FAP-targeting agent platforms and scaffolds

Abstract

The present invention is directed to scaffolds and compounds for targeting fibroblast activation protein (FAP) in cancer-associated fibroblasts (CAFs). The scaffolds are represented by formula (I): JPEG2025532147000048.jpg35170 (wherein X represents CH or O, and R 1 is H, Me, CH(CH3)C2H5, CH2CH(CH3)2, CH(CH3)2, CH2OH, CH2SH, CH(OH)CH3, CH2C(O)NH2, CH2CH2C(O)NH2, (CH2) m CO2H, (CH2) m NH2, where m is 1 to 4, and R 2 and R 3 each independently represents H or F. It comprises a (4-quinoynolyl)glycinyl-2-cyanopyrrolidine scaffold in which the 8-position of the quinoynolyl moiety is substituted with a bridging carbon or oxygen atom, according to

Description

[Technical Field]

[0001] The present invention relates to compounds for targeting fibroblast activation proteins (FAPs) in cancer-associated fibroblasts (CAFs), in particular FAP targeting agents based on a (4-quinoinolyl)glycinyl-2-cyanopyrrolidine scaffold. [Background technology]

[0002] Various FAP targeting agents based on the (4-quinolinolyl)glycinyl-2-cyanopyrrolidine scaffold have been developed and studied in the prior art for tumor imaging and treatment (see Lindner et al., Cancers 2021, 13, 5744). Early FAP targeting agents were disclosed by Jansen et al., ACS Med. Chem. Lett. 2013, 4, 491-496 and Jansen et al., J. Med. Chem. 2014, 57, 3053-3074, which describes UAMC-1110, a FAP inhibitor with a fluorinated (4-quinolinol)-glycyl-2-cyanopyrrolidine scaffold and nanomolar affinity for FAP.

[0003] For a FAP-targeting agent to be clinically meaningful, it must be functionalized with a payload, such as a radioisotope, fluorescent dye, or cytotoxic drug. For this purpose, the quinolinol moiety of the scaffold is commonly used. The current reference compound in clinical use is FAPI-46 (Loktev et al., Journal of Nuclear Medicine 2019 October, 60(10)1421-1429). FAPI-46 was developed after the development of FAPI-04 (Lindner et al., J. Nucl. Med. 2018;59:1415-1422) and showed an improved tumor-to-organ ratio compared to FAPI-04.

[0004] Other functionalized FAP-targeting compounds are described in WO 2021 / 160825 and Millul et al., PNAS 2021, Vol. 118, No. 16, e2101852118, and are referred to as OncoFAPs. These compounds are functionalized on the quinoinolyl using a bridging nitrogen extending to a carboxylic acid group that allows for connection to a payload. The drawback of this carboxylic acid group is that most metal chelating groups, prosthetic groups, optical dyes, etc. are not readily available as carboxylic acid-reactive substances. They are typically commercially available as amine-reactive substances. Therefore, in OncoFAP compounds, an additional linker (-NHCH2-CH2-NH-) is used to expose an amine that can react with a payload.

[0005] Other FAP targeting compounds are also described in WO2019 / 083990.

[0006] Despite recent advances, there remains a need to provide improved platforms and improved FAP targeting agents based on the (4-quinoinolyl)glycinyl-2-cyanopyrrolidine scaffold. However, previous research and development has shown that FAPs impose strict structural requirements on their inhibitors, and steric or electronic parameters are not sufficient to rationalize assay data (Jansen et al., J. Med. Chem. 2014, 57, 3053-3074). Therefore, developing new FAP targeting agents is difficult, because it is difficult, if not impossible, to predict which substituents and positions on the (4-quinoinolyl)glycinyl-2-cyanopyrrolidine scaffold will yield good inhibitors. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] WO2021 / 160825 [Patent Document 2] WO2019 / 083990 [Patent Document 3] WO2019 / 154859

Patent document 4

Patent document 5

Non-licensed literature

[0008] [Non-licensed document 1] Lindner et al., Cancers 2021, 13, 5744 [Non-licensed document 2] Jansen et al., ACS Med. Chem. Lett. 2013, 4, 491~496 [Non-licensed document 3] Jansen et al., J. Med. Chem. 2014, 57, 3053~3074

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Non-licensed literature 9

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Non-licensed Document 11

[0009] It is an object of the present invention to provide a platform of compounds and such compounds that exhibit improved FAP binding affinity and / or tumor uptake, including tumor-to-organ distribution. It is a further object of the present invention to provide a platform that allows access to such compounds. [Means for solving the problem]

[0010] The present inventors have surprisingly found that substituting a carbon or oxygen atom at position 8 of the (4-quinoinolyl)glycinyl-2-cyanopyrrolidine scaffold provides a variety of FAP targeting agents that exhibit good FAP binding affinity and / or tumor uptake, including good tumor-to-organ distribution. [Brief explanation of the drawings]

[0011] [Figure 1] Intermediate 10 was prepared according to the scheme in FIG. [Figure 2] FIG. 2 shows the LC / MS profile of eFAP-8. [Figure 3] Figure 3 shows the radio-TLC chromatogram of [In]In-eFAP-12, showing a radiolabeling yield (RCY) of 97.8%. [Figure 4] FIG. 4 shows radio-HPLC chromatograms of [ 111 In]In-eFAP-12 demonstrating the stability of the radiolabeled conjugate over time. [Figure 5] FIG. 5 shows the results of hFAP and mFAP inhibition experiments in the presence of eFAP compounds. [Figure 6] 1 shows the results of a competitive binding assay for eFAP-6 and eFAP-7 performed in HT-1080.hFAP cells. [Figure 7] 1 shows the results of a cytotoxicity assay performed with eFAP-18 in FAP+ U-87 cells. [Figure 8] 1 shows a comparison of the biodistribution in selected organs of [ 111 In]In-FAPI-46 and [ 111 In]In-eFAP-6 in HT-1080.hFAP tumor-bearing mice. [Figure 9] Evaluation of the targeting performance of eFAP-17 in near-infrared fluorescence imaging of U-87 tumor-bearing mice after intravenous injection of 2 nmol of the probe is shown. A) NIRF imaging of U-87 tumor-bearing mice 1 hour after intravenous injection of 2 nmol of CW800 (left, negative control) or eFAP-17 (right) and B) images of excised tumors 2.5 hours after administration of CW800 (left) or eFAP-17 (right). DETAILED DESCRIPTION OF THE INVENTION

[0012] scaffold Thus, the present invention is directed to scaffolds and compounds, particularly pharmaceutical compounds or pharmaceutically acceptable salts thereof, comprising a (4-quinoinolyl)glycinyl-2-cyanopyrrolidine scaffold (also referred to herein as a scaffold) in which the 8-position of the quinoinolyl moiety is substituted with a bridging carbon or oxygen atom. The bridging carbon or oxygen atom is attached to or allows attachment to a payload such as a therapeutic agent, a diagnostic agent, or a combination thereof, preferably a radioisotope, a fluorescent dye, a drug, or a combination thereof. The scaffold is depicted in the structure below, which also shows the numbering of the quinoinolyl moiety:

[0013] [ka]

[0014] It will be appreciated that this scaffold may contain additional substituents such as fluoride and others known for FAP inhibitors based on the (4-quinoinolyl)glycinyl-2-cyanopyrrolidine scaffold.

[0015] More particularly, the present invention provides a compound of formula (I):

[0016] [ka]

[0017] (In the formula, X represents CH2 or O, preferably O, R 1 is H, Me, CH(CH3)C2H5, CH2CH(CH3)2, CH(CH3)2, CH2OH, CH2SH, CH(OH)CH3, CH2C(O)NH2, CH2CH2C(O)NH2, (CH2) q CO2H, (CH2) q NH2, where q is 1 to 4; R 2 and R 3 each independently represents H or F. The present invention relates to a compound or a salt thereof comprising a scaffold of the formula:

[0018] In a preferred embodiment, the moiety of formula (I) is of formula (II)

[0019] [ka]

[0020] (In the formula, R 4 represents a spacer, preferably an optionally substituted hydrocarbon, preferably aliphatic, spacer, Q contains the payload) As exemplified by 4 The antibody is conjugated to a payload such as a therapeutic agent, a diagnostic agent, or a combination thereof via

[0021] Among the (4-quinoinolyl)glycinyl-2-cyanopyrrolidine scaffolds of the present invention, preferred are those in which R 2 and R 3 At least one of is F, more preferably at least R 2 is F (R 2 and R 3 is achiral), most preferably R 2 and R 3 are both F.

[0022] R 1 The substituent is preferably H.

[0023] Spacers and Linkers R 4 The spacer represented by the formula (IIIa) (wherein R 4' represents an optionally substituted hydrocarbylene or an optionally substituted alkylene ether, preferably alkylene, more preferably C1-C8 alkylene), contains a nitrogen atom, which can be used to connect Q to the (4-quinoinolyl)glycinyl-2-cyanopyrrolidine scaffold.

[0024] [ka]

[0025] Spacer (R 4 and R 4' ) can be substituted or interrupted with one or more heteroatoms such as one or more of OH, NH, SO, and halogens such as F, Cl, Br, and I. 4 and R 4'is preferably based on an aliphatic C1-C8 amino terminal spacer. Such spacers have been found to provide particularly good FAP binding affinity and tumor uptake. Thus, a particularly preferred embodiment of the compounds of the present invention is a compound of formula (IIIb):

[0026] [ka]

[0027] (wherein n is 1 to 6, preferably 1). It has a structure as follows.

[0028] Compounds of structure according to formula (IIIa) and (IIIb) containing a Q functional group can be represented by formula (VIIa) and (VIIb)

[0029] [ka]

[0030] (In the formula, X, R 1 ~R 4 , R 4' and n is as defined for formulas (IIIa) and (IIIb). Advantageously, this amine-terminated compound can be directly reacted and / or conjugated with most commercially available metal chelating groups, prosthetic groups, optical dyes, etc. Thus, this intermediate compound is highly versatile and is an aspect of the present invention.

[0031] The group represented by Q (also referred to herein simply as Q) comprises at least one payload, such as a therapeutic agent and / or a diagnostic agent. It will be understood that in addition to this payload, Q may also comprise a linking functional group (also referred to herein as a linker), one or more additional scaffolds, and / or a combination of at least two of a radioisotope, a fluorescent dye, and a drug.

[0032] In certain embodiments, Q is R4 or NHR 4' The linker may serve to link one or more payloads and one or more scaffolds. The linker may aid in the solubility of the compound. For example, 4-amino-3-hydroxybutanoic acid (GABOB) can be used to aid in water solubility and lower lipophilicity (as observed by logP values). The linker may be cleavable or non-cleavable (see, e.g., Kovtun et al., Cancer Letters 255 (2007) 232-240; Beck et al., Nature Reviews Drug Discovery 16 (2017) 315-337; and WO2021 / 160825), or a combination of both (i.e., one or more cleavable moieties linking one or more payloads and one or more other non-cleavable moieties linking one or more other payloads or scaffolds).

[0033] The compound containing the linker L has the formula (IV)

[0034] [ka]

[0035] (wherein L represents a linker, Q 1 comprises a payload, Z comprises an additional scaffold, r is 1 or greater, and s is 0 or greater). The additional scaffold can preferably be represented by the formula (Z)

[0036] [ka]

[0037] (Wherein, X and R 1 ~R 4 represents a group as defined for formula (II) It can be seen that the structure is as follows:

[0038] The linker may comprise a cleavable section, a non-cleavable section, and a combination thereof. As used herein, a section refers to a structural feature that connects a scaffold to one or more payloads and optional additional scaffolds. Because a linker can connect a scaffold to multiple payloads and additional scaffolds, the linker can contain multiple sections, each of which can be cleavable or non-cleavable. Whether a cleavable or non-cleavable section of the linker is preferred depends, inter alia, on the payload. It can be understood that when a compound comprises a single payload (i.e., r=1) and no additional scaffold (i.e., s=0), the linker may comprise a single linker section.

[0039] Typically, when the linker connects a fluorophore dye and / or a radioisotope and stability of the scaffold and payload is desired, the linker comprises a non-cleavable linker section, which is also preferred for attaching additional scaffolds.

[0040] Suitable non-cleavable linker sections in this regard may be based on or include straight-chain or branched amino acids, such as glycine, alanine, β-alanine, 3-aminopropionic acid, 4-aminobutyric acid, 4-amino-3-hydroxybutanoic acid, 5-aminovaleric acid, 6-aminohexanoic acid, 7-aminoheptanoic acid, 8-aminooctanoic acid, 9-aminononanoic acid, 10-aminodecanoic acid, 2-aminooctanoic acid, etc. In some embodiments, the linker comprises a peptide spacer (Xaa) 1~4 wherein each Xaa is independently a proteinogenic or non-proteinogenic amino acid residue, and wherein each peptide backbone amino group is independently optionally methylated. In certain embodiments, each non-proteinogenic amino acid residue is a D-amino acid, N-amino acid, or D-amino acid of a proteinogenic amino acid. ε ,N ε ,N ε-trimethyl-lysine, 2,3-diaminopropionic acid (Dap), 2,4-diaminobutyric acid (Dab), ornithine (Orn), homoarginine (hArg), 2-amino-4-guanidinobutyric acid (Agb), 2-amino-3-guanidinopropionic acid (Agp), β-alanine, 4-aminobutyric acid, 5-aminovaleric acid, 6-aminohexanoic acid, 7-aminoheptanoic acid, 8-aminooctanoic acid, 9-aminononanoic acid, 10-aminodecanoic acid, 2-aminooctanoic acid, 2-aminoadipic acid (2-Aad), 3-aminoadipic acid (3-Aad), 4-(aminomethyl)cyclohexane-1-carbonyl (Amcha), 4-amino-1-carboxymethyl-piperidinyl (Pip), cysteic acid, diglycolic acid, and NH2(CH2)2[O(CH2)2] t C(O)OH (where t=1 to 36). The linker may contain one or more of these aforementioned groups. For example, the linker may be based on a single GABOB unit, such as in eFAP-27 (see below), but it may also be based on two (or more), such as in eFAP-24 (see below).

[0041] In a further preferred embodiment, the linker L has the formula La to Ln;

[0042] [ka]

[0043] (In the formula, further, Y 1 is selected from the group consisting of C and N, preferably N for formulae La, Lg and Lh, and C for formula Ld, Y 2 is selected from the group consisting of C, N, and O, preferably N and C, more preferably C) The compound includes a moiety having a structure shown in any one of the following:

[0044] In still further preferred embodiments, the linker L comprises a moiety having a structure according to any of formulas Laa, Lfa, Lga, Lha, and Lma, which are specific versions of formulas La, Lf, Lg, Lh, and Lm, respectively.

[0045] [ka]

[0046] Unless the stereochemistry of an atom is specifically shown herein, it is understood that the stereochemistry of said atom is undefined, and that the structure represents all possible stereoisomers of said atom. For example, the structure Lfa represents at least two diastereoisomers: trans and cis isomers. However, preferably, the linker represented by this formula has a trans configuration, as shown by the following formula Lfa':

[0047] [ka]

[0048] Linkers having structures according to Formula IIaa and Lfa are based on 4-amino-1-carboxymethyl-piperidinyl (Pip) and 4-(aminomethyl)cyclohexane-1-carbonyl (Amcha), respectively. Pip-containing linkers, like any other amine-containing linkers disclosed herein, may be cationic under physiological conditions, thereby affecting the overall charge of the conjugate. Preferably, the linker comprises Amcha or Pip.

