Antitrypanosomal, antileishmanial, and antimalarial activities of quaternary arylalkylammonium 2-amino-4-chlorophenyl phenyl sulfides, a new class of trypanothione reductase inhibitor, and of N-acyl derivatives of 2-amino-4-chlorophenyl phenyl sulfide

Article abstract of DOI:10.1021/jm050819t

Quaternization of the nitrogen atom of 2-amino-4-chlorophenyl phenyl sulfide analogues of chlorpromazine improved inhibition ～40-fold (3′,4′-dichlorobenzyl-[5-chloro-2-phenylsulfanylphenylamino)-propyl] -dimethylammonium chloride inhibited trypanothione r

Full text of DOI:10.1021/jm050819t

J. Med. Chem. 2005, 48, 8087-8097
8087
Antitrypanosomal, Antileishmanial, and Antimalarial Activities of Quaternary
Arylalkylammonium 2-Amino-4-Chlorophenyl Phenyl Sulfides, a New Class of
Trypanothione Reductase Inhibitor, and of N-Acyl Derivatives of
2-Amino-4-Chlorophenyl Phenyl Sulfide
Seheli Parveen,† Mohammed O. F. Khan,† Susan E. Austin,† Simon L. Croft,‡,§ Vanessa Yardley,‡
Peter Rock,‡ and Kenneth T. Douglas*,†
School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Oxford Road, Manchester M13 9PL, U.K., and
London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, U.K.
Received August 17, 2005
Quaternization of the nitrogen atom of 2-amino-4-chlorophenyl phenyl sulfide analogues of
chlorpromazine improved inhibition ∼40-fold (3′,4′-dichlorobenzyl-[5-chloro-2-phenylsulfanyl-
phenylamino)-propyl]-dimethylammonium chloride inhibited trypanothione reductase from
Trypanosoma cruzi with a linear competitive Ki value of 1.7 ( 0.2 µM). Molecular modelling
explained docking orientations and energies by: (i) involvement of the Z-site hydrophobic pocket
(roughly bounded by F396′, P398′, and L399′), (ii) ionic interactions for the cationic nitrogen
with Glu-466′ or -467′. A series of N-acyl-2-amino-4-chlorophenyl sulfides showed mixed
inhibition (Ki, Ki′ ) 11.3-42.8 µM). The quaternized analogues of the 2-chlorophenyl phenyl
sulfides had strong antitrypanosomal and antileishmanial activity in vitro against T. brucei
rhodesiense STIB900, T. cruzi Tulahuan, and Leishmania donovani HU3. The N-acyl-2-amino-
4-chlorophenyl sulfides were active against Plasmodium falciparum. The phenothiazine and
diaryl sulfide quaternary compounds were also powerful antimalarials, providing a new
structural framework for antimalarial design.
Introduction
but rather the analogous enzyme, TR, which does not
accept GSSG as a substrate; conversely, host GR does
not reduce TSST.3,7 This mutually exclusive recognition
and rejection of substrates between host and parasite
enzymes suggested strongly that the selective-inhibitor
design could be achieved.3,4,7 TR has been shown geneti-
cally to be essential as TR-deficient T. brucei are
nonvirulent and have raised susceptibilities toward
oxidative stress.8 Efficient selective blockade of TR
would be expected to compromise the redox defenses of
the parasites, increasing their sensitivity to redox-
damage-based drugs, such as nifurtimox. Thus, a TR
inhibitor might be a drug in its own right or when
coadministered with a redox-active drug, such as nifur-
timox, or perhaps even with synergy.9
During the course of these studies, we discovered that
the target compounds not only showed marked antit-
rypanosomal and antileishmanial activities as was
reasonable to expect on the basis of the target rationale
used but that they were also active against the malarial
parasite Plasmodium falciparum. To investigate this a
little further, we initiated structure simplification to
begin to define the pharmacophore for this unantici-
pated biological activity.
Major Third World tropical diseases, including Afri-
can sleeping sickness, Chagas’ disease, and leishma-
niasis, are caused by pathogenic parasites of Trypano-
soma spp. and Leishmania spp. The relatively few
current treatments suffer from important drawbacks,
and the need for new drugs is desperate; millions are
currently at risk, and very many people die as a result
of the lack of adequate therapies. Moreover, resistance
against several of the available drugs has been reported.
A fundamental metabolic difference between mam-
malian host and trypanosomal or leishmanial parasite
is the trypanothione redox-defense system.1 In mam-
mals, potential redox damage is dealt with by the
glutathione (GSH)-based system during the course of
which glutathione disulfide (1, GSSG) is formed. Re-
generation of protective GSH from GSSG is catalyzed
by glutathione reductase (GR). In trypanosomes and
leishmanias, an analogous system has evolved2 based
on trypanothione, which as the disulfide (2, TSST)
differs from GSSG by the spermidine cross-link between
its two glycyl carboxyl groups. The enzyme trypan-
othione reductase (TR) reduces TSST to the dithiol form,
analogously to GR. With this discovery, TR was quickly
proposed as a target for the rational design of antitry-
panosomal drugs,1,3 a view supported by several sub-
sequent proposals.4-6 Trypanosomes do not contain GR,
Following the early discovery of tricyclic inhibitors
that are specific for TR over GR,5 several classes of
compounds have emerged to provide a framework on
which to build TR inhibitors, including several families
of tricyclics,9-12 polyamine derivatives,13-16 isoquino-
lines,17 peptides,18,19 and peptoids,20 and there have been
reviews of TR inhibition.21-23
* To whom correspondence should be addressed. Tel: 0161 275 2386.
Fax: 0161 275 2481. E-mail: Ken.Douglas@man.ac.uk.
† University of Manchester.
‡ London School of Hygiene and Tropical Medicine.
§ Present address: Drugs for Neglected Diseases Initiative, 1 Place
St. Gervais CH-1202, Geneva.
The tricyclic neuroleptic framework5,9,10 gives reason-
ably strong, selective inhibitors of TR, such as chlor-
10.1021/jm050819t CCC: $30.25 © 2005 American Chemical Society
Published on Web 11/09/2005

8088 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 25
Parveen et al.
of γ-glutamyl functions by hydrophobic structures,29 the
best being the benzyloxycarbonyl group.29,30 Molecular
modelling indicated9 that the Z group of (ZCG‚dmapa)2
might make use of a hydrophobic pocket (the Z-site,
approximately bounded by F396′, P398′, and L399′) not
involved in binding the γ-glutamyl analogue (ECG.d-
mapa)2. Up to 2 orders of magnitude improvement in
the binding strength of the parent phenothiazene tri-
cyclics5,9 could be achieved by a simple functionalization
of the ω-terminus of the side chain on the central bridge
nitrogen atom.31 The rationale that led to this design
improvement was to link the hydrophobic region puta-
tively occupied by the tricyclic nucleus (M113, W21)
with the Z-site. Appropriate hydrophobic quaternization
of the tertiary amine produced strong inhibitors of T.
cruzi TR (for example, Ki is 0.12 µM for 8 and 0.68 µM
for 9), and antiparasitic activity is retained.31
promazine (3), chlomipramine, amitryptyline, and tri-
fluperazine, with Ki values of 10.8, 6.5, 93.6, and 23 µM,
respectively. Ring-opening of the bridging site of the
central ring gave inhibitors (such as 49), some of which
were as powerful as more conformationally restricted
analogues.
