A Bispidol Chelator with a Phosphonate Pendant Arm: Synthesis, Cu(II) Complexation, and 64Cu Labeling

Article abstract of DOI:10.1021/acs.inorgchem.7b01731

Here we present the synthesis and characterization of a new bispidine (3,7-diazabicyclo[3.3.1]nonane) ligand with N-methanephosphonate substituents (L₂). Its physicochemical properties in water, as well as those of the corresponding Cu(II) and Zn(II) complexes, have been evaluated by using UV-visible absorption spectroscopy, potentiometry, ¹H and ³¹P NMR, and cyclic voltammetry. Radiolabeling experiments with ⁶⁴Cu^II have been carried out, showing excellent radiolabeling properties. Quantitative complexation was achieved within 60 min under stoichiometric conditions, at room temperature and in the nanomolar concentration range. It was also demonstrated that the complexation occurred below pH 2. Properties have been compared to those of the analogue bispidol bearing a N-methanecarboxylate substituent (L₁). Although both systems meet the required criteria to be used as new chelator for ^64/67Cu in terms of the kinetics of formation, thermodynamic stability, selectivity for Cu(II), and kinetic inertness regarding redox- or acid-assisted decomplexation processes, substitution of the carboxylic acid function by the phosphonic moiety is responsible for a significant increase in the thermodynamic stability of the Cu(II) complex (+2 log units for pCu) and also leads to an increase in the radiochemical yields with ⁶⁴Cu^II which is quantitative for L₂.

Full text of DOI:10.1021/acs.inorgchem.7b01731

Article
pubs.acs.org/IC
A Bispidol Chelator with a Phosphonate Pendant Arm: Synthesis,
6
4
Cu(II) Complexation, and Cu Labeling
†
†
y Brandel, Sandrine Huclier-Markai, Franck Camerel,⊥
‡
§,∥
Raphael
̈
Gillet, Amandine Roux, Jer
́
em
́
⊥
,†
,†
Olivier Jeannin, Aline M. Nonat,* and Loïc J. Charbonnier
̀
e*
†
Laboratoire d’Ingen
́
ierie Molec
́
ulaire Appliquee
es de Sep
GIP Arronax, 1 rue Aronnax, CS 10112, F-44817 Saint-Herblain, France
́
a
̀
l’Analyse, Universite
́
de Strasbourg, CNRS, IPHC UMR 7178, F-67000 Strasbourg,
France
‡
Laboratoire de Reconnaissance et Proced
́
́
́
aration Moleculaire, Universite de Strasbourg, CNRS, IPHC UMR 7178, F-67000
́
́
Strasbourg, France
§
∥
́
Subatech Laboratory, UMR 6457, Ecole des Mines de Nantes, IN2P3/CNRS, Universite de Nantes, 4 rue Alfred Kastler, F-44307
Nantes, France
⊥
́
̀ ́ ̀
e Condensee et Systemes Electroactifs, Institut des Sciences Chimiques de Rennes, UMR-CNRS 6226, 263
Laboratoire Matier
Avenue du General Leclerc, CS 74205, F-35042 Rennes Cedex, France
́
́
*
S Supporting Information
ABSTRACT: Here we present the synthesis and characterization of a new bispidine
(
(
3,7-diazabicyclo[3.3.1]nonane) ligand with N-methanephosphonate substituents
L2). Its physicochemical properties in water, as well as those of the corresponding
Cu(II) and Zn(II) complexes, have been evaluated by using UV−visible absorption
1
31
spectroscopy, potentiometry, H and P NMR, and cyclic voltammetry. Radio-
6
4
II
labeling experiments with Cu have been carried out, showing excellent
radiolabeling properties. Quantitative complexation was achieved within 60 min
under stoichiometric conditions, at room temperature and in the nanomolar
concentration range. It was also demonstrated that the complexation occurred below
pH 2. Properties have been compared to those of the analogue bispidol bearing a N-
methanecarboxylate substituent (L ). Although both systems meet the required criteria to be used as new chelator for 64/67Cu in
1
terms of the kinetics of formation, thermodynamic stability, selectivity for Cu(II), and kinetic inertness regarding redox- or acid-
assisted decomplexation processes, substitution of the carboxylic acid function by the phosphonic moiety is responsible for a
significant increase in the thermodynamic stability of the Cu(II) complex (+2 log units for pCu) and also leads to an increase in
6
4
II
the radiochemical yields with Cu which is quantitative for L .
2
INTRODUCTION
Chart 1. Structures of Acid-Functionalized Ligands (L
L ) Studied and the Related Bispidone (L )
and
1
■
2
3
Bispidine derivatives are highly preorganized ligands that can
accommodate metal ions with cis-octahedral, square-pyramidal,
or pentagonal geometries. They usually form thermodynami-
cally very stable metal complexes with transition-metal ions
which often show high kinetic inertness. Modification of the
coordinating pendant arms can be used to tune the ligand
denticity as well as all electronic, thermodynamic, and kinetic
parameters such as the ligand field, the metal selectivity, the
stability constants and the redox potentials. Such properties are
1
,2
3
−5
6
−12
very appealing for applications in catalysis,
in molecular
1
3−17
magnetism,
and in nuclear medicine and diagnosis as
chelators for 6 Cu.
4/67
18−21
a monoclonal antibody (mAb) (or a fragment) of interest. A
great deal of progress in antibody technologies as well as site-
This study focuses on the use of two bispidine derivatives (L1
23,24
specific conjugation methods
has been made in recent
and L , Chart 1) as chelators for radioactive copper for
2
years, and it is now within our grasp to find or design
engineered mAb fragments for almost any molecular target.
application in immuno-positron emission tomography (PET)
25
64
+
−
22
(
Cu, t1/2 = 12.7 h, β , 17.8%, 653 keV, β , 38.4%, 579 keV).
In this context, bifunctional chelators (BFCs) are needed,
providing a strong chelating site for radioactive copper
complexation as well as a reactive function for conjugation to
Received: July 7, 2017
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.inorgchem.7b01731
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
Chart 2. Structures of Other Ligands Discussed in This Work
Table 1. Radiolabeling Conditions (at Room Temperature), Half-Life (t1/2), pCu, and Reduction Potential (E ) for a Selection
red
of Ligands
a
radiolabeling conditions
t1/2
pCu
15.1
Ered (mV vs NHE)
−980 (irrev)
3
1,32
TETA
CB-TE2A
25 °C, 60 min, pH 5−7
95 °C, 60 min, pH 6−7
room temp, 15 min, pH 5
3.5 days (5 M HCl, 30 °C)
154 h (5 M HCl, 90 °C)
32 min (HCl 1 M, 25 °C)
5
1
−880 (E1/2
)
4
0,41
HTE1PA
18.64
1
44 min (5 M HClO , 25 °C)
4
9
4
CB-TE1PA
96 days (5 M HClO , 25 °C)
16.6
19.1
−620 (E1/2
)
4
6
3,35
PCTA
25 °C, 5 min, pH 5.5
5
5−57
DiamSar
25 °C, 5−30 min, pH 5.5
25 °C, 30−60 min, pH 5.5−6.5
room temp, 30 min, pH 6−7
25 °C, 5−10 min, pH 5.5
25 °C, 5−10 min, pH 5.5
room temp, 15 min, pH 2−6
room temp, 5−15 min, pH 3−6.6
room temp, 1 min, pH 6.5
room temp, 1 min, pH 5.5
50 °C, 60 min, pH 6.5
40 h (5 M HCl, 90 °C)
<3 min (5 M HCl, 30 °C)
204 min (3M, HCl, 90 °C)
<5 min (6 M HCl, 90 °C)
−900 (irrev)
−700 (irrev)
−518
3
0,64,32
NOTA
NO1PA2PY
18.4
17.75
18.5
4
4
5
3
H2DEDPA
−920 (irrev)
6
5
H2AZAPA
b21
L1
L2
L4
L5
L6
110 d (5 M HClO , 25 °C)
17.0
19.1
16.28
−560
−600
−303
4
b
>20 months (5 M HClO , 25 °C)
4
1
1
2
8,4
9
0
5
1
PCB-TEA1P
60 °C, 1 h, pH 8
8 days (12 M HCl, 90 °C)
−573
6
0,62
L7
15.5
a
Conditions: pCu = −log[Cu(II)free], [Cu] = 10−6 M, [L] = 10−5 M, pH 7.4. This work.
b
B
DOI: 10.1021/acs.inorgchem.7b01731
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Inorganic Chemistry
Article
Scheme 1. Synthesis of Ligand L2
Radiolabeled antibodies have been introduced in clinical
the radio-HPLC chromatogram of L , which were attributed to
4
2
6−29
30−32
use,
and a large range of BFCs are now available.
partial or total hydrolysis of the ester functions by the esterase
18
However, only a few chelators fulfill all of the very specific
criteria which are required to radiolabel antibody-BFC
conjugates under good conditions: i.e., (i) fast radiolabeling
in rat plasma. Dioxotetraaza macrocycles (L
6
, Chart 2) are
also very stable, although efficient labeling was observed only
20
after heating the samples at 50 °C.