[0049] In certain embodiments, the linker may comprise a cleavable linker section comprising a moiety that is cleavable in vivo. Such linker sections are particularly preferred when the payload comprises a drug that would benefit from delivery and release from the scaffold at a target site, such as near or within tumor cells (see, e.g., Kovtun et al., Cancer Letters 255 (2007) 232-240; Beck et al., Nature Reviews Drug Discovery 16 (2017) 315-337; and WO2021 / 160825).

[0050] Therefore, enzyme-sensitive cleavable linker sections are preferred linker sections, most preferred are cleavable sections that are sensitive to enzymes that are overexpressed in tumors, such as glutathione.

[0051] Examples of cleavable moieties that can be included in the cleavable linker section include amides, esters, carbamates, hydrazones, thiazolidines, methylenealkoxycarbamates, and disulfides. The cleavable moiety may comprise one or more of such moieties and may, for example, comprise a peptide, oligosaccharide, or another oligomeric sequence that can be selectively cleaved in vivo.

[0052] In a preferred embodiment, the cleavable moiety comprises a disulfide moiety or a terminal thiol that allows for disulfide linkage to a payload that also comprises a terminal thiol (e.g., mertansine or DM1).

[0053] Thus, in certain preferred embodiments, the cleavable linker section is based on or comprises a linear or branched mercaptocarboxylic acid, such as thioglycolic acid, thiolactic acid (also known as 2-mercaptopropionic acid), 3-mercaptopropionic acid (3-MPA), etc. Thus, the linker has the formula Lo

[0054] [ka]

[0055] (In the formula, R 5 represents an optionally present second spacer, preferably an optionally substituted second aliphatic spacer, more preferably C1-C6 alkylene, most preferably ethylene) The compound may include a moiety having a structure according to

[0056] The cleavable linker section may comprise a self-immolative moiety, which can release an unfunctionalized payload after activation by a cascade of reactions (see, e.g., RV Gonzaga et al., Journal of Pharmaceutical Sciences 109 (2020) 3262-3281). Examples of suitable self-immolative moieties include those in which p-aminobenzyl carbamate is coupled to either valine-citrulline (Val-Cit-PAB) or β-glucuronide, and those based on Grob cleavage (see, e.g., Ferhati et al., Org. Lett. 2021, 23, 21, 8580-8584).

[0057] The linker can be a dimeric linker linking the scaffold to one payload (i.e., r=1 and s=0 in Formula (IV)). In other embodiments, the linker is multimeric, meaning that it links the scaffold to a payload and at least one additional payload and / or scaffold (i.e., r≧1 and s+r≧2 in Formula (IV)). Thus, in certain embodiments, the linker links one or more additional scaffolds and / or at least two combinations of a radioisotope, a fluorescent dye, and a drug to the scaffold. Generally, if the linker links more than five entities, the compound is too large and renal excretion is adversely hindered. Thus, the linker typically links the scaffold to a payload and up to three additional payloads and / or scaffolds (i.e., s+r=1, 2, 3, or 4 in Formula (IV), while r≧1).

[0058] Depending on the payload, an appropriate linker can be selected. For example, when two payloads or one payload and one additional scaffold, one of which has a free carboxylic acid and the other has a free amine, are linked, the linker can be based on glutamic acid. Similarly, when two payloads, each having a free carboxylic acid, are linked, the linker can be based on or include lysine or its analog. Thus, certain embodiments can be, for example, a compound represented by formula (V):

[0059] [ka]

[0060] (wherein p is 1 to 6, preferably 3; Q 1' and Q 1'' each independently comprises a payload such as a radioisotope, a fluorescent dye, and / or a drug) It is a compound having the structure

[0061] In embodiments where a linker connects the scaffold to one additional scaffold, the linker may be represented by formula (Lp) to (Lv):

[0062] [ka]

[0063] and 1 The compound and the linker are the multimer moieties represented by formulas (Lp) to (Lv) and (Z) and Q 1 It can be understood that the scaffold (Z) may contain additional atoms and / or linking sections between X and R. Scaffold (Z) is structurally a scaffold as described hereinabove. Scaffolds are preferably structurally identical, but may also contain additional atoms and / or linking sections between X and R. 1 ~R4 The two may differ in this respect.

[0064] Diagnostic and Therapeutic Agents The present invention is not particularly limited to specific payloads, diagnostic and therapeutic agents. Advantageously, the present invention allows a wide variety of payloads to be used in combination with the scaffold without compromising the FAP affinity and / or tumor uptake of the compound. It can be understood that appropriate payloads and agents can be selected based on the desired use, such as diagnosis and treatment.

[0065] Known payloads for previously developed FAP targeting agents, for example those disclosed in WO2019 / 154859 and WO2021 / 160825, are also suitable for use with the present invention.

[0066] Chelating Agents and Radioisotopes In certain embodiments, the payload comprises a radioisotope complexed with a chelator. The radioisotope can be any suitable radioisotope. See, for example, Tornesello et al., Molecules 2017, 22(8), 1282, US2021 / 0402016A1, WO2021 / 005125A1, and Price and Orvig, Chem. Soc. Rev., 2014, 43, 260-290 and references cited therein. The radioisotope can be an α- or β-particle emitter, an Auger emitter, a positron emitter, and / or a γ-emitter. Alpha particle emitters and β-particle emitters, e.g., 90 Y, 212 Pb, 177 Lu, 188 Re, 186 Re, 67 Cu, 64 Cu, 195m Pt, 212 Bi, 213 Bi, 211 At, 225 Ac, 131 I, etc. can be used in therapy. Suitable gamma emitters, e.g. 99m Tc, 67 Ga, 111In, etc. can be used for SPECT imaging, while positron emitters, e.g. 68 Ga, 64 Cu, 18 F and the like are useful for PET imaging. Therefore, the radioisotope M of the present invention is preferably 18 F, 123 I, 124 I, 125 I, 131 I, 76 Br, 77 Br, 212 Pb, 203 Pb, 64 Cu, 67 Cu, 212 Bi, 68 Ga, 213 Bi, 225 Ac, 243 Am, 211 At, 217 At, 154 Dy, 148 Gd, 146 Sm, 147 Sm, 149 Tb, 152 Tb, 155 Tb, 161 Tb, 165 Er, 72 As, 77 As, 47 Sc, 188 Re, 186 Re, 105 Rh, 109 Pd, 199 Au, 175 Yb, 142 Pr, 114m In, 94m Tc, 99m Tc, 227 Th, 229 Th, 59 Fe, 60 Cu, 61 Cu, 62 Cu, 67 Ga, 44 Sc, 89 Zr, 90 Nb, 86 Y, 90 Y, 111 In, 177 Lu, 117mSn, 153 Gd, 153 Sm, and 166 From the group consisting of Ho, preferably 18 F, 64 Cu, 67 Ga, 68 Ga, 90 Y, 111 In, 177 Lu, 212 Pb and 225 From the group consisting of Ac, most preferably 18 F, 68 Ga, 111 In, 225 Ac and 177 Lu.

[0067] Various chelating agents are known in the art to bind radioisotopes. The chelating agents of the present invention are pharmaceutically acceptable chelating agents. Because not all chelating agents are equally suitable for all radioisotopes, the chelating agent is typically selected based on the radioisotope desired for diagnosis or treatment. Typically, radioisotope chelating agents include a cyclic or branched polyaminopolycarboxylic acid moiety or an amide derivative thereof. See, for example, Tornesello et al., Molecules 2017, 22(8), 1282; US2021 / 0402016A1; WO2021 / 005125A1; and Price and Orvig, Chem. Soc. Rev., 2014, 43, 260-290 and references cited therein.

[0068] In a preferred embodiment of the present invention, the radioisotope chelator is - derivatives such as DOTA (1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid, also known as tetraxetane) and p-SCN-Bn-DOTA (2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecanetetraacetic acid); - PSC (1,4,7,10-tetraazacyclododecane-7-acetamido-1,4,10-triacetic acid); - DO3A (1,4,7,10-tetraazacyclododecane-N,N',N''-triacetic acid); - DOTAGA (1,4,7,10-tetraazacyclododecane-1-(glutaric acid)-4,7,10-triacetic acid); - DO3AM (2,2',2''-(1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetamide); - derivatives such as DOTAM (2-[4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetamide) and p-SCN-Bn-TCMC (2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraaza-1,4,7,10-tetra(2-carbamoylmethyl)cyclododecane); - NOTA (1,4,7-triazacyclononane-N,N',N''-triacetic acid); - NODAGA (1-(1,3-carboxypropyl)-4,7-carboxymethyl-1,4,7-triazacyclononane); - NODASA (1,4,7-triazacyclononane-1-succinic acid-4,7-diacetic acid); - CB-DO2A (4,10-bis(carboxymethyl)-1,4,7,10-tetraazabicyclo[5.5.2]tetradecane); - 3p-C-DEPA (2-[(carboxymethyl)][5-(4-nitrophenyl-1-[4,7,10-tris-(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]pentan-2-yl)-amino]acetic acid); - TCMC (1,4,7,10-tetrakis(carbamoylmethyl)1,4,7,10-tetraazacyclododecane); - DTPA (diethylenetriaminepentaacetic acid) and DTPA derivatives such as CHX-A''-DTPA (2-(p-isothiocyanatobenzyl)cyclohexyldiethylenetriaminepentaacetic acid) and 1B4M-DTPA; - TETA (1,4,8,11-tetraazacyclotetradecane 1,4,8,11-tetraacetic acid) and analogues and derivatives such as C-NETA; - NE3TA (7-[2-[(carboxymethyl)amino]ethyl]-1,4,7-triazacyclononane-1,4-diacetic acid) and derivatives such as C-NE3TA; - CB-TE2A (4,11-bis(carboxymethyl)-1,4,8,11 tetraazabicyclo[6.6.2]hexadecane); NETA ({4-[2-(bis-carboxymethylamino)-ethyl]7-carboxymethyl-[1,4,7]triazonan-1-yl}-acetic acid); and NETA derivatives such as 3p-C-NETA (see Sun et al., ACS Omega 2020, 5, 44, 28615-28620). - H2azapa (N,N'-[1-benzyl-1,2,3-triazol-4-yl]methyl-N,N'-[6-(carboxy)pyridin-2-yl]-1,2-diaminoethane) and other picolinic acid derivatives such as H2dedpa (1,2-[[6-(carboxy)pyridin-2-yl]methylamino]ethane), H4octapa (N,N'-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,N'-diacetic acid), H4py4pa, H4Pypa, H6phospha, H4CHXoctapa, H5decapa (N,N''-[[6-(carboxy)pyridin-2-yl]methyl]-diethylenetriamine-N,N',N''-triacetic acid), and H4neunpa-p-Bn-NO2; - SHBED (N,N'-bis(2-hydroxy-5-sulfobenzyl)ethylenediamine-N,N'-diacetic acid); - HBED (N,N'-bis(2-hydroxybenzyl)-ethylenediamine-N,N'-diacetic acid); - H2-MACROPA(N,N'-bis[(6-carboxy-2-pyridyl)methyl]-4,13-diaza-18-crown-6); - PCTA (3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid); - Me-3,2-HOPO (see Ramdahl, Bioorganic & Medicinal Chemistry Letters 26 (2016) 17, 4318-4321); - CB-TE1A1P (1,4,8,11-tetraazacyclotetradecane-1-(methanephosphonic acid)-8-(methanecarboxylic acid)); - CB-TE2P (1,4,8,11-tetraazacyclotetradecane-1,8-di(methanephosphonic acid)); - MM-TE2A (N-monomethyl 1,8-N,N'-bis-(carboxymethyl)-1,4,8,11-tetraazacyclotetradecane); - DM-TE2A (N,N'-dimethyl 1,8-N,N'-bis-(carboxymethyl)-1,4,8,11-tetraazacyclotetradecane); - sarcofazine and its derivatives, such as SarAr (1-N-(4-aminobenzyl)-3,6,10,13,16,19-hexazabicyclo[6.6.6]-eicosane-1,8-diamine), diamSar, AmBaSar, and BaBaSar; - TRAP (1,4,7-triazacyclononane-1,4,7-tris[methyl(2-carboxyethyl)phosphinic acid]) and its analogues such as NOPO (1,4,7-triazacyclononane-1,4-bis[methylene(hydroxymethyl)phosphinic acid]-7-[methylene(2-carboxyethyl)phosphinic acid]); - AAZTA (1,4-bis(hydroxycarbonylmethyl)-6-[bis(hydroxylcarbonylmethyl)]amino-6-methylperhydro-1,4-diazepine); - DATA and DATA derivatives; - CP256 (4-acetamido-N1,N7-bis-[(3-hydroxy-1,6-dimethyl-4-methylene-1,4-dihydropyridin-2-yl)methyl]-4-(3-[(3-hydroxy-1,6-dimethyl-4-methylene-1,4-dihydropyridin-2-yl)methylamino]-3-oxopropyl)heptanediamide) and its derivative YM103 (4-(3-[3-(2,5-di oxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido]propanamido)-N1,N7-bis[(3-hydroxy-1,6-dimethyl-4-methylene-1,4-dihydropyridin-2-yl)methyl]-4-(3-[(3-hydroxy-1,6-dimethyl-4-methylene-1,4-dihydropyridin-2-yl)methylamino]-3-oxopropyl)heptanediamide; - PCTA (6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9,-triacetic acid); - BCPA (see Price and Orvig, Chem. Soc. Rev., 2014, 43, 260-290); - DFO (desferrioxamine) and DFO derivatives; - Trithiol chelate; - mercaptoacetyl; - Hydrazinonicotinamide; - Dimercaptosuccinic acid; - 1,2-ethylenediylbisL-cysteine ​​diethyl ester; - methylene diphosphonate; - hexamethylpropyleneamine oxime; - hexakis(methoxyisobutylisonitrile); and analogs thereof.

[0069] Most preferred are the DOTA and NOTA chelators because they: 18 F, 68 Ga, 111 In, 177 Lu and 225This is because it chelates particularly well with Ac.

[0070] Preferably, the chelator is bonded to the linker via one of the amide groups or one of the carboxylic acid groups of the chelator, resulting in an amide bond. However, it may also be bonded via one of its carbon atoms. For linking to a carbon atom, the chelator may suitably be equipped with an isothiocyanate functional group, such as isothiocyanatobenzyl. Examples of chelators equipped with such a functional group include p-SCN-Bn-DOTA and p-SCN-Bn-TCMC.

[0071] In a preferred embodiment, the linker is attached to one of the carboxylic acid groups of the chelator.

[0072] Thus, the radioisotope complexed to the chelator, in certain embodiments, has a structure according to any of the formulas Qa-Qh, preferably Qa, Qg, or Qh, more preferably Qa or Qg.

[0073] [ka]

[0074] (wherein m is individually 0 to 6, preferably 1; A 1 ~A 4 are independently selected from the group consisting of H, alkyl, aliphatic acids, e.g., carboxylic acids such as CHCOH and COH, and amides and esters thereof; A 5 is 1,4-butanesulton-1-yl(1,2λ 6 -oxathiane-2,2-dione-3-yl or 2,2-dioxide-1,2-oxathiane-3-yl), CH(SO3H)(CH2)3F and / or N(CH3)2)CH2BF3, or a precursor thereof, (M) represents a radioisotope; (L) represents an optional linker or an optional core as described herein; (Z) represents a scaffold as described herein).

