Radioactive drugs and imaging agents targeting FAP and related uses

Abstract

The tumor stroma, which occupies a large part of the tumor mass, represents an attractive target for the delivery of diagnostic and therapeutic compounds. Here, particular emphasis is on a subset of stromal cells, called cancer-associated fibroblasts, which are present in more than 90% of epithelial cancers, including pancreatic, colon, and breast cancers. Cancer-associated fibroblasts are characterized by high expression of FAP, which is not detectable in normal adult tissues but is associated with poor prognosis in cancer patients. The present invention provides small molecule radiopharmaceuticals and imaging agents based on FAP-specific inhibitors.

Description

[0001] Citation of Related Applications

[0002] This application claims the benefit of the priority of U.S. Provisional Patent Application No. 63 / 993,874, filed on March 24, 2020, which is hereby incorporated by reference in its entirety. Background Art

[0003] The growth and spread of tumors are determined not only by cancer cells, but also by the non-malignant components of the malignant lesion, which are subsumed under the term stroma. In tumors with a desmoplastic reaction such as breast cancer, colon cancer, and pancreatic cancer, the stroma can account for more than 90% of the mass. In particular, a subset of fibroblasts known as cancer-associated fibroblasts has been associated with tumor growth, migration, and progression. Thus, these cells represent attractive targets for diagnosis and anti-tumor therapy.

[0004] A distinguishing 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 has both dipeptidyl peptidase and endopeptidase activities. The endopeptidase activity distinguishes FAP from other members of the dipeptidyl peptidase 4 family. Substrates of the endopeptidase activity identified to date are denatured type I collagen, α1-antitrypsin, and several neuropeptides. FAP plays a role in normal development and tissue modeling during embryogenesis. Its expression in adult normal tissues is only insignificant or absent. However, high expression occurs in wound healing, arthritis, atherosclerotic plaques, fibrosis, and more than 90% of epithelial cancers.

[0005] The presence of FAP in cancer-associated fibroblasts (CAFs) in many epithelial tumors and the fact that overexpression in cancer patients is associated with a poor prognosis have led to the hypothesis that FAP activity is associated with cancer development, cancer cell migration, and cancer spread. Thus, targeting this enzyme for imaging and intraluminal radiotherapy can be considered a promising strategy for detecting and treating malignant tumors. Summary of the Invention

[0006] The tumor stroma, which occupies a large part of the tumor mass, represents an attractive target for delivering diagnostic and therapeutic compounds. Here, particular emphasis is on a subset of stromal cells, called cancer-associated fibroblasts (CAFs), which are present in more than 90% of epithelial cancers, including pancreatic cancer, colon cancer, and breast cancer. Cancer-associated fibroblasts are characterized by high expression of FAP, which is undetectable in adult normal tissues but is associated with a poor prognosis in cancer patients.

[0007] The present invention provides small molecule radiopharmaceuticals and imaging agents based on FAP-specific inhibitors. In certain embodiments, the FAP targeting agent has a structure represented by Formula I or a pharmaceutically acceptable salt thereof:

[0008]

[0009] in:

[0010] R represents a radioactive moiety, a chelator, a fluorescent moiety, a photoacoustic reporter, a Raman-active reporter, a contrast agent, a detectable nanoparticle, or an enzyme;

[0011] R1 represents a (C1-C6) alkyl group;

[0012] R2 represents -B(-Y 1 )(-Y 2 ) or -CN;

[0013] Y 1 and Y 2 are independently -OH, or together with the boron atom to which they are attached represent a group that can be hydrolyzed to boronic acid, or together with the boron atom to which they are attached form a 5- to 8-membered ring that can be hydrolyzed to boronic acid;

[0014] R3 represents H or (C1-C6) alkyl;

[0015] R4 is absent or represents 1, 2 or 3 substituents, each independently selected from (C1-C6)alkyl, -OH, -NH2 and halogen;

[0016] X represents O or S;

[0017] L represents a bond or linker.

[0018] In certain preferred embodiments, the compounds of Formula I contain one or more radioactive isotopes.

[0019] In certain preferred embodiments, the compounds of Formula I contain one or more therapeutic radioisotopes.

[0020] In certain preferred embodiments, the compounds of Formula I contain one or more diagnostic radioisotopes.

[0021] In certain preferred embodiments, R is a radioactive moiety.

[0022] In certain preferred embodiments, R is a chelating agent.

[0023] In another preferred embodiment, R is a chelating agent and the compound of formula I comprises one or more radioisotopes. In certain aspects of such embodiments, one or more of the radioisotopes may be therapeutic radioisotopes. In certain other aspects of such embodiments, one or more of the radioisotopes may be diagnostic radioisotopes.

[0024] In another preferred embodiment, R is a chelating agent comprising one or more complexed radioisotopes. In certain aspects of such embodiments, one or more of the radioisotopes may be therapeutic radioisotopes. In certain other aspects of such embodiments, one or more of the radioisotopes may be diagnostic radioisotopes.

[0025] In certain preferred embodiments, R1 represents -CH3 or -CH2CH3, and even more preferably represents -CH3.

[0026] In certain preferred embodiments, R2 represents -B(-Y 1 )(-Y 2 ), and even more preferably represents -B(OH)2.

[0027] In certain preferred embodiments, R3 represents H.

[0028] In certain preferred embodiments, R4 is absent.

[0029] In certain preferred embodiments, X represents O.

[0030] In certain preferred embodiments, the compound is represented by formula II or formula III or a pharmaceutically acceptable salt thereof:

[0031]

[0032]

[0033] wherein R and L are as defined above.

[0034] In certain preferred embodiments, the compound of formula II or III comprises one or more radioisotopes.

[0035] In certain preferred embodiments, the compound of formula I or III comprises one or more therapeutic radioisotopes.

[0036] In certain preferred embodiments, the compound of formula II or III comprises one or more diagnostic radioisotopes.

[0037] In certain preferred embodiments of the compound of formula II or III, R is a radioactive moiety.

[0038] In certain preferred embodiments of the compound of Formula II or III, R is a chelating agent.

[0039] In further preferred embodiments of the compound of Formula II or III, R is a chelating agent and the compound of Formula I comprises a radioisotope. In certain aspects of such embodiments, one or more of the radioisotopes may be therapeutic radioisotopes. In certain other aspects of such embodiments, one or more of the radioisotopes may be diagnostic radioisotopes.

[0040] In additional preferred embodiments, R is a chelating agent comprising one or more complexed radioisotopes. In certain aspects of such embodiments, one or more of the radioisotopes may be therapeutic radioisotopes. In certain other aspects of such embodiments, one or more of the radioisotopes may be diagnostic radioisotopes.

[0041] In certain embodiments, the FAP targeting agent comprises two or more FAP inhibitor moieties covalently linked to a radiopharmaceutical or imaging agent, such as having a structure represented by Formula IV or a pharmaceutically acceptable salt thereof:

[0042]

[0043] wherein

[0044] R, R1, R2, R3, R4, X and L are as defined above; and

[0045] n represents an integer between 2 and 6.

[0046] In certain preferred embodiments, the compound of Formula IV comprises one or more radioisotopes.

[0047] In certain preferred embodiments, the compound of Formula IV comprises one or more therapeutic radioisotopes.

[0048] In certain preferred embodiments, the compound of Formula IV comprises one or more diagnostic radioisotopes.

[0049] In certain preferred embodiments of the compound of Formula IV, R is a radioactive moiety.

[0050] In certain preferred embodiments of the compound of Formula IV, R is a chelating agent.

[0051] In further preferred embodiments of the compound of Formula IV, R is a chelating agent and the compound of Formula IV comprises a radioisotope. In certain aspects of such embodiments, one or more of the radionuclides may be therapeutic radioisotopes. In certain other aspects of such embodiments, one or more of the radioisotopes may be diagnostic radioisotopes.

[0052] In another preferred embodiment, R is a chelator comprising one or more complexed radionuclides. In certain aspects of such embodiments, one or more of the radionuclides may be therapeutic radionuclides. In certain other aspects of such embodiments, one or more of the radionuclides may be diagnostic radionuclides.

[0053] 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 the tumor distribution of the molecule. Such a PK / BD-modified FAP targeting agent may have a structure represented by Formula V or a pharmaceutically acceptable salt thereof:

[0054]

[0055] wherein

[0056] R, R1, R2, R3, R4, X, and L are as defined above;

[0057] R5 represents a moiety that modifies the pharmacokinetics and / or biodistribution of the molecule; and n represents an integer between 1 and 6.

[0058] In certain preferred embodiments, the compound of Formula V comprises one or more radioisotopes.

[0059] In certain preferred embodiments, the compound of Formula V comprises one or more therapeutic radioisotopes.

[0060] In certain preferred embodiments, the compound of Formula V comprises one or more diagnostic radioisotopes.

[0061] In certain preferred embodiments of the compound of Formula V, R is a radioactive moiety.

[0062] In certain preferred embodiments of the compound of Formula V, R is a chelator.

[0063] In another preferred embodiment of the compound of Formula V, R is a chelator and the compound of Formula I comprises a radioisotope. In certain aspects of such embodiments, one or more of the radioisotopes may be therapeutic radioisotopes. In certain other aspects of such embodiments, one or more of the radioisotopes may be diagnostic radioisotopes.

[0064] In another preferred embodiment, R is a chelator comprising one or more complexed radioisotopes. In certain aspects of such embodiments, one or more of the radionuclides may be therapeutic radioisotopes. In certain other aspects of such embodiments, one or more of the radionuclides may be diagnostic radioisotopes.

[0065] The present invention also provides a pharmaceutical composition, which comprises at least one compound of any one of Formulae I-V and optionally a pharmaceutically acceptable carrier and / or excipient. In certain embodiments, the pharmaceutical composition is intended for diagnosing or treating a disease characterized by overexpression of fibroblast activation protein (FAP) in an animal (preferably a human subject).

[0066] Yet another aspect of the present invention provides a kit, which comprises at least one compound of any one of Formulae I-V and instructions for diagnosing or treating a disease or consists of the same.

[0067] And yet another aspect of the present invention provides a method for diagnosing, imaging or reducing tissues overexpressing FAP in an animal (preferably a human patient), comprising administering to the animal at least one compound of any one of Formulae I-V.

[0068] Also provided is a method for treating a subject suffering from a tumor or cancer, which may comprise administering to a subject in need thereof an effective amount of one or more compounds disclosed herein, including one or more compounds of any one of Formulae I to V. The subject for treatment may include a human patient diagnosed with cancer such as a tumor (e.g., a solid tumor), including diagnosing and selecting a subject for treating prostate cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 Show 177 The tumor growth curve of Lu-6522.

[0070] Figure 2 Show 177 The survival curve of Lu-6522.

[0071] Figure 3 Show 68 The accumulation and retention of Ga-6522 over time after administration. DETAILED DESCRIPTION

[0072] Tumor masses are composed of cancer cells as well as vascular structures, inflammatory cells, fibroblasts and collagen, which together constitute the tumor stroma and account for up to 90% of the mass in highly desmoplastic cancers. Cancer cells induce fibroblast activation via TGFβ. CAFs have a supportive function in cancer growth and invasion. They contribute to the remodeling of the extracellular matrix (collagenolysis) and promote invasion and angiogenesis, and can induce epithelial-mesenchymal transition of epithelial cells via growth factor and cytokine secretion. CAFs are also involved in the immune interaction between the tumor and the host.

[0073] FAP-positive CAFs are found in over 90% of epithelial cancers and thus represent a potential pan-cancer target. Targeting FAP to deplete stromal CAFs can disrupt cancer-supportive functions and inhibit cancer growth. In addition, by breaking down the stromal barrier, the effectiveness of other pharmacological, immunological, radiation, or cell-based systemic therapies can thereby be enhanced.

[0074] Targeting CAFs with FAP radiopharmaceuticals is thought to have multiple anti-tumor modes of action, but mainly relies on inducing DNA damage in tumor cells by ionizing radiation locally emitted from adjacent CAFs targeted by the therapy. FAP-targeted radiotherapy can deliver ionizing radiation directly to CAFs, as well as to cancer cells, via the crossfire effect. Combining α- and β-emitters can improve these dual anti-tumor effects via short-range α-radiation to CAFs and medium- to long-range β-radiation to cancer cells.

[0075] Definition

[0076] In the following, some definitions of terms frequently used in this specification are provided. These terms will respectively have the defined meaning and the preferred meaning in each case where they are used in the remainder of the 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. In the following term definitions, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, alkenyl, and alkynyl are provided. These terms will respectively have the defined meaning and the preferred meaning in each case where they are used in the remainder of the specification.

[0077] The term "SPECT" as used herein is an abbreviation for single photon emission computed tomography.

[0078] The term "PET" as used herein is an abbreviation for positron emission tomography.

[0079] The term "CT" as used herein is an abbreviation for computed tomography.

[0080] The term "MRI" as used herein is an abbreviation for magnetic resonance imaging.

[0081] The term "SIRT" as used herein is an abbreviation for selective internal radiotherapy.

[0082] The term "EDTA" as used herein is an abbreviation for ethylenediaminetetraacetic acid.

[0083] The term "DOTA" as used herein is an abbreviation for 1,4,7,10-tetraazacyclododecane-1,4,7,10-N,N',N'',N'''-tetraacetic acid.

[0084] As used herein, the term "DOTAGA" is an abbreviation for 1,4,7,10-tetraazacyclododecane,1-(pentanedioic acid)-4,7,10-triacetic acid.

[0085] As used herein, the term "DTPA" is an abbreviation for diethylenetriaminepentaacetic acid.

[0086] As used herein, the term metal "chelating agent" or "chelator" refers to a multidentate ligand that forms two or more separate coordination bonds with a single central atom, particularly a radioisotope.

[0087] As used herein, the term "therapeutically effective amount" includes, within its meaning, a non-toxic but sufficient amount of a compound or composition for use in the present invention to provide the desired therapeutic effect. The exact amount required will vary from subject to subject and will depend on factors such as the species being treated, the age, weight and general condition of the subject, comorbidities, the severity of the condition being treated, the particular agent being administered and the mode of administration, etc. Thus, for any given situation, the appropriate "effective amount" can be determined by a person of ordinary skill in the art using only routine methods.

[0088] The term "alkyl" refers to a saturated straight or branched carbon chain. Preferably, the chain contains 1-10 carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, such as methyl, ethyl, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, pentyl or octyl. The alkyl is optionally substituted.

[0089] The term "heteroalkyl" refers to a saturated straight or branched carbon chain. Preferably, the chain contains 1-9 carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, pentyl, octyl, which is interrupted one or more times by the same or different heteroatoms, such as 1, 2, 3, 4, 5 times. Preferably, the heteroatoms are selected from O, S and N, such as -O-CH3, -S-CH3, -CH2-O-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-CH2-CH3, etc. The heteroalkyl is optionally substituted.

[0090] Unless otherwise indicated, the terms "cycloalkyl" and "heterocycloalkyl", alone or in combination with other terms, respectively denote the cyclic forms of "alkyl" and "heteroalkyl", wherein preferably 3, 4, 5, 6, 7, 8, 9 or 10 atoms form the ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc. The terms "cycloalkyl" and "heterocycloalkyl" also mean to include their bicyclic, tricyclic and polycyclic forms. The term "heterocycloalkyl" preferably refers to a five-membered saturated ring in which at least one ring member is an N, O or S atom and which optionally contains an additional O or an additional N; a six-membered saturated ring in which at least one ring member is an N, O or S atom and which optionally contains an additional O or an additional N or two additional N atoms; or a nine- or ten-membered saturated bicyclic ring in which at least one ring member is an N, O or S atom and which optionally contains one, two or three additional N atoms. "Cycloalkyl" and "heterocycloalkyl" are optionally substituted. Additionally, for heterocycloalkyl, the heteroatom can occupy the position where the heterocycle is connected to the rest 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, etc. Examples of heterocycloalkyl include 1-(1,2,5,6-tetrahydropyridinyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, 1,8-diaza-spiro-[4.5]decyl, 1,7-diaza-spiro-[4.5]decyl, 1,6-diaza-spiro-[4.5]decyl, 2,8-diaza-spiro[4.5]decyl, 2,7-diaza-spiro[4.5]decyl, 2,6-diaza-spiro[4.5]decyl, 1,8-diaza-spiro-[5.4]decyl, 1,7-diaza-spirotetrahydrofuran-3-yl, tetrahydrothiophen-2-yl, tetrahydrothiophen-3-yl, 1-piperazinyl, 2-piperazinyl, etc.

[0091] The term "aryl" preferably refers to an aromatic monocyclic ring containing 6 carbon atoms, an aromatic bicyclic system containing 10 carbon atoms or an aromatic tricyclic system containing 14 carbon atoms. Examples are phenyl, naphthyl or anthracenyl. Aryl is optionally substituted.

[0092] The term "aralkyl" refers to an alkyl moiety substituted by an aryl group, where the alkyl and aryl groups have the meanings as outlined above. An example is benzyl. Preferably, in this context, the alkyl chain contains 1 - 8 carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, or 8, such as methyl, ethylmethyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, pentyl, octyl. The aralkyl is optionally substituted in the alkyl and / or aryl moieties of the group. The term "heteroaryl" preferably refers to a five- or six-membered aromatic monocyclic ring in which at least one carbon atom is replaced by 1, 2, 3, or 4 (for a five-membered ring) or 1, 2, 3, 4, or 5 (for a six-membered ring) identical or different heteroatoms preferably selected from O, N, and S; an aromatic bicyclic system in which 1, 2, 3, 4, 5, or 6 of the 8, 9, 10, 11, or 12 carbon atoms are replaced by identical or different heteroatoms preferably selected from O, N, and S; or an aromatic tricyclic system in which 1, 2, 3, 4, 5, or 6 of the 13, 14, 15, or 16 carbon atoms are replaced by identical or different heteroatoms preferably selected from O, N, and S. Examples are 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, pyridyl, pyrimidinyl, pyrazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl, 1,3,5-triazinyl, 1-benzofuranyl, 2-benzofuranyl, indolyl, isoindolyl, benzothienyl, 2-benzothienyl, 1H-indazolyl, benzimidazolyl, benzoxazolyl, indoxazinyl, 2,1-benzisoxazolyl, benzothiazolyl, 1,2-benzisothiazolyl, 2,1-benzisothiazolyl, benzotriazolyl, quinolinyl, isoquinolinyl, quinoxalinyl, quinazolinyl, cinnolinyl, 1,2,3-benzotriazinyl, or 1,2,4-benzotriazinyl.

[0093] The term "heteroaralkyl" refers to an alkyl moiety substituted by a heteroaryl group, where the alkyl and heteroaryl groups have the meanings as outlined above. Examples are 2-alkylpyridyl, 3-alkylpyridyl, or 2-methylpyridyl. Preferably, in this context, the alkyl chain contains 1 - 8 carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, or 8, such as methyl, ethylmethyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, pentyl, octyl.

[0094] The heteroaralkyl is optionally substituted in the alkyl and / or heteroaryl moieties of the group.

[0095] The terms "alkenyl" and "cycloalkenyl" refer to a chain or a ring containing olefinically unsaturated carbon atoms having one or more double bonds. Examples are allyl and cyclohexenyl. Preferably, the alkenyl chain contains 2 - 8 carbon atoms, i.e., 2, 3, 4, 5, 6, 7 or 8 carbon atoms, such as vinyl, 1 - propenyl, 2 - propenyl, isopropenyl, 1 - butenyl, 2 - butenyl, 3 - butenyl, isobutenyl, sec - butenyl, 1 - pentenyl, 2 - pentenyl, 3 - pentenyl, 4 - pentenyl, hexenyl, pentenyl, octenyl. Preferably, the cycloalkenyl ring contains 3 - 8 carbon atoms, i.e., 3, 4, 5, 6, 7 or 8, such as 1 - cyclopropenyl, 2 - cyclopropenyl, 1 - cyclobutenyl, 2 - cyclobutenyl, 1 - cyclopentenyl, 2 - cyclopentenyl, 3 - cyclopentenyl, cyclohexenyl, cyclopentenyl, cyclooctenyl.

