Coupling of a competitive and an irreversible ligand generates mixed type inhibitors of Trypanosoma cruzi trypanothione reductase

Article abstract of DOI:10.1021/jm020885k

9-Aminoacridines and (terpyridine)platinum(II) complexes are competitive and irreversible inhibitors, respectively, of trypanothione reductase from Trypanosoma cruzi, the causative agent of Chagas' disease. Four chimeric compounds in which 2-methoxy-6-chl

Full text of DOI:10.1021/jm020885k

4524
J . Med. Chem. 2002, 45, 4524-4530
Cou p lin g of a Com p etitive a n d a n Ir r ever sible Liga n d Gen er a tes Mixed Typ e
In h ibitor s of Tr ypa n osom a cr u zi Tr yp a n oth ion e Red u cta se
Oliver Inhoff,† J onathan M. Richards,‡ J an Willem Brˆıet,‡ Gordon Lowe,‡ and R. Luise Krauth-Siegel*,†
Biochemie-Zentrum, Heidelberg University, Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany, and Dyson Perrins
Laboratory, Department of Chemistry, Oxford University, South Parks Road, Oxford OX1 3QY, United Kingdom
Received March 21, 2002
9-Aminoacridines and (terpyridine)platinum(II) complexes are competitive and irreversible
inhibitors, respectively, of trypanothione reductase from Trypanosoma cruzi, the causative agent
of Chagas’ disease. Four chimeric compounds in which 2-methoxy-6-chloro-9-aminoacridine
was covalently linked to the (2-hydroxyethanethiolate)(2,2′:6′,2′′-terpyridine)platinum(II)
complex were synthesized and studied as inhibitors of the parasite enzyme. The derivatives
differed by the nature and/or the length of the spacer connecting the two aromatic systems.
All four compounds were effective mixed type inhibitors of trypanothione reductase with Ki
and Ki′ values of 0.3-4 and 2-11 µM, respectively. The most potent inhibitor had an
ethylthioether linkage between the two aromatic ring systems, and the other compounds
contained an alkyl ether group with 4-6 methylene groups. In contrast to the parasite enzyme,
human glutathione reductase, the closest related host enzyme was not inhibited by these
compounds. The finding that the conjugation of a competitive and an irreversible inhibitor
can give rise to reversible mixed type inhibitors underlines the difficulties associated with
inhibitor design based on the three-dimensional structure of trypanothione reductase.
In tr od u ction
that the drug is fixed in the active site close to the
hydrophobic wall formed by Trp21 and Met113. The
residues in contact with the bound mepacrine are four
out of five residues in the active site, which are not
conserved between T. cruzi TR and human GR.5 The
fact that the activity of TR in bloodstream T. brucei has
to be lowered to 10% to cause an increased sensitivity
toward oxidative stress3 indicates that purely competi-
tive inhibitors may not become suitable drug candidates,
unless a ligand with a nanomolar inhibition constant
and/or slow binding behavior was discovered.6 However,
most competitive inhibitors of TR described so far have
inhibitor constants that are of the same order of
magnitude as the Km of the enzyme for trypanothione
disulfide.6-8 In contrast to competitive ligands, irrevers-
ible inhibitors may be effective at much lower concen-
trations and in addition, accumulation of substrate due
to blockage of the pathway cannot overcome inhibition.
This renders irreversible inhibitors promising drug
candidates. (2,2′:6′,2′′-Terpyridine)platinum(II) com-
plexes have been shown to be irreversible inhibitors of
T. cruzi TR but not of human GR. Most probably, Cys52
in the active site of TR is specifically modified.9
Acridines10,11 and (2,2′:6′,2′′-terpyridine)platinum(II)
complexes12 have trypanocidal activities.
The selective inhibition of TR by both 9-aminoacridines
and (2,2′:6′,2′′-terpyridine)platinum(II) complexes caused
us to synthesize chimeric ligands composed of (2-
hydroxyethanethiolate)(4′-substituted-2,2′:6′,2′′-terpy-
ridine)platinum(II) complexes and 2-methoxy-6-chloro-
9-aminoacridines. The two aromatic moieties were
connected by either an alkyl ether or an alkylthioether
group. It was assumed that the acridine moiety may
exert specificity for the parasite enzyme at the trypano-
thione binding site of TR (following mepacrine, see
above) whereas the (2,2′:6′,2′′-terpyridine)platinum(II)
Trypanosomes and Leishmania are the causative
agents of African sleeping sickness (Trypanosoma brucei
gambiense and Trypanosoma brucei rhodesiense), Cha-
gas’ disease (Trypanosoma cruzi), Nagana cattle disease
(Trypanosoma congolense and Trypanosoma brucei bru-
cei), Kala-azar (Leishmania donovani), and Oriental
sore (Leishmania tropica). All of these parasitic protozoa
lack the ubiquitous enzyme glutathione reductase. For
maintaining a reducing intracellular redox equilibrium,
Trypanosomes and Leishmania depend on the flavo-
enzyme trypanothione reductase (TR),1 which keeps
their main thiols, bisglutathionylspermidine (trypano-
thione, T(SH)2)2 and monoglutathionylspermidine (Gsp),
in the thiol state:
TS2 + NADPH + H+ f T(SH)2 + NADP+
TR shares many physical and chemical properties
with human GR, the closest related host enzyme. The
most important difference between parasite and host
enzyme is the mutually exclusive specificity toward
their disulfide substrates. Genetic approaches revealed
that trypanosomes lacking TR are avirulent and show
increased sensitivity to oxidative stress.3 The absence
of TR from mammalian cells and the essential role of
TR in the defense of oxidative stress render the enzyme
well-suited as a target molecule for rational drug
development (for reviews, see refs 6-8). 9-Aminoacridines
such as mepacrine are competitive inhibitors.4 The
crystal structure of the TR-mepacrine complex revealed
* To whom correspondence should be addressed. Tel: +49 6221 54
4187. Fax: +49 6221 54 55 86. E-mail: krauth-siegel@
urz.uni-heidelberg.de.
† Heidelberg University.
‡ Oxford University.
