# The antimalarial ferroquine: from bench to clinic.

Christophe Biot, F. Nosten, L. Fraisse, D. Ter-Minassian, Jamal Khalife, Daniel Dive

► To cite this version:

Christophe Biot, F. Nosten, L. Fraisse, D. Ter-Minassian, Jamal Khalife, et al.. The antimalarial ferroquine: from bench to clinic.. Parasite, 2011, 18 (3), pp.207-214. 10.1051/parasite/2011183207. hal-00642112

HAL Id: hal-00642112

https://hal.science/hal-00642112v1

Submitted on 16 Sep 2019

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons CC BY 4.0 - Attribution - International License
# THE ANTIMALARIAL FERROQUINE: FROM BENCH TO CLINIC

BIOT C.*, NOSTEN F.**, FRAISSE L.**, TER-MINASSIAN D.****, KHALIFE J.***** & DIVE D.*****

**Summary:**

Ferroquine (FQ, SSR97193) is currently the most advanced organo-metallic drug candidate and about to complete phase II clinical trials as a treatment for uncomplicated malaria. This ferrocene-containing compound is active against both chloroquine-susceptible and chloroquine-resistant *Plasmodium falciparum* and *P. vivax* strains and/or isolates. This article focuses on the discovery of FQ, its antimalarial activity, the hypothesis of its mode of action, the current absence of resistance in vitro and recent clinical trials.

**KEY WORDS:** malaria, bioorganometallics, drug candidate, ferroquine, mechanism of action, resistance.

**Résumé :** L'ACTIVITÉ ANTIPALUDIQUE DE LA FERROQUINE : DE LA RECHERCHE À LA CLINIQUE

La ferroquine (FQ, SSR97193) est actuellement le candidat médicament organométallique le plus avancé dans son développement industriel et, en tant que traitement du paludisme non compliqué, il devrait bientôt voir s'achever des études cliniques de phase II. Ce composé à base de ferrocène est extrêmement actif contre des isolats et/ou des clones de Plasmodium falciparum et de *P. vivax* sensibles ou résistants à la chloroquine. Cet article présente la découverte de la FQ, son activité antipaludique, les hypothèses liées à son mode d'action, l'absence actuelle de résistance in vitro et, enfin, les récents essais cliniques.

**MOTS-CLÉS :** paludisme, bio-organométallique, candidat médicament, ferroquine, mécanisme d'action, résistance.

## THE MALARIA PROBLEM

With approximately 243 million cases and more than 800,000 deaths reported in 2009, malaria remains the most important human parasitic disease. Among the five *Plasmodium* species able to infect human, *P. falciparum* is responsible for most cases of severe disease and death, mainly in African children below the age of five. The morbidity caused by *P. vivax* in tropical countries outside of Africa has long been underestimated (Anstey *et al*., 2009, Baird, 2009). Malaria is a factor of poverty

in endemic countries (Stratton *et al*., 2008). In the absence of an effective vaccine and reliable approaches for vector control, chemotherapy remains the corner stone of malaria control. Quinine has been the first widely used antimalarial drug. Synthetic derivatives of quinine were the 8-aminoquinoline primaquine and the 4-aminoquinoline chloroquine (CQ). When resistance to CQ emerged in the late 1950s, the strategy was to modify the chemical structure of the existing compounds. The synthesis of CQ-like drugs led to the discovery of amodiaquine (AQ) and later mefloquine (MQ), halofantrine in the United States and lumefantrine in China (Baird, 2005). But the pace of new drug development has been slow and no new antimalarial drugs have been introduced into clinical practice since artemether-lumefantrine registered in 1998 (Olliaro & Wells, 2009). For all new antimalarial drugs introduced the risk of resistance can be reduced by combination therapy (White, 1999; Nosten & White, 2007). In 2006, the WHO guidelines recommended new treatments combining two drugs with different mechanisms of action. Treatments containing an artemisinin derivative (artemisinin-combination therapies, ACTs) are now standard treatment for *falciparum* malaria. However, a decline of susceptibility to artesunate has been recently reported in the Thai-Cambodian border region (Dondorp *et al*., 2010). So the search for new molecules with antimalarial activity is more important than ever.

<footnote>* Unité de Catalyse et Chimie du Solide, CNRS UMR 8181, Université Lille Nord de France, Université Lille 1, BP 90108, 59652 Villeneuve d'Ascq Cedex, France. Present address: Unité de Glycobiologie Structurale et Fonctionnelle, CNRS UMR 8576, IFR 147, Université Lille Nord de France, Université de Lille 1, 59650 Villeneuve d'Ascq Cedex, France.</footnote>

<footnote>** Shoklo Malaria Research Unit, PO Box 46 Mae Sot Tak 63110, Thailand. Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand. Centre for Clinical Vaccinology and Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, United Kingdom.</footnote>

<footnote>*** Sanofi-Aventis, Centre de Toulouse, 195, route d'Espagne, BP 13669, 31036 Toulouse, France.</footnote>

<footnote>**** Sanofi-Aventis Research and Development, 1, avenue Pierre Brossolette, 91385 Chilly Mazarin Cedex, France.</footnote>

<footnote>***** CIIL, Inserm U1019, CNRS UMR 8024, Université Lille Nord de France, Institut Pasteur de Lille, 1, rue du Pr Calmette, 59019 Lille Cedex, France.
Correspondence: Christophe Biot.</footnote>

