Perspective

pubs.acs.org/jmc

Cell-Based Medicinal Chemistry Optimization of High-Throughput

Screening (HTS) Hits for Orally Active Antimalarials. Part 1:
Challenges in Potency and Absorption, Distribution, Metabolism,
Excretion/Pharmacokinetics (ADME/PK)
Miniperspectives Series on Phenotypic Screening for Antiinfective Targets
Arnab K. Chatterjee*
Calibr, 11119 North Torrey Pines Road, Suite 100, San Diego, California 92037, United States
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ABSTRACT: Malaria represents a significant health issue, and

novel and effective drugs are needed to address parasite
Publication Date (Web): September 13, 2013 | doi: 10.1021/jm400314m

resistance that has emerged to the current drug arsenal.

Antimalarial drug discovery has historically benefited from a
whole-cell (phenotypic) screening approach to identify lead
molecules. This approach has been utilized by several groups
to optimize weakly active antimalarial pharmacophores, such as
the quinolone scaffold, to yield potent and highly efficacious
compounds that are now poised to enter clinical trials. More
recently, GNF/Novartis, GSK, and others have employed the
same approach in high-throughput screening (HTS) of large
compound libraries to find novel scaffolds that have also been
optimized to clinical candidates by GNF/Novartis. This
perspective outlines some of the inherent challenges in cell-based medicinal chemistry optimization, including optimization of
oral exposure and hERG activity.

■ INTRODUCTION
After thousands of years, malaria is still one of the major
infectious diseases, affecting millions of people, especially those
in underdeveloped countries.1 In the absence of effective
antimalarial vaccines,2 low molecular weight antimalarial drugs
are important treatments against the disease. Quinine,
chloroquine, mefloquine, and artemisinin derivatives have
played an important role in the fight against malaria. However,
widespread drug resistance has made these drugs less effective,
with artemisinin derivatives being the only exception.
Artemisinin-based combination therapies (ACT) recommen-
ded by the WHO will likely delay the emergence of clinical
resistance against artemisinins, but reports of increased parasite
clearance times have emerged recently.3 Therefore, it is
important to discover antimalarials with novel mechanisms of
action that are effective against multidrug resistant parasite
strains. Several historical pharmacophores (including quino-
lones) have recently been optimized to provide potent
compounds. Additionally, scaffolds from recent large-scale
HTS campaigns have been utilized to generate several novel
antimalarial pharmacophores from GNF/Novartis and others.4
This miniperspective outlines the optimization of these
pharmacophores, highlighting the challenges and opportunities
faced in cell-based optimization.
■ QUINOLINE OPTIMIZATION
Atovaquone 1 (Figure 1) is an important napthoquinone
antimalarial drug that is used in combination therapy with the
dihydrofolate reductase inhibitor proguanil (Malarone, GSK).
After atovaquone was discovered in the early 1990s, it was
found to be a potent inhibitor of the mitochondrial electron
transport chain via inhibition of the cytochrome bc1 complex.
Atovaquone is a ubiquinone competitive inhibitor of bc1 and
serves as an excellent lead that has been investigated by several
groups. The majority of the optimization of pyridone and
quinolone-based compounds has been carried out based on
phenotypic cell-based chemical optimization, with these starting
points having deep roots in the literature as phenotypic hits.
For example, endochin (2),5−7 3 (ICI-56,780),8,9 and clopidol
(4)10 were discovered as having antimalarial activity several
decades ago (Figure 1). For example, 3 was shown to have
activity against blood- and liver-stage infections (including
dormant liver hypnozoites) by Ryley and Peters in the early
1970s.8 All these compounds have significant drawbacks
including high resistance frequencies, poor physiochemical
Special Issue: Miniperspectives Series on Phenotypic Screening for
Antiinfective Targets
Received: March 1, 2013
Published: August 8, 2013

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 Journal of Medicinal Chemistry
Figure 1. Quinone and pyridone antimalarials.
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Figure 2. Early optimization of endochin.
Publication Date (Web): September 13, 2013 | doi: 10.1021/jm400314m
properties, and low in vivo potency. Recent work from multiple
groups has focused on making very potent hybrid compounds
of atovaquone, clopidol, endochin, and 3 to address these
shortcomings and is discussed further in this miniperspective.
