Picolinate cross-bridged cyclams, chelates with metallic cations and use thereof

Abstract

Chelates resulting from the complexation of picolinate cross-bridged cyclams of formula (I), wherein n and the substituents L1-L4 and R1-R5 are as defined, with metallic cations. Picolinate cross-bridged cyclam ligands of formula (I), the use of chelates in nuclear medicine and the use of ligands in cations detection or epuration of effluents are also described.

Claims

1. A chelate resulting from the complexation of a ligand of formula (I) ##STR00054## wherein n is an integer selected from 1 and 2; R.sup.1 represents: a hydrogen atom; a picolinate arm of formula (II) ##STR00055## a coupling function, wherein the coupling function is selected from the group consisting of amine; isothiocyanate; isocyanate; activated ester carboxylic acid; activated carboxylic acid; alcohol; alkyne; halide; azide; siloxy; phosphonic acid; thiol; tetrazine; norbornen; oxoamine; aminooxy; thioether; haloacetamide; glutamate; glutaric anhydride, succinic anhydride, maleic anhydride; aldehyde; ketone; hydrazide; chloroformate and maleimide; or a vectorizing group, wherein the vectorizing group is selected from the group consisting of antibody; hapten; peptide; protein; sugar; nanoparticle; liposome; lipid; and polyamine; R.sup.2, R.sup.3, R.sup.4 and R.sup.7 each independently represent: a hydrogen atom; a coupling function, wherein the coupling function is selected from the group consisting of amine; isothiocyanate; isocyanate; activated ester; carboxylic acid; activated carboxylic acid; alcohol; alkyne; halide; azide; siloxy; phosphonic acid; thiol; tetrazine; norbornen; oxoamine; aminooxy; thioether; haloacetamide; glutamate; glutaric anhydride, succinic anhydride, maleic anhydride; aldehyde; ketone; hydrazide; chloroformate and maleimide; or a vectorizing group, wherein the vectorizing group is selected from the group consisting of antibody; hapten; peptide; protein; sugar; nanoparticle; liposome; lipid; and polyamine; R.sup.5 and R.sup.6 each independently represent: a hydrogen atom; an activating function, wherein the activating function is selected from the group consisting of N-hydroxysuccinimide, N-hydroxyglutarimide, maleimide; halide; and OCOR.sup.8 wherein R.sup.8 is selected from alkyl and aryl; or a vectorizing group, wherein the vectorizing group is selected from the group consisting of antibody; hapten; peptide; protein; sugar; nanoparticle; liposome; lipid; and polyamine; L.sup.1, L.sup.2, L.sup.3, L.sup.4 and L.sup.7 each independently represent: a bond; or a linker selected from the group consisting of alkyl, aryl, arylalkyl, alkylaryl, heteroaryl, heteroarylalkyl, alkylheteroaryl, alkenyl, and alkynyl, wherein alkyl moieties are optionally interrupted by one or more heteroatoms selected from O, N and S; with a metallic cation selected from the group consisting of 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) and holmium (III).

2. The chelate according to claim 1, wherein the ligand is of formula (Ia) or (Ia) ##STR00056## wherein -L.sup.1-R.sup.1 is selected from formulae (i), (ii), (iii), (iv) and (v): ##STR00057## where in m, p, q and r represent each independently an integer ranging from 0 to 10 and X represents an halogen.

3. The chelate according to claim 1, wherein the ligand is selected from 6-((11-(4-isothiocyanatophenethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecan-4-yl)methyl)picolinic acid; 6-((11-(4-isothiocyanatophenethyl)-1,4,8,11-tetraazabicyclo[6.6.3]heptadecan-4-yl)methyl)picolinic acid; methyl 6-((6-(4-aminobenzyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecan-4-yl)methyl)picolinate; 6-((6-(4-isothiocyanatobenzyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecan-4-yl)methyl)picolinic acid; 6-((6-(4-aminobenzyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecan-4-yl)methyl)picolinic acid; 6-((6-(2-hydroxyethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecan-4-yl)methyl)picolinic acid; methyl 6-((13-(4-aminobenzyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecan-4-yl)methyl)picolinate; 6-((13-(4-isothiocyanatobenzyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecan-4-yl)methyl)picolinic acid; 6-((13-(4-aminobenzyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecan-4-yl)methyl)picolinic acid; 6-((13-(2-hydroxyethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecan-4-yl)methyl)picolinic acid; 6-(1,4,8,11-tetraazabicyclo[6.6.2]hexadecan-4-ylmethyl)picolinic acid; 6-(1,4,8,11-tetraazabicyclo[6.6.3]heptadecan-4-ylmethyl)picolinic acid; 6,6-(1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diylbis(methylene))dipicolinic acid; and 6,6-(1,4,8,11-tetraazabicyclo[6.6.3]heptadecane-4,11-diylbis(methylene))dipicolinic acid.

4. The chelate according to claim 1, wherein the metallic cation is a radioisotope.

5. A pharmaceutical composition comprising the chelate according to claim 1, in association with at least one pharmaceutically acceptable excipient.

6. A ligand of formula (I) ##STR00058## wherein n is an integer selected from 1 and 2; R.sup.1 represents: a hydrogen atom; a picolinate arm of formula (II) ##STR00059## a coupling function, wherein the coupling function is selected from the group consisting of amine; isothiocyanate; isocyanate; activated ester; carboxylic acid; activated carboxylic acid; alcohol; alkyne; halide; azide; siloxy; phosphonic acid; thiol; tetrazine; norbornen; oxoamine; aminooxy; thioether; haloacetamide; glutamate; glutaric anhydride, succinic anhydride, maleic anhydride; aldehyde; ketone; hydrazide; chloroformate and maleimide; or a vectorizing group, wherein the vectorizing group is selected from the group consisting of antibody; hapten; peptide; protein; sugar; nanoparticle; liposome; lipid; and polyamine; R.sup.2, R.sup.3, R.sup.4 and R.sup.7 each independently represent: a hydrogen atom; a coupling function, wherein the coupling function is selected from the group consisting of amine; isothiocyanate; isocyanate; activated ester; carboxylic acid; activated carboxylic acid; alcohol; alkyne; halide; azide; siloxy; phosphonic acid; thiol; tetrazine; norbornen; oxoamine; aminooxy; thioether; haloacetamide; glutamate; glutaric anhydride, succinic anhydride, maleic anhydride; aldehyde; ketone; hydrazide; chloroformate and maleimide; or a vectorizing group, wherein the vectorizing group is selected from the group consisting of antibody; hapten; peptide; protein; sugar; nanoparticle; liposome; lipid; and polyamine; R.sup.5 and R.sup.6 each independently represent: a hydrogen atom; an activating function, wherein the activating function is selected from the group consisting of N-hydroxysuccinimide, N-hydroxyglutarimide, maleimide; halide; and OCOR.sup.8 wherein R.sup.8 is selected from alkyl and aryl; or a vectorizing group, wherein the vectorizing group is selected from the group consisting of antibody; hapten; peptide; protein; sugar; nanoparticle; liposome; lipid; and polyamine; L.sup.1, L.sup.2, L.sup.3, L.sup.4 and L.sup.7 each independently represent: a bond; or a linker selected from the group consisting of alkyl, aryl, arylalkyl, alkylaryl, heteroaryl, heteroarylalkyl, alkylheteroaryl, alkenyl and alkynyl, wherein alkyl moieties are optionally interrupted by one or more heteroatoms selected from O, N and S.

