Synthesis and solution thermodynamic study of rigidified and functionalised EGTA derivatives

Article abstract of DOI:10.1039/b804195d

The synthesis of a new series of EGTA (ethyleneglycol-bis(2- aminoethylether)-N,N,N′,N′-tetraacetic acid) derivatives incorporating aromatic and functionalized aromatic moieties into the oxoethylenic bridge is described. A solution thermodynamic study was

Full text of DOI:10.1039/b804195d

View Article Online / Journal Homepage / Table of Contents for this issue
PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis and solution thermodynamic study of rigidified and functionalised
EGTA derivatives†
Lorenzo Tei,a Zsolt Baranyai,b Mauro Botta,a Laura Piscopo,a Silvio Aimeb and Giovanni B. Giovenzana*c
Received 11th March 2008, Accepted 28th March 2008
First published as an Advance Article on the web 30th April 2008
DOI: 10.1039/b804195d
The synthesis of a new series of EGTA (ethyleneglycol-bis(2-aminoethylether)-N,N,Nꢀ,Nꢀ-tetraacetic
acid) derivatives incorporating aromatic and functionalized aromatic moieties into the oxoethylenic
bridge is described. A solution thermodynamic study was carried out to determine the influence of
structural modifications on the coordinating ability towards lanthanide and alkaline earth metal ions.
The presence of remote functional groups on the aromatic moiety would allow the conjugation of the
complexes to macromolecules or other biological targets.
Dermopathy, NSF/NFD) referred to the release of free
Introduction
Gd3+ after administration of the Gd-based contrast agent
Omniscan.8
The chelation of metal ions by highly polydentate ligands has
been widely investigated in the last thirty years. The interest in
such ligands is mainly driven by their ability (depending on the
nature of the donor atoms) to form metal complexes with tunable
physicochemical and functional properties engaged in a wide
range of applications as inorganic medicinal compounds,1 anion
or molecular receptors,2 catalysts for organic transformations,3
molecular sensors,4 and mimics for enzymes catalysing redox
and hydrolytic processes.5 Among them, polyaminocarboxylate
ligands have been extensively employed as chelators for lan-
thanide(III) ions. The ligands may be tailored to fine-tune the
kinetic and thermodynamic stabilities of the resulting complexes
according to the application in which they are to be used, e.g.
as sequestering agents or as diagnostic or therapeutic tools in
medicine.
Among the latter applications, the use as contrast-enhancing
agents (CAs) for magnetic resonance imaging (MRI) has attracted
much attention in recent years.6 In fact, CAs have been shown to
cause a dramatic variation of the water proton relaxation rates,
thus providing physiological information well beyond the im-
pressive anatomical resolution commonly obtained in the uncon-
trasted images. In the class of paramagnetic CAs, the attention has
been focused on Gd(III) complexes, which must have extremely
high kinetic and thermodynamic stabilities to preserve their
integrity for the time they stay in the patient body. A large database
has already been acquired concerning the stability constants of
Ln(III) polyaminocarboxylate complexes,7 and interest has been
recently renewed by the discovery of a new disease (named
as Nephrogenic Systemic Fibrosis/Nephrogenic Fibrosing
The specific application as CA for MRI, but also other
possible uses in biomedicine, prompted the search for the
relationships between the details of the solution structure
and the physico-chemical properties of the metal chelates.
More importantly, a rational modification of the ligand back-
bone with different donor groups or different structural moi-
eties can affect not only its coordinating ability but also
the size, the charge, and the lipophilicity of the correspond-
ing complexes, thus affecting their affinity for different bio-
logical targets.6 The poly(aminocarboxylate) ligands such as
the linear DTPA (diethylenetriamine-N,N,Nꢀ,Nꢀꢀ,Nꢀꢀ-pentaacetic
acid) or the macrocyclic DOTA (1,4,7,10-tetraazacyclododecane-
N,Nꢀ,Nꢀꢀ,Nꢀꢀꢀ-tetraacetic acid) and their derivatives represent the
most common and studied class of chelators for biomedical
applications, as they are octadentate and form highly stable Ln(III)
complexes with one additional water molecule coordinated by the
metal ion.6 In the search for new and efficient contrast agents
for MRI, a systematic investigation of the solution structure and
dynamics and relaxation properties of the Ln(III) complexes of the
simple acyclic ligand EGTA has been previously reported by our
group.9 Its Gd(III) complex is potentially a very efficient CA, as
it presents an optimal value of one of the key factors influencing
the efficacy, viz. the residence lifetime of the coordinated water
molecule. EGTA has also been reported to exhibit interesting
patterns of metal-binding selectivity, especially related with the
alkaline earth metal ions Ca2+ and Mg2+.10 In fact, it is highly
selective towards Ca2+ compared to the other alkaline earth metal
ions, with a stability constant six orders of magnitude higher for the
binding of Ca2+ over Mg2+, larger than that typically exhibited by
calcium-binding proteins. Despite the really interesting properties
of EGTA both as an MRI CA and as Ca2+ sequestering agent,
only a few papers have been published on EGTA derivatives in
the last twenty years.11 The reasons may be found in the fact that
EGTA lacks functional groups suitable for covalent linking to
macromolecules or biological targets, and that the corresponding
Gd(III) complex suffers from low thermodynamic stability and
selectivity, precluding practical and safe in vivo MRI applica-
tions.
aDipartimento di Scienze dell’Ambiente e della Vita, Universita` del Piemonte
Orientale “Amedeo Avogadro”, Via Bellini 25/G, I-15100, Alessandria, Italy
bDipartimento di Chimica IFM, Universita` degli Studi di Torino, Via
P. Giuria 7, I-10125, Torino, Italy
cDiSCAFF & DFB Center, Universita` degli Studi del Piemonte Orien-
tale “Amedeo Avogadro”, Via Bovio 6, I-28100, Novara, Italy. E-mail:
giovenzana@pharm.unipmn.it
† Electronic supplementary information (ESI) available: Experimental
details and procedures related to the synthetic protocol reported in
Scheme 2. See DOI: 10.1039/b804195d
This journal is
The Royal Society of Chemistry 2008
Org. Biomol. Chem., 2008, 6, 2361–2368 | 2361
©

View Article Online
Herein, we report synthesis and characterization of five deriva-
tives of EGTA where structural variations were introduced in order
to: i) investigate the effects on the stability of the corresponding
Ln(III) complexes by rigidification of key sites of the flexible
EGTA molecule or by functional group modification; and ii) allow
conjugation of the corresponding complexes to macromolecules
or biological targets, introducing additional remote functional
groups. In the new L1–L5 ligands (Scheme 1) the basic structure
of EGTA was modified in the central ethylenic moiety, rigidified
by fusion with an aromatic ring.
