Fibroblast Activation Protein Targeting Compositions and Methods of Use Thereof

Abstract

Disclosed are compounds, compositions, and methods useful for imaging in subjects and for treating tumor cells. Specifically, compounds having the structure of formula I are useful in the disclosed imaging methods: (I). Variables in formula (I) are defined herein. [Formula 1] TIFF2025526460000146.tif36148

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 392,899, filed July 28, 2022, the entire teachings of which are incorporated herein by reference.

[0002] The present technology relates to targeted imaging and therapeutic agents, more particularly compounds that are useful in disease diagnosis and treatment.For example, the compositions described herein can be used as radiopharmaceutical agents, or can be conjugated with optical dyes or fluorophores, or can be used as drug / toxin conjugates, which are useful for the diagnosis and treatment of cancer and fibrotic disease in tissue. [Background technology]

[0003] Fibroblast activation protein alpha ("FAP") is a 170 kDa type II membrane-bound enzyme that exhibits serine protease activity. A soluble form of FAP is present in plasma and lacks the intracellular and transmembrane sequences of the full-length protein. Other common names for FAP include prolyl endopeptidase and surface-expressed protease (seprase).

[0004] 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), alpha-2 antiplasmin, and denatured collagens I and III.

[0005] FAP is actively expressed in tissues undergoing wound healing and remodeling, but is not expressed or expressed at extremely low levels in otherwise healthy mature tissues.Tumor is a localized area of histological injury to the host, and is active in reconstructing local vasculature and endothelium, as well as in several other events that help hide tumors from immune surveillance and promote wound healing (the microenvironment that allows neoplastic cells to proliferate).Therefore, FAP expression correlates with the area of tumorigenic tissue, particularly tumor stroma, and therefore represents an excellent molecular target for the diagnosis and treatment of various cancers.Its expression has been confirmed in many cancers, such as pancreatic cancer, liver cancer, gallbladder cancer, neuroblastoma, breast cancer, ovarian cancer, esophageal cancer, renal cancer, melanoma, and many other fatal and aggressive tumor types and cancer types. FAP expression has also been detected in fibrotic tissue and may be a marker for a wide spectrum of clinical conditions, including systemic fibrotic diseases such as systemic sclerosis (SSc), graft-versus-host disease and renal fibrosis, as well as radiation-induced fibrosis, and numerous organ-specific disorders, including cardiac, pulmonary, hepatic (e.g., NAFLD: non-alcoholic fatty liver disease and NASH: non-alcoholic steatohepatitis), and renal fibrosis.

[0006] While several researchers have explored FAP as a cancer target, the development of agents that can act as diagnostic or even therapeutic agents has 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 that prior art compounds exhibit in vivo.What is needed is an improved FAP binder that demonstrates better binding kinetics and biodistribution, thereby providing a basis for improved FAP-targeted diagnostic and therapeutic agents, i.e., compounds that can accumulate to a higher degree in tumors without unacceptable uptake in normal non-target tissues and organs. Summary of the Invention

[0007] Disclosed herein are a series of compounds that bind to the extracellular domain of FAP, exhibiting "high" to "very high" affinity for the human FAP protein (see Example 12). 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 are 18 F, 68 Ga or 64 The compound may be radiolabeled with a positron emitter such as Cu and used for positron emission tomography (PET) (see Example 11). Alternatively, the compound may be radiolabeled with an alpha particle emitter, e.g. 225 Ac, beta particle emitters, e.g. 67 Cu or 177 Lu, or Auger electron emitters (e.g., 111 In, 67 Ga, 99 mTc, 195 mPt, 125 I and 123 I). The compounds can also be attached to cytotoxic agents for targeted delivery to tumors, such as gemcitabine or doxycycline, or conjugated to venom. Similarly, the compounds can be conjugated to compounds with physiological effects, such as TLR agonists that stimulate the recipient's immune response. The disclosed compounds have the advantage of extended circulation residence time, which has the effect of increasing target loading while reducing compound accumulation in non-target tissues (see Example 15). Thus, the favorable binding kinetics of the compounds disclosed herein reduces the "washout" effect (i.e., low residence time) seen with prior art FAP-targeting compounds.

[0008] One embodiment of the present invention is a compound of the following structural formula (I):

[0009] [ka]

[0010] [In the formula, n is 0 or 1; A is NH, O, S or CR 6 R 7 and; B comprises or is a branched, unbranched, or cyclic aliphatic group of up to 30 carbon atoms (e.g., 3 to 20 or 3 to 15 carbon atoms optionally interrupted by up to 6 heteroatoms or up to 5 amino acid residues), optionally interrupted by up to 10 heteroatoms or a peptidyl chain of up to 20 amino acid residues; wherein B is F, Cl, Br, I, ═O, OR 6 ,OCOR 6 , COOR 6 ,CN,=NR 6 , N.R. 6 R 7 , =S and SR 6 with the proviso that B contains at least 3 atoms in the chain between the D group and the A group; D is selected from the group consisting of OPO3H2, PO3H2, OSO3H, SO3H and COOH, and C1-C4 alkyl esters thereof; X is O or S; R 1 is a chelating group, an optical dye or fluorophore, a cytotoxic agent, an immunostimulatory agent, or R 5 is a benzoyl group optionally substituted with one or more groups represented by R 3 is C1-C8 alkyl or C1-C4 aralkyl, The alkyl and aryl portions of the aralkyl are each optionally and independently selected from F, Cl, Br, I, a branched, unbranched, or cyclic C1-C6 aliphatic group, OR 6 ,OCOR 6 , COOR 6 , CHO, COR 6 , CH2OR 6 , N.R. 6 R 7 , CH2NR 6 R 7 , S.R.6 , substituted with =O, =S and =NH; R 4 is CN or B(OH)2; Each R 5 are independently halo, cyano, halomethyl, N + (CH3)3W - (In the formula, W - is a pharmaceutically acceptable anion; R 6 and R 7 are independently selected from the group consisting of H or C1-C6 alkyl. or a pharmaceutically acceptable salt thereof.

[0011] Another embodiment of the present 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 containing a chelating group, the chelating group is preferably chelated with a radionuclide.

[0012] Another embodiment of the present invention is a method for treating a subject having diseased tissue that expresses fibroblast activation protein alpha. The diseased tissue may, in one aspect, be cancer or fibrotic tissue. The method includes administering to the subject an effective amount of a compound or pharmaceutically acceptable salt disclosed herein. Preferably, the compound used for treatment includes a chelating group having a cytotoxic agent, e.g., a radionuclide that emits beta, alpha, Auger, or other cytotoxic radiation capable of killing the diseased tissue.

[0013] Yet another embodiment of the present invention is a method of imaging an area in a subject having or suspected of having cancer or a fibrous tissue disease that expresses fibroblast activation protein alpha or fibrous tissue, comprising: a. administering to a subject a diagnostically effective amount of a compound disclosed herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition disclosed herein; b. exposing an area of the subject suspected of having diseased tissue to an imaging device; and c. Obtaining an image of the diseased tissue in the region.

[0014] Preferably, the compound used for imaging contains a chelating group with a radionuclide that emits gamma rays or positrons or other detectable radiation, hi another embodiment, the compound contains an optical dye or fluorophore, the emission of which can be detected.

[0015] Yet another embodiment of the present invention is a method of imaging a tumor, the method comprising: 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; and c. Detecting a signal from the bound and irradiated compound, thereby imaging the tumor and / or surrounding tissue.

[0016] Preferably, compounds used for imaging contain a chelating group bearing a radionuclide that emits gamma rays or positrons, or an optical dye or fluorophore, or other detectable radiation.

[0017] Yet another embodiment of the present invention is a method of treating diseased tissue, the method comprising: 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 affected tissue; b. irradiating the area of bound compound with one or more doses of external beam radiation using the compound as a location reference; Thereby treating the affected tissue with radiation.

[0018] Preferably, the compound used for location reference contains a chelating group with a radionuclide that emits gamma rays or positrons, or an optical dye or fluorophore, or other detectable radiation.

[0019] Yet yet another embodiment of the present invention is a method of treating diseased tissue, the method comprising: 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 affected tissue; b. using the compound as a location reference for guided surgical applications to remove areas of diseased tissue; This results in the removal of the stem tissue.

[0020] Preferably, the compound used for location reference contains a chelating group with a radionuclide that emits gamma rays or positrons, or an optical dye or fluorophore, or other detectable radiation. [Brief explanation of the drawings]

[0021] [Figure 1] Figure 1A shows the biodistribution of the [18F]RTX-1312S compound in BALB / c nude mice previously implanted with U-87 (human glioblastoma) cells expressing FAP. Figure 1B shows a graph showing the biodistribution of the [18F]RTX-1312S compound in BALB / c nude mice previously implanted with U-87 (human glioblastoma) cells expressing FAP. [Figure 2] 1 is a graph showing the organ biodistribution of [Cu-67]RTX-1363S in BALB / C nude mice that had been previously implanted with U-87MG cells. [Figure 3] 1 is a graph showing the organ biodistribution of [Lu-177]RTX-1354S in BALB / C nude mice that had been previously implanted with U-87MG cells. [Figure 4]1 is a graph showing the organ biodistribution of [Lu-177]RTX-1359R in BALB / C nude mice that had been previously implanted with U-87MG cells. [Figure 5] 1 is a graph showing a comparison of the biodistribution of [Ac-225]RTX-1399R in tumors compared to blood in tumor-bearing JAX nude mice. DETAILED DESCRIPTION OF THE INVENTION

[0022] Disclosed herein are a series of compounds that can bind with high affinity to the extracellular domain of FAPs and deliver a payload to tissues expressing the FAP. The compounds of the invention are described herein below.

[0023] A first embodiment of the present invention is a compound represented by structural formula (I), or a pharmaceutically acceptable salt thereof: wherein the variables in structural formula (I) are defined above in the Summary of the Invention.

[0024] A second embodiment of the present invention is a compound represented by structural formula (II):

[0025] [ka]

[0026] (wherein m is an integer of 0 to 12; o is 0 or 1; R 2 is H or C1-C4 alkyl; the remaining variables are as described in the first embodiment. or a pharmaceutically acceptable salt thereof.

[0027] A third embodiment of the present invention is a compound represented by structural formula (III):

[0028] [ka]

[0029] (wherein the variables are as described in the second embodiment) or a pharmaceutically acceptable salt thereof. A fourth embodiment of the present invention is a compound represented by structural formula (IV):

[0030] [ka]

[0031] (wherein the variables are as described in the second embodiment) or a pharmaceutically acceptable salt thereof. A fifth embodiment of the present invention is a compound represented by structural formula (I), (II), (III) or (IV): [In the formula, R 3 is C1-C8 alkyl optionally substituted with C1-C4 alkyl or C1-C4 aralkyl, and the remaining variables are as described in the first or second embodiment. 3 is methyl, propyl, pentyl, heptyl, (4-isobutylphenyl)methyl, (4-isobutylphenyl)propyl] or a pharmaceutically acceptable salt thereof.

[0032] A sixth embodiment of the present invention is a compound represented by structural formula (I), (II), (III) or (IV): wherein m is 3 to 12 and o is 1. Alternatively, m is 8 and o is 1. In another alternative embodiment, o is 0. The remaining variables are as described in the first, second, or fifth embodiment. or a pharmaceutically acceptable salt thereof.

[0033] A seventh embodiment of the present invention is a compound represented by structural formula (I), (II), (III) or (IV): wherein n is 1 and o is 0; and the remaining variables are as described for the first, second, fifth, or sixth embodiment. or a pharmaceutically acceptable salt thereof.

[0034] An eighth embodiment of the present invention is a compound represented by structural formula (I), (II), (III) or (IV): [In the formula, R 1 is a fluorophore or optical dye; the remaining variables are those described in the first, second, fifth, sixth, or seventh embodiment. or a pharmaceutically acceptable salt thereof. In one aspect, the fluorophore is a compound represented by:

[0035] [ka]

[0036] and the optical dyes are carbocyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, polymethine, coumarin, rhodamine, xanthene, fluorescein, borodipyrromethane (BODIPY), VivoTag-680, VivoTag-S750, AlexaFluor dyes (e.g., AlexaFluor660, AlexaFluor680, AlexaFluor700, AlexaFluor750, AlexaFluor790), and DylightFluor dyes.

[0037] A ninth embodiment of the present invention is a compound represented by structural formula (I), (II), (III) or (IV): [In the formula, R 1 is a chelating group that is the residue of a chelating agent; the remaining variables are as described in the first, second, fifth, sixth, or seventh embodiment. or a pharmaceutically acceptable salt thereof. Suitable chelating agents, and residues of chelating agents, are described below.

[0038] A tenth embodiment of the present invention is a compound represented by structural formula (I), (II), (III) or (IV): [In the formula, R1 is R 5 and each R is a benzoyl group optionally substituted with one or more groups represented by 5 are independently halo, cyano, halomethyl, N + (CH3)3W - Selected from; W - is a pharmaceutically acceptable anion; the remaining variables are those described in the first, second, fifth, sixth, or seventh embodiment. or a pharmaceutically acceptable salt thereof. 5 A halo group represented by 18 It's F.

[0039] An eleventh embodiment of the present invention is a compound represented by structural formula (I), (II), (III) or (IV): [In the formula, each R 5 are independently fluoro, cyano, trifluoromethyl, N + (CH3)3W - the remaining variables are those described in the first, second, fifth, sixth, seventh, or tenth embodiment. or a pharmaceutically acceptable salt thereof. 5 The fluoro group represented by 18 is.

[0040] A twelfth embodiment of the present invention is a compound represented by structural formula (I), (II), (III) or (IV): [In the formula, R 2 is H and R 4 is CN; and the remaining variables are those described in the first, second, fifth, sixth, seventh, eighth, ninth, tenth, or eleventh embodiment. or a pharmaceutically acceptable salt thereof.

[0041] Also included in the present invention are compounds whose preparation is described and illustrated in the Examples, both pharmaceutically acceptable salts and neutral forms thereof. For those compounds that contain a chelating group, chelation with a radionuclide is also included in the present invention.

[0042] Also included in the present invention are the compounds shown below, both pharmaceutically acceptable salts and neutralized forms thereof: Chelation with radionuclides is also included in the present invention.

[0043] [ka]

[0044] [ka]

[0045] [ka]

[0046] DO3A (DOTA chelating agent)

[0047] [ka]

[0048] [ka]

[0049] Examples of NOTA chelating agents

[0050] [ka]

[0051] Examples of N3O2 (NOTA) chelating agents

[0052] [ka]

[0053] Examples of DOTA chelators

[0054] [ka]

[0055] Examples of NCS-Macropa chelating agents

[0056] [ka]

[0057] Examples of NCO-Macropa Chelating Agents

[0058] [ka]

[0059] Examples of sarcophage chelators

[0060] [ka]

[0061] Examples of optical and near-infrared imaging compounds Thus, a nomenclature in which the compound name is preceded by an isotope indicates that the isotope is chelated to a chelating group of the compound. For example, "[Cu-67]RTX-1363S" indicates that the chelating group is 67 This refers to Cu-chelated RTX-1363S.

[0062] Exemplary compounds of the present invention (with their chelating groups) include [Ga]RTX-1339S; [Ga]RTX-1340S; [Ga]RTX-1363S; [Ga]RTX-1367S; [Cu]RTX-1339S; [Cu]RTX-1340S; [Cu]RTX-1363S; [Cu]RT [AlF]RTX-1367S; [AlF]RTX-1339S; [AlF]RTX-1340S; [AlF]RTX-1363S; [AlF]RTX-1367S; [AlF]RTX-1339S; [AlF]RTX-1340S; [AlF]RTX-1363S; and [AlF]RTX-1367S.

[0063] Other exemplary compounds of the present invention (with their chelating groups) include [68Ga]RTX-1350S; [68Ga]RTX-1352S; [68Ga]RTX-1353S; [68Ga]RTX-1354S; [68Ga]RTX-1355S; [68Ga]RTX-1356R; [68Ga]RTX-1357R; [68Ga] a]RTX-1358R;[68Ga]RTX-1359R;[68Ga]RTX-1360S;[68Ga]RTX-1360R;[177Lu]RTX-135 0S;[177Lu]RTX-1352S;[177Lu]RTX-1353S;[177Lu]RTX-1354S;[177Lu]RTX-1355S;[17 7Lu]RTX-1356R;[177Lu]RTX-1357R;[177Lu]RTX-1358R;[177Lu]RTX-1359R;[177Lu]R TX-1360S;[177Lu]RTX-1360R;[225Ac]RTX-1350S;[225Ac]RTX-1352S;[225Ac]RTX-135 [225Ac]RTX-1354S; [225Ac]RTX-1355S; [225Ac]RTX-1356R; [225Ac]RTX-1357R; [225Ac]RTX-1358R; [225Ac]RTX-1359R; [225Ac]RTX-1360S; and [225Ac]RTX-1360R.

[0064] Another exemplary compound of the present invention (with these chelating groups) includes [AL18F]RTX-1312S. Compounds with Macropa chelators are typically chelated with 225Ac. Thus, other compounds of the invention (with these chelating groups) include [Ac225]RTX-1399R.

[0065] "Aliphatic" means a saturated or unsaturated, straight-chain or branched, monovalent or divalent hydrocarbon group. Unless otherwise specified, an aliphatic group typically has 1 to 10 carbon atoms. "Alkyl" means a saturated aliphatic, straight-chain or branched, monovalent aliphatic group. Unless otherwise specified, an alkyl group typically has 1 to 10 carbon atoms (C 1~10 alkyl), or 1 to 6 carbon atoms (C 1~3 alkyl) (i.e., 1, 2 or 3).

[0066] "Cycloaliphatic" means a saturated or unsaturated, monovalent or divalent, cyclic hydrocarbon ring group. Unless otherwise specified, a cycloaliphatic group has 3 to 8 ring carbon atoms (C 3~8 "Cycloalkyl" means a saturated aliphatic cyclic aliphatic. Unless otherwise specified, a cycloalkyl has 3 to 8 ring carbon atoms.

[0067] "Aryl," alone or as a moiety or larger moiety, such as "aralkyl," is a carbocyclic aromatic group, such as phenyl or naphthyl. Compounds with one or more chiral centers can exist in various stereoisomeric forms; that is, each chiral center can have an R or S configuration, or 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 with two or more chiral centers that are not identical and are not mirror images of each other.

[0068] When the stereochemical configuration at one 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 "R" or "S") or structure (e.g., where the configuration is indicated by a "wedge" bond), the enrichment of the depicted configuration relative to the opposite configuration is greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 99%, or greater than 99.9%. "Enrichment of the depicted configuration relative to the opposite configuration" is a mole percent and is determined by dividing the number of compounds having the depicted stereochemical configuration at the chiral center by the total number of all compounds in the mixture having the same or opposite stereochemical configuration.

[0069] When a disclosed compound having a chiral center is depicted by a structure without indicating the configuration at that chiral center, the structure is meant to encompass compounds having the S configuration at that chiral center, compounds having the R configuration at that chiral center, or compounds having a mixture of R and S configurations at that chiral center. When a disclosed compound having a chiral center is depicted by its chemical name without indicating the configuration at that chiral center with an "S" or "R", the name is meant to encompass compounds having the S configuration at that chiral center, compounds having the R configuration at that chiral center, or compounds having a mixture of R and S configurations at that chiral center.

