Platform and scaffold for FAP targeting agents

Abstract

The present invention relates to compounds and scaffolds for targeting fibroblast activating protein (FAP) in cancer-associated fibroblasts (CAF). The scaffold comprises a (4-quinolylcarbonyl) glycyl-2-cyanopyrrolidine scaffold, the 8-position of the quinolylcarbonyl moiety of which is substituted by a bridging carbon atom or oxygen atom, as shown in formula (I). # imgabs0 # wherein X represents CH2 or O; r1 represents H, Me, CH (CH3) C2H5, CH2CH (CH3) 2, CH (CH3) 2, CH2OH, CH2SH, CH (OH) CH3, CH2C (O) NH2, CH2CH2C (O) NH2, (CH2) mCO2H, (CH2) mNH2, where m is from 1 to 4; and R2 and R3 each independently represent H or F.

Description

[0001] The present invention relates to compounds for targeting fibroblast activation protein (FAP) in cancer-associated fibroblasts (CAFs), and in particular, to FAP targeting agents based on a (4-quinolinylcarbonyl(quinoinolyl))glycyl-2-cyanopyrrolidine scaffold.

[0002] A variety of FAP targeting agents based on a (4-quinolinylcarbonyl)glycyl-2-cyanopyrrolidine scaffold have been developed and studied in the prior art for tumor imaging and therapy (see Lindner et al., Cancers 2021, 13, 5744). Early FAP targeting agents have been disclosed in Jansen et al., ACS Med. Chem. Lett. 2013, 4, 491-496 and Jansen et al., J. Med. Chem. 2014, 57, 3053–3074, where Jansen et al. described fluorinated (4-quinolinoyl)-glycyl-2-cyanopyrrolidine scaffolds and UAMC-1110, an FAP inhibitor with nanomolar affinity for FAP.

[0003] For FAP targeting agents to have clinical application value, they must be functionalized with payloads (such as radioisotopes, fluorescent dyes, cytotoxic drugs, etc.). For this purpose, the quinolinoyl moiety of the scaffold is typically used. The current clinical reference compound is FAPI-46 (Loktev et al., Journal of Nuclear Medicine, October 2019, 60(10)1421-1429). FAPI-46 was developed after FAPI-04 (see Lindner et al., J. Nucl. Med. 2018; 59:1415-1422), and has an improved tumor-to-organ ratio compared to FAPI-04.

[0004] Other functionalized FAP targeting compounds are described in WO2021 / 160825 and Millul et al., PNAS 2021, Vol. 118, No. 16, e2101852118, and are referred to as OncoFAP. Functionalization is carried out on the quinolinylcarbonyl of these compounds using a bridging nitrogen extending to a carboxylic acid group, thereby allowing attachment to the payload. The disadvantage of this carboxylic acid group is that most metal chelating groups, auxiliaries, optical dyes, etc. are not easily available in a carboxylic acid-reactive form. They are typically sold in an amine-reactive form on the market. Therefore, an additional linker (-NHCH2-CH2-NH-) is used in the OncoFAP compounds to expose an amine group that can react with the payload.

[0005] WO 2019 / 083990 describes other FAP targeting compounds.

[0006] Despite the advantages of the prior art, there is still a need to provide improved platforms and improved FAP targeting agents based on the (4 - quinolinylcarbonyl)glycyl - 2 - cyanopyrrolidine scaffold. However, early research and development have shown that FAP has strict requirements for the structure of its inhibitors, and steric or electronic parameters are insufficient to rationalize the experimental data (Jansen et al., J. Med. Chem. 2014, 57, 3053–3074). Therefore, the development of new FAP targeting agents is extremely challenging because it is difficult or even impossible to predict which substituents and at which positions on the (4 - quinolinylcarbonyl)glycyl - 2 - cyanopyrrolidine scaffold can produce good inhibitors.

[0007] One object of the present invention is to provide a platform for compounds that exhibit improved FAP binding affinity and / or tumor uptake (including tumor:organ distribution). In addition, one object of the present invention is to provide a platform that enables access to such compounds.

[0008] The inventors have surprisingly found that substituting the 8th position of the (4 - quinolinylcarbonyl)glycyl - 2 - cyanopyrrolidine scaffold with a carbon or oxygen atom can provide access to a variety of FAP targeting agents that exhibit good FAP binding affinity and / or tumor uptake (including good tumor:organ distribution).

[0009] Bracket

[0010] Accordingly, the present invention relates to scaffolds and compounds, particularly pharmaceutical compounds comprising a (4 - quinolinylcarbonyl)glycyl - 2 - cyanopyrrolidine scaffold or a pharmaceutically acceptable salt thereof, wherein the 8th position of the quinolinylcarbonyl moiety of the scaffold is substituted with a bridging carbon or oxygen atom (also referred to herein as the scaffold / this scaffold). The bridging carbon or oxygen atom is connected or allows connection to a payload, such as a therapeutic agent, a diagnostic agent, or a combination thereof, preferably connected to a radioisotope, a fluorescent dye, a drug, or a combination thereof. The structure of the scaffold is shown in the following figure, which also shows the numbering of the quinolinylcarbonyl moiety.

[0011]

[0012] It should be understood that the scaffold may also comprise other substituents, such as fluorides and other substituents known for FAP inhibitors based on the (4 - quinolinylcarbonyl)glycyl - 2 - cyanopyrrolidine scaffold.

[0013] More specifically, the present invention relates to compounds comprising a scaffold of formula (I), or a salt thereof

[0014]

[0015] wherein

[0016] X represents CH2 or O, preferably O;

[0017] R 1 represents H, Me, CH(CH3)C2H5, CH2CH(CH3)2, CH(CH3)2, CH2OH, CH2SH, CH(OH)CH3, CH2C(O)NH2, CH2CH2C(O)NH2, (CH2) q (CH2) q CO2H, (CH2)

[0018] R 2 and R 3 each independently represents H or F.

[0019] In a preferred embodiment, the moiety of formula (I) is conjugated to a payload (such as a therapeutic agent, a diagnostic agent, or a combination thereof) via at least one spacer R 4 as shown in formula (II)

[0020]

[0021] wherein

[0022] R 4 represents a spacer, preferably an optionally substituted hydrocarbon spacer, preferably an aliphatic spacer; and

[0023] Q comprises the payload.

[0024] In the (4 - quinolinylcarbonyl) glycinyl - 2 - cyanopyrrolidine scaffold of the present invention, preferably at least one of R 2 and R 3 is F, more preferably at least R 2 is F (such that the atoms to which R 2 and R 3 are attached have the (S) configuration or are achiral), and most preferably both R 2 and R 3 are F.

[0025] R 1 The substituent is preferably H.

[0026] Spacer and connector

[0027] R 4 The spacer represented preferably contains a nitrogen atom, as shown in formula (IIIa), where R 4 represents an optionally substituted alkylene or an optionally substituted alkylene ether, preferably an alkylene, more preferably a C1 - C8 alkylene, which can be used to link Q to the (4 - quinolinylcarbonyl) glycinyl - 2 - cyanopyrrolidine scaffold.

[0028]

[0029] Spacer (R 4 and R 4’ ) may be substituted or interrupted by one or more heteroatoms, such as one or more of OH, NH2, SO2, and halogens (e.g., F, Cl, Br, and I). R 4 and R 4’ represent a spacer which is preferably an amino-terminated aliphatic C1-C8 spacer. It has been found that such spacers result in excellent FAP binding affinity and tumor uptake. Thus, a particularly preferred embodiment of the compounds of the present invention has a structure according to formula (IIIb)

[0030]

[0031] where n is 1-6, preferably 1.

[0032] Compounds of formula (IIIa) and (IIIb) structures containing the Q functional group can be readily prepared from intermediates of formula (VIIa) and (VIIb).

[0033]

[0034] where X, R 1 -R 4 、R 4’ and n are as defined in formula (IIIa) and (IIIb). Advantageously, the amino-terminated compound can be directly reacted and / or linked with most commercially available metal chelating groups, prosthetic groups, optical dyes, etc. Thus, the intermediate compound has a wide range of uses and is an aspect of the present invention.

[0035] The group represented by Q (also simply referred to as Q herein) contains at least one payload, such as a therapeutic agent and / or a diagnostic agent. It should be understood that in addition to this payload, Q may also contain a linking functional group (also simply referred to as a linker herein), one or more additional scaffolds, and / or a combination of at least two of a radioisotope, a fluorescent dye, and a drug.

[0036] In a particular embodiment, Q contains a linker attached to R 4 or NHR 4’Linker L. This linker can be used to connect one or more payloads and one or more scaffolds. This linker can contribute to the solubility of the compound. For example, 4-amino-3-hydroxybutyric acid (GABOB) can be used to assist water solubility and reduce lipophilicity (observed by logP value). The linker can 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 connect one or more payloads, and one or more other non-cleavable moieties connect one or more other payloads or scaffolds).

[0037] The compound containing linker L can be represented by formula (IV)

[0038]

[0039] where L represents the linker, Q 1 includes the payload, Z includes additional scaffolds, r is 1 or greater, and s is 0 or greater. It should be understood that the additional scaffolds preferably have the structure of formula (Z).

[0040]

[0041] where X and R 1 -R 4 represents the group as defined in formula (II).

[0042] The linker can contain cleavable and non-cleavable regions and combinations thereof. The "section" herein refers to a structural feature that connects the scaffold to one or more payloads and optionally to additional scaffolds. Since the linker can connect the scaffold to multiple payloads and additional scaffolds, the linker can contain multiple sections, each of which can be cleavable or non-cleavable. For the linker, which type of cleavable or non-cleavable section is preferred depends on the payload. It should be understood that when the compound contains a single payload (i.e., r = 1) and does not contain additional scaffolds (i.e., s = 0), the linker can contain a single linker section.

[0043] Generally, when the linker connects a fluorophore dye and / or a radioisotope and it is necessary to ensure the stability of the scaffold and the payload, the linker contains a non-cleavable linker section. The non-cleavable linker section is also preferably used to connect additional scaffolds.

[0044] In this regard, suitable non-cleavable linker regions can be based on or include linear or branched amino acids 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. In some embodiments, the linker is a peptide spacer (Xaa) 1-4 , where each Xaa is independently a protein or non-protein amino acid residue. Herein, each peptide backbone amino group can be independently optionally methylated. In a specific embodiment, each non-protein amino acid residue is independently selected from the group consisting of: D-amino acids of protein amino acids, 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), sulfopropylalanine, diglycolic acid and NH2(CH2)2 t C(O)OH (where t = 1-36). The linker can contain one or more of the above groups. For example, the linker can be based on a single GABOB unit such as eFAP-27 (see below), or can be based on two (or even more) GABOB units such as eFAP-24 (see below).

[0045] In a further preferred embodiment, the linker L contains a moiety having any one of the structures shown in formulas La-Ln;

[0046]

[0047] And wherein further

[0048] Y 1 is selected from C and N, preferably N for formulas La, Lg and Lh, and preferably C for formula Ld; and

[0049] Y 2 is selected from the group consisting of C, N and O, preferably N and C; more preferably C.

