# Metallocene Antimalarials: The Continuing Quest

Authors: Margaret A. L. Blackie; Kelly Chibale
Journal: Metal-Based Drugs
 (2008)

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## Abstract

Over the last decade, a significant body of research has been developed around the inclusion of a metallocene moiety into known antimalarial compounds. Ferroquine is the most successful of these compounds. Herein, we describe our contribution to metallocene antimalarials. Our approach has sought to introduce diversity sites in the side chain of ferroquine in order to develop a series of ferroquine derivatives. The replacement of the ferrocenyl moiety with ruthenocene has given rise to ruthenoquine and a modest series of analogues. The reaction of ferroquine and selected analogues with Au(PPh3)NO3, Au(C6F5)(tht), and [Rh(COD)Cl2] has resulted in a series of heterobimetallic derivatives. In all cases, compounds have been evaluated for in vitro antiplasmodial activity in both chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum. Preliminary structure-activity relationships have been delineated.


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## Body

## 1. Introduction

The ongoing battle against malaria is far from over. In the 1950s and 1960s, there was a massive drive to try and eradicate malaria worldwide following the successful eradication of the disease in the United States. The population at risk from malaria was reduced to 10%. However, the banning of dicophane (DDT) and the concurrent emergence of chloroquine resistance led to the collapse of this campaign. A quarter of a century
later, over 300 million clinical cases of malaria occur annually and over 40% of the world’s population is at risk of contracting the disease. Of these cases, over one million will prove fatal [1]. The greatest tragedy of malaria
is that 90% of fatalities occur in sub-Saharan Africa,
and the overwhelming majority of those fatalities are children under the age of 5.
Malaria is still essentially a tropical disease, but continues to claim the
title of one of the leading killers among infectious diseases. The importance
of developing new antiplasmodial drugs cannot be overemphasised. The Roll Back
Malaria campaign which began in 1998 has yet to show a decrease in malaria
mortality rates [2].Over the last decade, there has been an increasing interest in
metal-containing antiplasmodials. This trend was in part initiated by the
successes of metal-containing antitumour drugs such as cisplatin. In
the 1980's and 1990’s, interest in coordination complexes of known
chemotherapeutic agents began to emerge [3, 4]. It was not long before coordination
complexes of chloroquine were synthesised and evaluated for efficacy against
both chloroquine-sensitive and chloroquine-resistant strains of P. falciparum [5–7]. It is not surprising that antiplasmodial efficacy
proved to be somewhat dependent on both metal and ligand.The incorporation of a metallocene into compounds with medicinal
application was rare prior to the 1980's. The low cytotoxicity of ferrocene,
coupled with its lipophilicity (logPoctanol/water = 3.28) and its
electrochemical behaviour (redox potential of the ferrocene/ferrocenium couple,
E0 = +0.400 V versus SCE (Saturated Calomel Electrode)), suggested that
this compound could yield interesting results if incorporated into a known drug
[8]. Indeed there are several reported successes of increased efficacy of
ferrocenyl analogues of known drugs. Ferrocifen, the ferrocenyl analogue of
tamoxifen,  has shown antiproliferation effects in both hormone-dependent and
hormone-independent tumours. Tamoxifen and other established chemotherapeutic
agents fail to exhibit such antiproliferative effects in both hormone-dependent
and hormone-independent cell lines [9].Ferroquine and other ferrocenyl chloroquine
analogues have been shown to be efficacious in vitro in both
chloroquine-sensitive and chloroquine-resistantP. falciparum across a variety of strains [10–12].The purpose of this paper is to draw together some of the research
carried out within our laboratories in the field of metal-containing
chloroquine derivatives. This research,
together with work published recently
by other researchers, will hopefully shed some light on the role of ferrocene in
ferroquine and related compounds.


