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