Discovery of a Plasmodium falciparum Glucose-6-phosphate Dehydrogenase 6-phosphogluconolactonase Inhibitor (R,Z)-N-((1-Ethylpyrrolidin-2-yl)methyl)-2- (2-fluorobenzylidene)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxamide (ML276) that reduces parasite growth in vitro

Article abstract of DOI:10.1021/jm300833h

A high-throughput screen of the NIH's MLSMR collection of ～340000 compounds was undertaken to identify compounds that inhibit Plasmodium falciparum glucose-6-phosphate dehydrogenase (Pf G6PD). PfG6PD is important for proliferating and propagating P. falciparum and differs structurally and mechanistically from the human orthologue. The reaction catalyzed by glucose-6-phosphate dehydrogenase (G6PD) is the first, rate-limiting step in the pentose phosphate pathway (PPP), a key metabolic pathway sustaining anabolic needs in reductive equivalents and synthetic materials in fast-growing cells. In P. falciparum, the bifunctional enzyme glucose-6-phosphate dehydrogenase-6- phosphogluconolactonase (Pf GluPho) catalyzes the first two steps of the PPP. Because P. falciparum and infected host red blood cells rely on accelerated glucose flux, they depend on the G6PD activity of PfGluPho. The lead compound identified from this effort, (R,Z)-N-((1-ethylpyrrolidin-2-yl)methyl)-2-(2- fluorobenzylidene)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carbox-amide, 11 (ML276), is a submicromolar inhibitor of PfG6PD (IC₅₀ = 889 nM). It is completely selective for the enzyme's human isoform, displays micromolar potency (IC₅₀ = 2.6 μM) against P. falciparum in culture, and has good drug-like properties, including high solubility and moderate microsomal stability. Studies testing the potential advantage of inhibiting Pf G6PD in vivo are in progress.

Full text of DOI:10.1021/jm300833h

Article
pubs.acs.org/jmc
Discovery of a Plasmodium falciparum Glucose-6-phosphate
Dehydrogenase 6‑phosphogluconolactonase Inhibitor (R,Z)‑N‑((1-
Ethylpyrrolidin-2-yl)methyl)-2-(2-fluorobenzylidene)-3-oxo-3,4-
dihydro‑2H‑benzo[b][1,4]thiazine-6-carboxamide (ML276) That
Reduces Parasite Growth in Vitro
Janina Preuss,†,‡,§,⊥ Patrick Maloney,∥,⊥ Satyamaheshwar Peddibhotla,∥,⊥ Michael P. Hedrick,§,⊥
Paul Hershberger,∥,⊥ Palak Gosalia,§ Monika Milewski,§ Yujie Linda Li,§ Eliot Sugarman,∥ Becky Hood,∥
Eigo Suyama,∥ Kevin Nguyen,∥ Stefan Vasile,∥ Eduard Sergienko,§ Arianna Mangravita-Novo,∥
Michael Vicchiarelli,∥ Danielle McAnally,∥ Layton H. Smith,∥ Gregory P. Roth,∥ Jena Diwan,§
Thomas D.Y. Chung,§ Esther Jortzik,‡ Stefan Rahlfs,‡ Katja Becker,*,‡ Anthony B. Pinkerton,*,§
and Lars Bode*,†
†Department of Pediatrics, University of California, San Diego, La Jolla, California 92037, United States
‡Biochemistry and Molecular Biology, Interdisciplinary Research Center, Justus Liebig University, Heinrich-Buff-Ring 26-32, 35392
Giessen, Germany
§Conrad Prebys Center for Chemical Genomics, Sanford−Burnham Medical Research Institute, La Jolla, California 92037, United
States
∥Conrad Prebys Center for Chemical Genomics, Sanford−Burnham Medical Research Institute, Orlando, Florida 32827, United
States
S
* Supporting Information
ABSTRACT: A high-throughput screen of the NIH’s MLSMR collection of ∼340000
compounds was undertaken to identify compounds that inhibit Plasmodium falciparum
glucose-6-phosphate dehydrogenase (Pf G6PD). Pf G6PD is important for proliferating and
propagating P. falciparum and differs structurally and mechanistically from the human
orthologue. The reaction catalyzed by glucose-6-phosphate dehydrogenase (G6PD) is the
first, rate-limiting step in the pentose phosphate pathway (PPP), a key metabolic pathway
sustaining anabolic needs in reductive equivalents and synthetic materials in fast-growing
cells. In P. falciparum, the bifunctional enzyme glucose-6-phosphate dehydrogenase-6-
phosphogluconolactonase (PfGluPho) catalyzes the first two steps of the PPP. Because P. falciparum and infected host red blood
cells rely on accelerated glucose flux, they depend on the G6PD activity of Pf GluPho. The lead compound identified from this
effort, (R,Z)-N-((1-ethylpyrrolidin-2-yl)methyl)-2-(2-fluorobenzylidene)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carbox-
amide, 11 (ML276), is a submicromolar inhibitor of Pf G6PD (IC50 = 889 nM). It is completely selective for the enzyme’s human
isoform, displays micromolar potency (IC50 = 2.6 μM) against P. falciparum in culture, and has good drug-like properties,
including high solubility and moderate microsomal stability. Studies testing the potential advantage of inhibiting PfG6PD in vivo
are in progress.
