Synthesis and biological properties of iron chelators based on a bis-2-(2-hydroxy-phenyl)-thiazole-4-carboxamide or -thiocarboxamide (BHPTC) scaffold

Article abstract of DOI:10.1016/j.bmc.2009.11.057

Bis-2-(2-hydroxy-phenyl)-thiazole-4-carboxamides and -thiocarboxamides (BHPTCs) form a family of gemini hexacoordinated bis-tridentate chelating scaffolds. Four molecules were synthesized and shown to chelate iron(III) efficiently with a 1:1 stoichiometry. A dithioamide BHPTC displayed promising antiproliferative activity in several cancerous cell lines, making this molecule an interesting lead compound for the design of new iron-chelating anticancer drugs. Conversely, diamide BHPTCs had significant cytoprotective activity against iron overload in HepaRG cells in vitro, and were as efficient as and less toxic than deferoxamine B (DFO).

Full text of DOI:10.1016/j.bmc.2009.11.057

Bioorganic & Medicinal Chemistry 18 (2010) 689–695
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological properties of iron chelators based
on a bis-2-(2-hydroxy-phenyl)-thiazole-4-carboxamide
or -thiocarboxamide (BHPTC) scaffold
David Rodriguez-Lucena a, François Gaboriau b, Freddy Rivault a, , Isabelle J. Schalk a,
Gérard Lescoat b, Gaëtan L. A. Mislin a,
*
a Métaux et Microorganismes: Chimie, Biologie et Applications, IREBS FRE3211-CNRS/Université de Strasbourg, ESBS, Boulevard Sébastien Brant, F-67400 Illkirch, France
b Fer et Foie: Aspects Physiologiques et Pathologiques U 991—INSERM/EA ‘MDC’/Université de Rennes 1, Hôpital Pontchaillou—2, rue Henri Le Guilloux, F-35033 Rennes Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 July 2009
Revised 17 November 2009
Accepted 28 November 2009
Available online 6 December 2009
Bis-2-(2-hydroxy-phenyl)-thiazole-4-carboxamides and -thiocarboxamides (BHPTCs) form a family of
gemini hexacoordinated bis-tridentate chelating scaffolds. Four molecules were synthesized and shown
to chelate iron(III) efficiently with a 1:1 stoichiometry. A dithioamide BHPTC displayed promising anti-
proliferative activity in several cancerous cell lines, making this molecule an interesting lead compound
for the design of new iron-chelating anticancer drugs. Conversely, diamide BHPTCs had significant cyto-
protective activity against iron overload in HepaRG cells in vitro, and were as efficient as and less toxic
than deferoxamine B (DFO).
Keywords:
Iron chelator
Cancer
Ó 2009 Elsevier Ltd. All rights reserved.
Iron chelation therapy
Thioamide
1. Introduction
Alternatively, from repeated transfusions may result in secondary
iron overload, as in sickle cell anemia7,8 and thalassemia major.8,9
Metals play a key role in the functional dynamics of living sys-
tems by promoting interactions and transformations of biomole-
cules. Iron is undoubtedly one of the most important metals in
biological systems. This metal is predominant in the active sites of
enzymes catalyzing many essential processes, such as the respira-
tory chain, metabolic transformations and deoxyribonucleotide bio-
synthesis. This predominance in such important systems makes iron
chelators potentially interesting bioactive agents for use in medi-
cine. The development of new iron chelators for medical purposes1
is opening up new possibilities for the treatment of severe diseases,
such as malaria,2 AIDS,3 and neurodegenerative diseases.4 This list is
far from exhaustive. However, two types of pathologies are histori-
cally more concerned with applications of iron chelators:iron chela-
tion therapy (ICT) for patients suffering of chronic iron overload and
pioneer approaches for the treatment of cancer by iron sequestra-
tion. In humans, iron homeostasis is regulated through the effects
of hepcidin on intestinal iron absorption and iron storage in the li-
ver.5 Some genetic diseases lead to iron overload, which may be
symptomatic (primary iron overload), as in hemochromatosis.6
Chronic iron overload causes progressive and severe liver damage.
Secondary iron overload is mostly treated by iron chelation therapy
with suitable chelators protecting sensitive organs and promoting
metal excretion by natural routes. Three compounds are used for
ICT.8 The oldest of these compounds, deferoxamine B (DFO or Des-
feral™), provides effective relief from symptoms in many patients.
However, the bioavailability of this compound is low, necessitating
overnight administration by perfusion.8,9 Severe side-effects have
also been reported in patients on long-term treatment.10 A second
molecule, 1,2-dimethyl-3-hydroxypyridin-4-one or deferiprone
(Feriprox™), is highly effective when administered per os,11 but is
thought to cause several rare but very severe side-effects.10b,12 More
recently, deferasirox (ICL670 or Exjade™), has been approved in sev-
eral countries.8,13 This oral chelator is administered daily but has not
been in use for long enough to draw any firm conclusions about its
toxicity, although some adverse effects are suspected.14
Iron chelators have several biological targets in humans,
accounting for their high degree of efficacy for treating certain dis-
eases and their toxicity when used over long periods. Cell division,
for example, is a complex process involving a large number of
metal-dependent enzymes and regulatory proteins.15,16 Some of
the proteins regulating the cell cycle, such as cyclins, are strongly
affected by chelator-mediated metal depletion. Iron is also an
essential component of the active sites of crucial enzymes involved
* Corresponding author. Tel.: +33 368854727; fax: +33 368854829.