Our previous studies on the methylene carboxylate
substituted bispidine L have shown that this ligand is another
(
a few minutes to 1 h) at room temperature and around
physiological pH; (ii) high in vivo stability and kinetic inertness
toward transmetalation, transchelation, and reduction, and (iii)
1
21
good candidate for PET applications. Fast complexation
occurs even at low pH values (pH 1), with a high binding
constant for Cu(II) versus competing metals (Co(II), Ni(II),
Zn(II)), and the complex is characterized by a strong stability
33,34
easy access to synthesis and bioconjugation.
Three classes of ligands with polyaza donor sets are
commonly used: macrocyclic, linear, and macrobicyclic.
3
5,36
37−39
in acidic medium (t1/2 = 110 days at 25 °C, 5 M HClO ) and
NOTA
and TETA
and their derivatives are easily
4
upon reduction (E = −430 mV vs NHE). Radiolabeling with
accessible but often suffer from low kinetic inertness. Variations
of the substituent are being explored in order to improve the in
1/2
64
CuCl2 is fast (<5 min) and easily performed at room
temperature and at micromolar concentrations of L in water (4
≤ pH ≤ 6). Under these conditions, ≥90% radiolabeling
yields were obtained. Moreover, the risk of enzymatic
degradation is suppressed since the ester functions have been
hydrolyzed prior to complexation. In this study, we report the
synthesis and physicochemical evaluation and the radiolabeling
4
0−43
vivo stability of the Cu complexes (see HTE1PA
and
1
58
4
4
45
NO1PA2PY and its derivatives, Chart 2 and Table 1). Very
stable and inert cyclen-based cross-bridged systems (such as
PycupBn and CB-TE2A, Chart 2) and macrobicycles such
as L7 are being developed, but their slow kinetics of
complexation is hampering their use for the labeling of
antibodies. Faster complexation is observed with N-phosphonic
acid analogues, although heating is still necessary for the
4
6
47
4
8
studies of the new methane phosphonate analogue L in water.
2
Substitution of the acetic acid pendant arm by a methane-
phosphonic acid moiety was expected to improve the ligand
selectivity for Cu(II) and to increase the thermodynamic and
kinetic stability of the complex. This expectation is corrobo-
4
9−52
moment.
New linear systems such as H DEDPA and
as well as other types of macrocyclic ligands and
2
5
3,54
H2AZAPA
cages (DiamSar
5
5−57
and its derivatives) offer good radio-
rated by literature data on phosphonate pendant-armed
labeling conditions at room temperature (Chart 2) and, for
some of them, a high degree of kinetic inertness. Bispidine
derivatives (L and L , Chart 2) also form particularly stable
51,59
tetraazamacrocyclic chelators such as PCB-TEA1P
as well
as on the podal pyridine derivatives developed in our group, L
8
60−62
4
5
(
Chart 2).
Cu(II) complexes in vitro and in vivo. L and L could be
4
5
radiolabeled with >95% radiolabeling yields within 1 min at
EXPERIMENTAL SECTION
4
,18
■
room temperature.
than 0.1 GBq/μmol were used for L4 and L5, and a specific
Nonoptimized specific activities of less
18
19
General Methods. Solvents and starting materials were purchased
from Aldrich, Acros, and Alfa Aesar and used without further
purification. IR spectra were recorded on a PerkinElmer Spectrum
One spectrophotometer as solid samples, and only the most significant
activity of 26 GBq/μmol was obtained for L after 90 min at 50
6
2
0
°
C. Preliminary in vivo studies in mice and rats for L and L6
4
−1
indicated rapid blood and tissue clearance as well as the absence
of demetalation. However, changes were observed over time in
absorption bands are given in cm . Elemental analyses and mass
spectrometry analysis were carried out by the Service Commun
C
DOI: 10.1021/acs.inorgchem.7b01731
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
d’Analyses of the University of Strasbourg. H and 13C NMR spectra
and 2D COSY, NOESY, HSQC, and HMBC experiments were
recorded on Bruker Avance 300 and Avance 400 spectrometers
operating at 300 and 400 MHz, respectively. Chemical shifts are
reported in ppm, relative to residual protonated solvent as internal
1
hydrogenocarbonate (143 mg, 1.7 mmol, 1.7 equiv). Piperidone P1
(396 mg, 1.0 mmol, 1 equiv) in 8 mL of MeOH was then added, as
well as formaldehyde (93.0 mg, 3.1 mmol, 3 equiv, 0.23 mL, 37%
solution in H O). The reaction mixture was heated to 60 °C for 5 h,
2
the reaction being monitored by TLC on (eluent DCM/MeOH 9/1,
Rf = 0.26). After completion of the reaction, the solvent was
evaporated under reduced pressure and the crude product was
suspended in EtOH (10 mL). Bispidone 2 was isolated by
66
reference. The pH values given are corrected for the deuterium
isotopic effects. Elemental analysis and monoisotopic masses were
67
68
calculated with the Chemcalc software.
1
X-ray Crystallography. Crystals of the intermediate 2 (Scheme 1)
centrifugation as a white powder (187 mg, 35%). H NMR (400
and L ligand suitable for X-ray diffraction were obtained by slow
MHz, CD OD): δ 1.87 (s, 3H, H3), 2.53 (d, J = 13.0 Hz, 2H, He),
2
3
evaporation of methanol solutions. The crystals were placed in oil, and
a single crystal was selected, mounted on a nylon loop, and placed in a
low-temperature N2 stream. X-ray diffraction data collection was
carried out on a Bruker APEX II Kappa-CCD diffractometer equipped
3.15 (AB system, δ = 2.66, δ = 3.64, J = 12.3 Hz, 4H, H6/H8),
A
B
AB
3.72 (s, 6H, OCH ), 4.68 (s, 2H, H2/H4), 7.35 (m, 2H, Hd), 7.42
3
(ddd, J = 7.7 Hz, J = 4.9 Hz, J = 0.9 Hz, 2H, Hb), 7.84 (td, J = 7.8
1
2
3
1
31
Hz, J = 1.5 Hz, 2H, Hc), 8.83 (dd, 2H, Ha). P NMR (162 MHz,
2
13
with an Oxford Cryosystem liquid N device, using Mo Kα radiation
CD OD): δ 15.29 ppm. C NMR (100 MHz, CD OD): δ 41.9
2
3
3
(
λ = 0.71073 Å) at 150(2) K (Centre de diffractomet
́
rie X, Universite
́
(CH ), 51.7 (2C, OCH ), 57.2 (d, Ce), 60.9 (2C, C6), 63.0 (2C, C1),
3 3
72.4 (2C, C2), 123.8 (2C, Cb), 124.7 (2C, Cd), 137.6 (2C, Cc), 150.6
de Rennes 1, France). The Bruker SMART program was used to refine
the values of the cell parameters. Data reduction and correction for
absorption (SADABS) were carried out using the Bruker SAINT
programs. The structures were solved by direct methods using the
SIR97 program and then refined with full-matrix least-squares
methods based on F (SHELX-97) with the aid of the WINGX
(2C, Ca), 156.3 (2C, Cpy), 167.5 (2C, CO Me), 202.3 (C9).
2
+
Electrospray ionization (ESI)/MS (CH OH): m/z 519.17 ([M +
H] , 100%). Anal. Calcd for C H N O PNa·0.5H O: C, 50.28; H,
4.95; N, 10.20. Found: C, 50.39; H, 4.75; N, 10.27.
3
+
23
26
4
8
2
69
2
70
Bispidol 3. Compound 2 (1.2 g, 2.3 mmol, 1 equiv) was dissolved in
80 mL of anhydrous MeOH by heating and using ultrasound. The
solution was then cooled to −78 °C, and sodium borohydride (107
mg, 2.8 mmol, 1.5 equiv) was gradually added. The reaction was
monitored by TLC on C18 (eluent H O/ACN 7/3, R = 0.28). After 5
71
program. All non-hydrogen atoms were refined with anisotropic
atomic displacement parameters.
For 2, all H atoms were included in their calculated positions,
whereas, for L , H atoms carried by heteroatoms were refined with
2
2
f
isotropic atomic displacement parameters. Crystallographic data for
h 30 min, the reaction was quenched at −78 °C by the addition of a
structural analysis of 2 and ligand L have been deposited with the
saturated NH Cl aqueous solution (5 mL). The solvent was
2
4
Cambridge Crystallographic Data Centre under CCDC nos. 1530206
and 1530205, respectively. Copies of this information may be obtained
free of charge from the Web site (www.ccdc.cam.ac.uk).
evaporated under vacuum, and the crude product was purified by
FPLC on a C18 reverse phase column (eluent system H O/ACN 0.1%
2
1
TFA), giving the bispidol 3 (0.79 g, 66%). H NMR (400 MHz,
Synthesis of the Ligands. Piperidinone dimethyl-1-methyl-4-oxo-
CD OD): δ 1.74 (s, 3H, H3), 3.35 (d, J = 11 Hz, 2H, He), 3.62 (s, 6H,
3
2
,6-bis(pyridin-2-yl)piperidine-3,5-dicarboxylate (P ) was synthesized
OCH ), 4.0 (AB system, δ 3.76, H6/8ax, δ 4.23, H6/8eq, J = 12.7
1
3
A
B
72
according to a previously reported procedure.
