [ka] [ka] wherein R and L are as defined above.

[0020] In certain preferred embodiments, the compounds of formula II or III contain one or more radioisotopes.

[0021] In certain preferred embodiments, the compounds of Formula I or III contain one or more therapeutic radioisotopes.

[0022] In certain preferred embodiments, the compounds of Formula II or III contain one or more diagnostic radioisotopes.

[0023] In certain preferred embodiments of compounds of Formula II or III, R is a radioactive moiety.

[0024] In certain preferred embodiments of compounds of formula II or III, R is a chelator.

[0025] In further preferred embodiments of the compound of Formula II or III, R is a chelator and the compound of Formula I comprises a radioisotope. In certain aspects of such embodiments, one or more radioisotopes can be therapeutic radioisotopes. In other specific aspects of such embodiments, one or more radioisotopes can be diagnostic radioisotopes.

[0026] In a further preferred embodiment, R is a chelating agent containing one or more complexed radioisotopes. In certain aspects of such embodiments, one or more radioisotopes can be therapeutic radioisotopes. In other specific aspects of such embodiments, one or more radioisotopes can be diagnostic radioisotopes.

[0027] In certain embodiments, the FAP targeting agent comprises two or more FAP inhibitor moieties covalently attached to a radiopharmaceutical or imaging agent, such as having the structure represented by Formula IV: [ka] [In the formula, R, R1, R2, R3, R4, X and L are as defined above; n represents an integer from 2 to 6.

[0028] In certain preferred embodiments, the compound of formula IV contains one or more radioisotopes.

[0029] In certain preferred embodiments, the compound of formula IV contains one or more therapeutic radioisotopes.

[0030] In certain preferred embodiments, the compound of formula IV contains one or more diagnostic radioisotopes.

[0031] In certain preferred embodiments of compounds of formula IV, R is a radioactive moiety.

[0032] In certain preferred embodiments of compounds of formula IV, R is a chelator.

[0033] In a further preferred embodiment of the compound of Formula IV, R is a chelator and the compound of Formula IV comprises a radioisotope. In certain aspects of such embodiments, one or more radionuclides can be therapeutic radioisotopes. In other specific aspects of such embodiments, one or more radioisotopes can be diagnostic radioisotopes.

[0034] In further preferred embodiments, R is a chelator containing one or more complexed radionuclides. In certain aspects of such embodiments, the one or more radionuclides can be therapeutic radionuclides. In other particular aspects of such embodiments, the one or more radionuclides can be diagnostic radionuclides.

[0035] In certain embodiments, the FAP targeting agent comprises a moiety that modifies the pharmacokinetics and / or biodistribution of the molecule, such as the serum half-life of the molecule and / or tumor distribution of the molecule. Such PK / BD-modified FAP targeting agents can have a structure represented by Formula V, or a pharmaceutically acceptable salt thereof: [ka] [In the formula, R, R1, R2, R3, R4, X and L are as defined above; R5 represents a moiety that modifies the pharmacokinetics and / or biodistribution of the molecule; n represents an integer from 1 to 6.

[0036] In certain preferred embodiments, the compound of formula V contains one or more radioisotopes.

[0037] In certain preferred embodiments, the compound of formula V contains one or more therapeutic radioisotopes.

[0038] In certain preferred embodiments, the compound of formula V contains one or more diagnostic radioisotopes.

[0039] In certain preferred embodiments of compounds of formula V, R is a radioactive moiety.

[0040] In certain preferred embodiments of compounds of formula V, R is a chelator.

[0041] In further preferred embodiments of the compound of Formula V, R is a chelator and the compound of Formula V comprises a radioisotope. In certain aspects of such embodiments, the one or more radioisotopes can be therapeutic radioisotopes. In other certain aspects of such embodiments, the one or more radioisotopes can be diagnostic radioisotopes.

[0042] In further preferred embodiments, R is a chelator containing one or more complexed radioisotopes. In certain aspects of such embodiments, one or more radionuclides can be therapeutic radioisotopes. In other specific aspects of such embodiments, one or more radionuclides can be diagnostic radioisotopes.

[0043] The present invention also provides pharmaceutical compositions comprising at least one compound of any of Formulas I-V and, optionally, a pharmaceutically acceptable carrier and / or excipient. In certain embodiments, the pharmaceutical composition is intended for use in the diagnosis or treatment of a disease characterized by overexpression of fibroblast activation protein (FAP) in an animal, preferably a human subject.

[0044] Yet another aspect of the present invention provides kits comprising or consisting of at least one compound of any of Formulas IV and instructions for diagnosing or treating disease.

[0045] Yet another aspect of the present invention provides a method for diagnosing, imaging, or reducing tissues that overexpress FAP in an animal (preferably a human patient), comprising administering to the animal at least one compound of any of Formulas I-V.

[0046] Also provided are methods of treating a subject suffering from a tumor or cancer, comprising administering to a subject in need thereof an effective amount of one or more compounds disclosed herein, including one or more compounds of any of Formulas I through V. Subjects to be treated can include human patients diagnosed with cancer, such as a tumor (e.g., a solid tumor), including subjects diagnosed and selected for treatment of prostate cancer. [Brief explanation of the drawings]

[0047] [Figure 1] 1 shows the tumor growth curve for 177Lu-6522. [Figure 2] 1 shows the survival curve for 177Lu-6522. [Figure 3] 6 shows the accumulation and retention of 68Ga-6522 over time after administration. DETAILED DESCRIPTION OF THE INVENTION

[0048] Tumor masses are composed not only of cancer cells but also of vasculature, inflammatory cells, fibroblasts, collagen, and other components that make up the tumor stroma, which can account for up to 90% of the mass in highly desmoplastic cancers. Cancer cells induce fibroblast activation via TGFβ. CAFs have a supportive function for cancer growth and invasion. They contribute to extracellular matrix remodeling (collagen degradation), promote invasiveness and angiogenesis, and can induce epithelial-mesenchymal transition through the secretion of growth factors and cytokines. CAFs also participate in immunological interactions between tumors and the host.

[0049] FAP-positive CAFs are found in over 90% of epithelial cancers and therefore represent a potential pan-cancer target. Targeting FAP to deplete stromal CAFs may disrupt cancer-supporting functions and inhibit cancer growth. Furthermore, disrupting the stromal barrier (or interstitial barrier) may enhance the efficacy of other pharmacological, immunological, radiation, or cell-based systemic therapies.

[0050] Targeting CAFs with FAP radiopharmaceuticals is thought to have multiple antitumor effects, primarily relying on the induction of DNA damage in tumor cells by ionizing radiation locally emitted from adjacent CAFs targeted for treatment. FAP-targeted radiotherapy can deliver ionizing radiation directly to CAFs and also to cancer cells via the crossfire effect. Combining α- and β-emitters can improve these dual antitumor effects through short-range α-emission to CAFs and medium- to long-range β-emission to cancer cells.

[0051] definition The following definitions provide definitions of some of the terms frequently used herein. These terms have their respective defined and preferred meanings at each instance of their use in the remainder of this specification. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Below, definitions are provided for the terms alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, alkenyl, and alkynyl. These terms have their respective defined and preferred meanings at each instance of their use in the remainder of this specification.

[0052] As used herein, the term "SPECT" is an abbreviation for Single Photon Emission Computed Tomography.

[0053] As used herein, the term "PET" is an abbreviation for positron emission tomography.

[0054] As used herein, the term "CT" is an abbreviation for computed tomography.

[0055] As used herein, the term "MRI" is an abbreviation for magnetic resonance imaging.

[0056] As used herein, the term "SIRT" is an abbreviation for selective internal radiation therapy.

[0057] As used herein, the term "EDTA" is an abbreviation for ethylenediaminetetraacetic acid.

[0058] As used herein, the term "DOTA" is an abbreviation for 1,4,7,10-tetraazacyclododecane-1,4,7,10-N,N',N",N'''-tetraacetic acid.

[0059] As used herein, the term "DOTAGA" is an abbreviation for 1,4,7,10-tetraazacyclododecane, 1-(glutaric acid)-4,7,10-triacetic acid.

[0060] As used herein, the term "DTPA" is an abbreviation for diethylenetriaminepentaacetic acid.

[0061] As used herein, the term metal "chelating agent" or "chelator" refers to a multidentate ligand that forms two or more separate coordinate bonds with a single central atom (especially a radioisotope).

[0062] The term "therapeutically effective amount" as used herein includes within its meaning a non-toxic but sufficient amount of the compound or composition used in the present invention to provide the desired therapeutic effect.The exact amount required varies from subject to subject, depending on factors such as the species being treated, the age, weight and general condition of the subject, coexisting diseases, the severity of the condition being treated, the specific drug being administered and the method of administration.Therefore, for any given case, the appropriate "effective amount" can be determined by those skilled in the art using only routine methods.

[0063] The term "alkyl" refers to a saturated straight or branched carbon chain. Preferably, the chain contains 1 to 10 carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, such as methyl, ethyl, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, pentyl, or octyl. Alkyl groups may be substituted.

[0064] The term "heteroalkyl" refers to a saturated straight or branched carbon chain. Preferably, the chain contains 1 to 9 carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, pentyl, octyl, etc., interrupted one or more times, e.g., 1, 2, 3, 4, 5 times, by the same or different heteroatoms. Preferably, heteroatoms are selected from O, S, and N, e.g., -O-CH3, -S-CH3, -CH2-O-CH2-CH3, -CH2-S-CH3, -CH2-S-CH2-CH3, -CH2-, CH2-O-CH3, -CH2-CH2-O-CH2-CH3, -CH2-CH2-S-CH3, -CH2-CH2-S-CH3, -CH2-CH2-CH3, etc. Heteroalkyl groups may be substituted.

[0065] The terms "cycloalkyl" and "heterocycloalkyl," alone or in combination with other terms, represent, unless otherwise stated, cyclic versions of "alkyl" and "heteroalkyl," respectively, preferably having 3, 4, 5, 6, 7, 8, 9, or 10 atoms forming the ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like. The terms "cycloalkyl" and "heterocycloalkyl" are also meant to include bicyclic, tricyclic, and polycyclic versions thereof. The term "heterocycloalkyl" preferably refers to a 5-membered saturated ring (at least one member is an N, O, or S atom and may contain one additional O or one additional N atom); a 6-membered saturated ring (at least one member is an N, O, or S atom and may contain one additional O, one additional N, or two additional N atoms); or a 9- or 10-membered saturated bicyclic ring (at least one member is an N, O, or S atom and may contain one additional N atom, two additional N, or three additional N atoms). "Cycloalkyl" and "heterocycloalkyl" groups can be optionally substituted. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, spiro[3,3]heptyl, spiro[3,4]octyl, spiro[4,3]octyl, spiro[3,5]nonyl, spiro[5,3]nonyl, spiro[3,6]decyl, spiro[6,3]decyl, spiro[4,5]decyl, spiro[5,4]decyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, adamantyl, and the like.Examples of heterocycloalkyl include 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, 1,8 diazo-spiro-[4,5]decyl, 1,7 diazo-spiro-[4,5]decyl, 1,6 diazo-spiro-[4,5]decyl, 2,8 diazo-spiro[4,5]decyl, 2,7 diazo-spiro[4,5]decyl, 2,6 diazo-spiro[4,5]decyl, 1,8 diazo-spiro-[5,4]decyl, 1,7 diazo-spirotetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like.

[0066] The term "aryl" preferably refers to an aromatic monocyclic ring containing 6 carbon atoms, an aromatic bicyclic ring system containing 10 carbon atoms, or an aromatic tricyclic ring system containing 14 carbon atoms. Examples include phenyl, naphthyl, or anthracenyl. Aryl groups may be substituted.

[0067] The term "aralkyl" refers to an alkyl moiety substituted by an aryl, where alkyl and aryl have the meanings outlined above. An example is the benzyl radical. Preferably, in this context, the alkyl chain contains 1 to 8 carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms, such as methyl, ethylmethyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butenyl, tert-butyl, pentyl, hexyl, pentyl, and octyl. The aralkyl group may be substituted in the alkyl and / or aryl portions of the group.

[0068] The term "heteroaryl" preferably refers to a 5- or 6-membered aromatic monocyclic ring in which at least one of the carbon atoms is replaced by 1, 2, 3, or 4 (in the case of a 5-membered ring) or 1, 2, 3, 4, or 5 (in the case of a 6-membered ring) heteroatoms, which may be the same or different, preferably selected from O, N, and S; an aromatic bicyclic ring system in which 1, 2, 3, 4, 5, or 6 of the 8, 9, 10, 11, or 12 carbon atoms are replaced by the same or different heteroatoms, preferably selected from O, N, and S; or an aromatic tricyclic ring system in which 1, 2, 3, 4, 5, or 6 of the 13, 14, 15, or 16 carbon atoms are replaced by the same or different heteroatoms, preferably selected from O, N, and S. Examples include oxazolyl, isoxazolyl, 1,2,5-oxadiazolyl, 1,2,3-oxadiazolyl, pyrrolyl, imidazolyl, pyrazolyl, 1,2,3-triazolyl, thiazolyl, isothiazolyl, 1,2,3-thiadiazolyl, 1,2,5-thiadiazolyl, pyridinyl, pyrimidinyl, pyrazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl, 1,3,5-triazinyl, 1-benzofuranyl, 2-benzofuranyl, indoyl, isoindoi. isoindoyl, benzothiophenyl, 2-benzothiophenyl, 1H-indazolyl, benzimidazolyl, benzoxazolyl, indoxazinyl, 2,1-benzoxazolyl, benzothiazolyl, 1,2-benzisothiazolyl, 2,1-benzisothiazolyl, benzotriazolyl, quinolinyl, isoquinolinyl, quinoxalinyl, quinazolinyl, quinolinyl, 1,2,3-benzotriazinyl or 1,2,4-benzotriazinyl.

[0069] The term "heteroaralkyl" refers to an alkyl moiety substituted by a heteroaryl, where alkyl and heteroaryl have the meanings outlined above. Examples include 2-alkylpyridinyl, 3-alkylpyridinyl, or 2-methylpyridinyl. Preferably, in this context, the alkyl chain contains 1 to 8 carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms, such as methyl, ethylmethyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butenyl, tert-butyl, pentyl, hexyl, pentyl, and octyl.

[0070] Heteroaralkyl groups may be optionally substituted on the alkyl and / or heteroaryl portions of the group.

[0071] The terms "alkenyl" and "cycloalkenyl" refer to an olefinically unsaturated carbon atom containing chain or ring with one or more double bonds. Examples include propenyl and cyclohexenyl. Preferably, the alkenyl chain contains 2 to 8 carbon atoms, i.e., 2, 3, 4, 5, 6, 7, or 8 carbon atoms, such as ethenyl, 1-propenyl, 2-propenyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl, isobutenyl, sec-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, hexenyl, pentenyl, and octenyl. Preferably, the cycloalkenyl ring contains 3 to 8, i.e., 3, 4, 5, 6, 7 or 8 carbon atoms, such as 1-cyclopropenyl, 2-cyclopropenyl, 1-cyclobutenyl, 2-cyclobutenyl, 1-cyclopentenyl, 2-cyclopentenyl, 3-cyclopentenyl, cyclohexenyl, cyclopentenyl, cyclooctenyl.

[0072] The term "alkynyl" refers to an unsaturated carbon atom containing chain or ring with one or more triple bonds. An example is the propargyl radical. Preferably, the alkynyl chain contains 2 to 8 carbon atoms, i.e., 2, 3, 4, 5, 6, 7, or 8 carbon atoms, such as ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, hexynyl, pentynyl, and octynyl.

[0073] In one embodiment, carbon or hydrogen atoms in the alkyl, heteroalkyl, cycloalkyl, aryl, aralkyl, alkenyl, cycloalkenyl, and alkynyl radicals may be substituted, independently of one another, with one or more elements selected from the group consisting of O, S, and N, or with a group containing one or more elements selected from the group consisting of O, S, and N.

[0074] Embodiments include alkoxy, cycloalkoxy, aryloxy, aralkoxy, alkenyloxy, cycloalkenyloxy, alkynyloxy, alkylthio, cycloalkylthio, arylthio, aralkylthio, alkenylthio, cycloalkenylthio, alkynylthio, alkylamino, cycloalkylamino, arylamino, aralkylamino, alkenylamino, cycloalkenylamino, alkynylamino radicals.

[0075] Other embodiments include hydroxyalkyl, hydroxycycloalkyl, hydroxyaryl, hydroxyaralkyl, hydroxyalkenyl, hydroxycycloalkenyl, hydroxyalinyl, mercaptoalkyl, mercaptocycloalkyl, mercaptoaryl, mercaptoaralkyl, mercaptoalkenyl, mercaptocycloalkenyl, mercaptoalkynyl, aminoalkyl, aminocycloalkyl, aminoaryl, aminoaralkyl, aminoalkenyl, aminocycloalkenyl, aminoalkynyl radicals.

[0076] In another embodiment, the hydrogen atoms in the alkyl, heteroalkyl, cycloalkyl, aryl, aralkyl, alkenyl, cycloalkenyl, and alkynyl radicals may be replaced, independently of one another, by one or more halogen atoms. One radical is the trifluoromethyl radical.

[0077] Where two or more radicals or two or more residues may be selected independently of each other, the term "independently" means that the radicals or residues may be the same or different.

[0078] As used herein, phrases defining a length range limit, such as "1 to 6," refer to any integer between 1 and 6, i.e., 1, 2, 3, 4, 5, and 6. In other words, any range defined by two explicitly stated integers is meant to include and disclose any integers defining said limit and any integers falling within said range.

[0079] The term "halo" as used herein refers to a halogen radical selected from the group consisting of F, Br, I and Cl. Preferably, the halogen is F.

[0080] The term "linker" as used herein refers to any chemically suitable linker. Preferably, the linker is not cleaved or is cleaved only slowly under physiological conditions. Therefore, preferably, the linker does not contain a recognition sequence for proteases or other degradative enzymes. Since the compounds of the present invention are preferably administered systemically, allowing broad access to all compartments of the body, and subsequently concentrating the compounds of the present invention wherever tumors are located in the body, it is preferable that the linker is selected so that it is not cleaved or is cleaved only slowly in the blood. Cleavage is considered slow if less than 50% of the linker is cleaved 2 hours after administration of the compound to a human patient. Suitable linkers include, for example, optionally substituted alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, alkenyl, heteroalkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, sulfonyl, amines, ethers, thioethers, phosphines, phosphoramidates, carboxamides, esters, imidoesters, amidines, thioesters, sulfonamides, 3-thiopyrrolidine-2,5-dione, carbamates, ureas, guanidines, thioureas, disulfides, oximes, hydrazines, hydrazides, hydrazones, diaza bonds, triazoles, triazolines, tetrazines, platinum complexes, and amino acids, or combinations thereof. Preferably, the linker includes or consists of 1,4-piperazine, 1,3-propane, and phenol ether, or combinations thereof.

[0081] The term "optionally substituted" refers to a group in which one, two, three or more hydrogen atoms may be replaced, independently of one another, by respective substituents.

[0082] As used herein, the term "amino acid" refers to any organic acid containing one or more amino substituents, for example, an α-, β-, or γ-amino derivative of an aliphatic carboxylic acid.

[0083] The term "conventional amino acids" refers to the 20 naturally occurring amino acids and includes all of their stereoisomers, i.e., D, L-, D- and L-amino acids.

[0084] The term "N-containing aromatic or non-aromatic monocyclic or bicyclic heterocycle" as used herein refers to a cyclic saturated or unsaturated hydrocarbon compound containing at least one nitrogen atom as a component of the cyclic chain.

[0085] The term "radioactive moiety" as used herein refers to a molecular assembly carrying a radioactive nuclide. The nuclide is attached by either a covalent or coordinate bond that remains stable under physiological conditions. Examples of radioactive moieties include [I]-3-iodobenzoic acid or GaDOTA.

[0086] As used herein, a "fluorescent isotope" emits electromagnetic radiation after excitation by electromagnetic radiation of a shorter wavelength.

[0087] As used herein, a "radioisotope" is a radioactive isotope of an element (subjected to the term "radionuclide") that emits α-, β-, or γ-radiation. Exemplary radioisotopes are discussed below and include, for example: 18 F, 43 K. 47 Sc, 51 Cr, 57 Co, 58 Co, 59 Fe, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 71 Ge, 72 As, 72 Se, 75 Br, 76 Br, 77 As, 77Br、 81 Rb、 88 THE, 90 THE, 97 Ru、 99m Tc、 100 Pd、 101m Rh、 103 Pb、 105 Rh、 109 Pd、 111 Ag、 111 See, 113 See, 119 Sb 121 Sn、 123 I、 124 I、 125 I、 127 Cs、 128 They、 129 Cs、 131 Cs、 131 I、 139 The、 140 The、 142 Pr、 143 Pr、 149 Pm、 151 Eu、 153 Eu、 153 Sm、 159 Gr、 161 Tb、 165 Of, 166 Ho、 169 Eu、 175 Yb、 177 Lu、 186 Re, 188 Re, 189 Re, 191 Horse、 193 Pt、 194 Is、 197 Hg、 198 I、 199 Ag、 199 I、 201 Tl、 203 Pb、 211 Available、 212 Wind、 212 Pb、 213 Wind、 225 Ac、 227 Th、Sc-44, Sc-47, As-77, In-110, Tb-152, Tb-149, Y-86, Sr-83, Sr-89, Zr-89, and 1-Dy

[0088] The term "radiopharmaceutical" is used in the context of this invention to refer to a biologically active compound modified with a radioisotope. Intercalating agents in particular can be used to deliver radioactivity directly into proximity with DNA (e.g., Hoechst-33258). 131 I-supported derivatives).

