Cyclic peptides and their conjugates for addressing alpha-V-beta-6-integrin in vivo

Abstract

The present invention provides conjugates of cyclic peptides as ligands for cell surface receptors, particularly for αββ-integrin. The conjugates further contain an effector moiety and are suitable for use as therapeutic agents, diagnostic agents, pharmaceutical agents for imaging, targeting moieties, and biomolecular research tools. The present invention particularly relates to the use of conjugates with signaling moieties or radionuclides to manipulate αββ-integrin in vivo.

Description

[Technical Field]

[0001] Technical Field The present invention relates to the field of cyclic peptides as ligands for cell surface receptors, in particular as ligands for αvβ6-integrin. The present invention further relates to conjugates of such peptides with effector moieties suitable for use as therapeutic agents, diagnostic agents, targeting moieties, and biomolecular research tools. The present invention particularly relates to the use of derivatives of such peptides bearing signaling moieties or radionuclides for in vivo addressing of αvβ6-integrin. [Background technology]

[0002] background Integrins are a family of 24 heterodimeric transmembrane receptors, all of which contain one of 18 α-subunits and one of eight β-subunits. Integrins mediate the selective binding of cells to various extracellular matrix proteins, such as vitronectin, fibronectin, collagen, or laminin, and are also involved in signal transduction pathways. 1 αvβ6 is one of eight integrin subtypes that recognize the arginine-glycine-aspartic acid (RGD) peptide sequence, in contrast to other common RGD-binding integrins such as αvβ3 and α5β1, which are expressed by a variety of cell types and have received considerable attention due to their involvement in the formation and sprouting of blood and lymphatic vessels (angiogenesis, vasculogenesis, and lymphangiogenesis). 2 , αvβ6 integrin levels in adult tissues are generally low. 3 Expression of αvβ6 integrin is restricted to epithelial cells. 4 Therefore, many tumors of epithelial origin (carcinomas), especially pancreatic 6 , as well as bile duct cells 7 ,stomach 8,9 ,breast 10 , ovaries 11,12 ,colon 13 , and tumors of the upper aerodigestive tract14 shows enhanced αvβ6 integrin expression. 5 αvβ6-integrin has also been described as a marker of increased invasiveness and malignancy, and therefore poor prognosis, for several carcinomas. 5,8,11,13 Thus, αvβ6-integrin has been proposed as a target for in vivo addressing of carcinoma tissues for the purposes of molecular imaging and targeted therapy. 15 Furthermore, αvβ6-integrin is involved in e.g. biliary fibrosis. 16 , renal fibrosis 17 , and pulmonary fibrosis 18 It is involved in epithelial-mesenchymal transition (EMT) during development and may therefore serve as a fibrosis marker.

[0003] technical level Several αvβ6-specific non-peptide 19 and peptide inhibitors 20,21,22,23 The linear peptide A20FMDV2 has been reported. 21 , H2009.1 22 , and cyclic peptide S02 23 By carrying a radioactive label, single photon emission computed tomography (SPECT) 25,26,27 and positron emission tomography (PET) 21,28,29,30,31,32 It has been applied to in vivo imaging of αvβ6-integrin expression by ELISA. 24 Recently, radiolabeled compounds targeting αvβ6-integrin have been tested for imaging carcinomas in humans. 33,34,35

[0004] Cyclic nonapeptide cyclo(FRGDLAFp(NMe)K) 36,37 (abbreviated as Phe2 herein) has been reported to exhibit high affinity for αvβ6-integrin (0.26 nM), remarkable selectivity over other integrins (αvβ3: 632 nM; α5β1: 73 nM; αvβ5 and αIIbβ3: >1 μM), and sufficient stability in human plasma for up to 3 hours. Phe2 derivatives were modified to have various chelators for radiometal binding. 38,39Their in vivo properties were determined in tumor-bearing mice. These studies showed that radiolabeled chelator conjugates containing only one Phe2 moiety (monomers) exhibited relatively low uptake in αvβ6-expressing tumor tissue. 39 Conjugates containing two, and especially three, Phe2 moieties (dimers and trimers, respectively) showed higher affinity for αββ-integrin, but due to their lipophilicity, they were also characterized by relatively high levels of nonspecific uptake in nontarget organs. This behavior of the trimers could not be attenuated by the introduction of pharmacokinetic modifiers, i.e., hydrophilic PEG linkers. 38 Summary of the Invention [Means for solving the problem]

[0005] Summary of the Invention In light of the above-described situation, there is a need to provide αββ-integrin-active functional compounds that exhibit improved pharmacokinetics and, in particular, increased target-specific tissue uptake and retention, while at the same time exhibiting low nonspecific uptake in αββ-integrin-negative tissues. In particular, low nonspecific uptake in liver and pancreatic tissues is desirable. Further objectives are rapid clearance from the blood pool and low nonspecific binding to blood components, as well as suitability for high-contrast in vivo imaging of such tissues, expressed as a high ratio of uptake in tumor lesions compared to other tissues.

[0006] The present invention solves this problem by providing conjugates containing specific cyclic nonapeptides that target αβ-integrin. These cyclic nonapeptides are characterized by the following amino acid sequences: cyclo(YRGDLAYp(NMe)K), hereafter referred to as Tyr2; cyclo(FRGDLAYp(NMe)K), hereafter referred to as FRGD; and cyclo(YRGDLAFp(NMe)K), hereafter referred to as YRGD. These abbreviations are also used to characterize each cyclic nonapeptide, which is covalently attached to an effector moiety via the terminal amino group of the (NMe)K side chain. This means that the abbreviations Tyr2, FRGD, and YRGD characterize not only the cyclopeptides cyclo(YRGDLAYp(NMe)K), cyclo(FRGDLAYp(NMe)K), and cyclo(YRGDLAFp(NMe)K), respectively, but also the same cyclopeptides in which one of the two hydrogens of the terminal amino group of the (NMe)K side chain is missing / replaced by a covalent bond to another moiety.

[0007] Tyr2, FRGD, and YRGD are structurally related to Phe2, and all of them are encompassed by the general teachings of WO 2017 / 046416A1. However, this patent application does not disclose Tyr2 in detail, nor does it disclose any specific conjugates with Tyr2, FRGD, and / or YRGD and / or any tissue-specific binding characteristics of conjugates comprising Tyr2, FRGD, and / or YRGD.

[0008] Surprisingly, it has been found that conjugates of Tyr2, FRGD, and / or YRGD, particularly those containing more than one Tyr2, FRGD, and / or YRGD moiety, exhibit high target-specific tissue uptake and retention, while at the same time exhibiting low nonspecific uptake in αβ-integrin-negative tissues (especially the liver) and rapid clearance from the blood pool, compared to, for example, structurally equivalent derivatives of Phe2. Thus, such conjugates enable selective and specific addressing of αβ-integrin-positive tissues in vivo, particularly for high-contrast in vivo imaging of such tissues.

[0009] The present invention therefore relates to conjugates of Tyr2, FRGD, and / or YRGD or at least one cyclic nonapeptide selected from Tyr2, FRGD, and YRGD, in which an effector moiety is covalently attached to the terminal amino group of an NMe-lysine residue. The present invention particularly relates to conjugates comprising more than one Tyr2, FRGD, and / or YRGD moiety, which exhibit higher affinity and integrin subtype selectivity than comparable compounds containing only one such moiety. These conjugates can be characterized by the following general formula (I): E(Cp) n (I) In the formula, Cp represents a cyclopeptide selected from Tyr2, FRGD, and / or YRGD, n is an integer selected from 1 to 4, and E represents an effector moiety.

[0010] According to the present invention, various types of effector moieties can be used, including moieties suitable for diagnostic use and pharmacologically active moieties for therapeutic use. Conjugates with moieties for diagnostic use are of particular interest. These include moieties containing radionuclides (for nuclear imaging or radio-guided surgery), fluorophores (for fluorescent imaging or fluorescent-guided surgery), or signaling units for magnetic resonance imaging (MRI). For therapeutic purposes, the effector moiety may contain, for example, radionuclides (internal radiotherapy) or chemotherapeutic agents (targeted drug delivery).

[0011] Yet another aspect of the present invention relates to the use of the conjugates described above in diagnostic or therapeutic methods.

[0012] The cyclopeptide Tyr2 is novel. Building blocks containing one of Tyr2, FRGD, and YRGD combined with a spacer element suitable for click chemistry coupling are also novel. Another aspect of the present invention therefore relates to the provision of these compounds.

[0013] Various aspects of the present application are described in further detail in the following detailed description and in the appended claims. [Brief explanation of the drawings]

[0014] DESCRIPTION OF THE DRAWINGS [Figure 1] Figure 1 shows illustrative positron emission tomography (PET) scans (maximum intensity projection) of the same H2009 tumor-bearing SCID mouse 75 minutes after injection of Ga-68-TRAP(Phe2)3 (left) and Ga-68-C-7 (right). The time between both scans was 24 hours. [Figure 2]Figure 2 shows the ex vivo biodistribution of Ga-68-TRAP(Phe2)3 (structured bars) and Ga-68-C-7 (solid bars) in H2009 tumor-bearing SCID mice at 90 min p.i. without (approximately 0.1 nmol, n = 5) and with (50 nmol, n = 3) blocker (data are expressed as mean ± standard deviation). [Figure 3] Figure 3 shows the disposition of Ga-68-TRAP(Phe2)3 (left) and Ga-68-C-7 (right) as determined by region-of-interest determination of a 90-minute dynamic PET scan. [Figure 4] Top: Ex vivo biodistribution in selected tissues of H2009 tumor-bearing SCID mice 90 min pi without blocker (approximately 0.1 nmol, n=5) and with blocker (50 nmol, n=3). Bottom: Tumor-to-tissue ratios derived from biodistribution data. All data are expressed as mean ± standard deviation. Legend for labels on vertical graph: a) Ga-68-TRAP(Phe2)3; b) Ga-68-TRAP(Phe2)3, blocker; c) Ga-68-C-11; d) Ga-68-C-11, blocker; e) Ga-68-C-9; f) Ga-68-C-9, blocker; g) Ga-68-C-8; h) Ga-68-C-8, blocker; i) Ga-68-C-10; k) Ga-68-C-10, blocker; l) Ga-68-C-7; m) Ga-68-C-7, blocker. [Figure 5] Figure 5 shows illustrative positron emission tomography (PET) scans (maximum intensity projection) of the same H2009 tumor-bearing SCID mouse 75 minutes after injection of Ga-68-TRAP(Phe2)3, Ga-68-C-9, Ga-68-C-8, and Ga-68-C-7 (from left to right). The time between scans was 24 hours. %IA / mL means percent injected activity per mL of tissue. [Figure 6]Figure 6 shows the disposition of Ga-68-TRAP(Phe2), Ga-68-C-9, Ga-68-C-8, and Ga-68-C-7 (from left to right) from region-of-interest based determination of a 90-minute dynamic PET scan. %IA / mL means percent of administered radioactivity per mL of tissue. DETAILED DESCRIPTION OF THE INVENTION

[0015] Detailed Description definition The term "derived from" indicates that the moiety contained in the conjugate has the same structure as the compound from which it is derived, the only difference being the replacement of a hydrogen atom with a covalent bond to attach the moiety to the remainder of the conjugate.

[0016] The term "heavy element" is used herein to characterize any atom other than hydrogen, deuterium, or any other isotope thereof. In the case of a divalent atom group, there must be at least one heavy element with at least two free valences. If a heavy element with more than two free valences is present, the remaining valences may be saturated with hydrogen or other heavy elements.

[0017] Standard amino acid nomenclature is used unless otherwise specified. Unless otherwise specified, amino acids are L-stereoisomers. Unless otherwise specified, amino acid components are linked to each other via peptide bonds. Unless otherwise specified, standard one-letter or three-letter abbreviations for amino acids are used. Unless otherwise specified, lowercase letters indicate that the amino acid is in the D-configuration and uppercase letters indicate that the amino acid is in the L-configuration.

[0018] Me refers to a methyl group. N-Me-amino acid refers to a group in which the α-amino group bears a methyl group.

