Targeted delivery of therapeutic diagnostic agents

Abstract

The present invention relates to compounds and methods for targeted delivery of diagnostic / therapeutic radionuclides to cancerous tissue. In particular, the present invention relates to the targeted delivery of diagnostic / therapeutic radionuclides to cholecystokin-2 receptor positive cancers, and to methods for diagnosing and treating cancer.

Description

Technical Field

[0001] The present invention relates to compounds and methods for targeted delivery of diagnostic / therapeutic radionuclides to cancer tissues. In particular, the present invention relates to targeted delivery of diagnostic / therapeutic radionuclides to cholecystokinin-2 receptor positive cancers, and to methods for diagnosing and treating cancer. Background Art

[0002] The basic principle of the field of theranostic radiopharmaceuticals is to develop well-targeted compounds (e.g., short peptides) that, after complexation with a radionuclide, can be used as treat or diagnosis Compounds. This rapidly developing field of personalized medicine has proven to be highly successful in treating patients with metastatic malignancies with remarkable efficacy and low toxicity (Hofman, MS, et al., Lancet, 2020, 395 (10231), 1208-1216; Hofman, MSP, et al., Lancet Oncology, 2018, 19 (6), 825-833; Kaltsas, G., et al., Neuroendocrinology 2017, 105 (3), 245-254). These remarkable results are due in part to the unique ability of clinicians to "see what they treat". In addition, the increased sensitivity and specificity of these compounds allow clinicians to more accurately stage patients before, during, and after treatment to ensure that patients receive the most appropriate disease management (Velikyan, I. Pharmaceuticals (Basel), 2020, 13, 39). Currently, the implementation of peptide-based therapeutic diagnostics in oncology is limited to a few cancers. These include somatostatin-2R-positive neuroendocrine tumors and prostate specific membrane antigen (PSMA)-positive prostate cancer.

[0003] The G protein-coupled receptor cholecystokinin-2 (CCK-2R), whose natural ligand is the gastrin hormone G17, is an important molecular target for theranostic applications and is overexpressed in a range of cancers, including but not limited to medullary thyroid carcinoma (MTC), somatostatin-2R-negative neuroendocrine tumors,

[0004] (Gotthardt,M.,etal.,

[0005] Endocrine-Related Cancer, 2006, 13(4), 1203-1211; Reubi, JC, Schaer, J.-C., Waser, B., Cancer Research, 1997, 57(7), 1377-1386; Béhé, M., Behr, TM, Peptide Science 2002, 66(6), 399-418: Reubi, JC, Maecke, HR, Journal of Nuclear Medicine 2017, 58(Supplement 2), 10S-16S), stromal ovarian cancer (100% incidence; Reubi, JC, Schaer, J.-C., Waser, B., Cancer Research, 1997, 57(7), 1377-1386) and small cell lung cancer

[0006] (56% incidence; Roy, J., et al., Oncotarget 2016, 7(12), 14605-14615: Reubi, JC, HR, Krenning, EP, Society of Nuclear Medicine 2005, 46Suppl1, 67s-75s; Copps, J., Murphy, RF, Lovas, S., Protein Pept Lett 2009, 16(12), 1504-1518; Zeng, Q., et al., Frontiers in Endocrinology 2020, 11(112))

[0007] etc.

[0008] Given the well-established link between CCK-2R and cancer, extensive research has been conducted to understand the key features of the G17 peptide in an attempt to establish modified CCK-2R peptides suitable for use as theranostic radiopharmaceuticals in nuclear medicine (Roosenburg, S., et al., Amino Acids, 2010, 41, 1049-1058). Several radiolabeled gastrin-derived peptide analogs have been developed, including DGlu-MG0, which is derived from human minigastrin, a truncated form of MG11, which lacks a five-glutamic acid sequence, and other derivatives of gastrin and cholecystokinin (Rottenburger, C., et al., J. Nucl. Med., 2020, 61, 520-526; Erba, P. A., et al., Pol. Arch. Intern. Med., 2018, 128, 791-795; Klinger, M., et al., Journal of Nuclear Medicine 2018). CP04 and its closely related analog PP-F11N containing an N-terminal six-D-glutamic acid sequence exhibit reduced renal uptake and retention (minimal nephrotoxicity) and enhanced metabolic stability, which are desirable properties required for therapeutic diagnostic compounds. In recent clinical trials, both CP04 and PP-F11N have been investigated for imaging and targeting CCK-2R in humans (Rottenburger, C., et al., J. Nucl. Med., 2020, 61, 520-526; Erba, PA, et al., Pol. Arch. Intern. Med., 2018, 128, 791-795). Although both peptides have been shown to accumulate in MTC lesions, they are not comparable to those used for the treatment of neuroendocrine tumors. 177 Suboptimal tumor radiation doses were observed compared to Lu]Lu-DOTA-TATE, indicating that improved agents are still needed. High tumor uptake and long residence time are critical for the successful delivery of peptide receptor radionuclide therapy (PRRT), thereby determining the tumor absorbed dose.

[0009] von Guggenberg and colleagues developed the peptide MGS5 using unnatural aromatic amino acids and N-methylation of the residue at the C-terminus of MG11. When compared to CP04 and PP-F11N, radiolabeled MGS5 exhibited improved in vitro and in vivo characteristics, with enhanced metabolic stability and improved tumor targeting.

[0010] (Klinger. M., et al., Journal of Nuclear Medicine 2018; Klinger, M., et al., Journal of Nuclear Medicine 2019.60(1), 1010-1016; Klinger, M., et al., Theranostics, 2018, 8, 2896-2908; Maximilian, K., Anton Amadeus, H., Guggenberg, Ev, Current Medicinal Chemistry, 2020.27, 1-21)

[0011] The results of these studies were recently reported in two patients. 68 The clinical translation of [Ga]Ga-DOTA-MG5S demonstrated the concept of CCK-2R PET imaging in patients with MTC (Guggenberg, EV, et al., Eur. J. Nucl. Med. Mol. Imaging, 2019, 46(S1), S268; Uprimny, C., et al., Eur. J. Nucl. Med. Mol. Imaging, 2020).

[0012] To date, there are only a limited number of studies that have elucidated the effects of amino acid modifications on the conformation of radiolabeled G17-based CCK-2R binding ligands (CP04, PP-F11N and MGS5), and therefore on receptor binding and bioactivity (Kolenc Peitl, P., et al., Bioconjugate Chemistry, 2015, 26, 1113-1119). Although it is known that the key sequence required for peptide-CCK-2R interaction is the C-terminal tetrapeptide Trp-Met-Asp-Phe-NH2, it only accounts for 10% of the total biopotency of G17 (measured by gastric acid secretion in rats). Interestingly, the gradual inclusion of polyglutamic acid residues at the N-terminus leads to conformational changes (as demonstrated by CD spectroscopy) and incrementally increases the biopotency to 90%.

[0013] (Peggion. E.. Foffani, MT, Mammi, S., Structure-Function Relationship of Gastrin Hormones: A Rational Approach to Drug Design. In Bioorganic Chemistry in Healthcare and Technology, Pandit, UK; Alderweireldt, FC, Eds. Springer US: Boston, MA, 1991, 121-133).

[0014] Peggion et al. concluded that although the N-terminus of G17 may adopt an α-helical conformation, there must be interactions between the "head" and "tail" of the peptide to explain the increased potency and conformational changes observed upon extension of the pentaglutamyl sequence. 11 -Trp 14 The β-turn at the center caps the α-helical portion of the N-terminus and brings the two ends of the peptide into close proximity. This study emphasizes the pharmacological significance of a suspected "U-shaped" hairpin fold, in which the α-helical N-terminus stabilizes the biologically active conformation of the C-terminus (Copps, J., Murphy, RF, Lovas, S., Protein Pept. Lett., 2009, 16, 1504-1518).

[0015] The three-dimensional structure of any receptor ligand is obviously important for binding specificity, affinity and functionality, and should be considered during the design and optimization of any targeting peptide intended as a therapeutic diagnostic compound. However, the design of short-medium peptides with limited secondary structures is difficult to implement because they are flexible in nature and are unlikely to form ordered structures similar to those present in larger proteins and biomolecules. The development of targeting peptides requires detailed simulation of interprotein contact points, which usually requires not only the primary structure of biomacromolecules to be reproduced, but also the secondary structure to be reproduced. Peptide cyclization and stapling have been used to limit peptide conformations by forming covalent bonds. However, cyclization and stapling do not produce a series of secondary structures, and are not very useful when functional linear peptides are required.

[0016] Therefore, there is a need to develop new theranostic compounds to expand their transformative effects in the management of other malignancies. Summary of the invention

[0017] Provided are novel compounds and methods for diagnosing and treating cancer in a subject. Therefore, in one aspect, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0018]

[0019] in:

[0020] R 1 is a side chain of an amino acid selected from phenylalanine, 3-(1-naphthyl)alanine, 3-(2-naphthyl)alanine or tyrosine, wherein the phenylalanine residue is optionally substituted with one or more halogen, -NH2, cyano, C 1-6 Alkyl, halogenated C 1-6 Alkyl or acetyl substitution;

[0021] R 2 Selected from H or C1-C3 alkyl;

[0022] A is a radionuclide binding ligand; and

[0023] -L- is the amino acid sequence: -D-Glu-D-Glu-D-Ala-D-Glu-D-Glu-D-Glu-

[0024] In one embodiment, the compound of formula (I) is represented by formula (Ia):

[0025]

[0026] In another embodiment, the compound of formula (I) is represented by formula (Ib):

[0027]

[0028] Each occurrence of R 3 independently selected from halogen, -NH2, cyano, -OH, C 1-6 Alkyl, halogenated C 1-6 alkyl or acetyl; and

[0029] n is 0 to 5.

