Scholarly article on tert-butyl 4-((2-(phenethylcarbamoyl)benzo[b]thiophen-3-yl)oxy)piperidine-1-carboxylate 1581303-42-5 from Journal of Medicinal Chemistry p. 2773

Article

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Design and Synthesis of High Aﬃnity Inhibitors of Plasmodium

falciparum and Plasmodium vivax N-Myristoyltransferases Directed

by Ligand Eﬃciency Dependent Lipophilicity (LELP)

Mark D. Rackham,^†James A. Brannigan,^‡Kaveri Rangachari,^§Stephan Meister,^∥Anthony J. Wilkinson,^‡

Anthony A. Holder,^§Robin J. Leatherbarrow,^†,⊥and Edward W. Tate*^,†

^†Department of Chemistry, Imperial College London, London SW7 2AZ, U.K.

^‡Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5DD, U.K.

^§MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, U.K.

^∥Department of Pediatrics, School of Medicine, University of California San Diego, La Jolla, California 92093, United States

S

* Supporting Information

ABSTRACT: 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.

compounding the urgent requirement for additional therapeutic

agents targeting Plasmodium parasites.

INTRODUCTION

■

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,¹primarily aﬀecting developing countries

and children under the age of 5.²Although ﬁve species of

Plasmodium parasite are known to infect humans,^2,3two species

are responsible for the majority of morbidity and mortality:

Plasmodium falciparum (Pf) and Plasmodium vivax (Pv). These

species are the focus of the work described in this paper.

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)^4,5target 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.⁶Furthermore, resistance to chloroquine is

long established⁷and signs of artemisinin resistance have been

detected along the eastern⁸and western borders⁹of Thailand,

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.¹⁰In 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 are required.

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. Myr-

Received: January 13, 2014

Published: March 5, 2014

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Figure 1. 2,3-Substituted benzo[b]thiophene PfNMT and PvNMT inhibitor 1 and the target proﬁle for the development of this series. Footnote a: K_i

values are quoted in place of IC₅₀values as a means of expressing the inhibitor aﬃnity while correcting for diﬀering Michaelis constants (K_m) between

enzymes. Enzyme K_ivalues are calculated from the IC₅₀values using the Cheng−Prusoﬀ equation, the deﬁnition of which is given in the Experimental

Section.²³IC₅₀values 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. Footnote c: LELP = cLogP/LE. LE = [−log(K_i)](1.374)/(no. of heavy atoms), with cLogP determined with ChemAxon, which

can be obtained from http://www.chemaxon.com/products/calculator-plugins/logp/.

istoylation is a widespread modiﬁcation that occurs co- and post-

translationally and has hundreds of putative substrates in vivo.¹¹

Furthermore, myristoylation of proteins is known to modulate a

variety of properties (such as protein localization¹²and

stability¹³) and is implicated in a variety of critical biological

pathways.^14,15

distribution.²⁵In 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).²⁶Furthermore lipophilicity tends to inﬂate

throughout drug development.²⁷Indeed, 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”.²⁸

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

molecules²⁹and has been shown to be a strong predictor of

druglikeness.³⁰It 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 < 10.²⁹In 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, all

LELP values quoted in this work refer to PfNMT LE.

Consideration of the LELP of 1 (13.5, Figure 1) emphasizes

the requirement for signiﬁcant improvement in the physico-

chemical proﬁle of this series.

Previous work showed that an appropriately placed 3-

methoxyphenyl substituent can form π−π and hydrogen bonding

interactions with Phe105 and Ser319 of PvNMT, respectively.³¹

Attempts to replicate these interactions within the benzothio-

phene 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.²¹A series of

phenethyl esters and amides were therefore synthesized and

assayed against a panel of NMTs (Scheme 1, Table 1).

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.

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,¹⁶⁻¹⁸

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.¹⁹Further-

more, 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.²⁰Herein, 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.

RESULTS AND DISCUSSION

■

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).²¹1 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.²²Although selectivity over HsNMT is desirable,

selectivity at the cellular level was considered the more critical

determinant for progression.

Further SAR mimicked that described previously for benzyl

esters:³¹adding 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

The metric ligand eﬃciency (LE) has been used extensively in

early stage drug development, and its utility has been well

documented.²⁴The 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

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a

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.

Scheme 1. Synthesis of Phenethyl Esters and Amides

Improvements in LELP: Capitalizing on Matched

Molecular Pairs Analysis. 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.²⁹The next stage in

development therefore focused on improving the LELP of the

series by improving aﬃnity while reducing lipophilicity and

molecular weight.

a

Reagents and conditions: (a) 1-ethyl-3-(3-dimethylaminopropyl)-

carbodiimide, hydroxybenzotriazole, N,N-diisopropylethylamine,

MeCN, rt, 18 h, 36−57%; (b) benzotriazo-1-yl-oxytripyrrolidino-

phosphonium hexaﬂuorophosphate, N,N-diisopropylethylamine,

DCM, rt, 18 h, 69−93%; (c) 10% TFA in DCM (v/v), rt, 2 h, 42−

97%. 2 was prepared as described previously.²¹

Several strategies for lowering lipophilicity have been

described in the recent literature; one of the most successful is

matched molecular pairs analysis,³³the 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.^34,35Because of the

contrasting dipole moment of each heterocycle, 1,3,4-oxadia-

zoles display on average 10-fold lower lipophilicity and more

favorable proﬁles with respect to metabolic stability, hERG

inhibition, and aqueous solubility.³⁵Switching 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,³⁶as well as a higher

aromatic stabilization energy.³⁷

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.

