Precursors and radiotracers targeting fibroblast activation protein

Abstract

Provided are a precursor compound and a radioactive tracer for targeting fibroblast activation protein. This precursor compound and radioactive tracer have a chelating agent (Ch) and a ligand (TV). Their respective structures are as follows. [Chemical Formula 1] JPEG2025521837000035.jpg108169

Description

Technical Field

[0001] The present invention relates to the expression of fibroblast activation protein (FAP) in relation to a precursor compound and a radiotracer derived therefrom. The structure of the precursor compound of the present invention is Ch-L1-B-L2-TV. Here,

Chemical Formula

Background Art

[0002] 68 Nuclear Medicine Diagnosis of Tumors Using Ga] A combination of positron emission tomography (PET) and computed tomography (CT) with gallium 68 (Ga-68 or 68 ​The use of [[Ga]]-68 is a well-established nuclear medicine imaging method today. The US Food and Drug Administration and the European Medicines Agency have approved [[octreotate (DOTATATE)]] of [[1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)]] labeled with [[Ga]]-68 and [[DOTA(0)-d-Phe(1)-Tyr(3)-octreotide (DOTATOC)]]. Its application in the US is for the localization of [[somatostatin receptor-positive neuroendocrine tumors (NETs)]] in adult and pediatric patients. In Europe, it is for the localization of [[somatostatin receptor-positive neuroendocrine tumors]] in adult patients with so-called well-differentiated [[gastroenteropancreatic neuroendocrine tumors (GEP-NETs)]]. Recently, [[PSMA-11]], a [[prostate-specific membrane antigen inhibitor]], has been approved for the application of imaging and staging evaluation of [[metastatic prostate cancer]]. The diagnostic value of [[PET / CT]] mainly depends on sensitivity, specificity, and accuracy. Sensitivity is the ratio of those correctly identified among the positive ones (true positive ÷ (true positive + false negative)). Specificity is the ratio of those correctly identified among the negative ones (true negative ÷ (true negative + false positive)). Diagnostic accuracy relates to the ability of a test to distinguish between the target condition and healthy individuals. The potential discriminatory ability can be quantified by measuring the sensitivity and specificity between the target condition and the background, or by measuring the area under the [[receiver operating characteristic curve (ROC curve)]].

[0003] Fibroblast activation protein (FAP) is a membrane-bound serine protease. Recently, this protein has attracted attention in the field of nuclear medicine for both diagnosis and treatment in order to target various types of cancer with FAP-specific radiolabeled agents. FAP is abundantly found in more than 90% of human epithelial tumors and is overexpressed in cancer-associated fibroblasts (CAFs). The phenotype of CAFs is myofibroblasts and they exist in the tumor stroma. Stromal cells account for up to 90% of the total tumor mass. FAP may also be expressed in fibroblasts in wound healing, chronic inflammation, liver cirrhosis, rheumatoid arthritis, pulmonary fibrosis, and bone and soft tissue sarcomas. However, FAP is not expressed in healthy tissues. Therefore, FAP-targeted radiopharmaceuticals are very suitable for molecular imaging by PET or SPECT and radiotherapy of cancer or metastatic cancer.

[0004] [FAP inhibitor: quinoline-glycine-4,4-difluoro-2-cyanopyrrolidine] A potent FAP inhibitor (FAPi) known as UAMC1110 is based on the quinoline-glycine-4,4-difluoro-2-cyanopyrrolidine skeleton. UAMC1110 has a low nanomolar affinity for FAP and is more selective than enzymes such as the dipeptidyl peptidase (DPP) family and the prolyl oligopeptidase (PREP) family. The high selectivity of UAMC1110 for FAP is particularly attractive for tumor targeting because DPP and PREP are expressed in almost all parts of the human body. PREP exhibits the same endopeptidase activity as FAP and is abundant in healthy organs and tissues, so high specificity for FAP is essential for a potent FAP antagonist ligand. The affinity of UAMC1110 for FAP is at the low nM level, which is higher than the μM level of PREP and DPP4. Therefore, in today's nuclear medicine world, FAPi radiopharmaceuticals based on UAMC1110 are being applied.

