Synthesis of N1-arylidene-N2-quinolyl- and N2-acrydinylhydrazones as potent antimalarial agents active against CQ-resistant P. falciparum strains

Article abstract of DOI:10.1016/j.bmcl.2006.07.060

A series of N1-arylidene-N2-quinolyl- and N2-acrydinylhydrazones were synthesized and tested for their antimalarial properties. These compounds showed remarkable anti-plasmodial activity in vitro especially against chloroquine-resistant strains. Their pot

Full text of DOI:10.1016/j.bmcl.2006.07.060

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5384–5388
Synthesis of N1-arylidene-N2-quinolyl- and N2-
acrydinylhydrazones as potent antimalarial agents
active against CQ-resistant P. falciparum strains
Sandra Gemma,a,b Gagan Kukreja,a,b Caterina Fattorusso,c,b Marco Persico,c,b
Maria P. Romano,a,b Maria Altarelli,a,b Luisa Savini,a,b Giuseppe Campiani,a,b,
Ernesto Fattorusso,c,b Nicoletta Basilico,d,b Donatella Taramelli,d,b
Vanessa Yardleye,b and Stefania Butinia,b
*
a
`
Dipartimento Farmaco Chimico Tecnologico, Universita di Siena, via Aldo Moro, 53100 Siena, Italy
`
b
European Research Centre for Drug Discovery and Development, Universita di Siena, via Aldo Moro, 53100 Siena, Italy
c
`
Dip. di Chimica delle Sostanze Naturali, Universita di Napoli Federico II, via D. Montesano 49, 80131 Napoli, Italy
d
`
Dip. di Sanita Pubblica-Microbiologia-Virologia, Universita’ di Milano, Via Pascal 36, 20133 Milano, Italy
eDepartment of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine,
Keppel Street, London WC1E 7HT, UK
Received 10 July 2006; revised 19 July 2006; accepted 20 July 2006
Available online 4 August 2006
Abstract—A series of N1-arylidene-N2-quinolyl- and N2-acrydinylhydrazones were synthesized and tested for their antimalarial
properties. These compounds showed remarkable anti-plasmodial activity in vitro especially against chloroquine-resistant strains.
Their potent biological activity makes them promising lead structures for the development of new antimalarial drugs.
Ó 2006 Elsevier Ltd. All rights reserved.
Malaria is a disease caused by parasitic protozoa of the
genus Plasmodium which afflicts more than 500 million
people worldwide, causing approximately 2 million
deaths each year. Despite significant advances in under-
standing the disease and the parasite, malaria still remains
one of the leading causes of morbidity and mortality, par-
ticularly in malaria-endemic regions of the world.1,2 For
decades, chloroquine (CQ, 1, Fig. 1) provided reliable
prophylaxis for travelers and therapy for those withestab-
lished infection. However, the emergence in the early
1960s and subsequent spread of CQ-resistant parasites
created a tremendous therapeutic void.3 As a result, there
is an urgent need for the rapid development of effective,
safe, and affordable chemotherapeutics.
Me
Me
NEt2
NEt2
HN
HN
N
MeO
Cl
N
CQ, 1
Cl
OH
NEt2
Mepacrine, 2
HN
N
Cl
Amodiaquine, 3
NH-N=CH-Ar
NH-N=CH-Ar
MeO
X
R
N
N
5a-c
Cl
The exact mode of action of CQ and other 4-amino
quinoline antimalarials, such as mepacrine 2 and amodi-
aquine 3, remains to be elucidated,4 but most investiga-
4a-g
Figure 1. Reference and title compounds.
tors accept that a crucial step in their mechanism of
action is the interference with the detoxification of free
heme,5–7 which is needed for the uninterrupted growth
Keywords: Malaria; Hydrazones; Hemozoin.
*
Corresponding author. Tel.: +39 0577 234172; fax: +39 0577
234333; e-mail: campiani@unisi.it
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.07.060