[0075] In the structure according to any of the formulae Qa to Qh, A1 to A4 are preferably H, (C1-C6)-alkyl, (C1-C6)-alkylene-(CO2A 6 ), (C1-C6)-alkylene-(C(O)NA 6 A 7 ), wherein A 6 and A 7 are independently selected from the group consisting of H and (C1-C6)-alkyl, preferably H. Most preferably, A 1 ~A 3 is CH2CO2H, and A 4 is COH to aid in the water solubility of the compound. 1 ~A 4 are all the same and are selected from the group consisting of —CH2CO2H and —CH2C(O)NH2.

[0076] It can also be understood that (L) may be absent.

[0077] In certain embodiments, the chelating agent is A 5 may be combined with a fluoride label, as exemplified by formula (Qh) where N(CH) represents CH(SO3H)(CH2)3F and / or N(CH3)2)CH2BF3. This fluoride is suitable for imaging, such as PET imaging. 18 The compound may also be an A isotope. 5 1,4-butane sultone (1,2λ 6 The compounds may include precursors for the preparation of such compounds, as exemplified by formula (Qh) which represents a 2,2-oxathiane-2,2-dione.

[0078] Fluorophores The payload can be a dye, preferably a fluorescent dye, such as a dye selected from the group consisting of cyanine, phthalocyanine, rhodamine, fluorescein, xanthene, coumarin, styryl, porphine, fluorescent organometallic complexes, oxanine, perylene, acridine, boron-dipyrromethene, and the like.

[0079] Particularly suitable fluorescent dyes include cyanines and / or phthalocyanines. A preferred dye is CY5 dye, especially sulfo-CY5.

[0080] drugs The payload can be a drug, preferably a cytotoxic or cytostatic drug. Examples include some of the radioisotopes and molecular drugs described hereinabove. Suitable drugs are also those described in WO2021 / 160825, which is incorporated herein in its entirety. Particular suitable drugs include geldanamycin analogs, radicicol analogs, zeravespib (PU-H71), onarespib (AT13387), SNX-0723, HSP990, YC-72-AB85, luminespib (AUY922), VER-49009, and Hsp90 inhibitors such as NMS-E973. Most preferably, the drug comprises mertansine (DM1).

[0081] Exemplary Compounds In particularly preferred embodiments, the pharmaceutical compound or pharmaceutically acceptable salt thereof has a structure according to any of the following formulas eFAP-6 to eFAP-42: Preferably, eFAP-6, eFAP-8, eFAP-9, eFAP-10, eFAP-11, eFAP-12, eFAP-13, eFAP-14, eFAP-15, eFAP-16, eFAP-19, eFAP-20, eFAP-25, eFAP-26, eFAP-28, eFAP-29, eFAP-30, eFAP-31, eFAP-34, eFAP-35, eFAP-36, eFAP-37, and eFAP-38 are radioisotopes, more preferably 111 In, 68 Ga, 177 Lu, Pb 212 or 225The compounds contain the Ac radioisotope. Compound eFAP-6 is particularly suitable for use in radioisotope medical imaging. Compounds eFAP-7 and eFAP-23 are particularly suitable for use in biological research or diagnostic screening. Compounds eFAP-8 to eFAP-16, eFAP-25, eFAP-20, eFAP-31, and eFAP-34 to eFAP-38 are particularly suitable for use in radioisotope-based tumor therapy and / or radionuclide-based tumor imaging, especially in view of their advantageous tumor retention. Compounds eFAP-17, eFAP-23, eFAP-24, and eFAP-27 are particularly suitable for use in image-guided surgery for the treatment of cancer. Compound eFAP-18 is particularly suitable for use in tumor treatment. Compound eFAP-19 inhibits the release of DM1 and, for example, 177 These compounds are biofunctionalized compounds suitable for the treatment of cancer by radioisotope therapy using Lu. Compounds eFAP-28 to eFAP-30 are: 18 eFAP-40, eFAP-41, and eFAP-42 are precursors for F-fluorination and can be used for positron emission tomography (PET) imaging. Compounds eFAP-40, eFAP-41, and eFAP-42 can be obtained from their respective precursors eFAP-28, eFAP-29, and eFAP-30, and therefore can be used for PET imaging.

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[0097] More than 90% of solid tumors contain cancer-associated fibroblasts that overexpress FAP. Thus, the compounds of the present invention can be used in the diagnosis and treatment of a wide variety of tumors, including sarcomas, cancers of the breast, lung, ovary, head and neck, prostate, colorectal, and the like, as well as rare or difficult-to-treat cancers such as pancreatic and / or brain cancer.

[0098] The compound or a pharmaceutically acceptable salt thereof may be for use as a pharmaceutical, more particularly for use in the medical treatment or diagnosis (including imaging) of tumors. Whether it can be used in treatment or diagnosis depends in large part on the payload used, as described herein.

[0099] Thus, a further aspect of the invention is directed to a method for treating or diagnosing tumors, particularly tumors that overexpress FAP, comprising administering to a patient a pharmaceutically acceptable dose of a compound or a pharmaceutically acceptable salt thereof.

[0100] For any moiety described herein that may be optionally substituted or interrupted, it may be understood that such moiety may also be unsubstituted or uninterrupted.

[0101] Additionally, when a compound is described herein, its salts, prodrugs, metabolites, etc. are also referenced, unless specifically stated otherwise.

[0102] When the stereochemistry of an atom in a structure is undefined (e.g., CR 1 ), the structure refers individually to all stereoisomers of said atoms.

[0103] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. The term "and / or" includes any and all combinations of one or more of the associated listed items. It will be understood that the terms "comprises" and / or "comprising" specify the presence of stated features but do not exclude the presence or addition of one or more other features.

[0104] Although, for purposes of clarity and concise description, features are described herein as part of the same or separate embodiments, it will be understood that the scope of the present invention may include embodiments having all or any combination of the described features.

[0105] The present invention can be illustrated by the following non-limiting examples and embodiments.

[0106] General Chemicals and solvents were purchased from commercial suppliers and used without further purification. Reactions were magnetically stirred and monitored by thin-layer chromatography (TLC) on precoated aluminum-backed Merck plates (silica gel 60 F254). Melting points were determined using a Stuart SMP20 column. Liquid chromatography-mass spectrometry (LC-MS) was performed on an Agilent 1260 Infinity II electrospray ionization (ESI) LC-MS system equipped with an Agilent® InfinityLab Poroshell 120 EC-C18 column (2.7 μm, 3.0 × 100 mm). Products were eluted using a gradient of acetonitrile (ACN; 5–100%) in HO containing 0.1% formic acid (FA) over 8 min at a flow rate of 0.5 mL / min and monitored at 220 nm, 254 nm, and 280 nm with a UV detector. Nuclear magnetic resonance (NMR) spectra were recorded at room temperature on a Bruker AVANCE 400 or Nanalysis 60 Pro in deuterated dimethyl sulfoxide (DMSO-d) and chloroform-d (CDCl). Chemical shifts are given as δ values ​​in ppm, and coupling constants J are given in Hz. Splitting patterns are reported as s (singlet), d (doublet), t (triplet), q (quartet), qt (quintet), m (multiplet), and br (broad signal). Raw NMR data were processed using MestreNova 13. Compound purification was performed on an Agilent preparative HPLC 1260 Infinity II system using a preparative column (50 × 21.2 mm, 5 μm) and gradient elution of ACN (10% to 95% in HO containing 0.1% FA) over 10 min at a flow rate of 10 mL / min.

[0107] Instant thin-layer chromatography (iTLC-SG) plates on silica gel-impregnated glass fiber sheets were eluted with sodium citrate (0.1 M, pH 5). Plates were analyzed using a Brightspec bSCAN radiochromatography scanner equipped with a sodium iodide detector. 7.4Radioactive samples used for the determination of β-amyloid, in vitro assays, and in vivo studies were counted using a Wizard 2480 gamma counter. Radioactivity measurements were performed using a VDC-405 dose calibrator. Quality control of radiolabeled compounds and their stability analysis were performed on a Waters ultra-high performance liquid chromatography (UHPLC) Acquity Arc system equipped with a diode array detector, a Canberra radiation detector, and an analytical C18 Gemini® column (250.0 × 4.6 mm, 5 μm) eluted with a gradient of ACN (5 to 95% in HO containing 0.1% trifluoroacetic acid, TFA) over 30 min at a flow rate of 1 mL / min.

[0108] Preparation of (S)-1-tert-butyl 2-methyl 4,4-difluoropyrrolidine-1,2-dicarboxylate

[0109] [ka]

[0110] Step 1: (S)-1-tert-butyl 2-methyl 4-oxopyrrolidine-1,2-dicarboxylate (2) 1,3,5-Trichloro-1,3,5-triazinane-2,4,6-trione (0.95 g, 4.07 mmol) was added to a cooled (0 °C) solution of (S)-1-tert-butyl 2-methyl 4-oxopyrrolidine-1,2-dicarboxylate (0.95 g, 3.88 mmol) in DCM (10 mL), followed by the addition of catalytic TEMPO (6 mg, 0.04 mmol). After 5 min, the mixture was allowed to reach room temperature, stirred for an additional 30 min, and filtered through Celite. The organic layer was washed with 10 mL of saturated potassium carbonate solution, sodium thiosulfate, brine, dried over anhydrous sodium sulfate, filtered, and evaporated. The crude compound 2 (0.65 g, 70%) was used without further purification. 1H NMR (400 M Hz, CDCl3): δ 4.77 (dd, 1H, J = 36.8, 8 Hz), 3.88 (br s, 2H), 3.75 (s, 3H), 2.90 (s, 1H), 2.57 (dd, 1H, J = 18.8, 2.4 Hz), 1.46 (s, 9H). ESI-MS: m / z 376.2 [M + MeOH + H] + .

[0111] Step 2: (S)-1-tert-butyl 2-methyl 4,4-difluoropyrrolidine-1,2-dicarboxylate (3) A solution of 2 (0.23 g, 0.946 mmol) in DCM (3 mL) was treated with a solution of diethylaminosulfur trifluoride (DAST, 0.197 mL, 1.607 mmol) in DCM (2 mL) at room temperature. Ethanol (0.011 mL, 0.189 mmol) was added, and the mixture was stirred at room temperature for 18 h. The solution was poured into saturated sodium bicarbonate, extracted with DCM (3 × 15 mL), dried (NaSO), filtered, and evaporated in vacuo. Chromatography on silica gel (100% DCM) afforded the product as a yellowish oil (0.150 g, 61%). 1 H NMR (400 MHz, CDCl3): δ 4.55-4.45 (m, 1H), 3.90-3.60 (m, 2H), 3.75 (s, 3H), 2.81-2.61 (m, 1H), 2.45 (dq, 1H, J = 13.6, 5.2 Hz,), 1.44 (br s, 9H). ESI-MS: m / z 266.1 [M + H] + .

[0112] Preparation of Intermediate 10 Intermediate 10 was prepared according to the scheme in FIG.

[0113] Step 1: (S)-1-(tert-butoxycarbonyl)-4,4-difluoropyrrolidine-2-carboxylic acid (4) 3 (2.06 g, 7.78 mmol) was dissolved in MeOH (10 mL) and a solution of NaOH (0.67 g, 17.18 mmol) in MeOH (15 mL) was added dropwise. The reaction mixture was stirred at room temperature for 30 min. Upon completion, the reaction mixture was acidified to pH 2 using 1 N HCl and the aqueous layer was extracted with DCM (3 × 50 mL). The combined organic layers were washed with brine, dried over MgSO4, and concentrated in vacuo. This gave an off-white solid product (1.62 g, 6.44 mmol, 86%). 1 H NMR (60 MHz, DMSO-d6): δ 4.33 (dd, 1H, J = 9.3, 4.4 Hz), 3.68 (t, 2H, J = 13.2 Hz), 2.76-2.12 (m, 2H), 1.34 (s, 9H). Melting point: 118-119℃. ESI-MS: m / z 152.1 [M + H - Boc] + .

[0114] Step 2: tert-butyl (S)-2-carbamoyl-4,4-difluoropyrrolidine-1-carboxylate (5) 4 (1.55 g, 6.15 mmol) was dissolved in DCM (dry, 30 mL), HONSu (0.88 g, 7.65 mmol) was added, and the mixture was stirred at room temperature until complete dissolution. DCC (1.55 g, 7.52 mmol) was added, and the mixture was stirred at room temperature for 30 min, causing insoluble material to slowly form in the reaction mixture. NH3 (14 mL, 4 M in MeOH) was added dropwise, and the reaction mixture was stirred at room temperature for 3 h. Upon completion, EtOAc (50 mL) was added, and the organic layer was washed with saturated aqueous NaHCO3 (5 × 25 mL), brine (25 mL), dried over MgSO4, and concentrated in vacuo. The product was obtained as an off-white solid (1.53 g, 6.02 mmol, 97%). 1H NMR (60 MHz, CDCl3): δ 6.50 (br, s, 1H), 5.60 (br, s, 1H), 4.48 (dd, 1H, J = 8.6, 5.8 Hz), 3.76 (td, 2H, J = 12.4, 5.2 Hz), 2.74 (td, 2H, J = 13.6, 4.6 Hz), 1.46 (s, 9H). Melting point: 127-128℃. ESI-MS: m / z 151.1 [M + H - Boc] + .

[0115] Step 3: (S)-4,4-difluoropyrrolidine-2-carboxamide (6) 5 (1.19 g, 4.69 mmol) was dissolved in DCM (10 mL) and TFA (5 mL). The reaction mixture was stirred at room temperature for 2.5 h. Upon completion, all volatiles were removed. Cold ether was added, and 6 was recovered as a white solid (0.98 g, 3.66 mmol, 78%). 1 H NMR (60 MHz, DMSO-d6): δ 7.82 (s, 1H), 7.70 (s, 1H), 4.55-4.17 (t, 1H, J = 7.8 Hz), 3.54 (d, 2H, J = 12.0 Hz), 2.45 (m, 2H). Melting point: > 130℃. ESI-MS: m / z 151.0 [M + H] + .

[0116] Step 4: tert-butyl (S)-(2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamate (7) 6 (0.93 g, 3.47 mmol) was dissolved in DMF (30 mL). Boc-Gly-OH (0.81 g, 4.62 mmol), HBTU (1.64 g, 4.32 mmol), and DIPEA (3.0 mL, 2.3 g, 17.6 mmol) were added, and the reaction mixture was stirred overnight. EtOAc (50 mL) was added, and the organic layer was washed with brine (5 × 25 mL), dried over MgSO, and concentrated in vacuo. The crude reaction mixture was redissolved in THF (30 mL) and cooled to −15 °C. Pyridine (1.00 mL, 12.4 mmol) and TFAA (1.00 mL, 7.19 mmol) were added, and the reaction mixture was stirred for 5 h and allowed to reach room temperature. EtOAc (50 mL) and HO (50 mL) were added. The layers were separated, and the aqueous layer was extracted with EtOAc (3 × 25 mL). The combined organic layers were washed with water and brine, dried over MgSO4, and concentrated in vacuo. The product was purified by flash chromatography (silica gel, hexane / EtOac 1:1). 7 was obtained as a yellow solid (200 mg, 0.69 mmol, 25%). 1 H NMR (60 MHz, CDCl3): δ 5.48 (s, 1H), 4.94 (t, 1H, J = 6.3 Hz), 3.70-4.15 (m, 4H), 2.42-2.94 (m, 2H), 1.40 (s, 9H). Melting point: 128-129℃. ESI-MS: m / z 190.1 [M + H - Boc] + .

