In vitro efficiency of new acridyl derivatives against Plasmodium falciparum

Article abstract of DOI:10.1016/j.bmc.2007.02.022

A series of new 9-substituted acridyl derivatives were synthesized and their in vitro antimalarial activity was evaluated against one chloroquine-sensitive strain (3D7) and three chloroquine-resistant strains [W2 (Indochina), Bre1 (Brazil) and FCR3 (Gambi

Full text of DOI:10.1016/j.bmc.2007.02.022

Bioorganic & Medicinal Chemistry 15 (2007) 3278–3289
In vitro efficiency of new acridyl derivatives against
Plasmodium falciparum
a
a
a
a
´ `
Lucie Guetzoyan, Florence Ramiandrasoa, Helene Dorizon, Christine Desprez,
a
b
b
a,
*
´
Alexandre Bridoux, Christophe Rogier, Bruno Pradines and Martine Perree-Fauvet
a
´
Equipe de Chimie Bioorganique et Bioinorganique, Institut de Chimie Mole´culaire et des Mate´riaux d’Orsay,
ˆ
Bat. 420, CNRS UMR 8182, Univ Paris-Sud, 91405 Orsay Cedex, France
bUnite´ de Recherche en Biologie et Epide´miologie Parasitaires, Institut de Me´decine Tropicale du Service des Arme´es,
Bd Charles Livon, Parc Le Pharo, 13998 Marseille Arme´es, France
Received 26 October 2006; revised 2 February 2007; accepted 9 February 2007
Available online 16 February 2007
Abstract—A series of new 9-substituted acridyl derivatives were synthesized and their in vitro antimalarial activity was evaluated
against one chloroquine-sensitive strain (3D7) and three chloroquine-resistant strains [W2 (Indochina), Bre1 (Brazil) and FCR3
(Gambia)] of Plasmodium falciparum. Some compounds inhibit the growth of malarial parasite with IC50 6 0.20 lM.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
Acridine compounds known to have antimalarial activ-
ity are essentially 9-aminoacridine derivatives such as
quinacrine and 9-anilinoacridine derivatives.7–11 It was
previously considered that the primary receptor was
DNA because acridines like chloroquine are able to
intercalate into DNA and consequently can inhibit
DNA transcription in parasites.12–14 Furthermore,
9-anilinoacridines that have good antimalarial activities
in vitro are potent inhibitors of parasite DNA topoiso-
merase II both in vitro and in situ.15,16 This relationship
is evidenced on 3,6-diamino-9-anilinoacridine that dis-
plays higher antiparasitic activity and increased inhibi-
tory effects on parasite topoisomerase II. It was first
suggested that low lipophilicity and high basicity were
important factors for the in vitro antimalarial activity
of 9-anilinoacridines.7 Even if it appeared that there
was no direct correlation between DNA binding and
antimalarial activity, one can consider that the increased
basicity and additional cationic charges resulting from
the 3,6-diamino substitution of the acridine ring should
greatly reinforce DNA binding. A model in which the
acridine ring intercalates into DNA and the 9-anilino
side group projects into the DNA minor groove where
it interacts with topoisomerase II could explain the
capacity of these compounds to inhibit the enzyme.17
More than 40% of the population in the world lives in
areas infected by malaria. Malaria is a disease that af-
fects hundreds of millions of people, causes more than
a million death per year, and is in continual increase.1
Quinine (QN), then chloroquine (CQ) were the drugs
of choice for malaria chemotherapy for over 50 years
because of their high efficacy, relatively low toxicity,
ease of use, and low cost.2 However, in recent years,
malaria parasites, particularly Plasmodium falciparum,
the most virulent of the human parasite, have developed
resistant strains to QN and CQ. Researches are in pro-
gress to elucidate the mechanism of action of those
drugs that contain a quinoline nucleus and much effort
is directed toward the synthesis of novel antimalarial
drugs containing an acridine ring.3–7
Abbreviations: b-Ala, b-alanine; Arg, arginine; Gly, glycine; Bn, ben-
zyl; Boc, tert-butoxycarbonyle; Fmoc, fluorenyl-9-methoxycarbonyle;
Pbf, 2,2,4,6,7-pentamethyldihydrobenzofurane-5-sulfonyle; BOP,
benzotriazol-1-yloxy-tris(dimethylamino)phosphonium
hexafluoro-
phosphate; DIEA, diisopropylethylamine; DBU, 1,8-diazabicy-
clo[5.4.0]undec-7-ene; DPPA, diphenylphosphoryl azide; TFA,
trifluoroacetic acid.
Keywords: Antimalarial; Plasmodium falciparum; Aminoacridine; Pep-
tidic synthesis.
However, it is recognized that inhibition of parasite
topoisomerase II may not be the only mechanism of ac-
tion of acridine derivatives. The antimalarial activity of
*
Corresponding author. Tel.: +33 1 69 15 47 20; fax: +33 1 69 15 72
81; e-mail: mperreef@icmo.u-psud.fr
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.02.022

L. Guetzoyan et al. / Bioorg. Med. Chem. 15 (2007) 3278–3289
3279
quinolines and acridine derivatives is actually believed
to be due to the inhibition of hemoglobin digestion by
the plasmodia, and subsequent blockade of the released
free heme polymerization into hemozoin, the malaria
pigment, in the parasite food vacuole.18–21 The structure
of hemozoin is thought to consist of a crystalline insol-
uble cyclic dimer of ferriprotoporphyrin IX.22 Antima-
larial drugs that contain an acridine nucleus can thus
bind strongly to heme and inhibit the crystallization
process.
function. It requires the preliminary protection of the
functional groups that are likely to react during the cou-
pling. In order to avoid secondary coupling reactions,
arginine used in this work was protected on its amine
function. It was also protected on its guanidinium
group, yielding derivatives easier to purify than the chlo-
ride salt and that protection was maintained until the
ultimate stage of the synthesis. We chose to protect
the
guanidinium
with
the
2,2,4,6,7-pentame-
(Pbf) protective
thyldihydrobenzofuran-5-sulfonyl
group, as it is cleaved by TFA24 while the arginine
amine function was protected by the Fmoc-group, one
of the very rare amine protective groups to be cleaved
by bases.25 Synthesis of the acridyl derivative bound to
two b-Ala units and one arginine (compound 22) was
achieved by two parallel ways using one bis-protected
b-Ala (O-Bn and N-Boc) and a bis-protected arginine
as starting materials, on one hand, and acridine and
one Boc-b-Ala moiety on the other hand (Scheme 2).
This strategy allows obtaining more soluble synthons
than the classical linear coupling method involved in
the synthesis of the other amino acid derivatives. Yield
of the final coupling and consequently, yield of com-
pound 22 is thus increased. All the acridine derivatives
were obtained as their TFA salts. As they were tested
under their Clꢀ form, the last step of the synthesis was
a trifluoroacetate–chloride ion exchange through a
Dowex Clꢀ resin column, followed by precipitation of
the product in H2O/acetone to remove the sodium salts
in excess.
As part of our research devoted to the synthesis of new
DNA intercalators,23 we have designed and synthesized
new acridine derivatives, bearing one or several cationic
charges and substituted at the 9-position by a chain of
various length and nature. An in vitro investigation of
the structure-antimalarial activity relationships will be
reported.
2. Chemistry
Two series of acridine derivatives have been investi-
gated. They are listed on Table 1. One is a series of
9-amino acridinium derivatives bearing several cationic
charges in order to increase its pKa: one on the acridine
ring and the other(s) on the chain grafted at the 9-amino
position. This chain can be a simple ammonium chain of
six carbon atoms (compound 2), or an amine-peptidic
chain of various length and various nature. Aminoacids
investigated in this work were Gly, b-Ala, alone or
bound to Arg. Synthesis of these derivatives is described
in Schemes 1 and 2. The second series of acridyl deriva-
tives is positively charged only on the peptidic chain
grafted on the acridine ring. Two derivatives (com-
pounds 30 and 31) have been synthesized. They differ
by the connection of the peptidic chain to the acridine
(NHCO vs CONH) and the total length of the chain.
Synthesis of compound 30 is described in Scheme 3.
Compound 31 was obtained by cleavage of the guanid-
inium protective group of the previously synthesized
9-acridinecarboxamide derivative.23
3. Antimalarial activity
The antimalarial activity of all the synthesized acridine
derivatives has been tested on P. falciparum 3D7, W2,
FCR3, and Bre1 strains (one chloroquine-sensitive and
three chloroquine-resistant strains, respectively), and
compared with that of chloroquine. Results are given
in Table 2.
The data emphasize two major points:
The general route of all the syntheses consists in a clas-
sical peptidic synthesis, using a series of peptidic cou-
plings with coupling reagents to activate the acidic
– Presence of a cationic charge on the acridine nucleus
is required to provide a significant antimalarial activ-
ity. Compounds 30 and 31 in which the chain is not
Table 1. Acridine derivatives tested in this work
R1
X
R2
R
Compound
X
R1
R2
R
2
6
NH+Clꢀ
NH+Clꢀ
NH+Clꢀ
NH+Clꢀ
NH+Clꢀ
NH+Clꢀ
N
Cl
Cl
Cl
Cl
Cl
Cl
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
NHðCH2Þ NH3þClꢀ
NHðCH2Þ6NHCOðCH2Þ NH þClꢀ
3
9
NHðCH2Þ6NHCOðCH2Þ3NHCOCH2NH þClꢀ
3
12
15
22
30
31
NHðCH2Þ6NHCOðCH2Þ3NHCOCH2NHArgþClꢀ
NHðCH2Þ6NHCOðCH2Þ3NH þClꢀ
6
2
3
NH(CH2)6NHCO(CH2)2NHCO(CH2)2NHArg+Clꢀ
NHCO(CH2)7NHCOCH2NHArg+Clꢀ
N
H
H
CONH(CH2)6NHCOCH2NHArg+Clꢀ
ArgþClꢀ@COCHðNH3þClꢀÞð CH2Þ3NHCð@NHÞðNH3þClꢀÞ.