[0096] The term "alkynyl" refers to a chain or a ring containing unsaturated carbon atoms having one or more triple bonds. An example is propargyl. Preferably, the alkynyl chain contains 2 - 8 carbon atoms, i.e., 2, 3, 4, 5, 6, 7 or 8, such as ethynyl, 1 - propynyl, 2 - propynyl, 1 - butynyl, 2 - butynyl, 3 - butynyl, 1 - pentynyl, 2 - pentynyl, 3 - pentynyl, 4 - pentynyl, hexynyl, pentynyl, octynyl.

[0097] In one embodiment, a carbon atom or a hydrogen atom in alkyl, heteroalkyl, cycloalkyl, aryl, aralkyl, alkenyl, cycloalkenyl, alkynyl may be independently substituted by one or more elements selected from O, S, N or a group containing one or more elements selected from O, S, N. Embodiments include alkoxy, cycloalkoxy, aryloxy, aralkyloxy, alkenyloxy, cycloalkenyloxy, alkynyloxy, alkylthio, cycloalkylthio, arylthio, aralkylthio, alkenylthio, cycloalkenylthiol, alkynylthio, alkylamino, cycloalkylamino, arylamino, aralkylamino, alkenylamino, cycloalkenylamino, alkynylamino.

[0098] Other embodiments include hydroxyalkyl, hydroxycycloalkyl, hydroxyaryl, hydroxyaralkyl, hydroxyalkenyl, hydroxycycloalkenyl, hydroxyalkynyl, mercaptoalkyl, mercapto - cycloalkyl, mercaptoaryl, mercaptoaralkyl, mercaptoalkenyl, mercapto - cycloalkenyl, mercaptoalkynyl, aminoalkyl, aminocycloalkyl, aminoaryl, aminoaralkyl, aminoalkenyl, aminocycloalkenyl, aminoalkynyl.

[0099] In another embodiment, a hydrogen atom in alkyl, heteroalkyl, cycloalkyl, aryl, aralkyl, alkenyl, cycloalkenyl, alkynyl may be independently substituted by one or more halogen atoms. One group is trifluoromethyl.

[0100] If two or more groups or two or more residues can be independently selected, the term "independently" means that the groups or residues can be the same or can be different.

[0101] As used herein, terms defining a length range limitation such as "1-6" mean any integer from 1 to 6, i.e., 1, 2, 3, 4, 5, and 6. In other words, any range defined by two explicitly mentioned integers means that it encompasses and discloses any integer that defines the limitation and any integer included within the range.

[0102] As used herein, the term "halo" refers to a halogen residue selected from F, Br, I, and Cl. Preferably, the halogen is F.

[0103] As used herein, the term "linker" refers to any chemically suitable linker. Preferably, the linker does not break or only slowly breaks under physiological conditions. Thus, it is preferred that the linker does not contain a recognition sequence for proteases or other recognition structures for degrading enzymes. Since it is preferred that the compounds of the present invention are administered systemically to allow wide access to all compartments of the body and then the compounds of the present invention are enriched at any location in the body where tumors are present, it is preferred that the linker is selected such that it does not break or only slowly breaks in the blood. If less than 50% of the linker breaks within 2 h after the compound is administered to a human patient, the breakage is considered slow. Suitable linkers include, for example, optionally substituted alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, alkenyl, heteroalkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, sulfonyl, amine, ether, thioether, phosphine, aminophosphate, carboxamide, ester, imidoester, amidine, thioester, sulfonamide, 3-thiopyrrolidine-2,5-dione, carbamate, urea, guanidine, thiourea, disulfide, oxime, hydrazine, hydrazide, hydrazone, diaza bond, triazole, triazoline, tetrazine, platinum complex, and amino acids or combinations or compositions thereof. Preferably, the linker contains 1,4-piperazine, 1,3-propane, and phenol ether or combinations or compositions thereof.

[0104] The expression "optionally substituted" means a group in which one, two, three, or more hydrogen atoms can be independently replaced by corresponding substituents.

[0105] As used herein, the term "amino acid" refers to any organic acid containing one or more amino substituents, such as α-, β-, or γ-amino derivatives of aliphatic carboxylic acids.

[0106] The term "conventional amino acid" refers to the 20 naturally occurring amino acids and includes all of their stereoisomeric homologs, i.e., their D,L-, D-, and L-amino acids.

[0107] As used herein, the term "N-containing aromatic or non-aromatic monocyclic or bicyclic heterocycle" refers to a cyclic saturated or unsaturated hydrocarbon compound containing at least one nitrogen atom as a ring chain constituent.

[0108] As used herein, the term "radioactive moiety" refers to a molecular assembly that carries a radionuclide. The nuclide is bound by a covalent or coordinate bond that remains stable under physiological conditions. Examples of radioactive moieties include [ 131 I]-3-iodobenzoic acid or 68 Ga-DOTA.

[0109] As used herein, a "fluorescent isotope" emits electromagnetic radiation upon excitation by electromagnetic radiation of a shorter wavelength.

[0110] As used herein, a "radioisotope" or "radioactive isotope" is a radioactive isotope of an element (including within the term "radionuclide") that emits α-, β-, or γ-radiation. Exemplary radioisotopes are described below and include, for example, e 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 Such as 99m Tc, 100 Pd, 101m Rh, 103 Pb, 105 Rh, 109 Pd, 111 Ag, 111 In, 113 In, 119 Sb, 121 Sn, 123 I. 124 I. 125 I. 127 Cs, 128 Ba, 129 Cs, 131 Cs, 131 I. 139 La, 140 La,142 Pr 143 Pr 149 Pm 151 Eu 153 Eu 153 Sm 159 Gr 161 Tb 165 Dy 166 Ho 169 Eu 175 Yb 177 Lu 186 Re 188 Re 189 Re 191 Os 193 Pt 194 Ir 197 Hg 198 Au 199 Ag 199 Au 201 Tl 203 Pb 211 At 212 Bi 212 Pb 213 Bi 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 Dy-166.

[0111] As used in the context of the present invention, the term "radiopharmaceutical" refers to a bioactive compound that is modified with a radioisotope. In particular, intercalating substances can be used to deliver radioactivity in direct proximity to DNA (e.g., 131 the I-carried derivative of Hoechst-33258).

[0112] The terms "chelating agent" or "chelate" are used interchangeably in the context of the present invention and refer to a molecule, typically an organic molecule and typically a Lewis base, having two or more unshared electron pairs available for a metal ion. The metal ion is typically coordinated to the chelating agent by two or more electron pairs. The terms "bidentate chelating agent", "tridentate chelating agent" and "tetradentate chelating agent" refer to chelating agents having 2, 3 and 4 electron pairs, respectively, that can be readily provided simultaneously to a metal ion coordinated through the chelating agent. Typically, the electron pairs of the chelating agent form coordination bonds with a single metal ion; however, in some instances, the chelating agent can form coordination bonds with more than one metal ion, where multiple binding modes are possible.

[0113] The term "fluorescent dye" (also "fluorescent moiety", "fluorophore" or "fluorochrome" herein) as used in the context of the present invention refers to a compound that emits visible or infrared light after excitation by electromagnetic radiation such as shorter and appropriate wavelengths. Those skilled in the art will understand that each fluorescent dye has a predetermined excitation wavelength. All fluorescent moieties are included within this term. Specific examples of fluorescent moieties given herein are illustrative and are not meant to limit the use of fluorescent moieties with the targeting molecules disclosed herein.

[0114] The term "contrast agent" as used in the context of the present invention refers to a compound that increases the contrast of a structure or fluid in medical imaging. Enhancement is achieved by absorption of electromagnetic radiation or alteration of the electromagnetic field.

[0115] The term "paramagnetic" as used herein refers to paramagnetism induced by unpaired electrons in a medium. If an external magnetic field is applied, a paramagnetic substance will induce a magnetic field. Different from diamagnetism, the direction of the induced field is the same as the external field; and different from ferromagnetism, the field cannot be maintained in the absence of an external field.

[0116] The term "nanoparticle" as used herein refers to preferably spherical particles with a diameter size between 1 - 100 nanometers. Depending on the composition, the nanoparticles may have magnetic, optical or physicochemical properties that can be evaluated. Additionally, for many types of nanoparticles, surface modification can be achieved. The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention. Suitable pharmaceutically acceptable salts of the compounds of the present invention include acid addition salts, which can be formed, for example, by mixing a solution of choline or its derivatives 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. Further, when the compounds of the present invention carry an acidic moiety, suitable pharmaceutically acceptable salts thereof may 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 using counter anions such as halide ions, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl sulfonate and aryl sulfonate).

[0117] Illustrative examples of pharmaceutically acceptable salts include, but are not limited to: acetates, adipates, alginates, ascorbates, aspartates, benzenesulfonates, benzoates, bicarbonates, bisulfates, bitartrates, borates, bromides, butyrates, calcium edetate, camphorates, camphorsulfonates, d-camphorsulfonates, carbonates, chlorides, citrates, clavulanates, cyclopentanepropionates, digluconates, dihydrochlorides, dodecyl sulfates, edetates, edisylates, ethoates, esylates (ethanesulfonates), formates, fumarates, gluceptates, glucoheptonates, glucuronates, glutamates, glycerophosphates, glycolylarsanilates, hemisulfates, heptanoates, hexanoates, hexylresorcinols, hydrabamines, hydrobromides, hydrochlorides, hydroiodides, 2-hydroxyethanesulfonates, hydroxynaphthoates, iodides, isothionates, lactates, lactobionates, laurates, lauryl sulfates, malates, maleates, malonates, mandelates, mesylates (methanesulfonates), methyl sulfates, mucates, 2-naphthalenesulfonates, naphthalenesulfonates, nicotinates, nitrates, N-methylglucamine ammonium salts, oleates, oxalates, pamoates (embonates), palmitates, pantothenates, pectates, persulfates, 3-phenylpropionates, phosphates / diphosphates, picrates, pivalates, polygalacturonates, propionates, salicylates, stearates, sulfates, basic acetates, succinates, tannates, tartrates, theoclorides, toluenesulfonates, triethyl iodide, undecanoates, valerates, etc. (see, e.g., Berge, S.M. 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, which allows the compound to be converted into base or acid addition salts.

[0118] The neutral form of the compound can be regenerated by contacting the salt with a base or acid and isolating the parent compound in a 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 for the purposes of the present invention, the salt is equivalent to the parent form of the compound.

[0119] In addition to the de-salted form, the present invention provides compounds in the form of prodrugs. Prodrugs of the compounds described herein are those compounds that are readily subject to chemical change under physiological conditions to provide the compounds of formula (I). A prodrug is an active or inactive compound that is chemically modified to the compound of the present invention by in vivo physiological actions such as hydrolysis, metabolism, etc. after the prodrug is administered to a patient. Additionally, a prodrug can be converted to the compound of the present invention by chemical or biochemical means in an ex vivo environment. For example, when a prodrug is placed in a transdermal patch reservoir together with a suitable enzyme, it can be slowly converted to the compound of the present invention. The suitability and techniques related to the preparation and use of prodrugs are well known to those skilled in the art. For a general discussion of prodrugs involving esters, see Svensson and Tunek Drug Metabolism Reviews 16.5 (1988) and Bundgaard Design of Prodrugs, Elsevier (1985).

[0120] Examples of masked carboxylate anions include a variety of esters such as alkyl (e.g., methyl, ethyl), cycloalkyl (e.g., cyclohexyl), aralkyl (e.g., benzyl, p-methoxybenzyl), and alkoxycarbonyloxyalkyl (e.g., pivaloyloxymethyl). Amines are masked as arylcarbonyloxymethyl-substituted derivatives, which are cleaved by esterases in vivo to release the free drug and formaldehyde (Bungaard J. Med. Chem. 2503 (1989)). Similarly, drugs containing acidic NH groups such as imidazoles, imides, indoles, etc. have been masked with N-acyloxymethyl (Bundgaard Design of Prodrugs, Elsevier (1985)).

[0121] Hydroxyl groups have been masked as esters and ethers. EP 0 039 051 (Sloan and Little, April 11, 1981) discloses Mannich base hydroxamic acid prodrugs, their preparation, and uses.

[0122] Certain compounds of the present invention can exist in unsolvated forms as well as solvated forms including hydrated forms. Generally, the solvated forms are equivalent to the unsolvated forms and are intended to be included within the scope of the present invention.

[0123] Certain compounds of the present invention can exist in multiple crystalline or amorphous forms. Generally, 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.

[0124] Certain compounds of the present invention have asymmetric carbon atoms (optical centers) or double bonds; racemates, diastereoisomers, geometric isomers, and individual isomers are all intended to be included within the scope of the present invention.

[0125] The compounds of the present invention may also contain non-natural proportions of atomic isotopes at one or more atoms constituting such compounds. For example, the compounds may be radiolabeled with radioactive isotopes such as 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 included within the scope of the present invention.

[0126] As used in this application, the term "pharmaceutical composition" refers to a substance and / or combination of substances for identifying, preventing, or treating a tissue condition or disease. The pharmaceutical composition is formulated to be suitable for administration to a patient for preventing and / or treating a disease. Further, a pharmaceutical composition refers to a combination of an active agent and an inert or active carrier that renders the composition suitable for therapeutic use. The pharmaceutical composition can be formulated for oral, parenteral, topical, inhalation, rectal, sublingual, transdermal, subcutaneous, or vaginal routes of administration according to its chemical and physical properties. The pharmaceutical composition includes solids, semi-solids, liquids, transdermal therapeutic systems (TTS). Solid compositions are selected from tablets, coated tablets, powders, granules, pills, capsules, effervescent tablets, or transdermal therapeutic systems. Also included are liquid compositions selected from solutions, syrups, infusions, extracts, solutions for intravenous use, solutions for infusion, or solutions of the carrier systems of the present invention. Semi-solid compositions that can be used in the context of the present invention include emulsions, suspensions, creams, lotions, gels, boluses, buccal tablets, and suppositories.

[0127] "Pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government or listed in the United States Pharmacopeia (U.S.Pharmacopeia) or other generally recognized pharmacopeias for animals (more particularly for humans).

[0128] As used herein, the term "carrier" 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 in water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. When the pharmaceutical composition is administered intravenously, a saline solution is a preferred carrier. Saline solutions as well as 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, skimmed milk powder, glycerol, propylene, ethylene 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 E.W. Martin.

[0129] As used herein, the term "fibroblast activation protein (FAP)" is also known under the term "seprase". The two terms are 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.

[0130] "Medical imaging" means any technique used to visualize internal regions of the human or animal body for purposes of diagnosis, research, or therapeutic treatment. For example, FAP targeting agents can be detected by radio-scintigraphy, magnetic resonance imaging (MRI), computed tomography (CT scan), nuclear imaging, positron emission with a metal tomography (PET) contrast agent, 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 that can be used to enhance such techniques are those materials that enable visualization of a specific site, organ, or diseased area in vivo and / or result in some improvement in the quality of the images generated by the imaging technique, provide an improvement in those images, or are more readily interpretable. Such agents are referred to herein as contrast agents, and their use facilitates the discrimination of different parts of the image by increasing the "contrast" between those different regions of the image. Thus, the term "contrast agent" includes agents that enhance the quality of an image that can be generated in the absence of such an agent (such as is the case in MRI), as well as agents that are a prerequisite for generating an image (such as is the case in nuclear imaging).

[0131] Compound

[0132] The different aspects of the present invention will be defined in more detail hereinafter. Each aspect so defined can be combined with any other one or more aspects, unless the contrary is expressly indicated. In particular, any feature indicated as preferred or advantageous can be combined with any other one or more features indicated as preferred or advantageous.

[0133] The present invention provides small molecule radiopharmaceuticals and imaging agents based on FAP specific inhibitors. In certain embodiments, the FAP targeting agent has a structure represented by Formula I or a pharmaceutically acceptable salt thereof:

[0134]

[0135] Wherein:

[0136] R represents a radioactive moiety, a chelating agent, a fluorescent moiety, a photoacoustic reporter, a Raman active reporter, a contrast agent, a detectable nanoparticle or an enzyme;

[0137] R1 represents (C1-C6) alkyl;

[0138] R2 represents -B(-Y 1 )(-Y 2 ) or -CN;

[0139] Y 1 and Y 2 independently represent -OH, or together with the boron atom to which they are attached represent a group that can be hydrolyzed to boric acid, or together with the boron atom to which they are attached form a 5- to 8-membered ring that can be hydrolyzed to boric acid;

[0140] R3 represents H or (C1-C6) alkyl;

[0141] R4 is absent or represents 1, 2 or 3 substituents, each independently selected from (C1-C6) alkyl, -OH, -NH2 and halogen;

[0142] X represents O or S;

[0143] L represents a bond or a linker.

[0144] In certain preferred embodiments, R1 represents -CH3 or -CH2CH3, and even more preferably represents -CH3.

[0145] In certain preferred embodiments, R2 represents -B(-Y 1 )(-Y 2 ) and even more preferably represents -B(OH)2.

[0146] In certain preferred embodiments, R3 represents H.

[0147] In certain preferred embodiments, R4 is absent.

[0148] In certain preferred embodiments, X represents O.

[0149] In certain preferred embodiments, R2 represents -CH3, R2 represents -B(OH)2, R3 represents H, and R4 is absent.

[0150] In certain preferred embodiments, the compound is represented by Formula II or Formula III below:

[0151]

[0152] In certain embodiments, R is a radioactive moiety, wherein the radioactive moiety comprises a fluorescent isotope, a radioactive isotope, a radiopharmaceutical, or a combination thereof. Preferably, the radioactive moiety comprises a radioactive isotope selected from an alpha-radiation emitting isotope, a beta-radiation emitting isotope, a gamma-radiation emitting isotope, an Auger electron emitting isotope, an X-ray emitting isotope, a fluorescence emitting isotope.

[0153] The radioactive isotopes of the present invention can be selected to achieve imaging and / or radiotherapy.

[0154] The radioactive isotopes of the present invention can comprise radioactive metal or metalloid isotopes. Preferably, the radioactive isotope is a water-soluble metal cation.

[0155] Exemplary radioactive isotopes 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 Ru, 99m Tc, 100 Pd, 101m Rh, 103 Pb, 105 Rh, 109 Pd, 111 Ag, 111 In,113 In, 119 Sb, 121 Sn, 123 I, 124 I, 125 I, 127 Cs, 128 Ba, 129 Cs, 131 Cs, 131 I, 139 La, 140 La, 142 Pr, 143 Pr, 149 Pm, 151 Eu, 153 Eu, 153 Sm, 159 Gr, 161 Tb, 165 Dy, 166 Ho, 169 Eu, 175 Yb, 177 Lu, 186 Re, 188 Re, 189 Re, 191 Os, 193 Pt, 194 Ir, 197 Hg, 198 Au, 199 Ag, 199 Au, 201 Tl, 203 Pb, 211 At, 212 Bi, 212 Pb, 213 Bi, 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 Dy-166.

[0156] Diagnostic radioisotopes can be suitably used for diagnostic imaging and can include 18 F, 123 I, 124 I, 125 I, 99m Tc, etc. Therapeutic radioisotopes can be suitably used for various therapies, including the treatment of cancer, and can include, for example 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 et al.

[0157] In certain embodiments, the radioisotope is intended to achieve 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 usually presented as a cross-sectional slice through the patient. Due to the gamma emission of the isotope, the accumulated position of the radioactive labeled material in the patient's body can be observed. This true 3D representation can help tumor imaging. Positron emission tomography (PET) is a nuclear medicine imaging technique that produces 3D images and has higher sensitivity than traditional SPECT imaging. The system detects gamma ray pairs indirectly emitted by positron-emitting radionuclides (tracers) introduced into the body. A 3D image of the tracer concentration in the body is then constructed by computer analysis, and 3D imaging is usually completed by means of a computed tomography (CT) X-ray scan performed on the patient in the same machine during the same period of time. Positron-emitting isotopes can also be used together with CT to provide 3D imaging of the anatomical distribution of labeled medical devices.

