Fibroblast activation protein-targeted compositions and methods of use thereof

Abstract

Disclosed is a compound represented by the following structural formula (I): or a pharmaceutically acceptable salt thereof. The variables of structural formula (I) are described herein. The compound of the inventon can be attached to chelating groups for radionuclide binding and are therefore suitable for radioimaging and / or radiotherapy applications, for example the disclosed compounds can be radiolabeled with a positron emitter such as 18P, 68Ga or MCu, and used for positron emission tomography (PET). Alternatively, the compounds can be radiolabeled with an alpha particle emitter such as 225 Ac, a beta particle emitter such as 67Cu or 177Lu, or an Auger electron emitter (e.g. niIn, 67Ga, "mTc, 195mPt, 125I and 1231). The compounds can also be attached to a cytotoxic agent for targeted delivery of the cytotoxic agent to tumors, for example conjugated to gemcitabine or doxycycline, or a venom. Likewise, the compounds can be conjugated to compounds having physiological effects, such as TLR agonists to stimulate the immune response of a recipient.

Description

[0001] Docket No. 137262-00320

[0002] FIBROBLAST ACTIVATION PROTEIN-TARGETED COMPOSITIONS AND METHODS OF USE THEREOF

[0003] RELATED APPLICATIONS

[0004] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 682,520, filed August 13, 2024, the entire teachings of which are incorporated herein by reference.

[0005] FIELD OF THE INVENTION

[0006] The present technology relates to targeted imaging and therapy agents, more particularly compounds useful in the diagnosis and treatment of disease. For example, the compositions described herein may be used as radiopharmaceutical agents, or conjugated to optical dyes or fluorophores, or drug / toxin conjugates, useful for the diagnosis and treatment of cancers and fibrotic disease in tissues.

[0007] BACKGROUND OF THE INVENTION

[0008] Fibroblast activation protein alpha (“FAP”) is a 170 kDa type-II membrane-bound enzyme that demonstrates serine protease activity. A soluble form of FAP is present in blood plasma, lacking the intracellular and transmembranal sequences of the full-length protein. Other common names for FAP include Prolyl Endopeptidase and Surface Expressed Protease (seprase).

[0009] FAP is one of several members of the S9B prolyl oligopeptidase subfamily, which includes among other proteins, DPP4, DPP8 and DPP9. Substrates for FAP include neuropeptide Y, peptide YY, substance P, B-type natriuretic peptide, fibroblast growth factor 21 (FGF-21), alpha2 antiplasmin, and denatured collagen I and III.

[0010] FAP is actively expressed in tissues that are undergoing wound healing and remodeling, but is otherwise not expressed, or expressed at extremely low levels in healthy, mature tissues. Tumors are localized regions of histological injury to the host, and are active at remodeling local vasculature and endothelium, as well as several other phenomena that serve to hide the tumor from immune surveillance and promote wound healing - a microenvironment that allows the neoplastic cells to proliferate. Accordingly, FAP expression is correlated with regions of tumorigenic tissues, particularly the tumor stroma and therefore presents an excellent molecular target for the diagnosis and treatment of various Docket No. 137262-00320 cancers. Its expression has been confirmed in numerous cancers, such as pancreatic, liver, gall bladder, neuroblastoma, breast, ovarian, esophageal, kidney, melanoma and many other deadly and aggressive tumors and cancer types. FAP expression is also detected in fibrotic tissues and can also be a marker for a wide spectrum of clinical conditions including systemic fibrotic diseases such as systemic sclerosis (SSc), sclerodermatous graft vs. host disease, and nephrogenic systemic fibrosis, as well as numerous organ- specific disorders including radiation-induced fibrosis and cardiac, pulmonary, liver (such as NAFLD: non-alcoholic fatty liver disease and NASH: non-alcoholic steatohepatitis), and kidney fibrosis.

[0011] While several researchers have explored FAP as a cancer target, the development of agents that can serve as diagnostics or even therapeutics have proven only marginally useful, due to their pharmacokinetic limitations. To date FAP has proven to be a difficult target due to the low and limited expression of the target and the limited residence time the prior art compounds display in vivo. What is needed are improved FAP-binding agents that demonstrate better binding kinetics and biodistribution, thereby providing the basis for improved FAP-targeted diagnostic and therapeutic agents, i.e., compounds that can accumulate to a greater degree in tumors without unacceptable uptake in normal non-target tissues and organs.

[0012] SUMMARY OF THE INVENTION

[0013] Disclosed herein are a series of compounds that bind to the extracellular domain of FAP, displaying high-to-very high affinities for the human FAP protein (see Example 2). They can be attached to chelating groups for radionuclide binding and are therefore suitable for radioimaging and / or radiotherapy applications, for example the disclosed compounds can be radiolabeled with a positron emitter such as18F,68Ga orMCu, and used for positron emission tomography (PET) (see Example 3). Alternatively, the compounds can be radiolabeled with an alpha particle emitter such as225Ac, a beta particle emitter such as67Cu or177LU, or an Auger electron emitter (e.g.n iIn,67Ga, "mTc,195mPt,125I and123I). The compounds can also be attached to a cytotoxic agent for targeted delivery of the cytotoxic agent to tumors, for example conjugated to gemcitabine or doxycycline, or a venom. Likewise, the compounds can be conjugated to compounds having physiological effects, such as TLR agonists to stimulate the immune response of a recipient. The disclosed compounds have the advantage of increased circulatory residence time, which has the effects of Docket No. 137262-00320 increasing target loading while reducing accumulation of the compound in non-target tissues (see Example 6). Accordingly, the favorable binding kinetics of the compounds disclosed herein reduce the “wash-out” effect (i.e., low residence time) seen with prior art FAP-targeted compounds.

[0014] One embodiment of the invention is a compound represented by the following structural formula (I):

[0015] (i); or a pharmaceutically acceptable salt thereof, wherein: n is 0 or 1 ;

[0016] Z is NH, O, S, CR6R7, NHCO, CONH or a 4-7 membered nitrogen containing heterocycle bonded to Y through a ring nitrogen atom of the heterocycle;

[0017] Y is a 5-6 membered nitrogen containing heteroaryl group:

[0018] A is NH, O, S or CR6R7;

[0019] B and B’ are independently a branched, unbranched or cyclic aliphatic group of up to 30 carbon atoms optionally interrupted by up to 10 heteroatoms or a peptidyl chain of up to 20 amino acid residues, wherein B is optionally substituted with 1-5 groups selected from F, Cl, Br, I, =0, OR6, OCOR6, COOR6, CN, =NR6, NR6R7, =S, and SR6;

[0020] X is O or S;

[0021] R1is a chelating group, an optical dye or fluorophore, a cytotoxic agent, an immune stimulant, or a benzoyl group optionally substituted by one or more groups represented by R5;

[0022] R3is Ci-Cs alkyl or C1-C4 aralkyl, wherein: the Ci-Cs alkyl and the aryl portions of the aralkyl are each optionally and independently substituted with F, Cl, Br, I, branched, unbranched or cyclic Ci-Ce aliphatic group, OR6, OCOR6, COOR6, CHO, COR6, CH2OR6, NR6R7, CH2NR6R7, SR6, =0, =S and =NH;

[0023] R4is CN or B(OH)2;each R5is independently selected from halo, cyano, halomethyl, N+(CHa)aW- wherein W is a pharmaceutically acceptable anion; Docket No. 137262-00320

[0024] R6and R7are independently selected from the group consisting of H or a Ci-Ce alkyl; and

[0025] R8and R9are independently H or C1-C4 alkyl or taken together with their intervening carbon atom form a C3-C6 cycloalkyl; or

[0026] B-A-C(=X)R3is N(R10), wherein each R10is independently H or Ci-Ce alkyl.

[0027] Another embodiment of the invention is a pharmaceutical composition comprising: i) a compound disclosed herein or a pharmaceutically acceptable salt thereof; and ii) a pharmaceutically acceptable carrier or diluent. For compounds comprising a chelating group, the chelating group is preferably chelated with a radionuclide.

[0028] Another embodiment of the invention is a method of treating a subject with diseased tissue that expresses fibroblast activation protein alpha. The diseased tissue in one aspect can be a cancer or a fibrotic tissue. The method comprises administering an effective amount of the compound disclosed herein or pharmaceutically acceptable salt to the subject. Preferably, the compound used for therapy comprises a cytotoxic agent, such as a chelating group having a radionuclide that emits beta, alpha, Auger or other cytotoxic radiation which can kill the diseased tissue.

[0029] Yet another embodiment of the invention is a method of imaging a region in a subject having or suspected of having a cancer or a fibrotic tissue disease which expresses fibroblast activation protein alpha or fibrotic tissue, comprising: a. administering to the subject a diagnostically effective amount of a compound disclosed herein or pharmaceutically acceptable salt thereof or a pharmaceutical composition disclosed herein; b. exposing a region in the subject to an imaging device, the region suspected of having diseased tissue; and c. obtaining an image of diseased tissue in the region.

[0030] Preferably, the compound used for imaging comprises a chelating group having a radionuclide that emits gamma-rays or positrons or other detectible radiation. In another aspect, the compound comprises an optical dye or a fluorophore, the emissions of which can be detected. Docket No. 137262-00320

[0031] Yet another embodiment of the invention is a method of imaging tumors. The method comprises: a. administering to a subject, a compound disclosed herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in an amount effective to contact and bind to the tumor and / or surrounding tissue; b. irradiating the tumor and / or surrounding tissue at a wavelength absorbed by the bound compound; c. and detecting a signal from the irradiated bound compound, thereby imaging the tumor and / or surrounding tissue.

[0032] Preferably, the compound used for imaging comprises a chelating group having a radionuclide that emits gamma-rays or positrons, or an optical dye or a fluorophore, or other detectible radiation.

[0033] Still another embodiment of the invention is a method of treating diseased tissue. The method comprises: a. administering to a subject, a compound disclosed herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in an amount effective to contact and bind to the diseased tissue; b. using the compound as a fiducial, irradiating the region of the bound compound with one or more doses of external beam radiation, thereby treating the diseased tissue with radiation.

[0034] Preferably, the compound used for the fiducial comprises a chelating group having a radionuclide that emits gamma-rays or positrons, or an optical dye or a fluorophore, or other detectible radiation.

[0035] Even still another embodiment of the invention is a method of treating diseased tissue. The method comprises: a. administering to a subject, a compound disclosed herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in an amount effective to contact and bind to the diseased tissue; b. using the compound as a fiducial for guided surgery applications, to resect the region of the diseased tissue Docket No. 137262-00320 thereby excising the diseased tissue.

[0036] Preferably, the compound used for the fiducial comprises a chelating group having a radionuclide that emits gamma-rays or positrons, or an optical dye or a fluorophore, or other detectible radiation.

[0037] BRIEF DESCRIPTION OF THE FIGURES

[0038] FIG. 1 shows the organ biodistribution of [Ga-68]RTX-1376S in BALB / C nude mice previously transplanted with U-87MG cells.

[0039] FIG. 2 shows the organ biodistribution of [Ga-68]RTX-1378R in BALB / C nude mice previously transplanted with U-87MG cells.

[0040] FIG. 3 shows the organ biodistribution of [Lu-177]RTX-1387S in BALB / C nude mice previously transplanted with U-87MG cells.

[0041] DETAILED DESCRIPTION

[0042] Disclosed herein are a series of compounds that bind with high affinity to the extracellular domain of FAP and are capable of delivering a pay load to a tissue expressing FAP. Compounds of the invention are described herein below.

[0043] A first embodiment of the invention is a compound represented by structural formula (I) or a pharmaceutically acceptable salt thereof. The definitions of the variables in structural formula (I) are provided above in the Summary of the Invention.

[0044] A second embodiment of the invention is a compound represented by structural formula (I) or a pharmaceutically acceptable salt thereof, wherein B and B’ are independently a branched or unbranched aliphatic group of 3 to 15 carbon atoms optionally interrupted by up to 3 heteroatoms or a peptidyl chain of up to 3 amino acid residues, wherein the aliphatic group is optionally substituted with F, Cl, Br, I, =0, OR6, OCOR6, COOR6, CN, =NR6, NR6R7, =S, or SR6, and the remainder of variables are as defined above for structural formula

[0045] (I).

[0046] A third embodiment of the invention is compound represented by structural formula

[0047] (II): Docket No. 137262-00320 or a pharmaceutically acceptable salt thereof, wherein the variables are as described in the first or second embodiment.

[0048] A fourth embodiment of the invention is a compound represented by structural formula (III): or a pharmaceutically acceptable salt thereof; wherein the variables are as described in the first or second embodiment.

[0049] A fifth embodiment of the invention is compound represented by structural formula

[0050] (IV): or a pharmaceutically acceptable salt thereof; wherein the variables are as described in the first or second embodiment.