7. The ligand according to claim 6, wherein at least one of -L.sup.1-R.sup.1, -L.sup.2-R.sup.2, -L.sup.3-R.sup.3 and -L.sup.4-R.sup.4 is selected from formulae (i), (ii), (iii), (iv), (v), (vi) and (vii): ##STR00060## wherein m, p, q, r, s and t represent each independently an integer ranging from 0 to 10 and X represents an halogen.

8. The ligand according to claim 6, of formula (Ia) or (Ia) ##STR00061## wherein R.sup.1 and L.sup.1 are as previously defined.

9. The ligand according to claim 6, of formula (Ib-R.sup.5), (Ic-R.sup.5), (Ib) or (Ic) ##STR00062## wherein R.sup.2, R.sup.3, L.sup.2 and L.sup.3 are as previously defined, and n is an integer selected from 1 or 2.

10. The ligand according to claim 6, selected from: 6-((11-(4-isothiocyanatophenethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecan-4-yl)methyl)picolinic acid; 6-((11-(4-isothiocyanatophenethyl)-1,4,8,11-tetraazabicyclo[6.6.3]heptadecan-4-yl)methyl)picolinic acid; methyl 6-((6-(4-aminobenzyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecan-4-yl)methyl)picolinate; 6-((6-(4-isothiocyanatobenzyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecan-4-yl)methyl)picolinic acid; 6-((6-(4-aminobenzyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecan-4-yl)methyl)picolinic acid; 6-((6-(2-hydroxyethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecan-4-yl)methyl)picolinic acid; methyl 6-((13-(4-aminobenzyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecan-4-yl)methyl)picolinate; 6-((13-(4-isothiocyanatobenzyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecan-4-yl)methyl)picolinic acid; 6-((13-(4-aminobenzyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecan-4-yl)methyl)picolinic acid; 6-((13-(2-hydroxyethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecan-4-yl)methyl)picolinic acid; 6-(1,4,8,11-tetraazabicyclo[6.6.2]hexadecan-4-ylmethyl)picolinic acid; 6-(1,4,8,11-tetraazabicyclo[6.6.3]heptadecan-4-ylmethyl)picolinic acid; 6,6-(1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diylbis(methylene))dipicolinic acid; and 6,6-(1,4,8,11-tetraazabicyclo[6.6.3]heptadecane-4,11-diylbis(methylene))dipicolinic acid.

11. A process for manufacturing a ligand according to claim 6, comprising: reacting compound of formula (i) ##STR00063## wherein L.sup.2,R.sup.2,L.sup.3 and R.sup.3 are as defined in formula (I) as previously defined; and M.sub.1 represents: a hydrogen atom, an amino-protecting group, or -L.sup.1-R.sup.1, wherein L.sup.1 and R.sup.1 are as defined in formula (I) as previously defined; with compound of formula (ii) ##STR00064## wherein L.sup.4 and R.sup.4 are as defined in formula (I) as previously defined, X represents an halogen atom; and M.sup.5 represents a protecting group selected from alkyl group, or R.sup.5, wherein R.sup.5 are as defined in formula (I) as previously defined, provided that it does not represents a hydrogen atom; to afford compound of formula (iii) ##STR00065## wherein L.sup.2, R.sup.2, L.sup.3, R.sup.3, L.sup.4 and R.sup.4 are as defined in formula (I) as previously defined and M.sup.1 and M.sup.5 are as defined above; and where needed conducting on (iii) one or more subsequent step selected from: deprotecting the acidic function protected by M.sup.5, to afford compound of formula (I) as previously defined, wherein R.sup.5 represents a hydrogen atom; introducing an activating function or a vectorizing group on the acidic function to afford compound of formula (I) as previously, wherein R.sup.5 represents an activating function or a vectorizing group; deprotecting the amine function protected by M.sup.1, to afford compound of formula (I) as previously defined, wherein -L.sup.1-R.sup.1 represents H; and introducing -L.sup.1-R.sup.1 on the amine function, wherein L.sup.1-R.sup.1 is as defined in in formula (I) as previously defined; to afford compound of formula (I).

12. The chelate according to claim 1, wherein the metallic cation is a radioisotope selected from the group consisting of .sup.64Cu(II), .sup.67Cu(II), .sup.68Ga(III), .sup.89Zr(IV), .sup.99mTc(III), .sup.111In(III), .sup.186Re(VI), .sup.188Re(VI), .sup.210At(III), .sup.212Bi (.sup.212Pb), .sup.213Bi(III), .sup.225Ac(III), .sup.90Y(III), .sup.177Lu(III), .sup.153Sm(III), .sup.149Tb(III) and .sup.166Ho(III).

13. The process according to claim 11, wherein in compound of formula (i) M represents an amino-protecting group selected from a carbobenzyloxy, a p-methoxybenzyl carbonyl, a tert-butoxy carbonyl, a 9-fluorenylmethyloxycarbonyl, a benzoyl, a benzyl, a carbamate group, a p-methoxybenzyl, a 3,4-dimethoxybenzyl, a p-methoxyphenyl, a tosyl and an arylsulphonyl.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a view of X-ray crystal structure of cb-te1pa(ClO.sub.4).sub.2 wherein perchlorate anions and hydrogen atoms bound to carbon atoms are omitted for clarity. The ORTEP plot is at the 30% probability level.

