Bisphosphonate sequestering agents. Synthesis and preliminary evaluation for in vitro and in vivo uranium(VI) chelation

Article abstract of DOI:10.1016/j.ejmech.2008.01.018

A library of bisphosphonate-based ligands was prepared using solution-phase parallel synthesis and tested for its uranium-binding properties. With the help of a screening method, based on a chromophoric complex displacement procedure, 23 dipodal and tripodal chelates bearing bisphosphonate chelating functions were found to display very high affinity for the uranyl ion and were selected for evaluation of their in vivo uranyl-removal efficacy. Among them, 11 ligands induced a huge modification of the uranyl biodistribution by deviating the metal from kidney and bones to liver. Among the other ligands, the most potent was the dipodal bisphosphonate 3C which reduced the retention of uranyl and increased its excretion by around 10% of the injected metal.

Full text of DOI:10.1016/j.ejmech.2008.01.018

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 2768e2777
http://www.elsevier.com/locate/ejmech
Original article
Bisphosphonate sequestering agents. Synthesis and preliminary
evaluation for in vitro and in vivo uranium(VI) chelation
a
a
b
Marcin Sawicki , Delphine Lecercle , Gerard Grillon , Beatrice Le Gall ,
b
´
Anne-Laure Serandour , Jean-Luc Poncy , Theodorine Bailly ,
´
´
b
b
c
´
´
Ramon Burgada c, Marc Lecouvey c, Vincent Challeix c, Antoine Leydier d,
d
e
a,
*
´ ´
Stephane Pellet-Rostaing , Eric Ansoborlo , Frederic Taran
a Service de Chimie Bio-organique et de Marquage, iBiTec-S, CEA-Saclay, 91191 Gif sur Yvette, France
b Laboratoire de Radiotoxicologie, DRR, CEA 91680 Bruye`res le Chatel, France
c Laboratoire de Chimie structurale Biomole´culaire, UMR 7033 CNRS, 74 rue Marcel-Cachin, 93017 Bobigny cedex, France
d Laboratoire de Catalyse et Synthe`se Organique, ICBMS UMR 5246, Universite´ Claude Bernard Lyon-1, 69622 Villeurbanne, France
e CEA/DEN/DRCP/CETAMA, VRH-Marcoule, BP 17171, 30207 Bagnols sur Ce`ze, France
Received 20 September 2007; received in revised form 12 December 2007; accepted 10 January 2008
Available online 30 January 2008
Abstract
A library of bisphosphonate-based ligands was prepared using solution-phase parallel synthesis and tested for its uranium-binding properties.
With the help of a screening method, based on a chromophoric complex displacement procedure, 23 dipodal and tripodal chelates bearing bi-
sphosphonate chelating functions were found to display very high affinity for the uranyl ion and were selected for evaluation of their in vivo
uranyl-removal efficacy. Among them, 11 ligands induced a huge modification of the uranyl biodistribution by deviating the metal from kidney
and bones to liver. Among the other ligands, the most potent was the dipodal bisphosphonate 3C which reduced the retention of uranyl and
increased its excretion by around 10% of the injected metal.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Actinide; Chelates; Ligand design; Uranium
1. Introduction
[1,2]. Although no final conclusions can yet be drawn regard-
ing cancer risks associated with uranium exposure, several
studies have shown the radiotoxic and chemotoxic properties
of this ‘‘heavy’’ metal. Renal injury is one of the major con-
cerns in the case of uranium contamination. In the kidneys,
uranium produces chemical damage to renal proximal tubules
[3,4], notably through inhibition of ATPase [5] and oxidative
stress [6]. Renal toxicogenomic responses to uranyl were
also recently studied [7,8]. Uranyl was also found to be cyto-
toxic to macrophages and CD4þ T-cells [9,10]. Finally, sev-
eral studies have shown the potential chemical genotoxicity
of uranyl [11,12].
The worldwide use of uranium notably as fuel in nuclear
power plants (enriched uranium) or for armor-piercing
weapons (depleted uranium) is expanding, thus providing in-
creasing opportunities for occupational and environmental ex-
posure. Whatever the chemical form or speciation of the
uranium compound incorporated, the uranyl ion, UO22þ, is
the most likely form of uranium species present in body fluids
after contamination. Once it is absorbed by the blood, 60% of
UO22þ is rapidly excreted in the urine, while tissues, especially
kidney and bones, accumulate uranium for months to years
Thus, in the case of accidental contamination, an appropri-
ate treatment may be necessary to reduce deposition in target
organs, therefore preventing manifestation of the above-
* Corresponding author. Tel.: þ33 (0)1 69 08 26 85; fax: þ33 (0)1 69 08 79 91.
E-mail address: frederic.taran@cea.fr (F. Taran).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.01.018

M. Sawicki et al. / European Journal of Medicinal Chemistry 43 (2008) 2768e2777
2769
mentioned adverse effects. Decorporation therapy that uses li-
2. Chemistry
gands with strong metal-binding capabilities remains the only
way to accelerate excretion of the poisoning metal. Stimulated
by the lack of effectiveness of existing treatments [13e19],
several specific ligands for UO22þ (i.e. uranophiles) were pre-
pared and tested for in vivo uranium removal. Most of this re-
search has been carried out by K.N. Raymond, P.W. Durbin
and co-workers and published in different reviews [2,20,21].
Several multidentate catecholate and hydroxypyridonate li-
gands were therefore synthesized [22e24] and some of these
chelates were found to display significant in vivo removal of
uranyl [25,26].
The bisphosphonate library was prepared by solution-phase
parallel synthesis employing Michael addition of amine scaf-
folds on tetraethyl ethenylidenebisphosphonic ester 26 as
a key step (Scheme 3).
A panel of dipodal as well as tripodal amines was first pre-
pared by classic methods (Scheme 2). The amine scaffolds to
be synthesized were designed in order to vary several struc-
tural features known to be important for chelation: denticity,
size of the spacer separating chelating functions and geometry.
Amines 1e16 were synthesized from bis- or tris-bromo-
methyl-aryl derivatives by (i) substitution of the bromine
atom by cyanide ions followed by reduction of the correspond-
ing nitrile with borane to afford amines 1e3 [34], (ii) treat-
ment with excess diamine to afford amines 4e15 [35], (iii)
substitution of the bromine atom by sodium azide and subse-
quent reduction by PPh3 to afford amine 16 [36]. The synthesis
of amines 17e25 was performed by treatment of the corre-
sponding aromatic esters with excess diamine [37]. All of
these procedures provided the desired amine scaffolds with
good to excellent yields.
