Synthesis and biological evaluation of novel ferrocene-naphthoquinones as antiplasmodial agents

Article abstract of DOI:10.1016/j.ejmech.2013.10.011

This work deals with the synthesis and evaluation of new compounds designed by combination of 1,4-naphthoquinone and ferrocene fragments in a 3-ferrocenylmethyl-2-hydroxy-1,4-naphthoquinone arrangement. A practical coupling reaction between 2-hydroxy-1,4-naphthoquinone and ferrocenemethanol derivatives has been developed. This procedure can be carried out "on-water", at moderate temperatures and without auxiliaries or catalysts, with moderate to high yields. The synthesized derivatives have shown significant in vitro antiplasmodial activity against chloroquine-sensitive and resistant Plasmodium falciparum strains and it has been shown that this activity is not related to the inhibition of biomineralization of ferriprotoporphyrin IX. Binding energy calculations and docking of these compounds to cytochrome b in comparison with atovaquone have been performed.

Full text of DOI:10.1016/j.ejmech.2013.10.011

Accepted Manuscript
Synthesis and biological evaluation of novel ferrocene-naphthoquinones as
antiplasmodial agents
Pedro M. García-Barrantes, Guy V. Lamoureux, Alice L. Pérez, Rory N. García-
Sánchez, Antonio R. Martínez, Arturo San Feliciano
PII:
S0223-5234(13)00652-1
10.1016/j.ejmech.2013.10.011
EJMECH 6474
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 31 March 2013
Revised Date: 2 October 2013
Accepted Date: 5 October 2013
Please cite this article as: P.M. García-Barrantes, G.V. Lamoureux, A.L. Pérez, R.N. García-
Sánchez, A.R. Martínez, A. San Feliciano, Synthesis and biological evaluation of novel ferrocene-
naphthoquinones as antiplasmodial agents, European Journal of Medicinal Chemistry (2013), doi:
10.1016/j.ejmech.2013.10.011.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of novel ferrocene-naphthoquinones as
antiplasmodial agents
a
a,
a
Pedro M. García-Barrantes , Guy V. Lamoureux *, Alice L. Pérez , Rory N. García-
b
b
c
Sánchez , Antonio R. Martínez , Arturo San Feliciano
a
Centro de Investigaciones en Productos Naturales and Escuela de Química, Universidad
de Costa Rica, San Pedro 2060, San José, Costa Rica pedro.garcia_b@ucr.ac.cr,
guy.lamoureux@ucr.ac.cr, alice.perez@ucr.ac.cr
b
Departamento de Parasitología, Facultad de Farmacia, Universidad Complutense de
Madrid, Madrid, España. rgbiounap@yahoo.es, arm@farm.ucm.es
c
Departamento de Química Farmacéutica, Facultad de Farmacia, CIETUS - IBSAL,
Universidad de Salamanca, Salamanca, España. asf@usal.es
Corresponding author
a
Guy V. LAMOUREUX , guy.lamoureux@ucr.ac.cr, phone: +(506)25114091, Fax: +(506)
2
2535020
a
Alice L. PEREZ , alice.perez@ucr.ac.cr, phone: +(506)25111405
a
Centro de Investigaciones en Productos Naturales and Escuela de Química, Universidad
de Costa Rica, San Pedro 2060, San José, Costa Rica.
1

ACCEPTED MANUSCRIPT
Graphical abstract
O
OH
Fe
O Me
IC = 1.3 ꢀM, SI = 150
5
0
2

ACCEPTED MANUSCRIPT
Highlights
1
1 new 3-(ferrocenylmethyl)-1,4-naphthoquinone derivatives were synthesized with an
“on-water” methodology.
3
compounds showed antiplasmodial activity (IC <2 µM) against chloroquine-sensitive
50
and resistant strains of Plasmodium falciparum.
3
compounds showed selective cytotoxicity (SI>100) towards the parasite strains relative to
a mammal cell line.
3

ACCEPTED MANUSCRIPT
Abstract
This work deals with the synthesis and evaluation of new compounds designed by
combination of 1,4-naphthoquinone and ferrocene fragments in a 3-ferrocenylmethyl-2-
hydroxy-1,4-naphthoquinone arrangement. A practical coupling reaction between 2-
hydroxy-1,4-naphthoquinone and ferrocenemethanol derivatives has been developed. This
procedure can be carried out “on-water”, at moderate temperatures and without auxiliaries
or catalysts, with moderate to high yields. The synthesized derivatives have shown
significant in vitro antiplasmodial activity against chloroquine-sensitive and resistant
Plasmodium falciparum strains and it has been shown that this activity is not related to the
inhibition of biomineralization of ferriprotoporphyrin IX. Binding energy calculations and
docking of these compounds to cytochrome b in comparison with atovaquone have been
performed.
4

ACCEPTED MANUSCRIPT
Keywords
Naphthoquinone, lawsone, ferrocene, antimalarial, on-water reactions
5

ACCEPTED MANUSCRIPT
Abbreviations
FBIT: Ferriprotoporphyrin IX Biomineralization Inhibition Test
SI: Selectivity Index
6

ACCEPTED MANUSCRIPT
1
. Introduction
Quinones are organic molecules that possess a cyclic six membered conjugated planar
system of two carbonyls and two double bonds. There are several types of quinones
depending on the size of the system, the arrangement of functional groups and substituents
and the possibility of heteroatoms involved. A quinone can be reduced reversibly to a
hydroquinone, passing through a stable semiquinone form [1]. Redox properties of
quinones can be modulated by their substituents. Electron-donating groups decrease the
reduction potential while the opposite occurs with electron-withdrawing substituents [2].
Quinones can act as nucleophiles or electrophiles, depending on electronic effects. As
electrophiles, attack occurs at the carbonyl group or at the β-position by a Michael-like
addition reaction; these reactions are favored by electron-withdrawing groups [3, 4].
Quinones acting as nucleophiles are less common and are favored by electron donors,
(hydroxy, methoxy, amino groups). For example, lawsone 1 can be substituted at the C-3
position by various electrophiles [5].
Quinones are known to be electron transporters (e.g. ubiquinone, vitamin K), and are
essential for many enzymatic processes [6]. They can act as anti- or pro-oxidants depending
on the conditions of the media. This chemical versatility gives them an important role in
different biochemical processes that are essential to living organisms. Also, exogenous
synthetic quinones (or quinones isolated from natural sources) have been shown to inhibit
different physio-pharmacological targets, to cause oxidative stress and cell death of
microbes [7]. Furthermore, different naphthoquinones have been reported and clinically
used as anticancer, antifungal, antiprotozoal, and antibacterial therapeutic agents, as well as
platelet antiaggregants and to prevent the myotoxicity of snake venom [8–14]. Their
biological activity has been ascribed to their electronic properties and, in some cases, to
specific interactions with defined targets such as DNA and dihydroorotate dehydrogenase
[
15]. Their readiness to stabilize radical species has been pointed out as the cause of their
cytotoxicity, producing oxidative stress and promoting cell death [4, 5, 7, 16, 17].
We are involved in the effort to explore the chemical space of naphthoquinones with
selective inhibition properties, with lesser cytotoxic effects, and to develop new derivatives
of naphthoquinones with more druglike characteristics [18]. A starting point for the
development of analogues of these kinds of compounds is the substitution at the C-3
position, such as the natural product lapachol 2 [19].
Malaria is one of the most important infectious diseases, causing the death of more than 1
million people in 2010 (the majority children under five years old) [20]. In 2011, there were
7

ACCEPTED MANUSCRIPT
estimated more than 250 million symptomatic cases. Incidence is greater in poorer regions
where control has not been effective and access to health services is scarce [21]. Efforts
have been made to combat widespread infection, such as vector control, opportune
diagnostics and the treatment of infected patients. However, the therapeutic arsenal is
limited and parasites are developing growing resistance to current medications; therefore,
new antimalarial drugs are needed. Naphthoquinones have received special attention due to
their redox and metal chelating properties [2]. The most successful quinone antimalarial
drug is atovaquone (ATV, 3). It was introduced by Glaxo as Malarone® in 2000, in
combination with proguanyl for the treatment and prevention of malaria when dealing with
strains resistant to chloroquine, mefloquine, amodiaquine and halofantrine. The main
mechanism of action proposed for atovaquone is the inhibition of the cytochrome b subunit
of the mitochondrial bc complex involved in the respiratory-chain of the parasite [22, 23].
1
Ferrocene is an organometallic compound formed by an iron(II) center surrounded by two
cyclopentadiene rings arranged in a sandwich-like structure. The iron(II) can be oxidized to
5
iron(III) generating the bis(η -cyclopentadienyl)iron(1+) ion, which possesses a similar
geometry [24]. Ferrocene is characterized by possessing aromatic behavior, each ring with
six electrons according to Hückel’s rule [25].
The use of ferrocene in Medicinal Chemistry has increased recently. As a substituent,
ferrocene presents high lipophilicity (π 2.46) and can act as an electron donor (σ -0.15; σ
m
p
-
0.18) similar to cyclohexane (π 2.51; σ -0.15; σ -0.22), but it presents high molar volume
m p
and refractivity [26]. Ferrocene has been proposed as an arene bioisostere to modulate the
pharmacokinetic or pharmacodynamic profiles of drugs [27, 28]. Moreover, ferrocene,
compared to other organometallic compounds, has many advantages for biological
applications, such as chemical modification capacity, low toxicity and stability to water and
oxygen [29, 30]. Compounds with ferrocene moieties have been evaluated as antimalarial,
anticancer, antifungal, antiviral [31–33], anti-inflammatory [34], anti-tuberculosis [35], and
as agonists to receptors like H3, D1, D2, D3, D4, alfa-1 adrenergic and 5-HT1A [36–38].
In malaria research, the incorporation of ferrocene led to ferroquine 4, a chloroquine (CQ)
analogue that advanced to phase II in clinical trials and effective against resistant
Plasmodium falciparum [39]. The advantages of this analogue have been attributed to the
increase in lipophilicity and the decrease in affinity towards the CQ exporter [40–42].
Many quinoline-ferrocene derivatives have been synthesized and tested as antimalarials
[
43, 44]. Also, ferrocene can act as a catalyst of Fenton-type reactions and promote the
generation of reactive oxygen species in the digestive vacuole of the parasite [27, 32].
8

