Synthesis and antileishmanial activity of 6-mono-substituted and 3,6-di-substituted acridines obtained by acylation of proflavine

Article abstract of DOI:10.1016/j.ejmech.2007.02.010

Two new series of diaminoacridinic derivatives obtained from proflavine and N-(6-amino-3-acridinyl)acetamide were synthesised and assessed for their cytotoxic and antileishmanial activities. Two compounds, N-[6-(acetylamino)-3-acridinyl]acetamide and N-[6-(benzoylamino)-3-acridinyl]benzamide demonstrated highly specific antileishmanial properties against the intracellular amastigote form of the parasite. Structure-activity relationships established that the antiproliferative activity against human cells was greatly enhanced by the presence of a benzoylamino group in 6-mono-substituted acridines, while the presence of two acetylamino or benzoylamino groups in 3,6-di-substituted acridines strongly increased the specificity of the molecules for Leishmania parasite, suggesting that symmetric conformations could preferentially interfere with Leishmania metabolism.

Full text of DOI:10.1016/j.ejmech.2007.02.010

European Journal of Medicinal Chemistry 42 (2007) 1277e1284
http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and antileishmanial activity of 6-mono-substituted and
,6-di-substituted acridines obtained by acylation of proflavine
3
a,*
b
b
a
Carole Di Giorgio , Kamal Shimi , Gerard Boyer , Florence Delmas , Jean-Pierre Galy
b
´
a
Laboratoire de Parasitologie, Hygiene et Zoologie, Facult e´ de Pharmacie, 27 Bd. Jean Moulin, 13385 Marseille Cedex 05, France
Laboratoire de Valorisation de la Chimie Fine, case 552, UMR CNRS SYMBIO 6178 e Universit e´ Paul C e´ zanne, Facult e´ St J e´ r oˆ me,
b
1
3397 Marseille Cedex 20, France
Received 29 September 2006; received in revised form 13 February 2007; accepted 13 February 2007
Available online 1 March 2007
Abstract
Two new series of diaminoacridinic derivatives obtained from proflavine and N-(6-amino-3-acridinyl)acetamide were synthesised and
assessed for their cytotoxic and antileishmanial activities. Two compounds, N-[6-(acetylamino)-3-acridinyl]acetamide and N-[6-(benzoyla-
mino)-3-acridinyl]benzamide demonstrated highly specific antileishmanial properties against the intracellular amastigote form of the parasite.
Structureeactivity relationships established that the antiproliferative activity against human cells was greatly enhanced by the presence of a ben-
zoylamino group in 6-mono-substituted acridines, while the presence of two acetylamino or benzoylamino groups in 3,6-di-substituted acridines
strongly increased the specificity of the molecules for Leishmania parasite, suggesting that symmetric conformations could preferentially inter-
fere with Leishmania metabolism.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Acridines; Cytotoxicity; Antileishmanial activity; DNA-binding activity
1
. Introduction
therapeutic solutions [4,5] for the treatment of visceral leish-
maniasis, however, they do not represent a safety treatment in
all clinical cases, rendering necessary the research of new anti-
leishmanial molecules.
For hundreds of years, leishmaniases have been the cause of
death among millions of people throughout the world [1].
Transmitted by the bite of a sand fly, they are due to an intracel-
lular protozoa belonging to the genus Leishmania which colo-
nizes human macrophages and produces a large spectrum of
clinical manifestations [1,2]. Among all leishmaniases, the
visceral form also known as kala-azar is the most severe [1e3].
Characterized by irregular bouts of fever, substantial weight
loss, swelling of the spleen and anaemia, this disease can pro-
duce, if untreated, a mortality rate of almost 100%, indepen-
dently on the immunological status of the patient [3].
Pentavalent antimony, Amphotericin B and the oral anticancer
drug miltefosine are considered at the moment as the best
Due to their rapid replication, Trypanosomatidae protozoa
have shown many similarities with tumour cells and various
anticancer drugs have been successfully tested for their
antileishmanial activity. Since the 1990s, various acridine
derivatives which have shown strong antiproliferative activ-
ities on human transformed cells, have been considered as
promising agents for anticancer and/or antiparasitic therapy
[6]. Among these compounds, proflavine, widely used as
a biochemical tool for studying the properties of DNA
and RNA in vivo and in vitro [7], showed some remarkable
antiproliferative effects on hamster melanoma [8], indicating
that synthesis of new derivatives of the 3,6-diaminoacridine
series would present new promising anticancer and/or anti-
parasitic agents.
*
Corresponding author. Tel.: þ33 4 91 83 55 44; fax: þ33 4 91 80 26 12.