[0158] In certain embodiments, the radioisotope is a transition metal, such as 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 Such as 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 Th. In some aspects, preferably the radioisotope is 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.

[0159] In some embodiments, the radioisotope is an s-block metal, such as 43 K, 81 Rb, 83 Sr, 89 Sr, 127 Cs, 128 Ba, 129 Cs, and 131 Cs.

[0160] In some embodiments, the radioisotope is in Groups 13-16 of the periodic table, such as 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. In some aspects, preferred radioisotopes include 68 Ga, 111 In, 212 Pb, or 213 Bi.

[0161] In some embodiments, the radioisotope is a halogen, such as 18 F, 75 Br,76 Br, 77 Br, 123 I, 124 I, 125 I, 131 I and 211 At. In some aspects, preferred radioisotopes include 18 F, 123 I, 124 I, 131 I or 211 At.

[0162] In some embodiments, the radioisotope is a lanthanide element, such as 139 La, 140 La, 142 Pr, 143 Pr, 149 Pm, 151 Eu, 153 Eu, 153 Sm, 159 Gr, 149 Tb, 152 Tb, [[ID=()]] 161 Tb, 165 Dy, 166 Dy, 166 Ho and 169 Eu, 175 Yb.

[0163] In some aspects, preferred radioisotopes include 149 Tb, 152 Tb or 161 Tb.

[0164] In some embodiments, the radioisotope is an actinide element, such as 225 Ac, 226 Th and 227T h . In some aspects, preferred radioisotopes include 225 Ac or 227 Th.

[0165] The radiolabeled material of the present invention may also suitably comprise a combination of at least two radioisotopes for imaging and / or therapy. Combinations of radioisotopes may be suitably 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.

[0166] The present invention may further comprise using at least one non-radioactive, non-toxic carrier metal. For example, the carrier metal may be selected from Bi and Fe. For example, the non-radioactive carrier metal may be a carrier metal capable of MRI imaging (such as Fe) or X-ray contrast imaging (such as Bi). Other examples of carrier metals include trivalent bismuth, which additionally provides X-ray contrast in the microspheres such that they can be imaged in CT.

[0167] In certain embodiments, R is a chelating agent or moiety, such as a chelating agent for a radioactive metal or paramagnetic ion, including a radioisotope.

[0168] The chelating agent may include any chelating agent known in the art, see for example 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.

[0169] Illustrative examples are shown in Table 1.

[0170] Table 1

[0171]

[0172]

[0173]

[0174]

[0175]

[0176] In certain preferred embodiments, R may be DOTA bonded through any one of its 4 carboxylic acid groups.

[0177] In certain embodiments, the chelating agent includes a radioisotope chelated thereto.

[0178] In certain embodiments, the chelating agent includes 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 combinations of these paramagnetic ions.

[0179] Where this portion is a detectable label, it can also be a fluorescent moiety.

[0180] In some embodiments, the fluorescent moiety is selected from: fluorescent proteins, fluorescent peptides, fluorescent dyes, fluorescent materials, or combinations thereof.

[0181] In certain embodiments, R is a fluorescent dye, such as, for example, selected from xanthene, acridine, oxazine, cyanine, styryl dyes, coumarins (such as coumarin 343, methoxycoumarin, and dialkylaminocoumarin), porphyrins, metal ligand complexes, fluorescent proteins, nanocrystals, perylene, boron-dipyrromethene, and phthalocyanines, as well as conjugates and combinations of dyes of these classes. Specific examples of fluorescent labels include, but are not limited to, organic dyes such as cyanines, fluorescein, and fluorescein derivatives, rhodamines and rhodamine derivatives, Alexa Fluors, Dylight fluors (such as DyLight547 and DyLight647), Hylight fluors (such as HiLyte Fluor 647, HiLyte Fluor 680, and HiLyte Fluor 750), IRDyes (e.g., IR Dye 800, IRDye 800CW, IRDye 800RS, and IRDye 700DX), Dy fluros (such as Dy677, Dy676, Dy682, Dy752, and Dy780), VivoTag fluors (such as VivoTag-680, VivoTag-S680, and VivoTag-S750), ATTO Dyes, BODIPY fluors (such as BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY581 / 591, BODIPY 630 / 650, and BODIPY 650 / 665), carbocyanines, indocyanines, oxacarbocyanines, thuicarbocyanines, phthalocyanines, polymethines, boron-dipyrromethene (BODIPY) dyes, ADS780WS, ADS830WS, and ADS832WS, and other fluorophores familiar to those skilled in the art.

[0182] For further illustration, the fluorescent moiety may be selected from 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'-tetrabromosulfone-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 fluors (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, RhodolGreen, tetramethyl-rhodamine, carboxytetramethylrhodamine, rhodamine derivative JA133, Alexa Fluorescent Dyes (such as 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, Alexa Fluor 750 and Alexa Fluor 790), 4',6-diamidino-2-phenylindole (DAPI), propidium iodide, AMCA, Spectrum Green, Spectrum Orange, Spectrum Aqua, Lissamine, and fluorescent transition metal complexes such as europium.Useful 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 WO 2008 / 142571, WO 2009 / 056282, and WO 99 / 22026, which are incorporated by reference in their entirety.

[0183] In certain embodiments, the detectable moiety is a biolumophore (such as a fluorescent polypeptide or peptide), including but not limited to GFP derivatives of the green fluorescent protein GFP (e.g., EBFP, EBFP2, Azurite, mKalamal, ECFP, Cerulean, CyPet, YFP, Citrine, Venus, Ypet), and R-phycoerythrin.

[0184] In certain embodiments, R is a photoacoustic reporter molecule. Exemplary photoacoustic reporter molecules include indocyanine green (ICG), Alexa Fluor 7, Evans Blue, BHQ3, QXL680, IRDye880CW, MMPSense680, methylene blue, PPCy-C8, and Cypate-C18.

[0185] 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.

[0186] In certain embodiments, the detectable moiety includes quantum dots.

[0187] In certain embodiments, the detectable moiety includes infrared-emitting quantum dots.

[0188] In certain embodiments, the detectable moiety is a Raman-active reporter molecule such as single-walled carbon nanotubes (SWNTs) or surface-enhanced Raman scattering (SERS) agents. Examples of SERS agents are metal nanoparticles labeled with Raman-active reporter molecules. In some cases, fluorescent dyes can also be used as Raman-active reporter molecules such as Cy3, Cy5, rhodamine, and chalcogenopyrylium dyes.

[0189] Examples of R as an enzyme label include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, and β-galactosidase.

[0190] In certain embodiments, the reagent of the present invention is an imaging agent, which is selected to be used as part of a method for performing image-guided surgery, such as for resection, dissection, ablation, removal, or for stent placement or placement of other in-situ devices. For example, a patient (human or veterinary subject) can be administered an amount of the reagent sufficient to become preferentially localized in the target tissue of the surgery, and the surgeon can detect the presence or absence of the imaging agent during the surgical procedure. In this regard, the detectable moiety can be preferentially optically detectable, such as fluorescence or the other optically active moieties described above. Such an imaging agent can be advantageously used in a surgical operating room, where the surgical area can be irradiated with electromagnetic radiation sufficient to render the detectable moiety (such as a fluorophore or quantum dot) detectable, which can be visualized by the surgeon directly or through monitoring means (such as a screen / monitor). Exemplary uses of such an imaging agent generally include endoscopic and lathroscopic surgical procedures, where the surgeon can optically observe the presence (or absence) of the imaging agent through an endoscope, laparoscope, or percutaneous means.

[0191] In certain embodiments, the image-guided surgery can be image-guided robot-assisted surgery.

[0192] 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 chelating agent.

[0193]

[0194] Exemplary FAP targeting agents include:

[0195]

[0196] 4613B

[0197]

[0198] 4536.X=N:DOTA-HyNic-D-Ala-boroPro

[0199] 4536B.X=C:DOTA-HyBz-D-Ala-boroPro

[0200] 6415

[0202] 6433.

[0204] 6481

[0206] In certain embodiments, the FAP targeting agent comprises two or more FAP inhibitor moieties covalently linked to a radiopharmaceutical or imaging agent, such as having a structure represented by Formula IV or a pharmaceutically acceptable salt thereof:

[0207]

[0208] wherein

[0209] R, R1, R2, R3, R4, X and L are as defined above; and

[0210] n represents an integer between 2 and 6.

[0211] In certain preferred embodiments of Formula IV, R is a chelating moiety.

[0212] In certain preferred embodiments of Formula IV, R is a chelating moiety and n is 2.

[0213] Particularly preferred compounds include the following:

[0214] 6555

[0216] 6952

[0218] 6522

[0220] Also particularly preferred are the above compounds 6555, 6952 and 6522 comprising a radionuclide. For example, preferred are the compounds of the following Formulas A, B and C:

[0221]

[0222] Formula A

[0223]

[0224] Formula B

[0225]

[0226] Formula C

[0227] wherein in each of those Formulas A, B and C, M is a radioisotope or metal.

[0228] In some aspects, in Formula A, B, and / or C, M is a diagnostic radioisotope.

[0229] In some aspects, in Formula A, B, and / or C, M is a therapeutic radioisotope.

[0230] In some aspects, in Formula A, B, and / or C, M is Ga-67, Ga-68, Lu-177, or Y-90.

[0231] In some aspects, in Formula A, B, and / or C, M is an s-block metal, such as 43 K, 81 Rb, 83 Sr, 89 Sr, 127 Cs, 128 Ba, 129 Cs, and 131 Cs.

[0232] In some aspects, in Formula A, B, and / or C, M is in Groups 13-16 of the Periodic Table of the Elements, such as 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, particularly 68 Ga, 111 In, 212 Pb, or 213 Bi.

[0233] In some aspects, in Formula A, B, and / or C, M is a halogen, such as 18 F, 75 Br, 76 Br, 77 Br, 123 I, 124 I, 125 I, 131 I, and 211 At, particularly 18 F, 123 I, 124 I, 131 I, or 211 At.

[0234] In some aspects, in Formulas A, B, and / or C, M is a lanthanide element, such as 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, particularly 149 Tb, 152 Tb or 161 Tb.

[0235] In some aspects, in Formulas A, B, and / or C, M is an actinide element, such as 225 Ac, 226 Th and 227 Th, particularly 225 Ac or 227 Th.

[0236] Also preferred is such a compound complexed with one or more radioisotopes comprising:

[0237]

[0238]

[0239] 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 the tumor distribution of the molecule. Such a PK / BD-modified FAP targeting agent may have a structure represented by Formula V or a pharmaceutically acceptable salt thereof: [[ID=Z8]]

[0240]

[0241] wherein

[0242] R, R1, R2, R3, R4, X, and L are as defined above;

[0243] R5 represents a moiety that modifies the pharmacokinetics and / or biodistribution of the molecule; and n represents an integer between 1 and 6.

[0244] In certain preferred embodiments of Formula V, R5 is a serum albumin-binding moiety.

[0245] In certain preferred embodiments of formula V, R5 is a serum albumin binding moiety and n is 1.

[0246] In certain preferred embodiments of formula V, R5 is a half-life extending moiety, such as a non-protein half-life extending moiety, such as a water-soluble polymer, such as polyethylene glycol (PEG) or discrete PEG, hydroxyethyl starch (HES), lipid, branched or unbranched acyl group, branched or unbranched C8-C 30 acyl group, branched or unbranched alkyl group, and branched or unbranched C8-C 30 alkyl group; and protein half-life extending moieties, such as serum albumin, transferrin, adnectins (e.g., albumin-binding or pharmacokinetic extension (PKE) adnectins), Fc domains, and unstructured polypeptides, such as XTEN and PAS polypeptides (e.g., conformationally disordered polypeptide sequences composed of the amino acids Pro, Ala, and / or Ser), and fragments of any of the foregoing.

[0247] The present compounds can be readily prepared in complex with a radioisotope or a metal, for example to provide a compound of any of the above formulas I-V, wherein R is a radioactive moiety or R is a chelator and a radioisotope or a metal complexed with a chelator. For example, 1) a radioisotope reagent such as a halide reagent of a radioisotope and 2) a precursor compound such as a compound having a chelating moiety are appropriately reacted in an aqueous mixture with agitation for a sufficient time and at a sufficient temperature to produce a radioisotope complexed with the precursor compound. Exemplary incorporation reaction times and temperatures are set forth in the subsequent examples and can appropriately include a reaction time of 5-60 minutes and a reaction temperature of up to 90 °C or higher.

[0248] The reaction mixture can appropriately include one or more stabilizer compounds, such as organic stabilizers, such as 2,5-dihydroxybenzoic acid or its salts, ascorbic acid or its salts, methionine, histidine, melatonin, N-acetylmethionine, or ethanol, where N-acetylmethionine is preferred in certain aspects. Preferred stabilizers can include those generally recognized as safe (GRAS) according to U.S. Food and Drug Administration standards. 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 inclusion in the radionuclide reagent / precursor compound mixture during the incorporation reaction.

[0249] A wide variety of macromolecular polymers and other molecules can be linked to the FAP targeting agents of the present disclosure to modulate the biological properties of the resulting FAP targeting agents and / or provide new biological properties to the FAP targeting agents. These macromolecular polymers can be linked to the FAP targeting agents via naturally encoded amino acids, via non-naturally encoded amino acids, or any functional substituents of natural or non-natural amino acids, or any substituents or functional groups added to natural or non-natural amino acids. The molecular weight of the polymer can have a wide range, including but not limited to from about 100 Da to about 100,000 Da or more. The molecular weight of the polymer can be between about 100 Da and about 100,000 Da, including but not limited to 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, 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 between about 100 Da and about 50,000 Da. In some embodiments, the molecular weight of the polymer is between about 100 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 1,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 5,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 10,000 Da and about 40,000 Da.

[0250] For this purpose, various methods have been developed, including polyethylene glycolylation, polysialylation, HESylation, glycosylation, or recombinant PEG analogues fused with flexible and hydrophilic amino acid chains (500 - 600 amino acids) (see Chapman, (2002) Adv Drug Deliv Rev. 54. 531 - 545; Schlapschy et al., (2007) ProtEng Des Sel. 20, 273 - 283; Contermann (2011) Curr Op Biotechnol. 22, 868 - 876; Jevsevar et al., (2012) Methods Mol Biol. 901, 233 - 246).

[0251] Examples of polymers include but are not limited to polyalkylethers and their alkoxy - terminated analogues (e.g., polyoxyethylene glycols, polyoxyethylene / propylene glycols and their methoxy - or ethoxy - terminated analogues, especially polyoxyethylene glycols, which are also known as polyethylene glycols or PEGs); discrete PEGs (dPEGs); polyvinylpyrrolidone; polyvinyl alkyl ethers; polyoxazolines, polyalkyl oxazolines and polyhydroxyalkyl oxazolines; polyacrylamides, polyalkyl acrylamides and polyhydroxyalkyl acrylamides (e.g., polyhydroxypropyl methacrylamide and its derivatives); polyhydroxyalkyl acrylates; polysialic acids and their analogues; hydrophilic peptide sequences; polysaccharides and their derivatives, including dextrans and dextran derivatives, such as carboxymethyl dextran, dextran sulfate, glucosamine; celluloses and their derivatives, such as carboxymethyl cellulose, hydroxyalkyl celluloses; chitins and their derivatives, such as chitosan, succinyl chitosan, carboxymethyl chitin, carboxymethyl chitosan; hyaluronic acid and its derivatives; starches; alginates; chondroitin sulfates; albumins; pullulan and carboxymethyl pullulan; polyamino acids and their derivatives, such as polyglutamic acid, polylysine, polyaspartic acid, polyasparagine; maleic anhydride copolymers such as styrene - maleic anhydride copolymer, divinyl ether - maleic anhydride copolymer; polyvinyl alcohol; its copolymers; its terpolymers; its mixtures; and derivatives of the foregoing.

[0252] The selected polymer can be water - soluble such that the attached FAP targeting agent does not precipitate in an aqueous environment such as a physiological environment. The water - soluble polymer can be of any structural form, including but not limited to linear, branched or dendritic. Generally, the water - soluble polymer is a poly(alkylene glycol), such as polyethylene glycol (PEG), but other water - soluble polymers can also be used. For example, PEG can be used to describe some embodiments of the present disclosure. For the therapeutic use of the FAP targeting agent, the polymer can be pharmaceutically acceptable.

[0253] The term "PEG" is used broadly to include any polyethylene glycol molecule, regardless of size or modification at the termini, and can be represented as attached to a FAP targeting agent by the following formula: XO-(CH2CH2O) n -CH2CH2-

[0254] or

[0255] XO-(CH2CH2O) n -

[0256] wherein n is 2-10,000 and X is H or a terminal modification, including but not limited to C 1-4 Alkyl, protecting group or terminal functional group. In some cases, the PEG used in the polypeptides of the present disclosure terminates at one end with a hydroxyl or methoxy group, ie, X is H or CH3 ("methoxy PEG").

[0257] The amount of water-soluble polymer attached to the FAP targeting agent (i.e., the degree of PEGylation or glycosylation) can be adjusted to provide altered (including but not limited to increased or decreased) pharmacological, pharmacokinetic, or pharmacodynamic characteristics, 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%, 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 that of the unmodified polypeptide.

[0258] Another variation on polymer systems that can be used to modify the PK or other biological properties of the resulting FAP-targeting agent is the use of unstructured, hydrophilic amino acid polymers as functional analogs of PEG. The inherent biodegradability of polypeptide platforms makes them attractive as potentially more benign alternatives to PEG. Another advantage, in contrast to the polydispersity of PEG, is the precise molecular structure of the recombinant molecule. Unlike HSA and Fc peptide fusions, where the three-dimensional fold of the fusion partner must be maintained, recombinant fusions with unstructured partners can, in many cases, be subjected to higher temperatures or harsh conditions, such as HPLC purification.

[0259] One of the more advanced such polypeptides is called XTEN (Amunix), is 864 amino acids in length, and is composed 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. Due to the biodegradable nature of the polymer, this is much larger than the commonly used 40KDa PEG and is accompanied by an extended half-life. The fusion of XTEN with an FAP targeting agent should result in a more than 60- to 130-fold increase in the half-life of the final FAP targeting agent over the unmodified polypeptide. 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. A random coil polymer is composed of an even more restricted set of only three uncharged small amino acids (proline, alanine, and serine).

[0260] In certain preferred embodiments of Formula V, R5 is a polyethylene glycol polymer.

[0261] In certain preferred embodiments of Formula V, R5 is a polyethylene glycol polymer and n is 1. The present invention also provides pharmaceutical compositions comprising at least one compound of any one of Formulas I-V and optionally a pharmaceutically acceptable carrier and / or excipient. In certain embodiments, the pharmaceutical compositions are intended for the diagnosis or treatment of diseases characterized by overexpression of fibroblast activation protein (FAP) in an animal (preferably a human subject).

[0262] Suitable pharmaceutically acceptable vehicles include, but are not limited to, non-toxic buffers such as phosphates, citrates and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives such as cetalkonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol or benzyl alcohol, alkyl esters of p-hydroxybenzoic acid (such as methyl or propyl p-hydroxybenzoate), catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol; low molecular weight polypeptides (e.g., fewer than about 10 amino acid residues); proteins such as serum albumin, 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 dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes such as zinc-protein complexes; and nonionic surfactants such as TWEEN or polyethylene glycol (PEG).

[0263] (Remington: The Science and Practice of Pharmacy, 22nd Edition, 2012, Pharmaceutical Press, London.)