These 25 polyamines and 16 additional commercially avail-
able amines were then used as starting material for the two-
step parallel synthesis of bisphosphonate-based chelates. After
reaction of the 41 amine scaffolds with the Michael acceptor
26, the resulting secondary amines were quenched by addition
of acetic anhydride to avoid the retro-Michael reaction as pre-
viously described [38]. The resulting protected chelates were
then purified by flash chromatography and the tetraethyl esters
were cleaved by treatment with TMSBr [39] affording the bi-
sphosphonate chelates 1Ae6A in moderate to high yields.
Each of the 41 products was fully characterized (see Section 6)
and their purities (established as >90%) were checked by
LCeMS.
In this context, we recently adopted a diversity-based ap-
proach that combines parallel synthesis [27] and in vitro
screening methods [28] to identify new powerful uranyl che-
lates. A collection of ligands bearing various chelating moieties
including polycarboxylates, hydroxamates, catecholates and
hydroxypyridonates, was thus screened for their uranyl-binding
capabilities. Among the tested candidates, ligands bearing bi-
sphosphonate subunits were found to be the best uranophiles.
The bisphosphonate moiety is a well-known stable ana-
logue of pyrophosphate that able to bind strongly to a range
of hard Lewis acid metals due to their high ionization at
physiological pH. Bisphosphonates are also known to target
bones and generally display low toxicity. These properties
make the bisphosphonate core an interesting chelating func-
tion for the design of new uranophiles. In vivo experiments
have shown that administration of simple bisphosphonates,
such as 1-hydroxyethane-1,10-diphosphonic acid (HEDP),
counteracts the effect of lethal doses of uranyl nitrate in
rats [29,30] and halves the kidney content [19]. The uranyl-
binding properties of HEDP and related compounds have
been studied in vitro in detail and induce the formation of
complexes as both 1:1 and 1:2 (metal:ligand) species in aque-
ous solution [31e33]. UO22þ is a hard Lewis acid adopting
unusual pseudo-planar penta- or hexa-coordinated complexes.
Exploiting the well-known chelate effect, our goal is to obtain
very powerful uranophiles by synthesizing a series of dipodal
and tripodal ligands bearing bisphosphonate functions. Such
ligands should be able to efficiently encapsulate the uranyl
ion (Scheme 1) and therefore should be valuable candidates
for chelation therapy.
Besides these 41 ligands we also collected 12 additional tri-
podal bisphosphonate ligands 6Be7E, whose synthesis was
previously published by our group [40e42], to complete our
library (Scheme 4). Some of these ligands such as 6H, 7A
and 7E possess rigid scaffolds in order to increase pre-
organization.
Herein we describe the parallel synthesis and metal-seques-
tering properties of a library of ligands bearing two or three
bisphosphonate chelating functions. Preliminary in vivo exper-
iments evaluating the efficiency of uranyl removal upon treat-
ment with some of these ligands are described.
3. U(VI)-binding properties of the bisphosphonate library
Before assessing the ability of ligands 1Ae7E to decorpo-
rate uranium in vivo, we first investigated the UO22þ-binding
properties of the chelate library. This was carried out using
O
P
HO
HO
O
P
O
P
OH
O
HO
O
O
P
O OH
HO O
O OH
O
O
O
U
O
P
O
OH
O
P
O
U
O
P
O
P
OH
P
OH
OH
O
O
O
U
O
O
O
P
P
O
O
O
P
O
P
O
O
O
P
O
O
HO
O
OH
HO
HO
OH
OH
O
P
O
P
O
Scheme 1. Hypothetical chelation mode of UO22þ by di- and tripodal bisphosphonates.

2770
M. Sawicki et al. / European Journal of Medicinal Chemistry 43 (2008) 2768e2777
1) NaCN, DMSO
NH2
1-3
80°C - 30 min.
Ar
2) BH3-THF
reflux - 5 days
m
H2N
NH2
excess
n
H2N
NH2
excess
n
HN
O
n
HN
n
Br
Ar
NH2
Ar CO2Me
4-15
Ar
NH2
m
Ar
m
MeOH - 25°C - 12h
THF-25°C - 12h
m
m
17-25
1) NaN3, DMF/H2O
80°C - 24h
2) PPh3, THF/H2O
25°C - 12h
NH2
16
Ar
m
NH2
NH2
n
NH2
NH2
n
H2N
NH2
NH2
N
H2N
N
H
N
H
n
N
n
N
H
H
(2, 77%)
(1, 72%)
(n = 2 : 7, 98%)
(n = 3 : 8, 98%)
(n = 4 : 9, 98%)
(n = 2 : 4, 98%)
(n = 3 : 5, 98%)
(n = 4 : 6, 98%)
NH2
OBn
NH2
n
NH2
O
O
NH2
NH2
O
O
NH2
n
N
H
N
H
(n = 2 : 20, 98%)
(n = 3 : 21, 98%)
(n = 4 : 22, 98%)
n
(n = 2 : 17, 98%)
(n = 3 : 18, 98%)
(n = 4 : 19, 98%)
N
N
n
N
H
n
N
n
N
H
(n = 2 : 10, 98%)
(n = 3 : 11, 98%)
(n = 4 : 12, 98%)
H
H
NH2
O
O
NH2
NH2
NH2
H2N
NH2
N
H
N
n
n
N
n
N
n
H2N
NH2
H
H
H
(n = 2 : 23, 98%)
(n = 3 : 24, 98%)
(n = 4 : 25, 98%)
(n = 2 : 13, 98%)
(n = 3 : 14, 98%)
(n = 4 : 15, 98%)
HN
H2N
O
HN
H2N
H2N
(3, 53%)
(16, 78%)
NH2
n
n
Scheme 2. Synthesis of amine scaffolds.
a displacement method of the preformed sulfochlorophenole
UO22þ chromogenic complex as we previously described [28].
This method allows a fast determination of the conditional
UO22þ-binding constants (Kcond) of candidate ligands by UVe
visible spectroscopy. The 53 bisphosphonate ligands were
thus screened in a parallel manner on microtiter plates for their
uranium-binding properties at pH 7.4 and 5.5 (physiological pH
in blood and kidneys) by following the disappearance of the
690 nm absorbance signal of the sulfochlorophenoleUO22þ
complex using a 96-well absorbance reader. All experiments
were carried out in duplicate and the results obtained at pH
7.4 are summarized in Fig. 1.