S. Gemma et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5384–5388
5385
and proliferation of the parasite.8 Although the molecu-
lar basis of CQ resistance is not fully understood, it is
clear from biochemical studies that the CQ-resistant
parasites accumulate fewer drugs than sensitive
strains.9,10 The fact that CQ resistance emerged after
decades of widespread use suggests that CQ activity is
not related to the interaction with a unique protein tar-
get.11–13 In fact, unlike inhibitors of parasite-encoded
enzymes and transporters that are currently under inves-
tigation and that may lead to the rapid appearance of
resistance under drug-pressure, the parasite has difficul-
ty in developing resistance to drugs that interfere with
the heme detoxification process as it seems not to be
dependent on any specific enzyme.14 Therefore, the met-
abolic functions related to hemoglobin digestion and
heme detoxification pathways still represent a valid tar-
get for the discovery of new antimalarial drugs.
These compounds were then heated under reflux for sev-
eral hours in the presence of hydrazine monohydrate with
methanol as the solvent.
Some of the 4-quinolylhydrazines were prepared using
an alternative procedure in which microwave radiation
was used as the heating source and the reaction was per-
formed in neat hydrazine monohydrate under pressure.
This latter method afforded corresponding hydrazines
in 80–90% yields, generally higher than that obtained
using the standard methods. In the next step of the syn-
thesis, hydrazines 8a–d and 9a,b were reacted with one
equivalent of the appropriate carboxaldehyde in boiling
ethanol and in the presence of an equimolar amount of
sodium acetate.22 Most of carboxaldehydes used for the
synthesis of final compounds were commercially
available except for aldehydes 12a and 12b which were
synthesized from the bromoester 10 as described in
Scheme 2. Alkylation of diethylamine or pyrrolidine
with bromide 10 was performed in refluxing acetone in
the presence of potassium carbonate and of a catalytic
amount of 18-crown-6. The resulting tertiary amines
11a,b were reacted with lithium aluminum hydride in
THF and the alcohols thus obtained were oxidized to
aldehydes 12a,b using manganese dioxide in refluxing
dioxane.
The compounds reported here were designed to investi-
gate novel chemical entities characterized by high activ-
ity against CQ-resistant Plasmodium falciparum (Pf)
strains and by a low resistance potential due to a mech-
anism of action that may be similar to that of 4-amino-
quinoline antimalarials. Accordingly, taking into
account that CQ analogues incorporating a hydrazone
or a hydrazine moiety in the side chain showed moder-
ate antimalarial activity,15 we developed a small series
of hydrazones characterized by the presence of different-
ly substituted quinolines (4a–g, Fig. 1) and acridines
(5a–c), structurally related to CQ and mepacrine, respec-
tively. These compounds were designed to interact with
iron III FPIX (hematin), possibly generating toxic radi-
cal species capable of interfering with the crucial reduc-
ing milieu of the parasite.16
All synthesized compounds were tested in vitro against a
series of Pf strains, namely the CQ-sensitive D10 and
3D7, and the CQ-resistant W2 and K1 strains of Pf.
The antimalarial activity (IC50, nM) was quantified as
inhibition of parasite growth, measured with the pro-
duction of parasite lactate dehydrogenase23 (D10 and
W2 strains) or the incorporation of [3H]-hypoxanthines
(3D7 and K1 strains).24
Compounds 4a–g and 5a–c were obtained starting from
suitable 4-chloroquinolines 6a–d,17–20 2,9-dichloro-6-
methoxyacridine 7a, and 7,10-dichloro-2-methoxyben-
zo[b][1,5]naphthyridine 7b,21 as described in Scheme 1.
The results are presented in Table 1. Among the new
compounds synthesized 4d–g and 5a–c showed an anti-
plasmodial activity against the CQ-sensitive D10 strain
in the same range of CQ. Similarly, 4f and 4g displayed
the same activity of CQ against CQ-sensitive 3D7 strain,
while compound 5b was 10 times more potent than CQ.
Additionally, the data showed that most of the tested
compounds were highly active against CQ-resistant
strains, two analogues (5b and 5c) being more active
against W2 CQ-resistant than D10 CQ-sensitive strains.
In the quinoline series (4a–g), the 8-OMe and 6,7-meth-
ylendioxy substituents gave rise to compounds generally
less active than the corresponding 6-OMe and 7-Cl
derivatives. These structure–activity relationships
Cl
N
Cl
N
MeO
X
R
Cl
6a, R=8-OMe
7a, X=CH
7b, X=N
6b, R=6,7-(OCH2O)
6c, R=6-OMe
6d, R=7-Cl
a
a
NH2
HN
NH2
HN
MeO
X
Br
R
R
R
N
9a,b
Cl
N
8a-d
a
b,c
Et2NH, for 11a
pyrrolidine, for 11b
b
CO2Me
b
CHO
CO2Me
10
11a, R= NEt2
12a,b
5a-c
4a-g
11b, R=
N
Scheme 1. Reagents and conditions: (a) NH2NH2ÆH2O, MeOH, reflux
or NH2NH2ÆH2O, MW radiation, 200 W, 5 min, sealed tube; (b)
ArCHO, EtOH, reflux.
Scheme 2. Reagents and condition: (a) K2CO3, 18-crown-6, acetone,
reflux; (b) LiAlH4, THF; (c) MnO2, dioxane.