classes, namely 4-aminoquinolines such as chloroquine and
amodiaquine, arylaminoalcohols such as quinine and meflo-
quine, 8-aminoquinolines such as primaquine, artemisinins such
as artemether and artesunate as well as synthetic peroxides such
as tri- and tetraoxanes,8 antifolates such as sulfadoxine and
pyrimethamine, inhibitors of the respiratory chain such as
atovaquone, and antibiotics.9,10 The occurrence of drug-
resistant P. falciparum strains11−13 as well as side effects from
drug administration14,15 resulted in the current WHO
INTRODUCTION
■
In 2010, around 1.2 million people died from malaria
infections.1 Severe forms of the disease are mainly caused by
Plasmodium parasites of the species falciparum, which are
transmitted to the human host by female Anopheles
mosquitoes.2 P. falciparum parasites have infected humans for
thousands to millions of years,3,4 and yet malaria eradication
has not been possible due to the parasites’ ability to develop
resistance to most antimalarial drugs.5 To date, no vaccine
against malaria is available,6 and infections are treated with
drugs commonly targeting the blood stage of the parasite.7 The
most common clinically used antimalarial drugs belong to seven
Received: June 13, 2012
Published: July 19, 2012
© 2012 American Chemical Society
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a
Scheme 1. Synthesis of 5a and Analogues
a
Conditions: (i) ethyl thioglycolate, NaOAc, H2O, 90 °C; (ii) ethyl thioglycolate, triethylamine, MeCN, 85 °C; (iii) Fe, HOAc, 80 °C; (iv) 2-
aminomethyl-1-ethylpyrrolidine, EDCI·HCl, HOBt, Et3N, CH2Cl2, 23 °C; (v) aldehyde, Ac2O, Et3N, reflux; (vi) aldehyde, Ac2O, Et3N, reflux;
LiOH, MeOH, H2O, 23 °C.
recommendation to use artemisinin-based combination thera-
pies to treat P. falciparum malaria.16 However, recent reports of
artemisinin resistances17−19 again create an urgent need for new
antimalarial drugs. On the basis of several findings, the enzyme
glucose-6-phosphate dehydrogenase 6-phosphogluconolacto-
nase of P. falciparum (Pf GluPho) has emerged as an intriguing
new target for antimalarial drug development.20 Deficiency in
human glucose-6-phosphate dehydrogenase (hG6PD), which
affects around 330 million people worldwide,21 is associated
with protection from severe malaria infections.22−24 It is
suggested that both hG6PD and PfGluPho play an important
role in Plasmodium development and survival.20,22,25 Pf GluPho
is the key enzyme of the parasitic pentose phosphate pathway
(PPP), which is supposed to be the major NADPH source
(reviewed by Preuss et al.26) for these parasites highly sensitive
to oxidative stress.27 A previous study showed that P.
falciparum-infected red blood cells (IRBC) present an increased
PPP activity compared to noninfected ones, 80% of which is
caused by Plasmodium’s PPP.25 Moreover, silencing of
PfGluPho with RNA interference (RNAi) stopped parasite
development at the trophozoite stage.28 Although Baum et al.
reported in 2009 that P. falciparum lacks an RNAi machinery,29
Lopez-Barragan et al. recently reported that antisense RNA
may be an important factor in P. falciparum’s regulation of gene
expression,30 strengthening the results of the previous RNAi-
based study. Additionally, PfGluPho presents considerable
structural differences compared to hG6PD, potentially allowing
selective targeting.31,32
Herein we describe the high-throughput screening (HTS) of
348911 compounds in the NIH’s Molecular Libraries Small
Molecule Repository (MLSMR) collection (http://mli.nih.
gov/mli/compound-repository/mlsmr-compounds) against
PfGluPho and a subsequent structure−activity relationship
(SAR) study of a class of benzothiazinone hits obtained from
the screen. Optimization of the screening hits resulted in the
discovery of (R,Z)-N-((1-ethylpyrrolidin-2-yl)methyl)-2-(2-flu-
orobenzylidene)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-
6-carboxamide, 11 (ML276),33 which inhibited Pf GluPho
selectively over hG6PD and inhibited P. falciparum growth in
vitro with an IC50 in the low micromolar range. Compound 11
represents the first reported selective Pf GluPho inhibitor and
therefore could provide a basis for future drug design of new
antimalarial therapeutics.
RESULTS AND DISCUSSION
■
High-Throughput Screening. In a primary screening
approach, the MLSMR compound library (∼350000 com-
pounds) was screened at a concentration of 20 μM against
Pf GluPho. The screening campaign resulted in the identi-
fication of 2709 compounds that inhibited Pf GluPho activity
≥59%, corresponding to a hit rate of 0.8%. Of these, 2429 were
available from the MLSMR collection and were retested in
triplicate at a concentration of 20 μM, resulting in 817
compounds inhibiting Pf GluPho ≥50%, about 34% of the
samples. To exclude samples that interfere with the coupled
resazurin/diaphorase system used in our study,34 active
compounds were run against a diaphorase inhibition assay at
a concentration of 20 μM. Of the compounds tested, 230
inhibited the diaphorase reaction and thus were discarded as
artifacts. The remaining 587 active compounds were further
tested in dose response in the PfGluPho assay and an hG6PD
counter-screen. As a result, 488 compounds presented IC50
values ≤20 μM vs Pf GluPho, and only 39 of the compounds
were active against hG6PD. For final confirmation, the
compounds were tested in an orthogonal assay that did not
rely on the resazurin and diaphorase coupled system. On the
basis of the initial slope of the reaction, around 50% of the
compounds were confirmed to inhibit PfGluPho with an IC50
≤ 80 μM. Several series of compounds were identified, of which
a group of benzothiazinones was most promising based on their
potency and selectivity. Detailed information is available in the
PubChem database (AIDs 504690, 504753, 504863, 504862,
504765, 504792, 540252, 540269).
Synthesis. The synthesis of the benzothiazinone-derived
compounds described herein was accomplished in 4−5 steps as
outlined in Scheme 1.35 Starting from 4-fluoro-3-nitrobenzoic
acid or the esters (1a−1c), nucleophilic substitution of the
fluorine substituent with ethyl thioglycolate under basic
conditions provided thioethers (2a−2c). Reductive cyclization
mediated by iron in acetic acid provided benzothiazinones
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a
Scheme 2. Synthesis of Compounds 10−15, 17, 22, 29, 33, 34, and 39−42 including 11 .
a
Conditions: (i) CoCl2, NaBH4, THF, 23°C, 53%, 2-aminomethyl-1-ethylpyrrolidine, EDCI·HCl, HOBt, Et3N, CH2Cl2, 23°C, 41%; (ii) RNH2,
EDCI·HCl, HOBt, Et3N, CH2Cl2, 23 °C, 13−61%.