The results offered a rapid readout of the ligand library and
highlighted some general trends. First, almost all of the tested
ligands displayed remarkable uranyl-binding properties, with
Kcond varying from 1015 to 1019.5 at pH 7.4. These properties
are conserved at pH 5.5, a pH that might be found in the kid-
ney (data not shown). As expected, tripodal bisphosphonate li-
gands appeared globally more powerful uranophiles than the
dipodal ones, the conditional constants being for 1 or 2 orders
of magnitude higher. This suggests that the third bisphospho-
nate subunit of the tripodal ligands participates in the coordi-
nation of UO22þ. Comparison of the results obtained with
dipodal bisphosphonates 1AeH showed the influence of the
spacer separating the two chelating moieties (12 atoms sepa-
rating the bisphosphonate moieties being the optimum). The
presence of phenol or pyridine moieties on the amine scaffold
did not improve the UO22þ-binding constants (compare 3EeG,
3H and 4AeB with 3BeD, Fig. 1). Flexible or rigid ramified
tripods were superior to ligands constructed on cyclic back-
bones (for example compare 6B and 6H with 6G, Fig. 1).
This screening assay allowed us to focus on interesting li-
gand candidates that can then be selected for in vivo experi-
ments. Thus, we selected dipodal bisphosphonates with
K
cond > 1017 and tripods with Kcond > 1018 at pH 7.4. Finally,
11 dipodal and 14 tripodal ligands were chosen for biological
evaluation on the basis of their UO22þ-binding properties.
4. Preliminary in vivo investigations
In order to limit the number of in vivo experiments, we first
investigated the biodistribution of uranyl after injection of the
preformed bisphosphonate chelateeuranyl complexes. Each of
25 UO22þebisphosphonate complexes was therefore prepared
in vitro by mixing 100 eq. of ligand with 5 kBq of 233U(VI)
for 30 min and was then intravenously injected into rats at
a concentration of 30 mmol kgꢀ1. Urine and feces were col-
lected daily and rats were sacrificed 5 days later. The different
organs and tissues were analyzed by liquid scintillation count-
ing, as were urine and feces.
This series of experiments highlighted complexes that can be
excreted and stable enough in vivo to modify the biodistribution
of UO22þ. Control experiments were carried out without ligand
or in the presence of HEDP used as reference chelate (Fig. 2).
As shown in Fig. 2, the tested complexes were globally sta-
ble in vivo. The injection of these complexes induced signifi-
cant changes in the biodistribution of UO22þ compared with
control experiments. Unfortunately, in many cases the UO22þ

M. Sawicki et al. / European Journal of Medicinal Chemistry 43 (2008) 2768e2777
2771
O
1) DMF - 25°C - 36h
then Ac2O-15 min.
P
P
OEt
OEt
PO3H2
PO3H2
1A-6A
2
2
N
NH2
+
n
=PO3H2
2) TMSBr, CH3CN
reflux - 2h
n
P
O
O
1-25
26
then MeOH
P
P
P
N
P
P
N
P
P
P
P
N
P
N
P
P
P
P
P
O
P
O
O
N
N
N
N
N
n
N
O
O
O
2C (71%)
O
O
O
N
O
O
n = 0 : 1A (86%)
n = 2 : 1B (93%)
n = 3 : 1C (84%)
n = 4 : 1D (95%)
n = 5 : 1E (66%)
n = 6 : 1F (50%)
n = 7 : 1G (58%)
n = 8 : 1H (86%)
2A (90%)
2B (71%)
P
P
N
P
P
N
P
P
P
P
P
P
P
P
N
O
N
N
N
O
N
O
O
O
O
O
2F (81%)
2D (50%)
O
2E (67%)
P
P
P
P
P
P
P
P
O
O
P
N
P
P
P
N
P
P
P
P
N
N
N
N
N
N
n
N
N
n
O
O
O
O
O
O
O
O
O
2H (88%)
2G (90%)
n = 2 : 3B (64%)
n = 3 : 3C (78%)
n = 4 : 3D (75%)
3A (68%)
P
P
P
N
P
P
P
P
P
N
P
N
O
O
P
N
P
P
O
O
O
P
P
P
P
N
OH
O
O
N
O
O
n
N
N
N
N
O
O
N
n
n
n
N
N
N
H
N
H
n
n
N
n
O
O
N
H
N
H
n
O
O
n = 2 : 4C(62%)
n = 3 : 4D(76%)
n = 4 : 4E (68%)
n = 2 : 3E (48%)
n = 3 : 3F (69%)
n = 4 : 3G (83%)
n = 2 : 3H (61%)
n = 3 : 4A (80%)
n = 4 : 4B (79%)
n = 2 : 4F (93%)
n = 3 : 4G (92%)
n = 4 : 4H (94%)
P
P
P
P
P
P
P
N
P
N
P
P
P
P
N
P
O
O
N
O
P
O
P
O
N
N
O
O
N
N
n
n
N
O
n
n
N
N
N
N
H
n
n
O
H
O
P
n = 2 : 5G (57%)
n = 3 : 5H (57%)
n = 4 : 6A (73%)
O
N
5A (48%)
n
O
n = 2 : 5D (52%)
n = 3 : 5E (34%)
n = 4 : 5F (60%)
N
P
P
N
HN
O
O
O
O
P
P
n = 1 : 5B (58%)
n = 2 : 5C (57%)
P
N
N
n
n
P
P
O
Scheme 3. Synthesis of a library of 41 bisphosphonate ligands.
biodistribution was not modified in the desired direction. For
many tripodal ligands, in particular, uranium deposition was
increased and partially rerouted from kidney and bones to
liver. This phenomenon is particularly important with com-
plexes formed with ligands 3G, 5C, 6A, 6C and 6H where
more than 40% of the injected uranyl is recovered in the liver.