10.1021/jm020885k CCC: $22.00 © 2002 American Chemical Society
Published on Web 08/27/2002

Trypanosoma cruzi Trypanothione Reductase
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 20 4525
Ta ble 1. Inhibition of T. cruzi TR by
Sch em e 1. Synthesis of
(2,2′:6′,2′′-Terpyridine)platinum(II) Acridine Conjugatesa
(2,2′:6′,2′′-Terpyridine)platinum(II) Acridine Conjugate
(1)a
Variation of the TS2 Concn (Mixed Type)
inhibitor concn
in the assay (µM)
Ki,slope
Ki,int
(µM)
compd
n
(µM)b
1
0.4
1
2
0.9
1.5
3
2
4
6
3
0.59
0.32
0.27
1.8
1.2
0.7
4
2.5
2.6
0.53
0.42
2.7
2.5
2
5.7
2.5
2.1
11.4
7.6
7.1
4
a
Reagents: (i) PhOH, NaOH. (ii) 2-Mercapto-ethylamine. (iii)
Heat in weakly acidic solution. (iv) (1,5-Cyclooctadiene)diiodo
Platinum(II).
2a
2b
2c
4
5
6
Sch em e 2. Synthesis of (2,2′:6′,2′′-Terpyridine)platinum
(II) Acridine Conjugate (2a -c)a
7.1
4.4
Variation of the NADPH Concn (Mixed Type)
inhibitor
concn (µM)
Ki,slope
Ki,int
(µM)
compd
(µM)b
1
1
2
2
0.8
2.8
2
a
The kinetics were measured at 25 °C in TR assay buffer (40
mM Hepes, 1 mM EDTA, pH 7.5). The TS2 and NADPH concen-
tration was varied in the presence of 100 µM NADPH and 110
µM TS2, respectively, and two or three inhibitor concentrations
as well as in the absence of inhibitor. Each set of kinetics was
measured twice whereby the single data points differed by e15%.
b
The inhibitor constants Ki,slope and Ki,int were calculated for each
inhibitor concentration from the slope and intersection values
derived from Lineweaver-Burk plots as described under the
Experimental Procedures. See text for details.
a
Reagents: (i) R,ω-Hydroxyalkylamines, NaH. (ii) Heat in
weakly acidic solution. (iii) (1,5-Cyclooctadiene)diiodo Platinum(II).
Alkoxide ions are known to displace chloride ion from
4′-chloro-2,2′:6′,2′′-terpyridine,12 and R,ω-hydroxyalkyl-
amines were found to react preferentially through the
alkoxide ion rather than the amine. By using a variety
of R,ω-hydroxyalkylamines of increasing chain length
under alkaline conditions, it was possible to make three
further acridine-terpyridine conjugates, which were
then converted into their 2-hydroxyethane-thiolate plat-
inum(II) complexes (Scheme 2).
(2-H yd r oxyet h a n et h iola t e)(4′-su b st it u t ed -2,2′:
6′,2′′-t er p yr id in e)p la t in u m (II)-Acr id in e Con ju -
ga tes Ar e Mixed Typ e In h ibitor s of T. cr u zi TR.
Four compounds (1 and 2a -c) in which a 9-aminoacri-
dine derivative was covalently linked to a (terpyridine)-
platinum(II) complex were studied as inhibitors of T.
cruzi TR (Table 1). The derivatives differed by the
nature and/or the length of the spacer connecting the
two aromatic systems. In compound 1, the spacer was
an alkylthioether, whereas the other compounds (2a -
c) contained alkyl ether groups with 4-6 methylene
moiety may lead to an irreversible modification of the
enzyme. Four (2,2′:6′,2′′-terpyridine)platinum(II)-acri-
dine conjugates were synthesized and studied as ir-
reversible and reversible inhibitors of T. cruzi TR and
as reversible inhibitors of human GR.
Resu lts
Syn t h esis of t h e Com p ou n d s. 2-Methoxy-6,9-
dichloro-acridine can be converted to the 9-phenoxy
derivative, which is more reactive toward nucleophilic
substitution at the 9-position.13 Acridine-terpyridine
conjugates can be made by reacting 2-methoxy-6-chloro-
9-phenoxy-acridine and 4′-chloro-2,2′;6′,2′′-terpyridine
with a suitable linker. Thus, 2-mercaptoethylamine
reacts with 4′-chloro-2,2′:6′,2′′-terpyridine to give 4′-(2-
aminoethylthio)-2,2′:6′,2′′-terpyridine, which reacts with
2-methoxy-6-chloro-9-phenoxyacridine to give the acri-
dine-terpyridine conjugate, which is then converted to
the 2-hydroxyethane-thiolate platinum(II) complex (1)
by established procedures (Scheme 1).14

4526 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 20
Inhoff et al.
F igu r e 1. Inhibition of T. cruzi TR by compound 1. The kinetics were measured at 25 °C as described under Experimental
Procedures at a constant concentration of 100 µM NADPH. Each set of kinetics was measured twice whereby the single data
points differed by e10%. (a) Lineweaver-Burk plot; the TS2 concentration was varied in the presence of (b) none, (O) 0.4, (2) 1,
and (4) 2 µM inhibitor. The data points were fitted to a straight line by linear regression analysis. (b) Dixon plot; 1/v is plotted
against the inhibitor concentration in the presence of fixed concentrations of (9) 154, (4) 110, (2) 66, (O) 44, and (b) 32 µM TS2.
(c) Replot of the slopes of the reciprocal plot a vs the inhibitor concentration. (d) Replot of the points of intersection of the reciprocal
plot a vs the inhibitor concentration. In panels c and d, standard errors based on the linear regression analysis of a are given.
groups. All four derivatives were effective inhibitors of
TR. The type of inhibition was derived from Lin-
eweaver-Burk plots conducted in the absence and
presence of at least two different inhibitor concentra-
tions. The double reciprocal plots were linear and
intersected to the left of the 1/v axis and above the
baseline in accordance with mixed type15,16 or noncom-
petitive17 inhibition (Figure 1a). The inhibitor constants
were calculated from the slope and intersection values
of the Lineweaver-Burk plots as described under the
Experimental Procedures. Table 1 gives the Ki,slope and
Ki,int values16 obtained at each inhibitor concentration.