[0019] Unless otherwise specified or dictated otherwise by context, references to "compounds of the invention," "conjugates of the invention," or the like, are to be understood as references not only to the compounds of the invention, conjugates, etc., as described below and / or as specified in the appended claims, but also to the pharmaceutically acceptable salts, esters, solvates, and polymorphs thereof.

[0020] References to "substituted" or "substituted by" include the implicit proviso that such substitution is pursuant to the substituted atom and the substitutable valence of the substituent, and that the substitution results in a stable compound that does not spontaneously undergo transformation, for example, by rearrangement, cyclization, elimination reactions, and the like. As used herein, the term "substituted" is intended to include all permissible substituents of organic compounds. Broadly construed, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. The permissible substituents can be one or more. Substituents include alkyl, preferably C 1~6 -Alkyl, alkenyl, preferably C 2~6 -Alkenyl, alkynyl, preferably C 2~6 -alkynyl, alkoxy, preferably C 1~6 -alkoxy, acyl, preferably C 2~6 -Acyl, amino (simple amino, mono and di-C 1~6 -Alkylamino, mono- and di-C 6~14 -arylamino, and C 1~6 -Alkyl-C 6~14 -arylamino), C 2~6 -acylamino (including carbamoyl and ureido), C 1~6 -Alkylcarbonyloxy, C 6~14 -arylcarbonyloxy, C 1~6 -alkoxycarbonyloxy, C 1~6 -Alkoxycarbonyl, carboxy, carboxylate, aminocarbonyl, mono and di-C 1~6-Alkylaminocarbonyl, cyano, azido, halogen, hydroxyl, nitro, trifluoromethyl, thio, C 1~6 -Alkylthio, arylthio, C 1~6 -Alkylthiocarbonyl, thiocarboxylate, C 4~8 -cycloalkyl, heterocycloalkyl having 4 to 8 ring members, C 6~14 -aryl, optionally heteroaryl having 5 to 6 ring atoms fused with 1 or 2 saturated, unsaturated, or aromatic carbocyclic or heterocyclic rings, each having 5 or 6 ring atoms; C 6~14 -aryloxy, C 6~14 -Aryloxycarbonyloxy, benzyloxy, benzyl, sulfinyl, C 1~6 -alkylsulfinyl, sulfonyl, sulfate, sulfonate, sulfonamido, phosphate, phosphonato, phosphinato, oxo, guanidine, imino, formyl, and the like. Any of the above substituents can be further substituted, where permissible, for example, with one or more of the listed substituents.

[0021] The terms "alkyl," "alkenyl," "alkynyl," "cycloalkyl," "carbocyclic," "heterocycloalkyl," "aryl," "heteroaryl," "heterocycle," "amine," "amide," "nitro," "halogen," "thiol," "hydroxyl" or "hydroxy," "alkylthio," "alkylcarboxyl," "carbonyl," "carboxy," "acyl," "solvate," "pharmaceutically acceptable salt," "pharmaceutically acceptable vehicle," "pharmaceutically acceptable carrier," and "pharmaceutical composition" may have the meanings defined in WO 2017 / 046416A.

[0022] Unless otherwise specified, all abbreviations are intended to have their commonly used meanings, e.g., as represented by the IUPAC-IUP Commission on Biochemical Nomenclature in Biochemistry 11, 1972, 942-944. For atoms contained in a conjugate, standard rules for valency apply, as described, for example, in the January 24, 2020, version of the Wikipedia entry "Atom Valency (Chemistry)." Unless otherwise specified or dictated by context, if an atom has more valencies than the indicated number of binding partners, the remaining valencies are saturated with hydrogen atoms.

[0023] Unless otherwise specified, the conjugates and other compounds of the present invention are "pharmaceutically acceptable," meaning that the respective compounds are suitable for use in humans and / or animals, do not cause side effects (such as irritation or toxicity), and are commensurate with a reasonable benefit / risk ratio.

[0024] The term "or" is generally used in its sense including "and / or" unless the context dictates otherwise.

[0025] "Room temperature" can be any temperature between 20°C and 25°C, and preferably room temperature is 22°C.

[0026] "Ga-68-TRAP(Phe2)3" was previously reported by Maltsev et al. 38 This refers to the compound described as "Ga-68-TRAP(AvB6)3" by

[0027] Unless otherwise specified, the term "chelating group," "chelator," or the like refers to a group capable of forming two or more, preferably 3, 4, 5, 6, 7, or 8, coordinate bonds with a metal ion.

[0028] Cyclopeptides The cyclopeptides used in the present invention are shown below: Tyr2: cyclo(YRGDLAYp(NMe)K), FRGD: cyclo(FRGDLAYp(NMe)K), YRGD: cyclo(YRGDLAFp(NMe)K)

[0029] Conjugates General structure The general structure of the conjugates of the present invention may be characterized by the following formula (I): E(Cp) n (I) wherein each Cp represents a cyclopeptide independently selected from Tyr2, FRGD, and / or YRGD; n is an integer selected from 1 to 4, preferably 2 to 4, more preferably 3 or 4; and E represents an effector moiety. According to a further embodiment, polymeric or resinous effector moieties can be used. In this case, n may be an integer selected from 2 to 100, preferably 10 to 30. Suitable polymeric scaffolds include polyethyleneimine, polysaccharides, polyamides, polypeptides, poly(amidoamine) (PAMAM) dendrimers, poly(propyleneimine) (PPI) dendrimers, polyether-copolyester (PEPE) dendrimers, polyether dendrimers, polyester dendrimers, and polyarylether dendrimers.

[0030] The one or more cyclopeptides are each covalently attached to the effector moiety via the terminal amino group in the side chain of an NMe-Lys residue.

[0031] In preferred embodiments, the conjugate of formula (I) contains 2, 3, or 4 cyclopeptide moieties. Most preferably, the conjugate of formula (I) contains 3 or 4 cyclopeptide moieties.

[0032] In conjugates of the invention containing two or more cyclopeptide moieties, these multiple cyclopeptide moieties may be the same or different from one another. All of the following specific conjugates are within the scope of the invention: E(Tyr2)1, E(Tyr2)2, E(Tyr2)3, E(Tyr2)4, E(FRGD)1, E(FRGD)2, E(FRGD)3, E(FRGD)4, E(YRGD)1, E(YRGD)2, E(YRGD)3, E(YRGD)4, E(Tyr2)1(FRGD)1, E(Tyr2)2(FRGD)1, E(Tyr2)1(FRGD)2, E(Tyr2)2(FRGD)2, E(Tyr2)1(FRGD)3, E(Tyr2)3(FRGD)1, E(Tyr2)1(YRGD)1, E(Tyr2)2(YRGD)1, E(Tyr2)1(YRGD)2, E(Tyr2)2(YRGD)2, E(Tyr2)1(YRGD)3, E(Tyr2)3(YRGD)1, E(FRGD)1(YRGD)1, E(FRGD)2(YRGD)1, E(FRGD)1(YRGD)2, E(FRGD)2(YRGD)2, E(FRGD)1(YRGD)3, E(FRGD)3(YRGD)1, E(Tyr2)1(FRGD)1(YRGD)1, E(Tyr2)2(FRGD)1(YRGD)1, E(Tyr2)1(FRGD)2(YRGD)1, E(Tyr2)1(FRGD)1(YRGD)2

[0033] In the case of a polymeric or resinous effector moiety, it is also possible to add multiple copies of the same cyclopeptide selected from Tyr2, YRGD, and FRGD. Alternatively, the polymeric or resinous effector may be attached to two or three different cyclopeptides, such that each of these two or three cyclopeptides is present more than once, provided that the total number of attached cyclopeptides is within the range specified above for n, i.e., the polymeric or resinous conjugate has the general formula E((Tyr2) n1 (FRGD) n2 (YRGD)n3 ) (wherein n1, n2, and n3 may each be in the range of 0 to n, provided that n1+n2+n3=n).

[0034] In principle, it is possible to obtain further compounds of the present invention by modifying the compounds of the present invention as specified above by covalently linking a cyclopeptide different from Tyr2, FRGD, and YRGD to the effector moiety. For example, one embodiment relates to the compound described above, but one, two, or three of the cyclopeptide moieties Tyr2, FRGD, and / or YRGD are replaced by the cyclopeptide moiety Phe2 described in the introduction, where Phe2 is linked to the remainder of the conjugate in the same way as the other cyclopeptide moieties, i.e., via the terminal amino group of the K(NMe) residue, and the number of Phe2 substitutions is such that at least one of the cyclopeptide moieties Tyr2, FRGD, and YRGD remains in the conjugate (i.e., if n is the number of cyclopeptide moieties, the number of Phe2 moieties is n-1 or less, and at least one cyclopeptide moiety is selected from Tyr2, FRGD, and YRGD). In another embodiment, no further cyclopeptides are present.

[0035] Effector component The effector moiety is an atomic group having 10 to 1000 heavy elements, preferably 20 to 200 heavy elements, more preferably 30 to 150 heavy elements. The effector moiety is further characterized by the following features: (a) the effector moiety has a number of free valences corresponding to the number of attached cyclopeptides, i.e., the number n in formula (I); (b) the effector moiety contains an active atom or group of atoms capable of exerting a desired effect, such as a radioisotope or chromophore for diagnostic purposes or a therapeutically active moiety for therapeutic purposes; (c) The effector moiety contains one or more groups of atoms that act as spacers to spatially separate one or more cyclopeptides from the active atom or group of active atoms, thereby reducing mutual interference.

[0036] The effector may, in some embodiments, be characterized by the following general formulas (II) and (II'): Aa(Cg)(S) n (II) Aa'(Cg) k (S) n (II') wherein Aa represents an active atom or active atomic group capable of being bonded via chelation, Aa' represents an active atom or active atomic group capable of being bonded via a covalent bond, Cg represents a chelating group, k is 0 or 1, S represents an atomic group acting as a spacer, and n is as described above for formula (I), with the proviso that n does not exceed the number of free valences of the chelating group, and n is 1 when k is 0, i.e., when there is no chelating group, a single spacer is directly bonded to the active atom or active atomic group. Combining formula (I) and formula (II) gives the following formula (Ia): Aa(Cg)(SCp) n (Ia) wherein Aa, Cg, S, Cp, and n have the same meanings as defined above for formulae (I) and (II).

[0037] In a related embodiment, the active atom or active atom group Aa' is covalently linked to a chelating group or to a spacer. The conjugate of this embodiment is characterized by the following formula (Ia'): Aa'(Cg) k (SCp) n (Ia') wherein Aa' is an active atom or group of atoms capable of forming a covalent bond, Cg, S, Cp, and n have the same meanings as defined above for formulae (I) and (II), k is 0 or 1, when k is 1, Aa' is covalently bonded to Cg, and when k is 0, Aa' is covalently bonded to S. In this case, n is 1, i.e., there is only one spacer that forms a covalent bond between Aa' and Cp.

[0038] In another embodiment, a second reactive group can be added to one of the spacers (instead of one of the cyclopeptides), and the conjugate is represented by the following formula (Ib): Aa(Cg)(SCp) n’ (SAa') (Ib) wherein Aa, Cg, S, and Cp have the same meaning as in formula (Ia) above, and wherein Aa' is an active atom or active atomic group different from Aa, so long as Aa' is covalently bonded to the spacer other than via a chelating group, and n' is 1, 2, or 3, provided that n'+1 is equal to or less than the number of free valences of the chelating group.

[0039] In yet another embodiment, the various linkers may be connected via a non-chelating central moiety. In these cases, the active atom or active atom group is covalently linked to another part of the molecule, which may be the central moiety, a spacer, or a cyclopeptide. The conjugates of this embodiment are characterized by the following formulae (Ic), (Id), (Ie), and (If): Aa'(Cm) k (SCp) n (I C) (Cm)(SCp) n-o (S(Aa') p (Cp) m ) o (Id) (Cm)(SCp) n-o (SCp(Aa') p ) o (Ie) Cp(Aa') p(If)

[0040] Formula (Ic) corresponds to formula (Ia') above, except that the chelating group is replaced by a central moiety, Cm. S, Cp, and n have the same meanings as defined above for formulae (I), (Ia), and (II); k is 0 or 1. Aa' is an active atom or active atomic group capable of forming a covalent bond. In formula (Ic), when k is 1, Aa' is bonded to Cm via a covalent bond, and when k is 0, Aa' is bonded to S. In the latter case, n must be 1, i.e., there is only one spacer bonded to both Aa' and Cp.

