malaria is an infectious disease caused 
 by parasites of the genus plasmodium and is a world 
 health crisis of paramount urgency .
malaria was responsible for over 
 200 million cases and 1 million deaths in 2010 alone , primarily affecting developing countries and children under 
 the age of 5 .
although five species of plasmodium parasite are known to infect humans , two species are responsible for the majority of morbidity and mortality : plasmodium falciparum ( pf ) and plasmodium vivax ( pv ) .
the current treatment for malaria is combination therapy , 
 typically comprising artemisinin derivatives and a companion drug 
 such as lumefantrine , mefloquine , or amodiaquine . these drugs ( and 
 the majority of antimalarials ) target only the symptomatic 
 blood - stage forms of the parasite ;
drugs that target additional life 
 stages ( such as asymptomatic liver stage parasites ) are in high demand .
furthermore , resistance to chloroquine is long 
 established and signs of artemisinin resistance 
 have been detected along the eastern and 
 western borders of thailand , compounding 
 the urgent requirement for additional therapeutic agents targeting plasmodium parasites .
although there has been a great 
 deal of funding and expertise directed toward antimalarial drug discovery 
 over the past decade , the majority of therapeutics in clinical development 
 are either elaborations of existing pharmacophores , reformulations / combinations 
 of existing drugs , or novel molecules that function by unknown mechanisms 
 of action . in order to combat resistance 
 and achieve the goal of malaria eradication , a range of therapies 
 targeting a variety of biological mechanisms and parasite life stages 

n - myristoylation is the covalent attachment of 
 myristate , a saturated 14-carbon fatty acid , to the n - terminal glycine 
 of target proteins from the acyl source myristoyl - coenzyme a ( coa ) . 

this transformation is catalyzed by n - myristoyltransferase 
 ( nmt ) , a monomeric enzyme found ubiquitously in eukaryotes .
myristoylation 
 is a widespread modification that occurs co- and post - translationally 
 and has hundreds of putative substrates in vivo .
furthermore , myristoylation of proteins is known to modulate 
 a variety of properties ( such as protein localization and stability ) and is implicated 
 in a variety of critical biological pathways . in the specific context of malaria , several important parasitic 
 proteins have been shown to require myristoylation in order to localize 
 correctly and to carry out their biological functions , and the genetic essentiality of nmt in p. berghei ( pb ) , the infectious species in the murine model of malaria , has 
 been confirmed by conditional knockdown experiments .
furthermore , we recently reported the validation of nmt 
 as an essential and chemically tractable drug target in p. 
 falciparum .
using an integrated chemical biology approach , 
 we identified the nmt substrate proteins in the blood stage of the 
 parasite and demonstrated with small molecule tools that on - target 
 inhibition of myristoylation disrupts the function of multiple specific 
 downstream pathways , resulting in rapid cell death .
herein , we describe the development of a previously described 
 series of benzo[b]thiophene inhibitors into 34c , a potent and selective parasite nmt inhibitor instrumental 
 to the validation of this drug target .
we also report further investigations 
 into the utility of nmt inhibitors in drug - resistant cell lines and 
 liver stage parasites .
previous 
 work reported discovery of a series of benzo[b]thiophene - based 
 inhibitors of p. falciparum ( pf)nmt and p. 
 vivax ( pv)nmt , exemplified by 1 ( figure 1).1 represents 
 a promising starting point for hit to lead development but has only 
 moderate enzyme affinity and high lipophilicity and contains a potentially 
 metabolically labile ester group .
further development therefore focused 
 on removal of this high - risk functionality combined with a 100-fold 
 improvement in enzyme affinity , reduced lipophilicity , and controlled 
 molecular weight .
little is currently known of the potential for toxicity 
 resulting from mammalian nmt inhibition , and previous data have shown 
 that a potent homo sapiens ( hs)nmt inhibitor is not 
 toxic to mice at high doses .
although 
 selectivity over hsnmt is desirable , selectivity at the cellular level 
 was considered the more critical determinant for progression . 2,3-substituted 
 benzo[b]thiophene pfnmt and pvnmt inhibitor 1 and the target profile for the development of this series . 

