Radiopharmaceuticals based on ((r)-1-((6-hydrazinylnicotinoyl)-d-alanyl)pyrrolidin-2-yl)boronic acid (hynic-ifap) for detecting overexpression of fibroblast activation protein

Abstract

To provide a new specific SPECT radiopharmaceutical with high sensitivity for the detection of FAP protein expression in the tumour microenvironment, which is based on boronPro-type inhibitors (molecular-target radiopharmaceuticals).SOLUTION: Provided is a new fibroblast activation protein (iFAP) inhibitory radiopharmaceutical based on ((R)-1-((6-hydrazinylnicotinoyl)-D-alanyl)pyrrolidin-2-yl)boronic acid (HYNIC-iFAP), wherein ethylenediamine diacetic acid is used to complete the coordination sphere of the radiometal, combining the conventional use of HYNIC as a chelating agent for the radiometal 99mTc, and wherein the nitrogens of the hydrazine of HYNIC act as chemical groups that promote the interaction of the HYNIC-iFAP molecule with phenylalanine, glutamic acid, and serine in the active centre of fibroblast activation protein (FAP).SELECTED DRAWING: None

Description

[Technical Field]

[0001] statement FIELD OF THE INVENTION The present invention describes a novel radiopharmaceutical based on the fibroblast activation protein (iFAP) inhibitor ((R)-1-((6-hydrazinylnicotinoyl)-D-alanyl)pyrrolidin-2-yl)boronic acid (HYNIC-iFAP), which is a radioactive metal 99m In conjunction with the conventional use of HYNIC as a chelator of Tc, the hydrazine nitrogen of HYNIC serves as a preferred chemical group for interaction of the HYNIC-iFAP molecule with the essential active centers of fibroblast activation protein (FAP), phenylalanine (Phe-350 and Phe-351), glutamic acid (Glu-203 and Glu-204), and serine (Ser-624), particularly with respect to radiopharmaceuticals. 99m The new Tc-labeled radiopharmaceutical HYNIC-iFAP detects FAP expressed in the tumor microenvironment with high affinity in vivo using SPECT molecular imaging technology in nuclear medicine. [Background technology]

[0002] background Fibroblast activation protein (FAP) is a type II serine protease that cleaves peptides downstream of proline residues through dipeptidyl peptidase and endopeptidase activity. FAP is highly expressed on the cell surface of activated stromal fibroblasts, which are present in most human epithelial tumors but not in normal fibroblasts. Cancer-associated fibroblasts account for up to 90% of the macroscopic tumor mass (Hamson et al., Understanding fibroblast activation protein (FAP): substrates, activities, expression, and targeting for cancer therapy. Proteomics Clin. Appl., 2014, Vol. 8, pp. 454–463).

[0003] Specific FAP inhibitors were first developed as potential anticancer drugs (Aertgeerts et al., Structural and kinetic analysis of the substrate specificity of human fibroblast activation protein α.J.Biol.Chem., 2005, vol. 280, 19441~19444; Edosada et al., Selective inhibition of fibroblast activation protein protease based on dipeptide substrate specificity. J.Biol.Chem. 2006, vol. 281, 7437~7444; Tran et al., Synthesis and structure-activity relationship of N-acyl-Gly-,N-acyl-Sar- and N-blocked-boroPro inhibitors of FAP, DPP4, and POP. Bioorg. Med. Chem. Lett., Vol. 17, 2007, pp. 1438-1442). Of these, the most relevant are two groups of highly selective compounds: one based on a quinoline-cyanopyrrolidine structure, and the other based on pyrrolidine-boronic acid, also known as proline-boronic acid (boronPro). Regarding the first group, Jansen et al. first reported the synthesis of 39 new FAP inhibitors and explored the structure-activity relationships of the 4-quinolinoyl-glycianopyrrolidine scaffold (Jansen et al., Extended structure (Jansen et al., "Selective inhibitors of fibroblast activation protein (FAP) with a (4-quinolinoyl)-glycyl-2-cyanopyrrolidin scaffold." ACS Med. Chem. Lett., 2013, 4, 491–496). The authors also discovered that N-pyridines yield highly selective FAP inhibitors over other post-proline cleaving enzymes, such as dipeptidyl peptidase (DPP) and prolyl oligopeptidase (PREP). The quinolinoyl fragment conferred higher affinity to FAP, but when this residue was replaced with another azaheteroaromatic substituent, affinity for FAP dramatically decreased. Furthermore, the addition of fluorine or difluoro to the cyanopyrrolidine ring fragment was found to improve affinity and selectivity for FAP. When the authors replaced the glycine residue with several other amino acids, affinity for PREP increased and FAP potency significantly decreased. As a result of this study, the authors identified the N-(4-quinolinoyl)-glycyl-(2-cyanopyrrolidine) scaffold as the best inhibitor of FAP. Pharmacokinetic studies in mice of selected FAP inhibitors demonstrated high bioavailability after oral administration, along with short plasma half-lives and prolonged in vivo FAP inhibition.

