Synthesis and in vitro evaluation of hydrazinyl phthalazines against malaria parasite, Plasmodium falciparum

Article abstract of DOI:10.1016/j.bmcl.2016.05.049

In this report, we describe the synthesis of 1-(Phthalazin-4-yl)-hydrazine using bronsted acidic ionic liquids and demonstrate their ability to inhibit asexual stage development of human malaria parasite, Plasmodium falciparum. Through computational studies, we short-listed chemical scaffolds with potential binding affinity to an essential parasite protein, dihydroorotate dehydrogenase (DHODH). Further, these compounds were synthesized in the lab and tested against P. falciparum. Several compounds from our library showed inhibitory activity at low micro-molar concentrations with minimal cytotoxic effects. These results indicate the potential of hydralazine derivatives as reference scaffolds to develop novel antimalarials.

Full text of DOI:10.1016/j.bmcl.2016.05.049

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and in vitro evaluation of hydrazinyl phthalazines against
malaria parasite, Plasmodium falciparum
Gowtham Subramanian a,y, C. P. Babu Rajeev b,y, Chakrabhavi Dhananjaya Mohan c, Ameya Sinha a,
Trang T. T. Chu a, Sebastian Anusha b, Huang Ximei d, Julian E. Fuchs e,f, Andreas Bender e,
b,
Kanchugarakoppal S. Rangappa c, Rajesh Chandramohanadas a, , Basappa
⇑
⇑
a Pillar of Engineering Product Development, Singapore University of Technology & Design, Singapore 487372, Singapore
b Laboratory of Chemical Biology, Department of Chemistry, Bangalore University, Central College Campus, Palace Road, Bangalore 560001, India
c Department of Studies in Chemistry, Manasagangotri, University of Mysore, Mysore 570006, India
d School of Biological Sciences, Nanyang Technological University, Singapore, Singapore
e Centre for Molecular Science Informatics, Department of Chemistry, University of Cambridge, Lensfield Road, CB2 1EW Cambridge, UK
f Institute of General, Inorganic and Theoretical Chemistry, University of Innsbruck, Innrain 82, 6020 Innsbruck, Austria
a r t i c l e i n f o
a b s t r a c t
Article history:
In this report, we describe the synthesis of 1-(Phthalazin-4-yl)-hydrazine using bronsted acidic ionic liq-
uids and demonstrate their ability to inhibit asexual stage development of human malaria parasite,
Plasmodium falciparum. Through computational studies, we short-listed chemical scaffolds with potential
binding affinity to an essential parasite protein, dihydroorotate dehydrogenase (DHODH). Further, these
compounds were synthesized in the lab and tested against P. falciparum. Several compounds from our
library showed inhibitory activity at low micro-molar concentrations with minimal cytotoxic effects.
These results indicate the potential of hydralazine derivatives as reference scaffolds to develop novel
antimalarials.
Received 23 February 2016
Revised 5 May 2016
Accepted 18 May 2016
Available online xxxx
Keywords:
Hydralazines
Antimalarials
Dihydroorotate dehydrogenase
P. falciparum
Ó 2016 Published by Elsevier Ltd.
Bronsted acidic ionic liquids
Malaria remains a major health burden to the developing world,
with an estimated death toll of half a million people every year.1
Despite concerted efforts, a malaria vaccine is not yet available.
Therefore, chemotherapy remains the primary mode of treat-
ment.2–5 However, rapidly increasing resistance against conven-
tional drugs such as chloroquine, artemisinin, mefloquine and
sulfadoxine/pyrimethamine is contributing to the decline in prog-
nosis and survival rate from malaria.6,7
Recent research highlights the significance of plasmodial dihy-
droorotate dehydrogenase (DHODH), a critical enzyme involved
in pyrimidine biosynthesis, as a therapeutic target.8 Generally,
pyrimidine biosynthesis occurs either de novo or by salvage path-
way. In contrast, Plasmodium species avail pyrimidines solely
through de novo synthesis which involves DHODH activity.9 Thus,
we set out to design and analyze potent small molecules that can
manipulate DHODH function and thereby survival and replication
of the parasites within human red blood cells.
In search of better antimalarials, various heterocycles with
structural similarity with quinolines have been studied extensively
including phthalazine derivatives. Several studies have demon-
strated the antimalarial potential of various phthalazine ana-
logues.10–14 Herein, we designed phthalazine based small
molecules, which are isomeric with quinazoline ring and tested
them for potential antimalarial activity. Piperidinyl phthalazine
derivatives have shown antimalarial properties in prior studies,
which reflect the possible use of these nuclei as antimalarials.
