Inhibition of Trypanosoma cruzi trypanothione reductase by acridines: Kinetic studies and structure-activity relationships

Article abstract of DOI:10.1021/jm990386s

Series of 9-amino and 9-thioacridines have been synthesized and studied as inhibitors of trypanothione reductase (TR) from Trypanosoma cruzi, the causative agent of Chagas' disease. The compounds are structural analogues of the acridine drug mepacrine (quinacrine), which is a competitive inhibitor of the parasite enzyme, but not of human glutathione reductase, the closest related host enzyme. The 9-aminoacridines yielded apparent K(i) values for competitive inhibition between 5 and 43 μM. The most effective inhibitors were those with the methoxy and chlorine substituents of mepacrine and NH₂ or NHCH(CH₃)(CH₂)₄N(Et)₂ at C9. Detailed kinetic analyses revealed that in the case of 9- aminoacridines more than one inhibitor molecule can bind to the enzyme. In contrast, the 9-thioacridine derivatives inhibit TR with mixed-type kinetics. The kinetic data are discussed in light of the three-dimensional structure of the TR-mepacrine complex. The conclusion that structurally very similar acridine compounds can give rise to completely different inhibition patterns renders modelling studies and quantitative structure-activity relationships difficult.

Full text of DOI:10.1021/jm990386s

5448
J . Med. Chem. 1999, 42, 5448-5454
In h ibition of Tr ypa n osom a cr u zi Tr yp a n oth ion e Red u cta se by Acr id in es:
Kin etic Stu d ies a n d Str u ctu r e-Activity Rela tion sh ip s
Susanne Bonse,† Christiane Santelli-Rouvier,‡ J acques Barbe,‡ and R. Luise Krauth-Siegel*,†
Biochemie-Zentrum Heidelberg, Heidelberg University, Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany, and
GERCTOP-UPRESA CNRS 6009, Faculte´ de Pharmacie, Universite´ de la Mediterranne´e, 27 Bd J ean-Moulin,
F-13385 Marseille, France
Received J uly 27, 1999
Series of 9-amino and 9-thioacridines have been synthesized and studied as inhibitors of
trypanothione reductase (TR) from Trypanosoma cruzi, the causative agent of Chagas’ disease.
The compounds are structural analogues of the acridine drug mepacrine (quinacrine), which
is a competitive inhibitor of the parasite enzyme, but not of human glutathione reductase, the
closest related host enzyme. The 9-aminoacridines yielded apparent Ki values for competitive
inhibition between 5 and 43 µM. The most effective inhibitors were those with the methoxy
and chlorine substituents of mepacrine and NH2 or NHCH(CH3)(CH2)4N(Et)2 at C9. Detailed
kinetic analyses revealed that in the case of 9-aminoacridines more than one inhibitor molecule
can bind to the enzyme. In contrast, the 9-thioacridine derivatives inhibit TR with mixed-type
kinetics. The kinetic data are discussed in light of the three-dimensional structure of the TR-
mepacrine complex. The conclusion that structurally very similar acridine compounds can give
rise to completely different inhibition patterns renders modelling studies and quantitative
structure-activity relationships difficult.
In tr od u ction
discriminating factor for specific inhibitors of TR versus
GR which should allow the design of inhibitors specific
for the parasite enzyme.8
The known sensitivity of trypanosomatids toward
oxidative stress and the absence of the enzyme from the
mammalian host render TR an attractive target mol-
ecule for the development of new antiparasitic drugs.
Several genetic approaches have unambiguously shown
that the enzyme is essential for growth and virulence
of the parasites.9-11
A prerequisite for a rational drug development is the
knowledge of the three-dimensional structure of the
target protein. The structures of TR4-6 and of complexes
with its substrates6,12,13 as well as with the reversible
inhibitor mepacrine have been elucidated.14 A variety
of lead compounds were revealed within the past few
years (for reviews, see refs 15-17). The large group of
reversible inhibitors include acridines, phenothiazines,
benzoazepines, and pyridoquinolines.18-22 None of these
tricyclic derivatives interfere with human glutathione
reductase, the closest related host enzyme.
Trypanothione reductase is a flavoenzyme which has
been found so far exclusively in trypanosomatid para-
sites1 and recentlystogether with glutathione reduc-
tasesin Euglena gracilis.2 Trypanosomes and leishma-
nia are the causative agents of African sleeping sickness
(Trypanosoma brucei gambiense, Trypanosoma brucei
rhodesiense), Chagas’ disease (Trypanosoma cruzi), Na-
gana cattle disease (Trypanosoma congolense, Trypa-
nosoma brucei brucei), Kala-azar (Leishmania dono-
vani), and oriental sore (Leishmania tropica). All these
parasitic protozoa have in common that they lack the
nearly ubiquitous enzyme glutathione reductase (GR).
