Dipicolinate Complexes of Gallium(III) and Lanthanum(III)

Article abstract of DOI:10.1021/acs.inorgchem.6b02357

Three dipicolinic acid amine-derived compounds functionalized with a carboxylate (H₃dpaa), phosphonate (H₄dppa), and bisphosphonate (H₇dpbpa), as well as their nonfunctionalized analogue (H₂dpa), were successful

Full text of DOI:10.1021/acs.inorgchem.6b02357

Article
pubs.acs.org/IC
Dipicolinate Complexes of Gallium(III) and Lanthanum(III)
David M. Weekes, Caterina F. Ramogida, Maria de Guadalupe Jaraquemada-Pelaez, Brian O. Patrick,
́
Chirag Apte, Thomas I. Kostelnik, Jacqueline F. Cawthray, Lisa Murphy, and Chris Orvig*
Medicinal Inorganic Chemistry Group, Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, British
Columbia V6T 1Z1, Canada
S
* Supporting Information
ABSTRACT: Three dipicolinic acid amine-derived com-
pounds functionalized with a carboxylate (H3dpaa), phospho-
nate (H4dppa), and bisphosphonate (H7dpbpa), as well as
their nonfunctionalized analogue (H2dpa), were successfully
synthesized and characterized. The 1:1 lanthanum(III)
complexes of H2dpa, H3dpaa, and H4dppa, the 1:2 lanthanum-
(III) complex of H2dpa, and the 1:1 gallium(III) complex of
H3dpaa were characterized, including via X-ray crystallography
for [La4(dppa)4(H2O)2] and [Ga(dpaa)(H2O)]. H2dpa,
H3dpaa, and H4dppa were evaluated for their thermodynamic
stability with lanthanum(III) via potentiometric and either
UV−vis spectrophotometric (H3dpaa) or NMR spectrometric
(H2dpa and H4dppa) titrations, which showed that the carboxylate (H3dpaa) and phosphonate (H4dppa) containing ligands
enhanced the lanthanum(III) complex stability by 3−4 orders of magnitude relative to the unfunctionalized ligand (comparing
log βML and pM values) at physiological pH. In addition, potentiometric titrations with H3dpaa and gallium(III) were performed,
which gave significantly (8 orders of magnitude) higher thermodynamic stability constants than with lanthanum(III). This was
predicted to be a consequence of better size matching between the dipicolinate cavity and gallium(III), which was also evident in
the aforementioned crystal structures. Because of a potential link between lanthanum(III) and osteoporosis, the ligands were
tested for their bone-directing properties via a hydroxyapatite (HAP) binding assay, which showed that either a phosphonate or
bisphosphonate moiety was necessary in order to elicit a chemical binding interaction with HAP. The oral activity of the ligands
and their metal complexes was also assessed by experimentally measuring log Po/w values using the shake-flask method, and these
were compared to a currently prescribed osteoporosis drug (alendronate). Because of the potential therapeutic applications of the
radionuclides 67/68Ga, radiolabeling studies were performed with 67Ga and H3dpaa. Quantitative radiolabeling was achieved at pH
6.5 in 10 min at room temperature with concentrations as low as 10−5 M, and human serum stability studies were undertaken.
La3+ is an approved orally administered treatment for
hyperphosphatemia, and the drug owes its effectiveness to its
INTRODUCTION
■
Osteoporosis, characterized by low bone mineral density, is one
of the most common diseases of the skeleton, predominantly
affecting the elderly, with 1 in 2 women and 1 in 4 men over
very low intestinal absorbance (estimated to be less than
0.002%).12 It was in this form that an effect of the metal ion on
bone histology was first noted;11 however, with such low
the age of 50 likely to suffer an osteoporotic fracture.1 The
bioavailability, vastly elevated quantities would need to be
most common medicinal agents designed to prevent bone loss
administered in order to generate an effective oral dose.
are bisphosphonates (BPs), a class of orally delivered drugs that
Fosrenol is known to have adverse effects on the GI tract,13 so
suppress the proliferation of cells responsible for bone
elevating the dose as a method to increase La3+ absorbed would
undoubtedly be met with extremely low compliance.
resorption.2,3 In spite of their wide use, BPs suffer from a
number of significant drawbacks, including side effects such as
musculoskeletal pain and gastrointestinal (GI) upset,4 as well as
growing uncertainty over their long-term efficacy.5 Conse-
quently, there is a strong drive to develop alternative
treatments.
By modification of the chemical environment around La3+
(through rational chelator design), our previous studies have
shown progress in the quest for a system that improves the oral
activity of the metal ion, without impeding its natural tendency
for targeting bone tissue upon absorbance.14−16 In 2007, Barta
et al. investigated a series of tris-bidentate lanthanide complexes
based on 3-hydroxy-4-pyrone and -pyridinone scaffolds and
found that the complex tris(1,2-dimethyl-3-oxy-4-pyridinone)-
Various studies over the past 15 years have suggested that, at
certain concentrations within bone, La3+ (which bears a number
of chemical similarities to Ca2+ including ionic radius, preferred
coordination number, and preference for hard donor ligands)
can invoke a cellular response that enhances bone building,
signifying a potential for La3+ to counter the effects of
osteoporosis.6−11 As a carbonate salt (known as Fosrenol),
Received: September 27, 2016
© XXXX American Chemical Society
A
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lanthanum(III) [La(dpp)3; Figure 1a] greatly improved
transport of the metal ion across the membrane of Caco-2
Chart 1. Structures of the Four dpa-Containing Ligands
Figure 1. Structures of the two lead compounds previously
investigated to improve the oral activity of trivalent lanthanum in
the development of novel treatments for osteoporosis: (a) La-
(dpp)3;14−16 (b) La(XT).15−17
administered compounds for delivering lanthanum to bone
tissue. The functionalized ligands include either a carboxylate
(6,6′-{[(carboxymethyl)azamediyl]dimethylene}dipicolinic
acid, H3dpaa), a phosphonate (6,6′-{[(2-phosphonoethyl)-
azanediyl]dimethylene}dipicolinic acid, H4dppa), or a BP
(6,6′-{[(4-hydroxy-4,4-diphosphonobutyl)azanediyl]-
dimethylene}dipicolinic acid, H7dpbpa) moiety. Also included
in these studies is the unfunctionalized version [6,6′-
(azanediyldimethylene)dipicolinic acid, H2dpa], which enables
a calibrated assessment of the effect of each of these functional
groups. We aimed to assess the bone-targeting ability of the
ligands through HAP binding studies, the contribution to La3+
complex stability from the carboxylate group in H3dpaa or the
phosphonate group in H4dppa through potentiometric titration,
and the expected cell permeability via lipophilicity measure-
ments (and the effect that lanthanum chelation has on these
values). The solid-state crystal structures of the 1:1 La3+
complex of H4dppa is also described.
In addition, because of its potential to form a charge-neutral
complex, the carboxylate-functionalized H3dpaa was inves-
tigated for its coordination properties, thermodynamic stability,
and in vitro kinetic inertness with gallium(III). Complexes of
H3dpaa with lutetium(III) and gadolinium(III) have been
reported previously as potential MRI contrast agents,19 and our
interest herein lies with gallium(III) because of the high-quality
positron emission tomography (PET) imaging potential of the
radionuclide 68Ga.20 The solid-state crystal structure of the 1:1
Ga3+ complex of H3dpaa was also obtained and is fully
described.
cells (an in vitro model for human epithelial cells).14 In 2013, a
similar result was obtained by Mawani et al. for the
phosphinate-containing 1:1 ethylenediaminetetraacetic acid
(EDTA)-type complex [bis[[bis(carboxymethyl)amino]-
methyl]phosphinate]lanthanum(III) [La(XT); Figure 1b],15,17
and in 2015, these two complexes were compared directly to
probe the thermodynamic and kinetic interactions between
each system and hydroxyapatite (HAP), an ex vivo model for
bone mineral, as well as the influence of a chelator on La3+ in
vivo biodistribution.16
Some key factors considered in the fundamental design of
these systems include synthetic accessibility, bone mineral
targeting, thermodynamic stability of the lanthanum complex,
and lipophilicity. In this regard, there are advantages and
drawbacks to both dpp and XT; in an effort to combine the
favorable attributes of each, we propose a ligand scaffold based
on a dipicolinic acid (dpa) binding motif (Figure 2).
Dipicolinate derivatives have been examined previously for
their chemistry with trivalent lanthanides, predominantly in the
context of magnetic resonance imaging (MRI) contrast
agents.18,19 Here, the ligand scaffold consists of two picolinic
acid (pa) groups connected via methylene bridges at the 6
position of each pa to a central nitrogen atom, resulting in a
metal binding cavity that is inherently five-coordinate, thereby
offering greater thermodynamic stability than bidentate dpp.
Furthermore, the central nitrogen atom provides a convenient
handle for functionalization, which allows tuning and
optimization of the ligand’s properties. Herein, we present a
set of N-functionalized dpa derivatives (Chart 1), as well as
their corresponding La3+ complexes, which have been
synthesized, characterized, and subjected to a number of
testing procedures to assess their suitability as orally
RESULTS AND DISCUSSION
■
Ligand Synthesis and Characterization. The synthetic
routes to all four dpa-based compounds, H2dpa, H3dpaa,
H4dppa, and H7dpbpa, rely on the alkylating agent methyl 6-
Figure 2. Solid-state ORTEP structures for crystals of (a) 2[H2dpa·H2O], (b) H3dpaa·H2O, and (c) H4dppa·3H2O. Full experimental details and
crystallographic data are presented in the Supporting Information (Table S1).