[0030] In another embodiment, the compound of formula (I) is represented by formula (Ic):

[0031]

[0032] In one embodiment, the compound according to the invention further comprises a radionuclide complexed with a radionuclide binding ligand.

[0033] In another aspect, the present invention provides a method for identifying CCK-2R positive cancer in a subject, comprising administering to the subject an effective amount of a compound according to the present invention or a pharmaceutically acceptable salt thereof, and detecting a radionuclide.

[0034] In another aspect, the present invention provides a compound according to the present invention or a pharmaceutically acceptable salt thereof for use in a method of identifying CCK-2R positive cancer in a subject, the method comprising administering an effective amount of the compound to the subject, and detecting a radionuclide.

[0035] In another aspect, a method for treating CCK-2R positive cancer in a subject in need thereof is provided, comprising administering to the subject a compound according to the present invention or a pharmaceutically acceptable salt thereof.

[0036] In yet another aspect of the present invention, there is provided a pharmaceutical composition comprising a compound according to the present invention or a pharmaceutically acceptable salt thereof.

[0037] These and other aspects of the present invention will become more apparent to the skilled person after reading the following detailed description in conjunction with the appended examples and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The present invention will herein be described by way of example only with reference to the following non-limiting drawings, in which:

[0039] Figure 1 Peptides A to F, Comparative Example-1 and CP04 in A431-CCK2R cells against [ 177 Lu] Lu-DOTA-CP04 IC 50 Graphical representation of determined binding curves. Curves are representative of 2 to 4 biological replicates performed in three technical replicates (n=3).

[0040] Figure 2 Compounds 1 to 4 and Comparative Example-2 were shown to be effective against [ 177 Lu] Lu-DOTA-CP04 IC 50 Graphical representation of determined binding curves. Curves are representative of 2 to 4 biological replicates performed in three technical replicates (n=3).

[0041] Figure 3 The compound is shown nat Ga-1 to 4 and nat Graphical representation of the CD spectra of Ga-Cmp ex-2 in water and 5 mM DPC micelles. Three spectra accumulated at 20 °C were averaged and baseline corrected.

[0042] Figure 4Compounds incubated in human serum, liver homogenate, and kidney homogenate are shown. 68 Ga]Ga-1 to 4 and [ 68 Graph showing the metabolic stability of Ga]Ga-Cmp ex-2. Samples were assayed at multiple time points over 90 minutes. Results are mean values ​​(n=2).

[0043] Figure 5 A) Compound [ 68 Ga]Ga-1 to 4 and [ 68 Distribution of compound [Ga]Ga-Cmp ex-2 in female BALB / c nu / nu mice (8 to 10 weeks old). b) 68 Ga]Ga-1 to 4 and [ 68 Tumor uptake analysis of compound [Ga]Ga-Cmp ex-2 at 1 hour, 2.5 hours after injection and co-administered with the blocker YM022, reported as %ID / g. c) At 1 hour 68 Ga]Ga-1 to 4 and [ 68 Quantitative analysis of the accumulation of Ga]Ga-Cmp ex-2 in selected organs, reported as %ID / g. d) Presented compounds [ 68 Ga]Ga-1 to 4 and [ 68 Quantitative analysis of tumor-to-organ ratios of Ga]Ga-Cmp ex-2 at 1 hour after injection. T:B; tumor-to-blood, T:Stom; tumor-to-stomach. DETAILED DESCRIPTION

[0044] Folding bodies offer several advantages, including the ability to design secondary structures, control of the orientation of side chain functional groups, and resistance to proteolytic degradation. In addition, folding bodies address the thermodynamic basis of protein-protein interactions (PPIs) by minimizing conformational freedom, thereby minimizing the entropic penalty paid during binding and resulting in higher binding affinity (Du, X., et al., International Journal of Molecular Sciences 2016, 17(2), 144).

[0045] Foldomers adopt an inherent tendency to form secondary and sometimes tertiary structures through hydrogen bonding, resulting in a wide variety of conformations, including all forms of β-turns, hairpins, and helices.

[0046] In one embodiment, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0047]

[0048] in:

[0049] R 1 is a side chain of an amino acid selected from phenylalanine, 3-(1-naphthyl)alanine, 3-(2-naphthyl)alanine or tyrosine, wherein the phenylalanine residue is optionally substituted with one or more halogen, -NH2, cyano, C 1-6 Alkyl, halogenated C 1-6 Alkyl or acetyl substitution;

[0050] R 2 Selected from H or C1-C3 alkyl;

[0051] A is a radionuclide binding ligand; and

[0052] -L- is the amino acid sequence: -D-Glu-D-Glu-D-Ala-D-Glu-D-Glu-D-Glu-.

[0053] It was found that replacing the amino acid residue on the N-terminal side of the C-terminal tetrapeptide Trp-Met-Asp-Phe-NH2 with the turn-inducing residue N-methylglycine increased the binding affinity of the resulting compound. It is believed that the inclusion of N-methylglycine restricts the compound to a β-hairpin conformation. The structure is believed to be further stabilized by interactions between the aromatic side chains and the glutamic acid side chains.

[0054] In another embodiment, the present invention provides a peptide of formula (II) or a pharmaceutically acceptable salt thereof:

[0055]

[0056] in:

[0057] R 1 is a side chain of an amino acid selected from phenylalanine, 3-(1-naphthyl)alanine, 3-(2-naphthyl)alanine or tyrosine, wherein the phenylalanine residue is optionally substituted with one or more halogen, -NH2, cyano, C 1-6 Alkyl, halogenated C 1-6 Alkyl or acetyl substitution;

[0058] R 2 is selected from H or C1-C3 alkyl; and

[0059] -L- is the amino acid sequence: -D-Glu-D-Glu-D-Ala-D-Glu-D-Glu-D-Glu-

[0060] In one embodiment, the peptide of formula (II) or a pharmaceutically acceptable salt thereof is represented by formula (IIa):

[0061]

[0062] In another embodiment, the peptide of formula (II) or a pharmaceutically acceptable salt thereof is represented by formula (IIb):

[0063]

[0064] Each occurrence of R 3 independently selected from halogen, -NH2, cyano, -OH, C 1-6 Alkyl, halogenated C 1-6 alkyl or acetyl; and

[0065] n is 0 to 5.

[0066] In another embodiment, the peptide of formula (II) or a pharmaceutically acceptable salt thereof is represented by formula (IIc):

[0067]

[0068] In another embodiment, the present invention provides a peptide of formula (II) selected from those listed in Table 1.

[0069] Table 1: Peptides of formula (II)

[0070]

[0071] -L- <![CDATA[R 1 ]]> <![CDATA[R 2 ]]> <![CDATA[-(D-Glu)2-D-Ala-(D-Glu)3-]]> <![CDATA[Nal 1 ]]> H <![CDATA[-(D-Glu)2-D-Ala-(D-Glu)3-]]> Phe methyl <![CDATA[-(D-Glu)2-D-Ala-(D-Glu)3-]]> Phe H <![CDATA[-(D-Glu)2-D-Ala-(D-Glu)3-]]> Nal methyl <![CDATA[-(D-Glu)2-D-Ala-(D-Glu)3-]]> <![CDATA[Phe(4-F) 2 ]]> H <![CDATA[-(D-Glu)2-D-Ala-(D-Glu)3-]]> <![CDATA[Phe(3,4-diF) 3 ]]> H <![CDATA[-(D-Glu)2-D-Ala-(D-Glu)3-]]> <![CDATA[Phe(3,4,5,-F) 4 ]]> H <![CDATA[-(D-Glu)2-D-Ala-(D-Glu)3-]]> <![CDATA[Phe(4-NH2) 5 ]]> H <![CDATA[-(D-Glu)2-D-Ala-(D-Glu)3-]]> <![CDATA[2-Nal 6 ]]> H <![CDATA[-(D-Glu)2-D-Ala-(D-Glu)3-]]> <![CDATA[Phe(4-CN) 7 ]]> H <![CDATA[-(D-Glu)2-D-Ala-(D-Glu)3-]]> <![CDATA[Phe(4-CF3) 8 ]]> H <![CDATA[-(D-Glu)2-D-Ala-(D-Glu)3-]]> Tyr H <![CDATA[-(D-Glu)2-D-Ala-(D-Glu)3-]]> <![CDATA[Phe(2,3,4,5,6-F) 9 ]]> H <![CDATA[-(D-Glu)2-D-Ala-(D-Glu)3-]]> <![CDATA[Phe(3-F) 10 ]]> H <![CDATA[-(D-Glu)2-D-Ala-(D-Glu)3-]]> <![CDATA[Phe(4-Ac) 11 ]]> H

[0072] 1. Nal = 3-(1-naphthyl)-L-alanine; 2. Phe(4-F) = 4-fluoro-L-phenylalanine; 3. Phe(3,4-diF) = 3,4-difluoro-L-phenylalanine; 4. Phe(3,4,5-F) 4 =3,4,5-trifluoro-L-phenylalanine; 5.Phe(4-NH2)=4-amino-L-phenylalanine; 6.2-Nal=3-(2-naphthyl)-L-alanine; 7.Phe(4-CN)=4-cyano-L-phenylalanine; 8.Phe(4-CF3)=4-(trifluoromethyl)-L-phenylalanine; 9.Phe(2,3,4,5,6-F)=2,3,4,5,6-pentafluoro-L-phenylalanine; 10.Phe(3-F)=3-fluoro-L-phenylalanine; 11.Phe(4-Ac)=4-(acetyl)-L-phenylalanine.