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.

Ester Bioisosteric Replacement. 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 π-system, eliminating this potential issue. Crystallography

suggested that the interactions of the ester with the enzyme were

conﬁned to long-distance π-interactions with Tyr211 and

Phe105. In this scenario oxadiazoles are common bioisosteric

replacements for methylene esters,³²displaying increased

biological stability but with similar size and shape and increased

potential for π-interactions. Both 1,2,4-regioisomers were

prepared using complementary routes (Scheme 2).

Replacement of the ester linker with a 1,2,4-oxadiazole

produced an increase in aﬃnity for all three enzymes, presumably

due to π-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

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

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Table 1. Enzyme Aﬃnity and Plasmodium falciparum LELP for Phenethyl Esters and Amides

a

b

K_ivalues are the mean value of two or more determinations, and standard deviation is within 20% of the K_i. LELP not calculated as enzyme aﬃnity

c

above measurable range. 3D7 EC₅₀values are determined using a Sybr Green ﬂuorometric assay.

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

1.²¹Furthermore, 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).

Improvements in LELP: Investigations of the Solvated

Pocket. The crystal structure of 20b bound to PvNMT indicated

that there was a voluminous solvent-ﬁlled pocket surrounding

the methylene group α 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).^38,39In addition, this position is a potential target for

oxidative metabolism and blocking with alternative substituents

may provide beneﬁts during subsequent lead development.

The abundance of readily available α-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.

The results show that a wide variety of substitutions at this

position produce only small changes in enzyme aﬃnity,

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Scheme 2. Synthesis of Two Regioisomers of 1,2,4-Oxadiazole

Bioisosteres

Scheme 3. Synthesis of 1,3,4-Oxadiazole and 1,2,4-Triazole

Linker Bioisosteres

a

Reagents and conditions: (a) NH₂NH₂·H₂O, EtOH, 78 °C, 24 h,

75%; (b) RCH₂C(O)Cl, N,N-diisopropylethylamine, DCM, rt, 15

min, 75−91%; (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, 48−65%; (f) TFA, DCM, rt, 2

h, 55−98%.

a

Reagents and conditions: (a) NH₂OH·H₂O, EtOH, rt, 5 h, 98−99%;

result in a stronger hydrogen bond with Ser319 and an

improvement in aﬃnity.

(b) 2, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, hydroxyben-

zotriazole, DMF, 140 °C, 3 h, 10−31%; (c) 10% TFA in DCM (v/v),

rt, 2 h, 13−99%; (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 Å molecular sieves, toluene, 18 h, 110 °C,

54−68% over two steps.

Brand et al. previously described the development of a series of

highly potent Trypanosoma brucei NMT inhibitors⁴²that 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 π-systems are typically excellent hydrogen

bond acceptors,⁴¹and 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.

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. Interest-

ingly, 34a displays 30-fold selectivity between PfNMT and

HsNMT, a signiﬁcant improvement over the 8- to 9-fold

observed in 34b/34c.

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 23g,

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 perme-

ability). 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.

Improvements in LELP: Optimization of Polar Con-

tacts. 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;⁴⁰(ii) aryl ethers are

notoriously poor H-bond acceptors,⁴¹and so replacement may

Addition of the N-methyl group in 34c produces a marked

increase in aﬃnity against all enzymes, resulting in the highest

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Table 2. Enzyme Aﬃnity and LELP of Bioisosteric Heterocycle-Linked Inhibitors

aﬃnity Plasmodium spp. NMT inhibitor reported to date with

single-digit nanomolar K_iagainst both enzymes. 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.²⁹

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The binding mode of 34c was elucidated by crystallography, as

brieﬂy described previously²⁰(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).

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.

Figure 2. X-ray crystal structure of 20b (blue) bound to PvNMT

(green). (A) 20b bound to PvNMT. 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.

Blood and Liver Stage in Vitro Cellular Activity. 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.

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.²⁰Pleasingly,

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

Figure 3. 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 CH₂occupies 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 Å of the benzylic position.

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.²⁰

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.

a

Scheme 4. Synthesis of α-Substituted Phenyl 1,3,4-Oxadiazole Inhibitors

a

Reagents and conditions: (a) Ph-CR₁R₂-CO₂H, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, hydroxybenzotriazole, THF, DMF,

rt, 18 h, 48−99%; (b) TsCl, 1,2,2,6,6pentamethylpiperidine, DCM, rt, 18 h; (c) 10% TFA in DCM (v/v), rt, 2 h, 4−40% over two steps.