(2) FIG. 2 is a ORTEP view of [Cu(cb-te1pa)](ClO.sub.4).sub.2 wherein perchlorate anions, water molecules and hydrogen atoms bound to carbon atoms are omitted for clarity.

EXAMPLES

(3) The present invention will be better understood with reference to the following examples. These examples are intended to representative of specific embodiments of the invention, and are not intended as limiting the scope of the invention.

(4) I. Materials and Methods

(5) Reagents were purchased from ACROS Organics and from Aldrich Chemical Co. Cross-bridged cyclam i-a was purchased from CheMatech (Dijon, France) and 6-chloromethyl-pyridine-2-carboxylic acid methyl ester ii-a was synthesized as previously described (Mato-Iglesias, M. Et al. Inorg. Chem. 2008, 47, 7840-7851). Elemental analyses were performed at the Service de Microanalyse, CNRS, 69360 Solaize, France. NMR and MALDI mass spectra were recorded at the Services communs of the University of Brest. .sup.1H and .sup.13C NMR spectra were recorded with Bruker Avance 400 (400 MHz) spectrometer. MALDI mass spectra were recorded with an Autoflex MALDI TOF III smartbeam spectrometer.

(6) When used hereafter, ca. stands for calculated.

(7) II. Synthesis of the Ligands

(8) 11.1. Synthesis of Hcb-te1pa

(9) ##STR00052##

Step i): Mono N-Functionalization of Cross-bridged Cyclam i-a Yielding Compound iii-a.

(10) A solution of 6-chloromethylpyridine-2-carboxylic acid methyl ester ii-a (0.180 g, 0.97 mmol) in 25 mL of distilled acetonitrile was added to a solution of cross-bridged cyclam i-a (0.200 g, 0.88 mmol) in 175 mL of distilled acetonitrile. The mixture was stirred at room temperature overnight. After evaporation of the solvent, the crude product was purified by column chromatography in silica gel (CHCl.sub.3/MeOH 8/2) to yield compound iii-a as a colorless oil (0.305 g, 92%).

(11) .sup.1H NMR (CDCl.sub.3, 400 MHz): 0.95-1.06 (m, 1 H); 1.41-1.55 (m, 1 H); 1.58-1.70 (m, 1 H); 1.83-1.94 (m, 1 H); 2.33-2.64 (m, 8 H); 2.65-2.79 (m, 3 H); 2.80-2.93 (m, 4 H); 2.93-3.06 (m, 3 H); 3.12-3.22 (m; 1 H); 3.46 (d, .sup.2J=13.2 Hz, 1 H); 3.48-3.59 (m; 1 H); 3.92 (s, 3 H); 4.08 (d, .sup.2J=12.8 Hz, 1 H); 7.52 (d, .sup.3J=7.6 Hz, 1 H); 7.90 (dd, .sup.3J=8.0 Hz, .sup.3J=7.6 Hz, 1 H); 7.98 (d, .sup.3J=8.0 Hz, 1 H). .sup.13C NMR (CDCl.sub.3, 100 MHz): 20.5; 25.2; 43.4; 45.7; 48.6; 50.6; 51.0; 52.0; 52.2; 52.3; 54.4; 54.9; 55.9; 62.7; 123.2; 127.9; 138.2; 145.5; 157.8; 164.1. MALDI-TOF (dithranol): m/z=376.25 (M+1). Elem. Anal. Calcd. for C.sub.20H.sub.33N.sub.5O.HCl.2.8H.sub.2O: C, 53.38; H, 8.96; N, 15.56%. Found: C, 53.62; H, 8.69; N, 15.35%.

Step ii): Hydrolysis of Compound 3 Yielding Hcb-te1pa

(12) Hydrochloric acid (20 mL, 6 M) was slowly added to compound iii-a (0.610 g, 1.62 mmol) and the mixture was refluxed overnight. After cooling to room temperature, the solvent was evaporated to yield Hcb-te1pa.4.5HCl3H.sub.2O in quantitative yield. Hcb-te1pa is then eluted through an ion-exchange resin with HClO.sub.4, preferably 0.1 M HClO.sub.4, followed by slow evaporation of the eluted solution to give crystals of H.sub.3cb-te1pa(ClO.sub.4).sub.2. These crystals are suitable for X-ray diffraction analysis.

(13) .sup.1H NMR (D.sub.2O, 400 MHz): 1.60 (d, .sup.2J=17.2 Hz, 1 H); 1.79 (d, .sup.2J=16.4 Hz, 1 H); 2.34-2.51 (m, 2 H); 2.59-2.76 (m, 4 H); 2.85-2.91 (m, 2 H); 3.10-3.70 (m, 13 H); 4.02 (dt, .sup.3J=7.6 Hz, .sup.4J=4.4 Hz, 1 H); 4.17 (d, .sup.2J=13.6 Hz, 1 H); 5.02 (d, .sup.2J=14.0 Hz, 1 H); 7.84 (d, .sup.3J=7.6 Hz 1 H); 8.17 (dd, .sup.3J=8.0 Hz, .sup.3J=7.6 Hz, 1 H); 8.34 (d, .sup.3J=8.0 Hz, 1 H). .sup.13C NMR (CDCl.sub.3, 100 MHz): 20.8; 21.1; 44.9; 49.9; 51.8; 52.2; 52.7; 56.3; 57.2; 58.1; 59.3; 60.5; 61.1; 129.5; 133.2; 143.0; 151.3; 154.0; 171.7. MALDI-TOF (dithranol): m/z=362.23 (M+1). Elem. Anal. Calcd. for C.sub.19H.sub.35N.sub.5O.sub.2.5HCl.4.5H.sub.2O: C, 39.52; H, 7.26; N, 11.21%. Found: C, 36.79; H, 7.22; N, 10.93%.

(14) An ORTEP view of the structure of H.sub.3cb-te1pa(ClO.sub.4).sub.2 is reported in FIG. 1.

(15) II.2. Synthesis of compound of formula (Ia-1)

(16) ##STR00053##

Step i): Trans-di-N-Functionalization of Cross-bridged Mono-methylpicolinate Cyclam iii-a Yielding Compound iv-b.

(17) 4-Nitrophenylethyl bromide (0.968 g, 4.20 mmol) and potassium carbonate (0.872 g, 6.31 mmol) were add to a solution of iii-a (0.865, 2.10 mmol) in 200 mL of distilled acetonitrile. The mixture was refluxed overnight. After evaporation of the solvent, the crude product was purified by column chromatography in silica gel (CHCl.sub.3/MeOH 8/2) to yield compound iv-b as a yellow oil (1.000 g, 85%).

