Macrocyclic ligands with picolinate group(s), complexes thereof and also medical uses thereof

Abstract

A macrocycle compound of general formula (X) ##STR00001##
in which Y.sub.1 represents a C(O)OH group or a group of formula (II) ##STR00002##

Claims

1. A compound of general formula (X) ##STR00045## in which: R.sub.1and R.sub.2 represent, independently of each other, one of the following: H, a (C.sub.1-C.sub.20)alkyl group or a (C.sub.1-C.sub.20)alkylene-(C.sub.6-C.sub.10)aryl group, wherein said alkyl, alkylene and aryl groups optionally are substituted with one or more organic acid functions; X.sub.1, X.sub.2 and X.sub.3 represent, independently of each other, one of the following: H, —C(O)N(Re)(Rd), a (C.sub.1-C.sub.20)alkyl, a (C.sub.2-C.sub.20)alkenyl, a (C.sub.2-C.sub.20)alkynyl and a (C.sub.6-C.sub.10)aryl, with Re and Rd being, independently of each other, H or a (C.sub.1-C.sub.20) alkyl group, wherein said alkyl, alkenyl and alkynyl groups optionally comprise one or more heteroatoms and/or one or more (C.sub.6-C.sub.10)arylenes in their chains, and wherein said alkyl, alkenyl and alkynyl groups optionally are substituted with a (C.sub.6-C.sub.10)aryl; wherein said alkyl, alkenyl, alkynyl and aryl groups optionally are substituted with one or more organic acid functions; and Y.sub.1 represents a C(O)OH group, a protected form of the C(O)OH group in which the hydrogen is replaced with an alkyl, or a group of formula (II) ##STR00046## in which the radicals Ri represent, independently of each other, one of the following: H, halogen, N.sub.3, (C.sub.1-C.sub.20)alkyl, (C.sub.2-C.sub.20)alkenyl, (C.sub.2-C.sub.20)alkynyl and (C.sub.6-C.sub.10)aryl, wherein said alkyl, alkenyl and alkynyl groups optionally comprise one or more heteroatoms and/or one or more (C.sub.6-C.sub.10)arylenes in their chains and, wherein said alkyl, akenyl, and alkynyl groups optionally are substituted with a (C.sub.6-C.sub.10) aryl; wherein said alkyl, alkenyl, alkynyl and aryl groups optionally are substituted with one or more organic acid functions.

2. The compound of claim 1, wherein X.sub.1, X.sub.2 and X.sub.3 represent, independently of each other, one of the following: H, (C.sub.1-C.sub.20)alkyl, (C.sub.2-C.sub.20)alkenyl and (C.sub.2-C.sub.20)alkynyl, wherein said alkyl, alkenyl and alkynyl groups optionally comprise one or more heteroatoms in their chain.

3. The compound of claim 2, wherein X.sub.1, X.sub.2 and X.sub.3 are H.

4. The compound of claim 1, wherein the organic acid functions optionally substituting the alkyl, alkenyl, alkynyl and aryl groups of the X.sub.1, X.sub.2 and X.sub.3 are, independently of each other, one of the following: —COOH, —SO.sub.2OH, —P(O)(OH).sub.2 and —O—P(O)(OH).sub.2.

5. The compound of claim 1, wherein R.sub.1 and R.sub.2 represent, independently of each other, H or (C.sub.1-C.sub.20)alkyl group.

6. The compound of claim 5, wherein R.sub.1 and R.sub.2 represent H.

7. The compound of claim 1, wherein the organic acid functions optionally substituting the alkyl, alkylene and aryl groups of R.sub.1 and R.sub.2 represent, independently of each other, one of the following: —COOH, —SO.sub.2OH, —P(O)(OH).sub.2 and —O—P(O)(OH).sub.2.

8. The compound of claim 1, wherein the radicals Ri represent, independently of each other, one of the following: H, (C.sub.1-C.sub.20)alkyl, (C.sub.2-C.sub.20)alkenyl and (C.sub.2-C.sub.20)alkynyl, wherein said alkyl, alkenyl and alkynyl groups optionally comprise one or more N, O and S heteroatoms.

9. The compound of claim 1, wherein the radicals Ri represent, independently of each other, H or (C.sub.2-C.sub.15)alkynyl.

10. The compound of claim 1, wherein the organic acid functions substituting the alkyl, alkenyl, alkynyl and aryl groups of the radicals Ri represent, independently of each other, one of the following: —COOH, —SO.sub.2OH, —P(O)(OH).sub.2 and —O—P(O)(OH).sub.2.

11. The compound of claim 10, wherein the organic acid functions substituting the alkyl, alkenyl, alkynyl and aryl groups of the radicals Ri represent —COOH.

12. The compound of claim 1, wherein the group of formula (II) is the following group: ##STR00047##

13. The compound of claim 1, wherein: R.sub.1 and R.sub.2 represent H; and Y.sub.1 represents C(O)OH or the following formula: ##STR00048##

14. A method of preparing the compound of claim 1, the method comprising: functionalizing a compound of general formula (IX) ##STR00049## by adding to the nitrogen atom of the NH group thereof a —C(R.sub.1)(R.sub.2)—Y.sub.1 group wherein: R.sub.1 and R.sub.2 represent, independently of each other, one of the following: H, a (C.sub.1-C.sub.20)alkyl group or a (C.sub.1-C.sub.20)alkylene-(C.sub.6-C.sub.10)aryl group, wherein said alkyl, alkylene and aryl groups optionally are substituted with one or more organic acid functions; Y.sub.1 represents a C(O)OH group, a protected form of the C(O)OH group in which the hydrogen is replaced with an alkyl, or a group of formula (II) ##STR00050## in which the radicals Ri represent, independently of each other, one of the following: H, halogen, N.sub.3, (C.sub.1-C.sub.20)alkyl, (C.sub.2-C.sub.20)alkenyl, (C.sub.2-C.sub.20)alkynyl and (C.sub.6-C.sub.10)aryl, wherein said alkyl, alkenyl and alkynyl groups optionally comprise one or more heteroatoms and/or one or more (C.sub.6-C.sub.10)arylenes in their chains and, wherein said alkyl, akenyl, and alkynyl groups optionally are substituted with a (C.sub.6-C.sub.10)aryl; wherein said alkyl, alkenyl, alkynyl and aryl groups optionally are substituted with one or more organic acid functions; to form the compound (X) of claim 1.

15. The compound of claim 1, wherein the alkyl that replaced the hydrogen of the protected form of the C(O)OH group of Y.sub.1 is selected from the group consisting of tert-butyl and methyl.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1: .sup.1H NMR spectra of the ligand P04213 and of its yttrium complex P04183 (300 MHz, 298 K, D.sub.2O)

(2) FIG. 2: Absorption spectra of the ligands and of their yttrium complexes recorded in water at pH 3.8 and 5.5 (acetate buffer). The absorption band corresponding to the π-π* transitions of pyridine extends from 240 to 300 nm for the ligands and the complexes.

(3) FIG. 3: Monitoring of the variation in absorbance at the Amax of the complex or of the ligand at pH 5.5 and 3.8.

(4) FIG. 4: .sup.1H NMR spectrum of the ligand P04330.

(5) FIG. 5: Percentage of extraction of the complex P04283 with .sup.90Y as a function of time, in hours (Re-SSS is a reference complex).

(6) FIG. 6: Ratio between the relaxivity of the gadolinium complexes of ligands P04218 and P04216 measured at a given time and that at t=0 min as a function of time in the presence of a solution of Zn and phosphates.

(7) The examples that follow are described as illustrations of the present invention.

EXAMPLES

(8) Summary table of the various names of the specific compounds of general formula (I) according to the invention:

(9) TABLE-US-00002 Yttrium .sup.90Yttrium Abbreviation Ligand complex complex Mono S Pc-2a1pa Sym P04218 P04219 Mono AS Pc-2a1pa Asym P04216 P04217 Di Sym Pc-1a2pa sym P04213 P04183 Di AS Pc-1a2pa Asym P04214 P04215 P04233 Tri Pc-3pa P04221 P04222 Di AS Pc-1a2pa Asym P04245 P04283 C12 Di AS Pc-1a2pa Asym P04330 C8

(10) A—Materials and Methods

(11) Yttrium-90 chloride is purchased from PerkinElmer Life Sciences. The activities involved were between 28 μCi and 8.51 mCi (1.04-314.87 MBq). The products (HPLC solvents, buffers, etc.) are used as furnished, without further purification. Unless otherwise specified, the ligand is dissolved in ethanol.

(12) The experiments were performed in crimped borosilicate glass bottles. The bottles were heated in a Bioblock heating block allowing up to 6 bottles to be heated. When stirring was necessary, a Lab Dancer S40 (VWR) vortex machine was used. The centrifugations were performed with an MF 20-R centrifuge (Awel).

(13) The activities were measured in a CRC-127R activimeter (Capintec), which was calibrated each morning.

(14) The quality controls were performed by TLC on Whatman 1 paper with an MeOH/0.1% Et.sub.3N mixture as eluent. The radiochemical purities are determined using a Cyclone phosphoimager (Perkin Elmer), with the aid of the Optiquant software.

(15) HPLC analyses were also performed, on a Dionex Ultimate 3000 HPLC line equipped with a diode array detector and an fLumo radiochromatographic detector (Berthold), run by the Chromeleon software.

(16) The analyses were performed on an Accucore C18 100×3 mm, 2.6μ column with the following program: 0.4 mL/min; A=H.sub.2O; B=ACN; 0-3 min: 100% A; 3-20 min: 0-90% B; 20-25 min: 10% A/90% B; 25-26 min: 90-0% B; 26-30 min: 100% A.

(17) Spectroscopic Studies

(18) The UV-visible spectra of the ligands and of the yttrium(III) complexes were measured in aqueous solution of acetate buffer (pH=5.5 or 3.8 without control of the ionic strength) at 298 K using a Jasco V-650 spectrophotometer.

(19) The NMR experiments (COSY, HMBC and HMQC) were recorded for the ligands and their complexes using a Brüker Avance 500 spectrometer (500 MHz) in D.sub.2O.

(20) Kinetic Studies

(21) The formation of the yttrium(III) complexes of do2pa sym, do2pa asym and do1pa sym were studied in an aqueous solution of acetate buffer (C=0.150 M) at 25° C. under pseudo-first-order conditions. The increase in intensity of the absorption band in the UV region was monitored at pH=3.8 and 5.5 with C.sub.L=C.sub.M=4×10.sup.−5 M and without control of the ionic strength.
Dissociation in acidic medium of the yttrium(III) complexes was studied under pseudo-first-order conditions without control of the ionic strength and by addition of aqueous solutions of HCl (0.5, 1, 2, 4 and 5M) to a solution of complex (C=4.10.sup.−5 M).
The dissociation was monitored by decrease of the intensity of the absorption band of the complex or increase of the absorption band of the ligand in the UV region. t.sub.1/2 was calculated by adjusting the curve A.sub.max=f (t) (A.sub.max=absorbance at λ.sub.max of the complex or of the ligand) with the following pseudo-first-order exponential equation: Abs(t)=Abs(eq)+(Abs(0)−Abs(eq))×exp (−x/t1).
Potentiometric Studies
Equipment:

(22) The experiments were performed under an inert atmosphere in aqueous solutions thermostatically maintained at 25.0±0.1° C. The protonation and complexation titrations were performed in a jacketed glass titrations cell using a Metrohm 702 SM Titrino automatic burette connected to a Metrohm 6.0233.100 combined glass electrode. The titrations were controlled automatically by software after selection of the appropriate parameters avoiding monitoring during long measurements.

(23) The titrant is an approximately 0.1 M KOH solution prepared from an analytical-grade commercial vial and its exact concentration is obtained by applying the Gran method by titrating with a standard HNO.sub.3 solution.

