Interactions of N-heteroalkylaminomethylenebisphosphonic acids with Cd(II) ions: Electrochemical and spectroscopic investigations

Article abstract of DOI:10.1016/j.ica.2015.05.034

Abstract The aminomethylenebisphosphonates are known to be excellent chelators for many metal ions. In this work, the coordination properties of three different aminomethylenebisphosphonates with N-substituted heteroalkyl moieties (L1, L2 and L3) and their N-pyridyl derivative (L4) toward cadmium(II) ions are described. Due to their coordination abilities over a broad range of pH, the compounds of this group are good candidates for heavy metal detoxification. To determine the stability constants and the coordination mode of formed aminomethylenebisphosphonate-cadmium(II) complexes, four analytical methods were employed: potentiometry, pulse polarography (DPP), nuclear magnetic resonance spectroscopy (NMR) and electrospray ionization mass spectrometry (ESI-MS). The studies performed revealed that ligands L1, L2 and L3 possessed similar coordination properties, with Cd(II) ions binding below pH 2 to form equimolar complexes and above pH 6 to form bis-complexes. The ligand L4 behaved in a different way, forming six complexes of varied stoichiometry: CdH₄L₃, CdH₃L₃, CdHL₂, CdL₂, CdH₂L and Cd₃H₄L₃. The hypothetical competition plot between common complexone d-penicillamine and the studied ligands showed better complexation properties for the aminomethylenebisphosphonates in the pH range 2-6 than for neutral pH, where above neutral pH d-penicillamine bound Cd(II) ions significantly better.

Full text of DOI:10.1016/j.ica.2015.05.034

Accepted Manuscript
Interactions of N-heteroalkylaminomethylenebisphosphonic acids with Cd(II)
ions: Electrochemical and spectroscopic investigations
Agnieszka Dobosz, Jan Spychała, Tomasz Ptak, Gabriela Maciejewska, Ewa
Chmielewska, Paweł Kafarski, Piotr Młynarz
PII:
S0020-1693(15)00300-X
http://dx.doi.org/10.1016/j.ica.2015.05.034
ICA 16568
DOI:
Reference:
Inorganica Chimica Acta
To appear in:
Received Date:
Revised Date:
Accepted Date:
5 February 2015
21 May 2015
28 May 2015
Please cite this article as: A. Dobosz, J. Spychała, T. Ptak, G. Maciejewska, E. Chmielewska, P. Kafarski, P.
Młynarz, Interactions of N-heteroalkylaminomethylenebisphosphonic acids with Cd(II) ions: Electrochemical and
spectroscopic investigations, Inorganica Chimica Acta (2015), doi: http://dx.doi.org/10.1016/j.ica.2015.05.034
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Interactions of N-heteroalkylaminomethylenebisphosphonic acids with Cd(II) ions:
Electrochemical and spectroscopic investigations
Agnieszka Dobosza*, Jan Spychałaa, Tomasz Ptaka, Gabriela Maciejewskab, Ewa
Chmielewskab, Paweł Kafarskib,c, Piotr Młynarzb
aWroclaw Medical University, Department of Basic Medical Sciences, Borowska 211 Str.,
50-556 Wroclaw, Poland
bDepartment of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of
Technology, Wybrzeże Wyspiańskiego 27, 50-370 Wrocław, Poland
cDepartment of Analytical and Ecological Chemistry, University of Opole, ul. Kopernika 11a,
45-040 Opole, Poland
*corresponding author: agnieszka.dobosz@umed.wroc.pl, phone: +48 717840482
Abstract:
The aminomethylenebisphosphonates are known to be excellent chelators for many
metal ions. In this work, the coordination properties of three different
aminomethylenebisphosphonates with N-substituted heteroalkyl moieties (L1, L2 and L3)
and their N-pyridyl derivative (L4) toward cadmium(II) ions are described. Due to their
coordination abilities over a broad range of pH, the compounds of this group are good
candidates for heavy metal detoxification.
To determine the stability constants and the coordination mode of formed
aminomethylenebisphosphonate-cadmium(II) complexes, four analytical methods were
employed: potentiometry, pulse polarography (DPP), nuclear magnetic resonance
spectroscopy (NMR) and electrospray ionisation mass spectrometry (ESI-MS). The studies
performed revealed that ligands L1, L2 and L3 possessed similar coordination properties,
with Cd(II) ions binding below pH 2 to form equimolar complexes and above pH 6 to form
bis-complexes. The ligand L4 behaved in a different way, forming six complexes of varied
stoichiometry: CdH4L3, CdH3L3, CdHL2, CdL2, CdH2L and Cd3H4L3. The hypothetical
competition plot between common complexone D-penicillamine and the studied ligands
showed better complexation properties for the aminomethylenebisphosphonates in the pH
range 2-6 than for neutral pH, where above neutral pH D-penicillamine bound Cd(II) ions
significantly better.
1

Keywords: aminobisphosphonates, metal ions, coordination, Cd(II) ions, polarography,
potentiometry, mass spectrometry MS, nuclear magnetic resonance NMR
Graphical Abstract:
Cd(D-Pen)2
Cd3H4(L4)3
Cd(D-Pen)
100
90
80
70
60
50
40
30
20
10
0
Cd(II)
CdH3(L4)3
Cd(L4)2
Cd3(D-Pen)4
CdH4(L4)3
CdH(L4)2
CdH2(L4)
2
3
4
5
6
7
8
9
10
11
pH
Highlights:
•
•
Aminobisphosphonic acids revealed strong chelating abilities for Cd(II) ions.
The potentiometric data were supported with differential pulse polarography, NMR
spectroscopy and ESI-MS.
• Binding abilities of aminobisphosphonates were compared to the common metal
chelator, penicillamine performing better in the acidic pH range, comparably in the
neutral range and worse in the basic pH range.
1.
Introduction
2