5386
S. Gemma et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5384–5388
Table 1. Anti-plasmodial activity of compounds 4a–g and 5a–c
b
b
Compound
R
Ar
X
Ionic forma
%
at
%
at D10c
W2d
3D7c
K1d
pH 7.2 pH 5.5 IC50 (nM) IC50 (nM) IC50 (nM) IC50 (nM)
e
e
e
e
4a
4b
4c
4d
4e
4f
8-OMe
8-OMe
6,7-(OCH2O)
6-OMe
6-OMe
6-OMe
7-Cl
—
—
—
—
—
—
—
P
3.50
96.48
0.02
64.54
35.46
—
519
337
210
83
254
356
163
104
128
5703
NT
155
NT
NT
11.0
19.1
112
1.0
NTf
NT
467
NT
NT
55.1
16.4
NT
4.5
N
A
NMe2
OMe
DP
P
—
2.08
64.02
33.90
99.37
0.63
—
3.74
N
P
96.26
75.97
24.03
—
N
A
NMe2
OMe
DP
P
0.05
3.20
95.86
0.94
98.73
1.27
—
67.07
32.88
60.77
39.22
0.01
N
P
99
N
A
NEt2
DP
P
59.11
40.68
0.21
98.64
1.36
—
39.2
79.0
N
DP
P
N
N
4g
5a
5b
4.00
95.41
0.59
67.63
32.37
—
28.8
97
58.3
N
CH DP
—
16.00
83.46
0.54
90.52
9.48
—
137
P
N
HN
HN
—
CH DP
0.05
12.64
79.59
7.77
—
62.6
30.8
26.9
N
N
P/ZW
N
16.90
83.00
0.05
A
5c
—
—
N
DP
P/ZW
ZW
A
—
1.03
93.29
5.68
—
72
283
198
258
24.87
74.35
0.78
CQ
—
—
DP
P
12.12
87.83
0.05
87.37
12.63
—
22.5
280
10.1
N
a DP, di-protonated form; P, protonated form; ZW, zwitterionic form; N, neutral form; A, anionic form.
b ACD/pKa DB verson 9.00 software (Advanced Chemistry Development Inc., Toronto, Canada).
c CQ-sensitive clone.
d CQ-resistant clone.
e IC50s are the mean of at least three determinations. Standard errors were all within 10% of the mean.
f NT, not tested.
(SARs) can be explained by taking into account the ste-
ric hindrance of the substituent at C-8 of the quinoline
ring (4a,b vs 4d–f) with respect to the capability of the
quinoline nitrogen to coordinate hematin.
tri-cyclic system, combined with the hydrazone moiety,
was used to investigate the effects of the 7-chloro substi-
tuent of 4g and of the 6-methoxy group of 4f. The acri-
dine analogues 5a–c were synthesized and tested. The
different activity against the panel of CQ-resistant
strains seems to be dependent upon the ionization state
of the molecule (Table 1). In fact, the introduction of an
extra basic nitrogen (4f and 4g), resulting into a di-pro-
tonated state at the Pf vacuole pH 5.5, increased activity
on CQ-resistant strains. However, when the extra basic
nitrogen was introduced in the acridine series (5a), the
resulting compound did not show antimalarial activity
comparable to the di-protonated analogues of the quin-
oline series (4f and 4g vs 5a). Interestingly, when the pyr-
rolidinylmethylphenyl moiety of 5a was replaced with an
imidazole ring, the resulting compound 5b showed an in-
creased potency profile against all tested Pf strains,
becoming particularly active against CQ-resistant
strains. Imidazoles are known to be excellent heme
Potency is also influenced by the arylidene substituent at
the hydrazine N1. Electron-donating groups, such as the
weakly basic dimethylamino (4b) (still not protonated at
vacuole pH of 5.5, Table 1), do not improve antimalarial
activity versus the unsubstituted aromatic ring (4a). On
the contrary, a significant increase of potency against
CQ-sensitive and CQ-resistant strains can be observed
when the aromatic ring presents a diethylaminomethyl
group, already protonated at pH 7.2 (4f vs 4d). The
extra basic group was introduced to mimic the distal
nitrogen of CQ and amodiaquine, critical for drug
sequestration into the acidic food vacuole of Pf.6 SARs
were also exploited by varying the bi-cyclic system of 4
introducing an acridine group (5a–c). The mepacrine