[0073] Reference to an amino acid "side chain" employs its standard meaning in the art. Some examples of amino acid side chains are shown below:

[0074]

[0075] As used herein, non-natural amino acids include any compound having both amino and carboxyl functionalities, derivatives thereof, or derivatives of naturally occurring amino acids. These amino acids form part of a peptide chain by bonding through their amino and carboxyl groups. Alternatively, these derivatives can be bonded to other naturally or non-naturally occurring amino acids to form a non-peptidyl connection.

[0076] It should be understood that in addition to the negatively charged side chains shown above, many side chains can also be protonated and thus become positively charged, such as the lysine side chains. The present invention also contemplates these protonated side chains within its scope.

[0077] It should be understood that the compounds of the present invention can exist in one or more stereoisomeric forms (e.g., diastereomers). The present invention includes within its scope all of these separated (e.g., enantiomeric separation) or combined (including racemic mixtures and diastereomeric mixtures) stereoisomeric forms. The present invention contemplates the use of amino acids in the form of L and D, including the use of amino acids independently selected from the form of L and D, for example, when the compound comprises two Glu residues, each Glu residue can have identical or opposite absolute stereochemistry.

[0078] The compounds of the present invention may also contain a radionuclide complexed or covalently bound to a radionuclide binding ligand. As described above, the compounds of the present invention can be used as therapeutic diagnostic compounds because the same peptide can be covalently coupled to a radionuclide binding ligand suitable for binding to a radionuclide that can be used to diagnose cancer and / or a radionuclide binding ligand suitable for binding to a radionuclide that can be used to treat cancer. In one embodiment, it is envisioned that the radionuclide binding ligand suitable for binding to a radionuclide that can be used to treat cancer is the same as the radionuclide binding ligand suitable for binding to a radionuclide that can be used to diagnose cancer. In another embodiment, the radionuclide binding ligand suitable for binding to a radionuclide used to treat cancer is different from the radionuclide binding ligand suitable for binding to a radionuclide used to diagnose cancer.

[0079] Reference to a "radionuclide binding ligand" is understood to mean a ligand or chelator that tightly binds a radionuclide (radioisotope). The radionuclide binding ligand is covalently bound to the compound so that when the compound of the invention is administered to a subject, the compound can deliver the radionuclide to the target site with no or minimal loss of the radionuclide, effectively providing a site-specific radiation source in vivo for imaging or treatment.Some examples of suitable radionuclide binding ligands include, but are not limited to, the following: DOTA (1,4,7,10-tetraazacyclododecane 1,4,7,10-tetraacetic acid), DOTA-NHS-ester, p-SCN-Bn-DOTA (C-DOTA), DOTAGA, DOTAGA-anhydride, CB-DO2A (4,10-bis(carboxymethyl)-1,4,7,10-tetraazabicyclo[5.5.2]tetradecane), TCMC (1,4,7,10-tetra(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane), p-SCN-Bn-TCMC, 3p-C-DEPA (2-[(carboxymethyl)]-[5-(4-nitrobenzene)] 1-[4,7,10-tris-(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl]pentan-2-yl)-amino]acetic acid), 3p-C-DEPA-NCS, p-NH2-Bn-oxo-DO3A, TETA (1,4,8,11-tetraazacyclotetradecane 1,4,8,11-tetraacetic acid), BAT, p-NH2-Bn-TE3A, C-TETA, CB-TE2A (4,11-bis-(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]-hexadecane), CB-TE1A1P, CB-TE2P, MM-TE2A, DM-TE2A, TE2A, Diamsar, SarAr (1-N-(4-aminobenzyl)-3,6,10,13,16,19-hexaazabicyclo[6.6.6]-eicosane-1,8-diamine), AmBaSar, BaBaSar, NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), p-SCN-Bn-NOTA, NODASA, NODAGA, NETA ({4-[2-(bis-carboxymethylamino)-ethyl]-7-carboxymethyl-[1,4,7]triazacyclononane (triazonan)-1-yl}-acetic acid), NETA-monoamide, C-NE3TA-NCS, C-NETA-NCS, 3p-C-NETA, TA CN-TM (N,N',N"-tris(2-mercaptoethyl)-1,4,7-triazacyclononane), DTPA (diethylenetriaminepentaacetic acid), p-SCN-Bn-1B-DTPA, p-SCN-Bn-1B4M-DTPA, CHX-A"-DTPA (2-(p-isothiocyanatebenzyl)-cyclohexyldiethylenetriaminepentaacetic acid), p-SCN-Bn-CHX-A"-DTPA, TRAP (1,4,7-triazacyclononane 1,4,7-tris[methyl(2-carboxyethyl)phosphinic acid]), AAZTA (1,4-bis(hydroxycarbonylmethyl)-6-[bis(hydroxycarbonylmethyl)]amino-6-methylperhydro-1,4-diazepine). )、NOPO、H2dedpa(1,2-[[6-(Carboxyl)-pyridin-2-yl]-methylamino]ethane)、H4octapa(N,N'-bis(6-carboxyl-2-pyridylmethyl)-ethylenediamine-N,N'-diacetic acid)、H2azapa(N,N'-[1-benzyl-1,2,3-triazol-4-yl]methyl-N,N'-[6-(carboxyl)pyridin-2-yl]-1,2-diaminoethane)、H5decapa(N,N"-[[6-(carboxyl)pyridin-2-yl]methyl]-diethylenetriamine-N,N',N"-triacetic acid)、p-SCN-Bn-H4octapa、HBED (N,N'-bis(2-hydroxybenzyl)-ethylenediamine-N,N'-diacetic acid), HBED-CC, (HBED-CC)TFP, SHBED (N,N'-bis(2-hydroxy-5-sulfobenzyl)-ethylenediamine-N,N'-diacetic acid), BPCA, CP256 (4-acetylamino-4-[2-[(3-hydroxy1,6-dimethyl-4-oxo-1,4-dihydro-pyridin-2-ylmethyl)-carbamoyl]-ethyl]-heptanedioic acid bis-[(3-hydroxy-1,6-dimethyl-4-oxo-1,4-dihydro-pyridin-2-ylmethyl)-amide]), PCTA (3,6,9,15-tetraazabicyclo[9.3.1]-pentadecan-1(15),11,13-triene-3,6,9,-triacetic acid), p-SCN-Bn-PCTA, DFO, p-SCN-Bn-DFO, H6phospha (N,N'-(methylenephosphonic acid)-N,N'-(6-(methoxycarbonyl)pyridin-2-yl)-methyl-1,2-diaminoethane), HEHA (1,4,7,10,13,16-hexaazacyclohexadecane-N,N'N",N"',N"",N""'-hexaacetic acid), p-SCN-Bn-HEHA, PEPA (1,4,7,10,13-pentazacyclopentadecan-N,N'N",N"'N""-pentaacetic acid), p-SCN-Bn-PEPA, Crown ether (Crown), MACROPA (4-amino-6-[ [16-[(6-carboxypyridin-2-yl)methyl]-1,4,10,13-tetraoxa-7,16-diazacyclooctadec-7-yl]methyl]pyridine-2-carboxylic acid), MACROPA-NCS, PYPA, PY4PA (6,6'-(((azanediylbis(ethane-2,1-diyl))bis((carboxymethyl)azanediyl))bis(methylene))pyridinedicarboxylic acid), noneunpa (6,6'-(((oxabis(ethane-2,1-diyl))bis((carboxymethyl)azanediyl))bis(methylene))pyridinedicarboxylic acid), DOTAM (2,2',2",2"'-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetamide), and derivatives thereof. In one embodiment, the radionuclide binding ligand is DOTA. .

[0080] Alternatively, the radionuclide binding ligand can be a peptide or a small organic part covalently bound to the radionuclide. As an example, the radionuclide binding ligand can be a short peptide or an organic part covalently bound to a radionuclide such as fluorine-18, iodine-124 or iodine-131 at an appropriate position. It should be understood that the radionuclide binding ligand should be selected so that it will not compete with the compound at the binding site at the CCK-2 receptor.

[0081] In one embodiment, the present invention provides a method for identifying CCK-2R positive cancer in a subject, comprising administering to the subject an effective amount of a compound according to the present invention or a pharmaceutically acceptable salt thereof, and detecting a radionuclide.

[0082] In another embodiment, the present invention provides a compound according to the present invention or a pharmaceutically acceptable salt thereof for use in a method of identifying CCK-2R positive cancer in a subject, the method comprising administering to the subject an effective amount of the compound, and detecting a radionuclide.

[0083] The compounds according to the invention localize to cancers overexpressing the CCK-2 receptor, such as medullary thyroid carcinoma (MTC), somatostatin-2R negative neuroendocrine tumors, stromal ovarian cancer (100% incidence) and small cell lung cancer (56% incidence).

[0084] After the compound is administered, for example by intravenous injection, the subject is placed on a scanner. When the injected radionuclide decays, it emits a positron, which annihilates with the electron, producing a photon or gamma ray pair traveling in opposite directions. Generally speaking, when the emitted photons reach a scintillator material in a scanning device, they are detected, producing a light pulse that is detected by a photomultiplier tube. Suitable scanning methods include computed tomography (CT), positron emission tomography (PET), and the combined procedure PET-CT.

[0085] Radionuclides used for diagnosis within the scope of the present invention include, but are not limited to, carbon-11, nitrogen-13, oxygen-15, fluorine-18, scandium-44, copper-64, gallium-67, gallium-68, yttrium-86, zirconium-89, technetium-99m, indium-111, iodine-124, iodine-125, and terbium-152.