Bisphosphonates are a group of pyrophosphate analogues containing a P-C-P scaffold
in place of the P-O-P fragment. Despite limitations resulting from their low uptake, they are
the drugs of choice in curing postmenopausal osteoporosis [1,2]. The most potent
bisphosphonates are those containing amino groups in their alkyl side chain.
Aminomethylenebisphosphonates are the fourth generation of these drugs, with zolendronate
the most successful example [3,4].
Aminomethylenebisphosphonates have been used in medicine as oncological
therapeutics because they are able to inhibit tumor cell migration, bone adhesion and
apoptosis-limiting properties [5]. These compounds have also been used in cancer
immunotherapy as activators of γδ T lymphocytes [6].
Cadmium, a heavy metal, is a significant environmental pollutant [7]. Small quantities
of this metal occur naturally in water, soil, air and unfortunately in food [8]. Due to their
ubiquity, cadmium ions often pass into the human body. They can be a serious health hazard,
not only to humans but to all living organisms, because of their ability to bind certain
metalloenzymes and enzyme substrates containing thiol groups [9]. Cd(II) ions are chemically
similar to Zn(II) and can replace them in apoproteins, inhibiting or distorting the catalytic
activity of the metalloenzymes [10]. The best example of Zn(II)ꢀCd(II) replacement occurs
in metallothioneins (MT) [11]. In the absence of cadmium(II) ions, metallothioneins contain
seven Zn(II) ions. When Cd(II) ions appear in the human body, Zn(II) ions are substituted
with Cd(II) in newly synthesized MT. Cd-MT and Zn-MT have identical stoichiometry [11].
Because of high cadmium toxicity, it is important to have coordinating agents, which
can bind cadmium ions to remove them from the environment and living organisms.
Bisphosphonates, which are well known to bind Zn(II) effectively and have short residence
times in the human body, seem to be obvious candidates for that function. There are numerous
literature data describing properties of Zn(II) complexes with aminobisphosphonates but only
a few studies demonstrating interaction of Cd(II) ions with these compounds [12,13]. For
example in the studies performed by Matczak-Jon et al. the same ligands as in this work (L1,
L2 and L3, Scheme 1) were tested towards coordination properties of Zn(II), Mg(II) and
Ca(II) ions [14,15].
3

S
N
O
N
N
HO
O
OH
O
HO
O
HO
O
OH
O
OH
O
P
P
P
P
P
P
OH
OH
OH
HO
HO
HO
L3
L1
L2
O
HO
P
P
OH
OH
NH
HO
L4
N
O
Scheme1. Studied ligands:
L1 – (piperyd-1-yl)aminomethanebisphosphonic acid;
L2–(thiomorpholin-1-yl)aminomethane-bisphosphonic acid;
L3- (morpholin-1-yl)aminomethanebisphosphonic acid;
L4 - (pyridin-2-yl)aminomethanebisphosphonic acid
There are some crystallographic data available, concerning Cd(II) binding into the
phosphonic group. One of them is the work of Li et al. [16] who showed that cadmium(II)
ions in triphosphonic-cadmium(II) complexes are mainly coordinated octahedrally by
phosphonate oxygen atoms from the chelating agents. On the other hand Sun et al.
characterized some cadmium(II) structures of aminodiphosphonates [17].
The main goal of this work was to find both the coordination models and the
formation constants of complexes formed between cadmium(II) ions and three
aminomethylenebisphosphonates (L1, L2 and L3) to determine the characteristics and
strength of the formed complexes. For comparison, the same properties of the intensively
studied N-pyridyl derivative (L4 - Scheme 1) have been determined [18,19,20].
4

2. Experimental
2.1. Materials and reagents:
General Information
All solvents and reagents were purchased from commercial suppliers, were of analytical grade
(A. R.) and were used without further purification. Unless otherwise specified, solvents were
1
31
removed with a rotary evaporator. H, P and 13C NMR experiments were performed on a
Bruker BioSpin operating at 500.14 MHz (1H), 202.46 MHz (31P) and 125.76 MHz (13C).
Measurements were made in D2O+ NaOD (99.9 at. % D). The solution temperature was 300
K, and the solvent was supplied by ARMAR AG (Dottingen, Switzerland). Chemical shifts
are reported in parts per million relative to TMS or 85% H3PO4 used as external standards,
and coupling constants are reported in Hertz. Melting points were determined on an
Electrothermal 9200 apparatus and are reported uncorrected. Elemental analyses were
performed at the Department of Chemistry at the University of Wroclaw on a Perkin Elmer
2400 CHN. Electrospray mass spectra were recorded at the Faculty of Chemistry at Wroclaw
University of Technology using a Waters LCT Premier XE mass spectrometer with ESI used
as the method of ionization.
2.1.1. Bisphosphonate synthesis
The diphosphonic acids were obtained according to the previously described
procedure [21]. The purity of the ligands was established by NMR spectroscopy.
General procedure of the synthesis:
A mixture of amine (0.03 mol), trialkyl (preferently triethyl) phosphite (0.062 mol) and
trialkyl (preferently triethyl) orthoformate (0.032 mol) was heated with stirring at elevated
temperature (80-120°C) for 14 - 18 h. Then, the volatile components of the reaction mixture
were evaporated and the resulting crude product was dissolved in chloroform (150 ml). The
organic layer was washed with water (150 ml), then with saturated sodium chloride (150 ml)
and again with water (150 ml) to remove any excess diethyl phosphite. The chloroform layer
was then dried over sodium sulfate. The drying agent was removed by filtration, and the
solvent was removed under reduced pressure. Crude esters were then subjected to hydrolysis.
Hydrolysis – general procedure
The obtained ester (0.030 M) was refluxed for 8-12 hours in 20 ml 6M aqueous hydrochloric
acid solution. Then, the volatile components were evaporated, and the resulting crude
5

