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  • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
  • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
  • C07D487/08—Bridged systems
• • A—HUMAN NECESSITIES
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  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
  • A61K47/6871—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting an enzyme
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/0474—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group
  • A61K51/0482—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group chelates from cyclic ligands, e.g. DOTA
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
  • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
  • A61K51/1075—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody the antibody being against an enzyme
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
  • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
  • A61K51/1093—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody conjugates with carriers being antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/04—Antineoplastic agents specific for metastasis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
  • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
  • C07B59/002—Heterocyclic compounds
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/32—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/04—X-ray contrast preparations
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
  • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
  • C07B2200/05—Isotopically modified compounds, e.g. labelled
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered

Definitions

• the present invention relates to chelates resulting from the complexation of picolinate cross-bridged cyclams with metallic cations, preferably copper (II) or gallium (III).
• the invention further relates to picolinate cross-bridged cyclam ligands.
• Another object of the invention is the use of chelates of the invention in nuclear medicine and the use of ligands of the invention in cations detection or epuration of effluents.
• Tetraazamacrocycles such as derivatives of cyclam (1,4,8,11-tetraazacyclotetradecane) generate an important interest in many fields such as medicine, especially nuclear medicine; epuration of effluents contaminated with radioactive elements or metals such as lead; catalysis; solid/liquid extraction and liquid/liquid extraction; or detection of traces of metallic cations.
• the present invention relates to all these fields of applications, especially nuclear medicine.
• radiopharmaceuticals used as therapeutic agents or as imaging agents often comprise chelates of radioelements.
• a targeting biomolecule may be appended on the chelating moiety in order to induce a site-specific delivery of the radiation, leading to a bifunctional chelating agent (BCA).
• BCA bifunctional chelating agent
• Obtaining a BCA requires the introduction of an appropriate conjugation group in the structure of the metal chelator, to allow for the bioconjugation prior or after labeling with the radioisotope.
• the targeting agent may be for example an antibody, an hapten or a peptide.
• the chelate should thus be bioconjugated to a biological vector while trapping the radionuclide to form a stable complex preventing the release of the metal in the organism.
• the kinetic constraint has to be considered because of the limited half-life of the radionuclide.
• Dota (1,4,7, 10-tetraazacyclododecane-l,4,7,10-tetraacetic acid) is a tetra N- functionalized cyclen (scheme 1).
• dota is referred to as "H 4 dota”
• the four hydrogen atoms specified before “dota” reflecting the fact that in order to have the four carboxylic acid functions in "COOH” form, the four amines of the macrocycle should be protonated.
• the same nomenclature is used along the description for macrocycles comprising carboxylic acid functions.
• Dota is the most used ligand to complex gadolinium (III) for MRI imaging. Dota also enables to complex other metals commonly used in nuclear medicine, such as for example m In, 68 Ga, 149 Tb, 213 Bi, 212 Bi, 212 Pb, ⁇ Cu or 67 Cu. Derivatives of the dota, are today widely studied (scheme 1).
• copper has been receiving much interest due to the existence of several radionuclides with different half- life times and emission properties suitable for diagnostic imaging or therapeutic applications.
• Copper exists predominantly as divalent metal cation that prefers donor groups such as amines and anionic carboxylates to form complexes with coordination numbers of 4-6.
• High coordination numbers are usually preferred, often providing square pyramidal, trigonal bipyramidal or octahedral geometries, so as to entirely surround the metal cation.
• the family of tetraaza macrocycles with N- appended coordinating arms stands out owing to the efficiency and versatility of its copper chelation.
• gallium prefers high coordination numbers, especially under the form of octahedral geometries and tetraaza macrocycles with N-appended coordinating arms may be used for its chelation.
• Metallation kinetics may be determined using UV-visible spectrometry by measuring the increasing intensity of the complex d-d transition band.
• metallation kinetics may also be determined by NMR. Suitable metallation kinetics depends on the half-life of the radionuclide used to form the chelate.
• Thermodynamic stability may be evaluated by determining the stability constants of the complexes, especially the association constant K and pK (or logK). Stability constants may be measured by potentiometry or spectroscopies. pM values may be calculated from pK in order to compare thermodynamic stability with corresponding values of other ligands of the prior art. Indeed, pM reflects the amount of ligand not chelated, taking into account the basicity of the ligand.
• a "very good thermodynamic stability” refers to a thermodynamic stability at least comparable, preferably better than that of the dota chelate formed with the same metal.
• Inertness with respect to other metals may be evaluated by determining and comparing the pCu 2+ versus pZn 2+ .
• Competitive experiments may also be conducted. Especially, excess of zinc necessary to lead to a transchelation may be determined in competitive experiments with zinc.
• a chelate is considered having a suitable inertness with respect to other metals when it has inertness at least comparable, preferably better than that of the dota chelate formed with the same metal.
• Kinetic inertness may be evaluated by measuring metal dissociation upon competition with H + , in acid medium. Especially, half-life of the complex may be determined in presence of H + at different concentrations and temperatures.
• a chelate is considered having a suitable kinetic inertness when it is at least comparable, preferably better than that of the dota chelate formed with the same metal.
• Stability upon reduction may be evaluated by determining the dissociation of the reduced metal. Dissociation may be measured with cyclic voltammetry in electrochemical experiments.
• a chelate is considered having a suitable stability upon reduction when it is at least comparable, preferably better than that of the dota chelate formed with the same metal. Chelates with a good thermodynamically stability and a kinetic inertness prevent possible transchelation of the metal when the complex is challenged with biological ligands or bioreductants.
• the chelate and the chelator display good water solubility.
• the commercially available dota is used to complex ⁇ Cu ⁇ I), 67 Cu(II) and 68 Ga(III).
• copper-dota chelates are far from meeting requirements of the above specifications.
• tetraazacycloalkanes derivatives of cyclam such as for example teta and te2a (scheme 1), were recently used to complex ⁇ Cu or 67 Cu for PET or RET applications.
• Their suitable N-functionalization can also give them a good affinity toward other metals such as heavy metal or lanthanides and extend their use in these applications with for example 99m Tc, 186 Re, 188 Re, U1 ln, 68 Ga, 89 Zr, 177 Lu, 149 Tb, 153 Sm, 212 Bi ( 212 Pb), 213 Bi and 225 Ac.