B
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a
(bromomethyl)picolinate (3), which is generated via a well-
established protocol from commercially available dimethylpyr-
idine-2,6-dicarboxylate (1; Scheme 1).20,21 The alkyl-protected
Scheme 3. Synthetic Route to H3dpaa as the HCl Salt
Scheme 1. Established Synthesis for the Alkylating Agent
320,21
a
Reagents and conditions: (i) 3 (2.1 equiv), K2CO3, CH3CN, 60 °C,
ligands are then generated from 3 via a double N-alkylation of
the primary amine synthon that corresponds to the desired
compound, followed by deprotection (for all except H7dpbpa,
in which the deprotected product is generated during
alkylation) in a strong acid to obtain the final ligands as the
HCl salts (Schemes 2−5).
12h; (ii) 6 M HCl, 110 °C, 12 h.
Scheme 4. Aza-Michael-Addition-Mediated Synthetic Route
a
to H4dppa as the HCl Salt
a
Scheme 2. Synthetic Route to H2dpa as the HCl Salt
a
Reagents and conditions: (i) excess NH4OH(aq), H2O, 0 °C to room
temperature, 12 h; (ii) 3 (2.1 equiv), K2CO3, 60 °C, 12 h; (iii) 12 M
†
HCl, 110 °C, 12 h. Yield over two steps.
a
Reagents and conditions: (i) 3 (2.1 equiv), K2CO3, CH3CN, 60 °C,
unwanted byproducts invariably forms and, given the oily
consistency of the alkyl phosphonates and the absence of a
chromophore, are very challenging to separate. This was
addressed by the direct alkylation of a crude reaction mixture
from step i in Scheme 4, followed by column separation to
isolate the desired dimethyl picolinate intermediate 11, which
was deprotected in strong acid in a manner similar to that for
the other compounds.
Finally, because of challenges in obtaining the necessary
alkyl-protected primary amine synthon, H7dpbpa was accessed
in a single step via a pH-controlled aqueous reaction of 3 with
alendronic acid (12; Scheme 5). Because of the basicity of the
24 h; (ii) PhSH (1.1 equiv), tetrahydrofuran, 70 °C, 48 h; (iii) 6 M
HCl, 110 °C, 12 h.
For H2dpa, because the final product is the secondary amine,
an additional 2-nitrobenzenesulfonamide (4)-mediated protec-
tion−deprotection step is required (Scheme 2). This chemistry
was optimized by Price et al. and enables good yields and
straightforward purification compared to other amine protect-
ing groups.21 The synthesis of H3dpaa is more straightforward
because it only requires N-alkylation of the commercially
available glycine ethyl ester (hydrochloride salt, 7) prior to
deprotection (Scheme 3), and a similar procedure has been
previously reported.19
a
Scheme 5. Synthetic Route to H7dpbpa as the HCl Salt
The primary amine synthon (10) required to make H4dppa
is not commercially available but can be synthesized in a single
step and under mild conditions via an aqueous-mediated
catalyst-free aza-Michael addition of diethyl vinylphosphonate
(9) with ammonium hydroxide (Scheme 4).22 One drawback of
this procedure is the tendency for overreaction to occur via
subsequent 1,4-additions into the vinylphosphonate Michael
acceptor from the more active amines that are formed, resulting
in bis- and trisphosphorylated byproducts; however, this can be
minimized by the very gradual addition of 9 into a large excess
of an ammonium hydroxide solution.
a
In spite of the optimized conditions, such is the activity of
the primary amine 10 relative to ammonia, a small quantity of
Reagents and conditions: (i) 3 (3.0 equiv), 3:1 water/tetrahydrofur-
an, 1 M NaOH (pH 12), 3 days (followed by acid workup).
C
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Figure 3. Solution depletion plots showing the [ligand] remaining in solution (unbound to HAP) over 48 h as % [ligand] at time = 0 (n = 3): (a)
H2dpa; (b) H3dpaa; (c) H4dppa; (d) H7dpbpa. Conditions for all experiments: 37 °C, pH 7.4, I = 0.16 M (NaCl), agitation at 220 rpm.
primary amine group of 12 (pKa 12.7),23 a pH between 12 and
13 was maintained throughout the course of the reaction by the
regular dropwise addition of 1 M NaOH. This has the
simultaneous effect of removing the alkyl-protecting groups of
the picolinate arms; however, unwanted bromide hydrolysis
obviated good yields despite efforts to optimize the reaction
conditions. A similar reaction procedure (with good yield) has
been previously reported in the synthesis of bone-seeking
chelating ligands for 99mTc, but in those cases the alkylating
agent was simply 2-(chloromethyl)pyridine (no carboxylate
functionality), which may explain the improved yield in that
case.24
Figure 3a shows the disappearance of H2dpa (R = H) from
solution to an approximate minimum of [H2dpa] = 80% after
48 h, with the other 20% presumably bound to HAP. Although
there is very little error associated with the data, the first few
time points are erratic and incoherent from a kinetic point of
view, and this, coupled with the fact that the majority of the
compound remains in solution, infers that there is little to no
chemical attraction between H2dpa and HAP. This is consistent
with other compounds that do not contain specific bone
binding groups (such as a phosphonate or BP), and the
minimal disappearance from solution that is observed is likely
due to physisorption in the form of weak van der Waals
interactions between solvated H2dpa and suspended HAP
particles. A similar argument can be made to explain the results
for H3dpaa (Figure 3b), which also does not contain any
functionality that specifically binds to bone mineral. As with
H2dpa, a slight depletion from solution is observed to a
minimum [H3dpaa] of approximately 85% after 48 h, which is
again likely only due to physical adsorption onto the surface of
HAP particles.
The observed depletion profile changes significantly with the
introduction of the phosphonate moiety in H4dppa (Figure 3c).
In this case, the concentration of the ligand in solution
disappears in a smooth and coherent manner to a minimum
[H4dppa] in solution of around 55% after 48 h. This is
indicative of a process involving chemical absorption, consistent
with the ability of phosphonates to bind to HAP with moderate
strength.25 With H7dpbpa (Figure 3d), the compound binds
rapidly to HAP and is almost undetectable in solution (<10%
remaining) after 48 h. This follows the notion that BP-based
systems exhibit a very high affinity for bone mineral by
mimicking the pyrophosphate network found in HAP,26 and of
the four systems tested, H7dpbpa is best suited to target bone.
From these results and from previous studies,3 it is clear that
BP-containing compounds have some of the greatest bone-
directing capability of any organic moiety; however, an ideal
1
The four compounds were characterized by H and 13C
NMR spectroscopy (as well as 31P{1H} NMR for the
phosphorus-containing compounds), low- and high-resolution
mass spectrometry, and elemental analysis (see the Supporting
Information). Crystals suitable for solid-state X-ray analysis for
2[H2dpa·H2O], H3dpaa·H2O, and H4dppa·3H2O (confirming
attainment of the desired products) were obtained as
zwitterions, and the structures are presented in Figure 2 (see
the Supporting Information, Table S1, for crystallographic
data).
Bone Targeting. With the dpa-based ligands in hand,
solution depletion experiments were performed to determine
the influence of the R appendage on bone mineral targeting and
binding (in the absence of a chelated metal ion) using HAP as a
model for the bone mineral. Ligand solutions (0.1 mM)
buffered to pH 7.4 were incubated and agitated (37 °C and 220
rpm) with a suspension of synthetic HAP (in 200-fold excess),
and at fixed time points over the course of 48 h, the
concentration of ligand remaining in solution (not bound to
HAP) was determined by ultraviolet−visible (UV−vis)
spectrometry. The results (Figure 3) are plotted as % [ligand]
remaining in solution relative to [ligand]t=0 versus time in
hours.
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Figure 4. ORTEP diagram of the crystal structure of [Ga(dpaa)(H2O)].
ligand here is one that directs lanthanum(III) to bone tissue
without itself impacting bone histology. One of the major
cautions associated with the use of BP-based pharmaceuticals is
their tendency to become incorporated into newly mineralized
tissue, where they can be retained with an elimination half-life
(t1/2) exceeding 10 years.27 This long-term retention is often
cited as the reason for some of the major side effects associated
with the chronic oral administration of BPs4 and serves as one
of the arguments for why alternative treatments (e.g.,
lanthanum-based compounds) should be explored. For this
reason, the weakly bone-directing monophosphonate com-
pound H4dppa may, in the context of this set of ligands, present
a more viable option for facilitating the inherent bone-targeting
ability of lanthanum(III), without itself impacting bone
histology through long-term ligand accumulation.
adjacent to the central nitrogen atom (see Supporting
Information, Figures S2−S10, for complete H NMR spectra).