Chemical formula

[0005] [Chelating Agents Contained in Radiopharmaceuticals Containing Radioactive Metals] The following properties are relevant to the precursor and the radioactive tracer derived therefrom. - To rapidly and efficiently complex a diagnostic or therapeutic radioisotope with a labeling group. - To have a high affinity and selectivity for tumor cells and metastatic cancers compared to healthy tissues. - To be stable in vivo. The biochemistry in serum under physiological conditions is persistent. - To be well taken up in tumors and metastatic cancers for accurate diagnosis and effective treatment. - To have rapid clearance from healthy tissues and blood, and to be able to minimize the dosage and toxicity upon systemic administration.

[0006] The following are the latest findings in this technical field. - Chelating agents and radioisotopes greatly influence the affinity and pharmacokinetics of radioactive tracers. - DOTA may have a significant adverse effect on the affinity of the targeting ligand. - The interaction between the chelating agent, radioisotope and targeting ligand cannot be predicted and can be synergistic or antagonistic.

[0007] Fani et al. reported that the chelating agent moiety and radioactive metal of a radioactive tracer can greatly affect the affinity with the targeting ligand. Reubi et al. showed that complexation of the targeting ligand with DOTA may substantially reduce the affinity with the target cell receptor. According to the data shown by Reubi et al., the combination of the chelating agent, radioactive metal and targeting ligand brings about a synergistic or antagonistic effect, and the result is hardly predictable. Generally, complexation of the targeting ligand with the linker and the large chelating agent moiety is not beneficial and greatly impairs the affinity with the target cell receptor.

[0008] Chelating agents such as DOTA are not suitable for complexing with relatively small (radioactive) metals such as gallium and require high reaction temperatures. However, such high reaction temperatures are disadvantageous for many antibodies and highly heat-sensitive biomolecules. 68 After the formation of a complex between Ga and the DOTA chelate, a cooling time is required before intravenous injection. 68 Since the half-life of Ga is short (67.7 minutes), this time has become a clinical constraint.

[0009] [The DATA and AAZTA, which are the chelating agent skeletons of 《Hybrid》] DATA (1,4-bis(carboxymethyl)-6-[methyl-carboxymethyl-amino]-1,4-diazepane) and AAZTA (1,4-bis(carboxymethyl)-6-[bis(carboxymethyl)-amino]-1,4-diazepane) exhibit hybrid characteristics of cyclic and acyclic forms 68 and have been found to have favorable labeling characteristics for Ga. In particular, DATA can rapidly and quantitatively 68 perform Ga labeling over a wide pH range and at room temperature. Furthermore, 68 the chelate of [Ga]Ga and DATA is resistant to chelate exchange (between DTPA and apotransferrin) and metal exchange (between Fe III and is stable in the physiological environment.

Brief Description of the Drawings

[0010]

Figure 1

Figure 2

Figure 3

Embodiments for Carrying out the Invention

[0011] An object of the present invention is to improve nuclear seranostics for endothelial carcinomas and metastatic cancers with increased expression of FAP.

[0012] This object is achieved by a precursor compound having the structure of Ch-L1-B-L2-TV. Here,

Chemical formula

[0013] A convenient embodiment of the precursor compound of the present invention is characterized by the following characteristic points or a combination of two or more thereof. However, the combination of two or more characteristic points shall not be mutually contradictory or inconsistent.

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[0014] A further object of the present invention is to provide a radioactive tracer that contributes to the advancement of seranostics by nuclei of endothelial carcinomas and metastatic carcinomas in which the expression of FAP is increased.

[0015] This object is achieved by a radioactive tracer composed of the above-described precursor compound and the radioisotope 68 Ga complexed therewith.