[0089] The terms "chelating agent" and "chelate" are used interchangeably in the context of the present invention and refer to molecules, often organic molecules, and often Lewis bases, that have two or more unshared electron pairs available to donate to a metal ion. The metal ion typically coordinates to the chelating agent with two or more electron pairs. The terms "bidentate chelating agent," "tridentate chelating agent," and "tetradentate chelating agent" refer to chelating agents that have two, three, and four electron pairs readily available to simultaneously donate to the metal ion coordinated by the chelating agent, respectively. Typically, the electron pairs of a chelating agent form coordinate bonds with a single metal ion, but in certain instances, a chelating agent may form coordinate bonds with multiple metal ions, allowing for a variety of binding modes.

[0090] The term "fluorescent dye" (also herein "fluorescent moiety," "fluorophore," or "fluorochrome") is used in the context of the present invention to refer to a compound that, after excitation with electromagnetic radiation, emits, for example, visible or infrared light of a shorter, appropriate wavelength. It will be understood by those skilled in the art that each fluorescent dye has a predetermined excitation wavelength. All fluorescent moieties are encompassed by this term. The specific examples of fluorescent moieties described herein are exemplary and are not meant to limit the fluorescent moieties for use with the targeting molecules disclosed herein.

[0091] The term "contrast agent" is used in the context of the present invention to refer to a compound that enhances the contrast of structures or fluids in medical imaging. Enhancement is achieved by absorbing electromagnetic radiation or by altering the electromagnetic field.

[0092] As used herein, the term "paramagnetism" refers to paramagnetism induced by unpaired electrons in a medium. Paramagnetic materials induce a magnetic field when an external magnetic field is applied. Unlike diamagnetism, the induced magnetic field has the same direction as the external magnetic field, and unlike ferromagnetism, the magnetic field is not maintained in the absence of an external magnetic field.

[0093] The term "nanoparticles" as used herein preferably refers to spherical particles with diameters ranging from 1 to 100 nanometers in size. Depending on their composition, nanoparticles can have appreciable magnetic, optical, or physicochemical properties. Furthermore, surface modification can be achieved for many types of nanoparticles. The term "pharmaceutically acceptable salts" refers to salts of the compounds of the present invention. Suitable pharmaceutically acceptable salts of the compounds of the present invention include, for example, acid addition salts that can be formed by mixing a solution of choline or a derivative thereof with a solution of a pharmaceutically acceptable acid, such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid, or phosphoric acid. Furthermore, when the compounds of the present invention contain an acidic moiety, suitable pharmaceutically acceptable salts thereof include alkali metal salts (e.g., sodium or potassium salts); alkaline earth metal salts (e.g., calcium or magnesium salts); and salts formed with suitable organic ligands (e.g., ammonium, quaternary ammonium, and amine cations formed with counteranions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyl sulfonates, and aryl sulfonates).

[0094] Illustrative examples of pharmaceutically acceptable salts include, but are not limited to, acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium edetate, camphorate, camphorsulfonate, camsylate, carbonate, chloride, citrate, clavulanate, cyclopentanepropionate, digluconate, dihydrochloride, dodecyl sulfate, edetate, Edisylate, estolate, esylate, ethanesulfonate, formate, fumarate, gluceptate, glucoheptonate, gluconate, glutamate, glycerophosphate, glycolylarsanilate, hemisulfate, heptanoate, hexanoate, hexylresorcinate e), hydrabamine, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonic acid, hydroxynaphthoate, iodide, isothiocyanate, lactate, lactobionate, laurate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methanesulfonate, methyl sulfate, mucate, 2-naphthalenesulfonate, napsylate, nicotinate, nitrate, N-methylglucamine ammonium salt, oleate, Oxalate, pamoate (embonate), palmitate, pantothenate, pectinate, persulfate, 3-phenylpropionate, phosphate / diphosphate, picrate, pivalate, polygalacturonate, propionate, salicylate, stearate, sulfate, acetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide, undecanoate, valerate, etc. (e.g., Berge, SM(See, e.g., Beck, et al., "Pharmaceutical Salts," Journal of Pharmaceutical Science, 1977, 66, 1-19.) Certain specific compounds of the present invention contain both basic and acidic functional groups that allow the compounds to be converted into either base or acid addition salts.

[0095] The neutral forms of the compounds can be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but the salts are otherwise equivalent to the parent form of the compound for purposes of this invention.

[0096] In addition to salt forms, the present invention provides compounds in prodrug form. Prodrugs of the compounds described herein are compounds that readily undergo chemical changes under physiological conditions to provide the compounds of Formula (I). Prodrugs are active or inactive compounds that are chemically modified into compounds of the present invention through in vivo physiological action, such as hydrolysis or metabolism, after administration of the prodrug to a patient. Furthermore, prodrugs can be converted to compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to compounds of the present invention when placed in a transdermal patch reservoir with a suitable enzyme. The properties and techniques involved in making and using prodrugs are well known to those skilled in the art. For a general discussion of prodrugs, including esters, see Svensson and Tunek, Drug Metabolism Reviews 16.5 (1988) and Bundgaard, Design of Prodrugs, Elsevier (1985).

[0097] Examples of masked carboxylate anions include various esters, such as alkyl (e.g., methyl, ethyl), cycloalkyl (e.g., cyclohexyl), aralkyl (e.g., benzyl, p-methoxybenzyl), and alkylcarbonyloxyalkyl (e.g., pivaloyloxymethyl). Amines have been masked as arylcarbonyloxymethyl-substituted derivatives, which are cleaved in vivo by esterases to release the free drug and formaldehyde (Bungaard J. Med. Chem. 2503 (1989)). Drugs containing an acidic NH group, such as imidazole, imide, and indole, have also been masked with N-acyloxymethyl groups (Bundgaard Design of Prodrugs, Elsevier (1985)).

[0098] Hydroxyl groups are masked as esters and ethers. EP 00039051 (Sloan and Little, April 11, 1981) discloses Mannich-base hydroxamic acid prodrugs, their preparation and use.

[0099] Certain compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to the unsolvated forms and are intended to be encompassed within the scope of the present invention.

[0100] Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.

[0101] Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, geometric isomers, and individual isomers are all intended to be encompassed within the scope of the present invention.

[0102] The compounds of the present invention may also contain unnatural abundance atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may contain isotopes of, for example, tritium ( 3 H), iodine-125( 125 I) or carbon-14 ( 14 C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.

[0103] The term "pharmaceutical composition" as used in this application refers to a substance and / or combination of substances used to identify, prevent, or treat a tissue condition or disease. A pharmaceutical composition is formulated to be suitable for administration to a patient to prevent and / or treat a disease. Furthermore, a pharmaceutical composition refers to a combination of an active agent and an inert or active carrier, making the composition suitable for therapeutic use. Depending on its chemical and physical properties, a pharmaceutical composition can be formulated for oral, parenteral, topical, inhalation, rectal, sublingual, transdermal, subcutaneous, or vaginal administration. Pharmaceutical compositions include solids, semisolids, liquids, and transdermal therapeutic systems (TTS). Solid compositions are selected from the group consisting of tablets, coated tablets, powders, granules, pellets, capsules, effervescent tablets, or transdermal therapeutic systems. Liquid compositions are also included, selected from the group consisting of solutions, syrups, infusions, extracts, solutions for intravenous application, solutions for infusion, or solutions of the carrier systems of the present invention. Semisolid compositions that can be used in the context of the present invention include emulsions, suspensions, creams, lotions, gels, globules, buccal tablets, and suppositories.

[0104] "Pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government or listed in the United States Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0105] The term "carrier," as used herein, refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as saline solutions and oils (including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like). Physiological saline is a preferred carrier when the pharmaceutical composition is administered intravenously. Physiological saline and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. If desired, the composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin.

[0106] As used herein, the term "fibroblast activation protein (FAP)" is also known as "seprase." Both terms can be used interchangeably herein. Fibroblast activation protein is a homodimeric integral protein with a dipeptidyl peptidase IV (DPPIV)-like fold, characterized by an α / β-hydrolase domain and an eight-bladed β-propeller domain.

[0107] "Medical imaging" refers to any technique used to visualize internal regions of the human or animal body for the purposes of diagnosis, research, or therapeutic treatment. For example, FAP targeting agents can be detected (and quantified) by radioscintigraphy, magnetic resonance imaging (MRI), computed tomography (CT scan), nuclear imaging, positron emission tomography (PET) with metallurgical tomography contrast agents, optical imaging (e.g., fluorescence imaging, including near-infrared fluorescence (NIRF) imaging), bioluminescence imaging, or a combination thereof. The functional moiety is optionally a contrast agent for X-ray imaging. Agents useful for enhancing such techniques are materials that enable visualization of specific loci, organs, or disease sites within the body and / or provide some improvement in the quality of images produced by image processing techniques, providing improved or easier interpretation of those images. Such agents are referred to herein as contrast agents, and their use facilitates differentiation of different parts of an image by increasing the "contrast" between different regions of the image. Thus, the term "imaging agent" encompasses agents used to enhance the quality of images that could be produced in the absence of such agents (e.g., as in MRI), as well as agents that are a prerequisite for the production of an image (e.g., as in nuclear imaging).

[0108] compound The compounds in the following different aspects of the present invention are defined in more detail. Each aspect so defined can be combined with any other one or more aspects, unless expressly indicated to the contrary. In particular, any feature indicated as preferred or advantageous can be combined with any other feature(s) indicated as preferred or advantageous.

[0109] The present invention provides small molecule radiopharmaceuticals and imaging agents based on FAP-specific inhibitors.

[0110] In certain embodiments, the FAP targeting agent has a structure represented by Formula I, or a pharmaceutically acceptable salt thereof: [ka] [In the formula, R represents a radioactive moiety, a chelator, a fluorescent moiety, a photoacoustic reporter molecule, a Raman-active reporter molecule, an imaging agent, a detectable nanoparticle, or an enzyme; R1 represents (C1-C6) alkyl; R2 is -B(-Y 1 )(-Y 2 ) or -CN; Y 1 and Y 2 are independently -OH or together with the boron atom to which they are attached represent a group hydrolyzable to boronic acids or together with the boron atom to which they are attached form a 5- to 8-membered ring hydrolyzable to boronic acids; R3 represents H or (C1-C6) alkyl; R4 is absent or represents one, two, or three substituents each independently selected from the group consisting of (C1-C6) alkyl, -OH, -NH2, and halogen; X represents O or S; L represents a bond or a linker.

[0111] In certain preferred embodiments, R1 represents -CH3 or -CH2CH3, and even more preferably represents -CH3.

[0112] In certain preferred embodiments, R2 is -B(-Y 1 )(-Y 2 ), and even more preferably represents —B(OH) 2 .

[0113] In certain preferred embodiments, R3 represents H.

[0114] In certain preferred embodiments, R4 is absent.

[0115] In certain preferred embodiments, X represents O.

[0116] In certain preferred embodiments, R2 represents -CH3, R2 represents -B(OH)2, R3 represents H, and R4 is absent.

[0117] In certain preferred embodiments, the compound has the following formula II or III: [ka] [ka]

[0118] In certain embodiments, R is a radioactive moiety, and the radioactive moiety comprises a fluorescent isotope, a radioisotope, a radioactive drug, or a combination thereof. Preferably, the radioactive moiety comprises a radioisotope selected from the group consisting of an α-emitting isotope, a β-emitting isotope, a γ-emitting isotope, an Auger electron-emitting isotope, an X-ray-emitting isotope, and a fluorescent-emitting isotope.

[0119] The radioisotopes of the present invention can be selected to allow for imaging and / or radiotherapy.

[0120] The radioisotopes of the present invention may include radioactive metal or metalloid isotopes. Preferably, the radioisotopes are water-soluble metal cations.

[0121] Exemplary radioisotopes include: 18 F, 43 K. 47 Sc, 51 Cr, 57 Co, 58 Co, 59 Fe, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 71 Ge, 72 As, 72 Se, 75 Br,76 Br、 77 As、 77 Br、 81 Rb, 88 Y、 90 Y、 97 Yes, 99m Tc, 100 Pd, 101m Rh, 103 Pb, 105 Rh, 109 Pd, 111 At, 111 In, 113 In, 119 Sb 121 Sn, 123 I, 124 I, 125 I, 127 Cs, 128 It was 129 Cs, 131 Cs, 131 I, 139 Day, 140 Day, 142 Mr. 143 Mr. 149 Pm, 151 Yes, 153 Yes, 153 Sm, 159 Gr、 161 Tb, 165 This, 166 Yes, 169 Yes, 175 Yb, 177 Hello, 186 Yes, 188 Yes, 189 Yes, 191 If, 193 Pt. 194 Yes, 197 Hg, 198 Oh, 199 At, 199 Oh, 201 Tl, 203 Pb, 211 At, 212 Be, 212 Pb, 213 Be, 225 And, 227These include Th, Sc-44, Sc-47, As-77, In-110, Tb-152, Tb-149, Y-86, Sr-83, Sr-89, Zr-89, and Dy-166.

[0122] Diagnostic radioisotopes can be suitably used for diagnostic imaging, in particular: 18 F, 123 I, 124 I, 125 I, 99m Therapeutic radioisotopes may be suitably used in a variety of therapies, including treating cancer, for example, among others: 225 Ac, 68 Ga, 177 Lu, 64 Cu, 67 Cu, 131 I, 32 P, 90 Sr, 90 Y, 186 Re, 188 Re, and 189 Re may be included.

[0123] In certain embodiments, the radioisotope is intended to enable imaging, such as by SPECT imaging and / or PET imaging. Single-photon emission computed tomography (SPECT) is a nuclear medicine tomography technique that uses gamma rays and can provide true 3D information. The information is often presented as cross-sectional slices across the patient. The gamma-ray emission of the isotope allows for the location of the radiolabeled substance's accumulation within the patient's body to be seen. Such true 3D representations can be useful in tumor imaging. Positron emission tomography (PET) is a nuclear medicine imaging technique that produces 3D images and has greater sensitivity than traditional SPECT imaging. The system detects pairs of gamma rays indirectly emitted by a positron-emitting radionuclide (tracer) introduced into the body. A 3D image of the tracer concentration within the body is then constructed by computer analysis, often achieved with the aid of a computed tomography (CT) X-ray scan performed on the patient during the same session on the same machine. Positron-emitting isotopes can also be used in conjunction with CT to provide 3D imaging of the anatomical distribution of labeled medical devices.

[0124] In certain embodiments, the radioisotope is 44 Sc, 47 Sc, 51 Cr, 51 Mn, 52 Mn, 57 Co, 58 Co, 59 Fe, 64 Cu, 67 Cu, 86 Y, 88 Y, 89 Zr, 90 Y, 97 Ru,, 99m Tc, 100 Pd, 101m Rh, 103 Pd 105 Rh, 109 Pd, 111 Ag, 177 Lu, 186 Re, 188 Re, 189 Re,191 Os, 193 Pt, 194 Ir, 197 Hg, 198 Au, 199 Ag and 199 Au, 225 Ac, 226 Th or 227 In certain embodiments, the radioisotope is preferably a transition metal such as Th. 44 Sc, 47 Sc, 64 Cu, 89 Zr, 90 Y, 99m Tc, 177 Lu, 186 Re, 188 Re, 225 Ac, 226 Th or 227 Th.

[0125] In certain embodiments, the radioisotope is 43 K. 81 Rb, 83 Sr, 89 Sr, 127 Cs, 128 Ba, 129 Cs and 131 s-block metals such as Cs.

[0126] In certain embodiments, the radioisotope is 67 Ga, 68 Ga, 71 Ge, 72 As, 72 Se, 77 As, 110 In, 111 In, 113 In, 119 Sb, 121 Sn, 201 Tl, 203 Pb, 212 Bi, 212 Pb, and 213 In certain embodiments, preferred radioisotopes include: 68 Ga, 111 In, 212 Pb or213 Contains Bi.

[0127] In certain embodiments, the radioisotope is 18 F, 75 Br, 76 Br, 77 Br, 123 I, 124 I, 125 I, 131 I and 211 In certain embodiments, preferred radioisotopes include: 18 F, 123 I, 124 I, 131 I or 211 At is included.

[0128] In certain embodiments, the radioisotope is 139 La, 140 La, 142 Pr, 143 Pr, 149 Pm, 151 EU, 153 EU, 153 Sm, 159 Gr, 149 Tb, 152 Tb, 161 Tb, 165 Dy, 166 Dy, 166 Ho and 169 EU, 175 In certain embodiments, preferred radioisotopes include: 149 Tb, 152 Tb or 161 Contains Tb.

[0129] In certain embodiments, the radioisotope is 225 Ac, 226 Th and 227 In certain embodiments, preferred radioisotopes include: 225 Ac or 227 Th is included.

[0130] The radiolabeled material of the present invention may also suitably comprise a combination of at least two radioisotopes to enable imaging and / or therapy. The combination of radioisotopes may suitably be selected from, for example, Ga-68 and Lu-177; F-18 and Lu-177; In-111 and Lu-177; Ga-68 and Y-90; F-18 and Y-90; In-111 and Y-90; Ga-68 and Ac-225; F-18 and Ac-225; In-111 and Ac-225.

[0131] The present invention can further include the use of at least one non-radioactive, non-toxic carrier metal.For example, the carrier metal can be selected from Bi and Fe.For example, the non-radioactive carrier metal can be one that allows MRI imaging (for example, Fe) or X-ray contrast imaging (for example, Bi).Another example of a carrier metal includes trivalent bismuth, which can also provide X-ray contrast to the microsphere and can be imaged by CT.

[0132] In certain embodiments, R is a chelator or chelating moiety, e.g., a chelator for radiometals or paramagnetic ions, including radioisotopes.

[0133] The chelating agent can include any chelating agent known in the art, see, e.g., Parus et al., "Chemistry and bifunctional chelating agents for binding (177)Lu," Curr Radiopharm. 2015; 8(2):86-94; Wangler et al., "Chelating agents and their use in radiopharmaceutical sciences," Mini Rev Med Chem. 2011 October; 11(11):968-83; Liu, "Bifunctional Coupling Agents for Radiolabeling of Biomolecules and Target-Specific Delivery of Metallic Radionuclides," Adv Drug Deliv Rev. 2008 September; 60(12): 1347-1370.

[0134] Specific examples are shown in Table 1. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0135] In certain preferred embodiments, R may be DOTA attached through any of its four carboxylic acid groups.

[0136] In certain embodiments, the chelating agent comprises a radioisotope chelated thereto.

[0137] In certain embodiments, the chelating agent comprises a paramagnetic substance chelated thereto. Examples of paramagnetic ions include chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), erbium (III), or a combination of these paramagnetic ions.

[0138] When the moiety is a detectable label, it may be a fluorescent moiety.

[0139] In some embodiments, the fluorescent moiety is selected from a fluorescent protein, a fluorescent peptide, a fluorescent dye, a fluorescent material, or a combination thereof.

[0140] In certain embodiments, R is a fluorescent dye such as may be selected from the group consisting of xanthenes, acridines, oxazines, cyanines, styryl dyes, coumarins (such as coumarin 343, methoxycoumarins, and dialkylaminocoumarins), porphines, metal-ligand complexes, fluorescent proteins, nanocrystals, perylenes, boron-dipyrromethenes, and phthalocyanines, as well as conjugates and combinations of these types of dyes. Examples of specific fluorescent labels include organic dyes, such as cyanines, fluorescein and fluorescein derivatives, rhodamine and rhodamine derivatives, Alexa Fluor, Dylight fluor (such as DyLight547 and Dylight647), Hylight fluor (such as HiLyte Fluor 647, HiLyte Fluor 680, and HiLyte Fluor 750), IRDye (such as IR Dye 800, IRDye 800CW, IRDye 800RS, and IRDye 700DX), Dy fluros (such as Dy677, Dy676, Dy682, Dy752, and Dy780), VivoTag fluor (such as VivoTag-680, VivoTag-S680, and VivoTag-S750), ATTO Dye, BODIPY fluor (BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, and BODIPY 650 / 665, etc.), carbocyanines, indocarbocyanines, oxacarbocyanines, thiacarbocyanines, merocyanines, polymethines, boron-dipyrromethane (BODIPY) dyes, ADS780WS, ADS830WS, and ADS832WS, and other fluorophores known to those of skill in the art.

[0141] By way of further example, fluorescent moieties include Cy3, Cy5, Cy5.5 (also known as Cy5++), CY2, CY7, CY7.5, fluorescein isothiocyanate (FITC), 4',5'-dichloro-2',7'-dimethoxy-fluorescein, naphthofluorescein, 2',4',5',7'-tetra-bromosulfone-fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin, Cy7, fluorescein (FAM), Cy3, Cy3.5 (also known as Cy3++), Texas Red, Texas Red-X, Marina Blue, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, PyMPO, AMCA, AMCA-S, Cascade Blue, Cascade Yellow, DM-NERF, eosin, erythrosin, FAM, LightCycler fluor (such as LightCycler-Red 640 and LightCycler Red 705), tetramethylrhodamine (TMR), rhodamine, rhodamine derivatives (ROX), hexachlorofluorescein (HEX), rhodamine 6G (R6G), carboxy-X-rhodamine, lissamine rhodamine B, pyrene, rhodamine B, rhodamine 6G, rhodamine green, rhodamine red, rhodamine green, tetramethyl-rhodamine, carboxytetramethylrhodamine, rhodamine derivative JA133, Alexa Fluorescent Dyes (Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 633, Alexa Fluor 555, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, AlexaFluor 750, and AlexaFluor 790), 4',The fluorescent compound may be selected from the group consisting of 6-diamidino-2-phenylindole (DAPI), propidium iodide, AMCA, Spectrum Green, Spectrum Orange, Spectrum Aqua, Lissamine, and fluorescent transition metal complexes such as europium. Usable fluorescent compounds also include fluorescent proteins such as GFP (green fluorescent protein), enhanced GFP (EGFP), blue fluorescent protein and derivatives (BFP, EBFP, EBFP2, Azurite, mKalama1), cyan fluorescent protein and derivatives (CFP, ECFP, Cerulean, CYPet), and yellow fluorescent protein and derivatives (YFP, Citrine, Venus, YPet). See also WO2008 / 142571, WO2009 / 056282, and WO99 / 22026 (all of which are incorporated by reference).

