Precursors and radiotracers for neuroendocrine theranostics

Abstract

DAZTA 5 -A radioisotope named PPA2 68 Ga and 177 Novel precursors for PET / CT diagnosis and nuclear therapy of SSR-active lesions using Lu improve affinity, specificity, and imaging of small metastases.

Description

Detailed Description of the Invention

[0001] 〔Technical Field〕 The present invention relates to a precursor named DAZTA , -PPA2, which contains the chelator DAZTA 5 and the peptide ligand PPA2 conjugated thereto, for radiolabeling and targeting the somatostatin receptor 2 (SSR2).

[0002] DAZTA 5 is 1,4-bis(carboxymethyl)-6-[methyl-carboxymethyl-amino]-6-[pentanoic acid]-1,4-diazepam or 1,4-bis(carboxymethyl)-6-[bis(carboxymethyl)-amino]-6-[pentanoic acid]-1,4-diazepam.

[0003] And PPA2 is Cpa-cyclo[DCys-Pal-DAph(Cbm)-Lys-Thr-Cys]DTyr-NH2, where Cpa is 4-chloro-phenylalanine, DAph(Cbm) is D-4-amino-carbamoyl-phenylalanine, and Pal is pyridylalanine. 〔Background Art〕

[0004] 〔Nuclear Medicine Diagnosis of Neuroendocrine Tumors〕 Positron emission tomography (PET) combined with computed tomography (CT) using gallium-68 (Ga-68 or 68 Ga) is a clinically established nuclear diagnostic technique today. The US Food and Drug Administration and the European Medicines Agency have 68 approved Ga-labeled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid ( 68 Ga-DOTA-octreotate or 68 Ga-DOTA-TATE) and 68 Ga-DOTA-d-Phe(1)-Tyr(3)-octreotide ( 68We have approved Ga-DOTA-TOC for localization diagnostics of somatostatin receptor (SSR)-positive neuroendocrine neoplasms (NETs) in adult and pediatric patients (USA), and for adult patients with signs of well-differentiated gastrointestinal pancreatic neuroendocrine neoplasms (GEP-NETs) (EU). DOTA-TOC and DOTA-TATE are DOTA-chelators to which an 8-amino acid cyclic peptide with high affinity for somatostatin receptor 2 (SSR2) is attached, and they act as agonists against SSR2.

[0005] The diagnostic value of PET / CT is determined by sensitivity, specificity, and accuracy. Sensitivity refers to the proportion of correctly identified positives (true positives divided by the sum of true positives and false negatives). Specificity refers to the proportion of correctly identified negatives (true negatives divided by the sum of true negatives and false positives). Diagnostic accuracy relates to the ability of the test to distinguish between target diseases and health conditions. This discriminative ability can be quantified by measures such as sensitivity and specificity, the target-to-background ratio, or the area under the receiver operating characteristic curve (ROC curve).

[0006] SSR imaging sensitivity can potentially be improved by increasing the affinity of the PET tracer to the target SSR, or by broadening the binding spectrum to include SSR3 and SSR5 in addition to SSR2. The latter approach results in higher tracer uptake in SSR-positive target tissue, but may also increase off-target uptake, resulting in a lower tumor-to-background ratio and reduced image contrast.

[0007] DOTA-NOC (DOTA-1-Nal(3)-octreotide) and HA-DOTA-TATE (DOTA-iodo-Tyr), which have high affinity for SSR2, SSR3, and SSR5, have been reported as somatostatin receptor ligands for PET / CT, offering improved diagnostic accuracy and other advantages. 3 There are SSR agonists like Octreotide.

[0008] DOTA-ST8951 (DOTA-(4-amino)-D-Phe-cyclo[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2) shows high affinity for SSR2 and SSR5, but it increases uptake into the liver, thus affecting the target / background ratio. 18 F-18-labeled SSR ligands such as F-FET-βAG-TOCA have been reported to have poor imaging characteristics.

[0009] [SSR Agonists and Antagonists] In nuclear medicine diagnostics, SSR agonists are complemented by SSR antagonists corresponding to multiple binding sites on target cells. This is due to the fact that the majority of SSRs exist in an inactive form and therefore correspond only to antagonist binding. Thus, compared to SSR2 agonist radiotracers, 68 Ga-DOTA-JR11 and 68 Complementary SSR2 antagonist radiotracers such as Ga-NODAGA-LM3 (JR11=Cpa-cyclo[D-Cys-Aph(Hor)-D-Aph(Cbm)-Lys-Thr-Cys]D-Tyr-NH2); NODAGA, i.e., 1,4,7-triazacyclononane, 1-glutaric acid-4,7-acetic acid; LM3, i.e., Cpa-cyclo[D-Cys-Tyr-D-4-amino-Phe(carbamoyl)-Lys-Thr-Cys]D-Tyr-NH2) show higher uptake in preclinical and clinical settings despite not having particularly high SSR2 affinity. In a direct comparison, 68 Ga-DOTA-JR11 is 68 While superior to Ga-DOTA-TATE in detecting liver metastases, its sensitivity for detecting bone metastases is significantly lower. This finding highlights the importance of image contrast in PET / CT diagnosis.