[0016] A further advantageous embodiment of the present invention is related to the following. - A kit for a radiopharmaceutical comprising any one of the above-described precursor compounds or a salt thereof. - A kit for a radiopharmaceutical comprising any one of the above-described precursor compounds or a salt thereof and a solvent. The solvent is selected from the group consisting of water, 0.45% aqueous NaCl solution, 0.9% aqueous NaCl solution, Ringer's solution (lactated Ringer's), 5% aqueous dextrose solution, and aqueous alcohol solution. - A kit for a radiopharmaceutical comprising a vial containing any one of the above-described precursor compounds or a salt thereof. - A kit for a radiopharmaceutical comprising a vial containing any one of the above-described precursor compounds or a salt thereof and a lyophilized buffer. Here, the buffer is selected from the group consisting of sodium acetate, gentiopic acid, ascorbic acid, succinic acid, HEPES, sodium carbonate, sodium bicarbonate, and salts thereof. - A kit for a radiopharmaceutical comprising the following (i) and (ii). (i) A first vial containing any one of the above-described precursor compounds or a salt thereof. (ii) A second vial containing a solvent or buffer solution. Here, the solvent is selected from the group consisting of water, 0.45% aqueous NaCl solution, 0.9% aqueous NaCl solution, Ringer's solution (lactated Ringer's), 5% aqueous dextrose solution, and aqueous alcohol solution. The buffer solution is configured to adjust the pH to 3 - 7.4.

[0017] In the present invention, the following expressions are adopted to represent the same compound.

Chemical formula

Examples

[0018] 〔Example 1: DATA 5m Synthesis of the prochelating agent〕 The synthesis of the DATA5m prochelating agent is shown in Formula 2 (see J. Seemann, B. Waldron, D. Parker, F. Roesch; DATATOC: a novel conjugate for kit-type 68 Ga labelling of TOC at ambient temperature; EJNMMI Radiopharmacy and Chemistry (2016) 1:4, DOI 10.1186 / s41181-016-0007-3).

[0019] Methyl 5-(1,4-dibenzyl-6-nitro-[1,4]diazepan-6-yl)-pentanoate ···(1) In EtOH, 2-nitrocyclohexanone (0.608 g, 4.3 mmol) was added to Amberlyst A21 (1.216 g, 2 equiv). The mixture was stirred at 60 °C for 2 h under argon. N,N'-Dibenzylethylenediamine (1.020 g, 4.3 mmol) and paraformaldehyde (0.446 g, 14.9 mmol) were added, and the reaction was carried out with stirring at 60 °C overnight. The mixture was filtered through Celite (R), and the solvent was removed under reduced pressure. The resulting residue was redissolved in CHCl3 (40 mL) and washed first with an aqueous K2CO3 solution (2 × 30 mL, 0.1 M) and then with H2O (30 mL). It was dried over MgSO4, filtered, and the solvent was removed under reduced pressure. Purification by silica gel column chromatography (DCM) gave the title compound as a yellow oil (1.607 g, 85%). Rf = 0.80 (DCM).

[0020] Methyl 5-(1,4-dibenzyl-6-nitro-[1,4]diazepan-6-yl)-pentanoate ··· (2) To the protected triamine compound 1 (0.10 g, 0.29 mmol) in MeOH (20 mL) were added a catalytic amount of Pd(OH)2 / C and acetic acid (50 μL, 0.87 mmol). The mixture was stirred for 3 h under a hydrogen atmosphere (1 atm of H2). The complete reduction of the nitro group and the cleavage of the benzyl N-substituent were confirmed using TLC (DCM). Pd(OH)2 / C was removed using a Celite (R) filter. The solvent was removed under reduced pressure to give a yellow oil (0.065 g, 97%).

[0021] Methyl 5-[1,4-bis-tert-butoxycarbonylmethyl-6-(tert-butoxycarbonylmethyl-amino)-[1,4]diazepan-6-yl]-pentanoate ··· (3) In MeCN (25 mL), to compound 2 (0.208 g, 0.91 mmol) and K₂CO₃ (0.377 g, 2.73 mmol), tert-butyl bromoacetate (0.567 g, 2.91 mmol) was added. The mixture was stirred at 368 K for 24 h under an argon atmosphere. The formation reaction of the tetraalkylation derivative was monitored by TLC (hexane / ethyl acetate; 1:1). The solvent was removed under reduced pressure, and the resulting oil was redissolved in CHCl₃ (25 mL) and washed first with an aqueous K₂CO₃ solution (2 × 25 mL, 0.1 M) and then with H₂O (25 mL). It was dried over MgSO₄, filtered, and the solvent was removed under reduced pressure. Purification by silica gel column chromatography (hexane / ethyl acetate; 2:1 → 1:1) gave a yellow oil (0.229 g, 44%). Rf = 0.35 (hexane / ethyl acetate; 2:1).