[0117] Step 5: 8-(3-((Tert-butoxycarbonyl)amino)propoxy)quinoline-4-carboxylic acid (9) 8 (0.25 g, 1.32 mmol) was dissolved in DMF (25 mL). 3-(Boc-amino)-propyl bromide (0.94 g, 4.0 mmol) and CsCO (1.31 g, 4.0 mmol) were added. The reaction mixture was stirred at room temperature for 2.5 h. Upon completion, EtOAc (50 mL) was added, and the organic layer was washed with brine (5 × 25 mL), dried over MgSO, and concentrated in vacuo. The residue was redissolved in MeOH (10 mL), and NaOH (78 mg, 2.0 mmol) was added. The reaction mixture was stirred at room temperature for 35 min. The reaction mixture was concentrated in vacuo, and the product was purified using preparative HPLC. 9 was obtained as a pale yellow solid (0.26 g, 0.75 mmol, 57%). 1 H NMR (60 MHz, DMSO-d6): δ 8.94 (d, 1H, J = 4.2 Hz), 8.16 (d, 1H, J = 8.3 Hz), 7.88 (d, 1H, J = 4.3 Hz), 7.55 (t, 1H, J = 8.1 Hz), 7.18 (d, Melting point: > 130℃. ESI-MS: m / z 346.7 [M + H] + .

[0118] Step 6: tert-butyl (S)-(3-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)carbamate (10) 7 (0.18 g, 0.61 mmol) was dissolved in DCM (3 mL) and TFA (3 mL). The reaction mixture was stirred at room temperature for 45 min. Upon completion, all volatiles were removed under a gentle stream of air. 9 (0.2 g, 0.58 mmol), HBTU (0.34 g, 0.87 mmol), and DIPEA (0.35 mL) were added to the residue, which was redissolved in DMF (5 mL). The reaction mixture was stirred at room temperature overnight. EtOAc (50 mL) was added, and the organic layer was washed with brine (5 × 25 mL), dried over MgSO4, and concentrated in vacuo. The product was purified using flash chromatography (silica gel, hexane / EtOac 1:1) to give 10 as a pale yellow sticky oil (0.23 g, 0.44 mmol, 72%). 1 H NMR (60 MHz, CDCl3): δ 8.69 (d, 1H, J = 4.2 Hz), 7.64 (m, 2H), 7.45-7.02 (m, 2H), 6.86 (d, 1H, J = 7.3 Hz), 6.30 (s, 1H), 4.82 (s, 1H), 4.02 (m, 4H), 3.22 (m, 2H), 2.55 (m, 4H), 2.04 (m, 2H), 1.35 (s, 9H). ESI-MS: m / z 618.0 [M + H] + . [Example]

[0119] Example 1 Preparation of (S)-2,2′,2″-(10-(2-((3-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (eFAP-6) 10 (0.02 g, 0.04 mmol) was dissolved in a mixture of DCM (200 μL), TIPS (20 μL), and TFA (200 μL). The reaction mixture was stirred at room temperature for 30 min. All volatiles were removed under a gentle stream of air. The residue was redissolved in DMF (1 mL). DOTA-NHS ester (0.035 g, 0.043 mmol) and DIPEA (45 μL) were added. The reaction mixture was stirred at room temperature for 30 min, and the solvent was removed under reduced pressure. The product was isolated by preparative HPLC to give eFAP-6 as a pale yellow powder (12.3 mg, 0.015 mmol, 38%). ESI-MS: m / z 804.4 [M+H + ].

[0120] Example 2 Preparation of 1-(6-((3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-6-oxohexyl)-3,3-dimethyl-5-sulfo-2-((1E,3E)-5-((E)-1,3,3-trimethyl-5-sulfoindolin-2-ylidene)penta-1,3-dien-1-yl)-3H-indol-1-ium (eFAP-7) 10 (0.01 g, 0.02 mmol) was dissolved in a mixture of DMC (100 μL), TIPS (10 μL), and TFA (100 μL). The reaction mixture was stirred at room temperature for 30 min. All volatiles were removed under a gentle stream of air. The residue was redissolved in DMF (0.5 mL). Sulfo-Cy5-NHS ester (2.5 mg, 3.2 μmol) and DIPEA (10 μL) were added. The reaction mixture was stirred at room temperature for 45 min, and the solvent was removed under reduced pressure. The product was isolated using preparative HPLC to give eFAP-7 as a bright blue powder (1.0 mg, 1 μmol). ESI-MS: m / z 1042.6 [M+H] + .

[0121] Example 3 Preparation of 2,2′,2″-(10-(1-carboxy-4-((3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (eFAP-8) 10 (10 mg, 9.6 μmol) was dissolved in a solution mixture of DCM / TFA (0.5 mL / 0.5 mL). The reaction mixture was stirred at room temperature for 30 min. All volatiles were removed, and the residue was redissolved in DMF (1 mL). DOTA-GA anhydride (5.5 mg, 0.012 mmol) and DIPEA (15 μL) were added, and the reaction mixture was stirred at room temperature for 2 h. Upon evaporation, eFAP-8 was purified using preparative HPLC to give a pale yellow powder (8 mg, 9.1 μmol, 95%). ESI-MS: m / z 876.4 [M+H] + .

[0122] Example 4 Preparation of 2,2′,2″-(10-(2-(((4-((3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)carbamoyl)cyclohexyl)methyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (eFAP-9) 10 (10 mg, 9.6 μmol) was treated with a solution of DCM / TFA (2 mL, 1:1) at room temperature for 30 min. All volatiles were removed under a gentle stream of air to give a crude brown solid. The crude was redissolved in DMF (1 mL), followed by the addition of Fmoc-Amcha-OH (2 equiv., 7.3 mg), HBTU (4 equiv., 14.6 mg), DIPEA (15 μL), and DMF (1 mL). The reaction was stirred at room temperature for 2 h, diluted with EtOAc (15 mL), washed with water (3 × 5 mL), dried over NaSO, and concentrated in vacuo to give a pale yellow solid. The crude product was used in the next step without further purification. This was redissolved in 20% piperidine in DMF (2 mL) and stirred at room temperature for 30 min. The solvent was removed under reduced pressure, followed by the addition of DMF (1.5 mL), DOTA-NHS ester (6.6 mg, 14.4 μmol), and DIPEA (15 μL). After 4 h, the solvent was removed in vacuo, and the crude product was purified by preparative HPLC to give eFAP-9 (4.1 mg, 2.4 μmol, 45%) as an off-white powder. ESI-MS: m / z 943.4 [M+H] + .

[0123] Example 5 Preparation of (S)-2,2′,2″-(10-(2-((1-(2-((3-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-2-oxoethyl)piperidin-4-yl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (eFAP-10) 10 (10 mg, 9.6 μmol) was treated with a solution of DCM / TFA (2 mL, 1:1) at room temperature for 30 min. All volatiles were removed under a gentle stream of air to give a crude brown solid. The crude was redissolved in DMF (1 mL), followed by the addition of Fmoc-Pip-OH (2 equiv., 7.5 mg), HBTU (4 equiv., 14.6 mg), DIPEA (15 μL), and DMF (1 mL). The reaction was stirred at room temperature for 2 h, diluted with EtOAc (15 mL), washed with water (3 × 5 mL), dried over NaSO, and concentrated in vacuo to give a pale yellow solid. The crude product was used in the next step without further purification. This was redissolved in 20% piperidine in DMF (2 mL) and stirred at room temperature for 30 min. The solvent was removed under reduced pressure, followed by the addition of DMF (1.5 mL), DOTA-NHS ester (6.6 mg, 14.4 μmol), and DIPEA (15 μL). After 4 h, the solvent was removed in vacuo, and the crude product was purified by preparative HPLC to give eFAP-10 (2.1 mg, 2.2 μmol, 23%) as an off-white powder. ESI-MS: m / z 944.4 [M+H] + .

[0124] Example 6 Preparation of (S)-2,2′,2″-(10-(2-((4-(2-(2-((3-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-2-oxoethoxy)acetamido)benzyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (eFAP-11) 10 (10 mg, 9.6 μmol) was treated with a solution of DCM / TFA (2 mL, 1:1) at room temperature for 30 min. All volatiles were removed under a gentle stream of air to give a crude brown solid. The crude was redissolved in DMF (1 mL), followed by the addition of Fmoc-p-Ada-OH (2 equiv., 8.7 mg), HBTU (4 equiv., 14.6 mg), DIPEA (15 μL), and DMF (1 mL). The reaction was stirred at room temperature for 4 h, diluted with EtOAc (15 mL), washed with water (3 × 5 mL), dried over NaSO, and concentrated in vacuo to give a pale yellow solid. The crude product was used in the next step without further purification. This was redissolved in 20% piperidine in DMF (2 mL) and stirred at room temperature for 30 min. The solvent was removed under reduced pressure, followed by the addition of DMF (1.5 mL), DOTA-NHS ester (4.5 mg, 9.6 μmol), and DIPEA (15 μL). After 4 h, the solvent was removed under vacuum, and the crude product was purified by preparative HPLC to give eFAP-11 (2.8 mg, 2.7 μmol, 56%) as an off-white powder. ESI-MS: m / z 1024.4 [M+H] + .

[0125] Example 7 Preparation of (S)-2,2′,2″-(10-(2-(4-(2-((3-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-2-oxoethyl)piperazin-1-yl)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (eFAP-12) 10 (10 mg, 9.6 μmol) was treated with a solution of DCM / TFA (2 mL, 1:1) at room temperature for 30 min. All volatiles were removed under a gentle stream of air to give a crude brown solid. The crude was redissolved in DMF (1 mL), followed by the addition of Boc-Pipa-OH (2 equiv., 4.7 mg), HBTU (4 equiv., 14.6 mg), DIPEA (15 μL), and DMF (1 mL). The reaction was stirred at room temperature for 4 h, diluted with EtOAc (15 mL), washed with water (3 × 5 mL), dried over NaSO, and concentrated in vacuo to give a pale yellow solid. The crude product was used in the next step without further purification. This was redissolved in 20% piperidine in DMF (2 mL) and stirred at room temperature for 30 min. The solvent was removed under reduced pressure, followed by the addition of DMF (1.5 mL), DOTA-NHS ester (6.6 mg, 14.4 μmol), and DIPEA (15 μL). After 4 h, the solvent was removed in vacuo, and the crude product was purified by preparative HPLC to give eFAP-12 (4.1 mg, 4.4 μmol, 49%) as an off-white powder. ESI-MS: m / z 930.3 [M+H] + .

[0126] Example 8 Preparation of (S)-2,2′,2″-(10-(2-((2-(4-(2-((3-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-2-oxoethyl)piperazin-1-yl)ethyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (eFAP-13) 10 (10 mg, 9.6 μmol) was treated with a solution of DCM / TFA (2 mL, 1:1) at room temperature for 30 min. All volatiles were removed under a gentle stream of air to give a crude brown solid. The crude was redissolved in DMF (1 mL), followed by the addition of Fmoc-ePipa-OH (2 equiv., 7.9 mg), HBTU (4 equiv., 14.6 mg), DIPEA (15 μL), and DMF (1 mL). The reaction was stirred at room temperature for 4 h, diluted with EtOAc (15 mL), washed with water (3 × 5 mL), dried over NaSO, and concentrated in vacuo to give a pale yellow solid. The crude product was used in the next step without further purification. This was redissolved in 20% piperidine in DMF (2 mL) and stirred at room temperature for 30 min. The solvent was removed under reduced pressure, followed by the addition of DMF (1.5 mL), DOTA-NHS ester (6.6 mg, 14.4 μmol), and DIPEA (15 μL). After 4 h, the solvent was removed in vacuo, and the crude product was purified by preparative HPLC to give eFAP-13 (3.8 mg, 4.1 μmol, 43%) as an off-white powder. ESI-MS: m / z 976.3 [M+H] + .

[0127] Example 9 Preparation of 2,2'-(4-(1-carboxy-5-(4-((2,2-dioxido-1,2-oxathian-3-yl)methyl)-1H-1,2,3-triazol-1-yl)pentyl)-10-(2-((3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,7-diyl)diacetic acid (eFAP-14) 10 (10 mg, 9.6 μmol) was dissolved in a solution mixture of DCM / TFA (0.5 mL / 0.5 mL). The reaction mixture was stirred at room temperature for 30 min. All volatiles were removed, and the residue was redissolved in DMF (1 mL). DOTA-K(N3)-NHS ester (5.5 mg, 12.0 μmol) and DIPEA (15 μL) were added, and the reaction mixture was stirred at room temperature for 4 h. EtOAc (15 mL) was added, washed with water (3 × 5 mL), dried over Na2SO4, and concentrated in vacuo to give a pale yellow solid. The crude product was used in the next step without further purification. A solution of 3-(prop-2-yn-1-yl)-1,2-oxathiane 2,2-dioxide (2.1 mg, 12.0 μmol), copper(II) acetate (0.22 mg, 1.2 μmol), sodium ascorbate (0.26 mg, 1.3 μmol), and acetonitrile and water (1 mL, 1:1) was added, and the reaction mixture was stirred at 60° C. for 12 hours. The mixture was concentrated under reduced pressure and purified by preparative HPLC to give eFAP-14 (5.0 mg, 4.7 μmol, 49%) as an off-white powder. ESI-MS: m / z 1076.2 [M+H] + .

[0128] Example 10 Preparation of 2,2'-(4-(1-carboxy-5-(4-(5-fluoro-2-sulfopentyl)-1H-1,2,3-triazol-1-yl)pentyl)-10-(2-((3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,7-diyl)diacetic acid (eFAP-15) 10 (10 mg, 9.6 μmol) was dissolved in a solution mixture of DCM / TFA (0.5 mL / 0.5 mL). The reaction mixture was stirred at room temperature for 30 min. All volatiles were removed, and the residue was redissolved in DMF (1 mL). DOTA-K(N3)-NHS ester (5.5 mg, 12.0 μmol) and DIPEA (15 μL) were added, and the reaction mixture was stirred at room temperature for 4 h. EtOAc (15 mL) was added, washed with water (3 × 5 mL), dried over Na2SO4, and concentrated in vacuo to give a pale yellow solid. The crude product was used in the next step without further purification. A solution of 7-(fluoro)hept-1-yne-4-sulfonic acid (2.2 mg, 12.0 μmol), copper(II) acetate (0.22 mg, 1.2 μmol), sodium ascorbate (0.26 mg, 1.3 μmol), and acetonitrile and water (1 mL, 1:1) was added, and the reaction mixture was stirred at 60° C. for 12 hours. The mixture was concentrated under reduced pressure and purified by preparative HPLC to give eFAP-14 (3.7 mg, 3.4 μmol, 35%) as an off-white powder. ESI-MS: m / z 1096.2 [M+H] + .