ACCEPTED MANUSCRIPT
A few reports about ferrocene-quinone conjugates have been published. These substances
have been synthesized mainly to explore their electrochemistry (5 and 6), but activity
against P. falciparum and Toxoplasma gondii has been reported for aminoquinone
derivatives 7 [16, 45, 46].
The aim of this work is to develop a synthesis (with a green perspective) of new 3-
(ferrocenylmethyl)-1,4-naphthoquinone derivatives, and to characterize these compounds in
terms of their antimalarial properties. We were inspired to design these new compounds
based on a hybrid molecule approach [47]. Similar to lapachol and atovaquone the 1,4-
naphthoquinone moiety was conserved, due to its role as a privileged structure. The
ferrocene moiety was introduced to explore its potentially positive influence, such as exists
in ferroquine, in the pharmacodynamic profile of the hybrid.
2
2
. Results and discussion
.1. Chemistry
For the synthesis of the desired ferrocenylnaphthoquinone derivatives, it was necessary to
obtain the ferrocenecarbinols 9. Their preparation was carried out in three steps, using
ferrocene and the corresponding carboxylic acid as starting materials (Scheme 1). Acyl
chlorides, obtained from the corresponding acids by treatment with thionyl chloride, were
used for the acylation of ferrocene, by the classical Friedel-Crafts procedure, using AlCl as
3
the Lewis acid and following a literature procedure to minimize diacylation subproducts
[
48, 49]. Yields for this reaction were moderate to good. Lower yields were attained when
9

ACCEPTED MANUSCRIPT
using aromatic acyl chlorides which can react with themselves [50]. For the reduction of
the ketones 8, LiAlH4 in THF was employed and we efficiently obtained the
ferrocenecarbinols 9 in high yields, with short reaction times and absence of subproducts. It
was observed that these ferrocenecarbinols (ferrocenyl alcohols) tend to oxidize slowly to
the corresponding ferrocenylketones when exposed to air. Therefore, these compounds
were used immediately after purification.
R:
H-
Cl(CH ) CH -
Ph-
2
2
2
CH3-
CH (CH ) CH -
cHex-
p-ClPh-
p-BrPh-
3
2 9
2
CH CH -
3
2
CH CH CH -
Bn-
3
2
2
Scheme 1.Synthesis of ferrocenecarbinols.
The coupling reaction between ferrocenecarbinols 9 and lawsone 1 to produce 3-
ferrocenylmethyl-1,4-naphthoquinones 10a-k utilized an heterogeneous “on-water”
procedure [51]. In general, ferrocenecarbinols are not reactive to nucleophilic substitution
and usually they need to be derivatized to form a better leaving group [52]. When dealing
with an S 1 type reaction, the stability of the generated carbocations should be considered
N
as it relates to its electrophilicity. In the Mayr scale of electrophilicity (E), the
ferrocenecarbinols generate more electrophilic carbocations than substances like
benzhydrol [53]. This reactivity can be justified because of the delocalization of the
positive charge [54] and electron density donation from iron d orbitals [55]. On the other
hand, 2-hydroxy-1,4-naphthoquinone represents a multidentate nucleophile similar to β-
dicarbonyl compounds [3]. Selectivity for C-3 alkylation is due to the irreversibility of C-
alkylation (thermodynamic product) and is consistent with the Pearson’s Hard Soft Acid
Base theory [56].
The solvent plays an important role for this reaction, as polar protic solvents better stabilize
carbocations and water’s capacity to form hydrogen bonds may promote the protonation of
the alcohol and the formation of these intermediates. The alcohol and the quinone have a
relatively low solubility in water, and they form heterogeneous mixtures while stirring
(“on-water” reaction). No addition of Brønsted or Lewis acid was necessary, so it is
assumed that water is acting as a catalyst, as demonstrated in other cases. Most alkylation
studies have been done in solution, but it was found recently that the rate enhancement
tends to be greater with “on-water” reactions [57]. The network of water molecules tends to
leave “dangling” protons in the water:oil interphase; it has been hypothesized that these
protons could stabilize possible transition states in the reaction [58]. Also, the reaction
could be favored by an hydrophobic effect that leads to a closer contact between the
alcohols and the quinone [59, 60]. This procedure has the advantage of green reaction
conditions and easy workup and purification of products.
1
0

ACCEPTED MANUSCRIPT
The reaction was tested with different ferrocenecarbinol derivatives 9a-k, producing
moderate to high yields (Table 1). Better yields were obtained when the substituent R was
a short (not bulky) aliphatic group. Competition between nucleophilic substitution and
elimination affected the yield in some cases; for instance in the synthesis of 10g and 10h,
elimination products were isolated and their structures confirmed by NMR.
Table 1. Reaction conditions and yields for the synthesis of ferrocene-naphthoquinones
1
0a-k
Compound
R
H-
Temperature / °C
Time / h
Yield / %
65
1
0a
0b
0c
0d
0e
0f
0g
0h
40
40
40
40
40
70
70
40
40
70
40
23
4
1
Me-
Et-
Pr-
79
1
18
18
18
6
75
1
83
1
ClCH
2
CH
2
CH
2
-
81
1
CH (CH
3
2
)
9
CH
2
-
28
1
cHex-
Bn-
24
6
11
1
53
1
0i
Ph-
18
6
68
1
0j
p-ClPh-
p-BrPh-
79
1
0k
15
73
All compounds were obtained and evaluated as racemic mixtures (when substituted at the
linking carbon). Structural characterization was made by spectroscopic methods and mass
spectrometry. Infrared spectra of the products show characteristic bands associated to O-H
-
1
-1
stretching signals (~3350-3400 cm ), C=O stretching signals (~1640-1670 cm ) and C=C
-
1
stretching signals (~1585-1595 cm ) in all cases. The spectrum of many derivatives of 2-
hydroxy-1,4-naphthoquinone shows a band of the intramolecular hydrogen bond between
the hydroxyl at C-2 and the carbonyl at C-1; this interaction was observed as a sharp O-H
-1
stretching signal (ν: 3350-3370 cm ) in the IR spectra, except for 10g and 10k that show a
1
wider band. The H-NMR spectra of the compounds can be subdivided in three regions.
The aromatic region related to the quinone is composed of four signals, two doublet of
doublets (8.00-8.15 ppm) and two triplets of doublets (7.6-7.8 ppm), and this region shows
1
1

ACCEPTED MANUSCRIPT
little variation between compounds. The ferrocene region can show from two to four
signals (around 4.0-4.5 ppm) that correspond to the substituted cyclopentadiene. The
number of signals depends on the heterotopicity of the ring protons, with one characteristic
singlet corresponding to the five protons of the non-substituted cyclopentadiene, around
4
.15 or 4.00 ppm for the cases of aliphatic or aromatic groups bound to the methane bridge,
respectively. The third region contains the signals of protons on the bridge and those of the
13
aliphatic substituents R, and it varies depending on the substituent. The C-NMR spectra
can be analyzed in an analogous manner to that of the proton spectra, with signals
corresponding to the naphthoquinone above 120 ppm and to the ferrocene unit between 65
and 90 ppm. Confirmation of the structural scaffold was obtained for 10b through X-Ray
crystallography (Supplementary material) and no significant deviations in angles or bonds
distances were observed with respect to those in ferrocene and 2-hydroxy-1,4-
naphthoquinone structures.
2
.2. Biological section
For the determination of antiplasmodial activity, in vitro tests were performed on two
cultured strains of P. falciparum (3d7, CQ-sensitive; Dd2, CQ-resistant). Most of the
compounds showed IC values under 15 ꢀM in both strains, 10j being the only exception
5
0
(
Table 2). For comparison, a IC for atovaquone of 0.0008 ꢀM has been measured against
50
cell lines Dd2 [61]. Maximum activity was observed for compounds 10a, 10b and 10c
which present an IC close to 1 ꢀM, and an important decrease in growth inhibition can be
5
0
observed when the substituent is a propyl or larger group. The use of bulky substituents in
this position does not favor antiplasmodial activity; however, compound 10f does not suffer
a dramatic change in its activity despite having an undecyl group as substituent, the most
voluminous of the series. As multiple effects could influence this behavior, an explanation
is hard to rationalize; the possibility that this compound has higher penetration in the cells
and more exposure to the parasite could be important, as well as the different
conformations that this lipophilic chain could achieve. Compounds 10a, 10b, 10c might be
considered as potential leads for further optimization. Moreover, these compounds have the
advantage of inhibiting both CQ-sensitive and CQ-resistant strains at a similar
concentration.
Table 2. In vitro antiplasmodial and biomineralization activities of the ferrocene-
naphthoquinone derivatives 10a-k.
P. falciparum strainIC (ꢀM)
FBIT test
5
0
Compound
IC (mg/mL)
5
0
3
D7
Dd2
1.48
1.19
2.10
13.3
13.4
Resistance index*
1
0a
0b
0c
0d
0e
1.40
1.30
1.60
13.2
12.1
1.1
0.9
1.3
1.0
1.1
>2
>2
>2
>2
>2
1
1
1
1
1
2