[0050] In yet another preferred embodiment, structure L comprises a portion having any one of the structures of formulae Laa, Lfa, Lga, Lha and Lma, which are specific forms of formulae La, Lf, Lg, Lh and Lm, respectively.

[0051]

[0052] It should be understood that unless the stereochemistry of an atom is explicitly indicated herein, the stereochemistry of that atom is undefined, indicating that the structure represents all possible stereoisomers of said atom. For example, 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 in formula Lfa’ below.

[0053]

[0054] Linkers having the structures of 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 linker disclosed herein, may be cationic under physiological conditions and may thus affect the overall charge of the conjugate. Preferably, the linker comprises Amcha or Pip.

[0055] In a specific embodiment, the linker may comprise a cleavable linker region that comprises a moiety cleavable in vivo. Such linker regions are particularly preferred when the payload comprises a drug that would benefit from delivery from a scaffold and release at the target location (e.g., 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).

[0056] Accordingly, an enzyme-sensitive cleavable linker region is a preferred linker region, most preferably a cleavable region sensitive to an enzyme overexpressed in tumors (such as glutathione).

[0057] Examples of cleavable moieties that may be included in the cleavable linker region include amides, esters, carbamates, hydrazones, thiazolidines, methylenealkoxycarbamates and disulfides. The cleavable moiety may comprise one or more such moieties and, for example, may include a peptide, an oligosaccharide or another oligomeric sequence that can be selectively cleaved in vivo.

[0058] In a preferred embodiment, the cleavable moiety comprises a disulfide moiety or a terminal thiol, which is capable of forming a disulfide bond with a payload (e.g., mertansine or DM1) that also contains a terminal thiol.

[0059] Thus, in a particularly preferred embodiment, the cleavable linker region is based on or comprises a linear or branched mercapto carboxylic acid, such as thioglycolic acid, thiolactic acid (also known as 2-mercaptopropionic acid), 3-mercaptopropionic acid (3-MPA), etc. Thus, the linker may comprise a moiety having the structure of Lo.

[0060]

[0061] Wherein R 5 represents an optionally present second spacer, preferably an optionally substituted second aliphatic spacer, more preferably a C1-C6 alkylene, most preferably ethylene.

[0062] The cleavable linker region may comprise a self-destructive moiety that, upon activation, can release an unfunctionalized payload through a series of reactions (see, e.g., R.V. Gonzaga et al., Journal of Pharmaceutical Sciences 109 (2020) 3262-3281). Examples of suitable self-destructive moieties include p-aminobenzyl carbamate conjugated to valine-citrulline (Val-Cit-PAB) or β-glucuronide, as well as self-destructive moieties based on Grob fragmentation (see, e.g., Ferhati et al., Org. Lett. 2021, 23, 21, 8580–8584).

[0063] The linker can be a dimer linker for attaching the scaffold to one payload (i.e., r = 1 and s = 0 in formula (IV)). In other embodiments, the linker is polymeric, meaning it attaches the scaffold to the payload and at least one additional payload and / or scaffold (i.e., r ≥ 1 and s + r ≥ 2 in formula (IV)). Thus, in a specific embodiment, the linker attaches one or more additional scaffolds and / or a combination of at least two of a radioisotope, a fluorescent dye, and a drug to the scaffold. Generally, when the linker attaches more than 5 entities, the compound is too large and renal excretion is adversely affected. Thus, the linker generally attaches the scaffold to the payload and at most three additional payloads and / or scaffolds (i.e., s + r = 1, 2, 3, or 4, and r ≥ 1 in formula (IV)).

[0064] A suitable linker can be selected according to the payload. For example, if two payloads or a payload and an additional scaffold are to be linked, one of which contains a free carboxylic acid and the other contains a free amine, the linker can be glutamate-based. Similarly, if two payloads each containing a free carboxylic acid are to be linked, the linker can be based on lysine or an analogue thereof or contain lysine or an analogue thereof. Thus, for example, one specific embodiment is a compound having the structure of formula (V).

[0065]

[0066] wherein p is 1 - 6, preferably 3; and

[0067] Q 1’ and Q 1” each independently contain a payload, such as a radioisotope, a fluorescent dye, and / or a drug.

[0068] In some embodiments, wherein the linker connects a scaffold to an additional scaffold, and the linker can comprise a polymeric moiety as shown in any one of formulas (Lp) to (Lv)

[0069]

[0070] The said formulas respectively show the relative positions of the scaffold (Z) and the payload Q 1 relative to the polymeric moiety. It should be understood that the compound and the linker can contain additional atoms and / or linking regions between the polymeric moiety and the (Z) and Q shown in formulas (Lp)-(Lv). The scaffold (Z) has the same structure as the scaffolds described above. These scaffolds are preferably of the same structure, but can differ, for example, in X and R 1 -R 1 -R 4 aspects.

[0071] Diagnostic agent and therapeutic agent

[0072] The present invention is not particularly limited to specific payloads, diagnostic agents, and therapeutic agents. Advantageously, the present invention allows for the combined use of a variety of payloads with scaffolds without compromising the FAP affinity and / or tumor uptake of the compound. It should be understood that suitable payloads and reagents can be selected according to the desired use (e.g., diagnosis and treatment).

[0073] For example, the known payloads of previously developed FAP-targeting agents disclosed in WO2019 / 154859 and WO2021 / 160825 can also be applicable to the present invention.

[0074] Chelating agents and radioisotopes

[0075] In one specific embodiment, the payload comprises a radioisotope complexed with a chelating agent. 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 the references cited therein. The radioisotope can be an α- or β-particle emitter, an Auger emitter, a positron emitter, and / or a γ-emitter. α-Particle emitters and β-particle emitters, such as 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 for therapy. Suitable γ-emitters, such as 99m Tc, 67 Ga, 111 In, etc., can be used for SPECT imaging; and positron emitters, such as 68 Ga, 64 Cu, 18 F, etc., can be used for PET imaging. Thus, the radioisotope M for use in the present invention is preferably selected from the group consisting of 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, 152Tb, 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, 117m Sn, 153 Gd, 153 Sm and 166 Ho, preferably selected from the group consisting of: 18 F, 64 Cu, 67 Ga, 68 Ga, 90 Y, 111 In, 177 Lu, 212 Pb and 225 Ac, most preferably selected from 18 F, 68 Ga, 111 In, 225 Ac and 177 Lu.

[0076] A variety of chelating agents are known in the art that can bind to radioisotopes. The chelating agent used in the present invention is a pharmaceutically acceptable chelating agent. The choice of chelating agent is generally based on the radioisotope required for diagnosis or treatment, as not all chelating agents are equally suitable for all radioisotopes. Typical radioisotope chelating agents contain cyclic or branched polyaminopolycarboxylic acid moieties or their amide derivatives. 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 the references cited therein.

[0077] In a preferred embodiment of the present invention, the radioisotope chelating agent is based on a compound selected from the group consisting of:

[0078] - DOTA (1,4,7,10 - tetraazacyclododecane - N,N′,N″,N″′ - tetraacetic acid, also known as tetraxetan) and its derivatives, such as p - SCN - Bn - DOTA (2 - (4 - isothiocyanatobenzyl) - 1,4,7,10 - tetraazacyclododecane tetraacetic acid);

[0079] - PSC (1,4,7,10 - tetraazacyclododecane - 7 - acetamide - 1,4,10 - triacetic acid);

[0080] - DO3A (1,4,7,10 - tetraazacyclododecane - N,N′,N″ - triacetic acid);

[0081] - DOTAGA (1,4,7,10 - tetraazacyclododecane, 1 - (pentanedioic acid) - 4,7,10 - triacetic acid);

[0082] - DO3AM (2,2′,2″ - (1,4,7,10 - tetraazacyclododecane - 1,4,7 - triyl) triacetamide);

[0083] - DOTAM (2 - [4,7,10 - tris(2 - amino - 2 - oxoethyl) - 1,4,7,10 - tetraazacyclododecane

[0084] - 1 - yl] acetamide) and its derivatives, such as pSCN - Bn - TCMC (2 - (4 - isothiocyanatobenzyl) - 1,4,7,10 - tetraaza - 1,4,7,10 - tetra(2 - carbamoylmethyl) cyclododecane);

[0085] - NOTA (1,4,7 - triazacyclononane - N,N′,N″ - triacetic acid);

[0086] -NODAGA (1-(1,3-carboxypropyl)-4,7-carboxymethyl-1,4,7-triazacyclononane);

[0087] -NODASA (1,4,7-triazacyclononane-1-succinic acid-4,7-diacetic acid);

[0088] -CB-DO2A (4,10-bis(carboxymethyl)-1,4,7,10-tetraazabicyclo[5.5.2]tetradecane);

[0089] -3p-C-DEPA (2-[(carboxymethyl)][5-(4-nitrophenyl-1-[4,7,10-tris-(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]pent-2-yl)-amino]acetic acid);

[0090] -TCMC (1,4,7,10-tetra(carbamoylmethyl)1,4,7,10-tetraazacyclododecane

[0091] -DTPA (diethylenetriaminepentaacetic acid) and DTPA derivatives such as CHX-A”-DTPA

[0092] (2-(p-isothiocyanatobenzyl)cyclohexyl diethylenetriaminepentaacetic acid and 1B4M-DTPA;

[0093] -TETA (1,4,8,11-tetraazacyclotetradecane 1,4,8,11-tetraacetic acid) and its analogs and derivatives such as C-NETA;

[0094] -NE3TA ((7-[2-[(carboxymethyl)amino]ethyl]-1,4,7-triazacyclononane-1,4-diacetic acid) and its derivatives such as C-NE3TA;

[0095] -CB-TE2A (4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane);

[0096] -NETA ({4-[2-(bis-carboxymethylamino)-ethyl]7-carboxymethyl-[1,4,7]triazacyclononane(triazonan)-1-yl)-acetic acid); and NETA derivatives such as

[0097] 3p-C-NETA (see, Sun et al. ACS Omega 2020, 5, 44, 28615–28620)

[0098] -H2azapa (N,N′-[1-benzyl-1,2,3-triazol-4-yl]methyl-N,N′-[6-(carboxy)pyridin-2-yl)-1,2-diaminoethane and other pyridine carboxylic acid derivatives, such as H2dedpa (1,2-[6-(carboxy)-pyridin-2-yl)methylamino)ethane, H4octapa (N,N′-bis(6-carboxy-2-pyridylmethyl)-

[0099] -ethylenediamino-N,N′-diacetic acid), H4py4pa, H4Pypa, H6phospha, H4CHXoctapa, H5decapa (N,N″-[[6-(carboxy)pyridin-2-yl]methyl]-

[0100] -diethylenetriamine-N,N′,N″-triacetic acid) and H4neunpa-p-Bn-NO2;

[0101] -SHBED (N,N′-bis(2-hydroxy-5-sulfobenzyl)ethylenediamino-N,N′-diacetic acid);

[0102] -HBED (N,N′-bis(2-hydroxybenzyl)-ethylenediamino-N,N′-diacetic acid);

[0103] -H2-MACROPA (N,N′-bis[(6-carboxy-2-pyridyl)methyl]-4,13-diaza-18-crown-6);