Plots of 1/v against [I] (Dixon) and replots of the slopes
and intercepts on the vertical axis of the reciprocal plots
vs the corresponding inhibitor concentration yield a
parabola when more than one inhibitor molecule can
bind to the same form of the enzyme. Linear mecha-
nisms in which more than one inhibitor molecule can
bind to different enzyme intermediates do not give rise
to curved inhibitor plots.15 With partially mixed inhibi-
tors, reciprocal plots of velocity against substrate con-
centration in the presence of a series of inhibitor
concentrations will also be linear, but plots against [I]
will curve downward to level off at a finite velocity.15
As shown for compound 1, the Dixon plot did not yield
straight lines but curved upward parabolically in ac-
cordance with the inhibitor constants not being inde-
pendent of the inhibitor concentration in the assay
(Figure 1b). The replot of the slopes of the reciprocal
plot against [I] also curved upward parabolically whereas
the replot of the intercepts against [I] is probably linear
(Figure 1c,d). This may indicate that two inhibitor
molecules can bind to the free enzyme but only one
molecule can bind to the ES complex.
To study a probable binding of the compounds at the
reduced nicotinamide adenine dinucleotide phosphate
(NADPH) binding site, kinetics were conducted at a
fixed concentration of 110 µM TS2 and varying concen-
trations of 8-102 µM NADPH in the absence and
presence of compound 1. Also in this case, strong mixed
type inhibition was observed (Table 1).
(2-H yd r oxyet h a n et h iola t e)(4′-su b st it u t ed -2,2′:
6′,2′′-t er p yr id in e)p la t in u m (II)-Acr id in e Con ju -
ga tes Do Not In h ibit TR Ir r ever sibly. TR was

Trypanosoma cruzi Trypanothione Reductase
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 20 4527
incubated with the chimeric compounds in the presence
and absence of NADPH as described under the Experi-
mental Procedures. At different time intervals, aliquots
of the incubation mixture were removed and assayed
for remaining activity in a standard TR assay. The
compounds inhibited neither the oxidized- nor the
NADPH-reduced enzyme in a time-dependent manner.
The partial inhibition observed remained constant over
time and corresponded to the reversible inhibition of TR
by the (terpyridine)platinum(II)-acridine conjugates.
(2-H yd r oxyet h a n et h iola t e)(4′-su b st it u t ed -2,2′:
6′,2′-ter pyr idin e)platin u m (II)-Acr idin e Con ju gates
Do Not In h ibit Hu m a n GR. Assays containing GR,
0.1-1 mM glutathione disulfide (GSSG), and 100 µM
NADPH were carried out in the presence and absence
of the respective chimeric compound as described under
the Experimental Procedures. Control assays contained
all components except that the inhibitor was replaced
by dimethyl sulfoxide (DMSO). After an initial lag
phase, the reaction rate slowly increased to give a final
activity nearly identical with that of the control. Obvi-
ously, the (terpyridine)platinum(II)-acridine conjugates
undergo a reversible interaction with the mammalian
enzyme, which is abolished under assay conditions. To
get an insight in the underlying mechanism, three
assays containing 81 µM 1, 100 µM NADPH, and 100
µM GSSG were conducted starting the reaction with
GSSG, GR, and NADPH, respectively. The lag phase
was most pronounced when starting the reaction with
GSSG. Independently of the order of additions, an initial
lag phase was observed that abolished during the course
of the assay resulting in a steady state velocity identical
with that in the absence of inhibitor.
gates, inhibit the enzyme irreversibly.9 The crystal
structure of the TR-mepacrine complex has shown a
single inhibitor molecule at the disulfide substrate
binding site close to a hydrophobic wall formed by Trp21
and Met113.5 Consistent with the X-ray crystal struc-
ture, mepacrine is a competitive inhibitor vs trypan-
othione disulfide with an apparent Ki value of about 19
µM. The detailed kinetic analysis of a series of 9-ami-
noacridines revealed the probable presence of more than
one binding site in the enzyme.4 The conjugation of
2-methoxy-6-chloro-9-aminoacridine with a (2,2′:6′,2′′-
terpyridine)platinum(II) complex lowers the inhibitor
constants of 9-aminoacridines by an order of magnitude
and leads to a mixed type inhibition pattern. In addition,
similar to the 9-aminoacridines, the chimeric conjugates
have probably more than one binding site in the enzyme.
One binding locus may be the cavity at the 2-fold axis
of the homodimeric protein. The respective pocket in
human GR has been shown to accommodate several
mixed type inhibitors such as xanthene,21 safranin,21,22
and isoalloxazines.23 No binding was observed in the
active site. Thus, it is tempting to speculate that the
respective cavity in TR, which is comparable in size and
in contrast to hGR, shows an overall negative charge24
can accommodate the compounds tested here. The
disulfide substrate binding site of TR is approximately
20 Å long, 15 Å wide, and 15 Å deep25 and thus much
more spacious than that of GR.26 Especially the outer
region of the active site of TR is much wider due to the
different orientation of two helices in the flavin adenine
dinucleotide (FAD) domain.25 In this region, a second
hydrophobic area formed by Phe396, Pro398, and Leu399,
called the Z-site, has been described.27 Modeling studies
on TR have suggested that tricyclics can assume dif-
ferent binding modes at the hydrophobic pockets in the
active site.19 Therefore, in addition to a possible binding
site at the 2-fold axis, the acridine moiety may interact
with one of the hydrophobic regions at the disulfide
binding site of TR with the (2,2′:6′,2′′-terpyridine)-
platinum(II) moiety pointing away from the active site
so that it is not in a position to interact with Cys52
directly. For compound 1, kinetics at varying concentra-
tions of NADPH also showed a mixed type inhibition
pattern. This finding supports the assumption that the
compounds do not exclusively bind at the trypanothione
disulfide binding site but probably have more than one
binding site.
Discu ssion
(2-Hydroxyethanethiolate)(4′-substituted-2,2′:6′,2′′-
terpyridine)platinum(II)-acridine conjugates are effec-
tive mixed type inhibitors of T. cruzi TR but do not
inhibit human GR. The complexes studied here are
bulky hydrophobic aromatic compounds, which are
positively charged under physiological conditions. They
contain a positive charge on the platinum ion, and N10
of the acridine ring is probably protonated.18 In addition
to the crucial hydrophobic nature of the compounds,19
the contribution of positive charges for ligands binding
in the active site of TR has been outlined by Faerman
et al.20 As shown previously, acridine itself and anionic
derivatives do not bind to the enzyme.5 (2-Hydroxy-
ethanethiolate)(4′-substituted-2,2′:6′,2′′-terpyridine)-
platinum(II) complexes are irreversible inhibitors of TR.
