,
, Absorption chromatogram of H-(Me)FGC(S t Bu)K(Cy5.0A)-NH2 (?abs = 650 nm
, Emission chromatogram of H-(Me)FGC(S t Bu)K(Cy5.0A)-NH2 (?exc = 610 nm and detected at ?em = 665 nm)
, -carboxypropyl)(methyl)amino)phenyl)diazenyl)-7-(diethylamino)-5-phenylphenazin-5-ium BHQ3A
, NOBF4 (51 mg, 0.44 mmol, 1.1 eq.) was added and the reaction mixture was stirred at 0 °C during 1 h, Then
, 92 mg, 0.48 mmol, 1.2 eq.) in dry ACN (2 ml) was slowly added and using methanol in DCM and 1% triethylamine as eluent (gradient of 0% to 20%) and semi-preparative RP-HPLC (system B) affording BHQ3A as a blue solid, A solution of functionalized tertiary aniline 9
, 300 MHz, DMSO-d6) ? 12.16 (s, 1H), 8.34 (d, J = 9.0 Hz, 1H), 8.16 -8.06 (m, 2H), 7.99 -7.89 (m, 4H), 7.75 (dd, J = 6.5, 3.0 Hz, 2H), 7.69 (m, 3H), 7.15 (d, J = 1.8 Hz, 1H), vol.6
,
, 0B)DEVDAK(Dde)-NH2
, 0B)DEVDAK(Dde)-NH2 was prepared according to the general procedure 4 from Cy5, vol.8
, DIPEA (7.2 µl, 41.2 µmol, 5 eq.) in NMP (1 ml) and Ac-KDEVDAK(Dde)-NH2 (10 mg, TSTU (2.7 mg, 9.1 µmol, 1.1 eq.), vol.9
, The coupling was monitored by RP-HPLC and 12 was precipitated in cold Et2O. The crude solid was isolated by centrifugation and dissolved in DMF
, Hydroxylamine hydrochloride (2.9 mg, 41.2 µmol, 5 eq.) and imidazole (2.8 mg, 41.2 µmol, 5 eq.) were added. The reaction mixture was stirred at RT for 3 hours and Dde removal was monitored by
, Selected fractions were lyophilised to give the TFA salt of targeted peptide affording Ac-K(Cy5.0B)DEVDAK(Dde)-NH2 as blue solid (5.2 mg, 3.9 µmol
, HRMS calculated for C68H99N12O15
, 0B)DEVDAK(BHQ3A)-NH2 P1
, NMP (150 µl) and Ac-K(Cy5.0B)DEVDAK(Dde)-NH2
, The coupling was monitored by RP-HPLC before purification by semi-preparative RP-HPLC (system A)
, HRMS calculated for C101H129Na3N18O16
,
, -methoxyethoxy)quinazolin-4-yl)amino)phenyl)-1H-1,2,3-triazol-1-yl)hexyl)-3,3-dimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-1-(5-carboxypentyl, vol.3, p.7
, DIPEA (33 µl, 190 µmol, 10 eq.), erlotinib (7.5 mg, 19 µmol, 1 eq.) and Cy5.0C (13 mg, 19 µmol, 1 eq.) in dry ACN (10 ml) affording 25 as a blue solid
, HRMS calculated for C56H65N8O10S2
,
, DIPEA (15 µl, 90 µmol, 10 eq.), erlotinib (3.5 mg, 9 µmol, 1 eq.) and MD130 (10 mg, 9 µmol, 1 eq.) in dry ACN (2 ml) affording 26 as a blue solid
, HRMS calculated for C84H111N14O9S2
,
, Absorption chromatogram of 26 (max plot mode) and corresponding absorption spectrum H-(Me)FGCK(Cy5.0A-erlotinib
, 300 µl of 0.1 M of aqueous solution of NaHCO3 affording the radiolabelable precursor 27 as a blue solid
, HRMS calculated for C80H103N14O9S
,
, sodium acetate (0.9 mg, 1 µmol, 1.5 eq.), ReOCl3(PPh3)2 (0.6 mg, 2.6 µmol, 1 eq.) were dissolved in MeOH (250 µl) and stirred at reflux (65 °C) overnight
, Formation of the complex was monitored by RP-HPLC before purification by semi-preparative RP-HPLC (system
, Imaging in the Era of Molecular Oncology, Nature, vol.452, issue.7187, pp.580-589, 2008.
