drug resistance to established antimalarials 
 such as chloroquine 
 is driving the rise in global mortality due to malaria . malaria is responsible 
 for roughly one million deaths annually , and as such novel inhibitors active against new parasite targets 
 are urgently required in order to sustain and develop treatments against 
 malaria . as described in the previous 
 companion paper in this issue ( doi : 10.1021/jm201179h ) , a program was undertaken to identify 
 hit compounds against nadh : ubiquinone oxidoreductase ( pfndh2 ) , a novel 
 enzyme target within the malaria parasite plasmodium falciparum . a number of these compounds were also 
 found to target the bc1 complex of p. falciparum giving these drugs a dual mode of action . pfndh2 has only one known inhibitor , n - hydroxy-2-dodecyl-4(1h)quinolone ( hdq ) , and this was used along with a 
 range of chemoinformatics methods in the rational selection of 17  000 
 compounds for high - throughput screening ( hts ) . several distinct chemotypes were identified and briefly examined 
 leading to the selection of the quinolone core as the key target for 
 structure  activity relationship ( sar ) development and subsequent 
 identification of ck-2 - 68 as a lead for further development . our initial studies focused on compounds with mono aryl groups 
 at the 2-position ; however it became rapidly apparent that activity below 
 500 nm against the 3d7 strain of p. falciparum was 
 not possible . a progression toward the close hdq analogues where a 
 longer biaryl / phenoxy biaryl replaced the metabolically vulnerable 
 hdq - side chain improved both antimalarial and pfndh2 activity . a series 
 of structural modifications including the introduction of a methyl 
 substituent at the 3-position led to the generation of over 60 compounds 
 as exemplified by 2 ( ck-2 - 68 ) with an activity of 31 nm 
 against 3d7 and 16 nm against pfndh2 ( figure 1 ) . it was apparent from preliminary animal studies that clogp needed 
 to be reduced and aqueous solubility needed to be enhanced in order 
 to administer the drug in a suitable vehicle without the need for 
 a pro - drug approach . introduction of various heterocycles into the 
 quinolone side chain led to the selection of a series of compounds 
 containing a pyridine group within the side chain . incorporation of 
 a pyridine group reduces clogp , improves aqueous solubility , and allows 
 the possibility of salt formation . further strategies investigated 
 included the use of polar heterocycles in the side chain , use of protonatable 
 groups within the side chain , extending the terminal group using polar 
 heterocycles and the placement of a polar group centrally in the side 
 chain with a lipophilic group at the terminal end . mono aryl quinolones 
 identified as hits from high - throughput screen 
 and initial sar work . investigations into possible 
 solutions to reduce clogp revealed 
 that the incorporation of a heterocycle into the side chain was imperative 
 to achieving this . it was apparent from 
 literature searches that the chemistry used to achieve this would 
 be more easily facilitated if there was no linker between the two 
 rings within the side chain . with this in mind , we undertook the synthesis 
 of some of the key bisaryl compounds known to have good activity ( see 
 previous companion paper in this issue ) 
 but with no linker between the aryl rings rather than a ch2 or o linker to check activity was maintained . the synthesis of these compounds is described in the following schemes 
 along with the heterocyclic compounds . initially , the incorporation of a pyridine ring into the side 
 chain 
 was targeted , and the optimal a ring and terminal aryl ring substituents 
 investigated . the methods used to synthesize these compounds can be 
 seen in schemes 13 . for measured solubility values of select compounds , please see 
 table s1 in supporting information . aldehyde 3 was used in a grignard 
 reaction to give 
 alcohol 4 in 6988% yields . where aldehyde 3 was not commercially available , the aldehydes were synthesized 
 in house ( see supporting information ) . oxazoline 7 was prepared from the respective isatoic anhydride 6 in yields of 3060% . in the majority of compounds the isatoic 
 anhydrides were commercially available ; when this was not the case 
 they were synthesized ( see supporting information ) . reaction of oxazoline 7 with ketone 5 in the presence of ptsa gave the desired 8a  z and 9a  c in 2369% yields . quinolones 9b and 9c were synthesized as precursors to quinolones 12a  f as seen in scheme 3 . as can be seen in table 1 this methodology was used not only to insert a pyridine into the side chain , it was 
 also used to incorporate a small number of bipyridyl , o - linked , c 
 linked , and other heterocyclic groups into the side chain . the presence of an alcohol both in the a ring of the 
 quinolone 
 core and in the terminal aryl group of the side chain was investigated . 