[0051] A sixth embodiment of the invention is a compound represented by structural formula (I), (II), (III) or (IV), or a pharmaceutically acceptable salt thereof, wherein B is Docket No. 137262-00320 alkylene- and the C2-C6 alkylene in the group represented by B is optionally substituted by COOR6; m is an integer from 0 to 12; o is 0 or 1; and B’ is *NH(C2-C6alkylene) optionally substituted by COOR6, represents the point of attachment to Y or the triazine and the remainder of the variables are as described in the first or second embodiment. .

[0052] A seventh embodiment of the invention is a compound represented by structural formula (I), (II), (III) or (IV), or a pharmaceutically acceptable salt thereof, pharmaceutically acceptable salt thereof, wherein o is 1 and m is 3-12. The remainder of the variables are as described in the sixth embodiment.

[0053] An eighth embodiment of the invention is a compound represented by structural formula (I), (II), (III) or (IV), or a pharmaceutically acceptable salt thereof, wherein n is 1 and the remainder of the variables are as described for the first, second, sixth or seventh embodiment.

[0054] A ninth embodiment of the invention is a compound represented by structural formula (I), (II), (III), or (IV), or a pharmaceutically acceptable salt thereof, wherein B and B’ are independently *NH(C2-C6 alkylene) optionally substituted by COOR6, represents the point of attachment to Y or the triazine and the remainder of the variables are as described for the first, second or eighth embodiment.

[0055] A tenth embodiment of the invention is a compound represented by structural formula (V): Docket No. 137262-00320 or a pharmaceutically acceptable salt thereof, wherein R10and R11are independently H or COOR6and the remainder of the variables are as described for the first, second embodiment or eighth embodiment.

[0056] An eleventh embodiment of the invention is a compound represented by the structural formula (VI): or a pharmaceutically acceptable salt thereof, wherein the remainder of the variables are as described in the first or tenth embodiment.

[0057] A twelfth embodiment of the invention is a compound represented by structural formula (VII):

[0058] (VII); or a pharmaceutically acceptable salt thereof, wherein the remainder of the variables are as described in the first or tenth embodiment. Docket No. 137262-00320

[0059] A thirteenth embodiment of the invention is a compound represented by structural formula (I), (II), (III), (IV), (V), (VI), (VII), or a pharmaceutically acceptable salt thereof, wherein Z is C(O)NH*, or NH and the remainder of the variables are as described in the first, second, sixth, seventh, eighth, ninth, or tenth embodiment.

[0060] A fourteenth embodiment of the invention is a compound represented by structural formula (VIII): or a pharmaceutically acceptable salt thereof, wherein: n is 0 or 1 ;

[0061] R1is a chelating group, an optical dye or fluorophore, a cytotoxic agent, an immune stimulant, or a benzoyl group optionally substituted by one or more groups represented by R5;

[0062] R3is Ci-Cs alkyl or C1-C4 aralkyl, wherein: the Ci-Cs alkyl and the aryl portion of the aralkyl are each optionally and independently substituted with F, Cl, Br, I, branched, unbranched or cyclic Ci-Ce aliphatic group, OR6, OCOR6, COOR6, CHO, COR6, CH2OR6, NR6R7, CH2NR6R7, SR6, =0, =S and =NH;

[0063] R4is CN or B(0H)2;and each R5is independently selected from halo, cyano, halomethyl, NVCHalaW- wherein W is a pharmaceutically acceptable anion;

[0064] R6and R7are independently selected from the group consisting of H or a Ci-Ce alkyl; and

[0065] R8and R9are independently H or C1-C4 alkyl or taken together with their intervening carbon atom form a C3-C6 cycloalkyl.

[0066] A fifteenth embodiment of the invention is a compound represented by structural formula (I), (II), (III), (IV), (V), (VI), (VII), or (VIII), or a pharmaceutically acceptable salt thereof, wherein R8and R9are independently H or methyl or take together with their intervening carbon atom are cyclopropyl or cyclobutyl; and the remainder of the variables are Docket No. 137262-00320 as described in the first, second, sixth, seventh, eighth, ninth, tenth, thirteenth or fourteenth embodiment.

[0067] A sixteenth embodiment of the invention is a compound represented by structural formula (I), (II), (III), (IV), (V), (VI), (VII), or (VIII), or a pharmaceutically acceptable salt thereof, wherein R3is Ci-Cs alkyl or C1-C4 aralkyl optionally substituted with C1-C4 alkyl; and the remainder of the variables are as described in the first, second, sixth, seventh, eighth, ninth, tenth, thirteenth, fourteenth or fifteenth embodiment.

[0068] A seventeenth embodiment of the invention is a compound represented by structural formula (I), (II), (III), (IV), (V), (VI), (VII), or (VIII), or a pharmaceutically acceptable salt thereof, wherein R1is a fluorophore or optical dye; and the remainder of the variables are as described in the first, second, sixth, seventh, eighth, ninth, tenth, thirteenth, fourteenth, fifteenth, or sixteenth embodiment. In one aspect, the fluorophore is indocarbocyanin, oxacarbocyanin, thiacarbocyanin, merocyanin, polymethine, coumarin, rhodamine, xanthene, fluorescein, Borodipyrromethane (BODIPY), VivoTag-680, VivoTag- S750, AlexaFluor dyes (e.g., AlexaFluor660, AlexaFluor680, AlexaFluor700, AlexaFluor750, AlexaFluor790) and DylightFluor dyes.

[0069] An eighteenth embodiment of the invention is a compound represented by structural formula (I), (II), (III), (IV), (V), (VI), (VII), or (VIII), or a pharmaceutically acceptable salt thereof, wherein R1is a chelating group that is the residue of a chelating agent; and the remainder of the variables are as described in the first, second, sixth, seventh, eighth, ninth, tenth, thirteenth, fourteenth, fifteenth, or sixteenth embodiment. Suitable chelating agents and residues of chelating agents are described below. Docket No. 137262-00320

[0070] A nineteenth embodiment of the invention is a compound represented by structural formula (I), (II), (III), (IV), (V), (VI), (VII), or (VIII), or a pharmaceutically acceptable salt thereof, wherein R1is a benzoyl group optionally substituted by one or more groups represented by R5; each R5is independently selected from halo, cyano, halomethyl, N+(CH3)3W"; and W is a pharmaceutically acceptable anion; and the remainder of the variables are as described in the first, second, sixth, seventh, eighth, ninth, tenth, thirteenth, fourteenth, fifteenth or sixteenth, embodiment. In one aspect, the halo group represented by R5is18F.

[0071] A twentieth embodiment of the invention is a compound represented by structural formula (I), (II), (III), (IV), (V), (VI), (VII), or (VIII), or a pharmaceutically acceptable salt thereof, wherein each R5is independently selected from fluoro, cyano, triflouoromethyl, N+(CH3)3W"; and the remainder of the variables are as described nineteenth embodiment. In one aspect, the fluoro group represented by R5is F18.

[0072] A twenty-first embodiment of the invention is a compound represented by structural formula (I), (II), (III), (IV), (V), (VI), (VII), or (VIII), or a pharmaceutically acceptable salt thereof, R2is H and R4is CN; and the remainder of the variables are as described in the first, second, sixth, seventh, eighth, ninth, tenth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, or twentieth embodiment.

[0073] Also included in the invention are the compounds whose preparation is described in the Exemplification and shown in the Figures, both pharmaceutically acceptable salts thereof and the neutral form. For those compounds comprising a chelating group, chelation with a radionuclide is also included in the invention.

[0074] Also included in the invention are the compounds shown below, both pharmaceutically acceptable salts thereof and the neutral form. Chelation with a radionuclide is also included in the invention.

[0075] RTX-1376S Docket No. 137262-00320

[0076] RTX-1384R Docket No. 137262-00320

[0077] RTX-1395S Docket No. 137262-00320 Docket No. 137262-00320

[0078] RTX-1406S

[0079] Docket No. 137262-00320

[0080] RTX-1445R

[0081] The nomenclature in which a compound name is preceded by an isotope indicates that the isotope is chelated to the chelating group of the compound. For example, “[68Ga]RTX- 1376S” refers to RTX-1376S in which its chelating group is chelated with68Ga.

[0082] Exemplary compounds of the invention (with their chelating group) include [68Ga]RTX-1376S; [68Ga]RTX-1378R; [68Ga]RTX-1380R; [68Ga]RTX-1382S; [68Ga]RTX-1387S; [68Ga]RTX-1395S; [68Ga]RTX-1396S; [68Ga]RTX-1397S; [68Ga]RTX-1398S; [68Ga]RTX-1404S; [68Ga]RTX-1405S; [68Ga]RTX-1406S; [68Ga]RTX-1445R; [177Lu]RTX-1376S; [177Lu]RTX-1378R; [177Lu]RTX-1380R; [177Lu]RTX-1382S; [177Lu]RTX-1387S; [177Lu]RTX-1395S; [177Lu]RTX-1396S; [177Lu]RTX-1397S; [177Lu]RTX-1398S; [177Lu]RTX-1404S; [177Lu]RTX-1405S; [177Lu]RTX-1406S; [177Lu]RTX-1445R [225Ac]RTX-1376S; [225Ac]RTX-1378R; [225Ac]RTX-1380R; [225Ac]RTX-1382S; [225Ac]RTX-1387S; [225Ac]RTX-1395S; [225Ac]RTX-1396S; [225Ac]RTX-1397S; [225Ac]RTX-1398S; [225Ac]RTX-1404S; [225Ac]RTX-1405S; [225Ac]RTX-1406S; and [225Ac]RTX-1445R.

[0083] “Aliphatic” means a saturated or unsaturated straight-chain or branched monovalent or bivalent hydrocarbon radical. Unless otherwise specified, an aliphatic group typically has 1 to 10 carbon atoms. “Alkyl” means a saturated aliphatic straight-chain or branched Docket No. 137262-00320 monovalent aliphatic radical. Unless otherwise specified, an alkyl group typically has 1 to 10 carbon atoms (Ci-io alkyl), alternatively, 1 to 6 carbon atoms (C1-3 alkyl) (i.e., 1, 2 or 3).

[0084] “Cyclic aliphatic” means a saturated or unsaturated, monovalent or bivalent, cyclic hydrocarbon ring radical. Unless otherwise specified, a cyclic aliphatic has 3 to 8 ring carbon atoms (C3-8 cycloalkyl). “Cycloalkyl” means a saturated aliphatic cyclic aliphatic. Unless otherwise specified, a cycloalkyl has 3 to 8 ring carbon atoms.

[0085] “Aryl”, alone or part or a larger moiety such as “aralkyl” is carbocyclic aromatic group such as phenyl or naphthyl.

[0086] “Aralkyl” refers to an alkyl group substituted with an aryl group. “Ci-Cxaralkyl” refers to an aralkyl group in which the alkyl portion has 1 to x carbon atoms.

[0087] “Nitrogen containing heteroaryl”, refers to a monocyclic aromatic group of 5-6 ring atoms comprising ring carbon atoms and 1-4 ring nitrogen heteroatoms. Five-membered nitrogen containing heteroaryls may optionally contain an oxygen or sulfur ring atom. Exemplary 5-6 membered heteroaryl groups include pyrrolyl, furanyl, thiophenyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, and tetrazinyl.

[0088] Compounds having one or more chiral centers can exist in various stereoisomeric forms, i.e., each chiral center can have an R or 5 configuration or can be a mixture of both. Stereoisomers are compounds that differ only in their spatial arrangement. Stereoisomers include all diastereomeric and enantiomeric forms of a compound. Enantiomers are stereoisomers that are non-superimposable mirror images of each other. Diastereomers are stereoisomers having two or more chiral centers that are not identical and are not mirror images of each other.

[0089] When the stereochemical configuration at a chiral center in a compound having one or more chiral centers is depicted by its chemical name (e.g., where the configuration is indicated in the chemical name by “7?” or “5”) or structure (e.g., the configuration is indicated by “wedge” bonds), the enrichment of the indicated configuration relative to the opposite configuration is greater than 50%, 60%, 70%, 80%, 90%, 99% or

[0090] 99.9%. “Enrichment of the indicated configuration relative to the opposite configuration” is a mole percent and is determined by dividing the number of compounds with the indicated Docket No. 137262-00320 stereochemical configuration at the chiral center(s) by the total number of all of the compounds with the same or opposite stereochemical configuration in a mixture.

[0091] When a disclosed compound having a chiral center is depicted by a structure without showing a configuration at that chiral center, the structure is meant to encompass the compound with the .S' configuration at that chiral center, the compound with the R configuration at that chiral center, or the compound with a mixture of the R and .S' configuration at that chiral center. When a disclosed compound having a chiral center is depicted by its chemical name without indicating a configuration at that chiral center with “5” or “7?”, the name is meant to encompass the compound with the .S' configuration at that chiral center, the compound with the R configuration at that chiral center or the compound with a mixture of the R and .S' configuration at that chiral center.