E-mail address: Carole.Digiorgio@Pharmacie.univ-mrs.fr (C. Di Giorgio).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.02.010

1278
C. Di Giorgio et al. / European Journal of Medicinal Chemistry 42 (2007) 1277e1284
In order to open a new way in antitumor drug research, an
3. Pharmacology
efficient synthetic route to monofunctional 6-substitued acri-
dine and bifunctional 3,6-di-substituted acridine derivatives
has been developed on the basis of direct electrophilic substi-
tution of acridinic moiety. This synthetic route led to a wide
range of acridinic derivatives which could be linked to various
side chains. On this basis we synthesised two new series of
diaminoacridinic derivatives obtained from proflavine and
N-(6-amino-3-acridinyl)acetamide and studied their antiproli-
ferative and antileishmanial activities as compared to their
capacity to bind with DNA.
The antiproliferative properties of acridine derivatives were
evaluated against transformed human monocytes (THP1 cells),
while antileishmanial activities were assessed against parasites
of the species Leishmania infantum responsible for kala-azar,
the most severe visceral form of leishmaniasis, on both its ex-
tracellular promastigote and intracellular amastigote forms. In
parallel, the capacity of the compounds to non-covalently in-
teract with DNA by intercalating into the DNA major groove
was evaluated by the DNAemethyl green bioassay.
4. Results and discussion
2
. Chemistry
Results are summarized in Table 1 and schematised in
Our synthetic pathway was based on the preparation of
Fig. 4. They showed that the pharmacological properties of
the compounds greatly depended on the number and the chem-
ical nature of the substituents. Compound 4 (N-(6-amino-3-
acridinyl)acetamide), the parent molecule of the 4aee series,
exerted an interesting antiproliferative activity on human
monocytes (IC50-monocytes ¼ 4.7 mM) while it was deprived
from toxicity toward Leishmania parasites on both their extra-
cellular and intracellular forms. Comparison with results ob-
served for proflavine indicated that the replacement of an
amino group by an acetylamino group slightly reduced the cy-
totoxicity of the compounds but deprived the derivatives from
antileishmanial activity.
symmetric and non-symmetric diaminoacridines by mono- or
di-acylation: proflavine 1, used as hemisulfate, was allowed
to stand with different anhydrides to yield the corresponding
amides 2aec as reported in Fig. 1.
The in situ neutralisation of 1 with NEt in pyridine was not
3
competitive to the acylation reaction because of the higher ki-
netic of amide toward acid formation. The formed hydrosul-
fate was then neutralized with excess base. No significant
yield augmentation was observed using neutral proflavine.
Compound 1 was also allowed to react with different benzoyl-
chlorides to yield the corresponding amides shown in Fig. 2
with medium to good yields. Moreover, a useful synthetic
route has been developed to yield selectively the mono-N-(6-
amino-3-acridinyl)acetamide 4 from proflavine 1: we used
a modified procedure from Albert and Linnell [9] using acetic
anhydride to give only one monomer with 84% yield. The re-
action was fully regioselective and did not require protection
of the second amino substituent. The reaction allowed the de-
symetrization of the molecule as the electrophilic substitution
only proceeded on one side of the molecule, because of the
strong withdrawing electronic effect of the acetylamino group.
Then, mono-acetyl 4 was reacted with different benzoylchlor-
ides to yield good yields of the corresponding mono-amides
Its bifunctional homologue 2a (N-[6-(acetylamino)-3-
acridinyl]acetamide) on the contrary, showed a weak
toxicity against human cells and Leishmania promastigotes
(IC50-monocytes > 500 mM and IC50-promastigotes > 300 mM) asso-
ciated with a strong anti-amastigote property (IC50-amastigotes
¼
4.5 mM). These values indicated that the replacement of the
amino group by a second acetylamino group on position 6
greatly modified the biological properties of the molecule, ren-
dering it less toxic for mammalian cells and highly specific
for the intracellular form of the parasite. Compounds 2b (N-
[6-(propionylamino)-3-acridinyl]propanamide) and 2c (N-[6-
(butyrilamino)-3-acridinyl]butanamide) were more efficient
than compound 2a against human cells and promastigotes,
4
aee, as reported in Fig. 3.
Pyridine
8
9
1
4
Et N
3
O
O
2
7
1h / 80°C
3
+
NH2
R
O
R
HN
N
NH
H N
N
2
6
5
O
2a-c
O
R
1
R
Comp
R
Yields
43 %
MW
293
321
321
Molecular formula
Mp °C
2a
CH3
C H
C17H15N O2
260
265
216
3
2b
65 %
C19H19N O2
2
5
3
2c
C H
32 %
C19H17N O2
3
7
3
Fig. 1. Synthesis of compounds 2a to 2c.