 in the case of the a ring of the quinolone core compounds with a hydroxyl 
 group at the 6(10a ) and 7(10b ) positions were synthesized from their respective methoxy compounds 
 using boron tribromide in 49% and 68% yields ( scheme 2 ) . hydroxyl groups at the 3(11a ) and 4(11b ) positions on the terminal phenyl ring were achieved using 
 a similar method in 91% and 81% yields , respectively . where synthesis of the bicyclic side chain was 
 not possible prior 
 to the quinolone formation step , it was often possible to carry out 
 the suzuki reaction at the final step ( scheme 3 ) . this was advantageous 
 when investigating the scope of the z group attached to the terminal 
 ring . quinolones 9a or 9b were reacted with 
 boronic acid under the suzuki conditions described below to give quinolones 12a  f in 5070% yields ( table 2 ) . the nature of the group at the 3-position was also 
 investigated 
 for both bisaryl no - linker compounds and heterocyclic quinolones . ester and methyl alcohol groups were synthesized by reaction of amine 
 ester 13 ( for synthesis , please see supporting information ) with oxalyl chloride . the product 
 from this reaction was used crude and heated in dowtherm to give iodo 
 quinolone 15 , and subsequent suzuki reaction gave bisaryl 
 ester quinolone compounds 16a and 16b in 
 53% and 75% yields . reduction of quinolone 16b to methyl 
 alcohol 17 was achieved using libh4 in 66% 
 yield . in the case of the heterocyclic side chain , 
 ester 19 cyclisation 
 to the quinolone 21 was achieved by reaction with nah 
 in dmf in 28% yield . reaction of 25 with aldehyde 5 in the presence of catalytic 
 piperidine gave alkene 26 in 93% yield . conversion to 
 quinolone 27 was achieved by reaction with bromine at 
 room temperature , followed by refluxing with sodium methoxide in meoh / dioxane , 
 and subsequently refluxing in concentrated hcl to give 3-methoxyquinolone 27 in 6% yield ( scheme 6 ) . incorporation of a morpholine group as the terminal 
 ring in side 
 chain was pursued to allow the possibility of salt formation . bromo benzyl compound 28 was reacted 
 with morpholine in the presence of potassium carbonate to give benzyl 
 morpholine 29 in 96% yield . reaction with etmgbr and 
 cui , then subsequently hcl , gave ethyl ketone 30 in 77% 
 yield . this was then coupled with oxazoline 7 in 30% 
 yields to give quinolones 31a and 31b ( scheme 7 ) . other functionality in the terminal ring has 
 been investigated 
 such as pyridine ring ( 8y ) , difluoro cyclohexyl ring 
 ( 8z ) , and a tetrahydropyran ( 9a ) . quinolone 35 was synthesized as a direct analogue of 8 containing 
 a nitrogen in the a ring . ethyl ketone 32 was reacted 
 with trimethyl orthoformate in the presence of ptsa to give dimethyl 
 acetal 33 in 70% yield . heating acetal 33 with pyridyl amine 34 in dowtherm a gave quinolone 35 in 32% yield ( scheme 8) . synthesis of quinolones with an extended morpholine 
 side chains 
 were also prepared using chemistry similar to that employed in scheme 3 . reaction of 9b / c with benzyl morpholine 
 borane ester gave morpholine quinolones 36a and 36b in 61% and 50% yields ( scheme 9 ) . a series of quinolones were then prepared containing 
 an aryl group 
 with an ethoxy linkage to a heterocyclic ring . this was to further 
 explore the side chain sar and provide us with further information 
 about the length of side chain tolerated . phenol 37 was reacted with chloro 
 ethyl amine 38 in the presence of potassium carbonate 
 to give ethoxy morpholine 39 . morpholine 39 was then reacted with oxazoline 7 as described previously 