[0092] The FAP-targeted compounds of the present invention are useful imaging agents for diagnostic applications. For example, these can be conjugated to various metals for magnetic resonance imaging applications or conjugated to an optical dye or a fluorophore or other detectable moiety (i.e., dyes, quantum dots, etc.) for histochemistry applications and luminescence imaging applications. Likewise, the compounds can be radiolabeled and used in nuclear medicine applications. Radionuclides that can be used for imaging applications are referred to herein as “imaging radionuclides”. Non-limiting examples of imaging radionuclides include18F,MCu or68Ga, which are suitable for use in PET imaging applications, and67Cu or177Lu, which are typically therapy nucleotides but are also suitable for use in SPECT imaging applications.

[0093] The FAP-targeting compounds of the present invention are useful therapy compounds. Such a therapy compound includes a FAP-targeted compound of the invention with a suitable therapeutic moiety. The FAP-targeted compound may be separated from the therapeutic moiety by a covalent linker. The separation between these (on the basis of a contiguous atom count) may be from about 4 atoms to about 100 atoms. Furthermore, by including additional targeting structures on the compound, the pharmacokinetics of the compound can be altered. For example, using a blood-targeting moiety it is possible to increase circulatory residence time, which has the effects of increasing tumor perfusion and loading while reducing accumulation of the radiotherapy compound in non-target tissues. See, for example U.S. Patent 11,285,277 which describes a trifunctional (“Trillium”) compound using various click chemistry and other synthesis techniques, forming a core to Docket No. 137262-00320 which three domains are affixed. The basic structure of an exemplary Trillium compound is illustrated in the structure below and includes a triazine core to which the first, second and third domains are attached. The first domain includes a targeting moiety that can bind with high affinity to a cellular protein expressed in a tumor or in the tumor microenvironment. The second domain includes another targeting moiety that can bind to a blood protein, such as albumin, with relatively low affinity. The third domain includes a moiety for detection or therapy, such as a chelator with a radionuclide; or a cytocidal or cytostatic agent such as a toxin, a venom, a metabolic poison, or a chemotherapy drug, for example gemcitabine. With radiopharmaceuticals, the third domain includes a chelator, and the radionuclide it can be loaded with may be an alpha-emitting radionuclide, a beta-emitting radionuclide, an Auger electron emitting radionuclide or one that emits a spectrum of radiation upon decay (including positron emissions, which are also suitable for diagnostic uses). For certain nonradioactive diagnostic applications, the third domain may include an optical dye.

[0094] Domain 1

[0095] Domain Domain 2

[0096] In a currently preferred embodiment, the FAP-binding compounds of the present invention are one of the targeting moieties that are adapted to the Trillium core by covalent linkage, and provide the tumor-targeting and binding function. Useful variations of the above may include bivalent compounds, where either the first and second domains include the same tumor- targeting moieties; or where each targets a different tumor / tumor microenvironment protein, in both cases the tumor-targeting moieties will display high affinity towards their targets.

[0097] For radiotherapy, the FAP-targeted compound is conjugated to a chelator, which is selected based on its suitability to hold an appropriate therapeutic radionuclide. A “therapeutic radionuclide” is a radionuclide that can be used for therapeutic purposes, e.g., for treating cancer or fibrotic tissue due to their radioactive emissions, which have cytotoxic effects on targeted tissues (i.e., FAP-expressing cancers and tumor microenvironments, malignancies, and fibrotic cells). Although targeted radiotherapy has been practiced for some time using macrocyclic complexes of radionuclides, the macrocycles currently in use (e.g., DOTA) generally form complexes with many therapeutic radionuclide metals, such as actinium, radium, bismuth, astatine, lutetium, and lead isotopes among others. Instability of Docket No. 137262-00320 many known macrocyclic-containing compounds can result in some dissociation of the radionuclide from the macrocycle, and this results in a lack of selective delivery to the intended targeted tissue, which can also result in toxicity to non-targeted tissue. Alphaemitting radionuclides such as 225 Ac can provide much greater cytotoxic effects, and thus for therapy are considered substantially more potent than beta-emitting radionuclides. But this toxicity requires a chelator with increased retention of the chelated metal. U.S. Patent 11,279,698 (see also, PCT / US2018 / 025488, and PCT / US2019 / 062479) describes a novel chelator (“Macropa”) and its’ use as a chelator for 225 Ac, including as a component of a Trillium PK-tuned, targeted radiotherapy agent. The ratio of tumor activity to kidney activity of 1 or greater may persist up to about 36 hours after administration of the radiotherapeutic, and in the case of an225Ac Trillium-based therapeutic may persist for 72, or even 128 hours, or longer, maximizing the therapeutic effects of the radiation on the target tissues.

[0098] Accordingly, an exemplary preferred FAP-targeted Trillium compound will have in its’ third (non-targeting) domain, a chelator. Macropa is the currently preferred chelator for225Ac-FAP-targeted Trillium compounds. Exemplary structures follow, (see also, PCT / CA2021 / 050226).

[0099] Docket No. 137262-00320

[0100] The radionuclide usable with the compounds disclosed herein depends on the application, radiation type desired and half-life as will be apparent to those of skill in the art. Exemplary radionuclides include:177Lu,175Lu,45Sc,64Cu,67Cu,68Cu,66Ga,67Ga,68Ga,69Ga,71Ga,90Y,89Y,86Y,89Zr,90Y, "mTc, "'in,113In,115In,139La,134Ce,136Ce,138Ce,140Ce,142Ce,151EU,153EU,152Dy,149Tb,159Tb,154Gd,155Gd,156Gd,157Gd,158Gd,160Gd,188Re,186Re,213Bi,21'At,217At,227Th,226Th,225Ac,233Ra,152Dy,213Bi,212Bi,21'Bi,203Pb,212Pb,255Fm, and Docket No. 137262-00320 uranium-230. The radionuclide of any embodiment herein may be both a therapeutic radionuclide, and a diagnostic radionuclide depending on its’ decay profile. Currently preferred alpha-emitting radionuclides for therapy applications include225Ac,233Ra, and212Pb. Currently preferred beta-emitting radionuclides for therapy applications include177Lu,90Y, and67Cu.

[0101] Chelating groups and polyaza polycarboxylic macrocycles useful in the present technology include, and refer to a group that can chelate, bind or otherwise deliver a radionuclide to a therapeutic or diagnostic target. A chelating group is the residue of a chelating agent after the chelating agent reacts with a nucleophilic group in a compound to form a targeted bivalent radio pharmaceutical or radio diagnostic agent that can bind and deliver a radionuclide. In the case of the disclosed compounds, the reactive group is the side chain amine of the lysyl group in the penultimate precursor that reacts with the chelating agent to form the disclosed compounds. Examples of chelating agents include, but are not limited to, a covalently conjugated substituted or unsubstituted member of the following group: l,4,7-triazacyclononane-l,4,7-triacetic acid (NOTA), p-SCN-Bn-NOTA, 1,4,7,10- tetraazacyclododecane-l,4,7,10-tetraacetic acid (DOTA), p-SCN-Bn-DOTA (also known as 2B-DOTA-NCS), PIP-DOTA, diethylenetriaminepentaacetic acid (DTPA), PIP-DTPA, AZEP-DTPA, ethylenediamine tetraacetic acid (EDTA), triethylenetetraamine- N,N,N',N",N'",N",-hexa-acetic acid (TTHA), 7-[2-(bis-carboxymethylamino)-ethyl]-4,10-bis- carboxymethyl-l,4,7,10-tetraaza-cyclododec-l-yl-acetic acid (DEPA), 2,2',2”-(10-(2- (bis(carboxymethyl)amino)-5-(4-isothiocyanatophenyl) pentylj-1,4,7,10- tetraazacyclododecane-l,4,7-triyl)triacetic acid (3p-C-DEPA-NCS), NETA,{4- carboxymethyl-7 - [2-(carboxymethylamino)-ethyl] -perhydro- 1 ,4,7 -tri azonin- 1 -yl } -acetic acid (NPTA) , diacetylpyridinebis(benzoylhydrazone) , 1 ,4,7 , 10, 13 , 16-hexaazacyclooctadecane N,N',N',,N'",N'",,N'""-hexaaceticacid (HEHA), octadentate terephthalamide ligands, 2,2'-(4- (2-(bis(carboxymethyl)amino)-5-(4-isothiocyanatophenyl)pentyl)-10-(2- (bis(carboxymethyl)amino)ethyl)- 1 ,4,7,10-tetraazacyclododecane- 1 ,7 -diyljdiacetic acid, N,N'-bis[(6-carboxy-2-pyridil)methyl]-4, 13-diaza- 18-crown-6 (H2macropa), 6-(( 16-((6- carboxypyridin-2-yl)methyl)- 1 ,4, 10,13-tetraoxa-7 , 16-diazacyclooctadecan-7 -yl)methyl)-4- isothiocyanatopicolinic acid (macropa-NCS), 6-((16-((6-carboxypyridin-2-yl)methyl)- 1 ,4, 10, 13-tetraoxa-7 , 16-diazacyclooctadecan-7 -yl)methyl)-4-isocyanatopicolinic acid (macropa-NCO), 3,9-carboxymethyl-6-(2-methoxy-5-isothiocyanatophenyl)carboxymethyl- 3,6,9,15- tetraazabicyclo-[9.3.1]pentadeca-l(15),l 1,13-triene and 2-[4,7,10-tris(2-amino-2- Docket No. 137262-00320 oxoethyl)- 1,4, 7, 10-tetrazacyclododec-l-yl] acetamide (TCMC or DOTAM). In a currently preferred embodiment, the chelator is the residue of a polyaza polycarboxylic macrocycle, such as Macropa NCS or NCO-Macropa. In another aspect, the chelator is the residue of a siderophores, In one aspect,225Ac is the radionuclide for Macropa NCS or NCO-Macropa. In another aspect, the chelating agent is the residue of p-SCN-Bn-DOTA, p-SCN-Bn-NOTA, NOTA or DOTA. In another embodiment, the chelating agent is sarcophagene chelator. In another aspect, the chelating agent is the residue of p-SCN-Bn-DOTA, p-SCN-Bn-NOTA, NOTA or DOTA chelated with68Ga.

[0102] As noted above, complexes of the disclosed compounds or pharmaceutically acceptable salts thereof may contain one or more radionuclides which are suitable for use as radio-imaging agents. Imaging methods include positron emission tomography (PET) or single photon emission comput ed tomography (SPECT). Accordingly in another aspect, the invention provides for theranostic applications, i.e., methods where a subject with a cancer, a tumor or a fibrotic disease is administered an effective amount of a disclosed compound (or a pharmaceutically acceptable salt thereof) having a chelator, which is complexed to an imaging radionuclide for imaging applications, and administered an effective amount of the compound complexed to a therapeutic radionuclide for treatment. Exemplary cancers which can be imaged and / or treated with the disclosed compounds or a pharmaceutically acceptable salt thereof include pancreatic cancer, liver cancer, gall bladder cancer, neuroblastoma, breast cancer, ovarian cancer, esophageal cancer, kidney cancer, prostate cancer, colorectal cancer, soft tissue sarcoma, bone sarcoma or melanoma.

[0103] A “subject” is a mammal in need of medical treatment or diagnosis, preferably a human, but can also be an animal in need of veterinary treatment, e.g., companion animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, sheep, pigs, horses, and the like) and laboratory animals (e.g., rats, mice, guinea pigs, and the like).

[0104] The disclosed compounds or pharmaceutically acceptable salts thereof (including chelation with a radionuclide) or pharmaceutical compositions thereof, may be administered orally or via a parenteral route, usually injection or infusion. A "parenteral administration route" means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, Docket No. 137262-00320 subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion.

[0105] “Effective amount” of the disclosed compounds or pharmaceutically acceptable salts thereof (including chelation with a radionuclide) means that amount of therapeutic agent alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment of prevention of a disease, that improves overall therapy, reduces or avoids symptoms or causes of disease, or enhances the therapeutic efficacy of or synergies with another therapeutic agent. With respect to imaging, “diagnostically effective amount” refers to the amount which will result in a useful image for diagnosing, e.g., the presence of a tumor. “Effective amount” of the disclosed compounds or pharmaceutically acceptable salt thereof, is determined by the physician on the basis of the patient- specific parameters, such as age, weight, sex, severity of the disease, etc. The dosage is preferably from 0.0001 mg / kg to 100 mg / kg body weight.

