Discovery of a Quinoline-4-carboxamide Derivative with a Novel Mechanism of Action, Multistage Antimalarial Activity, and Potent in Vivo Efficacy

The antiplasmodial activity, DMPK properties, and efficacy of a series of quinoline-4-carboxamides are described. This series was identified from a phenotypic screen against the blood stage of Plasmodium falciparum (3D7) and displayed moderate potency but with suboptimal physicochemical properties and poor microsomal stability. The screening hit (1, EC50 = 120 nM) was optimized to lead molecules with low nanomolar in vitro potency. Improvement of the pharmacokinetic profile led to several compounds showing excellent oral efficacy in the P. berghei malaria mouse model with ED90 values below 1 mg/kg when dosed orally for 4 days. The favorable potency, selectivity, DMPK properties, and efficacy coupled with a novel mechanism of action, inhibition of translation elongation factor 2 (PfEF2), led to progression of 2 (DDD107498) to preclinical development.


Plasmodium falciparum screening
Assays against P. falciparum were conducted as previously described. 1 Cultures of the widely-used malaria reference strain of chloroquine-sensitive Plasmodium falciparum strain 3D7 were maintained in a 5% suspension of human red blood cells (obtained from East of Scotland Blood Transfusion Service, Ninewells Hospital, Dundee) cultured in RPMI 1640 medium (pH 7.3) supplemented with 0.5% Albumax II (Gibco Life Technologies, San Diego, CA), 12 mM sodium bicarbonate, 0.2 mM hypoxanthine and 20 mg/L gentamicin at 37°C, in a humidified atmosphere of 1% O 2 , 3% CO 2 with a balance of nitrogen. Growth inhibition was quantified using a fluorescence assay utilising the binding of SYBR green to double stranded DNA, which emits a fluorescent signal at 528 nm after excitation at 485nm. 2 Mefloquine (potency range 30-60 nM) was used as a drug control to monitor the quality of the assay (Z' = 0.6 to 0.8, Signal to background >3, where Z' is a measure of the discrimination between the positive and negative controls on a screen plate). A 96-well [ 3 H]-Hypoxanthine incorporation assay was also developed as a secondary assay in order to validate compounds from the initial screen in an orthogonal platform. 3 Compound bioactivity from both assays was expressed as EC 50 , the effective concentration of compound causing 50% inhibition of parasite growth.

P. falciparum (panel of resistant strains) in vitro assay. (Swiss TPH)
All Plasmodium strains were cultured according to Trager and Jensen and are described at www.beiresources.org (resistant strains: K1, W2, 7G8, TM90C2A, D6 and V1/S, sensitive strain: NF54). EC 50 values were determined in vitro by measuring incorporation of the nucleic acid precursor [ 3 H]hypoxanthine. 1c

Mammalian Cell Growth Inhibition assay
A counter-screen against normal diploid human fibroblasts (MRC-5 cell line) was carried out to exclude non-selective, and toxic compounds. The assay was essentially carried out as described previously. 4 Cells were plated and incubated overnight to allow them to adhere as monolayers. A working stock of each test compound was transferred to an intermediate 384-well plate and prediluted with minimum essential media (MEM). The pre-diluted stocks were then transferred onto the cell monolayers, and the plates were incubated for 68 h. Resazurin, to a final concentration of 50 μM, was added to each well, after which plates were incubated for a further 3 h and measured for fluorescence (λ ex =528 nm, λ em =590 nm)

In vitro Cell Assay Data Analysis.
All data was processed using IDBS ActivityBase ® raw data was converted into per cent inhibition through linear regression by setting the high inhibition control as 100% and the no inhibition control as 0%. Quality control criteria for passing plates were as follows: Z'> 0. 5

Plasmodium falciparum late stage (IV-V) gametocyte high content imaging assay
Plasmodium falciparum NF54 -pfs16-LUC-GFP stage IV-V Gametocytes were produced as described previously. 5 Compounds were solubilized in DMSO and serially diluted in three concentrations per log standard curves in DMSO. One µl of compound was subsequently diluted in 25µl of water (4% DMSO) and 5µl transferred into the base of 384 well PerkinElmer CellCarrier plates. Forty-five µl of stage IV-V gametocytes (10%P in 0.1%H) were added to the imaging plates which were in turn sealed with a gas permeable membrane and incubated at 37°C, 5% CO 2 , 5% O 2 , 90% N 2 and 60% humidity for 72hours.
Post incubation, 5µl of MitoTracker® Red CM-H2Xros (MTR) solution (0.66ug/ml) was added to all wells and the plates incubated for a further 16hours. The assay plates were then imaged and the number of viable gametocytes determined for test compounds plus 0.4%DMSO and 5µM Puromycin. All data was normalized to % inhibition using the positive (5uM puromycin) and negative (0.4%DMSO) control values. Percent inhibition was plotted against log compound concentration using a 4 parameter log dose, non-linear regression analysis, with sigmoidal dose response (variable slope) curve fit using Prizm 4.0. IC50 values were obtained from this curve fit. The compound testing was performed in duplicate point within two separate experiments.

