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S.F.P. Braga et al. / European Journal of Medicinal Chemistry 71 (2014) 282e289
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To understand the mechanism of action of these compounds,
two possible molecular targets were investigated: trypanothione
reductase (TR) and cruzain.
Trypanothione Reductase (TR) [14,15] is a vital enzyme for the
antioxidant defenses in trypanosomatids. TR exerts a key role in
combating oxidative stress, regenerating the main antioxidant
present in these protozoa, trypanothione [16]. TR is involved in the
reduction of trypanothione disulfite T[S]2 to the dithiol T[SH]. This
enzyme also is considered an attractive due to its high conservation
between Leishmania and trypanosomes [17e19], since it is expected
that a TR inhibitor will be potentially active against both Trypano-
soma and Leishmania parasites.
Besides participating in an essential pathway for parasite sur-
vival, the remarkable structural differences with respect to the
human enzyme, Glutathione Reductase (GR), makes TR as potential
target for the development of selective inhibitors. The main dif-
ferences between TR and GR are related to the size, charge and
distribution of hydrophilic/hydrophobic regions in the active site of
the enzymes. The active site of TR is larger than that of GR and is
negatively charged with surrounding hydrophobic residues to
accommodate the positively charged spermidine portion of T[S]2
[20]. In contrast, the GR active site has three positively charged
arginine residues (Arg37, Arg38 and Arg347), required for interac-
tion with its substrate, GSSG, which has a formal charge of ꢂ2 at
physiological pH [21].
We hypothesized that bis-(arylmethylidene)-cycloalkanones
could interact strongly with the active site of TR due to the presence
of bulky and hydrophobic substituents. As human GR has a
hydrophilic and smaller binding site, low affinity would be
expected between these compounds and GR. To confirm this
hypothesis, enzyme inhibition assays were carried out against TR
and GR.
Cruzain, the major cysteine protease from T. cruzi, which is
among the most studied therapeutic targets for Chagas’ disease
[22e24], was also evaluated as a possible mechanism of trypano-
cidal activity. Cysteine proteases play a key role at various stages in
the life cycle of the parasites and are involved in host protein
degradation by the parasite, facilitating the evasion of host defense
mechanism, invasion cellular, replication and differentiation of the
parasite [23,25]. These enzymes contain a catalytic cysteine residue
in its active site responsible for the hydrolysis of peptide bonds
[26,27].
Scheme 1. General synthetic route to bis-(arylmethylidene)cycloalkanones.
performed under microwave irradiation in solvent-free condition
[33] and the product was obtained in low yield (17%), even after
optimization attempts (Scheme 2).
The first attempt to obtain compounds 17 and 18 (Scheme 3)
consisted of a five-step procedure. Initially, reaction of p-tolunitrile
with N-bromosuccinimide (NBS) in the presence of radical initiator
(PhCOO)2 provided the bromo derivative 19 in 58% yield [34].
Reduction of the nitrile group of 19 with diisobutylaluminum
hydride (DIBALH) followed by displacement of the derived benzylic
bromide 20 with sodium azide afforded 4-(azidomethyl)benzal-
dehyde 21 in 97% yield [34,35]. Cross-aldol condensation of 21 with
cyclopentanone led to diazide derivative 22 in quantitative yield.
However, the attempted copper-catalyzed 1,3-dipolar cycloaddi-
tions between azide derivative 22 and 3-butyn-1-ol or phenyl-
acetylene were unsuccessful, probably because of the low solubility
of the 21 in the reaction medium. To overcome this problem, we
decided to repeat the reaction replacing azide 22 by azide 21,
considerably more soluble in organic solvents. Thus, in the pres-
ence of the sodium ascorbate, CuSO4$5H2O and terminal alkyne,
the azide 21 was completely consumed, and the 1,4-disubstituted
triazole products 23 and 24 were formed in 61 and 86% yield,
respectively. Finally, the last step consisted in the aldol condensa-
tion of the aldehydes 23 and 24 with cyclopentanone, resulting in
the formation of the desired products 17 and 18 (Scheme 4).
There are several classes of cruzain inhibitors [24,28,29], but
typically cysteine protease inhibitors possess an electrophilic a,b-
unsaturated moiety, such as, vinyl ketone, vinyl sulfones, vinyl
amide, vinyl ester and vinyl nitrile, acting as a Michael acceptor for
the nucleophilic sulfhydryl groups of catalytic cysteine residue
[30,31]. Based on the potential of the bis-(arylmethylidene)-
cycloalkanones to act as Michael acceptor, the evaluation of the
ability of these compounds to inhibit cruzain is of interest.
3. Biological assays
The compounds were evaluated in vitro for their ability to inhibit
recombinant T. cruzi TR using the colorimetric assay described by
Hamilton et al. (2003) [36]. Inhibition of human GR activity was
carried out in parallel according to Carlberg and Mannervik (1985)
[37]. Cruzain inhibition was measured based on a fluorimetric
2. Chemistry
The bis-(arylmethylidene)cycloalkanones 2e16 were synthe-
sized by cross-aldol condensation of cycloalkanones (cyclo-
pentanone or cyclohexanone) with a variety of commercially
available aldehydes as outlined in Scheme 1. The main advantages
of this synthesis are that the compounds can be obtained by a
simple and rapid one-step procedure in good yield from readily
available starting materials. The only exception was the synthesis of
the dinitro derivative 8, which cannot be obtained using the usual
method under basic conditions [32]. The synthesis of 8 was
repeated several times varying the reaction temperature and time
and the ratio of the reagents, but in all cases, only the unreacted
starting materials were recovered. Then, the reaction was
Scheme 2. Synthetic route for the preparation of 8.