(3a−3c). 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide
(EDCI)-mediated coupling of 3a with 2-aminomethyl-1-
ethylpyrrolidine afforded amide 3d. The first point of diversity
was introduced by condensation of benzothiazinones (3a−3c)
with a range of aromatic aldehydes resulting in intermediates
(4a−4j). Coupling of intermediates (4a−4j) with 2-amino-
methyl-1-ethylpyrrolidine resulted in the corresponding amides
5a−5j (Scheme 1). The styryl moiety in the intermediate 4a
was reduced with sodium borohydride in the presence of cobalt
chloride, and the resulting acid was converted to amide 10
(Scheme 2). A second point of diversity resulted from amide
synthesis starting from 4a using diverse amines to provide the
amides 10−15, 17, 22, 29, 33, 34, and 39−42 (Scheme 2). All
final compounds were obtained in moderate yields and high
purity needed to support SAR studies.
Table 1. Structures of Active Benzothiazinone Hits from
HTS
Structure−Activity Relationships (SAR). Three main
assays were utilized to direct the SAR efforts. The primary SAR-
driving assay was the dose response, resazurin/diaphorase-
coupled Pf GluPho assay, with the hG6PD assay serving as the
counter-screen to ascertain specificity. None of the compounds
from this series showed any activity in the hG6PD counter-
screen (>80 μM) and thus were considered specific for the P.
falciparum enzyme. The orthogonal PfGluPho assay, which
relied on the direct detection of NADPH, was used to further
confirm activity in the absence of the coupled resazurin/
diaphorase system. Of the 156 substituted benzothiazinones
tested in the high-throughput screen, only five were confirmed
active by dose response. All were inactive (IC50 > 80 μM)
against hG6PD. These structures are represented in Table 1.
The structural features of these active benzothiazinones that
separate them from those that were either inactive or failed
confirmation in dose response include: (1) the amide side chain
contains a basic alkylamine, (2) the nitrogen in the
benzothiazinone ring is not alkylated, and (3) the sulfur exists
as a sulfide (Figure 1).
From this preliminary SAR, we focused on further
optimization of the scaffold. Specifically, we examined a wide
range of amide substitutions as well as substitution around the
pendant aryl ring. The general SAR strategy is depicted in
Figure 2.
Exploration of the styryl SAR was initiated by maintaining
the preferred 1-ethyl-2-amino-methylpyrrolidine moiety from
the HTS data and is summarized in Table 2. The primary hit
5a, identified during the HTS and subsequent hit confirmation,
was resynthesized as described in Scheme 1. It was equipotent
Figure 1. Structural requirements for activity.
1
to the purchased compound (entry 5a, Tables 1 and 2). H
NMR data of the purchased sample and the synthesized sample
of 5a were identical. The Z-geometry around the styryl portion
was assigned based on comparison to previously reported NMR
data.36
Figure 2. General SAR strategy.
A variety of substituted styryl analogues were synthesized;
however, the potency range was only affected by 7-fold. The 2-
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a
Table 2. SAR of Benzothiazinones Inhibiting Plasmodium
falciparum Glucose-6-phosphate Dehydrogenase
Table 3. Necessity of Styryl Moiety in SAR
more pronounced as the ring was expanded (entries 8, 9, 28−
39, Table 6). Examples of constrained tertiary or aromatic
amides proved substantially less active (entries 40−42, Table
6). All compounds represented in Tables 4, 5, and 6 exhibited
an IC50 > 80 μM against hG6PD.
Stereochemical evaluation of the 2-aminomethyl pyrrolidines
led us to synthesize each enantiomer of 5a from the
corresponding commercially available chiral amines. An 8-fold
preference for the R-enantiomer versus the S-enantiomer was
discovered (entries 11 and 12, Table 4). Compound 11, (R,Z)-
N-((1-ethylpyrrolidin-2-yl)methyl)-2-(2-fluorobenzylidene)-3-
oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxamide, was
the most potent compound identified and was therefore
declared a probe. Compound 11 was selective for PfGluPho
over hG6PD (Figure 3).
In Vitro ADME/T Profiling and Chemical Stability.
Compound 11 was evaluated in a detailed in vitro
pharmacology screen (absorption, distribution, metabolism,
excretion, toxicity (ADME/T)) as shown in Table 7.
Compound 11 is highly soluble, especially at lower pH,
which is likely due to the presence and protonation of the
alkylamino moiety.
Consistent with its solubility data, 11 exhibits high
permeability with increased pH of the donor compartment in
a standard parallel artificial membrane permeability assay
(PAMPA). When incubated with an artificial membrane that
models the blood−brain barrier (BBB), 11 was highly
permeable. Compound 11 had good stability in plasma, thus
increasing its exposure to blood-borne parasites such as P.
falciparum, and it was highly plasma protein bound, which may
contribute to its high plasma stability. Lastly, 11 shows
moderate stability in human and modest stability in mouse
liver homogenates and no toxicity (>50 μM) toward Fa2N-4
immortalized human hepatocytes.
Compound 11 contains an α,β-unsaturated amide and is a
potential Michael acceptor. To investigate this potential
chemical liability, 11 (10 μM, 1% DMSO) was incubated
with 5 equiv of glutathione (GSH) in PBS buffer (pH 7.4) at
23 °C. Loss of compound was monitored at different times over
18 h using RP-LCMS (Figure 4). Ethacrynic acid, a diuretic
containing an α,β-unsaturated ketone, was used as a positive
control because it has been previously reported to form a GSH-
adduct.37 Loss of ethacrynic acid was observed as early as 30
min and was ∼97% depleted in 24 h, whereas 11 remained
a
All compounds represented in this table exhibited an IC50 > 80 μM
b
against human G6PD. Screen hit.
fluoro substituent is important for activity, as the analogue
without any aryl substituent was ∼4-fold less active (entry 5a vs
5b, Table 2). Interestingly, neither the position nor the
electronics of the substituent dramatically affected the observed
potency. For example, analogues containing the 2-, 3-, or 4-
fluoro substitution (entries 5a, 5e, and 5f, Table 2) were
essentially equipotent. Replacement of the 2-fluoro substituent
with 2-chloro did not significantly affect the potency, but
replacement with electron-rich 2-methyl or 2-methoxy groups
decreased potency by 3-fold and 5-fold, respectively (entries 5a,
6, 5c, and 5d, Table 2). Fused ring systems such as 1-naphthyl
and 2-naphthyl (entries 5i and 5j, Table 2) also slightly lowered
potency. The unsubstituted phenyl and the 2-methoxy
analogues (entries 5b and 5d, Table 2) were the least preferred
of the synthesized analogues.
