A boronate prochelator built on a triazole framework for peroxide-triggered tridentate metal binding

Article abstract of DOI:10.1016/j.ica.2012.06.011

Iron chelating agents have the potential to minimize damage associated with oxidative stress in a range of diseases; however, this potential is countered by risks of indiscriminant metal binding or iron depletion in conditions not associated with systemic

Full text of DOI:10.1016/j.ica.2012.06.011

Inorganica Chimica Acta 393 (2012) 294–303
Contents lists available at SciVerse ScienceDirect
Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
A boronate prochelator built on a triazole framework for peroxide-triggered
tridentate metal binding
Filip Kielar a, Qin Wang a, Paul D. Boyle b, Katherine J. Franz a,
⇑
a Department of Chemistry, Duke University, Durham, NC 27708-0346, USA
b Department of Chemistry, North Carolina State University, Raleigh, NC 27695-820, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 23 June 2012
Iron chelating agents have the potential to minimize damage associated with oxidative stress in a range
of diseases; however, this potential is countered by risks of indiscriminant metal binding or iron deple-
tion in conditions not associated with systemic iron overload. Deferasirox is a chelator used clinically for
iron overload, but also is cytotoxic to cells in culture. In order to test whether a prodrug version of
deferasirox could minimize its cytotoxicity but retain its protective properties against iron-induced oxi-
dative damage, we synthesized a prochelator that contains a self-immolative boronic ester masking
group that is removed upon exposure to hydrogen peroxide to release the bis-hydroxyphenyltriazole
ligand deferasirox. We present here the synthesis and characterization of this triazole-based, self-imm-
olative prochelator: TIP (4-(5-(2-((4-boronobenzyl)oxy)phenyl)-3-(2-hydroxyphenyl)-1H-1,2,4-triazol-
1-yl)benzoic acid). TIP does not coordinate to Fe3+ and shows only weak affinity for Cu2+ or Zn2+, in stark
contrast to deferasirox, which avidly binds all three metal ions. TIP converts efficiently in vitro upon reac-
tion with hydrogen peroxide to deferasirox. In cell culture, TIP protects retinal pigment epithelial cells
from death induced by hydrogen peroxide; however, TIP itself is more cytotoxic than deferasirox in
unstressed cells. These results imply that the cytotoxicity of deferasirox may not derive exclusively from
its iron withholding properties.
Metals in Medicine Special Issue
Keywords:
Chelation therapy
Iron
Oxidative stress
Fenton chemistry
Reactive oxygen species
Prodrug
Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction
but for which iron-promoted oxidative stress contributes to dis-
ease. For example, neurodegenerative disorders, ischemia/reperfu-
Chelating agents have been used clinically for more than
40 years to reduce the iron overload that comes with chronic blood
transfusions of patients with thalassemia major, sickle cell disease,
and other rare anemias [1]. The excess iron accumulates particu-
larly in the heart, liver and endocrine organs, which eventually
experience significant tissue damage as a result of oxidative stress
from iron-catalyzed formation of highly reactive hydroxyl radicals
[1]. There are currently three chelating agents used worldwide for
the treatment of transfusion-induced iron overload, the gold stan-
dard being desferal (desferrioxamine B, DFO), which has been used
since the 1970s but must be administered parenterally via diffu-
sion pump [2]. The two oral alternatives to DFO now available
are ExjadeÒ (deferasirox, ICL670) and FerriproxÒ (deferiprone, L1)
[3]. While there remain drawbacks and side effects of each of these
drugs, they have certainly made dramatic improvements in the
quality of life and survival of these patients [4].
sion injury, and cardiovascular disease, among others, show signs
of localized iron accumulation or misappropriation that may con-
tribute to oxidative damage [5,6]. Recent attention is therefore
focusing on expanding the use of approved chelators for new indi-
cations, as well as designing novel chelating agents specifically tar-
geting conditions of localized metal imbalance [7–12].
There are many properties of deferasirox that make it an
attractive agent for use in other oxidative stress related diseases.
Its N-substituted bis-hydroxyphenyltriazole core, shown in Fig. 1,
positions two phenolate oxygen donors and one triazole nitrogen
donor for tridentate metal coordination with adjacent 5-mem-
bered chelate rings that favor small cations like Fe3+ over larger
divalent cations [13]. This construction provides a high affinity
binding pocket that has a favorable pFe3+ value of 22.5 and
negative redox potential that prevents reduction to Fe2+ under
physiological conditions [13]. The inhibition of redox cycling is
particularly attractive for preventing hydroxyl radical formation
via Fenton chemistry. With an octanol/water partition coefficient
of 3.8, the hydrophobicity of deferasirox predicts that it readily
accesses cells and accumulates in tissues. While this property is
advantageous for its oral availability and its ability to decrease car-
diac iron [14], it may be disadvantageous in diseases not associated
The success of using iron chelation to minimize tissue damage
due to iron overload has sparked an interest in exploring iron
chelation for diseases that are unrelated to systemic iron overload
⇑
Corresponding author. Tel.: +1 919 660 1541; fax: +1 919 660 1605.
E-mail address: katherine.franz@duke.edu (K.J. Franz).
0020-1693/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ica.2012.06.011

F. Kielar et al. / Inorganica Chimica Acta 393 (2012) 294–303
295
25-min gradient from 2–98% B into A, where mobile phase B is ace-
tonitrile and A is H2O), or method 2 (semipreparative XBridge col-
umn, 19 ꢀ 250 mm, 10 mL/min 2–98% B into A). UV–Vis spectra
were recorded on a Varian Cary 50 spectrophotometer.