(24) The ligand solutions were prepared at approximately 2.0×10.sup.−3 M and the Cu.sup.2+, Pb.sup.2+ and Y.sup.3+ solutions at approximately 0.04 M from analytical-grade chloride salts and standardized by complexometric titration with H.sub.4edta (ethylenediaminetetraacetic acid).sup.1. The solution to be titrated contains approximately 0.05 mmol of ligand in a volume of 30.00 mL, the ionic strength of which was maintained at 0.10 M using KNO.sub.3 as electrolyte. 1.2 equivalents of metal cation (Cu.sup.2+ or Pb.sup.2+) were added to the ligand (0.05 mmol) during the standardization titrations of the ligand solution.
0.9 equivalent of metal cation (Y.sup.3+) was added to the ligand during the complexation titration methods.
Measurements

(25) The electromotive force of the solution was measured after calibration of the electrode by titration of a standard 2.10.sup.−3 M HNO.sub.3 solution. The [H.sup.+] of the solutions was determined by measuring the electromotive force of the cell, E=E°′+Q log [H.sup.+]+Ej. The term “pH” is defined by −log[H.sup.+]. E°′ and Q are determined by the acidic region of the calibration curves. The liquid junction potential, Ej, is negligible under the experimental conditions used. The value of K.sub.e=[H.sup.+][OH.sup.−] is 10.sup.−13.78.

(26) Calculations

(27) The potentiometric data were refined with the Hyperquad software.sup.2 and the speciation diagrams were plotted using the HySS software.sup.3.

(28) The overall equilibrium constants β.sub.iH and βM.sub.mH.sub.hL.sub.l are defined by βM.sub.mH.sub.hL.sub.l=[M.sub.mH.sub.hL.sub.l]/[M].sub.m[H].sub.h[L].sub.l (β.sub.iH=[H.sub.hL.sub.l]/[H].sub.h[L].sub.l and βMH.sub.−1L=βML(OH)×Ke). The differences, in log units, between the protonation (or hydrolysis) values and the non-protonation constants give the intermediate reaction constants (log K) (with KM.sub.mH.sub.hL.sub.l=[M.sub.mH.sub.hL.sub.l]/[M.sub.mH.sub.h−1L.sub.l][H]). The errors indicated are the standard deviations calculated by the adjustment program from all of the experimental data for each system.

(29) B— Synthesis of the Compounds of General Formula (I)

(30) All the commercial reagents were used as received from the suppliers, unless otherwise indicated. The solvents were distilled before use, according to the procedures described in the literature. The purifications by semi-prep HPLC (high-performance liquid chromatography) were performed with a Prominence Shimadzu HPLC/LCMS-2020 machine equipped with an SPD-20 A UV detector. The HPLC chromatographic system uses a column (VisionHT C.sub.18 HL 5μ250×10 mm) eluted with an H.sub.2O (with 0.1% TFA or HCl)-MeCN isocratic gradient.
The .sup.1H and .sup.13C NMR spectra were recorded on a Brüker AMX3-300 MHz spectrometer operating at 300.17 and 75.47 MHz, respectively, for .sup.1H and .sup.13C. All the measurements were taken at 25° C. The signals are indicated as follows: δ chemical shift (ppm), multiplicity (s, singlet; d, doublet; t, triplet; m, multiplet; q, quartet), integration, coupling constants J in hertz (Hz).
The high-resolution mass spectrometry (HRMS-ESI) was performed in positive electrospray ionization mode (ESI+) by the mass spectrometry department of the Institut de Chimie Organique et Analytique (ICOA), Orleans, France.

(31) 1) Synthesis of the Ligands Pc1a2pa Sym P04213 of Formula (I) Via the “Direct” Route

(32) ##STR00024##

Example 1—Int.2

(33) A solution of 2-nitrobenzenesulfonyl chloride (4.3 g, 19.4 mmol) in freshly distilled THF is added at 0° C. to a mixture of diethylenetriamine (1.0 g, 9.69 mmol) and NaHCO.sub.3 (3.26 g, 38.8 mmol) in THF (200 ml). The medium is stirred at room temperature for 20 hours and the solid is then filtered off. The filtrate is concentrated to dryness to give a white solid. This compound is used in the following reaction without purification.

(34) .sup.1H NMR (300 MHz, DMSO-d.sub.6): δ 8.03-7.82 (m, 8H), 2.87 (t, 4H, .sup.3J=6.0 Hz), 2.47 (t, 4H, .sup.3J=6.0 Hz).

(35) .sup.13C NMR (75.47 MHz, DMSO-d.sub.6): δ 147.73, 133.99, 132.68, 132.60, 129.49, 124.39, 47.80, 42.65.

Example 1—Int.3

(36) A solution of tert-butyl bromoacetate (6.09 g, 31.2 mmol) in THF (50 ml) is added to a solution of the compound prepared previously (4.93 g, 10.4 mmol) and of triethylamine (6.31 g, 62.4 mmol) in THF (75 ml). The mixture is refluxed for 24 hours. After cooling the medium, 50 ml of saturated NH.sub.4Cl solution are added and the solvent is removed by evaporation under reduced pressure. The aqueous phase thus obtained is extracted three times with 50 ml of CH.sub.2Cl.sub.2. The chloromethylene fractions are combined and dried over MgSO.sub.4 and then filtered. After evaporating off the solvent, the white solid obtained is chromatographed on silica gel (5/5 to 8/2 ethyl acetate/pentane) to give a white solid after evaporating off the solvent (2.9 g, 51% calc. starting from 1).

(37) .sup.1H NMR (300 MHz, CDCl.sub.3): δ 8.09 (m, 2H), 7.82 (m, 2H), 7.72 (m, 4H), 5.94 (t, 2H, .sup.3J=5.7 Hz), 3.17 (s, 2H), 3.07 (m, 4H), 2.76 (t, 4H, .sup.3J=5.7 Hz), 1.41 (s, 9H).

(38) .sup.13C NMR (75.47 MHz, CDCl.sub.3): δ 170.78, 148.27, 133.71, 133.45, 132.78, 130.97, 125.52, 82.13, 55.99, 54.65, 41.90, 28.16.

Example 1—Int.4

(39) 4 g of K.sub.2CO.sub.3 are added to a solution in acetonitrile (20 ml) of the compound prepared previously (2.86 g, 4.87 mmol) and the mixture is brought to reflux. Dibromomethylpyridine (1.55 g, 5.84 mmol) dissolved in 10 ml of acetonitrile is then added. The medium is refluxed overnight and the solid is filtered off after cooling. The solvent is evaporated off under reduced pressure. The compound obtained is used in the following reaction without further purification.

(40) .sup.1H NMR (300 MHz, CDCl.sub.3): δ 8.1-7.6 (m, 9H), 7.42 (d, 2H, .sup.3J=7.8 Hz), 4.56 (s, 4H), 3.30 (m, 4H), 3.17 (s, 2H), 2.57 (m, 4H), 1.37 (s, 9H).

(41) .sup.13C NMR (75.47 MHz, CDCl.sub.3): δ 171.09, 154.75, 148.33, 139.17, 133.81, 132.74, 131.91, 130.86, 124.39, 124.27, 81.25, 57.51, 54.33, 51.18, 44.93, 28.18.

Example 1—Int.5

(42) The compound prepared previously (2.9 g, 4.29 mmol) is dissolved in DMF in the presence of Na.sub.2CO.sub.3 (3.64 g, 34.3 mmol). Thiophenol (1.88 g, 17.2 mmol) is then added and the medium is stirred at room temperature overnight. After evaporating off the solvent, the residue is taken up in CH.sub.2Cl.sub.2 (100 ml) and the solution obtained is washed with 3×40 ml of 0.5 M NaOH solution. After drying over MgSO.sub.4 and filtration, the organic solution is concentrated and the product obtained is chromatographed on neutral alumina (99/1 CH.sub.2Cl.sub.2/MeOH) to give a white solid (0.835 g, 54% calc. starting from 3).

(43) .sup.1H NMR (300 MHz, CDCl.sub.3): δ 7.4 (t, 1H, .sup.3J=7.5 Hz), 6.86 (d, 2H, .sup.3J=7.5 Hz), 3.83 (s, 4H), 3.22 (s, 2H), 2.48 (m, 4H), 2.40 (m, 4H), 1.28 (s, 9H).

(44) .sup.13C NMR (75.47 MHz, CDCl.sub.3): δ 171.06, 157.49, 136.65, 120.03, 80.98, 59.10, 56.00, 52.67, 47.41, 27.95.

Example 1—Int.6

(45) The methyl ester of 6-chloromethyl-2-pyridinecarboxylic acid (0.872 g, 4.70 mmol) is added to a solution of the compound prepared previously (0.835 g, 2.61 mmol) in acetonitrile (35 ml) in the presence of K.sub.2CO.sub.3 (1.4 g, 10.4 mmol). The medium is refluxed overnight and then filtered and concentrated. The residue is purified by chromatography on neutral alumina (98/2 CH.sub.2Cl.sub.2/MeOH) to give 1.09 g of a yellow oil (67%).

(46) .sup.13C NMR (75.47 MHz, CDCl.sub.3): δ 169.95, 164.98, 158.48, 145.96, 138.30, 137.54, 126.69, 123.11, 119.93, 81.07, 62.45, 61.81, 54.44, 53.13, 52.18, 51.19, 27.51.

Example 1—Int.7

(47) The compound prepared previously (1.09 g, 1.76 mmol) is dissolved in 6M hydrochloric acid solution and the medium is refluxed overnight. After concentration, the product is purified by HPLC on a C.sub.18 phase (100/0 to 10/90 H.sub.2O/acetonitrile) to give intermediate 7 in hydrochloride form (0.690 g, 57% calc. for 3 HCl).

(48) .sup.1H NMR (500.25 MHz, D.sub.2O): δ 8.21 (t, 2H, .sup.3J=7.8 Hz), 8.07 (d, 2H, .sup.3J=7.8 Hz), 7.88 (d, 2H, .sup.3J=7.8 Hz), 7.68 (t, 1H, .sup.3J=7.8 Hz), 7.07 (d, 2H, .sup.3J=7.8 Hz), 4.63 (s, 4H), 4.45 (s, br, 4H), 3.78 (s, 2H), 3.58 (m, 4H), 3.46 (s, br, 4H).

(49) .sup.13C NMR (125.79 MHz, D.sub.2O): δ 175.11, 170.44, 157.33, 153.78, 152.13, 145.76, 142.12, 131.02, 127.79, 124.63, 62.55, 60.72, 57.63, 56.20, 54.67.

(50) 2) Synthesis of Pc2a1pa Sym P04218 of Formula (I) Via the “Direct” Route

(51) ##STR00025##

Example 2—Int.8

(52) The methyl ester of 6-chloromethyl-2-pyridinecarboxylic acid (1.93 g, 10.42 mmol) is added to a solution of compound 2 (Example 1-int.2) (4.935 g, 10.42 mmol) in acetonitrile (60 ml) in the presence of K.sub.2CO.sub.3 (4.3 g, 31.26 mmol) and the mixture is stirred at room temperature for 4 days. The solvent is evaporated off and the residue is taken up in CH.sub.2Cl.sub.2, filtered, concentrated and purified by chromatography on silica gel (5/5 to 8/2 ethyl acetate/pentane). The product is recovered in the form of a yellow oil (2.78 g, 46% calc. starting from 1).

(53) .sup.1H NMR (300 MHz, CDCl.sub.3): δ 8.08-7.99 (m, 2H), 7.95 (d, 1H, .sup.3J=7.9 Hz), 7.82-7.61 (m, 7H), 7.47 (d, 1H, .sup.3J=7.9 Hz), 6.21 (m, 2H), 3.94 (s, 3H), 3.78 (s, 2H), 3.10 (m, 4H), 2.68 (t, 4H, .sup.3J=5.5 Hz).

(54) .sup.13C NMR (75.47 MHz, CDCl.sub.3): δ 165.64, 159.19, 148.11, 147.80, 138.01, 133.58, 132.71, 130.71, 126.16, 125.18, 123.99, 59.45, 54.83, 53.03, 41.58.