Hz, 4H), 4.55 (s, 1H, H9), 4.98 (s, 2H, H2/H4), 7.43 (dd, J = 7.0 Hz,
1
(
Aminomethyl)phosphonic Acid 1. 1 was obtained in three steps
J = 5.2 Hz, 2H, Hb), 7.65 (d, J = 7.6 Hz, 2H, Hd), 7.87 (td, J = 7.7
2
t
3
1
from diethyl phosphate by the Kabachnik−Fields reaction, according
to an adaptation of the procedure used in ref 73.
Hz, J = 1.6 Hz, 2H, Hc), 8.74 (d, J = 4.1 Hz, 2H, Ha). P NMR (162
d
13
MHz, CD OD): δ 8.24 ppm. C NMR (100 MHz, CD OD): δ 40.7
3 3
(
i) To a solution of diethyl phosphite (3.27 mL, 94%, 20.7 mmol,
.2 equiv) in THF (9 mL) were successively added dibenzylamine
3.39 mL, 98%, 17.27 mmol, 1 equiv) and formaldehyde (3.06 mL,
7% in water, 34.5 mmol, 2 equiv). The mixture was heated at 60 °C
(CH ), 51.2 (2C, C1), 51.8 (2C, OCH ), 53.2 (d, J = 133.9 Hz, Ce),
55.8 (2C, C6), 66.2 (2C, C2), 71.9 (C9), 123.9 (2C, Cb), 127.6 (2C,
3
3
1
(
3
Cd), 137.4 (2C, Cc), 149.3 (2C, Ca), 155.5 (2C, Cpy), 168.7 (2C,
+
CO Me). Electrospray ionization (ESI)/MS (CH OH): m/z 521.18
2
3
+
with stirring for 24 h, and the reaction was monitored by TLC. After
completion of the reaction, the mixture was taken to dryness under
vacuum and the as-obtained yellow oil was dissolved in cyclohexane
([M + H] , 100%). Anal. Calcd for C H N O P·0.5H O: C, 52.17;
23 29 4 8 2
H, 5.71; N, 10.58. Found: C, 51.94; H, 5.56; N, 10.41.
Ligand L . Compound 3 (514 mg, 1 mmol, 1 equiv) was dissolved
2
(
60 mL) and washed with water (3 × 15 mL). Diethyl
in a THF/H O (1/1) mixture (30 mL), and a solution of sodium
2
(
(dibenzylamino)methyl)phosphonate was obtained as a colorless oil
hydroxide (200 mg, 5 mmol, 5 equiv) in water (5 mL) was added. The
mixture was stirred at room temperature, and the reaction was
monitored by TLC (eluent system H O/ACN 8/2, 0.1% TFA, R =
after evaporation of the cyclohexane under reduced pressure (6 g,
1
quantitative). H NMR (400 MHz, CDCl ): δ 1.19 (t, J = 7.1 Hz, 6H,
3
2
f
CH CH ), 2.77 (d, J = 10.5 Hz, 2H, NCH P), 3.67 (s, 4H, NCH Φ),
0.65). After completion of the reaction, the mixture was evaporated to
dryness, redissolved in 1 M hydrochloric acid, and purified by flash
chromatography with a C18 reverse phase column (eluent system
H O/ACN 0.1% TFA), to give ligand L ·NaCl·4H O (621 mg,
2
3
2
2
3
.95 (qd, J = 7.1 Hz, J = 7.5 Hz, 4H, CH CH ), 7.11−7.28 (m, 10H,
1
2
2
3
31
Φ). P NMR (162 MHz, CDCl ): δ 25.7.
3
(
ii) Palladium over charcoal (10%, 600 mg) was added to a solution
2
2
2
1
of diethyl ((dibenzylamino)methyl)phosphonate (6 g, 17.27 mmol) in
EtOH (300 mL), and the mixture was refluxed under a flow of
hydrogen for 24 h. The crude mixture was filtered on a sintered-glass
filter funnel filled with Celite, and the solvent was removed under
quantitative). H NMR (300 MHz, CD OD): δ 1.78 (s, 3H, NCH ),
3
3
2.34 (d, J = 12.2 Hz, 2H, He), 2.67 (AB system, δ 2.09, H6/8ax, δB
A
3.24, H6/8eq, JAB = 12.4 Hz, 4H), 3.88 (s, 1H, H9), 4.61 (s, 2H, H2/
H4), 7.24 (m, 2H, Hb), 7.47 (d, J = 7.6 Hz, 2H, Hd), 7.65 (t, J = 7.3
3
1
vacuum to yield diethyl (aminomethyl)phosphonate (4.8 g,
Hz, Hc), 8.76 (d, J = 3.7 Hz, 2H, Ha). P NMR (162 MHz, CD OD):
3
1
13
quantitative). H NMR (400 MHz, CDCl ): δ 1.25 (t, J = 6.7 Hz,
δ 16.28 ppm (t, J = 12.0 Hz). C NMR (100 MHz, CD OD): δ 42.9
3
3
6
4
H, CH CH ), 1.97 (s, 2H, NH ), 2.94 (d, J = 10.2 Hz, 2H, NCH P),
(CH ), 51.7 (2C, C1), 59.2 (2C, C6), 60.5 (d, J = 145.7 Hz, C9), 68.3
(2C, C2), 74.8 (C9), 122.0 (Cb), 125.7 (Cd), 136.1 (Cc), 149.5 (Ca),
2
3
2
2
3
31
.05 (m, 4H, CH CH ). P NMR (162 MHz, CDCl ): δ 27.35 ppm
2
3
3
(
qd, J = 8.5 Hz, J = 9.2 Hz).
160.62 (2C, Cpy), 178.34 (2C, CO H). Electrospray ionization (ESI)/
1
2
2
+
+
(
iii) Diethyl (aminomethyl)phosphonate (4.81 g, 28.8 mmol) was
MS (CH OH): m/z 493.15 [M + H] , 100%). Anal. Calcd for
3
dissolved in 6 M hydrochloric acid (300 mL), and the mixture was
refluxed for 16 h with stirring. After evaporation to dryness under
C H N O P·NaCl·4H O: C, 40.49; H, 5.34; N, 8.99. Found: C,
2
1
25
4
8
2
40.32; H, 5.03; N, 8.92.
reduced pressure, (aminomethyl)phosphonic acid 1 was obtained as a
Physicochemical Studies. Materials. Distilled water was purified
by passing through a mixed bed of ion exchanger (Bioblock Scientific
R3−83002, M3-83006) and activated carbon (Bioblock Scientific
ORC-83005). All of the stock solutions were prepared by weighing
solid products using an AG 245 Mettler Toledo analytical balance
(precision 0.01 mg). Metal cation solutions were prepared from their
1
white powder (4.25 g, quantitative). H NMR (400 MHz, CDCl ): δ
3
3
.00 (d, J = 13.0 Hz, 2H). 31P NMR (162 MHz, CDCl ): δ 12.18 ppm.
Bispidone 2. (Aminomethyl)phosphonic acid 1 (126 mg, 1.13
3
mmol, 1.1 equiv) was dissolved in a H O/MeOH (3/7) mixture (11
2
mL) and stirred at room temperature in the presence of sodium
D
DOI: 10.1021/acs.inorgchem.7b01731
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Inorganic Chemistry
Article
perchlorate salts (Cu(ClO ) ·6H O, 98%, Fluka; Zn(ClO ) ·6H O,
CuL complex in 5 M HClO at 25 °C. Changes in the absorption
4
2
2
4
2
2
2
4
9
8.9%, Alfa Aesar), and their concentrations were determined by
spectra with time over a period of 20 months were monitored using a
−2
−4
colorimetric titrations with EDTA (10 M, Merck, Titriplex III)
PerkinElmer Lambda 950 spectrophotometer. A 1.98 × 10 mmol
74
II
according to standard procedures. Sodium hydroxide (NaOH) and
hydrochloric acid (HCl) were used to adjust pH during titrations. The
ionic strength of all the solutions was fixed to 0.1 M with potassium
chloride (KCl, Fluka, 99.0%). All of the experiments described were
repeated at least three times.
portion of Cu L complex was used to monitor the π−π* transition at
2
−3
262 nm and 8.78 × 10 mmol to follow the d−d transition at 680 nm.
