New benzimidazole derivatives as antiplasmodial agents and plasmepsin inhibitors: Synthesis and analysis of structure-activity relationships

Article abstract of DOI:10.1016/j.bmcl.2011.10.018

The newly synthesized benzimidazole compounds were suggested to be inhibitors of Plasmodium falciparum plasmepsin II and human cathepsin D by virtual screening of an internal library of synthetic compounds. This was confirmed by enzyme inhibition studies that gave IC₅₀ values in the low micromolar range (2-48 μM). Ligand docking studies with plasmepsin II predicted binding of benzimidazole compounds at the center of the extended substrate-binding cleft. According to the plausible mode of binding, the pyridine ring of benzimidazole compounds interacted with S1′ subsite residues whereas the acetophenone moiety was in contact with S1-S3 subsites of plasmepsin II active center. The benzimidazole derivatives were evaluated for capacity to inhibit the growth of intraerythrocytic P. falciparum in culture. Four benzimidazole compounds inhibited parasite growth at ≤3 μM. The most active compound 10, 1-(4-phenylphenyl)-2[2-(pyridinyl-2-yl)-1,3-benzdiazol-1-yl] ethanone showed an IC₅₀ of 160 nM. The substitution of a phenyl group and a chlorine atom at the para position of the acetophenone moiety were shown to be crucial for antiplasmodial activity.

Full text of DOI:10.1016/j.bmcl.2011.10.018

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1282–1286
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
New benzimidazole derivatives as antiplasmodial agents and plasmepsin
inhibitors: Synthesis and analysis of structure–activity relationships
Zafar Saied Saify a, , M. Kamran Azim a, Waseem Ahmad b, Mehrun Nisa a, Daniel E. Goldberg c,
⇑
Shaheen A. Hussain d, Shamim Akhtar d, Arfa Akram d, Arshad Arayne d, Anna Oksman c, Ishtiaq A. Khan b
a H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan
b Department of Biochemistry, Federal Urdu University, Karachi 75300, Pakistan
c Howard Hughes Medical Institute, Department of Medicine and Molecular Microbiology, Washington University School of Medicine, St. Louis, MO, USA
d Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Karachi, Karachi 75270, Pakistan
a r t i c l e i n f o
a b s t r a c t
Article history:
The newly synthesized benzimidazole compounds were suggested to be inhibitors of Plasmodium
falciparum plasmepsin II and human cathepsin D by virtual screening of an internal library of synthetic
compounds. This was confirmed by enzyme inhibition studies that gave IC50 values in the low micromo-
Received 24 May 2011
Revised 7 September 2011
Accepted 7 October 2011
Available online 20 October 2011
lar range (2–48 lM). Ligand docking studies with plasmepsin II predicted binding of benzimidazole com-
pounds at the center of the extended substrate-binding cleft. According to the plausible mode of binding,
the pyridine ring of benzimidazole compounds interacted with S10 subsite residues whereas the aceto-
phenone moiety was in contact with S1–S3 subsites of plasmepsin II active center. The benzimidazole
derivatives were evaluated for capacity to inhibit the growth of intraerythrocytic P. falciparum in culture.
Keywords:
Antimalarial
Aspartic protease
Cathepsin D
Four benzimidazole compounds inhibited parasite growth at 63 lM. The most active compound 10,
1-(4-phenylphenyl)-2[2-(pyridinyl-2-yl)-1,3-benzdiazol-1-yl]ethanone showed an IC50 of 160 nM. The
substitution of a phenyl group and a chlorine atom at the para position of the acetophenone moiety were
shown to be crucial for antiplasmodial activity.
FlexX docking
Flow cytometry
Ó 2011 Elsevier Ltd. All rights reserved.
As the most serious tropical parasitic disease, malaria accounts
for an estimated 300–500 million cases and up to 2.7 million
deaths annually, particularly among young children.1 Severe
malaria is a complex multisystem disorder involving adherence
of parasites to blood vessel endothelial cells and severe anemia.2
Four Plasmodium species are responsible for malaria in humans,
that is, P. vivax, P. ovale, P. malariae and P. falciparum.3 Infection
caused byP. falciparum is the only one normally lethal. P. falciparum
has developed resistance to nearly all available antimalarial drugs.4
Therefore, there is an urgent need for new antimalarials based on
novel mechanisms of action.5
The members of plasmepsin (Plm) family of Plasmodium aspar-
tic proteases are involved in hemoglobin degradation during the
intra-erythrocytic phase of infection; a process essential for prop-
agation carried out in food vacuole of malarial parasite.6 Ten
Plasmepsins were identified in the genome of P. falciparum, and
four of them, that is, Plm-I, -II, -III and –IV/(HAP) participate in
hemoglobin degradation.7 Previously, treatment of mouse malaria
using Pepstatin A suggested the potential of plasmepsin inhibitors
as antimalarial drugs.8 Recent knockout studies revealed a requi-
site inhibition of all the four plasmepsins to prevent proliferation
of parasite.9 Plasmepsins (in particular plasmepsin I and II) have
been considered as promising target for new antimalarial drugs
but Liu et al.10 have reported that inhibition of food vacuole plasm-
epsins does not appear to be a promising strategy, unless combined
with the inhibitors of malarial parasite cysteine proteases falci-
pains that are also involved in hemoglobin catabolism. Neverthe-
less, use of plasmepsin II as a target has led to the identification
of a number of potent antimalarial compounds.11 Therefore,
inhibition of this enzyme can be seen as a valuable proxy for
inhibition of other targets, most likely nonfood vacuole plasmep-
sins that have been hard to approach biochemically.10 Therefore,
search for inhibitors broadly active against all hemoglobin-
degrading plasmepsins (Plm-I, -II, -III and -IV) while remaining
inactive against the closely related human aspartic proteases
(cathepsins D and E) continues. Plasmepsin inhibitors with fair
selectivity versus human cathepsins have been reported.12
A number of chemical functionalities and structural units have
been utilized as noncleavable transition-state isosteres in
plasmepsin II inhibitors.11 These inhibitors include statine-derived
and reversed-statine based cores,13,14 tertiary amines based on
4-aminopiperidine-tert-butyl-carbamate,15 hydroxyethylamines,
dihydroxyethylenes and hydroxymethylcarbonyl,16,17 diphenyl-
urea,18 acridinyl hydrazides,19,20 achiral oligoamines21 and tetra-
hydro-azepine scaffold compounds.22
⇑
Corresponding author. Tel.: +9221 111222292x175; fax: +9221 4819018.
E-mail addresses: zssaify@gmail.com, zssaify@iccs.edu (Z.S. Saify), kamran.azi-
m@iccs.edu (M. Kamran Azim).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.10.018

