Synthesis and antileishmanial activities of 4,5-di-substituted acridines as compared to their 4-mono-substituted homologues

Article abstract of DOI:10.1016/j.bmc.2005.06.045

Newly synthesized 4,5-di-substituted acridines were assessed for in vitro antileishmanial activities as compared to those of their 4-mono-substituted homologues. Mono-substituted acridines exhibited a weak specificity for Leishmania parasites. Di-substitu

Full text of DOI:10.1016/j.bmc.2005.06.045

Bioorganic & Medicinal Chemistry 13 (2005) 5560–5568
Synthesis and antileishmanial activities of 4,5-di-substituted
acridines as compared to their 4-mono-substituted homologues
a,
b
Di Giorgio Carole, De M e´ o Michel, Chiron Julien, Delmas Florence, Nikoyan Anna,
c
a
b
*
b
Jean S e´ verine, Dumenil G e´ rard, Timon-David Pierre and Galy Jean-Pierre
b
a
c
a
Laboratoire de Parasitologie, Hygi e` ne et Zoologie, Facult e´ de Pharmacie, 27 Bd. Jean Moulin, 13385 Marseille Cedex 05, France
b
Laboratoire de Biog e´ notoxicologie et Mutagen e` se Environnementale, EA 1784, Facult e´ de Pharmacie,
2
7 Bd. Jean Moulin, 13385 Marseille Cedex 05, France
0
c
Laboratoire de M e´ thode et Valorisation de la Chimie Fine, Universit e´ d Aix-Marseille III, Site de Saint J e´ r oˆ me,
Avenue escadrille Normandie-Niemen, 13397 Marseille Cedex 20, France
Received 7 February 2005; revised 16 June 2005; accepted 22 June 2005
Available online 2 August 2005
Abstract—Newly synthesized 4,5-di-substituted acridines were assessed for in vitro antileishmanial activities as compared to those of
their 4-mono-substituted homologues. Mono-substituted acridines exhibited a weak specificity for Leishmania parasites. Di-substi-
tuted acridines, on the contrary, displayed interesting amastigote-specific activities through a mechanism of action that might not
involve intercalation to DNA. This antileishmanial property, associated with a low antiproliferative activity towards human cells,
led to the identification of a new class of promising acridine derivatives such as 4,5-bis(hydroxymethyl)acridine with a nonclassical
mechanism of action based on the inhibition of Leishmania internalization within macrophages. In the meantime, the effects of
experimental lighting on the biological properties of acridines were assessed: experimental lighting did not significantly improve
the antileishmanial activity of the compounds since it produced a greater toxicity against human cells.
ꢀ
2005 Elsevier Ltd. All rights reserved.
1
. Introduction
injected pentavalent antimony has been successfully
used, but since the 1990s, Leishmania parasites have
developed resistances. Amphotericin B and the oral
anticancer drug miltefosine are considered at the mo-
4
,5
For hundreds of years, leishmaniases have been the
cause of death among millions of people throughout
the world. Since the 1990s, as a consequence of agro-
industrial development, a significant expanse of Leish-
mania-endemic areas has been observed, correlated with
a sharp increase of cases. AIDS and other immuno-
suppressive conditions have also enhanced the risk of
Leishmania–HIV co-infected people and contributed to
the appearance of new severe clinical forms of the dis-
ease. Nowadays, leishmaniases are spread over five
continents and are endemic in the tropical and subtrop-
ical regions of 88 countries. Among all leishmaniases,
the visceral form also known as kala-azar is the most se-
vere, since it produces, if untreated, a mortality rate of
almost 100% independently to the immunological status
6
ment to be the best second-line therapeutic solutions.
Nevertheless, they do not represent a safe treatment in
all clinical cases and necessitate the research of new
1
,2
6
antileishmanial molecules.
It has been established for many years that the planar
structure of tricyclic rings conferred to acridine deriva-
tives the ability to intercalate in DNA and to interfere
with various metabolic processes in both prokaryotic
3
1
7,8
and eukaryotic cells. As a consequence, various natu-
ral and synthetic compounds of the acridine family have
been selected for antibacterial or anticancer chemother-
9
apy. Recent developments in the biology of protozoa
have also demonstrated that acridines could exert a
2
of the patient. For decades, treatment of kala-azar with
1
0
11
powerful toxicity toward Plasmodium, Trypanosoma
1
2
and Leishmania parasites. However, their mechanisms
of action toward protozoa are still poorly understood,
rendering necessary the synthesis of new acridine series
and the study of their antiparasitic profile.
Keywords: Leishmaniasis; Acridines; Bi-functional acridines; Photo-
activation.
*
Corresponding author. Tel.: +33 (0)4 91 83 55 44; fax: +33 (0)4 91 80
6 12; e-mail: carole.digiorgio@pharmacie.univ-mrs.fr
2
0
968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.06.045

D. G. Carole et al. / Bioorg. Med. Chem. 13 (2005) 5560–5568
5561
For this purpose, we investigated the antiparasitic prop-
erties of newly synthesized bifunctional acridines by
comparing their antileishmanial activities to those of
their mono-substituted homologues. In the meantime,
according to the statement that acridine dyes were
starting material. The best solvent for these syntheses
was CH Cl that permitted an optimal solubility for
all the components. However, with the addition of
TEA alone, weak reaction yields were obtained. In order
to resolve this problem, we added DMAP as an activa-
tor of acid function.
2
2
1
3
photosensitizers and demonstrated photo-enhanced
1
4
bactericidal and antitumoral activities, we explored
the effects of simulated sunlight on the biological prop-
erties of both the series of compounds.
Each compound structure was characterized by 1D and
2D NMR sequences and their purity was confirmed by
TLC, microanalysis, and HPLC.
2
. Results and discussion
2.2. Biological activity tests without irradiation
2
.1. Chemistry
Antileishmanial activity was assessed on the referenced
strain L. infantum (MHOM/FR/78/LEM75) in both
its promastigote and its intracellular amastigote
forms. Cytotoxicity was assessed on human monocytes
(THP1 cell line). Among 4-substituted acridines (A
and B series), compounds A66, A27, and A60 appeared
highly active toward human cells (IC50 = 3.2, 10.1–
13.4 lM); only compound A27 exerted a strong inhibito-
ry effect on the promastigote form of the parasite
(IC50 = 1.7 lM), while two compounds, A33 and A75,
produced an interesting selective antiamastigote activity
(SI > 30). Concerning di-substituted acridines, none of
the tested derivatives showed strong antiproliferative
properties on human monocytes and promastigotes.
Mono-substituted acridines (A and B series) were pre-
1
5
pared during a previous work. 4,5-Bis(aminoethyl)-
0
acridine A 12 was prepared using acridine as a starting
1
6
material according to a previously described method.
0
Compounds of the A series were synthesized according
0
to classical amide formation methods by reacting A 12
hydrochloride with various carboxylic acid and acyl
chloride derivatives as described in Figure 1. The parent
molecule was first solubilized into acetone and the mix-
ture was neutralized with concentrated NaOH. Under
these experimental conditions, the generation of amide
bindings was faster than the concomitant hydrolysis of
acyl chloride functions and the excess of NaOH neutral-
ized the HCl formed during the reaction.
0
0
Nevertheless, some of these compounds (B 52, A 12,
0
0
0
0
A 21, B 57, A 2, and A 11) expressed interesting amasti-
gote-specific activities, and more particularly compound
0
0
Compounds of the B series were prepared in a similar
0
B 52, 4,5-bis(hydroxymethyl)acridine, that displayed a
strong inhibitory activity on the intracellular amastigote
way by using 4,5-bis(hydroxymethyl)acridine B 52 as
Figure 1. Synthesis of 4,5-di-substituted acridines.