[0086] In one aspect, the compounds of the present invention can be applied in the application of tumor ablation therapy to detect the degree of damage occurring in the affected tissue. The compound is located in CCK-2R positive tumors, and indicates the tumor size and position to the medical practitioner, and then allows continuous monitoring to track the tumor size and indicate the effectiveness of the medical treatment method. The ability to monitor the effectiveness of ongoing therapeutic treatments allows the subject to avoid experiencing invalid medical treatments, and then helps to develop patient-specific treatments. This has specific value in a wide variety of potential treatments available (for example, in cancer treatment, there are a wide range of chemotherapy and radiotherapy available). By using the compound to continuously monitor the tumor size, the effectiveness of a specific treatment is assessed earlier, and then the subject is prevented from being exposed to invalid treatment lines for a long time. The ability of the compound to indicate that medical treatment is invalid enables medical practitioners to change or modify the course of medical treatment. Such diagnostic tools allow time-saving measures to be taken and improve overall patient outcomes.

[0087] In another embodiment, the present invention provides a method of treating CCK-2R positive cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound according to the present invention or a pharmaceutically acceptable salt thereof.

[0088] In another embodiment, the present invention provides a compound according to the present invention, or a pharmaceutically acceptable salt thereof, for use in treating a CCK-2R positive cancer in a subject in need thereof.

[0089] In yet another embodiment, the present invention provides the use of a compound according to the present invention or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for diagnosing and / or treating CCK-2R positive cancer in a subject in need thereof.

[0090] Generally speaking, the radionuclide suitable for cancer treatment is a beta-emitting radionuclide. After the compound is used and positioned at the CCK-2R positive cancer, the radionuclide complexed with the compounds of this invention directly interacts with the tumor cells. The radionuclide used to treat cancer within the scope of the present invention includes but is not limited to copper-67, yttrium-90, iodine-131, terbium-149, terbium-161, lutetium-177, rhenium-186, rhenium-188, bismuth-212, bismuth-213, astatine-211, lead-212 and actinium-225.

[0091] The term "subject" as used herein refers to an animal, such as a bird or a mammal. Specific animals include rats, mice, dogs, cats, cows, sheep, horses, pigs, or primates. The subject can be a human, or referred to as a patient. The subject can also be a rodent, such as a mouse or a rat.

[0092] In general, the techniques used to prepare the compounds of the present invention are well known in the art, for example, see:

[0093] a) Alewood, P.; Alewood, D.; Miranda, L; Love, S.; Meutermans, W.; Wilson, D. Meth. Enzymol., 1997, 289, 14-28;

[0094] b) Merrifield, RB, J. Am. Chem. Soc., 1964, 85, 2149;

[0095] c) Bodanzsky, "Principles of Peptide Synthesis", 2nd Ed., Springer-Verlag (1993); and

[0096] d) Houghten, Proc. Natl. Acad. Scci. USA, 1985, 82, 5131.

[0097] Known solid phase or solution phase techniques can be used for the synthesis of compounds of the invention, such as coupling the N-terminus or C-terminus to a solid support (usually a resin), followed by stepwise synthesis of linear peptides. Protective group chemistry for protecting amino acid residues (including side chains) is well known in the art and can be found in, for example, Theodora W. Greene and Peter GMWuts, Protecting Groups in Organic Synthesis (3rd edition, John Wiley & Sons, Inc, 1999), the entire contents of which are incorporated herein by reference.

[0098] Briefly, compounds of the present invention were synthesized according to the following General Scheme 1.

[0099] Scheme 1: General chemical synthesis of compounds of the invention.

[0100]

[0101] N-terminal free linear peptides A to I were synthesized using a general Fmoc-based solid phase peptide synthesis (SPPS) protocol using a microwave-assisted automated CEM Liberty peptide synthesis module. N-terminal free linear peptides A to I were collectively deprotected and cleaved from the resin, purified by HPLC, and subsequently conjugated in solution with a radionuclide binding ligand (shown as DOTA). The compounds were then coordinated with radionuclides or natural gallium in sodium ascorbate buffer (pH 4.5) for binding assays to provide coordinated compounds in quantitative yields.

[0102] When the compounds of the present invention need to be purified, chromatography techniques such as reverse phase high performance liquid chromatography (HPLC) can be used. The compounds can be characterized by mass spectrometry and / or other suitable methods.

[0103] When a compound contains one or more functional groups that can be protonated or deprotonated (e.g. at physiological pH), the compound can be prepared and / or isolated as a pharmaceutically acceptable salt. It should be understood that a compound can be zwitterionic at a given pH. As used herein, the expression "pharmaceutically acceptable salt" refers to a salt of a given compound, wherein the salt is suitable for administration as a drug. Such salts can be formed, for example, by reaction of an acid or base with an amine or carboxylic acid group, respectively. In the case of zwitterionic compounds, the salt can be an inner salt, wherein the compound contains suitable proton donating and accepting functional groups.

[0104] Pharmaceutically acceptable acid addition salts can be prepared from inorganic and organic acids. Some examples of inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc. Some examples of organic acids include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc.

[0105] Pharmaceutically acceptable base addition salts can be prepared from inorganic bases and organic bases. The corresponding counterions derived from inorganic bases include sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts and magnesium salts. Organic bases include primary amines, secondary amines and tertiary amines, substituted amines include naturally occurring substituted amines, and cyclic amines, including isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, tromethamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, N-alkylglucosamine, theobromine, purine, piperazine, piperidine and N-ethylpiperidine.

[0106] Acid / base addition salts tend to be more soluble in aqueous solvents than the corresponding free acid / base forms.

[0107] The compounds of the present invention may be in crystalline form or as solvates (e.g., hydrates), and it is intended that both forms are within the scope of the present invention. The term "solvate" is a complex of variable stoichiometry formed by a solute (in the present invention, a peptide of the present invention) and a solvent. Such a solvent should not interfere with the biological activity of the solute. For example, the solvent may be water, ethanol, or acetic acid. Methods of solvation are well known in the art.

[0108] The present invention also provides a pharmaceutical composition comprising a compound according to the present invention and at least one pharmaceutically acceptable carrier or diluent.

[0109] As will be readily understood by those skilled in the art, the properties of route of administration and pharmaceutically acceptable carriers will depend on the properties of the disease and the mammal to be treated. Radiolabeled compounds are usually administered intravenously, enterally or parenterally to a subject as therapeutic agents and / or diagnostic agents. It is believed that the selection of a specific carrier or delivery system and route of administration can be easily determined by those skilled in the art. When preparing any preparation containing a compound according to the present invention, it should be noted that the activity of the compound is not destroyed in the process, and the compound can reach its site of action without being destroyed. Similarly, the selected route of administration should allow the compound to reach its site of action.

[0110] Those skilled in the art can easily determine suitable formulations for the compounds of the present invention using conventional methods. The determination of preferred pH ranges and suitable excipients (e.g., antioxidants) is conventional in the art. Buffer systems are conventionally used to provide a pH value in a desired range, and include carboxylic acid buffers, such as acetate, citrate, lactate, and succinate. Various antioxidants can be used for such formulations, including phenolic compounds (e.g., BHT or vitamin E), reducing agents (e.g., methionine or sulfite), and metal chelators (e.g., EDTA).

[0111] It is envisioned that the compounds according to the present invention will be prepared in parenteral dosage forms (including those suitable for intravenous, intrathecal and intracerebral or epidural delivery). Pharmaceutical forms suitable for injectable use include sterile injectable solutions or dispersions and sterile powders for the temporary preparation of sterile injectable solutions. It should be stable under manufacturing and storage conditions and can be preserved to avoid contamination by reduction or oxidation and microorganisms (e.g., bacteria or fungi).

[0112] The solvent or dispersion medium for injectable solution or dispersion can include any conventional solvent or carrier system for active compound, and can include water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.), suitable mixture thereof and vegetable oil, for example. Suitable fluidity can be maintained, for example, by using coating (for example lecithin), by maintaining the required particle size in the case of dispersion and by using surfactant. The prevention of microbial action can be produced by including a variety of antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc., if necessary. In many cases, it is preferred to include substances that regulate osmotic volume, such as sugar or sodium chloride. Preferably, the preparation for injection is isotonic with blood. The extended absorption of injectable composition can be produced by using delayed absorption agents (for example, aluminum monostearate and gelatin) in the composition. The drug form suitable for injectable use can be delivered by any suitable route (including intravenous, intramuscular, intracerebral, intrathecal, epidural injection or infusion).

[0113] Sterile injectable solutions are prepared by: aqueous liquids of the present invention are incorporated into a suitable solvent in the desired amount with various other ingredients (such as those listed above) (as required), followed by filtration sterilization. Typically, dispersions are prepared by incorporating various sterilized active ingredients into a sterile carrier comprising a basic dispersion medium and other ingredients from those listed above as required.

[0114] Pharmaceutically acceptable carriers and / or diluents include any and all solvents, dispersion media, antibacterial and antifungal agents, isotonic agents and absorption delaying agents, etc. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the active ingredient, their use in therapeutic compositions is contemplated. Supplementary active ingredients may also be incorporated into the composition.

[0115] It is particularly advantageous to formulate the composition in unit dosage form for ease of administration and uniformity of dosage. As used herein, unit dosage form refers to physically discrete units suitable as unit dosages for mammalian subjects to be diagnosed, each unit containing a predetermined amount of nanoparticles calculated to produce the desired diagnosis in association with the required pharmaceutically acceptable carrier. The specifications of the novel unit dosage forms of the present invention are governed by and directly depend on: (a) the unique characteristics of the nanoparticles and the specific diagnosis to be achieved, and (b) the inherent limitations in the field of compounding the nanoparticles of the present invention in living subjects suffering from disease conditions in which physical health is impaired.

