Fibroblast-activating protein target composition and method of use thereof

Abstract

A compound represented by structural formula (I) or a pharmaceutically acceptable salt thereof is disclosed. The variables of structural formula (I) are described herein. The compounds of the present invention can be attached to a chelating group for the binding of a radionuclide and are thus suitable for use in radiotherapy imaging and / or radiotherapy applications. For example, the compounds of the present disclosure are F, 68 Ga, or 64 radiolabeled with a positron emitter such as Cu and can be used in positron emission tomography (PET). Alternatively, the compound can be 225 an alpha particle emitter such as Ac, 67 a beta particle emitter such as Cu or 177 Lu, or an Auger electron emitter (e.g., 111 In, 67 Ga, 99 mTc, 195 mPt, 125 I, and 123 I). The compound can also be attached to a cytotoxic agent for targeted delivery of the cytotoxic agent to a tumor, for example, gemcitabine or doxorubicin, or conjugated to a venom. Similarly, the compound can be conjugated to a compound having a physiological effect, such as a TLR agonist, to stimulate the recipient's immune response. TIFF2026527516000126.tif36152​

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits of U.S. Provisional Application No. 63 / 627,309, filed on 31 January 2024, and U.S. Provisional Application No. 63 / 529,522, filed on 28 July 2023, all of which are incorporated herein by reference.

[0002] This technology relates to targeted imaging and therapeutic agents, more specifically compounds useful in the diagnosis and treatment of diseases. For example, the compositions described herein may be used as radiopharmaceuticals, or conjugated with optical dyes or fluorophores, or as drug / toxin conjugates useful for the diagnosis and treatment of cancer and fibrous diseases in tissues. [Background technology]

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

[0004] FAP is one of several members of the S9B prolyl oligopeptidase subfamily, which includes DPP4, DPP8, and DPP9, among other proteins. Substrates for FAP include neuropeptide Y, peptide YY, substance P, type B natriuretic peptide, fibroblast growth factor 21 (FGF-21), alpha-2 antiplasmin, and denatured collagen I and III.

[0005] FAP is actively expressed in tissues undergoing wound healing and reconstruction, but otherwise, it is either absent or expressed at extremely low levels in healthy, mature tissues. Tumors are localized areas of histological wounds to the host and are active in reconstructing local vascular structures and endothelium, as well as in several other events (microenvironment that allows new cells to proliferate) that help conceal the tumor from immune surveillance and promote wound healing. Therefore, FAP expression correlates with areas of tumorigenic tissue, particularly tumor stroma, and thus presents an excellent molecular target for the diagnosis and treatment of various cancers. Its expression has been identified in numerous 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 tumor and cancer types. FAP expression has also been detected in fibrous tissues and may be a marker for a broad spectrum of clinical conditions, including systemic fibrotic diseases such as systemic scleroderma (SSc), graft-versus-host disease and renal fibrosis, as well as radiation-induced fibrosis, and numerous organ-specific disorders of the heart, lungs, and liver (e.g., NAFLD: non-alcoholic fatty liver disease and NASH: non-alcoholic steatohepatitis), as well as renal fibrosis.

[0006] While several researchers have explored FAP as a cancer target, the development of activators that could act as diagnostic or even therapeutic agents has proven to be only slightly useful due to their pharmacokinetic limitations. To date, FAP has proven to be a difficult target due to the target's low and limited expression, and the limited residence times exhibited in vivo by prior art compounds. What is needed are improved FAP-targeted diagnostic and therapeutic agents that exhibit better binding kinetics and biodistribution, thereby providing a base for compounds that can deposit to a greater extent in tumors without unacceptable uptake in normal non-target tissues and organs. [Overview of the project]

[0007] Therapeutic agents targeting FAP have emerged as a promising methodology for treating a variety of cancers that express FAP in the microenvironment and / or the tumor cells themselves. Several FAP-targeting molecules, such as FAPI-04, FAPI-46, FAP-2286, PNT6555, etc., have been studied in preclinical and early clinical trials. Although FAP-targeted therapy has shown some initial promise in humans, the relatively short tumor retention is a significant limitation. Current compounds targeting FAP are removed relatively rapidly from tumor tissue, resulting in limited radiation absorbed dose to the tumor. Efforts have been made to improve the tumor retention of FAP-targeted radiotracers, but the results have been limited.

[0008] This specification discloses a series of novel compounds that bind to the extracellular domain of FAP. Unlike existing compounds, the compounds of the present disclosure exhibit a unique chemical structure that enables a significant improvement in affinity for FAP (see Examples 2 and 3), resulting in improved tumor retention.

[0009] The compounds disclosed herein can be attached to a chelating group for binding a radionuclide and are thus suitable for use in radiation imaging applications and / or radiation therapy applications. For example, the disclosed compounds can be radiolabeled with a positron emitter such as 18 F, 68 Ga or 64 Cu and used for positron emission tomography (PET) (see Example 4). Alternatively, the compound can be an alpha particle emitter, such as 225 Ac, a beta particle emitter, such as 67 Cu or 177 Lu, or an Auger electron emitter (e.g., 111 In, 67 Ga, 99 mTc, 195 mPt, 125 I and 123The compounds may be radiolabeled as described in I). The compounds may also be conjugated to cytotoxic agents for targeted delivery to tumors, for example, gemcitabine or doxycycline, or to venom. Similarly, the compounds may be conjugated to compounds having physiological effects, such as TLR agonists that stimulate the recipient's immune response. The compounds of this disclosure have the advantage of remarkably high affinity for FAP. Many of these compounds also have albumin binding / extended circulating residence time, but the potency / affinity for FAP is thought to result in a significant tumor residence time. The extended circulating residence time has the effect of increasing target filling while reducing compound deposition in non-target tissues (see Example 6). Therefore, the preferred binding kinetics of the compounds disclosed herein reduce the “washout” effect (i.e., low residence time) seen in prior art FAP target compounds.

[0010] One embodiment of the present invention has the following structural formula (I): [ka] [In the formula, n is either 0 or 1; A is NH, O, S, or CR 6 R 7 and; B comprises or is an aliphatic group of up to 30 carbon atoms, branched, unbranched, or cyclic, which is optionally interrupted by up to 10 heteroatoms or by a peptidyl chain of up to 20 amino acid residues (e.g., 3 to 20 or 3 to 15 carbon atoms optionally interrupted by up to 6 heteroatoms or up to 5 amino acid residues), where B is F, Cl, Br, I, =O, OR 6 OCOR 6 COOR 6 ,CN,=NR 6 , NR 6 R 7 , =S and SR 6It is optionally substituted with 1 to 5 groups selected from, provided that B contains at least 3 atoms in the chain between the D group and the A group; D is selected from the group consisting of OPO3H2, PO3H2, OSO3H, SO3H, and COOH, and their C1-C4 alkyl esters; X is either O or S; R 1 This includes chelating groups, optical dyes or fluorophores, cytotoxic agents, immunostimulants, or R 5 A benzoyl group optionally substituted by one or more groups represented by; R 3 is a C1-C8 alkyl or C1-C4 aralkyl, in the formula, The alkyl and aryl portions of the aralkyl group are each optionally and independently F, Cl, Br, I, branched, unbranched, or cyclic C1-C6 aliphatic groups, 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 are substituted; R 4 is either CN or B(OH)2; Each R 5 These are independently halo, cyano, halomethyl, and N + (CH3)3W - (In the formula, W - (These are selected from anions that are acceptable as medicines; R 6 and R 7 These are independently selected from the group consisting of H or C1-C6 alkyl groups; R 8 C 1~4 It is alkyl, R 9 is selected from H and C1-C4 alkyl groups, or R 8 and R 9[These are compounds represented by or pharmaceutically acceptable salts thereof, in which the intervening carbon atoms together form a C3-C6 cycloalkyl group.]

[0011] Another embodiment of the present invention is a pharmaceutical composition comprising i) a compound disclosed herein or a pharmaceutically acceptable salt thereof, and ii) a pharmaceutically acceptable carrier or diluent. With respect to compounds containing a chelating group, the chelating group is preferably chelated with a radionuclide.

[0012] Another embodiment of the present invention is a method for treating a subject having affected tissue expressing fibroblast-activating protein alpha. In one embodiment, the affected tissue may be cancer or fibrous tissue. The method comprises the step of administering an effective amount of the compound or pharmaceutically acceptable salt disclosed herein to the subject. Preferably, the compound used for treatment comprises a cytotoxic agent, for example, a chelate group having a radionuclide that emits beta, alpha, Auger, or other cytotoxic radiation capable of killing the affected tissue.

[0013] Another embodiment of the present invention is a method for imaging a region in a subject having or suspected of having cancer or fibrous tissue disease that expresses fibroblast-activating protein alpha or fibrous tissue, and includes: a. A step of administering to a subject a diagnostically effective amount of a compound disclosed herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition disclosed herein; b. The step of exposing the area of ​​the subject suspected to have affected tissue to an imaging device; and c. Step of obtaining an image of the affected tissue in the region.

[0014] Preferably, the compound used for imaging comprises a chelate group having a radionuclide that emits gamma rays or positrons or other detectable radiation. In another embodiment, the compound comprises an optical dye or fluorophore whose emission may be detectable.

[0015] Another embodiment of the present invention is a method for imaging tumors. This method includes: a. The step of administering to a subject an amount effective to contact and bind to a tumor and / or surrounding tissue of a compound disclosed herein or a pharmaceutically acceptable salt thereof; b. The step of irradiating the tumor and / or surrounding tissue at wavelengths absorbed by the bound compound; and c. A step of detecting a signal from the bound and irradiated compound, thereby imaging the tumor and / or surrounding tissue.

[0016] Preferably, the compound used for imaging contains a chelate group having a radionuclide that emits gamma rays or positrons, or an optical dye or fluorophore, or other detectable radiation.

[0017] Another embodiment of the present invention is a method for treating affected tissue. This method includes: a. The step of administering to a subject an amount effective to contact and bind to the affected tissue of a compound disclosed herein or a pharmaceutically acceptable salt thereof; b. Using a compound as a positional reference, the region of the bound compound is irradiated with one or more doses of external beam radiation. This involves treating the affected tissue with radiation.

[0018] Preferably, the compound used for positional reference includes a chelate group having a radionuclide that emits gamma rays or positrons, or an optical dye or fluorophore, or other detectable radiation.

[0019] Yet another embodiment of the present invention is a method for treating affected tissue. The method includes: a. The step of administering to a subject an amount effective to contact and bind to the affected tissue of a compound disclosed herein or a pharmaceutically acceptable salt thereof; b. The compound is used as a positional reference for surgical applications to guide the removal of the affected tissue area. This is the step of removing the executive organization.

[0020] Preferably, the compound used for positional reference includes a chelate group having a radionuclide that emits gamma rays or positrons, or an optical dye or fluorophore, or other detectable radiation. [Brief explanation of the drawing]

[0021] [Figure 1] This shows the organ-specific intra vivo distribution of [Lu-177]RTX-1371R in BALB / C nude mice pre-transplanted with U-87 / MG cells. [Figure 2] This shows the organ-specific intra vivo distribution of [Lu-177]RTX-1391R in BALB / C nude mice pre-transplanted with U-87 / MG cells. [Figure 3] This shows the organ-specific intra vivo distribution of [Lu-177]RTX-1392R in BALB / C nude mice pre-transplanted with U-87 / MG cells. [Figure 4] This shows the organ-specific intra vivo distribution of [Lu-177]RTX-1356R and [Lu-177]RTX-1391R in BALB / C nude mice pre-transplanted with U-87 / MG cells. [Figure 5] This shows the organ-specific intra vivo distribution of [Lu-177]RTX-1357R and [Lu-177]RTX-1392R in BALB / C nude mice pre-transplanted with U-87 / MG cells. [Figure 6] This shows the organ-specific intra vivo distribution of [Lu-177]RTX-1359R and [Lu-177]RTX-1371R in BALB / C nude mice pre-transplanted with U-87 / MG cells. [Figure 7]This shows the organ-specific intra vivo distribution of [Lu-177]RTX-1427R and [Lu-177]RTX-1411R in BALB / C nude mice pre-transplanted with U-87 / MG cells. [Figure 8] This shows the organ-specific intra vivo distribution of [Lu-177]RTX-1418R in BALB / C nude mice pre-transplanted with U-87 / MG cells. [Figure 9] This shows the organ-specific intra vivo distribution of [Lu-177]RTX-1386S in BALB / C nude mice pre-transplanted with U-87 / MG cells. [Figure 10] This shows the organ vivo distribution of [ZW800-1]RTX-1384S in female Nu / J or NCr-Foxn1 nude mice pre-transplanted with U-87 / MG cells. [Figure 11] This shows the organ vivo distribution of muscle-normalized [ZW800-1]RTX-1384S in female Nu / J or NCr-Foxn1 nude mice pre-transplanted with U-87 / MG cells. [Figure 12] This study demonstrates the efficacy of [Ac-225]RTX-1359R at various doses in female Nu / J mice pre-transplanted with U-87 / MG cells. [Figure 13] This shows the survival rate of female Nu / J mice that were pre-transplanted with U-87 / MG cells after treatment with [Ac-225]RTX-1359R. [Figure 14] This study demonstrates the efficacy of [Ac-225]RTX-1392R at various doses in female Nu / J mice pre-transplanted with U-87 / MG cells. [Figure 15] This shows the survival rate of female Nu / J mice that were pre-transplanted with U-87 / MG cells after treatment with [Ac-225]RTX-1392R. [Figure 16] This paper compares the efficacy of [Ac-225]RTX-1392R and [Ac-225]RTX1411R in female Nu / J mice pre-transplanted with U-87 / MG cells. [Figure 17]This shows a comparison of the survival rates of female Nu / J mice pre-transplanted with U-87 / MG cells after treatment with [Ac-225]RTX-1392R or [Ac-225]RTX1411R. [Modes for carrying out the invention]

[0022] Disclosed herein is a series of compounds capable of binding with high affinity to the extracellular domain of FAP and delivering a payload to tissues expressing FAP. The compounds of the present invention are described below herein.

[0023] A first embodiment of the present invention is a compound represented by structural formula (I) or a pharmaceutically acceptable salt thereof, where the variables are those described above for structural formula (I).

[0024] A second embodiment of the present invention is a compound represented by structural formula (I) or a pharmaceutically acceptable salt thereof, where B is a branched or unbranched aliphatic group of 3 to 20 carbon atoms, which is optionally interposed independently by up to 10 heteroatoms or by a peptidyl chain of up to 5 amino acid residues, and the aliphatic group is F, Cl, Br, I, =O, OR 6 OCOR 6 COOR 6 ,CN,=NR 6 , NR 6 R 7 , =S, or SR 6 The variables have been substituted by choice, and the remaining variables are those listed above for structural formula (I).

[0025] A third embodiment of the present invention is structural formula (II): (II) [ka] (In the formula, m is an integer from 0 to 12; o is 0 or 1; R 2(wherein is H or C1-C4 alkyl; the remaining variables are those described in the first or second embodiment) is a compound or a pharmaceutically acceptable salt thereof.

[0026] A fourth embodiment of the present invention is structural formula (III): [ka] The compound represented by (wherein the formula, the variable is one described in the third embodiment) or a pharmaceutically acceptable salt thereof.

[0027] A fifth embodiment of the present invention is structural formula (IV): [ka] The compound represented by (wherein the formula, the variable is one described in the third embodiment) or a pharmaceutically acceptable salt thereof.

[0028] A sixth embodiment of the present invention is structural formula (V): [ka] The compound represented by (wherein the formula, the variable is one described in the third embodiment) or a pharmaceutically acceptable salt thereof.

[0029] A seventh embodiment of the present invention is a compound represented by structural formula (I), (II), (III), (IV), or (V), or a pharmaceutically acceptable salt thereof, where R 3 is F, Cl, Br, I, or C 1~4 C is optionally substituted with alkyl. 1~8 Alkyl or C 1~4 It is aralkyl; the remaining variables are those described in the first, second, or third embodiment.

[0030] An eighth embodiment of the present invention is a compound represented by structural formula (I), (II), (III), (IV), or (V), or a pharmaceutically acceptable salt thereof, where R 3 is I or C 1~4 C is optionally substituted with alkyl. 1~8 Alkyl or C 1~4 It is aralkyl; the remaining variables are those described in the first, second, third, or seventh embodiment.

[0031] A ninth embodiment of the present invention is a compound represented by structural formula (I), (II), (III), (IV), or (V), or a pharmaceutically acceptable salt thereof, where R 3 is a C1-C8 alkyl or C1-C4 aralkyl which is optionally substituted with a C1-C4 alkyl; the remaining variables are those described in the first, second, third, seventh, or eighth embodiment.

[0032] A tenth embodiment of the present invention is a compound represented by structural formula (I), (II), (III), (IV), or (V), or a pharmaceutically acceptable salt thereof, where R 3 is methyl, (4-isobutylphenyl)methyl, (4-isobutylphenyl)propyl, (4-iodophenyl)methyl, or (4-iodophenyl)propyl; the remaining variables are those described in the first, second, third, seventh, or eighth embodiment.

[0033] An eleventh embodiment of the present invention is a compound represented by structural formula (I), (II), (III), (IV), or (V), or a pharmaceutically acceptable salt thereof, where R 3 These are methyl, (4-isobutylphenyl)methyl, and (4-isobutylphenyl)propyl; the remaining variables are those described in the first, second, third, seventh, eighth, ninth, or tenth embodiment.

[0034] A twelfth embodiment of the present invention is a compound represented by structural formula (I), (II), (III), (IV), or (V), or a pharmaceutically acceptable salt thereof, where R 8 It is methyl, and R 9 is H; the remaining variables are those described in the first, second, third, seventh, eighth, ninth, tenth, or eleventh embodiment.

[0035] A thirteenth embodiment of the present invention is a compound represented by structural formula (II), (III), (IV), or (V), or a pharmaceutically acceptable salt thereof, where o is 1 and m is 3 to 12; the remaining variables are those described in the third, seventh, eighth, ninth, tenth, eleventh, or twelfth embodiment.