[0264] In a preferred aspect, the pharmaceutical composition may comprise one or more stabilizer compounds that inhibit degradation of the radiopharmaceutical after preparation and prior to administration. Preferred stabilizers may include those generally recognized as safe (GRAS) according to the standards of the U.S. Food and Drug Administration (U.S.Food and Drug Administration standards).

[0265] Exemplary stabilizer compounds include organic agents such as 2,5-dihydroxybenzoic acid or its salts, ascorbic acid or its salts, methionine, histidine, melatonin, N-acetylmethionine or ethanol, where N-acetylmethionine is a preferred stabilizer for inclusion in the present aqueous pharmaceutical composition. In certain aspects, sulfur-containing stabilizer compounds, including compounds containing one or more sulfide moieties such as reduced N-acetylmethionine and L-glutathione, are preferred stabilizers for the present pharmaceutical composition. Exemplary amounts of one or more stabilizers in the present pharmaceutical composition may be 5 - 120 mg stabilizer / mL fluid (e.g., aqueous formulation) of the pharmaceutical composition.

[0266] The pharmaceutical compositions of the present disclosure can be administered in any number of ways for local or systemic treatment. They can be administered topically through epidermal or transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, solutions, and powders; by inhalation or insufflation of powders or aerosols (including via nebulizers) into the lungs, intratracheally, and intranasally; orally; or parenterally, including intravenously, intraarterially, intratumorally, subcutaneously, intraperitoneally, intramuscularly (e.g., by injection or infusion), or intracranially (e.g., intrathecally or intraventricularly).

[0267] Typical administration of this radiopharmaceutical can be by intravenous injection or other parenteral administration.

[0268] Therapeutic formulations can exist in unit dosage forms. Such formulations include tablets, pills, capsules, powders, granules, solutions or suspensions in aqueous or non-aqueous media, or suppositories. In solid compositions such as tablets, the active ingredient is mixed with a pharmaceutical carrier. Conventional tableting ingredients include corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dibasic calcium phosphate, or gums, and diluents (such as water). These can be used to form solid preformulated compositions that are homogeneous mixtures containing the compounds of the present disclosure or their non-toxic pharmaceutically acceptable salts. The solid preformulated compositions are then subdivided into unit dosage forms of the above types. Tablets, pills, etc. of the formulation or composition can be coated or otherwise compounded to provide dosage forms having the advantage of extended action. For example, a tablet or pill can contain an inner composition covered by an outer component. Additionally, the two components can be separated by an enteric layer, which is used to resist disintegration and allow the inner component to pass intact through the stomach or for delayed release. A variety of materials can be used for such enteric layers or coatings, including many polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

[0269] Kit and Method

[0270] Yet another aspect of the present invention provides a kit that comprises at least one compound of any one of Formulas I-V and instructions for diagnosing or treating a disease or consists thereof.

[0271] And yet another aspect of the present invention provides a method for diagnosing, imaging, or reducing tissues overexpressing FAP in an animal (preferably a human patient), comprising administering to the animal at least one compound of any one of Formulas I-V.

[0272] In some embodiments, the tissue overexpressing FAP is a tumor, particularly a solid tumor. In some embodiments, the tumor is a tumor selected from the following: 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. For further illustration, the subject Affimer Agent can be used to treat patients suffering from cancer, such as osteosarcoma, rhabdomyosarcoma, neuroblastoma, kidney cancer, leukemia, renal transitional cell carcinoma, bladder cancer, Wilm's cancer, ovarian cancer, pancreatic cancer, breast cancer (including triple-negative breast cancer), prostate cancer, bone cancer, lung cancer (e.g., small cell 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, hepatoblastoma, hepatocellular carcinoma, melanoma, renal rhabdoid tumor, 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, thrombocytosis, idiopathic myelofibrosis, soft tissue sarcoma, thyroid cancer, endometrial cancer, carcinoid, or liver cancer, breast cancer, or gastric cancer. In some embodiments of the present disclosure, the cancer is a metastatic cancer such as the various ones described above.

[0273] In some embodiments, in addition to administering the FAP targeting agents described herein, the method or treatment further comprises administering at least one additional immune response stimulant. In some embodiments, the additional immune response stimulants include, but are not limited to, colony stimulating factors (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)), interleukins (e.g., IL-1, IL2, IL-3, IL-7, IL-12, IL-15, IL-18), checkpoint inhibitors, antibodies that block immunosuppressive functions (e.g., anti-CTLA-4 antibody, anti-CD28 antibody, anti-CD3 antibody), toll-like receptors (e.g., TLR4, TLR7, TLR9), or members of the B7 family (e.g., CD80, CD86). The additional immune response stimulant can be administered before, simultaneously with, and / or after the FAP targeting agent. Also provided are pharmaceutical compositions comprising an FAP targeting agent and an immune response stimulant. In some embodiments, the immune response stimulant comprises 1, 2, 3, or more immune response stimulants.

[0274] In some embodiments, in addition to administering the FAP targeting agents 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 FAP targeting agent. Also provided are pharmaceutical compositions comprising an FAP targeting agent and an additional therapeutic agent. In some embodiments, the at least one additional therapeutic agent comprises 1, 2, 3, or more additional therapeutic agents.

[0275] Combination therapies with two or more therapeutic agents typically use drugs that act by different mechanisms of action, although this is not required. Combination therapies using agents with different mechanisms of action can result in additive or synergistic effects. Compared to single-agent therapies, combination therapies can allow the use of lower doses of each drug, thereby reducing toxic side effects and / or increasing the therapeutic index of the FAP targeting agent. Combination therapies can reduce the likelihood of the development of resistant cancer cells. In some embodiments, the combination therapy comprises a therapeutic agent that affects the immune response (e.g., enhances or activates the response) and a therapeutic agent that affects (e.g., inhibits or kills) tumors / cancer cells.

[0276] In some embodiments of the methods described herein, the combination of the FAP targeting agent described herein and at least one additional therapeutic agent results in an additive or synergistic outcome. 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 a reduced toxicity and / or side effect of the FAP targeting agent. In some embodiments, the combination therapy results in a reduced toxicity and / or side effect of the additional therapeutic agent.

[0277] Useful classes of therapeutic agents include, for example, anti-tubulin agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cisplatin, mono(platinum), bis(platinum), and trinuclear platinum complexes and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, chemosensitizers, duocarmycins, etoposide, fluoropyrimidines, ionophores, lexitropsin, nitrosoureas, cisplatin, purine antimetabolites, puromycin, radiation sensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, and the like. In some embodiments, the second therapeutic agent is an alkylating agent, an antimetabolite, an anti-mitotic agent, a topoisomerase inhibitor, or an angiogenesis inhibitor.

[0278] Therapeutic agents that can be administered in combination with the FAP-targeting agents described herein include chemotherapeutic agents. Thus, in some embodiments, the method or treatment involves administering the FAP-targeting agent of the present disclosure in combination with a chemotherapeutic agent or in combination with a mixture of chemotherapeutic agents. Treatment with the FAP-targeting agent can occur before, simultaneously with, or after administration of the chemotherapy. The combination administration can include administration in a single pharmaceutical formulation or co-administration using separate formulations, or in either order but generally sequentially over a period of time such that all of the active agents can exert their biological activity simultaneously. The preparation and dosing schedule of such chemotherapeutic agents can be used according to the manufacturer's instructions or determined empirically by a skilled practitioner. The preparation and dosing schedule of such chemotherapy is also described in The Chemotherapy Source Book, 4th Edition, 2008, M.C. Perry, Editor, Lippincott, Williams & Wilkins, Philadelphia, Pa.

[0279] Chemotherapeutic agents useful in the present disclosure include, but are not limited to, alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN); alkyl sulfonates such as busulfan, prosulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;Antibiotics such as aclacinomysin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, 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 calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenal agents such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2”-trichloroethylamine; urethan;Vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (Ara-C); taxanes such as paclitaxel (TAXOL) and docetaxel (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; CPT11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicin; capecitabine (XELODA); and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. Chemotherapeutic agents also include antihormonal agents that act to regulate or inhibit the action of hormones on tumors such as antiestrogens including, for example, tamoxifen, raloxifene, 4(5)-imidazoles that inhibit aromatase, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone and toremifene (FARESTON); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. In some embodiments, the additional therapeutic agent is cisplatin. In some embodiments, the additional therapeutic agent is carboplatin.;

[0280] In some embodiments of the methods described herein, the chemotherapeutic agent is a topoisomerase inhibitor. A topoisomerase inhibitor is a chemotherapeutic agent that interferes with the action of a topoisomerase (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 of any of these. In some embodiments, the additional therapeutic agent is irinotecan.

[0281] In some embodiments, the chemotherapeutic agent is an antimetabolite. An antimetabolite is a chemical substance whose structure is similar to that of a metabolite required for normal biochemical reactions, yet different enough to interfere with one or more normal functions of a cell, 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 of any of these. In some embodiments, the additional therapeutic agent is gemcitabine.

[0282] In some embodiments of the methods described herein, the chemotherapeutic agent is an antimitotic agent, including but not limited to agents that bind to tubulin. In some embodiments, the drug is a taxane. In some embodiments, the drug is paclitaxel or docetaxel, or a pharmaceutically acceptable salt, acid, or derivative of paclitaxel or docetaxel. In some embodiments, the drug is paclitaxel (TAXOL), docetaxel (TAXOTERE), albumin-bound paclitaxel (albumin-bound 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 embodiments, the antimitotic agent is an inhibitor of kinesin Eg5 or an inhibitor of mitotic kinases such as Aurora A or Plk1. In some embodiments, the additional therapeutic agent is paclitaxel. In some embodiments, the additional therapeutic agent is albumin-bound paclitaxel.

[0283] In some embodiments of the methods described herein, the additional therapeutic agent includes an agent such as a small molecule. For example, treatment may involve administering a FAP targeting agent of the present disclosure in combination with a small molecule that acts as an inhibitor against a tumor-associated antigen, 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 includes an mTOR inhibitor.

[0284] In some embodiments of the methods described herein, the additional therapeutic agent is a small molecule that inhibits cancer stem cell pathways. 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.

[0285] In some embodiments of the methods described herein, the additional therapeutic agent includes biomolecules such as antibodies. For example, the treatment may involve co-administering an FAP-targeting agent of the present disclosure with an antibody against a tumor-associated antigen, including but not limited to antibodies that bind 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 Notch pathway component. In some embodiments, the additional therapeutic agent is an antibody that binds to a Wnt pathway component. In some embodiments, the additional therapeutic agent is an antibody that inhibits cancer stem cell pathways. 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 acts as 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).

[0286] In some embodiments of the methods described herein, the additional therapeutic agent is an antibody that modulates the immune response. In 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.

[0287] In addition, treatment with the FAP targeting agents described herein may include combination therapy with other biomolecules, such as one or more cytokines (e.g., lymphokines, interleukins, tumor necrosis factors, and / or growth factors), or may be accompanied by 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.

[0288] In some embodiments of the methods described herein, the FAP targeting agent may be combined with a growth factor selected from: 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, neurotrophin, 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.

[0289] In some embodiments of the methods described herein, the additional therapeutic agent is an immune response stimulator. In some embodiments, the immune response stimulator is selected from 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.

[0290] In some embodiments of the methods described herein, the immune response stimulator is selected from: PD-1 activity modulators, PD-L2 activity modulators, CTLA-4 activity modulators, CD28 activity modulators, CD80 activity modulators, CD86 activity modulators, 4-1BB activity modulators, OX40 activity modulators, KIR activity modulators, Tim-� activity modulators, LAG3 activity modulators, CD27 activity modulators, CD40 activity modulators, GITR activity modulators, TIGIT activity modulators, CD20 activity modulators, CD96 activity modulators, IDO1 activity modulators, cytokines, chemokines, interferons, interleukins, lymphokines, members of the tumor necrosis factor (TNF) family, and immunostimulatory oligonucleotides.

[0291] In some embodiments of the methods described herein, the immune response stimulant is selected from: PD-1 antagonists, PD-L2 antagonists, CTLA-4 antagonists, CD80 antagonists, CD86 antagonists, KIR antagonists, Tim-3 antagonists, LAG3 antagonists, TIGIT antagonists, CD20 antagonists, CD96 antagonists, and / or IDO1 antagonists.

[0292] 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 PCT Publication WO 2014 / 179664, such as the antibodies identified as APE2058, APE1922, APE1923, APE1924, APE 1950, or APE1963, or an antibody containing the CDR regions of any of these antibodies. In other embodiments, the PD-1 antagonist is a fusion protein comprising PD-L2, such as AMP-224. In other embodiments, the PD-1 antagonist is a peptide inhibitor, such as AUNP-12.

[0293] In some embodiments, the CTLA-4 antagonist is an antibody that specifically binds to CTLA-4.

[0294] 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.

[0295] 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.

[0296] 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.

[0297] In some embodiments, the immune response stimulant is selected from: CD28 agonists, 4-1BB agonists, OX40 agonists, CD27 agonists, CD80 agonists, CD86 agonists, CD40 agonists, and GITR agonists. 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 capable of expressing an OX40 ligand (e.g., an expression vector or a virus, such as an adenovirus). In some embodiments, the vector expressing OX40 is Delta-24-RGDOX or DNX2401.

[0298] In some embodiments, the 4-1BB (CD137) agonist is a binding molecule, such as an 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).

[0299] 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).

[0300] In some embodiments, the GITR agonist comprises a GITR ligand or a GITR-binding portion thereof. 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. In some embodiments, the immune response stimulant includes, but is not limited to, cytokines, such as chemokines, interferons, interleukins, lymphokines, and members of the tumor necrosis factor (TNF) family. In some embodiments, the immune response stimulant includes immune-stimulatory oligonucleotides, such as CpG dinucleotides.

[0301] In some embodiments, the immune response stimulators include, but are not limited to, anti-PD-1 antibodies, anti-PD-L2 antibodies, anti-CTLA-4 antibodies, anti-CD28 antibodies, anti-CD80 antibodies, anti-CD86 antibodies, anti-4-1BB antibodies, anti-OX40 antibodies, anti-KIR antibodies, anti-Tim-3 antibodies, anti-LAG3 antibodies, anti-CD27 antibodies, anti-CD40 antibodies, anti-GITR antibodies, anti-TIGIT antibodies, anti-CD20 antibodies, anti-CD96 antibodies, or anti-IDO1 antibodies.

[0302] In some embodiments, the FAP targeting agents disclosed herein can be used alone or in combination with radiotherapy.

[0303] In some embodiments, the FAP targeting agents disclosed herein can be used alone or in combination with a targeted therapy. Examples of targeted therapies include: hormonal 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 (such as imatinib (Gleevec) and dasatinib (Sprycel)); ALK inhibitors (such as crizotinib (Xalkori) and ceritinib (Zykadia)); BRAF inhibitors (such as 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)). In some embodiments of the present disclosure, the FAP targeting agents of the present disclosure are administered in combination with, for example, a STING agonist as part of a pharmaceutical composition. Cyclic dinucleotides (CDNs) 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), which bind to a pathogen recognition receptor (PRR) called 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 axes, leading to the induction of IFNβ and other gene products, thereby strongly activating innate immunity. It is now recognized that STING is a component of the host cytoplasmic surveillance pathway that senses infection by intracellular pathogens and, in response, induces the production of IFN-α, leading to the formation of an adaptive protective 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; PCT Publication Nos. WO2007 / 054279, WO2014 / 093936, WO2014 / 179335, WO2014 / 189805, WO2015 / 185565, WO2016 / 096174, WO2016 / 145102, WO2017 / 027645, WO2017 / 027646, and WO2017 / 075477 (each incorporated by reference); and Yan et al., Bioorg. Med. Chem Lett. 18:5631-4, 2008.

[0304] In some embodiments of the present disclosure, the FAP targeting agent of the present disclosure is administered in combination 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 (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]methyl]-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), ipatasertib ((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)-, AZD 5363 (4-piperidinecarboxamide, 4-amino-N-[(1S)-1-(4-chlorophenyl)-3-hydroxypropyl]-1-(7H-pyrazolo[2,3-d]pyrimidin-4-yl)), perifosine, GSK690693, GDC-0068, triciribine, 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] Triazine-10-one), Akt Inhibitor IX (CAS 98510-80-6). Additional Akt inhibitors include: ATP-competitive inhibitors such as isoquinoline-5-sulfonamide (e.g., H-8, H-89, NL-71-101), azepane derivatives (e.g., (-)-balanol derivatives), aminofurazan (e.g., GSK690693), heterocycles (e.g., 7-azaindole, 6-phenylpurine derivatives, pyrrolo[2,3-d]pyrimidine derivatives, CCT128930, 3-aminopyrrolidine, phenylaminotriazole derivatives, spiroindoline derivatives, AZD5363, A-674563, A-443654), phenylpyrazole derivatives (e.g., AT7867, AT13148), thiophenecarboxamide derivatives (e.g., Afuresertib (GSK2110183), 2-pyrimidinyl-5-acylaminothiophene derivatives (DC120), uprosertib (GSK2141795); allosteric inhibitors such as 2,3-diphenylquinoxaline analogs (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-glycerol-3-phosphocholine, ET-18-OCH3), ilmofosine (BM 41.440), miltefosine (hexadecylphosphocholine, HePC), perifosine (D-21266), brassidylphosphocholine (ErPC), erufosine (ErPC3, homobrassidylphosphocholine), indole-3-methanol analogs (e.g., indole-3-methanol, 3-chloroacetylindole, diindolylmethane, 6-methoxy-5,7-dihydroindolo[2,3-b]carbazole-2,10-dicarboxylic acid diethyl ester (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 analog (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-(cyclohex-1,3-dien-1-yl)-1H-pyrazol-3-yl]phenol, 3-oxo-tigliacic acid, 3α- and 3β-acetoxy-tigliacic acid, acetoxy-tigliacic acid; and irreversible inhibitors such as natural products, antibiotics, Lactoquinomycin, Frenolicin B, kalafungin, medermycin, Boc-Phe-vinyl ketone, 4-hydroxy nonenal (4-HNE), 1,6-naphthyridone derivatives and imidazo-1,2-pyridine derivatives.,

[0305] In some embodiments of the present disclosure, the FAP targeting agent of the present disclosure is administered in combination 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.

[0306] In some embodiments of the present disclosure, the FAP targeting agent of the present disclosure is administered in combination with anthracyclines such as doxorubicin and cyclophosphamide (including pegylated liposomal doxorubicin).

[0307] In some embodiments of the present disclosure, the FAP targeting agent of the present disclosure is administered in combination with both an anti-CD20 antibody and an anti-CD3 antibody or a bispecific CD20 / CD3 binding agent (including CD20 / CD3 BiTE).

[0308] In some embodiments of the present disclosure, the FAP targeting agent of the present disclosure is administered in combination with a CD73 inhibitor, a CD39 inhibitor, or both. These inhibitors can be CD73 binding agents or CD39 binding agents (such as antibodies, antibody fragments or antibody mimetics) that inhibit extracellular nucleotidase activity. The inhibitor can be a small molecule inhibitor of extracellular nucleotidase activity, such as 6-N,N-diethyl-β-γ-dibromomethylene-D-adenosine-5'-triphosphate trisodium hydrate, PSB069, PSB06126.

[0309] In some embodiments of the present disclosure, the FAP targeting agent of the present disclosure is administered in combination with an inhibitor of poly (ADP-ribose) polymerase (PARP). Exemplary PARP inhibitors include Olaparib, Niraparib, Rucaparib, Talazoparib, Veliparib, CEP9722, MK4827, and BGB-290. In some embodiments of the present disclosure, the FAP targeting agent of the present disclosure is administered in combination with an oncolytic virus. An exemplary oncolytic virus is laherparepvec.

[0310] In some embodiments of the present disclosure, the FAP targeting agent of the present disclosure is administered in combination 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.