3280
L. Guetzoyan et al. / Bioorg. Med. Chem. 15 (2007) 3278–3289
Scheme 1. Synthesis of compounds 2, 6, 9, and 12. Reagents and conditions: (a) TFA, 12 h, Dowex Clꢀ; (b) 1,10-carbonyldiimidazole (1 equiv),
CH2Cl2, rt, argon, 12 h; (c) CH2Cl2/TFA (1/1), rt, 12 h; (d) Dowex Clꢀ; (e) 1,10-carbonyldiimidazole (1.1 equiv), Et3N (2 equiv/5 + 1 equiv/Gly),
DMF, rt, argon, 72 h; (f) (Fmoc)Arg(Pbf)OH (1.1 equiv), BOP (1.2 equiv), DIEA (5 equiv/8 + 1.1 equiv/Arg), DMF, rt, argon, 72 h; (g) DBU
(4 equiv), THF, rt, 2 h; (h) TFA/H2O (95/5), Et3SiH (2 equiv), rt, 12 h.
attached to the acridine by an amino bond, have
pKa = 4 and consequently are not positively charged
on the ring in the in vitro assays. The IC50 values of
their antimalarial activity range between 27–30 lM
for 30 and 42–48 lM for 31, according to the strain.
– The most active molecules are those whose chain
attached to the acridine ring is shortest. Best results
are obtained for the acridine derivative 2, and IC50
values decrease in the order 9 > 12 > 6 > 2, whatever
the strains. However, the activity of 6 against Bre1 is
almost equal to that of 2, whereas it is about three
times lower against the two other CQ-resistant strains.
Antimalarial 9-anilinoacridines are potent inhibitors of
parasite DNA topoisomerase II both in vitro15 and
in situ.16 Nevertheless, such as chloroquine, the 9-anili-
noacridines can be considered inhibitors of heme poly-
merization or agents that act to divert heme from
participating in the crystallization process, leading to
the accumulation of free heme, which is toxic for the
parasite.26 Some of these compounds inhibit b-hematin
formation, form drug–hematin complexes, and enhance
hematin-induced lysis of erythrocytes. The nature of the
substitution in the anilino ring affects these abilities. In
addition, some 9-anilinoacridines show gametocytocidal
activity.27,28
In addition, compound 2 is between 2.5- and 3-fold
more efficient than CQ against CQ-resistant strains.
Compound 2, the most active acridine derivative pre-
sented in this work, has a structure similar to that of
mepacrine, a 9-aminoacridine synthetic anti-malarial
drug. Its higher activity may result from its facility,
due to its small size, to enter the vacuole.
The grafting of an arginine to compound 9 (compound
12) hardly increases its activity. In contrast, it improves
the activity against chloroquine-resistant strains of the
series encompassing b-ala linkers (22 > 15), but severely
decreases its activity against 3D7.
On the other hand, recent research has focused on acri-
dine derivatives as chemotherapeutic agents because of
the ability of the acridine chromophore to intercalate
into DNA.29 Based on this, the selection of 9-amino-6-
chloro-2-methoxyacridine (ACMA) derivatives as inter-
calating agents has been widely developed.30 As all the
present synthesized acridine derivatives that reveal good
inhibitors of P. falciparum are ACMA derivatives, it
4. Discussion
It is obvious that the mechanism of action of antimalar-
ial drugs is unclear and new data may help to better
understand the role played by DNA or hematin.

L. Guetzoyan et al. / Bioorg. Med. Chem. 15 (2007) 3278–3289
3281
Scheme 2. Synthesis of compounds 15 and 22. Reagents and conditions: (a) 1,10-carbonyldiimidazole (1 equiv), CH2Cl2, rt, argon, 12 h; (b) CH2Cl2/
TFA (1/1), rt, 12 h; (c) Dowex Clꢀ; (d) K2CO3 (1 equiv), DMF, BnBr (1 equiv), rt; (e) (Fmoc)Arg(Pbf)OH (0.83 equiv), DPPA (1 equiv), DMF,
DIEA (1.33 equiv), 0 ꢁC, 12 h; (f) H2 (10 bars), Pd/C, EtOAc, 48 h; (g) BOP (1 equiv), DIEA (2 equiv/14 + 2 equiv/19), DMF, rt, argon, 28 h; (h)
DBU (1 equiv), THF, rt, 72 h; (i) TFA/H2O (90/10), Et3SiH (2 equiv), rt, 12 h.
seems reasonable to consider that our molecules are also
good DNA intercalators. The positive charge on the
acridine ring favors the approach of the molecule to
DNA and reinforces DNA-interaction. This is sup-
ported by fluorescence experiments, that have demon-
strated that compound 22 is able to interact strongly
with poly(dAdT)2 and poly(dGdC)2, while compound
30 displays no detectable interaction with DNA.31 The
positively charged acridine side chain may lie in the min-
or groove and interacts with DNA phosphate groups. If
it contains an arginine moiety (compounds 12 and 22), it
projects rather in the major groove and the amino
groups of the guanidinium side chain of arginine can
form hydrogen bonds with guanines.32 So, the antima-
larial activity of 9-aminoacridine derivatives could be
related to their ability to intercalate into DNA.
In addition, the planarity of acridine nucleus favors the
formation of cofacial p–p complexes with the free
released heme. A NMR study reported that the interaction
between CQ and hematin consists of a close stacking
between the porphyrin ring and the quinoline nucleus
of the drug.33 The complexes may also be stabilized by
hydrogen bonding between the protonated terminal
amino groups of the acridine side chain and the propio-
nate carboxylate of the porphyrin. As these interactions
are hydrophobic and can occur between two neutral
rings, a positive charge on the acridine ring would not

3282
L. Guetzoyan et al. / Bioorg. Med. Chem. 15 (2007) 3278–3289
Scheme 3. Synthesis of compound 30. Reagents and conditions: (a) BOP (1.1 equiv), DIEA (1.2 equiv), DMF, rt, argon, 12 h; (b) CH2Cl2/TFA (1/1),
rt, 12 h; (c) BOP (1.2 equiv), DIEA (5 equiv/25 + 1.2 equiv/Gly), DMF, rt, argon, 12 h; (d) (Fmoc)Arg(Pbf)OH (1 equiv), BOP (1.2 equiv), DIEA
(5 equiv/27 + 1.2 equiv/Arg), DMF, rt, argon, 12 h; (e) DBU (4 equiv), THF, rt, 2 h; (f) TFA/H2O (95/5), Et3SiH (2 equiv), rt, 12 h.
Table 2. Comparative in vitro efficiency of chloroquine (CQ) and the acridine derivatives against chloroquine-sensitive and chloroquine-resistant
P. falciparum strains
Plasmodium falciparum strains
Chloroquine-sensitive
3D7
Chloroquine-resistant
FCR3
W2
Bre1
Compound IC50 (lM)
Compound IC50 (lM)
Compound IC50 (lM)
Compound IC50 (lM)
[confidence interval]
[confidence interval]
[confidence interval]
[confidence interval]
CQ
2
0.018 [0.016–0.020]
0.13 [0.09–0.19]
0.19 [0.14–0.27]
0.84 [0.76–0.93]
0.89 [0.75–1.05]
0.91 [0.71–1.16]
3.32 [3.07–3.58]
27.1 [24.5–30.0]
42.0 [38.7–45.5]
2
CQ
6
0.18 [0.13–0.25]
0.44 [0.38–0.51]
0.57 [0.47–0.68]
0.82 [0.73–0.91]
0.83 [0.69–1.01]
0.94 [0.79–1.13]
1.25 [0.97–1.61]
30.5 [26.9–34.7]
43.6 [36.7–51.7]
2
CQ
6
0.20 [0.18–0.23]
2
6
0.17 [0.13–0.21]
0.18 [0.12–0.19]
0.50 [0.42–0.58]
0.52 [0.42–0.66]
0.62 [0.53–0.72]
0.86 [0.79–0.92]
1.26 [1.13–1.41]
28.7 [24.5–33.6]
47.6 [42.8–53.1]
0.50 [0.39–0.64]
0.54 [0.50–0.59]
0.67 [0.63–0.71]
0.81 [0.65–1.01]
0.86 [0.77–0.95]
1.25 [0.98–1.60]
27.2 [23.0–32.2]
42.5 [32.3–56.0]
6
12
CQ
9
12
9
12
9
12
9
15
22
30
31
22
15
30
31
22
15
30
31
22
15
30
31
strengthen the stacking and improve the activity. Even if
antimalarial activity of our acridine derivatives should
result from the strength of their binding to heme, it must
also depend strongly on their ability to accumulate in
the food vacuole of the parasite. Fairly high basicity
as observed here for the active compounds (pKa > 8)
facilitates the accumulation in the malaria parasite
acidic food vacuole in which hemozoin formation takes
place.34
that, even if the sulfonyl group, in increasing the basicity
of the molecule, was essential for the antimalarial activ-
ity, the action was conferred by the acridine ring.
In conclusion, some of the acridine derivatives evaluated
in this study showed significant antimalarial activity
against P. falciparum in vitro. This activity is conferred
by the acridine ring, in its cationic form, and is depen-
dent on the nature of the chain linked to the ring. So,
all the mechanisms postulated to elucidate the antima-
larial activity of drugs that contain a quinoline ring
could be discussed in this work and research is in pro-
gress to optimize the structure of these derivatives. Sub-
sequent experiments will be performed in the laboratory
in order to find a correlation between the activity of our
compounds and their ability to bind to DNA and/or to
form complexes with ferriprotoporphyrin IX.
The role played both by the acridine chromophore and
the basicity of the molecule is highlighted by the fact
that most of the present compounds were found to be
more potent against P. falciparum parasites than aryl-
sulfonyl acridinyl derivatives.5 The antimalarial activity
of these sulfone compounds has been tested on P. falci-
parum 3D7 and W2 strains, and it has been concluded

