Terbium and dysprosium complexes with optimised antenna, used as luminescent markers
11649253 · 2023-05-16
Assignee
- Ecole Normale Superieure De Lyon (Lyons, FR)
- Centre National De La Recherche Scientifique (Paris, FR)
- Universite Claude Bernard Lyon 1 (Villeurbanne, FR)
Inventors
- Amandine Roux (Le Peage de Roussillon, FR)
- Anh-Thy Bui (Pessac, FR)
- Olivier Maury (Brindas, FR)
- Chantal Andraud (Genas, FR)
Cpc classification
C07F5/00
CHEMISTRY; METALLURGY
International classification
Abstract
The present technology relates to luminescent lanthanide complexes comprising a chelating agent, formed of a macrocycle or ligand, complexing a lanthanide ion Ln.sup.3+ selected from terbium and dysprosium, the chelating agent comprising at least one group of the structure (B) below; and a process for detecting a biomolecule using said lanthanide complex comprising coupling a luminescent lanthanide complex of the present technology having a reactive group with said biomolecule. ##STR00001##
Claims
1. A lanthanide complex comprising a chelating agent, formed of a macrocycle or a ligand, complexing a lanthanide ion Ln.sup.3+, wherein the lanthanide ion Ln.sup.3+ is selected from terbium and dysprosium and in that the chelating agent includes at least a group (B) of the following structure: ##STR00065## wherein: —R1 represents a hydrogen, a group —R6, or an electron-donating group -E1, —R2 represents a hydrogen, a group —R7, or an electron-donating group -E2, —R3 represents a hydrogen, a group —R8, or an electron-donating group -E3, —R4 and —R5, identical or different, independently represent a hydrogen or a group —R9 or —OR9, -E1, -E2 and -E3 are independently selected from the groups consisting of —OR10, —SR10, —NH(CO)R10, —NH(CO)NR10R′10, —NH(CS)NR10 R′10 and —NH(CS)NHR10, —R6, —R7, —R8, —R9, —R10 and R′10 identical or different, independently represent a (C1-C6) alkyl group, optionally substituted by a group —X1 or a group —Y, —X1 is a reactive group, —Y is a water-solubilizing group, with the proviso that: at least one of the substituents —R2 and —R3 is not hydrogen, only one of the groups —R1, —R2 and —R3 represents an electron-donating group, and said chelating agent comprises at most one reactive group.
2. The lanthanide complex according to claim 1 selected from the lanthanide complexes of formula (I): ##STR00066## wherein: Ln.sup.3+ is a lanthanide selected from Tb and Dy, -A- represents —CH.sub.2— or —CH(L.sub.A-X2)-, -L.sub.A- is a covalent bond, or a linear or branched (C1-C20) alkylene group, optionally containing one or more double or triple bonds, and optionally substituted by one to three groups —SO.sub.3H, or -L.sub.A- is a (C5-C8) cycloalkylene group or a (C6-C14) arylene group, —X2 is a reactive group, -Chrom1, -Chrom2 and -Chrom3 are identical or different and independently selected from the groups of formula (B1): ##STR00067## —R1, —R2, —R3, —R4, and —R5 are as defined in claim 1, —Z— represents —C— or —P(R.sub.Z)—, and —R.sub.Z represents a phenyl, benzyl, methyl, ethyl, propyl, n-butyl, sec-butyl, iso-butyl or tert-butyl group, with the proviso that if -A- is —CH(L.sub.A-X2)-, then none of groups -Chrom1, -Chrom2 and -Chrom3 of formula (B1) has group —X1.
3. The lanthanide complex according to claim 2, wherein -A- represents —CH.sub.2— and —Z— represents —C—.
4. The lanthanide complex according to claim 2, wherein -Chrom1, -Chrom2 and -Chrom3 are identical, wherein: —R1 represents an electron-donating group -E1 as defined in claim 1, —R2 is a group —R7 as defined in claim 1, and —R3 is a group —R8 as defined in claim 1.
5. The lanthanide complex according to claim 2 wherein: -A- represents —CH.sub.2—, -Chrom1 and -Chrom2 are identical and are of structure (B2): ##STR00068## with —R1.sup.1, —R2.sup.1, —R3.sup.1, —R4.sup.1, —R5.sup.1 and —Z.sup.1— as defined respectively for R1, —R2, —R3, —R4, —R5 and —Z— in claim 1, with the proviso that Chrom1 and -Chrom2 do not have a reactive group, and -Chrom3 is different from -Chrom1 and from -Chrom2 and is of structure (B3): ##STR00069## with —R1.sup.2, —R2.sup.2, —R3.sup.2, —R4.sup.2, —R5.sup.2 and —Z.sup.2— as defined respectively for —R1, —R2, —R3, —R4, —R5 and —Z— in claim 1, with the proviso that -Chrom3 has a reactive group.
6. The lanthanide complex according to claim 1 selected from the lanthanide complexes of formula (II): ##STR00070## wherein: Ln.sup.3+ is a lanthanide selected from Tb and Dy, —R11 and —R12, identical or different, are independently selected from —X1, —Y, a hydrogen atom or a (C1-C6) alkyl group, and -Chrom4-, -Chrom5-, and -Chrom6-, identical or different, independently represent a group of formula (B), as defined in claim 1.
7. The lanthanide complex according to claim 6, wherein -Chrom4-, -Chrom5- and -Chrom6- are identical, wherein: —R1 represents an electron-donating group -E1 as defined in claim 1, —R2 is a group —R7 as defined in claim 1, and —R3 is a group —R8 as defined in claim 1.
8. The lanthanide complex according to claim 6, wherein: either: Chrom5- and -Chrom6- are identical and are of structure (B10): ##STR00071## wherein —R1.sup.3, —R2.sup.3, —R3.sup.3, —R4.sup.3, and —R5.sup.3 are as defined respectively for —R1, —R2, —R3, —R4, and —R5 in claim 1, with the proviso that -Chrom5- and -Chrom6- do not have a reactive group, and Chrom4- is different from -Chrom5- and from -Chrom6- and is of structure (B11): ##STR00072## wherein —R1.sup.4, —R2.sup.4, —R3.sup.4, —R4.sup.4 and —R5.sup.4 are as defined respectively for —R1, —R2, —R3, —R4 and —R5 in claim 6, with the proviso that -Chrom4- has a reactive group —X1, or: -Chrom4- and -Chrom6- are identical and are of structure (B12): ##STR00073## wherein —R1.sup.5, —R2.sup.5, —R3.sup.5, —R4.sup.5 and —R5.sup.5 are as defined respectively for —R1, —R2, —R3, —R4 and —R5 in claim 1, with the proviso that -Chrom4- and -Chrom6- do not have a reactive group, and -Chrom5- is different from -Chrom4- and from -Chrom6- and is of structure (B13): ##STR00074## wherein —R1.sup.6, —R2.sup.6, —R3.sup.6, —R4.sup.6 and —R5.sup.6 are as defined respectively for —R1, —R2, —R3, —R4 and —R5 in claim 1, with the proviso that -Chrom5- has a reactive group —X1.
9. The lanthanide complex according to claim 1, wherein —R4 and —R5 represent hydrogens.
10. The lanthanide complex according to claim 1, comprising a single reactive group allowing its coupling by covalent bonding to a biomolecule ##STR00075##
11. The lanthanide complex according to claim 1, wherein the group Y is selected from the group consisting of —SO.sub.3.sup.−, —COO.sup.−, sulfobetaine and —O—[(CH.sub.2).sub.2—O].sub.m—CH.sub.3, m being an integer ranging from 1 to 10.
12. The lanthanide complex according to claim 1, wherein Ln.sup.3+ is terbium.
13. A chelating agent of formula (III): ##STR00076## with -Chrom1′, -Chrom2′, and -Chrom3′, identical or different, and independently selected from the groups of formula (B5): ##STR00077## with R1, R2, R3, R4 and R5 as defined in claim 1, and —R13 an acid-protecting group, or in the form of a salt.
14. A chelating agent of formula (IV): ##STR00078## with -Chrom4-, -Chrom5- and -Chrom6- as defined in claim 6, and —R13 an acid-protecting group, or in the form of a salt.
15. A method for detecting a biomolecule comprising the steps of coupling a luminescent complex according to claim 1, comprising a reactive group with said biomolecule on the reactive group of said luminescent complex, and detecting the luminescence of a conjugate of said biomolecule with said luminescent complex.
16. The lanthanide complex according to claim 2, wherein —RZ represents a phenyl or a methyl group.
17. The lanthanide complex according to claim 4, wherein: —R1 represents an electron-donating group -E1 selected from the groups consisting of —OR10 and —NH(CO)R10, —R10 being as defined in claim 1, —R2 is -Me, and —R3 is -Me or a hydrogen.
18. The lanthanide complex according to claim 4, wherein —R1 represents —OMe or —OPEG.
19. The lanthanide complex according to claim 5, wherein: —R1.sup.1 is a group —O(C1-C6) alkyl substituted by a group —Y, —R1.sup.2 is a group —O(C1-C6) alkyl substituted by a group —X1, —R2.sup.1 and —R2.sup.2, identical or different, represent a group —R7 as defined in claim 1, and —R3.sup.1, —R3.sup.2, —R4.sup.1, —R4.sup.2, —R5.sup.1, and —R5.sup.2 are hydrogens.
20. The lanthanide complex according to claim 5, wherein —R2.sup.1 and —R2.sup.2 are identical.
21. The lanthanide complex according to claim 5, wherein —R2.sup.1 and —R2.sup.2 represent a (C1-C6) alkyl group.
22. The lanthanide complex according to claim 10, wherein the reactive group is selected from the group consisting of —COOH, —NH.sub.2, an acrylamide, an activated amine, an activated ester, an aldehyde, an alkyl halide, an anhydride, an aniline, an azide, an aziridine, a carboxylic acid, a diazoalkane, a haloacetamide, a halotriazine, a hydrazine, an imido ester, an isocyanate, an isothiocyanate, a maleimide, a sulfonyl halide, a thiol, a ketone, an acid halide, a hydroxysuccinimidyl ester, a succinimidyl ester, a hydroxysulfosuccinimidyl ester, an azidonitrophenyl, an azidophenyl, a 3-(2-pyridyl dithio)-propionamide, a glyoxal, a triazine, and an acetylenic group, and the groups of formula: ##STR00079## wherein w is an integer belonging to the range from 0 to 8 and v is equal to 0 or 1, and Ar is a saturated or unsaturated 5- or 6-membered heterocycle, comprising 1 to 3 heteroatoms, optionally substituted by a halogen atom.