[0004] Concurrently, Poplawsky et al. designed and characterized over 20 proline boronic acid-based inhibitors of FAP and PREP (Poplawski et al., Identification of selective and potent inhibitors of fibroblast activation protein and prolyl oligopeptidase. J. Med. Chem. 2013, 56, 3467–3477). The authors reported that the affinity for FAP could be increased by using a protonated pyridinic nitrogen atom, which forms a hydrogen bond with the carbonyl oxygen of glutamic acid (Glu-204), present in FAP but absent in PREP. The authors also found that the endopeptidase activity of FAP has very stringent requirements, accepting only small amino acids such as glycine or d-alanine. d-alanine is preferred because this inhibitor retains FAP inhibitory potency in the nanomolar range and offers 360-fold selectivity for FAP over PREP. Based on these results, Poplawski et al. demonstrated that N-(pyridine-4-carbonyl)-D-Ala-boroPro was the best fibroblast activation protein inhibitor they had discovered.

[0005] Despite the large number of FAP inhibitors that have been synthesized and characterized, only a limited number of them have been radiolabeled for medical purposes. In 2015, the first boronic acid-based FAP inhibitor radiolabeled with iodine-125 ( 125Although I-125 (I-MIP-1232) has been reported for atherosclerotic plaque imaging, it is important to clarify that I-125 is not a useful radionuclide for single-photon emission computed tomography (SPECT) or positron emission tomography (PET) imaging (Meletta et al., Evaluation of the radiolabeled boronic acid-based FAP inhibitor MIP-1232 for atherosclerotic plaque imaging. Molecules, 2015, Vol. 20, pp. 2081–2099).

[0006] In 2018, Lindner and Loktev reported the conjugation of 2,2',2'',2''''-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid (DOTA) to a 4-quinolinoyl-Gly-cyanopyrrolidine scaffold for labeling with appropriate radionuclides for diagnostic imaging or therapeutic purposes (Lindner et al., Development of quinoline-based theranostic ligands for the targeting of fibroblast activation protein. J. Nucl. Med., 2018, 59, 1415-1422; Loktev et al., A tumor-imaging method targeting cancer-associated fibroblasts. J. Nucl. Med., 2018, 59, 1423-1429). To improve tumor uptake and retention of FAP inhibitor (FAPI) derivatives, the authors developed 15 derivatives in which DOTA was attached to a 4-quinolinoyl-Gly-cyanopyrrolidine scaffold using different positions. The 15 different FAPIs were: 177 Lu, 90 Y, or 68 Among all the radiolabeled FAPI conjugates reported, 68 Ga-FAPI-02 and 68Ga-FAPI-04 proved to be the most suitable agent for diagnostic applications. Subsequently, the same authors reported other derivatives of the same framework, numbered consecutively from FAPI-21 to FAPI-55 (Loktev et al., Development of Fibroblast Activation Protein-Targeted Radiotracers with Improved Tumor Retention. J. Nucl. Med., 2019, 60, 1421-1429). Replacement of DOTA with 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA) gave rise to the derivative FAPI-74, which is 68 Ga and 18 F( 18 F-AlF, in the presence of AlCl3) (Giesel et al., FAPI-74 PET / CT Using Either 18 F-AlF or Cold-Kit 68 Ga Labeling: Biodistribution, Radiation Dosimetry, and Tumor Delineation in Lung Cancer Patients. J.Nucl.Med., 2021, Vol. 62, pp. 201-207). In particular, FAPI-35 99m Tc and possibly 188 (Lindner et al., Design and Development of 99m Tc-Labeled FAPI Tracers for SPECT Imaging and 188 Re Therapy.J.Nucl.Med., 2020, Vol. 61, 1507-1513).