(18) .sup.1H NMR (CDCl.sub.3, 300 MHz): 1.63-1.70 (m, 4 H); 2.50-3.42 (m, 23H); 3.77-3.85 (m, 6 H); 7.41 (d, J=9 Hz, 2 H); 7.43 (d, J=6 Hz, 1 H); 7.72 (t, J=6 Hz, 1 H); 7.86 (d, J=6 Hz, 1 H); 7.86 (d, J=9 Hz, 2 H); 10.50 (S; 1 H). .sup.13C NMR (CDCl.sub.3, 75 MHz): 24.1; 24.5; 30.2; 50.0; 51.6; 51.7; 52.6; 52.8; 53.0; 53.6; 53.9; 54.0; 56.3; 57.7; 58.6; 123.5; 123.8; 127.2; 129.9; 137.5; 146.2; 147.4; 147.6; 157.7; 165.2. ESI-HRMS: calcd ml z=525.31838 [M+H].sup.+ for C.sub.28H.sub.41N.sub.6O.sub.4. found 525.31838.

Step ii): Reduction of Compound iv-b Yielding v-b.

(19) Tin chloride (1.810 g, 9.55 mmol) and iv-b (0.500 g, 0.95 mmol) were add to a 40 mL solution 1/9 of MeOH/HClaq 12M. The mixture was stirred at room temperature overnight then the excess of HCl was neutralized using potassium carbonate. The desired compound v-b was obtained by extraction with chloroform at pH=14 as yellow oil (420 mg, 83%).

(20) .sup.1H NMR (CDCl.sub.3, 300 MHz): 1.55 (b s, 4 H); 2.46-3.14 (m, 23 H); 3.69-3.84 (m, 8 H); 6.51 (d, J=9 Hz, 2 H); 6.77 (d, J=9 Hz, 2 H); 7.38 (d, J=6 Hz, 1 H); 7.69 (t, J=6 Hz, 1 H); 7.84 (d, J=6 Hz, 1 H); 10.46 (S; 1 H). .sup.13C NMR (CDCl.sub.3, 75 MHz): 24.0; 24.5; 30.2; 51.0; 51.6; 51.8; 52.2; 52.4; 52.6; 54.0; 54.2; 55.1; 56.3; 56.8; 58.4; 115.2; 123.7; 127.1; 128.5; 129.1; 137.5; 145.1; 147.3; 157.7; 165.3. ESI-HRMS: calcd ml z=495.34475 [M+H].sup.+ for C.sub.28H.sub.43N.sub.6O.sub.4. found 495.34420.

Step iii): Hydrolysis of Compound v-b Yielding vi-b.

(21) Hydrochloric acid (10 mL, 6 M) was slowly added to compound v-b (0.200 g, 0.38 mmol) and the mixture was refluxed overnight. After cooling to room temperature, the solvent was evaporated to yield vi-b as an off-white solid in quantitative yield.

(22) .sup.1H NMR (D.sub.2O, 300 MHz): 1.63-1.70 (m, 4 H); 2.11-3.68 (m, 28 H); 4.70 (d, J=15 Hz, 1 H); 5.03 (d, J=15 Hz, 1 H); 6.74 (d, J=9 Hz, 2 H); 6.93 (d, J=9 Hz, 2 H); 7.26-7.33 (m, 2 H); 7.56 (t, J=6 Hz, 1 H). .sup.13C NMR (D.sub.2O, 75 MHz): 20.9; 21.4; 28.5; 48.4; 50.9; 60.0; 52.2; 53.2; 55.6; 55.9; 57.4; 57.8; 58.0; 59.2; 60.2; 123.5; 123.8; 127.2; 129.9; 137.5; 146.2; 147.4; 147.6; 157.7; 165.2. ESI-HRMS: calcd ml z=481.32910 [M+H].sup.+ for C.sub.27H.sub.41N.sub.6O.sub.4. found 481.32855.

Step iv): Formation of the isothiocyanate Derivative of Compound vi-b Yielding cb-te1pa-N-EtPh-NCS.

(23) vi-b.5 HCl (100 mg, 0.15 mmol) was dissolved in hydrochloride acid (1 mL, 3 M) then a solution of thiophosgene (0.435 mg, 3.00 mmol) in 1 mL of chloroform was add to the reaction mixture. After an overnight stirring at room temperature, the reaction mixture was washed with chloroform (51 mL) by vigorous biphasic stirring followed by decanting of the organic phase to remove excess thiophosgene. Compound cb-te1pa-N-EtPh-NCS was obtained by an overnight lyophilisation as a fluffy off-white solid in quantitative yield.

(24) .sup.1H NMR (D.sub.2O, 300 MHz): 1.13 (t, J=7.5 Hz, 2 H); 1.64-1.81 (m, 2 H); 2.39-4.01 (m, 26 H); 4.31 (d, J=15 Hz, 1 H); 5.22 (d, J=15 Hz, 1 H); 7.52 (d, J=9 Hz, 2 H); 7.03 (d, J=9 Hz, 2 H); 7.50-7.57 (m, 2 H); 7.82 (t, J=6 Hz, 1 H). .sup.13C NMR (D.sub.2O, 75 MHz): 21.1; 21.5; 28.5; 48.5; 51.2; 52.2; 53.5; 55.7; 56.0; 57.9; 58.2; 59.3; 60.6; 128.4; 129.0; 130.2; 132.0; 132.2; 137.0. 138.1; 141.8; 149.0; 153.7; 169.5. ESI-MS: m/z=523.30 (M+1).

(25) II.3. Synthesis of C-Functionalized Compounds

(26) C-functionalized compounds, especially those of formula (Ib-R.sup.5-1), (Ib-1), (Ib-2), (Ib-3), (Ic-R.sup.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).

(27) II.4. Conjugation of Ib-1 to Trastuzumab

(28) Trastuzumab (4 mg) is added to a solution of Ib-1 (0.53 mg) in 0.1 M Na.sub.2CO.sub.3 (pH 9.0, 100 L). 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-1-trastuzumab conjugate is finely collected in 2 mL of PBS and stored at 20 C.

(29) III. Synthesis of the Chelates

(30) III.1. Complexation of Copper(II) by Hcb-te1pa

(31) Preparation of [Cu(cb-te1pa)]ClO.sub.4.