• chelates formed from these derivatives of cyclam do not meet all requirements of the above specifications.
• ligands potentially useful for radiopharmaceuticals should combine a high thermodynamic stability and kinetic inertness of the complexes with a fast metal complexation under mild conditions, as the latter is crucial to take full advantage of the short radioisotope half- life times and allow for use of heat- and pH- sensitive biomolecules.
• Picolinate arms have been demonstrated to induce strong coordination ability toward transition and post-transition metals when appended on macrocyclic ligands, as well as non macrocyclic ligands.
• picolinate moiety is bidentate: it has a nitrogen atom and an oxygen atom, both capable to participate to the coordination of a metal. Therefore, picolinate derivatives were recently used for the complexation of lanthanides, lead or bismuth (Rodrigez-Rodrigez A. et al. Inorg. Chem. 2012, 51, 13419-13429; Rodrigez-Rodrigez A. et al. Inorg. Chem. 2012, 51, 2509-2521). They were also recently used for the complexation of copper.
• Orvig et coll. disclosed a derivative of ethylenediamine grafted with two picolinate arms H 2 dedpa, represented on scheme 2 below for the chelation of copper (Boros et al., JACS, 2010 , 132, 15726-33; Boros et al. Nucl. Med. Biol. 2011, 38, 1165-1174).
• cross -bridged chelators Rigid tetraazamacrocycles, known as "cross -bridged chelators", are the subject of great interest due to the outstanding behavior of their complexes, especially their inertness.
• Examples of cross-bridged chelators are cross-bridged cyclam derivatives cb-te2a and side-bridged sb-telalp or cross-bridged cyclen derivative cb-do2a (scheme 1).
• Cross- bridged chelators are defined as containing an ethylene (or propylene) bridging unit connecting two nitrogen atoms of the macrocycle in trans position and they have originated some of the most inert copper (II) complexes ever reported.
• cross-bridged chelators and especially Hcb-te2a, meet the above mentioned specifications, especially inertness points d) and e), with the notable exception of a very slow metallation kinetics (point a).
• the Applicant expected a drastic decrease of metallation kinetics, leading to a ligand offering a compromise between good inertness and fast kinetics but not meeting all 5 requirements of the above specifications.
• the Applicant demonstrated that, as other cross-bridged derivatives, the Hcb-telpa ligand of the invention is a proton-sponge (see acido-basic studies - example 5, paragraph B.l).
• cross-bridging Htelpa to form Hcb-telpa and derivatives thereof did not lead to a decrease of metallation kinetic, compared to non-cross-bridged cyclams.
• the cross-bridged ligand of the invention Hcb-telpa surprisingly shows a very rapid metallation kinetic, even in acidic conditions.
• the invention relates to ligands of formula (I)
• n, R 1 , R 2 , R 3 , R 4 , R 5 , L 1 , L 2 , L 3 and L 4 are as defined below.
• the ligands of the invention lead to chelates meeting the 5 requirements of the above specifications.
• properties of copper(II) chelate of Hcb-telpa are reported in the experimental part below and compared to those of copper(II) chelates of the art, evidencing that the chelate of the invention entirely fulfills specifications.
• the invention also relates to chelates resulting from the complexation of a ligand of formula (I) with metallic cations.
• the ligand of formula (I) of the invention presents the advantage of being easily manufactured using a simple chemical synthesis.
• the ligand Hcb-telpa and derivatives thereof present a competency for diverse radioisotopes useful in nuclear medicine, such as for example 64 Cu, 67 Cu, 68 Ga, 89 Zr, 99m Tc, m In, 186 Re, 188 Re, 210 At, 212 Bi ( 212 Pb), 213 Bi, 225 Ac, 90 Y, 177 Lu, 153 Sm, 149 Tb or 166 Ho.
• Hcb-telpa enables the bio-vectorization of the chelate by the introduction of vectorizing groups on the cyclam core, through N-functionalization and/or C-functionalization.
• the cyclam core may be C-functionalized according to the method described in patent application WO2013/072491.
• the carboxylic function of the picolinate arm may also be functionalized.
• the invention thus encompasses Hcb-telpa ligand, functionalized and/or vectorized derivatives thereof and corresponding chelates with metallic cations, preferably copper(II) or gallium(III).
• the chelate of the invention is obtained in aqueous medium, contrary to what is currently done in the art, which is very advantageous for nuclear medicine applications.
• the ligand of formula (I) of the invention may be used for epuration of effluents contaminated with radioactive elements or metals such as lead; catalysis; solid/liquid extraction and liquid/liquid extraction; or detection of traces of metallic cations.
• complex or “chelate” refer to a molecule binding a metal ion. Chelation (or complexation) involves the formation or presence of two or more separate coordinate bonds between a polydentate (multiple bonded) molecule and a single central atom. Polydentate molecules are often organic compounds, and are called ligands, chelants, chelatants, chelators, chelating agents, or sequestering agents.
• ligand or "chelator” refer to a polydentate molecule able to form coordinate bonds with a metal ion to give a chelate.
• coupling function refers to a function capable to react with another function.
• the coupling function is selected from the group comprising amine; isothiocyanate; isocyanate; activated ester such as for example N-hydroxysuccinimide ester, N-hydroxyglutarimide ester or maleimide ester; carboxylic acid; activated carboxylic acid such as for example acid anhydride or acid halide; alcohol; alkyne; halide; azide; siloxy; phosphonic acid; thiol; tetrazine; norbornen; oxoamine; aminooxy; thioether; haloacetamide such as for example chloroacetamide, bromacetamide or iodoacetamide; glutamate; glutaric anhydride, succinic anhydride, maleic anhydride; aldehyde; ketone; hydrazide; chloroformate and maleimide.
• vectorizing group refers to a chemical group suitable to induce site-specific delivery of the compound once administered.
• the vectorizing group is selected from the group comprising antibody, preferably a monoclonal antibody; hapten, peptide, proteins, sugars, nanoparticle, liposomes, lipids, polyamines such as for example spermine.
• activating function refers to a chemical moiety capable to render reactive a chemical function.