footnote a : ki values are quoted in place 
 of ic50 values as a means of expressing the inhibitor affinity 
 while correcting for differing michaelis constants ( km ) between enzymes .
enzyme ki values are calculated from the ic50 values using the 
 cheng  prusoff equation , the definition of which is given in 
 the experimental section .
ic50 values are the mean value of two or more 
 determinations , and standard deviation is within 20% of the ic50 .
footnote b : no significant difference in inhibition between 
 hsnmt1 and hsnmt2 isoforms has been observed in this series ; therefore , 
 the hsnmt affinities reported in this work refer to hsnmt1 .
le = [ log(ki)](1.374)/(no . of heavy atoms ) , with clogp determined with chemaxon , 
 which can be obtained from http://www.chemaxon.com / products / calculator - plugins / logp/. the metric ligand efficiency ( le ) 
 has been used extensively in early stage drug development , and its 
 utility has been well documented .
the 
 main criticism of le is the omission of any reference to the lipophilicity 
 of the compound in question .
lipophilicity has been implicated as 
 one of the most important properties of a potential drug molecule ; 
 it is critical to many related properties including solubility , permeability , 
 and distribution . in vivo
, highly lipophilic 
 compounds will be more likely to partition from plasma to organic 
 components such as membranes and proteins , increasing biological promiscuity 
 ( and in turn toxicity ) .
indeed , a recent review on metrics in drug discovery has stated 
  optimizing le and lle ( ligand lipophilic efficiency , pic50  log  p ) in concert is an important 
 success factor for hit and lead optimization in drug discovery projects  .
ligand efficiency dependent lipophilicity 
 ( clogp / le , lelp ) has been proposed as a way to incorporate affinity , 
 lipophilicity , and molecular size into a single metric for the triage 
 of molecules and has been shown to be 
 a strong predictor of druglikeness .
it 
 is proposed that a low lelp will reduce the chance of later stage 
 failure due to nonspecific toxicity ; marketed drugs have an average 
 lelp of approximately 6 , and desirable leads ( le > 0.3 , clogp < 
 3 ) display lelp < 10 . in order to 
 control the lipophilicity and molecular weight of the current series , 
 lelp was used to determine the druglikeness or physicochemical quality 
 of the inhibitors tested . for clarity ,
consideration of the lelp of 1 ( 13.5 , figure 1 ) emphasizes the requirement 
 for significant improvement in the physicochemical profile of this 
 series .
previous work showed that an appropriately placed 3-methoxyphenyl 
 substituent can form  and hydrogen bonding interactions 
 with phe105 and ser319 of pvnmt , respectively .
attempts to replicate these interactions within the benzothiophene 
 series yielded only a small affinity enhancement , but previously reported 
 crystallographic evidence indicated that extension of the linker between 
 the ester and methoxyphenyl moieties might result in improved interactions .
a series of phenethyl esters and amides were 
 therefore synthesized and assayed against a panel of nmts ( scheme 1 , table 1 ) .
reagents 
 and conditions : ( a ) 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide , 
 hydroxybenzotriazole , n , n - diisopropylethylamine , 
 mecn , rt , 18 h , 3657% ; ( b ) benzotriazo-1-yl - oxytripyrrolidinophosphonium 
 hexafluorophosphate , n , n - diisopropylethylamine , 
 dcm , rt , 18 h , 6993% ; ( c ) 10% tfa in dcm ( v / v ) , rt , 2 h , 4297% .
ki values are the mean value 
 of two or more determinations , and standard deviation is within 20% 
 of the ki .
this hypothesis was successfully 
 validated , in that moving from 1 to 4b produced 
 a 6-fold improvement in pfnmt affinity , retained the high pvnmt affinity , 
 and pleasingly had little effect on hsnmt affinity . however , despite 
 this improvement , the accompanying lipophilic methylene unit resulted 
 in only a marginally improved lelp . furthermore , the antiparasitic 
 activity of 4b was reduced compared to 1 .
further sar mimicked that described previously for benzyl 
 esters : adding a second hydrogen bond 
 acceptor ( 4c ) significantly improved affinity but removed 
 all selectivity , resulting in a compound with high affinity against 
 all three enzymes .
esters are strongly preferred over amides ( 4a vs 6a , 4d vs 6b , 4e vs 6c ) , and the active site for pvnmt appears 
 to be more promiscuous than that of pfnmt and hsnmt . intriguingly , 
 increasing the linker length by a further methylene ( 4a vs 4d ) reduced affinity for pfnmt , maintained affinity 
 for pvnmt , and increased affinity for hsnmt .
this reinforces the requirement 
 for the ethyl spacer and indicates that selectivity is achievable 
 through interactions with residues in this area of the enzyme .
it 
 is notable that the antiparasitic activity of these compounds is slightly 
 reduced , with only the high affinity 4c displaying improved 
 activity compared to 1 .
it may be that the increased 
 lipophilicity of this series has reduced the free fraction available 
 for binding .
4c also displays the best lelp score of 
 any analogues tested ; further development shall continue to focus 
 on targeting a lelp of < 10 . the 
 increased linker length between the phenyl moiety and the ester presented 
 an excellent opportunity to investigate replacing the ester moiety 
 with a heterocycle . in earlier analogues
this modification was not 
 explored , since it would have resulted in four contiguous aromatic 
 rings , with likely deleterious effects on the solubility and toxicity 
 profile .
however , the incorporation of an extra methylene unit adds 
 an extra degree of freedom and interrupts the -system , eliminating 
 this potential issue .
crystallography suggested that the interactions 
 of the ester with the enzyme were confined to long - distance -interactions 
 with tyr211 and phe105 . in this scenario
oxadiazoles are common bioisosteric 
 replacements for methylene esters , displaying 
 increased biological stability but with similar size and shape and 
 increased potential for -interactions .
reagents and conditions : ( a ) nh2ohh2o , etoh , rt , 5 h , 9899% ; ( b ) 2 , 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide , hydroxybenzotriazole , 
 dmf , 140 c , 3 h , 1031% ; ( c ) 10% tfa in dcm ( v / v ) , rt , 
 2 h , 1399% ; ( d ) bromoacetonitrile , t - buok , 
 thf , 0 c to rt , 15 min , 88% ; ( e ) tert - butyl 
 4-hydroxypiperidine-1-carboxylate , diisopropyl azodicarboxylate , pph3 , thf , rt 1.5 h , 78% ; ( f ) rch2co2h , 
 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide , hydroxybenzotriazole , 
 mecn , rt , 18 h ; ( g ) 4  molecular sieves , toluene , 18 h , 110 
 c , 5468% over two steps
. replacement of the ester 
 linker with a 1,2,4-oxadiazole produced an increase in affinity for 
 all three enzymes , presumably due to -interactions within the 
 active site and/or increased rigidification of the linker with respect 
 to 4b .
a single methylene between the oxadiazole linker 
 and the phenyl moiety was preferred ( 9a vs 9b ) .
introduction of a m - methoxy substituent improved 
 affinity , although only by a small degree ( 9a vs 9c , 14a vs 14b ) , and replacement 
 of the methoxy group with a weakly hydrogen bonding and lipophilic 
 chlorine reduced affinity for all three enzymes ( 9c vs 9d ) .
interestingly , in 4c addition of the second 
 methoxy substituent produced an increase in affinity against pfnmt 
 and hsnmt , whereas in 9e selectivity is improved with 
 respect to 9c .
although this result was quite promising , 
 the solubility of 9e is very low ( data not shown ) , limiting 
 the potential of this substitution pattern in further development . 
 as with the ester extension ,
this optimization is accompanied by an 
 increase in heavy atom count resulting in only a small increase in 
 lelp , still above the target value of 10 . nonetheless , both isomers 
 of 1,2,4-oxadiazoles appear to be promising bioisosteres for the potentially 
 problematic ester functionality .
the developments detailed 
 above improved enzyme affinity and removed a potentially labile ester 
 group , making the compound series significantly more druglike .
a consideration 
 of lelp across the series highlights that the increase in enzyme affinity 
 from 1 to 9c has been achieved at the expense 
 of increased lipophilicity and molecular weight .
this results in only 
 a small improvement in lelp from 13.5 to 12.5 , still substantially 
 above the desired value of 10 for a promising lead .
the optimization 
 of biological potency at the expense of molecular properties is a 
 common pitfall in drug development that has been associated with clinical 
 attrition .
the next stage in development 
 therefore focused on improving the lelp of the series by improving 
 affinity while reducing lipophilicity and molecular weight .
several strategies for lowering lipophilicity have been described 
 in the recent literature ; one of the most successful is matched molecular 
 pairs analysis , the concept of replacing 
 a moiety with a  matched pair  , a structural analogue 
 that displays favorable physicochemical properties .
one of the most 
 studied ( and striking ) examples is the replacement of 1,2,4-oxadiazoles 
 with the symmetrical 1,3,4-oxadiazole isomer .
because of the contrasting dipole moment of each heterocycle , 1,3,4-oxadiazoles 
 display on average 10-fold lower lipophilicity and more favorable 
 profiles with respect to metabolic stability , herg inhibition , and 
 aqueous solubility .
switching to this 
 matched pair thus represented an attractive opportunity to lower lipophilicity 
 and improve physicochemical properties by making a minimal structural 
 change to the series , crucially without adding molecular weight .
the 
 mixed hydrazide intermediate 17a / b was synthesized 
 readily from ethyl ester 15 , and this was converted into 
 the 1,3,4-oxadiazole analogue 20a / b , and 
 additionally the 1,2,4-triazole analogue 18 from 17a ( scheme 3 ) . although less well 
 studied than 1,3,4-oxadiazoles , 1,2,4-triazoles are known to have 
 a lower log  d than 1,2,4-oxadiazoles , as well as a higher aromatic stabilization energy .
reagents and conditions : ( a ) nh2nh2h2o , etoh , 78 c , 24 h , 
 75% ; ( b ) rch2c(o)cl , n , n - diisopropylethylamine , dcm , rt , 15 min , 7591% ; ( c ) pocl3 , 100 c , 1 h ; ( d ) ammonium acetate , acetic acid , 140 
 c , 1.5 h , 6% over two steps ;
( e ) tscl , 1,2,2,6,6 pentamethylpiperidine , 
 dcm , rt , 3 h , 4865% ; ( f ) tfa , dcm , rt , 2 h , 5598% .
triazole 18 displayed a disappointing 
 affinity profile , with no change in affinity for hsnmt but a decrease 
 in affinity against the nmts of the two parasite species ( 9a , 14a vs 18 , table 2 ) .
nevertheless , an accompanying decrease in clogp resulted in a 
 significant improvement in the lelp for 18 , 9.5 compared 
 to 12.4 and 12.8 for 9a and 14a , respectively . 

this scaffold is therefore likely to have improved aqueous solubility 
 and druglike properties compared to the 1,2,4-oxadiazoles , which will 
 need to be considered in the future development of this series . as might be expected from the small difference in affinity between 
 the 1,2,4-oxadiazole regioisomers ( for example , 9c vs 14b ) ,
moving to the 1,3,4-oxadiazole 20a / b had only a small effect on pf / pv nmt affinity ( table 2 ) .
however , this modification unexpectedly decreased 
 hsnmt affinity ( 20b vs 9c , 14b ) , resulting in a 13-fold selectivity window between pfnmt and hsnmt , 
 24-fold between pvnmt and hsnmt .
in addition to the improved selectivity 
 profile , 20a / b has a significantly lower 
 clogp than 9a / c and 14a / b , resulting in a greatly improved lelp .
indeed , 18 , 20a , and 20b all display lelp within 
 the target range for promising lead compounds ( lelp < 10 ) .
crystallography confirmed the binding mode of 20b in 
 the peptide substrate pocket of pvnmt ( figure 2a , pdb entry 4cae ) , showing that the methoxyphenyl moiety makes hydrophobic contacts 
 with phe105 and a polar contact with ser319 , in addition to the interactions 
 observed previously for 1 .
furthermore , the oxadiazole is  sandwiched  between 
 the aromatic residues phe105 and tyr211 , potentially explaining the 
 affinity improvement observed when the ester in 4b is 
 replaced by an oxadiazole in 9c/14b ( figure 2b ) .
x - ray crystal structure of 20b ( blue ) bound 
 to pvnmt ( green ) .
the 3-methoxyphenyl 
 substituent forms the intended interactions with ser319 and phe105 , 
 in addition to the deeply buried hydrophobic scaffold and salt bridge 
 interaction observed in 1 .
( b ) the oxadiazole linker 
 is sandwiched between two aromatic residues , rationalizing the affinity 
 enhancement in moving to an aromatic heterocycle from the ester linker 
 in 4b .
dashed lines are drawn to highlight key interactions 
 between the enzyme and the ligand .
the crystal structure of 20b bound to pvnmt indicated 
 that there was a voluminous solvent - filled pocket surrounding the 
 methylene group  to the methoxyphenyl , inviting the introduction 
 of functionality that could either stabilize the water molecules in 
 this pocket or displace water to the bulk solvent ( figure 3 ) .
in addition , this position is 
 a potential target for oxidative metabolism and blocking with alternative 
 substituents may provide benefits during subsequent lead development .
x - ray 
 crystal structure of 20b ( blue ) bound to pvnmt ( green ) . 
 further inspection of the water molecules within the active site shows 
 that the benzylic ch2 occupies a heavily solvated pocket , 
 indicating that substitution may result in more favorable energetics 
 within the enzyme active site . dashed lines indicate water molecules 
 within 5  of the benzylic position .
the abundance of readily available -substituted phenylacetic 
 acids meant this strategy could be explored extensively , using previously 
 developed chemistry : a variety of substitutions were introduced as 
 shown in scheme 4 and table 3 .
reagents and conditions : ( a ) ph - cr1r2-co2h , 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide 
 hydrochloride , hydroxybenzotriazole , thf , dmf , rt , 18 h , 4899% ; 
 ( b ) tscl , 1,2,2,6,6pentamethylpiperidine , dcm , rt , 18 h ; ( c ) 10% tfa 
 in dcm ( v / v ) , rt , 2 h , 440% over two steps .
the results show that a wide variety of substitutions 
 at this position produce only small changes in enzyme affinity , reinforcing 
 the notion that there is flexibility in the binding pocket . of the 
 hydrophobic substituents , cyclopropyl results in the highest affinity : 

