Cyclic peptides and conjugates thereof for addressing α-v-β-6-integrin in vivo

Abstract

The present invention provides conjugates of cyclic peptides as ligands for cell surface receptors, particularly as ligands for αvβ6-integrin. The conjugates further comprise an effector moiety and are suitable for use as therapeutic agents, diagnostic agents, imaging agents, targeting moieties, and as biomolecular research tools. The present invention specifically relates to the use of conjugates with signaling moieties or radionuclides for in vivo targeting of αvβ6-integrin.

Description

Technical Field

[0001] The present invention relates to the field of cyclic peptides as ligands for cell surface receptors, in particular as ligands for αvβ6-integrin. Furthermore, the present invention relates to conjugates of such peptides with effector moieties, which are 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 with signaling moieties or radionuclides for in vivo targeting of αvβ6-integrin. Background Art

[0002] Integrins are a class of 24 heterodimeric transmembrane receptors, each of which contains 18 α-subunits and one of 8 β-subunits. They mediate the selective binding of cells to various extracellular matrix proteins (such as vitronectin, fibronectin, collagen or laminin) and are also involved in signaling pathways. 1 αvβ6 is one of eight integrin subtypes that recognize the arginine-glycine-aspartate (RGD) peptide sequence. Compared to other common RGD-binding integrins (such as αvβ3 and α5β1), they are expressed by different cell types and have received widespread attention due to their involvement in the formation and sprouting of blood and lymphatic vessels (angiogenesis, angiogenesis, and lymphangiogenesis). 2 αvβ6 integrin levels are generally low in adult tissues 3 αvβ6 integrin expression is restricted to epithelial cells 4 Thus, many tumors (carcinomas) of epithelial origin exhibit enhanced expression of αvβ6 integrin 5 , especially the pancreas 6 , and bile duct cells 7 ,Stomach 8,9 ,Chest 10 , ovaries 11,12 ,colon 13 , and those of the upper aerodigestive tract 14 Furthermore, αvβ6-integrin has been described as a marker for increased aggressiveness and malignancy in several cancers, and therefore a poor prognosis. 5,8,11,13 Therefore, αvβ6-integrin has been proposed as a target for in vivo addressing of cancer tissues for the purpose of molecular imaging and targeted therapy. 15 In addition, αvβ6-integrin is involved in epithelial-mesenchymal transition (EMT), for example, in biliary fibrosis 16 , renal fibrosis 17 , and pulmonary fibrosis 18During development, αvβ6-integrin can therefore be used as a fibrotic marker.

[0003] Current state of the art

[0004] Several αvβ6-specific, non-peptide 19 and peptide inhibitors 20,21,22,23 Linear peptide A20FMDV2 21 、H2009.1 22 and cyclic peptide S02 23 Radiolabeled and imaged by single-photon emission computed tomography (SPECT) 24,25,26 and positron emission tomography (PET) 21,27,28,29,30,31,32 Application to in vivo imaging of αvβ6-integrin expression. Recently, radiolabeled compounds targeting αvβ6-integrin have been tested for imaging of cancer in humans. 33,34,35 .

[0005] The reported cyclic nonapeptide ring (FRGDLAFp(NMe)K) 36,37 (Phe2) shows high affinity for αvβ6-integrin (0.26nM), significant selectivity compared to other integrins (αvβ3: 632nM; α5β1: 73nM; αvβ5 and αIIbβ3: >1μM), and is completely stable in human plasma for up to 3 hours. Phe2 derivatives are equipped with various chelators for radiometal binding. 38,39 , and their in vivo properties were evaluated in tumor-bearing mice. These studies showed that radiolabeled chelator conjugates containing only one Phe2 moiety (monomer) showed relatively low uptake in αvβ6-expressing tumor tissues. 39 Conjugates containing two and especially three Phe2 moieties (dimers and trimers, respectively) exhibited higher affinity for αvβ6-integrin, but due to their lipophilicity, they were also characterized by relatively high levels of nonspecific uptake in non-target organs. This behavior of trimers could not be mitigated by the introduction of pharmacokinetic modifiers (i.e., hydrophilic PEG linkers). 38 . Summary of the Invention

[0006] In view of the situation described above, it is desirable to provide αvβ6 integrin-active functionalized compounds with improved pharmacokinetics, particularly increased target-specific tissue uptake and retention, coupled with low nonspecific uptake in αvβ6 integrin-negative tissues. In particular, low nonspecific uptake in liver and pancreatic tissues is desirable. Other 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 those tissues that exhibit higher uptake in tumor lesions than in other tissues.

[0007] The present invention solves this problem by providing conjugates comprising specific cyclic nonapeptides that target αvβ6-integrin. These cyclic nonapeptides are characterized by the following amino acid sequences: cyclo(YRGDLAYp(NMe)K) (hereinafter referred to as Tyr2), cyclo(FRGDLAYp(NMe)K) (hereinafter referred to as FRGD), and cyclo(YRGDLAFp(NMe)K) (hereinafter referred to as YRGD). These abbreviations are also used to characterize the corresponding cyclic peptides that are covalently bound to the effector moiety via the terminal amino group of the (NMe)K side chain. In other words, this means that the abbreviations Tyr2, FRGD, and YRGD not only characterize the cyclic peptides cyclo(YRGDLAYp(NMe)K), cyclo(FRGDLAYp(NMe)K), and cyclo(YRGDLAFp(NMe)K), respectively, but also characterize the same cyclic peptide in the following form: wherein one of the two hydrogens at the terminal amino group of the (NMe)K side chain is absent / replaced by a covalent bond to another moiety.

[0008] Tyr2, FRGD, and YRGD are structurally related to Phe2 and are all covered by the general teachings of patent application WO 2017 / 046416A1. However, this patent application does not specifically disclose Tyr2, 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.

[0009] Surprisingly, the inventors have found that conjugates of Tyr2, FRGD and / or YRGD, in particular conjugates containing more than one Tyr2, FRGD and / or YRGD moiety, exhibit high target-specific tissue uptake and retention, combined with low nonspecific uptake in αvβ6 integrin-negative tissues (in particular the liver) and rapid clearance from the blood pool, compared to structurally equivalent derivatives of, for example, Phe2. Thus, these conjugates allow for selective and specific targeting of αvβ6-integrin-positive tissues in vivo, in particular for high-contrast in vivo imaging of these tissues.

[0010] Thus, the present invention relates to conjugates of Tyr2, FRGD and / or YRGD, wherein the effector moiety is covalently attached to the terminal amino group of an NMe-lysine residue, or to at least one cyclic nonapeptide selected from Tyr2, FRGD and YRGD. The present invention particularly relates to conjugates comprising more than one Tyr2, FRGD and / or YRGD moiety, which conjugates exhibit higher affinity and integrin subtype selectivity than comparable compounds comprising only one such moiety. These conjugates can be characterized by the following general formula (I):

[0011] E(Cp) n (I)

[0012] wherein Cp represents a cyclic peptide selected from Tyr2, FRGD and / or YRGD, n is an integer selected from 1 to 4, and E represents an effector moiety.

[0013] According to the present invention, various types of effector moieties can be used, including moieties suitable for diagnostic use, as well as pharmacologically active moieties for therapeutic use. Of particular interest are conjugates having moieties for diagnostic use. These include moieties comprising radionuclides (for nuclear imaging or radiation-guided surgery), fluorophores (for fluorescence imaging or fluorescence-guided surgery), or signal elements for magnetic resonance imaging (MRI). For therapeutic purposes, the effector moiety can, for example, comprise a radionuclide (internal radiation therapy) or a chemotherapeutic agent (targeted drug delivery).

[0014] Yet another aspect of the present invention relates to the use of the conjugate described above in a diagnostic or therapeutic method.

[0015] The cyclic peptide Tyr2 is novel. Structural units comprising Tyr2, one of FRGD and YRGD, combined with a spacer element suitable for click chemistry coupling are also novel. Therefore, another aspect of the present invention relates to providing these compounds.

[0016] Various aspects of the application are described in more detail in the following detailed description and appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 : Example 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 scans was 24 hours.

[0018] Figure 2: In vitro biodistribution of Ga-68-TRAP(Phe2)3 (structured bars) and Ga-68-C-7 (plain bars) in H2009 tumor-bearing SCID mice, 90 min pi, without blocking (approximately 0.1 nmol, n=5) and with blocking (50 nmol, n=3) (data are expressed as mean ± SD).

[0019] Figure 3 : Biokinetics of Ga-68-TRAP(Phe2)3 (left) and Ga-68-C-7 (right) obtained from evaluation of 90-min dynamic PET scans based on the target area.

[0020] Figure 4 Top: Ex vivo biodistribution in selected tissues of H2009 tumor-bearing SCID mice, 90 min p.i., without blocking (approximately 0.1 nmol, n=5) and with blocking (50 nmol, n=3); Bottom: Tumor-to-tissue ratios derived from biodistribution data. All data are expressed as mean ± standard deviation. Legend for bar labels: a) Ga-68-TRAP(Phe2)3; b) Ga-68-TRAP(Phe2)3, blocking; c) Ga-68-C-11; d) Ga-68-C-11, blocking; e) Ga-68-C-9; f) Ga-68-C-9, blocking; g) Ga-68-C-8; h) Ga-68-C-8, blocking; i) Ga-68-C-10; k) Ga-68-C-10, blocking; l) Ga-68-C-7; m) Ga-68-C-7, blocking.

[0021] Figure 5 Example 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 represents the percentage of injected activity per mL of tissue.

[0022] Figure 6 : Biokinetics of Ga-68-TRAP(Phe2)3, Ga-68-C-9, Ga-68-C-8, and Ga-68-C-7 (left to right) obtained from evaluation of a 90-minute dynamic PET scan of the target area. %IA / mL indicates the percentage of injected activity per mL of tissue. DETAILED DESCRIPTION

[0023] definition

[0024] The term "derived from" means that the radical contained in the conjugate has the same structure as the compound from which the radical is derived, the only difference being that a hydrogen atom is replaced by a covalent bond binding the radical to the rest of the conjugate.

[0025] As used herein, the term "heavy atom" refers to any atom other than hydrogen, deuterium, or any other isotope thereof. In the case of a divalent radical, there must be at least one heavy atom with at least two free valences. If there are heavy atoms with more than two free valences, the remaining valences may be saturated by hydrogen or other heavy atoms.

