Discovery and optimisation studies of antimalarial phenotypic hits

There is an urgent need for the development of new antimalarial compounds. As a result of a phenotypic screen, several compounds with potent activity against the parasite Plasmodium falciparum were identified. Characterization of these compounds is discussed, along with approaches to optimise the physicochemical properties. The in vitro antimalarial activity of these compounds against P. falciparum K1 had EC50 values in the range of 0.09–29 μM, and generally good selectivity (typically >100-fold) compared to a mammalian cell line (L6). One example showed no significant activity against a rodent model of malaria, and more work is needed to optimise these compounds.


Introduction
Malaria is a serious endemic disease and is a major threat to public health in more than 100 countries [1,2]. It affects about 200 million people per year, with approximately 580,000 associated deaths [3,4]. In addition malaria exerts a huge economic toll in endemic countries [3]. The need for a continual supply of new antimalarial therapeutics is still as relevant as ever.
Malaria is caused by protozoan parasites of the species Plasmodium [5], with Plasmodium falciparum being responsible for most malaria-related deaths. In many areas malaria parasites have developed resistance to chemotherapeutic agents such as chloroquine, mefloquine, and sulfadoxine/pyrimethamine. Therefore, an urgent need exists to develop new classes of antimalarial drugs that operate by novel mechanisms of action.
We have recently reported the identification of a hit (TDR32750) [6,7] from a screen of the ChemDiv5000 'maximally structurally diverse' compound collection against P. falciparum. This screen was carried out by the World Health Organisation Programme for Research and Training in Tropical Medicine (Fig. 1). TDR32750 showed potent activity against P. falciparum (EC 50 ¼ 9 nM), and good selectivity compared to L6 mammalian cells (>2000-fold). In order to follow up on the hit, other analogues from ChemDiv and PrincetonBio were screened. This led to identification of two more hits, TDR45024 and TDR45033 (Fig. 1), which shared the N-arylpyrrole found in TDR32750.
In this paper we report the follow up of these compounds, TDR45024 and TDR45033; systematic structure-activity relationship studies were undertaken with the aim of improving antiparasitic activity, and to generate compounds with drug-like physicochemical and pharmacokinetic properties. The studies encompassed variation of the phenyl ring attached to the pyrrole, modification of the pyrrole and modification of the thiazolidinedione ring. The activity of compounds against the chloroquine and pyrimethamine resistant (K1) strain of P. falciparum is reported, as well as a counter-screen (EC 50 ) against the L6 murine cell line, to provide an indication of selectivity (Table 1, Fig. 2).

Modification of the thiazolidinedione core
The lipophilic cyclohexyl and N-phenyl groups from the thiazolidinedione core were replaced with methyls (Scheme 3), in order to improve the physicochemical properties of the molecule.
We also investigated replacing the N-imino group on the thiazolidinedione with an oxygen and replacing the thiazolidinedione ring altogether with a barbituric acid moiety (Scheme 4).

Stereochemistry
There are two potential stereocenters; in compounds 20e42, the exocyclic double bond to the imine; the exocyclic double bond to the pyrrole. Literature precedent [13], suggests the stereochemistry of these double bonds is (Z,Z). In the case of the exocyclic double bond to the imine, an E-configuration is highly unlikely as there would be steric clashes between the substituent on the imine (in the case of compound 20, the phenyl), with the substituent on the ring nitrogen (in the case of compound 20, the cyclohexyl). In the case of the exocyclic double bond to the pyrrole, the 1 H NMR signal of the exocyclic alkenic proton is indicative of the Z-stereoisomer. As in the literature precedent [13], this proton (HC]C) is de-shielded by the carbonyl group and for most of the compounds (20e43) appeared with chemical shifts in the range of 7.65e8.35 ppm. Scheme 1. General Synthetic Approach to thiazolidin-4-ones: (a) sodium acetate, ethanol, 0 C, 30 min, 20%; (b) p-toluenesulfonic acid bound with silica gel, microwave (0e400 W at 2.45 GHz), 180 C, 15e20 min, 80e90%; or p-toluenesulfonic acid, toluene, 90 C, 3 h, DeaneStark Apparatus; (c) phosphorous oxychloride, DMF, 100 C, 3 h, 80e95%; (d) piperidine, ethanol, 3 h, reflux, 20%.