(32) Cu(ClO.sub.4).sub.2.6H.sub.2O (0.070 g, 0.19 mmol) was added to a solution of Hcb-te1pa.4.5HCl.3H.sub.2O (0.100 g, 0.17 mmol) in 10 mL of water, and the pH was adjusted to 7 with an aqueous KOH solution. The mixture was heated to 80 C. for 2 h and then stirred overnight at room temperature. Solid impurities were filtered off, and the solution was evaporated to dryness. After addition of acetonitrile, the grey powder was filtered off and the filtrate was evaporated to yield compound [Cu(cb-te1pa)]ClO.sub.4 as a blue powder (0.090 g, 83%).

(33) An ORTEP view of the structure of [Cu(cb-te1pa)](ClO.sub.4).sub.2 is shown in FIG. 2.

(34) Complexation of other metallic cation may be conducted by using the same protocol.

(35) III.2. Complexation of .sup.64Cu or .sup.68Ga by cb-te1pa

(36) Chelate .sup.64Cu radiolabeling was achieved by addition of 50 L .sup.64CuCl.sub.2 solution (40 to 60 MBq; metal composition: 10 ppm of copper for 60 ppm total metals) to a mixture of 50 L of 0.1 M sodium hydroxide and 500 L of 1 mM Hcb-te1pa solutions in 0.1 M ammonium acetate. Reaction mixtures were stirred at room temperature (r.t.) during 15 min for Hcb-te1pa. [.sup.64Cu]acetate was obtained by addition of 50 L .sup.64CuCl.sub.2 solution to a mixture containing 50 L of 0.1 M sodium hydroxide and 500 L of 0.1 M ammonium acetate. Reaction mixture was stirred at r.t. during 30 min. Radiochemical purity of [.sup.64Cu]cb-te1pa solution was controlled with both TLC and HPLC. [.sup.64Cu]acetate was taken as reference in the chromatographic system.

(37) Hcb-te1pa was successfully .sup.64Cu radiolabelled at r.t. in less than 15 min. Both TLC and HPLC chromatograms showed an overall of radiolabelled species of greater than 99% yield. This confirms the results obtained for the complexation of natural copper(II) by Hcb-te1pa. The tests carried out to optimize the labelling also showed that Hcb-te1pa could be radiolabelled even using a 0.01 mM ligand concentration. This demonstrates an important selectivity of Hcb-te1pa for copper(II) over contaminants divalent cations in solution (Fe.sup.2+, Mg.sup.2+, Ni.sup.2+ or Zn.sup.2+), since the ratio Hcb-te1pa/total metals was below 1.

(38) Chelate .sup.68Ga radiolabeling was achieved using the same method with appropriate reactants. Hcb-te1pa was successfully .sup.68Ga radiolabelled and an overall of radiolabelled species of greater than 99% yield was obtained.

(39) III.3. Complexation of .sup.64Cu by Ib-2

(40) Complexation of .sup.64Cu with Ib-2 can be achieved by a 30-min preincubation of Ib-2 (100 g) in EtOH with an excess of Cs.sub.2CO.sub.3 at 90 C. with constant stirring. Following centrifugation, .sup.64CuCl.sub.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 m Nylon Acrodisk 13 filter. Formation of .sup.64Cu-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 .sup.64Cu-Ib-2 can be accomplished using Xbridge C18 column (4.6150 mm, 5 m) with an isocratic method (0.1% TFA in water:MeOH (96:4), 1 mL/min flow rate).

(41) III.4. Complexation of .sup.64Cu by Ib-1-trastuzumab

(42) .sup.64Cu (0.5-2 mCi) in 0.1 M NH.sub.4OAc buffer (pH 8.0, 100 L) is added to 80 g of Ib-1-trastuzumab (cf paragraph 11.4 above) in 0.1 M NH.sub.4OAc buffer (pH 8.0, 100 L) or simple distilled water. 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 can be checked with instant thin layer chromatography (ITLC-SG, saline). The .sup.64Cu-labeled Ib-1-trastuzumab is purified by centrifugation using YM-50 filter to remove any .sup.64Cu-DTPA complexes. Radiochemical purity can be determined by size exclusion high-performance liquid chromatography (Bio Silect SEC 250-5 3007.8 mm; flow rate 1 mL/min, with the isocratic mobile phase consisting of PBS, pH 7.4).

(43) Specific Activity Determination of .sup.64Cu-Ib-1-Trastuzumab

(44) The fixed amount of .sup.64Cu (220 Ci) in 0.1 M NH.sub.4OAc buffer (pH 8.0, 100 L) is added to various concentrations (1-80 g) of Ib-1-trastuzumab in 0.1 M NH.sub.4OAc buffer (pH 8.0, 100 L). 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-1-trastuzumab showing 40-90% radiolabeling yield can be used to calculate the specific activity of .sup.64Cu-labeled Ib-1-trastuzumab.

(45) IV. Physicochemical Properties of Copper(II) Complex of Hcb-te1pa

(46) IV.A. Methods

(47) IV.A.1. Potentiometric Studies

(48) Equipment and work conditions. 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.sub.3 solution (Rossotti, F. J. and Rossotti, H. J. J. Chem. Educ. 1965, 42, 375-378). Ligand solutions were prepared at about 2.010.sup.3 M, and the Cu.sup.2+ and Zn.sup.2+ solutions at ca. 0.05 M from analytical grade nitrate salts and standardized by complexometric titrations with H.sub.4edta (ethylenediaminetetraacetic acid). Sample solutions for titration contained approximately 0.04 mmol of ligand in a volume of 30 mL where the ionic strength was kept at 0.10 M using KNO.sub.3 as background electrolyte. Metal cations were added at 0.9 equiv. of the ligand amount in complexation titrations. Batch titrations were prepared in a similar way but with each titration point corresponding to 1/10 of the amount of a conventional titration sample. Batch titration points were incubated in tightly closed vials at 25 C. until potential measurements attained complete stability, which happened within a week.

(49) Measurements. All measurements were carried out at 25.00.1 C. under inert atmosphere. The electromotive force of the sample solutions was measured after calibration of the electrodes by titration of a standard HNO.sub.3 solution at 2.010.sup.3 M in the work conditions. The [H.sup.+] of the solutions was determined by measurement of the electromotive force of the cell, E=E.sup.o+Q log [H.sup.+]+E.sub.j. The term pH is defined as log [H.sup.+]. E.sup.o and Q were determined from the acid region of the calibration curves. Deviations from the Nernst law at very low pH (pH<2.5) 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.sup.+] correction based on a very low pH calibration procedure. The liquid-junction potential, E.sub.j, was otherwise found to be negligible for pH >2.5 under the experimental conditions used. The value of K.sub.w=[H.sup.+][OH.sup.] was found to be equal to 10.sup.13.78 by titrating a solution of known [H.sup.+] at the same ionic strength in the alkaline pH region, considering E and Q valid for the entire pH range. Each titration consisted of 80-100 equilibrium points in the range of pH 2.5-11.5 (or 1.5-11.5 for Cu.sup.2+ complexations), and at least two replicate titrations were performed for each particular system.