OH
HO
N
N
N
2.2. Synthesis
OH
2.2.1. Methyl 4-(3,5-bis(2-hydroxyphenyl)-1H-1,2,4-triazol-1-
yl)benzoate (3)
A solution of 2 (760 mg, 4.6 mmol), 1 (1.01 g, 4.2 mmol), and
triethylamine (660 ll, 4.3 mmol) in ethanol (70 ml) was heated
O
deferasirox
(ICL670, Exjade)
Fig. 1. Structure of the metal-chelating agent deferasirox.
to 110 °C in a closed reaction vessel for 2 h. The reaction mixture
was diluted with water (100 ml) and the volume was reduced to
one half on a rotary evaporator. The pH of the remaining solution
was adjusted to 4 using glacial acetic acid, and it was extracted
with CH2Cl2 (3 ꢀ 50 ml). The combined organic layers were dried
with sodium sulfate and the solvent was removed in vacuo. The
residue was purified by column chromatography on silica (CH2Cl2
to 1% MeOH in CH2Cl2). The product was obtained as a yellowish
solid (1.03 g, 2.7 mmol, 63%). dH (CDCl3, 400 MHz): 3.99 (1H, s,
OCH3), 6.66 (1H, ddd, 3J = 6.8, 3J = 7.2, 4J = 1.2), 6.92 (1H, dd,
3J = 8.0, 4J = 1.6), 7.04 (1H, ddd, 3J = 7.2, 3J = 6.8, 4J = 1.2), 7.08 (1H,
dd, 3J = 8.4, 4J = 0.8), 7.15 (1H, dd, 3J = 8.4, 4J = 1.2), 7.35 (1H, m),
7.39 (1H, m), 7.61 (2H, d, 3J = 8.8), 8.13 (1H, dd, 3J = 8.0, 4J = 1.6),
8.23 (2H, d, 3J = 8.8), 9.58 (1H, s, OH), 11.32 (1H, s). dC (CDCl3,
125 MHz): 52.6, 109.8, 113.1, 117.1, 118.4, 119.0, 119.9, 126.0,
127.6, 127.7, 131.2, 131.6, 131.9, 133.1, 141.5, 152.1, 156.5,
158.0, 159.6, 165.7. HRMS (ES+) 388.1290 (C22H18N3O4 requires
388.1292) (MH+).
with systemic iron overload, as it increases the chances of undesir-
able metal binding or extraction from key metalloproteins. Indeed,
cell culture studies have shown that deferasirox is cytotoxic to sev-
eral cell lines [12,15,16]. While the mechanism of cytotoxicity has
not been firmly established, the removal or withholding of iron
from critical iron proteins is speculated [15]. Regardless of its
inherent cytotoxicity, deferasirox exerts significant protection of
cultured cardiac cells against oxidative stress induced by exposure
to catecholamines and tert-butyl hydroperoxide [12,16].
The previous results suggest that deferasirox has interesting
properties for protecting against oxidative damage via iron chela-
tion, especially if its cytotoxicity can be minimized. Our lab has
developed a prodrug, or prochelator, strategy that employs a
chemically reactive masking group to block adventitious metal
binding [17,18]. By choosing a masking group that is reactive
specifically under conditions unique to a pathological site, a chelat-
ing agent can in principle be released at the site of damage, thereby
minimizing its impact on the metal ion balance of normal cells. We
have found that this strategy indeed increases the protective win-
dow of a hexadentate chelator that is otherwise cytotoxic [19]. We
were therefore interested to test whether a prochelator version of
deferasirox could minimize its toxicity while retaining its protec-
tive properties against oxidative damage. Herein, we present a
prochelator of deferasirox that contains a self-immolative boronic
ester masking group that is removed upon exposure to hydrogen
peroxide to release deferasirox.
2.2.2. Methyl 4-(3-(2-hydroxyphenyl)-5-(2-((4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)benzyl)oxy)phenyl)-1H-1,2,4-triazol-1-
yl)benzoate (5)
A mixture of 3 (430 mg, 1.1 mmol), 4-bromomethylphenylbo-
ronic acid pinacol ester (4) (315 mg, 1.1 mmol), and potassium
carbonate (150 mg, 1.1 mmol) in acetonitrile was heated to reflux
for 20 h. The solvent was removed in vacuo and the residue was
purified by column chromatography on silica (CH2Cl2 to 2% MeOH
in CH2Cl2) to give the product as a yellowish solid (400 mg,
0.66 mmol, 60%). dH (CDCl3, 400 MHz): 1.32 (12H, s, CH3), 3.94
(3H, s, OCH3), 4.67 (2H, s, CH2), 6.84 (3H, m), 6.96 (1H, ddd,
3J = 6.8, 3J = 7.2, 4J = 1.2), 7.05 (1H, dd, 3J = 8.4, 4J = 1.2), 7.13 (1H,
ddd, 3J = 7.2, 3J = 7.6, 4J = 1.6), 7.32 (1H, m), 7.35 (2H, d, 3J = 8.8),
7.43 (1H, ddd, 3J = 7.2, 3J = 7.6, 4J = 1.6), 7.65 (2H, d, 3J = 8.0), 7.67
(1H, dd, 3J = 7.6, 4J = 1.6), 7.93 (2H, d, 3J = 8.4), 8.16 (1H, dd,
3J = 8.0, 4J = 1.6), 10.82 (1H, s, OH). dC (CDCl3, 125 MHz): 24.9,
52.4, 70.2, 83.9, 112.8, 113.9, 117.3, 117.4, 119.5, 121.5, 122.6,
126.1, 127.0, 129.5, 130.5, 131.4, 131.6, 132.7, 134.8, 138.8,
141.8, 151.4, 155.8, 157.1, 161.3, 166.1. HRMS (ES+) 604.2621
(C35H35BN3O6 requires 604.2619) (MH+).
2. Experimental
2.1. Materials and methods
Unless otherwise noted, chemicals were purchased commer-
cially from Sigma Aldrich and used without further purification.
Methyl 4-hydrazinylbenzoate (2), 2-(2-hydroxyphenyl)-4H-
benzo[e][1,3]oxazin-4-one (1) and 4-[3,5-bis(2-hydroxyphenyl)-
1,2,4-triazol-1-yl]benzoic acid (deferasirox) were synthesized by
following previously published procedures [13,20]. NMR spectra
were collected on a Varian Inova 400 or Varian Unity 500
spectrometer with chemical shifts reported in ppm and J values
in Hz. Liquid chromatography/mass spectrometry (LC/MS) was
performed using an Agilent 1100 Series apparatus with an LC/
MSD trap and a Daly conversion dynode detector. A Supelco Ascen-
tis C18 (50 ꢀ 1.0 mm) column was used and peaks were detected
by UV absorption at 280 nm. A linear gradient from 35% B in A to
95% B in A was run from 2 to 17 min, where A is water/2%
MeCN/0.1% formic acid and B is MeCN/2% water/0.1% formic acid.
