Trisubstituted Pyrimidines as Efficacious and Fast-Acting Antimalarials

In this paper we describe the optimization of a phenotypic hit against Plasmodium falciparum, based on a trisubstituted pyrimidine scaffold. This led to compounds with good pharmacokinetics and oral activity in a P. berghei mouse model of malaria. The most promising compound (13) showed a reduction in parasitemia of 96% when dosed at 30 mg/kg orally once a day for 4 days in the P. berghei mouse model of malaria. It also demonstrated a rapid rate of clearance of the erythrocytic stage of P. falciparum in the SCID mouse model with an ED90 of 11.7 mg/kg when dosed orally. Unfortunately, the compound is a potent inhibitor of cytochrome P450 enzymes, probably due to a 4-pyridyl substituent. Nevertheless, this is a lead molecule with a potentially useful antimalarial profile, which could either be further optimized or be used for target hunting.


Plasmodium falciparum screening
Assays against P. falciparum were conducted as previously described. [1][2][3][4] 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 528nm after excitation at 485nm. 5 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 key compounds from each hit series in an orthogonal platform. 6 Compound bioactivity from both assays was expressed as EC 50 , the effective concentration of compound causing 50% inhibition of parasite growth.

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. 7 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 384well plate and pre-diluted 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, S:B> 3, %CV (no inhibition control) < 15 . The formula used to calculate Z' is . All EC 50 Curve fitting was undertaken using XLFit version 4. ,where A=% inhibition at bottom, B=% inhibition at top, C= EC 50 , D= slope, x= inhibitor concentration and y= % inhibition. If curve did not reach 100% of inhibition, B was fixed to 100 only when at least 50% of inhibition was reached.

Aqueous solubility
The aqueous solubility of the test compounds was measured using laser nephelometry, as described previously. 8 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). Assays were run in triplicate.

Intrinsic Clearance (Cli) experiments
The procedure was carried out as reported previously. 8 Test compound (0.5 µM) was incubated with female CD1 mouse liver microsomes (Xenotech LLC TM ; 0.5 mg/mL 50 mM potassium phosphate buffer, pH 7.4) and the reaction started with 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 (50uL) 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 UPLCMSMS 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 52.5mg protein per g liver. Verapamil (0.5µM) was used as a positive control to confirm acceptable assay performance. Experiments were performed using a single timecourse experiment.

Plasma Protein Binding (PPB) experiments
This was based on a previously described method, except using NMRI mice. 9 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.

In vivo 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. 10 Pharmacokinetic parameters were derived from the blood concentration time curve 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 11 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 using standard flow cytometry techniques. Compounds 12 and 13 were prepared in 10% DMSO, 40% PEG 400 and 50% water prior to administration orally, once daily for 4 days. Blood samples were collected on day 4 (96 h after infection). 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.

In vivo antimalarial efficacy studies in P. falciparum. (GlaxoSmithKline)
Assays against P. falciparum were conducted as previously described. 12 Age-matched female immunodeficient NOD-scid IL-2Rγc-null mice (The Jackson Laboratory, Bar Harbor, ME) were engrafted with human erythrocytes (Red Cross Transfusion Blood Bank in Madrid, Spain) by daily injection with 1 mL of a 50% hematocrit erythrocyte suspension (RPMI 1640 medium, 25% (vol/vol) decomplemented human serum, 3.1 mM hypoxanthine) by intraperitoneal route throughout the experiment. When mice reached approximately 40% human erythrocytes in peripheral blood, they were intravenously infected with 2×10 7 P. falciparum Pf3D 70087/N9infected erythrocytes (day 0). Drug treatments were administered on day 3 after infection to mice randomly allocated to treatments once a day for 4 consecutive days by oral gavage at 10 mL/kg. Compound 13 was prepared in 10% DMSO; 40% PEG 400, 50% water before administration. Parasitaemia was measured by flow cytometry in samples of peripheral blood stained with the fluorescent nucleic acid dye SYTO-16 and anti-murine erythrocyte TER119 monoclonal antibody (Pharmingen, San Diego, CA, USA) in serial 2 µL blood samples taken every 24 h until assay completion.
The blood levels of 13 in the mice of the efficacy experiment were measured in serial samples of peripheral blood (25 µL) taken by tail puncture at 0.25, 0.5, 1, 3, 6, 8, 12 and 23 hours after the first administration. The blood samples were immediately lysed by mixing with 25 µL of distilled water, frozen on dry ice and stored at -80ºC until analysis. The compounds were extracted from 10 µL of each lysate by liquid-liquid extraction in the MultiScreen Solvinert 0.45µm Hydrophobic PTFE 96-well plate system (Millipore) and stored frozen at -80ºC until analysis by LC/MS/MS in AB Sciex API4000 (AB Sciex, Framingham, MA). The compound concentration versus time were analysed by non-compartmental analysis (NCA) using Phoenix® Version 6.3 (Pharsight Corporation, Mountain View, CA, USA). Additional statistical analysis was performed with GraphPad Prism® Version 6.02 (GraphPad Software Inc, San Diego CA, USA).
Efficacy was expressed as the daily exposure (AUC, µg·h/mL/day) of 13 in whole blood necessary to reduce parasitaemia at day 7 by 90 % with respect to vehicle-treated mice (AUC ED90 ). The AUC ED90 was estimated by fitting a four parameter logistic equation for the log10 [parasitaemia at day 7 for individual i] versus the AUC0-23h of 13 in blood for individual i using GraphPad Prism 6.0. (i = inhibitor concentration).

β-Haematin formation assay (GlaxoSmithKline)
The in vitro β-haematin formation assay was conducted as previously described. 13 Briefly, the compounds were dissolved in DMSO and 1/3 serial dilutions were tested in a final reaction volume of 0.4 mL using oleoyl glycerol as catalytic agent. Each assay was run at least in triplicate. Non-linear regression analysis was used to fit the normalized results of the dose response curves and IC 50 values determined using the GraFit5 software package (GraphFit program; Erithacus Software, Horley, Surrey, UK).

In vitro parasite reduction ratio (PRR) assay (GlaxoSmithKline)
The in vitro Parasite Rate Reduction assay (PRR) was conducted as previously described. 14 Briefly, parasites were exposed to 13 for 120 h at a concentration corresponding to 10 x EC 50 . Drug was renewed daily over the entire treatment period. Samples of parasites were taken from the treated culture at intervals (24, 48, 72, 96 and 120 h time points), drug was washed out and drug-free parasites were cultured in 96-well plates by adding fresh erythrocytes and new culture media. The number of viable parasites was determined by the serial dilution technique. Four independent serial dilutions were done with each sample to correct for experimental variation.
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.