[0041] The central moiety Cm can be any atom or group of atoms with a valence of at least n+1, so as to accommodate n spacer-cyclopeptide moieties and one active atom or group of atoms. Cm preferably has 1 to 30 atoms selected from C, N, O, S, and P. The remaining valences are saturated with hydrogen. Preferred Cm groups are aromatic groups such as phenyl, naphthyl, etc., or derived from larger fused aromatic groups containing three or four six-membered rings, e.g., anthracene, phenanthrene, benzpyrene, etc.; non-aromatic ring groups including C5-7 carbocyclic rings such as cyclopentane, cyclohexane, cycloheptane; fused groups containing two, three, or four rings, each consisting of five to seven ring atoms, such as fully or partially hydrogenated forms of naphthalene, anthracene, phenanthrene, benzpyrene, etc.; or bi- or tricyclic groups having 7 to 10 carbon atoms, such as norbornene or adamantane. More preferred central moieties may be heterocyclic groups containing one, two, three, or four fused rings, each having a ring size independently selected from five, six, or seven ring atoms. These groups may be aromatic and partially or fully saturated. Alternatively, the central moiety may be a single atom selected from C, N, and P.

[0042] Formula (Id) characterizes a conjugate in which the cyclopeptide component and the active atom or active group Aa' are all linked to the central component via a spacer. That is, the active atom or active group Aa' is covalently bonded to one of the spacers. The meanings of Cm, Aa', S, Cp, and n are the same as those explained above for formulae (I), (II), (Ia), and (Ic). Optionally, the spacer bearing the active atom or active group Aa' may bear an additional cyclopeptide Cp; thus, m may be 0 or 1. When an additional Cp is present, the active atom or active group Aa' and the position of its attachment should be selected so that adverse interactions with the cyclopeptide are avoided or at least minimized, for example, by attaching the two components to different atoms of the spacer that are at least five covalent bonds apart from each other. The number of spacers bearing active atoms or groups Aa' is characterized by o, which can be any integer from 1 to n. The number of active atoms Aa' bonded to separate spacers is characterized by p, which can be 1 or 2.

[0043] Formula (Ie) is characterized by the active atom or active atomic group Aa' attached to the cyclopeptide Cp. The meanings of Cm, Aa', S, Cp, and n are the same as those explained above for formulae (I), (II), (Ia), and (Ic). The variable o indicates the number of cyclopeptides Cp bearing the active atom or group Aa'. It can be any integer from 1 to n. The variable p characterizes the number of active atoms Aa' attached to separate cyclopeptides, and p can be 1 or 2.

[0044] Formula (If) characterizes the conjugate of the present invention, which does not contain any central component and / or spacer. Instead, the active atom or active group Aa' is directly bonded to the cyclopeptide. According to a preferred embodiment of formula (If), an iodine atom or radioisotope is added to the 3-position of one or both of the tyrosine residues present in Tyr2, FRGD, or YRGD, and the resulting cyclopeptides are cyclo(3-I-YRGDLAYp(NMe)K); cyclo(3-I-YRGDLA3-I-Yp(NMe)K); cyclo(YRGDLA3-I-Yp(NMe)K); cyclo(3-I-YRGDLAFp(NMe)K); cyclo(FRGDLA3-I-Yp(NMe)K); wherein 3-IY represents a Tyr residue having an iodine atom at the 3-position of the phenyl ring, said iodine atom being any non-radioactive or radioactive isotope of iodine.

[0045] Compounds of formula (1f) may have two features: they may serve as conjugates of the present invention as long as the binding of Aa' does not lead to a significant deterioration in affinity for αβ-integrin, i.e., the binding affinity of the cyclopeptide bearing Aa' is 5 nM or less, as determined according to the methods described in references 36 and 37. In addition, compounds of formula (1f) may also be incorporated into larger conjugates, for example of formula (1e), and thus may serve as building blocks of the present invention.

[0046] According to formulas (1a) to (1f), the binding modes of the active atoms and active groups Aa and Aa' described above may be freely combined. For example, the compound of formula (1a) or (1a') may have one or more cyclopeptides which themselves have one or more active atoms or active groups Aa'. In particular, the present invention also relates to conjugates of formula (1a) or (1a'), in which one or more cyclopeptides have one or two iodine atoms or radioisotopes bound to the 3-position of the tyrosine residue.

[0047] The active atoms or active groups Aa, Aa' may include: (b-1)La 3+ , Ce 3+ , Pr 3+ , Nd 3+ , Sm 3+ ,EU 2+ , Gd 3+ , Tb 3+ , Dy 3+ , Ho 3+ , Er 3+ , Tm 3+ , Yb 3+ , Lu 3+ ,Sc. 3+ , Y 3+ , Ga 3+ , Fe 3+ , Co 2+ , Co 3+ , Ge 4+ , In 3+ , Sn 2+ , Sn 4+ , Bi 3+ , Rh 3+ , Ru 3+ , Ru 4+ , Ag + , Au 3+ , Pb 2+ , Pd 2+ , Pd 4+ , Pm 3+ , Ac 3+ , Ti 4+ , Zr 4+ Al 3+ , Cr 3+ , Cu 2+ , Zn 2+ Non-radioactive or radioactive isotopes of metal ions selected from Ga 3+ , Gd 3+ , Cu 2+ ,Sc. 3+ , Y 3+ , and Lu 3+ Particularly preferred are metal ions selected from the group consisting of: 43 Sc, 44 Sc, 46 Sc, 47 Sc, 55 Co, 99m Tc, 203Pb, 212 Pb, 66 Ga, 67 Ga, 68 Ga, 72 As, 111 In, 113m In, 114m In, 97 Ru, 62 Zn, 61 Cu, 62 Cu, 64 Cu, 52 Fe, 52m Mn, 51 Cr, 186 Re, 188 Re, 77 As, 86 Y, 90 Y, 67 Cu, 169 Er, 117m Sn, 121 Sn, 127 Te, 142 Pr, 143 Pr, 198 Au, 199 Au, 149 Tb, 152 Tb, 155 Tb, 161 Tb, 109 Pd, 165 Dy, 149 Pm, 151 Pm, 153 Sm, 157 Gd, 166 Ho, 172 Tm, 169 Yb, 175 Yb, 177 Lu, 105 Rh, 111 Ag, 88 Zr, 89 Zr, 212 Bi, 213 Bi, 225 Ac, and mixtures thereof, and these radioisotopes are preferably used in the form of metal ions in the respective oxidation states listed above. Particularly preferably, the radioisotopes are 68 Ga, 44 Sc, 99m Tc, 111 In, 64 Cu,89 Zr, 90 Y, 177 Lu, 213 Bi, 225 Ac, and mixtures thereof. (b-2) 11 C. 13 N, 15 O. 18 F, 123 I, 124 I, 125 I, or 131 I, preferably 18 F or 123 A non-metallic radioisotope selected from I. In addition to its presence as Aa, Aa', or part thereof in the above formula, said non-metallic radioisotope may also be an active atom Aa' present anywhere else in the molecule, and atom Aa' may replace any other covalently bonded atom already present as part of the rest of the molecule, and atom Aa' has an appropriate number of binding partners. (b-3) chromophores of fluorescent or non-fluorescent dyes and preferably moieties derived from ThermoFisher's commercially available Cy® series, such as CY® 3, 5, 5.5, 7, 7.5, and AlexaFluor® series, such as AlexaFluor® 350, 405, 488, 532, 546, 555, 568, 594, 647, 680, and 750, as well as fluorescein, pyrene, rhodamine, BODIPY dyes, and analogs thereof; (b-4) Contrast agents for magnetic resonance imaging (MRI), preferably Gd, Fe, Mn, most preferably Gd in the form of Gd(III) in the form of a chelate complex; (b-5) An atom or atomic group suitable for imaging by X-ray-based techniques, preferably iodine or an atomic group containing iodine. (b-6) An atom or atomic group derived from a therapeutic agent, which may have therapeutic activity by itself or after cleavage of the cyclopeptide-containing moiety, thereby releasing the therapeutic agent. Preferably, the therapeutic agent is a therapeutic agent suitable for treating cancer or fibrosis.

[0048] When the indication for treatment is cancer, the therapeutic agent is preferably selected from alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other anti-tumor agents. More particularly, mention may be made of: platinum-based compounds, antibiotics with anticancer activity, anthracyclines, anthracenediones, alkylating agents, antimetabolites, mitotic inhibitors, taxanes, taxoids, microtubule inhibitors, vinca alkaloids, folate antagonists, topoisomerase inhibitors, antiestrogens, antiandrogens, aromatase inhibitors, GnRh analogues, inhibitors of 5α-reductase, bisphosphonates, metabolic inhibitors, preferably mTOR inhibitors; epigenetic inhibitors, preferably DNMT inhibitors; anthracycline antibiotics; camptotheca; anthracyclines; histone deacetylase (HDAC) inhibitors, proteasome inhibitors, JAK2 inhibitors, tyrosine kinase inhibitors (TKI), PI3K inhibitors, protein kinase inhibitors, inhibitors of serine / threonine kinases, inhibitors of intracellular signaling, inhibitors of Ras / Raf signaling, MEK inhibitors, AKT inhibitors, survival signaling proteins inhibitors of urokinase-type plasminogen activator receptor function, immunoconjugates, antibody-drug conjugates, antibody fragments, bispecific antibodies, bispecific T cell engagers (BiTEs).The anticancer drug is preferably selected from the group consisting of 5-fluorouracil, cisplatin, irinotecan hydrochloride, epirubicin, paclitaxel, docetaxel, camptothecin, doxorubicin, rapamycin, 5-azacytidine, doxorubicin irinotecan, topotecan (type 1 topoisomerase inhibitor), amsacrine, etoposide, etoposide phosphate, and teniposide (type 2 topoisomerase inhibitor); UFT, capecitabine, CPT-II, oxaliplatin, cyclophosphamide, methotrexate, navelbine, epirubicin, mitoxantrone, raloxifene, mitomycin, carboplatinum, gemcitabine, etoposide, and topotecan.

[0049] Further suitable therapeutic agents for the treatment of cancer are disclosed, for example, in "Cancer Drugs" by Judith Matray-Devoti, Chelsea House, 2006; "Physicians' Cancer Chemotherapy Drug Manual 2015" by Edward Chu, Vincent T DeVita, Jr., Jones & Bartlett Learning, 2015; "Cancer Chemotherapy and Biotherapy: Principles and Practice" by Bruce A. Chabner, Dan L. Longo, Wolters Kluwer, 2011; and "Drugs in Cancer Care" by Rachel Midgley, Mark R. Middleton, Andrew Dickman, David Kerr (Eds.), Oxford University Press, 2013. The agents disclosed in these works can be used as therapeutic agents when practicing the present invention. The disclosures of therapeutic agents in these references are hereby incorporated by reference.

[0050] When the indication for treatment is fibrosis, the therapeutic agent is preferably selected from therapeutic agents suitable for treating fibrosis. Such therapeutic agents are described, for example, in “Cystic Fibrosis in the 21st Century” by Andrew Bush (Ed.), S. Karger, 2006; “Liver Fibrosis: New Insights for the Healthcare Professional: 2013 Edition” by Q. Ahton Acton, ScholarlyEditions, 2013; “Idiopathic Pulmonary Fibrosis: A Comprehensive Clinical Guide” by Keith C. Meyer, Steven D. Nathan, Springer, 2014;“New Insights into the Pathogenesis and Treatment of Idiopathic Pulmonary Fibrosis: A Potential Role for Stem Cells in the Lung Parenchyma and Implications for Therapy”by M. Gharaee-Kermani et al. in Pharmaceutical Research, 2007, 24, 819-841;“Pulmonary Fibrosis: pathogenesis, etiology and regulation”by MS Wilson and TA Wynn in Mucosal Immunol. 2009, 2, 103-121. Certain preferred therapeutic agents are preferably selected from the agents and agent groups disclosed and listed in Table II of the review article by Gharaee-Kermani et al., cited above.

