Compositions targeting fibroblast activating proteins and methods of use thereof

Abstract

Disclosed herein are compounds, compositions, and methods for imaging and treating tumor cells in a subject. In particular, a compound having the structure of Formula I is used in the disclosed imaging method: # imgabs0 # formula (I) wherein the variables are as defined herein.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 392,899, filed on July 28, 2022, the entire teachings of which are incorporated herein by reference. Technical Field

[0003] The present technology relates to targeted imaging and therapeutic agents, and more specifically, to compounds that can be used for diagnosis and treatment of diseases. For example, the compositions described herein can be used as radiopharmaceuticals, or conjugated with optical dyes or fluorophores, or used as drug / toxin conjugates for diagnosis and treatment of cancer and fibrotic diseases in tissues. Background Art

[0004] Fibroblast activation protein alpha ("FAP") is a 170 kDa type II membrane-bound enzyme that exhibits serine protease activity. A soluble form of FAP that lacks the intracellular and transmembrane sequences of the full-length protein is present in plasma. Other common names for FAP include prolyl endopeptidase and surface-expressed protease (seprase).

[0005] FAP is one of several members of the S9B prolyl oligopeptidase subfamily, which includes proteins such as DPP4, DPP8, and DPP9. FAP substrates include neuropeptide Y, peptide YY, substrate P, B-type natriuretic peptide, fibroblast growth factor 21 (FGF-21), α2 antiplasmin, and denatured collagens I and III.

[0006] FAP is actively expressed in tissues undergoing wound healing and remodeling, but is absent or expressed at very low levels in healthy, mature tissues. Tumors are localized areas of host tissue damage and are active in remodeling the local vasculature and endothelium, among several other phenomena that help hide the tumor from immune surveillance and promote wound healing, a microenvironment that allows tumor cells to proliferate. Therefore, FAP expression is associated with areas of tumorigenic tissue, particularly the tumor stroma, and therefore provides an excellent molecular target for the diagnosis and treatment of various cancers. Its expression has been demonstrated in many cancers, such as pancreatic cancer, liver cancer, gallbladder cancer, neuroblastoma, breast cancer, ovarian cancer, esophageal cancer, kidney cancer, melanoma, and many other deadly and aggressive tumors and cancer types. FAP expression is also detected in fibrotic tissue and can also be used as a marker for a wide range of clinical conditions, including systemic fibrotic diseases such as systemic sclerosis (SSc), sclerosing skin graft-versus-host disease and nephrogenic systemic fibrosis as well as a variety of organ-specific disorders including radiation-induced fibrosis and fibrosis of the heart, lung, liver (such as NAFLD: non-alcoholic fatty liver disease and NASH: non-alcoholic steatohepatitis) and kidney.

[0007] Although some researchers have explored FAP as a cancer target, the development of agents that could exploit it as a diagnostic or even therapeutic has proven to be of only marginal benefit due to its 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 shown by prior art compounds in vivo. Improved FAP binders that exhibit better binding kinetics and biodistribution are needed to provide a basis for improved diagnostic and therapeutic agents targeting FAP, i.e., compounds that accumulate to a higher degree in tumors and are not unacceptably taken up in normal non-target tissues and organs. Summary of the invention

[0008] Disclosed herein is a series of compounds that bind to the extracellular domain of FAP and show high to very high affinity for 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 can be used with positron emitters such as 18 F. 68 Ga or 64 Cu is radiolabeled and used for positron emission tomography (PET) (see Example 11). Alternatively, the compounds can be labeled with alpha-particle emitters such as 225 Ac, beta particle emitters such as 67 Cu or 177 Lu or Auger electron emitters (e.g., 111 In, 67 Ga, 99 mTc, 195 mPt, 125 I and 123 1) radiolabeling. The compound can also be attached to a cytotoxic agent, which is used to target the cytotoxic agent to the tumor, for example, conjugated to gemcitabine or doxycycline or venom. Similarly, the compound can be conjugated to a compound with a physiological effect, such as a TLR agonist, to stimulate the immune response of the receptor. The disclosed compound has the advantage of increasing the circulation residence time, which has the effect of increasing the target load while reducing the accumulation of the compound in non-target tissues (see Example 15). Therefore, the favorable binding kinetics of the compounds disclosed herein reduce the "washout" effect (i.e., low residence time) of the prior art FAP targeting compounds.

[0009] One embodiment of the present invention is a compound represented by the following structural formula (I):

[0010]

[0011] or a pharmaceutically acceptable salt thereof, wherein:

[0012] n is 0 or 1;

[0013] A is NH, O, S or CR 6 R 7 ;

[0014] B comprises a branched, unbranched or cyclic aliphatic group of up to 30 carbon atoms, which is optionally interrupted by a peptidyl chain of up to 10 heteroatoms or up to 20 amino acid residues (e.g., 3-20 or 3-15 carbon atoms, which is optionally interrupted by up to 6 heteroatoms or up to 5 amino acid residues), wherein B is optionally substituted by 1-5 groups selected from the group consisting of F, Cl, Br, I, =O, OR 6 ,OCOR 6 、COOR 6 、CN、=NR 6 NR 6 R 7 , =S and SR 6 , provided that B contains at least 3 atoms in the chain between the group D and the group A;

[0015] D is selected from: OPO3H2, PO3H2, OSO3H, SO3H and COOH or C1-C4 alkyl esters thereof;

[0016] X is O or S;

[0017] R 1 is a chelating group, an optical dye or a fluorophore, a cytotoxic agent, an immunostimulant, or optionally substituted by one or more R 5 The group represented by is substituted benzoyl;

[0018] R 3 is a C1-C8 alkyl group or a C1-C4 aralkyl group, wherein:

[0019] The aryl portion of the alkyl and aralkyl groups are each optionally and independently replaced by F, Cl, Br, I, a branched, unbranched or cyclic C1-C6 aliphatic group, OR 6 ,OCOR 6 ,COOR 6 , CHO, COR 6 , CH2OR 6 , NR 6 R 7 , CH2NR 6 R 7 , SR 6 , =O, =S and =NH substituted;

[0020] R 4is CN or B(OH)2;

[0021] Each R 5 are independently selected from halogen, cyano, halomethyl, N + (CH3)3W - , where W - is a pharmaceutically acceptable anion; and

[0022] R 6 and R 7 Independently selected from: H or C1-C6 alkyl.

[0023] 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 comprising a chelating group, the chelating group is preferably chelated with a radionuclide.

[0024] Another embodiment of the present invention is a method for treating a subject with diseased tissue expressing fibroblast activation protein alpha. In one aspect, the diseased tissue can be cancer or fibrotic tissue. The method includes administering to the subject an effective amount of a compound disclosed herein or a pharmaceutically acceptable salt. Preferably, the compound for treatment includes a cytotoxic agent, such as a chelating group having a radionuclide that emits β, α, Auger or other cytotoxic rays that can kill diseased tissue.

[0025] Yet another embodiment of the present invention is a method of imaging a region in a subject having or suspected of having diseased tissue or fibrotic tissue expressing fibroblast activation protein alpha, comprising:

[0026] 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;

[0027] b. exposing an area in the subject to an imaging device, the area suspected of having diseased tissue; and

[0028] c. Obtain an image of the diseased tissue in the area.

[0029] Preferably, the compound used for imaging comprises a chelating group with a gamma-ray or positron emitting radionuclide or other detectable radiation. In another aspect, the compound comprises an optical dye or fluorophore whose emission can be detected.

[0030] Another embodiment of the present invention is a method for imaging a tumor. The method comprises:

[0031] 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 a tumor and / or surrounding tissue;

[0032] b. irradiating the tumor and / or surrounding tissue at a wavelength absorbed by the binding compound;

[0033] c. and detecting signals from the irradiated bound compound, thereby imaging the tumor and / or surrounding tissue.

[0034] Preferably, the compound used for imaging contains a chelating group with a gamma-ray or positron-emitting radionuclide, or an optical dye or fluorophore, or other detectable radiation.

[0035] Yet another embodiment of the present invention is a method for treating diseased tissue. The method comprises:

[0036] a. administering to a subject a compound disclosed herein or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof in an amount effective to contact and bind to the diseased tissue;

[0037] b. using the compound as a fiducial, irradiating the area of ​​the bound compound using one or more doses of external irradiation,

[0038] Irradiation is thereby used to treat diseased tissue.

[0039] Preferably, the compound used as a benchmark contains a chelating group with a gamma-ray or positron-emitting radionuclide, or an optical dye or fluorophore, or other detectable radiation.

[0040] In yet another embodiment of the present invention is a method for treating diseased tissue. The method comprises:

[0041] a. administering to a subject a compound disclosed herein or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof in an amount effective to contact and bind to the diseased tissue;

[0042] b. using the compound as a benchmark to guide surgical application to remove areas of diseased tissue,

[0043] This removes the diseased tissue.

[0044] Preferably, the compound used as a benchmark contains a chelating group with a gamma-ray or positron-emitting radionuclide, or an optical dye or fluorophore, or other detectable radiation. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1AB shows the expression of FAP in BALB / c nude mice that had been transplanted with U-87 (human glioblastoma) cells expressing FAP. 18 F] Compound biodistribution of RTX-1312S.

[0046] Figure 2 Shown is the organ biodistribution of [Cu-67]RTX-1363S in BALB / C nude mice previously transplanted with U-87MG cells.

[0047] Figure 3 Shown is the organ biodistribution of [Lu-177]RTX-1354S in BALB / C nude mice previously transplanted with U-87MG cells.

[0048] Figure 4 Shown is the organ biodistribution of [Lu-177]RTX-1359R in BALB / C nude mice previously transplanted with U-87MG cells.

[0049] Figure 5 Shown is a comparison of [Ac-225]RTX-1399R biodistribution in tumors compared to blood of tumor-bearing JAX nude mice. DETAILED DESCRIPTION

[0050] Disclosed herein is a series of compounds that bind to the extracellular domain of FAP with high affinity and are capable of delivering payloads to tissues expressing FAP. The compounds of the present invention are as described herein below.

[0051] The first embodiment of the present invention is a compound represented by structural formula (I) or a pharmaceutically acceptable salt thereof. The variables in structural formula (I) are as defined in the above summary of the invention.

[0052] The second embodiment of the present invention is a compound represented by structural formula (II):

[0053]

[0054] or a pharmaceutically acceptable salt thereof, wherein m is an integer from 0 to 12; o is 0 or 1; and R 2 is H or C1-C4 alkyl; and the remaining variables are as described in the first embodiment.

[0055] The third embodiment of the present invention is a compound represented by structural formula (III):

[0056]

[0057] or a pharmaceutically acceptable salt thereof; wherein the variables are as described in the second embodiment.

[0058] The fourth embodiment of the present invention is a compound represented by structural formula (IV):

[0059]

[0060] or a pharmaceutically acceptable salt thereof; wherein the variables are as described in the second embodiment.

[0061] The fifth embodiment of the present invention is a compound represented by structural formula (I), (II), (III) or (IV), or a pharmaceutically acceptable salt thereof, wherein R 3 is C1-C8 alkyl or C1-C4 aralkyl optionally substituted with C1-C4 alkyl, and the remaining variables are as described in the first or second embodiment. Alternatively, R 3 It is methyl, propyl, pentyl, heptyl, (4-isobutylphenyl)methyl, (4-isobutylphenyl)propyl.

[0062] A sixth embodiment of the present invention is a compound represented by structural formula (I), (II), (III) or (IV), or a pharmaceutically acceptable salt thereof, wherein m is 3 to 12 and o is 1. Alternatively, m is 8 and o is 1. In another alternative, o is 0. The remaining variables are as described in the first, second or fifth embodiment.

[0063] The seventh embodiment of the present invention is a compound represented by structural formula (I), (II), (III) or (IV), or a pharmaceutically acceptable salt thereof, wherein n is 1 and o is 0; and the remaining variables are as described in the first, second, fifth or sixth embodiment.

[0064] The eighth embodiment of the present invention is a compound represented by structural formula (I), (II), (III) or (IV), or a pharmaceutically acceptable salt thereof, wherein R 1 is a fluorophore or an optical dye; and the remaining variables are as described in the first, second, fifth, sixth or seventh embodiment. In the first aspect, the fluorophore is And the optical dyes are carbocyanine, indolecarbocyanine, oxycarbocyanine, thiocarbocyanine, merocyanine, polymethine, coumarin, rhodamine, xanthene, fluorescein, boron dipyrromethene (BODIPY), VivoTag-680, VivoTag-S750, AlexaFluor dyes (e.g., AlexaFluor660, AlexaFluor680, AlexaFluor700, AlexaFluor750, AlexaFluor790) and DylightFluor dyes.

[0065] The ninth embodiment of the present invention is a compound represented by structural formula (I), (II), (III) or (IV), or a pharmaceutically acceptable salt thereof, wherein R 1 is a chelating group which is the residue of a chelating agent; and the remaining variables are as described in the first, second, fifth, sixth or seventh embodiment. Suitable chelating agents and chelating agent residues are described below.

[0066] The tenth embodiment of the present invention is a compound represented by structural formula (I), (II), (III) or (IV), or a pharmaceutically acceptable salt thereof, wherein R 1 is a benzoyl group, which is optionally substituted by one or more R 5 The group represented by 5 are independently selected from halogen, cyano, halomethyl, N + (CH3)3W - ; and W - is a pharmaceutically acceptable anion; and the remaining variables are as described in the first, second, fifth, sixth or seventh embodiment. In the first aspect, R 5 The halogen group represented by 18 F.

[0067] The eleventh embodiment of the present invention is a compound represented by structural formula (I), (II), (III) or (IV), or a pharmaceutically acceptable salt thereof, wherein each R 5 are independently selected from fluorine, cyano, trifluoromethyl, N + (CH3)3W - ; and the remaining variables are as described in the first, second, fifth, sixth, seventh or tenth embodiment. In the first aspect, R 5 The fluorine group represented by 18 .

[0068] The twelfth embodiment of the present invention is a compound represented by structural formula (I), (II), (III) or (IV), or a pharmaceutically acceptable salt thereof, wherein R 2 It is H and R 4 is CN; and the remaining variables are as described in the first, second, fifth, sixth, seventh, eighth, ninth, tenth or eleventh embodiment.

[0069] The present invention also includes the compounds whose preparation is described in the examples and shown in the figures, including both pharmaceutically acceptable salts and neutral forms thereof. For those compounds containing a chelating group, chelation with a radionuclide is also included in the present invention.

[0070] The present invention also includes the compounds shown below, including both pharmaceutically acceptable salts and neutral forms thereof. Chelation with radionuclides is also included in the present invention.

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080] Examples of Optical and Near-IR Imaging Compounds

[0081] Nomenclature in which the isotope is preceded by the compound name indicates that the isotope is chelated to the chelating group of the compound. For example, "[Cu-67]RTX-1363S" refers to RTX-1363S in which the chelating group is chelated to 67Cu.