[0142] In certain embodiments, the detectable moiety is a biological fluorophore (such as a fluorescent polypeptide or peptide), including, but not limited to, green fluorescent protein (GFP) derivatives of GFP (e.g., EBFP, EBFP2, Azurite, mKalamal, ECFP, Cerulean, CYpet, YFP, Citrine, Venus, Ypet) and R-phycoerythrin.

[0143] In certain embodiments, R is a photoacoustic reporter molecule. Exemplary photoacoustic reporter molecules include indocyanine green (ICG), Alexa Fluor 750, Evans Blue, BHQ3, QXL680, IRDye880CW, MMPSense 680, methylene blue, PPCy-C8, and Cypate-C18.

[0144] In certain embodiments, the detectable moiety is a detectable nanoparticle selected from the group consisting of plasmonic nanoparticles, quantum dots, nanodiamonds, polypyrrole nanoparticles, copper sulfide nanoparticles, graphene nanosheets, iron oxide-gold core-shell nanoparticles, Gd2O3 nanoparticles, single-walled carbon nanotubes, dye-loaded perfluorocarbon nanoparticles, and superparamagnetic iron oxide nanoparticles.

[0145] In certain embodiments, the detectable moiety comprises a quantum dot.

[0146] In certain embodiments, the detectable moiety comprises an infrared-emitting quantum dot.

[0147] In certain embodiments, the detectable moiety is a Raman-active reporter molecule, such as a single-walled carbon nanotube (SWNT) or a surface-enhanced Raman scattering (SERS) agent. An example of a SERS agent is a metal nanoparticle labeled with a Raman-active reporter molecule. In certain examples, fluorescent dyes can also be used as Raman-active reporter molecules, such as Cy3, Cy5, rhodamine, and chalcogenopyrylium dyes.

[0148] Examples of R that are enzyme labels include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, and β-galactosidase.

[0149] In certain embodiments, the agents of the present invention are imaging agents selected to be useful as part of a method for performing image-guided surgery, such as for resection, dissection, ablation, removal, or stenting or placement of other in situ devices. For example, the agent can be administered to a patient (human or veterinary subject) in an amount sufficient to preferentially localize to the surgical target tissue, allowing the surgeon to detect the presence or absence of the imaging agent during the surgical procedure. In this regard, the detectable moiety may preferably be optically detectable, such as a fluorescent or other optically active moiety as described above. Such imaging agents can be advantageously used in the operating room, where the surgical field can be illuminated with electromagnetic radiation sufficient to render detectable a detectable moiety, such as a fluorophore or quantum dot, which can be visualized by the surgeon directly or via monitoring means (e.g., a screen / monitor). Exemplary uses of such imaging agents include endoscopic and laparoscopic surgical procedures, in which the surgeon can optically observe the presence (or absence) of the imaging agent, generally via endoscopic, laparoscopic, or percutaneous means.

[0150] In certain embodiments, the image-guided surgery may be image-guided robotic-assisted surgery.

[0151] In certain embodiments, the FAP targeting agent is represented by the general formula IIb, where R is as defined above and X is C or N. In certain preferred embodiments of IIb, R is a chelator. [ka]

[0152] Exemplary FAP targeting agents include: [ka] [ka]

[0153] In certain embodiments, the FAP targeting agent comprises two or more FAP inhibitor moieties covalently attached to a radiopharmaceutical or imaging agent, such as having the structure represented by Formula IV, or a pharmaceutically acceptable salt thereof: [ka] [In the formula, R, R1, R2, R3, R4, X and L are as defined above; n represents an integer from 2 to 6.

[0154] In certain preferred embodiments of Formula IV, R is a chelating moiety.

[0155] In certain preferred embodiments of Formula IV, R is a chelating moiety and n is 2.

[0156] Particularly preferred compounds include: [ka]

[0157] Also particularly preferred are the radionuclide-containing compounds 6555, 6952 and 6522. For example, the compounds of the following formulae A, B and C are preferred: [ka] wherein in each of Formulas A, B and C, M is a radioisotope or a metal.

[0158] In certain embodiments, in Formula A, B and / or C, M is a diagnostic radioisotope.

[0159] In certain embodiments, in Formula A, B and / or C, M is a therapeutic radioisotope.

[0160] In certain embodiments, in Formulas A, B and / or C, M is Ga-67, Ga-68, Lu-177 or Y-90.

[0161] In certain embodiments, in Formula A, B and / or C, M is 43 K. 81 Rb, 83 Sr, 89 Sr, 127 Cs, 128 Ba, 129 Cs and 131 s-block metals such as Cs.

[0162] In certain embodiments, in Formula A, B and / or C, M is in Groups 13-16 of the Periodic Table, e.g., 67 Ga, 68 Ga, 71 Ge, 72 As, 72 Se, 77 As, 110 In, 111 In, 113 In, 119 Sb, 121 Sn, 201 Tl, 203 Pb, 212 Bi, 212 Pb and 213 Bi, especially 68 Ga, 111 In, 212 Pb or 213 I'm Bi.

[0163] In certain embodiments, in formula A, B and / or C, M is halogen, e.g., 18 F, 75 Br, 76 Br, 77 Br, 123 I, 124 I, 125 I, 131 I and 211 At, especially 18 F, 123 I, 124 I, 131 I or 211 At.

[0164] In certain embodiments, in Formula A, B and / or C, M is a lanthanide, e.g., 139 La, 140 La, 142 Pr, 143 Pr, 149 Pm, 151 EU, 153 EU, 153 Sm, 159 Gr, 149 Tb, 152 Tb, 161 Tb, 165 Dy, 166 Dy, 166 Ho and 169 EU, 175 Yb, especially 149 Tb, 152 Tb or 161 Tb.

[0165] In certain embodiments, in Formula A, B and / or C, M is an actinide, e.g., 225 Ac, 226 Th and 227 Th, especially 225 Ac or 227 Th.

[0166] Also preferred are compounds complexed with one or more radioisotopes, including: [ka] [ka]

[0167] In certain embodiments, the FAP targeting agent comprises a moiety that modifies the pharmacokinetics and / or biodistribution of the molecule, such as the serum half-life of the molecule and / or tumor distribution of the molecule. Such PK / BD-modified FAP targeting agents can have a structure represented by Formula V, or a pharmaceutically acceptable salt thereof: [ka] [In the formula, R, R1, R2, R3, R4, X and L are as defined above; R5 represents a moiety that modifies the pharmacokinetics and / or biodistribution of the molecule; n represents an integer from 1 to 6.

[0168] In certain preferred embodiments of Formula V, R5 is a serum albumin binding moiety.

[0169] In certain preferred embodiments of Formula V, R5 is a serum albumin binding moiety and n is 1.

[0170] In certain preferred embodiments of Formula V, R5 is a half-life extending moiety, for example, a non-proteinaceous half-life extending moiety, such as a water-soluble polymer such as water-soluble polyethylene glycol (PEG) or discrete PEG, hydroxyethyl starch (HES), lipids, branched or unbranched acyl groups, branched or unbranched C8-C30 acyl groups, branched or unbranched alkyl groups, and branched or unbranched C8-C30 alkyl groups; and a proteinaceous half-life extending moiety, such as serum albumin, transferrin, Adnectins (e.g., albumin-binding or pharmacokinetics extending (PKE) Adnectins), Fc domains, and unstructured polypeptides (e.g., conformationally disordered polypeptide sequences composed of the amino acids Pro, Ala, and / or Ser) such as XTEN and PAS polypeptides, and fragments of any of the foregoing.

[0171] For example, compounds of the present invention complexed with a radioisotope or metal can be readily prepared to provide compounds of any of Formulas I-V above, where R is a radioactive moiety or R is a chelating agent, in which the radioisotope or metal is complexed with the chelating agent. For example, an aqueous admixture of 1) a radioisotope reagent, e.g., a halide reagent of a radioisotope, and 2) a precursor compound, such as a compound having a chelating moiety, is suitably reacted with stirring for a time and at a temperature sufficient to complex the radioisotope with the precursor compound. Exemplary incorporation reaction times and temperatures are described in the Examples below and may suitably include reaction times of 5 to 60 minutes and reaction temperatures of up to 90°C or higher.

[0172] The reaction mixture may suitably contain one or more stabilizer compounds, such as organic stabilizers, for example, 2,5-dihydroxybenzoic acid or its salts, ascorbic acid or its salts, methionine, histidine, melatonin, N-acetylmethionine, or ethanol, with N-acetylmethionine being preferred in certain embodiments. Preferred stabilizers include those that are generally regarded as safe (GRAS) under the standards of the U.S. Food and Drug Administration.

[0173] In certain embodiments, sulfur-containing stabilizer compounds, including compounds containing one or more sulfide moieties, such as N-acetylmethionine and reduced L-glutathione, are preferred stabilizers for inclusion in the radionuclide reagent / precursor compound mixture during the incorporation reaction.

[0174] A wide variety of macromolecular polymers and other molecules can be linked to the FAP targeting agents of the present disclosure to modulate and / or provide new biological properties to the resulting FAP targeting agents. These macromolecular polymers can be linked to the FAP targeting agents via naturally encoded amino acids, non-naturally encoded amino acids, or any functional substituent of a natural or non-natural amino acid, or any substituent or functional group added to a natural or non-natural amino acid. The molecular weight of the polymer can range widely, including, but not limited to, from about 100 Da to about 100,000 Da or more. The molecular weight of the polymer may be from about 100 Da to about 100,000 Da, and may be 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, or 30,000 Da. Examples of molecular weights include, but are not limited to, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, the molecular weight of the polymer is from about 100 Da to about 50,000 Da. In some embodiments, the molecular weight of the polymer is from about 100 Da to about 40,000 Da. In some embodiments, the molecular weight of the polymer is from about 1,000 Da to about 40,000 Da. In some embodiments, the molecular weight of the polymer is from about 5,000 Da to about 40,000 Da. In some embodiments, the molecular weight of the polymer is from about 10,000 Da to about 40,000 Da.

[0175] For this purpose, various methods have been developed, including PEGylation, polysialylation, hydroxylation, glycosylation, or recombinant PEG analogs fused to flexible, hydrophilic amino acid chains (500-600 amino acids) (see Chapman, (2002) Adv Drug Deliv Rev. 54, 531-545; Schlapschy et al., (2007) Prot Eng Des Sel. 20, 273-283; Contermann (2011) Curr Op Biotechnol. 22, 868-876; Jevsevar et al., (2012) Methods Mol Biol. 901, 233-246).

[0176] Exemplary polymers include polyalkyl ethers and their alkoxy-capped analogs (e.g., polyoxyethylene glycol, polyoxyethylene / propylene glycol, and their methoxy- or ethoxy-capped analogs, particularly polyoxyethylene glycol, the latter also known as polyethylene glycol or PEG); discrete PEG (dPEG); polyvinylpyrrolidone; polyvinyl alkyl ethers; polyoxazolines, polyalkyloxazolines, and polyhydroxyalkyloxazolines; polyacrylamides, polyalkylacrylamides, and polyhydroxyalkylacrylamides (e.g., polyhydroxypropylmethacrylamide and their derivatives); polyhydroxyalkylacrylates; polysialic acid and its analogs; hydrophilic peptide sequences; polysaccharides and their derivatives, including dextran and dextran derivatives. hydroxymethyl cellulose, hydroxymethyl cellulose, chitin, chitosan, succinyl chitosan, carboxymethyl chitin, carboxymethyl chitosan, hyaluronic acid, hyaluronic acid, starch, alginate, chondroitin sulfate, albumin, pullulan, and carboxymethyl pullulan, polyamino acids, and derivatives thereof, such as polyglutamic acid, polylysine, polyaspartic acid, polyasparagine amide, maleic anhydride copolymers, such as styrene-maleic anhydride copolymer, divinyl ethyl ether-maleic anhydride copolymer, polyvinyl alcohol, copolymers thereof, terpolymers thereof, mixtures thereof, and derivatives of the foregoing.

[0177] The selected polymer may be water-soluble so that the FAP targeting agent to which it is attached does not precipitate in an aqueous environment, such as a physiological environment. The water-soluble polymer may be of any structural form, including, but not limited to, linear, forked, or branched. Typically, the water-soluble polymer is a poly(alkylene glycol), such as poly(ethylene glycol) (PEG), although other water-soluble polymers may also be used. PEG is used as an example to illustrate some embodiments of the present disclosure. For therapeutic use of the FAP targeting agent, the polymer may be pharmaceutically acceptable.

[0178] The term "PEG" is used broadly to encompass any polyethylene glycol molecule, regardless of size or modification at the terminus of the PEG, and can be represented as linked to a FAP targeting agent by the following formula: XO-(CH2CH2O) n -CH2CH2- or XO-(CH2CH2O) n - where n is 2 to 10,000, and X is H or a terminal modification (including, but not limited to, C alkyl, a protecting group, or a terminal functional group). In some cases, PEG used in polypeptides of the present disclosure is terminated at one end with hydroxy or methoxy, i.e., X is H or CH ("methoxy PEG").

[0179] The number of water-soluble polymers linked to a FAP targeting agent (i.e., the degree of PEGylation or glycosylation) can be adjusted to provide changes (including, but not limited to, increases or decreases) in the pharmacological, pharmacokinetic, or pharmacodynamic properties, such as the in vivo half-life, of the resulting FAP targeting agent. In some embodiments, the half-life of the resulting FAP targeting agent is increased by at least about 10, 20, 30, 40, 50, 60, 70, 80, 90 percent, 2-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 50-fold, or at least about 100-fold over the unmodified polypeptide.

[0180] Another variation on polymer systems useful for modifying the PK or other biological properties of the resulting FAP targeting agent is the use of unstructured hydrophilic amino acid polymers, which are functional analogs of PEG. The inherent biodegradability of the polypeptide platform makes it attractive as a potentially more benign alternative to PEG. Another advantage is the precise molecular structure of the recombinant molecule, as opposed to the polydispersity of PEG. Unlike HSA and Fc peptide fusions, which require the three-dimensional folding of the fusion partner to be maintained, recombinant fusions to unstructured partners can often be subjected to harsh conditions, such as higher temperatures or HPLC purification.

[0181] One of the more advanced members of this class of polypeptides, called XTEN (Amunix), is 864 amino acids long and consists of six amino acids (A, E, G, P, S, and T). See Schellenberger et al. "A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner," 2009 Nat Biotechnol. 27(12):1186-90. Taking advantage of the biodegradable properties of the polymer, which is much larger than the typically used 40 kDa PEG, concomitantly confers a longer half-life extension. Fusion of XTEN to a FAP targeting agent should result in a 60- to 130-fold longer half-life extension of the final FAP targeting agent than the unmodified polypeptide.

[0182] A second polymer based on similar conceptual considerations is PAS (XL-Protein GmbH). Schlapschy et al. "PASYlation: a biological alternative to PEGylation for extending the plasma half-life of pharmaceutically active proteins" 2013 Protein Eng Des Sel. 26(8):489-501. Random coil polymers consist of an even more restricted set of only three small, uncharged amino acids: proline, alanine, and serine.

[0183] In certain preferred embodiments of Formula V, R5 is a polyethylene glycol polymer.

[0184] In certain preferred embodiments of Formula V, R5 is a polyethylene glycol polymer and n is 1.

[0185] The present invention also provides pharmaceutical compositions comprising at least one compound of any of Formulas I-V and, optionally, a pharmaceutically acceptable carrier and / or excipient. In certain embodiments, the pharmaceutical composition is intended for use in the diagnosis or treatment of a disease characterized by overexpression of fibroblast activation protein (FAP) in an animal, preferably a human subject.

[0186] Suitable pharmaceutically acceptable vehicles include non-toxic buffers such as phosphate, citrate, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol; low molecular weight polypeptides (e.g., less than about 10 amino acid residues); serum albumin; Examples of suitable surfactants include, but are not limited to, proteins such as amine, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; carbohydrates such as monosaccharides, disaccharides, glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes such as Zn-protein complexes; and nonionic surfactants such as TWEEN® or polyethylene glycol (PEG). (Remington: The Science and Practice of Pharmacy, 22nd Edition, 2012, Pharmaceutical Press, London.)

[0187] In preferred embodiments, the pharmaceutical composition may include one or more stabilizer compounds that can inhibit degradation of the radiopharmaceutical after preparation and before administration. Preferred stabilizers include those that are generally regarded as safe (GRAS) under U.S. Food and Drug Administration standards.

[0188] Exemplary stabilizer compounds include organic agents such as 2,5-dihydroxybenzoic acid or a salt thereof, ascorbic acid or a salt thereof, methionine, histidine, melatonin, N-acetylmethionine, or ethanol, with N-acetylmethionine being a preferred stabilizer for inclusion in the aqueous pharmaceutical compositions of the present invention.

[0189] In certain embodiments, sulfur-containing stabilizer compounds, including compounds containing one or more sulfide moieties, such as reduced N-acetylmethionine and L-glutathione, are preferred stabilizers for use in the present pharmaceutical compositions. Exemplary amounts of one or more stabilizers in the present pharmaceutical compositions can be 5 to 120 mg of stabilizer per mL of fluid (e.g., aqueous formulation) pharmaceutical composition.

[0190] The pharmaceutical compositions of the present disclosure can be administered in any number of ways for either local or systemic treatment.Administration can be topical administration with epidermal or transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders; pulmonary administration by inhalation or insufflation of powders or aerosols, including nebulizers, intratracheal, and intranasal; oral administration; or parenteral administration, including intravenous, intraarterial, intratumoral, subcutaneous, intraperitoneal, intramuscular (e.g., injection or infusion), or intracranial (e.g., intrathecal or intraventricular).

[0191] Typical administration of the radiopharmaceuticals of the present invention may be by intravenous injection or other parenteral administration.

[0192] Therapeutic formulations may be in unit dosage form. Such formulations include tablets, pills, capsules, powders, granules, solutions or suspensions in water or non-aqueous media, or suppositories. In solid compositions such as tablets, the primary active ingredient is mixed with a pharmaceutical carrier. Conventional tableting ingredients include corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, calcium diphosphate, or gums, and diluents (e.g., water). These can be used to form solid preformulation compositions containing a homogeneous mixture of the compound of the present disclosure or its non-toxic pharmaceutically acceptable salt. The solid preformulation composition is then subdivided into unit dosage forms of the type described above. Tablets, pills, etc. of the formulation or composition can be coated or otherwise compounded to provide a dosage form offering the advantage of prolonged action. For example, a tablet or pill can include an inner composition surrounded by an outer component. Moreover, the two components can be separated by an enteric layer which serves to resist disintegration and permits the inner component to pass intact through the stomach or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

[0193] Kits and Methods Yet another aspect of the present invention provides kits comprising or consisting of at least one compound of any of Formulas IV and instructions for diagnosing or treating disease.

[0194] Yet another aspect of the present invention provides a method for diagnosing, imaging, or reducing tissues that overexpress FAP in an animal (preferably a human patient), comprising administering to the animal at least one compound of any of Formulas I-V.

[0195] In some embodiments, the tissue that overexpresses a FAP is a tumor, particularly a solid tumor. In some embodiments, the tumor is selected from the group consisting of: colorectal tumor, pancreatic tumor, lung tumor, ovarian tumor, liver tumor, breast tumor, kidney tumor, prostate tumor, neuroendocrine tumor, gastrointestinal tumor, melanoma, cervical tumor, bladder tumor, glioblastoma, and head and neck tumor. In some embodiments, the tumor is a colorectal tumor. In some embodiments, the tumor is an ovarian tumor. In some embodiments, the tumor is a lung tumor. In some embodiments, the tumor is a pancreatic tumor. In some embodiments, the tumor is a melanoma. In some embodiments, the tumor is a bladder tumor. In some embodiments, the tumor is a prostate tumor. By way of further example, a subject Affimer drug The agent is effective against osteosarcoma, rhabdomyosarcoma, neuroblastoma, kidney cancer, leukemia, renal transitional cell carcinoma, bladder cancer, Wilms' cancer, ovarian cancer, pancreatic cancer, breast cancer (including triple-negative breast cancer), prostate cancer, bone cancer, lung cancer (e.g., small cell lung cancer or non-small cell lung cancer), gastric cancer, colorectal cancer, cervical cancer, synovial sarcoma, head and neck cancer, squamous cell carcinoma, multiple myeloma, renal cell carcinoma, retinoblastoma, hepatocellular carcinoma, and melanoma. The present invention can be used to treat patients suffering from cancers such as rhabdoid tumor of the kidney, Ewing's sarcoma, chondrosarcoma, brain cancer, glioblastoma, meningioma, pituitary adenoma, vestibular schwannoma, primitive neuroectodermal tumor, medulloblastoma, astrocytoma, anaplastic astrocytoma, oligodendroglioma, ependymoma, choroid plexus papilloma, polycythemia vera, thrombocythemia, idiopathic myelofibrosis, soft tissue sarcoma, thyroid cancer, endometrial cancer, carcinoid cancer, or liver cancer, breast cancer, or gastric cancer. In some embodiments of the present disclosure, the cancer is a metastatic cancer, such as those of the various types described above.

[0196] In some embodiments, in addition to administering a FAP targeting agent described herein, the method or treatment further comprises administering at least one additional immune response stimulator. In some embodiments, the additional immune response stimulator includes, but is not limited to, a colony-stimulating factor (e.g., granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), granulocyte-colony-stimulating factor (G-CSF), stem cell factor (SCF)), an interleukin (e.g., IL-1, IL-2, IL-3, IL-7, IL-12, IL-15, IL-18), a checkpoint inhibitor, an antibody that blocks immunosuppressive function (e.g., anti-CTLA-4 antibody, anti-CD28 antibody, anti-CD3 antibody), a Toll-like receptor (e.g., TLR4, TLR7, TLR9), or a member of the B7 family (e.g., CD80, CD86). The additional immune response stimulator can be administered before, simultaneously with, and / or after administration of the FAP targeting agent. Also provided are pharmaceutical compositions comprising a FAP targeting agent and an immune response stimulator. In some embodiments, the immune response stimulator comprises one, two, three, or more immune response stimulators.