[0051] When the indication for treatment is Covid-19 infection, the therapeutic agent may be any pharmaceutical agent with experimentally established or suspected activity in treating such infection, regardless of whether it is already in clinical use or is still under development. Pharmaceutical agents currently in use or under development for the treatment of Covid-19 infection include, for example, antiviral drugs, including the anti-Ebola drug remdesivir or the anti-influenza drug favilavir; kinase inhibitors, such as ATR-002; anti-inflammatory drugs, including glucocorticoids; IL-1 or IL-6 antagonists, such as anakinra and tocilizumab, respectively; anti-infective drugs, such as ivermectin; or pharmaceutical agents for the treatment of other pulmonary conditions, such as fibrosis. Accordingly, reference may be made to the literature and pharmaceutical agents mentioned above for fibrosis.

[0052] The active atom or group of atoms can be bonded to the cyclopeptide via an atomic group acting as a spacer. The atomic group acting as a spacer is typically a linear chain of 2 to 20 atoms, preferably 3 to 10 atoms, selected from C, N, O, P, and S, preferably an alkylene group optionally bearing one or more substituents, with the remaining valences saturated with hydrogen. This linear chain may be interrupted by one or more cyclic structures, preferably those having five ring atoms, more preferably triazole rings. The bond to the amino group of the side chain of N(Me)K is typically achieved by an amide bond. The bond of the spacer to the active atom or group Aa' in formula (1a'), (1b), (1c), or (1d) can also be achieved by an amide bond, although a direct covalent bond is also possible.

[0053] For example, the atomic group that acts as a spacer in the above formulas (Ia) to (If) is further described below. In one embodiment, it is represented by the following formula (IIIa): *-C(O)-(CH2) k -(taz) l -(CH2) m - (IIIa) (wherein taz represents a triazole ring in which all three nitrogen atoms are adjacent to one another, l may be 0 or 1, and each of k and m is an integer selected from 0 to 20 such that k+m=2 to 20. * ) indicates the position of attachment of the cyclopeptide.

[0054] In another embodiment, the additional divalent functional group is represented by the following formulae (IIIb) to (IIIf): *-C(O)-(CH2) k -NH-CO-(CH2) m - (IIIb) *-C(O)-(CH2) k -CO-NH-(CH2) m - (IIIc) *-C(O)-(CH2) k -(taz) l -(CH2) o -CO-NH-(CH2) m - (IIId) *-C(O)-(CH2) k -(taz) l -(CH2) o -NH-CO-(CH2) m - (IIIe) *-C(O)-(CH2) k -CO-NH-(CH2) o -(taz) l -(CH2) m - (IIIf) *-C(O)-(CH2) k -NH-CO-(CH2) o -(taz) l -(CH2) m - (IIIf) (wherein taz and l have the same meaning as set forth above for Formula (IIIa). k, m, and, if present, o are integers independently selected from the range of 0 to 20 such that k+m=2 to 20 and k+m+o=2 to 20, respectively. An asterisk ( *) may also be present as represented by ) indicating the position of attachment of the cyclopeptide.

[0055] According to one embodiment, one or more spacers may have one or more independently selected substituents. Each of these substituents is not particularly limited. According to a preferred embodiment, the substituent is itself a component containing a spacer and a cyclopeptide, preferably a spacer S and a cyclopeptide Cp described herein. The spacer portion of the substituent may even be further substituted to form a dendrimeric structure, thereby allowing up to three generations of substituents to be added to the zero-generation spacer represented by formulas (Ia) to (Ie).

[0056] In other embodiments, the spacer may be cleavable under physiological conditions, particularly in connection with an active atom or active atomic group suitable for therapeutic purposes. Such cleavable spacers may be selected from, but are not limited to, the spacers described in WO 2009 / 117531A, WO 2015 / 123679A, Younes et al. N. Engl. J. Med. 2010;363:1812-1821; Dorywalska et al. Mol. Cancer Ther. 2016;15(5):958-970, Jain et al., Pharm. Res. 2015;32(11):3526-3540, and references cited therein.

[0057] When the active atom is a metal ion, attachment is typically achieved via a chelating group, as described, for example, in Chem. Soc. Rev. 2011;40:3019-3049. 40The binding of the metal ion by the chelating group preferably occurs via a complex bond (Lewis acid / base interaction) caused by the N and O atoms of the chelating group. However, the chelating group is not particularly limited as long as it is capable of forming a chelate complex with the metal ion of interest, and the chelate complex is preferably stable under physiological conditions for a time long enough to carry out the intended diagnostic method. Preferred chelators or functional groups containing chelators include those described in Chem. Soc. Rev. 2014;43:260-290 (DOTA, B-DO2A, 3p-C-DEPA, TCMC, Oxo-DO3A, TETA, E2A, CB-TE2A, CB-TE1A1P, CB-TE2P, MM-TE2A, DM-TE2A, Diamsar, NOTA, NETA, and TACN-TM, DTPA, 1B4M-DTPA, CHX-A''-DTPA, AAZTA, DATA, H2dedpa, H4octapa, H2azapa, H5decapa, BCPA, CP256, YM103, DFO, PCTA, H6phospha, PCTA, HEHA, PEPA), bispidine (Dalton Trans. 2018;47: 9202-9220), radiohybrid ligands (described by Wurzer et al. in J. Nucl. Med. 2019, doi: 10.2967 / jnumed.119.234922), hydroxypyridinone ligands (described in Dalton Trans. 2019;48:4299-4313 or Bioconjugate Chem. 2015;26:2579-2591), picolinic acid-based chelators (described in Dalton Trans. 2017;46:14647-14658, Inorg. Chem. 2016;55:12544-12558, or Bioconjugate Chem. 2017;28:2145-2159), among others, fusarinine c (J. Label. Compd. Radiopharm. 2015;58:209-214), DOTPI (Chem. Eur. J.2013;19:7748-7757), DOTGA (described in Chem. Commun. 1998, 1381), NOTGA (described in Bioconjugate Chem. 2012;23:2229-2238), NODAPA (described in Bioorg. Med. Chem. Lett. 2008;18:5364-5367), DOTAZA (described in Chem. Asian J. 2014;9:2197-2204), HBED-CC (described in Eur. J. Nucl. Med. 1986;12:397-404), HBED-NN (described in J. Org. Chem. 2019;84:7501-7508), (NH2)2sar (Inorg. Chem. 2011;50:6701-6710), or TRAP (e.g., as described in Dalton Trans. 2015;44:11137). TRAP, its tetravalent homologs DOTPI and DOTAZA, and analogs and derivatives of these chelating groups are particularly preferred. Exemplary structures of these chelating groups are shown in formulae (IVa) to (IVd) below: [ka] (wherein the asterisk ( * ) indicates the position of attachment of the atomic group acting as a spacer. If the number of cyclopeptides and associated spacers (characterized by the variable n) is less than the number of valences of the chelating group, the remaining valences, represented by asterisks, are saturated with hydrogen or another atomic group, preferably a group selected from -CH2-COOH and -CH2-CH2-COOH.

[0058] Preparation of the conjugates of the present invention The conjugates of the present invention may be synthesized using standard materials and methods known in the art. When the conjugate is a chelate, the formation of the chelate is typically carried out as the final step. That is, a suitable procedure involves one or more steps to form a precursor, as described below, followed by reaction of the precursor with the atom, atomic group, or ion to be chelated. The final reaction is typically carried out under conventional conditions for such reactions known to those skilled in the art. In preferred circumstances, the reaction is carried out at ambient temperature (room temperature, e.g., 20-25°C). More preferably, the reaction is carried out at a temperature ranging from ambient temperature (room temperature) to 37°C.

[0059] The ions may be provided in the form of a salt, and the salt-forming counterion may be selected from the group consisting of sulfate, fluoride, chloride, bromide, nitrate, phosphate, carbonate, bicarbonate, sulfonate, acetate, and mixtures thereof. In another preferred embodiment, the ions are provided in the form of a solution.

[0060] The precursors are preferably prepared using a modular approach based on click chemistry to link a chelating group (or central moiety) to one or more cyclopeptide components. One or more spacers are formed in situ during the coupling reaction. The starting materials themselves contain precursors of the spacers at their termini, bearing functional groups suitable for click chemistry coupling.

[0061] Cyclopeptides bearing precursors of the spacer at the K(NMe) residue may be obtained by reacting the precursor with the respective precursor, which has a carboxyl group at the cyclopeptide bond terminus, and the precursor may be activated using, for example, HATU, HOBt, and DIPEA, and reacted with the respective cyclopeptide under standard amide coupling conditions, for example, as described in Maltsev OV, et al., Angew. Chem. Int. Ed. 2016;55:1535-1539 and / or WO 2017 / 046416 A1.

[0062] Cyclopeptides can be synthesized by applying appropriately adapted materials and procedures described in the literature, for example, in Maltsev OV, et al., Angew. Chem. Int. Ed. 2016;55:1535-1539 and / or WO 2017 / 046416 A1.

[0063] Specific Conjugates of the Invention Specific conjugates of the invention are shown below. The conjugates of the invention include non-radioactive metal ions or 68 These include both the corresponding conjugates that can be obtained by incorporating radionuclides such as Ga. [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0064] Constituent units of the present invention The present invention further relates to building blocks that can be used to obtain the conjugates of the present invention.

[0065] The first type of building blocks of the present invention are groups of compounds corresponding to the chelate complexes described above, but which do not have a coordination atom (such as Ga-68). These building blocks of the present invention have the following formula (IIa): Cg(SCp) n (IIa) wherein Cg represents a chelating group, S represents a group of atoms acting as a spacer, each Cp is a cyclopeptide independently selected from Tyr2, YRGD, and FRGD, and n is an integer from 1 to 4. All further information provided above for the corresponding coordination complexes applies equally to the building blocks of formula (IIa).

[0066] The present invention further relates to building blocks that are modified cyclopeptides that can be used in the early stages of a synthetic procedure for synthesizing the conjugates of the present invention and the building blocks described above by convenient click chemistry. Such building blocks include a cyclopeptide component selected from Tyr2, YRGD, and FRGD, a functional group that may participate in a click reaction (e.g., as specified, for example, in the January 24, 2020, version of the Wikipedia entry "Click Chemistry"), and an atomic group that links the cyclopeptide to the functional group via the terminal amino group of the side chain of the NMe-K residue.

[0067] These building blocks of the present invention have the following formula (V): Cp-L-Fg (V) wherein Cp represents a cyclopeptide selected from Tyr2, YRGD, and FRGD, L represents a linking group, and Fg represents a functional group for performing a click reaction.

[0068] The functional group is preferably an azide group, an alkyne group, especially containing a terminal ethyne group, a dibenzylcyclooctyne group, a trans-cyclooctene group, a tetrazine group, a dibenzocyclooctyne group, or a bicyclo[6.1.0]nonyne group.

[0069] The linking group typically contains a carbonyl group that forms an amide bond with the amino group on the side chain of the NMe-K residue. The linking group further contains a group of 1 to 15 atoms selected from C, N, and O that forms a linear chain between the amide bond and the functional group, which is optionally substituted with one or more substituents, and the remaining valences of the chain-forming atoms are saturated with hydrogen atoms. Preferably, the group is an alkylene group having 1 to 15, more preferably 2 to 6, methylene groups.

[0070] Formula BB-1 below illustrates this concept for a building block of the invention in which a Tyr2 cyclopeptide is linked to an azide functionality via a C4-alkylene group. [ka]

[0071] Further useful building blocks are represented by formulas BB-2 to BB-7 below. [ka]

[0072] BB-5a refers to structure BB-5, where X 1 and X 2 is hydrogen and n=2.

[0073] BB-6a refers to structure BB-6, where X is hydrogen and n=2.

[0074] BB-7a refers to structure BB-7, where X is hydrogen and n=2.

[0075] The Tyr2 cyclopeptide itself is novel and represents another building block of the present invention for obtaining the conjugates of the present invention described above and below. The same is true for the iodine-modified cyclopeptides Tyr2, FRGD, and YRGD. That is, further building blocks of the present invention are cyclo(3-I-YRGDLAYp(NMe)K); cyclo(3-I-YRGDLA3-I-Yp(NMe)K); cyclo(YRGDLA3-I-Yp(NMe)K); cyclo(3-I-YRGDLAFp(NMe)K); cyclo(FRGDLA3-I-Yp(NMe)K); where 3-IY represents a Tyr residue with an iodine atom at the 3-position of the phenyl ring, and the iodine atom can be any non-radioactive or radioactive isotope of iodine.