 to give quinolones 40a  g ( scheme 10 and table 3 ) . crystals of quinolones 40a ( see figure 2 , ccdc 833299 ) and 40c ( see figure 3 , ccdc 833897 ) were grown and their structures confirmed 
 by 
 x - ray crystallography . replacement of the phenyl group in the side chain 
 with a pyrazole 
 group provides a further dramatic reduction in clogp . compounds 42a and 42b were synthesized using chemistry 
 described previously ( scheme 11 ) . a series of compounds with an extended ethoxy 
 morpholine and piperazine 
 side chains were also synthesized . to incorporate the oxyl - linked 
 side chain bbr3 was the used to demethylate methoxy ketone 43 to give alcohol 44 in 6064% yields . addition of the ethyl morpholine / piperazine subunit was achieved using 
 potassium carbonate to give side chain 45 in 6977% 
 yields . reaction with oxazoline 7 in the presence of 
 triflic acid gave quinolones 46a  d in 4050% 
 yields ( scheme 12 ) . incorporation of protonatable nitrogens into the middle 
 of the 
 side chain with a lipophilic group at the terminal end ( a strategy 
 employed in gsks pyridone bc1 program ) has also briefly been investigated . 
 in house models of similar enzymatic systems ( e.g. , bc1 complex ) have 
 shown that charge is not tolerated close to the headgroup so this 
 would provide side chain extension and possibly improve binding . chloro , 
 bromo quinoline 47 was reacted with boronic ester 48 to give 2-substituted quinoline 49 in 79% 
 yield . bromination of the methyl group of quinolone 49 using nbs and aibn gave 50 in 44% yield . conversion to quinolone 53 was 
 achieved using formic acid in 70% yield ( scheme 13 ) . while our primary focus was to use medicinal 
 chemistry manipulation 
 of the core template to maximize solubility and activity , pro - drug 
 approaches were also briefly examined as detailed in the previous 
 paper . quinolone 8b was 
 reacted with tetrabenzyl pyrophosphate in the presence of nah to give 
 the phosphonate ester 54 in 80% yield . hydrogenation 
 using pd / c gave phosphate pro - drug 55 in 71% yield ( scheme 14 ) . morpholine carbamate pro - drug 56 was made by reacting quinolone 8b with morpholine 
 carbonyl chloride in the presence of potassium tert - butoxide to give the pro - drug in 78% yield ( scheme 15 ) . it was however found that the best activity was achieved 
 with the phosphoric acid salt of lead compounds 8b and 8h . tables 5 , 6 , and 7 show the 
 antimalarial activity of all quinolones synthesized against the 3d7 
 strain of p. falciparum . other sar details of note are that a 
 high degree of substitution in the side chain close to the quinolone 
 core is poorly tolerated as exemplified by 8a ( 940 nm ) 
 suggesting that flexibility of the side chain is key to the activity 
 of these compounds . as seen with previous side chain variations , the 
 4-ocf3 group is the optimal terminal group ( 12a , 59 nm ) . the methyl group is favored as a 3-substituent with me ( 12a , 59 nm ) > ch2oh ( 17 , 162 nm ) 
 > 
 co2et ( 16a , 347 nm ) in terms of activity . variations 
 in the x group show that smaller groups such as cl , f , and oh are 
 well tolerated with larger groups such as cf3 , ocf3 , and so2me not tolerated as exemplified by 8h ( 75 nm ) compared to 8l ( > 1000 nm ) . investigations 
 into the nature of the y group have shown that the presence of an 
 oh group results in loss of activity as seen with 11a and 11b . as a general rule substitution at the 4-position the methyl group again is found 
 to be optimal as a substituent at the 3-position with me ( 8b , 54 nm ) > ch2oh ( 21 , 103 nm ) > co2et ( 22 , 987 nm ) in terms of activity . table 7 shows 
 the antimalarial activity of the other heterocyclic quinolones investigated . 