[0007] The first FAP inhibitors used in humans for PET scans were 68 Ga-FAPI-02 and 68 Ga-FAPI-04 (Giesel et al., 68 Ga-FAPI PET / CT:biodistribution and preliminary dosimetry estimate of 2 DOTA-containing FAP-targeting agents in patients with various cancers.J.Nucl.Med., 2019, vol. 60, 386~392;Kratochwil et al. 68 Ga-FAPI PET / CT:Tracer Uptake in 28 Different Kinds of Cancer.J.Nucl.Med., 2019, vol. 60, 801~805).

[0008] Interestingly, 64 Cu- / 225 The Ac-FAPI-04 theranostic conjugate demonstrated its usefulness in treating pancreatic cancer overexpressing FAP in a mouse model. Therapy targeting fibroblast activation protein is effective in cancer treatment and may contribute to the establishment of new therapeutic strategies (Watabe et al., Theranostics Targeting Fibroblast Activation Protein in the Tumor Stroma: 64 Cu- and 22 Ac-Labeled FAPI-04 in Pancreatic Cancer Xenograft Mouth e Models.J.Nucl.Med., 2020, Volume 61, 563~569). Summary of the Invention [Problem to be solved by the invention]

[0009] However, before any radiation therapy, the uptake of the radiopharmaceutical in the tumor or its metastases must be assessed by nuclear imaging to confirm whether the treatment will be beneficial to the patient and to determine the activity required to deliver an ablative radiation dose to the tumor, i.e., personalized medicine is practiced. For this purpose, FAP inhibitor diagnostic radiopharmaceuticals must be used to acquire molecular images by PET or SPECT. Of these two techniques, PET has the highest spatial resolution and sensitivity. Therefore, as mentioned above, the diagnostic FAP inhibitor radiopharmaceuticals developed and applied to clinical studies to date are conjugated to 4-quinolinoyl-Gly-cyanopyrrolidine derivatives, which are radiopharmaceuticals for PET. 68 Ga and 18 F was used, similarly coupled to quinoline-cyanopyrrolidine 99m Only one study used a Tc derivative (see table below). Nevertheless, nationally and internationally, SPECT studies account for over 70% of all nuclear medicine studies due to their lower cost and greater availability of equipment and radionuclides, as they do not require a cyclotron to be installed in or near a hospital. For SPECT imaging, the most commonly used radionuclides are 99m There are no publications dedicated to studies on radiopharmaceuticals for FAP imaging based on Tc, pyrrolidine-boronic acid derivatives or boron-Pro.

[0010] [Table 1] [Brief explanation of the drawings]

[0011] DETAILED DESCRIPTION OF THE INVENTION

[0012] Detailed Description of the Invention For the purposes of the patent, the present invention provides a novel fibroblast activation protein (iFAP) inhibitory radiopharmaceutical based on the ((R)-1-((6-hydrazinylnicotinoyl)-D-alanyl)pyrrolidin-2-yl)boronic acid (HYNIC-iFAP) molecule, in which ethylenediaminediacetic acid (EDDA) is used to complete the coordination sphere of the radiometal. 99m In combination with the conventional use of HYNIC as a Tc chelator, HY A radiopharmaceutical is presented in which the hydrazine nitrogen of NIC serves as the preferred chemical group for interaction of the HYNIC-iFAP molecule with phenylalanine (Phe-350 and Phe-351), glutamic acid (Glu-203 and Glu-204), and serine (Ser-624) in the active center of fibroblast activation protein (FAP). 99m New radiopharmaceuticals of Tc-EDDA / HYNIC-iFAP ( 99m Tc-HYNIC-iFAP) uses nuclear medicine SPECT molecular imaging technology to detect FAP expressed in the microenvironment of malignant tumors of epithelial origin with high affinity in vivo. Figure 1 shows the patentable structure of HYNIC-iFAP. 99m The structure of a T radiopharmaceutical is shown schematically.