Scheme 1 Structure of L1–L5.
Scheme 2 Reagents and conditions: i) BrCH2COOMe, K2CO3, Me2CO,
reflux, 2 h; ii) NaBH4, EtOH, reflux, 3 h; iii) CH3SO2Cl, iPr2NEt, AcOEt,
0
EtOH, reflux, 2 h; vi) BrCH2COOtBu, K2CO3, CH3CN, r.t., 48 h;
vii) TFA/PhOCH3 (4 : 1), r.t., 24 h.
The modified ligands were prepared from 1,2-arenediols. L1
was obtained from 2,3-naphthalenediol, L2 from 1,2-benzenediol,
whereas L3, L4 and L5 were synthesised starting from 4-
nitrocatechol, in which the nitro group, once reduced, represents
a useful site for further functionalization. Two synthetic protocols
to access these molecules are described and compared. Moreover,
solution studies on the protonation of L2 and L3 and their stability
constants with lanthanide ions across the series and with Ca2+
and Mg2+ are reported in order to check the ability of these
ligands to form stable complexes with Ln3+ ions and/or to complex
selectively other metal ions.
◦C, 3 h; iv) PhtN−K+, K2CO3, DMF, 100 ◦C, 6 h; v) N2H4·H2O,
removal with TFA/anisole gave ligand 1 in overall 10–11% yield.
As multigram amounts of these ligands are needed to carry out a
full characterization of their complexes, a shorter synthesis with a
higher throughput was then sought. In order to reduce the number
of synthetic steps, we envisaged a convergent synthesis, outlined
in detail in Scheme 3.
Results and discussion
Synthesis of L1–L5
Two synthetic approaches were employed to synthesize the
EGTA-derivatives L1–L5. Commercially available 1,2-arenediols
(catechols) are the preferred starting materials for these syntheses.
The first strategy, outlined in Scheme 2, relies on a multistep
introduction of the aminoethyl group onto the phenolic oxygen
atoms.
2,3-Naphthalenediol 1 was used as a model compound to
explorethissyntheticprotocol. Alkylationofbothphenolicoxygen
atoms was performed with a slight excess of methyl bromoacetate
in refluxing acetone and in the presence of potassium carbonate.
Reduction of ester 2 with sodium borohydride in refluxing ethanol
afforded diol 3. The latter was converted to the dimesyl derivative
trying to follow the strategy employed by Brunet, Rodr´ıguez-
Ubis et al.12 to synthesize a fluorescent derivative of EGTA;
unfortunately, attempted alkylation of tert-butyl iminodiacetate
with this compound led to sluggish reactions and isolation
of only minute amounts of the monoalkylation product. The
dimesyl derivative was then redirected toward a more classical
Gabriel protocol, obtaining the overall conversion of diol 3 to
the diamine 4. Straightforward exhaustive alkylation with tert-
butyl bromoacetate/potassium carbonate and tert-butyl group
Scheme 3 Reagents and conditions: i) Ref. 13; ii) CH3SO2Cl, iPr2NEt,
AcOEt, 0 ◦C, 3 h; iii) K2CO3, 18-crown-6, PhCH3, r.t., 72 h; iv) TFA/
PhOCH3 (4 : 1), r.t., 24 h; v) H2, 10% Pd/C, MeOH, r.t., 2 h.
2362 | Org. Biomol. Chem., 2008, 6, 2361–2368
This journal is
The Royal Society of Chemistry 2008
©

View Article Online
In
this
alternative
strategy,
the
lateral
Scheme 5 shows the synthesis of L3–L5 starting from 10.
Reaction of 10 with isothiocyanates 13 and 15 was carried out
in dichloromethane, while acylation with stearoyl chloride was
performed under Schotten–Baumann conditions in a biphasic
mixture of dichloromethane and aqueous sodium carbonate.
Purification of the esters and deblocking of tert-butyl esters led
to the desired ligands L3–L5, subsequently used for metal ion
complexation.
bis(carboxymethyl)aminoethyl arms were preformed in two
steps and implanted onto the phenolic oxygen atoms through a
solid–liquid phase transfer catalyzed (PTC) alkylation. Removal
of tert-butyl esters completed the four-step synthesis. The
protocol was applied to 2,3-naphthalenediol, 1,2-benzenediol and
4-nitrocatechol, obtaining L1, L2 and the interesting tert-butyl
ester 9c, respectively; the latter was catalytically hydrogenated
to compound 10. This second synthetic pathway is shorter and
allows direct access from catechols to ligand esters in one single
step, using the alkylating agent 7. Overall yield is significantly
higher, being about 25% for L1 (from catechol 1), and hence
more than doubled with respect to the linear seven-step protocol.
Further optimization, especially of the PTC alkylation step,
may lead to an additional improvement of the overall yield of
the convergent strategy. Aminoester 10 is a key intermediate
as it allows the possibility to conjugate this class of EGTA
derivatives to specific targets through the free primary amine.
This nucleophilic reactive group is placed strategically on the
aromatic ring because: i) this position is remote with respect to the
coordinating group and should not disturb the coordination of
metal ions or the dynamic processes associated to the complex; ii)
it is directly and firmly bound to the ligand backbone, warranting
a rigid linkage with the target.
To demonstrate the conjugability and the potential improve-
ments in CA performance, compound 10 was converted into
ligands L3–L5. To this purpose, we selected three different
electrophiles to be reacted with compound 10 to impart specific
properties to the resulting ligand. We synthesized two isothio-
cyanates, 13 and 15, based on phenylalanine and dodecylamine,
respectively, according to Scheme 4.
Scheme 5 Reagents and conditions: i) CH2Cl2, r.t., 24–48 h; ii) TFA/
PhOCH3 (4 : 1), r.t., 24 h; iii) C17H35COCl, CH2Cl2–10% aq. Na2CO3, r.t.,
2 h.
Solution thermodynamic study
In the chemistry of lanthanides, it is generally accepted that non-
ionic donor atoms are more weakly bound than ionic donor atoms.