L. Guetzoyan et al. / Bioorg. Med. Chem. 15 (2007) 3278–3289
3283
5. Experimental
–
13C NMR (CDCl3, 63 MHz) d ppm: 24.8, 28.0, 30.9,
39.5, 78.9, 156.1, 177.2.
5.1. Chemistry
– MS (ES) m/z: 202.2 (MH+) (100%).
5.1.1. General methods. Coupling reactions were per-
formed under an argon atmosphere and in a commercial
acid- and base-free DMF (99.8% Aldrich). Most of the
reactions were carried out in the dark. THF was dried
over sodium and distilled prior to use. All other solvents
and reagents were pure grade and used without purifica-
tion, as well as the following commercially available
chemicals: 9-aminoacridine and 8-aminocaprylic acid
(Acros), 6,9-dichloro-2-methoxyacridine and 1,6-hexane-
diamine (Aldrich), c-aminobutyric acid, Boc-Gly-OH,
and Boc-b-Ala-OH (Fluka), and Fmoc-Arg(Pbf)-OH
(Senn Chemicals).
5.1.4. tert-Butyl 4-[6-(6-chloro-2-methoxyacridin-9-yl-
amino)hexylamino]-4-oxobutylcarbamate (4). A mixture
of compound 3 (788 mg, 3.88 mmol) and 1,10-carbon-
yldiimidazole (629 mg, 3.88 mmol) in CH2Cl2 (20 mL)
was stirred for 1 h. A solution of 1 (1.39 g, 3.88 mmol)
in CH2Cl2 (20 mL) was added dropwise and the reaction
mixture was stirred overnight, then concentrated in va-
cuo and purified by MPLC in CH2Cl2/MeOH (90/10)
to afford 4 as a yellow powder in 55% yield (1.16 g,
2.14 mmol).
1
– H NMR (CDCl3, 200 MHz) d ppm: 1.30 (m, 15H),
1.65 (m, J = 8 Hz, 4H), 2.15 (t, J = 8 Hz, 2H), 3.10
(m, 4H), 3.55 (t, J = 8 Hz, 2H), 3.90 (s, 3H), 4.90 (m,
1H), 6.45 (m, 1H), 7.15 (dd, J = 2.2 Hz, J2 = 9.7 Hz,
2H), 7.18 (dd, J1 = 2.2 Hz, J2 = 9.7 Hz, 1H), 7.35
(dd, J1 = 2.2 Hz, J2 = 9.7 Hz, 1H), 7.85 (s, 1H), 7.92
(dd, J1 = 2.2 Hz, J2 = 9.7 Hz, 2H), 7.95 (s, 1H).
Reactions were monitored by thin layer chromatography
(TLC) performed on silica gel sheets containing UV
fluorescent indicator (60 F254 Merck). 1H and 13C
NMR spectra were recorded on Bruker AC 200, Bruker
AC 250, Bruker AV 360, and Bruker AC 400 spectro-
meters (200, 250, 360, and 400 MHz for 1H, respectively,
and50, 63, 90, and100 MHzfor13C, respectively). Chemical
shifts, d, are reported in ppm taking residual CHCl3 or
CHD2OD as the reference. Mass spectra were recorded
on a Finnigan-MAT-95-S, using MeOH/CH2Cl2/H2O
(45/40/15, v/v) as solvent. Elemental analyses were per-
formed by the Service Central de Microanalyse du CNRS.
–
13C NMR (CDCl3, 50 MHz) d ppm: 26.2, 26.5, 28.3,
29.3, 31.4, 33.5, 39.0, 39.5, 50.2, 55.4, 65.0, 79.0,
99.2, 115.5, 117.7, 124.1, 124.2, 124.4, 127.8, 131.1,
134.6, 146.5, 148.2, 149.6, 149.8, 155.8, 172.6.
– MS (ES) m/z: 543.2 (MH+) (100%).
5.1.5.
9-[6-(4-Ammoniobutanamido)hexylamino]-6-
chloro-2-methoxyacridinium ditrifluoroacetate (5). Acri-
dine 4 (1.16 g, 2.14 mmol) was dissolved in TFA/CH2Cl2
(1/1) (13 mL). After stirring overnight in the dark, the
solvent was evaporated in vacuo. The TFA salt 5 was
obtained quantitatively and used without further
purification.
5.1.2. N1-(6-Chloro-2-methoxyacridin-9-yl)hexane-1,6-
diamine (1). This compound was synthesized from 6,9-
dichloro-2-methoxyacridine (1.1 g, 2.98 mmol) and
1,6-hexanediamine (14 mL), according to the procedure
described by Uno35, and obtained in 98% yield.
1
1
– H NMR (CD3OD, 200 MHz) d ppm: 1.20 (m, 6H),
– H NMR (CD3OD, 250 MHz) d ppm: 1.50 (m, 6H),
1.55 (qn, 2H), 2.40 (t, 2H), 3.55 (t, 2H), 3.80 (s, 3H),
7.05 (dd, 1H), 7.20 (dd, 1H), 7.25 (s, 1H), 7.60 (d,
1H), 7.65 (s, 1H), 8.00 (d, 1H).
1.98 (m, 4H), 2.35 (t, J = 6.2 Hz, 2H), 3.16 (t,
J = 6.2 Hz, 2H), 3.30 (t, J = 6.2 Hz, 2H), 3.32 (t,
J = 6.2 Hz, 2H), 3.99 (s, 3H), 4.11 (t, J = 7.2 Hz,
2H), 7.46 (dd, J1 = 2.5 Hz, J2 = 7.2 Hz), 7.48 (dd,
J1 = 2.5 Hz, J2 = 7.2 Hz, 1H), 7.60 (dd, J1 = 2.5 Hz,
J2 = 7.2 Hz, 1H), 7.65 (dd, J1 = 2.5 Hz, J2 = 7.2 Hz,
1H), 7.70 (dd, J1 = 2.5 Hz, J1 = 7.2 Hz, 1H), 8.20 (d,
J = 9.2 Hz, 1H).
–
13C NMR (CD3OD, 50 MHz) d ppm: 27.6, 27.8, 32.1,
33.6, 42.3, 50.9, 56.1, 101.0, 115.6, 118.2, 124.0, 125.9,
126.9, 130.3, 130.5, 136.3, 146.9, 149.2, 152.7, 157.0.
– MS (ES) m/z: 358.2 (MH+) (100%).
– Anal. Calcd for C20H24ClN3O, H2O: C, 63.91; H, 6.97;
N, 11.18. Found C, 64.02; H, 6.78; N, 11.02.
–
13C NMR (CD3OD, 63 MHz) d ppm: 22.7, 25.8, 28.3,
29.1, 32.3, 38.7, 48.7, 55.3, 62.4, 117.3, 119.9, 123.6,
127.1, 140.4, 152.0, 156.0, 172.6.
5.1.3. 4-(tert-Butoxycarbonylamino)butyric acid (3).
Di-tert-butyl dicarbonate [(Boc)2O] (3.27 g, 15 mmol)
in 15 mL dioxane was added dropwise to a solution of
c-aminobutyric acid (1.03 g, 10 mmol) in dioxane
(15 mL) and NaOH 1 N (15 mL) at 0 ꢁC. The reaction
mixture was allowed to stand overnight. The crude
product was concentrated under reduced pressure, HCl
1 N was added, and diluted with EtOAc. The organic
layer was washed with water and dried over Na2SO4.
The solvant was evaporated in vacuo. The compound
3 was obtained as a yellow oil in 85% yield (1.83 g,
9 mmol) and used without further purification.
– MS (ES) m/z: 443.2 (MH+) (100%). MS (HRMS) m/z
calcd for C24H32ClN4O2 (MH+): 443.2208; found:
443.2209.
5.1.6. tert-Butyl 2-{4-[6-(6-chloro-2-methoxyacridin-9-
ylamino)hexylamino]-4-oxobutylamino}-2-oxoethylcarba-
mate (7).
A
mixture of Boc-Gly-OH (376 mg,
2.14 mmol), BOP (1,14 g, 2.58 mmol), and DIEA
(450 lL, 2.58 mmol) in DMF (11 mL) was stirred for
15 min to allow the formation of the activated ester.
Compound
5
(946 mg, 2.14 mmol) and DIEA
(1.87 mL, 10.7 mmol) were dissolved in DMF (11 mL)
and added dropwise to the activated ester. The reaction
mixture was stirred overnight in the dark at room tem-
perature. DMF was removed under reduced pressure
– 1H NMR (CDCl3, 250 MHz) d ppm: 1.35 (s, 9H), 1.75
(qn, 2H), 2.35 (t, 2H), 3.10 (q, 2H), 4.85 (t, 1H), 6.05
(s, 1H).