[0311] In some embodiments of the present disclosure, the FAP targeting agent of the present disclosure is administered in combination with an anti-CD38 antibody. Exemplary anti-CD39 antibodies include Daratumumab and Isatuximab.

[0312] In some embodiments of the present disclosure, the FAP targeting agent of the present disclosure is administered in combination with an anti-CD40 antibody. Exemplary anti-CD40 antibodies include Selicrelumab and Dacetuzumab.

[0313] In some embodiments of the present disclosure, the FAP targeting agent of the present disclosure is administered in combination with an anaplastic lymphoma kinase (ALK) inhibitor. Exemplary ALK inhibitors include Alectinib, Crizotinib, and Ceritinib.

[0314] In some embodiments of the present disclosure, the FAP targeting agent of the present disclosure is administered in combination with a multi-kinase inhibitor or an anti-angiogenic inhibitor that inhibits one or more members selected from the VEGFR, PDGFR, and FGFR families. Exemplary inhibitors include Axitinib, Cediranib, Linifanib, Motesanib, Nintedanib, Pazopanib, Ponatinib, Regorafenib, Sorafenib, Sunitinib, Tivozanib, Vatalanib, LY2874455, or SU5402.

[0315] In some embodiments of the present disclosure, the FAP targeting agent of the present disclosure is administered together with one or more vaccines designed to stimulate an immune response against one or more predetermined antigens. The antigen can be administered directly to the individual or can be expressed in the individual from, for example, a tumor cell vaccine (such as GVAX), dendritic cell vaccine, DNA vaccine, RNA vaccine, virus-based vaccine, bacterial or yeast vaccine (such as Listeria monocytogenes or Saccharomyces cerevisiae), which can be autologous or allogeneic. 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 can also be a fragment or a fusion polypeptide containing an immunologically active portion of the antigens listed in the table.

[0316] In some embodiments of the present disclosure, the FAP targeting agents of the present disclosure are administered in combination with one or more antiemetics including but not limited to the following: casopitant (GlaxoSmithKline), netupitant (MGI-Helsinn), and other NK-1 receptor antagonists, palonosetron (sold by MGI Pharma as Aloxi), aprepitant (sold by Merck and Co. as Emend; Rahway, N.J.), diphenhydramine (sold by Pfizer as Benadryl; New York, N.Y.), hydroxyzine (sold by Pfizer as Atarax; New York, N.Y.), metoclopramide (sold by AH Robins Co. as Reglan; Richmond, Va.), lorazepam (sold by Wyeth as Ativan; Madison, N.J.), alprazolam (sold by Pfizer as Xanax; New York, N.Y.), haloperidol (sold by Ortho-McNeil as Haldol; Raritan, N.J.), droperidol (Inapsine), dronabinol (sold by Solvay Pharmaceuticals, Inc. as Marinol; Marietta, Ga.), dexamethasone (sold by Merck and Co. as Decadron; Rahway, N.J.), methylprednisolone (sold by Pfizer as Medrol; New York, N.Y.), prochlorperazine (sold by Glaxosmithkline as Compazine; Research Triangle Park, N.C.), granisetron (sold by Hoffmann-La Roche Inc. as Kytril; Nutley, N.J.), ondansetron (sold by Glaxosmithkline as Zofran; Research Triangle Park, N.C.), dolasetron (sold by Sanofi-Aventis as Anzemet; New York, N.Y.), tropisetron (marketed as Navoban by Novartis; East Hanover, NJ). Other side effects of cancer treatment include red blood cell and white blood cell deficiency. Therefore, in some embodiments of the present disclosure, FAP-targeting agents are administered in combination with agents that treat or prevent such deficiency, such as filgrastim, PEG-filgrastim, erythropoietin, epoetin alfa, or darbepoetin alfa. In some embodiments of the present disclosure, FAP-targeting agents of the present disclosure are administered in combination with anticancer radiation therapy. For example, in some embodiments of the present disclosure, the radiation therapy is external beam therapy (EBT): a method for delivering high-energy X-ray beams to the tumor site. The beams are generated outside the patient (e.g., by a linear accelerator) and targeted to the tumor site. These X-rays can destroy cancer cells, and careful treatment planning can spare surrounding normal tissue. No radiation source is placed in the patient's body. In some embodiments of the present disclosure, the radiation therapy is proton beam therapy: a type of conformal therapy that bombards diseased tissue with protons rather than 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: the temporary placement of radioactive material inside the body, usually to give an additional dose or boost of radiation to a region.

[0317] Example

[0318] Example 1: Synthesis of Compounds 4613B and 4613C

[0319]

[0320] Scheme 1. Reagents and conditions: i. L-boroPro-pn. HCl, HATU, DIEA; ii. 4N HCl in dioxane; iii. 6-(N'-Boc-hydrazino)-nicotinic acid (for 3a) or 6-(N'-Boc-hydrazino)-benzoic acid (for 3b), HATU, DIEA; iv. BCl3 in dichloromethane, -78°C; v. IRDye 800CW NHS ester, pH 7.8 buffer.

[0321] Experimental part

[0322] Reagents obtained from commercial sources were used without further purification. The synthesis of L-boroPro-pn was carried out using the 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 and a Discovery C18 569226-U RP-HPLC column. The mobile phase was generally prepared by mixing water (0.1% TFA) and acetonitrile (0.08% TFA) at gradient concentrations. The purity was determined to be greater than 95% by HPLC analysis. Mass spectra and HPLC retention times were recorded on a Hewlett-Packard HP LC / MSD system with an Eclipse Plus C18 RP-HPLC column (4.6x50 mm, 1.8 μm) and a solvent gradient of A) water (0.1% TFA) and B) acetonitrile (0.08% TFA) at a flow rate of 0.5 mL / min, monitored with a UV detector (at 215 nm). Unless otherwise indicated, all HPLC retention times were given for an elution gradient of 2% B for the first 3 min, then from 2% to 98% B over 6 min, followed by 5 min at 98% B. 1 1H NMR spectra were recorded on a Bruker Avance 300 MHz NMR spectrometer equipped with a 5 mm inverse multi-nuclear probe. Chemical shifts were reported in parts per million (δ) relative to DSS (in D2O).

[0323] Synthesis of Compound 2

[0324] Under ice-water bath cooling, L-boroPro-pn.HCl (3.0 g, 10.5 mmol), HATU (4.0 g, 10.5 mmol), and DIEA (4.0 mL, 23 mmol) were added to a stirred solution of N-Boc-D-Ala-OH (1, 1.9 g, 10 mmol) in anhydrous DMF (40 mL). 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 KHSO4 (3 x 40 mL), aqueous NaHCO3 (3 x 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 then added to a solution of 4 N HCl in dioxane (30 mL) under ice-water cooling. The resulting mixture was stirred at room temperature for 2 h and then concentrated in vacuo. The residue was co-evaporated with dichloromethane (3 x 30 mL) in vacuo to complete dryness. Compound 2 (3.3 g, 92% over two steps) was obtained as a white powder.

[0325] Synthesis of compound 3860

[0326] 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 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 NaHCO₃ (3 x 10 mL) and brine (10 mL). The organic phase was dried over anhydrous MgSO₃, filtered, and evaporated in vacuo to afford compound 3a, which was then dissolved in dry dichloromethane (5.0 mL) and cooled to -78°C while BCl₃ (1 M in dichloromethane, 5.0 mL) was added dropwise. The mixture was stirred at -78°C for 1 hour and then concentrated in vacuo. The residue was partitioned between diethyl ether (5 mL) and water (5 mL). The aqueous layer was washed twice with more 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=7.4min.

[0327] Synthesis of compound 4613B

[0328] 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 necessary. The resulting mixture was stirred at the same temperature for 3 hours and purified by semi-preparative RP-HPLC to give compound 4613B (11 mg, 84%) as a fluffy green powder. LC-MS (ESI + )m / z(relative intensity):1288.1([M-H2O+H] + ,25),635.8([(M-2x H2O) / 2+H] + ,100);tr=7.7min. 1H NMR(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),3.88-3.91 (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).

[0329] Synthesis of compound 4634

[0330] In a manner similar to the preparation of 3860, compound 4634 was obtained by reacting 6-(N'-Boc-hydrazino)-benzoic acid with compound 2. LC-MS (ESI + )m / z(relative intensity):605.5([2x(M-H2O)+H] + ,100),303.3([M-H2O+H] + ,67);tr=7.7min.

[0331] Synthesis of compound 4613C

[0332] Compound 4613C was obtained by reacting IRDye 800CW NHS ester with 4634 in a manner similar to the preparation of 4613B from 3860. LC-MS (ESI + )m / z(relative intensity):1287.6([M-H2O+H] + ,88),635.6([(M-2x H2O) / 2+H] + ,100);tr=7.9min.

[0333] Example 2: Synthesis of Compounds 4536B, 6481 and 5183

[0334]

[0335] Scheme 2. Reagents and conditions: i. Tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate, HBTU, HOBt, DIEA; ii. TFA-CH2Cl2 (1:4), then H2O; iii. CuCl2; iv. GdCl3.

[0336] Synthesis of compound 4536B

[0337] 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 cooling. The resulting mixture was stirred at room temperature for 15 min. Compound 4634 (40 mg, 0.11 mmol) was added to the above solution and stirred for another 2 hr. 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 removing TFA and dichloromethane, water (2 mL) was added and the resulting mixture was stirred at room temperature for 1 hr to obtain the crude product, which was directly purified by semi-preparative RP-HPLC to obtain 85 mg of compound 4536B as a white powder. LC-MS (ESI + )m / z(relative intensity):689.2([M-H2O+H] + ,100);tr=7.4min. 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).

[0338] Synthesis of compound 6481

[0339] 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%-50% B (solvent A: 0.05% TFA in water; solvent B: acetonitrile). The desired fractions were collected and lyophilized to obtain 4 mg of compound 6481 as a blue-green powder. LC-MS (ESI + )m / z(relative intensity):750.9([M-H2O+H] + ,49),745.7([M-H2O-H] + ,29),377.5([(M-2xH2O) / 2+H] + ,100);tr=7.4min.

[0340] Synthesis of compound 5183

[0341] 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 and stirred for half an hour, and then purified by semi-preparative RP-HPLC, eluting with 10%-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-H2O+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).

[0342] Example 3: Synthesis of Compounds 6486S - 6489S, 6486 - 6489

[0343]

[0344] Scheme 3. Reagents and conditions: i Boc-NH-(CH2) n -CO2H, HATU, DIEA; ii. 4N HCl in dioxane; iii. tris-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate, HBTU, HOBt, DIEA; iv. TFA-CH2Cl2 (1:4), then H2O; v. CuCl2.

[0345] Synthesis of compound 6487S

[0346] Compound 4634 was first coupled with N-Boc-Gly-OH, and then the Boc protection was removed under the same conditions as for the preparation of compound 2 from Boc-D-Ala-OH and boroPro-pn.HCl; and then it was coupled with tris-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate, and all -OtBu ester protections were removed under the same conditions as for 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-H2O+H] + , 100); tr = 7.4 min.

[0347] Synthesis of compound 6487

[0348] Compound 6487 was prepared as a blue-green powder from compound 6487 by the same method as that for preparing 6481 from 4536B. LC-MS (ESI + )m / z(relative intensity):807.5([M-H2O+H] + ,30),802.9([M-H2O-H] + ,100);tr=7.7min.

[0349] Synthesis of compound 6486S

[0350] Compound 6486S was prepared as a white powder by the same method as that for 6487S.

[0351] LC-MS (ESI + )m / z(relative intensity):803.3([M-H2O+H] + ,100);tr=7.6min.

[0352] Synthesis of compound 6486

[0353] Compound 6486 was prepared as a blue-green powder from compound 6486S by the same method as that used to prepare 6481 from 4536B. LC-MS (ESI + )m / z(relative intensity):862.7([M-H2O+H] + ,100);tr=7.7min.

[0354] Synthesis of compound 6488S

[0355] Compound 6488S was prepared as a white powder by the same method as that for 6487S.

[0356] LC-MS (ESI + )m / z(relative intensity):762.8([M-H2O+H] + ,100);tr=7.4min.

[0357] Synthesis of compound 6488

[0358] Compound 6488 was prepared as a blue-green powder from compound 6488S by the same method as that used to prepare 6481 from 4536B. LC-MS (ESI + )m / z(relative intensity):822.3([M-H2O+H] + ,100);tr=7.7min.

[0359] Synthesis of compound 6489S

[0360] Compound 6489S was prepared as a white powder by the same method as that for preparing 6487S.

[0361] LC-MS (ESI + ) m / z (relative intensity): 774.4 ([M - H2O + H] + , 100); tr = 7.5 min.

[0362] Synthesis of Compound 6489

[0363] Compound 6489 was prepared as a blue-green powder from Compound 6489S by the same method as that for preparing 6481 from 4536B. LC-MS (ESI + ) m / z (relative intensity): 836.8 ([M - H2O + H] + , 100), 832 ([M - H2O - H] + , 63); tr = 7.6 min.

[0364] Example 4: Synthesis of Compounds 6572 and 6572CU

[0365]

[0366] Scheme 4. Reagents and conditions: i. D-Ala-boroPro, HATU, DIEA; ii. 4N HCl in dioxane; iii. tris(tert-butyl) 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate, HBTU, HOBt, DIEA; iv. TFA-CH2Cl2 (1:4), then H2O; v. CuCl2.

[0367] Example 5: Synthesis of Compounds 6521 - 6522 and 6521CU - 6522CU:

[0368]

[0369] Scheme 5. Reagents and conditions: i. Gly-OMe, HBTU, HOBt, DIEA; ii. NaOH; iiia. Val-D-Ala-boroPro, HATU, DIEA;; iiib. Gly-Val-D-Ala-boroPro, HATU, DIEA; iv. TFA-CH2Cl2 (1:4), then H2O; v. CuCl2.

[0370] Example 6: Synthesis of Compounds 6549 and 6551:

[0371]

[0372] Scheme 6. Reagents and conditions: i. L-boroPro-pn, HATU, DIEA; ii. H2 / Pd-C; iii. Cbz-GABA-OH, HATU, DIEA; iv. H2 / Pd-C; v. tris(tert-butyl) 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate, HBTU, HOBt, DIEA; vi. TFA-CH2Cl2 (1:4), then H2O; vii. PhB(OH)2.

[0373] Example 7: Synthesis of Compounds 6555 and 6556:

[0374]

[0375] Scheme 7. Reagents and conditions: i. D-Ala-boroPro, HATU, DIEA; ii. 4N HCl in dioxane; iii. tris(tert-butyl) 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate, HBTU, HOBt, DIEA; iv. TFA-CH2Cl2 (1:4), then H2O.

[0376] Example 8: Synthesis of Compounds 6508 - 6509 and 6508CU - 6509CU:

[0377]

[0378] Scheme 8. Reagents and conditions: i. D-Ala-boroPro, HATU, DIEA; ii. TFA in dichloromethane; iii. tris(tert-butyl) 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate, HBTU, HOBt, DIEA; iv. TFA-CH2Cl2 (1:4), then H2O; v. CuCl2.

[0379] Table 2 shows the compounds in Examples 1-9.

[0380]

[0381]

[0382]

[0383]

[0384] Example 9: Synthesis of GHK Analogs 6415 and 6433

[0385] Synthesis of Compound 6415

[0386] As shown in Scheme 4, compound 6415, which is a white powder, was prepared from compound 5 in 7 steps.

[0387] LC-MS (ESI + ) m / z (relative intensity): 767.2 ([M - H2O + H] + , 100); tr = 7.6 min.

[0388] Synthesis of compound 6433

[0389] Compound 6415 (29 mg) was dissolved in water (0.2 mL). Cu(OAc)2 (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 (11 mg) as a green - blue powder. LC-MS (ESI + ) m / z (relative intensity): 829.2 ([M - H2O + H] + , 100); tr = 7.5 min (Note: The solvent used for this LCMS was ordinary. No TFA was added).

[0390] Example 10: In Vitro Assay

[0391] Biomaterials: For in vitro IC50 assays, recombinant human DPPIV, DPP9, FAP, and PREP were purchased from R&D Systems, and DPP8 was 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). The fluorescent substrates were Gly - Pro AMC, Z - GlyPro - AMC, or Suc - GlyPro AMC purchased from Bachem or an N - terminal blocked FAP - specific substrate. The cell culture medium was RPMI 1640 without phenol red and supplemented with 2 mM L - 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 adding 1.2 N HCl. The samples were analyzed by reversed - phase HPLC - MS on a Thermo Finnigan LCQ Duo, and the peaks in the resulting base - peak chromatogram were quantified. The relative cleavage values were determined by comparing the abundances of the intact peptides after quenching with those in the initial library.

[0392] 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 multimode microplate reader, monitoring fluorescence at an excitation wavelength of 380 nm and an emission wavelength of 460 nm. The substrates for DPPIV, DPP8, and DPP9 assays were H-Gly-Pro-AMC, and the substrates for FAP and PREP assays were Z-Gly-Pro-AMC. The reaction mixture contained 25 μM substrate, enzyme, buffer A (for DPPIV and DPP9), buffer B (for DPP8), buffer C (for FAP), or buffer D (for PREP), and an appropriate amount of inhibitor (ranging from 10 -4 -10 -11 M), with a total volume of 210 μL. The final enzyme concentrations of DPPIV, DPP8, DPP9, FAP, and PREP were 0.1, 0.8, 0.4, 1.2, and 0.6 nM, respectively. The IC50 value was defined as the inhibitor concentration required to reduce the enzyme activity by 50% after pre-incubating the enzyme with the inhibitor for 10 minutes at 25 °C before adding the substrate. Inhibitor stock solutions (100 mM) were prepared in pH 2.0 HCl solution or DMSO for compounds 1 and 20. Those prepared in pH 2.0 solution were pre-incubated at 25 °C for 4 h before dilution. Just before the experiment started, the 100 mM stock solutions were further diluted to 10 -3 M in the appropriate assay buffer, and 1:10 serial dilutions were prepared therefrom. All inhibitors were tested in triplicate.