We now report the synthesis of some quaternary
analogues (10-14 and 20) of open-chain compounds
based on 6 along with details of their inhibition of T.
cruzi TR and some preliminary antiparasite studies
with them against T. cruzi, T. b. rhodesiense, L. dono-
vani, and P. falciparum. The nature of some of the
parasite-inhibition data was such that we sought to
simplify the structures to begin to probe the minimal
framework necessary to obtain strong antiparasite
action. In doing so, we prepared compounds 15-19, and
their inhibition of T. cruzi TR is also reported.
A series of diphenyl sulfides24,25also showed reason-
ably strong inhibition of TR (Ki values of 27 µM for 5
and 66 µM for 6).26 Modifications of the side chain by
replacing the piperazine or dimethylamine with various
linear or branched amine chains including spermine and
spermidine yielded 7 (I50 ) 0.3 µM) as the most potent
TR inhibitor of this family.24-27 Dimers of the 2-amino-
diphenylsulfides also showed activity.25 Some of these
diphenyl sulfide open-chain analogues of chlorprom-
azine24,25 show considerably lowered neuroleptic activity26-
and at 10-8 M led to weak or nonsignificant displace-
ment in a binding study with the dopamine D2 receptor
using prochlorperazine as a control.26
Results
Synthesis. The quaternary alkylammonium deriva-
tives of 2-amino-4-chlorophenyl phenyl sulfide were
prepared (Scheme 1) similarly to the literature method26
with some modifications; the overall yield was 54-61%
based on the 2,5-dichloronitrobenzene or thiophenol.
Using equimolar NaOH to activate the thiophenol, in
contrast to metallic sodium,26 gave higher yields (90%)
and decreased the amounts of tarlike side products
formed. Reduction of the nitro group carried out by
means of iron/hydrochloric acid gave 86% yield (less
convenient reduction by hydrazine hydrate in the pres-
ence of Raney-nickel (Raney-Ni) afforded the amine in
TR can tolerate noncognate substrate architecture
such as noncross-linked structures28 and replacements

Novel Antitrypanosomal and Antimalarial Structures
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 25 8089
concentration range was increased to approximately 8
µM, the inhibition type was best analyzed as mixed
inhibition, as shown in Figure 1B, with values of Ki )
0.44 ( 0.01 µM and Ki′ ) 3.5 ( 0.09 µM.
Compound 9, studied only up to 20 µM because of
solubility, gave linear competitive inhibition kinetics
(Figure 2).
Inhibition by Quaternary Alkylammonium 2-
Amino-4-chlorophenyl Phenyl Sulfides. The quar-
ternary alkylammonium derivatives of 2-amino-4-chlo-
rophenyl phenyl sulfide (10-14) were linear competitive
inhibitors of T. cruzi TR, as can be seen in Figure 3 for
compound 10. Linear competitive inhibition kinetics
were found for all of them at the concentrations used,
and values of Ki using (ZCG‚dmapa)2 as a variable
substrate are given in Table 1.
Inhibition by N-Acyl-2-amino-4-chlorophenyl
Phenyl Sulfides. (N-Acyl-2-amino)phenyl sulfides (15-
19) were mixed inhibitors of T. cruzi TR. Figure 4 shows
a typical set of diagnostic plots for 16. Values of Ki and
Ki′ collected in Table 2.
Inhibition by 2-Amino-4-chlorophenyl Phenyl
Sulfide and by 20. 2-Amino-4-chlorophenyl phenyl
sulfide was a weak mixed inhibitor (Ki ) 0.15 ( 0.003
mM and Ki′ ) 0.86 ( 0.02 mM). Compound 20 was a
very weak inhibitor and showed an I50 value of 0.60 (
0.19 mM at 0.12 mM TSST under the standard assay
conditions.
96% yield). These methods are more convenient and
cheaper than the literature method (high-pressure
hydrogenation).
Inhibition by Quaternary Alkylammonium Chlo-
rpromazines. A typical set of diagnostic plots, based
on the combination of Lineweaver-Burk, Dixon, and
Cornish-Bowden plots,32 is shown for compound 8 in
Figure 1. In agreement with data in a previous study,31
at concentrations of inhibitor up to approximately 0.6
µM, the inhibition type appeared to be linear competi-
tive with a Ki value of 0.18 ( 0.03 µM; these data are
shown in Figure 1A. However, when the inhibitor
Biological Antiparasite Data. A number of the
compounds were tested against L. donovani, T. cruzi,
T. b. rhodesiense, and P. falciparum, and the results are
Figure 1. A: (a) Dixon plot (b) Lineweaver-Burk plot, and (c) Cornish-Bowden plot for inhibitor 8 at lower concentrations
(0.08-0.58 µM) at pH 7.25, 25 °C in 0.02 M HEPES buffer containing 0.15 M KCl, 1 mM EDTA, 0.1 mM NADPH. Points are
experimental, lines are theoretical, showing linear competitive inhibition with Ki ) 0.18 ( 0.03 µM, Vmax ) 6.58 × 10-3 ∆A/s, and
Km ) 55.3 ( 0.5 µM using TSST as variable substrate with S1 ) 30, S2 ) 60, S3 ) 120, and S4 ) 240 µM. Inhibitor concentrations
used were I1 ) 0, I2 ) 0.08, I3 ) 0.19, I4 ) 0.38, and I5 ) 0.58 µM. B: (a) Dixon plot (b) Lineweaver-Burk plot, and (c) Cornish-
Bowden plot for inhibitor 8 at pH 7.25, 25 °C in 0.02 M HEPES buffer containing 0.15 M KCl, 1 mM EDTA, 0.1 mM NADPH.
Points are experimental, lines are theoretical for mixed inhibition with Ki ) 0.44 ( 0.01 µM, Ki′ ) 3.5 ( 0.09 µM, Vmax ) 6.18 ×
10-3 ∆A/s, and Km ) 19.8 ( 0.02 µM using TSST as variable substrate as S1 ) 30, S2 ) 60, S3 ) 120, and S4 ) 240 µM. The
inhibitor concentrations used were I1 ) 0, I2 ) 0.38, I3 ) 1.1, I4 ) 1.9, I5 ) 3.8, and I6 ) 7.6 µM.

8090 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 25
Parveen et al.
Figure 2. (a) Dixon plot (b) Lineweaver-Burk plot, and (c) Cornish-Bowden plot for inhibitor 9 at pH 7.25, 25 °C in 0.02 M
HEPES buffer containing 0.15 M KCl, 1 mM EDTA, 0.1 mM NADPH. Points are experimental, lines are theoretical for linear
competitive inhibition with Ki ) 0.57 ( 0.03 µM, Vmax ) 9.08 × 10-3 ∆A/s, and Km ) 40.4 ( 0.02 µM using TSST as variable
substrate at S1 ) 30, S2 ) 60, S3 ) 120, and S4 ) 240 µM,. Inhibitor concentrations used were I1 ) 0, I2 ) 2, I3 ) 5, I4 ) 10, I5
) 15, and I6 ) 20 µM.