1
X-ray Crystal Structure of [Ga(dpaa)(H2O)]. Crystals
suitable for solid-state X-ray analysis were obtained by slow
evaporation from a water/methanol mixture. The neutral
complex crystallizes with two crystallographically independent
gallium complexes in the asymmetric unit (AU; Figure 4)
related to one another by 178° rotation about the (010)
reciprocal axis. At least six water molecules are present in the
AU, and any residual electron density that could not be
reasonably modeled was removed via the Olex2 solvent-mask
function.28 Both units exhibit a distorted octahedral structure
(N2O4), formed by the two pyridine nitrogen atoms and three
carboxylate oxygen donors, with the sixth site occupied by a
water molecule and not, as expected, the tertiary nitrogen atom
of the ligand backbone. Although the solid-state structure
shows that the central nitrogen atom is slightly puckered
between the two pa arms and pointed fittingly toward the metal
center, the Ga−Nt distances (Ga1−N3 = 2.485 Å; Ga2−N6 =
2.509 Å) are greater than the sum of the effective ionic radii for
gallium and nitrogen in their respective coordination environ-
ments. The two pa arms are in one plane, while the carboxylate
arm acts as a claw capping the top of the plane with the water
molecule capping the bottom of the plane. We predict that the
in vivo stability of this complex could suffer because of this,
given that the coordination site occupied by water is an obvious
point of attack for endogenous ligands, which compete with the
chelate for gallium(III). Selected interatomic distances are
presented in Table 1 and full crystallographic data in the
Supporting Information, Table S2.
Metal Complexes of the dpa-Based Ligands. The 1:1
lanthanum(III) complexes of H2dpa, H3dpaa, and H4dppa and
the 1:2 complex with H2dpa were successfully synthesized by
adjusting the pH to approximately 8−10 of an equimolar (or
1:2) mixture of the respective ligands and La(NO3)3·6H2O in
water using 1 M KOH. Attempts to perform an analogous
synthesis with H7dpbpa invariably resulted in the formation of a
precipitate at low pH, which was challenging to analyze because
of its poor solubility. This is likely due to the high affinity and
multiple possible binding modes between the BP moiety, the
intended chelate cavity, and trivalent lanthanum, which leads to
the incoherent and irregular formation of various polymeric
structures rather than the intended 1:1 complex. The
gallium(III) complex of H3dpaa was prepared via the addition
of 1 equiv of Ga(ClO3)3·6H2O to a stirred solution of the
ligand in water and methanol preadjusted to pH 3 (NaOH). In
X-ray Crystal Structure of [La4(dppa)4(H2O)2]. Crystals
suitable for solid-state X-ray analysis were obtained by the slow
diffusion of methanol into a concentrated solution of the
premade K[La(dppa)] complex in water. The residual electron
density was present in the lattice presumably because of the
1
all cases, successful metal coordination was confirmed by H
NMR spectra, which exhibit a clear and well-resolved upfield
shift and diastereotopic splitting of the methylene protons
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solvent and counterion, but unfortunately none of these sites
could be properly modeled as either water, methanol, or K+,
likely because of disorder. As a result, a data set free of solvent/
ions at those sites was generated using the PLATON/
SQUEEZE program.29 The material crystallized as a centrosym-
metric tetramer, with the AU comprising two unique
lanthanum ions (La1 and La2), two molecules of dppa
(dppa1 and dppa2), and one molecule of water bound to
La2 (Figure 5). La1 is eight-coordinate, forming bonds with the
five inherent donor atoms (N3O2) from the binding cavity of
dppa1. The coordination sphere is completed by three
phosphonate oxygen atoms: one from dppa1, one from
dppa2, and one from dppa1′ (from the other AU). A result
Table 1. Selected Interatomic Distances for
[Ga(dpaa)(H2O)]
bond
length (Å)
Ga1−N1
Ga1−N2
Ga1−N3
Ga2−N4
Ga2−N5
Ga2−N6
Ga1−O7
Ga2−14
2.219(4)
2.179(4)
2.485(3)
2.225(4)
2.183(4)
2.509(3)
1.909(4)
1.916(4)
a
a
a
Not shown as a bond in Figure 4.
Figure 5. ORTEP diagram of the crystal structure of [La4(dppa)4(H2O)2] modeled without a lattice solvent, showing the AU (upper) and the
complete unit cell (lower) consisting of two AUs. All hydrogen atoms (with the exception of those on water bound to La2), as well as the C−H
groups of the pyridine rings in the unit cell diagram, are omitted for clarity.
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of this third coordinating oxygen is the formation of an eight-
membered ring bridging the AUs and containing La1, La1′, and
an O−P−O linker from dppa1 and dppa1′. La2 is nine-
coordinate, with five sites similarly occupied by donor atoms
from the dpa scaffold of dppa2 and a phosphonate oxygen from
the same unit. La2 also bonds two oxygen atoms from dppa1,
which both stem from the same picolinate arm. The final
coordination site is occupied by a molecule of water, which is
the only well-resolved solvent molecule found within the lattice,
bound within the inner sphere of La2 at a La−O distance of
2.501 Å and stabilized by hydrogen-bonding interactions to
dppa1. As is often the case with nonrigid complexes of the
lanthanides, there is a lack of a clear and well-defined
coordinate geometry around either lanthanum center. For
both La1 and La2, the metal does not sit flush within the dpa
cavity of dppa but rather outside of it, with the ligand bent in
such a way as to push the two picolinate arms toward each
other [θ(NAr−La−NAr) < 90°], enforcing a facial binding
mode, and the (CH2)2PO(O)2 arm wrapping around the other
side of the metal enabling bridging between La1 and La2. The
La−Nt bond distances (La1−N2 = 2.863 Å; La2−N5 = 2.861
Å) are both slightly greater than the sum of the effective ionic
radii for lanthanum and nitrogen in their respective
coordination environments, implying only a partial bonding
interaction between these atoms. Similar phenomena have been
reported in which weak lanthanum(III)−tertiary amine
interactions (of a similar distance apart) are stabilized by
strong chelation across the rest of the molecule.30 Selected
interatomic distances and angles are presented in Table 2 and
full crystallographic data in the Supporting Information (Table
S2).
conventional methods and hindered elucidation of a fully
coherent charge-balanced structure.
As is always the case, a cautious approach should be taken
when attempting to extrapolate informative conclusions from
the solid-state structure of a material that is ultimately intended
to be used in solution (and, more importantly, in vivo).
Nonetheless, the crystal structures presented for Ga(dpaa) and
the La(dppa) tetramer provide a visually compelling insight
into the nature of the interaction between trivalent metal ions
and ligands based on the dpa motif and enable well-informed
guidance on the design of future compounds and insight into
the solution studies.
Thermodynamic Stability Measurements. The proto-
nation constants of the ligands (dpa, dpaa, and dppa) were
determined via potentiometric titrations (Table 3), carried out
at a ligand concentration [L] = 1.0 × 10−3 M at 298 K and I =
0.16 M (NaCl). For each species, log Ka1 is assigned to the
quaternization of the nonaromatic nitrogen (dpa = 8.15; dpaa =
7.23; dppa = 8.51). This is followed by the two carboxylate
groups for dpa (log Ka2 = 3.50; log Ka3 = 2.71), and these values
aid in assigning the protonation constants for the analogous
groups in dpaa (log Ka2 = 3.71; log Ka3 = 2.95) and dppa (log
Ka3 = 3.63; log Ka4 = 2.76). For dpaa, the final constant,
assigned to the acetic acid functionality, occurs at low pH (<2)
and was therefore not determinable by potentiometry. Overall,
these values are in reasonable agreement with those previously
reported.19 For dppa, the protonation constants for the
phosphonate functionality (log Ka2 = 6.33; log Ka5 = 0.78)
occur at either side of the pa groups. 31P{1H} NMR
spectrometric titrations were carried out in order to determine
the most acidic pKa5 in dppa. The protonation scheme for dppa
was confirmed by titration curves and basicity constants, which
were obtained by simultaneously refining 1H and 31P
experimental chemical shifts (42 batch solutions; see the
Supporting Information, Figure S11) using HypNMR2008.31
The values obtained by NMR titrations are presented in the
Supporting Information (Table S4) and are in agreement with
those determined by potentiometric titrations (Table 3).
Lanthanum Complex Stability with dpa, dpaa, and dppa.
The stability constants of the complexes formed between La3+
and either dpa, dpaa, or dppa were studied by direct
potentiometric titrations of 1:1 La/L ([dpa] = 8.43 × 10−4
M; [dpaa] = 8.00 × 10−4 M; [dppa] = 6.55 × 10−4 M) and 1:2
La/L ([dpa] = 8.88 × 10−4 M; [dpaa] = 8.47 × 10−4 M)
mixtures in a pH range of approximately 1.5−11.5 (298 K; I =
0.16 M NaCl). Because the potentiometric data showed that
Table 2. Selected Bond Lengths and Angles for
[La4(dppa)4(H2O)2]
bond
length (Å)
angle
θ (deg)
La1−N1
La1−N2
La1−N3
La2−N4
La2−N5
La2−N6
La2−O15
2.677(3)
2.863(3)
2.708(3)
2.679(4)
2.861(4)
2.673(3)
2.501(3)
N1−La1−N3
N4−La2−N6
85.68(10)
83.60(11)
Also obtained were crystals of a 1:2 complex La(dpa)2, with
two crystallographically independent 10-coordinate La3+
complexes present in the AU (see the Supporting Information,
Figure S1). Unfortunately, the material crystallized with a high
degree of unresolved residual electron density (likely solvents)
present in the crystal lattice, which was nonrefinable by
1
the metal was complexed at pH <2, H NMR titrations were
applied to confirm the stability constants associated with
lanthanum complex formation for the La3+-dpa and La3+-dppa
systems. These experiments were carried out by preparing a
D2O solution of approximately 1:1.5 La/L ratio and collecting
Table 3. Stepwise Protonation Constants for dpa, dpaa, and dppa Determined by Potentiometric Titrations (298 K, [L] = 1.0 ×
10−3 M, and I = 0.16 M NaCl) Using HyperQuad2013 Software32
species
log Ka
species
log Ka
species
log Ka
log Ka1
log Ka2
log Ka3
log Ka4
log Ka5
Hdpa−
H2dpa
H3dpa+
8.15(4)
3.50(3)
2.71(2)
Hdpaa2−
H2dpaa−
H3dpaa
7.232(6)
3.71(1)
2.95(1)
Hdppa3−
H2dppa2−
H3dppa−
H4dppa
8.51(2)
6.33(3)
3.63(4)
2.76(2)
0.78(4)
a
H4dpaa+
ND
b
H5dppa+
a
b
ND = not determined. Value determined by 31P{1H} NMR titrations.
