Antimalarial, antitrypanosomal, and antileishmanial activities and cytotoxicity of bis(9-amino-6-chloro-2- methoxyacridines): Influence of the linker

Article abstract of DOI:10.1021/jm990946n

Forty bis(9-amino-6-chloro-2-methoxyacridines), in which acridine moieties are joined by alkanediamines, polyamines, or polyamines substituted by a side chain, were synthesized and tested for their in vitro activity upon the erythrocytic stage of Plasmodi

Full text of DOI:10.1021/jm990946n

2
646
J . Med. Chem. 2000, 43, 2646-2654
Articles
An tim a la r ia l, An titr yp a n osom a l, a n d An tileish m a n ia l Activities a n d
Cytotoxicity of Bis(9-a m in o-6-ch lor o-2-m eth oxya cr id in es): In flu en ce of th e
Lin k er #
†
‡
⊥ ,†
§
‡
†
Sophie Girault, Philippe Grellier, Amaya Berecibar, Louis Maes, Elisabeth Mouray, Pascal Lemi e` re,
†
†
,†
Marie-Ange Debreu, Elisabeth Davioud-Charvet, and Christian Sergheraert*
UMR 8525 CNRS, Universit e´ de Lille II, Institut de Biologie et Institut Pasteur de Lille, 1 rue du Professeur Calmette, BP 447,
5
6
9021 Lille, France, Mus e´ um National d’Histoire Naturelle, Biologie et Evolution des Parasites, CNRS - EP1790,
1 rue Buffon, 75005 Paris, France, and Tibotec, L11 Gen. de Wittelaan, B-32800 Mechelen, Belgium
Received November 3, 1999
Forty bis(9-amino-6-chloro-2-methoxyacridines), in which acridine moieties are joined by
alkanediamines, polyamines, or polyamines substituted by a side chain, were synthesized and
tested for their in vitro activity upon the erythrocytic stage of Plasmodium falciparum,
trypomastigote stage of Trypanosoma brucei, and amastigote stage of Trypanosoma cruzi and
Leishmania infantum as well as for their cytotoxic effects upon MRC-5 cells. Results clearly
showed the importance of the nature of the linker and of its side chain for antiparasitic activity,
cytotoxicity, and cellular localization. Among several compounds devoid of cytotoxic effects at
2
5 µM upon MRC-5 cells, one displayed IC50 values ranging from 8 to 18 nM against different
P. falciparum strains while three others totally inhibited T. brucei at 1.56 µM.
In tr od u ction
Ch a r t 1. Structure of Antimalarial Molecules
Malaria, trypanosomiasis, and leishmaniasis are ma-
jor diseases in developing countries which continue to
infect several hundreds of millions of people and which
are responsible for a mortality rate in excess of 1 million
per year. Present chemotherapies are proving inad-
equate, are toxic, or are becoming ineffective due to an
increase in resistance. The trivalent arsenical drug
melarsoprol is still widely used against the second stage
of African trypanosomiasis in which Trypanosoma bru-
cei rhodesiense or gambiense has invaded the central
nervous system. The toxicity of this treatment is re-
sponsible for the death of approximately 5% of its
recipients. Benznidazole, the only commercial drug still
available for treatment during the chronic stage of
Chagas’ disease, is no longer efficient, while its safety
continues to be debated. Against Leishmania parasite,
amphotericin B and pentamidine are toxic in therapeu-
tic doses, while resistance to pentavalent antimonials
is increasing. The effectiveness of chloroquine (CQ,
Chart 1), the cheap, antimalarial mainstay for more
than 50 years, is also being undermined by the evolution
of resistant parasites.1 CQ is believed to exert its
activity by inhibiting hemozoin formation in the diges-
tive vacuole of the malaria parasite, though this has
recently been questioned by Ginsburg and co-workers
who have suggested that inhibition of the degradation
3,4
of ferriprotoporphyrin IX by glutathione-dependent re-
dox processes could be a second mode of action of CQ.5
,2
Biochemical studies indicate that isolates of the CQ-
resistant parasite accumulate less drug in the food
vacuole than their more sensitive counterparts. How-
ever, opinion remains divided upon the mechanistic
explanation for the reduction: (1) a rapid CQ efflux
#
Abbreviations: CQ, chloroquine; DAPI, 4,6-diamidino-2-phenyl-
indole; MF, mefloquine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphen-
yltetrazolium bromide (thiazolyl blue); PyBrop, bromotris(pyrrolidino-
phosphonium) hexafluorophosphate.
*
Corresponding author. Tel: (33) 3 20 87 12 11. Fax: (33) 3 20 87
6
mechanism by CQ-resistant parasites, (2) an elevated
1
2 33. E-mail: christian.sergheraert@pasteur-lille.fr.
pH in the food vacuole of the CQ-resistant parasites,7
†
Institut de Biologie et Institut Pasteur de Lille.
CNRS - EP1790.
Tibotec.
‡
(
3) resistance being linked to a carrier-mediated CQ
§
8
⊥
uptake, (4) resistance being linked to a reduced CQ
affinity to ferriprotoporphyrin. CQ resistance may
Present address: Institut de Recherche J ouveinal, Parke-Davis,
9
3
-9 rue de la Loge, 94265 Fresnes Cedex, France.