[0393] Table 3

[0394]

[0395]

[0396]

[0397]

[0398]

[0399]

[0400]

[0401]

[0402] Example 11: Exemplary Ligands Synthesized by Similar Methods

[0403]

[0404] 6591 DOTA-GABA-Aminomethyl-Bz-D-Ala-boroPro

[0405]

[0406] 6590 DOTA-APenA-PABA-D-Ala-boroPro (APenA = 5-Aminopentanoic acid)

[0407]

[0408] 6554 DOTA-HyNaph-D-Ala-boroPro

[0409]

[0410] 6645 DOTA-Lys(ABM)-GABA-HyBz-D-Ala-boroPro

[0411]

[0412] 6640 DOTA-Lys(ABM)-Gly-Gly-Val-D-Ala-boroPro

[0413]

[0414] 6644 DOTA-Lys(Piperazine-diacetyl

[0415] -GABA-HyBz-D-Ala-boroPro)-GABA-HyBz-Val-D-Ala-boroPro

[0416]

[0417] 6643 DOTA-Lys(Piperazine-diacetyl

[0418] -Gly-Gly-Val-D-Ala-boroPro)-Gly-Gly-Val-D-Ala-boroPro

[0419]

[0420] 6586 DOTA-MABA-D-Ala-boroPro [MABA = 4-Methylaminobenzoic acid]

[0421]

[0422] 6637 DOTA-Lys(ABM)-Gly-Gly-Val-D-Ala-boroPro

[0423]

[0424] 6638 DOTA-D-Lys(DOTA)-Gly-Gly-Val-D-Ala-boroPro

[0425]

[0426] 6619 DOTA-[GABA-HyBz-D-Ala-boroPro]4

[0427]

[0428] 6635 DOTA-Aminoethyl-Bz-D-Ala-boroPro

[0429]

[0430] 6636 DOTA-Aminopropyl-Bz-D-Ala-boroPro

[0431]

[0432] 6627 DOTA-Diaminobutane-dicarboxybenzene-D-Ala-boroPro

[0433]

[0434] 6628 DOTA-Diaminopropane-CMBA-D-Ala-boroPro[CMBA=4-(carboxymethyl)benzoic acid]

[0435]

[0436] 6634 DOTA-DAVA-Gly-D-Ala-boroPro[DAVA=5-aminovaleric acid]

[0437]

[0438] 6633 DOTA-betaAla-Gly-D-Ala-boroPro

[0439]

[0440] 6632 DOTA-Gly-Gly-D-Ala-boroPro

[0441]

[0442] 6631 DOTA-Gly-Ser-D-Ala-boroPro

[0443]

[0444] 6630 DOTA-Gly-Ala-D-Ala-boroPro

[0445]

[0446] 6629 DOTA-D-Ala-Gly-Val-D-Ala-boroPro

[0447]

[0448] 6626 DOTA-DAVA-PABA-D-Ala-boroPro [DAVA = 5-aminopentanoic acid]

[0449]

[0450] 6623 DOTA-GABA-aminomethyl-Nic-D-Ala-boroPro

[0451]

[0452] 6617 DOTA-EACA-Val-D-Ala-boroPro [EACA = ε-aminocaproic acid]

[0453]

[0454] 6618 DOTA-AEPA-Val-D-Ala-boroPro [AEPA = 3-(2-aminoethoxy)propionic acid]

[0455]

[0456] 6616 DOTA-AEAC-Val-D-Ala-boroPro [AEAC = (2-aminoethoxy)acetic acid]

[0457]

[0458] 6615 DOTA-betaAla-Val-D-Ala-boroPro

[0459]

[0460] 6613 DOTA-DAVA-Val-D-Ala-boroPro [DAVA = 5-aminovaleric acid]

[0461]

[0462] 6614 DOTA-GABA-Val-D-Ala-boroPro

[0463]

[0464] 6609 DOTA-GABA-HyNIC-D-Ala-boroPro

[0465]

[0466] 6601D DOTA-D-Lys(IRDye)-GABA-HyBz-D-Ala-boroPro

[0467]

[0468] 6589 DOTA-DAB-dcBn-D-Ala-boroPro

[0469]

[0470] 6581 DOTA-APenA-HyBz-D-Ala-boroPro (APenA = 5-aminovaleric acid)

[0471]

[0472] 6585 DOTA-AOA-HyBz-D-Ala-boroPro (AOA = 8-amino-octanoic acid)

[0473]

[0474] 6580 DOTA-AHepA-HyBz-D-Ala-boroPro (AHepA = 7-aminoheptanoic acid)

[0475]

[0476] 6575 DOTA-dimethyl-amino-Bz-D-Ala-boroPro

[0477]

[0478] 6566 DOTA-methylamino-Bz-D-Ala-boroPro

[0479]

[0480] 6574 DOTA-Vinyl-Bz-D-Ala-boroPro

[0481]

[0482] 6571 DOTASA-HyBz-D-Ala-boroPro

[0483]

[0484] 6563 DOTAGA-HyBz-D-Ala-boroPro

[0485]

[0486] 6583 NOTASA-GABA-HyBz-D-Ala-boroPro

[0487]

[0488] 6584 NOTAGA-GABA-HyBz-D-Ala-boroPro

[0489]

[0490] 6570 NOTAGA-HyBz-D-Ala-boroPro

[0491]

[0492] 6569 NOTASA-HyBz-D-Ala-boroPro

[0493]

[0494] 6565 NOTA-Aminomethyl-Bz-D-Ala-boroPro

[0495]

[0496] 6557 DO3A-Nic-D-Ala-boroPro

[0497]

[0498] 6558 DO3A-Bz-D-Ala-boroPro

[0499]

[0500] 6564 NOTA-HyBz-D-Ala-boroPro

[0501]

[0502] 6455 CB-TE2A-4613C

[0503]

[0504] 6523 FAPI-2D-Ala-boroPro derivative

[0505]

[0506] 6540 4536B with an albumin-binding moiety

[0507]

[0508] 6541 4536B with an albumin-binding moiety (Lys side chain)

[0509]

[0510] 6524 FAPI-46D-Ala-boroPro derivative

[0511]

[0512] 6456 DiAmSar-4613C derivative (C7)

[0513]

[0514] 6430 N2S2-(C7)-4613C derivative

[0515]

[0516] 6425 N-(4-BPA-C6-hydrazinobenzoyl)-D-Ala-boroPro click derivative

[0517]

[0518] 6432 DAHK-(4613C) derivative

[0519]

[0520] 6431 SAR-NH-(C7)-4613C

[0521]

[0522] 6419 AHK-(4613C) derivative (C7)

[0523]

[0524] 6418 GHK-(4613C) derivative (C5)

[0525]

[0526] 6417 GHK-(4613C) derivative (C4)

[0527]

[0528] 6416 GHK-(4613C) derivative (C6)

[0529] Example 12: Synthesis of DOTA - PNP

[0530] Scheme 10

[0531]

[0532] Synthesis Scheme 10. Reagents and conditions: i. DCC, PNP, Py-CAN-water, 30%.

[0533] Experimental Section

[0534] Reagents obtained from commercial sources were used without further purification. All target compounds were purified by RP-HPLC using a Varian semi-preparative system and a Discovery C18 569226-U RP-HPLC column. The mobile phase for semi-preparative HPLC was generally prepared 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 HP LC / MSD system using an Eclipse Plus C18 RP-HPLC column (4.6 x 50 mm, 1.8 μm) and a solvent gradient of A) water (0.1% TFA) and B) acetonitrile at 0.5 mL / min, monitored with a UV detector (at 215 nm). Unless otherwise noted, all HPLC retention times were given for an elution gradient of 2% B for the first 3 min, then from 2% to 98% B over 6 min, and then held at 98% B for 6 min.

[0535] Synthesis of DOTA-PNP

[0536] The synthesis of DOTA-PNP was carried out using the previously described synthetic method (Walter Mier, et al. Bioconjugate Chem., 2005, 16, 237-240. TS. 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 (TCIAmerica, 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 min and concentrated to dryness under reduced pressure. The residue was treated with 20% acetonitrile in water. The suspension was filtered to remove N,N'-dicyclohexylurea, and the filtrate was purified by a semi-preparative Discovery C18 569226-U RP-HPLC column (21.2 mm x 25 cm, 5 μm) with a UV detector (monitored at 215 nm). The gradient elution system used mobile phase A (4.8 mM HCl) and mobile phase B (acetonitrile). The gradient was carried out at a flow rate of 20 mL / min, starting with 98% A and 2% B for 5 min; and 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 (4x HCl salt, 250 mg, 30%) as a white powder. LC-MS (ESI + ) m / z (relative intensity): 526.1 ([M+H] + , 100); tr = 7.7 min.

[0537] Supporting material

[0538] Mass spectra and HPLC retention times were recorded on a Hewlett-Packard HP LC / MSD system with a UV detector (monitored at 215 nm) using a ZORBAX Eclipse Plus C18 RP-HPLC column (4.6 x 50 mm, 1.8 μm) and a solvent gradient of A) water (0.1% TFA) and B) acetonitrile at a rate of 0.5 mL / min. The elution gradient was 2% B for the first 3 min, then from 2% to 98% B over 6 min, and then held at 98% B for 5 min (0 - 3 min: 2% B; 3 - 9 min: 2 - 98% B; 9 - 15 min: 98% B). MS was run in the positive ion mode. The data were analyzed using Chemstation Software from Agilent.

[0539] Example 13: Synthesis of 6555 / 6555LU / 6555GA

[0540] Scheme 11

[0541]

[0542] Synthesis Scheme 11. Reagents and conditions: i. L-boroPro-pn.HCl, HATU, DIEA; ii. 4N HCl in dioxane, 92% for 2 steps; iii. 4-[(tert-butoxycarbonylamino)methyl]benzoic acid, HATU, DIEA; iv. 4N HCl in dioxane, 85% for 2 steps; Method I: v. DOTA-(OtBu)3, PyBOP, DIEA, DCM; vi. TFA-CH2Cl2 (4:1), then H2O; or Method II: vii. DOTA-PNP, TEA, DMF; viii. PhB(OH)2, H2O-TBME-ACN, 37% for 3 steps of Method I or 40% for 2 steps of Method II; ix. LuCl3, acetate buffer (0.23M, pH 5.2), 90 °C - 23 mins, 44%; ix. GaCl3, acetate buffer (0.23M, pH 5.2), 90 °C - 23 mins, 66%.

[0543] Solubility and Storage

[0544] After lyophilization, the target compounds 6555, 6555LU or 6555GA are readily soluble in water (solubility > 50 mg / ml). When in an aqueous solution with a pH of approximately 3, we did not observe any signs of degradation during HPLC purification and subsequent lyophilization. For long-term storage, the target compounds should be kept in a freezer at < -15 °C in solid form. For short-term storage, a refrigerator (+4 °C) will suffice.

[0545] Experimental Section

[0546] Reagents obtained from commercial sources were used without further purification. The synthesis of L-boroPro-pn was carried out using the 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 and a Discovery C18 569226-U RP-HPLC column. The mobile phase for semi-preparative HPLC was generally prepared by mixing water (0.1% TFA) with acetonitrile at gradient concentrations. Mass spectra and HPLC retention times were recorded on a Hewlett Packard HP LC / MSD system with an Eclipse Plus C18 RP-HPLC column (4.6x50 mm, 1.8 μm) and a solvent gradient of A) water (0.1% TFA) and B) acetonitrile at 0.5 mL / min, monitored with a UV detector (at 215 nm). Unless otherwise indicated, all HPLC retention times were given for an elution gradient of 2% B for the first 3 min, then from 2% to 98% B over 6 min, and then held at 98% B for 6 min.

[0547] Synthesis of Intermediate 1

[0548] Under ice-water bath cooling, L-boroPro-pn.HCl (3.0 g, 10.5 mmol), HATU (4.0 g, 10.5 mmol), and DIEA (4.0 mL, 23 mmol) were added to a stirred solution of N-Boc-D-Ala-OH (Aldrich, 15048-25G; 1.9 g, 10 mmol) in anhydrous DMF (40 mL). 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 successively with 0.1 N KHSO4 (3 x 40 mL), aqueous NaHCO3 (3 x 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 flash chromatography on silica gel, eluting with ethyl acetate / hexane, and then added to a solution of 4 N HCl in dioxane (30 mL) under ice cooling. The resulting mixture was stirred at room temperature for 2 h and then concentrated in vacuo. The residue was co-evaporated with dichloromethane (3 x 30 mL) under vacuum to complete dryness. Compound 1 (3.3 g, 92% over two steps) was obtained as a white powder.

[0549] Synthesis of Intermediate 2

[0550] Under ice-water bath cooling, to a stirred solution of 4-[(tert-butoxycarbonylamino)methyl]benzoic acid (TCI, B4305; 505 mg, 2 mmol) in anhydrous DMF (40 mL) was added compound 1 (750 mg, 2.1 mmol), HATU (800 mg, 2.1 mmol) and DIEA (0.80 mL, 4.6 mmol). 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 successively with 0.1 N KHSO4 (3 x 15 mL), aqueous NaHCO3 (3 x 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 flash chromatography on silica gel, eluting with ethyl acetate / hexane, and then added to a solution of 4 N HCl in dioxane (10 mL) under ice-water cooling. The resulting mixture was stirred at room temperature for 2 h and then concentrated in vacuo. The residue was co-evaporated with dichloromethane (3 x 20 mL) in vacuo to complete dryness. Compound 2 (830 mg, 85% over two steps) was obtained as a white powder. LC-MS (ESI + ) m / z (relative intensity): 453.7 ([M+H] + , 100); tr = 9.0 min.

[0551] Synthesis of Compound 6555 (Method I)

[0552] Under ice-water bath cooling, 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). The resulting mixture was stirred at room temperature for 3 hr and then diluted with more dichloromethane (30 mL) and washed sequentially with 5% citric acid (3 x 5 mL), aqueous NaHCO3 (3 x 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 removal of TFA and dichloromethane, water (9 mL) was added and the resulting mixture was stirred at room temperature for 1 hr, and 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 hr and the separated aqueous phase was washed with more TBME. The aqueous layer was concentrated in vacuo in small portions and purified by semi-preparative Discovery C18 569226-U RP-HPLC column (21.2 mm x 25 cm, 5 μm) equipped with a UV detector (monitored at 215 nm). The gradient elution system used 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 min; increasing to 70% A and 30% B over 20 min; then increasing to 2% A and 98% B over 1 min and holding for an additional 5 min. The combined fractions were directly lyophilized to give 6555 (4x TFA salt, 130 mg, 37% over 3 steps) as a white powder. LC-MS (ESI + ) m / z (relative intensity): 688.0 ([M-H2O+H] + , 100), 345.4 (63); tr = 7.6 min.

[0553] Synthesis of Compound 6555 (Method II)

[0554] Under ice-water bath cooling, TEA (360 μl, 2.07 mmol) was added to a stirred solution of DOTA-PNP (internally synthesized, 204 mg, 0.30 mmol) and intermediate compound 2 (189 mg, 0.33 mmol) in anhydrous DMF (4 mL). The resulting mixture was stirred overnight at room temperature. Then the reaction mixture was concentrated in vacuo. Water (9 mL) was added and the pH was adjusted to about 1.5 with 1N 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 hr and processed as described above to give 6555 (4x TFA salt, 140 mg, 40% over two steps) as a white powder.

[0555] Synthesis of Compound 6522LU

[0556] Compound 6555 (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 min and then purified by a semi-preparative Discovery C18 569226-U RP-HPLC column (21.2 mm x 25 cm, 5 μm) with a UV detector (monitored at 215 nm). The gradient elution system used 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 min; increasing to 70% A and 30% B over 20 min; then increasing to 2% A and 98% B over 1 min and holding for an additional 5 min. The combined fractions were lyophilized directly to give 6555LU (4x TFA salt, 5 mg, 44%) as a white powder. LC-MS (ESI + ) m / z (relative intensity): 859.5 (100); tr = 15.2 min (see attached LCMS and conditions).

[0557] Synthesis of Compound 6555GA

[0558] Compound 6555 (10 mg, 8.6 μmol) was added to a solution of GaCl3 (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 min and then purified by a semi-preparative Discovery C18 569226-U RP-HPLC column (21.2 mm x 25 cm, 5 μm) with a UV detector (monitoring at 215 nm). The gradient elution system used mobile phase A (0.05% TFA in water) and mobile phase B (acetonitrile). The gradient was carried out at a flow rate of 20 mL / min, starting with 95% A and 5% B for 5 min; increasing to 70% A and 30% B over 20 min; then increasing to 2% A and 98% B over 1 min and holding for an additional 5 min. The combined fractions were directly lyophilized to give 6555GA (4x TFA salt, 7 mg, 66%) as a white powder. LC-MS (ESI + ) m / z (relative intensity): 754.4 (100); tr = 16.9 min (see attached LCMS and conditions).

[0559] Support material

[0560] Compound 6555

[0561] LCMS spectrum of compound 6555:

[0562] The LCMS method was carried out using a Hewlett Packard HP LC / MSD system with a UV detector (monitoring at 215 nm) containing a ZORBAX Eclipse Plus C18 RP-HPLC column (4.6 x 50 mm, 1.8 μm). The gradient elution system used mobile phase A (0.1% TFA) and mobile phase B (acetonitrile). The gradient was carried out at a flow rate of 0.5 mL / min, starting with 98% A and 2% B for 3 min; increasing to 2% A and 98% B over 6 min; and holding for an additional 5 min. Finally, the gradient parameters were restored to the initial starting conditions. MS was run in the positive ion mode. Data was analyzed using Chemstation Software from Agilent.

[0563] Compound 6555LU

[0564] The LCMS method was carried out using a Hewlett Packard HP LC / MSD system with a UV detector (monitored at 215 nm) containing a Luna C18, 4.6 mm x 150 mm, 3.0 μm, 100A column. The gradient elution system used mobile phase A (50 mM Ammonium Acetate) and mobile phase B (acetonitrile). The gradient was carried out at a flow rate of 1.0 mL / min, starting with 98% A and 2% B for 5 min; and increasing to 74% A and 26% B over 15 min; then increasing to 2% A and 98% B over 5 min. Finally, the gradient parameters were restored to the initial starting conditions. The MS was operated in the negative ion mode. The data was analyzed using Chemstation Software from Agilent.

[0565] Compound 6555GA

[0566] The LCMS method was carried out using a Hewlett Packard HP LC / MSD system with a UV detector (monitored at 215 nm) containing a Luna C18, 4.6 mm x 150 mm, 3.0 μm, 100A column. The gradient elution system used mobile phase A (50 mM Ammonium Acetate) and mobile phase B (acetonitrile). The gradient was carried out at a flow rate of 1.0 mL / min, starting with 98% A and 2% B for 5 min; and increasing to 74% A and 26% B over 15 min; then increasing to 2% A and 98% B over 5 min. Finally, the gradient parameters were restored to the initial starting conditions. The MS was operated in the negative ion mode. The data was analyzed using Chemstation Software from Agilent.

[0567] Example 14: Synthesis of 6952 / 6952LU / 6952GA

[0568] Scheme 12

[0569]

[0570] Scheme 12. Reagents and conditions: i. L-boroPro-pn.HCl, HATU, DIEA; ii. 4N HCl in dioxane, 92% for 2 steps; iii. trans-4-(tert-butoxycarbonylaminomethyl)cyclohexanecarboxylic acid, HATU, DIEA; iv. 4N HCl in dioxane, 90% for 2 steps; Method I: v. DOTA-(OtBu)3, PyBOP, DIEA, DCM; vi. TFA-CH2Cl2 (4:1), then H2O; or Method II: vii. DOTA-PNP, TEA, DMF; viii. PhB(OH)2, H2O-TBME-ACN, 35% for 3 steps of Method I or 40% for 2 steps of Method II; ix. LuCl3, acetate buffer (0.23M, pH 5.2), 90 °C - 23 mins, 44%; ix. GaCl3, acetate buffer (0.23M, pH 5.2), 90 °C - 23 mins, 57%.

[0571] Solubility and Storage

[0572] After lyophilization, the target compounds 6952, 6952LU or 6952GA are highly soluble in water (solubility > 50 mg / ml). When in an aqueous solution at a pH of approximately 3, we did not observe any signs of degradation during HPLC purification and subsequent lyophilization. For long-term storage, the target compounds should be kept in a freezer at < -15 °C in solid form. For short-term storage, a refrigerator (+4 °C) will suffice.

[0573] Experimental Section

[0574] Reagents obtained from commercial sources were used without further purification. The synthesis of L-boroPro-pn was carried out using the 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 and a Discovery C18 569226-U RP-HPLC column. The mobile phase for semi-preparative HPLC was generally prepared by mixing water (0.1% TFA) and acetonitrile at gradient concentrations. Mass spectra and HPLC retention times were recorded on a Hewlett Packard HP LC / MSD system using an Eclipse Plus C18 RP-HPLC column (4.6 x 50 mm, 1.8 μm) and a solvent gradient of A) water (0.1% TFA) and B) acetonitrile at 0.5 mL / min, monitored with a UV detector (at 215 nm). Unless otherwise indicated, all HPLC retention times were given for an elution gradient of 2% B for the first 3 min, then from 2% to 98% B over 6 min, followed by 6 min at 98% B.

[0575] Synthesis of Intermediate 1

[0576] Under ice-water bath cooling, L-boroPro-pn.HCl (3.0 g, 10.5 mmol), HATU (4.0 g, 10.5 mmol), and DIEA (4.0 mL, 23 mmol) were added to a stirred solution of N-Boc-D-Ala-OH (Aldrich, 15048-25G; 1.9 g, 10 mmol) in anhydrous DMF (40 mL). 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 successively with 0.1 N KHSO4 (3 x 40 mL), aqueous NaHCO3 (3 x 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 flash chromatography on silica gel, eluting with ethyl acetate / hexane, and then added to a solution of 4 N HCl in dioxane (30 mL) under ice-water cooling. The resulting mixture was stirred at room temperature for 2 h and then concentrated in vacuo. The residue was co-evaporated with dichloromethane (3 x 30 mL) under vacuum to complete dryness. Compound 1 (3.3 g, 92% over two steps) was thus obtained as a white powder.