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1H NMR spectra (400 MHz, 298 K) at various pD values by
carefully adjusting with dilute NaOD or DCl (Figures 6 and 7).
stability constants obtained by potentiometric measurements
are reported in Table 4 and the related speciation plots in
Table 4. Lanthanum Complex Formation Constants for dpa,
dpaa, and dppa, Determined by Potentiometric Titrations
a
(298 K; I = 0.16 M NaCl)
dpa
log β
dpaa
log β
dppa
model
pKa
pKa
log β
pKa
d
c
MLH
ML
15.42(9)
18.26(6)
13.99(6)
b
,
c
d
b
9.72(6)
13.6(2)
1.82
4.27
b
13.22(8)
f
b
MLH−1
ML2
3.43(7)
10.56
b,c
17.01(7)
e
pLa
11.2
14.3
13.8
a
Standard deviations are reported in parentheses after the
b
experimentally determined values. Determined via potentiometric
titrations using HyperQuad2013.32 Determined via NMR titrations.
c
d
Determined via UV−vis spectrophotometry using HypSpec.32
e
Calculated from −log [La3+]free ([La3+] = 10−6 M; [L] = 10−5 M;
f
pH 7.4; 298 K; I = 0.16 M NaCl). H−n denotes a hydroxo complex.
Figure 8. Unfortunately, this technique was not possible for the
La3+-dpaa system because of interference between the
diagnostic signals associated with metal complexation, and the
residual solvent peak (H2O); therefore, UV−vis spectropho-
tometry was applied to determine the formation constant at pH
<2.5. This was achieved by following changes in the UV
absorbance band of the ligand in a 1:1 La/dpaa system at
different pH levels between 0.22 and 2.48 (see the Supporting
Information, Figure S16), and the refined constant is reported
in Table 4.
1
Figure 6. Portion of the H NMR spectra for the La3+-dpa system at
various pH levels (400 MHz, 298 K). Left: [La3+] = 0.010 M; [dpa] =
0.015 M. Right [La3+] = 0.010 M; [dpa] = 0.020 M. A: La(dpa). B:
La(dpa)2.
For the La3+-dpa system, the initial complexation is
evidenced by the diastereotopic character of the methylene
protons near the pyridine ring (A: δ 4.40 and 4.10) at
approximately pH 1.76 (Figure 6). Furthermore, the gradual
disappearance of the methylene protons associated with the
uncoordinated ligand (δ 4.50 ppm) across a broad pH range
agrees with the production of a rigid 1:1 structure in solution.
The emergence of new signals (B: δ 4.55 and 3.70) in the 1:1.5
1
system in the H NMR titrations above approximately pH 3
indicates the formation of the 1:2 complex La(dpa)2 (Figure 6,
left). The potentiometric titrations at 1:1 and 1:2 metal-to-
ligand molar ratios agree with the 1H NMR results. The related
stability constants and speciation diagram are presented in
1
Table 4 and Figure 8, respectively. The H NMR spectra of a
1:2 La/dpa mixture (Figure 6, right) show sharp signals
exclusively related to the 1:2 complex (δ 4.55 and 3.70) above
pH 3.79; indeed, its existence of this species was confirmed by
the isolation and full characterization of this complex, including
an X-ray crystal structure (see the Supporting Information,
Figure S1).
For the La3+-dppa system, complex formation is evidenced by
the emergence of two signals for the diastereotopic methylene
protons (δ 4.1 and 3.8) above pH 1.8 in the 1H NMR spectrum
of a 1:1.5 La/dppa solution (Figure 7). We expect that the
methylene signal resulting from uncoordinated dppa decreases
as the pH increases, as is the case with dpa; however, because of
the residual solvent peak (H2O) in the dppa spectra, these
signals are hidden. Further evidence of complex formation
includes the disappearance of the ethylene proton signals of the
phosphonate-linker arm of the uncoordinated ligand (δ 3.75
and 2.35) above pH 1.8 and the emergence of new signals (δ
Figure 7. Portion of the 1H NMR spectra for the La3+-dppa system at
various pH levels (400 MHz, 298 K). [La3+] = 0.010 M; [dppa] =
0.015 M.
The ratio of the integrals of the methylene protons associated
with either the free ligand or the complex were plotted against
the pH (pH = pD − 0.4)33 and subsequently overlaid with the
distribution diagrams calculated from potentiometric titrations
(see the Supporting Information, Figures S12 and S13). The
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Figure 8. Speciation diagrams for the La3+-ligand systems related to the values in Table 4 (298 K; I = 0.16 M NaCl). (a) [La3+] = 7.95 × 10−4 M;
[dpa] = 8.43 × 10−4 M. (b) [La3+] = 8.00 × 10−4 M; [dpaa] = 8.01 × 10−4 M. (c) [La3+] = 6.63 × 10−4 M; [dppa] = 6.55 × 10−4 M.
2.83 and 1.83) related to the same linker arm in the
coordinated complex. The relative integrals of these signals
allow evaluation of the formation constants, and it is implicit
from these spectra that the phosphonate moiety is involved in
lanthanum binding from the outset of complexation and
contributes significantly to the overall stability of the complex.
Potentiometric titrations fit this hypothesis (Table 4 and Figure
8c), where initially the LaHdppa species is formed and the
proton is lost with pKa = 4.17 (confirmed by 31P{1H} NMR
titrations of the same samples; see the Supporting Information,
Figures S13 and S14).
Gallium Complex Stability with dpaa. Determination of
the formation constants of dpaa with Ga3+ was performed using
potentiometric titrations of 1:1 ([dpaa] = 8.95 × 10−4 M) and
1:2 ([dpaa] = 9.26 × 10−4) metal-to-ligand solutions across a
pH range of approximately 1.5−11.5. These experiments show
complete formation of the GaLH complex at about pH 1
(Figure 9), and the associated formation constants are
Overall, from the speciation diagrams in Figure 8, favorable
lanthanum complexation occurs across a broad pH range for
dpa, dpaa, and dppa. At physiological pH, the 1:1 metal/ligand
species exist almost exclusively for all three systems. The log
βML values (Table 4) show that the carboxylate (dpaa) and
phosphonate (dppa)-containing ligands lead to a 1:1 La3+
complex approximately 3−4 orders of magnitude more stable
than the unfunctionalized ligand (dpa). The dpa ligand is also
capable of forming a 1:2 La/L complex; however, this species is
only dominant at very basic pH. Of greater biological
significance are the pM values, calculated from −log [La3+
]
Figure 9. Speciation diagram for the Ga(dpaa) system related to the
values given in Table 5. [Ga3+] = [dpaa] = 9.10 × 10−4 M, 298 K, and I
= 0.16 M NaCl.
free
([La3+total] = 10−6 M; [L] = 10−5 M) at pH 7.4 (Table 4), which
confirm the enhanced stability contribution from either a
carboxylate or phosphonate moiety. Comparing the pM values
for dpaa and dppa suggests that the stability contribution from
these moieties is of a similar magnitude. It is worth noting that,
because of the nature of the ligands, the 1:1 complexes consist
of either a positively charged [La(dpa)], neutral [La(dpaa)], or
negatively charged [La(dppa)] species, which could be of
relevance when predicting in vivo behavior.
presented in Table 5. Despite the low pH at which the
complex forms, this system is well determined because any
variation in log βGaLH produces a concurrent variation relative
to the formation of the hydroxo complex [Ga(OH)4]− (see the
Supporting Information, Figure S15). In other words, the
potentiometric study presented here can be considered a
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the metal. The least negative log Po/w value, and therefore most
likely to exhibit the greatest intestinal uptake, was observed for
the La(dppa) system, which also presented good thermody-
namic stability and bone-targeting ability in experiments
outlined previously.
