Design, synthesis, and structure-activity relationship studies of 4-quinolinyl- and 9-acrydinylhydrazones as potent antimalarial agents

Article abstract of DOI:10.1021/jm7012375

Malaria is a major health problem in poverty-stricken regions where new antiparasitic drugs are urgently required at an affordable price. We report herein the design, synthesis, and biological investigation of novel antimalarial agents with low potential to develop resistance and structurally based on a highly conjugated scaffold. Starting from a new hit, the designed modifications were performed hypothesizing a specific interaction with free heme and generation of radical intermediates. This approach provided antimalarials with improved potency against chloroquine-resistant plasmodia over known drugs. A number of structure-activity relationship (SAR) trends were identified and among the analogues synthesized, the pyrrolidinylmethylarylidene and the imidazole derivatives 5r, 5t, and 8b were found as the most potent antimalarial agents of the new series. The mechanism of action of the novel compounds was investigated and their in vivo activity was assessed.

Full text of DOI:10.1021/jm7012375

J. Med. Chem. 2008, 51, 1333–1343
1333
Design, Synthesis, and Structure–Activity Relationship Studies of 4-Quinolinyl- and
9-Acrydinylhydrazones as Potent Antimalarial Agents
Caterina Fattorusso,†, Giuseppe Campiani,*,†,‡ Gagan Kukreja,†,‡ Marco Persico,†, Stefania Butini,†,‡ Maria Pia Romano,†,‡
Maria Altarelli,†,‡ Sindu Ros,†,‡ Margherita Brindisi,†,‡ Luisa Savini,†,‡ Ettore Novellino,†,| Vito Nacci,†,‡ Ernesto Fattorusso,†,
Silvia Parapini,†,§ Nicoletta Basilico,†,§ Donatella Taramelli,†,§ Vanessa Yardley,†,¶ Simon Croft,†,¶ Marianna Borriello,†, and
Sandra Gemma†,‡
European Research Centre for Drug DiscoVery & DeVelopment and Dipartimento Farmaco Chimico Tecnologico, Via Aldo Moro 2, UniVersità
di Siena, 53100 Siena, Italy, Dipartimento di Chimica delle Sostanze Naturali and Dipartimento di Chimica Farmaceutica e Tossicologica,
UniVersità di Napoli Federico II, Via D. Montesano 49, 80131 Napoli, Italy, Dipartimento di Sanità Pubblica- Microbiologia- Virologia,
UniVersitá di Milano, Via Pascal 36, 20133, Milano, Italy, Department of Infectious and Tropical Diseases, London School of Hygiene and
Tropical Medicine, Keppel Street, London WC1E 7HT, U.K.
ReceiVed October 1, 2007
Malaria is a major health problem in poverty-stricken regions where new antiparasitic drugs are urgently
required at an affordable price. We report herein the design, synthesis, and biological investigation of novel
antimalarial agents with low potential to develop resistance and structurally based on a highly conjugated
scaffold. Starting from a new hit, the designed modifications were performed hypothesizing a specific
interaction with free heme and generation of radical intermediates. This approach provided antimalarials
with improved potency against chloroquine-resistant plasmodia over known drugs. A number of
structure-activity relationship (SAR) trends were identified and among the analogues synthesized, the
pyrrolidinylmethylarylidene and the imidazole derivatives 5r, 5t, and 8b were found as the most potent
antimalarial agents of the new series. The mechanism of action of the novel compounds was investigated
and their in vivo activity was assessed.
Chart 1. Reference and Title Compounds
Introduction
Malaria is endemic to the poorest countries in the world,
mainly in tropical and subtropical regions of Africa, Asia, and
the Americas. Over 300 million cases are reported annually and
1.5–2.5 million people die from this devastating disease.1
Although four species of the genus Plasmodium cause human
malaria, most fatal malaria cases are due to infection by P.
falciparum (Pf). Before the emergence of chloroquine-resistant
(CQ-R) parasites, CQ (1, Chart 1) was one of the most important
and effective antimalarial agents and has been used for decades
as drug of choice in several programs aimed toward the global
control of malaria. The spread of Pf strains resistant to CQ as
well as to halofantrine, antifolates, and 4-substituted quinolines,
such as amodiaquine (AQ, 2) and mefloquine (3), dramatically
reduced the therapeutic options to treat uncomplicated malaria.2
The natural endoperoxide artemisinin (4) and its semisynthetic
derivatives are increasingly being used because of their efficacy
on CQ-R strains, rapid mode of action, broad range of activity,
and low tendency to induce parasite resistance.3 However, the
thermal instability and short half-life of artemisinins (natural
and semisynthetic) and the high cost of therapy are the major
disadvantages for their widespread use.4,5 At the moment, two
promising drug combinations made of piperaquine or amodi-
aquine and artemisinin derivatives are under clinical develop-
ment,6 while isoquine and derivatives,7 a class of compounds
structurally related to AQ, have shown to be promising drug
candidates. However, despite the current efforts to develop new
antimalarials, the novel therapeutic options are far from being
ideal, so there is an urgent need for newer low-cost safer drugs
for the treatment of malaria, specifically designed to be active
against Plasmodium strains that have acquired resistance to the
most commonly prescribed antimalarials. The spread of resistant
parasites created a tremendous therapeutic void, and the rate at
* To whom correspondence should be addressed. Tel: 0039-0577-234172.