23. The lanthanide complex according to claim 10, wherein the reactive group is selected from —COOH, —NH.sub.2, succinimidyl esters, haloacetamides, azides, hydrazines, isocyanates and maleimides.
Description
(1) Finally, the invention relates to a process for detecting a biomolecule comprising detecting the luminescence of a conjugate of said biomolecule with a luminescent complex as defined in the context of the invention, comprising a reactive group, and obtained by coupling said biomolecule with said luminescent complex on its reactive group.
(2)
(3)
(4)
(5)
(6) The examples below are intended to illustrate the invention but are not restrictive.
EXAMPLES
(7) The abbreviations below are used in the following examples:
(8) DCM: dichloromethane; DMF: dimethylformamide; DMSO: dimethylsulphoxide; Ms: mesyl; n.d.: not determined; PBS: phosphate buffered saline; PEG: polyethylene glycol; PFA: paraformaldehyde; quant.: quantitative; TACN: triazacyclononane; THF: tetrahydrofuran; Ts: tosyl
(9) General Information:
(10) The NMR (.sup.1H, .sup.13C) spectra were recorded on a Bruker Avance device at a frequency of 300 MHz or 500.10 MHz, and 75 MHz or 125.75 MHz, for .sup.1H and .sup.13C respectively. Chemical shifts (δ) are expressed in parts per million (ppm) with respect to trimethylsilane used as an internal standard and using the solvents indicated. The coupling constants (J) are expressed in Hz and the following notations are used: s (singlet), br (broad), d (doublet), t (triplet), dd (doublet of doublets), m (multiplet). The high-resolution mass spectra were recorded at the Lyon Common Mass Spectrometry Centre (Claude Bernard Lyon University, Lyon, France) on a MicroTOFQII instrument equipped with a positive ESI source. Thin layer chromatography was performed on silica gel plates on aluminium foil (silica gel, Fluka) and revealed using a UV lamp (λ=254 or 365 nm) or by staining. Purifications were carried out by chromatographic column on silica gel (silica gel 0.035-0.070 mm, 60 A). The solvents used for the reactions were purchased from Aldrich or Acros Organics as dry or extra dry solvents and stored on 3 Å molecular sieves and the reagents used were purchased from Aldrich, Acros Organics or Alfa Aesar. The UV/Vis absorption spectra were recorded on a JASCO V670 spectrometer; the emission spectra on a JOBIN-YVON fluorolog 3 spectrofluorimeter. Retention times were performed on an Agilent Technologies 1260 device equipped with a Waters XBridge RP-C18 column (3.5 nm, 4.6×100 mm). The chromatographic system used is 0.25 M ammonium formate-MeCN (v/v) as eluents: isocratic 15% MeCN (2 min), linear gradient 15 to 100% MeCN (16 min), isocratic 100% MeCN (4 min), at a rate of 1 mL/min, UV detection at 210 and 252 nm. Cellular imaging tests were performed using a solution concentrated in complex in DMSO to obtain a final complex concentration in the order of 1.Math.10.sup.5 M in cellular medium (PBS) with <1% DMSO. The T24 cells are prefixed to the PFA. The biphotonic absorption spectra were performed with a confocal laser scanning microscope LSM710 NLO (Carl Zeiss).
A. Preparation of the Complexes
Example 1: Europium Complexes Ia-Ie
(11) ##STR00027##
(12) Compounds 1a (4-bromo-3-methylanisole), 1e (1-bromo-3,5-dimethoxybenzene) and 2e (2,4-dimethoxyphenylboronic acid) are commercially available. Compounds 1b and 1d are prepared from the corresponding phenol, while compound 1c is prepared from the corresponding aniline. Boronic acids 2a-2d are then obtained according to a procedure well known to the skilled person. The compounds 3a-e are obtained by a Suzuki coupling reaction with dimethyl-4-iododipicolinate. Then, a reduction and mesylation reaction is used to isolate the compounds 5a-e.
(13) ##STR00028##
(14) An alkylation reaction of TACN.3HCl with mesylate derivatives 5a-e under conventional alkylation conditions, followed by a step of deprotection of carboxylic acid functions, results in the lanthanide complexes Ia-Ie.
(15) ##STR00029##
(16) 4-Bromo-3-methylphenol (1.0 g, 5.3 mmol) and K.sub.2CO.sub.3 (3.7 g, 27 mmol) are dried under vacuum then solubilized in dry DMF (20 mL). After 30 minutes, triethylene glycol methyl ether tosylate (2.55 g, 8 mmol) is added to the Schlenk flask. The reaction mixture is stirred at 80° C. for 5 days. The solution is then filtered on sintered P3, rinsed with CH.sub.2Cl.sub.2 and concentred under vacuum. The milieu is diluted in AcOEt/Et.sub.2O and washed with H.sub.2O and NaCl.sub.sat. The organic phase is dried over Na.sub.2SO.sub.4, filtered and evaporated. The crude product is then purified on a chromatographic column (SiO.sub.2, CH.sub.2Cl.sub.2/AcOEt, 90/10 to 80/20) to give the pure product as colourless oil (1.62 g, 91%). R.sub.f (SiO.sub.2, CH.sub.2Cl.sub.2/AcOEt, 80/20)=0.53; .sup.1H NMR (300 MHz, CDCl.sub.3, δ): 7.38 (d, 1H, J=8.7 Hz, H.sup.6), 6.81 (d, 1H, J=2.9 Hz, H.sup.3), 6.62 (dd, 1H, J.sub.3=8.7 Hz, J.sub.4=2.9 Hz, H.sup.5), 4.08 (t, 2H, J=4.6 Hz, H.sup.8), 3.83 (t, 2H, J=4.6 Hz, H.sup.9), 3.75-3.53 (m, 8H, H.sup.10/H.sup.11/H.sup.12/H.sup.13), 3.38 (s, 3H, H.sup.14), 2.35 (s, 3H, H.sup.7); .sup.13C NMR (100 MHz, CDCl.sub.3, δ): 158.2 (C.sup.4), 138.9 (C.sup.2), 132.9 (C.sup.6), 117.4 (C.sup.3), 115.8 (C.sup.5), 113.8 (C.sup.5), 72.1 (C.sup.13), 71.0 (C.sup.10), 70.8 (C.sup.11), 70.8 (C.sup.12), 69.8 (C.sup.9), 67.8 (C.sup.8), 59.2 (C.sup.14), 23.3 (C.sup.7); HRMS (ESI) calculated for C.sub.14H.sub.21BrNaO.sub.4 355.0515. Exp 355.0520 [M+Na].sup.+.
(17) ##STR00030##
(18) In a round-bottom flask, acetic anhydride (305 μL, 3.22 mmol) is added to a solution of 4-bromo-3-methylaniline (500 mg, 2.69 mmol) in dry CH.sub.2Cl.sub.2 under argon. The mixture is stirred at room temperature under argon until the complete reaction (TLC monitoring, petroleum ether/ethyl acetate, 80/20). After 4 h, the mixture is washed with H.sub.2O (3×). The organic phase is dried over Na.sub.2SO.sub.4, filtered on cotton and evaporated to obtain a white solid (506 mg, 82%). R.sub.f (SiO.sub.2, CH.sub.2Cl.sub.2/AcOEt, 80/20)=0.46; .sup.1H NMR (300 MHz, CDCl.sub.3, δ): 7.45-7.42 (m, 2H, H.sup.6/H.sup.3), 7.19 (dd, 1H, J.sub.3=8.6 Hz, J.sub.4=2.4 Hz, H.sup.5), 2.36 (s, 3H, H.sup.7), 2.16 (s, 3H, H.sup.9); .sup.1C NMR (75 MHz, CDCl.sub.3, δ): 169.0 (C.sup.8), 138.5 (C.sup.4), 137.2 (C.sup.1), 132.6 (C.sup.3), 122.4 (C.sup.6), 119.6 (C.sup.2), 119.2 (C.sup.5), 24.5 (C.sup.9), 23.1 (C.sup.7); HRMS (ESI) calculated for C.sub.9H.sub.11BrNO 228.0024. Exp 228.0019 [M+H].sup.+.
(19) ##STR00031##
(20) 4-Bromo-3,5-dimethylphenol (1.30 g, 6.46 mmol) and K.sub.2CO.sub.3 (4.46 g, 9.68 mmol) are dried under vacuum then solubilized in dry DMF (12 mL). Argon bubbling is performed for 10 minutes and triethylene glycol methyl ether tosylate (3.08 g, 9.68 mmol) is added to the Schlenk flask. The reaction mixture is stirred at 80° C. for 3 days. A CH.sub.2Cl.sub.2/H.sub.2O extraction is then performed, then the organic phase is dried with NaCl.sub.sat and Na.sub.2SO.sub.4, filtered and evaporated. The crude product is then purified on a chromatographic column (SiO.sub.2, CH.sub.2Cl.sub.2/AcOEt, 100/0 to 80/20) to give an oil of the pure product (1.5 g, 67%). R.sub.f (SiO.sub.2, CH.sub.2Cl.sub.2/AcOEt, 95/5)=0.38; .sup.1H NMR (300 MHz, CDCl.sub.3, δ): 6.66 (s, 2H, H.sup.3), 4.07 (t, 2H, J=4.7 Hz, H.sup.8), 3.82 (t, 2H, J=4.7 Hz, H.sup.9), 3.74-3.52 (m, 8H, H.sup.10/H.sup.11/H.sup.12/H.sup.13), 3.37 (s, 3H, H.sup.14), 2.36 (s, 6H, H.sup.7); .sup.13C NMR (75 MHz, CDCl.sub.3, δ): 157.4 (C.sup.4), 139.2 (C.sup.1), 118.5 (C.sup.2/6), 114.7 (C.sup.3/5), 72.1 (C.sup.13), 71.0 (C.sup.10), 70.8 (C.sup.11), 70.7 (C.sup.12), 69.8 (C.sup.9), 67.7 (C.sup.8), 59.2 (C.sup.14), 24.1 (C.sup.7); HRMS (ESI) calculated for C.sub.15H.sub.23BrNaO.sub.4 369.0672. Exp 369.0664 [M+Na].sup.+.
(21) Compound 2a
(22) The compound 2a was prepared according to the protocol described in “Substituent Effects on Oxidation-Induced Formation of Quinone Methides from Arylboronic Ester Precursors”, Sheng Cao, Robin Christiansen, and Xiaohua Peng, Chem. Eur. J., 2013, 19, 9050-9058.