5562
D. G. Carole et al. / Bioorg. Med. Chem. 13 (2005) 5560–5568
form of the parasite (IC50 = 0.6 lM), leading to a selec-
tive index over than 200.
amastigote forms of the parasite than di-substituted acri-
dines (P < 0.01), and nitrogen-bearing derivatives were
significantly more active than their oxygen-bearing
homologues (P < 0.01).
2
.3. Biological activity tests after irradiation
0
Irradiation procedure was performed with a solar simula-
tor equipped with a xenon arc lamp. The results presented
in Tables 1 and 2 revealed that almost all the acridine
compounds exerted photo-inducible antiproliferative
properties; however, experimental illumination neither
enhanced antipromastigote abilities nor improved selec-
tive antiparasitic activity against Leishmania amastigotes.
2.5. Additional experiments on B 52
Complementary experiments were performed for study-
0
ing the mechanism of B 52 antileishmanial action. NO
production was analyzed in adherent macrophages and
inhibition of parasite internalization within macro-
phages was assessed by infecting adherent macrophages
with promastigotes previously treated with various con-
centrations of B 52. The results showed that B 52 did
not increase NO production by macrophages (data not
shown). On the contrary, the compound prevented mac-
rophage infection by inhibiting promastigote internali-
zation within human macrophages (Fig. 2).
0
0
2
.4. Statistical analysis
Multifactor ANOVA was used for evaluating the effects
of the light and the chemical structure of the compounds
on their biological properties (Statgraphics 5.0 plus soft-
ware, Statistical Graphics Corporation, USA). Cytotox-
icity towards human cells poorly depended on the
chemical nature of the substituted chains (P > 0.05); how-
ever, it was significantly enhanced by experimental light-
ing (P < 0.001). On the contrary, antileishmanial
activity greatly depended on the nature of the substituted
chains (P < 0.001), while it was poorly affected by lighting
3. Conclusion
8
,9
Due to their capacity to interact with macromolecules,
acridine chromophores have been widely synthesized
and assessed for their pharmacological activities
including antibacterial, anticancer, and antiparasitic
(P > 0.05): mono-substituted compounds were signifi-
cantly more efficient on both the promastigote and the
1
0–12
properties.
The results observed in the present study
Table 1. Structures and biological properties of mono-substituted acridines
R
H
Ring No.
Antiproliferative activity on human monocytes
Antileishmanial activity IC50 ± SD (lM)
IC50 ± SD (lM)
Without photo-activation With photo-activation
Without photo-activation
PRO AMA SI
1.7 ± 0.4 3.4 ± 0.5 2.9
>200
With photo-activation
PRO
AMA
SI
A
B
A27
B48
10.1 ± 1.2
18.4 ± 2.4
2.4 ± 0.8
5.3 ± 0.9
6.3 ± 1.1 4.8 ± 0.7
43.3 ± 4.6 Tox
—
—
32.7 ± 3.4
—
A
B
A66
B68
3.2 ± 0.5
28.6 ± 3.1
0.8 ± 0.1
13.2 ± 1.5
45.2 ± 6.4
158.4 ± 8.9
26.7 ± 3.8
153.1 ± 9.7
—
—
13.1 ± 2.1 Tox
0.7 ± 0.2 Tox
—
—
A
B
A60
B69
13.4 ± 2.1
16.5 ± 1.3
2.4 ± 0.6
175.8 ± 10.2
29.1 ± 2.6
6.8 ± 1.9
2.6 ± 0.7
1.9 42.7 ± 4.6 Tox
—
18.2 ± 1.9
6.3 22.1 ± 1.4 4.1 ± 0.4
4.4
A
B
A37
B50
20.8 ± 5.4
>200
69.6 ± 2.1
6.1 ± 1.6
45.1 ± 8.1 13.2 ± 6.1
76.7 ± 12.3 >200
1.5 29.4 ± 3.5 9.4 ± 2.1
2.8 ± 0.3 Tox
7.4
—
—
A
B
A32 >200
B61 124.7 ± 11.4
9.4 ± 2.4
1.3 ± 0.1
65.3 ± 6.7
55.1 ± 6.6
78.7 ± 8.9
>200
4.6 29.5 ± 3.4 Tox
2.7 ± 0.5 Tox
—
—
—
A
B
A31
B67
85.1 ± 9.4
>200
42.8 ± 6.1
8.1 ± 1.9
>200
61.6 ± 9.2
10.1 ± 2.1 8.4 22.9 ± 4.5 3.1 ± 0.2 13.8
27.1 ± 3.8 14.5 2.42 ± 0.4 3.3 ± 0.1 2.4
A
B
A33 >200
B59 136.1 ± 15.4
32.8 ± 5.1
8.7 ± 0.9
32.6 ± 8.5
29.6 ± 5.4
9.4 ± 2.3 42.8 26.4 ± 3.6 Tox
7.4 ± 2.2 18.4
—
6.3 ± 1.4 8.1 ± 3.2
1.1
A
B
A75 143.2 ± 20.1
B71 153.2 ± 22.4
4.8 ± 1.1
10.4 ± 1.7
34.6 ± 3.1
65.9 ± 3.5
4.7 ± 0.6 30.4 75.1 ± 5.6 9.4 ± 1.4
>200 9.6 ± 1.4 Tox
—
—
PRO: promastigotes; AMA: amastigotes; Tox: toxicity higher than antileishmanial activity; SI: selectivity index; SD: Standard deviations.

Table 2. Structures, physicochemical, and biological properties of di-substituted acridines
R
H
Ring
No.
Antiproliferative activity on human monocytes
Antileishmanial activity IC50 ± SD (lM)
IC50 ± SD (lM)
Without photo-activation
With photo-activation
Without photo-activation
AMA
3.4 ± 0.4
With photo-activation
AMA
0.6 ± 0.03
PRO
6.6 ± 1.9
>200
SI
PRO
1.4 ± 0.2
>200
SI
0
0
A
A 12
35.5 ± 2.1
>200
5.2 ± 0.9
10.4
>200
8.6
1.0
0
0
B
B 52
24.3 ± 3.1
0.6 ± 0.05
23.6 ± 4.1
0
0
0
0
A
A 21
B 84
48.2 ± 5.1
156.4 ± 12.4
4.5 ± 0.7
12.6 ± 2.7
4.6 ± 0.9
6.5 ± 0.9
7.4
7.3 ± 0.9
8.6 ± 1.1
Tox
2.3 ± 0.4
—
5.4
B
>200
>200
—
0
0
0
A
A 22
23.5 ± 3.7
34.1 ± 2.9
15.4 ± 1.8
18.6 ± 0.7
11.5 ± 2.1
11.6 ± 0.4
>200
>200
—
—
30.4 ± 5.4
11.2 ± 0.7
>200
Tox
—
—
0
B
B 81
0
0
0
0
A
A 1
B 57
98.1 ± 10.4
145.1 ± 9.9
65.3 ± 4.1
10.2 ± 2.5
3.6 ± 0.4
>200
17.2 ± 4.1
5.2 ± 1.4
5.6
27.9
41.2 ± 4.8
>200
46.5 ± 7.1
Tox
1.4
—
B
0
0
0
A
A 2
18.5 ± 9.9
26.6 ± 3.9
5.3 ± 1.2
5.7 ± 0.5
>200
1.8 ± 0.1
>200
10.2
—
2.7 ± 0.1
Tox
—
—
0
B
B 78
25.6 ± 4.7
155.2 ± 10.4
>200
0
0
0
0
A
A 3
B 63
94.1 ± 8.4
14.7 ± 2.1
75.3 ± 4.1
12.3 ± 0.4
29.2 ± 4.4
>200
36.5
>200
2.5
—
136.4 ± 9.8
>200
21.4 ± 1.5
Tox
3.5
—
B
0
0
0
0
A
A 4
B 62
124.1 ± 0.7
21.2 ± 4.1
86.5 ± 0.4
26.3 ± 0.7
62.6 ± 1.1
>200
>200
66.8 ± 1.4
—
—
>200
>200
>200
Tox
—
—
B
0
0
0
0
A
A 11
B 76
52.2 ± 6.7
31.4 ± 2.8
36.6 ± 2.4
18.2 ± 0.8
8.7 ± 1.4
>200
5.3 ± 0.1
46.5 ± 5.8
1.14
>200
>200
Tox
Tox
—
—
B
—
PRO: promastigotes; AMA: amastigotes; Tox: toxicity higher than antileishmanial activity; SI: selectivity index; SD : Standard deviations.