bisphosphonate was purified by recrystallized from water or water-ethanol mixture.
(Piperyd-1-yl)aminomethanebisphosphonic acid (L1) was obtained in 64% (4.97 g) yield as a
31
white solid; m.p. 242-243°C (lit. 250-255°C [22], 245-247°C [23]); PNMR (202.46 MHz,
1
D2O+NaOD, ppm): δ= 8.54; HNMR (500.14 MHz, D2O+ NaOD, ppm): δ= 1.47 (2H, d, J=
4.65), 1.68 (4H, d, J=4.90 Hz,), 2.94 (1H, t, J=18,86 Hz, PCHP), 3.43 (4H, t, J=6.35 Hz)
13
ppm; CNMR (125.76 MHz, D2O+NaOD, ppm): δ= 21.32, 24.90, 53.51, 66.95 & 66.99 (t,
Hz, J=114.62 Hz,PCP); HRMS (ESI-TOF) m/z: [M-H]+ Calcd for C6H15NO6P2 258.0294,
Found 258.0288.
(Thiomorpholin-1-yl)aminomethanebisphosphonic acid (L2) was obtained in 53% (4.41 g)
yield as a white solid; m.p. 239-240°C (lit. 250°C [24]); 31PNMR (202.46 MHz, D2O+NaOD,
ppm): δ= 16.39; 1HNMR (500.14 MHz, D2O+ NaOD, ppm): δ= 2.57 (4H, t, J=4.95 Hz), 2.66
(1H, t, J=22.41 Hz, PCHP),3.18 (4H, t, J=4.79 Hz,), ppm; 13CNMR (125.76 MHz,
D2O+NaOD, ppm): δ= 27.82 (t, J=5.72 Hz) 53.13, 68.44 & 68.49 (t, J=123.68 Hz, PCP);
HRMS (ESI-TOF) m/z: [M-H]+ Calcd for C5H13NO6P2S 275.9861, Found 275.9867.
(Morpholin-1-yl)aminomethanebisphosphonic acid (L3) was obtained in 59%(4.62 g) yield as
a white solid; m.p. 247°C (lit. 250-260°C [22], 248-250°C [23]); 31PNMR (202.46 MHz,
1
D2O+NaOD, ppm): δ= 16.39; HNMR (500.14 MHz, D2O+ NaOD, ppm): δ=2.71 (1H, t,
J=22.10 Hz, PCHP), 3.00 (4H, t, J=4.20 Hz,), 3.61 (4H, t, J=4.52 Hz) ppm; 13CNMR (125.76
MHz, D2O+NaOD, ppm): δ= 51.12 (t, J=4.85 Hz), 66.66 & 66.67 (t, J=123.62 Hz, PCP),
67.57; HRMS (ESI-TOF) m/z: [M-H]+ Calcd for C5H13NO7P2 260.0089, Found: 260.0081.
Anal. Calcd for C5H13NO7P2: C, 23.00; H, 5.02; N, 5.36; P, 23.72. Found: C, 22.98; H, 4.86;
N, 5.27; P, 22.10.
(Pyridin-2-yl)aminomethanebisphosphonic acid (L4) was obtained in 46% (3.71 g) yield as a
31
1
white solid; m.p. 280-282°C; PNMR (202.46 MHz, D2O+NaOD, ppm): δ= 14.03; HNMR
(500.14 MHz, D2O+ NaOD, ppm): δ=3.81 (1H, t, J=18.67 Hz, PCHP), 6.40 (1H, t, J=6.50
Hz, ArH), 6.83 (1H, d, J=9.20 Hz ArH), 7.23(1H, d, J=4,85 Hz ArH), 7.56 (1H, t, J=8,75 Hz,
ArH) ppm; 13CNMR (125.76 MHz, D2O+NaOD, ppm): δ= 51.74 (t, J=125.76 Hz, PCP),
1
31
13
111.47, 136.48, 142.74, 152.96; lit H, P, C NMR [18]; HRMS (ESI-TOF) m/z: [M-H]+
Calcd for C6H10N2O6P2 266.9936, Found: 66.9928. Anal. Calcd for C6H10N2O6P2x ½ H2O: C,
26.01; H, 4.00; N, 10.11; P, 22.35. Found: C, 26.18; H, 3.81; N, 10.035; P, 21.82.
2.1.2. Metal ions
Cd(NO3)2×4H2O was obtained from POCH S.A. (Poland). The metal ion stock solution was
prepared by dissolving the metal salt in distilled, deionized water. It was used as the source of
6

cadmium(II) ions in the potentiometric, polarographic and NMR studies. All other chemicals
used were A.R. grade quality and were obtained from Sigma-Aldrich Chem. Co.
2.2. Potentiometric studies:
Potentiometric measurements were made using a MOLSPIN 1000 automatic titration
system with a METTLER TOLEDO InLab 422 microcombined electrode. The titrator and the
electrode were calibrated daily using standard buffer solutions. All titrations were performed
3
in a purified argon atmosphere, using aqueous 0.098642 mol・dm− NaOH as the titrant.
Approximately 170 titration data points were measured, covering the pH range of 2.2--11.8 for
ligand and metal complexes. The pH meter readings were converted into hydrogen ion
3
concentration by titrating a standard acid solution (HNO3, c= 0.004 mol・dm− ) with a base
3
solution at 25°C and I =0.1 mol・dm− KNO . The concentration of ligands was 0.002
3
3
mol・dm− , and the molar ratio of M:L was 1:1, 1:2 and 1:3. All the solutions were prepared
using deionized, distilled water.
Calculations were performed using SUPERQUAD [25]. The model selected was the
one that gave the best statistical fit and was chemically consistent with the titration data. The
concentration distribution diagrams were obtained using SPECIES [26].
2.3. Polarographic studies:
Polarographic measurements were performed using a MTM M161 electrochemical
analyzer (MTM Anko, Kraków, Poland) interfaced with M164C cell stand (MTM Anko,
Kraków, Poland) in a three-electrode cell configuration. A hanging mercury drop electrode
(HMDE), an Ag/AgCl/KCl 3 M and a platinum wire served as the working, reference and
auxiliary electrodes, respectively. Experiments were carried out under an argon atmosphere at
25°C in 0.1 mol・dm-3 KNO3 as medium. The solutions were purged with argon (Ar-99%)
for 10 min before each experiment. The differential pulse polarographic (DPP) measurements
on HMDE were performed with a pulse amplitude of 25 mV, a scan range from -0.4 V to -0.8
V and at scan rate of 2 or 5 mV/s.
The cadmium ion concentration was approximately 2・10-4 mol·dm-3. The metal-to-
ligand mol ratios ranged from 1:2 to 1:40. All ligands were diluted in 0.1 mol·dm-3 KNO3. The
stability constants from the polarographic data were evaluated according to the Lingane
method modified by De Ford and Hume [27].
7