Their main low molecular mass thiols are conjugates
between glutathione and spermidine, namely monoglu-
tathionyl spermidine and bis(glutathionyl)spermidine
[trypanothione, T(SH)2].3 These compounds are kept
reduced by the flavoenzyme trypanothione reductase
(TR):
TS2 + NADPH + H+ f T(SH)2 + NADP+
Phenothiazines and acridines show trypanocidal ac-
tivities.21,23,24 Acridine derivatives are also well-known
antibacterial and antitumor drugs. In addition, they
have been considered for the treatment of several
protozoan infections.24 9-Amino- and 9-thioacridines
were highly active against African trypanosomes in
culture but many of them were also toxic to human
adenoma carcinoma cells.24 In a serial screening of
compounds licensed for human treatment, mepacrine
was one of the most active drugs against T. cruzi in
vitro.23
Mechanistically and structurally TR and human GR
are closely related. The most important difference
between parasite and host enzyme is their mutual
exclusive substrate specificity which is based on the
respective charge distributions of their active sites. The
binding site for trypanothione disulfide in TR is hydro-
phobic and negatively charged, but the GSSG binding
site in GR has an overall positive charge.4-7 This finding
has led to the elucidation of charge being a major
So far the TR-mepacrine structure is the only crystal
structure of trypanothione reductase in complex with
an inhibitor. Mepacrine is fixed in the active site close
to the hydrophobic wall formed by Trp21 and Met113.
* To whom correspondence should be addressed. Phone: +49 6221
54 41 87. Fax: +49 6221 54 55 86. E-mail: krauth-siegel@
urz.uni-heidelberg.de.
† Biochemie-Zentrum Heidelberg, Heidelberg University.
‡ Faculte´ de Pharmacie, Universite´ de la Mediterranne´e, Marseille.
10.1021/jm990386s CCC: $18.00 © 1999 American Chemical Society
Published on Web 12/04/1999

Inhibition of T. Cruzi Trypanothione Reductase
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 26 5449
Sch em e 2a
a
a: 8 R4 ) (CH2)2N(C2H5)2. b: 9 R4 ) (CH2)4N(C2H5)2.
F igu r e 1. Stereoview of the active site of T. cruzi TR with
bound mepacrine (after ref 14). The isoalloxazine ring of FAD
forms the center of the active site. The dashed line represents
the redox active disulfide. Mepacrine (bold lines) binds with
the acridine ring close to the hydrophobic region formed by
Trp21 and Met113. Specific pairwise interactions between
functional groups of the drug and side chains of the protein
include the ring nitrogen and Met113, the chlorine atom and
Trp21, and the methoxy group and Ser109. The alkylamino
chain is fixed to Glu18 via a solvent molecule (W2).
2-diethylaminoethyl chloride (8)29 or with 1,4-bromo-
chlorobutane followed by amination with diethylamine
(Scheme 2) (9).
9-Am in oa cr id in es Ar e Com p etitive In h ibitor s of
TR w ith Mor e Th a n On e Bin d in g Site. Acridine
derivatives with an amino substituent at position C9
of the aromatic ring inhibited the parasite enzyme
competitively (Table 1). The most effective compounds
were 2 and 7 which have in common the chlorine and
the methoxy substituents of mepacrine but differ in the
nature of their side chain at C9. The weakest inhibitor
was tacrine which represents 9-aminoacridine with one
of the outer rings saturated. The inhibitor constants
given in Table 1 were derived from Lineweaver Burk
plots conducted in the absence of inhibitor and in the
presence of at least two different inhibitor concentra-
tions. The double reciprocal plots were linear and
intersected in a common point on the 1/v axis in
accordance with a competitive type of inhibition. A
striking observation was that the calculated inhibitor
constants were not independent of the inhibitor con-
centration in the assay. In each case the value obtained
at the higher inhibitor concentration was (slightly) lower
than that calculated at the lower [I]. The inhibitor
constant determined from the slope of the reciprocal plot
is actually Ki,slope, which is not the Ki for the dissociation
of an EI complex but rather a more complex function of
Ki varying with [I].30 The Ki,apparent values given in Table
1 are the mean of these Ki,slope values at the respective
inhibitor concentrations (see below). Subsequent varia-
tion of the concentration of 9-aminoacridine as well as
of compounds 1 and 5, respectively, at fixed concentra-
tions of trypanothione disulfide resulted in Dixon plots
which curved upward parabolically (Figure 2a). These
forms of inhibition occur when more than one molecule
of inhibitor can bind to the same form of the enzyme.30,31
It should be emphasized that mechanisms in which
more than one inhibitor molecule can bind to different
enzyme intermediates do not give rise to curved inhibi-
tor plots.31
Sch em e 1a
a
1 R1 ) 3-Cl, R2 ) H, R3 ) H; 2 R1 ) 6-Cl, R2 ) 2-OCH3, R3
)
)
H; 3 R1 ) H, R2 ) 2-OCH3, R3 ) CH3CH(CH2)3N(C2H5)2; 4 R1
3-Cl, R2 ) H, R3 ) CH3CH(CH2)3N(C2H5)2; 5 R1 ) 6-Cl, R2)
2-OCH3, R3 ) CH3CH(CH2)3N(C2H5)2; 6 R1 ) 3-Cl, R2 ) H, R3
)
CH3CH(CH2)4N(C2H5)2; 7 R1 ) 6-Cl, R2 ) 2-OCH3, R3 ) CH3CH-
(CH2)4N(C2H5)2.
The acridine ring and its substituents are in direct
contact with side chains of the protein. The interactions
include the ring nitrogen and Met113, the chlorine atom
and Trp21, and the methoxy group and Ser109. The
alkylamino chain of mepacrine points into the inner
region of the active site and may undergo a water-
mediated hydrogen bond to Glu18.14 The residues in
contact with the bound mepacrine are four out of five
residues in the active site which are not conserved when
comparing T. cruzi TR and human GR. To get a deeper
insight in the relative contribution of each substituent
upon binding to the enzyme, several analogues of
mepacrine have been synthesized and studied as inhibi-
tors of T. cruzi TR. The kinetics will be discussed in light
of the three-dimensional structure of the TR-mepacrine
complex (Figure 1).