Table 5. Gallium Complex Formation Constants for dpaa
Determined by Potentiometric Titrations (298 K; I = 0.16 M
NaCl) Using HyperQuad2013 Software32
dpaa
Radiolabeling Experiments with 67Ga and dpaa. In
addition to the lanthanum complexation studies of this set of
pa-based chelators, dpaa was also tested as a gallium(III)
chelate candidate for 68Ga PET imaging agent elaboration. This
is because of the inherent ability of the fully deprotonated
dpaa3− to form a charge-neutral complex with Ga3+, and in
metal-based radiopharmaceutical design, it is often favorable for
the ligand to form a metal complex with an overall neutral or
near-neutral charge to facilitate clearance of the complex in
vivo. Furthermore, the high pGa value (22.0) implies good in
vivo stability.
model
MLH
log β
pKa
20.92(3)
18.70(1)
13.86(1)
22.0
ML
2.22
4.87
a
MLH−1
pGa
a
H−n denotes a hydroxo complex.
competition study in which the OH− anion acts as a competing
ligand for formation of the [Ga(OH)4]− complex, and the
accuracy of the Ga(dpaa) formation constants depends only on
the accuracy of log βGaH‑4. For this reason, a complementary
technique (e.g., NMR or UV−vis) was not required in order to
affirm the values. The calculated pGa value (22.0) classifies
dpaa as a strong chelating ligand for gallium complexation; for
comparison, this value lies between those reported for industry
“gold standards” 1,4,7-triazacyclononanetriacetic acid (NOTA)
and 1,4,7,10-tetraazacyclododecanetetraacetic acid (DOTA)
(pGa values 26.4 and 18.8, respectively).34,35 This implies
good thermodynamic stability, an ideal property for a
radiopharmaceutical; however, in vivo kinetic inertness
(addressed below) must also be considered when the Ga3+-
dpaa system is evaluated as a potential therapeutic.
Lipophilicity Measurements of dpa Ligands and Their
La3+ Complexes. Lipophilicity, quantified as a partition
coefficient (log Po/w), provides a measure by which to assess
the ability of a drug candidate to be absorbed across an
intestinal membrane, which, in turn, can be used to predict oral
bioavailability. According to Lipinski’s guidelines, a log Po/w of
between −0.5 and +5 is considered optimal for oral uptake;36
however, many approved drugs fall outside of this range,37
including alendronate (log Po/w = −4.49),38 which is routinely
prescribed to osteoporosis sufferers despite suffering from
extremely poor intestinal absorption. Presented in Table 6 are
The initial 67Ga (a longer lived t1/2 = 3.26 days γ-surrogate
for the positron emitter 68Ga; t1/2 = 67.7 min) radiolabeling
experiments with H3dpaa were tested at a pH range of 2−6.5 in
a NaOAc buffer (10 mM) or pure water. Quantitative labeling
(RCY > 99%, where RCY = radiochemical yield) was achieved
at an initial ligand concentration of 10−4 M at all pH values, in
10 min at room temperature; however, the labeling behavior of
the 67Ga(dpaa) complex varied as the pH was increased from 2
to 6.5 (Figure 10). Radiolabeling under acid conditions (pH 2,
no buffer added) resulted in two distinct peaks in the high-
performance liquid chromatography (HPLC) radiotrace
(retention times tR = 3.69 min and tR = 5.56 min) with peak
intensities of 77% and 23%, respectively. During radiolabeling
at pH 3, broadening and definite shoulders appeared on each of
the two distinct peaks, while the relative peak intensities
remained roughly constant (74% and 26%, respectively) with a
slight increase (3%) to the peak at later retention. This trend
continued as the labeling pH was increased from 3 to 4 to 5;
broadening and shoulders became more noticeable, and a small
increase in the labeling yield toward the set of peaks centered at
tR = 5.5 min was observed. At pH 6.5, one distinct peak in the
HPLC radiochromatogram was detected (tR = 5.54 min; RCY >
99%).
a
The appearance of multiple peaks in the HPLC radiotrace at
varying pH values may be indicative of multiple isomers in
solution, which is typically disadvantageous in radiolabeling
because different isomers may possess different stabilities in
vitro and in vivo. The speciation diagram presented in Figure 9
provides a rationale, where it is clear that only above pH 6.5
does a single metal/ligand species dominate.
Table 6. Experimentally Determined log Po/w Values
compound (L)
log Po/w (L)
log Po/w (La-L)
dpa
−2.19 0.16
−3.01 0.21
−2.38 0.21
−2.44 0.11
−4.49
−1.97 0.11
−2.01 0.01
−1.32 0.12
dpaa
dppa
dpbpa
b
Concentration-dependent labeling of the 67Ga(dpaa) system
was performed at pH 6.5 because it resulted in the formation of
one peak in the radiotrace (Figure 11). Quantitative 67Ga
labeling (RCY > 99%) in 10 min at room temperature was
achieved at ligand concentrations of 10−5 M, and radiochemical
yields decreased to 90% and 6% at ligand concentrations of
10−6 and 10−7 M, respectively.
alendronic acid
N/A
a
Shake-flask method; Po/w = [ ]n‑octanol/[ ]HEPES; pH 7.4; 298 K; n = 3;
b
concentration determined by UV−vis absorption. From ref 35.
the experimentally determined log Po/w values of the four dpa-
based ligands (dpa, dpaa, dppa, and dpbpa) and the 1:1 La3+
complexes of all but dpbpa, which were obtained in a biphasic
system (1-octanol/water) using the shake-flask method.39
Because of the ionizability of the compounds, and in an effort
to imitate physiological conditions, the water phase was
buffered to pH 7.4 (50 mM HEPES; I = 0.16 M NaCl).
All four ligands exhibit log Po/w values indicative of
predominantly hydrophilic character (−3 < log Po/w < −2);
however, all show increased lipophilic character relative to
alendronate. Of greater significance are the log Po/w values for
the La3+ complexes, which all show an expected increase in the
lipophilicity as the ligand’s polar functional groups chelate to
Human Serum Stability Studies with 67Ga(dpaa). In
vivo kinetic inertness of the 67Ga(dpaa) system was estimated
by a 2 h in vitro human serum competition assay and compared
to the industry “gold standards” in gallium(III) chelation
NOTA and DOTA (Table 7).40 Human serum contains
endogenous ligands, such as apo-transferrin and albumin, that
are well-known to form stable complexes with gallium(III);
thus, a radiopharmaceutical containing a gallium chelate
complex must be sufficiently inert to transchelation from
these proteins in vivo. This in vitro assay is a preferred method
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Figure 10. HPLC radiotraces of pH-dependent labeling (pH 2−6.5) of 67Ga with dpaa.
which results in an unsaturated coordination environment and
allows transchelation. Because of the modest stability of
67Ga(dpaa) in human serum, it is unlikely that H3dpaa would
make an appropriate ligand for use with 68Ga in vivo, despite
the considerable stability of [Ga(dpaa)(OH)]−, suggesting that
a stable complex should avoid an extra ligand such as OH−.
CONCLUSIONS
■
Four compounds based on a dpa scaffold were successfully
synthesized and characterized (H2dpa, H3dpaa, H4dppa, and
H7dpbpa) as HCl salts. Solution depletion studies with these
compounds and HAP under pseudophysiological conditions
showed that only the phosphonate- and BP-containing
compounds (H2dppa and H7dpbpa, respectively) demonstrated
an observable binding interaction with bone mineral. This
suggests that these ligands possess inherent bone-targeting
ability.
Figure 11. RCYs obtained from the concentration-dependent labeling
(10−4−10−7 M ligand) of 67Ga with H3dpaa at pH 6.5 in 10 min at
room temperature.
Table 7. In Vitro Human Serum Stability (37 °C, 2 h, and n
= 3) of 67Ga(dpaa) and Standards 67Ga(NOTA) and
67Ga(DOTA), with the Stability Shown as the Percentage of
Intact 67Ga Complex
The lanthanum complexes of all but H7dpbpa were also
synthesized, producing stable 1:1 metal/ligand complexes
1
shown by H NMR spectroscopy to be present as single static
67Ga complex
1 h (%)
2 h (%)
isomers in solution. Crystals suitable for solid-state X-ray
analysis of the 1:1 complex of lanthanum(III) with H4dppa
were obtained, which showed the material as a bridged
lanthanum tetramer with two water molecules included in the
lattice ([La4(dppa)4(H2O)2]). Speciation diagrams and for-
mation constants for H2dpa, H3dpaa, and H4dppa with
lanthanum were acquired by potentiometric, UV−vis, and
NMR titrations, which showed that the inclusion of a
carboxylate or a phosphonate moiety in the ligand scaffold
improved the lanthanum complex stability (log βML) and metal-
scavenging ability (pLa) at physiological pH. The modest
values obtained were concurrent with the fact that, in the solid-
state structures, lanthanum does not appear to be an ideal fit for
the dpa cavity present in the ligands; however, because the
intended function of an ideal system is to relinquish the metal
ion to bone mineral in vivo, it is possible that this metal-ligand
mismatch is ideal; further studies will be undertaken to confirm
this hypothesis. Lipophilicity measurements in the form of
partition coefficients (log Po/w) of the four free ligands and
three lanthanum complexes were experimentally determined
using the shake-flask method, which showed that La(dppa) was
likely to exhibit the greatest oral activity of all of the
[67Ga(dpaa)]
[67Ga(NOTA)]
[67Ga(DOTA)]
60.4 1.3
97.5 0.7
80.1 0.8
58.1 3.4
98.0 0.6
80.0 1.8
of predicting the in vivo kinetic inertness of the radiometal-ion
complexes.