1
0.1021/jm990946n CCC: $19.00 © 2000 American Chemical Society
Published on Web 06/17/2000

Bis(9-amino-6-chloro-2-methoxyacridines)
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 14 2647
Ch a r t 2. Bisacridines of the A, B, and C Series
involve several mechanisms; however its reversal by
molecules such as verapamil, desipramine, and chlor-
promazine suggests that an enhanced CQ efflux by a
6
,10
multidrug-resistant mechanism may be implicated.
One possibility to overcome this mechanism is to design
quinoline-based drugs which are not recognized by the
proteins involved in drug efflux. In this regard, bulky
bisquinolines were synthesized and suggested to be
extruded with difficulty by a proteinaceous trans-
porter.11 They were also discovered to inhibit the growth
of CQ-sensitive and CQ-resistant parasites with similar
efficiency though it was not proved that steric hindrance
was the deciding factor for this.11-14 Further develop-
1
3
ment of the most promising molecule Ro 47-7737
Chart 1) as that of other bisquinolines, described
(
earlier, piperaquine, hydroxypiperaquine, and dichloro-
quinazine,15 was suspended for reasons of toxicity.
Acridine derivatives have been considered as potential
antiprotozoal agents, e.g: 9-anilinoacridine topoisomerase
II inhibitors show high levels of antileishmanial and
antitrypanosomal activity.16 Furthermore, quinacrine,
the 9-amino-6-chloro-2-methoxyacridine analogue of CQ
or polyamines substituted by a side chain (Chart 2). In
the latter case, the length of the linker proved unfavor-
able to bisintercalation, while the nature of the side
chain could contribute to either a diminishing or cancel-
ing of the interaction with the phosphate groups of
human DNA and consequently to a decrease of cytotox-
icity. Antiparasitic efficacy of these compounds against
Trypanosoma cruzi, Trypanosoma brucei, and Leishma-
nia infantum, as well as the evaluation of their cyto-
toxicity, is also reported.
(Chart 1), which was used clinically before CQ, is still
an approved treatment of giardiasis. Sharing the same
features as a weak diprotic base, it accumulates in the
acidic food vacuole (pH ) 5) of Plasmodium and
4
prevents hematin polymerization. Until now, bisacri-
dines have been poorly studied for their antiparasitic
activity, yet they, along with bisquinolines, may repre-
sent an alternative method to avoiding the efflux
mechanism. In addition bisacridines offer the advantage
of being viewed precisely by cellular fluorescence imag-
ing. However, the presence of an acridine moiety in a
molecule can lead to stronger interactions with DNA
and generate cytotoxicity, especially in the case of
bisintercalation. Bisintercalation requires a minimal
distance between the acridine rings. Bis(9-amino-6-
chloro-2-methoxyacridines) corresponding to a spermine
or spermidine linker were found to bisintercalate, while
the removal of a single carbon (symmetrical linker
Ch em istr y
In preference to the generally adopted method, em-
ploying phenol, as described by Canellakis et al,24
bisacridines 1-9 (A and B series, Chart 2) were syn-
thesized according to the method described for 4-ami-
2
5
noquinolines preparation, by reacting polyamines with
3 equiv of 6,9-dichloro-2-methoxyacridine in DMF at
reflux, in the presence of potassium carbonate as an
inorganic base (Scheme 1). Chromatography on silica
gel columns was the selected method for purification.
In the C series (Chart 2), the side chain was fixed by
coupling various carboxylic groups with the central,
secondary amino group of compounds 6-8, using Py-
-
NH-(CH2)3-NH-(CH2)3-NH-) suppressed bisinter-
1
7
calation. It was also reported that acridine substitu-
1
8,19
ents play a role in intercalation and DNA recognition
2
6,27
and that an increase of rigidity can favor binding affinity
BroP reagent.
Thus, bisacridines 10-25 were syn-
in relation to the subsequent decrease of the loss in
thesized, as shown in Scheme 2, by coupling bisacridines
6-8 with appropriate N-Boc- and/or N-Fmoc-amino
acids. N-Boc-amino protecting groups of compounds 10
and 21 were removed by treatment with a 1:1 mixture
of TFA/CH Cl to give deprotected analogues 26 and 29,
entropy.2
0,21
Besides, for unsubstituted bisacridines, an
evolution from monofunctional to bifunctional interac-
tion was found by lengthening the methylene chain from
four carbons to six.1
9,22,23
2
2
We have therefore undertaken the synthesis and an
in vitro antiparasitic activity study upon Plasmodium
falciparum of three series of bisacridines in which
aromatic rings are joined by alkanediamines, polyamines,
respectively (Scheme 2). N-Fmoc-amino or Fmoc-ester
protecting groups of compounds 19, 20, and 25 were
removed by treatment with a 20:80 piperidine/DMF
mixture to give deprotected analogues 27, 28, and 30,
Sch em e 1. Synthesis of Bisacridines 1-9a
a
Reagents: K2CO3, DMF.

2
648 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 14
Girault et al.
Sch em e 2. Synthesis of Bisacridines 10-31a
a
Reagents: (a) PyBrop, DIEA, DMF; (b) TFA/CH2Cl2 (1:1); (c) piperidine/DMF (20:80); (d) K2CO3, H2O, MeOH.
Sch em e 3. Synthesis of Bisacridines 32-34a
a
Reagents: (a) succinic anhydride, pyridine; (b) Boc2O, NH4HCO3, pyridine, DMF; (c) morpholine, PyBrop, DIEA, DMF.
respectively (Scheme 2). Acetyl protection of compound
3 was removed by treatment with an aqueous solution
was previously reported to lead to a nonconvertible
pyridinium salt.