(50) Calculations. The potentiometric data were refined with the Hyperquad software, and speciation diagrams were plotted using the HySS software. The overall equilibrium constants .sub.i.sup.H and .sub.MmHhL1 are defined by .sub.MmHhL1=[M.sub.mH.sub.hL.sub.l]/[M].sup.m[H].sup.h[L].sup.l (.sub.i.sup.H=[H.sub.hL.sub.l]/[H].sup.h[L].sup.l and .sub.ML-1L=.sub.ML(OH)K.sub.w). Differences in log units between the values of protonated (or hydrolysed) and non-protonated constants provide the stepwise (log K) reaction constants (being K.sub.MmHhL1=[M.sub.mH.sub.hL.sub.l]/[M.sub.mH.sub.h-1L.sub.l][H]). The errors quoted are the standard deviations calculated by the fitting program from all the experimental data for each system.

(51) IV.A.2. Kinetics Studies

(52) Complex Formation. The formation of the copper(II) complex of Hcb-te1pa was studied in buffered aqueous solutions at 25 C. The increasing intensity of the complex d-d transition band in the visible range (600 nm) was followed at pH=5.0 (0.2 M potassium acetate buffer) and pH=7.4 (0.2 M HEPES buffer), with [Cu.sup.2+]=[Hcb-te1pa]=0.8 mM. Additionally, complex formation was also studied at pH=3.0 (0.2 M (K,H)Cl) under pseudo-first order conditions, by following the increasing charge transfer band in the UV range (at 310 nm) at [Cu.sup.2+]=10[Hcb-te1pa]=2 mM.

(53) Complex Dissociation. The acid-assisted dissociation of the copper(II) complex of Hcb-te1pa was studied under pseudo-first order conditions in 5 M HCl or 5 M HClO.sub.4 aqueous solutions containing the complex at 1.010.sup.3 M. Concentrated acid was added to sample solutions containing preformed complex without control of ionic strength, and the reaction was followed by the decreasing intensity of the complex d-d transition band, at the temperature of 20, 25, 37, 60, and 90 C. in HCl, and at 25 C. in HClO.sub.4.

(54) IV.A.3. Electrochemical Studies

(55) Cyclic voltammograms were measured using Autolab equipment at room temperature. All measurements were made using a three-electrode system: a glassy-carbon electrode as a working electrode, a platinum wire as a counter-electrode, and a saturated calomel reference electrode. All electrochemical experiments were performed in ca. 1 mM aqueous solutions of preformed complex under a N.sub.2 atmosphere containing 0.1 M NaClO.sub.4 as the supporting electrolyte. From the initial potential of the analysis (0 V), the voltage was ramped to 1.3 V, then to 0.2 V, and back to 0 V at a scan rate of 100 mV/s. All potentials are expressed relative to the saturated calomel electrode (SCE) except otherwise noted.

(56) IV.B. Results and Discussion

(57) IV.B.1. Acid-Base Properties of Hcb-te1pa

(58) The protonation constants of Hcb-te1pa 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-te1pa are compared with those of two other tetraazacycloalkalnes: te1pa and cb-cyclam.

(59) The proton-sponge behavior of cross-bridged tetraaza macrocyclic compounds is well known, corresponding to the very high value of the first protonation constant. For Hcb-te1pa, such behavior was verified by .sup.1H NMR spectroscopic titration in D.sub.2O in the basic pH range. While there are marked resonance shifts in the range of pD=8-12, corresponding undoubtedly to the second protonation constant of the compound (see below), there are no shifts of resonances in the range of pD=12-14, and minor shifts start to be visible only above pD=14. It is thus clear that only at pD >14 the last deprotonation step takes place. However, the spectroscopic data that could be obtained for the highest pH values do not allow for determination of the first protonation constant, as only the beginning of the deprotonation process was detected. Therefore, a value of 15 was postulated for the first protonation constant, which was subsequently used as a constant in all other thermodynamic equilibrium determinations. This particularly high protonation constant must correspond to protonation of one of the macrocyclic amines, and should be highly influenced by hydrogen bonding interactions as is usual in related compounds with relatively small and partially closed structural cavities.

(60) The remaining protonation constants of Hcb-te1pa were determined by conventional potentiometric titrations in aqueous solution and at 0.10 M KNO.sub.3 ionic strength. The second constant (log K=10.13) must correspond to the protonation of a second macrocyclic amine, while the third one (log K=2.43) should correspond to protonation of the carboxylate group, as observed in the solid state structure of H.sub.3cb-te1pa(ClO.sub.4).sub.2 described above. No other protonation constants could be calculated, meaning that additional protonation equilibrium may only happen at pH<2.

(61) TABLE-US-00003 TABLE 1 Overall (.sub.i.sup.H) and stepwise (K.sub.i.sup.H) protonation constants, in log units, for Hcb-te1pa and related compounds, at 25.0 C. in 0.10M KNO.sub.3. L = L = L = Equilibrium reaction .sup.a cb-te1pa.sup. b te1pa.sup. c cb-cyclam .sup.d log .sub.i.sup.H L + H.sup.+ HL >15 11.55 12.42 L + 2 H.sup.+ H.sub.2L 25.13(5) 21.66 22.61 L + 3 H.sup.+ H.sub.3L 27.56(5) 24.37 (20.23) L + 4 H.sup.+ H.sub.4L <29.56 26.07 24.00 log K.sub.i.sup.H L + H.sup.+ HL >15 11.55 12.42 HL + H.sup.+ H.sub.2L 10.13 10.11 10.20 H.sub.2L + H.sup.+ H.sub.3L 2.43 2.71 H.sub.3L + H.sup.+ H.sub.4L <2.0 1.7 1.39 .sup.a L denotes the ligand in general; charges are omitted for simplicity. .sup.b Values in parentheses are standard deviations in the last significant figures. .sup.c From Lima, L. M. P. Et al. Inorg. Chem. 2012, 51, 6916-6927. .sup.d From ref. Sun, X. et al. J. Med. Chem. 2002, 45, 469-477, with I = 0.1M in KCl.
IV.B.2. Thermodynamic Stability of the Metal Complexes of Hcb-te1pa

(62) This part corresponds to points b) and c) of the specifications mentioned above.