[0076] Peptide synthesis The cyclopeptides of the present invention can be synthesized using standard peptide methods, such as solid-phase peptide synthesis using Fmoc as a protecting group. Available techniques are described, for example, in J. Chatterjee, B. Laufer, H. Kessler, Nat. Protoc. 2012, 7, 432-444 and in WO 2017 / 046416A.

[0077] Cyclization of peptides can be achieved using standard techniques. For example, cyclization can be achieved on a solid support or in solution using HBTU / HOBt / DIEA, PyBop / DIEA, or PyClock / DIEA reagents. Available cyclization methods are described, for example, in WO 2017 / 046416A, J. Chatterjee, B. Laufer, H. Kessler, Nat. Protoc. 2012, 7, 432-444, and the references cited therein.

[0078] Synthesis of the conjugate The conjugates may be prepared by adapting methods described in the literature. 38,43,44,45

[0079] Diseases associated with cells with increased expression of αvβ6-integrin The conjugates of the present invention are useful for any disease associated with increased expression of αvβ6-integrin. Generally, the presence of αvβ6-integrin in tissue can be determined by immunohistochemistry (IHC). Application of this analytical technique to healthy adult tissue does not result in any αvβ6-integrin signal. Thus, for some embodiments of the present invention, tissue that produces a detectable IHC signal for αvβ6-integrin is considered to be tissue with increased expression of αvβ6-integrin. Any tissue that shows increased expression of αvβ6-integrin deviates from healthy adult tissue and may be due to diseases such as cancer, fibrosis, or Covid-19, or conditions such as early wounding that result in scar tissue formation. Any of these diseases and conditions may be confirmed using the conjugates of the present invention. Such diseases are described in the literature. 41,42

[0080] These diseases include cancers, especially non-small cell lung cancer (NSCLC), pancreatic cancer, cholangiocarcinoma, gastric cancer, breast cancer, head and neck squamous cell, basal cell, colon cancer, and ovarian cancer (Niu J, Li Z, Cancer Lett. 2017;403:128e137), and cancers of the upper aerodigestive tract, especially pancreatic ductal adenocarcinoma (PDAC) (Sipos et al., Histopathol. 2004;45:226, Reader CS, et al., J. Pathol. 2019;249:332, Steiger K, et al., Mol. Imaging 2017;16:1536012117709384). Of particular interest are lung adenocarcinoma, breast cancer, colon adenocarcinoma, pancreatic adenocarcinoma (PDAC), and head and neck squamous cell carcinomas, including oral squamous cell carcinoma, laryngeal squamous cell carcinoma, oropharyngeal squamous cell carcinoma, nasopharyngeal squamous cell carcinoma, and hypopharyngeal squamous cell carcinoma.

[0081] Using IHC, αvβ6 expression in fibrotic tissues has also been demonstrated (Munger CS, et al., Cell 1999;96:319). Additional diseases therefore include fibrosis, particularly biliary, renal, endomyocardial fibrosis, Crohn's disease, arthrofibrosis, and pulmonary fibrosis. Of particular interest is idiopathic pulmonary fibrosis (IPF).

[0082] Quantification of αvβ6-integrin in lung tissue (1) Stratification of patients eligible for inhaled therapy with αvβ6-blocking molecules (e.g., GSK3008348) and (2) assessing the therapeutic outcomes of such therapies (PT Lukey et al., European Journal of Nuclear Medicine and Molecular Imaging (2020) 47:967-979, https: / / doi.org / 10.1007 / s00259-019-04586-z; AE John et al., Nature Communications (2020)11:4659, https: / / doi.org / 10.1038 / s41467-020-18397-6, and TM Maher et al., Respiratory Research (2020) 21:75, https: / / doi.org / 10.1186 / s12931-020-01339-7) was identified as a potentially beneficial method. Thus, the present invention may be particularly suited to this and related fields of application.

[0083] Recent studies have suggested the expression of αβ in lung tissue affected by COVID-19 (Foster CC, et al., J. Nucl. Med. 2020;61:1717). The radiolabeled compounds of the present invention are therefore suitable for in vivo imaging of post-COVID-19 syndrome in patients.

[0084] Because αvβ6-integrin is an activator of transforming growth factor beta (TGF-beta), any disease associated with abnormal TGF-beta levels in the intracellular space or with impaired TGF-beta response in a particular cell type resulting from alterations in the TGF-beta signaling pathway may be associated with increased αvβ6-integrin expression. Such diseases may be diagnosed by determining the αvβ6-integrin expression status of cells in affected tissue. Of particular interest is the use of diagnostic procedures based on determining the density of αvβ6-integrin expression in tissues to determine therapeutic strategies involving the use of therapeutic agents, particularly antibodies, targeting the TGF-beta signaling pathway, particularly TGF-beta itself, either in its free form or in complex with latent-associated peptides.

[0085] Increased αvβ6 expression can be exploited for in vivo targeting using radiolabeled compounds such as those of the present invention.

[0086] Use for imaging and / or as a diagnostic agent The conjugate of the present invention is suitable for use as a diagnostic agent. The conjugate of the present invention is advantageously used, and the effector component contains an active atom or active atomic group suitable for the imaging / diagnostic method of interest, as described above. Depending on the selected imaging / diagnostic method, a suitable active atom or active atomic group is selected. The selected imaging / diagnostic method also determines the dosage, form, and administration timing of the conjugate of the present invention.

[0087] The conjugates of the present invention are suitable for virtually any analytical / diagnostic method involving the use of a diagnostic agent. The conjugates of the present invention are particularly suitable for imaging methods such as gamma scintigraphy, fluorescence-based imaging, positron emission tomography (PET), single photon emission computed tomography (SPECT), magnetic resonance tomography (MRT), optical or magnetic resonance imaging (MRI), X-ray-based CT imaging, scintigraphy, Cerenkov imaging, ultrasonography, thermography, and combinations thereof.

[0088] The conjugates of the present invention may be used by applying techniques described in the literature. 33,34,35,38 Accordingly, the present invention provides a method for imaging a patient, such as a cancer patient, a fibrosis patient, or a patient affected by a Covid-19 infection, including post-COVID-19 syndrome, the method comprising administering to the patient a conjugate of the invention, followed by subjecting the patient to an imaging method selected from gamma scintigraphy, fluorescence-based imaging, positron emission tomography (PET), single photon emission computed tomography (SPECT), magnetic resonance tomography (MRT), optical imaging or magnetic resonance imaging (MRI), X-ray based CT imaging, scintigraphy, Cerenkov imaging, ultrasonography, thermography, and combinations thereof, wherein the active atom or active atomic group is suitable for the selected imaging method, and the selected imaging method detects a signal attributable to the active atom or active atomic group.

[0089] Therapeutic use Conjugates of the invention having an effector moiety with an active atom or active atomic group derived from a drug can be used in the treatment of diseases associated with upregulation of αvβ6-integrin, for example, as listed above.

[0090] The conjugates of the present invention may be administered to a patient, for example, intravenously, transmucosally, transdermally, or intranasally. Suitable dosages may range from 0.1 to 1000 mg / day, preferably 0.1 to 10 mg / day. The conjugates of the present invention may be administered once daily, twice daily, three times daily, or for any period of time, which may be interrupted by one or more periods during which the compound of the present invention is not administered.

[0091] The conjugates of the present invention may also be used as components in combination therapy. The conjugates of the present invention may be combined with one or more other therapeutic agents effective in the treatment of cancer, such as those listed above and / or below. Such combination therapy may be carried out by administering the two or more therapeutic agents simultaneously or sequentially.

[0092] The conjugates of the present invention, in particular for targeted radiotherapy, e.g. 47 Sc, 67 Cu, 177 Lu, 90 Y, 213 Bi, 225 Ac, 161 Tb, 149 Tb, or 131 It is also possible to use conjugates that incorporate alpha- or beta-emitting radionuclides, such as I.

[0093] The conjugates of the present invention may also be used for the diagnosis or treatment of fibrosis. For such purposes, the conjugates of the present invention may be used by any suitable administration route, including intravenous, intraarterial, transmucosal, pulmonary, and intranasal administration. The dosage and administration schedule may be the same as those specified above for the treatment of cancer. Combination therapy is also possible, in which one or more other therapeutic agents are selected from other therapeutic agents suitable for the treatment of fibrosis, as cited above by cross-reference to the review article by Gharaee-Kermani et al., which is incorporated herein by reference. The conjugates of the present invention and one or more other therapeutic agents may be administered simultaneously or sequentially.

[0094] The conjugates of the present invention may also be used for the diagnosis or treatment of Covid-19 infection, including post-COVID-19 syndrome. For such purposes, the conjugates of the present invention may be administered by any suitable route, including intravenous, intraarterial, transmucosal, pulmonary, and intranasal administration. The dosage and administration schedule may be the same as those specified above for the treatment of cancer. Combination therapy is also possible, in which the one or more other therapeutic agents are selected from other therapeutic agents suitable for the treatment of Covid-19 infection, such as immunotherapy, such as dexamethasone or remdesivir. The conjugates of the present invention and the one or more other therapeutic agents may be administered simultaneously or sequentially.

[0095] Thus, the present invention provides a method for treating a patient suffering from a disease associated with increased αvβ6 integrin expression, in particular cancer, fibrosis or Covid-19 infection, the method comprising administering to the patient a conjugate of the present invention, wherein the active atom or active atom group is derived from a therapeutic agent selected to be suitable for treating the respective disease, for example as specified under item (b-6) in the effector moiety section above.

[0096] Uses for drug targeting and in biomolecular research The conjugates of the present invention may also be used for drug targeting and in biomolecular research. These uses may be carried out as described in the respective sections of WO 2017 / 046416A. In particular, the conjugates of the present invention, preferably containing a Tyr2 peptide sequence, can be covalently or noncovalently incorporated into nanocarriers such as nanoparticles, liposomes, or micelles to enable the peptide component to bind to target cells, thereby increasing the local concentration of the nanoparticles, typically containing a drug. This approach is particularly interesting for the treatment of cancer, particularly carcinomas, with chemotherapeutic drugs, as it may achieve "homing" to such αvβ6-expressing tissues.