[0070] The FAP target compounds of the present invention are useful imaging agents for diagnostic applications. For example, they can be conjugated to various metals for magnetic resonance imaging applications, or conjugated to optical dyes or fluorophores or other detectable moieties (i.e., dyes, quantum dots, etc.) for histochemistry and luminescence imaging applications. Similarly, 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 include: 18 F, 64 Cu or 68 Ga, which are suitable for use in PET imaging applications; 67 Cu or 177 Lu, which are typically therapeutic nucleotides, but are also suitable for use in SPECT imaging applications.

[0071] The FAP-targeting compounds of the present invention are useful therapeutic compounds. Such therapeutic compounds include FAP-targeting compounds of the present invention having a suitable therapeutic moiety. The FAP-targeting compound can be separated from the therapeutic moiety by a covalent linker. The separation between them (based on adjacent atom counting) can be from about 4 atoms to about 100 atoms. Furthermore, the inclusion of additional targeting structures on the compound can modify the pharmacokinetics of the compound. For example, the use of a blood-targeting moiety can extend circulation residence time, which has the effect of increasing tumor perfusion and loading while reducing deposition of the radiotherapeutic compound in non-target tissues. See, e.g., U.S. Pat. No. 11,285,277, which describes trifunctional ("trillium") compounds having a tumor-targeting domain, a blood protein-binding domain, and a third domain comprising a cytocidal or cytostatic therapeutic agent. In a currently preferred embodiment, the FAP-binding compounds of the present invention can be adapted to a trillium scaffold by a covalent linkage to constitute the tumor-targeting domain for such FAP-targeting trillium agents. Exemplary constructs include toxins, poisons, metabolic poisons, or chemotherapeutic agents; and radiotherapeutic compounds having alpha-emitting radionuclides, beta-emitting radionuclides, Auger electron-emitting radionuclides, or drug conjugates having those that emit a spectrum of radiation upon decay (including positron emission, which is also suitable for diagnostic uses).

[0072] For radiotherapy, FAP-targeting compounds are conjugated to chelators, which are selected based on their stability in retaining the appropriate therapeutic radionuclide. A "therapeutic radionuclide" is a radionuclide that can be used for therapeutic purposes, for example, to treat cancer or fibrotic tissue due to its radioactive emission, which has a cytotoxic effect on target tissues (i.e., FAP-expressing cancers, tumor microenvironments, malignancies, and fibrotic cells). While targeted radiotherapy has sometimes been performed using macrocyclic complexes of radionuclides, currently used macrocycles (e.g., DOTA) generally form complexes with many therapeutic radionuclide metals, including actinium, radium, bismuth, astatine, lutetium, and lead isotopes, among others. The instability of many known macrocycle-containing compounds can result in some dissociation of the radionuclide from the macrocycle, which results in a lack of selective delivery to the intended target tissue and can also result in toxicity to non-target tissues. Alpha-emitting radionuclides, e.g. 225 Ac can produce much greater cytotoxic effects and is therefore considered significantly more potent for therapy than beta-emitting radionuclides. However, this toxicity requires a chelating agent with extended retention of the chelated metal. U.S. Patent No. 11,279,698 (see also PCT / US Patent Application Publication Nos. 2018 / 025488 and 2019 / 062479) describes a novel chelating agent ("Macropa") and its use as a component of a radiotherapeutic agent synchronized and targeted with trillium K. 225 The present application describes its use as a chelating agent for Ac. A tumor activity to kidney activity ratio of 1 or greater can be sustained for up to about 36 hours after administration of the radiotherapeutic agent. 225 In the case of Ac trillium-based therapeutics, the duration can be as long as 72 hours, or even 128 hours, or longer, maximizing the therapeutic effect of the radiation on the target tissue.

[0073] Thus, an exemplary preferred FAP-targeted trillium compound will have a chelator in its third (non-targeting) domain. 225 Ac-FAP is the currently preferred chelator for targeting trilium compounds (see also PCT / CA2021 / 050226).

[0074] The radionuclides that can be used in the compounds disclosed herein depend on the application, the type of radiation desired, and the half-life, as will be apparent to one of skill in the art. Exemplary radionuclides include: 177 Lu, 175 Lu, 45 Sc, 64 Cu, 67 Cu, 68 Cu, 66 Ga, 67 Ga, 68 Ga, 69 Ga, 71 Ga, 90 Y, 89 Y, 86 Y, 89 Zr, 90 Y, 99m Tc, 111 In, 113 In, 115 In, 139 La, 134 Ce, 136 Ce, 138 Ce, 140 Ce, 142 Ce, 151 EU, 153 EU, 152 Dy, 149 Tb, 159 Tb, 154 Gd, 155 Gd, 156 Gd, 157 Gd, 158 Gd, 160 Gd, 188 Re, 186 Re, 213 Bi, 211 At, 217 At, 227 Th, 226 Th, 225 Ac, 233Ra, 152 Dy, 213 Bi, 212 Bi, 211 Bi, 203 Pb, 212 Pb, 255 Fm, and uranium-230. The radionuclides of any embodiment herein can be both therapeutic and diagnostic radionuclides, depending on their decay profile. Currently preferred alpha-emitting radionuclides for therapeutic use include: 225 Ac, 233 Ra and 212 Currently preferred beta-emitting radionuclides for therapeutic use include: 177 Lu, 90 Y and 67 Cu is an example.

[0075] The chelating groups and polyazapolycarboxylic acid macrocycles useful in the present technology include and refer to groups that can chelate, bind, or otherwise deliver radionuclides to therapeutic or diagnostic targets. The chelating group is the residue of a chelating agent after it reacts with a nucleophilic group in a compound to form a targeted, bivalent radiopharmaceutical or radiodiagnostic 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, covalently conjugated, substituted or unsubstituted members of the following group: 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), p-SCN-Bn-NOTA, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), p-SCN-Bn-DOTA (also known as 2B-DOTA-NCS), PIP-DOTA, diethylenetriaminepentaacetic acid (DTPA), PIP-DTPA, AZEP-DTPA, ethylenediaminetetraacetic acid (EDTA), triethylenetetraamine-N,N,N',N'',N''',N'''-hexaacetic acid (TTHA), 7-[2-(bis-carboxymethylamino)-ethyl]-4,10-bis-carboxymethyl 1,4,7,10-tetraazacyclododecyl-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-1,4,7-triazonin-1-yl}-acetic acid (NPTA), diacetylpyridine bis(benzoylhydrazone), 1,4,7,10,13,16-hexaazacyclooctadecane N,N',N'',N''',N'''',N'''''-hexaacetic acid (HEHA), octadentate terephthalamide ligand, 2,2'-(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-pyridyl)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-isothiocyanatopicolinic acid (macropa-NCO), 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-tetraazacyclododec-1-yl]acetamide (TCMC or DOTAM). In a currently preferred embodiment, the chelating agent is a residue of a polyazapolycarboxylic macrocycle, such as Macropa NCS or NCO-Macropa. In another embodiment, the chelator is a residue of a siderophore. 225 Ac is a radionuclide for Macropa NCS or NCO-Macropa. In another embodiment, the chelator is a residue of p-SCN-Bn-DOTA, p-SCN-Bn-NOTA, NOTA, or DOTA. In another embodiment, the chelator is a sarcophage chelator (compounds of the present invention having sarcophage chelators can be prepared according to the procedures disclosed in WO2021225760, the entire teachings of which are incorporated herein by reference). In another embodiment, the chelator is 68 A Ga-chelated residue of p-SCN-Bn-DOTA, p-SCN-Bn-NOTA, NOTA, or DOTA.

[0076] As noted above, conjugates of the disclosed compounds or pharmaceutically acceptable salts thereof may contain one or more radionuclides suitable for use as radioimaging agents. Imaging methods include positron emission tomography (PET) or single-photon emission computed tomography (SPECT). Thus, in another aspect, the present invention provides strategic uses, i.e., methods in which subjects with cancer, tumors, or fibrotic diseases are administered an effective amount of the disclosed compounds (or pharmaceutically acceptable salts thereof) with a chelating agent conjugated to an imaging radionuclide for imaging applications, and methods in which an effective amount of the compound conjugated to a therapeutic radionuclide for treatment is administered. Exemplary cancers that can be imaged and / or treated with the disclosed compounds or pharmaceutically acceptable salts thereof include pancreatic cancer, liver cancer, gallbladder cancer, neuroblastoma, breast cancer, ovarian cancer, esophageal cancer, renal cancer, prostate cancer, colon cancer, soft tissue sarcoma, osteosarcoma, or melanoma.

[0077] 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, such as pets (e.g., dogs, cats, etc.), farm animals (e.g., cows, sheep, pigs, horses, etc.), and laboratory animals (e.g., rats, mice, guinea pigs, etc.).

[0078] 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 by injection or infusion. "Parenteral administration route" means a method of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intraspinal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subcuticular, intraarticular, subcapsular, intrathecal, intrathecal, and intrasternal injection and infusion.

[0079] An "effective amount" of a disclosed compound or a pharmaceutically acceptable salt thereof (including chelation with a radionuclide) refers to an amount of a therapeutic agent, alone or in combination with other therapeutic agents, that provides a therapeutic benefit in the treatment of disease prevention, improves overall treatment, reduces or avoids the symptoms or causes of disease, or provides a therapeutic effect of another therapeutic agent or a synergistic effect with another therapeutic agent. In terms of imaging, a "diagnostically effective amount" refers to an amount that will result in an image useful for diagnosing, for example, the presence of a tumor. An "effective amount" of a disclosed compound or a pharmaceutically acceptable salt thereof is determined by a physician based on patient-specific parameters such as age, weight, sex, and severity of disease. The dosage is preferably 0.00001 mg / kg to 100 mg / kg of body weight.

[0080] Depending on the type of administration, the drugs are suitably formulated, for example, in the form of solutions or suspensions, plain or sugar-coated tablets, hard or soft gelatin capsules, suppositories, aveilles, or injection preparations, which are prepared according to the usual galenic methods.

[0081] When solutions for infusion or injection are used, they are preferably aqueous solutions or suspensions, which can be prepared before use, for example, from a freeze-dried preparation containing the active substance, either by itself or together with a carrier such as mannitol, lactose, glucose, albumin, etc. The ready-made solution is sterilized and, where appropriate, mixed with excipients such as preservatives, stabilizers, emulsifiers, solubilizers, buffers for adjusting osmotic pressure, and / or salts. Sterilization can be achieved, where appropriate, by aseptic filtration using a filter with a small pore size, according to which the composition can be freeze-dried. A small amount of antibiotics can also be added to ensure sterility.

[0082] According to another aspect, pharmaceutical compositions are provided, which are suitable for in vivo imaging and / or radiotherapy of target tissue. Suitable pharmaceutical compositions contain an element (i.e. 18 F), or diagnostic radioactive metal chelate complexes (e.g.64 Cu or 68 The radioactive imaging agent may contain a radionuclide, either as a radionuclide (e.g., a radioactive metal chelate complex), or a radiotherapeutic agent that is a radioactive metal chelate complex, in an amount sufficient to bind to the target tissue, along with a pharmaceutically acceptable radioactive vehicle. The radioactive vehicle should be suitable for injection or inhalation, such as human serum albumin; aqueous buffer solutions, such as tris(hydromethyl)aminomethane (and its salts), phosphate, citrate, bicarbonate, and the like; sterile water saline; and balanced ionic solutions containing chloride and / or dicarbonate salts or normal plasma precautions, such as calcium, potassium, sodium, and magnesium.

[0083] The concentration of the radiopharmaceutical agent in the radiopharmaceutical vehicle should be sufficient to provide reasonable binding to the target tissue, e.g., about 4% to 40% ID / gram. For example, when using an aqueous solution, the human dose can range from about 1.0 to 500 millicuries of activity. However, the actual dose administered to a patient for imaging or therapeutic purposes is determined by the physician administering the treatment. The imaging or treatment agent should be administered so that it remains in the patient for about 1 hour to 10 days, although both longer and shorter periods are acceptable. Therefore, convenient ampoules containing 1 to 10 mL of aqueous solution can be prepared.

[0084] Imaging can be performed in a conventional manner, for example, by injecting a sufficient amount of the imaging composition to provide adequate imaging, followed by scanning with a suitable imaging or scanning machine, such as a tomograph or gamma camera. In certain embodiments, a method for imaging a region in a patient includes the steps of (i) administering to the patient a diagnostically effective amount of a compound complexed with a radionuclide; exposing the region of the patient to a scanning device; and (ii) obtaining an image of the region of the patient. Thus, the present invention provides a method for obtaining an image of a mammalian subject, followed by administering a compound. Similarly, imaging can be performed after administration of a therapeutic agent or after a radiation therapy cycle to evaluate efficacy. Thus, obtaining an image after administration of a radiation therapy agent can occur 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 1 week, about 2 weeks, about 4 weeks, or after the completion of a cycle of therapeutic treatment. Thus, in some embodiments, a method of imaging tissue, such as FAP-expressing tumor tissue, is provided, comprising contacting the tissue with a conjugate synthesized by contacting an imaging radionuclide with the disclosed compounds.

[0085] According to another embodiment, complexes of the disclosed compounds or pharmaceutically acceptable salts thereof may contain one or more radionuclides suitable for use as radioimaging agents in the field of image-guided radiotherapy (IGRT). As described in U.S. Patent No. 1,068,832 (B2), IGRT uses images acquired prior to a treatment session to guide the application of therapeutic radiation during a treatment session. The concentration of the imaging or therapeutic agent in the radioactive material vehicle should be sufficient to provide satisfactory imaging. For example, when using an aqueous solution, the dosage is approximately 1.0 to 100 millicuries. Imaging can be performed to provide a location reference for guidance as to which target region receives a calculated radiation fluence from a therapeutic radiation source. Similar use of the compounds as location references can be used in guided surgery applications.

[0086] The amount of a compound of the invention or a formulation comprising a complex of a metal and a compound or a pharmaceutically acceptable salt thereof administered to a patient will depend on several physiological factors routinely used by physicians, including the nature of the procedure to be performed, the volume and body weight of the tissue to be targeted for imaging or treatment, and the medical history of the patient to be imaged or treated with the compound.

[0087] The examples herein are provided to illustrate the advantages of the present technology and to further assist those skilled in the art in preparing or using the compounds or salts, pharmaceutical compositions, derivatives, prodrugs, or tautomeric forms thereof of the present technology. The examples herein are also presented to more fully illustrate preferred aspects of the present technology. These examples should not be construed in any way to limit the scope of the present technology, which is defined by the appended claims. These 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 include or incorporate any or all other variations, aspects, or embodiment variations of the present technology, respectively. [Example]

[0088] Example 1 - Synthesis of RTX-1312S: (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-[3-(p-{[1,4,7-tris(carboxymethyl)-1,4,7-triazonan-2-yl]methyl}phenyl)thioureido]hexanoylamino]-6-acetylaminohexanoic acid. Synthesis of RTX-1312S from fragments:

[0089] [ka]

[0090] Synthetic scheme for intermediate 1:

[0091] [ka]

[0092] Preparation of Intermediate 1: (S)-6-acetylamino-2-[(S)-2-(3-{2-[2-(2-amino-ethoxy)-ethoxy]-ethoxy}-propionylamino)-6-tert-butoxycarbonylaminohexanoylamino]-hexanoic acid tert-butyl ester.

[0093] Procedure 1: Preparation of (S)-tert-butyl 2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((tert-butoxycarbonyl)amino)hexanamido)-6-(((benzoyloxy)carbonyl)amino)hexanoate: A solution of Fmoc-Lys(Boc)-OH (4.69 g), H-Lys(Z)-OtBu, HCl (4.48 g, 1.2 equiv.), EDC.HCl (3.82 g, 2.0 equiv.), and HOBt.HO (1.84 g, 1.2 equiv.) in DCM (100 mL) was stirred for 10 min, and DIEA (4 equiv.) was slowly added. The stirred reaction mixture was monitored by LC-MS RM at room temperature for 4 h. The mixture was diluted with DCM, and the organic layer was washed successively with water and brine, dried over MgSO, filtered, and the volatiles were removed under reduced pressure to give the crude product, which was purified by combi-flash silica gel chromatography (DCM and EtOAc eluent) to give the pure product (6.0 g, 77% yield). Procedure 2: Preparation of (S)-tert-butyl 2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((tert-butoxycarbonyl)amino)hexanamido)-6-aminohexanoate acetate: Activated palladium on carbon (5%, 1.0 g) was suspended in a solution of (S)-tert-butyl 2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((tert-butoxycarbonyl)amino)hexanoate (7.8 g, 10 mmol) in MeOH (100 mL) and acetic acid (1 mL). The suspension was subjected to hydrogenation (50 psi) at room temperature for 2 h. The mixture was then filtered through Celite, and the filtrate was concentrated under reduced pressure to give the product (6.05 g, 85% yield as the acetate); Fmoc deprotection was also observed during the hydrogenation (ca. 5%). The crude product was sufficiently pure to be used in the next step without further purification.

[0094] Step 3: Preparation of (S)-6-acetylamino-2-[(S)-6-tert-butoxycarbonylamino-2-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoylamino]-hexanoic acid tert-butyl ester: To a stirred solution of (S)-6-amino-2-[(S)-6-tert-butoxycarbonylamino-2-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoylamino]-hexanoic acid tert-butyl ester (3 g, 4.6 mmol) and DIEA (1.76 ml, 10.12 mmol) in DCM (24 ml) was added acetic anhydride (524 μl, 5.51 mmol). The mixture was stirred at room temperature for 90 minutes, at which time LCMS showed complete conversion. The reaction mixture was diluted with EtOAc, washed with water and brine, dried over anhydrous MgSO4, and filtered. Evaporation of the volatiles under reduced pressure left the target compound (2.9 g, 91%) as an off-white solid.

[0095] Step 4: Preparation of (S)-6-acetylamino-2-((S)-2-amino-6-tert-butoxycarbonylamino-hexanoylamino)-hexanoic acid tert-butyl ester: To a stirred solution of (S)-6-acetylamino-2-[(S)-6-tert-butoxycarbonylamino-2-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoylamino]-hexanoic acid tert-butyl ester (2.9 g, 4.2 mmol) in DCM (12 ml) was added diethylamine (6.51 ml, 62.59 mmol) at ambient temperature. Complete conversion was detected by LCMS after 45 min. The solvent was evaporated under reduced pressure, and the residue was dissolved in DCM and purified on a CombiFlash instrument (220 g silica gel column, gradient 0-50% MeOH / DCM over 100 min). The desired fractions were combined and evaporated to yield the target material (1.66 g, 84%) as an amorphous, colorless solid.