In EGTA derivatives, nitrogen atoms alone cannot coordinate to
the lanthanide ion to form an energetically stable five-membered
chelate ring, as typically found in other polyaminocarboxylate
complexes (e.g. [Ln(EDTA)]−). According to the literature, the
stability constants of the lanthanide(III) complexes formed with
diamino-tetracarboxylate ligands decrease with the increase of
the distance between the two nitrogen atoms. The value of the
stability constants of the [Ln(EGTA)]− complexes (logKLnL = 15–
18) are comparable with those of the corresponding [Ln(EDTA)]−
complexes (logKLnL = 15–20), but significantly higher than those
reported for the [Ln(HMDTA)]− chelates (logKLnL = 8–11)
(HMDTA= hexamethylenediamine-N,N,Nꢀ,Nꢀꢀ-tetraacetic acid;
the length of the ligand backbone between the two terminal
nitrogen atoms is similar to that of EGTA). These data suggest
that the coordination of the ethereal oxygen atoms contributes
to the thermodynamic stability of [Ln(EGTA)]− complexes.7a In
addition, the stability of the lanthanide complexes is influenced
Scheme 4 Reagents and conditions: i) tBuOAc, 70% aq HClO4, r.t., 24 h;
ii) CSCl2, CH2Cl2–sat. aq. NaHCO3, r.t., 1 h.
We selected these compounds in order to provide the EGTA-
like ligand with substructures with a known affinity for the
binding sites of Human Serum Albumin (HSA). Binding a
paramagnetic contrast agent to a slowly tumbling macromolecule
increases its relaxivity, extends its lifetime in the plasma and
its residence in blood vessels. HSA is known to bind reversibly
aromatic carboxylic acids and hydrophobic aliphatic chains and
this explains the choice of phenylalanine, dodecylamine and stearic
acid as recognition moieties conjugated to 10 in order to obtain
L3–L5. We have recently exploited the strong interaction with
HSA and the restricted local rotation upon HSA binding of the
Gd complex of L1 thanks to its naphthyl moiety. For this complex,
an unprecedented high relaxivity close to that predicted by theory
was observed.14
This journal is
The Royal Society of Chemistry 2008
Org. Biomol. Chem., 2008, 6, 2361–2368 | 2363
©

View Article Online
Table 1 Protonation constants of the ligands L2, L3, EGTA and EDTA
Stability and protonation constants of complexes. The stability
and protonation constants of the metal complexes formed with L2
and L3 are defined by eqns (2) and (3):
as determined by potentiometric titration at 25 ◦Ca
L2
L3
EGTAb
EDTAb
[ML]
logKH1
logKH2
logKH3
logKH4
logKH5
8.96 (0.02)
8.41 (0.01)
2.99 (0.02)
2.05 (0.01)
1.72 (0.02)
8.75 (0.01)
8.26 (0.01)
3.34 (0.02)
2.23 (0.02)
2.05 (0.02)
9.47
8.85
2.26
2.00
—
10.19
6.13
2.69
2.0
KML
=
(2)
(3)
[M][L]
[MHiL]
[MHi−1L][H+ ]
—
KMH,L
=
i =1,2,3
a Ionic strength 0.1 M KCl in all cases. b Ref. 7a.
The protonation and stability constants obtained by direct pH-
potentiometric titration are reported in Table 2. The protonation
and stability constants of [Ln(L2)]− and [Ln(L3)]− were calculated
from the titration curves, obtained with a 1 : 1 metal-to-ligand
concentration ratio. The best fitting was performed by using the
model that includes the formation of the ML, MHL and MH2L
species in the equilibrium.
The L2 and L3 ligands possess two amino nitrogens, four
carboxylate oxygens (or five in the case of L3) and two ethereal
oxygen donor atoms. The presence of the aromatic ring in the
ligands not only results in a decrease of the basicity of the oxygen
and nitrogen donor atoms, but it also increases the stereochemical
rigidity of the backbone, which in turn could affect the geometry
of the coordination polyhedron.
by their coordination geometry, which is dictated by the ligand
structure and flexibility. The presence of the aromatic ring
increases the stereochemical rigidity of the ligands and this could
affect their coordinating ability towards the Ln(III) cations.
Protonation constants. The protonation constants (logKH) of
i
L2 and L3 were determined by pH-potentiometry and are reported
in Table 1 with those of EGTA and EDTA for comparison
(standard deviations are shown in parentheses). The protonation
constants are defined as follows:
[HiL]
[Hi−1L][H+ ]
KiH
=
i =1,2,3…
(1)
A comparison of the protonation constants of L2, L3 and
EGTA, obtained in similar media, reveals that the logKH3 and
logKH4 values are quite similar, whereas the values of logKH1 and
logKH2 slightly differ. To explain these findings, we may assume
that, in analogy with the protonation scheme of EGTA,15,16 the
first and second equivalents of acid protonate the two nitrogen
atoms, while the third and fourth protonation processes involve
the carboxylate oxygens. The third protonation process in L3 is
likely to correspond to the protonation of the carboxylate oxygen
of the pendant phenylalanine moiety. The small differences found
between the corresponding protonation constants of L2 and L3
indicate a slightly different basicity of the nitrogen atoms imposed
by the substituent on the aromatic ring. On the other hand, the
protonation constants of the nitrogen atoms in L2 and L3 are
somewhat lower than those of EGTA, likely as a result of their
lower basicity arising from the electron- withdrawing character
of the aromatic group. Moreover, the similar values of logK1H
and logKH2 are simply accounted for in terms of two distinct
protonation processes originating from the large distance between
the two nitrogen atoms.
The values of the stability and protonation constants of the
complexes of Mg(II) with L2 and EGTA are rather similar
(Table 2), whereas the values of logKML and logKMHL for [CaL2]2−,
[LnL2]− and [LnL3]− are about one or two orders of magnitude
lower than those reported for [Ca(EGTA)]2− and [Ln(EGTA)]−.