While the SAR around the styryl moiety may appear to
minimally affect potency, its presence was required for activity.
When the styryl group was replaced with phenethyl or removed
entirely, a complete loss of activity resulted (5a vs 3d and 10,
Table 3).
The SAR of the alkylamino side chain proved to be much
more stringent than the styryl region, with a strong preference
for 2-aminomethyl pyrrolidines. Potency was maintained within
5-fold whether the nitrogen of the pyrrolidine ring was
substituted with ethyl, propyl, or was unsubstituted (entries
5a, 13 and 15, Table 4). The t-butoxycarbonyl derivative
proved inactive, consistent with observations from the HTS
(entry 14, Table 4). Less constrained examples such as
dimethylaminoethyl, diethylaminoethyl, and dipropylaminoeth-
yl demonstrated sharp decreases in potency (entries 16−27,
Table 5). Attachment of pyrrolidine through nitrogen via an
aminoethyl linker led to a 5-fold loss of potency, which became
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Table 4. SAR of Benzothiazinones Inhibiting Plasmodium falciparum Glucose-6-phosphate Dehydrogenase
Table 5. SAR of Benzothiazinones Inhibiting Plasmodium falciparum Glucose-6-phosphate Dehydrogenase
a
P: commercially purchased compounds.
intact for the duration of the study, indicating a lack of
reactivity with nucleophiles (Figure 4). Thus, 11 represents a
potential lead compound for the development of an in vivo
probe.
Activity against P. falciparum in Culture. Compound 11
was included in P. falciparum growth assays to assess its effects
on blood stage parasites in vitro. After incubation for 72 h, 11
gave IC50 values of 2.3 0.2 μM for the chloroquine sensitive
3D7 strain and 3.7
strain (Figure 5).
0.9 for the chloroquine resistant Dd2
Mechanism of Inhibition (MOI) Studies. The most likely
type of inhibition was determined by titrating compound and
substrate/cosubstrate at the same time. G6P and 11 titration
revealed increasing Km values and an α > 6.6 with increasing
compound concentration, suggesting that 11 inhibits PfGluPho
activity by competing with the substrate (Figure 6B). Increasing
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Table 6. SAR of Benzothiazinones Inhibiting Plasmodium falciparum Glucose-6-phosphate Dehydrogenase
a
b
P: commercially purchased compounds. Screen hit.
Km and decreasing Vmax values, and α between 1.4 and 4.7, were
found when compound and cosubstrate were titrated,
indicating mixed-type inhibition of 11 vs NADP+ (Figure 6A).
CONCLUSIONS
■
A series of benzothiazinones derived from an HTS of the NIH’s
MLSMR collection that are potent, fully selective inhibitors of
Pf GluPho has been reported. The most potent compound in
this series, 11, has antimalarial activity against both
chloroquine-sensitive and chloroquine-resistant strains. Com-
pound 11 has reasonable in vitro ADME/T properties,
Figure 3. Dose−response curve for 11 in the Plasmodium and human
G6PD assays, indicating full selectivity for Pf GluPho.
Table 7. Summary of in Vitro ADME/T Properties of P. falciparum G6PD Inhibitor Probe 11
a
aqueous solubility in pION’s buffer (μg/mL) [μM] pH 5.0/6.2/7.4
>126/>126/29.4
[>296/>296/69]
a
aqueous solubility in 1× PBS, pH 7.4 (μg/mL) [μM]
71.0 [166]
29/737/806
233
PAMPA permeability, Pe (× 10−6 cm/s) donor pH, 5.0/6.2/7.4; acceptor pH, 7.4
BBB−PAMPA permeability, Pe (× 10−6 cm/s) donor pH, 7.4; acceptor pH, 7.4
plasma protein binding (% bound)
human 1 μM/10 μM
mouse 1 μM/10 μM
98.39/97.98
98.95/98.34
90.98/80.84
60.53/37.57
>50
plasma stability (% remaining at 3 h) human/mouse
hepatic microsome stability (% remaining at 1 h) human/mouse
toxicity toward Fa2N-4 immortalized human hepatocytes LC50 (μM)
a
Solubility also expressed in molar units (μM) as indicated in italicized [bracketed values], in addition to more traditional μg/mL units.
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1−2 for positive control. The plates were centrifuged at 1500 rpm for
1 min and incubated in the dark for 2 h. Fluorescence of resorufin was
detected at excitation 530/emission 580 nm (ex530/em580) using a
Viewlux (PerkinElmer) plate reader. Data was analyzed with CBIS
(Chemical and Biological Information Systems, www.cheminnovation.
com), and compounds inhibiting PfGluPho for at least 59% were
retested as described before. Retested compounds inhibiting
Pf GluPho activity ≥50% were further investigated.