C. Di Giorgio et al. / European Journal of Medicinal Chemistry 42 (2007) 1277e1284
1279
O
Pyridine
Et N
3
R2
R1
1h / 80°C
Cl
+
HN
N
NH
N
NH2
H N
2
R2
R1
R2
R1
O
O
1
3a-f
Comp
R2
R1
Yields
MW
Molecular formula
Mp °C
3a
3b
3c
3d
3e
H
H
H
H
Cl
F
OMe
OMe
66 %
90 %
96 %
51 %
39 %
417
486
453
477
537
C27H19N3O2
C27H17N3O2Cl2
C27H17N3O2F2
C29H23N3O4
248
370
290
252
207
H
OMe
C31H27N3O6
Fig. 2. Synthesis of compounds 3a to 3e.
suggesting that their toxicity increased with the length of the
,6-di-substituted linear chains. Inversely, their anti-amasti-
IC50-promastigotes and IC50-amastigotes averaging 1.1 and 4.3 mM,
respectively, while it exhibited a lower antiproliferative activ-
ity against human monocytes. This suggested that replacement
of linear chains by aryl rings greatly increased the specificity
of the molecule for Leishmania metabolism. However, addi-
tion of a fluoro-, chloro- or methoxy-group on the 3,6-di-
substituted aryl chains considerably reduced the specificity
of the compounds for Leishmania parasite, rendering them
toxic against human cells.
3
gote activity proportionally decreased. These results implied
that such molecules could react with biological structures by
two different mechanisms. The first one, weakly specific for
the cell type, was highly dependent on the length of the 3,6-
di-substituted linear chains. The second one, strongly specific
of the amastigoteemacrophage system, was essentially due to
the combined action of the two 3,6-di-substituted acetylamino
groups. Compound 3a (N-[6-(benzoylamino)-3-acridinyl]ben-
zamide), bearing benzoylamino groups on positions 3 and 6,
demonstrated a strong affinity for both parasite forms with
Compounds 4ae4e obtained from N-(6-amino-3-acridinyl)
acetamide 4, exerted a strong antiproliferative activity against
human monocytes but were far less active on Leishmania
O
O
Me
O
Me
AcOH
H N
N
NH2
O
H2N
N
NH
2
1
4
Me
O
Pyridine
Et N
3
R2
1h / 80°C
Cl
+
NH
HN
N
NH
Me
H N
N
R1
2
R2
R1
O
4a-e
O
4
O
Me
Comp
R2
R1
Yields
MW
Molecular formula
Mp °C
4
a
H
H
H
H
OMe
H
Cl
F
OMe
OMe
63 %
83 %
84 %
74 %
72 %
355
390
373
385
415
C22H17N3O2
152
282
292
170
165
4b
4c
4d
4e
C27H16ClN3O2
C27H16FN3O2
C23H19N3O3
C24H21N3O4
Fig. 3. Synthesis of compounds 4a to 4e.

1280
C. Di Giorgio et al. / European Journal of Medicinal Chemistry 42 (2007) 1277e1284
Table 1
Cytotoxicity and antileishmanial activity of proflavine derivatives
a
Ref.
Cytotoxicity against monocytes
IC50-monocytes (mM)
Antileishmanial activity
IC50-promastigotes (mM)
90.8 ꢀ 5.5
341.5 ꢀ 15.4
159.8 ꢀ 12.4
46.7 ꢀ 3.2
DNA-binding activity
IC50-DNA (mM)
IC50-amastigotes (mM)
Specificity index (SI)
b
4
4.7 ꢀ 0.6
737.5 ꢀ 12.4
23.2 ꢀ 3.2
18.9 ꢀ 6.4
Toxic
e
59.1 ꢀ 5.2
>250
2a
2b
2c
4.5 ꢀ 1.2
7.7 ꢀ 1.4
12.8 ꢀ 5.1
163.8
3.0
1.47
170.9 ꢀ 8.1
102.5 ꢀ 6.6
3
3
3
3
3
a
b
c
110.3 ꢀ 15.2
2.49 ꢀ 0.3
0.77 ꢀ 0.1
13.4 ꢀ 2.1
26.8 ꢀ 4.2
5.4 ꢀ 0.8
1.9 ꢀ 0.1
1.1 ꢀ 0.1
1.76 ꢀ 0.1
2.1 ꢀ 0.3
4.31 ꢀ 1.2
0.11 ꢀ 0.03
1.7 ꢀ .7
1.1 ꢀ 0.2
0.2 ꢀ 0.01
0.03 ꢀ 0.01
1.1 ꢀ 0.2
100.2
11.3
23.3
12.1
32.7
>250
>250
>250
>250
>250
d
e
4.8 ꢀ 1.2
16.1 ꢀ 3.3
0.8 ꢀ 0.03
b
4a
4b
4c
4
4
186.2 ꢀ 10.4
11.5 ꢀ 2.7
Toxic
2.9 ꢀ 0.4
e
>250
>250
>250
>250
>250
e
132.7 ꢀ 14.1
86.9 ꢀ 5.4
0.67 ꢀ 0.04
e
b
d
e
Toxic
1.3 ꢀ 0.2
e
1.6
202.9 ꢀ 14.1
Amphotericin B
Doxorubicin
Proflavine
a
154.5 ꢀ 9.2
e
0.38 ꢀ 0.1
e
0.14 ꢀ 0.03
e
1103 ꢀ 26.4
e
e
1.38 ꢀ 0.8
>250
2.21 ꢀ 3.4
Ref.: compound references.