<footnote>Tel.: 33 (0)3 20 43 69 41.</footnote>

<footnote>E-mail: christophe.biot@univ-lille1.fr</footnote>
Many strategies can be used for the search of affor-
dable and efficient antimalarial drugs. These strategies
include ethnopharmacology (i.e. bio-evaluation of the
efficiency of traditional medicines), medicinal che-
mistry, combinatorial chemistry and chemical libraries
screening by high throughput screening, and drug
design. These strategies have led to the discovery of
potential antimalarials such as the synthetic endope-
roxides and others (Dhanawat *et al.*, 2009). But the
clinical development of new compounds is often
been stopped for various reasons: toxicity, chemistry,
pharmacology, or economics, and less than one in
ten promising molecules that have entered the pipe-
line reaches the stage of clinical studies. In the mid-
90s, we extended the strategy developed by Gérard
Jaouen (Vessieres *et al.*, 1988) in anticancer therapy
to antimalarial therapy (see Chavain & Biot, 2010 for
review). The main antimalarials in current use (CQ,
quinine, mefloquine, artemisinin, atovaquone) were
modified by introduction of a ferrocenyl moiety in
their chemical structure. More than 150 ferrocenic
analogues have been synthesized, by us and others
(Biot & Dive, 2010). The ferrocenic analogues were
systematically tested against *in vitro* cultures of *P. falciparum* with CQ-susceptible and CQ-resistant strains.
Ferroquine (FQ, SSR97193) was rapidly identified as a
lead compound to meet candidate nomination require-
ments (Biot *et al.*, 1997). The clinical phase IIb study
(efficacy/safety in adults, adolescents and children)
began in 2009 in Africa.

This mini-review will focus on the discovery of FQ,
its antimalarial activity, the hypothesis of its modes
of action and recent clinical trials.

THE ORGANOMETALLIC ANTIMALARIAL
COMPOUND SET

Since 1993, we and others have systematically
prepared organometallic versions of the anti-
malarials in current use such as CQ, primaquine,
mepacrine, mefloquine, quinine, artemisinin, and
atovaquone (see Dive & Biot, 2008 for review). New
sandwiches and half-sandwiches metal complexes
(Dunitz et al., 1956) have been synthesized and cha-
racterized. In vitro tests of their antimalarial activity
were performed. Other organometallic compounds
with a priori unknown antimalarial activity were still
screened. A collection of almost 150 compounds
was made available. Among the organometallic-
drug hybrids, the most interesting compounds were
the ferrocene-drug hybrids and among those the
ferrocene-chloroquine hybrids were the most promis-
sing (Fig. 1).

Note here that the ferrocene-artemisitene hybrids
showed also interesting properties with activities
equal to artemisinin (Delhaes et al., 2000, Dive & Biot,
2008). In the ferrocene-CQ hybrids series, we have
shown that the ferrocene moiety has to be covalently
flanked by a 4-aminoquinoline and an alkylamine
(Biot et al., 2006). Ferroquine (FQ, SSR97193) was
the first compound synthesized by us (Biot et al.,
1997). Later, a second generation of analogues of
FQ was designed and investigated. For example, we
synthesized dual molecules including a FQ analogue
conjugated with a glutathione reductase inhibitor
or a glutathione depletory (Chavain et al., 2009)
Nevertheless, this strategy failed to identify a "new"
lead for a further development. More interestingly,
amino-alcohols based on the FQ structure are active
against CQ-susceptible (CQ<sup>S</sup>) and CQ-resistant (CQ<sup>R</sup>)
clones of P. falciparum. In addition, in this second
generation of analogues the ferrocenic amino-alcohols
exert antiviral effects with some selectivity toward
SARS-CoV infection (Biot et al., 2006b).

ANTIMALARIAL ACTIVITY OF FERROQUINE

ANTIMALARIAL ACTIVITY ON LABORATORY CLONES

F
Q antimalarial activity was compared to that
of CQ with standard *in vitro* parasite growth
inhibition method, based on tritiated hypoxan-
thin incorporation in erythrocytes infected with *P.
falciparum*, incubated 48 hours (Desjardins *et al.*,
1978). Preliminary studies have shown that FQ was
equally active as a base, ditartrate or dichlorhydrate
salts (unpublished results).

Tests results available from 11 studies performed in different laboratories and using 19 CQ<sup>S</sup> and CQ<sup>R</sup> *P. falciparum* laboratory adapted clones are represented in Fig. 2. The results show that the response to CQ can be easily dissociated between susceptible and resistant clones, which are spread respectively on either sides of the 100 nM IC<sub>50</sub> for CQ. However, FQ is equally active on both types of clone and is at least equally active and often more active than CQ on CQ<sup>S</sup> parasites. No resistance to FQ occurred in CQ<sup>R</sup> clones and no correlation was found between susceptibility to FQ and polymorphism in transport proteins implicated in quinoline resistance (Henry *et al.*, 2008).