ACCEPTED MANUSCRIPT
1
0f
0g
0h
4.01
8.35
7.34
12.6
37.4
7.59
0.03
4.51
18.1
10.2
14.0
22.5
4.28
0.50
1.1
2.2
1.4
1.1
0.6
0.6
16.7
>2
>2
1
1
0.79
>2
1
0i
1
0j
>2
1
0k
>2
CQ
0.04
*
Values calculated by the equation RI = IC (DD2)/ IC (3D7).
50 50
To obtain more information about the mechanism of action of these substances, a
ferriprotoporphyrin IX biomineralization inhibition test (FBIT) was performed.
Ferriprotoporphyrin IX is the heme group product of the digestion of hemoglobin from
erythrocytes by the parasite. This substance is toxic to the parasite and its accumulation is
prevented by biomineralization, producing hemozoin. Ferriprotoporphyrin IX
biomineralization inhibition is the mechanism proposed for the chloroquine antimalarial
activity, and this is related to iron coordination [41, 62]. Therefore, as some quinones can
coordinate metals, a similar effect was expected for these derivatives; however, no
inhibition of biomineralization was observed except for 10h which showed low activity in
the full parasite tests.
Because of their quinonic nature, the antimalarial activity of these compounds might
involve the mitochondrial bc1 complex, as in the case of ATV. Malaria parasites contain
enzymatic electron-transfer complexes based on cytochromes in the mitochondria and in
the apicoplast (a non-photosynthetic plastid derived from alga). Experimentally it has been
suggested that the mitochondria and apicoplast are physically and functionally associated in
Plasmodium spp, both originating from the characteristic symbiont of this apicomplexan
group of protozoan [63, 64]. To clarify, ATV has been shown to bind to the mitochondrial
bc1 complex and, due to the redox system of the quinone, might disrupt the same electron-
transport system in the apicoplast. The proposed activity on the cytochrome bc1 complex is
probably extensible to the apicoplast. As a consequence, a pharmacological target based on
the plant-like apicoplast would be safer because of its phylogenic distance from the host.
The crystallized structure of this target is not available for P. falciparum and molecular
docking was carried out using the crystal structure of cytochrome b from Saccharomyces
cerevisiae; these structures have high homology and the protein from the yeast has been
used in the literature to simulate the plasmodial protein complex [23]. We used the LGA
search method with the standard parameters in AutoDock using a flexible ligand against a
rigid protein target in this study (no flexible side-chain docking was performed).
Interestingly, in these simulations and according to the values found, compounds 10a, 10b,
1
0d, 10g seem to display higher affinities (lower ꢁG values) than ATV (Table 3); two of
them, 10a and 10b showed IC values under 2 ꢀM (Table 2). It should be mentioned that
5
0
we do not expect a clear trend between the predicted binding energies and the experimental
activities for all the compounds. This lack of a clear correlation is obvious comparing the
1
3

ACCEPTED MANUSCRIPT
∆G with the results of inhibition for the anomalous compounds 10d and 10g. First, the
structures for P. falciparum and S. cerevisiae are not the same and until the former structure
is available we can only intuit the binding that occurs in the actual host. Second, the
docking studies were performed without the ability to include changes that occur in pKa
and entropy that are related to the change of the C3 group. Finally, our docking method
does not present a flexible protein during docking so that subtle changes in the backbone
and side-chains could affect the results.
Table 3. Free energy ꢁG values calculated for the simulated binding of ferrocene-
naphthoquinones 10a-k to cytochrome b.
-1
Compound
0a
0b
0c
0d
0e
0f
0g
0h
ꢁG (kcal•mol )
1
-9.12
-9.29
-8.85
-9.05
-8.16
-7.87
-9.25
-8.49
-8.10
-7.43
-6.95
-8.95
1
1
1
1
1
1
1
1
0i
1
0j
1
0k
ATV
When visualizing the predicted conformations, it is very important to note that only
compounds 10a, 10b and 10c share a similar binding mode with ATV (Figure 1A), unlike
the compounds 10d and 10g, which, when accommodated in the lowest energy according to
AutoDock, have a different binding orientation. We predict that the binding mode for
higher antimalarial activity would permit that the small substituent on the bridge can be
accommodated in a pocket of the cytochrome (Figure 1B). The substituent on the bridge
should not be larger than the ethyl group for proper interaction with the target. In terms of
future optimization, other positions should be explored to enhance binding, such as
substitutions on either cyclopentadiene ring or the introduction of a larger linker between
the ferrocene and the quinone fragments.
1
4

ACCEPTED MANUSCRIPT
A
B
Figure 1. A) Superimposition of the main conformers of 10a and atovaquone B) Docking
simulation of 10b in cytochrome b of the mitochondrial bc complex.
1
The binding of these compounds to the target site is predicted to have similar interactions
as atovaquone. It can be distinguished by a hydrogen-bond with His181 (1.998 Å, -4.149
kcal/mol) on the subunit that contains the iron-sulfur cluster of the cytochrome bc1
complex (Figure 2). This interaction could be especially important, as histidine could be
protonated and generate an electrostatic interaction with the deprotonated 2-
hydroxynaphthoquinone.
Figure 2. Hydrogen bond between the 2-OH group of 10a and His181 in the iron sulfur
cluster of the cytochrome bc complex.
1
Besides being toxic to the parasite, antiplasmodial agents should exert this toxicity
selectively, without significantly damaging human cells at therapeutic doses. The
cytotoxicity of the ferrocenequinones was tested in mammal cells (murine macrophages
JJ74) and CC values between 45 and 416 ꢀM were observed (Table 4). These values are
50
higher than those found for the parasite strains and represent selectivity indexes (SI)
between 2 and 260 (Table 4). It is remarkable that the most potent antiplasmodial
compounds 10a, 10b and 10c also show the highest selectivity indexes.
Table 4. Cytotoxicity of ferrocenenaphthoquinones for JJ74 mouse macrophages.
1
5

ACCEPTED MANUSCRIPT
Compound
0a
0b
0c
0d
0e
0f
0g
0h
CC (ꢀM)
Selectivity Index*
50
1
167
194
416
299
318
122
49.6
45.2
56.6
62.6
67.0
120
150
260
23
27
31
6
1
1
1
1
1
1
1
6
1
0i
5
1
0j
2
1
0k
9
*
Values calculated by the equation SI = CC (JJ74)/ IC (P. falciparum 3D7).
50 50
1
6

ACCEPTED MANUSCRIPT
3
. Conclusions
An “on-water” methodology was adapted for the synthesis of eleven new derivatives of 3-
ferrocenylmethyl)-2-hydroxynaphthoquinone. The structures of the compounds were
established by spectroscopic techniques. The compounds showed IC values under 20 ꢀM
(
5
0
to Plasmodium falciparum CQ-sensitive and CQ-resistant strains, without appreciable
inhibition of ferriprotoporphyrin IX biocrystallization. Most substances showed selective
toxicity for parasites with respect to mammal cells with SI values higher than 120.
Compounds 10a, 10b, 10c showed the best antimalarial activity with IC values under 2
5
0
ꢀ
M, as well as adequate ∆G values of in silico binding to the cytochrome b unit of the
mitochondrial bc complex; these compounds could be explored as a scaffold to new and
1
more potent analogues. Further work is needed to determine the in vivo efficacy of these
compounds and to confirm their mechanism of action.
1
7