Fax: 0039-0577-234333. E-mail: campiani@unisi.it.
†
European Research Centre for Drug Discovery & Development.
Dipartimento di Chimica delle Sostanze Naturali, University of Napoli
“Federico II”.
‡
University of Siena.
Dipartimento di Chimica Farmaceutica e Tossicologica, University of
|
Napoli “Federico II”.
§
Dipartimento di Sanità Pubblica- Microbiologia- Virologia, University
of Milano.
¶
London School of Hygiene and Tropical Medicine.
10.1021/jm7012375 CCC: $40.75
2008 American Chemical Society
Published on Web 02/16/2008

1334 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Fattorusso et al.
which resistance is growing outpaces the development of new
effective antimalarials.
for modulating the in vitro antimalarial activity, thus leading
to the development of a number of very potent antimalarials
mainly active against CQ-R parasites. The new analogues were
specifically designed to accumulate in the FV and interact with
free heme, possibly generating toxic radical species capable of
interfering with the antioxidant defense mechanisms of the
plasmodium. The most potent compounds against the CQ-R
strains were selected for further pharmacological investigation
aimed toward understanding their mechanism of action and their
in ViVo activity.
After infection, Pf establishes an initial asymptomatic liver
stage followed by an erythrocytic stage during which the
catabolism of hemoglobin (Hb) is a key source of food for the
parasite and probably helps to maintain the osmotic integrity
of the infected cell.8 Degradation of host Hb within the acidic
(pH ≈ 5.5) food vacuole (FV) is a central event for the
generation of oxidative stress, which must be controlled by the
parasite. Although Plasmodia are equipped with a range of
antioxidants to maintain redox equilibrium, they must detoxify
free heme. Indeed, Hb proteolysis in the acidic FV results in
the production of toxic free heme and promotes spontaneous
oxidation of oxyferro-protoporphyrin IX (FPIX-Fe(II)) to hy-
droxyferri-protoporphyrin IX (FPIX-Fe(III), hematin), with the
concomitant formation of superoxide anion, hydrogen peroxide,
and hydroxyl radicals (reactive oxygen species (ROS)). Parasite
heme detoxification represents a well specialized and efficient
process characterized by two main pathways: (i) crystallization
of hematin dimers (ꢀ-hematin) into an inert and insoluble
material referred to as the malaria pigment or hemozoin
(biomineralization)9 or (ii) diffusion through the vacuolar
membrane to the parasite cytoplasm with the subsequent
glutathione-dependent degradation.10 A number of drugs cur-
rently under clinical use exert their activity, exclusively or at
least in part, by increasing the oxidative stress in the parasitized
erythrocyte. Quinolines and artemisinins react with heme
moieties, preventing FP detoxification or forming cytotoxic
radicals, respectively. In particular, CQ and related 4-amino-
quinoline antimalarials are reported to interact with free heme,
to interfere with its detoxification to hemozoin and thus to impair
the plasmodium mechanisms of defense against oxidative
stress.11,12 This mechanism of action, characterized by the lack
of a specific parasite target protein, accounts for the delayed
onset and spreading of CQ-R Pf strains, which only occurred
after 40 years of heavy drug pressure.13 Although the molecular
bases of CQ resistance are still unclear, in CQ-R strains
mutations in pfcrt gene (Pf CQ-resistance transporter) always
occur.14,15 In particular, altered expression or mutations of
PfCRT is involved in ion-dependent FV transport processes that
could affect the CQ transport or the microenvironment of the
FV (e.g., pH) of resistant parasites.16 Variations of FV chemical
parameters may account for differences in efficacy against CQ-S
and CQ-R strains observed for drugs targeting free heme and
interfering with heme detoxification. On these bases, free heme
represents a privileged target for the design of antimalarials
active against CQ-R Pf strains. Thus, the aim of the present
work was the development of potent compounds, targeting free
heme and characterized by low potential of selecting resistant
parasites.
Chemistry
Compounds 5r, 6a,c,i,k,q,r, and 8a-c were synthesized as
previously described,19 and their detailed synthetic procedures
are reported therein. The general synthesis for the novel
hydrazones 5a-q,s-w, 6b,d-h,j,l-p,s,t, and 7a-d is depicted
in Scheme 1. Reaction of appropriate 4-hydrazinoquinolines,
9-hydrazino-1,2,3,4-tetrahydroacridines, 9-hydrazinoacridine,
and benzo[b][1,5]naphthyridin-10-yl-hydrazine (10a-n) with
the appropriate aromatic and heteroaromatic carboxaldehydes
in refluxing ethanol gave the desired hydrazones in good yields
(5–8).