2.4. Spectroscopic studies:
2.4.1. NMR studies
1
31
The H and P NMR spectra were collected at 600 MHz using a Bruker Avance
instrument at 298 K. The ligands were dissolved in D2O with trimethylsilyl propanoic acid
(TSP), using phosphoric acid as an external standard. NMR pD titrations were made using a
SevenMulti (Mettler Toledo) pH-meter with an InLab Micro Pro (Mettler Toledo) combined
electrode. The concentration of ligand was 4-5 mM.
2.4.2. ESI-MS measurements
The electrospray ionization mass spectra were recorded on a Waters GCT Premier
mass spectrometer. The ligand concentration was 1・10-3 mol·dm-3 and the metal-to-ligand
ratio was 1:3; 5% of MeOH was added to each sample. Each complex was measured at three
different pH (4, 7 and 9) reached using small amounts of formic acid and NH4OH. These pH
values were chosen to maximize species concentration in metal-to-ligand systems. The
desolvation temperature was 300°C, while the source temperature was 120°C. The spectra
were recorded in positive and negative ion mode.
3. Results and discussion:
3.1. Acid-base properties of compounds L1-L4
The studied compounds in their fully protonated form possess five dissociable protons
[H5L]+. For ligands L1, L2 and L3, four protons can be released from the two phosphonate
groups (PO3H2) and one from the heterocyclic nitrogen atom, whereas L4 has an additional
pyridine nitrogen.
Ligands L1, L2 and L3 have similar structures; thus, their pK values are similar to
each other (Table 1). Compound L1 has been previously studied by Matczak-Jon et al., and
only two dissociation constants were found for this compound, corresponding to the proton
released from the PO3H- groups (pK=8.16 and pK=4.60) [14]. Compounds L2 and L3 were
also studied by these authors [14], and their pK values were similar to those found in our
studies. Both phosphonate groups (PO3H-), have lower pK values than compound L4 [19].
The first dissociation step can be observed and roughly estimated (pK ~ 1.08, Table.1)
for only ligand L4, which corresponds to the first proton dissociation from PO3H2 group,
which usually lies in the range of pH 1-2. Measurement difficulty is due to the strong acidity
8

of the first protonation site in phosphonate groups, which is usually unmeasurable. A similarly
low pK for this ligand was also noted by Matczak-Jon et al. [18,20]. As shown in Table 1,
compound L4 releases two more protons in the measurable pH range. The proton with
pK=5.50 originated from the pyridine nitrogen NH+pyridine at a similar value to those shown in
literature data [18]. The second proton is released from a PO3H2 group (pK~ 1.08). For this
proton, there is also good agreement between the literature and measured dissociation
constant values [18,20].
Unlike ligand L4, the heterocyclic nitrogen atom of compounds L1, L2, and L3 is
directly bound to the methanebisphosphonate structural component of the molecule. The
strong basicity of the piperidyl nitrogen proton increases this pK. For our titration conditions,
it was measured for ligands L1 (pK ~12.63), L2 (pK ~12.17) and L3 (pK~11.63) (see
Table.1). Matczak et al. found this constant for compound L2 to be 11.91 and for L3 to be
11.65 [14]. The high basicity of the nitrogen can be explained by the presence of an
intermolecular hydrogen bond. Such an N-H…O (phosphonate) bond exists, for example, in
ligand L1 [28, 29]. However, protonation values for ligands L1-L3 seem to be correlated with
the electronegativity of the heteroatom in the piperydyl ring when S<O. The same trend in
protonation site acidity was also observed in other studies [14].
Table 1. Logarithmic protonation constants (logβ) and pK values for ligands L1-L4 at 25°C
and I=0.1 mol/dm-3 KNO3
Compound
L1
L2
L3
L4
S
O
O
HO
Assignment
P
P
OH
OH
N
NH
HO
N
N
N
HO
O
OH
O
HO
O
HO
O
OH
O
OH
O
P
P
P
P
P
P
O
OH
OH
OH
HO
HO
HO
log β1
log β2
log β3
log β4
12.63(5)
21.35(5)
26.26(5)
-
12.17(2)
20.62(3)
25.39(3)
-
11.63(2)
19.93(2)
24.68(2)
-
10.32(1)
18.13(1)
23.63(1)
24.71(6)
pK (NH+)
12.63
12.17
8.45
11.63
8.30
pK (PO3H-) 8.72
10.32
9

pK (PO3H-) 4.91
pK (PO3H2)
4.77
3.75
7.81
~1.08
5.50
pK (NH+
)
pyr
3.2. Cadmium(II) complexes
3.2.1. Potentiometric studies
Potentiometric measurements were used to determine the stability constants of
complexes formed between ligands L1-L4 and cadmium(II) ions (Table 2). Subsequently,
these stability constants were used to draw speciation diagrams indicating the species formed
in solution for a pH range from 2 to 11 (Figure 1).
Table 2. Stability constants (logβ) and pK values for Cd(II) complexes with measured
compounds
L1
L2
L3
L4
Species
Logβ
pK logβ
pK Logβ
pK Logβ
pK
CdH4L3
CdH3L3
-
-
-
-
-
-
53.47(2)
46.00(3)
-
CdH2L2 35.70(7)
34.75(7)
33.62(7)
10.70
9.48
9.12
CdHL2
25.00(7)
25.27(7)
24.50(7)
21.86(4)
11.71
11.38
10.59
CdL2
-
13.56(9)
24.28(2)
13.12(9)
23.68(2)
11.27(4)
22.72(2)
CdH2L
25.05(2)
5.20
4.86
4.79
CdHL
19.85(3)
-
19.42(3)
-
18.89(3)
-
-
Cd3H4L3
67.65(4)
L1
L2
10