Figure 3. (a) Dixon plot (b) Lineweaver-Burk plot, and (c) Cornish-Bowden plot of inhibitor 10 at pH 7.25, 25 °C in 0.02 M
HEPES buffer containing 0.15 M KCl, 1 mM EDTA, 0.1 mM NADPH using TSST as variable substrate at S1 ) 30, S2 ) 60, S3
) 120, and S4 ) 240 µM. Points are experimental, lines are theoretical for linear competitive inhibition with Ki ) 2.67 ( 0.09 µM,
Vmax ) 7.72 × 10-3 ∆A/s, and Km ) 21.34 ( 0.8 µM. Inhibitor 10 concentrations used were: I1 ) 0, I2 ) 12.02, I3 ) 19.84, I4 ) 24.8,
I5 ) 31.74, and I6 ) 39.7 µM.
Table 1. Linear Competitive Inhibition by Quaternary
Alkylammonium-Substituted-2-amino-4-chlorophenyl Phenyl
Sulfides of T. cruzi Trypanothione Reductase (3 µg/mL) in 0.02
M HEPES Buffer pH 7.25, 25 °C, Containing 0.15 M KCl, 1
mM EDTA, 0.1 mM NADPH, 0.24, 0.12, 0.06, and 0.03 mM
basis, the structurally simplified compounds 6 and 15-
18 were tested (see Table 3).
Discussion
a
(ZCG‚dmapa)2
The data for these open-chain quaternaries can be
compared with data for the parent compound, the open-
mean energy
of top cluster
% runs
in top 10
clusters
% runs
in top 20
clusters
26
TR inhibition
chain analogue of chlorpromazine 9, with a value of
cmpd
Ki (µM)
(kcal mol-1
)
Ki ) 66 ( 6 µM. The analogues in Table 1 provide
evidence of improvement in the TR inhibition potency
by 5-40-fold. Substitution with the simple aromatic ring
(cf. 12 with 9) gave ∼5-fold improvement over the parent
lead. For substitution by diphenylmethyl (14), benzy-
loxybenzyl (13), or 4-tert-butylbenzyl (10), the improve-
ments in TR inhibitory potency were about 10-15-fold.
The strongest inhibitor of this series was the 3,4-
dichlorobenzyl-quaternized derivative (11), which shows
an improvement of 39-fold over 9. This 3,4-dichloroben-
zyl substitution was also found to be optimal for the
chlorpromazine quaternary systems.31 This family of
compounds shows clear evidence of inhibitory potency
over the nonquaternized open-chain analogues of chlo-
rpromazines.
9
12
11
14
13
10
66 ( 6b
-66.04
-91.37
-70.04
-73.05
-70.47
-69.92
27
27
21
16
17
20
48
43
37
29
37
36
14.2 ( 0.12
1.69 ( 0.22
5.3 ( 0.92
6.6 ( 0.4
6.5 ( 0.7c
a For 11, no inhibition of human glutathione reductase was
detectable at 0.5 mM. Also indicated are the results of docking
calculations. b Literature value.26 c Using TSST as variable sub-
strate gave linear competitive Ki ) 2.67 ( 0.09 µM.
summarized in Table 3. Compounds 8 and 9, with the
parent phenothiazine quaternary structures, were se-
lected because these substitution patterns had been
shown in a previous study to confer strongest antitry-
panosomal activity. Compound 10, the ring-opened
version of 8, was also tested against this panel of
parasites. The antitrypanosomal and antileishmanial
activities of 8-10 were to be anticipated from previous
studies,31 but the powerful results in the test against
P. falciparum that has been introduced as a standard
part of this panel of tests were unexpected. On this
These simple ring-opened variants of the quaternized
compounds have two hydrophobic moieties. The major
one is the “tricyclic” parent region (here an opened
chain), which may interact with the major hydrophobic
region corresponding to W21 and M113 of the enzyme
active site in a way similar to that postulated9,10for
chlorpromazine and other tricyclics. The second hydro-

Novel Antitrypanosomal and Antimalarial Structures
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 25 8091
Figure 4. (a) Dixon plot (b) Lineweaver-Burk plot, and (c) Cornish-Bowden plot of inhibitor 16 at pH 7.25, 25 °C in 0.02 M
HEPES buffer containing 0.15 M KCl, 1 mM EDTA, 0.1 mM NADPH. Points are experimental, lines are theoretical for mixed
inhibition with Ki ) 11.3 ( 0.5 µM, Ki′ ) 42.6 ( 1.8 µM, Vmax ) 8.21 × 10-3 ∆A/s, and Km ) 42.08 ( 1.3 µM. The substrate (TSST)
concentrations were 30, 60, 120, and 240 µM, and inhibitor 16 concentrations used were: I1 ) 0, I2 ) 16.35, I3 ) 32.8, I4 ) 49.2,
and I5 ) 65.6 µM.
Table 2. Inhibition Data for Mixed Inhibition of T. cruzi
Trypanothione Reductase by N-Substituted
2-Amino-4-chlorophenyl Sulfides at pH 7.25, 25 °C in 0.02 M
HEPES Buffer Containing 0.15 M KCl, 1 mM EDTA, 0.1 mM
NADPH with TSST as Variable Substrate
times with the tricyclic or benzyl-substituted group near
the hydrophobic region of L17/W21/M113).
Compounds 6, 13, and 10 had roughly equal num-
bers of docked states in Modes I and II. For 9, ∼20% of
the total 100 runs finished in Mode I and 25% in Mode
II (the majority with the hydrophobic groups settling
near L17W21/M113). In some of the conformations for
both families, the NH group formed hydrogen bonds
with nearby residues, e.g., -OH of Y110, E466′, and
backbone carbonyl groups. For 13, ∼25% of the mem-
bers of the top 20 clusters had the usual N+-E465′/
E466′ interaction, but usually with their hydrophobic
groups lying in the open cavity of the active site.
Another ∼25% had the N+-S14 interaction, some with
the hydrophobic groups near W21/M113. Most of the
other conformations had the ligand situated in the
center of the cavity or toward the exterior away from
protein residues. For 10, a third of the conformations
showed the N+-E465′ interaction and one of the two
hydrophobic groups docking near the Z-site or M113/
L17/W21. About a third of the conformations had the
usual interaction of N+ with S14 and the benzyl group
positioned near M113/L17. Other conformations only
had the N+-S14 or -E465′ interaction.
Compound 11. Three times as many final docked
conformations were found with the N+-S14 interaction
as with the N+-E465′ interaction. Only one significant
cluster was seen (seven-members) with N+-S14 and the
hydrophobic sulfide atom in the Z-site. About the same
number of conformations in other clusters also had N+-
S14 interactions but with the sulfur atom near the
region containing W21/M113/Y110/L17. In some of these
structures, the chlorine substituents on the benzyl group
were within interacting distance of either E18 or E465′.