Scheme 2 describes the synthesis of 3-hydroxy-4-(tetrahydro-
1H-1-pyrrolylmethyl)benzaldehyde 16 and 4-(1H-pyrrolidin-1-
yl)benzaldehyde 18, required for the synthesis of compounds
5q,t and 6e. Esterification of commercially available 3-hydroxy-
4-methylbenzoic acid 11 with methanol under acid-catalyzed
conditions and subsequent protection of the phenolic hydroxyl
group as an acetate afforded compound 12. R,R′-Azobisisobu-
tyronitrile (AIBN)-catalyzed radical bromination of 12 using
N-bromosuccinimide (NBS) furnished the corresponding bromo
derivative 13 in good yield. This latter was treated with
pyrrolidine in alkaline medium to yield the alkylation product
14. Subsequent reduction with LiAlH4 afforded the correspond-
ing benzyl alcohol 15, which on oxidation with activated
manganese(IV) oxide resulted in the formation of aldehyde 16
in excellent overall yield.
The aldehyde 18 was synthesized in good yields following
the Buchwald synthesis, involving the reaction of 4-bromoben-
zaldehyde 17 with pyrrolidine in the presence of BINAP and
NaO-t-Bu with a catalytic amount of Pd2(dba)3.
The synthetic pathway for derivatives 9a-d is reported in
Scheme 3. EDCI-catalyzed coupling of hydrazine 10a with the
appropriate carboxylic acid in the presence of HOBt and
N-methylmorpholine provided the corresponding hydrazides
9a,b. Finally, the synthesis of compounds 9c,d was realized by
treatment of 10a with the suitable aromatic sulfonyl chloride
in dry pyridine. The experimental details for synthesis, molecular
modeling, and biology are given as Supporting Information.
Results and Discussion
Within a wide program aimed toward developing new
antimalarial drugs17 and taking into account our experience in
the synthesis of quinoline-based pharmaceuticals,18 we recently
designed and synthesized a small set of heteroarylhydrazones,
which exhibited exceptional potency toward CQ-R Plasmodia,
inhibiting ꢀ-hematin formation in the BHIA assay in vitro.19
Encouraged by the initial results,19 we performed a thorough
investigation of their structure–activity relationships (SARs).
The present paper describes the synthesis and the biological
investigation of novel 7-chloroquinolylhydrazones, methox-
yquinolylhydrazones, tricyclic derivatives, and finally, hy-
drazides and sulfonylhydrazides (5–9, Tables 1-3). Comparing
the new compounds with the original set of hydrazones,19 SAR
studies delineated a number of structural features responsible
1. In Vitro Antimalarial Activity and Structure–Acti
vity Relationships (SARs). The aim of this study was to
discover novel chemical entities characterized by high activity
against CQ-R Pf strains and by a low potential to induce
resistance. To reach this goal, we developed compounds
selectively targeting free heme and possibly interfering with the
redox equilibrium of the malaria parasite forming toxic radicals
and increasing oxidative stress in the parasitized erythrocytes.
It is known that heteroaroyl hydrazones and Schiff base
hydrazones are able to form complexes with different metals,20,21
and their metal coordination geometry has been elucidated by
X-ray analyses (Conquest 1.9, Cambridge Structural Database
System (CSDS)). Moreover, interaction with metal ions and

4-Quinolinyl and 9-Acrydinylhydrazones as Antimalarials
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1335
Table 1. Prevalent Ionic Forms and Antiplasmodial Activity of Compounds 5a-w
a ACD/pKa DB version 10.00 software (Advanced Chemistry Development Inc., Toronto, Canada). DP ) diprotonated form; P ) protonated form; ZW
) zwitterionic form; N ) neutral form. b IC50 values are the mean of at least three determinations; standard errors were all within 10% of the mean.
c CQ-sensitive clone. d CQ-resistant clone. e NT ) not tested. f Reference 19.
generation of radical intermediates has been proposed as the
mechanism of action of hydrazone antitumorals.22 Accordingly,
since the quinoline system is known to possess iron complex-
ation properties,21 we envisioned to combine quinolines with

1336 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Fattorusso et al.
Table 2. Prevalent Ionic Forms and Antiplasmodial Activity of Compounds 6a-t
a ACD/pKa DB version 10.00 software (Advanced Chemistry Development Inc., Toronto, Canada); DP ) diprotonated form; P ) protonated form; ZW
) zwitterionic form; N ) neutral form; A ) anionic form. b IC50 values are the mean of at least three determinations; standard errors were all within 10%
of the mean. c CQ-sensitive clone. d CQ-resistant clone. e NT ) not tested. f Reference 19.