The lower stability of [Mg(EGTA)]2− and [MgL2]2− compared
to [Mg(EDTA)]2− is explained in terms of the larger size of
EGTA and L2, which prevents the coordination of all the donor
atoms to the small Mg2+ ion due to the electrostatic repulsion
between the carboxylate groups. In the case of [LnL2]− and
[LnL3]−, coordination of all donor atoms of the ligands (except
the carboxylate group of the phenylalanine moiety in L3) is clearly
suggested by the results of solution NMR or X-ray diffraction
studies on the corresponding [Ln(EGTA)]− complexes.9,10 The
lower basicity of the amine N and ethereal O donor atoms
results in a lower stability of [CaL2]2−, [LnL2]− and [LnL3]−
compared to the complexes with EGTA (Table 2). The stability
constants of [LnL2]− complexes increase from La3+ to Er3+, where
a plateau is reached. This behaviour differs from that found
for the [Ln(EGTA)]− complexes whose logKML values increase
Table 2 The thermodynamic stability constants of complexes formed between L2, L3, EGTA, EDTA and selected lanthanide and alkaline earth metal
ions at 25 ◦Ca
L2
L3
EGTAb
EDTAc
logKML
logKMHL
logKML
logKMHL
logKMH
logKML
logKMHL
logKML
2 L
Mg2+
Ca2+
La3+
Ce3+
Nd3+
Gd3+
Er3+
5.12 (0.01)
9.99 (0.01)
—
14.62 (0.01)
15.26 (0.01)
15.76 (0.02)
16.21 (0.01)
16.23 (0.01)
7.35 (0.02)
4.64 (0.03)
—
3.09 (0.02)
3.05 (0.02)
2.33 (0.05)
2.34 (0.02)
2.31 (0.04)
—
—
—
—
—
—
5.28
10.86
15.55
15.70
16.28
16.97
17.40
17.81
7.62
3.79
—
—
—
—
—
—
8.69
10.61
15.46
15.95
16.56
17.35
18.83
19.80
14.07 (0.03)
—
—
15.25 (0.04)
—
15.43 (0.04)
3.20 (0.04)
—
—
3.03 (0.04)
—
3.12 (0.05)
3.04 (0.05)
—
—
2.09 (0.07)
—
—
Lu3+
a Ionic strength 0.1 M KCl for L2, L3 and EDTA; 0.1 M KNO3 for EGTA. b Ref. 17. c Ref. 7a.
2364 | Org. Biomol. Chem., 2008, 6, 2361–2368
This journal is
The Royal Society of Chemistry 2008
©

View Article Online
monotonically from La3+ to Lu3+. These data clearly indicate an
influence of the ligand “rigidity” on the trend of the logKML values
across the lanthanide series and the presence of an optimal size
match for the Er3+–Lu3+ cations. On the other hand, only a small
change in the stability constants for the complexes with L3 is
observed on passing from Gd to Lu. It could be hypothesized
that the lower stability of the [LnL3]− complexes is caused by the
presence of an intramolecular “stacking” interaction between the
aromatic moieties of L3. This interaction could further decrease
the flexibility of the chelator, with negative effects particularly in
the case of the coordination to the smaller Ln3+ ions.
7 was not characterized and was used as such for the following
steps.
Compound 5
2,3-Naphthalenediol (500 mg, 3.1 mmol), powdered potassium
carbonate (700 mg, 12.5 mmol) and 18-crown-6 (165 mg,
0.6 mmol) were mixed in toluene (40 mL), under a nitrogen
atmosphere. Compound 7 (2.82 g, 7.8 mmol) was slowly added
to the stirring suspension, previously cooled to 0–5 ◦C. After the
addition the stirring mixture was left to reach room temperature
and followed by HPLC. After 72 h the mixture was filtered and
evaporated in vacuo. The residue was dissolved in dichloromethane
(20 mL) and washed with 5% aq. K2CO3 (3 × 25 mL), aq. NaHSO3
(3 × 25 mL) and water (3 × 25 mL). The organic phase was then
dried over Na2SO4, filtered and evaporated. The oily crude product
was purified by flash chromatography (silica gel, eluant petroleum
ether–ethyl acetate–2-propanol 85 : 10 : 5), providing pure 5 as a
light yellow oil (899 mg, 41% yield). MS (ESI) 703.1 (MH+). Calc.
for C38H52N2O10: 702.1. 1H-NMR (CDCl3): 7.60 (m, 2H), 7.28 (m,
2H), 7.13 (s, 2H), 4.24 (t, 4H, J = 6.3 Hz), 3.58 (s, 8H), 3.24 (t, 4H,
J = 6.3 Hz), 1.44 (s, 36H). 13C-NMR (CDCl3): 170.8 (C), 148.9
(C), 129.2 (C), 126.2 (CH), 123.9 (CH), 107.7 (CH), 80.9 (C), 67.9
(CH2), 57.0 (CH2), 53.2 (CH2), 28.1 (CH3).
Conclusions
In this paper we have reported the synthesis of a new series of
EGTA derivatives incorporating an aromatic group into the ligand
backbone. Ligands L3–L5 feature a further functionalization on
the aromatic group, leading to bifunctional chelating agents of
potential utility for biomedical applications. Metal complexes
of L4 and L5 are expected to aggregate and form micelles as
well as to interact non-covalently with HSA: both these types of
macromolecular conjugates are of potential interest for contrast-
enhanced MRI applications.6 In fact, preliminary 1H and 17O
relaxometric data on the corresponding Gd(III) complexes of L1–
L5 are consistent with the presence of one coordinated water
molecule in fast exchange with the bulk.14 The relaxivity increases
as a function of the molecular weight (GdL1–L4) and it is strongly
enhanced in the presence of human serum albumin (GdL2–L5).
A full account of these results will be presented in a subsequent
paper. The structural modifications of the parent ligand do not
compromise the coordinating ability towards lanthanide and alka-
line earth metal ions, as demonstrated by solution thermodynamic
studies. Minor variations are explained in terms of increased
stereochemical rigidity imposed by the aromatic moiety. Future
work will be directed to further increasing the stability of the
complexes, which are still inadequate for medical applications.
Ligand L1
Compound 5 (350 mg, 0.49 mmol) was dissolved in a mixture
of anisole and trifluoroacetic acid (1 : 4, 5 mL) and stirred at
room temperature for 24 h. Volatiles were evaporated in vacuo
and the crude product was redissolved in methanol (1 mL); slow
addition of excess diethyl ether led to precipitation of L1 as a
white amorphous solid, isolated by centrifugation. Dissolution in
methanol and precipitation with diethyl ether was repeated thrice,
obtaining analytically pure L1. Yield 0.147 g. M.p. 151–152 ◦C
(dec.). MS (ESI) 479.0 (MH+). Calc. for C22H26N2O10: 478.1. 1H-
NMR (D2O): 7.65 (m, 2H), 7.25 (m, 2H), 7.23 (s, 2H), 4.25 (t, 4H,
J ∼5 Hz), 3.78 (s, 8H), 3.35 (t, 4H, J ∼5 Hz). 13C-NMR (D2O):
169.9 (C), 147.0 (C), 129.1 (C), 126.6 (CH), 125.0 (CH), 108.4
(CH), 63.1 (CH2), 57.5 (CH2), 54.6 (CH2).
Experimental
All chemicals were purchased from Sigma-Aldrich Co. and were
used without purification unless otherwise stated. NMR spectra
were recorded on a JEOL ECP 300 spectrometer (operating at
7.05 Tesla). ESI mass spectra were recorded on ThermoFinnigan
LCQ-Deca XP-Plus and melting points (uncorrected) with Stuart
Scientific SMP3 apparatus.