[0036] A fourteenth embodiment of the present invention is a compound represented by structural formula (II), (III), (IV), or (V), or a pharmaceutically acceptable salt thereof, where m is 8; the remaining variables are those described in the thirteenth embodiment.

[0037] A fifteenth embodiment of the present invention is a compound represented by structural formula (II), (III), (IV), or (V), or a pharmaceutically acceptable salt thereof, where o is 0; the remaining variables are described for the third, seventh, eighth, ninth, tenth, eleventh, or twelfth embodiment.

[0038] A sixteenth embodiment of the present invention is a compound represented by structural formula (I), (II), (III), (IV), or (V), or a pharmaceutically acceptable salt thereof, where n is 1; the remaining variables are described for the first, second, third, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, or fifteenth embodiment.

[0039] A 17th embodiment of the present invention is a compound represented by structural formula (I), (II), (III), (IV), or (V), or a pharmaceutically acceptable salt thereof, where R 1 is a fluorophore or optical dye; the remaining variables are those described in the first, second, third, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, or sixteenth embodiment. In one embodiment, the fluorophore is [ka] The optical dyes include carbocyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, polymethine, coumarin, rhodamine, xanthene, fluorescein, borodipyromethane (BODIPY), VivoTag-680, VivoTag-S750, AlexaFluor dyes (e.g., AlexaFluor660, AlexaFluor680, AlexaFluor700, AlexaFluor750, AlexaFluor790), and DylightFluor dyes.

[0040] An eighteenth embodiment of the present invention is a compound represented by structural formula (I), (II), (III), (IV), or (V), or a pharmaceutically acceptable salt thereof, where R 1 is a chelating group which is a residue of the chelating agent; the remaining variables are those described in the first, second, third, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, or sixteenth embodiment. Preferred chelating agents and chelating agent residues are described below.

[0041] A 19th embodiment of the present invention is a compound represented by structural formula (I), (II), (III), (IV), or (V), or a pharmaceutically acceptable salt thereof, where R 1 R 5 A benzoyl group optionally substituted by one or more groups represented by; each R 5 Halo, cyano, halomethyl, N +(CH3)3W - Selected independently from; W - is a pharmaceutically acceptable anion; the remaining variables are those described in the first, second, third, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, or sixteenth embodiment. In one embodiment, R 5 The halo group represented by is 18 It is F.

[0042] A 20th embodiment of the present invention is a compound represented by structural formula (I), (II), (III), (IV), or (V), or a pharmaceutically acceptable salt thereof, where R 5 Fluoro, cyano, trifluoromethyl, N + (CH3)3W - The variables are selected independently from the 19th embodiment, and the remaining variables are those described in the 19th embodiment. In one embodiment, R 5 The fluoro group represented by is F 18 That is the case.

[0043] A 21st embodiment of the present invention is a compound represented by structural formula (I), (II), (III), (IV), or (V), or a pharmaceutically acceptable salt thereof, where R 2 H is R 4 is B(OH)2; the remaining variables are those described in the first, second, third, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, or twentieth embodiment.

[0044] A 22nd embodiment of the present invention is a compound represented by structural formula (I), (II), (III), (IV), or (V), or a pharmaceutically acceptable salt thereof, R 2 H is R 4 is CN; the remaining variables are those described in the first, second, third, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, or twentieth embodiment.

[0045] Also included in the present invention are compounds whose preparations are described and illustrated in the examples, both of which are pharmaceutically acceptable salts and neutralized forms. Chelation of these compounds containing chelating groups with radionuclides is also included in the present invention.

[0046] Also included in the present invention are the compounds shown below, both of which are pharmaceutically acceptable salts and neutralized forms. Chelation with radionuclides is also included in the present invention.

[0047] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] Therefore, nomenclature in which the compound name is preceded by the isotope indicates that the isotope is chelated to the chelate group of the compound. For example, in "[68Ga]RTX-1371R", the chelate group is 68This refers to RTX-1371R chelated with Ga. Exemplary compounds of the present invention (having their chelating groups) include: [68Ga]RTX-1371R; [68Ga]RTX-1391R; [68Ga]RTX-1392R; [68Ga]RTX-1411R; [212Pb]RTX-1371R; [212Pb]RTX-1391R; [212Pb]RTX-1392R; [212Pb]RTX-1411R; [90Y]RTX-1371R; [90Y]RTX-1391R; [90Y]RTX-1392R; [90Y]RTX-1411R; [117Sn]RTX-1371R; [11 [188 [169Er]RTX-1371R;[169Er]RTX-1391R;[169Er]RTX-1392R;[169Er] [223Ra] RTX-1392R;[223Ra]RTX-1411R;[67Cu]RTX-1371R;[67Cu]RTX-1391R;[67Cu]RTX-1392R;[67Cu]RTX-1411R;[161Tb]RTX-1371R;[161Tb]RTX-13 91R;[161Tb]RTX-1392R;[161Tb]RTX-1411R;[213Bi]RTX-1371R;[213Bi]RTX-1391R;[213Bi]RTX-1392R;[213Bi]RTX-1411R;[166Ho]RTX-1371 R;[166Ho]RTX-1391R;[166Ho]RTX-1392R;[166Ho]RTX-1411R;[149Tb]RTX-1371R;[149Tb]RTX-1391R;[149Tb]RTX-1392R;[149Tb]RTX-1411R;Examples include [47Sc]RTX-1371R; [47Sc]RTX-1391R; [47Sc]RTX-1392R; [47Sc]RTX-1411R; [227Th]RTX-1371R; [227Th]RTX-1391R; [227Th]RTX-1392R; [227Th]RTX-1411R; [177Lu]RTX-1371R; [177Lu]RTX-1391R; [177Lu]RTX-1392R; [177Lu]RTX-1411R; [225Ac]RTX-1371R; [225Ac]RTX-1391R; [225Ac]RTX-1392R; and [225Ac]RTX-1411R.

[0048] Compounds containing a Macropa chelating agent are typically chelated with 225Ac. Therefore, other compounds of the present invention (having their chelating groups) include [Ac225]RTX-1400R, [Ac225]RTX-1401R, [Ac225]RTX-1402R, [Ac225]RTX-1407S, [Ac225]RTX-1413R, [Ac225]RTX-1414R, [Ac225]RTX-1415R, [134Ce]RTX-1400R, [134Ce]RTX-1401R, [134Ce]RTX-1402R, [134Ce]RTX-1407S, [134Ce]RTX-1413R, [134Ce]RTX-1414R, and [134Ce]RTX-1415R.

[0049] "Aliphatic" refers to a saturated or unsaturated, linear or branched, monovalent or divalent hydrocarbon group. Unless otherwise specified, aliphatic groups typically have 1 to 10 carbon atoms. "Alkyl" refers to a saturated aliphatic, linear or branched, monovalent aliphatic group. Unless otherwise specified, alkyl groups typically have 1 to 10 carbon atoms (C 1~10 Alkyl, or 1 to 6 carbon atoms (C 1~3 It has alkyl (i.e., 1, 2, or 3) elements.

[0050] "Cyclic aliphatic" means a saturated or unsaturated, monovalent or divalent, cyclic hydrocarbon ring group. Unless otherwise specified, cyclic aliphatic has 3 to 8 ring carbon atoms (C3-8 cycloalkyl). "Cycloalkyl" means a saturated aliphatic cyclic aliphatic. Unless otherwise specified, cycloalkyl has 3 to 8 ring carbon atoms.

[0051] "Aryl", alone or in part or in a larger part, such as "aralkyl", is a carbocyclic aromatic group, such as phenyl or naphthyl.

[0052] "Aralkyl" refers to an alkyl group substituted with an aryl group. "C1-C x "Aralkyl" refers to an aralkyl group in which the alkyl part has 1 to x carbon atoms.

[0053] A compound having one or more chiral centers can exist in various stereoisomeric forms, i.e., each chiral center can have an R or S configuration, or can be a mixture of both. Stereoisomers are compounds that differ only in their spatial arrangement. Stereoisomers include all diastereomeric forms and enantiomeric forms of the compound. Enantiomers are stereoisomers that are mirror images that cannot be superimposed on each other. Diastereomers are stereoisomers having two or more chiral centers that are not identical and not mirror images of each other.

[0054] When the stereochemical configuration at one of the chiral centers in a compound having one or more chiral centers is described by its chemical name (e.g., where the configuration is indicated by a chemical name with "R" or "S") or structure (e.g., where the configuration is indicated by a "wedge" bond), the degree of fulfillment of the indicated configuration relative to the opposite configuration is greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 99%, or greater than 99.9%. The "degree of fulfillment of the indicated configuration relative to the opposite configuration" is expressed in mole percent and is determined by dividing the number of compounds having the indicated stereochemical configuration at the chiral center by the total number of compounds in the mixture having the same or opposite stereochemical configurations.

[0055] When a disclosed compound having a chiral center is described by structure without indicating the stereochemistry at that chiral center, the structure is meant to encompass compounds having an S configuration at that chiral center, compounds having an R configuration at that chiral center, or compounds having a mixture of R and S configurations at that chiral center. When a disclosed compound having a chiral center is described by its chemical name without indicating the stereochemistry at the chiral center having "S" or "R", the name is meant to encompass compounds having an S configuration at that chiral center, compounds having an R configuration at that chiral center, or compounds having a mixture of R and S configurations at that chiral center.

[0056] The FAP target compounds of the present invention are useful imaging agents for diagnostic applications. For example, they can be conjugated to a variety of metals for magnetic resonance imaging applications, or to optical dyes or fluorophores or other detectable parts (i.e., dyes, quantum dots, etc.) for histochemical and luminescence imaging applications. Similarly, the compounds can be radiolabeled and used in nuclear medicine applications. Radionuclides that can be used for imaging applications are referred to herein as “imaging radionuclides.” Non-limiting examples of imaging radionuclides include:18 F, 64 Cu or 68 Ga is one example, and these are suitable for use in PET imaging applications. 67 Cu or 177 Lu is one example, and these are typically therapeutic nucleotides, but they are also suitable for use in SPECT imaging applications.

[0057] The FAP-targeted compounds of the present invention are useful therapeutic compounds. Examples of such therapeutic compounds include the FAP-targeted compounds of the present invention having a preferred therapeutic moiety. The FAP-targeted compounds can be separated from the therapeutic moiety by a covalent linker. The separation between these (based on the count of adjacent atoms) may be between approximately 4 and 100 atoms. Furthermore, the pharmacokinetics of the compound can be altered by incorporating additional target structures onto the compound. For example, the use of a blood-targeting moiety can extend the circulating residence time, which has the effect of increasing tumor perfusion and filling while reducing the deposition 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 cytodestructive or cell division-inhibiting therapeutic agent. In the currently preferred embodiments, the FAP-binding compounds of the present invention can be fitted to a trillium scaffold by a covalent linkage to constitute a tumor-targeting domain for such FAP-targeted trillium agents. Exemplary constructs include toxins, poisons, metabolites, or chemotherapeutic agents; and radiotherapeutic compounds having alpha-emitting radionuclides, beta-emitting radionuclides, Auger electron-emitting radionuclides, or drug conjugates having a spectrum of radiation at decay (including positron emission, which is also suitable for diagnostic use).

[0058] For radiotherapy, the FAP targeting compound is conjugated to a chelating agent, which is selected based on its stability to hold the appropriate therapeutic radionuclide. A "therapeutic radionuclide" is, for example, a radionuclide that can be used for therapeutic purposes to treat cancer or fibrotic tissue due to its radioactive emission, which has a cytotoxic effect on target tissues (i.e., FAP-expressing cancer, and the tumor microenvironment, malignancy and fibrotic cells). Targeted radiotherapy has sometimes been carried out using macrocyclic complexes of radionuclides, but the macrocycles currently in use (e.g., DOTA) generally form complexes with many therapeutic radionuclide metals, such as, among others, the respective isotopes of actinium, radium, bismuth, astatine, lutetium and lead. The instability of many known macrocycle-containing compounds can result in some dissociation of the radionuclide from the macrocycle, which leads to a lack of selective delivery to the intended target tissue, which can also result in toxicity to non-target tissues. Alpha-emitting radionuclides, such as 225 Ac, can have a much greater cytotoxic effect and are therefore considered to be significantly more potent than beta-emitting radionuclides for therapy. However, this toxicity requires a chelating agent with extended retention of the chelated metal. U.S. Patent No. 11,279,698 (see also PCT / US Patent Application Publication No. 2018 / 025488 and PCT / US Patent Application Publication No. 2019 / 062479) describes a novel chelating agent ("Macropa") and its use as a chelating agent for 225 Ac, included as a component of a thallium K-synchronized and targeted radiotherapy agent. The ratio of tumor activity to renal activity of 1 or more can persist for up to about 36 hours after administration of the radiotherapy agent and, in the case of a 225Ac thallium-based therapeutic agent, can persist for 72 hours, or even 128 hours, or more, maximizing the therapeutic effect of the radiation on the target tissue.

[0059] Thus, an exemplary preferred FAP-targeting thallium compound will have a chelating agent in its third (non-targeting) domain. Macropa is225 Ac-FAP is currently the preferred chelating agent for trillium-targeted compounds (see also PCT / CA2021 / 050226).

[0060] [ka] The radionuclides usable with the compounds disclosed herein depend on the application, the desired type of radiation, and the half-life, as will be apparent to those skilled in the art. Exemplary radionuclides include: 177 Lu, 175 Lu, 45 Sc, 47 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, 117 Sn, 153 Sm, 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, 161 Tb, 166 Ho, 169 Er, 188 Re, 186 Re, 213 Bi, 211At, 217 At, 227 Th, 226 Th, 225 Ac, 233 Ra, 152 Dy, 213 Bi, 212 Bi, 211 Bi, 203 Pb, 212 Pb, 223 Ra, 255 Fm, and uranium-230. The radionuclides of any embodiment herein may be both therapeutic and diagnostic radionuclides, depending on their decay profile. Currently preferred alpha-emitting radionuclides for therapeutic applications include: 225 Ac, 233 Ra and 212 Pb is one example. Currently preferred beta-emitting radionuclides for treatment purposes include: 177 Lu, 90 Y and 67 Cu is one example.

[0061] Useful chelating groups and polyazapolycarboxylic acid macrorings in this technology include, and refer to, groups that can chelate, bind, or otherwise deliver radionuclides to therapeutic or diagnostic targets. A chelating group is a residue of a chelating agent after it has reacted with a nucleophile in the compound to form a targeted divalent radiopharmaceutical or radiodiagnostic agent that can bind to and deliver radionuclides. In the case of the disclosed compounds, the reactive group is a side-chain amine of a lysyl group in the second-to-last precursor that reacts with the chelating agent to form the disclosed compounds. Examples of chelating agents include, but are not limited to, covalently conjugated, substituted, or unsubstituted members of the following group: 1,4,7-Triazacyclononane-1,4,7-triacetic acid (NOTA), p-SCN-Bn-NOTA, 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), p-SCN-Bn-DOTA (also known as 2B-DOTA-NCS), PIP-DOTA, diethylenetriaminepentaacetic acid (DTPA), PIP-DTPA, AZEP-DTPA, ethylenediaminetetraacetic acid (EDTA), triethylenetetraamine-N,N,N',N'',N''',N'''-hexaacetic acid (TTHA), 7-[2-(bis-carboxymethylamino)-ethyl]-4,10-bis-carboxymethyl Lu-1,4,7,10-tetraazacyclododecyl-1-ylacetic acid (DEPA), 2,2',2''-(10-(2-(bis(carboxymethyl)amino)-5-(4-isothiocyanatophenyl)pentyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (3p-C-DEPA-NCS), NETA, {4-carboxymethyl-7-[2-(carboxymethylamino)-ethyl]-perhydro-1,4,7-triazonin-1-yl}acetic acid (NPTA), diacetylpyridine bis(benzoylhydrazone), 1,4,7,10,13,16-hexaazacyclooctadecane N,N',N'',N''',N'''',N'''''-hexaacetic acid (HEHA), octadentate terephthalamide ligand, 2,2'-(4-(2-(bis(carboxymethyl)amino)-5-(4-isothiocyanatophenyl)pentyl)-10-(2-(bis(carboxymethyl)amino)ethyl)-1,4,7,10-tetraazacyclododecane-1,7-diyl)diacetic acid, N,N'-bis[(6-carboxy-2-pyridyl)methyl]-4,13-diaza-18-crown-6(H2macropa), 6-((16-((6-carboxypyridine-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)-4-isothiocyanatopicolinic acid (macropa-NCS), 6 -((16-((6-carboxypyridine-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)-4-isothiocyanatopicolinic acid (macropa-NCO), 3,9-carboxymethyl-6-(2-methoxy-5-isothiocyanatophenyl)carboxymethyl-3,6,9,15-tetraazabicyclo-[9.3.1]pentadeca-1(15),11,13-triene and 2-[4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecyl-1-yl]acetamide (TCMC or DOTAM). In currently preferred embodiments, the chelating agent is a residue of the macrocyclic polyazapylated polycarboxylic acid, e.g., Macropa NCS or NCO-Macropa. In another embodiment, the chelating agent is a siderophore residue. In one embodiment, 225 Ac is a radionuclide for Macropa NCS or NCO-Macropa. In another embodiment, the chelating agent is a residue of p-SCN-Bn-DOTA, p-SCN-Bn-NOTA, NOTA, or DOTA. In another embodiment, the chelating agent is a sarcophage chelating agent. In another embodiment, the chelating agent is 68 These are residues of p-SCN-Bn-DOTA, p-SCN-Bn-NOTA, NOTA, or DOTA that have been chelated with Ga.

[0062] As noted above, the disclosed compound or a pharmaceutically acceptable salt thereof may contain one or more radionuclides suitable for use as radioimaging agents. Imaging methods include positron emission tomography (PET) or single-photon emission computed tomography (SPECT). In other embodiments, the present invention provides a method in which a subject having cancer, tumor or fibrous disease is administered an effective amount of the disclosed compound (or a pharmaceutically acceptable salt thereof) having a chelating agent, compounded with an imaging radionuclide for imaging purposes, and a method in which a subject having a therapeutic radionuclide for treatment is administered an effective amount of the compound. Exemplary cancers that can be imaged and / or treated with 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.