Some other structures had a double N+ and NH- to
E465′/E466′ interaction, or only the N+-E465′ interac-
tion.
cmpd
Ki (µM)
Ki′(µM)
IC50 (µM)
15
16
17
18
19
20 ( 0.2
29.3 ( 0.13
42.6 ( 1.8
35.9 ( 0.09
37.7 ( 0.06
12.9 ( 1.7
31.6 ( 2.6
30.5 ( 0.14
36.8 ( 1.2
49.6 ( 3.4
44.5 ( 1.2
11.3 ( 0.5
24.6 ( 0.17
24.5 ( 0.09
42.8 ( 6
phobic moiety is formed by the benzyl or benzyl-
substituted groups via quaternization of the propylami-
no side chain. These two hydrophobic regions of the
inhibitor may be oriented in the TR active site by the
ionic interactions formed by the positive charge of the
inhibitor molecule (N+) and the side chains of the
glutamates (E466′ or 467′) of the TR active site. To probe
this further, quantitative docking methods were under-
taken.
Molecular Modelling for 10-14. Compounds 10-
14 have additional flexibility with three extra torsions
in each molecule compared to that of phenothiazines
such as 9, due to the open conformation of the main ring
structures. Docking energies and cluster data for ligands
of this family were determined by a protocol exactly
comparable to that published for 931(Table 1). The bound
conformations again revealed the patterns of interac-
tions previously detected31for phenothiazines such as 9
(viz. of the positive nitrogen (N+) of the ligand with Glu-
465′/466′ or Ser-14). However, the bridging NH group
also influences potential electrostatic interactions and
binding orientations in some cases, but the favored
conformations remain of energy similar to those of
phenothiazine docking. The modes of binding found31
for the cationic phenothiazines were: [Mode I(a)] The
N+ atom interacts electrostatically with E466′ (or
sometimes E467′), and the tricyclic moiety resides in
the hydrophobic Z-site (F396′/P398′/L399′). [Mode I(b)]
The N+ atom interacts electrostatically with E466′ (or
sometimes E467′), with the hydrophobic quaternary
aromatic group in the Z-site. [Mode I(c)] The N+ atom
interacts electrostatically with E466′ (or sometimes
E467′), with the hydrophobic quaternary aromatic
groups apparently not interacting with TR. [Mode II]
The N+ atom interacts electrostatically with S14 (usu-
ally showing no hydrophobic interactions, but some-
Compounds 12 and 14. A very commonly detected
docked conformation for these ligands was with the NH
of the central ring interacting with E465′/466′ and the
sulfide moiety (in the case of 12) or the benzhydryl
moiety (in the case of 14) in the Z-site. This conforma-
tion accounted for half of the conformations for 12. Many
others had N+-S14 interactions but no apparent hy-
drophobic interaction. Again, over half of the top low-
energy conformations of 14 were positioned thusly, but

8092 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 25
Parveen et al.
Table 3. Antiprotozoal Activities of 2-Amino-4-chlorophenyl Phenyl Sulfide, Its Quaternary Alkylammonium Analogues, and Its
N-Acyl Derivativesa,b,c
a KB screen for cytotoxicity. b L. donovani screen t/100 toxic to macrophages, no parasites present: t+0 toxic macrophages, parasites
present. c T. cruzi screen t/xx toxic to macrophages/percent inhibition of parasite.
the N+ was not seen to interact with any acidic residue
or with S14 in any top cluster. The PhCH2 group may
sterically hinder binding to S14. The phenothiazine
quaternized with the PhCH2 group displayed a strong
preference for the N+ to bind E465′/466′, but 14 did not;
the origin of this preference is not clear.
There was no correlation between the binding ener-
gies and the Ki values; for example, 11 is an 8-fold better

Novel Antitrypanosomal and Antimalarial Structures
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 25 8093
inhibitor than 12 on the basis of Ki values but binds
less strongly from the docking study. Compound 6 did
not bind differently with respect to docking energy or
orientation to its quaternary derivatives and was docked
in the N-protonated form.
systems on the basis of their effects as MDR modulators,
but they have also been reported to have intrinsic
antimalarial activity,33-36 which in some cases has been
discussed in relation to their effects on â-hematin
formation37 and falcipain action to destroy hemoglobin.38
The studies we report show that cationic versions of the
phenothiazine nucleus are even more potent and thus
may serve as leads. It has been suggested that, although
chlorpromazine itself is unlikely to serve as the base
for a new antimalarial, the thioridazine framework
might.35 The simplified nuclei based on diaryl sulfides
reported here may be considered in the light of alterna-
tive targets in malaria.
Antiparasitic Activities. Antitrypanosomal and
Antileishmanial Activities. Compound 5 at 27 µM
has been reported to reduce the T. cruzi parasite
number progressively over 12 h, whereas benznidazole
and nifurtimox did not.26 Compound 10 is an analogue
of one of the most active quaternary chlorpromazines
previously reported.31 The data in Table 3 show that
this ring-opened variant 10 is approximately as power-
ful against T. cruzi and L. donovani as the parent
quaternized chlorpromazine 9. However, it is twice as
powerful against T. b. rhodesiense, showing very strong
activity (IC50 ) 10 ng/mL). It showed some toxicity to
KB cells but with a much higher IC50 of 2.14 µg/mL,
giving an efficacy to (this) toxicity ratio of 214. Signs of
host macrophage toxicity also required similar higher
levels in the T. cruzi (∼1-3 µg/mL) and L. donovani
(∼10 µg/mL) tests. Thus, 10 represents a reasonable
lead structure.
Removing the tricyclic structure or the dirayl sulfide
moiety to give 20 effectively destroyed activity against
all the parasites tested. The diaryl sulfide framework
clearly had some inherent activity. Thus, against T.
cruzi, 15, 16, 18, 21, and 22 showed IC50 values in the
range 6.7-19.8 µg/mL. The Ki values against TR for 15-
19 were in the 10-40 µM range (mixed-type kinetics,
Table 2). In contrast, 10, with more powerful antipara-
sitic activity, showed a competitive Ki value of 6.5 µM
(Table 1). Against T. b. rhodesiense, the IC50 values of
15-18 and 21 and 22 ranged from 4.4 to 11.3 µg/mL,
with 19 being essentially inactive. Against L. donovani,
these compounds were inactive.
Antimalarial Activity. Unexpectedly, the ring-
closed (tricyclic) quaternaries, 8 and 9, clearly showed
reasonably strong activity against the malarial parasite
on the basis of the P. falciparum data in Table 3 (with
IC50 values against this strain of 0.47 and 0.13 µg/mL,
respectively). This is not greatly affected (only 4-fold)
by opening the central ring of the tricyclic to give 10,
for which the IC50 value is 0.6 µg/mL. The tert-benzyl
quaternary ammonium region of the molecule is clearly
not the main activity determinant as the ID50 value for
20 is >30 µg/mL. However, the other main region of the
starting framework of 8 and 9, viz. the amino-diaryl-
sulfide moiety, does exhibit antiplasmodial activity with
an ID50 value of 11.3 µg/mL for 22. Other evidence that
this region provides an important part of the architec-
ture of the pharmacophore comes from the activity of
compound 16 (ID50 ) 19.1 µg/mL). Acylation generally
leads to a loss of activity, but the N-propyl (16) is active.
The amino group of 22 is not essential, given the similar
activities of 21 and 22.