, A Molecular Imaging Primer: Modalities, Imaging Agents, and Applications, Physiol. Rev, vol.2012, issue.2, pp.897-965
, Radiopharmaceuticals as Probes to Characterize Tumour Tissue, Eur. J. Nucl. Med. Mol. Imaging, vol.42, issue.4, pp.537-561, 2015.
, Tissue-Specific Near-Infrared Fluorescence Imaging, Acc. Chem. Res, vol.49, issue.9, pp.1731-1740, 2016.
, Fluorescence Imaging of Tumors in Vivo, Curr. Med. Chem, vol.12, issue.7, pp.795-805, 2005.
, New Strategies for Fluorescent Probe Design in Medical Diagnostic Imaging, Chem. Rev, vol.110, issue.5, pp.2620-2640, 2010.
, Activatable Fluorescent Probes in Fluorescence-Guided Surgery: Practical Considerations, Bioorg. Med. Chem, vol.26, issue.4, pp.925-930, 2018.
, Fluorescence-Guided Surgery. Front. Oncol, vol.2017, issue.314, pp.1-16
, Optical Molecular Imaging for Tumor Detection and Image-Guided Surgery, Biomaterials, vol.157, pp.62-75, 2018.
,
, A Bright Future for Precision Medicine: Advances in Fluorescent Chemical Probe Design and Their Clinical Application, Cell Chem. Biol, vol.23, issue.1, pp.122-136, 2016.
, Fluorescent Chemical Probes for Accurate Tumor Diagnosis and Targeting Therapy, Chem. Soc. Rev, vol.2017, issue.8, pp.2237-2271
, IND-Directed Safety and Biodistribution Study of Intravenously Injected Cetuximab-IRDye800 in Cynomolgus Macaques, Mol. Imaging Biol, vol.17, issue.1, pp.49-57, 2015.
, Safety and Tumor Specificity of Cetuximab-IRDye800 for Surgical Navigation in Head and Neck Cancer, Clin. Cancer Res, vol.21, issue.16, pp.3658-3666, 2015.
, Activity-Based Profiling of Proteases, Annu. Rev. Biochem, vol.83, issue.1, pp.249-273, 2014.
, In Vivo Imaging of Intraperitoneally Disseminated Tumors in Model Mice by Using Activatable Fluorescent Small-Molecular Probes for Activity of Cathepsins, Bioconjug. Chem, issue.10, pp.1838-1846, 2014.
, Development of an Activatable Fluorescent Probe for Prostate Cancer Imaging, Bioconjug. Chem, issue.8, pp.2069-2076, 2017.
,
, Rapid Cancer Detection by Topically Spraying a -Glutamyltranspeptidase-Activated Fluorescent Probe, Sci. Transl. Med, vol.2011, issue.110, pp.1-10
,
, Intraoperative Imaging of Hepatic Cancers Using ?-Glutamyltranspeptidase-Specific Fluorophore Enabling Real-Time Identification and Estimation of Recurrence, Sci. Rep, vol.2017, issue.1, pp.1-10
, Rapid Detection of Metastatic Lymph Nodes of Colorectal Cancer with a Gamma-Glutamyl Transpeptidase-Activatable Fluorescence Probe, Sci. Rep, vol.8, issue.1, p.17781, 2018.
, Materials for Fluorescence Resonance Energy Transfer Analysis: Beyond Traditional Donor-Acceptor Combinations, Angew. Chem. Int. Ed, vol.45, issue.28, pp.4562-4589, 2006.
, FRET-Based and Other Fluorescent Proteinase Probes, Biotechnol. J, vol.2014, issue.2, pp.266-281
,
, Design of Protease Activated Optical Contrast Agents That Exploit a Latent Lysosomotropic Effect for Use in Fluorescence-Guided Surgery, ACS Chem. Biol, vol.10, issue.9, pp.1977-1988, 2015.
, Optimization of a Protease Activated Probe for Optical Surgical Navigation, Mol. Pharm, vol.15, issue.3, pp.750-758, 2018.
