A novel series of complexones with bis- or biazole structure as mixed ligands of paramagnetic contrast agents for MRI

Article abstract of DOI:10.1016/j.bmc.2003.07.002

We describe the syntheses, physicochemical properties and biological evaluation of a novel series of complexones containing bis- or biazoles moieties and two iminodiacetic acid units as novel ligands for paramagnetic lanthanides. The complexones were prepared by reaction of the corresponding 1,1′-bishaloethylbi- or bispyrazoles with methyl iminodiacetate and subsequent NaOH hydrolysis. 1,1′-Bisbromoethyl precursors were obtained by direct alkylation with an excess of 1,2-dibromoethane, or by heating the corresponding alcohol in HCl. Sigmoidal binding isotherms and MO calculations supported as most stable structures in solution, those containing two Gd(III) atoms bound per molecule of complexone with half saturation values S _0.5 (M^-1, 22 °C, pH 7.2) in the range 6.5 10 ^-60.5<36.1 10^-6. Relaxivity properties [r₁, r₂, s^-1 mM^-1 Gd(III)] determined at 1.5 Tesla gave values (12.01<17.7, 12.22<20), improving significantly the relaxivities of reference compounds such as Gd(III)EDTA (5.2, 5.6) or Gd(III)DTPA (4.30, 4.30). These improvements involve mainly increased hydration and slower rotational motions. In vitro toxicity experiments are reported.

Full text of DOI:10.1016/j.bmc.2003.07.002

Bioorganic & Medicinal Chemistry 11 (2003) 5555–5567
A Novel Series of Complexones with Bis- or Biazole Structure
as Mixed Ligands of Paramagnetic Contrast Agents for MRI
Elena P. Mayoral,a Marıa Garcıa-Amo,a Pilar Lopez,a
Elena Soriano,a Sebastian Cerdanb and Paloma Ballesterosa,*
a
´
Departamento Quımica Organica y Biologıa, Facultad de Ciencias, UNED, Senda del Rey 9, 28040 Madrid, Spain
´
´
Instituto de Investigaciones Biomedicas CSIC, c/ Arturo Duperier 4, 28029 Madrid, Spain
b
´
Received 22 August 2002; accepted 10 July 2003
Abstract—We describe the syntheses, physicochemical properties and biological evaluation of a novel series of complexones con-
taining bis- or biazoles moieties and two iminodiacetic acid units as novel ligands for paramagnetic lanthanides. The complexones
were prepared by reaction of the corresponding 1,10-bishaloethylbi- or bispyrazoles with methyl iminodiacetate and subsequent
NaOH hydrolysis. 1,10-Bisbromoethyl precursors were obtained by direct alkylation with an excess of 1,2-dibromoethane, or by
heating the corresponding alcohol in HCl. Sigmoidal binding isotherms and MO calculations supported as most stable structures in
solution, those containing two Gd(III) atoms bound per molecule of complexone with half saturation values S0.5 (MÀ1, 22 ꢀC, pH
7.2) in the range 6.5 10À6<S0.5<36.1 10À6. Relaxivity properties [r1, r2, sÀ1 mMÀ1 Gd(III)] determined at 1.5 Tesla gave values
(12.0<r1<17.7, 12.2<r2<20), improving significantly the relaxivities of reference compounds such as Gd(III)EDTA (5.2, 5.6) or
Gd(III)DTPA (4.30, 4.30). These improvements involve mainly increased hydration and slower rotational motions. In vitro toxicity
experiments are reported.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
mMÀ1, well below the optimal values of approximately
100 sÀ1 mMÀ1 predicted by theory.8 Reduced relaxivity
imposed the use of large doses of these agents, limiting
the possibilities to visualize successfully low concen-
tration molecular targets such as cell surface antigens,
receptors, enzymes or even genes.9
Even though Magnetic Resonance Imaging (MRI)
methods inherently provide high intrinsic tissue con-
trast, the use of extrinsic contrast agents has become a
routine in many diagnostic imaging procedures.1 Very
frequently, the paramagnetic lanthanide Gd(III) is used
to increase locally the longitudinal relaxation rate of
surrounding tissue water, highlighting the intensity of
specific tissue areas in T1 weighted images.2 However,
free Gd(III) is toxic in vivo and in vitro and Gd(III)
chelates must be used in the clinic for safety reasons.3,4
The first generation of Gd(III) ligands was derived from
linear polyaminopolycarboxylates such as diethylene-
triaminepentaacetic acid (DTPA) or from macrocycles
such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra-
acetic acid (DOTA).5 The corresponding Gd(III) com-
plexes depicted very high thermodynamic and kinetic
stability.6 However, their capacity to induce water
relaxation termed relaxivity,7 remained ca. 4–5 sÀ1
To overcome these limitations it became necessary to
increase the relaxivity of new generations of Gd(III)
chelates, maintaining simultaneously their high thermo-
dynamic stability. One approach towards this goal, is to
maintain the basic chemical structures of Gd(III)DTPA
or Gd(III)DOTA complexes but increase their relaxivity
by restricting their rotational dynamics through con-
jugation to linear polymers, dextrans and proteins or
through the production of dendrimeric derivatives.3,10
An extensive series of macromolecular DTPA and
DOTA derivatives were produced and characterized in
this way, increasing relaxivities to ca. 15 sÀ1 mMÀ1
per unit of bound Gd(III) chelate in linear polymers,
ca. 19 sÀ1 mMÀ1 in dendrimers and ca. 50 sÀ1 mMÀ1
in those bound to serum albumin.11 However, even
these values remained below the optimal relaxivities
predicted, suggesting that further improvements would
*Corresponding author. Tel.: +34-91-398-7320; fax: +34-91-398-
6697; e-mail: pballesteros@ccia.uned.es
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.07.002

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E. P. Mayoral et al. / Bioorg. Med. Chem. 11 (2003) 5555–5567
require additional modifications of the chemical struc-
ture of the ligands.
complexes per complexone molecule. The correspond-
ing Gd(III) complexes of 5a–d depicted remarkable
improvements in relaxivity of ca. 25<r1<37 and
25<r2<70 sÀ1 mMÀ1 complexone as compared to the
first series of heterocyclic complexones, and to Gd(III)
DTPA or Gd(III) DOTA. Thermodynamic stability is
also improved, being higher than the previous mono-
meric complexones but still lower than that of Gd(III)
DTPA or Gd(III) EDTA reference compounds.
With this aim, we proposed earlier the use of nitrogen
containing heterocycles as ligands for paramagnetic
contrast agents.12 The approach relied on the high cap-
ability of nitrogen as electron donor,13,14 a property
which allowed us previously to design and prepare a
series of pH sensitive probes for magnetic resonance
spectroscopy.15À17 However, few complexones had
incorporated earlier heterocycles in their structures.18À20
The first generation of azolic complexones 1–412 con-
tained only one iminodiacetic acid unit and one azole
ring (Fig. 1). These ligands were able to form tetra-
dentate complexes with Gd(III) involving two carboxy-
lates, one amine group, and the heterocyclic nitrogen
atom, showing improved relaxivity properties as com-
pared to Gd(III) DTPA or Gd(III)DOTA. However,
their thermodynamic stability was much lower than that
of these octadentated reference ligands.
Results
Chemical syntheses
Compounds 5a–d were prepared by reaction of the cor-
responding bi or bisazoles 6–8,21À23 with an excess of
1,2-dibromoethane using liquid-liquid phase transfer
catalysis (Scheme 1). In all cases, the corresponding
3,30-, 3,50-, 5,50-(2-bromoethylpyrazol-1yl) substituted
isomers were isolated, being the 3,30-regioisomers 9a, 10
and 11a the major products. The alkylation of 7 yielded
the dibromoderivative 10, the corresponding mono-
substituted compound 15, and a complex mixture of
polymeric material.
To improve the relaxivity and thermodynamic stability
of this type of compounds we approach here the syn-
theses and physicochemical characterization of the sec-
ond generation of heterocyclic complexones containing
two pyrazole rings and two iminodiacetic acid units per
molecule 5a–d (Fig. 1). These dimeric structures, con-
ceived initially to provide an octadentate geometry
around the Gd(III) ion are shown here to fold over
intramolecularly forming two tetradentadentate Gd(III)
Subsequently, the reaction of the 2-bromoethyl deriva-
tives 9a, 10 and 11a with four equivalents of methyl
iminodiacetate gave compounds 12–14. Basic hydrolysis
of 12–14 yielded the corresponding complexones 5a–c.
Bispyrazole 8 was synthesized from the aldehyde 16 via
tosylhydrazone intermediate, as reported by Lepage and
Lepage (Fig. 2).24 The formation of only one isomer
(E,E) of 16 was described then. However, we repro-
duced this reaction, in the same conditions and obtained
a mixture of E,E/E,Z isomers, both compounds being
separated by column chromatography on silica gel and
characterized by spectroscopic methods.
1H NMRof E,E-16 depicted a characteristic singlet at
9.52 ppm corresponding to both isochronous CHO
groups, while the spectrum of the E,Z-16 isomer showed
two singlets at 9.86 and 9.74 ppm derived from the two
anisochronous CHO groups. According to the synthetic
strategy employed to give pyrazole 6, the reaction of 16
(E,E/E,Z) with tosylhydrazine in MeOH yielded the
tosylhydrazone 17, as the only E,Z-isomer. The assign-
ment of 17 was based on NMRspectra. The 1H NMR
spectrum depicted two singlets at 2.26 and 2.09 ppm cor-
responding to the two different methyl groups. 13C NMR
confirmed this structure by showing the corresponding
methyl resonances at 20.9 and 20.7 ppm, respectively.
Compound 17 provides an interesting example where the
rotational barrier for cis–trans interconversion of the
isomers is low enough25 to yield only the compound most
stable thermodynamically. Indeed, theoretical conforma-
tional analysis (HF/6-31G*//HF/3-21G) of 17 showed a
higher stabilization of the E,Z isomer through the for-
mation of two intramolecular hydrogen bonds.
Figure 1. Structures of monoazolic complexones 1–412 and bis- or
biazolic complexones 5a–d.
The synthesis of 5d followed an alternative approach
(Scheme 2). 18a26 reacted with paraformadehyde in