(63) The stability constants of the complexes formed by Hcb-te1pa with Cu.sup.2+ and Zn.sup.2+ were determined by potentiometric titrations in aqueous solution at 25.0 C. in 0.10 M KNO.sub.3 ionic strength (Table 2). Results obtained for Hcb-te1pa are compared with those of two other tetraazacycloalkalnes: te1pa and cb-cyclam.

(64) The equilibrium of formation of the copper(II) and especially the zinc(II) complexes is slow in the acidic pH range. In the case of Cu.sup.2+, the complexation is almost complete from low pH but relatively slow up to pH=4. To overcome this double problem, conventional titrations were performed at pH values below 2 in order to observe a significant percentage of free metal ion (at least 18%) and thus allow for determination of the corresponding stability constant, while giving the solution enough time to reach equilibrium prior to the start of the titration. During titrations, each experimental point included a supplementary equilibration time in order to yield fully stabilized measurements. In the case of Zn.sup.2+, there is essentially no complexation below pH=4, and in the range of pH=4-6 the complexation is extensive but very slow, taking up to one week for reaching the final equilibrium. For this reason, batch titrations were prepared in the range of pH=4-6 and were left to equilibrate until full stabilization, while conventional titrations were used for the remaining pH regions.

(65) TABLE-US-00004 TABLE 2 Overall (.sub.MLHh) and stepwise (K.sub.MLHh) stability constants, in log units, for complexes of Hcb-te1pa and related ligands with Cu.sup.2+ and Zn.sup.2+ cations, at 25.0 C. in I = 0.10M KNO.sub.3. L = L = L = Equilibrium reaction .sup.a cb-te1pa.sup. b te1pa.sup. c cb-cyclam .sup.d log .sub.MLHh Cu.sup.2+ + L CuL .sup.26.00(5) 25.5 27.1 Cu.sup.2+ + H.sup.+ + L CuHL 27.67 Cu.sup.2+ + L CuLOH + H.sup.+ 14.35 Zn.sup.2+ + L ZnL .sup.18.83(6) 18.86 Zn.sup.2+ + H.sup.+ + L ZnHL 21.38 Zn.sup.2+ + L ZnLOH + H.sup.+ .sup.7.50(7) 7.84 log K.sub.MLHh Cu.sup.2+ + L CuL 26.00 25.5 27.1 CuL + H.sup.+ CuHL 2.17 CuLOH + H.sup.+ CuL 11.15 Zn.sup.2+ + L ZnL 18.83 18.86 ZnL + H.sup.+ ZnHL 2.52 ZnLOH + H.sup.+ ZnL 11.33 11.02 .sup.a L denotes the ligand in general; charges are omitted for simplicity. .sup.b Values in parentheses are standard deviations in the last significant figures. .sup.c From Lima, L. M. P. Et al. Inorg. Chem. 2012, 51, 6916-6927. .sup.d From ref. Sun, X. et al. J. Med. Chem. 2002, 45, 469-477, by spectrophotometric competition without ionic strength control.

(66) The speciation is notably simple with both Cu.sup.2+ and Zn.sup.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. For a correct comparison of the thermodynamic stability of the complexes of Hcb-te1pa with the corresponding values of other ligands from the literature, 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-te1pa. Importantly, they also show a very high selectivity of Hcb-te1pa for copper(II) complexation over zinc(II). Although the other two ligands taken for comparison exhibit larger pCu values, the value obtained for the copper(II) complex of Hcb-te1pa is still high enough for a very strong coordination of Cu.sup.2+ and to avoid potential transchelation. The 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.

(67) TABLE-US-00005 TABLE 3 Calculated pM .sup.a values for the complexes of Hcb-te1pa and related compounds. Metal ion Hcb-te1pa Hte1pa cb-cyclam Cu.sup.2+ 15.67 18.64 19.29 Zn.sup.2+ 8.50 12.00 .sup.a Values calculated at pH = 7.4 for 100% excess of ligand with [M.sup.2+].sub.tot = 1 10.sup.5 M, based on the presented stability constants.
IV.B.3. Formation and Dissociation of the Copper(II) Complex

(68) This part corresponds to points a) and d) of the specifications mentioned above.

(69) Rapid complexation kinetics are essential for a facile formation of the copper(II) complex. Therefore, some of the most inert cross-bridged complexes may be useless for medical applications given the rather harsh conditions (typically very high temperature and/or high pH) required to achieve near quantitative metal complexation within reasonable time with respect to the limited life time of the radioisotopes.

(70) The copper(II) complex formation with Hcb-te1pa was spectroscopically monitored in different buffered solutions from acidic to neutral pH. In equimolar metal-to-ligand ratio, the complex formation is instantaneous at physiological pH (7.4) and is extremely fast at pH=5, reaching completion (>99%) within a few seconds in the first case and within ca. 3 minutes in the latter case. 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. In this work such kinetic study was performed at pH=3, which is at the lower limit of the pH range in which the copper(II) complexation is approximately complete under equilibrium in equimolar metal-to-ligand conditions. The data obtained for this reaction under pseudo-first order conditions using an excess of 10 equivalents of metal cation resulted in a formation half-time (t.sub.1/2) of 1.7 minutes and showed that formation is quantitative (>99%) within ca. 10 minutes.

(71) According to these results, Hcb-te1pa 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 (FIG. 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.

(72) 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-te1pa 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 HClO.sub.4, or at 20, 25, 37, 60, and 90 C. in 5 M HCl. The half-life values determined are collected in Table 5 together with literature values for related compounds: te1pa, cb-te2a and cb-do2a.

(73) TABLE-US-00006 TABLE 4 Acid-assisted dissociation inertness for the copper(II) complexes of Hcb-te1pa and of selected literature ligands. half-life (t.sub.1/2), ligand conditions min 5M HCl, 90 C. 0.7 min 5M HCl, 60 C. 10.4 min 5M HCl, 37 C. 111 min 5M HCl, 25 C. 465 min 5M HCl, 20 C. 946 min Hcb-te1pa 5M HClO.sub.4, 25 C. >96 days Hte1pa 1M HCl, 25 C. 32 min 5M HClO.sub.4, 25 C. 144 min H.sub.2cb-te2a 5M HCl, 90 C. 9240 min H.sub.2cb-do2a 5M HCl, 30 C. <2 min

(74) A significant difference between the half-live values in HClO.sub.4 and HCl media, especially at 25 C., has been generally explained by the important role that anions sometimes play in the dissociation mechanism.