2
8
2
of K2CO3 to give deprotected compound 31 (Scheme 2).
Thick-layer chromatography was used for purification.
Compound 32 was synthesized, as shown in Scheme
Biologica l Resu lts
In Vitr o An tim a la r ia l Activity u p on P . fa lci-
pa r u m . Initially all of the compounds were tested for
their antimalarial activity upon the CQ-resistant strain
FcB1R (IC50 CQ ) 138.6 nM, IC50 MF ) 6.9 nM). In
the A series (Table 1), increasing the length of the
alkanediamine linker proved unfavorable toward activ-
ity (IC50 values increased from 16 to 176 nM from 6 to
12 carbons). Values in the B series (Table 1), were
consistent regardless of the distance between the ni-
trogen atoms of the linker (IC50 around 60 nM), except
for the piperazine derivative 9 which proved more
efficient (IC50 ) 17 nM). In comparison with all other
compounds of the A and B series, this derivative also
showed a unique and contrasting behavior at the
3
, by reaction of bisacridine 6 with succinic anhydride,
while its amide analogue 33 was prepared by treating
compound 32 with ammonium hydrogenocarbonate. The
morpholinoamide derivative 34 was synthesized by
coupling the carboxylic acid 32 with morpholine using
the coupling method described in Scheme 2.
In the case of bisacridines 35-40, fixation of the
amino side chain was accomplished in two steps: the
reaction of secondary amino compound 6 with 3-chlo-
ropropionyl chloride, using 1-ethylpiperidine as a base,
followed by substitution of the remaining chloro group
by the appropriate amine (Scheme 4).27 As a tertiary
amine 1-ethylpiperidine was preferred to pyridine which

Bis(9-amino-6-chloro-2-methoxyacridines)
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 14 2649
Sch em e 4. Synthesis of Bisacridines 35-40a
a
Reagents: 3-chloropropionyl chloride, 1-ethylpiperidine, THF, then R1R2NH, THF.
Ta ble 1. In Vitro Sensitivity of P. falciparum FcB1R Strain to
Bisacridines 1-9 (A and B Series)
infected erythrocytes incubated with 10 µM quinacrine
displayed a weak fluorescence associated with the
parasite food vacuole and the erythrocytic and parasitic
membranes (data not shown), a high fluorescence,
associated exclusively with the parasite, was observed
with 1 µM compound 9 (Figure 1A). No labeling of the
food vacuole and of the normal or infected erythrocyte
cytosols/membranes was recorded. Fluorescence was
mainly concentrated in structures which colocalized
with propidium iodide or DAPI labeling, suggesting a
concentration of compound 9 in the parasite nuclei (data
not shown). In murine muscle L-6 cells, which were used
as a control, no nuclear labeling was observed; the
fluorescence of compound 9 was only associated with
molecule
series
central amine
n
n′
IC50 (nM)a
1
2
3
4
5
6
7
8
9
A
A
A
A
A
B
B
B
B
4
6
8
10
12
3
2
3
3
18 ( 7b
16 ( 10b
b
19 ( 10
57 ( 30b
b
176 ( 96
NH
NH
NH
piperazine
3
2
4
3
55 ( 9c
b
63 ( 10
b
61 ( 3
17 ( 10d
a
b-d
IC50 CQ ) 138.6 ( 8.4 nM; IC50 MF ) 6.9 ( 0.3 nM.
n,
b
c
d
number of experiments: n ) 3; n ) 5; n ) 4.
cellular localization level. In confocal microscopy, while
F igu r e 1. Labeling of P. falciparum-infected erythrocytes (A, C) and mouse L-6 cells (B, D) using compounds 9 (A, B) and 1 (C,
D). Infected cultures of P. falciparum were incubated with 1 µM bisacridine derivative and then with 10 µg/mL nonexchangeable
lipid DIL C18(3) (see Experimental Section). DIL C18(3) permitted the visualization of the membranes of the infected and noninfected
erythrocytes (arrow). Bisacridine derivatives accumulated specifically in the intracellular parasites (arrowhead). Food vacuole
(FV) was not labeled. For compound 9, a high fluorescence was associated with a structure which colocalized with the parasite
nucleus (N). L-6 cells were only labeled with bisacridine derivatives. Bar scale is in µm. A and B are images of one section, C and
D are 3D reconstructions of a sequential image collection.

2
650 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 14
Girault et al.