[0197] In some embodiments, in addition to administering a FAP targeting agent described herein, the method or treatment further comprises administering at least one additional therapeutic agent. The additional therapeutic agent can be administered before, simultaneously with, and / or after the administration of the FAP targeting agent. Pharmaceutical compositions comprising a FAP targeting agent and an additional therapeutic agent are also provided. In some embodiments, the at least one additional therapeutic agent comprises one, two, three, or more additional therapeutic agents.

[0198] Combination therapy with two or more therapeutic agents often uses agents that act through different mechanisms of action, but this is not required. Combination therapy using agents with different mechanisms of action can result in additive or synergistic effects. Combination therapy can allow for lower doses of each agent than those used in monotherapy, thereby reducing toxic side effects and / or increasing the therapeutic index of the FAP targeting agent. Combination therapy can reduce the likelihood of resistant cancer cells developing. In some embodiments, combination therapy includes a therapeutic agent that affects the immune response (e.g., enhances or activates the response) and a therapeutic agent that affects tumor / cancer cells (e.g., inhibits or kills them).

[0199] In some embodiments of the methods described herein, the combination of a FAP targeting agent described herein with at least one additional therapeutic agent results in additive or synergistic results. In some embodiments, the combination therapy results in an increased therapeutic index of the FAP targeting agent. In some embodiments, the combination therapy results in an increased therapeutic index of the additional therapeutic agent. In some embodiments, the combination therapy results in reduced toxicity and / or side effects of the FAP targeting agent. In some embodiments, the combination therapy results in reduced toxicity and / or side effects of the additional therapeutic agent.

[0200] Useful classes of therapeutic agents include, for example, antitubulin agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., cisplatin, mononuclear (platinum), dinuclear (platinum), and trinuclear platinum complexes, and platinum complexes such as carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, chemotherapy sensitizers, duocarmycins, etoposide, fluorinated pyrimidines, ionophores, lexitropsin, nitrosoureas, platinol, purine antimetabolites, puromycin, radiosensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, etc. In some embodiments, the second therapeutic agent is an alkylating agent, antimetabolite, mitotic inhibitor, topoisomerase inhibitor, or angiogenesis inhibitor.

[0201] Therapeutic agents that can be administered in combination with the FAP targeting agents described herein include chemotherapeutic agents. Thus, in some embodiments, a method or treatment involves administering a FAP targeting agent of the present disclosure in combination with a chemotherapeutic agent or a cocktail of chemotherapeutic agents. Treatment with a FAP targeting agent can occur before, simultaneously with, or after the administration of the chemotherapeutic agent. Combined administration can include simultaneous administration in either a single pharmaceutical formulation or separate formulations, or sequential administration in any order, but within a period of time that allows all active agents to simultaneously exert their biological activity. The preparation and administration schedule of such chemotherapeutic agents can be used according to the manufacturer's instructions or as empirically determined by those skilled in the art. Preparation and administration schedules for such chemotherapy are also described in The Chemotherapy Source Book, 4th Edition, 2008, MC Perry, Editor, Lippincott, Williams & Wilkins, Philadelphia, PA.

[0202] Chemotherapeutic agents useful in the present disclosure include alkylating agents, such as thiotepa and cyclophosphamide (CYTOXAN); alkylsulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine. ); nitrogen mustards, for example, chlorambucil, chlornaphazine, colofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobuenbikinin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, for example, carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics, for example, aclacinomycin, actinomycin, ausramycin, azaserine, bleomycin, ka Cutinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tuberculin Lucidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytosine arabinoside, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU;Androgens such as calsterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; Antiadrenal drugs such as aminoglutethimide, mitotane, trilostane; Folic acid supplements such as folinic acid; Aceglatone; Aldophosphamide glycosides; Aminolevulinic acid; Amsacrine; Bestravcil; Bisantrene; Edatraxate; Defofamine; Demecolcine; Diaziquone; Elformitin; Elliptinium acetate; Etogluside ;Gallium nitrate;Hydroxyurea;Lentinan;Lonidamine;Mitoguazone;Mitoxantrone;Mopidamol;Nitracrine;Pentostatin;Fenamet;Pirarubicin;Podophyllic acid;2-Ethylhydrazide;Procarbazine;PSK;Razoxane;Sizofiran;Spirogermanium;Tenuazonic acid;Triazicon, 2,2',2"-Trichlorotriethylamine;Urethane;Vindesine;Dacarbazine;Mannomustine;Mitobronitol;Mitolactol;Pipobroman;Gasitosine;Arabinoside (Ara- C); taxoids, such as paclitaxel (TAXOL) and doxetaxel (TAXOTERE); chlorambucil; gemcitabine, 6-thioguanine; mercaptopurine; platinum analogs, such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; ibandronate; CPT-11; topoisomerase inhibitors R Chemotherapeutic agents include, but are not limited to, FS2000, difluoromethylolnitine (DMFO), retinoic acid, esperamicin, capecitabine (XELODA), and pharmaceutically acceptable salts, acids, or derivatives of any of the above. Chemotherapeutic agents also include antiestrogens, including, for example, tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (FARESTON);Also included are antihormonal agents that act to regulate or inhibit hormone action on tumors, such as antiandrogens, for example, flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the above. In certain embodiments, the additional therapeutic agent is cisplatin. In certain embodiments, the additional therapeutic agent is carboplatin.

[0203] In some embodiments of the methods described herein, the chemotherapeutic agent is a topoisomerase inhibitor. A topoisomerase inhibitor is a chemotherapeutic agent that inhibits the action of a topoisomerase enzyme (e.g., topoisomerase I or II). Topoisomerase inhibitors include, but are not limited to, doxorubicin HCl, daunorubicin citrate, mitoxantrone HCl, actinomycin D, etoposide, topotecan HCl, teniposide (VM-26), and irinotecan, as well as pharmaceutically acceptable salts, acids, or derivatives thereof. In some embodiments, the additional therapeutic agent is irinotecan.

[0204] In some embodiments, the chemotherapeutic agent is an antimetabolite. An antimetabolite is a chemical compound that resembles a metabolite required for normal biochemical reactions, but has a structure that is sufficiently different to interfere with one or more normal functions of cells, such as cell division. Antimetabolites include, but are not limited to, gemcitabine, fluorouracil, capecitabine, methotrexate sodium, ralitrexed, pemetrexed, tegafur, cytosine arabinoside, thioguanine, 5-azacytidine, 6-mercaptopurine, azathioprine, 6-thioguanine, pentostatin, fludarabine phosphate, and cladribine, as well as pharmaceutically acceptable salts, acids, or derivatives thereof. In some embodiments, the additional therapeutic agent is gemcitabine.

[0205] In some embodiments of the methods described herein, the chemotherapeutic agent is an antimitotic agent, including but not limited to, an agent that binds to tubulin. In some embodiments, the agent is a taxane. In some embodiments, the agent is paclitaxel or docetaxel, or a pharmaceutically acceptable salt, acid, or derivative of paclitaxel or docetaxel. In some embodiments, the agent is paclitaxel (TAXOL), docetaxel (TAXOTERE), albumin-bound paclitaxel (nab-paclitaxel; ABRAXANE), DHA-paclitaxel, or PG-paclitaxel. In certain alternative embodiments, the antimitotic agent comprises a vinca alkaloid, such as vincristine, vinblastine, vinorelbine, or vindesine, or a pharmaceutically acceptable salt, acid, or derivative thereof. In some aspects, the antimitotic agent is an inhibitor of kinesin Eg5 or an inhibitor of a mitotic kinase, such as Aurora A or Plk1. In some embodiments, the additional therapeutic agent is paclitaxel. In some embodiments, the additional therapeutic agent is nab-paclitaxel.

[0206] In some embodiments of the methods described herein, the additional therapeutic agent comprises an agent such as a small molecule. For example, treatment may include the combined administration of a FAP targeting agent of the present disclosure with a small molecule that acts as an inhibitor of tumor-associated antigens, including, but not limited to, EGFR, HER2 (ErbB2), and / or VEGF. In some embodiments, the FAP targeting agent of the present disclosure is administered in combination with a protein kinase inhibitor selected from the group consisting of gefitinib (IRESSA), erlotinib (TARCEVA), sunitinib (SUTENT), lapatanib, vandetanib (ZACTIMA), AEE788, CI-1033, cediranib (RECENTIN), sorafenib (NEXAVAR), and pazopanib (GW786034B). In some embodiments, the additional therapeutic agent comprises an mTOR inhibitor.

[0207] In some embodiments of the methods described herein, the additional therapeutic agent is a small molecule that inhibits a cancer stem cell pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the Notch pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the Wnt pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the BMP pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the Hippo pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the mTOR / AKR pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the RSPO / LGR pathway.

[0208] In some embodiments of the methods described herein, the additional therapeutic agent comprises a biological molecule such as an antibody. For example, treatment may include the combined administration of a FAP targeting agent of the present disclosure with an antibody against a tumor-associated antigen, including, but not limited to, an antibody that binds to EGFR, HER2 / ErbB2, and / or VEGF. In some embodiments, the additional therapeutic agent is an antibody specific for a cancer stem cell marker. In some embodiments, the additional therapeutic agent is an antibody that binds to a component of the Notch pathway. In some embodiments, the additional therapeutic agent is an antibody that binds to a component of the Wnt pathway. In some embodiments, the additional therapeutic agent is an antibody that inhibits a cancer stem cell pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the Notch pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the Wnt pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the BMP pathway. In some embodiments, the additional therapeutic agent is an antibody that inhibits β-catenin signaling. In some embodiments, the additional therapeutic agent is an antibody that is an angiogenesis inhibitor (e.g., an anti-VEGF or VEGF receptor antibody). In some embodiments, the additional therapeutic agent is bevacizumab (AVASTIN), ramucirumab, trastuzumab (HERCEPTIN), pertuzumab (OMNITARG), panitumumab (VECTIBIX), nimotuzumab, zalutumumab, or cetuximab (ERBITUX).

[0209] In some embodiments of the methods described herein, the additional therapeutic agent is an antibody that modulates the immune response, hi some embodiments, the additional therapeutic agent is an anti-PD-1 antibody, an anti-LAG-3 antibody, an anti-CTLA-4 antibody, an anti-TIM-3 antibody, or an anti-TIGIT antibody.

[0210] Additionally, treatment with the FAP targeting agents described herein can include combination treatment with other biological molecules, such as one or more cytokines (e.g., lymphokines, interleukins, tumor necrosis factors, and / or growth factors), or can involve surgical removal of tumors, removal of cancer cells, or any other therapy deemed necessary by the treating physician. In some embodiments, the additional therapeutic agent is an immune response stimulator.

[0211] In some embodiments of the methods described herein, the FAP targeting agent can be combined with a growth factor selected from the group consisting of adrenomedullin (AM), angiopoietin (Ang), BMP, BDNF, EGF, erythropoietin (EPO), FGF, GDNF, G-CSF, GM-CSF, GDF9, HGF, HDGF, IGF, migration stimulating factor, myostatin (GDF-8), NGF, neurotrophins, PDGF, thrombopoietin, TGF-α, TGF-β, TNF-α, VEGF, P1GF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, and IL-18.

[0212] In some embodiments of the methods described herein, the additional therapeutic agent is an immune response stimulator, hi some embodiments, the immune response stimulator is selected from the group consisting of granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), interleukin-3 (IL-3), interleukin-12 (IL-12), interleukin-1 (IL-1), interleukin-2 (IL-2), B7-1 (CD80), B7-2 (CD86), 4-1BB ligand, anti-CD3 antibody, anti-CTLA-4 antibody, anti-TIGIT antibody, anti-PD-1 antibody, anti-LAG-3 antibody, and anti-TIM-3 antibody.

[0213] In some embodiments of the methods described herein, the immune response stimulator is selected from the group consisting of a modulator of PD-1 activity, a modulator of PD-L2 activity, a modulator of CTLA-4 activity, a modulator of CD28 activity, a modulator of CD80 activity, a modulator of CD86 activity, a modulator of 4-1BB activity, a modulator of OX40 activity, a modulator of KIR activity, a modulator of Tim-3 activity, a modulator of LAG3 activity, a modulator of CD27 activity, a modulator of CD40 activity, a modulator of GITR activity, a modulator of TIGIT activity, a modulator of CD20 activity, a modulator of CD96 activity, a modulator of IDO1 activity, a cytokine, a chemokine, an interferon, an interleukin, a lymphokine, a member of the tumor necrosis factor (TNF) family, and an immunostimulatory oligonucleotide.

[0214] In some embodiments of the methods described herein, the immune response stimulator is selected from the group consisting of a PD-1 antagonist, a PD-L2 antagonist, a CTLA-4 antagonist, a CD80 antagonist, a CD86 antagonist, a KIR antagonist, a Tim-3 antagonist, a LAG3 antagonist, a TIGIT antagonist, a CD20 antagonist, a CD96 antagonist, and / or an IDO1 antagonist.

[0215] In some embodiments of the methods described herein, the PD-1 antagonist is an antibody that specifically binds to PD-1. In some embodiments, the antibody that binds to PD-1 is KEYTRUDA (MK-3475), pidilizumab (CT-011), nivolumab (OPDIVO, BMS-936558, MDX-1106), MEDI0680 (AMP-514), REGN2810, BGB-A317, PDR-001, or STI-A1110. In some embodiments, the antibody that binds to PD-1 is described in WO2014 / 179664, e.g., an antibody identified as APE2058, APE1922, APE1923, APE1924, APE1950, or APE1963, or an antibody comprising the CDR regions of any of these antibodies. In another embodiment, the PD-1 antagonist is a fusion protein that includes PD-L2, e.g., AMP-224. In another embodiment, the PD-1 antagonist is a peptide inhibitor, e.g., AUNP-12.

[0216] In some embodiments, the CTLA-4 antagonist is an antibody that specifically binds to CTLA-4. In some embodiments, the antibody that binds to CTLA-4 is ipilimumab (YERVOY) or tremelimumab (CP-675,206). In some embodiments, the CTLA-4 antagonist is a CTLA-4 fusion protein, such as KAHR-102.

[0217] In some embodiments, the LAG3 antagonist is an antibody that specifically binds to LAG3. In some embodiments, the antibody that binds to LAG3 is IMP701, IMP731, BMS-986016, LAG525, and GSK2831781. In some embodiments, the LAG3 antagonist comprises a soluble LAG3 receptor, such as IMP321.

[0218] In some embodiments, the KIR antagonist is an antibody that specifically binds to KIR. In some embodiments, the antibody that binds to KIR is lirilumab.

[0219] In some embodiments, the immune response stimulator is selected from the group consisting of a CD28 agonist, a 4-1BB agonist, an OX40 agonist, a CD27 agonist, a CD80 agonist, a CD86 agonist, a CD40 agonist, and a GITR agonist. In some embodiments, the OX40 agonist comprises an OX40 ligand or an OX40-binding portion thereof. For example, the OX40 agonist can be MEDI6383. In some embodiments, the OX40 agonist is an antibody that specifically binds to OX40. In some embodiments, the antibody that binds to OX40 is MEDI6469, MEDI0562, or MOXR0916 (RG7888). In some embodiments, the OX40 agonist is a vector (e.g., an expression vector such as an adenovirus or a virus) capable of expressing an OX40 ligand. In some embodiments, the OX40 expression vector is Delta-24-RGDOX or DNX2401.

[0220] In some embodiments, the 4-1BB (CD137) agonist is a binding molecule such as anticalin. In some embodiments, the anticalin is PRS-343. In some embodiments, the 4-1BB agonist is an antibody that specifically binds to 4-1BB. In some embodiments, the antibody that binds to 4-1BB is PF-2566 (PF-05082566) or urelumab (BMS-663513).

[0221] In some embodiments, the CD27 agonist is an antibody that specifically binds to CD27. In some embodiments, the antibody that binds to CD27 is varlilumab (CDX-1127).

[0222] In some embodiments, the GITR agonist comprises a GITR ligand or its GITR binding portion. In some embodiments, the GITR agonist is an antibody that specifically binds to GITR. In some embodiments, the antibody that binds to GITR is TRX518, MK-4166, or INBRX-110.

[0223] In some embodiments, immune response stimulators include, but are not limited to, cytokines such as chemokines, interferons, interleukins, lymphokines, and members of the tumor necrosis factor (TNF) family, hi some embodiments, immune response stimulators include immunostimulatory oligonucleotides, such as CpG dinucleotides.

[0224] In some embodiments, immune response stimulators include, but are not limited to, an anti-PD-1 antibody, an anti-PD-L2 antibody, an anti-CTLA-4 antibody, an anti-CD28 antibody, an anti-CD80 antibody, an anti-CD86 antibody, an anti-4-1BB antibody, an anti-OX40 antibody, an anti-KIR antibody, an anti-Tim-3 antibody, an anti-LAG3 antibody, an anti-CD27 antibody, an anti-CD40 antibody, an anti-GITR antibody, an anti-TIGIT antibody, an anti-CD20 antibody, an anti-CD96 antibody, or an anti-IDO1 antibody.

[0225] In some embodiments, the FAP targeting agents disclosed herein can be used alone or in conjunction with radiation therapy.

[0226] In some embodiments, the FAP targeting agents disclosed herein can be used alone or in conjunction with targeted therapy. Examples of targeted therapy include hormone therapy, signal transduction inhibitors (e.g., EGFR inhibitors such as cetuximab (Erbitux) and erlotinib (Tarceva)); HER2 inhibitors (e.g., trastuzumab (Herceptin) and pertuzumab (Perjeta)); BCR-ABL inhibitors (e.g., imatinib (Gleevec) and dasatinib (Sprycel)); ALK inhibitors (e.g., crizotinib (Xalkori) and ceritinib (Zykadia)); BRAF inhibitors (e.g., These include: anti-cancer drugs (e.g., vemurafenib (Zelboraf) and dabrafenib (Tafinlar)), gene expression modulators, apoptosis inducers (e.g., bortezomib (Velcade) and carfilzomib (Kyprolis)), angiogenesis inhibitors (e.g., bevacizumab (Avastin) and ramucirumab (Cyramza)), monoclonal antibodies conjugated to toxins (e.g., brentuximab vedotin (Adcetris) and ado-trastuzumab emtansine (Kadcyla)).

[0227] In some embodiments of the present disclosure, the FAP targeting agent of the present disclosure is administered in association with a STING agonist, e.g., as part of a pharmaceutical composition. The cyclic dinucleotide (CDN) cyclic di-AMP (produced by Listeria monocytogenes and other bacteria) and its analogs cyclic di-GMP and cyclic GMP-AMP are recognized by host cells as pathogen-associated molecular patterns (PAMPs) that bind to a pathogen recognition receptor (PRR) known as Stimulator of Interferon Genes (STING). STING is an adaptor protein in the cytoplasm of host mammalian cells that activates the TANK-binding kinase (TBK1)-IRF3 and NF-κB signaling axis, resulting in the induction of IFN-β and other gene products that potently activate innate immunity. STING is now recognized as a component of the host cytosolic surveillance pathway that senses infection by intracellular pathogens and responds by inducing the production of IFN-α, leading to the development of an adaptive defensive pathogen-specific immune response consisting of both antigen-specific CD4+ and CD8+ T cells and pathogen-specific antibodies. U.S. Patent Nos. 7,709,458 and 7,592,326; WO2007 / 054279, WO2014 / 093936, WO2014 / 179335, WO2014 / 189805, WO2015 / 185565, WO2016 / 096174, WO2016 / 145102, WO2017 / 027645, WO2017 / 027646, and WO2017 / 075477 (all of which are incorporated by reference); and Yan et al., Bioorg. Med. Chem Lett. 18:5631-4, 2008.