(23) ##STR00032##
(24) Dry potassium acetate (442 mg, 8.46 mmol) is solubilized in dry DMSO (15 mL) under argon then bis(dipicolinato)diboron (572 mg, 2.25 mmol) and 1b (500 mg, 1.50 mmol) are added. The solution is placed under argon bubbling for 20 minutes then PdCl.sub.2(dppf).sub.2 (77 mg, 0.11 mmol) is added. The reaction mixture is heated at 90° C. under argon and under stirring for 16 h. A serial extraction H.sub.2O/Et.sub.2O is then performed. The organic phases are combined, washed with NaCl.sub.sat, dried over Na.sub.2SO.sub.4, filtered and evaporated under vacuum. The crude product is then purified on a column (SiO.sub.2, CH.sub.2Cl.sub.2/AcOEt, 90/10 to 80/20) in order to obtain the pure product in the form of yellow oil (516 mg, 90%). R.sub.f (SiO.sub.2, CH.sub.2Cl.sub.2/AcOEt, 80/20)=0.57; .sup.1H NMR (500 MHz, CDCl.sub.3, δ): 7.69 (d, 1H, J=8.1 Hz, H.sup.6), 6.72-6.69 (m, 2H, H.sup.3/H.sup.5), 4.13 (t, 2H, J=4.7 Hz, H.sup.8), 3.85 (t, 2H, J=4.7 Hz, H.sup.9), 3.75-3.53 (m, 8H, H.sup.10/H.sup.11/H.sup.12/H.sup.13), 3.38 (s, 3H, H.sup.14), 2.50 (s, 3H, H.sup.7), 1.32 (s, 12H, H.sup.16); .sup.1C NMR (125 MHz, CDCl.sub.3, δ): 161.1 (C.sup.4), 147.3 (C.sup.2), 138.0 (C.sup.6), 120.6 (C.sup.1), 116.4 (C.sup.3), 110.9 (C.sup.5), 83.3 (C.sup.15), 72.1 (C.sup.13), 71.0 (C.sup.10), 70.8 (C.sup.11), 70.8 (C.sup.12), 69.9 (C.sup.9), 67.2 (C.sup.8), 59.2 (C.sup.14), 25.1 (C.sup.16), 22.6 (C.sup.7); HRMS (ESI) calculated for C.sub.20H.sub.33BNaO.sub.6 403.2262. Exp 403.2255 [M+Na].sup.+.
(25) ##STR00033##
(26) Potassium acetate (515 mg, 5.25 mmol) is dried under vacuum/argon for 1 h then bis(pinacolato)diboron (668 mg, 2.63 mmol), dry DMSO (6 mL) and 1c (400 mg, 1.75 mmol) are added. Argon bubbling in the mixture is carried out for 20 min then PdCl.sub.2(dppf).sub.2.CH.sub.2Cl.sub.2 (100 mg, 0.12 mmol) is added and the Schlenk flask is closed. The solution is heated at 90° C. under argon and under stirring for 19 h. After returning to room temperature, the mixture is filtered on sintered P3 and rinsed with Et.sub.2O. Next is a serial extraction with H.sub.2O/Et.sub.2O. The organic phases are combined, dried over Na.sub.2SO.sub.4, filtered on cotton and evaporated. Slow evaporation CH.sub.2Cl.sub.2 gives rise to the formation of crystals which are washed several times with CH.sub.2Cl.sub.2 and dried under vacuum. .sup.1H NMR shows that the expected pure product (240 mg, 50%) was obtained. R.sub.f (SiO.sub.2, CH.sub.2Cl.sub.2/AcOEt, 80/20)=0.61; .sup.1H NMR (300 MHz, CDCl.sub.3, δ): 7.72 (d, 1H J=8 Hz, H.sup.5), 7.33 (s, 1H, H.sup.3), 7.30 (d, 1H, J=8 Hz, H.sup.6), 7.18 (s, 1H, NH), 2.51 (s, 3H, H.sup.7), 2.16 (s, 3H, H.sup.9), 1.33 (s, 12H, H.sup.11); .sup.13C NMR (75 MHz, CDCl.sub.3, δ): 168.7 (C.sup.8), 146.4 (C.sup.2), 140.3 (C.sup.4), 137.1 (C.sup.6), 120.6 (C.sup.3), 115.9 (C.sup.5), 83.4 (C.sup.10), 25.1 (C.sup.9), 25.0 (C.sup.11), 22.4 (C.sup.7); HRMS (ESI) calculated for C.sub.15H.sub.23BNO.sub.3 276.1768. Exp 276.1762 [M+H].sup.+.
(27) ##STR00034##
(28) Dry potassium acetate (830 mg, 8.46 mmol) is solubilized in dry DMSO (10 mL) under argon then bis(dipicolinato)diboron (1.07 g, 4.23 mmol) and 1d (979 mg, 2.82 mmol) are added. The solution is placed under argon bubbling for 20 minutes then PdCl.sub.2(dppf).sub.2.CH.sub.2Cl.sub.2 (161 mg, 0.20 mmol) is added. The reaction mixture is heated at 90° C. under argon and under stirring for 40 h. A serial extraction H.sub.2O/Et.sub.2O is then performed. The organic phases are combined, washed with NaCl.sub.sat, dried over Na.sub.2SO.sub.4, filtered and evaporated under vacuum. The crude product is then purified on a chromatographic column (SiO.sub.2, CH.sub.2Cl.sub.2/AcOEt, 95/5 to 80/20) in order to obtain the pure product as oil (748 mg, 68%). R.sub.f (SiO.sub.2, CH.sub.2Cl.sub.2/AcOEt, 80/20)=0.43; .sup.1H NMR (300 MHz, CDCl.sub.3, δ): 6.51 (s, 2H, H.sup.3), 4.09 (t, 2H, J=4.7 Hz, H.sup.8), 3.82 (t, 2H, J=4.7 Hz, H.sup.9), 3.74-3.53 (m, 8H, H.sup.10/H.sup.11/H.sup.12/H.sup.13), 3.37 (s, 3H, H.sup.14), 2.37 (s, 6H, H.sup.7), 1.36 (s, 12H, H.sup.16); .sup.13C NMR (75 MHz, CDC.sub.3, δ): 159.7 (C.sup.4), 144.5 (C.sup.2/6), 113.2 (C.sup.3/5), 83.5 (C.sup.15), 72.1 (C.sup.13), 70.9 (C.sup.10), 70.8 (C.sup.11), 70.7 (C.sup.12), 69.9 (C.sup.9), 67.1 (C.sup.8), 59.1 (C.sup.14), 25.0 (C.sup.16), 22.7 (C.sup.7); HRMS (ESI) calculated for C.sub.21H.sub.35BNaO.sub.6 417.2423. Exp 417.2417 [M+Na].sup.+.
(29) ##STR00035##
(30) In a Schlenk flask, dimethyl-4-iododipicolinate (300 mg, 0.934 mmol) and 2-methyl,4-methoxyphenylboronic acid (186 mg, 1.121 mmol) are solubilized in dry DMF (15 mL) under argon. After 30 minutes of argon bubbling in the mixture, caesium fluoride (355 mg, 2.335 mmol) and Pd(PPh.sub.3).sub.4(cat.) are added. The reaction mixture is stirred at 90° C. under argon for 17 h. The mixture is then diluted in CH.sub.2Cl.sub.2 then extracted with H.sub.2O. The chlorinated phase is evaporated and the aqueous phase is again extracted with AcOEt. The organic phase is washed with NaCl.sub.sat, dried over Na.sub.2SO.sub.4, filtered and evaporated under vacuum. The crude product obtained is purified on 2 chromatographic columns (SiO.sub.2, CH.sub.2Cl.sub.2/acetone, 90/10 then SiO.sub.2, CH.sub.2Cl.sub.2/AcOEt 98/2 to 95/5) in order to obtain the pure product (159 mg, 54%). R.sub.f (SiO.sub.2, CH.sub.2Cl.sub.2/acetone, 80/20)=0.82; .sup.1H NMR (300 MHz, CDCl.sub.3, δ): 8.28 (s, 2H, H.sup.3/5), 7.24-7.20 (m, 1H, H.sup.12), 6.87-6.85 (m, 2H, H.sup.11/H.sup.9), 4.04 (s, 6H, H.sup.16), 3.86 (s, 3H, H.sup.14), 2.32 (s, 3H, H.sup.13); .sup.13C NMR (125 MHz, CDCl.sub.3, δ): 165.5 (C.sup.15), 160.5 (C.sup.10), 152.6 (C.sup.4), 148.4 (C.sup.2/6), 137.0 (C.sup.8), 131.0 (C.sup.12), 128.8 (C.sup.7), 121.7 (C.sup.35), 116.6 (C.sup.9), 112.1 (C.sup.11), 55.5 (C.sup.14), 53.4 (C.sup.16), 20.8 (C.sup.13); HRMS (ESI) calculated C.sub.17H.sub.17NNaO.sub.5 338.0999. Exp 338.0990 [M+Na].sup.+.