ACCEPTED MANUSCRIPT
4
. Experimental
4
.1.General
All commercial chemicals and solvents were reagent grade and were used without further
purification unless otherwise specified. Melting points were determined using a Mel-Temp
Thomas Scientific, Model IA9100 with a precision of ±1 °C. IR spectra were recorded on
-
Perkin Elmer Spectrum Paragon 1000 using KBr pellets and values were represented in cm
1
1
13
.
H-NMR and C-NMR were carried out on Varian-Mercury 400 BB (400 MHz and 100
MHz for H and C, respectively) and a Brucker Ascend 600 (600 and 150 MHz for H and
C, respectively), using TMS as an internal standard and chemical shifts were recorded in
1
13
1
1
3
ppm on a δ scale. Exact mass of the compounds was determined in a Waters Synapt high
resolution mass spectrometer in positive mode and introducing the sample by electrospray.
Thin layer chromatography (TLC) on silica gel plates containing UV indicator HF254 was
employed routinely to follow the course of reactions. When necessary, products were
purified by column chromatography using silica gel 230-400 mesh. High-performance
liquid chromatography was performed on a Dionex instrument: column, ProteCol GP C18
1
25 (250 x 4.6 mm, 5 ꢀm). Compounds were detected by UV at 254 nm. The mobile phase
was (water /0.1% acetic acid)/methanol (20:80 v/v) with flow rate of 1.2-2.0 mL/min. The
purity of all tested compounds was greater than 95% based on analytical HPLC.
4
.2. Synthesis of acyl chlorides
To 5 mL of previously distilled thionyl chloride were added 3 mmol of the carboxylic acid,
and immediately 3 drops of dimethylformamide (DMF). The reaction mixture was heated at
7
0 °C for approximately 90 minutes, forming a yellowish translucent solution. Excess of
thionyl chloride was eliminated from the mixture with a rotary evaporator connected to a
potassium hydroxide trap. Products obtained were pale yellow oils or solids. Acyl chlorides
were employed directly for following reactions without further purification.
4
.3. Synthesis of ferrocenylketones 8
A solution 3-5 mmol of ferrocene in dichloromethane (DCM) or dichloroethane (DCE) (3-5
mL) was prepared. A previously prepared solution of 1 equivalent of acyl chloride and 1
equivalent of aluminum chloride in DCM or DCE (3-5 mL) was transferred to the ferrocene
solution, while stirring and with positive nitrogen pressure. The mix of the two liquids
generated an intense purple or blue mixture. Reaction was stopped after 30-60 minutes of
stirring at room temperature. An equal quantity of water was added to the reaction mixture.
The organic phase was separated and the aqueous phase was extracted with methyl t-butyl
ether (MTBE), and the combined organic phase was dried over anhydrous sodium sulfate.
The solvent was removed and the crude product (red to orange solid) was purified by
column chromatography column on silica gel (230-400 mesh) with a gradient of
cyclohexane-benzene as eluent.
4
.4. Synthesis of ferrocenylcarbinol derivatives 9
To a clean and dry round bottom flask with a septum, 3-5 mmol of the ferrocenylketone
were added and approximately 20 mL of dimethoxyethane (DME) were transferred with a
positive nitrogen pressure; the mixture was stirred to obtain a reddish solution. 0.75
1
8

ACCEPTED MANUSCRIPT
equivalents of lithium aluminum hydride (1 M in tetrahydrofuran) were added, and a
change of color from red to yellow in the solution was observed. The reaction was stopped
after 30 minutes at room temperature; complete transformation was confirmed with TLC.
After this time, Glauber’s salt was added and the mixture was stirred until a formation of a
granular precipitate was observed. The mixture was filtered, and the solvent was eliminated
to obtain yellow to orange oils or solids. The compounds were employed directly for
following reactions without further purification.
4
.5. Synthesis of 3-(ferrocenylmethyl)-2-hydroxy-1,4-naphthoquinones derivatives 10
To a clean and dry round bottom flask containing 3-5 mmol of the ferrocenylcarbinol, an
equivalent quantity of 2-hydroxy-1,4-naphthoquinone and 20 mL of water were added. The
mixture was stirred and heated at 40 °C or 70 °C; the formation of a green solid in the walls
of the flask was observed. Reaction was stopped when no change was detected by TLC,
reaction time was variable depending on the product. The mixture was extracted with
MTBE and the combined organic phase was dried over anhydrous sodium sulfate. The
solvent was removed and the crude product (red to orange solid) was purified by column
chromatography column on silica gel (230-400 mesh) with a gradient of cyclohexane-
benzene as eluent, obtaining green solids.
4
.5.1. 3-(ferrocenylmethyl)-2-hydroxy-1,4-naphthoquinone 10a
1
6
2
0a was obtained from ferrocenemethanol 9a according to general procedure 4.5. Yield
5%, pale green powder. m.p. = 161-162 °C. IR (KBr) ν/cm : 3359 (s, sh), 3084 (w, b),
-1
928 (w, sh), 1656, 1592 (s, sh), 1457 (w, sh), 1426 (w, sh), 1368 (s, sh), 1344 (s, sh), 1273
1
(
(
(
m, sh), 1248 (m, sh). H-NMR (CDCl , 600 MHz) δ (ppm): 8.10 (1H, d, J=7.6 Hz), 8.03
1H, d, J=7.5 Hz), 7.73 (1H, t, J=7.5 Hz), 7.65 (1H, t, J=7.5 Hz), 7.25 (1H, s, -OH), 4.25
2H, s), 4.16 (5H, s), 4.02 (2H; s), 3.65 (2H, s). C-NMR (CDCl , 150 MHz) δ (ppm):
3
13
3
1
2
84.2, 182.2, 153.3, 134.9, 132.8, 132.8, 129.5, 126.8, 126.0, 123.0, 85.9, 69.1, 68.8, 67.4,
+
+
3.1. EI-HRMS (m/z) [M+H] Calcd for C H FeO 373.0527, [M+H] Found: 373.0522.
21
16
3
4
.5.2. 3-(1-ferrocenylethyl)-2-hydroxy-1,4-naphthoquinone 10b
1
7
2
1
0b was obtained from 1-ferroceneethanol 9b according to general procedure 4.5. Yield
9%, intense green powder. m.p. = 142-143 °C. IR (KBr) ν/cm : 3350 (s, sh), 3091 (w, sh),
-1
930 (w, b), 1667 (s, sh), 1646 (s, sh), 1635 (s, sh), 1561 (s, sh), 1413 (m, sh), 1361 (s, sh),
1
344 (s, sh), 1266 (s, sh). H-NMR (CDCl , 600 MHz) δ (ppm): 8.10 (1H, dd, J=7.7 Hz,
3
J=0.9 Hz), 8.03 (1H, dd, J=7.6 Hz, J=0.9 Hz), 7.74 (1H, td, J=7.6 Hz, J=7.6 Hz, J=1.3
Hz), 7.65 (1H, td, J=7.5 Hz, J=7.5 Hz, J=1.3 Hz), 7.37 (1H, s, -OH), 4.42 (2H, m), 4.16
13
(
5H, s broad), 4.08 (2H, m), 4.05 (1H, s broad), 1.67 (3H, d, J=7.3 Hz). C-NMR (CDCl ,
3
1
9
3
50 MHz) δ (ppm): 184.1, 181.9, 152.3, 135.0, 132.9, 132.8, 129.2, 127.2, 127.0, 125.9,
+
1.7, 68.7, 68.1, 67.2, 66.7, 29.2, 17.2. EI-HRMS (m/z) [M+H] Calcd for C H FeO
2
2
18
3
+
87.0684, [M+H] Found: 387.0674.
4
.5.3. 3-(1-ferrocenylpropyl)-2-hydroxy-1,4-naphthoquinone 10c
1
7
0c was obtained from 1-ferrocenepropanol 9c according to general procedure 4.5. Yield
5%, dark green powder. m.p. = 108-110 °C. IR (KBr) ν/cm : 3353 (s, sh), 3082 (w, b),
-1
1
9