High-resolution mass spectra (HRMS) were recorded on an Agilent
G6224 LCMS-TOF system. Thin layer chromatography was per-
formed on TLC sheets (silica gel 60 F254) from Merck (Darmstadt,
Germany). Column chromatography was performed on silicagel
(Silica gel 60) from Sigma Aldrich. HPLC analysis and purification
was performed on a Waters 600 system using method 1 (Waters
2.2.3. 4-(5-(2-((4-boronobenzyl)oxy)phenyl)-3-(2-hydroxyphenyl)-
1H-1,2,4-triazol-1-yl)benzoic acid (TIP, 6)
A mixture of 5 (150 mg, 0.25 mmol) and potassium hydroxide
(25 mg, 0.45 mmol) in water (10 ml) and MeOH (5 ml) was stirred
at r.t. for 48 h. The pH of the solution was adjusted to 4 by addition
of 1 M HCl. The solvent was removed in vacuo and the residue was
purified by semipreparative HPLC using method 2. The product
was obtained as a white solid (80 mg, 0.16 mmol, 64%). dH (CDCl3,
500 MHz): 4.59 (2H, s, CH2), 6.80 (3H, m), 6.87 (1H, ddd, 3J = 7.5,
3J = 7.5, 4J = 1.0), 6.9 (1H, dd, 3J = 8.5, 4J = 1.0), 7.59 (1H, ddd,
3J = 7.5, 3J = 7.5, 4J = 1.0), 7.23 (3H, m), 7.38 (1H, ddd, 3J = 6.5,
3J = 6.5, 4J = 1.5), 7.58 (1H, dd, 3J = 7.5, 4J = 2), 7.62 (2H, d, 3J = 8.0),
7.86 (2H, d, 3J = 8.5), 8.06 (1H, dd, 3J = 7.5, 4J = 2.0), 10.76 (1H, s,
OH). dC (CDCl3, 100 MHz): 70.0, 112.7, 113.7, 116.9, 117.0, 119.3,
121.2, 122.3, 126.0, 126.8, 130.4, 131.1,131.3, 132.5, 134.5, 137.5,
analytical XBridge column, 4.6 ꢀ 250 mm, 1 mL/min, with
a

296
F. Kielar et al. / Inorganica Chimica Acta 393 (2012) 294–303
141.2, 151.2, 155.7, 156.8, 160.7, 167.2. HRMS (ES+) 508.1684
(C28H23BN3O6 requires 508.1680) (MH+).
were designated as C544, C545, and C546 respectively. The occu-
pancy for this disorder refined to a value of 0.848(6) for the
predominant conformer. The structural model was fit to the data
using full matrix least-squares based on F2. The calculated struc-
ture factors included corrections for anomalous dispersion from
the usual tabulation. The structure was refined using the XL
program from SHELXTL [24], graphic plots were produced using the
NRCVAX crystallographic program suite [25]. Crystallographic data
of compound 5 has been deposited in the Cambridge Crystallo-
graphic Data center (CCDC ID: 880777).
2.2.4. 4-(3-(2-hydroxyphenyl)-5-(2-((4-(3a,5,5-trimethylhexahydro-
4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)benzyl)oxy)phenyl)-1H-
1,2,4-triazol-1-yl)benzoic acid (TIP-pd, 8)
A portion of (1R, 2R, 3S, 5R)-(-)-pinanediol (90 mg, 0.53 mmol)
was added to a solution of unpurified compound 6 dissolved in
DMF (10 ml) and toluene (90 ml). The mixture was stirred under
reflux in an apparatus equipped with Dean–Stark adaptor for
24 h. The solvent was removed in vacuo and the residue was puri-
fied by semipreparative HPLC using method 2 to yield the product
as a white solid (50 mg, 0.08 mmol, 31%). dH (CDCl3, 500 MHz):
0.90 (3H, s, CH3), 1.23 (1H, d, 3J = 8.0), 1.33 (3H, s, CH3), 1.50 (3H,
s, CH3), 1.97 (2H, m), 2.17 (1H, t, 3J = 5.0), 2.26 (1H, m), 2.42 (1H,
m), 4.46 (1H, m), 4.73 (2H, s, CH2), 6.94 (3H, m), 7.02 (1H, m),
7.10 (1H, d, 3J = 8.0), 7.20 (1H, m), 7.38 (1H, m), 7.42 (2H, m),
7.52 (1H, m), 7.72 (2H, d, 3J = 7.5), 7.75 (1H, m), 8.03 (2H, d,
3J = 9.0), 8.24 (1H, m). dC (CDCl3, 125 MHz): 24.0, 26.5, 27.1, 28.7,
35.5, 35.8, 38.2, 39.5, 51.4, 70.3, 78.3, 86.4, 112.9, 113.4, 117.4,
119.7, 121.7, 122.7, 126.4, 127.2, 128.7, 131.1, 131.7, 133.0,
134.0, 138.5, 142.4, 151.3, 155.8, 157.1, 160.9, 169.8. HRMS (ES+)
642.2779 (C38H37BN3O6 requires 642.2775) (MH+).
2.4. Metal binding studies
Metal salt solutions of Cu2+ (as CuSO4) and Zn2+ (as ZnSO4) were
freshly prepared as 30 mM stock solutions in nanopure water,
while 2 mM Fe3+ stock solutions were freshly prepared from FeCl3
in nanopure water or ferric ammonium citrate in PBS buffer, pH
7.4. Stock solutions of deferasirox (30.8 mM), TIP (10 mM), and
TIP-ctrl (10 mM) were freshly prepared in DMSO and further
diluted in PBS buffer with 1% DMSO, pH 7.4 to reach a final concen-
tration of 20
0.5 equiv (10
l
l
M. The solutions were equilibrated overnight with
M) of an indicated metal ion before UV–Vis spectra
were recorded.
2.2.5. 4-(5-(2-(benzyloxy)phenyl)-3-(2-hydroxyphenyl)-1H-1,2,4-
triazol-1-yl)benzoic acid (TIP-ctrl, 11)
2.5. Reaction with hydrogen peroxide
A mixture of 3 (150 mg, 0.43 mmol), benzyl bromide (75 mg,
0.44 mmol), and potassium carbonate (160 mg, 1.2 mmol) in DMF
was heated to 110 °C. The solvent was removed in vacuo and the
residue was purified by column chromatography on silica (CH2Cl2)
to yield the intermediate methyl 4-(3-(2-(benzyloxy)phenyl)-5-(2-
hydroxyphenyl)-1H-1,2,4-triazol-1-yl)benzoate (10), which was
treated with KOH (35 mg, 0.63 mmol) in water (10 ml) and MeOH
(5 ml) and stirred at r.t. for 48 h. The pH of the solution was adjusted
to 4 by addition of 1 M HCl. The solvent was removed in vacuo and
the residue was purified by semipreparative HPLC using method 2.