[0026] Unless otherwise noted, standard amino acid nomenclature is used. Unless otherwise noted, amino acids are L-stereoisomers. Unless otherwise noted, amino acid moieties are linked to each other by peptide bonds. Unless otherwise noted, the standard one-letter or three-letter amino acid codes apply. Unless otherwise noted, lowercase letters indicate an amino acid in the D-form, while uppercase letters indicate an amino acid in the L-form.

[0027] Me refers to a methyl group. N-Me-amino acid refers to a group in which the α-amino group bears a methyl group.

[0028] Unless otherwise indicated or the context dictates otherwise, references to "a compound of the invention," "a conjugate of the invention," etc., should be understood to refer not only to the compound, conjugate, etc. of the invention as described below and / or as specified in the appended claims, but also to pharmaceutically acceptable salts, esters, solvates, and polymorphs thereof.

[0029] References to "substituted" or "substituted with..." include the implicit proviso that the substitution is consistent with the permitted valencies of the substituted atom and substituent and that the substitution results in a stable compound, e.g., does not spontaneously undergo transformation, e.g., by rearrangement, cyclization, elimination, etc. As used herein, the term "substituted" is intended to include all permissible substituents of organic compounds. In a broad sense, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. The permissible substituents may be one or more. The substituents may be selected from the following: 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 (including simple amino, mono-C 1-6 -alkylamino and bis-C 1-6 -alkylamino, mono-C 6-14 -arylamino and bis-C 6-14 -arylamino and C1-6 -alkyl-C 6-14 -arylamino); C 2-6 -acylamino (including carbamoyl and urea); C 1-6 -alkylcarbonyloxy, C 6-14 -arylcarbonyloxy, C 1-6 -alkoxycarbonyloxy; C 1-6 -Alkoxycarbonyl; carboxyl; carboxylate; aminocarbonyl, mono-C 1-6 -alkylaminocarbonyl and bis-C 1-6 -alkylaminocarbonyl; cyano; azido; halogen; hydroxy; 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; heteroaryl having 5 to 6 ring members, which is optionally fused to 1 or 2 saturated, unsaturated or aromatic carbocyclic or heterocyclic rings, each having 5 or 6 ring members; C 6-14 -aryloxy; C 6-14 -aryloxycarbonyloxy; benzyloxy; benzyl; sulfinyl, C 1-6 -alkylsulfinyl; sulfonyl; sulfate; sulfonate; sulfonamide; phosphate; phosphonate; phosphinato; oxo; guanidine; imino; formyl, etc. Any of the above-described substituents may be further substituted, if permitted, for example, with one or more of the listed substituent groups.

[0030] The terms “alkyl”, “alkenyl”, “alkynyl”, “cycloalkyl”, “carbocycle”, “heterocycloalkyl”, “aryl”, “heteroaryl”, “heterocyclyl”, “amine”, “amide”, “nitro”, “halogen”, “thiol”, “hydroxy” or “hydroxyl”, “alkylthio”, “alkylcarboxy”, “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.

[0031] Unless otherwise indicated, all abbreviations are intended to have their usual meanings, for example, as indicated by the IUPAC-IUP Biochemistry Nomenclature Committee in Biochemistry 11, 1972, 942-944. For atoms contained in conjugates, the standard rules for valence are applied, for example, as described in the Wikipedia entry "Valence (Chemistry)" in its January 24, 2020 version. Unless otherwise indicated or the context dictates otherwise, if an atom has more valences than the number of bonding partners shown, the remaining valences are saturated with hydrogen atoms.

[0032] Unless otherwise indicated, the conjugates and other compounds of the invention are "pharmaceutically acceptable," meaning that the corresponding compound is suitable for use with humans and / or animals without causing adverse effects (e.g., irritation or toxicity), commensurate with a reasonable benefit / risk ratio.

[0033] The term "or" is generally employed in its sense including "and / or" unless the context dictates otherwise.

[0034] "Room temperature" can be any temperature from 20°C to 25°C, preferably 22°C.

[0035] "Ga-68-TRAP(Phe2)3" refers to the compound previously described by Maltsev et al. as "Ga-68-TRAP(AvB6)3" 38 .

[0036] Unless otherwise indicated, the terms "chelating group," "chelating agent," and the like refer to a group capable of forming two or more (preferably three, four, five, six, seven, or eight) coordinate bonds with a metal ion.

[0037] Cyclic peptides

[0038] The cyclic peptide used in the present invention is as follows:

[0039] Tyr2: cyclo(YRGDLAYp(NMe)K),

[0040] FRGD:ring(FRGDLAYp(NMe)K),

[0041] YRGD:Cyclo(YRGDLAFp(NMe)K)

[0042] Conjugate

[0043] General structure

[0044] The general structure of the conjugates of the present invention can be characterized by the following formula (I):

[0045] E(Cp) n (I)

[0046] wherein each Cp represents a cyclic peptide 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 other embodiments, a polymeric effector moiety or a dendritic effector moiety may be used. In this case, n may be an integer selected from 2 to 100, preferably 10 to 30. Suitable polymer scaffolds include polyethyleneimine, polysaccharides, polyamides, polypeptides, poly(amidoamine) (PAMAM) dendrimers, poly(propylene imine) (PPI) dendrimers, polyether-copolyester (PEPE) dendrimers, polyether dendrimers, polyester dendrimers, and polyarylether dendrimers.

[0047] The one or more cyclic peptides are each covalently bound to the Effector Moiety via the terminal amino group in the side chain of the NMe-Lys residue.

[0048] In a preferred embodiment, the conjugate of formula (I) comprises 2, 3 or 4 cyclic peptide moieties.Most preferably, the conjugate of formula (I) comprises 3 or 4 cyclic peptide moieties.

[0049] In the conjugates of the present invention comprising two or more cyclic peptide moieties, these multiple cyclic peptide moieties may be identical to or different from each other. All of the following specific conjugates are encompassed within the scope of the present invention:

[0050] E(Tyr2)1, E(Tyr2)2, E(Tyr2)3, E(Tyr2)4,

[0051] E(FRGD)1, E(FRGD)2, E(FRGD)3, E(FRGD)4,

[0052] E(YRGD)1, E(YRGD)2, E(YRGD)3, E(YRGD)4,

[0053] 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,

[0054] 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,

[0055] 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,

[0056] 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.

[0057] In the case of polymeric or dendritic effector moieties, multiple copies of the same cyclic peptide selected from Tyr2, YRGD and FRGD may also be attached. Alternatively, the polymeric or dendritic effector may be bound to two or three of these different cyclic peptides, such that each of the two or three cyclic peptides is present one or more times, provided that the total number of bound cyclic peptides is within the range defined above for n, i.e. the polymeric or dendritic conjugate is characterized by the general formula E((Tyr2) n1 (FRGD) n2 (N) n3 ), where each of n1, n2 and n3 can be in the range from 0 to n, provided that n1+n2+n3=n.

[0058] In principle, it is possible to obtain other compounds of the invention by modifying the compounds of the invention as described above by covalently attaching to the effector moiety a cyclic peptide other than Tyr2, FRGD and YRGD. For example, one embodiment relates to a compound as described above, but wherein one, two or three of the cyclic peptide moieties Tyr2, FRGD and / or YRGD are replaced by the cyclic peptide moiety Phe2 mentioned in the introduction, wherein Phe2 is linked to the rest of the conjugate in the same manner as the other cyclic peptide moieties (i.e., via the terminal amino group of the (NMe) K residue), wherein the number of substitutions by Phe2 is such that at least one of the cyclic peptide moieties Tyr2, FRGD and YRGD remains in the conjugate (i.e., if n is the number of cyclic peptide moieties, the number of Phe2 moieties does not exceed n-1, and at least one cyclic peptide moiety is selected from Tyr2, FRGD and YRGD). In another embodiment, no other cyclic peptides are present.

[0059] Effector part

[0060] The effector moiety is an atomic group having 10 to 1000 heavy atoms, preferably 20 to 200 heavy atoms, and more preferably 30 to 150 heavy atoms. It is characterized by the following features:

[0061] (a) it has a number of free valences corresponding to the number of bound cyclic peptides (i.e., the number n in formula (I));

[0062] (b) it contains a reactive atom or group of atoms capable of exerting the desired effect, such as a radioisotope or chromophore for diagnostic purposes or a therapeutically active moiety for therapeutic purposes;

[0063] (c) It contains one or more atomic groups that act as spacers to spatially separate the one or more cyclic peptides from the active atom or active atomic groups, thereby reducing mutual interference.

[0064] In some embodiments, the effector can be characterized by the following general formulas (II) and (II').

[0065] Aa(Cg)(S) n (II)

[0066] Aa'(Cg) k (S) n (II')

[0067] wherein Aa represents an active atom or group of active atoms capable of binding by chelation, Aa' represents an active atom or group of active atoms capable of binding by a covalent bond, Cg represents a chelating group, k is 0 or 1, S represents an atomic group serving as a spacer, and n is as defined above in relation to formula (I), with the proviso that n does not exceed the number of free valences of the chelating group and with the proviso that if k is 0, then n is 1, i.e., if no chelating group is present, then a single spacer is directly bound to the active atom or group of active atoms.

[0068] Combining formula (II) with formula (I) yields the following formula (Ia):

[0069] Aa(Cg)(SCp) n (Ia)

[0070] wherein Aa, Cg, S, Cp and n have the same meanings as defined above for formulae (I) and (II).

[0071] In a related embodiment, the active atom or group of active atoms Aa' is covalently bound to a chelating group or spacer. The conjugate of this embodiment is characterized by the following formula (Ia'):

[0072] Aa'(Cg) k (SCp) n (Ia')

[0073] wherein Aa' is an active atom or group of active atoms capable of forming a covalent bond, Cg, S, Cp and n have the same meanings as defined above with respect to formulae (I) and (II); k is 0 or 1; if k is 1, Aa' is covalently bonded to Cg; if 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 with Aa' and Cp.

[0074] In another embodiment, the second reactive group can be attached to one of the spacers (rather than one of the cyclic peptides) such that the conjugate is represented by the following formula (Ib):

[0075] Aa(Cg)(SCp) n '(SAa') (Ib)

[0076] wherein Aa, Cg, S and Cp have the same meanings as in formula (Ia) described above, and wherein Aa′ is an active atom or group of active atoms different from Aa, as long as it is covalently bound to the spacer rather than through a chelating group, and n′ is 1, 2 or 3, provided that n′+1 is the number of free valences of the chelating group or less.