Diaphorase Counter-Screen. First, 12 nL of 10 mM compounds in
DMSO were transferred into columns 5−48 of 1536-well plates using
the Echo Liquid Handler. Next, 12 nL of DMSO were transferred to
columns 1−4 for positive and negative controls. Subsequently, 3 μL of
substrate mix (50 mM Tris (pH 7.5), 0.005% Tween20, 1 mg/mL
BSA, 6.6 mM MgCl2, 0.06 mM/0.02 mM NADPH, 0.05 mM
resazurin) were added to columns 1−48 using the Multidrop Combi
reagent dispenser (Thermo Scientific). To initiate the reaction, 3 μL of
enzyme solution (50 mM Tris (pH 7.5), 0.005% Tween 20, 1 mg/mL
BSA, 6.6 mM MgCl2, 2 U/mL diaphorase) were added to columns 3−
48 with the Multidrop Combi dispenser. For the negative control, 3
μL of the enzyme solution without diaphorase were added to columns
1−2. The plates were centrifuged at 1500 rpm for 1 min, incubated in
the dark for 2 h, and read at ex530/em590 nm. Diaphorase inhibition
was analyzed using CBIS, and compounds exhibiting at least 50%
enzyme inhibition were assigned active status and thus not included in
further experiments.
Figure 4. Investigation of chemical reactivity/stability of 11 via GSH
adduct formation.
including excellent solubility, no toxicity, good permeability and
plasma stability, and moderate microsomal stability. Further
work in this series is in progress to optimize the potency and
ADME/T properties, which may lead to validation in in vivo
models.
Hit Confirmation in Dose Response. Varying volumes of
compounds in DMSO (starting final concentration of 20 μM) were
transferred to columns 5−48 of 1536-well plates using the Echo Liquid
Handler. DMSO was added to control and test compound wells to
equilibrate DMSO concentrations across plate (final concentration of
1.0%). The remaining assay procedures for the Pf GluPho assay were
performed as described in the Primary Screen section. Compounds
with an IC50 of ≤20 μM were further investigated.
hG6PD Counter-Screen. Using the Echo Liquid Handler, varying
volumes of compounds (starting final concentration of 20 μM) were
transferred to wells of columns 5−48 of 1536-well plates. DMSO was
added to control and test compound wells to equilibrate DMSO
concentrations across the plate (final concentration of 0.8%). Then 2.5
μL of enzyme solution (50 mM Tris (pH 7.5), 0.005% Tween 20, 1
mg/mL BSA, 0.05 μg/mL hG6PD) were transferred to all wells.
Following enzyme addition, 2.5 μL substrate solution (0.64 mM G6P,
50 mM Tris (pH 7.5), 0.005% Tween 20, 1 mg/mL BSA, 12 μM
NADP+, 6.6 mM MgCl2, 2 U/mL diaphorase, 0.28 mM resazurin)
were added to columns 3−48 and solution without G6P to columns
1−2 for positive control. After 1 min of centrifugation at 1500 rpm and
90 min incubation in the dark, fluorescence of resorufin was detected
at ex530/em590 by using the Viewlux plate reader. IC50 values were
calculated using CBIS.
EXPERIMENTAL SECTION
■
HTS/Biology. Materials. G6P, resazurin, diaphorase, Tween20,
MgCl2, and BSA were obtained from Sigma, St. Louis, MO. NADP+
was purchased from Amresco, Solon, OH, and 1536 well-plates from
Aurora, Poway, CA.
Compound Library. The Sanford−Burnham Center for Chemical
Genomics’ copy of the NIH Molecular Libraries Small Molecule
Repository (MLSMR) consisted of 348911 compounds when the
screen was run, which were present in 1536-well plates at a
concentration of 2 mM (in DMSO). The library was stored at −80
°C until first use, after which it was kept at room temperature under
desiccating conditions for a period not greater than six months.
HTS. Primary Screen. The previously developed HTS assay for
PfGluPho34 was used in a 384-well plate format. This assay was
slightly modified for use in 1536-well plates and performed in the
following way. A volume of 60 nL of the compounds (final 20 μM, 1%
DMSO) was transferred to columns 5−48 of 1536-well plates using
the Echo Liquid Handler (LabCyte). Then 60 nL of DMSO were
transferred to columns 1−4, which served as positive and negative
controls. A volume of 3 μL of enzyme mix (50 mM Tris (pH 7.5),
0.005% Tween 20, 1 mg/mL BSA, 0.1 μg/mL Pf GluPho) was added
to all wells using the Kalypsys (Kalypsys Systems). To start the
reaction, 3 μL of substrate mix (50 mM Tris (pH 7.5), 40 μM G6P,
0.005% Tween 20, 1 mg/mL BSA, 6 μM NADP+, 6.6 mM MgCl2, 2
U/mL diaphorase, 0.05 mM resazurin) were added to columns 3−48
using the Kalypsys. Substrate mix without G6P was added to columns
Orthogonal Assay. Using the Echo Liquid Handler, varying
volumes of compounds were transferred to wells of columns 5−48
of 1536-well plates. DMSO was added to control and test compound
wells to equilibrate DMSO concentrations across the plate. Following
Figure 5. P. falciparum growth inhibition by 11 of chloroquine-sensitive (A) and chloroquine-resistant (B) strains. One representative data set out of
two independent experiments is shown. Experiments were performed twice in triplicate, of which the average and standard deviation are given.
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Figure 6. Mechanistic characterization of 11 vs NADP+ (A) and G6P (B) for PfGluPho. The signal (RFU/s) was plotted against the compound and
NADP+ or G6P concentration. One representative data set from two independent experiments is shown. Data points are given as averages of
duplicates.
compound and DMSO transfer, 2.5 μL of enzyme solution (50 mM
Tris (pH 7.5), 0.005% Tween 20, 1 mg/mL BSA, 0.4 μg/mL
PfGluPho) were added to all wells using a Multidrop Combi
dispenser. Then 2.5 μL of substrate solution (40 μM G6P, 6 μM
NADP+, 50 mM Tris (pH 7.5), 0.005% Tween 20, 1 mg/mL BSA, 6.6
mM MgCl2) were added to columns 5−48, and 2.5 μL of solution
without G6P were added to columns 1−4 for positive control. The
plates were centrifuged and either incubated for 45 min to determine
end points of the reaction or kinetically read at ex 340 nm/em 450 nm
over 45 min. The reaction rate was calculated by dividing the relative
fluorescence units (RFU) by time in minutes. CBIS was used to
calculate IC50 values, and compounds with IC50 ≤ 80 μM were
designated active.