[0116] As described above, the main active ingredient can be compounded in an effective amount with a suitable pharmaceutically acceptable carrier in a unit dosage form for convenient and effective administration. For example, the unit dosage form can contain nanoparticles in an amount of 0.25 μg to about 2000 mg. In terms of ratio, the active compound can be present in a carrier of about 0.25 μg to about 2000 mg / mL. In the case where the composition includes a supplementary active ingredient, the dosage is determined by reference to the conventional dosage and administration of the ingredient.

[0117] Throughout this specification and the appended claims, unless the context requires otherwise, the words "comprises / comprising" and variations thereof, will be understood to imply the inclusion of stated integers or groups of integers or steps but not the exclusion of any other integers or groups of integers.

[0118] Reference in this specification to any previous publication (or information derived therefrom) or any known matter is not and should not be taken as an acknowledgement or approval or any form of indication that the previous publication (or information derived therefrom) or known matter forms part of the common general knowledge in the field of endeavor to which this specification relates.

[0119] The invention will now be described with reference to the following non-limiting examples:

[0120] Example 1. Study of D-alanine insertion in hexaglutamic acid sequence

[0121] A series of peptides were synthesized in which, for example, one of the glutamic acid residues in the hexa-glutamic acid sequence of CP04 was replaced by a D-glutamic acid residue. In addition, the Gly-Trp-Met-Asp-Phe-NH2 sequence of CP04 was replaced by NMe-Gly-Trp-NMe-Nle-Asp-Nal. The peptides with alanine substitutions were then evaluated for their ability to disrupt the binding of CCK-2 receptor (CCK-2R) to [ 177 The ability of Lu]Lu-DOTA-CP04 to bind.

[0122] 1.1. Peptide synthesis

[0123] The peptides were assembled using a standard Fmoc-based solid phase peptide synthesis (SPPS) procedure using Rink amide resin (0.8 mmol / g) on ​​an automatic CEM Liberty Blue microwave peptide synthesizer (John Morris Group, Victoria, Australia). The peptides were assembled on a 0.1 mmol reaction scale. Fmoc-deprotection was performed in two stages as follows. The peptide-resin was treated with 20% piperidine / DMF (v / v; 5 mL) containing oxyma (0.1 M) under microwave irradiation (40 W, 40 ° C) for 30 seconds. It was then filtered and the same deprotection mixture was treated a second time under microwave irradiation (45 W, 75 ° C; 3 minutes). The peptide resin was then rinsed with DMF (3×4 mL). The coupling of all standard Fmoc-amino acids was achieved by adding Fmoc-amino acids (5 equivalents, 0.5 mmol), DIC (5 equivalents) and oxyma (10 equivalents) in DMF (4 mL) to the Nα-deprotected peptide-resin, and the mixture was stirred for 3 minutes under microwave irradiation (30 W, 90 ° C). After the sequence assembly, the peptide-resin was manually rinsed with dichloromethane (DCM) (3×5 mL) and then subjected to the cleavage step. The peptide from the solid support was overall deprotected and cleaved using a mixture of TFA / TIPS / H2O:DODT:1,3-dimethoxybenzene (85:5:5:2.5:2.5, % v / v / v / v / v; 8 mL) for 2 hours. The cleavage mixture was then filtered, the TFA solution was evaporated under a stream of nitrogen, and the crude product was isolated by grinding and subsequently washed with diethyl ether (3×30 mL).

[0124] An Aeris 5um PEPTIDE XB-C18 column (10 μm, The peptides were purified using a microplate reader (250×21.2 mm).

[0125] 1.2. For in vitro ligand binding assays 177Radiosynthesis of Lu]Lu-DOTA-CP04

[0126]

[0127] [ 177 Lu]Lu-DOTA-CP04 was prepared similarly to the reported method with slight changes (Ocak, M. et al., Eur J Nucl Med Mol Imaging 2011, 38(8), 1426-35). Briefly, DOTA-CP04 (30 μg, 14.6 nmol) dissolved in sodium acetate (0.5 M, 100 μL (0.3 g / μL peptide solution)) was prepared in 0.4 M ammonium acetate / 0.24 M 2,5-dihydroxybenzoic acid (200 μL, pH 4.5) containing ethanol (50 μL), L-methionine (50 μL of 10 mg / mL solution in milliQ water) and sodium ascorbate (50 μL, 0.05 M in milliQ water). 500 to 1000 MBq of non-carrier in 0.04 M HCl (50 to 100 μL) was added to the solution. 177 Lu]LuCl3 was added to the mixture and the resulting solution was heated at 80°C for 20 minutes. The fraction containing about 200 MBq of this material was purified by using Proteo (250 × 4.6mm) was used for HPLC purification, eluting at 1 mL / min with a gradient of MeCN containing 0.1% TFA and 2% EtOH (v / v), starting at 25% MeCN for 1 minute and increasing to 65% within 80 minutes. The product eluted at 35 minutes was manually collected in a vial containing water: ethanol (9: 1 v / v, 10 ml) and then captured on a Strata-X SPE cartridge. The product on the cartridge was then eluted with ethanol (1 mL). The fractions of the purified peptide were then diluted in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 1% fetal bovine serum to obtain a final concentration of 4 MBq / mL suitable for ligand binding assays.

[0128] 1.3 Ligand binding assay

[0129] CCK-2 receptor binding experiments were performed using A431 (human epidermoid carcinoma) cells (A431-CCK-2R) stably transfected to overexpress human full-length CCK-2 receptors. As a negative control, A431 cells stably transfected with an empty vector (A431-EV) were analyzed simultaneously. A431 cells were maintained in DMEM (Gibco, Australia) culture medium supplemented with 10% fetal bovine serum and 250 μg / mL G418, as previously described (Aloj L, et al., J Nucl Med., 2004, Mar; 45 (3), 485-94).

[0130] In A431-CCK-2R cells, the 177 The competitive binding assay of Lu]Lu-DOTA-CP04 evaluated the affinity of the peptides for CCK-2R. Briefly, cells were plated in 6-well plates at a density of 650,000 cells / well 48 hours before the experiment. On the day of the experiment, the peptides were diluted to at least 7 different concentrations (0 to 400 nM) in DMEM supplemented with 1% fetal bovine serum, and approximately 50,000 cpm of [ 177 Lu]Lu-DOTA-CP04. The cells were washed twice in ice-cold binding buffer (DMEM supplemented with 1% fetal bovine serum) and resuspended in 1.5 mL of the pre-prepared compound mixture and then incubated at 37°C for 1 hour. After incubation, the medium was removed and saved for analysis (unbound fraction), and the cells were washed twice in ice-cold binding buffer and combined with the unbound fraction. Finally, 1M NaOH was added to the cells and allowed to incubate for 5 minutes before collecting the lysate (bound fraction). A small volume of the lysate was saved aside for use Protein concentration was determined using the BCA protein assay kit (Thermo Fisher, Australia). All samples were analyzed using the Perkin Elmer 2480 Wizard 2 TM The assay was performed using a γ counter (PerkinElmer, Massachusetts, USA) and normalized to protein concentration. The half-maximal inhibitory concentration (IC) was determined using nonlinear regression calculated using GraphPad Prism software (GraphPad Software, California, USA). 50 ).

[0131] Compared with Comparative Example-1 and CP04, the apparent binding affinity (IC 50 )(Table 2; Figure 1 ) shows the effect of D-Ala substitution on each glutamic acid residue in the key hexameric peptide sequence.177 When Lu]Lu-DOTA-CP04 was tested, CP04 showed a greater than 3-fold decrease in affinity for the CCK-2 receptor, from 0.76 nM to 2.33 nM. This loss of affinity very clearly demonstrates the importance of the linker and radiolabeled complex in improving the affinity of the peptide for the target receptor. Similarly, Comparative Example-1, which contains a hexa-glutamic acid sequence, has been previously evaluated. nat The Ga-DOTA-peptide form has an affinity of 0.05 nM for the CCK-2 receptor, but a 5-fold decrease in its affinity for the receptor was observed. Nevertheless, Comparative Example-1 establishes an important baseline for the D-Ala analog, IC 50 It is 0.32nM.

[0132] Table 2: Relative binding affinities (IC 50 )

[0133]

[0134] Peptides -L- <![CDATA[IC 50 (nM)]]> A -D-Ala-D-Glu-D-Glu-D-Glu-D-Glu-D-Glu- 3.071 B -D-Glu-D-Ala-D-Glu-D-Glu-D-Glu-D-Glu- 1.233 C -D-Glu-D-Glu-D-Ala-D-Glu-D-Glu-D-Glu- 0.204 D -D-Glu-D-Glu-D-Glu-D-Ala-D-Glu-D-Glu- 1.618 E -D-Glu-D-Glu-D-Glu-D-Glu-D-Ala-D-Glu- 2.822 F -D-Glu-D-Glu-D-Glu-D-Glu-D-Glu-D-Ala- 1.617 Cmp ex-1 <![CDATA[-(D-Glu)6-]]> 0.319 CP04 2.325

[0135] A significant loss of binding affinity for the receptor of interest was seen with peptide A replaced at the terminal position 1 with D-alanine. Substitution of a small D-alanine residue at position 1 clearly demonstrates the important role of the terminal glutamate in improving the binding affinity of the peptide sequence for the CCK-2 receptor, as observed by a greater than 10-fold loss in activity. Similarly, a considerable loss of activity for the receptor was observed with peptide E replaced at position 5 with D-alanine. Previous work on this scaffold has shown that peptides preferentially fold into a β-hairpin structure through backbone and residue intramolecular interactions, and that this secondary structure is critical for its high activity. The D-alanine substitutions indicate that the D-glutamate residues at positions 1 and 5 are critical in the formation of this secondary structure, and therefore damage to these key residues results in a loss of activity.