E-mail address: mislin@unistra.fr (G.L.A. Mislin).
Present address: QUIDD (IRCOF) 1, rue Lucien Tesnière. F-76130 Mont St Aignan,
France.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.11.057

690
D. Rodriguez-Lucena et al. / Bioorg. Med. Chem. 18 (2010) 689–695
in DNA biosynthesis and repair.15,16 Iron chelators could therefore
be used to decrease DNA synthesis, stop cell division and promote
apoptosis. They are therefore promising medicinal tools for con-
taining the proliferation of cancer cells and lowering the risk of
metastatic dissemination.16,17 The first molecules tested in this
context were originally developed for ICT.18,19 However, a new
generation of iron chelators specifically developed for cancer che-
motherapy, subsequently emerged.16,19 Triapine20 (or 3-AP), tach-
pyridine,21 di-2-pyridyl thiosemicarbazones like Dp44mT22 and
the pyridoxal isonicotinoyl hydrazone (PIH) family23 currently ap-
pear to be the most promising molecules for this application,
although none has yet been approved.
Continualefforts are thus required to develop new chelating mol-
ecules with therapeutic profiles, suitable for a specific application in
cancer treatment or ICT. Deferoxamine B (DFO), which is still consid-
ered the gold standard for ICT, is a siderophore synthesized by Strep-
tomyces pilosus.24 These secondary metabolites excreted by
microorganisms to facilitate the acquisition of iron, a crucial nutri-
ent, are highly diverse,25 providing an inexhaustible source of inspi-
ration for the design of new metal-chelating agents. Using this
strategy, Bergeron and coworkers have developed, over the last
20 years, several synthetic chelators derived from desferrithiocin,
a siderophore from Streptomyces antibioticus.26 These studies led to
the development of deferitrin (GT56-252), one of the most promis-
ing iron chelators currently in clinical trials.27 Previous studies of
the physical and chemical properties of compounds related to
pyochelin,28 a siderophore commonto several bacterial species, sug-
gested that 2-(2-hydroxyphenyl)-thiazolin-4-carboxamides and 2-
(2-hydroxyphenyl)-thiazole-4-carboxamides or -thiocarboxamides
would be efficient tridentate iron(III) chelators.29 The ICT drugs Exj-
ade™ and deferitrin and the anticancer chelators Dm44pT, triapine,
tachpyridine and the PIHs are all also tridentate chelators.30 Thus,
iron complexation by a bis-tridentate molecule appears to be a
promising avenue of research for the development of the next gen-
eration of chelators for treatment purposes. The chelators developed
to date1 includes only few rare examples of extensively studied bis-
tridentate chelators for therapeutic purposes.26a,b We report here
the synthesis and the biological data concerning four molecules of
the bis-2-(2-hydroxyl-phenyl)-thiazole-4-carboxamide and -thio-
carboxamide (BHPTC) family characterized by a gemini hexacoordi-
nate bis-tridentate chelating scaffold.
OH
OH
MeO
O
N
i (90%)
MeOOC
MeOOC
N
2
O
S
1
OH
ii
O
HN
O
S
S
MeOOC
N
N
HN
OH
O
O
OH
O
iii (71%)
MeOOC
OH
O
OH
S
HN
O
N
HN
O
N
MeOOC
S
3
4
Scheme 1. Synthesis of compound 4. Reagents and conditions: (i)
MeOH, 0.1 N phosphate buffer pH 6.4, 60 °C (90%); (ii) EDCI, NH2C2H4O-
C2H4OC2H4NH2, CH2Cl2, 25 °C; (iii) CBrCl3, DBU, CH2Cl2, 25 °C (71% for two steps).
L
-cysteine,
1 was condensed with L-cysteine in a buffered hydromethanolic
medium.26a The crude resulting thiazolin 2 isolated with a 90%
yield was coupled with 2,20-(ethylenedioxy)-bis-ethylamine in
the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDCI). The resulting crude mixture of diastereoisomers 3 was then
treated with CBrCl3 in the presence of DBU,32 generating the corre-
sponding dithiazole compound 4 isolated with a 71% overall yield
in two combined steps (Scheme 1). This method has been success-
fully used in the past for the synthesis of thiazole analogs of pyoch-
elin.33 Other methods tested for the conversion of dithiazoline 3
into the corresponding dithiazole compound 4 resulted in complex
mixtures in which unreacted starting material is predominant.
Diamide 4 was then converted into the corresponding dithioa-
mide 5, using Lawesson’s reagent.34 This reaction appears to be
fully regioselective, as we detected no side products resulting from
the thionation of one of the other carbonyl groups on the molecule.
