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

Abstract

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

Description

[Technical Field]

[0001] The present invention relates to a tetrapeptide that binds to the cholecystokinin 2 receptor (CCK-2R), the tetrapeptide being 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 are substituted with a substituent, independently for each H atom, selected from C1-C3 alkyl, -OH, -SH, -F, and -Cl, Phe, 1-Nal, 2-Nal, Tyr, or p-amino-Phe; Xaa2 is an N-methylamino acid, wherein the N-methyl group is at the α-carbon, preferably an N-methylamino 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.

[0002] Numerous documents, including patent applications and manufacturer's manuals, are cited herein. The disclosures of these documents, while not believed to be relevant to the patentability of this invention, are incorporated herein by reference in their entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document were specifically and individually indicated to be incorporated by reference. [Background technology]

[0003] Medullary thyroid carcinoma (MTC), a neuroendocrine tumor, is reported to account for 1–2% of all thyroid cancers in the United States [1]. Thyroid cancer generally accounts for approximately 3% of all new cancer cases in the United States, and therefore, the occurrence of MTC is considered rare [2]. Despite improved detection of MTC through various screening methods and biopsy techniques, median survival remains modest (8.6 ± 7.6 years), especially when metastasis has already begun [3–7].

[0004] Approximately 70% and 30% of MTC patients suffer from sporadic and hereditary / familial forms, respectively [8]. The diagnosis of hereditary MTC relies on the detection of receptor tyrosine kinase (RET) proto-oncogene mutations through DNA molecular analysis. This allows for regular testing of patients at risk of developing MTC, which in turn allows for early detection of MTC lesions [9,10].

[0005] The diagnosis of sporadic MTC is usually made after fine-needle aspiration. Given its neuroendocrine origin, calcitonin production is characteristic of this tumor. Infusion of calcium or the synthetic gastrin analog pentagestrin stimulates calcitonin secretion, particularly from MTC cells [9,11]. Therefore, elevated calcitonin levels are a strong indicator of MTC. However, biochemical tests only indicate the presence of tumor / metastasis, often precluding localization

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

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

[15] . In a retrospective analysis, [ 18 F]F-DOPA PET / CT is 18 F]FDG PET / CT (17%) or 68 showed an improved detection rate (72%) in patients with recurrent MTC compared with PET / CT performed with Ga-labeled somatostatin analogues (33%)

[16] . 18[F]F-DOPA PET has demonstrated good sensitivity in primary tumors (86%), but only moderate (57%) to poor (6%) sensitivity in lymph nodes (LNs) and distant metastases, respectively.

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

[0007] For patients with metastatic disease, treatment options remain limited. Neither external beam radiation nor conventional chemotherapy has demonstrated sufficient therapeutic efficacy and is therefore not recommended [1,19,20]. Unlike common forms of thyroid cancer, MTC originates from malignant dedifferentiation of parafollicular cells. Because these cells do not accumulate iodine, radioactive iodine therapy is not a treatment option [21,22]. For this reason, total thyroidectomy and central compartment neck dissection remain the most frequently performed treatments for MTC [1,23]. However, in general, the 10-year survival rates for patients with local and distant metastases were 76% and 40%, respectively [6]. Tyrosine kinase inhibitors, such as selpercatinib, vandetanib, or cabozantinib, have been shown to prolong survival (primarily progression-free survival) [1]. However, treatment is associated with various associated side effects, including hypertension, nephrotoxicity, and hepatotoxicity, among others [1,24].

[0008] Over the past few decades, the field of nuclear medicine has emerged as a powerful alternative due to its noninvasive imaging and therapeutic opportunities [13,14]. Therefore, as a complement to the aforementioned screening methods, tumor and metastasis localization can be determined via molecular imaging. In the 1990s, the diagnosis of MTC was first achieved via somatostatin receptor scintigraphy (SRS) using somatostatin receptor subtype 2 (sstr2)-specific ligands. Despite significant improvements in diagnosis, numerous MTC-negative scans are still reported, especially in patients with widespread metastases

[25] . For this reason, new options for targeting MTC, especially for advanced disease and metastases, are warranted.

[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 receptors or CCK-2 receptors (CCK-2R)

[26] . In 2006, Gotthardt et al. showed that CCK-2R expression was significantly higher 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]. Summary of the Invention

[0010] In view of the above, there is a continuing need for additional options for diagnosing and treating CCK-2R-expressing cancers, such as MTC. This need is addressed by the present invention. Thus, in a first aspect, the present invention relates to a tetrapeptide that binds to the 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 of the H atoms of 1H-indol-3-yl are, independently for each H atom, C1-C3 alkyl, - Xaa2 is an N-methyl amino acid, where the N-methyl group is at the α-carbon, preferably an N-methyl amino acid containing an aliphatic side chain, where 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 or an amide thereof having an aromatic side chain.

[0011] The term "tetrapeptide" as used herein relates to a peptide consisting of four amino acids covalently linked via peptide bonds. The salt of the tetrapeptide is preferably a pharmaceutically acceptable salt.

[0012] The first amino acid is designated Xaa1 and may be Trp, (β-(3-benzothienyl)-alanine, Trp, wherein one or more of the H atoms of 1H-indol-3-yl are substituted by a substituent independently selected for each H atom from C1-C3 alkyl, -OH, -SH, -F, and -Cl, Phe, 1-Nal, 2-Nal, Tyr, or p-amino-Phe. The one or more of the H atoms is preferably one or two H atoms, preferably one H atom. Among the substituents, C1-C3 alkyl is preferred, and the C1-C3 alkyl is preferably ethyl or methyl, and most preferably methyl.

[0013] The second amino acid, designated Xaa2, may be an N-methyl amino acid, where the N-methyl group is located at the α-carbon, and is preferably an N-methyl amino acid containing an aliphatic side chain, where the N-methyl group is located at the α-carbon. The N-methyl amino acid is preferably derived from the 22 proteinogenic or proteogenic 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 or proteogenic amino acids, N-methyl amino acids with an N-methyl group located at the α-carbon can be obtained by replacing the NH2 group of the α-C atom with an N-methyl group. Of the 22 proteinogenic or protein-producing 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 non-polar and hydrophobic.

[0014] 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 or protein-producing amino acids. Therefore, such amino acids are also called α-amino acids. The second carbon atom is called the beta carbon (Cβ), and the naming system follows the Greek alphabet.

[0015] The third amino acid, designated Xaa3, may be an amino acid with an acidic side chain at neutral pH. The amino acid Xaa3 is preferably an α-amino acid. Of the 22 proteinogenic or protein-producing amino acids, two have acidic side chains at neutral pH: aspartic acid (Asp) and glutamic acid (Glu).

[0016] The fourth amino acid, designated Xaa4, may be an amino acid with an aromatic side chain or its amide. Among the 22 proteinogenic or protein-producing amino acids, phenylalanine, tryptophan, and tyrosine are amino acids with aromatic side chains. Histidine, although containing an aromatic ring, is primarily classified in the art for its basic properties and is also classified 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 C-terminus of the tetrapeptide is typically -NH2. This amide is also referred to herein as Xaa4-NH2. 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-NH2.

[0017] The tetrapeptides of the present invention bind to the cholecystokinin 2 receptor (CCK-2R). CCK-2R is also known as the cholecystokinin B receptor. This protein is a G protein-coupled receptor for the regulatory peptides gastrin and cholecystokinin (CCK) in the brain and gastrointestinal tract. This protein is a type B gastrin receptor that has high affinity for both sulfated and non-sulfated CCK analogs and is found primarily in the central nervous system and gastrointestinal tract. Intron-containing, mis-spliced ​​transcript variants have been observed in cells of colon, pancreatic tumors, and thyroid cancers, particularly medullary thyroid carcinoma (MTC).

[0018] This tetrapeptide preferably specifically binds to CCK-2R. Specific binding means that the binding molecule essentially does not bind 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. This tetrapeptide is suitable, for example, for research purposes only. For example, this tetrapeptide can be used in immunoassays to capture CCK-2R, such as ELISA or Western blot. An immunoassay is a biochemical test that can measure the presence or concentration of CCK-2R in a sample (e.g., a solution). As further described herein below, this tetrapeptide is particularly suitable for medical and diagnostic applications for diseases associated with the expression of CCK-2R.

[0019] As can be seen from the accompanying examples, the tetrapeptide of the first aspect of the present invention is identical to or highly similar to the four N-terminal amino acids of the seven-amino acid prior art CCK-R2 binding agent minigastrin, i.e., the Trp-(N-Me)Nle-Asp-l-Nal motif. The surprising discovery that only the four N-terminal amino acids of minigastrin, H-Trp-(N-Me)Nle-Asp-l-Nal-NH2, are required for high affinity to CCK-R2 was advantageously found to result in a weaker CCK-R2 binder. It should be noted that many prior art CCK-R2 binding agents, including F11N, consist of seven amino acids. Although the four N-terminal amino acids of F11N are closely related to H-Trp-Nle-Asp-Phe-NH2, they did not exhibit high CCK-R affinity. Therefore, the (N-Me) amino acid at position Xaa2 of the tetrapeptide is believed to be particularly important for ensuring CCK-R2 binding. To the best of our knowledge, no high-affinity CCK-2R ligands consisting of a peptide consisting of only four amino acids are known in the prior art. Furthermore, the accompanying examples demonstrate that the high affinity of the tetrapeptide for CCK-2R is maintained when linked to a linker moiety, such as a PEG chain, along with a chelator capable of complexing with radioactive or non-radioactive cations. For this reason, this tetrapeptide can be used alone, but also as an important element in the design of additional CCK-2R-binding compounds with diagnostic or medical value.

[0020] According to a preferred embodiment of the first aspect of the present invention, Xaa1 is Trp. The accompanying examples reveal that Trp at position Xaa1 exhibits the highest binding affinity to CCK-2R. β-(3-benzothienyl)-alanine and Trp, in which one or more H atoms of 1H-indol-3-yl are substituted with a substituent independently selected from C1-C3 alkyl, -OH, -SH, -F, and -Cl for each H atom, are highly similar to Trp and are therefore believed to function similarly.

[0021] Furthermore, 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.

[0022] The accompanying examples show that tests using tetrapeptides identical to or very similar to the N-terminal four amino acids of minigastrin have revealed that (N-Me)Nle at position Xaa2 exhibits the highest binding affinity to CCK-2R. Since (N-Me)Ile, (N-Me)Leu, (N-Me)Val, (N-Me)Gly, (N-Me)Ala, and (N-Me)Glu are similar to (N-Me)Nle, and (N-Me)Met is very similar to (N-Me)Nle, these alternatives in particular are thought to be usable in place of (N-Me)Nle.

[0023] According to a further preferred embodiment of the first aspect of the present invention, Xaa3 is Asp, isoaspartic acid, Glu or isoglutamine, preferably Asp. Furthermore, the accompanying examples show that tests using various tetrapeptides identical to or closely similar to the N-terminal four amino acids of minigastrin have revealed that Asp at Xaa3 exhibits the highest binding affinity to CCK-2R. Because isoaspartic acid, Glu, or isoglutamine are highly similar to Asp, these alternatives are particularly considered to be usable in place of Asp. Among the alternatives, Glu is preferred because it is a proteinogenic or proteinogenic amino acid.

[0024] According to an even more 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 one of the foregoing, most preferably 1-Nal or Tyr or an amide of 1-Nal or Tyr.

[0025] Furthermore, the accompanying examples show that tests using tetrapeptides identical to or closely similar to the N-terminal four amino acids of minigastrin reveal that l-Nal or Tyr at Xaa4 exhibits the highest binding affinity to CCK-2R. Because Phe, Trp, 2-Nal, and amino-Phe are highly similar to l-Nal or Tyr, these alternatives are particularly considered to be usable in place of l-Nal or Tyr.

[0026] 1-Nal, Phe, Trp, Tyr, or 2-Nal and para-amino-Phe, as well as their amides, are all amino acids having a side chain with an H atom present as a substituent on the aromatic ring. Thus, Xaa4 may be a derivative of 1-Nal, Phe, Trp, Tyr, or 2-Nal, p-amino-Phe, or an amide thereof, in which one or more H atoms on the aromatic ring of the side chain are substituted with a substituent independently selected from C1-C3 alkyl, -OH, -SH, -F, and -Cl. The one or more H atoms are preferably one or two H atoms, and preferably one H atom. Among the substituents, C1-C3 alkyl is preferred, and the C1-C3 alkyl is preferably ethyl or methyl, most preferably methyl.

[0027] According to another preferred embodiment of the first aspect of the invention, the tetrapeptide has a half maximal inhibitory concentration (IC 50 ) has a CCK-2R binding affinity of 100 nM or less, preferably 20 nM or less, and most preferably 7.5 nM or less.

[0028] As shown in Table 2 of the accompanying Examples, a tetrapeptide identical to the N-terminal four amino acids of minigastrin exhibited IC activity against CCK-2R. 50 The tetrapeptide H-Trp-(N-Me)Nle-Asp- Phe -NH2 and H-Trp-(N-Me)Nle-Asp- Tyr -NH2 is the IC for CCK-2R 50 is slightly better, with IC of 4.5 ± 0.6 nM and 5.7 ± 0.5 nM, respectively. The tetrapeptide H-Trp-(N-Me)Nle-Asp-Trp-NH2 still has an IC of 18.8 ± 0.5 nM. 50 The tetrapeptide H-Trp-(N-Me)Nle-Asp-2-Nal-NH2 had an IC of 102 ± 9 nM. 50 maintained.

[0029] These particular test peptides fall within the scope of the preferred embodiment described above and are included in the IC of this preferred embodiment. 50 The values ​​shown are supported by the exemplified tetrapeptides.

[0030] In a second aspect, the present invention provides a method for producing a compound comprising, in a single molecule: (a) a tetrapeptide according to the first aspect of the invention, and (b) one or more chelating groups capable of forming a complex with a non-radioactive or radioactive cation, or one or more chelating groups comprising a chelated non-radioactive or radioactive cation. The present invention relates to a conjugate comprising:

[0031] The one or more chelating groups are preferably one or two chelating groups, and most preferably one chelating group. A chelating group capable of complexing with 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 which case the non-radioactive or radioactive cation is not part of the conjugate of the second aspect of the invention.

[0032] On the other hand, in the case of a chelating group that includes a chelated non-radioactive or radioactive cation, the non-radioactive or radioactive cation is complexed with the chelating group, and thus the non-radioactive or radioactive cation is part of the conjugate of the second aspect of the invention.

[0033] A variety of 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 or cation chelating agents, e.g., macrocyclic or acyclic compounds suitable for acting as chelating groups, are available from numerous manufacturers. It will be understood that many chelating agents can be used directly by those skilled in the art without further manipulation. Furthermore, it will be understood that a chelating group suitable for forming a chelate with a particular cation must be capable of providing a chelated ligand in a chelate complex containing that cation, but need not be capable of providing the sole ligand for the cation in the chelate complex. Thus, when a chelating group contains a chelated radioactive or non-radioactive cation, the cation may be a complex cation, e.g., a metal ion having an additional coordinating ligand other than the chelating group, e.g., an oxo ligand.

[0034] For example, the chelating group may comprise at least one of: (i) a macrocyclic ring structure having 8 to 20 ring atoms, of which at least two, preferably at least three, are selected from oxygen and nitrogen atoms; and (ii) an acyclic open-chain chelating structure having 8 to 20 main chain atoms, of which at least two, preferably at least three, are heteroatoms selected from oxygen and nitrogen atoms.

[0035] According to a preferred embodiment of the second aspect of the present invention, the chelating group is selected from the group consisting of bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CBTE2a), cyclohexyl-1,2-diaminotetraacetic acid (CDTA), 4-(1,4,8,11-tetraazacyclotetradec-1-yl)-methylbenzoic acid (CPTA), N'-[5-[acetyl(hydroxy)amino]pentyl]-N-[5-[[4-[5-aminopentyl-(hydroxy)amino]-4-oxobutanoic acid] [yl]amino]pentyl]-N-hydroxybutanediamide (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]-pentanedioic acid (DOTAGA or DOTA-GA), N,N'-dipyridoxylethylenediamine-N,N'- diacetate-5,5'-bis(phosphato) (DPDP), diethylenetriaminepentaacetic acid (DTPA), ethylenediamine-N,N'-tetraacetic acid (EDTA), ethylene glycol-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, 10-triacetate (HP-DOA3), 6-hydrazinyl-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-triazacyclononanetriacetic 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 (TETA), 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-pyridine)methyl]-4,13-diaza-18-crown-6 (H2 macropa), 4-amino-4-{2-[(3-hydroxy-1,6-dimethyl-4-oxo-1,4-dihydro-pyridin-2-ylmethyl)-carbamoyl]-ethyl}heptanedioate bis-[(3-hydroxy-1,6-dimethyl-4-oxo-1,4-dihydro-pyridin-2-ylmethyl)-carbamoyl]-ethyl}heptanedioate bis-[(3-hydroxy-1,6-dimethyl-4-oxo-1,4-dihydro-pyridin-2-ylmethyl)-carbamoyl]-ethyl}heptanedioate. and a group derivable from a chelating agent selected from S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecanetetraacetic acid, mercaptoacetyl-trycerin (mas3), hydrazinonicotinic acid (HYNIC), and an N4 chelating agent. The chelating group is typically provided using a functional group, such as a carboxyl group, of one of the chelating agents described above, which forms a bond, such as an amide bond, with the remainder of the conjugate containing the chelating group.

[0036] More preferred are DOTA, DOTAGA, DOTAM, DO3AM, NOTA, and NODAGA. As will be appreciated by those skilled in the art, the chelating group of the conjugates of the present invention can be conventionally derived from the chelating agents described above by providing a coupling group selected from, for example, -C(O)-, NH-, -S-, and -O-, using any of the functional groups contained in the chelating agent, such as a carboxylic acid group, an amide group, an amino group, a hydroxyl group, or a thiol group, to link the chelating group to the remainder of the compound. Preferably, a carboxylic acid group is used to provide the coupling group -C(O) (carbonyl) for forming an amide bond. This coupling group may then be covalently bonded to a further complementary coupling group contained in the conjugate, such as the tetrapeptide itself, linking the two coupling groups to form a linking unit such as the amide bond C(O)-NH-.

[0037] Alternatively, as will be appreciated by those skilled in the art, the chelating groups in the conjugates of the invention can be conventionally derived from the chelating agents described above by the introduction of additional functional groups, or chelating agents modified with additional groups having functional groups capable of forming chemical bonds with other moieties of the conjugate, preferably tetrapeptides, or linkers between the chelating group and the tetrapeptide, e.g., additional residues having an isothiocyanate capable of coupling with an amine. As will be appreciated by those skilled in the art, other conjugation strategies, generally collectively referred to as "bioconjugation strategies," can also be used to attach the chelating groups in the conjugates of the invention.

[0038] According to a further preferred embodiment of the second aspect of the invention, the chelating group is a group that can be derived from DOTA or DOTAGA, with or without a chelated non-radioactive or radioactive cation, and DOTA and DOTAGA preferably have one of their carboxyl groups attached to the remainder of the conjugate via an amide bond.

[0039] The amine may be an amine in the tetrapeptide (eg, an N-terminal amine group or an amine in the side chain of an amino acid) or an amine in a linker connecting the chelating group to the tetrapeptide.

[0040] According to another preferred embodiment of the second aspect of the present invention, the radioactive or non-radioactive cation is 43 Sc, 44 Sc, 47 Sc, 51 Cr, 52m Mn, 55 Co, 57 Co, 58 Co, 52 Fe, 56 Ni, 57 Ni, 62 Cu, 64 Cu, 67 Cu, 66 Ga, 68 Ga, 67 Ga, 89 Zr, 90 Y, 86 Y, 94m Tc, 99m Tc, 97 Ru, 105 Rh, 109 Pd, 111 Ag, 110m In, 111 In, 113m In, 114m In, 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 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 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 is selected from the cations of Th and non-radioactive isotopes thereof; 18 F or 19 Cationic molecules containing F, e.g. 18 F-[AlF] 2+ or 18 F-[ScF] 2+ is.

[0041] Of this group of cations, the cations are preferably 68 Ga, 90 Y, and 177 The radioactive cation is selected from the radioactive cations of Lu, and the non-radioactive cations of Ga, Y, or Lu.

[0042] 68 Ga is frequently used as a tracer for pharmaceutical molecules. 90 Y plays an important role in the treatment of hepatocellular carcinoma (HCC), leukemia, and lymphoma, but also has potential in the treatment of various tumors. 177Lu is used in the art for radiolabeling of pharmaceutical molecules for the purposes of either anti-cancer therapy or scintigraphy (medical imaging).

[0043] According to another preferred embodiment of the second aspect of the invention, the conjugate comprises in a single molecule: (c) the following: (i) a silicon atom and a fluorine atom, wherein the fluorine atom is directly bonded to the silicon atom via a covalent bond; 18 By F 19 F isotope exchange 18 may be labeled with F, or 18 (ii) a silicon-based fluoride acceptor moiety, labeled with F, comprising a silicon atom and a hydroxy group, wherein the hydroxy group is covalently bonded directly to the silicon atom; 18 by nucleophilic substitution of OH by F 18 a silicon-based fluoride acceptor moiety that can be labeled with F; and (iii) a silicon atom and a hydrogen atom, wherein the hydrogen atom is directly bonded to the silicon atom via a covalent bond, 18 by nucleophilic substitution of H by F 18 a silicon-based fluoride acceptor moiety R selected from silicon-based fluoride acceptor moieties that can be labeled with F; SiFA Further includes:

[0044] Silicon-based fluoride acceptor moieties R, also simply referred to as silicon fluoride acceptor moieties SiFA The use of 18 This is an attractive approach for introducing F labels. Silicon fluoride acceptor moieties are described, for example, in Lindner et al., Bioconjugate Chemistry 25, 738-749 (2014). In the literature, silicon-based fluoride acceptors have been used. 18 F-labeled compounds are 19 Against F 18 It can be produced by the isotope exchange reaction of F with OH. 18 F and even H 18It has also been demonstrated that silicon-fluoride bonds can be maintained by substitution reactions with F, for example, in Mu L et al., Angew Chem Int Ed Engl. 2008; 47(26): 4922-5 and Hohne A et al., Bioconjug Chem. 2008 Sep; 19(9): 1871-9, respectively. However, to maintain the silicon-fluoride bond, silicon-based fluoride acceptors, such as Si—F and Si- 18 Sterically demanding groups such as two tert-butyl groups in the F silicon group, e.g., Si(tert-butyl)2X (where X is F or 18F), are often used. This makes silicon fluoride acceptors highly hydrophobic. In terms of binding to target molecules, particularly target proteins such as CCK-2R, the hydrophobic moiety provided by silicon-based fluoride acceptors can potentially be used to establish interactions of radiodiagnostic or radiotherapeutic compounds with the hydrophobic pocket described in Zhang et al., Journal of the American Chemical Society 132, 12711-12716 (2010). However, the high lipophilicity introduced into the molecule prior to conjugation poses a serious problem in terms of developing radiopharmaceuticals with suitable in vivo biodistribution, namely, reduced non-specific binding in non-target tissues.

[0045] According to a more preferred embodiment of the second aspect of the present invention, the silicon-based fluoride acceptor moiety R SiFA is expressed by the following formula (S-1):

[0046] [ka]

[0047] wherein X S is F, OH or H, preferably F; R 1S and R 2S are independently linear or branched C3-C 10is an alkyl group, preferably R 1S and R 2S are independently selected from isopropyl and tert-butyl, more preferably R 1S and R 2S is tert-butyl. The wavy line indicates the bond where the group is attached to the remainder of the conjugate compound.

[0048] More preferably, R SiFA The portion is represented by formula (S-2):

[0049] [ka]

[0050] wherein X S is F, OH or H, preferably F; R 1S and R 2S are independently linear or branched C3-C 10 is an alkyl group, preferably R 1S and R 2S are independently selected from isopropyl and tert-butyl, more preferably R 1S and R 2S is tert-butyl; Ph is a phenylene group (-CH-) in which one or more of the hydrogen atoms, e.g., one, two, or three, may be replaced with an organic functional group as a substituent. The wavy line indicates the bond through which the group is attached to the remainder of the conjugate compound.

[0051] Silicon-based fluoride acceptor moiety R SiFA It is more preferable that the compound has 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):

[0052] [ka]

[0053] In the formula (S-3), R 1S and R 2S are each independently a linear or branched C3 to C10 alkyl group, preferably R 1S and R 2S is selected from isopropyl and tert-butyl, more preferably R 1S and R 2S is tert-butyl. The wavy line indicates the bond where the group is attached to the rest of the compound.

[0054] In the portion of formula (S-4), r is 1, 2 or 3, preferably 1, and —(CH) s -s is an integer of 1 to 6, preferably 1; the groups R are independently H or C1-C6 alkyl, preferably H or C1-C2 alkyl, more preferably both are methyl; R 1S and R 2S are each independently a linear or branched C3 to C10 alkyl group, preferably R 1S and R 2S is selected from isopropyl and tert-butyl, more preferably R 1S and R 2S is tert-butyl. The wavy line indicates the bond that attaches the group to the remainder of the compound. Exemplary counterions for the positively charged quaternary ammonium group having two substituents R, as shown in formula (S-4), include trifluoroacetate, acetate, or a halide anion, such as chloride.