Such two- or more than two-site competitive inhibi-
tion is not distinguishable from one-site pure competi-
tive or partial competitive inhibition by the usual v
versus S or 1/v versus 1/S plots. The best indication of
the nature of inhibition is obtained from the replots of
the slopes of the reciprocal plot versus the corresponding
[I].30 For two-site competitive inhibition, this replot
yields a parabola. One-site pure competitive and one-
site partial competitive systems would give linear and
hyperbolic replots, respectively. Figure 2b shows the
replot of slope1/S versus [I] of the Lineweaver Burk plot
(not shown) for compound 1. In the case of two inhibitor
Resu lts
Syn th esis of Acr id in e Der iva tives. The 9-ami-
noacridines 1-7 were prepared by heating the corre-
sponding 9-chloroacridines (Scheme 1) in phenol (a) with
ammonium carbonate (1, 2);25 (b) with 5-diethylamino-
2-aminopentane (3, 4, 5);26 and (c) with 6-diethylamino-
2-aminohexane (6, 7). These diamines were obtained by
reductive amination with NaBH3CN27 of the corre-
sponding diethylaminoalkane-2-one.28
The 9-thioacridine derivatives were obtained by alkyl-
ation of the corresponding 9-thioacridinone either with

5450 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 26
Ta ble 1. Inhibition of T. Cruzi TR by Acridine Derivativesa
Bonse et al.
9-Aminoacridines
substituent
compd
R1
R2
R3
Ki, apparent (µM)b (competitive)
9-aminoacridine
1
2
3
NH2
NH2
NH2
38 ( 10 [100; 200]
43 ( 5 [29; 58]
7 ( 2 [26; 58]
27 ( 2 [56; 92]
10 ( 1 [23; 60]
19 ( 5 [20; 46]
11 ( 1 [50; 100]
5.5 ( 1 [26; 60]
140 ( 18 [100; 200]
80 ( 20 [50; 100]
Cl
Cl
OCH3
OCH3
NHCH(CH3)(CH2)3N(Et)2
NHCH(CH3)(CH2)3N(Et)2
NHCH(CH3)(CH2)3N(Et)2
NHCH(CH3)(CH2)4N(Et)2
NHCH(CH3)(CH2)4N(Et)2
NH2
4
Cl
Cl
Cl
Cl
5 (mepacrine)
6
OCH3
OCH3
7
tacrinec
6-chlorotacrined
Cl
NH2
9-Thioacridinese
substituent
R2
compd
R1
R3
Ki (µM) (mixed-type)
Ki′ (µM)
829
9
OCH3
OCH3
Cl
Cl
S(CH2)2N(Et)2
S(CH2)4N(Et)2
37 ( 1
21 ( 3
83 ( 2 [46; 91]
67 ( 3 [21; 53]
a
The kinetics were measured at 25 °C in TR assay buffer (40 mM Hepes, 1 mM EDTA, pH 7.5) at 100 µM NADPH. The TS2 concentration
was varied in the presence of two different inhibitor concentrations as well as in the absence of inhibitor. Each data point has been
measured at least two times and differed by e5%. The inhibitor constants for competitive inhibition were derived from Lineweaver
b
Burk plots. They do not represent true dissociation constants for the EI complex since they were dependent on the respective inhibitor
concentration. In any case, the lower value resulted at the higher [I]. The Ki,apparent values given in the table are the mean of the kinetic
constants (actually Ki,slope values) calculated at the different inhibitor concentrations (given in brackets). For a detailed discussion, see
d
text. c 9-Amino-1,2,3,4-tetrahydroacridine. 9-Amino-6-chloro-1,2,3,4-tetrahydroacridine. e The inhibitor constants for mixed-type inhibition
were derived from Lineweaver Burk and Cornish-Bowden plots.
molecules binding to the enzyme, a replot of 1/Ki,slope
versus [I] yields a straight line from which Ki can be
derived. For compound 1 this plot was not perfectly
linear. In addition, for most other 9-aminoacridines the
kinetics were measured in the presence of only two
inhibitor concentrations. Therefore the Ki,apparent values
given in Table 1 are used for comparing the inhibitory
potency of the different derivatives.
1 with only an amino group at C9. On the other hand,
in the series of compounds 2, 5 (mepacrine), and 7 which
have a chlorine and a methoxy group, elongation of the
alkylamino chain by one methylene group (7) but also
replacement of this substituent by a single amino group
(2) improved inhibition relative to mepacrine. In the
TR-mepacrine complex the terminal diethylamino group
of the side chain may undergo a water-mediated hydro-
gen bond with the carboxylate of Glu18. The introduc-
tion of an additional methylene group could thus make
possible a direct contact with the protein. The efficient
interaction between 2 and the enzyme may indicate that
in the absence of the bulky alkylamino chain the
acridine derivative binds in the active site in a com-
pletely different orientation.