[0104] -PCTA (3,6,9,15-tetraazabicyclo[9.3.1]pentadec-1(15),11,13-triene-3,6,9-triacetic acid);

[0105] -Me-3,2-HOPO (see Ramdahl, Bioorganic & Medicinal Chemistry Letters 26(2016)17,4318 - 4321);

[0106] -CB-TE1A1P (1,4,8,11-tetraazacyclotetradecane-1-(methanephosphonic acid)-8-(methanecarboxylic acid));

[0107] -CB-TE2P (1,4,8,11-tetraazacyclotetradecane-1,8-bis(methanephosphonic acid);

[0108] -MM-TE2A (N-methyl-1,8-N,N′-bis-(carboxymethyl)-1,4,8,11-tetraazacyclotetradecane);

[0109] -DM-TE2A (N,N′-dimethyl 1,8-N,N′-bis-(carboxymethyl)-1,4,8,11-tetraazacyclotetradecane);

[0110] -sarcophagine and sarcophagine derivatives such as SarAr (1-N-(4-aminobenzyl)-3,6,10,13,16,19-hexaazabicyclo[6.6.6]-icosane-1,8-diamine), diamSar, AmBaSar, and BaBaSar;

[0111] -TRAP (1,4,7-triazacyclononane-1,4,7-tris[methyl(2-carboxyethyl)phosphinic acid]) and its analogs such as NOPO (1,4,7-triazacyclononane-1,4-bis[methylene(hydroxymethyl)phosphinic acid]-7-[methylene(2-carboxyethyl)phosphinic acid]);

[0112] -AAZTA (1,4-bis(hydroxycarbonylmethyl)-6-[bis(hydroxycarbonylmethyl)]amino-6-methylperhydro-1,4-diazine );

[0113] -DATA and DATA derivatives;

[0114] -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-dioxo-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);

[0115] -PCTA (6,9,15-tetraazabicyclo[9.3.1]pentadec-1(15),11,13-triene-3,6,9-triacetic acid);

[0116] -BCPA (see Price and Orvig, Chem. Soc. Rev., 2014, 43, 260 - 290);

[0117] - DFO (deferoxamine) and DFO derivatives;

[0118] - Trithiol chelates;

[0119] - Mercaptoacetyl;

[0120] - Hydrazinonicotinamide;

[0121] - Dimercaptosuccinic acid;

[0122] - 1,2 - Ethylene diyl bis - L - cysteine diethyl ester;

[0123] - Methylenediphosphonate;

[0124] - Hexamethylpropyleneamine oxime;

[0125] - Hexakis (methoxyisobutyl isonitrile);

[0126] And their analogs.

[0127] Most preferably, DOTA and NOTA chelators are preferred because they have particularly good chelating effects with 18 F, 68 Ga, 111 In, 177 Lu and 225 Ac.

[0128] Preferably, the chelator binds to the linker through one of its amide groups or one of its carboxylic acid groups to form an amide bond. However, it can also bind through one of its carbon atoms. To connect to a carbon atom, the chelator can be appropriately equipped with an isothiocyanate functional group, such as benzyl isothiocyanate. Examples of such equipped chelators include p - SCN - Bn - DOTA and p - SCN - Bn - TCMC.

[0129] In a preferred embodiment, the linker binds to one of the carboxylic acid groups of the chelator.

[0130] Therefore, in a specific embodiment, the radioisotope complexed with the chelator has a structure of any one of Qa - Qh, preferably Qa, Qg or Qh, more preferably Qa or Qg.

[0131]

[0132] Where m is independently 0 - 6, preferably 1;

[0133] A 1 - A 4 Independently selected from the group consisting of: H, alkyl, fatty acids (such as carboxylic acids like CH2CO2H and CO2H), and their amides and esters;

[0134] A 5represents a fluorine-containing group or its precursor, such as 1,4-butanesultone-1-yl(1,2 λ6 -oxathiacyclohexane-2,2-dione-3-yl or 2,2-dioxo-1,2-oxathiacyclohexane-3-yl), CH(SO3H)(CH2)3F and / or N(CH3)2)CH2BF3;

[0135] (M) represents a radioisotope;

[0136] (L) represents an optionally present linker or an optionally present core as described herein; and

[0137] (Z) represents a scaffold as described herein.

[0138] In any one of the structures of formula Qa-Qh, A1-A4 are preferably independently selected from the group consisting of: 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 CO2H to enhance the water solubility of the compound. In a further preferred embodiment, A 1 -A 4 are all the same and are selected from -CH2CO2H and -CH2C(O)NH2.

[0139] (L) may also be absent.

[0140] In a particular embodiment, the chelator can be combined with a fluoride label as shown in formula (Qh), wherein A 5 represents CH(SO3H)(CH2)3F and / or N(CH3)2)CH2BF3. The fluoride can be 18 an F isotope, suitable for imaging such as PET imaging. The compound may also contain a precursor for preparing such a compound as shown in formula (Qh), wherein A 5 represents 1,4-butanesultone(1,2λ 6 -oxathiacyclohexane-2,2-dione).

[0141] Fluorophore

[0142] The payload can be a dye, preferably a fluorescent dye, such as a dye selected from the group consisting of cyanines (cyanines), phthalocyanines, rhodamines, fluoresceins, xanthenes, coumarins, styryls, porphyrins, fluorescent organometallic complexes, oxanines, perylenes, acridines, boron-substituted methylene dipyrroles, and the like.

[0143] Particularly suitable fluorescent dyes include cyanines and / or phthalocyanines. Preferred dyes are CY5 dyes, especially sulfo-CY5.

[0144] Drug

[0145] The payload can be a drug, preferably a cytotoxic drug or a cell growth inhibitory drug. Examples thereof include some of the radioactive isotopes described herein and molecular drugs. Suitable drugs also include the drugs described in WO2021 / 160825, which is incorporated herein by reference in its entirety. Particularly suitable drugs include Hsp90 inhibitors, such as geldanamycin analogs, helvolic acid analogs, Zelavespib (PU-H71), Onalespib (AT13387), SNX-0723, HSP990, YC-72-AB85, Luminespib (AUY922), VER-49009, and NMS-E973. Most preferably, the drug includes maytansine (DM1).

[0146] Exemplary compound

[0147] In a particularly preferred embodiment, the drug compound or a pharmaceutically acceptable salt thereof has a structure of any one 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 contain radioactive isotopes, more preferably contain 111 In, 68 Ga, 177 Lu, Pb 212 or 225Ac radioisotope. The compound eFAP-6 is particularly suitable for radioisotope medical imaging. The compounds eFAP-7 and eFAP-23 are particularly suitable for biological research or diagnostic screening. The compounds eFAP-8 to eFAP-16, eFAP-25, eFAP-20, eFAP-31, and eFAP-34 to eFAP-38 are particularly suitable for radioisotope-based cancer therapy and / or radionuclide-based cancer imaging, especially considering their good tumor retention. The compounds eFAP-17, eFAP-23, eFAP-24, and eFAP-27 are particularly suitable for image-guided surgery for cancer treatment. The compound eFAP-18 is particularly suitable for cancer treatment. The compound eFAP-19 is a biofunctionalized compound suitable for treating cancer by releasing DM1 and using, for example 177 Lu for radioisotope therapy. The compounds eFAP-28 to eFAP-30 are 18 F-fluorinated precursors that can be used for positron emission tomography (PET) imaging. The compounds eFAP-40, eFAP-41, and eFAP-42 can be prepared from their respective precursors eFAP-28, eFAP-29, and eFAP-30 and can accordingly be used for PET imaging.

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161] More than 90% of solid tumors contain cancer-associated fibroblasts that overexpress FAP. Thus, the compounds of the present invention can be used for the diagnosis and treatment of a variety of tumors, including sarcoma, breast cancer, lung cancer, ovarian cancer, head and neck cancer, prostate cancer, colorectal cancer, etc., and even rare or refractory cancers such as pancreatic cancer and / or brain cancer.

[0162] The compound or a pharmaceutically acceptable salt thereof can be used as a drug. More specifically, for the medical treatment or diagnosis (including imaging) of tumors. Whether it can be used for treatment or diagnosis mainly depends on the payload used, as described herein.

[0163] Thus, another aspect of the present invention relates to a method for treating or diagnosing tumors, particularly tumors that overexpress FAP. The method includes administering to a patient the compound or a pharmaceutically acceptable salt thereof in a pharmaceutically acceptable dose.

[0164] It should be understood that for any optionally substituted or interrupted moiety described herein, such moiety can also be unsubstituted or uninterrupted.

[0165] Furthermore, when describing a compound herein, unless otherwise explicitly stated, it also refers to its salts, prodrugs, metabolites, etc.

[0166] When the stereochemistry of an atom in a structure is not defined (e.g., C–R 1 ), the structure refers to all stereoisomers of said atom.

[0167] As used herein, the singular forms "a", "an" and "the" also include the plural forms unless the context clearly dictates otherwise. The term "and / or" includes any combination and all combinations of one or more of the listed related items. It should be understood that the terms "comprises" and / or "comprising" specify the presence of the stated features, but do not preclude the presence or addition of one or more other features.

[0168] For the purposes of clarity and conciseness, features are described herein as part of the same or separate embodiments, but it should be understood that the scope of the present invention can include embodiments having combinations of all or some of the described features.

[0169] The present invention can be illustrated by the following non-limiting examples and embodiments.

[0170] Overview

[0171] Chemicals and solvents were obtained from commercial suppliers and used without further purification. Reactions were carried out under magnetic stirring and monitored by thin layer chromatography (TLC) on pre-coated plates (silica gel 60F254) on the back of Merck aluminum sheets. Melting points were determined using a Stuart SMP20. Equipped with Liquid chromatography - mass spectrometry (LC - MS) was performed on an Agilent 1260 Infinity II electrospray ionization (ESI) LC - MS system with an InfinityLab Poroshell 120 EC - C18 chromatographic column (2.7 μm, 3.0 x 100 mm). The product was gradient eluted with acetonitrile (ACN; 5–100%) in water containing 0.1% formic acid (FA) at a flow rate of 0.5 mL / min for 8 minutes and monitored using a UV detector at 220 nm, 254 nm, and 280 nm. Nuclear magnetic resonance (NMR) spectra were recorded in deuterated dimethyl sulfoxide (DMSO - d6) and chloroform - d (CDCl3) at room temperature on a Bruker AVANCE 400 or Nanalysis 60Pro. Chemical shifts were expressed as δ values (in ppm), and coupling constants J were expressed in Hz. The splitting patterns were reported as s (singlet), d (doublet), t (triplet), q (quartet), qt (quintet), m (multiplet), and br (broad signal). The raw NMR data were processed using MestreNova 13. The purification of the compound was carried out using an Agilent preparative HPLC 1260 Infinity II system with a preparative column (50×21.2 mm, 5 μm) and an ACN gradient elution method (10% to 95%, dissolved in water containing 0.1% FA, flow rate 10 mL / min, over 10 minutes).