thus , 23c retains the activity of 20a but 
 with a concomitant increase in lelp .
the most significant result is 23 g , which is approximately equipotent to 23a with the addition of a polar hydroxyl group .
this increases the 
 hydrophilicity and decreases the lelp but increases the molecular 
 complexity ( with the addition of a stereocenter ) and introduces a 
 further hydrogen bond donor ( which may adversely affect membrane permeability ) . 

weighing these detriments against the moderate improvements these 
 modifications provided , the decision was made to progress development 
 with the unsubstituted template as in 20a / b with the knowledge that functionality could be reintroduced during 
 future lead development should a solubilizing / metabolism blocking 
 group be required .
the next area of interest was modification of the 
 methoxyphenyl portion of the molecule .
the rationale for this change 
 was twofold : ( i ) the methoxyphenyl portion contributes significantly 
 to the lipophilicity of these molecules and , as such , presents a target 
 for improvement of lelp ; ( ii ) aryl ethers 
 are notoriously poor h - bond acceptors , and so replacement may result in a stronger hydrogen bond with ser319 
 and an improvement in affinity . brand et al .
previously described 
 the development of a series of highly potent trypanosoma brucei nmt inhibitors that form a hydrogen 
 bond to the conserved ser319 ( ser330 in leishmania major nmt ) residue , for example , via a 1,3,5-trimethylpyrazole moiety 
 ( pdb entry 2wsa ) .
building on this observation , we selected three distinct heterocycles 
 to replace the methoxyphenyl substituent ( scheme 5 ) on the basis that nitrogen atoms conjugated within -systems 
 are typically excellent hydrogen bond acceptors , and these moieties would have reduced lipophilicity relative 
 to the methoxyphenyl parent compound ( table 4 ) . the binding mode in pvnmt ( figure 2 ) indicated 
 that the heterocycles in molecules 34a  c ( bearing a methylene linker ) would not make direct contacts to the 
 desired residues . for this reason ,
compounds 35a  c with an extended two - carbon linker were also synthesized 
 in the expectation that this would place the heterocycle directly 
 adjacent to ser319 , albeit with the entropic and lipophilic penalty 
 associated with a longer alkyl chain .
reagents 
 and conditions : ( a ) nah , ethyl bromoacetate , thf , 0 c , 18 h , 
 78% ; ( b ) methyl 3-bromopropionate , k2co3 , dmf , 
 55 c , 18 h , 30% ; ( c ) nh2nh2h2o , meoh , rt , 3 h , 8399% ; ( d ) n = 
 1 , nh2ohhcl , k2co3 , etoh , 78 
 c , 3 h , 12% ; n = 2 , nh2ohhcl , 
 h2o , meoh , 60 c , 18 h , 89% ; ( e ) menhnh2 , acoh , 3 h , rt , 7395% ; ( f ) liohh2o , meoh , 
 rt , 18 h , 5195% ; ( g ) 16 , 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide 
 hydrochloride , hydroxybenzotriazole , thf , dmf , rt , 18 h , 4899% ; 
 ( h ) tscl , 1,2,2,6,6-pentamethylpiperidine , dcm , rt , 18 h ; ( i ) 10% 
 tfa in dcm ( v / v ) , rt , 2 h , 326% over two steps .
the sar obtained across these analogues was unexpected : 
 in all cases pvnmt and pfnmt had higher affinity for the methylene 
 linked 34 series , although this was not always the case 
 for hsnmt ( 34b vs 35b ) .
of the three heterocycles , 
 isoxazoles ( 34b and 35b ) and 1h - pyrazoles ( 34a and 35a ) had decreased 
 affinity compared to the methoxyphenyl parent 20b .
interestingly , 34a displays 30-fold selectivity between pfnmt and hsnmt , 
 a significant improvement over the 8- to 9-fold observed in 34b/34c .
addition of the n - methyl group in 34c produces a marked increase in affinity 
 against all enzymes , resulting in the highest affinity plasmodium spp .
furthermore , this compound 
 displays nanomolar efficacy against the parasite , allowing further 
 biological evaluation of this drug target ( vide infra ) . the affinity 
 enhancement imparted by the methylpyrazole
is accompanied by a reduction 
 in clogp ( relative to methoxyphenyl 20b ) resulting in 
 an excellent lelp of 5.5 , close to the reported average lelp of marketed 
 drugs .
consequently , this compound has 
 excellent druglike properties ; we have previously reported the excellent 
 aqueous solubility ( > 8 mm ) and in vitro pharmacokinetic properties 
 of this molecule .
although the selectivity 
 between pfnmt and hsnmt is 8-fold ( 30-fold vs pvnmt ) , comparison to 34a shows that significant changes in the selectivity profile 
 may potentially be achieved by altering the interactions with this 
 binding pocket .
the binding mode of 34c was elucidated 
 by crystallography , as briefly described previously ( figure 4 , pdb entry 2yne ) . as predicted during the compound design process
, the pyrazole 
 of 34c does not directly interact with the target ser319 , 
 indicating a subtler basis for the affinity improvement .
the pyrazole 
 n-2 interacts indirectly with ser319 via a water molecule , with the 
 methyl groups occupying hydrophobic regions of the pocket ( figure 4b ) .
the improved enzyme affinity is likely a result 
 of the strong pyrazole  water hydrogen bond combined with fit 
 complementarity within the binding site .
in addition , the compound 
 makes all the interactions observed with previous members of the series , 
 including the salt bridge interaction with leu410/tyr107 , a sandwiched 
 oxadiazole linker , and deeply buried benzothiophene scaffold ( figure 4a ) .
( a ) 34c ( gold ) bound to pvnmt ( green ) , showing piperidine 
leu410 
 salt bridge interaction , deeply buried benzothiophene scaffold , and 
 1,3,5-trimethylpyrazole heterocycle bound within the ser319 hydrophobic 
 pocket .
( b ) enlarged view of the 1,3,5-trimethylpyrazole of 34c ( gold ) with pvnmt ( green ) .
this shows the water - bridged 
 interaction between the pyrazole and ser319 , as well as multiple hydrophobic 
 contacts between the heterocycle and the binding pocket .
the binding mode of 34a in pvnmt was 
 also determined by crystallography ( figure 5a , pdb entry 4caf ) .
this compound displays a 40-fold lower binding affinity 
 to pvnmt than 34c ; however , the binding modes of these 
 two compounds are extremely similar ( figure 5b ) . the only point of differentiation is the pyrazole heterocycle 
 which forms a direct polar interaction with ser319 as opposed to the 
 water - bridged interaction observed in figure 4b . indeed
, this water molecule is excluded from the pocket occupied 
 by 34a , perhaps indicating that it is involved in stabilizing 
 ser319 and therefore that its expulsion to the bulk solvent is energetically 
 unfavorable .
( a ) 34a ( pink ) forms all previously observed interactions 
 with the enzyme .
( b ) comparison of the binding modes of 34a and 34c reaffirms this similarity , showing that the 
 only point of differentiation is in the phe105/ser319 binding site 
 occupied by the pyrazole .
( c ) enlarged view of the 1,3,5-trimethylpyrazole 
 of 34c ( gold ) and 3,5-dimethylpyrazole of 34a ( pink ) with pvnmt ( green ) .
the pyrazole of 34a forms 
 a direct interaction with ser319 ( 3.2  ) , and the water involved 
 in the bridged interaction in figure 4b has 
 been excluded from the pocket .
34c provides excellent enzyme affinity and lelp , and the cellular efficacy 
 enabled further biological evaluation of this inhibitor .
34c was tested against a variety of parasites in vitro , including both 
 drug - resistant cell lines and liver stage parasites , and for mammalian 
 cell toxicity against human hepatocellular carcinoma cell line hepg2 . 