Conclusion
The present work introduces the quaternized ana-
logues of the 2-chlorophenyl phenyl sulfides as having
strong antiparasite activity against trypansomes and
leishmanias. ID50 values as low as 10 ng/mL against T.
b. rhodesiense, <1.1 µg/mL against T. cruzi, and 3.9 µg/
mL against L. donovani were found. It also shows that
the very simple molecular skeleton of diaryl sulfides has
inherent activity against these parasites and this allows
a different set of approaches to analogue optimization.
The phenothiazine and diaryl sulfide quaternary com-
pounds are also very powerful antimalarials with 9
having an IC50 value against P. falciparum of 0.13 µg/
mL. Even simplified structures such as the N-acyl diaryl
sulfides retain intrinsic antiparasitic activities.
Experimental Section
Reagents were of the highest quality available and pur-
chased from the suppliers indicated. Benzyl bromide, diphe-
nylmethyl bromide, 4-(tert-butyl)benzyl bromide, borane-
tetrahydrofuran complex anhydrous tetrahydrofuran, and
anhydrous toluene, were purchased from Aldrich Chemical Co.
as were 3,4-dichlorobenzyl chloride, bromodiphenylmethane
and thiophenol. Fluka Chemicals Ltd. supplied 3-bromopro-
pionyl chloride and Raney-Ni ready for use. Lancaster Syn-
thesis supplied 2,5-dichloronitrobenzene and dimethylamine.
Trypanothione disulfide was from Bachem or synthesized by
a literature method,39 GSSG and NADPH were from Boe-
hringer, and N-benzyloxycarbonyl-L-cysteinylglycyl 3-dim-
ethylaminopropyl amide disulfide was synthesized as de-
scribed.30
Syntheses were monitored by thin-layer chromatography
(TLC) on plastic sheets precoated to 0.2 mm with aluminum
oxide (N/UV254) (Merck, Darmstadt). The TLC solvent mixture
used was A, chloroform:ethanol:acetic acid (10:9:1); B, diethyl
ether:petroleum ether 40-60 (1:1); C, diethyl ether:petroleum
ether 40-60 (1:9); D, diethyl ether:petroleum ether (2:8). RfX
values indicate the solvent system used (X) as superscript.
Spots were made visible by UV irradiation or by means of
iodine vapor. Melting points were determined on a Reichert
(Austria) melting point apparatus (Shandon Scientific Co. Ltd.,
London).
Values of pH were measured using a Corning 240 digital
pH meter calibrated with standard buffers (Sigma) at 20 °C
or using pH test strips (Sigma, range 0-14). All water used
was distilled and purified by ion-exchange and charcoal
filtering using a milliQ system (Millipore Ltd.). Phosphate,
HEPES, and tris buffers were prepared from analytical
reagent-grade materials according to standard procedures.
NMR spectra were recorded on a Jeol JNM EX 270 spec-
A full structure-variation study remains to be done,
but starting with the diaryl sulfide moiety, activity can
be improved by incorporating the alkylamino-quater-
nary chain (10), which gives a 20-fold improvement in
activity. Ring closure to give the tricyclic (9) improves
activity a further 4-fold.
1
trometer operating at 270 MHz for H NMR. Chemical shifts
(δ) are reported in parts per million (ppm) relative to Me4Si
(0.00 ppm) as an internal reference. Data are reported using
the following convention: chemical shift (integrated intensity,
splitting patterns, assignment). Splitting patterns are abbrevi-
Phenothiazines have been mostly studied in malaria
as adjuvants to chloroquine in chloroquine-resistant

8094 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 25
Parveen et al.
ated as: s, singlet; d, doublet; t, triplet; q, quartet; p, pentet;
m, unresolved multiplet.
5-6 h on a Gemini fluorescent plate reader (Softmax Pro. 3.1.1,
Molecular Devices, U.K.) at EX/EM 530/585 nm with a filter
cutoff at 550 nm. IC50 values were calculated with Msxlfit
(IDBS, U.K.)
Fast-atom bombardment mass spectra (FAB-MS) were
taken by means of a Kratos-Concept instrument operating in
the FAB mode (Xe-beam bombardment) using m-nitrobenzyl
alcohol (Aldrich Chemical Co.) as a matrix in the Department
of Chemistry, University of Manchester. Elemental analyses
were recorded on an EA 1108 Elemental Analyzer (Carlo Erba
Instruments) in the Department of Chemistry, University of
Manchester.
Plasmodium falciparum. 3D7 (a chloroquine-sensitive
strain) cultures were maintained in RPMI 1640 medium
(Sigma, U.K.) 37 °C, 5% CO2 in 5% hematocrit. Asynchronous
(65-75% ring stage) cultures were prepared at 0.5% para-
sitemia, and 50 L was added per well, the top test drug final
concentration being 30 g/mL. After 24 h incubation, 37 °C, 5%
CO2, 5 L of (3H)-hypoxanthine was added (0.2 Ci/well),45,46 and
plates were shaken for 1 min and then incubated for 48 h.
The plates were freeze/thawed rapidly, harvested, and dried.
Radioactive hypoxanthine uptake was measured by scintilla-
tion counter. IC50 values were calculated using Msxlfit.
Leishmania donovani. Peritoneal exudate macrophages
were harvested from CD1 mice 24 h after starch induction.
After washing, the macrophages were dispensed into Lab-tek
16-well tissue culture slides and maintained in RPMI1640 +
10% heat-inactivated fetal calf serum (HIFCS) at 37 °C, 5%
CO2/air mixture for 24 h. Leishmania donovani HU3 amastig-
otes were harvested from an infected golden hamster spleen
and were used to infect the macrophages at a ratio of 5
parasites:1 macrophage. Amastigotes were counted using
Thoma hemocytometer. Infected cells were left for a further
24 h and then exposed to the drug for a total of 5 days, with
the overlay being replaced on day 3.47 The top concentration
for the test compounds was 30 µg/mL, and all concentrations
were carried out in quadruplicate. The IC50 value for the
positive control drug, Pentostam, was usually 3-8 µg SbV/mL.
On day 5, the overlay was removed, and the slides were fixed
(100% methanol) and stained (10% Giemsa, 10 min) before
being evaluated microscopically. IC50 values were calculated
using Msxlfit.
Syntheses. The quaternary arylalkylammonium deriva-
tives of 2-amino-4-chlorophenyl phenyl sulfide (10-14) were
synthesized according to Scheme 1;25 the preparation of
4-chloro-2-nitrophenyl phenyl sulfide and 2-amino-4-chloro-
phenyl phenyl sulfide used variants of literature methods25
that are described here, whereas other steps followed literature
protocols.
4-Chloro-2-nitrophenyl Phenyl Sulfide. 2,5-Dichloroni-
trobenzene (19 g, 0.1 mol) was added to a solution of thiophenol
(11 g, 0.1 mol) in 5% ethanolic solution of NaOH (80 mL, 0.1
mol) and refluxed for 1 h, evaporated to dryness, and then
dissolved in a mixture of ethyl acetate and water. The organic
phase was dried with anhydrous Na2SO4 and evaporated to
dryness under reduced pressure to give the crude product,
which was then dissolved in a small amount of warm ethanol
and left overnight at 4 °C to give yellow crystals, which were
separated by filtration and washed with ethanol. The product
was recrystallized from AcOH (26 g, yield 90%), RfD 0.6; mp
82-83 °C (lit.49 mp 81.5-84.5 °C).