Example 2—Int.9

(55) The compound prepared previously (2.78 g, 4.46 mmol) and 3.7 g of K.sub.2CO.sub.3 in 30 ml of acetonitrile are brought to reflux and a solution of dibromomethylpyridine (1.42 g, 5.36 mmol) in 10 ml of acetonitrile is then added. The medium is stirred at the reflux point of the acetonitrile overnight, the solid is then filtered off and the filtrate is concentrated under reduced pressure. The compound obtained is used in the following step without further purification.

(56) .sup.1H NMR (300 MHz, CDCl.sub.3): δ 7.88-7.50 (m, 12H), 7.35 (d, 2H, .sup.3J=7.5 Hz), 4.52 (s, 4H), 3.89 (s, 3H), 3.77 (s, 2H), 3.27 (m, 4H), 2.52 (m, 4H).

(57) .sup.13C NMR (75.47 MHz, CDCl.sub.3): δ 165.23, 159.52, 153.98, 147.55, 146.63, 138.66, 137.10, 133.39, 131.81, 131.45, 130.08, 125.18, 123.78, 123.57, 123.25, 120.99, 120.56, 59.82, 53.62, 52.33, 48.61, 43.35.

Example 2—Int.10

(58) The compound prepared previously (3.9 g, 5.5 mmol) is dissolved in DMF in the presence of Na.sub.2CO.sub.3 (4.6 g, 43.9 mmol), thiophenol (2.42 g, 21.9 mmol) is then added and the medium is stirred at room temperature overnight. The solvent is then removed by distillation under reduced pressure and the residue is taken up in 100 ml of CH.sub.2Cl.sub.2. After washing the organic phase 3 times (3×40 ml) with 0.5M sodium hydroxide solution, drying over MgSO.sub.4 and evaporation of the solvent, the residue obtained is purified by chromatography on neutral alumina (99/1 CH.sub.2Cl.sub.2/MeOH) to give a yellow oil (0.297 g, 19% calc. starting from 8).

(59) .sup.1H NMR (300 MHz, CDCl.sub.3): δ 7.93 (d, 1H, .sup.3J=7.5 Hz), 7.76 (t, 1H, .sup.3J=7.5 Hz), 7.61 (t, 1H, .sup.3J=7.5 Hz), 7.40 (d, 1H, .sup.3J=7.5 Hz), 7.07 (d, 2H, .sup.3J=7.5 Hz), 4.13 (s, 4H), 4.00 (s, 2H), 3.79 (s, 3H), 2.73 (m, 8H).

(60) .sup.13C NMR (75.47 MHz, CDCl.sub.3): δ 165.32, 159.60, 155.26, 147.50, 137.96, 137.52, 125.85, 123.96, 120.60, 61.47, 55.96, 52.67, 51.96, 47.09.

Example 2—Int.11

(61) To a mixture of tert-butyl bromoacetate (0.294 g, 1.50 mmol) and K.sub.2CO.sub.3 (0.464 g, 3.4 mmol) in 10 ml of acetonitrile is added the compound prepared previously (0.297 g, 0.84 mmol). The medium is refluxed overnight, the solid is then filtered off and the solution obtained is concentrated. The residue obtained is purified by chromatography on neutral alumina (100/0 to 95/5 CH.sub.2Cl.sub.2/MeOH) to give compound 11 in the form of a yellow oil (0.185 g, 38%).

Example 2—Int.12

(62) The compound prepared previously (0.185 g, 0.32 mmol) is dissolved in 20 ml of 6M HCl and the medium is refluxed overnight. After evaporating off the solvent, the product is purified by HPLC on a C18 phase (100/0 to 10/90 H.sub.2O/acetonitrile) to give the expected product in hydrochloride form (0.050 g, 27% calc. for 3 HCl).

(63) .sup.1H NMR (500.25 MHz, D.sub.2O): δ 8.33 (t, 1H, .sup.3J=7.8 Hz), 8.25 (d, 1H, .sup.3J=7.8 Hz), 8.07 (d, 1H, .sup.3J=7.8 Hz), 8.00 (t, 1H, .sup.3J=7.8 Hz), 7.49 (d, 2H, .sup.3J=7.8 Hz), 4.81 (s, 4H), 4.20 (s, 2H), 3.76 (s, 4H), 3.63 (m, 4H), 2.99 (s, br, 4H).

(64) .sup.13C NMR (125.79 MHz, D.sub.2O): δ 172.17, 168.64, 157.78, 152.89, 150.25, 146.36, 142.80, 131.30,

(65) 3) Synthesis of a Preparation Intermediate Compound

(66) ##STR00026##

Example 3—Int.3′

(67) To a solution of compound 2 (Example 1-int.2) and of triethylamine (3.9 g, 38.8 mmol) in freshly distilled THF (150 ml) is added, at 0° C., a solution of di-tert-butyl dicarbonate (5.07 g, 23.3 mmol) in freshly distilled THF (50 ml). The medium is stirred at room temperature for 24 hours and then treated with saturated NH.sub.4Cl solution. The solvent is evaporated off under reduced pressure and the aqueous phase is washed with dichloromethane (3×80 ml). After drying over MgSO.sub.4, the organic solution is filtered and concentrated under vacuum. The residue is chromatographed on silica gel (3/7 to 7/3 ethyl acetate/heptane) to give the expected compound in the form of a yellow oil (7.0 g, 79%).

(68) .sup.1H NMR (300 MHz, CDCl.sub.3): δ 8.05-7.63 (m, 2H), 7.79-7.63 (m, 6H), 5.99 (s, br, 1H), 5.77 (s, br, 1H), 3.3 (m, 4H), 3.19 (m, 4H), 1.37 (s, 9H).

(69) .sup.13C NMR (75.47 MHz, CDCl.sub.3): δ 155.78, 147.75, 133.77, 133.09, 132.95, 130.71, 125.20, 80.85, 42.34, 28.16.

Example 3—Int.4′

(70) To a mixture composed of the product prepared previously (7.0 g, 12.2 mmol), Na.sub.2CO.sub.3 and DMF (200 ml) heated to 100° C. is added, under a nitrogen atmosphere, a solution of 2,6-bis(bromomethyl)pyridine (3.23 g, 12.2 mmol) in 100 ml of dry DMF. The medium is stirred at 100° C. for 24 hours and then cooled. The solvent is evaporated off under reduced pressure and the residue thus obtained is taken up in CH.sub.2Cl.sub.2. The organic phase is washed with 1M NaOH solution and dried with MgSO.sub.4. After filtration and concentration, the product is precipitated from acetone to give a white solid (3.76 g, 46%).

(71) .sup.1H NMR (300 MHz, DMSO-d.sub.6): δ 8.10-7.80 (m, 9H), 7.35 (m, 2H), 4.60 (s, 4H), 3.53 (s, 8H), 1.38 (s, 9H).

(72) .sup.13C NMR (75.47 MHz, DMSO-d.sub.6): δ 155.83, 155.75, 154.59, 147.95, 147.89, 138.40, 135.63, 134.57, 132.75, 132.62, 131.17, 130.97, 129.52, 129.19, 124.62, 124.63, 122.49, 122.44, 78.81, 55.17, 50.02, 49.79, 45.42, 44.72, 44.66, 43.09, 27.97.

Example 3-Int.5′

(73) To a suspension of Na.sub.2CO.sub.3 in 250 ml of DMF are added the compound prepared previously (3.59 g, 5.43 mmol) and then thiophenol (2.35 g, 21.3 mmol). The mixture is stirred at room temperature for 12 hours, the solvent is then evaporated off under reduced pressure and the residue obtained is taken up in CH.sub.2Cl.sub.2. The organic phase is washed with water, dried over MgSO.sub.4 and then filtered and concentrated. The residue thus obtained is purified by chromatography on silica gel (100/0 to 95/5 MeOH/32% NH.sub.3aq) to give the expected compound in the form of a yellow oil (1.06 g, 76%).

(74) .sup.1H NMR (300 MHz, CDCl.sub.3): δ 7.51 (t, 1H, .sup.3J=7.5 Hz), 6.94 (d, 2H, .sup.3J=7.5 Hz), 3.94 (s, 4H), 3.52 (t, 4H, .sup.3J=5.1 Hz), 3.04 (s, 2H), 2.61 (t, 4H, .sup.3J=5.65 Hz), 1.49 (s, 9H).

(75) .sup.13C NMR (75.47 MHz, CDCl.sub.3): δ 158.22, 157.53, 136.65, 120.34, 80.27, 52.06, 50.78, 48.88, 28.59.

(76) 4) Synthesis of the Ligand Pc2a1pa Sym P04218 of Formula (I) Via the “Boc” Route

(77) ##STR00027##

Example 4-Int.6′

(78) To a mixture composed of the product obtained previously (Example 3-int.5′) (0.803 g, 2.62 mmol) and K.sub.2CO.sub.3 in 150 ml of acetonitrile is added a solution of tert-butyl bromoacetate (1.022 g, 5.24 mmol) in 50 ml of acetonitrile and the mixture is stirred at room temperature for 24 hours. The solvent is evaporated off, the residue is taken up in CH.sub.2Cl.sub.2 and the solution obtained is then filtered and concentrated. The product is purified by chromatography on silica gel (100/0 to 98/2 CH.sub.2Cl.sub.2/MeOH) to give a yellow oil (1.06 g, 76%).

(79) .sup.1H NMR (300 MHz, CDCl.sub.3): δ 7.5 (t, 1H, .sup.3J=7.5 Hz), 7.08 (d, 2H, .sup.3J=7.5 Hz), 3.86 (s, br, 4H), 3.27 (d, 4H, .sup.3J=9.4 Hz), 3.01 (m, 4H), 2.75-2.55 (m, 4H), 1.34 (s, 18H), 1.24 (s, 9H).

(80) .sup.13C NMR (75.47 MHz, CDCl.sub.3): δ 170.45, 170.27, 157.44, 156.97, 155.26, 137.18, 122.67, 122.61, 80.75, 78.71, 59.99, 59.60, 59.02, 58.67, 51.77, 51.27, 45.04, 44.81, 28.21, 28.03.

Example 4-Int.7′

(81) The compound prepared previously (1.06 g, 9.5 mmol) is dissolved in 20 ml of 6M hydrochloric acid and the mixture is refluxed overnight. After cooling, the solvent is evaporated off and the expected product is obtained in the form of a brown solid (100%).

(82) .sup.1H NMR (300 MHz, D.sub.2O): δ 7.91 (t, 1H, .sup.3J=7.9 Hz), 7.36 (d, 2H, .sup.3J=7.9 Hz), 4.20 (s, 4H), 3.65 (s, 4H), 2.96 (m, 4H), 2.78 (m, 4H).

(83) .sup.13C NMR (75.47 MHz, D.sub.2O): δ 175.61, 154.59, 147.87, 127.29, 60.26, 59.52, 54.14, 46.69.

Example 4-Int.8′

(84) To a solution of the compound obtained previously in 30 ml of methanol is added 5 ml of concentrated H.sub.2SO.sub.4 and the mixture is then stirred and refluxed overnight. After cooling, the solvent is evaporated off, the residue is taken up in 10 ml of water and the pH is adjusted to 7 by adding K.sub.2CO.sub.3. The water is evaporated off and the residue is taken up in dichloromethane. The organic phase is then dried over MgSO.sub.4, filtered and concentrated. The expected product is obtained in the form of yellow oil (0.67 g, 98% calc. starting from 6′).

(85) .sup.1H NMR (300 MHz, CDCl.sub.3): δ 7.34 (t, 1H, .sup.3J=7.5 Hz), 6.84 (d, 2H, .sup.3J=7.5 Hz), 3.86 (s, 4H), 3.51 (s, 10H), 2.69 (m, 4H), 2.02 (m, 4H).

(86) .sup.13C NMR (75.47 MHz, CDCl.sub.3): δ 172.12, 159.24, 136.56, 120.66, 59.54, 57.73, 52.59, 50.95, 46.99.