IN VIVO ANTIMALARIAL ACTIVITY IN RODENT MODELS

Antimalarial activity of FQ was tested on various
rodent malaria strains (P. berghei, P. yoelii, P. vinckei)
by the standard four day test of Peters (1987) adapted
<figure><img src="image_2.png" /><figcaption>Fig 1. – Scheme of different strategies adopted in synthesis of ferrocene-CQ hybrids.</figcaption></figure>

to determine the curative dose. On *P. berghei* N and *P. yoelii* NS strains, FQ and CQ had a close EC₅₀ (treatment with a decrease in parasitaemia of 50 % at the end of assay) and the simple four days test could not lead to conclude to a better efficacy of FQ *versus* CQ. But the curative tests are more significant and showed that *P. berghei* and *P. vinckei* infections were cured in presence of 8.3 mg/kg/d of FQ for four days when with CQ 30 to 55 mg/kg/d were necessary to cure

CQS strains and the drug was unable to cure resistant strains, even at a toxic dose (Biot *et al.*, 1997, Delhaes *et al.*, 2001, Dive & Biot, 2008, Biot & Dive, 2010). Moreover, it has been shown that FQ was active not only by subcutaneous administration, but also by oral route, which was an interesting indication concerning the bioavailability of the drug by digestive tract. This was further confirmed by additional pharmacokinetic studies (Biot & Dive, 2010).
<figure><img src="image_3.png" /><figcaption>Fig 2. – Susceptibility of 19 laboratory *P. falciparum* clones to CQ and FQ compiled from 11 different published studies.</figcaption></figure>

$IC_{50}$ for CQ for each clone tested (●). + $IC_{50}$ for FQ for each clone tested (+). The dotted line indicate the threshold of resistance to CQ (Le Bras & Ringwald, 1990).

References associated to each clone tested: 3D7 (1, 6, 8, 9, 10); HB3 (1, 7, 9, 10); D10 (2, 3, 4, 5); W2 (1, 6, 8, 9, 10); K1 (2, 3, 4, 5); FCR3 (1, 6, 11); Dd2 (7, 10, 11); D6, 106/1, IMT8425, IMT10336, FCM39, IMT Bres, IMT K14, IMT K2, IMT K4, IMT L1, IMT Vol, Bre1 (1).

References: 1: Henry *et al.*, 2008; 2: Beagley *et al.*, 2002; 3: Beagley *et al.*, 2003, 4: Blackie *et al.*, 2007; 5: Blackie & Chibale 2008; 6 Biot *et al.*, 2006b; 7: Biot *et al.*, 1999; 8: Biot *et al.*, 2006a; 9: Daher *et al.*, 2006a; 10: Daher *et al.*, 2006b; 11: Delhaes *et al.*, 2001.

## ENANTIOMERS

As FQ is a racemic compound. The two stereoisomers were synthetized and showed an antimalarial activity similar to that of the parent compound *in vitro* (Delhaes *et al.*, 2002).

## METABOLIZATION AND ACTIVITY OF METABOLITES

It was first postulated that the metabolism of FQ may share a common pathway with that of CQ and potential metabolites (*N*-monodemethyl-FQ and *N*-didemethyl-FQ) were synthesized and tested (Biot *et al.*, 1999). The metabolism of FQ was then studied in details *in vitro* and enabled to determine its degradation pathway (Daher *et al.*, 2006a). *In vitro* FQ is mainly metabolized to a major *N*-monodemethylated metabolite, SSR97213 (EVT0233) and to a further potential metabolite that is an *N*-didemethylated compound. Antimalarial activity of *N*-monodemethyl-FQ was found to be comparable to that of parent compounds on two $CQ^S$ clones and remained much more active than CQ on two $CQ^R$ clones. On the another hand, *N*-didemethyl-FQ had a decreased activity on $CQ^R$ clones, mainly if $IC_{90}$ of compounds is taken into account (Daher *et al.*, 2006a).

## EFFICACY ON CLINICAL ISOLATES

Compounds were evaluated with standard *in vitro* parasite growth inhibition methods, in erythrocytes infected with *P. falciparum*, incubated at least 24 hours with the drugs. The antimalarial activity of FQ (SSR97193) on blood clinical isolates ($CQ^S$, $CQ^R$, and

multi-drug resistant isolates) infected by *P. falciparum* was assessed in seven different studies of African patients (Senegal, Gabon) (Pradines *et al.*, 2001 & 2002; Atteke *et al.*, 2003; Kreidenweiss *et al.*, 2006), or southeast Asian patients (Chim *et al.*, 2004; Barends *et al.*, 2007) in comparison with existing antimalarial drugs. Data on FQ, CQ, and artesunate are reported in Table I.

Taking all these studies together, FQ was evaluated on 534 clinical isolates, 220 from Southeast Asia and 314 from Africa. In all these studies, FQ, like artesunate, displayed a very potent antimalarial activity against *P. falciparum* (range $IC_{50}$ below 30 nM [13 ng/mL] for FQ and below 4 nM [1.5 ng/mL] for artesunate) with equal efficacy upon $CQ^S$ and $CQ^R$ clinical isolates (resistant isolates, with $IC_{50}$ over 100 nM, represented from 32 % to 100 % of samples).