[0096] Procedure 5: Preparation of (S)-6-acetylamino-2-[(S)-2-(3-{2-[2-(2-benzyloxycarbonylamino-ethoxy)-ethoxy]-ethoxy}-propionylamino)-6-tert-butoxycarbonylamino-hexanoylamino]-hexanoic acid tert-butyl ester: DIEA (1.34 ml, 1.72 mmol) was added to a stirred solution of (S)-6-acetylamino-2-((S)-2-amino-6-tert-butoxycarbonylamino-hexanoylamino)-hexanoic acid tert-butyl ester (1.66 g, 3.51 mmol), 3-{2-[2-(2-benzyloxy-carbonylamino-ethoxy)-ethoxy]-ethoxy}-propionic acid (1.25 g, 3.51 mmol), and HATU (1.47 g, 3.86 mmol) in DMF (10 ml) at ambient temperature. The reaction mixture was stirred at room temperature for 14 h, at which time complete conversion was detected by LCMS. The mixture was diluted with EtOAc, washed with water (×2) and brine, dried over anhydrous MgSO4, and filtered. The filtrate was evaporated to give the target material (2.84 g, 100%) as a colorless amorphous solid.

[0097] Procedure 6: Preparation of (S)-6-acetylamino-2-[(S)-2-(3-{2-[2-(2-amino-ethoxy)-ethoxy]-ethoxy}-propionylamino)-6-tert-butoxycarbonylamino-hexanoylamino]-hexanoic acid tert-butyl ester: To a solution of (S)-6-acetylamino-2-[(S)-2-(3-{2-[2-(2-benzyloxycarbonylamino-ethoxy)-ethoxy]-ethoxy}-propionylamino)-6-tert-butoxycarbonylamino-hexanoylamino]-hexanoic acid tert-butyl ester (2.84 mg, mmol) in methanol (50 ml) was added Pd / C (5%, 500 mg, 0.235 mmol). The mixture was subjected to hydrogenation at ambient temperature on a Parr apparatus (6 PSI H) for 1 h. The supported catalyst was removed by filtration through a pad of Celite, and the filtrate was evaporated to give the target material, Intermediate 1 (2.075 g, 88%), as a colorless amorphous solid. Preparation of Intermediate-2: {4-[2-((S)-2-cyano-pyrrolidin-1-yl)-2-oxoethylcarbamoyl]-quinolin-7-yloxy}-acetic acid.

[0098] [ka]

[0099] Step 7: Preparation of [2-((S)-2-cyano-pyrrolidin-1-yl)-2-oxo-ethyl]-carbamic acid tert-butyl ester: To a solution of (S)-pyrrolidine-2-carbonitrile hydrochloride (1 g, 7.5 mmol) in DCM (35 mL) were added DIEA (3.9 mL, 22.5 mmol) and Boc-Gly-OSu (2.26 g, 8.3 mmol). The reaction mixture was stirred at ambient temperature for 14 hours. The solvent was evaporated, and the residue was dissolved in DCM, washed with water, dried over anhydrous MgSO4, and filtered. The filtrate was concentrated to 20% of the original volume and loaded onto a CombiFlash™ instrument for elution (gradient 0-10% MeOH / DCM). The desired fractions were combined and evaporated to give the target product (1.1 g, 58%) as an amorphous solid.

[0100] Step 8: Preparation of (S)-1-(2-amino-acetyl)-pyrrolidine-2-carbonitrile: p-Toluenesulfonic acid monohydrate (1.25 g, 6.6 mmol) was added to a stirred solution of (2-((S)-2-cyano-pyrrolidin-1-yl)-2-oxo-ethyl)-carbamic acid tert-butyl ester (1.1 g, 4.4 mmol) in acetonitrile (20 ml), and the mixture was stirred at ambient temperature for 14 hours. The solvent was removed under reduced pressure to give the tosylate salt of the target product (1.8 g, yield in excess of theoretical). The compound was sufficiently pure to be carried through subsequent chemical transformations.

[0101] Procedure 9: Preparation of (S)—N-(2-(2-cyanopyrrolidin-1-yl)-2-oxoethyl)-7-hydroxyquinoline-4-carboxamide: A solution of 7-hydroxyquinoline-4-carboxylic acid (307 mg, 1.62 mmol), HOBt (221 mg, 1.62 mmol), and TBTU (521 mg, 1.62 mmol) in DMF (15 mL) was stirred at room temperature for 5 minutes. A solution of (S)-1-(2-amino-acetyl)-pyrrolidine-2-carbonitrile(1.5 tosylate) (1.78 mmol) and DIEA (0.74 mL, 4.86 mmol) in DMF (5 mL) was added to the activated ester solution, and the resulting mixture was stirred at room temperature for 2 hours, during which the progress of the reaction was monitored by LCMS. Upon complete conversion, the reaction mixture was concentrated under reduced pressure, dissolved in a small amount of dichloromethane, and filtered. The filtrate was evaporated, dissolved in DCM, loaded onto a CombiFlash™ silica gel column and purified (gradient 0-20% MeOH / DCM) to give the target product (422 mg, 80% yield) as an off-white solid.

[0102] Procedure 10: Preparation of (S)-tert-butyl 2-((4-((2-(2-cyanopyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-7-yl)oxy)acetate A flask containing 7-hydroxyquinoline-4-carboxylic acid [2-((S)-2-cyanopyrrolidin-1-yl)-2-oxo-ethyl]-amide (407 mg, 1.25 mmol), 2-tert-butyl glycolate (249 mg, 1.88 mmol), and triphenylphosphine (395 mg, 1.51 mmol) in DMF (15 mL) was quenched in an ice-water bath. Diisopropyl azodicarboxylate (300 μL, 1.51 mmol) was added dropwise to the quenched reaction mixture. The ice-water bath was removed, and the resulting solution was stirred at room temperature and monitored by LCMS. Upon completion, the solvent was removed under reduced pressure, and the residue was dissolved in DCM and loaded onto a CombiFlash™ silica gel column (gradient 0 to >10% MeOH / DCM) and purified to give the target product (404 mg, 61% yield) as a glassy solid.

[0103] Step 11: Preparation of {4-[2-((S)-2-cyano-pyrrolidin-1-yl)-2-oxo-ethylcarbamoyl]-quinolin-7-yloxy}-acetic acid: To a solution of {4-[2-((S)-2-cyano-pyrrolidin-1-yl)-2-oxo-ethylcarbamoyl]-quinolin-7-yloxy}-acetic acid tert-butyl ester (141 mg, 0.322 mmol) in DCM (1.5 ml) was added TFA (1.51 ml, 19.64 mmol) dropwise at 0° C. The temperature of the stirred reaction mixture was allowed to rise to room temperature over 1 h, at which time complete conversion was detected by LCMS. The solvent was evaporated at room temperature, and the residue was co-evaporated with toluene (×3) at 40° C. The crude compound, Intermediate 2, was sufficiently pure to be used for subsequent transformations, but it was hydrolytically unstable on prolonged standing. For this reason, the compound was prepared immediately before use.

[0104] Preparation of RTX-1312S from fragments: Procedure 12: Preparation of tert-butyl (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-(tert-butoxycarbonylamino)hexanoylamino]-6-acetylaminohexanoate:

[0105] [ka]

[0106] DIEA (1.64 ml, 9.43 mmol) was added to a stirred mixture of {4-[2-((S)-2-cyano-pyrrolidin-1-yl)-2-oxo-ethylcarbamoyl]-quinolin-7-yloxy}-acetic acid (1.57 mmol), (S)-6-acetylamino-2-[(S)-2-(3-{2-[2-(2-amino-ethoxy)-ethoxy]-ethoxy}-propionylamino)-6-tert-butoxycarbonylamino-hexanoylamino]-hexanoic acid tert-butyl ester (1.17 g, 1.73 mmol) and HATU (717 mg, 1.89 mmol) in DMF (10 ml). The stirred mixture was kept at room temperature for 30 minutes, at which time complete conversion was detected by LCMS. The mixture was diluted with EtOAc, washed with water and brine, and the collected aqueous phase was back-extracted with EtOAc. The combined organic extracts were washed with brine, dried over anhydrous MgSO4, filtered, and evaporated under reduced pressure. The residue was dissolved in DCM and loaded onto a CombiFlash silica gel column (220 g, gradient 0 to >30% MeOH / DCM, 60 min) for purification. The desired fractions were combined and evaporated to yield the target material (950 mg, 58%) as a glassy solid.

[0107] Procedure 13: Preparation of (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-aminohexanoylamino]-6-acetylaminohexanoic acid:

[0108] [ka]

[0109] To a solution of tert-butyl (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-(tert-butoxycarbonylamino)hexanoylamino]-6-acetylaminohexanoate (74 mg, 0.071 mmol) in anhydrous DCM (1 ml) was added trifluoroacetic anhydride (1 ml, 13 mmol) dropwise at 0° C. The temperature of the reaction mixture was allowed to rise to ambient temperature and stirring was continued for 90 minutes, at which time complete conversion was detected by LCMS. The solvent was evaporated at room temperature and the residue was co-evaporated with toluene. The compound thus obtained was used immediately in the subsequent chemical transformation.

[0110] Procedure 14: Preparation of RTX-1312S: (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-[3-(p-{[1,4,7-tris(carboxymethyl)-1,4,7-triazonan-2-yl]methyl}phenyl)thioureido]hexanoylamino]-6-acetylaminohexanoic acid:

[0111] [ka]

[0112] A solution of p-SCN-Bn-Nota 3HCl (Intermediate 3; 40 mg, 0.071 mmol) in water (500 μl) was added to a solution of (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-aminohexanoylamino]-6-acetylaminohexanoic acid×TFA (0.071 mmol) in DMSO (1300 μl), followed by the addition of a solution of sodium carbonate (188 mg, 1.78 mmol) in water (1 ml) at ambient temperature. The pH of the resulting solution mixture was approximately 9. The reaction mixture was stirred at ambient temperature for 14 hours and subjected to HPLC purification using a Clipeus C18, 10 μn, 250 × 20 mm column (CS-2520-C181 Higgins Analytical, Inc.); MP A: 0.1% TFA in HO; MP B: 0.1% TFA in 5% acetonitrile in Buffer B for 5 minutes, followed by 5% to 55% Buffer B for 40 minutes. Flow rate: 25 mL / min. UV detection: UV254. The desired fractions were combined and lyophilized to give the target compound RTX-1312S (84 mg, 88%) as an off-white solid. Synthesis of RTX-1317S:

[0113] [ka]

[0114] Prepared according to the procedure of Example 1 with the following modifications: Step 40 was used instead of step 3. In step 40, butyric acid was used instead of hexanoic acid. Synthesis of RTX-1318S:

[0115] [ka]

[0116] Prepared according to the procedure of Example 1 with the following modifications: Step 40 was used instead of step 3. Synthesis of RTX-1319R

[0117] [ka]

[0118] Prepared according to the procedure of Example 1 with the following modifications: Steps 52-54 were used instead of step 12. The chelating agent was introduced using procedures 13 & 14, and the intermediate was then subjected to procedures 52-54. Synthesis of RTX-1341S:

[0119] [ka]

[0120] Prepared according to the procedure of Example 1 with the following modifications: For conjugation of optical dyes or fluorophores, procedures 15 & 16 were used instead of procedure 14.

[0121] [ka]

[0122] Procedure 15: Synthesis of 2-((E)-2-((E)-3-((E)-2-(3,3-dimethyl-5-sulfonato-1-(3-(trimethylammonio)-propyl)-indolin-2-ylidene)-ethylidene)-2-(4-(3-((2,5-dioxopyrrolidin-1-yl)oxy)-3-oxopropyl)phenoxy)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(3-(trimethylammonio)propyl)-3H-indol-1-ium-5-sulfonate:

[0123] [ka]

[0124] To a solution of ZW800-1 (100 mg, 0.105 mmol) in anhydrous DMSO (10 mL) was added dipyrrolidino(N-succinimidyloxy)carbenium hexafluorophosphate (130 mg, 0.3 mmol), followed by dropwise addition of N,N-diisopropylethylamine (0.2 mL, 1.1 mmol) at room temperature. The reaction mixture was stirred at ambient temperature for 16 hours, at which time complete conversion was detected by LCMS. The mixture was treated with a solution of 1:1:1 ethanol:ethyl acetate:acetone (150 mL) and 0.1% trifluoroacetic acid (0.9 mL), mixed, and allowed to stand for 30 minutes. The solid was filtered and dried under vacuum to give 2-((E)-2-((E)-3-((E)-2-(3,3-dimethyl-5-sulfonato-1-(3-(trimethylammonio)-propyl)indolin-2-ylidene)ethylidene)-2-(4-(3-((2,5-dioxo-pyrrolidin-1-yl)oxy)-3-oxopropyl)phenoxy)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(3-(trimethylammonio)propyl)-3H-indol-1-ium-5-sulfonate as a green powder (51 mg, 46%), which was used directly for the next step without further purification. LCMS: C 46 H 61 N9O 12 :m / z:1040.32, actual value m / z=1040.5[M] + .

[0125] Step 16: Final step: Synthesis of RTX-1341S:

[0126] [ka]

[0127] To a solution of 3 (0.070 g, 0.067 mmol) in DMSO (1 mL) was added a solution of (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(R)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-aminohexanoylamino]-6-acetylaminohexanoic acid (0.040 g, 0.045 mmol) in DMSO (1 mL). N,N-Diisopropylethylamine (0.070 g, 0.067 mmol) was added at room temperature, and the mixture was allowed to stir for 16 hours. Complete conversion was detected by LCMS. After completion of the reaction, the volatiles were removed and the residue was purified by HPLC to give RTX-1341S (14.4 mg, 14%) as a green solid. LCMS: C 46 H 61 N9O 12 :m / z:1809.9, actual value m / z=1811.4[M+H] + . Synthesis of RTX-1370S:

[0128] [ka]

[0129] Prepared according to the procedure of Example 1 with the following modifications: Step 40 was used instead of step 3. In step 40, 2-(4-isobutylphenyl)acetic acid was used in place of hexanoic acid.

[0130] Alexa Fluor 568 was introduced using a procedure similar to procedure 16. Synthesis of RTX-1354S:

[0131] [ka]

[0132] Prepared according to the procedure of Example 1 with the following modifications: Step 40 was used instead of step 3. In step 40, 2-(4-isobutylphenyl)acetic acid was used in place of hexanoic acid.

[0133] For the introduction of the DOTA chelator, procedures 17 & 18 were used instead of procedure 14. Synthesis of RTX-1354S DOTA chelator:

[0134] [ka]

[0135] Procedure 17: To a solution of (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-aminohexanoylamino]-6-[4-(p-isobutylphenyl)butyrylamino]hexanoic acid trifluoroacetate (0.035 mmol) and DIEA (0.031 μL, 0.18 mmol) in DMSO (1 ml) was added DOTA-mono-NHS-tris(tBu ester) (31 mg, 0.046 mmol) and HO (1 ml). The pH of the reaction mixture was adjusted to 9 with DIEA (250 μL). The reaction mixture was stirred at ambient temperature overnight, at which time complete conversion was detected by LCMS. The reaction mixture was subjected to HPLC purification and the desired fractions were collected and evaporated to give 2 as a glassy solid. LCMS:C 80 H 123 N 13 O 19 :m / z:1569.91, actual value m / z=1571.5[M+H] + . Synthesis of 1354S:

[0136] [ka]

[0137] Procedure 18: To a solution of 2 (55 mg, 0.041 mmol) in DCM (1 ml) was added TFA (1 ml, 13 mmol) at 0° C. The reaction mixture was warmed to ambient temperature and stirring was continued for 1 h. Complete conversion was detected by LCMS. The mixture was evaporated and the residue was dissolved in DMSO (2 ml) and subjected to HPLC purification. The desired fractions were lyophilized to give 3 (16 mg, 33%) as an off-white solid. LCMS: C 68 H 99 N 13 O 19 :m / z:1402.59, actual value m / z=1403.0[M+H] + . Synthesis of 1350S:

[0138] [ka]

[0139] Prepared according to the procedure of Example 1 with the following modifications: Step 40 was used instead of step 3. In step 40, octanoic acid was used instead of hexanoic acid.

[0140] For the introduction of the DOTA chelator, procedures 17 & 18 were used instead of procedure 14. Synthesis of RTX-1352S:

[0141] [ka]

[0142] Prepared according to the procedure of Example 1 with the following modifications: Step 40 was used instead of step 3. In step 40, 2-(4-isobutylphenyl)butanoic acid was used in place of hexanoic acid.

[0143] For the introduction of the DOTA chelator, procedures 17 & 18 were used instead of procedure 14. Synthesis of RTX-1353S:

[0144] [ka]

[0145] Prepared according to the procedure of Example 1 with the following modifications: For the introduction of the DOTA chelator, procedures 17 & 18 were used instead of procedure 14. Synthesis of 1360S:

[0146] [ka]

[0147] Prepared according to the procedure of Example 1 with the following modifications: Step 40 was used instead of step 3. For the introduction of the DOTA chelator, procedures 17 & 18 were used instead of procedure 14.

[0148] Example 2 - Synthesis of RTX-1324S: (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-(3-cyano-4-fluorobenzoylamino)hexanoylamino]-6-acetylaminohexanoic acid.

[0149] [ka]

[0150] Procedure 19: Preparation of 3-cyano-4-fluoro-benzoyl chloride:

[0151] [ka]

[0152] To a suspension of 3-cyano-4-fluorobenzoic acid (500 mg, 3.03 mmol) in DCM (10 ml) was added oxalyl chloride (339 μl, 3.94 mmol) at ambient temperature, followed by DMF (12 μl, 0.151 mmol). After 1 hour, the solvent was evaporated and the residue was co-evaporated with toluene to give the target compound (607 mg, theoretical excess yield) as an orange solid. This material was used for the next step without further purification.

[0153] Procedure 20: Preparation of tert-butyl (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-aminohexanoylamino]-6-acetylaminohexanoate

[0154] [ka]

[0155] A solution of tert-butyl (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-(tert-butoxycarbonylamino)hexanoylamino]-6-acetylaminohexanoate (198 mg, 0.19 mmol) in 90% aqueous formic acid (2.17 ml, 57.17 mmol) was stirred at ambient temperature for 2.5 hours, at which time no starting material was detectable by LCMS. The solvent was removed under reduced pressure, and the residue was co-evaporated with ACN (×3) and toluene (×2). The residue was dissolved in a mixture of MeOH / water (1:1, 50 ml) and treated with ion exchange resin (Dowex 550A, OH form, 15 ml) at ambient temperature for 15 minutes. The resin was removed by filtration and washed with MeOH and water. The filtrate was evaporated to give the target material (166 mg, 93%) as an off-white solid (in the form of the free base).

[0156] Procedure 21: Preparation of tert-butyl (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-(3-cyano-4-fluorophenylamino)hexanoylamino]-6-acetylaminohexanoate

[0157] [ka]

[0158] To a solution of tert-butyl (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-aminohexanoylamino]-6-acetylaminohexanoate (83 mg, 0.088 mmol) and DIEA (31 ul, 0.176 mmol) in a mixture of DCM / DMF (1:1, 1 ml) was added 4-fluoro-3-trifluoromethyl-benzoyl chloride (16 mg, 0.088 mmol) in DCM (500 ul) at 0° C. under nitrogen. The temperature was allowed to rise to ambient temperature and the reaction mixture was stirred for 10 minutes. Complete conversion was detected by LCMS. The mixture was diluted with EtOAc, washed with water (x2) and brine, dried over anhydrous MgSO4, and filtered. The filtrate was evaporated to give the target material (37 mg, 39%) as a colorless glassy solid.