[0082] Exemplary compounds of the invention (having chelating groups thereof) include [68Ga]RTX-1339S; [68Ga]RTX-1340S; [68Ga]RTX-1363S; [68Ga]RTX-1367S; [64Cu]RTX-1339S; [64Cu]RTX-1340S; [64Cu]RTX-1363S; [64Cu]RTX-1367S; [67Cu]RTX-1339S; [67Cu]RTX-1340S; [67Cu]RTX-1363S; [67Cu]RTX-1367S; [Al18F]RTX-1339S; [Al18F]RTX-1340S; [Al18F]RTX-1363S; and [Al18F]RTX-1367S.

[0083] Other exemplary compounds of the invention (having chelating groups thereof) include [68Ga]RTX-1350S; [68Ga]RTX-1352S; [68Ga]RTX-1353S; [68Ga]RTX-1354S; [68Ga]RTX-1355S; [68Ga]RTX-1356R; [68Ga]RTX-1357R; [68Ga]RTX-1358R; [68Ga]RTX-1359R; [68Ga]RTX-1360S; [68Ga]RTX-1360R; [177Lu]RTX-1350S; [177Lu]RTX-1352S; [177Lu]RTX-1353S; [177Lu]RTX-1354S; [177Lu]RTX-1355S; [177Lu]RTX-1356R; [177Ga]RTX-1357R; [177Ga]RTX-1358R; [68Ga]RTX-1359R; [68Ga]RTX-1360S; [68Ga]RTX-1360R; [177Lu]RTX-1350S; [177Lu]RTX-1352S; [177Lu]RTX-1353S; [177Lu]RTX-1354S; [177Lu]RTX-1355S; [177 7Lu]RTX-1356R;[177Lu]RTX-1357R;[177Lu]RTX-1358R;[177Lu]RTX-1359R;[177Lu ]RTX-1360S;[177Lu]RTX-1360R;[225Ac]RTX-1350S;[225Ac]RTX-1352S;[225Ac]RTX -1353S;[225Ac]RTX-1354S;[225Ac]RTX-1355S;[225Ac]RTX-1356R;[225Ac]RTX-135 7R; [225Ac]RTX-1358R; [225Ac]RTX-1359R; [225Ac]RTX-1360S; and [225Ac]RTX-1360R.

[0084] Another exemplary compound of the invention (having a chelating group thereof) includes [AL18F]RTX-1312S.

[0085] Compounds with Macropa chelators are typically chelated with 225Ac. Thus, another compound of the invention (with its chelating group) includes [Ac225]RTX-1399R.

[0086] "Aliphatic" means a saturated or unsaturated straight or branched monovalent or divalent hydrocarbon radical. Unless otherwise specified, aliphatic groups generally have 1 to 10 carbon atoms. "Alkyl" means a saturated aliphatic straight or branched monovalent aliphatic radical. Unless otherwise specified, an alkyl group generally has 1 to 10 carbon atoms (C 1-10 alkyl), alternatively, 1 to 6 carbon atoms (C 1-3 alkyl) (ie, 1, 2 or 3).

[0087] "Cycloaliphatic" means a saturated or unsaturated monovalent or divalent cyclic hydrocarbon ring radical. Unless otherwise specified, a cycloaliphatic radical has 3 to 8 ring carbon atoms (C3-8 Cycloalkyl). "Cycloalkyl" means a saturated aliphatic cyclic aliphatic. Unless otherwise specified, a cycloalkyl group has 3 to 8 ring carbon atoms.

[0088] "Aryl", alone or as a portion or more of a moiety such as "aralkyl", is a carbocyclic aromatic group such as phenyl or naphthyl.

[0089] Compounds with one or more chiral centers can exist in various stereoisomeric forms, i.e. each chiral center can have the 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. Diastereoisomers are stereoisomers with two or more chiral centers that are not identical and are not mirror images of each other.

[0090] When the stereochemical configuration of a chiral center in a compound having one or more chiral centers is described by its chemical name (e.g., where the configuration is represented by "R" or "S" in the chemical name) or structure (e.g., where the configuration is represented by a "wedge-shaped" bond), the enrichment of the indicated configuration relative to the opposite configuration is greater than 50%, 60%, 70%, 80%, 90%, 99%, or 99.9%. The "enrichment of the indicated configuration relative to the opposite configuration" is a molar percentage determined by dividing the number of compounds having the indicated stereochemical configuration at the chiral center by the total number of all compounds in the mixture having the same or opposite stereochemical configuration.

[0091] When a disclosed compound having a chiral center is described by a structure that does not show the configuration at the chiral center, the structure is meant to encompass compounds having the S configuration at the chiral center, compounds having the R configuration at the chiral center, or compounds having a mixture of R and S configurations at the chiral center. When a disclosed compound having a chiral center is described by its chemical name, without using "S" or "R" to indicate the configuration of the chiral center, the name is meant to encompass compounds having the S configuration at the chiral center, compounds having the R configuration at the chiral center, or compounds having a mixture of R and S configurations at the chiral center.

[0092] The targeted FAP compounds of the present invention are useful imaging agents for diagnostic applications. For example, these can be conjugated to various metals for magnetic resonance imaging applications, or to optical dyes or fluorophores, or other detectable moieties (i.e., dyes, quantum dots, etc.) for histochemical applications and luminescent imaging applications. Likewise, these compounds can be radiolabeled and used in nuclear medicine applications. Radionuclides useful for imaging applications are referred to herein as "imaging radionuclides." Non-limiting examples of imaging radionuclides include 18 F. 64 Cu or68 Ga, which is suitable for PET imaging applications, and 67 Cu or 177 Lu, which is typically a therapeutic nucleotide, is also suitable for SPECT imaging applications.

[0093] The targeted FAP compounds of the present invention are useful therapeutic compounds. Such therapeutic compounds include targeted FAP compounds of the present invention having a suitable therapeutic portion. The targeted FAP compound can be separated from the therapeutic portion by a covalent linker. The interval between these (based on continuous atom counts) can be about 4 atoms to about 100 atoms. In addition, by including additional targeting structures on the compound, the pharmacokinetics of the compound can be changed. For example, the use of a blood-targeted portion can increase the circulation residence time, which has the effect of increasing tumor perfusion and load while reducing the accumulation of radiotherapeutic compounds in non-target tissues. See, for example, U.S. Patent No. 11,285,277, which describes a trifunctional ("Trillium") compound having a tumor-targeting domain, a blood protein binding domain, and a third domain constituting a cell-killing or cell growth-inhibiting therapeutic agent. In a currently preferred embodiment, the FAP-binding compound of the present invention can be bound to the Trillium scaffold by a covalent bond to constitute the tumor targeting domain of such a Trillium agent targeting FAP. Exemplary constructs include drug conjugates with toxins, venoms, metabolic poisons, or chemotherapeutic drugs, as well as radiotherapeutic compounds with alpha-, beta-, or Auger electron-emitting radionuclides or that emit a spectrum of radiation upon decay, including positron emission which is also suitable for diagnostic uses.

[0094] For radiotherapy, the targeted FAP compound is conjugated to a chelator, which is selected based on its suitability to accommodate 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 the target tissue (i.e., cancer and tumor microenvironment expressing FAP, malignant tumors, and fibrotic cells). Although targeted radiotherapy has been available for some time using macrocyclic complexes of radionuclides, the macrocycles currently used (e.g., DOTA) typically form complexes with many therapeutic radionuclide metals, such as actinium, radium, bismuth, astatine, lutetium, and lead isotopes. The instability of many known macrocyclic-containing compounds can result in the dissociation of the radionuclide from the macrocycle, which results in a lack of selective delivery to the intended target tissue, which can also result in toxicity to non-target tissues. α-emitting radionuclides, such as 225Ac, which can provide greater cytotoxic effects, is therefore considered more effective than beta-emitting radionuclides. However, this toxin requires a chelator to increase the retention of the chelated metal. U.S. Patent No. 11,279,698 (see also PCT / US2018 / 025488 and PCT / US2019 / 062479) describes a new chelator ("Macropa") and its use as a 225 Use of Ac chelators, including as components of Trillium PK-modulated targeted radiotherapeutics. A ratio of tumor activity to kidney activity of 1 or greater can be sustained for up to about 36 hours after administration of the radiotherapeutic, and in the case of 225Ac Trillium-based therapeutics, can be sustained for 72 hours or even 128 hours or longer to maximize the therapeutic effect of irradiation on target tissues.

[0095] Thus, an exemplary preferred Trillium compound targeting FAP will have a chelator in its third (non-targeting) domain. Macropa is currently targeted 225 Trillium compounds of Ac-FAP are preferred chelators. (See also, PCT / CA2021 / 050226).

[0096] The radionuclides that can be used in the compounds disclosed herein depend on the application, the type of radiation required, and the half-life, which will be apparent to those skilled 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, 152Dy, 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 Fm and uranium-230. Depending on their decay properties, the radionuclides of any embodiment herein can be either therapeutic radionuclides or diagnostic radionuclides. Currently, preferred α-emitting radionuclides for therapeutic applications include 225 Ac, 233 Ra and 212 Pb. Preferred beta-emitting radionuclides for therapeutic applications include 177 Lu, 90 Y and 67 Cu.

[0097] Chelating groups and azamacrocyclic polycarboxylic acids useful in the present technology include and refer to groups that are capable of chelating, binding or otherwise delivering a radionuclide to a therapeutic or diagnostic target. The chelating group is the residue after a chelating agent reacts with a nucleophilic group in a compound to form a targeted divalent 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 lysinyl group in the penultimate precursor, which 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 groups: 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), triethylenetetramine-N,N,N',N",N"',N"'-hexaacetic acid (TTHA), 7-[2- [(bis(carboxymethylamino)-ethyl]-4,10-bis(carboxymethylamino)-1,4,7,10-tetraazacyclododecane-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-triazacyclononane-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-diazacyclooctadecane-7-yl)methyl)-4 -isothiocyanatopicolinic acid (macropa-NCS), 6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)-4-isocyanatopicolinic acid (macropa-NCO), 3,9-carboxymethyl-6-(2-methoxy-5-isothiocyanatophenyl)carboxymethyl-3,6,9,15-tetraazabicyclo-[9.3.1]pentadeca-1(15),11,13-triene and 2-[4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1-yl]acetamide (TCMC or DOTAM).In a currently preferred embodiment, the chelating agent is the residue of a polyazapolycarboxylic acid macrocycle, such as Macropa NCS or NCO-Macropa. In another aspect, the chelating agent is the residue of a siderophore, in one aspect. 225 Ac is a radionuclide against MacropaNCS or NCO-Macropa. In another aspect, the chelator is a residue of p-SCN-Bn-DOTA, p-SCN-Bn-NOTA, NOTA or DOTA. In another embodiment, the chelator is a sarcophagene chelator (compounds of the invention having sarcophagene chelators can be prepared according to the procedures disclosed in WO2021225760, all of which are incorporated herein by reference). In another aspect, the chelator is 68 The residues of p-SCN-Bn-DOTA, p-SCN-Bn-NOTA, NOTA or DOTA chelated with Ga.

[0098] As described above, the disclosed compound or a pharmaceutically acceptable salt thereof may contain one or more radionuclides suitable for use as a radioactive imaging agent. Imaging methods include positron emission tomography (PET) or single photon emission computed tomography (SPECT). Therefore, in another aspect, the present invention provides therapeutic applications, i.e., administering an effective amount of a disclosed compound (or a pharmaceutically acceptable salt thereof) with a chelating agent to a subject suffering from cancer, a tumor or a fibrotic disease, which is complexed with an imaging radionuclide for imaging applications, and administering an effective amount of a compound complexed with a therapeutic radionuclide for treatment. Exemplary cancers that can be imaged and / or treated using the disclosed compound or a pharmaceutically acceptable salt thereof include pancreatic cancer, liver cancer, gallbladder cancer, neuroblastoma, breast cancer, ovarian cancer, esophageal cancer, kidney cancer, prostate cancer, colorectal cancer, soft tissue sarcoma, osteosarcoma or melanoma.

[0099] A "subject" is a mammal in need of medical treatment or diagnosis, preferably a human, but may also be an animal in need of veterinary treatment, for example, companion animals (e.g., dogs, cats, etc.), farm animals (e.g., cows, sheep, pigs, horses, etc.), and laboratory animals (e.g., rats, mice, guinea pigs, etc.).

[0100] The disclosed compounds or pharmaceutically acceptable salts thereof (including chelation with radionuclides) or pharmaceutical compositions thereof can be administered orally or by a parenteral route (usually injection or infusion). "Parenteral administration route" means modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0101] An "effective amount" of a disclosed compound or a pharmaceutically acceptable salt thereof (including chelation with a radionuclide) means an amount of a therapeutic agent used alone or in combination with other therapies that provides a therapeutic benefit in treating disease prevention, improves overall therapy, reduces or avoids disease symptoms or causes, or enhances the therapeutic effect of another therapeutic agent or synergizes with another therapeutic agent. With respect to imaging, a "diagnostally effective amount" refers to an amount that can produce a useful image for diagnosis, 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, severity of disease, etc. The dosage is preferably 0.00001 mg / kg to 100 mg / kg body weight.

[0102] Corresponding to the type of administration, the medicament is appropriately formulated, for example, in the form of solutions or suspensions, simple tablets or dragees, hard or soft gelatin capsules, suppositories, ovules, injection preparations, which are prepared according to conventional galenic methods.

[0103] When using infusion or injection solution, it is preferably an aqueous solution or suspension, which can be produced before use, for example, by a lyophilized preparation containing the active substance itself or with a carrier (such as mannitol, lactose, glucose, albumin, etc.). The pre-made solution is sterilized and mixed with an excipient, for example, with a preservative, stabilizer, emulsifier, solubilizer, buffer and / or salt, to adjust the osmotic pressure. Sterilization can be achieved by aseptic filtration using a filter with a pore size, and lyophilization can be performed in appropriate cases according to its composition. A small amount of antibiotic can also be added to ensure that a sterile state is maintained.

[0104] According to another aspect, a pharmaceutical composition suitable for in vivo imaging and / or radiotherapy of target tissue is provided. A suitable pharmaceutical composition may comprise a radioactive imaging agent together with a pharmaceutically acceptable radioactive carrier, the radioactive imaging agent having a radionuclide as an element (i.e., 18 F), or diagnostic radiometal chelate complexes (e.g., 64 Cu or 68Ga), or a radioactive therapeutic agent, which is a radioactive metal chelate complex in an amount sufficient to bind to the target tissue. The radioactive carrier should be suitable for injection or aspiration, such as human serum albumin; aqueous buffer solutions, for example, tris (hydroxymethyl) aminomethane (and its salts), phosphates, citrates, bicarbonates, etc.; sterile water saline; and balanced ion solutions containing chloride and or bicarbonate or normal plasma warnings such as calcium, potassium, sodium and magnesium.