E. P. Mayoral et al. / Bioorg. Med. Chem. 11 (2003) 5555–5567
5557
Scheme 1. Syntheses of complexones 5a–c.
following alkaline hydrolysis similar to that described
above.
Physicochemical characterization
Gd(III) binding. Binding isotherms (22 ꢀC) of complex-
ones 5a–d for Gd(III) were obtained spectro-
photometrically by competition with Arsenazo III
(ArsIII) as indicated in the Experimental.12 Figure 3
shows a representative example of the results obtained
with 5b, but similar isotherms were obtained for com-
plexones 5a, 5c and 5d.
Gd(III) chloride titrations of the model solutions
(0.15 M ionic strength, pH 6.5, 22 ꢀC) containing ArsIII
depicted typical hyperbolic saturation curves (solid
lines), providing a value for the apparent dissociation
constant of the Gd(III)–ArsIII complex of ca. 20 mM.
However, when 1 mM complexones 5a–d were present
in the titration mixtures, competitive binding of Gd(III)
to the complexone and to ArsIII caused a marked
deviation from the typically hyperbolic Gd(III)–ArsIII
titration curves. Under these conditions, Gd(III) did not
bind appreciably to the complexones below 0.03–0.05
mM, but evident binding occurred at higher Gd(III)
concentrations, as revealed by the sharp decrease in the
saturation value of the Gd(III)–ArsIII complex (dashed
line). This pattern suggested that the transition from
free to fully saturated complexones occurred in a nar-
row range of Gd(III) concentrations, following a sig-
moidal trend which indicated cooperative binding of
more than one Gd(III) ion per mole of complexone
(dotted lines). Non linear least squares fittings of the
Figure 2. Structures of the isomers of 16 and 17.
1,2-dichloroethane saturated with dry HCl to yield the
corresponding 4-chloromethylpyrazole.27 Subsequent
acid hydrolysis gave the alcohol 19 and a small amount
of the ether 20. Chloromethylation of 18b was carried
out under the same conditions used for 18a, to obtain a
mixture of products. The mass spectrum of this mixture
revealed the presence of the corresponding chloroethyl
derivatives produced through Br–Cl exchange.
Chloroethyl derivative 21 was obtained by refluxing
alcohol 19 in concd HCl. Compounds 22 and 5d were
obtained by reaction of 21 with methyl iminodiacetate

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E. P. Mayoral et al. / Bioorg. Med. Chem. 11 (2003) 5555–5567
Scheme 2. Synthesis of complexone 5d.
Table 1. Binding parameters obtained from titrations of solutions of
ArsIII with Gd(III) chloride in the absence and presence of complex-
ones 5a–d
na
a
Compd
Kda (mM)
S0.5 (mM)
ArsIII
5a
5b
5c
5d
23.3Æ8.3b
6.7Æ0.03b
9.0Æ0.02
6.5Æ1.0b
3.2Æ0.9
7.0Æ0.0
4.9Æ1.9
35.0Æ0.08
36.1Æ0.04
aBinding parameters for complexones 5a–d were determined by non
linear least squares fittings of Gd(III) titrations (Fig. 1), as described
in the Experimental.
Figure 3. Binding of Gd(III) to ArsIII in the absence (solid lines) or
presence (dashed line) of 5a. Sigmoidal Gd(III) binding to 5a (dotted
line) is calculated as the difference between the solid and dashed lines.
Binding parameters S0.5 and n reflecting the half maximal saturation
of the complexone and the degree of cooperativity can be estimated as
indicated in the Experimental (c.f. Table 1).
bResults are given as the meanÆSD of three independent determinations.
Gd(III) the complexone folded to create an internal
cavity in which an hexacoordinated metal (B,
E=À1800.990569 a.u.) was buried between the two
iminodiacetate units. In this folded form, the pyrazole
groups do not participate in chelation and the two
phenyl (5a, 5c) or methyl (5b, 5d) groups fall inside the
metal cavity causing steric hinderance and instability of
the complex. When two Gd(III) atoms are available, the
complexone folds into its most stable structure consisting
of two tetradentate complexes of Gd(III), each one
involving one azole ring, two carboxylates and one
amino group from the closest iminodiacetic acid moiety
(C, E=À1836.086424 a.u.). Under these conditions,
both phenyl groups in 5a and 5c or methyl groups in 5b
and 5d, remain outside the coordination centers avoiding
steric hindrance and resulting in a more stable chelate.
experimental data to an heuristic model consisting of a
single binding site in ArsIII and sigmoidal Gd(III)
binding in the complexone, provided optimal fits of the
results. This allowed to determine the corresponding
parameters S0.5 and cooperativity coefficients n for
every complexone (Table 1).
MO calculations. We used ab initio molecular orbital
calculations28 to investigate the geometries of the
Gd(III) complexes of 5a–d. This approach has pre-
viously provided reliable results in the study of some
lanthanide complexes with polyamino carboxylate and
phosphinic ligands.29À32 We calculated first the most
stable structures of representative complexone 5b in the
absence of Gd(III) and in the presence of one or two
Gd(III) atoms (Fig. 4). Similar calculations were per-
formed with complexones 5a, 5c and 5d.
Figure 5(A) illustrates in more detail the optimal geo-
metry calculated for one of the azole moieties of the
dimeric Gd(III) complex of 5a–d.
In the absence of Gd(III), the most stable structure of 5b
was the fully open conformation (A, E=À1764.731996
a.u.) with both iminodiacetate moieties located at
opposite sides of the molecule. In the presence of one
The Gd(III) atom is located above the O1–O2–N plane
by 0.703 A (5a), 0.641 A (5b), 0.689 A (5c) and 0.627 A
(5d). The phenyl and p-nitrophenyl rings of complex-
ones 5a and 5c deviate from coplanarity with the azole

E. P. Mayoral et al. / Bioorg. Med. Chem. 11 (2003) 5555–5567
5559
Figure 4. Optimized structures of biazolic complexone 5b calculated in the absence (A) and presence of one (B) or two (C) Gd(III) atoms. Calcu-
lations were performed using the HF/3-21G basis set for the ligand, and the 46+4f7 ECP with the [5s4p3d]-GTO valence basis set for the metal, as
indicated in the Experimental. The Gd(III) atoms are shown in green.
Figure 5. Coordination geometry around the metal in 5a–d complexes:
(a) distance from the metal to the N–O1–O2 plane of the ligand;
(b) effect of the phenyl ring.
ring by y=60.63ꢀ and y=58.42ꢀ, making the distance
from Gd(III) to the O1–O2–N plane to increase in those
complexones containing aromatic rings (Fig. 5B). This
effect is due to the participation of the p electrons of
the aromatic substituent in the complexation. Distances
from the phenyl carbons to Gd(III) were (5a; 5c, A);
(C1 3.091; 3.213, C2 3.023; 3.080, C3 3.604; 3.736, C4
4.155; 4.371, C5 4.218; 4.501, C6 3.734; 3.971). These
distances are longer in 5c than in 5a, because of the
electron withdrawal effect of the p-nitro group. Inter-
estingly, we have described previously metal-phenyl
interactions in complexes of Rh(I) with bis(pyrazol-1-
yl)phenylmethane.33
Because of the importance of hydration determining the
relaxivity properties, we calculated the structures of the
Figure 6. Calculated structures of 2 (A) and 5b (B) in the presence of
hydrated complexes of 5a–d Gd(III)–4(5)H2O, EDTA
Gd(III)–2(3)H2O and DTPA Gd(III)–H2O. Figure 6
five water molecules (A1–A5) in the inner coordination sphere of
Gd(III). Note that A5 is excluded from the coordination center. Cal-
culations were performed as in Figure 4.
shows optimized hydrated structures for 5b (B) and its