[0055] As will be understood by those skilled in the art, the fluorine atoms represented by formulas (S-3) and (S-4) are 18 F atom, or 19 F 18 F is exchanged by isotope exchange. 18 F can be provided 19 It can be an F atom.

[0056] 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"):

[0057] [ka]

[0058] During the ceremony, t Bu represents a tert-butyl group. The wavy line represents the bond that connects the group to the remainder of the compound. As noted above with respect to (S-4), exemplary counterions for the positively charged quaternary ammonium group of formula (S-4a) having two methyl substituents are trifluoroacetate, acetate, or a halide anion such as chloride.

[0059] [ka]

[0060] During the ceremony, R 1S and R 2S are independently linear or branched C3-C 10 is an alkyl group, preferably R 1S and R 2S are independently selected from isopropyl and tert-butyl, more preferably R 1S and R 2S is tert-butyl; R 3S teeth, (i) -OH or -O - , (ii) a sugar moiety or an amino sugar moiety; (iii) an amino acid moiety or an oligopeptide moiety; (iv) PEG moiety; and combinations of two or more of (ii), (iii) and (iv). The wavy line indicates the bond where the group is attached to the remainder of the conjugate compound.

[0061] R 3S -O - When the aromatic ring of the above formula represents 3S It will be understood that is a deprotonated carboxylate group, -C(=O)O-.

[0062] Among the formulae (S-5), (S-6) and (S-7), the formula (S-5) is preferred. R 3S The amino acid moiety as R is preferably derived from a hydrophilic amino acid which contains, in addition to the amino and carboxyl groups, an additional basic or acidic functional group, or 3S The oligopeptide moiety as such is preferably derived from an oligopeptide having at least one hydrophilic amino acid which, in addition to the amino and carboxyl groups, contains an additional basic or acidic functional group.

[0063] R 3S The oligopeptide moiety as such is preferably a linear or branched moiety containing 2 to 10, preferably 2 to 5, more preferably 2 or 3 amino acid moieties. The hydrophilic amino acids are preferably selected from lysine and glutamic acid.

[0064] R 3S The PEG moiety is preferably of the formula -NH-(CH-CH-O) X -R P1 where the nitrogen atom providing the open bond is R 3S forms an amide bond -NH-C(O) with the carbon atom to which R is bonded, where X is an integer of 2 to 10, preferably 4 to 10, more preferably 8, and R P1 is selected from -CH2-COOH and -CH2-CH2-COOH.

[0065] R 3SThe sugar or amino sugar moiety as is preferably a residue derived from 6-amino-6-deoxy-D-galactopyranose and the corresponding tautomers. R 3S is more preferably —OH and —O - Selected from: R 1S and R 2S represents a tert-butyl group, and R 3S The silicon-based fluoride acceptor moiety of formula (S-5), wherein represents —OH or —O—, is also referred to herein as “SiFA-ipa.”

[0066] According to a preferred embodiment of the second aspect of the present invention, the conjugate has formula (III):

[0067] [ka]

[0068] wherein: R L is one or more chelating groups as defined herein above, the chelating groups optionally comprising chelated non-radioactive or radioactive cations; L is a linking moiety; R H is a tetrapeptide according to the first aspect of the invention, R SiFA is present or absent and, if present, is a silicon-based fluoride acceptor moiety as defined hereinabove.

[0069] It is understood that the linking moiety can be any moiety that is capable of linking the chelating group to the tetrapeptide. The one or more chelating groups is preferably one chelating group.

[0070] According to a preferred embodiment of the second aspect of the present invention, the binding 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), and if present, the SiFA is conjugated to the D / L-Dap, D / L-Dab, D / L-Orn or D / L-Lys using the -NH2 group contained in these amino acids to provide a coupling group -NH-, wherein a bond to one hydrogen atom of the -NH2 group is replaced by a bond to the SiFA, and the D / L-Dap, D / L-Dap, D / L-Dab, D / L-Orn or D / L-Lys is preferably a binding moiety in which the D / L-Dap, D / L-Dab, D / L-Orn or Lys is R L and / or R SiFA is arranged to be directly connected to

[0071] Thus, the binding moiety according to this preferred embodiment is R SiFA Of the options D / L-Dap, D / L-Dab, D / L-Orn or D / L-Lys, the option D / L-Dap is most preferred.

[0072] According to a further preferred embodiment of the second aspect of the invention, the binding moiety is polyethylene glycol PEG, preferably (PEG) 3-11 optionally poly-L / D-hydroxyproline (L / D-Hyp), preferably (Hyp) 1-8 , and / or together with 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.

[0073] Thus, the moiety according to this preferred embodiment is R SiFAPreferably, L / D-Hyp is used in conjunction with a binding moiety according to the preferred embodiments described above, which can or does function as a linking site to a conjugate of L / D-Hyp. L / D-Hyp is preferably L-Hyp.

[0074] The binding moiety according to this preferred embodiment not only links one or more chelating groups and the tetrapeptide, but also provides distance between the one or more chelating groups and the tetrapeptide. As shown in the accompanying examples, adding distance by a linker between a sterically demanding chelator such as DOTA and the tetrapeptide results in high affinity for CCK-2R.

[0075] According to further preferred embodiments of the second aspect of the present invention, the conjugate is selected from the group consisting of 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, or DOTA-dap(SiFAlin)-y-glu-(PEG)4-y-glu-(PEG)3-Trp-(N-Me)Nle-Asp-1-Nal-NH2, wherein the DOTA chelator optionally comprises a chelated non-radioactive or radioactive cation, preferably natLu, natGa, 177Lu or 68Ga cation, as described herein above.

[0076] Of the four conjugates listed above, DOTA-dap(SiFA)-y-glu-(Hyp)6-y-glu-(PEG)3-Trp-(N-Me)Nle-Asp-l-Nal-NH2 is the most preferred, as this embodiment, with or without a silicon-based fluoride acceptor moiety, exhibits the best performance and is considered a highly suitable candidate for clinical applications, as shown in the accompanying examples.

[0077] DOTA-γ-glu-(PEG)3-Trp-(N-Me)Nle-Asp-l-Nal-NH2 consists of DOTA as the chelator, γ-glu-(PEG)3 as the linker, and the tetrapeptide-(N-Me)Nle-Asp-l-Nal-NH2. DOTA can be complexed with non-radioactive or radioactive cations, such as cations of natLu, natGa, 177Lu, or 68Ga, as described hereinabove. For the therapeutic and diagnostic applications described herein, DOTA is preferably complexed with non-radioactive or radioactive cations, as described hereinabove. DOTA-γ-glu-(PEG)3-Trp-(N-Me)Nle-Asp-l-Nal-NH2 is referred to in the Examples as DOTA-CCK-66, and the Examples demonstrate that DOTA-CCK-66 is particularly suitable as a therapeutic and diagnostic agent. When administered to tumor-bearing mice, it is specifically enriched in the tumor but in background in other tissues; see Figure 11. DOTA-CCK-66 is a compound that has been used clinically. 68 Ga]Ga- / [ 177 Compared with [Lu]Lu-DOTA-MGS5, in vivo data revealed improved tumor-to-background ratios, which were comparable or even improved, possibly due to the tetrapeptide design combined with the PEG linker.

[0078] DOTA-dap(SiFA)-γ-glu-(PEG)7-γ-glu-(PEG)3-Trp-(N-Me)Nle-Asp-l-Nal-NH2 consists of DOTA as a chelating agent, dap as a linking site to the SiFA moiety (i.e., the moiety of formula (S-3a) above), -glu-(PEG)7-γ-glu-(PEG)3 as a linker, and the tetrapeptide Trp-(N-Me)Nle-Asp-l-Nal-NH2. DOTA-dap(SiFA)-γ-glu-(PEG)7-γ-glu-(PEG)3-Trp-(N-Me)Nle-Asp-l-Nal-NH2 is referred to as DOTA-rhCCK-70 in the Examples. DOTA-rhCCK-70 also showed high levels of activity in tumors 24 hours after administration to tumor-bearing mice. The DOTA chelator may comprise a chelated non-radioactive or radioactive cation, preferably a cation of natLu, natGa, 177Lu or 68Ga, as described herein above.

[0079] DOTA-dap(SiFA)-y-glu-(Hyp)6-y-glu-(PEG)3-Trp-(N-Me)Nle-Asp-l-Nal-NH2 consists of DOTA as the chelating agent, dap as the linking moiety to the SiFA moiety (i.e., the moiety of formula (S-3a) above), -y-glu-(Hyp)6-y-glu-(PEG)3- as the linker, and the tetrapeptide Trp-(N-Me)Nle-Asp-l-Nal-NH2. DOTA-dap(SiFA)-y-glu-(Hyp)6-y-glu-(PEG)3-Trp-(N-Me)Nle-Asp-l-Nal is referred to as DOTA-rhCCK-84 in the Examples. As shown in the Examples, lipophilicity and CCK-2R affinity of many test compounds were evaluated via the poly-L-hydroxyproline linker. Among these test compounds, DOTA-rhCCK-84 advantageously exhibited the highest hydrophilicity (logD 7.4 =-2.14±0.06) and CCK-2R affinity (IC 50= 7.87 ± 0.3). DOTA-rhCCK-84 was also found to exhibit the highest level of activity in the tumor 24 hours after administration to tumor-bearing mice, while exhibiting lower levels of activity in the kidney. DOTA chelators, as described herein above, can include chelated non-radioactive or radioactive cations, preferably natLu, natGa, 177Lu, or 68Ga cations.

[0080] DOTA-dap(SiFAlin)-y-glu-(PEG)-y-glu-(PEG)-Trp-(N-Me)Nle-Asp-1-Nal-NH2 consists of DOTA as a chelating agent, dap as a linking site to the SiFAlin moiety (i.e., the moiety of formula (S-4a) above), -y-glu-(PEG)-y-glu-(PEG)- as a linker, and the tetrapeptide Trp-(N-Me)Nle-Asp-1-Nal-NH2. DOTA-dap(SiFAlin)-y-glu-(PEG)-y-glu-(PEG)-Trp-(N-Me)Nle-Asp-1-Nal is referred to as DOTA-rhCCK-91 in the examples. DOTA-rhCCK-91 is conveniently [ nat Lu] Lutetium (IC 50 =8.56±0.7) or [ nat Ga] Gallium (IC 50 = 8.24 ± 1.6), the DOTA chelating group can contain a chelated non-radioactive or radioactive cation, preferably a cation of natLu, natGa, 177Lu or 68Ga, as described herein above.

[0081] In a third aspect, the present invention relates to the use of a tetrapeptide or a conjugate of the invention in the in vitro or ex vivo diagnosis of cancer. In a related fourth aspect, the present invention relates to the use of a tetrapeptide or conjugate of the invention for detecting the presence of cholecystokinin 2 receptor (CCK-2R) in a sample in vitro or ex vivo, wherein the sample has preferably been obtained from a cancer patient or a subject suspected of having cancer.

[0082] 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 a subject suspected of having cancer, the method comprising (a) contacting the sample with a tetrapeptide or conjugate of the invention, and (b) detecting binding of the tetrapeptide or conjugate of the invention to CCK-2R.

[0083] In vitro or ex vivo cancer diagnosis is not performed on the human or animal body, but is usually performed on a sample obtained from a subject to be diagnosed with cancer. The sample may be a body fluid or a tissue sample. Preferably, the body fluid is a blood sample selected from whole blood, serum, or plasma. Preferably, the tissue sample is derived from a tissue suspected of being cancerous, such as breast, lung, liver, or colon tissue.

[0084] In a sixth aspect, the present invention relates to a pharmaceutical or diagnostic composition comprising a tetrapeptide or a conjugate of the invention. The term "composition" as used herein refers to a composition comprising at least a tetrapeptide or conjugate of the present invention, also hereinafter collectively referred to as a compound.

[0085] For all pharmaceutical and diagnostic applications described herein, it should be understood that the chelating agent in these aspects of the invention preferably complexes with a radioactive cation. The nature of the radioactive cation can be selected based on the intended pharmaceutical or diagnostic application. For example, for diagnostic applications, a radioactive cation with a relatively short half-life and a relatively weak emission that is still detectable is generally desirable, while for therapeutic applications, a radioactive cation with a relatively long half-life and a relatively strong emission is generally desirable to destroy tumor cells.

[0086] According to the present invention, the term "pharmaceutical composition" refers to a composition for administration to a patient, preferably a human patient, for the treatment or prevention of a disease. The pharmaceutical composition of the present invention comprises a compound of the present invention, as described above. Optionally, it may contain additional molecules that can alter the properties of the compound of the present invention, for example, stabilizing, regulating, and / or activating its function. The composition may be in solid, liquid, or gaseous form, particularly in the form of powder(s), tablet(s), liquid(s), or aerosol(s). The pharmaceutical composition of the present invention may optionally further comprise a pharmaceutically acceptable carrier. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate-buffered saline solution, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, and organic solvents such as DMSO. Compositions containing such carriers can be formulated by well-known, conventional methods. These pharmaceutical compositions can be administered to a subject at an appropriate dose. The administration regimen is determined by the attending physician and clinical factors. As is well known in the medical arts, the dosage administered to a patient depends on many factors, including the patient's size, body surface area, age, the specific compound being administered, sex, time and route of administration, health status, and other concurrently administered medications. The therapeutically effective amount for a given situation can be readily determined by routine experimentation and is within the skill and judgment of an ordinary clinician or physician. Generally, a regular administration regimen for a pharmaceutical composition should be in the range of 1 μg to 5 g units per day. However, more preferred dosages may be in the range of 0.01 mg to 100 mg per day, even more preferably in the range of 0.01 mg to 50 mg, and most preferably in the range of 0.01 mg to 10 mg.

[0087] According to the present invention, the term "diagnostic composition" relates to a composition for administration to a patient, preferably a human patient, to diagnose a disease. The diagnostic composition of the present invention comprises a compound of the present invention, as described above. Optionally, it may contain the same additional molecules as the pharmaceutical composition and may be in the same form as the pharmaceutical composition. The above-described preferred ingredients and formulations of the pharmaceutical composition also apply mutatis mutandis to the diagnostic composition.

[0088] In a seventh aspect, the present invention relates to the tetrapeptide, the conjugate or the pharmaceutical composition of the present invention for the treatment or prevention of cancer. In an eighth aspect, the present invention relates to a tetrapeptide or a conjugate or a pharmaceutical composition of the invention for use in a method for the in vivo diagnosis of cancer in a subject.

[0089] The subjects referred to herein are preferably mammals, and most preferably human. Cancer is an abnormal malignant new growth of tissues that has no physiological function, and usually results from uncontrolled rapid cell proliferation.The cancer is preferably selected from the group consisting of breast cancer, ovarian cancer, endometrial cancer, vaginal cancer, vulvar cancer, bladder cancer, salivary gland cancer, pancreatic cancer, thyroid cancer, kidney cancer, lung cancer, cancer related to the upper digestive tract, colon cancer, colorectal cancer, prostate cancer, squamous cell carcinoma of the head and neck, cervical cancer, glioblastoma, malignant ascites, lymphoma and leukemia.In this list of cancers, colorectal cancer, pancreatic cancer and thyroid cancer are preferred because abnormal CCK-2R expression has been found in these cancer types.Considering that these cancer types are illustrated by the accompanying examples, thyroid cancer, especially medullary thyroid carcinoma (MTC), is most preferred.

[0090] Preferably, the cancer is a solid tumor, which, in contrast to a liquid tumor, is an abnormal mass of tissue that usually does not contain cysts or areas of liquid. In a ninth aspect, the present invention provides a compound of formula (IV) R L -dap(R SiFA )-R GABA -LR H(IV) A conjugate compound that binds to the cholecystokinin 2 receptor (CCK-2R), wherein: 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 is a dap group having the formula: R GABA may or may not be present, and if present, one or more gamma-aminobutyric acids (GABA), preferably (GABA) 1-3 and; L is a linking moiety containing at least 5, preferably at least 6, α- or γ-glu, most preferably 6-9 α- or γ-glu, with γ-glu being preferred over α-glu; R H is or comprises Ala-Tyr-Glu ...

[0091] The above definitions and preferred embodiments with respect to the first to eighth aspects of the invention also apply mutatis mutandis to the ninth and further aspects of the invention described below, insofar as these definitions and preferred embodiments are combinable with the ninth and further aspects of the invention described herein below.

[0092] As can be seen from the accompanying examples, several chelator-minigastrin-silicon-based fluoride acceptor conjugates containing polyglutamic acid linkers have been synthesized and demonstrated in vitro. nat / 177 Lu-labeling was evaluated and is also a reference ligand of the prior art. nat / 177 Lu-DOTA-PP-F11N and nat / 177 Lu-CP04( nat / 177 Lu-DOTA-(glu)6-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2).

[0093] Surprisingly, we found that the highest CCK-2R affinity was observed when this group was separated by at least five or six α- / γ-glu moieties, and the addition of six more α- / γ-glu moieties did not result in any further improvement. This highest affinity for CCK-2R was observed for the CCK-2R binding sequence (Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH) followed by dap(R SiFA This is likely due to the separation of the α-glu and γ-glu moieties. The backbone of each peptide is significantly longer with the use of γ-glu rather than α-glu, so γ-glu is preferred over α-glu. Furthermore, different chelators were tested, and the best results in terms of CCK-2R affinity were obtained with DOTA or DOTAGA. DOTA and DOTAGA are preferred because one of their carboxyl groups is attached to the rest of the conjugate via an amide bond. Furthermore, the silicon-based fluoride acceptor moiety R SiFA Still other compounds and sites for introducing R into the conjugate were tested, and it was found that DAP was the compound of choice for introducing the silicon-based fluoride acceptor moiety, with the optimal site for DAP being between the chelator and the linker, and, if present, between GABA. As will be appreciated by those skilled in the art, DAP (diaminopropionic acid) is the compound of choice for introducing R into the conjugate of formula (IV). L and L(R GABA does not exist) or R L and R GABA (R GABA When present, it acts as a linking unit between the silicon-based fluoride acceptor moiety R and the three functional groups provided by the DAP. SiFA may further comprise:

[0094] One or more γ-aminobutyric acids (GABA) can optionally be present to reduce the negative charge of γglu by the γ-aminobutyric acid (GABA) moiety. At the same time, the amount of negative charge can be reduced while maintaining the length of the linker, resulting in four compounds. The addition of one or more γ-aminobutyric acids (GABA) can reduce the amount of conjugate found in the kidney, thereby enriching the specific accumulation of the conjugate in tumors even when kidney abundance is a concern (note that even in the absence of GABA, the conjugate of the ninth embodiment specifically accumulates in tumors upon administration).

[0095] Thus, a ninth aspect of the present invention relates to certain preferred cholecystokinin 2 receptor binding molecules useful for imaging and targeted radiotherapy. Tests with chelator-minigastrin-SiFA conjugates have shown that the conjugates of the ninth aspect are advantageously those with the highest CCK-2R affinity.

[0096] As mentioned above, the above definitions and preferred embodiments with respect to the first to eighth aspects of the present invention also apply mutatis mutandis to the ninth and further aspects described below, insofar as these definitions and preferred embodiments are combinable with the ninth and further aspects as described herein below.

[0097] Thus, according to a preferred embodiment of the ninth aspect of the present invention, the radioactive or non-radioactive cation of the chelating group is 43 Sc, 44 Sc, 47 Sc, 51 Cr, 52m Mn, 55 Co, 57 Co, 58 Co, 52 Fe, 56 Ni, 57 Ni, 62 Cu, 64 Cu, 67 Cu, 66 Ga,68 Ga、 67 Ga、 89 Zr、 90 Y、 86 Y、 94m Tc、 99m Tc、 97 Ru、 105 Rh、 109 Pd、 111 Ag、 110m In、 111 In、 113m In、 114m In、 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 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 Ir、 197 Hg、 198 Au、 199 Au、 212 Pb、 203 Pb、 211 At、 212 Bi、 213Bi, 223 Ra, 224 Ra, 225 Ac, 226 Th and 227 selected from the cations of Th, and non-radioactive isotopes; or 18 F or 19 Cationic molecules containing F, e.g. 18 F-[AlF] 2+ and preferably 68 Ga, 90 Y, or 177 The cation is selected from the cations of Lu, and Ga, Y, or non-radioactive isotopes of Lu.

[0098] Further details and preferred embodiments of the radioactive or non-radioactive cations are described herein above in relation to the second aspect of the invention, and these details and preferred embodiments apply mutatis mutandis to the ninth aspect of the invention.

[0099] Furthermore, according to a preferred embodiment of the ninth aspect of the present invention, the silicon-based fluoride acceptor moiety R SiFA is as defined herein above in relation to the second aspect of the invention.

[0100] In particular, silicon-based fluoride acceptor moieties include: (i) containing a silicon atom and a fluorine atom, wherein the fluorine atom is directly bonded to the silicon atom via a covalent bond; 18 By F 19 F isotope exchange 18 may be labeled with F, or 18 silicon-based fluoride acceptor moieties, labeled with F; (ii) comprising a silicon atom and a hydroxy group, wherein the hydroxy group is directly bonded to the silicon atom via a covalent bond; 18 by nucleophilic substitution of OH by F 18 silicon-based fluoride acceptor moieties that can be labeled with F, and (iii) containing a silicon atom and a hydrogen atom, wherein the hydrogen atom is directly bonded to the silicon atom via a covalent bond; 18 by nucleophilic substitution of H by F 18 Silicon-based fluoride acceptor moieties that can be labeled with F It is preferably selected from:

[0101] More preferably, the silicon-based fluoride acceptor moiety R SiFA is the formula (S-1) defined above:

[0102] [ka]

[0103] wherein X S is F, OH or H, preferably F; R 1S and R 2S are independently linear or branched C3-C 10 is an alkyl group, preferably R 1S and R 2S are independently selected from isopropyl and tert-butyl, more preferably R 1S and R 2S is tert-butyl.

[0104] More preferably, R SiFA is the formula (S-2) defined above:

[0105] [ka]

[0106] wherein X S is F, OH or H, preferably F; R 1S and R 2S are independently linear or branched C3-C 10 is an alkyl group, preferably R1S and R 2S are independently selected from isopropyl and tert-butyl, more preferably R 1S and R 2S is tert-butyl; Ph is a phenylene group (-C6H4-), and one or more, for example one, two, or three, of the hydrogen atoms may be substituted with an organic functional group as a substituent.

[0107] Silicon-based fluoride acceptor moiety R SiFA are the equations (S-3) and (S-4) defined above.

[0108] [ka]

[0109] It is more preferable that the compound has a structure represented by one of the following formulae (S-1) to (S-7): In the formula (S-3), R 1S and R 2S are each independently a linear or branched C3 to C10 alkyl group, preferably R 1S and R 2S is selected from isopropyl and tert-butyl, more preferably R 1S and R 2S is tert-butyl.

[0110] In the portion of formula (S-4), r is 1, 2 or 3, preferably 1, and —(CH) s where s is an integer of 1 to 6, preferably 1; the groups R are independently H or C1-C6 alkyl, preferably H or C1-C2 alkyl, more preferably both are methyl; R 1S and R 2S are each independently a linear or branched C3 to C10 alkyl group, preferably R 1S and R 2Sis selected from isopropyl and tert-butyl, more preferably R 1S and R 2S is tert-butyl.

[0111] 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"):

[0112] [ka]

[0113] During the ceremony, t Bu represents a tert-butyl group.

[0114] [ka]

[0115] During the ceremony: R 1S and R 2S are independently a linear or branched C3 to C10 alkyl group, preferably R 1S and R 2S are independently selected from isopropyl and tert-butyl, more preferably R 1S and R 2S is tert-butyl; R 3S teeth (i) -OH or -O - , (ii) a sugar moiety or an amino sugar moiety; (iii) an amino acid moiety or an oligopeptide moiety; (iv) PEG moiety; and combinations of two or more of (ii), (iii) and (iv).

[0116] The wavy line indicates the bond where the group is attached to the remainder of the conjugate compound. Among the formulae (S-5), (S-6) and (S-7), the formula (S-5) is preferred. Silicon-based fluoride acceptor moiety R SiFA Further details and preferred embodiments regarding 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 herein above in relation to the second aspect of the invention. These details and preferred embodiments also apply mutatis mutandis to the ninth aspect of the invention.

[0117] According to a preferred embodiment of the ninth aspect of the present invention, the conjugate comprises DOTAGA-dap(SiFA)-(γ-glu)6-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2, DOTAGA-dap(SiFA)-(α-glu)8-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2, It is represented by DOTA-dap(SiFA)-(γ-glu)8-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2.

[0118] The DOTA of the chelator DOTAGA optionally comprises a chelated non-radioactive or radioactive cation, as described herein above. The preferred embodiments described above relate to the three best performing conjugates among several chelator-minigastrin-SiFA conjugates produced and tested in the accompanying examples.

[0119] DOTAGA-dap(SiFA)-(γ-glu)6-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2 and DOTAGA-dap(SiFA)-(α-glu)8-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2 correspond to the compounds DOTAGA-MG-SiFA-19 and DOTAGA-MG-SiFA-21, respectively, mentioned in the Examples. Figure 21 shows improved tumor accumulation after 24 hours compared to the prior art reference DOTA-PP-F11N, with significant tumor accumulation already at 1 hour. In the context of DOTA-dap(SiFA)-(γ-glu)8-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2, the replacement of DOTAGA with DOTA in DOTAGA-MG-SiFA-23 was unexpectedly found to significantly increase CCK-2R affinity (DOTA-MG-SiFA-23: 4.71±0.62 nM), which was even greater than that of the prior art reference ligand; see Figure 22.