[0105] The concentration of the radiopharmaceutical in the radioactive carrier should be sufficient to provide reasonable binding to the target tissue, such as about 4% to 40% ID / gram. For example, when using an aqueous solution, a human dose can be in the range of about 1.0 to 500 millicuries of activity. However, the actual dose administered to the patient for imaging or therapeutic purposes is determined by the physician administering the treatment. The administration of the imaging agent or therapeutic agent should allow it to stay in the patient for about 1 hour to 10 days, but longer and shorter times are acceptable. Therefore, a convenient ampoule containing 1 to 10 mL of an aqueous solution can be prepared.

[0106] Imaging can be performed in the normal 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 instrument (such as a tomography scanner or a gamma camera). In certain embodiments, a method for imaging an area in a patient comprises the steps of: (i) administering to the patient a diagnostically effective amount of a compound complexed with a radionuclide; exposing an area of ​​the patient to a scanning device; and (ii) obtaining an image of the area of ​​the patient. Thus, the present invention provides a method for obtaining an image of a mammalian subject after administration of the compound. Similarly, imaging can be performed after administration of a therapeutic drug or a cycle of radiotherapy to assess efficacy. Thus, obtaining an image after administration of a radiotherapeutic agent can occur after about 1 hour, about 4 hours, about 9 hours, about 12 hours, about 16 hours, about 20 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 96 hours, about one week, about two weeks, about four weeks, or after completing a treatment cycle. Thus, in some embodiments, a method of imaging a tissue, such as a tumor tissue expressing FAP, is provided, comprising contacting the tissue with a complex synthesized by contacting an imaging radionuclide with a disclosed compound.

[0107] According to another aspect, the disclosed compound or a complex of a pharmaceutically acceptable salt thereof may contain one or more radionuclides suitable for use as a radioactive imaging agent in the field of image-guided radiation therapy (IGRT). As described in U.S. Pat. No. US10688320B2, IGRT uses images collected before a course of treatment to guide the application of therapeutic irradiation during the course of treatment. The concentration of the imaging agent or therapeutic agent in the radiation carrier should be sufficient to provide satisfactory imaging. For example, when an aqueous solution is used, the dose is about 1.0 to 100 millicuries. Imaging can be performed to provide a benchmark for guiding the target area to receive a calculated irradiation flux from a therapeutic irradiation source. Similar uses of compounds as benchmarks can be used to guide surgical applications.

[0108] The amount of the compounds of the invention or formulations comprising a complex of a metal with the compound or a pharmaceutically acceptable salt thereof administered to a patient will depend upon several physiological factors routinely used by the physician, including the nature of the procedure to be performed, the volume of target tissue to be imaged or treated, and the weight and medical history of the patient to be imaged or treated using these compounds.

[0109] The examples provided herein are to illustrate the advantages of the present technology and to further help those of ordinary skill in the art to prepare or use the compounds or salts thereof, pharmaceutical compositions, derivatives, prodrugs or tautomeric forms of the present technology. The examples herein are also to more fully illustrate the preferred aspects of the present technology. These embodiments should never be construed as limiting the scope of the present technology defined by the appended claims. These embodiments may include or be combined with any variant, aspect or embodiment of the above-mentioned present technology. The above-mentioned variants, aspects or embodiments may also further each include or be combined with any or all other variants, aspects or embodiments of the present technology.

[0110] Example

[0111] 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-quinolinyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-[3-(p-{[1,4,7-tris(carboxymethyl)-1,4,7-triazolin-2-yl]methyl}phenyl)thioureido]hexanoylamino]-6-acetylaminohexanoic acid.

[0112] Synthesize RTX-1312S from fragments:

[0113]

[0114] Synthesis scheme of intermediate-1:

[0115]

[0116] 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.

[0117] Procedure 1: Preparation of tert-butyl (S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((tert-butoxycarbonyl)amino)hexanamide)-6-(((benzyloxy)carbonyl)amino)hexanoate:

[0118] 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 added slowly. The stirred reaction mixture was monitored by LC-MS RM for 4 h at room temperature. The mixture was diluted with DCM and the organic layer was washed sequentially 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 combined flash silica gel chromatography (DCM and EtOAc eluent) to give the pure product (6.0 g, yield 77%).

[0119] Procedure 2: Preparation of (S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((tert-butoxycarbonyl)amino)hexanamide)-6-aminoacetic acid tert-butyl hexanoate:

[0120] 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)hexanamide)-6-(((benzyloxy)carbonyl)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, based on acetate), Fmoc deprotection was also observed during the hydrogenation (~5%). The crude product was pure enough to be used in the next step without further purification.

[0121] Procedure 3: Preparation of (S)-6-acetylamino-2-[(S)-6-tert-butoxycarbonylamino-2-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoylamino]-hexanoic acid tert-butyl ester:

[0122] 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 min, at which time LCMS indicated complete conversion. The reaction mixture was diluted with EtOAc, washed with water and brine, dried over anhydrous MgSO4 and filtered. The volatiles were evaporated under reduced pressure to give the title compound (2.9 g, 91%) as an off-white solid.

[0123] Procedure 4: Preparation of (S)-6-acetylamino-2-((S)-2-amino-6-tert-butoxycarbonylamino-hexanoylamino)-hexanoic acid tert-butyl ester:

[0124] To a stirred solution of (S)-6-acetylamino-2-[(S)-6-tert-butoxycarbonylamino-2-(9H-fluorene-9-ylmethoxycarbonylamino)-hexanoylamino]-tert-butyl hexanoate (2.9 g, 4.2 mmol) in DCM (12 ml) was added diethylamine (6.51 ml, 62.59 mmol) at ambient temperature. After 45 min, complete conversion was detected by LCMS. The solvent was evaporated under reduced pressure and the residue was dissolved in DCM and purified on a CombiFlash device (220 g silica gel column, gradient 0-50% MeOH / DCM for 100 minutes). The desired fractions were combined and evaporated to produce the target material (1.66 g, 84%) as an amorphous colorless solid.

[0125] 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:

[0126] DIEA (1.34 ml, 1.72 mmol) was added to a stirred solution of (S)-6-acetamido-2-((S)-2-amino-6-tert-butoxycarbonylamino-hexanoylamino)-tert-butyl hexanoate (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 point complete conversion was detected by LCMS. The mixture was purified with EtOAc, washed with water (x2) and brine, dried over anhydrous MgSO4 and filtered. The filtrate was evaporated to give the target material (2.84 g, 100%) as an amorphous colorless solid.

[0127] 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:

[0128] To a solution of (S)-6-acetamido-2-[(S)-2-(3-{2-[2-(2-benzyloxycarbonylamino-ethoxy)-ethoxy]-ethoxy}-propionamido)-6-tert-butoxycarbonylamino-hexanoyl-amino]-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 on a Parr apparatus (60 PSI H2) at ambient temperature 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 an amorphous colorless solid.

[0129] Preparation of Intermediate-2: {4-[2-((S)-2-Cyano-pyrrolidin-1-yl)-2-oxo-ethylcarbamoyl]-quinolin-7-yloxy}-acetic acid.

[0130]

[0131] Procedure 7: Preparation of [2-((S)-2-cyano-pyrrolidin-1-yl)-2-oxo-ethyl]-carbamic acid tert-butyl ester:

[0132] 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 reaction mixture was stirred at ambient temperature for 14 h. 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 initial volume and loaded onto a CombiFlash. TM The desired fractions were combined and evaporated to give the desired product (1.1 g, 58%) as an amorphous solid.

[0133] Procedure 8: Preparation of (S)-1-(2-amino-acetyl)-pyrrolidine-2-carbonitrile:

[0134] 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 h. The solvent was removed under reduced pressure to give the tosylate salt of the desired product (1.8 g, yield exceeded theoretical value). This compound was pure enough to carry out the following chemical transformations.

[0135] Procedure 9: Preparation of (S)-N-(2-(2-cyanopyrrolidin-1-yl)-2-oxoethyl)-7-hydroxyquinoline-4-carboxamide:

[0136] 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 min. 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 h while the progress of the reaction was monitored by LCMS. After 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 and loaded onto a CombiFlash. TM Purification on a silica gel column (gradient 0-20% MeOH / DCM) gave the desired product (422 mg, yield 80%) as an off-white solid.

[0137] Procedure 10: Preparation of (S)-tert-butyl 2-((4-((2-(2-cyanopyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-7-yl)oxy)acetate:

[0138] A flask containing a solution of 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 cooled in an ice-water bath. Diisopropyl azodicarboxylate (300 μL, 1.51 mmol) was added dropwise to the cooled 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 TM The residue was purified by silica gel column (gradient 0->10% MeOH / DCM) to afford the desired product (404 mg, yield 61%) as a glassy solid.

[0139] Procedure 11: Preparation of {4-[2-((S)-2-cyano-pyrrolidin-1-yl)-2-oxo-ethylcarbamoyl]-quinolin-7-yloxy}-acetic acid:

[0140] At 0 ° C, to {4- [2- ((S) -2- cyano - pyrrolidine -1- base) -2- oxo - ethylcarbamoyl] -quinoline -7- base oxygen} - tert-butyl acetate (141mg, 0.322mmol) in DCM (1.5ml) solution was added dropwise TFA (1.51ml, 19.64mmol). The temperature of the stirred reaction mixture was warmed to room temperature within 1h, and complete conversion was detected by LCMS. The solvent was evaporated at room temperature, and the residue was co-evaporated with toluene (x3) at 40 ° C. The crude compound intermediate 2 is pure enough for subsequent conversion, but it is unstable and hydrolyzed after long-term placement. For this reason, the compound is now used.

[0141] Preparation of RTX-1312S from fragments:

[0142] Procedure 12: Preparation of (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolinyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-(tert-butoxycarbonylamino)hexanoylamino]-6-acetylaminohexanoic acid tert-butyl ester:

[0143]

[0144] 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]-tert-butyl hexanoate (1.17 g, 1.73 mmol) and HATU (717 mg, 1.89 mmol) in DMF (10 ml). When complete conversion was detected by LCMS, the stirred mixture was kept at room temperature for 30 minutes. 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, loaded onto a CombiFlash silica column (220 g, gradient 0->30% MeOH / DCM over 60 min) and purified. The desired fractions were combined and evaporated to give the target material (950 mg, 58%) as a glassy solid.

[0145] Procedure 13: Preparation of (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolinyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-aminohexanoylamino]-6-acetylaminohexanoic acid:

[0146]

[0147] At 0°C, to a solution of (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolinyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-(tert-butoxycarbonylamino)hexanoylamino]-6-acetylaminohexanoic acid tert-butyl ester (74 mg, 0.071 mmol) in anhydrous DCM (1 ml) was added anhydrous trifluoroacetic acid (1 ml, 13 mmol) dropwise. The temperature of the reaction mixture was warmed to ambient temperature and stirred continuously for 90 minutes, at which point 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 immediately used for the next step of chemical transformation.

[0148] 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-quinolinyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-[3-(p-{[1,4,7-tris(carboxymethyl)-1,4,7-triazolin-2-yl]methyl}phenyl)thioureido]hexanoylamino]-6-acetylaminohexanoic acid.

[0149]

[0150] 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-quinolinyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-aminohexanoylamino]-6-acetylaminohexanoic acid x TFA (0.071 mmol) in DMSO (1300 μl) at ambient temperature, followed by a solution of sodium carbonate (188 mg, 1.78 mmol) in water (1 ml). The pH of the resulting solution mixture was approximately 9. The reaction mixture was stirred at ambient temperature for 14 h 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 / H2O; MP B: 0.1% TFA / buffer B containing 5% acetonitrile for 5 minutes, then 5% to 55% buffer B in 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.

[0151] Synthetic RTX-1317S:

[0152]

[0153] Prepared according to the procedure of Example 1 with the following changes:

[0154] Use program 40 instead of program 3.

[0155] In Procedure 40, butyric acid is used instead of caproic acid.

[0156] Synthetic RTX-1318S:

[0157]

[0158] Prepared according to the procedure of Example 1 with the following changes:

[0159] Use program 40 instead of program 3.

[0160] Synthetic RTX-1319R

[0161]

[0162] Prepared according to the procedure of Example 1 with the following changes:

[0163] Use procedures 52-54 instead of procedure 12.

[0164] Before subjecting the intermediate to procedures 52-54, the chelating agent was installed using procedures 13 and 14.

[0165] Synthetic RTX-1341S:

[0166]

[0167] Prepared according to the procedure of Example 1 with the following changes:

[0168] Use procedures 15 and 16 instead of procedure 14 for conjugation of optical dyes or fluorophores.

[0169]

[0170] 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:

[0171]

[0172] To a solution of ZW800-1 (100 mg, 0.105 mmol) in anhydrous DMSO (10 ml) was added dipyrrolidino (N-succinimidyloxy) carbonium hexafluorophosphate (130 mg, 0.3 mmol) at room temperature, followed by dropwise addition of N,N-diisopropylethylamine (0.2 mL, 1.1 mmol). 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 1:1:1 solution of ethanol:ethyl acetate:acetone (150 mL) and 0.1% trifluoroacetic acid (0.9 mL), and allowed to mix and stand for 30 min. 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 in the next step without further purification. LCMS: C 46 H 61 N9O 12 :m / z:1040.32,measured value m / z=1040.5[M] + .

[0173] Procedure 16: Final steps to synthesize RTX-1341S:

[0174]

[0175] 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-quinolinyloxy]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 stirred for 16 h. 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,measured value m / z=1811.4[M+H] + .

[0176] Synthetic RTX-1370S:

[0177]

[0178] Prepared according to the procedure of Example 1 with the following changes:

[0179] Use program 40 instead of program 3.

[0180] In Procedure 40, 2-(4-isobutylphenyl)acetic acid was used instead of hexanoic acid.

[0181] Install Alexa Fluor 568 using a procedure similar to Procedure 16.

[0182] Synthetic RTX-1354S:

[0183]

[0184] Prepared according to the procedure of Example 1 with the following changes:

[0185] Use program 40 instead of program 3.

[0186] In Procedure 40, 2-(4-isobutylphenyl)acetic acid was used instead of hexanoic acid.

[0187] Use procedures 17 and 18 instead of procedure 14 for installing the DOTA chelator.

[0188] Synthesis of RTX-1354SDOTA Chelating Agent

[0189]

[0190] Procedure 17: To a solution of (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbon-yl)-7-quinolinyloxy]acetamido}ethoxy)ethoxy]ethoxy}propionamido)-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-tri(tert-butyl ester) (31 mg, 0.046 mmol) and H2O (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,measured value m / z=1571.5[M+H] + .

[0191] Synthesis of 1354S:

[0192]

[0193] 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 hour. 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,measured value m / z=1403.0[M+H] + .