[0129] Example 11 Preparation of ((((1-(5-(4,10-bis(carboxymethyl)-7-(2-((3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)-5-carboxypentyl)-1H-1,2,3-triazol-4-yl)methyl)dimethylammonio)methyl)trifluoroborate (eFAP-16) 10 (10 mg, 9.6 μmol) was dissolved in a solution mixture of DCM / TFA (0.5 mL / 0.5 mL). The reaction mixture was stirred at room temperature for 30 min. All volatiles were removed, and the residue was redissolved in DMF (1 mL). DOTA-K(N3)-NHS ester (5.5 mg, 12.0 μmol) and DIPEA (15 μL) were added, and the reaction mixture was stirred at room temperature for 4 h. EtOAc (15 mL) was added, washed with water (3 × 5 mL), dried over Na2SO4, and concentrated in vacuo to give a pale yellow solid. The crude product was used in the next step without further purification. N-((difluoroboranyl)methyl)-N,N-dimethylprop-2-yn-1-aminium fluoride (2.2 mg, 12.0 μmol), copper(II) acetate (0.22 mg, 1.2 μmol), sodium ascorbate (0.26 mg, 1.3 μmol), and a solution of acetonitrile and water (1 mL, 1:1) were added, and the reaction mixture was stirred at 60° C. for 12 h. The mixture was concentrated under reduced pressure and purified by preparative HPLC to give eFAP-14 (2.3 mg, 2.2 μmol, 23%) as an off-white powder. ESI-MS: m / z 1066.2 [M+H] + .

[0130] Example 12 Preparation of 4-(2-((E)-2-((E)-3-(2-((E)-1-(6-((3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-6-oxohexyl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)ethylidene)-2-(4-sulfophenoxy)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-5-sulfo-3H-indol-1-ium-1-yl)butane-1-sulfonate (eFAP-17) 10 (8 mg, 7.7 μmol) was stirred in a solution of DCM / TFA (2 mL, 1:1) at room temperature for 30 min. All volatiles were then removed using a gentle stream of air to give a brown solid. The crude product was dissolved in DMF (1 mL) and treated with DIPEA (10 μL) and pre-activated CW800 dye. CW800 (5 mg, 5 μmol) was dissolved in DMF (1 mL) and activated by treatment with HONSu (5 mg) and DCC (10.3 mg) at room temperature for 1 h. The reaction mixture was dried and purified by preparative HPLC to give eFAP-17 (1.6 mg, 1.1 μmol, 22%) as a green powder. ESI-MS: m / z 1469.4 [M+H] + .

[0131] Example 13: (1 4 S,1 6 S,3 2 S,3 3 S, 2R, 4S, 10E, 12E, 14R)-8 6 -chloro-1 4 -hydroxy-8 5 ,14-dimethoxy-3 3 ,2,7,10-tetramethyl-1 2 Preparation of ,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl N-(3-((3-((3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-3-oxopropyl)disulfanayl)propanoyl)-N-methyl-L-alaninate (eFAP-18) 10 (12.1 mg, 23.4 μmol) was deprotected by treatment with TFA / DCM (1:1) for 1 h. The solvent was removed, and the residue was dissolved in DMF (4 mL) and then reacted with 3-(tritylthio)propionic acid (32.5 mg, 69.9 μmol), EDC (17.9 mg, 93.2 μmol), and DIPEA (16.3 μL, 93.2 μmol) for 2 h. DMF was removed under high vacuum. The crude product was dissolved in ACN / HO (1:1) and purified by preparative HPLC (11.6 mg, 15.5 μmol, 66%). This intermediate was treated with TFA / DCM (1:1) and TES (7.1 mg, 31.0 μmol) for 1 h. The solvent was removed by a gentle stream of air. To a solution of the product in DMF (1 mL) was added a solution of 2,2'-dithiodipyridine (17.1 mg, 77.5 μmol) in a mixture of DMF (0.5 mL) and acetic acid (0.1 mL). The solution was stirred for 15 minutes. Aqueous sodium acetate (1 mL, 0.2 M) was added dropwise to the stirred solution. The reaction was monitored by LCMS and stopped after 1.5 hours. The solvent was removed in vacuo, and the crude product was dissolved in ACN / HO (1:1) and then purified by preparative HPLC (5.7 mg, 9.3 μmol, 60%). The product was dissolved in DMF (2.5 mL), and a solution of DM1 (1.7 mg, 16.8 mmol) in PBS (1 mL, pH 7.4) was added. Aqueous sodium carbonate (0.5 mL, 3.5 mg / 10 mL) was added dropwise to the stirred solution. The reaction was monitored by LCMS. The reaction was stopped after 1 h. The crude product was dissolved in ACN / HO (1:1) and purified by preparative HPLC. eFAP-18 was obtained as an off-white solid (3.3 mg, 2.7 μmol, 28%). ESI-MS: m / z 1242.8 [M+H] + .

[0132] Example 14: 2,2',2''-(10-((2S,17S,22S)-22-carboxy-1-(((1 4 S,1 6 S,3 2 S,3 3 S, 2R, 4S, 10E, 12E, 14R)-8 6 -chloro-1 4 -hydroxy-85 ,14-dimethoxy-3 3 ,2,7,10-tetramethyl-1 2 Preparation of ,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl)oxy)-17-((3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)carbamoyl)-2,3-dimethyl-1,4,11,19-tetraoxo-7,8-dithia-3,12,18-triazadocosan-22-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (eFAP-19) 10 (11.9 mg, 23.1 μmol) was deprotected by treatment with TFA / DCM (1:1) for 1 h. The solvent was removed, and the residue was dissolved in DMF (4 mL) and then reacted with Fmoc-Lys(Boc)-OH (43.3 mg, 92.4 μmol), EDC (17.7 mg, 92.4 μmol), and DIPEA (16.1 μL, 92.4 μmol) for 5 h. DMF was removed under high vacuum. The crude product was dissolved in ACN / HO (1:1) and purified by preparative HPLC (24.2 mg, 16.4 μmol, 71%). This intermediate was treated with TFA / DCM (1:1) for 1 h. The solvent was removed by a gentle stream of air. The residue was dissolved in DMF (4 mL) and reacted with 3-(tritylthio)propionic acid (17.1 mg, 49.2 μmol), Oxyma Pure (9.3 mg, 65.6 μmol), HATU (24.9 mg, 65.6 μmol), and DIPEA (11.4 μL, 131.2 μmol). The reaction was monitored by LCMS and stopped after 1 h. Fmoc deprotection was carried out by treatment with 20% PIP in DMF for 1 h. DMF was removed under high vacuum, and the crude product was purified by preparative HPLC (2.8 mg, 3.2 μmol, 19%). DOTA-GA anhydride (1.9 mg, 4 μmol) and DIPEA (15 μL) were added to a solution of the intermediate in DMF (0.5 mL). The reaction mixture was stirred at room temperature for 2 h. Immediately after evaporation, a solution of 2,2'-dithiodipyridine (1.8 mg, 8.0 μmol) in a mixture of DMF (0.5 mL) and acetic acid (0.1 mL) was added. The solution was stirred for 15 minutes. Aqueous sodium acetate (1 mL, 0.2 M) was added dropwise to the stirred solution. The reaction was monitored by LCMS and stopped after 2.5 hours. The solvent was removed in vacuo, and the crude product was dissolved in ACN / HO (1:1) and then purified by preparative HPLC (1.4 mg, 2.0 μmol, 62%). The product was dissolved in DMF (0.5 mL), and a solution of DM1 (0.4 mg, 4.0 mmol) in PBS (0.5 mL, pH 7.4) was added. Aqueous sodium carbonate (0.25 mL, 3.5 mg / 10 mL) was added dropwise to the stirred solution. The reaction was monitored by LCMS. The reaction was stopped after 2 hours.The crude product was dissolved in ACN / HO (1:1) and purified by preparative HPLC to give eFAP-19 as an off-white solid (1.2 mg, 0.7 μmol, 34%). ESI-MS: m / z 1829.2 [M+H]. + .

[0133] Example 15 Preparation of 2,2′,2″-(10-(4-((3-((((1,4-bis((3-((3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-3-oxopropyl)thio)-5,6,7,8,9,10-hexahydrocycloocta[d]pyridazin-7-yl)oxy)carbonyl)amino)propyl)amino)-1-carboxy-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (eFAP-20) 10 (12.1 mg, 23.4 μmol) was deprotected by treatment with TFA / DCM (1:1) for 1 h. The solvent was removed, and the residue was dissolved in DMF (4 mL) and then reacted with 3-(tritylthio)propionic acid (32.5 mg, 69.9 μmol), EDC (17.9 mg, 93.2 μmol), and DIPEA (16.3 μL, 93.2 μmol) for 2 h. DMF was removed under high vacuum. The crude product was dissolved in ACN / HO (1:1) and purified by preparative HPLC (11.6 mg, 15.5 μmol, 66%). This intermediate was dissolved in a degassed solution of monosodium phosphate (50 mM, pH 5), followed by the addition of dichlorotetrazine (3 equiv.) in CHCl. ​​The two phases were vigorously stirred for 1 min. The aqueous phase was collected, and the organic layer was extracted with an additional portion of water. The aqueous fractions were combined and lyophilized. The crude mixture was then purified by preparative HPLC to yield an orange powder after lyophilization (5.1 mg, 4.7 μmol, 60%). eFAP-20 was prepared by reacting the above dimer with DOTA-GA-TCO (3.6 mg, 1.1 equiv.). Both starting materials were dissolved in HO / ACN (1:1) and incubated at 37°C for 10 min in an Eppendorf tube protected from light. The reaction mixture was purified by preparative HPLC to yield eFAP-20 (5.1 mg, 3.0 μmol, 63%). ESI-MS: m / z 1744.8 [M+H] + .

[0134] 2,2',2''-(10-(1-carboxy-4-((3-(((1-((3-((3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-3-oxopropyl)thio)-4-((3-((3-((4-((2-((S)-2-cyanopyrrolidin-1-yl) Preparation of -2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-3-oxopropyl)thio)-5,6,7,8,9,10-hexahydrocycloocta[d]pyridazin-7-yl)oxy)carbonyl)amino)propyl)amino)-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (eFAP-20) 10 (15 mg, 29 μmol) was treated with a solution of DCM / TFA (3 mL, 1:1) at room temperature for 30 min. All volatiles were removed under a gentle stream of air to give a crude brown solid. The crude was redissolved in DMF (1 mL), followed by the addition of 3-(tritylthio)propionic acid (1.5 equiv., 15 mg), HBTU (2 equiv., 23.6 mg), DIPEA (30 μL), and DMF (1.5 mL). The reaction was stirred at room temperature for 4 h, diluted with EtOAc (15 mL), washed with water (3 × 5 mL), dried over NaSO, and concentrated in vacuo to give a pale yellow solid. The crude product was treated with a solution of DCM / TFA (3 mL, 1:1) at room temperature for 30 min. The solvent was removed under a gentle stream of air, and the crude product was purified by preparative HPLC to give 8QCP-SH (4 mg, 7.9 μmol, 27%) as a pale yellow powder. ESI-MS: m / z 506.2 [M+H] + The powder was redissolved in DMF / DCM (1 mL, 1:1), followed by the addition of 3,6-dichloro-1,2,4,5-tetrazine (0.4 mg, 2.7 μmol) and DIPEA (10 μL). After 30 min, the solvent was removed in vacuo, and the crude product was purified by preparative HPLC to give (8QCP-SH)2-TZ (1.9 mg, 1.7 μmol, 43%) as a pale yellow powder. ESI-MS: m / z 1089.1 [M+H] +This powder was redissolved in ACN / HO (1 mL, 1:1), followed by the addition of TCO-DOTAGA (1.1 equiv., 1.3 mg). The reaction was stirred at 37 °C for 30 min, and the solvent was purified by preparative HPLC to give eFAP-20 (1 mg, 0.6 μmol, 34%) as an off-white powder. ESI-MS: m / z 1476.1 [M+H] + .

[0135] Example 16 Preparation of N-(2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)-8-(3-(4-(4-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-5-yl)thioureido)-3-hydroxybutanamido)-3-hydroxybutanamido)propoxy)quinoline-4-carboxamide (eFAP-23) 10 (10 mg, 9.6 μmol) was treated with a solution of DCM / TFA (2 mL, 1:1) at room temperature for 30 min. All volatiles were removed under a gentle stream of air to give a crude brown solid. The crude was redissolved in DMF (2 mL), followed by the addition of 4-((tert-butoxycarbonyl)amino)-3-hydrobutanoic acid (1.4 equiv., 7.6 mg), HBTU (2.3 equiv., 20.4 mg), and DIPEA (16.7 μL). After stirring at room temperature for 1.5 h, the reaction mixture was diluted with EtOAc (20 mL) and then washed with HO (1 × 10 mL) and brine (2 × 10 mL). The organic layer was dried over MgSO, filtered, and concentrated in vacuo to give an orange-yellow oil. To the crude was added a 1:1 mixture of TFA / DCM containing 10% TIPS (1.5 mL) and then stirred at room temperature for 30 min. Upon completion, all solvent was removed under a stream of air, followed by precipitation with ice-cold diethyl ether (2 x 20 mL). The precipitate was collected, dried under a stream of air, and used in subsequent reactions without further purification. The crude material was redissolved in DMF (2 mL) and subsequently treated with 4-((tert-butoxycarbonyl)amino)-3-hydrobutanoic acid (1.1 equiv., 4.6 mg), HBTU (2 equiv., 14.4 mg), and DIPEA (4 equiv., 13.2 μL). After stirring at room temperature for 2 h, the reaction mixture was concentrated in vacuo, and the product was purified using preparative HPLC to give an orange-yellow solid (5.8 mg, 0.008 mmol, 34% over three steps). ESI-MS: m / z 720.2 [M+H] +The product (5.8 mg, 0.008 mmol) was treated with a 1:1 mixture of TFA / DCM (1.5 mL) containing 10% TIPS, and the reaction mixture was then allowed to stir at room temperature for 30 minutes. Following completion, the reaction mixture was concentrated under airflow and then precipitated with ice-cold diethyl ether (2 × 20 mL). The precipitate was collected, dried under airflow, and used in subsequent reactions without further purification. This was redissolved in a solution of fluorescein isothiocyanate (1.2 equiv., 3.8 mg), DIPEA (5.6 μL) in DMF (1.5 mL) and stirred at room temperature for 2 hours under dark conditions. The reaction mixture was concentrated in vacuo, and the crude material was purified by preparative HPLC to yield eFAP-23 (4.2 mg, 0.004 mmol, 50.0%) as a yellow powder. ESI-MS: m / z 1009.1 [M+H] + .