A preformed 67Ga(dpaa) complex was incubated in excess
human serum (37 °C, pH 7.4), and aliquots of each reaction
mixture were removed at 1 or 2 h time points. The percentage
of intact 67Ga complex was determined by size-exclusion
chromatography to separate any 67Ga that may have trans-
chelated with serum proteins. The macrocycle NOTA is well-
known to form a gallium complex of exceptional kinetic
inertness, and this fact is reflected in the results obtained by the
serum stability assay; these suggest that only 2% of 67Ga
transchelated to serum proteins from the NOTA complex after
2 h. Conversely, 67Ga(dpaa) remained approximately 58%
intact, with 42% of 67Ga associated with serum proteins after 2
h. We hypothesize that this is due to the lack of metal
coordination from the tertiary nitrogen atom in the backbone
of dpaa (evidenced in the crystal structure of the complex),
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Potentiometric titrations were performed using a Metrohm Titrando
809 equipped with a Ross combination pH electrode and a Metrohm
Dosino 800 automatic buerette. Data were collected using PC Control
(version 6.0.91, Metrohm). Temperature control was maintained using
a Julabo water bath (25.0 0.1 °C).
compounds tested; however, further in vitro and in vivo studies
are required in order to verify this.
The gallium(III) complex of H3dpaa was also synthesized
and fully characterized, including the solid-state X-ray structure,
which revealed a six-coordinate structure in distorted octahedral
geometry with one site occupied by OH2 (and not the
backbone tertiary nitrogen, as predicted). The metal ion fits
flush within the dpa binding cavity, and potentiometric
titrations indicated good thermodynamic stability of the
Ga(dppa) complex (pGa = 22.0). Radiolabeling with 67Ga
was accomplished at ambient temperature and 10 min;
however, pH-dependent labeling suggested the presence of
multiple isomers in solution between pH 2 and 5. A single
isomer was observed at pH 6.5, and quantitative labeling was
achieved at ligand concentrations as low as 10−5 M; however,
kinetic inertness studies with the 67Ga(dpaa) complex [67Ga-
(dpaa)(OH)]−, through a human serum stability assay, revealed
that only 58% remained intact after 2 h at 37 °C, less stable
than the current hexadentate gold standards NOTA and
DOTA.
Human serum was purchased from Invitrogen. NOTA and DOTA
were purchased from Macrocylics. 67Ga(chelate) human serum
stability experiments were analyzed using GE Healthcare Life Sciences
PD-10 desalting columns (size exclusion for molecular weight MW <
5000 Da) and counted with a Capintec CRC 15R well counter. The
HPLC system used for the analysis and purification of nonradioactive
compounds consisted of a Waters 600 controller, a Waters 2487 dual
wavelength absorbance detector, and a Waters delta 600 pump.
Phenomenex Synergi Hydro-RP 80 Å columns (250 mm × 4.6 mm
analytical or 250 mm × 21.2 mm semipreparative) were used for the
purification of several of the deprotected ligands. The analysis of
radiolabeled complexes was carried out using a Phenomenex Synergi 4
μ Hydro-RP 80A analytical column (250 × 4.60 mm 4 μm) using a
Waters Alliance HT 2795 separation module equipped with a Raytest
Gabi Star NaI (Tl) detector and a Waters 996 photodiode array.
67GaCl3 was cyclotron-produced and provided by Nordion as a ∼0.1
M HCl solution.
Ligand and Metal Complex Synthesis and Characterization.
Refer to the Supporting Information.
EXPERIMENTAL SECTION
■
Bone Targeting. All time points were performed in triplicate.
Samples containing 10.0 mg of HAP were incubated and agitated (37
°C and 220 rpm) with 0.9 mL of HEPES (50 mM, pH 7.4 at 37 °C,
and I = 0.16 M NaCl) in 1.5 mL Eppendorf tubes for 24 h. Stock
solutions (1 mM) of each of the four dpa-containing compounds were
prepared in a buffer from their HCl salts. At the outset of the
experiment, 100 μL of either a H2dpa, H3dpaa, H4dppa, or H7dpbpa
solution was added to the HAP suspension and incubated and agitated
for a set period of time. When the desired time point was reached, the
sample tubes were centrifuged for 1 min, and the supernatant was
carefully extracted by a syringe and filtered through a 22 μm frit. An
aliquot (0.3 mL) of the supernatant was taken and diluted to 3.0 mL
with a HEPES buffer and analyzed by UV−vis spectrometry.
Compound concentrations were determined using the Beer−Lambert
relationship. See the Supporting Information, Table S3, for molar
extinction coefficients (ε) and absorbance maxima (λmax).
Thermodynamic Stability Studies. Potentiometry. All data
were collected in triplicate. The titration apparatus consisted of a 10
mL water-jacketed glass vessel maintained at 298 K. Nitrogen gas,
purified through a 10% NaOH solution to exclude any CO2, was
passed through the solution prior to and during the titrations. The
ionic strength was maintained at 0.16 M using NaCl. Prior to each
titration, the electrode was calibrated using a standard HCl solution.
Calibration data were analyzed by standard computer calculations to
obtain calibration parameters E0 and pKw. Equilibrium times for
titrations were 10 min for pKa titrations and 15 min for metal complex
titrations. Carbonate-free solutions of the titrant [NaOH(aq)] were
prepared by the dilution of 0.1 mol of NaOH (analytical standard)
with freshly boiled Milli-Q water (800 mL) under a stream of nitrogen.
The solution was standardized against freshly recrystallized potassium
hydrogen phthalate. Lanthanum and gallium ion solutions were
prepared by dilution of the AA standards. The exact amount of acid
present in the lanthanum and gallium standards was determined by the
titration of equimolar solutions of either lanthanum(III) or gallium-
(III) and Na2H2-EDTA using a Gran plot.42 The proton dissociation
constants corresponding to the hydrolysis of gallium(III) and
lanthanum(III) aqueous ions were taken from Baes and Mesmer.43
The ligand and metal concentrations were in the range of 0.8−1.0 mM
for potentiometric titrations. Determination of the acid dissociation
constants was achieved by the titration of 1.0 mM ligand solutions
with CO2-free NaOH at 25 °C and 0.16 M NaCl. The protonation
constants of dpa, dpaa, and dppa and the stability constants of
lanthanum(III) and gallium(III) complexes were calculated from the
experimental data using the HyperQuad2013 program.32
Materials, Reagents, and Instruments. Solvents used in all
experiments were of ACS grade or higher and were obtained from
Sigma-Aldrich or Fisher Scientific. HPLC-grade solvents were used in
all purification steps. Water used in all synthetic procedures was
deionized (DI; 15 MΩ-cm) and obtained from an Elga Purelab Option
water purifier. Deuterated solvents for NMR spectroscopy were
purchased from Cambridge Isotope Laboratories. H and 13C NMR
1
spectra were recorded at 25 °C unless otherwise noted on Bruker
AV300, AV400, or AV600 instruments; NMR spectra are expressed on
the δ scale and referenced to residual solvent peaks. 31P{1H} NMR
spectra were referenced externally to H3PO4. Low-resolution mass
spectrometry was performed using a Waters ZG spectrometer with an
ESCI electrospray/chemical-ionization source, and high-resolution
electrospray ionization mass spectrometry was performed on a
Micromass LCT time-of-flight instrument at the Department of
Chemistry, University of British Columbia. Microanalyses for carbon,
hydrogen, and nitrogen were performed on a Carlo Erba EA 1108
elemental analyzer. X-ray crystal structures were solved using a Bruker
APEX DUO diffractometer and Bruker SAINT software.
Concentrated hydrochloric acid (37%) and ammonium hydroxide
(30%) solutions and solid sodium hydroxide, potassium hydroxide,
ammonium chloride, and potassium carbonate were purchased from
Fisher Scientific or Sigma-Aldrich. The starting materials 1, 4, 7, 9, and
12 were obtained from commercial sources and used without further
purification, as were the reagents sodium borohydride, phosphorus
tribromide, and thiophenol. Lanthanum nitrate hexahydrate and
gallium perchlorate hydrate were purchased from Alfa Aesar. The
analytical thin-layer chromatography (TLC) plates used were
aluminum-backed ultrapure silica gel 60 Å of 250 μm thickness
(Merck, Germany).
Water used in all analytical experiments was of high-purity (Milli-Q,
18.2 MΩ-cm) and obtained from an Elga Purelab Ultra water purifier.
Buffered solutions of pH 7.4 and I = 0.16 M were prepared from solid
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; Fisher
Scientific) and trace metal-grade sodium chloride (Sigma-Aldrich) at
desired concentrations and temperatures using an online buffer recipe
calculator (Rob Beynon, University of Liverpool).41 Synthetic HAP
was purchased from Sigma-Aldrich. Sodium deuteroxide (30 wt % in
D2O) and deuterium chloride (35 wt % in D2O) for NMR titrations
were purchased from Sigma-Aldrich as was 1-octanol for log Po/w
experiments. UV−vis spectra were collected on a Varian Vary 100 Bio
UV−vis spectrometer.
Lanthanum-ion solutions for potentiometric titrations were
prepared from the atomic absorption (AA) standard solution
(Sigma-Aldrich, 1021 μg/mL lanthanum in 1 wt % HNO3).