Plasmodium berghei ookinete development assay
Assay was performed as previously reported. 6 Briefly blood containing P. berghei CTRPp-GFP gametocytes was rapidly harvested from infected mice and introduced to test compounds pre-diluted in ookinete medium (to induce gamete formation and support ookinete development) in 96-well plates. Plates were incubated in the dark at 21°C for 22 hr before GFP fluorescence intensity measured in a plate reader. % inhibition of ookinete formation was calculated with reference to the fluorescence intensity values of a positive control (20 µM cycloheximide) and a solvent-only negative control (DMSO). All assays were performed in triplicate independent experiments and EC 50 values calculated in Graphpad Prism.

Aqueous solubility
The aqueous solubility of the test compounds was measured using laser nephelometry, as described previously. 7 Compounds were subject to serial dilution from 10 mg/mL to 0.5 mg/mL in DMSO. An aliquot was then mixed with MilliQ water to obtain an aqueous dilution plate with a final concentration range of 100 -5 μg/mL, with a final DMSO concentration of 1.0%. Triplicate aliquots were transferred to a flat bottomed polystyrene plate which was immediately read on the NEPHELOstar (BMG Lab Technologies). The amount of laser scatter caused by insoluble particulates (relative nephelometry units, RNU) was plotted against compound concentration using a segmental regression fit, with the point of inflection being quoted as the compounds aqueous solubility (μg/mL; reported in µM).

Intrinsic Clearance (Cli) experiments
The procedure was carried out as reported previously. 7 Test compound (0.5 µM) was incubated with female CD1 mouse, male sprague-dawley rat or pooled human mixed gender liver microsomes (Xenotech LLC TM ; 0.5 mg/mL 50 mM potassium phosphate buffer, pH 7.4) and the reaction initiated by the addition of excess NADPH (8 mg/mL 50 mM potassium phosphate buffer, pH 7.4). Immediately, at time zero, then at 3, 6, 9, 15 and 30 min an aliquot (50 μL) of the incubation mixture was removed and mixed with acetonitrile (100 μL) to stop the reaction. Internal standard was added to all samples, the samples centrifuged to sediment precipitated protein and the plates then sealed prior to UPLC-MS/MS analysis using a Quattro Premier XE (Waters Corporation, USA).
XLfit (IDBS, UK) was used to calculate the exponential decay and consequently the rate constant (k) from the ratio of peak area of test compound to internal standard at each timepoint. The rate of intrinsic clearance (CLi) of each test compound was then calculated using the following calculation: CLi (mL/min/g liver) = k x V x Microsomal protein yield Where V (mL/mg protein) is the incubation volume/mg protein added and microsomal protein yield is taken as 52.5 mg protein per g liver. Verapamil (0.5 µM) was used as a positive control to confirm acceptable assay performance. Experiments were performed using a single time-course experiment.

Plasma Protein Binding (PPB) experiments
This was based on a previously described method, with the exception that NMRI mouse plasma was used here. 8 In brief, a 96 well equilibrium dialysis apparatus was used to determine the free fraction in plasma for each compound (HT Dialysis LLC, Gales Ferry, CT). Membranes (12-14 kDA cut-off) were conditioned in deionised water for 60 min, followed by conditioning in 80:20 deionised water:ethanol for 20 min, and then rinsed in isotonic buffer before use. Female CD1 mouse plasma was removed from the freezer and allowed to thaw on the day of experiment. Thawed plasma was then centrifuged (Allegra X12-R, Beckman Coulter, USA), spiked with test compound (final concentration 10 μg/mL), and 150 μL aliquots (n=6 replicate determinations) loaded into the 96-well equilibrium dialysis plate. Dialysis against isotonic buffer (150 µL) was carried out for 5 h in a temperature controlled incubator at ~37ºC (Barworld scientific Ltd, UK) using an orbital microplate shaker at 100 revolutions/minute (Barworld Scientific Ltd, UK). At the end of the incubation period, 50 μL aliquots of plasma or buffer were transferred to micronic tubes (Micronic B.V., the Netherlands) and the composition in each tube balanced with control fluid (50 μL), such that the volume of buffer to plasma is the same. Sample extraction was performed by the addition of 200 µL of acetonitrile containing an appropriate internal standard. Samples were allowed to mix for 1 min and then centrifuged at 3000 rpm in 96-well blocks for 15 min (Allegra X12-R, Beckman Coulter, USA) after which 150 μL of supernatant was removed to 50 μL of water. All samples were analysed by UPLC-MS/MS on a Quattro Premier XE Mass Spectrometer (Waters Corporation, USA). The unbound fraction was determined as the ratio of the peak area in buffer to that in plasma. Experiments were run in triplicate.