Interestingly, much of the medicinal chemistry optimization has
been based on whole cell screening data. So while biochemical
assays and in silico molecular modeling have been applied in
some of this work, most of the compounds reported have been
optimized based on whole-cell potency while minimizing their
toxicity to normal cells. For example, Riscoe and co-workers
have demonstrated excellent optimization results in a series of
quinolones derived from endochin 2. Figure 2 outlines some of
the important compounds from this work, including a
trifluoromethyl-substituted endochin 5 with good potency
(EC50 = 32 nM) and limited cross-resistance to atovaquone
resistant parasites (strain Tm90-C2B with an EC50 = 66 nM).
While many compounds from this series do not possess good
murine liver microsomal stability, limiting their use in patients,
recent work from the Roscoe group has shown a modest
improvement in metabolic stability by modification of the
quinolone aromatic ring. For example, 6 (ELQ-121) has a
microsomal t1/2 of ∼15 min (Figure 2).11 While this is a modest
improvement over endochin 2 (t1/2 ∼ 2 min), clearly more SAR
on the aromatic portion of the quinolone is warranted. It is
likely that improvement in log P/log D in this series can further
improve metabolic stability and will likely be reported in due
course. While several recent compounds were reported in the
patent literature in 2012, no peer-reviewed articles have been
published to further demonstrate the optimization of this
scaffold with improved oral efficacy in rodent models of
Plasmodium infection.
The groups at the University of South Florida (Dennis Kyle,
Roman Manetsch, and co-workers) have also done extensive
work in the last several years on the optimization from
endochin 2, and their SAR studies have identified similar
patterns to those reported by Riscoe and co-workers. Figure 3
highlights some of the SAR, including the introduction of a
phenyl ring at the 6-position of the quinolone (while removing
the long alkyl groups present in 2, 5, and 6) and addition of
chloro and methoxy groups at the distal aromatic group in their
first-generation compound 7.12 Compound 7 had an in vitro
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Figure 3. Optimization of quinolones.
EC50 against Plasmodium. falciparum of 26 nM and was
equipotent against the atovaquone-resistant strain TM90-C2B,
with an EC50 = 15.3 nM. In addition, this compound has
moderate aqueous solubility (between 5 and 10 μM at neutral
pH), good in vitro permeability (Pe = 48.3 × 10−6 cm/s) in an
artificial permeability assay (PAMPA), and good to moderate
liver microsomal stability (mouse half-life = 21.3 min and
human half-life =128.3 min) that are consistent with potential
use of this compound as an oral medication. Subsequent data
on the oral efficacy of such compounds have not been reported
yet. This team has also replaced the carboxylate group of
quinolone 3 to provide 2,4-disubstituted aryl analogue 8
(Figure 3) whose in vitro EC50 against W2 parasites
(atovaquone sensitive) is 28 nM and in close agreement
(EC50 = 31 nM) to atovaquone-resistant parasite line TM90-
C2B.13 No further details of in vivo efficacy or pharmacoki-
netics have been reported on this series of compounds.
Kip Guy and co-workers at St. Jude Children’s Research
Hospital along with several collaborators have also done
extensive work on quinolone esters related to 3, and the
resulting leads are exemplified by compounds 9 and 10 in
Figure 4. Initial optimization indicated that meta-substituted
aromatic rings, such as are present in 9, lead to moderate
potency on the parasite (EC50 = 100−130 nM) while balancing
excellent aqueous solubility (>100 μM) and good in vitro
permeability (172 cm−6/s) (Figure 4). Activity against
atovaquone resistant parasites was in a similar range (EC50 =
210 nM).14 A recent disclosure has reported improved potency
in analogue 10 (EC50 = 80 nM) with slightly lower aqueous
solubility (20 μM) by addition of a fluorine atom. This
substitution also affords good oral exposure (Cmax = 38 μM and
AUCinf = 130 μM·h after a 50 mg/kg oral dose).15 When tested
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 Journal of Medicinal Chemistry
Figure 4. Optimization of quinoline esters.