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E. P. Mayoral et al. / Bioorg. Med. Chem. 11 (2003) 5555–5567
monomeric analogue 2 (A) in the presence of five water
molecules.
Gd(III) complexes followed the order 5c>/
=5d>5b>5a>aqua>EDTA>DTPA>IDA. When
expressed per mol of Gd(III) ion, r1 and r2 relaxivity
values of complexes 5a–d, were significantly higher than
Gd(III) chloride solutions of the same concentration,
two to three fold higher than the Gd(III)EDTA and
Gd(III)DTPA reference complexes and 4- to 6-fold
higher than Gd(III)IDA. It should be noted here that
relaxivity of Gd(III)DTPA is strongly dependent of
ionic strength. While in the presence of 155 mM NaCl
solution the r1 and r2 values are 4.3, in absence of saline
solution, as it happens in distilled water solution, the
values decrease to 3.5 (see Table 3). Moreover, since the
Gd(III) complexes of 5a–d appear to contain two
Gd(III) atoms per mol of complexone, r1 and r2 relax-
avities may be doubled when expressed per mole of
complexone. These values remain to our knowledge,
among the largest reported for compounds of this
molecular weight3,10 and similar to those of the Gd(III)
chelates of DOTA and DTPA conjugated to synthetic
polymers of ca. 20 kDa35À37 or to those of dendrimeric
materials.38,39
In 5b, optimized Gd(III)–O water distances were:
Gd(III)–(OH2)A1 2.452 A; Gd(III)–(OH2)A2 2.434 A;
Gd(III)–(OH2)A3 2.391 A; Gd(III)–(OH2)A4 2.603 A;
Gd(III)–(OH2)A5 3.741 A. In the case of 2, Gd(III)–O
water distances were: Gd(III)–(OH2)A1 2.454 A;
Gd(III)–(OH2)A2 2.427 A; Gd(III)–(OH2)A3 2.393 A;
Gd(III)–(OH2)A4 2.628 A; Gd(III)–(OH2)A5 3.730 A.
Even though five water molecules were included in the
calculations, only four molecules (OH2)A1–(OH2)A4
became directly coordinated to Gd(III) in the optimized
structures, the fifth one (OH2)A5 being excluded from
the first coordination sphere in all cases. When only
four water molecules were included in the calculation,
all four water molecules showed very similar distances
to the metal ion (2.43<Gd(III)–O<2.53 A). Table 2
summarizes some relevant geometrical parameters of
these molecules.
The distances (A) calculated by ab initio methods match
well the experimental measurements for Gd(III)DT-
PA(H2O).34 Some examples include (A experimental, A
calculated) the Gd–Owater distance (2.490, 2.595) and
the distances from Gd to the oxygen atoms and nitrogen
atoms of the iminodiacetate and aminoacetate carbox-
ylates Gd(III)-O (2.363–2.437, 2.311–2.449) and
Gd(III)–N (2.629–2.710, 2.719–2.770). These results
provide a measure of confidence on the geometrical
parameters calculated for the Gd(III) complexes of 5a–d
for which no experimental X-ray diffraction measure-
ments are available yet.
Using the same methodology, we investigated the
relaxivities of some of these complexes in rat plasma
containing or not 5 mM complexones and 0.5 mM
Gd(III). The following values (T1, T2, s) were obtained;
rat plasma (2.01Æ0.03, 0.77Æ0.05), 5a (0.16Æ0.04,
0.10Æ0.04), 5b (0.14Æ0.06, 0.09Æ0.02) and 5d
(0.13Æ0.01, 0.08Æ0.01). These values correspond to
relaxivities (ion r1, ion r2 sÀ1 mMÀ1) of 5a (11.83Æ0.33,
18.12Æ0.01), 5b (13.63Æ0.08, 20.06Æ0.05) and 5d
(13.94Æ0.12, 20.84Æ0.013). The relaxivity values
obtained in plasma were similar than those found in
aqueous solution suggesting no significant enhance-
ment derived from binding of the complexes to plasma
components.
Relaxivity. Table 3 depicts the results of longitudinal
(T1) and transversal (T2) relaxation time measurements
and the corresponding r1 and r2 relaxivities of Gd(III)
chloride solutions and the Gd(III) complexes of 5a–d,
EDTA, DTPA and IDA performed at 1.5 Tesla as
indicated in the Experimental. A 10-fold excess of the
indicated ligand was used in these measurements to
minimize the effects of free Gd(III). As expected, the free
ligand did not contribute appreciably to the observed
relaxation times. r1 and r2 values of the corresponding
Biological evaluation
In vitro toxicity of complexones 5a and 5b was assayed
in cultures of C6 cells by monitoring the release of lactic
dehydrogenase (LDH) to the incubation medium (Fig.
7). Cells were incubated for 60 min with ligands 5a, 5b
Table 2. Optimized structural parameters (A) for the Gd(III) complexes of 5a–5d, EDTA and DTPA as refined by ab initio calculations
e
f
Ligand d(Gd–O1)a d(Gd–O2)a d(Gd–O3)a d(Gd–O4)a d(Gd–O5)b d(Gd–N1)c d(Gd–N2)c d(Gd–N3)d d(Gd–NAzol
)
d(Gd–Owater
)
(Lo/2)g
5a
5b
5c
5d
2.249
2.261
2.245
2.261
2.304
2.316
2.383
2.284
2.293
2.278
2.294
2.312
2.302
2.449
2.249
2.261
2.245
2.261
2.304
2.382
2.341
2.284
2.293
2.278
2.294
2.312
2.318
2.311
2.794
2.734
2.770
2.736
2.632
2.722
2.770
2.794
2.734
2.770
2.736
2.632
2.731
2.719
2.596
2.587
2.614
2.589
2.427–2.530
2.427–2.497
2.431–2.520
2.429–2.492
2.499
7.77
7.84
8.14
8.34
4.35i
EDTAh
2.482–2.858
2.595
DTPA
2.350
2.807
4.43
ad(Gd–O1À4), distance (A) from Gd to the oxygen atoms of the iminodiacetate carboxylate groups.
bd(Gd–O5), distance (A) from Gd to the oxygen atoms of the central aminoacetate carboxylate group.
cd(Gd–N1À2), distance (A) from Gd to the nitrogen atoms (N1,N2) of the iminodiacetate groups.
dd(Gd–N3), distance (A) from Gd to the nitrogen atom (N3) of the central aminoacetate group.
ed(Gd–Nazol), distance from Gd to the azolic nitrogen atom.
fd(Gd–Owater), distance from Gd(III) to the oxygen of the water molecule in complexes with q=1. In complexes where q>1, numbers indicate the
range of distances from Gd(III) to the different oxygens of water molecules.
gLo, molecular diameter.
hUpper numbers refer to Gd(III)EDTA–2H2O and lower numbers refer to Gd(III) EDTA–3H2O.
i4.89 A for HEDTA3À ligand.