Cyclic Voltammetry. Cyclic voltammetry (CV) was carried out on
the CuL2 complex at room temperature with a PC interfaced
Radiometer Analytical MDE150/PST50 instrument. The CV experi-
ments were performed using a glassy-carbon working electrode (0.071
Caution! Perchlorate salts combined with organic ligands are
2
potentially explosive and should be handled in small quantities and
cm , BASi). The electrode surface was polished routinely with 0.05 μm
75
with adequate precautions.
alumina−water slurry on a felt surface immediately before use. The
counter electrode was a Pt coil, and the reference electrode was a Ag/
Potentiometry. The protonated species of L2 and the stability
constants of L complexes with Cu(II) and Zn(II) complexes were
AgCl electrode. The CuL complex was measured in Ar-degassed
2
2
characterized and quantified by potentiometric titrations in water. All
of the solutions used in the potentiometric experiments were prepared
from boiled and degassed water. Titrations were performed using an
automated titrating system (DMS 716 Titrino, Metrohm) with a
combined glass electrode (Metrohm, 6.0234.100, Long Life) filled
with NaCl 0.1 M. The electrode was calibrated as a hydrogen
concentration probe by titrating known amounts of hydrochloric acid
water with ionic strength fixed at 0.1 M with NaClO , and the pHs of
4
the solutions were adjusted with NaOH and HClO solutions. Seven
4
different values of pH (pH 2.36, 4.04, 5.70, 7.2, 8.55, 10.23, 11.62) and
different scan rates (50−300 mV/s) were considered.
Radiolabeling. 64CuCl in 0.1 M hydrochloric acid was obtained
2
from the ARRONAX cyclotron (Saint-Herblain, France). Production
81
and purification procedures have already been described. Radio-
chemical purity was determined by γ spectroscopy, and chemical
purity was measured by ICP-AES. Water (18.2 MΩ cm) for aqueous
solutions was obtained from a Milli-Q gradient system (Millipore).
2−
with CO -free potassium hydroxide solutions. The GLEE pro-
3
76,77
gram
was used for the glass electrode calibration.
−3
In a typical experiment, an aliquot of 10 mL of L (2.10 M) or M/
2
64
L2 (M = Cu(II), Zn(II), [M]/[L] ≈ 1) was introduced into a
thermostated jacketed cell (25.0(2) °C, Metrohm) and kept under
argon during the titrations. The solutions were acidified with a known
volume of HCl, and the titrations were then carried out by addition of
known volumes of potassium hydroxide solution over the pH range
Radiolabeling of ligand L with Cu was performed, following the
2
58
same procedure as that previously described for ligand L .
1
Postprocessed 64Cu eluate diluted in 0.25 M ammonium acetate
buffer (pH 5.3) was mixed at room temperature with a ligand stock
−4
solution ([L2]stock = 4.1 × 10 M). Four radionuclide batches were
2
−12. The potentiometric data of L and its metal complexes were
used for radiolabeling purposes. Each batch was characterized with
2
78
64
refined with the Hyperquad 2008 program, which uses nonlinear
least-squares methods, taking into account the formation of metal
hydroxide species. The titration of each system was repeated at least in
duplicate, and the sets of data for each system were treated
independently and then merged together and treated simultaneously
to give the final stability constants. The distribution curves as a
function of pH of the protonated species of L and of L metal
regard to its specific activity (SA( Cu) per nmol of Cu) and the
content of cold metallic impurities. Briefly, the characteristics of each
2
+
−7
batch (1−4) are given: batch 1, 13.13 MBq/mL, [Cu ] = 4.02 × 10
−5
64
M, [M] = 2.48 × 10 M, SA( Cu) = 34.8 MBq/nmol; batch 2, 55.71
2+
−6
−6
64
MBq/mL, [Cu ] = 2.19 × 10 M, [M] = 8.93 × 10 M, SA( Cu) =
2+
−6
25.3 MBq/nmol; batch 3, 38.94 MBq/mL, [Cu ] = 1.53 × 10 M,
−6
64
2
2
[M] = 8.94 × 10 M, SA( Cu) = 25.3 MBq/nmol; batch 4, 9.74
2+ 64
7
9
−7
−6
complexes were calculated using the Hyss2009 program.
MBq/mL, [Cu ] = 3.8 × 10 M, [M] = 8.94 × 10 M, SA( Cu) =
25.3 MBq/nmol. Several parameters were scrutinized in repeated
experiments such as the pH of the reaction mixtures, the time, and the
ligand/metal molar ratio (where [metal] corresponds to the total
concentration in metal salts, including nonradioactive contaminants
from the source such as Co(II), Cu(II), Fe(II/III), Ni(II), and
Zn(II)). In typical experiments, 500 μL batch solutions were prepared
separately by mixing a known amount of 64Cu stock solution (5−40
μL, 0.5 MBq), a known volume of ligand stock solution, and a known
Spectrophotometry. The protonation constants of L2 and the
stability constants of M/L (M = Cu(II), Zn(II), [M]/[L] ≈ 1, [L] ≈
2
−5
5
× 10 M) were also determined by UV−visible spectrophotometric
titration versus pH. Since complexation started in very acidic medium,
the titrations were carried out in two different ways. Between pH −0.6
and pH 2, batch solutions were prepared. Each sample was prepared
separately by mixing a known amount of L stock solution, a known
2
+
amount of standardized HClO to adjust the pH (pH = −log [H ]),
4
and a known amount of Cu(II) stock solution in the case of the study
of the complexes ([Cu(II)]/[L] = 1). An absorption spectrum of each
sample was recorded in a 1 cm quartz Suprasil spectrophotometric cell
using a Varian (Cary 3) UV−visible spectrophotometer. Between pH
amount of AcONH buffer, the pH being previously adjusted to the
4
desired value (2 ≤ pH ≤ 7). The influence of temperature and
incubation time on the reaction yield was also investigated on L for an
2
L/M ratio of 0.25 by heating the samples for 1 h at 80 °C; the pH was
measured before and after the heating.
2
and 12.5, direct titrations were carried out. Typically, an aliquot of 10
mL of L solution was introduced into a thermostated jacketed titration
vessel (25.0(2) °C) with 1 equiv of metal (M) in the case of M/L
titrations. A known volume of hydrochloric acid solution was added to
adjust the pH to around 2, and the titrations were carried out by
addition of known volumes of potassium hydroxide solution. After
each addition, the pH was allowed to equilibrate, an aliquot was
transferred to a 1 cm quartz Suprasil spectrophotometric cell, a
spectrum was recorded using a Varian (Cary 3) spectrophotometer,
the aliquot was transferred back to the titration vessel, and a new
addition was made. The free hydrogen ion concentrations were
measured with a Mettler Toledo U402-S7/120 (pH 0−14) combined
glass electrode. Potential differences were given by a Tacussel
LPH430T millivoltmeter. Standardization of the millivoltmeter and
verification of the linearity of the electrode were performed with three
commercial buffer solutions (pH 4.01, 7.01, and 10.01, 25 °C). The
software Hypspec V1.1.33 was used to determine the coordination
model and calculate the stability constants (log β) of the formed
Radiolabeling was followed by spotting the reaction mixture onto a
TLC Flex Plate (silica gel 60A, lF-254, 200 μm, Merck) followed by
elution with concentrated aqueous NH /MeOH/H O 1/2/1 (v/v/v).
3
2
Quantitative distribution of radioactivity on TLC plates was measured
using an electronic autoradiography system (Cyclone, PerkinElmer).
6
4
Under these conditions the Cu complexes (R = 0.9) and the free
f
64
Cu (R < 0.1) are well separated. All yields are given with the
f
experimental uncertainties of the cyclone device of ±5%.
RESULTS AND DISCUSSION
■
Synthesis of Ligand L . Ligand L was obtained in three
2
2
steps from (aminomethyl)phosphonic acid 1 and the
piperidinone precursor P following a synthetic strategy similar
1
to that previously reported for the glycinate derivative L
1
21
(
Scheme 1). 1 was quantitatively obtained by a Kabachnik−
71,80
Fields reaction of diethyl phosphite, methanal, and dibenzyl-
amine. Hydrogenolysis of the benzyl protecting groups
followed by acid hydrolysis of the diethyl ester moieties was
species.
Acid Decomplexation Studies. Acid-decomplexation studies were
performed under pseudo-first-order conditions on two solutions of
E
DOI: 10.1021/acs.inorgchem.7b01731
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
performed by using standard conditions. Bispidone 2 was
obtained in 35% yield by a double Mannich reaction using P1,
1
, and methanal in the presence of NaHCO . By this method,
3
pure bispidone 2 could be obtained by precipitation from
ethanol. It can be noted that previous attempts using
diethyl(aminomethyl)phosphonate instead of the phosphonic
acid led to a mixture of products which was difficult to purify by
crystallizations or column chromatography. Selective reduction
of the central ketone of 2 was achieved in good yield by
addition of NaBH in cold methanol (−78 °C). A single
4
epimer, with H pointing toward N , was isolated after
9
7
purification by reverse phase flash chromatography (FPLC)
1
1
Figure 2. ORTEP drawing of ligand L
numbering. Thermal ellipsoids are drawn at the 50% probability level.