4-Quinolinyl and 9-Acrydinylhydrazones as Antimalarials
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1337
Table 3. Prevalent Ionic Forms and Antiplasmodial Activity of Compounds 7a-d, 8a-c, and 9a-d
a ACD/pKa DB version 10.00 software (Advanced Chemistry Development Inc., Toronto, Canada); DP ) diprotonated form; P ) protonated form; ZW
) zwitterionic form; N ) neutral form; A ) anionic form. c CQ-sensitive clone. d CQ-resistant clone. e IC50 values are the mean of at least three determinations;
standard errors were all within 10% of the mean. f NT ) not tested. g NA ) not active (IC50 > 10000). h Reference 19.
arylhydrazone moieties to obtain potent antimalarials.19 We
performed specific structural modifications of the original
diarylhydrazone class of antimalarials to reach a fine-tuning of
the antimalarial potency. Our design efforts were mainly dictated
by the hypothesis that antiplasmodial activity might be directly
related to the ability both to bind intracellular iron and to form
redox-active iron complexes that generate cytotoxic ligand-
centered radical species. To validate our hypothesis, we
exploited the effect on antimalarial potency of quinolyl, acridi-
nyl, and tetrahydroacridinyl hydrazone systems. Although the
exact mode of action of CQ has not been fully clarified, it is
generally accepted that CQ readily enters the parasite FV along
the pH gradient and complexes FPIX-Fe(III).10,23,24 The exact
structure of the complex intermediate formed between 4-ami-
noquinoline antimalarials and heme is still unclear. Most of the
modeling studies and interpretation of NMR data performed at
neutral or alkaline pH support heme/4-aminoquinolines interac-
tion as a face-to-face π-π alignment, although the X-ray
structure of the complex has never been obtained. A solid-state
NMR study of acid-precipitated CQ-FPIX-Fe(III) aggregates
provided the first direct evidence of a strong interaction,
probably a covalent complex, between the endocyclic quinoline
nitrogen (N1, Chart 1) and heme iron(III),25 similar to the
iron-nitrogen axial coordination bond of the quinoline ring of
metaquine to heme, later proposed.26 Accordingly, by specific
substituents, we modulated the electron density at N1 of our
polycyclic hydrazones, optimizing the interaction of the hydra-
zone-N1 with the heme iron center. As a consequence of this
binding, in the specific microenvironment of the plasmodium
FV, an electron transfer reaction could be facilitated leading to
the formation of radical intermediates, stabilized by delocal-
ization within the highly conjugated hydrazone scaffold. The
resulting radical could evolve either directly reacting with other
chemical entities in the Pf environment or undergoing the
reductive cleavage at the hydrazone linker, thus generating other
toxic species.21,27,28 Following this hypothesis, compounds

1338 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Fattorusso et al.
Scheme 1. Preparation of Final Compounds 5a-w, 6a-t,
7a-d, and 8a-c
Scheme 3. Synthesis of Final Compounds 9a-d
(i). Effect of the Structural and Electronic Properties
of 4-Hydrazinoquinoline Ring Substituents. To fully inves-
tigate the effect of different substituents at the 4-hydrazino-
quinoline system on antimalarial activity, we performed a
comprehensive computational analysis (see Experimental Sec-
tion), on all possible tautomers of compounds 5m, 6a, 6j, 6o,
6q, 6r, and 7a. In fact, all these compounds share the same
p-OMe-benzylidene moiety, while presenting diversely substi-
tuted 4-hydrazinoquinoline systems. Assuming an iron-nitrogen
axial coordination bond with heme, the steric hindrance and
the electron density at N1 were likely to affect the strength of
this interaction.
Scheme 2. Preparation of Intermediates 16 and 18
The results of our pKa calculations indicated that the
introduction of the 6-OMe (6a) and 6,7-methylendioxy (6r)
groups increased the protonatability of the 4-aminoquinoline
moiety with respect to the 7-Cl analogue (5m), affecting the
percentage of protonated forms at cytoplasmic pH (Tables 1
and 2) and, consequently, the antimalarial activity. Nevertheless,
our quantum-mechanical calculations revealed that all these
substituents show a comparable effect on the overall 4-amino-
quinoline ring electronic distribution, related to a similar
molecular dipole moment (Figure 1SIA,B, Supporting Informa-
tion). On the contrary, an 8-OMe group as in 6j decreased the
electron density/protonatability at N1 (Table 2), which affected
the dipole direction of the 4-aminoquinoline scaffold (Figure 1SI
panels A,B vs C,D). This effect, together with the steric
hindrance at C8 disfavored the interaction between free heme
iron and N1 and consequently decreased the antimalarial activity
(6a vs 6j, Table 2). According to our hypothesized mechanism
of action, the introduction of a methyl group at C2 (6q) increased
the electron density and protonatability at N1 (AM1 full
optimized partial charge ) -0.15) and, consequently, the
antimalarial activity (6q vs 6j, Table 2). Replacement of the
6-OMe group of 6q by a more hindered ethoxy group at C7
(6o), although maintaining a similar electronic effect at N1,
significantly lowered the activity against CQ-S and CQ-R strains
(6o vs 6q), due to unfavorable steric effects. Analogously,
introduction of a further saturated fused cycle (tetrahydroacri-
dine) as in 7a,d, with or without a chlorine substituent, dropped
down the potency against the strains tested (Table 3). The
resulting antiplasmodial activity of these compounds confirmed
the significant role played by the substituent on the 4-amino-
quinoline ring. As hypothesized, the antimalarial activity against
all tested strains varied according to the polarization of the
4-aminoquinoline moiety. In particular, potency is related to
the electron density at N1, which is crucial for the accumulation
inside the FV and for the interaction with iron. Moreover, the
presence of a strong molecular dipole moment, from the exo-
cyclic N2 to N1 (Chart 1), in the protonated form of the most
reported in Tables 1-3 were designed with the aim to define
the structural and electronic properties necessary to modulate
the antimalarial activity. Accordingly, specific 4-aminoquinoline
substitutents, coupled to different aromatic systems (i.e.,
arylidenes or heterocycles), were explored and the key role of
the hydrazone linker between the two aromatic moieties was
assessed.