In addition, in the study from Thailand the main FQ *in vivo* metabolite (SSR97213) was investigated (Barends *et al.*, 2007). SSR97213 was shown to be highly potent against *P. falciparum* (N = 64, $IC_{50}$ = 37 nM with 95 % confidence intervals [CIs] = 34.3 to 39.9 nM, or $IC_{50}$ = 16.0 ng/mL with 95 % CIs = 14.9 to 17.3 ng/mL) on all the clinical isolates. To investigate whether *P. vivax* was sensitive to FQ a study was conducted in north-western Thailand on 63 isolates collected from October 2006 to April 2009 to examine the effects of FQ and its demethylated metabolite (SSR97213) on the ring stage and the schizont maturation by microscopy. All samples were collected from patients with acute *P. vivax* who had mono-species parasitaemia of > 100/500 white blood cells. FQ was found to have a potent *ex*
<table><thead><tr><th rowspan="2">Country</th><th colspan="3">Ferroquine</th><th colspan="3">Chloroquine</th><th colspan="3">Artesunate</th><th rowspan="2">Reference</th></tr><tr><th>n</th><th>IC<sub>50</sub> (nM)<br>(ng/mL)</th><th>95 % CI</th><th>n</th><th>IC<sub>50</sub> (nM)<br>(ng/mL)</th><th>95 % CI</th><th>%<br>resistance</th><th>n</th><th>IC<sub>50</sub> (nM)<br>(ng/mL)</th><th>95 % CI</th></tr></thead><tbody><tr><td>Gabon</td><td>103</td><td>10.8<br>4.7</td><td>8.6-13.5<br>3.8-5.9</td><td>102</td><td>370<br>118.4</td><td>319-429<br>102-137</td><td>95 (a)</td><td>65</td><td>2.9<br>1.1</td><td>2.3-3.7<br>0.9-1.4</td><td>Biot et al.,<br>1999</td></tr><tr><td>Senegal</td><td>55</td><td>7.9<br>3.4</td><td>6.5-9.7<br>2.8-4.2</td><td>53</td><td>102<br>32.6</td><td>74-140<br>23.7-44.8</td><td>55 (a)</td><td>51</td><td>1.9<br>0.7</td><td>1.5-2.3<br>0.6-0.9</td><td>Biot et al.,<br>2006a</td></tr><tr><td>Gabon</td><td>56</td><td>16<br>6.9</td><td>14.4-17.8<br>6.3-7.8</td><td>56</td><td>141<br>45.1</td><td>70-285<br>22.4-91.2</td><td>52 (a)</td><td>nt</td><td>nt</td><td>nt</td><td>Daher et al.,<br>2006a</td></tr><tr><td>Gabon</td><td>60</td><td>27.9<br>12.1</td><td>2.3-33.2<br>1.0-14.5</td><td>60</td><td>398<br>127.3</td><td>166-956<br>53.1-306</td><td>97 (a)</td><td>nt</td><td>nt</td><td>nt</td><td>Daher et al.,<br>2006a</td></tr><tr><td>Thailand</td><td>65</td><td>9.3<br>4.0</td><td>8.7-10.0<br>3.8-4.4</td><td>62</td><td>341<br>109.1</td><td>304-382<br>87.2-122</td><td>100 (a)</td><td>56</td><td>4.0<br>1.5</td><td>3.1-6.3<br>1.2-2.4</td><td>Daher et al.,<br>2006b</td></tr><tr><td>Gabon</td><td>40</td><td>1.9<br>0.8</td><td>0.6-6.7<br>0.3-3.0</td><td>43</td><td>113<br>36.1</td><td>12.4-332<br>4.0-106</td><td>100 (b)</td><td>43</td><td>1.0<br>0.4</td><td>0.2-6.0<br>0.1-2.3</td><td>Delhaes et al.,<br>2001</td></tr><tr><td>Cambodia</td><td>155</td><td>29<br>12.6</td><td>26.3-31.6<br>11.6-13.8</td><td>155</td><td>135<br>43.1</td><td>121-151<br>38.7-48.2</td><td>32 (a)</td><td>150</td><td>1.1<br>0.4</td><td>1.0-1.2<br>0.4-0.5</td><td>EVT0231</td></tr></tbody></table>

n = number of clinical sites; nt = not tested; CI = confidence interval; IC₅₀ = inhibitory concentration decreasing a response by 50%; (a) = % of resistance using the threshold level of resistance IC₅₀ > 100 nM; (b) = % of resistance using the threshold of IC₉₉ > 30 nM for the HRP2 detection assay. For values in *italics*, units = ng/mL, calculated for translation to a free-base or free-acid gravimetric concentration.

<caption>Table I. – Effect of FQ (SSR97193 – IC₅₀ and 95 % confidence intervals) on *P. falciparum* clinical isolates from different studies.</caption>