Further saponification of compounds 4 and 5 converted these two
diesters into the corresponding diacids, 6 and 7, isolated with
yields of 65% and 59%, respectively (Scheme 2).
Compounds 4 and 6 were isolated in respectively 64% yield in
three steps and 41% yield in four steps, starting from 3-cyano-4-
hydroxy-methylbenzoate 1, with only one chromatographic purifi-
cation step. Chromatographic purification should be performed on
demetallated silica gel column in order to avoid a contamination
by the various metals present in commercial stationary phase.
The compounds 5 and 7 were isolated respectively in 40% yield
in four steps and 36% yield in five steps from the starting nitrile
compound 1.
2. Results and discussion
2.1. Synthesis
The BHPTC molecular scaffold is based on the chelating proper-
ties of two 2-(2-hydroxyphenyl)-thiazole-4-carboxylic acid mole-
cules. The heteroatoms involved in metal coordination are the
phenol oxygen, the thiazole nitrogen and the oxygen/sulfur atom
from the amide/thioamide group, as suggested by previous re-
sults.29 In a first approach, the aromatic ring was substituted with
a methylester function in para from phenol. This function may be
converted easily into many other organic functions. This strategy
make possible, at one and at the same time, to tune the acido-ba-
sic/chelating properties of the phenol function and the solubility of
our chelators in physiological medium. The two tridentate moieties
are connected to each other by a spacer arm with amide/thioamide
groups. We initially selected 2,20-(ethylenedioxy)-bis-ethylamine
as the spacer. Molecular modeling showed this spacer to be of an
ideal length and flexibility for optimal organization of the two tri-
dentate moieties in the iron(III) coordination shell. Starting from
commercially available 4-hydroxy-methylbenzoate, 3-cyano-4-hy-
droxy-methylbenzoate 1 was prepared in two steps, according to
an efficient, published protocol.31 The nitrile function of compound
O
S
S
S
ROOC
ROOC
ROOC
N
N
HN
HN
OH
OH
O
O
O
O
i
OH
S
OH
(96%)
HN
O
HN
S
N
N
ROOC
S
4 (R = CH3)
6 (R = H)
5 (R = CH3)
7 (R = H)
iii
(59%)
ii
(65%)
Scheme 2. Synthesis of compounds 5 to 7. Reagents and conditions: (i) Lawesson’s
reagent, toluene, reflux; (ii) 1 N NaOHaq, THF, 70 °C; (iii) 1 N KOHaq, dioxane, 60 °C.

D. Rodriguez-Lucena et al. / Bioorg. Med. Chem. 18 (2010) 689–695
691
2.2. Iron chelation properties
more efficient than DFO for iron chelation. Conversely, chelator 4
(IC50 = 1.0 M), which only partly restores calcein fluorescence in
l
Compounds 4, 5, 6 and 7 were treated with a hydromethanolic
solution of iron trichloride. The corresponding dark blue complexes
were isolated quantitatively and mass spectrometry showed that
ferric chelates with a 1:1 stoichiometry were the only products.
The desulfurization products which may be expected by the reaction
of compounds 5 and 7 with a Lewis acid such iron trichloride were
neither observed nor isolated.
this range of concentrations, seems to be less efficient that DFO for
iron chelation (Fig. 1).
Molecules 4–7 were found to be iron chelators of sufficient po-
tency to compete for this metal in physiological medium. We
therefore assessed the cytotoxicity of these molecules and their
antiproliferative activity in the human hepatoma cell line He-
paRG36 and several other cancer cell lines. In parallel, we evaluated
the cytoprotective effects of the chelators 4 and 6 against iron
overload.
The cellular labile iron pool (LIP) is a pool of chelatable and redox-
active iron, which plays a key role as a crossroad of cell iron metab-
olism. The ability of iron chelators to mobilize this temporary iron
pool bound to low-molecular weight and low-iron affinity chelators
(citrate, ascorbate, phosphate and adenosine triphosphate) is an
essential factor influencing their biological efficiency. So, in parallel,
an acellular calcein test was performed to compare the potential
ability of compounds 4, 5, 6, 7 and DFO to compete for this chelatable
iron pool in a physiological medium. This test uses calcein, a fluores-
ceinated analog of EDTA that binds both iron(II) and iron(III), with
the trivalent cation bound more slowly (Ka = 1024 Mꢀ1). This me-
tal-chelating dye, mainly used to estimate cellular iron level was
previously shown to be oxidatively degraded by iron(II) in a H2O2-
dependant pathway.35 Therefore we checked first that calcein-iro-
n(III) complex, which probably involves also ferric hydroxide and
ferric oxide interactions, is not degraded in our experiment condi-
tions (1 h in HEPES buffer at pH 7.3, data not shown). In solution,
the fluorescence of this metallosensor dye is quenched during its
interaction with iron and restored when iron is removed from the
calcein-iron complex by various chelators. The rate and extent of
fluorescence recovery (kExc = 485 nm, kEm = 520 nm) depend on che-
lator concentration, the kinetics and stoichiometry of iron binding
and the relative binding affinity. Increases in fluorescence may
therefore be correlated directly with iron sequestration following
the gradual addition of a competitive iron chelator to ferric calcein.