5564
D. G. Carole et al. / Bioorg. Med. Chem. 13 (2005) 5560–5568
4. Experimental
4
.1. General procedures
All reagents were of analytical grade, dried and purified
when necessary. Acridine derivatives were dissolved in
sterile dimethyl sulfoxide (analytical grade, Sigma, St
Louis, Mo, USA) and stored frozen at ꢀ70 ꢁC until
used.
4
4
.2. Chemistry
.2.1. 4,5-Bis(aminomethyl)acridine (A 12). 4,5-Bis(azi-
0
domethyl)acridine obtained from acridine according to
the methodology previously described by Hess and
1
6
Stewart was dissolved into dioxane/ethanol (30 mL,
:5, v:v) with Pd/C (100 mg, 10%). After a 4-h agitation
period at room temperature with H , the mixture was fil-
5
2
tered and washed with ethanol. The filtrate was evapo-
rated, dissolved into CHCl , and filtered until final
3
1
evaporation to give a brown oil (805 mg, 98%).
H
0
NMR (300 MHz,CDCl , 25 ꢁC): d = 2.03 (s, 4H, 2 H),
.50 (s, 4H, H-1 ), 7.42 (dd, J = 8.5, 6.7 Hz, 2H, H-2),
.64 (dd, J = 6.7, 0.7 Hz, 2H, H-3) 7.82 (dd, J = 8.5,
.2 Hz, 2H, H-1), 8.65 (s, 1H, H-9) ppm. C NMR
0
3
Figure 2. Effects of B 52 on macrophage infection by Leishmania
promastigotes (1.a.) and parasite proliferation within macrophages
0
4
7
1
(1.b.).
1
3
(
1
75 MHz, CDCl , 25 ꢁC): d = 44.42 (C-1), 125.56 (C-2),
3
26.45 (C-9a), 127.05 (C-1), 127.60 (C-3), 136.31 (C-9),
clearly confirmed that various 4-substituted and 4,5-di-
substituted acridines could exert interesting antileishma-
nial activities. Antiproliferative abilities towards human
cells were shown to vary according to the length of the
141.14 (C-4), 146.50 (C-4a) ppm. Anal. Calcd
(C H N ): C, 75.92; H, 6.37; N, 17.71. Found: C,
75.71; H, 6.35; N, 17.65.
1
5
15
3
0
0
4
- or 4,5-substituted groups rather than the nature of
A 12 hydrochloride was obtained by dissolving A 12
into 5 mL MeOH and incorporating the mixture into
the rings: mono-substituted acridines bearing a linear
side chain exerted stronger activity than compounds
bearing aryl chains, indicating that conformational
mechanisms could play an important role in the interac-
100 mL HCl saturated Et O. After an overnight incuba-
2
tion at room temperature under CaCl atmosphere, the
2
precipitate was filtered and washed with Et O to obtain
2
yellow powder (1.053 g).
1
7
tions of the compounds with macromolecules. Antile-
ishmanial activities, on the contrary, depended on both
the nature of the rings and the number of substituted
groups. Mono-substituted acridines demonstrated
stronger antipromastigote activities than their di-substi-
tuted homologues, and compounds bearing a nitrogen
bond were more active than their oxygen bond bearing
homologues. Di-substituted acridines were weakly ac-
tive on the promastigote form of the parasite. These
compounds appeared too cluttered on both sides to in-
volve a strong intercalative binding with DNA. As a
consequence, they did not demonstrate strong antipro-
liferative activity on tumoral cells. Nevertheless, some
4.2.2.
4,5-Bis[(4-chlorobutylamido)-N-methyl]acridine
0
0
(A 21). A 12 (500 mg, 1.60 mmol) was dissolved into
20 mL acetone at 0 ꢁC and neutralized with 5 mL NaOH
1 N. A solution of 4-chlorobutyl chloride (500 mg,
3.55 mmol) in Me CO (10 mL) was added at 0 ꢁC and
2
the mixture was incubated at room temperature for
3 h. Then, the solvent was evaporated and the residual
was dissolved into water/MeOH (20 mL, 8:2, v:v) under
ultrasounds. The precipitate was filtered and washed
with water to obtain white powder (681 mg, yield
1
95%). H NMR (300 MHz, DMSO-d , 25 ꢁC): d = 2.01
6
0
0
0
of these acridines, such as compounds B 52, A 12,
(quint, J = 6.10 Hz, 4H, H-4 ), 2.40 (t, 4H, J = 6.9 Hz,
0
0
0
0
0
0
A 21, B 57, A 2, and A 11, exhibited interesting amasti-
gote-specific properties, indicating that they could exert
another interaction mechanism with Leishmania parasite
that does not imply direct DNA intercalation. Comple-
4H, H-3 ), 3.67 (t, J = 6.11 Hz, 4H, H-5 ), 5.06 (d,
0
J = 5.6 Hz, 4H, H-1 ) 7.59 (t, J = 7.4 Hz, 2H, H-2),
7.69 (d, J = 6.3 Hz, 2H, H-3), 8.06 (d, J = 8.4 Hz, 2H,
0
H-1), 8.48 (t, J = 5.6 Hz, 2H, H-2 ), 9.09 (s, 1H, H-9)
0
13
mentary experiments concerning B 52 confirmed this
ppm. C NMR (75 MHz, DMSO-d , 25 ꢁC): d = 28.53
6
0
0
0
0
statement, since they established that subtoxic concen-
trations of the compound prevented macrophages from
Leishmania infection by a mechanism of action that sig-
nificantly reduced parasite internalization. On this basis,
additional in vitro and in vivo experiments should be
performed in order to confirm the antileishmanial prop-
erties of this new class of compounds and to better elicit
this mechanism of action.
(C-4 ), 32.62 (C-3 ), 39.08 (C-1 ), 45.27 (C-5 ), 125.78
(C-2), 126.05 (C-9a), 127.41 (C-1), 127.73 (C-3), 136.75
(C-9), 137.00 (C-4), 145.73 (C-4a), 171.58 (C-2a) ppm.
Anal. Calcd (C H Cl N O ): C, 61.89; H, 5.65; N,
2
3
25
2
3
2
9.41. Found: C, 62.11; H, 5.63; N, 9.37.
0
4.2.3. 4,5-Bis[(acrylamido)-N-methyl]acridine (A 22).