vivo effect on *P. vivax* schizont maturation (median IC₅₀ = 15 nM; 75 % CIs = 12 to 20 nM, n = 52) with SSR97213 being less active (IC₅₀ = 77 nM; 75 % CIs = 14 to 205 nM), and no significant cross-sensitivity between FQ and other antimalarials was detected; consequently FQ may be a suitable replacement for chloroquine in the treatment of drug-resistant *P. vivax* malaria (Leimanis *et al.*, 2010). In the Gabonese study (Kreidenweiss *et al.*, 2006), IC₉₉s were reported in comparison with IC₅₀s (Kreidenweiss *et al.*, 2006). For artesunate and FQ, the IC₉₉s were 5.76 nM (95 % CIs = 0.57 to 49.1 nM) or IC₅₀ = 2.21 ng/mL (95 % CIs = 0.22 to 18.9 ng/mL), and 5.75 nM (95 % CIs = 1.10 to 56.9 nM) or IC₅₀ = 2.50 ng/mL (95 % CIs = 0.48 to 24.8 ng/mL). These values are close to the reported IC₅₀s, indicating a strong potency and the ability to efficiently kill all parasites present in the field isolates. Finally, the susceptibility of *P. falciparum* isolates from Madagascar (n = 21), Guyana (n = 65) and Cambodia (n = 62) to FQ was measured at the local Pasteur Institutes using the [³H]-hypoxanthine incorporation method. The mean IC₅₀ (with minimum and maximum IC₅₀ values), were 5.96 nM (0.2-43.2), 8.68 nM (3.05-55.77) and 10.18 nM (2.53-43.43), respectively (Eric Legrand, personal communication).

In all studies, no cross-resistance was observed with CQ and other antimalarials, although weak occurrences could be attributed, in one study to fluctuations of initial inoculums used for test (Kreidenweiss *et al.*, 2006). This absence of cross-resistance is supported

by molecular studies, which showed that there was no association between polymorphims of resistance of *pfcrt* gene, the main molecular marker for CQ, and FQ susceptibility in field isolates (Daher *et al.*, 2006b). This last observation was then extended to other markers of quinoline resistance (Henry *et al.*, 2008) and to *pymdr* and *pycrt* genes of the rodent strain *P. yoelii* (Dive & Biot, 2008).

## RESISTANCE ACQUISITION UNDER FERROQUINE PRESSURE

An *in vitro* study on *P. falciparum* resistance acquisition under ferroquine pressure was performed on human red blood cells infected with the W2 clone. After two months of FQ pressure we were unable to obtain a viable resistant strain. During these experiments however, we observed very few parasites, which were unable to develop when transferred in drug-free medium (Daher *et al.*, 2006b).

An attempt to obtain a rodent FQ<sup>R</sup> strain starting from *P. yoelii* resulted in a phenotype that was not fixed genetically the resistance disappearing as soon as FQ pressure was removed. Moreover, the phenotype was emerging very slowly and was confined strictly to reticulocytes and easily cleared by the host (Dive & Biot, 2008).

These results clearly show that the fitness cost of FQ resistance is very high for the parasite and that it would be detrimental for them in competition with non-resistant clones.
<table><thead><tr><th>Properties</th><th>CQ</th><th>FQ</th></tr></thead><tbody><tr><td>Intramolecular hydrogen bond</td><td>Yes</td><td>Yes</td></tr><tr><td>Weak base properties (pK<sub>a1</sub> and pK<sub>a2</sub> values)</td><td>10.03 and 7.94</td><td><strong>8.19</strong> and <strong>6.99</strong></td></tr><tr><td>Neutral and protonated forms at vacuolar pH</td><td>One time</td><td>Ten times</td></tr><tr><td>Lipophilicity at pH 5.2</td><td>- 1.2</td><td>- 0.77</td></tr><tr><td>Lipophilicity at pH 7.4</td><td>0.85</td><td><strong>2.95</strong></td></tr><tr><td>Complex with hematin and stoichiometry</td><td>Yes (1:1)</td><td>Yes (1:1)</td></tr><tr><td>Interaction with monomeric hematin (log K)</td><td>Yes (5.52)</td><td>Yes (5.52)</td></tr><tr><td>Inhibition of β-hematin formation (IRS)</td><td>Yes</td><td>Yes</td></tr><tr><td>BHIA<sub>50</sub></td><td>1.9</td><td><strong>0.78</strong></td></tr><tr><td>Production of hydroxyl radicals</td><td>No</td><td><strong>Yes</strong></td></tr><tr><td>Activity on CQ<sup>R</sup> clones and isolates</td><td>No</td><td><strong>Yes</strong></td></tr><tr><td>Relation with specific molecular resistance markers</td><td>Yes</td><td><strong>No</strong></td></tr></tbody></table>

IRS = infrared spectroscopy. BHIA<sub>50</sub> = 50 % inhibitory concentration for β-hematin inhibition in equivalents of compounds to hemin (Biot *et al.*, 2005).

<caption>Table II. – Comparative properties of chloroquine (CQ) and ferroquine (FQ).</caption>

## MODES OF ACTION: HYPOTHESES

CQ is thought to act by interfering with the digestion of haemoglobin in the blood stages of the malaria life cycle. Even if CQ and FQ share some similarities in their activity, FQ clearly showed important and additional mechanisms of action when compared to CQ (Table II) (Biot *et al.*, 2005; Dubar *et al.*, 2011).

The weaker base properties of FQ compared to CQ combined with its higher lipophilicity at pH 7.4 and the peculiar conformation provided by the intra-molecular hydrogen bond present in non polar conditions result in a better potency for FQ to cross membranes and a higher accumulation in the digestive vacuole. At the pH in that organelle, the physicochemical properties of FQ evidenced a higher fraction of neutral and mono-protonated forms and suggested a more efficient inhibitory activity on hematin biocrystallization (Dubar *et al.*, 2011), which was verified *in vitro* in BHIA (β-Hematin Inhibition Assay). Moreover, preferential localization of FQ at the site of crystallization of hemozoin close to the membrane of acidic vacuole might induce two independent or concomitant behaviours: first FQ might inhibit the self assembly of the hemozoin crystal and second FQ might specifically generate reactive oxygen species (*per se*, or *via* destruction of the hemozoin crystal) and induce lipid peroxidation

and alteration of digestive vacuole (Chavain *et al.*, 2008; Dubar *et al.*, 2011).