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Figure 5. Optimized clopidol analogues.
in oral PK studies, this compound series shows less than
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proportional exposure when the dose is raised 4-fold from 50 to
200 mg/kg. For example, compound 9 results in only a 1.7-fold
increase in Cmax and a 2.2-fold increase in Cmax when the dose is
increased 4-fold.15 This might be due to low solubility but
appears to be consistent across the entire compound series
regardless of intrinsic aqueous solubility. This nonlinear and
less than proportional oral exposure could limit the multiples of
efficacious exposure needed to determine compound safety in
toxicity studies and make human dose calculation difficult.
Compound 10 was only able to suppress parasitemia by 57% in
a rodent model of malaria (dosed at 30 mg/kg po for three days
starting three days postinfection) and would require further
improvement to be considered a viable preclinical candidate.
Some recent work from O’Neill and co-workers at the
University of Liverpool has also demonstrated potent inhibitors
in the quinilone chemotype. For example, compound 11 has a
reported EC50 of 0.46 nM. The improved potency is attributed
by the authors to more favorable interactions with the Qo
binding site in the bc1 complex.16 These compounds likely
require optimization of physiochemical properties such as
solubility (clogP = 4.3) and must also be characterized for
cross-resistance to atovaquone. This group also optimized the
scaffold (originally found in the course of HTS) for activity
against the bc1 complex while also inhibiting NADH:ubiqui-
none oxidoreductase (Plasmodium falciparum NDH2 employed
in the HTS), allowing excellent in vivo efficacy in mice and
activity against liver-stage parasites in vitro to be demon-
strated.17 The lead compound from this optimization (12)
exhibits in vitro potencies in the 30−50 nM range (EC50) and
ED50 ∼ 2 mg/kg in a Plasmodium. berghei mouse model. This
compound has a good oral pharmacokinetic profile (t1/2 = 10.6
h) that could be useful for single-dose treatment, and further
development of these compounds is highly anticipated,
particularly in the area of improved solubility (clogP = 5.0
for 12).
Since the discovery of atovaquone, chemists at GSK have
conducted pioneering work on pyridones, and much of the
recent studies have centered on optimization of clopidol
(plasmodium EC50 ∼ 20 μM), leading to potent compounds
including one very recent report.18 Another such example from
Yeates and co-workers reported replacement of one of the
chloro group on the pyridine ring of clopidol to provide
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preclinical candidates 13 (GW844520) and 14 (GW308678)
(Figure 5).19 Introduction of an appropriately substituted
phenoxyphenyl group improves the in vitro activity to EC50 ∼5
nM and the ED50 in a suppressive animal model of infection
from 40 mg/kg for clopidol to 0.2 mg/kg from compounds 13
and 14. The SAR patterns are quite analogous to the SAR for
atovaquone, with a hydrophobic group proximal to the
carbonyl functionality. In addition, these compounds are
equipotent or more potent against an FCR3 strain of parasites
that are resistant to atovaquone. While these compounds also
act through the cytochrome bc1 complex (at the “Qo” binding
site), the lack of cross-resistance and lower resistance frequency
compared to atovaquone indicates that these compounds have
potential utility in man. From a medicinal chemistry
perspective, the increase in potency is truly remarkable, with
a >500-fold improvement in the EC50 against P. falciparum in
vitro and about 100-fold improvement with respect to the ED50
against Plasmodium yoelii in mice, showing that the relatively
weak hit clopidol could be rapidly optimized to viable lead
structures. A trifluoromethoxy analogue of lead compounds 13
and 14, compound 15 (GW932121) was advanced to first in
man studies, however, rat toxicology data on a phosphate
prodrug resulted in termination.20 Further optimization of this
quinolones toward a preclinical candidate in described below.