It is noteworthy that very small changes in ligand structure can
greatly alter uranyl biodistribution. For example, ligand 7B,
which is the only tripod that does not induce uranyl deposition
in the liver, is structurally very similar to ligand 6C, which ac-
counts for around 40% of hepatic retention. This result might
suggest that this hepatic deposition is avoided when N-methyl-
amide moieties are present.
significantly (arrows in Fig. 2) and not to disturb the normal
uranium distribution, and these were therefore selected for fur-
ther biological evaluation. Two additional tripods (5C and 6A)
were also chosen for the study of hepatic retention. HEDP and
5-LICAM(S), a well-known chelating agent for uranyl re-
moval [25,26], were included as reference chelates in this in
vivo experiment.
The evaluation of the selected ligands as decorporating
agents was carried out by prompt injection after uranyl con-
tamination. For each ligand a group of five rats was first con-
taminated by intraperitoneal injection of 233U(VI) followed
5 min later by injection of 100 eq of ligand (30 mmol kgꢀ1).
After 5 days, the animals were sacrificed and the radioactivity
associated with collected excreta and main organs was mea-
sured. The results are summarized in Table 1.
Globally, only three bisphosphonate ligands 2H, 3C, and
7B were found to increase excretion of the injected uranyl

2772
M. Sawicki et al. / European Journal of Medicinal Chemistry 43 (2008) 2768e2777
P
P
=PO3H2
P
P
P
O
P
O
P
P
P
P
HN
P
O
P
O
P
HN
O
N
O
N
O
H
N
P
N
H
P
n
P
HN
H
P
N
N
N
H
P
P
H
O
N
N
O
O
N
n
P
N
P
O
P
6B
HN
O
P
HN
n
O
P
6G
n = 1 : 6C
n = 2 : 6D
HN
O
O
6F
P
P
P
6E
P
P
P
P
P
OH
P
P
P
P
P
P
O
O
O
P
P
P
P
P
P
N
N
HN
n
P
P
NH
P
P
H
H
N
O
P
NH
HN
N
P
N
O
NH
P
OH
P
O
P
N
O
n
O
O
O
P
P
NH
P
P
7B
n = 1 : 7C
n = 2 : 7D
6H
7E
N
O
HN
P
O
P
n
7A
OH
P
P
Scheme 4. Other tripodal BP ligands under investigation.
Compared with the control, only dipodal ligand 3C signif-
icantly increased U(VI) excretion (Table 1). The administra-
tion of this ligand allowed a 13% increase in the injected
UO22þ and reduced uranyl deposition in the kidney by around
50%. Several other ligands, particularly tripod 7B, more effec-
tively reduced uranyl retention in the collected organs, but did
not increase excretion. This might be explained by uranyl re-
tention in soft tissues that were not collected. Whatever the li-
gand used, more than 75% of uranyl excretion occurred during
the first day after contamination (Fig. 3).
To be effective, a candidate ligand should have binding con-
stants at least close to those of biological targets. Unfortu-
nately, and despite the best efforts of researchers in this field
[43e45], little is known about the binding strength of the
UO22þ-biological ligands. Transferrin and carbonate ions are
known to bind UO22þ strongly in plasma, with thermodynamic
constants b1,1 ¼ 1016 and b1,3 ¼ 1021.5, respectively [46e48],
but information is still lacking about the natural ligands pres-
ent in kidney and bones.
The aim of our work was to synthesize new uranophiles
with very high binding constants and use them to compete
with the natural targets of UO22þ. Based on previous findings,
we focused our work on bisphosphonate-based ligands and we
succeeded in obtaining potent uranophiles. Tripodal bi-
sphosphonate ligands, in particular, were found to display
5. Discussion and conclusion
Chelation therapy for the removal of toxic metals present
in vivo is first, but not only, a matter of binding competition.
1020
1019
1018
1017
1016
1015
1014
Dipodal Ligands
Tripodal Ligands
Fig. 1. Conditional constants of BP ligands (pH 7.4).

M. Sawicki et al. / European Journal of Medicinal Chemistry 43 (2008) 2768e2777
2773
100
A
90
80
70
60
50
40
30
20
10
0
Retention
Excretion
80
70
60
50
40
30
20
10
0
B
Liver
Kidney
Skeleton
Dipodal Ligands
Tripodal Ligands
Fig. 2. Biodistribution of uranyl after injection of UO22þ/L complexes. (A) Global percentages of injected radioactivity on excreta and main organs, (B) detailed
percentages of the injected radioactivity in main organs. Complexes were prepared by mixing 100 eq. of ligand with 5 kBq of 233U(VI) and then injected iv
(30 mmol kgꢀ1); rats were sacrificed 5 days later.
remarkable UO22þ-binding capabilities with binding constants
up to Kcond ¼ 1019.5 at physiological pH. These compounds
are probably the most powerful uranophiles described to date.
However, while the UO22þ-binding properties of a metal-
sequestering agent candidate are necessary, they are not suffi-
cient to ensure successful chelation therapy. Many other
parameters, including the biodistribution and pharmacokinetics
of ligands and their metal complexes, are of prime importance
and, unfortunately, are difficult to predict. In our library, the li-
gands inducing the most significant changes in uranyl biodistri-
bution are the tripodal bisphosphonates, but many of them
seem to form uranyl complexes that cannot be eliminated
from the body. One possible explanation for the observed
accumulation of uranyl in liver after treatment with bisphosph-
onate tripods might be related to complex speciation of the
corresponding UO22þ complexes. Although time-resolved
Table 1
Effect of a prompt injection of bisphosphonate ligands on the biodistribution
and excretion of 233U(VI)a
Urine
50
Ligand
Percent of injected 233U(VI) ꢁ SD at 5 days
Control
HEDP
40
Retention
Excretion
5-LICAM(S)
Kidneys
Skeleton
Liver
Urine
Feces
2H
3C
5C
6A
7B
30
20
10
0
No (controls) 8.0 ꢁ 2.3 34.2 ꢁ 4.5
HEDP
5.5 ꢁ 0.5 30.2 ꢁ 1.2
0.4 ꢁ 0.0 40.7 ꢁ 3.4 1.8 ꢁ 1.0
0.4 ꢁ 0.1 50.1 ꢁ 4.3 2.9 ꢁ 1.7
0.5 ꢁ 0.1 48.0 ꢁ 5.7 2.2 ꢁ 1.2
2.8 ꢁ 0.5 31.6 ꢁ 7.8 4.9 ꢁ 1.3
0.3 ꢁ 0.0 53.5 ꢁ 2.7 2.3 ꢁ 0.5
5-LICAM(S) 6.3 ꢁ 0.8 22.8 ꢁ 4.7
2H
3C
5C
6A
7B
a
6.7 ꢁ 1.3 41.8 ꢁ 3.9
3.6 ꢁ 0.6 32.4 ꢁ 1.5
3.8 ꢁ 0.7 28.1 ꢁ 2.6 13.4 ꢁ 1.0 25.6 ꢁ 0.8 2.1 ꢁ 0.3
7.5 ꢁ 0.9 27.9 ꢁ 0.8 13.5 ꢁ 1.5 31.8 ꢁ 8.5 2.3 ꢁ 1.2
3.4 ꢁ 0.6 23.9 ꢁ 0.5
1.5 ꢁ 0.6 29.7 ꢁ 5.1 1.4 ꢁ 0.6
Ligands were injected ip (30 mmol kgꢀ1, molar ratio 100) 5 min after injec-
1
2
3
Days
4
5
tion of 9.2 kBq of 233U(VI); animals were sacrificed 5 days later. Data are ex-
pressed as % of injected radioactivity, discrepancies are due to rounding. Bold
means are significantly different than control mean.
