S. Abdelsayed et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3854–3860
3859
0,6
0,4
0,2
0,0
0,6
5-Fe
5
10-Fe
17-Fe
18-Fe
19-Fe
20-Fe
23-Fe
10
17
0,4
0,2
0,0
18
19
20
23
300
400
500
600
300
400
500
600
700
800
wavelength (nm)
wavelength (nm)
Figure 6. Absorption spectra of 5, 10, 17–20 and 23 (left) and their corresponding iron complexes (right) at pH 7.4, with ligand concentration (c0) of 5.0 ꢃ 10ꢂ5 M,
25 °C.
l
= 0.2 at
19–20 is probably different from that followed by the other com-
pounds synthesized, since in these cases partial suppression of
their inhibitory effect is induced by an excess of iron, even at high
inhibitor concentration. Moreover, the anti-chlamydial activity of
the molecules tested is related to inhibition of the development
of bacterial inclusions, as shown by immunofluorescence (Fig. 5).
In order to better understand the putative link between the already
observed bactericidal activity and iron-chelating properties, com-
pounds 5, 10, 17–20 and 23 were investigated by UV–visible spectro-
photometric titration (Table 1 and SI). The overall stability constants
(logb) and pFe values were determined from the proton dissociation
and the iron-binding constants. pFe is the negative logarithm of the free
Fe3+ concentration (ꢂlog[Fe3+]) at pH 7.4 for an analytical ligand and
Fe3+ concentrations of 10ꢂ5 M and 10ꢂ6 M, respectively. Unlike the sta-
bility constant logb, the pFe value takes into account the influence of
ligand proto-dissociation, the stoichiometries and the affinity con-
stants. It, therefore, provides a comparison between the efficiency of
different iron chelators under predefined standard conditions close
to those prevailing in physiological media.22,23
Finally, the expected absorption, distribution, metabolism and
excretion (ADME) properties of the tested compounds, evaluated
by means of the OCHEM predictor,26 are reported in the SI
(Table III). The selected properties are known to influence metabo-
lism, cell permeation, and bioavailability. The results show that all
compounds present a logP value and a molecular weight lower
than 5 and 500, respectively, values related to good absorption in
humans according to Lipinski’s rules.27 Moreover, except com-
pound 20, the tested compounds should not inhibit cytochrome
P450, a family of enzymes involved in the metabolism of a large
number of xenobiotics. The number of groups in each molecule
that can give hydrogen atoms to hydrogen bonds and the number
of groups that can accept hydrogen atoms to form hydrogen bonds
should be optimized for our compounds to improve their drug
potential.
In conclusion, we have shown that these 3-isoxazolidone
derivatives, with moderate iron-chelating properties, inhibit
Chlamydia trachomatis in a dose-dependent manner without any
toxicity at high concentration (200 lM). Among these, compound
Experiments were performed at an ionic strength of
l
= 0.2 in
19, an 8-hydroxyquinoline derivative, strongly inhibits Chlamydia
development at low concentrations. This bactericidal activity is
not related to iron chelation. It is most probably the result of either
an interference with fundamental bacterial metabolisms or carri-
ers, such as the T3SS bacterial secretion system, or peptidoglycan
synthesis. Further investigations are required to achieve a better
understanding of this particular anti-chlamydial activity. This
investigation provides a new example of the antibiotic activity of
the 8-hydroxyquinoline skeleton.
the presence of 10% DMSO to avoid ligand or complex precipita-
tion. At pH 7.4, the 3-isoxazolidone derivatives 5, 10, 17–20 and
23 show a strong absorption in the UV range (298–350 nm) due
to p–
p⁄ transitions (Fig. 6, left). Except for 19 and 20, the addition
of Fe3+ to the chelators in solution causes large variations in the
absorption spectra. The UV band red-shifts by 5–10 nm with
increase in absorption (Fig. 6, right). Furthermore, ligand-
to-metal-charge-transfer bands (LMCT) appear. On the other hand,
the addition of Fe3+ to 19 or 20 has no effect on the absorption
spectra. We, therefore, assume that these two molecules do not
complex Fe3+ under our conditions.
Acknowledgments
Our results show that the catechol moieties, and to a lesser
extent the imidazole group for compound 23, are responsible for
iron chelation because the acid dissociation constants (pKa) are
affected by the presence of iron (see SI).22,23 In contrast, the
remaining ligands form complexes with iron in 1 to 1 stoichiome-
try with, however, a moderate chelation efficacy that can be
explained by the low ligand denticity. Indeed, compound 23 is a
tricoordinate Fe3+ ligand, while biscatechol compounds 5, 10, 17
and 18 are tetradentate ligands. All these chelators have lower
pFe values than that of the multidentate deferoxamine (Table 1).
It should, moreover, be noted that several natural iron chelators,
such as pyochelin (pFe 16), also present iron-chelating properties
similar to those reported in Table 1.24 Finally, the absence of
chelating activity for 19 and 20 confirms the in cellulo assay and
implies that the bactericidal effect of these two compounds is
not related to chelation.
This work was supported by Ph.D. grants from the University
Paris Diderot (S. Abdelsayed) and the University of Langzhou in
China (J. Hai). The National Center for Scientific Research and the
University Paris Diderot are thanked for financial support. The
authors also gratefully thank Chang-Zhi Dong and Jérome Hénault,
for their help with HPLC experiments, and John Lomas for carefully
reading the manuscript, as well as the Small Molecule
Mass Spectrometry platform of IMAGIF (Centre de Recherche de
Supplementary data
Supplementary data (organic synthesis, physicochemical study
and antibacterial assay) associated with this article can be found,