activity was directly compared to chloroquine ( 36 ) and 
 atovaquone ( 37 ) where relevant .
nf54 , k1 , and dd2 
 ec50 values are determined using a [ h]hypoxanthine 
 incorporation assay ( performed by dr . sergio wittlin and dr .
standard deviation is within 20% of the ld50 . 3d7 and nf54 are chloroquine - sensitive 
 strains of the parasite , whereas k1 and dd2 are chloroquine - resistant 
 strains , as evidenced by the greatly reduced potency of this drug 
 against these strains ( table 5 ) .
34c was previously shown to act on - target in blood stage drug - sensitive 
 ( 3d7 ) parasites .
pleasingly , 34c is active against an additional drug - sensitive line ( nf54 ) and there 
 is very little loss of potency for drug - resistant cell lines ( less 
 than 4-fold between nf54 and dd2 ) , indicating that nmt inhibition 
 may be an effective mechanism to target drug - resistant parasites in 
 a clinical context .
in addition , 34c was tested 
 against a plasmodium berghei ( pb ) liver stage model 
 to determine whether nmt is likely to be a viable drug target for 
 clearance of liver stage parasites .
the pbnmt enzyme affinity of 34c was determined at 14 nm , validating an approximate comparison 
 with the pf blood stage models .
34c had a liver stage 
 ec50 of 372 nm , which , although significantly lower than 
 the atovaquone standard , is in excellent agreement with the blood 
 stage results .
this provides evidence that nmt inhibition is also 
 a promising mechanism for targeting liver stage parasites and may 
 therefore be an effective pathway for the disruption of multiple parasite 
 life stages .
lastly , 34c displays up to 40-fold 
 selectivity between parasitic and hepg2 cell lines ( 14-fold in the 
 case of dd2 ) .
this represents an encouraging window for a compound 
 with 10-fold enzyme selectivity : if hepg2 cytotoxicity is due to nmt 
 inhibition , then a more selective compound will have a larger window , 
 and if the lethality is nonspecific , then the cell potency and toxicity 
 appear sufficiently decoupled to be optimized independently .
the medicines 
 for malaria venture dictates that validated hit compounds should display 
 cellular selectivity of greater than 10-fold , whereas preclinical 
 candidates should be > 100-fold selective . with this target in mind ,
34c displays reasonable selectivity 
 for a lead compound : a further 3-fold improvement in efficacy without 
 accompanying toxicity will achieve the required window for a candidate 
 molecule . note
that the moderate selectivity is a functional of low 
 efficiacy rather than high toxicity ; the ld50 for 34c is approximately the top concentration tested ( 10 m ) 
 for the antimalarial standards . the metric lelp was used to guide the development 
 of this compound series , based on the hypothesis that compounds with 
 a low lelp ( ideally < 10 ) will have fewer off - target effects and 
 as a result are more druglike . in an 
 attempt to assess if lelp is a useful metric in this regard , the cellular 
 ec50 against the 3d7 parasite line was plotted against 
 nmt affinity for all tested members of this series .
( a ) plot of cellular potency vs enzyme 
 affinity for all members of this series tested in both assays .
( b ) 
 plot of cellular potency vs enzyme affinity for all compounds with 
 a lelp of > 10 .
( c ) plot of cellular potency vs enzyme affinity 
 for all compounds with a lelp of < 10 .
an initial assessment 
 of cellular potency vs enzyme affinity showed only a weak correlation 
 ( r = 0.47 ) between these two parameters 
 ( figure 6a ) and a relatively flat gradient 
 ( 0.40 ) .
however , when the compounds were separated into those with 
 a leadlike lelp score ( lelp < 10 ) and those without ( lelp > 
 10 ) , a qualitative assessment showed an improved correlation for those 
 compounds with a low lelp value ( figure 6c 
 vs figure 6b ) .
figure 6c shows that for those compounds with lelp < 10 there is a strong 
 correlation between cellular potency and enzyme affinity ( r = 0.82 ) and a gradient significantly closer 
 to 1 than in the full data set ( 0.75 vs 0.40 ) .
in contrast , for compounds 
 with a lelp of > 10 there is a very weak correlation between ec50 and ki ( r = 0.36 ) and a flat gradient ( 0.23 ) , indicating that antiparasitic 
 activity from these compounds derives predominantly from a mechanism 
 independent of nmt inhibition .
this observation is consistent 
 with the hypothesis that high - lelp compounds ( with relatively high 
 lipophilicity and low le ) are more promiscuous than low - lelp analogues and therefore may exhibit cytotoxic activity 
 by a variety of nonspecific mechanisms .
low - lelp analogues are predicted 
 to bind more specifically to the target enzyme , and this is supported 
 by the improved correlation between nmt affinity and antiparasitic 
 activity in this data set ; although wary of 
correlation inflation  , this analysis appears to provide a robust predictor 
 of cell efficacy when applied to these models .
it would be unwise 
 to invoke lelp < 10 as a rule rather than a guideline , as this 
 is unlikely to be a binary boundary , with some compounds in either 
 subset behaving not as predicted .
nevertheless , it provides encouraging 
 evidence that lelp is a useful metric for guiding the hit - to - lead 
 optimization process .
furthermore , this analysis highlights the danger 
 of assuming that weakly active ( le < 0.3 ) , lipophilic compounds 
 ( clogp > 3 ) function in cellular systems by the presumed mechanism 
 of action , without further validation .
previously 
 reported work described the discovery of 1 by a lead - hopping 
 approach , as a ligand efficient inhibitor of plasmodial nmt .
this work details lelp - guided development of 
 this series , optimizing enzyme affinity while retaining selectivity 
 over hsnmt to yield 34c , a high affinity druglike plasmodial 
 nmt inhibitor .
development from 1 to 34c achieved a 100-fold improvement in enzyme affinity coupled with 
 a 100-fold reduction in lipophilicity , at the expense of only two 
 additional heavy atoms .
this further demonstrates the utility of medicinal 
 chemistry strategies such as matched molecular pairs analysis , bioisosterism , 
 and metric - guided optimization in hit - to - lead development .
34c has antiparasitic activity in vitro , exhibiting similar 
 potency over four parasite strains including two drug - resistant ones . 