ACCEPTED MANUSCRIPT
2
967 (w, sh), 2919 (w, sh), 1652 (s, sh), 1590 (m, sh), 1458 (w, sh), 1372 (s, sh), 1269 (s,
1
sh), 1249 (m, sh). H-NMR (CDCl , 600 MHz) δ (ppm): 8.11 (1H, d, J=6.6 Hz), 8.05 (1H,
3
dd, J=7.6 Hz, J=0.9 Hz), 7.75 (1H, t, J=7.3 Hz), 7.67 (1H, td, J=7.5 Hz, J=7.5 Hz, J=1.6
Hz), 7.40 (1H, s, -OH), 4.36 (1H, s broad), 4.20 (1H, d broad), 4.12 (5H, s broad), 4.08
1
3
(
2H, m), 4.03 (1H, m), 2.3 (1H, m), 2.13 (1H, m), 0.93 (3H, t, J=7.5 Hz). C-NMR
(
1
CDCl , 150 MHz) δ (ppm): 184.4, 181.8, 152.6, 135.0, 132.9, 132.8, 129.2, 127.2, 126.0,
3
+
25.7, 91.3, 68.7, 67.9, 67.8, 67.1, 66.7, 29.7, 24.9, 12.9. EI-HRMS (m/z) [M+H] Calcd for
+
C H FeO 401.0840, [M+H] Found: 401.0819.
23
20
3
4
.5.4. 3-(1-ferrocenylbutyl)-2-hydroxy-1,4-naphthoquinone 10d
1
8
2
0d was obtained from 1-ferrocenebutanol 9d according to general procedure 4.5. Yield
3%, dark green powder. m.p. = 136-139 °C. IR (KBr) ν/cm : 3351 (s, sh), 3083 (w, b),
-1
955 (w, sh), 2932 (w, sh), 1657 (s, sh), 1648 (m, sh), 1592 (m, sh), 1460 (w, sh), 1377 (m,
1
sh), 1269 (s, sh). H-NMR (CDCl , 600 MHz) δ (ppm): 8.11 (1H, s broad), 8.05 (1H, dd,
3
J=7.6 Hz, J=0.8 Hz), 7.74 (1H, td, J=7.4 Hz, J=7.4 Hz, J=1.2 Hz), 7.66 (1H, td, J=7.5 Hz,
J=7.5 Hz, J=1.2 Hz), 7.41 (1H, s, -OH), 4.36 (1H, s broad), 4.30 (1H, s broad), 4.10 (5H, s
broad), 4.08 (2H, m), 4.03 (1H, s broad), 2.34 (1H, m broad), 2.07 (1H, m broad), 1.31 (2H,
1
3
sextet, J=7.3 Hz), 0,96 (3H, t, J=7.3 Hz). C-NMR (CDCl , 150 MHz) δ (ppm): 184.3,
3
1
3
81.8, 152.6, 135.0, 132.9, 132.8, 129.2, 127.1, 126.0, 91.4, 68.7, 68.0, 67.8, 67.1, 66.7,
+
+
4.0, 21.4, 21.4 14.2.EI-HRMS (m/z) [M+H] Calcd for C H FeO 415.0997, [M+H]
,
24 22
3
Found: 415.0989.
4
.5.5. 3-(4-chloro-1-ferrocenylbutyl)-2-hydroxy-1,4-naphthoquinone 10e
1
0e was obtained from 4-chloro-1-ferrocenebutanol 9e according to general procedure 4.5.
-1
Yield 81%, pale green powder. m.p. = 169-171 °C. IR (KBr) ν/cm : 3352 (s, sh), 3067 (w,
b), 2963 (w, b), 1655 (s, sh), 1634 (m, sh), 1589 (m, sh), 1458 (w, sh), 1376 (m, sh), 1265
1
(
s, sh). H-NMR (CDCl , 600 MHz) δ (ppm): 8.10 (1H, s broad), 8.06 (1H, dd, J=7.6 Hz,
3
J=1.0 Hz), 7.76 (1H, td, J=7.3 Hz, J=7.3 Hz, J=1.1 Hz), 7.68 (1H, td, J=7.5 Hz, J=7.5 Hz,
J=1.1 Hz), 7.45 (1H, s, -OH), 4.36 (1H, s broad), 4.29 (1H, s broad), 4.14 (5H, s), 4.09
13
(
2H, m), 4.05 (1H, m), 3,59 (2H, m), 2,46 (1H, m), 2,32 (1H, m), 1.78 (2H, m). C-NMR
(
1
CDCl , 150 MHz) δ (ppm): 184.3, 181.6, 152.7, 135.1, 133.0, 132.8, 129.2, 127.1, 126.1,
3
+
25.1, 90.8, 68.7, 68.0, 67.7, 67.2, 66.8, 44.8, 31.1, 28.8, 1.1. EI-HRMS (m/z) [M+H]
+
Calcd for C H ClFeO 449.0607, [M+H] Found: 449.0589.
2
4
21
3
4
.5.7. 3-(1-ferrocenyldodecyl)-2-hydroxy-1,4-naphthoquinone 10f
1
2
0f was obtained from 1-ferrocenedodecanol 9f according to general procedure 4.5. Yield
-1
8%, green oil. IR (KBr) ν/cm : 3354 (s, sh), 3092 (w, b), 2919 (s, sh), 1654 (s, sh), 1593
1
(m, sh), 1458 (w, sh), 1377 (s, sh), 1348 (m, sh), 1267 (s, sh). H-NMR (CDCl , 600 MHz)
3
δ (ppm): 8.11 (1H, s broad), 8.05 (1H, dd, J=7.6 Hz, J=1.0 Hz), 7.74 (1H, td, J=7.4 Hz,
J=7.4 Hz, J=1.0 Hz), 7.66 (1H, td, J=7.5 Hz, J=7.5 Hz, J=1.0 Hz), 7.41 (1H, s, -OH), 4.36
(
2
1H, s broad), 4.26 (1H, s broad), 4.11 (5H, s), 4.08 (2H, m), 4.03 (1H, m), 2.32 (1H, m),
1
3
.05 (1H, m), 1,15-1.40 (18H, m), 0.87 (3H, t, J=7.1 Hz). C-NMR (CDCl , 150 MHz) δ
3
(ppm): 184.3, 182.9, 152.6, 135.0, 132.9, 132.8, 129,25, 127.1, 126.1, 126,0, 91.5, 68.7,
2
0

ACCEPTED MANUSCRIPT
+
6
8.0, 67.8, 67.1, 66.7, 31.9, 28.4, 29.3, 29.6, 29.7, 22.7, 14.1. EI-HRMS (m/z) [M+H]
+
Calcd for C H FeO 527.2249, [M+H] Found: 527.2247.
3
2
38
3
4
.5.6. 3-(1-cyclohexyl-1-ferrocenylmethyl)-2-hydroxy-1,4-naphthoquinone 10g
0g was obtained from 1-cyclohexyl-1-ferrocenemethanol 9g according to general
1
-1
procedure 4.5. Yield 11%, green crystals. m.p. = 97-98 °C (dec). IR (KBr) ν/cm : 3404 (s,
b), 2925 (s, sh), 1654 (s, sh), 1592 (m, sh), 1364 (m, sh), 1267 (m, sh), 1222 (s, sh). H-
1
NMR (CDCl , 600 MHz) δ (ppm), mixture of major and minor rotameric compounds
3
(minor rotamer indicated as “rotamer”): 8.24 (1H, dd, J=7.7 Hz, J=1.3 Hz; rotamer 8.18,
1
7
H, d, J=7.7 Hz), 8.14 (1H, dd, J=7.6 Hz, J=1.3 Hz; rotamer 8.11, 1H, d, J=7.6 Hz), 7.83-
.77 (1H, m), 7.74-7.69 (1H, m), 7.59 (1H, s, -OH; rotamer 7.52, 1H, s), 4.33 (1H, s;
rotamer 4.28, 1H, s), 4.15 (1H, s; rotamer 4.11, 1H, s), 4.07 (2H, m broad), 3.93 (1H, d,
J=10.8 Hz; rotamer 3.60, 1H, d, J=10.6 Hz), 3.85 (5H, s; rotamer 3.86, 5H, s), 2.09 (1H, m;
1
3
rotamer 2.22, 1H, m), 1.56-1.61 (4H, m), 1.19-1.05 (4H, m), 0.76-0.94 (2H, m). C-NMR
CDCl , 150 MHz) δ (ppm): 184.9, 182.2, 152.1, 135.2, 133.1, 132.7, 129.2, 127.4, 126.4,
(
3
1
26.1, 89.5, 71.2, 68.4, 67.7, 67.1,66.0, 41.0, 39.8, 32.5, 32.2, 26.9, 26.4, 26.1. EI-HRMS
+
+
(m/z) [M+H] Calcd for C H FeO 455.1310, [M+H] Found: 455.1300.
27 26 3
4
1
.5.8. 3-(1-ferrocenyl-2-phenylethyl)-2-hydroxy-1,4-naphthoquinone 10h
0h was obtained from 2-phenyl-1-ferroceneethanol 9h according to general procedure 4.5.
-1
Yield 53%, dark green powder. m.p. = 68-70 °C (dec). IR (KBr) ν/cm : 3363 (m, b), 3084
w, sh), 2927 (w, b), 1659 (s, sh), 1648 (s, sh), 1592 (m, sh), 1494 (w, sh), 1458 (m, sh),
(
1
8
1
367 (s, sh), 1337 (m, sh), 1283 (s, sh), 1263 (m, sh). H-NMR (CDCl , 600 MHz) δ (ppm):
3
.03 (1H, d, J=7.2 Hz), 7.98 (1H, d, J=7.5 Hz), 7.70 (1H, td, J=7.6 Hz, J=7.6 Hz, J=1.2
Hz), 7.61 (1H, td, J=7.5 Hz, J=7.5 Hz, J=1.1 Hz), 7.19 (4H, m), 7.10 (1H, t, J=7.1 Hz),
.77 (1H, s broad), 4.40 (1H, s broad), 4.15 (5H, s broad), 4.11 (2H, m broad), 4.07 (1H, m
4
1
3
broad), 3.61 (1H, s broad), 3.49 (1H, s broad). C-NMR (CDCl , 150 MHz) δ (ppm): 183.9,
3
1
9
4
81.7, 152.4, 140.3, 134.9, 132.7, 132.5, 129.0, 128.6, 128.2, 127.0, 126.0, 125.9, 125.1,
+
0.6, 68.7, 68.1, 67.9, 67.3, 37.4, 35.5. EI-HRMS (m/z) [M+H] Calcd for C H FeO
28
22
3
+
63.0997, [M+H] Found: 463.1001.
4
.5.9. 3-(1-ferrocenyl-1-phenylmethyl)-2-hydroxy-1,4-naphthoquinone 10i
1
4
3
0i was obtained from 1-ferrocenyl-1-phenylmethanol 9i according to general procedure
.5. Yield 68%, bright green crystals. m.p. = 65-66 °C (dec). IR (KBr) ν/cm : 3364 (m, b),
-1
083 (w, b), 2928 (w, b), 1662 (s, sh), 1594 (m, sh), 1493 (w, sh), 1459 (w, sh), 1371 (s,
1
sh), 1337 (m, sh), 1266 (s, sh). H-NMR (CDCl , 600 MHz) δ (ppm): 8.18 (1H, dd, J=7.7
3
Hz, J=0.7 Hz), 8.09 (1H, dd, J=7.6 Hz, J=0.8 Hz), 7.78 (1H, td, J=7.6 Hz, J=7.6 Hz,
J=1.3 Hz), 7.70 (1H, td, J=7.6 Hz, J=7.6 Hz, J=1.2 Hz), 7.47 (1H, s, -OH), 7.36
(
impurity), 7.34 (2H, d, J=7.3 Hz), 7.24 (2H, d, J=7.8 Hz), 7.18 (1H, t, J=7.3 Hz), 5.79
1
3
(1H, s), 4.23 (1H, m), 4.19 (1H, m), 4.15 (2H, t, J=1.8 Hz), 4.03 (5H, s). C-NMR (CDCl ,
3
1
1
50 MHz) δ (ppm): 184.4, 181.8, 152.6, 135.2, 135.0, 132.9, 132.8, 129.2, 128.8, 127.9,
+
27.0, 126.3, 126.0, 125.7, 91.3, 68.6, 67.9, 67.1, 66.7, 29.7.EI-HRMS (m/z) [M+H] Calcd
+
for C H FeO 449.0840, [M+H] Found: 449.0827.
2
7
20
3
2
1