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L. Guetzoyan et al. / Bioorg. Med. Chem. 15 (2007) 3278–3289
1
and the residue was dissolved in acetone (14 mL) and
added dropwise to a stirred solution of 5% NaHCO3
(140 mL), according to the procedure previously devel-
oped by Kossanyi et al.36 The mixture was allowed to
stand for 24 h at room temperature. The resulting pre-
cipitate was filtered and dried, and acridine 7 was ob-
tained as an amorphous powder in 73% yield (940 mg,
1.57 mmol), after MPLC in CH2Cl2/MeOH (90/10).
– H NMR (CD3OD, 250 MHz) d ppm: 1.10–1.40 (m,
12H), 1.40 (m, 2H), 1.65 (m, 6H), 1.98 (s, 3H), 2.03
(t, 2H), 2.38 (s, 3H), 2.45 (s, 3H), 2.80 (s, 2H), 2.95
(t, 2H), 3.10 (m, 4H), 3.65 (t, 2H), 3.78 (m, 2H),
3.84 (s, 3H), 3.90 (t, 1H), 4.00 (q, 1H), 4.25 (t, 2H),
7.05–7.25 (m, 5H), 7.30 (dd, J1 = 2.2 Hz, J2 = 9.7 Hz,
1H), 7.40–7.50 (m, 2H), 7.63 (d, J = 7.5 Hz, 2H),
7.70 (dd, J1 = 9.7 Hz, 2H), 8.13 (d, J = 9.7 Hz, 1H).
–
13C NMR (CD3OD, 63 MHz) d ppm: 12.5, 18.4, 19.6,
26.7, 27.5, 28.6, 30.2, 31.0, 32.0, 34.2, 39.7, 40.0, 43.9,
44.0, 46.2, 56.4, 59.9, 63.5, 69.7, 87.6, 102.2, 109.4,
120.9, 124.4, 126.1, 126.7, 127.9, 128.1, 128.8, 133.4,
134.3, 139.0, 142.3, 145.0, 148.3, 151.6, 157.5, 158.0,
158.7, 171.5, 172.5, 175.0.
1
– H NMR (CDCl3, 250 MHz) d ppm: 1.41 (m, 15H),
1.80 (m, 4H), 2.18 (t, J = 5.7 Hz, 2H), 3.25 (t,
J = 5.7 Hz, 2H), 3.30 (t, J = 5.7 Hz, 2H), 3.75 (m,
4H), 3.87 (s, 3H), 5.40 (m, 1H), 6.50 (m, 1H), 6.61
(m, 1H), 7.08 (d, J = 2.4 Hz, 1H), 7.16 (dd,
J1 = 2.4 Hz, J2 = 9.3 Hz), 7.20 (d, J = 2.4 Hz, 1H),
7.28 (dd, J1 = 2.4 Hz, J2 = 9.3 Hz, 1H), 7.73 (d,
J = 9.3 Hz, 1H), 7.83 (s, 1H), 7.96 (d, J = 9.3 Hz, 1H).
– MS (ES) m/z: 1130.3 (MH+) (100%).
5.1.9. 2-Amino-N-(2-{4-[6-(6-chloro-2-methoxyacridin-9-
ylamino)hexylamino]-4-oxobutylamino}-2-oxoethyl)-5-[3-
(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-ylsulfo-
–
13C NMR (CD3OD, 63 MHz) d ppm: 26.7, 27.4, 28.6,
30.2, 30.6, 34.2, 39.6, 40.1, 44.7, 50.1, 56.6, 64.3, 80.6,
103.5, 110.8, 115.0, 118.6, 121.0, 121.4, 124.8, 128.3,
128.9, 135.5, 141.1, 141.4, 152.7, 157.8, 158.3, 175.2.
nyl)guanidine]-pentanamide
(11).
DBU
(340 lL,
2.28 mmol) was added to a solution of acridine 10
(644 mg, 0.57 mmol) in THF (5 mL) and the solution
was stirred at room temperature for 2 h to drive the
reaction to completion. The reaction mixture was
poured into diethyl ether (100 mL) whilst stirring. The
precipitate was filtered, washed with diethyl ether, and
dried. It was dissolved in CH2Cl2 (50 mL) and the solu-
tion was washed with water (3 · 50 mL), then brine, and
dried over Na2SO4. After evaporation in vacuo, acridine
11 was obtained in quantitative yield and used in the
next step without further purification.
– MS (ES) m/z: 600.3 (MH+) (100%).
5.1.7. 9-{6-[4-(2-Ammonioacetamido)butanamido]hexyl-
amino}-6-chloro-2-methoxyacridinium ditrifluoroacetate
(8). Dissolution of acridine 7 (940 mg, 1.57 mmol) in
TFA/CH2Cl2 (1/1) (5 mL) followed by the same treatment
as applied to 4 afforded the crude TFA salt 8, which was
used in the next step without further purification.
1
– H NMR (CD3OD, 250 MHz) d ppm: 1.50 (m, 6H),
1.79 (qn, J = 6.8 Hz, 2H), 1.98 (qn, 2H), 2.21 (t,
J = 6.8 Hz, 2H), 3.18 (t, J = 6.8 Hz, 2H), 3.26 (t,
J = 6.8 Hz, 2H), 3.65 (s, 2H), 3.99 (s, 3H), 4.12 (t,
J = 7.2 Hz, 2H), 7.50 (dd, J1 = 2.0 Hz, J2 = 7.9 Hz,
1H), 7.60 (dd, J1 = 2.0 Hz, J2 = 7.9 Hz, 1H), 7.70
(dd, J1 = 2.0 Hz, J2 = 7.9 Hz, 1H), 7.80 (dd,
J1 = 2.0 Hz, J2 = 7.9 Hz, 2H), 8.44 (d, J = 9.3 Hz, 1H).
1
– H NMR (CD3OD, 250 MHz) d ppm: 1.30 (m, 6H),
1.39 (s, 6H), 1.55 (m, 2H), 1.75 (m, 6H), 2.00 (s,
3H), 2.15 (t, J = 7.5 Hz, 2H), 2.46 (s, 3H), 2.54 (s,
3H), 2.91 (s, 2H), 3.15 (m, 6H), 3.80 (m, 4H), 3.93
(s, 3H), 7.24 (dd, J1 = 2.5 Hz, J2 = 9.3 Hz, 1H), 7.38
(dd, J1 = 2.5 Hz, J2 = 9.3 Hz, 1H), 7.49 (d,
J = 2.5 Hz, 1H), 7.82 (dd, J1 = 2.2 Hz, J2 = 9.3 Hz,
2H), 7.85 (s, 1H), 8.22 (d, J = 9.3 Hz, 1H).
–
13C NMR (CD3OD, 63 MHz) d ppm: 22.7, 25.8, 28.3,
29.1, 32.3, 38.7, 41.4, 48.7, 55.3, 62.4, 103.0, 117.3,
119.9, 123.6, 127.1, 140.4, 152.0, 156.0, 172.0.
–
13C NMR (CD3OD, 90 MHz) d ppm: 12.5, 14.5, 18.4,
19.6, 20.4, 20.9, 24.9, 26.7, 27.4, 27.6, 28.7, 30.3, 32.0,
33.1, 34.1, 39.3, 39.8, 40.2, 43.4, 43.9, 50.3, 50.9, 55.3,
55.7, 56.6, 87.6, 101.2, 115.6, 118.4, 124.1, 126.0,
126.1, 126.8, 127.2, 130.2, 133.4, 136.4, 139.3, 147.0,
149.3, 152.9, 157.2, 158.0, 159.8, 171.5, 175.3, 178.2.
– MS (ES) m/z: 500.2 (MH+) (100%). MS (HRMS): m/z
calcd for C26H35ClN5O3 (MH+): 500.2423; found:
500.2429.
5.1.8. (9H-Fluoren-9-yl)methyl 22-(6-chloro-2-methoxy-
acridin-9-ylamino)-1-imino-7,10,15-trioxo-1-(2,2,4,6,7-
pentamethyl-2,3-dihydrobenzofuran-5-sulfonamido)-
2,8,11,16-tetraazadocosan-6-ylcarbamate (10). A mixture
of Fmoc-Arg (Pbf)-OH (1.12 g, 1.73 mmol), BOP
(0.84 g, 1.90 mmol), and DIEA (330 lL, 1.90 mmol) in
DMF (10 mL) was stirred at room temperature for
30 min. Acridine 8 (1.57 mmol) was dissolved in DMF
(10 mL) with DIEA (1.36 mL, 7.85 mmol) and added
dropwise to the activated ester. The reaction mixture
was stirred for 3 days, then the solvent was evaporated.
Acetone (10 mL) was added to dissolve the crude residue
and the solution was poured dropwise into a solution of
5% NaHCO3 (100 mL). The mixture was allowed to
stand overnight and the resulting precipitate was fil-
tered, dried, and purified by MPLC using gradient of
CH2Cl2/MeOH from 2% to 15%. Acridine 10 was ob-
tained in 58% yield (1.03 g, 0.91 mmol).
– MS (ES) m/z: 454.6 (M+2H+)/2 (100%), 908.3 (MH+)
(45%).
5.1.10. 9-(1-Amino-6-ammonio-1-iminio-7,10,15-trioxo-
2,8,11,16-tetraazadocosan-22-ylamino)-6-chloro-2-meth-
oxyacridinium trichloride (12). Acridine 11 (250 mg,
0.275 mmol) was dissolved in TFA/H2O (95/5)
(25 mL),
triethylsilane
was
added
(88 lL,
0.550 mmol), and the resulting solution was stirred
at room temperature overnight. The solvents were re-
moved under reduced pressure. The crude residue was
dissolved in water (1 mL) and this solution was
passed through a column of ion exchange resin Dow-
ex Clꢀ. The aqueous layer was lyophilized and the
product was precipitated in H2O/acetone to afford
12 as
0.132 mmol).
a yellow powder in 48% yield (87 mg,