100
80
60
40
20
0
10
9
8
7
6
5
4
3
2
1
0
100
80
60
40
20
0
10
9
8
7
6
5
4
3
2
1
0
CdHL
Cd(II)
CdHL
Cd(II)
CdHL
2
CdH L
2
CdH L
2
2
CdH L
2
CdH L
2
2
CdHL
2
CdL
2
2
3
4
5
6
7
8
9
10
11
2
3
4
5
6
7
8
9
10
11
pH/pD
pH/pD
L3
L4
Cd H L
4 3
100
80
60
40
20
0
10
100
80
60
40
20
0
10
3
CdHL
Cd(II)
Cd(II)
CdH L
9
8
7
6
5
4
3
2
1
0
9
8
7
6
5
4
3
2
1
0
3
3
CdHL2
CdH L
2
CdL2
CdH L
4
3
CdHL
2
CdL2
CdH L
2
CdH L
2
2
2
3
4
5
6
7
8
9
10
11
2
3
4
5
6
7
8
9
10
11
pH/pD
pH/pD
Figure 1. The distribution of complexes for studied ligands and Cd(II) ions in a 3:1 ratio
31
(concentration of 4 mM) versus the chemical shift of bisphosphonate peaks in P NMR as a
function of pD for free (□) and bound ligands (●)
Titration curves for all studied ligands are present in Figure 2. Due to precipitation, L3
has only three titration curves present in the diagram (free ligand and 1:2 and 1:3 M:L molar
systems). For the remaining ligands, there was no precipitation in the 1:1 molar ratio system
but the solution was slightly opalescent between pH 7 and 8, which is not recorded in the
diagrams. Due to this opalescence, only the data obtained from the titrations at M:L 1:2 and
1:3 molar ratios were used in the calculations presented in Table 2.
11

300
250
200
150
100
50
300
250
200
150
100
50
L1
L1:Cd(II) 1:1
L2:Cd(II) 1:1
L1:Cd(II) 2:1
L2:Cd(II) 2:1
L2:Cd(II) 3:1
L2
L1:Cd(II) 3:1
0
0
-50
-50
-100
-150
-200
-250
-300
-100
-150
-200
-250
-300
0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20 0.22
0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20 0.22
VNaOH [µl]
VNaOH [µl]
300
300
250
200
150
100
50
250
200
150
100
50
L4:Cd(II) 1:1
L3:Cd(II) 2:1
L4
L4:Cd(II) 2:1
0
0
-50
-50
L3
-100
-150
-200
-250
-300
-100
-150
-200
-250
-300
L4:Cd(II) 3:1
L3:Cd(II) 3:1
0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20 0.22
0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20 0.22
VNaOH [µl]
VNaOH [µl]
Figure 2. Titration curves of free L1, L2, L3 and L4 aminobisphosphonic ligands and their
Cd(II) complexes with a metal-to-ligand ratio from 1:1 to 1:3.
The studied compounds represent a class of bisphosphonates in which the nitrogen
atom is attached directly to the α-carbon. Such compounds have a tendency to form
protonated multinuclear soluble complexes [20]. However, there are also some pyridine
bisphosphonates that form complexes with a 1:1 or 1:2 metal-to-ligand ratio with various
metal ions [18].
Although the compounds L1, L2, and L3 might be considered similar to each other,
metal complex speciation differs slightly among the studied ligands. According to the
potentiometric results, the Cd(II) ion coordination begins at pH below 2 for all compounds
(Figure 1). This result indicates the very strong binding ability of these compounds. The
complex formation processes for L1, L2 and L3 are very similar, and they form almost the
same types of complexes (excepting the CdL2 form for L1) (Table 2). All three heteroaliphatic
12

ligands (L1-L3) have a strong tendency to form equimolar species CdH2L and CdHL, which
predominate in the pH range of 2-8.5. (Figure 1, Table 2). These common complex forms are
the major species in acidic and neutral environments. The ligands may coordinate the Cd(II)
ion using oxygen donors originating from phosphonate groups, retaining one protonated
oxygen and nitrogen atom. Then, the subsequent deprotonation might lead to engagement of
the remaining oxygen in the complex formation or remain it not involved. At pH above 6, bis-
complexes begin to appear, forming the following species: CdH2L2, CdHL2 and CdL2 (see
Figure 1). However, L1 is the exception as it does not form a CdL2 complex. In the first
complex, CdH2L2, the Cd(II) ion has four oxygen donors, {4O}, and then consecutive
deprotonation occurs, most likely with the involvement of at least one nitrogen donor. This
phenomenon is justified by the significant reduction in the pKa of tertiary amine NH+ groups
leading to the CdL2 complexation form (Table 1,2). The final comparison of the binding
properties of ligands L1-L3 toward Cd(II) ions reveals that the lowest pKa values, probably
caused by the ring electronegative atom, have the most efficient coordination properties
(Figure 3).
L4 has quite different coordination behavior from those of L1, L2, and L3 because,
with the exception of equimolar and bis-complexes, this compound is the only one which
forms CdH4L3, CdH3L3 and the single trinuclear complex with stoichiometry Cd3H4L3. The
diverse nature of the resulting complexes is not surprising if one considers that L4 has a
substantially different N-substituent and rigid aromatic ring from those of L1, L2 and L3,
which can prompt the formation in acidic solution of trinuclear complexes similar to these
found for Cu(II) ions [30].
13

100
90
80
70
60
50
40
30
20
10
0
free Cd(II)
L4
L3
L2
L1
10
2
3
4
5
6
7
8
9
11
pH
Figure 3. Hypothetical competition plot for the Cd(II)–L1-L4 system assuming a 1:3:3 molar
3
ratio and the stability constants obtained for binary species; CL= 2×10-3mol・dm−
To compare the efficacy of cadmium(II) binding by the studied ligands (L1-L4), a
hypothetical competition plot was made (Figure 3). This competition plot shows that L4 is a
better chelator for Cd(II) ions than L1, L2 and L3.
It was previously established that the presence of a thiol group in the ligand structure
increases the binding ability of cadmium(II) ions [31]. The metal ions can be bound by
{S,N,O} donor sets with a strong tendency to form oligonuclear species. Therefore, it was
interesting to compare our ligands with a common drug containing a thiol group, D-
penicillamine (D-Pen). The data obtained in work performed by Kozlowski et. al. [31] was
used in a hypothetical competition plot to determine which ligand (thiolate or
aminomethylenebisphosphonic acids) is the better chelating agent for Cd(II) ions. Analysis of
the figure (Figure 4) showed that at acidic pH, L4 seems to be the better complexone for
Cd(II) ions and that at pH above 7 D-Pen is the more potent chelating agent.
14