[0097] Pharmaceutical Composition The conjugate of the present invention may be formulated as a pharmaceutical composition. This can be done using conventional means and methods for peptide-based medicines. Suitable literature is detailed, for example, in the section on pharmaceutical compositions of WO 2017 / 046416A. These disclosures are incorporated herein by reference. The pharmaceutical composition of the present invention may also comprise the nanoparticles described in the previous section. According to a preferred embodiment, such nanoparticles not only comprise the conjugate of the present invention and the nanoparticles themselves, but also additionally comprise a therapeutic agent, preferably a chemotherapeutic agent, within the nanoparticles. [Example]

[0098] Example Materials and Methods Abbreviation CuAAC = copper-catalyzed azide-alkyne cycloaddition, Dde = 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-ethyl, DIAD = diisopropyl azodicarboxylate, DIPEA = N,N-diisopropylamine, DMF = dimethylformamide, DPPA = diphenylphosphoryl azide, Fmoc = 9-fluorenylmethoxycarbonyl, HATU = N,N,N',N'-tetramethyluronium hexafluorophosphate, HFIP = 1,1,1,3,3,3-hexafluoro-2-propanol , HOBt = 1-hydroxybenzotriazole hydrate, NMP = N-methyl-2-pyrrolidone, NOTA = 1,4,7-triazacyclononane-1,4,7-triacetic acid, Pbf = 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl, PBS = phosphate-buffered saline, PPh3 = triphenylphosphine, tBu = tert-butyl, TFA = trifluoroacetic acid, THF = tetrahydrofuran, TIPS = triisopropylsilane, TRAP = 1,4,7-triazacyclononane-1,4,7-tris[methylene(2-carboxyethylphosphonic acid)]

[0099] overview Unless otherwise noted, all commercially available reagents and solvents were of analytical grade and used without further purification. Protected amino acids were purchased from IRIS Biotech (Germany). Cu(OAc)2·H2O, 4-pentynoic acid, diisopropylamine (DIPEA), and sodium ascorbate were purchased from Sigma Aldrich (Darmstadt, Germany). 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) was purchased from Chematech (Dijon, France). HATU was purchased from Bachem Holding AG (Bubendorf, Switzerland). HOBt hydrate was purchased from Carbolution (St. Ingbert, Germany). TRAP(azide)1 38 and TRAP(azide)3 43was synthesized as previously described. Semi-preparative reversed-phase HPLC was performed using Waters systems: Waters 2545 (Binary Gradient Module), Waters SFO (System Fluidics Organizer), Waters 2996 (Photodiode Array Detector), and Waters 2767 (Sample Manager). Separations were performed using a Dr. Maisch C18-column: Reprosil 100 C18, 5 μm, 150 × 30 mm (column 1), water (0.1% v / v trifluoroacetic acid) and acetonitrile (0.1% v / v trifluoroacetic acid) at a flow rate of 40 mL / min or a YMC C18-column: YMC-Pack ODS-A, 5 μm, 250 × 20 mm (column 2), water (0.1% v / v trifluoroacetic acid) and acetonitrile (0.1% v / v trifluoroacetic acid) at a flow rate of 16 mL / min. Analytical HESI-HPLC-MS (heated electrospray ionization mass spectrometry) was performed on an LCQ Fleet (Thermo Scientific) equipped with an UltiMate 3000 UHPLC focused (Dionex) connected to a C18-column: S1: Hypersil Gold aQ S1: 175 Å, 3 μm, 150 × 2.1 mm (for 8 or 20 min measurements); S2: Accucore C18, 80 Å, 2.6 μm, 50 × 2.1 mm (for 5 min measurements) (Thermo Scientific). A linear gradient (5% to 95% acetonitrile content) of water (0.1% v / v formic acid) and acetonitrile (0.1% v / v formic acid) was used as the eluent. The affinity and selectivity of the integrin ligands were determined by solid-phase binding assays, applying a previously described protocol. 44 Compounds containing metal-binding units (chelators, e.g., TRAP) can be converted to Ga by the addition of an equimolar amount of aqueous Ga(NO3)3. III It was previously converted to the complex.

[0100] Example 1: Peptide synthesis procedure The previously established protocol was followed, except that the synthesis was carried out in DMF instead of N-methyl-2-pyrrolidone (NMP). 44

[0101] Loading of CTC resin. Peptide synthesis was carried out using CTC resin (0.9 mmol / g) according to the standard Fmoc-protected peptide procedure. Fmoc-Xaa-OH (1.5 eq.) was attached to the CTC resin with N,N-diisopropylamine (DIPEA, 2.5 eq.) in anhydrous DCM (0.8 mL / g resin) for 1 h at rt. Capping of the remaining trityl-chloride groups was carried out by adding a solution of MeOH (1 mL / g resin) and DIPEA (5:1, v / v) for 15 min. The resin was filtered and washed with DCM (5x) and MeOH (3x).

[0102] On-resin Fmoc-deprotection. The Fmoc-protected peptidyl-resin was treated with 20% piperidine in DMF (v / v) for 10 min and an additional 5 min. The resin was washed with DMF (5x).

[0103] Standard amino acid coupling. A solution of Fmoc-Xaa-OH (2 eq.), HATU (2 eq.), HOBt (2 eq.), and DIEA (3 eq.) in DMF (1 mL / g resin) was added to the free aminopeptidyl-resin and shaken at rt for 1 h. The solution was washed with DMF (5x). All couplings were monitored by analytical RP-HPLC and MS. A small amount of resin was dissolved in a solution of 20% HFIP in DCM, followed by a small amount of MeOH and MeCN. The solution was filtered and analyzed by RP-HPLC and MS.

[0104] On-resin N-methylation. The linear Fmoc-deprotected peptide was treated with a solution of 2-nitrobenzenesulfonyl chloride (o-Ns-Cl, 4 eq.) and 2,4,6-collidine (10 eq.) for 20 min at rt. The resin was washed with DCM (3x) and THF (5x). A solution of triphenylphosphine (PPh3, 5 eq.) in anhydrous MeOH and a minimal amount of diisopropyl azodicarboxylate (DIAD, 5 eq.) in THF were prepared and added to the resin. The resin solution was shaken for 15 min before washing with THF (5x) and DMF (5x).

[0105] Cleavage of the linear peptide from the resin. The peptidyl-resin was treated with a solution of 20% HFIP in DCM (3 x 30 min) to ensure total cleavage of the peptide from the resin before solvent evaporation under pressure.

[0106] Cyclization of linear peptides. The peptides were dissolved in DMF (1 mM peptide concentration) before the addition of NaHCO (5 eq.) and DPPA (3 eq.). The reaction was carried out overnight at room temperature with stirring, and the cyclization was monitored by RP-HPLC and MS. The solvent was evaporated under pressure to a small volume, filtered through glass wool, and solvent evaporation was continued.

[0107] Cleavage of the Dde-protecting group. The cyclized peptide was dissolved in DMF before the addition of hydrazine hydrate (2% v / v). The reaction was carried out at rt for 30 min with stirring. Dde-deprotection was monitored by HPLC-MS.

[0108] Cleavage of acid-labile protecting groups. The cyclized peptide was dissolved in a 10:85:2.5:2.5 (DMF:TFA:TIPS:HO) solution for 1 h. Deprotection was monitored by HPLC-MS. [ka]

[0109] Synthesis of Y(tBu)R(tBu,Fmoc)GD(Pbf)LAY(tBu)p(NMe)K(Dde). The linear protected peptide Y(tBu)R(tBu,Fmoc)GD(Pbf)LAY(tBu)p(NMe)K(Dde) was synthesized according to the procedure described above. The formation of the entire linear sequence was monitored by HPLC-MS (m / z: 1903.00 [M+H + ] + ,952.08[M+2H + ] 2+ ). [ka]

[0110] Synthesis of cyclo(Y(tBu)R(Pbf)GD(tBu)LAY(tBu)p(NMe)K(Dde)). The cyclic protected peptide cyclo(Y(tBu)R(Pbf)GD(tBu)LAY(tBu)p(NMe)K(Dde)) was synthesized according to the procedure described above. The cyclization was carried out without any prior HPLC purification of the linear peptide. The formation of the cyclized peptide was monitored by HPLC-MS (m / z: 1663.17 [M+H + ] + ,832.08[M+2H + ] 2+ ). [ka]

[0111] Synthesis of Tyr2. Cleavage of the Dde protecting group from cyclo(Y(tBu)R(Pbf)GD(tBu)LAY(tBu)p(NMe)K(Dde)) was carried out as described above. cyclo(Y(tBu)R(Pbf)GD(tBu)LAY(tBu)p(NMe)K) was obtained as a white solid in 35% (508.7 mg, 339.4 μmol) yield (relative to the resin loading capacity). RP-HPLC (gradient: 20–60% MeCN in HO containing 0.1% TFA, 25 min): R= 10.35 min (column 1). Immediately after Dde-deprotection, 78 mg of the crude material was dissolved in toluene (50 mL) and rotary evaporated to remove any reagents from the Dde-deprotection. This resulted in an orange / brown oil, which was directly treated with 2 mL of the acid-labile deprotection solution described above. The cyclic peptide Tyr2[cyclo(YRGDLAYp(NMe)K)] was obtained as a colorless solid in 10.2% (5.75 mg, 5.33 μmol) yield (relative to the crude product). RP-HPLC (gradient: 20-70% MeCN in HO containing 0.1% TFA, 25 min): R = 10.07 min (column 1). m / z: 540.14 [M+2H + ] 2+ . [ka]

[0112] Synthesis of BB-5a. 4-Pentynoic acid (2.38 mg, 24.23 μmol, 1.2 eq), HATU (9.21 mg, 24.23 μmol, 1.2 eq), HOBt (3.3 mg, 24.23 μmol, 1.2 eq), and DIPEA (10.29 μL, 60.59 μmol, 3 eq) were dissolved in a minimum amount of DMF and reacted for 15 min before being added dropwise to a solution of the dissolved Dde-deprotected peptide bearing an acid-labile protecting group in DMF (30.27 mg, 20.19 μmol, 1 eq). The reaction was allowed to proceed with stirring for 1 h. Conjugation of the alkyne functionality was monitored by HPLC-MS. The solvent was evaporated under pressure, resulting in an orange / brown oil, which was directly treated with 2 mL of the acid-labile deprotection solution described above. Cyclo(YRGDLAYp(NMe)K(pentynoic acid)), BB-5a, was obtained as a colorless solid in 57% (13.26 mg, 11.45 μmol) yield. RP-HPLC (gradient: 30–50% MeCN in HO containing 0.1% TFA, 15 min): R = 7.67 min (column 1). m / z: 1737.30 [3M+2H + ] 2+ ,1158.51[M+H+ ] + ,580.05[M+2H + ] 2+ [ka]

[0113] Synthesis of BB-6a. 4-Pentynoic acid (7.63 mg, 77.79 μmol, 1.5 eq), HATU (23.66 mg, 62.23 μmol, 1.2 eq), HOBt (9.53 mg, 62.23 μmol, 1.2 eq), and DIPEA (27.1 μL, 155.58 μmol, 3 eq) were dissolved in a minimal amount of DMF and allowed to react for 15 min before being added dropwise to a solution of the dissolved Dde-deprotected YRGD peptide (73.99 mg, 51.86 μmol, 1 eq) bearing acid-labile protecting groups in DMF. The solvent was evaporated under pressure, resulting in an orange / brown oil, which was directly treated with 3 mL of the acid-labile deprotection solution described previously. C-9 was obtained as a colorless solid in 76% yield (45 mg, 39.39 μmol). RP-HPLC (gradient: 30–80% MeCN in HO containing 0.1% TFA, 20 min): R = 9.4 min (column 1). m / z: 1164.41 [M+Na + +H + ] + ,1142.46[M+H + ] + ,572.11[M+2H + ] 2+ . [ka]

[0114] Synthesis of BB-7a. 4-Pentynoic acid (3.05 mg, 31.12 μmol, 1.5 eq), HATU (9.47 mg, 24.9 μmol, 1.2 eq), HOBt (3.81 mg, 24.9 μmol, 1.2 eq), and DIPEA (10.84 μL, 62.24 μmol, 3 eq) were dissolved in a minimal amount of DMF and allowed to react for 15 min before being added dropwise to a solution of the dissolved Dde-deprotected FRGD peptide (29.6 mg, 20.75 μmol, 1 eq) bearing acid-labile protecting groups in DMF. The solvent was evaporated under pressure, resulting in an orange / brown oil, which was directly treated with 2 mL of the acid-labile deprotection solution described previously. C-8 was obtained as a colorless solid in 28.2% yield (6.68 mg, 5.85 μmol). RP-HPLC (gradient: 30–80% MeCN in HO containing 0.1% TFA, 20 min): R = 8.9 min (column 1). m / z: 1165.09 [M+Na + +H + ] + ,1142.47[M+H + ] + ,572.21[M+2H + ] 2+ .

[0115] Synthesis of C-1. Cyclo(YRGDLAYp(NMe)K(pentynoic acid)) (8.01 mg, 6.92 μmol, 1.5 eq) was added to a solution of TRAP(azide) 1 (3.05 mg, 4.61 μmol, 1 eq) and sodium ascorbate (45.7 mg, 230.5 μmol, 50 eq) in a minimum amount of HO. Copper(II) acetate (1.1 mg, 5.53 μmol, 1.2 eq) was added, and a brown precipitate immediately formed. Upon vortexing, the solution turned clear green. The solution was allowed to react at 60 °C for 1 h without stirring. After 1 h, Cu demetallation of the peptidyl-chelator compound was carried out by adding 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) (41.94 mg, 138.26 μmol, 30 eq.) dissolved in water (1 mL), the pH of which was adjusted to 2.2 by adding 1 M aqueous HCl. The mixture was reacted at 60 °C for 1 h. The synthesis of TRAP(Tyr2) was monitored by HPLC-MS. C-1 was obtained as a colorless solid in 5.7% yield (0.48 mg, 0.26 μmol). RP-HPLC (gradient: 20–70% MeCN in HO containing 0.1% TFA, 25 min): R = 12.3 min (column 1). m / z: 910.49 [M+2H + ] 2+ ,607.73[M+3H + ] 3+ .