[0172] The instant thin - layer chromatography (iTLC - SG) plates on silica - impregnated glass fiber sheets were eluted with sodium citrate (0.1 M, pH 5). The plates were analyzed using a bSCAN radiochromatography scanner equipped with a sodium iodide detector from Brightspec. Radioactive samples used for LogD 7.4 determination, in vitro assays, and in vivo studies were counted using a Wizard 2480 gamma counter. Activity measurements were performed using a VDC - 405 dose calibrator. Quality control and stability analysis of radiolabeled compounds were carried out using a Waters Acquity Arc ultra - high - performance liquid chromatography (UHPLC) system equipped with a diode array detector, a Canberra radioactive detector, and an analytical C18 column (250.0×4.6 mm, 5 μm) with an ACN gradient elution (5% to 95%, dissolved in water containing 0.1% trifluoroacetic acid TFA) at a flow rate of 1 mL / min for over 30 minutes.

[0173]

[0174] Preparation of (S) - 1 - tert - butyl 2 - methyl 4,4 - difluoropyrrolidine - 1,2 - dicarboxylate

[0175] Step 1: (S)-1-tert-butyl 2-methyl 4-oxopyrrolidine-1,2-dicarboxylate (2)

[0176] 1,3,5-Trichloro-1,3,5-triazine-2,4,6-trione (0.95 g, 4.07 mmol) was added to a solution of (S)-1-tert-butyl 2-methyl 4-oxopyrrolidine-1,2-dicarboxylate (0.95 g, 3.88 mmol) in DCM (10 mL) cooled to 0 °C, and then the catalyst TEMPO (6 mg, 0.04 mmol) was added. After 5 minutes, the mixture was allowed to warm to room temperature, stirred for an additional 30 minutes, and then filtered through Celite. The organic layer was washed successively with 10 mL of saturated potassium carbonate solution, sodium thiosulfate, and brine, dried over anhydrous sodium sulfate, filtered, and evaporated. The crude compound 2 (0.65 g, 70%) was used without further purification. 1 HNMR (400 MHz, 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] + .

[0177] Step 2: (S)-1-tert-butyl 2-methyl 4,4-difluoropyrrolidine-1,2-dicarboxylate (3)

[0178] A solution of compound 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 hours. The solution was poured into saturated sodium bicarbonate solution and extracted with DCM (3 x 15 mL), dried (Na2SO4), filtered, and evaporated in vacuo. Silica gel chromatography (100% DCM) gave the product as a yellow oil (0.150 g, 61%). 1 HNMR (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] + .

[0179] Preparation of Intermediate 10

[0180] According to Figure 1 the scheme shown, Intermediate 10 was prepared.

[0181] Step 1: (S)-1-(tert-butoxycarbonyl)-4,4-difluoropyrrolidine-2-carboxylic acid (4)

[0182] 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 minutes. After completion of the reaction, the reaction mixture was acidified to pH 2 with 1N HCl, and the aqueous layer was extracted with DCM (3 x 50 mL). The combined organic layers were washed with brine, dried over MgSO4 and concentrated in vacuo. A grayish-white solid product (1.62 g, 6.44 mmol, 86%) was obtained. 1 1H 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). mp: 118 - 119 °C. ESI-MS: m / z 152.1 [M + H - Boc] + .

[0183] Step 2: (S)-2-carbamoyl-4,4-difluoropyrrolidine-1-carboxylic acid tert-butyl ester (5)

[0184] 4 (1.55 g, 6.15 mmol) was dissolved in DCM (anhydrous, 30 mL), HONSu (0.88 g, 7.65 mmol) was added, and the mixture was stirred at room temperature until completely dissolved. DCC (1.55 g, 7.52 mmol) was added, and the mixture was stirred at room temperature for 30 minutes, during which an insoluble material slowly formed in the reaction mixture. NH3 (14 mL, 4M MeOH solution) was added dropwise, and the reaction mixture was stirred at room temperature for 3 hours. After completion of the reaction, EtOAc (50 mL) was added, and the organic layer was washed with saturated aqueous NaHCO3 (5 x 25 mL), brine (25 mL), dried over MgSO4 and concentrated in vacuo. A grayish-white solid product (1.53 g, 6.02 mmol, 97%) was obtained. 1 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). mp: 127–128 °C. ESI-MS: m / z 151.1 [M + H - Boc] + .

[0185] Step 3: (S)-4,4-difluoropyrrolidine-2-carboxamide (6)

[0186] 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. After completion of the reaction, all volatile substances 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). mp: >130 °C. ESI-MS: m / z 151.0 [M+H] + .

[0187] Step 4: (S)-(2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamic acid tert-butyl ester (7)

[0188] 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 x 25 mL), dried over MgSO4 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 to reach room temperature. EtOAc (50 mL) and H2O (50 mL) were added. The layers were separated, and the aqueous layer was extracted with EtOAc (3 x 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). mp: 128 - 129 °C. ESI-MS: m / z 190.1 [M+H-Boc] + .

[0189] Step 5: 8-(3-((tert-butoxycarbonyl)amino)propoxy)quinoline-4-carboxylic acid (9)

[0190] 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 Cs2CO3 (1.31 g, 4.0 mmol) were added. The reaction mixture was stirred at room temperature for 2.5 h. After completion of the reaction, EtOAc (50 mL) was added, and the organic layer was washed with brine (5 x 25 mL), dried over MgSO4 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 by prep-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, 1H, J = 7.4 Hz), 4.16 (t, 2H, J = 5.9 Hz), 3.39 - 2.91 (m, 2H), 1.93 (t, 2H, J = 6.1 Hz), 1.32 (s, 9H). mp: >130 °C. ESI-MS: m / z 346.7 [M+H] + .

[0191] Step 6: (S)-(3-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl yl)quinolin-8-yl)oxy)propyl)carbamic acid tert-butyl ester (10)

[0192] 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. After completion of the reaction, all volatile materials 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 dissolved 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 x 25 mL), dried over MgSO4 and concentrated in vacuo. The product was purified by flash chromatography (silica gel, hexane / EtOac 1:1) to give 10 as a pale yellow viscous oil (0.23 g, 0.44 mmol, 72%). 11H 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] + .

[0193] 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);

[0194] 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 minutes. All volatile substances 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 minutes and the solvent was removed under reduced pressure. The product was separated 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 + .

[0195] 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)

[0196] Dissolve 10 (0.01 g, 0.02 mmol) in a mixture of DMC (100 μL), TIPS (10 μL), and TFA (100 μL). Stir the reaction mixture at room temperature for 30 minutes. Remove all volatile substances under a gentle stream of air. Redissolve the residue in DMF (0.5 mL). Add Sulfo-Cy5-NHS ester (2.5 mg, 3.2 μmol) and DIPEA (10 μL). Stir the reaction mixture at room temperature for 45 minutes and remove the solvent under reduced pressure. Isolate the product by preparative HPLC to obtain eFAP-7 as a bright blue powder (1.0 mg, 1 μmol). ESI-MS: m / z 1042.6 [M+H] + .

[0197] 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)

[0198] Dissolve 10 (10 mg, 9.6 μmol) in a mixed solution of DCM / TFA (0.5 mL / 0.5 mL). Stir the reaction mixture at room temperature for 30 minutes. Remove all volatile substances and redissolve the residue in DMF (1 mL). Add DOTA-GA anhydride (5.5 mg, 0.012 mmol) and DIPEA (15 μL), and stir the reaction mixture at room temperature for 2 hours. After evaporation, purify eFAP-8 using prep-HPLC to obtain a pale yellow powder (8 mg, 9.1 μmol, 95%). ESI-MS: m / z 876.4 [M+H] + .

[0199] 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)

[0200] 10 (10 mg, 9.6 μmol) was treated with DCM / TFA (2 mL, 1:1) solution at room temperature for 30 minutes. All volatiles were removed under a gentle stream of air to afford a brown crude solid. The crude was redissolved in DMF (1 mL), then Fmoc-Amcha-OH (2 equiv, 7.3 mg), HBTU (4 equiv, 14.6 mg), DIPEA (15 μL) and DMF (1 mL) were added. The reaction was stirred at room temperature for 2 h, diluted with EtOAc (15 mL), washed with water (3 x 5 mL), dried over Na2SO4 and concentrated in vacuo to afford a pale yellow solid. The crude product was used for the next step without further purification. It was redissolved in a solution of 20% piperidine in DMF (2 mL) and stirred at room temperature for 30 minutes. The solvent was removed under reduced pressure, then DMF (1.5 mL), DOTA-NHS ester (6.6 mg, 14.4 μmol) and DIPEA (15 μL) were added. After 4 h, the solvent was removed in vacuo and the crude was purified by prep-HPLC to afford eFAP-9 (4.1 mg, 2.4 μmol, 45%) as an off-white powder. ESI-MS: m / z 943.4 [M+H] + .

[0201] 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)

[0202] Treat 10 (10 mg, 9.6 μmol) with DCM / TFA (2 mL, 1:1) solution at room temperature for 30 minutes. Remove all volatile substances under a gentle stream of air to obtain a brown crude solid. Redissolve the crude product in DMF (1 mL), then add Fmoc-Pip-OH (2 equivalents, 7.5 mg), HBTU (4 equivalents, 14.6 mg), DIPEA (15 μL) and DMF (1 mL). The reaction was stirred at room temperature for 2 hours, diluted with EtOAc (15 mL), washed with water (3 x 5 mL), dried over Na2SO4, concentrated under vacuum to obtain a pale yellow solid. The crude product was used for the next step without further purification. Redissolve it in a DMF solution of 20% piperidine (2 mL) and stir at room temperature for 30 minutes. Remove the solvent under reduced pressure, then add DMF (1.5 mL), DOTA-NHS ester (6.6 mg, 14.4 μmol) and DIPEA (15 μL). After 4 hours, remove the solvent under vacuum, and the crude product was purified by prep-HPLC to obtain eFAP-10 (2.1 mg, 2.2 μmol, 23%), as an off-white powder. ESI-MS: m / z 944.4 [M+H] + .

[0203] 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)

[0204] Compound 10 (10 mg, 9.6 μmol) was treated with DCM / TFA (2 mL, 1:1) solution at room temperature for 30 minutes. All volatile substances were removed under a gentle stream of air to give a brown crude solid. The crude product was redissolved in DMF (1 mL), then Fmoc-p-Ada-OH (2 equiv, 8.7 mg), HBTU (4 equiv, 14.6 mg), DIPEA (15 μL) and DMF (1 mL) were added. The reaction was stirred at room temperature for 4 hours, diluted with EtOAc (15 mL), washed with water (3 x 5 mL), dried over Na2SO4, concentrated under vacuum to give a pale yellow solid. The crude product was used for the next step without further purification. It was redissolved in a 20% piperidine in DMF solution (2 mL) and stirred at room temperature for 30 minutes. The solvent was removed under reduced pressure, then DMF (1.5 mL), DOTA-NHS ester (4.5 mg, 9.6 μmol) and DIPEA (15 μL) were added. After 4 hours, the solvent was removed under vacuum and the crude product was purified by prep-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] + .