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Table 3. Enzyme Aﬃnity and LELP of α-Substituted Phenyl 1,3,4-Oxadiazole Inhibitors

models. 34c had a liver stage EC₅₀of 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.⁴³With 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

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a

Scheme 5. Synthesis of Five-Membered Heterocyclic Methoxyphenyl Replacements

a

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) NH₂NH₂·H₂O, MeOH, rt, 3 h, 83−99%; (d) n = 1, NH₂OH·HCl, K₂CO₃, EtOH, 78 °C, 3 h, 12%; n = 2, NH₂OH·HCl, H₂O, MeOH, 60 °C, 18

h, 89%; (e) MeNHNH₂, AcOH, 3 h, rt, 73−95%; (f) LiOH·H₂O, MeOH, rt, 18 h, 51−95%; (g) 16, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

hydrochloride, hydroxybenzotriazole, THF, DMF, rt, 18 h, 48−99%; (h) TsCl, 1,2,2,6,6-pentamethylpiperidine, DCM, rt, 18 h; (i) 10% TFA in

DCM (v/v), rt, 2 h, 3−26% over two steps.

the moderate selectivity is a functional of low eﬃciacy rather than

high toxicity; the LD₅₀for 34c is approximately the top

concentration tested (10 μM) for the antimalarial standards.

Eﬀects of LELP on Cellular Activity. 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.²⁹In an attempt to assess if LELP is a useful metric in

this regard, the cellular EC₅₀against the 3D7 parasite line was

plotted against NMT aﬃnity for all tested members of this series.

Analysis of the EC₅₀/K_icorrelation is shown in Figure 6.

An initial assessment of cellular potency vs enzyme aﬃnity

showed only a weak correlation (R²= 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 (R²= 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 K_i(R²= 0.36) and a ﬂat gradient (0.23), indicating that

antiparasitic activity from these compounds derives predom-

inantly 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 analogues²⁹and 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”,⁴⁴

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.

CONCLUSION

■

Previously reported work described the discovery of 1 by a lead-

hopping approach, as a ligand eﬃcient inhibitor of plasmodial

NMT.²¹This 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.²⁰Taken 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.

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Table 4. Enzyme Aﬃnity and LELP of Heterocyclic Methoxyphenyl Replacements

EXPERIMENTAL SECTION

■

General. All chemicals were purchased from Sigma-Aldrich Ltd.

(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.) automated apparatus with SNAP silica cartridges

(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.

Figure 4. Binding mode of 34c (gold) bound to PvNMT. (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.

HPLC involved the following: Gilson semipreparative reverse phase

HPLC system equipped with a HICHROM C₁₈column (250 mm ×

21.2 mm), no. 306 pumps, and a Gilson UV/vis detector, detecting at

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following elution method was used: gradient of solvent A and solvent B

(as above) of 0−10 min 50−98% B, 10−12 min 98% B, 12−13 min 98−

50% B, 13−17 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 μm, 4.6 mm × 100 mm)

column, equipped with an XBridge C18 guard column (5 μm, 4.6 mm ×

20 mm). The following elution method was used: gradient of solvent A

and solvent B (as above) of 0−10 min 5−98% B, 10−12 min 98% B, 12−

13 min 98−5% B, 13−17 min 5% B. Flow rate was 1.2 mL/min. Purity of

tested compounds was ≥95% unless otherwise speciﬁed.

¹H and ¹³C NMR spectra were respectively recorded on 400 and 101

MHz Bruker AV instruments at room temperature unless speciﬁed

1

otherwise. In these cases H and ¹³C 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.

Prototypical Procedure for Preparation of Ester Family 4. 1

and 2 were prepared as described previously.²¹

Figure 5. Binding mode of 34a (pink) bound to PvNMT. (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-dimethylpyr-

azole of 34a (pink) with PvNMT (green). The pyrazole of 34a forms a

direct interaction with Ser319 (3.2 Å), and the water involved in the

bridged interaction in Figure 4B has been excluded from the pocket.

tert-Butyl 4-((2-(((3-Methoxybenzyl)oxy)carbonyl)benzo[b]-

thiophen-3-yl)oxy)piperidine-1-carboxylate (3b). 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 μL, 0.16 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)-

carbodiimide hydrochloride (30 mg, 0.16 mmol). The reaction mixture

was stirred at room temperature for 15 min. 2-(3-Methoxyphenyl)-

ethanol (22 μL, 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 (10g SNAP

cartridge, 6−50% B, R_f= 5.1 column volumes) to give 3b as a colorless

oil (24 mg, 36%). ¹H NMR (CDCl₃, δ, ppm) 7.86 (1H, d, J = 8.0), 7.75

(1H, d, J = 8.2), 7.51−7.46 (1H, m), 7.43−7.37 (1H, m), 7.32 (1H, dd, J

= 8.0, 7.8), 7.04 (1H, d, J = 7.8), 7.02−7.00 (1H, m), 6.90 (1H, dd, J =

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 0−2 min 50% B, 2−30 min 50−98% B, 30−32 min 98%, 32−32.5 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 μm, 19 mm × 100 mm) column, equipped

with an XBridge Prep C18 guard column (5 μm, 19 mm × 10 mm). The

Table 5. In Vitro Cellular Assay Data for 34c against Both Parasitic and Human Cells, Compared to Chloroquine (36) and