[0228] In some embodiments of the present disclosure, the FAP targeting agent of the present disclosure is administered in conjunction with an Akt inhibitor. Exemplary AKT inhibitors include GDC0068 (also known as GDC-0068, ipatasertib and RG7440), MK-2206, perifosine (also known as KRX-0401), GSK690693, AT7867, triciribine, CCT128930, A-674563, PHT-427, Akti-1 / 2, afuresertib ( GSK2110183 (also known as GSK2110183), AT13148, GSK2141795, BAY1125976, uprosertib (also known as GSK2141795), AKT inhibitor VIII (1,3-dihydro-1-[1-[[4-(6-phenyl-1H-imidazo[4,5-g]quinoxalin-7-yl)phenyl]m-ethyl]-4-piperidinyl]-2H-benzimidazol-2-one), Akt inhibitor X (2-chloro-N,N-diethyl -10H-phenoxazine-10-butanamine, monohydrochloride), MK-2206 (8-(4-(1-aminocyclobutyl)phenyl)-9-phenyl-[1,2,4]triazolo[3,4-f][-1,6]naphthyridin-3(2H)-one), uprosertib (N-((S)-1-amino-3-(3,4-difluorophenyl)propan-2-yl)-5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)furan-2-carboxamide), Patasertib ((S)-2-(4-chlorophenyl)-1-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-(isopropylamino)propan-1-one), AZD5363 (4-piperidinecarboxamide, 4-amino-N-[(1S)-1-(4-chlorophenyl)-3-hydroxypropyl]-1-(7H-pyrrolo[2,[3-d]p-irimidin-4-yl), perifosine, GSK690693, GDC-0068, tricirbine, CCT128930, A-674563, PF-04691502, AT7867, miltefosine, PHT-427, honokiol, triciribine phosphate, and KP372-1A (10H-indeno[2,1-e]tetrazolo[1,5-b][1,2,4]triazin-10-one), Akt inhibitor IX (CAS 98510-80-6). Additional Akt inhibitors include ATP-competitive inhibitors, such as isoquinoline-5-sulfonamides (e.g., H-8, H-89, NL-71-101), azepane derivatives (e.g., (-)-balanol derivatives), aminofurazans (e.g., GSK690693), heterocycles (e.g., 7-azaindole, 6-phenylpurine derivatives, pyrrolo[2,3-d]pyrimidine derivatives, CCT128930, 3-aminopyrrolidine, anilinotriazole derivatives, spiroindoline derivatives, AZD5363, A-674563 , A-443654), phenylpyrazole derivatives (e.g., AT7867, AT13148), thiophenecarboxamide derivatives (e.g., afuresertib (GSK2110183), 2-pyridimyl-5-amidothiophene derivatives (DC120), uprosertib (GSK2141795); allosteric inhibitors, for example, 2,3-diphenylquinoxaline analogues (e.g., 2,3-diphenylquinoxaline derivatives, triazolo[3,4-f][1,6] naphthyridin-3(2H)-one derivatives (MK-2206), alkylphospholipids (e.g., edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, ET-18-OCH3), ilmofosine (BM41.440), miltefosine (hexadecylphosphocholine, HePC), perifosine (D-21266), erucylphosphocholine (ErPC), erufosine (ErPC3, erucylphosphohomocholine), indole-3-cal Binol analogues (e.g., indole-3-carbinol, 3-chloroacetylindole, diindolylmethane, diethyl 6-methoxy-5,7-dihydroindolo[2,3-b]carbazole-2,10-dicarboxylate (SR13668), OSU-A9), sulfonamide derivatives (e.g., PH-316, PHT-427), thiourea derivatives (e.g., PIT-1, PIT-2, DM-PIT-1, N-[(1-methyl-1H-pyrazol-4-yl)carbonyl]-N'-(3-bromophenyl)-thiourea), purine derivatives (e.g., triciribine (TCN, NSC)), 154020), triciribine monophosphate active analogue (TCN-P), 4-amino-pyrido[2,3-d]pyrimidine derivative API-1, 3-phenyl-3H-imidazo[4,5-b]pyridine derivative, ARQ 092), BAY 1125976, 3-methyl-xanthine, quinoline-4-carboxamide, 2-[4-(cyclohexa-1,3-dien-1-yl)-1H-pyrazol-3-yl]phenol, 3-oxo-tirucaric acid, 3α- and 3β-acetoxy-tirucaric acid, acetoxy-tirucaric acid; and irreversible inhibitors, such as natural products, antibiotics, lactoquinomycin, frenolicin B, calafungin, medelmicin, Boc-Phe-vinyl ketone, 4-hydroxynonenal (4-HNE), 1,6-naphthyridinone derivatives, and imidazo-1,2-pyridine derivatives.

[0229] In some embodiments of the present disclosure, the FAP targeting agent of the present disclosure is administered in conjunction with a MEK inhibitor.Exemplary MEK inhibitors include AZD6244 (Selumetinib), PD0325901, GSK1120212 (Trametinib), U0126-EtOH, PD184352, RDEA119 (Rafametinib), PD98059, BIX02189, MEK162 (Binimetinib), AS-703026 (Pimasertib), SL-327, BIX02188, AZD8330, TAK-733, Cobimetinib and PD318088.

[0230] In some embodiments of the present disclosure, a FAP targeting agent of the present disclosure is administered in association with both an anthracycline, such as doxorubicin, and cyclophosphamide (including pegylated liposomal doxorubicin).

[0231] In some embodiments of the present disclosure, a FAP targeting agent of the present disclosure is administered in association with both an anti-CD20 antibody and an anti-CD3 antibody, or a bispecific CD20 / CD3 binding agent (including a CD20 / CD3 BiTE).

[0232] In some embodiments of the present disclosure, the FAP targeting agent of the present disclosure is administered in conjunction with a CD73 inhibitor, a CD39 inhibitor, or both. These inhibitors can be CD73 or CD39 binding agents (such as antibodies, antibody fragments, or antibody mimetics) that inhibit ectonucleosidase activity. The inhibitor can be a small molecule inhibitor of ectonucleosidase activity, such as 6-N,N-diethyl-β-γ-dibromomethylene-D-adenosine-5'-triphosphate trisodium salt hydrate, PSB069, or PSB06126.

[0233] In some embodiments of the present disclosure, the FAP targeting agent of the present disclosure is administered in conjunction with poly ADP-ribose polymerase (PARP) inhibitors. Exemplary PARP inhibitors include olaparib, niraparib, rucaparib, talazoparib, veliparib, CEP9722, MK4827, and BGB-290.

[0234] In some embodiments of the present disclosure, the FAP targeting agent of the present disclosure is administered in association with an oncolytic virus. An exemplary oncolytic virus is Talimogene laherparepvec.

[0235] In some embodiments of the present disclosure, the FAP targeting agent of the present disclosure is administered in conjunction with a CSF-1 antagonist, such as an agent that binds to CSF-1 or CSF1R and inhibits the interaction of CSF-1 with CSF1R on macrophages. Exemplary CSF-1 antagonists include emactuzumab and FPA008.

[0236] In some embodiments of the present disclosure, the FAP targeting agent of the present disclosure is administered in association with an anti-CD38 antibody. Exemplary anti-CD38 antibodies include Daratumumab and Isatuximab.

[0237] In some embodiments of the present disclosure, the FAP targeting agent of the present disclosure is administered in association with an anti-CD40 antibody. Exemplary anti-CD40 antibodies include Selicrelumab and Dacetuzumab.

[0238] In some embodiments of the present disclosure, the FAP targeting agent of the present disclosure is administered in conjunction with an inhibitor of anaplastic lymphoma kinase (ALK). Exemplary ALK inhibitors include alectinib, crizotinib, and ceritinib.

[0239] In some embodiments of the present disclosure, the FAP targeting agent of the present disclosure is administered in conjunction with a multikinase inhibitor that inhibits one or more selected from the group consisting of VEGFR, PDGFR and FGFR family members, or an anti-angiogenesis inhibitor.Exemplary inhibitors include axitinib, cediranib, linifanib, motesanib, nintedanib, pazopanib, ponatinib, regorafenib, sorafenib, sunitinib, tivozanib, vatalanib, LY2874455, or SU5402.

[0240] In some embodiments of the present disclosure, the FAP targeting agent of the present disclosure is administered in conjunction with one or more vaccines intended to stimulate an immune response to one or more predetermined antigens. The antigen may be administered directly to the individual or may be expressed in the individual from, for example, tumor cell vaccines (e.g., GVAX), which may be autologous or allogeneic, dendritic cell vaccines, DNA vaccines, RNA vaccines, virus-based vaccines, bacterial or yeast vaccines (e.g., Listeria monocytogenes or Saccharomyces cerevisiae), etc. See, for example, Guo et al., Adv. Cancer Res. 2013; 119: 421-475; Obeid et al., Semin Oncol. 2015 August; 42(4): 549-561. The target antigen may also be a fragment or fusion polypeptide containing an immunologically active portion of the antigen listed in the table.

[0241] In some embodiments of the present disclosure, the FAP targeting agent of the present disclosure is administered in association with one or more antiemetic drugs, including, but not limited to, casopitant (GlaxoSmithKline), netupitant (MGI-Helsinn) and other NK-1 receptor antagonists, palonosetron (sold as Aloxi by MGI Pharma), aprepitant (sold as Emend by Merck and Co.; Rahway, NJ), diphenhydramine (sold as Benadryl by Pfizer; New York, NY), hydroxyzine (sold as Atarax by Pfizer; New York, NY), metoclopramide (sold as Reglan by A.H. Robins Co.; Richmond, Va.), lorazepam (sold as Ativan by Wyeth; Madison, NJ), alprazolam (sold as Xanax by Pfizer; New York, NY), New York, NY), haloperidol (sold as Haldol by Ortho-McNeil; Raritan, NJ), droperidol (Inapsine), dronabinol (sold as Marinol by Solvay Pharmaceuticals, Inc.; Marietta, Ga.), dexamethasone (sold as Decadron by Merck and Co.; Rahway, NJ), methylprednisolone (sold as Medrol by Pfizer; New York, NY), prochlorperazine (sold as Compazine by Glaxosmithkline; Research Triangle Park, N.C.), granisetron (sold as Kytril by Hoffmann-La Roche Inc.; Nutley, NJ), ondansetron (sold as Zofran by Glaxosmithkline; Research Triangle Park, N.C.), dolasetron (sold as Anzemet by Sanofi-Aventis; New York, NY). York, NY), tropisetron (sold as Navoban by Novartis; East Hanover, NJ).

[0242] Other side effects of cancer treatment include red blood cell and white blood cell deficiency.Accordingly, in some embodiments of the present disclosure, the FAP targeting agent is administered in conjunction with an agent that treats or prevents such deficiency, such as filgrastim, PEG-filgrastim, erythropoietin, epoetin alfa, or darbepoetin alfa.

[0243] In some embodiments of the present disclosure, the FAP targeting agent of the present disclosure is administered in conjunction with anti-cancer radiation therapy. For example, in some embodiments of the present disclosure, the radiation therapy is external beam radiation therapy (EBT), a method for delivering a beam of high-energy X-rays to the location of a tumor. The beam is generated outside the patient's body (e.g., by a linear accelerator) and targeted to the tumor site. These X-rays can destroy cancer cells, and careful treatment planning allows surrounding normal tissue to be spared. No radioactive source is placed inside the patient's body. In some embodiments of the present disclosure, the radiation therapy is proton beam therapy, a type of conformal radiation therapy that bombards diseased tissue with protons instead of X-rays. In some embodiments of the present disclosure, the radiation therapy is conformal external beam radiation therapy, a procedure that uses advanced technology to tailor radiation therapy to an individual's anatomy. In some embodiments of the present disclosure, the radiation therapy is brachytherapy, a temporary placement of radioactive materials inside the body, usually used to give an area an extra dose or boost of radiation. [Example]

[0244] Example 1: Synthesis of Compounds 4613B and 4613C [ka]

[0245] Experimental Section Reagents obtained from commercial sources were used without further purification. The synthesis of L-boroPro-pn was carried out using a previously described synthetic method (TS. J. Coutts et al. J. Med. Chem. 1996, 39, 2087-2094). All target compounds were purified by RP-HPLC using a Varian semi-preparative system with a Discovery C18 569226-URP-HPLC column. The mobile phase was typically made by mixing water (0.1% TFA) with acetonitrile (0.08% TFA) in a gradient. Purity, as determined by HPLC analysis, was greater than 95%. Mass spectra and HPLC retention times were recorded on a Hewlett Packard HPLC / MSD system equipped with a UV detector (monitoring at 215 nm) using an Eclipse Plus C18 RP-HPLC column (4.6 × 50 mm, 1.8 μm) with a solvent gradient of A) water (0.1% TFA) and B) acetonitrile (0.08% TFA) at 0.5 mL / min. Unless otherwise noted, all HPLC retention times are given for an eluent gradient of 2% B for the first 3 minutes, then 2% to 98% B over 6 minutes, maintained there for the next 5 minutes. 1 1H NMR spectra were recorded on a Bruker Avance 300 MHz NMR spectrometer using a 5 mm inverse multinuclear probe. Chemical shifts are reported in ppm (δ) relative to DSS (in DO).

[0246] Synthesis of compound 2 To a stirred solution of N-Boc-D-Ala-OH (1, 1.9 g, 10 mmol) in anhydrous DMF (40 mL) was added L-boroPro-pn.HCl (3.0 g, 10.5 mmol), HATU (4.0 g, 10.5 mmol), and DIEA (4.0 mL, 23 mmol) under ice-water bath cooling. The resulting mixture was stirred at room temperature for 2 h and then concentrated in vacuo. The residue was dissolved in ethyl acetate (150 mL) and washed sequentially with 0.1 N KHSO (3 × 40 mL), aqueous NaHCO (3 × 40 mL), and brine (30 mL). The organic phase was dried over anhydrous MgSO, filtered, and evaporated in vacuo to give N-Boc-D-Ala-L-boroPro-pn, which was then added to a 4 N HCl solution in dioxane (30 mL) under ice-water cooling. The resulting mixture was stirred at room temperature for 2 hours and then concentrated in vacuo. The residue was coevaporated with dichloromethane (3×30 mL) in vacuo to complete dryness. Thus, compound 2 was obtained as a white powder (3.3 g, 92% over two steps).

[0247] Synthesis of compound 3860 To a stirred solution of 6-(N'-Boc-hydrazino)-nicotinic acid (253 mg, 1 mmol) in anhydrous DMF (4 mL) was added compound 2 (375 mg, 1.05 mmol), HATU (400 mg, 10.5 mmol), and DIEA (0.40 mL, 2.3 mmol) under ice-water bath cooling. The resulting mixture was stirred at room temperature for 2 hours and then concentrated in vacuo. The residue was dissolved in dichloromethane (50 mL) and washed sequentially with aqueous NaHCO (3 × 10 mL) and brine (10 mL). The organic phase was dried over anhydrous MgSO, filtered, and evaporated in vacuo to give compound 3a, which was then dissolved in dry dichloromethane (5.0 mL) and cooled to −78 °C while adding BCl (1 M in dichloromethane, 5.0 mL) dropwise. The mixture was stirred at −78 °C for 1 hour and then concentrated in vacuo. The residue was partitioned between ether (5 mL) and water (5 mL). The aqueous phase was washed twice with additional ether (2 x 5 mL), concentrated in vacuo, and further purified by semi-preparative RP-HPLC to give compound 3860 as a white powder (280 mg, 65%). LC-MS (ESI +) m / z (relative intensity): 322.1 ([M+H] + ,95);304.1([M-H2O+H] + ,100);tr=7.4 minutes.

[0248] Synthesis of compound 4613B To a stirred solution of IRDye 800CW NHS ester (11.7 mg, 0.01 mmol) in pH 7.8 phosphate buffer (10 mL) was added compound 3860 (11 mg, 0.03 mmol) at room temperature. The pH was adjusted with 5% NaHCO3 as needed. The resulting mixture was stirred at the same temperature for 3 h and purified by semi-preparative RP-HPLC to give compound 4613B as a fluffy green powder (11 mg, 84%). LC-MS (ESI) + ) m / z (relative intensity): 1288.1 ([M-HO+H] + ,25),635.8([(M-2xH2O) / 2+H] + ,100);tr=7.7 minutes. 1 HNMR(D2O):δ1.10-1.35(m,17H),1.50-2.02(m,14H),2.20-2.80(m,6H),2.88-2.93(m,3H),3.52-3.55(m,2H),3.88-3.9 1(m,4H),4.58-4.61(m,1H),6.00-6.09(m,1H),7.12-7.21(m,5H),7.67-7.76(m,9H),8.25(d,J=9.3Hz,1H),8.40(s,1H).

[0249] Synthesis of compound 4634 Compound 4634 was obtained by reacting 6-(N'-Boc-hydrazino)-benzoic acid with compound 2 in a manner similar to the preparation of 3860. LC-MS (ESI + ) m / z (relative intensity): 605.5 ([2x(M-HO)+H] + ,100),303.3([M-H2O+H] + ,67);tr=7.7 minutes.

[0250] Synthesis of compound 4613C Compound 4613C was obtained by reacting IRDye 800CW NHS ester with 4634 in a similar manner to the preparation of 4613B from 3860. LC-MS (ESI + ) m / z (relative intensity): 1287.6 ([M-HO+H] + ,88),635.6([(M-2xH2O) / 2+H] + ,100);tr=7.9 minutes.

[0251] Example 2: Synthesis of Compounds 4536B, 6481, and 5183 [ka]

[0252] Synthesis of compound 4536B To a stirred solution of tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate (57 mg, 0.10 mmol) in anhydrous DMF (1 mL) was added HBTU (40 mg, 0.105 mmol) and DIEA (40 μL, 0.23 mmol) under ice-water bath cooling. The resulting mixture was stirred at room temperature for 15 minutes. Compound 4634 (40 mg, 0.11 mmol) was added to the above solution and stirred for an additional 2 hours. The mixture was purified by semi-preparative RP-HPLC, dried, and then redissolved in dichloromethane (0.5 mL). TFA (2 mL) was added, and the reaction mixture was stirred at room temperature overnight. After removal of TFA and dichloromethane, water (2 mL) was added, and the resulting mixture was stirred at room temperature for 1 hour to give the crude product, which was directly purified by semi-preparative RP-HPLC to give 85 mg of compound 4536B as a white powder. LC-MS (ESI + ) m / z (relative intensity): 689.2 ([M-HO+H] + ,100);tr=7.4 minutes. 1 H NMR(D2O): δ1.43(d,J=7.0Hz,3H),1.65-1.71(m,1H),2.00-2.15(m,3H),2.85-3.90(m,26H),6.93-7.00(m,2H),7.71-7.78(m,2H).

[0253] Synthesis of compound 6481 Compound 4536B (6 mg) was dissolved in water (1.0 mL). CuCl2 (1.0 M in water, 20 μL) was added. The resulting mixture was stirred for half an hour and then purified by semi-preparative HPLC eluting with 10% to 50% B (solvent A: 0.05% TFA in water; solvent B: acetonitrile). The desired fractions were collected and lyophilized to give 4 mg of compound 6481 as a blue-green powder. LC-MS (ESI) + ) m / z (relative intensity): 750.9 ([M-HO+H] + ,49),745.7([M-H2O-H] + ,29),377.5([(M-2xH2O) / 2+H] + ,100);tr=7.4 minutes.

[0254] Synthesis of compound 5183 Compound 4536B (6 mg) was dissolved in water (1.0 mL). GdCl3 (1.0 M in water, 20 μl) was added. The resulting mixture was adjusted to pH 6 with 1N NH3.H2O, stirred for half an hour, and then purified by semi-preparative HPLC eluting with 10% to 50% B (solvent A: 0.05% TFA in water; solvent B: acetonitrile). The desired fractions were collected and lyophilized to give 4 mg of compound 5183 as a white powder. LC-MS (ESI) + ) m / z (relative intensity): 843.9 ([M-HO+H] + ,32),421.8([(M-2xH2O) / 2+H] + ,100);tr=9.1 min (0-3 min: 5% B; 3-9 min: 5-15% B; 9-14 min: 15-25% B).

[0255] Example 3: Synthesis of compounds 6486S to 6489S, 6486 to 6489 [ka]

[0256] Synthesis of compound 6487S Compound 4634 was first coupled with N-Boc-Gly-OH, followed by removal of the Boc protection under the same conditions as in the preparation of compound 2 from Boc-D-Ala-OH and boroPro-pn.HCl; this was then coupled with tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate, and all -OtBu ester protection was removed under the same conditions as in the preparation of compound 4536B from 4634, to give compound 6487S as a white powder. LC-MS (ESI) + ) m / z (relative intensity): 746.4 ([M-HO+H] + ,100);tr=7.4 minutes.

[0257] Synthesis of compound 6487 Compound 6487 was prepared from compound 6487 by the same method as for making 6481 from 4536B as a blue-green powder. + ) m / z (relative intensity): 807.5 ([M-HO+H] + ,30),802.9([M-H2O-H] + ,100);tr=7.7 minutes.

[0258] Synthesis of compound 6486S Compound 6486S was prepared by the same method to produce 6487S as a white powder. + ) m / z (relative intensity): 803.3 ([M-HO+H] + ,100);tr=7.6 minutes.

[0259] Synthesis of compound 6486 Compound 6486 was prepared from compound 6486S by the same method as for making 6481 from 4536B as a blue-green powder. + ) m / z (relative intensity): 862.7 ([M-HO+H] + ,100);tr=7.7 minutes.

[0260] Synthesis of compound 6488S Compound 6488S was prepared by the same method as 6487S as a white powder. + ) m / z (relative intensity): 762.8 ([M-HO+H] + ,100);tr=7.4 minutes.

[0261] Synthesis of compound 6488 Compound 6488 was prepared from compound 6488S by the same method as for making 6481 from 4536B. LC-MS (ESI + ) m / z (relative intensity): 822.3 ([M-HO+H] + ,100);tr=7.7 minutes.

[0262] Synthesis of compound 6489S Compound 6489S was prepared by the same method as 6487S as a white powder. + ) m / z (relative intensity): 774.4 ([M-HO+H] + ,100);tr=7.5 minutes.

[0263] Synthesis of compound 6489 Compound 6489 was prepared from compound 6489S by the same method as for making 6481 from 4536B. LC-MS (ESI + ) m / z (relative intensity): 836.8 ([M-HO+H] + ,100),832([M-H2O-H] + ,63);tr=7.6 minutes.

[0264] Example 4: Synthesis of Compounds 6572 and 6572CU [ka]

[0265] Example 5: Synthesis of Compounds 6521-6522 and 6521CU-6522CU : [ka]

[0266] Example 6: Synthesis of compounds 6549 and 6551: [ka]

[0267] Example 7: Synthesis of compounds 6555 and 6556: [ka]

[0268] Example 8: Synthesis of compounds 6508-6509 and 6508CU-6509CU: [ka]

[0269] Table 2 shows the compounds of Examples 1-9. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0270] Example 9: Synthesis of GHK analogs 6415 and 6433 [ka]

[0271] Synthesis of compound 6415 Compound 6415 was prepared as a white powder in seven steps from compound 5 as shown in Scheme 4. LC-MS (ESI + ) m / z (relative intensity): 767.2 ([M-HO+H] + ,100);tr=7.6 minutes.

[0272] Synthesis of compound 6433 Compound 6415 (29 mg) was dissolved in water (0.2 mL). Cu(OAc) (0.3 M in water, 103 μl) was added. The resulting mixture was stirred for half an hour and directly lyophilized to give 11 mg of compound 6433 as a green-blue powder (11 mg). LC-MS (ESI + ) m / z (relative intensity): 829.2 ([M-HO+H] + , 100); tr = 7.5 min (Note: Solvents for this LCMS were plain. No TFA was added).