Z. S. Saify et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1282–1286
1283
Table 1
Benzimidazole is a heterocyclic aromatic compound consists of
benzene and imidazole rings. The most prominent benzimidazole
compound in nature is N-ribosyl-dimethylbenzimidazole, which
serves as an axial ligand for cobalt in vitamin B12.23 Benzimid-
azole, in an extension of the well-elaborated imidazole system,
has been used as a carbon skeleton for N-heterocyclic carbenes
(NHC). The NHCs are usually used as ligands for transition metal
complexes. They are often prepared by deprotonating an N,
N0-disubstituted benzimidazolium salt at the two-position with
a base.24,25 Compounds bearing the benzimidazole moiety are
reported to have proton pump inhibiting,26 anticancer,27 antialler-
gic,28 antioxidant,29 antihistaminic30 and antimicrobial31 activi-
ties. We synthesized a new class of benzimidazole derivatives
(2-pyridine-2-yl-1H-benzimidazole) and evaluated their activity
against P. falciparum (Fig. 1).32 These compounds were also sug-
gested as potential plasmepsin II and human cathepsin D inhibi-
tors by virtual screening of an in-house compounds database. The
ligand docking simulation was confirmed by enzyme inhibition
studies.
Antiplasmodial and aspartic protease inhibitory activities of benzimidazole
derivatives
Compd.
IDs
Antiplasmodial
activity
Plasmepsin-II
inhibition
Cathepsin D
inhibition
IC50 in
20.4
19.6
21.2
30.6
30.5
10
lM
2
3
4
5
6
7
8
9
P3.0
P3.0
0.7
P3.0
2.1
21.2
28.5
31.9
39.2
17.8
31.9
44.5
39.2
2.0
3.0
P3.0
P3.0
0.16 0.4
63.0
63.0
48
10
14.7
37.7
34.0
10
11
12
14.2
21.2
proteins fixed. Among the two catalytic aspartates, Asp34 was
considered protonated and Asp214 negatively charged during
docking (plasmepsin II numberings). After each ligand
docking run, 10 top ranking docking solutions were saved and
considered for detailed analysis. The enzyme activities of
plasmepsin II and cathepsin D were measured as described
earlier.39,40
Molecular docking of the compounds present in the in-house
database predicted benzimidazole derivatives as potential inhibi-
tors of plasmepsin II and cathepsin D. Enzyme inhibition data
(Table 1) pointed out that (a) the IC50 values of benzimidazole
compounds against plasmepsin II and cathepsin D are in the ranges
Virtual screening was carried out by FlexX ligand docking
software (version 2.0)36 using an in-house library of >1000
synthetic compounds and crystal structural coordinate sets of
P. falciparum plasmepsin II (PDB id; 1M43)37 and human cathep-
sin
D
(PBD id; 1LYB).38 The in-house library compounds
corresponds to thirty different chemical scaffolds that have been
synthesized in our laboratory. 3D models of compounds in
SYBYL mol2 format were utilized for binding to the active sites
of both aspartic proteases. FlexX ligand docking was carried
out allowing full flexibility for the ligands, while keeping the
of 10–48 lM and 11–44 lM, respectively, [except cathepsin D
inhibition by compound 10] (Fig. 2A and B; Table 1); (b) com-
pounds 7 and 9 are three times more selective towards plasmepsin
O
H
NH2
NH2
II compared to cathepsin D with IC50 = 10
lM (Table 1) and (c)
N
HO
compound 10 is a comparably potent and selective inhibitor of
+
N
Poly-
cathepsin D with IC50 = 2.0
epsin II) (Fig. 2C; Table 1).
lM (compared to IC50 = 14.7 for plasm-
N
N
phosphoric
acid
compound 1
3D structural analysis of the predicted binding modes of
benzimidazole compounds provided the basis of inhibition from
a structural standpoint. The five top binders of benzimidazole
compounds were modeled into the active site of plasmepsin II
to examine interactions with protein residues. Analysis of FlexX
docking solutions predicted binding of benzimidazole com-
pounds at the center of substrate binding cleft (Fig. 3). The benz-
imidazole moiety of these compounds was docked in the middle
with pyridine and acetophenone side chains protruding in differ-
ent directions. The carbonyl group of the acetophenone side
chain is predicted to be pointing towards the side chain of cat-
alytic aspartate Asp34 (Fig. 3). Analysis of top five docking solu-
tions of 11 benzimidazole compounds indicated two binding
modes (Fig. 4A and B). In the ‘preferable’ mode of binding, 9
out of 11 compounds (i.e., except compounds 3 and 11) were
predicted to bind at the active center such that the pyridine
moiety interacted with S10 subsite binding residues whereas ace-
tophenone side chain interacted with S1–S3 subsite forming
residues (Fig. 4A). In contrast, compounds 3 and 11 were bound
in opposite fashion, that is, the pyridine moiety interacted with
S1–S3 subsite residues and the acetophenone side chain inter-
acted with the S10 subsite (Fig. 4B). Because the majority of
benzimidazole compounds (9 out of 11 compounds) were
docked in similar fashion, therefore the first mode of ligand
binding was considered more plausible.
R3
+
R2
R1
R4
R5
R1
R2
O
R3
O
Br
R5
R4
N
N
N
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
R1=H
R1=H
R1=H
R2= NO2
R2=OH
R2=H
R2=H
R2=H
R2=H
R2=H
R2=H
R2=H
R2=H
R2=H
R3=H
R4=H
R5=H
R5=H
R5=H
R5=H
R5=H
R5=H
R5=H
R5=H
R5=H
R5=H
R5=H
R3=OH
R3=Cl
R3=H
R4=H
R4=H
R4=H
R4=H
R4=H
R4=H
R4=H
R4=H
R4=H
R4=OMe
R1=NO2
R1=H
R1=H
R1=F
R3= NO2
R3=F
R3=F
R1=H
R3= H
R3= Ph
R3= OMe
R3=H
(10) R1=H
(11) R1=H
(12) R1=OMe
All antiplasmodial assays were carried out in P. falciparum
clone HB-3. EC50 values of the tested compounds were deter-
mined essentially as described earlier.41 The benzimidazole deriv-
atives with substitutions at the acetophenone ring showed potent
Figure 1. Synthesis of 2-pyridine-2-yl-1H-benzimidazole derivatives (2–12).