The product was obtained as a white solid (30 mg, 0.06 mmol,
15%). dH (CDCl3, 400 MHz): 4.69 (1H, s, CH2), 6.88 (2H, m), 6.93
(1H, d, 3J = 8.4), 6.98 (1H m), 7.07 (1H, d, 3J = 8.4), 7.18 (1H, m),
7.25 (3H, m), 7.35 (3H, m), 7.50 (1H, ddd, 3J = 6.8, 3J = 7.2, 4J = 1.2),
7.72 (1H, dd, 3J = 7.6, 4J = 1.6), 7.98 (2H, d, 3J = 7.6), 8.18 (1H, dd,
3J = 8.0, 4J = 1.6). dC (CDCl3, 100 MHz): 70.3, 112.8, 113.7, 117.2,
117.3 119.5, 121.5, 122.5, 127.0, 127.1, 128.1, 128.3, 128.4, 131.0,
131.4, 131.6, 132.7, 135.5, 142.6, 151.4, 155.8, 157.1, 181.3, 170.4.
HRMS (ES+) 464.1600 (C28H22N3O4 requires 464.1610) (MH+).
Stock solutions of TIP (10 mM) in DMSO-d6 or DMSO were
prepared for 1H NMR and LC/MS investigations of the TIP reaction
with H2O2, respectively. For NMR detection, 250 lM TIP in D2O
with 2.5% DMSO-d6 were prepared from the stock solutions. The
oxidation of the prochelator TIP was triggered by addition of con-
centrated H2O2 to give a final concentration of 50 mM. The species
of the reaction mixtures were then identified by 500 MHz NMR
spectrometer after 24 h of equilibration. For LC/MS analysis,
100 lM TIP in PBS buffer, pH 7.4 with 1% DMSO was prepared from
the stock solution followed by addition of H2O2 to get a 10 mM
final concentration. The products of the reaction were analyzed
by LC/MS.
The kinetics of TIP (20 lM) deprotection initiated by H2O2 were
also investigated by UV–Vis spectrophotometry under pseudo-
first-order conditions with 10- to 100-fold excess H2O2. The pseu-
do-first-order rate constant (kobs) at each H2O2 concentration (0.5,
1, 2, 4 and 6 mM) was determined in triplicate from plots of Abs at
273 nm versus time. Sigmaplot software was used to fit the data to
Eq. (1), where A is Abs273, k is kobs, A1 is the difference of the initial
minus Ainf, and Ainf is the absorbance at 273 at infinite reaction
time. A plot of kobs versus [H2O2] provides a linear plot where
the slope is the second-order rate constant, k.
2.3. Crystal structure determination
Single crystals of compound 5 were obtained by slow evapora-
tion from a solution of diethyl ether. The sample was mounted on a
Mitegen polyimide micromount with a small amount of Paratone N
oil. X-ray measurements were made on a Bruker-Nonius Kappa
Axis X8 Apex2 diffractometer at a temperature of 110 K. The unit
cell dimensions were determined from a symmetry constrained
fit of 9098 reflections with 4.78° < 2h < 61.5°. The data collection
A ¼ A1eꢁkt þ Ainf
ð1Þ
2.6. Cell culture
All cell culture reagents, including minimal essential medium
(MEM), Dulbecco’s modified eagle medium (DMEM), F12 Ham’s
nutrient mix (F12), fetal bovine serum (FBS), pencillin–streptomy-
strategy was a number of
x and / scans which collected data up
to 72.78° (2h). The frame integration was performed using SAINT
[21]. The resulting raw data was scaled and absorption corrected
using a multi-scan averaging of symmetry equivalent data using
SADABS [22]. The structure was solved by direct methods using the
SIR92 program [23]. All non-hydrogen atoms were obtained from
the initial solution. The hydrogen atoms were introduced at ideal-
ized positions and were allowed to ride on the parent atom. The
molecule exhibited a conformational disorder in the BO2C2 ring
and the attached methyl groups. Distinct alternate positions were
found for O6 (designated as O60), C444, C445, and C446, which
cin (pen-strep), L-glutamine, and trypsin–EDTA were purchased
from Gibco. The spontaneously immortalized human retinal
pigment epithelial cell line ARPE-19 was purchased from American
Type Culture Collection. The cells were grown in 1:1 DMEM and
F12 medium supplemented with FBS (10%), pen-strep (1%), and
glutamine (1%). Cells were cultured until confluent in 24-well
Falcon plates. CellTiter-Blue Cell Viability Assay was obtained from
Promega. Cell viability assays were performed on a Perkin Elmer
Victor3 1420 plate reader.

F. Kielar et al. / Inorganica Chimica Acta 393 (2012) 294–303
297
For cytotoxicity assays, the growth medium was removed when
cells reached confluence. Cells were then washed three times with
MEM and treated with deferasirox, TIP, TIP-pd, or TIP-ctrl at final
procedure for deferasirox [13]. The methyl ester of 4-hydrazino ben-
zoic acid was used in our procedure instead of the previously used
benzoic acid to afford the methyl ester of deferasirox (3). This route
was chosen to avoid alkylation of the free carboxylic acid of deferasi-
rox in later stages of the prochelator synthesis. The synthesis of the
prochelators was then achieved by alkylating the deferasirox methyl
ester (3) with 4-bromomethylphenylboronic acid pinacol ester (4)
to give the ester-protected prochelator 5. Basic hydrolysis of both
the carboxylic and boronate esters of 5 provides the triazole self-
immolative prochelator TIP (6). We chose to use the self-immolative
boronic acid protecting group because of synthetic ease and its
favorable masking and reaction properties, as we and others have
observed previously [26,27]. The free boronic acid of TIP was ester-
ified with pinanediol to afford a protected prochelator TIP-pd (8). A
control compound that masks one of the phenol groups but does not
contain a peroxide-reactive boronate group, TIP-ctrl (11), was syn-
thesized by alkylating the methyl ester version of deferasirox (3)
with benzyl bromide and hydrolyzing the methyl ester in a subse-
quent step. Remarkably, the alkylation steps in all cases proceeded
regioselectively on the phenol attached at position 5 of the triazole
ring. The regioselectivity of this reaction was confirmed by X-ray
crystallography, as described below.
concentrations ranging from 0–100 lM in MEM with up to 1%
DMSO. After 24 h of incubation, cell viability was determined by
the CellTiter-Blue assay. For cytoprotection assays, washed cells
were pre-treated for 5 h with deferasirox, TIP, or TIP-ctrl at the
indicated concentrations prior to addition of H2O2 (200 lM final
concentration). The cells were further incubated for 19 h before
determination of viability. The conditions compared in each exper-
iment were: positive control (cells treated only with MEM), nega-
tive control (cells treated with MEM containing H2O2), and cells
treated with H2O2 and a range of deferasirox/TIP/TIP-pd/TIP-ctrl
concentrations. Each condition was run in triplicate and variability
was determined as the standard deviation of the results.