All H atoms are omitted for the sake of clarity.
2
with the main atomic
on a C18 column, as evidenced by H− H NOESY
experiments. Interestingly, the same regioselectivity was
21
previously observed with acetate-substituted bispidol, suggest-
ing a facial regioselectivity, which is probably due to the
stabilization of the borohydride intermediate due to formation
of hydrogen bonds with the carbonyl and acid protons of the
in general positions (Figure 2). The ligand crystallizes in a
partially protonated form in the monoclinic space group P2 /n
(Table 2) and is characterized by the presence of four acidic
protons: one on the carboxylic acid (C9−O4H = 1.3150(16) Å
and C9 = O3 = 1.2035(16) Å), two on the phosphonic acid
1
phosphonic or carboxylic acid on N (see numbering in Scheme
7
1). Saponification of the methyl ester substituents was achieved
at room temperature in the presence of sodium hydroxide, and
the pure ligand could be isolated by reverse phase FPLC (see
Figures S1−S13 in the Supporting Information for the NMR
(
P1O5 = 1.4779(0) Å, P1O6H = 1.5598(10) Å, and P1
O7H = 1.5616(10) Å), and one localized on the tertiary amine
+
spectra of 2, 3 and L ).
2
R
NH (N
3
2
−H
2
= 0.894(17) Å). The carbon−oxygen distances
Structural Characterization of 2 and L . Single crystals
on the remaining carboxylate are almost identical (C −O =
2
8
1
of 2 (as the sodium salt 2Na) and L were obtained by slow
1.2534(15) Å and C −O 1.2642(15) Å), and the oxygen
2
8
2
evaporation of methanol solutions at room temperature. The
crystal structure refinement confirms the chemical structures of
atoms O and O are involved in hydrogen bonds with a
1 2
bidentate phosphonate of a neighboring molecule. Further-
2
and ligand L . The corresponding ORTEP view of the
more, the ammonium proton H is stabilized inside the cavity
2
2
asymmetric unit is shown in Figures 1 and 2, respectively, and
corresponding crystallographic data are presented in Tables 2
and 3.
by strong hydrogen bonds with the nitrogen atoms N (d
=
=
1
H−N1
2.1142(105) Å), N (d
= 2.3543(78) Å), and N (d
3
H−N3
4
H−N4
2.5267(86) Å). Deeper analysis of the Fourier transform of the
structure factors did not reveal any residual electron density
around nitrogen atom N1, meaning that, in the solid state, the
proton is exclusively well localized on the nitrogen atom N2.
This protonation scheme is typical of a Proton Sponge
behavior, which was confirmed by physicochemical titrations
(
see below). As a consequence, ligand L is highly preorganized
2
for metal complexation with (i) a chair−chair conformation of
the bispidine skeleton, (ii) a cis-symmetrical configuration of
the pyridine rings, and (iii) the lone pairs of N1 and N2
pointing toward the inside of the cavity. In addition, the N1···
N2 = 2.6935(13) and N3···N4 = 4.6913(16) Å distances are
significantly shorter than those for analogous bispidone in their
deprotonated form (N1···N2 = 2.888(2) and N3···N4 =
72
7
.186(2) Å for L3) and very close to the distances measured
Figure 1. ORTEP drawing of 2Na with the main atomic numbering.
Thermal ellipsoids are drawn at the 50% probability level. All H atoms
are omitted for the sake of clarity.
for 2Na and for another recent example of protonated structure
with Hbispa ligands (N1···N2 = 2.684(2) and N3···N4 =
83
4
.922(3) Å for Hbispa1a). This contraction of the cavity is
also likely attributed to the presence of the protonated
ammonium center, which more strongly attracts the electron-
rich surrounding nitrogen atoms. Moreover, the structure is
stabilized by a network of strong hydrogen bonds between the
phosphonic acids and the acetate moiety of a neighboring
molecule (O1−O6 = 2.5404(13) Å, O1−H6A−O6 =
169.8(2)°, O2−O7 = 2.5632(13) Å, O2−H7A−O7 =
177.8(2)° Å), which stabilizes this particular protonation
state. Finally, each ligand forms moderate H bonds (O2−O8
= 2.8016(18) Å, O2−H8−O8 = 157.2(1)°, O9−O002 =
2.6677(16) Å, O002−H002−O9 = 166.5(2)°) with two
methanol molecules, the first bridging the acetate group and
the hydroxyl at C9 and the second being linked to the
phosphonic acid. The rigid chair−chair conformation of the
The intermediate 2 was isolated as a sodium salt, and the
structure (Figure 1) confirms the chair−chair conformation of
1
the bispidone, as observed by H NMR studies (Figure S1 in
the Supporting Information). This intermediate was crystallized
̅
in the triclinic space group P1 with one sodium complex and
one methanol molecule in general positions. The Na(I) ion is
hexacoordinated by ligand 2 and one methanol molecule with a
distorted-octahedral coordination geometry (Table 2), which is,
as expected from the strong rigidity of the ligand backbone,
82
very similar to other hexacoordinated structures with Li(I) or
transition-metal ions such as Cr(III), Mg(II), Fe(II), Co(II),
5
Cu(I/II), and Zn(II). In the case of L , the asymmetric unit
2
contains one complete L ligand and two methanol molecules
2
F
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Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
72
Table 2. Crystallographic Data for the Structures of 2Na, L , and L
2
3
2
Na
L2
L3
formula
C H N NaO P, MeOH
C H N O P,2 MeOH
C H N O
7
2
4
30
4
9
21 25
4
8
26 30
4
mol wt (g mol−1)
temp (K)
cryst size (mm)
cryst syst
space group
unit cell dimens
a (Å)
604.52
150(2)
556.50
510.54
150(2)
173(2)
041 × 0.24 × 0.11
triclinic
0.55 × 0.49 × 0.43
monoclinic
0.30 × 0.25 × 0.20
monoclinic
P1
̅
P2 /n
1
P2 /c
1
8.3045(2)
13.5397(5)
14.3175(6)
113.495(2)
91.526(2)
93.495(2)
1471.37(9); 2
1.364
13.4531(5)
10.6527(3)
18.3216(7)
14.8091(4)
11.8613(4)
14.8551(4)
b (Å)
c (Å)
α (deg)
β (deg)
92.1580(10)
100.775(2)
γ (deg)
3
V (Å ); Z
2623.84(16);4
1.409
2563.37(13); 4
1.323
calcd density (g cm−3)
abs coeff (mm−1)
F(000)
0.168
0.167
0.097
636
1176
1080
θmax (deg)
27.48
27.52
27.46
no. of rflns collected
no. of indep rflns (I > 2σ(I))
no. of params
14303
23378
25582
4964
5409
5860
378
357
338
R1, wR2 (I > 2σ(I))
R1, wR2 (all data)
0.0511, 0.1307
0.0695, 0.1433
0.0367, 0.098
0.041, 0.1017
0.0566, 0.1418
0.0832, 0.1555
72
LH
n−1
+ H ↔ LHn
(1)
(2)
Table 3. Selected Bond Lengths and Angles in 2, L , and L
2
3
2
L2
L3
[
LHn]
Bond Lengths (Å)
K1H = [
n = 1−6
LH ][H]
Na−N1
Na−N2
Na−N3
Na−N4
Na−O8
Na−O9
N1···N2
N3···N4
2.6205(18)
n−1
2.4551(15)
2.4802(23)
2.4917(24)
2.3330(15)
2.3695(18)
2.9429(24)
4.6521(31)
2.6935(13)
4.6913(16)
2.888(2)
7.186(2)
Angles (deg)
Pyr1···Pyr2
144.05
116.8
159.8
1
bicycle was also observed in solution by H NMR (Figure S9 in
the Supporting Information) with a typical Overhauser effect
between the pyridyl protons H and the equatorial protons H
d
6
1
1
(
see Figure S13 in the Supporting Information for H− H
NOESY). Attempts to grow single crystals of the CuL complex
2
have also been carried out, unfortunately leading to crystals of
insufficient quality for X-ray diffraction.
Thermodynamic Studies. Protonation Constants of
Figure 3. Spectrophotometric titrations of L vs pH at −0.6 < p[H] <
2
1
.73 (batch titration) and 2.10 ≤ pH ≤ 11.93 (direct titration).
−5
Conditions: [L ] = 4.80 × 10 M; solvent, H O; I = 0.1 M KCl; T =
2
tot
2
Ligand L . Ligand L (Chart 1) has five protonatable sites in
25.0(2) °C.