The new hydrazones and their derivatives were tested in vitro
against a series of Pf strains, namely, the CQ-S D10 and 3D7
and the CQ-R W2 and K1 strains. The antimalarial activity (IC50,
nanomolar) was quantified as inhibition of parasite growth,
measured with the production of parasite lactate dehydrogenase
(D10 and W2; asynchronous culture) or the incorporation of
[3H]-hypoxanthines (3D7 and K1, synchronous culture).17a
Results are reported in Tables 1–3.

4-Quinolinyl and 9-Acrydinylhydrazones as Antimalarials
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1339
not significantly affect the antimalarial activity of the corre-
sponding compounds with respect to unsubstituted analogues
(6i vs 6j–l, Table 2). Even introducing different substituents
on the quinoline system, electron-donating groups at meta and
para positions of the arylidene system (5m,n,p,q,v,w or 6a-e,
6p, 6s, or 7b-d; Tables 1-3) provided compounds with a
similar trend in potency. Indeed, the antimalarial activity of this
set of compounds mainly depends upon the nature of the
4-aminoquinoline substituent.
On the contrary, a significant increase of potency against
CQ-S and CQ-R strains was observed when we introduced a
p-diethylaminomethyl or a p-pyrrolidinemethyl group on the
arylidene ring, protonated at pH 7.2 (6f19 vs 6d and 6g vs 6e in
the 6-MeO quinoline series, 5r19 vs 5q and 5s vs 5p in the
7-Cl quinoline series, or 6t vs 6r in the 6,7-methylendioxy
quinoline series and 8a,19 Tables 1-3). These extra protonatable
functions were introduced to mimic the basic aliphatic nitrogen
of CQ and AQ, critical for drug sequestration into the acidic
FV of Pf.29 Moreover, in the case of 5t, the introduction of an
extra basic group combined with an arylidene m-hydroxyl
provided a second iron coordination site in the molecule
(Conquest 1.9, CSDS) (Figure 1A), further improving potency
especially against 3D7 and W2 Pf strains (5t vs 5r, Table 1).
In general, analogues protonated at pH 7.2 (5r-t, 6f,g, and 6t)
are more potent than their unprotonated analogues (5m,n, 5p,q,
6a-e, and 6r,s) and represent some of the most potent
compounds of this series (Tables 1 and 2). Referring to the
protonation state of the molecule, it has to be underlined that,
when the extra aminomethyl basic chain was introduced in the
2-(benzylidene)-1-(6-methoxyquinolin-4-yl)hydrazine scaffold
the resulting compound was mainly diprotonated at cytoplasmic
pH (6f and 6g, Tables 1 and 2); consequently the antimalarial
potency against all tested CQ-R strains increased to a lesser
extent than in the corresponding 7-Cl derivatives (5r vs 6g and
5s vs 6f, Table 1), monoprotonated at the same value of pH
(probably due to different FV accumulation). This demonstrated
the importance of a fine-tuning of the electronic properties in
order to optimally balance the tropism for the FV and the
propensity to react with free heme.
Figure 1. Resulting conformers at pH 5.5 of 5t (A), 5f (B), 5g (C), 5l
(D), and 5o (E) were superimposed on the X-ray structure of iron
complexes containing the following moieties: (A-E) quinoline (white;
CSD code FOFROH); (A) 2-(aminomethyl)phenol (cyan; CSD code
DAMQAJ); (B) (1H-pyrrol-2-yl)methanimine (magenta; CSD
code WEFFUJ); (C) (1H-imidazol-2-yl)methanimine (orange; CSD code
FEJCAZ); (D) 3-phenyl-1H-pyrazole (pink; CSD code IDABIY), and
(E) 3-(iminomethyl)naphthalen-2-ol (yellow; CSD code QASSOT). The
superimposition was made fitting the putative iron coordinating atoms.
Iron atoms’ vdW volume (magenta) are scaled (0.3) for clarity.
Heteroatoms are colored: C, green; O, red; N, blue; Fe, magenta; Cl,
light green, and F, dark green. All hydrogens have been omitted for
clarity.