Th e Meth oxy Gr ou p . The crystal structure of the
TR-mepacrine complex revealed that the methoxy
group of the inhibitor is in hydrogen bonding distance
to the side chain oxygen of Ser109. The kinetic studies
presented here also indicate that the methoxy group
contributes to the interaction with the enzyme. Three
pairs of derivatives differing only by the presence/
absence of the methoxy group have been measured.
Comparison of compounds 6 and 7 as well as 1 and 2
shows that the derivatives with the methoxy group have
a significantly lower Ki value than those lacking the
substituent. Only in the case of 4 and 5 did introduction
of the methoxy group not improve the interaction with
the enzyme.
Th e Role of th e Su bstitu en ts for In h ibitor Bin d -
in g. In the three-dimensional structure of the TR-
mepacrine complex14 the chlorine, the methoxy, and the
alkylamino group have been shown to be in contact with
specific side chains of the protein (Figure 1). The newly
synthesized derivatives contain one or two of the sub-
stituents of mepacrine or have slightly modified ligands
(Table 1). As outlined in the previous section more than
one molecule of the 9-aminoacridine derivatives may
bind to the same form of the enzyme. Since the crystal-
lographic analysis revealed the binding mode of only one
mepacrine molecule in the active site of TR, quantitative
structure-activity relationships cannot be established.
In addition, the contributions of the substituents to the
binding strength of the inhibitor was not additive, an
observation also made for other tricyclic inhibitors of
TR.21,32 Subtle structural changes can obviously cause
significantly different binding modes of the compounds.
In the following section we will therefore compare
groups of two or three derivatives and discuss their
kinetics in light of the TR-mepacrine structure.
Th e Alk yla m in o Ch a in . The derivatives 1, 4, and
6 are 3-chloroacridines with a different substituent at
C9 (R3 in Table 1). The latter two compounds with long
alkylamino chains were more efficient inhibitors than
Th e Ch lor in e Atom . In the TR-mepacrine struc-
ture, the chlorine at C6 of the acridine ring is fixed in
the vicinity of the ring nitrogen of Trp21. The kinetic
results are in agreement with a positive effect of the

Inhibition of T. Cruzi Trypanothione Reductase
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 26 5451
F igu r e 3. Inhibition of TR by compound 9. The kinetics were
performed as described in the legend of Figure 2. (a) Lin-
eweaver Burk plot. The TS2 concentration was varied in the
presence of ([) none, (9) 21 µM, and (2) 53 µM inhibitor. (b)
Cornish-Bowden plot. The inhibitor concentration was varied
in the presence of ([) 18 µM, (9) 61 µM, (2) 105 µM, and (×)
210 µM TS2. The plot directly gives the inhibitor constants:
The lines intersect at a point where [I] ) - Ki′; Ki is calculated
from S/v ) Km[1 - (Ki′/ Ki)]/V.
F igu r e 2. Inhibition of T. cruzi trypanothione reductase by
compound 1. The kinetics were measured at 25 °C as described
under Experimental Procedures at a constant concentration
of 100 µM NADPH. (a) Dixon plot. The inhibitor concentration
was varied in the presence of fixed concentrations of ([) 200
µM, (9) 100 µM, (2) 50 µM, and (×) 20 µM TS2. The values
are the mean of two measurements which differed by less than
5%. The complete data set was measured at least in dublicate.
(b) Replot of the slopes of the reciprocal plot (a) versus [I].
Sch em e 3
halogen upon inhibitor binding. Derivatives with the
chlorine at C6 of the acridine ring, namely mepacrine
(5) and compound 1, show improved binding in com-
parison to compound 3 and 9-aminoacridine, respec-
tively. It should be kept in mind that the apparent Ki
values (Table 1) depend on the inhibitor concentration,
resulting in lower Ki values at higher concentrations of
inhibitor. For instance, at 58-116 µM the Ki,apparent
value for compound 1 is only 24 µM and thus signifi-
cantly lower than that for 9-aminoacridine (for details
see Figure 2 and the text below).
Th e Ar om a tic Rin g System . In the mepacrine-TR
structure, the aromatic ring is fixed close to the hydro-
phobic wall formed by Trp21, Met113, Leu17, and
Phe114. Tacrine (9-amino-1,2,3,4-tetrahydroacridine)
and 6-chlorotacrine are inhibitors of acetylcholine es-
terase where they bind in the vicinity of aromatic
residues.33 As shown in Table 1, tacrine and 6-chloro-
tacrine bind to TR much weaker than do the respective
acridines, whereby the presence of a chlorine atom again
improves binding. This finding indicates that the planar
acridine ring system is favorable for TR binding.
9-Th ioa cr id in es In h ib it TR w it h Mixed -Typ e
Kin etics. Compounds 8 and 9 (Table 1) are mepacrine
analogues which have an S-alkylamino chain at position
C9 of the acridine ring. In contrast to the 9-ami-
noacridines, they show mixed-type kinetics when stud-
ied as inhibitors of T. cruzi TR. Ki and Ki′ values (RKi)
have been obtained from the Lineweaver Burk plot
(Figure 3a) as well as graphically from the Cornish-
Bowden plots (Figure 3b).34 In addition, replots of the
primary reciprocal plot data, namely slope versus [I] and
intercept on the vertical axis versus [I], were linear,
showing that the ESI complex is catalytically inactive.