All these properties might explain why FQ is more active than CQ *in vitro* even in a susceptible *P. falciparum* clone. The *in vitro* assays emphasized the specific importance of the intra-molecular hydrogen bond in FQ. Indeed in our studies based on methyl-FQ (an analogue of FQ without the intra-molecular hydrogen bond due to the presence of a methyl group on the 4-amino group), we clearly showed that the presence of the intra-molecular hydrogen bond allows FQ to escape resistance mechanisms and avoid cross-resistance with the current antimalarials (Biot *et al.*, 2009; Dubar *et al.*, 2011).

## CLINICAL TRIALS

A total of 335 subjects, or patients have been administered with FQ (SSR97193) as of June 28 2010. In seven completed Phase 1/2 studies, 173 males subjects/patients were part of two trials performed in healthy Caucasian subjects, four trials conducted in asymptomatic African patients infected with *P. falciparum*, and one Phase IIa dose-escalation safety and activity (including adult African patients with mono-infection with *P. falciparum* and parasitemia within the 100 to 200,000/μL limits). Ongoing phase IIb dose-range study accounting for 440 patients conducted across seven African countries is currently assessing in four groups the safety and efficacy of an association of FQ-at a three dose level- with artesunate and FQ alone in patients with mono infection with *P. falciparum*. The first and second cohort consisting of adult/adolescent patients and children > 20 kg has been completed. Other potential combinations and indications are under evaluation at the time of writing this review.

## CONCLUSIONS AND PERSPECTIVES

In conclusion, FQ clinical trials will enable the definition of conditions of use of this new antimalarial drug, which appears to be well positioned in the pipeline. One remaining question is the cause of the potent activity of the drug, mainly towards CQ resistant parasites, and its relation with the structure of the molecule. Some clues (role of the hydrogen bond, role of redox activity, nature of the metal present in the metallophone moiety) are currently under examination to clarify the mechanisms of entry of FQ in the infected red blood cell, its site and mechanism of action and its relation with the transporters involved in resistance against different aminoquinolines, which appear ineffective to expel the molecule out of the
parasite. On the clinical front, it remains to determine
how this new drug will be best combined with a par-
tner to limit the risk of resistance.

ACKNOWLEDGEMENTS

All searches on FQ carried out in laboratories * and ***** were funded by Pierre Fabre Médicament and Sanofi-Aventis. The two labs are very grateful to all Ph.D. students who participated to this work (L. Delhaës, H. Abessolo, W. Daher, N. Chavain, and F. Dubar) and provided an excellent work. We acknowledge scientists who collaborated in the research: B. Pradines, T. Egan, K. Chibale, C. Slomianny, X. Trivelli, S. Bohic, E. Curis, and I. Forfar. F. Nosten is supported by the Wellcome Trust of Great Britain. We thank Ministère de l'Enseignement Supérieur, Université Lille Nord de France, CNRS and INSERM.

REFERENCES

ANSTEY N.M., RUSSELL B., YEO T.W. & PRICE R.N. The patho-
physiology of *vivax* malaria. *Trends Parasitol*, 2009, **25**,
220-227.

ATTEKE C., NDONG J.M., AUBOUY A., MACIEJEWSKI L., BROCARD J.,
LEBIBI J. & DELORON P. *In vitro* susceptibility to a new anti-
malarial organometallic analogue, ferroquine, of *Plasmo-
dium falciparum* isolates from the Haut-Ogooue region of
Gabon. *J Antimicrob Chemother*, 2003, **51**, 1021-1024.

BAIRD J.K. Effectiveness of antimalarial drugs. *N Engl J Med*, 2005, **352**, 1565-1577.

BAIRD J.K. Resistance to therapies for infection by *Plasmo*-  
*dium vivax. Clin Microbiol Rev*, 2009, **22**, 508-534.

BAREND S. M., JAIDEE A., KHAOHIRUN N., SINGHASIVANON P. & NOSTEN F. *In vitro* activity of ferroquine (SSR97193) against *Plasmodium falciparum* isolates from the Thai-Burmese border. *Malaria J*, 2007, **6**, 81.

BEAGLEY P., BLACKIE M.A.L., CHIBALE K., CLARKSON C., MOSS J.R. & SMITH P.J. Synthesis and antimalarial activity *in vitro* of new ruthenocene-chloroquine analogues *Dalton Trans*, 2002, *2002*, 4426-4433.

BEAGLEY P., BLACKIE M.A.L., CHIBALE K., CLARKSON C., MEIJBOOM R., MOSS J.R., SMITH P.J. & SU H. Synthesis and antiplasmodial activity *in vitro* of new ferrocene-chloroquine analogues. *Dalton Trans*, 2003, *2003*, 3046-3051.