Chart 2. Tautomers A and B for the Title Compounds and
Tautomers C for 5a,c,g,h,u and 6h,m,n
(iii). Heteroarylidene versus Arylidene Substituents. When
arylidene analogues were compared with their heteroarylidene
counterparts, a dramatic influence on the antimalarial activity
was observed (Tables 1 and 3). In particular, the introduction
of a furan ring (5d) led to a 2–5-fold drop in potency against
D10 and W2 strains when compared with the analogue bearing
a p-OMe benzylidene ring (5d vs 5m, Table 1) and to those
analogues bearing other five-membered heterocycles (5e-l vs
active compounds supported the hypothesis of a strong relation-
ship between potency (6a = 6q > 6r > 5m > 6o > 6j > 7a,
Tables 1–3) and ability to promote redox reactions after iron
binding. Finally, the presence of substitutions able to hinder
the hypothesized interaction with iron (i.e., at C2 (7a), C7 (6o),
and C8 (6j)) were detrimental for antimalarial activity.
Figure 2. Superimposition of the most stable conformers of all possible
tautomers of 5d (A), 5f (B), and 5g (C). The carbon atoms and the
hydrogens are colored: tautomer A (C green and H white); tautomer B
(C orange and H magenta), and tautomer C (C cyan and H yellow).
Heteroatoms are colored: O, red; N, blue; Cl, light green. All hydrogens,
except those involved in hydrogen bonds and those of protonable
nitrogens, have been omitted for clarity. Hydrogen bonds (dashed lines)
are colored according to the hydrogens color.
(ii). Effects of the Arylidene Substituents. The introduction
of electron-donating groups, such as ethers, dimethylamino, and
diethylamino at the para-position of the arylidene moiety did

1340 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Fattorusso et al.
Table 4. ꢀ-Hematin Inhibitory Activity Assay of Compounds 5d,g,l,r
quinoline N with a resulting modification of the electron density
at the hydrazone linker. In general, pyridine derivatives 5a-c,u
and the quinoline analogue 6h were slightly less than or equally
potent to the corresponding p-OMe arylidene derivatives
(5a-c,u vs 5m, 6h vs 6a, Tables 1 and 2). The quantum-
mechanical studies evidenced that these heterocycles improved
the electron density on the hydrazone linker similarly to the
p-OMe phenyl substituent of 5m and 6a.
and CQ
a
compd
IC50
5d
5g
5l
5r
CQ
1.13
0.65
0.83
0.98
1.68
a The IC50 represents the molar equivalents of compound, relative to
hemin, that inhibit ꢀ-hematin formation by 50%. Data are the mean of three
different experiments in triplicate. Standard errors were all within 10% of
the mean.
On these bases, we decided to introduce a 2-imidazolyl group
(Chart 2, 5g), which is also characterized by the presence of a
third tautomer but, compared with the other heterocycles, by a
higher degree of protonability (Table 1). Taking into account
the chemical-physical properties of the imidazole ring, our
conformational analysis was performed on the prevalent ionic
forms at pH 7.2 and 5.5 of all the three possible tautomers (A,
B, and C, Chart 2). The full optimization of the resulting
conformers by the quantum-mechanical method AM1 (see
Experimental Section, Supporting Information) revealed some
unique features of 5g, such as (i) a high electron density at N1,
(ii) a strong electron flow from N1 to the exocyclic quinoline
N2 in the protonated form, (iii) the presence of strongly
polarized hydrogen bonds between N2 and the imidazole
nitrogens, which stabilized the global minima of the three
tautomers in a planar conformation, thus favoring tautomeric
exchanges (Figure 2C), and finally, (iv) an electron flow from
the 4-aminoquinoline moiety to the imidazole ring, which
induced the polarization of the hydrazone linker. In particular,
as evidenced by pKa calculations and confirmed by computa-
tional studies, the imidazole ring drives the formation of an
intramolecular zwitterion due to the exchange of a proton from
the imidazole nitrogens to the N2. Moreover, in the most stable
conformers of all 5g tautomers, we uncovered an additional iron
coordination site (Conquest 1.9, CSDS; Figure 1C). These
electronic and conformational features allowed 5g to be one of
the most potent compounds of the series endowed with high
antimalarial activity against both CQ-R and CQ-S Pf strains
(Table 1).
5d). Our computational studies revealed that, although inducing
a polarization of the hydrazone linker similar to 5m, the furan
ring of 5d drove the formation of an intramolecular hydrogen
bond between the heterocyclic oxygen and the exocyclic
4-hydrazinoquinoline N2. Consequently, this hydrogen bonding
affected the tautomeric equilibrium, stabilizing one tautomer
(namely, tautomer A, Chart 2, Figure 2A) and thus reduced the
antimalarial potency (Table 1). It is well-known that 4-amino-
quinolines are present in equilibrium between two tautomeric
forms accounting for their peculiar properties, such as (i) the
increased electron density at N1 (dipole moment), (ii) the
consequent higher degree of protonatability with respect to
4-unsubstituted quinolines, and (iii) their chemical reactivity.30
Consequently, we hypothesized that the poor antimalarial
activity of 5d was related to its lower propensity to give an
electron transfer reaction with heme iron necessary for the
generation of toxic radical intermediates. On the other hand,
our data demonstrated that, although endowed with low anti-
malarial activity, 5d was still able to strongly interact with
Fe(III) heme, being highly active in the BHIA assay (Table 4).