[0120] DOTA-MG-SiFA-23 demonstrated the highest level of activity in tumors 24 hours after administration to tumor-bearing mice, with activity levels exceeding 8-fold that of the prior art reference DOTA-PP-F11N. Furthermore, after administration to tumor-bearing mice, both DOTA-MG-SiFA compounds demonstrated 3- to 6-fold increased activity in tumors despite having lower CCK2R affinity compared to the prior art reference DOTA-PP-F11N, likely due to slower clearance of the two DOTA-MG-SiFAs compared to the prior art reference.

[0121] Thus, the data in the examples demonstrate that the three best performers are particularly useful for imaging and targeted radiotherapy. According to a preferred embodiment of the ninth aspect of the present invention, the conjugate has a half maximal inhibitory concentration (IC 50 ) has a CCK-2R binding affinity of 100 nM or less, preferably 75 nM or less.

[0122] DOTAGA-MG-SiFA-19, DOTAGA-MG-SiFA-21, and DOTA-MG-SiFA-23 exhibited half-maximal inhibitory concentrations (IC) for binding to CCK-2R of 7.54 ± 0.26 nM, 55.3 ± 7.8 nM, and 4.71 ± 0.62 nM, respectively. 50 ) is shown.

[0123] Therefore, the IC50 of these three conjugates is 100 nM or less, and preferably 75 nM or less for the ninth conjugate. 50 This provides technical support for showing Additionally, medical and diagnostic applications such as those described herein above as aspects three to eight of the invention are suitable and contemplated for combination with the conjugate of the ninth aspect.

[0124] Thus, further aspects of the present invention relate to: Use of the conjugate of the ninth aspect in the in vitro or ex vivo diagnosis of cancer. Use of the conjugate of the ninth aspect for detecting the presence 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 a subject suspected of having cancer. An in vitro or ex vivo method for detecting the presence of cholecystokinin 2 receptor (CCK-2R) in a sample, the sample preferably being obtained from a cancer patient or a subject suspected of having cancer, the method comprising: (a) contacting the sample with a conjugate of the ninth aspect; and (b) detecting binding of the conjugate of the ninth aspect to CCK-2R. A pharmaceutical or diagnostic composition comprising the conjugate of the ninth aspect. A conjugate according to the ninth aspect, or a pharmaceutical composition comprising the conjugate according to the ninth aspect, for the treatment or prevention of cancer. The conjugate of the ninth aspect or the diagnostic composition of the ninth aspect for use in a method for in vivo diagnosis of cancer in a subject.

[0125] Further details and preferred embodiments relating to medical and diagnostic applications are described herein above in relation to the third to eighth aspects of the invention, and these details and preferred embodiments also apply mutatis mutandis to the further aspects of the invention described above, which use the conjugates of the ninth aspect.

[0126] With respect to the embodiments characterized in this specification, and in particular in the claims, it is intended that each embodiment mentioned in a dependent claim be combined with each embodiment of each claim (independent or dependent) on which said dependent claim depends. For example, if independent claim 1 recites three options A, B, and C, dependent claim 2 recites three options D, E, and F, and claim 3 is dependent on claims 1 and 2 and recites three options G, H, and I, the specification should be understood as expressly disclosing embodiments corresponding to the combinations 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.

[0127] Similarly, if a dependent claim refers to more than one preceding claim, any combination of the subject matter covered by those claims is understood to be explicitly disclosed, even if the independent and / or dependent claims do not recite alternatives. For example, given independent claim 1, dependent claim 2 which refers to claim 1, and dependent claim 3 which refers to both claims 2 and 1, the combination of the subject matter of claims 3 and 1 is clearly and explicitly disclosed, as is the combination of the subject matter of claims 3, 2, and 1. If there is also dependent claim 4 which refers to any one of claims 1 through 3, the combinations of claims 4 and 1, claims 4, 2 and 1, claims 4, 3 and 1, and claims 4, 3, 2 and 1 are clearly and explicitly disclosed. [Brief explanation of the drawings]

[0128] [Figure 1] Schematic diagram of the CCK-2R affinity (expressed as half-maximal inhibitory concentration, IC50) of CCK-2R-targeting ligands (H-Trp-(N-Me)Nle-Asp-l-Nal-NH2, CCK-49, [natLu]Lu-DOTA-γ-MGS5, H-Trp-Nle-Asp-Phe-NH2, CCK-32, and [natLu]Lu-DOTA-PP-γ-F11N). DOTA-MGS5 analogs are shown in black, and DOTA-PP-F11N analogs are shown in gray. [Figure 2] Schematic representation of the CCK-2R affinity (expressed as half-maximal inhibitory concentration, IC50) of CCK-2R-targeting ligands with engineered tetrapeptide binding motifs. [Figure 3] Schematic representation of CCK-2R affinity (half-maximal inhibitory concentration, IC50, represented by bars) and lipophilicity (n-octanol / PBS partition coefficient, pH = 7.4, logD7.4, represented by dots) of glycine scanning compounds (DOTA-CCK-55–-62) compared to the reference (DOTA-MGS5). [Figure 4] Schematic representation of the CCK-2R affinity (expressed as half-maximal inhibitory concentration, IC50) of natLu-, natGa-, and natCu-labeled DOTA-CCK-66 and -66.2 compared to DOTA-MGS5. [Figure 5] Schematic representation of the lipophilicity (expressed as n-octanol / PBS partition coefficient, pH = 7.4, logD7.4) of DOTA-CCK-66, -66.2, and DOTA-MGS5. The compounds tested were complexed with either [177Lu]lutetium, [67Ga]gallium, or [64Cu]copper. [Figure 6] HSA binding (%) of DOTA-CCK-66, -66.2, and DOTA-MGS5 complexed with either [natLu]lutetium, [natGa]gallium, or [natCu]copper. [Figure 7-1]Representative RP-HPLC chromatograms of 177Lu-labeled DOTA-MGS5, -CCK-66, and -CCK-66.2 after incubation in human serum for 72 ± 2 hours at 37 °C (10 → 30% MeCN in HO containing 0.1% TFA for 5 minutes and 30 → 60% MeCN in HO containing 0.1% TFA for 15 minutes) (black) compared to quality control chromatograms of the compounds immediately after 177Lu labeling (gray). [Figure 7-2] Representative RP-HPLC chromatograms of 177Lu-labeled DOTA-MGS5, -CCK-66, and -CCK-66.2 after incubation in human serum for 72 ± 2 hours at 37 °C (10 → 30% MeCN in HO containing 0.1% TFA for 5 minutes and 30 → 60% MeCN in HO containing 0.1% TFA for 15 minutes) (black) compared to quality control chromatograms of the compounds immediately after 177Lu labeling (gray). [Figure 8] Representative RP-HPLC chromatograms of the amount of intact [177Lu]Lu-DOTA-MGS5 and [177Lu]Lu-DOTA-CCK-66 (1 nmol, 35 MBq each) in mouse serum (black) and urine (dark gray) 30 min after injection (10 → 30% MeCN in HO with 0.1% TFA for 5 min and 30 → 60% MeCN in HO with 0.1% TFA for 15 min). The reference chromatogram for the compounds is shown in light gray. [Figure 9] Biodistribution and competition studies (%ID / g) of [67Ga]Ga-DOTA-CCK-66 in selected organs 1 h post-injection in AR4-2J tumor-bearing CB17-SCID mice (100 pmol each). For competition studies, an excess of [natGa]Ga-DOTA-MGS5 (40 nmol) was co-administered. Data are presented as mean ± SD (n = 4 for biodistribution studies, n = 2 for competition studies). For comparison, literature data for [68Ga]Ga-DOTA-MGS5 (20 pmol) are shown

[30] . Comparative analyses of tumor / blood, tumor / stomach, and tumor / kidney ratios of the two compounds are shown on the right. [Figure 10]A) Maximum intensity projection image of AR4-2J tumor-bearing CB17-SCID mice injected with [67Ga]Ga-DOTA-CCK-66 (100 pmol) 1 hour after injection; B) Representative μSPECT / CT image of a competition study of [67Ga]Ga-DOTA-CCK-66 (100 pmol) co-administered with [natGa]Ga-DOTA-MGS5 (40 nmol) in AR4-2J tumor-bearing CB17-SCID mice 1 hour after injection. [Figure 11] Biodistribution and competition studies (%ID / g) of [177Lu]Lu-DOTA-CCK-66 and [177Lu]Lu-DOTA-MGS5 in selected organs in AR4-2J tumor-bearing CB17-SCID mice (100 pmol each) 24 h after injection. In the competition study, an excess of [natLu]Lu-DOTA-MGS5 (40 nmol) was co-administered. Data are presented as mean ± SD (n = 4 for biodistribution study, n = 2 for competition study). Comparative analyses of tumor / blood, tumor / stomach, and tumor / kidney ratios of the two compounds are shown below. [Figure 12] Maximum intensity projection images of AR4-2J tumor-bearing CB17-SCID mice injected with A) [177Lu]Lu-DOTA-MGS5 and B) [177Lu]Lu-DOTA-CCK-66 (100 pmol each). C) Representative μSPECT / CT images 24 hours after injection of [177Lu]LuDOTA-CCK-66 (100 pmol) co-administered with [natLu]Lu-DOTA-MGS5 (40 nmol) in a competition study. [Figure 13] (A) PET / CT image of patient 1 with MTC using [68Ga]Ga-DOTA-CCK-66, which revealed (B) one left retroclavicular lymph node metastasis and (C) aortic arch mediastinal metastasis. [Figure 14] (A) PET / CT image of patient 2 with MTC using [68Ga]Ga-DOTA-CCK-66, which showed (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. [Figure 15](A) PET / CT image of patient 3 with MTC using [68Ga]-DOTA-CCK-66, which showed (B) lymph node metastasis in the left cervical vascular nerve sheath region, (C) local recurrence in the left thyroid bed, and (D) lymph node metastasis in the right upper mediastinum. [Figure 16] Schematic representation of the CCK-2R affinity (expressed as half-maximal inhibitory concentration, IC50) of natLu- and [19F]AlF-complexed DOTA-CCK-66 analogs, including various chelators (NOTA, NODAGA, (R)-DOTAGA, and (S)-DOTAGA). [Figure 17] Schematic representation of the CCK-2R affinity (expressed as half-maximal inhibitory concentration, IC50) and lipophilicity (expressed as n-octanol / PBS partition coefficient, pH 7.4, logD7.4) of [nat / 177Lu]Lu-DOTA-rhCCK-67–76, [nat / 177Lu]Lu-DOTA-rhCCK-83–86, and [nat / 177Lu]Lu-DOTA-rhCCK-90–91, containing different linker sections (light gray: (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: logD7.4 values), and negative charges (with and without γ-glu in the vicinity of dap(SiFA)). [Figure 18] Percent HSA binding of [natLu]Lu-DOTA-rhCCK-67–76, [natLu]Lu-DOTA-rhCCK-83–86, and [natLu]Lu-DOTA-rhCCK-90–91, containing different linker sections (light gray: (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: logD7.4 value), and negative charges (with and without γ-glu in the vicinity of dap(SiFA)). [Figure 19]Biodistribution (%ID / g) of [177Lu]Lu-DOTA-CCK-66 (n = 4), [177Lu]Lu-DOTA-rhCCK-70 (n = 4), [177Lu]Lu-DOTA-rhCCK-84 (n = 3), [177Lu]Lu-DOTA-rhCCK-91 (n = 3), and [177Lu]Lu-DOTA-MGS5 (n = 4) in selected organs in AR4-2J tumor-bearing CB17-SCID mice (100 pmol each) 24 hours after injection. Data are expressed as mean ± SD. [Figure 20] Maximum intensity projection images of AR4-2J tumor-bearing CB17-SCID mice 24 hours after injection of A) [177Lu]Lu-DOTA-CCK-66, B) [177Lu]Lu-DOTA-rhCCK-70, C) [177Lu]Lu-DOTA-rhCCK-84, and D) [177Lu]Lu-DOTA-rhCCK-91 (100 pmol each). [Figure 21] Biodistribution (%ID / g) of [18F]F-[natLu]Lu-DOTA-rhCCK-84 (1 h after injection) and [177Lu]Lu-DOTA-rhCCK-84 (2, 4, and 24 h after injection) in selected organs in AR4-2J tumor-bearing CB17-SCID mice (100 pmol each, n=4). Data are expressed as mean ± SD. [Figure 22] Biodistribution study of [177Lu]Lu-DOTA-rhCCK-84 administered in combination with [natLu]Lu-DOTA-MGS5 (40 nmol, n = 2, black) or [natLu]Lu-DOTA-MG-SiFA-23 (40 nmol, n = 1, gray) in selected organs (%ID / g) in AR4-2J tumor-bearing CB17-SCID mice (100 pmol each) 24 h post-injection. Data are presented as mean ± SD. [Figure 23]Representative RP-HPLC chromatograms of intact [177Lu]Lu-DOTA-rhCCK-84 (1 nmol, 35 MBq each, n=3) in mouse serum (black) and urine (light gray) 30 min after injection (10→30% MeCN in HO with 0.1% TFA for 5 min and 30→60% MeCN in HO with 0.1% TFA for 15 min). Quality control chromatograms of the compound are shown in dark gray. [Figure 24] Biodistribution (%ID / g) of [177Lu]Lu-DOTA-PP-F11N, [177Lu]Lu-DOTAGA-MG-SiFA-19, and [177Lu]Lu-DOTAGA-MG-SiFA-21 in selected organs in AR4-2J tumor-bearing CB17-SCID mice (100 pmol each) 24 hours after injection. Data are expressed as mean ± SD (n=4). [Figure 25] Biodistribution (%ID / g) of [177Lu]Lu-DOTA-MG-SiFA-23 at 1 and 24 hours after injection and [natLu]Lu-18F-DOTA-MG-SiFA-23 at 1 hour after injection (n=1) in selected organs in AR4-2J tumor-bearing CB17-SCID mice (100 pmol each) compared with [177Lu]Lu-DOTA-PP-F11N at 24 hours after injection. Data are expressed as mean ± SD (n=4). [Figure 26] Representative μSPECT / CT images 1, 4, and 24 hours after injection of A) [177Lu]Lu-DOTA-PP-F11N, B) [177Lu]Lu-DOTAGA-MG-SiFA-21, and C) [177Lu]Lu-DOTAGA-MG-SiFA-19 in AR4-2J tumor-bearing CB17-SCID mice (100 pmol each). The tumor (T) is indicated by the white arrow. [Figure 27] Representative μSPECT / CT images of A) [177Lu]Lu-DOTAGA-MG-SiFA-23 1 and 24 hours after injection, and B) [18F]F-[natLu]Lu-DOTA-MG-SiFA-23 1 hour after injection in AR4-2J tumor-bearing CB17-SCID mice (100 pmol each). The tumor (T) is indicated by the white arrow. [Figure 28] Representative μSPECT / CT images (n=1) of A) [177Lu]Lu-DOTAGA-MG-SiFA-19 and B) [177Lu]Lu-DOTA-MG-SiFA-23 co-injected with [natLu]Lu-DOTA-MGS5 (40 nmol each) in AR4-2J tumor-bearing CB17-SCID mice (100 pmol each) 24 hours after injection. C) Biodistribution (%ID / g) of [177Lu]Lu-DOTAGA-MG-SiFA-19 and [177Lu]Lu-DOTA-MG-SiFA-23 co-injected with [natLu]Lu-DOTA-MGS5 (40 nmol each) in selected organs in AR4-2J tumor-bearing CB17-SCID mice (100 pmol each, n=2) 24 hours after injection. Data are expressed as mean ± SD. [Figure 29] Example of a sigmoidal plot showing the correlation between human serum albumin (HSA) binding of selected reference substances and their retention time (tR) on a Chiralpak HSA column. HSA binding values ​​of the reference substances were previously published in the literature (lit. HSA [%]), and the logarithm of the respective affinity constants (log K HSA) was calculated [31, 32]. DETAILED DESCRIPTION OF THE INVENTION

[0129] The following examples are illustrative of the invention described herein. [Example]

[0130] Example 1 - N-terminal tetrapeptide of minigastrin 1.1. Overview of synthesized CCK-2R ligands reference compound

[0131] [ka]

[0132] DOTA-PP-F11N:RP-HPLC (10→90% MeCN in H2O containing 0.1% TFA, λ=220nm for 15 min):tR =7.6 minutes, K'=3.5;MS(ESI, positive):C 90 H 123 N 19 O 35 Calculated value: m / z = 2029.8, Measured value: m / z = 1016.4 [M+2H] 2+ .

[0133] [ka]

[0134] CP04:RP-HPLC (10→90% MeCN in H2O containing 0.1% TFA, 15 min, λ=220 nm):t R =7.2 minutes, K'=3.3;MS(ESI, positive):C 89 H 121 N 19 O 35 Calculated value of S: m / z = 2047.8, Measured value: m / z = 1024.2 [M+2H] 2+ .

[0135] [ka]

[0136] DOTA-MGS5:RP-HPLC (10→90% MeCN in H2O containing 0.1% TFA, 15 min, λ=220nm):t R =9.2 minutes;MS(ESI, positive):C 70 H 92 N 14 O 20 Calculated value: m / z = 1448.7, Measured value: m / z = 1449.4 [M+H] + , 725.3[M+2H] 2+ .

[0137] [ka]

[0138] DOTA-γ-MGS5:RP-HPLC (10→70% MeCN in H2O containing 0.1% TFA, 15 min, λ=220 nm):t R =10.9 minutes;MS(ESI, positive):C 70 H 92 N 14 O 20 Calculated value: m / z = 1448.7, Measured value: m / z = 1449.6 [M+H] + , 725.5[M+2H] 2+ . Compounds containing modified tetrapeptide motifs

[0139] [ka]

[0140] H-Trp-(N-Me)Nle-Asp-1-Nal-NH2:RP-HPLC (10→70% MeCN in H2O containing 0.1% TFA, 15 min, λ=220 nm):t R =11.5 minutes; MS(ESI, positive):C 35 H 42 Calculated value for N6O6: m / z = 642.8, Measured value: m / z = 643.2 [M+H] + .

[0141] [ka]

[0142] H-Trp-Nle-Asp-1-Nal-NH2:RP-HPLC (10→70% MeCN in H2O containing 0.1% TFA, 15 min, λ=220 nm):t R =10.6 minutes;MS(ESI, positive):C 34 H 40 Calculated value for N6O6: m / z = 628.7, Measured value: m / z = 628.3 [M+H] + , 1255.6[2M+H] + .

[0143] [ka]

[0144] H-Trp-Nle-Asp-Phe-NH2: RP-HPLC (0.1% TFA containing 10→70% MeCN in H2O, 15 min, λ=220 nm): t R =9.2 min; MS (ESI, positive): C 30 H 38 Calculated value of N6O6: m / z=578.7, measured value: m / z=579.2[M+H] + 、1157.3[2M+H] + .

[0145]

change

[0146] H-Trp-(N-Me)Nle-Asp-2-Nal-NH2: RP-HPLC (0.1% TFA containing 10→70% MeCN in H2O, 15 min, λ=220 nm): t R =11.5 min; MS (ESI, positive): C 35 H 42 Calculated value of N6O6: m / z=642.8, measured value: m / z=643.3[M+H] + 、1285.9[2M+H] + .

[0147]

change

[0148] H-Trp-(N-Me)Nle-Asp-Phe-NH2: RP-HPLC (0.1% TFA containing 10→70% MeCN in H2O, 15 min, λ=220 nm): t R =10.2 min; MS (ESI, positive): C 31 H 40 Calculated value of N6O6: m / z=592.7, measured value: m / z=593.3[M+H] + 、1186.3[2M+H] + .

[0149] [ka]

[0150] H-Trp-(N-Me)Nle-Asp-Trp-NH2:RP-HPLC (10→70% MeCN in H2O containing 0.1% TFA, 15 min, λ=220 nm):t R =10.3 minutes;MS(ESI, positive):C 33 H 41 Calculated value for N7O6: m / z = 631.7, Measured value: m / z = 632.3 [M+H] + , 1264.8[2M+H] + .

[0151] [ka]

[0152] H-Trp-(N-Me)Nle-Asp-Tyr-NH2:RP-HPLC (10→70% MeCN in H2O containing 0.1% TFA, 15 min, λ=220 nm):t R =8.79 minutes;MS(ESI, positive):C 31 H 40 Calculated value for N6O7: m / z = 608.7, Measured value: m / z = 609.3 [M+H] + , 1217.6[2M+H] + .

[0153] [ka]

[0154] H-Trp-(N-Me)Nle-Asp-p-amino-Phe-NH2:RP-HPLC (10→70% MeCN in H2O containing 0.1% TFA, 15 min, λ=220 nm):t R =7.34 minutes;MS(ESI, positive):C 31 H 41 Calculated value for N7O6: m / z = 607.3, Measured value: m / z = 608.2 [M+H] + , 1215.6[2M+H] + .

[0155]

change

[0156] H-Trp-(N-Me)Leu-Asp-1-Nal-NH2: RP-HPLC (0.1% TFA containing 10→70% MeCN in H2O, 15 min, λ=220 nm): t R =11.4 min; MS (ESI, positive): C 35 H 42 Calculated value of N6O6: m / z=642.3, measured value: m / z=643.7[M+H] + 、1285.1[2M+H] + .

[0157]

change

[0158] H-Trp-(N-Me)Val-Asp-1-Nal-NH2: RP-HPLC (0.1% TFA containing 10→70% MeCN in H2O, 15 min, λ=220 nm): t R =10.4 min; MS (ESI, positive): C 34 H 40 Calculated value of N6O6: m / z=628.3, measured value: m / z=629.3[M+H] + 、1257.0[2M+H] + .

[0159]

change

[0160] H-Trp-(N-Me)Gly-Asp-1-Nal-NH2: RP-HPLC (0.1% TFA containing 10→70% MeCN in H2O, 15 min, λ=220 nm): t R =9.48 min; MS (ESI, positive): C 31 H 34 Calculated value of N6O6: m / z=586.3, measured value: m / z=587.7[M+H] +.

[0161] [ka]

[0162] H-Trp-(N-Me)Ala-Asp-1-Nal-NH2:RP-HPLC (10→70% MeCN in H2O containing 0.1% TFA, 15 min, λ=220 nm):t R =9.92 minutes;MS(ESI, positive):C 32 H 36 Calculated value for N6O6: m / z = 600.3, Measured value: m / z = 601.7 [M+H] + , 1202.9[2M+H] + .

[0163] [ka]

[0164] H-Trp-(N-Me)Met-Asp-1-Nal-NH2:RP-HPLC (10→70% MeCN in H2O containing 0.1% TFA, 15 min, λ=220 nm):t R =11.0 minutes;MS(ESI, positive):C 34 H 40 Calculated value for N6O6S: m / z = 660.3, Measured value: m / z = 661.8 [M+H] + .

[0165] [ka]

[0166] H-Trp-(N-Me)Glu-Asp-1-Nal-NH2:RP-HPLC (10→70% MeCN in H2O containing 0.1% TFA, 15 min, λ=220 nm):t R =9.26 minutes;MS(ESI, positive):C 34 H 38 Calculated value for N6O8: m / z = 658.3, Measured value: m / z = 659.5 [M+H] + .

[0167]

change

[0168] H-Phe-(N-Me)Nle-Asp-1-Nal-NH2: RP-HPLC (0.1% TFA containing 10→70% MeCN in H2O, 15 min, λ=220 nm): t R =11.6 min; MS (ESI, positive): C 33 H 41 Calculated value of N5O6: m / z=603.3, measured value: m / z=604.6[M+H] + 、1208.6[2M+H] + .

[0169]

change

[0170] H-1-Nal-(N-Me)Nle-Asp-1-Nal-NH2: RP-HPLC (0.1% TFA containing 10→70% MeCN in H2O, 15 min, λ=220 nm): t R =12.9 min; MS (ESI, positive): C 37 H 43 Calculated value of N5O6: m / z=653.3, measured value: m / z=654.0[M+H] + .

[0171]

change

[0172] H-2-Nal-(N-Me)Nle-Asp-1-Nal-NH2: RP-HPLC (0.1% TFA containing 10→70% MeCN in H2O, 15 min, λ=220 nm): t R =12.8 min; MS (ESI, positive): C 37 H 43 Calculated value of N5O6: m / z=653.3, measured value: m / z=654.3[M+H] + .

[0173]

change

[0174] Hp-amino-Phe-(N-Me)Nle-Asp-1-Nal-NH2: RP-HPLC (0.1% TFA containing 10→70% MeCN in H2O, 15 min, λ=220 nm): t R =8.65 min; MS (ESI, positive): C 33 H 42 Calculated value of N6O6: m / z=618.3, measured value: m / z=619.2[M+H] + 、1237.5[2M+H] + .

[0175]

change

[0176] H-Tyr-(N-Me)Nle-Asp-1-Nal-NH2: RP-HPLC (0.1% TFA containing 10→70% MeCN in H2O, 15 min, λ=220 nm): t R =10.3 min; MS (ESI, positive): C 33 H 41 Calculated value of N5O7: m / z=619.3, measured value: m / z=620.3[M+H] + 、1240.2[2M+H] + .