[0010] To improve image contrast, and thus specificity, PET / CT tracers must have low affinity for off-target tissues and disease-unrelated receptors. Extending the binding spectrum to SSR1, SSR3, SSR4, and SSR5 receptor subtypes may increase off-target uptake, potentially reducing specificity and image contrast.

[0011] Furthermore, selecting appropriate targets that are specific to or overexpressed in each disease significantly impacts the diagnostic outcome. For example, the most commonly used PET tracer is a type of radiolabeled glucose. 18 F-2-fluoro-2-deoxy-D-glucose ( 18 It is F-FDG, which is absorbed by various tissues, and in non-malignant diseases, it is absorbed by tissues where systemic glucose consumption is increased.

[0012] Clinically approved 68 Ga-DOTA-TATE and 177 Theranostic dyads containing Lu-DOTA-TATE have significantly advanced the treatment of patients suffering from NETs, ​​embodying the advantages of nuclear medicine in fighting cancer. Further research to make improved therapeutic tools available for NET patients has revealed that radioisotope-labeled SSR2 antagonists offer significant advantages over their corresponding agonists, both at the preclinical and in vivo levels. Unlike radioactive agonists, SSR2 radioactive antagonists are not taken up by target cells via endocytosis. Nevertheless, SSR2 radioactive antagonists exhibit superior pharmacokinetics, strongly binding to and remaining in SSR2-positive tumor lesions for extended periods, while being rapidly eliminated from healthy tissues. The latter is also relevant to healthy organs that physiologically express SSR2, such as the stomach and pancreas. Studies at the molecular and cellular levels have shown that radioactive antagonists occupy a larger portion of the SSR2 population (consisting of both active and inactive receptors) on the target cell membrane, while agonists bind only to a subpopulation of active SSR2 on the cell membrane before being taken up into the cell.

[0013] In recent years, several types of SSR2 antagonists have been developed and are coupled with various chelators for complexing divalent and trivalent radiometals for the diagnosis and treatment of NETs. In particular, DOTA-LM3 (DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; LM3 = H-DPhe-cyclo[DCys-Tyr-DAph(Cbm)-Lys-Thr-Cys]-DTyr-NH2; DAph(Cbm)4 = D-4-amino-carbamoyl-phenylalanine, see Scheme 1) shows promise for the diagnosis and staging of NETs (RP Baum, J. Zhang, C. Schuchardt, D. Mueller, H. Maecke; First-in-human study of novel SSTR antagonist) 177 Lu-DOTA-LM3 for peptide receptor radionuclide therapy in patients with metastatic neuroendocrine neoplasms: dosimetry, safety and efficacy; Journal of Nuclear Medicine March 2021, jnumed.120.258889; see DOI: https: / / doi.org / 10.2967 / jnumed.120.258889). [Chelators that complexize metallic radioisotopes] According to current knowledge in this field - Chelates and radioisotopes significantly influence the affinity and pharmacokinetics of SSR radiotracers; -DOTA can have a serious impact on the ligand affinity of SSRs; - Chelates, radioisotopes, and SSR ligands interact in unpredictable ways, either synergistically or antagonistically.

[0014] For example, the chelator DOTA is not suitable for complex formation of relatively small (radioactive) metallic gallium molecules, requiring an increased reaction temperature, which is detrimental to many antibodies and heat-sensitive biomolecules. After complex formation, 68Ga-DOTA chelate requires cooling time before intravenous injection. 68 Because Ga has a short half-life of 67.7 minutes, its clinical use is limited.

[0015] EP2801582A1 (paragraphs 102, 129; Table 12) discloses a radiolabeled precursor having the structure DOTA-Cpa-cyclo[DCys-Pal-DAph(Cbm)-Lys-Thr-Cys]DTyr-NH2, which was not quantitatively uptaken in HEK293-SSR2 tumor cells and is clearly a reference example.

[0016] [ka]

[0017] [DATA as a "hybrid" chelator] Recently developed DATA-type chelators (see Scheme 2) exhibit cyclic, acyclic, and intermediate properties, and compared to existing chelators, 68 It possesses properties that are advantageous for Ga labeling. In particular, 68 Rapid and quantitative radioactive labeling using Ga is possible over a wide pH range at room temperature. Furthermore, 68 Ga-DATA chelates are transchelated (DTPA and apo-transferrin) and transmetallated (Fe III They are immune to )

[0018] The DAZTA of the present invention, which has a diazepam ring (1,4-bis(carboxymethyl)-6-[methyl-carboxymethyl-amino]-1,4-diazepam and 1,4-bis(carboxymethyl)-6-[bis(carboxymethyl)-amino]-1,4-diazepam, respectively) as its core, is shown in Scheme 2 below. 5 This shows a chelator.

[0019] [ka]

[0020] [Detailed explanation] The present invention aims to improve nuclear theranostics in diseases characterized by increased expression of somatostatin receptors (SSRs), particularly neuroendocrine carcinomas.

[0021] The purpose of this is DAZTA 5 -PPA2 is named and is achieved by a precursor or a salt thereof having the following structure.