As expected, compounds 5e and 5k, characterized by moieties
with similar electron properties to 5d but with heteroatoms less
prone to form hydrogen bonds, such as thiophene and 3,5-
dimethylisoxazole rings, respectively, resulted slightly more
potent than 5d (Table 1). In particular, our quantum-mechanical
calculations showed a very weak propensity of the sulfur bridged
atom (i.e., low electron density) of 5e to establish H-bond
interactions. On the contrary, our quantum-mechanical calcula-
tions indicated that the isoxazole (5k) could generate an
intramolecular H-bond; nevertheless, our conformational analy-
sis revealed that it is prevented by the steric hindrance provided
by the two methyl substituents.
Following the same rationale, we introduced a pyrrole
heterocycle obtaining compound 5f with improved antimalarial
activity especially against the 3D7 and K1 Pf strains (5f vs 5d,
5e, and 5m; Table 1). The pyrrole ring is a H-bond donating
group being able to establish a hydrogen bond either with the
exocyclic quinoline N2 (Figure 2B) or with the hydrazone linker
N3 (Chart 1). The formation of the hydrogen bond with N3 is
possible in both the neutral (24% at pH 5.5) and protonated
forms (most abundant at pH 5.5). Indeed, in the most stable
conformers, the H-bond donating properties of the pyrrole
nitrogen allowed the stabilization of both 4-aminoquinoline
tautomers (Figure 2B), without affecting the tautomeric equi-
libria. In addition, the intramolecular H-bond revealed the
presence of a further iron coordination site in the molecule, as
revealed by a fragment based search in the Cambridge Structural
Database (CSD) (Conquest 1.9, CSDS) and depicted in Figure
1B.
Introduction of a methyl group at the imidazole NH (5h),
which impaired the annular tautomerism, significantly decreased
the antimalarial potency (5h vs 5g, Table 1). Moreover, further
support to our hypothesis was provided by compounds 5i and
5j, characterized by the presence of a 4-imidazolyl or a
3-methyl-4-imidazolyl ring, respectively. In these isomers, the
formation of a third tautomer is not possible, switching the
electronic effect of the imidazole ring from withdrawing to
donating (5i and 5j vs 5g, Table 1). In agreement with the results
of our calculations, 5i and 5j displayed a drop in antimalarial
potency with respect to 5g (Table 1). Interestingly, the presence
of a methyl-substituted imidazole (5j) improved imidazole
protonatability at pH 5.5, 5j accumulation into the FV, and
consequently, its antimalarial potency compared to 5i
(Table 1).
On these bases, when the mepacrine-like system (mepacrine,
19, Chart 1) of derivatives 8a–c,19 characterized by the favorable
electronic effects of the 7-chloro and the 6-methoxy substituents,
was combined to the presence of a 2-imidazolyl group (8b),
we obtained one of the most potent compounds of the series,
especially against 3D7 and K1 strains (Table 3). The introduc-
tion of an extra N at the 5 position of the acridine system gave
an analogue (8c) that retained the activity against D10 and W2
strains while being weakly potent against 3D7 and K1 strains
(8c vs 8b, Table 3). This drop of activity could be explained
by comparing the conformational and electronic properties of
8b and 8c. In particular, the presence of the acridine nitrogen
When the five-membered heterocycles are substituted by
pyridines or quinolines (5a–c,u and 6h,m,n), we uncovered a
third tautomeric form (tautomer C, Tables 1 and 2 and Chart
2), which involves the hydrazone NH and the pyridine or

4-Quinolinyl and 9-Acrydinylhydrazones as Antimalarials
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1341
Table 5. In Vitro Cellular Toxicity of Compounds 5d-l,r,t, 8b, and
CQ (1)
Table 6. In Vivo Antimalarial Activity of Selected Compounds against
Plasmodium berghei ANKA in BALB/c Mice after i.p. Administration
compd
toxicity (KB cells), ED50 (µM)
dose
(mg/kg/day) i.p.
% suppression
on day 4a
mice alive
on day 4
compd
5d
5e
5f
5g
5h
5i
5j
5k
5l
5r
5t
19.9
10.4
4.8
19.5
17.1
29.1
73.8
171
1.2
5r
5t
6a
8a
8b
CQ (1)
30
30
30
30
30
10
83
71
38
19
40
98
5/5
5/5
4/5
5/5
5/5
5/5
a Percent suppression ) [(C - T)/C] × 100; where C ) parasitaemia in
control group and T ) parasitaemia in treated group.
19.5
152
5.1
compounds are endowed with in vitro toxicity ranging from 5
to 170 µM. With the exception of 8b, the most potent
compounds of the series displayed a toxicity vs antimalarial
activity ratio higher than 200 thus demonstrating good selectivity
indices.
8b
CQ(1)
260
(N5) in 8c altered the electronic distribution on the whole
system, reducing (i) the electron density at N1, possibly affecting
iron interaction properties, (ii) the protonatability of the acridine
system, critical for FV accumulation, and (iii) the capability to
promote intramolecular hydrogen exchanges between the imi-
dazole nitrogens and the exocyclic acridine N2, related to the
propensity to form radical intermediates.