D-Pen
free Cd(II)
L4
100
90
80
70
60
50
40
30
20
10
0
2
3
4
5
6
7
8
9
10
11
pH
Figure 4. Hypothetical competition plot for the Cd(II)–L4-D-Pen system assuming a 1:3:3
3
molar ratio and the stability constants obtained for binary species; CL= 2×10-3mol・dm−
The same study was conducted for the remainder of our ligands. The hypothetical
competition plot made for them and for D-panicillamine shows that they are better chelating
agents than thiols only at pH 2-5.5 (Figure 5).
15

D-Pen
100
90
80
70
60
50
40
30
20
10
0
free Cd(II)
L3
L2
L1
2
3
4
5
6
7
8
9
10
11
pH
Figure 5. Hypothetical competition plot for the Cd(II)–L1-L2-L3-D-Pen system assuming a
3
1:3:3 molar ratio and the stability constants obtained for binary species; CL= 2×10-3mol・dm−
According to the literature data, L2 and L3 form dinuclear complexes with Zn(II) ions
[14], but 1:1 and 1:2 metal-to-ligand complexes are also present in solution. These complexes
are similar to the ones we found for Cd(II) ions with the exception of CdH2L, which was
found in our coordination model. Even the stability constants are quite similar for Zn(II) and
Cd(II) systems with L2 and L3 [14].
The complexation behavior of some phosphonates toward Cd(II) ions has been
observed by Deluchat et al. [13]. This study observed complexes of 1-hydroxyethane-1,1’-
diphosphonic acid with cadmium(II) ions at a molar ratio M:L=1:1, so they could observe
only equimolar species. They found the same type of cadmium(II) complexes (CdH2L, CdHL,
CdL) but the stability constants were 3 orders of magnitude lower for CdH2L and CdHL than
in this work. Only the CdL species has a similar logβ [13]. This may be the result of the
involvement of hydroxy- groups present in the ligand in the complex formation.
According to Matczak-Jon et al., at acidic and neutral pH L4 forms three dinuclear
comlexes in which three or four ligand molecules are engaged in Zn(II) ion binding [20].
16

They also postulated the formation of three protonated bis-complexes and one equimolar
species under alkaline conditions. Our investigations with L4 have shown the formation of
1:1, 3:3, 1:3 and 1:2 metal-to-ligand ratio complexes with Cd(II) ions. However, the results of
potentiometric studies of L4 with Zn(II) ions revealed neither the trinuclear species nor the
1:3 metal-to-ligand ratio species [20].
Other previous results were achieved by Kowalik-Jankowska et al. who also studied
complexes of L4 ligand with copper(II) ions [30]. They suggested the formation of trinuclear
and mononuclear Cu(II) complexes. The formation of M3L3 species with 1-hydroxyethane-
1,1-diphosphonic acid was also suggested by Bouhsina et. al [32].
All of the ligands investigated in our work were studied previously as complexones of
Zn(II) ions [14,15,20]. Zn and Cd lie in the same column in the periodic table, and thus it
would be interesting to compare the stability constants of formed complexes. By using the
stability constants for Zn(II) complexes taken from the literature [14,15,20] and the obtained
results for Cd(II) complexes, such a comparison was made and the hypothetical competition
plots for each ligand were obtained (Figure 6). The mixture of each ligand and both metal ions
was fixed at Zn(II):Cd(II):L 1:1:2 molar ratio. These hypothetical systems revealed that L2
and L3 prefer Zn(II) binding in pH 6.5-9.5, but in very acidic pH, they prefer Cd(II) binding.
L4 is the only ligand that prefers Zn(II) binding even at pH 2. In basic solution
(approximately pH 8.5), L4 has a greater affinity to Cd(II) than Zn(II) ions. L1 prefers Cd(II)
binding in the entire pH range studied. The reason for this is probably that the stability
constants calculated for Zn(II) complexes of L1 [15] are significantly lower than the similar
Cd(II) complexes calculated in this work.
L1
L2
100
90
80
70
60
50
40
30
20
10
0
100
90
80
70
60
50
40
30
20
10
0
Zn(II) complexes
Cd(II) complexes
Cd(II) complexes
Zn(II) complexes
2
3
4
5
6
7
8
9
10
11
2
3
4
5
6
7
8
9
10
11
pH
pH
17

L3
100
90
80
70
60
50
40
30
20
10
0
100
90
80
70
60
50
40
30
20
10
0
L4
Zn(II) complexes
Zn(II) complexes
Cd(II) complexes
Cd(II) complexes
2
3
4
5
6
7
8
9
10
11
2
3
4
5
6
7
8
9
10
11
pH
pH
Figure 6. Hypothetical competition plot for 1:1:2 Cd(II):Zn(II):L systems assuming the
3
stability constants obtained for binary species; CL= 2×10-3mol・dm− .
3.2.2. Polarographic studies
The data obtained from potentiometry are reliable if they can be supported with studies
carried out using different technique. To confirm the coordination model calculated by
potentiometry, differential pulse polarography (DPP) was used as a supplementary method.
First, the titrations of the compounds L1-L4 with Cd(II) ions were made. The metal to ligand
ratio of 1:75 – 1:77 (cM=1.0122×10-4 mol·dm-3) was used to determine the dominant species
at each pH [33,34]. The cadmium(II) reduction potential was measured when each titrant
addition was made, followed by plotting the titration curve (Figure 7).
18

0.7
0.65
0.6
12
10
8
L1+Cd(II)
L2+Cd(II)
L3+Cd(II)
L4+Cd(II)
L1+Cd(II)
L2+Cd(II)
L3+Cd(II)
L4+Cd(II)
6
4
0.55
0.5
2
0
0
200
400
600
800
1000
1200
0
200
400
600
800
1000
1200
VNaOH [µL]
VNaOH[µL]
Figure 7. Polarographic titration curves, with the shift in the peak potential measured each
time the NaOH was added to a polarographic cell
The curves were analyzed to determine the pH range where the dominant complexes
were present (Figure 7) [12]. For every ligand (L1-L4) the proper pH values were found, and
the remaining DPP measurements were performed at these pH values. The plots in Figure 8
show the dependence of the reduction potential shifts of Cd(II) ions on the concentration of
the aminomethanebisphosphonates. When this dependence is linear it confirms that in the pH
range under investigation only one complex form is dominant.
19