L. Guetzoyan et al. / Bioorg. Med. Chem. 15 (2007) 3278–3289
3285
–
1H NMR (CD3OD, 400 MHz) d ppm: 1.46 (q,
J = 6.7 Hz, 2H), 1.54 (q, J = 6.7 Hz, 4H), 1.81 (m,
2H), 2.06 (q, J = 7.3 Hz, 4H), 2.26 (s, 2H), 3.20 (t,
2H), 3.21 (m, 2H), 3.26 (m, 2H), 3.78 (m, 2H), 3.96
(s, 2H), 4.03 (s, 3H), 4.10 (t, 1H), 4.19 (m, 2H), 7.50
(dd, J1 = 2.2 Hz, J2 = 9.5 Hz, 1H), 7.65 (dd, J1 =
2.2 Hz, J2 = 9.5 Hz, 1H), 7.82 (dd, J1 = 2.2 Hz, J2 =
9.5 Hz, 2H), 7.85 (s,1H), 8.50 (d, J = 9.5 Hz, 1H).
13C NMR (CD3OD, 100 MHz) d ppm: 19.0, 23.5,
23.9, 25.3, 26.1, 28.0, 28.6, 28.7, 29.2, 37.9, 38.5,
38.9, 40.4, 42.1, 47.0, 47.2, 47.4, 47.6, 47.8, 48.1,
48.3, 48.9, 52.8, 55.6, 59.6, 60.9, 67.2, 102.9, 117.1,
120.1, 123.7, 127.4, 140.4, 156.6, 156.8, 157.2, 166.1,
169.2, 169.8, 174.2, 177.7, 180.0.
temperature for 30 min. Then a solution of BnBr
(5.03 mL, 42.3 mmol) in DMF (10 mL) was added
dropwise at room temperature. The reaction mix-
ture was stirred overnight. After filtration and
evaporation, the crude product was purified by
chromatography over silica gel with pentane/diethyl
ether (90/10) to afford 16 in 70% yield (8.261 g,
29.6 mmol).
–
1
– H NMR (CDCl3, 250 MHz) d ppm: 1.40–1.55 (m,
9H), 2.61 (t, J = 7.2 Hz, 2H), 3.46 (q, J = 7.2 Hz,
2H), 5.24 (s, 2H), 7.39 (s, 5H).
–
13C NMR (CDCl3, 90 MHz) ppm: 28.2, 34.5, 36.0,
66.2, 79.1, 128.0, 128.1, 128.4, 135.5, 155.6, 172.1.
– UV–vis (H2O): kmax nm (e molꢀ1 L cmꢀ1) = 280
(55,560), 340 (5060), 422 (10,300), 439 (9560).
– MS (ES) m/z: 328.7 (M+2H+)/2 (100%), 656.4 (MH+)
(35%).
– Anal. Calcd for C32H49Cl4N9O4, NaCl, 2H2O: C,
44.69; H, 6.21; N, 14.66. Found C, 44.68; H, 6.11;
N, 14.18.
– MS (ES) m/z: 302.1 (MNa+) (100%).
5.1.14. 2-Benzyloxycarbonyl-ethyl-ammonium trifluoro-
acetate (17). The TFA salt 17 was obtained by the same
treatment as applied to 4 and was used in the next step
without further purification.
1
– H NMR (CDCl3, 400 MHz) d ppm: 2.81 (m, 2H),
3.30 (m, 2H), 5.15 (s, 2H), 7.37 (s, 5H), 7.93 (m, 3H).
5.1.11. tert-Butyl 3-[6-(6-chloro-2-methoxyacridin-9-yl-
amino)hexylamino]-3-oxopropylcarbamate (13). Synthesis
of acridine 13 was performed by coupling of 1 and Boc-
b-Ala-OH, using the procedure already applied to the
synthesis of 4. Acridine 13 was obtained in 54% yield, after
purification by chromatography using CH2Cl2/MeOH
(95/5).
–
13C NMR (CDCl3, 90 MHz) d ppm: 30.6, 35.7, 67.3,
128.2, 128.5, 128.6, 134.8, 171.6.
5.1.15. Benzyl 3-(2-{[(9H-fluoren-9-yl)methoxy]carbon-
ylamino}-5-[3-(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofu-
ran-5-ylsulfonyl)guanidino]pentanamido)propionate (18).
DPPA (4.07 mL, 18.76 mmol) was added to a solution
of Fmoc-Arg-(Pbf)-OH (10.14 g, 15.63 mmol) in DMF
(250 mL). The mixture was stirred for 1 h at 0 ꢁC.
Then a solution of 17 (5.25 g, 18.76 mmol) in DMF
(100 mL) and DIEA (4.35 mL, 25 mmol) was added
dropwise at 0 ꢁC. The reaction mixture was stirred
overnight. After evaporation of the solvent and chro-
matography with heptane/EtOAc (90/10), 18 was ob-
tained in 81% yield (relative to arginine) (10.2 g,
12.61 mmol).
1
– H NMR (CDCl3, 200 MHz) d ppm: 1.37 (m, 15H),
1.66 (qn, J = 5.8 Hz, 2H), 2.30 (t, J = 6 Hz, 2H),
3.22 (q, J = 6.3 Hz, 2H), 3.31 (q, J = 6 Hz, 2H), 3.60
(t, J = 6.9 Hz, 2H), 3.88 (s, 3H), 5.31 (t, 1H), 6.19 (t,
1H), 7.20 (m, 2H), 7.34 (dd, J1 = 2.4 Hz, J2 = 9.3 Hz,
1H), 7.90 (m, 2H), 7.94 (d, J = 9.5 Hz, 1H).
–
13C NMR (CD3OD, 50 MHz) d ppm: 26.2, 28.3, 29.3,
31.3, 36.3, 36.7, 38.9, 50.1, 55.5, 79.3, 99.4, 115.3,
117.6, 124.2, 124.3, 124.5, 127.4, 130.6, 134.9, 146.0,
147.0, 150.0, 155.8, 171.3.
– MS (ES) m/z: 529.2 (MH+) (100%).
– 1H NMR (CDCl3, 250 MHz) d ppm: 1.37 (s, 6H), 1.50
(m, 2H), 1.70 (m, 2H), 2.01 (s, 3H), 2.45 (s, 3H), 2.53
(s, 3H), 2.85 (s, 2H), 3.19 (m,2H), 3.47 (m, 2H), 4.10
(m, 2H), 4.29 (d, J = 7.3 Hz), 5.00 (s, 2H), 5.95, (m,
1H), 6.20 (m, 1H), 7.28 (m, 9H), 7.51 (d, J = 7.6 Hz,
2H), 7.68 (d, J = 7.6 Hz, 2H).
5.1.12.
9-[6-(3-Ammoniopropanamido)hexylamino]-6-
chloro-2-methoxyacridinium ditrifluoroacetate (14). The
TFA salt 14 was obtained by the same treatment as ap-
plied to 4 and was used in the next step without further
purification.
–
13C NMR (CDCl3, 63 MHz) d ppm: 12.4, 19.2, 25.2,
28.4, 46.9, 53.3, 86.3, 91.0, 108.2, 117.5, 119.8, 124.6,
127.0, 127.6, 128.1, 128.2, 128.4, 132.1, 135.6, 138.2,
141.1, 143.6, 156.4, 158.7, 172.0.
1
– H NMR (CD3OD, 250 MHz) d ppm: 1.40–1.58 (m,
8H), 1.97 (t, 2H), 3.22 (m, 2H), 3.92 (s, 3H), 4.01 (t,
2H), 7.34 (d, J = 9.3 Hz, 1H), 7.45 (d, J = 9.5 Hz,
1H), 7.56 (m, 2H), 8.24 (d, J = 9.5 Hz, 2H).
– MS (ES) m/z: 832.5 (MNa+) (100%), 810.5 (MH+)
(32%).
–
13C NMR (CD3OD, 90 MHz) d ppm: 26.2, 28.8, 29.3,
31.4, 35.7, 38.8, 49.0, 55.3, 114.0, 117.2, 120.1, 123.6,
127.3, 130.0, 132.0, 141.0, 159.0, 171.0.
5.1.16. 3-(2-{[(9H-Fluoren-9-yl)methoxy]carbonylamino}-
5-[3-(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl-
sulfonyl)guanidine]pentanamido)propionic acid (19). One
gram of Pd/C was added to a solution of benzylester
derivative 18 (10.2 g, 12.6 mmol) in EtOAc (200 mL).
Compound 19 was obtained by hydrogenolysis after
48 h under pressure (10 bars). Filtration over Celite
and evaporation afforded a white solid, which was crys-
tallized in heptane/EtOAc. Compound 19 was obtained
in 94% yield (8.56 g, 11.9 mmol).
– MS (ES) m/z: 429.2 (MH+) (100%). MS (HRMS): m/z
calcd for C23H30ClN4O2 (MH+): 429.2052; found:
429.2065.
5.1.13. Benzyl 3-(tert-butoxycarbonylamino)propionate
(16). Boc-b-Ala-OH (8 g, 42.3 mmol) was dissolved
in DMF (20 mL). K2CO3 (5.85 g, 42.3 mmol) was
added and the reaction mixture was stirred at room