in addition , this compound is equipotent against blood and liver stage 
 parasites and displays selectivity over human liver host cells .
it 
 has been previously shown that this molecule exerts its antiparasitic 
 activity via inhibition of nmt and moreover that it can reduce parasitemia 
 in vivo .
taken together , this biological 
 characterization shows that nmt is a highly promising target for the 
 development of a new generation of antimalarial drugs .
further work 
 will focus on optimization of cellular potency and in vivo pharmacokinetics , 
 with the aim of producing a candidate series for the treatment of 
 malaria .
( gillingham , uk ) , acros organics 
 ( geel , belgium ) and alfa aesar ( heysham , uk ) and used without further 
 purification .
moisture sensitive reactions were performed under nitrogen 
 atmosphere using dried glassware , anhydrous solvents , and standard 
 syringe / septa techniques .
silica gel normal phase column chromatography 
 was performed on an isolera ( biotage , u.k . )
mobile phase consisted of n - hexane ( solvent a ) and ethyl acetate ( solvent b ) , and 
 standard gradient consisted of x% solvent b for 1 
 column volumes , x% to y% b for 10 
 column volumes , and then y% b for 2 column volumes .
x and y are defined in the characterization 
 section of the compound of interest .
final compounds were purified 
 by one of two methods : either hplc or lc  ms .
postchromatography , 
 organic solvents were removed by partial evaporation under reduced 
 pressure and then compounds were dried by lyophilization overnight .
hplc involved the following : gilson semipreparative reverse phase 
 hplc system equipped with a hichrom c18 column ( 250 mm 
  21.2 mm ) , no . 306 pumps , and a gilson uv / vis detector , detecting 
 at 220 nm . the mobile phase consisted of h2o + 0.1% formic 
 acid ( solvent a ) and meoh + 0.1% formic acid ( solvent b ) , with an 
 elution method of 02 min 50% b , 230 min 5098% 
 b , 3032 min 98% , 3232.5 min 2% b at a flow rate of 
 12 ml / min .
lc  ms involved the following : rp - hplc / ms on 
 a waters 2767 system equipped with a photodiode array and an esi mass 
 spectrometer using a xbridge prep c18 ( 5 m , 19 mm  100 
 mm ) column , equipped with an xbridge prep c18 guard column ( 5 m , 
 19 mm  10 mm ) .
the following elution method was used : gradient 
 of solvent a and solvent b ( as above ) of 010 min 5098% 
 b , 1012 min 98% b , 1213 min 9850% b , 1317 
 min 50% b. flow rate was 20 ml / min .
the purity of the title 
 compounds was verified by reverse phase lc  ms on a waters 2767 
 system equipped with a photodiode array and an esi mass spectrometer 
 using a xbridge c18 ( 5 m , 4.6 mm  100 mm ) column , equipped 
 with an xbridge c18 guard column ( 5 m , 4.6 mm  20 mm ) . 

the following elution method was used : gradient of solvent a and solvent 
 b ( as above ) of 010 min 598% b , 1012 min 98% 
 b , 1213 min 985% b , 1317 min 5% b. flow rate 
 was 1.2 ml / min .
h and c nmr spectra 
 were respectively recorded on 400 and 101 mhz bruker av instruments 
 at room temperature unless specified otherwise . in these cases h and c nmr spectra
were respectively recorded 
 on 500 and 126 mhz bruker av instruments at room temperature and were 
 referenced to residual solvent signals .
data are presented as follows : 
 chemical shift in ppm , integration , multiplicity ( br = broad , app 
 = apparent , s = singlet , d = doublet , t = triplet , q = quartet , p 
 = pentet , m = multiplet ) , and coupling constants in hz .
mass 
 spectra were obtained from the mass spectrometry service of department 
 of chemistry , imperial college london .
1 and 2 were prepared as described previously . to a solution of 2 ( 50 mg , 0.13 mmol ) in 
 dry acetonitrile ( 2 ml )
were added hydroxybenzotriazole ( 27 mg , 0.20 
 mmol ) , n , n - diisopropylethylamine 
 ( 26 l , 0.16 mmol ) , and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide 
 hydrochloride ( 30 mg , 0.16 mmol ) .
2-(3-methoxyphenyl)ethanol ( 22 l , 
 0.15 mmol ) was then added and reaction mixture allowed to stir at 
 room temperature for 18 h. the reaction mixture was concentrated under 
 reduced pressure , dissolved in 10 ml of saturated ammonium chloride 
 solution , and 3b was extracted with 3  10 ml ethyl 
 acetate .
combined organic layers were then washed with brine ( 10 ml ) , 
 dried over magnesium sulfate , concentrated under reduced pressure 
 and crude product was purified by flash chromatography ( 10 g snap cartridge , 
 650% b , rf = 5.1 column volumes ) 
 to give 3b as a colorless oil ( 24 mg , 36% ) .
h nmr ( cdcl3 ,  , ppm ) 7.86 ( 1h , d , j = 8.0 ) , 7.75 ( 1h , d , j = 8.2 ) , 7.517.46 
 ( 1h , m ) , 7.437.37 ( 1h , m ) , 7.32 ( 1h , dd , j = 8.0 , 7.8 ) , 7.04 ( 1h , d , j = 7.8 ) , 7.027.00 
 ( 1h , m ) , 6.90 ( 1h , dd , j = 8.0 , 2.3 ) , 5.35 ( 2h , s ) , 
 4.744.66 ( 1h , m ) , 3.943.86 ( 2h , m ) , 3.84 ( 3h , s ) , 
 3.072.98 ( 2h , m ) , 1.981.88 ( 2h , m ) , 1.851.73 
 ( 2h , m ) , 1.48 ( 9h , s ) . to 
 a solution of 3b ( 23.0 mg , 0.05 mmol ) in dichloromethane 
 ( 1 ml ) was added trifluoroacetic acid ( 100 l ) , and the solution 
 was stirred at room temperature for 2 h. the reaction mixture was 
 concentrated under reduced pressure and purified by hplc , yielding 4b as a colorless oil ( 5 mg , 18% ) .
tr = 12.3 min ; h nmr ( cdcl3 ,  , 
 ppm ) 7.82 ( 1h , d , j = 8.0 ) , 7.76 ( 1h , d , j = 8.1 ) , 7.567.48 ( 1h , m ) , 7.477.40 ( 1h , 
 m ) , 7.33 ( 1h , app t , j = 7.9 ) , 7.04 ( 1h , d , j = 7.4 ) , 7.016.98 ( 1h , m ) , 6.91 ( 1h , dd , j = 8.2 , 2.4 ) , 5.34 ( 2h , s ) , 4.884.81 ( 1h , m ) , 3.84 
 ( 3h , s ) , 3.543.44 ( 2h , m ) , 3.113.01 ( 2h , m ) , 2.222.11 
 ( 4h , m ) ; c nmr ( cdcl3 ,  , ppm ) 161.48 , 
 159.84 , 153.90 , 138.23 , 136.98 , 134.06 , 129.82 , 128.39 , 125.07 , 123.14 , 
 122.61 ,
120.34 , 115.87 , 113.81 , 113.79 , 76.10 , 66.82 , 55.29 , 41.01 , 
 28.07 ; esi hrms , found 398.1425 ( c22h24no4s , [ m + h ] , requires 398.1426 ) .
to a solution of 2 ( 40 mg , 0.11 mmol ) in n , n - dimethylformamide ( 1 ml ) were added 
 hydroxybenzotriazole ( 16 mg , 0.12 mmol ) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide 
 hydrochloride ( 22 mg , 0.12 mmol ) , and the reaction mixture was stirred 
 at room temperature for 30 min .
n-hydroxy-2-phenylacetimidamide 
 was added ( 17.5 mg , 0.12 mmol ) , and the mixture was stirred at 140 
 c for 3 h. the reaction mixture was concentrated under reduced 
 pressure , dissolved in 20 ml of saturated ammonium chloride solution , 
 and 8a was extracted with 3  20 ml of ethyl acetate . 

the combined organic layers were washed with saturated potassium carbonate 
 solution ( 20 ml ) , brine ( 20 ml ) , dried over magnesium sulfate , concentrated 
 under reduced pressure and crude product was purified by flash chromatography 
 ( 10 g snap cartridge , 650% b , rf = 7.5 column volumes ) to give 8a as a clear yellow 
 oil ( 16 mg , 31% ) .
h nmr ( cdcl3 ,  , ppm ) 
 7.87 ( 1h , d , j = 7.8 hz ) , 7.80 ( 1h , d , j = 8.0 hz ) , 7.50 ( 1h , ddd , j = 8.0 , 7.1 , 1.2 hz ) , 
 7.467.28 ( 6h , m ) , 4.634.54 ( 1h , m ) , 4.16 ( 2h , s ) , 
 3.983.87 ( 2h , m ) , 3.002.91 ( 2h , m ) , 2.011.93 
 ( 2h , m ) , 1.901.78 ( 2h ,
9a was prepared as in 4b replacing 3b with 8a ( 16 mg , 0.03 mmol ) and purified by 
 lc 
tr = 4.22 min ; h nmr ( cdcl3 ,  , ppm ) 7.82 ( 2h , d , j = 8.1 hz ) , 7.52 ( 1h , 
 dd , j = 8.1 , 7.1 hz ) , 7.46 ( 1h , dd , j = 8.1 , 7.1 hz ) , 7.407.28 ( 5h , m ) , 4.804.72 ( 1h , 
 m ) , 4.16 ( 2h , s ) , 3.563.45 ( 2h , m ) , 3.042.95 ( 2h , 
 m ) , 2.222.15 ( 4h , m ) ; c nmr ( cdcl3 ,  , ppm ) 170.28 , 169.61 , 152.34 , 137.32 , 135.57 , 133.82 , 129.29 , 
 128.92 , 128.37 , 127.44 , 125.57 , 123.45 , 122.72 , 109.93 , 76.97 , 40.73 , 
 32.49 , 28.4l ; esi hrms , found 392.1425 ( c22h22n3o2s , [ m + h ] , requires 392.1433 ) . to a solution of methyl-2-mercaptobenzoate
( 1.64 
 ml , 11.9 mmol ) and 2-bromoacetonitrile ( 0.92 ml , 13.1 mmol ) in dry 
 tetrahydrofuran ( 100 ml ) at 0 c was added potassium tert - butoxide ( 5.14 g , 71.3 mmol ) gradually over 2 min . 