[0063] The "subjects" are mammals requiring medical treatment or diagnosis, preferably humans, but may also be animals requiring veterinary treatment, such as pets (e.g., dogs, cats, etc.), livestock (e.g., cattle, sheep, pigs, horses, etc.), and laboratory animals (e.g., rats, mice, guinea pigs, etc.).

[0064] The disclosed compounds or pharmaceutically acceptable salts thereof (including chelation with radionuclides) or pharmaceutically acceptable compositions thereof may be administered orally or by parenteral routes, usually by injection or infusion. "Pareral routes" means methods of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intra-arterial, intraspinal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intramedullary, and intrasternal injections and infusions.

[0065] The “effective dose” of a disclosed compound or pharmaceutically acceptable salt thereof (including chelation with radionuclides) means the amount of the therapeutic agent, alone or in combination with other therapeutic agents, that provides a therapeutic benefit in the treatment of disease prevention, improves overall treatment, reduces or avoids the symptoms or causes of disease, or provides the therapeutic effect or synergistic effect with another therapeutic agent. In terms of imaging, the “diagnostic effective dose” refers to the amount that would produce a useful image, for example, in diagnosing the presence of a tumor. The “effective dose” of a disclosed compound or pharmaceutically acceptable salt thereof is determined by a physician based on patient-specific parameters such as age, weight, sex, and severity of disease. The dosage is preferably 0.0001 mg / kg body weight to 100 mg / kg body weight.

[0066] Depending on the type of administration, the drug may be suitably formulated in the form of, for example, a solution or suspension, a simple tablet or sugar-coated tablet, a hard or soft gelatin capsule, a suppository, an avule, or an injectable preparation, which may be prepared according to the general methods for galenos formulations.

[0067] When solutions are used for infusion or injection, they are preferably aqueous solutions or suspensions, and it is possible to produce them either by themselves or together with carriers such as mannitol, lactose, glucose, or albumin, from, for example, lyophilized preparations containing the active substance before use. The prepared solutions are sterilized and, where appropriate, mixed with excipients, such as preservatives, stabilizers, emulsifiers, solubilizers, buffers and / or salts to adjust osmotic pressure. Sterilization can, where appropriate, be achieved by sterile filtration using a filter with a small pore size, accordingly allowing the composition to be lyophilized. Small amounts of antibiotics may also be added to ensure the maintenance of sterility.

[0068] In another embodiment, a pharmaceutical composition is provided which is suitable for in vivo imaging and / or radiotherapy of target tissue. A suitable pharmaceutical composition is an element (i.e. 18 F), or diagnostic radioactive metal chelate composites (e.g.,64 Cu or 68 The radioimaging agent may contain a radionuclide as one of the following (having Ga), or a radiotherapeutic agent which is a radioactive metal chelate complex may be contained together with a pharmaceutically acceptable radioactive vehicle in an amount sufficient to bind to the target tissue. The radioactive vehicle should be suitable for injection or aspiration and is, for example, human serum albumin; aqueous buffer solutions, such as tris(hydromethyl)aminomethane (and its salts), phosphates, citrates, bicarbonates, etc.; sterile water physiological saline; and chloride and / or dicarbonate salts or normal plasma cautions, such as calcium, potassium, sodium, and magnesium in a balanced ionic solution.

[0069] The concentration of the radiopharmaceutical agent in the radioactive vehicle should be sufficient to achieve reasonable binding to the target tissue, for example, about 4% to 20% ID / gram. For example, when using an aqueous solution, the human dose may range from 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 agent. The imaging or treatment agent should be administered so that it remains in the patient for about 1 hour to 10 days, although both longer and shorter periods may be acceptable. Therefore, convenient ampoules containing 1 to 10 mL of aqueous solution can be prepared.

[0070] Imaging may be carried out by conventional methods, for example, by injecting a sufficient amount of imaging composition to obtain appropriate imaging, followed by scanning with a suitable imaging or scanning machine, such as a tomograph or gamma camera. In certain embodiments, a method for imaging a region in a patient includes the steps of (i) administering a diagnostically effective amount of a compound combined with a radionuclide to the patient; exposing the patient's region to a scanning device; and (ii) obtaining an image of the patient's region. Thus, the present invention provides a method for obtaining an image of a mammalian subject, followed by the administration of a compound. Similarly, imaging may be performed after administration of a therapeutic agent or after a radiotherapy cycle to evaluate efficacy. Therefore, obtaining an image after administration of a radiotherapy agent may occur about 1 hour, about 4 hours, about 9 hours, about 12 hours, about 16 hours, about 20 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 96 hours, about 1 week, about 2 weeks, about 4 weeks, or after the completion of a cycle of therapeutic treatment. Therefore, in some embodiments, a method is provided for imaging tissue such as FAP-expressing tumor tissue, which includes contacting the tissue with a complex synthesized by contacting the imaging radionuclide with a compound disclosed therein.

[0071] In another embodiment, the disclosed compound or a complex of its pharmaceutically acceptable salt may contain one or more radionuclides suitable for use as radioimaging agents in the field of image-guided radiotherapy (IGRT). As described in U.S. Patent No. US 10,688,320B2, IGRT uses images acquired before the treatment session to guide the application of therapeutic radiation during the treatment session. The concentration of the imaging agent or therapeutic agent in the radioactive vehicle should be sufficient to produce satisfactory imaging. For example, when using an aqueous solution, the dose is about 1.0 to 100 millicuries. Imaging may be performed to provide a positional reference for guidance on how the target area receives the calculated radiation flux from the therapeutic radiation source. A similar use of the compound as a positional reference may be used in guided surgical applications.

[0072] The amount of a formulation containing the compound of the present invention, or a complex of a metal with the compound or a pharmaceutically acceptable salt thereof, administered to a patient depends on several physiological factors used as prescribed by the physician, including the nature of the procedure to be performed, the volume of tissue to be targeted for imaging or treatment, and body weight, as well as the medical history of the patient to be imaged or treated with the compound.

[0073] The examples provided herein are given to illustrate the advantages of the Art and to further assist those skilled in the art in preparing or using the compounds or salts of the Art, or their pharmaceutical compositions, derivatives, prodrugs, or tautomers. The examples provided herein are also presented to more fully illustrate preferred embodiments of the Art. These examples should never be construed as limiting the scope of the Art as defined by the appended claims. These examples may include or incorporate any of the variations, aspects, or embodiments of the Art described above. The variations, aspects, or embodiments described above may also each further include or incorporate any or all other variations, aspects, or embodiments of the Art. [Examples]

[0074] Example 1 - Compound synthesis Preparation of Int-1: [ka] Step 1: Synthesis of tert-butyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-aminohexanoate (2): [ka] 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), HCl in dioxane (4 M, 20 ml) was added dropwise at 0°C, and the reaction mixture was stirred at 0°C for 2 hours. The solvent was evaporated, the residue was co-evaporated with hexane, and the mixture was dried under vacuum to obtain the HCl salt of the product (1.62 g, 100%) as a white solid. The crude product was used for the next step without further purification. LCMS:C 25 H 32 N2O4: m / z: 424.53, measured value m / z = 425.3 [M + H] + .

[0075] Step 2: Synthesis of tert-butyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-(4-(4-isobutylphenyl)-butanamide)hexanoate (4): [ka] To a rapidly cooled solution of 4-(4-isobutylphenyl)butanoic acid (1.0 g, 4.57 mmol) in 10 ml of DCM, DIC (0.71 ml, 4.47 mmol) was added dropwise in an ice bath. The mixture was stirred for 30 minutes and filtered. The filtrate was added to a solution of tert-butyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-aminohexanoate (1.62 g, 3.81 mmol) in 5 ml of DCM, followed by the dropwise addition of DIEA (0.66 ml, 3.81 mmol). The mixture was stirred for a further 30 minutes. The solvent was evaporated under reduced pressure, and the crude product was purified by flash chromatography (eluted at 50% ethylethanol in hexane) to obtain the product (1.93 g, 82% yield) as a white solid. LCMS:C 39 H 50 N2O5: m / z: 626.82, measured value m / z = 627.4 [M + H] + .

[0076] Step 3: Synthesis of tert-butyl 2-amino-6-(4-(4-isobutylphenyl)butanamide)hexanoate (5): [ka] Diethylamine (8 ml) was added at room temperature to a solution of tert-butyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-(4-(4-isobutylphenyl)butanamide)-hexanoate (0.95 g, 1.51 mmol) in THF (8 ml). The reaction mixture was stirred at ambient temperature for 2 hours. The solvent was evaporated, the residue was washed with hexane (2 ×), and dried under vacuum to obtain the product (613 mg, 100%). The compound was used directly in the following chemical transformation. LCMS:C 24 H 40 N2O3: m / z: 404.59, measured value m / z = 405.4 [M + H] + .

[0077] Step 4: Synthesis of tert-butyl33-(4-(4-(4-isobutylphenyl)butanamide)butyl)-3,31-dioxo-1-phenyl-2,7,10,13,16,19,22,25,28-nonaoxa-4,32-diazatetratriacontane-34-oate (7): [ka] To a solution of 3-oxo-1-phenyl-2,7,10,13,16,19,22,25,28-nonaoxa-4-azahentriacontane-31-euic acid (872 mg, 1.51 mmol) in 10 ml of DCM, EDCI.HCl (434 mg, 2.27 mmol) and HOBt (203 mg, 1.51 mmol) were added, and the reaction mixture was stirred at room temperature for 5 minutes. Tert-butyl 2-amino-6-(4-(4-isobutylphenyl)butanamide)hexanoate (613 mg, 1.51 mmol) was sequentially added to DCM (10 ml) and DIEA (0.8 ml, 4.53 mmol), 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 dehydrated with Na2SO4 and concentrated under reduced pressure. The crude product was purified by flash chromatography (MeOH / DCM, eluted at 3-4% MeOH in DCM) to obtain the product (1.0 g, 69% yield) as a colorless liquid. LCMS:C 51 H 83 N3O 14 :m / z:962.22, actual value m / z=962.9[M+H] + .

[0078] Step 5: Synthesis of tert-butyl1-amino-29-(4-(4-isobutylphenyl)butanamide)butyl)-27-oxo-3,6,9,12,15,18,21,24-octaoxa-28-azatriacontane-30-oate(8): [ka] Carbon-supported palladium (10%, 0.2g, wet, 67%) was suspended in a solution of tert-butyl 33-(4-(4-(4-isobutylphenyl)butanamide)butyl)-3,31-dioxo-1-phenyl-2,7,10,13,16,19,22,25,28-nonaoxa-4,32-diazatetratriacontane-34-oate (1.0g, 1.03 mmol) in MeOH (25 ml). The suspension was stirred at room temperature for 2 hours under H2 balloon pressure. The mixture was filtered through Celite, and the filtrate was evaporated to obtain the product (850 mg, 100%) as a white solid. The crude product was used for the next step without further purification. LCMS:C 43 H 77 N3O 12 :m / z:828.08, actual value m / z=828.7[M+H] + .

[0079] Step 6: Synthesis of tert-butyl10-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-41-(4-(4-(4-isobutylphenyl)butanamide)butyl)-2,2-dimethyl-4,11,39-trioxo-3,15,18,21,24,27,30,33,36-nonaoxa-5,12,40-triazadotetracontane-42-oate(10): [ka] To a solution of 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((tert-butoxycarbonyl)amino)hexanoic acid (0.48 g, 1.02 mmol) in 10 ml of DCM, EDCI.HCl (0.29 g, 1.53 mmol) and HOBt (0.14 g, 1.02 mmol) were added, and the reaction mixture was stirred at room temperature for 5 minutes. Tert-butyl 1-amino-29-(4-(4-(4-isobutylphenyl)butanamide)butyl)-27-oxo-3,6,9,12,15,18,21,24-octaoxa-28-azatriacontane-30-oate (0.85 g, 1.02 mmol) was sequentially added to DCM (10 ml) and DIEA (0.53 ml, 3.07 mmol), 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 dehydrated with Na2SO4 and concentrated under reduced pressure. The crude product was purified by flash chromatography (MeOH / DCM, eluted at 5% MeOH in DCM) to obtain the product (0.66 g, 50% yield) as a white solid. LCMS:C 69 H 107 N5O 17 :m / z:1278.61, actual value m / z=1279.1[M+H] + .

[0080] Step 7: Synthesis of tert-butyl10-amino-41-(4-(4-(4-isobutylphenyl)butanamide)butyl)-2,2-dimethyl-4,11,39-trioxo-3,15,18,21,24,27,30,33,36-nonaoxa-5,12,40-triazadotetracontane-42-oate(11): [ka] Diethylamine (7 ml) was added at room temperature to a solution of tert-butyl10-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-41-(4-(4-(4-isobutylphenyl)-butanamide)butyl)-2,2-dimethyl-4,11,39-trioxo-3,15,18,21,24,27,30,33,36-nonaoxa-5,12,40-triazadotetracontane-42-oate (0.66 g, 0.51 mmol) in THF (7 ml). The reaction mixture was stirred at ambient temperature for 2 hours, and the solvent was evaporated. The residue was washed with hexane (2 ×) and dried under vacuum to obtain the product (700 mg, 100%). The compound was used for the next step without further purification. LCMS:C 54 H 97 N5O 15 :m / z:1056.37, actual value m / z=1057.1[M+H] + .

[0081] Step 8: Synthesis of tert-butyl18-(4-((tert-butoxycarbonyl)amino)butyl)-49-(4-(4-(4-isobutylphenyl)-butanamide)butyl)-3,16,19,47-tetraoxo-1-phenyl-2,7,10,13,23,26,29,32,35,38,41,44-dodecaoxa-4,17,20,48-tetraazapentacontane-50-oate(13): [ka] To a solution of 3-oxo-1-phenyl-2,7,10,13-tetraoxa-4-azahexadecane-16-euic acid (0.22 g, 0.62 mmol) in DCM (8 ml), EDCI.HCl (0.18 g, 0.93 mmol) and HOBt (0.08 g, 0.62 mmol) were added, and the reaction mixture was stirred at room temperature for 5 minutes. tert-butyl10-amino-41-(4-(4-(4-isobutylphenyl)-butanamide)butyl)-2,2-dimethyl-4,11,39-trioxo-3,15,18,21,24,27,30,33,36-nonaoxa-5,12,40-triazadotetracontane-42-oate (0.66 g, 0.62 mmol) was sequentially added to DCM (5 ml) and DIEA (0.32 ml, 1.87 mmol), 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 dehydrated with Na2SO4 and concentrated under reduced pressure. The crude product was purified by flash chromatography (MeOH / DCM, eluted at 8-10% MeOH in DCM) to obtain the product (0.70 g, 50% yield) as a white solid. LCMS:C 71 H 120 N6O 21 :m / z:1393.74, actual value m / z=1394.7[M+H] + .

[0082] Procedure 9: Synthesis of tert-butyl18-(4-aminobutyl)-49-(4-(4-(4-isobutylphenyl)butanamide)butyl)-3,16,19,47-tetraoxo-1-phenyl-2,7,10,13,23,26,29,32,35,38,41,44-dodecaoxa-4,17,20,48-tetraazapentacontane-50-oate (14): [ka] A solution of tert-butyl-18-(4-((tert-butoxycarbonyl)amino)butyl)-49-(4-(4-(4-isobutylphenyl)butanamide)butyl)-3,16,19,47-tetraoxo-1-phenyl-2,7,10,13,23,26,29,32,35,38,41,44-dodecaoxa-4,17,20,48-tetraazapentacontane-50-oate (0.70 g, 1.22 mmol) in 90% aqueous formic acid (10 ml) was stirred at ambient temperature for 2 hours. Complete conversion was detected by LC-MS. The solvent was evaporated, and the residue was co-evaporated with ACN (3×) and toluene (3×). The residue was dissolved in a MeOH / water mixture (1:1, 25 ml) and treated with an AmberLite HPR550 ion exchange resin (OH form) at ambient temperature for 15 minutes. The resin was filtered and washed with MeOH and water. The filtrate was evaporated to obtain the target material (free base, 0.62 g, 95% yield) as a colorless liquid. The crude amine was used for the next step without further purification. LCMS:C 66 H 112 N6O 19 :m / z:1293.62, actual value m / z=1294.1[M+H] + .

[0083] Procedure 10: 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-azahexadecanamide)-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazaoctatetracontyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (15): [ka] To a solution of tert-butyl-18-(4-aminobutyl)-49-(4-(4-(4-isobutylphenyl)butanamide)butyl)-3,16,19,47-tetraoxo-1-phenyl-2,7,10,13,23,26,29,32,35,38,41,44-dodecaoxa-4,17,20,48-tetraazapentacontane-50-oate (0.62 g, 0.48 mmol), DOTA-tris(t-Bu-ester) (0.27 g, 0.48 mmol), and PyBop (0.30 g, 0.56 mmol) in DMSO (8 ml), DIEA (0.20 ml, 1.15 mmol) was added, and the reaction mixture was stirred at ambient temperature for 16 hours. Water (20 ml) was added, and the mixture was extracted with SiO2 (30 ml). The separated organic layer was washed with water (7×) and brine (8×) to remove excess PyBOP. The organic layer was dehydrated with Na₂SO₄ and concentrated under reduced pressure to obtain the target product (820 mg, 93% yield) as a colorless liquid. The compound was used for the next step without further purification. LCMS:C 94 H 162 N 10 O 26 :m / z:1848.34, actual value m / z=1849.7[M+H] + .

[0084] Procedure 11: Synthesis of tri-tert-butyl 2,2',2''-(10-(8-(3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propanamide)-39-(tert-butoxycarbonyl)-48-(4-isobutylphenyl)-2,9,37,45-tetraoxo-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazaoctatetracontyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (16): [ka] Carbon-supported palladium (10%, 0.12g, wet, 67%) was suspended in a solution of tri-tert-butyl 2,2',2''-(10-(39-(tert-butoxycarbonyl)-48-(4-isobutylphenyl)-2,9,37,45-tetraoxo-8-(3-oxo-1-phenyl-2,7,10,13-tetraoxa-4-azahexadecanamide)-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazaoctatetracontyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl) triacetate (500mg, 0.270 mmol) in MeOH (15ml). The suspension was stirred at room temperature under H2 balloon pressure for 2 hours. The mixture was filtered through Celite, and the filtrate was evaporated to obtain the target compound (387 mg, 83% yield) as a colorless liquid. The crude product was used for the next step without further purification. LCMS:C 86 H 156 N 10 O 24 :m / z:1714.21, actual value m / z=1714.7[M+H] + .

