Lead Optimization of Anti-Malarial Propafenone Analogs

Abstract

Previously reported studies identified analogs of propafenone that had potent antimalarial activity, reduced cardiac ion channel activity, and properties that suggested the potential for clinical development for malaria. Careful examination of the bioavailability, pharmacokinetics, toxicology, and efficacy of this series of compounds using rodent models revealed orally bioavailable compounds that are non-toxic and suppress parasitemia in vivo. Although these compounds possess potential for further preclinical development, they also carry some significant challenges.

Introduction

In 2010 there were approximately 216 million cases of malaria worldwide with an estimated death toll of 655000.¹ Continued resistance to the currently available drugs^{2, 3} and recently observed slowing in patient responses to artemisinins^4–7 enforces a need for further research to discover new anti-malarial therapies. Screening of a small collections of known drug and bioactives revealed that propafenone, a Class 1c anti-arrhythmia drug⁸, potently inhibited growth of Plasmodium falciparum, and was selectively more potent against the multi-drug-resistant W2 and K1 strains.⁹ Use of propafenone, or any other antiarrhythmic drug as an anti-malarial was previously unreported and the increased activity against the multi-drug resistant strains is very unusual. Therefore, the SAR’s of this series were explored to establish if the mammalian ion channel activity could be separated from the antimalarial activity and if the unusual strain specificity was maintained. Preliminary hit-to-lead studies focused attention upon a particular subseries of related compounds in which the alkyl amine side chain of propafenone was replaced by a bulkier, less basic amine (Figure 1, 1-6). ¹⁰¹⁰

Figure 1.

Propafenone and analogues chosen for further studies

This hit-to-lead study carefully established structure activity relationships (SAR) and led to a set of analogues with improved in vitro efficacy, low cytotoxicity, and a reasonable therapeutic index with respect to the ion channels targeted by propafenone itself, as seen in Table 1. Using in vitro testing of the ion channels targeted by propafenone -- hERG, Nav1.5 and Kir6.2/SUR2A -- revealed that activity against NaV1.5 and Kir6.2 could be completely suppressed and that against hERG lessened while also increasing the efficacy of the analogs compared to propafenone (Table 1).¹⁰ This strongly suggests the resulting compounds would have significantly lessened cardiac effects relative to propafenone. With these two goals achieved it was hoped the analogs would maintain the favorable pharmacokinetic properties of the parent compound, despite the addition of the bulky side chain required to squelch ion channel activity. In the current study, six compounds from the lead optimization series were evaluated to determine their absorption, distribution, metabolism, and excretion (ADME), using both in vitro and in vivo methods in order to assess potential for development (Fig 1). The compounds were selected to include the 3 most potent analogs and the 3 with the best in vitro physiochemical properties which should translate to favorable in vivo pharmacokinetic behavior. Additionally the compounds were assessed in vitro and in vivo for toxicology. Based upon the examination of ADME and toxicology, two of the compounds were tested to determine in vivo efficacy in a rodent malaria model.

Table 1.

In vitro potency and in vitro ion channel effects of the tested propafenone analogues

						IC50 (μM)				TI (hERG IC50/ K1 EC50)
	3D7 EC50 (nM)	3D7 EC90 (nM)	K1 EC50 (nM)	K1 (nM)	EC90	hERG	Nav1.5 tonic	Nav1.5 phasic	Kir6.2
1	60±10	90±20	100±50	100±10		2.6	N/A	N/A	N/A	26
2	240±80	660±70	50±30	310±50		4.84	>5	>5	>5	895
3	160±20	450±60	100±40	210±20		N/A	N/A	N/A	N/A	N/A
4	340±90	750±50	120±40	240±40		3.46	>5	>5	>5	65
5	1330±880	2200±600	260±150	420±50		N/A	N/A	N/A	N/A	N/A
6	390±60	1300±80	190±90	640±120		4.31	>5	>5	>5	23

Results and Discussion

Chemistry

The synthesis of the targeted compounds (Figure 1, 1 – 6) proceeded smoothly and in accordance with previous work.¹⁰ The hydrogen transfer reductions, along with the microwave aminolysis of the epoxide rings, could be performed on up to a 1.5 g scale, in the Biotage^® Initiator 60. Removal of the palladium catalyst, post-reduction, was carried out using Celite filtration rather than through syringe filters, as previously reported. The final compounds were easily purified by recrystallization. The purity of all compounds was determined to be greater than 95% by UPLC/UV/ELSD/MS (Waters Affinity)¹¹ and the identity confirmed by NMR and MS.