Ta ble 2. In Vitro Sensitivity of P. falciparum FcB1R Strain to
Bisacridines 10-40 (C Series)
while their analogues 11 (n ) n′ ) 2) and 12 (n ) 3,
n′ ) 4) were less active (IC50 of 109 and 330 nM,
respectively). N-Boc-amino acids with longer hydropho-
bic side chains, leucine (compound 15) and valine
(compound 16), were the most active, while the polarity
of the side chains proved consistently unfavorable since
the protected derivatives 10 and 19-25 were more
active than their corresponding deprotected counter-
parts 26 and 27-31. Also, the succinimide derivative
33 revealed a better activity than its carboxylic analogue
molecule n n′
Ra
IC50 (nM)b
31 ( 2c
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
2
4
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
CO-CH(NHBoc)-CH3
CO-CH(NHBoc)-CH3
CO-CH(NHBoc)-CH3
CO-CH(NHBoc)-CH3 (D enantiomer)
CO-CH2-NHBoc
CO-CH(NHBoc)-CH2-CH(CH3)2
CO-CH(NHBoc)-CH(CH3)2
CO-CH(NHFmoc)-CH3
CO-CH2-NH-CO-CH(NHBoc)-CH3
CO-CH(NHBoc)-(CH2)4-NHFmoc
CO-CH(NHBoc)-CH2-NHFmoc
CO-CH(NHBoc)-CH2-NHBoc
CO-CH(NHBoc)-CH2OBn
CO-CH(NHBoc)-CH2OAc
CO-CH(NHBoc)-CH2-COOC6H11
CO-CH(NHBoc)-CH2-COOFmoc
CO-CH(NH2)-CH3
CO-CH(NHBoc)-(CH2)4-NH2
CO-CH(NHBoc)-CH2-NH2
CO-CH(NH2)-CH2-NH2
CO-CH(NHBoc)-CH2-COOH
CO-CH(NHBoc)-CH2OH
CO-(CH2)2-COOH
109 ( 3d
d
330 ( 59
33 ( 2d
92 ( 19
e
d
27 ( 4
25 ( 7d
232 ( 85
440f
d
d
3
2. Addition of a second amino acid residue (compound
273 ( 75
71 ( 3d
18) was unfavorable. Amino derivatives 35-40 dis-
played IC50 values between 26 and 91 nM, with the
exception of the piperazine derivative 39 (IC50 > 500
nM) which via its terminal nitrogen atom could play the
role of hydrogen-bond donor. In this series, the absence
of a protonable amino group led to the less active
morpholine compound 34. Five compounds among the
most active upon the CQ-resistant strain FcB1R (com-
pounds 9, 10, 13, 15, and 16) were subsequently
evaluated for their efficiency in inhibiting the growth
of seven strains showing different degrees of resistance
to CQ or MF (Table 3). An inverse correlation was
observed for compounds 10, 13, 15, and 16, as recently
reported for bisquinolines by Vennerstrom and co-
d
55 ( 3
138f
63 ( 15d
31 ( 4c
d
230 ( 16
>500d
d
>500
307f
>500d
>500d
d
247 ( 92
270f
CO-(CH2)2-CONH2
CO-(CH2)2-CO-morpholine
CO-(CH2)2-morpholine
CO-(CH2)2-piperidine
CO-(CH2)2-pyrrolidine
CO-(CH2)2-(4-methylpiperazine)
CO-(CH2)2-piperazine
CO-(CH2)2-NH(CH3)2
40 ( 10e
d
436 ( 56
e
26 ( 2
67 ( 11d
2
9
d
workers, the more the parasites appeared resistant to
CQ, the more they seemed sensitive to the compounds
under investigation. On the contrary, piperazine com-
pound 9 displayed quite similar efficiency (IC50 values
between 8 and 18 nM), whatever the CQ resistance of
the strain. No such correlation was observed with the
resistance to MF.
91 ( 19
d
62 ( 8
f
>500
39 ( 5d
a
Ac, acetyl; Bn, benzyl; Boc, tert-butoxycarbonyl; Fmoc, 9-fluo-
b
renylmethyloxycarbonyl. IC50 CQ ) 138.6 ( 8.4 nM; IC50 MF )
c-f
c
d
e
6
4
.9 ( 0.3 nM.
n, number of experiments: n ) 5; n ) 3; n )
f
; n ) 2.
In Vit r o An t it r yp a n osom a l Act ivit y u p on T.
cr u zi, T. br u cei, a n d L. in fa n tu m . Compounds of the
series A and B (Table 4), with the exception of the
piperazine derivative 9 and to a lesser extent com-
pounds 4 and 5, were found to be very cytotoxic upon
human diploid embryonic lung cells (MRC-5 cells) and
upon mouse primary peritoneal macrophages used in
the T. cruzi and L. infantum tests as well as upon T.
brucei which is known to be highly sensitive to toxic
molecules. In confocal imaging of T. cruzi epimastigotes,
compound 9 was found to be mainly associated with a
structure which colocalized with kinetoplast DNA (Fig-
ure 2), while compound 1 displayed a more diffuse
fluorescence associated with both the kinetoplast and
the cytosol (data not shown).
vesicles (Figure 1B), which co-localized with lysosome-
specific probe labeling (data not shown). The other
members of the A and B series displayed a specific
parasite labeling similar to that for compound 9 (Figure
1
C). However, a weaker fluorescence was associated
with the parasite nucleus while fluorescence was mainly
associated with the parasite cytoplasm. When compared
with compound 9, the A and B series revealed a marked
difference in L-6 cells localization, showing a diffuse
labeling within the cell cytosol and no concentration in
lysosome-like vesicles (Figure 1D).