1.3 ꢀ 0.5
0.12 ꢀ 0.06
18.1 ꢀ 7.3
b
Toxic: toxicity observed on human macrophages at concentrations that did not display antileishmanial activity.
parasites than their di-substituted homologues of the 3aee se-
ries. They indicated that a symmetric conformation was essen-
tial for a specific antileishmanial activity.
metabolism remain unclear and extensive additional studies
are necessary to complete current data.
Results observed in the present study clearly demonstrated
that some derivatives of the 6-substituted and 3,6-di-substituted
aminoacridine family could exert interesting anticancer and/or
antileishmanial activities. Among the 14 compounds tested,
compound 3c (4-fluoro-N-[6-(4-fluorobenzoyl)amino)-3-acri-
dinyl]benzamide exhibited the highest anticancer properties
against human monocytes, while compounds 2a N-[6-(acetyla-
mino)-3-acridinyl] acetamide and 3a N-[6-(benzoylamino)-3-
acridinyl]benzamide exerted the most specific antileishmanial
Due to their tricyclic structure, acridines have been largely
studied for their capacity to bind with DNA. Concerning our
series of proflavine derivatives, only four compounds demon-
strated this ability (Table 1): compound 4 (N-(6-amino-3-acri-
dinyl)acetamide) exhibited the highest affinity for DNA, while
compounds 2b (N-[6-(propionylamino)-3-acridinyl]propana-
mide) and 2c (N-[6-(butyrilamino)-3-acridinyl]butanamide)
were less active. Results established that the capacity of deriv-
atives to bind with DNA greatly depended on the presence of
the 6-amino group (compound 4), while linear chains in 3,6-
di-substituted derivatives were far less active. No correlation
could be observed by the rank correlation test of Spearman be-
tween cytotoxicity or antileishmanial activity and DNA-bind-
ing activity (P > 0.05), suggesting that binding to DNA could
not be considered as the main mechanism responsible for anti-
proliferative effects in human cells and Leishmania parasites.
6
5
4
3
2
1
0
0
0
0
0
0
0
Compounds with strong
antiproliferative activity
4
d
4a
4
5
. Conclusion
Acridine family includes a wide range of tricyclic mole-
Compounds with specific
antileishmanial activity
2c
cules with various biological properties. Considered as poten-
tial antiparasitic agents since the 1990s, numerous acridine
derivatives have been synthesised and successfully assessed
for their antimalarial, trypanocidal or antileishmanial proper-
ties [10e12]. On this basis, authors recently demonstrated
that diverse compounds of the 9-substituted [13], 4-substituted
and 4,5-di-substituted aminoacridine [14] series exerted potent
antileishmanial activities, however, the mechanisms by which
such molecules could specifically interact with parasite
2b
4e
2a
3a
4
b
4c
3b
3d
3e
3c
0
10
20
IC50 Human cells (µM)
30
40
50
60
Fig. 4. Schematic representation of antileishmanial and antiproliferative
activities.

C. Di Giorgio et al. / European Journal of Medicinal Chemistry 42 (2007) 1277e1284
1281
activities, suggesting that acridine compounds could interact
with mammalian and protozoan cells by different
mechanisms.
Structureeactivity relationships led to the identification of
some molecular structures responsible for the specificity of
the compounds for Leishmania parasites or for human cells.
Anticancer properties were shown to vary according to the
number and the quality of the substituents: they were greatly
enhanced by the presence of a single benzoylamino group in
6.2. Chemistry
Melting points were performed on a Electrothermal 9200
1
melting point apparatus and are uncorrected. The H and
1
3
C NMR spectra were measured on a BRUKER AC 300 spec-
1
trometer (300.13 MHz for H).
6.2.1. N-[6-(Acetylamino)-3-acridinyl]acetamide (2a)
Hemisulfate of 3,6-diaminoacridine 1 (1 g, 3.25 mmol) was
dissolved in acetic anhydride (1.9 ml, 20 mmol) and 0.25 g of
anhydrous sodium acetate. The mixture was warmed at 100 C
for 2 h. The obtained precipitate was filtered and the grey solid
was thoroughly washed with a solution of ammonium hydrox-
ide 10%. Recrystallization from ethanol yielded pure 2a as
6
-mono-substituted acridines as compared to their 3,6-di-
substituted homologues, or by the presence of a fluoro-,
a chloro- or a methoxy-substituent in both mono-substituted
or di-substituted derivatives.
Antiproliferative activities against Leishmania promasti-
gotes were preferentially related to the presence of two
benzoylamino groups in di-substituted derivatives. Moreover,
they appeared to depend on the presence of a chloro-, a fluoro-
or a methoxy-group in both mono-substituted and di-substituted
compounds.
ꢂ
ꢂ
orange crystalline powder. Yield 43%. M.p. 260 C. NMR
1
H (DMSO-d ): 10.35 (NH), 8.86 (9-H), 8.49 (4-H and 5-H),
6
0
7.59 (2-H and 7-H), 8.03 (1-H and 8-H), 2.15 (CH -1 ). Anal.