[0106] Corresponding to the kind of administration, the medicament is suitably formulated, e.g. in the form of solutions or suspensions, simple tablets or dragees, hard or soft gelatine capsules, suppositories, ovules, preparations for injection, which are prepared according to common galenic methods.

[0107] When solutions for infusion or injection are used, they are preferably aqueous solutions or suspensions, it being possible to produce them prior to use, e.g. from lyophilized preparations which contain the active substance as such or together with a carrier, such as mannitol, lactose, glucose, albumin and the like. The ready-made solutions are sterilized and, where appropriate, mixed with excipients, e.g. with preservatives, stabilizers, emulsifiers, solubilizers, buffers and / or salts for regulating osmotic pressure. The sterilization can be obtained by sterile filtration using filters having a small pore size according to which the composition can be lyophilized, where appropriate. Small amounts of antibiotics can also be added to ensure the maintenance of sterility.

[0108] According to another aspect, a pharmaceutical composition is provided, which is suitable for in vivo imaging and / or radiotherapy of a target tissue. Suitable pharmaceutical compositions may contain a radioimaging agent that has a radionuclide either as an element, (i.e.,18F), or a diagnostic radioactive metal chelate complex (e.g., with64Cu or68Ga) , or a radiotherapeutic agent which is radioactive metal chelate complex, in an amount sufficient Docket No. 137262-00320 for binding to the target tissue, together with a pharmaceutically acceptable radiological vehicle. The radiological vehicle should be suitable for injection or aspiration, such as human serum albumin; aqueous buffer solutions, e.g., tris(hydromethyl) aminomethane (and its salts), phosphate, citrate, bicarbonate, etc.; sterile water physiological saline; and balanced ionic solutions containing chloride and or dicarbonate salts or normal blood plasma cautions such as calcium potassium, sodium and magnesium.

[0109] The concentration of the radiopharmaceutical agent in the radiological vehicle should be sufficient to provide reasonable binding to the target tissue, such as about 4% to 20% ID / gram. For example, when using an aqueous solution, the human dosage can range from about 1.0 to 500 millicuries of activity. The actual dose administered to a patient for imaging or therapeutic purposes, however, is determined by the physician administering treatment. The imaging agent or therapeutic agent should be administered so as to remain in the patient for about 1 hour to 10 days, although both longer and shorter time periods are acceptable. Therefore, convenient ampoules containing 1 to 10 mL of aqueous solution may be prepared.

[0110] Imaging may be carried out in the normal manner, for example by injecting a sufficient amount of the imaging composition to provide adequate imaging and then scanning with a suitable imaging or scanning machine, such as a tomograph or gamma camera. In certain embodiments, a method of imaging a region in a patient includes the steps of: (i) administering to a patient a diagnostically effective amount of a compound complexed with a radionuclide; exposing a region of the patient to the scanning device; and (ii) obtaining an image of the region of the patient. Accordingly, the invention provides a method for obtaining an image of a mammalian subject following administration of the compound. Likewise, imaging can be performed after administration of a therapeutic drug or radiotherapy cycle to assess efficacy. Thus, obtaining an image after administration of the radiotherapeutic may occur after about 1 hour, about 4 hours, about 9 hours, about 12 hours, about 16 hours, about 20 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 96 hours, about one week, about two weeks, about four weeks, or after completion of a cycle of therapeutic treatments. Thus, in some embodiments, a method of imaging tissue such as FAP-expressing tumor tissue is provided including contacting the tissue with a complex synthesized by contacting an imaging radionuclide with a disclosed compound. Docket No. 137262-00320

[0111] According to another aspect, complexes of the disclosed compounds or pharmaceutically acceptable salts thereof may contain one or more radionuclides which are suitable for use as radio-imaging agents in the field of image guided radiation therapy (IGRT). As described in U.S. Patent No. US 10688320B2, IGRT uses images acquired before a treatment session to guide the application of therapeutic radiation during a treatment session. The concentration of the imaging agent or the therapeutic agent in the radiological vehicle should be sufficient to provide satisfactory imaging. For example, when using an aqueous solution, the dosage is about 1.0 to 100 millicuries. Imaging can be performed to provide a fiducial, for guidance for the target region to receive a calculated radiation fluence from a therapeutic radiation source. Similar uses of the compounds as fiducials can be used in guided surgery applications.

[0112] The amount of the compound of the present invention, or a formulation comprising a complex of a metal and a compound or pharmaceutically acceptable salt thereof that is administered to a patient depends on several physiological factors that are routinely used by the physician, including the nature of the procedure to be carried out, the volume of tissue to be targeted for imaging or therapy and the body weight and medical history of the patient to be imaged or treated using the compounds.

[0113] The examples herein are provided to illustrate advantages of the present technology and to further assist a person of ordinary skill in the art with preparing or using the compounds of the present technology or salts, pharmaceutical compositions, derivatives, prodrugs, or tautomeric forms thereof. The examples herein are also presented in order to more fully illustrate the preferred aspects of the present technology. The examples should in no way be construed as limiting the scope of the present technology, as defined by the appended claims. The examples can include or incorporate any of the variations, aspects or embodiments of the present technology described above. The variations, aspects or embodiments described above may also further each include or incorporate the variations of any or all other variations, aspects or embodiments of the present technology.

[0114] EXEMPLIFICATION

[0115] Example 1 - Preparation of Intermediates

[0116] Synthesis of tert-butyl (S)-2-amino-6-[2-(p-isobutylphenyl)acetylamino]hexanoate (Int-1) Docket No. 137262-00320 lnt-1

[0117] 7

[0118] Procedure 1: Synthesis of tert-butyl (S)-6-amino-2-[(9H-fluoren-9- yl)methoxycarbonylamino]hexanoate (6)

[0119] To a rt solution of tert-butyl (S)-2-[(9H-fluoren-9-yl)methoxycarbonylamino]-6-(tert- butoxycarbonylamino)hexanoate (100 mg, 0.190 mmol, 1 eq) in DCM was added the 4 N HCI in dioxane (95 pL, 0.38 mmol, 2 eq) and the mixture was stirred for 4 h. After LCMS analysis indicated consumption of starting material, the reaction was concentrated by rotary evaporation. The obtained solid was dried in vacuo overnight to yield the product (99 mg, 100%) as a white solid. The crude product was taken forward without further purification. LCMS: C25H32N2O4: m / z: 424.53, observed m / z = 425.56 [M+H]+.

[0120] Procedure 2: Synthesis of tert-butyl (S)-2-[(9H-fhioren-9-yl)methoxycarbonylamino]-6-[2-(p- isobutyl-phenyl) acetylamino] hexanoate (7 )

[0121] To a solution of tert-butyl (S)-6-amino-2-[(9H-fhioren-9-yl)methoxycarbonyl- amino]hexanoate (99 mg, 0.233 mmol, 1 eq) in DCM was added (p-isobutylphenyl)acetic acid (51mg, 0.233 mmol, 1 eq), DIPEA (125 pL, 0.699 mmol, 3 eq), HOBt (33 mg, 0.256 mmol, 1.1 eq), EDCI (40 mg, 0.256 mmol, 1.1 eq) and the mixture was stirred at rt for 16 h. DCM (10 ml) and water (10 ml) were added. The organic layer was separated, and the aqueous layer was further extracted with DCM (3x10 ml). The combined organic layers were dried over Na2SO4 and the solvent was evaporated to give a residue which was purified flash Docket No. 137262-00320 chromatography (0-10% MeOH in DCM) to yield the product (68 mg, 48%) as a light-yellow solid. LCMS: C37H46N2O5: m / z: 598.77, observed m / z = 599.93 [M+H]+.

[0122] Procedure 3: Synthesis of tert-butyl (S)-2-amino-6-[2-(p- isobutylphenyl)acetylamino]hexanoate (8)

[0123] Diethylamine (35 pL, 0.341 mmol, 3 eq) was added to a rt solution of tert-butyl (S)-2-[(9H- fluoren-9-yl)methoxycarbonylamino]-6-[2-(p-isobutyl-phenyl)acetylamino]hexanoate (68 mg, 0.113 mmol, 1 eq) in THF (1 ml) and the reaction mixture was stirred at room temperature for 1 h. The volatiles were removed, and the residue was purified by HPLC. The desired fractions were evaporated to yield the product (32 mg, 75%) as a colorless solid. LCMS: C22H36N2O3: m / z: 376.53, observed m / z = 377.40 [M+H]+.

[0124] The following compounds were prepared using procedures similar to those for Compound

[0125] Int-1 in procedures 1-3 above using the corresponding amine and carboxylic acid:

[0126] Synthesis of tert-butyl (S)-2-amino-6-{2-[4,7,10-tris(tert-butoxycarbonylmethyl)-l,4,7,10- tetraaza- 1 -cyclododecyl] acetylamino } hexanoate (Int-4) Docket No. 137262-00320

[0127] Procedure 4: Synthesis of tert-butyl (.S')-2-[(9 / / -fluorcn-9-yl)mcthoxycarbonylamino ]-6-{2-

[0128] [4,7, lO-tris(tert-butoxycarbonylmethyl)- 1 ,4,7, 10-tetraaza- 1 - cyclododecyl] acetylamino } hexanoate

[0129] 9 10

[0130] To a solution of tert-butyl (S)-6-amino-2-[(9H-fluoren-9- yl)methoxycarbonylamino]hexanoate (100 mg, 0.235 mmol, 1 eq) in DMSO (1 ml) was added [4,7, lO-tris(tert-butoxy-carbonylmethyl)- 1 ,4,7, 10-tetraaza- 1 -cyclododecyl] acetic acid (134 mg, 0.235 mmol, 1 eq), DIPEA (125 |1L, 0.705 mmol, 3 eq), and PyBOP (146 mg, 0.282 mmol, 1.2 eq). The reaction mixture was stirred at rt for 16 h. The reaction mixture was treated with brine (10 ml) and extracted with DCM (3x10 ml). The combined organic layers were dried over Na2SO4 and evaporated to give a crude compound that was purified by flash chromatography (0-20% MeOH in DCM) to yield the product (74 mg, 32%) as a colorless solid. LCMS: CsslfeNeOn: m / z: 979.25, observed m / z = 980.24 [M+H]+.

[0131] Procedure 5: Synthesis of tert-butyl (S)-2-amino-6-{2-[4,7,10-tris(tert- butoxycarbonylmethyl)- 1 ,4,7 , 10-tetraaza- 1 -cyclododecyl] acetylamino } hexanoate (Int-4) Docket No. 137262-00320

[0132] To a solution of tert-butyl (5')-2-[(977-fluoren-9-yl)methoxycarbonylamino]-6-{2-[4,7,10-tris- (tert-butoxycarbonylmethyl)- 1,4, 7, 10-tetraaza-l-cyclododecyl]acetylamino (hexanoate (74 mg, 0.075 mmol, 1 eq) in THF (1 ml) was added the diethylamine (23 pL, 0.225 mmol, 3 eq). The reaction mixture was stirred at rt for 1 h, the volatiles were removed, and the residue was purified by HPLC. The desired fractions were collected and evaporated to yield the product (44 mg, 77%) as a viscous oil. LCMS: C38H72N6O9: m / z: 757.01, observed m / z = 758.21 [M+H]+.

[0133] The following compounds were prepared using procedures similar to those for Compound Int-4 in procedures 4 and 5 using DOTA and the corresponding Fmoc protected diamine:

[0134] Synthesis of (S)- 1 - { 2- [(7 - { 2-oxo-2- [2- (2- { 2- [2-( 1 -piperazinyl)ethoxy] ethoxy } ethoxy )ethyl- amino]ethoxy } -4-quinolyl)carbonylamino] acetyl } -2-pyrrolidinecarbonitrile (Int-6)

[0135] Docket No. 137262-00320 lnt-6

[0136] Procedure 6: Synthesis of 2-{2-[2-(formylmethoxy)ethoxy]ethoxy}ethylamino phenylacetate (13)

[0137] To a solution of 2-{2-[2-(2-hydroxyethoxy)ethoxy]ethoxy}ethylamino phenylacetate (100 mg, 0.305 mmol, 1 eq) in DCM (1 ml) was added Dess Martin periodinane (140 mg, 0.335 mmol, 1.2 eq) and the mixture was stirred at room temperature for 36 h. The reaction mixture was diluted with brine (10 ml) and extracted with DCM (3x10 ml). The combined organic layers were dried over Na2SO4 and evaporated to give the crude product (70 mg, 70%) as a colorless oil. This material was used without further purification. LCMS: C16H23NO6: m / z: 325.36, observed m / z = 326.28 [M+H]+.