[0177]

change

[0178] H-Trp-(N-Me)Nle-Glu-1-Nal-NH2: RP-HPLC (0.1% TFA containing 10→70% MeCN in H2O, 15 min, λ=220 nm): t R =11.5 min; MS (ESI, positive): C 36 H 44Calculated value for N6O6: m / z = 656.3, Found value: m / z = 656.9 [M+H] + , 1314.3[2M+H] + .

[0179] [ka]

[0180] H-γ-glu-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2(CCK-32):RP-HPLC (10→70% MeCN in H2O containing 0.1% TFA, 15 min, λ=220nm):t R =9.7 minutes;MS(ESI, positive):C 49 H 62 N 10 O 13 Calculated value: m / z = 998.5, Measured value: m / z = 998.7 [M+H] + .

[0181] [ka]

[0182] H-γ-glu-Ala-Tyr-Gly-Trp-Nle-Asp-(homo-Phe)-NH2(CCK-33):RP-HPLC (10→70% MeCN in H2O containing 0.1% TFA, 15 min, λ=220nm):t R =10.2 minutes;MS(ESI, positive):C 50 H 64 N 10 O 13 Calculated value: m / z = 1012.5, Measured value: m / z = 1013.6 [M+H] + , 507.4[M+2H] 2+ .

[0183] [ka]

[0184] H-γ-glu-Ala-Tyr-Gly-Trp-Nle-Asp-(p-amino-Phe)-NH2(CCK-34):RP-HPLC(0.1% TFA containing 10→70% MeCN in H2O, 15 min, λ=220 nm):t R =7.5 min; MS (ESI, positive): C 49 H 63 N 11 O 13 Calculated value: m / z=1014.1, measured value: m / z=1014.5[M+H] + 、507.9[M+2H] 2+ .

[0185]

change

[0186] H-γ-glu-Ala-Tyr-Gly-Trp-Nle-Asp-Phg-NH2(CCK-35):RP-HPLC(0.1% TFA containing 10→70% MeCN in H2O, 15 min, λ=220 nm):t R =9.2 min; MS (ESI, positive): C 48 H 60 N 10 O 13 Calculated value: m / z=984.4, measured value: m / z=985.6[M+H] + 、1971.4[2M+H] + .

[0187]

change

[0188] H-γ-glu-Ala-Tyr-Gly-Trp-Nle-Asp-(α-Me)Phe-NH2(CCK-36):RP-HPLC(0.1% TFA containing 10→70% MeCN in H2O, 15 min, λ=220 nm):t R =9.2 min; MS (ESI, positive): C 50 H 64 N 10 O 13Calculated value: m / z = 1012.5, Measured value: m / z = 1013.3 [M+H] + .

[0189] [ka]

[0190] H-γ-glu-Ala-Tyr-Gly-(α-Me)Trp-Nle-Asp-Phe-NH2(CCK-37):RP-HPLC (10→70% MeCN in H2O containing 0.1% TFA, 15 min, λ=220nm):t R =9.9 minutes;MS(ESI, positive):C 50 H 64 N 10 O 13 Calculated value: m / z = 1013.2, Measured value: m / z = 1013.2 [M+H] + , 507.3[M+2H] 2+ .

[0191] [ka]

[0192] H-γ-glu-Ala-Tyr-Gly-Trp-Nle-Asp-Bip-NH2(CCK-38):RP-HPLC (10→70% MeCN in H2O containing 0.1% TFA, 15 min, λ=220nm):t R =11.3 minutes;MS(ESI, positive):C 55 H 66 N 10 O 13 Calculated value: m / z = 1074.5, Measured value: m / z = 1075.1 [M+H] + , 565.2[M+2H] 2+ .

[0193] [ka]

[0194] H-γ-glu-Ala-Tyr-Gly-Trp-Nle-Asp-Trp-NH2(CCK-39):RP-HPLC(0.1% TFA containing 10→70% MeCN in H2O, 15 min, λ=220 nm):t R =9.7 min; MS (ESI, positive): C 51 H 63 N 11 O 13 Calculated value: m / z=1037.5, measured value: m / z=1038.2[M+H] + 、519.8[M+2H] 2+ .

[0195]

change

[0196] H-γ-glu-Ala-Tyr-Gly-Trp-Nle-Asp-Dap(SiFA)-NH2(CCK-48):RP-HPLC(0.1% TFA containing 10→70% MeCN in H2O, 15 min, λ=220 nm):t R =14.2 min; MS (ESI, positive): C 58 H 80 FN 11 O 14 Si's calculated value: m / z=1201.6, measured value: m / z=1202.8[M+H] + 、602.2[M+2H] 2+ .

[0197]

change

[0198] H-γ-glu-Ala-Tyr-Gly-Trp-(N-Me)Nle-Asp-1-Nal-NH2(CCK-49):RP-HPLC(0.1% TFA containing 10→70% MeCN in H2O, 15 min, λ=220 nm):t R =11.4 min; MS (ESI, positive): C 54 H 66 N 10 O 13Calculated value: m / z = 1062.5, Measured value: m / z = 1062.8 [M+H] + . Glycine Scan Derivatives

[0199] [ka]

[0200] DOTA-(Gly)4-Trp-(N-Me)Nle-Asp-1-Nal-NH2(DOTA-CCK-55):RP-HPLC (10→70% MeCN in H2O containing 0.1% TFA, 15 min, λ=220 nm):t R =10.6 minutes;MS(ESI, positive):C 59 H 80 N 14 O 17 Calculated value: m / z = 1201.6, Measured value: m / z = 1257.0 [M+H] + , 629.0[M+2H] 2+ .

[0201] [ka]

[0202] DOTA-(Gly)2-Tyr-Gly-Trp-(N-Me)Nle-Asp-1-Nal-NH2(DOTA-CCK-56):RP-HPLC (10→70% MeCN in H2O containing 0.1% TFA, 15 min, λ=220 nm):t R =11.0 minutes;MS(ESI, positive):C 56 H 86 N 14 O 18 Calculated value: m / z = 1362.6, Measured value: m / z = 1363.1 [M+H] + , 682.0[M+2H] 2+ .

[0203] [ka]

[0204] DOTA-Gly-Ala-Tyr-Gly-Trp-(N-Me)Nle-Asp-1-Nal-NH2(DOTA-CCK-57):RP-HPLC (0.1% TFA containing 10→70% MeCN in H2O, 15 min, λ=220 nm):t R =11.0 min; MS (ESI, positive): C 57 H 88 N 14 O 18 Calculated value: m / z=1376.6, measured value: m / z=1377.1[M+H] + 、689.1[M+2H] 2+ .

[0205]

change

[0206] DOTA-Gly-Trp-(N-Me)Nle-Asp-1-Nal-NH2(DOTA-CCK-58):RP-HPLC (0.1% TFA containing 10→70% MeCN in H2O, 15 min, λ=220 nm):t R =10.9 min; MS (ESI, positive): C 53 H 71 N 11 O 14 Calculated value: m / z=1085.5, measured value: m / z=1086.6[M+H] + 、544.0[M+2H] 2+ .

[0207]

change

[0208] DOTA-γ-glu-Gly-Tyr-Gly-Trp-(N-Me)Nle-Asp-1-Nal-NH2(DOTA-CCK-59):RP-HPLC (0.1% TFA containing 10→70% MeCN in H2O, 15 min, λ=220 nm):t R =10.8 min; MS (ESI, positive): C 69 H 90 N 14 O20 Calculated value: m / z = 1434.7, Measured value: m / z = 1436.6 [M+H] + , 719.0[M+2H] 2+ .

[0209] [ka]

[0210] DOTA-γ-glu-Ala-(Gly)2-Trp-(N-Me)Nle-Asp-1-Nal-NH2(DOTA-CCK-60):RP-HPLC (10→70% MeCN in H2O containing 0.1% TFA, 15 min, λ=220 nm):t R =10.6 minutes;MS(ESI, positive):C 53 H 86 N 14 O 19 Calculated value: m / z = 1342.6, Measured value: m / z = 1343.9 [M+H] + , 672.7[M+2H] 2+ .

[0211] [ka]

[0212] DOTA-Gly-Ala-(Gly)2-Trp-(N-Me)Nle-Asp-1-Nal-NH2(DOTA-CCK-61):RP-HPLC (10→70% MeCN in H2O containing 0.1% TFA, 15 min, λ=220nm):t R =10.6 minutes;MS(ESI, positive):C 60 H 82 N 14 O 17 Calculated value: m / z = 1270.6, Measured value: m / z = 1272.4 [M+H] + , 636.9[M+2H] 2+ .

[0213] [ka]

[0214] DOTA-γ-glu-(Gly)3-Trp-(N-Me)Nle-Asp-1-Nal-NH2(DOTA-CCK-62):RP-HPLC (10→70% MeCN in H2O containing 0.1% TFA, 15 min, λ=220nm):t R =10.5 minutes; MS(ESI, positive):C 62 H 84 N 14 O 19 Calculated value: m / z = 1329.4, Measured value: m / z = 1330.5 [M+H] + , 665.8[M+2H] 2+ . Compounds containing PEG linkers

[0215] [ka]

[0216] DOTA-(PEG)4-Trp-(N-Me)Nle-Asp-1-Nal-NH2(DOTA-CCK-50):RP-HPLC (10→70% MeCN in H2O containing 0.1% TFA, 15 min, λ=220 nm):t R =11.0 minutes;MS(ESI, positive):C 64 H 93 N 11 O 19 Calculated value: m / z = 1319.7, Measured value: m / z = 1319.7 [M+H] + , 659.9[M+2H] 2+ .

[0217] [ka]

[0218] DOTA-γ-glu-(PEG)3-Trp-(N-Me)Nle-Asp-1-Nal-NH2(DOTA-CCK-66):RP-HPLC (10→70% MeCN in H2O containing 0.1% TFA, 15 min, λ=220nm):t R =10.8 minutes;MS(ESI, positive):C 67 H96 N 12 O 21 Calculated value: m / z = 1405.6, Measured value: m / z = 1405.3 [M+H] + , 702.8[M+2H] 2+ .

[0219] [ka]

[0220] NOTA-γ-glu-(PEG)3-Trp-(N-Me)Nle-Asp-1-Nal-NH2(NOTA-CCK-66):RP-HPLC (10→70% MeCN in H2O containing 0.1% TFA, 15 min, λ=220nm):t R =11.5 minutes; MS(ESI, positive):C 63 H 89 N 11 O 19 Calculated value: m / z = 1303.6, Measured value: m / z = 1304.0 [M+H] + , 652.9[M+2H] 2+ .

[0221] [ka]

[0222] NODAGA-γ-glu-(PEG)3-Trp-(N-Me)Nle-Asp-1-Nal-NH2(NODAGA-CCK-66):RP-HPLC (10→70% MeCN in H2O containing 0.1% TFA, 15 min, λ=220nm):t R =11.4 minutes;MS(ESI, positive):C 66 H 93 N 11 O 21 Calculated value: m / z = 1376.5, Measured value: m / z = 1375.9 [M+H] + , 689.2[M+2H] 2+ .

[0223] [ka]

[0224] (R)-DOTAGA-γ-glu-(PEG)3-Trp-(N-Me)Nle-Asp-1-Nal-NH2((R)-DOTAGA-CCK-66):RP-HPLC (0.1% TFA containing 10→70% MeCN in H2O, 15 min, λ=220 nm):t R =10.9 min; MS (ESI, positive): C 70 H 100 N 12 O 23 Calculated value: m / z=1477.6, measured value: m / z=1477.4[M+H] + 、739.7[M+2H] 2+ .

[0225]

change

[0226] (S)-DOTAGA-γ-glu-(PEG)3-Trp-(N-Me)Nle-Asp-1-Nal-NH2((S)-DOTAGA-CCK-66):RP-HPLC (0.1% TFA containing 10→70% MeCN in H2O, 15 min, λ=220 nm):t R =10.9 min; MS (ESI, positive): C 70 H 100 N 12 O 23 Calculated value: m / z=1477.6, measured value: m / z=1477.4[M+H] + 、739.6[M+2H] 2+ .

[0227]

change

[0228] DOTA-glu-(PEG)3-Trp-(N-Me)Nle-Asp-1-Nal-NH2(DOTA-CCK-66.2):RP-HPLC (0.1% TFA containing 10→70% MeCN in H2O, 15 min, λ=220 nm):t R=10.9 minutes;MS(ESI, positive):C 67 H 96 N 12 O 21 Calculated value: m / z = 1405.6, Measured value: m / z = 1405.9 [M+H] + , 703.6[M+2H] 2+ .

[0229] [ka]

[0230] DOTA-γ-glu-PEG-Trp-(N-Me)Nle-Asp-1-Nal-NH2(DOTA-CCK-77):RP-HPLC (10→70% MeCN in H2O containing 0.1% TFA, 15 min, λ=220nm):t R =10.8 minutes;MS(ESI, positive):C 62 H 86 N 12 O 19 Calculated value: m / z = 1303.4, Measured value: m / z = 1302.9 [M+H] + , 651.6[M+2H] 2+ .

[0231] [ka]

[0232] DOTA-γ-glu-(PEG)2-Trp-(N-Me)Nle-Asp-1-Nal-NH2(DOTA-CCK-63):RP-HPLC (10→70% MeCN in H2O containing 0.1% TFA, 15 min, λ=220nm):t R =10.9 minutes;MS(ESI, positive):C 65 H 92 N 12 O 20 Calculated value: m / z = 1361.5, Measured value: m / z = 1359.5 [M+H] + , 680.6[M+2H] 2+ .

[0233]

change

[0234] DOTA-γ-glu-(PEG)4-Trp-(N-Me)Nle-Asp-1-Nal-NH2(DOTA-CCK-78):RP-HPLC (0.1% TFA containing 10→70% MeCN in H2O, 15 min, λ=220 nm):t R =11.0 min; MS (ESI, positive): C 69 H 100 N 12 O 22 Calculated value: m / z=1449.6, measured value: m / z=1447.4[M+H] + 、724.3[M+2H] 2+ .

[0235]

change

[0236] DOTA-γ-glu-(PEG)5-Trp-(N-Me)Nle-Asp-1-Nal-NH2(DOTA-CCK-79):RP-HPLC (0.1% TFA containing 10→70% MeCN in H2O, 15 min, λ=220 nm):t R =11.1 min; MS (ESI, positive): C 71 H 104 N 12 O 23 Calculated value: m / z=1493.7, measured value: m / z=1491.1[M+H] + 、746.3[M+2H] 2+ .

[0237]

change

[0238] DOTA-γ-glu-(PEG)7-Trp-(N-Me)Nle-Asp-1-Nal-NH2(DOTA-CCK-80):RP-HPLC (0.1% TFA containing 10→70% MeCN in H2O, 15 min, λ=220 nm):t R=11.1 minutes;MS(ESI, positive):C 75 H 112 N 12 O 25 Calculated value: m / z = 1581.8, Measured value: m / z = 1582.4 [M+H] + , 792.0[M+2H] 2+ .

[0239] [ka]

[0240] DOTA-γ-glu-(PEG) 11 -Trp-(N-Me)Nle-Asp-1-Nal-NH2(DOTA-CCK-81):RP-HPLC (10→70% MeCN in H2O containing 0.1% TFA, 15 min, λ=220nm):t R =11.3 minutes;MS(ESI, positive):C 83 H 128 N 12 O 29 Calculated value: m / z = 1758.0, Measured value: m / z = 1759.0 [M+H] + , 880.0[M+2H] 2+ PEGylated rhCCK ligand

[0241] [ka]

[0242] DOTA-dap(SiFA)-(PEG)4-γ-glu-(PEG)3-Trp-(N-Me)Nle-Asp-1-Nal-NH2(DOTA-rhCCK-67):RP-HPLC (10→70% MeCN in H2O containing 0.1% TFA, 15 min, λ=220 nm):t R =14.1 min;MS(ESI, positive):C 98 H 148 FN 15 O 29 Si calculated value: m / z = 2047.4, measured value: m / z = 1022.9 [M+2H] 2+ .

[0243] [ka]

[0244] DOTA-dap(SiFA)-γ-glu-(PEG)4-γ-glu-(PEG)3-Trp-(N-Me)Nle-Asp-1-Nal-NH2(DOTA-rhCCK-68):RP-HPLC(10→70% MeCN in H2O containing 0.1% TFA, 15 min, λ=220nm):t R =13.3 minutes;MS(ESI, positive):C 103 H 155 FN 16 O 32 Si calculated value: m / z = 2175.1, measured value: m / z = 1087.3 [M+2H] 2+ .

[0245] [ka]

[0246] DOTA-dap(SiFA-ipa)-γ-glu-(PEG) 11 -γ-glu-(PEG)3-Trp-(N-Me)Nle-Asp-1-Nal-NH2(DOTA-rhCCK-69):RP-HPLC (10→70% MeCN in H2O containing 0.1% TFA, 15 min, λ=220nm):t R =12.7 minutes;MS(ESI, positive):C 118 H 183 FN 16 O 41 Si calculated value: m / z = 2528.9, measured value: m / z = 1264.9 [M+2H] 2+ .

[0247] [ka]

[0248] DOTA-dap(SiFA)-γ-glu-(PEG)7-γ-glu-(PEG)3-Trp-(N-Me)Nle-Asp-1-Nal-NH2(DOTA-rhCCK-70):RP-HPLC(10→70% MeCN in H2O containing 0.1% TFA, 15 min, λ=220nm):t R =13.6 minutes;MS(ESI, positive):C 103 H 155 FN 16 O 32 Si calculated value: m / z = 2175.1, measured value: m / z = 1153.8 [M+2H] 2+ .

[0249] [ka]

[0250] DOTA-dap(SiFAlin)-γ-glu-(PEG)7-γ-glu-(PEG)3-Trp-(N-Me)Nle-Asp-1-Nal-NH2(DOTA-rhCCK-71):RP-HPLC(10→70% MeCN in H2O containing 0.1% TFA, 15 min, λ=220nm):t R =13.2 minutes;MS(ESI, positive):C 113 H 177 FN 17 O 35 Si + Calculated value: m / z = 2380.8, Measured value: m / z = 1191.0 [M+2H] 2+ , 794.1[M+3H] 3+ .

[0251] [ka]

[0252] DOTA-dap(SiFA-ipa)-γ-glu-(PEG)7-γ-glu-(PEG)3-Trp-(N-Me)Nle-Asp-1-Nal-NH2(DOTA-rhCCK-72):RP-HPLC (10→70% MeCN in H2O containing 0.1% TFA, 15 min, λ=220nm):t R=12.8 minutes;MS(ESI, positive):C 110 H 167 FN 16 O 37 Si calculated value: m / z = 2352.7, measured value: m / z = 1177.8 [M+2H] 2+ , 785.4[M+3H] 3+ .

[0253] [ka]

[0254] DOTA-dap(SiFA)-(PEG)7-γ-glu-(PEG)3-Trp-(N-Me)Nle-Asp-1-Nal-NH2(DOTA-rhCCK-73):RP-HPLC (10→70% MeCN in H2O containing 0.1% TFA, 15 min, λ=220 nm):t R =14.1 min;MS(ESI, positive):C 104 H 160 FN 15 O 32 Si calculated value: m / z = 2179.6, Measured value: m / z = 1090.6 [M+2H] 2+ , 727.3[M+3H] 3+ .

[0255] [ka]

[0256] DOTA-dap(SiFA)-(PEG) 11 -γ-glu-(PEG)3-Trp-(N-Me)Nle-Asp-1-Nal-NH2(DOTA-rhCCK-74):RP-HPLC (10→70% MeCN in H2O containing 0.1% TFA, 15 min, λ=220nm):t R =14.0 minutes;MS(ESI, positive):C 112 H 176 FN 15 O 36 Si calculated value: m / z = 2355.8, measured value: m / z = 1176.8 [M+2H] 2+ , 784.8[M+3H]3+ .

[0257]

change

[0258] DOTA-dap(SiFAlin)-γ-glu-(PEG) 11 -γ-glu-(PEG)3-Trp-(N-Me)Nle-Asp-1-Nal-NH2(DOTA-rhCCK-75):RP-HPLC (0.1% TFA containing 10→70% MeCN in H2O, 15 min, λ=220 nm):t R =13.2 min; MS (ESI, positive): C 121 H 193 FN 17 O 39 Si + Calculated value m / z=2557.0, measured value: m / z=1279.5[M+2H] 2+ 、853.2[M+3H] 3+ .

[0259]

change

[0260] DOTA-dap(SiFA)-γ-glu-(PEG) 11 -γ-glu-(PEG)3-Trp-(N-Me)Nle-Asp-1-Nal-NH2(DOTA-rhCCK-76):RP-HPLC (0.1% TFA containing 10→70% MeCN in H2O, 15 min, λ=220 nm):t R =13.6 min; MS (ESI, positive): C 117 H 183 FN 16 O 39 Si's calculated value: m / z=2484.9, measured value: m / z=1241.4[M+2H] 2+ 、827.9[M+3H] 3+ .

[0261]

change

[0262] DOTA-dap(SiFAlin)-(PEG)4-γ-glu-(PEG)3-Trp-(N-Me)Nle-Asp-1-Nal-NH2(DOTA-rhCCK-90):RP-HPLC (0.1% TFA containing 10→70% MeCN in H2O, 15 min, λ=220 nm):t R =13.6 min; MS (ESI, positive): C 102 H 158 FN 16 O 29 Si + Calculated value: m / z=2119.6, measured value: m / z=1061.7[M+2H] 2+ 、708.2[M+3H] 3+ .

[0263]

change

[0264] DOTA-dap(SiFAlin)-γ-glu-(PEG)4-γ-glu-(PEG)3-Trp-(N-Me)Nle-Asp-1-Nal-NH2(DOTA-rhCCK-91):RP-HPLC (0.1% TFA containing 10→70% MeCN in H2O, 15 min, λ=220 nm):t R =13.1 min; MS (ESI, positive): C 107 H 165 FN 17 O 32 Si + Calculated value: m / z=2248.7, measured value: m / z=1125.6[M+2H] 2+ 、750.8[M+3H] 3+ .

[0265]

change

[0266] DOTA-dap(SiFA)-γ-glu-(Hyp)3-γ-glu-(PEG)3-Trp-(N-Me)Nle-Asp-1-Nal-NH2(DOTA-rhCCK-83):RP-HPLC (0.1% TFA containing 10→70% MeCN in H2O, 15 min, λ=220 nm):t R =13.2 min; MS (ESI, positive): C 105 H 151 FN 18 O 32 Si's calculated value: m / z=2224.5, measured value m / z=1113.8[M+2H] 2+ 、743.1[M+3H] 3+ .

[0267]

change

[0268] DOTA-dap(SiFA)-γ-glu-(Hyp)6-γ-glu-(PEG)3-Trp-(N-Me)Nle-Asp-1-Nal-NH2(DOTA-rhCCK-84):RP-HPLC (0.1% TFA containing 10→70% MeCN in H2O, 15 min, λ=220 nm):t R =12.6 min; MS (ESI, positive): C 120 H 172 FN 21 O 36 Si's calculated value: m / z=2563.9, measured value: m / z=1284.3[M+2H] 2+ 、856.5[M+3H] 3+ .

[0269]

change

[0270] DOTA-dap(SiOH)-γ-glu-(Hyp)6-γ-glu-(PEG)3-Trp-(N-Me)Nle-Asp-1-Nal-NH2(DOTA-rhCCK-84.2):RP-HPLC (0.1% TFA containing 10→70% MeCN in H2O, 15 min, λ=220 nm):tR =11.7 min, MS (ESI, positive): C 120 H 173 N 21 O 39 Si's calculated value: m / z=2561.9, measured value: m / z=1281.6[M+2H] 2+ 、854.2[M+3H] 3+ .

[0271]

change

[0272] DOTA-dap(SiFA)-γ-glu-(cit)3-(Hyp)3-γ-glu-(PEG)3-Trp-(N-Me)Nle-Asp-1-Nal-NH2(DOTA-rhCCK-85): RP-HPLC (0.1% TFA containing 10→70% MeCN in H2O, 15 min, λ=220 nm): t R =12.6 min; MS (ESI, positive): C 123 H 184 FN 27 O 38 Si's calculated value: m / z=2696.1, measured value: m / z=1349.8[M+2H] 2+ 、900.6[M+3H] 3+ .

[0273]

change

[0274] DOTA-dap(SiFA)-γ-glu-(Hyp)8-γ-glu-(PEG)3-Trp-(N-Me)Nle-Asp-1-Nal-NH2(DOTA-rhCCK-86):RP-HPLC (0.1% TFA containing 10→70% MeCN in H2O, 15 min, λ=220 nm):t R =12.2 min; MS (ESI, positive): C 130 H 186 FN 23 O 42 Si's calculated value: m / z=2790.1, measured value: m / z=1396.1[M+2H] 2+, 930.9[M+3H] 3+ . 1.2. In Vitro Results of Compounds with Modified Trp-(N-Me)Nle-Asp-l-Nal Motifs Although most CCK-2R targeting compounds consist of at least seven amino acids (plus a linker and chelator), several studies have suggested that the N-terminal four amino acids are important for high CCK-2R affinity [28,30,33]. nat All Lu-labeled reference compounds demonstrated high CCK-2R affinity (4.9–12.8 nm), whereas most compounds modified within the N-terminal (4) amino acid sequence exhibited slight or significant reductions in CCK-2R affinity (Table 1).