[0136] The biological activity of peptides B, D, and F with iterative D-alanine substitutions in positions 2, 4, and 6 was evaluated, and it was found that the removal of the D-glutamic acid moiety was well tolerated while maintaining a moderate affinity for the CCK-2 receptor. In all three peptides, the resulting activity showed an approximately 5-fold loss of binding affinity to the target receptor. This result provides some insight into what other possible substitutions can be made without severely compromising the affinity between the ligand and the receptor, unlocking many opportunities for further modification.

[0137] Peptide C, with D-alanine substituted at position 3, produced the most active ligand of the peptide library evaluated. 50At 0.18 nM, peptide C showed a greater than 10-fold improvement in activity over the established CP04. In addition, it exceeded the already excellent affinity of the comparative peptide by almost 2-fold. Thus, position 3 is not only highly tolerated for modification, but also replacement of a neutral hydrophobic moiety (e.g., D-alanine) greatly improves binding between the ligand and the receptor. Given the three-dimensional β-hairpin structure of the scaffold, the highly polar and bulky D-glutamate at this position is likely to repel the corresponding residue on the CCK-2 receptor binding site, while the small neutral hydrophobic D-alanine residue is better suited to the binding pocket of the receptor.

[0138] Further studies were conducted to evaluate the binding affinity of peptides based on Cmp ex-1, in which the 3-(1-naphthyl)alanine residue was replaced by various substituted phenylalanine residues or by 3-(2-naphthyl)-L-alanine (Table 3). Most of the resulting peptides showed comparable affinities to Cmp ex-1 or CP04. Notably, replacing 3-(1-naphthyl)alanine with 3,4,5-trifluoro-phenylalanine resulted in improved affinity compared to the comparative examples.

[0139] Table 3: Relative binding affinities (IC 50 )

[0140]

[0141] Peptides -L- <![CDATA[R 1 ]]> <![CDATA[IC 50 (nM)]]> G <![CDATA[-(D-Glu)6-]]> 4-Fluoro-L-Phe 0.92 H <![CDATA[-(D-Glu)6-]]> 3,4-Difluoro-L-Phe 0.64 I <![CDATA[-(D-Glu)6-]]> 3,4,5-Trifluoro-L-Phe 0.26 J <![CDATA[-(D-Glu)6-]]> 2,3,4,5,6-Pentafluoro-L-Phe 1.92 K <![CDATA[-(D-Glu)6-]]> 4-Amino-L-Phe 0.52 L <![CDATA[-(D-Glu)6-]]> 3-(2-Naphthyl)-L-Alanine 0.60 Cmp ex-1 <![CDATA[-(D-Glu)6-]]> 3-(1-Naphthyl)-L-Alanine 0.33 CP04 3.17

[0142] Finally, studies were performed to evaluate the binding affinity of peptides based on peptide C, which had a D-alanine substitution at position 3, in which the 3-(1-naphthyl)alanine residue was replaced by various substituted phenylalanine residues, by tyrosine, or by 3-(2-naphthyl)-L-alanine (Table 4). Most of the resulting peptides showed comparable affinities to peptide C.

[0143] Table 4: Relative binding affinities (IC) of modified peptides based on peptide C 50 )

[0144]

[0145] Peptides -L- <![CDATA[R 1 ]]> <![CDATA[IC 50 (nM)]]> C <![CDATA[-(D-Glu)2-D-Ala-(D-Glu)3-]]> 3-(1-Naphthyl)-L-Alanine 0.204±0.015 M <![CDATA[-(D-Glu)2-D-Ala-(D-Glu)3-]]> 4-Amino-L-Phe 0.741±0.064 N <![CDATA[-(D-Glu)2-D-Ala-(D-Glu)3-]]> 3,4,5-Trifluoro-L-Phe 0.444±0.032 O <![CDATA[-(D-Glu)2-D-Ala-(D-Glu)3-]]> 4-Fluoro-L-Phe 0.368±0.040 P <![CDATA[-(D-Glu)2-D-Ala-(D-Glu)3-]]> 4-Trifluoromethyl-L-Phe 1.143 Q <![CDATA[-(D-Glu)2-D-Ala-(D-Glu)3-]]> 4-Cyano-L-Phe 1.568±0.089 R <![CDATA[-(D-Glu)2-D-Ala-(D-Glu)3-]]> Tyr 1.146±0.096 S <![CDATA[-(D-Glu)2-D-Ala-(D-Glu)3-]]> 3,4-Difluoro-L-Phe 2.657±0.226 T <![CDATA[-(D-Glu)2-D-Ala-(D-Glu)3-]]> 2,3,4,5,6-Pentafluoro-L-Phe 0.607±0.052 U <![CDATA[-(D-Glu)2-D-Ala-(D-Glu)3-]]> 3-Fluoro-L-Phe 0.305±0.013 V <![CDATA[-(D-Glu)2-D-Ala-(D-Glu)3-]]> 4-Acetyl-L-Phe 0.948±0.052

[0146] Example 2. Study on C-terminal modification of peptide C

[0147] Following the D-alanine studies, a series of compounds were synthesized in which the sequence of peptide C was modified to NMe-Gly-Trp-NMe-Nle-Asp-Nal. The radionuclide binding ligand DOTA was then conjugated to the peptide and used in combination with nat Gallium chelate.

[0148] 2.1. Synthesis and purification of DOTA-conjugated compounds

[0149] The synthesis and purification of the peptide were performed as described above. The conjugation of free DOTA to the peptide was carried out in solution, similar to the previously reported procedure, with only minor modifications (Schottelius, M.; Schwaiger, M.; Wester, H.-J., Tetrahedron Letters, 2003, 44 (11), 2393-2396). Briefly, DOTA (1.5 equivalents relative to the peptide) was pre-activated with N-hydroxysuccinimide (NHS) (1.5 equivalents relative to NHS) using EDCI (1.5 equivalents relative to NHS) and DIPEA (2 equivalents relative to NHS) in anhydrous DMSO (usually 500 μL). The reaction mixture was sonicated at 50° C. for 30 minutes until all the starting materials were completely dissolved. The peptide was then added to a minimum volume of anhydrous DMSO (usually 200 to 500 uL). The resulting mixture was shaken for another 30 minutes at room temperature and the reaction progress was monitored by MS analysis. When the reaction was complete as indicated by the disappearance of the m / z peak of the starting peptide in MS analysis, distilled water was added to form a 20:80 mixture of DMSO:water and the crude peptide was purified using reverse phase HPLC. The HPLC conditions used to purify the DOTA-compounds were identical to those reported above for the purification of the corresponding compounds.

[0150] Table 4: Chemical structures of Compounds 1 to 4 and Comparative Example-2.

[0151]

[0152] Compound# -L- <![CDATA[R 1 ]]> <![CDATA[R 2 ]]> 1 <![CDATA[-(D-Glu)2-D-Ala-(D-Glu)3-]]> Nal H 2 <![CDATA[-(D-Glu)2-D-Ala-(D-Glu)3-]]> Phe methyl 3 <![CDATA[-(D-Glu)2-D-Ala-(D-Glu)3-]]> Phe H 4 <![CDATA[-(D-Glu)2-D-Ala-(D-Glu)3-]]> Nal methyl Cmp-ex 02 <![CDATA[-(D-Glu)6-]]> Nal methyl

[0153] 2.2. nat Synthesis and Purification of Gallium Chelated DOTA-Compounds

[0154] Follow published procedures nat Chelation of gallium with DOTA-compounds (Maurin, M., et al., Nucl Med Rev Cent East Eur, 2015, 18(2), 51-5). Briefly, the compound (2 to 8 μmol) was suspended in freshly prepared ascorbic acid buffer (3 to 5 mL, 50 mg / mL) neutralized to pH 4.5 with concentrated NaOH solution. Gallium nitrate (4 equivalents relative to DOTA-peptide) was then added and the resulting mixture was heated at 95°C for 15 minutes. The resulting mixture was cooled to room temperature, centrifuged to remove any precipitate, and purified using reverse phase HPLC using the same chromatographic conditions used to purify the corresponding N-terminal free peptide.

[0155] 2.3. Receptor binding assay

[0156] The above method was used to evaluate compounds for their ability to disrupt the binding of CCK-2 receptor (CCK-2R) to [ 177 Lu]Lu-DOTA-CP04, and compared it with Comparative Example-2 ( Figure 2 ; Table 5).

[0157] Table 5: Relative binding affinities (IC) of compounds based on peptide C 50 )

[0158]

[0159] Compound -L- <![CDATA[R 1 ]]> <![CDATA[R 2 ]]> <![CDATA[IC 50 (nM)]]> <![CDATA[ nat See-1]]> <![CDATA[-(D-Glu)2-D-Ala-(D-Glu)3-]]> Nal H 0.080 <![CDATA[ nat For-2]]> <![CDATA[-(D-Glu)2-D-Ala-(D-Glu)3-]]> Phe methyl 0.185 <![CDATA[ nat For-3]]> <![CDATA[-(D-Glu)2-D-Ala-(D-Glu)3-]]> Phe H 0.042 <![CDATA[ nat For-4]]> <![CDATA[-(D-Glu)2-D-Ala-(D-Glu)3-]]> Nal methyl 0.096 <![CDATA[ nat Ga-Cmp ex-2]]> <![CDATA[-(D-Glu)6-]]> Nal methyl 0.158

[0160] Comparative Example-2 (Cmp ex-2), previously evaluated in several preliminary studies, showed favorable biostability, selectivity for target receptors, and the highest tumor uptake in biodistribution mouse studies when evaluated with other potential ligand scaffolds. Unfortunately, however, this peptide also had the highest renal uptake of the tested compounds. This is largely due to the highly acidic hexameric terminal peptide chain, which is known to promote renal uptake. Cmp ex-2 serves as an important template to study the effects of D-alanine substitutions, reducing the overall acidic nature of the peptide, and exploring the effects of this structural change on the binding affinity of the peptide for the target receptor. Compounds 1 to 4 were designed by changing the key C-terminal recognition sequence residues.