E. P. Mayoral et al. / Bioorg. Med. Chem. 11 (2003) 5555–5567
5561
Table 3. Longitudinal and transversal relaxation times (T1 and T2) and relaxivities (r1 and r2) of aqueous solutions of 5a–d, EDTA, DTPA and
IDA and of the corresponding Gd(III) complexes determined at 1.5 Teslaa
Additions to
model solutionb
T1 (s)c
T2 (s)c
Ion r1 (sÀ1 mMÀ1
)
mol r1 (sÀ1 mMÀ1
)
Ion r2 (sÀ1 mMÀ1
)
Mol r2 (sÀ1 mMÀ1
)
None
Gd(III)
5a
5a+Gd(III)
5b
5b+Gd(III)
5c
5c+Gd(III)
5d
5d +Gd(III)
EDTA
EDTA+Gd(III)
DTPA
DTPA+Gd(III)
3.81Æ0.06
0.22Æ0.001
3.84Æ0.006
0.16Æ0.005
3.6Æ0.1
2.10Æ0.01
0.17Æ0.002
2.30Æ0.005
0.15Æ0.001
2.07Æ0.01
0.11Æ0.001
2.39Æ0.01
0.06Æ0.001
2.36Æ0.01
0.10Æ0.001
2.04Æ0.001
0.30Æ0.001
1.39Æ0.001
0.35Æ0.001
0.05Æ0.001
1.29 Æ0.001
0.36Æ0.002
8.3Æ0.02
12.0Æ0.4
14.6Æ0.08
18.5Æ0.1
17.7Æ0.1
5.2Æ0.02
8.3Æ0.02
24.0Æ0.8
29.2Æ0.1
37.0Æ0.2
35.4Æ0.2
5.2Æ0.02
10.8Æ0.1
12.2Æ0.1
16.9Æ0.03
35.0Æ0.01
20.0Æ0.02
5.6Æ0.01
10.8Æ0.1
24.4Æ0.2
0.14Æ0.001
3.26Æ0.08
0.10Æ0.005
3.80Æ0.02
0.11Æ0.001
3.81Æ0.01
0.35Æ0.001
3.61Æ0.03
0.41Æ0.001
0.06Æ0.01
3.60Æ0.2
33.80Æ0.06
70.0Æ0.01
40.0Æ0.04
5.6Æ0.01
4.3Æ0.01
4.3Æ0.01
4.3Æ0.01
4.3Æ0.01
3.5Æ0.03d
3.5Æ0.03d
3.6Æ0.02d
3.6Æ0.02d
IDA
IDA+Gd(III)
0.45Æ0.002
3.9Æ0.04
3.9Æ0.04
4.0Æ0.01
4.0Æ0.01
aDetermined in a Bruker Minispec 1.5 T using the inversion recovery (T1) and Carr Purcell Meiboom Gill (T2) sequences as described in the
Experimental.
bModel solutions contained 100 mM Tris/HCl (pH 6.5, 37 ꢀC), 155 mM NaCl and where indicated 5 mM complexone or 5 mM complexone and 0.5
mM Gd(III).
cResults are expressed as meanÆSD of at least three independent T1 or T2 measurements in each sample.
dSolution contained 5 mM pure complex Gd(III)DTPA in distilled water.
Solomon–Bloembergen–Morgan theory of paramagnetic
relaxation3,8,40 and MO calculations, respectively.32
Considering inner sphere effects only, the theory of
paramagnetic relaxation indicates that longitudinal (r1)
and transversal (r2) relaxivities of a Gd(III) complex are
described by the expressions;
r1 ¼ 1=T1IS ¼ q Á Pm=ðT1M þ ꢀmÞ
ð1Þ
À
Á
r2 ¼ 1=T2IS
ꢀ
ꢁ
À
Á À
Á2
¼ Pm=ꢀm T2MÀ2 þꢀmÀ1 þD!m2 = ꢀmÀ1 þT2MÀ1 þD!2m
ð2Þ
where q is the number of coordinated water mole-
cules to Gd(III), Pm represents the mole fraction of
bound water nuclei, T1M or T2M are the T1 or T2 of
bound water molecules, tm is the residence time of
the water molecules in the inner sphere of the
Gd(III) complex and Áo2m refers to the squared differ-
ence of chemical shifts between metal bound and bulk
water molecules.
Figure 7. Release of LDH to the incubation medium in cultures of C6
cells incubated with increasing concentrations of Gd(III) and com-
plexones 5a, 5c or EDTA.
and EDTA in the absence of Gd(III) or in the presence
of increasing concentrations of Gd(III) and complex-
ones. The release of LDH induced by the Gd(III) com-
plexes of 5a or 5b was smaller than that induced by
Gd(III)EDTA, indicating a lower toxicity.
Longitudinal relaxivities r1 of different Gd(III) com-
plexes are dominated normally, for the same Pm, by the
hydration number q and the value of T1M (ms or s
range), since tm values are much shorter (ns range). At
clinical fields, T1M is approximated by the expression;
Discussion
Causes of improved relaxivity
Â
À
ÁÃ
1=T1M ¼ K=r6 3ꢀc= 1 þ !i2ꢀc2
ð3Þ
The heterocyclic complexones prepared in this study
present important improvements in relaxivity as com-
pared to the earlier series.12 It is possible to understand
this on the basis of the dynamics and structure of the
corresponding Gd(III) complexes as described by the
where K is a constant, r is the distance between the
unpaired electrons of Gd(III) and the hydrogens of the
coordinated water molecule, oi is the observation fre-

5562
E. P. Mayoral et al. / Bioorg. Med. Chem. 11 (2003) 5555–5567
quency and tc is the reorientational correlation time of
dipolar interaction between the unpaired electron and
the water molecule described by
Therefore, from the known values of tr=51 ps of
Gd(III)DTPA3 and molecular radii of the Gd(III)
complexes of DTPA34 and 5a–d (Lo/2 in Table 2) it is
possible to calculate using 6 the values of tr (ps) for
complexes of 5a (303), 5b (310), 5c (345), 5d (370). These
calculated tr values of 5a–d Gd(III) chelates are rather
similar to those measured for much larger molecular
weight linear polymers of Gd(III)DTPA (232 ps),8,10,42
sugar derivatives as Gd(III)DTPA-BENGALAA (265
ps)43 and slightly smaller than those measured for some
Gd(III)DO3A dendrimers (580–870 ps).44 It is interest-
ing to note that the calculated tr values of the series
increase in the order 5a<5b<5c<5d in parallel with the
r1 relaxivities shown in Table 3. For the same hydration
value through the series, this trend suggests an impor-
tant contribution of tr to the observed r1. Indeed, mea-
sured r1 values of the heterocyclic complexones reported
here, are similar to those of ca. 16–18 sÀ1 mMÀ1 of tex-
aphyrins, a series of porphyrin analogues with similarly
high hydration number (q=3.5) and tr (ca. 295 ps).20,45
However, although the effects of q and tr appear to
dominate the observed increases in relaxivity, additional
contributions from more favorable tm or T1e values or
the formation of intermolecular Gd(III) complexes
involving more than one complexone molecule may also
be considered.11
1=ꢀc ¼ 1=T1e þ 1=ꢀm þ 1=ꢀr
ð4Þ
In this expression, T1e is the relaxation time of the
unpaired electron (1<T1e<10 ns), tm is the residence
time of water in the complex (100<tm<2500 ns) and tr
is the rotational correlation time of the complex
(50<tr<500 ps).3,8
Concerning the hydration number q, the Gd(III) che-
lates of 5a–d have q=4 (c.f. Fig. 6), as compared to
q=9 in the aqua Gd(III), q=5 in Gd(III)IDA, q=4 in
Gd(III)NOVAN41 or q=1 in Gd(III)DTPA or Gd(III)-
DOTA complexes.3,8 However, it is possible to show
here that the increased relaxivity observed in the Gd(III)
complexes of 5a–d as compared to Gd(III)DTPA or
Gd(III)DOTA is not exclusively due to their increased
hydration. Indeed, r1 values of bis- and bi-azolic Gd(III)
complexes vary importantly within the series
(12.0<r1<18.5 sÀ1 mMÀ1, Table 3), while the hydra-
tion number of the different complexes remains the
same. Moreover, the Gd(III) complexes 5a–d depict
higher r1 than those of molecules with even higher
hidration numbers such as the nonahydrated Gd(III)
aqua complex and the pentahydrated Gd(III)IDA com-
plex (Table 3). Thus, the increased r1 values observed
must include additional contributions of T1M, tm or
both (c.f. 1).
The particularly large values of transversal relaxivity r2
determined for the Gd(III) complexes 5a–d merit special
consideration. In addition to the circumstances affecting
r1, the squared Áo2 term containing the added differ-
ences in chemical shifts between the four bound water
molecules and the bulk solvent (c.f. eq 2), must con-
tribute importantly as described for the former series
1–4.12 In this respect, the particularly large r2 relaxivity
found in Gd(III)5c (ca. 70 sÀ1 mMÀ1) is remarkable.
This effect may involve; (i) a higher polarization of the
four water molecules bound to Gd(III) due to the elec-
tron withdrawal effects of the p-nitro group and (ii) a
more favorable water exchange in a less crowded coor-
dination cavity as revealed by the longer distances cal-
culated from Gd(III) to the benzene ring.
The possible changes in T1M may include modifications
in r or tc (c.f. 3). The value of r influences greatly r1
since it enters eq 3 as the sixth power. The calculated
distances r between Gd(III) and the oxygen atom of
the water molecules in the Gd(III) complexes 5a–d are
similar to those found in Gd(III)DTPA complex
(Table 2), suggesting that distances from Gd(III) to
the hydrogens of water molecules are in the same
range also. Therefore, a significant decrease in r can
be discarded as a relevant contribution to increased
relaxivity in these Gd(III) complexes. Consequently,
the dominant change in T1M must occur in tc (c.f. 3
and 4), which at 1.5 Tesla is normally controlled by
the value of the rotational correlation time tr expres-
sed as:
Considerations on Gd(III) binding
Present results reveal also some interesting aspects on
the relationship between chemical structure and ther-
modynamic stability of heterocyclic Gd(III) complexes.
In particular, cooperative Gd(III) binding to the same
complexone molecule represents to our knowledge, a
novel property of these ligands. The S0.5 values of 5a
and 5b are lower than those of 5c and 5d, suggesting
that the presence of the methylene bridge decreases the
stability of the Gd(III) complexes. Complexones 5a and
5c depicted higher cooperativity values than 5b and 5d,
indicating that participation of the phenyl rings in both
chelation centers favors an easier folding of the com-
plex. In general, compounds 5a–d show significantly
improved Gd(III) binding capacity as compared to the
earlier series of azolic complexones.12 However, their
S0.5 values remain well below the stability constants for
Gd(III) of classical complexones such as DTPA or
DOTA.3,4
ꢀr ¼ 4ꢁa3ꢂ=3kT
ð5Þ
where a is the molecular radius of gyration, Z the micro-
viscosity, k the Boltzman constant and T the absolute
temperature. It is possible to compare the rotational
correlation times of two molecules tr1 and tr2 with
molecular radii a1 and a2, using expression 6 under
the assumption that both molecules behave as rigid
rotors in a medium of identical microviscosity and
temperature;
ꢀr1=ꢀr2 ¼ a31=a32
ð6Þ

