Novel small gastrin-derived cholecystokinin 2 receptor binding molecules for imaging and targeted radiotherapy

Abstract

The present invention relates to a tetrapeptide binding to the cholecystokinin 2 receptor (CCK-2R) wherein the tetrapeptide is represented by the formula (I) Xaa1-Xaa2-Xaa3-Xaa4 (formula (I)) or a salt thereof wherein Xaa1 is Trp, (beta-(3-benzothienyl)-alanine, Trp wherein one or more H atoms of 1H-indol-3-yl are substituted with substituents, and wherein one or more H atoms of the 1H-indol-3-yl are substituted with substituents, and X is a hydrogen atom or a hydrogen atom of the (beta-(3-benzothienyl)-alanine. Wherein, for each H atom, the substituents are independently selected from C1 to C3 alkyl,-OH,-SH,-F and-Cl, Phe, 1-Nal, 2-Nal, Tyr or para-amino-Phe, Xaa2 is an N-methylamino acid wherein N-methyl is at a carbon alpha, and preferably an N-methylamino acid containing an aliphatic side chain wherein N-methyl is at a carbon alpha, Xaa3 is an amino acid having an acidic side chain at neutral pH, and X is an amino acid having an aliphatic side chain at neutral pH. And Xaa4 is an amino acid having an aromatic side chain or an amide thereof.

Description

[0001] The present invention relates to a tetrapeptide that binds to a cholecystokinin 2 receptor (CCK-2R), wherein the tetrapeptide is represented by formula (I) Xaa1-Xaa2-Xaa3-Xaa4 (formula (I)) or a salt thereof, wherein Xaa1 is Trp, (β-(3-benzothienyl)-alanine, Trp, wherein one or more H atoms of the 1H-indol-3-yl group are substituted by a substituent, and for each H atom the substituent is independently selected from C 1 To C 3 alkyl, -OH, -SH, -F and -Cl, Phe, 1-Nal, 2-Nal, Tyr or p-amino-Phe, Xaa2 is an N-methyl amino acid wherein the N-methyl group is at the alpha carbon, and preferably an N-methyl amino acid containing an aliphatic side chain wherein the N-methyl group is at the alpha carbon, Xaa3 is an amino acid having an acidic side chain at neutral pH, and Xaa4 is an amino acid having an aromatic side chain or its amide.

[0002] In this specification, many documents are cited, including patent applications and manufacturer's manuals. Although the disclosures of these documents are considered irrelevant to the patentability of the present invention, their entirety is hereby incorporated by reference. More specifically, all cited documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

[0003] Medullary thyroid carcinoma (MTC), a neuroendocrine tumor, reportedly accounts for 1–2% of all thyroid cancers in the United States [1]. In general, thyroid cancer accounts for approximately 3% of all new cancer cases in the United States, which is why the occurrence of MTC is considered rare [2]. Although the detection of MTC has improved through different screening methods and biopsy techniques, the average survival time remains short (8.6 ± 7.6 years), especially when metastasis has already begun [3–7].

[0004] Among MTC patients, approximately 70% and 30% have the sporadic form and the hereditary / familial form, respectively [8]. For the diagnosis of hereditary MTC, molecular analysis of DNA is used to detect receptor tyrosine kinase (RET) oncogene mutations. As a result, patients at risk of MTC can be regularly examined, enabling early detection of MTC lesions [9,10].

[0005] The diagnosis of sporadic MTC is usually performed after fine needle aspiration. Given its neuroendocrine origin, the production of calcitonin is a unique feature of this tumor. Injection of calcium or the synthetic gastrin analog pentagastrin stimulates the secretion of calcitonin, especially from MTC cells [9, 11]. Therefore, elevated calcitonin levels are a strong indicator of MTC. However, biochemical tests only indicate the presence of tumors / metastases, and localization is usually impossible

[12] . As a complement to these screening methods, better and earlier localization of tumors and metastases can be achieved through molecular imaging using positron emission tomography / computed tomography (PET / CT) [13, 14].

[0006] The recently published EANM practice guidelines for MTC imaging recommend 68 Ga-labeled somatostatin analogs, [ 18 F]fluorodeoxyglucose ([ 18 F]FDG) and [ 18 F] fluorodihydroxyphenylalanine ([ 18 F]F-DOPA)

[15] . 18 F]FDG PET / CT (17%) or 68 Compared with PET / CT performed with Ga-labeled somatostatin analogs (33%), 18 F]F-DOPA PET / CT showed an improved detection rate (72%) in patients with recurrent MTC

[16] . 18 F]F-DOPA PET shows good sensitivity (86%) in primary tumors but only moderate (57%) to low (6%) sensitivity in lymph node (LN) and distant metastases, respectively

[17] . In addition, as reported in a recent literature meta-analysis, in patients with suspected recurrent MTC, [ 18 The per-patient detection rate of F]F-DOPA PET or PET / CT was only 66% (95% confidence interval, 58–74%)

[18] .

[0007] For patients with metastatic disease, treatment options remain limited. Neither external beam radiotherapy nor conventional chemotherapy has shown adequate efficacy and is therefore not recommended [1, 19, 20]. Unlike common forms of thyroid cancer, MTC arises from the malignant dedifferentiation of parafollicular cells. Since these cells do not accumulate iodine, radioactive iodine therapy is not a treatment option [21, 22]. For this reason, total thyroidectomy and central neck dissection remain the most commonly performed procedures in cases of MTC [1, 23]. However, in general, the 10-year survival rates for patients with local and distant metastases are 76% and 40%, respectively [6]. Tyrosine kinase inhibitors such as seraceptinib, vastatinib, or cabozantinib have been shown to prolong (mainly progression-free) survival [1]. However, therapy is associated with a variety of side effects such as hypertension, nephrotoxicity, and hepatotoxicity [1, 24].

[0008] Over the past few decades, the field of nuclear medicine has emerged as a powerful alternative due to its non-invasive imaging and therapy opportunities [13, 14]. As a complement to the screening methods mentioned above, the localization of tumors and metastases can therefore be determined via molecular imaging. In the 1990s, the diagnosis of MTC was first achieved via somatostatin receptor scintigraphy (SRS) using a somatostatin receptor subtype 2 (sstr2)-specific ligand. Despite considerable improvements in diagnosis, a large number of MTC-negative scans are still reported, especially in patients with metastatic spread

[25] . For this reason, new options for MTC targeting are necessary, especially for progressive disease and metastasis.

[0009] As reported by Reubi et al. in 1997, MTC overexpresses cholecystokinin receptors A (CCK-A) and B (CCK-B), the latter also known as gastrin receptor or CCK-2 receptor (CCK-2R)

[26] . In 2006, Gotthardt et al. showed that CCK-2R expression was more prevalent than sstr2 expression in MTC-derived metastases (94% vs. 40%, respectively)

[27] . Since then, the development of CCK-2R-specific tracers based on the endogenous ligands CCK or minigastrin has increased[28,29].

[0010] In view of the above, there is a continuing need for further options for the diagnosis and treatment of cancers expressing CCK-2R, such as MTC. The present invention addresses this need.

[0011] Thus, in a first aspect, the present invention relates to a tetrapeptide that binds to a cholecystokinin 2 receptor (CCK-2R), wherein the tetrapeptide is represented by formula (I) Xaa1-Xaa2-Xaa3-Xaa4 (Formula (I)) or a salt thereof, wherein Xaa1 is Trp, (β-(3-benzothienyl)-alanine, Trp, wherein one or more H atoms of the 1H-indol-3-yl group are substituted with a substituent, and for each H atom the substituent is independently selected from C 1 To C 3 alkyl, -OH, -SH, -F and -Cl, Phe, 1-Nal, 2-Nal, Tyr or p-amino-Phe, Xaa2 is an N-methyl amino acid wherein the N-methyl group is at the alpha carbon, and preferably an N-methyl amino acid containing an aliphatic side chain wherein the N-methyl group is at the alpha carbon, Xaa3 is an amino acid having an acidic side chain at neutral pH, and Xaa4 is an amino acid having an aromatic side chain or its amide.

[0012] As used herein, the term "tetrapeptide" relates to a peptide consisting of 4 amino acids which are covalently linked via peptide bonds.

[0013] The salt of the tetrapeptide is preferably a pharmaceutically acceptable salt.

[0014] The first amino acid is designated as Xaa1 and may be Trp, (β-(3-benzothienyl)-alanine, Trp, wherein one or more H atoms of the 1H-indol-3-yl group are substituted with a substituent, wherein for each H atom the substituent is independently selected from C 1 To C 3 The one or more H atoms are preferably 1 or 2 H atoms, and preferably 1 H atom. In the substituent, C 1 To C 3 Alkyl groups are preferred, and C 1 To C 3 The alkyl group is preferably ethyl or methyl and most preferably methyl.

[0015] The second amino acid is designated as Xaa2 and may be an N-methyl amino acid (wherein the N-methyl group is at the α carbon), and is preferably an N-methyl amino acid (wherein the N-methyl group is at the α carbon) containing an aliphatic side chain. The N-methyl amino acid is preferably derived from 22 proteinogenic amino acids or proteinogenic amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, selenocysteine ​​and pyrrolysine (note that pyrrolysine is not used in human protein synthesis)). Based on these proteinogenic amino acids or proteinogenic amino acids, the NH at the αC atom can be replaced by an N-methyl group. 2 The N-methyl group is obtained by adding aliphatic groups to obtain N-methyl amino acids (wherein the N-methyl group is at the alpha carbon). Of the 22 proteinogenic amino acids or proteinogenic amino acids, alanine, isoleucine, leucine, proline, valine, and methionine have aliphatic side chains. Amino acids with aliphatic side chains are also referred to herein as aliphatic amino acids. Aliphatic amino acids are nonpolar and hydrophobic.

[0016] In organic chemistry, the alpha carbon (Cα) refers to the first carbon atom attached to a functional group (such as the COOH group in the 22 proteinogenic amino acids or proteinogenic amino acids). Therefore, such amino acids are also called alpha amino acids. The second carbon atom is called the beta carbon (Cβ), and the naming system continues in Greek alphabetical order.

[0017] The third amino acid is designated as Xaa3 and may be an amino acid having an acidic side chain at neutral pH. Amino acid Xaa3 is preferably an alpha amino acid. Among the 22 proteinogenic amino acids or proteinogenic amino acids, two amino acids have acidic side chains at neutral pH, namely aspartate (Asp) and glutamic acid or glutamate (Glu).

[0018] The fourth amino acid is designated as Xaa4 and may be an amino acid with an aromatic side chain or an amide thereof. Of the 22 proteinogenic amino acids or proteinogenic amino acids, phenylalanine, tryptophan and tyrosine are amino acids with aromatic side chains. Although histidine contains an aromatic ring, its basic properties make it primarily classified in the art and herein as a polar amino acid or an amino acid containing an aliphatic side chain. In the case of an amide of an amino acid with an aromatic side chain, the normal C-terminus of the tetrapeptide is -NH 2 This amide is also referred to herein as Xaa4-NH 2 The C-terminus of the tetrapeptide Xaa1-Xaa2-Xaa3-Xaa4 (Formula (I)) is preferably an amide. In this case, the tetrapeptide can be represented as Xaa1-Xaa2-Xaa3-Xaa4-NH 2 .

[0019] The tetrapeptide of the present invention binds to the cholecystokinin 2 receptor (CCK-2R). CCK-2R is also called cholecystokinin B receptor. The protein is a G protein-coupled receptor for gastrin and cholecystokinin (CCK), a regulatory peptide of the brain and gastrointestinal tract. This protein is a type B gastrin receptor, which has a high affinity for both sulfated and non-sulfated CCK analogs and is primarily present in the central nervous system and gastrointestinal tract. In cells of colorectal tumors, pancreatic tumors, and thyroid cancer, particularly medullary thyroid carcinoma (MTC), mis-splicing transcript variants including introns have been observed.

[0020] The tetrapeptide preferably specifically binds to CCK-2R. Specific binding means that the binding molecule does not substantially bind to or does not bind to other proteins or peptides other than CCK-2R. In particular, it is preferred that the binding molecule cannot bind to other cholecystokinin receptors other than CCK-2R. For example, the tetrapeptide is only suitable for research purposes. For example, the tetrapeptide can be used in immunoassays to capture CCK-2R, such as ELISA or Western blot. Immunoassays are biochemical tests that can measure the presence or concentration of CCK-2R in a sample (e.g., a solution). As will be further explained below, the tetrapeptide is particularly suitable for medical and diagnostic applications of diseases associated with the expression of CCK-2R.

[0021] It can be concluded from the attached examples that the tetrapeptide of the first aspect of the present invention is identical or very similar to the four N-terminal amino acids of the prior art CCK-R2 binder minigastrin having seven amino acids (i.e., Trp-(N-Me)Nle-Asp-1-Nal motif). Only the four N-terminal amino acids of minigastrin H-Trp-(N-Me)Nle-Asp-1-Nal-NH 2 The finding that F11N is required for high CCK-2R affinity is surprising and beneficially leads to smaller CCK-R2 binders. It is noteworthy that many prior art CCK-R2 binders (including F11N) consist of seven amino acids. The four N-terminal amino acids of F11N are bound to H-Trp-Nle-Asp-Phe-NH 2 Closely related, but did not show high CCK-2R affinity. Therefore, it is believed that the (N-Me) amino acid at the Xaa2 position in the tetrapeptide is particularly important for ensuring CCK-R2 binding. As far as the inventors know, peptide high-affinity CCK-2R ligands consisting of only four amino acids are not known in the prior art. It is also shown in the attached examples that when the tetrapeptide is connected to a linker moiety such as a PEG chain and a chelating agent capable of complexing radioactive or non-radioactive cations, the high CCK-2R affinity of the tetrapeptide is retained. Therefore, the tetrapeptide can not only be used alone, but can also become a key element in designing further CCK-2R binding compounds with diagnostic or medical value.

[0022] According to a preferred embodiment of the first aspect of the present invention, Xaa1 is Trp.

[0023] The attached examples reveal that Trp at position Xaa1 shows the highest binding affinity to CCK-2R. β-(3-benzothienyl)-alanine and wherein one or more H atoms of 1H-indol-3-yl are substituted with a substituent (for each H atom the substituent is independently selected from C 1 To C 3 Trp with alkyl, -OH, -SH, -F, and -Cl) is very similar to Trp and should therefore also work.

[0024] In addition, according to a preferred embodiment of the first aspect of the present invention, Xaa2 is (N-Me)Nle, (N-Me)Met, (N-Me)Ile, (N-Me)Leu, (N-Me)Val, (N-Me)Gly, (N-Me)Ala, (N-Me)Glu, preferably (N-Me)Nle or (N-Me)Met and most preferably (N-Me)Nle.

[0025] The attached examples reveal that (N-Me)Nle at position Xaa2 shows the highest binding affinity to CCK-2R through the test of tetrapeptides that are identical or very similar to the four N-terminal amino acids of minigastrin. Because (N-Me)Ile, (N-Me)Leu, (N-Me)Val, (N-Me)Gly, (N-Me)Ala, (N-Me)Glu are similar to (N-Me)Nle, and (N-Me)Met is very similar to (N-Me)Nle, it is believed that these substitutes in particular can be used to replace (N-Me)Nle.

[0026] According to another preferred embodiment of the first aspect of the present invention, Xaa3 is Asp, isoaspartic acid, Glu or isoglutamine, and is preferably Asp.

[0027] In addition, the attached examples reveal through various tetrapeptides and the four N-terminal amino acids of minigastrin that are identical or very similar to the test, and Asp at Xaa3 position shows the highest binding affinity to CCK-2R. Because isoaspartic acid, Glu or isoglutamine are very similar to Asp, it is believed that these substitutes can be used to replace Asp in particular. Among substitutes, Glu is preferred because it is a protein amino acid or a protein amino acid.

[0028] According to another preferred embodiment of the first aspect of the present invention, Xaa4 is 1-Nal, Phe, Trp, Tyr, 2-Nal or amino-Phe, preferably 1-Nal, Phe, Tyr or an amide of any of the foregoing, and most preferably 1-Nal or Tyr, or an amide of 1-Nal or Tyr.

[0029] The attached examples also reveal that 1-Nal or Tyr at position Xaa4 shows the highest binding affinity to CCK-2R by testing of tetrapeptides identical or very similar to the four N-terminal amino acids of minigastrin. Because Phe, Trp, 2-Nal and amino-Phe are very similar to 1-Nal or Tyr, it is believed that these substitutes in particular can be used to replace 1-Nal or Tyr.

[0030] 1-Nal, Phe, Trp, Tyr, or 2-Nal and p-amino-Phe and their amides are amino acids in which the aromatic ring of the side chain may be found to be substituted with H atoms. Thus, Xaa4 may also be a derivative of 1-Nal, Phe, Trp, Tyr, or 2-Nal, p-amino-Phe or their amides in which one or more H atoms of the aromatic ring in the side chain are substituted with a substituent, said substituent being independently selected from C 1 To C 3 The one or more H atoms are preferably 1 or 2 H atoms, and preferably 1 H atom. In the substituent, C 1 To C 3 Alkyl groups are preferred, and C 1 To C 3 The alkyl group is preferably ethyl or methyl and most preferably methyl.

[0031] According to another preferred embodiment of the first aspect of the present invention, the CCK-2R binding affinity of the tetrapeptide is characterized as a half maximal inhibitory concentration (IC) of 100 nM or less, preferably 20 nM or less and most preferably 7.5 nM or less. 50 ).

[0032] As shown in Table 2 of the attached Examples, a tetrapeptide identical to the four N-terminal amino acids of minigastrin showed an IC of 50 The IC value for CCK-2R is 6.4±1.0nM. 50 The tetrapeptide H-Trp-(N-Me)Nle-Asp- Phe -NH 2 and H-Trp-(N-Me)Nle-Asp- Tyr -NH2 Even slightly better. Tetrapeptide H-Trp-(N-Me)Nle-Asp-Trp-NH 2 Still maintains IC of 18.8±0.5nM 50 , and the tetrapeptide H-Trp-(N-Me)Nle-Asp-2-Nal-NH 2 Maintaining an IC of 102±9nM 50 .

[0033] These specifically tested peptides fall within the scope of the preferred embodiment above and show an IC of that preferred embodiment. 50 Values ​​are technically supported by exemplified tetrapeptides.

[0034] In a second aspect, the invention relates to a conjugate comprising within a single molecule:

[0035] (a) the tetrapeptide of the first aspect of the invention, and (b) one or more chelating groups capable of complexing non-radioactive or radioactive cations, or one or more chelating groups containing chelated non-radioactive or radioactive cations.

[0036] The one or more chelating groups are preferably one or two chelating groups, and most preferably one chelating group.

[0037] A chelating group capable of complexing a non-radioactive or radioactive cation refers to a state in which the chelating group is not complexed with a non-radioactive or radioactive cation but is still capable of complexing with a non-radioactive or radioactive cation. In this case, the non-radioactive or radioactive cation is not part of the conjugate of the second aspect of the invention.

[0038] On the other hand, in the chelating group containing a chelated non-radioactive or radioactive cation, the non-radioactive or radioactive cation is complexed to the chelating group such that the non-radioactive or radioactive cation is part of the conjugate of the second aspect of the invention.

[0039] Various chelating agents from which suitable chelating groups can be derived are well known in the art and can be used in the context of the present invention. Metal chelators or cationic chelators (e.g., macrocyclic or acyclic compounds) suitable for serving as chelating groups can be obtained from many manufacturers. It should be understood that the technician can use many chelating agents in a ready-made manner without difficulty. It should be further understood that the suitability of chelating groups to form chelates with given cations requires that the chelating groups can provide chelating ligands in the chelate complex containing the cationic being considered, but does not require that the chelating groups provide the only ligand of the cationic in the chelate complex. Therefore, if the chelating group contains a chelated radioactive or non-radioactive cation, the cation can be a complexing cation, such as a metal ion carrying an additional coordinating ligand (e.g., an oxo ligand) except the chelating group.

[0040] For example, the chelating group may comprise at least one of: (i) a macrocyclic structure having 8 to 20 ring atoms, wherein 2 or more, preferably 3 or more, of the ring atoms are selected from oxygen atoms and nitrogen atoms; and (ii) an acyclic open-chain chelating structure having 8 to 20 main chain atoms, wherein 2 or more, preferably 3 or more, of the main chain atoms are heteroatoms selected from oxygen atoms and nitrogen atoms.

[0041] According to a preferred embodiment of the second aspect of the present invention, the chelating group is a group that can be derived from a chelating agent selected from the following: bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CBTE2a), cyclohexyl-1,2-diaminetetraacetic acid (CDTA), 4-(1,4,8,11-tetraazacyclotetradecane-1-yl)-methylbenzoic acid (CPTA), N′-[5-[acetyl(hydroxy)amino]pentyl]-N-[5-[[4-[5-aminopentyl-(hydroxy)amino]-4-oxobutanoyl] amino]pentyl]-N-hydroxysuccinamide (DFO), 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (DO2A), 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA), 2-[1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid]-glutaric acid (DOTAGA or DOTA-GA), N,N'-dipyridoxal ethylenediamine-N,N'-diacetate-5,51-bis(phosphate) (N,N 1-dipyridoxylethylendiamine-N,N'-diacetate-5,5'-bis(phosphat))(DPDP), diethylenetriaminepentaacetic acid (DTPA), L-diamine-N,N'-tetraacetic acid (EDTA), L-diol-O,O-bis(2-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), N,N-bis(hydroxybenzyl)-ethylenediamine-N,N'-diacetic acid (HBED), hydroxyethyldiaminetriacetic acid (HEDTA), 1-(p-nitrobenzyl)-1,4,7,10-tetraazacyclodecane-4,7,1 0-triacetate (HP-DOA3), 6-hydrazino-N-methylpyridine-3-carboxamide (HYNIC), 1,4,7-triazacyclononane-1-succinic acid-4,7-diacetic acid (NODASA), 1-(1-carboxy-3-carboxypropyl)-4,7-(carboxy)-1,4,7-triazacyclononane (NODAGA), 1,4,7-triazacyclononane triacetic acid (NOTA), 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (TE2A), 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TET A), terpyridine-bis(methyleneamine)tetraacetic acid (TMT), 1,4,7,10-tetraazacyclotridecane-N,N',N",N"'-tetraacetic acid (TRITA), and triethylenetetraaminehexaacetic acid (TTHA), N,N'-bis[(6-carboxy-2-pyridyl)methyl]-4,13-diaza-18-crown-6 (H2macropa), 4-amino-4-{2-[(3-hydroxy-1,6-dimethyl-4-oxo-1,4-dihydro-pyridin-2-ylmethyl)-carbamoyl]-ethyl}pimelic acid, bis-[(3-hydroxy-1,6-dimethyl-4-oxo-1,4-dihydro- Hydrogen-pyridin-2-ylmethyl)-amide] (THP), 1,4,7-triazacyclononane-1,4,7-tri[methylene(2-carboxyethyl)phosphinic acid (TRAP), 2-(4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1-yl)acetic acid (DO3AM), and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra[methylene(2-carboxyethylphosphinic acid)] (DOTPI), S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecanetetraacetic acid, mercaptoacetyl-triserine (mass 3 ), hydrazinonicotinic acid (HYNIC) and N4 chelators. The chelating group is usually provided by utilizing a functional group of one of the above mentioned chelators (e.g., a carboxyl group) to form a bond (e.g., an amide bond) with the rest of the conjugate containing the chelating group.

[0042] More preferred are DOTA, DOTAGA, DOTAM, DO3AM, NOTA and NODAGA.

[0043] As will be appreciated by the skilled reader, the chelating group in the conjugate of the invention can be conveniently derived from the chelating agents listed above by utilizing a functional group contained in the chelating agent, such as a carboxylic acid group, an amide group, an amino group, a hydroxyl group, or a thiol functional group, to provide a coupling group (e.g., selected from -C(O)-, -NH-, -S-, and -O-) that connects the chelating group to the remainder of the compound. Preferably, the carboxylic acid group is used to provide a coupling group -C(O)- (carbonyl) to form an amide bond. The coupling group can be covalently linked to another complementary coupling group contained in the conjugate (e.g., the tetrapeptide itself), such that the two coupling groups combine to form a binding unit, such as an amide bond -C(O)-NH-.

[0044] Alternatively, as the skilled reader will appreciate, the chelating groups in the conjugates of the invention may be conveniently derived from the chelating agents listed above by introducing additional functional groups or introducing additional groups with functional groups capable of forming chemical bonds with other parts of the conjugate (preferably a tetrapeptide or a linker between the chelating group and the tetrapeptide), such as chelating agents modified with additional residues having isothiocyanate that can be linked to amines. As the skilled reader will appreciate, other conjugation strategies, generally summarized as "bioconjugation strategies", may also be used to attach the chelating groups in the conjugates of the invention.

[0045] According to another preferred embodiment of the second aspect of the invention, the chelating group is a group derivable from DOTA or DOTAGA with or without chelated non-radioactive or radioactive cation, wherein DOTA and DOTAGA preferably have one of their carboxylic acid groups bound to the rest of the conjugate via an amide bond.

[0046] The amine can be an amine within the tetrapeptide (eg, an N-terminal amine group or an amine in an amino acid side chain) or within a linker linking the chelating group to the tetrapeptide.

