Identification and in-vitro ADME assessment of a series of novel anti-malarial agents suitable for hit-to-lead chemistry

Article abstract of DOI:10.1021/ml300091c

Triage of a set of antimalaria hit compounds, identified through high throughput screening against the Chloroquine sensitive (3D7) and resistant (Dd2) parasite Plasmodium falciparum strains identified several novel chemotypes suitable for hit-to-lead chemistry investigation. The set was further refined through investigation of their in vitro ADME properties, which identified templates with good potential to be developed further as antimalarial agents. One example was profiled in an in vivo murine Plasmodium berghei model of malaria infection.

Full text of DOI:10.1021/ml300091c

Letter
pubs.acs.org/acsmedchemlett
Identification and In-Vitro ADME Assessment of a Series of Novel
Anti-Malarial Agents Suitable for Hit-to-Lead Chemistry
Chris D. Edlin,*,† Garreth Morgans,† Susan Winks,† Sandra Duffy,⊥ Vicky M. Avery,⊥ Sergio Wittlin,§
David Waterson,‡ Jeremy Burrows,‡ and Justin Bryans∥
†iThemba Pharmaceuticals, P.O. Box 21, Modderfontein 1645, South Africa
§Swiss Tropical and Public Health Institute, Socinstrasse 57, 4002 Basel, Switzerland
‡Medicines for Malaria Venture, ICCRoute de Pre-Bois 20, P.O. Box 1826, 1215 Geneva, Switzerland
⊥Discovery Biology Institute, Griffith University, Brisbane, Queensland 4111, Australia
∥Medical Research Council Technology, 1-3 Burtonhole Lane, Mill Hill, London, United Kingdom
S
* Supporting Information
ABSTRACT: Triage of a set of antimalaria hit compounds, identified through high
throughput screening against the Chloroquine sensitive (3D7) and resistant (Dd2)
parasite Plasmodium falciparum strains identified several novel chemotypes suitable for
hit-to-lead chemistry investigation. The set was further refined through investigation of
their in vitro ADME properties, which identified templates with good potential to be
developed further as antimalarial agents. One example was profiled in an in vivo murine
Plasmodium berghei model of malaria infection.
KEYWORDS: Screening, Plasmodium falciparum, phenotypic screening, hit-to-lead chemistry
bout 3.3 billion people, half of the world’s population, are
at risk of malaria. This leads to about 250 million malaria
retested against the 3D7 and resistant (Dd2) Plasmodium
falciparum strains, as well as a HEK-293 cell line for mammalian
cytotoxicity. After retesting, thirty compounds were found to be
equally active against both parasite strains, having IC50 values
<1 μM and no observable cytotoxicity at 4 μM.
The thirty most active compounds from the screen were
subdivided into twenty structural classes, which were then
reviewed and refined on the basis of potential for the
generation of structural analogues in addition to feedback
from MMV indicating which series were already being explored
by other organizations or had historically been unsuccessful
candidates for antimalaria drug discovery efforts.
As a result, the list was prioritized to thirteen active
compounds in eight distinct classes (see Figure 1), quinazolines
1, pyrazolopyridazines 2, benzoxathiazoles 3, sulfonamides 4,
thienopyrimidines 5, sulfamides 6, acyl guanidines 7, and
benzimidazoles 8.
Eleven of the thirteen active compounds were available for
repurchase as solid samples, and an additional forty-six
structural analogues (selected to establish rudimentary SAR
within each series) were also sourced from commercial
suppliers (see the Supporting Information for full details and
QC). The purity and structures of all 57 compounds were
confirmed and then assayed at the Swiss Tropical and Public
A
cases and nearly one million deaths annually.1 People living in
the poorest countries are the most vulnerable. Malaria is an
especially serious problem in Africa, where one in every five
childhood deaths is due to the effects of the disease. An African
child has on average between 1.6 and 5.4 episodes of malaria
fever each year. Worldwide, a child dies from malaria every 30
s.1 The disease is caused by parasites of the genus Plasmodium.
Plasmodium falciparum and Plasmodium vivax are predom-
inantly responsible for the highest mortality and highest
morbidity, respectively.2 Current therapy, while effective, is
suffering from increased incidence of drug resistance; plus,
there have been no new chemical classes of antimalarials
introduced into clinical practice since 1996.3 Taken together,
the discoveries of new antimalarial agents with novel modes of
action are desirable.