[0022] [ka]

[0023] The precursor of the present invention DAZTA 5 -A favorable embodiment of PPA2 has the following characteristics:

[0024] [ka]

[0025] The present invention further aims to provide a radiopharmaceutical for nuclear imaging of diseases associated with elevated SSR expression, particularly neuroendocrine carcinomas. [ka] DAZTA 5 -PPA2 and radioactive isotopes that complex with it 68 Radioactive tracer made of Ga 68 Ga-DAZTA 5 -Achieved by PPA2

[0026] The present invention further aims to provide a radiopharmaceutical for nuclear therapy of diseases associated with elevated SSR expression, particularly neuroendocrine carcinomas. This objective is to provide a radiopharmaceutical for nuclear therapy of diseases associated with elevated SSR expression, [ka] DAZTA 5-PPA2 and radioactive isotopes that are complexed with it 177 Radioactive tracer made of Lu 177 Lu-DAZTA 5 -Achieved by PPA2

[0027] Further preferred embodiments of the present invention relate to the following: [ka] DAZTA 5 - Radiopharmaceutical kits containing PPA2 or its salts; [ka] DAZTA 5 - A drug kit containing radioactive material including PPA2 or a salt thereof; [ka] DAZTA 5 - A radiopharmaceutical kit comprising PPA2 or a salt thereof, and a solvent selected from water, 0.45% NaCl aqueous solution, 0.9% NaCl aqueous solution, Ringer's solution (lactated Ringer's solution), 5% dextrose aqueous solution, and alcohol aqueous solution; [ka] DAZTA 5 A radiopharmaceutical kit comprising PPA2) or a salt thereof, and a solvent selected from water, 0.45% NaCl aqueous solution, 0.9% NaCl aqueous solution, Ringer's solution (lactated Ringer's solution), 5% glucose aqueous solution, and alcohol aqueous solution; A radiopharmaceutical kit containing [the specified substance]; [ka] DAZTA 5 - A first vial containing PPA2 or a salt thereof, and [ka] DAZTA 5 - A radiopharmaceutical kit containing a second vial containing PPA2 or a salt thereof; [ka] DAZTA 5 - A first vial containing PPA2 or a salt thereof, [ka] DAZTA 5 - Second vial containing PPA2 or a salt thereof, A third vial containing a solvent selected from water, 0.45% NaCl aqueous solution, 0.9% NaCl aqueous solution, Ringer's solution (lactated Ringer's), 5% glucose aqueous solution, and alcohol aqueous solution. Optionally, a fourth vial containing a solvent selected from water, 0.45% NaCl aqueous solution, 0.9% NaCl aqueous solution, Ringer's solution (lactated Ringer's solution), 5% glucose aqueous solution, and alcohol aqueous solution. A radiopharmaceutical kit containing [the specified ingredient].

[0028] The present invention 68 Ga-DOTA-TOC or 68 In PET / CT imaging using Ga-DOTA-TATE, when there are clinical signs of somatostatin receptor-positive neuroendocrine tumors, but the standardized uptake (SUV) is low or the results are difficult to interpret, 68 This enables the detection of somatostatin receptor expression using Ga-PET / CT.

[0029] DAZTA precursor having X=CH3 or X=CH2COOH 5 -PPA2 is a radioactive isotope for diagnostic purposes. 68 Ga or 44 Sc, and for therapeutic purposes 177 Lu,90 Y or 161 It may also be complexed with Tb. 68 Ga-DAZTA 5 -PPA2, 44 c-DAZTA 5 -PPA2, 177 Lu-DAZTA 5 -PPA2, 90 Y-DAZTA 5 -PPA2, and 161 Tb-DAZTA 5 The corresponding radiotracer, designated as -PPA2, exhibits an outstanding target-to-background ratio, with preferential uptake in tumor lesions and low uptake in healthy tissues, particularly liver and spleen tissue. Therefore, the radiotracer of the present invention provides high image contrast, sensitivity, and selectivity for the diagnosis and treatment of diseases associated with elevated somatostatin receptor expression.

[0030] Therefore, the present invention encompasses the following radioactive tracers: 68 Ga-DAZTA 5 -PPA2(X=CH3), that is, 68 Ga-DATA 5m -PPA2; 44 Sc-DAZTA 5 -PPA2(X=CH3), that is, 44 Sc-DATA 5m -PPA2; 68 Ga-DAZTA 5 -PPA2(X=CH2COOH), that is, 68 Ga-AAZTA-PPA2; 44 Sc-DAZTA 5 -PPA2(X=CH2COOH), that is, 44 Sc-AAZTA-PPA2; 177 Lu-DAZTA 5 -PPA2(X=CH2COOH), that is, 177 Lu-AAZTA-PPA2; 90 Y-DAZTA5 -PPA2 (X = CH2COOH), i.e., 90 Y-AAZTA-PPA2; 111 In-DAZTA 5 -PPA2 (X = CH2COOH), i.e., 111 In-AAZTA-PPA2; 161 Tb-DAZTA 5 -PPA2 (X = CH2COOH), i.e., 161 Tb-AAZTA-PPA2; and 225 Ac-DAZTA 5 -PPA2 (X = CH2COOH), i.e., 225 Ac-AAZTA-PPA2. DAZTA 5 -PPA2 is readily provided in lyophilized form and can be packaged as a point-of-use kit together with adjuvants such as a pH buffer, an antioxidant radical scavenger to prevent radiolysis, and a lyophilization bulking agent. The DAZTA 5 -PPA2-containing kit, respectively 68 GaCl3, 44 ScCl3, or 177 a European Pharmacopoeia-compliant hydrochloric acid solution containing LuCl3 is added at room temperature, and the reagent mixture is simply shaken to obtain the radiotracer 68 Ga-DAZTA 5 -PPA2, 44 Sc-DAZTA 5 -PPA2, or 177 Ga-DAZTA 5 -PPA2 for use in preparing. An automated module with a heating compartment is not required.