[0047] According to another preferred embodiment of the second aspect of the present invention, the radioactive or non-radioactive cation is selected from 43 Sc, 44 Sc, 47 Sc, 51 Cr, 52m Mn, 55 Co. 57 Co. 58 Co. 52 Fe, 56 You, 57 You, 62 Cu, 64 Cu, 67Cu、 66 Ga、 68 Ga、 67 Ga、 89 Zr、 90 Y、 86 Y、 94 mTc、 99m Tc、 97 Ru、 105 Rh、 109 Pd、 111 Ag、 110m In、 111 In、 113m In、 114m In、 117m Sn、 121 Sn、 127 For, 142 pr、 143 pr、 147 Nd、 149 Gd、 149 pm、 151 Pm、 149 Tb、 152 Tb、 155 Tb、 153 Sm、 156 Eu、 157 Gd、 155 Tb、 161 Tb、 164 Tb、 161 Ho、 166 Ho、 157 Dy、 165 Dc、 166 Dy、 160 Er、 165 Er、 169 Er、 171 Er、 166 Yb、 169 Yb、 175 Yb、 167 Tm、 172 Tm、 177 Lu、 186 Re, 186g Re, 188 Re, 188 W、 191 Pt、 195m pt、 194 Is、 197 Hg、 198 I、 199 I、 212 pb、 203 pb、 211 Assets,212 Bi, 213 Bi, 223 Ra, 224 Ra, 225 Ac, 226 Th and 227 Th cations, and cations of their non-radioactive isotopes, or containing 18 F or 19 F cationic molecules, such as 18 F-[AlF] 2+ or 18 F-[ScF] 2+ .

[0048] In this group of cations, the cation is preferably selected from 68 Ga, 90 Y and 177 The radioactive cations are Lu and the non-radioactive cations are Ga, Y or Lu.

[0049] 68 Ga is often used as a tracer for drug molecules. 90 Y plays an important role in the treatment of hepatocellular carcinoma (HCC), leukemia and lymphoma, but has the potential to treat a range of tumors. 177 Lu is used in the art for radiolabeling of drug molecules intended as anticancer therapy or for scintigraphy (medical imaging).

[0050] According to another preferred embodiment of the second aspect of the present invention, the conjugate further comprises in a single molecule: (c) a silicon-based fluoride acceptor moiety R selected from the group consisting of SiFA : (i) a silicon-based fluoride acceptor moiety comprising a silicon atom and a fluorine atom, wherein the fluorine atom is directly linked to the silicon atom via a covalent bond, and the moiety can be 18 F and 19 F isotope exchange 18 F mark, or the part is 18 F-labeled; (ii) a silicon-based fluoride acceptor moiety comprising a silicon atom and a hydroxyl group, wherein the hydroxyl group is directly attached to the silicon atom via a covalent bond, and the moiety can be 18 F nucleophilically replaces OH and uses 18 F label; and (iii) a silicon-based fluoride acceptor moiety comprising a silicon atom and a hydrogen atom, wherein the hydrogen atom is directly attached to the silicon atom via a covalent bond, and the moiety can be 18 F replaces H nucleophilically and uses 18 F mark.

[0051] Silicon-based fluoride acceptor moiety R SiFA(also referred to as the silicofluoride acceptor moiety) represents a method for introducing 18 An attractive method for F labeling is the silicon-fluoride acceptor moiety described, for example, in Lindner et al., Bioconjugate Chemistry 25, 738-749 (2014). The literature has shown that the use of silicon-based fluoride acceptors can 18 F-labeled compounds can be 18 F- 19 F isotope exchange reaction, and can also be produced by 18 F-OH and even 18 Substitution reactions of FH produce (e.g., in Mu L et al., Angew Chem Int Ed Engl. 2008; 47(26): 4922-5 and A et al., Bioconjug Chem. 2008 Sep;19(9):1871-9). However, in order to maintain the silicon-fluorine bond, silicon-based fluoride acceptors often rely on sterically demanding groups, such as Si-F and Si- 18 Two tert-butyl groups at the silicon group of F, such as Si(tert-butyl) 2 X, where X is F or 18 F. This in turn makes the silicon fluoride receptor highly hydrophobic. In terms of binding to target molecules (particularly to the target protein CCK-2R), the hydrophobic portion provided by the silicon-based fluoride receptor can be used for the purpose of establishing the interaction of radiodiagnostic or radiotherapeutic compounds with the hydrophobic pocket (which is described in Zhang et al., Journal of the American Chemical Society 132, 12711-12716 (2010)). However, before binding, the higher degree of lipophilicity introduced into the molecule brings serious problems to the development of radiopharmaceuticals with suitable in vivo biodistribution (i.e., lower nonspecific binding in non-target tissues).

[0052] According to a more preferred embodiment of the second aspect of the present invention, the silicon-based fluoride acceptor moiety R SiFA A group containing the following formula (S-1):

[0053]

[0054] in

[0055] X S is F, OH or H, preferably F;

[0056] R 1S and R 2S are independently linear or branched C 3 To C10 Alkyl, preferably R 1S and R 2S are independently selected from isopropyl and tert-butyl, and more preferably R 1S and R 2S It is tert-butyl.

[0057] The wavy line marks the bond connecting the group to the rest of the conjugate compound.

[0058] More preferably, part R SiFA A group containing formula (S-2):

[0059]

[0060] in

[0061] X S is F, OH or H, preferably F;

[0062] R 1S and R 2S are independently linear or branched C 3 To C 10 Alkyl, preferably R 1S and R 2S are independently selected from isopropyl and tert-butyl, and more preferably R 1S and R 2S is tert-butyl;

[0063] Ph is phenylene (-C 6 H 4 -), wherein one or more, such as one, two or three hydrogen atoms may be substituted by an organic functional group as a substituent. The wavy line marks the bond connecting this group to the rest of the conjugate compound.

[0064] Still further preferred are silicon-based fluoride acceptor moieties R SiFA Having a structure represented by one of the following formulae (S-3) and (S-4), or one of the following formulae (S-5) to (S-7):

[0065]

[0066] In the part of formula (S-3), R 1S and R 2S are independently linear or branched C3 to C10 alkyl groups, preferably R 1S and R 2S is selected from isopropyl and tert-butyl, and more preferably R 1S and R 2S The wavy line marks the bond connecting this group to the rest of the compound.

[0067] In the moiety of formula (S-4), r is 1, 2 or 3, preferably 1, -(CH 2 ) s - wherein s is an integer from 1 to 6 and preferably 1, the groups R are independently H or C1 to C6 alkyl, preferably H or C1 to C2 alkyl, and more preferably both are methyl, and

[0068] R 1S and R 2S are independently linear or branched C3 to C10 alkyl groups, preferably R 1S and R 2S is selected from isopropyl and tert-butyl, and more preferably R 1S and R 2S is tert-butyl. The wavy line marks the bond connecting this group to the rest of the compound. As exemplary counterions of the positively charged quaternary ammonium group carrying two substituents R indicated in formula (S-4), trifluoroacetate anion, acetate anion, or halide anion such as chloride ion can be mentioned.

[0069] As will be appreciated by the skilled reader, the fluorine atom indicated in formulas (S-3) and (S-4) may be 18 F atoms, or 19 F atoms (which can be 18 F and 19 F isotope exchange provides 18 F).

[0070] Furthermore, the moiety of formula (S-3) is preferably a moiety of formula (S-3a) (also referred to herein as "SiFA"), and the moiety of formula (S-4) is preferably a moiety of formula (S-4a) (also referred to herein as "SiFAlin"):

[0071]

[0072] in t Bu represents tert-butyl. The wavy line marks the bond connecting this group to the rest of the compound. As noted above for (S-4), exemplary counterions for the positively charged quaternary ammonium group carrying two methyl substituents represented in formula (S-4a) are trifluoroacetate anions, acetate anions, or halide anions such as chloride ions.

[0073]

[0074] in:

[0075] R 1S and R 2S are independently linear or branched C 3 To C 10 Alkyl, preferably R 1Sand R 2S are independently selected from isopropyl and tert-butyl, and more preferably R 1S and R 2S is tert-butyl; and

[0076] R 3S Selected from

[0077] (i) -OH or -O - ,

[0078] (ii) a sugar moiety or an amino sugar moiety,

[0079] (iii) an amino acid portion or an oligopeptide portion,

[0080] (iv) a PEG moiety;

[0081] and a combination of two or more of (ii), (iii) and (iv).

[0082] The wavy line marks the bond connecting the group to the rest of the conjugate compound.

[0083] It should be understood that if R 3S Indicates -O - , then the substituent -C(=O)R of the aromatic ring shown in the above formula 3S Deprotonated carboxylate group -C(=O)O - .

[0084] Among formulae (S-5), (S-6) and (S-7), formula (S-5) is preferred.

[0085] As R 3S The amino acid moiety is preferably derived from a hydrophilic amino acid which, in addition to the amino and carboxyl groups, contains a further basic or acidic functional group, or wherein as R 3S The oligopeptide moiety is derived from an oligopeptide having at least one hydrophilic amino acid which, in addition to the amino and carboxyl groups, also contains an additional basic or acidic functional group.

[0086] As R 3S The oligopeptide portion is preferably a linear or branched portion comprising 2 to 10, preferably 2 to 5, and more preferably 2 or 3 amino acid portions.

[0087] The hydrophilic amino acids are preferably selected from lysine and glutamic acid.

[0088] As R 3S The PEG moiety is preferably of the formula -NH-(CH 2 -CH 2 -O) X -R P1The part wherein the nitrogen atom providing the open bond is R 3S The carbon atom to which it is connected forms an amide bond -NH-C(O)-, x is an integer from 2 to 10, preferably from 4 to 10, and more preferably 8, and R P1 Selected from -CH 2 -COOH and -CH 2 -CH 2 -COOH.

[0089] As R 3S The sugar moiety or amino sugar moiety of is preferably a residue derived from 6-amino-6-deoxy-D-galactopyranose and its corresponding tautomers.

[0090] R 3S More preferably, it is selected from -OH and -O-. The silicon-based fluoride acceptor moiety of formula (S-5) is also referred to herein as "SiFA-ipa", wherein R 1S and R 2S represents tert-butyl and R 3S It represents -OH or -O-.

[0091] According to a preferred embodiment of the second aspect of the present invention, the conjugate is represented by formula (III):

[0092]

[0093] in:

[0094] RL is one or more chelating groups as defined above, wherein the chelating groups optionally contain chelated non-radioactive or radioactive cations, optionally contain chelated non-radioactive or radioactive cations;

[0095] L is the connecting part;

[0096] R H is the tetrapeptide of the first aspect of the invention, and

[0097] R SiFA is present or absent, and if present is a silicon-based fluoride acceptor moiety as defined above.

[0098] It should be understood that the linking moiety can be any moiety capable of linking the chelating group and the tetrapeptide.

[0099] The one or more chelating groups is preferably one chelating group.

[0100] According to a preferred embodiment of the second aspect of the present invention, the linking moiety comprises D / L-diaminopropionic acid (D / L-Dap), D / L-diaminobutyric acid (D / L-Dab), D / L-ornithine (D / L-Om) or D / L-lysine (D / L-Lys), wherein if present, the SiFA utilizes the -NH contained in these amino acids. 2 The group is conjugated with D / L-Dap, D / L-Dab, D / L-Om or D / L-Lys to provide a coupling group -NH-, wherein -NH 2 The bond of one hydrogen atom in the group is replaced by a bond of SiFA, wherein D / L-Dap, D / L-Dab, D / L-Om or D / L-Lys is preferably located in the linking portion so that D / L-Dap, D / L-Dab, D / LD / L-Om or Lys is directly linked to R L and / or R SiFA .

[0101] Thus, the linking moiety according to this preferred embodiment may be used as or serve to make R SiFA Site of attachment to the conjugate. Among the options D / L-Dap, D / L-Dab, D / L-Om or D / L-Lys, the option D / L-Dap is the most preferred.

[0102] According to another preferred embodiment of the second aspect of the present invention, the linking moiety comprises polyethylene glycol PEG, preferably (PEG) 3-11 , optionally together with poly-L / D-hydroxyproline (L / D-Hyp), preferably (Hyp) 1-8 , and / or α- or γ-glu, preferably γ-glu, and wherein the linker most preferably comprises y-glu-(Hyp) 6 -y-glu-(PEG) 3 or y-glu-(PEG) 4 -y-glu-(PEG) 3 .

[0103] Therefore, the moiety according to this preferred embodiment is preferably used together with a linking moiety according to the aforementioned preferred embodiment, which linking moiety can be used as or serve as a linking moiety to enable R SiFA The site of attachment to the conjugate. L / D-Hyp is preferably L-Hyp.

[0104] The linking moiety according to this preferred embodiment not only connects one or more chelating groups and the tetrapeptide, but also provides a distance between the one or more chelating groups and the tetrapeptide. In the attached examples, it is shown that increasing a distance between a sterically demanding chelating agent (such as DOTA) and the tetrapeptide by a linker is beneficial to high CCK-2R affinity.

[0105] According to another preferred embodiment of the second aspect of the present invention, the conjugate is represented by: DOTA-γ-glu-(PEG) 3 -Trp-(N-Me)Nle-Asp-1-Nal-NH 2 , DOTA-dap(SiFA)-γ-glu-(PEG) 7 -γ-glu-(PEG) 3 -Trp-(N-Me)N1e-Asp-1-Nal-NH 2 、DOTA-dap(SiFA)-y-glu-(Hyp) 6 -y-glu-(PEG) 3 -Trp-(N-Me)Nle-Asp-1-Nal-NH 2 , or DOTA-dap(SiFAlin)-y-glu-(PEG) 4 -y-glu-(PEG) 3 -Trp-(N-Me)N1e-Asp-1-Nal-NH 2 , wherein the DOTA chelator optionally contains a chelated non-radioactive or radioactive cation as described above, preferably a cation of natLu, natGa, 177Lu or 68Ga.

[0106] Among the above four conjugates, DOTA-dap(SiFA)-y-glu-(Hyp) 6 -y-glu-(PEG) 3 -Trp-(N-Me)Nle-Asp-1-Nal-NH 2 As shown in the appended examples, this embodiment involves the best performers with and without silicon-based fluoride acceptor moieties, and they are believed to be very good candidates for clinical applications.

[0107] DOTA-γ-glu-(PEG) 3 -Trp-(N-Me)Nle-Asp-1-Nal-NH 2 DOTA as a chelating agent and γ-glu-(PEG) as a linker 3 and tetrapeptide-(N-Me)Nle-Asp-1-Nal-NH 2DOTA can be complexed with a non-radioactive or radioactive cation as described above (such as a cation of natLu, natGa, 177Lu or 68Ga). For therapeutic and diagnostic applications as described herein, DOTA is preferably complexed with a non-radioactive or radioactive cation as described above. DOTA-Y-glu-(PEG)3-Trp-(N-Me)Nle-Asp-1-Nal-NH 2 In the examples, it is referred to as DOTA-CCK-66, and the examples show that DOTA-CCK-66 is particularly suitable as a theranostic agent. After administration to tumor-bearing mice, it is specifically enriched in tumors, while there is background in other tissues; see Fig.11 DOTA-CCK-66 and clinically applied compounds[ 68 Ga]Ga- / [ 177 The tumor / background ratio of the former was comparable or even improved, which can be attributed to the tetrapeptide design combined with the PEG linker.

[0108] DOTA-dap(SiFA)-Y-glu-(PEG) 7 -γ-glu-(PEG) 3 -Trp-(N-Me)Nle-Asp-1-Nal-NH 2 The chelating agent is composed of DOTA as a chelator, dap as a site for attachment of the SiFA moiety (i.e., the moiety of formula (S-3a), as shown above), -glu-(PEG) as a linker 7 -γ-glu-(PEG) 3 and tetrapeptide Trp-(N-Me)Nle-Asp-1-Nal-NH 2 composition. DOTA-dap(SiFA)-γ-glu-(PEG) 7 -γ-glu-(PEG) 3 -Trp-(N-Me)Nle-Asp-1-Nal-NH 2 In the examples, it is referred to as DOTA-rhCCK-70. In addition, DOTA-rhCCK-70 revealed high activity levels in tumors 24 hours after administration to tumor-bearing mice. The DOTA chelator may contain a chelated non-radioactive or radioactive cation as described above, preferably natL u , natGa, 177L u Or 68Ga cation.

[0109] DOTA-dap(SiFA)-y-glu-(Hyp) 6 -y-glu-(PEG)3 -Trp-(N-Me)Nle-Asp-1-Nal-NH 2 The chelating agent is composed of DOTA, dap, which is a site for attaching the SiFA moiety (i.e., the moiety of formula (S-3a), as shown above), and -y-glu-(Hyp) as a linker. 6 -y-glu-(PEG) 3 - and the tetrapeptide Trp-(N-Me)Nle-Asp-1-Nal-NH 2 composition. DOTA-dap(SiFA)-y-glu-(Hyp) 6 -y-glu-(PEG) 3 -Trp-(N-Me)Nle-Asp-1-Nal is referred to as DOTA-rhCCK-84 in the examples. In many of the tested compounds as shown in the examples, lipophilicity and CCK-2R affinity were addressed via a poly-L-hydroxyproline linker. Among the tested compounds, DOTA-rhCCK-84 advantageously showed the highest hydrophilicity (log D 74 =-2.14±0.06) and CCK-2R affinity (IC 50 =7.87±0.3). In addition, DOTA-rhCCK-84 revealed one of the highest activity levels in the tumor 24 hours after administration to tumor-bearing mice, while the activity level in the kidney was lower. The DOTA chelator may contain a chelated non-radioactive or radioactive cation as described above, preferably a cation of natLu, natGa, 177Lu or 68Ga.

[0110] DOTA-dap(SiFAlin)-y-glu-(PEG) 4 -y-glu-(PEG) 3 -Trp-(N-Me)Nle-Asp-1-Nal-NH 2 The poly(ethylene glycol)-containing peptide is composed of DOTA as a chelating agent, dap as a site for attaching the SiFAlin moiety (i.e., the moiety of formula (S-4a), as shown above), -y-glu-(PEG) as a linker 4 -y-glu-(PEG) 3 - and the tetrapeptide Trp-(N-Me)Nle-Asp-1-Nal-NH 2 composition. DOTA-dap(SiFAlin)-y-glu-(PEG)4-y-glu-(PEG) 3 -Trp-(N-Me)Nle-Asp-1-Nal is referred to as DOTA-rhCCK-91 in the examples. nat Lu]Lutetium (IC50 =8.56±0.7) or [ nat Ga] Gallium (IC 50 =8.24±1.6), DOTA-rhCCK-91 advantageously exhibits high CCK-2R affinity. The DOTA chelator group may contain a chelated non-radioactive or radioactive cation as described above, preferably a cation of natLu, natGa, 177Lu or 68Ga.

[0111] In a third aspect, the present invention relates to the use of the tetrapeptide or conjugate of the present invention in the diagnosis of cancer in vitro or ex vivo.

[0112] In a related fourth aspect, the invention relates to the use of a tetrapeptide or conjugate of the invention for detecting in vitro or ex vivo the presence of cholecystokinin 2 receptor (CCK-2R) in a sample, wherein the sample is preferably obtained from a cancer patient or an individual suspected of having cancer.

[0113] In a fifth aspect, the present invention relates to an in vitro or ex vivo method for detecting the presence of cholecystokinin 2 receptor (CCK-2R) in a sample, wherein the sample is preferably obtained from a cancer patient or an individual suspected of having cancer, and the method comprises (a) contacting the sample with the tetrapeptide or conjugate of the present invention, and (b) detecting the binding of the tetrapeptide or conjugate of the present invention to CCK-2R.

[0114] In vitro or ex vivo diagnosis of cancer is not performed on the human or animal body, but is usually based on a sample obtained from the individual to be diagnosed with cancer.

[0115] The sample may be a body fluid or a tissue sample. The body fluid is preferably a blood sample selected from whole blood, serum or plasma. The tissue sample is preferably from a tissue that is considered to be cancerous, such as breast, lung, liver or colon tissue.

[0116] In a sixth aspect, the present invention relates to a pharmaceutical or diagnostic composition comprising the tetrapeptide or conjugate of the present invention.

[0117] As used herein, the term "composition" refers to a composition comprising at least the tetrapeptide or conjugate of the present invention (which are also collectively referred to as compounds hereinafter).

[0118] With respect to all pharmaceutical and diagnostic applications as described herein, it will be understood that the chelating agents of these aspects of the invention are preferably complexed with radioactive cations. The nature of the radioactive cation can be selected based on the planned pharmaceutical or diagnostic application. For example, for diagnostic applications, it is generally desirable to have a relatively short half-life and relatively weak radiation but still detectable radioactive cations, while for therapeutic applications, it is generally desirable to have a relatively long half-life and relatively strong radiation radioactive cations in order to destroy tumor cells.

[0119] According to the present invention, the term "pharmaceutical composition" relates to a composition for use in a patient (preferably a human patient) to be administered to treat or prevent a disease. The pharmaceutical composition of the present invention comprises the compound of the present invention as described above. It may optionally comprise additional molecules capable of changing the characteristics of the compound of the present invention, such as stabilizing, regulating and / or activating their functions. The composition may be in solid, liquid or gaseous form, and may especially be in the form of a powder, tablet, solution or aerosol. The pharmaceutical composition of the present invention may optionally and additionally comprise a pharmaceutically acceptable carrier. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions (such as oil / water emulsions), various types of wetting agents, sterile solutions, organic solvents (including DMSO), etc. Compositions comprising such carriers may be formulated by well-known conventional methods. These pharmaceutical compositions may be administered to individuals at suitable doses. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical field, the dosage of any patient depends on many factors, including the patient's body shape, body surface area, age, specific compound to be administered, sex, time of administration and route, overall health status and other drugs administered simultaneously. The therapeutically effective amount for a given situation will be easily determined by routine experiments, and within the skill and judgment of a common clinician or physician. Usually, as a conventional application of a pharmaceutical composition, the regimen should be within the range of 1 μ g to 5 g units per day. However, the more preferred scope of dosage may be 0.01 mg to 100 mg per day, even more preferably 0.01 mg to 50 mg and most preferably 0.01 mg to 10 mg.

[0120] According to the present invention, the term "diagnostic composition" relates to a composition for administration to a patient (preferably a human patient) to be administered for diagnosis of a disease. The diagnostic composition of the present invention comprises the compound of the present invention as described above. It may optionally comprise the same other molecules as the pharmaceutical composition and may also be in the same form as the pharmaceutical composition. The above preferred ingredients and formulations of the pharmaceutical composition are applicable to the diagnostic composition mutatis mutandis.

[0121] In a seventh aspect, the present invention relates to a tetrapeptide or a conjugate or a pharmaceutical composition of the present invention for use in treating or preventing cancer.

[0122] In an eighth aspect, the present invention relates to a tetrapeptide or a conjugate or a pharmaceutical composition of the present invention for use in a method for diagnosing cancer in vivo in an individual.

[0123] As referred to herein, the individual is preferably a mammal, and most preferably a human.

[0124] Cancer is an abnormal malignant new growth tissue without physiological function, and is caused by uncontrolled usually rapid cell proliferation.Cancer is preferably selected from breast cancer, ovarian cancer, endometrial cancer, vaginal cancer, vulvar cancer, bladder cancer, salivary gland cancer, pancreatic cancer, thyroid cancer, kidney cancer, lung cancer, upper digestive tract cancer, colon cancer, colorectal cancer, prostate cancer, head and neck squamous cell carcinoma, cervical cancer, glioblastoma, malignant ascites, lymphoma and leukemia.Preferably in this cancer list is colorectal cancer, pancreatic cancer, and thyroid cancer is preferred because abnormal CCK-2R expression has been found in these cancer types.In view of the illustration of the attached embodiment to this cancer type, thyroid cancer, particularly medullary thyroid carcinoma (MTC) is most preferred.

[0125] The cancer is preferably a solid cancer. In contrast to liquid cancers, solid cancers are abnormal masses of tissue that usually do not contain cysts or areas of fluid.

[0126] In a ninth aspect, the present invention relates to a conjugate compound that binds to cholecystokinin 2 receptor (CCK-2R), which is represented by formula (IV): R L -dap(R SiFA )-RG ABA -LR H (IV) means, wherein:

[0127] R L is DOTA or DOTAGA optionally containing a chelated non-radioactive or radioactive cation;

[0128] dap(R SiFA ) is a silicon-based fluoride acceptor moiety R SiFA The dap group;

[0129] R GABA is present or absent, and if present is one or more gamma-aminobutyric acid (GABA), preferably (GABA) 1-3 :

[0130] L is a linking moiety comprising at least 5, preferably at least 6 α- or γ-glu and most preferably 6 to 9 α- or γ-glu, wherein γ-glu is preferred over α-glu; and

[0131] R H Is or contains Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH 2 .

[0132] The above definitions and preferred embodiments of the first to eighth aspects of the present invention apply mutatis mutandis to the ninth aspect and other aspects of the present invention to be followed hereinafter, provided that these definitions and preferred embodiments are suitable for combination with the ninth aspect and other aspects of the present invention to be followed hereinafter.

[0133] As can be seen in the accompanying examples, several chelator-minigastrin-silicon-based fluoride receptor conjugates containing polyglutamate linkers were synthesized and evaluated in vitro. nat / 177 Lu-labeled and also compared with prior art reference ligands nat / 177 Lu-DOTA-PP-F11N and nat / 1 77 Lu-CP04( nat / 177 Lu-DOTA-(glu) 6 -Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH 2 ) were compared.

[0134] It has surprisingly been found that the highest CCK-2R affinity is observed when the group is separated by at least five or six α- / γ-glu moieties, whereas a further six α- / γ-glu moieties do not provide further improvement. This highest CCK-2R affinity is believed to be due to the fact that dap(R SiFA ) and the chelating agent portion and the CCK-2R binding sequence (Ala-Tyr-Gly-Trp1-Nle-Asp-Phe-NH 2 ) separated by γ-glu. γ-glu is superior to α-glu because the corresponding peptide backbone is significantly extended by using γ-glu instead of α-glu. In addition, different chelators were tested, and the best results with respect to CCK-2R affinity were obtained by using DOTA or DOTAGA. DOTA and DOTAGA preferably bind one of their carboxylic acid groups to the rest of the conjugate via an amide bond. SiFA Several other different compounds and positions were introduced into the conjugate, and it was found that dap was the best compound for introducing the silicon-based fluoride receptor moiety, and the best position for dap was between the chelator and the linker and GABA (if present). As the skilled reader will appreciate, dap (diaminopropionic acid) acts as a linker between RL and L (if R GABA does not exist) or R L With R GABA Between (if R GABA The linker unit is present and can additionally carry a silicon-based fluoride acceptor moiety R due to the three functional groups provided by dap SiFA .