[0136] Example 17 Preparation of 2-((E)-2-((E)-3-(2-((E)-1-(6-((4-((4-((3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-2-hydroxy-4-oxobutyl)amino)-2-hydroxy-4-oxobutyl)amino)-6-oxohexyl)-3,3-dimethyl-5-sulfonatoindolin-2-ylidene)ethylidene)-2-(4-sulfonatophenoxy)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indol-1-ium-5-sulfonate (eFAP-24) 10 (10 mg, 9.6 μmol) was treated with a solution of DCM / TFA (2 mL, 1:1) at room temperature for 30 min. All volatiles were removed under a gentle stream of air to give a crude brown solid. The crude was redissolved in DMF (2 mL), followed by the addition of 4-((tert-butoxycarbonyl)amino)-3-hydrobutanoic acid (1.4 equiv., 7.6 mg), HBTU (2.3 equiv., 20.4 mg), and DIPEA (16.7 μL). After stirring at room temperature for 1.5 h, the reaction mixture was diluted with EtOAc (20 mL) and then washed with HO (1 × 10 mL) and brine (2 × 10 mL). The organic layer was dried over MgSO, filtered, and concentrated in vacuo to give an orange-yellow oil. To the crude was added a 1:1 mixture of TFA / DCM containing 10% TIPS (1.5 mL) and then stirred at room temperature for 30 min. Upon completion, all solvent was removed under a stream of air and then precipitated with ice-cold diethyl ether (2 × 10 mL). The precipitate was collected, dried under vacuum, and used in the next step without further purification. The crude material was redissolved in a solution of 4-((tert-butoxycarbonyl)amino)-3-hydrobutanoic acid (1.1 equiv., 4.6 mg), HBTU (2 equiv., 14.4 mg), and DIPEA (4 equiv., 13.2 μL) in DMF (2 mL) and stirred at room temperature. After 2 h, the reaction mixture was diluted with EtOAc (15 mL), washed with water (3 × 5 mL), dried over NaSO, and concentrated in vacuo to give a pale orange solid. The crude material was treated with a 1:1 mixture of TFA / DCM containing 10% TIPS (1.5 mL), and the reaction mixture was then allowed to stir at room temperature for 30 min. Following completion, the reaction mixture was concentrated under a gentle stream of air and the crude product was purified by preparative HPLC to give a pale yellow solid (4.4 mg, 7.1 μmol, 7% over four steps). ESI-MS: m / z 620.2 [M+H] + The product (2.4 mg, 3.9 μmol) was dissolved in DMF (1 mL), followed by the addition of 800CW-NHS ester (1.1 equiv., 5.0 mg, 4.3 μmol) and DIPEA (1.5 μL). After 2 h, the solvent was removed in vacuo and the crude product was purified by preparative HPLC to give eFAP-24 (5.2 mg, 3.2 μmol, 83%) as a dark green powder. ESI-MS: m / z 1605.4 [M+H]+ .

[0137] Example 18 Preparation of (S)-2,2′,2″-(10-(2-((3-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetamide (eFAP-25) 10 (5 mg, 4.8 μmol) was treated with a solution (1.5 mL) of DCM / TFA (1:1) containing 10% TIPS at room temperature for 20 min. All volatiles were removed under a gentle stream of air to give a crude dark yellow solid. The crude was redissolved in DMF (1.5 mL), followed by the addition of DO3AM-acetic acid (1.1 equiv., 2.1 mg) and DIPEA (15 μL). The reaction mixture was stirred overnight, the solvent removed in vacuo, and the crude product purified by preparative HPLC to give eFAP-25 (2.3 mg, 2.9 μmol, 60%) as a yellow powder. ESI-MS: m / z 801.4 [M+H] + .

[0138] Example 19 Preparation of 4-(2-((E)-2-((E)-3-(2-((E)-1-(6-((4-((3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-2-hydroxy-4-oxobutyl)amino)-6-oxohexyl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)ethylidene)-2-(4-sulfophenoxy)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-5-sulfo-3H-indol-1-ium-1-yl)butane-1-sulfonate (eFAP-27) 10 (10 mg, 9.6 μmol) was treated with a solution of 10% TIPS in DCM / TFA (2 mL, 1:1) at room temperature for 20 min. All volatiles were removed under a gentle stream of air to give a dark yellow solid. The crude material was redissolved in DMF (2 mL), followed by the addition of 4-((tert-butoxycarbonyl)amino)-3-hydrobutanoic acid (1.4 equiv., 7.6 mg), HBTU (2.3 equiv., 20.4 mg), and DIPEA (16.7 μL). After stirring at room temperature for 1.5 h, the reaction mixture was diluted with EtOAc (20 mL) and then washed with HO (1 × 10 mL) and brine (2 × 10 mL). The organic layer was dried over MgSO, filtered, and concentrated in vacuo to give an orange-yellow oil. The crude product was added with a 1:1 mixture of TFA / DCM containing 10% TIPS (1.5 mL) and stirred at room temperature for 30 minutes. Following completion, all solvent was removed under a stream of air and then precipitated with ice-cold diethyl ether (2 × 10 mL). The precipitate was collected, dried under vacuum, and used in the next step without further purification. It was dissolved in DMF (1 mL), followed by the addition of CW800-NHS ester (3.5 mg, 3.0 μmol) and DIPEA (1.5 μL). After 2 hours, the solvent was removed under vacuum, and the crude product was purified by preparative HPLC to give eFAP-27 (3.4 mg, 2.3 μmol, 24%) as a dark green powder. ESI-MS: m / z 1504.4 [M+H] + .

[0139] Example 20 Preparation of 4-(4-((4-((2,2-dioxido-1,2-oxathian-3-yl)methyl)-1H-1,2,3-triazol-1-yl)methyl)phenyl)-1-methyl-5,6,7,8,9,10-hexahydrocycloocta[d]pyridazin-7-yl(3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)carbamate (eFAP-28) 10 (12 mg, 0.023 mmol) was treated with a solution of 10% TIPS in DCM / TFA (2 mL, 1:1) at room temperature for 20 min. All volatiles were removed under a gentle stream of air to give a crude dark yellow solid. The crude was redissolved in DMF (1.5 mL), followed by the addition of TCO-NHS ester (1.2 equiv., 6.4 mg) and DIPEA (2 equiv., 7 μL). After stirring at room temperature for 30 min, the reaction mixture was concentrated in vacuo, and the crude product was purified by preparative HPLC to give TCO-8QCP as a pale yellow solid (12.2 mg, 0.021 mmol, 91%). ESI-MS: m / z 570.2 [M+H] + The product was dissolved in a solution of 3-((1-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)-1H-1,2,3-triazol-4-yl)methyl)-1,2-oxathiane 2,2-dioxide (1.1 equiv., 9.3 mg) in ACN / HO (3 mL, 1:1) and stirred at 37 °C. After 2 h, the reaction was concentrated in vacuo and the crude material purified by preparative HPLC to give eFAP-28 (14.0 mg, 0.015 mmol, 71%) as an off-white powder. ESI-MS: m / z 941.2 [M+H] + .

[0140] Example 21 Preparation of 4-(4-((4-((2,2-dioxido-1,2-oxathian-3-yl)methyl)-1H-1,2,3-triazol-1-yl)methyl)phenyl)-1-methyl-5,6,7,8,9,10-hexahydrocycloocta[d]pyridazin-7-yl(19-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)-15-oxo-3,6,9,12-tetraoxa-16-azanonadecyl)carbamate (eFAP-29) 10 (16 mg, 0.039 μmol) was dissolved in DMF (3 mL), followed by the addition of TCO-PEG4-NHS ester (1.3 equiv., 0.05 mg) and DIPEA (2 equiv., 13.5 μL). After stirring at room temperature for 30 min, the reaction mixture was concentrated in vacuo, and the crude product was purified by preparative HPLC to give TCO-PEG4-8QCP as a pale yellow solid (22 mg, 0.027 mmol, 69%). ESI-MS: m / z 817.3 [M+H] + The product was dissolved in a solution of 3-((1-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)-1H-1,2,3-triazol-4-yl)methyl)-1,2-oxathiane 2,2-dioxide (1.1 equiv., 11.9 mg) in ACN / HO (3 mL, 1:1) and stirred at 37 °C. After 4 h, the reaction was concentrated in vacuo and the crude material was purified by preparative HPLC to give eFAP-29 (16.4 mg, 0.014 mmol, 52%) as an off-white powder. ESI-MS: m / z 1188.4.

[0141] Example 22 Preparation of N-(2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)-8-(3-(5-(4-((2,2-dioxido-1,2-oxathian-3-yl)methyl)-1H-1,2,3-triazol-1-yl)pentanamido)propoxy)quinoline-4-carboxamide (eFAP-30) 10 (22 mg, 0.052 μmol) was dissolved in DMF (3 mL), followed by the addition of 5-azidopentanoic acid (2.7 equiv., 20 mg), HBTU (1.5 equiv., 29.6 mg), and DIPEA (2 equiv., 21.5 μL). After stirring at room temperature for 2 h, the reaction mixture was concentrated in vacuo, and the crude product was purified by preparative HPLC to give azido-8QCP as a pale yellow solid (17.3 mg, 0.032 mmol, 61.5%). ESI-MS: m / z 543.2 [M+H] +The product was dissolved in acetone and treated with CuSO4·5H2O (0.2 equiv., 1.6 mg) and sodium ascorbate (0.22 equiv., 1.47 mg). The reaction mixture was heated to 70 °C and stirred for 2 h. The reaction was concentrated under reduced pressure, and the crude product was purified by preparative HPLC to give eFAP-30 (17.7 mg, 0.025 mmol, 78%) as an off-white powder. ESI-MS: m / z 717.2 [M+H] + .

[0142] Example 23 Preparation of 2,2′,2″-(10-(4-((3-((((1,4-bis((6-((3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-6-oxohexyl)thio)-5,6,7,8,9,10-hexahydrocycloocta[d]pyridazin-7-yl)oxy)carbonyl)amino)propyl)amino)-1-carboxy-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (eFAP-31) 10 (15 mg, 29 μmol) was treated with a solution of DCM / TFA (3 mL, 1:1) at room temperature for 30 min, and all volatiles were removed under a gentle stream of air to give a crude brown solid. The crude was redissolved in DMF (1 mL), followed by the addition of TrtS-Hx-OH (1.5 equiv., 17.5 mg), HBTU (2 equiv., 23.6 mg), DIPEA (30 μL), and DMF (1.5 mL). The reaction was stirred at room temperature for 6 h, diluted with EtOAc (15 mL), washed with water (3 × 5 mL), dried over NaSO, and concentrated in vacuo to give a pale yellow solid. The crude product was treated with a solution of DCM / TFA (3 mL, 1:1) at room temperature for 30 min, the solvent was removed under a gentle stream of air, and the crude product was purified by preparative HPLC to give 8QCP-SHx (4.5 mg, 8.2 μmol, 35%) as a pale yellow powder. ESI-MS: m / z 548.3 [M+H] +The powder was redissolved in DMF / DCM (1 mL, 1:1), followed by the addition of 3,6-dichloro-1,2,4,5-tetrazine (0.4 mg, 2.7 μmol) and DIPEA (10 μL). After 30 min, the solvent was removed in vacuo, and the crude product was purified by preparative HPLC to give (8QCP-SHx)2-Tz (2 mg, 1.7 μmol, 43%) as a pale yellow powder. ESI-MS: m / z 1173.4 [M+H] + The powder was redissolved in ACN / HO (1 mL, 1:1), followed by the addition of TCO-DOTAGA (1.1 equiv., 1.2 mg). The reaction was stirred at 37 °C for 30 min, and the solvent was purified by preparative HPLC to give eFAP-31 (2 mg, 1 μmol, 63%) as an off-white powder. ESI-MS: m / z 1830.4 [M+H] + .

[0143] Example 24: 2,2',2''-(10-(4-((2-((((5aR,6S,6aS)-1-(2-(3,5-bis(2-((3-((3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-3-oxopropyl)thio)acetyl)-1,3,5-triazinium Preparation of nan-1-yl)-2-oxoethyl)-1,4,5,5a,6,6a,7,8-octahydrocyclopropa[5,6]cycloocta[1,2-d][1,2,3]triazol-6-yl)methoxy)carbonyl)amino)ethyl)amino)-1-carboxy-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (eFAP-34) 8QCP-SH (5.5 mg, 10.8 μmol) was dissolved in DMF (1 mL), followed by the addition of TADB-N3 (1.3 mg, 3.1 μmol) and DIPEA (10 μL). After 30 min, the solvent was removed in vacuo, and the crude product was purified by preparative HPLC to give (8QCP-SH)2-TADB (2.9 mg, 2.2 μmol, 41%) as an off-white powder. ESI-MS: m / z 1346.3 [M+H] +The powder was redissolved in ACN / HO (1 mL, 1:1), followed by the addition of BCN-DOTAGA (1.1 equiv., 1.7 mg). The reaction was stirred at 37 °C for 30 min, and the solvent was purified by preparative HPLC to give eFAP-34 (1.4 mg, 0.7 μmol, 31%) as an off-white powder. ESI-MS: m / z 1956.5 [M+H] + .

[0144] Example 25: 2,2',2''-(10-(4-((2-((((5aS,6R,6aR)-1-(2-(3,5-bis(2-((6-((3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-6-oxohexyl)thio)acetyl)-1,3,5-triazinium Preparation of nan-1-yl)-2-oxoethyl)-1,4,5,5a,6,6a,7,8-octahydrocyclopropa[5,6]cycloocta[1,2-d][1,2,3]triazol-6-yl)methoxy)carbonyl)amino)ethyl)amino)-1-carboxy-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (eFAP-35) 8QCP-SHx (4.1 mg, 7.5 μmol) was dissolved in DMF (1 mL), followed by the addition of TADB-N3 (1 mg, 2.4 μmol) and DIPEA (10 μL). After 30 min, the solvent was removed in vacuo, and the crude product was purified by preparative HPLC to give (8QCP-SHx)2-TADB (2.2 mg, 1.6 μmol, 44%) as an off-white powder. ESI-MS: m / z 1346.3 [M+H] + The powder was redissolved in ACN / HO (1 mL, 1:1), followed by the addition of BCN-DOTAGA (1.1 equiv., 1.3 mg). The reaction was stirred at 37 °C for 30 min, and the solvent was purified by preparative HPLC to give eFAP-35 (1.8 mg, 0.9 μmol, 54%) as an off-white powder. ESI-MS: m / z 2040.8 [M+H] + .

[0145] Example 26: 2,2',2''-(10-(4-((2-((((5aR,6S,6aS)-1-(2-(3,5-bis(16-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)-12-oxo-6,9-dioxa-3,13-diazahexadecanoyl)-1,3,5-triazinyl Preparation of (1-phenyl)-2-oxoethyl)-1,4,5,5a,6,6a,7,8-octahydrocyclopropa[5,6]cycloocta[1,2-d][1,2,3]triazol-6-yl)methoxy)carbonyl)amino)ethyl)amino)-1-carboxy-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (eFAP-36) 10 (15 mg, 29 μmol) was treated with a solution of DCM / TFA (3 mL, 1:1) at room temperature for 30 min, and all volatiles were removed under a gentle stream of air to give a crude brown solid. The crude was redissolved in DMF (1 mL), followed by the addition of PEG2-TADB-PEG2 (1.5 equiv., 26 mg), HBTU (2 equiv., 23.6 mg), DIPEA (30 μL), and DMF (1.5 mL). The reaction was stirred at room temperature for 1 h, diluted with EtOAc (15 mL), washed with water (3 × 5 mL), dried over MgSO4, and concentrated in vacuo to give an off-white solid. The crude product was redissolved in ACN / HO (1 mL, 1:1), followed by the addition of BCN-DOTAGA (1.1 equiv., 2.6 mg). The reaction was stirred at 37° C. for 30 min, and the solvent was purified by preparative HPLC to give eFAP-36 (2.7 mg, 1.2 μmol, 34%) as an off-white powder. ESI-MS: m / z 2099.2 [M+H] + .