The kinetics are in accordance with linear mixed-type
inhibition (Scheme 3). Originally, compound 8 had been
reported to inhibit TR competitively with a Ki value of
70 µM.20 Since the inhibitor constant was derived from
a Dixon plot, which does not allow to distinguish
between competitive and mixed-type kinetics, the true
inhibition type was overlooked.

5452 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 26
Bonse et al.
Discu ssion
Lineweaver Burk plot versus [I] show that the ESI
complex is not active. Such linear mixed-type (intersect-
ing linear noncompetitive) inhibition pattern has also
been observed for human GR complexed with xanthene
and safranin.36 Isoalloxazines are another class of GR
inhibitors with mixed-type kinetics as revealed by
plotting the published Dixon data40 into a Cornish-
Bowden diagram. The crystal structures of GR in
complex with xanthene, safranin, as well as two isoal-
loxazine derivatives showed, in any case, one inhibitor
molecule fixed in the cavity at the 2-fold axis of the
homodimeric protein.36,37 No binding was observed in
the active site. One may therefore speculate that the
respective cavity in TR is the binding site for the
9-thioacridines.
All acridine derivatives studied here as inhibitors of
T. cruzi trypanothione reductase are positively charged
under physiological conditions. The crucial role of a
positive charge for binding in the active site of TR has
been outlined by Faerman et al.8 As shown previously,
acridine itself and anionic derivatives do not bind to the
enzyme.14 The 9-aminoacridine derivatives inhibit TR
competitively, consistent with a localization at the
disulfide substrate binding site.13 This region is mainly
formed by Glu18, Trp21, Ser109, and Met113 and is the
binding site for mepacrine (Figure 1).14 As indicated by
the parabolic Dixon plots observed with three different
9-aminoacridine derivatives, more than one inhibitor
molecule can bind to the enzyme. The crystal structure
of the TR-mepacrine complex has visualized a single
inhibitor molecule in the active site. No other localiza-
tion was observed, but this may be due to the low
occupancy for mepacrine and the relatively low resolu-
tion of 2.8 Å.14 Therefore, we can only speculate about
probable additional binding sites. One site 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 ligands such as menadione35 as
well as the tricyclic compounds safranin,35,36 xanthene,36
and isoalloxazine derivatives.37 In the complex of GR
with dinitrophenylglutathione (DNPG), two binding
sites for the inhibitor have been revealed.38 The main
localization was in the active site coinciding with the
binding site for glutathione disulfide; an additional
minor binding was observed in the cavity at the 2-fold
axis. Interestingly, the kinetics of GR inhibition by
DNPG are very similar to those found here for the
9-aminoacridines and TR. In the former case, the
reciprocal plot also showed competitive inhibition and
the Dixon plot was parabolic.38
Con clu sion s
Inhibition of T. cruzi TR by 9-aminoacridines and
9-thioacridines, respectively, follows different types of
kinetics. 9-Aminoacridines are competitive inhibitors
with more than one binding site in the enzyme whereas
the 9-thioacridines give a mixed-type inhibition pattern.
These findings were surprising and showed that the
binding mode of even structurally very similar com-
pounds can be unpredictible. From the structural analy-
ses of several inhibitor complexes of TR and GR and
the kinetic results presented here for TR, one may
conclude that competitive inhibitors with multiple bind-
ing sites are fixed at the disulfide binding site with
additional localizations in the cavity at the 2-fold axis
and/or the outer region of the active site. On the other
hand, compounds with mixed-type inhibition pattern
preferably bind at the 2-fold axis.36 For drug develop-
ment approaches based on inhibitors of TR it would be
very helpful if the binding locus in the enzyme could be
deduced from the kinetic data. Clearly, some more
kinetic and crystallographic data of TR inhibitors have
to be available before a general correlation can be
established.
Another possibility is that two inhibitor molecules
bind in the active site. Modeling approaches on TR have
suggested a second hydrophobic pocket in the outer
region of the active site formed by Phe396, Pro398, and
Leu399.21,39
Exp er im en ta l P r oced u r es
Ma ter ia ls. T. cruzi trypanothione reductase was purified
from recombinant Escherichia coli SG5 cells as described.41
Trypanothione disulfide (TS2) was purchased from Bachem,
Heidelberg, Germany. Tacrine was obtained from Sigma,
Deisenhofen. 6-Chlorotacrine was kindly provided by Parke-
Davis, Ann Arbor, MI. 9-Aminoacridine was purchased from
Aldrich, Steinheim, Germany.
The finding that the 9-aminoacridine derivatives can
bind at more than one site in the enzyme makes
quantitative structure-activity relationships based on
the TR-mepacrine structure difficult. In addition, the
calculated inhibitor constants for competitive inhibition
depend on the inhibitor concentration in the assay and
are therefore not directly comparable. All 9-ami-
noacridines are inhibitors of TR with apparent Ki values
ranging from 5 to 43 µM. The most potent inhibitors
are compounds 2 and 7 which have a methoxy group
and a chlorine atom at C2 and C6, respectively, of the
acridine ring. A general statement about the contribu-
tion of each individual substituent upon binding to TR
cannot be made since the effects were not additive
(Table 1). An important result of the studies presented
here is that the often-used reciprocal plot at one
inhibitor concentration is not sufficient to elucidate the
real type of inhibition.