[0135] One or more gamma-aminobutyric acid (GABA) may be optionally present to partially reduce the negative charge of γ-glu by the gamma-aminobutyric acid (GABA). At the same time, the linker length may be retained, but with a smaller amount of negative charge, which results in four compounds. If the amount in the kidney should be concerned, due to the addition of one or more gamma-aminobutyric acid (GABA), the kidney can reduce the amount of the conjugate that may be present in the kidney, thereby enriching the specific accumulation of the conjugate in the tumor (note that the conjugate of the ninth aspect in the absence of GABA is also specifically accumulated in the tumor after administration).

[0136] Thus, a ninth aspect of the invention relates to particularly preferred cholecystokinin 2 receptor binding molecules useful for imaging and targeted radiotherapy. Testing of chelator-minigastrin-SiFA conjugates revealed that the conjugate of the ninth aspect is advantageously the one with the highest CCK-2R affinity.

[0137] As mentioned, the above definitions and preferred embodiments of the first to eighth aspects of the present invention apply mutatis mutandis to the ninth aspect and further aspects of the present invention to follow as follows, provided that these definitions and preferred embodiments are suitable for combination with the ninth aspect and further aspects of the present invention to follow as follows.

[0138] It follows that, also according to a preferred embodiment of the ninth aspect of the present invention, the radioactive or non-radioactive cation of the chelating group is selected from 43 Sc, 44 Sc, 47 Sc, 51 Cr, 52m Mn, 55 Co. 57 Co. 58 Co. 52 Fe, 56 You, 57 You, 62 Cu, 64 Cu, 67 Cu, 66 Ga, 68 Ga, 67 Ga, 89 Zr, 90 Y. 86 Y. 94m Tc, 99m Tc, 97 Such as 105 Rh, 109 pd, 111 Ag, 110m In, 111 In, 113m In, 114mIn, 117m Sn, 121 Sn, 127 Te, 142 pr, 143 Pr, 147 Nd, 149 Gd, 149 Pm, 151 Pm, 149 Tb, 152 Tb, 155 Tb, 153 Sm, 156 Eu, 157 Gd, 155 Tb, 161 Tb, 164 Tb, 161 Ho, 166 Ho, 157 Dy, 165 Dy, 166 Dy, 160 2. 165 2. 169 2. 171 2. 166 Yb, 169 Yb, 175 Yb, 167 Tm, 172 Tm, 177 Lu, 186 Re, 186 gRe, 188 Re, 188 W. 191 Pt, 195m pt, 194 Ir, 197 Hg, 198 Au, 199 Au, 212 pb, 203 pb, 211 At 212 Bi, 213 Bi, 223 Ra, 224 Ra, 225 Ac, 226 Th and 227 Th cations, and cations of their non-radioactive isotopes, or containing 18 F or 19 F cationic molecules, such as 18 F-[AlF] 2+ , and preferably selected from 68 Ga, 90 Y, or 177Cations of Lu and cations of non-radioactive isotopes of Ga, Y or Lu.

[0139] Further details and preferred embodiments of the radioactive or non-radioactive cations are described above in conjunction with the second aspect of the invention. These details and preferred embodiments apply mutatis mutandis to the ninth aspect of the invention.

[0140] Furthermore, it follows that, also according to a preferred embodiment of the ninth aspect of the invention, the silicon-based fluoride acceptor moiety R SiFA As defined above in conjunction with the second aspect of the invention.

[0141] In particular, the silicon-based fluoride acceptor moiety is preferably selected from:

[0142] (i) a silicon-based fluoride acceptor moiety comprising a silicon atom and a fluorine atom, wherein the fluorine atom is directly linked to the silicon atom via a covalent bond, and the moiety can be 18 F and 19 F isotope exchange 18 F mark, or the part is 18 F marked;

[0143] (ii) a silicon-based fluoride acceptor moiety comprising a silicon atom and a hydroxyl group, wherein the hydroxyl group is directly attached to the silicon atom via a covalent bond, and the moiety can be 18 F nucleophilically replaces OH and uses 18 F mark; and

[0144] (iii) a silicon-based fluoride acceptor moiety comprising a silicon atom and a hydrogen atom, wherein the hydrogen atom is directly attached to the silicon atom via a covalent bond, and the moiety may be 18 F replaces H nucleophilically and uses 18 F mark.

[0145] More preferably, the silicon-based fluoride acceptor moiety R SiFA A group containing the formula (S-1) as defined above:

[0146]

[0147] in

[0148] X S is F, OH or H, preferably F;

[0149] R 1S and R 2S are independently linear or branched C 3 To C 10 Alkyl, preferably R 1S and R 2Sare independently selected from isopropyl and tert-butyl, and more preferably R 1S and R 2S It is tert-butyl.

[0150] More preferably, part R SiFA A group containing the formula (S-2) as defined above:

[0151]

[0152] in

[0153] X S is F, OH or H, preferably F;

[0154] R 1S and R 2S are independently linear or branched C 3 To C 10 Alkyl, preferably R 1S and R 2S are independently selected from isopropyl and tert-butyl, and more preferably R 1S and R 2S is tert-butyl;

[0155] Ph is phenylene (-C 6 H 4 -), wherein one or more, such as one, two or three hydrogen atoms may be substituted by an organic functional group as a substituent.

[0156] Still further preferred are silicon-based fluoride acceptor moieties R SiFA Having a structure represented by one of formulae (S-3) and (S-4) as defined above, or one of formulae (S-5) to (S-7):

[0157]

[0158] In the part of formula (S-3), R 1S and R 2S are independently linear or branched C3 to C10 alkyl groups, preferably R 1S and R 2S is selected from isopropyl and tert-butyl, and more preferably R 1S and R 2S It is tert-butyl.

[0159] In the moiety of formula (S-4), r is 1, 2 or 3, preferably 1, -(CH 2 ) s - wherein s is an integer from 1 to 6 and preferably 1, the groups R are independently H or C1 to C6 alkyl, preferably H or C1 to C2 alkyl, and more preferably both are methyl, and

[0160] R 1S and R 2S are independently linear or branched C3 to C10 alkyl groups, preferably R 1S and R 2S is selected from isopropyl and tert-butyl, and more preferably R 1S and R 2S It is tert-butyl.

[0161] Furthermore, the moiety of formula (S-3) is preferably a moiety of formula (S-3a) (also referred to herein as "SiFA"), and the moiety of formula (S-4) is preferably a moiety of formula (S-4a) (also referred to herein as "SiFAlin"):

[0162]

[0163] in t Bu indicates tert-butyl.

[0164]

[0165]

[0166] in:

[0167] R 1S and R 2S are independently linear or branched C 3 To C 10 Alkyl, preferably R 1S and R 2S are independently selected from isopropyl and tert-butyl, and more preferably R 1S and R 2S is tert-butyl; and

[0168] R 3S Selected from

[0169] (i) -OH or -O-,

[0170] (ii) a sugar moiety or an amino sugar moiety,

[0171] (iii) an amino acid portion or an oligopeptide portion,

[0172] (iv) a PEG moiety;

[0173] and a combination of two or more of (ii), (iii) and (iv).

[0174] The wavy line marks the bond connecting the group to the rest of the conjugate compound.

[0175] Among formulae (S-5), (S-6) and (S-7), formula (S-5) is preferred.

[0176] Silicon-based fluoride acceptor moiety R SiFA Further details and preferred embodiments of the groups and moieties of formula (S-1), (S-2), (S-3), (S-3a), (S-4), (S-4a), (S-5), (S-6) and (S-7) are described above in conjunction with the second aspect of the invention. These details and preferred embodiments apply mutatis mutandis to the ninth aspect of the invention.

[0177] According to a preferred embodiment of the ninth aspect of the present invention, the conjugate is represented by:

[0178] DOTAGA-dap(SiFA)-(γ-glu) 6 -Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH 2 ,

[0179] DOTAGA-dap(SiFA)-(α-glu) 8 -Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH 2 ,

[0180] DOTA-dap(SiFA)-(γ-glu) 8 -Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH 2 .

[0181] The DOTA or DOTAGA chelator optionally contains a chelated non-radioactive or radioactive cation as described above.

[0182] The above preferred embodiments relate to the three best performers among several chelator-minigastrin-SiFA conjugates prepared and tested in the appended examples.

[0183] DOTAGA-dap(SiFA)-(γ-glu) 6 -Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH 2 and DOTAGA-dap(SiFA)-(α-glu) 8 -Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH 2 Corresponding to compounds DOTAGA-MG-SiFA-19 and DOTAGA-MG-SiFA-21 as respectively mentioned in the examples. Fig.21Compared with the prior art reference DOTA-PP-F11N, the tumor accumulation was improved after 24 hours, and a significant amount was accumulated in the tumor after 1 hour. 8 -Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH 2 , unexpectedly found that replacing DOTAGA with DOTA in DOTAGA-MG-SiFA-23 resulted in a substantial increase in CCK-2R affinity (DOTA-MG-SiFA-23: 4.71 ± 0.62 nM), which even exceeded the prior art reference ligand, see Fig. 22 .

[0184] DOTA-MG-SiFA-23 revealed the highest activity level in tumors 24 hours after administration to tumor-bearing mice, which was more than 8 times higher than the activity level of the prior art reference DOTA-PP-F11N. In addition, although the two DOTAGA-MG-SiFA compounds had lower CCK2R affinity than the prior art reference DOTA-PP-F11N, they showed a three- to six-fold increase in activity level in tumors after administration to tumor-bearing mice, which should be due to the slower clearance of activity of the two DOTAGA-MG-SiFA compared to the prior art reference.

[0185] Thus, the data in the Examples demonstrate that the three top performers are particularly useful for imaging and targeted radiotherapy.

[0186] According to a preferred embodiment of the ninth aspect of the present invention, the CCK-2R binding affinity of the conjugate is characterized by a half maximal inhibitory concentration (IC) of 100 nM or less, and preferably 75 nM or less. 50 ).

[0187] DOTAGA-MG-SiFA-19, DOTAGA-MG-SiFA-21, and DOTA-MG-SiFA-23 showed half-maximal inhibitory concentrations (IC 50 ) were 7.54±0.26nM, 55.3±7.8 and 4.71±0.62nM.

[0188] Therefore, the IC of these three conjugates 50 The conjugates of the ninth aspect are technically supported to show an IC of 100 nM or less, and preferably 75 nM or less. 50 .

[0189] Additionally, the medical and diagnostic applications as described above as the third to eighth aspects of the invention are also suitable and intended to be combined with the conjugate of the ninth aspect.

[0190] Therefore, other aspects of the present invention relate to:

[0191] The conjugate of the ninth aspect is used for in vitro or ex vivo diagnosis of cancer.

[0192] The conjugate of the ninth aspect is used for detecting the presence or absence of cholecystokinin 2 receptor (CCK-2R) in a sample in vitro or ex vivo, wherein the sample is preferably obtained from a cancer patient or an individual suspected of having cancer.

[0193] An in vitro or ex vivo method for detecting the presence of cholecystokinin 2 receptor (CCK-2R) in a sample, wherein the sample is preferably obtained from a cancer patient or an individual suspected of having cancer, the method comprising (a) contacting the conjugate of the ninth aspect with the sample, and (b) detecting the binding of the conjugate of the ninth aspect to CCK-2R.

[0194] A pharmaceutical or diagnostic composition comprising the conjugate of the ninth aspect.

[0195] The conjugate of the ninth aspect or a pharmaceutical composition comprising the conjugate of the ninth aspect is used for treating or preventing cancer.

[0196] The conjugate of the ninth aspect or the diagnostic composition conjugate of the ninth aspect for use in a method of diagnosing cancer in vivo in an individual.

[0197] Further details and preferred embodiments for medical and diagnostic applications are described above in connection with the third to eighth aspects of the invention. These details and preferred embodiments apply mutatis mutandis to the above other aspects of the invention using the conjugate of the ninth aspect.

[0198] With regard to the embodiments characterized in the present description, in particular in the claims, it is intended that every embodiment mentioned in the dependent claims be combined with every embodiment of every claim (independent or dependent) to which said dependent claims are dependent. For example, in the case of independent claim 1 recording three alternatives A, B and C, dependent claim 2 recording three alternatives D, E and F, and claim 3 dependent on claims 1 and 2 and recording three alternatives G, H and I, unless explicitly mentioned otherwise, it should be understood that the specification unambiguously discloses embodiments corresponding to the combination A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I.

[0199] Similarly, and in those cases where the independent and / or dependent claims do not recite alternatives, it is to be understood that if a dependent claim refers to multiple preceding claims, any combination of subject matter covered thereby is deemed to be explicitly disclosed. For example, in the case of independent claim 1, dependent claim 2 referring to claim 1, and dependent claim 3 referring to both claims 2 and 1, it follows that the combination of the subject matter of claims 3 and 1 is as clearly and explicitly disclosed as the combination of the subject matter of claims 3, 2, and 1. In the presence of another dependent claim 4 referring to any one of claims 1 to 3, it follows that the combination of the subject matter of claims 4 and 1, claims 4, 2, and 1, claims 4, 3, and 1, and claims 4, 3, 2, and 1 is clearly and explicitly disclosed.

[0200] The attached figure shows:

[0201] Figure 1 :CCK-2R targeting ligand (H-Trp-(N-Me)Nle-Asp-1-Nal-NH 2 、CCK-49、[ nat Lu]Lu-DOTA-γ-MGS5, H-Trp-Nle-Asp-Phe-NH2, CCK-32 and [ nat CCK-2R affinity of α-Lu-DOTA-PP-γ-F11N) (depicted as half maximal inhibitory concentration, IC 50 ). The DOTA-MGS5 analog is shown in black and the DOTA-PP-F11N analog is shown in grey.

[0202] Figure 2 : CCK-2R affinity of CCK-2R targeting ligands with modified tetrapeptide binding motifs (depicted as half maximal inhibitory concentration, IC 50 ) schematic diagram.

[0203] Figure 3 : CCK-2R affinity of glycine scanning compounds (DOTA-CCK-55 to -62) compared to reference (DOTA-MGS5) (depicted as half maximal inhibitory concentration, IC 50 , bars) and lipophilicity (depicted as n-octanol / PBS partition coefficient, pH = 7.4, logD 7.4 , dotted) schematic diagram.

[0204] Figure 4 :Compared with DOTA-MGS5 nat Lu-, nat Ga-and natCCK-2R affinity of Cu-labeled DOTA-CCK-66 and -66.2 (depicted as half maximal inhibitory concentration, IC 50 ) schematic diagram.

[0205] Figure 5 : Lipophilicity of DOTA-CCK-66, -66.2 and DOTA-MGS5 (depicted as n-octanol / PBS partition coefficient, pH = 7.4, log D 74 ). The compounds tested were similar to [ 177 Lu] Lutetium, [ 67 Ga] Gallium and [ 64 Cu] copper complex.

[0206] Figure 6 :and[ nat Lu] Lutetium, [ nat Ga] Gallium or [ nat HSA binding (%) of copper-complexed DOTA-CCK-66, -66.2 and DOTA-MGS5.

[0207] Figure 7 : With direct 177 Quality control chromatograms of each compound after Lu-labeling (grey) compared to that of the sample in human serum incubated at 37°C for 72 ± 2 hours. 177 Representative RP-HPLC chromatograms of Lu-labeled DOTA-MGS5, -CCK-66, and -CCK-66.2 (10→30% MeCN / H 2 O, 0.1% TFA, 5 min, and 30→60% MeCN / H 2 O, with 0.1% TFA, 15 min) (black).

[0208] Figure 8 :30 minutes after injection, the serum (black) and urine (dark gray) of mice [ 177 Lu]Lu-DOTA-MGS5 and [ 177 Representative RP-HPLC chromatogram of the amount of intact compound of Lu]Lu-DOTA-CCK-66 (1 nmol each, 35 MBq) (10→30% MeCN / H 2 O, 0.1% TFA, 5 min, and 30→60% MeCN / H 2 O, with 0.1% TFA, 15 min). The reference chromatogram of each compound is depicted in light grey.

[0209] Fig. 9 : In AR4-2J tumor-bearing CB17-SCID mice (100 pmol each) 1 hour after injection, [ 67Biodistribution and competition studies of [Ga]Ga-DOTA-CCK-66 (in %ID / g). For competition studies, excess [ nat Ga]Ga-DOTA-MGS5 (40 nmol). Data are presented as mean ± SD (n = 4 for biodistribution studies and n = 2 for competition studies). 68 Literature data for Ga]Ga-DOTA-MGS5 (20 pmol) were used for comparison

[30] . Comparative analysis of tumor / blood, tumor / stomach, and tumor / kidney ratios for the two compounds is depicted on the right.

[0210] Fig.10 :A) Injection [ 67 Maximum density projection of AR4-2J tumor-bearing CB17-SCID mice 1 hour after co-administration of [Ga]Ga-DOTA-CCK-66 (100 pmol). B) Maximum density projection of AR4-2J tumor-bearing CB17-SCID mice 1 hour after co-administration of [ 67 Ga]Ga-DOTA-CCK-66 (100 pmol) and [ nat Representative μSPECT / CT images of competition studies with Ga]Ga-DOTA-MGS5 (40 nmol) (1 hour after injection).

[0211] Fig.11 : In AR4-2J tumor-bearing CB17-SCID mice (100 pmol each) 24 hours after injection, [ 177 Lu]Lu-DOTA-CCK-66 and [ 177 Biodistribution and competition studies of Lu]Lu-DOTA-MGS5 (in %ID / g). For competition studies, excess [ nat Lu]Lu-DOTA-MGS5 (40 nmol). Data are presented as mean ± SD (n = 4 for biodistribution studies and n = 2 for competition studies). Comparative analysis of tumor / blood, tumor / stomach and tumor / kidney ratios of the two compounds is depicted at the bottom.

[0212] Fig.12 :Injection A)[ 177 Lu]Lu-DOTA-MGS5 and B)[ 177 Maximum density projection of AR4-2J tumor-bearing CB17-SCID mice treated with [Lu]Lu-DOTA-CCK-66 (100 pmol each). C) 177 Lu]LuDOTA-CCK-66 (100 pmol) and [ natRepresentative μSPECT / CT images of competition studies of Lu]Lu-DOTA-MGS5 (40 nmol) (24 h after injection).

[0213] Fig.13 :(A) Use [ 68 PET / CT images of MTC patient 1 treated with [Ga]Ga-DOTA-CCK-66, revealing (B) a left retroclavicular lymph node metastasis and (C) a mediastinal metastasis in the aortic arch.

[0214] Fig.14 :(A) Use [ 68 PET / CT images of MTC patient 2 treated with [Ga]Ga-DOTA-CCK-66, showing (B) right retroclavicular lymph node metastasis, (C) bilateral hilar lymph node metastasis, (D) two liver metastases, and (E) bone metastases in the right femur and right ischium.

[0215] Fig.15 :(A) Use [ 68 PET / CT images of MTC patient 3 treated with [Ga]Ga-DOTA-CCK-66, which showed (B) lymph node metastasis in the left carotid nerve sheath area, (C) local recurrence in the left thyroid bed, and (D) lymph node metastasis in the right upper mediastinum.

[0216] Fig.16 : Contains various chelating agents (NOTA, NODAGA, (R)-DOTAGA and (S)-DOTAGA) nat Lu- and 19 CCK-2R affinity of [F]AlF-complexed DOTA-CCK-66 analogs (depicted as half maximal inhibitory concentration, IC 50 ) schematic diagram.

[0217] Fig.17 : Contains the following [ nat / 177 Lu]Lu-DOTA-rhCCK-67 to -76, [ nat / 177 Lu]Lu-DOTA-rhCCK-83 to -86 and [ nat / 177 CCK-2R affinity of Lu]Lu-DOTA-rhCCK-90 to -91 (depicted as half maximal inhibitory concentration, IC 50 ) and lipophilicity (depicted as n-octanol / PBS partition coefficient, pH = 7.4, logD 7.4 ) Schematic diagram of different linker moieties (light grey: (PEG) 4 , Gray: (PEG) 7 , dark gray: (PEG) 11 , white: (cit) 0-3-(Hyp) 3-8 ), silicon-based fluoride acceptor moieties (SiFA, SiFA-ipa, SiFAlin, black: logD 7.4 value) and negative charge (with and without Y-glu near dap(SiFA)).

[0218] Fig.18 : Contains the following [ nat Lu]Lu-DOTA-rhCCK-67 to -76, [ nat Lu]Lu-DOTA-rhCCK-83 to -86 and [ nat HSA binding (%) of Lu]Lu-DOTA-rhCCK-90 to -91: different linker moieties (light grey: (PEG) 4 , Gray: (PEG) 7 , dark gray: (PEG) 11 , white: (cit) 0-3 -(Hyp) 3-8 ), silicon-based fluoride acceptor moieties (SiFA, SiFA-ipa, SiFAlin, black: logD 7.4 value) and negative charge (with and without Y-glu near dap(SiFA)).

[0219] Fig.19 : AR4-2J tumor-bearing CB17-SCID mice (100 pmol each) in selected organs 24 hours after injection 177 Lu]Lu-DOTA-CCK-66(n=4), [ 177 Lu]Lu-DOTA-rhCCK-70 (n=4), [ 177 Lu]Lu-DOTA-rhCCK-84(n=3),[ 177 Lu]Lu-DOTA-rhCCK-91 (n=3) and [ 177 Biodistribution (in %ID / g) of Lu]Lu-DOTA-MGS5 (n=4). Data are presented as mean±SD.

[0220] Fig. 20 :Injection A)[ 177 Lu]Lu-DOTA-CCK-66, B)[ 177 Lu]Lu-DOTA-rhCCK-70, C)[ 177 Lu]Lu-DOTA-rhCCK-84 and D)[ 177Maximum intensity projection of AR4-2J tumor-bearing CB17-SCID mice 24 hours after injection of [Lu]Lu-DOTA-rhCCK-91 (100 pmo1 each).

[0221] Fig.21 : AR4-2J tumor-bearing CB17-SCID mice (100 pmol each, n=4) [ 18 F]F-[ nat Lu]Lu-DOTA-rhCCK-84 (1 hour after injection) and [ 177 Biodistribution (in %ID / g) of Lu]Lu-DOTA-rhCCK-84 (2, 4, 24 hours after injection). Data are presented as mean ± SD.

[0222] Fig. 22 : AR4-2J tumor-bearing CB17-SCID mice (100 pmol each) were injected 24 hours after injection into selected organs with [ nat Lu]Lu-DOTA-MGS5 (40 nmol, n=2, black) or [ nat Lu]Lu-DOTA-MG-SiFA-23 (40 nmol, n=1, gray) co-administered 177 Biodistribution study of Lu]Lu-DOTA-rhCCK-84 (in %ID / g). Data are expressed as mean ± SD.

[0223] Fig.23 :30 minutes after injection, the serum (black) and urine (light gray) of mice [ 177 Representative RP-HPLC chromatogram of the amount of intact compound of Lu]Lu-DOTA-rhCCK-84 (1 nmol each, 35 MBq, n=3) (10→30% MeCN / H 2 O, 0.1% TFA, 5 min, and 30→60% MeCN / H 2 O, with 0.1% TFA, 15 min). Quality control chromatograms of each compound are depicted in dark grey.

[0224] Fig.24 : AR4-2J tumor-bearing CB17-SCID mice (100 pmol each) in selected organs 24 hours after injection 177 Lu]Lu-DOTA-PP-F11N, [ 177 Lu]Lu-DOTAGA-MG-SiFA-19 and [ 177 Biodistribution of Lu]Lu-DOTAGA-MG-SiFA-21 (in %ID / g). Data are presented as mean±SD (n=4).

[0225] Fig.25 : Compared with 24 hours after injection [ 177 Lu]Lu-DOTA-PP-F11N in selected organs 1 and 24 h after injection in AR4-2J tumor-bearing CB17-SCID mice (100 pmol each) 177 Lu]Lu-DOTA-MG-SiFA-23 and [ nat Lu]Lu- 18 Biodistribution (in %ID / g) of F-DOTA-MG-SiFA-23 (n=1). Data are presented as mean±SD (n=4).

[0226] Fig.26 : AR4-2J tumor-bearing CB17-SCID mice (100 pmol each) 1, 4 and 24 hours after injection (A) [ 177 Lu]Lu-DOTA-PP-F11N, B)[ 177 Lu]Lu-DOTAGA-MG-SiFA-2l and C)[ 177 Representative μSPECT / CT images of Lu]Lu-DOTAGA-MG-SiFA-19. The tumor (T) is indicated by a white arrow.

[0227] Fig. 27 :AR4-2J tumor-bearing CB17-SCID mice (100 pmol each) A) [ 177 Lu]Lu-DOTAGA-MG-SiFA-23 1 and 24 hours after injection and B) [ 18 F]F-[ nat Representative μSPECT / CT images of Lu]Lu-DOTA-MG-SiFA-23 at 1 hour after injection. The tumor (T) is indicated by a white arrow.

[0228] Fig.28 : Co-injection [ nat A) 24 hours after injection of [Lu]Lu-DOTA-MGS5 (40 nmol each) in AR4-2J tumor-bearing CB17-SCID mice (100 pmol each, n=1) 177 Lu]Lu-DOTAGA-MG-SiFA-19 and B)[ 177 Representative μSPECT / CT images of Lu]Lu-DOTA-MG-SiFA-23. C) Co-injection of [ nat Lu]Lu-DOTA-MGS5 (40 nmol each) in AR4-2J tumor-bearing CB17-SCID mice (100 pmol each, n=2) in selected organs 24 hours after injection177 Lu]Lu-DOTAGA-MG-SiFA-19 and [ 177 Biodistribution of Lu]Lu-DOTA-MG-SiFA-23 (in %ID / g). Data are presented as mean ± SD.