Atovaquone (37)

a

NF54, K1, and Dd2 EC₅₀values are determined using a [³H]hypoxanthine incorporation assay (performed by Dr. Sergio Wittlin and Dr. Christian

b

Scheurer, Swiss Tropical and Public Health Institute). P. berghei liver stage EC₅₀values determined using a luciferase bioluminescence assay. EC₅₀

value is the mean of two determinations. Standard deviation is within 50% of the EC₅₀. LD₅₀values determined using an MTS cellular viability assay.

c

LD₅₀value is the mean of six determinations. Standard deviation is within 20% of the LD₅₀.

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(40 mg, 0.11 mmol) in N,N-dimethylformamide (1 mL) were added

hydroxybenzotriazole (16 mg, 0.12 mmol) and 1-ethyl-3-(3-dimethyl-

aminopropyl)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, 6−

50% B, R_f= 7.5 column volumes) to give 8a as a clear yellow oil (16 mg,

31%). ¹H NMR (CDCl₃, δ, 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), 7.46−7.28 (6H, m), 4.63−

4.54 (1H, m), 4.16 (2H, s), 3.98−3.87 (2H, m), 3.00−2.91 (2H, m),

2.01−1.93 (2H, m), 1.90−1.78 (2H, m), 1.49 (9H, s).

3-Benzyl-5-(3-(piperidin-4-yloxy)benzo[b]thiophen-2-yl)-1,2,4-ox-

adiazole (9a). 9a was prepared as in 4b replacing 3b with 8a (16 mg,

0.03 mmol) and puriﬁed by LC−MS, yielding 9a as a white solid (12 mg,

94%). t_R= 4.22 min; ¹H NMR (CDCl₃, δ, 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), 7.40−7.28

(5H, m), 4.80−4.72 (1H, m), 4.16 (2H, s), 3.56−3.45 (2H, m), 3.04−

2.95 (2H, m), 2.22−2.15 (4H, m); ¹³C NMR (CDCl₃, δ, 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).

3-Hydroxybenzo[b]thiophene-2-carbonitrile (10). To a solution of

methyl-2-mercaptobenzoate (1.64 mL, 11.9 mmol) and 2-bromoaceto-

nitrile (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. 10 was immediately extracted with 3 ×

75 mL portions of ethyl acetate. 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%). ¹H NMR (CDCl₃, δ, ppm) 8.54 (1H,

brs), 7.91 (1H, d, J = 8.1 Hz), 7.73 (1H, d, J = 8.2 Hz), 7.62−7.50 (1H,

m), 7.51−7.40 (1H, m).

tert-Butyl 4-((2-Cyanobenzo[b]thiophen-3-yl)oxy)piperidine-1-

carboxylate (11). 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, 2−18% B, R_f=

Figure 6. (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.

1

8.5 column volumes) to give 11 as a white solid (800 mg, 78%). H

8.0, 2.3), 5.35 (2H, s), 4.74−4.66 (1H, m), 3.94−3.86 (2H, m), 3.84

(3H, s), 3.07−2.98 (2H, m), 1.98−1.88 (2H, m), 1.85−1.73 (2H, m),

1.48 (9H, s).

NMR (CDCl₃, δ, 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), 3.85−3.74 (2H, m), 3.46−3.36 (2H,

m), 2.18−2.07 (2H, m), 1.98−1.87 (2H, m), 1.49 (9H, s).

3-Methoxyphenethyl 3-(Piperidin-4-yloxy)benzo[b]thiophene-2-

carboxylate (4b). To a solution of 3b (23.0 mg, 0.05 mmol) in

dichloromethane (1 mL) was added triﬂuoroacetic acid (100 μL), and

the solution was stirred at room temperature for 2 h. The reaction

mixture was concentrated under reduced pressure and puriﬁed by

(Z)-tert-Butyl 4-((2-(N′-Hydroxycarbamimidoyl)benzo[b]-

thiophen-3-yl)oxy)piperidine-1-carboxylate (12). To a solution of

11 (100 mg, 0.28 mmol) in ethanol (2 mL) was added hydroxylamine as

50% aqueous solution (170 μL, 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

1

HPLC, yielding 4b as a colorless oil (5 mg, 18%). t_R= 12.3 min; H

NMR (CDCl₃, δ, ppm) 7.82 (1H, d, J = 8.0), 7.76 (1H, d, J = 8.1), 7.56−

7.48 (1H, m), 7.47−7.40 (1H, m), 7.33 (1H, app t, J = 7.9), 7.04 (1H, d,

J = 7.4), 7.01−6.98 (1H, m), 6.91 (1H, dd, J = 8.2, 2.4), 5.34 (2H, s),

4.88−4.81 (1H, m), 3.84 (3H, s), 3.54−3.44 (2H, m), 3.11−3.01 (2H,

m), 2.22−2.11 (4H, m); ¹³C NMR (CDCl₃, δ, 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).