[0205] 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)piperazine-1-yl)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (eFAP-12)

[0206] 10 (10 mg, 9.6 μmol) was treated with DCM / TFA (2 mL, 1:1) solution at room temperature for 30 minutes. All volatile substances were removed under a gentle stream of air to give a brown crude solid. The crude product was redissolved in DMF (1 mL), then Boc-Pipa-OH (2 equiv, 4.7 mg), HBTU (4 equiv, 14.6 mg), DIPEA (15 μL) and DMF (1 mL) were added. The reaction was stirred at room temperature for 4 hours, diluted with EtOAc (15 mL), washed with water (3 x 5 mL), dried over Na2SO4 and concentrated in vacuo to give a pale yellow solid. The crude product was used for the next step without further purification. It was redissolved in a 20% piperidine in DMF solution (2 mL) and stirred at room temperature for 30 minutes. The solvent was removed under reduced pressure, then DMF (1.5 mL), DOTA-NHS ester (6.6 mg, 14.4 μmol) and DIPEA (15 μL) were added. After 4 hours, the solvent was removed in vacuo and the crude product was purified by prep-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] + .

[0207] 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)

[0208] Compound 10 (10 mg, 9.6 μmol) was treated with DCM / TFA (2 mL, 1:1) solution at room temperature for 30 minutes. All volatile substances were removed under a gentle stream of air to afford a brown crude solid. The crude product was redissolved in DMF (1 mL), then Fmoc-ePipa-OH (2 equiv, 7.9 mg), HBTU (4 equiv, 14.6 mg), DIPEA (15 μL) and DMF (1 mL) were added. The reaction was stirred at room temperature for 4 h, diluted with EtOAc (15 mL), washed with water (3 x 5 mL), dried over Na2SO4 and concentrated in vacuo to give a pale yellow solid. The crude product was used for the next step without further purification. It was redissolved in a 20% piperidine in DMF solution (2 mL) and stirred at room temperature for 30 minutes. The solvent was removed under reduced pressure, then DMF (1.5 mL), DOTA-NHS ester (6.6 mg, 14.4 μmol) and DIPEA (15 μL) were added. After 4 h, the solvent was removed in vacuo and the crude product was purified by prep-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] + .

[0209] Example 9: Preparation of 2,2'-(4-(1-carboxy-5-(4-((2,2-dioxido-1,2-oxathiolan-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)

[0210] 10 (10 mg, 9.6 μmol) was dissolved in a mixed solution of DCM / TFA (0.5 mL / 0.5 mL). The reaction mixture was stirred at room temperature for 30 minutes. All volatile substances 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 hours. EtOAc (15 mL) was added, washed with water (3 x 5 mL), dried over Na2SO4, concentrated under vacuum to give a pale yellow solid. The crude product was used for the next step without further purification. 3-(Prop-2-yn-1-yl)-1,2-oxathiolane-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 a solution of acetonitrile and water (1 mL, 1:1) were added, and the reaction mixture was stirred at 60 °C for 12 hours. The mixture was concentrated under reduced pressure and purified by pre-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] + .

[0211] Example 10: Preparation of 2,2'-(4-(1-carboxy-5-(4-(5-fluoro-2-sulfonatopentyl)-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)

[0212] 10 (10 mg, 9.6 μmol) was dissolved in a mixed solution of DCM / TFA (0.5 mL / 0.5 mL). The reaction mixture was stirred at room temperature for 30 minutes. All volatile substances 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 hours. EtOAc (15 mL) was added, washed with water (3 x 5 mL), dried over Na2SO4, concentrated under vacuum to give a pale yellow solid. The crude product was used for the next step without further purification. 7-(Fluoro)hept-1-yn-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 a solution of acetonitrile and water (1 mL, 1:1) were added, and the reaction mixture was stirred at 60 °C for 12 hours. The mixture was concentrated under reduced pressure and purified by pre-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] + .

[0213] 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-tetraazacyclododecane-1-yl)-5-carboxypentyl)-1H-1,2,3-triazol-4-yl)methyl)dimethylammonio)methyl)trifluoroborate (eFAP-16)

[0214] Dissolve 10 (10 mg, 9.6 μmol) in a mixed solution of DCM / TFA (0.5 mL / 0.5 mL). Stir the reaction mixture at room temperature for 30 minutes. Remove all volatile substances and redissolve the residue in DMF (1 mL). Add DOTA-K(N3)-NHS ester (5.5 mg, 12.0 μmol) and DIPEA (15 μL), and stir the reaction mixture at room temperature for 4 hours. Add EtOAc (15 mL), wash with water (3 x 5 mL), dry over Na2SO4, concentrate under vacuum to obtain a pale yellow solid. The crude product is used for the next step without further purification. Add 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), and stir the reaction mixture at 60 °C for 12 hours. Concentrate the mixture under reduced pressure and purify by prep-HPLC to obtain eFAP-14 (2.3 mg, 2.2 μmol, 23%), as an off-white powder. ESI-MS: m / z 1066.2 [M+H] + .

[0215] 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)

[0216] Stir 10 (8 mg, 7.7 μmol) in a DCM / TFA (2 mL, 1:1) solution at room temperature for 30 minutes. Then, remove all volatile substances with a gentle stream of air to obtain a brown solid. Dissolve the crude product in DMF (1 mL) and treat it with DIPEA (10 μL) and the pre-activated CW800 dye. Dissolve CW800 (5 mg, 5 μmol) in DMF (1 mL) and treat it with HONSu (5 mg) and DCC (10.3 mg) at room temperature for 1 hour for activation. Dry the reaction mixture and purify by prep-HPLC to obtain eFAP-17 (1.6 mg, 1.1 μmol, 22%), as a green powder. ESI-MS: m / z 1469.4 [M+H] + .

[0217] Example 13: (1 4 S,1 6 S,3 2 S,3 3 S,2R,4S,10E,12E,14R)-8 6 -Chloro-14-hydroxy-8 5 ,14-dimethoxy-3 3 ,2,7,10-tetramethyl-1 2 ,6-dioxo-7-aza-1(6,4)-oxazino-3(2,3)-epoxyethane-8(1,3)-benzenacyclotetracontane-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)dithioalkyl)propionyl)-N-methyl-L-alaninate (eFAP-18) Preparation

[0218] 10 (12.1 mg, 23.4 μmol) was treated with TFA / DCM (1:1) for 1 h for deprotection. The solvent was removed, and the residue was dissolved in DMF (4 mL), 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 / H2O (1:1) and purified by prep-HPLC (11.6 mg, 15.5 μmol, 66%). The intermediate was treated with TFA / DCM (1:1) and TES (7.1 mg, 31.0 μmol) for 1 h. The solvent was removed by gentle airflow. To a solution of the product in DMF (1 mL) was added a solution of 2,2'-dithiopyridine (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 min. An aqueous solution of 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 h. The solvent was removed under vacuum, and the crude product was dissolved in ACN / H2O (1:1) and then purified by prep-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. An aqueous solution of 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 / H2O (1:1) and purified by prep-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] + .

[0219] 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-8 5 ,14-dimethoxy-3 3 ,2,7,10-tetramethyl-1 2Preparation of (2S,3S,6S,9S,12S,15S,18S,21S)-6,12-dioxo-7-aza-1(6,4)-oxazine-3(2,3)-epoxy-8(1,3)-benzocyclotetracosan-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)

[0220] 10 (11.9 mg, 23.1 μmol) was treated with TFA / DCM (1:1) for 1 h for deprotection. The solvent was removed, and the residue was dissolved in DMF (4 mL), 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 / H2O (1:1) and purified by prep-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 gentle airflow. 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 treating with a 20% PIP solution in DMF for 1 h. DMF was removed under high vacuum, and the crude product was purified by prep-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. After evaporation, a solution of 2,2'-dipyridyl disulfide (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 min. An aqueous sodium acetate solution (1 mL, 0.2 M) was added dropwise to the stirred solution. The reaction was monitored by LCMS and stopped after 2.5 h. The solvent was removed under vacuum, and the crude product was dissolved in ACN / H2O (1:1) and then purified by prep-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. An aqueous sodium carbonate solution (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 h. The crude product was dissolved in ACN / H2O (1:1) and purified by prep-HPLC. eFAP-19 was obtained as an off-white solid (1.2 mg, 0.7 μmol, 34%). ESI-MS: m / z 1829.2 [M+H] + .

[0221] 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)

[0222] 10 (12.1 mg, 23.4 μmol) was treated with TFA / DCM (1:1) for 1 h for deprotection. The solvent was removed and the residue was dissolved in DMF (4 mL), 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 / H2O (1:1) and purified by prep-HPLC (11.6 mg, 15.5 μmol, 66%). The intermediate was dissolved in degassed sodium dihydrogen phosphate solution (50 mM, pH 5), then dichlorotetrazine (3 equiv) dissolved in CHCl3 was added. 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 phases were combined and lyophilized. Subsequently, the crude mixture was purified by prep-HPLC and orange powder was obtained 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). The two starting materials were dissolved in water / acetonitrile (1:1) and incubated at 37 °C for 10 min in the dark in an Eppendorf tube. The reaction mixture was purified by preparative HPLC to give eFAP-20 (5.1 mg, 3.0 μmol, 63%). ESI-MS: m / z 1744.8 [M+H] + .

[0223] Preparation of 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)-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)

[0224] Treat 10 (15 mg, 29 μmol) with DCM / TFA (3 mL, 1:1) solution at room temperature for 30 minutes. Remove all volatile substances under a gentle stream of air to obtain a brown crude solid. Redissolve the crude product in DMF (1 mL), then add 3-(tritylthio)propionic acid (1.5 equiv, 15 mg), HBTU (4 equiv, 23.6 mg), DIPEA (30 μL) and DMF (1.5 mL). Stir the reaction at room temperature for 4 hours, dilute with EtOAc (15 mL), wash with water (3 x 5 mL), dry over Na2SO4 and concentrate under vacuum to obtain a pale yellow solid. The crude product is treated with DCM / TFA (3 mL, 1:1) solution at room temperature for 30 minutes, the solvent is removed under a gentle stream of air, and the crude product is purified by prep-HPLC to obtain 8QCP-SH (4 mg, 7.9 μmol, 27%), as a pale yellow powder. ESI-MS: m / z 506.2 [M+H] + . Redissolve the powder in DMF / DCM (1 mL, 1:1), then add 3,6-dichloro-1,2,4,5-tetrazine (0.4 mg, 2.7 μmol) and DIPEA (10 μL). After 30 minutes, remove the solvent under vacuum, and the crude product is purified by prep-HPLC to obtain (8QCP-SH)2-TZ (1.9 mg, 1.7 μmol, 43%), as a pale yellow powder. ESI-MS: m / z 1089.1 [M+H] + . Redissolve the powder in ACN / H2O (1 mL, 1:1), then add TCO-DOTAGA (1.1 equiv, 1.3 mg). Stir the reaction solution at 37 °C for 30 minutes, and the solvent is purified by prep-HPLC to obtain eFAP-20 (1 mg, 0.6 μmol, 34%), as an off-white powder. ESI-MS: m / z 1476.1 [M+H] + .