As previously reported¹⁰ the diphenylmethyl-piperazine compounds 1, 2, and 3 were the most potent of the compounds, had the weakest activity in the ion channels targeted by the parent compound and consistently proved to be more active against the K1 strain of P. falciparum. In order to allow for better pharmacological performance, the lower molecular weight, but less potent, compounds 4, 5, and 6 were tested as well.

ADME Studies – In Vitro

In vitro studies were aimed at identifying potential liabilities of the compounds including potential for drug-drug interactions, predicted bioavailability, and metabolism.

Drug-drug interactions (DDI’s) are a serious concern with antimalarial drugs due to their use in areas where co-treatment of HIV is common.¹² Propafenone is extensively metabolized by CYP2D6⁸ and therefore has potential for adverse drug-drug interactions with currently used malaria drugs that inhibit CYP2D6, such as chloroquine,¹³ quinine and quinidine,¹⁴ along with HIV protease inhibitors such as indinavir, ritonavir, and saquinavir¹². Additionally, the new propafenones may bind to other common cytochrome p450’s. In order to provide a preliminary assessment of the potential for DDI’s an in vitro CYP inhibition assay was carried out. Compounds 1-6 were incubated with control CYP substrates (indicated in parentheses in column headings, see S1), human liver microsomes and cofactors for 20 minutes at 37 °C. Known CYP inhibitors were used as controls. Formation of metabolites was measured by LC-MS/MS and compared to control incubations (incubation of substrates with microsomes and cofactors, no test articles or inhibitors). None of the propafenones inhibited CYP1A2, CYP2B6, CYP2C9, CYP2C19, or CYP3A4 (See S1, Table 1). However compounds 4 and 5 were weak CYP2D6 inhibitors (Fig. 2). Compounds 4 and 5 also showed some oxidation of the phenyl ring of phenyl-piperidine moiety that is consistent with interaction with 2D6 in the microsomal models.

Figure 2.

Heat map of CYP2D6 % activity in presence of propafenones. Propafenone significantly inhibits 2D6 at both concentrations tested whereas the new propafenone analogs do not.

In earlier SAR studies, it was decided to incorporate fluorine into the propafenones at the points of metabolism of propafenone itself, which vary between mice and humans.^{15, 16} In humans, the primary metabolite of propafenone is 5-hydroxy-propafenone, whereas in rodents it is 4′-hydroxypropafenone. Incorporation of the fluorine at the 4′- and 5-positions did not affect potency but generally led to decreased solubility relative to unsubstituted analogs (Table 2). Likewise, permeability was generally lowered for all of the analogs relative to propafenone. Thus the substitutions that allowed modulation of the cardiac ion channel activities of propafenone while retaining antimalarial potency introduced liabilities with respect to physiochemical properties that were exacerbated by fluorination.

Table 2.

Calculated physical properties, measured permeability (PAMPA), and measured stability in microsomal models

Compound	calc LogD7.4*	Avg. Pe/cm/s)	Sol./(uM)	Mouse Microsomes Clint’ (mL/min/kg)	Human Microsomes Clint’ (mL/min/kg)
Propafenone	2.5	1500±360	70±20	46±5	4±1
1	6.5	2.3±1	0.1±0	70±10	14±1
2	6.7	0.8±0.1	0.2±0.1	34±3	5±2
3	6.7	unda	0.6±0.2	43±3	13±1
4	5.5	490±70	7.3±1	27±2	11±1
5	5.7	unda	0.2±0.1	29±2	19±1
6	5.7	unda	2.2±	30±6	38±4

^*calculated using pipeline pilot ^aunder the detectable limit

Fluorination of the 5-position of the compounds slowed human microsomal metabolism for diphenylmethyl-piperazines 1 and 2. However, fluorination of the 4′-postion failed to slow metabolism of any analog in the mouse microsome models. Overall, the mouse microsomes caused more oxidative cleavage at the β nitrogen of the piperazine ring whereas the human microsomes afforded more hydroxylation of the A ring. At this site, the enzymes could accommodate fluorination by moving the site of oxidation around the ring to “avoid” the fluorinated position if present.