E. P. Mayoral et al. / Bioorg. Med. Chem. 11 (2003) 5555–5567
5563
Concluding remarks
7.44 (s, 1H, CH¼C), 7.40 (s, 1H, CH¼C), 3.66 (s, 2H,
CH2).
In summary, we described the syntheses, physicochem-
ical properties and toxicological evaluation of a novel
series of heterocyclic complexones. These ligands appear
to form two intramolecular Gd(III) complexes depicting
large r1 and r2 relaxivity values but their unfavorable
binding properties for Gd(III) do not advise their use as
Gd(III) chelators in the clinic. However, they may
become useful clinical chelators of less toxic metals
similar to dipyridoxaldiphosphate DPDP,46,47 a ligand
currently employed in the clinic to provide delayed
To a solution of tosylhydrazine (1.55 g; 8.36 mmol) in
MeOH (10 mL) was added 16 (1.53 g; 4.18 mmol, mix-
ture of E,E/E,Z isomers) and the reaction mixture was
refluxed for 1 h and 30 min. After cooling, the solid was
filtered to give 17 (1.71 g; 58%, only E,Z isomer) as a
yellow solid (mp 208–210 ꢀC, MeOH). IR(KBr): n 3480,
3200, 1590, 1505, 1340, 1160, 1065 cmÀ1 1H NMR
.
(200 MHz, DMSO-d6, d): 8.15 (d, 2H, J=7.6 Hz,
AA0XX0 system, aromatics), 8.10 (d, 2H, J=7.6 Hz,
AA0XX0 system, aromatics), 7.91 (s, 1H, CH¼C), 7.73
(s, 1H, CH¼C), 7.69 (d, 2H, J=8.2 Hz, AA0XX0 sys-
tem, aromatics), 7.48–7.40 (2 d, 4H, AA0XX0 system,
aromatics), 7.32 (d, 2H, J=8.3 Hz, AA0XX0 system,
aromatics), 7.17 (s, 1H, CH¼N), 7.13–7.06 (2 d, 4H,
AA0XX0 system, aromatics), 6.31 (s, 1H, CH¼N), 3.48
(s, 2H, CH2), 2.26 (s, 3H, CH3), 2.09 (s, 3H, CH3).
release and organ selectivity of Mn2+
.
Experimental
General
Melting points were obtained on a microscope hot stage
and are uncorrected. Elemental analyses were per-
formed with a Perkin-Elmer 240 apparatus. Mass spec-
tra were carried out on a GC/mass spectrometer
Schimadzu QP-5000 at 70 eV. IRspectra were deter-
mined on a Philips PU-9700 spectrophotometer. NMR
A mixture of 17 (3.17 g; 4.51 mmol), sodium methoxide
(1.1 g; 20.3 mmol) and triethylen glycol (7 mL) was
heated at 100 ꢀC for 2 h. After cooling, the reaction
crude was poured into ice-water and AcOH (3 mL). The
solid obtained was filtered, and washed with saturated
solution of NaHCO3, H2O and MeOH. Finally, the
solvent of the filtrate was removed in vacuo to give 8
(0.56 g; 33%) as a brown solid (mp 129–140 ꢀC,
decomp) and 1.2 g of polymeric material. IR(KBr): n
3390, 3210, 1595, 1505, 1335, 1855 cmÀ1. MS m/z (%):
spectra were obtained with
a Bruker DRX-400
(400.13 MHz for 1H, and 100.03 MHz for 13C) and
Bruker AC-200 (200.13 MHz for 1H, and 50.33 MHz for
1
13C). H and 13C chemical shifts (d) in CDCl3 are given
from internal tetramethylsylane and 13C chemical shifts
1
(d) in D2O are given from external DMSO-d6 with an
390 (M+, 100), 168 (95). H NMR(400 MHz, DMSO-
accuracy ofÆ0.01 ppm for H and Æ0.1 ppm for 13C.
d6, d): 8.21 (d, 4H, J=8.8 Hz, AA0XX0 system, aro-
matics), 7.87 (d, 4H, J=8.8 Hz, AA0XX0 system, aro-
matics), 7.46 (s, 2H, H3), 4.09 (s, 2H, CH2).
1
1
The residual water signal in H NMRspectra obtained
in D2O solution was suppressed when necessary using a
1 s (low power, 0.5 watts) presaturating pulse applied
1
with decoupler. H–1H coupling constants (J) are accu-
Alkylation of bi and bispyrazoles with 1,2-dibromoethane
1
rate to Æ0.2 Hz for H NMRspectra. TLC chromato-
graphy was performed on DC-Aulofolien/Kieselgel 60
F245 (Merck) and column chromatography through
silica gel Merck 60 (230–400 mesh). Methyl iminodia-
cetate hydrochloride was basified with solid Na2CO3 in
the minimum amount of water prior to use. D2O (99.9
D) was purchased from Appollo Scientific (Stockport,
UK). The rest of the products were obtained from
Aldrich.
General procedure. A mixture of bi- or bispyrazole (1
equivalent), (40%) NaOH (3 equivalents), BTBA (0.025
equivalents) and dibromoethane (10 equivalents) was
refluxed until the consumption of the starting material
was detected by TLC. The organic layer was then sepa-
rated and the aqueous layer was extracted with CH2Cl2.
The combined organic extracts were washed with water,
dried over MgSO4 and evaporated in vacuo. The residue
was purified by column chromatography on silica gel.
4,40-Methylenebis[5-(4-nitrophenyl)-1H-pyrazole] (8). A
mixture of p-nitrobenzaldehyde (4.53 g; 30 mmol),
2-methoxy-2,3-dihydro-4H-pyrane (1.82 g; 16 mmol),
AcOH (2 mL), H2O (3 mL) and piperidine (two drops)
was refluxed for 24 h. After cooling, the aqueous layer
was decanted and the solvent was evaporated in vacuo.
The residue was purified by column chromatography on
silica gel. Elution with hexane/AcOEt (85:15) gave 16
(1.02 g; 19 %) as a white solid (E,E;24 E,Z isomers). E,Z
3,30-Diphenyl-1,10-bis(2-bromoethyl)-4,40-bipyrazole (9a).
According to the general procedure we used bipyrazole
6 (2.2 g; 7.69 mmol), NaOH (0.92 g; 23.07 mmol),
BTBA (62 mg; 0.19 mmol), 1,2-dibromoethane (23.13 g;
123.04 mmol) and H2O (1.5 mL) and the mixture was
refluxed for 1 h and 30 min. Elution with CH2Cl2/
MeOH (98:2) gave 9a (1.17 g; 30%) as a white solid (mp
137–139 ꢀC, EtOH), 9b (0.69 g; 18%) as a yellow oil and
9c (0.25 g; 6%) as a white solid (mp 170–175 ꢀC,
CH2Cl2/hexane). 9a: IR(KBr): n 1600, 1515, 1510,
1440, 1415, 1350, 1320, 1270, 1180, 950, 775, 720, 695
cmÀ1. MS m/z (%): 502 (M++2, 31), 500 (M+, 68), 498
(M+À2, 32), 233 (100), 77 (10), 63 (51). 1H NMR
(200 MHz, CDCl3, d): 7.61–7.48 (m, 4H, aromatics),
7.28 (m, 2H, H5), 7.25–7.16 (m, 6H, aromatics), 4.46 (t,
4H, J=6.2 Hz, CH2–N(Azole)), 3.74 (t, 4H, J=6.2 Hz,
isomer (mp 143–145 ꢀC AcOEt/hexane): IR(KBr):
n
1685, 1605, 1510 cmÀ1. MS m/z (%): 366 (M+, 12), 338
(32), 321 (24), 215 (53), 202 (91), 189 (21), 161 (31), 128
(27), 115 (100), 89 (34), 77 (31), 63 (30). 1H NMR
(200 MHz, CDCl3, d): 9.86 (s, 1H, CHO), 9.74 (s, 1H,
CHO), 8.32 (d, 2H, J=8.8 Hz, AA0XX0 system, aro-
matics), 8.24 (d, 2H, J=8.8 Hz, AA0XX0 system, aro-
matics), 7.68–7.60 (2 d, 4H, AA0XX0 system, aromatics),