(31) ##STR00036##
(32) Dry dimethyl 4-iodo-2,6-pyridinedicarboxylate (344 mg, 1.07 mmol) is solubilized in dry DMF (12 mL) and 2b (448 mg, 1.18 mmol) is added under argon. The solution is placed under argon bubbling for 30 minutes then potassium fluoride (205 mg, 3.53 mmol) and Pd(dba).sub.3tBu.sub.3PH.BF.sub.4 (43 mg, 0.08 mmol) are added. The reaction mixture is placed at 80° C. under stirring and under argon for 40 h. A serial extraction AcOEt/H.sub.2O is then performed. The organic phases are washed with NaCl.sub.sat, dried over Na.sub.2SO.sub.4, filtered and evaporated under vacuum. The crude product is then purified on a chromatographic column (SiO.sub.2, CH.sub.2Cl.sub.2/acetone, 95/5 to 90/10) to obtain the pure product as white solid (343 mg, 72%). R.sub.f (SiO.sub.2, CH.sub.2Cl.sub.2/acetone, 90/10)=0.45; .sup.1H NMR (500 MHz, CDCl.sub.3, δ): 8.26 (s, 2H, H.sup.15), 7.19 (d, 1H, J=8.3 Hz, H.sup.12), 6.87 (d, 1H, J=2.3 Hz, H.sup.9), 6.86 (dd, 1H, J.sub.3=8.3 Hz, J.sub.4=2.3 Hz, H.sup.11), 4.17 (t, 2H, J=4.7 Hz, H.sup.14), 4.03 (s, 6H, H.sup.22), 3.86 (t, 2H, J=4.7 Hz, H.sup.15), 3.76-3.54 (m, 8H, H.sup.16/H.sup.17/H.sup.18/H.sup.19), 3.38 (s, 3H, H.sup.20), 2.30 (s, 3H, H.sup.13); .sup.13C NMR (125 MHz, CDCl.sub.3, δ): 165.5 (C.sup.21), 159.7 (C.sup.10), 152.6 (C.sup.4), 148.3 (C.sup.2/6), 136.9 (C.sup.8), 130.9 (C.sup.12), 130.1 (C.sup.7), 128.7 (C.sup.3/5), 117.3 (C.sup.9), 112.7 (C.sup.11), 72.1 (C.sup.19), 71.0 (C.sup.16), 70.9 (C.sup.17), 70.8 (C.sup.18), 69.8 (C.sup.15), 67.7 (C.sup.14), 59.2 (C.sup.20), 53.4 (C.sup.22), 20.8 (C.sup.13); HRMS (ESI) calculated for C.sub.23H.sub.30NO.sub.8 448.1966. Exp 448.1959 [M+H].sup.+.
(33) ##STR00037##
(34) 2c (200 mg, 0.73 mmol) and dimethyl 4-iodo-2,6-pyridinedicarboxylate (212 mg, 0.66 mmol) are first dried under vacuum/argon then dry DMF (15 mL) is added. The solution is degassed for 20 min under argon and potassium fluoride (126 mg, 2.18 mmol) and Pd(dba).sub.3tBu.sub.3PH.BF.sub.4 (27 mg, 0.05 mmol) are added. The mixture is placed at 80° C. under argon for 3 days. After returning to room temperature, CH.sub.2Cl.sub.2 is added and the organic phase is washed with H.sub.2O (3×) and saturated NaCl (lx). The organic phase is dried over Na.sub.2SO.sub.4, filtered and evaporated under vacuum. After slow evaporation of a DCM/MeOH mixture, crystals are obtained and rinsed with a minimum of CH.sub.2Cl.sub.2 in order to obtain the pure product (107 mg, 47%). R.sub.f (SiO.sub.2, CH.sub.2Cl.sub.2/MeOH, 95/5)=0.55; .sup.1H NMR (300 MHz, CDCl.sub.3, δ): 8.25 (s, 2H, H.sup.3/5), 7.96 (s, 1H, NH), 7.55 (s, 1H, H.sup.9), 7.48 (d, 1H, J=8.3 Hz, H.sup.11), 7.19 (d, 1H, J=8.3 Hz, H.sup.12), 4.00 (s, 6H, H.sup.17), 2.26 (s, 3H, H.sup.13), 2.19 (s, 3H, H.sup.15); .sup.13C NMR (75 MHz, CDCl.sub.3, 6): 168.9 (C.sup.14), 165.3 (C.sup.16), 152.3 (C.sup.4), 148.3 (C.sup.2/6), 139.2 (C.sup.10), 136.2 (C.sup.8), 133.0 (C.sup.7), 130.2 (C.sup.12), 128.6 (C.sup.3/5), 121.9 (C.sup.9), 117.8 (C.sup.11), 53.3 (C.sup.17), 24.7 (C.sup.15), 20.6 (C.sup.13); HRMS (ESI) calculated for C.sub.18H.sub.19N.sub.2O.sub.5 343.1288. Exp 343.1289 [M+H].sup.+.
(35) ##STR00038##
(36) Dry dimethyl 4-iodo-2,6-pyridinedicarboxylate (144 mg, 0.45 mmol) is solubilized in dry DMF (5 mL) and 2d (195 mg, 0.50 mmol) is added under argon. The solution is placed under argon bubbling for 20 minutes then caesium fluoride (171 mg, 0.11 mmol) and Pd(PPh.sub.3).sub.4 (52 mg, 0.05 mmol) are added. The reaction mixture is placed at 80° C. under stirring and under argon for 5 days. A serial extraction AcOEt/H.sub.2O is then performed. The organic phases are washed with NaCl.sub.sat, dried over Na.sub.2SO.sub.4, filtered and evaporated under vacuum. The crude product is then purified on a chromatographic column (SiO.sub.2, CH.sub.2Cl.sub.2/acetone, 100% to 80/20) to obtain the pure product as oil (85 mg, 39%). R.sub.f (SiO.sub.2, CH.sub.2Cl.sub.2/acetone, 95/5)=0.41; .sup.1H NMR (300 MHz, CDCl.sub.3, δ): 8.08 (s, 2H, H.sup.15), 6.66 (s, 2H, H.sup.8), 4.11 (t, 2H, J=4.6 Hz, H.sup.14), 3.99 (s, 6H, H.sup.22), 3.83 (t, 2H, J=4.6 Hz, H.sup.15), 3.72-3.50 (m, 8H, H.sup.16/H.sup.17/H.sup.18/H.sup.19), 3.34 (s, 3H, H.sup.20), 1.94 (s, 6H, H.sup.13); .sup.13C NMR (75 MHz, CDCl.sub.3, δ): 165.2 (C.sup.21), 158.7 (C.sup.10), 152.5 (C.sup.4), 148.7 (C.sup.2/6), 136.5 (C.sup.8/12), 130.0 (C.sup.7), 129.6 (C.sup.3/5), 114.0 (C.sup.9/11), 72.0 (C.sup.19), 70.9 (C.sup.16), 70.7 (C.sup.17), 70.6 (C.sup.18), 69.7 (C.sup.15), 67.4 (C.sup.14), 59.0 (C.sup.20), 53.2 (C.sup.22), 21.0 (C.sup.13).
(37) ##STR00039##
(38) In a Schlenk flask, dimethyl-4-iododipicolinate (200 mg, 0.623 mmol) and 2,4-dimethoxyphenylboronic acid (125 mg, 0.685 mmol) are solubilized in dry DMF (7 mL) under argon. After 30 minutes of argon bubbling in the mixture, potassium fluoride (119 mg, 2.056 mmol) and Pd(dba).sub.3tBu.sub.3PH.BF.sub.4 (36 mg, 0.062 mmol) are added. The reaction mixture is stirred at 80° C. under argon for 19 h. The mixture is then diluted in a mixture AcOEt/Et.sub.2O then extracted with H.sub.2O then NaCl.sub.sat. The organic phase is then dried over Na.sub.2SO.sub.4, filtered and evaporated under vacuum. The brown solid obtained is finally purified on a chromatographic column (SiO.sub.2, AcOEt/EP, 20/80 to 60/40, solid deposition) in order to obtain the pure product in the form of a beige solid (131 mg, 63%). R.sub.f (SiO.sub.2, EP/AcOEt, 60/40)=0.73; .sup.1H NMR (300 MHz, CDCl.sub.3, δ): 8.50 (s, 2H, H.sup.35), 7.40 (d, 1H, J=8.5 Hz, H.sup.12), 6.63 (dd, 1H, J.sub.3=8.4 Hz, J.sub.4=2.4 Hz, H.sup.11), 6.58 (d, 1H, J=2.4 Hz, H.sup.9), 4.04 (s, 6H, H.sup.16), 3.88 (s, 3H, H.sup.13), 3.87 (s, 3H, H.sup.14); .sup.1C NMR (100 MHz, CDC.sub.3, δ): 165.8 (C.sup.15), 162.5 (C.sup.10), 158.2 (C.sup.8), 149.2 (C.sup.4), 148.1 (C.sup.2/6), 131.6 (C.sup.12), 128.4 (C.sup.3/5), 118.6 (C.sup.7), 105.5 (C.sup.9), 99.2 (C.sup.11), 55.8 (C.sup.13), 55.7 (C.sup.14), 53.4 (C.sup.16); HRMS (ESI) calculated for C.sub.17H.sub.17NNaO.sub.6 354.0948. Exp 354.0937 [M+Na].sup.+.
(39) ##STR00040##
(40) 3a (180 mg, 0.571 mmol) is solubilized in a CH.sub.2Cl.sub.2/MeOH mixture (2/3 mL) then cooled to 0° C. NaBH.sub.4 (42 mg, 1.1 mmol) is then added and after 30 minutes, the reaction mixture is placed at room temperature for 4 h. The reaction is then quenched with a 1 M HCl solution. The organic phase is washed with H.sub.2O (2×) then NaCl.sub.sat, dried over Na.sub.2SO.sub.4, filtered and evaporated. The crude product is then purified on a chromatographic column (SiO.sub.2, CH.sub.2Cl.sub.2/acetone 80/20 to 65/35) in order to obtain the pure product as pale yellow crystals (136 mg, 83%). R.sub.f (SiO.sub.2, CH.sub.2Cl.sub.2/acetone, 80/20)=0.25; .sup.1H NMR (500 MHz, CDCl.sub.3, δ): 8.01 (s, 1H, H.sup.3), 7.46 (s, 1H, H.sup.5), 7.17 (d, 1H, J=8.8 Hz, H.sup.12), 6.85-6.81 (m, 2H, H.sup.11/H.sup.9), 4.89 (d, 2H, J=4.6 Hz, H.sup.17), 4.01 (s, 3H, H.sup.16), 3.85 (s, 3H, H.sup.14), 3.31 (t, 1H, J=4.6 Hz, OH), 2.29 (s, 3H, H.sup.13); .sup.13C NMR (125 MHz, CDCl.sub.3, δ): 165.9 (C.sup.15), 160.1 (C.sup.6, C.sup.10), 151.8 (C.sup.4), 147.1 (C.sup.2), 136.9 (C.sup.8), 130.9 (C.sup.7), 130.8 (C.sup.12), 125.0 (C.sup.3), 124.6 (C.sup.5), 116.5 (C.sup.9), 111.9 (C.sup.11), 64.9 (C.sup.17), 55.5 (C.sup.14), 53.1 (C.sup.16), 20.8 (C.sup.13); HRMS (ESI) calculated for C.sub.16H.sub.8NO.sub.4 288.1230. Exp 288.1220 [M+H].sup.+. Calc for C.sub.16H.sub.17NNaO.sub.4 310.1050. Exp 310.1038 [M+Na].sup.+.