3. Results and discussion
3.1. Synthesis
As outlined in Scheme 1, the synthesis of the triazole prochelators
was achieved by following a modification of a published synthetic
O
B
O
O
B
OH
HO
O
OH
O
O
O
O
N
O
Br
4
N
N
N
N
NEt3
EtOH, 100oC
N
N
K2CO3
ACN
NH
HO
H2N
O
O
1
2
3
5
O
O
O
OH
B
B
OH
HO
HO
O
OH
O
OH
O
N
N
7
KOH, pH 12
H2O, MeOH
5
N
N
N
N
Toluene, DMF
OH
OH
O
O
6
TIP
8
TIP-pd
OH
OH
O
O
Br
N
N
9
KOH, pH 12
H2O, MeOH
3
N
N
N
K2CO3
ACN
N
OH
O
O
O
10
11
TIP-ctrl
Scheme 1. Synthetic scheme for triazole prochelators. The two prochelators TIP and TIP-pd along with the control compound TIP-ctrl that are used in cell culture studies are
highlighted in boxes.

298
F. Kielar et al. / Inorganica Chimica Acta 393 (2012) 294–303
Table 1
Summary of crystal data for 5.
Formula
C35H34BN3O6
Formula weight (g/mol)
Crystal dimensions (mm)
Crystal color and habit
Crystal system
Space group
T (K)
603.46
0.38 ꢀ 0.33 ꢀ 0.09
colorless prism
triclinic
ꢀ
P1
110
a (Å)
8.704(3)
b (Å)
13.004(5)
c (Å)
14.806(4)
a
(°)
70.487(11)
b (°)
77.924(8)
c
(°)
85.695(15)
1544.6(9)
V (Å3)
Number of reflections to determine final unit cell
9098
Minimum and Maximum 2h for cell determination (°)
4.78, 61.5
Z
2
F(000)
636
q
(g/cm)
k (Å), (Mo K
(cmꢁ1
1.297
0.71073
0.089
a)
l
)
Diffractometer type
Bruker-Nonius Kappa Axis X8
Apex2
Scan type(s)
x and / scans
Maximum 2h for data collection (°)
Measured fraction of data
Number of reflections measured
Unique reflections measured
Rmerge
Number of reflections included in refinement
Cut off threshold expression
Structure refined using
72.78
0.986
79918
13015
0.0394
13015
>2r(I)
full matrix least-squares using F2
Weighting scheme
calca
Number of parameters in least-squares
442
b
R1
wR2
0.0552
0.1328
0.1065
0.1532
1.046
0.001
c
R1 (all data)
wR2 (all data)
Goodness-of-fit (GOF)d
Maximum shift/error
Minimum and Maximum peak heights on final dF Map ꢁ0.274, 0.831
(e-/Å)
a
b
c
w = 1/[
R1
wR2 = [
r
2(Fo2) + (0.0676P)2 + 0.2020P] where P = (Fo2 + 2Fc2)/3.
=
R
(|Fo| ꢁ |Fc|)/
RFo.
2
R
(w(Fo ꢁ Fc2)2)/
R
(wFo4)]½.
d
2
½
GOF = [
R
(w(Fo ꢁ Fc2)2)/No. of reflections ꢁ No. of parameters)]
.
3.2. Structure of prochelator 5
phenol ring and the triazole is 43.9° while the methyl ester
containing ring off of N3 has a torsion angle of 32.9° with respect
Initial investigation of 1H NMR spectra of the TIP precursor 5
provided evidence for the regioselective formation of a mono-
alkylated product, as the spectra contained a single species, and
confirmed that the alkylation proceeded on a phenolic oxygen.
X-ray crystallography was used to further elucidate the structure
of this molecule. Suitable crystals were grown by slow evaporation
from a solution of diethyl ether. A summary of crystal data is
provided in Table 1, with full details provided in cif format in the
Supplemental information. The structure in Fig. 2 shows that the
boron containing self-immolative group is attached to the phenolic
ring connected to position 5 (C1) of the triazole ring. The unmasked
phenol maintains a hydrogen bond with N1 of the triazole, with an
O1–N1 distance of 2.653(1) Å and an O1–Hꢂ ꢂ ꢂN1 hydrogen bond
angle of 148.2°. Intramolecular hydrogen bonds between the
phenols and triazole nitrogens to form 6-membered rings are well
documented for ligands of the bis-hydroxyphenyltriazole family
[13,28]. In the existing structures of such ligands, 2 of the 3 aro-
matic rings are roughly coplanar with the central triazole ring. In
the case of 5, only 1 of the 3 aromatic rings is roughly coplanar,
with the torsion angle between the phenolic ring and the triazole
ring being 6.3°. In contrast, the torsion angle between the masked
to the triazole. Some of this torsion is likely due to an offset p-
stacking interaction between the masking aromatic ring (defined
by C41–C46) and the methyl ester-containing ring (defined by
C31–C36), where the distance from C41 and C35 to the center of
the corresponding opposite ring is 3.4 Å.
3.3. Differential metal binding of TIP versus deferasirox
An effective prochelator should have a significantly diminished
affinity for metal ions in comparison to its unmasked chelator. We
therefore compared the interaction of deferasirox and our triazole
prochelator TIP with biologically important transition metal ions
Fe3+, Cu2+, and Zn2+. The limited aqueous solubility of the pinane-
diol-modified prochelator TIP-pd precluded similar studies of this
compound.
As shown in the UV–Vis spectra in Fig. 3, no significant spectral
change occurs after addition of Fe3+ to a buffered aqueous solution
of TIP. Conversely, addition of Fe3+ to a solution of deferasirox
induces a significant change in spectrum indicating formation of
the 1:2 [Fe(deferasirox)2]3ꢁ complex [13].