,
, Introduction to Concepts and Strategies for Molecular Imaging, Chem. Rev, vol.110, issue.5, pp.2575-2578, 2010.
, Two Is Better than One'-Probes for Dual-Modality Molecular Imaging, Chem. Commun, issue.24, pp.3511-3524, 2009.
, Multimodal Radio-(PET/SPECT) and Fluorescence Imaging Agents Based on Metallo-Radioisotopes: Current Applications and Prospects for Development of New Agents, Dalton Trans, vol.40, issue.23, pp.6129-6143, 2011.
, Multimodality Molecular Imaging with Combined Optical and SPECT/PET Modalities, J. Nucl. Med, vol.49, issue.2, pp.169-172, 2008.
, Bimodal Imaging Probes for Combined PET and OI: Recent Developments and Future Directions for Hybrid Agent Development, BioMed Res. Int, pp.1-13, 2014.
, Development of a Clickable Bimodal Fluorescent/PET Probe for in Vivo Imaging, EJNMMI Res, vol.2015, issue.1
, Universal Molecular Scaffold for Facile Construction of Multivalent and Multimodal Imaging Probes, Bioconjug. Chem, vol.27, issue.3, pp.515-520, 2016.
, Click Chemistry and Radiochemistry: The First 10 Years, Bioconjug. Chem, issue.12, pp.2791-2807, 2016.
, Cyanine Polyene Reactivity: Scope and Biomedical Applications, Org Biomol Chem, vol.13, issue.28, pp.7584-7598, 2015.
, Recent Development of Chemosensors Based on Cyanine Platforms, Chem. Rev, vol.116, issue.14, pp.7768-7817, 2016.
, Productive Manipulation of Cyanine Dye ?-Networks, Angew. Chem. Int. Ed, vol.59, issue.2-5, 2019.
, Cyanine Dye Labeling Reagents: Sulfoindocyanine Succinimidyl Esters, Bioconjug. Chem, vol.4, issue.2, pp.105-111, 1993.
, Synthesis and Post-Synthetic Derivatization of a Cyanine-Based Amino Acid. Application to the Preparation of a Novel Water-Soluble NIR Dye, Tetrahedron Lett, issue.47, pp.8279-8284, 2006.
, Standards for Photoluminescence Quantum Yield Measurements in Solution (IUPAC Technical Report), Pure Appl. Chem, vol.83, issue.12, pp.2213-2228, 2011.
, Radioactive Transition Metals for Imaging and Therapy, Chem. Rev, vol.2019, issue.2, pp.870-901
, Single Amino Acid Chelates (SAAC): A Strategy for the Design of Technetium and Rhenium Radiopharmaceuticals, Chem Commun, issue.5, pp.493-512, 2009.
, Kit Formulation for 99mTc-Labeling of Recombinant Anti-HER2 Affibody Molecules with a C-Terminally Engineered Cysteine, Nucl. Med. Biol, vol.37, issue.5, pp.539-546, 2010.
, Me)FGCDEVD, a Potential Tracer for Apoptosis Detection, Bioorg. Med. Chem. Lett, vol.23, issue.5, pp.1375-1378, 2013.
URL : https://hal.archives-ouvertes.fr/hal-00937472
, Formation of Carboxamides with N,N,N?,N?-Tetramethyl (Succinimido) Uronium Tetrafluoroborate in Aqueous / Organic Solvent Systems, Tetrahedron Lett, vol.32, issue.9, pp.1157-1160, 1991.
, Piperazine and DBU: A Safer Alternative for Rapid and Efficient Fmoc Deprotection in Solid Phase Peptide Synthesis, RSC Adv, vol.2015, issue.126, pp.104417-104425
, Apoptosis: A Basic Biological Phenomenon with Wideranging Implications in Tissue Kinetics, Br. J. Cancer, vol.26, issue.4, pp.239-257, 1972.
, Caspases: Keys in the Ignition of Cell Death, Chem. Rev, vol.102, issue.12, pp.4489-4500, 2002.
, Hallmarks of Cancer: The Next Generation, Cell, vol.144, issue.5, pp.646-674, 2011.
, Biomarkers and Molecular Probes for Cell Death Imaging and Targeted Therapeutics, Bioconjug. Chem, vol.2012, issue.10, pp.1989-2006
, Caspases: Enemies Within, Science, vol.281, issue.5381, pp.1312-1316, 1998.