[0085] Procedure 12: Synthesis of 2,2',2''-(10-(8-(3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propanamide)-39-carboxy-48-(4-isobutylphenyl)-2,9,37,45-tetraoxo-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazaoctatetracontyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (17): [ka] A solution of tri-tert-butyl 2,2’,2’’-(10-(8-(3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propanamide)-39-(tert-butoxycarbonyl)-48-(4-isobutylphenyl)-2,9,37,45-tetraoxo-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazatetracontyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (387 mg, 0.225 mmol) in DCM (4 ml) at 0 °C was treated with 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 (2×) and ether (2×) to give the product (340 mg, 100%) as a white powder. The crude product was used for the next step without further purification. LCMS: C 70 H 124 N 10 O 24 : m / z: 1489.79, found m / z = 1490.5 [M+H] + 。

[0086] Preparation of Int2:

Chem.

Chem.

[0087] Step 14: Synthesis of tert-butyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-(4-(4-isobutylphenyl)-butanamide)hexanoate (4): [ka] To a rapidly cooled solution of 4-(4-isobutylphenyl)butanoic acid (1.0 g, 4.57 mmol) in 10 ml of DCM, DIC (0.71 ml, 4.47 mmol) was added dropwise in an ice bath. The mixture was stirred for 30 minutes and filtered. The filtrate was added to a solution of tert-butyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-aminohexanoate (1.62 g, 3.81 mmol) in 5 ml of DCM, followed by the dropwise addition of DIEA (0.66 ml, 3.81 mmol). The mixture was stirred for a further 30 minutes. The solvent was evaporated under reduced pressure, and the crude product was purified by flash chromatography (eluted at 50% ethylethanol in hexane) to obtain the product (1.93 g, 82% yield) as a white solid. LCMS:C 39 H 50 N2O5: m / z: 626.82, measured value m / z = 627.4 [M + H] + .

[0088] Step 15: Synthesis of tert-butyl 2-amino-6-(4-(4-isobutylphenyl)butanamide)hexanoate (5): [ka] Diethylamine (8 ml) was added at room temperature to a solution of tert-butyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-(4-(4-isobutylphenyl)butanamide)-hexanoate (0.95 g, 1.51 mmol) in THF (8 ml). The reaction mixture was stirred at ambient temperature for 2 hours. The solvent was evaporated, the residue was washed with hexane (2 ×), and dried under vacuum to obtain the product (613 mg, 100%). The compound was used directly in the following chemical transformation. LCMS:C 24 H 40 N2O3: m / z: 404.59, measured value m / z = 405.4 [M + H] + .

[0089] Step 16: Synthesis of tert-butyl 2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((tert-butoxycarbonyl)-amino)hexanoamide)-6-(4-(4-isobutylphenyl)butanamide)hexanoate (7): [ka] To a solution of 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((tert-butoxycarbonyl)amino)hexanoic acid (1.50 g, 3.19 mmol) in 15 ml of DCM, EDCI.HCl (0.92 g, 4.78 mmol) and HOBt (0.43 g, 3.19 mmol) were added, and the reaction mixture was stirred at room temperature for 5 minutes. Tert-butyl 2-amino-6-(4-(4-isobutylphenyl)butanamide)hexanoate (1.29 g, 3.19 mmol) in DCM (10 ml) and DIEA (1.66 ml, 9.57 mmol) were added sequentially, and the mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with DCM (20 ml) and washed with water. The organic layer was dehydrated with Na2SO4 and concentrated under reduced pressure. The crude product was purified by flash chromatography (HCl / hexane, eluted in 75% HCl in hexane) to obtain the product (1.18 g, yield 43%) as a glassy solid. LCMS:C 12 H 24N2O3: m / z: 855.11, measured value m / z = 245.4 [M + H] + .

[0090] Procedure 17: Synthesis of tert-butyl 2-(2-amino-6-((tert-butoxycarbonyl)amino)hexanoamide)-6-(4-(4-isobutylphenyl)butanamide)hexanoate (8): [ka] To a solution of tert-butyl 2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((tert-butoxycarbonyl)-amino)hexaneamide)-6-(4-(4-isobutylphenyl)butanamide)hexanoate (1.18 g, 1.38 mmol) in THF (10 ml), diethylamine (10 ml) was added at room temperature. The reaction mixture was stirred at ambient temperature for 2 hours, the solvent was evaporated, the residue was washed with hexane (2x), and dried under vacuum to obtain the product (900 mg, 100%). The compound was used for the next step without further purification. LCMS:C 35 H 60 N4O6: m / z: 632.87, measured value m / z = 633.9 [M+H] + .

[0091] Procedure 18: Synthesis of tert-butyl18-(4-((tert-butoxycarbonyl)amino)butyl)-21-(4-(4-(4-isobutylphenyl)butanamide)butyl)-3,16,19-trioxo-1-phenyl-2,7,10,13-tetraoxa-4,17,20-triazadocosane-22-oate(10): [ka] To a solution of 3-oxo-1-phenyl-2,7,10,13-tetraoxa-4-azahexadecane-16-euic acid (0.50 g, 1.42 mmol) in 8 ml of DCM, EDCI.HCl (0.41 g, 2.13 mmol) and HOBt (0.19 g, 1.42 mmol) were added, and the reaction mixture was stirred at room temperature for 5 minutes. Tert-butyl 2-(2-amino-6-((tert-butoxycarbonyl)amino)hexane-amide)-6-(4-(4-isobutylphenyl)butanamide)hexanoate (0.90 g, 1.42 mmol) in DCM (10 ml) and DIEA (0.72 ml, 4.27 mmol) were sequentially added, 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 dehydrated with Na2SO4 and concentrated under reduced pressure. The crude product was purified by flash chromatography (MeOH / DCM, eluted at 3-4% MeOH in DCM) to obtain the product (0.95 g, yield 69%) as a colorless oil. LCMS:C 52 H 83 N5O 12 :m / z:970.24, actual value m / z=970.8[M+H] + .

[0092] Procedure 19: Synthesis of tert-butyl1-amino-14-(4-((tert-butoxycarbonyl)amino)butyl)-17-(4-(4-(4-isobutylphenyl)butanamide)butyl)-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecane-18-oate (11): [ka] Carbon-supported palladium (10%, 0.040 g, wet, 67%) was suspended in a solution of tert-butyl-18-(4-((tert-butoxycarbonyl)amino)butyl)-21-(4-(4-(4-isobutylphenyl)butanamide)butyl)-3,16,19-trioxo-1-phenyl-2,7,10,13-tetraoxa-4,17,20-triazadocosan-22-oate (160 mg, 0.165 mmol) in MeOH (5 ml). The suspension was stirred at room temperature for 2 hours under H2 balloon pressure. The mixture was then filtered through Celite, and the filtrate was evaporated to obtain the target compound (150 mg, 100%) as a white solid. The crude product was used for the next step without further purification. LCMS:C 44 H 77 N5O 10 :m / z:836.11, actual value m / z=836.80[M+H] + .

[0093] Procedure 20: Synthesis of tert-butyl18-(4-((tert-butoxycarbonyl)amino)butyl)-1-(9H-fluoren-9-yl)-21-(4-(4-(4-isobutylphenyl)butanamide)butyl)-3,16,19-trioxo-2,7,10,13-tetraoxa-4,17,20-triazadocosan-22-oate(12): [ka] To a solution of tert-butyl 1-amino-14-(4-((tert-butoxycarbonyl)amino)butyl)-17-(4-(4-(4-isobutylphenyl)butanamido)butyl)-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecane-18-oate (150 mg, 0.179 mmol) in DCM (4 ml) at room temperature was added FmocOSu (73 mg, 0.215 mmol) and DIPEA (0.1 ml, 0.538 mmol), and the reaction was stirred at room temperature for 16 h. Water was added and the mixture was extracted with DCM. The organic layer was dried over Na2SO4, filtered, and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography (eluting with EtOAc / hexane, 50% EtOAc in hexane) to give the product (145 mg, 77% yield) as a white solid. LCMS: C 59 H 87 N5O 12 : m / z: 1058.35, found m / z = 1059.0 [M+H] + .

[0094] Procedure 21: Synthesis of 18-(4-aminobutyl)-1-(9H-fluoren-9-yl)-21-(4-(4-(4-isobutylphenyl)butanamido)butyl)-3,16,19-trioxo-2,7,10,13-tetraoxa-4,17,20-triazadocosan-22-oic acid (13): [Chemical formula] To a solution of tert-butyl18-(4-((tert-butoxycarbonyl)amino)butyl)-1-(9H-fluoren-9-yl)-21-(4-(4-(4-isobutylphenyl)butanamide)butyl)-3,16,19-trioxo-2,7,10,13-tetraoxa-4,17,20-triazadocosan-22-oate (145 mg, 0.137 mmol) at 0°C in DCM (3 ml), TFA (1 ml) was added, and the reaction mixture was stirred at room temperature for 3 hours. The solvent was evaporated under reduced pressure, and the crude product was subjected to HPLC purification. The desired fractions were combined and lyophilized to obtain the product (65 mg, 53%) as an off-white solid. LCMS:C 50 H 71 N5O 10 :m / z:902.13, actual value m / z=902.9[M+H] +

[0095] Procedure 22: Synthesis of 6-((16-((6-carboxypyridine-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)-4-(4-isothiocyanatophenethoxy)picolinic acid (16): [ka] 4-(4-aminophenethoxy)-6-((16-((6-carboxypyridine-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)picolinic acid 2×TFA (120 mg, 0.14 mmol) was dissolved in DCM (3.5 ml) and Na2CO3 (44 mg, 0.42 mmol) was added. The reaction mixture was stirred at 40°C for 15 minutes (until a homogeneous solution was obtained). The reaction mixture was cooled to room temperature and O,O-di(pyridine-2-yl)carbonothioate (36 mg, 0.15 mmol) was added to 0.5 ml of DCM. After LCMS analysis showed consumption of the amine, the solid was removed by filtration and volatiles were removed under reduced pressure to obtain product 16 (100 mg, 100%) as a yellow oil. LCMS:C 35 H 43N5O9S: m / z: 709.81, measured value m / z = 710.4 [M+H] + .

[0096] Procedure 23: Synthesis of 4-(4-(3-(18-((1-carboxy-5-(4-(4-isobutylphenyl)butanamide)pentyl)carbamoyl)-1-(9H-fluoren-9-yl)-3,16-dioxo-2,7,10,13-tetraoxa-4,17-diazadocosan-22-yl)thioureido)phenethoxy)-6-((16-((6-carboxypyridine-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)picolinic acid (17): [ka] A solution of 18-(4-aminobutyl)-1-(9H-fluoren-9-yl)-21-(4-(4-(4-isobutylphenyl)butanamide)butyl)-3,16,19-trioxo-2,7,10,13-tetraoxa-4,17,20-triazadocosan-22-euic acid (21 mg, 0.023 mmol) in DMSO:H2O (0.5 ml:0.5 ml) is mixed with Na2CO3 (8 mg, 0.069 mmol) and 6-((16-((6-carboxypyridine-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)-4-(4-isothiocyanatophenethoxy)picolinic acid (20 mg, 0.027 mmol, DMSO) (dissolved in 0.5 ml) was added, and the reaction mixture was stirred at room temperature for 2 hours. After LC-MS 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 obtain the product (28 mg, 76%) as an off-white solid. LC-MS:C 85 H 114 N 10 O 19 S:m / z:1611.94, actual value m / z=1612.4[M+H] + .

[0097] Procedure 24: Synthesis of 4-(4-(3-(1-amino-14-((1-carboxy-5-(4-(4-isobutylphenyl)butanamide)pentyl)carbamoyl)-12-oxo-3,6,9-trioxa-13-azaoctadecane-18-yl)thioureido)phenethoxy)-6-((16-((6-carboxypyridine-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)picolinic acid (18): [ka] Diethylamine (0.5 ml) was added to a room temperature solution of 4-(4-(3-(18-((1-carboxy-5-(4-(4-isobutylphenyl)butanamide)pentyl)carbamoyl)-1-(9H-fluoren-9-yl)-3,16-dioxo-2,7,10,13-tetraoxa-4,17-diazadocosanate-22-yl)thioureido)phenethoxy)-6-((16-((6-carboxypyridine-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)picolinic acid (28 mg, 0.017 mmol) in THF (0.5 ml). The reaction mixture was stirred at ambient temperature for 2 hours, the solvent was evaporated, and the crude residue was subjected to HPLC purification. The desired fractions were combined and freeze-dried to obtain the product (13 mg, 54%) as an off-white solid. LC-MS:C 70 H 104 N 10 O 17 S:m / z:1389.70, actual value m / z=1390.2[M+H] + .

[0098] Synthesis of Int-3 [ka] Procedure 25: Preparation of tert-butyl-(S)-2-[(S)-2-(3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propionylamino)-6-(tert-butoxycarbonylamino)hexanoylamino]-6-(hexanoylamino)hexanoate:Fmoc-L-Lys(Boc)-OtBu [ka] Fmoc-L-Lys(Boc)-OH (47 g, 100 mmol) was dissolved in DCM (470 ml), and diisopropyl ethylamine (25 ml) was added at -30°C. The reaction mixture was stirred at -30°C for 5 minutes, and then Boc anhydrous (30.1 g, 140 mmol) was slowly added at -30°C. The reaction mixture was stirred at -30°C for a further 30 minutes, and then N,N-dimethylaminopyridine (1.7 g, 14 mmol) was added at -30°C. The reaction mixture was slowly heated to 0°C over 2 hours and stirred at 0°C for a further 2 hours. At this point, LCMS analysis of the reaction mixture showed complete consumption of the starting carboxylic acid. The reaction mixture was cooled to -30°C, and 1 M aqueous HCl was slowly added (while maintaining the temperature of the mixture below -20°C) to obtain a pH of approximately 3. The resulting two-phase mixture was separated, the organic layer was washed with water (200 ml) and 0.1 M NaHCO3 (200 ml), dehydrated with MgSO4, filtered, and evaporated. The residue was diluted with 30 ml of ethyl acetate, heated under reflux, and diluted with 300 ml of hexane. The mixture was stirred at room temperature for 2 hours and filtered. The precipitate was washed with 100 ml of 20% ethyl acetate in hexane, and the remaining solvent was removed under vacuum overnight. Yield = 23 g (44%).

[0099] Step 26: Fmoc-L-Lys(Boc)-OtBu [ka] To a solution of Fmoc-L-Lys(Boc)-OtBu (23 g, 44 mmol) in 130 ml of DCM, 4 M HCl in 275 ml of dioxane was added at -5°C. The mixture was stirred at -5°C to 0°C for 2.5 hours (until LCMS no longer showed the starting material). The solvent was evaporated under vacuum without heating, re-evaporated with 150 ml of DCM, and the remaining solvent was removed overnight under vacuum at room temperature. The product was obtained as a white amorphous solid (22.6 g, 110%).

[0100] Step 27: tert-butyl-(S)-2-[(9H-fluoren-9-yl)methoxycarbonylamino]-6-(hexanoylamino)hexanoate [ka] Diisopropylcarbodiimide (DIC, 16.7 g, 132 mmol) was added at 0°C to a solution of hexanoic acid (30.7 g, 264 mmol) in DCM (120 ml), 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 the addition of DIPEA (7.7 ml, 1 equivalent); the temperature was maintained between 0°C and 5°C during the addition. The reaction mixture was stirred at 5°C for 30 minutes, during which time LCMS showed that no starting material remained. The mixture was diluted with 150 ml of DCM, the organic layer was washed with 0.5 M HCl solution (100 ml), washed with 0.1 M NaHCO3 solution (100 ml), dehydrated with MgSO4, and evaporated. The residue was purified by flash chromatography (330g silica gel column, 0%-50% ethyl acetate in hexane) to obtain the target compound (16.8g, 73%) as a white glass.

[0101] Step 28: tert-butyl(S)-2-amino-6-(hexanoylamino)hexanoate [ka] Diethylamine (92 ml, 900 mmol) was added at 10°C to a solution of tert-butyl-(S)-2-[(9H-fluoren-9-yl)methoxycarbonylamino]-6-(hexanoylamino)hexanoate (16 g, 30 mmol) in THF (200 ml). The reaction mixture was stirred for 1 hour and evaporated under vacuum without heating. The residue was re-evaporated with toluene (100 ml) at 25-30°C and subjected to silica gel purification (220 g column, 0%-15% MeOH in DCM) to obtain the product (7.1 g, 79%) as a viscous yellow oil.

[0102] Step 29: tert-butyl-(S)-2-[(S)-2-amino-6-(tert-butoxycarbonylamino)hexanoylamino]-6-(hexanoylamino)hexanoate [ka] To a solution of CBZ-Lys(Boc)-OH (9.7 g, 25.5 mmol) in dry DCM (100 ml), HOBt (3.45 g, 25.5 mmol) was added, and the mixture was stirred at ambient temperature for 10 minutes. Solutions of tert-butyl(S)-2-amino-6-(hexanoylamino)hexanoate (6.9 g, 23 mmol) in DCM, DIPEA (12.2 ml), and EDC (5.35 g) were added sequentially. The reaction mixture was stirred at ambient temperature for 4 hours (until LCMS indicated that the tert-butyl(S)-2-amino-6-(hexanoylamino)hexanoate had been consumed), diluted with DCM (200 ml), washed with water (150 ml) and saturated NaCl solution (100 ml), and dehydrated with 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 hexane) to obtain the product (11.4 g, 75%) as an amorphous yellow solid.