In the C series (Table 2), activity proved to be highly
dependent upon the nature of the Boc- and/or Fmoc-
protected amino acid residue fixed to the central nitro-
gen atom of the linker and upon the distance between
the terminal nitrogen atoms. A high level of activity
In the C series (Table 5), only five compounds (25,
29-32) were devoid of cytotoxicity toward MRC-5 cells
and mouse macrophages. Four of these molecules pos-
sess a polar group (amine, alcohol, or carboxylic acid)
(IC50 values about 30 nM) was observed for the alanine
derivatives 10 and 13 (n ) n′ ) 3), for both enantiomers,
Ta ble 3. Efficiency of Five of the Most Active Compounds against FcB1R, To Inhibit Growth of Parasites Expressing Different
Degrees of Resistance to CQ
IC50 (nM)a
P. falciparum
strain
CQ
MF
9
10
13
15
16
3
D7
20 ( 4
22 ( 2
40 ( 10
62 ( 6
17 ( 3
36 ( 15
8 ( 3
30 ( 3
12 ( 1
13 ( 3
8 ( 2
11 ( 5
18 ( 6
13 ( 6
12 ( 2
16 ( 5
22 ( 10
66 ( 5
57 ( 6
89 ( 11
66 ( 5
32 ( 8
11 ( 4
18 ( 8
23 ( 5
48 ( 6
52 ( 4
63 ( 6
17 ( 6
13 ( 5
15 ( 7
14 ( 7
49 ( 10
57 ( 2
83 ( 6
22 ( 8
16 ( 6
40 ( 11
36 ( 6
F32a
GP1
FCR3
FCM29
W2
39 ( 2
35 ( 12
b
102 ( 6
216 ( 25
219 ( 16
267 ( 42
nd
11 ( 4
10 ( 5
12 ( 2
K1
a
Parasites were considered resistant to CQ for IC50 > 100 nM. IC50 values are the mean ( standard deviation of three independent
b
experiments. nd, not determined.

Bis(9-amino-6-chloro-2-methoxyacridines)
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 14 2651
For compounds 1-9, no clear relationship appears
between potential bisintercalation and cytotoxic effects
upon MRC-5 cells. Among the compounds unable to
bisintercalate (compounds 1, 6, and 7), cytotoxicity of 1
upon MRC-5 cells cannot be related to a definite target
since this compound shows a diffuse labeling in murine
L-6 cell cytosol (Figure 1D) while that of 6 and 7 might
be explained by the possible inhibition of the polyamine
3
2
metabolism. With compounds capable of bisintercala-
tion (2-5 and 8-9), surprisingly no localization in the
nucleus of murine L-6 cells was observed by confocal
analysis. As with compound 1, a diffuse labeling of the
cytoplasm was observed for compounds 2-5 and 8, in
contrast to mepacrine which accumulated in the nucleus
(data not shown). Compounds 4 and 5 displayed the
lowest cytotoxic effects upon MRC-5 cells, while an
absence of cytotoxicity was noted for compound 9. This
difference in cytotoxicity could reflect differences in their
cellular distribution. In fact, introduction of a piperazine
leads to the capture of compound 9 in lysosome-like
vesicules of L-6 cells (Figure 1B). The high level of
activity of compound 9 upon P. falciparum could result
from its greater accumulation in the parasite nucleus
(Figure 1A) and its bisintercalating properties. The
difference in composition of plasmodial and mammalian
DNA may contribute to such a sizable accumulation.3
However, cytoplasmic labeling in the parasite was also
observed for compound 9, as well as for compounds 2-5
and 8, suggesting that these compounds might also
interfere with cytoplasmic targets.
F igu r e 2. Labeling of T. cruzi epimastigotes with compound
9
: A, phase contrast; B, labeling with 1 µM compound 9.
Fluorescence was mainly associated with a structure which
colocalized with kinetoplast DNA (K). Bar ) 3 µm.
in the side chain linked to the spacer. They were highly
active against T. brucei (IC50 < 1.56 µM or close to 1.56
µM for compound 29) and only displayed a marginal
activity against P. falciparum, T. cruzi, and L. infantum.
Compound 29 inhibited trypanothione reductase (TR)
from T. cruzi (IC50 ) 20 µM), while IC50 values for the
other compounds were equal or superior to 50 µM in
the presence of 50 µM trypanothione disulfide (data not
shown); this result was not really surprising since
compound 29 displays the structural requirements for
TR recognition, e.g. the presence of aromatic rings and
of a protonable side chain.2
7,28
3,34
Discu ssion
Bisquinolines were used to overcome CQ resistance
since they were likely to be extruded with difficulty by
a proteinaceous transporter.11 They were also discovered
to inhibit the growth of CQ-sensitive and CQ-resistant
parasites. However, further development was suspended
for reasons of toxicity. Monoacridine derivatives were
Each of the compounds 1-8 shows a high level of
activity upon T. brucei, yet none of which can be
retained due to their toxicity found upon murine mac-
rophages and of the known sensitivity of T. brucei to
toxic compounds. Therapeutic indexes of compounds 4
and 5 proved to be too weak for potential drug use in
large numbers of patients benefitting only from a
minimal medical supervision. Thus from series A and
B, compound 9 is the sole representative worthy of note
as a trypanocidal lead and especially as an antimalarial
lead whose interest is enhanced by its consistent ef-
ficiency whatever the CQ sensitivity of the strain tested
reported to display high levels of antileishmanial and
antitrypanosomal activity16 while quinacrine, the acri-
dine analogue of CQ, was in clinical use before CQ.
Bisacridines could constitute an alternative to bisquino-
lines and avoid the efflux mechanism related to CQ
resistance. Compared with bisquinolines, bisacridines
are sterically more hindering, yet they have similar pKa
values (pKa of acridine rings ) 6.5) which permit
ionization and consequent accumulation in the food
vacuole of Plasmodium. However their potential inter-
action with DNA is greater, especially in the case of
bisintercalation which can generate cytotoxicity. Bisin-
tercalation requires a minimal distance between the
(Table 3).