[0161] It has been previously determined ( nat The importance of (Ga)-DOTA conjugated peptides in improving the binding affinity of linear peptides to receptors. Therefore, variants of six-D-glutamic acid replacement of compounds 1, 3 and 4 have been previously studied and shown to have subnanomolar affinity for the CCK-2 receptor. The high aromaticity in the C-terminal position promotes the improvement of receptor binding. The peptides were further restricted by introducing N-methylation at key residues, which stabilized the peptides, but a slight decrease was indeed observed in affinity. In order to determine the impact of D-alanine replacement at position 3 of these peptides, the ligands were determined and showed very good tolerance to the replacement, with minimal loss of binding affinity among all tested compounds.

[0162] Example 3. Radiosynthesis of DOTA-compounds labeled with Ga-68

[0163]

[0164] 3.1. Radiolabeling of DOTA-compounds

[0165] DOTA-compounds 1 to 4 and comparative example-2 were radiolabeled according to published procedures (Haskali, MB et al., EJNMMI Radiopharmacy and Chemistry, 2019, 4 (1), 23). Briefly, a mixture of the corresponding DOTA-peptide precursor (30 μg) in 0.5 M sodium acetate solution (800 μL), ethanol (200 μL), 0.05 M sodium ascorbate (200 μL), 0.05 M 2,5-dihydroxybenzoic acid sodium salt (200 μL) and 10 mg / ml methionine (100 μL) was freshly prepared before radiosynthesis. The reaction mixture was then transferred to the reactor of the MultiSyn radiochemistry module. ITG was then eluted by using 0.05 M HCl (4 mL) 68 Ge / 68 The Ga generator delivers gallium-68 to the reaction vessel. The reaction mixture is heated to 90°C for 480 seconds (pH of the reaction mixture is 4.5). The reaction mixture is then diluted with water (5 mL) and the gallium-68 labeled peptide is captured on a Strata-X SPE cartridge. The captured product is rinsed with water (5 mL), eluted with ethanol (about 0.5 mL), and diluted with saline (9 mL) to obtain a compound with high radiochemical purity [ 68 Ga]Ga-1 to 4 and [ 68 Ga]Ga-Cmp ex-2.

[0166] 3.2. Quality Control of Ga-68 Labeled DOTA Compounds

[0167] The final formulation of each radiolabeled compound was evaluated for appearance, pH, radionuclide identity (half-life testing), purity, and radiochemical properties (Table 6). 68 Ga]Ga-1 to 4 and [ 68 Radiochemical properties and purity were assessed by radio-HPLC analysis of Ga]Ga-Cmp ex-2. The compounds labeled with Ga-68 were matched to their corresponding non-radioactive reference standards (with nat The radiochemical identity was determined by the retention time (and co-mobility) of the radioactive peaks observed (GaCl). The radiochemical purity was determined by integrating all observed radioactive peaks and comparing their relative area %.

[0168] Table 6: Compounds [ 68 Ga]Ga-1 to 4 and [ 68Quality control analysis of Ga]Ga-Cmp ex-2. The specific activity of the radiopharmaceuticals used in our experiments varies according to the amount of Ga-68 eluted from the ITG 68Ge / 68Ga generator, which depends on its age.

[0169]

[0170] Example 4. LogD 7.4 Determination and serum protein binding assay

[0171] Radiolabeled compounds [ 68 Ga]Ga-1 to 4 and [ 68 The distribution coefficient (LogD 7.4 ) is measured by measuring the aqueous phase (phosphate buffer pH 7.4 ) and the organic phase (n-octanol) to determine the distribution of radioactivity. To determine the distribution coefficient (Log D), the compound labeled with Ga-68 radioactivity was diluted to 20 pmol / mL in PBS (pH 7.4) and an equal volume of n-octanol was added. The mixture was vortexed vigorously for 10 cycles at room temperature, each cycle for 1 minute, and then centrifuged for 6 minutes to separate the two phases. Equal volumes from each phase were aliquoted into separate counting tubes and counted using a Perkin Elmer 2480 Wizard 2 TM The samples were analyzed for 1 min using a gamma counter (PerkinElmer, Massachusetts, USA). The partition coefficient was calculated using the following equation:

[0172]

[0173] Ga-68 labeled compounds 68 Ga]Ga-1 to 4 and [ 68 Ga]Ga-Cmp ex-2 shows high hydrophilicity and low LogD 7.4 Values ​​ranged from -4.1 to -2.5 (Table 7). 7.4 Consistent with other peptide-based theranostics successfully employed in clinical studies, rapid renal excretion and low background uptake resulted. In addition, metabolic stability and bioavailability are regulated in part by serum protein binding, which reduces glomerular filtration and enzymatic degradation and increases retention for tumor degradation (Smith, D., Di, L & Kerns, E., Nat Rev Drug Discov. 2010, 9, 929-939). Compound [ 68 Ga]Ga-1 to 4 showed moderate to high plasma protein binding (59% to 84%).

[0174] Table 7: 68 Ga]Ga-1 to 4 and [ 68 LogD of Ga]Ga-Cmp ex-2 7.4 LogD obtained after incubation in PBS / octanol mixture 7.4 Values ​​(n=5). Serum protein binding results after incubation of peptides with pooled human serum at 37°C for 1 hour.

[0175] <![CDATA[LogD 7.4 ]]> Serum protein binding (%) <![CDATA[[ 68 Ga]Ga-Cmp ex-2]]> -2.97±0.13 82.43±0.38 <![CDATA[[ 68 Ga]Ga-1]]> -2.88±0.09 78.69±2.11 <![CDATA[[ 68 Ga]Ga-2]]> -3.56±0.01 59.75±1.04 <![CDATA[[ 68 Ga]Ga-3]]> -4.06±0.14 63.62±0.45 <![CDATA[[ 68 Ga]Ga-4]]> -2.59±0.02 83.16±0.13

[0176] Example 5. Circular Dichroism

[0177] Circular dichroism (CD) measurements were obtained on a Chirascan-plus polarimeter (Applied photophysics, United Kingdom). Samples were prepared from diluted NMR samples where applicable. CD experiments were performed in triplicate between 195nm and 260nm with a 1nm step, 1nm bandwidth, 1 second per point time, and a 1mm quartz cell (Starna, United Kingdom). The signal was recorded as millidegrees, zeroed at 260nm, and normalized to obtain mean-residue ellipticity (MRE) units according to the following equation:

[0178]

[0179] where θ is the recorded ellipticity in millidegrees, C is the peptide concentration in dmol / L, l is the cell path-length in cm, and N is the number of residues per peptide.

[0180] Peptides A to F, compounds 1 to 4 and comparative examples 1 and 2 were analyzed by CD spectroscopy to investigate any changes in the secondary structure ( Figure 3 ). Peptides C and D showed a minimum at about 228 nm in water, indicating an α-helical structure, which was resolved in DPC micelles. The overall linear shape of Comparative Example-1 and peptides A and B in water showed a random coil structure.

[0181] The CD spectra of the remaining peptides and compounds showed two maxima at 200 nm and 230 nm and a minimum at about 220 to 227 nm.

[0182] Example 6. In vitro metabolic assay

[0183] Drug detoxification, drug metabolism, and metabolic stability are important determinants of the potency of theranostic candidates in the clinical setting. To assess metabolic stability, radiolabeled compounds were incubated in human serum, mouse liver or kidney homogenates, and HEPES buffer as a negative control. 68 Ga]Ga-1 to 4 and Comparative Example-2 ([ 68 Ga]Ga-Cmp ex-2) was attacked (challenge). Repeated samples (750 to 900pmol / mL) were incubated together with combined human serum, 15% mouse kidney homogenate, 30% mouse liver homogenate or 20mM HEPES (pH 7.3) (control) at 37°C. Human blood was collected and serum was prepared using SSTIIadvance vacuum blood collection tubes according to manufacturer's instructions (Becton Dickinson, New Jersey, USA). Mouse kidney and liver homogenate were prepared by washing the tissue in ice-cold HEPES buffer (pH 7.3) and placing it in a precooled tube containing HEPES buffer and 2.38mm metal beads (Mo-Bio Laboratories, Hilden, Germany). Tissue was homogenized using PowerLyzer24 homogenizer (Qiagen, Hilden, Germany) according to manufacturer's instructions. The peptides radiolabeled with Ga-68 were incubated with various lysates at 37°C and samples were collected at 15, 30, 60 and 90 minutes. The reaction was terminated by adding an equal volume of acetonitrile and centrifuging at 15,000 rpm for 2 minutes. The supernatant was diluted in water and then analyzed by HPLC using a Kinetex C18 XB column (5 μm 4.6×150 mm) with a gradient elution of 20% to 60% MeCN in water containing 0.1% formic acid at 1.5 mL / min in 7 minutes.

[0184] The experiments showed that the tested compounds remained intact when incubated with human serum for a time course of 90 minutes at 37°C ( Figure 4 a). Throughout the experiment, [ 68 Ga]Ga-1 and [ 68 Both Ga]Ga-3 underwent significant metabolism in liver and kidney homogenates ( Figure 4 b, 4c). [ 68 Ga]Ga-3 was particularly susceptible to degradation, with approximately 35% of the peptide intact in liver microsomes and only 20% of the peptide intact in kidney homogenates after 90 minutes. 68 Ga]Ga-1 showed a more stable metabolic profile, with 85% of the peptide intact and 75% of the parent peptide intact at 90 min in incubation with liver microsomes. 68Ga]Ga-2,[ 68 Ga]Ga-4 and [ 68 Ga]Ga Cmp ex-2 was extremely stable in liver and kidney homogenates, with no observable metabolites detected at the 90 min time point ( Figure 4 ).