Overall the in vitro studies predicted that the new compounds would not have significantly worse potential for drug interactions or metabolism than propafenone itself. However, they also predicted that the bioavailability may be worse. Since the compounds were active in cellular models that required permeation through two membranes, it was decided to proceed with in vivo pharmacokinetic assessment even in light of modest to low permeability of the compounds.

ADMET - In vivo

The in vivo pharmacokinetic behavior of all 6 compounds was established at oral doses of 20 mg/kg and 200 mg/kg in female CD-1 mice (Table 3). Compounds 1, 3, and 4 were also tested at 500 mg/kg (See S1, Table 2). The C_max of methyl-diphenylpiperidine compounds rose approximately 10-fold when increasing dosing from 20 mg/kg to 200 mg/kg. Interestingly, phenyl-piperidine compound 4 had a supra-stoichiometric increase in C_max, almost 25-fold, with increasing the dose from 20 mg and 200 mg/kg. One possible reason for this could be saturation of the metabolism mechanism. In contrast, phenyl-piperazine 5 showed only a 2-fold increase in C_max when increasing dosing from 20 mg and 200 mg/kg. None of the compounds tested at 500 mg/kg showed a significant increase in C_max when ranging dosing from 200 mg/kg to 500 mg/kg dosing, indicating that an absorption limit had been reached. Compound 4 also showed the highest value for AUC_inf at the higher concentrations, with approximately 5-fold improvement over compound 1 at 500 mg/kg. Overall compounds 4 and 6 exhibited the best overall profiles.

Table 3.

Pharmacokinetic Profile in Mice

Test Article	Dose (mg/kg)	Cmax (nM)	Tmax (hr)	AUCinf (nM/ml)	V/F (L/kg)	Cl/F (ml/hr/kg)
1	20	73.7	1	248.7	318.2	141107
	200	667	0.5	3103.1	602.9	113136
	500	597.7	1	3368.5	2020	260562
2	20	98.7	1	NDa	NDa	NDa
	200	907.5	1	6166.5	275.8	55189
3	20	92.4	0.5	298.1	369.6	114161
	200	1140.1	1	5665.8	287.6	60066
	500	860.6	0.5	4078.8	1758	208599
4	20	69.6	0.3	224.0	1093.3	186546
	200	1622.9	0.5	6799.7	277.5	61460
	500	1745.4	0.5	15332.9	623	68135
5	20	434.4	1	1596.1	223.8	25233
	200	480.7	1	3748.5	981.7	107444
6	20	301.8	8	NDa	NDa	NDa
	200	866.5	0.5	6486.8	271.8	60268

^aND- Not determined - due to limited # of time-points in the elimination phase

In choosing which compounds to examine in efficacy models, we compared the plasma exposure of compounds (as a marker for the target tissue, erythrocytes) and the in vitro potencies against P. falciparum. Unfortunately, the species of malaria that infects rodents, P. berghei, cannot be cultured in vitro so potency against this organism could not be used. Assessing the 6 compounds, the non-fluorinated methyldiphenyl-piperazine 1, showed the greatest in vitro efficacy and reasonable PK parameters. The second compound chosen was piperidine compound 4, which gave much improved PK parameters at the cost of lower potency (Fig. 3). Compound 1 was expected to maintain plasma levels above the measured EC90 for P. falciparum for over ten hours after a single oral dose of 200 mg/kg, whereas compound 4 was expected to maintain appropriate levels for roughly 5 hours. Therefore efficacy experiments were justified with compounds 1 and 4 in order to determine if correlations could be made between in vitro activity and in vivo efficacy.