2-Amino-4-chloro-phenyl Phenyl Sulfide. 4-Chloro-2-
nitro-1-phenylsulfanyl-benzene (3 g, 11.3 mmol) was dissolved
in warm ethanol (45 mL), and reduced iron powder (3.15 g,
56.5 mmol) was added. Concentrated hydrochloric acid (14 mL,
140 mmol) was added gradually and the reaction mixture
refluxed for 1 h for the first two occasions (in the third
instance, the reaction mixture turned colorless after adding
concentrated hydrochloric acid, so reflux was omitted). Water
was added to the reaction mixture, which was extracted twice
with dichloromethane (2 × 10 mL). The combined organic
layers were washed with dilute potassium carbonate solution
(2 × 10 mL). The dichloromethane layer was collected, dried
over anhydrous sodium sulfate, and evaporated to give a thick
oily liquid from which the product was crystallized from its
solution in cold methanol by the careful addition of cold water
(2.3 g, 86% yield). An alternative procedure with Raney-Ni and
hydrazine was as follows. A solution of 4-chloro-2-nitrophenyl
phenyl sulfide (14.25 g, 0.05 mol) and 100% hydrazine hydrate
(8 mL, 0.257 mol) in 225 mL of ethanol was heated to 50 °C,
and about 2 g of Raney-Ni was added carefully (frothing
occurs). When the initial frothing had subsided (after about 1
Enzyme Isolation, Assay, and Inhibition Studies. Try-
panothione reductase from T. cruzi was isolated by means of
overexpression of the gene in Escherichia coli JM109 cells
bearing the expression vector PBSTNAV40 as previously
described.5 The enzyme was homogeneous by the criterion of
SDS-PAGE and had a specific activity identical to that of wild-
type TR.7 Enzyme activity was assayed at 25 °C in 0.02 M
HEPES buffer, pH 7.25, containing 0.15 M KCl, 1 mM EDTA,
0.12 mM TSST, and 0.1 mM NADPH7 at an enzyme concen-
tration of approximately 0.3 µg/mL. For inhibition studies, TR
assays were measured in 0.02 M HEPES buffer pH 7.25, 25
°C, containing 0.15 M KCl, 1 mM EDTA, 0.1 mM NADPH, 3
µg/mL TR, and varying concentrations of substrate ((ZCG‚
dmapa)2) and inhibitor as indicated. For I50 determination, a
single concentration of substrate (120 µM) was used. Human
GR was isolated from an overexpression system for GR in E.
Coli SG5 cells as described41 and assayed following literature
conditions42 in 0.1 M potassium phosphate buffer, pH 7.00,
containing 0.2 M KCl and 1 mM EDTA in the presence of 1
mM GSSG and 0.1 mM NADPH using approximately 15 µg/
mL of GR per assay. Enzyme rate assays were conducted
spectrophotometrically at 340 nm using a Peltier-thermostated
cuvette holder maintained at 30.0 °C in a Cary 1E UV-visible
spectrophotometer and using the Cary Enzyme Kinetics
Software. Enzyme rate assays, spectral scans, and absorbance
readings at fixed wavelengths were conducted spectrophoto-
metrically using a Peltier-thermostated cuvette holder in a
Cary 1E UV-visible spectrophotometer and using the Cary
Enzyme Kinetics Software or using a Perkin-Elmer λ3B
spectrophotometer thermostated with a Haake GH water
circulator and attached to a Perkin-Elmer R100A chart
recorder. Quartz cuvettes were used for readings in the UV
range, for UV scanning of enzyme samples and compounds,
and for determining extinction coefficients; otherwise, dispos-
able plastic cuvettes were used.
Values of I50, the concentration required to give 50%
inhibition under the assay conditions described above, were
determined by interpolation from a plot of assay velocity versus
inhibitor concentration, fitting to the equation corresponding
to linear competitive inhibition by nonlinear regression analy-
sis written into Grafit. Inhibition type was assessed by
analyzing the patterns of three diagnostic classes of plot: 1/vo
versus 1/[So] for various [1]; 1/vo versus [1] for various [So];
[So]/vo versus [1] at various [So]. Values of Ki for competitive
inhibition and of Ki and Ki′ for mixed inhibition were deter-
2
mined by direct weighted (1/vo for weighting) least-squares
nonlinear regression analysis of the raw data using the
appropriate equation using the Grafit program (Erithacus
Software, distributed by Sigma Chemical Co.), viz., for linear
competitive inhibition, v ) Vmax[So]/([So] + Km(1 + [1]/Ki)), and
for mixed inhibition, v ) Vmax[So]/([So](1 + [1]/Ki′) + Km(1 +
[1]/Ki)). Values of Vmax and Km were obtained by least-squares
nonlinear regression analysis using Grafit.
Parasitology. Trypanosoma brucei rhodesiense. STIB900
blood stream form (bsf) trypomastigotes were maintained in
HMI-18 medium43 with 15% heat-inactivated fetal calf serum
(HIFCS) [Harlan-SeraLab, U.K.] at 37 °C, 5% CO2/air mixture.
Trypomastigotes were washed and resuspended in fresh
medium at a concentration of 2 × 105/mL, and 100 µL was
added to the drug dilutions. The top concentration for the test
compounds was 30 µg/mL. The IC50 for pentamidine is usually
between 1.0 and 0.1 pg/mL. Plates were incubated for 72 h at
37 °C, 5% CO2. At 72 h, the plates were assessed microscopi-
cally before Alamar blue was added.44 Plates were read after

Novel Antitrypanosomal and Antimalarial Structures
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 25 8095
Scheme 1. Synthesis of Quaternary Alkylammonium (10) and N-Acyl (15-19) Derivatives of 2-Amino-4-chlorophenyl
Sulfidea
a
(i) PhSH, 5% ethanolic NaOH, reflux, (ii) Fe/HCl in EtOH, (iii) 3-bromopropionyl chloride, dry CHCl3, reflux, 30 min, (iv) BH3‚THF,
(v) Me2NH, Et3N, toluene, reflux 16 H, (vi) alkyl halide, Me2CO, overnight, (vii) RCOX/ Et3N in CHCl3
h), an additional 1 g of Raney-Ni was added and the mixture
refluxed for 1 h. The catalyst was removed by filtration
through Celite, solvent was evaporated under reduced pres-
sure, and the product crystallized (12.7 g, yield 96%), RfD 0.75;
mp 61-63 °C (lit.49 mp 63-65 °C).
N-(3-Bromopropionyl)-5-chloro-2-phenylthiophenyl-
amide). The crude product was recrystallized from ethanol
to yield the product quantitatively (7.8 g, 100%) as a colorless
solid; RfB 0.5, mp 85-86 °C (lit.25 mp 91 °C).