F. Kielar et al. / Inorganica Chimica Acta 393 (2012) 294–303
299
Fig. 2. ORTEP drawing of 5 showing naming and numbering scheme. Ellipsoids are at the 50% probability level and the hydroxyl hydrogen atom was drawn with an arbitrary
radius while all other hydrogen atoms were omitted for clarity. The disordered portion of the molecule is depicted by ‘‘hollow’’ bonds and atoms.
0.6
0.7
Deferasirox
TIP
Deferasirox + Fe
TIP + Cu
0.5
0.4
0.3
0.2
0.1
0
0.6
0.5
0.4
0.3
0.2
0.1
0
TIP
TIP + Fe
TIP + Cu + NTA
250 300 350 400 450 500 550 600
Wavelength (nm)
240 260 280 300 320 340 360 380 400
Wavelength (nm)
Fig. 3. UV–Vis spectra of TIP and deferasirox (20
lM in PBS with 1% DMSO at pH
Fig. 5. UV–Vis spectra showing interaction of TIP (20
l
M) with CuSO4 (10
lM) and
7.4) before and after addition of Fe3+ (10
lM).
the effect of NTA (20
lM) in PBS with 1% DMSO at pH 7.4.
1
0.8
0.6
0.4
0.2
0
1
0.8
0.6
0.4
0.2
0
Deferasirox
Deferasirox + Cu
Deferasirox
Deferasirox + Zn
TIP
Deferasirox + Cu + NTA
Deferasirox + Cu + EDTA
TIP + Zn
250
300
350
400
250
300
350
400
Wavelength (nm)
Wavelength (nm)
Fig. 6. UV–Vis spectra showing interaction of deferasirox (20
(10 M) and the effect of NTA (20 M) and EDTA (20 M) in PBS with 1% DMSO at
pH 7.4.
lM) with CuSO4
Fig. 4. UV–Vis spectra of TIP and deferasirox (20
7.4) before and after addition of ZnSO4 (10 M).
lM in PBS with 1% DMSO at pH
l
l
l
l

300
F. Kielar et al. / Inorganica Chimica Acta 393 (2012) 294–303
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
TIP-ctrl
TIP-ctrl + Fe
TIP-ctrl + Zn
TIP-ctrl + Cu
TIP-ctrl + Cu + NTA
240 260 280 300 320 340 360 380 400
Wavelength (nm)
Fig. 8. The aromatic regions of 1H-NMR spectra of (a) TIP, (b) the reaction mixture
of TIP with 50 mM H2O2 after 24 h, and (c) deferasirox at the concentration of
250
lM in D2O with 2.5% DMSO-d6. The arrows indicate the peaks corresponding to
Fig. 7. UV–Vis spectra of TIP-ctrl (20
after addition of FeCl3, ZnSO4 and CuSO4 (10
effect of NTA (20
M) on the interaction of TIP-ctrl with Cu2+
l
M in PBS with 1% DMSO at pH 7.4) before and
the aromatic protons of 4-hydroxybenzyl alcohol.
lM). The orange dash line indicates the
l
.
Similar to what was observed for Fe3+, addition of Zn2+ to a
lM buffered aqueous solution of TIP results in virtually no
20
spectral change, whereas significant change is observed when
Zn2+ is added to solutions of deferasirox (Fig. 4). We note that addi-
tion of ZnSO4 to higher concentrations of TIP results in visible pre-
cipitation, indicating that Zn2+ likely does complex with TIP to
form insoluble species, similar to what has been noted for deferasi-
rox [29].
In contrast to the results for zinc and iron, addition of Cu2+ to
solutions of either TIP or deferasirox results in spectral changes
in both cases, not just the deferasirox case (Figs. 5 and 6). The
change in spectrum of TIP upon the addition of Cu2+ suggests that
even in its prochelator form, TIP retains binding capacity for cupric
ions. Notable also is a weak band centered ꢃ650 nm that can be
observed at higher concentrations of TIP plus Cu2+ (not shown),
which is very similar to the d-d band observed for Cu2+ complexed
to deferasirox [30].
In order to gauge the relative strength of the TIP-Cu interaction,
we used nitrilotriacetic acid (NTA) as a competitive chelator that
has overall stability constants for Cu2+ of 12.7 and 17.4 for log b1
and log b2, respectively [31]. As shown in Fig. 5, the addition of
one equivalent of NTA to a Cu–TIP solution at pH 7.4 results in full
restoration of the original TIP spectrum, indicating that NTA read-
ily competes with TIP for complete extraction of Cu2+. In contrast,
one equivalent of NTA does not change the spectrum of a solution
of Cu–deferasirox at all, indicating that deferasirox is significantly
stronger than NTA for Cu2+ binding (Fig. 6), which is consistent
with the known stability constants of deferasirox for Cu2+ of 18.8
and 23.9 for log b1 and log b2, respectively [30]. Additionally, we
tested the strength of binding of deferasirox to copper using EDTA,
Fig. 9. LC trace of (a) TIP (100 lM in PBS with 1% DMSO), and (b) reaction mixture
of TIP with H2O2 (10 mM) after 12 h. The peak eluting at 9 min in (a) has a
corresponding m/z value of 508, consistent with intact TIP, while the peak eluting at
8.5 min in (b) has a corresponding m/z value of 374, matching that of authentic
deferasirox.
OH
OH
OH
HO
B
OH
OH
OH
O
O
N
OH
O
N
N
H2O2
N
H2O
N
N
+
N
N
N
B(OH)3
OH
OH
OH
O
OH
O
O
Scheme 2. Reaction of TIP with hydrogen peroxide generates deferasirox and 4-hydroxybenzyl alcohol.

F. Kielar et al. / Inorganica Chimica Acta 393 (2012) 294–303
301
3.4. Hydrogen peroxide triggered conversion of TIP to deferasirox
The unmasking reaction of TIP with H2O2 is anticipated to occur
as shown in Scheme 2, where oxidation of the boronic acid by H2O2
gives a phenol intermediate that undergoes spontaneous 1,6
elimination to release deferasirox and quinone methide, which
converts to 4-hydroxybenzyl alcohol in water [27].
Fig. 8 shows the aromatic region of a 1H NMR spectrum of a
250-lM sample of TIP in D2O after reaction with excess H2O2.