Compound 9b
Prepared in 44% yield following the procedure reported for
compound 5, starting from catechol 8b. Light yellow oil. MS (ESI)
653.2 (MH+). Calc. for C34H56N2O10: 652.1. H-NMR (CDCl3):
1
6.84–6.75 (m, 4H), 4.05 (t, 4H, J ∼6.0 Hz), 3.48 (s, 8H), 3.09 (t,
4H, J ∼6.1 Hz), 1.45 (s, 36H). 13C-NMR (CDCl3): 170.7 (C), 148.7
(C), 121.7 (CH), 113.8 (CH), 80.9 (C), 68.2 (CH2), 56.8 (CH2), 53.4
(CH2), 28.1 (CH3).
The synthesis and characterisation of compounds 1–4
(Scheme 2) are reported in the ESI†.
Compound 7
Compound 9c
Compound 613 (5.0 g, 17.3 mmol) was dissolved in dry ethyl
acetate (30 mL); ethyldiisopropylamine (3.23 mL, 19.0 mmol)
was added to the solution and the mixture was cooled to
0 ◦C. Methanesulfonyl chloride (1.18 mL, 17.3 mmol) was slowly
added dropwise and the mixture stirred at 0 ◦C for 3 h. The solvent
was removed in vacuo and the oily product was purified by flash
chromatography (eluant petroleum ether–ethyl acetate 7 : 3) giving
compound 7 (4.88 g, 77% yield). Due to its instability, compound
Prepared in 27% yield following the procedure reported for
compound 5, starting from 4-nitrocatechol 8c. Light yellow oil.
MS (ESI) 698.4 (MH+). Calc. for C34H55N3O12: 697.1. H-NMR
1
(CDCl3): 7.86 (dd, 1H, J1 = 8.8 Hz, J2 = 2.6 Hz), 7.76 (d, 1H,
J = 2.6 Hz), 6.93 (d, 1H, J = 8.9 Hz), 4.24 (t, 2H, J = 6.1 Hz),
4.21 (t, 2H, J = 5.8 Hz), 3.57 (s, 4H), 3.56 (s, 4H), 3.22 (t, 2H,
J = 5.9 Hz), 3.21 (t, 2H, J = 6.1 Hz), 1.46 (s, 18H), 1.44 (s, 18H).
This journal is
The Royal Society of Chemistry 2008
Org. Biomol. Chem., 2008, 6, 2361–2368 | 2365
©

View Article Online
13C-NMR (CDCl3): 170.6 (2 × C), 154.2 (C), 148.3 (C), 141.3 (C),
117.8 (CH), 111.1 (CH), 107.9 (CH), 81.1 (C), 81.0 (C), 68.7 (2 ×
CH2), 57.0 (CH2), 56.9 (CH2), 53.1 (2 × CH2), 28.1 (2 × CH3).
(68%). MS (ESI) 264.2 (MH+). Calc. for C14H17NO2S: 263.1. 1H-
NMR (CDCl3): 7.37–7.23 (m, 5H), 4.33 (dd, 1H, J1 = 7.9 Hz, J2 =
5.2 Hz), 3.20 (dd, 1H, J1 = 13.8 Hz, J2 = 5.2 Hz), 3.11 (dd, 1H,
J1 = 13.8 Hz, J2 = 7.9 Hz), 1.47 (s, 9H). 13C-NMR (CDCl3): 166.7
(C), 137.8 (C), 135.3 (C), 129.5 (CH), 128.6 (CH), 127.5 (CH),
83.7 (C), 61.3 (CH), 39.5 (CH2), 27.9 (CH3).
Ligand L2
Compound 9b (950 mg, 1.46 mmol) was dissolved in a mixture
of anisole and trifluoroacetic acid (1:4, 10 mL) and stirred at
room temperature for 24h. Volatiles were evaporated in vacuo
and the crude product was redissolved in methanol (2 mL); slow
addition of excess diethyl ether led to precipitation of L2 as a
white amorphous solid, isolated by centrifugation. Dissolution in
methanol and precipitation with diethyl ether was repeated thrice,
obta◦ining analytically pure L2. Yield 0.485 g. M.p. 132 ◦C (sint.),
180 C (dec.). MS (ESI) 451.1 (MNa+), 429.2 (MH+). Calc. for
C18H24N2O10: 428.1. 1H-NMR (D2O): 7.04 (m, 4H), 4.43 (bt, 4H),
4.18 (s, 8H), 3.85 (bt, 4H). 13C-NMR (D2O): 169.2 (C), 146.8 (C),
122.6 (CH), 113.7 (CH), 63.1 (CH2), 57.5 (CH2), 56.7 (CH2), 55.3
(CH2).
Compound 15
Obtained as off-white amorphous solid following the same pro-
cedure adopted for compound 13, starting from dodecylamine 14
(1.98 g, 10.8 mmol). Yield 1.86 g (76%). MS (ESI) 228.0 (MH+).
Calc. for C13H25NS: 227.1. 1H-NMR (CDCl3): 3.50 (t, 2H, J = 6.6
Hz), 1.70 (quint, 2H, J = 6.8 Hz), 1.41 (m, 2H), 1.27 (m, 16H), 0.88
(bt, 3H, J = 6.7 Hz). 13C-NMR (CDCl3): 129.7 (C), 45.1 (CH2),
31.8 (CH2), 30.0 (CH2), 29.6 (2 × CH2), 29.5 (CH2), 29.4 (CH2),
29.3 (CH2), 28.8 (CH2), 26.5 (CH2), 22.7 (CH2), 14.1 (CH3).