[0194] SYNTHETIC 1350S:

[0195]

[0196] Prepared according to the procedure of Example 1 with the following changes:

[0197] Use program 40 instead of program 3.

[0198] In Procedure 40, octanoic acid was used instead of hexanoic acid.

[0199] Use procedures 17 and 18 instead of procedure 14 for installing the DOTA chelator.

[0200] Synthetic RTX-1352S

[0201]

[0202] Prepared according to the procedure of Example 1 with the following changes:

[0203] Use program 40 instead of program 3.

[0204] In Procedure 40, 2-(4-isobutylphenyl)butanoic acid was used instead of hexanoic acid.

[0205] Use procedures 17 and 18 instead of procedure 14 for installing the DOTA chelator.

[0206] Synthetic RTX-1353S:

[0207]

[0208] Prepared according to the procedure of Example 1 with the following changes:

[0209] Use procedures 17 and 18 instead of procedure 14 for installing the DOTA chelator.

[0210] Synthetic 1360S:

[0211]

[0212] Prepared according to the procedure of Example 1 with the following changes:

[0213] Use program 40 instead of program 3.

[0214] Use procedures 17 and 18 instead of procedure 14 for installing the DOTA chelator.

[0215] 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-quinolinyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-(3-cyano-4-fluorobenzoylamino)hexanoylamino]-6-acetylaminohexanoic acid.

[0216]

[0217] Procedure 19: Preparation of 3-cyano-4-fluoro-benzoyl chloride

[0218]

[0219] 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) followed by DMF (12 μl, 0.151 mmol) at ambient temperature. After 1 h, the solvent was evaporated and the residue was co-evaporated with toluene to give the title compound (607 mg, yield exceeded theoretical value) as an orange solid. The material was used in the next step without further purification.

[0220] Procedure 20: Preparation of (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolinyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-aminohexanoylamino]-6-acetylaminohexanoic acid tert-butyl ester:

[0221]

[0222] A solution of (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolinyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-(tert-butoxycarbonylamino)hexanoylamino]-6-acetylaminohexanoic acid tert-butyl ester (198 mg, 0.19 mmol) in 90% aqueous formic acid (2.17 ml, 57.17 mmol) was stirred at ambient temperature for 2.5 h when no starting material was detected by LCMS. The solvent was removed under reduced pressure and the residue was co-evaporated with ACN (x3) and toluene (x2). The residue was dissolved in a mixture of MeOH / water (1:1, 50 ml) and treated with an ion exchange resin (Dowex 550A, OH-form, 15 ml) for 15 mins at ambient temperature. 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 free base form).

[0223] Procedure 21: Preparation of (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolinyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-(3-cyano-4-fluorophenylamino)hexanoylamino]-6-acetylaminohexanoic acid tert-butyl ester:

[0224]

[0225] To a mixture of (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolinyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-aminohexanoylamino]-6-acetylaminohexanoic acid tert-butyl ester (83 mg, 0.088 mmol) and DIEA (31 μl, 0.176 mmol) in DCM / DMF (1:1, 1 ml) at 0°C under nitrogen was added DCM (500 μl) containing 4-fluoro-3-trifluoromethyl-benzoyl chloride (16 mg, 0.088 mmol). The temperature was allowed to warm to ambient temperature and the reaction mixture was stirred for 10 min. Complete conversion was detected by LCMS. The mixture was purified with EtOAc, washed with water (x2) and brine, dried over anhydrous MgSO4 and filtered. The filtrate was evaporated to afford the target material (37 mg, 39%) as a colorless glassy solid.

[0226] 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-quinolinyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-(3-cyano-4-fluorobenzoylamino)hexanoylamino]-6-acetylaminohexanoic acid:

[0227]

[0228] To a solution of (S)-tert-butyl 2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolinyloxy]acetamido}ethoxy)ethoxy]ethoxy}propionamido)-6-(3-cyano-4-fluorophenylamino)hexanoylamino]-6-acetamidohexanoate (37 mg, 0.034 mmol) in anhydrous DCM (1 ml) at 0°C was added TFA (1 ml, 13 mmol) dropwise. The temperature of the reaction mixture was allowed to warm to ambient temperature and stirring was continued for 90 minutes. Complete conversion was detected by LCMS. The solvent was evaporated at room temperature and the residue was co-evaporated with toluene. The residue thus obtained was subjected to HPLC purification. The desired fractions were combined and lyophilized to give the target material RTX-1324S (15.8 mg, 45%) as an off-white solid.

[0229] 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-quinolinyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-[4-fluoro-3-(trifluoromethyl)benzylamino]hexanoylamino]-6-acetylaminohexanoic acid.

[0230]

[0231] Procedure 23: Preparation of (S)-tert-butyl 2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolinyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propanoylamino)-6-[4-fluoro-3-(trifluoromethyl)benzoylamino]hexanoylamino]-6-acetylaminohexanoate.

[0232] To a solution of (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolinyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-aminohexanoylamino]-6-acetylaminohexanoic acid tert-butyl ester (83 mg, 0.088 mmol) and DIEA (31 μl, 0.176 mmol) in a mixture of DCM / DMF (1:1, 1 ml) at 0°C under nitrogen was added 4-fluoro-3-trifluoromethyl-benzoyl chloride (20 mg, 0.088 mmol) in DCM (500 μl). The temperature of the reaction mixture was allowed to warm to ambient temperature and stirring was continued for 10 min. 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 afford the target material (57 mg, 58%) as a colorless glassy solid.

[0233] 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-quinolinyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-[4-fluoro-3-(trifluoromethyl)benzoylamino]hexanoylamino]-6-acetylaminohexanoic acid (RTX-1325S):

[0234]

[0235] To a solution of (S)-tert-butyl 2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolinyloxy]acetamido}ethoxy)ethoxy]ethoxy}propionamido)-6-(3-trifluoromethyl-4-fluorophenylamino)hexanoamido]-6-acetamidohexanoate (57 mg, 0.051 mmol) in anhydrous DCM (1 ml) at 0°C was added anhydrous TFA (1 ml, 13 mmol) dropwise. The temperature of the reaction mixture was allowed to warm 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 thus obtained was subjected to HPLC purification. The desired fractions were combined and lyophilized to give the target material RTX-1325S (21.8 mg, 40%) as an off-white solid.

[0236] Example 4 - Synthesis of RTX-1326S: 7-Methoxy-quinoline-4-carboxylic acid [2-((S)-2-cyano-pyrrolidin-1-yl)-2-oxo-ethyl]-amide.

[0237]

[0238] Procedure 25: Preparation of [2-((S)-2-cyano-pyrrolidin-1-yl)-2-oxo-ethyl]-carbamic acid tert-butyl ester:

[0239]

[0240] 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 concentrated under vacuum and the residue was dissolved in DCM, washed with water, dried over anhydrous MgSO4 and filtered. The filtrate was evaporated and flash purified on 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, yield 58%) as an amorphous solid.

[0241] Procedure 26: Preparation of (S)-1-(2-amino-acetyl)-pyrrolidine-2-carbonitrile hydrochloride:

[0242]

[0243] 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) was added p-toluenesulfonic acid monohydrate (1.25 g, 6.6 mmol). The reaction mixture was stirred at ambient temperature for 14 h. The solvent was evaporated to dryness to give the desired product (1.8 g, yield exceeded theoretical value). The material was used without further purification.

[0244] Procedure 27: Preparation of 7-methoxy-quinoline-4-carboxylic acid [2-((S)-2-cyano-pyrrolidin-1-yl)-2-oxo-ethyl]-amide:

[0245]

[0246] 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 hours, followed by evaporation of the solvent. 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 fraction was collected and evaporated to give the target material (400 mg, 75% yield) as an off-white solid.

[0247] Example 5 -Synthesis of RTX-1335S: (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolinyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-(3-cyano-4-fluorobenzoylamino)hexanoylamino]-6-acetylaminohexanoic acid tert-butyl ester.

[0248]

[0249] Procedure 28: To a mixture of (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolinyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-aminohexanoylamino]-6-acetylaminohexanoic acid tert-butyl ester (0.072 mmol) and DIEA (25 μl, 0.144 mmol) in DCM / DMF (1:1, 1 ml) at 0°C under nitrogen was added 4-fluoro-3-cyano-benzoyl chloride (13 mg, 0.072 mmol) in DCM (500 μl). The temperature of the reaction mixture was allowed to warm to ambient temperature and the reaction was stirred for 10 min. 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 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.

[0250] Example 6 -Synthesis of RTX-1336S: (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolinyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-(3-cyano-4-trimethylammoniumbenzamido)hexanoylamino]-6-acetylaminohexanoic acid tert-butyl ester.

[0251]

[0252] Procedure 29: Preparation of 3-cyano-4-dimethylamino-benzoic acid methyl ester:

[0253]

[0254] 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) at ambient temperature and the resulting mixture was stirred for 14 h. The mixture was concentrated using a high vacuum rotary evaporator at 65 °C. The residual solution was diluted with DCM and extracted with water (x2). 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 title compound (1.13 g, 99%) as an off-white solid.

[0255] Procedure 30: Preparation of (2-cyano-4-methoxycarbonylphenyl)-trimethylammonium trifluoromethanesulfonate:

[0256]

[0257] Methyl trifluoromethanesulfonate (6.26 ml, 55.3 mmol) was slowly added (dropwise) to a stirred solution of methyl 3-cyano-4-dimethylamino-benzoate (1.13 g, 5.53 mmol) in DCM (20 ml). The reaction mixture was stirred for 14 h, followed by the addition of diethyl ether. After evaporation of one third of the solvent volume, the desired compound was precipitated from the solution and the remaining solvent was decanted. The solid was extensively washed with copious amounts of ether. The residual ether was removed in vacuo and the solid was purified by HPLC. The desired fractions were collected and evaporated, and the residue was dried in vacuo to give the target compound (1.033 g, 51%) as an off-white solid.

[0258] Procedure 31: Preparation of (4-carboxy-2-cyanophenyl)-trimethylammonium trifluoromethanesulfonate:

[0259]

[0260] 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 under vacuum at ambient temperature for 14 h. The residue was treated with diethyl ether and the precipitate formed was filtered, washed with diethyl ether and dried under vacuum to give the title compound (562 mg, 83%) as an off-white solid.

[0261] Procedure 32: Preparation of RTX-1336S: (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolinyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-(3-cyano-4-trimethylammoniumbenzoylamino)hexanoylamino]-6-acetylaminohexanoic acid tert-butyl ester

[0262]

[0263] To a solution of (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolinyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-aminohexanoylamino]-6-acetylaminohexanoic acid tert-butyl ester (0.125mmol), (4-carboxy-2-cyano-phenyl)-trimethylammonium trifluoromethanesulfonate (0.51mg, 0.150mmol) and HOAt (1-hydroxy-7-azabenzotriazole, 24mg, 0.175mmol) in DMSO (3ml) was added DIC (24μl, 0.150mmol). The reaction mixture was stirred at ambient temperature for 14h. 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 trifluoroacetic acid salt of the title compound (56 mg, 35%) as an off-white solid.

[0264] Example 7 -Synthesis of RTX-1337S: (S)-tert-butyl 2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolinyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-[4-fluoro-3-(trifluoromethyl)benzoylamino]hexanoylamino]-6-acetylaminohexanoate.

[0265]

[0266] Procedure 33: To a solution of (S)-tert-butyl 2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolinyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-aminohexanoylamino]-6-acetylaminohexanoate (0.072 mmol) and DIEA (25 μl, 0.144 mmol) in a mixture of DCM / DMF (1:1, 1 ml) at 0°C under nitrogen was added 4-fluoro-3-trifluoromethyl-benzoyl chloride (16 mg, 0.072 mmol) in DCM (500 μl). The temperature of the reaction mixture was allowed to warm to ambient temperature and the reaction was stirred for 10 min. 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 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.

[0267] Example 8 : Synthesis of RTX-1338S: (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolinyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-(3-trifluoromethyl-4-trimethylammoniumbenzoylamino)hexanoylamino]-6-acetylaminohexanoic acid tert-butyl ester.

[0268]

[0269] Procedure 34: Preparation of 3-trifluoromethyl-4-dimethylamino-benzoic acid methyl ester.

[0270]

[0271] 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) was added potassium carbonate (1.62 g, 11.72 mmol) at ambient temperature, and the reaction mixture was stirred for 14 h. The reaction mixture was concentrated at 65 °C using a high vacuum rotary evaporator. The residual solution was diluted with DCM and extracted with water (x2). The combined aqueous layer was back extracted with DCM. The combined organic layer was washed with a diluted sodium bicarbonate aqueous solution, dried over anhydrous MgSO4, filtered and evaporated to give the target compound (1.38 g, quant.) as a colorless amorphous solid.

[0272] Procedure 35: Preparation of (2-trifluoromethyl-4-methoxycarbonylphenyl)-trimethylammonium trifluoromethanesulfonate:

[0273]

[0274] Methyl trifluoromethanesulfonate (9.51 ml, 84.2 mmol) was slowly added (dropwise) to a stirred solution of methyl 4-dimethylamino-3-trifluoromethylbenzoate (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 was precipitated and the solvent was removed by decantation. The solid was repeatedly washed with diethyl ether and dried in vacuo. The resulting solid was purified by HPLC. The desired fractions were collected and evaporated, and the residue was dried in vacuo to give the target compound, which was isolated as a trifluoromethanesulfonate (1.35 g, 59%) as an off-white solid.

[0275] Procedure 36: Preparation of (4-carboxy-2-trifluoromethylphenyl)-trimethylammonium trifluoromethanesulfonate:

[0276]

[0277] 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 h. The residue was treated with diethyl ether and the resulting precipitate was filtered, washed with diethyl ether and dried in vacuo to give the title compound (668 mg, 58%) as a white solid.

[0278] Procedure 37: Preparation of RTX-1338S: (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolinyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-(3-trifluoromethyl-4-trimethylammoniumbenzoylamino)hexanoylamino]-6-acetylaminohexanoic acid tert-butyl ester

[0279]

[0280] To a solution of (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolinyloxy]acetamido}ethoxy)ethoxy]ethoxy}propionamido)-6-aminohexanoylamino]-6-acetamidohexanoic acid tert-butyl ester (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 time complete conversion was detected by LCMS. The reaction mixture was purified by HPLC and the desired fractions were combined and lyophilized to give the target compound RTX-1338S trifluoroacetic acid salt (56 mg, 35%) as an off-white solid.