[0077] In yet another embodiment, different linkers can be connected via a non-chelating central moiety. In these cases, the active atom or group of active atoms is covalently bound to another part of the molecule, which can be a central moiety, a spacer, or a cyclic peptide. The conjugates of this embodiment are characterized by the following formulae (Ic), (Id), (Ie), and (If):

[0078] Aa'(Cm) k (SCp) n (Ic)

[0079] (Cm)(SCp) n-o (S(Aa') p (Cp) m ) o (Id)

[0080] (Cm)(SCp) n-o (SCp(Aa') p ) o (Ie)

[0081] Cp(Aa') p (If)

[0082] Formula (Ic) corresponds to Formula (Ia') above, but in which the chelating group is replaced by a central moiety, Cm. S, Cp, and n have the same meanings as defined above for Formulas (I), (Ia), and (II); k is 0 or 1. Aa' is an active atom or group of active atoms capable of forming a covalent bond. In Formula (Ic), if k is 1, it is covalently bonded to Cm, and if k is 0, it is bonded to S. In the latter case, n must be 1, i.e., there is only one spacer binding Aa' and Cp.

[0083] The central portion Cm can have any atom or atomic group of at least n+1 valence to accommodate n spacer-cyclic peptide moieties and 1 active atom or active atomic group. 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 the following groups: aromatic groups (such as phenyl, naphthyl), or derived from larger fused aromatic groups containing 3 or 4 6-membered rings (such as anthracene, phenanthrene, benzopyrene, etc.); non-aromatic cyclic groups, which include C5-7 carbocyclic rings (such as cyclopentane, cyclohexane, cycloheptane), fused groups containing 2, 3 or 4 rings, each ring including 5 to 7 ring members (such as naphthalene, anthracene, phenanthrene, benzopyrene, etc., fully or partially hydrogenated forms), bicyclic or tricyclic groups with 7 to 10 carbon atoms (such as norbornene or adamantane). Further preferred central moieties may be heterocyclic groups containing 1, 2, 3 or 4 fused rings, each fused ring having a ring size independently selected from 5, 6 or 7 membered rings. These groups may be partially or fully saturated aromatics. Alternatively, the central moiety may be a single atom selected from C, N and P.

[0084] Formula (Id) characterizes a conjugate in which the cyclic peptide portion and the active atom or active atom group Aa' are both linked to the central portion via a spacer. That is, the active atom or active atom group Aa' is covalently bound to one of the spacers. The meanings of Cm, Aa', S, Cp, and n are the same as those described above for formulas (I), (II), (Ia), and (Ic). Optionally, the spacer with the active atom or active atom group Aa' may additionally carry the cyclic peptide Cp; therefore, m may be 0 or 1. If an additional Cp is present, the active atom or active atom group Aa' and its attachment point must be selected so as to avoid or at least minimize adverse interactions with the cyclic peptide, for example by attaching the two parts to different atoms of the spacer, the two parts being separated from each other by at least 5 covalent bonds. The number of spacers with the active atom or active atom group Aa' is characterized by o, which can be any integer from 1 to n. The number of active atoms Aa' bound to a single spacer is characterized by p, which can be 1 or 2.

[0085] Formula (Ie) is characterized in that the active atom or active atom group Aa' is bound to the cyclic peptide Cp. Cm, Aa', S, Cp and n have the same meanings as described above for formulas (I), (II), (Ia) and (Ic). The variable o represents the number of cyclic peptides Cp carrying active atoms or active atom groups Aa'. This can be any integer from 1 to n. The variable p represents the number of active atoms Aa' bound to a single cyclic peptide, which can be 1 or 2.

[0086] Formula (If) characterizes the conjugates of the present invention, which do not contain any central part and / or spacer. Instead, the active atom or group of active atoms Aa' is directly bound to the cyclic peptide. According to a preferred embodiment of formula (If), the iodine atom or radioisotope is attached to the 3-position of one or both of the tyrosine residues present in Tyr2, FRGD or YRGD, so that the resulting cyclic peptide is 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);

[0087] Wherein 3-IY represents a Tyr residue having an iodine atom at the 3-position of the benzene ring, wherein the iodine atom may be any non-radioactive isotope or radioactive isotope of iodine.

[0088] The compound of formula (1f) may have a dual feature: as long as the binding of Aa' does not lead to a significant decrease in affinity for αvβ6-integrin, that is, when according to reference 36 and 37 If the binding affinity of cyclic peptides with Aa' is 5 nM or less when determined by the method described in , they can be used as conjugates of the present invention. In addition, they can also be incorporated into larger conjugates, such as the conjugates of formula (1e), and thus serve as building blocks of the present invention.

[0089] The patterns of binding of active atoms and active groups Aa and Aa' described above by formulae (1a) to (1f) can be freely combined. For example, the compound of formula (1a) or (1a') may carry one or more cyclic peptides, which themselves carry one or more active atoms or active groups Aa'. In particular, the present invention also relates to conjugates of formula (1a) or (1a'), wherein one or more cyclic peptides carry one or two iodine atoms or radioactive isotopes bound to the 3-position of a tyrosine residue.

[0090] Active atoms or active atomic groups Aa, Aa' may include the following:

[0091] (b-1) Non-radioactive isotopes or radioactive isotopes of metal ions selected from the group consisting of 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+ , and mixtures thereof. Particularly preferred are metal ions selected from the group consisting of: Ga 3+ 、Gd 3+ 、Cu 2+ Sc 3+ 、Y 3+ , and Lu 3+ , and mixtures thereof. The radioactive isotope can be specifically selected from: 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 Such as 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 Second, 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, wherein these radioactive isotopes are preferably used in the form of metal ions in the corresponding oxidation states listed above. Particularly preferably, the radioactive isotope is selected from the group consisting of: 68 Ga, 44 Sc, 99m Tc, 111 In, 64 Cu, 89 Zr, 90 Y. 177 Lu, 213 Bi, 225Ac, and mixtures thereof.

[0092] (b-2) non-metallic radioisotopes selected from the following: 11 C. 13 N. 15 O. 18 F. 123 I. 124 I. 125 I, or 131 1, preferably 18 F or 123 I. In addition to being present as Aa, Aa' or a portion thereof in the above formula, the non-metallic radioisotope may also be the active atom Aa' present at any other position within the molecule, where it may replace any other covalently bound atom that is already present as part of the rest of the molecule and has an appropriate number of binding partners.

[0093] (b-3) a chromophore of a fluorescent or non-fluorescent dye, and preferably derived from a moiety commercially available from ThermoFisher series (such as, )and series (such as,

[0094] and ), as well as fluorescein, pyrene, rhodamine, BODIPY dyes and their analogs;

[0095] (b-4) contrast agents for magnetic resonance imaging (MRI), preferably Gd, Fe, or Mn, most preferably Gd in the form of a Gd(III) chelate complex;

[0096] (b-5) Atoms or atomic groups suitable for imaging by X-ray based techniques, preferably iodine or iodine-containing atomic groups.

[0097] (b-6) an atom or group of atoms derived from a therapeutic agent. The atom or group of atoms may have therapeutic activity itself or after cleavage of the cyclic peptide moiety to release the potential therapeutic agent. Preferably, the therapeutic agent is a therapeutic agent suitable for treating cancer or fibrosis.

[0098] If the therapeutic indication is cancer, the therapeutic agent is preferably selected from alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors and other antitumor drugs. More specifically, the following may be mentioned: platinum compounds, antibiotics with anticancer activity, anthracyclines, anthracenediones, alkylating agents, antimetabolites, antimitotics, taxanes, taxanes, microtubule inhibitors, vinca alkaloids, folic acid antagonists, topoisomerase inhibitors, antiestrogens, antiandrogens, aromatase inhibitors, GnRh analogs, 5α-reductase inhibitors, bisphosphonates, metabolic inhibitors, preferably mTOR inhibitors; epigenetic inhibitors, preferably DNMT inhibitors; anthracyclines; camptotheca; anthracyclines; histone deacetylase (HDAC) inhibitors, proteasome inhibitors, JAK2 inhibitors, tyrosine kinase inhibitors inhibitor (TKI), PI3K inhibitors, protein kinase inhibitors, serine / threonine kinase inhibitors, intracellular signaling inhibitors, Ras / Raf signaling inhibitors, MEK inhibitors, AKT inhibitors, survival signaling protein inhibitors, cyclin-dependent kinase inhibitors, therapeutic monoclonal antibodies, TRAIL pathway agonists, anti-angiogenic agents, metalloproteinase inhibitors, cathepsin inhibitors, urokinase plasminogen activator receptor function inhibitors, immunoconjugates, antibody drug conjugates, antibody fragments, bispecific antibodies, bispecific T cell engagers (BiTE). 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 (topoisomerase type 1 inhibitor), amsacrin, etoposide, etoposide phosphate and teniposide (topoisomerase type 2 inhibitor); UFT, capecitabine, CPT-II, oxaliplatin, cyclophosphamide, methotrexate, vinorelbine, epirubicin, mitoxantrone, raloxifene, mitomycin, carboplatin, gemcitabine, etoposide and topotecan.

[0099] Other suitable therapeutic agents for treating cancer are disclosed in, for example, "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 and Dan L. Longo, Wolters Kluwer, 2011; and "Drugs in Cancer Care" by Rachel Midgley, Mark R. Middleton, Andrew Dickman, and David Kerr (eds.), Oxford University Press 2013. When practicing the present invention, the drugs disclosed in these books can be used as therapeutic agents. Therefore, the disclosures of the therapeutic drugs in these references are incorporated herein.

[0100] If the therapeutic indication is fibrosis, the therapeutic agent is preferably selected from therapeutic drugs suitable for the treatment of fibrosis. For example, in "Cystic Fibrosis in the 21st Century" by Andrew Bush (Editor-in-Chief), S. Karger, 2006; "Liver Fibrosis: New Insights for the Healthcare Professional: 2013 Edition" by Q. Ahton Acton, Scholarly Editions, 2013; "Idiopathic Pulmonary Fibrosis: A Comprehensive Clinical Practice" by Keith C. Meyer, Steven D. Nathan, Springer Guide", 2014; "New Insights into the Pathogenesis and Treatment of Idiopathic Pulmonary Fibrosis: A Potential Role for Stem Cells in the LungParenchyma 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 TAWynn, in Mucosal Immunol. 2009, 2, 103-121. Particularly preferred therapeutic agents are preferably selected from the drugs and drug classes listed in Table II of the review article by Gharaee-Kermani et al. cited above.