4. In Vivo Antimalarial Activity. Selected compounds that
displayed the best in vitro activities were administered intrap-
eritoneally to infected mice to determine whether the antimalarial
activities were retained in vivo. Compounds 5r,t and 8b were
submitted to the full suppressive 4-day Peters’ test32 against P.
berghei ANKA in BALB/c mice, and the results are reported
in Table 6. At the dose of 30 mg/kg, none of the compounds
tested was able to completely clear parasitemia in the 4 days of
administration. Compound 8b, which displayed the higher in
vitro activity against 3D7 and K1 strains, acheived only 40%
reduction in parasitemia with respect to untreated control mice.
Compounds 5r and 5t gave 83% and 71% reduction of
parasitemia, respectively, after i.p. administration at the same
dose. Further tests are ongoing to clarify whether the reduced
in vivo antimalarial activity of the compounds tested, especially
of 8b, compared with the high in vitro activity, is due to low
bioavailability and hence to the pharmacokinetic properties of
these compounds or whether activity is different between rodent
and human strains of Plasmodium.
Finally, encouraged by our previous results17b and by the
potency of multisite iron complexing compounds 5g and 5t
(Table 1, Figure 1A,C), we introduced specific aromatic moieties
potentially able to coordinate iron (ConQuest 1.9, CSDS) such
as 3-(4-trifluoromethylphenyl)1H-pyrazol-4-yl (5l) and 2-meth-
oxynaphthalen-1-yl (5o) (Figure 1D,E). This led to the discovery
of two compounds endowed with potent antimalarial activity,
especially against 3D7 and K1 strains (Table 1). The lower
activity of 5o with respect to 5l could be due to the formation
of an intramolecular hydrogen bond between the methoxy group
and the exocyclic quinoline N2, which similarly to what was
observed for 5d (Figure 2A) stabilized tautomer A, thus altering
the electronic properties of the compound.
(iv). The Effect of the Hydrazone Linker. The hydrazone
tether allows the electronic conjugation between the quinoline/
acridine system and the (hetero)arylidene moiety. The critical
role played by this molecular fragment in the antimalarial
activity of our series is demonstrated by the comparison of the
pharmacological profile of compounds 9a and 9b (Table 3) with
that of their analogues 5g and 5i (Table 1), respectively. Indeed,
our data confirmed the complete loss of activity displayed by
derivatives 9a-d, characterized by the presence of an acylhy-
drazino (9a,b) or a sulfonylhydrazino linker (9c,d).
2. ꢀ-Hematin Inhibitory Activity Assay. Selected com-
pounds (5d,g,l,r) were screened for inhibition of ꢀ-hematin
formation by using the ꢀ-hematin inhibitory activity (BHIA)
assay (hemin (FeIII) in dimethylsulfoxide/acetate buffer at pH
5.0, 37 °C, 18 h).31 All tested compounds showed a dose-
dependent inhibition in the BHIA assay (Table 4) and were
found to be more potent than CQ in inhibiting hemozoin
formation, suggesting that the quinolyl hydrazones described
are able to interact with free hematin possibly with a higher
affinity than CQ. Since 5d inhibited ꢀ-hematin formation
similarly to 5g (Table 4) but showed a reduced antimalarial
activity (Table 1), these results suggested that, besides the
interaction with FPIX-Fe(III), a further mechanism of action is
required for the antimalarial activity of this class of CQ
analogues.
Conclusions
In summary, a series of novel and highly potent antimalarial
agents were designed and synthesized. In particular, compounds
5g,r–t, 6f–g, and 8b showed remarkable antimalarial potency
in vitro, especially against CQ-R Pf strains. Accumulation inside
the FV and interaction with free heme possibly followed by
their activation to form toxic radical intermediates able to
overcome the antioxidant defense mechanisms of the parasite
seem to be the bases of their mechanism of action. In fact,
oxidative stress represents one of the most promising rationales
for the development of antimalarial chemotherapy. The low
antimalarial activity of 5d together with its potent effect on the
BHIA assay seems to confirm our hypothesis. Compounds 5r,t
showed a promising antiplasmodial activity in vivo against P.
berghei ANKA after i.p. administration at a dose of 30 mg/kg.
Their further pharmacological and pharmacokinetic development
is currently under progress. Notably, starting from commercially
available starting materials, the development of these novel
antimalarials requires few synthetic steps and is characterized
by low cost of goods.
Acknowledgment. Authors thank Sigma-Tau, Industrie
Farmaceutiche Riunite (Campiani et Al. EP06005306.3) for
financial support. Marco Persico - Italian Malaria Network was
funded by Compagnia di San Paolo, Torino, Italy. This
investigation received financial support from the UNICEF/
UNDP/World Bank/WHO Special Programme for Research and
Training in Tropical Diseases (TDR).
3. In Vitro Cytotoxicity. Cytotoxicity of compounds
5d-l,r,t, 8b, and CQ (1) was assessed by using KB cells, a
cell line derived from a human carcinoma of the nasopharynx
system, and results are reported in Table 5. The tested

1342 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
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