3286
L. Guetzoyan et al. / Bioorg. Med. Chem. 15 (2007) 3278–3289
–
1H NMR (CDCl3, 250 MHz) d ppm: 1.17 (m, 6H), 1.42
–
1H NMR (CD3OD, 400 MHz) d ppm: 1.30–1.64 (m,
(m, 2H), 1.45 (m, 2H), 1.94 (s, 3H), 2.37–2.46 (m, 8H),
2.80 (s, 2H), 3.15 (m, 2H), 3.43 (m, 1H), 4.00 (t, 1H),
4.17 (m, 2H), 6.41 (m, 2H), 7.20 (m, 4H), 7.56 (d,
J = 10.4 Hz, 2H), 7.63 (d, J = 10.4 Hz, 2H).
8H), 1.40 (s, 6H), 1.77 (m, 4H), 2.02 (s, 3H), 2.34–
2.38 (m, 4H), 2.47 (s, 3H), 2.54 (s, 3H), 2.90 (s, 2H),
3.08–3.15 (m, 4H), 3.38–3.46 (m, 6H), 3.77 (t,
J = 7 Hz, 2H), 3.91 (s, 3H), 7.20 (dd, J1 = 2.0 Hz,
J2 = 9.6 Hz, 1H), 7.36 (dd, J1 = 2.8 Hz, J2 = 9.2 Hz,
1H), 7.44 (d, J = 2.8 Hz, 1H), 7.72 (d, J = 9.6 Hz,
1H), 7.75 (d, J = 2.0 Hz, 1H), 8.15 (d, J = 9.2 Hz, 1H).
13C NMR (CD3OD, 63 MHz) d ppm: 12.3, 19.3, 20.0,
27.2, 28.7, 30.3, 31.7, 35.1, 35.6, 38.7, 42.4, 48.2, 49.3,
53.9, 54.8, 86.0, 100.0, 116.9, 122.7, 124.4, 125.7,
128.8, 131.8, 132.3, 132.9, 137.9, 138.3, 155.2, 156.0,
157.8, 158.7, 172.0, 175.2.
–
13C NMR (CDCl3, 63 MHz) d ppm: 12.4, 17.9, 19.2,
25.1, 28.4, 29.6, 34.0, 35.4, 40.2, 43.0, 46.8, 50.4, 54.5,
67.0, 86.4, 117.5, 119.8, 123.9, 124.7, 125.1, 126.8,
127.0, 127.6, 132.2, 138.3, 141.1, 143.5, 143.7, 156.6,
158.8, 173.0, 175.5.
–
– MS (ES) m/z: 720.3 (MH+) (100%).
5.1.17. (9H-Fluoren-9-yl)methyl 22-(6-chloro-2-methoxy-
acridin-9-ylamino)-1-imino-7,11,15-trioxo-1-(2,2,4,6,7-
pentamethyl-2,3-dihydrobenzofuran-5-sulfonamido)-
2,8,12,16-tetraazadocosan-6-ylcarbamate (20). DIEA
(393 lL, 2.26 mmol) was added to a solution of 19
(0.81 g, 1.13 mmol) in DMF (10 mL) and BOP (0.5 g,
1.13 mmol). The resulting solution was stirred for
30 min, and a solution of acridine derivative 14
(1.13 mmol) and DIEA (393 lL, 2.26 mmol) in DMF
(20 mL) was added dropwise. The reaction mixture
was stirred for 48 h at room temperature. The DMF
was evaporated, the residue was dissolved in acetone
(10 mL) and added dropwise to a solution of 5% NaH-
CO3 (100 mL). The mixture was allowed to stand for
24 h at room temperature. This afforded a precipitate,
which was filtered, washed with water, and dried to give
20 in 33% yield (380 mg, 0.369 mmol) after purification
by chromatography using gradient of CH2Cl2/MeOH
from 98/2 to 85/15.
– MS (ES) m/z: 454.7 (M+2H+)/2 (100%), 908.2 (MH+)
(94%).
5.1.19. 9-(1-Amino-6-ammonio-1-iminio-7,11,15-trioxo-
2,8,12,16-tetraazadocosan-22-ylamino)-6-chloro-2-meth-
oxyacridinium trichloride (22). Et3SiH (15 lL,
0.08 mmol) was added in the dark to a solution of acri-
dine 21 (37 mg, 0.04 mmol) in TFA/H2O (90/10) (4 mL).
The resulting solution was stirred at room temperature
overnight. Deprotection was monitored by reverse phase
TLC in AcOH/MeOH/H2O (2/2/4). The solvents were
removed under reduced pressure and the crude residue
was washed with methanol, evaporated in vacuo, and
dissolved in water (1 mL). This solution was passed
through a column of ion exchange resin Dowex Clꢀ.
The aqueous layer was lyophilized and the product
was precipitated in H2O/acetone to afford acridine 22
as a yellow powder in 70% yield (18 mg, 0.028 mmol).
–
1H NMR (CD3OD, 250 MHz) d ppm: 1.22–1.45 (m,
10H), 1.34 (s, 6H), 1.86 (m, 2H), 1.99 (s, 3H), 2.32
(t, J = 6.4 Hz, 4H), 2.43 (s, 3H), 2.45 (s, 3H), 2.86
(s, 2H), 3.12 (t, J = 6.9 Hz, 4H), 3.38 (m, 6H), 3.88
(s, 3H), 3.94 (m, 5H), 4.20 (t, 1H), 7.22–7.45 (m,
6H), 7.50 (dd, J1 = 2.2 Hz, J2 = 9.3 Hz 1H), 7.62 (m,
4H), 7.73 (d, J = 7.4 Hz, 2H), 8.24 (d, J = 9.3 Hz,
1H).
–
1H NMR (CD3OD, 250 MHz): 1.50–1.67 (m, 4H),
2.40 (m, 4H), 3.17 (m, 4H), 3.40 (m, 4H), 3.97 (t,
1H), 4.10 (s, 3H), 4.12 (t, J = 2.2 Hz, 2H), 7.53 (dd,
J1 = 2.8 Hz, J2 = 9.2 Hz, 1H), 7.64 (dd, J1 = 2.8 Hz,
J2 = 7.2 Hz, 1H), 7.80 (d, J = 7.2 Hz, 1H), 7.89 (dd,
J1 = 2.8 Hz, J2 = 10.8 Hz, 2H), 8.50 (d, J = 9.2 Hz,
1H ).
–
13C NMR (CD3OD, 50 MHz) d ppm: 26.6, 26.7, 27.4,
30.1, 30.6, 34.3, 39.8, 39.9, 40.0, 40.1, 41.7, 56.9, 57.2,
58.4, 110.7, 118.5, 121.4, 121.5, 125.0, 128.6, 128.7,
128.8, 129.3, 129.5, 141.7, 141.8, 158.1, 169.0.
–
13C NMR (CD3OD, 50 MHz) d ppm: 12.5, 17.2,
18.4, 19.6, 27.0, 27.4, 28.7, 30.2, 30.7, 36.8, 37.2,
40.1, 41.3, 43.9, 56.2, 56.7, 67.8, 87.6, 104.1,
111.3, 111.8, 115.4, 118.6, 120.8, 121.5, 125.0,
126.3, 126.4, 127.0, 128.1, 128.7, 129.2, 133.4,
135.9, 139.3, 141.4, 141.7, 142.4, 144.9, 145.1,
157.7, 158.1, 159.8, 173.7, 174.7.
– UV–vis (H2O): kmax nm (e molꢀ1 L cmꢀ1) = 278
(52,810), 340 (4800), 422 (9920), 440 (9260).
– MS (ES) m/z: 328.8 (M+2H+)/2 (100%), 656.4 (MH+)
(39%).
– Anal. Calcd for C32H49Cl4N9O4, NaCl, 3H2O: C,
43.77; H, 6.31; N, 14.36. Found C, 43.80; H, 6.41;
N, 14.78.
– MS (ES) m/z: 1130.5 (MH+) (100%).
5.1.18. 2-Amino-N-(3-{3-[6-(6-chloro-2-methoxyacridin-
9-ylamino)hexylamino]-3-oxopropylamino}-3-oxopropyl)-
5-[3-(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl-
sulfonyl)guanidino]-pentanamide (21). DBU (27 lL,
0.18 mmol) was added to a solution of 20 (200 mg,
0.18 mmol) in THF (10 mL) and the solution was stirred
at room temperature for 3 days. The reaction mixture
was poured into diethyl ether (100 mL) whilst stirring.
The precipitate was filtered, washed with diethyl ether,
and dried. It was dissolved in CH2Cl2 (100 mL) and
the solution was washed with saturated aqueous NaCl
(50 mL). After preparative TLC in CH2Cl2/MeOH (90/
10), acridine 21 was obtained in 32% yield (53 mg,
0.058 mmol).
5.1.20. 9-(6-Ammoniohexylamino)-6-chloro-2-methoxy-
acridinium dichloride (2). Compound 1 (100 mg) was dis-
solved in TFA (2 mL). The mixture was stirred
overnight at room temperature, then TFA was evapo-
rated. The solid was dissolved in distilled water and
passed over an ion exchange resin Dowex Clꢀ. The chlo-
ride salt 2 was obtained quantitatively, after precipita-
tion in a water-acetone mixture and lyophilization.
– UV–vis (H2O): kmax nm (e molꢀ1 L cmꢀ1) = 276
(30,700), 340 (2375), 422 (5920), 442 (5510).
– MS (ES) m/z: 358.2 (MH+) (100%).