Parallel Artificial Membrane Permeability Assay (PAMPA)
PAMPA was performed using a 96-well pre-coated BD Gentest™ PAMPA plate (BD Biosciences, U.K.). Each well was divided into two chambers; donor and acceptor, separated by a lipid-oil-lipid trilayer constructed in a porous filter. The effective permeability, P e , of the compound was measured at pH 7.4. Stock solutions (5 mM) of the compound were prepared in DMSO. The compound was then further diluted to 10 µM in phosphate buffered saline at pH 7.4. The final DMSO concentration did not exceed 5 % v/v. The compound dissolved in phosphate buffered saline was then added to the donor side of the membrane and phosphate buffered saline without compound was added to the acceptor side. The PAMPA plate was left at room temperature for 5 h. After which time, an aliquot (100 µl) was then removed from both acceptor and donor compartments and mixed with acetonitrile (80 µl) containing an internal standard. The samples were centrifuged (10 min, 5⁰C, 3270 g) to sediment precipitated protein and sealed prior to UPLC-MS/MS analysis using a Quattro Premier XE (Waters Corp, USA). P e was calculated as shown in the below equation: Where: C A (t) = peak area of compound present in acceptor well at time t = 18000 sec = acceptor well volume t = incubation time C D (t) = peak area of compound present in donor well at time t = 18000 sec Recovery of compound from donor and acceptor wells was calculated and data was only accepted when recovery exceeded 70 %.

Human ether-à-go-go related gene (hERG) K + assay (Outsourced)
Compounds were tested for inhibition of the human ether-à-go-go-related gene (hERG) K + channel using IonWorks patch clamp electrophysiology. Eight-point concentration-response curves were generated on 2 occasions using 3-fold serial dilutions from the maximum final assay concentration.

Mouse pharmacokinetics
Compound was dosed as a bolus solution intravenously (12) at 3mg free base/kg (dose volume: 5 mL/kg; dose vehicle: 10% DMSO, 90% saline) to female NMRI mice (n=3) or dosed orally (12 and 13) by gavage as a solution at 10 mg free base/kg (dose volume: 10mL/kg; Dose vehicle: 5 or 10% DMSO; 40% PEG400; 50 or 55% distilled water) to female NMRI mice (n=3/dose level). Female NMRI mice were chosen as these represent the sex and strain used for the P.berghei mouse model of malaria. Blood samples (10 µl) were taken from each mouse at 5, 15 and 30 minutes, 1, 2, 4, 6, and 8 hours post-dose, mixed with two volumes of distilled water and stored frozen until UPLC-MS/MS analysis. The level of each compound in mouse blood was determined by UPLC-MS/MS as previously reported. 9 Pharmacokinetic parameters were derived from the blood concentration time curve using PKsolutions software v 2.0 (Summit Research Services, USA).
Pharmacokinetic parameters were derived from the blood concentration time curve for each compound using PKsolutions software v 2.0 (Summit Research Services, USA).

In vivo antimalarial efficacy studies in P. berghei. (Swiss TPH)
In vivo efficacy was conducted as previously described 10 with the modification that female NMRI mice (n = 3) were infected with a GFP-transfected P. berghei ANKA strain (donated by A. P. Waters and C. J. Janse, Leiden University, The Netherlands), and parasitaemia determined (96 h after infection) using standard flow cytometry techniques . Compounds 1, 25,  days. Blood samples were collected on day 4 (96 h after infection) in two volumes water. Animals were observed for signs of overt toxicity/poor tolerability every 15 min for the first hour post dosing and then hourly up to 4 h after dosing each day. The animals were selected randomly for each group but were not blinded.

1-(3-(Bromomethyl)phenyl)ethanone.
To 1-(m-tolyl)ethanone (2 g, 14.9 mmol) and NBS (2.9 g, 16.4 mmol) in dichlorobenzene (100 mL), benzoyl peroxide (wet with 25% water, 0.2 g, 0.7 mmol) was added and the reaction was refluxed overnight. The reaction was allowed to cool down to room temperature and filtered through Celite. The filtrated was concentrated under reduced pressure and the product was purified by column chromatography using 40 g silica cartridge, A: hexane, B: ethyl acetate and the following gradient: 1 min hold 100% A, 18 min ramp to 30% B, 3 min hold 100% B. Fractions containing product were pooled together and the solvent was removed to obtain the desired product as a yellow oil with a purity of 64% by LC-MS. Product was used for the next step without further purification. Yield, 49% (2.44 g); ¹H NMR (500 MHz, CDCl 3