ACCEPTED MANUSCRIPT
4
.5.10. 3-(1-(4-chlorophenyl)-1-ferrocenylmethyl)-2-hydroxy-1,4-naphthoquinone 10j
0j was obtained from 1-(4-chlorophenyl)-1-ferrocenemethanol 9j according to general
1
-1
procedure 4.5. Yield 79%, bright green crystals. m.p. = 83-84 °C (dec). IR (KBr) ν/cm :
367 (m, b), 3088 (w, b), 1654 (s, sh), 1647 (s, sh), 1593 (m, sh), 1489 (m, sh), 1459 (w,
3
1
sh), 1370 (s, sh), 1338 (s, sh), 1265 (s, sh). H-NMR (CDCl , 600 MHz) δ (ppm): 8.18 (1H,
3
dd, J=7.7 Hz, J=0.9 Hz), 8.10 (1H, dd, J=7.6 Hz, J=1.0 Hz), 7.80 (1H, td, J=7.6 Hz, J=7.6
Hz, J=1.3 Hz), 7.71 (1H, td, J=7.6 Hz, J=7.6 Hz, J=1.3 Hz), 7.49 (1H, s, -OH), 7.25 and
7
.20 (4H, AA’BB’, J=8.3 Hz), 5.74 (1H, s) H , 4.21 (1H, m), 4.16 (3H, m), 4.03 (5H;
1’
1
3
m). C-NMR (CDCl , 150 MHz) δ (ppm): 183.9, 181.9, 152.8, 141.2, 135.4, 133.2, 132.7,
3
1
32.1, 130.1, 129.2, 128.0, 127.3, 126.3, 125.3, 87.4, 69.8, 68.8, 68.6, 67.7, 67.6, 40.2. EI-
+
+
HRMS (m/z) [M+H] Calcd for C H ClFeO 483.0450, [M+H] Found: 483.0431.
2
7
19
3
4
.5.11. 3-(1-(4-bromophenyl)-1-ferrocenylmethyl)-2-hydroxy-1,4-naphthoquinone 10k
1
0k was obtained from 1-(4-bromophenyl)-1-ferrocenemethanol 9k according to general
-1
procedure 4.5. Yield 73%, bright green crystals. m.p. = 77-78 °C (dec). IR (KBr) ν/cm :
3
1
368 (m, b), 2923 (w, b), 1654 (s, sh), 1594 (w, sh), 1484 (w, sh), 1375 (s, sh), 1339 (s, sh),
1
266 (s, sh). H-NMR (CDCl , 600 MHz) δ (ppm): 8.19 (1H, dd broad, J=7.7 Hz, J=0.9
3
Hz), 8.10 (1H, dd, J=7.6 Hz, J=0.9 Hz), 7.80 (1H, td, J=7.6 Hz, J=7.6 Hz, J=1.3 Hz), 7.7
1H, td, J=7.6 Hz, J=7.6 Hz, J=1.2 Hz), 7.49 (1H, s, -OH), 7.36 and 7.20 (4H, AA’BB’,
(
1
3
J=8.4 Hz), 5.73 (1H, s), 4.20 (1H, m broad), 4.16 (3H, m broad), 4.03 (5H; s). C-NMR
CDCl , 150 MHz) δ (ppm): 183.9, 181.9, 152.7, 141.7, 135.4, 133.2, 132.7, 130.9, 130.6,
(
3
1
29.1, 127.3, 126.2, 125.2, 120.2, 87.3, 69.8, 68.8, 68.6, 67.7, 67.5, 40.2. EI-HRMS (m/z)
+
+
[
M+H] Calcd for C H BrFeO 526.9945, [M+H] Found: 526.9928.
27 19 3
4
4
.6. Biological evaluation
.6.1. In vitro antimalarial activity screening against Plasmodium falciparum [61]
The SYBR® GreenI-based micromethod was followed for testing the antimalarial activity
of the compounds. Erythrocytic stages of P. falciparum 3D7 strain, CQ-sensitive, and Dd2
strain, CQ-resistant, were maintained in RPMI 1640 culture medium supplemented with
0
.5% Albumax II at 37 ºC in an atmosphere with 5% CO . An erythrocyte suspension, with
2
initial 1% parasitemia and 4% hematocrit, was prepared using the aforementioned culture
and then distributed into a 96-well plate, 50 ꢀL per well. Next, stock solutions of each
compound were prepared in DMSO and diluted in RPMI medium. The final DMSO
concentration was never higher than 0.1%. 50 ꢀL of each prepared concentration were
added per well. DMSO and chloroquine were included as negative and positive controls,
respectively. All compounds and controls were placed in triplicate. The plate was incubated
under the same conditions and after 48 h, the plate was removed from the incubator and
frozen for at least 1 h at -70 °C and then thawed. Finally, 100 ꢀL of SYBR®GreenI in lysis
buffer (0.2 ꢀL/mL) was added per well and shaken for 5 minutes or until no precipitated
erythrocytes were observed. The plate was left to stand in the dark for 1 h at room
temperature. Fluorescence intensity (FI) for each well was measured (exciting wavelength
2
2

ACCEPTED MANUSCRIPT
4
85 nm, detecting wavelength 535 nm). The background of the nonparasitized erythrocytes
was subtracted from each well tested. Percentage inhibition of the parasite growth for each
concentration was calculated by using the following formula: % inhibition = 100 ×
[
(F.I.control – F.I.comp)/(F.I.control)]. IC values were estimated by plotting drug
50
concentration versus percentage inhibition.
4
.6.2. Nonspecific cytotoxicity tests [62]
Murine J774 macrophages were maintained in RPMI 1640 medium supplemented with
0% FBS at 37 °C in a 5% CO atmosphere. First, in a flat bottom 96-well microplate, 100
1
ꢀ
2
4
L of macrophage suspension in RPMI medium, containing 7 × 10 cells, were distributed
per well. The cells were allowed to attach for 24 h at 37 °C. Next, the medium was replaced
by different concentrations of the compounds in 200 ꢀL of medium, or DMSO at the same
concentration as growth control, and the cells were exposed to the compounds solutions for
another 24 h. Each concentration was assayed three times. Afterwards, the medium was
eliminated and 100 ꢀL/well of 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) solution, 0.4 mg/mL in PBS, was added and the plates were returned to
incubator for 1 h. The suspension was removed and the toxic effect of the compounds was
assessed by the reduction of MTT to formazan crystals (as a cell viability indicator); said
crystals were solubilized by adding 100 ꢀL of DMSO. Finally, the absorbance (A) was
measured at 595 nm and the viability percentage was calculated as follows: % viability =
[
(Acontrol - Acomp)/(Acontrol)] × 100. IC50 values were estimated by plotting drug
concentration versus percentage of viability.
4
.6.3.Ferriprotoporphyrin IX biomineralization inhibition test (FBIT) [65]
The procedure for testing FP biomineralization was carried out according to the method
described by Deharo [62]. A mixture containing 50 ꢀL of a 10 mg/mL drug solution or
solvent (for control) or chloroquine, 50 ꢀL of 0.5 mg/mL of hemin chloride freshly
dissolved in dimethylsulfoxide (DMSO) and 100 ꢀL of 0.5 M sodium acetate buffer pH 4.4
was incubated in anon-sterile flat bottom 96-well plate at 37 °C for 24 hrs. After
incubation, the plate was centrifuged at 1,600 G for 5 min and the supernatant was
discarded. The remaining pellet was re-suspended with 200 ꢀL of DMSO in order to
remove unreacted FP. The plate was then centrifuged once again and the supernatant
discarded. The pellet (precipitate of ß-hematin) was dissolved in 150 ꢀL of 0.1 M NaOH
and the absorbance (A) read at 405 nm. The data was expressed as the percentage of
inhibition of FP biomineralization, calculated using the following equation: % inhibition =
1
00 × [(Acontrol -Acomp)/(Acontrol)]. IC50 values were estimated by plotting drug
concentration versus percentage inhibition.
4
.6.4. Molecular docking simulation in the cytochrome b of the cytochrome b-c1 complex
The ligands were docked using a regular rigid receptor-flexible ligand docking approach.
Molecular docking simulations was performed in the software AutoDockv4.2.3, using
PyRx v0.8 as virtual screening tool and AutoDock Tools v1.5.4 for visualization. The
crystallized structure of Saccharomyces cerevisiae cytochrome b-c1 complex bound to its
2
3