[0116] Synthesis of C-7. Cyclo(YRGDLAYp(NMe)K(pentynoic acid)) (24.96 mg, 21.55 μmol, 3.3 eq) was added to a solution of TRAP(azide) 3 (5.39 mg, 6.53 μmol, 1 eq) and sodium ascorbate (64.7 mg, 326.6 μmol, 50 eq) in a minimal amount of HO. Upon addition of copper(II) acetate (1.56 mg, 7.84 μmol, 1.2 eq), a brown precipitate immediately formed. Upon vortexing, the solution turned clear green. The solution was allowed to react at 60 °C for 1 h without stirring. After 1 h, Cu demetallation of the peptidyl-chelator compound was carried out by adding 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) (39.6 mg, 130.6 μmol, 20 eq.) dissolved in water (1 mL), the pH of which was adjusted to 2.2 by adding 1 M aqueous HCl. The mixture was reacted at 60 °C for 1 h. The synthesis of TRAP(Tyr2)3 was monitored by HPLC-MS. C-7 was obtained as a colorless solid in 36.1% yield (10.11 mg, 2.35 μmol). RP-HPLC (gradient: 20–40% MeCN in HO containing 0.1% TFA, 15 min, followed by a 6 min wash phase (100% MeCN):t R = 17.35 min (column 2). m / z: 1434.01 [M+3H + ] 3+ ,1075.97[M+4H + ] 4+ ,861.03[M+5H + ] 5+ .

[0117] Synthesis of C-8. BB-7a (6 mg, 5.25 μmol, 3.3 eq) was added to a solution of TRAP(azide) 3 (1.3 mg, 1.6 μmol, 1 eq) and sodium ascorbate (15.8 mg, 79.6 μmol, 50 eq) in a minimal amount of 4:1 HO:tBuOH. Upon addition of copper(II) acetate (381.3 μg, 1.91 μmol, 1.2 eq), a brown precipitate immediately formed. Upon vortexing, the solution turned clear green. The solution was allowed to react at 60 °C for 1 h without stirring. After 1 h, the formation of C-8 was monitored by HPLC-MS. Cu removal of the peptidyl-chelator compound was achieved by the addition of 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) (14.5 mg, 47.8 μmol, 30 eq.) dissolved in water (0.5 mL) adjusted to pH 2.2. The mixture was reacted at 60 °C for 1 h. C-8 was obtained as a colorless solid in 42.9% yield (2.9 mg, 0.7 μmol). RP-HPLC (gradient: 10–70% MeCN in HO containing 0.1% TFA, 20 min): R = 19.2 min (column 1). m / z: 1426.38 [M+Na + +3H + ] 3+ ,1070.15[M+Na + +4H + ] 4+ ,856.34[M+Na + +5H + ] 5+ ,713.74[M+Na + +6H + ] 6+ .

[0118] Synthesis of C-9. BB-6a (45 mg, 39.39 μmol, 3.3 eq) was added to a solution of TRAP(azide) 3 (9.86 mg, 11.94 μmol, 1 eq) and sodium ascorbate (118.24 mg, 596.9 μmol, 50 eq) in a minimal amount of 4:1 HO:tBuOH. Upon addition of copper(II) acetate (2.86 mg, 14.32 μmol, 1.2 eq), a brown precipitate immediately formed. Upon vortexing, the solution turned clear green. The solution was allowed to react at 60 °C for 1 h without stirring. After 1 h, the formation of C-9 was monitored by HPLC-MS. Cu removal of the peptidyl-chelator compound was achieved by the addition of 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) (110.7 mg, 365 μmol, 30 eq.) dissolved in water (1 mL) adjusted to pH 2.2. The mixture was reacted at 60 °C for 1 h. C-9 was obtained as a colorless solid in 24.7% yield (12.78 mg, 3.01 μmol). RP-HPLC (gradient: 10–70% MeCN in HO containing 0.1% TFA, 20 min): R = 19.5 min (column 1). m / z: 1426.11 [M+Na + +3H + ] 3+ ,1070.11[M+Na + +4H + ] 4+ ,856.38[M+Na + +5H + ] 5+ ,713.68[M+Na + +6H + ] 6+

[0119] Synthesis of C-10 and C-11 building blocks AvB6 (Maltsev et al. 38To a solution of TRAP(azide)3 (4.46 mg, 5.4 μmol, 1 eq) and sodium ascorbate (53.47 mg, 269.90 μmol, 50 eq) in a minimum amount of HO, copper(II) acetate (1.29 mg, 6.48 μmol, 1.2 eq) was added, and a brown precipitate immediately formed. Upon vortexing, the solution turned clear green. The solution was reacted at 60 °C for 1 h without stirring. BB-5a (13.75 mg, 11.87 μmol, 2.2 eq) was added directly to the reaction mixture, and the reaction was continued at 60 °C for an additional 1 h without stirring. After 1 h, Cu demetallation of the peptidyl-chelator compound was carried out by adding 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) (48.62 mg, 160.31 μmol, 30 eq.) dissolved in water (1 mL) adjusted to pH = 2.2. The mixture was reacted at 60 °C for 1 h. The formation of C-11 and C-10 was monitored by HPLC-MS.

[0120] C-10 was obtained as a colorless solid in 6.8% (1.55 mg, 0.37 μmol) yield. RP-HPLC (gradient: 40–95% MeCN in HO containing 0.1% TFA, 30 min): R = 10.6 min (column 1). m / z: 1424.0 [M+3H + ] 3+ ,1067.9[M+2H + ] 4+ ,854.8[M+4H + ] 5+ .

[0121] C-11 was obtained as a colorless solid in 8.75% (2 mg, 0.47 μmol) yield. RP-HPLC (gradient: 40–95% MeCN in HO containing 0.1% TFA, 30 min): R = 14.9 min (column 1). m / z: 1413.2 [M+3H + ] 3+ ,1059.9[M+2H + ] 4+ ,848.2[M+4H + ]5+ .

[0122] Radiochemistry Radiometal incorporation and radiochemical purity of labeled compounds were determined by radio-TL on ITLC silica-impregnated chromatography paper (Agilent, Santa Clara, USA; eluent: 1:1 (v / v) mixture of 0.1 M trisodium citrate or 1 M ammonium acetate and methanol) and analyzed using a scan-RAM radio-TLC detector from LabLogic systems Inc. (Brandon, USA). 68 Ga-labeling was performed using a fully automated on-site system (GallElut from Scintomics, Lindach, Germany) as previously described. + ). 45 In short, it has a SnO2 matrix 68 Ge / 68 Eluent for Ga generator (IThemba LABS, SA; 1.25 mL, eluent: approximately 500 MBq 68 A 1 M aqueous HCl solution containing Ga was adjusted to pH 2 by adding HEPES buffer (450 μL, 2.7 M) and applied for labeling of 5 nmol of chelator conjugate at 95°C for 2 min. Radiolabeled peptide was captured on a Sep Pak® C8 light solid-phase extraction (SPE) cartridge purged with water (10 mL). The product was eluted with 2 mL aqueous EtOH (50%). After evaporation of the ethanol, purity was determined by radio-TLC and was always found to be ≥ 98%.

[0123] Example 2: Radioactivity assessment Determination of log D values n-octanol-PBS partition coefficient (log D 7.4For the determination of ), 500 μL 1-octanol and 500 μL phosphate-buffered saline were combined in a 1.5 mL Eppendorf tube. Approximately 1 MBq of radiolabeled compound was added and vigorously vortexed for 3 minutes. The sample was centrifuged (13,000 rpm, 5 minutes), and the radioactivity in 200 μL of the organic phase and 20 μL of the aqueous phase was quantified in a γ-counter.

[0124] Cell lines and animal models All animal experiments were performed in accordance with general animal welfare regulations in Germany and the institutional guidelines for the care and use of animals. H2009 human lung adenocarcinoma cells (CRL-5911; American Type Culture Collection) were cultured as recommended by the vendor. To generate tumor xenografts, 6- to 8-week-old female CB17 SCID mice (Charles River) were cultured with 1000 cells / mL of Matrigel (CultrexBME, type 3 PathClear; Trevigen, GENTAUR GmbH). 7 Mice were inoculated with H2009 cells. Mice were used for biodistribution or PET studies once tumors had grown to a diameter of 10–12 mm (4–6 weeks after inoculation).

[0125] PET imaging Mice were anesthetized with isoflurane for intravenous administration of radiolabeled compounds. The administered radioactivity per mouse ranged between 10 and 15 MBq (100–200 pmol, depending on the production and timing of administration). PET imaging was performed on a Siemens Inveon small-animal PET system under isoflurane anesthesia for 90 min either dynamically or as a single frame at 75 min pi with a 20 min acquisition time. Data were reconstructed using the Siemens Inveon Research Workspace software with the three-dimensional ordered subset expectation maximum (OSEM3D) algorithm without scatter correction or attenuation correction. For kinetic analysis, regions of interest (ROIs) were manually defined.

[0126] Distribution in the body For biodistribution studies, 3–6 MBq (between 70–140 pmol) of radiolabeled compound was injected into the tail vein. Mice were sacrificed 90 min after injection, blood samples were collected, and organs of interest were dissected. Quantification of radioactivity in weighed tissue samples was performed using the 2480 WIZARD. 2 The radioactivity administered per gram of tissue (%ID / g) was calculated from organ weights and counted radioactivity using an automatic γ counter (PerkinElmer, Waltham, USA).

[0127] result The novel peptide compounds and conjugates were synthesized and characterized as described above.

[0128] Phe2 and Tyr2 68 Ga-labeled trimeric conjugate, Ga-68-TRAP(Phe2)3 38and Ga-68-C-7 were evaluated in H2009 tumor-bearing mice. Comparison of PET images (Figure 1) shows that low background radioactivity and clear tumor delineation are achieved with Ga-68-C-7 but not with Ga-68-TRAP(Phe2)3, primarily due to robust uptake in the liver. Corresponding ex vivo biodistribution data (Figure 2) demonstrate high levels of accumulation of Ga-68-TRAP(Phe2)3 in the liver. This uptake is not reduced by co-injection of a large excess (50 nmol) of unlabeled TRAP(Phe2)3 (blocker), demonstrating that Ga-68-TRAP(Phe2)3 is not target-specific. Surprisingly, substitution of Phe with Tyr in Ga-68-C-7 reduced this nonspecific uptake to insignificant levels and also reduced nonspecific uptake in other compartments and tissues, i.e., blood, heart, spleen, and tumor, ultimately resulting in excellent PET image contrast, as shown in Figure 1.

[0129] Although pharmacokinetic analysis (Figure 3) shows good tumor retention of both compounds, Ga-68-C-7 is cleared from the blood pool much more rapidly, ultimately resulting in lower background in PET images, as depicted in Figure 1.

[0130] In summary, Ga-68-C-7 exhibits significantly improved biodistribution and imaging properties compared to the corresponding state-of-the-art compound, Ga-68-TRAP(Phe2)3. 38 , demonstrating that Tyr2 is advantageously used in αvβ6-integrin targeting compounds for in vivo applications.

[0131] Contains various combinations of Phe2, FRGD, YRGD, and Tyr2 68The biodistribution of Ga-labeled trimeric TRAP conjugates, i.e., Ga-68-TRAP(Phe2)3, Ga-68-C-7, Ga-68-C-8, Ga-68-C-9, Ga-68-C-10, and Ga-68-C-11, was determined in H2009 tumor-bearing mice. Figure 4 shows that the exchange of one Phe2 for a Tyr2 in the structure of Ga-68-TRAP(Phe2)3, resulting in Ga-68-C-11, significantly reduced nonspecific liver uptake (as evidenced by the similarity of the control vs. blocker experiments), reduced residual radioactivity in the blood, and reduced pancreatic uptake, while Ga-68-C-10 still exhibited high tumor uptake. Replacement of two Phe2 residues in the Ga-68-TRAP(Phe2)3 structure with Tyr2 residues, yielding Ga-68-C-10, had a similar but more pronounced effect. Similarly, replacement of all Phe2 residues in the Ga-68-TRAP(Phe2)3 structure with FRGD or YRGD residues, yielding Ga-68-C-8 and Ga-68-C-9, respectively, demonstrated that cyclopeptides containing only one tyrosine also exhibited superior properties. Of all the investigated trimeric conjugates, Ga-68-C-7 exhibited the best tumor-to-liver and especially tumor-to-pancreas ratios, suggesting that it should be most suitable for imaging αββ-integrin-positive lesions in organs, such as pancreatic adenocarcinoma metastases or primary tumors.