Example 4-Int.9′

(87) To a mixture composed of the product obtained previously (0.67 mg, 1.9 mmol) and K.sub.2CO.sub.3 (0.524 g, 3.8 mmol) in 50 ml of acetonitrile is added 0.353 g (1.9 mmol) of the methyl ester of 6-chloromethyl-2-pyridinecarboxylic acid and the medium is stirred for two days at room temperature. The solvent is evaporated off and the residue is taken up in CH.sub.2Cl.sub.2 and then filtered. The solution obtained is concentrated and the product is used directly in the following step without further purification.

Example 4-Int.12

(88) To the compound prepared previously are added 20 ml of 6M hydrochloric acid and the mixture is refluxed overnight. The solvent is evaporated off and the residue obtained is purified by HPLC on a C18 phase (100/0 to 10/90 H.sub.2O 0.1% HCl/acetonitrile) to give the expected product in the form of a colorless oil (0.237 g, 22% calc. starting from 8′ for 3 HCl).

(89) .sup.1H NMR (500.25 MHz, D.sub.2O): δ 8.33 (t, 1H, .sup.3J=7.8 Hz), 8.25 (d, 1H, .sup.3J=7.8 Hz), 8.07 (d, 1H, .sup.3J=7.8 Hz), 8.00 (t, 1H, .sup.3J=7.8 Hz), 7.49 (d, 2H, .sup.3J=7.8 Hz), 4.81 (s, 4H), 4.20 (s, 2H), 3.76 (s, 4H), 3.63 (m, 4H), 2.99 (s, br, 4H).

(90) .sup.13C NMR (125.79 MHz, D.sub.2O): δ 172.17, 168.64, 157.78, 152.89, 150.25, 146.36, 142.80, 131.30, 128.33, 125.60, 62.35, 60.08, 59.47, 56.09, 52.88.

(91) 5) Synthesis of the Ligand Pc1a2pa Sym P04213 of Formula (I) Via the “Boc” Route

(92) ##STR00028##

Example 5-Int.11′

(93) To a mixture composed of the product obtained previously (Example 3-int.5′) (0.326 g, 1.06 mmol) and K.sub.2CO.sub.3 (0.587 g, 4.3 mmol) in 50 ml of acetonitrile is added a solution of the methyl ester of 6-chloromethyl-2-pyridinecarboxylic acid (0.395 g, 2.13 mmol) in 20 ml of acetonitrile. The mixture is stirred at room temperature for 5 days and the solvent is evaporated off. The residue is taken up in dichloromethane and the suspension is filtered. The chloromethylene solution is concentrated and the residue is purified by chromatography on neutral alumina (100/0 to 98/2 CH.sub.2Cl.sub.2/MeOH) to give a yellow oil (0.407 g, 63%).

(94) .sup.1H NMR (300 MHz, CDCl.sub.3): δ 8.05-7.95 (m, 2H), 7.87-7.73 (m, 4H), 7.66 (t, 1H, .sup.3J=7.2 Hz), 7.2 (m, 2H), 4.10-3.80 (m, 14H), 3.46-3.31 (m, 4H), 2.75-2.50 (m, 4H), 1.17 (s, 9H).

(95) .sup.13C NMR (75.47 MHz, CDCl.sub.3): δ 165.91, 160.82, 160.70, 156.80, 156.54, 155.48, 147.44, 137.66, 137.55, 137.38, 126.14, 126.07, 123.83, 23.14, 122.96, 79.03, 62.90, 62.71, 59.96, 58.78, 53.00, 51.59, 51.27, 45.14, 44.75, 28.30.

Example 5-Int.12′

(96) To a solution of the compound prepared previously (0.407 g, 0.67 mmol) in 20 ml of methanol is added 1 ml of concentrated sulfuric acid. The mixture is stirred at reflux for 2 days. After cooling, the solvent is evaporated off, the residue is taken up in water (10 ml) and the pH of the medium is adjusted to 7 by adding K.sub.2CO.sub.3. The water is evaporated off and the residue is taken up in dichloromethane. The organic phase is dried over magnesium sulfate, filtered and concentrated. The product is purified by chromatography on neutral alumina (100/0 to 98/2 CH.sub.2Cl.sub.2/MeOH) to give a yellow oil (0.214 g, 63%).

(97) .sup.13C NMR (75.47 MHz, CDCl.sub.3): δ 165.60, 159.30, 159.24, 146.64, 137.17, 127.10, 123.58, 119.79, 61.92, 57.51, 52.72, 52.56, 46.12.

Example 5-Int.6

(98) To a mixture of the compound prepared previously (0.214 g, 0.423 mmol) and K.sub.2CO.sub.3 (0.117 g, 0.85 mmol) in 20 ml of acetonitrile is added a solution of tert-butyl bromoacetate (0.083 g, 0.423 mmol) in 10 ml of acetonitrile. The mixture is stirred at room temperature for 24 hours and then concentrated. The residue is taken up in CH.sub.2Cl.sub.2 and the salts are filtered off. After evaporating off the solvent, the residue is purified by chromatography on neutral alumina (100/0 to 98/2 CH.sub.2Cl.sub.2/MeOH) to give the expected product in the form of a yellow oil (0.155 g, 60%).

Example 5-Int.7

(99) The compound obtained previously is dissolved in 20 ml of 6M hydrochloric acid and the mixture is refluxed overnight. After evaporating off the water, the residue is purified by HPLC on a C.sub.18 phase (100/0 to 90/10 H.sub.2O/ACN) to give a colorless oil (0.089 g, 55% calc. for 3 HCl).

(100) .sup.1H NMR (500.25 MHz, D.sub.2O): δ 8.21 (t, 2H, .sup.3J=7.8 Hz), 8.07 (d, 2H, .sup.3J=7.8 Hz), 7.88 (d, 2H, .sup.3J=7.8 Hz), 7.68 (t, 1H, .sup.3J=7.8 Hz), 7.07 (d, 2H, .sup.3J=7.8 Hz), 4.63 (s, 4H), 4.45 (s, br, 4H), 3.78 (s, 2H), 3.58 (m, 4H), 3.46 (s, br, 4H).

(101) .sup.13C NMR (125.79 MHz, D.sub.2O): δ 175.11, 170.44, 157.33, 153.78, 152.13, 145.76, 142.12, 131.02, 127.79, 124.63, 62.55, 60.72, 57.63, 56.20, 54.67.

REFERENCES

(102) 1. Schwarzenbach, G.; Flaschka, W. Complexometric Titrations; Methuen & Co.: London, 1969. 2. Gans, P.; Sabatini, A.; Vacca, A. Talanta 1996, 43, 1739-1753. 3. Alderighi, L.; Gans, P.; Ienco, A.; Peters, D.; Sabatini, A.; Vacca, A. Coord. Chem. Rev. 1999, 184, 311-318. 6) Synthesis of the Ligand Pc1a2pa Asym P04214 of Formula (I) Via the “Oxalate” Route

(103) ##STR00029##
solution of diethyl oxalate (2.02 g, 13.8 mmol) in EtOH (100 mL) was added to a solution of pyclene (2.37 g, 11.5 mmol) in EtOH (200 mL). The mixture was stirred at room temperature overnight and then concentrated. The residue obtained was purified by chromatography on a column of alumina (98/2 CH.sub.2Cl.sub.2/MeOH). The final product was obtained in the form of a white solid (0.548 g, 19%).

(104) .sup.1H NMR (300 MHz, CDCl.sub.3): δ 7.52 (t, 1H, .sup.3J=7.7 Hz), 7.02 (d, 1H, .sup.3J=7.9 Hz), 6.93 (d, 1H, .sup.3J=7.5 Hz), 5.59 (d, 1H, .sup.2J=16.2 Hz), 4.62 (ddd, 1H, .sup.2J=13.9 Hz, .sup.3J=11.1 Hz, .sup.3J=2.5 Hz), 4.08 (d, 1H, .sup.2J=16.6 Hz), 3.95 (d, 1H, .sup.2J=17.3 Hz), 3.77 (ddd, 1H, .sup.2J=13.9 Hz, .sup.3J=10.6 Hz, .sup.3J=4.52 Hz), 3.70 (d, 1H, .sup.2J=17.3 Hz), 3.5 (ddd, 1H, .sup.2J=12.4 Hz, .sup.3J=10.6 Hz, .sup.3J=4.5 Hz), 3.24 (dt, 1H, .sup.2J=13.9 Hz, .sup.3J=4.4 Hz), 3.13 (dt, 1H, .sup.2J=12.4 Hz, .sup.3J=4.1 Hz), 3.01 (dt, 1H, .sup.2J=12.2 Hz, .sup.3J=3.2 Hz), 2.83 (dt, 1H, .sup.2J=13.9 Hz, .sup.3J=3.0 Hz), 2.74 (td, 1H, .sup.2J=11.7 Hz, .sup.3J=2.3 Hz).

(105) .sup.13C NMR (75.47 MHz, CDCl.sub.3): δ 162.96, 161.23, 159.10, 153.42, 136.83, 120.58, 119.44, 55.40, 52.53, 47.89, 47.66, 44.61, 44.20

(106) Synthesis of the Pyclene Oxalate Intermediate 2″:

(107) Various tests were performed to obtain the “pyclene oxalate” intermediate 2″, as indicated below.

(108) ##STR00030## ##STR00031##

(109) TABLE-US-00003 Route Test used Conditions Purification Yield 1 3 DIPEA, EtOH, 2 Precipitation 22% days, RT 2 1 EtOH, 1.5 days, Chromatography on 33% RT alumina 3 1 EtOH, 2 days, RT Chromatography on 36% alumina 4 2 EtOH, 41 h, RT Chromatography on 37% alumina 5 1 MeOH, 48 h, RT Precipitation 54% 6 1 MeOH, 23 h, RT Precipitation 93% Summary of the tests on the synthesis of the pyclene oxalate intermediate 2″

(110) It is observed that the “pyclene oxalate” intermediate 2″ is obtained according to the various operating conditions and routes tested. In particular, a very good yield is observed, greater than 90%, in the presence of methanol.

(111) Continuation of the Synthesis:

(112) ##STR00032##
A solution of tert-butyl bromoacetate (0.668 g, 3.42 mmol) in acetonitrile (100 mL) was added to a solution of 2″ (0.890 g, 3.42 mmol) and K.sub.2CO.sub.3 (1.42 g, 10.3 mmol) in acetonitrile (150 mL). The mixture was stirred at room temperature for 24 hours. The solvent was evaporated off and the residue was taken up in dichloromethane and then filtered and concentrated. The desired product was obtained in the form of a yellow oil and used in the following steps without further purification (1.25 g).

(113) .sup.1H NMR (300 MHz, CDCl.sub.3): δ 7.28 (t, 1H, .sup.3J=7.7 Hz), 6.8 (d, 1H, .sup.3J=7.5 Hz), 6.63 (d, 1H, .sup.3J=7.5 Hz), 5.26 (d, 1H, .sup.2J=16.6 Hz), 4.08 (m, 1H), 3.89 (d, 1H, .sup.2J=16.6 Hz), 3.68 (m, 4H), 3.0 (m, 4H), 2.77 (m, 2H), 2.52 (m, 1H), 1.18 (m, 9H).

(114) .sup.13C NMR (75.47 MHz, CDCl.sub.3): δ 170.65, 162.42, 159.73, 158.43, 153.60, 136.48, 119.67, 119.11, 80.37, 61.08, 56.45, 52.59, 52.07, 46.64, 46.07, 44.76, 27.61.

(115) Compound 3″ was dissolved in MeOH (100 mL) and concentrated sulfuric acid (10 mL) was added slowly. The mixture was refluxed for 24 hours. After cooling to room temperature, the solvent was evaporated off. 20 mL of water were added and the pH was adjusted to 7 with K.sub.2CO.sub.3. The water was evaporated off and the residue was taken up in dichloromethane. Magnesium sulfate was added and the organic phase was filtered and then concentrated. The crude product was purified by chromatography on a column of alumina (98/2 to 95/5 CH.sub.2Cl.sub.2/MeOH). Compound 4″ is in the form of a white solid (0.939 g, 99% calculated starting from 2″).