Calc. for C H N O : C, 69.61; H, 5.15; N, 14.33. Found: C,
3
1
7 15 3 2
69.59; H, 5.10; N, 14.36.
Strong anti-amastigote activities could be observed with
almost all the acridine compounds, nevertheless the most
specific properties were obtained for compounds bearing
6
.2.2. N-[6-(Propionylamino)-3-acridinyl]propanamide (2b)
Hemisulfate of 3,6-diaminoacridine (300 mg, 1 mmol) was
ꢂ
3
,6-acetylamino groups or 3,6-benzoylamino groups, indicat-
dissolved in pyridine (6 ml) at 25 C and NEt (0.5 ml)
3
ing that a symmetric conformation with acetylamino or
benzoylamino substituents was essential for such specific anti-
leishmanial activity.
was added. Then, propionic anhydride (2.1 equiv, 0.27 ml)
ꢂ
was added dropwise at 50 C and stirring was continued for
ꢂ
5
0 min more at 80 C. The resulting mixture was poured
Cytotoxicity and antileishmanial activity were not corre-
lated with the properties of the derivatives to bind with
DNA, indicating that other mechanisms of action should be in-
vestigated. Nevertheless the interaction with DNA observed
with compound 4 confirmed previous studies concerning the
capacities of acridines to bind with DNA [15,16] and illus-
trated the important role of the amino group in acridinee
DNA interactions. However, methyl green is a major groove
binding molecule which does not detect the entire DNA-bind-
ing agents and more particularly acridines capable of forming
minor groove adducts [17]. Consequently, complementary ex-
periments should be performed in order to better analyse the
interactions of 6-substituted and 3,6-di-substituted acridines
with macromolecules.
On the other hand, results observed on human macrophages
indicated that some of the acridine derivatives could have an
indirect antileishmanial activity by interfering with Leishma-
niaemacrophage interactions. They also suggested that acri-
dines could react with cellular structures by a wide range of
mechanisms and justified the constant need of extended bio-
logical studies.
into water (80 ml). The obtained precipitate was filtered and
washed with water. The resulting solid was recrystallized
from ethanol to give an orange powder 2b. Yield 65%. M.p.
ꢂ
65 C. NMR H (DMSO-d ): 10.28 (NH), 8.81 (9-H), 8.48
6
1
2
(
2
4-H and 5-H), 8.01 (1-H and 8-H), 7.60 (2-H and 7-H),
0 0
.43 (CH -1 ), 1.13 (CH -2 ). Anal. Calc. for C H N O :
2 3 19 19 3 2
C, 71.01; H, 5.96; N, 13.08. Found: C, 71.04; H, 5.92; N,
13.03.
6.2.3. N-[6-(Butyrilamino)-3-acridinyl]butanamide (2c)
As reported for 2b but with (0.35 ml) of butyric anhydride.
After work up the recovered solid was recrystallized from eth-
ꢂ
anol to give an orange powder 2c. Yield 32%. M.p. 216 C.
1
NMR H (DMSO-d ): 10.27 (NH), 8.80 (9-H), 8.50 (4-H
6
and 5-H), 7.98 (1-H and 8-H), 7.58 (2-H and 7-H), 2.38
0 0 0
CH -1 ), 1.67 (CH -2 ), 1.09 (CH -3 ). Anal. Calc. for
2 2 3
(
C H N O : C, 72.18; H, 6.63; N, 12.03. Found: C, 72.21;
3 2
21 23
H, 6.65; N, 12.05.
6.2.4. N-[6-(Benzoylamino)-3-acridinyl]benzamide (3a)
Hemisulfate of 3,6-diaminoacridine 1 (300 mg, 1 mmol)
ꢂ
was dissolved in pyridine (6 ml) at 25 C and NEt (0.5 ml)
3
6
. Experimental
was added. Then, benzoylchloride (2.1 equiv., 0.25 ml) was
ꢂ
added dropwise at 40 C and the stirring was left for 50 min
ꢂ
6
.1. General procedures
more at 80 C. The mixture was poured into water (80 ml).
The precipitate was filtered and washed with water. The result-
ing solid was recrystallized from ethanol to give an orange
All reagents were of analytical grade, dried and purified
when necessary. Proflavine derivatives were dissolved in ster-
ile dimethyl sulfoxide (analytical grade, Sigma) and stored
frozen at ꢁ70 C until used.
ꢂ
1
powder 3a. Yield 66%. M.p. 248 C. NMR H (DMSO-d ):
6
10.69 (NH), 8.95 (9-H), 8.73 (4-H and 5-H), 8.13 (1-H and
0 0
ꢂ
8-H), 8.05 (CH-2 ), 7.92 (2-H and 7-H), 7.65 (CH-4 ), 7.58

1282
C. Di Giorgio et al. / European Journal of Medicinal Chemistry 42 (2007) 1277e1284
0
(
CH-3 ). Anal. Calc. for C H N O : C, 77.68; H, 4.59; N,
3
Calc. for C H N O: C, 71.70; H, 5.21; N, 16.72. Found:
15 13 3
2
7
19
2
1
0.07. Found: C, 77.71; H, 4.55; N, 10.03.
C, 71.68; H, 5.19; N, 16.70.