The expected products deferasirox and 4-hydroxybenzyl alcohol
are clearly identified. The clean conversion of TIP to deferasirox
was also observed by LC/MS, as shown by the chromatograms in
Fig. 9.
The rate of the unmasking reaction was assessed by UV–Vis
spectrophotometry. As can be seen in Fig. 10, the UV–Vis spectrum
of 20 lM TIP in PBS buffer pH 7.4 changes upon exposure to H2O2.
The increase in absorbance at 273 nm versus time for solutions of
TIP under pseudo first-order conditions of excess of H2O2 was used
to obtain observed rate constants kobs. The second-order rate
constant for this reaction was then obtained from the data shown
in Fig. 10 to be 1.4 Mꢁ1sꢁ1, a value that is similar to that found for
prochelator BHAPI, which also contains a self-immolative boronate
protecting group [27].
Fig. 10. UV–Vis spectra of TIP (50
reaction with H2O2 (5 mM) for indicated time intervals. Inset shows a plot of kobs at
273 nm versus H2O2 concentration for its reaction with TIP (20 M).
lM in PBS with 1% DMSO at pH 7.4) upon
l
which has a higher affinity for Cu2+ than NTA, with a log b1 value of
18.78 [31]. Addition of one equivalent of EDTA weakens the signal
of the Cu–deferasirox spectrum but does not fully restore the free
deferasirox spectrum, indicating comparative binding strength.
TIP-ctrl shows the same trends in metal binding as observed for
TIP. As shown in Fig. 7, TIP-ctrl does not interact with Fe3+ or Zn2+
under these conditions, but does show an obvious interaction with
Cu2+. The spectrum of TIP-ctrl in the presence of Cu2+ shown in
Fig. 7 is notably very similar to that of TIP-Cu in Fig. 5. Addition
of NTA to a Cu–(TIP-ctrl) mixture results in competitive removal
of Cu2+ from this complex (Fig. 7), again reminiscent of the result
found for TIP. These combined results indicate that the bidentate
chelating moiety formed from the phenol oxygen and triazole
nitrogen, which is preserved in both TIP and TIP-ctrl, is capable
of binding to copper, even though relatively weakly.
3.5. Cell experiments
Cell culture experiments to test the toxicity and protective
effect of the new triazole prochelators were conducted on retinal
pigment epithelial ARPE-19 cells because of their implication in
macular degeneration. Prior studies have shown iron chelation to
be effective at reducing oxidative stress in both cell and animal
models of macular degeneration [9,17,32]. The cytotoxicity data
in Fig. 11 shows that deferasirox is cytotoxic to this cell line,
especially at higher doses, where a 100 lM dose leaves only
ꢃ50% viable cells after a 24 h treatment. This result is consistent
with other data showing deferasirox toxicity in myocardial and
CHO cells in culture [12,15,16].
1.2
1
Deferasirox
TIP
TIP-ctrl
TIP-pd
0.8
0.6
0.4
0.2
0
0
5
10
25
50
75
100
Concentration (µM)
Fig. 11. Viability of ARPE-19 cells exposed to various concentrations of deferasirox, TIP, TIP-ctrl and TIP-pd for 24 h.

302
F. Kielar et al. / Inorganica Chimica Acta 393 (2012) 294–303
1.2
1
4. Conclusion
Deferasirox
TIP
The new boronate-masked compounds presented here fulfill
TIP-ctrl
many of the design properties that are desireable for a prochelator,
specifically: a significantly decreased affinity for metal ions in the
masked form, and a reactive handle that enables conversion from
the prochelator to the chelator under specific conditions. Indeed,
0.8
0.6
0.4
0.2
0
the prochelator TIP has weak to no affinity for Fe3+, Zn2+ or Cu2+
,
while its chelator version deferasirox has well-established metal
binding properties. Furthermore, H2O2 activates the boronate
reactive handle, which results in clean and efficient conversion to
deferasirox. Unfortunately, masking one of the phenol groups of
deferasirox does not alleviate the cytotoxicity of this family
of compounds. While TIP shows similar ability as deferasirox to
protect cells from oxidative stress caused by H2O2 exposure, all
of the phenol-masked compounds tested were more cytotoxic than
deferasirox. The trends in cytotoxicity observed for this small set of
triazole compounds suggest that the source of cytotoxicity
of deferasirox is likely not due to adventitious iron chelation.
H2O2 (200 µM)
-
+
+
+
+
+
+
+
0
0
5
10
25
50
75
100
Concentration (µM)
Acknowledgement
Fig. 12. Viability of ARPE-19 cells stressed with 200 lM H2O2 in the presence of
deferasirox, TIP and TIP-ctrl. Cell viability was measured 19 h after peroxide
treatment.
We thank the National Institutes of Health (grant GM084176)
for supporting this work. We also thank Dr. George Dubay for
measuring high-resolution mass spectra, which were obtained on
an instrument funded by NSF grant 0923097, and the Department
of Chemistry of North Carolina State University and the State of
North Carolina for funding the purchase of the Apex2
diffractometer.
One of the motivators for creating the current triazole prochela-
tors was to test the hypothesis that masking the iron chelation site
of deferasirox might minimize its inherent cytotoxicity. However,
the data in Fig. 11 show that the prochelator TIP is actually more
cytotoxic than deferasirox itself. We speculated that part of the
reason for enhanced cytotoxity of TIP could be the ability of boro-
nic acids to inhibit proteolytic enzymes. To test this hypothesis, we
synthesized the pinanediol-protected prochelator, TIP-pd. In our
experience, we have found that pinanediol capping groups retain
their boronic ester forms even in aqueous solution, whereas the
pinacol ester versions (such as the one in 5) readily hydrolyze to
their boronic acid derivatives [27]. TIP-pd is not readily soluble
and was therefore added to cells as a suspension. As shown in
Fig. 11, TIP-pd was the most cytotoxic of the molecules tested, sug-
gesting that the boronic acid group is not likely the source of
toxicity.
To further explore the structure–activity relationship among
these molecules and their cytoxicity, we tested the benzyl-contain-
ing control compound TIP-ctrl, which does not have a boronate
functionality, does not have a reactive handle so is unlikely to con-
vert to deferasirox intracellularly, and lacks a strong tridentate
iron-binding pocket. As shown in Fig 11, the cytotoxicity of TIP-ctrl
tracks very closely with TIP and shows a dose-dependent increase
in cytotoxicity.