TR Assa y. TR activity was measured at 25 °C in TR assay
buffer (40 mM Hepes, 1 mM EDTA, pH 7.5) as described.42
The standard assay mixture (1 mL) contained 100 µM NADPH
and 7-10 mU TR. The reaction was started by adding 105
µM TS2, and the absorption decrease at 340 nm was followed.
Vmax is calculated using a Km value of 18 µM for TS2.
Deter m in a tion of In h ibitor Con sta n ts. The structural
formula of the compounds tested are given in Table 1. Stock
solutions (3-5 mM) of 9-aminoacridine and tacrine were
prepared in TR assay buffer. Stock solutions of the other
acridine derivatives and 6-chlorotacrine were made in DMSO.
Control assays containing the respective amount of DMSO
were carried out where appropriate. Inhibitor constants were
derived from Lineweaver Burk, Dixon, and Cornish-Bowden
plots as described.
The 9-thioacridine derivatives 8 and 9 inhibit TR with
mixed-type kinetics, the Ki values being comparable to
those for the 9-aminoacridines. Ki′ values of about 70
µM show that binding of the inhibitor to the enzyme
substrate complex is weaker than that to the free
enzyme. The linearity of the Dixon plots as well as of
the replots of slopes and intercepts, respectively, of the
Ch em istr y. 1H and 13C NMR spectra were recorded on a
Bruker ARX 200 spectrometer with TMS as internal reference;
chemical shifts are given on the δ (ppm) scale with J values
in hertz. The compounds were purified by liquid chromatog-
raphy on silica gel 60 (70-230 mesh).
Syn th esis of Am in oa cr id in es 1 a n d 2. Ammonium
carbonate (1.5 g, 17 mmol) was added to the corresponding

Inhibition of T. Cruzi Trypanothione Reductase
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 26 5453
9-chloroacridine25 (10 mmol) and mixed with phenol (8 g). The
mixture was stirred and heated to 120 °C for 45 min and then
poured into an excess of 5 N NaOH to give pH 12. The solid
was filtered, washed with 1 N NaOH and water, and dried
(yield: 70%).
(CDCl3): 156.2 (s); 149.3 (s); 148.2 (s); 147 (s); 134.7 (s); 131.7
(d); 128.5 (d); 125.1 (d); 124.7 (d); 123.8 (d); 119.4 (s); 117.4
(s); 99.3 (d); 56.1 (d); 55.5 (q); 52.7 (t); 46.9 (t); 39.1 (t); 27.2
(t); 24.5 (t); 22.2 (q); 11.6 (q). Anal. (C24H32N3OCl) C, H, N.
P r ep a r a tion of Am in oth ioa cr id in e 8 a n d 9. 6-Ch lor o-
9-Am in o-3-ch lor oa cr id in e (1).43 Mp: 267 °C. 1H NMR
(DMSO-d6): 8.44 (d, J ) 9.3, 1 H); 8.39 (d, J ) 8.8, 1 H); 7.97
(b s, 2 H); 7.82 (d, J ) 2.1, 1 H); 7.81 (d, J ) 7.8, 1 H); 7.67
(dd, J ) 7.8, J ) 6.6, 1 H); 7.32 (m, 2 H). 13C NMR (DMSO-
d6): 150.6 (s); 149.4 (s); 149.2 (s); 134.8 (s); 130.6 (d); 128.7
(d); 126.8 (d); 125.8 (d); 123.4 (d);122.1 (d); 121.9 (d); 113.1
(s); 111.4 (s). Anal. (C13H9N2Cl) C, H, N.
2-m eth oxy-9-(4′-d ieth yla m in oeth yl)-th ioa cr id in e (8).29
A
mixture of 6-chloro-2-methoxy-9-thioacridinone (2.7 g, 10
mmol), 2-diethylaminoethyl chloride hydrochloride (1.9 g, 11
mmol), toluene (150 mL), and 5 N KOH solution (50 mL) was
refluxed 4 h. After cooling, the organic phase was separated,
the aqueous phase was extracted with chloroform, and the
organic phases were dried over drierite. The solvent was
removed under reduced pressure, and the product was purified
by chromatography on SiO2 and eluted with ether and ether-
methanol (70/30) (yield: 75%) as an oil. By addition of HCl
saturated ether, the hydrochloride was obtained. Mp: 188-
190 °C. 1H NMR (D2O) (hydrochloride): 8.40 (d, J ) 9.4, 1 H);
7.82 (d, J ) 2.0) and 7.80 (d, J ) 8.7) (2 H); 7.66-7.57 (m, 2
H); 7.46 (d, J ) 2.5, 1 H); 3.92 (s, 3 H); 3.27-3.19 (m, 2 H);
3.24 (m) and 2.91 (q, J ) 7.3) (2 H + 4 H); 0.92 (t, J ) 7.3, 6
H). 13C NMR (D2O): 159.9 (s); 150.2 (s); 142.2 (s); 138.3 (s);
137.4 (s); 132.3 (d); 131 (d); 130.5 (s); 129 (d); 127.4 (s); 123.8
(d); 120.7 (d); 103.6 (d); 57 (q); 51.5 (t); 48.2 (t); 31.6 (t); 8.7
(q). Anal. (C20H23N2OSCl) (HCl)2 (H2O) C, H, N.
9-Am in o-6-ch lor o-2-m eth oxya cr id in e (2).44 Mp: 274 °C.