[0031] 〔Example〕 〔Synthesis method〕 tert-Butyl-protected and carboxylated DAZTA 5 -PPA2 prochelator is synthesized as described below in connection with Schemes 4 and 5.

[0032] The SSR2 peptide ligand PPA2 shown in Scheme 3 is prepared by general solid-phase peptide synthesis (SPPS) using Fmoc as a protecting group and a deprotection / binding cycle (Scheme 6), purified by reverse-phase chromatography, and then characterized by HPLC and MS.

[0033] [ka]

[0034] [Reagents and Analysis] Reagents were purchased from Sigma-Aldrich® or Merck® and used without purification. Purite® water was filtered through a Millex® Millipore filter membrane (0.54 μm). The reaction progress was monitored using a silica TLC plate (Silica 60F 254 4.5 × 4.5 cm, Merck) and UV absorbance and / or KMnO4 titration at a wavelength of 254 nm. Column chromatography was performed using silica gel 60 (Fisher Scientific®, 0.04–0.063 nm).

[0035] The synthesized compound is 1 H-, 13 Chemically identified by C-NMR and HRMS, but DAZTA 5 -Only the PPA2 conjugate was identified by HPLC and HRMS. 1 H-, 13 1C-NMR and HRMS data should be described in SI units.

[0036] NMR spectrum ( 1 H, 13C, HSQC, HMBC) were recorded using an Avance III HD400 spectrometer (Bruker, USA). Chemical shifts are shown in ppm. MS (ESI) was performed using a Thermo Quest Navigator Instrument (Thermo Electron). Mass spectrometry results are given in m / z in g / mol units. HPLC was performed using a metal-free Dionex ICS-5000 system equipped with a quaternary pump, AS-50 autosampler, UV / Vis detector, and AFC-3000 automated fraction collector.

[0037] [DAZTA 5 (Synthesis of X=CH3 prochulator) 5-(1,4-dibenzyl-6-nitro-[1,4]diazepam-6-yl)-methyl pentanoate (1) 2-Nitrocyclohexanone (0.608 g, 4.3 mmol) was added to Amberlist A21 (1.216 g, 2 mass equivalents) in EtOH and stirred at 60°C for 2 hours under argon. N,N′-Dibenzyl-ethylenediamine (1.020 g, 4.3 mmol) and paraformaldehyde (0.446 g, 14.9 mmol) were added and the reaction was stirred overnight at 60°C. The mixture was filtered through Celite® and the solvent was removed under reduced pressure. The resulting residue was redissolved in CHCl3 (40 mL), washed sequentially with aqueous K2CO3 (2 × 30 mL, 0.1 M) and H2O (30 mL), dried on MgSO4, filtered, and the solvent was removed under reduced pressure. The compound was purified by silica gel column chromatography (DCM) to obtain the title compound as a yellow oil (1.607 g, 85%). Rf = 0.80 (DCM).

[0038] [5-(1,4-dibenzyl-6-nitro-[1,4]diazepam-6-yl)-methyl pentanoate (2)] A catalytic amount of Pd(OH)2 / C and acetic acid (50 μL, 0.87 mmol) were added to protected triamine 1 (0.10 g, 0.29 mmol) in MeOH (20 mL), and the mixture was stirred under a hydrogen atmosphere for 3 hours (1 atm H2). Complete reduction of the nitro group and cleavage of the benzyl N-substituent were confirmed by TLC (DCM). Pd(OH) / C was removed using a Celite® filter. The solvent was removed under reduced pressure to obtain a yellow oil (0.065 g, 97%).

[0039] [5-[1,4-bis-tert-butoxycarbonylmethyl-6-(tert-butoxycarbonylmethyl-amino)-[1,4]diazepam-6-yl]-methyl pentanoate (3)] tert-butyl-bromoacetate (0.567 g, 2.91 mmol) was added to 2 (0.208 g, 0.91 mmol) and K2CO3 (0.377 g, 2.73 mmol) in MeCN (25 mL), and the mixture was stirred at 368 K for 24 hours under an argon atmosphere. The reaction was monitored by TLC (hexane / ethyl acetate; 1:1) to confirm the formation of the tetraalkylated derivative. The solvent was removed under reduced pressure, the resulting oil was redissolved in CHCl3 (25 mL), and successively washed with aqueous K2CO3 solution (2 × 25 mL, 0.1 M) and H2O (25 mL), dried on MgSO4, filtered, and the solvent was removed under reduced pressure. Purification by silica gel column chromatography (hexane / ethyl acetate, 2:1 → 1:1) yielded a yellow oil (0.229 g, 44%). Rf = 0.35 (hexane / ethyl acetate; 2:1).