S. Gemma et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5384–5388
5387
and in vivo studies to evaluate bioavailability are cur-
rently in progress.
Acknowledgments
Authors thank EU Commission for financial support
Antimal-LSHP-CT-2005-18834. This investigation re-
ceived financial support from the UNICEF/UNDP/
World Bank/WHO Special Programme for Research
and Training in Tropical Diseases (TDR).
Figure 2. b-Hematin inhibitory activity assay of compounds 4g, CQ,
and 3 (AQ).
References and notes
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confirmed by computational studies (data not shown),
the imidazole ring drives the formation of an intramo-
lecular zwitterion due to the exchange of a proton from
the imidazole nitrogen to the hydrazine nitrogen. Con-
trastingly, introduction of an extra nitrogen atom at po-
sition 5 of the acridine nucleus gave an analogue (5c)
that retained the activity against D10 and W2 strains,
being poorly active on 3D7 and K1 strains.
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Compound 4g was screened for inhibition of b-hematin
(hemozoin) formation by using the BHIA (b-hematin
inhibitory activity) assay (hemin in dimethylsulfoxide-
acetate buffer at pH 5.0, 37 °C, 18 h).25 Compound 4g
showed a dose-dependent inhibition in the BHIA assay
(IC50 = 0.53 0.09) and became more potent than CQ
(IC50 = 0.91 0.23) and amodiaquine (IC50 = 0.79
0.01) in inhibiting hemozoin formation (Fig. 2), suggest-
ing that its antimalarial activity was related to the inhibi-
tion of the heme detoxification process.
Compounds 5b and 5c, bearing an imidazole ring known
to have heme binding properties (e.g., azoles antifungal),
are among the most promising analogues of the series.
Further studies are in progress to understand the mech-
anism of action of these compounds and in particular to
know whether the activity profile of 5b and 5c could be
related to improved heme binding. Cytotoxicity on mur-
ine fibrosarcoma cells WEHI, clone 13, was assayed on
the most active compounds 4g and 5b using the MTT
test.23 While 5b showed an ED50 of 5.1 lM similar to
that one of CQ (ED50 = 9.7 lM), compound 4g
(ED50 = 19.4 lM) proved to be much less toxic than
5b and CQ.
18. Gall-Istok, K.; Sterk, L.; Deak, G. Acta Chim. Hung.
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19. Khan, M. A.; da Rocha, J. F. J. Heterocycl. Chem. 1978,
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To summarize, a series of novel and highly active anti-
malarial agents were synthesized. In particular, com-
pounds 4f–g and 5b–c showed remarkable antimalarial
activity especially against CQ-resistant Pf strains and
they represent promising lead structures for the develop-
ment of new antimalarial drugs. Inhibition of the heme
detoxification process seems to be the basis of their
mechanism of action. Notably, the synthesis of these
compounds involves few steps with commercially avail-
able products and has low production costs. Stability of
4g (over 2 h) in acidic conditions was proven by NMR
techniques.26 Further pharmacological characterization
22. In a typical procedure: a mixture of the appropriate
carboxaldehyde (1 equiv) and hydrazine derivative (1 equiv)
was heated under reflux in ethanol for 2–3 h. After cooling
and diluting with H2O, the respective hydrazones precipi-
tated from the reaction mixture. They were filtered and
washed with ice-cold ethanol. The purification was carried
out by recrystallization and/or by flash column chromato-
graphy (70–90% yields). The structures assigned to the
synthesized compounds were in good agreement with their
1
analytical and spectral data (elemental analyses, MS, H
NMR).

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S. Gemma et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5384–5388
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26. Samples of 4g in D2O-DCl (pH 4.0) were monitored at 300
intervals for 4 h by NMR and HPLC-MS.