Thus, compounds 4–7 were compared with deferoxamine (DFO) in
titration experiments using the calcein test. DFO, which is still con-
sidered to be the gold standard in the field of iron chelation for treat-
2.3. Cytotoxicity and antiproliferative activity in HepaRG cells
We evaluated the effects of compounds 4–7 and DFO on cell
viability in an MTT test assessing succinate dehydrogenase (SDH)
activity in proliferating HepaRG cells. Mitochondrial SDH converts
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide
(MTT) into formazan, a compound detectable at 535 nm. The resid-
ual SDH activities of the cells after treatment with the new chelators
and DFO are expressed as a percentage of the control value. Enzyme
activity was assessed in the absence of iron(III) or in the presence of
20
decreased cell viability more strongly than DFO (IC50 = 50
and compounds 4 (IC50 = 64 11 M), 6 (IC50 = 120 M) and 7
(IC50 = 114 M) were less cytotoxic than DFO (Fig. 2).
This effect was only partly reversed in the presence of 20
exogenous iron(III) for DFO (IC50 = 70 10 M), chelators
(IC50 = 78 M) and 5 (IC50 = 50
l
M iron(III). In absence of iron(III), chelator 5 (IC50 = 42
7
lM)
4
lM),
l
6 l
8
l
lM
l
4
6
l
8
lM), whereas no significant
difference was observed for ligands 6 and 7 (data not shown).
These results are consistent with the increase in extracellular lac-
tate dehydrogenase (LDH) activity observed only for concentra-
tions of compounds 4, 6, 7 and DFO greater than 100 lM. LDH is
a cytoplasmic protein, so this increase in the extracellular LDH
activity is a marker of cell membrane disruption correlated to cyto-
toxicity. Conversely, compound 5 was highly cytotoxic to HepaRG
cells at concentrations above 50 lM (data not shown). We there-
ment purposes, with an IC50 of 4.4
of chelator 6. With IC50 values of 0.07
mide compounds 7 and 5, respectively, these compounds are much
l
M, has efficiency similar to that
fore assessed the antiproliferative activity of the four BHPTC
ligands in a wider range of cancer cell lines. These compounds
were tested on 13 human cancer cell lines representative of various
tissues or organs. We compared the four chelators tested with DFO.
l
M and 0.50 M for dithioa-
l
All five compounds were tested at two concentrations—10
lM and
1
l
M—in the following cell lines: KB (epidermoid carcinoma),
80 000
70 000
60 000
50 000
40 000
30 000
120
110
100
90
80
70
60
50
40
30
20
10
0
ol
RFUalcein
20 000
10 000
0
%MT/contr
DFO
4
5
6
7
0,01
0,1
1
10
100
[Chelator] (µM)
1
10
100
1000
[Chelator] (µM)
Figure 1. Comparison of the iron-chelating efficiency of compounds 4 (ꢀ), 5 (d), 6
(N) and 7 (j), with DFO (s), by calcein fluorescence measurements in a cell-free
system. Fluorescence of 100 nM calcein (kExc = 485 nm, kEm = 520 nm) in the
Figure 2. Effect of ligands
4 to 7 and DFO on cell viability (MTT assay) in
presence of 1
l
M Fe(III) in Hepes buffer (20 mM HEPES, 150 mM NaCl, pH 7.3)
proliferating HepaRG cell cultures. HepaRG cells at D4 were maintained in culture
in the absence of iron(III) for 72 h, with various concentrations of 4 (ꢀ), 5 (d), 6 (N),
7 (j) and DFO (s).
was detected in a microplate fluorescence reader (free calcein). Values are means of
three independent experiments.

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D. Rodriguez-Lucena et al. / Bioorg. Med. Chem. 18 (2010) 689–695
HCT116, HT29 and HCT15 (colon adenocarcinoma cells), MCF7
(breast adenocarcinoma), MCF7R (doxorubicin-resistant MCF7),
SK-OV-3 (ovary adenocarcinoma), HepG2 (hepatocarcinoma), PC-
ative activity and large capacity to penetrate membranes is re-
quired for chelators for anticancer treatments. Compounds 4, 5, 6
and 7 have a similar molecular architecture but differ in lipophilic-
ity and in the heteroatoms involved in the iron coordination shell
(amides vs thioamides). These slight chemical differences strongly
affected the biological properties of our chelators. The diamide
compounds 4 and 6 protected cells against iron overload as effi-
ciently as DFO. However, the hydrosoluble diamide chelator 6
exhibits in vitro a lower cytotoxicity, independent from iron(III),
than the reference ICT drug. Our results suggest that hydrophilic
BHPTCs bearing amide groups are interesting lead compounds for
the development of a new generation of safer chelators for ICT.