BIOT C., GLORIAN G., MACIEJEWSKI L.A., BROCARD J.S., MILLET P., GEORGES A.J., ABESSOLO H., DIVE D. & LEBIBI J. Synthesis and antimalarial activity *in vitro* and *in vivo* of a new ferrocene-chloroquine analogue. *J Med Chem*, 1997, *40*, 3715-3718.

BIOT C., DELHAËS L., N'DIAYE C.M., MACIEJEWSKI L.A., CAMUS D., DIVE D. & BROCARD J.S. Synthesis and antimalarial activity *in vitro* of potential metabolites of ferrochloroquine and related compounds. *Bioorg Med Chem*, 1999, 7, 2843-2847.

BIOT C., TARAMELLI D., FORFAR-BARES I., MACIEJEWSKI L.A., BOYCE M., NOWOGROCKI G., BROCARD J.S., BASILICO N., OLLIARO P. & EGAN T.J. Insights into the mechanism of action of ferroquine. Relationship between physicochemical properties and antiplasmodial activity. *Mol Pharm*, 2005, **2**, 185-193.

BIOT C., DAHER W., JARRY C., NDIAYE C.H., PELINSKI L., KHALIFE J., FRAISSE L., BROCARD J., MELNYK P., FORFAR-BARES I. & DIVE D. Probing the role of the covalent linkage of ferrocene into a chloroquine template. *J Med Chem*, 2006a, **49**, 4707-4714.

BIOT C., DAHER W., CHAVAIN N., FANDEUR T., KHALIFE J., DIVE D. & DE CLERCQ E. Design and synthesis of hydroxyferroquine derivatives with antimalarial and antiviral activities. *J Med Chem*, 2006b, **49**, 2845-2849

BIOT C., PRADINES B., SERGEANT M.H., GUT J., ROSENTHAL P.J. & CHIBALE K. Design, synthesis, and antimalarial activity of structural chimeras of thiosemicarbazone and ferroquine analogues. *Bioorg Med Chem Lett*, 2007, **17**, 6434-6438.

BIOT C., CHAVAIN N., DUBAR F., PRADINES B., BROCARD J., FORFAR I. & DIVE D. Structure-activity relationships of 4-N-substituted ferroquine analogues. Time to re-evaluate the mechanism of action of ferroquine. *J Organomet Chem*, 2009, **694**, 845-854.

BIOT C. & DIVE D. Bioorganometallic chemistry and malaria.
*Top Organomet Chem*, 2010, **32**, 155-193.

BLACKIE M.A., BEAGLEY P., CROFT S.L., KENDRICK H., MOSS J.R. & CHIBALE K. 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*, 2007, **15**, 6510-6516.

BLACKIE M.A. & CHIBALE K. Metallocene antimalarials: the continuing quest. *Met Based Drugs*, 2008, 2008, 495123.

CHAVAIN N., VEZIN H., DIVE D., TOUATI N., PAUL J.F., BUISINE E. & BIOT C. Investigation of the redox behavior of ferroquine, a new antimalarial. *Mol Pharm*, 2008, **5**, 710-716.

CHAVAIN N., DAVIOUD-CHARVET E., TRIVELLI X., MBEKI L., ROTTMANN M., BRUN R. & BIOT C. Antimalarial activities of ferroquine conjugates with either glutathione reductase inhibitors or glutathione depletors via a hydrolyzable amide linker. *Bioorg Med Chem*, 2009, **17**, 8048-8059.

CHAVAIN N. & BIOT C. Organometallic complexes: new tools for chemotherapy. *Curr Med Chem*, 2010, **17**, 2729-2745.

CHIM P., LIM P., SEM R., NHEM S., MACIEJEWSKI L. & FANDEUR T. The *in-vitro* antimalarial activity of ferrochloroquine, measured against Cambodian isolates of *Plasmodium falciparum*. *Ann Trop Med Parasitol*, 2004, **98**, 419-424.

DAHER W.E., PELINSKI L., KLIEBER S., SADOUN F., MEUNIER V., BOURRIE M., BIOT C., GUILLOU F., FABRE G., BROCARD J., FRAISSE L., MAFFRAND J.P., KHALIFE J. & DIVE D. *In vitro* metabolism of ferroquine (SSR97193) in animal and human hepatic models and antimalarial activity of major metabolites on *Plasmodium falciparum*. *Drug Metab Dispos*, 2006a, **34**, 667-682.

DAHER W., BIOT C., FANDEUR T., JOUIN H., PELINSKI L., VISCO-GLIOSI E., FRAISSE L., PRADINES B., BROCARD J., KHALIFE J. & DIVE D. Assessment of *P. falciparum* resistance to ferro-
quine in field isolates and in W2 strain under pressure. *Malaria J*, 2006b, **5**, 11.

DELHAËS L., BIOT C., BERRY L., MACIEJEWSKI L.A., CAMUS D., BROCARD J.S. & DIVE D. Novel ferrocenic artemisinin derivatives: synthesis, *in vitro* antimalarial activity and affinity of binding with ferroprotoporphyrin IX. *Bioorg Med Chem*, 2000, **8**, 2739-2745.

DELHAËS L., ABESSOLO H., BIOT C., DELORON P., KARBWANG J., MORTUAIRE M., MACIEJEWSKI L.A., CAMUS D., BROCARD J. & DIVE D. Ferrochloroquine, a ferrocenyl analogue of chloroquine, retains a potent activity against resistant *Plasmodium falciparum* *in vitro* and *P. vinckei* *in vivo*. *Parasitol Res*, 2001, **87**, 239-244.