[0225] 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)

[0226] 10 (10 mg, 9.6 μmol) was treated with DCM / TFA (2 mL, 1:1) solution at room temperature for 30 minutes. All volatiles were removed under a gentle stream of air to afford a brown crude solid. The crude was redissolved in DMF (2 mL), and then 4-((tert-butoxycarbonyl)amino)-3-hydroxybutyric acid (1.4 equiv, 7.6 mg), HBTU (2.3 equiv, 20.4 mg), and DIPEA (16.7 μL) were added. After stirring at room temperature for 1.5 h, the reaction mixture was diluted in EtOAc (20 mL) and then washed with H2O (1 x 10 mL) and brine (2 x 10 mL). The organic layer was dried over MgSO4, filtered, and concentrated in vacuo to afford an orange-yellow oil. To the crude, a 1:1 TFA / DCM mixture containing 10% TIPS (1.5 mL) was added, and then stirred at room temperature for 30 minutes. Upon completion, all solvents were removed under a stream of air and then precipitated with ice-cold diethyl ether (2 x 20 mL). The precipitate was collected and dried under a stream of air and used in the subsequent reaction without further purification. The crude was redissolved in DMF (2 mL) and then treated with 4-((tert-butoxycarbonyl)amino)-3-hydroxybutyric 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 by prep-HPLC to afford an orange-yellow solid (5.8 mg, 0.008 mmol, 34% yield 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 TFA / DCM mixture containing 10% TIPS (1.5 mL), and then the reaction mixture was stirred at room temperature for 30 minutes. After completion, the reaction mixture was concentrated under a stream of air and then precipitated with ice-cold diethyl ether (2 x 20 mL). The precipitate was collected, dried under a stream of air, and used in the subsequent reaction without further purification. It was redissolved in a solution of DMF (1.5 mL) containing fluorescein isothiocyanate (1.2 equiv, 3.8 mg) and DIPEA (5.6 μL), and stirred at room temperature for 2 hours in the dark. The reaction mixture was concentrated in vacuo, and the crude product was purified by prep-HPLC to give the yellow powder eFAP-23 (4.2 mg, 0.004 mmol, 50.0%). ESI-MS: m / z 1009.1 [M+H] + .

[0227] 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-sulfonyloxyindolin-2-ylidene)ethylidene)-2-(4-sulfonyloxyphenoxy)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(4-sulfonyloxybutyl)-3H-indol-1-ium-5-sulfonate (eFAP-24)

[0228] Compound 10 (10 mg, 9.6 μmol) was treated with DCM / TFA (2 mL, 1:1) solution at room temperature for 30 minutes. All volatile substances were removed under a gentle stream of air to afford a brown crude solid. The crude product was redissolved in DMF (2 mL), and then 4-((tert-butoxycarbonyl)amino)-3-hydroxybutyric acid (1.4 equiv, 7.6 mg), HBTU (2.3 equiv, 20.4 mg), and DIPEA (16.7 μL) were added. After stirring at room temperature for 1.5 h, the reaction mixture was diluted in EtOAc (20 mL) and then washed with H2O (1 x 10 mL) and brine (2 x 10 mL). The organic layer was dried over MgSO4, filtered, and concentrated in vacuo to give an orange-yellow oil. To the crude product was added a 1:1 TFA / DCM mixture containing 10% TIPS (1.5 mL), and then the mixture was stirred at room temperature for 30 minutes. After completion, all solvents were removed under a stream of air, and then the product was precipitated with ice-cold diethyl ether (2 x 10 mL). The precipitate was collected and dried in vacuo and used for the next step without further purification. The crude product was redissolved in a solution of DMF (2 mL) containing 4-((tert-butoxycarbonyl)amino)-3-hydroxybutyric acid (1.1 equiv, 4.6 mg), HBTU (2 equiv, 14.4 mg), and DIPEA (4 equiv, 13.2 μL) and stirred at room temperature. After 2 h, it was diluted with EtOAc (15 mL), washed with water (3 x 5 mL), dried over Na2SO4, and concentrated in vacuo to give a pale orange solid. The crude product was treated with a 1:1 TFA / DCM mixture containing 10% TIPS (1.5 mL), and then the reaction mixture was stirred at room temperature for 30 minutes. After completion, the reaction mixture was concentrated under a gentle stream of air and the crude product was purified by prep-HPLC to give a pale yellow solid (4.4 mg, 7.1 μmol, 7% yield 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), and then 800CW-NHS ester (1.1 equiv, 5.0 mg, 4.3 μmol) and DIPEA (1.5 μL) were added. After 2 h, the solvent was removed in vacuo and the crude product was purified by prep-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] + .

[0229] 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)

[0230] 10 (5 mg, 4.8 μmol) was treated with a solution of DCM / TFA (1:1) containing 10% TIPS (1.5 mL) at room temperature for 20 minutes. All volatile substances were removed under a gentle stream of air to give a dark yellow crude solid. The crude product was redissolved in DMF (1.5 mL), then DO3AM-acetic acid (1.1 equiv, 2.1 mg) and DIPEA (15 μL) were added. The reaction mixture was stirred overnight, the solvent was removed in vacuo, and the crude product was purified by prep-HPLC to give eFAP-25 (2.3 mg, 2.9 μmol, 60%) as a yellow powder. ESI-MS: m / z 801.4 [M+H] + .

[0231] 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)

[0232] Treat 10 (10 mg, 9.6 μmol) with a DCM / TFA solution (2 mL, 1:1) containing 10% TIPS at room temperature for 20 minutes. Remove all volatile substances under a gentle stream of air to obtain a dark yellow solid. Redissolve the crude product in DMF (2 mL), then add 4-((tert-butoxycarbonyl)amino)-3-hydroxylbutyric acid (1.4 equivalents, 7.6 mg), HBTU (2.3 equivalents, 20.4 mg), and DIPEA (16.7 μL). After stirring at room temperature for 1.5 hours, dilute the reaction mixture in EtOAc (20 mL), and then wash it with H2O (1 x 10 mL) and brine (2 x 10 mL). Dry the organic layer over MgSO4, filter, and concentrate it in vacuo to obtain an orange-yellow oil. Add a 1:1 TFA / DCM mixture containing 10% TIPS (1.5 mL) to the crude product, and then stir at room temperature for 30 minutes. After completion, remove all solvents under a stream of air, and then precipitate with ice-cold diethyl ether (2 x 10 mL). Collect the precipitate and dry it in vacuo, which can be used for the next step without further purification. Dissolve it in DMF (1 mL), then add CW800-NHS ester (3.5 mg, 3.0 μmol) and DIPEA (1.5 μL). After 2 hours, remove the solvent in vacuo, and purify the crude product by prep-HPLC to obtain eFAP-27 (3.4 mg, 2.3 μmol, 24%), as a dark green powder. ESI-MS: m / z 1504.4 [M+H] + .

[0233] Example 20: Preparation of 4-(4-((4-((2,2-dioxido-1,2-oxathiolan-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)

[0234] Treat 10 (12 mg, 0.023 mmol) with a DCM / TFA solution (2 mL, 1:1) containing 10% TIPS at room temperature for 20 minutes. Remove all volatile substances under a gentle stream of air to obtain a dark yellow crude solid. Redissolve the crude product in DMF (1.5 mL), then add TCO-NHS ester (1.2 equivalents, 6.4 mg) and DIPEA (2 equivalents, 7 μL). After stirring at room temperature for 30 minutes, concentrate the reaction mixture in vacuo, and purify the crude product by prep-HPLC to obtain a pale yellow solid TCO-8QCP (12.2 mg, 0.021 mmol, 91%). ESI-MS: m / z 570.2 [M+H]+ . Dissolve the product 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-oxathiolane 2,2-dioxide (1.1 eq., 9.3 mg) in ACN / H2O (3 mL, 1:1), and stir at 37 °C. After 2 hours, concentrate the reaction mixture in vacuo, and purify the crude product by prep-HPLC to obtain eFAP-28 (14.0 mg, 0.015 mmol, 71%), as an off-white powder. ESI-MS: m / z 941.2 [M+H] + .

[0235] Example 21: Preparation of 4-(4-((4-((2,2-dioxido-1,2-oxathiolan-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-aza-nonadecyl)carbamate (eFAP-29)

[0236] Dissolve 10 (16 mg, 0.039 μmol) in DMF (3 mL), then add TCO-PEG4-NHS ester (1.3 eq., 0.05 mg), DIPEA (2 eq., 13.5 μL). After stirring at room temperature for 30 minutes, concentrate the reaction mixture in vacuo, and purify the crude product by prep-HPLC to obtain pale yellow solid TCO-PEG4-8QCP (22 mg, 0.027 mmol, 69%). ESI-MS: m / z 817.3 [M+H] + . Dissolve the product 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-oxathiolane 2,2-dioxide (1.1 eq., 11.9 mg) in ACN / H2O (3 mL, 1:1), and stir at 37 °C. After 4 hours, concentrate the reaction mixture in vacuo, and purify the crude product by prep-HPLC to obtain eFAP-29 (16.4 mg, 0.014 mmol, 52%), as an off-white powder. ESI-MS: m / z 1188.4.

[0237] 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-oxathiolan-3-yl)methyl)-1H-1,2,3-triazol-1-yl)valeramido)propoxy)quinoline-4-carboxamide (eFAP-30)

[0238] 10 (22 mg, 0.052 μmol) was dissolved in DMF (3 mL), and then 5-azidovaleric acid (2.7 equivalents, 20 mg), HBTU (1.5 equivalents, 29.6 mg), and DIPEA (2 equivalents, 21.5 μL) were added. After stirring at room temperature for 2 h, the reaction mixture was concentrated in vacuo, and the crude product was purified by prep-HPLC to give the pale yellow solid azido-8QCP (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 equivalent, 1.6 mg) and sodium ascorbate (0.22 equivalent, 1.47 mg). The reaction mixture was heated to 70 °C and stirred for 2 h. The reactant was concentrated under reduced pressure, and the crude product was purified by pre-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] + .

[0239] 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)

[0240] Treat 10 (15 mg, 29 μmol) with DCM / TFA (3 mL, 1:1) solution at room temperature for 30 minutes. Remove all volatile substances under a gentle stream of air to obtain a brown crude solid. Redissolve the crude product in DMF (1 mL), then add TrtS-Hx-OH (1.5 equiv, 17.5 mg), HBTU (4 equiv, 23.6 mg), DIPEA (30 μL), and DMF (1.5 mL). Stir the reaction at room temperature for 6 hours, dilute with EtOAc (15 mL), wash with water (3 x 5 mL), dry over Na2SO4, and concentrate under vacuum to obtain a pale yellow solid. Treat the crude product with DCM / TFA (3 mL, 1:1) solution at room temperature for 30 minutes, remove the solvent under a gentle stream of air, and purify the crude product by prep-HPLC to obtain 8QCP-SHx (4.5 mg, 8.2 μmol, 35%), as a pale yellow powder. ESI-MS: m / z 548.3 [M+H] + . Redissolve the powder in DMF / DCM (1 mL, 1:1), then add 3,6-dichloro-1,2,4,5-tetrazine (0.4 mg, 2.7 μmol) and DIPEA (10 μL). After 30 minutes, remove the solvent under vacuum, and purify the crude product by prep-HPLC to obtain (8QCP-SHx)2-Tz (2 mg, 1.7 μmol, 43%), as a pale yellow powder. ESI-MS: m / z 1173.4 [M+H] + . Redissolve the powder in ACN / H2O (1 mL, 1:1), then add TCO-DOTAGA (1.1 equiv, 1.2 mg). Stir the reaction solution at 37 °C for 30 minutes, and purify the solvent by prep-HPLC to obtain eFAP-31 (2 mg, 1 μmol, 63%), as an off-white powder. ESI-MS: m / z1830.4 [M+H] + .