[0138] Procedure 7: Synthesis of tert-butyl 4-[2-(2-{2-[2-

[0139] (benzyloxycarbonylamino)ethoxy ] ethoxy } ethoxy )-ethyl] - 1 -piperazinecarboxylate (14) Docket No. 137262-00320

[0140] To a solution of 2-{2-[2-(formylmethoxy)ethoxy]ethoxy]ethylamino phenylacetate (70 mg, 0.215 mmol, 1 eq) and tert-butyl 1 -piperazinecarboxylate (70 mg, 0.237 mmol, 1.1 eq) in DCE (0.7 ml) was added DIPEA (115 pL, 0.645 mmol, 3 eq) and sodium triacetoxyborohydride (54 mg, 0.258 mmol, 1.2 eq) The mixture was stirred at room temperature for 3 h before it was treated with water (10 ml) and extracted with DCM (3x10 ml). The combined organic layers were dried over Na2SO4 and evaporated to give a residue that was purified by flash chromatography (0-5% MeOH in DCM) to yield the product (62 mg, 58%) as a light-yellow solid. LCMS: C25H41N3O7: m / z: 495.61, observed m / z = 496.84 [M+H]+.

[0141] Procedure 8: Synthesis of tert-butyl 4-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethyl)-l- piperazinecarbo-xylate (15)

[0142] To a solution of tert-butyl 4-[2-(2-{2-[2-(benzyloxycarbonylamino)ethoxy]ethoxy}ethoxy)- ethyl]- 1 -piperazinecarboxylate (62 mg, 0.125 mmol, 1 eq) in EtOH (2 ml) was added Pd / C (10% wet) (13 mg, 0.125 mmol). The mixture and stirred under H2 balloon pressure for 2 h. The mixture was filtered through celite and concentrated. The obtained solid was dried in vacuo for 16 h to yield the product (39 mg, 86%) as a viscous oil. This material was used without further purification. LCMS: C17H35N3O5: m / z: 361.48, observed m / z = 362.45 [M+H]+.

[0143] Procedure 9: Synthesis of tert-butyl 4-(2-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-l-pyrrolidinyl]-2- oxoethylamino } carbonyl)-7 -quinolyloxy] acetylamino } ethoxy )ethoxy] ethoxy } ethyl)- 1 - piperazinecarboxylate (16)

[0144] To a solution of tert-butyl 4-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethyl)-l- piperazinecarboxylate (39 mg, 0.108 mmol, 1 eq) in DCM was added [4-({2-[(S)-2-cyano-l- Docket No. 137262-00320 pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetic acid (41 mg, 0.108 mmol, 1 eq), DIPEA (58 pL, 0.324 mmol, 3 eq), HOBt (16 mg, 0.118 mmol, 1.1 eq), EDCI (18 mg, 0.118 mmol, 1.1 eq). The mixture was stirred for 16 h at rt before it was diluted with DCM (10 ml) and water (10 ml). The aqueous layer was extracted with DCM (3x10 ml) and the combined organic layers were dried over Na2SO4 and evaporated to give a residue.

[0145] Purification by flash chromatography (0-10% MeOH in DCM) yielded the product (31 mg, 40%) as a white solid. (LCMS: C36H51N7O9: m / z: 725.83, observed m / z = 726.90 [M+H]+.

[0146] Procedure 10: Synthesis of (S)-l-{2-[(7-{2-oxo-2-[2-(2-{2-[2-(l- piperazinyl)ethoxy] ethoxy] ethoxy )ethyl-amino] ethoxy }-4-quinolyl)carbonylamino] acetyl }-

[0147] 2-pyrrolidinecarbonitrile (Int-6)

[0148] To a solution of tert-butyl 4-(2-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-l-pyrrolidinyl]-2- oxoethylamino } carbonyl)-7 -quinolyloxy] acetylamino } ethoxy )ethoxy] ethoxy } ethyl)- 1 - piperazinecarboxylate (31 mg, 0.042 mmol, 1 eq), in DCM (1 ml) was added TFA (64 pL, 0.84 mmol, 20 eq). The mixture was stirred at rt for 4 h. The volatiles were evaporated to give the product (26 mg, 97%) as an orange solid. This material was used without further purification. LCMS: C31H43N7O7: m / z: 625.72, observed m / z = 626.54 [M+H]+.

[0149] The following compounds were prepared using procedures similar to those for Compound

[0150] Int-6 in procedures 6-10 above using the corresponding reagents: Docket No. 137262-00320

[0151] Synthesis of (4-{[2-(2-{(lR,2S,8R)-2,9,9-trimethyl-3,5-dioxa-4-boratricyclo[6.1.1.02,6]dec-

[0152] 4-yl}- l-pyrrolidinyl)-2-oxoethylamino]carbonyl}-7-quinolyloxy)acetic acid (Int-12): Docket No. 137262-00320

[0153] Int-12 was prepared according to the procedures described in WO 2024 / 026072 and WO 2025 / 029608.

[0154] Synthesis of ((R)- l-((R)-2-(7-(2-oxo-2-(2,3,5,6-tetrafluorophenoxy)ethoxy)quinoline-4- carboxami-do)propanoyl)pyrrolidin-2-yl)boronic acid (Int-13):

[0155] Int-13 was prepared according to the procedures described in WO 2025 / 029608.

[0156] Synthesis of tri-tert-butyl 2,2',2"-(10-(2-(((S)-5-((4-(4-(2-(2-(2-(2- aminoethoxy )ethoxy)ethoxy)ethyl)piperazin-l-yl)-6-(((S)-l-(tert-butoxy)-6-(2-(4- isobutylphenyl)acetamido)-l-oxohexan-2-yl)amino)-l,3,5-triazin-2-yl)amino)-6-(tertbutoxy)-6-oxohexyl)amino)-2-oxoethyl)- 1 ,4,7, 10-tetraazacyclododecane- 1 ,4,7-triyl)triacetate Docket No. 137262-00320

[0157] Step 1: Under nitrogen atmosphere, a clean and dried microwave vial was charged with 3 (1 eq) and benzyl (2-(2-(2-(2-(piperazin-l-yl)ethoxy)ethoxy)ethoxy)ethyl)carbamate (1 eq), ACN (0.53 ml) and DIPEA (5 eq) (measured pH = 9-10). The vial was sealed and heated to 90 °C for 4 h. The volatiles were removed and the residue was purified by HPLC to yield the desired product.

[0158] Step 2: To a solution of tri-tert-butyl 2,2',2"-(10-(2-(((S)-6-(tert-butoxy)-5-((4-(((S)-l-(tert- butoxy)-6-(2-(4-isobutylphenyl)acetamido)-l-oxohexan-2-yl)amino)-6-(4-(3-oxo-l -phenyl- 2,7,10,13-tetraoxa-4-azapentadecan-15-yl)piperazin-l-yl)-l,3,5-triazin-2-yl)amino)-6- oxohexyl)amino)-2-oxoethyl)- 1 ,4,7, 10-tetraazacyclododecane- 1 ,4,7-triyl)triacetate (0.125 mmol, 1 eq) in EtOH (2 ml) was added Pd / C (10% wet) (13 mg, 0.125 mmol). The mixture and stirred under H2 balloon pressure for 2 h. The mixture was filtered through celite and concentrated. The obtained solid was dried in vacuo for 16 h to yield the Int-14. This material was used without further purification.

[0159] Synthesis of 2-((E)-2-((E)-2-(4-(l-amino-13-oxo-3,6,9-trioxa-12-azapentadecan-15- yl)phenoxy)-3-(2-((E)-3,3-dimethyl-5-sulfonato-l-(3-(trimethylammonio)propyl)indolin-2- ylidene)ethylidene)cyclohex-l-en-l-yl)vinyl)-3,3-dimethyl-l-(3-(trimethylammonio)propyl)-

[0160] 3H-indol-l-ium-5-sulfonate (Int-16): Docket No. 137262-00320

[0161] Step 1: Int-15 was prepared according to the procedures described in WO 2024 / 026072. To the solution of Int-15 (1.5 eq.) in DMSO (1 mL) was added a solution of (9H-fluoren-9- yl)methyl (2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)carbamate (1.0 eq.) in DMSO (1 mL). N,N-Diisopropylethylamine (1.5 eq.) was added at room temperature, and the mixture was allowed to stir for 16 h. Full conversion was detected by LCMS. After the reaction completion, the volatiles were removed and the residue was purified by HPLC to yield the desired product.

[0162] Step 2: The resulting product was dissolved in THF and diethylamine was added (10 eq.) at rt. The reaction mixture was stirred for 1 hour and evaporated to dryness under vacuum without heating. The residue was subjected to silica gel purification to afford Int-16.

[0163] Synthesis of 2-(2-{2-[2-(chloroformyl)ethoxy]ethoxy}ethoxy)ethylaminophenylacetate (Int- 17):

[0164] To a solution of 3-(2-{2-[2-(benzyloxycarbonylamino)ethoxy]ethoxy}ethoxy)propionic acid (100 mg, 0.280 mmol) in toluene (1.0 mL) was added SOCh (0.030 mL, 0.422 mmol). The mixture was stirred at 30 °C for 7 h and subsequently concentrated and co-evaporated with toluene (3 x 5 mL) to yield the title compound (136 mg, 0.364 mmol, quantitative yield) as a light-yellow oil. This material was used without further purification. LCMS: C18H27NO7 (Mass with MeOH addition): m / z: 369.41, observed m / z = 370.20 [M+H]+.

[0165] Synthesis of benzyl (2-(2-(2-(3-((4-chloro-6-(dimethylamino)-l,3,5-triazin-2-yl)amino)-3- oxopropoxy )ethoxy)ethoxy)ethyl)carbamate (Int-18):

[0166] A solution of 2 (602 mg, 1.20 mmol) in ACN (7.5 mL) was stirred for 5 min in an ice-water bath. Dimethyl amine (0.136 mL, 1 mmol, 33% aqueous solution) was added in portions over 20 min. K2CO3 (0.166 g, 1.2 mmol) was added and the reaction mixture was stirred at 0 °C for 3 h. Upon completion the mixture was concentrated, treated with brine (50 mL) and extracted with EtOAC (3 x 50 mL). The volatiles were removed to obtain a crude material Docket No. 137262-00320 which was purified by flash chromatography (0-40% EtOAc in hexanes) to yield Int-18 (373 mg, 61%), isolated as a colorless solid. LCMS: C22H31CIN6O6: m / z: 510.20, observed m / z = 511.48 [M+H]+.

[0167] Synthesis of tri-tert-butyl 2,2',2"-(10-(2-((6-(tert-butoxy)-5-((4-(dimethylamino)-6-(3-oxo-l- phenyl-2,7,10,13-tetraoxa-4-azahexadecan-16-amido)-l,3,5-triazin-2-yl)amino)-6- oxohexyl)amino)-2-oxoethyl)- 1 ,4,7, 10-tetraazacyclododecane- 1 ,4,7-triyl)(S)-triacetate (Int-

[0168] 19):

[0169] To a solution Int-18 (118 mg, 0.233 mmol) in ACN (0.45 mL) and DMSO (0.2 mL) was cooled to -20 °C and treated with a solution of DIPEA (50 pL, 0.29 mmol) and amine (44 mg, 0.058 mmol) in ACN (0.2 mL). The mixture was stirred at 0 °C for 30 min and then slowly warmed to room temperature and stirred for 3 h. The mixture was concentrated to obtain a crude product which was purified by flash chromatography (0-10% MeOH in DCM) to yield Int-19 (32%), LCMS: C60H102N12O15: m / z: 1230.76, observed m / z = 1231.50 [M+H]+.