[0229] Fig.29 : Example of a sigmoid plot showing the correlation between human serum albumin (HSA) binding of selected reference substances and their retention time (tR) on a Chiralpak HSA column. The HSA binding values ​​of the reference substances have been previously published in the literature (Literature HSA [%]) and the corresponding logarithmic values ​​of the affinity constants (log K HSA) were calculated [31,32].

[0230] The examples illustrate the invention.

[0231] Example 1 - N-terminal tetrapeptide of minigastrin

[0232] 1.1. Overview of synthetic CCK-2R ligands

[0233] Reference compound

[0234]

[0235] DOTA-PP-F11N: RP-HPLC(10→90%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R =7.6 min, K'=3.5; MS (ESI, positive ion): C 90 H 123 N 19 O 35 m / z calculated value: 2029.8, found value: m / z = 1016.4 [M + 2H] 2+ .

[0236]

[0237] CP04: RP-HPLC(10→90%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R =7.2 min, K'=3.3; MS (ESI, positive ion): C 89 H 121 N 19 O 35 m / z calculated for S: 2047.8, found: m / z = 1024.2 [M+2H] 2+ .

[0238]

[0239] DOTA-MGS5: RP-HPLC(10→90%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R =9.2 minutes; MS (ESI, positive ion): C 70 H 92 N 14 O 20 m / z calculated value: 1448.7, found value: m / z = 1449.4 [M+H] + ,725.3[M+2H] 2+ .

[0240]

[0241] DOTA-γ-MGS5: RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 10.9 min; MS (ESI, positive ion): C 70 H 92 N 14 O 20 m / z calculated value: 1448.7, found value: m / z = 1449.6 [M+H] + ,725.5[M+2H] 2+ .

[0242] Compounds containing modified tetrapeptide motifs

[0243]

[0244] H-Trp-(N-Me)Nle-Asp-1-Nal-NH 2 : RP-HPLC (10→70% MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 11.5 min; MS (ESI, positive ion): C 35 H 42 N 6 O 6 m / z calculated value: 642.8, found value: m / z = 643.2 [M+H] + .

[0245]

[0246] H-Trp-Nle-Asp-1-Nal-NH2 : RP-HPLC (10→70% MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 10.6 min; MS (ESI, positive ion): C 34 H 40 N 6 O 6 m / z calculated value: 628.7, found value: m / z = 628.3 [M + H] + , 1255.6[2M+H] + .

[0247]

[0248] H-Trp-Nle-Asp-Phe-NH 2 : RP-HPLC (10→70% MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R =9.2 minutes; MS (ESI, positive ion): C 30 H 38 N 6 O 6 m / z calculated value: 578.7, found value: m / z = 579.2 [M + H] + ,1157.3[2M+H] + .

[0249]

[0250] H-Trp-(N-Me)Nle-Asp-2-Nal-NH 2 : RP-HPLC (10→70% MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 11.5 min; MS (ESI, positive ion): C 35 H 42 N 6 O 6 m / z calculated value: 642.8, found value: m / z = 643.3 [M + H] + , 1285.9[2M+H] + .

[0251]

[0252] H-Trp-(N-Me)Nle-Asp-Phe-NH 2: RP-HPLC (10→70% MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 10.2 min; MS (ESI, positive ion): C 31 H 40 N 6 O 6 m / z calculated value: 592.7, found value: m / z = 593.3 [M + H] + ,1186.3[2M+H] + .

[0253]

[0254] H-Trp-(N-Me)Nle-Asp-Trp-NH 2 : RP-HPLC (10→70% MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 10.3 min; MS (ESI, positive ion): C 33 H 41 N 7 O 6 m / z calculated value: 631.7, found value: m / z = 632.3 [M + H] + ,1264.8[2M+H] + .

[0255]

[0256] H-Trp-(N-Me)Nle-Asp-Tyr-NH 2 : RP-HPLC (10→70% MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R =8.79 min; MS (ESI, positive ion): C 31 H 40 N 6 O 7 m / z calculated value: 608.7, found value: m / z = 609.3 [M + H] + ,1217.6[2M+H] + .

[0257]

[0258] H-Trp-(N-Me)Nle-Asp-p-amino-Phe-NH 2 : RP-HPLC (10→70% MeCN / H2 O, containing 0.1% TFA, 15 min, λ=220nm): t R =7.34 min; MS (ESI, positive ion): C 31 H 41 N 7 O 6 m / z calculated value: 607.3, found value: m / z = 608.2 [M+H] + ,1215.6[2M+H] + .

[0259]

[0260] H-Trp-(N-Me)Leu-Asp-1-Nal-NH 2 : RP-HPLC (10→70% MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 11.4 min; MS (ESI, positive ion): C 35 H 42 N 6 O 6 m / z calculated value: 642.3, found value: m / z = 643.7 [M+H] + ,1285 . 1[2M+H] + .

[0261]

[0262] H-Trp-(N-Me)Val-Asp-1-Nal-NH 2 : RP-HPLC (10→70% MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 10.4 min; MS (ESI, positive ion): C 34 H 40 N 6 O 6 m / z calculated value: 628.3, found value: m / z = 629.3 [M + H] + ,1257.0[2M+H] + .

[0263]

[0264] H-Trp-(N-Me)Gly-Asp-1-Nal-NH 2 : RP-HPLC (10→70% MeCN / H2 O, containing 0.1% TFA, 15 min, λ=220nm): t R =9.48 min; MS (ESI, positive ion): C 31 H 34 N 6 O 6 Calculated value of m / z: 586.3, found value: m / z = 587 . 7[M+H] + .

[0265]

[0266] H-Trp-(N-Me)Ala-Asp-1-Nal-NH 2 : RP-HPLC (10→70% MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R =9.92 min; MS (ESI, positive ion): C 32 H 36 N 6 O 6 m / z calculated value: 600.3, found value: m / z = 601.7 [M + H] + ,1202.9[2M+H] + .

[0267]

[0268] H-Trp-(N-Me)Met-Asp-1-Nal-NH 2 : RP-HPLC (10→70% MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 11.0 min; MS (ESI, positive ion): C 34 H 40 N 6 O 6 m / z calculated for S: 660.3, found: m / z = 661.8 [M+H] + .

[0269]

[0270] H-Trp-(N-Me)Glu-Asp-1-Nal-NH 2 : RP-HPLC (10→70% MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R=9.26 min; MS (ESI, positive ion): C 34 H 38 N 6 O 8 m / z calculated value: 658.3, found value: m / z = 659.5 [M+H] + .

[0271]

[0272] H-Phe-(N-Me)Nle-Asp-1-Nal-NH 2 : RP-HPLC (10→70% MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 11.6 min; MS (ESI, positive ion): C 33 H 41 N 5 O 6 m / z calculated value: 603.3, found value: m / z = 604.6 [M + H] + ,1208.6[2M+H] + .

[0273]

[0274] H-1-Nal-(N-Me)Nle-Asp-1-Nal-NH 2 : RP-HPLC (10→70% MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 12.9 minutes; MS (ESI, positive ion): C 37 H 43 N 5 O 6 m / z calculated value: 653.3, found value: m / z = 654.0 [M+H] + .

[0275]

[0276] H-2-Nal-(N-Me)Nle-Asp-1-Nal-NH 2 : RP-HPLC (10→70% MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 12.8 min; MS (ESI, positive ion): C 37 H 43 N 5 O6 m / z calculated value: 653.3, found value: m / z = 654.3 [M + H] + .

[0277]

[0278] H-p-amino-Phe-(N-Me)Nle-Asp-1-Nal-NH 2 : RP-HPLC (10→70% MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 8.65 min; MS (ESI, positive ion): C 33 H 42 N 6 O 6 m / z calculated value: 618.3, found value: m / z = 619.2 [M+H] + ,1237.5[2M+H] + .

[0279]

[0280] H-Tyr-(N-Me)Nle-Asp-1-Nal-NH 2 : RP-HPLC (10→70% MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 10.3 min; MS (ESI, positive ion): C 33 H 41 N 5 O 7 m / z calculated value: 619.3, found value: m / z = 620.3 [M + H] + , 1240.2[2M+H] + .

[0281]

[0282] H-Trp-(N-Me)Nle-Glu-1-Nal-NH 2 : RP-HPLC (10→70% MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 11.5 min; MS (ESI, positive ion): C 36 8 44 N 6 O 6m / z calculated value: 656.3, found value: m / z = 656.9 [M + H] + ,1314.3[2M+H] + .

[0283]

[0284] H-γ-glu-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH 2 (CCK-32): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R =9.7 minutes; MS (ESI, positive ion): C 49 H 62 N 10 O 13 m / z calculated value: 998.5, found value: m / z = 998.7 [M + H] + .

[0285]

[0286] H-γ-glu-Ala-Tyr-Gly-Trp-Nle-Asp-(homo-Phe)-NH 2 (CCK-33): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 10.2 min; MS (ESI, positive ion): C 50 H 64 N 10 O 13 Calculated value of m / z: 1012.5, found value: m / z = 1013.6 [M+H] + ,507.4[M+2H] 2+ .

[0287]

[0288] H-γ-glu-Ala-Tyr-Gly-Trp-Nle-Asp-(p-amino-Phc)-NH 2 (CCK-34): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R =7.5 min; MS (ESI, positive ion): C 49 H 63 N11 O 13 Calculated value of m / z: 1014.1, found value: m / z = 1014.5 [M+H] + ,507.9[M+2H] 2+ .

[0289]

[0290] H-γ-glu-Ala-Tyr-Gly-Trp-Nle-Asp-Phg-NH 2 (CCK-35): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R =9.2 minutes; MS (ESI, positive ion): C 48 H 60 N 10 O 13 m / z calculated value: 984.4, found value: m / z = 985.6 [M + H] + ,1971.4[2M+H] + .

[0291]

[0292] H-γ-glu-Ala-Tyr-Gly-Trp-Nle-Asp-(a-Me)Phe-NH2(CCK-36): RP-HPLC (10→70% MeCN / H2O, containing 0.1% TFA, 15 min, λ=220 nm): t R =9.2 minutes; MS (ESI, positive ion): C 50 H 64 N 10 O 13 m / z calculated value: 1012.5, found value: m / z = 1013.3 [M+H] + .

[0293]

[0294] H-γ-glu-Ala-Tyr-GIy-(a-Me)Trp-Nle-Asp-Phe-NH 2 (CCK-37): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R =9.9 minutes; MS (ESI, positive ion): C 50 H 64 N10 O 13 Calculated value of m / z: 1013.2, found value: m / z = 1013.2 [M + H] + ,507.3[M+2H] 2+ .

[0295]

[0296] H-γ-glu-Ala-Tyr-Gly-Trp-Nle-Asp-Bip-NH 2 (CCK-38): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 11.3 min; MS (ESI, positive ion): C ss H 66 N 10 O 13 Calculated value of m / z: 1074.5, found value: m / z = 1075.1 [M+H] + ,565.2[M+2H] 2+ .

[0297]

[0298] H-γ-glu-Ala-Tyr-Gly-Trp-Nle-Asp-Trp-NH 2 (CCK-39): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R =9.7 minutes; MS (ESI, positive ion): C 51 H 63 N 11 O 13 m / z calculated value: 1037.5, found value: m / z = 1038.2 [M+H] + ,519.8[M+2H] 2+ .

[0299]

[0300] H-γ-glu-Ala-Tyr-Gly-Trp-Nle-Asp-Dap(SiFA)-NH 2 (CCK-48): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R= 14.2 minutes; MS (ESI, positive ion): C 58 H 80 FN 11 O 14 Calculated m / z of Si: 1201.6, found: m / z = 1202.8 [M+H] + ,602.2[M+2H] 2+ .

[0301]

[0302] H-γ-glu-Ala-Tyr-Gly-Trp-(N-Me)Nle-Asp-1-Nal-NH 2 (CCK-49): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 11.4 min; MS (ESI, positive ion): C 54 H 66 N 10 O 13 m / z calculated value: 1062.5, found value: m / z = 1062.8 [M + H] + .

[0303] Glycine scanning derivatives

[0304]

[0305] DOTA-(Gly) 4 -Trp-(N-Me)Nle-Asp-1-Nal-NH 2 (DOTA-CCK-55): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 10.6 min; MS (ESI, positive ion): C 59 H 80 N 14 O 17 m / z calculated value: 1201.6, found value: m / z = 1257.0 [M+H] + ,629.0[M+2H] 2+ .

[0306]

[0307] DOTA-(Gly)2-Tyr-Gly-Trp-(N-Me)Nle-Asp-1-Nal-NH 2(DOTA-CCK-56): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 11.0 min; MS (ESI, positive ion): C 56 H 86 N 14 O 18 m / z calculated value: 1362.6, found value: m / z = 1363.1 [M+H] + ,682.0[M+2H] 2+ .

[0308]

[0309] DOTA-Gly-Ala-Tyr-Gly-Trp-(N-Me)Nle-Asp-1-Nal-NH 2 (DOTA-CCK-57): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 11.0 min; MS (ESI, positive ion): C 57 H 88 N 14 O 18 m / z calculated value: 1376.6, found value: m / z = 1377.1 [M+H] + ,689.1[M+2H] 2+ .

[0310]

[0311] DOTA-Gly-Trp-(N-Me)Nle-Asp-1-Nal-NH 2 (DOTA-CCK-58): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 10.9 min; MS (ESI, positive ion): C 53 H 71 N 11 O 14 m / z calculated value: 1085.5, found value: m / z = 1086.6 [M + H] + ,544.0[M+2H] 2+ .

[0312]

[0313] DOTA-γ-glu-Gly-Tyr-Gly-Trp-(N-Me)Nle-Asp-1-Nal-NH 2 (DOTA-CCK-59): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 10.8 min; MS (ESI, positive ion): C 69 H 90 N 14 O 20 m / z calculated value: 1434.7, found value: m / z = 1436.6 [M + H] + ,719.0[M+2H] 2+ .

[0314]

[0315] DOTA-γ-glu-Ala-(Gly) 2 -Trp-(N-Me)Nle-Asp-1-Nal-NH 2 (DOTA-CCK-60): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 10.6 min; MS (ESI, positive ion): C 53 H 86 N 14 O 19 m / z calculated value: 1342.6, found value: m / z = 1343.9 [M+H] + ,672.7[M+2H] 2+ .

[0316]

[0317] DOTA-Gly-Ala-(Gly) 2 -Trp-(N-Me)Nle-Asp-1-Nal-NH 2 (DOTA-CCK-61): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 10.6 min; MS (ESI, positive ion): C 60 H 82 N 14 O 17m / z calculated value: 1270.6, found value: m / z = 1272.4 [M+H] + ,636.9[M+2H] 2+ .

[0318]

[0319] DOTA-γ-glu-(Gly) 3 -Trp-(N-Me)Nle-Asp-1-Nal-NH 2 (DOTA-CCK-62): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 10.5 min; MS (ESI, positive ion): C 62 H 84 N 14 O 19 m / z calculated value: 1329.4, found value: m / z = 1330.5 [M + H] + ,665.8[M+2H] 2+ .

[0320] Compounds containing PEG linkers

[0321]

[0322] DOTA-(PEG) 4 -Trp-(N-Me)Nle-Asp-1-Nal-NH 2 (DOTA-CCK-50): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 11.0 min; MS (ESI, positive ion): C 64 H 93 N 11 O 19 m / z calculated value: 1319.7, found value: m / z = 1319.7 [M + H] + ,659.9[M+2H] 2+ .

[0323]

[0324] DOTA-γ-glu-(PEG) 3 -Trp-(N-Me)Nle-Asp-1-Nal-NH 2(DOTA-CCK-66): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 10.8 min; MS (ESI, positive ion): C 67 H 96 N 12 O 21 m / z calculated value: 1405.6, found value: m / z = 1405.3 [M + H] + ,702.8[M+2H] 2+ .

[0325]

[0326] NOTA-γ-glu-(PEG) 3 -Trp-(N-Me)Nle-Asp-1-Nal-NH 2 (NOTA-CCK-66): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 11.5 min; MS (ESI, positive ion): C 63 H 89 N 11 O 19 m / z calculated value: 1303.6, found value: m / z = 1304.0 [M+H] + ,652.9[M+2H] 2+ .

[0327]

[0328] NODAGA-γ-glu-(PEG) 3 -Trp-(N-Me)Nle-Asp-1-Nal-NH 2 (NODAGA-CCK-66): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 11.4 min; MS (ESI, positive ion): C 66 H 93 N 11 O 21 Calculated value of m / z: 1376.5, found value: m / z = 1375.9 [M+H] + ,689.2[M+2H] 2+ .

[0329]

[0330] (R)-DOTAGA-γ-glu-(PEG) 3 -Trp-(N-Me)Nle-Asp-1-Nal-NH 2 ((R)-DOTAGA-CCK-66): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 10.9 min; MS (ESI, positive ion): C 70 H 100 N 12 O 23 m / z calculated value: 1477.6, found value: m / z = 1477.4 [M+H] + ,739.7[M+2H] 2+ .

[0331]

[0332] (S)-DOTAGA-γ-glu-(PEG) 3 -Trp-(N-Me)Nle-Asp-1-Nal-NH 2 ((S)-DOTAGA-CCK-66): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 10.9 min; MS (ESI, positive ion): C 70 H 100 N 12 O 23 m / z calculated value: 1477.6, found value: m / z = 1477.4 [M+H] + ,739.6[M+2H] 2+ .

[0333]

[0334] DOTA-glu-(PEG) 3 -Trp-(N-Me)Nle-Asp-1-Nal-NH 2 (DOTA-CCK-66.2): ​​RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 10.9 min; MS (ESI, positive ion): C 67 H96 N 12 O 21 m / z calculated value: 1405.6, found value: m / z = 1405.9 [M + H] + ,703.6[M+2H] 2+ .

[0335]

[0336] DOTA-γ-glu-PEG-Trp-(N-Me)Nle-Asp-1-Nal-NH 2 (DOTA-CCK-77): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 10.8 min; MS (ESI, positive ion): C 62 H 86 N 12 O 19 m / z calculated value: 1303.4, found value: m / z = 1302.9 [M + H] + ,651.6[M+2H] 2+ .

[0337]

[0338] DOTA-γ-glu-(PEG) 2 -Trp-(N-Me)Nle-Asp-1-Nal-NH 2 (DOTA-CCK-63): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 10.9 min; MS (ESI, positive ion): C 65 H 92 N 12 O 20 m / z calculated value: 1361.5, found value: m / z = 1359.5 [M+H] + ,680.6[M+2H] 2+ .

[0339]

[0340] DOTA-γ-glu-(PEG) 4 -Trp-(N-Me)Nle-Asp-1-Nal-NH 2(DOTA-CCK-78): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 11.0 min; MS (ESI, positive ion): C 69 H 100 N 12 O 22 m / z calculated value: 1449.6, found value: m / z = 1447.4 [M+H] + ,724.3[M+2H] 2+ .

[0341]

[0342] DOTA-γ-glu-(PEG) 5 -Trp-(N-Me)Nle-Asp-1-Nal-NH 2 (DOTA-CCK-79): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 11.1 min; MS (ESI, positive ion): C 71 H 104 N 12 O 23 m / z calculated value: 1493.7, found value: m / z = 1491.1 [M+H] + , 746-3[M+2H] 2+ .

[0343]

[0344] DOTA-γ-glu-(PEG) 7 -Trp-(N-Me)Nle-Asp-1-Nal-NH 2 (DOTA-CCK-80): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 11.1 min; MS (ESI, positive ion): C 75 H 112 N 12 O 25 Calculated value of m / z: 1581.8, found value: m / z = 1582.4 [M+H] + ,792.0[M+2H] 2+ .

[0345]

[0346] DOTA-γ-glu-(PEG) 11 -Trp-(N-Me)Nle-Asp-1-Nal-NH 2 (DOTA-CCK-81): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 11.3 min; MS (ESI, positive ion): C 83 H 128 N 12 O 29 m / z calculated value: 1758.0, found value: m / z = 1759.0 [M+H] + ,880.0[M+2H] 2+ .

[0347] PEGylated rhCCK ligand

[0348]

[0349] DOTA-dap(SiFA)-(PEG) 4 -γ-glu-(PEG) 3 -Trp-(N-Me)Nle-Asp-1-Nal-NH 2 (DOTA-rhCCK-67): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 14.1 min; MS (ESI, positive ion): C 98 H 148 FN 15 O 29 Calculated m / z of Si: 2047.4, found: m / z = 1022.9 [M+2H] 2+ .

[0350]

[0351] DOTA-dap(SiFA)-γ-glu-(PEG) 4 -γ-glu-(PEG) 3 -Trp-(N-Me)Nle-Asp-1-Nal-NH 2 (DOTA-rhCCK-68): RP-HPLC (10→70%MeCN / H 2O, containing 0.1% TFA, 15 min, λ=220nm): t R = 13-3 minutes; MS (ESI, positive ion): C 103 H 155 FN 16 O 32 Calculated m / z of Si: 2175.1, found: m / z = 1087.3 [M+2H] 2+ .

[0352]

[0353] DOTA-dap(SiFA-ipa)-γ-glu-(PEG) 11 -γ-glu-(PEG) 3 -Trp-(N-Me)Nle-Asp-1-Nal-NH 2 (DOTA-rhCCK-69): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 12.7 minutes; MS (ESI, positive ion): C 118 H 183 FN 16 O 41 Calculated m / z of Si: 2528.9, found: m / z = 1264.9 [M+2H] 2+ .

[0354]

[0355] DOTA-dap(SiFA)-γ-glu-(PEG) 7 -γ-glu-(PEG) 3 -Trp-(N-Me)Nle-Asp-1-Nal-NH 2 (DOTA-rhCCK-70): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 13.6 min; MS (ESI, positive ion): C 103 H 155 FN 16 O 32 Calculated m / z for Si: 2175.1, found: m / z = 1153.8 [M+2H] 2+ .

[0356]

[0357] DOTA-dap(SiFAlin)-γ-glu-(PEG) 7 -γ-glu-(PEG) 3 -Trp-(N-Me)Nle-Asp-1-Nal-NH 2 (DOTA-rhCCK-71): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 13.2 minutes; MS (ESI, positive ion): C 113 H 177 FN 17 O 35 Si + m / z calculated value: 2380.8, found value: m / z = 1191.0 [M + 2H] 2+ ,794.1[M+3H] 3+ .

[0358]

[0359] DOTA-dap(SiFA-ipa)-γ-glu-(PEG) 7 -γ-glu-(PEG) 3 -Trp-(N-Me)Nle-Asp-1-Nal-NH 2 (DOTA-rhCCK-72): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 12.8 min; MS (ESI, positive ion): C 110 H 167 FN 16 O 37 Calculated m / z of Si: 2352.7, found: m / z = 1177.8 [M+2H] 2+ ,785.4[M+3H] 3+ .

[0360]

[0361] DOTA-dap(SiFA)-(PEG) 7 -γ-glu-(PEG) 3 -Trp-(N-Me)Nle-Asp-1-Nal-NH 2 (DOTA-rhCCK-73): RP-HPLC (10→70%MeCN / H 2O, containing 0.1% TFA, 15 min, λ=220nm): t R = 14.1 min; MS (ESI, positive ion): C 104 H 160 FN 15 O 32 Calculated m / z of Si: 2179.6, found: m / z = 1090.6 [M+2H] 2+ ,727.3[M+3H] 3+ .

[0362]

[0363] DOTA-dap(SiFA)-(PEG) 11 -γ-glu-(PEG) 3 -Trp-(N-Me)Nle-Asp-1-Nal-NH 2 (DOTA-rhCCK-74): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 14.0 min; MS (ESI, positive ion): C 112 H 176 FN 15 O 36 Calculated m / z of Si: 2355.8, found: m / z = 1176.8 [M+2H] 2+ ,784.8[M+3H] 3+ .

[0364]

[0365] DOTA-dap(SiFAlin)-γ-glu-(PEG) 11 -γ-glu-(PEG) 3 -Trp-(N-Me)Nle-Asp-1-Nal-NH 2 (DOTA-rhCCK-75): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 13.2 minutes; MS (ESI, positive ion): C 121 H 193 FN 17 O 39 Si + m / z calculated value: 2557.0, found value: m / z = 1279.5 [M + 2H] 2+,853.2[M+3H] 3+ .

[0366]

[0367] DOTA-dap(SiFA)-γ-glu-(PEG) 11 -γ-glu-(PEG) 3 -Trp-(N-Me)Nle-Asp-1-Nal-NH 2 (DOTA-rhCCK-76): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 13.6 min; MS (ESI, positive ion): C 117 H 183 FN 16 O 39 Calculated m / z of Si: 2484.9, found: m / z = 1241.4 [M+2H] 2+ ,827.9[M+3H] 3+ .

[0368]

[0369] DOTA-dap(SiFAlin)-(PEG) 4 -γ-glu-(PEG) 3 -Trp-(N-Me)Nle-Asp-1-Nal-NH 2 (DOTA-rhCCK-90): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 13.6 min; MS (ESI, positive ion): C 102 H 158 FN 16 O 29 Si + m / z calculated value: 2119.6, found value: m / z = 1061.7 [M + 2H] 2+ ,708.2[M+3H] 3+ .

[0370]

[0371] DOTA-dap(SiFAlin)-γ-glu-(PEG) 4 -γ-glu-(PEG) 3-Trp-(N-Me)Nle-Asp-1-Nal-NH 2 (DOTA-rhCCK-91): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 13.1 min; MS (ESI, positive ion): C 107 H 165 FN 17 O 32 Si + m / z calculated value: 2248.7, found value: m / z = 1125.6 [M + 2H] 2+ ,750.8[M+3H] 3+ .