[0159] Procedure 22: Preparation of RTX-1324S: (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-(3-cyano-4-fluorobenzoylamino)hexanoylamino]-6-acetylaminohexanoic acid:

[0160] [ka]

[0161] To a solution of tert-butyl (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-(3-cyano-4-fluorophenylamino)hexanoylamino]-6-acetylaminohexanoate (37 mg, 0.034 mmol) in anhydrous DCM (1 ml) was added dropwise anhydrous TFA (1 ml, 13 mmol) at 0° C. The temperature of the reaction mixture was allowed to rise to ambient temperature and stirring was continued for 90 min. Complete conversion was detected by LCMS. The solvent was evaporated at room temperature and the residue was co-evaporated with toluene. The residue so obtained was subjected to HLPC purification. The desired fractions were combined and lyophilized to give the target material RTX-1324S (15.8 mg, 45%) as an off-white solid.

[0162] Example 3 - Synthesis of RTX-1325S: ((S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-[4-fluoro-3-(trifluoromethyl)benzoylamino]hexanoylamino]-6-acetylaminohexanoic acid.

[0163] [ka]

[0164] Procedure 23: Preparation of tert-butyl (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-[4-fluoro-3-(trifluoromethyl)benzoylamino]hexanoylamino]-6-acetylaminohexanoate To a solution of tert-butyl (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-aminohexanoylamino]-6-acetylaminohexanoate (83 mg, 0.088 mmol) and DIEA (31 ul, 0.176 mmol) in a DCM / DMF mixture (1:1, 1 ml), 4-fluoro-3-trifluoromethyl-benzoyl chloride (20 mg, 0.088 mmol) in DCM (500 ul) was added at 0° C. under nitrogen. The temperature of the reaction mixture was allowed to rise to ambient temperature and stirring was continued for 10 minutes. Complete conversion was detected by LCMS. The reaction mixture was diluted with EtOAc, washed with water (x2) and brine, dried over anhydrous MgSO4, and filtered. The filtrate was evaporated to give the target material (57 mg, 58%) as a colorless glassy solid. Procedure 24: Preparation of RTX-1325S: (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-[4-fluoro-3-(trifluoromethyl)benzoylamino]hexanoylamino]-6-acetylaminohexanoic acid (RTX-1325S):

[0165] [ka]

[0166] To a solution of tert-butyl (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-(3-trifluoromethyl-4-fluorophenylamino)hexanoylamino]-6-acetylaminohexanoate (57 mg, 0.051 mmol) in anhydrous DCM (1 ml) was added dropwise anhydrous TFA (1 ml, 13 mmol) at 0° C. The temperature of the reaction mixture was allowed to rise to ambient temperature and stirring was continued for 90 min. Complete conversion was detected by LCMS. The solvent was evaporated at room temperature and the residue was co-evaporated with toluene. The residue so obtained was subjected to HLPC purification. The desired fractions were combined and lyophilized to give the target material RTX-1325S (21.8 mg, 40%) as an off-white solid.

[0167] Example 4 - Synthesis of RTX-1326S: 7-Methoxy-quinoline-4-carboxylic acid [2-((S)-2-cyano-pyrrolidin-1-yl)-2-oxoethyl]-amide.

[0168] [ka]

[0169] Procedure 25: Preparation of [2-((S)-2-cyano-pyrrolidin-1-yl)-2-oxo-ethyl]-carbamic acid tert-butyl ester:

[0170] [ka]

[0171] To a solution of (S)-pyrrolidine-2-carbonitrile hydrochloride (1 g, 7.5 mmol) in DCM (35 mL) was added DIEA (3.9 mL, 22.5 mmol) and Boc-Gly-OSu (2.26 g, 8.3 mmol). The resulting solution was stirred at ambient temperature for 14 h. The reaction mixture was stirred under vacuum, and the residue was dissolved in DCM, washed with water, dried over anhydrous MgSO4, and filtered. The filtrate was evaporated and subjected to flash purification on a CombiFlash (silica gel, gradient 0-10% MeOH / DCM, 30 min). The desired fractions were collected and evaporated to give the target material (1.1 g, 58% yield) as an amorphous solid. Procedure 26: Preparation of (S)-1-(2-amino-acetyl)-pyrrolidine-2-carbonitrile hydrochloride:

[0172] [ka]

[0173] To a solution of (2-((S)-2-cyano-pyrrolidin-1-yl)-2-oxo-ethyl)-carbamic acid tert-butyl ester (1.1 g, 4.4 mmol) in acetonitrile (20 ml) (1.1 g, 4.4 mmol) was added p-toluenesulfonic acid monohydrate (1.25 g, 6.6 mmol). The reaction mixture was stirred at ambient temperature for 14 hours. The solvent was evaporated to dryness to give the target product (1.8 g, in excess of theoretical yield). This material was used without further purification.

[0174] Procedure 27: Preparation of 7-Methoxy-quinoline-4-carboxylic acid [2-((S)-2-cyano-pyrrolidin-1-yl)-2-oxo-ethyl]-amide:

[0175] [ka]

[0176] To a solution of 7-methoxy-4-quinolinecarboxylic acid (303 mg, 1.5 mmol) in DMF (5 mL) was added HOAt (205 mg, 1.5 mmol) and TBTU (482 mg, 1.5 mmol), followed by (S)-1-(2-amino-acetyl)-pyrrolidine-2-carbonitrile tosylate (789 mg, 1.8 mmol) and DIEA (0.7 mL, 4.5 mmol). The reaction mixture was stirred at ambient temperature for 4 h, after which the solvent was evaporated. The residue was suspended in DCM, and insoluble material was removed by centrifugation. The DCM solution was loaded onto a CombiFlash silica gel column and purified by flash chromatography (gradient 0–10% MeOH / DCM, 30 min). The desired fractions were collected and evaporated to give the target material (400 mg, 75% yield) as an off-white solid.

[0177] Example 5 - Synthesis of RTX-1335S: tert-butyl (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-(3-cyano-4-fluorobenzoylamino)hexanoylamino]-6-acetylaminohexanoate.

[0178] [ka]

[0179] Procedure 28: To a solution of tert-butyl (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-aminohexanoylamino]-6-acetylaminohexanoate (0.072 mmol) and DIEA (25 ul, 0.144 mmol) in a DCM / DMF mixture (1:1, 1 ml) was added 4-fluoro-3-cyano-benzoyl chloride (13 mg, 0.072 mmol) in DCM (500 ul) at 0° C. under nitrogen. The temperature of the reaction mixture was allowed to rise to ambient temperature and the reaction was stirred for 10 minutes. Complete conversion was detected by LCMS. The reaction mixture was diluted with EtOAc, washed with water (×2) and brine, dried over anhydrous MgSO4, and filtered. The filtrate was evaporated, and the residue was subjected to HPLC purification. The desired fractions were collected and lyophilized to give the target material RTX-1335S (25.4 mg, 33%) as an off-white solid.

[0180] Example 6 - Synthesis of RTX-1336S: tert-butyl (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-(3-cyano-4-trimethylammoniumbenzoylamino)hexanoylamino]-6-acetylaminohexanoate.

[0181] [ka]

[0182] Procedure 29: Preparation of 3-cyano-4-dimethylamino-benzoic acid methyl ester:

[0183] [ka]

[0184] To a solution of methyl 3-cyano-4-fluorobenzoate (1 g, 5.58 mmol) and dimethylamine hydrochloride (546 mg, 6.7 mmol) in DMSO (12 ml) was added potassium carbonate (1.62 g, 11.72 mmol), and the resulting mixture was stirred at ambient temperature for 14 hours. The mixture was concentrated using a high-vacuum rotary evaporator at 65 °C. The remaining solution was diluted with DCM and extracted with water (×2). The combined aqueous layers were back-extracted with DCM. The combined organic layers were washed with dilute sodium bicarbonate solution, dried over anhydrous MgSO4, filtered, and evaporated to give the target compound (1.13 g, 99%) as an off-white solid. Procedure 30: Preparation of (2-cyano-4-methoxycarbonylphenyl)-trimethylammonium trifluoromethanesulfonate:

[0185] [ka]

[0186] Methyl triflate (6.26 ml, 55.3 mmol) was added slowly (dropwise) to a stirred solution of 3-cyano-4-dimethylamino-benzoic acid methyl ester (1.13 g, 5.53 mmol) in DCM (20 ml). The reaction mixture was stirred for 14 hours, after which diethyl ether was added. After evaporation of one-third of the solvent volume, the desired compound precipitated from the solution and the remaining solvent was decanted. The solid was washed thoroughly with a large amount of ether. The remaining ether was removed in vacuo, and the solid was purified by HPLC. The desired fractions were collected and evaporated, and the residue was dried under vacuum to give the target compound (1.033 g, 51%) as an off-white solid. Procedure 31: Preparation of (4-carboxy-2-cyanophenyl)-trimethylammonium trifluoromethanesulfonate:

[0187] [ka]

[0188] A solution of (2-cyano-4-methoxycarbonylphenyl)-trimethylammonium trifluoromethanesulfonate (680 mg, 1.85 mmol) in water (20 ml) and TFA (20 ml, 260 mmol) was heated at reflux for 2 days. The solvent was evaporated and the residue was dried in vacuo at ambient temperature for 14 hours. The residue was treated with diethyl ether, and the precipitate formed was filtered, washed with ether, and dried in vacuo to give the target compound (562 mg, 83%) as an off-white solid.

[0189] Procedure 32: Preparation of RTX-1336S: tert-butyl (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-(3-cyano-4-trimethylammonium benzoylamino)hexanoylamino]-6-acetylaminohexanoate:

[0190] [ka]

[0191] To a solution of tert-butyl (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-aminohexanoylamino]-6-acetylaminohexanoate (0.125 mmol), (4-carboxy-2-cyano-phenyl)-trimethylammonium trifluoromethanesulfonate (0.51 mg, 0.150 mmol), and HOAt (1-hydroxy-7-azabenzotriazole, 24 mg, 0.175 mmol) in DMSO (3 ml) was added DIC (24 μl, 0.150 mmol). The reaction mixture was stirred at ambient temperature for 14 hours. Complete conversion was observed by LCMS. The reaction mixture was subjected to HPLC purification and the desired fractions were isolated, combined and lyophilized to give the trifluoroacetate salt of the target compound (56 mg, 35%) as an off-white solid.

[0192] Example 7 - Synthesis of RTX-1337S: tert-butyl (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-[4-fluoro-3-(trifluoromethyl)benzoylamino]hexanoylamino]-6-acetylaminohexanoate.

[0193] [ka]

[0194] Procedure 33: To a solution of tert-butyl (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-aminohexanoylamino]-6-acetylaminohexanoate (0.072 mmol) and DIEA (25 ul, 0.144 mmol) in a DCM / DMF mixture (1:1, 1 ml) was added 4-fluoro-3-trifluoromethyl-benzoyl chloride (16 mg, 0.072 mmol) in DCM (500 ul) at 0° C. under nitrogen. The temperature of the reaction mixture was allowed to rise to ambient temperature and the reaction was stirred for 10 minutes. Complete conversion was detected by LCMS. The reaction mixture was diluted with EtOAc, washed with water (×2) and brine, dried over anhydrous MgSO4, and filtered. The filtrate was evaporated, and the residue was purified by HPLC (Method 4). The desired fractions were collected and lyophilized to give the target material RTX-1337S (19.5 mg, 24%) as an off-white solid.

[0195] Example 8 - Synthesis of RTX-1338S: tert-butyl (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-(3-trifluoromethyl-4-trimethylammoniumbenzoylamino)hexanoylamino]-6-acetylaminohexanoate.

[0196] [ka]

[0197] Procedure 34: Preparation of 3-trifluoromethyl-4-dimethylamino-benzoic acid methyl ester:

[0198] [ka]

[0199] To a solution of methyl 4-fluoro-3-(trifluoromethyl)benzoate (1.24 g, 5.58 mmol) and dimethylamine hydrochloride (546 mg, 6.7 mmol) in DMSO (12 ml), potassium carbonate (1.62 g, 11.72 mmol) was added, and the reaction mixture was stirred at ambient temperature for 14 hours. The reaction mixture was concentrated at 65 °C using a high-vacuum rotary evaporator. The remaining solution was diluted with DCM and extracted with water (×2). The combined aqueous layers were back-extracted with DCM. The combined organic layers were washed with dilute aqueous sodium bicarbonate, dried over anhydrous MgSO4, filtered, and evaporated to give the target compound (1.38 g, arbitrary amount) as a colorless amorphous solid. Procedure 35: Preparation of (2-trifluoromethyl-4-methoxycarbonylphenyl)-trimethylammonium trifluoromethanesulfonate:

[0200] [ka]

[0201] Methyl triflate (9.51 ml, 84.2 mmol) was added slowly (dropwise) to a stirred solution of 4-dimethylamino-3-trifluoromethylbenzoic acid methyl ester (1.38 g, 5.59 mmol) in DCM (25 ml). The reaction mixture was heated at reflux for 2 days, cooled to ambient temperature, and treated with diethyl ether. The desired compound precipitated and was decanted to remove the solvent. The solid was washed repeatedly with diethyl ether and dried in vacuo. The remaining solid was purified by HPLD. The desired fractions were collected, evaporated, and the residue was dried in vacuo to give the target compound, isolated as the trifluoromethanesulfonate salt (1.35 g, 59%) as an off-white solid. Procedure 36: Preparation of (4-carboxy-2-trifluoromethylphenyl)-trimethylammonium trifluoromethanesulfonate:

[0202] [ka]

[0203] A solution of (2-trifluoromethyl-4-methoxycarbonyl-phenyl)trimethylammonium trifluoromethanesulfonate (760 mg, 2.9 mmol) in a mixture of water (20 ml) and TFA (20 ml, 260 mmol) was heated at reflux for 2 days. The solvent was evaporated, and the residue was dried in vacuo at ambient temperature for 14 hours. The residue was treated with diethyl ether, and the resulting precipitate was filtered, washed with diethyl ether, and dried in vacuo to give the target compound (668 mg, 58%) as a white solid.

[0204] Procedure 37: Preparation of RTX-1338S: tert-butyl (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-(3-trifluoromethyl-4-trimethylammonium benzoylamino)hexanoylamino]-6-acetylaminohexanoate

[0205] [ka]

[0206] To a solution of tert-butyl (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-aminohexanoylamino]-6-acetylaminohexanoate (77 mg, 0.082 mmol), (4-carboxy-2-trifluoromethylphenyl)-trimethylammonium trifluoromethanesulfonate (35 mg, 0.098 mmol), and HATU (62 mg, 0.164 mmol) in DMF (2 mL) was added 2,6-lutidine (22 μL, 0.205 mmol). The reaction mixture was stirred at ambient temperature for 14 h, at which point complete conversion was detected by LCMS. The reaction mixture was subjected to HPLC purification and the desired fractions were combined and lyophilized to give the trifluoroacetate salt of the target compound RTX-1338S (56 mg, 35%) as an off-white solid.

[0207] Example 9 - Synthesis of RTX1363S: 2,2'-(7-((S)-17-(((S)-1-carboxy-5-hexamidopentyl)carbamoyl)-1-((4-((2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-7-yl)oxy)-2,15,23-trioxo-6,9,12-trioxa-3,16,22-triazatetracosan-24-yl)-1,4,7-triazonane-1,4-diyl)diacetic acid

[0208] [ka]

[0209] For synthesis purposes, the RTX-1363S molecule was broken down into three fragments as follows: Preparation of Intermediate #1:

[0210] [ka]

[0211] Preparation of RTX-1363S from fragments:

[0212] [ka]

[0213] Procedure 38: Preparation of Intermediate #1. Tert-butyl-(S)-2-[(S)-2-(3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propionylamino)-6-(tert-butoxycarbonylamino)hexanoylamino]-6-(hexanoylamino)hexanoate: Intermediate 1A, Fmoc-L-Lys(Boc)-OtBu, SCR448-17

[0214] [ka]

[0215] Fmoc-L-Lys(Boc)-OH (47 g, 100 mmol) was dissolved in DCM (470 ml) and diisopropyl-ethylamine (25 ml) was added at −30° C. The reaction mixture was stirred at −30° C. for 5 minutes, followed by the slow addition of Boc anhydride (30.1 g, 140 mmol) at −30° C. The reaction mixture was stirred at −30° C. for an additional 30 minutes, and then N,N-dimethylaminopyridine (1.7 g, 14 mmol) was added at −30° C. The reaction mixture was slowly warmed to 0° C. over 2 hours and stirred at 0° C. for an additional 2 hours, at which time LCMS analysis of the reaction mixture indicated complete consumption of the starting carboxylic acid. The reaction mixture was cooled to −30° C. and 1 M aqueous HCl was slowly added (keeping the temperature of the mixture below −20° C.) to obtain a pH of approximately 3. The resulting biphasic mixture was separated, and the organic layer was washed with water (200 ml) and 0.1 M NaHCO3 (200 ml), dried over MgSO4, filtered, and evaporated to dryness. The residue was diluted with 30 ml of ethyl acetate, heated to reflux, and diluted with 300 ml of hexane. The mixture was stirred at room temperature for 2 hours and filtered. The precipitate was washed with 100 ml of 20% ethyl acetate in hexane, and the remaining solvent was removed in vacuo overnight. Yield = 23 g (44%).

[0216] Step 39: Intermediate 2A, Fmoc-L-Lys(Boc)-OtBu

[0217] [ka]

[0218] To a solution of Fmoc-L-Lys(Boc)-OtBu (23 g, 44 mmol) in 130 ml of DCM was added 4 M HCl in dioxane (275 ml) at −5° C. The mixture was stirred at −5° C. to 0° C. for 2.5 hours (until LCMS showed no starting material). The solvent was evaporated in vacuo without heating, re-evaporated with 150 ml of DCM, and the remaining solvent was removed under vacuum at room temperature overnight. The product was obtained as a white amorphous solid (22.6 g, 110%).

[0219] Step 40: Intermediate #3A, tert-butyl-(S)-2-[(9H-fluoren-9-yl)methoxycarbonylamino]-6-(hexanoylamino)hexanoate

[0220] [ka]

[0221] Diisopropylcarbodiimide (DIC, 16.7 g, 132 mmol) was added to a solution of hexanoic acid (30.7 g, 264 mmol) in DCM (120 ml) at 0°C, and the reaction mixture was stirred at 0°C for 30 minutes. The precipitated solid was removed by filtration. The filtrate was added to a suspension of Fmoc-L-Lys-OtBu hydrochloride (20.3 g, 44 mmol) in DCM (30 ml), followed by DIPEA (7.7 ml, 1 equiv.); the temperature was maintained between 0°C and 5°C during the addition. The reaction mixture was stirred at 5°C for 30 minutes, at which time LCMS indicated no starting material remained. The mixture was diluted with 150 ml of DCM, and the organic layer was washed with 0.5 M HCl solution (100 ml), 0.1 M NaHCO3 solution (100 ml), dried over MgSO4, and evaporated. The residue was purified by flash chromatography (330 g silica gel column, 0% to 50% ethyl acetate in hexanes) to give the target compound (16.8 g, 73%) as a white glass.