[0275] [Table 1-1]

[0276] [Table 1-2]

[0277] Interestingly, DOTA-PP-F11N (CCK-32), which does not contain the DOTA chelator or the six D-glutamates, showed lower CCK-2R affinity than the parent peptide. Substitution of the C-terminal Phe of CCK-32 with similar aromatic amino acids (CCK-33 to -39) completely abolished CCK-2R affinity for most compounds. Substitution of Trp by (α-Me)Trp in CCK-32 reduced the IC 50 The IC value was three-fold higher, while the substitution of p-amino-Phe for Phe increased the IC 50 The values ​​were slightly decreased. All these observations point to a conserved region at the C-terminus.

[0278] Interestingly, DOTA-MGS5(CCK-49), which does not contain the DOTA chelator, differs from CCK-32 at only two positions ((N-Me)Nle instead of Nle, and 1-Nal instead of Phe) and [ nat Lu]Lu-DOTA-MGS5 equivalent IC 50 values, approximately 4-fold lower than CCK-32 50 Surprisingly, evaluation of a truncated DOTA-PP-F11N motif (H-Trp-Nle-Asp-Phe-NH2) consisting of only the four N-terminal amino acids showed low CCK-2R affinity, whereas a truncated DOTA-MGS5 motif (H-Trp-(N-Me)Nle-Asp-1-Nal-NH2) showed high affinity for CCK-2R. nat Lu]Lu-DOTA-MGS5 and relatively high CCK-2R affinity were revealed (Fig. 1).

[0279] These results led to the hypothesis that the N-terminal four amino acids of DOTA-MGS5 (H-Trp-(N-Me)Nle-Asp-1-Nal-NH2) are sufficient for high CCK-2R affinity, whereas for DOTA-PP-F11N and its analogs, only the complete peptide structure exhibited CCK-2R affinity in the low nanomolar range.

[0280] Further investigation of the tetrapeptide structure H-Trp-(N-Me)Nle-Asp-l-Nal-NH2 (derived from DOTA-MGS5) showed that substituting l-Nal with Phe further increased CCK-2R affinity, whereas substituting l-Nal with Phe in H-Trp-Nle-Asp-Phe-NH2 (derived from DOTA-PP-F11N) significantly reduced CCK-2R affinity. Therefore, high CCK-2R affinity for CCK-2-targeting tetrapeptides was observed only when a methyl group was present at the amide nitrogen between the Trp and Nle bonds (Figure 2). Maintaining this (N-Me)Nle motif within the tetrapeptide also tolerated C-terminal substitutions (Trp, Tyr, and p-amino-Phe instead of l-Nal in H-Trp-(N-Me)Nle-Asp-l-Nal-NH2). However, this position remains conserved, as substitution of 1-Nal with 2-Nal resulted in a significant loss of CCK-2R affinity.

[0281] 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 did not tolerate high CCK-2R affinities. Therefore, it is speculated that Nle can only be replaced with aliphatic amino acids of similar length to maintain high CCK-2R affinity. Neither negatively charged nor smaller aliphatic side chains can be successfully installed at this position. Substitution of Trp with various aromatic amino acids, such as Phe, 1-Nal, 2-Nal, p-amino-Phe, or Tyr, did not improve CCK-2R affinity, leading to the hypothesis that the Trp moiety at this position is essential for the compound's high binding affinity. Furthermore, substitution of Ala with Glu significantly reduced CCK-2R affinity. 1.3. In vitro results of glycine scan derivatives To verify our hypothesis that the N-terminal four amino acids of DOTA-MGS5 alone would provide sufficient CCK-2R affinity, we performed a glycine scan of the remaining four amino acids of the parent peptide. Therefore, we evaluated the receptor affinity (IC) of the novel conjugates in AR4-2J cells. 50 ) and lipophilicity (logD 7.4 ) was determined (Table 2).

[0282] [Table 2]

[0283] These studies demonstrated that the amino acids γ-glu / glu, Ala, and Tyr are identical to those in the parent peptide ([ nat Compared with [Lu]Lu-DOTA-MGS5), the substitution of glycine residues without significant loss of CCK-2R affinity revealed that our hypothesis was largely correct. These data support the hypothesis that the N-terminal motif H-Trp-(N-Me)Nle-Asp-l-Nal is sufficient for high CCK-2R affinity and that the N-terminal amino acid can be considered as a linker moiety. However, [ nat The significantly reduced CCK-2R affinity of [Lu]Lu-DOTA-CCK-50 indicates that sterically demanding chelators such as DOTA require a distinct distance for high CCK-2R affinity. Therefore, the PEG-10 ... x It has been suggested that a simple linker such as a chain is both necessary and sufficient.

[0284] Furthermore, these studies showed that the presence of the γ-glu moiety in the linker section is beneficial not only for CCK-2R affinity but also for overall lipophilicity in particular, since all ligands containing this moiety exhibited a logD 7.4 The logD values ​​were -2.66 to -2.56, whereas the compounds that did not contain this moiety had logD values ​​of -2.66 to -2.56. 7.4 This is because the values ​​ranged from -2.09 to -1.26 (Table 2, Figure 3).

[0285] As mentioned above, no effective strategy has been reported to address the metabolically unstable Gly-Trp bond that is widely present in CCK-2R agonists. Based on the results described in this chapter, the N-terminal amino acid of H-Trp-(N-Me)Nle-Asp-1-Nal-NH2 (H-glu-Ala-Tyr-Gly-) can be easily substituted with a simple PEG. x The PEG-Trp bond is thought to be more metabolically stable than the Gly-Trp bond, and it is hypothesized that this substitution may also stabilize the entire peptide. Therefore, this PEG-containing design was evaluated and is described in the next chapter. 1.4. In Vitro and In Vivo Results of CCK-2R Targeting Compounds Containing PEG Linkers Previous studies have shown that the four amino acids in DOTA-MGS5, γ-glu-Ala-Tyr-Gly, can be combined with PEGs of different lengths. x The γ-glu moiety was substituted with a γ-glu chain. Since several major metabolic instabilities have been reported at the Gly-Trp site, it was assumed that these modifications would further improve the metabolic stability of the DOTA-MGS5 derivatives. Furthermore, some compounds also contain a γ-glu moiety, which has been observed to be beneficial for both CCK-2R affinity and lipophilicity. All data were compared with DOTA-MGS5 (Table 3).

[0286] [Table 3]

[0287] PEG x Regardless of chain length and chelator-metal chelate used, all compounds had IC in the low nanomolar range (3.5–9.0 nM). 50However, the CCK-2R affinity gradually decreased as the PEG chain length increased, and thus the highest CCK-2R affinity was observed for DOTA-CCK-77, -63, -66, and -66.2. Furthermore, the insertion of the N-terminal γ-glu moiety positively affected the overall CCK-2R affinity, and DOTA-CCK-78 exhibited a significantly higher affinity for CCK-50 (IC), despite the same PEG chain length. 50 =8.84±1.25) 50 The values ​​were shown.

[0288] Furthermore, the logD of these compounds complexed with lutetium-177, gallium-67, or copper-64 7.4 The values ​​were evaluated (Table 4).

[0289] [Table 4]

[0290] Generally, 177 Among all the Lu-labeled compounds, those containing short PEG chains showed low lipophilicity. Furthermore, the addition of the γ-glu moiety significantly reduced the logD 7.4 The values ​​were slightly reduced (DOTA-CCK-78 vs. -50). 7.4 [ with a value less than -3 67 Ga]Ga-DOTA-CCK-66,[ 177 Lu]Lu-DOTA-CCK-63, and [ 177 [Lu]Lu-DOTA-CCK-77 was the most hydrophilic compound in this series. However, all compounds had log D values ​​ranging from -3.1 to -2.3, which is considered ideal for in vivo applications. 7.4 The values ​​were shown.

[0291] CCK-2R affinity is highest, logD 7.4 Among the compounds with the lowest values ​​(DOTA-CCK-77, -63, -66, and -66.2), [ nat / 67 Ga] gallium and [ nat / 64We decided to further investigate DOTA-CCK-66 and -66.2, along with DOTA-MGS5, for their CCK-2R affinity and lipophilicity (Figures 4-5) as well as human serum albumin (HSA) binding and metabolic stability when labeled with [Cu] copper.

[0292] Klingler et al. 68 reported a favorable biodistribution profile and rapid clearance kinetics for Ga]Ga-DOTA-MGS5

[30] . nat Lu] lutetium, [ nat Ga] gallium, or [ nat HSA binding was assessed for DOTA-CCK-66 and -66.2 and DOTA-MGS5 complexed with copper [Cu] (Table 5).

[0293] [Table 5]

[0294] A trend was observed for the different metal chelates of the three PEGylated CCK derivatives evaluated (Figure 6). nat All three Lu-labeled compounds were found to have the highest binding to HSA, while nat The Cu-complexed peptide was shown to have less interaction with HSA. nat Each of the Ga-labeled ligands was observed to bind least strongly to HSA. Comparative analysis revealed that DOTA-MGS5 bound best to HSA, while DOTA-CCK-66 and DOTA-CCK-66.2 had lower affinities for HSA. This suggests that, regardless of the radiometal used, both DOTA-CCK-66 and -66.2 are cleared more rapidly than DOTA-MGS5.

[0295] moreover, 177Stability studies of the Lu-labeled compounds DOTA-MGS5, DOTA-CCK-66, and DOTA-CCK-66.2 were performed in vitro in human serum by incubation at 37°C for 72 ± 2 hours. The more promising compounds in vitro were 177 The metabolic stability of [Lu]Lu-DOTA-CCK-66 was also evaluated in vivo in mouse serum and urine 30 min after injection. 177 Lu]Lu-DOTA-MGS5 (Table 6).

[0296] [Table 6]

[0297] In vitro, all three compounds showed high metabolic stability in human serum after 72 ± 2 hours of incubation at 37 °C. 177 Lu]Lu-DOTA-MGS5 showed a slightly higher intact fraction than the PEGylated compound (Figure 7).

[0298] In vivo, a slightly higher proportion of the parent compound was still present intact in mouse serum 30 minutes after injection. 177 The intact fraction of [Lu]Lu-DOTA-CCK-66 was measured in mouse urine 30 min after injection. 177 Lu]Lu-DOTA-MGS5 was almost three times higher than that of [ 177 Lu]Lu-DOTA-CCK-66 was cleared almost intact, while [ 177 It is assumed that [Lu]Lu-DOTA-MGS5 is primarily metabolized and cleared (Figure 8, Table 6), and since metabolites tend to accumulate nonspecifically, this may be beneficial in reducing accumulation and retention of activity in non-target organs, particularly the kidney.

[0299] To investigate the pharmacokinetic profile of the novel PEGylated CCK-2R targeting compound of the present invention, 67Biodistribution studies of [Ga]Ga-DOTA-CCK-66 were performed 1 hour after injection, and the same time points were compared with [ 68 The data were compared with those of Ga]Ga-DOTA-MGS5

[30] (Fig. 9).

[0300] Comparative analyses should be made with caution, as each animal was injected with 100 pmol, whereas Klingler et al. injected only 20 pmol

[30] . However, despite the five-fold higher dose of peptide administered, [ 67 One hour after injection, the activity level in the tumor was [ 68 The tumor-to-background (T / B) ratio was only slightly lower than that of [Ga]Ga-DOTA-MGS5. As expected, the former showed lower activity levels in all non-tumor organs, likely due to improved metabolic stability and clearance kinetics. Even if higher activity levels in all organs are expected at lower peptide doses, tumor uptake is likely also increased. Therefore, the tumor-to-background (T / B) ratio suggests efficacy regardless of the administered peptide dose. 67 Ga]Ga-DOTA-CCK-66 68 Compared with [Ga]Ga-DOTA-MGS5, the T / B ratio was higher in all organs, especially in the blood, stomach, and kidney. 68 Ga]Ga-DOTA-CCK-66 67 It is chemically identical to [Ga]Ga-DOTA-CCK-66 and is therefore a legitimate candidate for clinical translation. 67 This is supported by μSPECT / CT images of Ga]Ga-DOTA-CCK-66 ( Figure 10 ).

[0301] The accumulation in CCK-2R-positive tumors and the stomach is specific, which is due to the excessive amount of [ nat This was demonstrated by a competition study using co-administration of [Ga]Ga-DOTA-MGS5. To further explore the therapeutic and diagnostic potential of DOTA-CCK-66, 177 Lu labeling was assessed and [177 Lu]Lu-DOTA-MGS5 (Figure 11).

[0302] In a biodistribution study 24 hours after injection, 177 The intratumoral activity level of Lu]Lu-DOTA-CCK-66 was [ 177 The activity of [Lu]Lu-DOTA-MGS5 was found to be slightly lower than that of [Lu]Lu-DOTA-MGS5 (8.56 ± 1.08 vs. 11.0 ± 1.15 %ID / g). However, the tumor / blood ratio was improved for the former due to improved clearance of activity from non-tumor organs (2009 ± 469 vs. 1480 ± 329), which is beneficial for therapeutic applications. The tumor / stomach and tumor / kidney ratios were comparable for both compounds. Competition studies revealed significantly reduced activity levels in both the stomach and tumor, which led to [Lu]Lu-DOTA-MGS5. 177 The CCK-2R specificity of [Lu]Lu-DOTA-CCK-66 was confirmed. These observations are supported by μSPECT / CT images (Figure 12).

[0303] For all these reasons, a first-in-human study was conducted in MTC patients with elevated serum tumor markers and a short calcitonin doubling time. 68 [Ga]Ga-DOTA-CCK-66 PET / CT demonstrated tumor lesions and CCK-2R at 2 hours post-injection. + High accumulation in the stomach was observed, demonstrating good pharmacokinetics (Figures 13A, 14A, and 15A). Except for the bladder and ureter due to physiological excretion, no significant activity was observed in other organs. 68 Multiple MTC-derived lesions were detected by [Ga]Ga-DOTA-CCK-66 PET / CT in each patient. Patient 1 had one left retroclavicular lymph node metastasis and additional mediastinal metastases in the aortic arch. Patient 2 had one right retroclavicular lymph node metastasis, bilateral hilar lymph node metastases, two liver metastases, and bone metastases in the right femur and right ischium. Patient 3 had one lymph node metastasis in the left cervical vascular nerve sheath, a local recurrence in the left thyroid bed, and lymph node metastases in the right upper mediastinal region.

[0304] All patients were 68 We greatly benefited from these findings with [Ga]Ga-DOTA-CCK-66 PET / CT. When surgical resection was not possible, identified metastases were 177 [Lu]Lu-DOTA-CCK-66 may be amenable to radioligand therapy because this compound exhibited a favorable biodistribution profile in animals, with sustained high activity levels in tumors for extended periods and rapid clearance of activity from the blood. Further studies in humans are needed to confirm these promising results in smaller cohorts.

[0305] In conclusion, a novel PEGylated CCK-2R targeting compound, [ 67 / 68 Ga]Ga- / [ 177 Lu]Lu-DOTA-CCK-66 is a compound that has been used clinically. 68 Ga]Ga- / [ 177 Compared with [Lu]Lu-DOTA-MGS5, [Lu]Lu-DOTA-MGS5 showed improved in vivo data. The T / B ratio was comparable or even improved in the former, which is likely due to the tetrapeptide design of the PEG linker combination. This allows the N-terminal amino acid to be PEG. x These promising preclinical properties have led to the development of a novel steroid hormone, [ 68 The first human application in MTC patients was performed using [Ga]Ga-DOTA-CCK-66, confirming the favorable biodistribution profile of this compound already observed in animals. Rapid clearance from the blood resulted in high tumor-to-background contrast, allowing the detection of multiple tumor manifestations in three MTC patients. Further trials in humans are needed to clearly elucidate whether this compound will have a beneficial impact on the management of MTC. However, initial results suggest that [ 68 These results suggest that [Ga]Ga-DOTA-CCK-66 may indeed be a viable option for PET imaging of MTC-derived lesions. 177This compound is a very promising therapeutic and diagnostic agent, as the Lu-labeled analogue may also be an effective therapeutic option. 1.5. In Vitro and In Vivo Results of CCK-2R Targeting Compounds Containing PEG Linkers and Various Chelators Previous studies have demonstrated the suitability of combining the optimized tetrapeptide binding motif of the present invention with various chelators (NOTA, NODAGA, (S)-DOTAGA, and (R)-DOTAGA) by replacing the DOTA chelator in DOTA-CCK-66 (Table 7).

[0306] [Table 7]

[0307] These studies revealed that the DOTA chelator in DOTA-CCK-66 could be replaced with NODAGA without compromising CCK-2R affinity (Figure 16). nat Ga- and [ 19 Both [F]AlF-complexed derivatives lost CCK-2R affinity. Furthermore, the introduction of (R)- and (S)-DOTAGA chelators in place of DOTA resulted in IC 50 Although most DOTA-CCK-66 analogs had low CCK-2R affinity, NOTA and NODAGA in particular [ nat Ga] gallium or [ 19 Combination with [F]AlF can be considered for future applications. 1.6. In Vitro and In Vivo Results of First Generation PEGylated rhCCK Ligands Several PEGylated rhCCK compounds were synthesized and evaluated for CCK-2R affinity (IC) in AR4-2J cells. 50 ) and lipophilicity (logD 7.4 ) nat / 177 Lu / nat Ga labeling was evaluated (Table 8).

[0308] [Table 8-1]

[0309] [Table 8-2]

[0310] nat Both Lu-labeled rhCCK ligands were nat Lu]Lu-DOTA-MGS5 or [ nat As expected, the [Lu]Lu-DOTA-CCK-66 did not exhibit the same affinity for CCK-2R as the [Lu]Lu-DOTA-CCK-66 (Figure 17). 177 Lu]Lu-DOTA-rhCCK-67,[ 177 Lu]Lu-DOTA-rhCCK-73, and [ 177 Lu]Lu-DOTA-rhCCK-74 showed enhanced lipophilicity, which is due to the lipophilic SiFA moiety not being fully compensated.

[0311] Therefore, when another γ-glu moiety was introduced, the lipophilicity was significantly improved while maintaining the CCK-2R affinity ([ nat / 177 Lu]Lu-DOTA-rhCCK-68,[ nat / 177 Lu]Lu-DOTA-rhCCK-70, and [ nat / 177 Lu]Lu-DOTA-rhCCK-76). To further improve lipophilicity, SiFA was converted to a 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 The compounds containing the SiFA-ipa moiety were replaced with the [Lu]Lu-DOTA-rhCCK-91) building block. 7.4It was found that the value could be improved by about half an order of magnitude, but the CCK-2R affinity was significantly reduced. When SiFA was replaced with a SiFAlin moiety, the same CCK-2R affinity was observed for the (PEG)7 linker-containing derivative. However, no improvement was observed in lipophilicity. nat / 177 Lu]Lu-DOTA-rhCCK-75 (PEG) 11 (PEG) comprising a linker and a SiFAlin moiety 11

[0043] including 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) 50 The values ​​of the hydroxyl groups (19.2±1.6) and lipophilicity (-1.15±0.05) were also observed. nat / 177 Lu]Lu-DOTA-rhCCK-67,[ nat / 177 The SiFAlin moiety ([ Lu]Lu-DOTA-rhCCK-68) nat / 177 Lu]Lu-DOTA-rhCCK-90,[ nat / 177 Exchange of DOTA-rhCCK-91 with [Lu]Lu-DOTA-rhCCK-91 had little effect on lipophilicity but improved CCK-2R affinity. nat Lu] Lutetium (IC 50 =8.56±0.7) and [ nat Ga] Gallium (IC 50 = 8.24 ± 1.6) and showed high CCK-2R affinity in contrast to previously evaluated rhCCK derivatives.

[0312] Another approach was to investigate the lipophilicity and CCK-2R affinity of rhCCK derivatives via a polyhydroxyproline linker. To this end, compounds containing three to eight hydroxyproline moieties in the linker structure (DOTA-rhCCK-83, -84, and -86) were evaluated in vitro. Furthermore, a peptide containing three hydroxyproline and three D-citrulline moieties (DOTA-rhCCK-85) was also tested in vitro. Of this series, [ nat / 177 Lu]Lu-DOTA-rhCCK-84 exhibited the highest hydrophilicity (logD 7.4 =-2.14±0.06) and CCK-2R affinity (IC 50 =7.87±0.3).

[0313] Not surprisingly, HSA binding (84.7–94.5%) was high for all rhCCK derivatives tested (Figure 18), which was expected since increased HSA binding had already been observed for compounds containing other silicon-based fluoride acceptors, regardless of the type of target molecule used.

[0314] Overall, the most compelling in vitro data was [ 177 Lu]Lu-DOTA-rhCCK-70,[ 177 Lu]Lu-DOTA-rhCCK-84, and [ 177 Lu]Lu-DOTA-rhCCK-91 was evaluated in vivo 24 hours after injection and [ 177 Lu]Lu-DOTA-CCK-66 and [ 177 Lu]Lu-DOTA-MGS5 (Figure 19). [ 177 Lu]Lu-DOTA-rhCCK-84 showed the highest activity level in tumors 24 hours after injection ([ 177 Lu]Lu-DOTA-CCK-66 and [ 177[Lu]Lu-DOTA-MGS5 (approximately 2-fold higher, respectively). Kidney activity levels were slightly higher (<10%ID / g) compared to non-silicon-based fluoride acceptor-containing references. The overall biodistribution pattern was promising, supported by μSPECT / CT imaging, particularly in the context of previously developed silicon-based fluoride acceptor-containing minigastrin derivatives, where the presence of multiple negative charges in close proximity to the silicon-based fluoride acceptor component resulted in significantly increased retention of activity in the kidney (>80%ID / g 24 hours after injection) (Figure 20). Furthermore, it should be mentioned that activity accumulation and retention in the mouse kidney are not directly comparable to the human situation. Therefore, whether this slowdown in kidney clearance is also observed in humans and whether it is of concern requires further investigation in future patient studies. Apart from the significant increase in intratumoral activity levels at 24 h after injection, DOTA-rhCCK-84 (over DOTA-CCK-66 and DOTA-MGS5) has the advantage of being easily labeled with fluorine-18, which is beneficial for high-contrast positron emission tomography.

[0315] In addition to good tumor uptake (36.3±3.7%ID / g), the structure is identical [ 18 F]F-[ nat Biodistribution studies of [Lu]Lu-DOTA-rhCCK-84 revealed high activity levels in the blood (11.3±1.3%ID / g) 1 hour after injection (FIG. 21). However, biodistribution studies at 2 and 4 hours after injection revealed significant loss of [Lu]Lu from the blood over time. 177 A good clearance of the activity of [Lu]Lu-DOTA-rhCCK-84 was confirmed (3.71±0.46%ID / g and 1.14±0.18%ID / g, respectively), while the activity 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 A competition study using co-administration of [Lu]Lu-DOTA-MG-SiFA-23 significantly reduced the activity levels in both the stomach and tumor, thereby177 The CCK-2R specificity of [Lu]Lu-DOTA-rhCCK-84 was confirmed (Figure 22).

[0316] moreover,[ 177 The in vivo stability of [Lu]Lu-DOTA-rhCCK-84 was high in both serum (93.5±2.1%) and urine (55.4±9.0%) at 30 min after injection (Figure 23), especially compared with the reference compound DOTA-MGS5 (Table 6). In all aspects described above, this compound may be a promising candidate for clinical translation.

[0317] In addition to DOTA-rhCCK-84, [ 177 Lu]Lu-DOTA-rhCCK-70 also showed promising in vivo data. At 24 hours post-injection, the activity level in the tumor was [ 177 Lu] was slightly higher than Lu-DOTA-MGS5, but [ 177 The activity level in the kidney was significantly lower than that of [Lu]Lu-DOTA-rhCCK-84 (Figure 19). 177 [Lu]Lu-DOTA-rhCCK-84, but still higher than the non-silicon-based fluoride acceptor-containing reference. 177 The increased activity level of [Lu]Lu-DOTA-rhCCK-70 was due, in part, to the 177 This may be due to incomplete Lu labeling (approximately 5% of the free lutetium-177 injected into the animals), but also to the increased lipophilicity of this compound compared to non-silicon-based fluoride acceptor-containing ligands. 177 Lu]Lu-DOTA-rhCCK-91 had the lowest activity level in the kidney 24 hours after injection among rh-based CCK-2R-targeting compounds, but also reduced activity levels in the tumor.

[0318] in conclusion,[ 177 The biodistribution profiles of [Lu]Lu-DOTA-rhCCK-70, -84, and -91 were 177The results confirmed the present hypothesis that the high uptake and retention in the kidney (e.g., compared to [Lu]Lu-DOTA-MG-SiFA-23) can be addressed by reducing the negative charge in the vicinity of the silicon-based fluoride acceptor moiety. Furthermore, a novel design of PEGylated rhCCK ligands yielded a promising peptide, DOTA-rhCCK-84, which exhibits [ 177 Lu]Lu-DOTA-MGS5 or [ 177 Lu]Lu-DOTA-CCK-66 showed significantly higher levels of activity in tumors 24 hours after injection. Furthermore, this rh-based compound 18 It offers the possibility of easy and rapid F-labeling and showed significantly increased activity accumulation in tumors 1, 2, and 4 hours after injection, making this ligand a viable candidate for therapeutic and diagnostic applications with fluorine-18 and lutetium-177 in humans.