[0281] Example 9 - Synthesis of RTX 1363S: 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-triazacyclohexan-24-yl)-1,4,7-triazolin-1,4-diyl)diacetic acid

[0282]

[0283] For synthesis, the RTX-1363S molecule was broken down into the following three fragments:

[0284] Preparation of Int#1:

[0285]

[0286] Synthesize RTX-1363S from fragments:

[0287]

[0288] Procedure 38: Preparation of Int#1. (S)-tert-butyl 2-[(S)-2-(3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propionylamino)-6-(tert-butoxycarbonylamino)hexanoylamino]-6-(hexanoylamino)hexanoate: Int 1A, Fmoc-L-Lys(Boc)-OtBu, SCR448-17

[0289]

[0290] 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 min, 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 another 30 min, followed by the addition of N,N-dimethylaminopyridine (1.7 g, 14 mmol) at -30°C. The reaction mixture was slowly warmed to 0°C over a 2 h period and stirred at 0°C for another 2 h, at which time LCMS analysis of the reaction mixture showed complete consumption of the initial carboxylic acid. The reaction mixture was cooled to -30°C and 1 M aqueous HCl was slowly added to obtain a pH of ~3 (keeping the temperature of the mixture below -20°C). The resulting two-phase mixture was separated and the organic layer was washed with water (200 ml) and 0.1 M NaHCO (200 ml), dried over MgSO, filtered and evaporated to dryness. The residue was diluted with 30 ml ethyl acetate, heated to reflux and diluted with 300 ml hexane. The mixture was stirred at room temperature for 2 h and filtered. The precipitate was washed with 100 ml 20% ethyl acetate / hexane and the residual solvent was removed overnight in vacuo. Yield = 23 g (44%).

[0291] Procedure 39: Int 2A, Fmoc-L-Lys(Boc)-OtBu

[0292]

[0293] To a solution of Fmoc-L-Lys(Boc)-OtBu (23 g, 44 mmol) in 130 ml DCM was added 4 M HCl / 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 DCM, and the residual solvent was removed in vacuo at room temperature overnight. The product was obtained as a white amorphous solid (22.6 g, 110%).

[0294] Procedure 40: Int#3A, (S)-tert-butyl 2-[(9H-fluoren-9-yl)methoxycarbonylamino]-6-(hexanoylamino)hexanoate

[0295]

[0296] Diisopropylcarbodiimide (DIC, 16.7 g, 132 mmol) was added dropwise 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 eq); the temperature was maintained at 0°C to 5°C during the addition. The reaction mixture was stirred at 5°C for 30 minutes, at which time LCMS showed no starting material remaining. The mixture was diluted with 150 ml of DCM, and the organic layer was washed with 0.5 M HCl solution (100 ml), washed with 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 afford the title compound (16.8 g, 73%) as a white glass.

[0297] Procedure 41: Int#4A, (S)-tert-butyl 2-amino-6-(hexanoylamino)hexanoate

[0298]

[0299] To a solution of (S)-2-[(9H-fluorene-9-yl)methoxycarbonylamino]-6-(hexanoylamino)hexanoic acid tert-butyl ester (16 g, 30 mmol) in THF (200 ml) at 10°C was added diethylamine (92 ml, 900 mmol). The reaction mixture was stirred for 1 hour and evaporated to dryness under vacuum without heating. The residue was evaporated again with toluene (100 ml) at 25-30°C and purified using silica gel (220 g column, 0% to 15% MeOH in DCM) to give the product (7.1 g, 79%) as a viscous yellow oil.

[0300] Procedure 42: Int#5A, (S)-tert-butyl 2-[(S)-2-amino-6-(tert-butoxycarbonylamino)hexanoylamino]-6-(hexanoylamino)hexanoate

[0301]

[0302] To a solution of CBZ-Lys(Boc)-OH (9.7 g, 25.5 mmol) in anhydrous DCM (100 ml) was added HOBt (3.45 g, 25.5 mmol) and the mixture was stirred at ambient temperature for 10 min. To a solution of (S)-tert-butyl 2-amino-6-(hexanoylamino)hexanoate (6.9 g, 23 mmol) in DCM was added DIPEA (12.2 ml) and EDC (5.35 g) in sequence. The reaction mixture was stirred at ambient temperature for 4 hours (until LCMS showed that (S)-tert-butyl 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 afford the product (11.4 g, 75%) as an amorphous yellow solid.

[0303] Procedure 43: Int#6A, (S)-tert-butyl 2-[(S)-2-amino-6-(tert-butoxycarbonylamino)hexanoylamino]-6-(hexanoylamino)hexanoate

[0304]

[0305] To a solution of (S)-2-[(S)-2-amino-6-(tert-butoxycarbonylamino)hexanoylamino]-6-(hexanoylamino)hexanoic acid tert-butyl ester (11 g, 16.6 mmol) in methanol (400 ml) was added 10% palladium (50% humidity) on activated carbon (2 g). The suspension was subjected to catalytic hydrogenation (H2, 1 atm, overnight) at ambient temperature (after 12 h, LCMS indicated complete consumption of the starting material). The reaction mixture was filtered through diatomaceous earth. The filtrate was concentrated under reduced pressure, evaporated again twice with 200 ml of acetonitrile, and dried under high vacuum for 4 hours to give the product (8.96 g, 100%) as a colorless glass.

[0306] Procedure 44: Int#7A, (S)-tert-butyl 2-[(S)-2-[3-(2-{2-[2-(benzyloxycarbonylamino)ethoxy]ethoxy}ethoxy)propionylamino]-6-(tert-butoxycarbonylamino)hexanoylamino]-6-(hexanoylamino)hexanoate

[0307]

[0308] To a solution of 3-(2-{2-[2-(benzyloxycarbonylamino)ethoxy]ethoxy}ethoxy)propanoic acid (5.8 g, 16.6 mmol) in anhydrous DCM (50 ml) was added HOBt (2.53 g, 18.7 mmol) and the mixture was stirred at ambient temperature for 10 min. To a solution of (S)-tert-butyl 2-[(S)-2-amino-6-(tert-butoxycarbonylamino)hexanoylamino]-6-(hexanoylamino)hexanoate (8.9 g, 16.6 mmol) in DCM (50 ml) was added EDC (5.35 g) and DIPEA (12.2 ml) in sequence. 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 MgSO4. 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 afford the desired product (9.63 g, 67%) as a colorless glass.

[0309] Procedure 45: (Int#1), (S)-tert-butyl 2-[(S)-2-(3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propionylamino)-6-(tert-butoxycarbonylamino)hexanoylamino]-6-(hexanoylamino)hexanoate

[0310]

[0311] To a solution of (S)-2-[(S)-2-[3-(2-{2-[2-(benzyloxycarbonylamino)ethoxy]ethoxy}ethoxy)propionamido]-6-(tert-butoxycarbonylamino)hexanoamido]-6-(hexanoamido)tert-butyl hexanoate (9.5 g) in methanol (100 ml) was added 10% palladium on activated carbon (50% humidity, 2 g). The suspension was catalytically hydrogenated at room temperature overnight (1 atm of H2, then LCMS control for the absence of starting material). The reaction mixture was filtered through celite. The filtrate was concentrated under reduced pressure, evaporated twice more using 100 ml of acetonitrile and dried in high vacuum for 4 hours to give the desired product (8.15 g, 100%) as a colorless glass.

[0312] Preparation of RTX-1363S from fragments:

[0313] Procedure 46: Int#1C, scaled up, (S)-tert-butyl 2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolinyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propanoyl-amino)-6-(tert-butoxycarbonylamino)hexanoylamino]-6-(hexanoylamino)hexanoate

[0314] 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 minutes (LCMS checked for the absence of SM) and then diluted with EtOAc (500 ml), 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 ~7.2 g of a yellow glass. The crude was dissolved in DCM (40 ml) and flash purified 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.

[0315] Int#2C, (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolinyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-aminohexanoylamino]-6-(hexanoylamino)hexanoic acid tert-butyl ester, and

[0316] Int#3C, (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolinyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-{2-[4,7-bis(tert-butoxycarbonylmethyl)-1,4,7-triazolin-1-yl]acetylamino}hexanoylamino]-6-(hexanoylamino)hexanoic acid tert-butyl ester

[0317]

[0318] Procedure 47: A solution of (S)-tert-butyl 2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolinyloxy]acetamido}ethoxy)ethoxy]ethoxy}propionamido)-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 h and then the solvent was evaporated. The residue was co-evaporated with acetonitrile (60 ml x 3) and toluene (60 ml x 2), dissolved in 180 ml of 2-methyltetrahydrofuran and treated with 1% aqueous K2CO3 (30 ml). The organic layer was separated, dried over MgSO4 and evaporated to give the target material as a free base. The material was used as is in the next step.

[0319] Procedure 48: To acetonitrile (120 ml) containing the material from the previous step (~5.96 mmol), NOTA-bis(tBu-ester) (3.66 g, 8.82 mmol) and PyBop (3.10 g, 5.96 mmol) was added DIPEA (2.3 ml, 12.3 mmol) and the reaction mixture was stirred at ambient temperature for 2 hours (LCMS control). The reaction mixture was evaporated, diluted with ethyl acetate (600 ml) and extracted with water (2*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 desired product as a light yellow glass.

[0320] RTX-1363S, (S)-2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolinyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-{2-[4,7-bis(carboxymethyl)-1,4,7-triazolin-1-yl]acetylamino}hexanoylamino]-6-(hexanoylamino)hexanoic acid

[0321]

[0322] Procedure 49: To a solution of (S)-tert-butyl 2-[(S)-2-(3-{2-[2-(2-{2-[4-({2-[(S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7quinolinyloxy]acetamido}ethoxy)ethoxy]ethoxy}propionamido)-6-{2-[4,7-bis(tert-butoxycarbonylmethyl)-1,4,7-triazolin-1-yl]acetamido}hexanoamido]-6-(hexanoylamino)hexanoate (1 g, 0.717 mmol) in DCM (10 ml) at 0°C was added TFA (15 ml, 195 mmol) dropwise. The temperature of the reaction mixture was allowed to warm to ambient temperature and stirring was continued for 1 h (~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, 50x300 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 mixture was purified again by HPLC (5 mM NH4OAc / ACN) and lyophilized to give two fractions of the target material (fr#23: 243 mg, 98.5% purity and fr#24: 80 mg, 96.8% purity) as white powders (total yield 36.8%).

[0323] Synthetic RTX-1339.

[0324]

[0325] Prepared according to the procedure of Example 9 with the following changes:

[0326] Use program 3 instead of program 40.

[0327] Synthetic RTX-1340S.

[0328]

[0329] Prepared according to the procedure in Example 9, using butyric acid instead of hexanoic acid in Procedure 40.

[0330] Synthesis of RTX-1363S-Cu

[0331]

[0332] Prepared according to the procedure of Example 9. The resulting product was subjected to procedure 50 for installation of the radionuclide.

[0333] Procedure 50: 20 μg of precursor (unless otherwise stated) was combined with the indicated radioisotope. The radioisotope was obtained from a generator (for 68 Ga) or commercially available in HCl solutions buffered with varying amounts of 3N NaOAc to obtain a final pH of 4 to 6. Reactions were run using C18 Sep-Pak Lite cartridges as needed to obtain additional purity and / or reconstitution for injection.

[0334] Synthetic RTX-1359

[0335]

[0336] Prepared according to the procedure of Example 9 with the following changes:

[0337] In Procedure 40, 2-(4-isobutylphenyl)acetic acid was used instead of hexanoic acid.

[0338] The product of procedure 44 was used in procedures 13, 17 and 18 for the installation of the DOTA chelator.

[0339] The resulting product was subjected to procedures 51-54 for the installation of the borylated proline derivative.

[0340]

[0341] Synthesis of 2-[2-(3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propionylamino)-6-{2-[4,7,10-tris(carboxymethylmethyl)-1,4,7,10-tetraaza-1-cyclododecyl]acetylamino}hexanoylamino]-6-[2-(p-isobutylphenyl)-acetylamino]hexanoic acid:

[0342]

[0343] 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 H2 balloon (1 atm) for 2 h. The reaction was filtered through celite and concentrated. The obtained solid was dried in vacuo for 16 h to give tert-butyl 2-[2-(3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propionamido)-6-{2-[4,7,10-tris(tert-butoxycarbonylmethyl)-1,4,7,10-tetraaza-1-cyclododecyl]acetamido}hexanoamido]-6-[2-(p-isobutylphenyl)acetamido]hexanoate as a viscous oil. The isolated material was used without further purification. LCMS: C 65 H 115 N9O 15 :m / z:1262.66, found m / z=1263.5[M+H] + .

[0344] Procedure 52: To a solution of tert-butyl 2-[2-(3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propionamido)-6-{2-[4,7,10-tris(tert-butoxycarbonylmethyl)-1,4,7,10-tetraaza-1-cyclododecyl]acetamido}hexanoamido]-6-[2-(p-isobutylphenyl)acetamido]hexanoate (0.129 mmol) in DCM (4 mL) at 0° C. was added TFA (2 mL) 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 diethyl 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 in the next step without further purification. LCMS: C 49 H 83 N9O 15 :m / z:1038.23, measured value m / z=1039.2[M+H] + .

[0345] Synthesis of 2-[2-(3-{2-[2-(2-{2-[4-({2-[(R)-2-{(1R,2R,6S,8R)-6,9,9-trimethyl-3,5-dioxa-4-boratricyclo[6.1.1.02,6]dodecan-4-yl}-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolinyloxy]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

[0346]

[0347] Procedure 53: To a solution of 2-[2-(3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propionylamino)-6-{2-[4,7,10-tris(carboxymethylmethyl)-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) at -78 °C was added DIPEA (82 μL) and the reaction mixture was stirred at -78 °C for 5 min. Next, ((R)-1-((R)-2-(7-(2-oxo-2-(2,3,5,6-tetrafluorophenoxy)ethoxy)quinoline-4-carboxamide)propanol-yl)pyrrolidin-2-yl)boronic acid 3 (158 mg, 0.230 mmol) in DMF (0.2 mL) was added at -78°C, and the temperature of the mixture was raised to 20°C and stirred for 15 min. After the amine was completely consumed as shown by LCMS, the crude product was submitted to 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-boronatricyclo[6.1.1.0 2,6 ] dodecan-4-yl}-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolinyloxy]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 4 (55 mg, 31%), as an off-white solid. LCMS: C 77 H 115 BN 12 O 21 :m / z:1554.84, measured value m / z=778.2[(1 / 2)M+H]+ .

[0348] Synthesis of 2-[2-(3-{2-[2-(2-{2-[4-({2-[(R)-2-(1,2-dihydroxyboron)-1-pyrrolidinyl]-2-oxoethylamino}-carbonyl)-7-quinolinyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionylamino)-6-{2-[4,7,10-tris(carboxymethylmethyl)-1,4,7,10-tetraaza-1-cyclododecyl]acetylamino}hexanoylamino]-6-[2-(p-isobutylphenyl)-acetylamino]hexanoic acid

[0349]

[0350] Procedure 54: To 2-[2-(3-{2-[2-(2-{2-[4-({2-[(R)-2-{(1R,2R,6S,8R)-6,9,9-trimethyl-3,5-dioxa-4-boranetricyclo[6.1.1.0 2,6 To a solution of 6-{2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraaza-1-cyclodecyl]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 2 drops of 1M HCl. The reaction mixture was stirred at ambient temperature for 10 min. 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-dihydroxyboron)-1-pyrrolidinyl]-2-oxoethylamino}carbonyl)-7-quinolinyloxy]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, measured value m / z=1403.8[M-OH] + .