[0577] Synthesis of Intermediate 2

[0578] Under ice-water bath cooling, 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). 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 successively with 0.1 N KHSO4 (3 x 15 mL), aqueous NaHCO3 (3 x 15 mL), and brine (10 mL). The organic phase was dried over anhydrous MgSO4, filtered and evaporated in vacuo to give N-Boc-protected 2, which was purified by flash chromatography on silica gel, eluting with ethyl acetate / hexanes, and then added to a solution of 4 N HCl in dioxane (10 mL) under ice-water cooling. The resulting mixture was stirred at room temperature for 2 h and then concentrated in vacuo. The residue was co-evaporated with dichloromethane (3 x 20 mL) in vacuo to 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 min.

[0579] Synthesis of Compound 6952 (Method I)

[0580] Under ice-water bath cooling, 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). The resulting mixture was stirred at room temperature for 3 hr and then diluted with more dichloromethane (30 mL) and washed sequentially with 5% citric acid (3 x 5 mL), aqueous NaHCO3 (3 x 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 removal of TFA and dichloromethane, water (9 mL) was added and the resulting mixture was stirred at room temperature for 1 hr, and 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 hr and the separated aqueous phase was washed with more TBME. The aqueous layer was concentrated in vacuo in small portions and purified by semi-preparative Discovery C18 569226-U RP-HPLC column (21.2 mm x 25 cm, 5 μm) with a UV detector (monitored at 215 nm). The gradient elution system used 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 min; increasing to 70% A and 30% B over 20 min; then increasing to 2% A and 98% B over 1 min and holding for an additional 5 min. The combined fractions were lyophilized directly to give 6952 (4x TFA salt, 123 mg, 35% over 3 steps) as a white powder. LC-MS (ESI + ) m / z (relative intensity): 694.1 ([M-H2O+H] + , 100), 348.9 (29); tr = 7.5 min.

[0581] Synthesis of Compound 6952 (Method II)

[0582] Under ice-water bath cooling, TEA (360 μl, 2.07 mmol) was added to a stirred solution of DOTA-PNP (internally synthesized, 204 mg, 0.30 mmol) and intermediate compound 2 (162 mg, 0.33 mmol) in anhydrous DMF (4 mL). The resulting mixture was stirred overnight at room temperature. Then the reaction mixture was concentrated in vacuo. Water (9 mL) was added and the pH was adjusted to about 1.5 with 1N 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 hr and processed as described above to give 6952 (4x TFA salt, 140 mg, 40% over two steps) as a white powder.

[0583] Synthesis of Compound 6952LU

[0584] 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 min and then purified by a semi-preparative Discovery C18 569226-U RP-HPLC column (21.2 mm x 25 cm, 5 μm) equipped with a UV detector (monitored at 215 nm). The gradient elution system used 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 min; increasing to 70% A and 30% B over 20 min; then increasing to 2% A and 98% B over 1 min and holding for an additional 5 min. The combined fractions were lyophilized directly to give 6952LU (4x TFA salt, 5 mg, 44%) as a white powder. LC-MS (ESI + ) m / z (relative intensity): 865.5 (100); tr = 14.9 min (see attached LCMS and conditions).

[0585] Synthesis of Compound ##

[0586] Compound 6952 (10 mg, 8.6 μmol) was added to a solution of GaCl3 (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 min and then purified by a semi-preparative Discovery C18 569226-U RP-HPLC column (21.2 mm x 25 cm, 5 μm) with a UV detector (monitored at 215 nm). The gradient elution system used mobile phase A (0.05% TFA in water) and mobile phase B (acetonitrile). The gradient was carried out at a flow rate of 20 mL / min, starting with 95% A and 5% B for 5 min; and increased to 70% A and 30% B over 20 min; then increased to 2% A and 98% B over 1 min and held for an additional 5 min. The combined fractions were directly lyophilized to give 6952GA (4x TFA salt, 6 mg, 57%) as a white powder. LC-MS (ESI + ) m / z (relative intensity): 760.9 (100); tr = 16.1 min (see the attached LCMS and conditions).

[0587] Support material

[0588] Compound 6952:

[0589] The LCMS method was carried out using a Hewlett Packard HP LC / MSD system with a UV detector (monitored at 215 nm) containing a ZORBAX Eclipse Plus C18 RP-HPLC column (4.6 x 50 mm, 1.8 μm). The gradient elution system used mobile phase A (0.1% TFA) and mobile phase B (acetonitrile). The gradient was carried out at a flow rate of 0.5 mL / min, starting with 98% A and 2% B for 3 min; and increased to 2% A and 98% B over 6 min; which was held for an additional 5 min. Finally, the gradient parameters were restored to the initial starting conditions. MS was run in the positive ion mode. The data was analyzed using Chemstation Software from Agilent.

[0590] Compound 6952LU

[0591] The LCMS method was carried out using a Hewlett Packard HP LC / MSD system with a UV detector (monitored at 215 nm) containing a Luna C18, 4.6 mm x 150 mm, 3.0 μm, 100A column. The gradient elution system used mobile phase A (50 mM AcONH4) and mobile phase B (acetonitrile). The gradient was carried out 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; then increasing to 2% A and 98% B over 5 min. Finally, the gradient parameters were restored to the initial starting conditions. The MS was operated in the negative ion mode. The data was analyzed using Chemstation Software from Agilent.

[0592] Compound 6952GA

[0593] The LCMS method was carried out using a Hewlett Packard HP LC / MSD system with a UV detector (monitored at 215 nm) containing a Luna C18, 4.6 mm x 150 mm, 3.0 μm, 100A column. The gradient elution system used mobile phase A (50 mM AcONH4) and mobile phase B (acetonitrile). The gradient was carried out 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; then increasing to 2% A and 98% B over 5 min. Finally, the gradient parameters were restored to the initial starting conditions. The MS was operated in the negative ion mode. The data was analyzed using Chemstation Software from Agilent.

[0594] Example 15: In Vitro Assay of Dipeptidyl Peptidase IV, Fibroblast Activation Protein and Prolyl Oligopeptidase

[0595] The purpose of this assay was to determine the IC 50 .

[0596] The assay was carried out as follows:

[0597] 1. The compound was dissolved in DMSO to a final concentration of 100 mM. Thus, a 1 mM stock solution in 50 mM Tris, 140 mM NaCl buffer (FAP) at pH 7.5 / 25 mM Tris, 250 mM NaCl buffer at pH 7.5 / 140 mM NaCl buffer (PREP) at pH 8.0 was prepared.

[0598] 2. Serial dilute (1:10) a 1 mM previously prepared compound stock solution 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) onto a row of 96-well plates.

[0599] 3. Prepare 20x substrate solutions (FAP and PREP: 2.5 mM Z-Gly-Pro-AMC (VWR, Cat. No. I-1145.0050BA) in DMSO / DPPIV: 100 mM Gly-Pro-AMC (VWR, Cat. No. 100042-646) in DMSO) by diluting the DMSO stock solution into the appropriate assay buffer.

[0600] 4. Dilute the enzyme into its appropriate assay buffer. The final enzyme concentrations for DPPIV, FAP, and PREP should be 0.1, 1.2, and 0.6 nM, respectively. Add 180 μL to each well required in columns 2-10. Column 1 (A, B, C) should be prepared as a control with 200 ul of the appropriate assay buffer. Column 1 (D, E, F, G, H) should be prepared as an inhibitor-free control with 20 ul of the appropriate assay buffer and 180 ul of the enzyme.

[0601] 5. When appropriate, add 20 μL of the compound of interest from the dilution plate prepared in step 2 to columns 2-10 of the assay plate. Each sample should be tested in triplicate. Incubate it at room temperature for 10 minutes, shaking the plate continuously for the first 2 minutes.

[0602] 6. Add 10 μL of the 20x substrate prepared in step 3 to each well and incubate it at room temperature for 15 minutes, shaking the plate continuously for the first 2 minutes.

[0603] 7. Read the fluorescence at λ ex : 380, λ em : 460.

[0604] Compounds with DOTA|DOTAGA-[XXaa]n-DPcore (Group I) and their in vivo assay results are summarized in Table 4. (DPcore = [dAla|dSer|Gly]-[boroPro|Pro-nitrile], XXaa = α-amino acid).

[0605] Table 4: Group I Compounds with DOTA|DOTAGA-[XXaa]n-DPcore

[0606]

[0607]

[0608]

[0609]

[0610]

[0611]

[0612]

[0613]

[0614]

[0615]

[0616]

[0617]

[0618]

[0619]

[0620]

[0621]

[0622]

[0623]

[0624] Compounds with DOTA|DOTAGA-alkyl-[XXaa]n-DPcore (Group IA) and the in vivo assay results are summarized in Table 5.

[0625] Table 5: Group IA Compounds with DOTA|DOTAGA-alkyl-[XXaa]n-DPcore

[0626]

[0627]

[0628]

[0629]

[0630]

[0631] Compounds of Group II having DOTA|DOTAGA-[XXaa]n-[aromatic]-DPcore and their in vivo assay results are summarized in Table 6.

[0632] Table 6: Group II Compounds having DOTA|DOTAGA-[XXaa]n-[aromatic]-DPcore

[0633]

[0634]

[0635]

[0636]

[0637]

[0638]

[0639]

[0640]

[0641]

[0642]

[0643]

[0644] Compounds of Group IIA having DOTA|DOTAGA-alkyl-[aromatic]-DPcore and their in vivo assay results are summarized in Table 7.

[0645] Table 7: Group IIA Compounds having DOTA|DOTAGA-alkyl-[aromatic]-DPcore

[0646]

[0647]

[0648]

[0649]

[0650]

[0651]

[0652]

[0653]

[0654]

[0655] Compounds (Group III) having DOTA|DOTAGA-[XXaa]n-[cycloalkyl]-DPcore and their in vivo assay results are summarized in Table 8.

[0656] Table 8: Group III Compounds Having DOTA|DOTAGA-[XXaa]n-[cycloalkyl]-DPcore

[0657]

[0658]

[0659]

[0660]

[0661]

[0662] Compounds (Group IIIA) having DOTA|DOTAGA-alkyl-[cycloalkyl]-DPcore and their in vivo assay results are summarized in Table 9.

[0663] Table 9: Group IIIA Compounds Having DOTA|DOTAGA-alkyl-[cycloalkyl]-DPcore

[0664]

[0665]

[0666] Other compounds and their in vivo assay results are summarized in Table 10.

[0667] Table 10: Compounds

[0668]

[0669]

[0670]

[0671] [[ID=]]

[0672]

[0673]

[0674]

[0675]

[0676]

[0677]

[0678]

[0679]

[0680]

[0681]

[0682]

[0683]

[0684]

[0685]

[0686] Example 16: 68 Preparation of [[Ga]-6522

[0687]

[0688] The above radiopharmaceutical 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 of GaCl3 (total volume 7.875 mL, pH 4.0) are heated with shaking at 90 °C for 20 min. The reaction mixture is diluted with 40 mL of water and purified using a C18 solid phase extraction column pretreated with ethanol and water. The product is eluted with 2 mL of ethanol and the ethanol is evaporated. The evaporated product is diluted in 0.6 mL of 0.9% saline and 70 μL of 1 M NaOH is added to adjust the pH to 5.0. The product is sterile filtered (Millex-GV, 0.22 μm).

[0689] The labeling efficiency is analyzed by instant thin layer chromatography (iTLC) and is generally >90%. For iTLC analysis, 1 μL of the product is applied to a strip of iTLC-SG chromatography paper (Agilent, P / N SGI0001, 114 cm x 2.5 cm) and developed with 30% CH3CN / 70% 1 M NH4OAc (6.5 cm solvent migration) to evaluate free 68 Ga and68 Ga-colloid (Rf ~ 0) and 68 Ga]-6522 and its related impurities (Rf ~ 0.7). The 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 generally > 98%. Briefly, the product was analyzed using a Phenomenex Luna 3.0 μm C18(2), 150 mm x 4.6 mm column. Eluent A: 50 mM ammonium acetate in water, 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, radioactive HPLC detector: NaI (Eckert & Zeigler FC-1000), UV: 215 nm.

[0690] Example 17: 177 Preparation of

[0691]

[0692] The above radiopharmaceutical 177 Lu]-6522 can be prepared under the following conditions: 73 nmol / mL of the radiochemical precursor 6522 (Example 5 above), 80 mM sodium acetate, 0.4 M N-acetylmethionine, and 7.8 GBq / mL 177 LuCl3 (total volume 0.26 mL, pH 4) was heated at 70 °C with shaking for 15 min. The reaction mixture was diluted with 2.34 mL of buffer to obtain these final conditions: 8 mM sodium acetate, 0.2 M N-acetylmethionine, 6.5 mg / mL sodium ascorbate, and 0.1 mg / mL DTPA, at pH 5. The product was sterile filtered (Millex-GV, 0.22 μm). The labeling efficiency was analyzed by instant thin layer chromatography (iTLC) and was generally > 98%. For iTLC analysis, 1 μL of the diluted labeling solution was applied to a strip of iTLC-SA chromatographic paper (Agilent P / N A120B12, 114 x 2.5 mm) and developed with 0.1 M citrate buffer (8 cm solvent migration) to evaluate free 177 Lu (Rf > 0.5) and 177Lu]-6522 (Rf ~ 0). The 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 generally > 70%. Briefly, the products were analyzed using a Phenomenex Luna 3.0 μm C18(2), 150 mm x 4.6 mm column. Eluent A: 50 mM ammonium acetate in water, 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, radioactive HPLC detector: NaI (Eckert & Zeigler FC-1000), UV: 215 nm.

[0693] Example 18: 177 Additional Preparation of

[0694]

[0695] The above radiopharmaceutical 177 Lu]-6522 can be prepared under the following conditions: about 58 μg / mL of compound 6522 (Example 5 above), 70 mM sodium acetate, 0.2 M N-acetylmethionine and 7.8 GBq / mL 177 LuCl3 (total volume of 1.27 mL, pH 4) was heated at 90 °C with shaking for about 15 min. The reaction mixture was diluted with 17.43 mL of buffer to obtain these final conditions: 0.2 M sodium acetate, 0.2 M N-acetylmethionine, at pH 6.

[0696] Radiochemical purity was analyzed by high performance liquid chromatography (HPLC) and was generally > 85%. Briefly, 20 μL of the diluted product was analyzed using a Luna C18(2) column. Eluent A: 50 mM ammonium acetate in water, Eluent B: acetonitrile, gradient 2% B (5 min), from 2% to 26% B in 15 min and to 98% B in 5 min, flow rate 1.1 mL / min, detector: NaI radioactive detector (Eckert & Zeigler), UV / Vis at 215 nm.

[0697] Example 19: 177 Lu]-6555

[0698]

[0699] The above radiopharmaceutical177 Lu]-6555 can be prepared under the following conditions: 73 nmol / mL of the radiochemical 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 177 LuCl3 (total volume 0.5 mL, pH 4.5) is heated with shaking at 50 °C for about 40 min. The reaction mixture is diluted with 4.5 mL of buffer to obtain 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, at pH 5.

[0700] The labeling efficiency is analyzed by instant thin-layer chromatography (iTLC), and generally >98%. For iTLC analysis, 1 μL of the diluted labeling solution is applied to a strip of iTLC-SA chromatographic paper (Agilent P / N A120B12, 114 x 2.5 mm) and developed with 0.1 M citrate buffer (8 cm solvent migration) to evaluate free 177 Lu (Rf > 0.5) and 177 Lu]-6555 (Rf ~ 0). The iTLC strips are analyzed using an Eckert & Ziegler AR-2000 Radio-TLC imaging scanner. The radiochemical purity is analyzed by high-performance liquid chromatography (HPLC), and generally >90%. Briefly, the product is analyzed using a Phenomenex Luna 3.0 μm C18(2), 150 mm x 4.6 mm column. Eluent A: 50 mM ammonium acetate in water, 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, radioactive HPLC detector: NaI (Eckert & Zeigler FC-1000), UV: 215 nm. The radiochemical purity remains >90% at room temperature for 3 days.

[0701] Example 20: 177 Lu]-6952

[0702]

[0703] The above radiopharmaceutical 177Lu]-6952 can be prepared using the radiochemical precursor 6952 as described in Example 19 (Example 14 above). The labeling efficiency was analyzed by instant thin layer chromatography (iTLC), and generally >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 x 2.5 mm) and developed with 0.1 M citrate buffer (8 cm solvent migration) to evaluate free 177 Lu (Rf > 0.5) and 177 Lu]-6952 (Rf ~ 0). The iTLC strip was analyzed using an Eckert & Ziegler AR-2000 Radio-TLC imaging scanner. The radiochemical purity was analyzed by high performance liquid chromatography (HPLC), and generally >90%. Briefly, the product was analyzed using a Phenomenex Luna 3.0 μm C18(2), 150 mm x 4.6 mm column. Eluent A: 50 mM ammonium acetate in water, 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, radioactive HPLC detector: NaI (Eckert & Zeigler FC-1000), UV: 215 nm. The radiochemical purity remained >90% at room temperature for 3 days.

[0704] Example 21: 68 Ga]-6555

[0705]

[0706] The above radiopharmaceutical 68Ga]-6555 can be prepared under the following conditions: 73 nmol of the radiochemical precursor 6555 (Example 7 above), 0.5 M sodium acetate, 0.4 M N-acetylmethionine, and approximately 1200 MBq of GaCl3 (total volume 7.875 mL, pH 4.0) are heated with shaking at 90 °C for 20 min. The reaction mixture is diluted with 40 mL of water and purified using a C18 solid-phase extraction column pretreated with ethanol and water. The product is eluted with 3 mL of ethanol and the ethanol is evaporated. The evaporated product is diluted in 0.5 mL of phosphate-buffered saline and 70 μL of 1 M NaOH is added to adjust the pH to 5.0. The product is sterile filtered (Millex-GV, 0.22 μm). The labeling efficiency is analyzed by instant thin-layer chromatography (iTLC), and generally >95%. For iTLC analysis, 1 μL of the product is applied to a strip of iTLC-SG chromatography paper (Agilent, P / N SGI0001, 114 cm x 2.5 cm) and developed with 30% CH3CN / 70% 1 M NH4OAc (6.5 cm solvent migration) to evaluate free 68 Ga and 68 Ga-colloid (Rf ~ 0) and 68 Ga]-6555 and its related impurities (Rf ~ 0.7). The iTLC strip is analyzed using an Eckert & Ziegler AR-2000 Radio-TLC imaging scanner. The radiochemical purity is analyzed by high-performance liquid chromatography (HPLC), and generally >95%. Briefly, the product is analyzed using a Phenomenex Luna 3.0 μm C18 (2), 150 mm x 4.6 mm column. Eluent A: 50 mM ammonium acetate in water, 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, radioactive HPLC detector: NaI (Eckert & Zeigler FC-1000), UV: 215 nm. The radiochemical purity remains >95% at room temperature for 4 hours.