General Procedure for the Synthesis of Compounds 5a−j and
10−42 as Illustrated by Synthesis of (R,Z)-N-((1-Ethylpyrrolidin-2-
yl)methyl)-2-(2-fluorobenzylidene)-3-oxo-3,4-dihydro-2H-benzo[b]-
[1,4]thiazine-6-carboxamide (11). 4-(2-Ethoxy-2-oxoethylthio)-3-
nitrobenzoic Acid (2a). 4-Fluoro-3-nitrobenzoic acid (1a) (5.55 g,
30 mmol) and sodium acetate (2.71 g, 33 mmol) were combined in a
round-bottom flask. Ethyl mercaptoacetate (3.62 mL, 33 mmol) and
water (30 mL) were then added, and the mixture was heated under
nitrogen atmosphere at 90 °C for 24 h. The reaction mixture was
cooled to 23 °C and then placed in an ice bath for 10−20 min. The
precipitate formed was collected by filtration, washed with water, and
1
dried to afford 2a as a tan solid (8.66 g, quant). H NMR (500 MHz,
CDCl3) δ 9.02−8.91 (m, 1H), 8.34−8.20 (m, 1H), 7.64 (d, J = 8.6 Hz,
1H), 4.26 (q, J = 7.1 Hz, 2H), 3.84 (s, 2H), 1.31 (t, J = 7.1 Hz, 3H).
LRMS (ESI-ve): calculated for C11H11NO6S, [M − H] = 284.02;
observed, [M − H] = 283.99.
Chemistry. All reactions involving air- and moisture-sensitive
reagents and solvents were performed under a nitrogen atmosphere
using standard chemical techniques. Anhydrous solvents were
purchased and freshly used from Sigma-Aldrich or EMD Biosciences.
All organic reagents were used as purchased. (R)-(1-ethylpyrrolidin-2-
yl)methanamine (catalogue no. 357000010, CAS 22795−97−7) and
(S)-(1-ethylpyrrolidin-2-yl)methanamine (catalogue no. 367750010,
CAS 22795−99−9) were purchased from Acros Organics and used
without further purification. Analytical thin-layer chromatography was
performed on Partisil K6F silica gel 60 Å, 250 μm. Microwave-assisted
3-Oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxylic Acid
(3a). 2a (3.94 g, 13.8 mmol) and iron powder (3.08 g, 55 mmol)
were suspended in glacial acetic acid (33 mL) in a round-bottom flask
equipped with an air condenser under nitrogen atmosphere. The
mixture was heated to 80 °C in an oil bath, during which the
suspension turned reddish−brown, and the temperature of the bath
rose to 92 °C. After 30 min, the temperature of the oil bath was
lowered to 80 °C and maintained for 2 h. The mixture was then
allowed to cool to 23 °C and stirred for about 20 h and then was
poured into 1N HCl solution (150 mL). After stirring the mixture for
30 min, the precipitate was filtered, washed extensively with water and
diethyl ether, and dried to afford 3a as a light-gray solid (2.56 g, 89%).
1H NMR (500 MHz, DMSO-d6) δ 12.99 (s, 1H), 10.72 (s, 1H), 7.55
1
reactions were performed using a CEM Discover system. H and 13C
chemical shifts are reported in δ values in ppm in the corresponding
solvent. All solvents used for chromatography on the synthetic
materials were Fisher Scientific HPLC grade, and the water was
Millipore Milli-Q PP filtered. All NMR spectra for the synthetic
materials were recorded on a Bruker Avance II 400 or DRX-500 MHz
instrument. High resolution mass spectrometry (HRMS) spectra were
carried out on an Agilent 6224A Accurate-Mass time-of-flight (TOF)
LC/MS system with electron spray ionization (ESI). LCMS analysis of
synthetic materials was completed on a Waters Autopurification
system, which consists of a 2767 sample manager, a 2545 binary
gradient module, a system fluidics organizer, a 2489 UV/vis detector,
and a 3100 mass detector, all controlled with MassLynx software. A
Sunfire Analytical C18 5 μm column (4.6 mm × 50 mm) and stepwise
gradient {10% [(MeCN + 0.1% TFA) in (water +0.1% TFA)] to 98%
[(MeCN + 0.1% TFA) in (water +0.1% TFA)] for 9 min} was used
for analytical LCMS of final compounds. The final compounds were
purified by RP-HPLC on a SunFire preparative C18 column (5 μm; 19
mm × 50 mm) using a stepwise gradient {10% [(MeCN + 0.1% TFA)
in (water + 0.1% TFA)] for 1 min; 30% for 1 min; 50% for 4 min; 70%
for 1.5 min and 98% for 1.5 min}. All synthesized final compounds
5a−j and 10−42 were determined to be ≥95% pure by LC-MS.
Compound identity was verified by 1H NMR and HRMS, and
additionally by 13C NMR for 11. The MestReNova program was used
to interpret NMR spectra.
(s, 1H), 7.50 (d, J = 7.9 Hz, 1H), 7.42 (d, J = 7.9 Hz, 1H), 3.31 (s,
2H). LRMS (ESI-ve): calculated for C9H6NO3S, [M − H] = 208.01;
observed, [M − H] = 207.95.(Z)-2-(2-Fluorobenzylidene)-3-oxo-3,4-
dihydro-2H-benzo[b][1,4]thiazine-6-carboxylic Acid (4a)
3a (1.2 g, 5.7 mmol), was suspended in acetic anhydride (20 mL) in a
40 mL vial. Triethylamine (2.39 mL, 17.2 mmol) and 2-
fluorobenzaldehyde (1.21 mL, 11.5 mmol) were added, and the
mixture was heated at 145 °C for 15 h. The reaction mixture was
cooled to 23 °C, poured into 100 mL of diethyl ether, and cooled at
4 °C for 20 min. The precipitate was collected by filtration, washed
with ether (25 mL), and dried to afford 4a as a yellowish−brown solid
(1.28 g, 71%). 1H NMR (500 MHz, DMSO-d6) δ 11.30 (s, 1H), 7.86
(s, 1H), 7.79 (t, 1H), 7.69 (d, J = 1.7 Hz, 1H), 7.55 (dd, J = 8.3, 1.7
Hz, 1H), 7.53−7.48 (m, 1H), 7.43 (d, J = 8.2 Hz, 1H), 7.40−7.33 (m,
2H). LRMS (ESI-ve): calculated for C16H10FNO3S, [M − H] =
314.03; observed, [M − H] = 314.0.