Acridones (the tricyclic version of pyridones) have also been
used to incorporate other interesting pharmacophores and have
been an area of investigation in the Riscoe laboratories.21
Starting from xanthones, replacement of the xanthone oxygen
with a nitrogen atom was investigated to provide another area
for modification to improve potency/cross-resistance to
atovaquone. Recently, Riscoe and co-workers have combined
the heme-targeting functionality of the acridone (due to π-
stacking with porphyrin) with a basic amine side chain that
concentrates compounds in the food vacuole of the parasite
(common to quinoline drugs chloroquine and mefloquine). In
addition, the Riscoe acridone 16 (T3.5)22 incorporates a
chemosensitizing substituent on the acridone nitrogen to aid in
overcoming chloroquine resistance (caused by gene mutations
in the chloroquine resistant transporter termed PfCRT)
(Figure 6). Acridone/pyridone hybrid structures from Kyle,
Manetsch, and co-workers at the University of South Florida
have also demonstrated some interesting activities, especially
compound 17, where one of the aromatic rings is saturated
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 Journal of Medicinal Chemistry
Figure 6. Novel acridones/quinolines.
(Figure 6).23 Compound 17 has shown in vitro potency in the
30−60 nM range against P. falciparum as well as having
equivalent activity on the atovaquone resistant TM90-C2B
strain. Compound 17 has poor aqueous solubility at pH = 7.4
(2−3 μM), moderate in vitro permeability in the PAMPA assay,
and good stability in mouse liver microsomes (half-life >3 h).23
While no in vivo PK data has been described thus far, these data
clearly indicate that these compounds (particularly those that
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have aryl substitutions on the pyridone ring) warrant further
investigation.
The tremendous amount of work by these groups based on
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the quinolone scaffolds has culminated in the discovery and
selection of a preclinical candidate 18 (ELQ300) for advanced
formulation and preclinical safety studies.24 18 is potent in vitro
on P. falciparum (EC50 ∼ 1−15 nM on P. falciparum strains
including clinical isolates) and efficacious at low doses in vivo
on liver and blood stages of infection (ED90 ∼ 0.15 mg/kg)
(Figure 7). The exposure at the efficacious dose is quite low in
Figure 7. Novel quinoline preclinical candidate.
mice (1.1 ug/mL·h), indicating that reaching sufficient
multiples of exposures in toxicity studies should be attainable.
However, while 18 has a remarkably clean in vitro ADMET
profile (Cyp P450 inhibition, hERG inhibition, human target
panel, mutagenicity), the scaffold does have low solubility (<1
μM at pH 6.8) due to high crystallinity (mp >300 °C). The low
intrinsic solubility has not hampered the oral exposure of the
compounds at this point but may be a concern as the
compound progresses toward clinical trials.
The work around the quinolones scaffold illustrates the
excellent whole-cell potency-based medicinal chemistry opti-
mization. While these pharmacophores have been known in the
literature for quite some time, effective optimization has only
recently been reported to generate preclinical candidates. It is
Figure 8. Benzamide optimization from a kinase template.
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particularly interesting that the SAR of this scaffold does
diverge somewhat from atovaquone itself, which may explain
the differences in the resistance profile of these compounds and
their interactions with the cytochrome bc1 complex. While 18
is less potent in vivo than atovaquone, laboratory generation of
parasites resistant to 18 has not been successful to date. These
results suggest that clinical resistance to 18 may be lower than
that of atovaquone, where lab-evolved resistance and clinical
resistance are well precedented. As 18 progresses through pre-
IND studies it will be interesting to see the clinical effects of the
extensive SAR and SPR studies performed to date on this
privileged antimalarial template.
■
WHOLE-PARASITE HTS
As we began the Novartis/GNF antimalarial medicinal
chemistry program in 2007 based on target-based optimization,
we observed a significant disconnect between enzymatic and
cellular potency with human kinase inhibitor scaffolds that also
inhibited P. falciparum calcium-dependent kinase cdpk1.25 This
observation led us to ask a more general question that
eventually moved us away from our original goal to work on P.