By contrast, the dithioamide chelator 5 had higher levels of cyto-
toxic/antiproliferative activity than both chelator 4 and DFO,
whereas compounds 6 and 7 were totally ineffective, probably be-
cause they were not lipophilic enough to cross the cell membrane
and compete for the intracellular iron pool.37 Thus, liphophilic
BHPTC dithioamide derivatives may be considered a promising
synthetic platform for the design of new iron chelators for antican-
cer chemotherapy.
3
(prostate adenocarcinoma), A549 (lung carcinoma), HL60
(promyeocytic leukemia), K562 (chronic myelogenous leukemia)
and SF268 (glioblastoma) (Table 1). DFO displayed moderate anti-
proliferative activity at 10
lM and had only a very slight impact on
proliferation at a concentration of 1
lM. Compounds 4, 6 and 7
were less active than DFO, with little or no antiproliferative activity
at either of the concentrations and in any of the cells studied. Con-
sistent with our findings for HepaRG cells, compound 5 appeared
to be the most interesting molecule, as it was more active, at a con-
centration of 10 lM, than DFO for almost all the cell lines tested,
and had a particularly strong impact on the proliferation of A549
lung carcinoma cells. However, the effect of this compound was
much weaker at a concentration of 1 lM.
Based on the results of the MTT test, diamide compounds 4 and
6, which were less toxic than DFO, were tested for cytoprotective
activity against iron overload.
2.4. Cytoprotective activity
3. Conclusion
We assessed the cytoprotective effect against iron overload tox-
icity of compounds 4 and 6 and DFO on differentiating HepaRG
cells cultured in the presence of ferric nitriloacetate (FeNTA), as
an in vitro model of the hepatocytes of a patient presenting iron
overload. The cytotoxicity of iron overload was evaluated by mea-
suring extracellular lactate dehydrogenase (LDH) activity. As men-
tioned before, this enzyme is a cytoplasmic protein, an increase in
the extracellular activity of LDH reflect cell membrane disruption
We have described the synthesis of a new family of gemini bis-
tridentate hexacoordinated iron chelators, BHPTCs—bis-hydroxy-
phenyl-thiazole-carboxamides or -thiocarboxamides. BHPTCs 4–7
were efficiently synthesized and formed one-to-one complexes
with iron(III). These chelators were also able to sequester iron in
physiological media. Chelators 4 and 6 significantly protected
hepatocytes against iron overload and were as efficient as deferox-
amine B (DFO). Three of these four compounds (4, 6 and 7) were
not more cytotoxic than DFO, the gold standard for iron chelation
therapy (ICT). By contrast, chelator 5 had higher levels of cytotoxic/
antiproliferative activity than DFO, demonstrating the considerable
potential of the BHPTC scaffold for the development of therapeutic
iron chelators.38 We are currently synthesizing new chelating mol-
ecules for cancer treatment or ICT, based on the results reported
here.
correlated with the cytotoxicity. The addition of 50 lM FeNTA to
the culture medium increased extracellular LDH activity by a factor
of 6.5 times (643 18%) over that recorded in the control experi-
ment (100%), demonstrating the toxicity of iron overload in hepa-
tocytes. In the presence of deferoxamine (DFO), the impact of iron
overload was greatly decreased, with extracellular LDH activity
only 1.6 times higher (162 30%) than in the control experiment.
With increases of LDH activity of 148 55% and 179 20%, respec-
tively, chelators 4 and 6 proved to be as efficient as DFO for
protecting hepatic cells from iron overload. The differences in iron
sequestration from calcein observed between 4 and 6 in an
acellular test seem to have been equaled in the hepatoma cell cyto-
protection test.
4. Experimental
4.1. Chemistry
Iron chelators are highly effective drugs, but have the downside
of being cytotoxic, often due to a lack of selectivity for the iron
pools targeted. This aspect is particularly crucial for chelators
developed for ICT, whereas a high level of cytotoxicity/antiprolifer-
All the reactions were carried out under an inert argon atmo-
sphere. Analytical grade solvents were used. Reactions were moni-
tored by thin-layer chromatography (TLC), using Merck precoated
silica gel 60F254 (0.25 mm). Column chromatographies were per-
formed with demetallated Merck kieselgel 60 (63–200 l
m).39 Melt-
Table 1
ing points were determined with a Stuart Scientific Bibby SMP3
apparatus. NMR spectra were recorded on a Bruker Avance 300
(300 MHz for 1H and 75 MHz for 13C). Mass spectra were recorded
in the Service Commun d’Analyse (SCA) de la Faculté de Pharmacie
de l’Université de Strasbourg and were measured after calibration in
In vitro antiproliferative activity in 13 cancer cell lines after 72 h of exposure to
compounds 4–7 and DFO at concentrations of 10 lM and 1 lM
Cell lines
4a,b
5a,b
6a,b
7a,b
DFOa,b
KB
28/0
13/0
4/0
14/11
4/0
19/0
11/0
37/8
19/0
22/10
0/0
58/5
69/6
3/0
2/0
0/0
1/0
0/0
0/0
2/0
0/1
8/0
0/0
7/4
0/0
9/0
0/0
0/0
0/0
0/0
8/0
0/0
1/0
7/0
0/0
5/0
2/3
0/0
2/0
0/0
16/0
20/0
42/0
46/0
0/0
HCT116
HT29
HCT15
MCF7
MCF7R
A549
ES-TOF experiments on
spectrometer.