L2+Cd(II)
L1 + Cd(II)
6
5
4
3
2
1
0
6
5
4
3
2
1
0
pH 4.70
pH 7.45
pH 9.92
pH 4.5
n=1.97
r2=0.9947
pH 7.46
pH 9.07
pH 11.56
n=1.77
r2=0.9969
n=2.01
r2=0.9958
n=1.00
r2=0.9948
n=0.95
r2=0.9837
n=0.16
r2=0.6777
n=0.74
r2=0.9855
−2.8
−2.6
−2.4
−2.2
−2
−3
−2.8
−2.6
−2.4
−2.2
−2
log CL
log CL
L3 + Cd(II)
L4 + Cd(II)
6
5
4
3
2
1
0
7
6
5
4
3
2
1
0
pH 3.45
pH 4.58
pH 4.7
pH 7.45
pH 9.21
pH 11.25
pH 5.03
pH 8.27
pH 9.60
n =2.73
r2 = 0.9971
n=2.06
r2=0.9954
n = 1.68
r2 = 0.9977
n=1.66
r2=0.9961
n = 2.62
r2 = 0.9949
n = 3.09
r2 = 0.9898
n = 3.05
r2 = 0.9893
n=0.91
r2=0.9865
n = 1.09
r2 =0.9797
−3.2
−3
−2.8
−2.6
−2.4
−2.2
−2
−3.4 −3.2
−3
−2.8 −2.6 −2.4 −2.2
−2
log CL
log CL
Figure 8. Dependence of the reduction potential shifts of Cd(II) ions on the concentration of
the compounds L1-L4 taken from DPP measurements. Values inside the frames describe the
complex stoichiometry (n) and fitting of the measuring points to the regression curves (r2).
According to the equation of Lingane, modified by De Ford and Hume [27], the
stoichiometry of the complexes (n) was determined:
ꢀ
ꢊꢋꢄ
ꢍ ꢏ
= ꢌꢇꢈꢉ ꢎ + ꢇꢈꢉꢐꢑ
ꢁ
ꢆ
ꢂꢃꢄ − ꢂꢃꢄꢅ + ꢇꢈꢉ
0.059
ꢊꢋꢄꢅ
20

(y = ax + b)
Using the series of the measurements where the concentration of metal is constant and
the concentration of ligand increases, the above relationship appears in the form of a diagram.
The only condition required is the selection of concentrations where CM<<CL (in this case [L]
≈ CL). In the case where only one dominant complex is present in the solution, this
relationship is linear (Figure 8). Analysis of the slope and the intersection with the y-axis
enables determination of n and βn [27,35].
It was found that all bisphosphonates studied (L1-L4) form different complexes in the
pH range selected (Figure 8). The first complex present in the solution has the stoichiometry
of M:L 1:1. L1 and L3 form these complexes in the pH range of 4.5-5 (n=0.74 for L1 and
n=0.91 for L3). L2 behaves slightly differently from L1 and L3, and its equimolar complex
1:1 can be observed only in pH above 7 (n=1). At approximately pH 4, the adsorption process
disturbs the reduction potential and no complexes can be observed (n=0.15). This
phenomenon may be the result of the presence of the sulfur atom in the pyrimidine ring,
which may interact with the hanging mercury drop electrode. In Cd(II)-L1 and Cd(II)-L2
systems, when the pH of the solutions increases (pH 7.45), equimolar complexes are still
present (n=0.95 and n=1 respectively). In the case of L3 at pH 8.27, the mixture of equimolar
and bis-complexes can be observed (n=1.66). At pH above 9, the stoichiometry of the
complexes changes to a 1:2 metal-to-ligand ratio (n=2.01 for L1, n=1.77 for L2, n=2.06 for
L3).
The structure of L4 differs from the rest of the ligands and behaves differently in the
solution. The ligand was investigated at different pH values, chosen on the basis of
polarographic titration (Figure 7). For this species, the equimolar complexes start to form at a
pH one order of magnitude lower than for the other ligands. This complex is dominant and
stable, n=1.05 (pH 3.45). At pH above 4.58, the new complex with M:L 1:3 (n=2.91) appears.
When the pH increases, this complex is still present (n=2.62 in pH 9.21) but the decrease of n
means that a new complex is being formed (n=1.68 in pH 11.25). According to the
potentiometric data, this species has a metal-to-ligand stoichiometry of 1:2 and starts to form
at pH above 10 (Figure 1).
Potentiometric studies suggest the presence of a polynuclear species in the Cd(II)-L4
system between pH 3 and 9.5. The polarographic studies can confirm the presence of such
species in the solution qualitatively. It is known that the presence of polynuclear species in
solution depends on the metal ion concentration. When the concentration of metal ions is very
21

low, there are no polynuclear species in the solution [36]. To remove polynuclear species, one
may dilute the complex solution where the metal-to-ligand ratio is constant [37]. When there
are polynuclear species in the initial solution, the dilution will cause their rearrangement into
mononuclear complexes [36]. Polarography can document such a process by increasing the
cadmium ion reduction potential on the hanging drop mercury electrode.
An experiment of this type was performed for the Cd(II)-L4 system, where the metal-
to-ligand ratio was kept at a constant 1:3 and the total concentration was changed up to 40×
(Figure 9). The potential of metal ion reduction (-Ep) in the monomeric species is independent
of the dilution process, whereas the increase of reduction potential is characteristic of the
destabilization of the oligomeric species [31]. The results obtained clearly indicate that the
dilution of the solution increases the reduction potential from -640 mV to -602 mV (Figure 9).
It confirms the presence of a polynuclear species suggested by the potentiometric data
(Cd3H4L3). The experiment was performed at pH approximately 6.5, where this trinuclear
species is present in high concentration.
L4 + Cd(II)
pH6.5-6.9
645
640
635
630
625
620
615
610
605
600
0
20
40
60
80
100
120
Vdod[ml]
Figure 9. The dilution of Cd(II)-L4 system up to 40× by 0.1 mol/dm3 KNO3. Constant metal-
to-ligand ratio 1:3, initial metal concentration cCd(II)=1.69×10-3 mol/dm3.
Similar experiments were performed for the remaining ligands (L1-L3), but no
changes in the metal ion reduction potential were observed. These results confirm the model
22