[0372]

[0373] DOTA-dap(SiFA)-γ-glu-(Hyp) 3 -γ-glu-(PEG) 3 -Trp-(N-Me)Nle-Asp-1-Nal-NH 2 (DOTA-rhCCK-83): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 13.2 minutes; MS (ESI, positive ion): C 105 H 151 FN 18 O 32 m / z calculated for Si: 2224.5, found: m / z = 1113.8 [M+2H] 2+ ,743.1[M+3H] 3+ .

[0374]

[0375] DOTA-dap(SiFA)-γ-glu-(Hyp) 6 -γ-glu-(PEG) 3 -Trp-(N-Me)Nle-Asp-1-Nal-NH 2 (DOTA-rhCCK-84): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 12.6 min; MS (ESI, positive ion): C 120 H172 FN 21 O 36 m / z calculated for Si: 2563.9, found: m / z = 1284-3 [M+2H] 2+ ,856.5[M+3H] 3+ .

[0376]

[0377] DOTA-dap(SiOH)-γ-glu-(Hyp) 6 -γ-glu-(PEG) 3 -Trp-(N-Me)Nle-Asp-1-Nal-NH 2 (DOTA-rhCCK-84.2): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 11.7 minutes; MS (ESI, positive ion): C 120 H 173 N 21 O 39 Calculated m / z of Si: 2561.9, found: m / z = 1281.6 [M+2H] 2+ ,854.2[M+3H] 3+ .

[0378]

[0379] DOTA-dap(SiFA)-γ-glu-(cit) 3 -(Hyp) 3 -γ-glu-(PEG) 3 -Trp-(N-Me)Nle-Asp-1-Nal-NH 2 (DOTA-rhCCK-85): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 12.6 min; MS (ESI, positive ion): C 123 H 184 FN 27 O 38 m / z calculated for Si: 2696.1, found: m / z = 1349.8 [M+2H] 2+ ,900.6[M+3H] 3+ .

[0380]

[0381] DOTA-dap(SiFA)-γ-glu-(Hyp) 8 -γ-glu-(PEG) 3 -Trp-(N-Me)Nle-Asp-1-Nal-NH 2 (DOTA-rhCCK-86): RP-HPLC (10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 12.2 min; MS (ESI, positive ion): C 130 H 186 FN 23 O 42 Calculated m / z of Si: 2790.1, found: m / z = 1396.1 [M+2H] 2+ ,930.9[M+3H] 3+ .

[0382] 1.2. In vitro results of compounds with modified Trp-(N-Me)Nle-Asp-1-Nal motif

[0383] Although most CCK-2R targeting compounds consist of at least seven amino acids (plus linker and chelator), several publications indicate that the four N-terminal amino acids are essential for high CCK-2R affinity [28,30,33]. nat The Lu-labeled reference compounds all revealed high CCK-2R affinity (4.9-12.8 nM), while most of the compounds modified within the N-terminal (four) amino acid sequence showed slightly or significantly reduced CCK-2R affinity (Table 1).

[0384] Table 1: nat Lu-labeled reference DOTA-PP-F11N (DOTA-glu 1 -glu 2 -glu 3 -glu4-glu 5 -glu 6 -Ala 7 -Tyr 8 -Gly 9 -Trp 10 -Nle 11 -Asp 12 -Phe 13 -NH 2 ), DOTA-PP-y-F11N(DOTA-γ-glu 1 -γ-glu 2-γ-glu 3 -γ-glu 4 -γ-glu 5 -y-glu 6 -Ala 7 -Tyr 8 -Gly 9 -Trp 10 -Nle 11 -Asp 12 -Phe 13 -NH 2 )、DOTA-MGS5(DOTA-glu 6 -Ala 7 -Tyr 8 -Gly 9 -Trp 10 -(N-Me)Nle 11 -Asp 12 -1-Nal 13 -NH 2 )、DOTA-γ-MGS5(DOTA-y-glu 6 -Ala 7 -Tyr 8 -Gly 9 -Trp 10 -(N-Me)Nle 11 -Asp 12 -1-Nal 13 -NH 2 ) and CP04 (DOTA-glu 1 -glu 2 -glu 3 -glu 4 -glu 5 -glu 6 -Ala 7 -Tyr 8 -Gly 9 -Trp 10 -Met 11 -Asp 12 -Phe 13 -NH 2 ) compared with the CCK-2R affinity of compounds containing modifications within the tetrapeptide binding motif for AR4-2J cells (depicted as half maximal inhibitory concentration, IC 50 ).

[0385]

[0386]

[0387] Interestingly, DOTA-PP-F11N (CCK-32), which lacks the DOTA chelator and its six D-glutamates, exhibited lower CCK-2R affinity than its parent peptide. Substitution of the C-terminal Phe in CCK-32 (CCK-33 to -39) with similar aromatic amino acids resulted in a complete loss of CCK-2R affinity for most compounds. Substitution of Trp in CCK-32 with (a-Me)Trp resulted in a decrease in IC 50 3 times higher, while substitution of p-amino-Phe for Phe resulted in an IC 50 All these observations point to a conserved site at the C-terminus.

[0388] Interestingly, DOTA-MGS5 without DOTA chelator (CCK-49, which differs from CCK-32 in only two positions ((N-Me)Nle instead of Nle, and 1-Nal instead of Phe)) showed similar nat Lu] Lu-DOTA-MGS5 equivalent IC 50 value, and IC 50 The value was about four times lower than that of CCK-32. Surprisingly, the truncated DOTA-PP-F11N motif (H-Trp-Nle-Asp-Phe-NH 2 ) showed low CCK-2R affinity, while the truncated DOTA-MGS5 motif (H-Trp-(N-Me)Nle-Asp-1-Nal-NH 2 ) reveals that nat Lu]Lu-DOTA-MGS5 has a relatively high CCK-2R affinity ( Figure 1 ).

[0389] These results led to the hypothesis that the four N-terminal amino acids of DOTA-MGS5 (H-Trp-(N-Me)Nle-Asp-1-Nal-NH 2 ) is sufficient for high CCK-2R affinity, whereas for DOTA-PP-F11N and its analogs, only the complete peptide structure revealed CCK-2R affinity in the low nanomolar range.

[0390] Tetrapeptide structure H-Trp-(N-Me)Nle-Asp-1-Nal-NH 2 Further examination of the 5-mercaptoethanol (derived from DOTA-MGS5) showed that substitution of 1-Nal with Phe resulted in even higher CCK-2R affinity, whereas H-Trp-Nle-Asp-Phe-NH 2Substitution of Phe with 1-Nal in (derived from DOTA-PP-F11N) resulted in a significantly reduced CCK-2R affinity. Thus, it was shown that high CCK-2R affinity of the CCK-2 targeting tetrapeptide could only be observed when a methyl group was present on the amide nitrogen between the Trp-Nle bonds ( Figure 2 Maintaining this (N-Me)Nle motif within the tetrapeptide even allows for substitutions at the C-terminus (in H-Trp-(N-Me)Nle-Asp-1-Nal-NH 2 However, this position is still conservative, as the replacement of 1-Nal with 2-Nal resulted in a significant loss of CCK-2R affinity.

[0391] Furthermore, substitution of Nle with Met and Leu resulted in tetrapeptides with CCK-2R affinities in the low nanomolar range, whereas substitution with Val, Gly, Ala, and Glu was not permissive for high CCK-2R affinities. Therefore, we speculate that Nle can only be exchanged with aliphatic amino acids of similar length to maintain high CCK-2R affinity. Neither negatively charged nor amino acids with smaller aliphatic side chains could be successfully placed in this position. Substitution of Trp with various aromatic amino acids (such as Phe, 1-Nal, 2-Nal, p-amino-Phe, or Tyr) did not reveal improvements in CCK-2R affinity, which led to the assumption that the Trp moiety at the position is inevitable for the high binding affinity of the compound. Furthermore, when Ala was substituted with Glu, a significant loss of CCK-2R affinity was observed.

[0392] 1.3. In vitro results of glycine scanning derivatives

[0393] To confirm our hypothesis that only the four N-terminal amino acids of DOTA-MGS5 would have sufficient CCK-2R affinity, glycine scanning of the remaining four amino acids of the parent peptide was performed. Thus, the receptor affinity (IC 50 ) and lipophilicity (logD 7.4 )(Table 2).

[0394] Table 2: Affinity of glycine scanning derivatives for CCK-2R in AR4-2J cells (depicted as half maximal inhibitory concentration, IC 50 ) and lipophilicity (depicted as n-octanol / PBS partition coefficient, pH = 7.4, logD 7.4 All compounds in this series share the same C-terminal sequence (H-Trp-(N-Me)Nle-Asp-1-Nal-NH 2 ). Structural differences at the N-terminus are given in parentheses.

[0395]

[0396] These studies revealed that our hypothesis was largely correct, as the parent peptide ([ nat Compared to [Lu]Lu-DOTA-MGS5), the amino acids γ-glu / glu, Ala, and Tyr can be replaced by Gly residues without significant loss of CCK-2R affinity. These data support the hypothesis that the N-terminal motif H-Trp-(N-Me)Nle-Asp-1-Nal is sufficient for high CCK-2R affinity and that the additional N-terminal amino acids can be considered as part of the linker. However, [ nat The dramatic loss of CCK-2R affinity of Lu]Lu-DOTA-CCK-50 suggests that a significant distance of a sterically demanding chelator such as DOTA is necessary for high CCK-2R affinity. x Chain) is necessary, but also sufficient.

[0397] Furthermore, these studies revealed that the presence of the γ-glu moiety within the linker moiety was beneficial not only for CCK-2R affinity but also, in particular, for overall lipophilicity, as all ligands containing this moiety exhibited logD values ​​between −2.66 and −2.56. 7.4 values, while those compounds that did not contain this moiety showed logD values ​​ranging from -2.09 to -1.26 7.4 Values ​​(Table 2, Figure 3 ).

[0398] As mentioned previously, no successful strategy has been described to address the metabolically labile Gly-Trp bond that is prevalent in CCK-2R agonists. Based on the results described in this chapter, it is hypothesized that H-Trp-(N-Me)Nle-Asp-1-Nal-NH 2 The amino acid at the N-terminal end (H-glu-Ala-Tyr-Gly-) can be simply PEGylated x Chain substitution, which may also stabilize the entire peptide, since PEG-Trp bonds are thought to be metabolically more stable than Gly-Trp bonds. Therefore, this PEG-containing design was evaluated and is described in the next chapter.

[0399] 1.4. In vitro and in vivo results of CCK-2R targeting compounds containing PEGylated groups

[0400] Due to the results of previous studies, the four amino acids γ-glu-Ala-Tyr-Gly in DOTA-MGS5 are replaced by PEGx chains of different lengths. Because several reports have been made that the main metabolic instability at the Gly-Trp site, it is speculated that these modifications can further enhance the metabolic stability of these DOTA-MGS5 derivatives. In addition, some compounds also include γ-glu moieties, which are observed to be beneficial to both CCK-2R affinity and lipophilicity. All data are compared with DOTA-MGS5 (Table 3).

[0401] Table 3: Affinity of CCK-2R targeting compounds containing PEG linkers for AR4-2J cells compared to the reference compound DOTA-MGS5 (depicted as half maximal inhibitory concentration, IC 50 The compounds tested were quantified using [ nat Lu] Lutetium, [ nat Ga] Gallium or [ nat Cu] copper labeling.

[0402]

[0403] With PEG x Independent of the chain length and the chelator-metal chelate used, all compounds revealed ICs in the low nanomolar range (3.5-9.0 nM). 50 However, the CCK-2R affinity gradually decreased with the application of extended PEG chains, which is why the highest CCK-2R affinity was observed for DOTA-CCK-77, -63, -66, and -66.2. In addition, the N-terminal insertion of the y-glu moiety had a positive impact on the overall CCK-2R affinity, as DOTA-CCK-78 showed a higher affinity than CCK-50 (IC 50 =8.84±1.25) lower IC 50 value.

[0404] In addition, the logD values ​​of these compounds complexed with lutetium-177, gallium-67, or copper-64 were evaluated. 7.4 Values ​​(Table 4).

[0405] Table 4: Lipophilicity of CCK-2R targeting compounds containing a PEG linker compared to DOTA-MGS5 (depicted as n-octanol / PBS partition coefficient, pH=7.4, log D 7.4 The compounds tested were [ 177 Lu] Lutetium, [ 67 Ga] Gallium and [ 64 Cu] copper labeling.

[0406]

[0407] Generally speaking, in all 177 Among the Lu-labeled compounds, those containing shorter PEG chains all showed lower lipophilicity. In addition, the addition of the γ-glu moiety resulted in a decrease in logD 7.4 The value decreased slightly (DOTA-CCK-78 vs. -50). 7.4 Value < -3, [ 67 Ga]Ga-DOTA-CCK-66, [ 177 Lu]Lu-DOTA-CCK-63 and [ 177 Lu]Lu-DOTA-CCK-77 is the most hydrophilic compound in the series. However, all compounds revealed logD in the range of -3.1 to -2.3. 7.4 values, which is considered ideal for in vivo applications.

[0408] The highest CCK-2R affinity and lowest logD 7.4 Among the compounds with the highest value (DOTA-CCK-77, -63, -66 and -66.2), it was decided to further study the effects of DOTA-CCK-66 and -66.2 as well as DOTA-MGS5 in [ nat / 67 Ga] Gallium and [ nat / 64 CCK-2R affinity and lipophilicity when labeled with copper (Cu) Figure 4-5 ) and human serum albumin (HSA) binding and metabolic stability.

[0409] As reported by Klingler et al. 68 Ga]Ga-DOTA-MGS5 has favorable biodistribution characteristics and rapid clearance kinetics

[30] , so it was evaluated with [ nat Lu] Lutetium, [ nat Ga] Gallium or [ nat Cu] copper-complexed DOTA-CCK-66 and -66.2 and DOTA-MGS5 for HSA binding (Table 5).

[0410] Table 5: nat Lu] Lutetium, [ nat Ga] Gallium or [ nat HSA binding (%) of copper-complexed DOTA-CCK-66, -66.2 and DOTA-MGS5.

[0411]

[0412] For the different metal chelates of the three PEGylated CCK derivatives evaluated, certain trends could be observed ( Figure 6).

[0413] All three nat Lu-labeled compounds all revealed the highest HSA binding, while nat The Cu-complexed peptides showed less interaction with HSA. nat Ga-labeled ligand, the least HSA binding was observed. In a comparative analysis, DOTA-MGS5 bound most to HSA, while DOTA-CCK-66 and DOTA-CCK-66.2 revealed lower HSA affinity. It is therefore believed that DOTA-CCK-66 and -66.2 will be cleared faster than DOTA-MGS5 regardless of which radiometal is used.

[0414] In addition, the in vitro culture of human serum was performed by incubation at 37°C for 72 ± 2 hours. 177 Stability studies of the Lu-labeled compounds DOTA-MGS5, DOTA-CCK-66, and DOTA-CCK-66.2. The more promising in vitro compounds were also evaluated in vivo. 177 The metabolic stability of Lu]Lu-DOTA-CCK-66 in mouse serum and urine 30 minutes after injection was compared with [ 177 Lu]Lu-DOTA-MGS5 were compared (Table 6).

[0415] Table 6: [ 177 Lu]Lu-DOTA-MGS5, [ 177 Lu]Lu-DOTA-CCK-66 and [ 177 Evaluation of the metabolic stability of Lu]Lu-DOTA-CCK-66.2.

[0416]

[0417] In vitro, all three compounds revealed high metabolic stability in human serum after incubation at 37°C for 72 ± 2 h. 177 Lu]Lu-DOTA-MGS5 showed a slightly higher intact fraction than the PEGylated compound ( Figure 7 ).

[0418] In vivo, slightly higher amounts of the parent compound were still intact in the mouse serum 30 min after injection. However, it could be shown that, in contrast to [ 177 Compared with Lu]Lu-DOTA-MGS5, in the urine of mice 30 minutes after injection, [ 177 The intact fraction of Lu]Lu-DOTA-CCK-66 is almost three times higher. Therefore, it is speculated that [177 Lu]Lu-DOTA-CCK-66 was mostly cleared, while [ 177 Lu]Lu-DOTA-MGS5 was mostly metabolized and cleared ( Figure 8 , Table 6), which may be beneficial in reducing the accumulation and retention of activity in non-target organs, as metabolites tend to accumulate nonspecifically, particularly in the kidney.

[0419] To examine the pharmacokinetic characteristics of our novel PEGylated CCK-2R targeting compounds, we performed a 4-hour pharmacokinetics test on [ 67 The biodistribution of Ga]Ga-DOTA-CCK-66 was studied and compared with the [ 68 The corresponding data of Ga]Ga-DOTA-MGS5 at the same time point

[30] were compared ( Fig. 9 ).

[0420] Comparative analyses must be made with caution because 100 pmol were injected per animal, whereas Klingler et al. injected only 20 pmol

[30] . However, despite the fivefold higher amount of peptide administered, 1 h after injection, 67 Ga]Ga-DOTA-CCK-66 showed activity in tumors at a level only slightly lower than that of [ 68 Ga]Ga-DOTA-MGS5. As expected, the former revealed lower activity levels in all non-tumor organs, which may be due to its improved metabolic stability and clearance kinetics. Even if it is speculated that the activity levels in all organs are higher when lower peptide amounts are administered, tumor uptake may also be increased. Therefore, this suggests that the relative tumor to background (T / B) ratio is effective regardless of the peptide amount administered. 68 Compared with Ga]Ga-DOTA-MGS5, [ 67 Ga]Ga-DOTA-CCK-66 had a higher T / B ratio in all organs (especially in relation to blood, stomach, and kidney). 67 Ga]Ga-DOTA-CCK-66 same [ 68 Ga]Ga-DOTA-CCK-66 is a reasonable candidate for clinical translation. 67 Support for μSPECT / CT images of Ga]Ga-DOTA-CCK-66 ( Fig.10 ).

[0421] Accumulation in CCK-2R-positive tumors and the stomach was specific, as demonstrated by co-administration of excess [ nat Competition studies of Ga]Ga-DOTA-MGS5 showed.

[0422] To investigate the potential of DOTA-CCK-66 as a theranostic agent, additional evaluation was performed 24 h after injection. 177 Lu-labeled and with [ 177 Lu]Lu-DOTA-MGS5 for comparison ( Fig.11 ).

[0423] Biodistribution studies 24 hours after injection revealed that [ 177 The activity level of Lu]Lu-DOTA-CCK-66 in tumors is related to 177 Lu]Lu-DOTA-MGS5 (8.56±1.08 vs. 11.0±1.15% ID / g). However, the tumor / blood ratio of the former was improved (2009±469 vs. 1480±329), which is due to the improved clearance of activity from non-tumor organs, which is beneficial for therapeutic applications. The tumor / stomach and tumor / kidney ratios of the two compounds were comparable. Competition studies resulted in significantly reduced activity levels in both the stomach and tumor, confirming the [ 177 These observations are supported by μSPECT / CT images ( Fig.12 ).

[0424] For all these reasons, a first-in-human study was performed in patients with MTC who showed elevated serum tumor markers and a shorter calcitonin doubling time. In all three MTC patients evaluated, [ 68 Ga]Ga-DOTA-CCK-66 PET / CT showed favorable pharmacokinetics, with significant increases in tumor lesions and CCK-2R expression 2 h after injection. + Highly accumulated in the stomach Fig.13 A, 14A and 15A). No significant activity levels were found in organs other than the bladder and ureters as a result of physiological excretion. 68 In Ga]Ga-DOTA-CCK-66PET / CT, multiple MTC-derived lesions were detected in each patient. Patient 1 had a left retroclavicular lymph node metastasis and another mediastinal metastasis in the aortic arch. Patient 2 had a right retroclavicular lymph node metastasis, bilateral hilar lymph node metastases, two liver metastases, and bone metastases in the right femur and right sciatic bone. Patient 3 had a lymph node metastasis in the left carotid nerve sheath, a local recurrence in the left thyroid bed, and lymph node metastases in the right upper mediastinum.

[0425] All patients benefited significantly from these 68 Ga]Ga-DOTA-CCK-66PET / CT findings. If surgical resection is not possible, [ 177Lu]Lu-DOTA-CCK-66 was used for radioligand therapy of identified metastases because the compound exhibited a favorable biodistribution profile—high levels of activity in tumors over time and rapid clearance of activity from the blood of the animals. Further studies in humans are necessary to confirm these promising results in this small population.

[0426] In conclusion, the novel PEGylated CCK-2R targeting compounds [ 67 / 68 Ga]Ga- / [ 177 Lu]Lu-DOTA-CCK-66 reveals compounds with clinical applications [ 68 Ga]Ga- / [ 177 The in vivo data of the PEG-linked Lu-DOTA-MGS5 were improved compared to the Lu-DOTA-MGS5. The T / B ratio of the former was comparable or even improved, which can be attributed to the tetrapeptide design combined with the PEG linker. To this end, it was demonstrated that the increase in metabolic stability at the Gly-Trp site is a result of the N-terminal amino acid being PEGylated. x chain substitution results. Due to these promising preclinical properties, the use of [ 68 The first human application of [Ga]Ga-DOTA-CCK-66 in MTC patients was performed, which confirmed the favorable biodistribution characteristics of this compound already observed in animals. Due to its rapid clearance from the blood, the tumor-to-background contrast was high and multiple tumor manifestations could be detected in three MTC patients. Further studies in humans are needed to clearly elucidate whether this compound has a beneficial effect on MTC management. However, preliminary results suggest that [ 68 Ga]Ga-DOTA-CCK-66 may indeed be a viable option for PET imaging of MTC-derived lesions. 177 Lu-labeled analogs may also be an effective therapeutic option, making this compound a very promising theranostic agent.

[0427] 1.5. In vitro and in vivo results of CCK-2R targeting compounds containing PEG linkers and different chelators

[0428] As a result of previous studies, the DOTA chelator of DOTA-CCK-66 was replaced with various chelators (NOTA, NODAGA, (S)-DOTAGA, and (R)-DOTAGA) to demonstrate the applicability of combining different chelators with our optimized tetrapeptide binding motif (Table 7).

[0429] Table 7: Affinity of DOTA-CCK-66, NOTA-CCK-66, NODAGA-CCK-66, (R)-DOTAGA-CCK66 and (S)-DOTAGA-CCK-66 for CCK-2R in AR4-2J cells (depicted as half maximal inhibitory concentration, IC 50 The compounds tested were [ nat Ga] Gallium or [ 19 F]AlF labeling.

[0430]

[0431] These studies revealed that the DOTA chelator of DOTA-CCK-66 could be replaced by NODAGA without any loss of CCK-2R affinity ( Fig.16 ). In contrast, for nat Ga-and[ 19 For both of the A1F-complexed derivatives, replacing DOTA with NOTA was accompanied by a loss of affinity for CCK-2R. In addition, the introduction of (R)- and (S)-DOTAGA chelators instead of DOTA resulted in a decrease in IC 50 Although most DOTA-CCK-66 analogs have lower CCK-2R affinity, NOTA and NODAGA in particular have a similar affinity to [ nat Ga] Gallium or [ 19 The use of F]AlF together is conceivable for future applications.

[0432] 1.6. In vitro and in vivo results of first-generation PEGylated rhCCK ligands

[0433] Several PEGylated rhCCK compounds were synthesized and compared with the CCK-2R affinity (IC 50 ) and lipophilicity (logD 7.4 ) nat / 177 Lu / nat Ga-labeling (Table 8).

[0434] Table 8: Affinity of PEGylated CCK-2R ligands containing SiFA building blocks for CCK-2R in AR4-2J cells (depicted as half maximal inhibitory concentration, IC 50 ), lipophilicity (depicted as n-octanol / PBS partition coefficient, pH = 7.4, logD 7.4 ) and human serum albumin (HSA). All compounds share the C-terminal sequence Hy-glu-(PEG) 3 -Trp-(N-Me)Nle-Asp-1-Nal-NH 2(=X). The tested compounds and [ nat Lu / 177 Lu] lutetium or [ nat DOTA-MGS5 and DOTA-CCK-66 are depicted as reference ligands without silicon-based fluoride acceptors.

[0435]

[0436]

[0437] None nat Lu-labeled rhCCK ligand showed similar nat Lu]Lu-DOTA-MGS5 or [ nat Lu]Lu-DOTA-CCK-66 has comparable CCK-2R affinity ( Fig.17 As expected, the linker moiety carries only one negative charge [ 177 Lu]Lu-DOTA-rhCCK-67, [ 177 Lu]Lu-DOTA-rhCCK-73 and [ 177 Lu]Lu-DOTA-rhCCK-74 revealed increased lipophilicity because the lipophilic SiFA moiety was not fully compensated.

[0438] Therefore, an additional γ-glu moiety was introduced, which significantly improved the lipophilicity ([ nat / 177 Lu]Lu-DOTA-rhCCK-68, [ nat / 177 Lu]Lu-DOTA-rhCCK-70 and [ nat / 177 Lu]Lu-DOTA-rhCCK-76) while maintaining CCK-2R affinity. To further improve lipophilicity, SiFA was replaced with the more hydrophilic SiFA-ipa ([ nat / 177 Lu]Lu-DOTA-rhCCK-69, [ nat / 177 Lu]Lu-DOTA-rhCCK-72) or SiFAlin([ nat / 177 Lu]Lu-DOTA-rhCCK-71, [ nat / 177 Lu]Lu-DOTA-rhCCK-75, [ nat / 177 Lu]Lu-DOTA-rhCCK-90, [ nat / 177 Lu]Lu-DOTA-rhCCK-91) structural unit replacement. Compounds containing SiFA-ipa part revealed that logD 7.4The value can be improved by half an order of magnitude, but the CCK-2R affinity is significantly reduced. SiFA is partially replaced by SiFAlin, for proteins containing (PEG) 7 Comparable CCK-2R affinity was observed for the derivatives containing the linker. However, no improvement in lipophilicity was observed. 11 The linker and SiFA moiety [ nat / 177 Lu]Lu-DOTA-rhCCK-76(IC 50 :14.2±0.9,logD 7.4 :-1.82±0.09) compared to (PEG) 11 The linker and SiFAlin part [ nat / 177 Lu]Lu-DOTA-rhCCK-75 revealed a higher IC 50 Value (19.2±1.6) and lipophilicity (-1.15±0.05). 4 In the rhCCK derivatives connected, the SiFAlin part ([ nat / 177 Lu]Lu-DOTA-rhCCK-90, [ nat / 177 Lu]Lu-DOTA-rhCCK-91) replaces SiFA ([ nat / 177 Lu]Lu-DOTA-rhCCK-67, [ nat / 177 Lu]Lu-DOTA-rhCCK-68) had only a slight effect on lipophilicity, but resulted in increased CCK-2R affinity. Surprisingly, DOTA-rhCCK-91 revealed that [ nat Lu]Lutetium (IC 50 =8.56±0.7) and [ nat Ga] Gallium (IC 50 =8.24±1.6) with high CCK-2R affinity, which is in contrast to previously evaluated rhCCK derivatives.