5564
E. P. Mayoral et al. / Bioorg. Med. Chem. 11 (2003) 5555–5567
CH2-Br); 13C NMR(100 MHz, CDCl , d): 150.6, 133.2,
identified as compound 11b and 13 mg of product 11c.
Further purification of these products was not pursued.
3
131.5, 128.2, 127.5, 110.6, 53.6, 30.4. Anal. calcd for
C22H20Br2N4: C, 52.81; H, 4.04; N, 11.20. Found: C,
53.03; H, 4.23; N, 11.08. 9b: IR(KBr): n 1495, 1445,
1305, 1215, 940, 760, 700 cmÀ1. MS: m/z (%): 502
Reaction of haloethyl derivatives of bi- or bispyrazole
with methyl iminodiacetate
(M++2, 47), 500 (M+, 100), 498 (M+À2, 48), 392 (20),
1
286 (39), 107 (26), 77 (41). H NMR(200 MHz, CDCl ,
3
General procedure. A mixture of the corresponding
haloethyl derivative (1 equivalent) and methyl imino-
diacetate (2 equivalents) was heated at 110 ꢀC until the
consumption of the starting material was detected by
TLC. After cooling, the mixture was extracted with
CH2Cl2 and the organic layer was dried over MgSO4.
Organic solvent was evaporated in vacuo and the resi-
due was purified by column chromatography on silica
gel.
d): 7.45 (s, 1H, H3), 7.44–7.38 (m, 2H, aromatics), 7.31
(m, 6H, aromatics), 7.13–7.08 (m, 3H, aromatics and
H5), 4.36 (t, 2H, J=6.4 Hz, CH2–N(Azol-H5)), 4.35 (t,
2H, J=6.8 Hz, CH2–N(Azol-H3)), 3.67 (t, 2H, J=6.8
Hz, BrCH2CH2–N(Azol-H3)), 3.65 (t, 2H, J=6.4 Hz,
BrCH2CH2–N(Azol-H5)); 13C NMR(100 MHz, CDCl ,
3
d): 150.6, 143.6, 141.5, 139.7, 133.3, 130.6, 129.9, 129.3,
128.6, 128.1, 127.7, 127.4, 112.2, 110.6, 53.4, 50.3, 30.3,
29.7. 9c: IR(KBr): n 1485, 1440, 1395, 1325, 1285, 1230,
970, 935, 860, 790, 750, 700, 630 cmÀ1. MS m/z (%): 502
[(2-{10 -[2-(Bismethoxycarbonylmethylamino)ethyl]-3,30 -
diphenyl-10H-[4,40]bipyrazolyl-1-yl}ethyl)methoxycarbo-
nylmethylamino] acetic acid methyl ester (12). Accord-
ing to the general procedure we used 9a (1 g; 2 mmol)
and methyl iminodiacetate (1.29 g; 8 mmol) the mixture
being heated at 110 ꢀC for 8 h and 15 min. Elution with
CH2Cl2/MeOH (98:2) gave 12 (0.844 g; 65%) as a col-
orless oil. IR(KBr): n 1750, 1450, 1210, 1190, 1160
cmÀ1. MS m/z (%): 660 (M+, 15), 601 (12), 587 (23),
286 (25), 174 (100), 128 (43), 116 (35). 1H NMR
(200 MHz, CDCl3, d): 7.52–7.47 (m, 4H, aromatics),
7.34 (s, 2H, H5), 7.20–7.13 (m, 6H, aromatics), 4.20 (t,
4H, J=6.2 Hz, CH2–N(Azole)), 3.61 (s, 12H, CH3),
3.45 (s, 8H, CH2–CO2Me), 3.22 (t, 4H, J=6.2 Hz,
(M++2, 46), 500 (M+, 100), 498 (M+À2, 46), 392 (17),
1
286 (68), 107 (49), 77 (37). H NMR(200 MHz, CDCl ,
3
d): 7.33–7.21 (m, 8H, aromatics and H3), 7.03–6.99 (m,
4H, aromatics), 4.23 (t, 4H, J=6.9 Hz, CH2–N(Azole)),
3.56 (t, 4H, J=6.9 Hz, CH2-Br); 13C NMR(100 MHz,
CDCl3, d): 140.8, 138.7, 129.9, 129.6, 128.7, 128.6,
112.2, 50.1, 29.3.
1,10-Bis(2-bromoethyl)-3,30,5,50 -tetramethyl-4,40 -bipyra-
zole (10). According to the general procedure, we
employed bipyrazole 7 (0.96 g; 5.05 mmol), NaOH (0.6 g;
15.2 mmol), BTBA (40 mg; 0.26 mmol), 1,2-dibromo-
ethane (15.03 g; 80.4 mmol) and H2O (1.5 mL) and the
mixture was refluxed for 1 h. Elution with CH2Cl2/
MeOH (98:2) gave 10 (0.22 g; 11%) as a yellow solid
(mp 112–114 ꢀC, hexane) and 15 (0.15 g; 8%) as a yel-
low oil. 10: IR(KBr): n 1735, 1620, 1540, 1500, 1460,
1435, 1400, 1310, 1270, 1130, 955, 900, 825, 720, 640
cmÀ1. MS m/z (%): 404 (M+, 36), 296 (15), 229 (14),
190 (100), 107 (29). 1H NMR(400 MHz, CDCl 3, d):
4.27 (t, 4H, J=6.7 Hz, CH2–N(Azole)), 3.62 (t, 4H,
J=6.7 Hz, CH2–Br), 1.98 (s, 6H, CH3), 1.93 (s, 6H,
CH3); 13C NMR(100 MHz, CDCl 3, d): 147.6, 137.6,
109.7, 49.6, 30.2, 11.9, 9.7. Anal. calcd for
C14H20Br2N4: C, 41.60; H, 5.00; N, 13.86. Found: C,
42.08; H, 5.11; N, 13.77. 15: 1H NMR(200 MHz,
CDCl3, d): 4.42 (t, 2H, J=6.4 Hz, CH2–N(Azole)), 3.76
(t, 2H, J=6.5 Hz, CH2–Br), 2.15 (s, 6H, CH3), 1.98 (s,
3H, CH3), 1.93 (s, 3H, CH3).
CH2–N); 13C NMR(100 MHz, CDCl , d): 171.6, 149.7,
3
133.6, 131.7, 128.1, 127.3, 127.2, 110.8, 55.6, 54.9, 51.7,
.
.
51.5. Anal. calcd for C34H40N6O8 C12H6N6O14 (12 2
picric acid): C, 49.47; H, 4.16; N, 15.05. Found: C,
50.08; H, 4.54; N, 14.58.
[(2 - {10 - [2 - (Bismethoxycarbonylmethylamino)ethyl] -
3,5,30,50-tetramethyl-10H-[4,40]bipyrazolyl-1-yl}ethyl)me-
thoxycarbonylmethylamino] acetic acid methyl ester
(13). According to the general procedure we used 10
(0.31 g; 0.76 mmol) and methyl iminodiacetate (0.39 g;
3.03 mmol) and the mixture was heated at 110 ꢀC for 2
h. Elution with AcOEt/hexane (1:1) gave 13 (0.22 g;
51%) as a colorless oil. IR(film): n 1735, 1420, 1200,
1020, 745 cmÀ1. MS m/z (%): 564 (M+, 20), 187 (68),
174 (100), 146 (54), 128 (91). 1H NMR(400 MHz,
CDCl3, d): 4.08 (t, 4H, J=6.7 Hz, CH2–N(Azole)), 3.61
(s, 12H, OCH3), 3.41 (s, 8H, CH2CO2Me), 3.09 (t, 4H,
J=6.7 Hz, CH2–N), 2.00 (s, 6H, CH3), 1.95 (s, 6H,
CH3); 13C NMR(100 MHz, CDCl 3, d): 171.5, 147.1,
138.0, 110.0, 55.5, 54.5, 51.5, 48.4, 12.2, 9.8.
4,40-Methylenebis[1-(2-bromoethyl)-3-(4-nitrophenyl)pyr-
azole] (11a). According to the general procedure we
employed bispyrazole 8 (575 mg; 1.47 mmol), NaOH
(176 mg; 4.41 mmol), BTBA (12 mg; 0.037 mmol),
1,2-dibromoethane (2 mL; 23.52 mmol) and the mixture
was refluxed for 2 h. Elution with hexane/AcOEt (98:2)
gave 11a (115 mg; 13%) as a white solid (mp
270–272 ꢀC, CH2Cl2/hexane). IR(KBr): n 1595, 1500,
1330, 1110, 855, 705 cmÀ1. MS m/z (%): 606 (M++2,
25), 604 (M+, 49), 602 (M+À2, 24), 107 (58), 80 (100).
1H NMR(400 MHz, CDCl 3, d): 8.23 (d, 4H, J=8.9 Hz,
AA0XX0 system, aromatics), 7.80 (d, 4H, J=8.9 Hz,
AA0XX0 system, aromatics), 7.28 (s, 2H, H5), 4.49 (t,
4H, J=6.0 Hz, CH2–N(Azole)), 4.04 (s, 2H, CH2), 3.76
(t, 4H, J=6.0 Hz, CH2–Br). One further elution with
the same eluent gave 64 mg of a product which could be
({2-[10 -[2-(Bismethoxycarbonylmethylamino)ethyl]-3,30 -
bis(4 -nitrophenyl) -10H -[4,40]bipyrazolyl-1-yl]ethyl}me-
thoxycarbonylmethylamino) acetic acid methyl ester
(14). According to the general procedure we employed
11a (33 mg; 0.055 mmol) and methyl iminodiacetate (39
mg; 0.24 mmol) and the mixture was heated at 110 ꢀC
for 5 h. Elution with CH2Cl2/EtOH (99:1) gave 14 (28
1
mg; 67%) as a yellow oil. H NMR(200 MHz, CDCl ,
3
d): 8.21 (d, 4H, J=8.9 Hz, AA0XX0 system, aromatics),
7.79 (d, 4H, J=8.7 Hz, AA0XX0 system, aromatics),
7.38 (s, 2H, H5), 4.22 (t, 4H, CH2–N(Azole)), 4.02 (s,