1H NMR (DMSO-d6): 8.40 (d, J ) 9.2, 1 H); 7.81 (d, J ) 2.2,
1 H); 7.76 (d, J ) 9.3, 1 H); 7.68 (sh, 2 H); 7.67 (d, J ) 2.5, 1
H); 7.38 (dd, J ) 9.3, J ) 2.5, 1 H); 7.30 (dd, J ) 9.2, J ) 2.2,
1 H); 3.91 (s, 3 H). 13C NMR (DMSO-d6): 154.8 (s); 148.9 (s);
147.8 (s); 146 (s); 133.7 (s); 130.5 (d); 126.9 (d); 125.4 (d); 124.3
(d); 122.2 (d); 113.3 (s); 111.3 (s); 100.4 (d); 55.8(q). Anal.
(C14H11N2OCl) C, H, N.
P r ep a r a tion of Su bstitu ted Am in oa cr id in es 3, 4, 5, 6,
7. The corresponding 9-chloroacridine (10 mmol) was reacted
with 5-diethylamino-2-aminopentane (1.58 g, 10 mmol) (3, 4,
5) or 6-diethylamino-2-aminohexane (1.72 g, 10 mmol,) (6, 7)
in phenol (15 g), heated at 110 °C for 2 h. The reaction mixture
was poured into 5 N NaOH solution, and the product was
extracted with chloroform. The solvent was removed under
reduced pressure and the amine purified by chromatography
on SiO2 in ether-methanol (90/10 to 80/20) (yield: 45-55%).
2-Met h oxy-9-(4′-d iet h yla m in o-1′-m et h ylb u t yla m in o)-
a cr id in e (3).45 1H NMR (CDCl3): 8.11 (d, J ) 9.3, 1 H); 8.05
(b d, J ) 9.4, 2 H); 7.64 (td, J ) 6.7, J ) 1.3, 1 H); 7.42 (m, 2
H); 7.26 (b s, 1 H); 4.41 (d, J ) 10.5, 1 H); 4.03 (m, 1 H); 3.96
(s, 3 H); 2.6-2.3 (m, 6 H); 1.66 (m, 4 H); 1.27 (d, J ) 6.3, 3 H);
0.95 (t, J ) 7.1, 6 H). 13C NMR (CDCl3): 155.9 (s); 148.9 (s);
148 (s); 146.5 (s); 131.6 (d); 130 (d); 128.8 (d); 124.2 (d); 124.1
(d); 122 (d); 119.3 (s); 119.3 (s); 99.4 (d); 55.7 (d); 55.4 (q); 52.8
(t); 46.8 (t); 37 (t); 24 (t); 22.2 (q); 11.5 (q). Anal. (C23H31N3O)
C, H, N.
3-Ch lor o-9-(4′-dieth ylam in o-1′-m eth ylbu tylam in o)-acr i-
d in e (4).26 1H NMR (CDCl3): 8.05-7.96 (m, 4 H); 7.65 (dd, J
) 7.1, J ) 7.6, 1 H); 7.35 (dd, J ) 7.5, J ) 7.7, 1 H); 7.23 (m,
1 H); 4.90 (m, 1 H); 4.11 (m, 1 H); 2.40 (q, J ) 7.1), 2.31 (t, J
) 6.2) (6 H); 1.7-1.5 (m, 4 H); 1.26 (d, J ) 6.3, 3 H); 0.91 (t,
J ) 7.1, 6 H). 13C NMR (CDCl3): 151.2 (s); 149.9 (s); 149.7 (s);
135.7 (s); 130.3 (d); 128.9 (d); 128.1 (d); 124.5 (d); 124.2 (d);
123.7 (d); 122.4 (d); 117.9 (s); 116 (s); 56.1 (d); 52.5 (t); 46.6
(t); 36.8 (t); 23.8 (t); 22.1 (q); 11.4 (q). Anal. (C22H28N3Cl)
(H2O)1.5 C, H, N.
6-Ch lor o-2-m eth oxy-9-(4′-d ieth yla m in o-1′-m eth ylbu tyl-
a m in o)-a cr id in e (5).46 Mp: 248-250 °C. 1H NMR (D2O)
(hydrochloride): 7.63 (d, J ) 9.3, 1 H); 7.24-7.19 (m, 3 H);
7.11 (dd, J ) 9.3, J ) 1.6, 1 H); 6.92 (b s, 1 H); 4.03 (q, J )
6.3, 1 H); 3.78 (s, 3 H); 2.75 (q, J ) 7.3, 4 H + 2 H); 1.62 (m,
2 H); 1.40 (d, J ) 6.2, 3 H); 1.28 (m, 2 H); 0.88 (t, J ) 7.3, 6
H). 13C NMR (D2O): 156.2 (s); 154.9 (s); 141.8 (s); 139.7 (s);
127 (d); 126.7 (d); 125.1 (d); 123.6 (d); 120.3 (d); 114.9 (s); 111.6
(s); 102.2 (d); 56.7 (d or q); 56 (q or d); 51.5 (t); 47.9 (t); 34.9
(t); 21.4 (q); 21 (t); 8.7 (q). Anal. (C23H30N3OCl) (HCl)2(H2O)1.5
C, H, N.