L. Guetzoyan et al. / Bioorg. Med. Chem. 15 (2007) 3278–3289
3287
– Anal. Calcd for C20H26Cl3N3O, 1.1NaCl, 1.1H2O: C,
46.65; H, 5.52; N, 8.16. Found C, 46.59; H, 5.49; N,
7.51.
5% NaHCO3 (60 mL). The mixture was allowed to
stand for 24 h at room temperature. This afforded
a precipitate, which was filtered and purified by
MPLC on silica gel (CH2Cl2/MeOH, 98/2). Acridine
24 was obtained as an amorphous powder in 64%
yield (1.78 g, 4.09 mmol).
5.1.21. 9-[6-(4-Ammoniobutanamido)hexylamino]-6-
chloro-2-methoxyacridinium dichloride (6). Compound
5 was dissolved in distilled water and passed over an
ion exchange resin Dowex Clꢀ. The chloride salt 6 was
obtained quantitatively, after precipitation in a water-
acetone mixture and lyophilization. Same treatment
applied to compounds 8 and 14 afforded the chloride
salts 9-{6-[4-(2-ammonioacetamido)butanamido]hexyla-
mino}-6-chloro-2- methoxyacridinium dichloride (9)
and 9-[6-(3-ammoniopropanamido)hexylamino]-6-chloro-
2-methoxyacridinium dichloride (15), respectively.
1
– H NMR (CDCl3, 250 MHz) d ppm: 1.35 (m, 17H),
1.75 (m, 2H), 2.40 (t, 2H), 3.10 (m, 3H), 4.70 (s,
1H), 6.95 (t, J = 7.4 Hz, 2H), 7.25 (t, J = 7.4 Hz,
2H), 7.50 (d, J = 8.6 Hz, 2H), 7.90 (d, J = 8.6 Hz, 2H).
–
13C NMR (DMSO, 63 MHz) d ppm: 25.3, 26.3, 28.3,
28.5, 28.6, 28.9, 29.6, 36.0, 77.9, 121.7, 122.6, 123.4,
127.9, 130.3, 147.9, 148.9, 150.8, 156.0.
– MS (ES) m/z: 458.3 (MNa+) (100%).
Acridine 6
5.1.24. 8-(Acridin-9-ylamino)-8-oxooctan-1-ammonium
trifluoroacetate (25). Treatment of 24 with TFA (same
procedure as for 4) afforded acridine 25 in quantitative
yield.
– UV–vis (H2O): kmax nm (e molꢀ1 L cmꢀ1) = 276
(45,980), 340 (3875), 422 (8540), 442 (8080).
– MS (ES) m/z: 443.2 (MH+) (100%).
1
– Anal. Calcd for C24H33Cl3N4O2, HCl, H2O: C, 50.54;
H, 6.36; N, 9.82. Found C, 50.88; H, 6.29; N, 9.89.
– H NMR (CD3OD, 250 MHz) d ppm: 1.40 (m, 6H),
1.70 (m, 2H), 1.90 (m, 2H), 2.80 (t, 2H), 2.90 (m,
2H), 7.20 (t, 1H), 7.50 (t, 1H), 7.70 (dd, 2H), 8.10
(dd, 2H), 8.30 (d, 1H).
Acridine 9
–
13C NMR (CD3OD, 50 MHz) d ppm: 26.3, 27.3, 28.5,
29.9, 30.1, 37.4, 40.7, 112.3, 119.5, 120.7, 122.7, 124.9,
125.1, 127.2, 128.5, 136.6, 138.3, 140.2, 141.5, 153.4,
175.5.
– UV–vis (H2O): kmax nm (e molꢀ1 L cmꢀ1) = 278
(36,750), 340 (3010), 420 (7070), 440 (6755).
– MS (ES) m/z: 500.2 (MH+) (100%).
– Anal. Calcd for C26H36Cl3N5O3, 0.7NaCl, 3H2O: C,
46.75; H, 6.34; N, 10.49. Found C, 46.74; H, 6.31;
N, 11.27.
– MS (ES) m/z: 336.3 (MH+) (100%).
5.1.25. tert-Butyl 2-[8-(acridin-9-ylamino)-8-oxooctyl-
amino]-2-oxoethylcarbamate (26). Coupling of 25
(324 mg, 0.967 mmol) with Boc-Gly-OH was performed
as described for synthesis of 7 and afforded acridine 26
in 38% yield (181 mg, 0.367 mmol).
Acridine 15
– UV–vis (H2O): kmax nm (e molꢀ1 L cmꢀ1) = 278
(35,265), 340 (2640), 422 (6780), 442 (6535).
– MS (ES) m/z: 429.2 (MH+) (100%).
– 1H NMR (CD3OD, 250 MHz ) d ppm: 1.35 (m, 17H),
1.75 (qn, J = 8 Hz, 2H), 2.60 (t, J = 8 Hz, 2H), 3.10 (t,
2H), 3.55 (d, 2H), 7.50 (t, J = 7.4 Hz, 2H), 7.75 (t,
J = 7.4 Hz, 2H), 8.00 (d, J = 8.6 Hz, 2H), 8.04 (d,
J = 8.6 Hz, 2H).
– Anal. Calcd for C23H31Cl3N4O2, 4.3H2O: C, 47.68; H,
6.89; N, 9.67. Found C, 47.62; H, 7.16; N, 11.55.
5.1.22. 8-(tert-Butoxycarbonylamino)caprylic acid (23).
Boc-protection of 8-aminocaprylic acid was performed
as described for 3. Compound 23 was obtained as a yel-
low oil in 90% yield (2.33 g, 9 mmol) and used without
further purification.
–
13C NMR (CD3OD, 50 MHz) d ppm: 25.4, 26.0, 28.2,
28.3, 28.8, 29.2, 36.4, 39.0, 44.4, 80.3, 122.6, 123.4,
125.9, 126.2, 129.5, 130.2, 149.1, 155.0, 169.7, 173.1.
– MS (ES) m/z: 515.2 (MNa+) (100%), 493.2 (MH+)
(32%).
1
– H NMR (CDCl3, 250 MHz) d ppm: 1.30 (m, 8H),
1.40 (s, 9H), 1.60 (m, 2H), 2.30 (t, J = 6.6 Hz, 2H),
3.00 (q, 2H), 4.55 (s, 1H), 5.75 (s, 1H).
5.1.26. 2-[8-(Acridin-9-ylamino)-8-oxooctylamino]-2-oxo-
ethanammonium trifluoroacetate (27). Acridine 27 was
obtained from 26 quantitatively according to the proce-
dure described for the preparation of 5.
–
13C NMR (CDCl3, 50 MHz) d ppm: 23.9, 26.4, 28.4,
28.8, 28.9, 29.9, 34.0, 40.3, 84.0, 155.9, 169.4.
– MS (ES) m/z: 258.3 (MH+) (100%).
1
– H NMR (CD3OD, 250 MHz) d ppm: 1.50 (m, 8H),
5.1.23. tert-Butyl 8-(acridin-9-ylamino)-8-oxooctylcar-
bamate (24). A mixture of 23 (1.65 g, 6.37 mmol),
BOP (3.1 g, 7 mmol), and DIEA (1.33 mL,
7.64 mmol) in DMF (25 mL) was stirred at room
temperature for 15 min. Then, 9-aminoacridine
(6.37 mmol) was added and the reaction mixture
was stirred overnight in the dark. The solvent was
evaporated and the residue was dissolved in acetone
(6 mL), and added dropwise to a stirred solution of
1.88 (m, 2H), 2.88 (t, J = 7 Hz, 2H), 3.25 (t,
J = 6.7 Hz, 2H), 3.70 (s, 2H), 7.86 (t, J = 7.4 Hz,
2H), 8.25 (m, 4H), 8.41 (d, J = 8.5 Hz, 2H).
–
13C NMR (CD3OD, 50 MHz) d ppm: 26.3, 27.7, 30.0,
30.1, 30.3, 37.4, 40.5, 41.4, 112.3, 119.5, 120.7, 122.6,
122.7, 125.1, 127.1, 128.7, 136.6, 138.3, 140.2, 141.6,
151.5, 152.9, 175.5.
– MS (ES) m/z: 393.2 (MH+) (100%), 415.2 (MNa+)
(72%).