E. P. Mayoral et al. / Bioorg. Med. Chem. 11 (2003) 5555–5567
5565
2H, CH2), 3.66 (s, 12H, OCH3), 3.46 (s, 8H,
CH2CO2CH3), 3.22 (t, 4H, CH2–Br).
AA0XX0 system, aromatics), 7.52 (s, 2H, H5), 7.39 (d,
4H, J=8.7 Hz, AA0XX0 system, aromatics), 4.11 (t, 4H,
CH2–N(Azole)), 3.98 (s, 2H, CH2), 3.08 (s, 8H,
CH2CO2Na), 2.84 (t, 4H, CH2N).
{[2-(4-{1-[2-(Bismethoxycarbonylmethylamino)ethyl]-3,5-
dimethyl-1H-pyrazol-4-ylmethyl}3,5-dimethylpyrazol-1-
yl)ethyl]methoxycarbonylmethylamino}
acetic
acid
{[2-(4-{1-[2-(Biscarboxymethylamino)ethyl]-3,5-dimethyl
-1H-pyrazol-4-ylmethyl}-3,5-dimethylpyrazol-1-yl)ethyl]-
carboxymethylamino} acetic acid tetrasodium salt (5d).
According to the general procedure we used 14 (81 mg;
0.14 mmol) and NaOH (22.5 mg; 0.56 mmol). The
reaction mixture was stirred at room temperature for 18
methyl ester (22). According to the general procedure
we used 20 (340 mg; 1.033 mmol) and methyl iminodia-
cetate (733 mg; 4.55 mmol) and the mixture was heated
at 110 ꢀC for 24 h. Elution with CH2Cl2/EtOH (98:2)
gave 22 (118 mg; 20%) as a yellow oil. IR(film): n 1725,
1420, 1210, 915, 735 cmÀ1. MS m/z (%): 578 (M+, 2),
h to give 5d (80 mg; 94%) as a white solid. IR(KBr): n
1
505 (3), 187 (24), 174 (60), 146 (40), 128 (62), 114 (43),
1
3440, 1600, 1410 cmÀ1. H NMR(200 MHz, D O, d):
2
86 (38). H NMR(400 MHz, CDCl , d): 3.95 (t, 4H,
4.02 (t, 4H, J=7.4 Hz, CH2–N(Azole)), 3.36 (s, 2H,
CH2), 3.14 (s, 8H, CH2CO2Na), 2.79 (t, 4H, J=7.3 Hz,
CH2–N), 2.05 (s, 6H, CH3), 1.84 (s, 6H, CH3); 13C
3
J=6.5 Hz, CH2–N(Azole)), 3.57 (s, 12H, OCH3), 3.41
(s, 8H, CH2CO2Me), 3.27 (s, 2H, CH2), 2.90 (t, 4H,
J=6.6 Hz, CH2–N), 2.05 (s, 6H, CH3), 1.86 (s, 6H,
CH3); 13C NMR(100 MHz, CDCl 3, d): 171.5, 146.0,
136.1, 114.0, 55.4, 54.6, 51.4, 47.8, 18.1, 11.9, 9.3.
NMR(100 MHz, D O, d): 178.3, 146.2, 137.6, 113.5,
2
57.7, 52.7, 45.4, 16.1, 9.8, 7.7.
[1-(2-Chloroethyl)-3,5-dimethyl-1H-pyrazol-4-yl]metha-
nol (19). A stream of dry hydrogen chloride was passed
through a solution of 1812 (1.0 g; 6.3 mmol), paraf-
ormaldehyde (0.23 g) in 1,2-dichloroethane (5 mL) for 2
h. Subsequently, the reaction mixture was refluxed for 2
h. Concd HCl (5 mL) was added and the water layer
was made alkaline with Na2CO3 and extracted with
CH2Cl2. The combined organic extracts were washed
with water, dried over MgSO4 and evaporated in vacuo.
The residue was purified by recrystallization to give 19
(763 mg; 67%) as a white solid (mp 95–97 ꢀC, CH2Cl2/
Hexane). IR(KBr): n 3260, 1565, 1475, 1450, 1325,
1300, 995, 765 cmÀ1. MS m/z (%): 190 (M++2, 18),
188 (M+, 54), 171 (52), 139 (100), 126 (49), 109 (60), 97
(19), 56 (23). 1H NMR(400 MHz, CDCl 3, d): 4.47 (s br,
2H, CH2O), 4.25 (t, 2H, J=6.4 Hz, CH2–N(Azole)),
3.84 (t, 2H, J=6.3 Hz, CH2Cl), 2.28 (s, 3H, CH3), 2.24
Basic hydrolysis
General procedure. A mixture of corresponding ester (1
equivalent) and (0.6%; H2O MQ) NaOH (4 equivalents)
was stirred at room temperature or heated at 50–60 ꢀC
until consumption of the starting material was detected
by TLC. After cooling, the reaction mixture was washed
with CH2Cl2 and the water was removed in vacuo.
[(2-{10 -[2-(Biscarboxymethylamino)ethyl]-3,30 -diphenyl-
10H - [4,40]bipyrazolyl - 1 - yl} - ethyl)carboxymethylamino]
acetic acid tetrasodium salt (5a). According to the gen-
eral procedure we used 12 (800 mg; 1.21 mmol) and
NaOH (194 mg; 4.85 mmol). The mixture was stirred at
room temperature for 48 h to give 5a (772 mg; 92%) as
a white solid. IR(KBr): n 2150, 1575, 1405 cmÀ1. H
NMR(200 MHz, D O, d): 7.62 (s, 2H, H5), 7.12 (s br,
1
(s, 3H, CH3); 13C NMR(100 MHz, CDCl , d): 147.6,
2
3
10H, aromatics), 4.20 (t, 4H, CH2–N(Azole)), 3.12 (s,
8H, CH2–CO2Na), 3.01 (t, 4H, CH2–N); 13C NMR
(100 MHz, D2O, d): 177.4, 149.4, 131.8, 131.3, 127.4,
126.9, 126.6, 109.6, 57.5, 53.4, 48.8, 48.6.
138.4, 116.0, 54.7, 49.7, 42.7, 11.5, 9.4. Anal. calcd for
C8H13ClN2O: C, 50.92; H, 6.95; N, 14.85. Found: C,
51.12; H, 6.85; N, 14.91. Traces of ether 20 were isolated
in this reaction. MS m/z (%): 358 (M+, 2), 200 (25), 171
(100), 109 (29).
[(2-{10 -[2-(Biscarboxymethylamino)ethyl]-3,5,30,50 -tetra-
methyl-10H-[4,40]bipyrazolyl-1-yl}ethyl)carboxymethyla-
mino] acetic acid tetrasodium salt (5b). According to the
general procedure we used 13 (800 mg; 1.4 mmol) and
NaOH (224 mg; 5.6 mmol). The mixture was stirred at
room temperature for 72 h to give 5b (768 mg; 90%) as
4,40-Methylenebis[1-(2-chloroethyl)-3,5-dimethylpyrazole]
(20). A suspension of 19 (990 mg; 5.25 mmol) in concd
HCl (0.5 mL) was refluxed for 1 h. After cooling was
added of H2O (10 mL), neutralized with Na2CO3,
extracted with CH2Cl2 and organic layer was dried over
MgSO4. The organic solvent was evaporated in vacuo to
give 10 (680 mg; 79%) as a white solid (mp 99–101 ꢀC,
CH2Cl2/hexane). IR(KBr): n 1555, 1465, 1425, 1380,
1310, 1280, 745, 655 cmÀ1. MS m/z (%): 328 (M+, 19),
313 (88), 279 (4), 171 (100), 135 (21), 108 (15). 1H NMR
(200 MHz, CDCl3, d): 4.23 (t, 4H, J=6.2 Hz, CH2–N),
3.80 (t, 4H, J=6.2 Hz, CH2Cl), 3.39 (s, 2H, CH2), 2.10
(s, 6H, CH3), 2.03 (s, 6H, CH3); 13C NMR(100 MHz,
CDCl3, d): 147.0, 136.5, 114.0, 49.6, 43.0, 18.1, 12.0, 9.5.
Anal. calcd for C15H22Cl2N4: C, 54.70; H, 6.75; N,
17.02. Found: C, 54.64; H, 6.66; N, 16.94.
1
a white solid. IR(KBr): n 1590, 1310 cmÀ1. H NMR
(400 MHz, D2O, d): 3.93 (t, 4H, J=7.5 Hz, CH2–
N(Azole)), 2.91 (s, 8H, CH2CO2Na), 2.68 (t, 4H, J=7.5
Hz, CH2–N), 1.77 (s, 6H, CH3), 1.69 (s, 6H, CH3); 13C
NMR(100 MHz, D O, d): 180.4, 149.2, 141.2, 111.2,
2
60.4, 55.3, 48.2, 12.7, 10.8.
({2-[10-[2-(Biscarboxymethylamino)ethyl]-3,30-bis(4-nitro-
phenyl)-10H-[4,40]bipyrazolyl-1-yl]ethyl}carboxymethyla-
mino) acetic acid tetrasodium salt (5c). According to the
general procedure we used 14 (28 mg; 0.037 mmol) and
NaOH (6 mg; 0.148 mmol). The reaction mixture was
heated at 60 ꢀC for 31 h to give 5c (23 mg; 78%) as a
Gd(III) binding studies. Binding isotherms (22 ꢀC, pH
6.5) of Gd(III) to ArsIII and to the different complex-
ones were determined spectrophotometrically at 680 nm
yellow solid. IR(KBr): n 3440, 1600, 1390, 1340 cmÀ1
1H NMR(200 MHz, D O, d): 8.06 (d, 4H, J=8.7 Hz,
.
2