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Z. S. Saify et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1282–1286
Figure 2. (A) Plasmepsin-II inhibition plots as a function of benzimidazole compounds 2–12 concentration; (B) Cathepsin D inhibition plots as a function of benzimidazole
compounds 2–12 concentration (except compound 10); (C) Cathepsin D inhibition plot as a function of compound 10 concentration.
antiplasmodial activity. Among the compounds tested, compound
10 had the highest antiplasmodial activity with IC50
0.16 0.4 (Fig. 5A and B) whereas compounds 4 and 6
showed IC50 values of 0.7 M and 2.1 M, respectively, (Fig. 5C
=
lM
l
l
and D). Assessment of killing with two different well-established
methods, flow cytometry (Fig. 5A) and hypoxanthine (Fig. 5B)
gave similar results. The remaining compounds were considered
to be inactive with IC50 P 3.0 lM (Table 1). Structure activity
relationship (SAR) analysis indicated that the higher antiplasmo-
dial activity of compound 10 was due to the extra phenyl group
present at the para position of acetophenone ring (Fig. 1). Fur-
thermore, the presence of a chlorine atom in compound 4 and a
nitro group in compound 6 pointed out a role of these substitu-
tions at the para position acetophenone in antiplasmodial activity
(Fig. 1).
Our data show that these benzimidazole compounds are clearly
aspartic protease inhibitors. However, antiplasmepsin activity did
not correlate well with parasite killing. There are 10 aspartic pro-
teases encoded by the P. falciparum genome and not all are well
characterized. It is likely that the antiparasitic action of these com-
pounds is against one of the less characterized aspartic proteases.
The potency of these compounds, especially compound 10 suggests
that this is a new class of inhibitors with promise for further anti-
malarial drug development.
Figure 3. Docking of a benzimidazole derivative (compound 2) in the substrate
binding cleft of plasmepsin II. The compound 2 is shown in ball-stick, plasmepsin-II
in solid ribbon and Catalytic aspartates (Asp 34 and Asp214) in stick representation.