(75) But more important are the overall very good half-life values obtained for the copper(II) complex of Hcb-te1pa. The experimental kinetic data was used to determine the temperature dependence of the observed rate constants from fitting to the Arrhenius equation. Although an important decrease of the kinetic inertness was found for higher temperatures, the complex half-life is still nearly 2 hours at 37 C. and 5 M HCl.

(76) IV.B.4. Electrochemistry of the Copper(II) Complex

(77) This part corresponds to point e) of the specifications mentioned above.

(78) One of the explanations for the dissociation of copper(II) complexes of macrocyclic ligands in biological media is the metal reduction to copper(I) followed by the demetallation of the complex. It is thus important to ensure the electrochemical inertness as well as the reversibility of the redox system. To determine the redox behavior of the copper complex of Hcb-te1pa, cyclic voltammetry experiments were performed in aqueous solution at pH values of 2.3 and 6.8. The experiments were carried out with a glassy-carbon working electrode in solutions containing 0.1 M NaClO.sub.4 as supporting electrolyte.

(79) At neutral pH, a quasi-reversible system at E.sub.1/2=0.86 V.sub.SCE (E.sub.p=160 mV) was observed with a negligible oxidation peak of free Cu.sup.+ ions to Cu.sup.2+ at 0 V.sub.SCE. This study indicates that the complex is stable on the CV time scale. Furthermore, the reduction process observed for the copper(II) complex of Hcb-te1pa (E.sub.pc=0.696 V versus NHE, upon conversion) is well below the estimated 0.400 V (NHE) threshold for typical bioreductants.

(80) IV.C. Properties Overview and Comparative Data

(81) Specifications for an optimized chelate intended to be used in nuclear medicine are recalled with associated parameters:

(82) TABLE-US-00007 Specifications Related parameters a metallation kinetics time required for complete (>99%) complex formation b thermodynamic association constant metal-ligand: K.sub.MLHh stability (log K.sub.MLHh) and calculated pM c inertness with respect association constant with other metals (log to other metals K.sub.MLHh and pM) to be compared with log K.sub.MLHh and pM d kinetic inertness half-life (t.sub.1/2) (acid-assisted dissociation assay) e stability upon cyclic voltammetry assays results reduction

(83) Values for the copper(II) complex of cb-te1pa are summarized in the table below. Data are compared with those of copper chelates formed with ligands of the prior art.

(84) Especially, properties of copper chelates of cb-te1pa are compared with those of te1pa. The copper chelate of te1pa gives good results relative to the requirements a)-c) of the specifications. However, inertness in acidic medium, (point d) of the specifications, and inertness with regard to reduction (point e) are not optimized, contrary to copper chelate of cb-te1pa.

(85) Data relative to dota and cb-do2a are also provided, as well as for teta and cb-te2a. Introducing a cross-bridge in dota and teta drastically slowers the metallation kinetics, which was surprisingly not observed when cross-bridging te1pa to afford cb-te1pa.

(86) Thermodynamic stability of dota and teta is much lower than that of te1pa and cb-te1pa. Cross-bridging of teta to afford cb-te2a improves thermodynamic stability.

(87) Kinetic inertness in HClO.sub.4, 5M at 25 C. is drastically improved for copper chelate of cb-te1pa compared to other chelates.

(88) Moreover, copper chelate of cb-te1pa is the only chelate displaying suitable stability to reduction among those compared in the table below.

(89) Therefore, cb-te1pa 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.

(90) TABLE-US-00008 e) stability upon reduction a) metallation kinetics b) thermodynamic c) inertness d) kinetic inertness Stability Cu(II) -> Cu (I) time required for stability vs Zn half-life (t.sub.1/2) reduction complete (>99%) log log HClO.sub.4, HCl, HCl, potential Cu complexation K.sub.[CuL] pCu K.sub.[ZnL] pZn 5M, 25 C. 5M, 30 C. 5M, 90 C. reversibility (V) te1pa 3 min 25.50 18.64 18.86 12.00 144 min \ \ quasi-reversible 1.05 cb-te1pa 3 min 26.00 15.67 18.83 8.50 96 days \ 0.7 min reversible 0.696 dota fast 22.21 15.19 21.01*** 15.01 about 5 min \ <1 min irreversible 0.74 cb-do2a too slow \ \ \ \ \ <2 min <3 min irreversible 0.72 teta fast 21.60 15.19 15.81** 10.08 about 8 min 3.5 days 4.5 min irreversible 0.98 cb-te2a too slow 27.10* \ \ \ \ \ 154 hours quasi-reversible 0.88 *estimation by C. Anderson and Ferdani, Cancer Biother. Radiopharm., 2009, 24(4), 379-393 **Delgado and Da Silva, Talanta, 1982, 29, 815-822 ***Chaves et al., Talanta, 1992, 39(3), 249-254
V. Biological Studies
V.I. In Vitro Serum Stability of .sup.64Cu-Ib-2

(91) In vitro serum stability of .sup.64Cu-Ib-2 (cf part 111.3 above) can be carried out by adding 50 L of .sup.64Cu-Ib-2 (1-2 mCi) to 500 L 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.

(92) V.2. In Vivo Tests of .sup.64Cu-Ib-1-Trastuzumab

(93) Animal Models

(94) Xenograft tumor models of NIH3T6.7 cell lines can be prepared using 6-week-old BALB/c nu/nu female nude mice. 5106 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.

(95) Biodistribution

(96) The NIH3T6.7 tumor-bearing BALB/c nude mice (n=4) are injected via tail-vein with .sup.64Cu-Ib-1-trastuzumab (ca. 20 Ci in 200 L saline per mouse). Animals are sacrificed at 1 and 2 days postinjection. Organs and tissues of interest (blood, muscle, bone, spleen, kidney, intestine, liver, and tumor) are then removed, weighed, and counted using gamma-counter. The percent of injected dose per gram (% ID/g) can be calculated by comparison to a weighted, counted standard.

(97) MicroPET Imaging in NIH3T6.7 Tumor Bearing Nude Mice

(98) 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 .sup.64Cu-TE2A-Bn-NCS-trastuzumab (200 Ci). 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.