ACCEPTED MANUSCRIPT
inhibitor Stigmatellin A (PDB: 1KYO) was used for the study. Preparation of the protein
(
elimination of hydration waters, merge non-polar, add polar hydrogens and charges) was
performed in Discovery Studio Visualizer v3.1.1.11517. Docking was performed in a grid
18.75 Å x 18.75 Å x 22.5 Å) that enclosed Stigmatellin A binding site generated with
(
Autogrid v4.2.3. After simulation, predicted conformations were analyzed by visual
inspections and by their binding free energy.
Acknowledgements
We are thankful to Dr. Grettel Valle (School of Chemistry, Universidad de Costa Rica) and
Dr. Thomas Weyhermueller (Max-Planck-Institute für Bioanorganische Chemie) for
collaboration on X-Ray Crystallography; to the Vice Presidency of Research, Universidad
de Costa Rica and to the National Rectors Council, CONARE, (projects No. 809-A8-001
and FEES-UCR No. 809-A8-518) and to Spanish FIS-ISCIII Program (project No.
PI060782) for the financial support. We also wish to thank the referees for their helpful
suggestions.
2
4

ACCEPTED MANUSCRIPT
References
[
[
[
1]
2]
3]
H.G. Viehe, R. Merenyi, Z. Janousek, Captodative substituent effects in radical
chemistry, Pure App. Chem. 60 (1988) 1635-1644.
M. Uchimiya, A.T. Stone, Reversible redox chemistry of quinones: impact on
biogeochemical cycles, Chemosphere 77 (2009) 451-458.
S. Spyroudis, Hydroxyquinones: synthesis and reactivity, Molecules 5 (2000) 1291-
1
330.
[
[
4]
5]
C. Asche, Antitumour quinones, Mini-Reviews Med. Chem. 5 (2005) 449-467.
H. Hussain, K. Krohn, V.U. Ahmad, G.A. Miana, I. R. Greend, Lapachol: an
overview, ARKIVOC ii (2007) 145-171.
[
[
6]
7]
G. Lenaz, R. Fato, A. Baracca, M.L. Genova, Mitochondrial quinone reductases:
complex I, Methods Enzymol. 382 (2004) 3-20.
M.N. da Silva, V.F. Ferreira, and M.C. de Souza, Um panorama atual da química e
da farmacologia de naftoquinonas, com ênfase na β-lapachona e derivados, Quim.
Nova 26 (2003) 407-416.
[
8]
9]
E. Pérez-Sacau, R.G. Díaz-Peñate, A. Estévez-Braun, A.G. Ravelo, J.M. García-
Castellano, L. Pardo, M. Campillo, Synthesis and pharmacophore modeling of
naphthoquinone derivatives with cytotoxic activity in human promyelocytic
leukemia HL-60 cell line, J. Med. Chem. 50 (2007) 696-706.
[
[
[
K. Sasaki, H. Abe, F. Yoshizaki, In vitro antifungal activity of naphthoquinone
derivatives, Biol. Pharm. Bull 25 (2002) 669-670.
10] K.H. Xu, D.P. Lu, Plumbagin induces ROS-mediated apoptosis in human
promyelocytic leukemia cells in vivo, Leuk. Res. 34 (2010) 658-665.
11] S.C. Cho, M.Z. Sultan, S.S. Moon, Anti-acne activities of pulsaquinone,
hydropulsaquinone, and structurally related 1,4-quinone derivatives, Arch. Pharm.
Res. 32 (2009) 489-494.
[
[
12] Y.R. Jin, K.A. Hwang, M.R. Cho, S.Y. Kim, J.H. Kim, C.K. Ryu, D.J. Son, Y.H.
Park, Y.P. Yun, Antiplatelet and antithrombotic activities of CP201, a newly
synthesized 1,4-naphthoquinone derivative, Vascul. Pharmacol. 41 (2004) 35-41.
13] G.J.
Kapadia,
M.A.
Azuine,
V.
Balasubramanian,
R.
Sridhar,
Aminonaphthoquinones - a novel class of compounds with potent antimalarial
activity against Plasmodium falciparum, Pharmacol. Res. 43 (2001) 363-367.
2
5

ACCEPTED MANUSCRIPT
[
14] A.J. da Silva, C.D. Buarque, F.V. Brito, L. Aurelian, L.F. Macedo, L.H. Malkas, R.J.
Hickey, D.V. Lopes, F. Noel, Y.L. Murakami, N.M Silva, P.A. Melo, R.R. Carusso,
N.G. Castro, P.R. Costa, Synthesis and preliminary pharmacological evaluation of
new (+/-) 1,4-naphthoquinones structurally related to lapachol, Bioorg. Med. Chem.
1
0 (2002) 2731-2738.
[
[
15] W. Knecht, J. Henseling, M. Löffler, Kinetics of inhibition of human and rat
dihydroorotate dehydrogenase by atovaquone, lawsone derivatives, brequinar
sodium and polyporic acid, Chem. Biol. Interact. 124 (2000) 61-76.
16] L. Pelinski, A. Baramee, A. Coppin, S. Tomavo, J. Brocard, Synthesis and in vitro
activities of ferrocenic aminohydroxynaphthoquinones against Toxoplasma gondii
and Plasmodium falciparum, Bioorg. Med. Chem. 14 (2006) 1294-1302.
[
[
[
17] A.V. Pinto, S. Castro, The trypanocidal activity of naphthoquinones: a review,
Molecules (2009) 4570-4590.
18] J.L. Bolton, M.A. Trush, T.M. Penning, G. Dryhurst, T.J. Monks, Role of quinones
in toxicology, Chem. Res. Tox. 13 (2000) 135-160.
19] D. Marzin, D. Kirkland, 2-Hydroxy-1,4-naphthoquinone, the natural dye of Henna,
is non-genotoxic in the mouse bone marrow micronucleus test and does not produce
oxidative DNA damage in chinese hamster ovary cells, Mutation. Res. 560 (2004)
4
1–47.
[
20] G. Kokwaro, Ongoing challenges in the management of malaria, Malaria J. 8 (2009)
S2-S7.
[
[
21] World Health Organization, World malaria report, (2012).
22] I.K. Srivastava, J.M. Morrisey, E. Darrouzet, F. Daldal, A.B. Vaidya, Resistance
mutations reveal the atovaquone-binding domain of cytochrome b in malaria
parasites, Mol. Microbiol. 33 (1999) 704-712.
[
23] J. J. Kessl, B.B. Lange, T. Merbitz-Zahradnik, K. Zwicker, P. Hill, B. Meunier, H.
Palsdottir, C. Hunte, S. Meshnick, B.L. Trumpower, Molecular basis for atovaquone
binding to the Cytochrome bc1 Complex, J. Biol. Chem. 278 (2003) 31312-31318.
[
[
[
24] G.B. Kauffman, The discovery of ferrocene: the first sandwich compound, J. Chem.
Ed. 60 (1983) 185-186.
25] M. Laskoski, W. Steffen, M.D. Smith, U.H. Bunz, Is ferrocene more aromatic than
benzene?, Chem. Commun. 2001 (2001) 691-692.
26] X. Wu, M. Go, Antimalarial activity of ferrocenyl chalcones, Bioorg. Med. Chem.
Lett. 12 (2002) 2299-2302.
2
6