6-Ch lor o-2-m eth oxy-9-(4′-ch lor obu tyl)-th ioacr idin e. This
intermediate compound was synthesized by mixing 6-chloro-
2-methoxy-9-thioacridinone (2.7 g, 10 mmol), excess 1-bromo-
4-chlorobutane (2.6 g, 15 mmol), TEBAC (triethylbenzylam-
monium chloride, 0.5 g), toluene (100 mL), and NaOH (8 g) in
H2O (20 mL) and refluxing for 4 h.47 After cooling, the organic
phase was washed to pH 7 and dried over MgSO4. The product
was purified by chromatography on SiO2 using ether-
petroleum ether (50/50) as eluent. The yield of pure product
1
was 60%. H NMR (CDCl3): 8.67 (d, J ) 9.3, 1 H); 8.19 (d, J
) 2.0, 1 H); 8.09 (d, J ) 9.4, 1 H); 7.51 (m, 2 H); 4.04 (s, 3 H);
3.43 (t, J ) 6.4, 2 H); 2.94 (t, J ) 7.0, 2 H); 1.90 (m, 2 H); 1.64
(m, 2 H).
6-Ch lor o-2-m eth oxy-9-(4′-d ieth yla m in obu tyl)-th ioa cr i-
d in e (9). 6-Chloro-2-methoxy-9-(4′-chlorobutyl)-thioacridine
(3.6 g, 5 mmol), excess diethylamine (2 g, 27 mmol), and
Na2CO3 (1 g, 10 mmol) were refluxed in acetone (40 mL) for 4
days. The solvent was removed under vacuum, and NaOH was
added to give pH 12. The solution was extracted with chloro-
form, and the organic phase was dried over drierite. Chroma-
tography on SiO2 and elution with ether and ether-methanol
(70/30) yielded pure compound 9 as an oil. The hydrochloride
was obtained as described for compound 8. Mp: 185-187 °C.
1H NMR (D2O) (hydrochloride): 8.38 (d, J ) 9.3, 1 H); 7.81
(m, 2 H); 7.65 (d, J ) 9.3, 1 H); 7.58 (d, J ) 9.7, 1 H); 7.44 (b
s, 1 H); 3.91 (s, 3 H); 3.03 (t, J ) 6.7) and 2.94 (q, J ) 7.3) (6
H); 2.79 (m, 2 H); 1.49 (m, 2 H); 1.31 (m, 2 H); 1.03 (t, J ) 7.3,
6 H). 13C NMR (D2O): 159.6 (s); 155.4 (s); 142.7 (s); 137.4 (s);
135.9 (s); 132.5 (d); 130.6 (d); 130.2 (s); 129.5 (d); 127.2 (s);
123 (d); 120 (d); 104.2 (d); 57.1 (q); 51.5 (t); 48 (t); 38.3 (t);
27.5 (t); 23 (t); 8.9 (q). Anal. (C22H27N2OSCl) (HCl)2 (H2O) C,
H, N.
Ack n ow led gm en t. Heike Lu¨demann and Edith
Ro¨ckel are acknowledged for excellent technical as-
sistance. We thank Dr. Heiner Schirmer for many
helpful discussions. Our work is supported by the
Deutsche Forschungsgemeinschaft (DFG Kr 1242/1-3
and SFB 544) and the Bundesministerium fu¨r Bildung
und Forschung (BMBF Schwerpunkt Tropical Medicine
Heidelberg, TMH).
3-Ch lor o-9-(5′-d iet h yla m in o-1′-m et h ylp en t yla m in o)-
1
a cr id in e (6). H NMR (CDCl3): 8.09-7.99 (m, 4 H); 7.69 (m,
1 H); 7.40 (m, 1 H); 7.30 (dd, J ) 9.2, J ) 2.0, 1 H); 4.72 (b s,
1 H); 4.11 (m, 1 H); 2.46 (q, J ) 7.2, 4 H); 2.33 (m, 2 H); 1.67
(m, 2 H); 1.41 (m, 4 H); 1.32 (d, J ) 6.3, 3 H); 0.97 (t, J ) 7.2,
6 H). 13C NMR (CDCl3): 151.3 (s); 149.7 (s); 149.5 (s); 135.8
(s); 130.4 (d); 129.5 (d); 127.9 (d); 124.4 (d); 124.3 (d); 123.7
(d); 122.3 (d); 117.8 (s); 115.9 (s); 56.2 (d); 52.4 (t); 46.6 (t);
38.8 (t); 26.6 (t) 24.2 (t); 22.1 (q); 11.2 (q). Anal. (C23H30N3Cl)
C, H, N.
Refer en ces
6-Ch lor o-2-m eth oxy-9-(5′-d ieth yla m in o-1′-m eth ylp en -
tyla m in o)-a cr id in e (7).25 1H NMR (CDCl3): 8.0 (d, J ) 2.0,
1 H); 7.92 (d, J ) 9.4, 1 H); 7.88 (d, J ) 9.5, 1 H); 7.34 (dd, J
) 9.4, J ) 2.6, 1 H); 7.23 (dd, J ) 9.5, J ) 1.7, 1 H); 7.11 (d,
J ) 2.6, 1 H); 4.22 (d, J ) 10.5, 1 H); 3.9 (m, 1 H); 3.87 (s, 3
H); 2.35 (q, J ) 7.1, 4 H); 2.25 (m, 2 H); 1.58 (m, 2 H); 1.33 (m,
4 H); 1.17 (d, J ) 6.3, 3 H); 0.88 (t, J ) 7.1, 6 H). 13C NMR
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J M990386S