[0707] Example 22: 68 Ga]-6952

[0708]

[0709] The above radiopharmaceutical 68Ga]-6952 can be formed under the following conditions: 73 nmol of the radiochemical precursor 6952 (Example 14 above), 0.5 M sodium acetate, 0.4 M N-acetylmethionine, and approximately 1200 MBq of GaCl3 (total volume of 7.875 mL, pH 4.0) were heated with shaking at 90 °C for 20 min. The reaction mixture was diluted with 40 mL of water and purified using a C18 solid-phase extraction column pretreated with ethanol and water. The product was eluted with 2 mL of ethanol and the ethanol was evaporated. The evaporated product was diluted in 0.5 mL of phosphate-buffered saline and 65 μL of 1 M NaOH was added to adjust the pH to 5.0. The product was sterile filtered (Millex-GV, 0.22 μm). The labeling efficiency was analyzed by instant thin-layer chromatography (iTLC), and generally >95%. For iTLC analysis, 1 μL of the product was applied to a strip of iTLC-SG chromatography paper (Agilent, P / N SGI0001, 114 cm x 2.5 cm) and developed with 30% CH3CN / 70% 1 M NH4OAc (6.5 cm solvent migration) to evaluate free 68 Ga and 68 Ga-colloid (Rf ~ 0) and 68 Ga]-6952 and its related impurities (Rf ~ 0.7). The iTLC strip was analyzed using an Eckert & Ziegler AR-2000 Radio-TLC imaging scanner. The radiochemical purity was analyzed by high-performance liquid chromatography (HPLC), and generally >95%. Briefly, the product was analyzed using a Phenomenex Luna 3.0 μm C18 (2), 150 mm x 4.6 mm column. Eluent A: 50 mM ammonium acetate in water, 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, radioactive HPLC detector: NaI (Eckert & Zeigler FC-1000), UV: 215 nm. The radiochemical purity remained >95% at room temperature for 4 hours.

[0710] Example 23: In Vivo Biodistribution Study

[0711] Using a gamma counter, biodistribution studies were performed with 177 Lu]-6522 (also known as Compound #2) in a group of tumor-bearing male Fox Chase SCID mice inoculated with the HEK mFAP cell line. The study design used 15 mice injected with 177Mice (average body weight 22.7 ± 1.4 g) of Lu]-6522. Each mouse was injected intravenously (I.V.) with 177 Lu]-6522, receiving 175 μL, 9.05 ± 0.70 MBq. Animals in each group (n = 3 - 5) were sacrificed at specific time points, cardiac puncture was performed to collect blood, and organs were collected at 4 h, 24 h, 48 h, and 168 h after injection. Organs were excised, weighed, and their activity was measured using a gamma counter (165.6 - 364.3 keV). Tumor and normal tissue uptake was expressed as %ID / g.

[0712] Materials and Methods

[0713] Animals and Animal Husbandry

[0714] This study obtained the Fox Chase SCID mouse strain code 236 from Charles River Laboratories (Kingston, NY, USA). Before the start of the experiment, animals were housed in groups of 5. Before the start of the study, animals were acclimated for 7 days. All animal experiments were approved by the Animal Care Committee of the University Health Network (UHN) and complied with the ethical guidelines of the Canadian Council on Animal Care. Animals were housed at a constant temperature (20 °C) and 40% relative humidity, on a 12 h light / 12 h dark schedule, and given free access to food and water. Animal body weights were measured and recorded 4 days after inoculation with the HEK mFAP cell line and monitored until the day of radioactive tracer injection. Animals were not fasted before dosing. The body weights on the day of tracer administration are provided in Table 11.

[0715] Table 11. Body weights on the study date.

[0716]

[0717]

[0718] Cell Culture and Seeding

[0719] HEK-mFAP cells were cultured in RPMI 1640 (VWR, Cat. No. 45000 - 404) supplemented with the following:

[0720] 1.2 mM L-glutamine (VWR, Cat. No. 45000 - 676)

[0721] 2.10 mM HEPES (VWR, Cat. No. 45000 - 690)

[0722] 3.1 mM sodium pyruvate (VWR, Cat. No. 45000 - 710)

[0723] 4.4500 mg / L glucose (VWR, Cat. No. 45001 - 116)

[0724] 5. 1x Penicillin - Streptomycin (VWR, Cat. No. 45000 - 652)

[0725] 6. 10% FBS (Thermo Fisher Scientific, Cat. No. 10082147)

[0726] Cells were cultured in a 5% CO2 atmosphere at 37 °C. Tumor xenografts were established in male Fox Chase SCID (Charles River Laboratories, strain code 236) at 7 - 9 weeks of age by subcutaneous injection of 4×10 6 cells in 100 μL of RPMI 1640 (VWR, Cat. No. 45000 - 410) without phenol red, supplemented as described for the growth medium but without antibiotics or FBS, into the right side of the peritoneal cavity. Cells were seeded at passage 9 with a viability > 90%. 16 mice were seeded in the first batch and 15 mice in the second batch.

[0727] Mouse Tumor Volume and Randomization

[0728] Biodistribution studies were performed 30 days after tumor cell inoculation in mice injected with compound #2 ( 177 Lu]-6522), at which time the average tumor volume was 51.8 ± 44.4 mm 3 . Tumor volume was calculated using V = length × width 2 × 0.5. Table 13 shows the randomization of animals according to tumor volume.

[0729] Table 13. Tumor volumes of mice injected with compound #2 at the time of administration

[0730]

[0731] 177 Batch usage and quality of Lu]-6522​

[0732] A vial containing 177 Lu]-6522 with a radiochemical purity of 85.98% (prepared according to Example 17a) was used.

[0733] The prepared dose in the syringe was 9.05 ± 0.70 MBq 177Lu]-6522 (Compound #2). The injection dose was calculated as the decay-corrected activity in the syringe before injection minus the decay-corrected residual activity in the syringe after injection. The injection doses for each animal and each group are summarized in Table 15.

[0734] Anesthesia, Dose Administration

[0735] Mice were anesthetized with isoflurane (Fresenius Kabi Canada Ltd.) (5% induction, 1.5 - 2% maintenance). A 27Ga catheter (27G wings Infusion Set, length 15 cm, SAI Infusion Technologies) was placed in the tail vein, and ~145 - 175 μL of tracer was injected manually. The actual dose administered to each animal is shown in Table 15. After injection, the catheter was flushed with 30 μL of saline.

[0736] Table 15. Injection Doses (MBq) of Compound #2 ( 177 Lu]-6522)

[0737]

[0738]

[0739] Biodistribution Study

[0740] Biodistribution studies were performed at 4, 24, 48 h, and 168 h (p.i.). At each time point, 3 - 5 mice were sacrificed, and tumor, blood, and normal tissue samples were collected, weighed, and the radioactivity of each was measured with a gamma counter. Tumor and normal tissue uptake was expressed as the mean ± SEM (%ID / g) of the percentage of the injected dose per gram.

[0741] γ - Count Data Acquisition

[0742] Organ / tissue radioactivity was measured using a gamma counter (1480WIZARD 3”, Perkin Elmer; 60 sec counting time per vial). Depending on the animal lot number used, each time an 177 Lu]-6522 sample was used to measure an organ, a conversion factor obtained from a standard sample of known volume and known radioactivity (MBq) that was counted was used to convert the counts to activity. By this method, all activity values were corrected for inherent decay according to the injection time.

[0743] The percentage of the injected dose per organ (%ID) was calculated using the following formula:

[0744] %ID = Decay-corrected organ activity [MBq] / Injection dose [MBq] x 100%

[0745] Calculate the percentage of injected dose per gram of organ weight (%ID / g) for each organ using the following formula: %ID / g = %ID / organ weight [g]

[0746] Results and Discussion

[0747] A batch of lot number (1) 177 Lu]-6522 formulation was administered to a total of 15 male Fox Chase SCID mice. Ex vivo γ counting of various organs was performed at 4 h, 24 h, 48 h, and 168 h (n = 3 - 5) after tracer administration.

[0748] The uptake results of Compound #2 expressed as %ID / g are summarized in Table 19 below.

[0749] For Compound #2, the highest tumor uptake and the lowest radioactive concentration in blood and other normal tissues were observed at 4 h p.i. Compound #2 showed high tumor uptake as early as 4 h p.i., at 33.04 ± 5.29 %ID / g. The kidneys showed similar uptake to Compound #1, at 2.35 ± 0.51 %ID / g at 4 h, and the uptake measured at 7 days p.i. decreased over time to 0.17 ± 0.02 %ID / g, as shown in Table 19.

[0750] Compared to all other organs, higher uptake was found in the kidneys, indicating that the main route of excretion is through the kidneys. It was observed that the mouse skin showed high radioactivity at 4 h after injection, which may be due to the excretion of the compound in urine and the contamination of the mouse skin by radioactive urine.

[0751] Table 19. %ID / g of Compound #2( 177 Lu]-6522) for each organ / tissue of interest by group

[0752]

[0753] Conclusion

[0754] Up to 168 h p.i., Compound #2( 177 Lu]-6522) showed high localization in tumor xenografts and low uptake in normal tissues.

[0755] Example 24: Efficacy and Survival Study

[0756] The purpose of this study was to evaluate the therapeutic efficacy of a single injection of 177 Lu-PNT2004( 177 Lu]-6522) by assessing tumor growth delay and median survival.

[0757] Three concentrations of 177 Lu-PNT2004( 177 Lu]-6522) were prepared for injection (80 μL / mouse), and the injection dose was determined using a micro-counter (Capintec calibrated) #430x10.

[0758] Provided as described in Example 17 above 177 Lu]-6522. The following therapeutic compositions were prepared:

[0759] 1. Vehicle (selected formulation, 100 μL)

[0760] 2. Precursor (6522 compound) (80 μL)

[0761] 3. 177 Lu]-6522 15 MBq (80 μL)

[0762] 4. 177 Lu]-6522 30 MBq (80 μL)

[0763] 5. 177 Lu]-6522 60 MBq (80 μL)

[0764] A total of 30 HEK mFAP tumor-bearing mice were used for 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 side of the abdomen. Throughout the study, the mice were examined for health weekly, including body weight measurement. Tumor growth was monitored weekly by measuring with calipers (tumor volume = length × width 2 × 0.5). Study endpoints included any tumor dimension > 2 cm, tumor ulceration, the mouse being moribund, and a weight loss > 15% from the last measurement. The mice were housed 5 per cage and had free access to food and water at an environmental temperature of 20°C, humidity of 40% - 50%, and a 12-hour light / 12-hour dark cycle.

[0765] The mice were randomly divided into 5 groups, with n = 6 mice per group. The therapeutic compositions (1 - 5 above) were injected via tail IV using a catheter (fitted with a 30Ga needle). The injection dose was determined using a micro-counter (Capintec calibrated). Tumor growth was monitored weekly by measuring with calipers, and the survival of the mice was tracked.

[0766] Results:

[0767] Data were collected for tumor volume and survival analysis.

[0768] · No weight loss was observed in any of the treatment groups.

[0769] · Compared with the vehicle or precursor groups, only 177 [[Lu]-6522 at a dose of 60 MBq showed a statically significant survival benefit (see Figure 2 ). 177 All mice in the [[[Lu]-6522 60 MBq group remained alive for more than 50 days after treatment (see Figure 2 ).

[0770] · In 177 [[Lu]-6522 15 and 30 MBq groups, a delay in tumor growth was observed ( Figure 1 ), although the delay in tumor growth was not interpreted as a survival benefit ( Figure 2 ).

[0771] · In 177 [[Lu]-6522 60 MBq group, tumor regression occurred until ~43 days after treatment and then began to regrow (see Figure 1 ).

[0772] The study was terminated 57 days after the start of treatment.

[0773] Example 25: 68 [[Ga-6555 PET Imaging and Biodistribution

[0774] Part 1: Dynamic PET Imaging. The purpose of this study was to perform 68 [[Ga-6555 PET / CT dynamic imaging on HEK-mFAP tumor-bearing mice to evaluate the uptake and retention of the tumor over time and the non-specific uptake. The study was performed using HEK-mFAP tumor-bearing mice (N = 3). 68 [[Ga-6555 (prepared according to Example 20) PET imaging was performed on 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 the injection of the radiotracer and throughout the scan. During anesthesia, warming was used to maintain the healthy core body temperature of the mice. During the intravenous bolus (via the lateral tail vein) 68After injection of Ga-6555 (mean 8 MBq, range 7.7 - 8.1 MBq), dynamic emission scans were acquired in list mode for 60 min. The acquired data were then binned into 0.5-mm sinograms and 19 time frames for image reconstruction using FORE / 3D-OSEM-MAP. After PET acquisition, low-dose CT scans (80 kVp, 0.5 mA) were obtained for anatomical reference and to guide delineation of the selected volumes of interest (VOIs) of tissue. The reconstructed PET / CT images were analyzed using Siemens Inveon Research Workplace software. Radioactivity retention in the selected tissues was obtained from the mean voxel intensity values within the VOIs and was then converted to megabecquerels per milliliter using the calibration factor determined for the Inveon PET system. These values were then divided by the administered activity in megabecquerels and the animal body weight to obtain the standardized uptake values (SUVs) derived from the image VOIs. We used the maximum SUV value (SUV max ) within the VOI as a quantitative imaging metric, which is independent of tissue intrinsic variations. The representative PET images are axial, coronal, and sagittal sections with the mouse placed in the prone position.

[0775] Ga-6555 uptake was observed in the tumors and the eliminating organs (kidneys and bladder) and was consistent among the 3 mice. 68 Ga-6555 tumor time-versus-activity curves showed rapid accumulation (<5 min) and retention in the tumors, reaching a plateau at 60 min. 68 Data from one mouse are shown. Figure 3 One mouse's data are shown.

[0776] Part 2. Biodistribution. The aim of this study was to evaluate the 68 biodistribution of Ga-6555 in HEK-mFAP tumor-bearing mice. Studies were performed using HEK-mFAP tumor-bearing mice (N = 3). The mice were injected with ~8 MBq (range 7.3 - 8.5 MBq) of 68 Ga-6555 (prepared according to Example 20; IV injection via the tail vein using a catheter fitted with a 30G needle). After a 50-min uptake time (injection was performed under anesthesia using isoflurane inhalant and the mice were kept under anesthesia for 50 min), the mice were euthanized (using CO2) and tissues were harvested (blood, heart, lung, liver, spleen, pancreas, stomach, small intestine, kidney, muscle, femur, bone, skin, brain, tumor by cardiac puncture). After excision, the tissue samples were counted for gallium-68 radioactivity on a Cobra-II Auto-Gamma counter (Packard Instruments, Meriden, CT, USA), weighed and the data were expressed as % injected dose per gram (% ID / g).

[0777] The majority of activity was localized in tumors (mean %ID / g was 10.1). Kidney activity was second highest (mean %ID / g was 1.37). Uptake in all other selected tissues was low compared to muscle and considered background.

[0778] % ID / g #005 #009 #002 Mean SEM Blood 0.38 0.64 0.67 0.57 0.09 Heart 0.17 0.43 0.41 0.34 0.08 Lung 0.39 0.67 0.62 0.56 0.08 Liver 0.79 1.23 1.00 1.01 0.13 Spleen 0.74 1.34 0.23 0.77 0.32 Pancreas 0.93 0.42 1.41 0.92 0.29 Stomach 0.21 0.32 0.07 0.20 0.07 Small Intestine 0.19 0.35 0.39 0.31 0.06 Kidney 1.03 1.58 1.50 1.37 0.17 Muscle 0.21 1.92 0.09 0.74 0.59 Bone (Femur) 0.50 0.20 0.31 0.33 0.09 Skin 0.27 0.25 0.31 0.28 0.02 Brain 0.07 0.10 0.03 0.07 0.02 Tumor 8.47 11.83 10.01 10.10 0.97

[0779] Example 26: 68 Ga-6952 PET imaging and biodistribution

[0780] Using the 68 Ga-6952 was prepared according to Example 24.

[0781] Part 1 Dynamic PET imaging. Mice were injected with ~8.6 MBq (range 7.6-10.0 MBq) 68 Ga-6555 was observed in tumors and elimination organs (kidney and bladder) 68 Ga-6952 uptake was consistent among the three mice. 68 The Ga-6952 tumor time-relative-activity curves demonstrated rapid accumulation (<5 min) and retention in the tumor, reaching a plateau at 60 min.

[0782] Biodistribution. Mice were injected with ~8.6 MBq (range 7.6-10.0 MBq). The majority of activity was localized in the tumor (mean %ID / g 8.8). The kidney showed the next highest level of activity (mean %ID / g 2.18). Uptake in all other selected tissues was low compared to muscle and considered background. The data are shown below.

[0783] % ID / g #008 #016 #007 Mean SEM Blood 1.22 0.95 1.59 1.25 0.19 Heart 0.42 0.60 0.76 0.59 0.10 Lung 0.92 0.72 1.49 1.04 0.23 Liver 0.67 0.57 0.80 0.68 0.07 Spleen 0.44 0.33 0.42 0.40 0.03 Pancreas 0.41 0.44 0.36 0.40 0.02 Stomach 0.39 0.29 0.37 0.35 0.03 Small Intestine 0.34 0.19 0.75 0.43 0.17 Kidney 2.03 1.66 2.84 2.18 0.35 Muscle 0.21 0.26 0.24 0.23 0.02 Bone (Femur) 0.44 0.54 0.29 0.42 0.07 Skin 0.75 0.60 0.92 0.76 0.09 Brain 0.05 0.05 0.04 0.05 0.00 Tumor 13.30 7.13 5.97 8.80 2.27

[0784] Example 26: Treatment Plan

[0785] A human patient is selected for treatment after being diagnosed with metastatic cancer.

[0786] [ 177 Lu]-6522 is administered intravenously in a sterile aqueous solution. The dosing regimen may include four infusions of 6.8 GBq each, administered four weeks apart.

Claims

1. Compounds selected from the following: or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1, wherein the compound is: or a pharmaceutically acceptable salt thereof.

3. The compound according to claim 1, wherein the compound is: or a pharmaceutically acceptable salt thereof.

4. The compound according to claim 1, wherein the compound is: or a pharmaceutically acceptable salt thereof.

5. The compound according to claim 1, further comprising a radionuclide complexed with the compound.

6. The compound according to claim 5, wherein the radionuclide is a diagnostic radionuclide.

7. The compound according to claim 5, wherein the radionuclide is a therapeutic radionuclide.

8. The compound according to claim 5, wherein 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, 203 Pb, 18 F, 76 Br, 77 Br, 123 I, 124 I or 125 I.

9. The compound according to claim 5, wherein 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, 203 Pb, 18 F, 76 Br, 77 Br, 123 I, 124 I or 125 I.

10. The compound according to claim 5, wherein the radionuclide is 64 Cu, 68 Ga, 89 Zr, 99m Tc, 111 In, 18 F, 123 I or 124 I.

11. The compound according to claim 5, wherein 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, 131 I or 211 At.

12. The compound according to claim 5, wherein the radionuclide is 47 Sc, 67 Cu, 90 Y, 161 Tb, 177 Lu, 188 Re, 212 Pb, 213 Bi, 225 Ac, 227 Th, 131 I or 211 At.

13. The compound according to claim 5, wherein the radionuclide is 90 Y, 161 Tb, 177 Lu, 225 Ac, 227 Th, 131 I or 211 At.

14. The compound according to claim 5, wherein the radionuclide is 177 Lu.

15. The compound according to claim 5, wherein the radionuclide is 68 Ga.

16. The compound according to claim 5, wherein the radionuclide is 225 Ac.

17. The compound according to claim 5, wherein the radionuclide is 64 Cu.

18. A pharmaceutical composition comprising the compound of any one of claims 1-17, and a pharmaceutically acceptable carrier or excipient.

19. The pharmaceutical composition according to claim 18, wherein the composition further comprises N-acetylmethionine.

20. Use of the compound of any one of claims 1-17 in the manufacture of a medicament for the treatment of cancer, wherein the cancer is a solid tumor overexpressing fibroblast activation protein (FAP).

21. The use according to claim 20, wherein the cancer is a metastatic cancer of the solid tumor.

22. The use according to claim 20, wherein the cancer is prostate cancer.

23. The use according to claim 20, wherein the cancer is metastatic prostate cancer.

24. Use of the compound of any one of claims 5-17 in the manufacture of a medicament for image-guided surgery.

Images