(41) ##STR00041##
(42) 3b (320 mg, 0.72 mmol) is solubilized in a CH.sub.2Cl.sub.2/MeOH mixture (2.5 mL/4.5 mL). After having placed the mixture at 0° C., NaBH.sub.4 (30 mg, 0.79 mmol) is added and after 30 minutes, the solution is placed at room temperature under stirring for 3 h. The reaction is quenched by adding 1 M HCl solution. Then a CH.sub.2Cl.sub.2/H.sub.2O extraction is carried out. The organic phase is washed with NaCl.sub.sat, dried over Na.sub.2SO.sub.4, filtered and evaporated. A purification on a chromatographic column (SiO.sub.2, CH.sub.2Cl.sub.2/MeOH, 98/2 to 97/3) is carried out in order to obtain the pure product as colourless oil (270 mg, 90%). R.sub.f (SiO.sub.2, CH.sub.2Cl.sub.2/MeOH, 97/3)=0.22; .sup.1H NMR (500 MHz, CDCl.sub.3, δ): 7.99 (d, 1H, J=0.9 Hz, H.sup.3), 7.47 (s, 1H, H.sup.5), 7.15 (d, 1H, J=8.4 Hz, H.sup.12), 6.85 (d, 1H, J=2.3 Hz, H.sup.9), 6.83 (dd, 1H, J.sub.3=8.4 Hz, J.sub.4=2.3 Hz, H.sup.11), 4.89 (d, 2H, J=3.9 Hz, H.sup.23), 4.16 (t, 2H, J=4.7 Hz, H.sup.14), 3.99 (s, 3H, H.sup.2), 3.87 (t, 2H, J=4.7 Hz, H.sup.15), 3.72-3.54 (m, 8H, H.sup.16/H.sup.17/H.sup.18/H.sup.19), 3.51 (m, 1H, OH), 3.37 (s, 3H, H.sup.20), 2.27 (s, 3H, H.sup.13); .sup.13C NMR (125 MHz, CDCl.sub.3, δ): 165.9 (C.sup.21), 160.3 (C.sup.6), 159.3 (C.sup.10), 151.8 (C.sup.4), 147.1 (C.sup.2), 136.8 (C.sup.8), 131.0 (C.sup.7), 130.8 (C.sup.12), 125.0 (C.sup.3), 124.6 (C.sup.5), 117.2 (C.sup.9), 112.5 (C.sup.11), 72.1 (C.sup.19), 71.0 (C.sup.16), 70.9 (C.sup.17), 70.8 (C.sup.18), 69.9 (C.sup.15), 67.6 (C.sup.14), 64.9 (C.sup.23), 59.2 (C.sup.20), 53.1 (C.sup.22), 20.8 (C.sup.13); HRMS (ESI) calculated for C.sub.22H.sub.30NO.sub.7 420.2017. Exp 420.2015 [M+H].sup.+.
(43) ##STR00042##
(44) 3c is solubilized in a CH.sub.2Cl.sub.2/MeOH mixture and NaBH.sub.4 is added at 0° C. After 5 minutes, the solution is left at room temperature and the monoreduction is monitored by TLC (SiO.sub.2, DCM/MeOH, 95/5). At the end of 2.5 h, the reaction is quenched with 1 M HCl. The organic phase is then washed with H.sub.2O and NaCl.sub.sat, then dried over Na.sub.2SO.sub.4, filtered and evaporated. The crude product is purified on a chromatographic column (SiO.sub.2, DCM/MeOH, 95/5) and a white powder is obtained (56 mg, 58%). R.sub.f (SiO.sub.2, CH.sub.2Cl.sub.2/MeOH, 95/5)=0.30; .sup.1H NMR (300 MHz, CDCl.sub.3, δ): 7.98 (s, 1H, H.sup.3), 7.48 (m, 2H, H.sup.4/H.sup.9/NH), 7.42 (d, 1H, J=8.3 Hz, H.sup.11), 7.17 (d, 1H, J=8.3 Hz, H.sup.12), 4.90 (s, 2H, H.sup.18), 4.00 (s, 6H, H.sup.17), 3.57 (s, 1H, OH), 2.26 (s, 3H, H.sup.13), 2.20 (s, 3H, H.sup.15); .sup.13C NMR (75 MHz, CDCl.sub.3, δ): 168.7 (C.sup.14), 165.8 (C.sup.16), 160.4 (C.sup.6), 151.4 (C.sup.4), 147.1 (C.sup.2), 138.6 (C.sup.10), 136.2 (C.sup.8), 134.2 (C.sup.7), 130.2 (C.sup.12), 124.7 (C.sup.3), 124.4 (C.sup.5), 121.9 (C.sup.9), 117.7 (C.sup.11), 64.9 (C.sup.18), 53.1 (C.sup.17), 24.8 (C.sup.15), 20.6 (C.sup.13); HRMS (ESI) calculated for C.sub.17H.sub.19N.sub.2O.sub.4 315.1339. Exp 315.1342 [M+H].sup.+.
(45) ##STR00043##
(46) 3d (136 mg, 0.29 mmol) is solubilized in a CH.sub.2Cl.sub.2/MeOH mixture (50/50, 4 mL). After having placed the mixture at 0° C., NaBH.sub.4 (42 mg, 1.11 mmol) is added (brown colouring of the solution) and after 20 minutes, the solution is placed at room temperature under stirring for 6 h. The reaction is quenched by adding 1 M HCl solution (4 mL). Then a CH.sub.2Cl.sub.2/H.sub.2O extraction is carried out. The organic phase is washed with NaCl.sub.sat, dried over Na.sub.2SO.sub.4, filtered and evaporated. A purification on a chromatographic column (SiO.sub.2, CH.sub.2Cl.sub.2/AcOEt, 70/30 to 50/50 then 10% MeOH) is carried out in order to obtain the pure product (123 mg, 98%). R.sub.f (SiO.sub.2, CH.sub.2Cl.sub.2/AcOEt, 80/20)=0.32; .sup.1H NMR (300 MHz, CDCl.sub.3, δ): 8.08 (s, 1H, H.sup.3), 7.79 (s, 1H, H.sup.5), 6.63 (s, 2H, H.sup.9), 4.87 (s, 2H, H.sup.23), 4.09 (t, 2H, J=4.6 Hz, H.sup.14), 3.93 (s, 3H, H.sup.2), 3.81 (t, 2H, J=4.6 Hz, H.sup.15), 3.72-3.49 (m, 8H, H.sup.16/H.sup.17/H.sup.18/H.sup.19), 3.33 (s, 3H, H.sup.2), 1.93 (s, 6H, H.sup.13); .sup.13C NMR (75 MHz, CDCl.sub.3, δ): 165.6 (C.sup.21), 161.0 (C.sup.6), 158.2 (C.sup.10), 151.4 (C.sup.4), 147.1 (C.sup.2), 136.4 (C.sup.8/12), 130.9 (C.sup.7), 125.2 (C.sup.3/5), 113.6 (C.sup.9/11), 71.8 (C.sup.19), 70.7 (C.sup.16), 70.5 (C.sup.17), 70.4 (C.sup.18), 69.6 (C.sup.15), 67.2 (C.sup.14), 64.6 (C.sup.23), 58.9 (C.sup.20), 52.7 (C.sup.22), 20.8 (C.sup.13); HRMS (ESI) calculated for C.sub.23H.sub.32NO.sub.7 434.2173. Exp 434.2158 [M+H].sup.+.
(47) ##STR00044##
(48) 3e (120 mg, 0.36 mmol) is solubilized in a CH.sub.2Cl.sub.2/MeOH mixture (2/3 mL) then cooled to 0° C. NaBH.sub.4 (15 mg, 0.40 mmol) is then added and after 15 minutes, the reaction mixture is placed at room temperature for 4 h. The reaction is then quenched with a 1 M HCl solution. The organic phase is washed with H.sub.2O (2×) then NaCl.sub.sat, dried over Na.sub.2SO.sub.4, filtered and evaporated. The crude product is then purified on a chromatographic column (SiO.sub.2, CH.sub.2Cl.sub.2/acetone 80/20) in order to obtain the pure product in the form of a beige solid (53 mg, 56%). R.sub.f (SiO.sub.2, CH.sub.2Cl.sub.2/acetone, 80/20)=0.17; .sup.1H NMR (300 MHz, CDCl.sub.3, δ): 8.21 (s, 1H, H.sup.3), 7.67 (s, 1H, H.sup.5), 7.33 (d, 1H, J=8.3 Hz, H.sup.12), 6.60 (dd, 1H, J.sub.3=8.3 Hz, J.sub.4=2.4 Hz, H.sup.11), 6.57 (d, 1H, J=2.4 Hz, H.sup.9), 4.87 (d, 2H, J=4.5 Hz, H.sup.17), 4.00 (s, 3H, H.sup.16), 3.87 (s, 3H, H.sup.13), 3.84 (s, 3H, H.sup.14), 3.42 (br, 1H, OH).
(49) ##STR00045##
(50) 4a (126 mg, 0.439 mmol) is solubilized in dry CH.sub.2Cl.sub.2 (4 mL) and triethylamine (180 μL, 1.317 mmol) is added. After having placed the mixture at 0° C., mesyl chloride (50 μL, 0.702 mmol) is added and after 5 minutes, the reaction mixture is placed at room temperature for 30 minutes. The organic phase is washed with H.sub.2O, then dried over Na.sub.2SO.sub.4, filtered and evaporated to give a yellow oil used with purification for the following step (137 mg, 85%). R.sub.f (SiO.sub.2, CH.sub.2Cl.sub.2/MeOH, 95/5)=0.83; .sup.1H NMR (300 MHz, CDCl.sub.3, δ): 8.08 (s, 1H, H.sup.3), 7.62 (s, 1H, H.sup.5), 7.18 (m, 1H, H.sup.12), 6.86-6.82 (m, 2H, H.sup.11/H.sup.9), 5.47 (s, 2H, H.sup.17), 4.02 (s, 3H, H.sup.16), 3.85 (s, 3H, H.sup.14), 3.16 (s, 3H, H.sup.18), 2.30 (s, 3H, H.sup.13).