(116) .sup.1H NMR (300 MHz, CDCl.sub.3): δ 7.52 (t, 1H, .sup.3J=7.5 Hz), 6.96 (m, 2H), 3.96 (d, 4H, .sup.3J=9.8 Hz), 3.63 (m, 5H), 3.28 (m, 2H), 3.18 (m, 2H), 2.86 (dt, 4H, .sup.2J=11.3, .sup.3J=5.7 Hz).

(117) .sup.13C NMR (75.47 MHz, CDCl.sub.3): δ 172.17, 161.02, 159.09, 137.57, 120.07, 119.97, 57.69, 57.04, 52.35, 51.72, 51.54, 46.80, 46.29, 46.23.

(118) The methyl ester of chloromethyl-2-pyridinecarboxylic acid was added to a solution of compound 4″ (0.939 g, 3.38 mmol) in acetonitrile (150 mL) in the presence of K.sub.2CO.sub.3 (1.8 g, 13.5 mmol). The mixture was stirred at room temperature for one week and then filtered and concentrated. The crude product was purified by chromatography on a column of alumina (98/2 CH.sub.2Cl.sub.2/MeOH) to give compound 5″ in the form of a yellow oil (0.822 g, 42%).
Hydrochloric acid (20 mL, 6M) was added slowly to compound 5″. The mixture was refluxed for 24 hours and then concentrated. The crude product was purified using C18 HPLC (90/10 to 5/95 H.sub.2O 0.1% HCl/acetonitrile) and the ligand 6″ was obtained in the form of a colorless oil (0.310 g, 35% calculated for 3 HCl).

(119) .sup.1H NMR (500 MHz, D.sub.2O): δ 7.98-7.87 (m, 5H), 7.65 (d, 1H, .sup.3J=7.3 Hz), 7.47 (m, 1H), 7.43 (d, 1H, .sup.3J=7.9 Hz), 7.31 (d, 1H, .sup.3J=7.9 Hz), 4.78 (s, 2H), 4.74 (s, br, 2H), 4.54 (s, 2H), 4.20 (s, 2H), 3.78 (s, br, 2H), 3.63 (s, br, 2H), 3.55 (s, 2H), 3.12 (m, 4H).

(120) .sup.13C NMR (125.77 MHz, D.sub.2O): 172.25, 171.82, 170.66, 158.41, 153.72, 153.30, 152.78, 152.14, 151.95, 143.56, 142.61, 142.36, 130.33, 129.50, 127.63, 127.24, 125.33, 125.16, 61.91, 61.78, 61.72, 60.08, 56.17, 56.12, 53.57, 53.37

(121) 7) Synthesis of the Ligand Pc2a1pa Asym P04216 of Formula (I) Via the “Oxalate” Route

(122) ##STR00033##
The methyl ester of 6-chloromethyl-2-pyridinecarboxylic acid (711 g, 3.85 mmol) was added to a solution of compound 2″ (1.0 g, 3.85 mmol) in acetonitrile (300 mL) in the presence of K.sub.2CO.sub.3 (1.5 g, 12 mmol). The mixture was refluxed for 4 days and then filtered and concentrated. The crude product was purified by chromatography on a column of alumina (98/2 CH.sub.2Cl.sub.2/MeOH) to give compound 7″ in the form of a yellow oil (1.56 g, 99%).
Compound 7″ (1.56 g, 3.81 mmol) was dissolved in MeOH (40 mL) and concentrated sulfuric acid (1 mL) was added slowly. The mixture was refluxed for 24 hours. After cooling to room temperature, the solvent was evaporated off. 20 mL of water were added and the pH was adjusted to 7 with K.sub.2CO.sub.3. The water was evaporated off and the residue was taken up in dichloromethane. Magnesium sulfate was added and the organic phase was filtered and then concentrated. The crude product was purified by chromatography on a column of alumina (98/2 to 95/5 CH.sub.2Cl.sub.2/MeOH) to give 8″ in the form of a yellow oil (1.24 g, 92%).
A solution of tert-butyl bromoacetate (1.36 g, 6.98 mmol) in acetonitrile (150 mL) was added to a solution of 8″ (1.24 g, 3.49 mmol) and K.sub.2CO.sub.3 (1.93 g, 14 mmol) in acetonitrile (150 mL). The mixture was refluxed for 2 days. The solvent was evaporated off and the residue was taken up in dichloromethane, filtered and concentrated. Compound 9″ was obtained in the form of a yellow oil and used in the following step without further purification.
Hydrochloric acid (20 mL, 6M) was added slowly to compound 9″. The mixture was refluxed for 24 hours and then concentrated. The crude product was purified using C18 HPLC (90/10 to 5/95 H.sub.2O 0.1% HCl/acetonitrile) and the ligand 10″ was obtained in the form of a colorless oil.

(123) 8) Synthesis of the Ligand Pc3pa P04221 of Formula (I):

(124) ##STR00034##
The methyl ester of chloromethyl-2-pyridinecarboxylic acid (1.35 g, 7.28 mmol) was added to a solution of compound 1″ (0.50 g, 2.43 mmol) in acetonitrile (350 mL) in the presence of K.sub.2CO.sub.3 (1 g, 7.28 mmol). The mixture was refluxed for two days and then filtered and concentrated. The crude product was purified by chromatography on a column of alumina (98/2 CH.sub.2Cl.sub.2/MeOH). Compound 11″ is obtained in the form of a yellow oil (862 mg, 54%).
Hydrochloric acid (20 mL, 6M) was added to compound 11″ (862 mg, 1.32 mmol). The mixture was refluxed for 48 hours and then concentrated. The crude product was purified by precipitation from acetone. Compound 12″ was obtained in hydrochloride salt form (0.574 g, 57% calculated for 4 HCl).

(125) .sup.1H NMR (300 MHz, D.sub.2O): δ 7.5-7.25 (m, 8H), 7.12-7.09 (m, 2H), 6.75 (d, 2H), 4.17 (s, 4H), 4.09 (s, 4H), 3.86 (s, 2H), 3.29 (m, 4H), 2.83 (m, 4H).

(126) .sup.13C NMR (75.47 MHz, D.sub.2O): δ 170.11, 168.95, 158.31, 154.51, 153.79, 150.12, 149.48, 145.95, 144.50, 143.67, 132.57, 132.24, 129.55, 126.76, 62.60, 62.03, 60.78, 57.15, 54.14. 9-1) Synthesis of a Picolinate Bromide Derivative of Formula (II)

(127) ##STR00035##
Chelidamic acid monohydrate 1′″ (5 g, 24.9 mmol) and phosphorus pentabromide (34 g, 79.0 mmol) were heated at 90° C. Once a liquid mixture was obtained, heating was continued for 2 hours at 90° C. After cooling the mixture with ice, chloroform (100 mL) and MeOH (100 mL) were added. The solution is mixed for 20 hours at room temperature and the pH is adjusted to 7 with saturated NaHCO.sub.3 solution. The solvents were evaporated off and the aqueous phase was extracted using dichloromethane (3×100 mL). The organic phase was dried over MgSO.sub.4, filtered and concentrated to give compound 2′″ in the form of a white solid (6.43 g, 94%).

(128) .sup.1H NMR (300 MHz, CDCl.sub.3): δ 8.42 (s, 2H), 3.99 (s, 6H).

(129) .sup.13C NMR (300 MHz, CDCl.sub.3): δ 164.02, 149.12, 135.13, 131.33, 53.52.

(130) Compound 2′″ (6.43 g, 23.5 mmol) was dissolved in dichloromethane (50 mL) and methanol (70 mL). NaBH.sub.4 (1.02 g, 28.2 mmol) was added portionwise to the mixture at 0° C., under nitrogen. After stirring for 4 hours, hydrochloric acid was added to adjust the pH to 5. The solvents were evaporated off and the pH of the aqueous phase was adjusted to 12 with Na.sub.2CO.sub.3. The aqueous phase was extracted with dichloromethane (3×100 mL) and the organic phase was dried with MgSO.sub.4, filtered and concentrated under vacuum. After purification on alumina, compound 3′″ was obtained in the form of a white solid (3.92 g, 68%).

(131) .sup.1H NMR (300 MHz, CDCl.sub.3): δ 8.12 (s, 1H), 7.76 (s, 1H), 4.82 (s, 2H), 3.95 (s, 3H).

(132) .sup.13C NMR (300 MHz, CDCl.sub.3): δ 164.55, 162.33, 147.96, 134.66, 127.31, 127.21, 64.49, 53.29.

(133) Under an inert atmosphere, Pd(Ph.sub.3).sub.2Cl.sub.2 (232 mg, 0.33 mmol) and Cul (124.2, 0.65 mmol) were added to a degassed solution of 1-dodecyne (651 mg, 3.92 mmol) in triethylamine (10 mL) and 3′″ (800 mg, 3.26 mmol) in freshly distilled THF. The mixture was stirred at 40° C. for 20 hours. After cooling to room temperature, the suspension was filtered and triturated with Et.sub.2O (40 mL). The filtrate was washed with saturated NH.sub.4Cl solution (2×50 mL) and brine (40 mL). Finally, the organic phase was dried over MgSO.sub.4, filtered and concentrated under vacuum. The crude product was purified on silica gel (7/3 to 4/6 hexane/ethyl acetate) to give compound 4′″ in the form of a white solid (727 mg, 67%).

(134) .sup.1H NMR (300 MHz, CDCl.sub.3): δ 7.93 (s, 1H), 7.48 (s, 1H), 4.80 (s, 2H), 3.95 (s, 3H), 2.41 (t, 2H), 1.58 (m, 2H), 1.5-1.1 (m, 14H), 0.85 (t, 3H).

(135) .sup.13C NMR (300 MHz, CDCl.sub.3): δ 165.37, 160.62, 147.05, 134.54, 126.15, 125.98, 97.83, 78.05, 64.62, 53.02, 31.99, 29.67, 29.59, 29.40, 29.21, 29.01, 28.39, 22.77, 19.60, 14.20.

(136) Compound 4′″ (727 mg, 2.15 mmol) was dissolved in dichloromethane (80 mL) and triethylamine (653 mg, 6.45 mmol). Mesyl chloride (369 mg, 3.23 mmol) was added and the mixture was stirred at room temperature for 30 minutes. The organic phase was washed with saturated aqueous sodium hydrogen carbonate solution (100 mL) and then dried over MgSO.sub.4, filtered and concentrated. Compound 5′″ is obtained in the form of a white solid (896 mg, quantitative yield). 9-2) Synthesis of the Ligand Pc1a2pa Asym C12 P04245 of Formula (I):

(137) ##STR00036##
A solution of compound 5′″ (712 mg, 1.75 mmol) in acetonitrile (50 mL) was added to a solution of compound 4″ (243 mg, 0.87 mmol) in refluxing acetonitrile (100 mL) in the presence of K.sub.2CO.sub.3 (361 g, 2.6 mmol). The mixture was refluxed for one week. After cooling to room temperature, the suspension was filtered and the solvent was evaporated off. The crude product was purified by chromatography on a column of alumina to give compound 7′″ in the form of a yellow oil.
Production and Purification of the Ligand Pc1a2pa Asym C12 P04245: Saponification Step
A solution of KOH (5 mL, 1M) was added to a solution of compound 7′″ (91 mg, 0.10 mmol) in THF (6 mL). The mixture was stirred vigorously for 5 hours at room temperature. The organic phase was evaporated and the residue was then purified by exclusion chromatography (Sephadex LH20, 100/0 to 90/10 CH.sub.2Cl.sub.2/MeOH). Product 8′″ was obtained in the form of a colorless solid (48 mg, 56%).

(138) 10) Synthesis of the Analog Pc1a2pa Asym C8 P04330: 10-1) Synthesis of a C8 Picolinate Bromide Derivative

(139) ##STR00037##
Under an inert atmosphere, Pd(Ph.sub.3).sub.2Cl.sub.2 (246 mg, 0.35 mmol) and Cul (134, 0.70 mmol) were added to a degassed solution of 1-octyne (464 mg, 4.21 mmol) in triethylamine (10 mL) and 3′″ (863 mg, 3.51 mmol) in freshly distilled THF (20 mL). The mixture was stirred at 40° C. for 20 hours. After cooling to room temperature, the suspension was filtered and triturated with Et.sub.2O (40 mL). The filtrate was washed with saturated NH.sub.4Cl solution (2×20 mL) and brine (20 mL). Finally, the organic phase was dried over MgSO.sub.4, filtered and concentrated under vacuum. The crude product was purified on silica gel (7/3 to 4/6 hexane/ethyl acetate) to give compound 4″″ in the form of a white solid (573 mg, 59%).