## 2. Ferroquine Analogues and Derivatives

Brocard and colleagues have developed a number of ferrocene-containing
chloroquine analogues [10]. Ferrocene was
incorporated as an integral part of the side chain; as a terminal
component of the side chain; and bonded through the quinoline nitrogen [11–13].
They have shown that incorporation of a ferrocenyl moiety as an integral part
of the side chain of chloroquine between the two nitrogens had superior
efficacy to other analogues in which the moiety was terminal on the side chain
or bonded to the quinoline nitrogen. Some analogues of the compound were
produced bearing different alkyl groups on the terminal tertiary nitrogen. They
established that the dimethylamino terminal group was superior in efficacy
[10].Building on this foundation, we decided to explore the possibility of
developing other analogues of ferroquine in which a reactive secondary amine
group between the quinoline and ferrocenyl moieties would serve as a site for
introducing further chemical diversity. We reasoned that this would facilitate
the exploration of structure-activity relationships. The role of the length of
the methylene spacer between the two nitrogens in chloroquine analogues has
been shown to have an influence on efficacy in chloroquine-resistant strains ofP. falciparum [14]. Krogstad
and coworkers have shown that aminoquinolines with short (2-3 carbons) and long
(10–12 carbons) methylene side chains are equipotent against chloroquine-sensitive,
chloroquine-resistant, and multidrug-resistant strains of P. falciparum. Whilst aminoquinolines with side chains of
intermediate length (4–8 carbons) showed efficacy against chloroquine-sensitive
strains of P. falciparum, they showed
a significant decrease in efficacy against chloroquine-resistant strains of P. falciparum  [15]. For this reason, we decided to explore the influence of chain
length on ferroquine analogues. The parallel series of urea derivatives was
also synthesised and evaluated at the same time. The introduction of structural
diversity via the urea functionality is a well-established strategy in the
development of new biologically active compounds [16].Synthesis of the ferroquine-type compounds was achieved via the
synthetic strategy shown in Schemes1–3. The reaction of 5 with n-butyllithium
gave the desired product in a reasonable yield (70%) [10]. However, the use of tert-butyllithium had the effect of
reducing the reaction time and improving the yield [17]. The presence of the
dimethylaminomethyl substituent on the ferrocene results in a high regioselectivity
for the 1,2-disubstituted product 6.Scheme 1
Synthesis of [(N,N-dimethylamino)methyl] ferrocenecarboxaldehyde
(6),  and 2-[(N,N-dimethylamino)methyl] ferrocenemethylamine (8).Structure of ferroquine analogues.
(a)(b)(c)(d)Scheme 2
Synthesis of
ferroquine (2).Ferroquine (2) was synthesised from 2-[(N,N-dimethylamino)methyl] ferrocenemethylamine (8) and 4,7-di- chloroquinoline. The
published procedure uses dichloromethane and brine for the workup, in order to
extract the product from 1-methylpyrrolidinone [10]. We favoured the use of
ethyl acetate rather than dichloromethane as it was more time efficient. This
had little impact on the moderate yield of the reaction (53%).Synthesis of
compound10 was achieved from
4,7-dichloroquinoline and the appropriate 1,n-alkyldiamine. The two starting
materials were simply reacted together in the melt to form the product [18].
Compound 10 was then reacted with
the ferrocenecarboxaldehyde (6) in
methanol, to form the imine (Schiff base) which was then reduced in situ with sodium borohydride to
deliver compound 3 which was in turn
reacted with phenyl isocyanate in dichloromethane at room temperature (see Scheme 3).Consistent with previous observations [15, 16], the length of the
methylene spacer was found to have an influence on antiplasmodial activity. In
the chloroquine-sensitive D10 strain, the longer the methylene spacer, the
lower the efficacy. A distinguishable pattern was not quite so clear in the
chloroquine-resistant K1 strain, but the methylene spacer length appeared to
have an impact on efficacy with compound 3b (3-carbon spacer) showing the greatest efficacy [19]. For the urea derivatives,
the chain length made no significant difference to efficacy in D10, but a
decrease in efficacy with an increase in the length of the methylene spacer was
observed in the K1 strain [19].Given the distinctive redox chemistry of ferrocene, we decided to
establish the electrochemical behaviour of the analogues we had synthesised (see Table2). It was postulated that the half-wave potential E1/2 or ΔE value could provide a cheap and quick method of screening for potential
biological activity if this could be correlated to the in vitro antiplasmodial
activity. Unfortunately, no discernable trend was observed relating either of
these values with in vitro
efficacy in either strain of P.
falciparum. However, it was noted that the presence of the reactive
secondary amine centre had a marked effect on the redox chemistry of the
ferrocenyl moiety. Ferroquine exhibits a fully reversible one-electron
oxidation. The curve is similar to that of ferrocene (see Figure 2) although
the ferrocenyl moiety in ferroquine is significantly more difficult to oxidise (E1/2 for FQ = 147;
E1/2 for Fc = 79).Table 1
In vitro antiplasmodial activities of
ferroquine analogues.Table 2
Cyclic voltametry measurements on antimalarial ferroquine analogues.Table 3
Results of in vitro antimalarial tests conducted on the chloroquine-sensitive (D10) and
chloroquine-resistant (K1) strains ofP. falciparum.
CompoundD10K1IC50/nMIC50/nMCQ·2H3PO4233522Fe18142Ru19132′Fe19492′Ru25293aFe33373aRu20203a′Fe16653a′Ru34127Cyclic voltammograms of selected
ferrocene, ferroquine,3a, and 4a.
(a)(b)(c)(d)Compounds3a–d showed, at
best, a quasireversible oxidation. The cathodic peak current was significantly
smaller than the anodic peak current (see Figure 2). The ease of reversibility
of oxidation increases with an increase in carbon chain length, as demonstrated
by the decrease in ΔEp values (see Table 2). A fully reversible one-electron
oxidation requires a peak separation, ΔEp, of 70–90 mV. In compounds 4a–d, the reversibility of the one-electron
oxidation event in the ferrocenyl moiety was restored. As with ferroquine,
these compounds are significantly more difficult to
oxidise than ferrocene, as demonstrated
by the increase in half-wave potential E1/2 values (see  Table 2).