Prototypical Procedure for Preparation of 1,2,4-Oxadiazole

(9/14b). tert-Butyl 4-((2-(3-Benzyl-1,2,4-oxadiazol-5-yl)benzo[b]-

thiophen-3-yl)oxy)piperidine-1-carboxylate (8a). To a solution of 2

1

mg, 99%). H NMR (CDCl₃, δ, ppm) 7.77−7.73 (1H, m), 7.72−7.67

(1H, m), 7.41−7.37 (2H, m), 6.25 (1H, brs), 5.55 (2H, brs), 4.50−4.41

(1H, m), 4.17−3.99 (2H, m), 2.87 (2H, ddd, J = 14.2, 11.7, 2.6 Hz),

2.12−2.00 (2H, m), 1.87−1.74 (2H, m), 1.48 (9H, s).

tert-Butyl 4-((2-(5-(3-Methoxybenzyl)-1,2,4-oxadiazol-3-yl)benzo-

[b]thiophen-3-yl)oxy)piperidine-1-carboxylate (13b). 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

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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 Å 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 (10g SNAP cartridge,

5−40% B, R_f= 8.0 column volumes) to give 13b as a clear orange oil (66

mg, 68%). ¹H NMR (CDCl₃, δ, 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), 7.33−7.28 (1H,

m), 6.97 (1H, d, J = 8.0 Hz), 6.95−6.92 (1H, m), 6.87 (1H, dd, J = 8.0,

2.4 Hz), 4.60−4.50 (1H, m), 4.28 (2H, s), 4.00−3.89 (2H, m), 3.83 (3H,

s), 3.02−2.93 (2H, m), 2.01−1.92 (2H, m), 1.89−1.77 (2H, m), 1.48

(9H, s).

5-(3-Methoxybenzyl)-3-(3-(piperidin-4-yloxy)benzo[b]thiophen-

2-yl)-1,2,4-oxadiazole (14b). 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%). t_R= 9.7 min; ¹H NMR (CDCl₃, δ, ppm) 8.03

(1H, dd, J = 7.0, 1.3 Hz), 7.92−7.84 (1H, m), 7.58−7.47 (2H, m), 7.31

(1H, app t, J = 7.9 Hz), 7.04−7.00 (1H, m), 6.99−6.94 (1H, m), 6.94−

6.87 (1H, m), 4.66 (1H, tt, J = 7.7, 3.6 Hz), 4.43 (2H, s), 3.76 (3H, s),

3.44−3.27 (2H, m), 3.03−2.90 (2H, m), 2.13−2.03 (2H, m), 2.01−1.89

(2H, m); ¹³C NMR (CDCl₃, δ, 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).

Prototypical Procedure for Preparation of 1,3,4-Oxadiazole

(23a). tert-Butyl 4-((2-(Hydrazinecarbonyl)benzo[b]thiophen-3-yl)-

oxy)piperidine-1-carboxylate (16). To a solution of 15 (300 mg, 0.74

mmol) in ethanol (1 mL) was added hydrazine monohydrate (145 μL,

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%). ¹H NMR (CDCl₃, δ, ppm)

7.79 (1H, d, J = 7.6 Hz), 7.74 (1H, d, J = 7.2 Hz), 7.48−7.37 (2H, m),

4.58 (1H, tt, J = 9.8, 4.1 Hz), 4.13−4.04 (2H, m), 2.95−2.82 (2H, m),

2.14−2.05 (2H, m), 1.92−1.78 (2H, m), 1.48 (9H, s).

Procedure for Preparation of 34c. Ethyl 3-Acetyl-4-oxopenta-

noate (24). 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

1

CDCl₃at room temperature. Diketone H NMR (CDCl₃, δ, ppm)

4.20−4.12 (3H, m), 2.90 (2H, d, J = 7.3 Hz), 2.29 (6H, s), 1.31−1.25

(3H, m); enol ¹H NMR (CDCl₃, δ, ppm) 4.22−4.08 (2H, m), 3.25 (2H,

s), 2.17 (6H, s), 1.32−1.23 (3H, m).

Ethyl 2-(1,3,5-Trimethyl-1H-pyrazol-4-yl)acetate (26c). To a

solution of 24 (400 mg, 2.15 mmol) in acetic acid (3 mL) was added

methylhydrazine (125 μL, 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%). ¹H NMR (CDCl₃, δ, 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).