(51) ##STR00046##
(52) 4b (282 mg, 0.67 mmol) is solubilized in CH.sub.2Cl.sub.2 (8 mL) then triethylamine (280 μL, 2.02 mmol) is added. The mixture is placed at 0° C. then mesyl chloride (80 μL, 1.08 mmol) is added. After 5 minutes, the reaction mixture is placed at room temperature for 30 minutes. The organic phase is washed with H.sub.2O then the aqueous phases are re-extracted with CH.sub.2Cl.sub.2. The organic phases are combined, dried over Na.sub.2SO.sub.4, filtered and evaporated under vacuum. The crude product is purified on a chromatographic column (SiO.sub.2, CH.sub.2Cl.sub.2/MeOH, 98/2 to 96/4) in order to obtain the pure compound as a pale yellow oil (304 mg, 91%). R.sub.f (SiO.sub.2, CH.sub.2Cl.sub.2/MeOH, 95/5)=0.49; .sup.1H NMR (500 MHz, CDCl.sub.3, δ): 8.08 (d, 1H, J=1.5 Hz, H.sup.3), 7.62 (d, 1H, J=1.5 Hz, H.sup.5), 7.16 (d, 1H, J=8.3 Hz, H.sup.12), 6.86 (d, 1H, J=2.6 Hz, H.sup.9), 6.84 (dd, 1H, J.sub.3=8.3 Hz, J.sub.4=2.6 Hz, H.sup.11), 5.46 (s, 2H, H.sup.2), 4.17 (t, 2H, J=4.7 Hz, H.sup.14), 4.01 (s, 3H, H.sup.2), 3.88 (t, 2H, J=4.7 Hz, H.sup.15), 3.76-3.55 (m, 8H, H.sup.16/H.sup.17/H.sup.18/H.sup.19), 3.38 (s, 3H, H.sup.20), 3.16 (s, 3H, H.sup.24), 2.28 (s, 3H, H.sup.13); .sup.13C NMR (125 MHz, CDCl.sub.3, δ): 165.6 (C.sup.21), 159.5 (C.sup.10), 154.4 (C.sup.6), 152.4 (C.sup.4), 147.8 (C.sup.2), 136.9 (C.sup.8), 130.8 (C.sup.12), 130.5 (C.sup.7), 125.90 (C.sup.3), 125.88 (C.sup.5), 117.3 (C.sup.9), 112.6 (C.sup.11), 72.1 (C.sup.19), 71.2 (C.sup.23), 70.9 (C.sup.16), 70.8 (C.sup.17), 70.1 (C.sup.18), 69.9 (C.sup.15), 67.8 (C.sup.14), 59.2 (C.sup.20), 53.3 (C.sup.22), 38.3 (C.sup.24), 20.8 (C.sup.13); HRMS (ESI) calculated for C.sub.23H.sub.32NO.sub.9S 498.1792. Exp 498.1779 [M+H].sup.+.
(53) ##STR00047##
(54) 4c (56 mg, 0.18 mmol) is solubilized in CH.sub.2Cl.sub.2 (4 mL) then triethylamine 75 μL, 0.53 mmol) is added. The mixture is placed at 0° C. and mesyl chloride (21 μL, 0.27 mmol) is added. After 5 minutes, the reaction is placed at room temperature under stirring for 40 minutes. A washing of the organic phase with H.sub.2O is then carried out and the aqueous phases are re-extracted with CH.sub.2Cl.sub.2. The organic phases are combined, dried over Na.sub.2SO.sub.4, filtered and evaporated under vacuum. .sup.1H NMR shows the obtaining of the pure product quantitatively (70 mg) and is used without further purification for the following step. R.sub.f (SiO.sub.2, CH.sub.2Cl.sub.2/MeOH, 95/5)=0.64; .sup.1H NMR (300 MHz, CDCl.sub.3, δ): 8.04 (s, 1H, H.sup.3), 7.89 (s, 1H, NH), 7.59 (s, 1H, H.sup.45), 7.51 (s, 1H, H.sup.9), 7.46 (d, 1H, J=8.3 Hz, H.sup.11), 7.16 (d, 1H, J=8.3 Hz, H.sup.12), 5.43 (s, 2H, H.sup.18), 3.99 (s, 6H, H.sup.17), 3.14 (s, 3H, H.sup.19), 2.25 (s, 3H, H.sup.13), 2.19 (s, 3H, H.sup.15).
(55) ##STR00048##
(56) 4d (123 mg, 0.28 mmol) is solubilized in CH.sub.2Cl.sub.2 (5 mL) then triethylamine (192 μL, 1.38 mmol) is added. The mixture is placed at 0° C. then mesyl chloride (53 μL, 0.68 mmol) is added. After 5 minutes, the reaction mixture is placed at room temperature for 30 minutes. The organic phase is washed with H.sub.2O then the aqueous phases are re-extracted with CH.sub.2Cl.sub.2. The organic phases are combined, dried over Na.sub.2SO.sub.4, filtered and evaporated under vacuum. The product obtained is used without further purification for the following step. R.sub.f (SiO.sub.2, CH.sub.2Cl.sub.2/MeOH, 95/5)=0.76; .sup.1H NMR (300 MHz, CDC.sub.3, δ): 7.90 (s, 1H, H.sup.3), 7.45 (s, 1H, H.sup.5), 6.66 (s, 2H, H.sup.9), 5.44 (s, 2H, H.sup.23), 4.12 (t, 2H, J=4.6 Hz, H.sup.14), 3.97 (s, 3H, H.sup.2), 3.83 (t, 2H, J=4.6 Hz, H.sup.15), 3.74-3.51 (m, 8H, H.sup.16/H.sup.17/H.sup.18/H.sup.19), 3.35 (s, 3H, H.sup.20), 3.13 (s, 3H, H.sup.24), 1.95 (s, 6H, H.sup.13); .sup.13C NMR (75 MHz, CDCl.sub.3, δ): 165.3 (C.sup.21), 158.6 (C.sup.10), 154.7 (C.sup.6), 152.3 (C.sup.4), 148.0 (C.sup.2), 136.5 (C.sup.8/12), 130.4 (C.sup.7), 126.6/126.5 (C.sup.3/C.sup.5), 113.9 (C.sup.9/11), 71.9 (C19), 70.9 (C.sup.16), 70.7 (C.sup.17), 70.6 (C.sup.18), 69.7 (C.sup.15), 67.4 (C.sup.14), 59.0 (C.sup.20), 53.1 (C.sup.22), 38.1 (C.sup.23), 31.6 (C.sup.24), 21.0 (C.sup.13).
(57) ##STR00049##
(58) 4e (60 mg, 0.198 mmol) is solubilized in CH.sub.2Cl.sub.2 (3 mL) and triethylamine (83 μL, 0.593 mmol) is added. After having placed the mixture at 0° C., mesyl chloride (24 μL, 0.317 mmol) is added and after 5 minutes, the reaction mixture is placed at room temperature for 30 minutes. The organic phase is washed with H.sub.2O, then dried over Na.sub.2SO.sub.4, filtered and evaporated to give a yellow oil used with purification for the following step (76 mg, quant). R.sub.f (SiO.sub.2, CH.sub.2Cl.sub.2/MeOH, 95/5)=0.76; .sup.1H NMR (300 MHz, CDCl.sub.3, δ): 8.29 (s, 1H, H.sup.3), 7.86 (s, 1H, H.sup.5), 7.35 (d, 1H, J=8.4 Hz, H.sup.12), 6.61 (dd, 1H, J.sub.3=8.4 Hz, J.sub.4=2.3 Hz, H.sup.11), 6.57 (d, 1H, J=2.3 Hz, H.sup.9), 5.46 (s, 2H, H.sup.17), 4.02 (s, 3H, H.sup.16), 3.87 (s, 3H, H.sup.13), 3.85 (s, 3H, H.sup.14), 3.14 (s, 3H, H.sup.18).
(59) ##STR00050##
(60) Triazacyclononane (27 mg, 0.115 mmol) and sodium carbonate (122 mg, 1.15 mmol) are solubilized in dry acetonitrile (7 mL). After 10 minutes at room temperature, 5a (130 mg, 0.356 mmol) is added and the reaction mixture is heated to 60° C. under stirring for 24 h then at room temperature for 24 h. The mixture is then filtered on sintered P3, rinsed with dry CH.sub.3CN and the mother liquors are evaporated. The crude product is purified on a chromatographic column (Al.sub.2O.sub.3, CH.sub.2Cl.sub.2/MeOH, 99/1 to 98/2) in order to obtain the pure product in the form of a pale yellow solid (81 mg, 75%). R.sub.f (Al.sub.2O.sub.3, CH.sub.2Cl.sub.2/MeOH, 95/5)=0.58; .sup.1H NMR (500 MHz, CDCl.sub.3, δ): 7.94 (s, 3H, H.sup.3), 7.68 (s, 3H, H.sup.5), 7.09 (d, 3H, J=8.2 Hz, H.sup.12), 6.79-6.75 (m, 6H, H.sup.9/H.sup.11), 3.98 (s, 9H, H.sup.16), 3.97 (s, 6H, H.sup.17), 3.83 (s, 9H, H.sup.14), 2.92 (s, 12H, H.sup.18), 2.23 (s, 9H, H.sup.13); .sup.13C NMR (125 MHz, CDCl.sub.3, δ): 166.2 (C.sup.15), 161.1 (C.sup.6), 159.9 (C.sup.10), 151.2 (C.sup.4), 147.3 (C.sup.2), 136.7 (C.sup.8), 131.1 (C.sup.7), 130.8 (C.sup.12), 127.0 (C.sup.5), 124.7 (C.sup.3), 116.3 (C.sup.9), 111.8 (C.sup.11), 64.8 (C.sup.17), 56.0 (C.sup.18), 55.4 (C.sup.14), 53.1 (C.sup.16), 20.9 (C.sup.13); HRMS (ESI) calculated for C.sub.54H.sub.62N.sub.6O.sub.9 469.2284. Exp 469.2273 [M+2H].sup.++.