[0146] Example 27: 2,2',2''-(10-(4-((2-((((5aR,6S,6aS)-1-(3-((4,6-bis((2-(2-(3-((3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-3-oxopropoxy)ethoxy)ethyl)amino)-1,3, Preparation of 5-triazin-2-yl)amino)propyl)-1,4,5,5a,6,6a,7,8-octahydrocyclopropa[5,6]cycloocta[1,2-d][1,2,3]triazol-6-yl)methoxy)carbonyl)amino)ethyl)amino)-1-carboxy-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (eFAP-37) 10 (15 mg, 29 μmol) was treated with a solution of DCM / TFA (3 mL, 1:1) at room temperature for 30 min, and all volatiles were removed under a gentle stream of air to give a crude brown solid. The crude was redissolved in DMF (1 mL), followed by the addition of PEG2-triazine-PEG2 (1.5 equiv., 23 mg), HBTU (2 equiv., 23.6 mg), DIPEA (30 μL), and DMF (1.5 mL). The reaction was stirred at room temperature for 1 h, diluted with EtOAc (15 mL), washed with water (3 × 5 mL), dried over Na2SO4, and concentrated in vacuo to give a pale white solid. The crude product was redissolved in ACN / HO (1 mL, 1:1), followed by the addition of BCN-DOTAGA (1.1 equiv., 2.6 mg). The reaction was stirred at 37° C. for 30 min and the solvent was purified by preparative HPLC to give eFAP-37 (3 mg, 1.5 μmol, 39%) as an off-white powder. ESI-MS: m / z 2024.1 [M+H] + .

[0147] Example 27 Preparation of 2,2′,2″-(10-(4-((3-(1-(2-(3,5-bis(2-((3-((3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-3-oxopropyl)thio)acetyl)-1,3,5-triazinan-1-yl)-2-oxoethyl)-1,9-dihydro-8H-dibenzo[b,f][1,2,3]triazolo[4,5-d]azocin-8-yl)-3-oxopropyl)amino)-1-carboxy-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (eFAP-38) (8QCP-SH)2-TADB (0.8 equiv., 1 mg) was dissolved in ACN / HO (1 mL, 1:1), followed by the addition of DBCO-DOTAGA (1.1 equiv., 0.6 mg). The reaction was stirred at 37 °C for 30 min, and the solvent was purified by preparative HPLC to give eFAP-38 (0.5 mg, 0.25 μmol, 31%) as an off-white powder. ESI-MS: m / z 1996.7 [M+H] + .

[0148] [ka]

[0149] Example 28 Preparation of 4-(2-((E)-2-((E)-3-(2-((E)-1-(6-((3-((4-((carboxymethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-6-oxohexyl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)ethylidene)-2-(4-sulfophenoxy)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-5-sulfo-3H-indol-1-ium-1-yl)butane-1-sulfonate (eFAP-39) 8-(3-((tert-Butoxycarbonyl)amino)propoxy)quinoline-4-carboxylic acid (7 mg, 0.020 mmol) was dissolved in DMF (1 mL), followed by the addition of methyl glycinate (2 equiv., 3.6 mg), HBTU (2 equiv., 14.4 mg), DIPEA (5 μL), and DMF (1 mL). The reaction was stirred at room temperature for 1 h, diluted with EtOAc (15 mL), washed with water (3 × 5 mL), dried over NaSO, and concentrated in vacuo to give a pale yellow solid. The crude product was used in the next step without further purification. It was treated with a mixture of DCM / TFA (2 mL, 1:1) at room temperature for 30 min, followed by precipitation with ice-cold diethyl ether (2 × 10 mL). The precipitate was redissolved in a solution of NaOH (0.5 M) in MeOH for 20 min. The reaction mixture was concentrated under reduced pressure to give a crude pale yellow solid. The crude product was redissolved in DMF (1 mL), followed by the addition of CW800-NHS ester (3.5 mg, 3.0 μmol) and DIPEA (1.5 μL). After 1 h, the solvent was removed in vacuo, and the crude product was purified by preparative HPLC to give eFAP-39 (3.7 mg, 2.9 μmol, 14.5%) as a dark green powder. ESI-MS: m / z 1289.3 [M+H] + .

[0150] Example 29 Preparation of 1-(1-(5-((3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-5-oxopentyl)-1H-1,2,3-triazol-4-yl)-5-fluoropentane-2-sulfonate (eFAP-42) A mixture of eFAP-30 (2.5 mg, 3.5 μmol), potassium fluoride (1.2 equivalents, 0.24 mg), and 18-crown-6 (1 equivalent, 0.9 mg) in acetonitrile (1 mL) was heated to 100° C. After 2 h, the reaction mixture was cooled to room temperature and subsequently purified using preparative HPLC to give eFAP-42 (0.8 mg, 1.1 μmol, 32%) as an off-white solid. ESI-MS: m / z 737.3 [M+H] + .

[0151] Analysis and Results radioactive label 111 InCl3 or 177 Labeling was performed by adding LuCl to eFAP compound (1 nmol) dissolved in a mixture of Gz / Asc (50 mM, 10 μL), NaOAc (2.5 M, 1 μL), EtOH (10 μL), and MQ-HO (adjusting the final volume to a total of 140 μL). The reaction mixture was heated at 90°C for 20 min. After cooling, a solution of DTPA (5 μL) was added to complex any remaining free radiometal.

[0152] FIG. 2 shows the LC / MS profile of eFAP-8.

[0153] Figure 3 shows a radiolabeling yield (RCY) of 97.8%. 111 1 shows the radio-iTLC chromatogram of In]In-eFAP-12.

[0154] in vitro stability Radiolabeled compounds (approximately 2 MBq) were mixed with 300 μL of PBS (0.1 M, pH 7.4) or mouse serum at 37°C for 1, 4, and 24 hours. At each time point, proteins in the serum samples were precipitated by adding 300 μL of acetonitrile, and the solution was centrifuged at 13,000 rpm for 5 minutes (twice). The supernatant was loaded onto a radio-HPLC system for analysis. Samples in PBS were analyzed directly by radio-HPLC without any pretreatment. All experiments were performed in triplicate.

[0155] Figure 4 shows the stability of the radiolabeled conjugate over time. 111 Radio-HPLC chromatograms of [In]In-eFAP-12 are shown. A) t=0 97.4% radiochemical purity (RCP) was obtained, B) RCP=97% at 2 hours, C) RCP=95.8% at t=4 hours, and D) RCP=93.6% at t=24 hours. E) The final chromatogram is nat corresponds to the UV signal obtained after injection of In-eFAP-12, [ 111The identity of In]In-eFAP-12 is confirmed.

[0156] LogD 7.4 Partition coefficient (LogD 7.4 values) were determined by the shake flask method. 203 A sample containing Pb-labeled compound was added to a vial containing 600 μL of PBS (pH 7.4) and 600 μL of n-octanol. The vial was vortexed vigorously and then centrifuged for 10 minutes for phase separation. Samples of the octanol phase (200 μL) and aqueous phase (200 μL) were taken and counted in a PerkinElmer WIZARD 2480 gamma counter. LogD 7.4 The value is calculated using the formula: LogD 7.4 = log [(counts in octanol phase) / (counts in aqueous phase)]. All experiments were performed in triplicate.

[0157] [Table 1]

[0158] In vitro inhibition assays for human and mouse FAP The enzymatic activity of human FAP (hFAP) and mouse FAP (mFAP) on the Z-GP-AMC substrate was measured at room temperature by monitoring fluorescence at an excitation wavelength of 360 nm and an emission wavelength of 465 nm in a Hidex microplate reader. The reaction mixture contained 20 μM substrate, 20 nM FAP, assay buffer (50 mM Tris, 100 mM NaCl, 1 mM EDTA, pH 7.4), and test compound (10 -6 From 10 -12 The IC (serial dilutions up to IC) was contained in a total volume of 50 μL. 50 The value is defined as the concentration of inhibitor required to reduce enzyme activity by 50% after addition of substrate.

[0159] Figure 5 shows the results of hFAP and mFAP inhibition experiments in the presence of eFAP compounds. All eFAP compounds tested had an IC of less than 6 nM against human FAP. 50 Values ​​are shown. Results for FAPI-46 obtained in the same assay are included for comparison.

[0160] [Table 2]

[0161] In vitro inhibition assay for PREP The enzymatic activity of prolyl endopeptidase (PREP) on the Z-GP-AMC substrate (CAS number CAS 68542-93-8) was measured at room temperature by monitoring fluorescence at an excitation wavelength of 360 nm and an emission wavelength of 465 nm in a Hidex microplate reader. The reaction mixture contained 20 μM substrate, 2 nM PREP, assay buffer (50 mM Tris (tris(hydroxymethyl)aminomethane), 100 mM NaCl, 1 mM EDTA (ethylenediaminetetraacetic acid), pH 7.4), and test compound (10 -6 From 10 -12 The IC (serial dilutions up to IC) was contained in a total volume of 50 μL. 50 The value is defined as the concentration of inhibitor required to reduce enzyme activity by 50% after addition of substrate.

[0162] The results are shown in Table 3. Results of FAPI-46 obtained in the same assay are included for comparison.

[0163] In vitro inhibition assay for DPP4 The enzymatic activity of PREP on the Z-GP-pNA substrate was measured at room temperature by monitoring fluorescence at an excitation wavelength of 380 nm and an emission wavelength of 425 nm in a Hidex microplate reader. The reaction mixture contained 20 μM substrate, 0.5 nM PREP, assay buffer (50 mM Tris, 100 mM NaCl, 1 mM EDTA, pH 7.4), and test compound (10-6 From 10 -12 The samples contained 100 μL of IC (serial dilutions up to IC 50 The value is defined as the concentration of inhibitor required to reduce enzyme activity by 50% after addition of substrate.

[0164] The results are shown in Table 3. Results of FAPI-46 obtained in the same assay are included for comparison.

[0165] [Table 3]

[0166] Competitive binding assays in cell culture U-87 MG glioblastoma cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v / v) fetal bovine serum at 37°C and 5% CO. HT-1080.hFAP cells were maintained in DMEM supplemented with fetal bovine serum (10% FBS) and antimycotic antibiotics (1% AA) at 37°C and 5% CO. For passage, upon reaching 90% confluence, cells were detached using trypsin-EDTA 0.05% and replated at a 1:4 dilution.

[0167] [ 111 Competitive binding experiments were performed by incubating [In]In-FAPI-46 radioligand with increasing concentrations of eFAP compounds in HT-1080 hFAP cells for 1 hour at 37°C. Cells were then washed three times with binding buffer and lysed. Cell-associated radioactivity was measured in a γ-counter. IC was calculated by fitting the data by nonlinear regression using GraphPad Prism. 50 values ​​were calculated.

[0168] The results of competitive binding assays for eFAP-6 and eFAP-7 performed in HT-1080 hFAP cells are shown in Figure 6 and Table 4. Both compounds exhibited low nanomolar binding affinity for FAP. Results for FABI-46 obtained in the same assay are included for comparison.

[0169] [Table 4]

[0170] Cytotoxicity assay In a 96-well plate, U-87 cells (3 × 10 3 Cells were seeded into each well and incubated at 37°C and 5% CO2 for 24 hours. The medium was removed, and the wells were washed once with PBS. Cytotoxic drugs (positive control) or drug-containing eFAP conjugates were prepared at various concentrations (i.e., 1, 10, 100, and 1000 nM) in DMEM culture medium supplemented with 2 mM L-glutamine, 10% FBS, 50 units / mL penicillin, and 50 μg / mL streptomycin. Culture medium was used as a negative control. Cells were incubated with 100 μL of test compound for 1, 24, 48, and 72 hours. After incubation, the medium was removed, and each well was washed once with PBS. Next, 100 μL of a 10% resazurin solution in culture medium was added to each well and incubated for 2.5 hours. Fluorescence measurements were performed using a microplate reader at excitation of 544 nm and emission of 590 nm.

[0171] FAP + The results of a cytotoxicity assay performed with eFAP-18 in U-87 cells are shown in Figure 7. When cells were treated with a single dose of eFAP-18 (1 μM), cell viability was reduced to approximately 25% compared to 40% when cells were incubated with the parent drug, DM1.

[0172] Biodistribution studies in animal models Six-week-old male Balb / c nu / nu specific and opportunistic pathogen-free (SOPF) mice were housed in individually ventilated cages with four mice per cage. Upon arrival, mice were allowed to acclimate for one week with free access to food and water. U-87 or HT-1080.hFAP cells (1 × 10) were implanted into the right shoulder of the mice. 6 Xenografts were grown subcutaneously to reach an appropriate size at the start of the study. All animal experiments were approved by the Animal Welfare Committee of Erasmus MC and performed in accordance with institutional guidelines (license number: AVD101002017867).

[0173] Biodistribution studies were performed to determine tumor and organ uptake of radiolabeled eFAP compounds. Animals (n = 4 per time point and per compound) were intravenously injected with an average of 100 kBq / 0.5 nmol at t = 0. At three selected time points (1, 4, and 24 h) post-injection, blood was collected by cardiac puncture under isoflurane / O2 anesthesia, and the mice were then sacrificed. Tumors and organs of interest (prostate, pancreas, spleen, gallbladder, liver, stomach, small intestine, cecum, colon, adrenal glands, kidneys, lungs, heart, salivary glands, muscle, bone, and brain) were excised, washed in PBS, and blotted dry. Blood, tumors, and relevant organs were weighed and measured in a gamma counter. Calibration curves using indium-111 and lutetium-177 were established to determine the total injected radioactivity per animal. The percentage of injected dose per gram (%ID / g) was determined for each tissue sample and corrected for both the injected volume and the %ID present at the injection site (tail).

[0174] In HT-1080.hFAP tumor-bearing mice, 111 In]In-FAPI-46 and [ 111A comparison of the biodistribution of [In]In-eFAP-6 in selected organs is shown in Figure 8. In vivo biodistribution studies using indium-111-labeled eFAP-6 demonstrated high and rapid tumor uptake and low background uptake in healthy organs, resulting in high-contrast images. The tumor-targeting performance was comparable to that of FAPI-46, the clinical gold standard for FAP-targeted tracers. Rapid clearance from the body was observed with eFAP-6, making it ideal for imaging purposes.

[0175] Evaluation of the targeting performance of eFAP-17 in near-infrared fluorescence imaging of U-87 tumor-bearing mice after intravenous injection of 2 nmol of the probe is shown in Figure 9. Figure 9 shows A) NIRF imaging of U-87 tumor-bearing mice 1 hour after intravenous injection of 2 nmol of CW800 (left, negative control) or eFAP-17 (right), and B) images of excised tumors 2.5 hours after administration of CW800 (left) or eFAP-17 (right). Tumors are indicated by open circles. eFAP-17 showed high uptake in FAP-overexpressing tumors and low accumulation in healthy tissues.

Claims

1. Formula (I) 【Chemical 1】 (In the formula, X is CH 2 or O, R 1 are H, Me, CH(CH 3 )C 2 H 5 , C.H. 2 CH(CH 3 ) 2 , CH(CH 3 ) 2 , C.H. 2 OH, CH 2 SH, CH(OH)CH 3 , C.H. 2 C(O)NH 2 , C.H. 2 CH 2 C(O)NH 2 , (CH 2 ) m CO 2 H, (CH 2 ) m NH 2 where m is 1 to 4; R 2 and R 3 each independently represents H or F. or a pharmaceutically acceptable salt thereof, comprising a scaffold according to

2. Formula (II) 【Chemistry 2】 (In the formula, R 4 represents a spacer, preferably an aliphatic spacer, Q comprises a payload such as a therapeutic agent, a diagnostic agent, or a combination thereof, preferably a radioisotope, a fluorescent dye, a drug, or a combination thereof.