2-(1,3,5-Trimethyl-1H-pyrazol-4-yl)acetic Acid (28c). 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%). ¹H NMR (CDCl₃,

δ, ppm) 3.80 (3H, s), 3.41 (2H, s), 2.24 (3H, s), 2.23 (3H, s).

tert-Butyl 4-((2-(2-(2-(1,3,5-Trimethyl-1H-pyrazol-4-yl)acetyl)-

hydrazinecarbonyl)benzo[b]thiophen-3-yl)oxy)piperidine-1-carbox-

ylate (30c). 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%). ¹H NMR (CDCl₃, δ, ppm) 9.75 (1H, d, J = 6.2 Hz), 7.89−7.72

(3H, m), 7.53−7.37 (2H, m), 4.75−4.65 (1H, m), 4.12−4.06 (2H, m),

3.76 (3H, s), 3.46 (2H, s), 2.86−2.82 (2H, m), 2.26 (6H, s), 2.14−2.09

(2H, m), 1.99−1.87 (2H, m), 1.48 (9H, s).

2-(3-(Piperidin-4-yloxy)benzo[b]thiophen-2-yl)-5-((1,3,5-trimeth-

yl-1H-pyrazol-4-yl)methyl)-1,3,4-oxadiazole (34c). 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%). t_R= 7.1 min; ¹H

NMR (CD₃OD, δ, ppm) 8.47 (1H, brs), 7.99−7.88 (2H, m), 7.61−7.45

(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), 2.21−2.04 (4H, m); ¹³C NMR (CD₃OD, δ, 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).

Enzyme Inhibition Assay. All IC₅₀determinations were carried out

using a 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin

(CPM) ﬂuorescence assay, as described previously for HsNMT1,⁴⁵

PvNMT,⁴⁶and PfNMT.²¹

IC₅₀values are the mean value of two or more determinations, and

standard deviation is within 20% of the IC₅₀unless otherwise speciﬁed.

Data were elaborated using Microsoft Oﬃce Excel 2010, and IC₅₀values

were determined using GraFit 7.0 (Erithacus Software Ltd., U.K.) by

nonlinear regression ﬁtting, which were then quoted as K_ias deﬁned

below.

tert-Butyl 4-((2-(2-(2-Phenylpropanoyl)hydrazinecarbonyl)benzo-

[b]thiophen-3-yl)oxy)piperidine-1-carboxylate (21a). 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 μL, 0.15 mmol).

Reaction mixture was stirred at room temperature for 18 h and then

diluted with 1.0 M NaOH_(aq)(4 mL). 21a was extracted with EtOAc (2

× 5 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%). ¹H NMR (CDCl₃, δ, ppm)

7.82−7.76 (2H, m), 7.49−7.20 (7H, m), 4.68 (1H, tt, J = 10.0, 4.1 Hz),

4.19−4.06 (2H, m), 3.85 (1H, q, J = 7.2 Hz), 2.88−2.79 (2H, m), 2.16−

2.05 (2H, m), 2.02−1.88 (2H, m), 1.60 (3H, d, J = 7.2 Hz), 1.48 (9H, s).

2-(1-Phenylethyl)-5-(3-(piperidin-4-yloxy)benzo[b]thiophen-2-yl)-

1,3,4-oxadiazole (23a). To a solution of 21a (63 mg, 0.12 mmol) and

1,2,2,6,6-pentamethylpiperidine (47 μL, 0.26 mmol) in dichloro-

methane (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

K_iCalculations. K_ivalues quoted are the K_icalculated from the

experimentally determined IC₅₀values, the substrate concentration

([S]), and the Michaelis−Menten constant (K_m) as described by the

Cheng−Prusoﬀ equation:²³

1

yielding 23a as a yellow oil (7 mg, 14%). t_R= 11.0 min; H NMR

(CDCl₃, δ, ppm) 8.44 (1H, brs), 7.85−7.73 (2H, m), 7.54−7.30 (7H,

m), 4.65 (1H, brs), 4.44 (1H, q, J = 6.6 Hz), 3.53−3.35 (2H, m), 3.06−

2.86 (2H, m), 2.20−2.01 (4H, m), 1.84 (3H, d, J = 6.6 Hz); ESI HRMS,

found 406.1587 (C₂₃H₂₄N₃O₂S, [M + H]⁺, requires 406.1589).

IC₅₀

K_i=

[S]

1 +

K_m

(1)

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For example, 34c had an experimentally determined PfNMT IC₅₀of

0.017 0.002 μM. The Michaelis constant (K_m) was 3.64 μM and the

substrate concentration was 4.00 μM, resulting in a K_iof 0.008 μM. K_m

values were calculated as described previously.⁴⁵

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. IC₅₀values were

obtained using the measured bioluminescence intensity and a nonlinear

variable slope four-parameter regression curve ﬁtting model in Prism 6

(GraphPad Software Inc.).

Plasmodium falciparum Sybr Green Viability Assay. Plasmo-

dium falciparum Culture. Synchronous Plasmodium falciparum 3D7

late stage trophozoites at 33−36 h were used. Final parasitemia and

hematocrit were 0.1−0.2% and 2%, respectively. 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 g/L ^L-glutamine.