Fig. 3. Kinetic of uranyl excretion.

2774
M. Sawicki et al. / European Journal of Medicinal Chemistry 43 (2008) 2768e2777
laser-induced fluorescence (TRLIF) as well as electrospray
mass spectroscopy (ES-MS) measurements conducted on
selected UO22þetripodal bisphosphonate complexes confirmed
a 1:1 stoichiometry [28], one cannot exclude the possibility of
in vivo formation of high-molecular-weight polymetallic com-
plexes. Preliminary experiments on tripods 5A [28], 5H and
5C (data not shown) highlighted a high level of metal specific-
ity toward UO22þ over Ca2þ, Mg2þ and Zn2þ, but a very
low specificity over Fe3þ. All of these critical factors might
explain the low level of in vivo efficacy of tripodal
bisphosphonates.
MeOH, precipitated by addition of ether and fi-
nally dried over P2O5 to afford amines 1e3.
Method II To 1,3-dibromo or 1,3,5-tribromomethyl-aryl de-
rivatives (1.5 mmol, 1 eq.) in 45 mL of dry THF
were added ethane, propane or butane-diamine
(60 mmol, 40 eq.). The mixture was stirred at
25 ꢂC for 12 h. Solvent and excess of diamine
were removed under reduced pressure. The re-
sulting oil was solubilized in MeOH and KOH
(3 mmol, 2 eq.) was added. Inorganic salts were
precipitated by addition of ether and removed
by filtration. The filtrate was evaporated to afford
quantitative yields of amine scaffolds 4e15.
Method III NaN3 (36 mmol, 2.34 g) was added to a solution
of 2,6-dibromomethylpyridine (3 mmol, 795 mg)
in 7 mL CH2Cl2/DMF (2/5) and the mixture was
warmed at 80 ꢂC for 24 h. Water was added and
the hoped 2.6-di(azidomethyl)-pyridine was ex-
tracted by CH2Cl2, dried over MgSO4 and puri-
fied by flash chromatography (CH2Cl2:hexane,
1:1).
Dipodal ligand 3C is the only compound that induces a sig-
nificant benefit after UO22þ contamination. Further investiga-
tions are under way to define better protocols (increased
doses or repeated injection of 3C).
6. Experimental protocols
6.1. Chemistry
6.1.1. General experimental procedures
2.6-Di(azidomethyl)-pyridine
(2.65 mmol,
Unless otherwise stated, starting materials were obtained
from commercial suppliers and used without purification.
THF was distilled from sodium/benzophenone ketyl before
use. 1H NMR (400 MHz), 13C NMR (100 MHz) and 31P
NMR (160 MHz) were measured on a Brucker Avance
400 MHz spectrometer. Chemical shifts are reported in parts
per million (ppm, d) downfield from residual solvents peaks
and coupling constants are reported as Hertz (Hz). Splitting
patterns are designated as singlet (s), doublet (d), triplet (t).
Splitting patterns that could not be interpreted or easily visu-
alized are designated as multiplet (m). Electrospray mass spec-
tra were obtained using an ESI/TOF Mariner Mass
Spectrometer.
500 mg) was reacted with PPh3 (13 mmol,
3.57 g) and water (52 mmol, 0.5 mL) in 10 mL
THF. After 24 h at room temperature, THF was
removed, water was added and the aqueous phase
was washed by CH2Cl2 and then evaporated to
afford 300 mg of amine 16.
Method IV Isophthalic acid dimethyl ester, pyridine-2,6-di-
carboxylic acid dimethyl ester or benzene-1,3,5-
tricarboxylic acid trimethyl ester (2.5 mmol)
were reacted with ethane, propane or butane-di-
amine (0.1 mol, 40 eq.) in 45 mL MeOH at
25 ꢂC for 12 h. Solvent and excess of diamine
were removed under reduced pressure to afford
quantitative yields of amine scaffolds 17e25.
6.1.2. General procedures for the synthesis
of amine scaffolds 1e25
6.1.3. General procedures for the synthesis
Method I 1,3-Bis-bromomethyl-benzene, 2,4-bis-bromo-
methyl-1,3,5-triethyl-benzene or 1,3,5-tris-bro-
momethyl-2,4,6-trimethyl-benzene (3.5 mmol)
were reacted with NaCN (10 mmol, 2.8 eq.) in
dry DMSO. The mixture was stirred at 80 ꢂC
for 30 min. After cooling, ether was added and
the organic phase was washed with water and
then dried on MgSO4.
of bisphosphonate ligands 1Ae6A
To a solution of amine 1e25 (0.025 mmol, 1 eq.) in 2 mL
of dry DMF, was added tetraethyl ethylidenebisphosphonic es-
ter (60 mg, 0.2 mmol, 8 eq. for dipodal amines or 135 mg,
0.45 mmol, 18 eq. for tripodal amine). After reaction at
room temperature for 48 h, acetic anhydride (40 mL,
0.43 mmol, 17 eq. for dipodal amines or 60 mL, 0.63 mmol,
25 eq. for tripodal amine) was added. The solution was stirred
for 15 min at room temperature, evaporated and then directly
filtered through a silica gel (5 g, hexane/acetone 1:1 to remove
the excess of acrylate, then acetone/MeOH 7:3) to yield pro-
tected chelate. After removal of the solvents, protected che-
lates were then dissolved in dry CH3CN and TMSBr (3 eq.