[0103] Step 30: tert-butyl-(S)-2-[(S)-2-amino-6-(tert-butoxycarbonylamino)hexanoylamino]-6-(hexanoylamino)hexanoate [ka] To a solution of tert-butyl-(S)-2-[(S)-2-amino-6-(tert-butoxycarbonylamino)hexanoylamino]-6-(hexanoylamino)hexanoate (11 g, 16.6 mmol) in methanol (400 ml), 10% palladium (50% wet) supported on activated carbon (2 g) was added. The suspension was subjected to catalytic hydrogenation (H2, 1 atm, overnight) at ambient temperature (after 12 hours, LC-MS showed complete consumption of the starting material). The reaction mixture was filtered through Celite. The filtrate was concentrated under reduced pressure, re-evaporated twice with 200 ml of acetonitrile, and dried under high vacuum for 4 hours to obtain the product (8.96 g, 100%) as colorless glass.

[0104] Step 31: tert-butyl-(S)-2-[(S)-2-[3-(2-{2-[2-(benzyloxycarbonylamino)ethoxy]ethoxy}ethoxy)propionylamino]-6-(tert-butoxycarbonylamino)hexanoylamino]-6-(hexanoylamino)hexanoate [ka] To a solution of 3-(2-{2-[2-(benzyloxycarbonylamino)ethoxy]ethoxy}ethoxy)propionic acid (5.8 g, 16.6 mmol) in dry DCM (50 ml), HOBt (2.53 g, 18.7 mmol) was added, and the mixture was stirred at ambient temperature for 10 minutes. Solutions of tert-butyl-(S)-2-[(S)-2-amino-6-(tert-butoxycarbonylamino)hexanoylamino]-6-(hexanoylamino)hexanoate (8.9 g, 16.6 mmol) in DCM (50 ml), EDC (5.35 g), and DIPEA (12.2 ml) were added sequentially. The reaction mixture was stirred for 4 hours (under LC-MS monitoring), diluted with DCM (200 ml), washed with water (150 ml) and saturated NaCl solution (100 ml), and dehydrated with MgSO4. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (220 g column, 0%-10% MeOH in DCM) to obtain the target product (9.63 g, 67%) as a colorless glass.

[0105] Step 32: Synthesis of tert-butyl18-(4-aminobutyl)-49-(4-(4-(4-isobutylphenyl)butanamide)butyl)-3,16,19,47-tetraoxo-1-phenyl-2,7,10,13,23,26,29,32,35,38,41,44-dodecaoxa-4,17,20,48-tetraazapentacontane-50-oate [ka] A solution of tert-butyl-18-(4-((tert-butoxycarbonyl)amino)butyl)-49-(4-(4-(4-isobutylphenyl)butanamide)butyl)-3,16,19,47-tetraoxo-1-phenyl-2,7,10,13,23,26,29,32,35,38,41,44-dodecaoxa-4,17,20,48-tetraazapentacontane-50-oate (0.70 g, 1.22 mmol) in 90% aqueous formic acid (10 ml) was stirred at ambient temperature for 2 hours. Complete conversion was detected by LC-MS. The solvent was evaporated, and the residue was co-evaporated with ACN (3×) and toluene (3×). The residue was dissolved in a MeOH / water mixture (1:1, 25 ml) and treated with an AmberLite HPR550 ion exchange resin (OH form) at ambient temperature for 15 minutes. The resin was filtered and washed with MeOH and water. The filtrate was evaporated to obtain the target material (free base, 0.62 g, 95% yield) as a colorless liquid. The crude amine was used for the next step without further purification. LCMS:C 66 H 112 N6O 19 :m / z:1293.62, actual value m / z=1294.1[M+H] + .

[0106] Procedure 33: 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-azahexadecanamide)-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazaoctatetracontyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (15): [ka] To a solution of tert-butyl-18-(4-aminobutyl)-49-(4-(4-(4-isobutylphenyl)butanamide)butyl)-3,16,19,47-tetraoxo-1-phenyl-2,7,10,13,23,26,29,32,35,38,41,44-dodecaoxa-4,17,20,48-tetraazapentacontane-50-oate (0.62 g, 0.48 mmol), DOTA-tris(t-Bu-ester) (0.27 g, 0.48 mmol), and PyBop (0.30 g, 0.56 mmol) in DMSO (8 ml), DIEA (0.20 ml, 1.15 mmol) was added, and the reaction mixture was stirred at ambient temperature for 16 hours. Water (20 ml) was added, and the mixture was extracted with SiO2 (30 ml). The separated organic layer was washed with water (7×) and brine (8×) to remove excess PyBOP. The organic layer was dehydrated with Na₂SO₄ and concentrated under reduced pressure to obtain the target product (820 mg, 93% yield) as a colorless liquid. The compound was used for the next step without further purification. LCMS:C 94 H 162 N 10 O 26 :m / z:1848.34, actual value m / z=1849.7[M+H] + .

[0107] Procedure 34: Synthesis of tri-tert-butyl 2,2',2''-(10-(8-(3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propanamide)-39-(tert-butoxycarbonyl)-48-(4-isobutylphenyl)-2,9,37,45-tetraoxo-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazaoctatetracontyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (16): [ka] Carbon-supported palladium (10%, 0.12g, wet, 67%) was suspended in a solution of tri-tert-butyl 2,2',2''-(10-(39-(tert-butoxycarbonyl)-48-(4-isobutylphenyl)-2,9,37,45-tetraoxo-8-(3-oxo-1-phenyl-2,7,10,13-tetraoxa-4-azahexadecanamide)-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazaoctatetracontyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl) triacetate (500mg, 0.270 mmol) in MeOH (15ml). The suspension was stirred at room temperature under H2 balloon pressure for 2 hours. The mixture was filtered through Celite, and the filtrate was evaporated to obtain the target compound (387 mg, 83% yield) as a colorless liquid. The crude product was used for the next step without further purification. LCMS:C 86 H 156 N 10 O 24 :m / z:1714.21, actual value m / z=1714.7[M+H] + .

[0108] Procedure 35: Synthesis of 2,2',2''-(10-(8-(3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propanamide)-39-carboxy-48-(4-isobutylphenyl)-2,9,37,45-tetraoxo-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazaoctatetracon-tyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (17): [ka] Tri-tert-butyl 2,2',2''-(10-(8-(3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propanamide)-39-(tert-butoxycarbonyl)-48-(4-isobutylphenyl)-2,9,37,45-tetraoxo-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazaoctatetracontyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl) triacetate (387 mg, 0.225 mmol) was added to a solution of tri-tert-butyl 2,2',2''-(10-(8-(3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propanamide)-39-(tert-butoxycarbonyl)-48-(4-isobutylphenyl)-2,9,37,45-tetraoxo-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazaoctatetracontyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl) triacetate (387 mg, 0.225 mmol) in DCM (4 ml) at 0°C, to which TFA (2 ml) was added. The reaction mixture was stirred at 40°C for 2 hours, and the solvent was evaporated under reduced pressure. The residue was washed with hexane (2×) and ether (2×) to obtain the product (340 mg, 100%) as a white powder. The crude product was used for the next step without further purification. LCMS:C 70 H 124 N 10 O 24 :m / z:1489.79, actual value m / z=1490.5[M+H] + .

[0109] Synthesis of RTX-1392R [ka] RTX-1392R was prepared using Int-1 as the starting material, following the procedure below.

[0110] Procedure 36: 2,2',2''-(10-(39-carboxy-48-(4-isobutylphenyl)-2,9,37,45-tetraoxo-8-(2-oxo-1-((4-(((R)-1-oxo-1-((R)-2-((3aR,4R,6R,7aS)-5,5,7a-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxabolol-2-yl)pyrrolidine Synthesis of (19) quinolin-1-yl)propan-2-yl)carbamoyl)quinoline-7-yl)oxy)-6,9,12-trioxa-3-azapentadecanamide)-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazaoctatetracontyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid: [ka] To a solution of 2,2',2''-(10-(8-(3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propanamide)-39-carboxy-48-(4-isobutylphenyl)-2,9,37,45-tetraoxo-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazaoctatetracontyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (200 mg, 0.134 mmol) at 0°C in DMF (2 ml), DIPEA (0.19 ml) was added, and the reaction mixture was stirred at 0°C for 5 minutes. 2,3,5,6-tetrafluorophenyl 2-((4-(((2R)-1-oxo-1-((2R)-2-((3aS,4R,6R)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxabolol-2-yl)pyrrolidine-1-yl)propan-2-yl)carbamoyl)quinoline-7-yl)oxy)acetate (121 mg, 0.174 mmol) was added to DMF (1 ml), and the reaction mixture was stirred at 0°C for 0.5 hours. After complete consumption of the amine was shown by LC-MS, the crude product was subjected to HPLC purification. The desired fractions were combined and lyophilized to obtain the product (72 mg, 26%) as an off-white solid. LC-MS:C 99 H158 BN 13 O 30 :m / z:2021.19, actual value m / z=1011.7[M / 2+H] + .

[0111] Procedure 37: Synthesis of 2,2',2''-(10-(8-(1-((4-(((R)-1-((R)-2-Boronopyrrolidine-1-yl)-1-oxopropane-2-yl)carbamo-yl)quinoline-7-yl)oxy)-2-oxo-6,9,12-trioxa-3-azapentadecanamide)-39-carboxy-48-(4-isobutylphenyl)-2,9,37,45-tetraoxo-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazaoctatetracontyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (RTX-1392R(20)): [ka] 2,2',2''-(10-(39-carboxy-48-(4-isobutylphenyl)-2,9,37,45-tetraoxo-8-(2-oxo-1-((4-(((R)-1-oxo-1-((R)-2-((3aR,4R,6R,7aS)-5,5,7a-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxabolol-2-yl)pyrrolidine-1-yl)propan-2-yl)cal in acetone (1.5 ml) and 0.2 N HCl (1.5 ml) To a solution of (bamoyl)quinoline-7-yl)oxy)-6,9,12-trioxa-3-azapentadecanamide)-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazaoctatetracontyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (70 mg, 0.034 mmol) at room temperature, MeB(OH)2 (10 mg, 0.173 mmol) was added, and the reaction mixture was stirred at ambient temperature for 30 minutes. After complete consumption of the starting material was shown by LC-MS, the crude product was subjected to HPLC purification. The desired fractions were combined and lyophilized to obtain the product (31 mg, 48%) as an off-white solid. LC-MS:C 89 H 144 BN 13 O 30 :m / z:1886.98, measured value m / z=943.9[M-OH / 2] + .

[0112] Synthesis of RTX-1401R: [ka] RTX-1401R was prepared using Int-2 as the starting material, following the procedure below.

[0113] Step 38: Synthesis of tert-butyl(R)-2-[(7-hydroxy-4-quinolyl)carbonylamino]propionate [ka] 2.0 g of 7-hydroxy-4-quinoline carboxylic acid (10.5 mmol, 1 equivalent), (R)-Ala-t-butyl hydrochloride (2.0 g, 11.5 mmol, 1.1 equivalents), and HATU (5.98 g, 15.75 mmol, 1.5 equivalents) were added to 40 ml of DMF with DIPEA (4.6 ml, 2.5 equivalents) at 5°C, and the mixture was stirred at room temperature for 2 hours. Upon completion (indicated by LC-MS), H2O was added, and the product was extracted with n-butanol. The organic layer was separated, dehydrated with Na2SO4, and evaporated under reduced pressure. The resulting residue was purified by flash column (0-10% MeOH in DCM) to obtain 3 (2.01 g, yield 61%) as an off-white powder. ESI-MS m / z calculated value 316.351, measured value 317.0 [M+H] + .

[0114] Step 39: Synthesis of tert-butyl(R)-2-({7-[(benzyloxycarbonyl)methoxy]-4-quinolyl}carbonylamino)propionate [ka] Benzyl bromoacetate (4.4 g, 19.2 mmol, 3 equivalents) was added at room temperature to a suspension of tert-butyl(R)-2-[(7-hydroxy-4-quinolyl)carbonylamino]propionate (2.0 g, 6.4 mmol, 1 equivalent) and K2CO3 (2.7 g, 19.2 mmol, 3 equivalents) in DMF, and the mixture was stirred at 60°C for 16 hours. At completion, H2O was added, and the product was extracted with SiO2. The organic layer was separated, dehydrated with Na2SO4, and evaporated under reduced pressure. The resulting residue was purified by flash column chromatography (0-80% ethyl acetate in hexane) to obtain 5 (2 g, yield 67%) as an off-white powder. ESI-MS m / z calculated value 464.509, measured value 465.1 [M+H] + .

[0115] Procedure 40: Synthesis of (R)-2-({7-[(benzyloxycarbonyl)methoxy]-4-quinolyl}carbonylamino)-propionic acid [ka] 20 ml of TFA / DCM (DCM:TFA, 1:2) was added to tert-butyl(R)-2-({7-[(benzyloxycarbonyl)methoxy]-4-quinolyl}carbonylamino)propionate (2.0 g, 4.3 mmol) in DCM at room temperature, and the mixture was stirred for 4 hours. Upon completion, the solvent was evaporated under reduced pressure to obtain 6 (1.75 g, 100%) as an off-white solid. This was used in the next step without further purification. ESI-MS m / z calculated value 408.403, measured value 409.3 (M+1)+.

[0116] Procedure 41: Synthesis of benzyl(4-{[(R)-2-(2-{(1R,2S,8S)-2,9,9-trimethyl-3,5-dioxa-4-boratricyclo[6.1.1.02,6]deca-4-yl}-1-pyrrolidinyl)-1-methyl-2-oxoethylamino]carbonyl}-7-quinolyloxy) acetate [ka] To a solution of (R)-2-({7-[(benzyloxycarbonyl)methoxy]-4-quinolyl}carbonylamino)propionic acid (1.75 g, 4.3 mmol, 1 equivalent), (R)-boropro-(+)-pinanediol hydrochloride (1.35 g, 4.73 mmol, 1.1 equivalents), and HATU (2.45 g, 6.45 mmol, 1.5 equivalents) in DMF (15 ml), DIPEA (1.9 ml, 10.75 mmol, 2.5 equivalents) was added at 5°C. The mixture was stirred at room temperature for 2 hours, at which point completion of the reaction was indicated by LC-MS. H2O was added, and the product was extracted with SiO2. The organic layer was separated, dehydrated with Na2SO4, and evaporated under reduced pressure. The resulting residue was purified by flash chromatography (0-10% MeOH in DCM) to obtain 8 (1.75 g, 64% yield) as an off-white powder. ESI-MS m / z calculated value: 639.547, measured value: 640.2 [M+H] + .

[0117] Procedure 42: Synthesis of (4-{[(R)-2-(2-{(1R,2S,8S)-2,9,9-trimethyl-3,5-dioxa-4-boratricyclo[6.1.1.02,6]deca-4-yl}-1-pyrrolidinyl)-1-methyl-2-oxoethylamino]carbonyl}-7-quinolyloxy)acetic acid [ka] Activated carbon-supported palladium (5%, 0.4g) was suspended in a solution of 8 (1.75g, 2.75 mmol) in MeOH (150mL). The suspension was stirred at room temperature for 1 hour under an H2 atmosphere (40 psi). The mixture was then filtered through Celite, and the filtrate was concentrated under reduced pressure to obtain 1.45g of crude product. This material was purified by reverse-phase HPLC (30-100% gradient, 0.1% formic acid / ACN) to obtain 9 (200mg, yield 14%) as an off-white solid. ESI-MS m / z calculated value 549.425, measured value 550.3 [M+H] + .

[0118] Procedure 43: Synthesis of 2-((4-(((2R)-1-(2-Boronopyrrolidine-1-yl)-1-oxopropan-2-yl)carbamoyl)quinoline-7-yl)oxy)acetic acid (20): [ka] To a solution of 2-((4-(((R)-1-oxo-1-((R)-2-((3aR,4R,6R,7aS)-5,5,7a-trimethylhexahydro-4,6-methano-benzo[d][1,3,2]dioxabolol-2-yl)pyrrolidine-1-yl)propan-2-yl)carbamoyl)quinoline-7-yl)oxy)acetic acid (5 mg, 0.009 mmol) in a mixture of acetone (1.5 ml) and 0.2 N HCl (1.5 ml), MeB(OH)2 (10 mg, 0.173 mmol) was added at room temperature, and the reaction mixture was stirred at ambient temperature for 30 minutes. Water was added, and the reaction mixture was extracted with DCM. The organic layer was separated, dehydrated with Na2SO4, and concentrated under reduced pressure to obtain the product (8 mg, 100%) as a colorless oil. The crude product was used for the next step without further purification. LCMS:C 19 H 22 BN3O7: m / z: 415.20, measured value m / z = 416.2 [M+H] + .

[0119] Procedure 44: Synthesis of ((R)-1-((R)-2-(7-(2-oxo-2-(2,3,5,6-tetrafluorophenoxy)ethoxy)quinoline-4-carboxamide)propanoyl)pyrrolidine-2-yl)boronic acid (21): [ka] To a solution of 2-((4-(((2R)-1-(2-voronopyrrolidine-1-yl)-1-oxopropan-2-yl)carbamoyl)quinoline-7-yl)-oxy)acetic acid (8 mg, 0.019 mmol) and 2,3,5,6-tetrafluorophenol (5 mg, 0.028 mmol) in 0.5 ml of DCM, EDC.HCl (5.5 mg, 0.028 mmol) was added, and the reaction mixture was stirred at room temperature for 0.5 hours. Water was added to the reaction mixture, and it was extracted with DCM. The organic layer was separated, dehydrated with Na2SO4, and concentrated under reduced pressure to obtain the activated ester (10 mg, 100%) as a white powder. The crude product was used for the next step without further purification. LCMS:C 25 H 22BF4N3O7: m / z: 563.26, measured value m / z = 563.9 [M+H] + .