[0351] Synthetic RTX-1355R:

[0352]

[0353] Prepared according to the procedure of Example 9 with the following changes:

[0354] In Procedure 40, octanoic acid was used instead of hexanoic acid.

[0355] The product of procedure 44 was subjected to procedures 13, 17 and 18 for installation of the DOTA chelator.

[0356] The resulting product was subjected to procedures 51-54 for the installation of the borylated proline derivative.

[0357] Synthetic RTX-1356R:

[0358]

[0359] Prepared according to the procedure of Example 9 with the following changes:

[0360] In Procedure 40, 2-(4-isobutylphenyl)butanoic acid was used instead of hexanoic acid.

[0361] The product of procedure 44 was subjected to procedures 13, 17 and 18 for installation of the DOTA chelator.

[0362] The resulting product was subjected to procedures 51-54 for the installation of the borylated proline derivative.

[0363] Synthesis of 1357R:

[0364]

[0365] Prepare according to the following procedure:

[0366] Procedure 55: Synthesis of tert-butyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-aminohexanoate (2):

[0367]

[0368] 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 HCl in dioxane (4M, 20 ml) dropwise at 0°C and the reaction mixture was stirred at 0°C for 2 h. The solvent was evaporated and the residue was co-evaporated with hexanes 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 in the next step without further purification. LCMS: C 25 H 32N2O4: m / z: 424.53, measured value m / z = 425.3 [M+H] + .

[0369] Procedure 56: Synthesis of tert-butyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-(4-(4-isobutylphenyl)-butyrylamino)hexanoate (4):

[0370]

[0371] In an ice bath, DIC (0.71 ml, 4.47 mmol) was added dropwise to a cooled solution of 4-(4-isobutylphenyl)butanoic acid (1.0 g, 4.57 mmol) in 10 ml of DCM. The mixture was stirred for 30 min and filtered. The filtrate was added to a solution of tert-butyl 2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-6-aminohexanoate (1.62 g, 3.81 mmol) in 5 ml of DCM, and then additional DIEA (0.66 ml, 3.81 mmol) was added dropwise. The mixture was stirred for another 30 min. The solvent was evaporated under reduced pressure, and the crude product was purified by flash chromatography (EtOAc / hexanes, eluted in 50% EtOAc / hexanes) to give the product (1.93 g, 82% yield) as a white solid. LCMS: C 39 H 50 N2O5: m / z: 626.82, found m / z = 627.4 [M+H] + .

[0372] Procedure 57: Synthesis of tert-butyl 2-amino-6-(4-(4-isobutylphenyl)butyrylamino)hexanoate (5):

[0373]

[0374] To a solution of tert-butyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-(4-(4-isobutylphenyl)butyrylamino)-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 h. The solvent was evaporated and the residue was washed with hexane (2x) and dried in vacuo to give the product (613 mg, 100%). The compound was used as is for the following chemical transformations. LCMS: C 24 H 40 N2O3: m / z: 404.59, found m / z = 405.4 [M+H] + .

[0375] Procedure 58: Synthesis of tert-butyl 33-(4-(4-(4-isobutylphenyl)butyrylamino)butyl)-3,31-dioxo-1-phenyl-2,7,10,13,16,19,22,25,28-nonyloxy-4,32-diazatriatriacont-34-ate (7):

[0376]

[0377] EDCI.HCl (434 mg, 2.27 mmol) and HOBt (203 mg, 1.51 mmol) were added to a solution of 3-oxo-1-phenyl-2,7,10,13,16,19,22,25,28-nonyloxy-4-azatriacontane-31-acid (872 mg, 1.51 mmol) in 10 ml DCM, and the reaction mixture was stirred at room temperature for 5 min. DCM (10 ml) and DIEA (0.8 ml, 4.53 mmol) containing 2-amino-6-(4-(4-isobutylphenyl)butyrylamino)hexanoic acid tert-butyl ester (613 mg, 1.51 mmol) were added successively, and the mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with 20 ml DCM and then washed with water. The organic layer was dried with Na2SO4 and concentrated under reduced pressure. The crude product was purified by flash chromatography (MeOH / DCM, eluting with 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,measured value m / z=962.9[M+H] + .

[0378] Procedure 59: Synthesis of 1-amino-29-(4-(4-(4-isobutylphenyl)butyrylamino)butyl)-27-oxo-3,6,9,12,15,18,21,24-octaoxa-28-azatriacontane-30-oic acid tert-butyl ester (8):

[0379]

[0380] Palladium on carbon (10%, 0.2 g, 67% moisture) was suspended in a solution of tert-butyl 33-(4-(4-(4-isobutyl-phenyl)butyrylamino)butyl)-3,31-dioxo-1-phenyl-2,7,10,13,16,19,22,25,28-nonoxy-4,32-diazatriacontane-34-acid (1.0 g, 1.03 mmol) in MeOH (25 ml). The suspension was stirred at room temperature under H2 and balloon pressure for 2 h. 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 in the next step without further purification. LCMS: C 43 H 77 N3O 12 :m / z:828.08,measured value m / z=828.7[M+H] + .

[0381] Procedure 60: Synthesis of 10-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-41-(4-(4-(4-isobutylphenyl)butyrylamino)butyl)-2,2-dimethyl-4,11,39-trioxo-3,15,18,21,24,27,30,33,36-nonyloxy-5,12,40-triazatetradecane-42-oic acid tert-butyl ester (10):

[0382]

[0383] 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 was added EDCI.HCl (0.29 g, 1.53 mmol) and HOBt (0.14 g, 1.02 mmol), and the reaction mixture was stirred at room temperature for 5 min. DCM (10 ml) and DIEA (0.53 ml, 3.07 mmol) containing 1-amino-29-(4-(4-(4-isobutylphenyl)butyrylamino)butyl)-27-oxo-3,6,9,12,15,18,21,24-octaoxa-28-azatriacontane-30-acid tert-butyl ester (0.85 g, 1.02 mmol) were added successively, 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 in DCM containing 5% MeOH) to give the product (0.66 g, yield 50%) as a white solid. LCMS: C 69 H 107 N5O 17:m / z:1278.61,measured value m / z=1279.1[M+H] + .

[0384] Procedure 61: Synthesis of 10-amino-41-(4-(4-(4-isobutylphenyl)butyrylamino)butyl)-2,2-dimethyl-4,11,39-trioxo-3,15,18,21,24,27,30,33,36-nonyloxy-5,12,40-triazatetradecane-42-oic acid tert-butyl ester (11):

[0385]

[0386] To a solution of 10-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-41-(4-(4-(4-isobutylphenyl)-butyrylamino)butyl)-2,2-dimethyl-4,11,39-trioxo-3,15,18,21,24,27,30,33,36-nonyloxy-5,12,40-triazatetradecane-42-acid tert-butyl ester (0.66 g, 0.51 mmol) in THF (7 ml) at room temperature was added diethylamine (7 ml). The reaction mixture was stirred at ambient temperature for 2 hours and the solvent was evaporated. The residue was washed with hexanes (2x) and dried in vacuo to give the product (700 mg, 100%). The compound was used in the next step without further purification. LCMS: C 54 H 97 N5O 15 :m / z:1056.37,measured value m / z=1057.1[M+H] + .

[0387] Procedure 62: Synthesis of 18-(4-((tert-butoxycarbonyl)amino)butyl)-49-(4-(4-(4-isobutylphenyl)-butyrylamino)butyl)-3,16,19,47-tetraoxo-1-phenyl-2,7,10,13,23,26,29,32,35,38,41,44-dodecane-4,17,20,48-tetraazapentacontan-50-oic acid tert-butyl ester (13):

[0388]

[0389] To a solution of 3-oxo-1-phenyl-2,7,10,13-tetraoxa-4-azahexadecane-16-oic acid (0.22 g, 0.62 mmol) in DCM (8 ml) were 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 min. DCM (5 ml) and DIEA (0.32 ml, 1.87 mmol) containing 10-amino-41-(4-(4-(4-isobutylphenyl)-butyrylamino)butyl)-2,2-dimethyl-4,11,39-trioxo-3,15,18,21,24,27,30,33,36-nonyloxy-5,12,40-triazatetradecane-42-acid tert-butyl ester (0.66 g, 0.62 mmol) were added successively, and the mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with 20 ml 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, washed in DCM containing 8-10% MeOH) to give the product (0.70 g, yield 50%) as a white solid. LCMS: C 71 H 120 N6O 21 :m / z:1393.74,measured value m / z=1394.7[M+H] + .

[0390] Procedure 63: Synthesis of 18-(4-aminobutyl)-49-(4-(4-(4-isobutylphenyl)butyrylamino)butyl)-3,16,19,47-tetraoxo-1-phenyl-2,7,10,13,23,26,29,32,35,38,41,44-dodecane-4,17,20,48-tetraazapentacontan-50-oic acid tert-butyl ester (14):

[0391]

[0392] A solution of 18-(4-((tert-butoxycarbonyl)amino)butyl)-49-(4-(4-(4-isobutylphenyl)butyrylamino)butyl)-3,16,19,47-tetraoxo-1-phenyl-2,7,10,13,23,26,29,32,35,38,41,44-dodecane-4,17,20,48-tetraazapentacontan-50-acid tert-butyl ester (0.70 g, 1.22 mmol) in 90% aqueous formic acid (10 ml) was stirred at ambient temperature for 2 h. Complete conversion was detected by LCMS. The solvent was evaporated and the residue was co-evaporated 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, yield 95%) as a colorless liquid. The crude amine was used in this step without further purification. LCMS: C 66 H 112 N6O 19 :m / z:1293.62,measured value m / z=1294.1[M+H] + .

[0393] 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-azahexadecylamino)-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazaoctadecanoyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (15):

[0394]

[0395] To a solution of 18-(4-aminobutyl)-49-(4-(4-(4-isobutylphenyl)butyrylamino)butyl)-3,16,19,47-tetraoxo-1-phenyl-2,7,10,13,23,26,29,32,35,38,41,44-dodecane-4,17,20,48-tetraazapentacontan-50-oic acid tert-butyl ester (0.62 g, 0.48 mmol), DOTA-tri(tert-butyl ester) (0.27 g, 0.48 mmol) and PyBop (0.30 g, 0.56 mmol) in DMSO (8 ml) was added DIEA (0.20 ml, 1.15 mmol) and the reaction mixture was stirred at ambient temperature for 16 h. Water (20 ml) was added and the mixture was extracted with EtOAc (30 ml). The separated organic layer was washed with water (7x) and brine (8x) to remove excess PyBOP. The organic layer was dried over Na2SO4 and concentrated under reduced pressure to give the desired product (820 mg, yield 93%) as a colorless liquid. The compound was used in the next step without further purification. LCMS: C 94 H 162 N 10 O 26 :m / z:1848.34,measured value m / z=1849.7[M+H] + .

[0396] Procedure 65: Synthesis of tri-tert-butyl 2,2',2"-(10-(8-(3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propionamide)-39-(tert-butoxycarbonyl)-48-(4-isobutylphenyl)-2,9,37,45-tetraoxo-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazaoctadecanoyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (16):

[0397]

[0398] Palladium / carbon (10%, 0.12 g, humidity 67%) was suspended in 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-azahexadecylamino)-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazaoctadecanoyl) )-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)tri-tert-butyl triacetate (500 mg, 0.270 mmol) in MeOH (15 ml). The suspension was stirred under H2 and balloon pressure for 2 h at room temperature. The mixture was filtered through celite and the filtrate was evaporated to give the title compound (387 mg, yield 83%) as a colorless liquid. The crude product was used in the next step without further purification. LCMS: C 86 H 156 N 10 O 24 :m / z:1714.21,measured value m / z=1714.7[M+H] + .

[0399] Procedure 66: Synthesis of 2,2',2"-(10-(8-(3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propionamide)-39-carboxy-48-(4-isobutylphenyl)-2,9,37,45-tetraoxo-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazaoctadecanoyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (17):

[0400]

[0401] 2,2',2"-(10-(8-(3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propionamide)-39-(tert-butoxycarbonyl)-48-(4-isobutylphenyl)-2,9,37,45-tetraoxo-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazaoctadecanoyl)-1,4,7,10-tetraazacyclododecanand To a solution of tri-tert-butyl triacetate (387 mg, 0.225 mmol) in DCM (4 ml) was added TFA (2 ml). The reaction mixture was stirred at 40 °C for 2 h and the solvent was evaporated under reduced pressure. The residue was washed with hexane (2x) and diethyl ether (2x) to give the product (340 mg, 100%) as a white powder. The crude product was used in the next step without further purification. LCMS: C70 H 124 N 10 O 24 :m / z:1489.79,measured value m / z=1490.5[M+H] + .

[0402] Procedure 67: Synthesis of 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]dioxaborolane -2-yl)-pyrrolidin-1-yl)ethyl)carbamoyl)quinolin-7-yl)oxy)-6,9,12-trioxa-3-azapentadecanylamino)-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazaoctadecanoyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (4):

[0403]

[0404] To a solution of 2,2',2"-(10-(8-(3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propionamide)-39-carboxy-48-(4-isobutylphenyl)-2,9,37,45-tetraoxo-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazaoctadecanoyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (120 mg, 0.080 mmol) in DMF (2 mL) at 0°C was added DIPEA (0.11 mL, 0.645 mmol) and the reaction mixture was stirred at 0°C for 5 min. Next, , 2-((4-((2-oxo-2-((R)-2-((3aR,4R,6R,7aS)-5,5,7a-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborolan-2-yl)pyrrolidin-1-yl)ethyl)carbamoyl)quinolin-7-yl)oxy)2,3,5,6-tetrafluorophenylacetate (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 monitored by LCMS, the crude product was purified by HPLC. 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,measured value m / z=1004.80[M / 2+H] + .

[0405] Procedure 68: Synthesis of 2,2',2"-(10-(8-(1-((4-((2-((R)-2-borylpyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-7-yl)oxy)-2-oxo-6,9,12-trioxa-3-azapentadecanylamino)-39-carboxy-48-(4-isobutylphenyl)-2,9,37,45-tetraoxo-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazaoctadecanoyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (RTX-1357R(5)):

[0406]

[0407] At room temperature, 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]dioxaborolan-2-yl)pyrrolidin-1-yl) To a solution of (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)2 (5 mg, 0.087 mmol) and the reaction mixture was stirred at ambient temperature for 30 min. After complete consumption of the starting material as monitored by LCMS, the volatiles were evaporated under reduced pressure and the crude product was subjected to HPLC purification. The desired fractions were combined and lyophilized to afford the title 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,measured value m / z=1873.80[M+H] + .