Figure 3.

Plasma drug concentrations of compounds 1 and 4 versus time after oral dosing of 200 mg/kg. The in vitro EC₉₀ efficacies against 3D7 and K1 P. falciparum are shown for reference.

Toxicity Studies in Mice

Toxicity studies were carried out in female CD-1 mice on all 6 propafenone compounds to determine the maximum tolerated dose (MTD). This was done in parallel with the pharmacokinetics studies described above in order to establish dose-toxicity relationships. The mice were treated with a single oral dose at 10, 100, 200 and 500 mg/kg and observed for 48 hr. No adverse clinical signs were observed in this time period at any of the dosage concentrations for any of the compounds. This finding can be explained by the relatively low C_max values and the poor dose linearity that were observed, together with the lack of cytotoxicity observed during in vitro testing.⁸ Propafenone has an LD₅₀ of 605 mg/kg in female mice when administered orally so no toxic effects should be expected.⁸ Therefore, the modifications made to propafenone did not manifest new patent toxicology and efficacy experiments could be justified with any of the lead compounds.

In Vivo Efficacy of 1 and 4

The studies summarized above provided justification for carrying efficacy modeling with two compounds (1 and 4) that should have sufficient exposure to lead to significant suppression of parasitemia and were non-toxic at doses expected to be efficacious. Propafenone analogues 1 and 4 were tested against P. berghei (NK-65) infected ICR mice using the modified Thompson’s test¹⁷ to establish oral efficacy (See S1, Table 3). Chloroquine was used as a control. Both compounds were tested at 100 mg/kg and 200 mg/kg, administered orally on days 3, 4, and 5 after initial inoculation with parasites (Fig. 4).

Figure 4.

In Vivo Efficacy of compounds 1 and 4.

Dosing compound 1 orally at 100 mg/kg gave a mean survival rate of 7 days. One mouse died on day 6, four on day 7 and one on day 8, post inoculation. The mean parasitemia for the group of the 5 surviving mice was 53.6%. At 200 mg/kg the mean survival rate was 7 days. One mouse died on day 6, 4 died on day 7 and 2 on day 9. The mean parasitemia for the 4 surviving mice was 55.3%. Comparatively at 50 mg/kg chloroquine showed a mean survival time of 17 days and at 100 mg/kg, 22 days. Thus, the performance of compound 1 was not significantly better than that of the negative controls, despite the fact that it had sufficient exposure to ensure that parasites should have been killed.

For compound 4 dosed at 100 mg/kg, the mean survival time was 9 days. Two mice died on day 6, 4 on day 7, and one on days 13 and 18. The mean parasitemia on day 6 for the surviving mice was 33.3%. At 200 mg/kg, the mean survival time was 10 days. Three mice died on day 6, 3 on day 7, and one on day 22. The average parasitemia of the surviving mice on day 6 was 44.5% and the lone surviving mouse had 28.4% parasitemia on day 13 and 50.4% on day 20. Thus, compound 4 had some efficacy in suppressing parasitemia and life extension, but did not provide durable cures.

Although methyldipheny-piperazine compound 1 was more potent in vitro, piperidine compound 4 showed slightly improved in vivo efficacy, especially at higher doses. The slightly improved activity of 4 could be due to differing sensitivity of the P. berghei to the compounds relative to P. falciparum, or the increased plasma concentration on initial administration as seen above. Overall the efficacy and potency of the compounds agree qualitatively with the pharmacokinetic behaviors and potencies observed in earlier studies.

Conclusions

Evaluation of the compounds from the propafenone hit-to-lead campaign and comparison of the in vitro and in vivo properties has led to some insights in to how to proceed with this compound series. Although in vitro potency is acceptable, at a level comparable to early leads from other programs, there is poor in vivo efficacy. This is attributable mostly to the poor drug-like properties of the series that limit exposure. Use of the piperidine moiety appears to improve physical properties although it leads to modest CYP2D6 inhibition. Further exploration of both the lower part of the molecule and the linker between the A and B rings could lead to greater increases in potency and compounds with improved permeability and solubility, while retaining the reduced ion channel activity already achieved. Clearly the most important challenge will be to raise exposure and/or increase potency while maintaining acceptable toxicology.