per functions) was added under argon. The mixture was stirred
under reflux for 3 h and then quenched with water (3 eq. per
functions). After refluxing for additional 15 min, the solvents
were evaporated and co-evaporated with MeOH. Resulting
residues were dissolved in a minimum of MeOH and
The expected di- or tri-cyano compounds were
purified by flash chromatography (hexane:ether)
and then dissolved in 20 mL of BH3/THF (1 M,
20 mmol, 10 eq.). The corresponding mixture
was refluxed under argon for 3 days. Two millili-
ter of an aqueous solution of HCl (1 N) were
added and the mixture was refluxed for 2 h. The
aqueous solution was washed by dichlorome-
thane and then evaporated under reduced pres-
sure. The recovered solid was dissolved in

M. Sawicki et al. / European Journal of Medicinal Chemistry 43 (2008) 2768e2777
2775
precipitated with ether. The resulting slurry was decanted,
washed with ether and dried under high vacuum in the pres-
ence of P2O5 to afford the target chelates.
Ligands 1A, 1B, 1D, 1F, 1H, 2G and 5A are described in
our previous work [28].
(2s, 4P); MS m/z 871 (100%, [M þ 23]þ); HRMS: Calcd for
C18H33N2O14P4: m/z 625.0882. Found: m/z 4625.0908.
6.1.3.11. Ligand 3A. 1H NMR (D2O): 1.22 (m, 9H), 2.21 and
2.36 (2s, 6H), 2.70 (m, 6H), 3.02 (m, 7H), 3.47 (m, 3H), 3.89
(m, 2H), 3.92 (m, 2H), 4.03 (m, 2H),7.06 (s, 1H); 31P NMR
(D2O): 18.83 and 19.54 (2s, 4P); MS m/z 709 (100%,
[M þ 23]þ); HRMS: Calcd for C24H45N2O14P4: m/z
709.1821. Found: m/z 709.1842.
6.1.3.1. Ligand 1C. 1H NMR (D2O): 1.38 (m, 2H), 1.71 (m,
4H), 2.19 (s, 3H), 2.29 (s, 3H), 3.06 (m, 2H), 3.54 (m, 4H),
3.90 (m, 3H), 4.01 (m, 1H); 31P NMR (D2O): 21.10 and
21.80 (2s, 4P); MS m/z 585 (100%, [M þ 23]þ).
6.1.3.2. Ligand 1E. 1H NMR (D2O): 1.40 (m, 6H), 1.67 (m,
4H), 2.08, 2.20 and 2.30 (3s, 6H), 3.07 (m, 2H), 3.53 (m,
4H), 3.90 (m, 3H), 4.02 (m, 1H); 31P NMR (D2O): 19.03
and 19.64 (2s, 4P); MS m/z 613 (100%, [M þ 23]þ).
6.1.3.12. Ligand 3B. 1H NMR (D2O): 2.12e2.35 (m, 12H),
2.77 (m, 1H), 3.10 (m, 1H), 3.66 (m, 8H), 3.84 (m, 3H)
4.02 (m, 1H), 4.75 (m, 4H), 7.18 (s, 1H), 7.35 (m, 2H), 7.55
(m, 1H); 31P NMR (D2O): 18.53 and 19.23 (2s, 4P); MS m/z
767 (100%, [M þ 1]þ).
6.1.3.3. Ligand 1G. 1H NMR (D2O): 1.40 (m, 10H), 1.70 (m,
4H), 2.22 and 2.33 (2s, 6H), 3.10 (m, 6H), 3.50 (m, 4H), 3.95
(m, 3H), 4.07 (m, 1H); 31P NMR (D2O): 18.93 and 19.64 (2s,
4P); MS m/z 641 (100%, [M þ 23]þ).
6.1.3.4. Ligand 2A. 1H NMR (D2O): 2.25 (m, 6H), 2.75 (m,
1H), 3.13 (m, 1H), 3.45 (m, 4H), 3.64 (m, 4H), 3.80 (m,
2H), 3.96 (m, 2H); 31P NMR (D2O): 16.42, 18.73 and 19.44
(3s, 4P); MS m/z 565 (100%, [M þ 1]þ).
6.1.3.5. Ligand 2B. 1H NMR (D2O): 2.14e2.39 (m, 9H),
2.14e2.39 (m, 9H), 2.86 (m, 1H), 3.13 (m, 1H), 3.59 (m,
2H), 3.71 (m, 4H), 3.81 (m, 2H), 3.93 (m, 3H), 4.08 (m,
1H); 31P NMR (D2O): 18.63 and 19.34 (2s, 4P); MS m/z
606 (100%, [M þ 1]þ).
6.1.3.6. Ligand 2C. 1H NMR (D2O): 1.60 (m, 4H), 1.87 (m,
2H); 2.19 (m, 9H), 3.06 (m, 2H), 3.43 (m, 8H), 3.82 (m,
3H), 3.97 (m, 1H); 31P NMR (D2O): 19.74 and 20.45 (2s,
4P); MS m/z 648 (100%, [M þ 23]þ).
6.1.3.7. Ligand 2D. 1H NMR (D2O): 2.10 (s, 6H); 2.62e2.74
(m, 1H), 2.83e3.00 (m, 1H), 3.38e3.47 (m, 4H), 3.51e3.62
(m, 8H), 3.64e3.79 (m, 2H), 3.82e3.98 (m, 2H); 31P NMR
(D2O): 16.02, 18.42 and 19.05 (3s, 4P); MS m/z 631 (100%,
[M þ 23]).
6.1.3.8. Ligand 2E. 1H NMR (D2O): 1.83 (m, 4H), 1.90, 2.00
and 2.12 (3s, 6H), 2.20 (m, 4H), 2.85 (m, 2H), 3.37 (m, 8H),
3.56 (m, 4H), 3.77 (m, 4H); 31P NMR (D2O): 19.22 and 19.82
(2s, 4P); MS m/z 661 (100%, [M þ 1]þ).
6.1.3.9. Ligand 2F. 1H NMR (D2O): 1.30 (m, 2H), 1.46e1.83
(m, 6H), 2.03 (m, 2H), 2.01, 2.12 and 2.24 (3s, 6H), 3.06 (m,
2H), 3.33 (m, 4H), 3.84 (m, 3H), 3.96 (m, 1H); 31P NMR
(D2O): 18.83 and 19.54 (2s, 4P); MS m/z 625 (100%,
[M þ 23]þ).