A 12 hydrochloride (500 mg, 1.60 mmol) was dissolved
0

D. G. Carole et al. / Bioorg. Med. Chem. 13 (2005) 5560–5568
5565
1
9.14 (s, 1H, H-9) ppm. C NMR (50 MHz, DMSO-
3
into 5 mL water, neutralized with NaOH 5 N and
extracted with CH Cl (3 · 10 mL). The organic phase
0
d , 25 ꢁC): d = 39.69 (C-1 ), 115.29 (d, J = 22.4 Hz, C-
2
2
6
0
was dried with MgSO and evaporated. The residue
4
4 ) 125.28 (C-2), 125.99 (C-9a), 126.84 (C-3), 127.10
0
was dissolved into 20 mL CH Cl and TEA (0.7 mL,
2
(C-1), 130.09 (d, J = 9.7 Hz, C-3 ), 131.06 (d,
0
2
4
(
98 mmol) at 0 ꢁC. Then, a solution of acryloyl chloride
579 mg, 6.40 mmol) was incorporated into 5 mL
CH Cl under N atmosphere and the mixture was incu-
J = 4.2 Hz, C-3 a), 136.74 (C-4), 136.74 (C-9), 145.68
0
0
(C-4a), 163.97 (d, J = 246.5 Hz, C-4 a), 165.59 (C-2a )
ppm. Anal. Calcd (C H F N O ): C, 72.34; H, 4.40;
N, 8.73. Found: C, 72.45; H, 4.28; N, 8.72.
2
2
2
29 21
2
3
2
bated for 24 h at room temperature. The solvent was
evaporated, and the residue was dissolved into 5 mL
Me CO and placed into 30 mL water. The precipitate
4.2.6.
4,5-Bis[(4-chlorobenzamido)-N-methyl]acridine
2
0
0
was filtered and washed with H O/MeOH (9:1, v:v) to
2
(A 3). A 12 hydrochloride (200 mg, 0.65 mmol) was dis-
solved into 25 mL acetone at 0 ꢁC and neutralized with
5 mL NaOH 1 N. 4-Chlorobenzoyl chloride (237 mg,
1.36 mmol) in 5 mL acetone was added at 0 ꢁC and the
mixture was incubated at room temperature for 3 h.
The solvent was evaporated and the residue was mixed
1
obtain white powder (300 mg, yield 57%). H NMR
(
4
300 MHz, DMSO-d , 25 ꢁC): d = 5.15 (d, J = 5.70 Hz,
6
0
0
H, H-1 ), 5.64 (dd, J = 10.1, 2.2 Hz, 2H, H-4 ), 6.16
0
(
1
dd, J = 17.2, 2.2 Hz, 2H, H-5 ), 6.40 (dd, J = 17.2,
0
0.1 Hz, 2H, H-3 ), 7.59 (t, J = 7.4 Hz, 2H, H-2), 7.70
d, J = 6.3 Hz, 2H, H-3), 8.08 (d, J = 8.4 Hz, 2H, H-1),
(
with H O/MeOH (20 mL, 5:5, v:v) under ultrasounds.
2
0
8
.67 (t, J = 5.7 Hz, 2H, H-2 ), 9.11 (s, 1H, H-9) ppm.
C NMR (75 MHz, DMSO-d , 25 ꢁC): d = 39.22
The precipitate was filtered and washed successively
with water and MeOH to obtain white powder
1
3
6
0
0
1
(652 mg, yield 92%). H NMR (300 MHz, DMSO-d ,
(
1
C-1 ), 125.55 (C-4 ), 125.83 (C-2), 126.14 (C-9a),
0
6
0
27.62 (C-1), 128.25 (C-3), 131.98 (C-3 ), 136.72 (C-9),
0
25 ꢁC): d = 5.29 (d, J = 5.5 Hz, 4H, H-1 ), 7.56 (m, 4H,
H-4 ), 7.60 (dd, J = 6.2, 8.3 Hz, 2H, H-2), 7.73 (d,
J = 6.2 Hz, 2H, H-3), 7.97 (m, 4H, H-3 ), 8.10 (d,
J = 8.3 Hz, 2H, H-1), 9.15 (s, 1H, H-9), 9.18 (t,
0
136.90 (C-4), 145.81 (C-4a), 165.08 (C-2a ) ppm. Anal.
Calcd (C H N O ): C, 73.03; H, 5.54; N, 12.17.
Found: C, 72.75; H, 5.56; N, 12.22.
0
2
1
19
3
2
0
13
J = 5.7 Hz, 2H, H-2 ) ppm.
DMSO-d , 25 ꢁC): d = 39.97 (C-1 ), 125.82 (C-2),
C NMR (50 MHz,
0
0
0
4.2.4. 4,5-Bis[(benzamido)-N-methyl]acridine (A 1). A 12
hydrochloride (500 mg, 1.60 mmol) was dissolved into
6
126.12 (C-9a), 127.49 (C-3), 127.78 (C-1), 128.61
0
0
0
5
1
0 mL acetone at 0 ꢁC and neutralized with 7 mL NaOH
N. Benzoyl chloride (492 mg, 3.35 mmol) in 5 mL ace-
(C-3 ), 129.45 (C-4 ), 133.45 (C-3a ), 136.25 (C-4),
0
136.25 (C-4 a), 136.68 (C-9), 145.80 (C-4a), 165.75
0
tone was added at 0 ꢁC and the mixture was incubated at
room temperature for 3 h. The solvent was evaporated
(C-2a ) ppm. Anal. Calcd (C H Cl N O ): C, 67.71;
3
2
9
21
2
2
H, 4.11; N, 8.17. Found: C, 67.82; H, 4.03; N, 8.05.
and the residue was mixed with H O/MeOH (20 mL,
2
5:5, v:v) under ultrasounds. The precipitate was filtered
and washed successively with water and MeOH to ob-
4.2.7. 4,5-Bis[(4-methoxybenzamido)-N-methyl]acridine
0
0
(A 4). A 12 hydrochloride (200 mg, 0.65 mmol) was dis-
solved into 25 mL acetone at 0 ꢁC and neutralized with
5 mL NaOH 1 N. 4-Methoxybenzoyl chloride (232 mg,
1.36 mmol) in 5 mL acetone was added at 0 ꢁC and the
mixture was incubated at room temperature for 3 h.
The solvent was evaporated and the residue was mixed
1
tain white powder (652 mg, yield 92%). H NMR
(
300 MHz, DMSO-d , 25 ꢁC): d = 5.31 (d, J = 5.70 Hz,
6
0
0
0
4
H, H-1 ), 7.49 (m, 4H, H-4 ), 7.52 (m, 2H, H-5 ), 7.61
dd, J = 8.1, 6.8 Hz, 2H, H-2), 7.73 (d, J = 6.8 Hz, 2H,
(
H-3), 8.00 (d, J = 6.8 Hz, 4H, H-3 ), 8.10 (d,
0
0
J = 8.1 Hz, 2H, H-1), 9.12 (t, J = 5.7 Hz, 2H, H-2 ),
9
d , 25 ꢁC): d = 39.92 (C-1 ), 125.78 (C-2), 126.09 (C-
9
with H O/MeOH (20 mL, 5:5, v:v) under ultrasounds.
2
1
.15 (s, 1H, H-9) ppm. C NMR (50 MHz, DMSO-
3
The precipitate was filtered and washed successively
with water and MeOH to obtain white powder
0
6
0
1
(300 mg, yield 95%). H NMR (200 MHz, DMSO-d ,
a), 127.39 (C-3*), 127.51 (C-3 ), 127.65 (C-1*), 128.54
0
6
0
0
0
(
C-4 ), 131.41 (C-5 ), 134.75 (C-3a ), 136.86 (C-4),
0
25 ꢁC): d = 3.81 (s, 6H, H-5 ), 5.29 (d, J = 5.5 Hz, 4H,
H-1 ), 7.02 (m, 4H, H-4 ), 7.59 (dd, J = 6.2, 8.3 Hz,
2H, H-2), 7.72 (d, J = 6.2 Hz, 2H, H-3), 7.97 (m, 4H,
0
0
136.86 (C-9) 145.79 (C-4a), 166.81 (C-2a ) ppm. Anal.
Calcd (C H N O ): C, 78.18; H, 5.20; N, 9.43. Found:
C, 78.25; H, 5.15; N, 9.51.