Experimental

Potency

Growth of parasites and IC₅₀ determinations

The two P. falciparum strains were used in this study, 3D7 and K1, were provided by the MR4 Unit of the American Type Culture Collection (ATCC, Manassas, VA). Asynchronous parasites were maintained in culture based on the method of Trager.¹⁸ Parasites were grown in presence of fresh group O-positive erythrocytes (Lifeblood Memphis, TN) in Petri dishes at a hematocrit of 4-6% in media consisted of RPMI 1640 supplemented with 0.5% AlbuMAX II, 25 mM HEPES, 25 mM NaHCO₃ (pH 7.3), 100 μg/mL hypoxanthine, and 5 μg/mL gentamycin. Cultures were incubated at 37° C in a gas mixture of 90% N₂, 5% O₂, and 5% CO₂. For IC₅₀ determinations, 20 μL of RPMI 1640 with 5 μg/mL gentamycin were dispensed per well in an assay plate (Corning 8807BC 384-well microtiter plate). 40 nL of each compound, previously serial diluted in a separate assay plate (Corning 3657 384-well white polypropylene plate), were dispensed in the assay plate followed by 20 μL of a synchronized culture suspension (2% rings, 10% hematocrit), thus giving a final hematocrit and parasitemia of 5% and 1%, respectively. Assay plates were incubated for 72 h and the parasitemia were determined by a method previously described.¹⁹ Briefly, 10 μL of the development solution (10X Sybr Green I, 0.5% v/v triton, 0.5 mg/ml saponin, in RPMI) was added per well, assay plates were shaken for 30 s, incubated in the dark for 4 h, then read with the Envision spectrophotomer at Ex/Em 485nm/535nm. EC₅₀’s were calculated with the RISE (Robust Investigation of Screening Experiments) software.

Cytotoxicity

BJ, HEK293, Hep G2, and Raji cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and were cultured according to recommendations. Cell culture media were purchased from ATCC. Cells were routinely tested for mycoplasma contamination using the MycoAlert Mycoplasma Detection Kit (Lonza). Exponentially growing cells were plated in Corning 384-well white assay plates, and incubated overnight at 37 ºC in a humidified incubator with atmosphere controlled at 5% CO₂ and 100% humidity. DMSO inhibitor stock solutions were added the following day to a final concentration of 25 μM, 0.25% DMSO, and then diluted 1/3 for a total of ten testing concentrations. Cytotoxicity was determined following a 72 h incubation using Promega Cell Titer Glo Reagent according to the manufacturer’s recommendation. Luminescence was measured on an Envision plate reader (Perkin Elmer)

Solubility

Solubility assays were carried out on a Biomek FX lab automation workstation (Beckman Coulter, Inc., Fullerton, CA) using μSOL Evolution software (pION Inc., Woburn, MA). The detailed method is as follows: 10 μL of compound stock was added to 190 μL 1-propanol to make a reference stock plate. 5 μL from this reference stock plate was mixed with 70 μL 1-propanol and 75 μL phosphate buffered saline (PBS, pH 7.4 and 4) to make the reference plate, and the UV spectrum (250 nm – 500 nm) of the reference plate was read. 6 μL of 10 mM test compound stock was added to 600 μL PBS in a 96-well storage plate and mixed. The storage plate was sealed and incubated at room temperature for 18 hours. The suspension was then filtered through a 96-well filter plate (pION Inc., Woburn, MA). 75 μL of filtrate was mixed with 75 μL 1-propanol to make the sample plate, and the UV spectrum of the sample plate was read. Calculation was carried out by μSOL Evolution software based on the AUC (area under curve) of UV spectrum of the sample plate and the reference plate. All compounds were tested in triplicate.