, Functional Imaging of Proteases: Recent Advances in the Design and Application of Substrate-Based and Activity-Based Probes, Curr. Opin. Chem. Biol, vol.15, issue.6, pp.798-805, 2011.
, Detection of Active Caspases During Apoptosis Using Fluorescent Activity-Based Probes, In Programmed Cell Death, vol.1419, pp.27-39, 2016.
, Noninvasive Optical Imaging of Apoptosis by Caspase-Targeted Activity-Based Probes, Nat. Med, vol.15, issue.8, pp.967-973, 2009.
, A Novel Quenched Fluorescent Activity-Based Probe Reveals Caspase-3 Activity in the Endoplasmic Reticulum during Apoptosis, Chem. Sci, vol.2016, issue.2, pp.1322-1337
,
, Synthesis and Characterization of a Small, Membrane-Permeant, Caspase-Activatable Far-Red Fluorescent Peptide for Imaging Apoptosis, J. Med. Chem, issue.17, pp.5404-5407, 2005.
, Piwnica-Worms, D. Biochemical and in Vivo Characterization of a Small, Membrane-Permeant, Caspase-Activatable Far-Red Fluorescent Peptide for Imaging Apoptosis ?, Biochemistry, vol.46, issue.13, pp.4055-4065, 2007.
, An Improved Cell-Penetrating, Caspase-Activatable, Near-Infrared Fluorescent Peptide for Apoptosis Imaging, Bioconjug. Chem, vol.20, issue.4, pp.702-709, 2009.
, In Vitro and Ex Vivo Evaluation of Smart Infra-Red Fluorescent Caspase-3 Probes for Molecular Imaging of Cardiovascular Apoptosis, Int. J. Mol. Imaging, pp.1-13, 2011.
URL : https://hal.archives-ouvertes.fr/inserm-02296580
, Biochemical Characterization of a Caspase-3 Far-Red Fluorescent Probe for Non-Invasive Optical Imaging of Neuronal Apoptosis, J. Mol. Neurosci, vol.54, issue.3, pp.451-462, 2014.
URL : https://hal.archives-ouvertes.fr/hal-01196843
, Evaluation of [18F]-CP18 as a PET Imaging Tracer for Apoptosis, Mol. Imaging Biol, vol.15, issue.6, pp.739-747, 2013.
, In Vitro and In Vivo Evaluation of the Caspase-3 Substrate-Based Radiotracer [18F]-CP18 for PET Imaging of Apoptosis in Tumors, Mol. Imaging Biol, vol.15, issue.6, pp.748-757, 2013.
, Multimodality Molecular Imaging of Apoptosis in Oncology, Am. J. Roentgenol, vol.197, issue.2, pp.308-317, 2011.
, Cell-Permeable 99mTc(CO3)-Labeled Fluorogenic Caspase 3 Substrate for Dual-Modality Detection of Apoptosis, vol.15, pp.8979-8984
, Complementary Optical and Nuclear Imaging of Caspase-3 Activity Using Combined Activatable and Radio-Labeled Multimodality Molecular Probe, J. Biomed. Opt, vol.14, issue.4, pp.405071-0405073, 2009.
, Eliminating Caspase-7 and Cathepsin B Cross-Reactivity on Fluorogenic Caspase-3 Substrates, Mol BioSyst, vol.12, issue.3, pp.693-696, 2016.
, Selective Detection and Inhibition of Active Caspase-3 in Cells with Optimized Peptides, J. Am. Chem. Soc, vol.135, issue.34, pp.12869-12876, 2013.
, Discovery of a Highly Selective Caspase-3 Substrate for Imaging Live Cells, ACS Chem. Biol, vol.2014, issue.10, pp.2199-2203
, Kondrat'eva Ligation: Diels-Alder-Based Irreversible Reaction for Bioconjugation, J. Org. Chem, issue.21, pp.10353-10366, 2014.
URL : https://hal.archives-ouvertes.fr/hal-01144842
, Azo-Based Fluorogenic Probes for Biosensing and Bioimaging: Recent Advances and Upcoming Challenges, Chem. -Asian J, vol.12, issue.16, pp.2008-2028, 2017.