2
9
23
3
2
0
H-3 ), 8.08 (d, J = 8.3 Hz, 2H, H-1), 8.99 13(t,
0
J = 5.7 Hz, 2H, H-2 ), 9.11 (s, 1H, H-9) ppm.
C
0
4
(
.2.5.
0
4,5-Bis[(4-fluorobenzamido)-N-methyl]acridine
A 2). A 12 hydrochloride (500 mg, 1.60 mmol) was dis-
NMR (75 MHz, DMSO-d , 25 ꢁC): d = 39.80 (C-1 ),
6
0
0
0
55.52 (C-5 ), 113.75 (C-4 ), 125.82 (C-2), 126.12 (C-9a),
0
solved into 50 mL acetone at 0 ꢁC and neutralized with
126.92 (C-3a ), 127.37 (C-3), 127.66 (C-1), 129.31 (C-
0
7
3
mL NaOH 1 N. 4-Fluorobenzoyl chloride (533 mg,
.36 mmol) in 5 mL acetone was added at 0 ꢁC and the
3 ), 137.11 (C-4), 137.11 (C-9), 145.84 (C-4a), 161.79
0
0
(C-4 a),
166.24
(C-2a )
ppm.
Anal.
Calcd
(C H N O ): C, 73.65; H, 5.38; N, 8.31. Found: C,
mixture was incubated at room temperature for 3 h.
The solvent was evaporated and the residue was mixed
with H O/MeOH (20 mL, 5:5, v:v) under ultrasounds.
3
1
27
3
4
73.73; H, 5.28; N, 8.42.
2
The precipitate was filtered and washed successively
with water and MeOH to obtain white powder
4.2.8. 4,5-Bis[(4-dimethylaminobenzamido)-N-methyl]ac-
0
0
ridine (A 11). A 12 hydrochloride (500 mg, 1.63 mmol)
was dissolved into 20 mL acetone at 0 ꢁC and neutral-
ized with 5 mL NaOH 1 N. 4-Dimethylaminobenzoyl
chloride (618 mg, 3.37 mmol) in 10 mL acetone was add-
ed at 0 ꢁC and the mixture was incubated at room tem-
perature for 3 h. The solvent was evaporated and the
1
740 mg, yield 96%). H NMR (300 MHz, DMSO-d ,
(
2
4
6
0
5 ꢁC): d = 5.31(d, J = 5.60 Hz, 4H, H-1 ), 7.32 (m,
H, H-4 ), 7.61 (t, J = 7.60 Hz, 2H, H-2), 7.73 (d,
0
0
J = 6.8 Hz, 2H, H-3), 8.04 (m, 4H, H-3 ), 8.09 (d,
0
J = 8.3 Hz, 2H, H-1), 9.13 (t, J = 5.6 Hz, 2H, H-2 ),

5566
D. G. Carole et al. / Bioorg. Med. Chem. 13 (2005) 5560–5568
residue was mixed with H O/MeOH (20 mL, 5:5, v:v)
2
pickle and dried with MgSO . The solvent was evaporated
4
under ultrasounds. The precipitate was filtered and
washed successively with water and MeOH to obtain
and the residue was purified by silicagel chromatography
(CH Cl /EtOAc, 10:0 and 9:1, v:v) to obtain brown oil
(504 mg, yield 90%). H NMR (300 MHz, CDCl ,
2
2
1
1
white powder (542 mg, yield 64%).
(
H
NMR
0
3
0
300 MHz, DMSO-d , 25 ꢁC): d = 2.96 (s, 12H, H-5 ),
25 ꢁC): d = 2.18 (quint, J = 6.8 Hz, 5H, H-3 ), 2.65 (t,
J = 7.2 Hz, 4H, H-2 ), 3.64 (t, J = 6.3 Hz, 4H, H-4 ),
6
0
0
0
0
5
.27 (d, J = 5.7 Hz, 4H, H-1 ), 6.71 (m, 4H, H-4 ), 7.59
dd, J = 8.4, 6.3 Hz, 2H, H-2), 7.71 (d, J = 6.3 Hz, 2H,
0
(
H-3), 7.84 (m, 4H, H-3 ), 8.08 (d, J = 8.4 Hz, 2H, H-
5.97 (s, 4H, H-1 ), 7.52 (dd, J = 8.5, 6.9 Hz, 2H, H-2),
7.76 (d, J = 6.9 Hz, 2H, H-3), 7.94 (d, J = 8.5 Hz, 2H,
0
0
13
H-1), 8.72 (s, 1H, H-9) ppm. C NMR (75 MHz, CDCl ,
1
ppm.
), 8.76 (t, J = 5.7 Hz, 2H, H-2 ), 9.13 (s, 1H, H-9)
NMR (100 MHz, DMSO-d , 25 ꢁC):
3
1
3
0
0
0
C
25 ꢁC): d = 27.69 (C-3 ), 31.29 (C-2 ), 44.14 (C-4 ), 63.09
(C-1 ), 125.44 (C-2), 126.24 (C-9a), 128.13 (C-3), 128.48
6
0
0
0
0
d = 39.58 (C-1 ), 39.82 (C-5 ), 110.97 (C-4 ), 121.26 (C-
0
3
a ), 125.71 (C-2), 126.02 (C-9a), 127.18 (C-3*), 127.65
0
(C-1), 134.37 (C-4), 136.14 (C-9), 145.97 (C-4a), 172.53
0
(
C-1*), 128.71 (C-3 ), 137.35 (C-4), 136.76 (C-9),
0
(C-2a ) ppm. Anal. Calcd (C H Cl NO ): C, 61.62; H,
2
3
23
2
4
0
145.78 (C-4a), 152.24 (C-4a ), 166.55 (C-2a ) ppm. Anal.
Calcd (C H N O ): C, 74.55; H, 6.26; N, 13.17.
Found: C, 74.26; H, 6.28; N, 13.12.
5.17; N, 3.12. Found: C, 61.38; H, 5.19; N, 3.13.
3
3
33
5
2
0
0
4.2.12. Acridin-4,5-yldimethyl-bis-acrylate (B 81). B 52
(300 mg, 1.25 mmol) was dissolved into 20 mL Me CO.
2
4
4
1
.2.9. 4,5-Bis(bromomethyl)acridine. BMME (6.08 g,
4.64 mmol) was added to a solution of acridine (2 g,
1.16 mmol) in H SO (25 mL) at 50 ꢁC. The mixture
TEA (0.5 mL, 3.60 mmol), DMAP (383 mg, 3.13 mmol),
and acryloyl chloride (249 mg, 2.75 mmol) diluted into
20 mL Me CO were added at 0 ꢁC under drying atmo-
2
4
2
was maintained under nitrogen for 12 h and refreshed
in ice for an hour. The precipitate was filtered and dis-
solved into CHCl , and the organic phase was dried with
MgSO . After evaporation of the solvent, the residue
4
was recrystallized into anhydrous EtO to obtain yellow
2
powder (2.49 g, yield 64%).
3
J = 8.5, 6.8 Hz, H-2), 7.89 (d, 2H, J = 6.8 Hz, H-3),
sphere (CaCl ), and the mixture was incubated for
2
24 h at room temperature. Then, the mixture was trans-
ferred into 100 mL basic water (5 mL NaOH, 5 N) for
1 h. The precipitate was filtered and washed with water.