SYBR Green Assay. 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 μM in a ﬁnal volume of 100 μL 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 μL of

SYBR Green I dye (SYBR Green I nucleic acid gel stain 10000×, in

DMSO from Invitrogen) in lysis buﬀer (1 μL 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).

HepG2 Toxicity Assay. 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. TB169.

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 μL of medium was added to wells 2−7 of a 12-well reservoir

per replicate, along with 598 μL of medium in well 1. Compound was

dissolved in DMSO at a top concentration of 50 mM, and 1.8 μL of this

stock was added to column 1. Well 1 was mixed thoroughly with a 1 mL

pipet, and 200 μL was transferred to well 2 and mixed thoroughly. This

was repeated until well 7. Furthermore, a puromycin control was

prepared by diluting 3 μL of puromycin stock (1 mg/mL) in 1497 μL of

medium.

Cells were added to a 96-well plate at a concentration of 5000 cells per

well (50 μL), excluding the exterior wells. To wells B2−G2 and B11−

G11 is added 100 μL of 0.2% DMSO/medium (positive control wells).

To wells B3−G3 was added 100 μL of puromycin/medium (negative

control wells). To wells B4−B10, C4−C10, and D4−D10 was added

100 μL of each of the seven drug dilutions. This was repeated with a

second compound if required in wells E4−E10, F4−F10, and G4−G10.

The plate was placed in an incubator at 37 °C and cell growth/

morphology inspected at 24, 48, and 72 h.

Plates were warmed to room temperature, and ﬂuorescence intensity

was measured with a FLUOstar Omega microplate ﬂuorescence reader

(BMG Labtech). Values were expressed in relative ﬂuorescence units.

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:

At 72 h, 20 μL of MTS/PMS solution (1.9 mg/mL MTS, 43.8 μg/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 LD₅₀values were

determined using GraFit 7.0 (Erithacus Software Ltd., U.K.) by

nonlinear regression ﬁtting.

(culture under drug) − (uninfected RBC)

(culture with no drug) − (uninfected RBC)

% growth =

× 100

Crystallography. Crystals of ternary complexes of PvNMT, a

nonhydrolyzable myristoyl-CoA analogue (NHM),⁴⁸and the inhibitors

20b and 34a were prepared as described previously.²¹

X-ray diﬀraction data were collected on synchrotron beamlines at the

Diamond Light Source, Harwell, U.K., and processed using XDS⁴⁹and

SCALA⁵⁰implemented within xia2.⁵¹

Model reﬁnement was by maximum likelihood methods imple-

mented in REFMAC⁵²using the protein chains of 4A95.pdb⁴⁶as a

starting model interspersed with cycles of model building and

adjustment using COOT.⁵³A summary of data collection and

reﬁnement statistics is in Supporting Information.

Plasmodium falciparum [³H]Hypoxanthine Assay. This assay

was performed by Dr. Sergio Wittlin and 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.:⁴⁷

Serial drug dilutions were prepared with a multichannel pipet,

transferring 100 μL in a 2-fold serial dilution. Wells of rows A served as

controls without drug. An amount of 100 μL of infected blood

(parasitemia of 0.3%, 2.5% hematocrit) was added to all wells with a

multipipette. The control wells (A9−A12) received uninfected blood of

2.5% hematocrit. 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 μL of [³H]hypoxanthine (=0.5 μCi) 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.

Plasmodium berghei Liver Stage Assay. This assay is a slightly

modiﬁed version of the assay previously described:⁵

HepG2-A16-CD81EGFP cells stably transformed to express a GFP-

CD81 fusion protein were cultured at 37 °C in 5% CO₂in DMEM

(Invitrogen, Carlsbad, CA, USA) supplemented with 10% FCS, 0.29

mg/mL glutamine, 100 units of penicillin, and 100 μg/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

ASSOCIATED CONTENT

■

S

* Supporting Information

Experimental procedures and characterization of all intermedi-

ates and target compounds, full biological results, and X-ray

crystallographic collection data and reﬁnement statistics. This

material is available free of charge via the Internet at http://pubs.

acs.org.

Accession Codes

The coordinates and structure factor ﬁles have been deposited in

the Protein Data Bank under the accession codes 4CAE

(PvNMT-NHM-20b) and 4CAF (PvNMT-NHM-34a).

AUTHOR INFORMATION

■

Corresponding Author

*Phone: +44 (0) 20 7594 3752. E-mail: e.tate@imperial.ac.uk.

2786

dx.doi.org/10.1021/jm500066b | J. Med. Chem. 2014, 57, 2773−2788

Journal of Medicinal Chemistry

Article

PfCRT and evidence for their role in chloroquine resistance. Mol. Cell

2000, 6, 861−871.

Present Address

^⊥R.J.L.: Liverpool John Moores University, Egerton Court, 2

Rodney Street, Liverpool L1 2UA, U.K.