(140) .sup.1H NMR (300 MHz, CDCl.sub.3): δ 7.69 (s, 1H), 7.39 (s, 1H), 4.63 (s, 2H), 3.74 (s, 3H), 2.22 (t, 2H), 1.40 (m, 2H), 1.24 (m, 2H), 1.35-1.15 (m, 4H), 0.69 (t, 3H).

(141) .sup.13C NMR (300 MHz, CDCl.sub.3): δ 164.88, 161.27, 146.43, 133.96, 125.53, 125.35, 97.05, 77.74, 64.26, 52.45, 30.96, 28.25, 27.94, 22.18, 19.12, 13.66.

(142) A solution of compound 4″″ (573 mg, 2.08 mmol) in anhydrous CH.sub.2Cl.sub.2 (50 mL) was cooled to 0° C. under an inert atmosphere. PBr.sub.3 (676 mg, 2.5 mmol) was added and the mixture was then refluxed for 2 hours. After cooling to room temperature, the reaction medium was neutralized with 50 mL of water and K.sub.2CO.sub.3 to pH 7. The organic phase was dried over MgSO.sub.4, filtered and then concentrated under vacuum. After purification on silica gel (9/1 to 4/6 hexane/ethyl acetate), product 5′″ was obtained in the form of a white solid (289 mg, 41%).

(143) .sup.1H NMR (300 MHz, CDCl.sub.3): δ 7.91 (s, 1H), 7.54 (s, 1H), 4.52 (s, 2H), 3.93 (s, 3H), 2.37 (t, 2H), 1.54 (m, 2H), 1.37 (m, 2H), 1.3-1.2 (m, 4H), 0.85 (t, 3H).

(144) 13C NMR (300 MHz, CDCl.sub.3): δ 165.00, 157.39, 147.63, 134.85, 128.82, 126.58, 98.23, 77.60, 53.04, 32.80, 31.25, 28.55, 28.18, 22.48, 19.48, 14.01. 10-2) Synthesis of the Ligand Pc1a2pa Asym C8 P04330

(145) ##STR00038##
Compound 5″″ (289 mg, 0.85 mmol) was added to a solution of compound 4″ (106 mg, 0.38 mmol) in anhydrous acetonitrile (30 mL) in the presence of K.sub.2CO.sub.3 (158 mg, 1.1 mmol). The mixture was refluxed for one week. After cooling to room temperature, the suspension was filtered and the solvent was evaporated off. The crude product was dissolved in a minimum amount of ethyl acetate and pentane was then added until the solution became cloudy. The oil formed was rinsed with pentane and precipitated again. Compound 7″″ was obtained in the form of a yellow oil (155 mg, 51%).
A solution of KOH (2 mL, 1M) was added to a solution of compound 7″″ (55 mg, 0.069 mmol) in THF (5 mL). The mixture was stirred vigorously for 5 hours at room temperature. The organic phase was evaporated and the residue was then purified by exclusion chromatography (Sephadex LH20, 100/0 to 90/10 CH.sub.2Cl.sub.2/MeOH). Product 8″″ was obtained in the form of a colorless solid (30 mg, 58%).

(146) C— Study of the Compounds of Formula (I) and of the Complexes According to the Invention

(147) C-1 Synthesis of the Yttrium Complexes

(148) 1) The procedure for synthesizing the complex Y-Pc1a2pa sym P04183 is described below and is applicable to all of the ligands of general formula (I):

(149) The ligand P04213 is dissolved in ultra-pure water and the pH is adjusted to 5 with 1M sodium hydroxide solution. The salt YCl.sub.3.6H.sub.2O (1.5 eq) is dissolved in ultra-pure water. The yttrium solution is added to the ligand solution with stirring. After adjusting the pH to 5, the solution is refluxed overnight. The complex is then purified by HPLC on C18 (100/0 to 10/90 H.sub.2O/ACN) according to the scheme below:

(150) ##STR00039##
2) Synthesis of an yttrium-90 complex, P04233:

(151) The ligand P04214 was used in a complexation reaction with yttrium-90 in order to confirm the complexation results obtained with non-radioactive natural yttrium. A radiolabeling study was performed.

(152) The parameters studied are as follows:

(153) TABLE-US-00004 Parameters Study range Optimum pH 1-9 6.5-9 Temperature 20-100° c. 80° c. Ligand concentration Mol/L 10.sup.−5-10.sup.−2 mol/L 10.sup.−4-10.sup.−2 mol/L Time Min. 5-60 min. 15 min

(154) In summary, the optimum conditions for labeling P04214 are yttrium-90 in acetate medium pH=6.5-9, the ligand P04214 between 10.sup.−4 and 10.sup.−2 M in EtOH; 15 min at 80° C. The radiolabeling yield obtained is >90% (P04233).

(155) 3) Procedure for and result of labeling of the ligand P04245 with yttrium-90, production of the complex P04283:

(156) The parameters studied are as follows:

(157) TABLE-US-00005 Parameters Study range Optimum pH 4.65-9 6.5-9 Temperature 20-90° c. 50° c. Ligand concentration Mol/L 10.sup.−5-10.sup.−3 mol/L 10.sup.−3 mol/L Time Min. 5-60 min. 15 min
The optimum labeling conditions are:

(158) yttrium-90 in acetate medium;

(159) pH=4.65-9;

(160) ligand P04245 at 10.sup.−3 M in EtOH;

(161) for 15 min at 50° C.

(162) 4) Procedure for and result of extraction of the complex P04283 with Lipiodol, production of P04284:

(163) The solution containing the complex P04283 was made up to 2 mL with 1 mL of saline, and an equivalent volume of Lipiodol (2 mL) was added to the solution containing the complex. After stirring and centrifugation, the phases are separated and counted. The yield for extraction into Lipiodol is 89.8±5.0% (n=3).

(164) 5) Procedure for and results of the stability tests in human physiological saline:

(165) Procedure for Preparing the Radiotracer P04284

(166) 1 mL of yttrium-90 acetate at pH=7 is added to 1 mL of ligand P04245 dissolved in ethanol at a concentration of 10.sup.−3 mol/L to form the complex P04283. The solution is heated for 30 minutes at 90° C. 2 mL of Lipiodol are added and the mixture is stirred vigorously. The phases are separated by centrifugation (3500 rpm, 15 minutes). The lipiodol-based phase is collected and made up to 2 mL with Lipiodol to give the expected radiotracer P04284.

(167) 1 mL of freshly prepared radiotracer is taken and then deposited in a 12 mL flat-bottomed glass flask. The activity is measured with an activimeter, and the time is noted. 10 mL of 0.9% saline solution (physiological saline) are added and the mixture is stirred. The flask is then placed in the incubator set at 37° C., equipped with a stirrer set at 30 rpm (revolutions per minute).

(168) The system is left stirring for several days. The aqueous phase is sampled at various times to assay the yttrium-90 released. Each sample was taken in triplicate.

(169) The results are given in FIG. 5. The complexes formed according to the invention and vectorized with Lipiodol are stable in physiological saline.

(170) C-2 Synthesis of the Lanthanide Complexes:

(171) 1) The complexation reactions of gadolinium with the ligands P04218 and P04216 and also with ligand P04213 are performed in water in the presence of one equivalent of GdCl.sub.3 at pH 5-6 at reflux overnight.

(172) ##STR00040##

Example: Complexation of the Ligand P04216 with Gadolinium

(173) Purification of the complexes is performed by preparative HPLC so as to remove the remaining salts.

(174) 2) Study of the Relaxivity of the Gadolinium Complexes:

(175) The relaxivity studies were performed on the gadolinium complexes of the ligands P04218, P04216 and P04213, on Minispec Mq-20 and Minispec Mq-60 machines (Brüker, Karlsruhe, Germany) at 20 MHz (0.47 T) and 60 MHz (1.4 T) in water at 37° C.

(176) For each complex prepared previously, a [Gd]concentration range extending from 0.5 to 5 mM was prepared and the values T1 and T2 of each of these solutions were then measured to determine the relaxivity values r1 and r2 by means of equation 1. For each of the ligands, a trend curve whose correlation coefficient was equal to or very close to 1 was obtained, which made it possible to check equation 1 and to validate the quality of the measurements taken. The curves plotted make it possible to determine the relaxivity value “r” which corresponds to coefficient “a” of the equation for the straight line “ax+b”.

(177) $r=\frac{1}{\left[{\mathrm{Gd}}^{3+}\right]}\left(\frac{1}{T_{\mathrm{obs}}}-\frac{1}{T_{H2O}}\right)$

Equation 1: General Formula for Calculating the Relaxivity Values r1 and r2

(178) TABLE-US-00006 Relaxivity (mmol.sup.−1s.sup.−1) 20 MHz 60 MHz Pc2a1pa sym r1 = 3.9 r1 = 3.2 P04218 r2 = 4.5 r2 = 3.9 Pc2a1pa asym r1 = 3.7 r1 = 3.2 P04216 r2 = 4.1 r2 = 3.7 Pc1a2pa sym r1 = 1.9 r1 = 1.6 P04213 r2 = 2.1 r2 = 1.8

(179) It is found that the relaxivities observed are of the same order of magnitude as those obtained with the gadolinium-based contrast agents used clinically, for example Dotarem®. 3) Stability of the Gd Complexes in Competitive Medium:

(180) To a solution comprising the gadolinium complex of ligands P04218 and P04216 at 2.5 mM in phosphate buffer at 333 mM is added a solution of ZnCl.sub.2 at 2.5 mM. The relaxivity value of these solutions is measured regularly. The relationship between the relaxivity measured at a given time and that at t=0 min as a function of the time in the presence of the Zn solution is given in FIG. 6. The complexes according to the invention are stable over time. 4) Synthesis and Characterization of the Complexes

(181) General procedure for preparing the lanthanide complexes (Ln=Y.sup.3+, Gd.sup.3+, Eu.sup.3+, Tb.sup.3+, Yb.sup.3+, Lu.sup.3+).

(182) The ligand is dissolved in water and the pH is adjusted to 5 with 1M KOH solution and a solution of the metal chloride (M=Y.sup.3+, Gd.sup.3+, Eu.sup.3+, Tb.sup.3+, Yb.sup.3+, Lu.sup.3+) is then added (1.2 equivalents). The mixture is refluxed overnight and the solution obtained is concentrated.

(183) The complex is purified by preparative chromatography on a column of C-18 grafted silica, eluting with a water/acetonitrile mixture.