• an activating function may be N-hydroxysuccinimide, N-hydroxyglutarimide maleimide, halide or anhydride moieties.
• alkyl refers to any saturated linear or branched hydrocarbon chain, with 1 to 12 carbon atoms, preferably 1 to 6 carbon atoms, and more preferably methyl, ethyl, n- propyl, i-propyl, n- butyl, i-butyl, s-butyl and t-butyl, pentyl and its isomers (e.g. n- pentyl, iso-pentyl), and hexyl and its isomers (e.g. n-hexyl, iso-hexyl).
• alkene or “alkenyl” refer to any linear or branched hydrocarbon chain having at least one double bond, of 2 to 12 carbon atoms, and preferably 2 to 6 carbon atoms.
• alkenyl groups are ethenyl, 2-propenyl, 2-butenyl, 3-butenyl, 2- pentenyl and its isomers, 2-hexenyl and its isomers, 2,4-pentadienyl and the like.
• alkyne or “alkynyl” refer to refer to any linear or branched hydrocarbon chain having at least one triple bond, of 2 to 12 carbon atoms, and preferably 2 to 6 carbon atoms.
• alkynyl groups are ethynyl, 2- propynyl, 2-butynyl, 3-butynyl, 2-pentynyl and its isomers, 2-hexynyl and its isomers-and the like.
• aryl refers to refers to a polyunsaturated, aromatic hydrocarbyl group having a single ring (i.e. phenyl) or multiple aromatic rings fused together (e.g. naphtyl) or linked covalently, typically containing 5 to 12 atoms; preferably 6 to 10, wherein at least one ring is aromatic.
• the aromatic ring may optionally include one to two additional rings (either cycloalkyl, heterocyclyl or heteroaryl) fused thereto.
• Non- limiting examples of aryl comprise phenyl, biphenylyl, biphenylenyl, 5- or 6- tetralinyl, naphthalen-1- or -2-yl, 4-, 5-, 6 or 7-indenyl, 1- 2-, 3-, 4- or 5- acenaphtylenyl, 3-, 4- or 5-acenaphtenyl, 1- or 2-pentalenyl, 4- or 5-indanyl, 5-, 6- , 7- or 8-tetrahydronaphthyl, 1,2,3,4-tetrahydronaphthyl, 1,4-dihydronaphthyl, 1-, 2-, 3-, 4- or 5-pyrenyl.
• arylalkyl refers to an alkyl group substituted by an aryle group: aryl-alkyl-.
• alkylaryl refers to an aryl group substituted by an alkyl group: alkyl-aryl-.
• heteroaryl refers but is not limited to 5 to 12 carbon-atom aromatic rings or ring systems containing 1 to 2 rings which are fused together or linked covalently, typically containing 5 to 6 atoms; at least one of which is aromatic, in which one or more carbon atoms in one or more of these rings is replaced by oxygen, nitrogen and/or sulfur atoms where the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quaternized.
• Such rings may be fused to an aryl, cycloalkyl, heteroaryl or heterocyclyl ring.
• Non-limiting examples of such heteroaryl include: furanyl, thiophenyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, oxatriazolyl, thiatriazolyl, pyridinyl, pyrimidyl, pyrazinyl, pyridazinyl, oxazinyl, dioxinyl, thiazinyl, triazinyl, imidazo[2, 1 -b] [ 1 ,3] thiazolyl, thieno [3 ,2- b] furanyl, thieno [3 ,2-b] thiophenyl, thieno[2,3-d][l,3]thiazolyl, thieno[2,3- d]imidazoly
• heteroarylalkyl refers to an alkyl group substituted by an aryle group: heteroaryl- alkyl-.
• alkylheteroaryl refers to an aryl group substituted by an alkyl group: alkyl- heteroaryl-.
• thioether refers to a functional group with the connectivity C-S-C.
• halide refers to fluoro, chloro, bromo, or iodo. Preferred halo groups are fluoro and chloro.
• aminooxy refers to a -0-NH 2 group
• hapten refers to a small molecule that can elicit an immune response only when attached to a large carrier.
• Radiopharmaceutical refers to a radioactive medicinal product. Radiopharmaceuticals are used in the field of nuclear medicine for the treatment of many diseases and/or as tracers for their diagnosis.
• patient refers a warm-blooded animal, more preferably a human, who/which is awaiting the receipt of, or is receiving medical care or is/will be the object of a medical procedure.
• treat are meant to include alleviating, attenuating or abrogating a condition or disease and/or its attendant symptoms.
• prevent refers to a method of delaying or precluding the onset of a condition or disease and/or its attendant symptoms, barring a patient from acquiring a condition or disease, or reducing a patient's risk of acquiring a condition or disease.
• therapeutically effective amount means the amount of active agent or active ingredient that is sufficient to achieve the desired therapeutic or prophylactic effect in the patient to which/whom it is administered.
• administration means providing the active agent or active ingredient, alone or as part of a pharmaceutically acceptable composition, to the patient in whom/which the condition, symptom, or disease is to be treated or prevented.
• pharmaceutically acceptable is meant that the ingredients of a pharmaceutical composition are compatible with each other and not deleterious to the patient thereof.
• pharmaceutical vehicle as used herein means a carrier or inert medium used as solvent or diluent in which the pharmaceutically active agent is formulated and/or administered.
• pharmaceutical vehicles include creams, gels, lotions, solutions, and liposomes.
