malaria is a disease caused by parasites from the genus plasmodium and is transmitted to a human host when a vector , an infected female anopheles mosquito , bites the host . p. falciparum and p. vivax are responsible for most malaria infections , which has become one of the major public health problems in many regions of africa , asia , and latin america . the world health organization ( who ) estimated 198 million cases of malaria and 584 000 deaths occurred globally in 2013 with the most occurrence in africa . resistance to antimalarials has been reported in both p. falciparum and p. vivax , but their resistance pattern differs and requires the development of new candidates of effective antimalarial drugs . younis et al . [ 6 , 7 ] identified 3,5-diaryl-2-aminopyridines as potential antimalarial drug candidates based on in vivo efficacy data obtained from a p. berghei infected mouse model . these compounds were identified from an image - based high - throughput screening of a biofocus dpi softfocus kinase library of promising selective in vitro antiplasmodial hits . a number of active compounds with varying aryl groups at positions 3 and 5 of the aminopyridine core were identified and analogues were synthesized in order to explore the structure - activity relationship ( sar ) around aryl substitution . some of these analogues showed good oral bioavailability in a pharmacokinetic study performed in rats , identifying them as potential clinical candidates . this led to the synthesis of new derivatives of 3,5-diaryl-2-aminopyridines with the aim of overcoming liabilities such as the human ether -  - go - go - related gene ( herg ) activity , which was identified in an earlier study as a property that is not ideal for a clinical candidate [ 5 , 6 ] . compounds 1 and 2 ( figure 1 ) are derivatives of compound 3 , an active 3,5-diaryl-2-aminopyridine antimalarial ( figure 1 ) . studies conducted by gonzalez cabrera et al .   presented data that showed the potential antimalarial activity of these derivatives when tested in vitro against chloroquine - sensitive and chloroquine - resistant p. falciparum strains displayed activity in a p. berghei infected mouse model . these compounds also showed metabolic stability and reduced herg activity compared to the parent , that is , compound 3 . dimethyl sulfoxide ( merck ) , phosphate buffered saline tablets ( sigma ) , and heparin - coated minicollect plasma tubes ( lasec sa ) were obtained . male and female c57bl/6 mice ( 2030  g ) were obtained from the university of cape town medical school animal unit . the mice were housed in ventilated cages at room temperature ( approximately 22c ) with a constant supply of food and water and were monitored twice daily . the study was authorized by the university of cape town 's faculty of health science animal research ethics committee before its commencement ( reference number 012/020 ) . the compounds ( figure 1 ) were dissolved in dmso and diluted further with phosphate buffered saline ( pbs ) to give a final dmso concentration of 1% . comprehensive pk studies were performed on groups of five animals to determine the pharmacokinetic properties of the test compounds . this involved the oral dosing ( 200  l ) of 10-week - old c57bl/6 male mice at 20  mg / kg and intravenous dosing ( 100  l ) under anesthesia at 4  mg / kg via the dorsal penile vein . for the oral dosage a gavage needle was used for the administration of test compounds directly into the lower esophagus or stomach . blood samples ( approximately 30  l ) were collected serially by needle prick on the tail vein near to the tip of the tail at 0 , 0.17 , 0.5 , 1 , 2 , 3 , 5 , 7 , and 9 hours after dosing . the collected blood samples were placed on ice immediately after sampling and were frozen at 80c until analyzed . the blood samples were brought to room temperature and then mixed by vortexing to ensure homogeneity . twenty microliters of blood was mixed with 50  l milli - q water and 150  l acetonitrile . the sample was vortexed for 15 seconds , sonicated for 10 minutes , and centrifuged at 13000  g for 5 minutes . the supernatant was transferred to a flat - bottom glass insert and placed in a glass vial for analysis . the liquid chromatography ( lc ) system employed was an ultrafast liquid chromatography ( uflc ) system ( shimadzu ) and the separation of the compounds was performed on a phenomenex , luna 5  m pfp(2 ) , 100   , and 50  mm  2  mm analytical column . for method development , the compounds were prepared at 1  mg / ml stock in methanol and were further diluted with 50% acetonitrile : 0.1% formic acid to make a final concentration of 1  g / ml . analyst software version 1.5.2 ( ab sciex ) was employed for data acquisition , peak - area integration , and quantitation of compounds in blood samples . the mobile phase a consisted of 0.1% formic acid in water ( v / v ) and mobile phase b consisted of acetonitrile . the flow rate was set at 500  l / min and the temperature of the column was maintained at 40c . for the separation of the compounds the mobile phase was increased from 5% to 95% b over 2 minutes and returned to 5% b within 0.1 minutes and then equilibrated for 3 minutes . calibration curves were derived in each analytical run in duplicate and were employed to extrapolate the concentration of the compounds in the blood samples . the ms system consisted of an ab sciex 3200 q - trap mass spectrometer which was operated at unit resolution in the multiple reaction monitoring ( mrm ) mode . the calibration range for all the compounds was between 7.8 and 1000  ng / ml and the accuracy ( % nom ) for the calibration curves was between 90.31 and 104.0% . noncompartmental analysis was performed on each individual set of data using pk solutions 2.0 pharmacokinetic analysis software ( summit research services , montrose , usa ) , which uses an automated excel - based program . the following pk parameters were calculated using pk solutions equations listed in program ( http://www.summitpk.com/equations/equations.htm ) : maximum blood concentration ( cmax [ m ] ) and corresponding time ( tmax [ min ] ) , apparent terminal half - life ( t1/2 [ min ] ) , total exposure ( auc0 [ mmin ] ) , volume of distribution ( vss [ l / kg ] ) , blood clearance ( cl [ l / min / kg ] ) , and percentage oral bioavailability ( % ba ) . a pharmacokinetic study was performed on two 2-aminopyridine compounds , namely , compounds 1 and 2 . five mice were used for each route of administration , where each compound was dosed at 20  mg / kg orally and 4  mg / kg intravenously ( iv ) . the mean concentration versus time profiles of compounds 1 and 2 obtained after the mice were dosed is given in figure 2 . the pharmacokinetic parameters for the oral and iv groups are presented in table 2 . after administering 20  mg / kg of compound 1 orally , a maximum concentration of 1.915  0.03  m was reached within 1 hour with a moderate terminal half - life of 2.53  0.1 hours , which was longer than the iv half - life of 0.87  0.03 hours . the oral auc0 was 359.3  97 and the iv auc was 103.4  11  mmin , respectively . a high volume of distribution of 9.51  1  l / kg and a high clearance rate of 83.6  12  ml / min / kg were observed . compound 2 reached a maximum concentration of 3.34  0.2  m within 30 minutes and had moderate terminal half - life of 2.96  0.2 hours , which was longer than a half - life of 0.68  0.6 hours for the intravenous dose . auc0 for the mice dosed orally was 313.6  30 and 105.1  23  mmin for mice dosed intravenously . the volume of distribution was very high at 27  1.3  l / kg with a high clearance rate of 92.6  19  ml / min / kg . the two compounds evaluated in this study are potent antimalarials derived from 3,5-diaryl-2-aminopyridine ( compound 3 ) that was identified as a potential clinical trial candidate for antimalarial treatment in a previous study [ 5 , 6 ] . compounds 1 and 2 were synthesized in order to minimise the liabilities such as herg activity associated with compound 3 . the activity of the derivatives was evaluated in another study and improved ic50 values were observed in the range from 7 to 14  nm for p. falciparum chloroquine - sensitive strains ( d10 and 3d7 ) and chloroquine - resistant strains ( dd2 and k1 ) when comparing to the parent compound 3 with ic50 values in the range from 16 to 194  nm . this study was designed , therefore , to evaluate the pharmacokinetic properties of these two derivatives in a mouse model . both compounds 1 and 2 data in table 2 showed a rapid absorption with maximum concentrations reached within 1 hour after drug administration . the high volume of distribution and a high clearance rate may have influenced the short half - life of these compounds , which range between 2.5 and 3 hours for mice dosed orally and between 0.68 and 0.87 hours for mice dosed intravenously . even though the bioavailability of compounds 1 and 2 was high , it is a parameter that determines the amount of compound reaching the systemic circulation , which in turn may affect the pharmacological response . these parameters include an increased clearance rate and a significant reduction in their half - life . a pharmacokinetic study of compound 3 was performed on male sprague dawley rats with a clearance rate of 18.3  ml / min / kg and apparent t1/2 values of 6.5 and 8 hours for rats dosed orally and intravenously , respectively . the oral and intravenous auc values of compound 3 were 6880 and 789  mmin , respectively , with an oral bioavailability of > 100% . unfortunately , the pk parameters presented in this study for compounds 1 and 2 could not be compared to those of the parent compound 3 because of the physiological differences between the two species . therefore , the data presented in this study can provide a baseline for further exploration of more derivatives in order to optimize the pharmacokinetic properties while retaining high potency in vitro and metabolic stability .
malaria caused by plasmodium falciparum is responsible for approximately 80% of the incidence and 90% of deaths which occur in the world health organization ( who ) african region , with children and pregnant women having the highest incidence . 
 p. falciparum has developed resistance , and therefore new effective candidate antimalarial drugs need to be developed . 
 previous studies identified 3,5-diaryl-2-aminopyridines as potential antimalarial drug candidates ; therefore , derivatives of these compounds were synthesized in order to improve their desired properties and pharmacokinetic ( pk ) properties of the derivatives were investigated in a mouse model which was dosed orally and intravenously . 
 collected blood samples were analyzed using liquid chromatography coupled to mass spectrometer ( lc - ms / ms ) . 
 the mean peak plasma level of 1.9  m was obtained at 1 hour for compound 1 and 3.3  m at 0.5 hours for compound 2 . a decline in concentration 
 was observed with a half - life of 2.53 and 0.87 hours for compound 1 in mice dosed orally and intravenously , respectively . for compound 2 a half - life of 2.96 and 0.68 hours was recorded . 
 the bioavailability was 69% and 59.7% for compound 1 and compound 2 , respectively .