[0101] If the treatment indication is Covid-19 infection, the therapeutic agent can be any medicament with experimental determination or suspected activity in treating these infections, regardless of whether they have been used in clinical practice or are still in the development stage. Currently used for or being developed, for treating Covid-19 infection, the medicament is, for example, an antiviral agent, including, for example, anti-Ebola virus agent Redcivir or anti-influenza agent Favipiravir, kinase inhibitors (such as ATR-002), anti-inflammatory agents (including glucocorticoids), antagonists of IL-1 or IL-6 (such as anakinra and tocilizumab, respectively), anti-infective agents (such as ivermectin) or medicaments for the treatment of other lung diseases (such as fibrosis). Therefore, reference can be made to the literature and medicaments mentioned above about fibrosis.

[0102] The active atom or group of atoms can be bound to the cyclic peptide (or multiple cyclic peptides) through an atomic group used as a spacer. The atomic group used as a spacer is usually a straight chain of 2 to 20, preferably 3 to 10 atoms selected from C, N, O, P and S, preferably an alkylene group, which optionally carries one or more substituents, and the remaining valences are saturated with hydrogen. This straight chain can be interrupted by one or more cyclic structures, which preferably have 5 ring atoms, more preferably a triazole ring. The amino group bound to the N(Me)K side chain is usually achieved through an amide bond. The binding of the spacer to the active atom or atomic group Aa' in formula (1a'), (1b), (1c) or (1d) can also be achieved through an amide bond, but can also be a direct covalent bond.

[0103] For example, the atomic groups used as spacers in the above-described formulas (Ia) to (If) will be further described below. In one embodiment, the atomic group can be characterized by the following formula (IIIa):

[0104] *-C(O)-(CH2) k -(taz) l -(CH2) m -(IIIa)

[0105] wherein taz represents a triazole ring with all three nitrogen atoms adjacent to each other, l can be 0 or 1, and k and m are each an integer selected from 0 to 20 such that k+m=2-20. An asterisk (*) marks the point of attachment of the cyclic peptide.

[0106] In another embodiment, additional divalent functional groups may be present, as shown below in formulas (IIIb) to (IIIf'):

[0107] *-C(O)-(CH2) k -NH-CO-(CH2) m - (IIIb)

[0108] *-C(O)-(CH2) k -CO-NH-(CH2) m - (IIIc)

[0109] *-C(O)-(CH2) k -(taz) l -(CH2) o -CO-NH-(CH2) m - (IIId)

[0110] *-C(O)-(CH2) k -(taz) l -(CH2) o -NH-CO-(CH2) m - (IIIe)

[0111] *-C(O)-(CH2) k -CO-NH-(CH2) o -(taz) l -(CH2) m - (IIIf)

[0112] *-C(O)-(CH2) k -NH-CO-(CH2) o -(taz) l -(CH2) m - (IIIf')

[0113] wherein taz and l have the same meanings as 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=2-20 and k+m+o=2-20, respectively. The asterisk (*) again marks the point of attachment of the cyclic peptide.

[0114] According to one embodiment, one or more spacers may carry one or more independently selected substituents. Each of these substituents is not particularly limited. According to a preferred embodiment, the substituent itself is a portion containing a spacer and a cyclic peptide, preferably a spacer S and a cyclic peptide Cp as described herein. The spacer portion of the substituent may even be further substituted to form a dendritic structure having up to 3 generations of substituents attached to the 0th generation spacer shown in formulas (Ia) to (Ie).

[0115] In other embodiments, particularly those related to active atoms or groups of active atoms suitable for therapeutic purposes, the spacer can be cleavable under physiological conditions. Such cleavable spacers are not particularly limited and can be selected from 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.

[0116] If the active atom is a metal ion, binding is usually accomplished via a chelating group, for example as described in Chem. Soc. Rev. 2011; 40: 3019–3049 40. The binding of the metal ion by the chelating group preferably occurs through a coordination bond (Lewis acid / base interaction) influenced by the N and O atoms of the chelating group. However, the chelating group is not particularly limited as long as it can form a chelate complex with the target metal ion, and the chelating group is preferably stable under physiological conditions for a long enough time to carry out the intended diagnostic method. Preferred chelating agents or chelating agent-containing functional groups are: those mentioned 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, H2dedp a, H4octapa, H2azapa, H5decapa, BCPA, CP256, YM103, DFO, PCTA, H6phospha, PCTA, HEHA, PEPA), bispidines (as described in Dalton Trans. 2018; 47:9202–9220), radiohybrid ligands (as described by Wurzer et al. in J. Nucl. Med. 2019 doi: 10.2967 / jnumed.119.234922), hydroxypyridone ligands (as described in Dalton Trans. 2019; 48: 4299–4313 or Bioconjugate Chem. 2015; 26: 2579-2591), picolinic acid chelators (as described in Dalton Trans. 2017; 46: 14647–14658, Inorg. Chem. 2016; 55: 12544–12558 or Bioconjugate Chem. 2017; 28: 2145–2159), and in particular chelating groups that allow conjugation of more than one peptide without additional branched linkers (such as, for example, falcarine c) (as described in J. Label. Compd. Radiopharm. 2015; 58: 209–214), DOTPI (as described in Chem. Eur. J. 2013; 19: 7748–7757), DOTGA (as described in Chem. Commun. 1998, 1381), NOTGA (as described in Bioconjugate Chem. 2012; 23: 2229–2238), NODAPA (as described in Bioorg. Med. Chem. Lett.2008;18:5364–5367), DOTAZA (as described in Chem. Asian J. 2014;9:2197–2204), HBED-CC (as described in Eur. J. Nucl. Med. 1986;12:397–404), HBED-NN (as described in J. Org. Chem. 2019;84:7501–7508), (NH2)2sar (as described in Inorg. Chem. 2011;50:6701–6710), or TRAP (for example, as mentioned in Dalton Trans. 2015;44:11137). Particularly preferred are TRAP, its tetravalent homologues DOTPI and DOTAZA, and analogs and derivatives of these chelating groups. Typical structures of these chelating groups are represented by the following formulas (IVa) to (IVd):

[0117]

[0118]

[0119] Wherein the asterisk (*) marks the point of attachment of the atomic group serving as the spacer. If the number of cyclic peptides and associated spacers (as characterized by the variable n) is less than the number of valences of the chelating group, the other valences indicated by the asterisk are saturated by hydrogen or another atomic group, preferably by a group selected from -CH2-COOH and -CH2-CH2-COOH.

[0120] Preparation of the conjugate of the present invention

[0121] The conjugates of the present invention can be synthesized using standard materials and methods known in the art. If the conjugate is a chelate, the formation of the chelate is usually performed as the last step. That is, a suitable process includes one or more steps for forming a precursor as described below, followed by the reaction of the precursor with the atom, atomic group or ion to be chelated. The final reaction is usually carried out under the usual conditions known to those skilled in the art for such reactions. In a preferred arrangement, the reaction is carried out at ambient temperature (room temperature, e.g., 20-25°C). Also preferably, the reaction is carried out in a temperature range of ambient temperature (room temperature) to 37°C.

[0122] The ions may be provided in the form of a salt, wherein the counter ions forming the salt may be selected from the group consisting of sulfate, fluoride, chloride, bromide, nitrate, phosphate, carbonate, bicarbonate, sulfonate, acetate, and mixtures thereof. In other preferred embodiments, the ions are provided in the form of a solution.

[0123] The precursor is preferably prepared using a modular approach based on click chemistry, linking the chelating group (or central part) to the cyclic peptide part / parts. The spacer / spacers are formed in situ during the coupling reaction. The starting material itself contains a precursor of the spacer, with functional groups suitable for click chemistry coupling at their ends.

[0124] Cyclic peptides carrying a spacer precursor at their (NMe)K residue can be obtained by reacting the corresponding precursor, which carries a carboxyl group at the cyclic peptide binding end and can be activated using, for example, HATU, HOBt and DIPEA, with the corresponding cyclic peptide under standard amide coupling conditions, as described in Maltsev OV et al., Angew. Chem. Int. Ed. 2016; 55: 1535–1539 and / or WO 2017 / 046416A1.

[0125] Cyclic peptides can be synthesized by applying appropriately adapted materials and processes described in the literature, such as Maltsev OV et al., Angew. Chem. Int. Ed. 2016; 55: 1535–1539 and / or WO 2017 / 046416A1.

[0126] Specific conjugates of the present invention

[0127] In the following, specific conjugates of the present invention are shown. The conjugates of the present invention include the conjugates shown below, and the conjugates obtained by incorporating non-radioactive metal ions or radionuclides (such as, 68 Ga) to obtain the corresponding conjugate.

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149] Structural units of the present invention

[0150] The present invention further relates to building blocks that can be used to obtain the conjugates of the present invention.

[0151] The first type of structural unit of the present invention is a group of compounds corresponding to the chelate complexes described above, but without a coordinating atom (such as Ga-68). These structural units of the present invention can be characterized by the following formula (IIa):

[0152] Cg(SCp) n (IIa)

[0153] wherein Cg represents a chelating group, S represents an atomic group serving as a spacer, each Cp is a cyclic peptide independently selected from Tyr2, YRGD and FRGD, and n is an integer from 1 to 4. All other information provided above for the corresponding coordination complex applies in an analogous manner to the structural unit of formula (IIa).

[0154] The present invention also relates to a structural unit, which is a modified cyclic peptide that can be used in the early stages of a synthetic method for synthesizing the conjugates of the present invention and the structural units mentioned above by convenient click chemistry. These structural units comprise a cyclic peptide portion selected from Tyr2, YRGD and FRGD, a functional group that can participate in a click reaction (e.g., as defined in the Wikipedia entry "Click Chemistry" in its January 24, 2020 version) and an atomic group that links the cyclic peptide to the functional group via the terminal amino group of the side chain of the NMe-K residue.

[0155] These structural units of the present invention can be represented by the following formula (V):

[0156] Cp-L-Fg (V)

[0157] wherein Cp represents a cyclic peptide selected from Tyr2, YRGD, and FRGD, L represents a linker group, and Fg represents a functional group for performing a click reaction.