[0120] Procedure 45: Synthesis of 4-(4-(3-(1-((4-(((R)-1-((R)-2-Boronopyrrolidine-1-yl)-1-oxopropane-2-yl)carbamoyl)quinoline-7-yl)oxy)-17-((1-Carboxy-5-(4-(4-isobutylphenyl)butanamide)pentyl)carbamoyl)-2,15-Dioxo-6,9,12-Trioxa-3,16-Diazahenicosan-21-yl)thioureido)phenethoxy)-6-((16-((6-Carboxypyridine-2-yl)methyl)-1,4,10,13-Tetraoxa-7,16-Diazahenicosan-7-yl)methyl)Picolinic Acid (RTX-1401R(22): [ka] To a solution of 4-(4-(3-(1-amino-14-((1-carboxy-5-(4-(4-isobutylphenyl)butanamide)pentyl)carbamoyl)-12-oxo-3,6,9-trioxa-13-azaoctadecane-18-yl)thioureido)phenethoxy)-6-((16-((6-carboxypyridine-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)picolinic acid (6 mg, 0.0043 mmol) at 0°C in DMF (0.3 ml), DIPEA (6 μL) was added, and the reaction mixture was stirred at 0°C for 5 minutes. ((R)-1-((R)-2-(7-(2-oxo-2-(2,3,5,6-tetrafluorophenoxy)ethoxy)quinoline-4-carboxamide)propano-yl)pyrrolidine-2-yl)boronic acid (3 mg, 0.0051 mmol) was added to DMF (0.1 ml), and the reaction mixture was stirred at 0°C for 0.5 hours. After complete consumption of the amine was shown by LC-MS analysis, the crude product was subjected to HPLC purification. The desired fractions were combined and lyophilized to obtain the product (2.5 mg, 32%) as an off-white solid. LC-MS:C 89 H 124 BN 13 O23 S: m / z: 1786.89, measured value m / z = 1769.4 [M-OH] + .

[0121] Synthesis of RTX-1407S: [ka] RTX-1407S was prepared using Int-3 as the starting material, following the procedure below. Int-3 was prepared with the following modifications.

[0122] In step 24, 2-(4-isobutylphenyl)acetic acid was used instead of hexanoic acid.

[0123] Procedure 46: Preparation of [2-((S)-2-cyanopyrrolidine-1-yl)-2-oxo-ethyl]-carbamate tert-butyl ester: [ka] To a solution of (S)-pyrrolidine-2-carbonitrile hydrochloride (1 g, 7.5 mmol) in DCM (35 ml), DIPEA (3.9 ml, 22.5 mmol) and Boc-Gly-OSu (2.26 g, 8.3 mmol) were added. The reaction mixture was stirred at ambient temperature for 14 hours. The solvent was evaporated, the residue was dissolved in DCM, washed with water, dehydrated with anhydrous Mg2SO4, and filtered. The filtrate was concentrated to 20% of the initial volume and eluted on a CombiFlash® instrument (gradient 0-10% MeOH / DCM). The desired fractions were combined and evaporated to obtain the target product (1.1 g, 58%) as an amorphous solid.

[0124] Procedure 47: Preparation of (S)-1-(2-amino-acetyl)-pyrrolidine-2-carbonitrile: [ka] p-toluenesulfonic acid monohydrate (1.25 g, 6.6 mmol) was added to a stirred solution of (2-((S)-2-cyano-pyrrolidine-1-yl)-2-oxo-ethyl)-carbamate tert-butyl ester (1.1 g, 4.4 mmol) in acetonitrile (20 ml), and the mixture was stirred at ambient temperature for 14 hours. The solvent was removed under reduced pressure to obtain the tosylate salt of the target product (1.8 g, theoretically in excess yield). The compound was pure enough to be retained through the following chemical transformation.

[0125] Procedure 48: Preparation of (S)-N-(2-(2-cyanopyrrolidine-1-yl)-2-oxoethyl)-7-hydroxyquinoline-4-carboxamide: [ka] Solutions 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) were stirred at room temperature for 5 minutes. Solutions of (S)-1-(2-amino-acetyl)-pyrrolidine-2-carbonitrilate (1.5 tosylate) (1.78 mmol) and DIPEA (0.74 mL, 4.86 mmol) in DMF (5 mL) were added to the activated ester solution, and the resulting mixture was stirred at room temperature for 2 hours, during which time the progress of the reaction was monitored by LC-MS. Upon complete conversion, the reaction mixture was concentrated under reduced pressure, dissolved in a small amount of dichloromethane, and filtered. The filtrate was evaporated, dissolved in DCM, and placed on a CombiFlash® silica gel column for purification (gradient 0-20% MeOH / DCM) to obtain the target product (422 mg, 80% yield) as an off-white solid.

[0126] Procedure 49: Preparation of (S)-tert-butyl 2-((4-((2-(2-cyanopyrrolidine-1-yl)-2-oxoethyl)carbamoyl)quinoline-7-yl)oxy)acetate: [ka] Flasks containing 7-hydroxyquinoline-4-carboxylic acid [2-((S)-2-cyanopyrrolidine-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) were rapidly cooled in an ice bath. Diisopropyl azodicarboxylate (300 μL, 1.51 mmol) was added dropwise to the rapidly cooled reaction mixture. The ice bath was removed, and the resulting solution was stirred at room temperature and monitored by LC-MS. Upon completion, the solvent was removed under reduced pressure, the residue was dissolved in DCM, loaded onto a CombiFlash® silica gel column (gradient 0-10% MeOH / DCM), and purified to obtain the target product (404 mg, 61% yield) as a glassy solid.

[0127] Procedure 50: Preparation of {4-[2-((S)-2-cyano-pyrrolidine-1-yl)-2-oxo-ethylcarbamoyl]-quinoline-7-yloxy}acetic acid: [ka] To a solution of {4-[2-((S)-2-cyano-pyrrolidine-1-yl)-2-oxo-ethylcarbamoyl]-quinoline-7-yloxy}-tert-butyl acetate (141 mg, 0.322 mmol) in DCM (1.5 ml), TFA (1.51 ml, 19.64 mmol) was added dropwise at 0°C. The temperature of the stirred reaction mixture was raised to room temperature over 1 hour, during which time complete conversion was detected by LC-MS. The solvent was evaporated at room temperature, and the residue was co-evaporated with toluene (×3) at 40°C. The crude compound and intermediate 2 were pure enough to be used for subsequent conversions, but they were unstable to hydrolysis when left standing for a long time. For this reason, the compounds were prepared immediately before use.

[0128] Procedure 51: Synthesis of tert-butyl 2-[2-(3-{2-[2-(2-{2-[4-({(R)-2-[(S)-2-cyano-1-pyrrolidinyl]-1-methyl-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionyl-amino)-6-{2-[4,7,10-tris(tert-butoxycarbonylmethyl)-1,4,7,10-tetraaza-1-cyclododecyl]-acetylamino}hexanoylamino]-6-[2-(p-isobutylphenyl)acetylamino]hexanoate (3) [ka] DCM contains tert-butyl 2-[2-(3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propionylamino)-6-{2-[4,7,10-tris(tert-butoxycarbonylmethyl)-1,4,7,10-tetraaza-1-cyclododecyl]acetylamino}hexanoylamino]-6-[2-(p-isobutylphenyl)acetylamino]hexanoate (59 mg, 0.046 mmol, 1 equivalent) To a solution of ), [4-({(R)-2-[(S)-2-cyano-1-pyrrolidinyl]-1-methyl-2-oxoethylamino}carbonyl)-7-quinolyl-oxy]acetic acid (18 mg, 0.046 mmol, 1 equivalent), DIPEA (25 μL, 0.699 mmol, 3 equivalents), HOBt (6.8 mg, 0.050 mmol, 1.1 equivalents), and EDCI (7.8 mg, 0.050 mmol, 1.1 equivalents) were added. The mixture was stirred at room temperature for 16 hours, and then diluted with DCM (5 ml) and water (5 ml). The aqueous layer was extracted with DCM (3 x 5 ml), and the combined organic layers were dehydrated with Na2SO4 and concentrated. The residue was purified by flash chromatography (0-20% MeOH in DCM) to obtain the product (62 mg, 82%) as a colorless solid. LCMS:C 85 H 133 N 13 O 19 :m / z:1641.04, actual value m / z=1642.32[M+H] + .

[0129] Procedure 52: Synthesis of 2-[2-(3-{2-[2-(2-{2-[4-({(R)-2-[(S)-2-cyano-1-pyrrolidinyl]-1-methyl-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionyl-amino)-6-{2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraaza-1-cyclododecyl]acetylamino}-hexanoylamino]-6-[2-(p-isobutylphenyl)acetylamino]hexanoic acid (RTX 1407S) [ka] To a solution of tert-butyl 2-[2-(3-{2-[2-(2-{2-[4-({(R)-2-[(S)-2-cyano-1-pyrrolidinyl]-1-methyl-2-oxoethylamino}carbonyl)-7-quinolyloxy]acetylamino}ethoxy)ethoxy]ethoxy}propionyl-amino)-6-{2-[4,7,10-tris(tert-butoxycarbonylmethyl)-1,4,7,10-tetraaza-1-cyclododecyl]acetyl-amino}hexanoylamino]-6-[2-(p-isobutylphenyl)acetylamino]hexanoate (62 mg, 0.037 mmol, 1 equivalent), TFA (283 μL, 3.70 mmol) was added. The resulting mixture was stirred for 10 hours. After removing volatile substances and purifying the residue by HPLC, the product (35.8 mg, 68%) was obtained as a colorless solid. LCMS:C 69 H 101 N 13 O 19 :m / z:1416.62, actual value m / z=1417.60[M+H] +

[0130] Step 53: Synthesis of tert-butyl N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-acetyllignate [ka] Acetic anhydride (524 µl, 5.51 mmol) was added to a stirred solution of 5-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-(tert-butoxy)-6-oxohexane-1-aminium chloride (3 g, 4.6 mmol) and DIEA (1.76 ml, 10.12 mmol) in DCM (24 ml). The mixture was stirred at room temperature for 90 minutes, during which time LCMS showed complete conversion. The reaction mixture was diluted with ethyl acetate, washed with water and brine, dehydrated with anhydrous MgSO4, and filtered. After evaporation of volatiles under reduced pressure, the target compound (2.9 g, 91%) remained as an off-white solid.

[0131] Procedure 54: Synthesis of 2-((E)-2-((E)-3-((E)-2-(3,3-dimethyl-5-sulfonate-1-(3-(trimethylammonio)propyl)-indoline-2-ylidene)-ethylidene)-2-(4-(3-((2,5-dioxopyrrolidine-1-yl)oxy)-3-oxopropyl)phenoxy)cyclohexe-1-en-1-yl)vinyl)-3,3-dimethyl-1-(3-(trimethylammonio)propyl)-3H-indole-1-ium-5-sulfonate: [ka] To a solution of ZW800-1 (100 mg, 0.105 mmol) in anhydrous DMSO (10 ml), dipyrrolidino(N-succinimidyloxy)carbenium hexafluorophosphate (130 mg, 0.3 mmol) was added, followed by the dropwise addition of N,N-diisopropylethylamine (0.2 mL, 1.1 mmol) at room temperature. The reaction mixture was stirred at ambient temperature for 16 hours, during which time complete conversion was detected by LC-MS. 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), mixed, and allowed to stand for 30 minutes. The solid was filtered and dried under vacuum to obtain 2-((E)-2-((E)-3-((E)-2-(3,3-dimethyl-5-sulfonate-1-(3-(trimethylammonio)-propyl)indoline-2-ylidene)ethylidene)-2-(4-(3-((2,5-dioxo-pyrrolidine-1-yl)oxy)-3-oxopropyl)phenoxy)cyclohexe-1-en-1-yl)vinyl)-3,3-dimethyl-1-(3-(trimethylammonio)propyl)-3H-indly-1-ium-5-sulfonate as a green powder (51 mg, 46%), which was used directly for the next step without further purification. LCMS:C 46 H 61 N9O 12 :m / z:1040.32, actual value m / z=1040.5[M] + .

[0132] Step 55: [ka] DMSO (1 mL) contains 2-((E)-2-((E)-3-(2((E)-3,3-dimethyl-5-sulfonato-1-(3-(trimethylammonio)propyl)indoline-2-ylidene)ethylidene)-2-(4-(3-((2,5-dioxopyrrolidine-1-yl)oxy)-3-oxopropyl)phenoxy)cyclohexa-1-en-1-yl)vinyl)-3,3-dimethyl-1-(3-(trimethylammonio)propyl)-3H-indole-1-yl To a solution of um-5-sulfonate (0.070 g, 0.067 mmol), a solution of N6-acetyl-N2-((1-((4-(((R)-1-((S)-2-cyanopyrrolidine-1-yl)-1-oxopropan-2-yl)carbamoyl)quinoline-7-yl)oxy)-2-oxo-6,9,12-trioxa-3-azapentadecane-15-oil)-L-lysyl)-L-lysine (0.040 g, 0.045 mmol) in DMSO (1 mL) was added. N,N-diisopropylethylamine (0.070 g, 0.067 mmol) was added at room temperature, and the mixture was stirred for 16 hours. Complete conversion was detected by LC-MS. After the reaction was complete, volatiles were removed, and the residue was purified by HPLC to obtain the desired product.

[0133] Synthesis of RTX-1371R: [ka] Int-3 was used as the starting material and prepared following a procedure similar to that of RTX-1401R. Int-3 was prepared with the following modifications.

[0134] In step 27, 2-(4-isobutylphenyl)acetic acid was used instead of hexanoic acid.

[0135] Synthesis of RTX-1384S [ka] The following modifications were made, and the preparation was carried out using a procedure similar to that used for preparing Int-2.

[0136] I used step 53 instead of step 14.

[0137] The product from step 19 was used for further synthesis of RTX-1384S by introducing quinoline in step 51 and removing the protecting group in step 52. Subsequently, fluorophores were introduced in steps 54 and 55.

[0138] Synthesis of RTX-1391R: [ka] Int-3 was used as the starting material and prepared following a procedure similar to that of RTX-1401R. Int-3 was prepared with the following modifications.

[0139] In step 27, 2-(4-isobutylphenyl)butanoic acid was used instead of hexanoic acid.

[0140] RTX-1400R synthesis [ka] Int-2 was used as the starting material and prepared following a procedure similar to that of RTX-1401R. Int-2 was prepared with the following modifications.

[0141] In step 14, 2-(4-isobutylphenyl)acetic acid was used instead of 2-(4-isobutylphenyl)butanoic acid.

[0142] Synthesis of RTX-1402R [ka] Int-1 was used as the starting material and prepared following a procedure similar to that of RTX-1392. Int-1 was prepared with the following modifications:

[0143] Step 23 was used in place of step 10 to introduce the Macropa chelating agent.

[0144] Synthesis of RTX-1411R [ka] Procedure 56: (tert-butyl N6-((benzyloxy)carbonyl)-N2-(2,2-dimethyl-4-oxo-3,8,11,14,17,20,23,26,29-nonaoxa-5-azadotricontane-32-oil)lignate)(3): [ka] DIPEA (2.2 equivalents) was added dropwise to a mixture of 1 (1 equivalent), 2 (1.1 equivalents), and HATU (1.2 equivalents) in DMF at 0°C. The resulting reaction mixture was stirred at room temperature for 3 hours. Completion of the reaction was observed by LC-MS. The solvent was evaporated to dryness, and the resulting crude reaction mixture was purified by Combi Flash using 0-100% SiO in hexane (quantitative yield) LC-MS:C 42 H 73 N3O 15 :m / z:859.50, actual value m / z=860.6[M+H] + .

[0145] Procedure 57: tert-butyl(2,2-dimethyl-4-oxo-3,8,11,14,17,20,23,26,29-nonoxa-5-azadotricontane-32-oil)lignate(4): [ka] 10% palladium (100 mg) supported on activated carbon was added to a solution of 3 (0.5 g) in methanol (10 ml). The suspension was subjected to catalytic hydrogenation (H2, under balloon pressure) at ambient temperature for 3 hours (LCMS control - complete consumption of starting material). The reaction mixture was filtered through Celite, and the filtrate was concentrated under reduced pressure. The residue was dried under high vacuum to obtain the target product (quantitatively) as a colorless liquid. LCMS:C 34 H 67 N3O 13 :m / z:825.92, actual value m / z=826.9[M+H]+ .

[0146] Procedure 58: 2,5-Dioxopyrrolidine-1-yl 4-(4-iodophenyl)butanoate (5): [ka] To a stirred solution of iodophenylbutanoic acid (2.0 g, 1 equivalent) in DCM (20 ml), N-hydroxysuccinimide (1.3 equivalents) was added at 0°C. The reaction mixture was stirred at the same temperature for 5 minutes, and EDC.HCl (1.3 equivalents) was added. The resulting solution was stirred at room temperature for 1 hour (the progress of the reaction was monitored by TLC). The reaction mixture was diluted with DCM (20 ml), washed with water (50 ml), separated, and the organic layer was dehydrated with Na2SO4 and evaporated. The residue was washed with diethyl ether (2 × 20 ml) to obtain compound 5 (2.0 g) as an off-white solid.

[0147] Procedure 59: tert-butyl N2-(2,2-dimethyl-4-oxo-3,8,11,14,17,20,23,26,29-nononaoxa-5-azadotricontane-32-oil)-N6-(4-(4-iodophenyl)butanoyl)lignate(6): [ka] To a solution of 4 (2.0 g, 1 equivalent) in DCM (25 ml), 2,5-dioxopyrrolidine-1-yl-4-(4-iodophenyl)butanoate (5) (1.0 equivalent) was added at room temperature, followed by the addition of DIPEA (1.2 equivalents). The reaction mixture was stirred for 30 minutes. After the reaction was complete (the progress of the reaction was monitored by LC-MS), the solvent was evaporated under reduced pressure, and the crude residue was purified by Combi Flash using MeOH:DCM (0-20%) to obtain compound 6 as a light brown, viscous oil (1.8 g). LC-MS:C 44 H 76 IN3O 14 m / z: 997.44, measured value (tert-butyl group loss) m / z = 471.45 [M / 2 + H] + .

[0148] Procedure 60: N2-(1-amino-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-oil)-N6-(4-(4-iodophenyl)butanoyl)lysine(7): [ka] To a stirred solution of 6 (1.8 g, 1 equivalent) in DCM (10 ml), TFA (10 ml) was added at room temperature, and the reaction mixture was stirred for 30 minutes. After the reaction was complete (the progress of the reaction was monitored by LC-MS), the volatiles were evaporated under reduced pressure, co-distilled with acetonitrile (3 × 30 ml), and dried under high vacuum for 30 minutes. The crude product (2.0 g) was used in the next step of the reaction sequence without further purification. LC-MS:C 35 H 60 IN3O 12 :m / z:841.32, actual value m / z=842.32[M+H] + .