(184) TABLE-US-00007 Abbreviation Ligand Mono S Pc-2a1pa Sym P04218 L1 Mono AS Pc-2a1pa Asym P04216 L3 Di Sym Pc-1a2pa sym P04213 L2 Di AS Pc-1a2pa Asym P04214 L4
Synthesis of [ML1(H.sub.2O)]

(185) ##STR00041##
[YL1(H.sub.2O)]
L1.3HCl (27.2 mg, 0.048 mmol), YCl.sub.3.6H.sub.2O (25.0 mg, 0.082 mmol)
Yield: 24.5 mg, 91%
ESI-HR-MS (positive, H.sub.2O) m/z calc. [C.sub.22H.sub.25YN.sub.5O.sub.6].sup.+, 544.0858; measured 544.0858 [M+H].sup.+, calc. [C.sub.22H.sub.26YN.sub.5O.sub.6].sup.2+, 272.5465; measured 272.5469 [M+2H].sup.2+.
[GdL1(H.sub.2O)]
L1.3HCl (36.5 mg, 0.064 mmol), GdCl.sub.3.6H.sub.2O (27.5 mg, 0.074 mmol)
Yield: 39.8 mg, 98%
ESI-HR-MS (positive, H.sub.2O) m/z calc. [C.sub.22H.sub.25GdN.sub.5O.sub.6].sup.+, 613.1040; measured 613.1031 [M+H].sup.+, calc. [C.sub.22H.sub.26GdN.sub.5O.sub.6].sup.2+, 307.0557; measured 307.0560 [M+2H].sup.2+.
[EuL1(H.sub.2O)]
L1.3HCl (22.0 mg, 0.039 mmol), EuCl.sub.3.6H.sub.2O (17.1 mg, 0.047 mmol)
Yield: 22.1 mg, 91%
ESI-HR-MS (positive, H.sub.2O) m/z calc. [C.sub.22H.sub.25EuN.sub.5O.sub.6].sup.+, 608.1012; measured 608.1004 [M+H].sup.+, calc. [C.sub.22H.sub.26EuN.sub.5O.sub.6].sup.2+, 304.5542; measured 304.5544 [M+2H].sup.2+:
[TbL1(H.sub.2O)]
L1.3HCl (22.0 mg, 0.039 mmol), TbCl.sub.3.6H.sub.2O (17.4 mg, 0.047 mmol)
Yield: 22.6 mg, 95%
ESI-HR-MS (positive, H.sub.2O) m/z calc. [C.sub.22H.sub.25TbN.sub.5O.sub.6].sup.+, 614.1053; measured 614.1048 [M+H].sup.+, calc. [C.sub.22H.sub.26TbN.sub.5O.sub.6].sup.2+, 307.5563; measured 307.5565 [M+2H].sup.2+.
[YbL1(H.sub.2O)]
L1.3HCl (25.0 mg, 0.044 mmol), YbCl.sub.3.6H2O (20.5 mg, 0.053 mmol)
Yield: 27.3 mg, 96%
ESI-HR-MS (positive, H.sub.2O) m/z calc. [C.sub.22H.sub.25YbN.sub.5O.sub.6], 629.1188; measured 629.1187 [M+H].sup.+, calc. [C.sub.22H.sub.26YbN.sub.5O.sub.6].sup.2+, 315.0630; measured 315.0635 [M+2H].sup.2+.
[LuL1(H.sub.2O)]
L1.3HCl (25.0 mg, 0.044 mmol), LuCl.sub.3.6H.sub.2O (20.6 mg, 0.053 mmol)
Yield: 26 mg, 91%
ESI-HR-MS (positive, H.sub.2O) m/z calc. [C.sub.22H.sub.25LuN.sub.5O.sub.6].sup.+, 630.1207; found 630.1196 [M+H].sup.+, calc. [C.sub.22H.sub.26LuN.sub.5O.sub.6].sup.2+, 315.5640; found 315.5641 [M+2H].sup.2+.
Synthesis of [ML2]

(186) ##STR00042##
[YL2]
L2.3HCl (100.0 mg, 0.155 mmol), YCl.sub.3.6H2O (89.0 mg, 0.293 mmol)
Yield: 84.8 mg, 88%
ESI-HR-MS (positive, H.sub.2O) m/z calc. [C.sub.27H.sub.28YN.sub.6O.sub.6].sup.+, 621.1123; measured 621.1116 [M+H].sup.+, calc. [C.sub.27H.sub.29YN.sub.6O.sub.6].sup.2+, 311.0598; measured 311.0603 [M+2H].sup.2+.
[GdL2]
L2.3HCl (39.0 mg, 0.061 mmol), GdCl.sub.3.6H2O (27.0 mg, 0.073 mmol)
Yield: 41.1 mg, 99%
ESI-HR-MS (positive, H.sub.2O) m/z calc. [C.sub.27H.sub.28GdN.sub.6O.sub.6].sup.+, 690.1306; measured 690.1313 [M+H].sup.+, calcd. for [C.sub.27H.sub.29GdN.sub.6O.sub.6].sup.2+, 345.5689; measured 345.5690 [M+2H].sup.2+.
[EuL2]
L2.3HCl (25.0 mg, 0.039 mmol), EuCl.sub.3.6H.sub.2O (17.1 mg, 0.047 mmol)
Yield: 25.3 mg, 96%
ESI-HR-MS (positive, H.sub.2O) m/z calc. [C.sub.27H.sub.28EuN.sub.6O.sub.6].sup.+, 685.1277; measured 685.1279 [M+H].sup.+, calc. [C.sub.27H.sub.29EuN.sub.6O.sub.6].sup.2+, 343.0675; measured 343.0680 [M+2H].sup.2+.
[TbL2]
L2.3HCl (20.0 mg, 0.031 mmol), TbCl.sub.3.6H.sub.2O (13.9 mg, 0.037 mmol)
Yield: 19.6 mg, 92%
ESI-HR-MS (positive, H.sub.2O) m/z calc. [C.sub.27H.sub.28TbN.sub.6O.sub.6].sup.+, 691.1318; measured 691.1314 [M+H].sup.+, calc. [C.sub.27H.sub.29TbN.sub.6O.sub.6].sup.2+, 346.0696; measured 346.0697 [M+2H].sup.2+.
[YbL2]
L2.3HCl (22.0 mg, 0.034 mmol), YbCl.sub.3.6H.sub.2O (15.9 mg, 0.041 mmol)
Yield: 22.1 mg, 92%
ESI-HR-MS (positive, H.sub.2O) m/z calc. [C.sub.27H.sub.28YbN.sub.6O.sub.6].sup.+, 706.1453; measured 706.1454 [M+H].sup.+, calc. [C.sub.27H.sub.29YbN.sub.6O.sub.6].sup.2+, 353.5763; measured 353.5764 [M+2H].sup.2+.
[LuL2]
L2.3HCl (22.0 mg, 0.034 mmol), LuCl.sub.3.6H.sub.2O (16.0 mg, 0.041 mmol)
Yield: 22.8 mg, 95%
ESI-HR-MS (positive, H.sub.2O) m/z calc. [C.sub.27H.sub.28LuN.sub.6O.sub.6].sup.+, 707.1473; measured 707.1476 [M+H].sup.+, calc. [C.sub.27H.sub.29LuN.sub.6O.sub.6].sup.2+, 354.0773; measured 354.0776 [M+2H].sup.2+.
Synthesis of [ML3(H.sub.2O)]

(187) ##STR00043##
[YL3(H.sub.2O)]
L3.3HCl (30.0 mg, 0.053 mmol), YCl.sub.3.6H.sub.2O (24.0 mg, 0.079 mmol)
Yield: 28.0 mg, 94%
ESI-HR-MS (positive, H.sub.2O) m/z calc. [C.sub.22H.sub.25YN.sub.5O.sub.6].sup.+, 544.0858; measured 544.0859 [M+H].sup.+ calc. [C.sub.22H26YN.sub.5O.sub.6].sup.2+, 272.5465; measured 272.5469 [M+2H].sup.2+.
[GdL3(H.sub.2O)]
L3.3HCl (53.0 mg, 0.093 mmol), GdCl.sub.3.6H2O (41.3 mg, 0.111 mmol)
Yield: 58.7 mg, 99%
ESI-HR-MS (positive, H.sub.2O) m/z calc. [C.sub.22H.sub.25GdN.sub.5O.sub.6].sup.+, 613.1040; measured 613.1030 [M+H].sup.+, calc. [C.sub.22H.sub.26GdN.sub.5O.sub.6].sup.2+, 307.0557; measured 307.0568 [M+2H].sup.2+.
[EuL3(H.sub.2O)]
L3.3HCl (28.5 mg, 0.050 mmol), EuCl.sub.3.6H.sub.2O (22.1 mg, 0.060 mmol)
Yield: 29.0 mg, 92%
ESI-HR-MS (positive, H.sub.2O) m/z calc. [C.sub.22H.sub.25EuN.sub.5O.sub.6].sup.+, 608.1012; measured 608.1007 [M+H].sup.+, calc. [C.sub.22H.sub.26EuN.sub.5O.sub.6].sup.2+, 304.5542; measured 304.5544 [M+2H].sup.2+.
[TbL3(H.sub.2O)]
L3.3HCl (24.0 mg, 0.042 mmol), TbCl.sub.3.6H2O (19.0 mg, 0.051 mmol)
Yield: 23.2 mg, 89%
ESI-HR-MS (positive, H.sub.2O) m/z calc. [C.sub.22H.sub.25TbN.sub.5O.sub.6].sup.+, 614.1053; measured 614.1049 [M+H].sup.+, calc. [C.sub.22H.sub.26TbN.sub.5O.sub.6].sup.2+, 307.5563; measured 307.5563 [M+2H].sup.2+.
[YbL3(H.sub.2O)]
L3.3HCl (25.0 mg, 0.044 mmol), YbCl.sub.3.6H2O (20.5 mg, 0.053 mmol)
Yield: 27.8 mg, 98%
ESI-HR-MS (positive, H.sub.2O) m/z calc. [C.sub.22H.sub.25YbN.sub.5O.sub.6].sup.+, 629.1188; measured 629.1182 [M+H].sup.+, calc. [C.sub.22H.sub.26YbN.sub.5O.sub.6].sup.2+, 315.0630; measured 315.0634 [M+2H].sup.2+.
[LuL3(H.sub.2O)]
L3.3HCl (28.0 mg, 0.049 mmol), LuCl.sub.3.6H.sub.2O (23.1 mg, 0.059 mmol)
Yield: 29 mg, 91%
ESI-HR-MS (positive, H.sub.2O) m/z calc. [C.sub.22H.sub.25LuN.sub.5O.sub.6].sup.+, 630.1207; measured 630.1204 [M+H].sup.+, calc. [C22H26LuN5O6].sup.2+, 315.5640; measured 315.5642 [M+2H].sup.2+.
Synthesis of [ML4(H.sub.2O)]

(188) ##STR00044##
[YL4]
L4.3HCl (30.0 mg, 0.047 mmol), YCl.sub.3.6H.sub.2O (24.0 mg, 0.079 mmol)
Yield: 24.8 mg, 92%
ESI-HR-MS (positive, H.sub.2O) m/z calc. [C.sub.27H.sub.28YN.sub.6O.sub.6].sup.+, 621.1123; measured 621.1121 [M+H].sup.+, calc. [C.sub.27H.sub.29YN.sub.6O.sub.6].sup.2+, 311.0598; measured 311.0601 [M+2H].sup.2+.
[GdL4]
L4.3HCl (36.2 mg, 0.056 mmol), GdCl.sub.3.6H.sub.2O (25.1 mg, 0.068 mmol)
Yield: 38.1 mg, 98%
ESI-HR-MS (positive, H.sub.2O) m/z calc. [C.sub.27H.sub.28GdN.sub.6O.sub.6].sup.+, 690.1306; measured 690.1321 [M+H].sup.+, calc. [C.sub.27H.sub.29GdN.sub.6O.sub.6].sup.2+, 345.5698; measured 345.5690 [M+2H].sup.2+.
[EuL4]
L4.3HCl (23.5 mg, 0.036 mmol), EuCl.sub.3.6H.sub.2O (16.0 mg, 0.044 mmol)
Yield: 21.8 mg, 87%
ESI-HR-MS (positive, H.sub.2O) m/z calc. [C.sub.27H.sub.28EuN.sub.6O.sub.6].sup.+, 685.1277; measured 685.1277 [M+H].sup.+, calc. [C.sub.27H.sub.29EuN.sub.6O.sub.6].sup.2+, 343.0675; measured 343.0680 [M+2H].sup.2+.
[TbL4]
L4.3HCl (24.0 mg, 0.037 mmol), TbCl.sub.3.6H.sub.2O (16.4 mg, 0.044 mmol)
Yield: 25.3 mg, 98%
ESI-HR-MS (positive, H.sub.2O) m/z calc. [C.sub.27H.sub.28TbN.sub.6O.sub.6].sup.+, 691.1318; measured 691.1316 [M+H].sup.+ calc. [C.sub.27H.sub.29TbN.sub.6O.sub.6].sup.2+, 346.0696; measured 346.0699 [M+2H].sup.2+.
[YbL4]
L4.3HCl (30.0 mg, 0.047 mmol), YbCl.sub.3.6H.sub.2O (21.7 mg, 0.056 mmol)
Yield: 30.4 mg, 93%
ESI-HR-MS (positive, H.sub.2O) m/z calc. [C.sub.27H.sub.28YbN.sub.6O.sub.6].sup.+, 706.1453; measured 706.1454 [M+H].sup.+, calc. [C.sub.27H.sub.29YbN.sub.6O.sub.6].sup.2+, 353.5763; measured 353.5768 [M+2H].sup.2+.
[LuL4]
L4.3HCl (30.0 mg, 0.047 mmol), LuCl.sub.3.6H.sub.2O (21.8 mg, 0.056 mmol)
Yield: 30.4 mg, 92%
ESI-HR-MS (positive, H.sub.2O) m/z calc. [C.sub.27H.sub.28LuN.sub.6O.sub.6].sup.+, 707.1473; measured 707.1470 [M+H].sup.+, calc. [C.sub.27H.sub.29LuN.sub.6O.sub.6].sup.2+, 354.0773; measured 354.0776 [M+2H].sup.2+.
C-3 Study in Solution

(189) 1) Study by Nuclear Magnetic Resonance:

(190) By way of example, the .sup.1H NMR spectra of the ligand P04213 and of its yttrium complex P04183 recorded in D.sub.2O are shown in FIG. 1. Relative to the spectrum of the ligand, the presence of the metal cation generates dissymmetry and thus a larger number of signals (cf. FIG. 1).