[0158] The functional group is preferably an azide group, an alkynyl group, especially including a terminal ethynyl group, a dibenzylcyclooctynyl group, a trans-cyclooctenyl group, a tetrazinyl group, a dibenzocyclooctynyl group or a bicyclo[6.1.0]nonynyl group.

[0159] The linking group typically comprises a carbonyl group that forms an amide bond with the amino group of the side chain of the NMe-K residue. It also includes a group of 1 to 15 atoms selected from C, N, and O, forming a linear chain between the amide bond and the functional group, the group optionally being substituted with one or more substituents, with the remaining valences of the chain atoms being saturated with hydrogen atoms. Preferably, the group is an alkylene group having 1 to 15, more preferably 2 to 6, methylene groups.

[0160] Formula BB-1 below illustrates this concept of the building block of the present invention, wherein the Tyr2 cyclic peptide is linked to the azide functionality via a C4-alkylene group.

[0161]

[0162] Formulas BB-2 through BB-7 below describe further useful building blocks.

[0163]

[0164] BB-5a refers to the structure BB-5, where X 1 and X 2 It is hydrogen, n=2.

[0165] BB-6a refers to structure BB-6, wherein X is hydrogen and n=2.

[0166] BB-7a refers to structure BB-7, wherein X is hydrogen and n=2.

[0167] The Tyr2 cyclic peptide itself is novel and represents another structural unit of the present invention for obtaining the conjugates of the present invention described above and below. The same is true for the iodine-modified cyclic peptides Tyr2, FRGD and YRGD. That is, the other structural units 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);

[0168] Wherein 3-IY represents a Tyr residue having an iodine atom at the 3-position of the benzene ring, wherein the iodine atom may be any non-radioactive isotope or radioactive isotope of iodine.

[0169] Peptide synthesis

[0170] The cyclic peptides 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 WO 2017 / 046416A.

[0171] Peptide cyclization can be achieved using standard techniques. For example, cyclization can be performed on a solid support or in solution using HBTU / HOBt / DIEA, PyBop / DIEA, or PyClock / DIEA reagents. Useful cyclization methods are described, for example, in WO 2017 / 046416A, J. Chatterjee, B. Laufer, H. Kessler, Nat. Protoc. 2012, 7, 432-444, and references therein.

[0172] Synthesis of conjugates

[0173] The conjugate can be prepared by using the 38 ,43,44 ,45 Prepared similarly to the method described in .

[0174] Diseases associated with cells with increased expression of αvβ6-integrin

[0175] The conjugates of the present invention are effective against any disease associated with increased expression of αvβ6-integrin. Typically, the presence of αvβ6-integrin in tissue can be determined by immunohistochemistry (IHC). Application of this analytical technique to healthy adult tissue does not produce any αvβ6-integrin signal. Therefore, in the context of 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 is a tissue that deviates from healthy adult tissue and may be due to diseases such as cancer, fibrosis or Covid-19, or it may be due to scar tissue formation due to conditions such as early wounds. Any of these diseases and conditions can be identified using the conjugates of the present invention. These diseases are described in the literature 41,42 Described in .

[0176] These diseases include cancer, especially non-small-cell lung cancer (NSCLC), pancreatic cancer, cholangiocarcinoma, gastric cancer, breast cancer, head and neck squamous cell carcinoma, basal cell carcinoma, colon cancer, ovarian cancer (Niu J, Li Z, Cancer Lett. 2017; 403: 128e137), and upper aerodigestive tract cancer, 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 adenocarcinoma, colon adenocarcinoma, pancreatic adenocarcinoma (PDAC), head and neck squamous cell carcinomas (such as oral squamous cell carcinoma, laryngeal squamous cell carcinoma, oropharyngeal squamous cell carcinoma, nasopharyngeal squamous cell carcinoma, hypopharyngeal squamous cell carcinoma).

[0177] Using IHC, expression of αvβ6 has also been demonstrated in fibrotic tissue (Munger CS, et al., Cell 1999; 96:319). Thus, other diseases include fibrosis, particularly biliary fibrosis, renal fibrosis, endomyocardial fibrosis, Crohn's disease, arthrofibrosis, and pulmonary fibrosis. Of particular interest is idiopathic pulmonary fibrosis (IPF).

[0178] Quantification of αvβ6-integrin in lung tissue has been identified as a potentially valuable method for,

[0179] (1) stratify patients eligible for inhaled αvβ6-inhibitor molecule therapy (e.g., GSK3008348), and

[0180] (2) Evaluate the therapeutic success rate of this treatment (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

[0181] Communications (2020) 11:4659, https: / / doi.org / 10.1038 / s41467-020-18397-6and TM;Maher et al., Respiratory Research (2020) 21:75, https: / / doi.org / 10.1186 / s12931-020-01339-7). Therefore, the present invention may be particularly suitable for this application field and related application fields.

[0182] A recent study showed that the expression of αvβ6 in lung tissue is affected by COVID-19 (Foster CC, et al., J. Nucl. Med. 2020; 61: 1717). Therefore, the radiolabeled compounds of the present invention are suitable for in vivo imaging of post-COVID-19 syndrome in patients.

[0183] Because αvβ6-integrin is an activator of transforming growth factor β (TGF-β), any disease associated with abnormal levels of TGF-β in the intracellular space, or any disease associated with a disturbed TGF-β response in certain cell types resulting in altered TGF-β signaling, may be associated with increased αvβ6-integrin expression. These diseases can be diagnosed by determining the αvβ6-integrin expression status of cells in affected tissues. Of particular interest is the use of diagnostic procedures based on determining the density of αvβ6-integrin expression in tissues for treatment decisions related to the use of therapeutic agents, particularly antibodies targeting the TGF-β signaling pathway, and particularly targeting TGF-β itself, either in free form or in complex with potentially relevant peptides.

[0184] Increased αvβ6 expression can be targeted in vivo using radiolabeled compounds such as those of the present invention.

[0185] For imaging and / or as a diagnostic agent

[0186] The conjugates of the present invention are suitable for use as diagnostic agents. Advantageously, the conjugates of the present invention are used wherein the effector moiety comprises an active atom or group of active atoms suitable for the imaging method / diagnostic method of interest, as described above. Depending on the imaging / diagnostic method selected, an appropriate active atom or group of active atoms is selected. The selected imaging / diagnostic method also determines the dosage, form, and timing of administration of the conjugates of the present invention.

[0187] The conjugates of the present invention are suitable for almost any analytical / diagnostic method involving the use of diagnostic agents. 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 imaging or magnetic resonance imaging (MRI), X-ray-based CT imaging, scintigraphy, Cerenkov imaging, ultrasound scanning, thermal imaging, and combinations thereof.

[0188] The conjugates of the present invention can be prepared by applying the literature 33,34,35,38 Thus, the present invention provides a method of imaging a patient (such as a cancer patient, a fibrosis patient, or a patient affected by a Covid-19 infection, including a patient with post-COVID-19 syndrome), the method comprising administering to the patient a conjugate of the invention and then subjecting the patient to an imaging method selected from the group consisting of 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, ultrasound scanning, thermal imaging, and combinations thereof, wherein the reactive atom or reactive group of atoms is suitable for the selected imaging method, and wherein the selected imaging method detects a signal generated by the reactive atom or reactive group of atoms.

[0189] Use as a therapeutic agent

[0190] Conjugates of the invention having an effector moiety with an active atom or group of active atoms derived from a drug can be used in the treatment of diseases associated with upregulation of αvβ6-integrin, such as those listed above.

[0191] The conjugates of the present invention can be administered to a patient, for example, by intravenous, transmucosal, transdermal, or intranasal administration. Suitable dosages can range from 0.1 mg / day to 1000 mg / day, preferably from 0.1 mg / day to 10 mg / day. The conjugates of the present invention can be administered once a day, twice a day, three times a day, etc., over any period of time, wherein multiple periods can be interrupted by one or more periods in which the compound of the present invention is not administered.

[0192] The conjugates of the present invention can also be used as components in combination therapies. They can 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 therapies can be performed by administering two or more therapeutic agents simultaneously or sequentially.

[0193] The conjugates of the invention may also be used for targeted radiotherapy, particularly conjugates incorporating alpha- or beta-radiation emitting radionuclides, such as 47 Sc, 67 Cu, 177 Lu, 90 Y. 213 Bi, 225 Ac, 161 Tb, 149 Tb or 131 I.

[0194] The conjugates of the present invention can also be used for the diagnosis and treatment of fibrosis. The conjugates of the present invention can be used for these purposes by any suitable form of administration, including intravenous, intra-arterial, transmucosal, pulmonary and intranasal administration. The dosage and administration regimen can be the same as those defined above for cancer treatment. Combination therapy is also possible, wherein one or more other therapeutic agents are selected from other therapeutic agents suitable for treating fibrosis, such as those cited above by cross-referencing 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 can be administered simultaneously or sequentially.

[0195] The conjugates of the present invention can also be used for the diagnosis or treatment of Covid-19 infection, including the diagnosis or treatment of post-COVID-19 syndrome. The conjugates of the present invention can be used for these purposes by any suitable form of administration, including intravenous, intra-arterial, transmucosal, pulmonary and intranasal administration. The dosage and administration regimen may be the same as those defined above for cancer treatment. Combination therapy is also possible, in which one or more other therapeutic agents are selected from other therapeutic agents suitable for treating Covid-19 infection, such as immunotherapy, dexamethasone or remdesivir. The conjugates of the present invention and one or more other therapeutic agents can be administered simultaneously or sequentially.

[0196] Thus, the present invention provides a method for treating a patient suffering from a disease associated with increased expression of αβ integrin, in particular cancer, fibrosis or Covid-19 infection, which method comprises administering to the patient a conjugate of the invention, wherein the active atom or group of active atoms is derived from a therapeutic agent selected as being suitable for treating the respective disease, for example as defined in item (b-6) of the Effector section above.

[0197] For drug targeting and biomolecular research

[0198] The conjugates of the present invention can also be used for drug targeting and biomolecular research. These uses can be carried out as described in the corresponding parts of WO2017 / 046416A. In particular, the conjugates of the present invention (preferably comprising the Tyr2 peptide sequence) can be covalently or non-covalently incorporated into nanocarriers (such as nanoparticles, liposomes or micelles) to bind the peptide portion to target cells, thereby increasing the local concentration of nanoparticles, which typically contain drugs. This approach is particularly meaningful for treating cancer (especially tumors) with chemotherapeutic agents because it can achieve "homing" in such tissues expressing αvβ6.