6.1.3.10. Ligand 2H. 1H NMR (D2O): 1.86, 2.09 and 2.27 (3s,
6H), 2.95 (m, 4H), 3.10 (m, 2H), 3.84 (m, 4H), 3.78 (m, 4H),
7.20 (m, 2H), 7.38 (m, 1H); 31P NMR (D2O): 18.93 and 19.64
6.1.3.13. Ligand 3C. 1H NMR (D2O): 1.85 (m, 4H), 2.04e
2.31 (m, 12H), 2.82 (m, 1H), 3.06 (m, 1H), 3.44 (m, 8H),
3.80 (m, 3H) 3.96 (m, 1H), 4.71 (m, 4H), 7.14 (s, 1H), 7.32
(m, 2H), 7.50 (m, 1H); 31P NMR (D2O): 18.63 and 19.24
(2s, 4P); MS m/z 795 (100%, [M þ 1]þ); HRMS: Calcd for
C26H47N4O16P4: m/z 795.1938. Found: m/z 795.1937.
6.1.3.14. Ligand 3D. 1H NMR (D2O): 1.58 (m, 8H), 2.10e
2.32 (m, 12H), 3.12 (m, 2H), 3.49 (m, 8H), 3.84 (m, 3H)
3.98 (m, 1H), 4.70 (m, 4H), 7.12 (s, 1H), 7.30 (m, 2H), 7.47
(m, 1H); 31P NMR (D2O): 18.93 and 19.64 (2s, 4P); MS m/z
823 (100%, [M þ 23]þ).
6.1.3.15. Ligand 3E. 1H NMR (D2O): 2.26 (m, 12H), 2.86 (m,
2H), 3.81 (m, 8H), 3.97 (m, 2H), 4.09 (m, 2H), 5.21 (m, 4H),
7.93 (m, 2H), 8.60 (m, 1H); 31P NMR (D2O): 18.53 and 19.13
(2s, 4P); MS m/z 768 (100%, [M þ 1]þ).
6.1.3.16. Ligand 3F. 1H NMR (D2O): 2.14 (m, 4H), 2.28 (m,
12H), 3.09 (m, 2H), 3.67 (m, 8H), 3.99 (m, 2H), 4.11 (m, 2H),
5.09 (m, 4H), 7.96 (m, 2H), 8.59 (m, 1H); 31P NMR (D2O):
18.63 and 19.24 (2s, 4P); MS m/z 796 (100%, [M þ 1]þ).
6.1.3.17. Ligand 3G. 1H NMR (D2O): 1.73 (m, 8H), 2.17,
2.30 and 2.34 (3s, 12H), 2.82 (m, 1H), 3.05 (m, 1H), 3.40e
3.70 (m, 8H), 3.87 (m, 3H), 4.02 (m, 1H), 4.99 (s, 4H), 7.90
(m, 2H), 8.55 (m, 1H); 31P NMR (D2O): 19.64 and 21.85
(2s, 4P); MS m/z 824 (100%, [M þ 1]þ).
6.1.3.18. Ligand 3H. 1H NMR (D2O): 1.73e2.07 (m, 15H),
2.81 (m, 2H), 3.34 (m, 4H), 3.51 (m, 4H), 3.67 (m, 3H),
3.88 (m, 1H), 4.38 (m, 4H), 6.92 (m, 2H); 31P NMR (D2O):
17.84 (s, 4P); MS m/z 796 (100%, [M þ 1]þ).
6.1.3.19. Ligand 4A. 1H NMR (D2O): 1.58 (m, 4H), 1.73e
2.07 (m, 15H), 2.81 (m, 2H), 3.15 (m, 8H), 3.50 (m, 4H),
4.22 (m, 4H), 6.73 (m, 2H); 31P NMR (D2O): 19.54 and
21.58 (2s, 4P); MS m/z 825 (100%, [M þ 1]þ).
6.1.3.20. Ligand 4B. 1H NMR (D2O): 1.32 (m, 8H; H2,3),
1.81e2.08 (m, 15H; Hc,7), 2.80 (m, 2H; Ha), 3.20 (m, 8H;

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M. Sawicki et al. / European Journal of Medicinal Chemistry 43 (2008) 2768e2777
H1,4), 3.62 (m, 3H; Hb), 3.89 (m, 1H; Hb), 4.30 (m, 4H; H5),
6.81 (m, 2H; H6); 31P NMR (D2O): 18.92 and 19. (2s, 4P);
MS m/z 853 (100%, [M þ 1]þ).
3.31 (m, 3H), 3.57 (m, 4H), 3.77 (m, 2H), 4.91 (m, 6H); 31P
NMR (D2O): 18.73 and 19.44 (2s, 4P); MS m/z 1195
(100%, [M þ 1]þ).
1
6.1.3.21. Ligand 4C. H NMR (D2O): 1.83, 1.92 and 2.04 (3s,
6.1.3.31. Ligand 5F. 1H NMR (D2O): 1.41 (m, 12H), 2.11e
2.44 (m, 27H), 2.95 (m, 3H), 3.74 (m, 3H), 3.11 (m, 12H),
3.94 (m, 3H), 4.91 (m, 6H); 31P NMR (D2O): 18.83 and
19.44 (2s, 4P); MS m/z 1238 (100%, [M þ 1]þ).
6H), 2.73 (m, 2H), 3.30 (m, 4H), 3.55 (m, 4H), 3.69 (m, 2H),
3.86 (m, 2H), 7.42 (s, 1H), 7.70 (s, 2H), 8.00 (s, 1H); 31P NMR
(D2O): 17.96 and 19.79 (2s, 4P); MS m/z 711 (100%, [M þ 1]þ).
6.1.3.22. Ligand 4D. 1H NMR (D2O): 1.77 (m, 4H), 1.93 and
2.04 (2s, 6H), 2.85 (m, 2H), 3.25 (m, 4H), 3.61 (m, 2H), 3.84
(m, 4H), 3.92 (m, 2H), 7.42 (m, 1H), 7.72 (s, 2H), 7.89 (s, 1H);
31P NMR (D2O): 18.93, 19.54, 21.65 and 21.85 (4s, 4P); MS
m/z 739 (100%, [M þ 1]þ).