 from the results below it can be seen that one pyridine ring 8b ( 54 nm ) is favored over two pyridine rings 8v ( 370 nm ) . again 4-ocf3 is the optimal terminal substituent 
 as seen with 8w ( 40 nm ) vs 8x ( 279 nm ) . incorporation of a morpholine / piperazine group generally leads to 
 a loss of activity . having established the whole cell activity of all 
 quinolone compounds 
 they were then tested against the pfndh2 enzyme ( see tables 5 and 6 ) . because of the time - consuming 
 nature of the assay and large volume of parasites 
 needed only a small selection of the most active compounds were then 
 tested against pfbc1 ( see 
 footnote , tables 5 and 6 ) . a large number of the quinolones tested demonstrate nanomolar 
 activity against both pfndh2 and pfbc1 confirming the dual mechanism of action that these compounds possess . from these compounds a selection was tested against the atovaquone 
 resistant tm90c2b strain of p. falciparum ( ic50 for atovaquone is 12 m in this strain ) containing 
 the cytochrome b mutation y268s . additionally a more select range of compounds were 
 tested against 
 the chloroquine resistant strain of p. falciparum , w2 . the sar trends identified from the 3d7 data largely hold true 
 for the w2 data with the presence of a group other than a para ocf3 group causing a reduction in activity 12e ( 88 nm ) and 12f ( 120 nm ) groups reducing 
 activity when compared to 8b ( 50 nm ) . 8b , the phosphate salt of 8b , and both the morpholine and phosphate pro - drugs were tested 
 for 
 in vivo activity using peters standard 4-day test . with 8b some solubility problems 
 were encountered with the use of standard suspension vehicle ( ssv ) 
 as the vehicle ; the compound had to be dosed as a suspension and a 
 reduced % parasite clearance was seen . the use of 5% dmso and 5% etoh 
 in tetraglycol ( det ) , where 8b fully dissolved provided 
 proof of concept that 8b clears the parasite in vivo 
 with 100% parasite kill being achieved at 20 mg / kg . however , for full 
 ed50/ed90 determination the use of ssv was preferred . 
 with both the phosphate salt of 8b and the morpholine 
 prodrug 56 100% parasite kill was seen in ssv . as salt 
 formulation was preferable over a pro - drug approach , formulation as 
 the phosphate salt was employed for full ed50/ed90 determination . the phosphate pro - drug of 8b , compound 55 was successfully dosed in a sodium carbonate solution and 
 100% parasite kill was also seen at 20 mg / kg . four - day suppressive activity of 
 key compounds in male cd-1 mice infected with plasmodium berghei ( ns strain ) mice were exposed to the infection via intraperitoneal 
 injection and then orally dosed with the relevant compound . compound 8b was selected to determine 
 the in vivo 
 activity in terms of ed50/ed90 . formulation as the phosphate salt 
 offers greater in vivo activity as is demonstrated by the marked improvement 
 in activities when comparing the parent compound 8b ( ed50/ed90 = 12.75/27.30 mg / kg ) to its phosphate salt 
 ( ed50/ed90 = 1.87/4.72 mg / kg ) . against the same 
 strain , chloroquine had an ed50/ed90 of 3.3 
 mg / kg/4.6 mg / kg and artemether was active at 3.1 mg / kg/5.8 mg / kg in 