DELHAËS L., BIOT C., BERRY L., DELCOURT P., MACIEJEWSKI L.A., CAMUS D., BROCARD J.S. & DIVE D. Synthesis of ferroquine enantiomers. First investigation of metalloenic, chirality upon antimalarial activity and cytotoxicity. *ChemBioChem*, 2002, **3**, 101-106.

DESJARDIN R.E., CANFIELD C., HAYNES J., & CHULAY J. Quantitative assessment of antimalarial activity *in vitro* by a semiautomated microdilution technique. *Antimicrob Agents Chemother*, 1979, **16**, 710-718.

DHANAWAT M., DAS N., NAGARWAL R.C. & SHRIVASTAVA S.K. Antimalarial drug development: past to present scenario. *Mini Rev Med Chem*, 2009, **9**, 1447-1469.

DIVE D. & BIOT C. Ferrocene conjugates of chloroquine and other antimalarials: the development of ferroquine, a new antimalarial. *ChemMedChem*, 2008, **3**, 383-391.

DONDORP A.M., YEUNG S., WHITE L., NGUON C., DAY N.P., SOCHEAT D. & VON SEIDLEIN L. Artemisinin resistance: current status and scenarios for containment. *Nat Rev Microbiol*, 2010, **8**, 272-280.

DUBAR F., EGAN T.J., PRADINES B., KUTER D., NCOKAZI K.K., FORGE D., PAUL J.P., PIERROT C., KALAMOU H., KHALIFE J., BUISINE E., ROGIER C., VEZIN H., FORFAR I., SLOMIANNY C., TRIVELLI X., KAPISHNIKOV S., LEISEROWITZ L., DIVE D. & BIOT C. The antimalarial ferroquine: role of the metal and intramolecular hydrogen bond in activity and resistance. *ACS Chem Biol*, 2011, **6** (3), 275-287.

DUNITZ J., ORGEL L. & RICH A. The crystal structure of ferroquine. *Acta Cryst*, 1956, **9**, 373-375.

HENRY M., BRIOLANT S., FONTAINE A., MOSNIER J., BARET E., AMALVICT R., FUSAÍ T., FRAISSE L., ROGIER C. & PRADINES B. *In vitro* activity of ferroquine is independent of polymorphisms in transport protein genes implicated in quinoline resistance in *Plasmodium falciparum*. *Antimicrob Agents Chemother*, 2008, **52**, 2755-2759.

KREIDENWEISS A., KREMSNER P.G., DIETZ K. & MORDMUELLER B. *In vitro* activity of ferroquine (SSR97193) is independent of chloroquine resistance in *Plasmodium falciparum*. *Amer J Trop Med Hyg*, 2006, **75**, 1178-1181.

LEIMANIS M.L., JAIDEE A., SRIPRAWAT K., KAEWPONGSRI S., SUWANARUSK R., BARENDS M., PHYO A.P., RUSSELL B., RENIA L. & NOSTEN F. *Plasmodium vivax* susceptibility to ferroquine. *Antimicrob Agents Chemother*, 2010, **54**, 2228-2230.

NOSTEN F. & WHITE N.J. Artemisinin-based combination treatment of *falciparum* malaria. *Am J Trop Med Hyg*, 2007, **77**, 181-192.

OLLIARO P. & WELLS T.N. The global portfolio of new antimalarial medicines under development. *Clin Pharmacol Ther*, 2009, **85**, 584-595.

PETERS W. *In*: Chemotherapy, and drug resistance in malaria. *Peters W.* (ed.), LiverpoolSchool of Tropical Medicine, Liverpool, 1987, Vol. 1, 145-273.

PRADINES B., FUSAÍ T., DARIES W., LALOGE V., ROGIER C., MILLET P., PANCONI E., KOMBILA M. & PARZY D. Ferrocene-chloroquine analogues as antimalarial agents: *in vitro* activity of ferrochloroquine against 103 Gabonese isolates of *Plasmodium falciparum*. *J Antimicrob Chemother*, 2001, **48**, 179-184.

PRADINES B., TALL A., ROGIER C., SPIEGEL A., MOSNIER J., MARRAMA L., FUSÍ T., MILLET P., PANCONI E., TRAPE J.F. & PARZY D. *In vitro* activities of ferrochloroquine against 55 Senegalese isolates of *Plasmodium falciparum* in comparison with those of standard antimalarial drugs. *Trop Med Int Health*, 2002, **7**, 265-270.

STRATTON L., O'NEILL M.S., KRUK M.E. & BELL M.L. The persistent problem of malaria: addressing the fundamental causes of a global killer. *Social Sci Med*, 2008, **67**, 854-862.

VESSIERES A., JAOUEN G., GRUSELLE M., ROSSIGNOL J.L., SAVIGNAC M., TOP S. & GREENFIELD S. Synthesis and receptor binding of polynuclear organometallic estradiol derivatives. *J Steroid Biochem*, 1988, **30**, 301-316.

WHITE N.J. Antimalarial drug resistance and combination therapy. *Philos Trans R Soc London B Biol Sci*, 1999, **354**, 739-749.

Received on January 10th, 2011

Accepted on March 28th, 2011