## 3. Ruthenoquine Analogues

The chemistry of ferrocene is similar to the chemistry of ruthenocene.
In order to begin to probe the role of ferrocene in ferroquine derivatives, we reasoned
that it would be of interest to synthesise the ruthenocene analogues of
selected derivatives (see Figure3). The antiproliferative properties of ferrocifen and its ruthenocene analogue have
proven quite different.
This difference in efficacy has been attributed to the
stability of the ferrocenium ion relative to the ruthenocenium ion. The
presence of the ferrocenium ion allows for a change in chemical reactivity at
another point in the molecule as a result of the highly conjugated system
present in the ferrocifen
molecule. The instability of the ruthenocenium ion does not allow for this
change in chemical reactivity [9]. It is clear that a similar
through-bond effect is not available in ferroquine or ruthenoquine as there is
no conjugation accessible to the metallocene moiety in these compounds.
However, it was thought that some insight into the role of ferrocene could be
gained by examining the biological activity of the ruthenocene analogues.Ferrocene and
ruthenocene analogues of ferroquine.
(a)(b)(c)(d)Scheme 3
Synthesis of
ferroquine analogues(3a–d) and urea
derivatives (4a–d).It was envisioned
that a similar synthetic strategy could be employed to synthesise the compounds2Ru and 3aRu, as had been used
for the ferrocene analogues and
described in Schemes 1–3. However, the ruthenocene system proved more
complicated [20]. Firstly, (N,N-dimethylaminomethyl) ruthenocene is not
commercially available and had to be synthesised using Eschenmoser's salt ([CH2=NMe2]I)
[21]. Secondly, in the ferrocenyl system, high regioselectivity for the 1,2-disubstituted
product 6 is observed. In the
ruthenocenyl system, when using n-butyllithium to
achieve deprotonation, significant quantities of the 1,1′-monoaldehyde 11, and the dialdehyde 12 were formed
(see Scheme 4).
Isolation of 11 led to the synthesis
of isoruthenoquine, 2′Ru, and 3a′Ru. It was later discovered that the
use of tert-butyllithium affords 6-Ru exclusively. However, by this
stage, 2′Ru and 3a′Ru had already been synthesised and found to show antiplasmodial
activity [21]. In order to create a better comparison of the efficacies of the
ferrocene and ruthenocene compounds, the 1,1′-ferrocenyl derivatives were also
synthesised. The synthesis of the 1,1′-ferrocene compounds required a fairly
lengthy synthetic procedure which is discussed in full elsewhere [22].All compounds tested exhibited a good efficacy in both chloroquine-sensitive
and chloroquine-resistant strains. Consistent with previous findings, the
addition of the metallocene was advantageous in overcoming chloroquine
resistance. These results indicate that the 1,2-disubstituted compounds (2Fe, 2Ru, 3aFe, and 3aRu) are more efficacious than the 1,1′-disubstituted analogues (2′Fe, 2′Ru, 3a′Fe, and 3a′Ru) in the chloroquine-resistant K1
strain. It has not been established why this trend should be observed, but the
crystal structures of these compounds clearly show that there is a significant
increase in distance between the 4-aminoquinoline nitrogen and the terminal
nitrogen when moving from the 1,2- to 1,1′-disubstituted product [21, 23]. It
has been previously established that this distance is significant in the
observed efficacy of 4-aminoquinolines [15, 16]. It is noteworthy that barring
compounds 3a′Fe and 3a′Ru, there is no significant
difference in efficacy between ferrocenyl and ruthenocenyl analogues. This may
suggest that the difference in redox chemistry and chemical
reactivity of these moieties is not a factor in the efficacy of these
compounds.
The observed efficacy may be associated with the lipophilicity or
physical bulk of the metallocene group.Comparative studies on the behaviour of chloroquine and ferroquine have
led to the conclusion that the effect of shape, volume, lipophilicity, basicity,
and electronic profiles
of the ferrocenyl moiety leads to a modification of the pharmacological
behaviour of the analogue [23]. It has yet to be established which of these
factors is most significant. In chloroquine-resistant strains of P. falciparum, a transporter
protein, P. falciparum chloroquine
resistance transporter (PfCRT), has
been identified which allows the efflux of chloroquine from the food vacuole. It has been noted that ferroquine resistance cannot be induced under drug
pressure in the W2 strain of P.
falciparum. This has led to the conclusion that the bulky lipophilic
ferrocene moiety overcomes PfRCT resistance, thereby maintaining ferroquine in
the food vacuole. In addition, ferroquine retains all the features that have
been identified as necessary in the structure of chloroquine (see Figure 4) [23].
The similarity of behaviour of the ruthenoquine and ferroquine molecules opens
an interesting avenue of research. Ruthenium is known to be a good contrasting
agent in electron microscopy [24]. It is reasonable, in the absence of data to
the contrary, to postulate that the mechanism of action of ruthenoquine and
ferroquine is similar. The sites of accumulation of ruthenoquine can be
established easily using scanning electron microscopy. In mice infected with Plasmodium berghei N and treated with
ruthenoquine, the drug has been found to accumulate close to the malaria pigment
and in the parasitic
membrane. Chloroquine exhibits no such accumulation in the parasitic membrane
[25]. It is possible that ferroquine exhibits a similar pattern of accumulation
to ruthenoquine, although this has yet to be established. The reason for this
accumulation of ruthenoquine in the membrane has not been ascertained, but it
may well be associated with the increase in lipophilicity afforded by the
metallocene moiety.Figure 4
Proposed structure-activity
relationships for ferroquine [23].Scheme 4