a Bruker Daltonic MicroTOF mass
42/1
10/0
64/0
98/8
54/10
62/0
45/10
52/0
53/1
43/0
4.1.1. 2-(2-Hydroxy-5-methoxycarbonyl-phenyl)-4,5-dihydro-
thiazole-4-carboxylic acid (2)
9/0
50/2
44/1
33/0
52/5
0/0
A
solution of 3-cyano-4-hydroxy-methylbenzoate 131 (1 g,
PC3
SF268
SK-OV-3
HL60
K562
HepG2
5.65 mmol) and L-cysteine (1.59 g, 13.11 mmol) in a mixture of
MeOH (32 mL) and phosphate buffer (pH 6.4, 0.1 N, 20 mL) was
heated at 60 °C, with stirring, for 16 h. The mixture was then
cooled down to room temperature before being evaporated to dry-
ness under reduced pressure. The resulting yellow foamy residue
was dissolved in water (100 mL) and washed with a 2:1 mixture
10/2
26/0
0/0
0/0
a
Percentage inhibition of cell proliferation at 10ꢀ5 M/10ꢀ6 M.
Values are means of triplicate experiments.
b

D. Rodriguez-Lucena et al. / Bioorg. Med. Chem. 18 (2010) 689–695
693
of cyclohexane and Et2O (75 mL). The pH of the aqueous phase was
4.1.4. Benzoic acid, 3,30-[(1,12-dioxo-5,8-dioxa-2,11-diazad-
odecane-1,12-diyl)bis(4,2-thiazolediyl)]bis-[4-hydroxy] (6)
adjusted to 2.0–3.0 by addition of solid citric acid. The solution was
then extracted with CH2Cl2 (3 ꢁ 75 mL). The organic layers were
collected and dried over Na2SO4 before filtering and evaporation
under reduced pressure. The expected thazoline compound 2
(1.43 g, 5.10 mmol, yield: 90%) was isolated as a yellow crystalline
powder and was used in this form for subsequent steps. Mp: 184–
186 °C. 1H NMR (300 MHz, DMSO-d6) d 3.69–3.81 (m, 1H), 3.84 (s,
3H), 5.51 (dd, J = 7.5, 9.6 Hz, 1H), 7.10 (d, J = 9.3 Hz, 1H), 7.98–8.02
(m, 2H). 13C NMR (75Mz, DMSO-d6) d 33.72, 52.07, 76.10, 115.46,
117.48, 120.58, 131.69, 134.26, 162.16, 165.08, 170.99, 171.98.
Diester 4 (257 mg, 0.38 mmol) was suspended in a mixture of
THF (30 mL) and an aqueous 1 N solution of NaOH (30 mL). The
suspension was heated at 70 °C for 4 h. The crude mixture was
cooled to room temperature, diluted in water (50 mL) and Et2O
(100 mL) and vigorously stirred for few minutes. The aqueous
phase was then acidified to pH 1 by adding aqueous HCl 1 N. The
resulting precipitate was filtered off, washed with ice-cold milliQ
water and dried under reduced pressure. The expected diacid 6
(160 mg, 0.25 mmol, yield: 65%) was isolated as a white powder.
1H NMR (300 MHz, DMSO-d6) d 3.45–3.60 (m, 12H), 7.12 (d,
J = 8.4 Hz, 2H), 7.90 (dd, J = 2.1, 8.4 Hz, 2H), 8.27 (s, 2H), 8.50 (t,
J = 5.7 Hz, 2H), 8.88 (d, J = 2.1 Hz, 2H). 13C NMR (75 MHz, DMSO-
d6) d 38.20, 68.65, 69.26, 116.07, 118.97, 122.02, 124.37, 129.67,
132.32, 148.89, 158.58, 160.66, 161.66, 166.82. MS: m/z 643
(M+H+), 665 (M+Na+), 681 (M+K+). HRMS m/z found: 665.099
(M+Na+), m/z calcd for C28H26N4NaO10S2: 665.071.