• This invention relates to a picolinate cross-bridged cyclam derivative ligand of formula (I):
• n is an integer selected from 1 and 2;
• R 1 represents:
• a coupling function wherein the coupling function is selected from the group comprising amine; isothiocyanate; isocyanate; activated ester such as for example N-hydroxysuccinimide ester, N-hydroxyglutarimide ester or maleimide ester; carboxylic acid; activated carboxylic acid such as for example acid anhydride or acid halide; alcohol; alkyne; halide; azide; siloxy; phosphonic acid; thiol; tetrazine; norbornen; oxoamine; aminooxy; thioether; haloacetamide such as for example chloroacetamide, bromacetamide or iodoacetamide; glutamate; glutaric anhydride, succinic anhydride, maleic anhydride; aldehyde; ketone; hydrazide; chloroformate and maleimide;
• activated ester such as for example N-hydroxysuccinimide ester, N-hydroxyglutarimide
• a vectorizing group wherein the vectorizing group is selected from the group comprising antibody, preferably monoclonal antibody; hapten; peptide; protein; sugar; nanoparticle; liposome; lipid; polyamine such as spermine;
• R 4 and R 7 each independently represent:
• a coupling function wherein the coupling function is selected from the group comprising amine; isothiocyanate; isocyanate; activated ester such as for example N-hydroxysuccinimide ester, N-hydroxyglutarimide ester or maleimide ester; carboxylic acid; activated carboxylic acid such as for example acid anhydride or acid halide; alcohol; alkyne; halide; azide; siloxy; phosphonic acid; thiol; tetrazine; norbornen; oxoamine; aminooxy; thioether; haloacetamide such as for example chloroacetamide, bromacetamide or iodoacetamide; glutamate; glutaric anhydride, succinic anhydride, maleic anhydride; aldehyde; ketone; hydrazide; chloroformate and maleimide; a vectorizing group, wherein the vectorizing group is selected from the group comprising antibody, preferably monoclonal antibody
• R 5 and R 6 each independently represent:
• activating function is selected from the group comprising N-hydroxysuccinimide, N-hydroxyglutarimide and maleimide; halide; -OCOR 8 wherein R 8 is selected from alkyl, aryl;
• a vectorizing group wherein the vectorizing group is selected from the group comprising antibody, preferably monoclonal antibody; hapten; peptide; protein; sugar; nanoparticle; liposome; lipid; polyamine such as spermine;
• L 1 , L 2 , L 3 , L 4 and L 7 each independently represent:
• linker selected from the group comprising alkyl, aryl, arylalkyl, alkylaryl, heteroaryl, heteroarylalkyl, alkylheteroaryl, alkenyl, alkynyl, wherein alkyl moieties are optionally interrupted by one or more heteroatoms selected from O, N and S.
• At least one of -I ⁇ -R 1 , -L 2 -R 2 , -L 3 -R 3 and -L 4 -R 4 is selected from
• m, p, q and r represent each independently an integer ranging from 0 to 10, preferably 0, 1, 2, 3 or 4 and X represents an halogen, preferably CI.
• X represents an halogen, preferably CI.
• at least one of -I ⁇ -R 1 , -L 2 -R 2 , -L 3 -R 3 and -L 4 -R 4 is selected from formulae (i), (ii); (iii), (iv), (v), (vi) and (vii):
• m, p, q, r, s and t represent each independently an integer ranging from 0 to 10, preferably 0, 1, 2, 3 or 4 and X represents an halogen, preferably CI.
• the ligand of the invention is of formula ( ⁇ ) or (I")
• the ligand of the invention is of formula (la') or (la")
• -I ⁇ -R 1 in formula (la') or (la") is preferably selected from formulae (i), (ii); (iii) and (iv):
• the ligand of the invention is of formula (Ia'-l) or (Ia"-1)
• the ligand of the invention is of formula (Ib-R 5 ) or (Ic-R 5 )
• the ligand of the invention is of formula (lb)
• R 2 and L 2 are as defined in formula (I), and n is an integer selected from 1 or 2, preferably n is equal to 1.
• the ligand of the invention is of formula (Ic)
• R 3 and L 3 are as defined in formula (I), and n is an integer selected from 1 or 2, preferably n is equal to 1.
• -L 2 -R 2 in formula (lb) and -L 3 -R 3 in formula (Ic) are preferably selected from formulae (ii) and (v):
• m and r represent each independently an integer ranging from 0 to 10, preferably 0, 1, 2, 3 or 4 and X represents an halogen, preferably CI.
• m is preferably equal to 1 in formula (ii).
• -L 2 -R 2 in formula (lb) and -L 3 -R 3 in formula (Ic) are preferably of formula (vi) or (vii):
• the ligand of the invention is of formula (Id') or (Id")
• -L 4 -R 4 in formula (Id' (Id") is preferably of formula (iv):
• the ligand of the invention is of formula (Ie') or (Ie")
• R 5 is as defined in formula (I), preferably R 5 is an activating function, wherein the activating function is selected from the group comprising N- hydroxysuccinimide, N-hydroxyglutarimide and maleimide; halide; -OCOR wherein R is selected from alkyl, aryl.
• the ligand of the invention is of formula "Hcb-telpa” or "Hpcb-telpa”:
• the ligand of the invention is of formula (If)
• R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , L 1 , L 2 , L 3 , L 4 and L 7 are as defined in formula (I).
• the ligand of the invention is of formula (If- ) or (If- 1")
• the ligand of the invention is of formula "cb-te2pa" or "pcb- te2pa”:
• the ligand of formula (I) of the invention is grafted on nanoparticles.
• Particularly preferred compounds of formula (I) of the invention are those listed in Table 1 hereafter.
• the present invention further relates to a chelate resulting from the complexation of a ligand of the invention of formula (I) and a metallic cation selected from the group comprising copper (II), copper (I), gallium (III), zirconium (IV), technetium (III), indium (III), rhenium (VI), astatine (III), bismuth (III), lead (II), actinium (III), yttrium (III), lutetium (III), samarium (III), terbium (III) or holmium (III).