(R,Z)-N-((1-Ethylpyrrolidin-2-yl)methyl)-2-(2-fluorobenzylidene)-
3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxamide (11).
4a (300 mg, 0.95 mmol), HOBt (193 mg, 1.43 mmol), and EDCI·HCl
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(274 mg, 1.43 mmol) were combined in an 8 mL vial. Dry
dichloromethane (4 mL) and triethylamine (0.4 mL, 2.85 mmol)
were added, and the mixture was stirred at 23 °C for 5 min. (R)-(1-
Ethylpyrrolidin-2-yl)methanamine (0.2 mL, 1.43 mmol) was added,
and within a few minutes the solution became homogeneous and was
allowed to stir for about 15 h. The reaction mixture was then diluted
with 20% methanol/dichloromethane (5 mL) and extracted with water
(4 mL, 3×). The organic layer was dried over anhydrous sodium
sulfate and evaporated to afford a brown residue. The residue was
purified by RP-HPLC on a SunFire preparative C18 column (5 μm; 19
mm × 50 mm) using a stepwise gradient {10% [(MeCN + 0.1% TFA)
in (water + 0.1% TFA)] for 1 min, 30% for 1 min, 50% for 4 min, 70%
for 1.5 min, and 98% for 1.5 min}. The fractions containing the
expected molecular weight were collected and freeze-dried to afford a
formate salt of the product as a pale-yellow solid. The solid was
dissolved in dichloromethane and extracted with 25% K2CO3 solution
(3×). The organic layer was dried over anhydrous sodium sulfate and
evaporated to afford 11 as a yellowish−brown solid (0.13 g, 32%); mp
= 194−197 °C; [α]D = (+)-12.9 (c = 0.001, MeOH, 23 °C). 1H NMR
(500 MHz, acetic acid-d4) δ 8.05 (s, 1H), 7.83 (t, J = 7.3 Hz, 1H), 7.60
(dd, J = 8.3, 1.7 Hz, 1H), 7.52 (d, J = 1.6 Hz, 1H), 7.44 (dd, J = 13.3,
6.3 Hz, 1H), 7.36 (d, J = 8.3 Hz, 1H), 7.31 (t, J = 7.5 Hz, 1H), 7.20 (t,
J = 7.5 Hz, 1H), 3.98−3.76 (m, 4H), 3.54−3.41 (m, 1H), 3.23−3.08
(m, 2H), 2.37−2.22 (m, 1H), 2.12−2.05 (m, 3H), 1.33 (t, J = 7.5 Hz,
3H). For 1H NMR in methanol-d4, see Supporting Information. LRMS
(ESI+ve): calculated for C23H24FN3O2S, [M + H] = 426.17; observed,
[M + H] = 426.26. HRMS (ESI+ve): calculated for C23H24FN3O2S,
[M + H] = 426.1646; observed, [M + H] = 426.1631.
If Lineweaver−Burk plots intersected at the y axis, Kampp increased,
app
and V was constant with increasing inhibitor concentrations, α = ∞,
max
then compounds were assigned to inhibit competitively. Lineweaver−
app
max
Burk plots intersecting at the x axis, constant Kampp and decreasing V
values with increasing inhibitor concentrations, and α = 1 indicated
noncompetitive inhibition. Parallel lines in Lineweaver−Burk plots,
app
decreasing Kampp and V
values with increasing inhibitor concen-
max
tration, and α < 1 was characteristic for uncompetitive inhibition.42
Were parameters found to be between those for competitive and
noncompetitive inhibitors, compounds were assigned to follow mixed-
type inhibition.
Protocol for Chemical Stability (Reactivity/GSH Adduct For-
mation). Protocol: 10 μL of compound (1 mM stock in DMSO) was
added to 985 μL of PBS buffer (1×, pH = 7.4) in a capped glass vial.
Then 5 μL of GSH solution (10 mM stock in PBS) was added to the
solution, and the mixture was gently mixed on a Grant-Bio PS-M3D
platform shaker at 23 °C. Then 20 mL aliquots were analyzed at 0, 0.5,
1, 2, 4, 6, 18, and 25 h on a Waters HPLC-MS system using a SunFire
analytical C18 column (5 μm; 4.6 mm × 50 mm) using a stepwise
gradient [11 {10% [(MeCN + 0.1% TFA) in (water + 0.1% TFA)] for
1 min, 30% for 1 min, 50% for 4 min, 70% for 1.5 min, and 98% for 1.5
min}; ethacrynic acid {10% [(MeCN + 0.1% TFA) in (water + 0.1%
TFA)] for 1 min, 30% for 1 min, 50% for 4 min, 70% for 1.5 min, and
98% for 1.5 min}]. The percentage remaining for each compound
relative to the starting amount of compound at 0 h was calculated from
the area under the curve (AUC) at different time points and plotted.