[0013] Based on molecular docking studies, we designed and synthesized a HYNIC-iFAP derivative [((R)-1-((6-hydrazinylnicotinoyl-)-dalanyl)-pyrrolidin-2-yl)boronic acid], which is compatible with the two most representative structures of boronPro reported in the literature: N-acyl-Gly-boroPro (Tran et al., Synthesis and structure-activity relationship of N-acyl-Gly-, N-acyl-Sar-, and N-blocked-boroPro inhibitors of FAP, DPP4, and POP. Bioorg. Med. Chem. Lett., Vol. 17, 2007, pp. 1438-1442) and N-(pyridine-4-carbonyl)-D-Ala-boroPro (Poplawski et al., Identification of selective and potent inhibitors of fibroblast activation protein and prolyl The affinity and inhibition constant (Ki) of the HYNIC-iFAP molecule conjugated to another derivative, S-2-(4-isothiocyanatobenzyl)-DOTA (DOTA-Bz-NCS-HYNIC-iFAP), was also compared. 68 Ga, 177 Lu, 64 Cu, 225 These structures were obtained to study their suitability for labeling with other DOTA-like radiometals, such as Ac. For this purpose, all structures were created in ChemDraw (.cdx format), and the 3D structures were exported in .pdb format via Chem3D software. Using the molecular editor AVOGADRO 1.2.0, due to the presence of boron atoms in each structure, the molecular geometries were optimized using a universal force field (UFF) for a total of 10,000 steps. Subsequently, a second geometric optimization was performed using the semi-empirical quantum chemistry software MOPAC 2016 at the PM7 level of theory, and the resulting spatial configurations were exported to .pdb format.

[0014] The crystal structure of the alpha subunit of fibroblast activation protein (FAP) was obtained from the RSCB Protein Data Bank database (PDB ID: 1Z68). For use as the receptor macromolecule in molecular docking calculations, this molecule was edited in BIOVIA Discovery Studio 2021 to remove water molecules and residues indicated by X-ray diffraction, leaving only the major amino acid chain in the .pdb file. The A chain of the dimer represented in the model was also removed, leaving only the B chain. Because a three-dimensional model of the macromolecule required a resolution of 2.60 Å, a homology modeling step was performed using the SWISS-MODEL online platform. The resulting structure was saved in .pdb format for use as the receptor. Both the receptor and HYNIC-iFAP-derived structures were prepared using the OPEN BABEL GUI 2006 library, which included the addition of missing hydrogens and molecular optimization for physiological pH (pH = 7.4), again generating the structures in .pdb format. The AutoDock tool 1.5.6 software package was used to organize the receptor as a macromolecule and each ligand in a separate file, exporting the files with a .pdbqt extension. The search box was used to search for the hydrophobic site around Ser-624 of the receptor, as proposed by Poplawsky and coworkers in 2013. The box was placed in S1 and centered at XYZ coordinates 18.948, 10.676, and 28.989, respectively, with a size of 90 for each axis. For the ligand, add the boron atom parameters available at http: / / mgldev.scripps.edu / pipermail / autodock / 2009-March / 005439.html using<AD4_parameters.dat> I had to modify the file.

[0015] Protein-ligand molecular docking was performed using the AutoDock 4.2.6 package. The necessary .map grids were calculated beforehand using the AutoGrid 4.2.6 tool. The log files containing the molecular docking results were visualized with the AutoDock tool by exporting the complex with the best affinity score to .pdb format. Visualization and analysis of distances and interactions were performed using BIOVA Discovery. Implemented by Studio Visualizer 2021.