the reaction mixture was stirred and allowed to warm to room temperature 
 over 15 min , quenched with 2 m hydrochloric acid to ph 2 , and diluted 
 with 75 ml of water .
the organic layers were 
 combined , washed with 75 ml of brine , dried over magnesium sulfate , 
 and concentrated under reduced pressure to give desired product 10 as a dark brown solid ( 2.03 g , 88% ) .
h nmr 
 ( cdcl3 ,  , ppm ) 8.54 ( 1h , brs ) , 7.91 ( 1h , d , j = 8.1 hz ) , 7.73 ( 1h , d , j = 8.2 hz ) , 
 7.627.50 ( 1h , m ) , 7.517.40 ( 1h , m ) . to a solution of 10 ( 500 mg , 
 2.85 mmol ) in tetrahydrofuran ( 7.5 ml )
was added tert - butyl 4-hydroxypiperidine-1-carboxylate ( 1.15 g , 5.71 mmol ) and 
 triphenylphosphine ( 1.50 g , 5.71 mmol ) .
the reaction mixture was stirred 
 under nitrogen for 20 min and cooled to 0 c , and diisopropyl 
 azodicarboxylate ( 1.12 ml , 5.71 mmol ) in tetrahydrofuran ( 10 ml ) was 
 added dropwise over 5 min .
reaction mixture was allowed to warm to 
 room temperature and stirred for 1.5 h , then concentrated under reduced 
 pressure and the crude product purified by flash chromatography ( 100 
 g snap cartridge , 218% b , rf = 
 8.5 column volumes ) to give 11 as a white solid ( 800 
 mg , 78% ) .
h nmr ( cdcl3 ,  , ppm ) 7.85 
 ( 1h , dd , j = 8.2 , 0.9 hz ) , 7.72 ( 1h , d , j 
 = 8.2 hz ) , 7.55 ( 1h , ddd , j = 8.2 , 7.1 , 
 1.1 hz ) , 7.44 ( 1h , ddd , j = 8.2 , 7.2 , 0.9 hz ) , 5.25 
 ( 1h , tt , j = 7.3 , 3.6 hz ) , 3.853.74 ( 2h , 

m ) , 3.463.36 ( 2h , m ) , 2.182.07 ( 2h , m ) , 1.981.87 
 ( 2h , m ) , 1.49 ( 9h , s ) . to a solution of 11 ( 100 mg , 0.28 mmol ) in 
 ethanol ( 2 ml ) was added hydroxylamine as 50% aqueous solution ( 170 
 l , 2.79 mmol ) , and reaction mixture was stirred under refluxing 
 conditions for 4 h. reaction mixture was concentrated under reduced 
 pressure to give 12 as a white solid ( 109 mg , 99% ) .
h nmr ( cdcl3 ,  , ppm ) 7.777.73 ( 1h , 
 m ) , 7.727.67 ( 1h , m ) , 7.417.37 ( 2h , m ) , 6.25 ( 1h , 
 brs ) , 5.55 ( 2h , brs ) , 4.504.41 ( 1h , m ) , 4.173.99 ( 2h , 
 m ) , 2.87 ( 2h , ddd , j = 14.2 , 11.7 , 2.6 hz ) , 2.122.00 
 ( 2h , m ) , 1.871.74 ( 2h , m ) , 1.48 ( 9h , s ) .
to a solution of 3-methoxyphenylacetic acid ( 31 mg , 0.19 
 mmol ) in acetonitrile ( 1 ml ) were added hydroxybenzotriazole ( 27.5 
 mg , 0.21 mmol ) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide 
 hydrochloride ( 39 mg , 0.21 mmol ) , and reaction mixture was stirred 
 at room temperature for 15 min .
12 was added ( 80 mg , 
 0.21 mmol ) and the mixture stirred at room temperature for 18 h. the 
 reaction mixture was diluted with 20 ml of 0.5 m naoh(aq ) solution , 
 and 13b was extracted with 3  20 ml of ethyl acetate . 

the combined organic layers were washed with saturated potassium carbonate 
 solution ( 20 ml ) , brine ( 20 ml ) , dried over sodium sulfate , and concentrated 
 under reduced pressure .
crude product ( 120 mg ) and 4  molecular 
 sieves ( 200 mg ) were dissolved in toluene ( 3 ml ) , and reaction mixture 
 was stirred at 110 c for 18 h. the reaction mixture was filtered , 
 concentrated under reduced pressure and crude product purified by 
 flash chromatography ( 10 g snap cartridge , 540% b , rf = 8.0 column volumes ) to give 13b as a clear orange oil ( 66 mg , 68% ) .
h nmr ( cdcl3 ,  , ppm ) 7.84 ( 1h , dd , j = 7.1 , 1.2 
 hz ) , 7.79 ( 1h , dd , j = 7.1 , 1.2 hz ) , 7.43 ( 2h , dd , j = 7.1 , 1.2 hz ) , 7.337.28 ( 1h , m ) , 6.97 ( 1h , d , j = 8.0 hz ) , 6.956.92 ( 1h , m ) , 6.87 ( 1h , dd , j = 8.0 , 2.4 hz ) , 4.604.50 ( 1h , m ) , 4.28 ( 2h , s ) , 
 4.003.89 ( 2h , m ) , 3.83 ( 3h , s ) , 3.022.93 ( 2h , m ) , 
 2.011.92 ( 2h , m ) , 1.891.77 ( 2h , m ) , 1.48 ( 9h , s ) .
14b was prepared as in 4b replacing 3b with 13b ( 66 mg , 0.13 mmol ) and purified by hplc , yielding 14b as a white solid ( 21 mg , 40% ) .
tr = 9.7 min ; h nmr ( cdcl3 ,  , 
 ppm ) 8.03 ( 1h , dd , j = 7.0 , 1.3 hz ) , 7.927.84 
 ( 1h , m ) , 7.587.47 ( 2h , m ) , 7.31 ( 1h , app t , j = 7.9 hz ) , 7.047.00 ( 1h , m ) , 6.996.94 ( 1h , m ) , 6.946.87 
 ( 1h , m ) , 4.66 ( 1h , tt , j = 7.7 , 3.6 hz ) , 4.43 ( 2h , 
 s ) , 3.76 ( 3h , s ) , 3.443.27 ( 2h , m ) , 3.032.90 ( 2h , 
 m ) , 2.132.03 ( 2h , m ) , 2.011.89 ( 2h , m ) ; c nmr ( cdcl3 ,  , ppm ) 178.57 , 162.84 , 159.46 , 149.67 , 
 136.80 , 135.25 , 133.88 , 129.86 , 127.34 , 125.28 , 123.55 , 122.15 , 121.40 , 
 115.13 , 112.72 , 112.35 , 76.88 , 55.09 , 40.82 , 31.85 , 28.20 ; esi hrms , 
 found 422.1534 ( c23h24n3o3s , [ m + h ] , requires 422.1538 ) . to a solution of 15 ( 300 mg , 0.74 mmol ) in 
 ethanol ( 1 ml ) was added hydrazine monohydrate ( 145 l , 2.96 
 mmol ) .
the reaction mixture was heated under refluxing conditions 
 for 24 h and then concentrated under reduced pressure , yielding 16 as a yellow oil ( 217 mg , 75% ) .
h nmr ( cdcl3 ,  , ppm ) 7.79 ( 1h , d , j = 7.6 hz ) , 
 7.74 ( 1h , d , j = 7.2 hz ) , 7.487.37 ( 2h , m ) , 
 4.58 ( 1h , tt , j = 9.8 , 4.1 hz ) , 4.134.04 
 ( 2h , m ) , 2.952.82 ( 2h , m ) , 2.142.05 ( 2h , m ) , 1.921.78 
 ( 2h , m ) , 1.48 ( 9h , s ) .
to a solution of 16 ( 48 mg , 0.12 
 mmol ) in tetrahydrofuran / n , n - dimethylformamide 
 ( 4:1 v / v , 0.6 ml ) were added hydroxybenzotriazole ( 8 mg , 0.06 mmol ) , 
 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride 
 ( 28 mg , 0.15 mmol ) and 2-phenylpropanoic acid ( 20 l , 0.15 mmol ) . 