(8) Dondorp, A. M.; Nosten, F.; Yi, P.; Das, D.; Phyo, A. P.; Tarning, J.;

Lwin, K. M.; Ariey, F.; Hanpithakpong, W.; Lee, S. J.; Ringwald, P.;

Silamut, K.; Imwong, M.; Chotivanich, K.; Lim, P.; Herdman, T.; An, S.

S.; Yeung, S.; Singhasivanon, P.; Day, N. P. J.; Lindegardh, N.; Socheat,

D.; White, N. J. Artemisinin resistance in Plasmodium falciparum malaria.

N. Engl. J. Med. 2009, 361, 455−467.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

(9) Phyo, A. P.; Nkhoma, S.; Stepniewska, K.; Ashley, E. A.; Nair, S.;

McGready, R.; ler Moo, C.; Al-Saai, S.; Dondorp, A. M.; Lwin, K. M.;

Singhasivanon, P.; Day, N. P.; White, N. J.; Anderson, T. J.; Nosten, F.

Emergence of artemisinin-resistant malaria on the western border of

Thailand: a longitudinal study. Lancet 2012, 379, 1960−1966.

(10) Medicines for Malaria Venture. Interactive R&D Portfolio.

http://www.mmv.org/research-development/rd-portfolio (accessed

Dec 8, 2013).

(11) Wright, M. H.; Heal, W. P.; Mann, D. J.; Tate, E. W. Protein

myristoylation in health and disease. J. Chem. Biol. 2010, 3, 19−35.

(12) Tull, D.; Heng, J.; Gooley, P. R.; Naderer, T.; McConville, M. J.

Acylation-dependent and independent membrane targeting and distinct

functions of small myristoylated proteins (SMPs) in Leishmania major.

Int. J. Parasitol. 2012, 42, 239−247.

The authors are grateful to Andrew Bell, Jennie Hutton, and

Zhiyong Yu for valuable discussions. We thank Sergio Wittlin

and Christian Scheurer for performing assays on NF54, K1, and

Dd2 Pf cell lines, and Elizabeth A. Winzeler and colleagues for

enabling access to the liver stage assay. We also thank the

Medicines for Malaria Venture (MMV) for funding Elizabeth A.

Winzeler to support liver assays at University of California San

Diego, and Sergio Wittlin to support cell-line testing at Swiss

TPH. We thank William Heal and Zhiyong Yu for performing the

HepG2 toxicity assay. We thank Munira Grainger for providing

the parasites used in the in vitro assay, and Barbara Clough for

performing the assay. We acknowledge the Diamond Light

Source, Harwell, U.K., for the provision of data collection

facilities and Shirley Roberts for expert crystal handling. This

work was supported by the Engineering and Physical Sciences

Research Council (Doctoral Prize Fellowship) and Medical

Research Council (MRC Grants 0900278 and U117532067).

(13) Patwardhan, P.; Resh, M. D. Myristoylation and membrane

binding regulate c-Src stability and kinase activity. Mol. Cell. Biol. 2010,

30, 4094−4107.

(14) Resh, M. D. Targeting protein lipidation in disease. Trends Mol.

Med. 2012, 18, 206−214.

(15) Zhao, H.; Sun, J.; Deschamps, A. M.; Kim, G.; Liu, C.; Murphy, E.;

Levine, R. L. Myristoylated methionine sulfoxide reductase A protects

the heart from ischemia−reperfusion injury. Am. J. Physiol.: Heart Circ.

Physiol. 2011, 301, H1513−H1518.

ABBREVIATIONS USED

■

nd, not determined; Pb, Plasmodium berghei; Pv, Plasmodium

vivax; Pf, Plasmodium falciparum; Ld, Leishmania donovani;

NMT, N-myristoyltransferase; Hs, Homo sapiens; LE, ligand

eﬃciency; LELP, cLogP/LE; MTS, 3-(4,5-dimethylthiazol-2-yl)-

5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazo-

lium, inner salt; DMEM, Dulbecco’s modiﬁed Eagle medium;

FBS, fetal bovine serum; PMS, phenazine methosulfate

(16) Poulin, B.; Patzewitz, E. M.; Brady, D.; Silvie, O.; Wright, M. H.;

Ferguson, D. J.; Wall, R. J.; Whipple, S.; Guttery, D. S.; Tate, E. W.;

Wickstead, B.; Holder, A. A.; Tewari, R. Unique apicomplexan IMC sub-

compartment proteins are early markers for apical polarity in the malaria

parasite. Biol. Open 2013, 2, 1160−1170.

(17) Moskes, C.; Burghaus, P. A.; Wernli, B.; Sauder, U.; Durrenberger,

̈

M.; Kappes, B. Export of Plasmodium falciparum calcium-dependent

protein kinase 1 to the parasitophorous vacuole is dependent on three

N-terminal membrane anchor motifs. Mol. Microbiol. 2004, 54, 676−

691.

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Design and synthesis of high affinity inhibitors of Plasmodium falciparum and Plasmodium vivax N-myristoyltransferases directed by ligand efficiency dependent lipophilicity (LELP)

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