With the exception of 11 and 12, compounds 10-40
are derived from compound 6 with their side chain
bound by an amide bond. As expected, the nature of
their respective side chains had a strong influence on
both cytotoxicity and activity against parasites. When
bisacridines 10-40 were compared with starting com-
pound 6, a global decrease in cytotoxic effects upon
MRC-5 cells was observed while activity upon macro-
phages remained high. Only compounds 25 and 29-32
cause no or low levels of cytotoxicity upon both cell lines,
and from among them, four include the presence of a
polar group (alcohol, amino, or carboxylic). The relative
polarity of this group renders likely a decreased cellular
availability that would be responsible for this low
cytotoxicity and also for the weak activity against all
parasites, with the exception of T. brucei. T. brucei
differs from P. falciparum, T. cruzi, and L. infantum
by an extracellular growth that confers differences in
biochemical pathways, especially in polyamine metabo-
3
0,31
acridine rings, different if the “excluded site model”
two aromatic rings have to be separated by at least two
(
base pairs) is observed: g10.2 Å or not (two aromatic
rings on either side of a base pair): g6.8 Å. These
minimal distances were compared by Le Pecq and co-
workers, with the distances calculated for bis(9-amino-
6
-chloro-2-methoxyacridines) bound with amino link-
1
7
ers. Compounds corresponding to a spermine or
spermidine linker displayed distances respectively of
1
6.1 and 11.2 Å and were found to bisintercalate, while
the removal of a single carbon (symmetical linker
NH-(CH2)3-NH-(CH2)3-NH-) reduced the distance
-
to 9.9 Å and suppressed bisintercalation. The latter
named linker was therefore introduced into the majority
of compounds 10-40 and was substituted by a variety
of side chains in order to decrease possible interaction
with DNA. Bisacridines 1-9, differing by the length,
the nature of the linker, and the absence of a side chain,
were also prepared and studied in comparison.
3
5
lism. Trypanothione reductase from T. cruzi, which
is inhibited by compound 29 (IC50 ) 20 µM), cannot be

2
652 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 14
Girault et al.
Ta ble 4. In Vitro Cytotoxicity of Compounds 1-9 upon MRC-5 Cells and in Vitro Activities toward Trypomastigote Forms of T.
brucei and Amastigote Forms of T. cruzi and L. infantum
molecule
cytotoxicity (%)
T. brucei (%)
T. cruzi (%)a
L. infantum (%)a
concn (µM)
25
12.5
6.25
3.13
12.5
6.25
3.13
1.56
12.5
6.25
3.13
1.56
12.5
6.25
3.13
1.56
1
2
3
4
5
6
7
8
9
100
100
100
90
100
100
100
100
0
100
90
100
80
100
80
100
0
100
20
20
0
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
90
100
99
100
90
100
100
70
100
100
100
60
T
70
T
99
90
T
T
T
0
T
40
98
70
40
T
T
T
0
T
0
90
40
0
T
T
T
0
90
0
70
0
0
60
T
T
40
T
T
T
T
T
T
0
T
0
95
90
80
T
T
T
0
T
0
90
70
40
T
T
T
0
T
0
70
20
0
40
T
T
100
100
100
100
100
100
80
20
0
0
100
100
100
0
100
100
100
0
100
100
100
0
T
0
0
b
Mel
Benz
Amp B
CC50 ) 12.5 µM
CC50 > 50 µM
CC50 ) 25 µM
IC50 ) 200 nM
b
IC50 ) 3130 nM
b
IC50 ) 50 nM
a
T, toxic upon mouse primary peritoneal macrophages used in the test. b Mel, melarsoprol; Benz, benznidazole; Amp B, amphotericin
B. These reference drugs are included in each primary in vitro screening batch.
Ta ble 5. In Vitro Cytotoxicity of Compounds 10-40 upon MRC-5 Cells and in Vitro Activities toward Trypomastigote Forms of T.
brucei and Amastigote Forms of T. cruzi and L. infantum
molecule
cytotoxicity (%)
T. brucei (%)a
T. cruzi (%)b
L. infantum (%)b
concn (µM)
25
12.5
6.25
3.13
12.5
6.25
3.13
1.56
12.5
6.25
3.13
1.56
12.5
6.25
3.13
1.56
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
100
100
100
100
100
55
100
0
100
0
0
0
0
0
81
0
42
43
100
31
20
55
43
100
20
40
0
20
0
0
0
0
0
0
100
0
100
100
100
nd
100
nd
nd
100
nd
100
nd
nd
100
100
100
100
100
100
nd
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
90
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
60
100
100
80
100
100
100
100
100
100
99
100
99
T
60
90
T
80
T
T
T
T
T
T
T
T
70
98
40
0
99
T
90
40
70
T
40
T
40
0
20
95
0
80
90
0
0
0
0
60
0
40
70
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
95
0
40
40
0
0
0
T
90
70
T
80
T
T
T
T
T
T
T
T
90
T
20
0
40
T
0
0
40
0
T
0
T
T
T
T
T
95
60
40
T
60
T
T
T
T
40
95
99
90
80
80
0
0
0
T
0
60
20
20
99
40
95
99
40
0
40
0
0
95
0
90
95
0
100
84
100
49
59
100
71
90
60
50
100
90
100
0
100
100
37
0
0
0
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
99
84
20
47
54
100
41
80
57
48
100
40
100
0
40
40
0
0
0
0
0
100
0
60
T
T
100
100
100
68
40
80
90
80
80
40
70
0
0
90
T
0
0
20
0
T
0
T
T
T
T
0
0
0
0
20
20
40
40
0
20
0
0
40
40
0
0
0
0
T
0
60
T
T
T
0
T
0
70
95
70
60
60
0
0
0
0
0
0
0
0
T
0
T
T
T
20
80
40
0
20
0
0
0
0
0
0
0
0
T
0
T
T
T
T
0
70
100
100
100
0
100
100
39
0
0
0
0
100
60
100
78
76
80
nd
20
0
100
100
100
100
100
100
nd
nd
nd
nd
nd
100
100
95
100
100
60
100
100
100
100
100
100
95
80
80
0
T
60
T
T
T
T
90
T
0
0
0
T
0
T
T
T
0
100
20
100
71
65
74
0
100
100
100
100
100
100
100
100
100
57
39
57
0
80
42
37
40
0
T
0
T
T
0
T
15
54
40
39
30
100
100
T
40
T
T
a
nd, not determined. b T, toxic upon mouse primary peritoneal macrophages used in the test.