3-Bromo(N-(2-phenylthio-5-chloro)phenyl)propyl-
amine was isolated (4.3 g, 79%) as a colorless semisolid. RfD
0.65; m/z (ES-MS): 355; δH (CDCl3): 6.6-7.4 (8H, m, Ar), 6.6*
(1H, t, broad, NH), 3-3.5 (4H, m, 2 × CH2, attached to ring N
and Br), 2.0 (2H, m, -CH2-).
3-(N-(2-Phenylthio-5-chloro)phenyl) Propyldimethyl-
amine was isolated quantitatively (3.1 g, 99%) as a yellowish
oil. RfC 0.8; m/z (FAB-MS): 320; δH (CDCl3): 7.4-6.6 (8H, m,
Ph), 5.63* (1H, t, broad, NH), 3.15 (2H, q, CH2-NHPh), 2.2
(2H, t, CH2-NMe2) 2.1 (6H, (CH3)2N), 1.7 (2H, p, -CH2-).
Quaternary Arylalkylammonium 4-Chlorophenyl
Phenyl Sulfides, (10-14). 3-Dimethylamino(N-(2-phenylthio-
5-chloro)phenyl)propylamine (450 mg, 1.41 mmol) was dis-
solved in acetone (5 mL), and an equimolar amount of alkyl
halide in acetone (5 mL) was added. The reaction mixture was
stirred at room temperature for 18 h (for compound 10, 3-4
days were needed to obtain a reasonable yield of product). The
product precipitated (or ether was added to induce precipita-
tion) and was collected by filtration, dried, and recrystallized
from acetone-ether (yields were typically 80-90%). NMR
spectral data and analytical data for the compounds 10-14
were satisfactory. The molecular weights of the compounds
were estimated using fast atom-bombardment (Xe-beam) mass
spectrometry, which gave the molecular ion (M+) peak, usually
as the most abundant peak (100%), with very low abundance
peaks (5-25%) corresponding to the 3-dimethylamino(N-(2-
phenylthio-5-chloro)phenyl)propylamine nucleus, the propy-
lammonium side chain, and the 2-amino-4-chlorophenyl phenyl
sulfide nucleus.
C, H, N; 11, Anal. (C24H26N2Cl3BrS‚1/2H2O) C, H, N; 12, Anal.
(C24H28N2ClBrS) C, H, N; 13, Anal. (C31H34Cl2N2SO‚H2O) C,
H, N; 14, Anal. (C30H32ClBrN2S) C, H, N.
For N-acyl derivatives of 2-amino-4-chlorophenyl phenyl
sulfide 15-19: 15, Anal. (C15H14ClNOS) C, H, N; 16, Anal.
(C16H16ClNOS) C, H, N; 17, Anal. (C20H16ClNOS) C, H, N; 18,
Anal. (C19H14ClNOS) C, H, N; 19, Anal. (C19H13ClN2O3S) C,
H, N.
Synthesis of Amide Derivatives of 5-Chloro-2-phenyl-
sulfanyl-phenylamine. Amide derivatives of 5-chloro-2-
phenylsulfanyl-phenylamine were synthesized according to
Scheme 1 by standard acylation using the appropriate acid
chloride.
Molecular Modelling Quantative Docking. Potential
inhibitors were designed to bind via specific interactions:
hydrogen bond, charge-charge, and hydrophobic. The pub-
lished Chamaecrista fasciculata6,50,51 and T. cruzi52 X-ray
diffraction coordinates were obtained from the Brookhaven
database. Molecular modelling was carried out using Sybyl
6.4.2 software53 and an R10000 or R4000 Silicon Graphics
machine.
Docking searches, performed on a Silicon Graphics Origin
2000, took 12-76 hours CPU time using the program AU-
TODOCK 2.4,54 which uses a Metropolis Monte Carlo simulated-
annealing for positional and configurational exploration with
grids of molecular affinity potentials55 based on the AMBER
force field,56 allowing the inclusion of terms for the Van der
Waals’, electrostatic, and hydrogen-bonding interactions. The
X-ray structure of TR complexed with mepacrine57 was used
for docking studies. Mepacrine and all water molecules were
removed, charged amines and carboxyl groups were capped,
and essential hydrogens were added using the Kollman united-
atom library. The X-ray structure was very briefly relaxed (25
steps Simplex) to remove any bad geometry and short contacts
present, but not to allow any heavy atoms to move significantly
from the calculated crystal structure positions. All ligands to
be docked (Table 2) were built in SYBYL 6.4.253 with a formal
charge of +1 added to the quaternary nitrogen atom. Partial
atom-centered charges were calculated using the semi-empiri-
cal PM3 method implemented in MOPAC,58 and after an initial
For quaternary alkylammonium derivatives of 2-amino-4-
chlorophenyl phenyl sulfides 10-14: 10, Anal. (C28H38Cl2N2S)

8096 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 25
Parveen et al.
(3) Shames, S. L.; Fairlamb, A. H.; Cerami, A.; Walsh, C. T.
Purification and Characterization of Trypanothione Reductase
from Crithidia fasciculata, a New Member of the Family of
Disulfide-Containing Flavoprotein Reductases. Biochemistry
1986, 25, 3519-3526.
(4) Schirmer, R. H.; Mu¨ller, J. G.; Krauth-Siegel, R. L. Disulfide-
Reductase Inhibitors as Chemotherapeutic Agents: The Design
of Drugs for Trypanosomiasis and Malaria. Angew. Chem., Int.
Ed. Engl. 1995, 34, 141-154.
(5) Benson, T. J.; McKie, J. H.; Garforth, J.; Borges, A.; Fairlamb,
A. H.; Douglas, K. T. Rationally Designed Selective Inhibitors
of Trypanothione Reductase. Biochem. J. 1992, 286, 9-11.
(6) Hunter, W. N.; Bailey, S.; Habash, J.; Harrop, S. J.; Helliwell,
J. R.; Aboagye-Kwarteng, T.; Smith, K.; Fairlamb, A. H. Active
Site of Trypanothione Reductase. A Target for Rational Drug
Design. J. Mol. Biol. 1992, 227, 322-333.
(7) Krauth-Siegel, R. L.; Enders, B.; Henderson, G. B.; Fairlamb,
A. H.; Schirmer, R. H. Trypanothione Reductase from Trypa-
nosoma cruzi: Purification and Characterization of the Crystal-
line Enzyme. Eur. J. Biochem. 1987, 164, 123-128.
(8) Krieger, S.; Schwarz, W.; Ariyanayagam, M. R.; Fairlamb, A.
H.; Krauth-Siegel, R. L.; Clayton, C. Trypanosomes Lacking
Trypanothione Reductase are Avirulent and Show Increased
Sensitivity to Oxidative Stress. Mol. Microbiol. 2000, 35, 542-
552.
(9) Chan, C.; Yin, H.; Garforth, J.; McKie, J. H.; Jaouhari, R.;
Speers, P.; Douglas, K. T.; Rock, P. J.; Yardley, V.; Croft, S. L.;
Fairlamb, A. H. Phenothiazine Inhibitors of Trypanothione
Reductase as Potential Antitrypanosomal and Antileishmanial
Drugs. J. Med. Chem. 1998, 41, 148-156.
(10) Garforth, J.; Yin, H.; McKie, J. H.; Douglas, K. T.; Fairlamb, A.