[0408] Synthetic RTX-1358R:

[0409]

[0410] Prepared according to the procedure of Example 9 with the following changes:

[0411] Use program 3 instead of program 40.

[0412] The product of procedure 44 was subjected to procedures 13, 17 and 18 for installation of the DOTA chelator.

[0413] The resulting product was then subjected to procedures 51-54 for the installation of the borylated proline derivative.

[0414] Synthetic RTX-1361R:

[0415]

[0416] Prepared according to the procedure of Example 9 with the following changes:

[0417] The product of procedure 44 was subjected to procedures 13, 17 and 18 for installation of the DOTA chelator.

[0418] The resulting product was subjected to procedures 51-54 for the installation of the borylated proline derivative.

[0419] Example 10 -Synthetic RTX-1389

[0420] Synthesis of compound 10:

[0421]

[0422] Procedure 69: Synthesis of tert-butyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-aminohexanoate (2):

[0423]

[0424] 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 HCl in dioxane (4M, 20 ml) dropwise at 0°C and the reaction mixture was stirred at 0°C for 2 h. The solvent was evaporated and the residue was co-evaporated with hexanes 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 in the next step without further purification. LCMS: C 25 H 32 N2O4: m / z: 424.53, measured value m / z = 425.3 [M+H] + .

[0425] Procedure 70: Synthesis of tert-butyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-acetamidohexanoate (3):

[0426]

[0427] 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) at 0°C was added DIEA (1.64 ml, 9.43 mmol). The reaction mixture was stirred at room temperature for 1 h. 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 / hexane, eluted with 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, measured value m / z = 467.3 [M+H] + .

[0428] Procedure 71: Synthesis of tert-butyl 6-acetamido-2-aminohexanoate (4):

[0429]

[0430] 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 (2x) and dried in vacuo to give the product (900 mg, 99%). The compound was used in the next step without further purification. LCMS: C 12 H 24 N2O3: m / z: 244.33, measured value m / z = 245.4 [M+H] + .

[0431] Product 72: Synthesis of tert-butyl 2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((tert-butoxycarbonyl)amino)hexanamide)-6-acetamidohexanoate (6):

[0432]

[0433] To a solution of 2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-6-((tert-butoxycarbonyl)amino)hexanoic acid (1.72 g, 3.68 mmol) in 15 ml of DCM was added EDCI.HCl (1.05 g, 5.53 mmol) and HOBt (0.50 g, 3.68 mmol), and the reaction mixture was stirred at room temperature for 5 min. Next, DCM (10 ml) and DIEA (1.92 ml, 11.06 mmol) containing tert-butyl 6-acetamide-2-aminohexanoate (0.90 g, 3.68 mmol) were added in sequence, 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, eluting with 75% EtOAc in hexanes) to give the product (2.20 g, 77% yield) as a white solid. LCMS: C 38 H 54 N4O8: m / z: 694.86, found m / z = 695.3 [M+H] + .

[0434] Procedure 73: Synthesis of tert-butyl 6-acetamide-2-(2-amino-6-((tert-butoxycarbonyl)amino)hexanamide)-hexanoate (7):

[0435]

[0436] To a solution of tert-butyl 2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((tert-butoxycarbonyl)-amino)hexanamide)-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 (2x) and dried under vacuum to give the product (1.0 g, yield 92%). The compound was used in the next step without further purification. LCMS: C 23 H 44 N4O6: m / z: 472.62, found m / z = 473.5 [M+H] + .

[0437] Procedure 74: Synthesis of 21-(4-acetamidobutyl)-18-(4-((tert-butoxycarbonyl)amino)butyl)-3,16,19-trioxo-1-phenyl-2,7,10,13-tetraoxa-4,17,20-triazadocosan-22-oic acid tert-butyl ester (9):

[0438]

[0439] EDCI.HCl (0.67 g, 3.49 mmol) and HOBt (0.31 g, 2.33 mmol) were added to a solution of 3-oxo-1-phenyl-2,7,10,13-tetraoxa-4-azahexadecane-16-acid (0.83 g, 2.33 mmol) in 10 ml of DCM, and the reaction mixture was stirred at room temperature for 5 min. Next, DCM (10 ml) and DIEA (1.2 ml, 6.99 mmol) containing 6-acetamide-2-(2-amino-6-((tert-butoxycarbonyl)amino)hexanamide)hexanoic acid tert-butyl ester (1.0 g, 2.33 mmol) were added in sequence, 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 with Na2SO4 and concentrated under reduced pressure. The crude product was purified by flash chromatography (MeOH / DCM, eluting in 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, measured value m / z = 810.7 [M+H] + .

[0440] 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-ate (10):

[0441]

[0442] Palladium / carbon (10%, 0.03 g, 67% moisture) was suspended in a solution of 21-(4-acetamidobutyl)-18-(4-((tert-butoxycarbonyl)amino)butyl)-3,16,19-trioxo-1-phenyl-2,7,10,13-tetraoxa-4,17,20-triazadocosa-22-oic acid tert-butyl ester (130 mg, 0.160 mmol) in MeOH (3 ml). The suspension was stirred for 2 h at room temperature under H2 and balloon pressure. The mixture was filtered through celite and the filtrate was evaporated to give the title compound (106 mg, 98% yield) as a white solid. The crude product was used in the next step without further purification. LCMS: C 32 H 61 N5O 10 :m / z: 675.85, found m / z = 676.5 [M+H] + .

[0443]

[0444] Procedure 76: Synthesis of tert-butyl [(7-hydroxy-4-quinolyl)carbonylamino]acetate

[0445]

[0446] At 5°C, a solution of 7-hydroxy-4-quinolinecarboxylic acid (1.0 g, 5.3 mmol, 1 eq), glycine tert-butyl ester hydrochloride (1.2 g, 5.78 mmol, 1.1 eq) and HATU (3.02 g, 7.95 mmol, 1.5 eq) in DMF (20 ml) was added with DIPEA (2.2 ml, 2.5 eq) and stirred at room temperature for 2 h. After completion, H2O was added and the product was extracted with n-butanol. The organic layer was separated, dried over Na2SO4 and evaporated under reduced pressure. The resulting residue was purified by flash chromatography (0-10% MeOH in DCM) to give 3 (1.1 g, yield 67%) as an off-white powder. ESI-MS m / z calculated value: 302.324, found value 303.0 [M+H]+.

[0447] Procedure 77: Synthesis of benzyl (4-{[(tert-butoxycarbonylmethyl)amino]carbonyl}-7-quinolinyloxy)acetate

[0448]

[0449] To a solution of tert-butyl [(7-hydroxy-4-quinolyl)carbonylamino]acetate (1.1 g, 3.55 mmol, 1 eq) and K2CO3 (1.5 g, 10.7 mmol, 3 eq) in DMF (20 ml) was added benzyl bromoacetate (2.4 g, 10.7 mmol, 3 eq) at room temperature. The mixture was stirred at 60 °C for 16 h. Upon completion, H2O was added and the product was extracted with EtOAc. The organic layer was separated, dried over Na2SO4 and evaporated under reduced pressure. The resulting residue was purified by flash chromatography (0-80% ethyl acetate in hexane) to give 5 (1 g, yield 61%) as an off-white powder. ESI-MS m / z, calculated: 450.482, found: 451.0 [M+H] + .

[0450] Procedure 78: Synthesis of ({7-[(benzyloxycarbonyl)methoxy]-4-quinolyl}carbonylamino)acetic acid

[0451]

[0452] To a solution of benzyl (4-{[(tert-butoxycarbonylmethyl)amino]carbonyl}-7-quinolinyloxy)acetate (1 g, 2.2 mmol) in DCM was added 10 ml of TFA / DCM (DCM:TFA, 1:2) 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 complete the reaction. The solvent was evaporated under reduced pressure to give 6 in quantitative yield as an off-white solid. ESI-MS m / z, calculated: 394.376, found: 395.0 [M+H] + .

[0453] Procedure 79: Synthesis of benzyl (4-{[2-(2-{(1R,2S,8R)-2,9,9-trimethyl-3,5-dioxa-4-boratricyclo-[6.1.1.02,6]dodecan-4-yl}-1-pyrrolidinyl)-2-oxoethylamino]carbonyl}-7-quinolinyloxy)acetate

[0454]

[0455] To a solution of ({7-[(benzyloxycarbonyl)methoxy]-4-quinolyl}carbonylamino)acetic acid (0.87 g, 2.2 mmol, 1 eq), 2-{(1R,2R,8S)-2,9,9-trimethyltricyclo[6.1.1.02,6]dodecan-4-yl}pyrrolidine (0.6 g, 2.4 mmol, 1.1 eq) and HATU (1.25 g, 3.3 mmol, 1.5 eq) in DMF (5 ml) was added DIPEA (0.8 ml, 5.5 mmol, 2.5 eq) at 5°C. The mixture was stirred at room temperature for 2 h, H2O was added and the product was extracted with EtOAc. The organic layer was separated, dried over Na2SO4 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: 25.520, found: 626.1 [M+H] + .

[0456] Procedure 80: Synthesis of (4-{[2-(2-{(1R,2S,8R)-2,9,9-trimethyl-3,5-dioxa-4-boratricyclo[6.1.1.02,6]dodecan-4-yl}-1-pyrrolidinyl)-2-oxoethylamino]carbonyl}-7-quinolinyloxy)acetic acid

[0457]

[0458] Activated palladium / carbon (5%, 0.2 g) was suspended in a solution of 8 (1.1 g, 1.8 mmol) in MeOH (50 mL). The suspension was stirred at room temperature for 1 h under H2 atmosphere (40 psi). The mixture was filtered through celite and the filtrate was concentrated under reduced pressure to give 11 in quantitative yield (950 mg, yield 99%). ESI-MS m / z, calculated value: 535.398, found value: 536.1 [M+H] + .

[0459] Synthetic RTX-1389R(16):

[0460]

[0461] Procedure 81: Synthesis of 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]dioxaborolan-2-yl)pyrrolidin-1-yl)ethyl)carbamoyl)quinolin-7-yl)oxy)-6,9,12-trioxa-3,16,19-triazaheneicosane-21-oic acid tert-butyl ester (12):

[0462]

[0463] To a solution 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-ate (86 mg, 0.127 mmol) and 2-((4-((2-oxo-2-(2-((3aS,4R,6R)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborolan-2-yl)pyrrolidin-1-yl)ethyl)carbamoyl)quinolin-7-yl)oxy)acetic acid (68 mg, 0.127 mmol) in 2 ml of DMF were 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 h. The progress of the reaction was monitored by LCMS. DMF was removed under reduced pressure. The crude product was purified by flash chromatography (MeOH / DCM, eluted in DCM containing 3-4% MeOH) to give the product (75 mg, yield 50%) as a colorless liquid. LCMS: C 60 H 93 BN8 16 :m / z:1193.24,measured value m / z=1194.0[M+H]+ .

[0464] 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-tetraazaheptacosanyl)oxy)quinoline-4-carboxamide)acetyl)pyrrolidin-2-yl)boronic acid (13):

[0465]

[0466] 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]dioxaborolan-2-yl)-pyrrolidin-1-yl)ethyl)carbamoyl)quinolin-7-yl)oxy)-6,9,12-trioxa-3,16,19-triazaheneicosane-21-oate (65 mg, 0.054 mmol) and MeB(OH)2 (16 mg, 0.272 mmol) in acetone (1 ml) was added 0.2 N HCl (1 ml) at room temperature and the reaction mixture was stirred at ambient temperature for 30 min. 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 in the next step without further purification. LCMS: C 50 H 79 BN8 16 :m / z:1059.02,measured value m / z=1059.8[M+H] + .

[0467] Procedure 83: Synthesis of 20-(4-acetamidobutyl)-17-(4-aminobutyl)-1-((4-((2-(2-borylpyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-7-yl)oxy)-2,15,18-trioxo-6,9,12-trioxa-3,16,19-triazaheneicosane-21-oic acid (14):

[0468]

[0469] 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-tetraazaheptacosanoyl)oxy)quinoline-4-carboxamide)acetyl)pyrrolidin-2-yl)boronic acid (84 mg, 0.079 mmol) in DCM (1.5 ml) at 0°C was added TFA (0.5 ml) and the reaction mixture was stirred at ambient temperature for 30 min. 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 BN8 14 : m / z: 902.80, found value m / z = 885.5 [M-OH] +.

[0470] Procedure 84: Synthesis of 2-((E)-2-((E)-2-(4-((17S)-17-(((S)-5-acetamido-1-carboxypentyl)carbamoyl)-1-((4-((2-(2-borylpyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-7-yl)oxy)-2,15,23-trioxo-6,9,12-trioxa- 3,16,22-triazolopentacosan-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

[0471]

[0472] To the reaction mixture was added 20-(4-acetamidobutyl)-17-(4-aminobutyl)-1-((4-((2-(2-borylpyrrolidin-1-yl)-2-oxo-ethyl)carbamoyl)quinolin-7-yl)oxy)-2,15,18-trioxo-6,9,12-trioxa-3,16,19-triazaheneicosane-21-oic acid (5 mg, 0.0055 mmol) and 2-((E)-2-((E)-3-((E)-2-(3,3-dimethyl-5-sulfonyl-1-(3-(trimethylammonio)-propyl) To a solution of 1-(4-((2-((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, measured value m / z=906.1[M-OH / 2] + .

[0473] Synthetic RTX-1403

[0474]

[0475] 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.

[0476] Synthetic RTX-1399R

[0477] Compounds of the present invention having Macropa chelators can be prepared according to the procedures disclosed in PCT / US2019 / 062479, the entire teachings of which are incorporated herein by reference.

[0478]

[0479] Prepared according to Example 10 with the following changes:

[0480] Use program 40 instead of program 70.

[0481] In Procedure 40, 2-(4-isobutylphenyl)acetic acid was used instead of hexanoic acid.

[0482] Macropa chelators were prepared using procedures 85 and 86 and installed in place of the fluorophore chelator.

[0483] Procedure 85: Synthesis of 6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)-4-(4-isothiocyanatophenethoxy)picolinic acid (16):

[0484]

[0485] To a solution of 4-(4-aminophenethoxy)-6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)picolinic acid 2xTFA (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 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 the product 16 (100 mg, 100%) as a yellow oil. LCMS: C 35 H 43 N5O9S: m / z: 709.81, found m / z = 710.4 [M+H] + .