Z. S. Saify et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1282–1286
1285
Figure 4. (A) Docking of compound 2 (thick stick) in the active site cleft of plasmepsin-II representing the benzimidazole derivatives binding with prefered mode of binding;
(B) Docking of compound 3 (thick stick) in the active site of plasmepsin-II with alternative mode of binding. Enzyme residues involved in binding of inhibitors are shown.
Locations of different subsites (S1, S2, S3, S10 and S20) are indicated.
Figure 5. In vitro antiplasmodial activities of compounds 4, 6 and 10 (designated as #4, #6 and #10). Graphs are log compound concentration versus% inhibition of parasite
growth compared to no inhibitor control. (A) Parasite growth measured by flow cytometry; (B–D) parasite growth measured by 3H-hypoxanthine incorporation.

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Z. S. Saify et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1282–1286
25. Huynh, H. V.; Ho, J. H. H.; Neo, T. C.; Koh, L. L. J. Organomet. Chem. 2005, 16,
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Z.S.S. and M.K.A. are grateful to Higher Education Commission,
Islamabad, Ministry of Health, Govt. of Pakistan, Islamabad and
Pakistan Science Foundation for financial support.
Supplementary data
30. Goker, H.; Ayhan-Kilcigil, G.; Tun-cbilek, C.; Kus, R.; Ertan Kendi, E.; Ozbey, S.;
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32. The 2-pyridine-2-yl-1H-benzimidazole nucleus (compound 1) was synthesized
by using equimolar quantities of picolinic acid and o-phenylenediamine, added
to polyphosephoric acid (PPA) in 1:10 ratio and stirred under N2 at 150 °C for
8 h.33–35 The resulting yellow solution was poured into water, the produced
solid filtered and washed with 0.5 M sodium carbonate solution. Compounds
2–12 were synthesized by treating compound 1 with equimolar quantities of
each of the other halogenated acetophenones with various substitutions
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Briefly, 200 microliter aliquots of late ring stage cultures at 0.5% parasitemia
were incubated with various concentrations of benzimidazole compounds in
hypoxanthine-free rich medium for 42 h. 0.5 lCi of [3H]hypoxanthine
(178.7 Ci/mmol; PerkinElmer) was added to the culture and the incubation
continued for 24 h. Cultures were harvested on glass fiber paper, immersed in
UltimaGold scintillation counting mixture (PerkinElmer, USA) and counted in a
scintillation counter. The percentage of the inhibition of [3H] hypoxanthine
uptake was plotted against the drug concentration and the curve was fitted
using the modified dose–response logistic equation in KaleidaGraph software.
Flow cytometry was carried out as has been described.10