[0132] Figure 5 confirms that the peptides FRGD and YRGD, featuring Ga-68-C-8 and Ga-68-C-9, respectively, are also suitable for the synthesis of targeted radiolabeled molecules with significantly lower liver uptake than Ga-68-TRAP(Phe2)3. Accordingly, Figure 6 shows that the blood clearance of Ga-68-C-8 and Ga-68-C-9 is significantly faster than Ga-68-TRAP(Phe2)3 and similar to Ga-68-C-10.

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Claims

1. A conjugate represented by formula (I): E(Cp) ｎ (I) wherein each Cp is Tyr ２ wherein n is an integer selected from 1 to 4, and E represents an effector moiety, said effector moiety being covalently attached to the cyclopeptide via the terminal amino group of the (NMe)K residue, said effector moiety containing an active atom or active atomic group suitable for the diagnosis, imaging, or treatment of a medical indication associated with increased expression of αvβ6-integrin. or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

2. The conjugates may be of the following structural group: E (Tyr ２ ) １ , E(Tyr ２ ) ２ , E(Tyr ２ ) ３ , E(Tyr ２ ) ４ 2. The conjugate of claim 1, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, selected from:

3. The conjugates of formula (I) may be represented by the following formulae (Ia), (Ia'), (Ib) to (If): Aa(Cg)(SCp) ｎ (Ia) Aa’(Cg) ｋ (SCp) ｎ (Ia’) Aa (Cg) ｋ (SCp) ｎ’ (SAa') (Ib) Aa’(Cm)(SCp) ｎ (Ic) (Cm)(SCp) ｎ－ｏ (S(Aa’) ｐ (Cp) ｍ ) ｏ (Id) (Cm)(SCp) ｎ－ｏ (SCp(Aa’) ｐ ) ｏ (Ie) Cp(Aa’) ｐ (If) wherein Aa represents an active atom or an active atomic group capable of forming a chelate complex, Aa' represents an active atom or an active atomic group capable of forming a covalent bond, Cg represents a chelating group, k is 1 or 0, S represents an atomic group acting as a spacer, n is as defined above with respect to formula (I) with the proviso that n is 1 when k is 0, o is any integer from 1 to n, p is 1 or 2, m is 0 or 1, n' is 1, 2, or 3 with the proviso that n'+1 is equal to or less than the number of free valences of the chelating group, and Cm is a central moiety comprising 1 to 30 atoms selected from C, N, O, S, and P.

3. The conjugate of claim 1 or 2, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, characterized by a formula selected from:

4. 4. The conjugate of claim 1, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein the active atom or active group is selected from: a radioisotope suitable for scintigraphy, SPECT, or PET imaging, or targeted radiotherapy; a chromophore of a fluorescent dye, a contrast agent for magnetic resonance imaging, an atom or group suitable for imaging by X-ray-based techniques, or an atom or group derived from a therapeutic agent suitable for treating a medical indication associated with increased expression of αvβ6-integrin, wherein the term "derived from" indicates that the group contained in the conjugate has the same structure as the compound from which it is derived, differing only by the replacement of a hydrogen atom with a covalent bond to attach the group to the remainder of the conjugate.

5. The active atom or active atomic group is La ３＋ , Ce ３＋ , Pr ３＋ , Nd ３＋ , Sm ３＋ , Eu ２＋ , Gd ３＋ , Tb ３＋ , Dy ３＋ , Ho ３＋ , Er ３＋ , Tm ３＋ , Yb ３＋ , Lu ３＋ , Sc ３＋ , Y ３＋ , Ga ３＋ , Fe ３＋ , Co ２＋ , Co ３＋ , Ge ４＋ , In ３＋ , Sn ２＋ , Sn ４＋ , Bi ３＋ , Rh ３＋ , Ru ３＋ , Ru ４＋ , Ag ＋ , Au ３＋ , Pb ２＋ , Pd ２＋ , Pd ４＋ , Pm ３＋ , Ac ３＋ , Ti ４＋ , Zr ４＋ Al ３＋ , Cr ３＋ , Cu ２＋ , Zn ２＋ 5. The conjugate according to claim 1, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein the metal ion is selected from the group consisting of:

6. The active atom or active atomic group is ４３ Sc, ４４ Sc, ４６ Sc, ４７ Sc, ５５ Co, ９９ｍ Tc, ２０３ Pb, ２１２ Pb, ６６ Ga, ６７ Ga, ６８ Ga, ７２ As, １１１ In, １１３ｍ In, １１４ｍ In, ９７ Ru, ６２ Zn, ６１ Cu, ６２ Cu, ６４ Cu, ５２ Fe, ５２ｍ Mn, ５１ Cr, １８６ Re, １８８ Re, ７７ As, ８６ Y. ９０ Y. ６７ Cu, １６９ Er, １１７ｍ Sn, １２１ Sn, １２７ Te, １４２ Pr, １４３ Pr, １９８ Au, １９９ Au, １４９ Tb, １５２ Tb, １５５ Tb, １６１ Tb, １０９ Pd, １６５ Dy, １４９ Pm, １５１ Pm, １５３ Sm, １５７ Gd, １６６ Ho, １７２ Tm, １６９ Yb, １７５ Yb, １７７ Lu, １０５ Rh, １１１ Ag, ８８ Zr, ８９ Zr, ２１２ Bi, ２１３ Bi, ２２５ 6. The conjugate according to any one of claims 1 to 5, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein the radioactive isotope is selected from the group consisting of N, N-Ac ...

7. The active atom or active atomic group is １１ C. １３ N. １５ O. １８ F. １２３ I, １２４ I, １２５ I, １３１ 5. The conjugate according to claim 1, wherein the non-metallic radioisotope is selected from I, or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

8. 5. The conjugate or a pharmaceutically acceptable salt, hydrate, or solvate thereof according to any one of claims 1 to 4, wherein the active atom or active atomic group is a contrast agent for magnetic resonance imaging selected from Gd, Fe, and Mn.

9. 5. The conjugate of claim 1, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein the active atom or active group is a therapeutic group derived from an agent for the treatment of fibrosis or an anti-cancer agent selected from alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other anti-tumor agents, wherein the term "derived from" indicates that the group contained in the conjugate has the same structure as the compound from which the group is derived, differing only in the replacement of a hydrogen atom by a covalent bond to attach the group to the remainder of the conjugate.

10. 10. The conjugate of any one of claims 3 to 9, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein the atomic group acting as a spacer is a linear chain of 2 to 20 atoms selected from C, N, O, P, and S, with the remaining valences being saturated with hydrogen.

11. 11. The conjugate of claim 10, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein the linear chain consists of 3 to 10 atoms.

12. 11. The conjugate of claim 10, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein said atom bears one or more substituents.

13. The atomic group acting as a spacer is represented by the following formulae (IIIa) to (IIIf): *-C(O)-(CH ２ ) ｋ -(taz) ｌ -(CH ２ ) ｍ - (IIIa) *-C(O)-(CH ２ ) ｋ -NH-A-(CH ２ ) ｍ - (--Iｬ) *-C(O)-(CH ２ ) ｋ -CO-NH-(CH ２ ) ｍ - (IIIIc) *-C(O)-(CH ２ ) ｋ -(taz) ｌ -(CH) ２ ) ｏ -CO-NH-(CH ２ ) ｍ - (IIIId) -C(O)-(32 ２ ) ) ｋ -(taz) ｌ -(32) ２ ) ) ｏ -NH-C-((H ２ ) ) ｍ - (999e) -C(O)-(32 ２ ) ) ｋ -CO-8((3H ２ ) ) ｏ -(taz) ｌ -(32) ２ ) ) ｍ - (999ff) -C(O)-(32 ２ ) ) ｋ -NH-C-((H ２ ) ) ｏ -(taz) ｌ -(32) ２ ) ) ｍ - (999ff) wherein taz represents a triazole ring in which all three nitrogen atoms are adjacent to one another; l can be 0 or 1; k, m, and, if present, o are integers independently selected from the range of 0 to 20 such that k+m=2 to 20 and k+m+o=2 to 20, respectively; and an asterisk ( ＊ ) indicates the position of attachment of the cyclopeptide. The conjugate according to any one of claims 3 to 12, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, selected from:

14. The chelating group has the following formulae (IVa) to (IVd): 【Chemistry 1】 【Chemistry 2】 【Transformation 3】 【Chemistry 4】 (wherein the asterisk ( ＊ ) indicates the position of attachment of the atom group acting as a spacer, provided that if the number of cyclopeptides and associated spacers (characterized by the variable n) is less than the number of valences of the chelating group, the remaining valences represented by the asterisks are hydrogen or -CH ２ -COOH and -CH ２ -CH ２ -COOH), provided that it is saturated by another atomic group, including a group selected from The conjugate according to any one of claims 3 to 13, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, selected from:

15. The conjugate may be a compound as set forth below: 【Transformation 5】 【Transformation 6】 【Transformation 7】 【Transformation 8】 【Chemistry 9】 【Chemistry 10】 【Chemistry 11】 【Chemistry 12】 【Chemistry 13】 【Chemistry 14】 【Chemistry 15】 【Chemistry 16】 【Chemistry 17】 [Chemistry 18] The conjugate of any one of claims 1 to 14, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, comprising a structure selected from:

16. Fibrosis or cancer. The conjugate of any one of claims 1 to 8 and 10 to 15, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, for use in a method for diagnosing or imaging a disease associated with increased expression of αvβ6-integrin.

17. 16. The conjugate of any one of claims 1 to 4 and 9 to 15, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, for use in a method for treating a disease associated with increased expression of αvβ6-integrin, including fibrosis or cancer.

18. 16. A method for in vitro localization of cells with increased expression of αvβ6-integrin in a sample obtained from a patient, wherein the conjugate of any one of claims 1 to 8 and 10 to 15, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, is added to the sample, the method comprising subjecting the sample to an imaging method selected from PET, SPECT, MRI, and X-ray computed tomography, wherein the conjugate contains an active atom or atomic group that matches the imaging method to be performed.

19. A compound of any one of the following 1) to 5): 1)Cg(SCp) ｎ (IIa) wherein Cg represents a chelating group, S represents an atomic group acting as a spacer, and each Cp is a cyclopeptide of the formula cyclo(YRGDLAYp(NMe)K), and n is an integer from 1 to 4. a compound of 2) cyclo(YRGDLAYp(NMe)K); cyclo(3-I-YRGDLAYp(NMe)K); cyclo(3-I-YRGDLA3-I-Yp(NMe)K); or cyclo(YRGDLA3-I-Yp(NMe)K) wherein 3-I-Y represents a Tyr residue having an iodine atom at the 3-position of the phenyl ring, said iodine atom being any non-radioactive or radioactive isotope of iodine. a compound of 3) A compound of the following structural formula: 【Chemistry 19】 4) a compound of the following structural formula: 【Chemistry 20】 5) A compound of the following structural formula: 【Chemistry 21】 A structural unit compound selected from:

20. A pharmaceutical composition comprising the conjugate of any one of claims 1 to 15 or a pharmaceutically acceptable salt, hydrate, or solvate thereof and one or more pharmaceutically acceptable excipients.

21. 21. The pharmaceutical composition of claim 20, further comprising one or more other therapeutic agents.

22. 22. The pharmaceutical composition of claim 20 or 21, wherein the conjugate or a pharmaceutically acceptable salt, hydrate, or solvate thereof is administered to a patient and the patient is subjected to an imaging method selected from PET, SPECT, MRI, and X-ray computed tomography, and the conjugate contains an active atom or atomic group that matches the imaging method being performed.

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