URL : https://hal.archives-ouvertes.fr/hal-01562215
, Bioconjugatable Azo-Based Dark-Quencher Dyes: Synthesis and Application to Protease-Activatable Far-Red Fluorescent Probes, Chem. -Eur. J, vol.19, issue.5, pp.1686-1699, 2013.
URL : https://hal.archives-ouvertes.fr/hal-00996530
, Symmetrical and Asymmetrical Cyanine Dyes. Synthesis, Spectral Properties, and BSA Association Study, J. Org. Chem, issue.12, pp.5511-5520, 2014.
, Vitro Evaluation, and In Vivo Metabolism of Fluor/Quencher Compounds Containing IRDye 800CW and Black Hole Quencher-3 (BHQ-3), pp.1287-1297, 2011.
, A Novel 4-Aminobenzyl Ester-Based Carboxy-Protecting Group for Synthesis of Atypical Peptides by Fmoc-But Solid-Phase Chemistry, J. Chem. Soc. Chem. Commun, vol.21, pp.2209-2210, 1995.
, Efficient Use of the Dmab Protecting Group: Applications for the Solid-Phase Synthesis of N-Linked Glycopeptides, Org. Biomol. Chem, vol.7, issue.11, pp.2255-2258, 2009.
, Selectively Activatable Latent Thiol and Selenolesters Simplify the Access to Cyclic or Branched Peptide Scaffolds, Org. Lett, issue.14, pp.3636-3639, 2015.
, Problem of Aspartimide Formation in Fmoc-Based Solid-Phase Peptide Synthesis Using Dmab Group to Protect Side Chain of Aspartic Acid, J. Pept. Sci, vol.14, issue.3, pp.335-341, 2008.
, Amino Acid-Protecting Groups, Chem. Rev, vol.109, issue.6, pp.2455-2504, 2009.
, Bioorthogonal Chemistry: Fishing for Selectivity in a Sea of Functionality, Angew. Chem. Int. Ed, vol.48, issue.38, pp.6974-6998, 2009.
, Copper-Catalyzed Azide-Alkyne Click Chemistry for Bioconjugation, Current Protocols in Chemical Biology
, , vol.110148, 2011.
, Kinase Inhibitors in Cancer, Reference Module in Chemistry, 2014.
, Rôle de l'EGFR dans le cancer pulmonaire non à petites cellules, Rev. Médicale Suisse, vol.5, pp.1096-1100, 2009.
, In Synthesis of Best-Seller Drugs, pp.495-547, 2016.
, Nat. Rev. Drug Discov, vol.4, issue.1, pp.13-14, 2005.
, Rôle du récepteur à l'EGF dans la survenue et le traitement des cancers bronchiques non à petites cellules -EGFR plays a key-role in the pathogenesis and treatment of non-small-cell lung cancer, p.7, 2007.
, Indications des inhibiteurs de tyrosine kinase de l'EGFR dans le cancer bronchique non à petites cellules : pratiques actuelles et perspectives, vol.8
, Imaging with Small-Molecule Tyrosine Kinase Inhibitors: TKI-PET, Drug Discov. Today, vol.2012, pp.1175-1187
, Positron Emission Tomography (PET) Imaging with [11C]-Labeled Erlotinib: A Micro-PET Study on Mice with Lung Tumor Xenografts, Cancer Res, vol.69, issue.3, pp.873-878, 2009.
, Development of [11C]Erlotinib Positron Emission Tomography for In Vivo Evaluation of EGF Receptor Mutational Status, Clin. Cancer Res, vol.19, issue.1, pp.183-193, 2013.
, Dash, A. 68 Ga Labeled Erlotinib: A Novel PET Probe for Imaging EGFR over-Expressing Tumors, Bioorg. Med. Chem. Lett, vol.2017, issue.19, pp.4552-4557
, In-and Ex-Vivo Molecular Imaging of Apoptosis to Assess Sensitivity of Non-Small Cell Lung Cancer to EGFR Inhibitors Using Probe-Based Confocal Laser Endomicroscopy, PLOS ONE, vol.2017, issue.7
URL : https://hal.archives-ouvertes.fr/hal-01643963
, Real-Time Molecular Optical Micro-Imaging of EGFR Mutations Using a Fluorescent Erlotinib Based Tracer, BMC Pulm. Med, vol.19, issue.3, pp.1-9, 2019.