Thus, in continuation of our research to synthesize and explore
the pharmacological properties of various heterocycles15–24, we
designed and evaluated novel phthalazines against malaria para-
sites. Our results revealed that, some of the compounds exhibited
inhibition of parasite development at concentrations comparable
to previously tested DHODH inhibitors.
General synthesis of phthalazine derivatives: Initially, we synthe-
sized 1-(Phthalazin-4-yl) hydrazine (4) by a two-step simple and
efficient synthetic route shown in Scheme 1A. The phthalazine
⇑
Corresponding authors.
E-mail addresses: rajesh@sutd.edu.sg (R. Chandramohanadas), salundibasappa@
gmail.com (Basappa).
y
Contributed equally.
http://dx.doi.org/10.1016/j.bmcl.2016.05.049
0960-894X/Ó 2016 Published by Elsevier Ltd.
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G. Subramanian et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Scheme 1. (A and B) Schematic representation for the synthesis of the novel hydralazine reported here.
Table 1
Physical data of the newly prepared hydrazinyl phthalazines
Entry
Aldehyde (5a–l)
Product
Yield
87a
OHC
O2N
OCH3
N
N
N
6a
OCH3
N
H
NO2
N
HN
O
O
N N
O
O
6b
6c
6d
86a
91b
90b
O
H
N
N
N
N
N
N
HN
HN
N
O
H
H
N
N
N
H
O
H
N
N
N
6e
6f
88b
N
O
NH
HN
O
S
O
N
N
N
81a
N
H
O
S
O
O
N
N
N
N
N
6g
6h
88b
83a
N
O
N
O
N
H
N
N
O
O
N
N
H
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G. Subramanian et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
Table 1 (continued)
Entry
Aldehyde (5a–l)
OH
Product
Yield
91a
OH
N
N
N
O
N
H
6i
6j
Cl
Cl
O
N
N
77a
N
N
H
a
Novel compounds.
Literature reported compounds.
b
Figure 1. Predicted protein-ligand interactions of DHODH and hydralazine-derived compounds: A cut through the binding site of DHODH is shown as green cartoon (PDB:
4CQ9), interaction centers Arg-265 and Phe-227 are highlighted as lines. (A) Co-crystallized ligand IDI-6253 is shown as sticks in atomic coloring (white: carbon, blue:
nitrogen). (B) The predicted docking pose of compound 6b shows high similarity in orientation and anchor points in the binding pocket.
(1) was selectively chlorinated by trichloroisocyanuric acid at
40 °C–50 °C in DMF to get 1-chloro phthalazine (2) as product
and 1,4-dichlorophthalazine (3) as byproduct. The 1-chlorophtha-
lazine was allowed to react with excess of 99% hydrazine hydrate
in ethanol to get the required product (4) and it was recrystallized
in ethanol.
the number of hydrogen bond acceptors is very similar with on
average 4.94 (SD = 1.08) versus 5 in IDI-6253.
After successfully redocking the co-crystallized ligand to the
binding site with an all atom RMSD of 0.25 Å, we docked the full
series of hydralazine compounds to DHODH. We found a consistent
binding mode for the set of compounds. The phthalazine anchor
group is involved in hydrogen bonding with Arg-265, thereby
replacing the triazole of IDI-6523 (see Fig. 1). The apolar tail groups
form several hydrophobic and p-contacts involving Phe-227, thus
occupying a similar region as the tetrahydro-isoquinoline of IDI-
6523.
The use of ionic liquids as a green solvent in organic transfor-
mations has gained significant attention due to their wide liquid
range, essentially limited vapor pressure and good solvating ability
which contributes to the replacement of organic solvents.25,26 In
the second step, we condensed compound 4 with various substi-
tuted aldehydes to prepare Schiff base derivatives of 1-(Phtha-
lazin-4-yl)-hydrazine. The using of bronsted acids (H2SO4, HCl,
PPA, AcOH) as dehydrating agents resulted in the formation of
impure products with poor yield. Therefore, we used bronsted
acidic ionic liquids (1,2,3-trimethylimidazoliummethylsulphate)
as a dehydrating agent in the new approach to get desired product
with high purity and good yield (Table 1). The reaction is outlined
in Scheme 1B. The obtained Schiff base derivatives of 1-(Phtha-
lazin-4-yl)-hydrazine (6a–j) of hydralazine were characterized by
LC–MS, 1H NMR, 13C NMR, IR and elemental analysis and details
are provided as Supplementary information.