[0149] Procedure 61: 2,5-Dioxopyrrolidine-1-yl N2-(((9H-Fluoren-9-yl)methoxy)carbonyl)-N6-(tert-butoxycarbonyl)lignate (8): [ka] EDC.HCl was added at 0°C to a stirred solution of 8a (2.0 g, 1 equivalent) and NHS (1.3 equivalents) in DCM (10 ml), and the mixture was stirred at room temperature for 30 minutes. The reaction mixture was diluted with 20 ml of DCM, washed with 50 ml of water, dehydrated with Na2SO4, and evaporated. The residue was washed with diethyl ether (2 × 25 ml) to obtain compound 8 (2.0 g) as an off-white solid. LCMS:C 30 H 35 N3O8: m / z: 565.24, measured value m / z = 588.19 [M + Na] + .

[0150] Procedure 62: N2-(10-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2,2-dimethyl-4,11-dioxo-3,15,18,21,24,27,30,33,36-nonoxa-5,12-diazanonatricontan-39-oil)-N6-(4-(4-iodophenyl)butanoyl)lysine(9): [ka] To a solution of 7 (2.0 g, 1 equivalent) in DMF (10 ml), DIPEA (3.0 equivalents) was added, followed by the addition of a solution of 8 (1.0 equivalent) in DMF at room temperature, and the mixture was stirred for 30 minutes. After the reaction was complete (the progress of the reaction was monitored by LC-MS), the solvent was evaporated under reduced pressure, and the crude residue was purified by silica gel column chromatography eluting in MeOH:DCM (0-40%) to obtain compound 9 as a light brown, viscous oil (1.7 g). LC-MS:C 61 H 90 IN5O 17 :m / z:1291.54, actual value m / z=647.46[M / 2+H] +

[0151] Procedure 63: N2-(10-amino-2,2-dimethyl-4,11-dioxo-3,15,18,21,24,27,30,33,36-nonoxa-5,12-diazanonatricontane-39-oil)-N6-(4-(4-iodophenyl)butanoyl)lysine(10): [ka] Diethylamine (4 ml) was added to a stirred solution of 9 (0.4 g, 1 equivalent) in DCM (4 ml) at room temperature, and the reaction mixture was stirred for 30 minutes. After the reaction was complete (the progress of the reaction was monitored by LC-MS), the solvent was evaporated under reduced pressure, co-distilled with acetonitrile (3 × 30 ml), and dried under high vacuum for 30 minutes. Crude product 10 (0.5 g) was used in the next step as obtained, without any further purification. LC-MS:C 46 H 80 IN5O 15:m / z:1069.47, actual value m / z=535.98[M / 2+H] + .

[0152] Procedure 64: N2-(10-(1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13-tetraoxa-4-azahexadecane-16-amide)-2,2-dimethyl-4,11-dioxo-3,15,18,21,24,27,30,33,36-nononaoxa-5,12-diazanonatriacontane-39-oil)-N6-(4-(4-iodophenyl)butanoyl)lysine(12): [ka] To a solution of 10 (0.5 g, 1 equivalent of crude) in DMF (5 ml), DIPEA (3.0 equivalents) was added, followed by the addition of 11 (1.0 equivalent) in DMF (5 ml) at room temperature. The reaction mixture was stirred for 30 minutes. After the reaction was complete (the progress of the reaction was monitored by LC-MS), the solvent was evaporated. The crude residue was purified by silica gel column chromatography (MeOH:DCM, 0-30%) to obtain compound 9 as a light brown, viscous oil (0.7 g, crude). LC-MS:C 70 H 107 IN6O 21 :m / z:1494.65, actual value m / z=499.24[M / 3+H] + , 521.5[M / 3+Na] + .

[0153] Procedure 65: N2-(30-(1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13-tetraoxa-4-azahexadecane-16-amide)-34-amino-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azatetratriacontanoyl)-N6-(4-(4-iodophenyl)butanoyl)lysine(13): [ka] To a stirred solution of 12 (0.7 g, 1 equivalent of crude product) in DCM (4 ml), TFA (6 ml) was added at room temperature, and the mixture was stirred for 30 minutes. After the reaction was complete (the progress of the reaction was monitored by LC-MS), the solvent was evaporated under reduced pressure, co-distilled with acetonitrile (3 × 10 ml), and dried under high vacuum for 30 minutes. The crude product (0.6 g) was used in the next step as obtained, without any further purification. LC-MS:C 65 H 99 IN6O 19 :m / z:1394.65, actual value m / z=698.85[M / 2+H] + .

[0154] Procedure 66: 2,2',2''-(10-(8-(1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13-tetraoxa-4-azahexadecane-16-amide)-39-carboxy-48-(4-iodophenyl)-2,9,37,45-tetraoxo-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazaoctatetracontyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (15): [ka] To a stirred solution of Solution 13 (0.6 g, 1 equivalent of crude) in DMF (5 ml), DIPEA (2 equivalents) was added, followed by the addition of DOTA NHS ester (14, 1 equivalent). The reaction mixture was stirred for 30 minutes. After the reaction was complete (the progress of the reaction was monitored by LC-MS), the solvent was evaporated, and the crude residue was used in the next step in its isolated state without further purification (0.65 g, crude). LC-MS:C 81 H 125 IN 10 O 26 :m / z:1780.78, actual value m / z=892.12[M / 2+H] + .

[0155] Procedure 67: 2,2',2''-(10-(8-(3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propanamide)-39-carboxy-48-(4-iodophenyl)-2,9,37,45-tetraoxo-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazaoctatetracontyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid(16): [ka] Diethylamine (5 ml) was added to 15 (0.65 g) at room temperature, and the reaction mixture was stirred at the same temperature for 30 minutes. Upon completion (the progress of the reaction was monitored by LC-MS), the solvent was evaporated, and the crude residue was purified by preparative HPLC (ACN:H2O, 5-95%, 40 min) to obtain pure compound 16 as a colorless solid (240 mg). LC-MS:C 66 H 115 IN 10 O 24 :m / z:1558.71, actual value m / z=780.46[M / 2+H] + .

[0156] Procedure 68: 2,2',2''-(10-(39-carboxy-48-(4-iodophenyl)-2,9,37,45-tetraoxo-8-(2-oxo-1-((4-((1-oxo-1-((R)-2-((3aR,4R,6R,7aS)-5,5,7a-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxabolol-2-yl)pyrrolidine- 1-yl)propan-2-yl)carbamoyl)quinoline-7-yl)oxy)-6,9,12-trioxa-3-azapentadecane-15-amide)-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazaoctatetracontyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate(18): [ka] To a stirred solution of compound 16 (20 mg, 1 equivalent) in DMF (5 ml), DIPEA (2 equivalents) was added, followed by compound 17 (1.5 equivalents). The reaction mixture was stirred for 30 minutes. Upon completion (the progress of the reaction was monitored by LC-MS), the solvent was evaporated, and the crude residue was purified by preparative HPLC (ACN:H2O, 5-95%, 40 mins) to obtain pure compound 18 as a white solid (10 mg). LC-MS:C 66 H 115 IN 10 O 24 :m / z:2089.97, actual value m / z=698.31[M / 3+H] + .

[0157] Procedure 69: 2,2',2''-(10-(8-(1-((4-((1-((R)-2-Boronopyrrolidine-1-yl)-1-oxopropane-2-yl)carbamoyl)quinoline-7-yl)oxy)-2-oxo-6,9,12-trioxa-3-azapentadecane-15-amide)-39-carboxy-48-(4-iodophenyl)-2,9,37,45-tetraoxo-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazaoctatetracontyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate(1411R): [ka] To a stirred solution of compound 16 (10 mg, 1 equivalent) in acetone (1 ml), 1 N HCl (1 ml) was added, followed by the addition of methylboronic acid (5 equivalents). The reaction mixture was stirred at room temperature for 12 hours. After the reaction was complete (monitored by LC-MS), the compound was purified by preparative HPLC (ACN:H2O, 5-95%, 40 minutes (60% at 30 minutes, then 95% at 40 minutes)) to obtain the pure compound as a white solid (4 mg). LC-MS:C 85 H 135 BIN 13 O 30 :m / z:1955.86, actual value m / z=647.31[M-18 / 3+H] + .

[0158] Synthesis of RTX-1409R [ka] With the following modifications, Boc-L-Lys(H)-OtBu was used as the starting material and prepared according to a procedure similar to that of RTX-1411R.

[0159] In step 58, 2-(4-iodophenyl)acetic acid was used instead of iodophenylbutanoic acid.

[0160] Synthesis of RTX-1410R [ka] Using Boc-L-Lys(H)-OtBu as the starting material, it was prepared following a procedure similar to that of RTX-1411R.

[0161] Synthesis of RTX-1413R [ka] With the following modifications, Boc-L-Lys(H)-OtBu was used as the starting material and prepared according to a procedure similar to that of RTX-1411R.

[0162] In step 58, 2-(4-iodophenyl)acetic acid was used instead of iodophenylbutanoic acid.

[0163] The Macropa chelating agent was introduced using procedure 23 instead of procedure 66.

[0164] Synthesis of RTX-1414R [ka] With the following modifications, Boc-L-Lys(H)-OtBu was used as the starting material and prepared according to a procedure similar to that of RTX-1411R.

[0165] The Macropa chelating agent was introduced using procedure 23 instead of procedure 66.

[0166] Synthesis of RTX-1415R [ka] The following modifications were made, and the sample was prepared following a procedure similar to that of the RTX-1411R.

[0167] The Macropa chelating agent was introduced using procedure 23 instead of procedure 66.

[0168] Example 2 - Compounds of the present invention that bind to FAP with high affinity The protease reaction was constructed in a 384-well plate (Greiner) in a total volume of 20 μL, as described below.

[0169] Recombinant proteins were pre-diluted in assay buffer consisting of 100 mM HEPES, pH 7.5, 0.1% BSA, 0.01% Triton X-100, and 1 mM DTT, and dispensed into 384-well plates (10 μL per well). The test compounds were serially pre-diluted in DMSO and added to the assay wells by acoustic dispensing (Labcyte Echo 550). Control samples (0% inhibition in the absence of the inhibitor, DMSO only) and 100% inhibition (in the absence of the enzyme) were constructed in four replicates and used to calculate the % inhibition rate in the presence of the compound. The concentration of DMSO was equal to 1% in all samples.

[0170] The compound was 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 2× FAM-labeled substrate peptide (FAM-GPRPFNYLAKK-NH2) and prepared in the same assay buffer. The final enzyme concentration was 0.5 nM. The final substrate peptide concentration was 1 μM.

[0171] The reaction was carried out at room temperature. The incubation time was 3 hours for human FAP and 0.5 hours for mouse FAP. After incubation, the termination buffer (100 × IC) was used. 50 The kinase reaction was quenched by adding 50 μL of assay buffer (supplemented with a reference inhibitor).

[0172] The final plates were analyzed using a microfluidic electrophoresis apparatus (Caliper LabChip® 3000, Caliper Life Sciences / Perkin Elmer). The change in relative intensity between the peptide substrate and the cleaved product was used as the measured parameter. The activity in each test sample was determined as the product ratio relative to the sum (PSR): P / (S+P) (where P is the peak height of the product and S is the peak height of the substrate). Percentage inhibition rate (P inh ) was determined using the following equation: P inh =(PSR 0%inh -PSR compound ) / (PSR 0%inh -PSR 100%inh )×100, in the formula, PSR compound This is the product / sum ratio in the presence of the compound, and is PSR. 0%inh This is the product / sum ratio in the absence of the compound, and is PSR. 100%inh This is the product / sum ratio in the absence of the enzyme. IC of a compound. 50 To determine the (50% inhibition rate), %inh data (P relative to compound concentration) inh The values ​​are fixed using a four-parameter S-shaped dose-response model with XLfit software (IDBS). These values ​​are compiled and grouped in Table 1, where AA is IC. 50 <250 pM represents IC 50 <0.1nM; B is for ICs with a capacitance of 0.1~0.5nM 50 ;C is an IC with a capacitance of 0.5~5.0nM 50 ;D is an IC with a impedance of 5.0~100nM 50 ;E represents IC50 > 100 nM. Some of the compounds in this disclosure have an IC50 greater than the detection limit of this assay. 50A value (i.e., less than 250 pM) was given.

[0173] [Table 1]

[0174] In pyrrolidine, the S stereoisomer with the S configuration is considerably more potent than the corresponding R stereoisomer. The inhibition rate study described above also showed 1.5 nM LC 50 Having, [ka] We revealed that extending the quinolinyl core chain significantly improves its activity. Specifically, progressively lengthening the chain resulted in improvements with each extension, and the addition of the final amino acid (lysine) led to a further increase in activity.

[0175] Greater differentiation was observed between certain compounds of the present invention and FAPI-46 in surface plasmon resonance assay data (FAP-46 was measured to have an affinity of 255 pM, while certain disclosed compounds had an affinity stronger than 20 pM).

[0176] Example 3 - Surface plasmon resonance studies to investigate the binding affinity of the compound of the present invention As discussed in Example 2, some compounds of the present invention have binding affinities exceeding the detection limit of the assay (i.e., about 250 pM). Surface plasmon resonance gives a remarkably low detection limit of around 1–10 pM. Both the non-metallated compounds of the present invention and the non-radioactive lanthanum (La) and lutetium (Lu) labeled compounds were subjected to surface plasmon resonance assays to analyze their binding affinities to human, mouse, and rat FAP.

[0177] SPR method: SPR experiments were performed at 37°C using Biacore 8K (Cytiva Europe GmbH). Ligands were immobilized in flow cell 2 for all 8 channels by SA-biotin capture on SA tips (Cytiva #BR100531) (mouse FAP, Biosystems Acro / catalog number FAP-M82Q8 and human FAP, Biosystems Acro / catalog number FAP-H82Q6) or by amide coupling on CM5 tips (Cytiva #29149603) (rat FAP, Biosystems Acro / catalog number FAP-R5246). For immobilization, ligand solutions with concentrations of 10–20 micrograms / mL were prepared and DMSO-free running buffer HBS-P+ (Cytiva #BR100671) was used on the surface of the selected flow cell at a temperature of 25°C at a flow rate of 5 microliters / min. Each of the eight channels' flow cell 1 was left blank and used as a reference surface.

[0178] K of the compound D The determination was made in a single-cycle experiment. HBS-P+ containing 1% DMSO was used as the running buffer for the experiment. A 1:4 dilution series with six concentrations (100 nM, 25 nM, 6.25 nM, 1.56125 nM, 0.390625 nM, 0.0976525 nM, or 400 nM, 100 nM, 25 nM, 6.25 nM, 1.56125 nM, 0.390625 nM) and a 0 value as a blank were used. The experiment was conducted at a flow rate of 100 microliters / min, with a contact time of 80 seconds for each solution and a final dissociation time of 2500 seconds. Solvent correction was performed at the start and end of the run.

[0179] All data were analyzed using Biacore Insight Evaluation Software (version 5.0.18.22102 Cytiva2022). For all channels, flow cell 1 was used as the reference, and solvent corrections were applied to all experiments via the measured calibration curve. Single-cycle data were analyzed using a 1:1 coupling kinetic model. All measurements were performed in double or triple configurations.

[0180] Average dissociation constant (K D The values ​​were determined from repeated experiments with n=2 or n=3 and are summarized in Table 2 below. Some of the compounds of the present invention gave binding affinity above the detection limit of this assay (i.e., <1 pM). For the compounds tested, labeling with non-radioactive La or Lu did not significantly alter the FAP binding affinity when evaluated within the limits of the SPR assay.

[0181] [Table 2]

[0182] Example 4 - 18 Radiolabeling of FAP target compounds with direct F labeling 18 F is received from the manufacturer, loaded onto a Sep Pak QMA Light Plus cartridge, and eluted into a glass reaction vial using a Cs2CO3 / K222 solution. 18 F is azeotropically dried at 95°C under a flow of N2 using acetonitrile (3 × 1 ml). 2.5 mg of the FAP target compound is dissolved in 0.3 ml of DMSO, and then dried. 18Add F to the vial containing F. Seal the vial and heat to 90°C for 15 minutes. Cool the vial, add a 1N HCl solution to the vial, then seal it and heat to 95°C for 10 minutes. Cool the reaction mixture to neutralize it, then load it onto a Semi Prep HPLC column for purification and prepare it appropriately for use. Alternatively, a suitable FAP target compound having a chelating agent can be used. 18 F can be used for AlF labeling, where AlCl3 stock in acetate buffer (22.5 μL, 45 nmol, 0.9 equivalents) is mixed with sodium acetate (200 μL) 18 Add to solution F and let the reaction vial stand at room temperature for 5 minutes. Next, add 12.5 μL of FAP target compound solution (50 nmol scale) from the precursor stock to the vial. Correct the pH to approximately 4.0 by adding 1 v / v% acetic acid in 15 μL of water. Add 200 μL of cosolvent, EtOH, seal the reaction vial, and then heat at 100°C for 15 minutes. Dilute the reaction mixture to 9.5 ml and load it onto a C18 Sep Pak cartridge. Elute the product with 300 μL of acidified EtOH and prepare for use.

[0183] Example 5 - Radiation labeling method and results General procedure: Combine 20 μg of precursor (unless otherwise specified) with the indicated radioisotope. The radioisotope is eluted from the generator. 68 Either Ga (or a commercially available HCl solution) is obtained and buffered to a final pH of 4-6 with varying amounts of 3N NaOAc. The reactant is reacted in a C18 Sep-Pak Lite cartridge as needed for further purity and / or reconstitution for injection. The labeling results are shown in Table 3.

[0184] [Table 3-1] [Table 3-2]

[0185] Example 6 - Detection of tumors in mouse models using the compound of the present invention Approximately 10 BALB / c nude mice 7 U-87 (human glioblastoma) cells are transplanted to induce a tumor. A certain amount of the present invention 18 A fluorine-labeled FAP-targeting agent is administered to mice by intravenous injection (e.g., tail vein), and the mice are sacrificed one hour later. The mice are then imaged. 18 This study evaluates tumor binding of F-labeled FAP target compounds. The in vivo distribution of these compounds is assessed through analysis of excised tissue samples from various organs.