6
.2.5. 4-Chloro-N-[6-(4-chlorobenzoyl)amino)-3-
6.2.10. N-[6-(Acetylamino)-3-acridinyl]benzamide (4a)
N-(6-Amino-3-acridinyl)acetamide (0.2 g, 0.8 mmol)) was
dissolved in pyridine (4 ml) at room temperature, then NEt3
(0.3 ml) was added. Benzoylchloride (2.1 equiv, 0.2 ml,
acridinyl]benzamide (3b)
As reported for 3a but with 4-chlorobenzoylchloride,
(0.24 ml). After work up the resulting solid was recrystallized
from ethanol to give an orange powder 3b. Yield 90%. M.p.
ꢂ
1.68 mmol) was added dropwise at 40 C under stirring; the
ꢂ
ꢂ
1
3
70 C. NMR H (DMSO-d ): 10.71 (NH), 8.92 (9-H), 8.68
stirring was maintained for 45 min at 80 C. The mixture
6
0
(4-H and 5-H), 8.11 (1-H and 8-H), 8.06 (CH-2 ), 7.89 (2-H
0
and 7-H), 7.66 (CH-3 ). Anal. Calc. for C H N O Cl : C,
was poured into water (80 ml). The obtained precipitate was
filtered and washed with water. The solid was finally recrystal-
lized from ethanol to yield a yellow powder 4a. Yield 63%.
2
7
17
3
2
2
6
6.68; H, 3.52; N, 8.64. Found: C, 66.65; H, 3.49; N, 8.61.
ꢂ
1
M.p. 152 C. NMR H, recorded as hydrochloride, (DMSO-
d ): 11.41 (NH ), 11.29 (NH), 9.46 (9-H), 8.96 (5-H), 8.78
6
.2.6. 4-Fluoro-N-[6-(4-fluorobenzoyl)amino)-3-
6
2
0
acridinyl]benzamide (3c)
As reported for 3a but with 4-fluorobenzoylchloride
(4-H), 8.26 (8-H), 8.24 (1-H), 8.10 (7-H), 8.08 (CH-2 ), 7.83
0
0
0
(2-H), 7.65 (CH-4 ), 7.56 (CH-3 ), 2.22 (CH -1 ). Anal.
3
(
as an orange powder. Yield 96%. M.p. 290 C. NMR H
0.25 ml). After recrystallization from ethanol 3c was obtained
1
Calc. for C H N O : C, 74.35; H, 4.82; N, 11.82. Found:
2
C, 74.32; H, 4.84; N, 11.79.
2 17 3 2
ꢂ
(
8
DMSO-d ): 10.77 (NH), 9.07 (9-H), 8.76 (4-H and 5-H),
6
0
.16 (1-H and 8-H), 8.13 (CH-2 ), 7.91 (2-H and 7-H), 7.41
0
6.2.11. N-[6-(Acetylamino)-3-acridinyl]-4-
chlorobenzamide (4b)
As described for 4a, but with 4-chlorobenzoylchloride
(CH-3 ). Anal. Calc. for C H N O F : C, 71.52; H, 3.78;
N, 9.27. Found: C, 71.49; H, 3.80; N, 9.30.
2
7 17 3 2 2
(0.2 ml). After recrystallization from ethanol 4b was obtained
as a yellow powder. Yield 83%. M.p. 282 C. NMR H
ꢂ
1
6
acridinyl]benzamide (3d)
.2.7. 4-Methoxy-N-[6-(4-methoxybenzoyl)amino)-3-
(DMSO-d ): 10.69 (NH), 10.36 (NH), 8.86 (9-H), 8.64 (5-
6
0
As described for 3a, but with 4-methoxybenzoylchloride
0.29 ml). After recrystallization from ethanol 3d was ob-
H), 8.48 (4-H), 8.08 (8-H), 8.07 (CH-2 ), 8.02 (1-H), 7.86
0
0
(
tained as a yellow powder. Yield 71%. M.p. 252 C. NMR
(7-H), 7.65 (CH-3 ), 7.60 (2-H), 2.13 (CH -1 ). Anal. Calc.
3
ꢂ
for C H ClN O : C, 67.78; H, 4.14; N, 10.78. Found: C,
3 2
2
2
16
1
H (DMSO-d ): 10.50 (NH), 8.91 (9-H), 8.65 (4-H and 5-
6
67.76; H, 4.17; N, 10.76.
0
H), 8.10 (1-H and 8-H), 8.03 (CH-2 ), 7.89 (2-H and 7-H),
0
7
7
.09 (CH-3 ), 3.85 (OCH ). Anal. Calc. for C H N O : C,
29 23 3 4:
6.2.12. N-[6-(Acetylamino)-3-acridinyl]-4-
fluorobenzamide (4c)
3
2.94; H, 4.85; N, 8.80. Found: C, 72.91; H, 4.84; N, 8.83.