In silico analysis to identify possible hydralazines with binding
affinity to Plasmodium falciparum dihydroorotate dehydrogenase
(DHODH): Initially, we searched the Protein Data Bank to identify
protein-ligand co-crystal structures involving PfDHODH with
known chemical scaffolds and detailed methodology is provided
in Supplementary information. From these analyses, we observed
that our hydrophobic ligands show similar chemical scaffolds to
the co-crystallized ligand IDI-6253 involving several nitrogen-
containing aromatic rings.27 Average calculated logPs for our com-
pounds is 3.38 (standard deviation SD = 0.72) and therefore very
similar to logP = 3.00 for the template ligand IDI-6253. Moreover,
In vitro validation of new hydralazines against P. falciparum: First,
we evaluated the effect of newly synthesized hydralazines against
common laboratory strains (3D7) of P. falciparum at trophozoite
stage (ꢀ30 h post-invasion (hpi)) at three different concentrations
(1, 20, and 100 lM) and detailed methodology is provided in
Supplementary information. DMSO-treated parasites were
included as negative control. After 48 h, parasitemia was estimated
by flow cytometry.28 Five of the compounds tested, namely, 6c, 6d,
6e, 6g, and 6i displayed significant inhibitory potential against
P. falciparum (Fig. 2A) with estimated IC50 less than 20 lM, which
is comparable to previously reported PfDHODH inhibitors synthe-
sized by Pavadai et al.29 These compounds were selected for
further experiments.
To document the precise inhibitory activity of these com-
pounds, trophozoite stage parasites (3D7, ꢀ24–26 hpi) were incu-
bated at concentrations ranging from 1.6 lM to 100 lM for 48 h.
Parasites treated with DMSO and chloroquine were included in
the assay as negative and positive controls, respectively. After the
assay was completed, parasites were harvested and counted
through flow cytometry. Compounds 6c, 6d, 6e, 6g, and 6i had
IC50 values of 13.7, 12.1, 3.4, 3.4, and 1.6
lM, respectively against
3D7 parasites (Fig. 2B). In a parallel experiment performed using
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G. Subramanian et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Figure 2. Inhibitory activity of hydrazinyl phthalazine compounds used in this study against P. falciparum (A). Dose response curve for the 5 most effective compounds are
shown in the figure. These compounds were screened against laboratory strain of malaria parasite, 3D7 (B) and chloroquine resistant parasite variant, K1 (C). Screening was
performed at concentrations ranging from 0 lM to 100 lM. Both parasite strains showed comparable sensitivity to most inhibitors (all except 6c and 6d). Microscopic
examination of Giemsa-stained smears indicated a general inhibition of parasite maturation at schizont stage. Representative phenotypes observed for compounds 6g and 6i
are shown in D.
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G. Subramanian et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
5
Table 2
Overall susceptibility to each molecules of P. falciparum strains 3D7 and K1. Sensitivity of parasites (3D7 and K1) within a 0–100
l
M compound concentration range was
evaluated. Further, cytotoxicity of the same compounds was tested on the MDCK cell line, and the resulting selectivity is evaluated as 3D7 IC50/MDCK IC50
Entry
IC50 3D7 (
lM)
95% Confidence interval
IC50 K1 (lM)
95% Confidence interval
Cytotoxicity IC50 MDCK (
lM)
Selectivity index
6c
6d
6e
6g
6i
13.74
12.18
3.49
3.495
1.646
12.91–14.63
11.00–13.48
3.002–4.058
3.246–3.764
1.513–1.791
6.66
5.98
2.43
2.23
1.69
5.51–8.05
5.08–7.04
1.77–3.34
1.85–2.69
1.60–1.80
>200
>200
>200
>200
>100
14.56
16.42
57.31
57.22
60.75
Figure 3. Cytotoxicity assays (MTT) was performed against Madin-Darby Canine Kidney (MDCK) cell lines, using the compounds 6c, 6d, 6e, 6g and 6i, together with
appropriate controls. None of the compounds showed significant cytotoxicity up to 200 lM.
chloroquine-resistant K1 strain, IC50 values of 6.6, 5.9, 2.4, 2.2 and
1.6 lM were obtained for the same compounds (Fig. 2C). Giemsa-
growth and development before the formation of multinucleated
schizont stage.32
stained images prepared from representative experiments were
also tested to examine possible phenotypes (Fig. 2D). These images
demonstrated a general inhibition of merozoite maturation at sch-
izont stage.