The irreversible inhibition implies the coordinative
replacement of 2-mercaptoethanol by Cys52 in the
active site of TR.9 The surprising finding that combina-
tion of an irreversible with a competitive ligand did not
conserve the irreversible type of inhibition indicates that
the acridine moiety determines the binding mode.
Obviously, conjugation with the acridine moiety pre-
vents the platinum complex from binding in the vicinity
of Cys52 thus not allowing the replacement of the fourth
ligand by the protein thiol group. Increase in size and
loss of flexibility cannot be responsible for the lack of
irreversible inhibition, since the most bulky (2,2′:6′,2′′-
terpyridine)platinum(II) complexes, comparable with
the (2,2′:6′,2′′-terpyridine)platinum(II)-acridine conju-
The (2,2′:6′,2′′-terpyridine)platinum(II)-acridine con-
jugates do not inhibit human GR. An initial lag phase
is completely abolished within the first minutes of the
assay. A fast recovery of GR activity after an initial
strong “inhibition” has also been observed with (2,2′:
6′,2′′-terpyridine)platinum(II) complexes.9 A possible
explanation is that the disulfide substrate displaces the
inhibitor from the active site and that the establishment
of the equilibrium (EI T E T ES) is a relatively slow
process.9 In accordance with this assumption is the
observation that starting the reaction with GSSG
showed a slightly longer initial lag phase than the
assays started with GR or NADPH. In the assays
started with substrate, EI can be formed before GSSG
is added, whereas in assays started with enzyme or
NADPH, no preformed EI complex is present. Another
explanation is that the DMSO present as solvent in the

4528 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 20
Inhoff et al.
concentration. The type of inhibition was derived from Lin-
eweaver-Burk plots, and the inhibitor constants were calcu-
lated from the expressions for the slopes and the intercepts
on the vertical axis. For mixed type inhibition, the equations
are15
assay is responsible for a slow conformational change
as reported earlier for yeast GR.28
Con clu sion s
The (2,2′:6′,2′′-terpyridine)platinum(II)-acridine con-
jugates studied here are mixed type inhibitors of T. cruzi
TR but do not interact with human GR. From the
structural analyses of several complexes of GR with
linear mixed type inhibitors and the kinetic results
presented here for TR, one may conclude that the (2,2′:
6′,2′′-terpyridine)platinum(II)-acridine conjugates can
bind in the cavity at the 2-fold axis of the homodimeric
enzyme with a probable additional binding site in the
active site. Unexpectedly, the combination of an ir-
reversible inhibitor [(2-hydroxyethanethiolate)(4′-sub-
stituted-2,2′:6′,2′′-terpyridine)platinum(II) complexes]
and a competitive ligand (9-aminoacridine) created
reversible mixed type inhibitors. These findings under-
line the difficulty to set up structure-activity relation-
ships based on known kinetic and crystallographic data
on inhibitors of TR.
(1 + [I]/Ki)
slope ) Ks
and
V
(1 + [I]/Ki′)
intercept on vertical axis )
V
where Ks is equated with Km assuming equilibrium conditions.
Ch em ica l Syn th esis. Chemicals were obtained from Ald-
rich, Lancaster, and Sigma and were used without further
purification. NMR spectra were taken on a Bruker DPX 250
spectrometer with a 5 mm multinuclear probe. The tempera-
ture was kept constant (298 K) by a variable unit. Mass spectra
were recorded by Dr. Robin Aplin either by electrospray
(complexes) or chemical ionization techniques (ligands and
organic compounds).
6-Ch lor o-2-m eth oxy-9-p h en oxya cr id in e. 2-Methoxy-6,9-
dichloroacridine (2 g, 7 mmol) was added to a solution of NaOH
(400 mg, 10 mmol) in phenol (10 g, 106 mmol), and the mixture
was stirred at 100 °C for 2 h and then poured into NaOH
solution (2 M, 50 mL). The solution was extracted with CHCl3
(3 × 50 mL), and the organic phase was dried (MgSO4) and
concentrated in vacuo. Recrystallization of the residue from
hot MeOH afforded the product as yellow needles (2.02 g, 84%).
mp: measured 154 °C; literature value 155 °C.13 1H NMR (CD2-
Cl2): δ ) 3.79 [s, 3 H, OCH3], 6.86 [d, 2 H, J ) 10 Hz, phenoxy
H 2], 7.06 [t, 1 H, J ) 7 Hz, phenoxy H 4], 7.16 [d, 1 H, J )
2.75 Hz, H1′′ or H5′′], 7.26 [d, 2 H, J ) 2.80 Hz, phenoxy H 3],
7.37 [dd, 1 H, J ) 9.25, 2 Hz, H3′′ or H7′′], 7.45 [dd, 1 H, J )
2.75, 9.5 Hz, H3′′ or H7′′], 7.97 [d, 1 H, J ) 9.25 Hz, H4′′ or
H8′′], 8.08 [d, 1 H, J ) 9.5 Hz, H4′′ or H8′′], and 8.17 [d, 1 H,
J ) 2 Hz, H1′′ or H5′′]. DCI-MS m/z (%): 336 (100) [MH+]
Exp er im en ta l P r oced u r es
Ma ter ia ls. Recombinant T. cruzi TR29 and human GR30
were prepared according to published procedures. Trypano-
thione disulfide was purchased from Bachem, Heidelberg,
Germany. Stock solutions (1-4 mM) of the (2,2′:6′,2′′-terpy-
ridine)platinum(II)-acridine conjugates were made in DMSO
and stored at -20 °C.
TR In h ibition Stu d ies Assa y. TR activity was measured
spectrophotometrically at 25 °C in TR assay buffer (40 mM
Hepes, 1 mM ethylenediaminetetraacetic acid (EDTA), pH 7.5)
as described.31 In the assay mixture (1 mL) containing 7-10
mU of T. cruzi TR, the enzyme activity was followed in the
absence and presence of different fixed concentrations of
inhibitor varying the trypanothione disulfide (TS2) concentra-
tion (24-186 µM). The reaction was started by adding 100 µM
NADPH, and the absorption decrease at 340 nm due to
NADPH consumption was followed. In the presence of com-
pound 1, starting the reaction with TS2 led to a slight initial
lag phase. Therefore, all assays were started with NADPH
where no lag phase was observed. In addition, for compound
1, T. cruzi TR activity was followed in the presence of a fixed
concentration of 110 µM TS2 and varying concentrations of
8-102 µM NADPH in the absence and presence of inhibitor.