[0273] Example 10: In vitro assay Biological Materials: For in vitro IC50 determination assays, recombinant human DPPIV, DPP9, FAP, and PREP were purchased from R&D Systems, and DPP8 was purchased from Biomol International. The buffer systems used were A (25 mM Tris, pH 8.0), B (50 mM Tris, pH 7.5), C (50 mM Tris, 140 mM NaCl, pH 7.5), D (25 mM Tris, 250 mM NaCl, pH 7.5), and E (20 mM Tris, 20 mM KCl, pH 7.4). Fluorogenic substrates were Gly-Pro-AMC, Z-Gly-Pro-AMC, or Suc-Gly-Pro-AMC, purchased from Bachem, or an N-terminally blocked FAP-specific substrate. Cell culture medium was RPMI 1640 without phenol red, supplemented with 2 mM glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4500 mg / L glucose, 100 IU / mL penicillin, and 100 μg / mL streptomycin. Substrate specificity assay. The peptide library (0.21 mM) was incubated with 1 nM FAP in buffer E at 37°C for 24 h. The reaction was quenched by the addition of 1.2 N HCl. Samples were analyzed by reverse-phase HPLC-MS on a Thermo Finnigan LCQ Duo, and peaks in the resulting base-peak chromatogram were quantified. Relative cleavage values ​​were determined by comparing the post-quench abundance of intact peptides with those in the initial library.

[0274] In vitro enzyme IC50 assay. The enzyme activities of DPPIV, DPP8, DPP9, FAP, and PREP were measured at 25°C on a Molecular Devices M2e multidetection microtiter plate reader, monitoring fluorescence at an excitation wavelength of 380 nm and an emission wavelength of 460 nm. The substrate was either H-Gly-Pro-AMC for the DPPIV, DPP8, and DPP9 assays or Z-Gly-Pro-AMC for the FAP and PREP assays. The reaction mixture contained 25 μM substrate, enzyme, buffer A (DPPIV and DPP9), buffer B (DPP8), buffer C (FAP), or buffer D (PREP), and an appropriate amount of inhibitor (10 -4 ~10 -11 The assay contained 100 mM of each inhibitor (range: 0.1, 0.8, 0.4, 1.2, and 0.6 nM) in a total volume of 210 μL. The final enzyme concentrations were 0.1, 0.8, 0.4, 1.2, and 0.6 nM for DPPIV, DPP8, DPP9, FAP, and PREP, respectively. IC50 values ​​are defined as the concentration of inhibitor required to reduce enzyme activity by 50% after 10 min of preincubation with the enzyme at 25 °C before adding the substrate. Inhibitor stock solutions (100 mM) were prepared in either pH 2.0 HCl solution for compounds 1 and 20, or in DMSO. The pH 2.0 solutions were preincubated for 4 h at 25 °C before dilution. Just before the start of the experiment, the 100 mM stocks were diluted to 100 mM in the appropriate assay buffer. -3 Further dilutions were made in M ​​from which 1:10 serial dilutions were prepared. All inhibitors were tested in triplicate.

[0275] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] Table 3-5 Table 3-6 Table 3-7 Table 3-8

[0276] Example 11: Exemplary Ligands Synthesized by Similar Methods

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[0277] Example 12: Synthesis of DOTA-PNP Scheme 10 [ka]

[0278] Experimental Section Reagents obtained from commercial sources were used without further purification. All target compounds were purified by RP-HPLC using a Varian semi-preparative system with a Discovery C18 569226-U RP-HPLC column. The mobile phase for semi-preparative HPLC was typically made by mixing water (4.8 mM HCl) with acetonitrile at a gradient concentration. Mass spectra and HPLC retention times were recorded on a Hewlett Packard HPLC / MSD system equipped with an Eclipse Plus C18 RP-HPLC column (4.6 × 50 mm, 1.8 μm) and a solvent gradient of A) water (0.1% TFA) and B) acetonitrile at 0.5 mL / min, with UV detection (monitoring at 215 nm). Unless otherwise noted, all HPLC retention times are given for an eluent gradient of 2% B for the first 3 minutes, then 2% to 98% B over 6 minutes, maintained there for the next 6 minutes.

[0279] Synthesis of DOTA-PNP DOTA-PNP was synthesized using a previously described synthetic method (Walter Mier, et al., Bioconjugate Chem., 2005, 16, 237-240; T.S. J. Coutts et al., J. Med. Chem., 1996, 39, 2087-2094). DOTA (AstaTech, BN21603; 500 mg, 1.24 mmol) was dissolved in 10 mL of water. A solution of 1.24 mmol of 4-nitrophenol (TCI America, N022025G) in 8 mL of acetonitrile was added. A solution of 255 mg (1.24 mmol) of N,N'-dicyclohexylcarbodiimide in 8 mL of pyridine was added dropwise with vigorous stirring. The reaction mixture was stirred for 90 minutes and concentrated to dryness under reduced pressure. The residue was dissolved in 20% aqueous acetonitrile. The suspension was filtered to remove N,N'-dicyclohexylurea, and the filtrate was purified using a semi-preparative Discovery C18 569226-U RP-HPLC column (21.2 mm x 25 cm, 5 μm) equipped with a UV detector (monitoring at 215 nm). A gradient elution system utilized mobile phase A (4.8 mM HCl) and mobile phase B (acetonitrile). The gradient was run at a flow rate of 20 mL / min, starting from 98% A and 2% B for 5 min; increasing to 70% A and 30% B over 15 min; and holding for an additional 5 min. The combined fractions were directly lyophilized to give DOTA-PNP as a white powder (4×HCl salt, 250 mg, 30%). LC-MS (ESI) + ) m / z (relative intensity): 526.1 ([M+H] + ,100);tr=7.7 minutes.

[0280] supporting material Mass spectra and HPLC retention times were recorded on a Hewlett Packard HPLC / MSD system equipped with a ZORBAX Eclipse Plus C18 RP-HPLC column (4.6 x 50 mm, 1.8 μm) and a UV detector (monitoring at 215 nm) using a solvent gradient of A) water (0.1% TFA) and B) acetonitrile at 0.5 mL / min. The eluent gradient was 2% B for the first 3 min, then 2% to 98% B over 6 min and maintained there for the next 5 min (0–3 min: 2% B; 3–9 min: 2–98% B; 9–15 min: 98% B). MS was run in positive mode. Data were analyzed using Agilent Chemstation software.

[0281] Example 13: Synthesis of 6555 / 6555LU / 6555GA Scheme 11 [ka]

[0282] Solubility and Storage After lyophilization, the target compounds 6555, 6555LU, or 6555GA are readily soluble in water (solubility >50 mg / ml). When in aqueous solution at a pH of approximately 3, the inventors observed no signs of decomposition during HPLC purification and the subsequent lyophilization process. For long-term storage, the target compounds should be kept in solid form in a freezer at temperatures below -15°C. For short-term storage, a refrigerator (+4°C) will suffice.

[0283] Experimental Section Reagents obtained from commercial sources were used without further purification. The synthesis of L-boroPro-pn was carried out using a previously described synthetic method (T.S. J. Coutts et al. J. Med. Chem. 1996, 39, 2087-2094). All target compounds were purified by RP-HPLC using a Varian semi-preparative system with a Discovery C18 569226-U RP-HPLC column. The mobile phase for semi-preparative HPLC was typically prepared by mixing water (0.1% TFA) with acetonitrile at a gradient concentration. Mass spectra and HPLC retention times were recorded on a Hewlett Packard HPLC / MSD system equipped with an Eclipse Plus C18 RP-HPLC column (4.6 × 50 mm, 1.8 μm) and a solvent gradient of A) water (0.1% TFA) and B) acetonitrile at 0.5 mL / min, with a UV detector (monitoring at 215 nm). Unless otherwise stated, all HPLC retention times are given for an eluent gradient of 2% B for the first 3 min, then 2% to 98% B over 6 min, which was maintained for the next 6 min.

[0284] Synthesis of intermediate 1 To a stirred solution of N-Boc-D-Ala-OH (Aldrich, 15048-25G; 1.9 g, 10 mmol) in anhydrous DMF (40 mL) was added L-boroPro-pn.HCl (3.0 g, 10.5 mmol), HATU (4.0 g, 10.5 mmol), and DIEA (4.0 mL, 23 mmol) under ice-water bath cooling. The resulting mixture was stirred at room temperature for 2 h and then concentrated in vacuo. The residue was dissolved in ethyl acetate (150 mL) and washed sequentially with 0.1 N KHSO (3 × 40 mL), aqueous NaHCO (3 × 40 mL), and brine (30 mL). The organic phase was dried over anhydrous MgSO4, filtered, and evaporated in vacuo to give N-Boc-D-Ala-L-boroPro-pn, which was purified by silica gel flash chromatography eluting with ethyl acetate / hexane; then, under ice-water cooling, added to a solution of 4N HCl in dioxane (30 mL). The resulting mixture was stirred at room temperature for 2 hours and then concentrated in vacuo. The residue was coevaporated with dichloromethane (3 x 30 mL) in vacuo to complete dryness. Thus, compound 1 was obtained as a white powder (3.3 g, 92% for two steps).

[0285] Synthesis of intermediate 2 To a stirred solution of 4-[(tert-butoxycarbonylamino)methyl]benzoic acid (TCI, B4305; 505 mg, 2 mmol) in anhydrous DMF (8 mL), compound 1 (750 mg, 2.1 mmol), HATU (800 mg, 2.1 mmol), and DIEA (0.80 mL, 4.6 mmol) were added under ice-water bath cooling. The resulting mixture was stirred at room temperature for 2 hours and then concentrated in vacuo. The residue was dissolved in dichloromethane (100 mL) and washed sequentially with 0.1 N KHSO (3 × 15 mL), aqueous NaHCO (3 × 15 mL), and brine (10 mL). The organic phase was dried over anhydrous MgSO4, filtered, and evaporated in vacuo to give 4-(N-Boc-aminomethyl-PhCO-D-Ala-L-boroPro-pn), which was purified by silica gel flash chromatography eluting with ethyl acetate / hexane; then added to a solution of 4N HCl in dioxane (10 mL) under ice-water cooling. The resulting mixture was stirred at room temperature for 2 hours and then concentrated in vacuo. The residue was coevaporated with dichloromethane (3 x 20 mL) in vacuo to complete dryness. In this way, compound 2 was obtained as a white powder (830 mg, 85% for two steps). LC-MS (ESI + ) m / z (relative intensity): 453.7 ([M+H] + ,100);tr=9.0 minutes.

[0286] Synthesis of Compound 6555 (Method I) To a stirred solution of DOTA-(OtBu)3 (AstaTech, 67012, CAS: 137076-54-1; 172 mg, 0.3 mmol) in anhydrous DCM (3 mL) was added intermediate compound 2 (162 mg, 0.33 mmol), PyBOP (172 mg, 0.33 mmol), and DIEA (0.12 mL, 0.69 mmol) under ice-water bath cooling. The resulting mixture was stirred at room temperature for 3 hours, then diluted with additional dichloromethane (30 mL) and washed sequentially with 5% citric acid (3 × 5 mL), aqueous NaHCO3 (3 × 5 mL), and brine (5 mL). The organic phase was dried over anhydrous MgSO4, filtered, and evaporated in vacuo to give the crude intermediate, which was redissolved in dichloromethane (1.5 mL) and TFA (6 mL). The resulting mixture was stirred at room temperature overnight. After removing the TFA and dichloromethane, water (9 mL) was added, and the resulting mixture was stirred at room temperature for 1 hour. Then, phenylboronic acid (48 mg, 0.39 mmol), acetonitrile (3 mL), and TBME (18 mL) were added. The resulting mixture was stirred at room temperature for 3 hours, and the separated aqueous phase was washed with additional TBME. The aqueous phase was partially concentrated in vacuo and purified using a semi-preparative Discovery C18 569226-U RP-HPLC column (21.2 mm × 25 cm, 5 μm) equipped with a UV detector (monitoring at 215 nm). The gradient elution system utilized mobile phase A (0.1% TFA) and mobile phase B (acetonitrile). The gradient was run at a flow rate of 20 mL / min, starting with 95% A and 5% B for 5 minutes, increasing to 70% A and 30% B over 20 minutes, and then increasing to 2% A and 98% B over 1 minute and holding for an additional 5 minutes. The combined fractions were directly lyophilized to give 6555 as a white powder (4× TFA salt, 130 mg, 37% over three steps). LC-MS (ESI + ) m / z (relative intensity): 688.0 ([M-HO+H] + ,100),345.4(63);tr=7.6 minutes.

[0287] Synthesis of Compound 6555 (Method II) To a stirred solution of DOTA-PNP (synthesized in-house, 204 mg, 0.30 mmol) and intermediate compound 2 (189 mg, 0.33 mmol) in anhydrous DMF (4 mL), TEA (360 μL, 2.07 mmol) was added under ice-water bath cooling. The resulting mixture was stirred at room temperature overnight. The reaction mixture was then concentrated in vacuo. Water (9 mL) was added, and the pH was adjusted to approximately 1.5 with 1 N TFA. Phenylboronic acid (48 mg, 0.39 mmol), acetonitrile (3 mL), and TBME (18 mL) were added. The resulting mixture was stirred at room temperature for 3 h and worked up as described above to give 6555 as a white powder (4× TFA salt, 140 mg, 40% over two steps).

[0288] Synthesis of compound 6522LU Compound 6555 (10 mg, 8.6 μmol) was added to a solution of LuCl (18 mg, 64 μmol) in acetate buffer (0.23 M, pH 5.2, 3 mL). The resulting mixture was stirred at 90 °C for 23 minutes and then purified on a semi-preparative Discovery C18 569226-U RP-HPLC column (21.2 mm × 25 cm, 5 μm) using a UV detector (monitoring at 215 nm). A gradient elution system utilized mobile phase A (0.05% TFA in water) and mobile phase B (acetonitrile). The gradient was run at a flow rate of 20 mL / min, starting with 95% A and 5% B for 5 minutes, increasing to 70% A and 30% B over 20 minutes, then increasing to 2% A and 98% B over 1 minute and holding for an additional 5 minutes. The combined fractions were directly lyophilized to give 6555 LU as a white powder (4× TFA salt, 5 mg, 44%). + ) m / z (relative intensity): 859.5 (100); tr = 15.2 min (see attached LCMS and conditions).

[0289] Synthesis of compound 6555GA Compound 6555 (10 mg, 8.6 μmol) was added to a solution of GaCl (12 mg, 66 μmol) in acetate buffer (0.23 M, pH 5.2, 4 mL). The resulting mixture was stirred at 90 °C for 23 minutes and then purified on a semi-preparative Discovery C18 569226-U RP-HPLC column (21.2 mm × 25 cm, 5 μm) using a UV detector (monitoring at 215 nm). A gradient elution system utilized mobile phase A (0.05% TFA in water) and mobile phase B (acetonitrile). The gradient was run at a flow rate of 20 mL / min, starting with 95% A and 5% B for 5 minutes, increasing to 70% A and 30% B over 20 minutes, then increasing to 2% A and 98% B over 1 minute and holding for an additional 5 minutes. The combined fractions were directly lyophilized to give 6555GA as a white powder (4×TFA salt, 7 mg, 66%). + ) m / z (relative intensity): 754.4 (100); tr = 16.9 min (see attached LCMS and conditions).

[0290] supporting material Compound 6555 LCMS spectrum of compound 6555: The LCMS method was performed using a Hewlett Packard HPLC / MSD system equipped with a ZORBAX Eclipse Plus C18 RP-HPLC column (4.6 × 50 mm, 1.8 μm) and a UV detector (monitoring at 215 nm). The gradient elution system utilized mobile phase A (0.1% TFA) and mobile phase B (acetonitrile). The gradient started at 98% A and 2% B for 3 min, increased to 2% A and 98% B over 6 min, and maintained this for an additional 5 min at a flow rate of 0.5 mL / min. Finally, the gradient parameters were returned to the initial starting conditions. The MS was run in positive mode. Data were analyzed using Agilent's Chemstation software.

[0291] Compound 6555LU The LCMS method was performed using a Hewlett Packard HPLC / MSD system equipped with a Luna C18, 4.6 mm x 150 mm, 3.0 μm, 100 A column and a UV detector (monitoring at 215 nm). The gradient elution system utilized mobile phase A (50 mM AcONH4) and mobile phase B (acetonitrile). The gradient was run at a flow rate of 1.0 mL / min, starting with 98% A and 2% B for 5 min; increasing to 74% A and 26% B over 15 min; and then increasing to 2% A and 98% B over 5 min. Finally, the gradient parameters were returned to the initial starting conditions. The MS was run in negative mode. Data were analyzed using Agilent's Chemstation software.

[0292] Compound 6555GA The LCMS method was performed using a Hewlett Packard HPLC / MSD system equipped with a Luna C18, 4.6 mm x 150 mm, 3.0 μm, 100 A column and a UV detector (monitoring at 215 nm). The gradient elution system utilized mobile phase A (50 mM AcONH4) and mobile phase B (acetonitrile). The gradient was run at a flow rate of 1.0 mL / min, starting with 98% A and 2% B for 5 min; increasing to 74% A and 26% B over 15 min; and then increasing to 2% A and 98% B over 5 min. Finally, the gradient parameters were returned to the initial starting conditions. The MS was run in negative mode. Data were analyzed using Agilent's Chemstation software.

[0293] Example 14: Synthesis of 6952 / 6952LU / 6952GA Scheme 12. [ka]

[0294] Solubility and Storage After lyophilization, the target compounds 6952, 6952LU, or 6952GA are readily soluble in water (solubility >50 mg / ml). When in aqueous solution at a pH of approximately 3, the inventors did not observe any signs of decomposition during HPLC purification and the subsequent lyophilization process. For long-term storage, the target compounds should be kept in solid form in a freezer at temperatures below -15°C. For short-term storage, a refrigerator (+4°C) would be sufficient.

[0295] Experimental Section Reagents obtained from commercial sources were used without further purification. The synthesis of L-boroPro-pn was carried out using a previously described synthetic method (T.S. J. Coutts et al. J. Med. Chem. 1996, 39, 2087-2094). All target compounds were purified by RP-HPLC using a Varian semi-preparative system with a Discovery C18 569226-U RP-HPLC column. The mobile phase for semi-preparative HPLC was typically prepared by mixing water (0.1% TFA) with acetonitrile at a gradient concentration. Mass spectra and HPLC retention times were recorded on a Hewlett Packard HPLC / MSD system equipped with an Eclipse Plus C18 RP-HPLC column (4.6 × 50 mm, 1.8 μm) and a solvent gradient of A) water (0.1% TFA) and B) acetonitrile at 0.5 mL / min, with a UV detector (monitoring at 215 nm). Unless otherwise stated, all HPLC retention times are given for an eluent gradient of 2% B for the first 3 min, then 2% to 98% B over 6 min, which was maintained for the next 6 min.

[0296] Synthesis of intermediate 1 To a stirred solution of N-Boc-D-Ala-OH (Aldrich, 15048-25G; 1.9 g, 10 mmol) in anhydrous DMF (40 mL) was added L-boroPro-pn.HCl (3.0 g, 10.5 mmol), HATU (4.0 g, 10.5 mmol), and DIEA (4.0 mL, 23 mmol) under ice-water bath cooling. The resulting mixture was stirred at room temperature for 2 h and then concentrated in vacuo. The residue was dissolved in ethyl acetate (150 mL) and washed sequentially with 0.1 N KHSO (3 × 40 mL), aqueous NaHCO (3 × 40 mL), and brine (30 mL). The organic phase was dried over anhydrous MgSO4, filtered, and evaporated in vacuo to give N-Boc-D-Ala-L-boroPro-pn, which was purified by silica gel flash chromatography eluting with ethyl acetate / hexane; then, under ice-water cooling, added to a solution of 4N HCl in dioxane (30 mL). The resulting mixture was stirred at room temperature for 2 hours and then concentrated in vacuo. The residue was coevaporated with dichloromethane (3 x 30 mL) in vacuo to complete dryness. Thus, compound 1 was obtained as a white powder (3.3 g, 92% for two steps).

[0297] Synthesis of intermediate 2 To a stirred solution of trans-4-(tert-butoxycarbonylaminomethyl)cyclohexanecarboxylic acid (TCI, B3253; 515 mg, 2 mmol) in anhydrous DMF (8 mL) was added compound 1 (750 mg, 2.1 mmol), HATU (800 mg, 2.1 mmol), and DIEA (0.80 mL, 4.6 mmol) under ice-water bath cooling. The resulting mixture was stirred at room temperature for 2 h and then concentrated in vacuo. The residue was dissolved in dichloromethane (100 mL) and washed sequentially with 0.1 N KHSO (3 × 15 mL), aqueous NaHCO (3 × 15 mL), and brine (10 mL). The organic phase was dried over anhydrous MgSO, filtered, and evaporated in vacuo to give N-Boc-protected 2, which was purified by silica gel flash chromatography eluting with ethyl acetate / hexane; then, to a solution of 4 N HCl in dioxane (10 mL) under ice-water cooling, the resulting mixture was added. The resulting mixture was stirred at room temperature for 2 hours and then concentrated in vacuo. The residue was coevaporated with dichloromethane (3 x 20 mL) in vacuo to complete dryness. Compound 2 was thus obtained as a white powder (890 mg, 90% over two steps). LC-MS (ESI + ) m / z (relative intensity): 459.9 ([M+H] + ,100);tr=8.9 minutes.