(61) ##STR00051##
(62) Triazacyclononane (93 mg, 0.39 mmol) and sodium carbonate (414 mg, 3.91 mmol) are solubilized in dry acetonitrile (24 mL) under argon. After 10 minutes at room temperature, 5b (289 mg, 1.21 mmol) is added and the reaction mixture is heated to 60° C. under stirring for 24 h then at room temperature for 24 h. The mixture is then filtered on sintered P3, rinsed with dry CH.sub.3CN and the mother liquors are evaporated. The crude product is purified on a chromatographic column (Al.sub.2O.sub.3, CH.sub.2Cl.sub.2/MeOH, 99/1 to 95/5) in order to obtain the pure product in the form of a yellow oil (176 mg, 34%). .sup.1H NMR (500 MHz, CDC.sub.3, δ): 7.92 (d, 3H, J=1.1 Hz, H.sup.3), 7.67 (s, 3H, H.sup.5), 7.07 (d, 1H, J=8.4 Hz, H.sup.12), 6.81 (d, 1H, J=2.4 Hz, H.sup.9), 6.77 (dd, 1H, J.sub.3=8.4 Hz, J.sub.4=2.4 Hz, H.sup.11), 4.14 (t, 6H, J=4.6 Hz, H.sup.4), 3.96 (br s, 15H, H.sup.23/H.sup.22), 3.86 (t, 6H, J=4.6 Hz, H.sup.15), 3.74-3.53 (m, 24H, H.sup.16/H.sup.17/H.sup.18/H.sup.19), 3.37 (s, 9H, H.sup.20), 2.91 (br s, 12H, H.sup.24), 2.21 (s, 9H, H.sup.13); .sup.13C NMR (125 MHz, CDCl.sub.3, δ): 166.2 (C.sup.21), 161.0 (C.sup.6), 159.2 (C.sup.10), 151.1 (C.sup.4), 147.3 (C.sup.2), 136.7 (C.sup.8), 131.3 (C.sup.7), 130.7 (C.sup.12), 127.0 (C.sup.5), 124.7 (C.sup.3), 117.0 (C.sup.9), 113.8 (C.sup.11), 72.1 (C.sup.19), 71.0 (C.sup.16), 70.8 (C.sup.17), 70.7 (C.sup.18), 69.8 (C.sup.15), 67.6 (C.sup.14), 64.7 (C.sup.23), 59.2 (C.sup.20), 55.9 (C.sup.24), 53.0 (C.sup.22), 20.8 (C.sup.13); HRMS (ESI) calculated for C.sub.72H.sub.98N.sub.6O.sub.18 667.3463 Exp 667.34558 [M+2H].sup.2+.
(63) ##STR00052##
(64) 5c dry (70 mg, 0.18 mmol) is solubilized in dry acetonitrile (3 mL) and Na.sub.2CO.sub.3 (67 mg, 0.63 mmol) is added as well as triazacyclononane (13.6 mg, 0.06 mmol). After argon bubbling in the mixture for 5 min, the reaction mixture is heated at 60° C. for 5 days. The mixture is then filtered on sintered P3, rinsed with dry CH.sub.3CN and the mother liquors are evaporated. The crude product is purified on a chromatographic column (Al.sub.2O.sub.3, CH.sub.2Cl.sub.2/MeOH, 96/4) in order to obtain the pure product in the form of a white powder (47 mg, 81%). .sup.1H NMR (300 MHz, CDCl.sub.3, δ): 8.94 (s, 3H, NH), 7.88 (s, 3H, H.sup.3), 7.54-7.45 (m, 9H, H.sup.8/H.sup.9/H.sup.10), 7.07 (d, 1H, J=8.3 Hz, H.sup.12), 3.98 (m, 15H, H.sup.18/H.sup.17), 2.87 (s, 12H, H.sup.19), 2.25 (s, 9H, H.sup.13), 2.09 (s, 9H, H.sup.15); .sup.13C NMR (75 MHz, MeOD, δ): 171.8 (C.sup.14), 166.9 (C.sup.16), 162.4 (C.sup.6), 152.8 (C.sup.4), 148.3 (C.sup.2), 140.7 (C.sup.10), 136.9 (C.sup.8), 134.9 (C.sup.7), 131.0 (C.sup.12), 128.6 (C.sup.3), 124.4 (C.sup.5), 123.0 (C.sup.9), 118.9 (C.sup.11), 65.0 (C.sup.18), 57.0 (C.sup.19), 53.3 (C.sup.17), 24.05 (C.sup.15), 20.9 (C.sup.13); HRMS (ESI) calculated for C.sub.57H.sub.64N.sub.9O.sub.9 1018.4822. Exp 1018.4782 [M+H].sup.+: t.sub.R=8.33 min (method HPLC-MM-ACN, H.sub.2O/CH.sub.3CN 85/15 to 0/100 in 16 min).
(65) ##STR00053##
(66) Triazacyclononane (21.5 mg, 0.09 mmol) and sodium carbonate (95 mg, 0.90 mmol) are solubilized in dry acetonitrile (5 mL). After 5 minutes at room temperature, 5d (143 mg, 0.28 mmol) is added, argon bubbling is carried out for 5 minutes and the reaction mixture is heated to 60° C. under stirring for 4 days. The mixture is then filtered on sintered P3, rinsed with dry CH.sub.3CN and the mother liquors are evaporated. The crude product is purified on a chromatographic column (Al.sub.2O.sub.3, CH.sub.2Cl.sub.2/MeOH, 100% to 97/3) in order to obtain the pure product in the form of a white powder (82 mg, 66%). .sup.1H NMR (300 MHz, CDCl.sub.3, δ): 7.77 (s, 3H, H.sup.3), 7.47 (s, 3H, H.sup.5), 6.64 (s, 6H, H.sup.9), 4.12 (t, 6H, J=4.7 Hz, H.sup.14), 3.94 (s, 9H, H.sup.2), 3.91 (s, 6H, H.sup.3), 3.84 (t, 6H, J=4.7 Hz, H.sup.15), 3.74-3.52 (m, 24H, H.sup.16/H.sup.17/H.sup.18/H.sup.19), 3.35 (s, 9H, H.sup.20), 2.82 (s, 12H, H.sup.24), 1.91 (s, 18H, H.sup.13); .sup.13C NMR (75 MHz, CDCl.sub.3, δ): 166.0 (C.sup.21), 161.4 (C.sup.6), 158.3 (C.sup.10), 151.0 (C.sup.4), 147.5 (C.sup.2), 136.5 (C.sup.8/12), 131.3 (C.sup.7), 127.8 (C.sup.5), 125.3 (C.sup.3), 113.8 (C.sup.9/11), 72.0 (C.sup.19), 70.9 (C.sup.16), 70.7 (C.sup.17), 70.6 (C.sup.18), 69.8 (C.sup.15), 67.4 (C.sup.14), 64.6 (C.sup.23), 59.1 (C.sup.20), 55.6 (C.sup.24), 52.9 (C.sup.22), 21.0 (C.sup.13); HRMS (ESI) calculated for C.sub.75H.sub.104N.sub.6O.sub.18 688.3698. Exp 688.3686 [M+2H].sup.2+; t.sub.R=13.09 min (method HPLC-MM-ACN, H.sub.2O/CH.sub.3CN 85/15 to 0/100 in 16 min).
(67) ##STR00054##
(68) Triazacyclononane (15 mg, 0.064 mmol) and sodium carbonate (68 mg, 0.64 mmol) are solubilized in dry acetonitrile (4 mL). After 10 minutes at room temperature, 5e (76 mg, 0.198 mmol) is added and the reaction mixture is heated to 60° C. under stirring for 24 h then at room temperature for 5 days. The mixture is then filtered on sintered P3, rinsed with dry CH.sub.3CN and the mother liquors are evaporated. The crude product is purified on a chromatographic column (Al.sub.2O.sub.3, CH.sub.2C.sub.2/MeOH, 100% to 97/3) in order to obtain the pure product in the form of a white powder (41 mg, 65%). R.sub.f (Al.sub.2O.sub.3, CH.sub.2C.sub.2/MeOH, 95/5)=0.54; .sup.1H NMR (500 MHz, CDCl.sub.3, δ): 8.15 (s, 3H, H.sup.3), 7.92 (s, 3H, H.sup.5), 7.25 (d, 3H, J=8.0 Hz, H.sup.12), 6.54-6.49 (m, 6H, H.sup.9/H.sup.11), 3.99 (s, 6H, H.sup.17), 3.97 (s, 9H, H.sup.16), 3.84 (s, 9H, H.sup.13), 3.75 (s, 9H, H.sup.14), 2.99 (s, 12H, H.sup.18); .sup.13C NMR (125 MHz, CDCl.sub.3, δ): 166.5 (C.sup.15), 161.9 (C.sup.10), 160.9 (C.sup.6), 158.0 (C.sup.8), 147.8 (C.sup.4), 147.2 (C.sup.2), 131.4 (C.sup.12), 126.6 (C.sup.5), 124.4 (C.sup.3), 119.9 (C.sup.7), 105.3 (C.sup.9), 99.2 (C.sup.11), 65.0 (C.sup.17), 56.2 (C.sup.18), 55.7 (C.sup.13), 55.6 (C.sup.14), 53.0 (C.sup.16).
(69) ##STR00055##
(70) 6c (47 mg, 0.05 mmol) is dissolved in THF (3 mL) then 1 M NaOH (3 mL) is added to pH>12. The reaction mixture is stirred at room temperature for 24 h. LC-MS monitoring is used to observe the complete deprotection of the methyl esters. The product is used for the complexation without further purification. HRMS (ESI) calculated for C.sub.54H.sub.55N.sub.9O.sub.9 976.4352. Exp 976.4306 [M+H].sup.+; t=6.24 min (method HPLC-MM-ACN, H.sub.2O/CH.sub.3CN 85/15 to 0/100 in 16 min).
(71) ##STR00056##
(72) 6d (38.7 mg, 0.028 mmol) is dissolved in tetrahydrofuran (3 mL) and 1 M NaOH (3 mL) is added to pH 14. The reaction mixture is stirred at room temperature for 6.5 h. Mass spectrometric monitoring shows the disappearance of the starting material. The mixture is used for the complexation step without further purification. HRMS (ESI) calculated for C.sub.75H.sub.98N.sub.6O.sub.18 667.3463. Exp 667.3461 [M+2H].sup.2+; t.sub.R=9.57 min (method HPLC-ESI-ACN, H.sub.2O/CH.sub.3CN 85/15 to 0/100 in 16 min).
(73) ##STR00057##
(74) The ligand 6a (70 mg, 0.075 mmol) is dissolved in THF (10 mL) then 1 M NaOH (5 mL) is added and the solution is stirred at room temperature for 1.5 h. The pH is then adjusted to pH 4 with 1 M HCl (5 mL) and the mixture is concentrated under vacuum. The mixture is diluted with MeOH then the pH is adjusted to pH 8-9 aided by a solution of Na.sub.2CO.sub.3sat. Finally, TbCl.sub.3.6H.sub.2O (84 mg, 0.224 mmol) is added and the reaction mixture is stirred at room temperature for 72 h. The mixture is concentrated under reduced pressure then the complex is precipitated by adding H.sub.2O (2×) and centrifuged to obtain a white solid corresponding to the terbium(III) complex (79 mg, quant). HRMS (ESI) calculated for C.sub.51H.sub.53N.sub.6O.sub.9Tb 526.1558. Exp 526.1557 [M+2H].sup.++. Calc for C.sub.51H.sub.52N.sub.6O.sub.9Tb 1051.3044. Exp 1051.3011 [M+H].sup.+.