[0022] Methyl 5-[1,4-bis-tert-butoxycarbonylmethyl-6-(tert-butoxycarbonylmethyl-methyl-amino)-[1,4]diazepan-6-yl]pentanoate ··· (4) While cooling in an ice bath, in DCM / MeCN (3:1), to compound 3 (0.104 g, 0.18 mmol) and K₂CO₃ (0.025 g, 0.18 mmol), iodomethane (0.023 g, 0.16 mmol) was added. The reaction mixture was warmed to room temperature and left overnight. The solvent was removed under reduced pressure, and the resulting oil was redissolved in CHCl₃ (20 mL). After filtration, it was washed first with an aqueous K₂CO₃ solution (2 × 20 mL, 0.1 M) and then with H₂O (20 mL). It was dried over MgSO₄, filtered, and the solvent was removed under reduced pressure. Purification by silica gel column chromatography (hexane / ethyl acetate; 3:1 → 2:1) gave a yellow oil (0.043 g, 46%). Rf = 0.38 (hexane / ethyl acetate; 2:1).

[0023] 5-[1,4-bis-tert-butoxycarbonylmethyl-6-(tert-butoxycarbonyl-methyl-methyl-amino)-[1,4]diazepan-6-yl]pentanoic acid ··· (5) In THF (0.5 mL), to compound 4 (0.010 g, 0.023 mmol), LiOH (0.009 g, 0.039 mmol) dissolved in H2O (0.5 mL) was added, and the mixture was stirred at 298 K. The cleavage reaction of the ester was monitored by LC-ESI MS. After completion of the reaction, the solvent was removed by lyophilization. The step of adding H2O (5 mL) and removing it by lyophilization was repeated twice. The obtained solid was washed with ice-cooled DCM (0.5 mL) and dried under reduced pressure to obtain a waxy yellow solid (0.009 g, 70%).

[0024]

Chemical formula

[0025] 〔Example 2: Synthesis of FAP-targeting ligand〕 The synthesis of the FAP-targeting ligand is shown in Schemes 3 - 5 (see K. Jansen, L. Heirbaut, R. Verkerk, J.D. Cheng, J. Joossens, P. Cos, L. Maes, A.-M. Lambeir, I. De Meester, K. Augustyns, P. Van der Veken; Extended Structure-Activity Relationship and Pharmacokinetic Investigation of (4-Quinolinoyl)glycyl-2-cyanopyrrolidine Inhibitors of Fibroblast Activation Protein (FAP); J. Med. Chem. 2014 Apr 10; 57(7): 3053-74, DOI 10.1021 / jm500031w).

[0026]

Chemical formula

[0027] [Example 3: Affinity for FAP and PREP] The precursor compounds and radiotracers of the present invention have high affinity for the FAP target and excellent selectivity (low binding to PREP). Therefore, by using the precursor compounds and radiotracers, the contrast of images can be improved in the diagnosis of endothelial tumors and metastatic cancers using gallium 68 PET / CT.

[0028] The measured values of the affinity of the precursor compounds 1 to 4 of the present invention and the reference compound 5 for FAP and PREP are shown in Table 1 below. In addition, the measured values of UAMC1110, a prior art FAP inhibitor, are also shown in Table 1 as the latest reference values.

[0029] [Table 1]

[0030] The precursor compounds (A), (B), (D), and (E) of the present invention had higher affinity for FAP than the reference compound (F) and the reference UAMC1110 (G). Compounds (A), (D), and (E) had lower binding to the off-target PREP than the reference values (F) and (G). The affinity of compound (B) for PREP was comparable to that of (F) and (G). All of compounds (A), (B), (D), and (E) had discriminably higher selectivity for FAP than for PREP compared to the reference values (F) and the reference (G). The selectivity of compound (B) was comparable to that of (G). The selectivity of compound (E) was twice that of (G). The selectivity of compound (D) was more than three times that of (G). The selectivity of compound (A) was six times that of (G). DATA 5m The reference compound (F) complexed with [substance] had lower affinity and selectivity for FAP than (G), as expected. This is due to chemical modification.

[0031] According to the radioactive tracer of the present invention, an immediate-use kit can be easily provided. The kit includes a lyophilized product of one of the above-mentioned precursor compounds or a salt thereof, a buffer for pH adjustment, an antioxidant for capturing radicals and preventing radiolysis, and a lyophilized filler. 68 Each radioactive tracer is prepared by adding it to a hydrochloric acid solution of [Ga]GaCl3. In this case, it complies with the monograph "Gallium (68Ga) chloride solution" in the European Pharmacopoeia.