reaction mixture was stirred at room temperature for 18 h and then 
 diluted with 1.0 m naoh(aq ) ( 4 ml ) .
combined organic layers were 
 washed with brine ( 5 ml ) , dried over sodium sulfate , and concentrated 
 under reduced pressure , yielding 21a as a colorless oil 
 ( 63 mg , 98% ) .
h nmr ( cdcl3 ,  , ppm ) 7.827.76 
 ( 2h , m ) , 7.497.20 ( 7h , m ) , 4.68 ( 1h , tt , j = 10.0 , 4.1 hz ) , 4.194.06 ( 2h , m ) , 3.85 ( 1h , q , j 
 = 7.2 hz ) , 2.882.79 ( 2h ,
m ) , 2.162.05 ( 2h , 
 m ) , 2.021.88 ( 2h , m ) , 1.60 ( 3h , d , j = 7.2 
 hz ) , 1.48 ( 9h , s ) . to a solution of 21a ( 63 mg , 0.12 mmol ) and 1,2,2,6,6-pentamethylpiperidine 
 ( 47 l , 0.26 mmol ) in dichloromethane ( 1 ml ) was added m - toluenesulfonyl chloride ( 25 mg , 0.13 mmol ) , and the reaction 
 mixture was stirred at room temperature for 18 h. the reaction mixture 
 was then diluted with a further 2 ml of dichloromethane , washed with 
 water ( 2 ml ) , washed with 1.0 m naoh(aq ) ( 2 ml ) , washed 
 with brine ( 2 ml ) , dried over magnesium sulfate , and concentrated 
 under reduced pressure .
crude reaction mixture was boc - deprotected 
 without further purification as in 4b , replacing 3b with 22a ( 10 mg , 0.02 mmol ) , and purified 
 by hplc yielding 23a as a yellow oil ( 7 mg , 14% ) .
tr = 11.0 min ; h nmr ( cdcl3 ,  , ppm ) 8.44 ( 1h , brs ) , 7.857.73 ( 2h , m ) , 7.547.30 
 ( 7h , m ) , 4.65 ( 1h , brs ) , 4.44 ( 1h , q , j = 6.6 hz ) , 
 3.533.35 ( 2h , m ) , 3.062.86 ( 2h , m ) , 2.202.01 
 ( 4h , m ) , 1.84 ( 3h , d , j = 6.6 hz ) ; esi hrms , found 
 406.1587 ( c23h24n3o2s , 
 [ m + h ] , requires 406.1589 ) .
to a solution of sodium hydride ( 576 mg , 
 24.0 mmol ) in anhydrous tetrahydrofuran ( 30 ml ) cooled to 0 c 
 was added pentane-2,4-dione ( 2.05 ml , 20.0 mmol ) in anhydrous tetrahydrofuran 
 ( 40 ml ) , and the mixture was stirred for 1 h. ethyl bromoacetate ( 2.66 
 ml , 24.0 mmol ) in anhydrous tetrahydrofuran ( 30 ml ) was then added , 
 and the reaction mixture was stirred for 18 h. the reaction mixture 
 was then washed with saturated nh4cl(aq ) ( 100 
 ml ) , and aqueous layer was back - extracted with etoac ( 100 ml ) .
combined 
 organic layers were washed with brine ( 100 ml ) , dried over magnesium 
 sulfate , and concentrated under reduced pressure , yielding 24 as a yellow oil ( 2.90 g , 78% ) .
mixture of diketone / enol tautomers 
 2:1 was observed by nmr in cdcl3 at room temperature .
diketone h nmr ( cdcl3 ,  , ppm ) 4.204.12 ( 3h , 
 m ) , 2.90 ( 2h , d , j = 7.3 hz ) , 2.29 ( 6h , s ) , 1.311.25 
 ( 3h , m ) ; enol h nmr ( cdcl3 ,  , ppm ) 4.224.08 
 ( 2h , m ) , 3.25 ( 2h , s ) , 2.17 ( 6h , s ) , 1.321.23 ( 3h , m ) .
to a solution of 24 ( 400 mg , 2.15 
 mmol ) in acetic acid ( 3 ml ) was added methylhydrazine ( 125 l , 
 2.37 mmol ) dropwise , and reaction mixture was stirred at room temperature 
 for 3 h. reaction mixture was concentrated under reduced pressure , 
 yielding 26c as a colorless oil ( 349 mg , 73% ) .
h nmr ( cdcl3 ,  , ppm ) 4.15 ( 2h , q , j = 7.1 hz ) , 3.74 ( 3h , s ) , 3.35 ( 2h , s ) , 2.22 ( 6h , s ) , 1.28 ( 3h , t , j = 7.1 hz ) .
to a solution of 26c ( 300 mg , 1.53 mmol ) in methanol ( 3 ml ) was added lithium hydroxide 
 monohydrate ( 642 mg , 15.3 mmol ) , and the mixture was stirred at room 
 temperature for 18 h. reaction mixture was diluted with water ( 20 
 ml ) and acidifed with 2.0 m hcl(aq ) to ph 4 . then 28c was extracted with etoac ( 3  20 ml ) .
combined organic 
 layers were then dried over sodium sulfate and concentrated under 
 reduced pressure , yielding 28c as a pink crystalline 
 solid ( 130 mg , 51% ) .
h nmr ( cdcl3 ,  , 
 ppm ) 3.80 ( 3h , s ) , 3.41 ( 2h , s ) , 2.24 ( 3h , s ) , 2.23 ( 3h , s ) .
30c was prepared as in 21a replacing 2-phenylpropanoic acid with 28c ( 25 mg , 0.15 
 mmol ) , yielding 30c as an orange oil ( 43 mg , 66% ) .
h nmr ( cdcl3 ,  , ppm ) 9.75 ( 1h , d , j = 6.2 hz ) , 7.897.72 ( 3h , m ) , 7.537.37 
 ( 2h , m ) , 4.754.65 ( 1h , m ) , 4.124.06 ( 2h , m ) , 3.76 
 ( 3h , s ) , 3.46 ( 2h , s ) , 2.862.82 ( 2h , m ) , 2.26 ( 6h , s ) , 2.142.09 
 ( 2h , m ) , 1.991.87 ( 2h , m ) , 1.48 ( 9h , s ) .
34c was prepared as in 23a replacing 21a with 30c ( 66 mg , 0.12 mmol ) and purified 
 by lc 
ms , yielding 34c as a yellow solid ( 9 mg , 
 17% ) .
tr = 7.1 min ; h nmr 
 ( cd3od ,  , ppm ) 8.47 ( 1h , brs ) , 7.997.88 
 ( 2h , m ) , 7.617.45 ( 2h , m ) , 4.81 ( 1h , tt , j = 7.2 , 4.1 hz ) , 4.12 ( 2h , s ) , 3.72 ( 3h , s ) , 3.56 ( 2h , ddd , j = 12.1 , 7.5 , 4.1 hz ) , 3.11 ( 2h , ddd , j = 12.5 , 7.5 , 4.1 hz ) , 2.29 ( 3h , s ) , 2.22 ( 3h , s ) , 2.212.04 
 ( 4h , m ) ; c nmr ( cd3od ,  , ppm ) 170.17 , 
 160.26 , 147.99 , 134.99 , 139.01 , 134.93 , 134.27 , 128.98 , 126.59 , 124.49 , 
 123.45 , 120.38 , 109.98 , 77.73 , 45.42 , 42.37 , 36.01 , 29.56 , 20.79 , 
 9.52 ; esi hrms , found 424.1801 ( c22h26n5o2s , [ m + h ] , requires 424.1807 ) .
all ic50 determinations 
 were carried out using a 7-diethylamino-3-(4-maleimidylphenyl)-4-methylcoumarin 
 ( cpm ) fluorescence assay , as described previously for hsnmt1 , pvnmt , and pfnmt .
ic50 values are the mean value 
 of two or more determinations , and standard deviation is within 20% 
 of the ic50 unless otherwise specified .
data were elaborated 
 using microsoft office excel 2010 , and ic50 values were 
 determined using grafit 7.0 ( erithacus software ltd .
, u.k . ) by nonlinear 
 regression fitting , which were then quoted as ki as defined below .
ki values quoted are the ki calculated from the experimentally determined ic50 values , the substrate concentration ( [ s ] ) , and the michaelis 
prusoff 
 equation:1for example , 34c had an experimentally determined pfnmt ic50 of 0.017 
  0.002 m .
the michaelis constant ( km ) was 3.64 m and the substrate concentration was 4.00 
 m , resulting in a ki of 0.008 m .
red blood cells used for 
 the assay were centrifuged to remove the buffy coat and washed twice 
 in roswell park memorial institute ( rpmi ) medium 1640 so that no white 
 blood cells were present .
the culture medium contained rpmi 1640 with 
 5 g / l albumax , 0.025 g / l gentamycin , and 0.292
sterile 96-well black tissue culture plates 
 ( costar ) were used routinely for every assay .
drugs were diluted in 
 culture medium and used in duplicate wells for each dilution of 10.0 , 
 3.333 , 1.111 , 0.370 , 0.123 , 0.041 , and 0.014 m in a final volume 
 of 100 l per well .
chloroquine was used as a standard with 
 10 times reduced concentration range as above .
two sets of control 
 were used in duplicate wells , one set with no added drugs ( positive 
 control ) and one with uninfected red blood cells ( negative control ) .
the plates were incubated at 37 c for 48 h in a gas chamber 
 flushed with 5% co2 , 5% o2 , and 90% n2 . after 48 h supernatants were taken from each well and replaced 
 with fresh drug and incubated for a further 48 h in the same manner . 
 at the end of the 96 h incubation , 25 l of sybr green
i dye 
 ( sybr green i nucleic acid gel stain 10000 , in dmso from invitrogen ) 
 in lysis buffer ( 1 l dye to 1 ml of lysis buffer ) was added 
 to each well and stored overnight at 20 c .
the lysis 
 buffer contained tris - hcl ( 20 mm , ph 8.0 ) , edta ( 2 mm ) , saponin ( 0.16% ) , 
 and triton x-100 ( 1.6% v / v ) .
plates were warmed to room temperature , 
 and fluorescence intensity was measured with a fluostar omega microplate 
 fluorescence reader ( bmg labtech ) .
binding of sybr green is specific for parasite 
 dna , as mature erythrocytes lack dna and rna .
fluorescence intensity 
 unit was converted to percentage ( % ) of growth as follows : this assay was performed 
 by dr .
christian scheurer at the swiss tropical 
 and public health institute and is a modified version of the original 
 hypoxanthine assay published by desjardins et al .
: serial drug dilutions were prepared with a multichannel 
 pipet , transferring 100 l in a 2-fold serial dilution .
an amount of 100 l 
 of infected blood ( parasitemia of 0.3% , 2.5% hematocrit ) was added 
 to all wells with a multipipette .
the plates were incubated 
 in an incubation chamber at 37 c in an atmosphere containing 
 93% n2 , 4% co2 , 3% o2 . after 48 h
, 
 an amount of 50 l of [ h]hypoxanthine ( = 0.5 ci ) 
 solution was added to each well of the plate .
the plates were incubated 
 for another 24 h. the plates were then harvested with a betaplate 
 cell harvester ( wallac , zurich , switzerland ) .
the dried filters were 
 inserted into a plastic foil with 10 ml of scintillation fluid and 
 counted in a betaplate liquid scintillation counter ( wallac , zurich , 
 switzerland ) .
the results were recorded as counts per minute ( cpm ) 
 per well at each drug concentration .
data were analyzed using 
 a graphic program ( e.g. , excel ) and expressed as percentage of the 
 untreated controls . the 50% inhibitory concentration ( ic50 ) value was evaluated by logit regression analysis .
this assay is a slightly 
 modified version of the assay previously described : hepg2-a16-cd81egfp cells stably transformed to express 
 a gfp - cd81 fusion protein were cultured at 37 c in 5% co2 in dmem ( invitrogen , carlsbad , ca , usa ) supplemented with 
 10% fcs , 0.29 mg / ml glutamine , 100 units of penicillin , and 100 g / ml 
 streptomycin .
the cells were seeded 24 h prior to infection into 1536-well 
 plates at 3000 cells / well .
the cells were pretreated for 12 h with 
 the drug in a 12-point dilution series , and the cells were then infected 
 with freshly dissected p. berghei sporozoites expressing 
 luciferase ( 1000 sporozoites / well ) .
after 48 h of incubation , the 
 viability of p. berghei exoerythrocytic forms ( eef ) 
 was measured by bioluminescence .
ic50 values were obtained 
 using the measured bioluminescence intensity and a nonlinear variable 
 slope four - parameter regression curve fitting model in prism 6 ( graphpad 
 software inc . ) .
measurement 
 of the ability of 34c to kill human cells ( hepg2 , human 
 hepatocellular carcinoma ) was performed using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h - tetrazolium , inner salt ( mts ) , cell viability assay , modified 
 from the promega corporation technical bulletin no .
the cells 
 were cultured in dulbecco s modified eagle medium ( dmem ) plus 
 10% fetal bovine serum ( fbs ) plus 1% penicillin / streptomycin , incubated 
 at 37 c in humidified atmosphere with 5% co2 .
then 200 l of medium was added to wells 27 of a 12-well 
 reservoir per replicate , along with 598 l of medium in well 
 1 .
compound was dissolved in dmso at a top concentration of 50 mm , 
 and 1.8 l of this stock was added to column 1 .
well 1 was mixed 
 thoroughly with a 1 ml pipet , and 200 l was transferred to 
 well 2 and mixed thoroughly .
furthermore , 
 a puromycin control was prepared by diluting 3 l of puromycin 
 stock ( 1 mg / ml ) in 1497 l of medium .
cells were added 
 to a 96-well plate at a concentration of 5000 cells per well ( 50 l ) , 
 excluding the exterior wells . to wells b2g2 and b11g11 
 is added 100 l of 0.2% dmso / medium ( positive control wells ) . 
 to wells
b3g3 was added 100 l of puromycin / medium ( negative 
 control wells ) . to wells b4b10 , c4c10 , and d4d10 