2
2
the usual 2 ≤ pH ≤ 12 window in water: two tertiary amines,
two carboxylic acids, and one phosphonic acid. The crystal
structure of L suggests that only one of the two tertiary amines
Because of the known strong stability of the metal complexes
4,20
2
is protonated. However, three potential additional protonation
constants have to be taken into account at lower pH values,
which account for the second protonation of the phosphonic
of bispidine derivatives,
ligand L and its metal complexes
2
were also studied under strongly acidic conditions (−0.59 < pH
< 1.73) by means of spectrophotometric titrations vs pH. As
such a low pH cannot be measured with an electrode, the batch
titration technique was used and the pH of the solutions was
21
acid and for the protonations of the two pyridine rings.
Protonation constants, as defined by eqs 1 and 2, were
determined by a combination of potentiometric titrations
fixed by adding known volumes of standardized HClO (see the
4
(
Figure S14 in the Supporting Information) and UV−visible
absorption titrations versus pH between pH −0.6 and 12
Figure 3).
Experimental Section for details). It has to be noted that the
ionic strength was not fixed below pH 1 in the batch titrations
and that no decomposition of the ligand was observed, even
(
G
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Inorganic Chemistry
Article
under strongly acidic conditions. The spectral variations of L2
observed at −0.59 < pH < 1.73 (batch titration) and 2.10 < pH
From these values, the electronic spectra of the protonated
species of L (Figure 4a) and their distribution diagram (Figure
2
89
<
11.93 (direct titration) were combined in Figure 3. L2
4b) were calculated. The distribution curves showed that, due
H
2
showed one band, centered at 263 nm, attributed to the
π−π* transition of the pyridine rings, which underwent a
hypochromic variation with the appearance of shoulders upon
to the presence of the phosphonate moiety (pK = 7.2(1)),
3−
2−
the ligand exists in its L H and L H
forms under
2
2
2
physiological conditions (pH 7.4).
60
an increase in the pH. The hypochromic variation is typical of
the deprotonation of pyridinium nitrogens, while the shoulders
appearing under basic conditions suggest the existence of
hydrogen bonding with at least one pyridine nitrogen lone
Stability Constants of the Cu(II) and Zn(II) Complexes.
Spectrophotometric titrations versus pH of solutions of L and
2
Cu(II) were carried out between pH −0.59 and 2 (batch
titration) to ensure complete decomplexation of Cu(II) and
between pH 2 and 12 (direct titration) both on the ligand
bands and on the Cu(II) d−d bands (Figure 5). The position
8
4
pair.
The statistical analysis of the potentiometric and spectro-
photometric data versus pH was achieved with Hyper-
of the Cu(II) d−d bands at pH 2 (λ 682 nm) shows that the
max
8
5
78
quad2008 and Hypspec software, respectively, and led to
Cu(II) complex is already formed at this pH and suggests a
90
the determination of five protonation constants of ligand L in
square-pyramidal geometry. Significant changes were seen in
2
the pH range from −0.59 to 11.93 (Table 4). The first
the UV−visible absorption spectra of CuL as a function of pH,
2
where the hypo- and hypsochromic shifts of the main band at
260 nm together with the appearance of a small charge transfer
a
Table 4. Successive Protonation Constants of L and L
2
1
61
band (Figure 5a) indicated the complete formation of the
complex at pH 0.2. Further spectral variations were observed
between pH 2 and 12, suggesting the successive formation of
pKnH
L2
L1
pK1H
pK2H
pK3H
pK4H
pK5H
pK6H
Ntert
11.5(3)
7.2(1)
3.8(3)
2.4(4)
0.5(1)
<0.5
10.6(6)
HPO3−
COOH
COOH
PO32−/Npyr
Npyr
different species.
Data analysis
4.5(1)
2.0(2)
85,78
suggested that the best model involves the
successive formation of CuL H , CuL H, and CuL over the
2
2
2
2
0.82(1)
<0.82
entire studied pH range (Table 5) with high stability constants.
This model was confirmed by potentiometric titrations between
a
Conditions: solvent, H O; I = 0.1 M (KCl); T = 25.0 °C. The values
2
pH 2 and 12 in which the CuL H2 stability constant,
2
in parentheses correspond to the standard deviations expressed as the
last significant digit.
determined from the batch titration, was fixed.
We then focused our attention on the study of the
complexation of Zn(II), since it is a common metallic impurity
81
in no-carrier-added radiocopper solutions. Potentiometric
and spectrophotometric titrations versus pH of L with
protonation constant (log K H = 11.5(3)) was assigned to the
1
2
82,86−88
tertiary amine of the bispidine skeleton.
From crystallo-
stoichiometric amounts of Zn(II) showed the same model of
complexation as for Cu(II),: i.e., the successive formation of
graphic data, it was postulated that simultaneous protonation of
the two tertiary amines does not occur. The second
protonation constant (log K2H = 7.2(1)) was attributed to
the first protonation of the phosphonic acid. The third and
ZnL
H , ZnL H, and ZnL species.
2 2 2
2
In order to compare the chelating ability of L
for Cu(II) and
2
Zn(II) with other ligands, their pM (M = Cu(II), Zn(II))
values at physiological pH (pH 7.4) were calculated (Tables 1
and 5). By representing the amount of free metal in solution at
physiological pH, the pM allows a comparison of the chelation
power of ligands having different denticities and protonation
properties for various metals. These results indicated a very
strong stability of the Cu(II) complex bearing a phosphonate
arm, with a pCu value 2 orders of magnitude larger than that of
H
H
fourth protonation constants (K , K ) were attributed to the
3
4
two carboxylic acid oxygens. The two most acidic protonation
constants belong to the pyridine nitrogens and/or to the
second protonation of the phosphonic acid. Only one of them
could be determined from our spectrophotometric batch
titrations (log K5H = 0.5(1)). These protonation constants
are in good agreement with those determined previously for
1
ligand L and for an analogue of L bearing a thiophene group
the previously studied ligand L with a carboxylate arm and
2
1
21
64
in place of the phosphonic acid.
among the highest observed for Cu PET ligands (Table 1).
Figure 4. (a) Electronic spectra and (b) distribution diagram of the protonated species of L . Conditions: [L ] = 5.0 × 10− M; solvent, H O; I =
5
2
2
tot
2
0.1 M KCl; T = 25.0(2) °C.
H
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Inorganic Chemistry
Article
Figure 5. Spectrophotometric titration of Cu L vs pH: (a) [L ] = 4.79 × 10− M, [Cu(II)] /[L ] = 0.97, − 0.60 < pH < 11.99, H O, I = 0.1 M
5
2
2
tot
tot
2
tot
2
−3
(
KCl), T = 25.0(2) °C; (b) [L2]tot = 2.50 × 10 M, [Cu(II)] /[L ] = 0.97, −0.61 < pH < 12.07, H O, I = 0.1 M (KCl), T = 25.0(2) °C.
tot
2
tot
2
Table 5. Overall Stability Constants (log β) of the ML and
experiments (Figure S16 in the Supporting Information).
Significant variations are observed in the chemical shifts of the
2
a
ML Complexes
1
protons H6/8 (Δδ = 0.2 ppm) and H (Δδ = 0.29 ppm), as
e
Cu(II)
Zn(II)
well as of the phosphorus atom (Δδ = 4.63 ppm) (Figure 8; see
L1
L2
L1
L2
31
numbering in Scheme 1). The strong variations in P and He
log βML
pKa1
19.2(3)
22.5(1)
4.9(3)
3.1(3)
19.1
14.45(2)
18.8(1)
5.3(2)
3.1(2)
15.4
suggest that the first protonation occurs on the phosphonate
31
function and values of pK ( P) = 5.4 and pK (H6) = 5.3
a1
a1
pKa2
could be determined from the analysis of the chemical shift of
pM (pH 7.4)
17.0
12.2
31
the P atom and proton H6, respectively (Figure 7c). These
a
Conditions: M = Cu(II), Zn(II); solvent, H O; I = 0.1 M; T = 25.0
C; βMLH = [MLH]/([M][L][H]). Charges are omitted for clarity. log
2
results are in very good agreement with the pK value obtained
a1
°
from potentiometric and spectrophotometric titrations. Proto-
nation of the phosphonate also influences the chemical shift of
the H6/8 protons in the 4.8 ≤ pH ≤ 7.2 range. Moreover, the
KCu(OH) = −6.29. log K
= −13.1. log K
= −7.89. log
Cu(OH)2
Zn(OH)
K
1
= −14.92 (from ref 91). pM = −log[M(II) ] with [M] =
Zn(OH)2
free
−
6
−5
0
M and [L] = 10 M, pH 7.4.
protonation curve of H6/8 clearly indicates that a second
eq
protonation reaction takes place below pH 4, which could be
assigned to the protonation of a carboxylate function.
Electrochemical Studies of the Cu(II) Complexes with
Moreover, ligand L showed a good selectivity for Cu(II) in
2
comparison to Zn(II), with a more than 3 orders of magnitude
increase in pM values between the Cu(II) and Zn(II)
complexes.