[0222] Step 41: Intermediate #4A, tert-butyl (S)-2-amino-6-(hexanoylamino)hexanoate

[0223] [ka]

[0224] To a solution of tert-butyl-(S)-2-[(9H-fluoren-9-yl)methoxycarbonylamino]-6-(hexanoylamino)hexanoate (16 g, 30 mmol) in THF (200 mL) was added diethylamine (92 mL, 900 mmol) at 10°C. The reaction mixture was stirred for 1 hour and evaporated to dryness under vacuum without heating. The residue was re-evaporated with toluene (100 mL) at 25-30°C and subjected to silica gel purification (220 g column, 0%-15% MeOH in DCM) to give the product (7.1 g, 79%) as a viscous yellow oil.

[0225] Step 42: Intermediate #5A, tert-butyl-(S)-2-[(S)-2-amino-6-(tert-butoxycarbonylamino)hexanoylamino]-6-(hexanoylamino)hexanoate:

[0226] [ka]

[0227] To a solution of CBZ-Lys(Boc)-OH (9.7 g, 25.5 mmol) in dry DCM (100 ml) was added HOBt (3.45 g, 25.5 mmol), and the mixture was stirred at ambient temperature for 10 minutes. A solution of tert-butyl (S)-2-amino-6-(hexanoylamino)hexanoate (6.9 g, 23 mmol) in DCM, DIPEA (12.2 ml), and EDC (5.35 g) was added sequentially. The reaction mixture was stirred at ambient temperature for 4 hours (until LCMS showed that tert-butyl (S)-2-amino-6-(hexanoylamino)hexanoate was consumed), diluted with DCM (200 ml), washed with water (150 ml) and saturated NaCl solution (100 ml), and dried over anhydrous MgSO4. The organic solution was concentrated under reduced pressure and the residue was purified by silica gel chromatography (220 g column, 15% to 80% ethyl acetate in hexanes) to give the product (11.4 g, 75%) as an amorphous yellow solid.

[0228] Step 43: Intermediate #6A, tert-butyl-(S)-2-[(S)-2-amino-6-(tert-butoxycarbonylamino)hexanoylamino]-6-(hexanoylamino)hexanoate

[0229] [ka]

[0230] To a solution of tert-butyl-(S)-2-[(S)-2-amino-6-(tert-butoxycarbonylamino)hexanoylamino]-6-(hexanoylamino)hexanoate (11 g, 16.6 mmol) in methanol (400 mL) was added activated 10% palladium on carbon (2 g) (50% wet). The suspension was subjected to catalytic hydrogenation (H, 1 atm, overnight) at ambient temperature (after 12 h, LCMS indicated complete consumption of the starting material). The reaction mixture was filtered through Celite. The filtrate was concentrated under reduced pressure, re-evaporated twice with 200 mL of acetonitrile, and dried under high vacuum for 4 h to give the product (8.96 g, 100%) as a colorless glass. Step 44: Intermediate #7A, tert-butyl-(S)-2-[(S)-2-[3-(2-{2-[2-(benzyloxycarbonylamino)ethoxy]ethoxy}ethoxy)propionylamino]-6-(tert-butoxycarbonylamino)hexanoylamino]-6-(hexanoylamino)hexanoate

[0231] [ka]

[0232] To a solution of 3-(2-{2-[2-(benzyloxycarbonylamino)ethoxy]ethoxy}ethoxy)propionic acid (5.8 g, 16.6 mmol) in dry DCM (50 ml) was added HOBt (2.53 g, 18.7 mmol), and the mixture was stirred at ambient temperature for 10 minutes. A solution of tert-butyl-(S)-2-[(S)-2-amino-6-(tert-butoxycarbonylamino)hexanoylamino]-6-(hexanoylamino)hexanoate (8.9 g, 16.6 mmol) in DCM (50 ml), EDC (5.35 g), and DIPEA (12.2 ml) was added sequentially. The reaction mixture was stirred for 4 h (LCMS monitoring), diluted with DCM (200 ml), washed with water (150 ml) and saturated NaCl solution (100 ml), and dried over MgSO. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (220 g column, 0% to 10% MeOH in DCM) to give the target product (9.63 g, 67%) as a colorless glass.

[0233] Step 45: (Intermediate #1), tert-butyl-(S)-2-[(S)-2-(3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propionylamino)-6-(tert-butoxycarbonylamino)hexanoylamino]-6-(hexanoylamino)hexanoate

[0234] [ka]

[0235] To a solution of tert-butyl-(S)-2-[(S)-2-[3-(2-{2-[2-(benzyloxycarbonylamino)ethoxy]ethoxy}ethoxy)propionylamino]-6-(tert-butoxycarbonylamino)hexanoylamino]-6-(hexanoylamino)hexanoate (9.5 g) in methanol (100 ml) was added activated 10% palladium on carbon (50% wet, 2 g). The suspension was subjected to catalytic hydrogenation overnight at room temperature (1 atm H, followed by LCMS to control the absence of starting material). The reaction mixture was filtered through Celite. The filtrate was concentrated under reduced pressure, re-evaporated twice with 100 ml of acetonitrile, and dried under high vacuum for 4 hours to give the target product (8.15 g, 100%) as a colorless glass. Preparation of RTX-1363S from fragments:

[0236] Step 46: Intermediate #1C, scaled up, tert-butyl-(S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionyl-amino)-6-(tert-butoxycarbonylamino)hexanoylamino]-6-(hexanoylamino)hexanoate To a mixture of {4-[2-((S)-2-cyano-pyrrolidin-1-yl)-2-oxo-ethylcarbamoyl]-quinolin-7-yloxy}-acetic acid (2.7 g, 5.2 mmol, crude TFA salt), (S)-2-[(S)-2-(3-{2-[2-(2-amino-ethoxy)-ethoxy]-ethoxy}-propionylamino)-6-tert-butoxycarbonylamino-hexanoylamino]-6-hexanoylamino-hexanoic acid tert-butyl ester (4.20 g, 5.72 mmol) and HATU (2.40 g, 6.24 mmol) in DMF (85 ml) was added DIEA (5.45 ml, 31.2 mmol) at ambient temperature. The mixture was stirred for 30 min (LCMS check for the absence of SM), then diluted with EtOAc (500 ml) and washed with water (2 × 100 ml) and brine (100 ml). The aqueous phase was back-extracted with EtOAc (250 ml). The EtOAc extract was washed with brine (100 ml). The organics were combined, dried over anhydrous MgSO4, and filtered. The filtrate was evaporated to give approximately 7.2 g of a yellow glass. The crude product was dissolved in DCM (40 ml) and subjected to flash purification on CombiFlash (0% to 15% MeOH in DCM, 2 × 220 g silica gel columns). The desired fractions were combined and evaporated to give the target material (5.42 g, 95%) as a yellow glass. Intermediate #2C, tert-butyl-(S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-aminohexanoylamino]-6-(hexanoylamino)hexanoate, and Intermediate #3C, tert-butyl-(S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-{2-[4,7-bis(tert-butoxycarbonylmethyl)-1,4,7-triazonan-1-yl]acetylamino}hexanoylamino]-6-(hexanoylamino)hexanoate

[0237] [ka]

[0238] Procedure 47: A solution of tert-butyl-(S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-(tert-butoxycarbonylamino)hexanoylamino]-6-(hexanoylamino)hexanoate (5.4 g, 5.9 mmol) in 90% aqueous formic acid (180 ml) was stirred at ambient temperature for 2.5 hours, and then the solvent was evaporated. The residue was co-evaporated with acetonitrile (60 ml × 3) and toluene (60 ml × 2), dissolved in 180 ml of 2-methyltetrahydrofuran, and treated with 1% aqueous KCO solution (30 ml). The organic layer was separated, dried over MgSO4, and evaporated to give the target material in free base form, which was used as obtained for the next step.

[0239] Step 48: To the material from the previous step (approximately 5.96 mmol), NOTA-bis(tBu-ester) (3.66 g, 8.82 mmol), and PyBop (3.10 g, 5.96 mmol) in acetonitrile (120 ml), DIPEA (2.3 ml, 12.3 mmol) was added, and the reaction mixture was stirred at ambient temperature for 2 h (LCMS control). The reaction mixture was evaporated, diluted with ethyl acetate (600 ml), and extracted with water (2 x 120 ml). The organic fraction was evaporated and purified by silica gel column chromatography (220 g column, 3% to 40% MeOH in DCM with 5% DIPEA added). The desired fractions were combined and evaporated to give 6.2 g (4.63 mmol, 75%) of the target product as a bright yellow glass. RTX-1363S, (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-{2-[4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl]acetylamino}hexanoylamino]-6-(hexanoylamino)hexanoic acid

[0240] [ka]

[0241] Procedure 49: To a solution of tert-butyl-(S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-{2-[4,7-bis(tert-butoxycarbonylmethyl)-1,4,7-triazonan-1-yl]acetylamino}hexanoylamino]-6-(hexanoylamino)hexanoate (1 g, 0.717 mmol) in DCM (10 ml) was added TFA (15 ml, 195 mmol) dropwise at 0° C. The temperature of the reaction mixture was allowed to rise to ambient temperature and stirring was continued for 1 h (approximately 60% conversion). TFA (10 ml) was added, and the reaction mixture was stirred at ambient temperature for another hour. Complete conversion was detected by LCMS. The solvent was evaporated at ambient temperature. The residue was dissolved in DMSO (10 ml) and subjected to HPLC purification (Nanosyn-Pack, Silicycle, C-18, 100-10, 50 × 300 mm; 0.1% TFA / ACN / water). The desired fractions were combined and lyophilized to give the target compound (550 mg, 59%; 96% purity) as an off-white powder. The compound was repurified by HPLC (5 mM NH4OAc / ACN) and lyophilized to give two fractions of the target material (fraction #23: 243 mg, 98.5% purity, and fraction #24: 80 mg, 96.8% purity) as a white powder (total 36.8% yield). Synthesis of RTX-1339

[0242] [ka]

[0243] Prepared according to the procedure of Example 9 with the following modifications: Step 3 was used instead of step 40. Synthesis of RTX-1340S

[0244] [ka]

[0245] Prepared according to the procedure in Example 9, substituting butyric acid for hexanoic acid in step 40. Synthesis of RTX-1363S-Cu

[0246] [ka]

[0247] Prepared according to the procedure of Example 9. The resulting product was subjected to Procedure 50 for the incorporation of radionuclides.

[0248] Step 50: Combine 20 μg of precursor (unless otherwise specified) with the indicated radioisotope. The radioisotope is eluted from the generator ( 68 The reaction mixture was either obtained commercially as a HCl solution (for GA) or buffered with various amounts of 3N NaOAc to a final pH of 4-6. The reaction mixture was transferred to a C18 Sep-Pak Lite cartridge as needed for further purification and / or reconstitution for injection. Synthesis of RTX-1359

[0249] [ka]

[0250] Prepared according to the procedure of Example 9 with the following modifications: In step 40, 2-(4-isobutylphenyl)acetic acid was utilized in place of hexanoic acid.

[0251] The product of step 44 was subjected to steps 13, 17 and 18 for the introduction of the DOTA chelator. The resulting product was subjected to steps 51-54 for the introduction of boronated proline derivatives.

[0252] [ka]

[0253] Synthesis of 2-[2-(3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propionylamino)-6-{2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraaza-1-cyclododecyl]acetylamino}hexanoylamino]-6-[2-(p-isobutylphenyl)-acetylamino]hexanoic acid:

[0254] [ka]

[0255] Procedure 51: To a solution of tert-butyl 2-{2-[3-(2-{2-[2-(benzyloxycarbonylamino)ethoxy]ethoxy}ethoxy)-propionylamino]-6-{2-[4,7,10-tris(tert-butoxycarbonylmethyl)-1,4,7,10-tetraaza-1-cyclododecyl-]acetylamino}hexanoylamino}-6-[2-(p-isobutylphenyl)acetylamino]hexanoate 1 (180 mg, 0.129 mmol) in MeOH (2 mL) was added Pd / C (10% wet) (23 mg, 0.217 mmol). The heterogeneous mixture was stirred under a H balloon (1 atm) for 2 h. The reaction was filtered through Celite and concentrated. The resulting solid was dried in vacuo for 16 hours to give tert-butyl 2-[2-(3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propionylamino)-6-{2-[4,7,10-tris(tert-butoxycarbonylmethyl)-1,4,7,10-tetraaza-1-cyclododecyl]acetylamino}hexanoylamino]-6-[2-(p-isobutylphenyl)acetylamino]hexanoate as a viscous oil. The isolated material was used without further purification. LCMS: C 65 H 115 N9O 15 :m / z:1262.66, actual value m / z=1263.5[M+H] + .

[0256] Procedure 52: To a solution of tert-butyl 2-[2-(3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propionylamino)-6-{2-[4,7,10-tris(tert-butoxycarbonylmethyl)-1,4,7,10-tetraaza-1-cyclododecyl]acetylamino}hexanoylamino]-6-[2-(p-isobutylphenyl)acetylamino]hexanoate (0.129 mmol) in DCM (4 mL) was added TFA (2 mL) at 0° C., and the reaction mixture was stirred at ambient temperature for 2 h. The solvent was evaporated under reduced pressure, and the residue was washed with hexane (2×) and ether (2×) to give 2 (120 mg) as a colorless powder. The crude product, 2-[2-(3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propionylamino)-6-{2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraaza-1-cyclododecyl]acetylamino}hexanoylamino]-6-[2-(p-isobutylphenyl)acetylamino]hexanoic acid, was used for the next step without further purification. LCMS: C 49 H 83 N9O 15 :m / z:1038.23, actual value m / z=1039.2[M+H] + . 2-[2-(3-{2-[2-(2-{2-[4-({2-[(R)-2-{(1R,2R,6S,8R)-6,9,9-trimethyl-3,5-dioxa-4-boratricyclo[6.1.1.0 2,6 Synthesis of ]dec-4-yl}-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-{2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraaza-1-cyclododecyl]acetylamino}hexanoylamino]-6-[2-(p-isobutylphenyl)acetylamino]hexanoic acid

[0257] [ka]

[0258] Procedure 53: To a solution of 2-[2-(3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propionylamino)-6-{2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraaza-1-cyclododecyl]acetylamino}hexanoylamino]-6-[2-(p-isobutylphenyl)acetylamino]hexanoic acid 2 (120 mg, 0.115 mmol) in DMF (0.3 mL) was added DIPEA (82 uL) at −78° C. and the reaction mixture was stirred at −78° C. for 5 minutes. Next, ((R)-1-((R)-2-(7-(2-oxo-2-(2,3,5,6-tetrafluorophenoxy)ethoxy)quinoline-4-carboxamido)propanoyl)pyrrolidin-2-yl)boronic acid 3 (158 mg, 0.230 mmol) in DMF (0.2 mL) was added at −78° C., and the mixture was allowed to warm to 20° C. and stirred for 15 min. After complete consumption of the amine was evident by LCMS, the crude product was submitted for HPLC purification. The desired fractions were combined and lyophilized to give 2-[2-(3-{2-[2-(2-{2-[4-({2-[(R)-2-{(1R,2R,6S,8R)-6,9,9-trimethyl-3,5-dioxa-4-boratotricyclo[6.1.1.0 2,6 ]dec-4-yl}-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-{2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraaza-1-cyclodecyl]acetylamino}hexanoylamino]-6-[2-(p-isobutylphenyl)acetylamino]hexanoic acid 4 (55 mg, 31%) was obtained as an off-white solid. LCMS: C 77 H 115 BN 12 O 21 :m / z:1554.84, actual value m / z=778.2[(1 / 2)M+H] + . Synthesis of 2-[2-(3-{2-[2-(2-{2-[4-({2-[(R)-2-(1,2-dihydrooxaboro)-1-pyrrolidinyl]-2-oxoethylamino}-carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-{2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraaza-1-cyclododecyl]acetylamino}hexanoylamino]-6-[2-(p-isobutylphenyl)-acetylamino]hexanoic acid

[0259] [ka]

[0260] Procedure 54: 2-[2-(3-{2-[2-(2-{2-[4-({2-[(R)-2-{(1R,2R,6S,8R)-6,9,9-trimethyl-3,5-dioxa-4-boratricyclo[6.1.1.0 2,6To a solution of 6-{2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraaza-1-cyclododecyl]acetylamino}hexanoylamino]-6-[2-(p-isobutylphenyl)acetylamino]hexanoic acid (55 mg, 0.035 mmol) in acetonitrile (1 mL) was added a mixture of water and diethyl ether (1 mL) containing phenylboronic acid (8.6 mg, 0.070 mmol) at room temperature, followed by the addition of two drops of 1 M HCl. The reaction mixture was stirred at ambient temperature for 10 minutes. The crude reaction mixture was purified by HPLC. The desired fractions were combined and lyophilized to give 2-[2-(3-{2-[2-(2-{2-[4-({2-[(R)-2-(1,2-dihydrooxaboro)-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-{2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraaza-1-cyclododecyl]acetylamino}hexanoylamino]-6-[2-(p-isobutylphenyl)acetylamino]hexanoic acid 5 (26.3 mg, 27%) as a white solid. LCMS: C 67 H 101 BN 12 O 21 : m / z: 1420.73, actual value m / z = 1403.8 [M-OH] + . Synthesis of RTX-1355R:

[0261] [ka]

[0262] Prepared according to the procedure of Example 9 with the following modifications: In step 40, octanoic acid was utilized instead of hexanoic acid. The product of step 44 was subjected to steps 13, 17 and 18 for the introduction of the DOTA chelator.

[0263] The resulting product was subjected to steps 51-54 for the introduction of boronated proline derivatives. Synthesis of RTX-1356R:

[0264] [ka]

[0265] Prepared according to the procedure of Example 9 with the following modifications: In step 40, 2-(4-isobutylphenyl)butanoic acid was utilized in place of hexanoic acid.

[0266] The product of step 44 was subjected to steps 13, 17 and 18 for the introduction of the DOTA chelator. The resulting product was subjected to steps 51-54 for the introduction of boronated proline derivatives. Synthesis of 1357R:

[0267] [ka]

[0268] It was prepared according to the following procedure:

[0269] Procedure 55: Synthesis of tert-butyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-aminohexanoate (2):

[0270] [ka]

[0271] To a solution of tert-butyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((tert-butoxycarbonyl)-amino)hexanoate (2.0 g, 3.81 mmol) in DCM (10 ml) was added dropwise HCl in dioxane (4 M, 20 ml) at 0° C. and the reaction mixture was stirred at 0° C. for 2 hours. The solvent was evaporated and the residue was co-evaporated with hexane and dried in vacuo to give the HCl salt of the product (1.62 g, 100%) as a white solid. The crude product was used for the next step without further purification. LCMS: C 25 H 32 N2O4: m / z: 424.53, actual value m / z = 425.3 [M+H] + .