(75) ##STR00058##
(76) The ligand 6b (50 mg, 0.038 mmol) is solubilized in MeOH (5 mL) then 1 M NaOH (2 mL) is added and the solution is left under stirring at room temperature for 1 h. Then the pH is adjusted to pH 3˜4 with 1 M HCl, then to pH ˜7 with Na.sub.2CO.sub.3sat. TbCl.sub.3.6H.sub.2O (21 mg, 0.056 mmol) is added and the solution is left at room temperature overnight. After concentration under reduced pressure, the mixture is purified by extractions/washes with DCM/H.sub.2O, and the organic phase evaporated to give the product in the form of a pale yellow solid (55 mg, quant.). HRMS (ESI) calculated for C.sub.69H.sub.87N.sub.6Na.sub.2O.sub.18Tb 746.2557. Exp 746.2565 [M+2Na].sup.2+; t.sub.R=9.65 min (method HPLC-MM, H.sub.2O/CH.sub.3CN 85/15 to 0/100 in 16 min).
(77) ##STR00059##
(78) The pH of the solution of 7c is adjusted to pH b by adding solution of 1 M HCl then TbCl.sub.3.6H.sub.2O is added at room temperature. The reaction mixture is stirred for 48 h then THF is evaporated under reduced pressure. The precipitate formed is centrifuged in H.sub.2O (3×) to remove excess salts present then the white solid is dried under vacuum (43 mg, 83%). HRMS (ESI) calculated for C.sub.54H.sub.55N.sub.9O.sub.9Tb 1132.3371. Exp 1132.3323 [M+H].sup.+, C.sub.54H.sub.54N.sub.9O.sub.9TbNa 1154.3190, Exp 1154.3133 [M+Na].sup.+; t.sub.R=6.67 min (method HPLC-MM-ACN, H.sub.2O/CH.sub.3CN 85/15 to 0/100 in 16 min).
(79) ##STR00060##
(80) The pH of the solution 7d is adjusted to pH 6 with 1 M HCl then TbCl.sub.3.6H.sub.2O (12 mg, 0.031 mmol) is added and the solution is left under stirring at room temperature for 6 days. THF is then evaporated and a CH.sub.2Cl.sub.2/H.sub.2O extraction is carried out. The organic phases are combined and evaporated to obtain the pure terbium(III) complex (38 mg, 90%). HRMS (ESI) calculated for C.sub.2H.sub.95N.sub.6O.sub.18Tb 745.2973. Exp 745.2966 [M+2H].sup.2+, C.sub.72H.sub.94N.sub.6O.sub.18TbNa 756.2882. Exp 756.2882 [M+H+Na]+; t.sub.R=9.64 min (method HPLC-ESI-ACN-365, H.sub.2O/CH.sub.3CN 85/15 to 0/100 in 16 min).
(81) ##STR00061##
(82) The ligand 6e (31 mg, 0.032 mmol) is dissolved in MeOH (5 mL) then 1 M NaOH (2 mL) is added and the solution is stirred at room temperature for 2 h. The pH is then adjusted to pH 3-4 with 1 M HCl (2 mL) then to pH 7 aided by a solution of Na.sub.2CO.sub.3sat. ThCl.sub.3.6H.sub.2O (18 mg, 0.047 mmol) is finally added and the reaction mixture is stirred at room temperature for 14 h. The mixture is concentrated under reduced pressure then the complex is precipitated by adding H.sub.2O (2×) and centrifuged. Then the solid obtained is again precipitated in Et.sub.2O and centrifuged to obtain a new white precipitate corresponding to the terbium(III) complex (30 mg, 85%). MS Calc for C.sub.51H.sub.53N.sub.6O.sub.12Tb 550.1488. Exp 550.1482 [M+2H].sup.2+; calc for C.sub.51H.sub.52N.sub.6NaO.sub.12Tb 561.1392. Exp 561.1405 [M+H+Na].sup.2+; calc for C.sub.51H.sub.51N.sub.6Na.sub.2O.sub.12Tb 572.1301. Exp 572.1323 [M+2Na].sup.2+.
Example 2: Dysprosium Complex Ia.5 and Ia.6
(83) ##STR00062##
(84) The ligand 6b (50 mg, 0.038 mmol) is solubilized in MeOH (5 mL) then 1 M NaOH (2 mL) is added and the solution is left under stirring at room temperature for 1 h. Then the pH is adjusted to pH 3˜4 with 1 M HCl, then to pH ˜7 with Na.sub.2CO.sub.3sat. Dy(NO.sub.3).sub.3.5H.sub.2O (20 mg, 0.056 mmol) is added and the solution is left at room temperature overnight. After concentration under reduced pressure, the mixture is purified by extractions/washes with DCM/H.sub.2, and the organic phase evaporated to give the product in the form of a pale yellow solid (50 mg, 92%). HRMS (ESI) calculated for C.sub.69H.sub.87DyN.sub.6Na.sub.2O.sub.18 748.7577. Exp 748.7576 [M+2Na].sup.2+.
(85) ##STR00063##
(86) 6d (9 mg, 0.007 mmol) is dissolved in tetrahydrofuran (2 mL) and 1 M NaOH (2 mL) is added to pH 14. The reaction mixture is stirred at room temperature for 24 h. The pH of the solution is then adjusted to pH 6 with 1 M HCl then DyCl.sub.3.6H.sub.2O (3.2 mg, 0.008 mmol) is added and the solution is left under stirring at room temperature for 3 days. THF is then evaporated and a CH.sub.2Cl.sub.2/H.sub.2O extraction is carried out. The organic phases are combined and evaporated to obtain the pure dysprosium(III) complex (8 mg, 77%). HRMS (ESI) calculated for C.sub.72H.sub.93N.sub.6O.sub.18DyNa.sub.2 769.7811. Exp 769.7818 [M+2Na].sup.2+, C.sub.72H.sub.93N.sub.6O.sub.18DyNa.sub.3 520.8505. Exp 520.8521 [M+3Na].sup.3+; t.sub.R=9.57 min (method HPLC-ESI-ACN-365, H.sub.2O/CH.sub.3CN 85/15 to 0/100 in 16 min).
B. Evaluation of the Complexes
Example 3: Spectroscopic Properties
(87) The properties of complexes Ia.1-Ia.6 and 8 were evaluated in methanol (Table 1) and water (Table 2). These properties were compared to those of complex 9, described in the document, Dalton Trans. 2015, 44, 4918, and of the following structure:
(88) ##STR00064##
(89) Photophysical measurements of the complexes show that terbium complexes Ia.1-Id.4 according to the invention have an intense absorption in methanol (Table 1).
(90) Moreover, these complexes have very high quantum yields, and much higher than that of the complex 9 outside the invention, which has no substituent other than alpha hydrogen of the pyridine-phenyl bond (positions R2 and R3). The strong limitation, or even suppression, of internal rotation around the pyridine-phenyl bond thus significantly improves quantum efficiency.
(91) The brightness of the complexes according to the invention is then higher than that of complex 9.
(92) Different electron-donating groups were tested: methoxy, PEG or amide. Poorer results were observed with an amide group (complex Ia.3), due to a lower quantum yield, but still satisfactory in terms of brightness.
(93) On the other hand, the presence of a second methoxy-type electron donor group on the phenyl group (complex 8) causes a drop in quantum efficiency, compared to a methyl group (complex Ia.1).
(94) The introduction of a second methyl group has a major effect on the molar extinction coefficient with a decrease of more than 50%. Thus, despite excellent quantum efficiency, Ia.4 has a brightness at 330 nm lower than its counterpart Ia.2.
(95) The spectroscopic properties of complexes Ia.2 and Ia.4 are stored in water (Table 2).
(96) The brightness of complex Ia.2 is very high, and comparable to that of complex TbLumi-4, resulting from the work of the group of K. Raymond (J. Xu et al. J. Am. Chem. Soc. 2011, 133, 19900-19910), which is among the best complexes currently on the market (Φ=59%, ε of the order of 25000 L.Math.mol.sup.−1.Math.cm.sup.−1 and a brightness around 15000 L.Math.mol.sup.−1.Math.cm.sup.−1).
(97)
(98) TABLE-US-00001 TABLE 1 Photophysical properties of complexes Ia.1-Ia.6, 8 and 9 measured in methanol λ max ε T B at λ max B at 330 nm complex (nm) (L .Math. mol.sup.−1 .Math. cm.sup.−1) Φ (%) (ms) (L .Math. mol.sup.−1 .Math. cm.sup.−1) (L .Math. mol.sup.−1 .Math. cm.sup.−1) Ia.1 306 21000 74 1.33 15500 n.d. Ia.2 305 34800 74 1.32 26000 10000 Ia.3 305 34800 35 0.79 12000 5600 Ia.4 274 19000 65 1.59 12000 3400 302 13000 8500 8 (outside 325 51000 13 0.22 6600 6500 invention) Ia.5 306 n.d. 2.8 0.021 n.d. n.d. Ia.6 301 n.d. 2.6 0.020 n.d. n.d. 9 (outside 308 33000 12 n.d. 3900 n.d. invention)
(99) TABLE-US-00002 TABLE 2 Photophysical properties of complexes Ia.2 and Ia.4-Ia. 6 measured in water λ max ε T B at λ max B at 330 nm complex (nm) (L .Math. mol.sup.−1 .Math. cm.sup.−1) Φ (%) (ms) (L .Math. mol.sup.−1 .Math. cm.sup.−1) (L .Math. mol.sup.−1 .Math. cm.sup.−1) Ia.2 302 30400 74 1.36 23000 8900 Ia.4 275 21000 66 1.46 14000 2500 300 11800 7800 Ia.5 302 n.d. 1.8 0.017 n.d. n.d. Ia.6 300 n.d. 2.5 0.028 n.d. n.d.
(100) Dysprosium complexes Ia.5 and Ia.6 also have remarkable photophysical properties in methanol and water. These complexes are among the most brilliant complexes reported in the literature.
(101)
Example 4: Cellular Imaging
(102) Complex Ia.2 has shown significant interest in single- or two-photon fluorescence microscopy imaging.
(103)