The assays lacking inhibitor contained the respective amount
of DMSO.
GR In h ibition Stu d ies Assa y. GR activity was measured
in GR assay buffer (20.5 mM KH2PO4, 26.5 mM K2HPO4, 200
mM KCl, 1 mM EDTA, pH 6.9) at 25 °C as described.30 The
assay mixture (1 mL) contained 100 µM NADPH, 5-10 mU
GR, 1 mM or 0.1 mM GSSG and 54-81 µM 1, 19.2 µM 2a , 23
µM 2b, and 14.2 µM 2c, respectively. The reaction was started
by adding either GSSG, GR, and NADPH, respectively, and
the absorption decrease at 340 nm was followed. Control
samples contained all components except that the inhibitor
was replaced by DMSO.
C
20H14ClNO2 (335.5).
4′-(2-Am in o-eth a n eth io)-2,2′:6′,2′′-ter p yr id in e. 4′-Chloro-
2,2′:6′,2′′-terpyridine (1.34 g, 5 mmol) and 2-mercaptoethyl-
amine hydrochloride (6.82 g, 60 mmol) were suspended in 50
mL of 2-methylpropan-1-ol, and the mixture was stirred at
reflux for 54 h. The crude product was isolated by filtration of
the hot suspension and redissolved in 20 mL of water, and
the acidic solution (pH 2.5) was washed with 2 × 20 mL
dichloromethane. The aqueous phase was heated to 90 °C, and
1 M aqueous sodium hydroxide solution was added dropwise
until no further solid was seen to precipitate. The suspension
was cooled slowly to 5 °C, and the precipitated solid was
isolated by filtration, washed with aqueous base, and dried
under vacuum to yield the product as a white solid (821.8 mg,
53.3%); mp 93.5-94.5 °C. δ1H (250 MHz, CDCl3)/ppm: 3.12
(2H, t, J ) 5.8 Hz, CH2N), 3.31 (2H, t, J ) 6.1 Hz, CH2S), 7.36
(2H, dd, J ) 6.6, 4.7 Hz, H5,5′′), 7.87 (2H, t, J ) 7.0 Hz, H4,4′′),
8.39 (2H, s, H3′,5′), 8.63 (2H, d, J ) 7.9 Hz, H3,3′′), 8.71 (2H,
d, J ) 3.2 Hz, H6,6′′); δ13C (62.9 MHz, CDCl3)/ppm: 35.4 (CN),
41.0 (CS), 118.5 (C3′,5′), 121.8 (C3,3′′), 124.3 (C5,5′′), 137.3
(C4,4′′), 149.5 (C6,6′′), 151.1 (C4′), 155.5 (C2′,6′), 156.2 (C2,2′′)
(assigned from HMQC and HMBC spectra); m/z (APCI+) 309
(80%, [MH]+), 331 (100%, [MNa]+). HR-CI: 309.1174 (C17H16
N4S requires 309.1174).
-
Test of Ir r ever sible In h ibition of TR by (2-Hyd r oxy-
eth a n eth iola te)(4′-su bstitu ted -2,2′:6′,2′′-ter p yr id in e)p la t-
in u m (II)-2-m eth oxy-6-ch lor o-a cr id in e Con ju ga tes. In a
total volume of 0.4 mL of TR assay buffer, pH 7.5, 0.55 µM
TR was incubated at 25 °C with 54 µM 1, 19 µM 2a , 5 µM 2b,
and 50 µM 2c, respectively, in the presence and absence of
100 µM NADPH and varying concentrations of inhibitor.
Control samples contained all components except that the
inhibitor was replaced by DMSO. At different time intervals
(0-120 min), aliquots of 4-20 µL were removed and assayed
for remaining activity in a standard TR assay.31
4′[N-(6-Ch lor o-2-m eth oxyacr idin e-9-am in oeth an eth io)]-
2,2′:6′,2′-ter p yr id in e. 4′-(2-Amino-ethanethio)-2,2′:6′,2′′-terpy-
ridine (500 mg, 1.62 mmol) and 6-chloro-2-methoxy-9-phe-
noxyacridine (1.0 g, 2.98 mmol) were suspended in 30 mL of
acetonitrile, and 1 mL of glacial acetic acid was added. The
resulting suspension was heated at reflux, and the progress
of the reaction was followed by TLC (normal phase silica,
eluting with 10% MeOH in CHCl3). After 2.5 h, no terpyridine
starting material could be detected. The solvent was removed
under vacuum, and the residual gum was resuspended in 25
mL of chloroform and 25 mL of aqueous sodium hydroxide
solution. The solid material was removed by filtration, the
phases were separated, and the aqueous layer was extracted
with chloroform (2 × 10 mL). The combined organic fractions
Deter m in a tion of In h ibitor Con sta n ts. The activity of
T. cruzi TR was followed in the absence and presence of
different fixed concentrations of inhibitor varying the substrate

Trypanosoma cruzi Trypanothione Reductase
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 20 4529
1
were dried over K2CO3, and the solvent was removed in vacuo.
The residual gum was redissolved in chloroform and purified
by column chromatography (eluting with a gradient of 1-4%
MeOH in CHCl3). Removal of solvent in vacuo left a gum,
which was triturated with chloroform and hexane. The result-
ing solid was dried under vacuum to leave the product as a
11.5%). H NMR (CD2Cl2): δ 1.60 [m, 4 H, CH2CH2CH2CH2],
1.91 [m, 4 H, CH2CH2CH2CH2], 3.82 [t, 2 H, J ) 7 Hz, CH2-
NH2], 4.01 [s, 3H, OCH3], 4.27 [t, 2 H, J ) 7 Hz, CH2O], 7.37
[ddd, 2 H, J ) 7.5, 5.0, 1.0 Hz, H5,5′′], 7,40 [m, 3 H, acridine],
7.92 [dt, 2 H, J ) 8.0, 7.6, 1.8 Hz, H4,4′′], 8.04 [s, 2 H, H3′,5′],
8,08 [m, 3 H, acridine], 8.67 [ddd, 2 H, J ) 8.0, 2.0, 1.0 Hz,
H3,3′′], 8.73 [ddd, 2 H, J ) 5.0, 2.0, 1.0 Hz, H6,6′′] DCI-MS;
m/z (%): 590 (100) [MH+] C35H33ClN5O2 (590.0).