As described for 4a, but with 4-fluorobenzoylchloride
(0.2 ml). The mixture was poured into water (80 ml). The pre-
cipitate was filtered and washed with water. After recrystalli-
zation from ethanol 4c was obtained as a yellow powder.
6
3
.2.8. 3,4-Dimethoxy-N-[6-(3,4-dimethoxybenzoyl)amino)-
-acridinyl]benzamide (3e)
As described for 3a, but with 3,4-dimethoxybenzoyl chlo-
ꢂ
1
ride (0.42 g). After recrystallization from ethanol 3e was ob-
ꢂ
Yield 84%. M.p. 292 C. NMR H (DMSO-d ): 10.64 (NH),
6
tained as a yellow powder. Yield 61%. M.p. 207 C. NMR
1
10.35 (NH), 8.87 (9-H), 8.63 (5-H), 8.51 (4-H), 8.08 (8-H),
0
H (DMSO-d ): 10.51 (NH), 8.92 (9-H), 8.67 (4-H and 5-
6
8.11 (CH-2 ), 8.05 (1-H), 7.87 (7-H), 7.60 (2-H), 7.41 (CH-
0
0 0
3 ), 2.17 (CH -1 ). Anal. Calc. for C H FN O : C, 70.77;
3 22 16 3 2
H), 8.12 (1-H and 8-H), 7.91 (2-H and 7-H), 7.93 (CH-6 ),
0
0
7.65 (CH-2 ), 7.08 (CH-5 ), 3.86 (OCH ), 3.85 (OCH ).
Anal. Calc. for C H N O : C, 69.26; H, 5.06; N, 7.82.
H, 4.32; N, 11.25. Found: C, 70.79; H, 4.35; N, 11.23.
3
3
3
1 27 3 6
Found: C, 69.28; H, 5.09; N, 7.79.
6.2.13. N-[6-(Acetylamino)-3-acridinyl]-4-
methoxybenzamide (4d)
6
.2.9. N-(6-Amino-3-acridinyl)acetamide (4)
,6-Diaminoacridine 1 (2.5 g, 12 mmol) was dissolved in
a mixture of glacial acetic acid (10 ml) and acetic anhydride
2.5 ml). The mixture was warmed until solidification in a wa-
As described for 4a, but with 4-methoxybenzoylchloride
(0.23 ml). After recrystallization from ethanol 4d was ob-
3
ꢂ
tained as a yellow powder. Yield 74%. M.p. 170 C. NMR
1
(
H (DMSO-d ): 10.46 (NH), 10.35 (NH), 8.86 (9-H), 8.64
6
0
ter bath, heating was continued 15 min more and the resulting
solid turned yellow, then acetic acid (40 ml) was added. The
mixture was warmed for 15 min under stirring at 120 C.
(5-H), 8.51 (4-H), 8.08 (8-H), 8.05 (CH-2 ), 8.03 (1-H), 7.89
0
0
(7-H), 7.61 (2-H), 7.10 (CH-3 ), 2.42 (OCH ), 2.15 (CH-1 ).
3
ꢂ
Anal. Calcd for C H N O : C, 71.67; H, 4.97; N, 10.90.
3 19 3 3
2
The resulting precipitate was filtered and thoroughly washed
with 10% ammonium hydroxide. The grey compound was re-
crystallized from ethanol to yield pure 4 as a yellow powder.
Found: C, 71.65; H, 4.99; N, 10.88.
6.2.14. N-[6-(Acetylamino)-3-acridinyl]-3,4-
dimethoxybenzamide (4e)
ꢂ
1
Yield 80%. M.p. 260 C. NMR H (DMSO-d ): 10.97 (NH),
6
9
7
.05 (9-H), 8.62 (4-H), 8.10 (1-H), 7.97 (8-H), 7.82 (NH ),
2
.58 (2-H), 7.17 (7-H), 6.89 (5-H), 2.19 (CH -1 ). Anal.
3
As described for 4a, but with 3,4-dimethoxybenzoyl chlo-
ride (0.34 g). 4e was obtained as a yellow powder. Yield
0

C. Di Giorgio et al. / European Journal of Medicinal Chemistry 42 (2007) 1277e1284
1283
ꢂ
1
7
(
(
7
(
6
2%. M.p. 165 C. NMR H (DMSO-d ): 10.48 (NH), 10.37
suspended in RPMI medium containing stationary-phase pro-
mastigotes (cells/promastigotes ratio ¼ 1/10). After a 24 h in-
6
NH), 8.86 (9-H), 8.65 (5-H), 8.52 (4-H), 8.08 (8-H), 8.05
0
0
0
ꢂ
CH-2 ), 8.05 (1-H), 7.91 (CH-6 ), 7.89 (7-H), 7.66 (CH -2 ),
2
cubation period at 37 C (5% CO ), promastigotes were
2
0
0
0
.61 (2-H), 7.10 (CH -3 ), 6.98 (CH -5 ), 2.15 (CH-1 ), 3.86
2
OCH ), 3.85 (OCH ). Anal. Calc. for C H N O : C,
3 3 24 21 3 4
9.39; H, 5.10; N, 10.11. Found: C, 69.41; H, 5.13; N, 10.09.