[0241] Example 24: Preparation of 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-triazin-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)

[0242] Dissolve 8QCP-SH (5.5 mg, 10.8 μmol) in DMF (1 mL), then add TADB-N3 (1.3 mg, 3.1 μmol) and DIPEA (10 μL). After 30 minutes, remove the solvent under vacuum, and purify the crude product by prep-HPLC to obtain (8QCP-SH)2-TADB (2.9 mg, 2.2 μmol, 41%), which is an off-white powder. ESI-MS: m / z 1346.3 [M+H] + . Redissolve the powder in ACN / H2O (1 mL, 1:1), then add BCN-DOTAGA (1.1 eq, 1.7 mg). Stir the reaction solution at 37 °C for 30 minutes, and purify the solvent by prep-HPLC to obtain eFAP-34 (1.4 mg, 0.7 μmol, 31%), which is an off-white powder. ESI-MS: m / z 1956.5 [M+H] + .

[0243] Example 25: Preparation of 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-triazin-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)

[0244] Dissolve 8QCP-SHx (4.1 mg, 7.5 μmol) in DMF (1 mL), then add TADB-N3 (1 mg, 2.4 μmol) and DIPEA (10 μL). After 30 minutes, remove the solvent under vacuum, and purify the crude product by prep-HPLC to obtain (8QCP-SHx)2-TADB (2.2 mg, 1.6 μmol, 44%), which is an off-white powder. ESI-MS: m / z 1346.3 [M+H] +. The powder was redissolved in ACN / H2O (1 mL, 1:1), and then BCN-DOTAGA (1.1 equiv, 1.3 mg) was added. The reaction mixture was stirred at 37 °C for 30 min, and the solvent was purified by prep-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] + .

[0245] Example 26: Preparation of 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-diazapentadecanoyl)-1,3,5-triazine-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)

[0246] 10 (15 mg, 29 μmol) was treated with a DCM / TFA (3 mL, 1:1) solution at room temperature for 30 min. All volatiles were removed under a gentle stream of air to give a brown crude solid. The crude product was redissolved in DMF (1 mL), and then PEG2-TADB-PEG2 (1.5 equiv, 26 mg), HBTU (2 equiv, 23.6 mg), DIPEA (30 μL), and DMF (1.5 mL) were added. The reaction was stirred at room temperature for 1 h, diluted with EtOAc (15 mL), washed with water (3 x 5 mL), dried over MgSO4, and concentrated in vacuo to give an off-white solid. The crude product was redissolved in ACN / H2O (1 mL, 1:1), and then BCN-DOTAGA (1.1 equiv, 2.6 mg) was added. The reaction mixture was stirred at 37 °C for 30 min, and the solvent was purified by prep-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] + .

[0247] Example 27: Preparation of 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)

[0248] Compound 10 (15 mg, 29 μmol) was treated with a DCM / TFA (3 mL, 1:1) solution at room temperature for 30 minutes. All volatiles were removed under a gentle stream of air to give a brown crude solid. The crude product was redissolved in DMF (1 mL), then PEG2-triazine-PEG2 (1.5 equiv, 23 mg), HBTU (2 equiv, 23.6 mg), DIPEA (30 μL) and DMF (1.5 mL) were added. The reaction was stirred at room temperature for 1 hour, diluted with EtOAc (15 mL), washed with water (3 x 5 mL), dried over Na2SO4 and concentrated in vacuo to give a pale white solid. The crude product was redissolved in ACN / H2O (1 mL, 1:1), then BCN-DOTAGA (1.1 equiv, 2.6 mg) was added. The reaction mixture was stirred at 37 °C for 30 minutes and the solvent was purified by prep-HPLC to give eFAP-37 (3 mg, 1.5 μmol, 39%), as an off-white powder. ESI-MS: m / z 2024.1 [M+H] + .

[0249] 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-triazin-1-yl)-2-oxoethyl)-1,9-dihydro-8H-dibenzo[b,f][1,2,3]triazolo[4,5-d]azacyclooctatetraene-8-yl)-3-oxopropyl)amino)-1-carboxy-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (eFAP-38);

[0250] (8QCP-SH)2-TADB (0.8 eq., 1 mg) was dissolved in ACN / H2O (1 mL, 1:1), and then DBCO-DOTAGA (1.1 eq., 0.6 mg) was added. The reaction mixture was stirred at 37 °C for 30 min, and the solvent was purified by prep-HPLC to obtain eFAP-38 (0.5 mg, 0.25 μmol, 31%), as an off-white powder. ESI-MS: m / z 1996.7 [M+H] + .

[0251]

[0252] 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)

[0253] 8-(3-((tert-Butoxycarbonyl)amino)propoxy)quinoline-4-carboxylic acid (7 mg, 0.020 mmol) was dissolved in DMF (1 mL), and then glycine methyl ester (2 eq., 3.6 mg), HBTU (2 eq., 14.4 mg), DIPEA (5 μL), and DMF (1 mL) were added. The reaction was stirred at room temperature for 1 h, diluted with EtOAc (15 mL), washed with water (3 x 5 mL), dried over Na2SO4, and concentrated under vacuum to obtain a pale yellow solid. The crude product was used for the next step without further purification. It was treated with a DCM / TFA mixture (2 mL, 1:1) at room temperature for 30 min and then precipitated with ice-cold diethyl ether (2 x 10 mL). The precipitate was redissolved in a NaOH (0.5 M) solution in MeOH for 20 min. The reaction mixture was concentrated under reduced pressure to obtain a pale yellow crude solid. The crude product was redissolved in DMF (1 mL), and then CW800-NHS ester (3.5 mg, 3.0 μmol) and DIPEA (1.5 μL) were added. After 1 h, the solvent was removed under vacuum, and the crude product was purified by prep-HPLC to obtain eFAP-39 (3.7 mg, 2.9 μmol, 14.5%), as a dark green powder. ESI-MS: m / z 1289.3 [M+H] + .

[0254] 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)

[0255] eFAP-30 (2.5 mg, 3.5 μmol), potassium fluoride (1.2 equiv., 0.24 mg), and 18-crown-6 (1 equiv., 0.9 mg) were dissolved in 1 mL of acetonitrile and heated to 100 °C. After 2 h, the reaction mixture was cooled to room temperature and then purified by prep-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] + .

[0256] Analysis and Results

[0257] Radioactive Labeling

[0258] To 111 InCl3 or 177 LuCl3 was added to an 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-H2O (final volume adjusted to 140 μL) for labeling. The reaction mixture was heated at 90 °C for 20 min. After cooling, DTPA (5 μL) solution was added to complex the remaining free radioactive metal.

[0259] Figure 2 The LC / MS chromatogram of eFAP-8 is shown.

[0260] Figure 3 Shown is 111 the radioactivity-iTLC chromatogram of [111In]In-eFAP-12 with a radioactive labeling yield (RCY) of 97.8%.

[0261] In Vitro Stability

[0262] The radiolabeled compound (approx. 2 MBq) was mixed with 300 μL of PBS (0.1 M, pH 7.4) or mouse serum at 37 °C and allowed to stand for 1, 4, and 24 hours respectively. At each time point, acetonitrile (300 μL) was added to precipitate the proteins in the serum samples, and then 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 directly subjected to radio-HPLC analysis without any pretreatment. All experiments were repeated three times.

[0263] Figure 4 shows 111 The radio-HPLC chromatogram of In-eFAP-12 shows the stability of the radiolabeled conjugate over time. A) The radiochemical purity (RCP) obtained at t = 0 was 97.4%; B) RCP = 97% at 2 hours; C) RCP = 95.8% at t = 4 hours; D) RCP = 93.6% at t = 24 hours. E) The last chromatogram corresponds to the UV signal obtained after injecting nat In-eFAP-12, confirming 111 the identity of In-eFAP-12.

[0264] LogD 7.4

[0265] The partition coefficient (LogD 7.4 value) was determined by the shake-flask method. Samples containing 203 the Pb-labeled compound were 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 the aqueous phase (200 μL) were taken and counted using a PerkinElmer WIZARD 2480 gamma counter. The LogD 7.4 value was calculated as: LogD 7.4 = log[(counts in octanol phase) / (counts in aqueous phase)]. All experiments were repeated three times.

[0266] Table 1.

[0267] Partition coefficients (LogD 7.4 values) of eFAP-8 to eFAP-13. The eFAP compounds showed hydrophilicity.

[0268]

[0269]

[0270] In vitro inhibition assays against human and mouse FAP

[0271] At room temperature, on a Hidex microplate reader, the enzymatic activities of human FAP (hFAP) and mouse FAP (mFAP) towards the Z-GP-AMC substrate were determined by monitoring fluorescence at an excitation wavelength of 360 nm and an emission wavelength of 465 nm. 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 the test compound (serially diluted from 10 -6 to 10 -12 ), with a total volume of 50 μL. The IC 50 value was defined as the concentration of inhibitor required to reduce the enzymatic activity by 50% after addition of the substrate.

[0272] Figure 5 The results of the hFAP and mFAP inhibition experiments in the presence of eFAP compounds are shown. The IC 50 values of all tested eFAP compounds for human FAP were below 6 nM. The results obtained for FAPI-46 in the same assay were also included for comparison.

[0273] Table 2.

[0274] Determination of the IC 50 values of eFAP drugs for human FAP based on enzymatic assays.

[0275]

[0276]

[0277] PREP In Vitro Inhibition Assay

[0278] At room temperature, on a Hidex microplate reader, the enzymatic activity of prolyl endopeptidase (PREP) towards the Z-GP-AMC substrate (CAS number: CAS 68542-93-8) was determined by monitoring fluorescence at an excitation wavelength of 360 nm and an emission wavelength of 465 nm. 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 the test compound (serially diluted from 10 -6 to 10 -12 ), with a total volume of 50 μL. The IC 50 value was defined as the concentration of inhibitor required to reduce the enzymatic activity by 50% after addition of the substrate.

[0279] The results are shown in Table 3. The results obtained for FAPI-46 in the same assay were also included for comparison.

[0280] DPP4 In Vitro Inhibition Assay

[0281] At room temperature, on a Hidex microplate reader, the enzymatic activity of PREP towards the Z-GP-pNA substrate was determined by monitoring fluorescence at an excitation wavelength of 380 nm and an emission wavelength of 425 nm. 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 the test compound (serial dilution, from 10 -6 to 10 -12 ), with a total volume of 100 μL. The IC 50 value was defined as the inhibitor concentration required to reduce the enzymatic activity by 50% after the addition of the substrate.