calculated by potentiometry (Table 2, Figure 1) where only monomeric species are present in
the solution.
Polarographic studies confirmed that all of the studied ligands form stable complexes
with Cd(II) ions. These are ML and ML2 complexes with metal-to-ligand ratios of 1:1 and
1:2. According to the polarographic data L1, L2 and L3 form complexes that have a high
degree of similarity (Figure 7). These data confirm the results received from potentiometry,
where these ligands represent the same type of metal binding (Table 2).
L4 has a different chemical structure from the other ligands. It has an additional
pyridine nitrogen atom, which is able to bind metal ions. It is possible that the presence of this
atom is the reason why the ligand is able to form M3L3 and ML3 complexes. The presence of
such complexes in the solution was confirmed by both potentiometry and polarography.
3.2.3. NMR studies
To confirm the aforementioned models of complexation, additional studies were
undertaken to relate 31P NMR chemical shift changes of free ligand versus the mixture of 1:3
metal-ligand ratio as a function of pH.
L1 forms four complexes (see 31P NMR chemical shift presented versus
potentiometric distribution curves in Figure 1). The CdH2L complex (1:1 molar ratio) is
observed at very low pH, which, upon increase of pH, yields the CdHL species. At the
maximal appearance of this species all Cd(II) ions are complexed. At pH over 6.5, only
complexes of M:L of 1:2 are observed (CdH2L2 and CdHL2). This is supported by NMR
titrations, which indicate complexation at low pH. The greatest change in chemical shift is
observed at pH 6.5, where CdH2L2 species begin to be formed. This may result from the
change in type of interaction between the ligand and Cd(II) ions in CdHL and CdH2L2
complexes. L2 and L3 are similar to L1, however the changes in chemical shifts between free
and bound ligands are less pronounced (Figure 1).
L4 yields the most complicated distribution of complexes (Figure 1). As in all of the
previous cases, they are formed at pH as low as 2. Total disappearance of Cd(II) ions is
observed at a pH of 4.5. 31P NMR spectra clearly support the potentiometric data with the
presence of 3:3, 1:3 and 1:2 complexes in the studied pH range.
3.2.4. ESI-MS studies
23

ESI method is a technique widely used to analyze compounds in the solution [38].
Although, this method is not perfect when using solely it can confirm the type of metal
complexes formed in the solution because of the presence of characteristic isotopic
distribution pattern. Thus, it allows to elucidate the metal-to-ligand stoichiometry.
ESI-MS measurements were made to confirm the stoichiometries of Cd(II) complexes
calculated from the studied by means of potentiometry. Both positive and negative spectra
were made but because of the ligands acidities the negative ionization method was more
effective one. All measurements for all ligands taken into investigation were performed in
three different pH values (pH 4, 7 and 9) in order to find major species present in the solution.
During analysis of MS spectra the most abundant signals originated from metal free
ligands, their alkali ion adducts and ligand fragments, additionally their dimeric and trimeric
forms were found. The less intense peaks were related to Cd(II) complexes which are present
in the solution.
Spectra recorded for L1, L2 and L3 were investigated in order to confirm the lack of
presence of polynuclear Cd(II) complexes in the solution. According to potentiometric
studies ligands L1, L2 and L3 form only mononuclear complexes with dominant 1:1 metal-
to-ligand ratio at pH about 4 (Figure 10 a). Such complexes were found for all ligands as
mono charged [CdH2LCH3OH]- adducts appearing in a range of 405 to 424 m/z in the spectra.
The presence of methanol molecule in the complex is not a surprise and results from its
addition to the solution during MS measurements [39].
a
24

b
Figure 10. Experimental and calculated isotopic distribution patterns in the ESI-MS spectra
performed for Cd(II)-L2 system in pH 4 (a - [CdH2LCH3OH]- complex) and pH 9 (b -
[CdHL2K]- complex).
Also metal-to-ligand 1:2 molar ratio species appear in higher pH values (Fig. 10b).
They are detected in the spectra as mono charged potassium adducts at the range m/z 666-706
(Fig. 10b). The signals rising from polynuclear species were not observed which is consistent
with results obtained by other analytical methods used in this study.
The only ligand which forms polynuclear complexes is L4, which is documented by
finding of the methanol adduct of CdHL species in the sample at pH 4. In higher pH signals
corresponding to polynuclear potassium adduct of Cd3H4L3 complex were observed at the m/z
1220 (Figure 11). Additionally CdH3L3 - 952 m/z (Figure 12) and CdL2 (669 m/z) complexes
as potassium adduct forms were also detected. Thus, the performed MS experiments confirm
the results obtained by other applied techniques.
25

Figure 11. Experimental (a) and calculated (b) isotopic distribution patterns in the ESI-MS
spectra performed for Cd(II)-L4 system in pH 7 for [Cd3H4L3K2]- complex
Figure 12. Experimental (a) and calculated (b) isotopic distribution patterns in the ESI-MS
spectra performed for Cd(II)-L4 system in pH 9 for [CdH3L3K]- complex
4. Conclusions
26

As shown by the results presented in this paper, compounds L1, L2, L3 and L4 are
effective ligands for Cd(II) ions. They bind Cd(II) ions even more readily then Zn(II) ions.
Their binding properties were demonstrated with solution studies in potentiometry,
differential pulse polarography and NMR and ESI-MS spectroscopy. The model of
coordination reported in this paper is one which is consistent with all the experimental
techniques used.
Acknowledgement
The Project was financially supported by Wroclaw Research Centre EIT+ under the project
”Biotechnologies and advanced medical technologies” – BioMed (POIG.01.01.02-02-003/08)
financed from the European Regional Development Fund (Operational Programme Innovative
Economy, 1.1.2). AD would also like to thank Wroclaw Medical University Grant ST-697.
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