6.1.3.23. Ligand 4E. 1H NMR (D2O): 1.45 (m, 8H), 1.93 and
2.04 (2s, 6H), 2.83 (m, 2H), 3.22 (m, 4H), 3.37 (m, 4H), 3.64
(m, 3H) 3.77 (m, 1H), 7.39 (m, 1H), 7.68 (s, 2H), 7.85 (s, 1H);
31P NMR (D2O): 18.35 and 19.26 (2s, 4P); MS m/z 767
(100%, [M þ 1]þ).
6.1.3.24. Ligand 4F. 1H NMR (D2O): 2.05, 2.13 and 2.31 (3s,
6H), 3.12 (m, 2H), 3.65 (m, 4H), 3.78 (m, 4H), 3.90 (m, 3H)
4.08 (m, 1H), 8.20 (m, 3H); 31P NMR (D2O): 19.54 and 21.85
(2s, 4P); MS m/z 712 (100%, [M þ 1]þ).
6.1.3.32. Ligand 5G. 1H NMR (D2O): 1.25 (m, 36H), 2.09,
2.22 and 2.32 (3s, 9H), 3.08 (m, 3H), 3.77 (m, 12H), 3.98
(m, 6H), 8.29 (m, 3H); 31P NMR (D2O): 18.63 and 19.44
(2s, 4P); MS m/z 1027 (100%, [M þ 1]þ).
6.1.3.33. Ligand 5H. 1H NMR (D2O): 1.95 (m, 6H), 2.19 and
2.31 (2s, 9H), 3.08 (m, 3H), 3.55 (m, 12H), 3.90 (m, 4H), 4.03
(m, 2H); 31P NMR (D2O): 19.93 and 19.54 (2s, 4P); MS m/z
1087 (100%, [M þ 1]þ); HRMS: Calcd for C30H55N6O24P6:
m/z 1069.1694. Found: m/z 1069.1691.
6.1.3.34. Ligand 6A. 1H NMR (D2O): 1.68 (m, 12H), 2.18 and
2.29 (2s, 9H), 3.08 (m, 3H), 3.50 (m, 12H), 3.88 (m, 4H), 4.01
(m, 2H);31P NMR (D2O): 18.80 and 19.49 (2s, 4P); MS m/z
1134 (100%, [M þ 23]þ).
6.2. In vivo experiments
6.1.3.25. Ligand 4G. 1H NMR (D2O): 21.98 (m, 4H), 2.10,
2.19 and 2.35 (3s, 6H), 3.12 (m, 2H), 3.43 (m, 4H), 3.56
(m, 4H), 3.91 (m, 2H), 4.06 (m, 2H), 8.20 (m, 2H); 31P
NMR (D2O): 19.53 and 21.84 (2s, 4P); MS m/z 740 (100%,
[M þ 1]þ); HRMS: Calcd for C21H38N5O16P4: m/z 740.1264.
Found: m/z 740.1287.
Adult male Sprague Dawley rats weighing 250 ꢁ 50 g were
used for these experiments.
Animal experimentation was run according to national eth-
ical and veterinary guidelines.
6.2.1. Biodistribution after injection
of uranyl/ligand complexes
6.1.3.26. Ligand 4H. 1H NMR (D2O): 1.74 (m, 8H), 2.19 and
2.32 (2s, 6H), 3.12 (m, 2H), 3.59 (m, 8H), 3.92 (m, 2H), 4,03
(m, 2H), 8.17 (m, 3H); 31P NMR (D2O): 19.64 and 21.85 (2s,
4P); MS m/z 768 (100%, [M þ 1]þ).
6.1.3.27. Ligand 5B. 1H NMR (D2O): 1.93e2.27 (m, 18H),
2.39 (m, 9H), 2.98 (m, 3H), 3.24 (m, 3H), 3.32 (m, 3H),
4.91 (m, 6H); 31P NMR (D2O): 18.73 and 19.43 (2s, 4P);
MS m/z 898 (100%, [M þ 23]þ).
6.1.3.28. Ligand 5C. 1H NMR (D2O): 1.88 and 1.93 (2s, 9H),
2.17 (m, 9H), 2.50e2.78 (m, 9H), 3.14 (m, 3H), 3.39 (m, 3H),
3.62 (m, 3H), 3.81 (m, 3H); 31P NMR (D2O): 18.73 and 19.34
(2s, 4P); MS m/z 940 (100%, [M þ 1]þ); HRMS: Calcd for
C27H52N3O21P6: m/z 940.1519. Found: m/z 940.1536.
Each rats was given a single 30 mmol kgꢀ1 injection
(200 mL, 5 kBq, iv) of uranyl/bisphosphonate complexes pre-
pared as followed: to 1 mL of a stock solution of 233U
(50 kBq/mL) in HNO3 1 N was added 100 eq. of bisphospho-
nate ligand, pH was adjusted to 7.4 in a final volume of 2 mL
and the mixture was stirred for 30 min.
Urine and feces samples were periodically collected from
individual metabolic cages and rats were killed at day 5.
The different organs, tissues as well as urines and feces sam-
ples were analyzed by liquid scintillation counting.
6.2.2. Removal of uranyl
Groups of five rats injected ip with 9.2 kBq of 233U(VI) were
treated 5 min later by ip injection of bisphosphonates ligands
(30 mmol kgꢀ1, molar ratio 100). Urine and feces samples were
periodically collected from individual metabolic cages and rats
werekilled atday5. Thedifferent organs, tissuesaswellasurines
and feces samples were analyzed by liquid scintillation counting.
6.1.3.29. Ligand 5D. 1H NMR (D2O): 1.90e2.43 (m, 27H),
2.95 (m, 3H), 3.32 (m, 12H), 3.80 (m, 4H), 4.05 (m, 2H),
4.91 (m, 6H); 31P NMR (D2O): 18.53 and 19.33 (2s, 4P);
MS m/z 1153 (100%, [M þ 1]þ); HRMS: Calcd for
C36H67N6O24P6: m/z 1153.2629. Found: m/z 1153.2632.
Acknowledgements
6.1.3.30. Ligand 5E. 1H NMR (D2O): 1.98 (m, 6H), 2.28e
2.45 (m, 27H), 2.98 (m, 3H), 3.08 (m, 6H), 3.20 (m, 3H),
This work was supported by a program of interdisciplinary
research called ‘‘Nuclear and Environmental Toxicology,

M. Sawicki et al. / European Journal of Medicinal Chemistry 43 (2008) 2768e2777
2777
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