[0282] The results are shown in Table 3. The results obtained for FAPI-46 in the same assay are also included for comparison.

[0283] Table 3.

[0284] IC 50 values of eFAP compounds against murine FAP, PREP, and DPP4 were determined based on the enzymatic assay. The binding of eFAP-6 to FAP was specific, as the IC 50 value of eFAP-6 against PREP was 68-fold higher than that against human FAP.

[0285]

[0286]

[0287] Competitive Binding Assay in Cell Culture

[0288] U-87MG glioblastoma was cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% (v / v) fetal bovine serum at 37 °C and 5% CO2. HT-1080.hFAP cells were cultured in DMEM medium supplemented with fetal bovine serum (10%, FBS) and antibiotic-antimycotic (1%, AA) at 37 °C and 5% CO2. When the cells reached 90% confluence, the cells were detached using 0.05% trypsin-EDTA and re-seeded at a dilution of 1:4.

[0289] Add 111The In-FAPI-46 radioligand was incubated with increasing concentrations of the eFAP compound in HT-1080.hFAP cells at 37 °C for 1 hour for a competitive binding experiment. Subsequently, the cells were washed three times with binding buffer and lysed. The cell-associated radioactivity was measured using a gamma counter. The data were non-linearly regression fitted using GraphPad Prism software to calculate the IC 50 value.

[0290] Figure 6 Table 3 and Table 4 show the results of the competitive binding assays of eFAP-6 and eFAP-7 conducted in HT-1080.hFAP cells. Both compounds exhibited low nanomolar binding affinities for FAP. The results obtained for FABI-46 in the same assay were also included for comparison.

[0291] Table 4.

[0292] The binding affinities of eFAP-6 and eFAP-7 for FAP were determined in a cell-based assay. The IC 50 values of both compounds for FAP were lower than those of the reference FAP inhibitor FAPI-46.

[0293]

[0294] Cytotoxicity assay

[0295] In a 96-well plate, U-87 cells (3 x 10 3 ) 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. The cytotoxic drug (positive control) or the eFAP conjugate containing the drug was dissolved in DMEM medium supplemented with 2 mM L-glutamine, 10% FBS, 50 units / mL penicillin, and 50 μg / mL streptomycin at different concentrations (i.e., 1, 10, 100, 1000 nM). The medium served as the negative control. The cells were incubated with 100 μL of the 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 medium containing 10% resazurin solution was added to each well and incubated for 2.5 hours. Fluorescence measurements were performed using a microplate reader at an excitation wavelength of 544 nm and an emission wavelength of 590 nm.

[0296] Figure 7 Table 5 shows the results of the cytotoxicity assay of eFAP-18 in FAP + U-87 cells. When the cells were treated with a single dose of eFAP-18 (1 μM), the cell viability decreased to approximately 25%, compared to 40% when the cells were incubated with the parent drug DM1.

[0297] Biodistribution studies in animal models

[0298] 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. After arrival, the mice were allowed to acclimatize for 1 week and had free access to food and water. U-87 or HT-1080.hFAP cells (1×10 6 cells) were inoculated subcutaneously into the right shoulder of the mice. The xenografts were allowed to grow until they reached an appropriate size at the start of the study. All animal experiments were approved by the Animal Welfare Committee of the Erasmus MC and were conducted in accordance with institutional guidelines (permit number: AVD101002017867).

[0299] Biodistribution studies were performed to determine the uptake of radiolabeled eFAP compounds in tumors and organs. At t = 0, animals (n = 4 per compound per time point) were injected intravenously with an average dose of 100 kBq / 0.5 nmol. Under isoflurane / O2 anesthesia, blood was collected by cardiac puncture p.i. at three selected time points (1, 4, and 24 hours), and then the mice were sacrificed. Tumors and organs of interest (prostate, pancreas, spleen, gallbladder, liver, stomach, small intestine, cecum, colon, adrenal gland, kidney, lung, heart, salivary gland, muscle, bone, and brain tissue) were excised, washed with PBS, and blotted dry. Blood, tumors, and relevant organs were weighed and measured using a gamma counter. To determine the total radioactivity injected per animal, calibration curves were drawn using indium-111 and lutetium-177. The percentage of injected dose per gram (%ID / g) of each tissue sample was determined and corrected for the %ID of the injection volume and injection site (tail).

[0300] Figure 8 The biodistributions of 111 [[In]In-FAPI-46 and 111 [[In]In-eFAP-6 in selected organs of HT-1080.hFAP tumor-bearing mice were compared. In vivo biodistribution studies using indium-111-labeled eFAP-6 showed that eFAP-6 had high and rapid uptake in tumors, while having lower background uptake in healthy organs, thus obtaining high-contrast images. Its tumor-targeting performance was comparable to that of FAPI-46 (the clinical reference of FAP-targeted tracers). eFAP-6 could be rapidly cleared from the body and was very suitable for imaging.

[0301] Figure 9Demonstrated the evaluation of the targeting performance of eFAP-17 in near-infrared fluorescence imaging after intravenous injection of 2 nmol of the probe into U-87 tumor-bearing mice. 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); B) Images of excised tumors 2.5 hours after administration of CW800 (left) or eFAP-17 (right). The tumors are indicated by white circles. eFAP-17 showed high uptake in tumors with overexpressed FAP, while low accumulation in healthy tissues.

Claims

1. A compound, or a pharmaceutically acceptable salt thereof, which comprises a scaffold represented by formula (I) wherein X represents CH2 or O; R 1 represents 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 - 4; and R 2 and R 3 each independently represents H or F.

2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, which has a structure represented by formula (II) wherein R 4 represents a spacer, preferably an aliphatic spacer; and Q includes 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.

3. The compound according to claim 2, or a pharmaceutically acceptable salt thereof, which has a structure represented by formula (IIIa), preferably as shown in (IIIb) wherein R 4’ represents an optionally substituted alkylene or an optionally substituted alkylene ether, preferably an alkylene, more preferably a C1-C8 alkylene; and n is from 1 to 6, preferably 1.

4. The compound according to any one of claims 2-3, or a pharmaceutically acceptable salt thereof, wherein Q includes one or more of a radioisotope, a fluorescent dye and a drug complexed with a chelating agent, and the drug is, for example, a cytotoxic agent or a cell growth inhibitor.

5. The compound according to any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein the compound has a structure represented by formula (IV) (IV) where L represents a joint, Q 1 includes at least one load, Z includes an additional bracket as shown in formula (I), r is 1 or greater, and s is 0 or greater.

6. A compound as claimed in the preceding claim, wherein the linker comprises a cleavable region, a non-cleavable region or a combination thereof, preferably wherein the linker is based on or comprises linear or branched amino acids 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., peptide spacer (Xaa) 1-4 , wherein each Xaa is independently a protein amino acid residue or a non-protein amino acid residue selected from the group consisting of: D-amino acids of protein amino acids, 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), sulfopropionic acid, diglycolic acid and NH2(CH2)2 q C(O)OH (where q = 1 - 36), more preferably wherein the linker comprises a moiety having a structure as shown in any one of formulas La - Ln; wherein Y 1 selected from C and N, preferably N for formulas La, Lg and Lh, and preferably C for formula Ld; and Y 2 Selected from C, N, and O, preferably N and C; more preferably C, and even more preferably, wherein the linker comprises a moiety having a structure represented by any one of the formulas Laa, Lfa, Lga, Lha, and Lma 7. The compound according to any one of claims 5-6, or a pharmaceutically acceptable salt thereof, wherein the linker L comprises a cleavable linker region, which comprises an in vivo cleavable moiety and an optional self-destructive moiety, preferably the in vivo cleavable moiety is selected from amides, esters, carbamates, hydrazones, thiazolidines, methylene alkoxy carbamates, disulfides and combinations thereof, more preferably wherein the in vivo cleavable moiety comprises a disulfide moiety or a terminal thiol capable of forming a disulfide bond with a payload that also contains a terminal thiol, and most preferably, wherein the linker comprises a moiety having the structure of formula Lo wherein R 5 represents an optionally present second spacer, preferably an optionally substituted second aliphatic spacer, more preferably a C1-C6 alkylene group, and most preferably an ethylene group.

8. The compound according to any one of claims 2-7, or a pharmaceutically acceptable salt thereof, wherein Q comprises at least two of a radioisotope, a fluorescent dye and a drug, preferably comprises at least a radioisotope and a fluorescent dye, or at least a radioisotope and a drug.

9. The compound according to any one of claims 2-8, or a pharmaceutically acceptable salt thereof, which has a structure represented by formula (V) wherein p is 1-6, preferably 3; Q 1’ and Q 1” each independently comprises a payload, such as a radioisotope, a fluorescent dye or a drug.

10. The compound according to any one of claims 5-8, or a pharmaceutically acceptable salt thereof, wherein the linker connects the scaffold represented by formula (I) to an additional scaffold, and the linker comprises a polymeric moiety represented by any one of formulas (Lp) to (Lv) The said formulas respectively show the support (Z) and the load Q 1 The relative positions with respect to the polymer part.

11. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, is suitable for preparing any compound according to any one of claims 2-10, and the compound has a structure represented by formula (VIIa), preferably a structure represented by formula (VIIb) Among them, R 4’ represents an optionally substituted alkylene or an optionally substituted alkylene ether; and n is from 1 to 6, preferably 1.

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

13. The compound according to any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein X represents O, R 1 represents H; and / or R 2 and R 3 at least one of which represents F, preferably R 2 and R 3 both represent F.

14. The compound according to any one of the preceding claims, or a pharmaceutically acceptable salt thereof, which is selected from the following group: -(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)pent-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-oxathiolan-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-fluoropentylsulfonyl)-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)-oxazine-3(2,3)-epoxyethane-8(1,3)-benzenacyclotetratetracontane-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)dithio)propyl)carbamoyl)-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)-oxazine-3(2,3)-epoxyethane-8(1,3)-benzenacyclotetracontane-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) -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-sulfonyloxyindolin-2-ylidene)ethylidene)-2-(4-sulfonyloxyphenoxy)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(4-sulfonyloxybutyl)-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); -1-yl)butane-1-sulfonate (eFAP-27); -4-(4-((4-((2,2-dioxido-1,2-oxathiolan-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-oxathiolan-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-oxathiolan-3-yl)methyl)-1H-1,2,3-triazole -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-triazin-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-triazin-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-diazapalmitoyl)-1,3,5-triazin-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-triazin-1-yl)-2-oxoethyl)-1,9-dihydro-8H-dibenz[b,f][1,2,3]triazolo[4,5-d]azacyclooctatetraen-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)amino)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-triazole -4-yl)-5-fluoropentane-2-sulfonate (eFAP-42); - and - radioisotope-labeled, preferably, 111 In, 68 Ga, 177 Lu, 225 Ac, Pb 212 radioisotope-labeled 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, preferably selected from the group consisting of eFAP-6, eFAP-8, eFAP-17 and eFAP-24, and most preferably selected from eFAP-8 and eFAP-24.

15. A compound as claimed in any one of the preceding claims, or a pharmaceutically acceptable salt thereof, for use in medicine, preferably for the diagnosis and / or treatment of tumours.

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