[0170] Example 2 - Synthesis of Products

[0171] Triazine Compounds

[0172] Synthesis of (S)-2-(6-{(S)-l-carboxy-5-[2-(4-isobutylphenyl)acetylamino]pentylamino}-4- [4-(2- { 2- [2-(2- { 2- [4-( { 2- [(S)-2-cyano- 1 -pyrrolidinyl]-2-oxoethylamino }carbonyl)-7- quinolyloxy] acetylamino }ethoxy)ethoxy]ethoxy Jethyl)- 1-piperazinyl]- 1 ,3,5-triazin-2- ylamino)-6- { 2- [4,7 , 10-tris(carboxymethyl)- 1 ,4,7 , 10-tetraaza- 1 - cyclododecyl] acetylamino} hexanoic acid (RTX-1406S) Docket No. 137262-00320

[0173] Procedure 11: Synthesis of tert-butyl (5)-2-(4,6-dichloro-l,3,5-triazin-2-ylamino)-6-[2-(4- isobutylphenyl)acetylamino]hexanoate (2):

[0174] A -20 °C solution of 2,4,6-trichloro-l,3,5-triazine (65 mg, 0.685 mmol, 4 eq) in acetonitrile (0.65 ml) was treated with DIPEA (76 pL, 0.850 mmol, 5 eq),) and Int-1 (32 mg, 0.170 mmol, 1 eq) in acetonitrile / DMSO (0.3 ml). The mixture was warmed to -10 °C over 20 min, and then to 0 °C over 1 h. The progress of the reaction was monitored by LCMS. After starting material was consumed, the mixture was diluted with cold EtOAc (5 ml, chilled at - 10 °C) and poured carefully in a mixture of ice and 10 % citric acid buffer (5 ml, pH = 4). The organic layer was separated, washed with brine (3x5 ml), dried over Na2SO4, and concentrated to obtain a residue that was purified by flash chromatography (0-10% MeOH in DCM) to yield the product (62 mg, 34%) as a colorless solid. LCMS: C25H35C12N5O3: m / z: 524.48, observed m / z = 525.50 [M+H]+. Docket No. 137262-00320

[0175] Procedure 12: Synthesis of tert-butyl (S)-2-(6-{(S)-5-[2-(4-isobutylphenyl)acetylamino]-l- tert-butoxycarbonylpentylamino]-4-chloro-l,3,5-triazin-2-ylamino)-6-{2-[4,7,10-tris(tert- butoxycarbonylmethyl)- 1 ,4,7, 10-tetraaza- 1 -cyclododecyl] acetylamino } hexanoate (3)

[0176] A cold (-20 °C) solution of tert-butyl (S)-2-(4,6-dichloro-l,3,5-triazin-2-ylamino)-6-[2-(4- isobutylphenyl)acetylamino]hexanoate (62 mg, 0.118 mmol, 4 eq) in ACN (0.62 ml) and DMSO (0.31 ml) was treated with a solution of DIPEA (105 pL, 0.59 mmol, 5 eq),) and Int- 4 (89 mg, 0.118 mmol, 1 eq) in DCM. The mixture was stirred at 0 °C for 30 min and slowly warmed to rt over 3 h. The volatiles were evaporated to obtain a residue that was purified by flash chromatography (0-10% MeOH in DCM) to yield the product (53 mg, 33%) as a colorless solid. LCMS: C63H106CIN11O12: m / z: 1245.04, observed m / = 1246.20 [M+H]+.

[0177] Procedure 13: Synthesis of tert-butyl (S)-2-{4-[4-(2-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-l- pyrrolidinyl] -2-oxoethylamino } carbonyl)-7 - quinolyloxy] acetylamino }ethoxy)ethoxy]ethoxy } ethyl)- 1 -piperazinyl] -6- { (S)-5-[2-(4- isobutylphenyl)acetylamino]-l-tert-butoxycarbonylpentylamino]-l,3,5-triazin-2-ylamino}-6- {2-[4,7,10-tris(tert-butoxycarbonylmethyl)-l,4,7,10-tetraaza-l- cyclododecyl] acetylamino [hexanoate Docket No. 137262-00320

[0178] Under nitrogen atmosphere, a clean and dried microwave vial was charged with tert-butyl (S)-2-(6-{(S)-5-[2-(4-isobutylphenyl)acetylamino]-l-tert-butoxycarbonylpentylamino}-4- chloro- 1 ,3 ,5-triazin-2-ylamino)-6- { 2- [4,7 , 10-tris(tert-butoxycarbonylmethyl)- 1,4,7,10- tetraaza-1 -cyclododecyl] acetylamino [hexanoate (53 mg, 0.042 mmol, 1 eq) and (S)-l-{2-[(7- { 2-oxo-2- [2- (2- { 2- [2-( 1 -piperazinyl)ethoxy] ethoxy } ethoxy )ethyl-amino] ethoxy } -4- quinolyl)carbonylamino] acetyl }-2-pyrrolidinecarbonitrile (26 mg, 0.042 mmol, 1 eq), ACN (0.53 ml) and DIPEA (37 pL, 0.21 mmol, 5 eq) (measured pH = 9-10). The vial was sealed and heated to 90 °C for 4 h. The volatiles were removed, and the residue was purified by HPLC to yield the product (42 mg, 47%) as a colorless solid. LCMS: C94H148N18O19: m / z: 1834.29, observed m / z = 1835.40 [M+H]+.

[0179] Procedure 14: Synthesis of (S)-2-(6-{(S)-l-carboxy-5-[2-(4- isobutylphenyl)acetylamino]pentylamino } -4-[4-(2- { 2- [2- (2- { 2- [4-( { 2- [(S)-2-cyano- 1 - pyrrolidinyl] -2-oxoethylamino } carbonyl)-? - quinolyloxy] acetylamino }ethoxy)ethoxy]ethoxy (ethyl)- 1-piperazinyl]- 1 ,3,5-triazin-2- ylamino)-6- { 2- [4,7 , 10-tris(carboxymethyl)- 1 ,4,7 , 10-tetraaza- 1 - cyclododecyl] acetylamino] hexanoic acid (RTX-1406S) Docket No. 137262-00320

[0180] TFA (225 pL, 2.94 mmol) in DCM (0.5 ml) was added to a solution of tert-butyl (S)-2-{4-[4- (2- { 2- [2- (2- { 2- [4-( { 2- [(S )-2-cy ano- 1 -pyrrolidinyl] -2-oxoethylamino } carbonyl)-? - quinolyloxy] acetylamino }ethoxy)ethoxy]ethoxy } ethyl)- 1 -piperazinyl] -6- { (S)-5-[2-(4- isobutylphenyl)acetylamino] - 1 -tert-butoxy carbonylpentylamino } - 1 ,3 ,5-triazin-2-ylamino } -6- {2-[4,7,10-tris(tert-butoxycarbonylmethyl)-l,4,7,10-tetraaza-l- cyclododecyl] acetylamino (hexanoate (36 mg, 0.019 mmol, 1 eq) and the mixture was stirred for 10 h. The volatiles were removed, and the residue was purified by HPLC to yield the product (12.3 mg, 41%) as a colorless solid. LCMS: C74H108N18O19: m / z: 1553.76, observed m / z = 1554.50 [M+H]+.

[0181] The following compounds were prepared using procedures similar to those for RTX-1406S in procedures 11-14 above: Docket No. 137262-00320

[0182] Synthesis of RTX-1397S

[0183] RTX-1397S

[0184] Step 1: To a solution of 4,6-dichloro-l,3,5-triazin-2-ylamine (60 mg, 0.064 mmol) in ACN (0.6 mL) and toluene (0.3 mL) was added 2 M aq. NaOH (32 pL, 0.064 mmol), Int-17 (136 mg, 0.064 mmol). The mixture was stirred at rt for 10 min. After completion of the reaction, the mixture was concentrated to obtain a crude product which was purified by flash Docket No. 137262-00320 chromatography (0-10% MeOH in DCM) to yield 2 (68 mg, 0.135 mmol, 37%) as a lightyellow solid. LCMS: C20H25CI2N5O6: m / z: 502.35, observed m / z = 503.50 [M+H]+.

[0185] Step 2: A solution of 2-[3-(2-{2-[2- (benzyloxycarbonylamino)ethoxy]ethoxy } ethoxy )propionyl amino]-4,6-dichloro- 1,3,5- triazine (68 mg, 0.135 mmol) in ACN (0.68 mL) and DMSO (0.34 mL) was cooled to -20 °C and treated with a solution of DIPEA (117 pL, 0.675 mmol, 5 eq),) and amine (50 mg, 0.135 mmol) in ACN (0.34 mL). The mixture was stirred at 0 °C for 30 min, and then slowly warmed to rt over 3 h. The mixture was concentrated to obtain a crude product that was purified by flash chromatography (0-10% MeOH in DCM) to yield 3 (49 mg, 0.058 mmol, 43%) as a colorless solid. LCMS: C42H60CIN7O9: m / z: 842.42, observed m / z = 843.90 [M+H]+.

[0186] Step 3: A clean and dried microwave vial was charged with tert-butyl (S)-2-{4-[3-(2-{2-[2- (benzyloxycarbonylamino)ethoxy]ethoxy]ethoxy)propionylamino]-6-chloro-l,3,5-triazin-2- ylamino}-6-[2-(4-isobutylphenyl)acetylamino]hexanoate (49 mg, 0.058 mmol), amine (37 mg, 0.058 mmol), ACN (0.5 mL) and DIPEA (50 pL, 0.29 mmol under nitrogen. The vial was sealed and its contents were heated to 90 °C for 4 h. The volatiles were removed and the remainder was purified by HPLC to yield 4 (53 mg, 0.036 mmol, 63%) as a colorless solid. LCMS: C74H121N13O16: m / z: 1448.83, observed m / z = 1449.40 [M+H]+.

[0187] Step 4: To a solution of tert-butyl (S)-2-{4-[3-(2-{2-[2-(benzyloxycarbonylamino)ethoxy] ethoxy } -ethoxy )propiony lamino] -6-(4- { 2- [4,7 , 10-tris(tert-butoxycarbonylmethyl)- 1 ,4,7 , 10- tetraaza-l-cyclodode-cyl]acetylamino}butylamino)-l,3,5-triazin-2-ylamino}-6-[2-(p- isobutylphenyl)acetylamino]hexanoate (53 mg, 0.036 mmol) in EtOH (2 mL) was added Pd / C (10% wet, 38 mg, 0.036 mmol). The mixture was stirred under H2 (balloon pressure, 1 atm) for 2 h. The mixture was filtered through celite and the filtrate was concentrated. The obtained product was dried un vacuo for 16 h to yield 5 (29 mg crude, 0.022 mmol, 61%) as a colorless, viscous oil. This material crude was used without further purification. LCMS: C66H115N13O14: m / z: 1314.70, observed m / z = 1315.40 [M+H]+.

[0188] Step 5: To a solution of tert-butyl (S)-2-[4-(3-{2-[2-(2- aminoethoxy)ethoxy] ethoxy (propionyl amino)-6-(4-{2-[4,7,10-tris(tert- butoxycarbonylmethyl)- 1 ,4,7, 10-tetraaza- 1 -cyclododecyl] acetyl amino Jbutylamino)- 1,3,5- triazin-2-ylamino]-6-[2-(4-isobutylphenyl)acetylamino]hexanoate (29 mg, 0.022 mmol) was added acid (7.9 mg, 0.022 mmol), DIPEA (11 pL, 0.066 mmol), HOBt (3.2 mg, 0.024 mmol), and EDCI (3.7 mg, 0.024 mmol) in DCM (0.5 ml). The mixture was stirred for 16 h at room temperature. The mixture was diluted with DCM (5 mL) and water (5 mL). The Docket No. 137262-00320 aqueous layer was extracted with DCM (3 x 5 mL), and the combined organic extracts were dried over Na2SO4 and concentrated using rotavapor. The crude product was purified by flash chromatography (0-20% MeOH in DCM) to yield 6 (16 mg, 0.009 mmol, 43%) as a colorless solid. LCMS: CssH iNnOis: m / z: 1679.05, observed m / z = 1670.20 [M+H]+.

[0189] Step 6: TFA (103 pL, 1.35 mmol) was added to a solution of tert-butyl (S)-2-[4-(3-{2-[2-(2- { 2- [4-( { 2- [(S)-2-cy ano- 1 -pyrrolidinyl] -2-oxoethylamino } carbonyl)-7 - quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-(4-{2-[4,7,10-tris(tert- butoxycarbonylmethyl)- 1 ,4,7, 10-tetraaza- 1 -cyclododecyl] acetylamino } -butylamino)- 1,3,5- triazin-2-ylamino]-6-[2-(4-isobutylphenyl)acetylamino]hexanoate (16 mg, 0.009 mmol) in DCM (0.2 mL) and the mixture was stirred for 10 h. The volatiles were removed under reduced pressure and the crude product was purified by HPLC to yield RTX-1397S (2.8 mg, 0.0019 mmol, 21%, 96.4 % HPLC purity) as a colorless solid. LCMS: C69H99N17O18: m / z: 1454.63, observed m / z = 1455.20 [M+H]+.

[0190] The following compounds were prepared using procedures similar to those for RTX-1397S above: Docket No. 137262-00320

[0191] Synthesis of RTX-1445R

[0192] Step 1: To a solution Int-13 (26 mg, 1.5 Eq, 37 pmol) in DMF was added solution of Int-14 (30 mg, 1 Eq, 25 pmol) in DMF followed by DIPEA (6.4 mg, 8.6 pL, 2 Eq, 49 pmol). After completion of the reaction, crude mixture was purified using reverse phase chromatography (C18 column, 0.1% FA in water / 0.1% FA in MeCN, 5-95 ACN in water) to give the desired product as white foam (22 mg, 48%).

[0193] Step 2: The resulting product (12 mg, 1 Eq, 6.9 pmol) was dissolved in acetonitrile (2 mL) and 0.5N HC1 (1 mL), methyl boronic acid (10 eq) were added and allowed to stir at rt for 1 hr. Completion of the reaction was observed by LCMS and solvent was evaporated to obtain the target compound in crude, which was purified using combi flash (reverse phase, C-18, 5- 100% gradient of 0.1% FA in acetonitrile and 0.1% FA in water)to give RTX-1445 (8 mg, 5 pmol, 70%) as a white solid.