The electronic spectra of the Cu(II) and Zn(II) complexes of
L2 (Figure 6a) and their species distribution profiles (Figure
Ligand L . In order to verify that the reduction potential of
2
CuL is below the threshold for in vivo reduction, estimated to
2
92,93
−
0.4 V (vs NHE),
we performed cyclic voltammetry
studies at different pH (Figure 8).
6
b) were calculated from their thermodynamic stability
At physiological pH where the CuL species predominates,
constants. The distribution curves show that, for both Cu(II)
and Zn(II), the ML complex is the major species at
physiological pH (pH 7.4).
2
quasi-reversible processes were observed in both reduction and
oxidation. A single redox couple was identified, corresponding
to Cu(II)/Cu(I) (Ered = −0.81 V vs Ag/AgCl, i.e. E = −0.60
pK s of the Zn(II) complexes have been confirmed by
red
a
1
31
V vs NHE), clearly indicating the absence of demetalation and
variable-pH H and P NMR spectroscopy of a stoichiometric
mixture of L and ZnCl in D O (Figure 7). At pD 12.6, the H
1
suggesting that L
is able to stabilize both Cu(II) and Cu(I) in
2
2
2
2
NMR spectrum is consistent with the formation of a rigid 1:1
complex (Figure S15 in the Supporting Information) and all
this pH range. Similar behaviors were also observed for ligand
2
1,4
L and other bispidone derivatives and for NO1PA2PY (Ered
1
1
1
44
protons could be assigned by H− H COSY and NOESY
= −0.518 V vs NHE) and CB-TE1PA (E1/2 = −0.62 vs
Figure 6. (a) Electronic spectra and (b) distribution diagram of the different Cu(II)-L species. Conditions: [L ] = 5.05 × 10− M; [Cu] /[L ]
5
2
2
tot
tot
2
tot
=
0.98; H O, I = 0.1 M KCl; T = 25.0(2) °C).
2
I
DOI: 10.1021/acs.inorgchem.7b01731
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
31
1
Figure 7. Variable pH NMR of a 1:1 L :ZnCl solution in D O at 25 °C: (a) P NMR spectra; (b) enlargement of the H NMR spectra in the
2
2
2
2
.25−2.75 ppm region; (c) protonation curves f(pD) for P, H , and H6/8.
e
good selectivity, but more importantly, high kinetic inertness
95
toward dissociation. The kinetic inertness of a complex is
commonly evaluated by following its acid-assisted dissociation
under strongly acidic conditions under pseudo-first-order
conditions. Providing all other criteria were satisfied, the
obtained half-life was shown to be a good gauge of the in vivo
stability of 64Cu-labeled chelates. The decomplexation of the
CuL H complex in 5 M HClO aqueous solutions at 25 °C
96
2
2
4
was followed by UV−visible absorption spectrophotometry on
both the ligand π−π* transitions and the Cu(II) d−d bands
over a period of 20 months. Very minor decomplexation is
observed within the period, as assessed by the slight decrease of
the absorption spectrum of the d−d transitions (only 6.4% at
6
70 nm; Figure S19 in the Supporting Information), which
indicates a higher degree of inertness of the complex in
comparison to the preciously studied CuL (t = 110 days)
1
1/2
Figure 8. Cyclic voltammograms of CuL at different pHs (V = 200
under the same conditions.
2
−
3
mV/s). Conditions: [CuL ] = 1.09 × 10 M; solvent, H O; I = 0.1 M
Radiolabeling Studies. The high stability of the Cu(II)
2
2
(
NaClO ); T = 25.0(2) °C.
4
complex prompted us to study the radiolabeling efficiency of
64
ligand L with CuCl in 0.1 M ammonium acetate buffer at
2
2
64
9
4
room temperature (Figure 9). Cu sources of different specific
NHE). Quasi-reversibility was also verified by measuring the
peak anodic (i ) and cathodic (i ) currents with varying scan
pa
pc
1/2
speed (v) at fixed pH. The linear plots of i or i = f(v ) are
pa
pc
shown in Figure S17 in the Supporting Information. The Ered
value is similar to the value obtained for ligand L (Ered = −0.56
1
V) and is well below the estimated −0.40 V (vs NHE)
30
threshold for typical bioreductants and below those of Cu(II)
complexes with bispidones L (E = −0.323 V vs NHE) and
0
red
4
HZ2 (Ered = −0.225 V vs NHE). More stable bispidine ligands
have been reported more recently; however, redox potentials
were measured in organic solvents such as DMF (E = −1.17
red
+
1b
+
V vs fc/fc , i.e. E = −0.72 V vs NHE for [Cu(bispa )] ) and
red
+
acetonitrile (Ered = −0.66 V vs fc/fc , i.e. E = −0.26 V vs
red
Figure 9. Thin-layer radiochromatogram of 64Cu-L after 30, 45, and
2
+
NHE for [Cu(N2py4)] ) and therefore cannot be compared
2
8
3
60 min reaction time at pH 5.4 at room temperature in 0.1 M
NH OAc (SiO , aqueous NH /MeOH/H O 1/2/1).
with our system. With such a low redox potential, the CuL2
complex should not be subject to reduction, demetalation, or
dismutation under physiological conditions. Below pH 5 and
above pH 8.5 more complex phenomena were observed
4
2
3
2
(
Figure S18 in the Supporting Information), in accordance
activities and cold metal impurities were used. For each
experiment, the total concentration in metals ([M]) of the
batch used was taken into account. Two series of measurements
were performed in order to optimize the radiolabeling
conditions. On the one hand, the influence of the ligand to
metal ratio was investigated at a fixed pH of 5.4, and on the
with the distribution curves showing the presence of other
chemical species.
Kinetic Inertness of CuL H in Strongly Acidic Media.
2
2
Good candidates for radiopharmaceutical applications exhibit
strong stability at physiological pH and in reductive medium
J
DOI: 10.1021/acs.inorgchem.7b01731
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
other hand, the influence of pH (i.e., pH 2−7) was studied for a
fixed L/M ratio.
Table 7. Time Dependence of the Radiolabeling Yields of L
at Different pH Values
2
a
Quantitative radiolabeling was achieved for ligand L after 30
2
t (min)
min at room temperature with L/M ratios of 1.25 and above
pH
5
10
40
70
15
49
30
45
60
(Figure 10). In the presence of a substoichiometric amount of
2.1
3.3
4.6
5.4
6.6
36
60
61
87
70
89
74
90
b
b
b
b
b
≥95
≥95
≥95
≥95
≥95
≥95
≥95
b
b
b
b
≥95
≥95
95
96
94
100
100
100
a
64
Conditions: room temperature; 0.1 M NH OAc; Cu from batch 1
pH 2.1−4.6), n(ligand) = 0.6 nm, n(Cu) = 8.04 pmol, Cu from
batch 2 (pH 4.6-6.6), n(ligand) = 0.11 nmol, n(Cu) = 22.4 pmol; L/M
4
6
4
(
b
=
1.25. No free Cu(II) was seen on radio-TLCs.
Figure 10. Radiolabeling yields of L (red ▲) at different ligand/metal
2
ratios. Conditions: 0.1 M NH OAc; pH 5.4; room temperature; 30
4
min; 64Cu from batch 1; 0.12 nmol ≤ n(ligand) ≤ 12.0 nmol; n(Cu) =
8.04 pmol.
ligand, radiolabeling did not occur at room temperature (Table
). This might be explained by a slower kinetics of the
6
Table 6. Time Dependence of the 64Cu Radiolabeling Yields
a
for L at Different Metal/Ligand Ratios
2
Figure 11. Radiolabeling yields of L (red ▲) at different pH values.
2
t (min)
64
Conditions: room temperature; 0.1 M NH OAc; 15 min; Cu from
4
6
4
L/M
5
10
15
30
45
60
batch 1 (pH 2.1−4.6), n(ligand) = 0.6 nm, n(Cu) = 8.04 pmol; Cu
from batch 2 (pH 4.6−6.6), n(ligand) = 0.11 nmol, n(Cu) = 22.4
pmol; L/M = 1.25. No free Cu(II) was seen on radio-TLCs.
0
0
0
1
2
.25
0
0
0
0
0
0
0
0
0
.5
0
12
0
11
0
11
.75
.25
.5
0
b
0
b
0
b
significantly slower under acidic conditions, probably due to the
≥95
92
≥95
93
≥95
95
96
94
competition with the protonated species L H , as emphasized
93
94
92
100
100
100
2
3
b
b
b
by potentiometric and X-ray crystallographic data. However,
above pH 5.4, an efficient and quantitative radiolabeling was
achieved at room temperature. With quantitative radiolabeling
12.5
≥95
100
≥95
100
≥95
100
100
100
100
100
25
a
Conditions: 0.1 M NH OAc; pH 5.4; room temperature; 64Cu from
batch 1; 0.12 nmol ≤ n(ligand) ≤ 12.0 nmol; n(Cu) = 8.04 pmol. No
free Cu(II) was seen on radio-TLCs.