During the last five years there has been a significant increase
in activity in the antimalarial discovery arena with Novartis,4 St
Judes Children’s Research Hospital Memphis,5 and Glax-
oSmithKline6 making major contributions to the body of
publicly available compound data.
The Medical Research Council Technology’s compound
library of 48,000 compounds was screened using a phenotypic
image based antimalarial high-throughput screening (HTS)
assay developed and performed within the Discovery Biology
group at Griffith University.7 From the primary screening, 389
compounds were identified with >40% inhibition against the
Chloroquine sensitive 3D7 strain. These hits were subsequently
Received: April 12, 2012
Accepted: May 28, 2012
Published: May 28, 2012
© 2012 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
Table 1. SAR Pyrazolopyridazines 9−18
Figure 1. Eight distinct classes of active anti-malarials after HTS and
review.
Health Institute against the Plasmodium falciparum strain NF54
(3D7 is a clone of NF54 and is Chloroquine sensitive) using
3
incorporation of H-Hypoxanthine as the primary readout.
Of the eight classes of compounds rescreened, four classes
(benzoxathiazoles 3, sulfonamides 4, quinazolines 1, and
sulfamides 6) were abandoned due to either weak or lack of
inhibitory activity of the original hits or close analogues or poor
whole cell ligand efficiency.8
From the four classes selected for progression, the
pyrazolopyridazines (Table 1) demonstrate reasonable SAR.
SAR data clearly indicates that the presence of the chloro
substituent in the 4 position is essential for activity and
replacement with isosteric methyl, ethoxy, or morpholine
substituents, resulting in loss of all activity. The presence of the
seemingly essential chlorine atom adjacent to the pyridazine
nitrogen represented a potential downstream development risk,
and as such, this series of molecules was down-prioritized.
The guanadinyl substituted quinazoline was investigated
(Table 2), and although the potency of this series of molecules
was modest, the whole cell ligand efficiency values indicated
that they may be reasonable starting points for a hit-to-lead
effort, with all examples displaying ligand efficiency indices over
0.3. However, this series was down prioritized due to the
presence of the guanidine functionality and the associated likely
permeability risk (even though heteroaryl guanidine has lower
pKa) and relatively weak potency.9
a
Values are a mean of ≥2 experiments in all tables unless otherwise
b
stated. Reference 8.
Table 2. SAR of Acyl Guanidines 19−22
The benzimidazole containing hit molecules represents a
good starting point for hit-to-lead chemistry due to the
expected relative ease of synthesis of the hits and close
analogues. The series shows good activity in the original HTS
(1−100 μM) and clear SAR with simple substituted amines in
the 4-position preferred (Table 3). Acylation and sulfonylation
(compounds 23, 24, and 28) of the 4-amino substituent results
in loss of activity. However, molecules with simple substituted
benzyl groups have respectable whole cell ligand efficiencies
(LE). A search of the literature and a robust discussion with
MMV indicated that there was adequate freedom to operate for
further exploration.
a
Values are a mean of ≥2 experiments in all tables unless otherwise
b
stated. Reference 8.
position of the thienopyrmidine ring including monocyclic and
fused bicyclic derivatives (see Table 4).
However, there appears to be a decrease in potency with
substitution on the 2- and 3-positions of the thienopyrimidine
The thienopyrimidine subclass of molecules was examined.
This series demonstrates clear SAR with a relatively wide level
of diversity tolerated in the heterocyclic substituent in the 4-
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On the basis of the literature searches, potency, demon-
stration of SAR, and whole cell ligand efficiency, the
benzimidazoles and thienopyrimidines were selected for further
investigation and an example from each series was chosen for
determination of some critical in vitro ADME parameters
(Table 5).
Table 3. SAR of Benzimidazoles 23−28
Table 5. Selected ADME Data
33
20
27
<5
a
b
solubility (μM)
log D (pH 7.4)
>3.8
50
2.0
19
c
human microsomal stab. (% turnover, 40 min)
permeability (A > B [10−6 cm/s])
d
50
1.5
0.43
efflux ratio
0.28
a
Kinetic solubility in phosphate buffered solution (PBS) at pH 7.4.
b
Partition coefficient determined between ocatan-1-ol and phosphate
c
buffered PBS at pH 7.4. 1 μM test concentration was carried out with
pooled microsomes (0.25 mg protein/mL). Apical and basal
d
chambers were prepared in Hanks Balanced Salt Solution (HBSS) at
pH 7.4. Compounds (10 μM) were added to the donor at time zero.