Permeability assay

The Parallel Artificial membrane Permeability Assay (PAMPA) was conducted on a Biomek FX lab automation workstation (Beckman Coulter, Inc., Fullerton, CA) using the PAMPA Evolution software (pION Inc., Woburn, MA). The detailed method is described as follows: 3 μL 10 μM test compound stock was mixed with 600 μL of SSB (system solution buffer, pH 7.4 or 4, pION Inc., Woburn, MA) to make diluted test compound. 150 μL of diluted test compound was transferred to a UV plate (pION Inc., Woburn, MA) and the UV spectrum was read as the reference plate. The membrane on pre-loaded PAMPA sandwich (pION Inc., Woburn, MA) was painted with 4 μL GIT lipid (pION Inc., Woburn, MA). The acceptor chamber was then filled with 200 μL ASB (acceptor solution buffer, pION Inc., Woburn, MA), and the donor chamber was filled with 180 μL diluted test compound. The PAMPA sandwich was assembled, placed on the Gut-box and stirred for 30 minutes. The aqueous Boundary Layer was set to 40 μm for stirring. The UV spectrum (250-500 nm) of the donor and the acceptor were read. The permeability coefficient was calculated using PAMPA Evolution software (pION Inc., Woburn, MA) based on the AUC of the reference plate, the donor plate, and the acceptor plate. All compounds were tested in triplicate.

Liver microsomal stability

0.633 mL of mouse liver microsomes (20 mg/mL, female CD9 mice, Fisher Scientific, #NC9567486) or human liver microsomes (20 mg/mL, 200 pooled mixed gender, Fisher Scientific #50-722-552) was mixed with 0.051 mL of 0.5 M EDTA solution and 19.316 mL potassium phosphate buffer (0.1M, pH 7.4, 37°C) to make 20 mL of liver microsome solution. 1 part of 10 mM DMSO compound stock was mixed with 4 part of acetonitrile to make 2 mM diluted compound stock in DMSO and acetonitrile. 29.1 μL of the diluted compound stock was added to 2.3 mL of liver microsomal solution and vortexed to make a microsomal solution with compound. 180 μL of the microsomal solutions with different compounds were dispensed into respective rows of a 96-well storage plate (pION Inc., MA, #110323). For 0 hour time point, 450 μL pre-cooled (4 ºC) internal standard (10 μM warfarin in methanol) was added to the first three columns before the reaction starts. 1.25 mlL of microsome assay solution A (Fisher Scientific, #NC9255727) was combined with 0.25 mL of solution B (Fisher Scientific, #NC9016235) in 3.5 mL of potassium phosphate buffer (0.1 M, pH 7.4). 45 μL of this A+B solution was added to each well of the 96-well storage plate (reaction plate). Liquid in the first 3 columns was moved to another storage plate (quenched plate). The reaction plate was then sealed and incubated at 37 ºC, shaken at a speed of 60 rpm. The solutions were sampled at 0.5 hr, 1 hr, and 2 hr time points. At each time point, 450 μL of pre-cooled internal standard was added to 3 rows in the reaction plate, and the liquid was then transferred to the quenched plate. The quenched plate was then centrifuged (model 5810R, Eppendorf, Westbury, NY) at 4000 rpm for 20 minutes. 200 μL supernatant was then transferred to a 96-well plate and analyzed by UPLC-MS (Waters Inc., Milford, MA). The compounds and internal standard were detected by SIR. The log peak area ratio (compound peak area / internal standard peak area) was plotted against time and the slope was determined to calculate the elimination rate constant [k = (-2.303) * slope]. The half life (hour) was calculated as t (1/2) = 0.693 / k.