[0186] Example 7 - Biomedical distribution research Female BALB / C nude mice were subcutaneously injected with U87MG cells in a Matrigel:PBS 1:1 mixture into the right shoulder. When the tumor reached a volume of 150–500 mm³, a radiolabeled ligand was administered intravenously (IV) via the tail vein. At various time points after injection, the mice were humanely euthanized by phlebotomy, and tissue samples (bladder, blood, urine, bone (femur), heart, lung, liver, both kidneys, small intestine (including contents), large intestine (including contents), muscle (quadriceps femoris), tumor, and tail) were collected, calibrated, and counted using a gamma counter. The activity of each collected tissue was measured in counts per minute (CPM). Triple alicots of radioactive tracers were also assayed using a gamma counter to calculate a function (μCi / CPM) for converting counts to units of activity. The values ​​were attenuated for the number of injections and corrected for background radiation. The in vivo distribution of compounds [177Lu]RTX-1371R, [177Lu]RTX-1391R, and [177Lu]RTX-1392R demonstrated localization of the compounds within tumors and minimum concentrations in other organs (see Figures 1-3). This in vivo distribution demonstrated a time-dependent increase in tumor selectivity, clearly indicating high affinity for FAP. Incorporation of D-alanine significantly increased tumor retention at 72 and 168 hours (see Figures 4-6). The compounds disclosed herein show a significant increase in tumor retention at later time points (48-168 hours post-injection) compared to the existing compounds [177Lu]PNT5555 ([177Lu]RTX-1418R) and [177Lu]3BP-2286 ([177Lu]RTX-1386S) (see Figures 7 and 8).

[0187] Example 8 - Cryofluorescence tomography (CFT) experiment U-87MG cells in Matrigel:PBS 1:1 were subcutaneously inoculated into the right shoulder of female Nu / J or NCr-Foxn1 nude mice. Tumors were at least 150 mm in size. 3Upon reaching the specified volume, 1.5 μg of ligand diluted in sterile PBS was administered intravenously (IV) via tail injection. At various points after injection, the mice were humanely euthanized by CO2 inhalation and cervical dislocation, and frozen for 10 minutes in a hexane bath, which had been cooled by surrounding a metal container filled with hexane with dry ice for 30 minutes prior to freezing. The mice were stored at -80°C, and at the time of use, the mice were embedded in a block of the frozen OCT compound, and the distribution of ligand in the mice was imaged using cryo-tomography (CFT) with Xerra (Emit Imaging). The top of the block was sectioned with a razor blade to separate 35 μm thick cuts, which were then subjected to fluorescence excitation and subsequent 500 ms fluorescence emission reading at wavelengths suitable for the ligand-conjugated fluorophores. In addition to fluorescence images, RGB images were also taken after each sectioning. Sectioning and subsequent imaging were performed throughout the entire block. Data were processed using the Xerra Recon application (Emit Imaging) and analyzed via Vivoquant (Invicro). For the heart, liver, lungs (one ROI each), kidneys (one ROI each), joints (right hind leg knee), muscles (right hind leg quadriceps), and brain, a single spherical ROI within the boundaries of the organ of interest was visualized. Whole-body regions of interest (ROIs) were also visualized. 2D ROIs were visualized through tumors and then interpolated to form 3D ROIs capturing the entire tumor volume. Mean fluorescence values ​​(in arbitrary units) of ROIs in mice injected with different compounds at different time points were compared. See Figure 10. Mean organ ROIs were normalized to whole-body or muscle ROI values ​​by dividing the values ​​for all other organs by the whole-body or muscle values. See Figure 11. These normalized mean ROI values ​​were compared across compounds and time points to evaluate the signal-to-noise ratio.

[0188] Example 9 - Study on the efficacy and tolerability of U-87 xenotransplantation U-87MG cells in Matrigel:PBS 1:1 were subcutaneously inoculated into the right flank of female Nu / J mice. The tumor was 120 mm. 3 ~250mm3 volume (

number

[0189] Equivalents While certain embodiments are illustrated and described, those skilled in the art, after reading the prior specification, can perform modifications, substitutions of equivalents, and other types of modifications to the compounds or salts of the Art described herein, their pharmaceutical compositions, derivatives, prodrugs, metabolites, tautomers, or racemic mixtures thereof, as described herein. Each of the embodiments described above could also include, or incorporate, any or all of, such modifications or embodiments disclosed in any or all of the other embodiments.

[0190] The Art is not limited in any particular aspect to the embodiments described herein, and these are intended to be examples of individual embodiments of the Art. Many modifications and variations of the Art can be made in a manner that will be apparent to those skilled in the art without departing from its spirit and scope. In addition to those enumerated herein, functionally equivalent methods within the scope of the Art will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to be included within the scope of the attached Claims. It will be understood that the Art is not limited to any particular method, reagent, compound, composition, labeled compound or biological system, and that these can naturally be diverse. It will also be understood that the terms used herein are intended to describe only specific aspects and are not intended to be limiting. Thus, it is intended that this specification is merely illustrative, with respect to the breadth, scope and spirit of the Art as shown only by the attached Claims, the definitions therein, and their equivalents.

[0191] All publications, patents, and other documents relating to this specification are incorporated herein by reference in their entirety.

Claims

1. The following structural formula: 【Chemistry 1】 [In the formula, n is either 0 or 1; Z is NH, O, S, CR ６ R ７ , NHCO, CONH, or the heterocycle bonded to Y via a cyclic nitrogen atom of a 4- to 7-membered nitrogen-containing heterocycle, A is NH, O, S or CR ６ R ７ And; B contains a branched, unbranched or cyclic aliphatic group of up to 30 carbon atoms optionally interrupted by up to 10 heteroatoms, or of a peptidyl chain of up to 20 amino acid residues, wherein B is selected from F, Cl, Br, I, =O, OR ６ , OCOR ６ , COOR ６ , CN, =NR ６ , NR ６ R ７ , =S and SR ６ and is optionally substituted with 1 to 5 groups selected from, provided that B contains at least 3 atoms in the chain between the D group and the A group; D is OPO ３ H ２ , PO ３ H ２ OSO ３ H, SO ３ H and COOH, and their C １ ~C ４ Selected from the group consisting of alkyl esters; X is either O or S; R １ This includes chelating groups, optical dyes or fluorophores, cytotoxic agents, or immunostimulants, or R ５ A benzoyl group optionally substituted by one or more groups represented by; R ３ C １ ~C ８ Alkyl or C １ ~C ４ It is Aralquil, and in the formula, The alkyl and aryl portions of the aralkyl group are each optionally and independently of F, Cl, Br, I, branched, unbranched, or cyclic C. １ ~C ６ aliphatic group, OR ６ ,OCOR ６ COOR ６ ,CHO,COR ６ ,CH ２ OR ６ , NR ６ R ７ ,CH ２ NR ６ R ７ , SR ６ ,=O,=S and=NH are substituted; R ４ is CN or B(OH) ２ And; Each R ５ These are independently halo, cyano, halomethyl, and N ＋ (CH ３ ) ３ W － (In the formula, W － (These are selected from anions that are acceptable as medicines; R ６ and R ７ H or C １ ~C ６ Selected from the group consisting of alkyl groups; R ８ C １ ~C ４ It is alkyl, R ９ H and C １ ~C ４ Selected from alkyl groups, or R ８ and R ９ Together with those intervening carbon atoms, C ３ ~C ６ A compound represented by [forming a cycloalkyl group] or a pharmaceutically acceptable salt thereof.

2. B is a branched or unbranched aliphatic group of 3 to 20 carbon atoms, which is independently and optionally intercepted by up to 10 heteroatoms or by a peptidyl chain of up to 5 amino acid residues, wherein the aliphatic group is F, Cl, Br, I, =O, OR ６ ,OCOR ６ COOR ６ ,CN,=NR ６ , NR ６ R ７ , = S, or SR ６ The compound according to claim 1, which is optionally substituted with [the specified compound].

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

4. The following structural formula: 【Transformation 3】 A compound or pharmaceutically acceptable salt thereof as described in claim 3, represented by [the specified method].

5. The following structural formula: 【Chemistry 4】 A compound or pharmaceutically acceptable salt thereof as described in claim 3, represented by [the specified figure].

6. The following structural formula: 【Transformation 5】 A compound or pharmaceutically acceptable salt thereof as described in claim 3 or 4.

7. R ３ However, F, Cl, Br, I, or C １～４ C is optionally substituted with alkyl. １～８ Alkyl or C １～４ A compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, which is aralkyl.

8. R ３ However, I or C １～４ C is optionally substituted with alkyl. １～８ Alkyl or C １～４ A compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, which is aralkyl.

9. R ３ However, C １ ~C ４ C is optionally substituted with alkyl. １ ~C ８ Alkyl or C １ ~C ４ A compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 8, which is aralkyl.

10. R ３ The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 8, wherein the compound is methyl, (4-isobutylphenyl)methyl, (4-isobutylphenyl)propyl, (4-iodophenyl)methyl, or (4-iodophenyl)propyl.

11. R ３ The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 10, wherein the compound is methyl, (4-isobutylphenyl)methyl, and (4-isobutylphenyl)propyl.

12. R ８ is methyl and R ９ is H, a compound according to any one of claims 1 to 11 or a salt thereof acceptable as a medicament.

13. A compound or pharmaceutically acceptable salt thereof according to any one of claims 3 to 12, wherein o is 1 and m is 3 to 12.

14. A compound or pharmaceutically acceptable salt thereof according to claim 13, wherein m is 8.

15. A compound or pharmaceutically acceptable salt thereof according to any one of claims 3 to 12, wherein o is 0.

16. A compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 15, wherein n is 1.

17. R １ A compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 16, wherein the compound is a fluorophore or an optical dye.

18. The fluorophore is, 【Transformation 6】 The compound or pharmaceutically acceptable salt thereof according to claim 17, wherein the optical dye is selected from the group consisting of carbocyanin, indocarbocyanin, oxacarbocyanin, thiacarbocyanin, merocyanine, polymethine, coumarin, rhodamine, xanthene, fluoroceine, borodipyromethane (BODIPY), VivoTag-680, VivoTag-S750, AlexaFluor dyes (e.g., AlexaFluor 660, AlexaFluor 680, AlexaFluor 700, AlexaFluor 750, AlexaFluor 790) and DylightFluor dyes.

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

20. The chelating group is 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), p-SCN-Bn-NOTA, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), p-SCN-Bn-DOTA (also known as 2B-DOTA-NCS), PIP-DOTA, diethylenetriaminepentaacetic acid (DTPA), PIP-DTPA, AZEP-DTPA, ethylenediaminetetraacetic acid (EDTA), triethylenetetraamine-N,N,N',N'',N''',N'''-hexaacetic acid ( TTHA), 7-[2-(bis-carboxymethylamino)-ethyl]-4,10-bis-carboxymethyl-1,4,7,10-tetraazacyclododecyl-1-yl-acetic acid (DEPA), 2,2',2''-(10-(2-(bis(carboxymethyl)amino)-5-(4-isothiocyanatophenyl)pentyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (3p-C-DEPA-NCS), NETA, {4-carboxymethyl-7-[2-(carboxymethylamino)-ethyl]- Perhydro-1,4,7-triazonin-1-yl}-acetic acid (NPTA), diacetylpyridine bis(benzoylhydrazone), 1,4,7,10,13,16-hexaazacyclooctadecane N,N',N'',N''',N'''',N'''''-hexaacetic acid (HEHA), octadentate terephthalamide ligand, 2,2'-(4-(2-(bis(carboxymethyl)amino)-5-(4-isothiocyanatophenyl)pentyl)-10-(2-(bis(carboxymethyl)amino)ethyl)-1,4,7,10-teto Raazacyclododecane-1,7-diyl)diacetic acid, N,N'-bis[(6-carboxy-2-pyridyl)methyl]-4,13-diaza-18-crown-6(H2macropa),6-((16-((6-carboxypyridine-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)-4-isothiocyanatopicolinic acid (macropa-NCO),6-((16-((6-carboxypyridine-2-yl)methyl)-1,4,10,13-tetraoxa-7,The compound according to claim 19 or a pharmaceutically acceptable salt thereof, wherein the residue is a chelating agent selected from 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-tetraazabicyclododecyl-1-yl]acetamide (TCMC or DOTAM).

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

22. The compound according to claim 19 or a pharmaceutically acceptable salt thereof, wherein the residue of the chelating agent is a residue of p-SCN-Bn-NOTA, p-SCN-Bn-DOTA, NOTA, or DOTA.

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

24. R １ However, R ５ A benzoyl group that is optionally substituted by one or more groups represented by Each R ５ is independently selected from halo, cyano, halomethyl, N ＋ (CH ３ ) ３ W － ; and W － A compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 16, wherein the compound is a pharmaceutically acceptable anion.

25. Each R ５ However, independently, fluoro, cyano, trifluoromethyl, N ＋ (CH ３ ) ３ W － A compound or a pharmaceutically acceptable salt thereof, selected from the compounds described in claim 24.

26. R ５ The halo or fluoro group represented by １８ F is the compound according to claim 24 or 25, or a pharmaceutically acceptable salt thereof.

27. R ２ However, H is R ４ However, B(OH) ２ A compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 26.

28. R ２ However, H is R ４ A compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 26, wherein the compound is CN. 【Request Item 29】 【Chemistry 7-1】 【Chemistry 7-2】 【Transformation 7-3】 A compound according to claim 1 or a pharmaceutically acceptable salt thereof, represented by a structural formula selected from the following. 【Request Item 30】 【Chemistry 8-1】 【Chemistry 8-2】 A compound according to claim 1 or a pharmaceutically acceptable salt thereof, represented by a structural formula selected from the following.

31. A compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 16, 19 to 23, or 27 to 30, wherein a residue of the chelating agent is chelated with a radionuclide.

32. The aforementioned radioactive nuclide is １７７ Lu, １７５ Lu, ４５ Sc, ６４ Cd, ６７ Cd, ６８ Cd, 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 A compound or pharmaceutically acceptable salt thereof, selected from Fm and uranium-230, according to claim 31.

33. The aforementioned radioactive nuclide is ２２５ Ac, ２３３ Ra and ２１２ A compound according to claim 31 or a pharmaceutically acceptable salt thereof, which is an alpha-emitting radionuclide such as Pb.

34. The radioactive nuclide is an Auger electron beam emitting radionuclide, or １７７ Lu, ９０ Y and ６７ A compound according to claim 31 or a pharmaceutically acceptable salt thereof, which is a beta-emitting radionuclide such as Cu.

35. Residues of macropa-NCS or macropa-NCO, ２２５ A compound according to claim 21 or a pharmaceutically acceptable salt thereof, chelated with Ac.

36. a. A compound or pharmaceutically acceptable salt thereof as described in any one of claims 1 to 35; and b. A pharmaceutical composition comprising a carrier or diluent that is pharmaceutically acceptable.

37. A method for treating affected tissue in a subject, comprising the step of administering to the subject an effective amount of a compound or pharmaceutically acceptable salt according to any one of claims 31 to 35, or a pharmaceutical composition according to claim 36, wherein the affected tissue expresses fibroblast-activating protein alpha, and the radionuclide is a therapeutic radionuclide.

38. The method according to claim 37, wherein the affected tissue is cancerous.

39. The method according to claim 38, 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.

40. The method according to claim 38, wherein the affected tissue is fibrous.

41. A method for imaging a region in a subject that has or is suspected to have affected tissue or fibrous tissue expressing fibroblast-activating protein alpha, a. A step of administering to a subject a diagnostically effective amount of a compound or pharmaceutically acceptable salt thereof according to any one of claims 16 to 19, 25 to 30, or 32 to 35, or a pharmaceutical composition according to claim 36, wherein the radionuclide is a diagnostic radionuclide; b. The step of exposing the area in the object to an imaging device; and c. A method comprising the step of obtaining an image of the affected tissue in the region.

42. The method according to claim 41, wherein the region has or is suspected to have affected tissue including primary cancer or metastasis of cancer.

43. The method according to claim 41, wherein the region has, or is suspected to have, affected tissue including fibrous tissue.

44. A method for imaging tumors, a. The step of bringing a compound or a pharmaceutically acceptable salt thereof according to any one of claims 18 to 19 into contact with the tumor and / or surrounding tissue in an amount sufficient to bind to the tumor; b. Irradiating the tumor and / or surrounding tissue at wavelengths absorbed by the compound; and c. A method comprising the step of detecting a signal from the compound, thereby imaging the tumor and / or surrounding tissue.

45. A method for treating the affected tissue, a. The step of administering to a subject an amount effective to contact and bind to the affected tissue of a compound or a pharmaceutically acceptable salt thereof according to any one of claims 18 to 19; b. A method comprising the step of using the compound as a positional reference and irradiating a region of the compound-bound with one or more doses of external beam radiation, thereby treating the affected tissue with radiation.

46. The method according to claim 45, wherein the compound comprises a chelate group having a radionuclide that emits gamma rays or positrons, or an optical dye or fluorophore, or other detectable radiation.

47. A method for treating affected tissue, comprising the steps of: administering to a subject an amount effective to contact and bind to the affected tissue, a compound or pharmaceutically acceptable salt thereof as described in any one of claims 18 to 19; and using the compound as a positional reference for a surgical application to guide the removal of an area of ​​the affected tissue, thereby excising the affected tissue.

48. The method according to claim 47, wherein the compound comprises a chelate group having a radionuclide that emits gamma rays or positrons, or an optical dye or fluorophore, or other detectable radiation.

49. The method according to claim 37, further comprising administering a chemotherapy drug, administering an immunotherapy drug, or irradiating the affected tissue with one or more doses of external beam radiation.

50. The method according to claim 38, wherein the compound or a pharmaceutically acceptable salt thereof is administered intravenously, subcutaneously, intramuscularly, topically, or directly into the bladder to an object in need thereof.

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