retained as the target since this compound is totally
inactive upon T. cruzi. Compounds 10, 13, and 15-16
display high levels of activity upon T. cruzi and L.
infantum at 1.56 µM. They also show an intriguing
inverse correlation toward P. falciparum growth inhibi-
tion for the seven strains studied and varying in their
sensitivity to CQ. This might suggest either the pres-
ence of more sensitive target(s) in CQ-resistant para-
sites compared with CQ-sensitive ones or a higher
accumulation inside the CQ-resistant parasites due to
mechanism(s) which might be involved in CQ resistance.
However, interest in these four compounds is rendered
questionable by their cytotoxic effects upon MRC-5 cells
and macrophages.
Con clu sion s
From the hypothesis that bulky structures are ex-
truded with difficulty by CQ-resistant strains of P.
falciparum, a series of bisacridine derivatives (aliphatic
di-, tri-, and tetramine) was prepared. A side chain with
a variety of amino acid residues was attached to the
polyamine linker, both to improve the weak solubility
of this type of compound and to reduce the possible
interaction with human DNA. The introduction of a
piperazine moiety to the linker led to a unique behav-
ior: a strong and selective activity upon Plasmodium,
a localization outside of the food vacuole and associated
mainly with the parasite nucleus along with a total
absence of cytotoxic effects upon MRC-5 cells and

Bis(9-amino-6-chloro-2-methoxyacridines)
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 14 2653
murine macrophages. Although no structure-activity
relationship was evident in the C series with respect to
bisintercalation, the presence of a polar group in the
side chain attached to the linker afforded a reduction
in antimalarial activity while favoring a specific activity
upon T. brucei.
Compounds 9, 25, 30, and 31, which satisfy both of
the conditions required for antiparasitic drugs, e.g.
safety and low cost, can be considered as good, new lead
structures against P. falciparum and T. brucei.
the total parasite load (free trypomastigotes and intracellular
amastigotes) compared with untreated control cultures. Scor-
ing was performed microscopically.
In Vit r o Act ivit y a ga in st In t r a cellu la r L. in fa n tu m
Am a stigotes. Primary mouse peritoneal macrophages were
seeded in 16-well Lab-Tek culture slides at 30 000 cells/well.
After 24 h, amastigotes of L. infantum (derived from the spleen
of an infected donor animal) were added at an infection ratio
of 10/1 together with 2-fold dilutions of the drug. The cultures
were incubated at 37 °C in 5% CO -95% air for 7 days.
2
Treatment of uninfected control cultures was also included to
determine a selectivity index. Drug activity was semiquanti-
tatively scored as a percent (%) reduction of the total parasite
load or the number of infected macrophages in Wright stained
preparations. Scoring was performed microscopically.
Exp er im en ta l Section
In Vitr o P . fa lcipa r u m Cu ltu r e a n d Dr u g Assa ys. P.
falciparum strains were maintained in continuous culture on
human erythrocytes as described by Trager and J ensen.36 In
vitro antiplasmodial activity was determined using a modifica-
tion of the semiautomated microdilution technique of Desjar-
Con foca l Micr oscop y. P. falciparum-infected red blood
cells were maintained for 1 h under normal culture conditions,
at 37 °C, in the presence of 1 µM bisacridine derivative.
Following two washes (2000g, 5 min), cells were incubated in
culture medium without serum for 15 min in the presence of
3
7
dins et al. P. falciparum CQ-sensitive (3D7/unknown origin,
F32a/Tanzania, GP1/Thailand), moderately CQ-resistant (FCR3/
Gambia), and CQ-resistant (FcB1R/Colombia, FCM29/Cam-
eroun, K1/Thailand, W2/Indochina) strains were used in
sensitivity testing. FcB1R, F32a, GP1, and FCM29 were
strains obtained by limit dilution. Stock solutions of chloro-
quine diphosphate and test compounds were prepared in
sterile distilled water and DMSO, respectively. Drug solutions
were serially diluted with culture medium and added to
asynchronous parasite cultures (0.5% parasitemia and 1% final
hematocrite) in 96-well plates for 24 h, at 37 °C, prior to the
1
0 µg/mL DIL C18(3) (1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-
indocarbocyanine perchlorate; Molecular Probes, Inc., Eugene,
OR). After three further washes, cells were mounted on poly-
L-lysine-coated slides and immediately observed using a con-
focal laser scanning microscope (MRC 1024, Bio-Rad). In some
experiments, following incubation with a bisacridine deriva-
tive, cells were fixed with 2% (v/v) formaldehyde in phosphate-
buffered saline (PBS), washed twice, and incubated for 15 min
with 20 µg/mL propidium iodide or 2 µg/mL DAPI (Sigma).