[0040] [5-[1,4-bis-tert-butoxycarbonylmethyl-6-(tert-butoxycarbonylmethyl-amino)-[1,4]diazepam-6-yl]-methyl pentanoate (4)] Iodomethane (0.023 g, 0.16 mmol) was added to 3 (0.104 g, 0.18 mmol) and K2CO3 (0.025 g, 0.18 mmol) in DCM / MeCN (3:1) cooled in an ice bath. The reaction mixture was warmed to room temperature and left overnight. The solvent was removed under reduced pressure, the resulting oil was redissolved in CHCl3 (20 mL), filtered, and successively washed with K2CO3 aqueous solution (2 × 20 mL, 0.1 M) and H2O (20 mL). The mixture was dried on MgSO4, filtered, and the solvent was removed under reduced pressure. Purification by silica gel column chromatography (hexane / ethyl acetate, 3:1 → 2:1) yielded yellow oil (0.043 g, 46%). Rf = 0.38 (hexane / ethyl acetate; 2:1).

[0041] [5-[1,4-bis-tert-butoxycarbonylmethyl-6-(tert-butoxycarbonylmethyl-methyl-amino)-[1,4]diazepam-6-yl]-pentanoic acid (5)] Add 0.009 g, 0.039 mmol of LiOH dissolved in 0.5 mL of H2O to 0.010 g, 0.023 mmol of 4 in 0.5 mL of THF, and stir the mixture at 298 K. Monitor the ester cleavage using LC-ESIMS. After the reaction is complete, remove the solvent by freeze-drying. Add 5 mL of H2O, remove by freeze-drying, and repeat this procedure twice. Wash the resulting solid with ice-cold DCM (0.5 mL) and dry in vacuum to obtain a waxy yellow solid (0.009 g, 70%).

[0042] [ka]

[0043] [DAZTA 5 (Synthesis of the X=CH2COOH prochator) Prochulator DAZTA 5(X = CH2COOH), which is generally also referred to as AAZTA, can be prepared by the method described by Manzoni et al. (L. Manzoni, L. Belvisi, D. Arosio, M. P. Bartolomeo, A. Bianchi, C. Brioschi, F. Buonsanti, C. Cabella, C. Casagrande, M. Civera, M. De Matteo, L. Fugazza, L. Lattuada, F. Maisano, L. Miragoli, C. Neira, M. Pilkington-Miksa, C. Scolastico; Synthesis of Gd and 68 Ga Complexes in Conjugation with a Conformationally Optimized RGD Sequence as Potential MRI and PET Tumour-Imaging Probes; ChemMedChem 2012, 7, 1084 - 1093)).

[0044] [Compound 6] N,N′-Dibenzylethylenediamine diacetate (14.67 g; 40.7 mmol) is suspended in EtOH (50 mL), and the mixture is heated at 50°C until a clear solution is obtained. Paraformaldehyde (3.67 g; 122.1 mmol) is added, and the suspension is heated at 80°C for 1.5 hours to obtain a clear, dark orange solution. A solution of methyl 6-nitrohexanoate (R. Ballini, M. Petrini, V. Polzonetti, Synthesis 1992, 355-357) (7.13 g; 40.7 mmol) in EtOH (10 mL) is added dropwise. The resulting solution is cooled to room temperature, stirred at room temperature for 18 hours, and then stirred at 50°C for 4.5 hours. The mixture is evaporated, the residue is dissolved in SiO2 (100 mL), and the solution is washed with aqueous Na2CO3 solution and brine. The aqueous phase is separated and extracted with ethyl acetate (1 × 50 mL; 1 × 30 mL). The organic phase is collected, dried (Na₂SO₄), filtered, and evaporated. The crude product is purified by flash chromatography (silica gel column, 90:10 petroleum ether / ethyl acetate) to obtain compound 23 as a pale yellow oil (10.8 g; 24.6 mmol). (60%). 1 H-NMR (CDCl3,400MHz): δ0.80(m,2H),1.32(m,2H),1.58(m,2H),2.12(t,2H,J=7.5Hz),2.62(m,4H),2.96(d, 2H,J=14.2Hz), 3.52(d, 2H, J=14.2Hz), 3.59(d, 2H, J=13Hz), 3.66(s, 3H), 3.75(d, 2H, J=13Hz), 7.28(m, 10H). 13 C-NMR(CDCl3,100.6MHz):δ174.0,139.5,129.5,128.7,127.6,95.2,64.4,62.0,59.2,51.9,36.9,33.9,25.0,23.0.MS(ESI + )m / z:(M+H + ),440.5.