[0199] Pharmaceutical composition

[0200] The conjugates of the present invention can be formulated into pharmaceutical compositions. This can be accomplished using conventional means and methods for peptide drugs. Suitable literature is described, for example, in the pharmaceutical composition section of WO 2017 / 046416A. These disclosures are incorporated herein by reference. The pharmaceutical compositions of the present invention may also contain the nanoparticles mentioned in the previous section. According to a preferred embodiment, these nanoparticles contain not only the conjugates of the present invention and the nanoparticles themselves, but also a therapeutic agent (preferably a chemotherapeutic agent) within the nanoparticles.

[0201] Example

[0202] Materials and methods

[0203] Abbreviations:

[0204] CuAAC = copper-catalyzed azide-alkyne cycloaddition, Dde = 1-(4,4-dimethyl-2,6-dioxocyclohexylidene-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-carboxyethylphosphinic acid)]

[0205] General

[0206] Unless otherwise stated, all commercial 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-pentenoic 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 obtained from Bachem Holding AG (Bubendorf, Switzerland). HOBt hydrate was obtained from Carbolution (St. Ingbert, Germany). TRAP (azide) 1 38 and TRAP(azide)3 43Synthesized as described above. Semi-preparative reversed-phase HPLC was performed using a Waters system: Waters 2545 (binary gradient module), Waters SFO (system fluid manager), Waters 2996 (photodiode array detector), and Waters 2767 (sample manager). Separation was performed using the following columns: Dr. Maisch C18 Reprosil 100C18, 5 μm, 150×30 mm (column 1), with a flow rate of 40 mL / min of water (0.1% v / v trifluoroacetic acid) and acetonitrile (0.1% v / v trifluoroacetic acid); or YMC C18 column: YMC-Pack ODS-A, 5 μm, 250×20 mm (column 2), with a flow rate of 16 mL / min of water (0.1% v / v trifluoroacetic acid) and acetonitrile (0.1% v / v trifluoroacetic acid). Analytical HESI-HPLC-MS (heated electrospray ionization mass spectrometry) was performed on an LCQ Fleet (Thermo Scientific) equipped with an UltiMate 3000 UHPLC focusing (Dionex) connected to a C18 column: S1: Hypersil Gold aQ 3μm, 150×2.1mm (measured for 8 or 20 minutes); S2: Accucore C18, 2.6 μm, 50 × 2.1 mm (measured for 5 minutes) (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 integrin ligands were determined by solid phase binding assays using the protocol described above. 44 , wherein the compound containing a metal binding unit (chelating agent, such as TRAP) is pre-converted into Ga by adding an equimolar amount of Ga(NO3)3 solution III complex.

[0207] Example 1: Peptide Synthesis Process

[0208] The previously established protocol was followed except that the synthesis was performed in DMF instead of N-methyl-2-pyrrolidone (NMP). 44 .

[0209] CTC resin was loaded. Peptide synthesis was performed using CTC resin (0.9 mmol / g) according to the standard Fmoc protected peptide strategy. Fmoc-Xaa-OH (1.5 eq.) was attached to CTC-resin with N,N-diisopropylamine (DIPEA, 2.5 eq.) in anhydrous DCM (0.8 mL / g (resin)) for 1 hour at room temperature. The remaining trityl chloride groups were capped for 15 minutes by adding a solution of MeOH (1 mL / g (resin)) and DIPEA (5:1, v / v). The resin was filtered and washed with DCM (5×) and MeOH (3×).

[0210] The resin was Fmoc-deprotected. The Fmoc-protected peptide resin was treated with 20% piperidine in DMF (v / v) for 10 minutes and then for an additional 5 minutes. The resin was washed with DMF (5×).

[0211] 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 amino peptide resin and shaken at room temperature for 1 hour. The solution was washed with DMF (5×). The completion of the coupling was monitored by analytical RP-HPLC and MS. A small amount of resin was dissolved in 20% HFIP in DCM, followed by small amounts of MeOH and MeCN. The solution was filtered and analyzed by RP-HPLC and MS.

[0212] The resin was N-methylated. 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.) at room temperature for 20 minutes. The resin was washed with DCM (3×) and THF (5×). 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 minutes and then washed with THF (5×) and DMF (5×).

[0213] Cleavage of linear peptides from the resin The peptide resin was treated with 20% HFIP in DCM (3 x 30 min) to ensure complete cleavage of the peptide from the resin under pressure prior to evaporation of the solvent.

[0214] Cyclization of the linear peptide. The peptide was dissolved in DMF (1 mM peptide concentration) before adding NaHCO (5 eq.) and DPPA (3 eq.). The reaction was stirred overnight at room temperature, and the degree of cyclization was monitored by RP-HPLC and MS. The solvent was evaporated under pressure to a small volume, filtered through glass wool, and the solvent was further evaporated.

[0215] Cleavage of the Dde-protecting group. The cyclized peptide was dissolved in DMF before adding hydrazine hydrate (2% v / v). The reaction was stirred at room temperature for 30 minutes. The degree of Dde deprotection was monitored by HPLC-MS.

[0216] Cleavage of acid-labile protecting groups: The cyclized peptide was dissolved in a 10:85:2.5:2.5 (DMF:TFA:TIPS:H2O) solution for 1 hour. The degree of deprotection was monitored by HPLC-MS.

[0217]

[0218] Structural formula of the linear peptide Y(tBu)R(tBu,Fmoc)GD(Pbf)LAY(tBu)p(NMe)K(Dde).

[0219] Synthesis of Y(tBu)R(tBu,Fmoc)GD(Pbf)LAY(tBu)p(NMe)K(Dde). The linear protective peptide Y(tBu)R(tBu,Fmoc)GD(Pbf)LAY(tBu)p(NMe)K(Dde) was synthesized according to the above process. The formation of the complete linear sequence was monitored by HPLC-MS (m / z: 1903.00 [M+H + ] + ,952.08[M+2H + ] 2+ ).

[0220]

[0221] Structural formula of the protected cyclic peptide ring (Y(tBu)R(Pbf)GD(tBu)LAY(tBu)p(NMe)K(Dde)).

[0222] 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 above procedure. Cyclization was performed 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+ ).

[0223]

[0224] The structural formula of Tyr2[cyclo(YRGDLAYp(NMe)K)].

[0225] Synthesis of Tyr2. Cleavage of the Dde protecting group from cyclo(Y(tBu)R(Pbf)GD(tBu)LAY(tBu)p(NMe)K(Dde)) was performed as described above. Cyclo(Y(tBu)R(Pbf)GD(tBu)LAY(tBu)p(NMe)K) was obtained as a white solid in 35% yield (508.7 mg, 339.4 μmol) (depending on the resin loading). RP-HPLC (gradient: 20–60% MeCN in H2O containing 0.1% TFA, 25 min): t R =10.35 min (column 1). After Dde-deprotection, 78 mg of the crude material was dissolved directly in toluene (50 mL) and rotary evaporated to remove any reagent from the Dde-deprotected material. This gave an orange / brown oil that was directly treated with 2 ml of the acid-labile deprotection solution described above. This gave the cyclic peptide Tyr2 [cyclo(YRGDLAYp(NMe)K)] as a colorless solid in a yield of 10.2% (relative to the crude product) (5.75 mg, 5.33 μmol). RP-HPLC (gradient: 20-70% MeCN in H2O containing 0.1% TFA over 25 min): t R =10.07 min (column 1). m / z: 540.14 [M+2H + ] 2+ .

[0226]

[0227] 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 allowed to react for 15 min before being added dropwise to a DMF solution of a dissolved Dde-deprotected peptide (30.27 mg, 20.19 μmol, 1 eq) containing an acid-labile protecting group. The reaction was stirred for 1 hour. The degree of conjugation of the alkyne functional group was monitored by HPLC-MS. The solvent was evaporated under pressure to give an orange / brown oil, which was directly treated with 2 mL of the acid-labile deprotection solution described above. This afforded cyclo(YRGDLAYp(NMe)K(pentynoic acid), BB-5a, as a colorless solid in 57% yield (13.26 mg, 11.45 μmol). RP-HPLC (gradient: 30-50% MeCN in H2O containing 0.1% TFA over 15 min): R =7.67min (column 1). m / z: 1737.30[3M+2H + ] 2+ ,1158.51[M+H + ] + ,580.05[M+2H + ] 2+ .

[0228]

[0229] 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. The mixture was then added dropwise to a DMF solution of YRGD peptide (73.99 mg, 51.86 μmol, 1 eq) with an acid-labile protecting group. The solvent was evaporated under pressure to yield an orange / brown oil, which was directly treated with 3 mL of the acid-labile deprotection solution described above. This afforded C-9 as a colorless solid in 76% yield (45 mg, 39.39 μmol). RP-HPLC (gradient: 30-80% MeCN in H2O containing 0.1% TFA in 20 min): t R =9.4min (column 1). m / z: 1164.41[M+Na+ +H + ] + ,1142.46[M+H + ] + ,572.11[M+2H + ] 2+ .

[0230]

[0231] 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. The mixture was then added dropwise to a DMF solution of FRGD peptide (29.6 mg, 20.75 μmol, 1 eq) with an acid-labile protecting group. The solvent was evaporated under pressure to yield an orange / brown oil, which was directly treated with 2 mL of the acid-labile deprotection solution described above. This afforded C-8 as a colorless solid in 28.2% yield (6.68 mg, 5.85 μmol). RP-HPLC (gradient: 30-80% MeCN in H2O containing 0.1% TFA in 20 min): t R =8.9min (column 1). m / z: 1165.09[M+Na + +H + ] + ,1142.47[M+H + ] + ,572.21[M+2H + ] 2+ .

[0232] Synthesis of C-1. Cyclo(YRGDLAYp(NMe)K(pentynoic acid)) (8.01 mg, 6.92 μmol, 1.5 eq) was added to a minimal amount of H2O solution of TRAP(azide) 1 (3.05 mg, 4.61 μmol, 1 eq) and sodium ascorbate (45.7 mg, 230.5 μmol, 50 eq). Copper(II) acetate (1.1 mg, 5.53 μmol, 1.2 eq) was added, and a brown precipitate immediately formed. After vortexing, the solution turned clear green. The solution was reacted at 60°C for 1 hour without stirring. After 1 hour, the copper demetallation reaction of the peptide chelator compound was completed 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) and adjusting the pH to 2.2 by adding 1 M aqueous HCl. The mixture was reacted at 60°C for 1 hour. The synthesis of TRAP(Tyr2) was monitored by HPLC-MS. C-1 was obtained as a colorless solid in a yield of 5.7% (0.48 mg, 0.26 μmol). RP-HPLC (gradient: 20-70% MeCN in H2O containing 0.1% TFA over 25 min): t R =12.3 min (column 1). m / z: 910.49 [M+2H + ] 2+ ,607.73[M+3H + ] 3+ .