NMR. Protonation constants for the dppa ligand were determined
by 1H and 31P{1H} NMR titrations using the HypNMR2008
L
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Article
program.31 Solutions of the ligand (10 mM) in D2O were prepared at
different pD levels, adjusting with dilute NaOD or DCl. The pD values
were measured via an electrode (Mettler Toledo) and corrected for
the deuterium isotopic effect by the relation pD = pH + 0.4.33 The pKa
was calculated using the HypNMR2008 program.31 Complex
formation constants of La(dpa) and La(dppa) were determined by
1H NMR titrations. Spectra of 15 mM lanthanum-ligand solutions
were collected in approximately a 1:1.5 ratio in D2O at 298 K at
various pD levels, and the concentrations of La3+ and Ga3+ ions were
achieved by using a stock deuterated aqueous acidic solution of the
LaCl3 and GaCl3 salts.
UV−Vis. The formation constants log βMLH and log βML for the
La3+-dpaa system were determined by measuring changes in the
absorbance spectra of an approximately 0.07 mM La3+-dpaa solution at
different pH values (0.22−2.5) in the 240−330 nm spectral range
using a 1 cm cuvette. Complex formation data were analyzed using the
HypSpec program.32
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.6b02357.
■
S
Synthesis and characterization of the ligands and their
metal complex and associated spectra, crystallographic
data for the ligands and metal complexes, molar
extinction coefficients of the dpa ligands and lanthanum
complexes, and potentiometric and NMR titration
supporting data (PDF)
X-ray crystallographic data in CIF format (CIF)
X-ray crystallographic data in CIF format (CIF)
X-ray crystallographic data in CIF format (CIF)
Lipophilicity Measurements. The shake-flask method was used
to experimentally determine the log Po/w values for four ligands
(H2dpa, H3dpaa, H4dppa, and H7dpbpa) and three lanthanum
complexes [La(dpa), La(dpaa), and La(dppa)]. All measurements
were run in triplicate. The aqueous (50 mM HEPES, pH 7.4 at 25 °C,
and I = 0.16 M) and 1-octanol solvents were presaturated with one
another prior to use. To a 1.5 mL Eppendorf tube was added 1-octanol
(0.750 mL) and either 1.0 mM ligand or metal complex aqueous
solution (0.750 mL). Samples were vortexed manually (1.0 min),
inverted on an automixer (5.0 min), and centrifuged (1.0 min and
6000 rpm). The upper and lower layers were carefully separated, and
an aliquot (0.500 mL) of either the 1-octanol or HEPES layer was
diluted to 5.0 mL with ethanol or aqueous buffer, respectively. Each
layer was analyzed by UV−vis spectrometry, applying the Beer−
Lambert law to determine either the ligand or metal/ligand complex
concentration (ε and λmax values in the Supporting Information, Table
S3).
Radiolabeling Experiments. The chelator H3dpaa was made up
as a stock solution (1 mg/mL, ∼10−3 M) in DI water. A 100 μL
aliquot of a chelator stock solution was transferred to screw-cap mass
spectrometry vials and diluted with a pH 3 NaOAc (10 mM) buffer
such that the final volume was 1 mL after the addition of 67GaCl3, to a
final chelator concentration of ∼10−4 M for each sample. An aliquot of
67GaCl3 (∼1 mCi for labeling studies and ∼3−6 mCi for serum
competitions) was added to the vials containing the chelator and
allowed to radiolabel at ambient temperature for 10 min and then
analyzed by reversed phase-HPLC (RP-HPLC) to confirm radio-
labeling and calculate yields. Areas under the peaks observed in the
HPLC radiotrace were integrated to determine radiolabeling yields.
Elution conditions used for RP-HPLC analysis were gradient: A, 0.1%
trifluoroacetic acid in water; B, CH3CN; 0−100% B linear gradient 20
min (tR = 3.3 min for free 67Ga).
AUTHOR INFORMATION
Corresponding Author
*E-mail: orvig@chem.ubc.ca. Phone: +16048224449.
ORCID
Chris Orvig: 0000-0002-2830-5493
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We acknowledge funding from Nordion (Canada), the Natural
Sciences and Engineering Research Council (NSERC) of
Canada, for grant support (CR&D, Discovery, USRA), NSERC
for CGS-M/CGS-D fellowships (to T.I.K. and C.F.R.), and the
University of British Columbia for a 4YF fellowship (to C.F.R.).
We also acknowledge funding from NSERC and the Canadian
Institutes of Health Research in two Collaborative Health
Research Projects. C.O. thanks the Canada Council for the Arts
for a Killam Research Fellowship (2011−2013), and the
authors profusely thank Prof. Dr. Guido Crisponi for his
guidance and support.
REFERENCES
■
(1) National Osteoporosis Foundation, https://www.nof.org/
patients/what-is-osteoporosis/ (accessed July 11, 2016).
(2) Drake, M. T.; Clarke, B. L.; Khosla, S. Bisphosphonates:
mechanism of action and role in clinical practice. Mayo Clin. Proc.
2008, 83, 1032−1045.
(3) Zhang, S.; Gangal, G.; Uludag, H. ‘Magic bullets’ for bone
̆
diseases: progress in rational design of bone-seeking medicinal agents.
Chem. Soc. Rev. 2007, 36, 507−531.
(4) Kennel, K. A.; Drake, M. T. Adverse effects of bisphosphonates:
implications for osteoporosis management. Mayo Clin. Proc. 2009, 84,
632−638.
(5) Whitaker, M.; Guo, J.; Kehoe, T.; Benson, G. Bisphosphonates
for osteoporosis − where do we go from here? N. Engl. J. Med. 2012,
366, 2048−2051.
(6) Takenaka, T.; Yamanaka, T.; Yoshida, M.; Suzuki, H. Unexpected
action of lanthanum carbonate. Osteoporosis Int. 2015, 26, 2385−2386.
(7) Weekes, D. M.; Orvig, C. Harness the bone-seeking ability of
Ca(II)-like metal ions in the treatment of metastatic cancer and
resorption disorders. Chem. Soc. Rev. 2016, 45, 2024−2031.
(8) Atherton, N. D.; Totten, J. W.; Gaitonde, M. D. Use of
lanthanum compounds for the treatment of bone diseases. WO Patent
WO2002000227 A2, 2002.
Human Serum Stability Studies. The 67Ga(dpaa) compound
was prepared with the radiolabeling protocol as described above.
Human serum was removed from the freezer and allowed to thaw at
ambient temperature for 1 h. In triplicate, solutions were prepared in
vials with 750 μL of human serum, 500 μL of 67Ga complex, and 250
μL of phosphate-buffered saline (PBS) and incubated at 37 °C in a
water bath. At time points 1 and 2 h, 500 μL of the human serum
competition mixture was removed from each vial, diluted to a total
volume of 2.5 mL with PBS, and then counted in a Capintec CRC 15R
well counter to obtain a value for the total activity to be loaded on the
PD-10 column. The 2.5 mL of diluted human serum mixture was
loaded onto a PD-10 column that had previously been conditioned via
elution with 20 mL of PBS, and the empty vial was counted in a well
counter to determine the residual activity left in the vial. The 2.5 mL of
loading volume was allowed to elute into a waste container, and then
the PD-10 column was eluted with 3.5 mL of PBS and collected into a
separate vial. The eluent that contained 67Ga bound/associated with
serum proteins (size exclusion for MW < 5000 Da) was counted in a
well counter and then compared to the total amount of activity that
was loaded on the PD-10 column to obtain the percentage of 67Ga that
was bound to serum proteins and therefore no longer chelate-bound.
M
DOI: 10.1021/acs.inorgchem.6b02357
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
(9) Behets, G. J.; Dams, G.; Varcauteren, S. R.; Damment, S. J.;
Bouillon, R.; de Broe, M. E.; D’Haese, P. C. Does the phosphate
binder lanthanum carbonate affect bone in rats with chronic renal
failure? J. Am. Soc. Nephrol. 2004, 15, 2219−2228.
(10) Wang, X.; Yuan, L.; Huang, J.; Zhang, T.-L.; Wang, K.
Lanthanum enhances in vitro osteoblast differentiation via pertussis
toxin-sensitive Gi protein and ERK signaling pathway. J. Cell. Biochem.
2008, 105, 1307−1315.
(11) von Rosenberg, S. J.; Wehr, U. A. Lanthanum salts improve
bone formation in a small animal model of post-menopausal
osteoporosis. J. Anim. Physiol. Anim. Nutr. 2012, 96, 885−894.
(12) Pennick, M.; Dennis, K.; Damment, S. J. Absolute bioavailability
and disposition of lanthanum in healthy human subjects administered
lanthanum carbonate. J. Clin. Pharmacol. 2006, 46, 738−746.
(13) Drugs.com: Fosrenol Side Effects, http://www.drugs.com/sfx/
fosrenol-side-effects.html (accessed July 11, 2016).
nanoparticles targeted to damaged bone tissue. J. Nanopart. Res. 2012,
14, 1175.
(26) Leu, C.-T.; Luegmayr, E.; Freedman, L. P.; Rodan, G. A.;
Reszka, A. A. Relative binding affinities of bisphosphonates for human
bone and relationship to antiresorptive efficacy. Bone 2006, 38, 628−
636.
(27) Rodan, G.; Reszka, A. A.; Golub, E.; Rizzoli, R. Bone safety of
long-term bisphosphonate treatment. Curr. Med. Res. Opin. 2004, 20,
1291−1300.