 terms of ed50/ed90 indicating that these molecules 
 have potency similar to proven antimalarials in this model . measured 
 solubility values as depicted in table s1 ( supporting 
 information ) confirm that while solubility is a challenge we 
 still face , the incorporation of a heterocyclic ring does improve 
 solubility at low ph and that solubility is further enhanced when 
 compounds are formulated as the phosphate salt . no significant cytotoxicity was observed 
 for 8b at any concentration ( cc50 > 50 m ) in 
 hepg2 
 cells . 8b was 
 incubated at a concentration of 1 m with human liver microsomes 
 ( 1 mg / ml ) in the presence of nadph for 0 , 10 , 30 , and 60 min . greater 
 than 60% of the parent compound was present after 60 min incubation . the in vitro half - life for 8b was shown to be 96.7 min , 
 with an intrinsic clearance value of 1.78 ml / min / kg . upon oral 
 dosing in the rat of the morpholine pro - drug ( 20 mg / kg ) , the observed 
 pharmacokinetic parameters in plasma for the parent compound , 8b , were cmax 8.1 g / ml , tmax 7.0 h , half - life ( t1/2 ) of 20.3 h , a volume of distribution vd of 2875.6 ml / kg , an area under the curve of auc0-t 167.2 
 g.h / ml and a calculated total clearance clt was 98.0 ml / h / kg 
 ( see supporting information ) . the pharmacokinetic 
 features of the 8b salt were also favorable ; the observed 
 parameters for the parent compound , 8b following oral 
 administration ( 20 mg / kg ) were cmax 3.7 
 g / ml , tmax 7.0 h , t of 9.9 h , vd 3970.8 ml / kg , auc0-t 69.3 g.h / ml and 
 a clt of 276.3 ml / h / kg ( see supporting information ) . these pharmacokinetic parameters are consistent with a once - daily 
 oral dosing target product profile . to conclude , a 46 step synthesis 
 of a range of heterocylic 
 quinolones with potent antimalarial activity both in vitro and in 
 vivo have been reported . several compounds within this series have 
 been proven to be potent against the novel pfndh2 enzymatic target . representative analogue sl-2 - 25 ( 8b ) demonstrates outstanding 
 antimalarial activity , reduced clogp , and improved solubility . 8b has antimalarial activity against the 3d7 strain of p. falciparum of 54 nm , pfndh2 activity of 15 nm and an 
 ed50/ed90 of 1.87/4.72 mg / kg when formulated 
 as the phosphoric acid salt . all reactions that employed moisture 
 sensitive reagents were performed 
 in dry solvent under an atmosphere of nitrogen in oven - dried glassware . all reagents were purchased from sigma aldrich or alfa aesar chemical 
 companies , and were used without purification . thin layer chromatography 
 ( tlc ) was carried out on merck silica gel 60 f-254 plates and u.v . flash column chromatography was performed on icn ecochrom 60 ( 32 - 63 
 mesh ) silica gel eluting with various solvent mixtures and using an 
 air line to apply pressure . nmr spectra were recorded on a bruker 
 amx 400 ( h , 400 mhz ; c , 100 mhz ) spectrometer . chemical shifts are described in parts per million (  ) downfield 
 from an internal standard of trimethylsilane . mass spectra were recorded 
 on a vg analytical 7070e machine and fisons trio spectrometer using 
 electron ionization ( ei ) and chemical ionization ( ci ) . all compounds 