[0319] Example 2 - CCK2R targeting compounds including SiFA 2.1. Overview of synthesized CCK-2R-targeting ligands

[0320] [ka]

[0321] DOTA-PP-F11N(3):RP-HPLC (10→90% MeCN in H2O containing 0.1% TFA, 15 min, λ=220nm):t R =7.6 minutes, K'=3.5;MS(ESI, positive):C 90 H 123 N 19 O 35 Calculated value: m / z = 2029.8, Measured value: m / z = 1016.4 [M+2H] 2+ .

[0322] [ka]

[0323] CP04(4): RP-HPLC (10→90% MeCN in H2O containing 0.1% TFA, 15 min, λ=220 nm): t R =7.2 minutes, K'=3.3;MS(ESI, positive):C 89 H 121 N 19 O 35 Calculated value of S: m / z = 2047.8, Measured value: m / z = 1024.2 [M+2H] 2+ .

[0324] In the following, the array 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 -NH2 is defined as F11N. MG-SiFA compounds containing polyglutamic acid linkers In the following, the array 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 -NH2 is defined as F11N.

[0325] [ka]

[0326] DOTAGA-glu-[dap(SiFA) 6]F11N(DOTAGA-MG-SiFA-5):RP-HPLC(0.1% TFA containing 10→90% MeCN in H2O, 15 min, λ=220 nm):t R =10.2 min, K'=5.0 MS (ESI, positive): C 111 H 154 FN 21 O 39 Si's calculated value: m / z=2452.0, measured value: m / z=1226.8[M+2H] 2+ .

[0327]

change

[0328] DOTAGA-glu-[dap(SiFA) 5 ]F11N(DOTAGA-MG-SiFA-6):RP-HPLC (0.1% TFA containing 10→90% MeCN in H2O, 15 min, λ=220 nm)t R =9.6 minutes, K'=4.7; MS (ESI, positive): C 111 H 154 FN 21 O 39 Si's calculated value: m / z=2452.0, measured value: m / z=1226.4[M+2H] 2+ .

[0329]

change

[0330] DOTAGA-glu-[dap(SiFA) 4 ]F11N(DOTAGA-MG-SiFA-7):RP-HPLC(0.1% TFA containing 10→90% MeCN in H2O, 15 min, λ=220 nm):t R =9.3 minutes, K'=5.5; MS (ESI, positive): C 111 H 154 FN 21 O 39 Si's calculated value: m / z=2452.0, measured value: m / z=1226.4[M+2H] 2+ .

[0331] [ka]

[0332] DOTAGA-glu-[dap(SiFA) 3 ]F11N(DOTAGA-MG-SiFA-8):RP-HPLC (10→90% MeCN in H2O containing 0.1% TFA, 15 min, λ=220nm):t R =9.5 minutes, K'=4.6;MS(ESI, positive):C 111 H 154 FN 21 O 39 Si calculated value: m / z = 2452.0, measured value: m / z = 1226.7 [M+2H] 2+

[0333] [ka]

[0334] DOTAGA-glu-[dap(SiFA) 2 ]F11N(DOTAGA-MG-SiFA-9): Analytical RP-HPLC (10→90% MeCN in H2O containing 0.1% TFA, 15 min, λ=220 nm): t R =9.1 min, K'=4.4;MS(ESI, positive):C 111 H 154 FN 21 O 39 Si calculated value: m / z = 2452.0, measured value: m / z = 1227.3 [M+2H] 2+ .

[0335] [ka]

[0336] DOTAGA-glu-[dap(SiFA) 1]F11N(DOTAGA-MG-SiFA-10):RP-HPLC (10→90% MeCN in H2O containing 0.1% TFA, 15 min, λ=220nm):t R =9.2 minutes, K'=4.5;MS(ESI, positive):C 111 H 154 FN 21 O 39 Si calculated value: m / z = 2452.0, measured value: m / z = 1227.4 [M+2H] 2+

[0337] [ka]

[0338] DOTAGA-dap(SiFA)-F11N(DOTAGA-MG-SiFA-11):RP-HPLC(10→90% MeCN in H2O containing 0.1% TFA, 15 min, λ=220nm):t R =9.6 minutes, K'=5.6;MS(ESI, positive):C 111 H 154 FN 21 O 39 Si calculated value: m / z = 2452.0, measured value: m / z = 1227.0 [M+2H] 2+ .

[0339] 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 -NH2 is defined as γ-F11N.

[0340] [ka]

[0341] DOTA-PP-γ-F11N: RP-HPLC (0.1% TFA containing 10→90% MeCN in H2O, 15 min, λ=220 nm): t R =7.1min, K'=3.2; MS (ESI, positive): C 90 H 123 N 19 O 35 Calculated value: m / z=2029.8, measured value: m / z=1015.7[M+2H] 2+ .

[0342]

change

[0343] DOTAGA-γ-glu-[dap(SiFA) 6 ]γ-F11N(DOTAGA-MG-SiFA-13): Analytical by RP-HPLC (0.1% TFA containing 10→90% MeCN in H2O, 15 min, λ=220 nm): t R =10.1min, K'=6.1MS(ESI, positive):C 111 H 154 FN 21 O 39 Si's calculated value: m / z=2452.0, measured value: m / z=1227.2[M+2H] 2+ .

[0344]

change

[0345] DOTAGA-γ-glu-[dap(SiFA) 5 ]γ-F11N(DOTAGA-MG-SiFA-14):RP-HPLC (0.1% TFA containing 10→90% MeCN in H2O, 15 min, λ=220 nm)t R =9.6 minutes, K'=5.7; MS (ESI, positive): C 111 H 154 FN 21 O 39 Si's calculated value: m / z=2452.0, measured value: m / z=1227.3[M+2H]2+ .

[0346]

change

[0347] DOTAGA-γ-glu-[dap(SiFA) 4 ]γ-F11N(DOTAGA-MG-SiFA-15):RP-HPLC(0.1% TFA containing 10→90% MeCN in H2O, 15 min, λ=220 nm):t R =9.3 minutes, K'=5.5; MS (ESI, positive): C 111 H 154 FN 21 O 39 Si's calculated value: m / z=2452.0, measured value: m / z=1226.4[M+2H] 2+ .

[0348]

change

[0349] DOTAGA-γ-glu-[dap(SiFA) 3 ]γ-F11N(DOTAGA-MG-SiFA-16):RP-HPLC(0.1% TFA containing 10→90% MeCN in H2O, 15 min, λ=220 nm):t R =9.1min, K'=5.4; MS (ESI, positive): C 111 H 154 FN 21 O 39 Si's calculated value; m / z=2452.0, measured value: m / z=1226.7[M+2H] 2+ .

[0350]

change

[0351] DOTAGA-γ-glu-[dap(SiFA) 2]γ-F11N(DOTAGA-MG-SiFA-17):RP-HPLC(0.1% TFA containing 10→90% MeCN in H2O, 15 min, λ=220 nm):t R =9.5min, K'=5.6; MS (ESI, positive): C 111 H 154 FN 21 O 39 Si's calculated value: m / z=2452.0, measured value: m / z=1226.6[M+2H] 2+ .

[0352]

change

[0353] DOTAGA-γ-glu-[dap(SiFA) 1 ]γ-F11N(DOTAGA-MG-SiFA-18):RP-HPLC(0.1% TFA containing 10→90% MeCN in H2O, 15 min, λ=220 nm):t R =9.6 minutes, K'=5.7; MS (ESI, positive): C 111 H 154 FN 21 O 39 Si's calculated value: m / z=2452.0, measured value: m / z=1227.2[M+2H] 2+ 、817.8[M+3H] 3+ .

[0354]

change

[0355] DOTAGA-dap(SiFA)-γ-F11N(DOTAGA-MG-SiFA-19): RP-HPLC (0.1% TFA containing 10→90% MeCN in H2O, 15 min, λ=220 nm): t R =9.5min, K'=5.6; MS (ESI, positive): C 111 H 154 FN 21 O 39 Si's calculated value: m / z=2452.0, measured value: m / z=1227.3[M+2H]2+ 、818.7[M+3H] 3+ .

[0356]

change

[0357] DOTA-MG-SiFA-19: RP-HPLC (0.1% TFA containing 10→90% MeCN in H2O, 15 min, λ=220 nm): t R =10.0 min; MS (ESI, positive): C 108 H 150 FN 21 O 57 Si's calculated value: m / z=2380.0, measured value: m / z=1190.9[M+2H] 2+ 、794.4[M+3H] 3+

[0358]

change

[0359] DOTAGA-dap(SiFA)-glu-F11N(DOTAGA-MG-SiFA-20):RP-HPLC(0.1% TFA containing 10→90% MeCN in H2O, 15 min, λ=220 nm):t R =9.6 minutes, K'=4.7; MS (ESI, positive): C 116 H 161 FN 22 O 42 Si's calculated value: m / z=2581.1, measured value: m / z=1292.0[M+2H] 2+ .

[0360]

change

[0361] DOTAGA-dap(SiFA)-(glu)2-F11N (DOTAGA-MG-SiFA-21): analyzed by RP-HPLC (0.1% TFA containing 10→90% MeCN in H2O, 15 min, λ=220 nm): t R =9.4 minutes, K'=4.6; MS (ESI, positive): C 122 H 169 FN 22 O 45 Si's calculated value: m / z=2710.1, measured value: m / z=1355.9[M+2H] 2+ .

[0362]

change

[0363] DOTAGA-dap(SiFA)-γ-glu-γ-F11N(DOTAGA-MG-SiFA-22): analyzed by RP-HPLC (0.1% TFA containing 10→90% MeCN in H2O, 15 min, λ=220 nm): t R =9.4 minutes, K'=4.6; MS (ESI, positive): C 116 H 161 FN 22 O 42 Si's calculated value: m / z=2581.1, measured value: m / z=1291.8[M+2H] 2+ 、861.6[M+3H] 3+ .

[0364]

change

[0365] DOTAGA-dap(SiFA)-(γ-glu)2-γ-F11N (DOTAGA-MG-SiFA-23): analyzed by RP-HPLC (0.1% TFA containing 10→90% MeCN in H2O, 15 min, λ=220 nm): t R =9.3 minutes, K'=4.5; MS (ESI, positive): C 121 H 168 FN 23 O 45Si's calculated value: m / z=2710.1, measured value: m / z=1356.1[M+2H] 2+ 、904.2[M+3H] 3+ .

[0366]

change

[0367] DOTA-MG-SiFA-23: RP-HPLC (0.1% TFA containing 10→90% MeCN in H2O, 15 min, λ=220 nm): t R =9.8 min; MS (ESI, positive): C 118 H 164 FN 23 O 43 Si's calculated value: m / z=2638.1, measured value: m / z=1320.3[M+2H] 2+ 、880.5[M+3H] 3+ .

[0368]

change

[0369] DOTAGA-(γ-glu)2-[dap(SiFA) 2 ]γ-F11N(DOTAGA-MG-SiFA-24): Analytical by RP-HPLC (0.1% TFA containing 10→90% MeCN in H2O, 15 min, λ=220 nm): t R =9.4 minutes, K'=4.6; MS (ESI, positive): C 116 H 161 FN 22 O 42 Si's calculated value: m / z=2581.1, measured value: m / z=1292.5[M+2H] 2+ 、862.0[M+3H] 3+ .

[0370]

change

[0371] DOTAGA-(γ-glu)3-[dap(SiFA) 2 ]γ-F11N(DOTAGA-MG-SiFA-25): Analytical by RP-HPLC (0.1% TFA containing 10→90% MeCN in H2O, 15 min, λ=220 nm): t R =9.3 minutes, K'=4.5; MS (ESI, positive): C 122 H 169 FN 22 O 45 Si's calculated value: m / z=2710.1, measured value: m / z=1357.1[M+2H] 2+ 、905.1[M+3H] 3+ .

[0372]

change

[0373] DOTAGA-(γ-glu)4-[dap(SiFA) 1 ]γ-F11N(DOTAGA-MG-SiFA-26):RP-HPLC(0.1% TFA containing 10→90% MeCN in H2O, 15 min, λ=220 nm):t R =9.2 minutes, K'=4.5; MS (ESI, positive): C 126 H 175 FN 24 O 48 Si's calculated value: m / z=2839.2, measured value: m / z=1422.4[M+2H] 2+ 、948.3[M+3H] 3+ .

[0374]

change

[0375] DOTA-dap(SiFA)-γ-orn-(γ-glu)5-F11N(DOTA-MG-SiFA-27): RP-HPLC (0.1% TFA containing 10→90% MeCN in H2O, 15 min, λ=220 nm): t R =9.5 min; MS (ESI, positive): C 108 H 153FN 23 O 35 Calculated value of Si: m / z = 2366.1, Measured value m / z: 1183.8 [M+2H] 2+ , 789.6[M+3H] 3+ .

[0376] [ka]

[0377] DOTA-dap(SiFA)-γ-orn-(γ-glu)7-F11N(DOTA-MG-SiFA-28):RP-HPLC (10→70% MeCN in H2O containing 0.1% TFA, 15 min, λ=220nm):t R =11.5 minutes; MS(ESI, positive):C 118 H 167 FN 24 O 41 Si calculated value: m / z = 2623.2, measured value: m / z = 1312.6 [M+2H] 2+ .

[0378] [ka]

[0379] DOTA-dap(SiFA)-glutamol-(γ-glu)7-F11N(DOTA-MG-SiFA-29):RP-HPLC (10→70% MeCN in H2O containing 0.1% TFA, 15 min, λ=220nm):t R =11.5 minutes; MS(ESI, positive):C 118 H 166 FN 23 O 42 Si calculated value: m / z = 2625.8, measured value: m / z = 1313.6 [M+2H] 2+ , 876.1[M+3H] 3+ .

[0380] [ka]

[0381] DOTA-dap(SiFA)-γ-lys-(γ-glu)7-F11N(DOTA-MG-SiFA-30): RP-HPLC (0.1% TFA containing 10→70% MeCN in H2O, 15 min, λ=220 nm): t R =11.4 min; MS (ESI, positive): C 119 H 169 FN 24 O 41 Si's calculated value: m / z=2637.2, measured value: m / z=1319.5[M+2H] 2+ 、880.1[M+3H] 3+ .

[0382]

change

[0383] DOTA-γ-lys-dap(SiFA)-γ-lys-(γ-glu)7-F11N(DOTA-MG-SiFA-31):RP-HPLC (0.1% TFA containing 10→70% MeCN in H2O, 15 min, λ=220 nm):t R =11.0 min; MS (ESI, positive): C 125 H 181 FN 26 O 42 Si's calculated value: m / z=2767.1, measured value: m / z=1383.7[M+2H] 2+ 、922.9[M+3H] 3+ .

[0384]

change

[0385] DOTA-dap(SiFA)-(γ-glu)8-MGS5(DOTA-MG-SiFA-40):RP-HPLC (0.1% TFA containing 10→70% MeCN in H2O, 15 min, λ=220 nm):t R =12.5 min; MS (ESI, positive): C 123 H 168 FN 23 O 43Si calculated value: m / z = 2702.1, measured value: m / z = 1352.8 [M+2H] 2+ .

[0386] [ka]

[0387] DOTA(-dap(SiFA)-GABA-(γ-glu)7-F11N(DOTA-MG-SiFA-44):RP-HPLC(10→70% MeCN in H2O containing 0.1% TFA, 15 min, λ=220nm):t R =11.8 minutes;MS(ESI, positive):C 117 H 164 FN 23 O 41 Si calculated value: m / z = 2594.1, measured value: m / z = 1298.9 [M+2H] 2+ , 866.3[M+3H] 3+ .

[0388] [ka]

[0389] DOTA-dap(SiFA)-(GABA)2-(γ-glu)6-F11N(DOTA-MG-SiFA-45):RP-HPLC (10→70% MeCN in H2O containing 0.1% TFA, 15 min, λ=220nm):t R =11.8 minutes;MS(ESI, positive):C 116 H 164 FN 23 O 39 Si calculated value: m / z = 2550.1, measured value: m / z = 1277.0 [M+2H] 2+ , 851.6[M+3H] 3+ .

[0390] [ka]

[0391] DOTA-dap(SiFA)-(GABA)3-(γ-glu)5-F11N(DOTA-MG-SiFA-46):RP-HPLC (10→70% MeCN in H2O containing 0.1% TFA, 15 min, λ=220nm):t R =11.9 minutes;MS(ESI, positive):C 115 H 164 FN 23 O 37 Si calculated value: m / z = 2507.8, measured value: m / z = 1254.5 [M+2H] 2+ , 836.7[M+3H] 3+ .

[0392] [ka]

[0393] DOTA-dap(SiFA)-(GABA-γ-glu)4-F11N(DOTA-MG-SiFA-47):RP-HPLC (10→70% MeCN in H2O containing 0.1% TFA, 15 min, λ=220nm):t R =11.8 minutes;MS(ESI, positive):C 114 H 164 FN 23 O 35 Si calculated value: m / z = 2462.2, measured value: m / z = 1232.5 [M+2H] 2+ , 822.0[M+3H] 3+ . 2.2. In Vitro and In Vivo Results of MG-SiFA Compounds Containing Polyglutamic Acid Linkers Several MG-SiFA compounds have been synthesized and shown to be nat / 177 Lu labeling was assessed. CCK-2R affinity (IC) in AR4-2J cells 50 ) and lipophilicity (logD 7.4 ) was determined. nat / 177 Lu-DOTA-PP-F11N and nat / 177 Lu-CP04( nat / 177 Lu-DOTA-(glu)6-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2) was also synthesized and evaluated similarly (Table 2).

[0394] [Table 9-1]

[0395] [Table 9-2]

[0396] Generally, minigastrin analogs containing poly-γ-glu linkers ([ nat Lu]Lu-DOTAGA-MG-SiFA13-19) is a nucleotide analogue of the counterpart containing the exact amount of α-glu residues in the linker ([ nat Lu]Lu-DOTAGA-MG-SiFA5-11), which showed improved CCK-2R affinity. nat This was also observed in [Lu]Lu-DOTA-PP-γ-F11N. nat 1.7-fold lower IC than Lu]Lu-DOTA-PP-F11N 50 This is because the binding sequence (Ala 7 -Tyr 8 -Gly 9 -Trp 10 -Nle 11 -Asp 12 -Phe 13 For the MG derivatives containing the dap(SiFA) moiety, the enhanced separation of the chelator moiety as well as the dap(SiFA) moiety was suggested to be due to the significantly longer backbone of each peptide due to the use of γ-glu instead of α-glu. For compounds containing the dap(SiFA) moiety, the highest CCK-2R affinity was observed when this group was separated by at least five or six α / γ-glu residues. Not surprisingly, the SiFA-containing MG derivatives, which additionally contain the DOTAGA chelator [ nat [Lu]Lu-DOTAGA-MG-SiFA-19 showed the highest CCK-2R affinity. Interestingly, the γ-glu moiety ([ natAddition of [Lu]Lu-DOTAGA-MG-SiFA-22 / -23) did not improve CCK-2R affinity. nat When DOTAGA in [Lu]Lu-DOTAGA-MG-SiFA-23 was replaced with DOTA, the CCK-2R affinity was significantly increased ([ nat Lu]Lu-DOTA-MG-SiFA-23: 4.71 ± 0.62 nM), outperforming the reference ligand. It is assumed that the receptor prefers a neutral overall charge on the chelate at each site. nat Similar observations were made for the substitution of DOTAGA with DOTA in [Lu]Lu-DOTAGA-MG-SiFA-19, which resulted in IC 50 The value decreased threefold.

[0397] Naturally, 177 All Lu-labeled MG-SiFA ligands also exhibited higher lipophilicity compared to non-SiFA-containing reference ligands. However, as mentioned above, this higher hydrophilicity has been suggested to prevent high tumor accumulation, resulting in rapid clearance of the administered active agent into the bladder. For this reason, 177 It is assumed that the reduced hydrophilicity of the Lu-labeled MG-SiFA conjugates leads to a longer circulation time in vivo, thereby promoting tumor uptake. 177 Most of the Lu-labeled MG-SiFA compounds exhibited logD values ​​in the range of -3 to -2. 7.4 reported a pharmacophore-modified CCK-2R targeting ligand, which showed log D values ​​in a similar range to the MG-SiFA conjugates of the present invention. 68 Ga]Ga- / [ 111 In]In- / [ 177 Lu]Lu-DOTA-MGS5 and [ 99m [Tc]Tc-HYNIC-MGS11 showed excessively high tumor accumulation [30,34]. Therefore, this lipophilicity range seems perfectly suitable for derivatives directed against CCK-2R.

[0398] For better comparison, the most promising DOTAGA-containing conjugates, containing poly-α-glutamic acid and poly-γ-glutamic acid linkers, respectively, were selected for further testing in vivo. 177 Lu]Lu-DOTAGA-MG-SiFA-21 and [ 177 Biodistribution studies were performed using both [Lu]Lu-DOTAGA-MG-SiFA-19 at 24 hours after injection, and the reference [ 177 Lu]Lu-DOTA-PP-F11N (Figure 24).

[0399] Furthermore, it showed higher CCK-2R affinity than the reference compound [ 177 Lu]Lu-DOTA-MG-SiFA-23 was evaluated in vivo at 1 and 24 hours after injection (Figure 25).

[0400] As mentioned above, [ nat Compared with [Lu]Lu-DOTA-PP-F11N, [ nat Lu]Lu-DOTAGA-MG-SiFA-21 was approximately 5 times, [ nat Lu]Lu-DOTAGA-MG-SiFA-19 increased IC by approximately 2-fold 50 indicates the value, while [ nat Lu]Lu-DOTA-MG-SiFA-23 is [ nat Lu]Lu-DOTA-PP-F11N than IC 50 The values ​​were almost three times lower. All three MG-SiFA derivatives showed significantly higher lipophilicity than the reference ligand. Nevertheless, 177 Lu]Lu-DOTA-MG-SiFA-23 showed the highest activity level (>25%ID / g) in tumors 24 hours after injection, which was 177 Lu]Lu-DOTA-PP-F11N was over 8-fold higher. Interestingly, despite its lower affinity for CCK2R, the DOTAGA-MG-SiFA compound showed a 3- to 6-fold increase in activity in tumors 24 hours after injection, supporting our hypothesis of the benefit of slowed activity clearance

[35] . The second reference ligand, [ 177Lu]Lu-CP04 is 177 Structurally closely related to [Lu]Lu-DOTA-PP-F11N, which has been reported to exhibit 0.63±0.08% ID / g in tumor tissue 24 hours after injection in A431-CCK-2R(+) tumor-bearing mice, further supporting our data

[36] . Improved tumor uptake and slower clearance over time were also observed for the DOTAGA-conjugated MG-SIFA compound at 1, 4, and 24 hours after injection (Figure 26) and [Lu]Lu-DOTA-PP-F11N. 177 The effect of [Lu]Lu-DOTA-MG-SiFA-23 was highlighted by μSPECT / CT imaging at 1 and 24 hours after injection (Figure 27). 177 Lu]Lu-DOTAGA-MG-SiFA-19 and [ 177 The accumulation of both [Lu]Lu-DOTA-MG-SiFA-23 in CCK-2R-positive tumors and the stomach was specific, and was due to excessive [ nat This was demonstrated by a competition study in which [Lu]Lu-DOTA-MGS5 was co-administered (Figure 28).

[0401] Furthermore, all MG-SiFA ligands demonstrated significantly higher tumor accumulation at 1 hour post-injection compared to the reference, which is important for the potential use of these compounds for fluorine-18 labeling and imaging. The presence of the chelator and SiFA moieties allows for nat Lu / 18 F label and 177 Lu / 19 The biodistribution profiles of the F-labeled MG-SiFA ligands are expected to be very similar or identical. Activity levels in the stomach are also higher for all MG-SiFA ligands compared to the reference, as expected since the stomach is a CCK-2R-positive organ. Interestingly, activity levels in the liver are significantly higher than those in the control, despite increased lipophilicity. 177The activity of the SiFA moiety was relatively low compared to that of the [Lu]Lu-DOTA-PP-F11N. This suggests that the introduction of the SiFA moiety into the minigastrin analogs is appropriate. However, it is worth noting that all three MG-SiFA compounds showed significantly elevated kidney activity levels (>80% ID / g) 24 hours after injection, which was not observed with the reference compounds. While this observation remains unexplained, it is speculated that an unknown synergistic effect of the SiFA moiety and the negative charge in the linker may have increased kidney uptake and retention. However, 18 In the case of F-labeling, this issue is less important due to the short physical half-life of this isotope.

[0402] Nevertheless, we attempted to address this issue by reducing or separating the negative charge in the vicinity of the dap (SiFA) component. The N-terminal γ-glu moiety was replaced with either γ-orn (DOTA-MG-SiFA-27 / -28), glutamol (DOTA-MG-SiFA-29), or γ-lys (DOTA-MG-SiFA-30 / -31). Interestingly, these substitutions significantly reduced both CCK-2R affinity and lipophilicity. These compounds ([ 177 Each of [Lu]Lu-DOTA-MG-SiFA-27-31) 177 Lu]Lu-DOTA-MG-SiFA-23 7.4 The values ​​were found to be almost one order of magnitude higher (Table 9). 177 IC of [Lu]Lu-DOTA-MG-SiFA-27 and -29 50 The value improved slightly, but 177 The charge distribution in the N-terminal region is very delicate, and even small changes can have a significant effect.