falciparum kinase inhibitors: Can cellular potency, namely
antimalarial activity, be derived from a repurposed human
kinase pharmacophore? Eliminating the kinase pharmacophore
early in hit-to-lead optimization would also reduce molecular
weight, improve ligand efficiency, and potentially reduce off-
target toxicity. Figure 8 outlines one such example reported that
has been reported in work from our laboratories.26 Compound
19, originally designed as a pan-kinase Bcr-Abl inhibitor,
displays moderate activity against the chloroquine sensitive 3D7
strain of P. falciparum (EC50 ∼ 200 nM). The primary issues
with this compound series included potent human kinase
activity as well as reduced potency against multidrug resistance
strains (such as W2). A scan of aqueous solubility-enhancing
groups quickly identified a methylpiperidinylpiperidine group
with similar 3D7 potency but improved potency on W2. Small
changes on the distal phenyl ring were also tolerated, resulting
in equipotent compounds, and led to a complete loss of human
kinase activity as measured in a Ba/F3 cell line panel of
receptor tyrosine kinases. From this observation, we concluded
that the pharmacophore responsible for kinase activity might
not be essential for antimalarial activity, and thus removing this
moiety from the scaffold could yield the minimum
pharmacophore. As expected, the loss of activity on human
kinases also resulted in a better safety profile for the series on
various mammalian cell lines (CC50 > 10 μM). Optimization of
the substituent on both aromatic rings led to 20, displaying an
EC50 = 58 nM against the 3D7 strain of P. falciparum and an
EC50 = 211 nM vs the multidrug resistant W2 strain. This
represents a ∼4-fold improvement in potency from the starting
point, a nearly 2-fold reduction in molecular weight (which
provided an orally bioavailable compound), and removal of
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 Journal of Medicinal Chemistry
Figure 9. Optimization of imidazolopyrimidine kinase library hit.
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Figure 10. Enantiospecific potency of spiroindolone hit.
potential off-target effects due to human kinase inhibition.
When compound 20 was screened against 15 different P.
falciparum wild-type and drug-resistant strains, the in vitro
EC50s were between 100 and 300 nM. On the basis of
comparison to known antimalarials, we predicted this series
would lack the in vitro potency required for in vivo efficacy and
did not progress the series further. Despite this, we concluded
that hit compounds that are human kinase inhibitors can be
useful starting points for lead optimization, providing that the
intrinsic kinase activity can be separated from cellular activity
early on and that positive SAR could be obtained from cell-
based optimizations.
The imidazolopyrimidines represent another class of
antimalarial hits previous optimized from a human kinase
program (Figure 9). The hit compound 21 possessed moderate
activities on the 3D7 parasite strain (EC50 = 420 nM) and did
not exhibit significant activity against a panel of 40 mammalian
kinases, which suggested that it was not a pan-kinase
inhibitor.27 The primary liability of 21 is low aqueous solubility
(36 μM at pH 6.8), which is likely driven by the high clogD
(7.4) of the compound. We initially sought to address this by
the introduction of hydrophilic or ionizable functionality into
the series. An SAR study of the optimal site(s) on the molecule
to introduce hydrophilic groups that did not have a large
negative effect on potency led us to the C2 pyrimidine position.
We generated a focused library which identified the N-
methylpiperazine derivative 22, which displayed an EC50 of
34 nM against the P. falciparum strain 3D7. Compound 22 was
also evaluated against a panel of 15 drug resistant strains of P.
falciparum. EC50 values across all strains were lower than 140
nM and many of which were below 100 nM, suggesting that the
compound might be a good lead to address drug resistance.
The incorporation of the basic functionality on the central ring
that is in close proximity to the presumed kinase binding
pharmacophore also resulted in a clean human kinase profile.
This was done through testing the compound on a larger panel
of 190 kinases in both biochemical and cellular assay formats.
Unfortunately, despite the greater than 10-fold improvement in
potency and the addition of an ionizable group, both the
aqueous solubility and the clogD of 22 were not improved
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compared to those of the hit compound 21. In addition, a
potential hERG channel blocker pharmacophore was intro-
duced by incorporation of a basic lipophilic group. Because we
were unable to improve physiochemical properties and in vitro
potency, we decided to focus on other scaffolds.