Biology. In vitro activity
The compounds (20e32) were assayed against P. falciparum K1 strain [14], and counter-screened in mammalian L6-cells [15]. Changes to the pyrrole ring also had little effect on potency: including removal of the methyl groups (28, EC 50 ¼ 2.3 mM); or changing the pyrrole to a pyrazole (32, EC 50 ¼ 1.9 mM).

3.2.
Modifications to the cyclohexyl-2-(phenylimino)-4thiazolidinedione core (33e52; Table 2) Replacing the cyclohexyl ring on the thiazolidinedione with methyl (33e38) gave a drop in potency of between 2 and 10-fold (EC 50 ¼ 0.99e4.6 mM). However, given the drop in molecular weight and logP, this is a relatively small loss in activity, but gives an improvement in physicochemical properties. Replacement of the N-phenyl imine with an N-methyl imine (39e43) also led to a small drop in activity (EC 50 ¼ 3.6->13 mM), compared to the parent analogues. Replacement of the imine with an oxygen 44e46, 49, 50 with or without a methyl group on the thiazolidinedione nitrogen (45) gave a similar level in activity (EC 50 ¼ 1.9e13 mM).  In addition, some of these compounds showed reduced selectivity based in the L-6 assay.

In vivo efficacy studies in Plasmodium berghei mouse model
To establish proof of concept, compound 20 was taken forward to the P. berghei mouse model [16]. Compound 20 as a suspension in aqueous DMSO was dosed intraperitoneally at 50 mg/kg for 4 days but resulted in no significant reduction in parasitaemia or increase in survival time (Table 3).

Physiochemical properties and in vitro DMPK
The physicochemical properties of 20 were evaluated using a combination of in silico and experimental techniques, and the metabolic stability was assessed in vitro using human and mouse liver microsomes, (Table 4). Compound 20 meets the Lipinski criteria, except for the high lipophilicity with a logD of 7.1, which explains the poor aqueous solubility at pH 2 and 6.5.
Compound 20 underwent rapid NADPH-dependent degradation upon incubation with human and mouse liver microsomes, (Table 5). There was no species difference in the relative rate of metabolism between human and mouse liver microsomes, and no obvious metabolites were detected using the analytical conditions employed for the parent compound. The rapid microsomal clearance and low solubility probably explains the lack of activity of compound 20 in the mouse model.

Conclusion
As a result of a phenotypic screen, some antimalarial compounds were identified. Despite activity against P. falciparum in vitro, an example compound was not active in a rodent model of disease, most likely due to a combination of high metabolic turnover and low aqueous solubility arising from their relatively high lipophilicity. To address these issues a number of analogues were prepared with lower lipophilicity, measured by cLogP, but unfortunately these showed no significant improvement in potency against the parasite. In order to progress this series further, compounds need to be identified with improved potency and reduced lipophilicity. A further impetus would come from an identification of the molecular target(s) of these compounds.

General experimental information
Chemicals and solvents were purchased from Aldrich Chemical Co. or Fluka, and were used as received unless otherwise stated. Airand moisture-sensitive reactions were carried out under an inert atmosphere of argon in oven-dried glassware. Analytical thin-layer chromatography (TLC) was performed on pre-coated TLC plates (layer 0.20 mm silica gel 60 with fluorescent indicator UV254, from Merck). Developed plates were air-dried and analysed under a UV    conducted with a Waters Xbridge C18 column, 50 mm Â 2.1 mm, 3.5 mm particle size; mobile phase, water/acetonitrile þ0.1% HCOOH, or water/acetonitrile þ 0.1% NH 3 ; linear gradient from 80:20 to 5:95 over 3.5 min and then held for 1.5 min; flow rate of 0.5 mL min À1 . All assay compounds had a measured purity of !95% (by total ion current (TIC) and UV) as determined using this analytical LCÀMS system. High resolution electrospray measurements were performed on a Bruker DaltonicsMicrOTOF mass spectrometer.