The solid layer was purified by silicagel chromatography
(CH Cl /EtOAc, 10:0 and 9:1, v:v) to obtain solid yel-
3
1
H
NMR (CDCl3,
2
2
0
1
00 MHz): d = 5.39 (s, 4H, H-1 ), 7.46 (dd, 2H,
low crystal (295 mg, yield 68%). H NMR (300 MHz,
CDCl , 25 ꢁC): d = 5.86 (dd, J = 10.3, 1.5 Hz, 2H, H-
3 ), 6.07 (s, 4H, H-1 ), 6.24 (dd, J = 17.4, 10.3 Hz, 2H,
3
0
0
7
.93 (d, 2H, J = 8.5 Hz, H-1), 8.70 (s, 1H, H-9) ppm.
0
1
3
0
0
C NMR (CDCl , 75 MHz): d = 27.01 (C-1 ), 126.94
H-2 ), 6.50 (dd, J = 17.4, 1.5 Hz, 2H, H-4 ), 7.52 (dd,
J = 8.4, 6.9 Hz, 2H, H-2), 7.78 (dd, J = 6.9, 1.1 Hz,
2H, H-3), 7.96 (d, J = 8.4 Hz, 2H, H-1), 8.76 (s, 1H,
3
(
(
(
C-2), 127.59 (C-9a), 128.81 (C-1), 131.55 (C-3), 137.83
C-4), 139.45 (C-9), 151.22 (C-4a) ppm. Anal. Calcd
C H N ): C, 49.35; H, 3.04; N, 3.84. Found: C,
1
3
H-9) ppm.
C
NMR (75 MHz, CDCl3, 25 ꢁC)
1
5
11
7
0
4
9.49; H, 3.03; N, 3.83.
d = 63.04 (C-1 ), 125.48 (C-2), 126.27 (C-9a), 128.05
(C-2 *), 128.36 (C-3*), 128.54 (C-1*), 130.97 (C-3 ),
134.52 (C-4), 136.11 (C-9), 146.02 (C-4a), 166.15 (C-
0
0
0
4.2.10. 4,5-Bis(hydroxymethyl)acridine (B 52). 4,5-
Bis(bromomethyl)acridine (2 g, 5.48 mmol) was dis-
0
2a ) ppm. Anal. Calcd (C H NO ): C, 72.61; H, 4.93;
2
1
17
4
solved into 60 mL dioxane and CaCO (10 g, 99.91 mmol)
3
N, 4.03. Found: C, 72.91; H, 4.89; N, 4.00.
in 60 mL water was added. The mixture was filtered, the
solvent was evaporated, the residue was dissolved into
0
0
4.2.13. Acridin-4,5-yldimethyl-bis-benzoate (B 57). B 52
(300 mg, 1.25 mmol) was dissolved into 20 mL CH Cl .
2
MgSO . The solid residue was purified by silicagel chro-
00 mL CH Cl and the organic phase was dried with
2
2
2
2
TEA (0.5 mL, 3.60 mmol), DMAP (383 mg, 3.13 mmol),
and benzoyl chloride (370 mg, 2.63 mmol) diluted into
10 mL CH Cl were added at 0 ꢁC under drying atmo-
4
matography (CH Cl /EtOAc, 5:5, v:v) to obtain a yellow
2
2
1
powder (1.21 g, yield 92%). H NMR (CDCl , 300 MHz):
3
2
2
0
0
d = 4.10 (s, 2H, H-2 ), 5.33 (s, 4H, H-1 ), 7.50 (dd, 2H,
J = 8.5, 6.8 Hz, H-2), 7.73 (d, 2H, J = 6.8 Hz, H-3), 7.92
sphere (CaCl ). Then, the mixture was incubated for
2
3 h at room temperature. The solvent was evaporated,
the residual was dissolved into 10 mL Me CO, and
1
3
(
d, 2H, J = 8.5 Hz, H-1), 8.78 (s, 1H, H-9) ppm.
0
C
2
NMR (75 MHz, CDCl , 25 ꢁC): d = 64.02 (C-1 ), 125.71
transferred into 80 mL of water. After 1 h, the precipi-
tate was filtered and washed with water. The resulting
dried solid was dissolved into 10 mL CHCl , clarified
3
(
(
(
C-2), 126.50 (C-9a), 127.80 (C-1), 128.49 (C-3), 137.23
C-4), 137.75 (C-9), 146.40 (C-4a) ppm. Anal. Calcd
C H NO ): C, 75.30; H, 5.48; N, 5.85. Found: C,
3
by filtration and evaporated. The residue was recrystal-
lized in MeOH to obtain white powder (441 mg, yield
79%). H NMR (300 MHz, CDCl , 25 ꢁC): d = 6.26 (s,
2H, H-1 ), 7.42 (m, 4H, H-3 ), 7.51 (m, 2H, H-2), 7.54
1
5
13
2
7
5.19; H, 5.47; N, 5.86.
1
3
0
0
4
(
.2.11.
0
Acridin-4,5-yldimethyl-bis-4-chlorobutyrate
B 84). B 52 (300 mg, 1.25 mmol) was dissolved into
0
0
(m, 4H, H-4 ), 7.86 (dd, 2H, J = 6.8, 1.1 Hz, H-3), 7.95
0
2
0 mL Me CO. TEA (0.5 mL, 3.60 mmol), DMAP
2
(d, 2H, J = 8.5 Hz, H-1), 8.14 (m, 4H, H-2 ), 8.76 (s,
1
3
(
(
383 mg, 3.13 mmol) and 4-chlorobutyryl chloride
388 mg, 2.75 mmol) diluted into 20 mL Me CO were
1H, H-9) ppm. C NMR (75 MHz, CDCl , 25 ꢁC):
3
0
d = 63.44 (C-1 ), 125.53 (C-2), 126.33 (C-9a), 128.01
(C-3), 128.27 (C-1), 128.37 (C-3 ), 129.78 (C-2 ), 130.47
2
0
0
added at 0 ꢁC under drying atmosphere (CaCl ), and the
2
0
0
mixture was incubated for 24 h at room temperature.
Then, the mixture was transferred into 100 mL basic
water (5 mL NaOH, 5 N) for 1 h and extracted with
CH Cl (3 · 20 mL). The organic layer was washed with
(C-2a ), 132.87 (C-4 ), 134.82 (C-4), 136.14 (C-9),
0
146.10 (C-4a), 166.52 (C-2b ) ppm. Anal. Calcd
(C H NO ): C, 77.84; H, 4.73; N, 3.13. Found: C,
78.12; H, 4.71; N, 3.11.
2
9
21
4
2
2

D. G. Carole et al. / Bioorg. Med. Chem. 13 (2005) 5560–5568
5567
4
(
.2.14.
0
Acridin-4,5-yldimethyl-bis-4-fluorobenzoate
B 78). B 52 (400 mg, 1.67 mmol) was dissolved into
2H, H-3), 7.95 (d, J = 8.5 Hz, 2H, H-1), 8.08 (m, 4H,
0
0
13
H-2 ), 8.76 (s, 1H, H-9) ppm. C NMR (75 MHz,
0
0
2
0 mL of anhydrous Me CO with 0.5 mL TEA
2
CDCl , 25 ꢁC): d = 55.37 (C-4 ), 63.15 (C-1 ), 113.62
3
0
0
(
3.60 mmol) at 0 ꢁC under drying atmosphere (CaCl ).
(C-3 ), 122.93 (C-2a ), 125.51 (C-2), 126.30 (C-9a),
0
2
4-Fluorobenzoyl chloride (557 mg, 3.51 mmol) diluted
into 10 mL Me CO were added and the mixture was
127.89 (C-3), 128.18 (C-1), 131.79 (C-2 ), 135.07 (C-4),
0
136.05 (C-9), 146.09 (C-4a), 163.32 (C-3a ), 166.26
0
2
incubated for 5 h at room temperature. Then, the mix-
ture was transferred into 100 mL of basic water (5 mL
NaOH, 5 N). After 1 h, the precipitate was filtered and
washed with water. The resulting solid was dissolved into
(C-2b ) ppm. Anal. Calcd (C H NO ): C, 73.36; H,
3
0
25
6
4.96; N, 2.76. Found: C, 73.07; H, 5.00; N, 2.77.