[0439] In another approach, the lipophilicity and CCK-2R affinity of rhCCK derivatives were addressed via a polyhydroxyproline linker. Thus, compounds containing three to eight hydroxyproline moieties in their linker structure (DOTA-rhCCK-83, -84, and -86) were evaluated in vitro. In addition, a peptide containing three hydroxyproline and three D-citrulline moieties (DOTA-rhCCK-85) was tested in vitro. In this series, [ nat / 177 Lu]Lu-DOTA-rhCCK-84 combines the highest hydrophilicity (logD 7.4 =-2.14±0.06) and CCK-2R affinity (IC 50=7.87±0.3).

[0440] As expected, HSA binding was high (84.7 to 94.5%) for all rhCCK derivatives tested ( Fig.18 ), which was expected since increased HSA binding has been observed for other compounds containing silicon-based fluoride receptors, regardless of which target molecule was used.

[0441] The most attractive in vitro data were evaluated in vivo at 24 hours post-injection. 177 Lu]Lu-DOTA-rhCCK-70, [ 177 Lu]Lu-DOTA-rhCCK-84 and [ 177 Lu]Lu-DOTA-rhCCK-91, and with [ 177 Lu]Lu-DOTA-CCK-66 and [ 177 Lu]Lu-DOTA-MGS5 compares the two ( Fig.19 ).

[0442] [ 177 Lu]Lu-DOTA-rhCCK-84 revealed the highest activity level in the tumor 24 h after injection (compared to [ 177 Lu]Lu-DOTA-CCK-66 and [ 177 The activity levels in the kidney were slightly increased (<10% ID / g) compared to the reference without silicon-based fluoride receptors. The overall biodistribution pattern was promising, which was supported by μSPECT / CT images ( Fig. 20 ), especially in the context of previously developed minigastrin derivatives containing silicon-based fluoride receptors - due to the presence of several negative charges in the direct vicinity of the silicon-based fluoride receptor building block, these derivatives showed a greatly increased retention of activity in the kidney (>80% ID / g at 24 hours after injection). In addition, it must be mentioned that the accumulation and retention of activity in the rat kidney is not directly comparable to the human situation. Therefore, it must be explored in future patient studies whether this slowed clearance of activity from the kidney also exists in humans and whether it is a cause for concern. In addition to the significantly increased activity levels in the tumor 24 hours after injection, DOTA-rhCCK-84 also has the advantage (relative to DOTA-CCK-66 and DOTA-MGS5) that it can be easily labeled with fluorine-18, which is beneficial for high-contrast positron emission tomography.

[0443] In addition to favorable tumor uptake (36.3 ± 3.7% ID / g), the structurally identical [ 18 F]F-[nat The biodistribution study of Lu]Lu-DOTA-rhCCK-84 also revealed a high activity level (11.3±1.3%ID / g) in the blood 1 hour after injection ( Fig.21 ). However, biodistribution studies at 2 and 4 hours after injection confirmed that over time, [ 177 The active clearance of Lu]Lu-DOTA-rhCCK-84 from the blood was good (3.71±0.46%ID / g and 1.14±0.18%ID / g, respectively), while the active level in the tumor remained high (35.0±2.9 and 34.0±3.6%ID / g, respectively). nat Lu]Lu-DOTA-MGS5 or [ nat Competition studies with Lu]Lu-DOTA-MG-SiFA-23 resulted in significantly reduced activity levels in both the stomach and tumors, confirming that [ 177 CCK-2R specificity of Lu]Lu-DOTA-rhCCK-84 ( Fig. 22 ).

[0444] In addition, 30 minutes after injection, in serum (93.5±2.1%) and urine (55.4±9.0%), [ 177 The in vivo stability of Lu]Lu-DOTA-rhCCK-84 is high ( Fig.23 ), especially compared with the reference compound DOTA-MGS5 (Table 6). For all mentioned aspects, this compound may be a promising candidate for clinical translation.

[0445] In addition to DOTA-rhCCK-84, [ 177 Lu]Lu-DOTA-rhCCK-70 also showed promising in vivo data. The activity level in the tumor was slightly higher than that of [ 177 Lu]Lu-DOTA-MGS5, but significantly lower than [ 177 Lu]Lu-DOTA-rhCCK-84 ( Fig.19 ). Activity levels in the kidneys are slightly lower than [ 177 Lu]Lu-DOTA-rhCCK-84, but still increased compared to the reference without silicon-based fluoride receptor. 177 The increase in the activity level of Lu]Lu-DOTA-rhCCK-70 may be partly attributed to the incomplete 177Lu-labeling (approximately 5% free Lu-177 injected into animals) and is also attributed to the increased lipophilicity of the compound compared to ligands that do not contain silicon-based fluoride receptors. Among all rh-based CCK-2R targeting compounds, [ 177 Lu]Lu-DOTA-rhCCK-91 showed the lowest activity level in the kidney at 24 hours after injection, but its activity level in tumors was also reduced.

[0446] The conclusion is that 177 The biodistribution characteristics of Lu]Lu-DOTA-rhCCK-70, -84, and -91 confirmed our hypothesis that high activity uptake and retention in the kidney could be addressed by reducing the negative charge in the neighborhood of the silicon-based fluoride receptor moiety (when combined with [ 177 In addition, the novel design of PEGylated rhCCK ligands led to the promising peptide DOTA-rhCCK-84, which is similar to [ 177 Lu]Lu-DOTA-MGS5 or [ 177 Lu]Lu-DOTA-CCK-66 showed significantly higher activity levels in tumors 24 hours after injection. In addition, this rh-based compound provides easy and rapid 18 The potential for F-labeling was investigated and revealed a significantly elevated accumulation of activity in tumors at 1, 2, and 4 h after injection, which is why this ligand could be a valid candidate for theranostic applications of fluorine-18 and lutetium-177 in humans.

[0447] Example 2 - CCK2R Targeting Compounds Comprising SiFA

[0448] 2.1. Overview of synthetic CCK-2R targeting ligands

[0449]

[0450] DOTA-PP-F11N(3): RP-HPLC(10→90%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R =7.6 min, K'=3.5; MS (ESI, positive ion): C 90 H 123 N 19 O 35 m / z calculated value: 2029.8, found value: m / z = 1016.4 [M + 2H] 2+ .

[0451]

[0452] CP04(4): RP-HPLC(10→90%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R =7.2 min, K'=3.3; MS (ESI, positive ion): C 89 H 121 N 19 O 35 m / z calculated for S: 2047.8, found: m / z = 1024.2 [M+2H] 2+ .

[0453] In the following, the sequence H-glu 1- glu 2 -glu 3 -glu 4 -glu 5 -glu 6 -Ala 7 -Tyr 8 -Gly 9 -Trp 10 -Nle 11 -Asp 12 -Phe 13 -NH 2 Defined as F11N.

[0454] MG-SiFA compounds containing polyglutamate linkers

[0455] In the following, the sequence H-glu 1 -glu 2 -glu 3 -glu 4 -glu 5 -glu 6 -Ala 7 -Tyr 8 -Gly 9 -Trp 10 -Nle 11 -Asp 12 -Phe 13 -NH 2 Defined as F11N.

[0456]

[0457] DOTAGA-glu-[dap(SiFA) 6 ]F11N(DOTAGA-MG-SiFA-5): RP-HPLC(10→90%MeCN / H 2O, containing 0.1% TFA, 15 min, λ=220nm): t R = 10.2 min K' = 5.0 MS (ESI, positive ion): C 111 H 154 FN 21 O 39 m / z calculated for Si: 2452.0, found: m / z = 1226.8 [M+2H] 2+ .

[0458]

[0459] DOTAGA-glu-[dap(SiFA) 5 ]F11N(DOTAGA-MG-SiFA-6): RP-HPLC(10→90%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm), t R =9.6 min, K'=4.7; MS (ESI, positive ion): C 111 H 154 FN 21 O 39 m / z calculated for Si: 2452.0, found: m / z = 1226.4 [M+2H] 2+ .

[0460]

[0461] DOTAGA-glu-[dap(SiFA) 4 ]F11N(DOTAGA-MG-SiFA-7): RP-HPLC(10→90%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R =9.3 min, K'=5.5; MS (ESI, positive ion): C 111 H 154 FN 21 O 39 m / z calculated for Si: 2452.0, found: m / z = 1226.4 [M+2H] 2+ .

[0462]

[0463] DOTAGA-glu-[dap(SiFA) 3 ]F11N(DOTAGA-MG-SiFA-8): RP-HPLC(10→90%MeCN / H 2O, containing 0.1% TFA, 15 min, λ=220nm): t R =9.5 min, K'=4.6; MS (ESI, positive ion): C 111 H 154 FN 21 O 39 m / z calculated for Si: 2452.0, found: m / z = 1226.7 [M+2H] 2+ .

[0464]

[0465] DOTAGA-glu-[dap(SiFA) 2 ]F11N(DOTAGA-MG-SiFA-9): analytical RP-HPLC (10→90%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R =9.1 min, K'=4.4; MS (ESI, positive ion): C 111 H 154 FN 21 O 39 m / z calculated for Si: 2452.0, found: m / z = 1227.3 [M+2H] 2+ .

[0466]

[0467] DOTAGA-glu-[dap(SiFA) 1 ]F11N(DOTAGA-MG-SiFA-10): RP-HPLC(10→90%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R =9.2 min, K'=4.5; MS (ESI, positive ion): C 111 H 154 FN 21 O 39 Calculated m / z of 8i: 2452.0, found: m / z = 1227.4 [M+2H] 2+ .

[0468]

[0469] DOTAGA-dap(SiFA)-F11N(DOTAGA-MG-SiFA-11): RP-HPLC(10→90%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): tR =9.6 min, K'=5.6; MS (ESI, positive ion): C 111 H 154 FN 21 O 39 m / z calculated for Si: 2452.0, found: m / z = 1227.0 [M+2H] 2+ .

[0470] In the following, the sequence H-γ-glu 1 -γ-glu 2 -γ-glu 3 -γ-glu 4 -γ-glu 5 -γ-glu 6 -Ala 7 -Tyr 8 -Gly 9 -Trp 10 -Nle 11 -Asp 12 -Phe 13 -NH 2 It is defined as γ-F11N.

[0471]

[0472] DOTA-PP-γ-F11N: RP-HPLC (10→490%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R =7.1 min, K'=3.2; MS (ESI, positive ion): C 90 H 123 N 19 O 35 m / z calculated value: 2029.8, found value: m / z = 1015.7 [M + 2H] 2+ .

[0473]

[0474] DOTAGA-γ-glu-[dap(SiFA) 6 ]γ-FllN(DOTAGA-MG-SiFA-13): analytical RP-HPLC (10→90% MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 10.1 min K' = 6.1 MS (ESI, positive ion): C 111 H 154 FN 21 O39 m / z calculated for Si: 2452.0, found: m / z = 1227.2 [M+2H] 2+ .

[0475]

[0476] DOTAGA-γ-glu-[dap(SiFA) 5 ]γ-F11N (DOTAGA-MG-SiFA-14): RP-HPLC (10→90%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm), t R =9.6 min, K'=5.7; MS (ESI, positive ion): C 111 H 154 FN 21 O 39 m / z calculated for Si: 2452.0, found: m / z = 1227.3 [M+2H] 2+ .

[0477]

[0478] DOTAGA-7-glu-[dap(SiFA) 4 ]γ-F11N (DOTAGA-MG-SiFA-15): RP-HPLC (10→90%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R =9.3 min, K'=5.5; MS (ESI, positive ion): C 111 H 154 FN 21 O 39 Calculated m / z of 8i: 2452.0, found: m / z = 1226.4 [M+2H] 2+ .

[0479]

[0480] DOTAGA-γ-glu-[dap(SiFA) 3 ]γ-F11N (DOTAGA-MG-SiFA-16): RP-HPLC (10→90%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R =9.1 min, K'=5.4; MS (ESI, positive ion): C 111 H 154 FN 21 O39 Calculated m / z of 8i: 2452.0, found: m / z = 1226.7 [M+2H] 2+ .

[0481]

[0482] DOTAGA-γ-glu-[dap(SiFA) 2 ]γ-F11N (DOTAGA-MG-SiFA-17): RP-HPLC (10→90%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R =9.5 min, K'=5.6; MS (ESI, positive ion): C 111 H 154 FN 21 O 39 m / z calculated for Si: 2452.0, found: m / z = 1226.6 [M+2H] 2+ .

[0483]

[0484] DOTAGA-γ-glu-[dap(SiFA) 1 ]γ-F11N (DOTAGA-MG-SiFA-18): RP-HPLC (10→90%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R =9.6 min, K'=5.7; MS (ESI, positive ion): C 111 H 154 FN 21 O 39 m / z calculated for Si: 2452.0, found: m / z = 1227.2 [M+2H] 2+ ,817.8[M+3H] 3+ .

[0485]

[0486] DOTAGA-dap(SiFA)-γ-F11N(DOTAGA-MG-SiFA-19): RP-HPLC (10→90%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R =9.5 min, K'=5.6; MS (ESI, positive ion): C 111 H 154 FN21 O 39 m / z calculated for Si: 2452.0, found: m / z = 1227.3 [M+2H] 2+ ,818.7[M+3H] 3+ .

[0487]

[0488] DOTA-MG-SiFA-19: RP-HPLC (10→90%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 10.0 min; MS (ESI, positive ion): C 108 H 150 FN 21 O 57 m / z calculated for Si: 2380.0, found: m / z = 1190.9 [M+2H] 2+ ,794.4[M+3H] 3+ .

[0489]

[0490] DOTAGA-dap(SiFA)-glu-F11N(DOTAGA-MG-SiFA-20): RP-HPLC (10→90%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R =9.6 min, K'=4.7; MS (ESI, positive ion): C 116 H 161 FN 22 O 42 m / z calculated for Si: 2581.1, found: m / z = 1292.0 [M+2H] 2+ .

[0491]

[0492] DOTAGA-dap(SiFA)-(glu) 2 -F11N (DOTAGA-MG-SiFA-21): analytical RP-HPLC (10→90% MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R =9.4 min, K'=4.6; MS (ESI, positive ion): C 122 H 169 FN 22 O45 m / z calculated for Si: 2710.1, found: m / z = 1355.9 [M+2H] 2+ .

[0493]

[0494] DOTAGA-dap(SiFA)-γ-glu-γ-F11N(DOTAGA-MG-SiFA-22): Analytical RP-HPLC (10→90%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R =9.4 min, K'=4.6; MS (ESI, positive ion): C 116 H 161 FN 22 O 42 Calculated m / z of Si: 2581.1, found: m / z = 1291.8 [M+2H] 2+ ,861.6[M+3H] 3+ .

[0495]

[0496] DOTAGA-dap(SiFA)-(γ-glu) 2 -γ-F11N (DOTAGA-MG-SiFA-23): analytical RP-HPLC (10→90% MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R =9.3 min, K'=4.5; MS (ESI, positive ion): C 121 H 168 FN 23 O 45 m / z calculated for Si: 2710.1, found: m / z = 1356.1 [M+2H] 2+ ,904.2[M+3H] 3+ .

[0497]

[0498] DOTA-MG-SiFA-23: RP-HPLC (10→90%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R =9.8 min; MS (ESI, positive ion): C 118 H 164 FN 23 O 43m / z calculated for Si: 2638.1, found: m / z = 1320.3 [M+2H] 2+ ,880.5[M+3H] 3+ .

[0499]

[0500] DOTAGA-(γ-glu) 2 -[dap(SiFA) 2 ]γ-F11N(DOTAGA-MG-SiFA-24): analytical RP-HPLC (10→90%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R =9.4 min, K'=4.6; MS (ESI, positive ion): C 116 H 161 FN 22 O 42 m / z calculated for Si: 2581.1, found: m / z = 1292.5 [M+2H] 2+ ,862.0[M+3H] 3+ .

[0501]

[0502] DOTAGA-(γ-glu) 3 -[dap(SiFA) 2 ]γ-F11N(DOTAGA-MG-SiFA-25): analytical RP-HPLC (10→90%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R =9.3 min, K'=4.5; MS (ESI, positive ion): C 122 H 169 FN 22 O 45 m / z calculated for Si: 2710.1, found: m / z = 1357.1 [M+2H] 2+ ,905.1[M+3H] 3+ .

[0503]

[0504] DOTAGA-(γ-glu) 4 -[dap(SiFA) 1 ]γ-F11N (DOTAGA-MG-SiFA-26): RP-HPLC (10→90%MeCN / H2 O, containing 0.1% TFA, 15 min, λ=220nm): t R =9.2 min, K'=4.5; MS (ESI, positive ion): C 126 H 175 FN 24 O 48 m / z calculated for Si: 2839.2, found: m / z = 1422.4 [M+2H] 2+ ,948.3[M+3H] 3+ .

[0505]

[0506] DOTA-dap(SiFA)-γ-orn-(γ-glu) 5 -F11N(DOTA-MG-SiFA-27): RP-HPLC(10→90%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 9.5 minutes; MS (ESI, positive ion): C 108 H 153 FN 23 O 35 Calculated m / z of Si: 2366.1, found: m / z = 1183.8 [M+2H] 2+ ,789.6[M+3H] 3+ .

[0507]

[0508] DOTA-dap(SiFA)-γ-orn-(γ-glu) 7 -F11N(DOTA-MG-SiFFA-28): RP-HPLC(10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 11.5 min; MS (ESI, positive ion): C 118 H 167 FN 24 O 41 m / z calculated for Si: 2623.2, found: m / z = 1312.6 [M+2H] 2+ .

[0509]

[0510] DOTA-dap(SiFA)-Glutamol-(γ-glu) 7-FllN(DOTA-MG-SiFA-29): RP-HPLC(10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 11.5 min; MS (ESI, positive ion): C 118 H 166 FN 23 O 42 Calculated m / z of Si: 2625.8, found: m / z = 1313.6 [M+2H] 2+ ,876.1[M+3H] 3+ .

[0511]

[0512] DOTA-dap(SiFA)-γ-lys-(γ-glu) 7 -F11N(DOTA-MG-SiFA-30): RP-HPLC(10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 11.4 min; MS (ESI, positive ion): C 119 H 169 FN 24 O 41 Calculated m / z of 8i: 2637.2, found: m / z = 1319.5 [M+2H] 2+ ,880.1[M+3H] 3+ .

[0513]

[0514] DOTA-y-lys-dap(SiFA)-y-lys-(γ-glu) 7 -F11N(DOTA-MG-SiFA-31): RP-HPLC(10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 11.0 min; MS (ESI, positive ion): C 125 H 181 FN 26 O 42 m / z calculated for Si: 2767.1, found: m / z = 1383.7 [M+2H] 2+ ,922.9[M+3H] 3+ .

[0515]

[0516] DOTA-dap(SiFA)-(γ-glu) 8 -MGSS(DOTA-MG-SiFA-40): RP-HPLC(10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 12.5 min; MS (ESI, positive ion): C 123 H 168 FN 23 O 43 Calculated m / z of 8i: 2702.1, found: m / z = 1352.8 [M+2H] 2+ .

[0517]

[0518] DOTA(-dap(SiFA)-GABA-(γ-glu) 7 -FllN(DOTA-MG-SiFA-44): RP-HPLC(10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 11.8 minutes; MS (ESI, positive ion): C 117 H 164 FN 23 O 41 Calculated m / z of 8i: 2594.1, found: m / z = 1298.9 [M+2H] 2+ ,866.3[M+3H] 3+ .

[0519]

[0520] DOTA-dap(SiFA)-(GABA) 2 -(γ-glu) 6 -FllN(DOTA-MG-SiFA-45): RP-HPLC(10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 11.8 minutes; MS (ESI, positive ion): C 116 H 164 FN 23 O 39 m / z calculated for Si: 2550.1, found: m / z = 1277.0 [M+2H] 2+ ,851.6[M+3H] 3+.

[0521]

[0522] DOTA-dap(SiFA)-(GABA) 3 -(γ-glu) 5 -F11N(DOTA-MG-SiFA-46): RP-HPLC(10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 11.9 minutes; MS (ESI, positive ion): C 115 H 164 FN 23 O 37 Calculated m / z of Si: 2507.8, found: m / z = 1254.5 [M+2H] 2+ ,836.7[M+3H] 3+ .

[0523]

[0524] DOTA-dap(SiFA)-(GABA-γ-glu) 4 -F11N(DOTA-MG-SiFA-47): RP-HPLC(10→70%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 11.8 minutes; MS (ESI, positive ion): C 114 H 164 FN 23 O 35 Calculated m / z of Si: 2462.2, found: m / z = 1232.5 [M+2H] 2+ ,822.0[M+3H] 3+ .

[0525] 2.2. In vitro and in vivo results of MG-SiFA compounds containing polyglutamate linkers

[0526] Several MG-SiFA compounds were synthesized and evaluated in vitro nat / 177 Lu-labeled. The affinity of CCK-2R to AR4-2J cells (IC 50 ) and lipophilicity (logD 7.4 In addition, reference ligands were synthesized and evaluated. nat / 177 Lu-DOTA-PP-F11N and nat / 177 Lu-CP04( nat / 177 Lu-DOTA-(glu)6 -Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH 2 )(Table 2).

[0527] Table 9: Affinity of CCK-2R targeting ligands with dap (SiFA) building blocks at different positions within the poly-D-α / γ-glutamate chain for CCK-2R in AR4-2J cells (depicted as half maximal inhibitory concentration, IC 50 ) and lipophilicity (depicted as n-octanol / PBS partition coefficient, pH = 7.4, logD 7.4 ). nat Lu / 177 Lu complex and compared with two reference ligands. Sequence H-glu 1 -glu 2 -glu 3 -glu 4 -glu 5 -glu 6 -Ala 7 -Tyr 8 -Gly 9 -Trp 10 -Nle 11 -Asp 12 -Phe 13 -NH 2 Defined as F11N. Sequence Hy-glu 1 -γ-glu 2 -γ-glu 3 -y-glu 4 -γ-glu 5 -γ-glu 6 -Ala 7 -Tyr 8 -Gly 9 -Trp 10 -Nle 11 -Asp 12 -Phe 13 -NH 2 Defined as y-F11N. MGS5: H-Ala 7 -Tyr 8 -Gly 9 -Trp 10 -(N-Me)Nle 11 -Asp 12 -1-Nal 13 -NH 2 , GABA: gamma-aminobutyric acid.

[0528]

[0529]

[0530] In general, the counterparts ([ nat Compared with the small gastrin analogs containing a poly-γ-glu linker ([ nat Lu]Lu-DOTAGA-MG-SiFA13-19) showed improved CCK-2R affinity. nat Lu]Lu-DOTA-PP-y-F11N (which shows a ratio of [ nat Lu]Lu-DOTA-PP-F11N has 1.7 times lower IC 50 This is believed to be due to the interaction of dap (SiFA) and the chelating agent with the binding sequence (Ala 7 -Tyr 8 -Gly 9 -Trp 10 -Nle 11 -Asp 12 -Phe 13 -NH 2 ) was enhanced, since the use of γ-glu instead of α-glu significantly prolonged the corresponding peptide backbone. In the case of compounds containing dap(SiFA) moieties, the highest CCK-2R affinity was observed when this group was separated by at least five or six α- / γ-glu moieties. As expected, in the SiFA-containing MG derivatives that additionally included the DOTAGA chelator, [ nat Lu]Lu-DOTAGA-MG-SiFA-19 revealed the highest CCK-2R affinity. Interestingly, further addition of the γ-glu moiety ([ nat Lu]Lu-DOTAGA-MG-SiFA-22 / -23) did not lead to improved CCK-2R affinity. nat The substitution of DOTA for DOTAGA in Lu]Lu-DOTAGA-MG-SiFA-23 resulted in a significant increase in the affinity for CCK-2R ([ nat Lu]Lu-DOTA-MG-SiFA-23: 4.71±0.62nM), which even exceeded the reference ligand. It is speculated that the receptor prefers the chelate to have a neutral overall charge at the corresponding site. nat Similar observations were made by replacing DOTAGA with DOTA in Lu]Lu-DOTAGA-MG-SiFA-19, which resulted in IC 50 The value is reduced by three times.

[0531] As expected, all 177 Lu-labeled MG-SiFA ligands also revealed increased lipophilicity. However, as mentioned previously, this highly hydrophilic nature has been shown to hinder enhanced tumor accumulation as a result of rapid clearance of the administered active into the bladder. For this reason, it is speculated that 177 The reduced hydrophilicity of the Lu-labeled MG-SiFA conjugates results in a prolonged in vivo circulation time and thus enhanced tumor uptake. 177 Lu-labeled MG-SiFA compounds showed logD ranging from -3 to -2 7.4 values. Klingler et al. reported pharmacophore-modified CCK-2R targeting ligands that exhibited logD values ​​in a similar range to our MG-SiFA conjugates. Notably, their ligands [ 68 Ga]Ga- / [ 111 In]In- / [ 177 Lu]Lu-DOTA-MGS5 and [ 99m The tumor accumulation of Tc]Tc-HYNIC-MGS11 was too high [30,34]. Therefore, it was inferred that this lipophilicity range might be well suited for CCK-2R-targeting derivatives.