1
yellow powder (370.6 mg, 41.6%); mp 68-71 °C. δ H (400 MHz,
CD2Cl2)/ppm: 3.50 (2H, t, J ) 6.2 Hz, CH2S), 3.95 (3H, s, CH3),
4.06 (2H, t, J ) 6.2 Hz, CH2N), 7.3-7.4 (5H, m, H5,5′′ + HA3
+ HA1 + HA7), 7.9-8.0 (3H, m, H4,4′′ + HA4), 8.02 (1H, d, J
) 2.0 Hz, HA5), 8.17 (1H, d, J ) 9.2 Hz, HA8), 8.42 (2H, s,
[6-Ch lor o-2-m eth oxy-9-[4′-(6-a m in oh exyloxy)-2,2′:6′,2′′-
ter p yr id in e P la tin u m (II) Hyd r oxyeth a n eth iola te]a cr i-
d in e Bisn itr a te (2c). Silver nitrate (35.7 mg, 0.21 mmol) in
aqueous acetone (80% acetone, 25 mL) was added to a
suspension of Pt(COD)I2 (55.7 mg, 0.1 mmol) in aqueous
acetone (0.75 mL). Silver iodide was removed by centrifugation.
The supernatant was added to a suspension of ligand (53 mg,
0.09 mmol) in MeCN (5 mL). After 10 min, the solid was
isolated, and the supernatant was discarded. The solid was
washed with ether:acetone 3:1 and taken up in DMF, and
mercaptoethanol (20 µL, 0.20 mmol) was added. The solid was
precipitated with ether, isolated by centrifugation, washed
with ether several times, and dried in vacuo; yield (23 mg, 30%)
(found: C, 41.5; H, 4.7; N, 8.5. Calcd for C37H38ClSN7O9Pt: C,
41.3; H, 4.4; N, 9.1%). 1H NMR (DMSO): δ 1.58 [m, 4 H,
CH2CH2CH2CH2], 1.92 [m, 4 H, CH2CH2CH2CH2], 2.50 [t, 2
H, J ) 7 Hz, SCH2], 3.64 [t, 2 H, J ) 7 Hz, HOCH2], 3.92 [s,
3H OCH3], 4.10 [t, 2 H, J ) 7 Hz, CH2NH], 4.35 [t, 2 H, J )
7 Hz, CH2O], 7.73 [m, 7 H, acridine + terpy], 8.21 [s, 2 H,
H3′,5′], 8,58 [m, 5 H, acridine + terpy], 9.31 [s, 2 H, H6,6′′],
9.58 [s, 1 H, NH (acridine)]. 13C NMR (DMSO): δ 32.4, 33.3
[CH2CH2CH2CH2], 35.5, 36.5 [CH2CH2CH2CH2], 40.4 [CH2-
NHAcr], 72.8 [CH2OTerpy], 77.9 [CH3O], 56.3, 63.7, [mercap-
toethanol]. ESI-MS; m/z (%): 862 (100) [MH+] C37H38ClSN5O3-
Pt (862.0).
4′-(5-Am in op en tyloxy)-2,2′:6′,2′′-ter p yr id in e. The com-
pound was prepared as for 4′-(6-aminohexyloxy)-2,2′:6′,2′′-
terpyridine; yield (0.916 g, 70%). 1H NMR (CDCl3): δ 1.38 [m,
2 H, CH2CH2CH2], 1.98 [t, 4 H, J ) 7 Hz, CH2CH2CH2], 2.75
[t, 2 H, J ) 7 Hz, CH2NH2], 4.31 [t, 2 H, J ) 7 Hz, CH2O],
7.41 [ddd, 2 H, J ) 7.5, 5.0, 1.0 Hz, H5,5′′], 7.92 [dt, 2 H, J )
8.0, 7.6, 1.8 Hz, H4,4′′], 8.09 [s, 2 H, H3′,5′], 8.70 [ddd, 2 H, J
) 8.0, 2.0, 1.0 Hz, H3,3′′], 8.73 [ddd, 2 H, J ) 5.0, 2.0, 1.0 Hz,
H6,6′′] DCI-MS; m/z (%): 334 (100) [MH+] C21H24N4O (334.0).
6-Ch lor o-2-m eth oxy-9-[4′-(5-a m in op en tyloxy)-2,2′:6′,2′′-
ter pyr idin e]acr idin e. The compound was prepared as 6-chlo-
ro-2-methoxy-9-[4′-(6-aminohexyloxy)-2,2′:6′,2′′-terpyridine]-
acridine; yield (0.792 g, 58%) (found: C, 69.9; H, 5.4; N,12.3.
H3′,5′), 8.64 (2H, d, J ) 8.0 Hz, H3,3′′), 8.69 (2H, d, J ) 4.8
13
Hz, H6,6′′). δ
(100.6 MHz, CD2Cl2)/ppm: 33.1 (CS), 49.0
C
(CN), 56.2 (CO), 99.3 (CA3 or CA1), 117.9 (CA13), 118.8
(C3′,5′), 120.0 (CA10), 121.8 (C3,3′′), 124.7 (C5,5′′), 124.9 (CA8),
125.4 (CA1 or CA3), 125.6 (CA7), 128.8 (CA5), 132.1 (CA4),
135.0 (CA12), 137.5 (C4,4′′), 147.4 (CA11), 148.7 (CA6), 149.2
(CA9), 149.7 (C6,6′′ + C4′), 156.0 (C2,2′′ + C2′,6′), 157.2 (CA2)
(assigned from HMQC and HMBC spectra); m/z (APCI+): 551
(100%, [M.H]+), 573 (25%, [M.Na]+). HR-CI: 550.1456,
552.1434 (C31H25N5OSCl requires 550.1468, 552.1439).