[0272] Procedure 56: Synthesis of tert-butyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-(4-(4-isobutylphenyl)-butanamido)hexanoate (4):

[0273] [ka]

[0274] To a quenched solution of 4-(4-isobutylphenyl)butanoic acid (1.0 g, 4.57 mmol) in 10 mL of DCM was added DIC (0.71 mL, 4.47 mmol) dropwise in an ice bath. The mixture was stirred for 30 minutes and filtered. The filtrate was added to a solution of tert-butyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-aminohexanoate (1.62 g, 3.81 mmol) in 5 mL of DCM, followed by the dropwise addition of DIEA (0.66 mL, 3.81 mmol). The mixture was stirred for an additional 30 minutes. The solvent was evaporated under reduced pressure, and the crude product was purified by flash chromatography (eluted with 50% EtOAc in hexane) to give the product (1.93 g, 82% yield) as a white solid. LCMS: C 39 H 50 N2O5: m / z: 626.82, actual m / z = 627.4 [M+H]+ .

[0275] Procedure 57: Synthesis of tert-butyl 2-amino-6-(4-(4-isobutylphenyl)butanamido)hexanoate (5):

[0276] [ka]

[0277] To a solution of tert-butyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-(4-(4-isobutylphenyl)butanamido)-hexanoate (0.95 g, 1.51 mmol) in THF (8 ml) was added diethylamine (8 ml) at room temperature. The reaction mixture was stirred at ambient temperature for 2 hours. The solvent was evaporated and the residue was washed with hexane (2×) and dried in vacuo to give the product (613 mg, 100%). The compound was used as is for the next chemical transformation. LCMS: C 24 H 40 N2O3: m / z: 404.59, actual value m / z = 405.4 [M+H] + .

[0278] Procedure 58: Synthesis of tert-butyl 33-(4-(4-(4-isobutylphenyl)butanamido)butyl)-3,31-dioxo-1-phenyl-2,7,10,13,16,19,22,25,28-nonaoxa-4,32-diazatetratriacontan-34-oate (7):

[0279] [ka]

[0280] To a solution of 3-oxo-1-phenyl-2,7,10,13,16,19,22,25,28-nonaoxa-4-azahentriacontan-31-oic acid (872 mg, 1.51 mmol) in 10 mL of DCM, EDCI.HCl (434 mg, 2.27 mmol) and HOBt (203 mg, 1.51 mmol) were added, and the reaction mixture was stirred at room temperature for 5 minutes. tert-Butyl 2-amino-6-(4-(4-isobutylphenyl)butanamido)hexanoate (613 mg, 1.51 mmol) in 10 mL of DCM and DIEA (0.8 mL, 4.53 mmol) was added sequentially, and the mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with 20 mL of DCM and then washed with water. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by flash chromatography (MeOH / DCM, eluted at 3-4% MeOH in DCM) to give the product (1.0 g, 69% yield) as a colorless liquid. LCMS: C 51 H 83 N3O 14 :m / z:962.22, actual value m / z=962.9[M+H] + .

[0281] Procedure 59: Synthesis of tert-butyl 1-amino-29-(4-(4-(4-isobutylphenyl)butanamido)butyl)-27-oxo-3,6,9,12,15,18,21,24-octaoxa-28-azatriacontan-30-oate (8):

[0282] [ka]

[0283] Palladium on carbon (10%, 0.2 g, wet, 67%) was suspended in a solution of tert-butyl 33-(4-(4-(4-isobutyl-phenyl)butanamido)butyl)-3,31-dioxo-1-phenyl-2,7,10,13,16,19,22,25,28-nonaoxa-4,32-diazatetratriacontan-34-oate (1.0 g, 1.03 mmol) in MeOH (25 ml). The suspension was stirred under H balloon pressure at room temperature for 2 hours. The mixture was filtered through Celite, and the filtrate was evaporated to give the product (850 mg, 100%) as a white solid. The crude product was used for the next step without further purification. LCMS: C 43 H 77 N3O 12 :m / z:828.08, actual value m / z=828.7[M+H] + .

[0284] Procedure 60: Synthesis of tert-butyl 10-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-41-(4-(4-(4-isobutylphenyl)butanamido)butyl)-2,2-dimethyl-4,11,39-trioxo-3,15,18,21,24,27,30,33,36-nonaoxa-5,12,40-triaza-dotetracontan-42-oate (10):

[0285] [ka]

[0286] To a solution of 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((tert-butoxycarbonyl)amino)hexanoic acid (0.48 g, 1.02 mmol) in 10 mL of DCM, EDCI.HCl (0.29 g, 1.53 mmol) and HOBt (0.14 g, 1.02 mmol) were added, and the reaction mixture was stirred at room temperature for 5 minutes. tert-Butyl 1-amino-29-(4-(4-(4-isobutylphenyl)butanamido)butyl)-27-oxo-3,6,9,12,15,18,21,24-octaoxa-28-azatriacontan-30-oate (0.85 g, 1.02 mmol) in DCM (10 mL) and DIEA (0.53 mL, 3.07 mmol) was added sequentially, and the mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with 20 ml of DCM and washed with water. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by flash chromatography (MeOH / DCM, eluted with 5% MeOH in DCM) to give the product (0.66 g, 50% yield) as a white solid. LCMS: C 69 H 107 N5O 17 :m / z:1278.61, actual value m / z=1279.1[M+H] + .

[0287] Procedure 61: Synthesis of tert-butyl 10-amino-41-(4-(4-(4-isobutylphenyl)butanamido)butyl)-2,2-dimethyl-4,11,39-trioxo-3,15,18,21,24,27,30,33,36-nonaoxa-5,12,40-triazadtetracontan-42-oate (11):

[0288] [ka]

[0289] To a solution of tert-butyl 10-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-41-(4-(4-(4-isobutylphenyl)-butanamido)butyl)-2,2-dimethyl-4,11,39-trioxo-3,15,18,21,24,27,30,33,36-nonaoxa-5,12,40-triazadtetracontan-42-oate (0.66 g, 0.51 mmol) in THF (7 ml) was added diethylamine (7 ml) at room temperature. The reaction mixture was stirred at ambient temperature for 2 hours and the solvent was evaporated. The residue was washed with hexane (2×) and dried in vacuo to give the product (700 mg, 100%). The compound was used for the next step without further purification. LCMS: C 54 H 97 N5O 15 :m / z:1056.37, actual value m / z=1057.1[M+H] + .

[0290] Procedure 62: Synthesis of tert-butyl 18-(4-((tert-butoxycarbonyl)amino)butyl)-49-(4-(4-(4-isobutylphenyl)-butanamido)butyl)-3,16,19,47-tetraoxo-1-phenyl-2,7,10,13,23,26,29,32,35,38,41,44-dodecaoxa-4,17,20,48-tetraazapentacontan-50-oate (13):

[0291] [ka]

[0292] To a solution of 3-oxo-1-phenyl-2,7,10,13-tetraoxa-4-azahexadecan-16-oic acid (0.22 g, 0.62 mmol) in DCM (8 ml) was added EDCI.HCl (0.18 g, 0.93 mmol) and HOBt (0.08 g, 0.62 mmol) and the reaction mixture was stirred at room temperature for 5 minutes. tert-Butyl 10-amino-41-(4-(4-(4-isobutylphenyl)-butanamido)butyl)-2,2-dimethyl-4,11,39-trioxo-3,15,18,21,24,27,30,33,36-nonaoxa-5,12,40-triazadtetracontan-42-oate (0.66 g, 0.62 mmol) in DCM (5 ml) and DIEA (0.32 ml, 1.87 mmol) were added sequentially, and the mixture was stirred at room temperature for 16 h. The reaction mixture was diluted with 20 ml of DCM and washed with water. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by flash chromatography (MeOH / DCM, eluted with 8-10% MeOH in DCM) to give the product (0.70 g, 50% yield) as a white solid. LCMS: C 71 H 120 NO 21 :m / z:1393.74, actual value m / z=1394.7[M+H] + .

[0293] Procedure 63: Synthesis of tert-butyl 18-(4-aminobutyl)-49-(4-(4-(4-isobutylphenyl)butanamido)butyl)-3,16,19,47-tetraoxo-1-phenyl-2,7,10,13,23,26,29,32,35,38,41,44-dodecaoxa-4,17,20,48-tetraazapentacontan-50-oate (14):

[0294] [ka]

[0295] A solution of tert-butyl 18-(4-((tert-butoxycarbonyl)amino)butyl)-49-(4-(4-(4-isobutylphenyl)butanamido)butyl)-3,16,19,47-tetraoxo-1-phenyl-2,7,10,13,23,26,29,32,35,38,41,44-dodecaoxa-4,17,20,48-tetraazapentacontan-50-oate (0.70 g, 1.22 mmol) in 90% aqueous formic acid (10 ml) was stirred at ambient temperature for 2 hours. Complete conversion was detected by LCMS. The solvent was evaporated and the residue was coevaporated with ACN (3x) and toluene (3x). The residue was dissolved in a mixture of MeOH / water (1:1, 25 ml) and treated with ion exchange resin AmberLite HPR550 (OH form) at ambient temperature for 15 minutes. The resin was filtered and washed with MeOH and water. The filtrate was evaporated to give the target material (free base, 0.62 g, 95% yield) as a colorless liquid. The crude amine was used for the next step without further purification. LCMS: C 66 H 112 NO 19 :m / z:1293.62, actual value m / z=1294.1[M+H] + .

[0296] Procedure 64: Synthesis of tri-tert-butyl 2,2',2''-(10-(39-(tert-butoxycarbonyl)-48-(4-isobutylphenyl)-2,9,37,45-tetraoxo-8-(3-oxo-1-phenyl-2,7,10,13-tetraoxa-4-azahexadecanamido)-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazaoctatetracontyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (15):

[0297] [ka]

[0298] To a solution of tert-butyl 18-(4-aminobutyl)-49-(4-(4-(4-isobutylphenyl)butanamido)butyl)-3,16,19,47-tetraoxo-1-phenyl-2,7,10,13,23,26,29,32,35,38,41,44-dodecaoxa-4,17,20,48-tetraazapentacontan-50-oate (0.62 g, 0.48 mmol), 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 hours. 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 give the target product (820 mg, 93% yield) as a colorless liquid. The compound was used for the next step without further purification. LCMS: C 94 H 162 N 10 O 26 :m / z:1848.34, actual value m / z=1849.7[M+H] + .

[0299] Procedure 65: Synthesis of tri-tert-butyl 2,2',2''-(10-(8-(3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propanamido)-39-(tert-butoxycarbonyl)-48-(4-isobutylphenyl)-2,9,37,45-tetraoxo-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazaoctatetracontyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (16):

[0300] [ka]

[0301] Palladium on carbon (10%, 0.12 g, wet, 67%) was suspended in a solution of tri-tert-butyl 2,2',2''-(10-(39-(tert-butoxycarbonyl)-48-(4-isobutylphenyl)-2,9,37,45-tetraoxo-8-(3-oxo-1-phenyl-2,7,10,13-tetraoxa-4-azahexadecanamido)-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazaoctatetracontyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (500 mg, 0.270 mmol) in MeOH (15 ml). The suspension was stirred under H balloon pressure at room temperature for 2 h. The mixture was filtered through celite and the filtrate was evaporated to give the target compound (387 mg, 83% yield) as a colorless liquid. The crude product was used for the next step without further purification. LCMS: C 86 H 156 N 10 O 24 :m / z:1714.21, actual value m / z=1714.7[M+H] + .

[0302] Procedure 66: Synthesis of 2,2',2''-(10-(8-(3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propanamido)-39-carboxy-48-(4-isobutylphenyl)-2,9,37,45-tetraoxo-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazaoctatetracontyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (17):

[0303] [ka]

[0304] To a solution of tri-tert-butyl 2,2',2''-(10-(8-(3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propanamido)-39-(tert-butoxycarbonyl)-48-(4-isobutylphenyl)-2,9,37,45-tetraoxo-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazaoctatetracontyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (387 mg, 0.225 mmol) in DCM (4 ml) at 0°C was added TFA (2 ml). The reaction mixture was stirred at 40°C for 2 hours and the solvent was evaporated under reduced pressure. The residue was washed with hexane (2x) and ether (2x) to give the product (340 mg, 100%) as a white powder. The crude product was used for the next step without further purification. LCMS: C 70 H 124 N 10 O 24 :m / z:1489.79, actual value m / z=1490.5[M+H] + .

[0305] Procedure 67: 2,2',2''-(10-(39-carboxy-48-(4-isobutylphenyl)-2,9,37,45-tetraoxo-8-(2-oxo-1-((4-((2-oxo-2-((R)-2-((3aR,4R,6R,7aS)-5,5,7a-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)-pi Synthesis of (roridin-1-yl)ethyl)carbamoyl)quinolin-7-yl)oxy)-6,9,12-trioxa-3-azapentadecanamido)-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazaoctatetracontyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (4):

[0306] [ka]

[0307] To a solution of 2,2',2''-(10-(8-(3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propanamido)-39-carboxy-48-(4-isobutylphenyl)-2,9,37,45-tetraoxo-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazaoctatetracontyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (120 mg, 0.080 mmol) in DMF (2 mL) was added DIPEA (0.11 mL, 0.645 mmol) at 0°C, and the reaction mixture was stirred at 0°C for 5 minutes. Then, 2,3,5,6-tetrafluorophenyl 2-((4-((2-oxo-2-((R)-2-((3aR,4R,6R,7aS)-5,5,7a-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)pyrrolidin-1-yl)ethyl)carbamoyl)quinolin-7-yl)oxy)acetate (66 mg, 0.096 mmol) in DMF (0.5 mL) was added at 0° C., and the reaction mixture was stirred at the same temperature for 0.5 h. After complete consumption of the amine by LCMS, the crude product was submitted for HPLC purification. The desired fractions were combined and lyophilized to give 4 (50 mg, 31%) as an off-white solid. LCMS: C 98 H 156 BN 13 O 30 :m / z:2006.12, actual value m / z=1004.80[M / 2+H] + .

[0308] Procedure 68: Synthesis of 2,2',2''-(10-(8-(1-((4-((2-((R)-2-boronopyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-7-yl)oxy)-2-oxo-6,9,12-trioxa-3-azapentadecanamido)-39-carboxy-48-(4-isobutylphenyl)-2,9,37,45-tetraoxo-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazaoctatetracontyl)-1,4,7,10-tetraazacyclo-dodecane-1,4,7-triyl)triacetic acid (RTX-1357R(5)):

[0309] [ka]

[0310] 2,2',2''-(10-(39-carboxy-48-(4-isobutylphenyl)-2,9,37,45-tetraoxo-8-(2-oxo-1-((4-((2-oxo-2-((R)-2-((3aR,4R,6R,7aS)-5,5,7a-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)pyrrolidin-1-yl)ethyl)carba To a solution of (6,9,12-trioxa-3-azapentadecanamido)-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazaoctatetracontyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (35 mg, 0.017 mmol) in a mixture of acetone (0.5 mL) and 0.2 N HCl (0.5 mL) was added MeB(OH) (5 mg, 0.087 mmol) at room temperature, and the reaction mixture was stirred at ambient temperature for 30 minutes. After complete consumption of the starting material by LCMS, the volatiles were evaporated under reduced pressure, and the crude product was submitted for HPLC purification. The desired fractions were combined and lyophilized to give the target compound (15 mg, 46%, HPLC purity 95%) as an off-white solid. LCMS: C 88 H 142 BN 13 O 30 :m / z:1872.95, actual value m / z=1873.80[M+H] + . Synthesis of RTX-1358R:

[0311] [ka]

[0312] Prepared according to the procedure of Example 9 with the following modifications: Step 3 was used instead of step 40. The product of step 44 was subjected to steps 13, 17 and 18 for the introduction of the DOTA chelator.

[0313] The resulting product was then subjected to steps 51-54 for the introduction of boronated proline derivatives. Synthesis of RTX-1361R:

[0314] [ka]

[0315] Prepared according to the procedure of Example 9 with the following modifications: The product of step 44 was subjected to steps 13, 17 and 18 for the introduction of the DOTA chelator.

[0316] The resulting product was subjected to steps 51-54 for the introduction of boronated proline derivatives. Example 10 – Synthesis of RTX-1389 Synthesis of compound 10:

[0317] [ka]

[0318] Procedure 69: Synthesis of tert-butyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-aminohexanoate (2):

[0319] [ka]

[0320] To a solution of tert-butyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((tert-butoxycarbonyl)amino)hexanoate (2.5 g, 4.77 mmol) in DCM (10 ml) was added dropwise HCl in dioxane (4 M, 20 ml) at 0° C. and the reaction mixture was stirred at 0° C. for 2 hours. The solvent was evaporated and the residue was co-evaporated with hexane and dried in vacuo to give the HCl salt of the target material (2.02 g, 100%) as a white solid. The crude product was used for the next step without further purification. LCMS: C 25 H 32 N2O4: m / z: 424.53, actual value m / z = 425.3 [M+H] + .

[0321] Procedure 70: Synthesis of tert-butyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-acetamidohexanoate (3):

[0322] [ka]

[0323] To a solution of tert-butyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-aminohexanoate (2.02 g, 4.71 mmol) and acetic anhydride (2.22 ml, 23.58 mmol) in DCM (20 ml) was added DIEA (1.64 ml, 9.43 mmol) at 0° C. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was diluted with 20 ml of DCM and then washed with water. The organic layer was dried over NaSO and concentrated under reduced pressure. The crude product was purified by flash chromatography (eluted in EtOAc / hexane, 50% EtOAc in hexane) to give the product (1.60 g, 73% yield) as a white solid. LCMS: C 27 H 34 N2O5: m / z: 466.57, actual value m / z = 467.3 [M+H] + .

[0324] Procedure 71: Synthesis of tert-butyl 6-acetamido-2-aminohexanoate (4):

[0325] [ka]

[0326] To a solution of tert-butyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-acetamidohexanoate (1.60 g, 3.43 mmol) in THF (10 ml) was added diethylamine (10 ml) at room temperature. The reaction mixture was stirred at ambient temperature for 2 hours. The solvent was evaporated and the residue was washed with hexane (2×) and dried in vacuo to give the product (900 mg, 99%). The compound was used for the next step without further purification. LCMS: C 12 H 24 N2O3: m / z: 244.33, actual value m / z = 245.4 [M+H] + .

[0327] Procedure 72: Synthesis of tert-butyl 2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((tert-butoxycarbonyl)amino)hexanamido)-6-acetamidohexanoate (6):

[0328] [ka]

[0329] To a solution of 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((tert-butoxycarbonyl)amino)hexanoic acid (1.72 g, 3.68 mmol) in 15 mL of DCM, EDCI.HCl (1.05 g, 5.53 mmol) and HOBt (0.50 g, 3.68 mmol) were added, and the reaction mixture was stirred at room temperature for 5 minutes. Next, tert-butyl 6-acetamido-2-aminohexanoate (0.90 g, 3.68 mmol) in DCM (10 mL) and DIEA (1.92 mL, 11.06 mmol) was added sequentially, and the mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with 20 mL of DCM and then washed with water. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by flash chromatography (EtOAc / hexanes, eluted at 75% EtOAc in hexanes) to give the product (2.20 g, 77% yield) as a white solid. LCMS: C 38 H 54 NO: m / z: 694.86, actual m / z = 695.3 [M+H] + .