(28) Dolomanov, O. V.; Bourhis, L. J.; Gildea, R. J.; Howard, J. A. K.;
Puschmann, H. OLEX2: a complete structure solution, refinement and
analysis program. J. Appl. Crystallogr. 2009, 42, 339−341.
(29) van der Sluis, P.; Spek, A. L. BYPASS: an effective method for
the refinement of crystal structures containing disordered solvent
regions. Acta Crystallogr., Sect. A: Found. Crystallogr. 1990, 46, 194−
201.
(14) Barta, C. A.; Sachs-Barrable, K.; Jia, J.; Thompson, K. H.;
Wasan, K. M.; Orvig, C. Lanthanide containing compounds for
therapeutic care in bone resorption disorders. Dalton Trans. 2007,
5019−5030.
(15) Mawani, Y.; Cawthray, J. F.; Chang, S.; Sachs-Barrable, K.;
Weekes, D. M.; Wasan, K. M.; Orvig, C. In vitro studies of lanthanide
complexes for the treatment of osteoporosis. Dalton Trans. 2013, 42,
5999−6011.
(16) Cawthray, J. F.; Weekes, D. M.; Sivak, O.; Creagh, A. L.;
Ibrahim, F.; Iafrate, M.; Haynes, C. A.; Wasan, K. M.; Orvig, C. In vivo
study and thermodynamic investigation of two lanthanum complexes,
La(dpp)3 and La(XT), for the treatment of bone resorption disorders.
Chem. Sci. 2015, 6, 6439.
(17) Xu, L.; Rettig, S. J.; Orvig, C. Lanthanide chemistry with
(bis[[bis(carboxymethyl) amino]methyl]phosphinate: what does an
extra phosphinate group do to EDTA? Inorg. Chem. 2001, 40, 3734−
3738.
(30) Smith, P. H.; Reyes, Z. E.; Lee, C.-W.; Raymond, K. N.
Characterization of a series of lanthanide amine cage complexes. Inorg.
Chem. 1988, 27, 4154−4165.
(31) Alderighi, L.; Gans, P.; Ienco, A.; Peters, D.; Sabatini, A.; Vacca,
A. Hyperquad simulation and speciation (HySS): a utility program for
the investigation of equilibria involving soluble and partially soluble
species. Coord. Chem. Rev. 1999, 184, 311−318. Frassineti, C.; Ghelli,
S.; Gans, P.; Sabatini, A.; Moruzzi, M. S.; Vacca, A. Nuclear magnetic
resonance as a tool for determining protonation constants of natural
polyprotic bases in solution. Anal. Biochem. 1995, 231, 374−382.
(32) HyperQuad2013: Equilibrium Constants from Potentiometric
Data, http://www.hyperquad.co.uk/HQ2013.htm (accessed Septem-
ber 13, 2016). HypSpec: Equilibrium Constants from Solution
Spectrophotometric Data, http://www.hyperquad.co.uk/HypSpec.
htm (accessed September 15, 2016).
(33) Covington, A. K.; Paabo, M.; Robinson, R. A.; Bates, R. G. Use
of the glass electrode in deuterium oxide and the relation between the
standardized pD scale and the operational pH in heavy water. Anal.
Chem. 1968, 40, 700−706.
(18) Platas-Iglesias, C.; Mato-Iglesias, M.; Djanashvili, K.; Muller, R.
N.; Vander Elst, L.; Peters, J. A.; de Blas, A.; Rodríguez-Blas, T.
Lanthanide chelates containing pyridine units with potential
applications as contrast agents in magnetic resonance imaging.
Chem. - Eur. J. 2004, 10, 3579−3590. Balogh, E.; Mato-Iglesias, M.;
(34) Clarke, E. T.; Martell, A. E. Stabilities of the Fe(III), Ga(III) and
In(III) chelates of N,N′,N″-triazacyclononanetriacetic acid. Inorg.
Chim. Acta 1991, 181, 273−280. Wadas, T. J.; Wong, E. H.;
Weisman, G. R.; Anderson, C. J. Coordinating radiometals of copper,
gallium, indium, yttrium and zirconium for PET and SPECT imaging
of disease. Chem. Rev. 2010, 110, 2858−2902.
́
Platas-Iglesias, C.; Toth, E.; Djanashvili, K.; Peters, J. A.; de Blas, A.;
Rodríguez-Blas, T. Pyridine and phosphonate containing acyclic
ligands for stable lanthanide complexation. Extremely fast water
exchange on the on the GdIII chelates. Inorg. Chem. 2006, 45, 8719−
8728.
(35) Clarke, E. T.; Martell, A. E. Stabilities of trivalent metal ion
complexes of the tetraacetate derivatives of 12-, 13- and 14-membered
tetraazamacrocycles. Inorg. Chim. Acta 1991, 190, 37−46. Chaves, S.;
(19) Nonat, A.; Fries, P. H.; Pecaut, J.; Mazzanti, M. Structure,
stability, dynamic, high-field relaxivity and ternary-complex formation
of a new tris(aquo) gadolinium complex. Chem. - Eur. J. 2007, 13,
8489−8506.
(20) Boros, E.; Ferreira, C. L.; Cawthray, J. F.; Price, E. W.; Patrick,
B. O.; Wester, D. W.; Adam, M. J.; Orvig, C. Acyclic chelate with ideal
properties for 68Ga PET imaging agent elaboration. J. Am. Chem. Soc.
2010, 132, 15726−15733.
(21) Price, E. W.; Zeglis, B. M.; Cawthray, J. F.; Ramogida, C. F.;
Ramos, N.; Lewis, S. J.; Adam, M. J.; Orvig, C. H4octapa-trastuzumab:
versatile acyclic chelate system for 111In and 177Lu imaging and
therapy. J. Am. Chem. Soc. 2013, 135, 12707−12721. Price, E. W.;
Cawthray, J. F.; Adam, M. J.; Orvig, C. Modular syntheses of H4octapa
and H2dedpa, and yttrium coordination chemistry relevant to 86Y/90Y
radiopharmaceutical. Dalton Trans. 2014, 43, 7176−7190.
(22) Matveeva, E. V.; Petrovskii, P. V.; Odinets, I. L. Efficient
synthesis of racemic β-aminophosphonates via-Michael reaction in
water. Tetrahedron Lett. 2008, 49, 6129−6133.
Mendonca̧ , A. C.; Marques, S. M.; Prata, M. I.; Santos, A. C.; Martins,
A. F.; Geraldes, C. F. G. C.; Santos, M. A. A gallium complex with a
new tripodal tris-hydroxypyridinone for potential diagnostic imaging:
solution and in vivo studies of 67Ga-labeled species. J. Inorg. Biochem.
2011, 105, 31−38.
(36) Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J.
Experimental and computational approches to estimate solubility and
permeability in drug discovery and development settings. Adv. Drug
Delivery Rev. 2001, 46, 3−26.
(37) Zhang, M.-Q.; Wilkinson, B. Drug discovery beyond the ‘rule of
five’. Curr. Opin. Biotechnol. 2007, 18, 478−488.
(38) Ananchenko, G.; Novakovic, J.; Tikhomirova, A. In Profiles of
Drug Substances, Excipients and Related Methodology, 1st ed.; Brittain,
H. G., Eds.; Academic Press: San Diego, 2013; Vol. 38, Chapter 1, p 5.
(39) Test 107: Partition Coefficient (n-octanol/water): Shake Flask
Method. In OECD Guidelines for the Testing of Chemicals; Section 1:
Physical−Chemical Properties; OECD Publishing: Paris, 1995; pp 1−4.
(40) Price, E. W.; Orvig, C. Matching chelators to radiometals for
́ ́
(23) Kubicek, V.; Kotek, J.; Hermann, P.; Lukes, I. Aminoalkylbis-
(phosphonates): their complexation properties in solution and in the
solid state. Eur. J. Inorg. Chem. 2007, 2007, 333−344.
radiopharmaceuticals. Chem. Soc. Rev. 2014, 43, 260−290. Kubíce
̌
k, V.;
́
Havlíck
̌
ova,
́
J.; Kotek, J.; Tircso,
́
G.; Hermann, P.; Tot
́
h, E.; Lukes, I.
̌
(24) Torres Martin de Rosales, R.; Finucane, C.; Mather, S. J.;
Blower, P. J. Bifunctional bisphosphonate complexes for the diagnosis
and therapy of bone metastases. Chem. Commun. 2009, 4847−4849.
(25) Ross, R. D.; Cole, L. E.; Roeder, R. K. Relative binding affinity
of carboxylate-, phosphonate-, and bisphosphonate-functionalized gold
Gallium(III) complexes of DOTA and DOTA-monoamide: kinetic
and thermodynamic studies. Inorg. Chem. 2010, 49, 10960−10969.
(41) A recipe calculator for thermodynamically correct buffers for pH
control, https://www.liverpool.ac.uk/buffers/buffercalc.html (accessed
Aug 3, 2016).
N
DOI: 10.1021/acs.inorgchem.6b02357
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
(42) Gran, G. Determination of the equivalence point in
potentiometric tirations, Part II. Analyst 1952, 77, 661−671.
(43) Baes, C. F.; Mesmer, R. E.Hydrolysis of cations; Wiley: New
York, 1976.
O
DOI: 10.1021/acs.inorgchem.6b02357
Inorg. Chem. XXXX, XXX, XXX−XXX