[0045] [Compound 7] Add 10% Pd / C (1.5 g) to the solution of compound 23 (10 g; 22.8 mmol) in MeOH (400 mL), and stir the suspension under a hydrogen atmosphere at 40°C for 5 hours. Filter the suspension (Millipore® filter FT 0.45 μm) and evaporate the solution. Dissolve the residue in MeCN (100 mL), and add freshly ground K2CO3 (16.8 g; 122 mmol) and Na2SO4 (3 g; 21 mmol). Add t-bromobutyl acetate (20.8 g; 107 mmol), stir the orange mixture, and heat at 80°C for 7 hours. Filter the mixture, add K2CO3 (18 g; 122 mmol), Na2SO4 (3 g; 21 mmol) and t-bromobutyl acetate (0.88 g; 4.5 mmol), and heat the fresh mixture at 80°C for 9.5 hours. The mixture was filtered and evaporated, and the residue was purified by chromatography (silica gel column, 3:2n-hexane / siRNA) to obtain 24 as a pale yellow oil (7.8 g; 11.4 mmol). (50%). 1 H-NMR(CDCl3,400MHz):δ1.46(s,36H),1.62-1.48(br,6H),2.33(t,2H,J=7.5Hz),2.65(d, 2H,J=14.2Hz),2.83(m,4H),3.00(d,2H,J=14.2Hz),3.24(s,4H),3.62(s,4H),3.67(s,3H). 13 C-NMR (CDCl3,400MHz): δ173.1,171.2,81.1,80.6,65.5,63.4,62.9,60.8,52.3,51.8,37.6,34.5,28.5,26.1,22.1.MS(ESI + )m / z:(M+H + ),686.5,(M+Na + ), 708.5.

[0046] [DAZTA 5 (X=CH3COOH) / AAZTA(8) To a solution of compound 24 (8.17 g; 11.9 mmol) in THF (200 mL) cooled to 0°C, a 1 M solution of LiOH (95.4 mL; 95.4 mmol) is added dropwise. The solution is then stirred at room temperature for 28 hours. The pH of the solution is adjusted to pH 7 by adding AcOH (4 mL). Water (50 mL) is added and the THF is evaporated. The aqueous residue is extracted with siRNA (3 × 75 mL). The organic phase is collected, dried (Na₂SO₄), filtered, and evaporated. The crude product is purified by flash chromatography (silica gel column, 3:2n-hexane / siRNA) to obtain compound 4 as a pale yellow oil (3.76 g; 5.6 mmol) (47%). 1 H-NMR (CDCl3,400MHz): δ1.48(s,36H),1.66-1.57(br,6H),2.38(t,2H,J=7.5Hz),2.79-2.67(br,6H),3.03(d,2H,J=14.2Hz),3.05(s,4H),3.63(s,S-4 4H). 13 C-NMR(CDCl3,100.6MHz):δ178.8,173.1,171.0,81.3,80.8,65.4,63.3,62.7,59.4,37.4,34.4,28.4,28.3,22.1.MS(ESI + )m / z:(M+H + ), 672.6.

[0047] [ka]

[0048] [PPA2 peptide synthesis] The PPA2 peptide can be prepared by classical solution synthesis, or preferably by established solid-phase techniques as depicted in Scheme 6 and described in U.S. Patents 7,019,109 and 5,874,227. The contents of U.S. Patents 7,019,109 and 5,874,227 are incorporated in whole hereby as reference. Side-chain protecting groups are known in the art and are particularly used as part of any amino acid having a reactive side chain, and can optionally be used in the case of other amino acids such as Trp (tryptophan), such amino acids being bound to the chain constructed on the resin. Such synthesis yields a fully protected intermediate peptide resin. The protecting group is generally cleaved, and the peptide is cleaved from the resin support before oxidation, forming disulfide bonds between the Cys side chains.

[0049] [ka]

[0050] Alternatively, peptide PPA2 can be obtained from various commercial providers, such as Peptide Specialty Laboratories GmbH (https: / / www.peptid.de / ).

[0051] [Latest PET / CT imaging technology] Figure 1 shows established radioactive tracers 68 Ga-NODAGA-LM3 (Figure 1a) and 68 These are PET / CT images of liver cancer patients using Ga-DOTA-TATE (Figures 1b and 1c). 68 Ga-NODAGA-LM3 improved the visualization of metastatic lesions.

[0052] [ 68 Ga-DAZTA 5 - Disease staging using PET / CT imaging with PPA2(X=CH)3] Figure 2 shows the radioactive tracer of the present invention. 68 Ga-DAZTA 5 -PPA2(X=CH3, i.e., 68Ga-DATA 5m These are five images of a patient acquired at different times using PET / CT with PPA2), distinguished by highly sensitive visualization of liver metastases, sharp contrast, and detection of small metastases and affected lymph nodes.

[0053] [ 68 Ga-DAZTA 5 -PET / CT imaging of bone metastases using PPA2(X=CH3) Figure 3 shows PET / CT images of a patient with multiple bone metastases that cannot be detected by CT scans because there are no changes in osteoblasts. Figures 3a and 3b show the CT image and a fusion of the CT and PET images, respectively.