Compound 16
Compound 10 (435 mg, 0.65 mmol) was dissolved in
dichloromethane (5.0 mL) and stirred at 0–5 ◦C under nitrogen. A
solution of 13 (175 mg, 0.66 mmol) in dichloromethane (5.0 mL)
was added dropwise and the resulting solution was stirred for
48 h. The solvent was removed in vacuo and the oily residue
was purified by flash chromatography (petroleum ether–ethyl
acetate–2-propanol 85 : 10 : 5) to provide the conjugate 16 as
a yellow oil. Yield 314 mg (52%). MS (ESI) 931.5 (MH+). Calc. for
C48H74N4O12S: 930.5. 1H-NMR (CDCl3): 7.63 (bs, 1H), 7.30–6.40
(m, 9H), 5.19 (m, 1H), 4.10 (t, 2H, J = 5.6 Hz), 4.00 (bt, 2H), 3.55
(s, 4H), 3.54 (s, 4H), 3.29 (dd, 1H, J1 = 13.9 Hz, J2 = 6.6 Hz),
3.20–3.14 (m, 5H), 1.44 (s, 36H), 1.37 (s, 9H). 13C-NMR (CDCl3):
170.6 (2 × C), 170.2 (C), 149.7 (C), 148.0 (C), 136.0 (C), 129.5
(CH), 128.6 (C), 128.3 (CH), 126.9 (CH), 117.9 (CH), 113.9 (CH),
111.0 (CH), 82.3 (C), 81.0 (2 × C), 68.5 (2 × CH2), 58.9 (CH),
56.9 (CH2), 56.7 (CH2), 53.4 (CH2), 53.2 (CH2), 37.5 (CH2), 28.1
(2 × CH3), 27.9 (CH3).
Compound 10
Compound 9c (697 mg, 1.00 mmol) was dissolved in methanol
(20 mL) and Pd/C (10%, 100 mg) was added. The mixture was
introduced into a hydrogenation bottle, purged with nitrogen and
then stirred under hydrogen (1 atm) until HPLC analysis showed
complete reduction of the substrate (∼2 h). The catalyst was
R
removed by filtration on Celiteꢀ and the solvent by evaporation
in vacuo leaving the aminoester 5 as a light orange oil. Yield 510 mg
(76% yield). MS (ESI) 668.4 (MH+). Calc. for C34H57N3O10: 667.1.
1H-NMR (CD3OD): 6.74 (d, 1H, J = 8.5 Hz), 6.44 (d, 1H, J =
2.4 Hz), 6.27 (dd, 1H, J1 = 8.9 Hz, J2 = 2.4 Hz), 4.07 (t, 2H, J =
5.4 Hz), 4.01 (t, 2H, J = 5.4 Hz), 3.55 (s, 4H), 3.54 (s, 4H), 3.12
(t, 2H, J = 5.4 Hz), 3.07 (t, 2H, J = 5.4 Hz), 1.46 (s, 36H). 13C-
NMR (CD3OD): 171.0 2x(C), 149.7 (C), 142.7 (C), 141.1 (C), 116.4
(CH), 107.5 (CH), 102.5 (CH), 80.94 (C), 80.90 (C), 69.1 (CH2),
67.5 (CH2), 56.4 (CH2), 56.3 (CH2), 53.7 (CH2), 53.6 (CH2), 27.1
(2 × CH3).
Compound 17
L-Phenylalanine tert-butyl ester (12)
Obtained as a yellow oil following the same procedure adopted for
compound 16, starting from aminoester 10 (450 mg, 0.67 mmol)
and isothiocyanate 15 (161 mg, 0.71 mmol). Yield 341 mg (57%).
MS (ESI) 895.5 (MH+). Calc. for C47H82N4O10S: 894.5. 1H-NMR
(CDCl3): 6.97–6.68 (m, 3H), 7.73 (bs, 1H), 6.38 (bs, 1H), 4.09 (bt,
4H), 3.58 (s, 4H), 3.56 (s, 4H), 3.48 (bt, 2H), 3.18 (bt, 4H), 1.58
(m, 2H), 1.41 (s, 36H), 1.33–1.21 (m, 18H), 0.88 (bt, 3H, J = 6.3
Hz). 13C-NMR (CDCl3): 170.5 (C), 170.3 (C), 148.6 (C), 146.6 (C),
134.7 (C), 117.8 (CH), 113.8 (CH), 111.8 (CH), 81.9 (C), 81.1 (C),
67.8 (2 × CH2), 56.6 (2 × CH), 53.7 (CH2), 53.6 (CH2), 44.9 (CH2),
31.8 (CH2), 29.7 (CH2), 29.6 (CH2), 29.3 (CH2), 29.0 (CH2), 28.0
(4 × CH2), 27.9 (2 × CH3), 22.6 (CH2), 14.0 (CH3).
L-Phenylalanine (3.00 g, 18.2 mmol) was suspended in tert-
butyl acetate (100 mL) and 70% aq. HClO4 (2.0 g, 20 mmol)
and the mixture stirred at room temperature. After 24 h the
reaction mixture was washed with 5% aq. Na2CO3 (50 mL), brine
(50 mL) and water (50 mL), then dried over Na2SO4, filtered and
evaporated in vacuo to provide 12 as a colorless oil (2.18 g, 54%),
used without further purification in the following step.
Compound 13
A
solution of thiophosgene (1.37 g, 11.8 mmol) in
dichloromethane (5.0 mL) was slowly added dropwise to a cooled
(0–5 ◦C) flask containing a stirring mixture of phenylalanine tert-
butyl ester (12, 2.37 g, 10.7 mmol), dichloromethane (5 mL) and
sat. aq. NaHCO3 (10 mL). Stirring was maintained for 1 h and then
the organic layer was separated, washed thrice with water (10 mL),
dried over Na2SO4, filtered and evaporated in vacuo. Compound
13 was obtained as an amorphous off-white solid. Yield 1.93 g
Compound 18
Aminoester 10 (520 mg, 0.78 mmol) was dissolved in a stirring
mixture of dichloromethane (10.0 mL) and 10% aq. Na2CO3,
cooled in an ice bath under an N2 atmosphere. A solution
of stearoyl chloride (245 mg, 0.80 mmol) in dichloromethane
2366 | Org. Biomol. Chem., 2008, 6, 2361–2368
This journal is
The Royal Society of Chemistry 2008
©

View Article Online
(5.0 mL) was slowly added dropwise and the resulting suspension
was stirred for 2 h. The organic layer was separated and washed
with 10% aq. Na2CO3 (3 × 20 mL), dried over Na2SO4, filtered
and evaporated in vacuo. The conjugate 18 was obtained as a light
yellow oil. Yield 662 mg (91%). MS (ESI) 934.5 (MH+). Calc. for
C52H91N3O11: 933.8. 1H-NMR (CDCl3): 7.52 (bs, 1H), 7.28 (s, 1H),
7.10 (d, 1H, J = 8.3 Hz), 6.81 (d, 1H, J = 8.7 Hz), 4.12 (t, 2H, J =
6.0 Hz), 4.09 (t, 2H, J = 6.0 Hz), 3.54 (s, 4H), 3.53 (s, 4H), 3.14 (t,
4H, J = 5.9 Hz), 2.30 (bt, 2H, J = 7.2 Hz), 1.69 (quint, 2H, J =
6.9 Hz), 1.44 (s, 18H), 1.42 (s, 18H), 1.40–1.25 (m, 28H), 0.86 (bt,
3H, J = 6.4 Hz). 13C-NMR (CDCl3): 171.3 (C), 170.7 (C), 170.6
(C), 148.8 (C), 145.1 (C), 132.3 (C), 114.3 (CH), 112.2 (CH), 107.0
(CH), 81.0 (C), 80.9 (C), 68.5 (CH2), 68.4 (CH2), 56.7 (CH2), 56.6
(CH2), 56.5 (CH2), 53.4 (CH2), 37.7 (CH2), 31.9 (CH2), 29.6–29.3
(12 × CH2), 28.11 (CH3), 28.07 (CH3), 25.3 (CH2), 22.6 (CH2),
14.1 (CH3).