To evaluate the toxic effect of the lead compounds against
mammalian cells and thereby to estimate selectivity, we per-
formed cytotoxicity assay (MTT) using Madin-Darby Canine Kidney
(MDCK) cell line.30,31 None of the compounds induced detectable
Structural activity relationship analyses of the tested com-
pounds revealed that the presence of electron donating hydroxyl
or amino groups attached to gamma position to the phthalazine
ring like phthalazineANHAN@CHAC@CAOH CHACHAOH (or
NH) was responsible for profound inhibitory activity of the com-
pounds. On the other hand, the presence of electron withdrawing
groups like nitro, carbonyl, or sulphonyl moiety at the same posi-
tion reduced the inhibitory potential.
cytotoxicity at concentrations up to 200
l
M (Table 2 and Fig. 3).
In conclusion, herein we report the synthesis and antimalarial
activity determination of novel hydralazine derivatives. The anti-
malarial activity of new compounds is likely to be mediated
through the inhibition of DHODH, an important enzyme in de novo
biosynthesis of pyrimidines and therefore nuclear division in P.
falciparum.
From these observations, it was apparently clear that most com-
pounds are capable of selectively inhibiting the malaria parasite
and possibly not the host cells.
We further performed stage specific assays on P. falciparum. To
do this, compounds were introduced at distinct time points/life
stages (ring, trophozoite and schizont) and general growth/matu-
ration and morphological characteristics were assessed. In brief,
compounds were added to rings (2–4 h)/trophozoite (24–26 h)/
Acknowledgments
schizonts (36–38 h) at a range of concentrations (4 * IC50, 2 * IC50
,
This research was supported by University Grants Commission
(41-257-2012-SR), Vision Group Science and Technology, Depart-
ment of Science and Technology (NO. SR/FT/LS-142/2012) to Bas-
appa. GS, AS, TC and RC acknowledge the following grants to RC:
SRLS13049, SUTD-ZJU/RES/02/2013. RC acknowledges laboratory/
instrument support provided by the SUTD-MIT International
Design Centre (IDC) and Singapore-MIT Alliance for Research &
Technology (SMART) Center funded by the National Research
Foundation, Singapore. KSR thanks DST Indo-Korea [INT/Indo-
Korea/122/2011-12] and Institution of Excellence, University of
Mysore for financial support. CDM thanks the University of Mysore
for Department of Science and Technology-Promotion of University
Research and Scientific Excellence (DST-PURSE) Research Associate
IC50 and 0.25 * IC50—estimated from Table 2). Roughly 12 h later,
samples were harvested for each treatment. Flow cytometry and
microscopy-based examination of parasites was conducted to
monitor any stage specific inhibitory effect induced by the com-
pounds. All compounds showed a potential inhibitory effect on
schizont to ring stage transition (Fig. 4A–C), which indicates that
these compounds act by interfering with late stage schizont devel-
opment (Table 3). This is consistent with prior studies where inhi-
bitors of PfDHODH were tested against malaria parasites.
Furthermore, the observed phenotypes are similar to the anti-
malarial activities of DSM265, the first DHODH inhibitor to reach
clinical development for malaria treatment, arresting the parasite
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G. Subramanian et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Figure 4. Effect of the selected compounds on distinct stages of parasite development. To document stage-specificity of the compounds tested, experiments were carried out
using parasites at early stage during ring-trophozoite transition (A), trophozoite-schizont transition (B) and schizont-ring transition (C), at four different compound
concentrations (4 * IC50, 2 * IC50, IC50 and 0.25 * IC50). Majority of the compounds showed an effect during the later stages of parasite development, and appeared to inhibit
merozoite formation.
Table 3
Stage-specific effects of the compounds presented here on Plasmodium falciparum. ‘ꢁ’ represents no effect; ‘+’ indicates effect at 4 * IC50; ‘++’ indicates
effect at 4 * IC50 and 2 * IC50; ‘+++’ indicates effect at 4 * IC50, 2 * IC50 and IC50. It can be seen that most compound are active on the later stages of
parasite development
Entry
Ring to trophozoite
Trophozoite to schizont
Schizont to ring
6c
6d
6e
6g
6i
Control
ꢁ
+
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
+
++
++
ꢁ
++
++
+++
++
ꢁ
ꢁ
ꢁ
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Please cite this article in press as: Subramanian, G.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.05.049