4.1.2. Benzoic acid, 3,30-[(1,12-dioxo-5,8-dioxa-2,11-diazado-
decane-1,12-diyl)-bis(4,2-thiazolediyl)]bis-[4-hydroxy], 1,10-
dimethyl ester (4)
A
solution of 2,20-(ethylenedioxy)-bisethylamine (134 mg,
0.93 mmol) in CH2Cl2 (20 mL) was added dropwise at room tem-
perature (25–27 °C) to a solution of 2-(2-hydroxy-5-methoxycar-
bonyl-phenyl)-4,5-dihydro-thiazole-4-carboxylic acid 2 (565 mg,
2.01 mmol) and EDCI (463 mg, 2.41 mmol) in CH2Cl2 (30 mL). The
mixture was then stirred for 16 h at 25 °C and washed with
0.5 M aqueous HCl solution (50 mL). The organic phase was col-
lected, dried over Na2SO4, filtered and the solvent was evaporated
under reduced pressure. The crude mixture was dissolved in
4.1.5. Benzoic acid, 3,30-[(1,12-dithioxo-5,8-dioxa-2,11-diaza-
dodecane-1,12-diyl)bis(4,2-thiazolediyl)]bis[4-hydroxy] (7)
Diester 5 (63 mg, 89 lmol) was suspended in a mixture of diox-
ane (5 mL) and an aqueous 1 N solution of KOH (5 mL). The suspen-
sion was heated at 60 °C for 2 h. The crude mixture was cooled to
room temperature, diluted with water (50 mL) and CH2Cl2 (30 mL)
and vigorously stirred for a few minutes. The aqueous phase was
then acidified to pH 1 by adding aqueous HCl 1 N. The resulting
precipitate was filtered off, washed with ice-cold water and dried
CH2Cl2 (50 mL) and DBU (546
lL, 555 mg, 3.65 mmol) was added,
followed by CBrCl3 (315 L, 634 mg, 3.20 mmol). The resulting
l
solution gradually turned brown and was stirred for 14 h at
25 °C. The mixture was then adsorbed and purified onto demetal-
lated silica gel (30 g of SiO2, CH2Cl2 then CH2Cl2/EtOH: 95/5). The
pale yellow solid obtained was washed with boiling ethanol, fil-
tered and dried under reduced pressure. Chelator 4 (444 mg,
0.66 mmol, overall yield over two steps: 71%) was isolated as a
white powder. 1H NMR (300 MHz, DMSO-d6) d 3.44–3.61 (m,
12H), 3.83 (s, 6H), 7.12 (d, J = 8.6 Hz, 2H), 7.90 (dd, J = 2.3, 8.6 Hz,
2H), 8.28 (s, 2H), 8.50 (t, J = 5.7 Hz, 2H), 8.88 (d, J = 2.3 Hz, 2H).
13C NMR (75 MHz DMSO-d6) d 38.35, 51.95, 68.99, 69.61, 116.61,
119.25, 121.00, 124.56, 129.54, 131.95, 149.00, 158.97, 160.75,
161.50, 165.80. MS m/z 671 (M+H+), 693 (M+Na+), 709 (M+K+).
HRMS m/z found: 671.1359 (M+H+), m/z calcd for C30H31N4O10S2:
671.1476.
under reduced pressure. The expected diacid 7 (35 mg, 52 lmol,
yield: 59%) was isolated as a pale yellow powder. 1H NMR
(300 MHz, DMSO-d6) d 3.67 (s, 4H), 3.73 (t, J = 5.8 Hz, 4H), 3.94
(q, J = 5.8 Hz, 4H), 7.12 (d, J = 8.7 Hz, 2H), 7.91 (dd, J = 2.1, 8.7 Hz,
2H), 8.45 (s, 2H), 8.91 (d, J = 2.1 Hz, 2H); 10.38 (t, J = 5.7 Hz, 2H).
13C NMR (75 MHz, DMSO-d6)
d 44.44, 67.23, 69.68, 116.34,
118.98, 122.03, 127.00, 129.72, 132.43, 152.61, 158.86, 161.33,
166.87, 186.13. HRMS m/z found 675.070 ([M+H]+), calcd for
C28H27N4O8S4: 675.071.
4.1.6. Ferric complexes of chelators (4), (5), (6) and (7)
To solutions of compounds 4, 5, 6 and 7 (7.11
(2.5 mL), we added 0.22 M FeCl3 (prepared by dilution of a commer-
cial aqueous 2.2 M solution, 32 L, 7.11 mol). A dark blue suspen-
lmol) in MeOH
4.1.3. Benzoic acid, 3,30-[(1,12-dithioxo-5,8-dioxa-2,11-diaza-
dodecane-1,12-diyl)bis(4,2-thiazolediyl)]bis-[4-hydroxy], 1,10-
dimethyl ester (5)
l
l
sion was obtained, which was stirred for 16 h at 20 °C. The solvent
was evaporated and the residue suspended in water and sonicated
for two minutes. Water was eliminated by freeze-drying, to obtain
the corresponding ferric complexes in quantitative yield. Ferric
complex of chelator 4: MS m/z found 724.05 ([M(4)ꢀ2H+Fe]+).
HRMS m/z found 724.0595 ([M(4)ꢀ2H+Fe]+), calcd for C30H28Fe-
N4O10S2: 724.0591. Ferric complex of chelator 5: MS m/z found
756.0 ([M(5)ꢀ2H+Fe]+). Ferric complex of chelator 6: MS m/z found
696.0 ([M(6)ꢀ2H+Fe]+). Ferric complex of chelator 7 MS m/z found
727.9 ([M(7)ꢀ2H+Fe]+).