Potentiometric studies
The chemicals used for the experiments were of the highest analyt-
ical grade. The LnCl3 solutions were prepared from LnCl3·xH2O
(x = 5–7). The concentration of the MgCl2, CaCl2, and LnCl3
solutions were determined by complexometric titration with
standardized Na2H2EDTA and xylenol orange (LnCl3), Patton &
Reeder (CaCl2) and eriochrome black T (MgCl2) as indicator. The
concentration of L2 ligand was determined by pH-potentiometric
titration in the presence and absence of a large excess (40-fold) of
CaCl2. The protonation and the stability constants of the metal
complexes formed with L2 were determined by pH-potentiometric
titration. The metal-to-ligand concentration ratios were 1 : 1 with
a concentration of ligand generally of 0.002 M.
pH measurements and titrations were performed on a CRISON
micro pH 2002 pH-meter, a CRISON micro BU2030 autoburette
and a Metrohm-6.0233.100 combined electrode. Equilibrium
measurements were carried out at a constant ionic strength (0.1 M
◦
KCl) in 10 mL sample at 25 C. The solutions were stirred with
Ligand L3
N2 bubbling. The titrations were carried out in the pH range 1.7–
11.7. For the calibration of the pH meter, buffer standard solution,
color-coded “pink” (pH = 4.010) and buffer standard solution,
color coded “yellow” (pH = 7.000) buffers were used. For the
calculation of [H+] from the measured pH values, the method
proposed by Irving et al. was used.18 A 0.01 M HCl solution
was titrated with the standardized KOH solution. The differences
between the measured and calculated pH values were used to
obtain the H+ concentration from the pH values, measured in
the titration experiments. The protonation and stability constants
were calculated with the program PSEQUAD.19
Obtained as an amorphous light yellow powder following the same
procedure adopted for the tert-butyl group removal giving L1,
starting from ester 16 (126 mg, 0.13 mmol). Yield 94 mg. M.p.
158–159 ◦C (dec.). MS (ESI, negative ion mode) 649.2 (M − H+).
1
Calc. for C28H34N4O12S: 650.2. H-NMR (DMSO-d6): 10.52 (bs,
4H), 9.69 (bs, 1H), 7.31–6.88 (m, 8H), 6.33 (bd, 1H), 6.13 (bs,
1H), 5.22 (m, 1H), 4.03–3.84 (m, 4H), 3.55 (s, 8H), 3.46 (m, 1H),
3.17–2.93 (m, 5H). 13C-NMR (D2O): 173.9 (C), 173.2 (C), 148.6
(C), 148.2 (CH), 134.8 (C), 130.3 (CH), 129.5 (CH), 128.6 (CH),
121.4 (CH), 113.8 (CH), 113.0 (CH), 68.4 (CH2), 68.1 (CH2), 60.4
(CH), 55.9 (CH2), 53.2 (CH2), 36.7 (CH2).
Acknowledgements
Ligand L4
Financial support from PRIN 2005 is gratefully acknowledged.
This work was carried out under the auspices of CIRCMSB and
COST Action D38.
Obtained as an amorphous yellow powder following the same
procedure adopted for the tert-butyl group removal giving L1,
starting from ester 17 (243 mg, 0.27 mmol). Yield 147 mg. MS
(ESI) 671.5 (MH+). Calc. for C31H50N4O10S: 670.5. 1H-NMR
(D2O): 6.89 (m, 2H), 6.78 (d, 1H, J = 8.8 Hz), 4.03 (bt, 4H),
3.47 (bt, 2H), 3.17 (s, 8H), 2.92 (bt, 4H), 1.54 (m, 2H), 1.24 (m,
18H), 0.85 (bt, 3H), J = 6.1 Hz).
References
1 Z. J. Guo and P. J. Sadler, Adv. Inorg. Chem., 2000, 49, 183; T. Storr,
K. H. Thompson and C. Orvig, Chem. Soc. Rev., 2006, 35, 534; E.
Meggers, Curr. Opin. Chem. Biol., 2007, 11, 287; S. M. Cohen, Curr.
Opin. Chem. Biol., 2007, 11, 115.
2 Supramolecular Chemistry of Anions, ed. A. Bianchi, K. Bowman-James
and E. Garcia-Espana, Wiley-VCH, New York, 1997; J. L. Sessler,
P. A. Gale and W.-S. Cho, Anion Receptor Chemistry, RSC Publishing,
Cambridge, 2006; K. Bowman-James, Acc. Chem. Res., 2005, 38, 671;
K. Wichmann, B. Antonioli, T. Sohnel, M. Wenzel, K. Gloe, K. Gloe,
J. R. Price, L. F. Lindoy, A. J. Blake and M. Schroder, Coord. Chem.
Rev., 2006, 250, 2987; P. Gamez, T. J. Mooibroek, S. J. Teat and J.
Reedijk, Acc. Chem. Res., 2007, 40, 435.
3 Recent reviewed examples: S. F. Liu and J. L. Xiao, J. Mol. Catal. A:
Chem., 2007, 270, 1; A. Fihri, P. Meunier and J. C. Hierso, Coord.
Chem. Rev., 2007, 251, 2017; S. Gladiali and E. Alberico, Chem. Soc.
Rev., 2006, 35, 226; V. Dragutan, I. Dragutan, L. Delaude and A.
Demonceau, Coord. Chem. Rev., 2007, 251, 765.
4 Selected books and reviews: B. Valeur and I. Leray, Coord. Chem.
Rev., 2000, 205, 3; Chemosensors of Ion and Molecule Recognition,
ed. J. P. Desvergne and A. W. Czarnik, Kluwer Academic Publishers,
Dordrecht, The Netherlands, 1997; A. P. de Silva, H. Q. N. Gunaratne,
T. Gunnlaugsson, A. J. M. Huxley, C. P. McCoy, J. T. Rademacher
and T. E. Rice, Chem. Rev., 1997, 97, 1515; L. Fabbrizzi, F. Foti, S.