[0330] Procedure 73: Synthesis of tert-butyl 6-acetamido-2-(2-amino-6-((tert-butoxycarbonyl)amino)hexanamido)-hexanoate (7):

[0331] [ka]

[0332] To a solution of tert-butyl 2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((tert-butoxycarbonyl)-amino)hexanamido)-6-acetamidohexanoate (1.60 g, 2.30 mmol) in THF (10 ml) was added diethylamine (10 ml) at room temperature. The reaction mixture was stirred at ambient temperature for 2 hours. The solvent was evaporated and the residue was washed with hexane (2×) and dried under vacuum to give the product (1.0 g, 92% yield). The compound was used for the next step without further purification. LCMS: C23 H 44 N4O6: m / z: 472.62, actual m / z = 473.5 [M+H] + .

[0333] Procedure 74: Synthesis of tert-butyl 21-(4-acetamidobutyl)-18-(4-((tert-butoxycarbonyl)amino)butyl)-3,16,19-trioxo-1-phenyl-2,7,10,13-tetraoxa-4,17,20-triazadocosane-22-oate (9):

[0334] [ka]

[0335] To a solution of 3-oxo-1-phenyl-2,7,10,13-tetraoxa-4-azahexadecan-16-oic acid (0.83 g, 2.33 mmol) in 10 mL of DCM, EDCI.HCl (0.67 g, 3.49 mmol) and HOBt (0.31 g, 2.33 mmol) were added, and the reaction mixture was stirred at room temperature for 5 minutes. Next, tert-butyl 6-acetamido-2-(2-amino-6-((tert-butoxycarbonyl)amino)hexanamido)hexanoate (1.0 g, 2.33 mmol) in 10 mL of DCM and DIEA (1.2 mL, 6.99 mmol) was added sequentially, and the mixture was stirred at room temperature for 16 hours. The mixture was diluted with 20 mL of DCM and washed with water. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by flash chromatography (MeOH / DCM, eluted at 3-4% MeOH in DCM) to give the product (1.20 g, 70% yield) as a white solid. LCMS: C 12 H 24 N2O3: m / z: 809.99, actual value m / z = 810.7 [M+H] + .

[0336] Procedure 75: Synthesis of tert-butyl 17-(4-acetamidobutyl)-1-amino-14-(4-((tert-butoxycarbonyl)amino)butyl)-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecane-18-oate (10):

[0337] [ka]

[0338] Palladium on carbon (10%, 0.03 g, wet, 67%) was suspended in a solution of tert-butyl 21-(4-acetamidobutyl)-18-(4-((tert-butoxycarbonyl)amino)butyl)-3,16,19-trioxo-1-phenyl-2,7,10,13-tetraoxa-4,17,20-triazadocosane-22-oate (130 mg, 0.160 mmol) in MeOH (3 ml). The suspension was stirred under H balloon pressure at room temperature for 2 h. The mixture was then filtered through Celite and the filtrate was evaporated to give the target compound (106 mg, 98% yield) as a white solid. The crude product was used for the next step without further purification. LCMS: C 32 H 61 N5O 10 :m / z:675.85, actual value m / z=676.5[M+H] + .

[0339] [ka]

[0340] Procedure 76: Synthesis of tert-butyl [(7-hydroxy-4-quinolyl)carbonylamino]acetate:

[0341] [ka]

[0342] To 7-hydroxy-4-quinolinecarboxylic acid (1.0 g, 5.3 mmol, 1 equiv.), glycine t-butyl ester hydrochloride (1.2 g, 5.78 mmol, 1.1 equiv.), and HATU (3.02 g, 7.95 mmol, 1.5 equiv.) in DMF (20 ml) was added DIPEA (2.2 ml, 2.5 equiv.) at 5 °C and stirred at room temperature for 2 h. Upon completion, HO was added and the product was extracted with n-butanol. The organic layer was separated, dried over NaSO, and evaporated under reduced pressure. The resulting residue was purified by flash chromatography (0–10% MeOH in DCM) to give 3 (1.1 g, 67% yield) as an off-white powder. ESI-MS m / z calculated 302.324, found 303.0 [M+H]. + .

[0343] Procedure 77: Synthesis of benzyl (4-{[(tert-butoxycarbonylmethyl)amino]carbonyl}-7-quinolyloxy)acetate:

[0344] [ka]

[0345] To a suspension of tert-butyl [(7-hydroxy-4-quinolyl)carbonylamino]acetate (1.1 g, 3.55 mmol, 1 equiv.) and KCO (1.5 g, 10.7 mmol, 3 equiv.) in DMF (20 ml) was added benzyl bromoacetate (2.4 g, 10.7 mmol, 3 equiv.) at room temperature. The mixture was stirred at 60 °C for 16 h. Upon completion, HO was added and the product was extracted with EtOAc. The organic layer was separated, dried over NaSO, and evaporated under reduced pressure. The resulting residue was purified by flash chromatography (0–80% ethyl acetate in hexanes) to give 5 (1 g, 61% yield) as an off-white powder. ESI-MS m / z calculated 450.482, found 451.0 [M+H]. + .

[0346] Procedure 78: Synthesis of ({7-[(benzyloxycarbonyl)methoxy]-4-quinolyl}carbonylamino)acetic acid:

[0347] [ka]

[0348] To a solution of benzyl (4-{[(tert-butoxycarbonylmethyl)amino]carbonyl}-7-quinolyloxy)acetate (1 g, 2.2 mmol) in DCM, 10 ml of TFA / DCM (DCM:TFA, 1:2) was added at room temperature, and the mixture was stirred for 4 h. The progress of the reaction was monitored by LCMS, and additional TFA solution was added as needed to drive the reaction to completion. The solvent was evaporated under reduced pressure to give 6 as an off-white solid in quantitative yield. ESI-MS m / z calculated 394.376, found 395.0 [M+H] + .

[0349] Step 79: Benzyl (4-{[2-{(1R,2S,8R)-2,9,9-trimethyl-3,5-dioxa-4-boratocyclo-[6.1.1.0 2,6 Synthesis of ]dec-4-yl}-1-pyrrolidinyl)-2-oxoethylamino]carbonyl}-7-quinolyloxy)acetate:

[0350] [ka]

[0351] ({7-[(benzyloxycarbonyl)methoxy]-4-quinolyl}carbonylamino)acetic acid (0.87 g, 2.2 mmol, 1 equiv.), 2-{(1R,2R,8S)-2,9,9-trimethyltricyclo[6.1.1.0 2,6To a solution of ]dec-4-yl}pyrrolidine (0.6 g, 2.4 mmol, 1.1 equiv.) and HATU (1.25 g, 3.3 mmol, 1.5 equiv.) in DMF (5 ml) was added DIPEA (0.8 ml, 5.5 mmol, 2.5 equiv.) at 5 °C. The mixture was stirred at room temperature for 2 h, HO was added, and the product was extracted with EtOAc. The organic layer was separated, dried over NaSO, and evaporated under reduced pressure. The resulting residue was purified by flash chromatography (0–10% MeOH in DCM) to give 8 (1.1 g, 82% yield) as an off-white solid. ESI-MS m / z calculated 625.520, found 626.1 [M+H]. + .

[0352] Procedure 80: (4-{[2-(2-{(1R,2S,8R)-2,9,9-trimethyl-3,5-dioxa-4-boratricyclo[6.1.1.0 2,6 Synthesis of ]dec-4-yl}-1-pyrrolidinyl)-2-oxoethylamino]carbonyl}-7-quinolyloxy)acetic acid:

[0353] [ka]

[0354] Activated palladium on carbon (5%, 0.2 g) was suspended in a solution of 8 (1.1 g, 8 mmol) in MeOH (50 mL). The suspension was stirred under H atmosphere (40 psi) at room temperature for 1 h. The mixture was filtered through Celite, and the filtrate was concentrated under reduced pressure to give 11 in quantitative yield (950 mg, 99% yield). ESI-MS m / z calculated 535.398, found 536.1 [M+H]. + . Synthesis of RTX-1389R (16):

[0355] [ka]

[0356] Procedure 81: Synthesis of tert-butyl 20-(4-acetamidobutyl)-17-(4-((tert-butoxycarbonyl)amino)butyl)-2,15,18-trioxo-1-((4-((2-oxo-2-(2-((3aS,4R,6R)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)pyrrolidin-1-yl)ethyl)carbamoyl)quinolin-7-yl)oxy)-6,9,12-trioxa-3,16,19-triazahenicosan-21-oate (12):

[0357] [ka]

[0358] tert-Butyl 17-(4-acetamidobutyl)-1-amino-14-(4-((tert-butoxycarbonyl)amino)butyl)-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecane-18-oate (86 mg, 0.127 mmol) and 2-((4-((2-oxo-2-(2-((3aS,4R,6R)-3a,5,5-trimethylhexyl) To a solution of 4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)pyrrolidin-1-yl)ethyl)carbamoyl)quinolin-7-yl)oxy)acetic acid (68 mg, 0.127 mmol) in 2 mL of DMF was added HATU (53 mg, 0.140 mmol) and DIEA (0.11 mL, 0.637 mmol), and the reaction mixture was stirred at room temperature for 1 hour. The reaction progress was monitored by LCMS. DMF was removed under reduced pressure. The crude product was purified by flash chromatography (MeOH / DCM, eluted with 3-4% MeOH in DCM) to give the product (75 mg, 50% yield) as a colorless liquid. LCMS: C 60 H 93 BN8O 16 :m / z:1193.24, actual value m / z=1194.0[M+H] + .

[0359] Procedure 82: Synthesis of (1-(2-(7-((20-(tert-butoxycarbonyl)-17-(4-((tert-butoxycarbonyl)amino)butyl)-2,15,18,26-tetraoxo-6,9,12-trioxa-3,16,19,25-tetraazaheptacosyl)oxy)quinoline-4-carboxamido)acetyl)pyrrolidin-2-yl)boronic acid (13):

[0360] [ka]

[0361] To a solution of tert-butyl 20-(4-acetamidobutyl)-17-(4-((tert-butoxycarbonyl)amino)butyl)-2,15,18-trioxo-1-((4-((2-oxo-2-(2-((3aS,4R,6R)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)-pyrrolidin-1-yl)ethyl)carbamoyl)quinolin-7-yl)oxy)-6,9,12-trioxa-3,16,19-triazahenicosan-21-oate (65 mg, 0.054 mmol) and MeB(OH) (16 mg, 0.272 mmol) in acetone (1 ml), 0.2 N HCl (1 ml) was added at room temperature and the reaction mixture was stirred at ambient temperature for 30 minutes. The solvent was evaporated and the residue was dried in vacuo to give the product (84 mg, 100%) as a colorless liquid. The crude product was used for the next step without further purification. LCMS: C 50 H 79 BN8O 16 :m / z:1059.02, actual value m / z=1059.8[M+H] + .

[0362] Procedure 83: Synthesis of 20-(4-acetamidobutyl)-17-(4-aminobutyl)-1-((4-((2-(2-boronopyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-7-yl)oxy)-2,15,18-trioxo-6,9,12-trioxa-3,16,19-triazahenicosan-21-oic acid (14):

[0363] [ka]

[0364] To a solution of (1-(2-(7-((20-(tert-butoxycarbonyl)-17-(4-((tert-butoxycarbonyl)amino)butyl)-2,15,18,26-tetraoxo-6,9,12-trioxa-3,16,19,25-tetraazaheptacosyl)oxy)quinoline-4-carboxamido)acetyl)pyrrolidin-2-yl)boronic acid (84 mg, 0.079 mmol) in DCM (1.5 ml) was added TFA (0.5 ml) at 0° C. and the reaction mixture was stirred at ambient temperature for 30 minutes. The solvent was evaporated and the crude product was subjected to HPLC purification. The desired fractions were combined and lyophilized to give the product (28 mg, 48%) as an off-white solid. LCMS: C 41 H 63 BN8O 14 : m / z: 902.80, actual value m / z = 885.5 [M-OH] + .

[0365] Procedure 84: 2-((E)-2-((E)-2-(4-((17S)-17-(((S)-5-acetamido-1-carboxypentyl)carbamoyl)-1-((4-((2-(2-boronopyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-7-yl)oxy)-2,15,23-trioxo-6,9,12-trioxa-3,16,22-triaza Synthesis of pentacosan-25-yl)phenoxy)-3-((E)-2-(3,3-dimethyl-5-sulfonato-1-(3-(trimethylammonio)propyl)indolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(3-(trimethylammonio)propyl)-3H-indol-1-ium-5-sulfonate:

[0366] [ka]

[0367] 20-(4-acetamidobutyl)-17-(4-aminobutyl)-1-((4-((2-(2-boronopyrrolidin-1-yl)-2-oxo-ethyl)carbamoyl)quinolin-7-yl)oxy)-2,15,18-trioxo-6,9,12-trioxa-3,16,19-triazahenicosan-21-oic acid (5 mg, 0.0055 mmol) and 2-((E)-2-((E)-3-((E)-2-(3,3-dimethyl-5-sulfonato-1-(3-(trimethylammonio)-propyl) To a solution of (indolin-2-ylidene)ethylidene)-2-(4-(3-((2,5-dioxopyrrolidin-1-yl)oxy)-3-oxopropyl)phenoxy)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(3-(trimethylammonio)propyl)-3H-indol-1-ium-5-sulfonate (5.8 mg, 0.0055 mmol) in DMSO (0.2 ml) was added TEA (1.7 mg, 0.0166 mmol) and the reaction mixture was stirred at ambient temperature for 15 min. Complete conversion was detected by LCMS. The crude product was subjected to HPLC purification. The desired fractions were combined and lyophilized to give the product (3 mg, 30%) as a dark blue solid. LCMS: C 92 H 128 BN 12 O 22 S2 + : m / z: 1829.01, actual value m / z = 906.1 [M-OH / 2] + . Synthesis of RTX-1403

[0368] [ka]

[0369] Prepared according to example 10, using Alexa Fluor 488 instead of 2-((E)-2-((E)-3-((E)-2-(3,3-dimethyl-5-sulfonato-1-(3-(trimethylammonio)-propyl)indolin-2-ylidene)ethylidene)-2-(4-(3-((2,5-dioxopyrrolidin-1-yl)oxy)-3-oxopropyl)phenoxy)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(3-(trimethylammonio)propyl)-3H-indol-1-ium-5-sulfonate in procedure 84. Synthesis of RTX-1399R Compounds of the invention having Macropa chelators can be prepared according to the procedures described in PCT / US2019 / 062479, the entire teachings of which are incorporated herein by reference.

[0370] [ka]

[0371] Prepared according to the procedure of Example 10 with the following modifications: Step 40 was used instead of step 70. In step 40, 2-(4-isobutylphenyl)acetic acid was used in place of hexanoic acid.

[0372] Macropa chelators are chelators prepared and introduced in place of fluorophores using procedures 85 and 86.

[0373] Procedure 85: Synthesis of 6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)-4-(4-isothiocyanatophenetoxy)picolinic acid (16):

[0374] [ka]

[0375] To a solution of 4-(4-aminophenethoxy)-6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinic acid 2×TFA (120 mg, 0.14 mmol) in DCM (3.5 ml) was added Na2CO3 (44 mg, 0.42 mmol). The reaction mixture was stirred at 40 °C for 15 min (until a homogeneous solution was obtained). The reaction mixture was cooled to room temperature and O,O-di(pyridin-2-yl)carbonothioate (36 mg, 0.15 mmol) in 0.5 ml of DCM was added to the reaction mixture. After LCMS analysis showed consumption of the amine, the solids were removed by filtration and the volatiles were removed under reduced pressure to give product 16 (100 mg, 100%) as a yellow oil. LCMS: C 35 H 43 N5O9S: m / z: 709.81, actual m / z = 710.4 [M+H] + .

[0376] Procedure 86: Synthesis of 4-(4-(3-((S)-1-((4-((2-((R)-2-boronopyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-7-yl)oxy)-17-(((S)-1-carboxy-5-(2-(4-isobutylphenyl)acetamido)pentyl)carbamoyl)-2,15-dioxo-6,9,12-trioxa-3,16-diazahenicosan-21-yl)thioureido)phenetoxy)-6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinic acid):

[0377] [ka]

[0378] N 2-((1-((4-((2-((R)-2-boronopyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-7-yl)oxy)-2-oxo-6,9,12-trioxa-3-azapentadecan-15-oyl)-L-lysyl)-N 6 To a solution of -(2-(4-isobutylphenyl)acetyl)-L-lysine (21 mg, 0.023 mmol) in DMSO:HO (0.5 ml:0.5 ml) was added NaCO (8 mg, 0.069 mmol) and 6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)-4-(4-isothiocyanatophenetoxy)picolinic acid (20 mg, 0.027 mmol, dissolved in DMSO 0.5 ml) and the reaction mixture was stirred at room temperature for 2 hours. After LCMS analysis showed complete consumption of the amine, the solid was removed by filtration and the crude product was submitted for HPLC purification. The desired fractions were combined and lyophilized to give the product (28 mg, 76%) as an off-white solid. LCMS: C 86 H 118 BN 13 O 23 S: m / z: 1743.83, measured value m / z = 855.6.

[0379] Example 11 – Radiolabeling Methods and Results General procedure: 20 μg of precursor (unless otherwise specified) was combined with the indicated radioisotope. The radioisotope was eluted from the generator ( 68 The reaction mixture was either obtained commercially as a HCl solution (for Ga) or buffered with various amounts of 3N NaOAc to a final pH of 4-6. The reaction mixture was then transferred to a C18 Sep-Pak Lite cartridge for further purification and / or reconstitution for injection as needed. The labeling results are shown in Table 1.

[0380] [Table 1-1]

[0381] [Table 1-2]

[0382] Example 12 - Compounds of the invention bind to FAP with high affinity Protease reactions were assembled in 384-well plates (Greiner) in a total volume of 20 uL as described below.

[0383] Recombinant proteins were prediluted in assay buffer consisting of 100 mM HEPES, pH 7.5, 0.1% BSA, 0.01% Triton X-100, and 1 mM DTT and dispensed into 384-well plates (10 μL per well). Test compounds were serially prediluted in DMSO and added to 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 constructed in four replicates and used to calculate the % inhibition in the presence of compound. The concentration of DMSO was equal to 1% in all samples.

[0384] Compounds were pre-incubated with the enzyme for 15 minutes. Human FAP was obtained from Enzo, catalog number BML-SE409-0010. The reaction was initiated by the addition of 10 uL of 2x FAM-labeled substrate peptide (FAM-GPRPFNYLAKK-NH2), prepared in the same assay buffer. The final enzyme concentration was 0.5 nM. The final substrate peptide concentration was 1 uM.

[0385] The reaction proceeded at room temperature. The incubation time was 3 hours for human FAP and 0.5 hours for mouse FAP. After incubation, termination buffer (100x IC 50 The kinase reaction was quenched by adding 50 uL of assay buffer (assay buffer supplemented with reference inhibitor at RT).