[0403] Based on these results, it was decided to reduce the amount of negative charge by substituting one or more γ-glu moieties with γ-aminobutyric acid (GABA) moieties to reduce the negative charge while maintaining the backbone length, resulting in four compounds: DOTA-MG-SiFA-44-47. 177 The Lu-labeled compounds each have a similar logD 7.4 value, which is 177 The CCK-2R affinity was more than half an order of magnitude higher than that of [Lu]Lu-DOTA-MG-SiFA-23 (Table 9). 177 Lu]Lu-DOTA-MG-SiFA-23, which was surprising because the negative charge was thought to be important for high CCK-2R affinity. Nevertheless, due to increased lipophilicity, GABA-containing MG-SiFA compounds have not been evaluated in vivo.

[0404] In conclusion, the functionality of adding a silicon-based fluoride acceptor moiety to the linker section of a minigastrin derivative has been demonstrated. The resulting MG-SiFA compound is a promising candidate for imaging MTC by PET. 18 This allows for easy and rapid F-labeling. Furthermore, the addition of the lipophilic SiFA moiety significantly improved overall pharmacokinetics by slowing down activity clearance, resulting in significantly increased activity levels in the tumor at 1 and 24 hours after injection. Although assessed at 24 hours after injection, these MG-SiFA ligands are not recommended for targeted radiotherapy with lutetium-177 due to their high renal retention. However, 18 When F-labeled, this increased renal uptake is less of a concern because PSMA inhibitors generally show higher uptake values ​​1 hour after injection. The diagnostic value of DOTA-MG-SiFA-23 is 177 Lu- and 18Both F-labeling were confirmed by biodistribution and imaging studies (Figures 22 and 24) at 1 hour after injection, making this compound highly promising for future clinical use for MTC imaging.

[0405] Example 3 - Materials and Methods General Fmoc-(9-fluorenylmethoxycarbonyl-) and other protected amino acid analogs were all purchased from Bachem Inc. (Bubendorf, Switzerland), Sigma-Aldrich GmbH (Munich, Germany), or Iris Biotech GmbH (Marktredwitz, Germany). H-Rink amide ChemMatrix® resin (35-100 mesh particle size, 0.4-0.6 mmol / g loading) was purchased from Sigma-Aldrich GmbH (Munich, Germany). CheMatech (Dijon, France) purchased the chelating agent DOTA ( t Bu)3 and DOTAGA( t Peptide syringes were obtained from VWR International GmbH (Bruchsal, Germany).

[0406] All necessary solvents and other organic reagents were purchased from either Alfa Aesar (Karlsruhe, Germany), Sigma-Aldrich GmbH (Munich, Germany), or VWR International GmbH (Bruchsal, Germany). Solid-phase peptide synthesis was performed manually using a Scilogex MX-RL-E Analog Rotisserie Tube Rotator (Scilogex, Rocky Hill, CT, USA). HO was used after purification using a Barnstead MicroPure system (Thermo Fisher Scientific Inc., Waltham, MA, USA).

[0407] Analytical and preparative reversed-phase high-performance liquid chromatography (RP-HPLC) were performed using a Shimadzu gradient system (Shimadzu Deutschland GmbH, Neufahrn, Germany) equipped with an SPD-20A UV / Vis detector (220 nm, 254 nm), respectively. Different gradients of MeCN (0.1% TFA) in HO (0.1% TFA) were used as eluents for all RP-HPLC runs.

[0408] For analytical measurements, a Multokrom 100-5 C18 (5 μm, 125 × 4.6 mm) column (CS Chromatographie GmbH, Langerwehe, Germany) was used at a flow rate of 1 mL / min. Specific gradients and corresponding retention times t R , as well as the capacity factor K′, are quoted in the text.

[0409] Preparative RP-HPLC purification was performed using a MultoKrom 100-5 C18 (5 μm, 250 × 10 mm) column (CS Chromatographie GmbH, Langerwehe, Germany) at a constant flow rate of 10 mL / min.

[0410] Analytical and preparative radioactive RP-HPLC were performed using a MultoKrom 100-5 C18 (5 μm, 125 × 4.6 mm) column (CS Chromatographie GmbH, Langerwehe, Germany). Electrospray ionization mass spectrometry for the characterization of the materials was performed using the expression L Acquired on a CMS mass spectrometer (Advion Ltd., Harlow, UK).

[0411] Radioactive labels include [ 177 Lu]LuCl3 (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 UV-photometer outlet to an AceMate 925-Scint NaI(Tl) well-type scintillation counter from EG&G Ortec (Oak Ridge, TN, USA). The radioactive probe was WIZARD 2 The IC was measured using a 2480 Automatic γ-Counter (Perkin Elmer, Waltham, MA, USA). 50 Values ​​were determined using GraphPad Prism 6 (GraphPad Software Inc., San Diego, CA, USA). Radio-TLC was performed using a Scan-RAM™ scanner equipped with Laura™ software (LabLogic Systems Ltd., Broomhill, Sheffield, United Kingdom). NMR spectra were recorded on a Bruker (Billerica, United States) AVHD-300 or AVHD-400 spectrometer at 300 K. Freeze-drying was achieved using an Alpha 1-2 LDplus freeze dryer (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany) in combination with an RZ-2 vacuum pump (Vacuubrand GmbH & Co KG, Olching, Germany).

[0412] For in vitro and in vivo studies, RPMI 1640 medium (supplemented with 5 mM L-Gln, 5 mL of non-essential amino acids (100x), and 10% FCS) was used as the nutrient medium. For subculture, phosphate-buffered saline (PBS) containing 0.1% EDTA (v / v) was used. 2+ / Mg 2+Cells were detached using a solution containing 100% ethanol (free of ethanol). All mentioned solutions and fetal bovine serum (FBS Superior) were obtained from Biochrom GmbH (Berlin, Germany). Tracepur® H2O (Merck KGaA, Darmstadt, Germany) was used to dissolve the purified product. Bovine serum albumin (BSA) was purchased from Biowest (Nuaille, France).

[0413] Cells were cultured in CELLSTAR® cell culture flasks and counted using a Neubauer hemocytometer (Paul Marienfeld, Lauda-Konigschofen, Germany) using trypan blue (0.4% in 0.81% NaCl and 0.06% potassium phosphate) solution (Sigma-Aldrich GmbH, Munich, Germany) before seeding into 24-well plates (Greiner Bio-One GmbH, Kremsmunster, Austria). Cells were handled in a laminar flow hood at MSC Advantage and maintained at 37°C in a humidified 5% CO2 atmosphere in a Heracell 150i incubator (Thermo Fisher Scientific Inc., Waltham, MA, USA). CCK-2R-expressing rat pancreatic tumor cells, AR4-2J, were obtained from ECACC (European Collection of Cell Cultures, Salisbury, UK). 3.2. Synthesis Protocol 3.2.1 Solid-phase peptide synthesis using the Fmoc strategy On-resin peptide formation using DIPEA Each side-chain protected Fmoc-AA-OH (1.5 equiv.) was dissolved in NMP and preactivated by adding TBTU (1.5 equiv.), HOAt (1.5 equiv.), and DIPEA (4.5 equiv.). After 10 min of activation, the solution was added to the resin-bound free amine peptide and shaken at room temperature (rt) for 1.5 h. The resin was then washed with NMP (6 × 20 mL / g resin), and after Fmoc deprotection, the next amino acid was coupled in the same manner. On-resin peptide formation using 2,4,6-collidine For coupling of amino acids to the free N-terminus of peptides containing a dap (Dde) moiety or for coupling of dap (Dde) amino acids, 2,4,6-collidine was used as the base. Therefore, the respective side-chain-protected Fmoc-AA-OH (1.5 equiv.) was dissolved in NMP and preactivated by adding TBTU (1.5 equiv.), HOAt (1.5 equiv.), and 2,4,6-collidine (6.0 equiv.). After 10 min of activation, the solution was added to the resin-bound free amine peptide and shaken at RT for 1.5 h. The resin was then washed with NMP (6 × 20 mL / g resin), and after Fmoc deprotection, the next amino acid was coupled in the same manner. On-resin Fmoc deprotection The resin-bound Fmoc-peptide was treated with 20% piperidine / NMP (v / v) for 5 min, followed by 15 min, after which the resin was washed with NMP (6 x 20 mL / g resin). On-resin Fmoc deprotection of Fmoc-D-Cit-OH The resin-bound Fmoc-peptide was treated with 20% piperidine / NMP (v / v) for 15 min, and the solvent was filtered off. This procedure was repeated twice. The resin was then washed with NMP (6 × 20 mL / g resin). Dde deprotection on resin Dde deprotection was carried out by adding a solution of imidazole (75 equiv.), hydroxylamine hydrochloride (100 equiv.) in NMP (7 mL) and DCM (3 mL) to the resin and shaking for 2 h at rt. After deprotection was complete, the resin was washed with NMP (6 × 20 mL / g resin). Conjugation of chelating agents or SiFA moieties Protected chelating agent DOTA( t Bu)3 or DOTAGA( tThe (Bu)4 or SiFA moiety (1.5 equiv.) was dissolved in NMP and preactivated by adding TBTU (1.5 equiv.), HOAt (1.5 equiv.), and 2,4,6-collidine (6.0 equiv.). After 10 min of activation, the solution was added to the resin-bound N-terminally deprotected peptide (1.0 equiv.) and shaken at RT for 3 h. The resin was then washed with NMP (3 × 20 mL / g resin) and DCM (3 × 20 mL / g resin). Conjugation of SiFA-Br To generate the SiFAlin moiety, the resin was swollen with DCM. A solution (2 mL) of DIPEA (6.0 equiv.) and SiFA-Br (3.0 equiv.) in DCM was added to the resin and shaken overnight. The resin was washed with DCM (5 × 5 mL) and dried in a desiccator. SiFA-IPA conjugation After preactivation at rt for 10 min, the resin-bound peptide was treated with a solution of SiFA-IPA (3.0 equiv.) and HOAt (3.0 equiv.) in NMP (4 mL / g resin) and a solution of TBTU (3 equiv.) and 2,4,6-collidine (11.0 equiv.) in NMP (4 mL / g resin). The reaction mixture was shaken at rt for 3 h and then filtered. The resin was then washed with NMP (6 × 10 mL / g resin). Cleavage of the peptide from the resin and concomitant removal of acid-labile protecting groups The fully protected resin-bound peptide was washed with DCM and then dissolved in a mixture of TFA / TIPS / HO (v / v / v; 95 / 2.5 / 2.5) and shaken for 60 min. The solution was filtered off, and the resin was treated similarly for another 60 min. Both filtrates were combined and stirred at rt for another 6 h. After removing the TFA under a stream of nitrogen, the crude product was obtained. p-Nosyl protection The resin-bound peptide was dissolved in a solution of p-nosyl chloride (5.0 equiv.) and 2,4,6-collidine (10.0 equiv.) in NMP (10 mL / g resin) and shaken at RT for 30 min. After deprotection, the resin was washed with NMP (6 × 10 mL / g resin). p-Nosyl deprotection A solution of β-mercaptoethanol (10 equiv.) and DBU (5.0 equiv.) in NMP (10 mL / g resin) was added to the p-nosyl-protected resin-bound peptide and shaken twice for 15 min at RT, after which the resin was washed with NMP (6 × 10 mL / g resin). N-methylation The resin was dissolved in a solution of PPh3 (0.3 M in anhydrous THF, 5.0 equiv.) and MeOH (10 equiv.), and the resin was shaken at RT for 1 min. DIAD (5.0 equiv.) was then added, and the reaction mixture was shaken at RT for 30 min. This procedure was repeated, and the resin was then washed with NMP (6 × 10 mL / g resin). 3.2.2. Synthesis and Characterization of Silicon-Based Fluoride Acceptor Building Blocks The synthesis of each silicon-based fluoride acceptor building block (iv, v, and vii) was carried out according to a previously published procedure, slightly modified by our group. All water- and oxygen-sensitive reactions were carried out in a dry reaction vessel under an argon atmosphere using a vacuum gas manifold. ((4-bromobenzyl)oxy)(tert-butyl)dimethylsilane(i) To a stirred solution of 4-bromobenzyl alcohol (4.68 g, 25.0 mmol, 1.0 equiv) in anhydrous DMF (70 mL) was added imidazole (2.04 g, 30.0 mmol, 1.2 equiv) and TBDMSCl (4.52 g, 30.0 mmol, 1.2 equiv), and the resulting mixture was stirred at rt for 16 h. The mixture was then poured into ice-cold HO (250 mL) and extracted with EtO (5 × 50 mL). The combined organic fractions were washed with saturated aqueous NaHCO (2 × 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 / petroleum) to give i as a colorless oil (7.18 g, 95%).

[0414] RP HPLC (50→100% MeCN in H2O containing 0.1% TFA, 15 min, λ=220 nm):t R =15 min. K'=7.43. Di-tert-butyl[4-((tert-butyldimethylsilyloxy)methyl)phenyl]fluorosilane(II) With magnetic stirring at −78° C., 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 anhydrous THF (15 mL). After stirring the reaction mixture at −78° C. for 30 min, the resulting suspension was added dropwise over 30 min to a cooled (−78° C.) solution of di-tert-butyldifluorosilane (1.12 g, 6.23 mmol, 1.2 equiv.) in anhydrous THF (10 mL). The reaction mixture was warmed to rt over 12 h and then hydrolyzed with saturated aqueous NaCl (100 mL). The organic layer was separated, and the aqueous layer was extracted with diethyl ether (3×50 mL). The combined organic layers were dried over magnesium sulfate and filtered. The filtrate was concentrated in vacuo to give ii as a yellowish oil (1.88 g, 95%), which was used in the subsequent reaction without further purification. RP HPLC (50→100% MeCN in H2O containing 0.1% TFA, 15 min, λ=220 nm):t R =19 minutes. K'=9.67. 4-(di-tert-butylfluorosilanyl)benzyl alcohol (iii) 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 equiv) in methanol (50 mL). The reaction mixture was stirred at rt for 18 h, after which 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 aqueous NaHCO3. The aqueous layer was extracted with diethyl ether (3 × 50 mL). The combined organic layers were dried over magnesium sulfate and filtered. The filtrate was concentrated in vacuo to give iii as a yellowish oil (1.29 g, 98%) that solidified. This product was used without further purification. RP-HPLC (50→100% MeCN in H2O containing 0.1% TFA, 15 min, λ=220 nm):t R =8.2 minutes. K'=3.61. 4-(Di-tert-butylfluorosilyl)benzoic acid ((4-SiFA)Bz-OH, iv) At rt, 5.8 g of KMnO4 (36.7 mmol, 1.5 equiv.) was dissolved in HO and added to a solution of iii (6.61 g, 24.6 mmol, 1.0 equiv.), tert-butanol (65 mL), dichloromethane (9 mL), and 1.25 M NaH2PO4·HO buffer (36 mL) at pH 4.0–4.5. The mixture was stirred for 25 min, then cooled on ice for 10 min, and excess KMnO4 (7.8 g, 49.2 mmol, 2.0 equiv.) was added. The reaction mixture was stirred on ice for 2 h and then warmed to rt for 30 min. The reaction was then quenched by the addition of saturated aqueous Na2SO3 (50 mL). The addition of 2 M aqueous HCl dissolved all of the MnO2. The resulting solution was extracted with diethyl ether (3 × 100 mL). The combined organic layers were washed with saturated aqueous NaHCO3, dried over MgSO4, filtered and concentrated under reduced pressure to give a white solid, which was purified by recrystallization from Et2O / n-hexane (1:3, 12 h) to give iv (2.57 g, 37%). 1 H NMR (300 MHz, CDCl3): d [ppm] = 8.10 (d, 2H 3 J( 1 H, 1 H) = 8.1 Hz; H m ), 7.74 (d, 2H, 3 J( 1 H, 1 H) = 8.1 Hz; H o ), 1.07 (s, 18H; CCH3). 13 C{ 1 H} NMR (101 MHz, DMSO-D6): d [ppm] = 167.2 (s; COOH), 138.3 (d, 2 J( 13 C, 19 F) = 14 Hz; C p ), 133.8 (d, 3 J( 13 C, 19 F) = 4 Hz; Cm ), 132.1 (s; C i ), 128.3 (s; C o ), 26.9 (s; CCH3), 19.7 (d, 2 J( 13 C, 19 F) = 12 Hz; CCH3). 19 F{ 29 Si} NMR (376 MHz, DMSO-D6): d [ppm] = -187.2. 29 Si{ 1 H}INEPT NMR (79 MHz, DMSO-D6): d [ppm] = 14.1 (d, 1 J( 19 F, 29 Si) = 299 Hz). RP HPLC (50→100% MeCN in H2O containing 0.1% TFA, 15 min, λ=220 nm):t R = 8.5 min. K' = 3.78. ESI-MS (positive): monoisotopic mass (C 15 H 23 FOSi) calculated: 282.15; found: m / z = 283.2 [M+H] + , 265.2 [M-H2O+H] + .

[0415] [ka]

[0416] Scheme 1: Synthesis of (4-SiFA)Bz-OH(iv): a) TBDMSCl, imidazole, rt, 16 h (DMF); b) tBuLi, di-tert-butyldifluorosilane, -78 °C to rt, overnight (THF); c) HCl, rt, 18 h (MeOH); d) KMnO, 0 °C to rt, 2.5 h (DCM / tBuOH / NaH2PO4·H2O buffer). 4-(Di-tert-butylfluorosilanyl)benzyl bromide (4-SiFA)Bz-Br(v) 4-(Di-tert-butylfluorosilanyl)benzyl bromide was synthesized similarly to (4-SiFA)Bz-OH (iv). Only in the final step, 4-(di-tert-butylfluorosilanyl)benzyl alcohol (iii) (2.41 g, 8.96 mmol, 1.0 equiv.) was dissolved in anhydrous DCM (40 mL) and CBr4 (2.59 g, 9.86 mmol, 1.1 equiv.) was added under argon. The reaction mixture was then cooled to 0 °C, and PPh3 (3.27 g, 9.86 mmol, 1.1 equiv., dissolved in 30 mL of anhydrous DCM) was added dropwise over 30 min. The reaction mixture was then stirred at RT for 2 h, and the solvent was removed in vacuo. The crude product was purified by flash column chromatography (silica, 100% pentane). RP HPLC (50→100% MeCN in H2O containing 0.1% TFA, 15 min, λ=220 nm):t R =17.3 minutes.

[0417] [ka]

[0418] Scheme 2: Synthesis of (4-SiFA)Bz-Br(iv): a) TBDMSCl, imidazole, rt, 16 h (DMF); b) tBuLi, di-tert-butyldifluorosilane, -78 °C to rt, overnight (THF); c) HCl, rt, 18 h (MeOH); d) CBr4, PPh3, 0 °C to rt, 2.5 h (DCM). Di-tert-butyl(3,5-dimethylphenyl)fluorosilane(vi) A solution of 4.54 g of 1-bromo-3,5-dimethylbenzene (25.1 mmol, 1.0 equiv.) in 73.1 mL of anhydrous THF was cooled to -78 °C, and 34.7 mL of tBuLi (55.5 mmol, 1.6 M pentane solution, 2.2 equiv.) was added dropwise and stirred at -78 °C for 30 min. This reaction mixture was then added to a solution of 5.0 g of di-tert-butyldifluorosilane (27.7 mmol, 1.1 equiv.) in 49.1 mL of THF at -78 °C and stirred overnight while warming to rt under pressure control. The reaction was quenched by the addition of 100 mL of brine. The aqueous layer was extracted with EtO (3 × 100 mL), the combined organic phases were dried over MgSO, and the solvent was removed under reduced pressure to give 6.6 g of di-tert-butyl(3,5-dimethylphenyl)fluorosilane (vi, 24.8 mmol, 99%) as a colorless solid. 1 H NMR (500 MHz, CDCl3): d [ppm] = 7.19 (s, 2H; H o ), 7.04 (s, 1H; H p ), 2.33 (s, 6 H; CH3), 1.06 (s, 18 H; CCH3). RP HPLC (50–100% B for 15 min, 100% B for 10 min):t R =16.4 minutes. K'=8.21. 5-(di-tert-butylfluorosilyl)isophthalic acid ((5-SiFA)Ip-OH, vii) 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 NaH2PO4·HO solution (40.0 mmol, 2.5 M in HO, 10.0 equiv.). To this solution was added 7.6 g of KMnO4 (48.0 mmol, 12.0 equiv.) at rt, and the reaction was carefully heated in stages to 75 °C and stirred for 24 h. The reaction was quenched by adding saturated aqueous NaSO3 (50 mL). Concentrated aqueous HCl (10 mL) was added to completely dissolve the MnO2. The solution was extracted with Et2O (3 x 100 mL), the combined organic phases were dried over MgSO4, and the solvent was removed under reduced pressure to give ii (1.3 g, 4.0 mmol, 100%) as a colorless solid. 1 H NMR (400 MHz, DMSO-D6): d ppm = 8.53 (t, 1 H, 4 J( 1 H, 1 H) = 1.7 Hz; H Ar-2 ), 8.32 (d, 2 H, 4 J( 1 H, 1 H) = 1.6 Hz; H Ar-4,-6 ), 1.03 (s, 18 H; CH3). 13 C{ 1 H} NMR (101 MHz, DMSO-D6): d [ppm] = 166.5 (s; COOH), 137.9 (d, 3 J( 13 C, 19 F) = 4 Hz; C Ar-4,-6 ), 134.0 (d, 2 J( 13 C, 19 F) = 14 Hz; C Ar-5 ), 131.4 (s; C Ar-2 ), 130.8 (s; C Ar-1,-3 ), 26.8 (s; CCH3), 19.7 (d, 2 J( 13 C, 19F) = 12 Hz; CCH3). 19 F{ 29 Si} NMR (376 MHz, DMSO-D6): d [ppm] = -187.1. 29 Si{ 1 H}INEPT NMR (79 MHz, DMSO-D6): d [ppm] = 13.8 (d, 1 J( 19 F, 29 Si) = 299 Hz). RP-HPLC (50-100% B for 15 min): t R = 5.7 min. K' = 2.20. ESI-MS (positive): monoisotopic mass (C 16 H 23 FO4Si) calculated: 326.13; found: m / z = 327.2 [M+H] + , 309.2 [M-H2O+H] + .

[0419] [ka]

[0420] Scheme 3: Synthesis of (5-SiFA)Ip-OH(vii): a) tBu2SiF2, tBuLi, -78 °C to rt, overnight (THF) b) KMnO4, 75 °C, 24 h (DCM / tBuOH / NaH2PO4·H2O buffer solution) 3.3. Labeling experiments nat Lu-labeled: Purified chelator-containing ligand (10 -3 M, Tracepur® HO, 1.0 equivalent) and nat [Lu]LuCl3 (20 mM, Tracepur® HO, 2.5 equivalents) was diluted with Tracepur® HO to a final concentration of 10 -4 M and heated to 95° C. for 30 min. After cooling to rt, the crude product was obtained. nat Ga-labeled: natGa-complexation was achieved by stirring a solution of Ga(NO3)3 (20 μL, 10 mM, 2.0 equiv.), peptide precursor (80 μL, 1 mM), and Tracepur® HO (710 μL) at 70 °C for 30 min. nat Cu-labeled: CCK-2R ligands were prepared by incubating the CCK-2R ligands with Cu(OAc)2 solution (24 μL, 10 mM, 3.0 equiv.), peptide precursor (80 μL, 1 mM), and Tracepur® HO (694 μL) at 80 °C for 30 min. nat Cu] to form a complex with copper. [ 19 [F]AlF-labeling: AlCl (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) for 10 min at rt. The solution was then mixed with the labeled precursor (700 μL, 700 nmol, 10 -3 M 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 [ 19 F]AlF-NOTA-labeled peptides were purified by RP-HPLC. 177 Lu-label: 177 Lu-labeling was performed according to a procedure developed in our group. Therefore, purified chelator-containing ligand solution (10 -3 M, Tracepur® HO, 1 μL), NaOAc buffer (1 M, pH = 5.50, 10 μL) and approximately 10-30 MBq of [ 177 [Lu]LuCl3 (0.04 M, 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. 177Lu incorporation was determined by radio-TLC (ITLC-SG chromatography paper, mobile phase: 0.1 M trisodium citrate). The radiochemical purity of the labeled compounds was determined by radio-RP-HPLC. 67 Ga-labeling: A solution of the labeled precursor (1 μL, 1 nmol, 1 mM, DMSO) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (7 μL, 2.5 M, HEPES) was added to [ 67 [Ga]GaCl3 (10–30 MBq, Curium Germany GmbH, Berlin, Germany) was added and the reaction mixture was heated to 90 °C for 15 min. Radiochemical purity was determined using radio-RP-HPLC and radio-TLC. 64 Cu-labeling: A solution of the labeling precursor (1 μL, 1 nmol, 1 mM, DMSO), NaOAc buffer (10 μL, 1 M, pH = 5.5), and [ 64 [Cu]CuCl2 (5-20 MBq, 925 MBq / mL, DSD Pharma, Purkersdorf, Austria) was heated to 80 °C for 10 min. After the reaction solution was cooled, sodium ascorbate (10 µL, 1 M) was added. The radiochemical purity was then determined using radio-RP-HPLC and radio-TLC. 18 F-labeling: On the day of the experiment, 100–3000 MBq of [ 18 [F] fluoride was loaded onto a SEP-Pak® Light (46 mg) Accell™ Plus QMA cartridge pretreated with HO (10 mL). 18 [F] fluoride was extracted with anhydrous DMSO (8 mL) and reverse-eluted with ammonium formate (100–300 μL, 0.3 μM). Then, a solution of the eluate (30–100 μL) and peptide precursor (5–15 nmol) was heated (40–60 °C) for 5 min, and the reaction mixture was diluted with PBS (10 mL, pH = 3). 18The F-labeled peptide was loaded onto an Oasis® HLB (30 mg) light cartridge. After washing the cartridge with PBS (10 mL, pH = 7.4), the peptide was back-eluted with 300 μL of HO / EtOH (1:7). Radiochemical purity was determined using radioactive RP-HPLC and radioactive TLC. 3.4. In vitro experiments n-octanol-PBS distribution coefficient (logD 7.4 ) Approximately 1 MBq of labeled tracer was dissolved in 1 mL of a 1 / 1 (v / v) mixture of phosphate-buffered saline (PBS, pH 7.4) and n-octanol in an Eppendorf tube (1.5 mL). After vigorously mixing the suspension for 3 min at room temperature, the vial was centrifuged at 9,000 rpm for 5 min (Biofuge 15, Heraeus Sepatech, Osterode, Germany), and 200 μL aliquots of both layers were measured in a γ-counter. Experiments were repeated at least five times. I C 50 Decision AR4-2J cells were harvested 24 ± 2 hours before the experiment and seeded into 24-well plates (2.0 × 10 cells per well, 1 mL). 5 After removing the nutrient medium (RPMI 1640, 5 mM L-Gln, 5 mL of non-essential amino acids (100x), 10% FCS), the cells were washed once with 500 μL of PBS. Then, 200 μL of nutrient medium (5% BSA, v / v) was added. Next, the cells were incubated with nutrient medium (5% BSA, v / v) as a control or with increasing concentrations (10 -10 ~10 -4 M) 25 μL of a solution containing one of the compounds was added to each well, followed by 25 μL of [ 177 Lu]Lu-DOTA-PP-F11N (0.3 pmol / well) was added.