Patroni, P. Pallavicini and A. Taglietti, Angew. Chem., Int. Ed., 2004, 43,
5073.
Ligand L5
Obtained as an amorphous white powder following the same
procedure adopted for tert-butyl group removal giving L1, starting
from ester 18 (402 mg, 0.43 mmol). Yield 249 mg. M.p. 160 ◦C
(dec.). MS (ESI) 711.0 (MH+). Calc. for C36H59N3O11: 709.8. 1H-
NMR (DMSO-d6): 11.30 (bs, 4H), 9.67 (bs, 1H), 7.28 (d, 1H, J =
1.7 Hz), 7.07 (dd, 1H, J1 = 8.6 Hz, J2 = 1.9 Hz), 6.86 (d, 1H,
J = 8.7 Hz), 3.98 (t, 2H, J = 5.7 Hz), 3.97 (t, 2H, J = 6.0 Hz),
3.53 (bs, 8H), 3.05 (t, 2H, J = 5.7 Hz), 3.02 (t, 2H, J = 5.7 Hz),
2.24 (t, 2H, J = 7.4 Hz), 1.56 (m, 2H), 1.31–1.15 (m, 28H), 0.86
(bt, 3H, J = 6.4 Hz). 13C-NMR (DMSO-d6): 173.2 (2 × C), 171.2
(C), 148.5 (C), 144.3 (C), 133.8 (C), 114.5 (CH), 111.6 (CH), 106.0
(CH), 68.4 (CH2), 68.0 (CH2), 56.0 (2 × CH2), 53.3 (2 × CH2),
36.8 (CH2), 31.7 (CH2), 29.5–29.1 (12 × CH2), 25.2 (CH2), 22.5
(CH2), 14.4 (CH3).
This journal is
The Royal Society of Chemistry 2008
Org. Biomol. Chem., 2008, 6, 2361–2368 | 2367
©

View Article Online
5 P. Chaudhuri, K. Wieghardt, T. Weyhermuller, T. K. Paine, S. Mukher-
jee and C. Mukherjee, Biol. Chem., 2005, 386, 1023; P. Chaudhuri and
K. Wieghardt, Prog. Inorg. Chem., 2001, 50, 151; D. Fiedler, D. H.
Leung, R. G. Bergman and K. N. Raymond, Acc. Chem. Res., 2005,
38, 349.
6 P. Caravan, J. J. Ellison, T. J. McMurry and R. B. Lauffer, Chem.
Rev., 1999, 99, 2293; S. Aime, S. Geninatti Crich, E. Gianolio, G. B.
Giovenzana, L. Tei and E. Terreno, Coord. Chem. Rev., 2006, 250,
1562; S. Aime, M. Botta and E. Terreno, Adv. Inorg. Chem., 2005,
57, 173; The Chemistry of Contrast Agents in Medical Magnetic
Resonance Imaging, ed. E. Toth and A. E. Merbach, Wiley, New
York, 2001; S. Aime, M. Botta, M. Fasano and E. Terreno, Chem.
Soc. Rev., 1998, 27, 19; R. Artali, M. Botta, C. Cavallotti, G. B.
Giovenzana, G. Palmisano and M. Sisti, Org. Biomol. Chem., 2007, 5,
2441.
10 C. K. Schauer and O. P. Anderson, Acta Crystallogr., Sect. C: Cryst.
Struct. Commun., 1986, 42, 760; C. K. Schauer and O. P. Anderson,
J. Am. Chem. Soc., 1987, 109, 3646.
11 E. Brunet, M. T. Alonso, O. Juanes, O. Velasco and J. C. Rodriguez-
Ubis, Tetrahedron, 2005, 57, 3105; S. R. Adams, J. P. Y. Kao, G.
Grynkiewicz, A. Minta and R. Y. Tsien, J. Am. Chem. Soc., 1988,
110, 3212; G. C. R. Ellis-Davies and J. H. Kaplan, Proc. Natl. Acad.
Sci. U. S. A., 1994, 91, 187; G. C. R. Ellis-Davies, Tetrahedron Lett.,
1998, 39, 953; W. Li, S. Fraser and T. Meade, J. Am. Chem. Soc., 1999,
121, 1413; R. Y. Tsien, Biochemistry, 1980, 19, 2396.
12 E. Brunet, M. T. Alonso, O. Juanes, O. Velasco and J. C. Rodr´ıguez-
Ubis, Tetrahedron, 2000, 57, 3105.
13 M. A. Williams and H. Rapoport, J. Org. Chem., 1993, 58, 1151.
14 S. Avedano, L. Tei, A. Lombardi, G. B. Giovenzana, S. Aime, D. Longo
and M. Botta, Chem. Commun., 2007, 4726.
7 (a) A. E. Martell and R. M. Smith, Critical Stability Constants, vol. 4,
Plenum Press, New York, 1974; (b) A. Bianchi, L. Calabi, F. Corana,
S. Fontana, P. Losi, A. Maiocchi, L. Paleari and B. Valtancoli, Coord.
Chem. Rev., 2000, 204, 309.
8 See for example: J.-M. Idee, M. Port, I. Raynal, M. Schaefer, S. Le
Greneur and C. Corot, Fundam. Clin. Pharmacol., 2006, 20, 563; S. K.
Morcos, Br. J. Pharmacol., 2007, 80, 73.
15 S. Aime, A. Barge, M. Botta, L. Frullano, U. Merlo and K. I.
Hardcastle, J. Chem. Soc., Dalton Trans., 2000, 3435.
16 J. Felcman and J. da Silva, Talanta, 1983, 301, 565.
17 J. L. Mackey, M. A. Hiller and J. E. Powell, J. Phys. Chem., 1962, 66,
311.
18 H. M. Irving, M. G. Miles and L. Pettit, Anal. Chim. Acta, 1967, 28,
475.
9 S. Aime, A. Barge, A. Borel, M. Botta, S. Chemerisov, A. E.
Merbach, U. Mu¨ller and D. Pubanz, Inorg. Chem., 1997, 36,
5104.
19 L. Ze´ka´ny and I. Nagypa´l, in Computational Methods for Determination
of Formation Constants, ed. D. J. Legett, Plenum, New York, 1985,
p. 291.
2368 | Org. Biomol. Chem., 2008, 6, 2361–2368
This journal is
The Royal Society of Chemistry 2008
©