this was 
 repeated with a second compound if required in wells e4e10 , 
 f4f10 , and g4g10 .
the plate was placed in an incubator 
 at 37 c and cell growth / morphology inspected at 24 , 48 , and 
 72 h. at 72 h , 20 l of mts / pms solution ( 1.9 mg / ml mts , 
 43.8 g / ml pms ) was added to each assay well , and the plate 
 returned to the incubator for 4 h. fluorescence was then read at 490 
 nm on a spectramax m2e microplate reader from molecular devices .
data 
 were elaborated using microsoft office excel 2011 , and ld50 values were determined using grafit 7.0 ( erithacus software ltd . , 
 u.k . ) by nonlinear regression fitting .
crystals of ternary complexes of pvnmt , a nonhydrolyzable myristoyl - coa 
 analogue ( nhm ) , and the inhibitors 20b and 34a were prepared as described previously .
x - ray diffraction data were collected 
 on synchrotron beamlines at the diamond light source , harwell , u.k . , 
 and processed using xds and scala implemented within xia2 .
model refinement was by maximum likelihood methods 
 implemented in refmac using the protein 
 chains of 4a95.pdb as a starting model 
 interspersed with cycles of model building and adjustment using coot .
 n - myristoyltransferase ( nmt ) is an essential eukaryotic 
 enzyme and an attractive drug target in parasitic infections such 
 as malaria . 
 we have previously reported that 2-(3-(piperidin-4-yloxy)benzo[b]thiophen-2-yl)-5-((1,3,5-trimethyl-1h - pyrazol-4-yl)methyl)-1,3,4-oxadiazole ( 34c ) is a high 
 affinity inhibitor of both plasmodium falciparum and p. vivax nmt and displays activity in vivo against a rodent malaria model . 
 here 
 we describe the discovery of 34c through optimization 
 of a previously described series . 
 development , guided by targeting 
 a ligand efficiency dependent lipophilicity ( lelp ) score of less than 
 10 , yielded a 100-fold increase in enzyme affinity and a 100-fold 
 drop in lipophilicity with the addition of only two heavy atoms . 
 34c was found to be equipotent on chloroquine - sensitive and 
 -resistant cell lines and on both blood and liver stage forms of the 
 parasite . 
 these data further validate nmt as an exciting drug target 
 in malaria and support 34c as an attractive tool for 
 further optimization . 