4
−7
b
at ligand concentrations as low as 2 × 10 M, we believe that
6
4
L is a promising system for the complexation of Cu and that
2
it is worth investigating its in vivo behavior in further studies.
radiolabeling reaction, due to the presence of competitive metal
salts (in particular, Fe(II/III) was present at a concentration of
CONCLUSION
In conclusion, the phosphonate pendant-armed bispidol ligand
■
6
.38 ppm in batch 1, which is relatively high for such
production and purification processes). However, after
incubation of the samples at 80 °C for 1 h, the Cu-L2
complex was formed in 4%, 5%, and 22% yields for L /M ratios
L
2
was easily synthesized in three steps from piperidinone and
64
(aminomethyl)phosphonic acid. The evaluation of the
physicochemical properties of the corresponding Cu(II) and
Zn(II) complexes in water using UV−visible absorption
2
of 0.25, 0.5, and 0.75, respectively, at 80 °C, which indicate a
1
31
thermodynamic selectivity of L for Cu(II) over the other
spectrophotometry, potentiometry, H and P NMR, and
cyclic voltammetry as well as radiolabeling experiments with
2
cations. Moreover, very efficient radiolabeling (up to 95%) was
64
II
achieved within 30 min at room temperature and with L /M
Cu has been reported. Fast complexation of Cu(II) occurs in
the pH 4.6−6.6 range, and the complex demonstrates a strong
thermodynamic stability (log β = 22.5, pCu = 19.1 at pH
2
ratios of only 1.25. In the presence of a larger excess of ligand
(
5
25 equiv), quantitative radiolabeling was achieved within only
min. Under similar conditions, the radiochemical yield of
ligand L leveled at about 90%. In the case of L and L (Chart
CuL2
7.4) and a good selectivity for Cu(II) vs. Zn(II) (pZn = 15.4 at
pH 7.4), as well as a very good kinetic inertness regarding
reduction (with a reversible redox potential of Ered = −0.60 vs
NHE) and acid-assisted dissociation (t1/2 ≫ 20 months).
Quantitative radiolabeling (100% ± 5%) was achieved within 5
min at room temperature at pH above 5.4 at ligand
concentrations as low as 2 × 10− M. From a coordination
1
4
5
2
), radiochemical yields of 95% were obtained at room
−4
temperature with a large excess of ligand (10 M). These
results indicate a better radiolabeling capacity of the
phosphonate derivative L over the carboxylate and the pyridyl
2
7
analogues, which is partially explained by the stronger
thermodynamic stability of the copper(II) complex with L2
chemistry point of view, we believe that L meets all the
2
(
Table 6).
required criteria to be used as a new chelator for PET imaging
64
The influence of the pH was also investigated in the range
.1−6.6 (Table 7 and Figure 11). The results indicate that pH
with Cu. Although it is out of the scope of the present paper,
in vitro and in vivo stability studies as well as biodistribution
2
6
4
control is mandatory for ligand L , the reaction kinetics being
studies are still needed to validate the potential of L for Cu-
2
2
K
DOI: 10.1021/acs.inorgchem.7b01731
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
immuno PET imaging. Several functionalization strategies are
currently being studied in order to obtain bifunctional ligands
conjugated to antibodies.
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Remenyi, R.; Schiek, W.; Xiong, Y. Structural Variation in Transition-
̈
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Metal Bispidine Compounds. Chem. - Eur. J. 2002, 8 (24), 5750−5760.
(6) Bukowski, M. R.; Comba, P.; Lienke, A.; Limberg, C.; Lopez de
́
Laorden, C.; Mas-Balleste, R.; Merz, M.; Que, L. Catalytic Epoxidation
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.7b01731.
■
and 1,2-Dihydroxylation of Olefins with Bispidine−Iron(II)/H O
2
2
*
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Systems. Angew. Chem., Int. Ed. 2006, 45 (21), 3446−3449.
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Catecholase Activity of Dicopper(II)-Bispidine Complexes: Stabilities
and Structures of Intermediates, Kinetics and Reaction Mechanism.
JBIC, J. Biol. Inorg. Chem. 2006, 12 (1), 36−48.
1D and 2D NMR spectra, plots of potentiometric
(8) Comba, P.; Haaf, C.; Lienke, A.; Muruganantham, A.; Wadepohl,
titration data, plot showing the influence of scan speed
on current intensity at pH 7.4, cyclic voltammograms of
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Aziridination with a Tetraaza-Bispidine Ligand. Chem. - Eur. J. 2009,
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2
absorption spectra of the d−d transition on CuL H in 5
2
2
M HClO over 20 months at 25 °C (PDF)
4
2
(
012, 31 (7), 2841−2853.
Accession Codes
10) Scharnagel, D.; Muller, A.; Prause, F.; Eck, M.; Goller, J.; Milius,
̈
CCDC 1530205−1530206 contain the supplementary crystal-
lographic data for this paper. These data can be obtained free of
charge via www.ccdc.cam.ac.uk/data_request/cif, or by email-
ing data_request@ccdc.cam.ac.uk, or by contacting The
Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
W.; Breuning, M. The First Modular Route to Core-Chiral Bispidine
Ligands and Their Application in Enantioselective Copper(II)-
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AUTHOR INFORMATION
Corresponding Authors
E-mail for A.M.N.: aline.nonat@unistra.fr.
E-mail for L.J.C.: l.charbonn@unistra.fr..
ORCID
Aline M. Nonat: 0000-0003-0478-5039
̀
Loïc J. Charbonniere: 0000-0003-0328-9842
Notes
■
(
7), 2095−2099.
(
12) Ang, W. J.; Chng, Y. S.; Lam, Y. luorous Bispidine: A
*
Bifunctional Reagent for Copper-Catalyzed Oxidation and Knoevena-
gel Condensation Reactions in Water. RSC Adv. 2015, 5 (99), 81415−
81428.
*
(
13) Bautz, J.; Comba, P.; Que, L. Spin-Crossover in an Iron(III)−
Bispidine−Alkylperoxide System. Inorg. Chem. 2006, 45 (18), 7077−
082.
14) Atanasov, M.; Busche, C.; Comba, P.; El Hallak, F.; Martin, B.;
Rajaraman, G.; van Slageren, J.; Wadepohl, H. Trinuclear {M1}CN-
M2}2 Complexes (M1 = CrIII, FeIII, CoIII; M2 = CuII, NiII, MnII).
Are Single Molecule Magnets Predictable? Inorg. Chem. 2008, 47 (18),
112−8125.
7
(
The authors declare no competing financial interest.
{
ACKNOWLEDGMENTS
This work was supported by the by the French Centre National
de la Recherche Scientifique and the University of Strasbourg
UMR7178). The authors thank Mourad Elhabiri (Laboratoire
■
8
(
15) Atanasov, M.; Comba, P.; Helmle, S. Cyanide-Bridged FeIII−
CuII Complexes: Jahn−Teller Isomerism and Its Influence on the
Magnetic Properties. Inorg. Chem. 2012, 51 (17), 9357−9368.
(
(
de Chimie Medicinale et Bioorganique, UMR 7509 CNRS/
́
16) Kolanowski, J. L.; Jeanneau, E.; Steinhoff, R.; Hasserodt, J.
UdS) for providing the facilities for cyclic voltammetry
experiments as well as for thoughtful discussions. R.G. thanks
the French Ministry of research and higher education for
financial support for his Ph.D. The ARRONAX cyclotron is a
project promoted by the Regional Council of Pays de la Loire,
financed by local authorities, the French government, and the
European Union. This work has been, in part, supported by the
French National Agency for Research (“Investissements
d’Avenir” research grant Equipex Arronax-Plus no. ANR-11-
EQPX-0004 and Labex no. ANR-11-LABX-0018-01) and Labex
IRON.
Bispidine Platform Grants Full Control over Magnetic State of Ferrous
Chelates in Water. Chem. - Eur. J. 2013, 19 (27), 8839−8849.
(17) Comba, P.; Rudolf, H.; Wadepohl, H. Synthesis and Transition
Metal Coordination Chemistry of a Novel Hexadentate Bispidine
Ligand. Dalton Trans. 2015, 44 (6), 2724−2736.
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18) Juran, S.; Walther, M.; Stephan, H.; Bergmann, R.; Steinbach, J.;
Kraus, W.; Emmerling, F.; Comba, P. Hexadentate Bispidine
Derivatives as Versatile Bifunctional Chelate Agents for Copper(II)
Radioisotopes. Bioconjugate Chem. 2009, 20 (2), 347−359.
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Wadepohl, H. Optimization of Pentadentate Bispidines as Bifunctional
64
Chelators for Cu Positron Emission Tomography (PET). Inorg.
Chem. 2013, 52 (14), 8131−8143.
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