5566
E. P. Mayoral et al. / Bioorg. Med. Chem. 11 (2003) 5555–5567
1
using
a
microplate spectrophotometer (Molecular
Determination of relaxivities. H NMRrelaxation times
T1 and T2 (37 ꢀC, pH=6.5) of the water protons in
aqueous solutions of complexones 5a–d containing or
not Gd(III), were measured at 1.5 Tesla in a Bruker
Minispec NMRspectrometer. T1 values were deter-
mined by the inversion-recovery method (d1-p-t-p/2-aq)
and T2 values were determined by the Carr–Purcell–
Maiboom–Gill sequence (d1-p/2-[t-p-t]n-aq) using in
both cases not less than 13 different t values. Three dif-
ferent measurements of T1 or T2 were performed in
every sample. Typically, 5 mM complexones were dis-
solved in 100 mM Tris/HCl, 150 mM NaCl containing or
not 0.5 mM Gd(III). In addition, some determinations
were performed in rat plasma. In these cases, blood was
drained from the inferior caval vein of well fed, anesthe-
tized (Nembutal 50 mg/kg), female Wistar rats (250–300
g) using an heparinized syringe. Plasma was recovered as
the supernatant of blood centrifugation at 5000g (4 ꢀC, 5
min) and added 5 mM complexone or 5 mM complexone
and 0.5 mM Gd(III) chloride. Relaxivities r1(2) were cal-
culated according to the expression:
Devices Spectramax, Sunnyvale, CA, USA). Gd(III)
chloride (up to 0.35 mM in 5-mM steps) was added into
model solutions containing 20 mM HEPES (pH 6.7),
100 mM NH4Cl and 0.1 mM ArsIII in the absence and
presence of 1 mM complexones 5a–d, monitoring at 680
nm the increase in concentration of the ArsIII–Gd(III)
complex (e=1.1 10 M cmÀ1).12,48,49 Binding simula-
tions and not linear least squares regressions were pro-
grammed using the Mathematica 4.1 program (Wolfram
Research Inc., Campaign, IL, USA) implemented on a
Pentium IV platform. Binding parameters were
obtained by non linear least squares fittings of the
titration curves to a mathematical model which con-
sidered the competition between hyperbolic binding to
ArsIII (y1) and sigmoidal binding to the complexone
(y2). Hyperbolic binding of Gd(III) to ArsIII is given by
the equation y1=A*[Gd(III)]/([Gd(III)]+Kd) where Kd
and A represent the apparent dissociation constant and
saturation values of ArsIII. Sigmoidal binding of
Gd(III) to the complexone is described by the equation
0
4
À1
.
y2=B [Gd(III)]n/([Gd(III)]n+S0.5 n)] where S
is the
0.50
À
Á
apparent Gd(III) concentration for half maximal
saturation of the complexone in the presence of com-
peting ArsIII and n is a parameter reflecting the degree
of cooperativity in the binding. The amount of Gd(III)
bound to ArsIII in the presence of complexone is given
by y=y1Ày2. It is possible to calculate the apparent
Gd(III) concentration for half maximal saturation of
the complexone in the abscence of the competing
ArsIII, S0.5 by the expression S0.5=S0.50/(1+[ArsIII]/
Kd).
r1ð2Þ ¼ D 1=T1ð2Þ =½GdðIIIÞꢀ
where, Á is the difference in longitudinal or transversal
relaxation rates (1/T1(2)) of the water protons in the
presence and absence of Gd(III), and [Gd(III)] the con-
centration of Gd(III) expressed in mM.
In vitro toxicity studies. Toxicity was investigated by
measuring the amount of intracellular LDH released to
the medium in cultures of glioma C6 cells.12 Cells were
grown to confluence in DMEM medium containing 5%
fetal calf serum and incubated for one h in the absence
and presence of increasing concentrations of Gd(III)
and 5a, 5c and EDTA (Fig. 7). The amount of LDH
released to the medium was measured spectro-
photometrically in a microplate reader (Molecular
Devices Spectramax, Sunnyvale, CA, USA) at 340 nm,
using an incubation mixture containing 50 mM HEPES
pH 7.2, 5 mM sodium pyruvate and 0.35 mM NADH.
Results are the mean of three independent experiments.
Ab initio calculations. Ab initio molecular orbital calcu-
lations were performed with the Gaussian 98 package.28
The 46+4f7 core electrons of the gadolinium atoms
were described by the quasi-relativistic pseudopotential
of Dolg et al.50,51 and the valence electrons by a
(7s6p5d)/[5s4p3d] Gaussian basis set. For the ligands
3-21G and 6-31G(d,p) basis sets were applied. The geo-
metries of the systems were obtained as follows. Initi-
ally, we performed a conformational analysis of each
free ligand with the molecular mechanics MMFF94
model implemented in the Spartan suite of programs
(PC Spartan Pro v1.03, Wavefunction Inc., Irvine, CA,
USA) in order to select the lowest energy conformer.
This conformer was then fully optimized at the RHF/
3-21G level. As none of the empirical (MMFF94, Sybyl)
or semiempirical (AM1, PM3) available models is con-
veniently parametrized for lanthanide atoms, we studied
the coordination modes and conformations of the
ligand groups starting from the optimized structure of
the Gd(III)-iminodiacetate complex at the HF level. So,
we performed full optimizations of molecular systems
without symmetry constraints for different conforma-
tions of the –CH2–CH2-azole arm at the RHF level,
with the 46+4f7 core electrons ECP and the [5s4p3d]-
GTO valence basis set for the metal and 3-21G basis set
for the ligands. It has been tested that geometry opti-
mizations using the 3-21G basis set for the ligand, fol-
lowed by single point energy calculation with higher
basis set provide a satisfactory representation of ener-
getic properties.29À31,51,52
Acknowledgements
This work was supported in part by strategic group grant
2000-3 from the Community of Madrid to P.B., grant
SAF 2001-2245 from the Ministry of Science and Tech-
nology to S.C. and ROVI Pharmaceutical Laboratories.
E.P.M. and P.L.L. received postdoctoral fellowships
from the Community of Madrid and from U.N.E.D.,
respectively. E.S. and M.G.A. obtained predoctoral fel-
lowships from M.E.C. and ROVI Pharmaceutical
Laboratories, respectively. The expert technical assis-
tance of Mrs. Susana Garrido is gratefully acknowledged.
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