Appendix A. Supplementary material
CCDC 880777 contains the supplementary crystallographic data
for this paper. These data can be obtained free of charge from The
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ica.2012.06.011.
References
[1] R. Ward, Hematology 15 (2010) 311.
[2] P.V. Bernhardt, Dalton Trans. (2007) 3214.
[3] E. Vichinsky, Am. J. Hematol. 83 (2008) 398.
[4] E.J. Neufeld, ASH Education Program Book, 2010 (2010) 451.
[5] D. Kell, BMC Med. Genet. 2 (2009) 2.
[6] R.R. Crichton, D.T. Dexter, R.J. Ward, Coord. Chem. Rev. 252 (2008) 1189.
[7] N. Boddaert, K. Sang, A. Rotig, A. Leroy-Willig, S. Gallet, F. Brunelle, D. Sidi, J.C.
Thalabard, A. Munnich, Z.I. Cabantchik, Blood 110 (2007) 401.
[8] T. Zhou, Y. Ma, X. Kong, R.C. Hider, Dalton Trans. (2012), http://dx.doi.org/
10.1039/c2dt12159j.
[9] M. Hadziahmetovic, Y. Song, N. Wolkow, J. Iacovelli, S. Grieco, J. Lee, A.
Lyubarsky, D. Pratico, J. Connelly, M. Spino, Z.L. Harris, J.L. Dunaief, Invest.
Ophthalmol. Vis. Sci. 52 (2011) 959.
While deferasirox itself can be cytotoxic at elevated doses,
others have found that it retains a protective effect against oxida-
tive insult [12,16]. In order to test whether the prochelators might
also show a protective effect against oxidative stress, we pre-
treated cells with the compounds then exposed them to a toxic
[10] L.R. Perez, K.J. Franz, Dalton Trans. 39 (2010) 2177.
[11] D.S. Kalinowski, D.R. Richardson, Pharmacol. Rev. 57 (2005) 547.
[12] P. Bendova, E. Mackova, P. Haskova, A. Vavrova, E. Jirkovsky, M. Sterba, O.
Popelova, D.S. Kalinowski, P. Kovarikova, K. Vavrova, D.R. Richardson, T.
Simunek, Chem. Res. Toxicol. 23 (2010) 1105.
[13] S. Steinhauser, U. Heinz, M. Bartholoma, T. Weyhermuller, H. Nick, K.
Hegetschweiler, Eur. J. Inorg. Chem. (2004) 4177.
200-lM bolus of H2O2. The results in Fig. 12 show that cells are
protected from death by both deferasirox and TIP, but not by the
non-chelating control TIP-ctrl. While deferasirox is more cytopro-
tective than TIP, being maximally effective at concentrations of
[14] R.M. Al-Rousan, S. Paturi, J.P. Laurino, S.K. Kakarla, A.K. Gutta, E.M. Walker, E.R.
Blough, Am. J. Hematol. 84 (2009) 565.
5 versus 25 lM, respectively, it should be noted that the protective
effect of TIP is observed well below the concentration of added
H2O2. These results imply that metal chelation is the probable
mode of protective action for both TIP and deferasirox, the
consumption of H2O2 in reaction with TIP being unlikely to provide
much supplementary protection. The decreased protection for def-
erasirox and its prochelators at higher concentrations is likely the
result of the inherent cytotoxicity of these compounds, as observed
in the cytotoxicity studies of Fig. 11.
[15] B.B. Hasinoff, D. Patel, X. Wu, Free Radical Biol. Med. 35 (2003) 1469.
[16] P. Haskova, L. Koubkova, A. Vavrova, E. Mackova, K. Hruskova, P. Kovarikova, K.
Vavrova, T. Simunek, Toxicology 289 (2011) 122.
[17] L.K. Charkoudian, T. Dentchev, N. Lukinova, N. Wolkow, J.L. Dunaief, K.J. Franz,
J. Inorg. Biochem. 102 (2008) 2130.
[18] L.K. Charkoudian, D.M. Pham, K.J. Franz, J. Am. Chem. Soc. 128 (2006) 12424.
[19] M.G.D. Leed, N. Wolkow, D.M. Pham, C.L. Daniel, J.L. Dunaief, K.J. Franz, J. Inorg.
Biochem. 105 (2011) 1161.
[20] F. Stieber, U. Grether, H. Waldmann, Chem. Eur. J. 9 (2003) 3270.
[21] Bruker-Nonius SAINT version 2009.9, Madison, WI 53711, USA, 2009.

F. Kielar et al. / Inorganica Chimica Acta 393 (2012) 294–303
303
[22] Bruker-Nonius SADABS version 2009.9, Madison, WI 53711, USA, 2009.
[23] A. Altomare, G. Cascarano, C. Giacovazzo, A. Guagliardi, M.C. Burla, G. Polidori,
M. Camalli, J. Appl. Crystallogr. 27 (1994) 435.
[24] Bruker-AXS XL version 2009.9, Madison, WI 53711, USA, 2009.
[25] E.J. Gabe, Y. Le Page, J.P. Charland, F.L. Lee, P.S. White, J. Appl. Crystallogr. 22
(1989) 384.
[26] J. Major Jourden, S. Cohen, Angew. Chem., Int. Ed. 49 (2010) 6795.
[27] F. Kielar, M.E. Helsel, Q. Wang, K.J. Franz, Metallomics (2012), http://
dx.doi.org/10.1039/C1032MT20069D.
[29] U. Heinz, K. Hegetschweiler, P. Acklin, B. Faller, R. Lattmann, H.P. Schnebli,
Angew. Chem., Int. Ed. 38 (1999) 2568.
[30] S. Steinhauser, U. Heinz, J. Sander, K. Hegetschweiler, Z. Anorg, Allg. Chem. 630
(2004) 1829.
[31] A.E. Martell, R.M. Smith, NIST Critically Selected Stability Constants of Metal
Complexes, in: R.J. Motekaitis (Ed.) NIST Standard Reference Database 46,
NIST, Gaithersburg, MD, 2001.
[32] N. Lukinova, J. Iacovelli, T. Dentchev, N. Wolkow, A. Hunter, D. Amado, G.S.
Ying, J.R. Sparrow, J.L. Dunaief, Invest. Ophthalmol. Vis. Sci. 50 (2009) 1440.
[28] U. Heinz, J. Sander, K. Hegetschweiler, Z. Kristallogr. New Crystallogr. Struct.
216 (2001) 113.