removed by four successive washes with fresh medium. Adap-
ted dilutions of chemical compounds were added in duplicate
2
ꢂ
chambers and cultures were incubated for 96 h at 37 C (5%
CO ). Negative controls treated by solvent (DMSO) and pos-
2
6
6
.3. Biology
itive controls containing a range of amphotericin B (Sigma,
St Louis, Mo, USA) concentrations were added to each set
of experiments. At the end of the incubation period, cells
were harvested with analytical grade methanol (Sigma) and
stained with 10% Giemsa stain (Eurobio, Paris, France). The
percentage of infected macrophages in each assay was deter-
mined microscopically at 1000 times magnification. The anti-
leishmanial activity of acridines derivatives against the
intracellular amastigote form of the parasite was expressed
by IC50-amastigotes corresponding to the concentration of deriv-
atives required to induce a 50% decrease of infected macro-
phages. The specificity index (SI) corresponding to the ratio
between toxicity and antileishmanial activity was expressed
.3.1. Antiproliferative activity toward human
transformed monocytes
The effects of acridines on the growth of human monocytes
were assessed by colorimetric determination of cell viability
using the oxidationereduction indicator Alamar Blue . Cyto-
toxicity was assessed on the THP1 human monocyte cell line
Ò
(ATCC, Manassas VA, USA) by colorimetric determination of
cell viability using the oxidationereduction indicator Alamar
Ò
Blue [18]. Late log-phase human monocytes were incubated
Ò
in RPMI medium containing 10% Alamar Blue supple-
mented with 10% foetal calf serum (Eurobio, Paris, France)
and a range of acridine concentrations was incorporated in du-
plicate cultures (final DMSO concentration less than 5%). Af-
ter a 72 h incubation period at 37 C with 5% CO , reduction
2
of Alamar Blue from blue to red was measured by absor-
bance monitoring at 570 and 630 nm. IC50-monocytes was de-
fined as the concentration of acridine required to induce
a 50% decrease of cell growth, corresponding to a 50% reduc-
as follows: SI ¼ IC50-monocytes/IC50-amastigotes
.
ꢂ
6.3.4. Intercalation to DNA
Ò
The capacities of proflavine derivatives to bind with DNA
were assessed by the DNAemethyl green bioassay [21], using
triphenyl methane methyl green dye that may reversibly inter-
act with DNA according to a reaction that can be followed
spectrophotometrically. Twenty milligrams of DNAemethyl
green (Sigma, St. Louis, MO, USA) were suspended in
100 mL of 0.05 M TriseHCl buffer, pH 7.5, containing
Ò
tion of alamar Blue as compared to the control culture.
6
.3.2. Antileishmanial activity against promastigotes
The effects of acridines on the growth of Leishmania pro-
ꢂ
7.5 mM of magnesium sulfate and stirred at 37 C for 24 h.
mastigotes were assessed by colorimetric determination of
parasite viability using the oxidationereduction indicator Ala-
mar Blue [19]. Antileishmanial activity was assessed on the
referenced strain L. infantum (MHOM/FR/78/LEM75). Pro-
Two hundred microlitres of the above solution were added
to each well and various concentrations of acridine derivatives
were added. The initial absorbance of each sample was mea-
sured at 630 nm. After a 24-hour incubation period in the
dark at room temperature, the final absorbance of the samples
was measured as above. The readings were corrected for the
initial absorbance and normalized as percentages of the un-
treated DNAemethyl green absorbance value. The IC50-DNA
corresponded to the concentration of chemical compounds
that induced a 50% decrease of DNA-related absorbance.
Ò
mastigotes in late log-phase were incubated in RPMI medium
Ò
(
without phenol red) containing 10% Alamar Blue supple-
mented with 12% foetal calf serum and a range of acridine
concentrations was aseptically incorporated into duplicate cul-
tures (final DMSO concentration less than 5%). Following
ꢂ
Ò
a 48 h incubation period at 25 C, Alamar Blue reduction
from blue to red was assessed by absorbance monitoring at
5
70 and 630 nm. IC50-promastigotes was determined as the con-
centration of acridine necessary to inhibit 50% of parasite
6.3.5. Statistical analysis
Correlations between cytotoxicity observed on human
monocytes or antileishmanial activity and DNA-binding
were performed by the rank correlation test of Spearman by
using the Statgraphic plus (V5) software (Manugistics Inc.,
USA).
Ò
growth, corresponding to a 50% reduction of alamar Blue
as compared to the control culture.
6
.3.3. Antileishmanial activity against intracellular
amastigotes
Intracellular amastigote cultures were performed in human
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