• a metallic cation selected from the group comprising copper (II), copper (I), gallium (III), zirconium (IV), technetium (III), indium (III), rhenium (VI), astatine (III), bismuth (III), lead (II), actinium (III), yttrium (III), lutetium (III), samarium (III), terbium (III) or holmium
• the present invention relates to a chelate resulting from the complexation of a ligand of formula (I)
• n is an integer selected from 1 and 2;
• R 1 represents:
• a coupling function wherein the coupling function is selected from the group comprising amine; isothiocyanate; isocyanate; activated ester such as for example N-hydroxysuccinimide ester, N-hydroxyglutarimide ester or maleimide ester; carboxylic acid; activated carboxylic acid such as for example acid anhydride or acid halide; alcohol; alkyne; halide; azide; siloxy; phosphonic acid; thiol; tetrazine; norbornen; oxoamine; aminooxy; thioether; haloacetamide such as for example chloroacetamide, bromacetamide or iodoacetamide; glutamate; glutaric anhydride, succinic anhydride, maleic anhydride; aldehyde; ketone; hydrazide; chloroformate and maleimide;
• activated ester such as for example N-hydroxysuccinimide ester, N-hydroxyglutarimide
• a vectorizing group wherein the vectorizing group is selected from the group comprising antibody, preferably monoclonal antibody; hapten; peptide; protein; sugar; nanoparticle; liposome; lipid; polyamine such as spermine;
• R 2 , R 3 , R 4 and R 7 each independently represent:
• a coupling function wherein the coupling function is selected from the group comprising amine; isothiocyanate; isocyanate; activated ester such as for example N-hydroxysuccinimide ester, N-hydroxyglutarimide ester or maleimide ester; carboxylic acid; activated carboxylic acid such as for example acid anhydride or acid halide; alcohol; alkyne; halide; azide; siloxy; phosphonic acid; thiol; tetrazine; norbornen; oxoamine; aminooxy; thioether; haloacetamide such as for example chloroacetamide, bromacetamide or iodoacetamide; glutamate; glutaric anhydride, succinic anhydride, maleic anhydride; aldehyde; ketone; hydrazide; chloroformate and maleimide;
• activated ester such as for example N-hydroxysuccinimide ester, N-hydroxyglutarimide
• vectorizing group is selected from the group comprising antibody, preferably monoclonal antibody; hapten; peptide; protein; sugar; nanoparticle; liposome; lipid; polyamine such as spermine;
• R 5 and R 6 each independently represent:
• activating function is selected from the group comprising N-hydroxysuccinimide, N-hydroxyglutarimide and maleimide; halide; -OCOR 8 wherein R 8 is selected from alkyl, aryl;
• a vectorizing group wherein the vectorizing group is selected from the group comprising antibody, preferably monoclonal antibody; hapten; peptide; protein; sugar; nanoparticle; liposome; lipid; polyamine such as spermine;
• L 1 , L 2 , L 3 , L 4 and L 7 each independently represent:
• linker selected from the group comprising alkyl, aryl, arylalkyl, alkylaryl, heteroaryl, heteroarylalkyl, alkylheteroaryl, alkenyl, alkynyl, wherein alkyl moieties are optionally interrupted by one or more heteroatoms selected from O, N and S;
• a metallic cation selected from the group comprising copper (II), copper (I), gallium (III), zirconium (IV), technetium (III), indium (III), rhenium (VI), astatine (III), bismuth (III), lead (II), actinium (III), yttrium (III), lutetium (III), samarium (III), terbium (III) or holmium (III).
• the metallic cation is a radioisotope, preferably a radioisotope selected from the group comprising 64 Cu(II), 67 Cu(II), 68 Ga(III), 89 Zr(IV), 99m Tc(III), m In(III), 186 Re(VI), 188 Re(VI), 210 At(III), 212 Bi ( 212 Pb), 213 Bi(III), 225 Ac(III), yu Y(III), 'hu(m), 13J Sm(III), 14y Tb(III) or 10 ⁇ ( ⁇ ), more preferably 04 Cu(II), 67 Cu(II) or 68 Ga(III).
• a radioisotope selected from the group comprising 64 Cu(II), 67 Cu(II), 68 Ga(III), 89 Zr(IV), 99m Tc(III), m In(III), 186 Re(VI), 188 Re(VI), 210 At(III), 212 Bi ( 212 Pb), 213 Bi(III),
• the chelate of the invention is a radiopharmaceutical.
• Preferred embodiments relative to the ligand of formula I described above apply to the chelate of the invention.
• the ligand of the chelate of the invention is of formula (la') or (la")
• -L ⁇ R 1 is selected from formulae (i), (ii); (iii), (iv) and (v):
• m, p, q and r represent each independently an integer ranging from 0 to 10, preferably 0, 1, 2, 3 or 4 and X represents an halogen, preferably CI.
• the ligand of the chelate of the invention is of formula Hcb-telpa:
• the ligand of the chelate of the invention is of formula (Ia'-l) S
• the ligand of the chelate of the invention are those listed in Table 1 above.
• the chelate of the invention meets all the requirements of the specifications described in the introduction of the present application. Evidences are provided in the experimental part below.
• the present invention further relates to a process for manufacturing the ligand of the invention.
• the process for manufacturing the ligand of formula (I) of the invention comprises: reacting compound of formula (i)
• an amino-protecting group such as for example a carbobenzyloxy, a p- methoxybenzyl carbonyl, a tert-butoxy carbonyl, a 9- fluorenylmethyloxycarbonyl, a benzoyl, a benzyl, a carbamate group, a /7-methoxybenzyl, a 3,4-dimethoxybenzyl, a p-methoxyphenyl, a tosyl, an arylsulphonyl, or any other suitable amino-protecting group known by those skilled in the art,
• X represents an halogen atom, preferably CI
• a protecting group selected from alkyl group, preferably methyl or ethyl, more preferably methyl;
• R 5 wherein R 5 are as defined in formula (I) provided that it does not represents a hydrogen atom;
• L 2 , R 2 , L 3 , R 3 , L 4 and R 4 are as defined in formula (I) and M 1 and
• M 5 are as defined above; and where needed conducting on (iii) one or more subsequent step selected from: o deprotecting the acidic function protected by M 5 , to afford compound of formula (I) wherein R 5 represents a hydrogen atom;
• compound of formula (iii) corresponds to compound of formula (I).
• the synthetic protocol used for the preparation of the Hcb-telpa ligand of the invention is described in scheme 4 and consists in two steps starting from the previously described cross-bridged cyclam (i-a) (Wong et al. J. Am. Chem. Soc. 2000, 122, 10561-10572) and 6-chloromethylpyridine methyl ester (ii-a) (Mato-Iglesias et al. Inorg. Chem. 2008, 47, 7840-7851). Alkylation of the constrained cyclam with the electrophilic derivative in absence of a base affords compound (iii-a). Ester derivative (iii-a) is subsequently hydrolyzed with an aqueous acidic solution to lead uantitatively to Hcb-telpa, preferably in its hydrochloride salt form.
• the present invention further relates to a process of manufacturing of the chelate of the invention.