ACCEPTED MANUSCRIPT
[
[
[
27] N. Chavain, H. Vezin, D. Dive, N. Touati, J.F. Paul, E. Buisine, C. Biot,
Investigation of the redox behavior of ferroquine, a new antimalarial, Mol. Pharm. 5
(2008) 710-716.
28] J. Manosroi, K. Rueanto, K. Boonpisuttinant, W. Manosroi, C. Biot, Novel
ferrocenic steroidal drug derivatives and their bioactivities, J. Med. Chem. 53 (2010)
3
937-3943.
29] C.L. Ferreira, C.B. Ewart, C.A. Barta, S. Little, V. Yardley, C. Martins, E.
Polishchuk, P.J. Smith, J.R. Moss, M. Merkel, M.J. Adam, C. Orvig Synthesis,
structure, and biological activity of ferrocenyl carbohydrate conjugates, Inorg.
Chem. 45 (2006) 659-667.
[
30] B. Long, S. Liang, D. Xin, Y. Yang, J. Xiang, Synthesis, characterization and in
vitro antiproliferative activities of new 13-cis-retinoyl ferrocene derivatives, Eur. J.
Med. Chem. 44 (2009) 2572-2576.
[
[
31] M.F.R. Fouda, M.M. Abd-elzaher, R.A. Abdelsamaia, A.A. Labib, On the medicinal
chemistry of ferrocene, App. Organometal. Chem. 21 (2007) 613-625.
32] N. Chavain, E. Davioud-Charvet, X. Trivelli, L. Mbeki, M. Rottmann, R. Brun, C.
Biot, Antimalarial activities of ferroquine conjugates with either glutathione
reductase inhibitors or glutathione depletors via a hydrolyzable amide linker, Bioorg.
Med. Chem. 17 (2009) 8048-8059.
[
33] M. Soares, J. Oliveira, M. Santos, R. Ribeiro, P. Pigeon, G. Jaouen, A.E. Santana,
M. Goulart, E. Hillard, Antiparasitic and Immunomodulatory Activities of 1,1-bis(4-
hydroxyphenyl)-2-phenyl-but-1-ene and its protected and free 2-ferrocenyl
derivatives, Drug Develop. Res. 71 (2010) 69–75.
[
[
[
[
34] B. Maryanoff, S. Keeley, F. Persico, Replacement of aromatic or heteroaromatic
groups in nonsteroidal antiinflammatory agents with the ferrocene group, J. Med.
Chem. 26 (1983) 226-229.
35] A. Mahajan, L. Kremer, S. Louw, Y. Guéradel, K. Chibale, C. Biot, Synthesis and in
vitro antitubercular activity of ferrocene-based hydrazones, Bioorg. Med. Chem.
Lett. 21 (2011) 2866-2868.
36] D. Huber, H. Hubner, P. Gmeiner, 1,1’-Disubstituted ferrocenes as molecular hinges
in mono- and bivalent dopamine receptor ligands, J. Med. Chem. 52 (2009) 6860-
6
870.
37] K. Schlotter, F. Boeckler, H. Hubner, P. Gmeiner, Fancy Bioisosteres
:
Metallocene-derived G-protein-coupled receptor ligands with subnanomolar binding
affinity and novel selectivity profiles, J. Med. Chem. 48 (2005) 3696-3699.
2
7

ACCEPTED MANUSCRIPT
[
[
[
38] K. Sander, T. Kottke, C. Hoffend, M. Walter, L. Weizel, J.C. Camelin, X. Ligneau,
E.H. Scheneider, R. Seifert, J.C. Schwartz, H. Stark, First metal-containing
histamine H3 receptor ligands, Org. Lett. 12 (2010) 2578-2581.
39] A. Kreidenweiss, P.G. Kremsner, K. Dietz, B. Mordmüller, In vitro activity of
ferroquine (SAR97193) is independent of chloroquine resistance in Plasmodium
falciparum, Am. J. Trop. Med. Hyg. 75 (2006) 1178-1181.
40] M.A. Blackie, P. Beagley, S.L. Croft, H. Kendrick, R. Moss, K. Chibale,
Metallocene-based antimalarials
: An exploration into the influence of the
ferrocenyl moiety on in vitro antimalarial activity in chloroquine-sensitive and
chloroquine-resistant strains of Plasmodium falciparum, Bioorg. Med. Chem. 15
(2007) 6510-6516.
[
[
41] C. Biot, D. Taramelli, I. Forfar-Bares, L.A. Maciejewski, M. Boyce, G. Nowogrocki,
J.S. Brocard, N. Basilico, P. Olliaro, T.J. Egan, Insights into the mechanism of action
of ferroquine. Relationship between physicochemical properties and antiplasmodial
activity, Mol. Pharm. 2 (2001) 185-193.
42] F. Dubar, T.J. Egan, B. Pradines, D. Kuter, K.K. Ncozaki, D. Forge, C. Pierrot, H.
Kalamou, J. Khalife, E. Buisine, C. Rogier, I. Forfar, C. Slomianny, O.X. Trivelli, S.
Kapishnikov, L. Leiserowitz, D. Dive, C. Biot, The antimalarial ferroquine: role of
the metal and intramolecular hydrogen bond in activity and resistance, ACS Chem.
Biol. 6 (2011) 275-287.
[
43] J. Guillon, S. Moreau, E. Mouray, V. Sinou, I. Forfar, S. Belisle, V. Desplat, P.
Millet, D. Parzy, C. Jarry, P. Grellier, New ferrocenic pyrrolo [1,2-a] quinoxaline
derivatives
: Synthesis, and in vitro antimalarial activity, Bioorg. Med. Chem. 16
(2008) 9133-9144.
[
[
[
44] C. Biot, W. Daher, N. Chavain, T. Fandeur, J. Khalife, D. Dive, E. Clercq, Design
and synthesis of hydroxyferroquine derivatives with antimalarial and antiviral
activities, J. Med. Chem. 49 (2006) 2845-2849.
45] S.B. Colbran, S.T. Lee, D.G. Lonnon, F.J.D Maharai, A.M. Mcdonagh, K.A.
Walker, R.D. Young, Covalently linked ferrocenyl quinones
: proton-dependent
redox behavior and charge redistribution, Organometallics 25 (2006) 2216-2224.
46] K. Okamoto, H. Imahori, S. Fukuzumi, Metal ion-promoted intramolecular electron
transfer in a ferrocene-naphthoquinone linked dyad. Continuous change in driving
force and reorganization energy with metal ion concentration, J. Am. Chem. Soc. 4
(2003) 7014-7021.
[
47] L. Vendier, J. Brocard, B. Meunier, A. Robert, Trioxaferroquines as new hybrid
antimalarial drugs, J. Med. Chem. 53 (2010) 4103-4109.
2
8

ACCEPTED MANUSCRIPT
[
[
48] N. Srouji, T. L. Newirth, Acetylation of ferrocene, J. Chem. Educ. 72 (1995) 454-
4
56.
49] R.M. Janet, N. Kora, S. Allen, L. Cannon, J. Coughlan, L. Cusumano, B. Nolan,
Friedel-Crafts acylation and alkylation with acid chlorides, J. Chem. Educ. 66 (1989)
1
056-1057.
[
[
[
50] P.H. Groggins, S.B. Detwiler, Friedel-Crafts reactions, Ind. Eng. Chem. 43 (1951)
970-1974.
1
51] P.G. Cozzi, L. Zoli, A rational approach towards the nucleophilic substitutions of
alcohols “on water”, Angew. Chem. Int. Ed. 47 (2008) 4162 –4166.
52] G.W. Gokel, D. Marquarding, I.K. Ugi, Stereoselective synthesis. VIII. Retentive
nucleophilic displacements of alpha-substituted alkylferrrocenes, J. Org. Chem. 37
(1972) 3052-3057.
[
[
53] H. Mayr, B. Kempf, A.R. Ofial, π-Nucleophilicity in carbon-carbon bond-forming
reactions, Acc. Chem. Res. 36 (2003) 66-77.
54] C. Schindele, K.N. Houk, H. Mayr, Relationships between carbocation stabilities and
electrophilic reactivity parameters, E: quantum mechanical studies of benzhydryl
cation structures and stabilities, J. Am. Chem. Soc. 124 (2002) 11208-11214.
[
[
[
55] G.K. Prakash, Electrophilic intermediates and their reactions in superacids, J. Org.
Chem. 71 (2006) 3661-3676.
56] H. Tse-Lok, The hard soft acid bases (HSAB) principle in organic chemistry,
Chemical Reviews 75 (1975) 1-20.
57] S. Narayan, J. Muldoon, M.G. Finn, V.V. Fokin, H.C. Kolb, K.B. Sharpless, “On
water”: unique reactivity of organic compounds in aqueous suspension, Angew.
Chem. Int. Ed. 44 (2005) 3275-3279.
[
[
[
[
58] Y. Jung, R.A. Marcus, On the theory of organic catalysis “on water”, J. Am. Chem.
Soc. 129 (2007) 5492-5502.
59] T. P. Silverstein, The real reason why oil and water don’t mix, J. Chem. Educ. 75
(1998) 116-118.
60] R. Breslow, Hydrophobic effects on simple organic reactions in water, Acc. Chem.
Res. 24 (1991) 159-164.
61] M. Rottmann, C. McNamara, B.K. Yeung, M.C. Lee, B. Zou, B. Russell, P. Seitz,
D.M. Plouffe, N.V. Dharia, J. Tan, S.B. Cohen, K.R. Spencer, G.E. González-Páez,
S.B. Lakshminarayana, A. Goh, R. Suwanarusk, T. Jegla, E.K. Schmitt, H.P. Beck,
2
9