3288
L. Guetzoyan et al. / Bioorg. Med. Chem. 15 (2007) 3278–3289
5.1.27. (9H-Fluoren-9-yl)methyl 1-{2-[8-(acridin-9-ylami-
no)-8-oxooctylamino]-2-oxoethylamino}-1-oxo-5-[3-(2,2,
4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-ylsulfonyl)gu-
anidino]pentan-2-ylcarbamate (28). A mixture of Fmoc-
Arg(Pbf)-OH (238 mg, 0.367 mmol), BOP (195 mg,
0.44 mmol), and DIEA (76 lL, 0.44 mmol) in DMF
(15 mL) was stirred at room temperature for 45 min,
then cooled to 0 ꢁC. Acridine 27 (144 mg, 0.367 mmol)
and DIEA (320 lL, 1.835 mmol) were dissolved in
DMF (5 mL) and added dropwise at 0 ꢁC to the acti-
vated ester. The reaction mixture was stirred for
20 min at 0 ꢁC, then overnight at room temperature.
After usual work-up, the precipitate was purified by
MPLC using gradient of CH2Cl2/MeOH from 99/1 to
90/10. Acridine 28 was obtained in 45% yield (170 mg,
0.166 mmol).
0.025 mmol) was dissolved in TFA/H2O (95/5) (2 mL)
and triethylsilane was added (8 lL, 0.05 mmol). The
reaction mixture was stirred overnight at room temper-
ature and the solvent was evaporated. The crude residue
was dissolved in water (1 mL) and this solution was
passed through a column of ion exchange resin Dowex
Clꢀ. The aqueous layer was lyophilized and afforded
acridine 30 as a yellow powder in quantitative yield
(13.8 mg, 0.025 mmol).
1
– H NMR (CD3OD, 400 MHz) d ppm: 1.58 (m, 4H),
1.75 (m, 4H), 1.91 (q, J = 8 Hz, 4H), 1.99 (q,
J = 5.6 Hz, 2H), 2.93 (m, 2H), 3.29 (t, J = 14 Hz,
4H), 3.85 (s, 2H), 4.05 (t, J = 6 Hz, 1H), 7.89 (t,
J = 7.2 Hz, 2H), 8.25 (t, J = 7.2 Hz, 2H), 8.35 (d,
J = 7.2 Hz, 2H), 8.45 (d, J = 7.2 Hz, 2H).
–
13C NMR (CD3OD, 50 MHz) d ppm: 25.1, 26.3, 27.7,
29.4, 30.0, 30.2, 37.6, 40.4, 41.8, 43.3, 54.0, 120.9,
123.1, 127.4, 128.8, 138.6, 141.9, 158.5, 170.3, 175.5.
1
– H NMR (CD3OD, 400 MHz) d ppm: 1.40–1.92 (m,
16H), 2.13 (m, 4H), 2.17 (s, 3H), 2.40 (s, 3H), 2.45
(s, 3H), 2.58 (t, 2H), 2.80 (s, 2H), 3.02 (q, J= 5.7 Hz,
4H), 3.22 (q, J = 14 Hz, 2H), 3.77 (t, 1H), 4.07 (t,
1H), 4.27 (t, 2H), 7.14 (t, J = 7.4 Hz, 2H), 7.24 (t,
J = 7.1 Hz, 2H), 7.48 (t, J = 7.6 Hz, 4H), 7.65 (d,
J = 7.2 Hz, 2H), 7.71 (t, J = 7.3 Hz, 2H), 7.98 (d,
J = 8.7 Hz, 2H), 8.04 (d, J = 8.8 Hz, 2H).
– UV–vis (H2O): kmax nm (e molꢀ1 L cmꢀ1) = 251
(33,900), 358 (4200).
– MS (ES) m/z: 549.4 (MH+) (100%), 275.4 (M+2H+)/2
(56%).
– Anal. Calcd for C29H42Cl2N8O3, 1.5 NaCl, 3H2O: C,
45.63; H, 6.34; N, 14.68. Found C, 45.52; H, 6.51;
N, 13.87.
–
13C (NMR CD3OD, 100 MHz) d ppm: 12.5, 18.4, 19.6,
25.5, 26.3, 28.0, 28.6, 28.7, 28.9, 29.3, 35.8, 39.1, 42.2,
42.5, 46.9, 48.2, 53.4, 66.6, 86.2, 117.0, 122.9, 124.6,
132.1, 132.8, 138.0, 140.4, 141.2, 143.7, 148.8, 156.7,
157.4, 158.4, 169.9, 173.9, 174.7.
5.1.30. Compound 31
– UV–vis (H2O): kmax nm (e molꢀ1 L cmꢀ1) = 251
(88,300), 358 (7600).
– MS (ES) m/z: 1023.4 (MH+) (100%).
– MS (ES) m/z: 535.3 (MH+) (100%), 268.2 (M+2H+)/2
(90%). MS (HRMS) m/z calcd for C28H39N8O3
(MH+): 535.3145; found: 535.3151.
5.1.28. 1-{2-[8-(Acridin-9-ylamino)-8-oxooctylamino]-2-
oxoethylamino}-1-oxo-5-[3-(2,2,4,6,7-pentamethyl-2,3-
dihydrobenzofuran-5-ylsulfonyl)guanidine]pentan-2-
ammonium chloride (29). DBU (100 lL, 0.66 mmol) was
added to a solution of acridine 28 (170 mg, 0.166 mmol)
in THF (5 mL) and the mixture was stirred at room tem-
perature for 2 h. It was then poured into diethyl ether
(100 mL) whilst stirring. The precipitate was filtered,
washed with diethyl ether, and dried. It was then dis-
solved in CH2Cl2 (50 mL) and the solution was washed
with water (3 · 50 mL), then with saturated aqueous
NaCl solution (50 mL), and dried over Na2SO4. After
evaporation in vacuo, acridine 29 was obtained in quan-
titative yield.
– Anal. Calcd for C28H40Cl2N8O3, NaCl, 2H2O: C,
47.90; H, 6.32; N, 15.96. Found C, 47.78; H, 6.29;
N, 15.46.
5.2. Biology
5.2.1. P. falciparum strains. Both CQ-sensitive (3D7)
and CQ-resistant (W2, FCR3, and Bre1) P. falciparum
strains maintained continuously in culture were used.
Synchronous parasites were diluted with uninfected
erythrocytes (A-positive human blood) and completed
RPMI 1640 medium (Invitrogen, Paisley, United King-
dom), supplemented with 10% human serum Abcys S.A.
(Paris, France) and buffered with 25 mM HEPES and
25 mM NaHCO3 to achieve 0.2 parasitemia and 1.5
hematocrit.
1
– H NMR (CD3OD, 250 MHz) d ppm: 1.20–1.80 (m,
20H), 1.93 (s, 3H), 2.36 (s, 3H), 2.43 (s, 3H), 2.57 (t,
J = 7.8 Hz, 2H), 2.85 (s, 2H), 3.02 (m, 4H), 3.69 (t,
J = 4.3 Hz, 1H), 3.85 (q, J = 18.4 Hz, 2H), 7.45 (t,
J = 8 Hz, 2H), 7.70 (t, J = 7.4 Hz, 2H), 8.00 (d,
J = 8.6 Hz, 2H), 8.07 (d, J = 8.6 Hz, 2H).
5.2.2. Measurement of in vitro antimalarial activity. Solu-
tions of drugs were prepared in RPMI 1640 medium and
distributed in triplicate into Falcon 96-well flat-bottomed
plates (Becton Dickinson, Franklin Lakes, NJ) to
achieve concentrations ranging from 0.06 lM to 200 lM.
–
13C NMR (CD3OD, 50 MHz) d ppm: 12.5, 18.4, 19.6,
26.5, 26.8, 27.7, 28.7, 30.0, 30.3, 33.1, 37.1, 40.4, 41.6,
43.3, 43.9, 55.6, 87.6, 118.4, 124.3, 125.1, 125.4, 125.9,
127.3, 129.4, 132.1, 133.4, 134.3, 139.3, 141.7, 150.1,
158.1, 159.8, 171.3, 176.1, 177.8.
For in vitro isotopic microtests, 200 lL/well of the sus-
pension of parasitized erythrocytes was distributed in
96-well plates predosed with antimalarial agents. Para-
site growth was assessed by adding 1 lCi of [3H]hypo-
xanthine with a specific activity 14.1 Ci/mmol (NEN
Products, Dreiech, Germany) to each well. Plates were
incubated for 42 h at 37 ꢁC in an atmosphere of 10%
– MS (ES) m/z: 801.5 (MH+) (100%).
5.1.29. 1-{2-[8-(Acridin-9-ylamino)-8-oxooctylamino]-2-
oxoethylamino}-5-[amino(iminio)methylamino]-1-oxopen-
tan-2-ammonium dichloride (30). Acridine 29 (20 mg,

L. Guetzoyan et al. / Bioorg. Med. Chem. 15 (2007) 3278–3289
3289
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were collected on standard filter microplates (UnifilterTM
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23. Far, S.; Kossanyi, A.; Verchere-Beaur, C.; Gresh, N.;
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The authors thank R. Amalvict, E. Baret, and J. Mos-
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nier (Institut de Medecine Tropicale du Service de Sante
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´
2004, 1781–1797.
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