Both the donor and acceptor chamber were sampled following 60 min
of incubation at 37 °C.
a
Values are a mean of ≥2 experiments in all tables unless otherwise
b
As expected, the triazole-thienopyrimidine 33 had moderate
solubility (20 μM) and the benzimidazole 27 showed poor
solubility (<10 μM; which may be addressed by decreasing the
lipophilicity of the compounds). The triazole-thienopyrimidine
had moderate microsomal turnover (50% remaining after 40
min exposure), possibly due to the sulfur linker, but the
benzimidazole was metabolically stable, with a metabolic
turnover in human liver microsomes of only 19% after 40
min. The triazole-thienopyrimidine showed good permeability
while the benzimidazole showed poor permeability and low
mass balance, which may be reflective of the low compound
solubility.
stated. Reference 8.
ring 34 and 35. In all cases, the LE supports the fact that this
series of molecules represent a good starting point for further
optimization. Literature searches indicated excellent freedom to
operate for this series which was confirmed by MMV.
Table 4. SAR of Thienopyrimidines 29−35
The most attractive compound from this initial study in
terms of antiplasmodial activity and in vitro ADME properties,
33, was assessed in vivo in the Plasmodium berghei infected
mouse model. The ability of the compound to reduce the
parasitemia was evaluated by dosing compound 33 at 50 mg/kg
subcutaneously (sc) once a day for 4 days (Peters test). This
dosing regimen resulted in a 34% reduction in parasitemia over
the course of the experiment. Also, no gain in survival days
compared to the case of infected, untreated control animals was
observed. This level of activity is marginal in terms of statistical
significance but is of sufficient interest to warrant further
investigation into this series. Specifically, given that the in vitro
ADME properties of both compounds studied are moderate,
there is sufficient justification to suppose that optimization of
these parameters will lead to an improved in vivo DMPK profile
and hence improved efficacy of these compound classes when
assessed in vivo. Also, both series demonstrate SAR, which
appears not to be linked to lipophilicity, giving further
confidence that solubility and microsomal stability can be
optimized. Future optimization plans will focus on improving
the in vitro ADME profile compounds focusing on reducing the
cLogP and improving the solubility of the series.
In summary, a HTS campaign identified 389 novel
antiplasmodial compounds with 30 examples demonstrating
sub-micromolar IC50’s against resistant and nonresistant strains
of Plasmodium falciparum with good selectivity indices against
cytotoxicity. The genesis of the hit identification; hit
a
Values are a mean of ≥2 experiments in all tables unless otherwise
b
stated. Reference 8.
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ACS Medicinal Chemistry Letters
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Figure 2. Schematic representation of compound progression to in vivo assessment.
S.; Brown, J. R.; Peishoff, C. E.; Cardon, L. R.; Garcia-Bustos, J. F.
Thousands of chemical starting points for antimalarial lead
identification. Nature 2010, 465, 305−310.
confirmation; resistance and cytotoxicity profiling; analogue
sourcing; SAR analysis; ADME studies; and finally in vivo
assessment is summarized diagrammatically in Figure 2.
Preliminary SAR refined the hits to two promising series of
compounds with good freedom to operate and the potential for
further follow-up through optimization of their ADME
properties.
(7) Duffy, S.; Avery, V. M. Development and Optimization of a
Novel 384-Well Anti-Malarial Imaging Assay Validated for High-
Throughput Screening. Am. J. Trop. Med. Hyg. 2012, 86 (1), 84−92.
(8) Alex, A.; Hopkins, A. L.; Groom, C. R. Ligand efficiency: A useful
metric for lead selection. Drug Discovery Today 2004, 9, 430−431.
(9) Humphreys, W. G.; Obermeier, M. T.; Chong, S.; Kimball, S. D.;
Das, J.; Chen, P.; Moquin, R.; Han, W.-C.; Gedamke, R.; White, R. E.;
Morrison, R. A. Oxidative activation of acylguanidine prodrugs:
intestinal presystemic activation in rats limits absorption and can be
inhibited by co-administration of ketoconazole. Xenobiotica 2003, 33
(1), 93−106.
ASSOCIATED CONTENT
* Supporting Information
■
S
Spectral data and vendors for compounds 9−35 and details
regarding biological assays. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: cedlin@ithembapharma.com.
■
Author Contributions
All authors have given approval to the final version of the
manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors thank M.M.V. for advice on compound selection.
■
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■
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