Cells were observed after two further washes; no difference
in labeling with bisacridine derivatives was noted between
3
addition of 0.5 µCi of [ H]hypoxanthine (1-5 Ci/mmol; Amer-
sham, Les Ulis, France) per well, for 24 h. The growth
inhibition for each drug concentration was determined by a
relative comparison of the radioactivity incorporated in the
treated culture with that in the control culture (without drug)
maintained on the same plate. The concentration causing 50%
inhibition (IC50) was obtained from the drug concentration-
response curve, and the results are expressed as the mean
determined from several independent experiments. The DMSO
concentration never exceeded 0.1% and did not inhibit the
parasite growth.
Cytotoxicity Test u p on MRC-5 Cells. A human diploid
embryonic lung cell line (MRC-5, Bio-Whittaker 72211D) and
primary peritoneal mouse macrophages were used to assess
the cytotoxicity for host cells. The peritoneal macrophages were
collected from the peritoneal cavity 48 h after stimulation with
potato starch and seeded in 96-well microplates at 30 000 cells/
well. MRC-5 cells were seeded at 5 000 cells/well. After 24 h,
the cells were washed and 2-fold dilutions of the drug were
added in 200 µL of standard culture medium (RPMI + 5%
FCS). The final DMSO concentration in the culture remained
below 0.5%. The cultures were incubated with four concentra-
tions of compounds (25, 12.5, 6.25, and 3.13 µM) at 37 °C in
either living or fixed cells, or cells not incubated with DIL C18
-
(3). For the labeling with mepacrine, infected erythrocytes
were incubated with 10 µM drug for 1 h, washed twice, and
immediately observed. Murine muscle L-6 cells were grown
in 8-well Lab-Tek culture slides (Nunc, Inc., Naperville, IL)
in DMEM supplemented with 5% (v/v) fetal calf serum, at 37
°
2
C, in 5% CO -95% air. Cells were incubated for 1 h in the
presence of 1 µM bisacridine derivative and washed three
times prior to observation. For double-labeling experiments
using a lysosome-specific probe, cells were incubated simul-
taneously with a bisacridine derivative and LysoTracker Red
DND-99 diluted according to the manufacturer’s recommenda-
tions (Molecular Probes). T. cruzi epimastigotes (Y strain) were
maintained and grown in liver infusion medium, containing
40
1
0% (v/v) fetal calf serum, at 28 °C. Cells were washed twice
in PBS and incubated with 1 µM bisacridine derivative in PBS,
for 1 h. After three washes with PBS, cells were fixed with
1
2
% (v/v) formaldehyde in PBS and incubated for 15 min with
µg/mL DAPI. Cells were observed following three further
washes.
Assa ys for TR In h ibition . Recombinant T. cruzi trypano-
thione reductase was produced from the SG5 Escherichia coli
strain with the overproducing expression vector pIBITczTR.
TR activity was measured at 21 °C in 0.02 M Hepes buffer,
pH 7.25, containing 0.15 M KCl, 1 mM EDTA, and 0.2 mM
5
% CO
2
-95% air for 7 days. Untreated cultures were included
as controls. For MRC-5 cells, the cytotoxicity was determined
3
8
using the colorimetric MTT assay and scored as a percent
%) reduction of absorption at 540 nm of treated cultures
(
-
1
NADPH with an enzyme concentration of 0.02 U mL . The
reaction was promoted by the addition of the enzyme, and the
subsequent NADPH oxidation was followed at 340 nm. IC50
of the different compounds was evaluated in the presence of
versus untreated control cultures. For macrophages, scoring
was performed microscopically.
In Vitr o Activity a ga in st T. br u cei Tr yp om a stigotes.
Bloodstream forms of T. brucei were cultivated in HMI-9
3
9
2
50 µM T(S) and 1% DMSO.
medium. In a 96-well microplate, 10 000 hemoflagellates
were incubated at different drug concentrations (12.5, 6.25,
3
.13, and 1.56 µM) for 4 days. Parasite multiplication was
Ack n ow led gm en t. We express our thanks to G e´ -
rard Montagne for NMR experiments, Val e´ rie Landry
for IC50 measurements upon TR, and Dr. Steve Brooks
and Sandrine Delarue for proofreading. This work was
supported by CNRS (GDR 1077, EP CNRS 1790, UMR
CNRS 8525) and Universit e´ de Lille II.
measured colorimetrically (490 nm) following addition of MTT,
which converts to an aqueous soluble, formazan product.38
In Vitr o Activity again st In tr acellu lar T. cr u zi Am astig-
otes. Primary mouse peritoneal macrophages were seeded in
9
1
6-well microplates at 30 000 cells/well. After 24 h, about
0 0000 trypomastigotes of T. cruzi were added per well
together with 2-fold dilutions of the drug. The cultures were
incubated at 37 °C in 5% CO
fixation in methanol and Giemsa staining, the drug activity
was semiquantitatively scored as a percent (%) reduction of
2
-95% air for 4 days. Following
Su p p or tin g In for m a tion Ava ila ble: Details of chemical
procedures and analytical data. This material is available free
of charge via the Internet at http://pubs.acs.org.

2
654 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 14
Girault et al.
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