4′[N-(6-Ch lor o-2-m eth oxyacr idin e-9-am in oeth an eth io)]-
2,2′:6′,2′′-ter p yr id in e P la tin u m (II)-2-h yd r oxyeth a n eth i-
ola te (1). The title compound was prepared by the published
method14 from 4′[N-(6-chloro-2-methoxyacridine-9-amino-
ethanethio)]-2,2′:6′,2′′-terpyridine (0.2 mmol) and mercapto-
ethanol (32 µL 36.5 mg, 0.47 mmol). The title compound was
1
a brown solid (27.8 mg, 14.7%); mp >230 °C. δ H (400 MHz,
d6-DMSO)/ppm: 2.66 (2H, t, J ) 7.5 Hz, CH2SPt), 3.69 (2H, t,
J ) 7.5 Hz, CH2O), 3.73 (3H, s, CH3), 3.87 (2H, br (t), CH2-
Sterpy), 4.65 (2H, br (t), CH2N), 7.36 (2H, s, HA3, HA4), 7.41
(1H, d, J ) 9.0 Hz, HA7), 7.47 (1H, d, J ) 2.0 Hz, HA5), 7.61
(1H, s, HA1), 8.03 (2H, m, H5,5′′), 8.19 (2H, s, H3′,5′), 8.30
(2H, d, J ) 8.0 Hz, H3,3′′), 8.5 (3H, m, HA8, H4,4′′), 9.40 (2H,
13
br d, J ) 5.0 Hz, H6,6′′), 9.56 (1H, br (t), NH). δ C (100.6 MHz,
d6-DMSO)/ppm: 33.5 (CH2Sterpy), 34.0 (CH2SPt), 49.0 (CH2N),
57.0 (CH2O), 66.0 (CH3), 103.5 (CA1), 110.5 (CA13), 118.0
(CA7), 122.0 (CA3), 123.0 (C3′,5′), 125.0 (CA12), 126.5 (C3,3′′),
128.0 (CA8, CA10), 129.5 (CA4), 130.5 (C5,5′′), 134.5 (CA11),
140.5 (CA6), 143.5 (C4,4′′), 152.0 (C2′,6′), 153.5 (C6,6′′), 155.5
(C4′), 157.0 (CA2), 159.0 (C2,2′′) (assigned from HMQC and
HMBC spectra; no cross-peaks for CA9 or CA5). m/z (ESI+):
411.6 (100%, [MH]2+), 822.2 (60%, [M]+). HR-ESI of [M]2+
411.62 (C33H31N5S2O2ClPt requires 411.56).
:
4′-(6-Am in oh exyloxy)-2,2′:6′,2′′-ter p yr id in e. A solution
of NaH (120 mg, 3 mmol) and 6-amino-1-hexanol (585 mg, 5
mmol) in 10 mL of dimethyl formamide (DMF) was stirred for
30 min at room temperature. 4′-Chloro-2,2′:6′,2′′-terpyridine
(267 mg, 1 mmol) was added, and the mixture was stirred
overnight at 80 °C under argon. A few drops of water were
added, and DMF was evaporated. The product was taken up
in dichloromethane and washed with basic NaOH/H2O solution
for several times, and the organic layer was dried (MgSO4)
and concentrated in vacuo to give the product (0.301 g, 87%).
1H NMR (CDCl3): δ 1.38 [m, 6 H, (CH2)n], 1.91 [t, 2 H, J ) 7
Hz, CH2CH2O], 2.75 [t, 2 H, J ) 7 Hz, CH2NH2], 4.27 [t, 2 H,
J ) 7 Hz, CH2O], 7.37 [ddd, 2 H, J ) 7.5, 5.0, 1.0 Hz, H5,5′′],
7.89 [dt, 2 H, J ) 8.0, 7.6, 1.8 Hz, H4,4′′], 8.04 [s, 2 H, H3′,5′],
8.65 [ddd, 2 H, J ) 8.0, 2.0, 1.0 Hz, H3,3′′], 8.73 [ddd, 2 H, J
) 5.0, 2.0, 1.0 Hz, H6,6′′] DCI-MS; m/z (%): 348 (100) [MH+]
1
Calcd for C34H31ClN5O2: C, 70.8; H, 5.2; N, 12.2%). H NMR
(CD2Cl2): δ 1.69 [m, 2 H, CH2CH2CH2], 1.95 [m, 4 H, CH2-
CH2CH2], 3.82 [t, 2 H, J ) 7 Hz, CH2NH2], 4.01 [s, 3H, OCH3],
4.29 [t, 2 H, J ) 7 Hz, CH2O], 7.37 [ddd, 2 H, J ) 7.5, 5.0, 1.0
Hz, H5,5′′], 7,40 [m, 3 H, acridine], 7.92 [dt, 2 H, J ) 8.0, 7.6,
1.8 Hz, H4,4′′], 8.04 [s, 2 H, H3′,5′], 8,08 [m, 3 H, acridine],
8.67 [ddd, 2 H, J ) 8.0, 2.0, 1.0 Hz, H3,3′′], 8.73 [ddd, 2 H, J
) 5.0, 2.0, 1.0 Hz, H6,6′′] DCI-MS; m/z (%): 576 (100) [MH+]
C
34H31ClN5O2 (576.0).
6-Ch lor o-2-m eth oxy-9-[4′-(5-a m in op en tyloxy)-2,2′:6′,2′′-
ter pyr idin e P latin u m (II)h ydr oxyeth an eth iolate]acr idin e
Bisn itr a te (2b). This was prepared as [6-chloro-2-methoxy-
9-[4′-(6-aminohexyloxy)-2,2′:6′,2′′-terpyridine platinum(II)hy-
droxyethanethiolate]acridine bisnitrate; yield (22 mg, 30%)
(found: C, 42.3; H, 4.1; N, 9.5. Calcd for C36H36ClSN7O9Pt‚
3H2O: C, 42.1; H, 4.1; N, 9.5%). 1H NMR (DMSO): δ 1.68 [m,
2 H, (CH2CH2CH2)], 2.00 [m, 4 H, (CH2CH2CH2)], 2.50 [t, 2 H,
J ) 7 Hz, SCH2], 3.64 [t, 2 H, J ) 7 Hz, HOCH2], 3.92 [s, 3H
OCH3], 4.10 [t, 2 H, J ) 7 Hz, CH2NH2], 4.35 [t, 2 H, J ) 7
Hz, CH2O], 7,73 [m, 7 H, acridine + terpy], 8.21 [s, 2 H, H3′,5′],
8.58 [m, 5 H, acridine + terpy], 9.31 [s, 2 H, H6,6′′], 9.58 [s, 1
C
21H24N4O (348.0).