[0486] Procedure 86: Synthesis of 4-(4-(3-((S)-1-((4-((2-((R)-2-borylpyrrolidin-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-diazaheneicostan-21-yl)thioureido)phenethoxy)-6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinic acid):

[0487]

[0488] To N 2 -((1-((4-((2-((R)-2-borylpyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-7-yl)oxy)-2-oxo-6,9,12-trioxa-3-azapentadecan-15-yl)-L-lysyl)-N- 6 To a solution of -(2-(4-isobutylphenyl)acetyl)-L-lysine (21 mg, 0.023 mmol) in DMSO:H2O (0.5 ml:0.5 ml) was added Na2CO3 (8 mg, 0.069 mmol) and 6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)-4-(4-isothiocyanatophenethoxy)picolinic acid (20 mg, 0.027 mmol, dissolved in 0.5 ml DMSO) and the reaction mixture was stirred at room temperature for 2 h. After LCMS, analysis showed complete consumption of the amine, the solid was removed by filtration and the crude product was subjected to 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.

[0489] Embodiment 11 - Radiolabeling methods and results

[0490] General procedure: 20 μg of precursor (unless otherwise stated) was combined with the indicated radioisotope. The radioisotope in the HCl solution was obtained from a generator (for 68Ga), or commercially available, HCl solutions were buffered with varying amounts of 3N NaOAc to obtain a final pH of 4-6. Reactions were run using C18 Sep-Pak Lite cartridges as needed to obtain additional purity and / or reconstitution for injection. The labeling results are shown in Table 1.

[0491] Table 1: Results of representative labeling reactions

[0492]

[0493]

[0494] Example 12 - The compounds of the present invention bind to FAP with high affinity

[0495] Protease reactions were assembled in a total volume of 20 μL in 384-well plates (Greiner) as described below.

[0496] Recombinant protein was pre-diluted in a determination buffer comprising 100mM HEPES, pH 7.5, 0.1% BSA, 0.01% Triton X-100, 1mM DTT, and distributed to 384 well plates (10 μL / well). Test compounds were pre-diluted continuously in DMSO and added to the assay wells by ultrasonic distribution (Labcyte Echo 550). Control samples (0% inhibition in the absence of inhibitor, DMSO only) and 100% inhibitor (in the absence of enzyme) were assembled into four replicates for calculating inhibition % in the presence of compounds. The concentration of DMSO was 1% in all samples.

[0497] The 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 adding 10 μL of 2x FAM-labeled substrate peptide (FAM-GPRPFNYLAKK-NH2) prepared in the same assay buffer. The final concentration of the enzyme was 0.5 nM. The final concentration of the substrate peptide was 1 μM.

[0498] The reaction was allowed to proceed at room temperature. The incubation time was three hours for human FAP and 0.5 hours for mouse FAP. After incubation, the kinase reaction was quenched by adding 50 μL of stop buffer: 100x IC 50 Supplement assay buffer with reference inhibitor.

[0499] Using a microfluidic electrophoresis instrument (Caliper 3000, Caliper Life Sciences / PerkinElmer) analysis terminated plates. The relative intensity change of the peptide substrate and the cleavage product is the measured parameter. The activity in each test sample is determined as the product to total ratio (PSR): P / (S+P), where P is the peak height of the product and S is the peak height of the substrate. The percentage of inhibition (P) is determined using the following equation inh ): P inh =(PSR 0％inh -PSR 化合物 ) / (PSR 0％inh -PSR 100％inh )*100, where: PSR 化合物 is the product / total ratio in the presence of the compound, PSR 0％inh is the ratio of product / total in the absence of the compound and the PSR 100％inh is the ratio of product / total in the absence of enzyme. To determine the IC 50 (50% inhibition), %-inh data (P inh These values ​​are summarized and grouped in Table 2, where A represents IC 50 <0.1nM; B is IC 50 From 0.1 to 0.5 nM; C is IC 50 From 0.5 to 5.0 nM; D is IC 50 From 5.0 to 100 nM; E is IC 50 >100nM.

[0500] Table 2: Binding affinity study results and quality characterization data

[0501]

[0502]

[0503] From the above inhibition studies, it was found that the S stereoisomer was much more potent than the R stereoisomer. The above inhibition studies also showed that the prolonged (It has a LC of 1.5 nM 50 ) significantly improved the activity. Specifically, each extension of the chain brought improvements, and the addition of the terminal amino acid (lysine) provided further improvement in activity.

[0504] In the surface plasmon resonance assay data, a greater distinction was observed between certain compounds of the invention and FAPI-46 (the affinity of FAP-46 was measured to be 255 pM, while the affinity of certain disclosed compounds was 20 pM or stronger). This is also reflected in the U87 bioD study described below, where certain compounds of the invention exhibited significantly greater retention in vivo compared to FAPI-46.

[0505] Example 13 -use 18 Radiolabeling of FAP-targeting compounds by direct F labeling

[0506] Received from the manufacturer 18 F was loaded onto a Sep Pak QMA Light Plus cartridge and precipitated using Cs2CO3 / K 222 The solution was eluted into a glass reaction vial. 18 F was azeotropically dried using acetonitrile (3 x 1 ml) at 95°C under N2 flow. 2.5 mg of the targeted FAP compound was dissolved in 0.3 ml of DMSO and then added to the dried 18 The vial was sealed and heated to 90°C for 15 minutes. The vial was cooled and 1N HCl solution was added to the vial, which was then sealed and heated to 95°C for 10 minutes. The reaction mixture was cooled and neutralized, then loaded onto a Semi Prep HPLC column for purification and appropriately formulated for use. Alternatively, a suitable FAP targeting compound with a chelating agent may be used 18 F AlF labeling, where an AlCl3 stock in acetate buffer (22.5 μL, 45 nmol, 0.9 eq) was added to 18 F solution (200 μL), and the reaction vial was placed at room temperature for 5 min. Then the targeted FAP compound solution from the precursor reserve (50nmol scale, 12.5 μL) was added to the above vial. The pH was corrected to about 4.0 by adding 1% v / v acetic acid aqueous solution (15 μL). 200 μL EtOH co-solvent was added and the reaction vial was sealed, 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 prepared for use.

[0507] Embodiment 14 - Detection of tumors in mouse models using the compounds of the invention

[0508] Transplanted into BALB / c nude mice 7U-87 (human glioblastoma) cells and allowed tumor development. 1 μg of the invention was administered to mice by intravenous injection (e.g., tail vein) 18 F-labeled FAP-targeting agent RTX-1312S (prepared according to Example 13) and then sacrificed one hour later. Figure 1 a shows that in mice 18 F-labeled tumor binding of FAP-targeted compounds. Figure 1 b shows the biodistribution of the compounds in various mouse tissues.

[0509] Embodiment 15 -Biodistribution studies

[0510] U87MG cells were implanted subcutaneously into the right shoulder of female BALB / C nude mice in 1:1 matrigel:PBS. When tumors reached 150-500 mm 3 1 μg of radiolabeled ligand was administered intravenously (IV) via the tail vein at 4 °C volume. At various time points after injection, mice were humanely euthanized by exsanguination and tissue samples (bladder, blood, urine, bone (femur), heart, lung, liver, both kidneys, small intestine (including contents), large intestine (including contents), muscle (quadriceps), tumor, and tail) were removed, weighed, and counted using a gamma counter. The activity of each collected tissue was measured in counts per minute (CPM). Three aliquots of the radiotracer were also measured in the gamma counter to calculate the factor (μCi / CPM) for converting counts to units of activity. Decay values ​​were corrected for injection time and 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 Figure 2-4 ). This biodistribution showed increasing selectivity towards tumors over time, demonstrating high affinity for FAP.

[0511] Example 16 -[Ac-225] RTX-1399 biodistribution study

[0512] Radiolabeling procedure: The salt of 225Ac was dissolved in 600 μl 1M NHOAc. 100 μl of 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 saline (0.9%) was added and the reaction was shaken at 25°C for 30 minutes. The yield and purity of the radiolabeling were measured by TLC using Macherey-Nagel aluminum plates (ALUGRAM SILG / UV254, 4x8 cm, spotted with 2-5 μl of solution, placed in a saline chamber with 50 mM EDTA) and quantitatively analyzed using a Packard phosphorimager. 1 μg of the radiolabeled compound was administered intravenously (IV) to tumor-bearing JAX nude mice. Over the course of 120 hours, it was apparent that the target compound was localized in the tumor and filtered out of the blood (see Figure 5 ).

[0513] Equivalent

[0514] Although certain embodiments have been illustrated and described by those skilled in the art, after reading the above description, the compounds of the present technology or their salts, pharmaceutical compositions, derivatives, prodrugs, metabolites, tautomers or racemic mixtures described herein may be altered, replaced with equivalents, and other types of changes. Each of the above aspects and embodiments may also include or combine these variations or aspects disclosed with respect to any or all other aspects and embodiments.

[0515] The present technology is also not limited to the specific aspects described herein, which are intended to be a single illustration of various aspects of the present technology. As will be apparent to those skilled in the art, many modifications and changes can be made to the present technology without departing from its spirit and scope. In addition to those listed herein, functional equivalent methods within the scope of the present technology are apparent to those skilled in the art from the above description. Such modifications and changes are intended to fall within the scope of the appended claims. It should be understood that the present technology is not limited to specific methods, reagents, compounds, compositions, labeled compounds or biological systems, and of course, they can vary. It should also be understood that the terms used herein are only used to describe specific aspects and are not intended to be limited. Therefore, this specification is only considered as exemplary under the breadth, scope and spirit of the present technology indicated by the appended claims, the definitions therein and any equivalents thereof.

[0516] All publications, patents, and other documents cited in this specification are incorporated herein by reference in their entirety.

Claims

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

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein B is a branched, unbranched or cyclic aliphatic group of up to 30 carbon atoms, which is optionally interrupted by a peptidyl chain of up to 10 heteroatoms or up to 20 amino acid residues (e.g., 3-20 carbon atoms, which is optionally interrupted by up to 6 heteroatoms or up to 5 amino acid residues), wherein B is optionally substituted by 1-5 groups selected from the group consisting of F, Cl, Br, I, =O, OR 6 ,OCOR 6 、COOR 6 、CN、=NR 6 NR 6 R 7 , =S and SR 6 , provided that B contains at least 3 atoms in the chain between the group D and the group A.

3. The compound according to claim 1 or 2, which is represented by the following structural formula: or a pharmaceutically acceptable salt thereof, wherein m is an integer from 0 to 12; o is 0 or 1; and R 2 It is H or C1-C4 alkyl.

4. The compound according to claim 3, which is represented by the following structural formula: or a pharmaceutically acceptable salt thereof.

5. The compound according to claim 3, which is represented by the following structural formula: or a pharmaceutically acceptable salt thereof.

6. The compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, wherein R 3 It is a C1-C8 alkyl group or a C1-C4 aralkyl group which is optionally substituted by a C1-C4 alkyl group.

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

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

9. The compound according to claim 6 or a pharmaceutically acceptable salt thereof, wherein m is 8. 10 . The compound according to claim 3 , or a pharmaceutically acceptable salt thereof, wherein o is 0. 11 . The compound according to any one of claims 1 to 10 , or a pharmaceutically acceptable salt thereof, wherein n is 1.

12. The compound according to any one of claims 1 to 11 or a pharmaceutically acceptable salt thereof, wherein R 1 are fluorophores or optical dyes.

13. The compound according to claim 12 or a pharmaceutically acceptable salt thereof, wherein the fluorophore is And the optical dye is selected from: carbocyanine, indolecarbocyanine, oxycarbocyanine, thiocarbocyanine, merocyanine, polymethine, coumarin, rhodamine, xanthene, fluorescein, boron dipyrromethene (BODIPY), VivoTag-680, VivoTag-S750, AlexaFluor dye (e.g., AlexaFluor660, AlexaFluor680, AlexaFluor700, AlexaFluor750, AlexaFluor790) and DylightFluor dye.

14. The compound according to any one of claims 1 to 11 or a pharmaceutically acceptable salt thereof, wherein R 1 is a chelating group which is a residue of a chelating agent.

15. The compound according to claim 14 or a pharmaceutically acceptable salt thereof, wherein the chelating group is a residue of a chelating agent selected from the group consisting of 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), triethylenetetramine-N,N,N',N",N"',N"'-hexadecane Acetic acid (TTHA), 7-[2-(bis(carboxymethylamino)-ethyl]-4,10-bis-carboxymethyl-1,4,7,10-tetraazacyclododecane-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-triazacyclononane-1-yl}-acetic acid (NPTA), diacetylpyridinium 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-diazacyclooctadecane-7-yl) methyl)-4-isocyanatopicolinic acid (macropa-NCO), 6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-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-tetraazacyclododecane-1-yl]acetamide (TCMC or DOTAM). 16 . The compound or a pharmaceutically acceptable salt thereof according to claim 14 , wherein the residue of the chelating agent is a residue of macropa-NCS or macropa-NCO.

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

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

19. The compound according to any one of claims 1 to 11 or a pharmaceutically acceptable salt thereof, wherein: R 1 is optionally represented by one or more R 5 The group represented by is substituted benzoyl; Each R 5 are independently selected from halogen, cyano, halomethyl, N + (CH3)3W - ;and W - It is a pharmaceutically acceptable anion.

20. The compound according to claim 19 or a pharmaceutically acceptable salt thereof, wherein each R 5 are independently selected from fluorine, cyano, trifluoromethyl, N + (CH3)3W - .

21. The compound according to claim 19 or 20 or a pharmaceutically acceptable salt thereof, wherein R 5 The halogen or fluorine group represented by 18 F.

22. A compound according to any one of claims 1 to 21 or a pharmaceutically acceptable salt thereof, wherein R 2 It is H and R 4 It is CN.

23. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, which is represented by a structural formula selected from the following:

24. The compound according to 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 to a radionuclide.

25. The compound according to claim 24 or a pharmaceutically acceptable salt thereof, wherein the radionuclide is selected from 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 Fm and uranium-230.

26. The compound according to claim 24 or a pharmaceutically acceptable salt thereof, wherein the radionuclide is an α-emitting radionuclide such as 225 Ac, 233 Ra and 212 Pb.

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

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

29. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is represented by the following structural formula:

30. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is represented by the following structural formula: Where Cu is 64 Cu.

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

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

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

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

36. A method of imaging an area in a subject having or suspected of having diseased or fibrotic tissue expressing fibroblast activation protein alpha, comprising: a. administering to the subject a diagnostically effective amount of a compound of any one of claims 12-13, 19-22 or 24-28 or a pharmaceutically acceptable salt thereof, 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 the diseased tissue in the region.

37. The method of claim 33, wherein the area has or is suspected of having diseased tissue including a primary cancer or a metastasis of the cancer.

38. The method of claim 33, wherein the region has or is suspected of having a diseased area comprising fibrotic tissue.

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

40. 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 diseased tissue; b. using the compound as a reference, irradiating the area of ​​the bound compound using one or more doses of external irradiation, The diseased tissue is thereby treated using radiation.

41. The method of claim 38, wherein the compound comprises a radionuclide that emits gamma rays or positrons, or an optical dye or fluorophore, or other chelating group that detects 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 benchmark to guide surgical application to remove the area of ​​the diseased tissue, thereby removing the diseased tissue.

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

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