[0054] [ 68 Ga-DAZTA 5 -PET / CT imaging of lymph nodes using PPA2(X=CH3) Figure 4 shows a PET / CT image (Figure 4a) of a small abdominal lymph node metastasis originating from neuroendocrine carcinoma with a diameter of 6 mm or less that could not be detected by a CT scan (Figure 4b).

[0055] [ 68 Ga-NODAGA-LM3 and 68 Ga-DAZTA 5 -PET / CT imaging using PPA2 (X=CH3) Figure 5 shows the radioactive tracer. 68 GaNODAGA-LM3- and 68 Ga-DAZTA 5 -PPA2(X=CH3, i.e., 68 Ga-DATA 5m These are PET / CT images of liver cancer patients using PPA2. 68 Ga-DATA 5m -PPA2 significantly reduces background signals from healthy liver and spleen tissue, while also providing better visualization of metastatic lesions.

[0056] [ 68 Ga-DAZTA 5-PET / CT imaging of breast metastases using PPA2(X=CH3) Figure 6 shows patients in whom no lesions were found when examined with magnetic resonance imaging (MRI) and CT, compared to a standard CT (a) and... 68 Ga-DAZTA 5 -PPA2(X=CH3, i.e., 68 Ga-DATA 5m This is a comparison with images obtained by PET / CT(b) using -PPA2). Unlike MRI and CT imaging, 68 Ga-DAZTA 5 - By using PPA2 PET / CT, it is possible to detect metastatic lesions as small as 2 mm in diameter.

[0057] [Uptake and binding into cells] Figure 7 shows the agonist radioactive tracer using the cell line HEK293-SSR2. 68 Ga-DATA 5m -TOC and antagonist radioactive tracers 68 Ga-DAZTA 5 -PPA2 (i.e., X=CH3) 68 Ga-DATA 5m The results of a comparison of the uptake of PPA2 into in vitro cells are shown. 68 Ga-DATA 5m -PPA2 shows excellent overall uptake, with a high ratio of intracellular uptake (endocytosis) to membrane binding.

[0058] [ 68 Ga-DAZTA 5 -PPA2(X=CH3) radiolabeling dynamics] 50 μg of the present invention prochator DAZTA 5 -PPA2(X=CH3, i.e., DATA 5m -PPA2) at room temperature (RT) and 95°C, 68 The galvanic acid (GA) is added to 500 μL of sodium acetate buffer (pH 4.5). A radiochemical yield (RCY) of over 95% is obtained within 5-10 minutes (see Figure 8).

[0059] [In vitro stability] Figure 9 shows 68 Ga-DAZTA 5 - Demonstrates the in vitro stability of PPA2. The present invention's radioactive tracer DAZTA has X=CH3. 5 -PPA2 (i.e., DATA 5m -PPA2) was suspended in human serum, phosphate-buffered saline (PBS), and physiological NaCl solution, respectively, at 37°C for 120 minutes. No measurable degradation was detected during the 2-hour period.

[0060] [Affinity analysis] Table 1 shows the [ 125 I | Leu 8 DTrp 22 ,I-Tyr 25 ]SS28([ 125 Data on non-metallated, Ga-, In-, and Lu-complexed precursors based on substitution analysis using [I]I-[LTT]SS28) 5m - Relative IC of comparative combined analysis of PPA2 and AAZTAPPA2 50 The values ​​are shown (at 22°C for 1 hour). Figures 10a and 10b show the corresponding measurement curves.

[0061] [Table 1]

[0062] The p-values ​​corresponding to the data in Table 1 are as follows: DATA 5 -PPA2 vs. Ga-DATA 5 For -PPA2 and In-AAZTA-PPA2 vs. Lu-AAZTA-PPA2, P > 0.05; for AAZTA-PPA2 vs. In-AAZTA-PPA2 or Lu-AAZTA-PPA2, P < 0.01.

[0063] [Distribution of organs outside the body] Figure 11 shows the [ ] in male SCID mice with HEK293-SST2R-positive (+) tumors. 68 Ga]Ga-DAZTA 5-The in vitro organ distribution of PPA2 is shown. Organs were extracted 1 hour and 4 hours after injection. Furthermore, tumor specificity was analyzed by blocking with 100 μg octreotide (TATE) administration 4 hours after injection.

[0064] Figure 12 shows the results in male SCID mice with HEK293-SST2R-positive (+) and negative (-) tumors. 111 In vitro organ distribution of In-AAZTA-PPA2, 111 This is shown in comparison with In]In-DOTA-LM3. Organs were extracted 4 hours and 24 hours after injection. [Brief explanation of the drawing]