[0386] The finished plate was analyzed using a microfluidic electrophoresis instrument (Caliper LabChip® 3000, Caliper Life Sciences / Perkin Elmer). The change in relative intensity of the peptide substrate and the cleaved product was the measured parameter. Activity in each test sample was determined as the product to sum ratio (PSR):P / (S+P), where P is the product peak height and S is the substrate peak height. Percent inhibition (P inh ) was determined using the following equation: P inh =(PSR 0%inh -PSR compound ) / (PSR 0%inh -PSR 100%inh )×100, in the formula, PSR compound is the product / sum ratio in the presence of the compound, and PSR 0%inh is the product / sum ratio in the absence of the compound, and PSR 100%inh is the product / sum ratio in the absence of enzyme. IC of the compound 50 To determine the 50% inhibition rate, the %inh data (P vs. compound concentration) inh ) are fitted by a four-parameter sigmoidal dose-response model using XLfit software (IDBS). These values are compiled and grouped in Table 2, where A is the IC 50 <0.1 nM; B is IC of 0.1–0.5 nM 50 ; C is IC of 0.5-5.0 nM 50 ;D is IC50 between 5.0 and 100 nM 50 ;E is IC 50 represents >100nM.

[0387] [Table 2-1]

[0388] [Table 2-2]

[0389] From the inhibition studies described above, it was found that the S stereoisomer was significantly more potent than the R stereoisomer. The inhibition studies also showed an LC of 1.5 nM. 50 having

[0390] [ka]

[0391] demonstrated that extending the chain of the quinolinyl core of α-glucan significantly improved activity. Specifically, progressively longer chains resulted in improvements with each extension, and addition of the final amino acid (lysine) resulted in a further increase in activity.

[0392] Greater discrimination was observed between certain compounds of the invention and FAPI-46 in surface plasmon resonance assay data (FAP-46 was measured to have an affinity of 255 pM, while certain disclosed compounds had affinities greater than 20 pM). This is also reflected in the U87 bioD study described below in comparison with FAPI-46, in which certain compounds of the invention exhibited significantly greater in vivo retention.

[0393] Example 13 18 Radiolabeling of FAP target compounds with F direct labels received from the manufacturer 18 F was loaded onto a Sep Pak QMA Light Plus cartridge and Cs2CO3 / K 222 The solution was used to elute the compound into a glass reaction vial. 18 F was azeotropically dried using acetonitrile (3 x 1 ml) under a stream of N2 at 95 °C. 2.5 mg of the FAP target compound was dissolved in 0.3 ml of DMSO, which was then dried. 18The reaction mixture was cooled and neutralized, then loaded onto a SemiPrep HPLC column for purification and appropriately formulated for use. Alternatively, a suitable FAP target compound with a chelator can be prepared by: 18 F can be used for AlF labeling, where AlCl stock in acetate buffer (22.5 μL, 45 nmol, 0.9 equiv.) is dissolved in sodium acetate (200 μL) 18 The F solution was added, and the reaction vial was left at room temperature for 5 minutes. Then, FAP target compound solution (50 nmol scale, 12.5 μL) from precursor stock was added to the vial. The pH was adjusted to approximately 4.0 by adding 15 μL of 1% v / v acetic acid in water. 200 μL of cosolvent, EtOH, was added, and the reaction vial was sealed and then heated at 100°C for 15 minutes. The reaction mixture was diluted to 9.5 ml and loaded onto a tC18 Sep Pak cartridge. The product was eluted with 300 μL of acidified EtOH and formulated for use.

[0394] Example 14 - Detection of tumors in mouse models with compounds of the present invention BALB / c nude mice were incubated for approximately 10 7 U-87 (human glioblastoma) cells were transplanted and the tumor was allowed to progress. 18 1 μg of the F-labeled FAP-targeting agent TX-1312S (prepared according to Example 13) was administered to mice by intravenous injection (e.g., tail vein), which were then sacrificed 1 hour later. 18 Figure 1B illustrates tumor binding of the F-labeled FAP-targeted compound. Figure 1B shows the biodistribution of the compound in various mouse tissues.

[0395] Example 15 – Biodistribution studies Female BALB / C nude mice were injected subcutaneously in the right shoulder with U87MG cells in a 1:1 mixture of Matrigel and PBS. Tumors grew to a volume of 150–500 mm. 3When the IL-10 receptor agonist (RIA) reached 1000kJ / mL, 1 μg of radiolabeled ligand was administered intravenously (IV) via the 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 intestine (with contents), large intestine (with contents), muscle (quadriceps), tumor, and tail) were removed, weighed, and counted in a gamma counter. The activity of each recovered tissue was measured in counts per minute (CPM). Triplicate aliquots of the radiotracer were also assayed in a gamma counter to calculate the function for converting counts to units of activity (μCi / CPM). Values were decay-corrected for the number of injections and corrected for background radiation. The biodistribution of compounds [Cu-67]RTX-1363S, [177Lu]RTX-1354S, and [177Lu]1359R showed localization of the compounds within the tumor and minimal concentrations in other organs (see Figures 2-4). This biodistribution demonstrated increasing selectivity for tumors over time, demonstrating high affinity for FAP.

[0396] Example 16 – Biodistribution studies of [Ac-225]RTX-1399 Radiolabeling procedure: The 225Ac salt was dissolved in 600 μl of 1 M NHOAc. 100 μl of the 225Ac stock solution was added to a reaction vial. 50 μl (50 μg) of the precursor was dissolved in DMSO and then added to the reaction vial. 3 mL of normal saline (0.9%) was added, and the reaction was shaken at 25°C for 30 minutes. The radiolabeling yield and purity were measured by TLC using Macherey-Nagel aluminum sheets (ALUGRAM SIL G / UV254, spotted 4 × 8 cm with 2–5 μl of solution and placed in a chamber of 50 mM EDTA in normal saline) and analyzed using a Packard Phosphorimager for quantification. 1 μg of the radiolabeled compound was administered intravenously (IV) into tumor-bearing JAX nude mice. Over a 120-hour period, the target compound was evidently localized in the tumor and filtered from the blood (see Figure 5).

[0397] equivalent While certain embodiments have been illustrated and described, those skilled in the art, after reading the foregoing specification, can make modifications, equivalent substitutions, and other types of modifications to the compounds or salts, pharmaceutical compositions, derivatives, prodrugs, metabolites, tautomers, or racemic mixtures of the present technology described herein. Each of the above-described aspects and embodiments could also include or incorporate such variations or aspects disclosed in any or all respects of the other aspects and embodiments.

[0398] The present technology is also not limited in terms of the specific embodiments described herein, which are intended to be illustrative of individual embodiments of the technology. Many modifications and variations of the present technology may be made, as will be apparent to those skilled in the art, without departing from its spirit and scope. In addition to those enumerated herein, functionally equivalent methods within the scope of the present technology will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to be included within the scope of the appended claims. It will be understood that the present technology is not limited to particular methods, reagents, compounds, compositions, labeled compounds, or biological systems, which may, of course, vary. It will also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Therefore, it is intended that the specification be considered exemplary only, with the breadth, scope, and spirit of the present technology being indicated only by the appended claims, the definitions therein, and equivalents thereof.

[0399] All publications, patents, and other documents related to this specification are incorporated herein by reference in their entirety.

Claims

1. The following structural formula: 【Chemical 1】 [In the formula, n is 0 or 1; A is NH, O, S or CR ６ R ７ and B comprises 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, where B is F, Cl, Br, I, ═O, OR ６ , O.C.O.R. ６ , COOR ６ , CN, = NR ６ , N.R. ６ R ７ , = S and SR ６ with the proviso that B contains at least 3 atoms in the chain between the D group and the A group; D is OPO ３ H ２ , P.O. ３ H ２ , OSO ３ H, SO ３ H and COOH, and their C １ ~C ４ alkyl esters; X is O or S; R １ is a chelating group, an optical dye or fluorophore, a cytotoxic agent, an immunostimulatory agent, or R ５ is a benzoyl group optionally substituted with one or more groups represented by R ３ is C １ ~C ８ Alkyl or C １ ~C ４ aralkyl, wherein The alkyl and aryl portions of the aralkyl are each optionally and independently selected from F, Cl, Br, I, branched, unbranched, or cyclic C １ ~C ６ aliphatic group, OR ６ , O.C.O.R. ６ , COOR ６ , CHO, COR ６ , C.H. ２ OR ６ , N.R. ６ R ７ , C.H. ２ NR ６ R ７ , S.R. ６ , substituted with =O, =S and =NH; R ４ is CN or B(OH) ２ and Each R ５ are independently halo, cyano, halomethyl, N ＋ (CH ３ ) ３ W － (In the formula, W － is a pharmaceutically acceptable anion; R ６ and R ７ are independently H or C １ ~C ６ alkyl] or a pharmaceutically acceptable salt thereof.

2. B is a branched, unbranched, or cyclic aliphatic group of up to 30 carbon atoms (e.g., 3-20 carbon atoms optionally interrupted by up to 6 heteroatoms or up to 5 amino acid residues), optionally interrupted by up to 10 heteroatoms or by a peptidyl chain of up to 20 amino acid residues; where B is F, Cl, Br, I, ═O, OR ６ , O.C.O.R. ６ , COOR ６ , CN, = NR ６ , N.R. ６ R ７ , = S and SR ６ or a pharmaceutically acceptable salt thereof, wherein B comprises at least 3 atoms in the chain between the D and A groups;

3. The following structural formula: 【Chemistry 2】 (wherein m is an integer from 0 to 12; o is 0 or 1; R ２ is H or C １ ~C ４ alkyl) 3. The compound of claim 1 or 2, represented by: or a pharmaceutically acceptable salt thereof.

4. The following structural formula: 【Chemistry 3】 4. The compound of claim 3, represented by: or a pharmaceutically acceptable salt thereof.

5. The following structural formula: 【Chemistry 4】 4. The compound of claim 3, represented by: or a pharmaceutically acceptable salt thereof.

6. R ３ But C １ ~C ４ C optionally substituted with alkyl １ ~C ８ Alkyl or C １ ~C ４ 6. The compound of any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, which is aralkyl.

7. R ３ 7. The compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, wherein is methyl, propyl, pentyl, heptyl, (4-isobutylphenyl)methyl, (4-isobutylphenyl)propyl.

8. 6. The compound according to any one of claims 3 to 5, wherein o is 1 and m is 3 to 12, or a pharmaceutically acceptable salt thereof.

9. 7. The compound of claim 6, or a pharmaceutically acceptable salt thereof, wherein m is 8.

10. 6. The compound of any one of claims 3 to 5, or a pharmaceutically acceptable salt thereof, wherein o is 0.

11. 11. The compound of any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof, wherein n is 1.

12. R １ 12. The compound of any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, wherein is a fluorophore or an optical dye.

13. The fluorophore is 【Chemistry 5】 13. The compound of claim 12, or a pharmaceutically acceptable salt thereof, wherein the optical dye is selected from the group consisting of a carbocyanine, an indocarbocyanine, an oxacarbocyanine, a thiacarbocyanine, a merocyanine, a polymethine, a coumarin, a rhodamine, a xanthene, a fluorescein, a borodipyrromethane (BODIPY), VivoTag-680, VivoTag-S750, an AlexaFluor dye (e.g., AlexaFluor 660, AlexaFluor 680, AlexaFluor 700, AlexaFluor 750, AlexaFluor 790), and a DylightFluor dye.

14. R １ 12. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein: is a chelating group that is a residue of a chelating agent.

15. The chelating group may be 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), p-SCN-Bn-NOTA, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), p-SCN-Bn-DOTA (also known as 2B-DOTA-NCS), PIP-DOTA, diethylenetriaminepentaacetic acid (DTPA), PIP-DTPA, AZEP-DTPA, ethylenediaminetetraacetic acid (EDTA), triethylenetetraamine-N,N,N',N'',N''',N'''-hexaacetic 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-1,4,7-triazonin-1-yl}-acetic acid (NPTA), diacetylpyridine bis(benzoylhydrazone), 1,4,7,10,13,16-hexaazacyclooctadecane N,N',N'',N''',N'''',N''''-hexaacetic acid (HEHA), octadentate terephthalamide ligand, 2,2'-(4-(2-(bis(carboxymethyl)amino)-5-(4-isothiocyanatophenyl)pentyl)-10-(2-(bis(carboxymethyl)amino)ethyl)-1,4,7,10-tetradecanoic acid N,N'-bis[(6-carboxy-2-pyridyl)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-NCO), 6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)-4-isothiocyanatopicolinic acid (macropa-NCO),The compound of claim 14, or a pharmaceutically acceptable salt thereof, is a residue of a chelating agent selected from (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-tetraazacyclododec-1-yl]acetamide (TCMC or DOTAM).

16. 15. The compound of claim 14, or a pharmaceutically acceptable salt thereof, wherein the residue of the chelating agent is a residue of macropa-NCS or macropa-NCO.

17. 15. The compound of claim 14, or a pharmaceutically acceptable salt thereof, wherein the residue of the chelating agent is a residue of p-SCN-Bn-NOTA, p-SCN-Bn-DOTA, NOTA, or DOTA.

18. 15. The compound of any one of claims 1 to 11 or 14, or a pharmaceutically acceptable salt thereof, wherein the chelating group is a residue of a siderophore.

19. R １ But, R ５ is a benzoyl group optionally substituted with one or more groups represented by Each R ５ are independently halo, cyano, halomethyl, N ＋ (CH ３ ) ３ W － is selected from: W － is a pharmaceutically acceptable anion; 12. A compound according to any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof.

20. Each R ５ are independently fluoro, cyano, trifluoromethyl, N ＋ (CH ３ ) ３ W － 20. The compound of claim 19, or a pharmaceutically acceptable salt thereof, selected from:

21. R ５ a halo or fluoro group represented by １８ 21. The compound of claim 19 or 20, wherein R is F, or a pharmaceutically acceptable salt thereof.

22. R ２ is H and R ４ 22. The compound of any one of claims 1 to 21, or a pharmaceutically acceptable salt thereof, wherein is CN. 【Request 23】 【Chemical 6-1】 【Chemistry 6-2】 【Chemistry 6-3】 【Chemistry 6-4】 【Chemistry 6-5】 【Chemistry 6-6】 【Chemistry 6-7】 【Chemistry 6-8】 【Chemistry 6-9】 2. The compound of claim 1, represented by a structural formula selected from:

24. 24. The compound of any one of claims 1 to 11, 14 to 18, or 23, or a pharmaceutically acceptable salt thereof, wherein the residue of the chelator is chelated with a radionuclide.

25. The radionuclide is １７７ Lu, １７５ Lu, ４５ Sc, ６４ Cu, ６７ Cu, ６８ Cu, ６６ Ga, ６７ Ga, ６８ Ga, ６９ Ga, ７１ Ga, ９０ Y. ８９ Y. ８６ Y. ８９ Zr, ９０ Y. ９９ｍ Tc, １１１ In, １１３ In, １１５ In, １３９ La, １３４ Ce, １３６ Ce, １３８ Ce, １４０ Ce, １４２ Ce, １５１ EU, １５３ EU, １５２ Dy, １４９ Tb, １５９ Tb, １５４ Gd, １５５ Gd, １５６ Gd, １５７ Gd, １５８ Gd, １６０ Gd, １８８ Re, １８６ Re, ２１３ Bi, ２１１ At, ２１７ At, ２２７ Th, ２２６ Th, ２２５ Ac, ２３３ Ra, １５２ Dy, ２１３ Bi, ２１２ Bi, ２１１ Bi, ２０３ Pb, ２１２ Pb, ２５５ 25. The compound of claim 24, or a pharmaceutically acceptable salt thereof, selected from: Fm, and uranium-230.

26. The radionuclide is ２２５ Ac, ２３３ Ra and ２１２ 25. The compound of claim 24, or a pharmaceutically acceptable salt thereof, which is an alpha-emitting radionuclide such as Pb.

27. the radionuclide is an Auger electron-emitting radionuclide, or １７７ Lu, ９０ Y and ６７ 25. The compound of claim 24, or a pharmaceutically acceptable salt thereof, which is a beta-emitting radionuclide such as Cu.

28. The residue of macropa-NCS or mcaropa-NCO is ２２５ 17. The compound of claim 16, or a pharmaceutically acceptable salt thereof, chelated with Ac.

29. The following structural formula: 【Chemistry 7】 2. The compound of claim 1, represented by: or a pharmaceutically acceptable salt thereof.

30. The following structural formula: 【Chemistry 8】 (Wherein Cu is ６４ Cu) 2. The compound of claim 1, represented by: or a pharmaceutically acceptable salt thereof.

31. 30. A pharmaceutical composition comprising: i) a compound according to any one of claims 1 to 28 or a pharmaceutically acceptable salt thereof; and ii) a pharmaceutically acceptable carrier or diluent.

32. 30. A method of treating diseased tissue in a subject, comprising administering to the subject an effective amount of a compound or pharmaceutically acceptable salt of any one of claims 24 to 28, or a pharmaceutical composition of claim 29, wherein the diseased tissue expresses fibroblast activation protein alpha and the radionuclide is a therapeutic radionuclide.

33. 31. The method of claim 30, wherein the diseased tissue is cancer.

34. 32. The method of claim 31, wherein the cancer is pancreatic cancer, liver cancer, gallbladder cancer, neuroblastoma, breast cancer, ovarian cancer, esophageal cancer, renal cancer, prostate cancer, colorectal cancer, soft tissue sarcoma, osteosarcoma, or melanoma.

35. 32. The method of claim 31, wherein the diseased tissue is fibrotic.

36. 1. A method of imaging an area in a subject having or suspected of having diseased or fibrotic tissue that expresses fibroblast activation protein alpha, comprising: a) administering to a subject a diagnostically effective amount of a compound or a pharmaceutically acceptable salt thereof of any one of claims 12-13, 19-22, or 24-28, or a pharmaceutical composition of claim 29, wherein the radionuclide is a diagnostic radionuclide; b. exposing the area in the subject to an imaging device; and c. obtaining an image of said diseased tissue in said region. A method comprising:

37. 34. The method of claim 33, wherein the area contains or is suspected of containing diseased tissue containing a primary cancer or a metastasis of a cancer.

38. 34. The method of claim 33, wherein the area has or is suspected of having diseased tissue, including fibrous tissue.

39. 1. A method of imaging a tumor, comprising: a. contacting a tumor and / or surrounding tissue with a compound of claim 12 or 13, or a pharmaceutically acceptable salt thereof, in an amount sufficient to bind to the tumor; b. irradiating the tumor and / or surrounding tissue at a wavelength absorbed by the compound; and c. Detecting a signal from said compound, thereby imaging said tumor and / or surrounding tissue. A method comprising:

40. 1. A method of treating diseased tissue, comprising: 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 affected tissue; b. irradiating the area of bound compound with one or more doses of external beam radiation using said compound as a location reference; thereby treating said diseased tissue with radiation; A method comprising:

41. 39. The method of claim 38, wherein the compound comprises a chelating group having a radionuclide that emits gamma rays or positrons, or an optical dye or fluorophore, or other detectable radiation.

42. 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 location reference for guided surgical applications to remove a region of the diseased tissue, thereby excising the diseased tissue.

43. 40. The method of claim 39, wherein the compound comprises a chelating group having a radionuclide that emits gamma rays or positrons, or an optical dye or fluorophore, or other detectable radiation.

Images