## 4. Coordination Complexes

A number of transition metals have been used to form coordination
complexes of chloroquine. These coordination complexes have been shown to
exhibit improved efficacy in both chloroquine-sensitive and chloroquine-resistant
strains ofP. falciparum compared to
chloroquine [6, 7, 26]. The [Ru(Cl)2(CQ)]2 dimeric complex showed considerably better in vivo efficacy than was expected on the basis of the in vitro
results [6]. The same study reports that both [Rh(COD)Cl]2 and RuCl2(DMSO)4 failed to exhibit any antiplasmodial activity. As the metal complexes of
chloroquine showed improved efficacy against both chloroquine-sensitive and
chloroquine-resistant strains of the parasite, it was concluded that the
coordination of a metal to the quinoline nitrogen enhanced the efficacy of
chloroquine in vitro. However,
a decade after the advent of these and other chloroquine-metal coordination
complexes, the mode of action of these complexes is still poorly understood. It
has not been established whether these complexes reach the site of action   intact or not.
Furthermore, it has not been established whether the inhibition of β-haematin
formation is possible with the presence of a coordinated metal. This question
is relevant as it has been established that the 7-chloro-4-aminoquinoline
structure is crucial for this mode of action in chloroquine [27].Given that the ferroquine derivatives could give rise to an analogous
series of ferroquine coordination complexes, we thought it would be interesting
to examine the effect of coordination of a second metal. To our knowledge, no
such heterobimetallic coordination complexes of chloroquine had been
synthesised and tested for antiplasmodial efficacy [28]. Four ligands
(1–4), in Figure 5, were selected for preliminary
comparative studies.Ligands for
coordination.
(a)(b)(c)(d)The results shown in Table4 indicate that the chloroquine complexes [Au(CQ)(PPh3)]NO3,
[Au(C6F5)(CQ)], and [Rh(Cl)(COD)(CQ)] all show comparable
efficacy to chloroquine in the chloroquine-sensitive strain. In the chloroquine-resistant
strain, these complexes exhibit an efficacy 7 to 9 times better than
chloroquine. However, it is noteworthy that efficacy of these compounds dropped up to three times
in moving from sensitive to resistant strain suggesting that some
cross-resistance may occur. Unfortunately, the metal complexes of the
ferrocenyl-4-aminoquinolines did not perform so well. At best, addition of the
second metal had little effect on the efficacy of the parent ligand (as in
pentafluorophenyl gold complexes of L2, L3, and L4), whilst at worst, there appeared to be a significant
antagonistic effect (as in the cyclooctadiene rhodium complexes of L3 and L4). Whilst the reason for this antagonistic effect was not
explored, it was noted that the presence of the second metal made the
ferrocenyl moiety far more difficult to oxidise. This was evidenced by the
increase in half-wave potential, E1/2, in the complexes of L2 and L4 when compared with the free ligand. In the case of L3, where no fully reversible
one-electron oxidation was observed, the significant increase in cathodic peak
potential suggests that the ferrocenyl moiety is more difficult to oxidise.
Once again we found no discernible correlation between redox activity and
antiplasmodial efficacy.Table 4
In vitro activity of coordination complexes and the free 4-aminoquinoline ligands in chloroquine-sensitive (D10) and chloroquine-resistant (K1) strains ofP. falciparum.
CompoundD10 (CQ sensitive)K1 (CQ resistant)IC50(nM)IC50 (nM)L137.10570.0[Au(L1)(PPh3)]NO321.0961.53[Au(C6F5)(L1)]18.0761.09[Rh(Cl)(COD)(L1)]21.5081.31L216.304.96[Au(L2)(PPh3)]NO310.505.68[Au(C6F5)(L2)]10.083.96[Rh(Cl)(COD)(L2)]15.8010.55L311.3012.56[Au(L3)(PPh3)]NO333.2733.89[Au(C6F5)(L3)]13.7014.65[Rh(Cl)(COD)(L3)]202.00597.59L416.5415.54[Au(L4)(PPh3)]NO330.3231.09[Au(C6F5)(L4)]23.3612.92[Rh(Cl)(COD)(L4)]56.1047.70Table 5
Cyclic voltametry of gold triphenylphosphine complexes and the parent ligands.
Compound(a)Epa(mV)(b)Epa(mV)(c)E1/2(mV)Epa-Epc(mV)L218111314770[Au(L2)(PPh3)]NO325216220790L315030—120[Au(L3)(PPh3)]NO3294186—108L419811515883[Au(L4)(PPh3)]NO321914118078(a)anodic potential;
(b)cathodic potential; (c)half wave potential
(Epa + Epc)/2.