ASSOCIATED CONTENT
* Supporting Information
■
Activity against P. falciparum in Culture. The chloroquine-
sensitive P. falciparum 3D7 and the resistant Dd2 strains were cultured
as described elsewhere.38 Effects of Pf GluPho inhibitors on
Plasmodium parasites were tested using isotopic drug sensitivity
S
Detailed experimental procedures for the synthesis of 1b, 1c,
2b, 2c, 3b−3d, 4b−4j, 5a−5j, 10, 12−15, 17, 22, 29, 33, 34,
and 39−42 as well as NMR spectra of 2a, 3a, 3d, 4a, 5a−5j, 10,
11 (acetic acid-d4), 11 (methanol-d4), 12−15, 17, 22, 29, 33,
34, and 39−42. This material is available free of charge via the
Internet at http://pubs.acs.org.
assays,39,40 which are based on the incorporation of radioactive H-
3
hypoxanthine. Hypoxanthine is taken up by Plasmodium parasites
because it is needed for nucleic acid synthesis. In brief, P. falciparum-
infected RBCs were seeded in 96-well plates (Nunc) at a parasitemia
of 0.25% (>70% ring forms) and 1.25% hematocrit in hypoxanthine-
free medium. Varying concentrations of the test compounds and the
control chloroquine (Sigma) were added to the wells. The parasites
AUTHOR INFORMATION
Corresponding Author
■
3
were incubated for 48 h, after which 0.5 μCi H-hypoxanthine was
*For K.B.: phone, +49-641-9939121; fax, +49-641-9939129; E-
mail, katja.becker@uni-giessen.de. For A.B.P.: phone, 01-858-
695-5320; E-mail, apinkerton@sanfordburnham.org. For L.B.:
phone, 01-619-543-7545; fax, 01-619-543-7537; E-mail,
lbode@ucsd.edu.
added for a further 24 h. The cells of each well were harvested on a
glass fiber filter (Perkin-Elmer), washed, and dried. Finally, the
radioactivity of each condition was detected, which is supposed to be
proportional to the parasites’ growth. IC50 values were calculated using
nonlinear regression of the GraphPad Prism software.
Author Contributions
MOI Studies. Pf GluPho inhibitors were mechanistically charac-
terized as previously described.34 In brief, varying G6P or NADP+
concentrations were prepared in a substrate solution (final assay
concentration (FAC) 3.3 mM MgCl2, 1 mM TCEP, 0.05 M Tris; for
G6P titration, 5 μM NADP+; for NADP+ titration, 25 μM G6P) and
transferred to 384-well plate. Varying compound concentrations
(starting at around 8× IC50) titrated in DMSO were added to the
wells using the Echo Liquid Handler. Addition of an enzyme mix
(FAC 0.2 μg/mL PfGluPho, 0.005% Tween 20) started the reaction.
Fluorescence of NADPH production was monitored over 10 min at
ex340/em460 with a SpectraMax M5 (Molecular Devices) multiwell
plate reader, and the initial slope was determined with the SoftMax Pro
5.2 software (Molecular Devices). On the basis of the data,
Lineweaver−Burk plots were created,41 and Km and Vmax values were
calculated with the GraphPrad Prism software. Additionally, α from
the following rate equation was calculated using SigmaPlot (Systat
Software) (eq 1; v = velocity, S = substrate, I = inhibitor).
⊥These authors contributed equally to this work
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Michaela Stumpf, Beate Hecker, and Elisabeth
Fischer for their excellent technical assistance. The study was
supported by the NIH (1R21AI082434-01) to L.B., the
Deutsche Forschungsgemeinschaft (BE 1540/18-1) to K.B.,
and an NIH Molecular Libraries grant (U54 HG005033-03) to
the Conrad Prebys Center for Chemical Genomics at the
Sanford Burnham Medical Research Institute, one of the
comprehensive centers of the NIH Molecular Libraries Probe
Production Centers Network (MLPCN).
Vmapapx[S]
v =
ABBREVIATIONS USED
■
Kmapp + [S]
ADME/T, absorption distribution metabolism excretion
toxicity; AUC, area under the curve; BBB, blood-brain barrier;
CBIS, Chemical and Biological Information Systems; EDCI, 1-
ethyl-3-(3-dimethylaminopropyl) carbodiimide; ESI, electron
spray ionization; ex/em, excitation/emission; FAC, final assay
Vmax
(1 + [I]/Ki)
app
where V
=
and Kmapp = K
max
(1 + [I]/αKi)
m (1 + [I]/αKi)
(1)
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Ringwald, P.; Silamut, K.; Imwong, M.; Chotivanich, K.; Lim, P.;
Herdman, T.; An, S. S.; Yeung, S.; Singhasivanon, P.; Day, N. P.;
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(18) Noedl, H.; Se, Y.; Sriwichai, S.; Schaecher, K.; Teja-Isavadharm,
P.; Smith, B.; Rutvisuttinunt, W.; Bethell, D.; Surasri, S.; Fukuda, M.
M.; Socheat, D.; Chan Thap, L. Artemisinin resistance in Cambodia: a
clinical trial designed to address an emerging problem in Southeast
Asia. Clin. Infect. Dis. 2010, 51, e82−e89.
(19) Carrara, V. I.; Zwang, J.; Ashley, E. A.; Price, R. N.; Stepniewska,
K.; Barends, M.; Brockman, A.; Anderson, T.; McGready, R.;
Phaiphun, L.; Proux, S.; van Vugt, M.; Hutagalung, R.; Lwin, K. M.;
Phyo, A. P.; Preechapornkul, P.; Imwong, M.; Pukrittayakamee, S.;
Singhasivanon, P.; White, N. J.; Nosten, F. Changes in the treatment
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concentration; G6P, glucose-6-phosphate; G6PD, glucose-6-
phosphate dehydrogenase; hG6PD, human glucose-6-phos-
phate dehydrogenase; HRMS, high resolution mass spectrom-
etry; HTS, high-throughput screening; IRBC, infected red
blood cells; MLSMR, Molecular Libraries Small Molecule
Repository; MOI, mechanism of inhibition; PAMPA, parallel
artificial membrane permeability assay; PfG6PD, Plasmodium
falciparum glucose-6-phosphate dehydrogenase; Pf GluPho,
Plasmodium falciparum glucose-6-phosphate dehydrogenase 6-
phosphogluconolactonase; PPP, pentose phosphate pathway;
RNAi, RNA interference; SAR, structure−activity relationship;
TOF, time-of-flight
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