[0421] All experiments were performed in triplicate for each concentration. After 3 hours of incubation at 37°C, the experiment was terminated by removing the medium and rinsing successively with 300 μL of PBS. The medium from both steps was combined into one fraction, representing the amount of free radiolabeled reference. Cells were then lysed with 300 μL of 1 M NaOH for at least 15 minutes and mixed with 300 μL of NaOH from the next washing step. Quantification of the bound and free radiolabeled reference was achieved with a γ-counter. The IC of each conjugate was calculated. 50 The measurements were repeated twice. Determining internalization kinetics To determine the internalization kinetics of various peptides, AR4-2J cells (3.0 × 10 5 Cells were seeded onto polylysine-coated 24-well plates and supplemented with 1 mL of nutrient medium (RPMI 1640, 5 mM L-Gln, 5 mL of non-essential amino acids (100x), 10% FCS). Cells were then incubated at 37°C in a humidified atmosphere (5% CO) for 24 ± 2 hours.

[0422] On the day of the experiment, the medium was removed and each well was washed with 300 μL of incubation medium (RPMI 1640, 5 mM L-Gln, 5 mL non-essential amino acids (100x)). Then, 200 μL of nutrient medium (RPMI 1640, 5 mM L-Gln, 5 mL non-essential amino acids (100x), 10% FCS) was added to each well. Subsequently, 25 μL of nutrient medium (RPMI 1640, 5 mM L-Gln, 5 mL non-essential amino acids (100x)) (n = 3) or 25 μL of DOTA-PP-F11N (10 μmol) (n = 3) was added for blocking. 17725 μL of Lu-labeled peptide (0.3 pmol / well) was added to each well (n=6). The assay was then incubated at 37°C in a humidified atmosphere (5% CO2) for various times (1, 2, 4, and 6 hours). After incubation, the cells were placed on ice, and the supernatant was collected. The cells were then washed with 300 μL of ice-cold incubation medium (RPMI 1640, 5 mM L-Gln, 5 mL of non-essential amino acids (100x)), and both fractions were combined. To release the peptide from the cell membrane, 300 μL of ice-cold glycine buffer (1 M, pH=2.2) was added, and the cells were incubated on ice for 15 minutes. The supernatant was then collected, and the cells were washed with 300 μL of ice-cold glycine buffer (1 M, pH=2.2). Both fractions were combined. After lysing the cells with NaOH (300 μL, 1 M) for 15 min, each well was washed with NaOH (300 μL, 1 M), and both fractions were combined. The radioactivity in the supernatant, acid wash, and lysate fractions was quantified using a γ-counter. Stability test in human serum 177 Lu-labeled CCK-2R ligand (1 nmol, approximately 5 MBq) was incubated in human serum (200 μL) at 37 °C for 72 ± 2 h. After incubation, ice-cold EtOH (125 μL) and MeCN (375 μL) were added, and the suspension was centrifuged at 5,000 rpm for 2 min. The supernatant was then transferred to an ultracentrifuge vial and centrifuged at 5,000 rpm for an additional 2 min. After separating the precipitate from the solution, the stability of the ligand was determined by RP-HPLC chromatography. Determination of HSA binding As reported by Valko et al.

[31] , human serum albumin (HSA) binding of CCK-2R-targeting compounds was determined at RT using a Chiralpak HSA column (50 x 3 mm, 5 μm, H13H-2433, Daicel, Tokyo, Japan) at a constant flow rate of 0.5 mL / min. Therefore, an aqueous solution of NHOAc (pH = 6.9, 50 mM) was freshly prepared as mobile phase A, and isopropanol as mobile phase B. To calibrate the column before the experiment, the retention times of nine reference substances with HSA binding rates ranging from 13 to 99% were determined using a gradient of 100% A (0–3 min) followed by 80% A (3–40 min)

[32] . Furthermore, all tested substances were dissolved in a mixture of A and B (1 / 1, v / v) to achieve a final concentration of 0.5 mg / mL. HSA binding of all test rhCCK ligands was determined using the same gradient as the calibration probe. Nonlinear regressions of calibration experiments and CCK-2R ligands (Figure 29) were generated using OriginPro 2016G software (Nothampton, United States). In vivo experiments All animal experiments were performed in accordance with the German general animal welfare regulations (Notification dated May 18, 2006, German Animal Protection Act, amended by Article 280 dated June 19, 2020, approval number ROB-55.2-1-2532.Vet_02-18-109 by the General Administration of Upper Bavaria) and the institutional guidelines for animal care and use. CB17-SCID mice of both sexes (Charles River Laboratories International Inc., Sulzfeld, Germany) were acclimatized in the in-house animal care facility for at least 1 week before inoculation. To establish tumor xenografts, AR4-2J cells (5.0 × 10 per 200 μL) were cultured in 200 μL of 5% CO2. 6The cells were suspended in a 1 / 1 mixture (v / v) of Dulbecco's modified Eagle's medium / nutrient mixture F-12 (1 / 1) supplemented with Glutamax-1 (1 / 1) and Cultrex® Basement Membrane Matrix Type 3 (Trevigen, Gaithersburg, MD, USA) and inoculated subcutaneously into the right shoulder of 6- to 10-week-old CB17-SCID mice (Charles River, Sulzfeld, Germany). Mice were cultured until tumors reached 150–300 mm. 3 The animals were used for the experiment when they had grown to a volume of 1,500 mm (1-2 weeks after inoculation). The criteria for excluding animals from the experiment were a weight loss rate of more than 20% and a tumor size of 1,500 mm. 3 The following were signs of morbidity: hypertension, tumor ulceration, respiratory distress, or behavioral changes. None of these criteria were present in the animals used in the study. No randomization or blinding was applied to experimental assignment. Health status was SPF according to FELASA criteria. Biodistribution Female CB17-SCID mice bearing AR4-2J tumors were injected via the tail vein with approximately 1–5 MBq (100 pmol) of radiolabeled minigastrin analogs and sacrificed 1–24 h postinjection (n = 4–5). Selected organs were removed, weighed, and measured in a γ-counter. Competitive Exam In the competition test, radiolabeled compounds (approximately 1–5 MBq, 100 pmol) were added to [ nat AR4-2J tumor-bearing CB17-SCID mice were injected with [Lu]Lu-DOTA-MGS5 (40 nmol) into the tail vein and sacrificed 1-24 h after injection (n = 2). Selected organs were removed, weighed, and measured in a γ-counter. μSPECT / CT imaging Imaging tests were performed using MILabs VECTor 4Small-animal SPECT / PET / OI / CT (MILabs, Utrecht, the Netherlands) was used. Data were reconstructed using MILabs-Rec software (version 10.02) and a pixel-based Similarity-Regulated Ordered Subsets Expectation Maximization (SROSEM) algorithm with window-based scatter correction (20% below and 20% above the photon peak, respectively). Further data analysis was accomplished using PMOD 4.0 software (PMOD TECHNOLOGIES LLC, Zurich, Switzerland) with the specified settings: 80 μm voxel size for CT, 0.8 mm voxel size for SPECT, 1.6 mm (FWHM) Gaussian blur postprocessing filter, calibration factor in kBq / mL, with and without attenuation correction. For SPECT studies, mice were anesthetized with isoflurane and 2–4 MBq (100 pmol) of radiolabeled tracer was injected into the tail vein. Still images were recorded 1, 4, and 24 h after injection using a HE-GP-RM collimator and stepwise multiplanar bed movement with acquisition times of 45–60 min. Stability testing Stability testing is 177 Lu-labeled peptide (approximately 35 MBq, 1 nmol) was injected into the tail vein of anesthetized (2% isoflurane) CB17-SCID mice (n = 3). Thirty 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 5,000 rpm for 2 minutes. Serum was then collected, and ice-cold MeCN (1:1, v / v) and EtOH (1:2, v / v) were added. Samples were centrifuged again at 5,000 rpm for 2 minutes, and the supernatant was analyzed by RP-HPLC (10% to 30% for 5 minutes, 30% to 60% for 15 minutes, MeCN / HO + 0.1% TFA). Patient Testing All patients were [ 68Written informed consent was signed before undergoing [Ga]Ga-DOTA-CCK-66 PET / CT. The retrospective analysis of imaging results was approved by the ethics committee of the University of Munich. The anonymized analysis was performed in accordance with the Declaration of Helsinki and its subsequent amendments, as well as legal considerations of clinical guidelines.

[0423] The patient [ 68 120 min after injection of 150–200 MBq of ]Ga-DOTA-CCK-66 using a Biograph mCT 40 (Siemens Healthineers, Erlangen, Germany) [ 68 Patients underwent whole-body PET / CT with [Ga]Ga-DOTA-CCK-66. Whole-body CT imaging was performed as an adjunct CT (120 kVp, 40 mAs). PET datasets were reconstructed using standard protocols and corrected for randomization, scatter, attenuation, and attenuation (using whole-body adjunct CT). References 1. Wells, SA, Jr.; Asa, SL; Dralle, H.; Elisei, R.; Evans, DB; Gagel, RF; Lee, N.; Machens, A.; Moley, JF; Pacini, F.; et al. Revised American Thyroid Association guidelines for the management of medullary thyroid carcinoma. Thyroid 2015, 25, 567-610, doi:10.1089 / thy.2014.0335. 2. Society, AC Cancer Facts & Figures 2020. 2020, 2020. 3. Kebebew, E.; Kikuchi, S.; Duh, Q.-Y.; Clark, O.H. Long-term Results of Reoperation and Localizing Studies in Patients With Persistent or Recurrent Medullary Thyroid Cancer. 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Treglia, G.; Castaldi, P.; Villani, M.F.; Perotti, G.; de Waure, C.; Filice, A.; Ambrosini, V.; Cremonini, N.; Santimaria, M.; Versari, A.; et al. Comparison of 18F-DOPA, 18F-FDG and 68Ga-somatostatin analogue PET / CT in patients with recurrent medullary thyroid carcinoma. Eur J Nucl Med Mol Imaging 2012, 39, 569-580, doi:10.1007 / s00259-011-2031-6. 17. Brammen, L.; Niederle, M.B.; Riss, P.; Scheuba, C.; Selberherr, A.; Karanikas, G.; Bodner, G.; Koperek, O.; Niederle, B. Medullary Thyroid Carcinoma: Do Ultrasonography and F-DOPA-PET-CT Influence the Initial Surgical Strategy? Ann Surg Oncol 2018, 25, 3919-3927, doi:10.1245 / s10434-018-6829-3. 18. Treglia, G.; Cocciolillo, F.; Di Nardo, F.; Poscia, A.; de Waure, C.; Giordano, A.; Rufini, V. Detection rate of recurrent medullary thyroid carcinoma using fluorine-18 dihydroxyphenylalanine positron emission tomography: a meta-analysis. Acad Radiol 2012, 19, 1290-1299, doi:10.1016 / j.acra.2012.05.008. 19. Stamatakos, M.; Paraskeva, P.; Stefanaki, C.; Katsaronis, P.; Lazaris, A.; Safioleas, K.; Kontzoglou, K. Medullary thyroid carcinoma: The third most common thyroid cancer reviewed. Oncol Lett 2011, 2, 49-53, doi:10.3892 / ol.2010.223. 20. Hadoux, J.; Schlumberger, M. Chemotherapy and tyrosine-kinase inhibitors for medullary thyroid cancer. Best Practice & Research Clinical Endocrinology & Metabolism 2017, 31, 335-347, doi:10.1016 / j.beem.2017.04.009. 21. Hazard, J.B. The C cells (parafollicular cells) of the thyroid gland and medullary thyroid carcinoma. A review. Am J Pathol 1977, 88, 213-250. 22. Ljungberg, O. Medullary carcinoma of the human thyroid gland. Autoradiographic localization of radioiodine. Acta Pathol Microbiol Scand 1966, 68, 476-480. 23. Fussey, J.M.; Vaidya, B.; Kim, D.; Clark, J.; Ellard, S.; Smith, J.A. The role of molecular genetics in the clinical management of sporadic medullary thyroid carcinoma: A systematic review. Clin Endocrinol (Oxf) 2019, doi:10.1111 / cen.14060. 24. Resteghini, C.; Cavalieri, S.; Galbiati, D.; Granata, R.; Alfieri, S.; Bergamini, C.; Bossi, P.; Licitra, L.; Locati, L.D. Management of tyrosine kinase inhibitors (TKI) side effects in differentiated and medullary thyroid cancer patients. Best Pract Res Clin Endocrinol Metab 2017, 31, 349-361, doi:10.1016 / j.beem.2017.04.012. 25. Behr, T.M.; Gratz, S.; Markus, P.M.; Dunn, R.M.; Hufner, M.; Becker, H.; Becker, W. Enhanced bilateral somatostatin receptor expression in mediastinal lymph nodes ("chimney sign") in occult metastatic medullary thyroid cancer: a typical site of tumour manifestation? Eur J Nucl Med 1997, 24, 184-191. 26. Reubi, J.C.; Schaer, J.C.; Waser, B. Cholecystokinin(CCK)-A and CCK-B / gastrin receptors in human tumors. Cancer Res. 1997, 57, 1377-1386. 27. Gotthardt, M.; Behe, M.P.; Beuter, D.; Battmann, A.; Bauhofer, A.; Schurrat, T.; Schipper, M.; Pollum, H.; Oyen, W.J.; Behr, T.M. Improved tumour detection by gastrin receptor scintigraphy in patients with metastasised medullary thyroid carcinoma. Eur J Nucl Med Mol Imaging 2006, 33, 1273-1279, doi:10.1007 / s00259-006-0157-8. 28. Roosenburg, S.; Laverman, P.; van Delft, F.L.; Boerman, O.C. Radiolabeled CCK / gastrin peptides for imaging and therapy of CCK2 receptor-expressing tumors. Amino Acids 2011, 41, 1049-1058, doi:10.1007 / s00726-010-0501-y. 29. Fani, M.; Peitl, P.K.; Velikyan, I. Current Status of Radiopharmaceuticals for the Theranostics of Neuroendocrine Neoplasms. Pharmaceuticals (Basel) 2017, 10, doi:10.3390 / ph10010030. 30. Klingler, M.; Summer, D.; Rangger, C.; Haubner, R.; Foster, J.; Sosabowski, J.; Decristoforo, C.; Virgolini, I.; von Guggenberg, E. DOTA-MGS5, a New Cholecystokinin-2 Receptor-Targeting Peptide Analog with an Optimized Targeting Profile for Theranostic Use. J Nucl Med 2019, 60, 1010-1016, doi:10.2967 / jnumed.118.221283. 31. 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Cholecystokinin-2 Receptor Targeting with Novel C-terminally Stabilized HYNIC-Minigastrin Analogs Radiolabeled with Technetium-99m. Pharmaceuticals (Basel) 2019, 12, doi:10.3390 / ph12010013. 35. Rottenburger, C.; Nicolas, G.P.; McDougall, L.; Kaul, F.; Cachovan, M.; Vija, A.H.; Schibli, R.; Geistlich, S.; Schumann, A.; Rau, T.; et al. Cholecystokinin 2 Receptor Agonist (177)Lu-PP-F11N for Radionuclide Therapy of Medullary Thyroid Carcinoma: Results of the Lumed Phase 0a Study. J Nucl Med 2020, 61, 520-526, doi:10.2967 / jnumed.119.233031. 36. Kolenc-Peitl, P.; Tamma, M.; Kroselj, M.; Braun, F.; Waser, B.; Reubi, J.C.; Sollner Dolenc, M.; Maecke, H.R.; Mansi, R. Stereochemistry of amino acid spacers determines the pharmacokinetics of (111)In-DOTA-minigastrin analogues for targeting the CCK2 / gastrin receptor. Bioconjug Chem 2015, 26, 1113-1119, doi:10.1021 / acs.bioconjchem.5b00187.

Claims

1. A tetrapeptide that binds to the cholecystokinin 2 receptor (CCK-2R), comprising the 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 of the hydrogen atoms of the 1H-indol-3-yl is independently C for each H atom; １ ~C ３ substituted by substituents selected from alkyl, —OH, —SH, —F, and —Cl, Phe, 1-Nal, 2-Nal, Tyr, or p-amino-Phe; Xaa2 is an N-methyl amino acid, where the N-methyl group is at the α carbon; Xaa3 is an amino acid with an acidic side chain at neutral pH, Xaa4 is an amino acid having an aromatic side chain or an amide thereof. A tetrapeptide represented by the formula:

2. 2. The tetrapeptide of 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, or (N-Me)Glu.

3. 2. The tetrapeptide of claim 1, wherein Xaa3 is Asp, isoaspartic acid, Glu, or isoglutamine.

4. 2. The tetrapeptide of claim 1, wherein Xaa4 is 1-Nal, Phe, Trp, Tyr, 2-Nal, p-amino-Phe, or an amide of any one of the foregoing.

5. Xaa2 is an N-methyl amino acid containing an aliphatic side chain, wherein the N-methyl group is at the α-carbon; or Xaa2 is (N-Me)Nle or (N-Me)Met, or Xaa3 is Asp, or Xaa4 is 1-Nal, Phe, Tyr, or an amide of any one of the foregoing; or Xaa4 is 1-Nal or Tyr, or an amide of 1-Nal or Tyr; The tetrapeptide of claim 1.

6. Within a single molecule: (a) the tetrapeptide of claim 1, and (b) one or more chelating groups capable of complexing with non-radioactive or radioactive cations, or one or more chelating groups containing chelated non-radioactive or radioactive cations; , a conjugate comprising:

7. 7. The conjugate of claim 6, wherein the chelating group is DOTA or DOTAGA, with or without a chelated non-radioactive or radioactive cation.

8. The conjugate of claim 7, wherein DOTA and DOTAGA are linked at one of their carboxyl groups to the remainder of the conjugate via an amide bond.

9. Within a single molecule: (c) Below: (i) comprising a silicon atom and a fluorine atom, wherein the fluorine atom is directly bonded to the silicon atom via a covalent bond; １８ By F １９ By isotope exchange of F １８ F, or １８ silicon-based fluoride acceptor moieties, labeled with F; (ii) comprises a silicon atom and a hydroxy group, wherein the hydroxy group is directly bonded to the silicon atom via a covalent bond; １８ by nucleophilic substitution of OH by F １８ a silicon-based fluoride acceptor moiety that can be labeled with F; and (iii) comprising a silicon atom and a hydrogen atom, wherein the hydrogen atom is directly bonded to the silicon atom via a covalent bond; １８ by nucleophilic substitution of H by F １８ Silicon-based fluoride acceptor moieties that can be labeled with F a silicon-based fluoride acceptor moiety R selected from ＳｉＦＡ 7. The conjugate of claim 6, further comprising:

10. Silicon-based fluoride acceptor moiety R ＳｉＦＡ but The following formula (S-1): 【Chemistry 1】 [In the formula, X Ｓ is F, OH or H; R １Ｓ and R ２Ｓ are independently linear or branched C ３ ~C １０ alkyl group] 10. The conjugate compound of claim 9, comprising the group:

11. Silicon-based fluoride acceptor moiety R ＳｉＦＡ Formulas (S-3) to (S-7): 【Chemistry 2】 [In the formula, R １Ｓ and R ２Ｓ are each independently a linear or branched C3 to C10 alkyl group. 【Transformation 3】 [In the formula, r is 1, 2 or 3, and —(CH2) ｓ where s is an integer from 1 to 6, The groups R are independently H or C1-C6 alkyl; R １Ｓ and R ２Ｓ are independently a linear or branched C3 to C10 alkyl group. 【Chemistry 4】 [In the formula, R １Ｓ and R ２Ｓ are independently linear or branched C ３ ~C １０ is an alkyl group, R ３Ｓ teeth (i) —OH or —O—, (ii) a sugar moiety or an amino sugar moiety; (iii) an amino acid moiety or an oligopeptide moiety; (iv) PEG moiety; and combinations of two or more of (ii), (iii) and (iv). The conjugate compound according to claim 9, having a structure represented by any one of the following:

12. Formula (III): 【Transformation 5】 [In the formula: R Ｌ is one or more chelating groups according to any one of claims 7 to 11, which may contain chelated non-radioactive or radioactive cations; L is a linking moiety; R Ｈ is a tetrapeptide according to any one of claims 1 to 5; R ＳｉＦＡ is present or absent, and if present is a silicon-based fluoride acceptor moiety according to any one of claims 10 to 12. The conjugate according to any one of claims 6 to 11, represented by:

13. If the binding moiety includes 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), the SiFA, when present, includes the -NH ２ The group is used to conjugate to D / L-Dap, D / L-Dab, D / L-Orn or D / L-Lys to provide the coupling group -NH-, where -NH ２ 13. The conjugate of claim 12, wherein a bond to one hydrogen atom of the group is replaced by a bond to a SiFA.

14. The conjugate of claim 13, wherein D / L-Dap, D / L-Dab, D / L-Orn or D / L-Lys is arranged in the linking moiety such that D / L-Dap, D / L-Dab, D / L-D / L-Orn or Lys is directly linked to R L and / or R SiFA .

15. The conjugation moiety may be polyethylene glycol (PEG), poly-L / D-hydroxyproline (L / D-Hyp), and / or Optionally, together with α- or γ-glu, The conjugate of claim 12.

16. PEG is (PEG) 3-11 , and / or poly-L / D-hydroxyproline (L / D-Hyp) is (Hyp) 1-8 , and / or the linker comprises y-glu-(Hyp) 6 -y-glu-(PEG) 3 or y-glu-(PEG) 4 -y-glu-(PEG) 3 ; The conjugate of claim 15.

17. The conjugate is 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)Nle-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 、 DOTA-dap(SiFAlin)-y-glu-(PEG) ４ -y-glu-(PEG) ３ -Trp-(N-Me)Nle-Asp-1-Nal-NH ２ is expressed as In the formula, SiFA represents the moiety of formula (S-3a), and SiFAlin represents the moiety of formula (S-4a): 【Transformation 6】 wherein DOTA may contain a chelated non-radioactive or radioactive cation. The conjugate according to any one of claims 6 to 11, wherein

18. Formula (IV) R Ｌ -dap(R ＳｉＦＡ )-R ＧＡＢＡ -L-R Ｈ (IV) [In the formula: R Ｌ is DOTA or DOTAGA, which may contain a chelated non-radioactive or radioactive cation; dap (R ＳｉＦＡ ) is a silicon-based fluoride acceptor moiety R ＳｉＦＡ is a dap group having the formula: R ＧＡＢＡ is present or absent, and if present, is one or more gamma-aminobutyric acids (GABA); L is a linking moiety containing at least five α- or γ-glu; R Ｈ is Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH ２ is or contains A conjugate compound represented by the formula:

19. R GABA is (GABA) 1-3 ; and / or L is a linking moiety containing at least 6 α- or γ-glu, or 6 to 9 α- or γ-glu; 19. The conjugate compound of claim 18.

20. The conjugate is - ６ - ２ 、 DOTAGmdap(3i00｡)-(α-glu) ８ -lamTyrmolymTrp--lemAspm-hee ２ 、 DOTA-dap(SiFA)-(γ-glu) ８ -Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH ２ is expressed as SiFA represented by formula (S-3a): 【Transformation 7】 wherein DOTA or DOTAGA may contain a chelated non-radioactive or radioactive cation.

20. The conjugate compound of claim 18 or 19, wherein the silicon-based fluoride acceptor moiety is

Images