URL : https://hal.archives-ouvertes.fr/hal-02353026
, Les inhibiteurs de tyrosine kinase en oncologie -Tyrosine kinase inhibitors in oncology, vol.14
, Nuclear and Optical Bimodal Imaging Probes Using Sequential Assembly: A Perspective, Cancer Biother. Radiopharm, vol.33, issue.8, pp.308-315, 2018.
, Modular Assembly of Multimodal Imaging Agents through an Inverse Electron Demand Diels-Alder Reaction, Bioconjug. Chem, vol.2019, issue.3, pp.888-897
, Review on Near-Infrared Heptamethine Cyanine Dyes as Theranostic Agents for Tumor Imaging, Targeting, and Photodynamic Therapy, J. Biomed. Opt, vol.21, issue.5, pp.509011-05090111, 2016.
, Hydroxycoumarin?Hemicyanine Hybrids: A New Class of Far-Red Emitting Fluorogenic Dyes, pp.4175-4178, 2008.
, A Convenient Synthesis of Cyanine Dyes: Reagents for the Labeling of Biomolecules, Eur. J. Org. Chem, issue.12, pp.2107-2117, 2008.
, Solid Phase Synthesis of Functionalised SAM-Forming Alkanethiol-Oligoethyleneglycols, J Mater Chem B, vol.2014, issue.24, pp.3741-3744
, Base-Promoted Reactions of Bridged Ketones and 1,3-and 1,4-Haloalkyl Azides: Competitive Alkylation vs Azidation Reactions of Ketone Enolates, J. Org. Chem, vol.69, issue.5, pp.1720-1722, 2004.
,
, La qualité du diagnostic oncologique, déterminant la thérapie et le pronostic du patient, repose sur différentes techniques d'imagerie ou modalités, associées à des molécules de contraste afin de générer une image représentative
, L'imagerie moléculaire, incluant l'imagerie optique de fluorescence et l'imagerie nucléaire, est couramment utilisée pour la prise en charge oncologique des patients
, MOnomolecular Multimodal Imaging Agent) a émergé, associant plusieurs modalités au sein d'une structure moléculaire unique. Cette combinaison performante cumule les forces de chacune des modalités combinées en palliant leur limitation respective
, ces travaux de thèse portent sur le développement et l'exploitation d'une plateforme polyvalente bimodale (fluorescente et radioactive) composée d'un synthon peptidique, radiomarquable par chélation du 99m Tc, couplé à fluorophore présentant des propriétés spectroscopiques d'émission dans le proche infra-rouge, gamme spectrale privilégiée pour l'imagerie in vivo. Cette plateforme est finalement clickable à toute (bio)molécule d'intérêt
, L'exploitation de cette plateforme bimodale clickable a été explorée dans le contexte du diagnostic oncologique via deux cibles enzymatiques principales : la caspase-3 et les tyrosine-kinases
, Les étapes de conception, synthèse, optimisation, développement et validation biologique préliminaire in vitro de la plateforme et des sondes dérivées bimodales sont présentés
, Mots clés : imagerie moléculaire, multimodalité, fluorescence, SPECT, caspase-3, erlotinib Abstract Oncologic healthcare and remission prognosis rely on a reliable and accurate diagnosis. Molecular imaging, including optical and nuclear imaging, is currently used for the management cancer therapy
, Recently, a new class of bio-imaging agent called MOMIA (MOnomolecular Multimodal Imaging Agent) emerged by combining the synergistic strengths of several modalities on the same molecular structure
, We envisioned to merge optical and nuclear imaging in order to develop a molecular tool offering a non-invasive, highly resolutive and sensitive detection
, Our approach relies on a universal bimodal clickable scaffold with a selected targeting ligand. Two distinct enzymatic targets have been explored in the oncologic context: caspase-3 as a key component in an apoptotic program and tyrosine kinase inhibitors involved in lung cancer therapy
, These multimodal sensors have a promising potential in translational clinical applications. Key words: molecular imaging, multimodality, fluorescence, SPECT, p.3