[0532] For better comparison, the most promising DOTAGA-containing conjugates containing poly-α-glutamate and poly-γ-glutamate linkers were selected for further in vivo studies. 177 Lu]Lu-DOTAGA-MG-SiFA-21 and [ 177 The biodistribution of Lu]Lu-DOTAGA-MG-SiFA-19 was studied and compared with the reference [ 177 Lu]Lu-DOTA-PP-F11N for comparison ( Fig.24 ).

[0533] In addition, the [ 177 Lu]Lu-DOTA-MG-SiFA-23( Fig.25 ).

[0534] As discussed earlier, [ nat Lu]Lu-DOTAGA-MG-SiFA-21 Display IC 50 Value ratio[ nat Lu]Lu-DOTA-PP-F11N increases by about five times and is natLu]Lu-DOTAGA-MG-SiFA-19 increased by about two times, while [ nat Lu]Lu-DOTA-MG-SiFA-23 Display IC 50 Value ratio[ nat Lu]Lu-DOTA-PP-F11N was nearly three times lower. All three MG-SiFA derivatives also revealed significantly higher lipophilicity than the reference ligand. However, [ 177 Lu]Lu-DOTA-MG-SiFA-23 revealed the highest activity level in tumors 24 h after injection (>25% ID / g), which was higher than [ 177 Interestingly, despite the lower CCK2R affinity of the two DOTAGA-MG-SiFA compounds, they showed a three- to six-fold increase in activity levels in the tumor 24 h after injection, supporting our hypothesis of a benefit of slower clearance of activity

[35] . The second reference ligand [ 177 Lu]Lu-CP04 (which is structurally similar to [ 177 Lu]Lu-DOTA-PP-F11N) showed 0.63±0.08%ID / g in tumor tissue 24 h after injection in A431-CCK-2R(+) tumor-bearing mice, which further strengthened our data

[36] . DOTAGA-conjugated MG-SIFA compounds showed 0.63±0.08%ID / g in tumor tissue 1, 4 and 24 h after injection ( Fig.26 )as well as[ 177 Lu]Lu-DOTA-MG-SiFA-23 1 and 24 hours after injection ( Fig. 27 ) also highlighted improved tumor uptake and slower clearance over time. 177 Lu]Lu-DOTAGA-MG-SiFA-19 and [ 177 The accumulation of both Lu]Lu-DOTA-MG-SiFA-23 was specific in CCK-2R positive tumors and the stomach, which was confirmed by co-administration of excess [ nat Lu]Lu-DOTA-MGS5 competition study shows that ( Fig.28 ).

[0535] Furthermore, it is evident that tumor accumulation at 1 h post-injection was also significantly higher for all MG-SiFA ligands compared to the reference, which is important for the potential use of these compounds for imaging when labeled with fluorine-18. nat Lu / 18 F-mark and 177 Lu / 1 9F-labeled MG-SiFA ligands each had very similar or even identical biodistribution profiles. All MG-SiFA ligands also had higher levels of activity in the stomach compared to the reference, which was expected since the stomach is a CCK-2R positive organ. Interestingly, despite their increased lipophilicity, the levels of activity in the liver were relative to [ 177 Lu]Lu-DOTA-PP-F11N were relatively low. Therefore, it is considered very suitable to introduce the SiFA part into the small gastrin analogs. However, it must be mentioned that all three MG-SiFA compounds experienced a substantial increase in activity levels in the kidney 24 hours after injection (>80% ID / g), which was not seen in the reference compound. Although no explanation has been given for this observation, it is speculated that there seems to be an unknown synergistic effect of the SiFA part and the negative charge within the linker, which leads to this increased renal uptake and retention. However, when 18 With F-labeling, this should be a minor problem, since the physical half-life of this isotope is very short.

[0536] Nevertheless, attempts were made to address this problem by reducing or isolating the negative charge near the dap(SiFA) building block. The N-terminal γ-glu moiety was replaced with γ-om (DOTA-MG-SiFA-27 / -28), Glutamol (DOTA-MG-SiFA-29), or γ-lys (DOTA-MG-SiFA-30 / -31). Interestingly, both CCK-2R affinity and lipophilicity were significantly impaired by those substitutions. These compounds ([ 177 Each of the Lu]Lu-DOTA-MG-SiFA-27-31) revealed a higher 177 Lu]Lu-DOTA-MG-SiFA-23 has a logD value nearly one order of magnitude higher 7.4 Values ​​(Table 9). In addition, [ 177 IC of Lu]Lu-DOTA-MG-SiFA-27 and -29 50 The value is slightly increased, but [ 177 IC of Lu]Lu-DOTA-MG-SiFA-28, -30 and especially -31 50 Therefore, it is speculated that the charge distribution at the N-terminal region is very subtle, as small changes appear to have major effects.

[0537] Based on these results, it was decided to reduce the negative charge by replacing one or more γ-glu moieties with γ-aminobutyric acid (GABA) to retain the backbone length but reduce the amount of negative charge, which resulted in four compounds, DOTA-MG-SiFA-44-47. 177Each of the Lu-labeled compounds revealed similar logD 7.4 Value, ratio 177 Lu]Lu-DOTA-MG-SiFA-23 was more than half an order of magnitude higher (Table 9). 177 The results are comparable or even higher for Lu]Lu-DOTA-MG-SiFA-23, which is surprising since negative charge is presumed to be important for high CCK-2R affinity. However, due to increased lipophilicity, no in vivo evaluation of GABA-containing MG-SiFA compounds was performed.

[0538] In conclusion, the functionality of adding silicon-based fluoride acceptor building blocks to the linker moiety of minigastrin derivatives was demonstrated. The resulting MG-SiFA compounds can be easily and rapidly 18 F-labeling was performed to image MTC via PET. Furthermore, the addition of the lipophilic SiFA moiety also greatly improved the overall pharmacokinetics due to a slowed clearance of the activity, which resulted in a clear increase in the activity levels in the tumor at 1 and 24 h after injection. Although the evaluation was performed at 24 h after injection, it must be mentioned that the use of these MG-SiFA ligands for targeted radiotherapy of Lutetium-177 is not recommended, as these compounds suffer from increased renal retention. However, when 18 This increased renal uptake is not worrisome when F-labeled, as PSMA inhibitors typically reveal higher uptake values ​​1 hour after injection. 177 Lu-and 18 F-labeling was confirmed by biodistribution and imaging studies 1 hour after injection in both cases ( Fig. 22 and 24 ), which is why this compound holds great promise for future clinical use in MTC imaging.

[0539] Example 3 - Materials and Methods

[0540] 3.1. General

[0541] Fmoc-(9-fluorenylmethoxycarbonyl-) and all other protected amino acid analogs were purchased from Bachem Inc. (Bubendorf, Switzerland), Sigma-Aldrich GmbH (Munich, Germany) or Iris Biotech GmbH (Marktredwitz, Germany). The resin (35-100 mesh size, 0.4-0.6 mmol / g loading) was purchased from Sigma-Aldrich GmbH (Munich, Germany). CheMatech (Dijon, France) provided the chelating agent DOTA ( t Bu) 3 and DOTAGA( t Bu) 4 The peptide syringe was obtained from VWR International GmbH (Bruchsal, Germany).

[0542] All necessary solvents and other organic reagents were purchased from Alfa Aesar (Karlsruhe, Germany), Sigma-Aldrich GmbH (Munich, Germany) or VWR International GmbH (Bruchsal, Germany). Solid phase synthesis of peptides was performed manually using a Scilogex MX-RL-E Analog Rotisserie tube rotator (Scilogex, Rocky Hill, CT, USA). 2 O was used after purification via the Barnstead MicroPure system (Thermo Fisher Scientific Inc., Waltham, MA, USA).

[0543] Analytical and preparative reverse phase high performance liquid chromatography (RP-HPLC) were performed using a Shimadzu gradient system (Shimadzu Deutschland GmbH, Neufahrn, Germany), each equipped with a SPD-20A UV / Vis detector (220 nm, 254 nm). In all RP-HPLC runs, different gradients of H 2 MeCN (0.1% TFA) in O (0.1% TFA) was used as eluent.

[0544] For analytical measurements, a MultoKrom 100-5C18 (5 μm, 125×4.6 mm) column (CS Chromatographie GmbH, Langerwehe, Germany) was used with a flow rate of 1 mL / min. Specific gradients and corresponding retention times t are quoted in the text. R and capacity factor K'.

[0545] Preparative RP-HPLC purification was performed with a MultoKrom 100-5C18 (5 μm, 250×10 mm) column (CS Chromatographie GmbH, Langerwehe, Germany) at a constant flow rate of 10 mL / min.

[0546] Analytical and preparative radioactive RP-HPLC were performed using a MultoKrom 100-5C18 (5 μm, 125×4.6 mm) column (CS Chromatographie GmbH, Langerwehe, Germany). L Electrospray ionization-mass spectra for characterization of the species were acquired on a CMS mass spectrometer (Advion Ltd., Harlow, UK).

[0547] For radiolabeling, use [ 177 Lu]LnCl 3 (Molar activity (A M )>3,000 GBq / mg, 740 MBq / mL, 0.04 M HCl, ITG GmbH, Garching, Germany). Radioactivity was detected by connecting the outlet of the UV photometer to an AceMate 925-Scint NaI(T1) well-type scintillation counter from EG&G Ortec (Oak Ridge, TN, USA). The radioactive probe was measured using a 2480 automated γ-counter (Perkin Elmer, Waltham, MA, USA), and IC was analyzed using GraphPad Prism 6 (GraphPad Software Inc., San Diego, CA, USA). 50 For radioactive TLC, a Laura TM Scan-RAM software TM Scanner (LabLogic Systems Ltd., Broomhill, Sheffield, UK).

[0548] NMR spectra were recorded on a Bruker (Billerica, USA) AVHD-300 or AVHD-400 spectrometer at 300 K. Lyophilization was achieved using an Alpha 1-2 LDplus freeze dryer (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany) in combination with a RZ-2 vacuum pump (Vacuubrand GmbH & CoKG, Olching, Germany).

[0549] For in vitro and in vivo studies, RPMI 1640 medium (supplemented with 5 mM L-Gln, 5 mL non-essential amino acids (100×) and 10% FCS) was used as the nutrient medium. For culture passages, phosphate buffered saline (PBS Dulbecco, Ca-free) containing 0.1% EDTA (v / v) was used. 2+ / Mg 2+ ) solution was used to separate the cells. All solutions mentioned and fetal bovine serum (FBS Superior) were obtained from Biochrom GmbH (Berlin, Germany). H 2 Solubilization of the purified product was performed by BioQ® O (Merck KGaA, Darmstadt, Germany). Bovine serum albumin (BSA) was purchased from Biowest (Nuaillé, France).

[0550] exist Cells were cultured in cell culture flasks and counted using a Neubauer hemacytometer (Paul Marienfeld, The cells were counted and seeded into 24-well plates (Greiner Bio-One GmbH, Kremsmünster, Austria). The cells were processed in a MSC Advantage laminar flow cabinet and maintained at 37°C and a humidified atmosphere of 5% CO 2 The cells were maintained in a Heracell 150i incubator (Thermo Fisher Scientific Inc., Waltham, MA, USA) in a 4% CO atmosphere. Rat pancreatic tumor cells AR4-2J expressing CCK-2R were obtained from ECACC (European Collection of Cell Cultures, Salisbury, UK).

[0551] 3.2. Synthesis scheme

[0552] 3.2.1 Solid-phase peptide synthesis following the Fmoc strategy

[0553] Use DIPEA to form peptides on resin

[0554] The corresponding side chain protected Fmoc-AA-OH (1.5 eq) was dissolved in NMP and pre-activated by adding TBTU (1.5 eq), HOAt (1.5 eq) and DIPEA (4.5 eq). After activation for 10 min, the solution was added to the resin-bound free amine peptide and shaken for 1.5 h at room temperature (rt). Subsequently, the resin was washed with NMP (6×20 mL / g resin) and, after Fmoc deprotection, the next amino acid was similarly coupled.

[0555] Formation of peptides on resin using 2,4,6-trimethylpyridine

[0556] For the coupling reaction of amino acids to the free N-terminus of peptides containing dap(Dde) moieties or the coupling of dap(Dde) amino acids, 2,4,6-collidine was used as a base. Therefore, the corresponding side-chain protected Fmoc-AA-OH (1.5 equiv) was dissolved in NMP and pre-activated by adding TBTU (1.5 equiv), HOAt (1.5 equiv) and 2,4,6-collidine (6.0 equiv). After activation for 10 min, the solution was added to the resin-bound free amine peptide and shaken at room temperature for 1.5 h. Subsequently, the resin was washed with NMP (6×20 mL / g resin) and the next amino acid was coupled similarly after Fmoc deprotection.

[0557] Fmoc deprotection on resin

[0558] The resin-bound Fmoc-peptide was treated with 20% piperidine in NMP (v / v) for 5 min and then for 15 min. Afterwards, the resin was washed with NMP (6 x 20 mL / g resin).

[0559] On-resin Fmoc deprotection of Fmoc-D-Cit-OH

[0560] The resin-bound Fmoc-peptide was treated with 20% piperidine in NMP (v / v) for 15 min and the solvent was filtered off. This process was repeated twice. Afterwards, the resin was washed with NMP (6×20 mL / g resin).

[0561] Dde deprotection on resin

[0562] Dde deprotection was performed by adding a solution of imidazole (75 eq.), hydroxylamine hydrochloride (100 eq.) in NMP (7 mL) and DCM (3 mL) to the resin and shaking at room temperature for 2 hours. After complete deprotection, the resin was washed with NMP (6×20 mL / g resin).

[0563] Conjugation of chelator or SiFA moiety

[0564] The protected chelating agent DOTA( tBu) 3 or DOTAGA( t Bu) 4 Or SiFA part (1.5 eq) was dissolved in NMP and pre-activated by adding TBTU (1.5 eq), HOAt (1.5 eq) and 2,4,6-trimethylpyridine (6.0 eq). After activation for 10 min, the solution was added to the resin-bound N-terminal deprotected peptide (1.0 eq) and shaken at room temperature for 3 h. Subsequently, the resin was washed with NMP (3×20 mL / g resin) and DCM (3×20 mL / g resin).

[0565] Conjugation of SiFA-Br

[0566] To generate the SiFAlin moiety, the resin was swollen in DCM. A solution of DIPEA (6.0 eq.) and SiFA-Br (3.0 eq.) in DCM (2 mL) was added to the resin and shaken overnight. The resin was washed with DCM (5 x 5 mL) and dried in a desiccator.

[0567] Conjugation of SiFA-ipa

[0568] After pre-activation at room temperature for 10 minutes, the resin-bound peptide was treated with SiFA-ipa (3.0 eq.) and HOAt (3.0 eq.) in NMP (4 mL / g resin), and TBTU (3 eq.) and 2,4,6-trimethylpyridine (11.0 eq.) in NMP (4 mL / g resin). The reaction mixture was shaken at room temperature for 3 hours before being filtered. The resin was then washed with NMP (6×10 mL / g resin).

[0569] Cleavage of peptide from resin with concomitant deprotection of acid-labile protecting groups

[0570] The fully protected resin-bound peptide was washed with DCM and then dissolved in TFA / TIPS / H 2 The mixture of 4-nitropropene (2-nitropropene) and 4-nitropropene (2-nitropropene) was stirred for 60 minutes. The mixture was stirred for 60 minutes in a mixture of 1% 4-nitropropene (v / v / v; 95 / 2.5 / 2.5) and vibrated for 60 minutes. The solution was filtered out and the resin was treated in the same way for another 60 minutes. The two filtrates were combined and stirred for another 6 hours at room temperature. After removing TFA under a nitrogen stream, a crude product was obtained.

[0571] p-Nitrobenzenesulfonyl protection

[0572] The resin-bound peptide was dissolved in a solution of p-nitrobenzenesulfonyl chloride (5.0 eq.) and 2,4,6-collidine (10.0 eq.) in NMP (10 mL / g resin) and shaken at room temperature for 30 min. After deprotection, the resin was washed with NMP (6 x 10 mL / g resin).

[0573] Deprotection of p-nitrobenzenesulfonyl group

[0574] β-Mercaptoethanol (10 eq.) and DBU (5.0 eq.) in NMP (10 mL / g resin) were added to the p-nitrobenzenesulfonyl protected resin-bound peptide and shaken at room temperature for 2 x 15 min. Afterwards, the resin was washed with NMP (6 x 10 mL / g resin).

[0575] N-methylation

[0576] Dissolve the resin in PPh 3 (0.3 M in anhydrous THF, 5.0 eq.) and MeOH (10 eq.) were added and the resin was shaken at room temperature for 1 min. After that, DIAD (5.0 eq.) was added and the reaction mixture was shaken at room temperature for 30 min. After repeating the previous procedure, the resin was washed with NMP (6×10 mL / g resin).

[0577] 3.2.2. Synthesis and characterization of silicon-based fluoride acceptor building blocks

[0578] The synthesis of the corresponding silicon-based fluoride acceptor building blocks (iv, v and vii) was performed according to previously published procedures with slight modifications by our group. All water- and oxygen-sensitive reactions were performed in dry reaction vessels under argon atmosphere using a vacuum gas manifold.

[0579] ((4-bromobenzyl)oxy)(tert-butyl)dimethylsilane (i)

[0580] To a stirred solution of 4-bromobenzyl alcohol (4.68 g, 25.0 mmol, 1.0 eq) in anhydrous DMF (70 mL) was added imidazole (2.04 g, 30.0 mmol, 1.2 eq) and TBDMSCl (4.52 g, 30.0 mmol, 1.2 eq) and the resulting mixture was stirred at room temperature for 16 h. The mixture was then poured into an ice-cold HO solution. 2 O (250 mL) and washed with Et 2 O (5 × 50 mL). The combined organic fractions were washed with saturated NaHCO 3 The solution was washed with aqueous solution (2 x 100 mL) and brine (100 mL), dried, filtered and concentrated in vacuo to give the crude product which was purified by flash column chromatography (silica, 5% EtOAc / petrol) to give i (7.18 g, 95%) as a colorless oil.

[0581] RP-HPLC (50→100%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 15 minutes. K' = 7.43.

[0582] Di-tert-butyl[4-((tert-butyldimethylsilyloxy)methyl)phenyl]fluorosilane (ii)

[0583] A solution of tBuLi in pentane (7.29 mL, 1.7 mol / L, 12.4 mmol, 2.4 equiv) was added to a solution of ((4-bromobenzyl)oxy)(tert-butyl)dimethylsilane (i) (1.56 g, 5.18 mmol, 1.0 equiv) in dry THF (15 mL) at -78 °C with magnetic stirring. After the reaction mixture was stirred at -78 °C for 30 minutes, the obtained suspension was added dropwise to a cooled (-78 °C) solution of di-tert-butyldifluorosilane (1.12 g, 6.23 mmol, 1.2 equiv) in dry THF (10 mL) over a 30 minute period. The reaction mixture was allowed to warm to room temperature over a 12 hour period and then hydrolyzed with saturated aqueous NaCl solution (100 mL). The organic layer was separated and the aqueous layer was extracted with diethyl ether (3 x 50 mL). The combined organic layers were dried over magnesium sulfate and filtered. The filtrate was concentrated in vacuo to give ii (1.88 g, 95%) as a light yellow oil, which was used in the subsequent reaction without further purification.

[0584] RP-HPLC (50→100%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 19 minutes. K' = 9.67.

[0585] 4-(Di-tert-butylfluorosilyl)benzyl alcohol (iii)

[0586] A catalytic amount of concentrated aqueous HCl (0.5 mL) was added to a suspension of ii (1.88 g, 4.92 mmol, 1.0 eq) in methanol (50 mL). The reaction mixture was stirred at room temperature for 18 h, and then the solvent and volatiles were removed under reduced pressure. The residue was redissolved in diethyl ether (40 mL), and the solution was washed with saturated NaHCO 3 The aqueous solution was washed. The aqueous layer was extracted with diethyl ether (3 x 50 mL). The combined organic layers were dried over magnesium sulfate and filtered. The filtrate was concentrated in vacuo to give solid iii (1.29 g, 98%) as a light yellow oil. The product was used without further purification.

[0587] RP-HPLC (50→100%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R =8.2 minutes. K' =3.61.

[0588] 4-(Di-tert-butylfluorosilyl)benzoic acid ((4-SiFA)Bz-OH, iv)

[0589] At room temperature, 5.8 g KMnO 4 (36.7 mmol, 1.5 eq.) was dissolved in H 2 O and added to iii (6.61 g, 24.6 mmol, 1.0 eq.), tert-butyl alcohol (65 mL), dichloromethane (9 mL) and 1.25 M NaH 2 PO 4 ·H 2 O buffer (36 mL). After the mixture was stirred for 25 min, it was cooled on ice for 10 min, and then an excess of KMnO was added. 4 (7.8 g, 49.2 mmol, 2.0 equiv). The reaction mixture was stirred on ice for 2 h and then allowed to warm to room temperature for 30 min. 2 SO 3 The reaction was quenched with aqueous solution (50 mL). After the addition of 2 M aqueous HCl, all the MnO 2 The resulting solution was extracted with diethyl ether (3×100 mL). The combined organic layers were washed with saturated NaHCO 3 The aqueous solution was washed, dried over MgSO 4 , filtered, and concentrated under reduced pressure to provide a white solid which was purified by purification from Et 2 The product was purified by recrystallization from O / n-hexane (1:3, 12 hours) to obtain iv (2.57 g, 37%).

[0590] 1 H NMR (300 MHz, CDCl 3 ): d [ppm] = 8.10 (d, 2H 3 J( 1 H, 1 H)=8.1Hz; H m ), 7.74(d, 2H, 3 J( 1 H, 1 H)=8.1Hz; H o ), 1.07 (s, 18H; CCH3).

[0591] 13 C{ 1 H}NMR (101 MHz, DMSO-D 6 ): d[ppm]=167.2(s;COOH), 138.3(d, 2 J( 13 C, 19F)=14Hz; C p ), 133.8(d, 3 J( 13 C, 19 F)=4Hz; C m ), 132.1(s; C i ), 128.3(s;C o ), 26.9(s; CCH 3 ), 19.7(d, 2 J( 13 C, 19 F)=12Hz;CCH 3 ).

[0592] 19 F{ 29 Si}NMR (376 MHz, DMSO-D 6 ): d[ppm]=-187.2.

[0593] 29 Si 1 H}INEPT NMR (79 MHz, DMSO-D 6 ): d[ppm]=14.1(d, 1 J( 19 F 29 Si)=299Hz).

[0594] RP-HPLC (50→100%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R = 8.5 min. K' = 3.78. ESI-MS (positive ion): calculated monoisotopic mass (C 15 H 23 FO 2 Si): 282.15; Found: m / z = 283.2 [M+H] + , 265.2[MH 2 O+H] + .

[0595]

[0596] Route 1: Synthesis of (4-SiFA)Bz-OH (iv): a) TBDMSCl, imidazole, room temperature, 16 hours (DMF); b) tBuLi, di-tert-butyldifluorosilane, -78 °C to room temperature, overnight (THF); c) HCl, room temperature, 18 hours (MeOH); d) KMnO 4 , 0°C to room temperature, 2.5 hours (DCM / tBuOH / NaH 2PO 4 ·H 2 (O buffer).

[0597] 4-(Di-tert-butylfluorosilyl)benzyl bromide (4-SiFA) Bz-Br(v)

[0598] 4-(Di-tert-butylfluorosilyl)benzyl bromide was synthesized similarly to (4-SiFA)Bz-OH (iv). For the last step only, 4-(di-tert-butylfluorosilyl)benzyl alcohol (iii) (2.41 g, 8.96 mmol, 1.0 eq) was dissolved in anhydrous DCM (40 mL) and CBr was added under argon countercurrent. 4 (2.59 g, 9.86 mmol, 1.1 eq.) Afterwards, the reaction mixture was cooled to 0°C and PPh was added dropwise over a period of 30 minutes. 3 (3.27 g, 9.86 mmol, 1.1 eq., dissolved in 30 mL of anhydrous DCM). The reaction mixture was then stirred at room temperature for 2 hours, and the solvent was removed in vacuo. The crude product was purified by flash column chromatography (silica, 100% pentane).

[0599] RP-HPLC (50→100%MeCN / H 2 O, containing 0.1% TFA, 15 min, λ=220nm): t R =17.3 minutes.

[0600]

[0601] Route 2: Synthesis of (4-SiFA)Bz-Br(iv): a) TBDMSCl, imidazole, room temperature, 16 hours (DMF); b) tBuLi, di-tert-butyldifluorosilane, -78°C to room temperature, overnight (THF); c) HCl, room temperature, 18 hours (MeOH); d) CBr4, PPh 3 , 0°C to rt, 2.5 h (DCM).

[0602] Di-tert-butyl(3,5-dimethylphenyl)fluorosilane (vi)

[0603] A solution of 4.54 g 1-bromo-3,5-dimethylbenzene (25.1 mmol, 1.0 eq) in 73.1 mL dry THF was cooled to -78 ° C, and 34.7 mL of tBuLi (55.5 mmol, 1.6 M in pentane, 2.2 eq) was added dropwise and stirred at -78 ° C for 30 minutes. Then, at -78 ° C, the reaction mixture was added to a solution of 5.0 g of di-tert-butyldifluorosilane (27.7 mmol, 1.1 eq) in 49.1 mL THF and stirred overnight while allowing it to warm to room temperature under pressure control. The reaction was terminated by adding 100 mL of brine. Et 2 O (3 × 100 mL), and the combined organic phases were purified by MgSO 4 After drying and removal of the solvent under reduced pressure, 6.6 g of di-tert-butyl(3,5-dimethylphenyl)fluorosilane (vi, 24.8 mmol, 99%) was obtained as a colorless solid.