2. The compound of claim 1 having the structure:

3. Formula (IIIa), preferably (IIIb) 【Chemistry 3】 (In the formula, R 4' is an optionally substituted hydrocarbylene or an optionally substituted alkylene ether, preferably alkylene, more preferably C 1 ~C 8 represents alkylene, and n is 1 to 6, preferably 1.

3. The compound of claim 2 having the structure:

4. 4. The compound of claim 2 or 3, or a pharmaceutically acceptable salt thereof, wherein Q comprises one or more of a radioisotope, a fluorescent dye, and a drug such as a cytotoxic or cytostatic agent complexed with a chelating agent.

5. Formula (IV) 【Chemistry 4】 (wherein L represents a linker, Q 1 comprises at least one payload, Z comprises an additional scaffold according to formula (I), r is 1 or greater, and s is 0 or greater.

5. The compound of any one of claims 1 to 4, having a structure according to:

6. The linker may comprise a cleavable section, a non-cleavable section, or a combination thereof, and preferably the linker is a linear or branched amino acid, such as glycine, alanine, β-alanine, 3-aminopropionic acid, 4-aminobutyric acid, 4-amino-3-hydroxybutyric acid, 5-aminovaleric acid, 6-aminohexanoic acid, 7-aminoheptanoic acid, 8-aminooctanoic acid, 9-aminononanoic acid, 10-aminodecanoic acid, 2-aminooctanoic acid, etc., a peptide spacer (Xaa) 1～4 wherein each Xaa is independently a D-amino acid of a proteinogenic amino acid, N ε ,N ε ,N ε -trimethyl-lysine, 2,3-diaminopropionic acid (Dap), 2,4-diaminobutyric acid (Dab), ornithine (Orn), homoarginine (hArg), 2-amino-4-guanidinobutyric acid (Agb), 2-amino-3-guanidinopropionic acid (Agp), β-alanine, 4-aminobutyric acid, 5-aminovaleric acid, 6-aminohexanoic acid, 7-aminoheptanoic acid, 8-aminooctanoic acid, 9-aminononanoic acid, 10-aminodecanoic acid, 2-aminooctanoic acid, 2-aminoadipic acid (2-Aad), 3-aminoadipic acid (3-Aad), 4-(aminomethyl)cyclohexane-1-carbonyl (Amcha), 4-amino-1-carboxymethyl-piperidinyl (Pip), cysteic acid, diglycolic acid, and NH 2 (CH 2 ) 2 [O(CH 2 ) 2 ] q C(O)OH, where q=1 to 36, more preferably the linker is a proteinogenic or non-proteinogenic amino acid residue selected from the group consisting of: 【Chemistry 5】 (In the formula, Y 1 is selected from the group consisting of C and N, preferably N for formulae La, Lg and Lh, and C for formula Ld, Y 2 is selected from the group consisting of C, N, and O, preferably N and C, more preferably C) and even more preferably, the linker comprises a moiety having a structure shown in any of the formulas Laa, Lfa, Lga, Lha, and Lma 【Chemistry 6】 6. The compound of claim 5, comprising a moiety having a structure according to any of the following:

7. the linker L comprises a cleavable linker section comprising an in vivo cleavable moiety and optionally a self-immolative moiety, preferably an in vivo cleavable moiety selected from the group consisting of an amide, an ester, a carbamate, a hydrazone, a thiazolidine, a methylenealkoxycarbamate, a disulfide, and combinations thereof, more preferably the in vivo cleavable moiety comprises a disulfide moiety or a terminal thiol that allows disulfide linkage to a payload that also comprises a terminal thiol, and most preferably the linker has the formula Lo 【Chemistry 7】 (In the formula, R 5 is an optionally present second spacer, preferably an optionally substituted second aliphatic spacer, more preferably C 1 ~C 6 alkylene, most preferably ethylene) 7. The compound of claim 5 or 6, comprising a moiety having the structure:

8. 8. The compound or pharmaceutically acceptable salt thereof according to claim 2, wherein Q comprises at least two of a radioisotope, a fluorescent dye, and a drug, preferably at least a radioisotope and a fluorescent dye or at least a radioisotope and a drug.

9. Formula (V) 【Chemistry 8】 (wherein p is 1 to 6, preferably 3; Q 1' and Q 1'' each independently comprises a payload such as a radioisotope, a fluorescent dye, or a drug) 9. The compound of any one of claims 2 to 8, having a structure according to:

10. A linker connects the scaffold according to formula (I) with one additional scaffold, the linker being selected from the formulae (Lp) to (Lv): 【Chemistry 9】 wherein the formulas each represent a scaffold (Z) and a payload Q for the multimeric moiety. 1 9. The compound according to any one of claims 5 to 8, or a pharmaceutically acceptable salt thereof, showing the relative positions of:

11. Suitable for the preparation of any of the compounds according to any one of claims 2 to 10, said compounds having formula (VIIa), preferably formula (VIIb) 【Chemistry 10】 (In the formula, R 4' represents an optionally substituted hydrocarbylene or an optionally substituted alkylene ether, and n is 1 to 6, preferably 1.

2. The compound of claim 1 having the structure:

12. 12. The compound of any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, wherein X represents O.

13. X represents O, and R 1 represents H and / or R 2 and R 3 At least one of R represents F, preferably R 2 and R 3 and R 1 and R 2 represent F, or a pharmaceutically acceptable salt thereof.

14. - (S)-2,2',2''-(10-(2-((3-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (eFAP-6); - 1-(6-((3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-6-oxohexyl)-3,3-dimethyl-5-sulfo-2-((1E,3E)-5-((E)-1,3,3-trimethyl-5-sulfoindolin-2-ylidene)penta-1,3-dien-1-yl)-3H-indol-1-ium (eFAP-7) 2,2',2''-(10-(1-carboxy-4-((3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (eFAP-8) - 2,2',2''-(10-(2-(((4-((3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)carbamoyl)cyclohexyl)methyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (eFAP-9) - (S)-2,2',2''-(10-(2-((1-(2-((3-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-2-oxoethyl)piperidin-4-yl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (eFAP-10) - (S)-2,2',2''-(10-(2-((4-(2-(2-((3-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-2-oxoethoxy)acetamido)benzyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (eFAP-11) - (S)-2,2',2''-(10-(2-(4-(2-((3-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-2-oxoethyl)piperazin-1-yl)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (eFAP-12) - (S)-2,2',2''-(10-(2-((2-(4-(2-((3-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-2-oxoethyl)piperazin-1-yl)ethyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (eFAP-13) 2,2'-(4-(1-carboxy-5-(4-((2,2-dioxido-1,2-oxathian-3-yl)methyl)-1H-1,2,3-triazol-1-yl)pentyl)-10-(2-((3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,7-diyl)diacetic acid (eFAP-14); 2,2'-(4-(1-carboxy-5-(4-(5-fluoro-2-sulfopentyl)-1H-1,2,3-triazol-1-yl)pentyl)-10-(2-((3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,7-diyl)diacetic acid (eFAP-15), - ((((1-(5-(4,10-bis(carboxymethyl)-7-(2-((3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)-5-carboxypentyl)-1H-1,2,3-triazol-4-yl)methyl)dimethylammonio)methyl)trifluoroborate (eFAP-16) - 4-(2-((E)-2-((E)-3-(2-((E)-1-(6-((3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-6-oxohexyl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)ethylidene)-2-(4-sulfophenoxy)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-5-sulfo-3H-indol-1-ium-1-yl)butane-1-sulfonate (eFAP-17) - (1 4 S,1 6 S,3 2 S,3 3 S, 2R, 4S, 10E, 12E, 14R)-8 6 -chloro-1 4 -hydroxy-8 5 ,14-dimethoxy-3 3 ,2,7,10-tetramethyl-1 2 ,6-Dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl N-(3-((3-((3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-3-oxopropyl)disulfanayl)propanoyl)-N-methyl-L-alaninate (eFAP-18) - 2,2',2''-(10-((2S,17S,22S)-22-carboxy-1-(((1 4 S,1 6 S,3 2 S,3 3 S, 2R, 4S, 10E, 12E, 14R)-8 6 -chloro-1 4 -hydroxy-8 5 ,14-dimethoxy-3 3 ,2,7,10-tetramethyl-1 2 ,6-Dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl)oxy)-17-((3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)carbamoyl)-2,3-dimethyl-1,4,11,19-tetraoxo-7,8-dithia-3,12,18-triazadocosane-22-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (eFAP-19) 2,2',2''-(10-(4-((3-((((1,4-bis((3-((3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-3-oxopropyl)thio)-5,6,7,8,9,10-hexahydrocycloocta[d]pyridazin-7-yl)oxy)carbonyl)amino)propyl)amino)-1-carboxy-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (eFAP-20); - N-(2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)-8-(3-(4-(4-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-5-yl)thioureido)-3-hydroxybutanamido)-3-hydroxybutanamido)propoxy)quinoline-4-carboxamide (eFAP-23); 2-((E)-2-((E)-3-(2-((E)-1-(6-((4-((4-((3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-2-hydroxy-4-oxobutyl)amino)-2-hydroxy-4-oxobutyl)amino)-6-oxohexyl)-3,3-dimethyl-5-sulfonatoindolin-2-ylidene)ethylidene)-2-(4-sulfonatophenoxy)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indol-1-ium-5-sulfonate (eFAP-24); - (S)-2,2',2''-(10-(2-((3-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetamide (eFAP-25); 4-(2-((E)-2-((E)-3-(2-((E)-1-(6-((4-((3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-2-hydroxy-4-oxobutyl)amino)-6-oxohexyl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)ethylidene)-2-(4-sulfophenoxy)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-5-sulfo-3H-indol-1-ium-1-yl)butane-1-sulfonate (eFAP-27); 4-(4-((4-((2,2-dioxido-1,2-oxathian-3-yl)methyl)-1H-1,2,3-triazol-1-yl)methyl)phenyl)-1-methyl-5,6,7,8,9,10-hexahydrocycloocta[d]pyridazin-7-yl(3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)carbamate (eFAP-28); 4-(4-((4-((2,2-dioxido-1,2-oxathian-3-yl)methyl)-1H-1,2,3-triazol-1-yl)methyl)phenyl)-1-methyl-5,6,7,8,9,10-hexahydrocycloocta[d]pyridazin-7-yl(19-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)-15-oxo-3,6,9,12-tetraoxa-16-azanonadecyl)carbamate (eFAP-29); - N-(2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)-8-(3-(5-(4-((2,2-dioxido-1,2-oxathian-3-yl)methyl)-1H-1,2,3-triazol-1-yl)pentanamido)propoxy)quinoline-4-carboxamide (eFAP-30); 2,2',2''-(10-(4-((3-((((1,4-bis((6-((3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-6-oxohexyl)thio)-5,6,7,8,9,10-hexahydrocycloocta[d]pyridazin-7-yl)oxy)carbonyl)amino)propyl)amino)-1-carboxy-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (eFAP-31); - 2,2',2''-(10-(4-((2-(((((5aR,6S,6aS)-1-(2-(3,5-bis(2-((3-((3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-3-oxopropyl)thio)acetyl)-1,3,5-triazinane -1-yl)-2-oxoethyl)-1,4,5,5a,6,6a,7,8-octahydrocyclopropa[5,6]cycloocta[1,2-d][1,2,3]triazol-6-yl)methoxy)carbonyl)amino)ethyl)amino)-1-carboxy-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (eFAP-34); - 2,2',2''-(10-(4-((2-((((5aS,6R,6aR)-1-(2-(3,5-bis(2-((6-((3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-6-oxohexyl)thio)acetyl)-1,3,5-triazinane -1-yl)-2-oxoethyl)-1,4,5,5a,6,6a,7,8-octahydrocyclopropa[5,6]cycloocta[1,2-d][1,2,3]triazol-6-yl)methoxy)carbonyl)amino)ethyl)amino)-1-carboxy-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (eFAP-35); - 2,2',2''-(10-(4-((2-((((5aR,6S,6aS)-1-(2-(3,5-bis(16-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)-12-oxo-6,9-dioxa-3,13-diazahexadecanoyl)-1,3,5-triazinane- 1-yl)-2-oxoethyl)-1,4,5,5a,6,6a,7,8-octahydrocyclopropa[5,6]cycloocta[1,2-d][1,2,3]triazol-6-yl)methoxy)carbonyl)amino)ethyl)amino)-1-carboxy-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (eFAP-36); - 2,2',2''-(10-(4-((2-((((5aR,6S,6aS)-1-(3-((4,6-bis((2-(2-(3-((3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-3-oxopropoxy)ethoxy)ethyl)amino)-1,3,5- triazin-2-yl)amino)propyl)-1,4,5,5a,6,6a,7,8-octahydrocyclopropa[5,6]cycloocta[1,2-d][1,2,3]triazol-6-yl)methoxy)carbonyl)amino)ethyl)amino)-1-carboxy-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (eFAP-37); 2,2',2''-(10-(4-((3-(1-(2-(3,5-bis(2-((3-((3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-3-oxopropyl)thio)acetyl)-1,3,5-triazinan-1-yl)-2-oxoethyl)-1,9-dihydro-8H-dibenzo[b,f][1,2,3]triazolo[4,5-d]azocin-8-yl)-3-oxopropyl)amino)-1-carboxy-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (eFAP-38); 1-(1-(4-(8-(((3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)carbamoyl)oxy)-4-methyl-5,6,7,8,9,10-hexahydrocycloocta[d]pyridazin-1-yl)benzyl)-1H-1,2,3-triazol-4-yl)-5-fluoropentane-2-sulfonate (eFAP-40); 1-(1-(4-(8-(((19-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)-15-oxo-3,6,9,12-tetraoxa-16-azanonadecyl)carbamoyl)oxy)-4-methyl-5,6,7,8,9,10-hexahydrocycloocta[d]pyridazin-1-yl)benzyl)-1H-1,2,3-triazol-4-yl)-5-fluoropentane-2-sulfonate (eFAP-41); and 1-(1-(5-((3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)oxy)propyl)amino)-5-oxopentyl)-1H-1,2,3-triazol-4-yl)-5-fluoropentane-2-sulfonate (eFAP-42); and - radiolabeled, preferably 111 In, 68 Ga, 177 Lu, 225 Ac, Pb 212 Radiolabeled eFAP-6, eFAP-8, eFAP-9, eFAP-10, eFAP-11, eFAP-12, eFAP-13, eFAP-14, eFAP-15, eFAP-16, eFAP-19, eFAP-20, eFAP-25, eFAP-31, eFAP-34, eFAP-35, eFAP-36, eFAP-37, and eFAP-38 14. The compound of any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, selected from the group consisting of: eFAP-6, eFAP-8, eFAP-17 and eFAP-24, preferably selected from the group consisting of eFAP-8 and eFAP-24.

15. 15. A compound according to any one of claims 1 to 14 or a pharmaceutically acceptable salt thereof for medical use, preferably for use in the diagnosis and / or treatment of tumors.

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