• the process for manufacturing a chelate according to the invention comprises reacting a ligand of formula (I) according to the invention with a metallic cation selected from the group comprising copper (II), copper (I), gallium (III), zirconium (IV), technetium (III), indium (III), rhenium (VI), astatine (III), bismuth (III), lead (II), actinium (III), yttrium (III), lutetium (III), samarium (III), terbium (III) or holmium (III).
• a metallic cation selected from the group comprising copper (II), copper (I), gallium (III), zirconium (IV), technetium (III), indium (III), rhenium (VI), astatine (III), bismuth (III), lead (II), actinium (III), yttrium (III), lutetium (III), samarium (III), terbium (III) or holmium (III
• the process of manufacturing the chelate of the invention comprises reacting the ligand of formula (I) of the invention with a metallic cation in a aqueous medium, preferably by adjusting the pH around neutrality with KOH.
• the process of the invention is preferably conducted at a temperature ranging from room temperature to reflux, preferably from at room temperature. Chelation process is generally performed for a period ranging from few minutes to 24 hours.
• the metallic cation used in the process the invention is under the form of a salt, preferably perchlorate, chloride, bromide, nitrates, sulfates, acetate, triflate salts.
• the process of manufacturing a copper(II) chelate according to the invention comprises reacting the ligand of formula (I) of the invention with a copper cation in an aqueous solution.
• the copper cation is selected from the group comprising Cu(C10 4 ) 2 -6H 2 0, Cu 2 (OAc) 4 , CuCl 2 , Cu(N0 3 ) 2 , Cu(OS0 2 CF ) 2 .
• the complexation of the copper cation is performed at a pH ranging from 2 to 12, preferably from 2 to 7, more preferably a pH of about 7.
• the invention is further directed to the use of the chelates of the invention in nuclear medicine, preferably as imaging agents or medicaments, preferably as radiopharmaceuticals .
• the chelates of the invention are useful as imaging agents.
• chelates of radioisotopes preferably chelates of 64 Cu, 68 Ga, 89 Zr, 99m Tc, m In, 186 Re, 177 Lu, 153 Sm or 1 66 Ho may be used in PET imaging and/or in SPECT imaging.
• Chelates of gadolinium (III) may be used in MRI imaging.
• Chelates of lanthanides, preferably chelates of Eu(III), Tb(III) or Yb(III) may be used for imaging by luminescence.
• the chelates of the invention are also useful as medicaments.
• chelates of radioisotopes preferably chelates of 67 Cu, 89 Zr, 188 Re, 210 At, 212 Bi ( 212 Pb), 213 Bi, 225 Ac,
• Y, Sm or Tb may be used in RET.
• a broad variety of diseases may be targeted.
• the following diseases may be targeted using specified vectorizing groups:
• the invention thus provides methods of treatment and/or prevention of diseases, comprising the administration of a therapeutically effective amount of a chelate of the invention, preferably a chelate of a radioisotope, to a patient in need thereof.
• a chelate of the invention preferably a chelate of a radioisotope
• the invention further provides the use of a chelate of the invention, preferably a chelate of a radioisotope, for the manufacture of a medicament, preferably a radiopharmaceutical.
• the chelates of the invention may be administered as part of a combination therapy.
• a combination therapy comprising coadministration of a compound of the present invention as active ingredient and additional therapeutic agents and/or active ingredients.
• the present invention further relates to a pharmaceutical composition
• a pharmaceutical composition comprising the chelate of the invention in association with at least one pharmaceutically acceptable excipient.
• the present invention further relates to a medicament comprising the chelate of the invention.
• the chelates of the invention may be formulated as a pharmaceutical preparation comprising at least one chelate of the invention and at least one pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant, and optionally one or more further pharmaceutically active compounds.
• such a formulation may be in a form suitable for oral administration, for parenteral administration (such as by intravenous, intramuscular, intradermic or subcutaneous injection or intravenous infusion), for intralesional administration, for submucosal administration, for intra- articular administration, for intra-cavitary administration, for topical administration (including ocular), for artery embolization, for administration by inhalation, by a skin patch, by an implant, by a suppository, etc.
• parenteral administration such as by intravenous, intramuscular, intradermic or subcutaneous injection or intravenous infusion
• intralesional administration for submucosal administration
• intra- articular administration for intra-cavitary administration
• topical administration including ocular
• artery embolization for administration by inhalation, by a skin patch, by an implant, by a suppository, etc.
• Such suitable administration forms - which may be solid, semi-solid or liquid, depending on the manner of administration - as well as methods and carriers, diluents and excipients for use in the preparation thereof, will be clear to the skilled person; reference is made to the latest edition of Remington's Pharmaceutical Sciences.
• Such preparations include tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, ointments, cremes, lotions, soft and hard gelatin capsules, suppositories, drops, sterile injectable solutions and sterile packaged powders (which are usually reconstituted prior to use) for administration as a bolus and/or for continuous administration, which may be formulated with carriers, excipients, and diluents that are suitable per se for such formulations, such as salts (especially NaCl), glucose, lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, polyethylene glycol, cellulose, (sterile) water,
• salts especially NaCl
• the formulations can optionally contain other substances that are commonly used in pharmaceutical formulations, such as buffers, antioxidants, lubricating agents, wetting agents, emulsifying and suspending agents, dispersing agents, desintegrants, bulking agents, fillers, preserving agents, sweetening agents, flavoring agents, flow regulators, release agents, etc..
• the compositions may also be formulated so as to provide rapid, sustained or delayed release of the active compound(s) contained therein.
• the pharmaceutical preparations of the invention are preferably in a unit dosage form, and may be suitably packaged, for example in a box, blister, vial, bottle, sachet, ampoule or in any other suitable single-dose or multi-dose holder or container (which may be properly labeled); optionally with one or more leaflets containing product information and/or instructions for use.
• the ligand of the invention is used for the synthesis of a chelate according to the present invention.
• the ligand of the invention may be used as chelating agent to for chelates which may be used as imaging agents or medicaments in nuclear medicine. According to an embodiment, the ligand of the invention may be used as scavenging agent.
• the ligand of the invention is used for depollution of liquid medium by trapping of metallic cations.