[0065] [Figure 1] Figure 1 shows established radioactive tracers 68 Ga-NODAGA-LM3 (Figure 1a) and 68 These are PET / CT images of liver cancer patients using Ga-DOTA-TATE (Figures 1b and 1c). [Figure 2] Figure 2 shows the radioactive tracer of the present invention. 68 Ga-DAZTA 5 -PPA2(X=CH3, i.e., 68 Ga-DATA 5m These are five images of a patient acquired at different times using PET / CT with PPA2. [Figure 3] Figure 3 shows PET / CT images of a patient with multiple bone metastases that cannot be detected by CT scans because there are no changes in osteoblasts. Figures 3a and 3b show the CT image and the fusion of the CT and PET images, respectively. [Figure 4] Figure 4 shows a PET / CT image (Figure 4a) of a small abdominal lymph node metastasis originating from neuroendocrine carcinoma with a diameter of 6 mm or less that could not be detected by a CT scan (Figure 4b). [Figure 5] Figure 5 shows a radioactive tracer 68 GaNODAGA-LM3- and 68 Ga-DAZTA 5 -PPA2(X=CH3, i.e., 68 Ga-DATA 5m These are PET / CT images of liver cancer patients using PPA2. [Figure 6] Figure 6 shows patients in whom no lesions were found when examined with magnetic resonance imaging (MRI) and CT, compared to a normal CT (a) and... 68 Ga-DAZTA 5 -PPA2(X=CH3, i.e., 68 Ga-DATA 5m This is a comparison with images obtained by PET / CT(b) using PPA2). [Figure 7] Figure 7 shows the agonist radioactive tracer using the cell line HEK293-SSR2. 68 Ga-DATA 5m -TOC and antagonist radioactive tracers 68 Ga-DAZTA 5 -PPA2 (i.e., X=CH3) 68 Ga-DATA 5m The results of a comparison of the uptake of PPA2 into in vitro cells are shown. [Figure 8] 50 μg of the present invention's Prokerator DAZTA 5 -PPA2(X=CH3, i.e., DATA 5m -PPA2) at room temperature (RT) and 95°C, 68 This shows the relationship with the radiochemical yield (RCY) when Ga is added to 500 μL of sodium acetate buffer (pH 4.5) in which Ga is dissolved. [Figure 9] Figure 9 shows, 68 Ga-DAZTA 5 - Demonstrates the in vitro stability of PPA2. [Figure 10] Figures 10a and 10b show the [ 125 I | Leu 8 DTrp 22 ,I-Tyr 25 ]SS28([ 125 Data on non-metallated, Ga-, In-, and Lu-complexed precursors based on substitution analysis using [I]I-[LTT]SS28) 5m - Relative IC of comparative combined analysis of PPA2 and AAZTAPPA2 50 The measurement curve corresponding to the value is shown. [Figure 11] Figure 11 shows the [ ] in male SCID mice with HEK293-SST2R-positive (+) tumors. 68Ga]Ga-DAZTA 5 - Shows the in vitro organ distribution of PPA2. [Figure 12] Figure 12 shows the results in male SCID mice with HEK293-SST2R positive (+) and negative (-) tumors. 111 In vitro organ distribution of In-AAZTA-PPA2, 111 This is shown in comparison with In-DOTA-LM3.

Claims

1. DAZTA, a precursor for neuroendocrine theranostics, has the following structure. ５ - PPA2 or a salt thereof. 【Chemistry 1】 【Request Item 2】 【Chemistry 2】 The precursor DAZTA according to claim 1, having ５ - PPA2 and radioactive isotopes that complex with it ６８ A radioactive tracer made of Ga ６８ Ga-DAZTA ５ - PPA2. 【Request Item 3】 【Chemistry 3】 The precursor DAZTA according to claim 1 having ５ -PPA2 and a radioisotope complexing with the same １７７ A radioactive tracer consisting of Lu １７７ Lu-DAZTA ５ -PPA2. 【Request Item 4】 【Chemistry 4】 The precursor DAZTA according to claim 1, having ５ - A radiopharmaceutical kit containing PPA2 or a salt thereof. 【Request Item 5】 【Chemistry 5】 The precursor DAZTA according to claim 1, having ５ - A radiopharmaceutical kit containing PPA2 or a salt thereof.

6. A radiopharmaceutical kit according to claim 4 or 5, comprising a solvent selected from water, a 0.45% NaCl aqueous solution, a 0.9% NaCl aqueous solution, Ringer's solution (lactated Ringer's solution), a 5% dextrose aqueous solution, and an alcohol aqueous solution. 【Request Item 7】 【Transformation 6】 The precursor DAZTA according to claim 1, having ５ - A first vial containing PPA2 or a salt thereof, and 【Transformation 7】 The precursor DAZTA according to claim 1, having ５ - A second vial containing PPA2 or a salt thereof A radiopharmaceutical kit containing [the specified ingredient].

8. The radiopharmaceutical kit according to claim 7, comprising one or two solvents independently selected from water, a 0.45% NaCl aqueous solution, a 0.9% NaCl aqueous solution, Ringer's solution (lactated Ringer's solution), a 5% glucose aqueous solution, and an alcohol aqueous solution.

9. The precursor according to claim 1 for use in PET imaging, SPECT imaging, or internal radiotherapy of somatostatin-expressing tissue.

10. A radiotracer according to claim 2 or 3 for use in PET imaging, SPECT imaging, or internal radiotherapy of somatostatin-expressing tissue.

11. A radiopharmaceutical kit according to claim 4 or 5 for use in PET imaging, SPECT imaging, or internal radiotherapy of somatostatin-expressing tissue.

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