■ SPIROINDOLONES AND IMIDAZOLOPIPERAZINES
OPTIMIZATION OF ADMET PROPERTIES
Natural product derived antimalarials represent the mainstay of
antimalarial chemotherapies that were primarily optimized by
whole-cell screening approaches, including quinine and
artemisinin. On the basis of this premise, the Novartis natural
product library of ∼10K compounds, containing both pure
natural products and natural product-like synthetic compounds,
was screened. Hit compound 23 was identified as a racemic
mixture of unknown configuration (Figure 9) and displayed
moderate potency on both on the drug susceptible NF54 and
chloroquine-resistant K1 P. falciparum strains (EC50 ∼ 90
nM).28 This compound was unique compared to many other
natural product hits in that most of the hits were complex high
molecular weight structures with many rotatable bonds, making
them less interesting structures for this indication where cheap
oral compounds are needed. Compound 23 is a fully synthetic
molecule inspired by the privileged tetrahydrobetacarboline
moiety.29 The compound displayed moderate aqueous
solubility (75 μM at pH 6.8) and good oral exposure, as a 25
mg/kg oral dose in mice resulted in a high Cmax (1.4 μM), good
oral bioavailability (F = 59%), and an oral t1/2 of nearly 4 h.
Compound 23 also has a favorable safety profile for a screening
hit when evaluated against a panel of mammalian cell lines, in
hERG inhibition assays, against cytochrome P450 isoforms, and
in a human off-target panel (IC50 >10−30 μM), making it an
attractive hit compound. On the basis of the favorable
physicochemical properties and in vivo PK of 23, it was
evaluated in the P. berghei mouse model where compound is
administered 24 h after infection. Remarkably, a single oral dose
of 100 mg/kg resulted in a 96% reduction in parasitemia
measured two days after treatment. Given this exciting profile,
chiral separation followed by X-ray crystal structure determi-
nation unambiguously defined the configuration of both
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 Journal of Medicinal Chemistry
stereocenters. Interestingly, only one enantiomer is active with
the (1R,3S) enantiomer 24 responsible for biological activity.
Enantiomer 25 (1S,3R) is inactive against the parasite at >5000
nM EC50. Even more interesting was the discovery that the
active enantiomer had significantly different ADME properties
with regard to metabolic stability in microsomes (Figure 10).
Transitioning to the six-member spirocycle 26 (primarily due
to an improvement in potency relative to the seven-member
congener), the more active enantiomer was also the more
rapidly metabolized (t1/2 = 1.8 min). In contrast, the 1S,3R
enantiomer of 26 had a t1/2 = 103 min in mouse microsomes
(structure not shown). This significant difference in potency
and microsomal stability between enantiomers led the team to
improve metabolic stability in the active 1S,3R series using MS/
MS based metabolite ID as a guiding tool for optimization.
The phenyl ring of indole 26 is metabolically susceptible
(Figure 10). Small halogen substituents were introduced to
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hinder oxidation at these sites. By systematic substitutions of
the indole moiety of the spiroindolones, it was discovered that
blocking the C7 position had the most marked effect on
increasing the half-life determined in the presence of liver
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microsomes. Fortuitously, an added halogen at C6 also
provides a 3−10-fold gain in potency. The 6,7-disubstituted
derivatives display the most additive effects, providing current
phase 2 clinical candidate 27 (NITD609, Figure 11).30 The
Figure 11. Optimization of metabolic stability of spiroindolones.
improvements in the metabolic stability and potency of 27
translated remarkably well to in vivo efficacy in the P. berghei
infected mouse model. Orally administered 27 displayed a long
half-life (T1/2 = 10 h in mice) and excellent oral bioavailability
(F =100%). The 10-fold improvement in potency and reduced
clearance led to demonstration that a single oral dose of 27 (30
Table 1. Imidazolopiperazines in EBI Public Database
compd
ID HTS 3D7 EC50 (μM) HTS W2 EC50 (μM) HTS Huh7 CC50 (μM)
28 0.063 0.097 >10
29 0.235 0.271 >10
30 0.116 0.119 >10
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mg/kg) reduced parasitemia by 99.6% and has the potential to
be used as a single-dose cure in man.