 were found to be > 95% pure by hplc unless specified below . the appropriately substituted oxazoline 7 ( 4 
 mmol , 1.0 equiv ) was added to a solution of ketone 5 ( 4 
 mmol , 1.0 equiv ) and para - toluenesulfonic acid ( 20 
 mol % ) in n - butanol ( 10 ml ) . the reaction mixture 
 was heated to 130 c under nitrogen and stirred for 24 h. the 
 solvent was removed under a vacuum and water ( 20 ml ) was added . the 
 aqueous solution was extracted with etoac ( 3  20 ml ) , dried 
 over mgso4 , and concentrated under a vacuum . the product 
 was purified by column chromatography ( eluting with 20% 80% 
 etoac in n - hexane ) to give quinolone 8 . 8b : white solid ( yield 69% ) ; mp 277278 
 c ; h nmr ( 400 mhz , dmso )  11.76 ( s , 1h ) , 
 8.90 ( d , j = 2.1 hz , 1h ) , 8.37  8.31 ( m , 
 2h ) , 8.25 ( d , j = 8.2 hz , 1h ) , 8.17 ( dd , j = 8.2 , 2.2 hz , 1h ) , 8.15 ( dd , j = 7.0 , 
 1.5 hz , 1h ) , 7.65 ( ddd , j = 8.2 , 6.8 , 1.4 hz , 1h ) , 
 7.60 ( d , j = 7.7 hz , 1h ) , 7.55 ( d , j = 8.2 hz , 2h ) , 7.33 ( ddd , j = 8.0 , 6.8 , 1.2 hz , 
 1h ) , 1.96 ( s , 3h ) ; c nmr ( 101 mhz , dmso )  176.98 , 
 155.52 , 149.75 , 144.79 , 139.95 , 138.49 , 137.46 , 131.83 , 130.30 , 129.34 , 
 129.17 ( c-8 ) , 125.37 , 123.50 , 123.22 , 121.70 , 120.35 , 119.18 , 
 118.51 , 115.59 , 12.39 ; hrms ( esi ) c22h16n2o2f3 [ m + h ] requires m / z 397.1164 , 
 found 397.1173 . c22h15n2o2f3 requires c 66.67% , h 3.81% , n 7.07% , found c 
 66.77% , h 3.73% , n 6.98% . 8h : pale yellow solid 
 ( yield 30% ) ; mp 317319 
 c ; h nmr ( 400 mhz , dmso )  11.83 ( s , 1h ) , 
 8.90 ( d , j = 1.8 hz , 1h ) , 8.398.31 ( m , 2h ) , 
 8.24 ( t , j = 8.7 hz , 1h ) , 8.22  8.11 ( m , 
 2h ) , 7.55 ( d , j = 8.2 hz , 2h ) , 7.30 ( dd , j = 10.1 , 2.4 hz , 1h ) , 7.20 ( td , j = 8.8 , 
 2.5 hz , 1h ) , 1.95 ( s , 3h ) ; c ; hrms ( esi ) c22h14n2o2f4na [ m + na ] requires m / z 437.0889 , found 437.0905 . sodium hydride ( 0.57 mmol , 2.5 equiv ) was added at 0 c to a 
 stirred solution of quinolone ( 0.23 mmol , 1.0 equiv ) in dry thf ( 10 
 ml ) . after 1 h , tetrabenzyl pyrophosphate ( 0.19 mmol , 0.8 equiv ) was 
 added and the stirring continued for 20 min . the mixture was filtered 
 and the filtrate concentrated under a vacuum at a temperature below 
 35 c . the residue was dissolved in dcm , washed with nahco3 aq , dried over mgso4 , and concentrated under vacuum 
 to give phosphonate 55 . where necessary the product was 
 purified by flash column chromatography ( eluting with 10% ethyl acetate 
 in n - hexane ) . 55 : pale yellow 
 solid ( yield 70% ) ; mp 257258 c ; h nmr ( 400 
 mhz , dmso )  11.77 ( s , 2h ) , 8.91 ( s , 1h ) , 8.378.27 ( m , 
 3h ) , 8.17 ( dd , j = 6.9 , 1.7 hz , 2h ) , 8.02 ( d , j = 8.4 hz , 1h ) , 7.76 ( t , j = 7.6 hz , 1h ) , 
 7.687.59 ( m , 1h ) , 7.54 ( m , 2h ) , 2.46 ( s , 3h ) ; p nmr ( 162 mhz , dmso )  1.17 , 5.89 . 8b ( 124 mg , 0.31 mmol ) in anhydrous thf was added buok ( 52.7 mg , 0.47 mmol)at room temperature . the mixture 
 was stirred for 1/2 h. 4-morpholinecarbonyl chloride ( 0.05 ml , 0.41 
 mmol ) was added . the mixture was stirred for a further 2 h ( followed 
 by tlc ) . the reaction was quenched with brine and was extracted with 