[0016] The inhibition constant (Ki) is calculated by the formula:

number

[0017] Ki values ​​are given in molar units (M). Table 2 shows the affinity score, Ki inhibition constant, and distance to Ser624 for each ligand. As can be seen in Table 2, the HYNIC-iFAP molecule exhibited a 26-fold lower inhibition constant (Ki = 0.536 nM) than the N-(pyridine-4-carbonyl)-D-Ala-boroPro derivative (Ki = 14.07), indicating a higher inhibitory ability of HYNIC-iFAP by FAP.

[0018] [Table 2]

[0019] Consistent with the interaction map between HYNIC-iFAP and amino acid residues in the FA active center obtained by molecular docking studies, the increased affinity of HYNIC-iFAP over N-(pyridine-4-carbonyl)-D-Ala-boroPro is due to the presence of hydrazine nitrogen in HYNIC, which favors van der Waals interactions and hydrogen bonds between the HYNIC-iFAP molecule and the FAP active center, mainly with residues Glu-203, Glu-204, Phe-350, and Phe-351, as well as with the more proximal residue Ser-624 (interaction map between FAP amino acid residues and the HYNIC-iFAP ligand obtained by the molecular docking methodology described above). The high affinity and FAP inhibitory ability of the DOTA-Bz-NCS-HYNIC-iFAP ligand were confirmed. have shown the potential use in the preparation of new diagnostic and therapeutic radiopharmaceuticals, since this ligand is capable of chelating radionuclides, e.g. 68 Ga, 64 Cu, 177 Lu, and 225 This is because it is a useful ligand that can be labeled with Ac.

[0020] Methods for preparing radiopharmaceuticals of the present invention To synthesize the HYNIC-iFAP molecule, Boc-pyrrolidine was first dissolved in ethyl ether / TMEDA at -40 °C under a nitrogen atmosphere. It was then reacted with a solution of s-BuLi (in cyclohexane) at 5 °C. B(OMe)3 was then added, and an extractive purification procedure (first extracted with 2 M NaOH, then acidified with 2 M HCl, and finally extracted with EtOAc, followed by evaporation of the solvent) gave Boc-pyrrolidine-boronic acid, to which pinanediol was added. HPLC separation (microporasil column) afforded the R diastereomer. After Boc deprotection, D-alanine coupling was followed by succinimidyl-N-boc-HYNIC coupling using HATU / DIPEA. Finally, the compound was deprotected with TFA, purified by reverse-phase HPLC, and lyophilized. The final product was ((R)-1-((6-hydrazinylnicotinoyl)-dalanyl)pyrrolidin-2-yl)boronic acid (HYNIC-iFAP) and showed the expected mass spectrum: m / z 322 (calculated 321) [M+H]. + m / z 642 (calculated value 321) × [M+H] + . 1 H-NMR (300MHz, DMSO), δ(ppm):8.5-6.7(s,1H,-CH 6 -,arom.pyridine), 9.7 (s,1H,-NH-NH-), 8.3 (s,1H,-CH2-NHCO), 3.1-3.3 (m,1H,CH2CHB).

[0021] Reverse phase HPLC analysis of the lyophilized white solid indicated that the compound was 95% chemically pure.

[0022] HYNIC-iFAP (30 μg) was formulated as a lyophilized dosage form containing 10 mg EDDA, ​​20 mg tricine, 20 μg stannous chloride, and 50 mg mannitol. The formulation was prepared by dissolving 1 mL of a 0.2 M, pH 7 solution in 1 mL of ... 99 Mo / 99m Sodium pertechnetate solution obtained in situ from the Tc generator ( 99m When reconstituted with 1 mL of TcO4Na, the compound is patented. 99mTc-EDDA / HYNIC-iFAP( 99m Tc-HYNIC-iFAP) is produced with a radiochemical purity of greater than 98% as determined by reverse-phase HPLC.