4.2.17.
0
Acridin-4,5-yldimethyl-dimethylaminobenzoate
(B 76). B 52 (500 mg, 2.09 mmol) was dissolved into
0
1
The residue was recrystallized in MeOH to obtain white
0 mL CHCl , clarified by filtration, and evaporated.
3
20 mL anhydrous Me CO containing 1 mL TEA
2
1
powder (614 mg, yield 76%). H NMR (300 MHz,
(7.20 mmol) at 0 ꢁC under drying atmosphere (CaCl ).
2
0
CDCl , 25 ꢁC): d = 6.22 (s, 4H, H-1 ), 7.07 (m, 4H, H-
3
J = 6.8, 1.2 Hz, H-3), 7.98 (d, 2H, J = 8.5 Hz, H-1),
4-Dimethylaminobenzoyl chloride (806 mg, 4.39 mmol)
diluted into 40 mL Me CO was added and the mixture
3
0
), 7.54 (dd, 2H, J = 8.5, 6.8 Hz, H-2), 7.84 (dd, 2H,
2
was incubated for 5 h at room temperature. Then, the
mixture was transferred into 100 mL basic water
(5 mL NaOH, 5 N). After 1 h, the precipitate was fil-
tered and washed with water. The solid layer was dis-
0
13
.12 (m, 4H, H-2 ), 8.79 (s, 1H, H-9) ppm. C NMR
8
0
(75 MHz, CDCl , 25 ꢁC): d = 63.57 (C-1 ), 115.48 (d,
J = 21.8 Hz, C-3 ), 125.50 (C-2), 126.31 (C-9a), 126.63
3
0
0
(
(
(
d, J = 2.3 Hz, C-2a ) 128.18 (C-3), 128.54 (C-1), 132.26
0
solved into 10 mL CHCl , clarified by filtration, and
3
d, J = 9.2 Hz, C-2 ), 134.56 (C-4), 136.18 (C-9), 146.10
0
evaporated. The residue was recrystallized in MeOH
to obtain brown powder (680 mg, yield 61%).
0
1
C-4a), 165.53 (C-2b ), 165.72 (d, J = 253.6 Hz, C-3a )
H
ppm. Anal. Calcd (C H F NO ): C, 72.04; H, 3.96;
2
N, 2.90. Found: C, 72.33; H, 3.94; N, 2.89.
NMR (300 MHz, CDCl , 25 ꢁC): d = 3.02 (s, 12H, H-
9
19
2
4
3
0
0
0
4 ), 6.25 (s, 4H, H-1 ), 6.65 (m, 4H, H-3 ), 7.50 (dd,
J = 8.5, 7.0 Hz, 2H, H-2), 7.84 (dd, J = 7.0, 1.2 Hz,
2H, H-3), 7.93 (d, J = 8.5 Hz, 2H, H-1), 8.03 (m, 4H,
4
(
.2.15.
0
Acridin-4,5-yldimethyl-bis-4-chlorobenzoate
B 63). B 52 (300 mg, 1.25 mmol) was dissolved into
0
0
13
H-2 ), 8.75 (s, 1H, H-9) ppm. C NMR (75 MHz,
0
0
2
chlorobenzoyl chloride (614 mg, 3.51 mmol) diluted into
0 mL Me CO with 0.5 mL TEA (3.60 mmol) and 4-
CDCl , 25 ꢁC): d = 40.07 (C-4 ), 62.69 (C-1 ), 110.77
2
3
0
0
(C-3 ), 117.34 (C-2a ), 125.53 (C-2), 126.26 (C-9a),
0
1
sphere (CaCl ). The mixture was incubated for 5 h at
0 mL Me CO was added at 0 ꢁC under drying atmo-
127.53 (C-3*), 127.62 (C-1*), 131.50 (C-2 ), 135.64
0
2
(C-4), 135.93 (C-9), 146.04 (C-4a), 153.33 (C-3a ),
0
2
room temperature and transferred into 100 mL basic
water (5 mL NaOH, 5 N) for 1 h. The precipitate was fil-
tered and washed with water and the solid layer was dis-
166.87 (C-2b ) ppm. Anal. Calcd (C H N O ): C,
3
3
31
3
4
74.28; H, 5.86; N, 7.87. Found: C, 74.58; H, 5.82; N, 7.82.
solved into 10 mL CHCl , clarified and evaporated to
3
4.3. Biology
1
obtain a yellow powder (640 mg, yield 75%). H NMR
0
(
(
(
300 MHz, CDCl , 25 ꢁC): d = 6.21 (s, 4H, H-1 ), 7.35
4.3.1. Irradiation procedure. Irradiation was performed
with a solar simulator Suntest CPS+ (Atlas Material
Testing Technology BV, Moussy le Neuf, France)
equipped with a xenon arc lamp, a treated quartz filter
to block infrared light, and a glass filter to block UVC
light and to reduce the UVB light (UVA/UVB: 0.5/
3
0
m, 4H, H-3 ), 7.53 (dd, J = 8.5, 6.9 Hz, 2H, H-2), 7.84
dd, J = 6.9, 0.8 Hz, 2H, H-3), 7.97 (dd, J = 8.5,
.8 Hz, 2H, H-1), 8.02 (m, 4H, H-2 ), 8.77 (s, 1H, H-9)
0
0
1
3
ppm. C NMR (75 MHz, CDCl , 25 ꢁC): d = 63.67
3
0
(
1
2
C-1 ), 125.50 (C-2), 126.33 (C-9a), 128.26 (C-3),
0
0
2
28.67 (C-3 ), 128.73 (C-1), 128.85 (C-2 a), 131.10 (C-
0
0.001 mW/cm , visible light 800 lx). The average light
incident dose was 2 mJ/cm for a period of 1 min.
0
2
), 134.46 (C-4), 136.18 (C-9), 139.31 (C-3 a), 146.13
0
(
C-4a),
165.61
C H Cl NO ): C, 67.45; H, 3.71; N, 2.71. Found: C,
(C-2 b)
ppm.
Anal.
Calcd
(
4.3.2. Antileishmanial activity against promastigotes.
Leishmania infantum promastigotes in late log phase
were incubated in RPMI medium supplemented with
2
9
19
2
4
6
7.70; H, 3.69; N, 2.70.
5
4
(
.2.16.
0
Acridin-4,5-yldimethyl-bis-4-methoxybenzoate
B 62). B 52 (300 mg, 1.25 mmol) was dissolved into
12% fetal calf serum at an average of 10 cells/mL and
0
a range of acridine concentrations was aseptically incor-
porated into duplicate cultures (final DMSO concentra-
tion was <5%). Following a 48-h incubation period at
25 ꢁC, promastigote growth was estimated by counting
parasites with a hemacytometer. IC was defined as
the drug concentration necessary to inhibit 50% of
parasite growth.
2
0 mL CH Cl . TEA (0.5 mL, 3.60 mmol), DMAP
2 2
(
(
383 mg, 3.13 mmol) and 4-methoxybenzoyl chloride
471 mg, 2.75 mmol) diluted into 10 mL CH Cl were
2
2
added at 0 ꢁC under drying atmosphere (CaCl ). Then,
2
50
the mixture was incubated for 5 h at room temperature.
The solvent was washed with 70 mL NaOH 1 N, the
organic layer was dried with MgSO and the solvent
4
was evaporated. The residue was purified by silicagel
chromatography (CH Cl /EtOAc, 99:1, v:v) to obtain
4.3.3. Antileishmanial activity against intracellular
amastigotes. Intracellular amastigote cultures were per-
formed in human monocyte-derived macrophages
according to the methodology previously described
by us. Maturation of monocytes into adherent macro-
phages was performed by treating exponentially growing
2
2
1
yellow powder (515 mg, yield 81%).