[0233] Synthesis of C-7. Cyclo(YRGDLAYp(NMe)K(pentynoic acid)) (24.96 mg, 21.55 μmol, 3.3 eq) was added to a minimal amount of H2O solution of TRAP(azide) 3 (5.39 mg, 6.53 μmol, 1 eq) and sodium ascorbate (64.7 mg, 326.6 μmol, 50 eq). Copper(II) acetate (1.56 mg, 7.84 μmol, 1.2 eq) was added, and a brown precipitate immediately formed. After vortexing, the solution turned clear green. The solution was reacted at 60°C for 1 hour without stirring. After 1 hour, the copper demetallation reaction of the peptide chelator compound was completed 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) and adjusting the pH to 2.2 by adding 1 M aqueous HCl. The mixture was reacted at 60°C for 1 hour. The synthesis of TRAP(Tyr2)3 was monitored by HPLC-MS. C-7 was obtained as a colorless solid in a yield of 36.1% (10.11 mg, 2.35 μmol). RP-HPLC (gradient: 20-40% MeCN in H2O containing 0.1% TFA over 15 min): t R =17.35 min (column 2). m / z: 1434.01 [M+3H + ] 3+ ,1075.97[M+4H + ] 4+ ,861.03[M+5H + ] 5+ .

[0234] Synthesis of C-8. BB-7a (6 mg, 5.25 μmol, 3.3 eq) was added to a minimal amount of HO:tBuOH (4:1) solution of TRAP (azide) 3 (1.3 mg, 1.6 μmol, 1 eq) and sodium ascorbate (15.8 mg, 79.6 μmol, 50 eq). Copper(II) acetate (381.3 μg, 1.91 μmol, 1.2 eq) was added, and a brown precipitate immediately formed. After vortexing, the solution turned transparent green. The solution was reacted at 60°C for 1 hour without stirring. After 1 hour, the formation of C-8 was monitored by HPLC-MS. Copper removal by the peptide chelator compound was performed by adding 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) (14.5 mg, 47.8 μmol, 30 eq.) dissolved in water (0.5 mL) and adjusting the pH to 2.2. The mixture was reacted at 60°C for 1 h. This afforded C-8 as a colorless solid in a yield of 42.9% (2.9 mg, 0.7 μmol). RP-HPLC (gradient: 10-70% MeCN in H2O containing 0.1% TFA over 20 min): R =19.2min (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+ .

[0235] 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 minimum amount of H2O:tBuOH (4:1). Copper(II) acetate (2.86 mg, 14.32 μmol, 1.2 eq) was added, and a brown precipitate immediately formed. After vortexing, the solution turned clear green. The solution was reacted at 60°C for 1 hour without stirring. After 1 hour, the formation of C-9 was monitored by HPLC-MS. Copper removal of the peptide chelator compound was performed by adding 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) (110.7 mg, 365 μmol, 30 eq.) dissolved in water (1 mL) and adjusting the pH to 2.2. The mixture was reacted at 60°C for 1 h. This afforded C-9 as a colorless solid in a yield of 24.7% (12.78 mg, 3.01 μmol). RP-HPLC (gradient: 10-70% MeCN in H2O containing 0.1% TFA over 20 min): t R =19.5min (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+ .

[0236] Synthesis of C-10 and C-11. The structural unit AvB6 (as in Maltsev et al. 38To a minimal amount of H2O solution of TRAP(azide)3 (4.46 mg, 5.4 μmol, 1 eq) and sodium ascorbate (53.47 mg, 269.90 μmol, 50 eq) was added 1.29 mg, 6.48 μmol, 1.2 eq) of copper(II) acetate, which immediately formed a brown precipitate. After vortexing, the solution turned transparent green. The solution was reacted at 60°C for 1 hour without stirring. BB-5a (13.75 mg, 11.87 μmol, 2.2 eq) was added directly to the reaction mixture and reacted at 60°C for another 1 hour without stirring. After 1 hour, copper demetallation of the peptide chelator compound was performed 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) and adjusting the pH to 2.2. The mixture was reacted at 60°C for 1 hour. The formation of C-11 and C-10 was monitored by HPLC-MS.

[0237] C-10 was obtained as a colorless solid in a yield of 6.8% (1.55 mg, 0.37 μmol). RP-HPLC (gradient: 40-95% MeCN in H2O containing 0.1% TFA over 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+ .

[0238] C-11 was obtained as a colorless solid in 8.75% yield (2 mg, 0.47 μmol). RP-HPLC (gradient: 40-95% MeCN in H2O containing 0.1% TFA over 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+ .

[0239] radiochemistry

[0240] The radiometal incorporation and radiochemical purity of the labeled compounds were determined by radio-TL on ITLC silica gel impregnated chromatographic paper (Agilent, Santa Clara, USA; eluent: 0.1 M trisodium citrate or 1 M ammonium acetate and methanol in a 1:1 (v / v) mixture) and analyzed using a scan-RAM radio-TLC detector from LabLogic Systems (Brandon, USA). 45 As stated, 68 Ga labeling was performed using a fully automated on-site system (Scintomics GallElut + , Lindach, Germany). Briefly, the SnO2 matrix was prepared by adding HEPES buffer (450 μL, 2.7 M) in water. 68 Ge / 68 Ga-generator (IThemba LABS, South Africa; 1.25 mL, eluent: 1 M aqueous HCl solution containing approximately 500 MBq 68 The eluent of Ga) was adjusted to pH 2 and used to label 5 nmol of chelate conjugate at 95°C for 2 minutes. The radiolabeled peptide was The product was captured on a C8 light solid phase extraction (SPE) column, which was washed with water (10 mL). The product was eluted with 2 mL of 50% ethanol in water. After evaporation of the ethanol, the purity was determined by radioactive TLC, which always yielded a value of ≥98%.

[0241] Example 2: Activity Evaluation

[0242] Determination of logD value

[0243] In order to determine the n-octanol-PBS partition coefficient (log D 7.4 ), 500 μL of 1-octanol and 500 μL of phosphate-buffered saline were combined in a 1.5 mL microcentrifuge tube. Approximately 1 MBq of radiolabeled compound was added and vortexed vigorously for 3 minutes. The sample was centrifuged (13,000 rpm, 5 minutes), and the activity in 200 μL of the organic phase and 20 μL of the aqueous phase was quantified in a gamma counter.

[0244] Cell lines and animal models

[0245] All animal studies were performed in accordance with the general animal welfare regulations of 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 according to the distributor's recommendations. To generate tumor xenografts, 10 μg of ... 7Female CB17 SCID mice (Charles River) aged 6 to 8 weeks were inoculated with H2009 cells (Cultrex BME, PathClear Type 3; Trevigen, GENTAURG MBH). When tumors grew to 10-12 mm in diameter (4-6 weeks after inoculation), the mice were used for biodistribution or PET studies.

[0246] PET imaging

[0247] Mice were anesthetized with isoflurane and radiolabeled compounds were administered intravenously. The range of administration activity for each mouse was between 10MBq and 15MBq (100-200pmol, depending on the variation of production and administration time). PET imaging was performed on a Siemens Inveon small animal PET system and was performed dynamically for 90 minutes under isoflurane anesthesia, or as a single frame with pi (intraperitoneal) for 75 minutes, with an acquisition time of 20 minutes. Data were reconstructed using Siemens Inveon Research Workspace software using the three-dimensional ordered subset expectation maximum (OSEM3D) algorithm without scattering and attenuation correction. For kinetic analysis, regions of interest (ROI) were manually defined.

[0248] Biodistribution

[0249] For biodistribution studies, 3-6 MBq (between 70-140 pmol) of radiolabeled compound was injected into the tail vein. Mice were sacrificed 90 minutes after injection, blood samples were taken, and target organs were dissected. 2 The activity in the weighed tissue samples was quantified using an automated gamma counter (PerkinElmer, Waltham, MA, USA). The injected dose per gram of tissue (% ID / g) was calculated from the organ weight and the counted activity.

[0250] result

[0251] As described above, novel peptide compounds and conjugates were synthesized and characterized.

[0252] Phe2 and Tyr2, Ga-68-TRAP(Phe2)3 38 and Ga-68-C-7 68 The Ga-labeled trimer conjugates were evaluated in H2009 tumor-bearing mice. Comparison of PET images ( Figure 1) showed that Ga-68-C-7 (rather than Ga-68-TRAP(Phe2)3) achieved low background activity and clear delineation of tumors, primarily due to strong uptake in the liver. The corresponding in vitro biodistribution data ( Figure 2 ) demonstrated high levels of accumulation of Ga-68-TRAP(Phe2)3 in the liver. Because this uptake was not reduced by co-injection of a high excess (50 nmol) of unlabeled TRAP(Phe2)3 (blocking), it was demonstrated that it was not target specific. Surprisingly, substitution of Tyr for Phe in Ga-68-C-7 reduced this nonspecific uptake to insignificance and also reduced nonspecific uptake in other compartments and tissues (i.e., blood, heart, spleen, and tumors), ultimately leading to the following: Figure 1 Excellent PET image contrast shown.

[0253] Although biokinetic analysis ( Figure 3 ) showed that both compounds had good tumor retention, but Ga-68-C-7 was cleared faster from the blood pool, ultimately leading to Figure 1 The background is lower in the PET image shown.

[0254] In summary, compared with the corresponding prior art compound Ga-68-TRAP(Phe2)3 38 In comparison, Ga-68-C-7 showed significantly improved biokinetics and imaging properties, confirming that Tyr2 is advantageous for the in vivo application of αvβ6-integrin targeting compounds.