• the ligand of the invention may be used in epuration of effluents contaminated with metals.
• the ligand of the invention may be used to trap lead or radioactive elements.
• the ligand of the invention is used for ultrapurification of liquids.
• ultrapurification refers to the purification of a contaminated solution to a level of contaminant which is much less than 1 ppm (part per million), and generally in the range of ppb (part per billion), ppt (part per trillion), or lower i.e. an ultrapure solution.
• the ligand of the invention may be used in cation detection, preferably in detection of traces of metallic cations.
• the ligand and/or the chelate of the invention may be grafted on solid support, such as for example nanoparticules, preferably gold nanoparticles or iron nanoparticles.
• the ligand and/or the chelate of the invention may be linked to other ligands/chelates, such as for example porphyrines, cyclodextrines, calixarenes or azacycloalkanes.
• Figure 1 is a view of X-ray crystal structure of cb-telpa(C10 4 )2 wherein perchlorate anions and hydrogen atoms bound to carbon atoms are omitted for clarity.
• the ORTEP plot is at the 30% probability level.
• Figure 2 is a ORTEP view of [Cu(cb-telpa)](ClO 4 ) 2 wherein perchlorate anions, water molecules and hydrogen atoms bound to carbon atoms are omitted for clarity.
• Hcb-telpa is then eluted through an ion-exchange resin with HCIO 4 , preferably 0.1 M HCIO 4 , followed by slow evaporation of the eluted solution to give crystals of 3 ⁇ 4cb- telpa(C10 4 ) 2 . These crystals are suitable for X-ray diffraction analysis.
• C-functionalized compounds especially those of formula (Ib-R 5 -1), (Ib- 1), (Ib-2), (Ib- 3), (Ic-R 5 -1), (Ic- 1), (Ic-2) and (Ic-3), may be prepared as described in WO2013/072491, especially as described for compounds of type XVI, and more precisely as described in example 3 for compound (10) (page 30 of WO2013/072491).
• Trastuzumab (4 mg) is added to a solution of Ib-1 (0.53 mg) in 0.1 M Na 2 C0 3 (pH 9.0, 100 ⁇ >. The resulting solution is gently agitated at room temperature overnight. The following day, this solution is then placed on a centricon YM-50 (Millipore), and spun down to reduce the volume and washed with PBS (pH 7.4, 2 mL) three times to remove unreacted Ib-1 chelator. The purified Ib-l-trastuzumab conjugate is finely collected in 2 mL of PBS and stored at -20 °C. III. Synthesis of the chelates
• Chelate 64 Cu radiolabeling was achieved by addition of 50 ⁇ L ⁇ 64 CuCl 2 solution (40 to 60 MBq; metal composition: 10 ppm of copper for 60 ppm total metals) to a mixture of 50 ⁇ ⁇ of 0.1 M sodium hydroxide and 500 ⁇ ⁇ of 1 mM Hcb-telpa solutions in 0.1 M ammonium acetate. Reaction mixtures were stirred at room temperature (r.t.) during 15 min for Hcb-telpa. [ 64 Cu] acetate was obtained by addition of 50 ⁇ L ⁇ 64 CuCl 2 solution to a mixture containing 50 ⁇ ⁇ of 0.1 M sodium hydroxide and 500 ⁇ ⁇ of 0.1 M ammonium acetate. Reaction mixture was stirred at r.t. during 30 min. Radiochemical purity of [ 64 Cu]cb-telpa solution was controlled with both TLC and HPLC. [ ⁇ Cu] acetate was taken as reference in the chromatographic system.
• Hcb-telpa was successfully ⁇ Cu radiolabeled at r.t. in less than 15 min. Both TLC and HPLC chromatograms showed an overall of radiolabeled species of greater than 99 % yield. This confirms the results obtained for the complexation of natural copper(II) by Hcb-telpa. The tests carried out to optimize the labelling also showed that Hcb-telpa could be radiolabeled even using a 0.01 mM ligand concentration. This demonstrates an important selectivity of Hcb-telpa for copper(II) over contaminants divalent cations in solution (Fe 2+ , Mg 2+ , Ni 2+ or Zn 2+ ), since the ratio Hcb-telpa/total metals was below 1.
• Chelate 68 Ga radiolabeling was achieved using the same method with appropriate reactants. Hcb-telpa was successfully 68 Ga radiolabeled and an overall of radiolabeled species of greater than 99 % yield was obtained.
• Complexation of ⁇ Cu with Ib-2 can be achieved by a 30-min preincubation of Ib-2 (100 ⁇ g) in EtOH with an excess of CS 2 CO 3 at 90 °C with constant stirring. Following centrifugation, 64 CuCl 2 is added to the isolated supernatant. The mixture is vortexed and incubated at 90 °C for 30 min. The mixture is centrifuged, and the isolated supernatant is evaporated. The dried mixture is dissolved in water, and passed through the 0.2 ⁇ Nylon Acrodisk 13 filter. Formation of 64 Cu-Ib-2 complexes can be verified by radio- TLC using a mobile phase consisting of MeOH: 10% ammonium acetate (1: 1) on silica plates.
• Radio-HPLC analysis of 64 Cu-Ib-2 can be accomplished using Xbridge CI 8 column (4.6 x 150 mm, 5 ⁇ ) with an isocratic method (0.1% TFA in watenMeOH (96:4), 1 mL/min flow rate).
• the ⁇ Cu-labeled Ib-l-trastuzumab is purified by centrifugation using YM-50 filter to remove any 64 Cu-DTPA complexes. Radiochemical purity can be determined by size exclusion high-performance liquid chromatography (Bio Silect SEC 250-5 300 x 7.8 mm; flow rate 1 mL/min, with the isocratic mobile phase consisting of PBS, pH 7.4). Specific Activity Determination of Cu-Ib-l-trastuzumab
• the fixed amount of ⁇ Cu (220 ⁇ (3 ⁇ 4 in 0.1 M NH 4 OAc buffer (pH 8.0, 100 ⁇ .) is added to various concentrations (1-80 ⁇ g) of Ib-l-trastuzumab in 0.1 M NH 4 OAc buffer (pH 8.0, 100
• the reaction mixture is incubated at 25 °C for 10 min, then 50 ⁇ g of DTPA is added and the reaction mixture is further incubated for 20 min at 30 °C.