(191) 2) Study by UV-Visible Spectroscopy:

(192) The absorption spectra of the ligands and of their yttrium complexes were recorded in water at pH 3.8 and 5.5 (acetate buffer). The absorption band corresponding to the π-π* transitions of pyridine extend from 240 to 300 nm for the ligands and the complexes (cf. FIG. 2).

(193) C-4 Complexation Kinetics

(194) The complexation kinetics of the ligands Pc1a2pa sym P04213, Pc1a2pa asym P04214 and Pc2a1pa sym P04218 with yttrium were studied at pH 3.8 and pH 5.5 in acetate buffer medium by UV-visible spectroscopy. Placed at the absorption maximum of the complex, the increase in absorbance intensity is measured every two seconds until the maximum absorbance is reached. The decrease in intensity of the absorbance at the absorbance maximum of the ligand is monitored when the absorption band of the complex is masked by that of the ligand. For this study, the concentration of the ligands Pc1a2pa sym and Pc1a2pa asym is 4×10.sup.−5 M and 8×10.sup.−5 M for the ligand Pc2a1pa sym. At pH 5.5 and 3.8, the ligand Pc1a2pa sym has the fastest complexation kinetics, with total complexation in 30 and 400 seconds, respectively. For the ligands Pc1a2pa asym and Pc2a1pa sym, the complexation is complete in 1100 seconds at pH 3.8 and 100 seconds at pH 5.5.

(195) Complexation is thus rapid for all of the ligands under the conditions studied. (cf FIG. 3).

(196) C-5 Kinetic Inertia in Competitive Medium

(197) The dissociation kinetics of the complexes in concentrated acidic medium makes it possible to determine the behavior of the complexes in highly competitive medium. The rate of decomplexation is monitored by UV-visible spectroscopy, with C.sub.YL=4×10.sup.−5 M for the complexes Y-Pc1a2pa sym P04183, Y-Pc1a2pa asym P04215 and Y-Pc2a1pa sym P04219, in 0.5, 1, 2, 4 and 5M HCl medium. The absorption band of the complex disappears more or less rapidly to reveal the absorption band of the ligand at shorter wavelengths. The plot of the increase in intensity of the absorbance at the absorption maximum of the ligand as a function of time (A=f(t)) makes it possible to determine the half-life times t.sub.1/2. The t.sub.1/2 values for the various complexes are listed in the table below. The complexes may be classified in the following manner from the most inert to the least inert: Y-Pc1a2pa asym>>Y-Pc1a2pa sym>Y-PCTA>Y-PCTMB>Y-Pc2a1pa sym. The presence of two picolinate arms on the pyclene macrocycle increases the inertia of the yttrium complex in acidic medium. Furthermore, the inertia of the complex Y-Pc1a2pa asym is greater than that of its symmetrical analog, with, respectively, a t.sub.1/2 of 433 minutes in 5 M HCl medium, as opposed to 8.5 minutes.

(198) TABLE-US-00008 Ligands PCTMB Pc1a2pa sym Pc1a2pa asym Pc2a1pa sym PCTA C.sub.HCl t.sub.1/2 (min) 0.5M   37 347 >1 week 55 95 (in progress) 1M 20 140 (in progress) 27 39 2M 9 51 2745 10.6 17 4M 3.2 13 907 2.7 6.7 5M 2.6 8.5 433 0.8 3.1
C-6 Studies of Thermodynamic Stability by Potentiometry

(199) 1) Protonation Constants of the Liqands

(200) Four protonation constants were determined for the ligands Pc1a2pa sym P04213, Pc1a2pa asym P04214, Pc2a1pa sym P04216 and Pc3pa P04221. These values are coherent with those determined for PCTMB (phosphonic acid, P,P′,P″-[3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triyltris(methylene)]tris-, P, P′, P″-tributyl ester) and also with those described in the literature especially for PCTA (3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid), EDTA (ethylenediaminetetraacetic acid) and DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid).

(201) TABLE-US-00009 TABLE 13 Ligands PCTMB EDTA .sup.2 PCTA .sup.3 DOTA .sup.4 Pc1a2pa sym Pc1a2pa asym Pc2a1pa sym I 0.1 KNO.sub.3 0.1 KNO.sub.3 1.0 KCl 0.1 Me.sub.4NNO.sub.3 0.1 KNO.sub.3 0.1 KNO.sub.3 0.1 KNO.sub.3 log K.sub.t.sup.H [HL]/[L][H] 11.16 10.22 11.36 12.09 11.30 10.50 10.43 [H.sub.2L]/[HL][H] 5.28 6.16 7.35 9.76 5.58 6.73 7.38 [H.sub.3L]/[H.sub.2L][H] 1.72 2.71 3.83 4.56 4.23 3.86 3.95 [H.sub.4L]/[H.sub.3L][H] — 2.0 2.12 4.09 3.01 2.98 2.15 [H.sub.5L]/[H.sub.4L][H] — — 1.29 — — — — Σlog K.sub.i 18.15 21.09 25.95 30.50 24.12 24.06 23.90
For the derivative Pc3pa P04221, the values obtained are as follows:

(202) TABLE-US-00010 TABLE 14 Pc3pa P04221 logK.sup.H.sub.i [HL]/[L][H] 10.03 [H.sub.2L]/[HL][H] 5.95 [H.sub.3L]/[H.sub.2L][H] 3.82 [H.sub.4L]/[H.sub.3L][H] 2.98

(203) 2) Stability Constants of the Complexes

(204) The thermodynamic protonation and stability constants of the complexes were determined by potentiometry at 25° C. with control of the ionic strength (I=0.1 M KNO.sub.3). Refinement of the titration curves with the HyperQuad software makes it possible to determine the overall constants (log β), from which the partial constants (log K) are calculated.

(205) The stability constants of the ligands Pc1a2pa sym P04213, Pc1a2pa asym P04214 et Pc2a1pa sym P04218 and P04221 with yttrium were determined by direct potentiometric titration. The log K.sub.YL constant values for the ligands Pc1a2pa sym, Pc1a2pa asym and Pc2a1pa sym are, respectively, 19.78, 19.49 and 19.28, and the log K.sub.YLH.sup.−1 constant values are 11.84, 11.79 and 10.60.

(206) TABLE-US-00011 Reaction equilibrium PCTMB .sup.b EDTA .sup.c PCTA .sup.f DOTA .sup.g Pc1a2pa sym Pc1a2pa asym Pc2a1pa sym Log K.sub.MHiL Y.sup.3+ [ML]/[M][L] 19.49 18.5 .sup.d 20.28 24.9 .sup.h 19.78 19.49 19.28 [MHL]/[ML][H] 3.45 — 1.81 — — — — [ML]/[MLOH][H] 9.10 — 11.10 — 11.84 11.79 10.60 .sup.a For the sake of claritiy, the charges are not indicated. .sup.b Values determined by competition with EDTA. 0.1M KNO.sub.3. .sup.c Ref 2, 0.1M KNO.sub.3. .sup.d Ref .sup.5, 0.1M NMe.sub.4Cl. .sup.e Ref .sup.6, 0.1M KNO.sub.3. .sup.f Ref 3, 1.0M KCl. .sup.g Ref 4, 0.1M NMe.sub.4Cl. .sup.h Ref .sup.7, 0.1M NMe.sub.4NO.sub.3.

(207) TABLE-US-00012 TABLE 16 Pc3pa + Y.sup.3+P04222 logK.sub.MHiL ML]/[M][L] 16.42 [MHL]/[ML][H] 3.11 [ML]/[MLOH][H] 11.02
These stability constants are not comparable as such: the basicity of the ligands needs to be taken into account. The constant pM=−log[M] is used for this purpose. It is calculated from the protonation constants of the ligands and the stability constants of the complexes with CL=10×CM=10.sup.−5 M at pH 7.4. The ligand Pc1a2pa asym P04214 has a p(Y) of 17.3, which is higher than that of PCTA (p(Y)=17.0), of Pc1a2pa sym P04213 (p(Y)=16.8) and of Pc2a1pa sym P04218 (p(Y)=16.9). The highest p(Y) nevertheless remains that of DOTA, with a value of 18.8.

(208) TABLE-US-00013 TABLE 17 pM = −log[M] .sup.a Ligands PCTMB EDTA PCTA DOTA Pc1a2pa sym Pc1a2pa asym Pc2a1pa sym pY 16.7 16.6 17.0 18.8 16.8 17.3 16.9 .sup.a Values calculated from the constants of the preceding tables. with C.sub.L = 10 × C.sub.M = 10.sup.−5 M à pH 7.4.
For Pc3pa P04222, the pM calculated is 14.7.
The speciation diagrams, plotted from the thermodynamic stability constants of the yttrium complexes, indicate that the complexes exist exclusively in the form YL over a wide pH range, including at pH 7.4.

REFERENCES

(209) 1 Aime, S.; Botta, M.; Geninatti Crich, S.; Giovenzana, G. B.; Jommi, G.; Pagliarin, R.; Sisti, M. Inorg. Chem.
1997, 36, 2992-3000. 2 Delgado, R.; Figueira, C.; Quintino, S. Talanta 1997, 45, 451. 3 Tircsó, G.; Kovacs, Z.; Dean Sherry, A. Inorg. Chem. 2006, 45, 9269. 4 Chaves, S.; Delgado, R.; Frausto da Silva, J. J. R. Talanta 1992, 39, 249. 5 Kumar, K.; Chang C. A.; Francesconi, L. C.; Dischino, D. D.; Malley, M. F.; Gougoutas, J. Z.; Tweedle,
M. F. Inorg. Chem. 1994, 33, 3567. 6 Delgado, R.; Frausto da Silva, J. J. R. Talanta 1982, 29, 815. 7 Cox, J. P. L.; Jankowski, K. J.; Kataky, R.; Parker, D.; Beeley, N. R. A.; Boyce, B. A.; Eaton, M. A. W.; Millar,
K.; Millican, A. T.; Harrison, A.; Walkerc, C. J. Chem. Soc. Chem. Commun. 1989, 797.
C-7 Solid-State Study

(210) The yttrium complex P04183 crystallizes in water. The structure obtained by x-ray diffraction is presented below. The metal is coordinated to the four nitrogen atoms of the macrocycle, the two nitrogen atoms of the picolinate arms and the three oxygen atoms of the carboxylic acids. The coordination sphere of the metal is N6O3, i.e. 9 coordinant atoms. The helicities Δ and Λ derived from the orientation of the picolinate and acetate arms are both present: the complex thus crystallizes as a racemic mixture.