CYP Inhibition

Test articles were incubated with model CYP substrates (indicated in parentheses in column headings), human liver microsomes and cofactors for 20 minutes at 37° C. Incubations containing known CYP inhibitors (listed in the first column) were also included. Formation of metabolites was measured by LC-MS/MS and compared to control incubations (incubation of substrates with microsomes and cofactors, no test articles or inhibitor 

In Vivo PK

Female CD-1 mice (Charles River Laboratories, Wilmington, MA) aged 8 weeks (25.0 ± 1.2 g) were housed in hanging polycarbonate cages. Animals were provided food and water ad libitum. The dose formulations were a suspension in 0.5% methylcellulose in sterile water and were administered by oral gavage (10 mL/kg). Blood was collected from three mice at each time point (5 min to 24 hr) and two blood collections were made per mouse. Blood samples were mixed with K3EDTA anticoagulant, processed to plasma using standard methods, and stored frozen (–70°C) until analysis. Protocols for all animal studies were approved by the Institutional Animal Care and Use Committee. The studies were in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals (U.S. National Institutes of Health).

Sample Processing and Analysis

Plasma sample aliquots (50 μL) aliquots were transferred to microfuge tubes containing acetonitrile (200 μL) to precipitate the plasma proteins. These mixtures were vortexed for 10 min and centrifuged (18,000 g for 5 min). The resulting supernatants were transferred to new tubes, evaporated under vacuum, and reconstituted with 50 μL of 10 mM Na2EDTA in water containing 50 ng/mL of the internal standard for the assay (warfarin). The reconstituted samples were centrifuged (18,000 g for 5 min), and transferred to HPLC vials fitted with glass inserts for liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis.

Pharmacokinetics Analysis

The plasma pharmacokinetics of the test compounds 1-6 were determined using WinNonlin Professional (v 5.2) with noncompartmental analysis. The curve fitting of the terminal phase slope used uniform weighting and area under the concentration-time curve (AUC) was calculated using the linear up/log down trapezoidal method. Reported parameters were observed maximum plasma concentration (Cmax), time to Cmax (Tmax), AUC extrapolated to infinity (AUCinf), clearance (Cl/F), and volume of distribution (V/F). Time points from the terminal elimination phase were selected manually for curve fitting analysis and a minimum of three non-zero plasma concentrations after the Tmax were required.

In Vivo Efficacy of 1 and 4

Animals and Parasites

All experiments were conducted in Swiss outbreed (ICR) female mice weighing 15–20 g, purchased form Harlan (N. America). The animals were maintained in cages in an animal facility with alternative light and dark cycles in pellet food and tap water ad libitum. Plasmodium berghei (NK-65) were maintained in mice by serial passaging of infected blood from female donors until the experiment was initiated. Animal Experiment protocols were approved by University of South Florida IACUC and experiments were conducted in accordance to animal care policies.

In Vivo Efficacy Study and Treatment

The oral antimalarial efficacy of selected propafenone analogues 1 and 4 compounds were tested in P. berghei (NK-65) infected ICR mice by the Thompson’s test¹⁷. Briefly, the experiment was initiated by inoculating 1 X 10⁶ infected red blood cells from donor mice in plasma at Day 0. Patent infection was confirmed by making tail vein blood smears on Day 3, before dosing the test compounds. The animals were treated with single oral dose of either the test compound or reference drug chlorquine with a concentration ranging from 50–500 mg/kg on days 3, 4 and 5 post infection. The drugs were dissolved in 0.5% hydroxyethylcellulose (HEC) and the control mice received only the vehicle. The condition of the mice was monitored daily and the survival was recoded. Parasitemia was monitored by preparing thin blood films on Day 3 and 6 post inoculation, then at weekly intervals (days 13, 20 and 27) through day 30. Parasitemia was determined by microscopically observing at least 1000 Giema stained cells (magnification, X1,000).

Abbreviations

WHO

World Health Organization

MMV

Medicines for Malaria Venture

HTS

High Throughput Screening

SAR

Structure Activity Relationship

hERG

human Ether-à-go-go Related Gene

TI

Therapeutic Index

MTD

Maximum Tolerated Dose

PK

Pharmacokinetic

CYP

Cytochrome P450

ADMET

Absorption, Distribution, Metabolism, Excretion, and Toxicity

AUC

Area Under The Curve

C_max

Maximum Plasma Concentration

t_1/2

elimination half-life

t_max

time to maximum concentration