6-Ch lor o-2-m eth oxy-9-[4′-(6-a m in oh exyloxy)-2,2′:6′,2′′-
ter p yr id in e]a cr id in e. A solution of the phenoxyacridine (220
mg, 0.655 mmol) and 4′-(6-aminohexyloxy)-2,2′:6′,2′′-terpyri-
dine (152 mg, 0.437 mmol) in 10 mL of acetonitrile and 1 mL
of acetic acid was refluxed for 3 h. The MeCN was evaporated,
and the product was taken up in CH2Cl2 and washed with basic
NaOH/H2O solution. The organic phase was dried (MgSO4) and
flash-chromatographed over silica with dichloromethane-
methanol-ammonia 9:1:0.1. The fractions containing acridone
and product were combined and flash-chromatographed over
silica again with dichloromethane-ammonia 90:1 yielding the
product as a yellow solid (170 mg, 66%) (found: C, 68.9; H,
5.5; N,11.6. Calcd for C35H32ClN5O2‚H2O: C, 69.1; H, 5.6; N,
H, NH (acridine)] ESI-MS; m/z (%): 848 (100) [MH+] C36H36
ClSN5O3Pt (848.0).
4′-(4-Am in obu t yloxy)-2,2′:6′,2′′-t er p yr id in e. This was
prepared as for 4′-(6-aminohexyloxy)-2,2′:6′,2′′-terpyridine;
yield (1.082 g, 85%). 1H NMR (CDCl3): δ 1.38 [m, 2 H, (CH2)],
1.98 [t, 2 H, J ) 7 Hz, CH2], 2.81 [t, 2 H, J ) 7 Hz, CH2NH2],
4.30 [t, 2 H, J ) 7 Hz, CH2O], 7.41 [ddd, 2 H, J ) 7.5, 5.0, 1.0
-

4530 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 20
Inhoff et al.
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and structure relationship of acridine derivatives against African
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L.; Pratt, J . M.; Rock, P.; Yardley, V.; Croft, S. L. Cytotoxicity
of (2,2′:6′,2′′-terpyridine)platinum(II) complexes to Leishmania
donovani, Trypanosoma cruzi, and Trypanosoma brucei. J . Med.
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conjugates of the bis(phenanthroline) ligand “Clip-Phen” and
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complexes. Eur. J . Inorg. Chem. 1999, 557-563.
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1996, 386-387.
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Hz, H5,5′′], 7.92 [dt, 2 H, J ) 8.0, 7.6, 1.8 Hz, H4,4′′], 8.09 [s,
2 H, H3′,5′], 8.70 [ddd, 2 H, J ) 8.0, 2.0, 1.0 Hz, H3,3′′], 8.73
[ddd, 2 H, J ) 5.0, 2.0, 1.0 Hz, H6,6′′] DCI-MS; m/z (%): 320
(100) [MH+] C20H22N4O (320.0).
6-Ch lor o-2-m eth oxy-9-[4′-(4-a m in obu tyloxy)-2,2′:6′,2′′-
ter p yr id in e]a cr id in e. This was prepared as 6-chloro-2-
methoxy-9-[4′-(6-aminohexyloxy)-2,2′:6′,2′′-terpyridine]acri-
dine; yield (1.230 g, 65%) (found: C, 69.2; H, 6.3; N,12.5. Calcd
for C33H28ClN5O2‚H2O: C, 68.4; H, 5.3; N, 12.1%). 1H NMR
(CD2Cl2): δ 1.70 [m, 2 H, (CH2)], 2.11 [m, 2 H, (CH2)], 3.89 [t,
2 H, J ) 7 Hz, CH2NH2], 4.01 [s, 3H OCH3], 4.32 [t, 2 H, J )
7 Hz, CH2O], 7.37 [ddd, 2 H, J ) 7.5, 5.0, 1.0 Hz, H5,5′′], 7,40
[m, 3 H, acridine], 7.92 [dt, 2 H, J ) 8.0, 7.6, 1.8 Hz, H4,4′′],
8.04 [s, 2 H, H3′,5′], 8,08 [m, 3 H, acridine], 8.67 [ddd, 2 H, J
) 8.0, 2.0, 1.0 Hz, H3,3′′], 8.73 [ddd, 2 H, J ) 5.0, 2.0, 1.0 Hz,
H6,6′′] DCI-MS; m/z (%): 562 (100) [MH+] C33H29ClN5O2
(562.0).
6-Ch lor o-2-m eth oxy-9-[4′-(4-a m in obu tyloxy)-2,2′:6′,2′′-
ter pyr idin e P latin u m (II)h ydr oxyeth an eth iolate]acr idin e
Bisn itr a te (2a ). This was prepared as [6-chloro-2-methoxy-
9-[4′-(6-aminohexyloxy)-2,2′:6′,2′′-terpyridine platinum(II)hy-
droxyethanethiolate]acridine bisnitrate; yield (25 mg, 36%)
(found: C, 42.1; H, 4.1; N, 9.7. Calcd for C35H34ClSN7O9Pt ‚
1
3H2O: C, 41.5; H, 4.0; N, 9.7%). H NMR (DMSO): δ 2.05[m,
2 H, (CH2)], 2.15 [m, 2 H, (CH2)], 2.50 [t, 2 H, J ) 7 Hz, SCH2],
3.64 [t, 2 H, J ) 7 Hz, HOCH2], 3.92 [s, 3H OCH3], 4.10 [t, 2
H, J ) 7 Hz, CH2NH2], 4.35 [t, 2 H, J ) 7 Hz, CH2O], 4.81 [s,
1 H, NH (acridine)], 7,73 [m, 7 H, acridine + terpy], 8.21 [s, 2
H, H3′,5′], 8,58 [m, 5 H, acridine + terpy], 9.31 [s, 2 H, H6,6′′].
ESI-MS; m/z (%): 834 (100) [MH+] C35H33ClSN5O3Pt (834.0).
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Ack n ow led gm en t. This work was supported by the
Deutsche Forschungsgemeinschaft (DFG Kr 1242/1-4
and SFB544 “Kontrolle tropischer Infektionskrank-
heiten”). We thank the BBSRC for a research student-
ship (J .M.R.) and the EU for an Erasmus scholarship
(J .W.B.).
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M.-A.; Landry, V.; Sergheraert, C.; Croft, S. L.; Krauth-Siegel,
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quinone derivatives as subversive substrates of trypanothione
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N. J .; Field, H.; Cerami, A.; Henderson, G. B. X-ray structure of
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A. H. Phenothiazine inhibitors of trypanothione reductase as
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