 ethyl acetate , dried over na2so4 , filtered , 
 and concentrated to an oil . the crude product was purified by column 
 chromatography using 20% ethyl acetate in hexane to give the title 
 compound . 56 : white solid ( 125 mg , yield 78% ) ; 
 mp 150151 c ; h nmr ( 400 mhz , cdcl3 )  8.96 ( dd , j = 2.2 , 0.8 hz , 1h ) , 8.15 ( d , j = 8.4 hz , 1h ) , 8.138.10 ( m , 2h ) , 8.08 ( dd , j = 8.1 , 2.3 hz , 1h ) , 7.917.81 ( m , 2h ) , 7.73 ( ddd , j = 8.4 , 6.9 , 1.4 hz , 1h ) , 7.60 ( ddd , j = 8.2 , 6.9 , 1.1 hz , 1h ) , 7.36 ( d , j = 8.0 hz , 2h ) , 
 3.91 ( m , 2h ) , 3.89 ( m , 2h ) , 3.85 ( m , 2h ) , 3.67 ( m , 2h ) , 2.40 ( s , 3h ) ; 
 hrms ( esi ) c27h23n3o4f3 [ m + h ] requires m / z 510.1641 , found 510.1637 . ic50s 
 ( 50% inhibitory concentrations ) were calculated by using the four - parameter 
 logistic method ( grafit program ; erithacus software , united kingdom ) pfndh2 activity was 
 measured using an end - point assay in a 384-well plate format . final 
 assay concentrations used were 200 m nadh , 10 mm kcn , 1 g / ml 
 f571 membrane , and 20 m decylubiquinone 
 ( dq ) . a pre - read at 340 nm was obtained prior to the addition of dq 
 to initiate the reaction followed by a post - read at 1 min . the agreed qc pass criteria 
 was z > 0.6 and signal / background > 10 . compounds were 
 selected 
 by the described chemoinformatics algorithms from the biofocus dpi 
 compound library ( galapagos company ) . p. falciparum cell - free 
 extracts were prepared from erythrocyte - freed parasites as described 
 previously , and recombinant pfndh2 was 
 prepared from the escherichia coli heterologous expression 
 strain f571 . pfndh2 and bc1 activities 
 were measured as described previously . in vivo efficacy studies were measured 
 against p. berghei ( ns - strain ) in the standard 4-day test . all in vivo studies were 
 approved by the appropriate institutional animal care and use committee 
 and conducted in accordance with the international conference on harmonization 
 ( ich ) safety guidelines .
following a program undertaken to identify hit compounds 
 against 
 nadh : ubiquinone oxidoreductase ( pfndh2 ) , a novel enzyme target within 
 the malaria parasite plasmodium falciparum , hit to 
 lead optimization led to identification of ck-2 - 68 , a molecule suitable 
 for further development . in order to reduce clogp and improve solubility 
 of ck-2 - 68 incorporation of a variety of heterocycles , within the 
 side chain of the quinolone core , was carried out , and this approach 
 led to a lead compound sl-2 - 25 ( 8b ) . 8b has 
 ic50s in the nanomolar range versus both the enzyme and whole cell p. falciparum ( ic50 = 15 nm pfndh2 ; ic50 = 54 nm ( 3d7 strain 
 of p. falciparum ) with notable oral activity of ed50/ed90 of 1.87/4.72 mg / kg versus plasmodium 
 berghei ( ns strain ) in a murine model of malaria when formulated 
 as a phosphate salt . 
 analogues in this series also demonstrate nanomolar 
 activity against the bc1 complex of p. falciparum providing the potential added benefit of a 
 dual mechanism of action . 
 the potent oral activity of 2-pyridyl quinolones 
 underlines the potential of this template for further lead optimization 
 studies .