[0298] Synthesis of Compound 6952 (Method I) To a stirred solution of DOTA-(OtBu)3 (AstaTech, 67012, CAS: 137076-54-1; 172 mg, 0.3 mmol) in anhydrous DCM (3 mL) was added intermediate compound 2 (162 mg, 0.33 mmol), PyBOP (172 mg, 0.33 mmol), and DIEA (0.12 mL, 0.69 mmol) under ice-water bath cooling. The resulting mixture was stirred at room temperature for 3 hours, then diluted with additional dichloromethane (30 mL) and washed sequentially with 5% citric acid (3 × 5 mL), aqueous NaHCO3 (3 × 5 mL), and brine (5 mL). The organic phase was dried over anhydrous MgSO4, filtered, and evaporated in vacuo to give the crude intermediate, which was redissolved in dichloromethane (1.5 mL) and TFA (6 mL). The resulting mixture was stirred at room temperature overnight. After removing the TFA and dichloromethane, water (9 mL) was added, and the resulting mixture was stirred at room temperature for 1 hour. Phenylboronic acid (48 mg, 0.39 mmol), acetonitrile (3 mL), and TBME (18 mL) were then added. The resulting mixture was stirred at room temperature for 3 hours, and the separated aqueous phase was washed with additional TBME. The aqueous phase was partially concentrated in vacuo and purified on a semi-preparative Discovery C18 569226-U RP-HPLC column (21.2 mm × 25 cm, 5 μm) using a UV detector (monitoring at 215 nm). The gradient elution system utilized mobile phase A (0.1% TFA) and mobile phase B (acetonitrile). The gradient was run at a flow rate of 20 mL / min, starting with 95% A and 5% B for 5 minutes, increasing to 70% A and 30% B over 20 minutes, and then increasing to 2% A and 98% B over 1 minute and holding for an additional 5 minutes. The combined fractions were directly lyophilized to give 6952 as a white powder (4× TFA salt, 123 mg, 35% over three steps). LC-MS (ESI + ) m / z (relative intensity): 694.1 ([M-HO+H] + ,100),348.9(29);tr=7.5 minutes.

[0299] Synthesis of Compound 6952 (Method II) To a stirred solution of DOTA-PNP (synthesized in-house, 204 mg, 0.30 mmol) and intermediate compound 2 (162 mg, 0.33 mmol) in anhydrous DMF (4 mL), TEA (360 μL, 2.07 mmol) was added under ice-water bath cooling. The resulting mixture was stirred at room temperature overnight. The reaction mixture was then concentrated in vacuo. Water (9 mL) was added, and the pH was adjusted to approximately 1.5 with 1 N TFA. Phenylboronic acid (48 mg, 0.39 mmol), acetonitrile (3 mL), and TBME (18 mL) were added. The resulting mixture was stirred at room temperature for 3 h and worked up as described above to give 6952 as a white powder (4× TFA salt, 140 mg, 40% over two steps).

[0300] Synthesis of compound 6952LU Compound 6952 (10 mg, 8.6 μmol) was added to a solution of LuCl3 (18 mg, 64 μmol) in acetate buffer (0.23 M, pH 5.2, 3 mL). The resulting mixture was stirred at 90 °C for 23 minutes and then purified on a semi-preparative Discovery C18 569226-U RP-HPLC column (21.2 mm × 25 cm, 5 μm) using a UV detector (monitoring at 215 nm). A gradient elution system utilized mobile phase A (0.05% TFA in water) and mobile phase B (acetonitrile). The gradient was run at a flow rate of 20 mL / min, starting with 95% A and 5% B for 5 minutes, increasing to 70% A and 30% B over 20 minutes, then increasing to 2% A and 98% B over 1 minute and holding for an additional 5 minutes. The combined fractions were directly lyophilized to give 6952 LU as a white powder (4× TFA salt, 5 mg, 44%). + ) m / z (relative intensity): 865.5 (100); tr = 14.9 min (see attached LCMS and conditions).

[0301] Synthesis of compound 6952GA Compound 6952 (10 mg, 8.6 μmol) was added to a solution of GaCl (12 mg, 66 μmol) in acetate buffer (0.23 M, pH 5.2, 4 mL). The resulting mixture was stirred at 90 °C for 23 minutes and then purified on a semi-preparative Discovery C18 569226-U RP-HPLC column (21.2 mm × 25 cm, 5 μm) using a UV detector (monitoring at 215 nm). A gradient elution system utilized mobile phase A (0.05% TFA in water) and mobile phase B (acetonitrile). The gradient was run at a flow rate of 20 mL / min, starting with 95% A and 5% B for 5 minutes, increasing to 70% A and 30% B over 20 minutes, then increasing to 2% A and 98% B over 1 minute and holding for an additional 5 minutes. The combined fractions were directly lyophilized to give 6952GA as a white powder (4×TFA salt, 6 mg, 57%). + ) m / z (relative intensity): 760.9 (100); tr = 16.1 min (see attached LCMS and conditions).

[0302] supporting material Compound 6952 : The LCMS method was performed using a Hewlett Packard HPLC / MSD system equipped with a ZORBAX Eclipse Plus C18 RP-HPLC column (4.6 × 50 mm, 1.8 μm) and a UV detector (monitoring at 215 nm). The gradient elution system utilized mobile phase A (0.1% TFA) and mobile phase B (acetonitrile). The gradient started at 98% A and 2% B for 3 min, increased to 2% A and 98% B over 6 min, and maintained this for an additional 5 min at a flow rate of 0.5 mL / min. Finally, the gradient parameters were returned to the initial starting conditions. The MS was run in positive mode. Data were analyzed using Agilent's Chemstation software.

[0303] Compound 6952LU The LCMS method was performed using a Hewlett Packard HPLC / MSD system equipped with a Luna C18, 4.6 mm x 150 mm, 3.0 μm, 100 A column and a UV detector (monitoring at 215 nm). The gradient elution system utilized mobile phase A (50 mM AcONH4) and mobile phase B (acetonitrile). The gradient was run at a flow rate of 1.0 mL / min, starting with 98% A and 2% B for 5 min; increasing to 74% A and 26% B over 15 min; and then increasing to 2% A and 98% B over 5 min. Finally, the gradient parameters were returned to the initial starting conditions. The MS was run in negative mode. Data were analyzed using Agilent's Chemstation software.

[0304] Compound 6952GA The LCMS method was performed using a Hewlett Packard HPLC / MSD system equipped with a Luna C18, 4.6 mm x 150 mm, 3.0 μm, 100 A column and a UV detector (monitoring at 215 nm). The gradient elution system utilized mobile phase A (50 mM AcONH4) and mobile phase B (acetonitrile). The gradient was run at a flow rate of 1.0 mL / min, starting with 98% A and 2% B for 5 min; increasing to 74% A and 26% B over 15 min; and then increasing to 2% A and 98% B over 5 min. Finally, the gradient parameters were returned to the initial starting conditions. The MS was run in negative mode. Data were analyzed using Agilent's Chemstation software.

[0305] Example 15: In Vitro Assays Dipeptidyl Peptidase IV, Fibroblast Activation Protein, and Prolyl Oligopeptidase The purpose of this assay is to determine the IC of various inhibitors against recombinant human dipeptidyl peptidase IV (DPPIV), fibroblast activation protein (FAP) or prolyl oligopeptidase (PREP). 50 The purpose is to determine:

[0306] The assay is carried out in the following steps: 1. Compounds are dissolved in DMSO to a final concentration of 100 mM. From this, 1 mM stocks are prepared in 50 mM Tris, 140 mM NaCl buffer, pH 7.5 (FAP) / 25 mM Tris, 250 mM NaCl buffer, pH 7.5 / 140 mM NaCl buffer, pH 8.0 (PREP). 2. Serially dilute (1:10) previously prepared 1 mM compound stocks into the appropriate assay buffer (FAP: 50 mM Tris, 140 mM NaCl, pH 7.5 / PREP: 25 mM Tris, 0.25 M NaCl, pH 7.5 / DPPIV: 25 mM Tris, pH 8.0) in one row of a 96-well plate. 3. Prepare 20x substrate solution (FAP and PREP: 2.5mM Z-Gly-Pro-AMC (VWR, Cat. No. I-1145.0050BA) in DMSO / DPPIV: 100mM Gly-Pro-AMC (VWR, Cat. No. 100042-646) in DMSO) by diluting the DMSO stock in the appropriate assay buffer. 4. Dilute the enzyme in the appropriate assay buffer. The final enzyme concentrations should be 0.1, 1.2, and 0.6 nM for DPPIV, FAP, and PREP, respectively. Add 180 μL to each required well in columns 2 through 10. Column 1 (A, B, C) should be prepared with 200 μL of the appropriate assay buffer as a control. Column 1 (D, E, F, G, H) should be prepared with 20 μL of the appropriate assay buffer and 180 μL of enzyme as a no-inhibitor control. 5. Add 20 μL of the compound of interest from the dilution plate prepared in step 2 to columns 2-10 of the assay plate as needed. Each sample should be tested in triplicate. Incubate at room temperature for 10 minutes, shaking the plate for the first 2 minutes. 6. Add 10 μL of 20× substrate prepared in step 3 to each well and incubate this at room temperature for 15 minutes, shaking the plate for the first 2 minutes. 7.λ ex :380, λ emRead fluorescence at 460.

[0307] The compounds with the DOTA / DOTAGA-[XXaa]n-DP core (Group I) and the results of their in vivo assays are summarized in Table 4. (DP core = [dAla / dSer / Gly]-[boroPro / Pro-nitrile], XXaa = α-amino acid) [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7] [Table 4-8] [Table 4-9] [Table 4-10] [Table 4-11] [Table 4-12] [Table 4-13] [Table 4-14] [Table 4-15] [Table 4-16] [Table 4-17]

[0308] Compounds with a DOTA / DOTAGA-alkyl-[XXaa]n-DP core (Group IA) and the results of their in vivo assays are summarized in Table 5. [Table 5-1] [Table 5-2] [Table 5-3]

[0309] Compounds with a DOTA / DOTAGA-[XXaa]n-[aromatic]-DP core (Group II) and the results of their in vivo assays are summarized in Table 6. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6] [Table 6-7] [Table 6-8] [Table 6-9]

[0310] Compounds with a DOTA / DOTAGA-alkyl-[aromatic]-DP core (Group IIA) and the results of their in vivo assays are summarized in Table 7. [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6] [Table 7-7] [Table 7-8]

[0311] Compounds with a DOTA / DOTAGA-[XXaa]n-[cycloalkyl]-DP core (Group III) and the results of their in vivo assays are summarized in Table 8. [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4]

[0312] Compounds with a DOTA / DOTAGA-alkyl-[cycloalkyl]-DP core (Group IIIA) and the results of their in vivo assays are summarized in Table 9. [Table 9]

[0313] Other compounds and their in vivo assay results are summarized in Table 10. [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4] [Table 10-5] [Table 10-6] [Table 10-7] [Table 10-8] [Table 10-9] [Table 10-10] [Table 10-11] [Table 10-12] [Table 10-13] [Table 10-14]

[0314] Example 16: 68 Preparation of Ga]-6522 [ka] The above radiopharmaceuticals [ 68 [Ga]-6522 can be prepared under the following conditions: 73 nmol of radiochemical precursor 6522 (Example 5 above), 0.5 M sodium acetate, 0.4 M N-acetylmethionine, and approximately 400 MBq GaCl were heated at 90 °C for 20 min with shaking in a total volume of 7.875 mL at pH 4.0. The reaction mixture was diluted with 40 mL of water and purified using a C18 solid-phase extraction cartridge preconditioned with ethanol and water. The product was eluted with 2 mL of ethanol, and the ethanol was evaporated. The evaporated product was diluted with 0.6 mL of 0.9% saline, and the pH was adjusted to 5.0 by adding 70 μL of 1 M NaOH. The product was sterile filtered (Millex-GV, 0.22 μm).

[0315] Labeling efficiency was analyzed by instant thin-layer chromatography (iTLC) and was typically >90%. For iTLC analysis, 1 μL of product was applied to a strip of iTLC-SG chromatography paper (Agilent, P / NSGI0001, 114 cm × 2.5 cm) and developed with 30% CH3CN / 70% 1 M NH4OAc (6.5 cm solvent transfer) to obtain the free methyl group. 68 Ga and 68 Ga-colloid (Rf approx. 0) and [ 68The purity of [Ga]-6522 and related impurities (Rf ∼0.7) was assessed. iTLC strips were analyzed using an Eckert & Ziegler AR-2000 Radio-TLC Imaging Scanner. Radiochemical purity was analyzed by high-performance liquid chromatography (HPLC) and was typically >98%. Briefly, the product was analyzed using a Phenomenex Luna 3.0 μm C18(2), 100 Å, 150 mm × 4.6 mm column. Eluent A: 50 mM aqueous ammonium acetate; Eluent B: acetonitrile. Gradient: 0–5 min, 2% B; 5–20 min, 2%–26% B; 20–25 min, 26%–98% B; 25–26 min, 98%–2% B; 26–30 min, 2% B. Flow rate: 1.0 mL / min, Radio-HPLC detector: NaI (Eckert & Zeigler FC-1000), UV: 215 nm.

[0316] Example 17: 177 Preparation of [Lu]-6522 [ka] The above radiopharmaceuticals [ 177 Lu]-6522 can be prepared under the following conditions: 73 nmol / mL of labeled precursor 6522 (Example 5 above), 80 mM sodium acetate, 0.4 M N-acetylmethionine, and 7.8 GBq / mL of 177 LuCl3 was heated at 70°C for 15 minutes with shaking in a total volume of 0.26 mL at pH 4. The reaction mixture was diluted with 2.34 mL of buffer to these final conditions: 8 mM sodium acetate, 0.2 M N-acetylmethionine, 6.5 mg / mL sodium ascorbate, and 0.1 mg / mL DTPA (pH 5). The product was sterile filtered (Millex-GV, 0.22 μm).

[0317] Labeling efficiency was analyzed by instant thin-layer chromatography (iTLC) and was typically >98%. For iTLC analysis, 1 μL of the diluted labeling solution was applied to a strip of iTLC-SA chromatography paper (Agilent P / N A120B12, 114 × 2.5 mm) and developed in 0.1 M citrate buffer (8 cm solvent displacement) to obtain the released 177 Lu (Rf>0.5) and [ 177 The product was characterized as [Lu]-6522 (Rf ∼0). iTLC strips were analyzed using an Eckert & Ziegler AR-2000 Radio-TLC Imaging Scanner. Radiochemical purity was analyzed by high-performance liquid chromatography (HPLC) and was typically >70%. Briefly, the product was analyzed using a Phenomenex Luna 3.0 μm C18(2), 100 Å, 150 mm × 4.6 mm column. Eluent A: 50 mM aqueous ammonium acetate; Eluent B: acetonitrile. Gradient: 0–5 min, 2% B; 5–20 min, 2%–26% B; 20–25 min, 26%–98% B; 25–26 min, 98%–2% B; 26–30 min, 2% B. Flow rate: 1.0 mL / min, Radio-HPLC detector: NaI (Eckert & Ziegler FC-1000), UV: 215 nm.

[0318] Example 18: 177 Additional preparation of Lu]-6522 [ka] The above radiopharmaceuticals [ 177 Lu]-6522 can be prepared under the following conditions: approximately 58 μg / mL of compound 6522 (Example 5 above), 70 mM sodium acetate, 0.2 M N-acetylmethionine, and 7.8 GBq / mL of 177 LuCl3 was heated at 90°C for approximately 15 minutes with shaking, in a total volume of 1.27 mL, at pH 4. The reaction mixture was diluted with 17.43 mL of buffer to give these final conditions: 0.2 M sodium acetate, 0.2 M N-acetylmethionine, pH 6.

[0319] Radiochemical purity was analyzed by high-performance chromatography (HPLC) and was typically >85%. Briefly, 20 μL of diluted product was analyzed using a Luna C18(2) column. Eluent A: 50 mM aqueous ammonium acetate; Eluent B: acetonitrile; gradient: 2% B (5 min), 2% to 26% B in 15 min, 98% B in 5 min; flow rate: 1.1 mL / min; detector: NaI radio detector (Eckert & Ziegler); UV / Vis at 215 nm.

[0320] Example 19: 177 Lu]-6555 [ka] The above radiopharmaceuticals [ 177 Lu]-6555 can be prepared under the following conditions: 73 nmol / mL of labeled precursor 6555 (Example 13 above), 0.2 M sodium acetate, 10 mg / mL sodium ascorbate, 5 mg / mL gentisic acid, 0.1 M N-acetylmethionine, and 4.0 GBq / mL of 177 LuCl3 was heated at 50°C for 40 minutes with shaking at pH 4.5 in a total volume of 0.5 mL. The reaction mixture was diluted with 4.5 mL of buffer to give these final conditions: 20 mM sodium acetate, 0.2 M N-acetylmethionine, 6.5 mg / mL sodium ascorbate, 0.5 mg / mL gentisic acid, and 0.1 mg / mL DTPA, pH 5.

[0321] Labeling efficiency was analyzed by instant thin-layer chromatography (iTLC) and was typically >98%. For iTLC analysis, 1 μL of the diluted labeling solution was applied to a strip of iTLC-SA chromatography paper (Agilent P / N A120B12, 114 × 2.5 mm) and developed in 0.1 M citrate buffer (8 cm solvent displacement) to obtain the released 177 Lu (Rf>0.5) and [ 177Lu]-6555 (Rf ∼0) was evaluated. iTLC strips were analyzed using an Eckert & Ziegler AR-2000 Radio-TLC Imaging Scanner. Radiochemical purity was analyzed by high-performance liquid chromatography (HPLC) and was typically >90%. Briefly, products were analyzed using a Phenomenex Luna 3.0 μm C18(2), 100 Å, 150 mm × 4.6 mm column. Eluent A: 50 mM aqueous ammonium acetate; Eluent B: acetonitrile. Gradient: 0–5 min, 2% B; 5–20 min, 2%–26% B; 20–25 min, 26%–98% B; 25–26 min, 98%–2% B; 26–30 min, 2% B. Flow rate: 1.0 mL / min, Radio-HPLC detector: NaI (Eckert & Ziegler FC-1000), UV: 215 nm. Radiochemical purity remained >90% for 3 days at room temperature.

[0322] Example 20: 177 Lu]-6952 [ka] The above radiopharmaceuticals [ 177 Lu]-6952 can be prepared as described in Example 19 using the labeled precursor 6952 (Example 14 above). Labeling efficiency was analyzed by instant thin layer chromatography (iTLC) and was typically >98%. For iTLC analysis, 1 μL of the diluted labeling solution was applied to a strip of iTLC-SA chromatography paper (Agilent P / N A120B12, 114 × 2.5 mm) and developed in 0.1 M citrate buffer (8 cm solvent displacement) to obtain the released 177 Lu (Rf>0.5) and [ 177The product was characterized as [Lu]-6952 (Rf ∼0). iTLC strips were analyzed using an Eckert & Ziegler AR-2000 Radio-TLC Imaging Scanner. Radiochemical purity was analyzed by high-performance liquid chromatography (HPLC) and was typically >90%. Briefly, the product was analyzed using a Phenomenex Luna 3.0 μm C18(2), 100 Å, 150 mm × 4.6 mm column. Eluent A: 50 mM aqueous ammonium acetate; Eluent B: acetonitrile. Gradient: 0–5 min, 2% B; 5–20 min, 2%–26% B; 20–25 min, 26%–98% B; 25–26 min, 98%–2% B; 26–30 min, 2% B. Flow rate 1.0 mL / min, Radio-HPLC detector: NaI (Eckert & Ziegler FC-1000), UV 215 nm. Radiochemical purity remained >90% for 3 days at room temperature.

[0323] Example 21: 68 Ga]-6555 [ka] The above radiopharmaceuticals [ 68 [Ga]-6555 can be prepared under the following conditions: 73 nmol of labeled precursor 6555 (Example 7 above), 0.5 M sodium acetate, 0.4 M N-acetylmethionine, and approximately 1200 MBq GaCl3 were heated at 90 °C for 20 min with shaking in a total volume of 7.875 mL at pH 4.0. The reaction mixture was diluted with 40 mL of water and purified using a C18 solid-phase extraction cartridge preconditioned with ethanol and water. The product was eluted with 3 mL of ethanol, and the ethanol was evaporated. The evaporated product was diluted with 0.5 mL of phosphate-buffered saline, and the pH was adjusted to 5.0 by adding 70 μL of 1 M NaOH. The product was sterile filtered (Millex-GV, 0.22 μm).

[0324] Labeling efficiency was analyzed by instant thin-layer chromatography (iTLC) and was typically >95%. For iTLC analysis, 1 μL of product was applied to a strip of iTLC-SG chromatography paper (Agilent, P / NSGI0001, 114 cm × 2.5 cm) and developed in 30% CH 3 CN / 70% 1 M NH 4 OAc (6.5 cm solvent transfer) to obtain the free methyl group. 68 Ga and 68 Ga-colloid (Rf approx. 0) and [ 68 The purity of [Ga]-6555 and related impurities (Rf ∼0.7) was assessed. iTLC strips were analyzed using an Eckert & Ziegler AR-2000 Radio-TLC Imaging Scanner. Radiochemical purity was analyzed by high-performance liquid chromatography (HPLC) and was typically >95%. Briefly, the product was analyzed using a Phenomenex Luna 3.0 μm C18(2), 100 Å, 150 mm × 4.6 mm column. Eluent A: 50 mM aqueous ammonium acetate; Eluent B: acetonitrile. Gradient: 0–5 min, 2% B; 5–20 min, 2%–26% B; 20–25 min, 26%–98% B; 25–26 min, 98%–2% B; 26–30 min, 2% B. Flow rate: 1.0 mL / min, Radio-HPLC detector: NaI (Eckert & Ziegler FC-1000), UV: 215 nm. Radiochemical purity remained >95% for 4 hours at room temperature.