Diamide
4 (350 mg, 0.52 mmol) and Lawesson’s reagent
(422 mg, 1.04 mmol) were suspended in anhydrous toluene
(20 mL). The resulting suspension was refluxed (110 °C) for 2 h.
The mixture was cooled to room temperature and the solvent
was evaporated under reduced pressure. The oily residue was dis-
solved in CH2Cl2 (50 mL) and this organic phase was then washed
successively with water (30 mL), a saturated aqueous solution of
NaHCO3 (30 mL) and finally brine (30 mL). The organic phase was
then dried over Na2SO4, filtered and the solvent removed by evap-
oration under reduced pressure. Chromatography on a demetallat-
ed silica gel column (30 g of SiO2, MeOH/CH2Cl2: 1/99) resulted in
the purification of the expected dithioamide 5 (351 mg, 0,50 mmol,
yield: 96%) isolated as a bright yellow powder. 1H NMR (300 MHz,
DMSO-d6) d 3.68–3.97 (m, 12H), 3.83 (s, 6H), 7.12 (d, J = 8.6 Hz,
2H), 7.90 (dd, J = 2.2; 8.6 Hz, 2H), 8.45 (s, 2H), 8.90 (d, J = 2.2 Hz,
2H), 10.36 (t, J = 5.7 Hz, 2H). 13C NMR (75 MHz, DMSO-d6) d
44.57, 51.94, 67.30, 69.70, 116.55, 119.23, 121.03, 127.16, 129.55,
132.32, 152.68, 159.11, 161.14, 165.78, 186.14. MS m/z 703
(M+H+), 725 (M+Na+), 741 (M+K+). HRMS m/z found: 703.1013
(M+H+), m/z calcd for C30H31N4O8S4: 703.1019. Anal. Calcd for
C30H30N4O8S4: C, 51.27; H, 4.30; N, 7.97. Found: C, 51.05; H,
4.55; N, 7.68.
4.2. Calcein fluorescence measurements
The fluorescence (kExc = 485 nm, kEm = 520 nm) of calcein
(100 mM) in Hepes buffer (20 mM HEPES, 150 mM NaCl, pH
7.3) was measured as a function of time, at room temperature
(20 °C), in a microplate fluorescence reader (Packard, Fusion™),
equipped with an orbital stirrer. Iron(III) (1 lM) reacted slowly
with calcein and maximal fluorescence quenching was observed
after 6 h. The kinetics of fluorescence recovery was monitored
over a period of one hour in the presence of various chelator
concentrations, and the initial rate of dequenching was deduced
from the kinetics.

694
D. Rodriguez-Lucena et al. / Bioorg. Med. Chem. 18 (2010) 689–695
4.3. HepaRG cell cultures
treated 24 h later with compounds dissolved in DMSO at concentra-
tions of 1 and 10 M, using a Biomek 3000 (Beckman-Coulter). Con-
l
HepaRG cells were obtained from a liver tumor (hepatocarci-
noma) from a female patient.36 Small pieces of the tumor were di-
trols received the same volume of DMSO (1% final volume). After
72 h of incubation, MTS reagent (Promega) was added and the plates
incubated for 3 h at 37 °C. Absorbance was monitored at 490 nm and
the results are expressed as the inhibition of cell proliferation, calcu-
lated as the ratio [(1 ꢀ (OD490 treated/OD490 control)) ꢁ 100] in trip-
licate experiments.
gested with 0.025% collagenase
D diluted in Hepes buffer
supplemented with 0.075% CaCl2. The cell population was sus-
pended in William’s E medium supplemented with 10% fetal calf
serum (FCS), 5
l
g/mL insulin and 5 ꢁ 10ꢀ7 M hydrocortisone hemi-
succinate and dispensed into several dishes. The cell populations
most closely resembling hepatocytes were selected and passaged
by gentle trypsin treatment. After three passages, cell aliquots were
preserved by freezing. The cells from a frozen aliquot were defrosted
and used to seed, at low density (2.5 ꢁ 104/cm2), a growth medium
consisting of William’s E medium supplemented with 10% fetal calf
Acknowledgments
G.L.A.M., D.R.L., F.R. and I.J.S. would like to thank the Centre
National de la Recherche Scientifique (C.N.R.S.), l’Agence Nationale
pour la Recherche (ANR-05-JCJC-0181-01) and Région Alsace for
financial support. G.L. and F.G. would like to thank la Ligue contre
le Cancer (Ille et Vilaine/Loire Atlantique).
serum (FCS), 100 units/mL penicillin, 100 lg/mL streptomycin,
5
l
g/mL insulin, 2 mM glutamine and 5 ꢁ 10ꢀ5 M hydrocortisone
hemisuccinate. After two weeks of culture, the cells reached conflu-
ence and began to differentiate. A higher state of differentiation was
attained by shifting the cells to the same culture medium supple-
mented with 1.5% DMSO and incubating for two weeks.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.11.057.
4.4. Lactate dehydrogenase (LDH) activity
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Germany). Extracellular LDH activity was measured with the man-
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