The imidazolopiperazines represent the latest scaffold
optimized from the whole-cell screen, which resulted in the
identification of a new chemotype with promising blood- and
liver-stage activity. Imidazolopiperazine hits 28−30 represented
an attractive series displaying favorable potency on both drug-
susceptible and -resistant strains while maintaining good
selectivity in Huh7 cells (Table 1). Potency was retained
upon resynthesis, adding confidence to the HTS data set. These
three hit compounds, along with ∼5K other HTS hits from a
1+ million compound screen at Novartis/GNF, have been
deposited in the EBI public database.31 Data from other large
screens have also been made available in this database and have
been used as a source of additional potential hit compounds for
optimization.32−35
Compound 28 displayed excellent aqueous solubility (>175
μM at pH 6.8) and did not inhibit a panel of cytochrome P450
isoforms (IC50 >10 μM). The SAR from the screening
collection indicated some flexibility in the amino acid
substitutions off the piperazine nitrogen. Early issues associated
with the scaffold included metabolic instability and a moderate
hERG binding signal (IC50 = 19 μM). Figure 12 outlines the
early optimization results based on ∼100 initial analogues
made. Compound 31 (GNF776) was identified as our lead
molecule. Potency was improved 15-fold, exposure was
improved significantly (AUC, Cmax and high bioavailability),
and efficacy in mice was achieved (ED90 ∼20 mg/kg).
However, hERG binding activity also increased to an IC50 ∼
4 μM.
Early toxicity profiling identified a potential hERG liability
which we associated with the primary amine of the glycine
moiety. A hERG liability has also been observed for other
antimalarial scaffolds, including quinolines and more recently in
aminopyridines.36 A significant effort was spent on under-
standing the SAR of hERG activity and differentiating it from in
vitro potency. As the terminal amine was required for favorable
solubility and PK properties, we found that glycine turned out
to be the optimal substituent in terms of lower hERG activity.
The key was to incorporate the improved hERG inhibition
properties of the glycine group in 28 (presumably due to less
lipophilicity in the vicinity of the primary amine) with
improved PK of the dimethylglycine analogues (31).37 The
breakthrough result was in the incorporation of the dimethyl
powder Huh7 CC50
powder 3D7 EC50 (μM) powder W2 EC50 (μM) (μM)
0.46 0.473 >100
0.119 0.122 >79
0.030 0.029 42.82
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Figure 12. Hit to lead optimization of an imidazolopiperazine scaffold.
Figure 13. Lead optimization summary for imidazolopiperazine.
groups into the piperazine rings from the amino acid group of
31 as shown in Figure 13. Our primary motivation for making
this substitution was data suggesting chemical instability of the
benzylic methylene functionality and was confirmed by
independent synthesis as being an inactive degradation product.
Subsequent substitution of the benzylic carbon of the
piperazine present in 32 led to identification of compound
33 (GNF179) that significantly improves oral exposure and
potency against parasites raised to resistance of the parent HTS
hit 28.38 This result might have importance because the in vivo
activity of substituted piperazine derivatives such as 33 are quite
remarkable. Compound 33 displays an ED90 of ∼1 mg/kg (a
20× improvement from 31) that is superior to any of the
compounds discussed in this perspective. A close analogue of
33 is now progressing as a clinical candidate for both blood-
stage and liver-stage infections according to cell-based
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optimization of an HTS hit.39 We anticipate several more
reports of cell-based optimization of HTS hits to yield novel
drug candidates that can also provide new insight into parasite
biology.
■ AUTHOR INFORMATION
Corresponding Author
*Phone: +1 858 242-1016. E-mail: achatterjee@calibr.org.
Notes
The authors declare no competing financial interest.
Biography
Arnab K. Chatterjee was born in Calcutta, India, in 1975. After
completing a Bachelor of Arts in Chemistry from Northwestern
University in 1997, he proceeded on to Caltech in 1998 in the
laboratory of Professor Robert H. Grubbs. His doctoral research
focused on understanding the selectivity patterns of olefin cross-
dx.doi.org/10.1021/jm400314m | J. Med. Chem. 2013, 56, 7741−7749
 Journal of Medicinal Chemistry
metathesis reactions using catalytic ruthenium alkylidenes. Upon
completion of his doctoral research in September 2002, Arnab joined
the Genomics Institute of the Novartis Research Foundation. His work
over the last 10+ years has been focused on optimization in several
medicinal chemistry projects ranging a variety of therapeutic areas
(neuroscience, oncology, respiratory disease, and infectious diseases).
He is currently at the newly formed nonprofit California Institute for
Biomedical Research (Calibr) where he leads the chemistry efforts.
■
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