[0032] 〔Example 4: 68 [Ga]Ga-DATA 5m .Pip.FAPi for PET MIP〕 Figure 1 is an image obtained by applying the radioactive tracer [Ga]Ga-DATA 68 [Ga]Ga-DATA 5m .Pip.FAPi of the present invention to a male patient (52 years old) suffering from radioiodine-refractory differentiated thyroid cancer and obtained by PET MIP. Multiple metastases in the pancreas (1), lung (2) and lymph nodes (3) were detected.

[0033] 〔Example 5: 68 [Ga]Ga-DATA 5m .NH.FAPi for PET MIP〕 Figure 2 is an image obtained by applying the radioactive tracer [Ga]Ga-DATA 68 [Ga]Ga-DATA 5m .NH.FAPi. of the present invention to a male patient (40 years old) suffering from radioiodine-refractory differentiated thyroid cancer and obtained by PET MIP. Multiple lesions in the liver (1), lung (2) and lymph nodes (3) were detected.

[0034] 〔Example 6: Imaging by Coronal PET / CT〕 Figure 3 is a coronal PET / CT image taken by applying the radioactive tracer [Ga]Ga-DATA 68 [Ga]Ga-DATA 5m .NH-Pyr.FAPi. of the present invention to a male patient suffering from metastatic gastric and esophageal cancer. Multiple bone metastases were detected.

[0035] The explanations of the symbols described in FIG. 3 are as follows. Li: Liver Ki: Kidney Bl: Bladder L1: Bone tumor lesion (pelvis) L2: Bone tumor lesion (pelvis) L3: Bone tumor lesion (rib) L4: Tumor lesion L5: Tumor lesion L6: Bone tumor lesion (vertebra) L7: Bone tumor lesion (shoulder) L8: Multiple bone tumor lesions (pelvis) L9: Tumor lesion (pelvis) L10: Multiple bone tumor lesions (vertebra) L11: Multiple bone tumor lesions (pelvis) L12: Bone tumor lesion (rib)

Claims

1. A precursor compound for a radioactive tracer having the structure Ch-L1-B-L2-TV, the precursor compound represented below: 【Chemistry 1】 L1 is a dual-function linker portion; L2 is a dual-function linker section; B is either non-existent or a dual-function linker portion; L1 is composed of a covalent bond between Ch and L2, or, if B is present, a covalent bond between Ch and B; If present, B consists of a covalent bond between L1 and L2; L2 is composed of a covalent bond between L1 and TV, or, if B is present, a covalent bond between TV and B.

2. The precursor compound according to claim 1, characterized as follows. 【Chemistry 2】

3. The precursor compound according to claim 1 or 2, characterized by the following: 【Transformation 3】

4. The precursor compound according to claim 1 or 2, characterized by the following: 【Chemistry 4】

5. The precursor compound according to claim 4, characterized in that m = 4.

6. The precursor compound according to claim 1 or 2, characterized by the following: 【Transformation 5】

7. The precursor compound according to claim 6, characterized in that n = 4.

8. The precursor compound according to claim 1 or 2, characterized by the following: 【Transformation 6】

9. The precursor compound according to claim 1 or 2, characterized by the following: 【Transformation 7】

10. The precursor compound according to claim 1 or 2, characterized by the following: 【Transformation 8】

11. The precursor compound according to claim 1 or 2, characterized by the following: 【Chemistry 9】

12. The precursor compound according to claim 1, characterized by having the following structure. 【Chemistry 10】

13. The precursor compound according to claim 1, characterized by having the following structure. 【Chemistry 11】

14. The precursor compound according to claim 1, characterized by having the following structure. 【Chemistry 12】

15. The precursor compound according to claim 1, characterized by having the following structure. 【Chemistry 13】

16. The precursor compound according to claim 1, characterized by having the following structure. 【Chemistry 14】

17. A precursor compound according to any one of claims 1, 2, 12 to 16, Radioactive isotopes that form a complex with the above precursor compound ６８ Ga and, A radioactive tracer composed of [components].

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