H
NMR
0
(
(
300 MHz, CDCl , 25 ꢁC): d = 3.84 (s, 6H, H-4 ), 6.23
3
0
0
17
s, 4H, H-1 ), 6.89 (m, 4H, H-3 ), 7.52 (dd, 2H,
J = 8.5, 6.8 Hz, 2H, H-2), 7.84 (dd, J = 6.8, 1.0 Hz,

5568
D. G. Carole et al. / Bioorg. Med. Chem. 13 (2005) 5560–5568
5
0
monocytes (10 cells/mL) with 1 lM phorbol myristate
acetate (Sigma). After a 48-h incubation period at 37 ꢁC
4.3.6. Effects of compound B 52 on the phagocytic
capacities of human macrophages. Assays were per-
formed on human monocyte-derived macrophages.
Maturation of monocytes into adherent macrophages
was performed by treating exponentially growing mono-
(
5% CO ) in chamber slides (Fisher, Paris, France), cells
2
were rinsed with fresh medium and suspended in RPMI
medium containing stationary-phase promastigotes
5
(
cells/promastigotes ratio = 1:10). After a 24-h incuba-
cytes (10 cells/ml) with 1 lM phorbol myristate acetate
tion period at 37 ꢁC (5% CO ), promastigotes were re-
(Sigma). After a 48-h incubation period at 37 ꢁC (5%
2
moved by four successive washes with fresh medium.
Adapted dilutions of chemical compounds were added
in duplicate chambers and cultures were incubated for
CO ) in chamber slides (Fisher, Paris, France), cells
were rinsed with fresh medium and various concentra-
2
0
tions of compound B 52 were incorporated into dupli-
9
6 h at 37 ꢁC (5% CO ). Negative controls treated by sol-
cate cultures. After a 48-h incubation period at 37 ꢁC,
cells were rinsed with fresh medium and infected with
RPMI medium containing stationary-phase promasti-
gotes (cells/promastigotes ratio = 1:10). After a 4-h incu-
2
vent (DMSO) and positive controls containing a range of
amphotericin B (Sigma, St Louis, Mo, USA) concentra-
tions were added to each set of experiments. At the end
of the incubation period, cells were harvested with analyt-
ical grade methanol (Sigma) and stained with 10% Giem-
sa stain (Eurobio, Paris, France). The percentage of
infected macrophages in each assay was determined
microscopically at 1000· magnification. IC was defined
bation period at 37 ꢁC (5% CO ), promastigotes were
2
removed by four successive washes with fresh medium,
fixed with methanol and stained with 10% Giemsa stain.
The percentage of macrophages containing adherent or
intracellular parasites was analyzed microscopically at
1000· magnification.
5
0
as the concentration of drug necessary to produce a 50%
decrease of infected macrophages.
4
.3.4. Toxicity against human monocytes. In vitro toxic-
References and notes
ity of acridine derivatives was assessed on human mono-
cytes maintained in RPMI medium (Eurobio, Paris,
France) supplemented with 10% fetal calf serum (Euro-
1
. Choi, C. M.; Lerner, E. A. J. Invest. Derm. Symp. P. 2001,
, 175–182.
6
bio, Paris, France) at 37 ꢁC in 5% CO and replicated
2. Herwalt, B. L. Lancet 1999, 354, 1191–1199.
3. Harms, G.; Feldmeier, H. Trop. Med. Int. Health 2002, 7,
479–488.
2
every week. A range of concentrations was incorporated
in late log-phase monocytes (10 cells/ml) and cultures
5
4
. Croft, S. L.; Yardley, Y. Curr. Pharm. Design 2002, 8,
19–342.
were incubated at 37 ꢁC with 5% CO . After a 72-h incu-
2
3
bation period, cell growth was measured by counting
monocytes in a hemacytometer. IC50 was defined as
the concentration of drug required to induce a 50% de-
crease of cell growth. An in vitro selective index (SI),
corresponding to the ratio between antiparasitic and
cytotoxic activities, was calculated according to the fol-
5
6
7
. Sundar, S. Trop. Med. Int. Health 2001, 6, 849–854.
. Murray, H. W. Int. J. Infect. Dis. 2001, 4, 158–177.
. Adams, A.; Guss, J. M.; Denny, W. A.; Wakelin, L. P.
Nucleic Acids Res. 2002, 30, 719–725.
8
. Alberti, P.; Ren, J.; Teulade-Fichou, M. P.; Guittat, L.;
Riou, J. F.; Chaires, J.; Helene, C.; Vigneron, J. P.; Lehn, J.
M.; Mergny, J. L. J. Biomol. Struct. Dyn. 2001, 19, 505–513.
. Werbovetz, K. A.; Spoors, P. G.; Pearson, R. D.; Macdon-
ald, T. L. Mol. Biochem. Parasit. 1994, 65, 1–10.
lowing formula: SI = IC5
/IC
.
0ꢀmonocytes
50ꢀamastigotes
9
0
4.3.5. Effects of compound B 52 on nitric oxide production
by macrophages. Maturation of human monocytes into
adherent macrophages was performed by treating expo-
1
1
1
0. Girault, S.; Grellier, P.; Berecibar, A.; Maes, L.; Mouray,
E.; Lemiere, P.; Debreu, M. A.; Davioud-Charvet, E.;
Sergheraert, C. J. Med. Chem. 2000, 43, 2646–2654.
1. Gamage, S. A.; Figgit, D. P.; Wojcik, S. J.; Ralph, R. K.;
Ransijn, A.; Mauel, J.; Yardley, V.; Snowdon, D.; Croft,
S. L.; Denny, W. J. Med. Chem. 1997, 40, 2634–2642.
2. Mrozek, A.; Karolak-Wojciechowska, J.; Bsiri, N.; Barbe,
J. Acta. Pol. Pharm. 2000, 57, 345–351.
13. Wainwright, M. J. Antimicrob. Chemother. 2001, 47, 1–13.
14. Iwamoto, Y.; Ferguson, L. L.; Pearson, A.; Baguley, B. C.
Mutat. Res. 1992, 268, 35–41.
5. Chiron, J.; Galy, J. P. Heterocycles 2003, 60, 45–49.
16. Hess, F. K.; Stewart, P. B. J. Med. Chem. 1975, 18, 320–
21.
5
nentially growing monocytes (10 cells/ml) with 1 lM
phorbol myristate acetate (Sigma). After a 48-h incuba-
tion period at 37 ꢁC (5% CO ) in chamber slides (Fisher,
2
Paris, France), cells were rinsed with fresh medium and
suspended in RPMI medium containing various concen-
0
trations of compound B 52 in the presence or absence of
1
4
0 U/mL human recombinant interferon gamma. After
8 h at 37 ꢁC, NO production was measured by assess-
1
ing the nitrite content of culture supernatants by the
1
8
method described by Ding et al. : 100lL of fresh Griess
reagent was added to equal volumes of culture superna-
tants and the optical density at 540 nm was measured
after 15 min of incubation at room temperature. Nitrite
3
1
7. Di Giorgio, C.; Delmas, F.; Filloux, N.; Robin, M.;
Seferian, L.; Azas, N.; Gasquet, M.; Timon-david, P.; Galy,
J. P. Antimicrob. Agents Chemother. 2003, 47, 174–180.
18. Ding, A. H.; Nathan, C. F.; Stuehr, D. J. J. Immunol.
1988, 141, 2407–2412.
concentrations were determined by using NaNO dilut-
2
ed in DMEM as the standard.