H. Rational Design of Selective Ligands for Trypanothione
Reductase from Trypanosoma cruzi. Structural Effects on the
Inhibition by Dibenzazepines Based on Imipramine. J. Enzyme
Inhib. 1997, 12, 161-173.
(11) Bonse, S.; Santelli-Rouvier, C.; Barbe, J.; Krauth-Siegel, R. L.
Inhibition of Trypanosoma cruzi Trypanothione Reductase by
Acridines: Kinetic Studies and Structure-Activity Relation-
ships. J. Med. Chem. 1999, 42, 5448-5454.
Simplex optimization, ligands were minimized to convergence
(Powell method, gradient 0.05 kcal/mol).
The number of rotatable bonds of the ligands varies from 9
to 12; nonpolar hydrogens were united automatically using
AUTOTORS. Using AUTOGRID, a grid (29.625 Å × 29.625 Å
× 30.375 Å, with grid spacing 0.375 Å) was centered on the
active site for each test case, with 40, 40, and 41 grid points
in each Cartesian direction (80 points × 80 points × 82 points
total per grid). Appropriate Lennard-Jones 12-10 parameters
for H atoms bonded to O, N, or S in the ligand, and to O or S
in the protein, were specified in the AUTOGRID parameter
file (N atoms of TR were all assumed to participate in backbone
amide bonds, making their lone pairs unavailable for hydrogen
bonds).
As 50 docking runs of AUTODOCK performed for each
ligand gave practically no clustering, 100 docking runs were
carried out per docking. At the beginning of each simulated
annealing run, the ligand was positioned randomly within the
grid, and movement began with a random translation, random
rigid body rotation, and random torsions. Each run consisted
of 100 annealing cycles, a cycle terminating if the ligand made
either 30 000 sequentially accepted or rejected moves. The
annealing RT (616 cal mol-1 in the first cycle) was reduced
linearly at the end of each cycle by 0.95-fold. The minimum
energy state of the current cycle was used to start the next
cycle. All cycles had a maximum translational step size of 0.2
Å and maximum torsional rotation of 5° per step.
Following docking, cluster analysis of all structures gener-
ated for a single compound was performed. Once sorted by total
energy of interaction, the structures were sequentially as-
signed to cluster families, represented by top-of-cluster struc-
tures. Structures not satisfying a tolerance of 1.5 Å in the RMS
deviation for all atoms to each top-of-cluster structure found
defined new clusters. Each final docked position of mepacrine
was referenced to that in the crystal structure. Ligand internal
energy results were referenced to the initial undocked energy,
calculated using AMBER. The calculated differences between
the undocked and docked energies were used for analyses.
(12) Bonse, S.; Krauth-Siegel, R. L.; Schlichting, I.; Lowe, G. Ir-
reversible Inhibitors of T. cruzi Trypanothione Reductase:
Kinetic and Crystallographic Studies. In Flavins and Flavopro-
teins 1999, Proc. 13th Int. Symp Konstanz, Germany, August 29-
September 4, 1999; Ghisla, S., Kroneck, P., Macheroux, P., Sund,
H., Eds; Agency for Scientific Publ.: Berlin, 1999.
(13) O’Sullivan, M. C.; Zhou, Q.; Li, Z.; Durham, T. B.; Rattendi, D.;
Lane, S.; Bacchi, C. J. Polyamine Derivatives as Inhibitors of
Trypanothione Reductase and Assessment of Their Trypanocidal
Activities. Bioorg. Med. Chem. 1997, 5, 2145-2155.
Acknowledgment. We are grateful to Professors
Alan Fairlamb and Heiner Schirmer for generous gifts
of the overproducing clones for TR and human GR,
respectively, for the support of the Wellcome Trust
(D.M.) and Association of Commonwealth Universities
(M.O.F.K.), and for financial support (C.C.) from the
UNDP/World Bank/WHO Special Program for Research
and Training in Tropical Diseases.
(14) Li, Z.; Fennie, M. W.; Ganem, B.; Hancock, M. T.; Kobaslija, M.;
Rattendi, D.; Bacchi, C. J.; O’Sullivan, M. C. Polyamines with
N-(3-Phenylpropyl) Substituents Are Effective Competitive In-
hibitors of Trypanothione Reductase and Trypanocidal Agents.
Bioorg. Med. Chem. Lett. 2001, 11, 251-254.
(15) Ponasik, J. A.; Strickland, C.; Faerman, C.; Savvides, S.; Karplus,
P. A.; Ganem, B. Kukoamine
A and Other Hydrophobic
Acylpolyamines: Potent and Selective Inhibitors of Crithidia
fasciculata Trypanothione Reductase. Biochem. J. 1995, 311,
371-375.
Appendix
(16) Chitkul, B.; Bradley, M. Optimizing Inhibitors of Trypanothione
Reductase using Solid-Phase Chemistry. Bioorg. Med. Chem.
Lett. 2000, 10, 2367-2369.
(17) Fournet, A.; Inchausti, A.; Yaluff, G.; Royas De Arias, A.;
Guinaudeau, H.; Bruneton, J.; Breidenbach, M. A.; Karplus, P.
A.; Faerman, C. H. Trypanocidal Bisbenzylisoquinoline Alkaloids
Are Inhibitors of Trypanothione Reductase. J. Enzyme Inhib.
1998, 13, 1-9.
(18) Garforth, J.; McKie, J. H.; Jaouhari, R.; Benson, T. J.; Fairlamb,
A. H.; Douglas, K. T. Rational Design of Peptide-Based Inhibitors
of Trypanothione Reductase as Potential Antitrypanosomal
Drugs. Amino Acids 1994, 6, 295-300.
(19) McKie, J. H.; Garforth, J.; Jaouhari, R.; Chan, C.; Yin, H.;
Besheya, T.; Fairlamb, A. H.; Douglas, K. T. Specific Peptide
Inhibitors of Trypanothione Reductase with Backbone Structures
Unrelated to That of Substrate: Potential Rational Drug Design
Lead Frameworks. Amino Acids 2001, 20, 145-153.
(20) Chan, C.; Yin, H.; McKie, J. H.; Fairlamb, A. H.; Douglas, K. T.
Peptoid Inhibition of Trypanothione Reductase as a Potential
Antitrypanosomal and Antileishmanial Drug Lead. Amino Acids
2002, 22, 297-308.
Abbreviations. DMSO, dimethyl sulfoxide; GR, glu-
tathione reductase; GSSG, glutathione disulfide; GSH,
reduced glutathione; T[S]2, trypanothione disulfide;
T[SH]2, reduced trypanothione as dithiol; TR, trypan-
othione reductase; HEPES, N-2-hydroxyethylpipera-
zine-N’-2-ethanesulfonic acid; THF, tetrahydrofuran;
TEA, triethylamine; Z, benzyloxycarbonyl; (ZCG‚dmapa)2,
N,N-bis-(benzyloxycarbonyl)-L-cysteinylglycyl-3-dime-
thylamino)propylamide disulfide.
Supporting Information Available: Analytical and spec-
tral characterization data. This material is available free of
charge via the Internet at http://pubs.acs.org.
References
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