[0194] Linear Compounds

[0195] Synthesis of RTX-1376S Docket No. 137262-00320

[0196] Step 1: To a solution of Int-7, DOTA-tris(t-Bu-ester) (0.27 g, 0.48 mmol) and PyBop (0.30 g, 0.56 mmol) in DMSO (8 ml) was added DIEA (0.20 ml, 1.15 mmol) and the reaction mixture was stirred at ambient temperature for 16 h. Water (20 ml) was added, and the mixture was extracted with EtOAc (30 ml). The separated organic layer was washed with water (7x) and brine (8x) to remove excess PyBOP. The organic layer was dried over Na2SO4 and concentrated under reduced pressure to obtain the target product. The compound was used for the next step without further purification.

[0197] Step 2: To a 0 °C solution of the resulting residue in DCM (4 ml), was added TFA (2 ml). The reaction mixture was stirred at 40 °C for 2 h and the solvents were evaporated under reduced pressure. The residue was washed with hexane (2x) and ether (2x) to obtain RTX- 1376S.

[0198] The following compounds were prepared using procedures similar to those for RTX-1376S above: Docket No. 137262-00320

[0199] Step 1: To a stirred solution of 2,2',2"-(10-(14-amino-2-oxo-6,9,12-trioxa-3-azatetradecyl)- l,4,7,10-tetraazacyclododecane-l,4,7-triyl)triacetic acid (1 eq.) in DMF (5 ml) was added DIPEA (2 eq.) followed by the addition of compound Int-12 (1.5 eq.) and the reaction mixture was stirred for 30 minutes. Upon completion (the reaction progress was monitored by LCMS), the solvent was evaporated, and the crude residue was purified by prep HPLC to afford the desired product. Docket No. 137262-00320

[0200] Step 2: The resulting product (1 eq.) was dissolved in acetonitrile (2 mL) and 0.5N HC1 (1 mL), methyl boronic acid (10 eq) were added and allowed to stir at rt for 1 hr. Completion of the reaction was observed by LCMS and solvent was evaporated to obtain the target compound in crude, which was purified using silica gel chromatography to give RTX-1378R.

[0201] The following compounds were prepared using procedures similar to those for RTX-1376S above:

[0202] Example 3 - Compounds of the Invention Bind to FAP With High Affinity Docket No. 137262-00320

[0203] Protease reactions were assembled in 384 well plates (Greiner) in a total volume of 20 uL as described below.

[0204] Recombinant proteins were pre-diluted in assay buffer comprising of lOOmM HEPES, pH 7.5, 0.1% BSA, 0.01% Triton X-100, ImM DTT, and dispensed into 384 well plate (10 uL per well). Test compounds were serially pre-diluted in DMSO and added to the assay wells by acoustic dispensing (Labcyte Echo 550). Control samples (0%-inhibition in the absence of inhibitor, DMSO only) and 100%-inhibition (in the absence of enzyme) were assembled in replicates of four and used to calculate the %-inhibition in the presence of compounds. Concentration of DMSO was equalized to 1% in all samples.

[0205] Compounds were pre-incubated with enzymes for 15 minutes. Human FAP was obtained from Enzo, catalogue number BML-SE409-0010.The reactions are initiated by addition of 10 uL of 2x FAM-labeled substrate peptide (FAM-GPRPFNYLAKK-NH2) prepared in the same assay buffer. Final concentration of enzymes were 0.5 nM. Final concentration of substrate peptides was 1 uM.

[0206] The reactions were allowed to proceed at room temperature. Incubation time was three hours for human FAP, 0.5 hours for mouse FAP. After incubation, kinase reactions were quenched by addition of 50 uE of termination buffer: assay buffer supplemented with reference inhibitor at 100 x IC50.

[0207] Terminated plates were analyzed using a microfluidic electrophoresis instrument (Caliper EabChip® 3000, Caliper Fife Sciences / Perkin Elmer). A change in relative intensity of the peptide substrate and cleaved product was the parameter measured. Activity in each test sample was determined as the product to sum ratio (PSR): P / (S+P), where P is the peak height of the product, and S is the peak height of the substrate. Percent inhibition (Pinh) was determined using the following equation: Pinh = (PSRo%inh - PSRCompound) / (PSRo%in - PSRioo%inh)* 100 , in which: PSRcompound is the product / sum ratio in the presence of compound, PSRo%in is the product / sum ratio in the absence of compound and the PSRioo%in is the product / sum ratio in the absence of the enzyme. To determine IC50 of compounds (50%-inhibition) the %-inh data (Pinh versus compound concentration) are fitted by a 4 parameter sigmoid dose-response model using XEfit software (IDBS). These values are compiled in Table 1 and grouped, where A represents an IC50 <0.1nM; B is an IC50 from 0.1 to 0.5 nM; C is an IC50 from 0.5 to 5.0 nM; D is an IC50 from 5.0 to 100 nM; E an IC50 >100 nM. Docket No. 137262-00320

[0208] Table 1. Results for Binding Affinity Studies

[0209] It has been found from the inhibition studies described above that the S stereoisomers are considerably more potent than the R stereoisomers. The inhibition studies above also revealed that extending the chain off of the quinolinyl core of Docket No. 137262-00320 , which has an LC50 of 1.5 nM, significantly improved activity. Specifically, the lengthening the chain piecemeal provided an improvement with each extension, and the addition of the final amino acid (lysine) provided an additional increase in activity.

[0210] Example 4 - Radiolabeling Methods and Results

[0211] General procedure: 20 pg of precursor (unless otherwise noted) are combined with the indicated radioisotopes The radioisotopes are either eluted from a generator (for 68Ga) or obtained commercially in HC1 solutions that were buffered with varying amounts of 3 N NaOAc to obtain a final pH of 4-6. Reactions with a C18 Sep-Pak Lite cartridge as needed for additional purity and / or reformulation for injection. Labeling results are shown in Table 2.

[0212] Table 2. Results for representative labeling reactions

[0213] Example 5 - Radiolabeling of a EAP-targeted compound with18E direct labelling Docket No. 137262-00320

[0214] 18F is received from the manufacturer, is loaded on to a Sep Pak QMA Light Plus cartridge, and is eluted into a glass reaction vial using a CS2CO3 / K222 solution. Eluted18F is azeotropic ally dried using acetonitrile (3 x 1 ml) at 95 °C under a stream of N2. 2.5mg of the FAP-targeted compound is dissolved in 0.3 ml DMSO which is then added to the above vial containing the dried18F. The vial is sealed and heated to 90 °C for 15 minutes. The vial is cooled and a solution of 1 N HC1 is added to vial, which is then sealed and heated to 95 °C for 10 minutes. The reaction mixture is cooled, neutralized, and then loaded on to a Semi Prep HPEC column for purification and formulated appropriately for use. Alternatively, a suitable FAP-targeted compound having a chelator can be used for18F A1F labelling, wherein AlCh stock in acetate buffer (22.5 pF, 45 nmol, 0.9 eq) is added to a18F solution in sodium acetate (200uE) and the reaction vial is left at room temperature for 5 min. From precursor stock, a FAP-targeted compound solution (50 nmol scale, 12.5 pF) is then added to the above vial. pH is corrected to about 4.0 by addition of 1% v / v acetic acid in water (15 pF). Co- solvent, 200 pF of EtOH, is added and the reaction vial is sealed, then heated at 100°C for 15 mins. Reaction mixture is diluted to 9.5 ml and loaded on to a tC18 Sep Pak cartridge. Product is eluted with 300uE acidified EtOH and formulated for use.

[0215] Example 6 - Detection of Tumors in Murine Models with Compounds of the Invention BAEB / c nude mice are transplanted with approximately 107U-87 (human glioblastoma) cells and the tumors are allowed to develop. A quantity of an18F-labeled FAP- targeted agent of the invention is administered to the mice by intravenous injection (e.g., tail vein), which are then sacrificed after one hour. The mouse is then imaged to evaluate the tumor binding of the18F-labeled FAP targeted compound. Biodistribution of the compound is evaluated by analysis of resected tissue samples from each organ

[0216] Example 7 - Biodistribution studies

[0217] Female BAEB / C nude mice were inoculated subcutaneously on the right shoulder with U- 87MG cells in 1:1 matrigekPBS. When the tumors reached a volume of 150-500 mm3, radiolabeled ligand was administered intravenously (IV) via tail vein. At various time points post-injection, mice were humanely euthanized via exsanguination and tissue samples (bladder, blood, urine, bone (femur), heart, lungs, liver, both kidneys, small intestines (including contents), large intestines (including contents), muscle (quadriceps), tumor, and tail) were resected, weighed and counted with a gamma counter. The activity of each Docket No. 137262-00320 collected tissue was measured in units of counts per minute (CPM). Triplicate aliquots of the radiotracer were also assayed in the gamma counter to calculate a factor for converting counts to units of activity (pCi / CPM). Values were decay corrected to the time of injection and corrected for background radiation. The biodistribution of compounds [68Ga]RTX-1376S, [68Ga]RTX-1378S, and [177Lu]RTX-1387R showed localization of the compounds within the tumor and minimal concentrations within other organs (See FIG. 1-3). This biodistribution demonstrated increased selectivity for the tumor over time demonstrating the high affinity for FAP.

[0218] EQUIVALENTS

[0219] While certain embodiments have been illustrated and described a person with ordinary the art, after reading the foregoing specification, can effect changes, substitutions of equivalents and other types of alterations to the compounds of the present technology or salts, pharmaceutical compositions, derivatives, prodrugs, metabolites, tautomers or racemic mixtures thereof as set forth herein. Each aspect and embodiment described above can also have included or incorporated therewith such variations or aspects as disclosed in regard to any or all of the other aspects and embodiments.

[0220] The present technology is also not to be limited in terms of the particular aspects described herein, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. It is to be understood that this present technology is not limited to particular methods, reagents, compounds, compositions, labeled compounds or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Thus, it is intended that the specification be considered as exemplary only with the breadth, scope and spirit of the present technology indicated only by the appended claims, definitions therein and any equivalents thereof. Docket No. 137262-00320

[0221] All publications, patents, and other documents referred to in this specification are herein incorporated by reference in its entirety.

Claims

Docket No. 137262-00320CLAIMSWhat is claimed is:1 . A compound represented by the following structural formula:or a pharmaceutically acceptable salt thereof, wherein: n is 0 or 1 ;Z is NH, O, S, CR6R7, NHCO, CONH or a 4-7 membered nitrogen containing heterocycle bonded to Y through a ring nitrogen atom of the heterocycle;Y is a 5-6 membered nitrogen containing heteroaryl group;A is NH, O, S or CR6R7;B and B’ are independently a branched, unbranched or cyclic aliphatic group of 3 to 30 carbon atoms optionally interrupted by up to 10 heteroatoms or a peptidyl chain of up to 20 amino acid residues, wherein B is optionally substituted with 1-5 groups selected from F, Cl, Br, I, =0, OR6, OCOR6, COOR6, CN, =NR6, NR6R7, =S, and SR6;X is O or S;R1is a chelating group, an optical dye or fluorophore, a cytotoxic agent, an immune stimulant, or a benzoyl group optionally substituted by one or more groups represented by R5;R3is Ci-Cs alkyl or C1-C4 aralkyl, wherein: the Ci-Cs alkyl and the aryl portion of the aralkyl are each optionally and independently substituted with F, Cl, Br, I, branched, unbranched or cyclic Ci- C6aliphatic group, OR6, OCOR6, COOR6, CHO, COR6, CH2OR6, NR6R7, CH2NR6R7, SR6, =0, =S and =NH;R4is CN or B(OH)2;and each R5is independently selected from halo, cyano, halomethyl, NhCHaJaW’ wherein W is a pharmaceutically acceptable anion;R6and R7are independently selected from the group consisting of H or a Ci-Ce alkyl; andDocket No. 137262-00320R8and R9are independently H or C1-C4 alkyl or taken together with their intervening carbon atom form a C3-C6 cycloalkyl; orB-A-C(=X)R3is N(R10), wherein each R10is independently H or Ci-Ce alkyl.

2. The compound of claim 1, wherein B and B’ are independently a branched or unbranched aliphatic group of 3 to 15 carbon atoms optionally interrupted by up to 3 heteroatoms or a peptidyl chain of up to 3 amino acid residues, wherein the aliphatic group is optionally substituted with F, Cl, Br, I, =0, OR6, OCOR6, COOR6, CN, =NR6, NR6R7, =S, or SR6.