[0255] The results were evaluated in H2009 tumor-bearing mice. 68 Biodistribution of Ga-labeled trimeric TRAP conjugates. 68 Ga-labeled trimeric TRAP conjugates include different combinations of Phe2, FRGD, YRGD, and Tyr2, 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. Figure 4They showed that even exchanging a single Phe2 in the Ga-68-TRAP(Phe2)3 structure with Tyr2 to produce Ga-68-C-11 significantly reduced nonspecific liver uptake (demonstrated by the similarity of control and blocking experiments), reduced residual activity in the blood, and reduced pancreatic uptake, while Ga-68-C-10 still showed high tumor uptake. Exchanging both Phe2 in the Ga-68-TRAP(Phe2)3 structure with Tyr2 to produce Ga-68-C-10 had similar, albeit even more pronounced, effects. Similarly, exchanging all Phe2 in the Ga-68-TRAP(Phe2)3 structure with FRGD or YRGD to produce Ga-68-C-8 and Ga-68-C-9, respectively, demonstrated that cyclic peptides containing only one tyrosine also exhibit excellent properties. Of all the trimeric conjugates studied, Ga-68-C-7 showed the best tumor-to-liver ratio and, in particular, tumor-to-pancreas ratio, suggesting that it should be best suited for imaging αvβ6-integrin-positive lesions in these organs, such as metastases or primary tumors of the pancreatic adenocarcinoma type.

[0256] Figure 5 It was demonstrated that the peptides FRGD and YRGD, characterized by 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. Figure 6 The results showed that the blood clearance of Ga-68-C-8 and Ga-68-C-9 was much faster than that of Ga-68-TRAP(Phe2)3 and was similar to that of Ga-68-C-10.

[0257] References

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[0259] 2Avraamides CJ, Garmy-Susini B, Varner JA. Integrins in angiogenesis and lymphangiogenesis. Nat Rev Cancer. 2008:8; 604-617.

[0260] 3Breuss JM,Gillett N,Lu L,Sheppard D,Pytela R.Restricted distributionof integrinβ6mRNA in primate epithelial tissues.J Histochem Cytochem.1993:41;1521-1527.

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Claims

1. A conjugate represented by the following formula (I) or a pharmaceutically acceptable salt thereof: E(Cp) n (THE) in, Each Cp represents a cyclic peptide of the formula cyclo(YRGDLAYp(NMe)K), "Tyr2", n is an integer selected from 1 to 4, and E represents an effector moiety, wherein the effector moiety is covalently bound to the cyclic peptide through the terminal amino group of the (NMe)K residue, and wherein the effector moiety comprises an atom or group of atoms suitable for diagnosing, imaging, or treating a medical indication associated with increased expression of αvβ6-integrin selected from cancer and fibrosis.

2. The conjugate according to claim 1 or a pharmaceutically acceptable salt thereof, wherein The conjugate of formula (I) is characterized by a formula selected from 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 group of atoms capable of forming a chelate complex, Aa' represents an active atom or group of atoms capable of forming a covalent bond, Cg represents a chelating group, k is 1 or 0, S represents an atomic group used as a spacer, and n is as defined above with respect to formula (I), provided that if k is 0 then n is 1, o can be any integer from 1 to n, p can be 1 or 2, m is 0 or 1, n' is 1, 2 or 3, provided that n'+1 is the number of free valences of the chelating group or less, and Cm is a central portion comprising 1 to 30 atoms selected from C, N, O, S and P.

3. The conjugate according to claim 1 or a pharmaceutically acceptable salt thereof, wherein The atom or group of atoms suitable for diagnosing, imaging, or treating a medical indication associated with increased expression of αvβ6-integrin selected from cancer and fibrosis is selected from the group consisting of: a radioisotope suitable for scintigraphy, SPECT imaging or PET imaging, or targeted radiation therapy; a chromophore of a fluorescent dye; a contrast agent for magnetic resonance imaging; an atom or group of atoms suitable for imaging by X-ray-based techniques; or an atom or group of atoms derived from a therapeutic agent suitable for treating a medical indication associated with increased expression of αvβ6-integrin selected from cancer or fibrosis, wherein the term "derived from" means that the atom contained in the conjugate has the same structure as the compound from which the atom is derived, except that a hydrogen atom is replaced by a covalent bond connecting the atom to the remainder of the conjugate.

4. The conjugate according to claim 2 or a pharmaceutically acceptable salt thereof, wherein The reactive atom or reactive group of atoms is selected from the group consisting of a radioisotope suitable for scintigraphy, SPECT imaging or PET imaging, or targeted radiotherapy; a chromophore of a fluorescent dye; a contrast agent for magnetic resonance imaging; an atom or a group of atoms suitable for imaging by X-ray based techniques; or an atom or a group of atoms derived from a therapeutic agent suitable for treating a medical indication associated with increased expression of αvβ6-integrin selected from cancer or fibrosis, wherein the term "derived from" means that the radical contained in the conjugate has the same structure as the compound from which the radical is derived, the only difference being that a hydrogen atom is replaced by a covalent bond connecting the radical to the rest of the conjugate.

5. The conjugate according to claim 2 or 4, or a pharmaceutically acceptable salt thereof, wherein: The active atom or active atom group is a metal ion selected from the following: 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+ , and mixtures thereof.

6. The conjugate according to claim 2 or 4, or a pharmaceutically acceptable salt thereof, wherein: The active atom or active atom group is a radioactive isotope selected from the following: 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 Such as 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 Second, 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.

7. The conjugate according to claim 2 or 4, or a pharmaceutically acceptable salt thereof, wherein: The active atom or active atom group is a non-metallic radioactive isotope selected from the following: 11 C. 13 N. 15 O. 18 F. 123 I. 124 I. 125 I. 131 I.

8. The conjugate according to claim 6 or a pharmaceutically acceptable salt thereof, wherein The radioisotope is selected from 68 Ga, 44 Sc, 99m Tc, 111 In, 64 Cu, 89 Zr, 90 Y. 177 Lu, 213 Bihe 225 Ac.

9. The conjugate according to claim 2 or 4, or a pharmaceutically acceptable salt thereof, wherein: The active atom or active atom group is a contrast agent for magnetic resonance imaging, and the contrast agent is selected from Gd, Fe and Mn.

10. The conjugate according to claim 2 or 4, or a pharmaceutically acceptable salt thereof, wherein: The active atom or active atomic group is a therapeutic group derived from a drug for treating fibrosis or an anticancer drug, wherein the drug for treating fibrosis or the anticancer drug is selected from alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors and other antitumor drugs, wherein the term "derived from" means that the atomic group contained in the conjugate has the same structure as the compound from which the atomic group is derived, with the only difference being that a hydrogen atom is replaced by a covalent bond that binds the atomic group to the rest of the conjugate.

11. The conjugate according to claim 2 or a pharmaceutically acceptable salt thereof, wherein: The atomic group as a spacer is a linear chain of 2 to 20 atoms selected from C, N, O, P and S, the linear chain optionally carrying one or more substituents, the remaining valences being saturated with hydrogen.

12. The conjugate according to claim 11 or a pharmaceutically acceptable salt thereof, wherein The linear chain has 3 to 10 atoms selected from C, N, O, P and S.

13. The conjugate according to claim 2 or a pharmaceutically acceptable salt thereof, wherein The atomic group serving as a spacer is selected from the following formulae (IIIa) to (IIIf'): *-C(O)-(CH2) k -(taz) l -(CH2) m -(IIIa) *-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 represents a triazole ring in which all three nitrogen atoms are adjacent to each other, l can be 0 or 1, k, m and, if present, o are each independently selected from integers ranging from 0 to 20 such that k+m=2-20 and k+m+o=2-20, and wherein an asterisk (*) marks the point of attachment of the cyclic peptide.

14. The conjugate according to any one of claims 1 to 2 or a pharmaceutically acceptable salt thereof, wherein: n is 3.

15. The conjugate according to claim 2 or a pharmaceutically acceptable salt thereof, wherein The chelating group is selected from the following formulae (IVa) to (IVd): wherein the asterisk * marks the point of attachment of the atomic group serving as a spacer, provided that if the number of the cyclic peptides and associated spacers is less than the number of valences of the chelating group, wherein the number of the cyclic peptides and associated spacers is characterized by the variable n, the remaining valences indicated by the asterisks are saturated with hydrogen or another atomic group.

16. The conjugate according to claim 15 or a pharmaceutically acceptable salt thereof, wherein The chelating group is selected from formula (IVa) and (IVb).

17. The conjugate according to claim 15 or a pharmaceutically acceptable salt thereof, wherein The another atomic group is a group selected from -CH2-COOH and -CH2-CH2-COOH.

18. The conjugate according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein: The conjugate comprises a compound selected from the group consisting of:

19. Use of the conjugate according to any one of claims 1 to 9 and 11 to 18 or a pharmaceutically acceptable salt thereof in the preparation of a diagnostic agent for use in a method for diagnosing or imaging a disease associated with increased αvβ6-integrin expression, wherein The disease associated with increased expression of αvβ6-integrin is fibrosis or cancer.

20. Use of the conjugate according to any one of claims 1 to 4 and 10 to 18 or a pharmaceutically acceptable salt thereof in the preparation of a pharmaceutical composition for treating a disease associated with increased αvβ6-integrin expression, wherein The disease associated with increased expression of αvβ6-integrin is fibrosis or cancer.

21. Use of the conjugate according to any one of claims 1 to 9 and 10 to 18 or a pharmaceutically acceptable salt thereof for preparing a pharmaceutical composition for use in a method for localizing cells with increased αvβ6-integrin expression in a patient, wherein The conjugate, or a pharmaceutically acceptable salt thereof, has been administered to a patient, wherein the method comprises subjecting the patient to an imaging method selected from PET, SPECT, MRI, and X-ray computed tomography, wherein the conjugate comprises an active atom or group of atoms that is compatible with the imaging method to be performed.

22. A structural unit compound selected from the following compounds 1) to 5): 1)Cg(SCp) n (IIa) wherein Cg represents a chelating group, S represents an atomic group used as a spacer, each Cp is a cyclic peptide of the formula cyclo(YRGDLAYp(NMe)K), and n is an integer from 1 to 4; 2) Ring (YRGDLAYp(NMe)K); Ring (3-I-YRGDLAYp(NMe)K); Ring (3-I-YRGDLA3-I-Yp(NMe)K); Ring (YRGDLA3-I-Yp(NMe)K); wherein 3-IY represents a Tyr residue having an iodine atom at position 3 of the phenyl ring, wherein the iodine atom can be any non-radioactive isotope or radioactive isotope of iodine; 3) 4) as well as 5) 23. A pharmaceutical composition comprising: The conjugate according to any one of claims 1 to 18, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, and optionally one or more other therapeutic agents.

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