[0185] Corlett and colleagues have previously demonstrated the metabolic instability of the terminal Phe residue present in compound 3 (Corlett, A. et al., J. Med. Chem 2021, 64, 4841-4856). The results shown here are consistent with those of previous studies, in which compound 3 showed significant degradation at 90 minutes. Further analysis of metabolites determined the cleavage site at position 13 (terminal Phe residue). As shown in compound 2, enzymatic cleavage at Phe residues can be prevented by replacing non-natural N-methylated Phe with natural Phe moieties. Incorporating N-methylated residues is a known strategy for enhancing protease resistance. This is demonstrated again in compound 1 and its N-methylated variant compound 4 at position 13. Although not as susceptible to metabolic degradation as compound 3, N-methylation of the terminal naphthylalanine residue prevents enzymatic degradation of the peptide, leaving 100% of the parent peptide intact.

[0186] Example 7. Small Animal PET Imaging and Biodistribution Studies

[0187] The compounds were evaluated using static small animal PET imaging and quantitative biodistribution studies. 68 Ga]Ga-1 to 4 and [ 68 Female BALB / c nu / nu mice (8 to 10 weeks old) were inoculated subcutaneously in the right flank with 3×10 6 A431CCK2R cells. Mice were weighed and tumor volumes (mm 3 ) was calculated as length × width × height × π / 6. Mice were assigned to imaging and / or biodistribution groups (tumor volume: 50 to 500 mm 3). The corresponding tracer labeled with Ga-68 (4 to 5MBq, 100 μL, 13.7 to 14.2pmol) was mixed with a vehicle solution (75 μL, DMSO: Tween 80: water, 2:2:6v / v / v) and then administered intravenously to 6 mice by tail vein injection. Three mice were euthanized 1 hour after injection for biodistribution, and the selected tissues were cut, weighed, and counted using a Capintec (Captus 4000e) gamma counter. Three mice were anesthetized using 1.5% isoflurane and imaged using a G8 small animal PET / CT scanner (Perkin Elmer / sofiBiosciences) at 1 and 2 hours. A 10-minute static PET scan was obtained, followed by a CT scan. PET images were obtained using the G8 acquisition engine software and reconstructed using a 3D maximum likelihood and expectation maximization (maximal likelihood andexpectation maximization, ML-EM) algorithm. PET images were analyzed using VivoQuant software (version 3.0) (inviCRO Imaging Services and Software) to quantify the maximum standardised uptake value (SUVmax) in the region of interest. Following 2 hour time point imaging, mice were harvested for biodistribution analysis as above.

[0188] To block CCK2R binding, N-[(3R)-2,3-dihydro-1-[2-(2-methylphenyl)-2-oxoethyl]-2-oxo-5-phenyl-1H-1,4-benzodiazepine was used. -3-yl]-N'-(3-methylphenyl)-urea solution (YM022; SigmaAldrich). YM022 (75 μL of a 1 mg / mL solution consisting of DMSO: Tween 80: water, 2:2:6 v / v / v) was added to the corresponding Ga-68 labeled tracer (4 to 5 MBq, 100 μL, 13.7 to 14.2 pmol), and the mixture (175 μL in total) was intravenously administered to three mice via tail vein injection, and organs were harvested 1 hour after injection for biodistribution analysis, as above. All animal studies were approved by the Peter MacCallum Cancer Centre Animal Experimentation Ethics Committee and conducted in accordance with the Australian code for the care and use of animals for scientific purposes (2013, 8th edition).

[0189] The compound [ 68 The imaging and biodistribution characteristics of Ga]Ga-1 to 4 are similar to those of [ 68 Ga]Ga-Cmp ex-2 was compared ( Figure 5 ). [ 68 Ga]Ga-Cmp ex-2 showed the best tumor uptake. However, disadvantageously for this compound, it also showed the highest renal uptake. 68 Compared with Ga]Ga-Cmp ex-2, compound [ 68 Ga]Ga-1 to 4 had almost half or less renal uptake. Compounds [ 68 Ga]Ga-4 had the best tumor to kidney ratio. Biodistribution of all tested compounds was low in blood, lung, heart, liver and spleen.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof: in: R 1 is a side chain of an amino acid selected from phenylalanine, 3-(1-naphthyl)alanine, 3-(2-naphthyl)alanine or tyrosine, wherein the phenylalanine residue is optionally substituted with one or more halogen, -NH2, cyano, C 1-6 Alkyl, halogenated C 1-6 Alkyl or acetyl substitution; R 2 Selected from H or C1-C3 alkyl; A is a radionuclide binding ligand; and -L- is the amino acid sequence: -D-Glu-D-Glu-D-Ala-D-Glu-D-Glu-D-Glu-.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof represented by the following formula (Ia): Where R 2 As defined in claim 1.

3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof represented by the following formula (Ib): Where R 2 As defined in claim 1; Each occurrence of R 3 independently selected from halogen, -NH2, cyano, -OH, C 1-6 Alkyl, halogenated C 1-6 alkyl or acetyl; and n is 0 to 5.

4. The compound according to claim 3, or a pharmaceutically acceptable salt thereof, wherein n is 0.

5. The compound according to claim 3 or a pharmaceutically acceptable salt thereof, wherein R 3 is F and n is 1 to 3.

6. The compound according to claim 5, or a pharmaceutically acceptable salt thereof, wherein n is 3.

7. The compound according to claim 5, or a pharmaceutically acceptable salt thereof, wherein n is 1.

8. The compound according to claim 3 or a pharmaceutically acceptable salt thereof, wherein R 3 is -NH2 and n is 1.

9. The compound according to claim 1 or a pharmaceutically acceptable salt thereof represented by the following formula (Ic): Where R 2 As defined in claim 1.

10. The compound according to any one of claims 1 to 9, or a pharmaceutically acceptable salt thereof, wherein R2 is methyl.

11. The compound according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof, wherein A is a radionuclide binding ligand selected from the following: DOTA, DOTA-NHS-ester, p-SCN-Bn-DOTA, DOTAGA, DOTAGA-anhydride, CB-DO2A, TCMC, p-SCN-Bn-TCMC, 3p-C-DEPA, 3p-C-DEPA-NCS, p-NH2-Bn-oxo-DO3A, TETA.BAT, p-NH2-Bn-TE3A, C-TETA, CB-TE2A, CB-TE1A1P, CB-TE2P, MM-TE2A, DM-TE2A, TE2A, Diamsar, SarAr, AmBaSar, BaBaSar, NOTA, p-SCN-Bn-NOTA, NODASA, NODAGA, NETA, NETA-monoamide, C-NE3TA-NCS, C-NETA-NCS, 3p-C-NETA.TACN-TM.DTPA, p- SCN-Bn-1B-DTPA, p-SCN-Bn-1B4M-DTPA.CHX-A″-DTPA, p-SCN-Bn-CHX-A″-DTPA,TRAP,AAZTA,NOPO,H2dedpa,H4octapa,H2azapa,H5decapa,p-SCN-Bn-H4octapa,HBED,HBED-CC,(HBED-CC)T FP, SHBED, BPCA, CP256, PCTA, p-SCN-Bn-PCTA, DFO, H6phospha, p-SCN-Bn-DFO, HEHA, p-SCN-Bn-HEHA, PEPA, p-SCN-Bn-PEPA, crown ether, MACROPA, MACROPA-NCS, pypa, py4pa, noneunpa, DOTAM, and its derivatives.

12. The compound according to any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, wherein A is DOTA.

13. The compound according to any one of claims 1 to 12, or a pharmaceutically acceptable salt thereof, further comprising a radionuclide complexed with the radionuclide binding ligand.

14. A pharmaceutical composition comprising the compound according to claim 13 or a pharmaceutically acceptable salt thereof.

15. A method for identifying CCK-2R positive cancer in a subject, comprising administering to the subject an effective amount of the compound according to claim 13 or a pharmaceutically acceptable salt thereof, and detecting the radionuclide.

16. The method of claim 15, wherein the radionuclide is detected using positron emission tomography (PET).

17. The method of claim 15 or 16, wherein the radionuclide is selected from carbon-11, nitrogen-13, oxygen-15, fluorine-18, scandium-44, copper-64, gallium-67, gallium-68, yttrium-86, zirconium-89, technetium-99m, indium-111, iodine-124, iodine-125 and terbium-152.

18. A method for treating CCK-2R positive cancer in a subject in need thereof, comprising administering to the subject an effective amount of a compound according to claim 13 or a pharmaceutically acceptable salt thereof.

19. The method of claim 18, wherein the CCK-2R positive cancer is selected from medullary thyroid carcinoma, somatostatin-2R negative neuroendocrine tumors, stromal ovarian cancer or small cell lung cancer.

20. The method of claim 18 or 19, wherein the radionuclide is selected from copper-67, yttrium-90, iodine-131, terbium-149, terbium-161, lutetium-177, rhenium-186, rhenium-188, bismuth-212, bismuth-213, astatine-211, lead-212, and actinium-225.

21. The compound according to claim 13, or a pharmaceutically acceptable salt thereof, for use in a method for identifying CCK-2R positive cancer in a subject, the method comprising administering an effective amount of the compound to the subject, and detecting the radionuclide.

22. The compound of claim 13, or a pharmaceutically acceptable salt thereof, for use in treating CCK-2R positive cancer in a subject in need thereof.

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