[0604] 1 H NMR (500MHz, CDCl3): d[ppm]=7.19 (s, 2H; H o ), 7.04(s, 1H; H p ), 2.33 (s, 6H; CH3), 1.06 (s, 18H; CCH 3 RP-HPLC (50 to 100% B within 15 minutes, 100% B within 10 minutes): t R = 16.4 minutes. K' = 8.21.

[0605] 5-(Di-tert-butylfluorosilyl)isophthalic acid ((5-SiFA)Ip-OH, vii)

[0606] To a solution of 1.1 g of di-tert-butyl(3,5-dimethylphenyl)fluorosilane (i) (4.0 mmol, 1.0 equiv.) in 16.8 mL of tBuOH / DCM (v / v=3.5 / 1) was added 16.0 mL of NaH 2 PO 4 ·H 2 O solution (40.0 mmol, 2.5 M in H 2 O, 10.0 equivalents). At room temperature, 7.6 g KMnO was added to the solution. 4 (48.0 mmol, 12.0 equiv), the reaction was carefully heated stepwise to 75 °C and stirred for 24 h. 3 The reaction was quenched with aqueous solution (50 mL). Concentrated aqueous HCl (10 mL) was added to completely dissolve the MnO 2 . Use Et 2O (3 × 100 mL) and the combined organic phases were purified by MgSO 4 Drying and removal of the solvent under reduced pressure afforded ii (1.3 g, 4.0 mmol, 100%) as a colorless solid.

[0607] 1 H NMR (400 MHz, DMSO-D 6 ): dppm = 8.53 (t, 1H, 4 J( 1 H, 1 H)=1.7Hz; H Ar-2 ), 8.32(d, 2H, 4 J( 1 H, 1 H)=1.6Hz; H Ar-4,-6 ), 1.03(s,18H;CH 3 ).

[0608] 13 C{ 1 H}NMR (101 MHz, DMSO-D 6 ): d[ppm]=166.5(s;COOH), 137.9(d, 3 J( 13 C, 19 F)=4Hz; C Ar-4,-6 ), 134.0(d, 2 J( 13 C, 19 F)=14Hz; C Ar-5 ), 131.4(s;C Ar-2 ), 130.8(s;C Ar-1，-3 ), 26.8(s; CCH 3 ), 19.7(d, 2 J( 13 C 19 F)=12Hz;CCH 3 ).

[0609] 19 F{ 29 Si}NMR (376 MHz, DMSO-D 6 ): d [ppm] = -187.1.

[0610] 29 Si 1 H}INEPT NMR (79 MHz, DMSO-D 6 ): d[ppm]=13.8(d, 1 J( 19 F, 29Si)=299 Hz). RP-HPLC (50 to 100% B in 15 min): t R = 5.7 min. K' = 2.20. ESI-MS (positive ion): calculated monoisotopic mass (C 16 H 23 FO 4 Si): 326.13; Found: m / z = 327.2 [M+H] + , 309.2[MH 2 O+H] + .

[0611]

[0612] Route 3: Synthesis of (5-SiFA)Ip-OH (vii): a) tBu 2 SiF 2 , tBuLi, -78°C to room temperature, overnight (THF); b) KMnO 4 , 75°C, 24 hours (DCM / tBuOH / NaH 2 PO 4 ·H 2 (O buffer).

[0613] 3.3. Labeling experiment

[0614] nat Lu-labeled: purified ligand containing chelating agent (10 -3 M H 2 O, 1.0 equivalent) and [ nat Lu]LuCl 3 (20mM in H 2 O, 2.5 equivalents) with H 2 Dilute to 10 -4 The mixture was stirred for 30 min at 95 °C and the final concentration was 100 M. After cooling to room temperature, the crude product was obtained.

[0615] nat Ga-mark: convert Ga(NO 3 ) 3 (20 μL, 10 mM, 2.0 equiv), peptide precursor (80 μL, 1 mM) and H 2 A solution of O (710 μL) was stirred at 70 °C for 30 min to achieve nat Ga-complex.

[0616] nat Cu-labeling: Cu(OAc) at 80°C2 (24 μL, 10 mM, 3.0 equiv), peptide precursor (80 μL, 1 mM) and H 2 O (694 μL) solution, so that CCK-2R ligand and [ nat Cu] copper complex for 30 minutes.

[0617] [ 19 F]AlF-labeling: AlCl 3 (3.70 mg, 2.80 μmol, 4 equiv) and NaF (1.20 mg, 2.80 μmol, 4 equiv) were preactivated in NaOAc buffer (pH = 4.2, 80 / 20 HoAc / NaOAc, v / v, 0.5 M) at room temperature for 10 min. Afterwards, the solution was added to the labeled precursor (700 μL, 700 nmol, 10 -3 M in DMSO, 1 equivalent), and DMSO was added to the reaction solution (1 / 1, v / v, DMSO / NaOAc buffer). The reaction mixture was heated to 110°C for 30 minutes and purified by RP-HPLC [ 19 F]AlF-NOTA-labeled peptide.

[0618] 177 Lu-marking: 177 Lu-labeling was done using a procedure developed in-house. -3 M H 2 O, 1 μL), NaOAc buffer (1 M, pH = 5.50, 10 μL), and approximately 10-30 MBq [ 177 Lu]LuCl 3 A solution of (0.04 M in HCl, Isotope Technologies Munich SE, Garching, Germany) was diluted with HCl (0.04 M) to a total volume of 90 μL and heated to 95° C. for 10 min. Sodium ascorbate (0.1 M, 10 μL) was added immediately after labeling to prevent radiolysis. 177 The incorporation of Lu was determined by radioactive TLC (ITLC-SG chromatography paper, mobile phase: 0.1 M trisodium citrate). The radiochemical purity of the labeled compound was determined by radioactive RP-HPLC.

[0619] 67 Ga-labeling: To a solution of the labeling precursor (1 μL, 1 nmol, 1 mM in DMSO) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (7 μL, 2.5 M, HEPES) was added [ 67 Ga]GaCl3 (10-30 MBq, Curium Germany GmbH, Berlin, Germany) and the reaction mixture was heated to 90°C for 15 min. Radiochemical purity was determined using radio-RP-HPLC and radio-TLC.

[0620] 64 Cu-labeling: The labeling precursor (1 μL, 1 nmol, 1 mM in DMSO), NaOAc buffer (10 μL, 1 M, pH = 5.5) and [ 64 Cu]CuCl 2 A solution of (5-20 MBq, 925 MBq / mL, DSD Pharma, Purkersdorf, Austria) was heated to 80°C for 10 minutes. After the reaction solution was cooled, sodium ascorbate (10 μL, 1 M) was added. Subsequently, the radiochemical purity was determined using radio-RP-HPLC and radio-TLC.

[0621] 18 F-labeling: On the day of the experiment, add 100 to 3000 MBq of [ 18 F] fluoride H 2 O (2.5 mL) was loaded into the 2 O (10 mL) pre-conditioned Light(46mg)Accell TM Plus QMA column. Then, dry with anhydrous DMSO (8 mL) [ 18 F] fluoride and reverse eluted with ammonium formate (100-300 μL, 0.3 μM). Afterwards, the solution of the eluate (30 to 100 μL) and the peptide precursor (5 to 15 nmol) was heated (40 to 60°C) for 5 minutes, the reaction mixture was diluted in PBS (10 mL, pH = 3), and 18 F-labeled peptides were loaded into HLB (30 mg) was loaded onto a Light column. After washing the column with PBS (10 mL, pH = 7.4), 300 μL of H 2 The peptide was eluted in the reverse direction with O / EtOH (1:7). The radiochemical purity was determined using radio-RP-HPLC and radio-TLC.

[0622] In vitro experiments

[0623] n-octanol-PBS partition coefficient (logD 7.4 )

[0624] In an Eppendorf tube (1.5 mL), approximately 1 MBq of labeled tracer was dissolved in 1 mL of a 1 / 1 mixture (v / v) of phosphate buffered saline (PBS, pH=7.4) and n-octanol. After vigorous mixing of the suspension for 3 minutes at room temperature, the vial was centrifuged at 9,000 rpm for 5 minutes (Biofuge 15, Heraeus Sepatech, Osterode, Germany), and 200 μL aliquots of the two layers were measured in a γ-counter. The experiment was repeated at least five times.

[0625] IC 50 Determination of

[0626] AR4-2J cells were harvested 24 ± 2 hours before the experiment and seeded into 24-well plates (2.0 × 10 5 After removing the nutrient medium (RPMI 1640, 5 mM L-Gln, 5 mL non-essential amino acids (100×), 10% FCS), the cells were washed once with 500 μL PBS. Then, 200 μL nutrient medium (5% BSA, v / v) was added. Next, 25 μL of nutrient medium containing a control (5% BSA, v / v) or increasing concentrations (10 -10 -10 -4 M) solution of the corresponding compound, followed by the addition of 25 μL [ 177 Lu] Lu-DOTA-PP-F11N (0.3 pmol / well).

[0627] All experiments were performed in triplicate for each concentration. After incubation for 3 h at 37C, the experiments were terminated by removing the culture medium and washing with 300 μL PBS successively. The culture medium from both steps was combined into one fraction and represents the amount of free radiolabeled reference. Afterwards, the cells were lysed with 300 μL of 1 M NaOH for at least 15 min and combined with 300 μL NaOH of the subsequent washing steps. Quantification of bound and free radiolabeled reference was done in a γ-counter. The IC of each conjugate was 50 The assay was repeated twice.

[0628] Determination of internalization kinetics

[0629] To determine the internalization kinetics of various peptides, AR4-2J cells (3.0×10 5 ) were seeded into a 24-well plate coated with polylysine, and 1 mL of nutrient medium (RPMI 1640, 5 mM L-Gln, 5 mL of non-essential amino acids (100×), 10% FCS) was added. Afterwards, the cells were incubated at 37°C in a humidified atmosphere (5% CO 2 ) and incubate for 24 ± 2 hours.

[0630] On the day of the experiment, the culture medium was removed and each well was washed with incubation medium (RPMI 1640, 5 mM L-Gln, 5 mL non-essential amino acids (100×)) (300 μL). Then, 200 μL of nutrient medium (RPMI 1640, 5 mM L-Gln, 5 mL non-essential amino acids (100×), 10% FCS) was added to each well, followed by 25 μL of nutrient medium (RPMI 1640, 5 mM L-Gln, 5 mL non-essential amino acids (100×) (n=3) or 25 μL of DOTA-PP-F11N (10 μmol) for blocking (n=3). Then, 25 μL of the corresponding 177 Lu-labeled peptide (0.3 pmol / well) was added to each well (n=6). Afterwards, the cells were incubated at 37°C in a humidified atmosphere (5% CO 2 ) and incubate the assay at different time points (1, 2, 4 and 6 hours). After incubation, the cells were placed on ice and the supernatant was collected. Then, the cells were washed with ice-cold incubation medium (RPMI 1640, 5mM L-Gln, 5mL non-essential amino acids (100×)) (300 μL), and the two fractions were merged. In order to displace the peptide from the cell membrane, 300 μL of ice-cold glycine buffer (1M, pH=2.2) was added and the cells were incubated on ice for 15 minutes. Then, the supernatant was collected and the cells were washed with ice-cold glycine buffer (300 μL, 1M, pH=2.2). The two fractions were merged. After lysing the cells with NaOH (300 μL, 1M) for 15 minutes, the corresponding wells were washed with NaOH (300 μL, 1M), and the two fractions were merged. The radioactivity of the supernatant, the acid wash and the lysate fractions was quantified using a γ-counter.

[0631] Stability studies in human serum

[0632] Will 177 Lu-labeled CCK-2R ligand (1 nmol, about 5 MBq) was incubated in human serum (200 μL) at 37C for 72 ± 2 hours. After incubation, ice-cold EtOH (125 μL) and MeCN (375 μL) were added, and the suspension was centrifuged at 5,000 rpm for 2 minutes. Then, the supernatant was transferred to an ultracentrifugation vial and centrifuged at 5,000 rpm for another 2 minutes. After separating the precipitate from the solution, the stability of the ligand was determined via RP-HPLC chromatography.

[0633] Determination of HSA binding

[0634] Human serum albumin (HSA) binding of CCK-2R targeting compounds was determined as reported by Valko et al.

[31] using a Chiralpak HSA column (50×3 mm, 5 μm, H13H-2433, Daicel, Tokyo, Japan) at room temperature at a constant flow rate of 0.5 mL / min. 4 OAc (pH = 6.9, 50 mM) was used as mobile phase A and isopropanol was used as aqueous solution as mobile phase B. To calibrate the column before the experiment, a gradient of 100% A (0 to 3 min) followed by 80% A (3 to 40 min) was used to determine the retention times of nine reference substances with HSA binding ranging from 13 to 99%

[32] . In addition, all tested substances were dissolved in a mixture of A and B (1 / 1, v / v) to a final concentration of 0.5 mg / mL. The HSA binding of all tested rhCCK ligands was determined using the same gradient as the calibration probe. The calibration experiment and nonlinear regression of CCK-2R ligands were set up using OriginPro 2016G software (Northampton, USA). Fig.29 ).

[0635] In vivo experiments

[0636] All animal experiments were performed in accordance with the general German animal welfare regulations (German animal protection act, May 18, 2006, revised on June 19, 2020, Section 280, approved by the General Administration of Upper Bavaria, No. ROB-55.2-1-2532.Vet_02-18-109) and institutional guidelines for animal care and use. CB17-SCID mice of both sexes (Charles River Laboratories International Inc., Sulzfeld, Germany) were acclimated in the in-house animal facility for at least one week before inoculation. To establish tumor xenografts, AR4-2J cells (5.0 × 10 6 cells / 200 μL) were suspended in a solution containing Glutamax-I (1 / 1) and The cells were inoculated subcutaneously in the right shoulder of 6-10 week old CB17-SCID mice (Charles River, Sulzfeld, Germany) in a 1 / 1 mixture (v / v) of basement membrane matrix type 3 (Trevigen, Gaithersburg, MD, USA) of Dulbecco's modified Eagle's medium / Nutrient mixture F-12. 3The mice were used for the experiment when the tumor size was greater than 1,500 mm (1-2 weeks after inoculation). The exclusion criteria for the animals in the experiment were weight loss greater than 20%, tumor size greater than 1,500 mm 3 , tumor ulceration, respiratory distress or behavioral changes. None of these criteria were applied to any of the animals in the experiment. Neither randomization nor blinding was applied in the allocation of the experiment. The health status was SPF according to FELASA.

[0637] Biodistribution

[0638] Approximately 1-5 MBq (100 pmol) of radiolabeled minigastrin analogs were injected into the tail vein of AR4-2J tumor-bearing female CB17-SCID mice and sacrificed 1 to 24 hours after injection (n=4-5). Selected organs were removed, weighed and measured in a gamma-counter.

[0639] Competition Research

[0640] For competition studies, radiolabeled compounds (approximately 1-5 MBq, 100 pmol) were mixed with [ nat Lu]Lu-DOTA-MGS5 (40 nmol) was co-injected into the tail vein of AR4-2J tumor-bearing CB17-SCID mice and sacrificed 1 to 24 hours after injection (n=2). Selected organs were removed, weighed and measured in a gamma-counter.

[0641] μSPECT / CT imaging

[0642] Imaging Research at MILabs VECTor 4 Small animal SPECT / PET / OI / CT (MILabs, Utrecht, The Netherlands) was performed. The data were reconstructed using MILabs-Rec software (version 10.02) with a pixel-based similarity-regulated ordered subsets expectation maximization (SROSEM) algorithm and window-based scatter correction (20% below and 20% above the light peak, respectively). Further data analysis was completed using PMOD4.0 software (PMOD TECHNOLOGIES LLC, Zurich, Switzerland) under defined settings (CT voxel size: 80 μm, SPECT voxel size: 0.8 mm, 1.6 mm (FWHM) Gaussian blur post-processing filter, with calibration factors in kBq / mL and decay correction and no decay correction).

[0643] For SPECT studies, mice were anesthetized with isoflurane and 2-4 MBq (100 pmol) of radiolabeled tracer was injected into the tail vein. Using a HE-GP-RM collimator and step-wise multi-planar bed movement, static images were recorded at 1, 4, and 24 hours after injection with an acquisition time of 45-60 minutes.

[0644] Stability studies

[0645] By 177 Lu-labeled peptides (approximately 35MBq, 1nmol) were injected into the tail vein of anesthetized (2% isoflurane) CB17-SCID mice (n=3) for stability studies. 30 minutes after injection, mice were euthanized and urine and blood samples were collected. Urine samples were analyzed without further processing. Blood samples were centrifuged at 5,000rpm for 2 minutes. Afterwards, serum was collected and ice-cold MeCN (1:1, v / v) and EtOH (1:2, v / v) were added. The samples were centrifuged again at 5,000rpm for 2 minutes and analyzed by RP-HPLC (10→30% in 5 minutes, 30→60% in 15 minutes, MeCN / H 2 The supernatant was analyzed by ELISA (0 ± 0.1% TFA).

[0646] Patient studies

[0647] All patients received [ 68 All patients signed written informed consent before undergoing Ga]Ga-DOTA-CCK-66 PET / CT. The retrospective analysis of imaging results was approved by the responsible ethics committee of the University of Munich. The anonymous analysis was performed in accordance with the legal considerations of the Declaration of Helsinki and its later amendments and clinical guidelines.

[0648] Injection of 150-200MBq [ 68 120 minutes after the infusion of Ga]Ga-DOTA-CCK-66, patients underwent [ 68 Ga]Ga-DOTA-CCK-66 whole-body PET / CT. Whole-body CT imaging was performed as an adjunct to CT (120 kVp, 40 mAs). The PET data sets were reconstructed using standard protocols and corrected for random, scatter, decay, and attenuation (using whole-body adjunct CT).

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Claims

1. A tetrapeptide that binds to cholecystokinin 2 receptor (CCK-2R), wherein the tetrapeptide is represented by formula (I) or a salt thereof: Xaa1-Xaa2-Xaa3-Xaa4 (Formula (I)) in Xaa1 is Trp, (β-(3-benzothienyl)-alanine, Trp, wherein one or more H atoms of the 1H-indol-3-yl group are substituted by substituents, wherein the substituents for each H atom are independently selected from C1 to C3 alkyl, -OH, -SH, -F and -Cl, Phe, 1-Nal, 2-Nal, Tyr or p-amino-Phe, Xaa2 is an N-methyl amino acid, wherein the N-methyl group is at the α carbon; and preferably is an N-methyl amino acid containing an aliphatic side chain, wherein the N-methyl group is at the α carbon, Xaa3 is an amino acid having an acidic side chain at neutral pH, and Xaa4 is an amino acid having an aromatic side chain or an amide thereof.

2. The tetrapeptide according to claim 1, wherein Xaa2 is (N-Me)Nle, (N-Me)Met, (N-Me)Ile, (N-Me)Leu, (N-Me)Val, (N-Me)Gly, (N-Me)Ala, (N-Me)Glu, preferably (N-Me)Nle or (N-Me)Met, and most preferably (N-Me)Nle.

3. The tetrapeptide according to claim 1 or 2, wherein Xaa3 is Asp, isoaspartic acid, Glu or isoglutamine, and preferably Asp.

4. The tetrapeptide according to any one of claims 1 to 3, wherein Xaa4 is 1-Nal, Phe, Trp, Tyr, 2-Nal, p-amino-Phe or an amide of any of the foregoing, preferably 1-Nal, Phe, Tyr or an amide of any of the foregoing, and most preferably 1-Nal or Tyr, or an amide of 1-Nal or Tyr.

5. A conjugate comprising in a single molecule: (a) a tetrapeptide according to any one of claims 1 to 4, and (b) one or more chelating groups capable of complexing non-radioactive or radioactive cations, or one or more chelating groups containing chelated non-radioactive or radioactive cations.

6. The conjugate according to claim 5, wherein the chelating group is DOTA or DOTAGA with or without chelated non-radioactive or radioactive cation, wherein DOTA and DOTAGA preferably have one of their carboxylic acid groups bound to the rest of the conjugate via an amide bond.

7. The conjugate according to claim 5 or 6, further comprising in the single molecule: (c) a silicon-based fluoride acceptor moiety R selected from the following SiFA : (i) a silicon-based fluoride acceptor moiety comprising a silicon atom and a fluorine atom, wherein the fluorine atom is directly attached to the silicon atom via a covalent bond, and the moiety can be 18 F and 19 F isotope exchange 18 F mark, or the part is 18 F marked; (ii) a silicon-based fluoride acceptor moiety comprising a silicon atom and a hydroxyl group, wherein the hydroxyl group is directly attached to the silicon atom via a covalent bond, and the moiety can be 18 F nucleophilically replaces OH and uses 18 F mark; and (iii) a silicon-based fluoride acceptor moiety comprising a silicon atom and a hydrogen atom, wherein the hydrogen atom is directly attached to the silicon atom via a covalent bond, and the moiety can be 18 F replaces H nucleophilically and uses 18 F mark.

8. The conjugate compound of claim 7, wherein the silicon-based fluoride acceptor moiety R SiFA A group containing the following formula (S-1): in X S is F, OH or H; R 1S and R 2S are independently linear or branched C3 to C 10 alkyl.

9. The conjugate compound of claim 7, wherein the silicon-based fluoride acceptor moiety R SiFA Having a structure as represented by any one of formulae (S-3) to (S-7): in R 1S and R 2S are independently a linear or branched C3 to C10 alkyl group; Where r is 1, 2 or 3, -(CH2) s - in which s is an integer from 1 to 6, The groups R are independently H or C1 to C6 alkyl, and R 1S and R 2S are independently a linear or branched C3 to C10 alkyl group; in: R 1S and R 2S are independently linear or branched C3 to C 10 Alkyl; and R 3S Selected from (i) -OH or -O - , (ii) a sugar moiety or an amino sugar moiety, (iii) an amino acid portion or an oligopeptide portion, (iv) a PEG moiety; and a combination of two or more of (ii), (iii) and (iv).

10. The conjugate according to any one of claims 5 to 9, which is represented by formula (III): in: R L One or more chelating groups as defined in any one of claims 6 to 9, which may contain chelated non-radioactive or radioactive cations; L is the connecting part; R H is a tetrapeptide according to any one of claims 1 to 5, and R SiFA Present or absent, and if present is a silicon-based fluoride acceptor moiety as defined in any one of claims 10 to 12.

11. The conjugate of claim 10, wherein the linking moiety comprises D / L-diaminopropionic acid (D / L-Dap), D / L-diaminobutyric acid (D / L-Dab), D / L-ornithine (D / L-Orn) or D / L-lysine (D / L-Lys), wherein if present, the SiFA is conjugated to D / L-Dap, D / L-Dab, D / L-Orn or D / L-Lys using the -NH2 groups contained in these amino acids to provide a coupling group -NH-, wherein the bond of one hydrogen atom in the -NH2 group is replaced by a bond to the SiFA, Wherein D / L-Dap, D / L-Dab, D / L-Orn or D / L-Lys is preferably located in the linking portion such that D / L-Dap, D / L-Dab, D / LD / L-Orn or Lys is directly linked to R L and / or R SiFA .

12. The conjugate according to claim 10 or 11, wherein the linking moiety comprises polyethylene glycol (PEG), preferably (PEG) 3-11 , optionally together with poly-L / D-hydroxyproline (L / D-Hyp), preferably (Hyp) 1-8 , and / or α- or γ-glu, preferably γ-glu, and The linker most preferably comprises y-glu-(Hyp)6-y-glu-(PEG)3 or y-glu-(PEG)4-y-glu-(PEG)3.

13. The conjugate according to any one of claims 5 or 9, wherein the conjugate is represented by: DOTA-γ-glu-(PEG)3-Trp-(N-Me)Nle-Asp-1-Nal-NH2, DOTA-dap(SiFA)-γ-glu-(PEG)7-γ-glu-(PEG)3-Trp-(N-Me)Nle-Asp-1-Nal-NH2, DOTA-dap(SiFA)-y-glu-(Hyp)6-y-glu-(PEG)3-Trp-(N-Me)Nle-Asp-1-Nal-NH2, DOTA-dap(SiFAlin)-y-glu-(PEG)4-y-glu-(PEG)3-Trp-(N-Me)Nle-Asp-1-Nal-NH2, wherein SiFA represents a portion of formula (S-3a), and SiFAlin represents a portion of formula (S-4a): and wherein DOTA optionally contains chelated non-radioactive or radioactive cations.

14. A conjugate compound that binds to cholecystokinin 2 receptor (CCK-2R) represented by formula (IV): R L -dap(R SiFA )-R GABA -L-R H (IV) in: R L is DOTA or DOTAGA optionally containing a chelated non-radioactive or radioactive cation; dap(R SiFA ) is a silicon-based fluoride acceptor moiety R SiFA The dap group; R GABA is present or absent, and if present is one or more gamma-aminobutyric acid (GABA), preferably (GABA) 1-3 ; L is a linking moiety comprising at least 5, preferably at least 6 α- or γ-glu and most preferably 6 to 9 α- or γ-glu, wherein γ-glu is preferred over α-glu; and R H Is or contains Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2.

15. The conjugate according to claim 14, wherein the conjugate is represented by: DOTAGA-dap(SiFA)-(γ-glu)6-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2, DOTAGA-dap(SiFA)-(α-glu)8-Al a-Tyr-Gly-Trp-Nle-Asp-Phe-NH2, DOTA-dap(SiFA)-(γ-glu)8-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2, wherein SiFA represents the silicon-based fluoride acceptor moiety of formula (S-3a): And wherein DOTA or DOTAGA optionally contains a chelated non-radioactive or radioactive cation.

Images