• the radiochemical yield is checked with instant thin layer chromatography (ITLC-SG, saline). Three concentrations of Ib-l-trastuzumab showing 40-90% radiolabeling yield can be used to calculate the specific activity of 64 Cu-labeled Ib-l-trastuzumab.
• the potentiometric setup has been described in Roger, M. et al. Inorg. Chem. 2013, 52, 5246-5259.
• the titrant was a KOH solution prepared at ca. 0.1 M from a commercial ampoule of analytical grade, and its accurate concentration was obtained by application of the Gran's method upon titration of a standard HNO 3 solution (Rossotti, F. J. and Rossotti, H. J. /. Chem. Educ. 1965, 42, 375-378).
• Ligand solutions were prepared at about 2.0 x 10 ⁇ 3 M, and the Cu 2+ and Zn 2+ solutions at ca.
• Deviations from the Nernst law at very low pH were corrected with the VLpH software (Calibration software from the maker of Hyperquad available for free at http://www.hyperquad.co.uk/), which performs a [H + ] correction based on a very low pH calibration procedure.
• the liquid-junction potential, E j was otherwise found to be negligible for pH > 2.5 under the experimental conditions used.
• Each titration consisted of 80-100 equilibrium points in the range of pH 2.5-11.5 (or 1.5-11.5 for Cu 2+ complexations), and at least two replicate titrations were performed for each particular system.
• Hcb-telpa The protonation constants of Hcb-telpa were studied in aqueous solution at 25.0 °C.
• the compound has five basic centers consisting of the four amines and the carboxylate function, from which only two could be accurately determined by potentiometric titrations (Table 1). Results obtained for Hcb-telpa are compared with those of two other tetraazacycloalkalnes: telpa and cb-cyclam.
• the remaining protonation constants of Hcb-telpa were determined by conventional potentiometric titrations in aqueous solution and at 0.10 M KN0 3 ionic strength.
• a L denotes the ligand in general; charges are omitted for simplicity. * Values in parentheses are standard deviations in the last significant figures. c From Lima, L.
• Hcb-telpa The stability constants of the complexes formed by Hcb-telpa with Cu 2+ and Zn 2+ were determined by potentiometric titrations in aqueous solution at 25.0 °C in 0.10 M KN0 3 ionic strength (Table 2). Results obtained for Hcb-telpa are compared with those of two other tetraazacycloalkalnes: telpa and cb-cyclam.
• the speciation is notably simple with both Cu 2+ and Zn 2+ ; the fully deprotonated complex is the single species in the intermediate pH range, and a zinc(II) hydroxo complex can only be found at very basic pH.
• the pM values that take into account the variable basicity properties of different ligands were also calculated (Table 3). Both the stability constants obtained and the pM values calculated demonstrate a very high thermodynamic stability of the copper(II) complex of Hcb-telpa.
• thermodynamic stability is not the only important criterion to determine the efficiency of metal complexation because, depending on the application, other factors such as kinetic inertness or in vivo stability can be more important.
• Table 3. Calculated pM a values for the complexes of Hcb-telpa and related compounds.
• the copper(II) complex formation with Hcb-telpa was spectroscopically monitored in different buffered solutions from acidic to neutral pH.
• the reaction becomes progressively slower because of the increase of the acidity of the reaction media, enabling a kinetic study under pseudo-first order conditions using conventional UV-vis spectroscopic methods.
• Hcb-telpa is, to the best of the Applicant' s knowledge, the cross-bridged ligand endowed with the fastest complexation ability for copper(II) under very mild conditions. Without willing to be linked by a theory, this performance might be, at least partly, explained by analysis of the crystallographic structure of the free ligand ( Figure 1). Indeed, the pre-organization of the ligand is favored by a hydrogen bond between the acid function of the picolinate and the secondary amine of the macrocycle. The nitrogen atom of the picolinate arm is located just outside of the macrocyclic pocket in favorable position for the coordination to copper(II), which should thus be easily chelated by the five amine functions of the ligand.
• the slow dissociation of complexes is probably the most important feature to be taken in consideration when selecting compounds to be used in medical applications.
• the kinetics of acid-assisted dissociation of the copper(II) complex of Hcb-telpa were studied under pseudo-first order conditions in acidic aqueous solutions. The dissociation was monitored by following the changes in the visible absorption band of the complex at 25°C in 5 M HC10 4 , or at 20, 25, 37, 60, and 90°C in 5 M HC1.
• the half-life values determined are collected in Table 5 together with literature values for related compounds: telpa, cb-te2a and cb-do2a.
• cb-telpa provides chelates meeting all requirements of the specifications for an optimized chelate intended to be used in nuclear medicine, which was never achieved with chelates from ligands of the prior art.
• In vitro serum stability of 64 Cu-Ib-2 (cf part III.3 above) can be carried out by adding 50 ⁇ . of ⁇ Cu-Ib ⁇ (1-2 mCi) to 500 ⁇ . of FBS (Fetal Bovine Serum). The solution is then incubated at 37 °C, and samples is analyzed by radio-TLC at 0, 10, 30, 60 min, and 2, 4, 10, 24, 48, and 72 h postadministration to FBS.
• FBS Fetal Bovine Serum
• Xenograft tumor models of NIH3T6.7 cell lines can be prepared using 6-week-old BALB/c nu/nu female nude mice. 5x106 NIH3T6.7 cells were inoculated subcutaneously into left shoulder and right flank of mice. Tumors of appropriate size usually grew within 15 d after the implantation.
• Small animal PET scans and image analysis can be performed using a microPET R4 rodent model scanner. Imaging studies is carried out on female nude mice bearing NIH3T6.7 tumors. The mice are injected via the tail vein with 64 Cu-TE2A-Bn-NCS- trastuzumab (200 ⁇ ). At 1, 2, and 3 days after injection, the mice are anesthetized with 1% to 2% isoflurane, positioned in prone position, and imaged. The images can be reconstructed by a two-dimensional ordered-subsets expectation maximum (OSEM) algorithm.
• OSEM ordered-subsets expectation maximum

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