[0325] Example 22: 68 Ga]-6952 [ka] The radiolabeled product [ 68[Ga]-6952 was formed under the following conditions: 73 nmol of labeled precursor 6952 (Example 14 above), 0.5 M sodium acetate, 0.4 M N-acetylmethionine, and approximately 1200 MBq GaCl were heated at 90 °C for 20 min with shaking in a total volume of 7.875 mL at pH 4.0. The reaction mixture was diluted with 40 mL of water and purified using a C18 solid-phase extraction cartridge preconditioned with ethanol and water. The product was eluted with 2 mL of ethanol, and the ethanol was evaporated. The evaporated product was diluted with 0.5 mL of phosphate-buffered saline, and the pH was adjusted to 5.0 by adding 65 μL of 1 M NaOH. The product was sterile filtered (Millex-GV, 0.22 μm).

[0326] Labeling efficiency was analyzed by instant thin-layer chromatography (iTLC) and was typically >95%. For iTLC analysis, 1 μL of product was applied to a strip of iTLC-SG chromatography paper (Agilent, P / NSGI0001, 114 cm × 2.5 cm) and developed with 30% CH3CN / 70% 1 M NH4OAc (6.5 cm solvent transfer) to obtain the free methyl group. 68 Ga and 68 Ga-colloid (Rf approx. 0) and [ 68The purity of [Ga]-6952 and related impurities (Rf ∼0.7) was evaluated. iTLC strips were analyzed using an Eckert & Ziegler AR-2000 Radio-TLC Imaging Scanner. Radiochemical purity was analyzed by high-performance liquid chromatography (HPLC) and was typically >95%. Briefly, the product was analyzed using a Phenomenex Luna 3.0 μm C18(2), 100 Å, 150 mm × 4.6 mm column. Eluent A: 50 mM aqueous ammonium acetate; Eluent B: acetonitrile. Gradient: 0–5 min, 2% B; 5–20 min, 2%–26% B; 20–25 min, 26%–98% B; 25–26 min, 98%–2% B; 26–30 min, 2% B. Flow rate: 1.0 mL / min, Radio-HPLC detector: NaI (Eckert & Ziegler FC-1000), UV: 215 nm. Radiochemical purity remained >95% for 4 hours at room temperature.

[0327] Example 23: In vivo biodistribution study Biodistribution studies were performed using a gamma counter in groups of tumor-bearing male Fox Chase SCID mice inoculated with the HEK-mFAP cell line. 177 Lu]-6522 (also referred to as Compound #2). 177 Fifteen mice (average weight 22.7±1.4 g) injected with [Lu]-6522 were used. 177 [Lu]-6522 was administered intravenously (IV) in 175 μL at a dose of 9.05 ± 0.70 MBq. Animals (n = 3-5) in each group were sacrificed at specific time points, and blood was collected by cardiac puncture at 4, 24, 48, and 168 h after injection, and organs were harvested. Organs were excised, weighed, and their activity was measured using a γ counter (165.6-364.3 keV). Tumor and normal tissue uptake was expressed as %ID / g.

[0328] material and method Animals and husbandry Fox Chase SCID mice strain code 236 were obtained for this study from Charles River Laboratories (Kingston, NY, USA). Animals were housed in groups of five until the start of the experiment. Animals were allowed to acclimate for 7 days before the start of the study. All animal experiments were approved by the University Health Network (UHN) Animal Care Committee and conformed to the ethical guidelines of the Canadian Council on Animal Care. Animals were housed under a 12-hour light / 12-hour dark schedule at a constant temperature (20°C) and relative humidity of 40%, with free access to food and water.

[0329] The body weights of the animals were measured and recorded 4 days after inoculation with the HEK-mFAP cell line and monitored until the day of injection of the radioactive tracer. The animals were not fasted prior to administration. The body weights on the day of administration of the tracer are shown in Table 11. [Table 11]

[0330] Cell culture and inoculation HEK-mFAP cells were cultured in RPMI 1640 (VWR, Cat. No. 45000-404) supplemented with: 1. 2 mM L-glutamine (VWR, catalog number 45000-676) 2. 10 mM HEPES (VWR, Cat. No. 45000-690) 3. 1 mM Sodium Pyruvate (VWR, Cat. No. 45000-710) 4. 4500 mg / L glucose (VWR, catalog number 45001-116) 5. 1x Penicillin-Streptomycin (VWR, Cat. No. 45000-652) 6. 10% FBS (Thermo Fisher Scientific, Catalog No. 10082147)

[0331] Cells were cultured at 37°C in a 5% CO atmosphere. Seven- to nine-week-old male Fox Chase SCIDs (Charles River Laboratories, strain code 236) were cultured at 4 × 10 cells in 100 μL of phenol red-free RPMI 1640 (VWR, catalog no. 45000-410) supplemented as described for growth medium, but without antibiotics or FBS. 6 Tumor xenografts were established by subcutaneous injection of cells into the right flank. Cells were inoculated at passage #9 with a viability of >90%. Sixteen mice were inoculated from batch 1 and 15 mice were inoculated from batch 2.

[0332] Tumor volume and mouse randomization Compound #2([ 177 The mean tumor volume of mice injected with α-Lu-6522 was 51.8 ± 44.4 mm 3 Biodistribution studies were performed 30 days after tumor cell inoculation. Tumor volume was calculated as V = length × width. 2 The calculation was performed using a ×0.5. Table 13 shows the randomization of animals by tumor volume.

[0333] TIFF0007784142000133.tif133169

[0334] [ 177 Use and quality of Lu]-6522 batches It has a radiochemical purity of 85.98% 177 One vial containing [Lu]-6522 was used (prepared according to Example 17a).

[0335] 9.05±0.70MBq [ 177 Syringes were prepared with a dose of [Lu]-6522 (Compound #2). The injected dose was calculated by subtracting the decay-corrected residual activity in the syringe after injection from the decay-corrected activity in the syringe before injection. The administered radioactivity per animal and per group is summarized in Table 15.

[0336] Anesthesia, dosage management(dose administration) Mice were anesthetized using isoflurane (Fresenius Kabi Canada Ltd.) anesthesia (5% induction, 1.5–2% maintenance). A 27 Ga catheter (27 Ga winged infusion set, 15 cm long, SAI Infusion Technologies) was placed in the tail vein, and approximately 145–175 μL of tracer was manually injected. The actual dose administered to each animal is shown in Table 15. After injection, the catheter was flushed with 30 μL of saline.

[0337] TIFF0007784142000134.tif183164

[0338] Biodistribution studies Biodistribution studies were performed at 4, 24, 48, and 168 hours post-injection (pi). Three to five mice were sacrificed at each time point, and tumors, blood samples, and normal tissues were collected and weighed, and the radioactivity in each was measured using a γ-counter. Tumor and normal tissue uptake was expressed as the mean ± SEM of the percentage of administered radioactivity per gram (%ID / g).

[0339] Gamma counting data collection Organ / tissue radioactivity was measured using a gamma counter (1480 WIZARD 3", Perkin Elmer; counting for 60 seconds per vial). Depending on the animal batch used, [ 177 Counts were converted to activity using a conversion factor obtained from a standard sample of known volume and known radioactivity (MBq) that was counted each time an organ was measured using [Lu]-6522 samples. Through this method, all activity values ​​are essentially decay-corrected for the time of injection.

[0340] The percentage of administered radioactivity per organ (%ID) was calculated using the following formula: %ID = Decay-corrected organ activity [MBq] / administered radioactivity [MBq] × 100%

[0341] The percentage of administered radioactivity per gram organ weight (%ID / g) for each organ was calculated using the following formula: %ID / g=%ID / organ weight[g]

[0342] Results and Discussion One batch of 177 A total of 15 male Fox Chase SCID mice were administered [Lu]-6522 formulations. Ex vivo gamma counting of various organs was performed at 4, 24, 48, and 168 hours (n = 3–5) after tracer administration.

[0343] The uptake results expressed as %ID / g for Compound #2 are summarized in Table 19 below.

[0344] For Compound #2, the highest tumor uptake and the lowest radioactivity concentration in blood and other normal tissues were observed 4 hours after injection. Compound #2 showed high tumor uptake as early as 4 hours after injection, which was 33.04±5.29% ID / g. As shown in Table 19, the kidney showed uptake of 2.35±0.51% ID / g after 4 hours, similar to that of Compound #1, and this uptake decreased over time, decreasing to 0.17±0.02% ID / g at 7 days after injection.

[0345] Higher uptake was found in the kidney compared to all other organs, suggesting that the primary excretion route was via the kidney. Mouse skin was observed to exhibit high radioactivity 4 hours after injection, which may be due to excretion of the compound in the urine and contamination of mouse skin with radioactive urine. TIFF0007784142000135.tif108170

[0346] conclusion Compound #2([ 177 Lu]-6522) showed high localization in tumor xenografts and low normal tissue uptake up to 168 hours after administration.

[0347] Example 24: Efficacy and survival studies The purpose of this study was to evaluate tumor growth delay and median survival. 177 Lu-PNT2004([ 177 The objective of this study was to evaluate the therapeutic efficacy of a single injection of [Lu]-6522.

[0348] 177 Lu-PNT2004([ 177 Lu]-6522) was provided in three concentrations, ready for injection (80 μL / mouse), and administered radioactivity was determined using a well counter (Capintec calibrated) #430x10.

[0349] [ 177 Lu]-6522 is provided as described above in Example 17. The following treatment compositions were prepared: 1. Vehicle (selected formulation, 100 μL) 2. Precursor (6522 compound) (80 μL) 3. 177 Lu]-6522 15MBq (80μL) 4. 177 Lu]-6522 30MBq (80μL) 5. 177 Lu]-6522 60MBq (80μL)

[0350] A total of 30 HEK-mFAP tumor-bearing mice were used in the study. Tumor xenografts were established in male Fox Chase SCID mice (6–8 weeks old, Charles River Laboratories) by subcutaneous injection of 5 million HEK-mFAP cells in 100 μL of PBS into the right flank.

[0351] Health checks of the mice were performed weekly throughout the study, including weight measurements. Tumor growth was monitored weekly by caliper measurements (tumor volume = length x width). 2× 0.5). Study endpoints included tumor size >2 cm in any dimension, tumor ulceration, moribund mice, and loss of >15% body weight since the last measurement. Mice were housed five per cage with free access to food and water at an ambient temperature of 20°C, 40%-50% humidity, and a 12-hour light / 12-hour dark cycle.

[0352] Mice were randomized into five groups, with n=6 mice per group. Treatment compositions (1-5 above) were injected intravenously through the tail vein using a catheter (fitted with a 30 Ga needle). Administered radioactivity was determined using a well counter (Capintec calibrated). Tumor growth was monitored weekly by caliper measurement, and mice were followed for survival.

[0353] result: Data were collected as tumor volume and survival analysis. No weight loss was observed in any of the treatment groups. ·[ 177 Only treatment with the 60MBq dose of [Lu]-6522 demonstrated a statistically significant survival benefit compared to the vehicle or precursor groups (see Figure 2). 177 All mice from the [Lu]-6522 60 MBq group survived for more than 50 days after treatment (see Figure 2). ·[ 177 Tumor growth delay was observed in the [Lu]-6522 15MBq and 30MBq groups (Fig. 1), but this tumor growth delay did not translate into a survival benefit (Fig. 2). ·[ 177 In the [Lu]-6522 60MBq group, tumors regressed until approximately day 43 after treatment, after which they began to regrow (see Figure 1). The study ended 57 days after treatment initiation.

[0354] Example 25: 68 PET imaging and biodistribution of Ga-6555 Part 1. Dynamic PET imaging. The purpose of this study is to investigate the dynamics of HEK-mFAP tumor-bearing mice. 68Ga-6555 PET / CT dynamic imaging will be performed to assess tumor uptake and retention over time, as well as nonspecific uptake. 68 PET imaging of Ga-6555 (prepared according to Example 20) was performed using a dedicated small animal PET / CT scanner (Siemens Multimodality Inveon, Siemens Medical Solutions USA, Inc.). Mice were anesthetized with 3% isoflurane / medical air inhalation before radiotracer injection and throughout the scan. During the unconscious period, warming was used to maintain healthy core body temperatures in the mice. 68 After a bolus intravenous injection (via the lateral tail vein) of Ga-6555 (mean 8 MBq, range 7.7–8.1 MBq), dynamic emission scans were acquired in list-mode format over 60 min. The acquired data were then sorted into 0.5 mm sinogram bins and 19 time frames for image reconstruction using FORE / 3D-OSEM-MAP. After PET acquisition, a low-dose CT scan (80 kVp, 0.5 mA) was acquired for anatomical reference and to provide guidance for delineation of selected tissue volumes of interest (VOIs). Reconstructed PET / CT images were analyzed using Siemens Inveon Research Workplace software. Radioactivity retention within selected tissues was obtained from the mean voxel intensity values ​​within the VOI and then converted to megabecquerels per milliliter using calibration factors determined for the Inveon PET System. These values ​​were then divided by the administered activity in megabecquerels and the animal's body weight to obtain the image VOI-derived standardized uptake value (SUV). We used the maximum SUV value (SUVmax) within the VOI as a quantitative imaging metric independent of tissue-specific variations. PET images shown are axial, coronal, and sagittal sections with the mouse placed in the prone position.

[0355] 68Ga-6555 uptake was observed in the tumor and elimination organs (kidneys and bladder) in three mice. The time-activity curve of 68Ga-6555 in the tumor showed rapid accumulation (<5 min) and retention in the tumor, reaching a plateau at 60 min. Data from one mouse are shown in Figure 3.

[0356] Part 2: Biodistribution. The purpose of this study was to evaluate the biodistribution of 100% β-lactam serogroup A in HEK-mFAP tumor-bearing mice. 68 The purpose of this study was to evaluate the biodistribution of Ga-6555. HEK-mFAP tumor-bearing mice were used in the study (N=3). Approximately 8 MBq (range 7.3-8.5 MBq) of Ga-6555 was administered to the mice. 68 Ga-6555 (prepared as in Example 20; administered IV into the tail vein using a catheter equipped with a 30 Ga needle) was injected. After a 50-minute uptake period (injections were performed under anesthesia with isoflurane inhalant, and anesthesia was maintained for 50 minutes), mice were euthanized (with CO2) and tissues were harvested (blood via cardiac puncture, heart, lung, liver, spleen, pancreas, stomach, small intestine, kidney, muscle, femur, bone, skin, brain, tumor). After resection, tissue samples were counted for gallium-68 radioactivity using a Cobra-II Auto-Gamma counter (Packard Instruments, Meriden, CTA), weighted, and data expressed as a percentage of administered radioactivity per gram (%ID / g).

[0357] The majority of the activity was in the tumor (mean %ID / g of 10.1). The kidney had the next highest activity (mean %ID / g of 1.37). All other selected tissues had low uptake levels similar to muscle and were considered background levels. TIFF0007784142000136.tif84170

[0358] Example 26: 68 PET imaging and biodistribution of Ga-6952 Prepared according to Example 22 68 Performed according to Example 24 using Ga-6952.

[0359] Part 1: Dynamic PET imaging. Approximately 8.6 MBq (range 7.6–10.0 MBq) of PET was administered to mice. 68 Ga-6952 was injected. 68 Ga-6952 uptake was observed in the tumor and in the absent organs (kidney and bladder), which was consistent in three mice. 68 The time versus activity curve of Ga-6952 in tumors showed rapid accumulation (<5 min) and retention in tumors, reaching a plateau at 60 min.

[0360] Biodistribution. Mice were injected with approximately 8.6 MBq (range 7.6-10.0 MBq). The majority of the activity was in the tumor (mean %ID / g of 8.8). The kidney had the next highest activity (mean %ID / g of 2.18). All other selected tissues had low uptake levels similar to muscle and were considered background levels. The data are shown below. TIFF0007784142000137.tif84170

[0361] Example 26: Treatment Protocol Human patients are selected for treatment after being diagnosed with metastatic cancer.

[0362] in sterile aqueous solution 177 Lu]-6522 is administered by intravenous injection. The dosing regimen may include four infusions of 6.8 GBq each administered four weeks apart. Finally, preferred embodiments of the present invention are described in sections. [Embodiment 1] A compound represented by formula I below, or a pharmaceutically acceptable salt thereof: [ka] [In the formula, R represents a radioactive moiety, a chelator (optionally having a metal ion chelated thereto), a fluorescent moiety, a photoacoustic reporter molecule, a Raman-active reporter molecule, an imaging agent, a detectable nanoparticle, or an enzyme; R 1 is (C 1 -C 6 ) represents alkyl; R 2 is -B(-Y1 )(-Y 2 ) or -CN; Y 1 and Y 2 are independently -OH or together with the boron atom to which they are attached represent a group hydrolyzable to boronic acids or together with the boron atom to which they are attached form a 5- to 8-membered ring hydrolyzable to boronic acids; R 3 is H or (C 1 -C 6 ) represents alkyl; R 4 are absent or, independently, (C 1 -C 6 ) Alkyl, -OH, -NH 2 and halogen; X represents O or S; L represents a bond or a linker. [Embodiment 2] R 1 Ha-CH 3 or -CH 2 CH 3 2. The compound of embodiment 1, wherein [Embodiment 3] R 1 Ha-CH 3 2. The compound of embodiment 1, wherein [Embodiment 4] R 2 is -B(-Y 1 )(-Y 2 4. The compound according to any one of embodiments 1 to 3, wherein [Embodiment 5] R 2 -B(OH) 2 4. The compound according to any one of embodiments 1 to 3, wherein [Embodiment 6] R 3 6. The compound of any one of embodiments 1 to 5, wherein represents H. [Embodiment 7] R 4 7. The compound of any one of embodiments 1 to 6, wherein is absent. [Embodiment 8] The compound according to any one of embodiments 1 to 7, wherein X represents O. [Embodiment 9] A compound represented by the following formula II or III, or a pharmaceutically acceptable salt thereof:

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Claims

1. A compound of the formula: 【Chemistry 1】 【Chemistry 2】 and 【Transformation 3】 or a pharmaceutically acceptable salt thereof.

2. A compound of the formula: 【Chemistry 4】 or a pharmaceutically acceptable salt thereof.

3. A compound of the formula: 【Transformation 5】 or a pharmaceutically acceptable salt thereof.

4. A compound of the formula: 【Transformation 6】 or a pharmaceutically acceptable salt thereof.

5. A complex comprising a compound according to any one of claims 1 to 4 and a radionuclide complexed to said compound.

6. The radionuclide is ４３ Sc, ４４ Sc, ５１ Mn, ５２ Mn, ６４ Cu, ６７ Ga, ６８ Ga, ８６ Y. ８９ Zr, ９４ｍ Tc, or ９９ｍ 6. The complex of claim 5, wherein the complex is Tc.

7. The radionuclide is １１１ In, １４９ Tb, １５２ Tb, １５５ Tb, ２０１ Tl, or ２０３ 6. The complex of claim 5, wherein the metal is Pb.

8. The radionuclide is ４３ Sc, ４４ Sc, ５１ Mn, ５２ Mn, ６４ Cu, ６７ Ga, ６８ Ga, ８６ Y. ８９ Zr, ９４ｍ Tc, ９９ｍ Tc, １１１ In, １４９ Tb, １５２ Tb, １５５ Tb, ２０１ Tl, or ２０３ 6. The complex of claim 5, wherein the metal is Pb.

9. The radionuclide is ４３ Sc, ４４ Sc, ６４ Cu, ６７ Ga, ６８ Ga, ８６ Y. ８９ Zr, ９９ｍ Tc, １１１ In, １５２ Tb, １５５ Tb, or ２０３ 6. The complex of claim 5, wherein the metal is Pb.

10. The radionuclide is ６４ Cu, ６８ Ga, ８９ Zr, ９９ｍ Tc, or １１１ 6. The complex of claim 5, wherein In is In.

11. The radionuclide is ４７ Sc, ６７ Cu, ８９ Sr, ９０ Y. １５３ Sm, １４９ Tb, １６１ Tb, １７７ Lu, １８６ Re, １８８ Re, ２１２ Pb, ２１３ Bi, ２２３ Ra, ２２５ Ac, ２２６ Th, ２２７ Th, or ２１１ 6. The complex of claim 5, wherein At is At.

12. The radionuclide is ４７ Sc, ６７ Cu, ９０ Y. １６１ Tb, １７７ Lu, １８８ Re, ２１２ Pb, ２１３ Bi, ２２５ Ac, ２２７ Th, or ２１１ 6. The complex of claim 5, wherein At is At.

13. The radionuclide is ９０ Y. １６１ Tb, １７７ Lu, ２２５ Ac, ２２７ Th, or ２１１ 6. The complex of claim 5, wherein At is At.

14. 6. The complex of claim 5, wherein said radionuclide is 177-Lu.

15. 6. The complex of claim 5, wherein the radionuclide is 68-Ga.

16. 6. The complex of claim 5, wherein said radionuclide is 225-Ac.

17. 6. The complex of claim 5, wherein said radionuclide is 64-Cu.

18. A pharmaceutical composition comprising a compound according to any one of claims 1 to 4 or a complex according to any one of claims 5 to 17.

19. 19. The pharmaceutical composition of claim 18, further comprising a pharmaceutically acceptable carrier and / or excipient.

20. 20. The pharmaceutical composition of claim 18 or 19, formulated for use in a human subject.

21. A pharmaceutical composition for use in the treatment of cancer, comprising a complex according to any one of claims 5 to 17.

22. 22. The pharmaceutical composition of claim 21, wherein the cancer is prostate cancer.

23. 23. The pharmaceutical composition of claim 21 or 22, wherein the cancer is a metastatic cancer.

24. A pharmaceutical composition for use in image-guided surgery, comprising a complex according to any one of claims 5 to 17.

25. 25. The pharmaceutical composition of any one of claims 18 to 24, further comprising one or more stabilizer compounds.

26. 26. The pharmaceutical composition of claim 25, wherein the one or more stabilizer compounds comprise a sulfur-containing compound.

27. 26. The pharmaceutical composition of claim 25, wherein the one or more stabilizer compounds comprise one or more sulfide moieties.

28. 26. The pharmaceutical composition of claim 25, wherein the one or more stabilizer compounds comprises N-acetylmethionine.

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