3. The compound of claim 1 or 2 represented by the following structural formula:or a pharmaceutically acceptable salt thereof.

4. The compound of claim 3 represented by the following structural formula:or a pharmaceutically acceptable salt thereof.

5. The compound of claim 3 represented by the following structural formula:Docket No. 137262-00320or a pharmaceutically acceptable salt thereof.

6. The compound of any one of claims 1-5 or pharmaceutically acceptable salt thereof, whereinthe C2-C6 alkylene in the group represented by B is optionally substituted by COOR6; m is an integer from 0 to 12; o is 0 or 1; and B’ is *NH(C2-Ce alkylene) optionally substituted by COOR6, whereinrepresents the point of attachment to Y or the triazine.

7. The compound of claim 6, wherein or pharmaceutically acceptable salt thereof, wherein o is 1 and m is 3-12.

8. The compound of any one of claims 1-7 or a pharmaceutically acceptable salt thereof, wherein n is i.

9. The compound of any one of claims 1-5 or 8 or pharmaceutically acceptable salt thereof, wherein B and B’ are independently *NH(C2-Ce alkylene) optionally substituted by COOR6whereinrepresents the point of attachment to Y or the triazine.

10. The compound of claim 3 or 8 represented by the following structural formula:Docket No. 137262-00320 or pharmaceutically acceptable salt thereof; and R10and R11are independently H or COOR6.

11. The compound of claim 10 represented by the following structural formula:or pharmaceutically acceptable salt thereof.

12. The compound of claim 10 represented by the following structural formula:or pharmaceutically acceptable salt thereof13. The compound of any one of claims 1-12, or a pharmaceutically acceptable salt thereof, wherein14. A compound represented by the following structural formula:or a pharmaceutically acceptable salt thereof, wherein:Docket No. 137262-00320 n is 0 or 1 ;R1is a chelating group, an optical dye or fluorophore, a cytotoxic agent, an immune stimulant, or a benzoyl group optionally substituted by one or more groups represented by R5;R3is Ci-Cs alkyl or C1-C4 aralkyl, wherein: the Ci-Cs alkyl and the aryl portion of the aralkyl are each optionally and independently substituted with F, Cl, Br, I, branched, unbranched or cyclic Ci- C6aliphatic group, OR6, OCOR6, COOR6, CHO, COR6, CH2OR6, NR6R7, CH2NR6R7, SR6, =0, =S and =NH;R4is CN or B(0H)2;and each R5is independently selected from halo, cyano, halomethyl, N+fCHaJaW- wherein W is a pharmaceutically acceptable anion;R6and R7are independently selected from the group consisting of H or a Ci-Ce alkyl; andR8and R9are independently H or C1-C4 alkyl or taken together with their intervening carbon atom form a C3-C6 cycloalkyl.

15. The compound of any one of claims 1-14, or a pharmaceutically acceptable salt thereof, wherein R8and R9are independently H or methyl or take together with their intervening carbon atom are cyclopropyl or cyclobutyl.

16. The compound of any one of claims 1-15, or a pharmaceutically acceptable salt thereof, wherein R3is Ci-Cs alkyl or C1-C4 aralkyl optionally substituted with C1-C4 alkyl.

17. The compound of any one of claims 1 to 16, or a pharmaceutically acceptable salt thereof, wherein R1is a fluorophore or an optical dye.Docket No. 137262-0032018. The compound of claim 17, or a pharmaceutically acceptable salt thereof, wherein theselected from the group consisting of: a carbocyanin, indocarbocyanin, oxacarbocyanin, thiacarbocyanin, merocyanin, polymethine, coumarin, rhodamine, xanthene, fluorescein, Borodipyrromethane (BODIPY), VivoTag-680, VivoTag-S750, AlexaFluor dyes (e.g., AlexaFluor660, AlexaFluor680, AlexaFluor700, AlexaFluor750, AlexaFluor790) and DylightFluor dyes.

19. The compound of any one of claims 1 to 16 or a pharmaceutically acceptable salt thereof, wherein R1is a chelating group that is the residue of a chelating agent.

20. The compound of claim 19 or a pharmaceutically acceptable salt thereof, wherein the chelating group is the residue of a chelating agent selected from 1,4,7- triazacyclononane-l,4,7-triacetic acid (NOTA), p-SCN-Bn-NOTA, , 1,4,7,10- tetraazacyclododecane-l,4,7,10-tetraacetic acid (DOTA), p-SCN-Bn-DOTA (also known as 2B-DOTA-NCS),PIP-DOTA, diethylenetriaminepentaacetic acid (DTPA), PIP-DTPA, AZEP-DTPA, ethylenediamine tetraacetic acid (EDTA), triethylenetetraamine-N,N,N',N",N"',N"'-hexa- acetic acid (TTHA), 7-[2-(bis- carboxymethylamino)-ethyl]-4, 10-bis-carboxymethyl- 1 ,4,7, 10-tetraaza-cyclododec- 1 - yl-acetic acid (DEPA), 2,2',2"-(10-(2-(bis(carboxymethyl)amino)-5-(4- isothiocyanatophenyl) pentyl)- 1 ,4,7, 10-tetraazacyclododecane- 1 ,4,7-triyl)triacetic acid (3p-C-DEPA-NCS), NETA,{4-carboxymethyl-7-[2-(carboxymethylamino)- ethyl]-perhydro-l,4,7-triazonin-l-yl}-acetic acid (NPTA), diacetylpyridinebis(benzoylhydrazone) , 1,4, 7, 10, 13,16-hexaazacyclooctadecane N,N',N",N'",N'"',N'""-hexaaceticacid (HEHA), octadentate terephthalamide ligands,Docket No. 137262-003202,2'-(4-(2-(bis(carboxymethyl)amino)-5-(4-isothiocyanatophenyl)pentyl)-10-(2- (bis(carboxymethyl)amino)ethyl)- 1 ,4,7, 10-tetraazacyclododecane- 1 ,7-diyl)diacetic acid, N,N'-bis[(6-carboxy-2-pyridil)methyl]-4,13-diaza-18-crown-6 (H2macropa), 6- (( 16-((6-carboxypyridin-2-yl)methyl)- 1 ,4, 10, 13-tetraoxa-7 , 16-diazacyclooctadecan-7 - yl)methyl)-4-isocyanatopicolinic acid (macropa-NCO), 6-((16-((6-carboxypyridin-2- yl)methyl)-l,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)-4- isothiocyanatopicolinic acid (macropa-NCS), 3,9-carboxymethyl-6-(2-methoxy-5- isothiocyanatophenyl)carboxymethyl-3,6,9,15- tetraazabicyclo-[9.3.1]pentadeca- 1 ( 15) , 11 , 13-triene and 2- [4,7,10-tris(2-amino-2-oxoethyl)- 1,4,7,10- tetrazacyclododec-l-yl] acetamide (TCMC or DOTAM).

21. The compound of claim 19 or a pharmaceutically acceptable sat thereof, wherein the residue of the chelating agent is the residue of macropa-NCS or macropa-NCO.

22. The compound of claim 19 or a pharmaceutically acceptable salt thereof, wherein the residue of a chelating agent is the residue of p-SCN-Bn-NOTA, p-SCN-Bn-DOTA, NOTA or DOTA,23. The compound of any one of claims 1-16 or 19 or a pharmaceutically acceptable salt thereof, wherein the chelating group is the residue of a siderophore.

24. The compound of any one of claims 1-16 or 19, or a pharmaceutically acceptable salt thereof, wherein:R1is a benzoyl group optionally substituted by one or more groups represented by R5; each R5is independently selected from halo, cyano, halomethyl, NACHaJaW-; and W is a pharmaceutically acceptable anion.

25. The compound of claim 24 or a pharmaceutically acceptable salt thereof, wherein each R5is independently selected from fluoro, cyano, triflouoromethyl, N+(CHa)aW’.

26. The compound of claim 24 or 25, or a pharmaceutically acceptable salt thereof, wherein the halo or flouro group represented by R5is18F.Docket No. 137262-0032027. The compound of any one of claims 1-26 or a pharmaceutically acceptable salt thereof, wherein R6is H and R4is CN.

28. The compound of claim 1 or pharmaceutically acceptable salt thereof, represented by a structural formula selected from:Docket No. 137262-00320Docket No. 137262-0032029. The compound of claim 1 or pharmaceutically acceptable salt thereof, represented by a structural formula:

30. The compound of any one of claims 1-16, 19-23, 28 or 29, or a pharmaceutically acceptable salt thereof, wherein the residue of the chelating agent is chelated with a radionuclide.

31. The compound of claim 30 or a pharmaceutically acceptable salt thereof, wherein the radionuclide is selected from177Lu,175Lu,45Sc,64Cu,67Cu,68Cu,66Ga,67Ga,68Ga, 69Ga,71Ga,90Y,89Y,86Y,89Zr,90Y, "mTc,n iIn,113In,115In,139La,134Ce,136Ce,138Ce, 140Ce,142Ce,151EU,153EU,152Dy,149Tb,159Tb,154Gd,155Gd,156Gd,157Gd,158Gd,160Gd, 188Re,186Re,213Bi,211At,217At,227Th,226Th,225Ac,233Ra,152Dy,213Bi,212Bi,211Bi, 203Pb,212Pb,255Fm, and uranium-230.

32. The compound of claim 30 or a pharmaceutically acceptable salt thereof, wherein the radionuclide is an alpha-emitting radionuclide such as225Ac,233Ra, and212Pb.

33. The compound of claim 30 or a pharmaceutically acceptable salt thereof, wherein the radionuclide is an Auger electron emitting radionuclide or a beta-emitting radionuclide such as177Lu,90Y, and67Cu.Docket No. 137262-0032034. The compound of claim 21 or a pharmaceutically acceptable salt thereof, wherein the residue of macropa-NCS or mcaropa-NCO is chelated with225Ac.

35. A pharmaceutical composition comprising: i) the compound of any one of claims 1-34 or a pharmaceutically acceptable salt thereof; and ii) a pharmaceutically acceptable carrier or diluent.

36. A method of treating diseased tissue in a subject, wherein the diseased tissue expresses fibroblast activation protein alpha, comprising administering an effective amount of the compound or pharmaceutically acceptable salt of any one of claims 29- 34 or the pharmaceutical composition of claim 27 to the subject and wherein the radionuclide is a therapeutic radionuclide.

37. The method of claim 36, the diseased tissue is a cancer.

38. The method of claim 37, wherein the cancer is pancreatic cancer, liver cancer, gall bladder cancer, neuroblastoma, breast cancer, ovarian cancer, esophageal cancer, kidney cancer, prostate cancer, colorectal cancer, soft tissue sarcoma, bone sarcoma or melanoma.

39. The method of claim 36, wherein the diseased tissue is fibrotic.

40. A method of imaging a region in a subject having or suspected of having diseased tissue which expresses fibroblast activation protein alpha or fibrotic tissue, comprising:(i) administering to the subject a diagnostically effective amount of a compound or pharmaceutically acceptable salt thereof of any one of claims 17-18, 24-27, or 29- 34 or the pharmaceutical composition of claim 35 and wherein the radionuclide is a diagnostic radionuclide;(ii) exposing the region in the subject to an imaging device; and(iii) obtaining an image of the diseased tissue in the region.

41. The method of claim 40, wherein the region has or is suspected of having diseased tissue that includes a primary cancer or a metastasis of the cancer.Docket No. 137262-0032042. The method of claim 40, wherein the region has or is suspected of having diseased tissue that includes fibrotic tissue.

43. A method of imaging tumors, the method comprising:(i) contacting the tumor and / or surrounding tissue with a compound or pharmaceutically acceptable salt thereof of any one of claims 17-18 in an amount sufficient to bind to the tumor;(ii) irradiating the tumor and / or surrounding tissue at a wavelength absorbed by the compound;(iii)and detecting a signal from the compound, thereby imaging the tumor and / or surrounding tissue.

44. A method of treating diseased tissue, comprising:(i) administering to a subject, a compound disclosed herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in an amount effective to contact and bind to the diseased tissue;(ii) using the compound as a fiducial, irradiating the region of the bound compound with one or more doses of external beam radiation, thereby treating the diseased tissue with radiation.

45. The method of claim 44, wherein the compound comprises a chelating group having a radionuclide that emits gamma-rays or positrons, or an optical dye or a fluorophore, or other detectible radiation.

46. A method of treating diseased tissue, comprising: administering to a subject, a compound disclosed herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in an amount effective to contact and bind to the diseased tissue; and using the compound as a fiducial for guided surgery applications, to resect the region of the diseased tissue thereby excising the diseased tissue.

47. The method of claim 46, wherein the compound comprises a chelating group having a radionuclide that emits gamma-rays or positrons, or an optical dye or a fluorophore, or other detectible radiation.

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