## 5. Discussion

The ferrocenyl moiety has several characteristics which make it a good
addition to known drug molecules. Its lipophilicity, electron density, relative
thermal and chemical stability, and interesting redox behaviour are all
favourable in this respect. We have shown that altering the structure of the
ferroquine analogue can have a significant effect on the redox behaviour of the
ferrocenyl moiety. However, there is no discernible correlation between the
differences in redox behaviour, whether ease of oxidation or reversibility of
oxidation, and the efficacy of a compound against either chloroquine-sensitive
or chloroquine-resistant strain ofP.
falciparum. It is worth noting that ferrocene and ruthenocene have quite
different redox chemistry. The ruthenocenyl analogue of the antitumour agent ferrocifen has been shown
to have a significantly different range of efficacy which is attributed
principally to this difference in redox chemistry and the effect on the highly
conjugated molecule to which the metallocene is bonded. No such difference in
antiplasmodial efficacy is observed here between ferrocenyl and ruthenocenyl
analogues of ferroquine. This, together with our cyclic voltammetric studies
and the lack of correlation with antiplasmodial efficacy, suggests that the
redox behaviour of the metallocene is not a significant factor in the efficacy
of these compounds. Some study of the redox chemistry of the ruthenoquine
analogues would be useful to determine the accuracy of this supposition.The incorporation of a ferrocenyl moiety into chloroquine has yielded
fruitful results in terms of overcoming chloroquine resistance. Ferroquine
continues to undergo testing to ascertain its suitability as a candidate for
full-scale human clinical trials [8]. However, the results of incorporation of
a ferrocenyl moiety into other known antimalarial drugs such as mefloquine,
quinine [29], or artemisinin [30] have proved far less rewarding. This raises a
question as to whether there is any inherent antiplasmodial toxicity associated
with the ferrocenyl moiety. It may be that the changes in lipophilicity and pKa values and other
physicochemical effects of the incorporation of the ferrocenyl moiety into
chloroquine are the primary factor in the enhanced efficacy of ferroquine. This
may also explain the antagonistic effects exhibited by the heterobimetallic
complexes of ferroquine and its analogues. The presence of the complexed metal and
associated ligands may have adverse effects on the influence of the ferrocenyl
moiety.The role of the ferrocenyl moiety in the ability of ferroquine to
overcome chloroquine resistance has still not been fully determined, although
its role in overcoming PfCRT resistance seems supported by experimental
evidence [24]. The continuing biological success of ferroquine means that this
avenue of research must remain open and active. If we are to overcome the
problem of malaria in sub-Saharan Africa, a
potent, cheap alternative to chloroquine must be found.



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