[0023] The radiopharmaceutical is stable with a radiochemical purity of >95% even after 24 hours of labeling. In vitro stability studies in human serum show serum protein binding of 2.1±0.3% and high radiochemical stability (>95%). 99m To evaluate the in vitro specificity of TcHYNIC-iFAP, we used tumor stroma (patient biopsies) from two different molecular subtypes of breast cancer: luminal B, characterized by high expression of estrogen receptor (ER) and human epidermal growth factor receptor type 2 (HER2), and triple-negative (TNBC), characterized by zero expression of ER, progesterone receptor (PR), and HER2. We selected these tumor stroma, similar to previous studies of different breast tumor phenotypes, which showed that TNBC tumors expressed the highest levels of FAP, while luminal B tumors expressed the lowest levels of FAP (Park et al., Differential expression of cancer-associated fibroblast-related proteins according to molecular subtype and stromal histology in breast cancer. Breast. Cancer Res. Treat., 2015, vol. 149, pp. 727–741). Results showed that the radioactivity added to the stroma of TNBC tumors significantly increased the expression of TNBC tumors. 99m The uptake of Tc HYNIC-iFAP was 7.8 ± 1.2%, which corresponds to the radioactivity taken up by the stroma of luminal B tumors (2.3 ± 0.0 of the added radioactivity). 3%) was significantly higher (P<0.05, t-Student).

[0024] This compound showed no toxicity or side effects when administered at a dose of 40 mg / kg to BALB-C mice.99m Micro-SPECT / CT imaging of Tc-HYNIC-iFAP showed that 9.2 ± 1.4% of the administered activity per gram of tissue (%ID / g) was taken up into the tumor (Figure 2 ) and rapidly excreted, primarily via the kidney.

[0025] Pharmacokinetic and dosimetry studies in healthy volunteers show rapid blood clearance due to increased renal uptake and excretion and an effective dose of 2.0 ± 0.5 mSv per 740 MBq administered. Figure 3 shows the radiopharmaceutical dose obtained 1 hour after administration in healthy volunteers. 99m Figure 4 shows a SPECT image of Tc-HYNIC-iFAP. 18 F-FDG (PET, control radiopharmaceutical, gold standard for tumor metabolism detection) and 99m Figure 5 shows PET and SPECT images of the same patient with triple-negative breast cancer who received both Tc-HYNIC-iFAP(SPECT), demonstrating that both radiopharmaceuticals detect breast cancer tumors with high sensitivity. 18 F-FDG (PET, control radiopharmaceutical, gold standard for tumor metabolism detection) and 99m PET and SPECT images of the same lung cancer patient (poorly differentiated lung adenocarcinoma with a predominantly solid pattern) who received both Tc-HYNIC-iFAP(SPECT) demonstrate that both radiopharmaceuticals detect lung tumors with high sensitivity. 99m In the case of Tc-HYNIC-iFAP, high uptake correlates with recognition of FAP expression in the tumor microenvironment. These images are due to the FAP activity inhibitory properties enhanced by the nitrogen present in HYNIC hydrazine. 99m This provides primary evidence that Tc-HYNIC-iFAP can detect tumor lesions in a specific manner.

[0026] in conclusion, 99m Tc-HYNIC-iFAP is obtained with the following characteristics: - Greater than 98% radiochemical purity. - The ability of the radiopharmaceutical to specifically detect in vivo the tumor microenvironment expressing fibroblast activation protein by nuclear medicine single photon emission computed tomography (SPECT). - In addition to molecular recognition of boronPro residues, 99m The Tc-based patented radiopharmaceutical has the ability to significantly capture and detect the FAP-expressing tumor microenvironment with high sensitivity (low Ki value; Ki=0.536) due to the increased affinity conferred by the presence of a hydrazine nitrogen within the HYNIC molecule, which allows it to dock and efficiently interact with the active site of the FAP enzyme for detection by SPECT imaging.

[0027] Having fully described the invention, the following clauses are considered new and are therefore claimed as exclusive features.

Claims

1. structure: 【Chemistry 1】 Chemical formula containing ９９ｍ Tc-EDDA / HYNIC-iFAP ( ９９ｍ Tc-HYNIC-iFAP) radiopharmaceutical.

2. A radiopharmaceutical composition comprising the radiopharmaceutical of claim 1.

3. 10. The radiopharmaceutical of claim 1 for use as a radiodiagnostic agent.

4. structure: 【Chemistry 2】 A radiopharmaceutical precursor ligand comprising:

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