Exploring beta amyloid cleavage enzyme-1 inhibition and neuroprotective role of benzimidazole analogues as anti-alzheimer agents

Article abstract of DOI:10.1002/jccs.201900200

Beta amyloid cleavage enzyme-1 (BACE1) is the key enzyme involved in Aβ peptide formation in Alzheimer's disease pathogenesis. We intend to target this enzyme by exploring benzimidazole analogues against BACE1 as potential anti-Alzheimer agents. Docking studies were performed to determine the hydrogen bond interactions between the designed molecules and the target protein's active site. Research indicates the relationship between oxidative stress and Aβ effect in precipitating neurodegeneration; hence, the series was also studied in vitro to ascertain its neuroprotective role by performing the lipid peroxidation assay. In silico absorption, distribution, metabolism, and excretion studies were undertaken to assess the drug-like suitability of the analogues. To judge the effect of the synthesized analogues on central nervous system (CNS), toxicity and memory model studies were conducted on mice. Thus, overall results showcase analogues 11 and 14 as the most promising ones with the dual role of BACE1 inhibition and neuroprotection, along with memory retention.

Full text of DOI:10.1002/jccs.201900200

Received: 21 May 2019
Revised: 4 October 2019
Accepted: 13 October 2019
DOI: 10.1002/jccs.201900200
A R T I C L E
Exploring beta amyloid cleavage enzyme-1 inhibition
and neuroprotective role of benzimidazole analogues
as anti-alzheimer agents
Archana S. Gurjar
| Vivek S. Solanki | Ankita R. Meshram |
Suchita S. Vishwakarma
Department of Pharmaceutical Chemistry,
Principal K. M. Kundnani College of
Pharmacy, Mumbai, India
Abstract
Beta amyloid cleavage enzyme-1 (BACE1) is the key enzyme involved in Aβ
peptide formation in Alzheimer's disease pathogenesis. We intend to target this
enzyme by exploring benzimidazole analogues against BACE1 as potential
anti-Alzheimer agents. Docking studies were performed to determine the
hydrogen bond interactions between the designed molecules and the target
protein's active site. Research indicates the relationship between oxidative
stress and Aβ effect in precipitating neurodegeneration; hence, the series was
also studied in vitro to ascertain its neuroprotective role by performing the
lipid peroxidation assay. In silico absorption, distribution, metabolism, and
excretion studies were undertaken to assess the drug-like suitability of the ana-
logues. To judge the effect of the synthesized analogues on central nervous sys-
tem (CNS), toxicity and memory model studies were conducted on mice. Thus,
overall results showcase analogues 11 and 14 as the most promising ones with
the dual role of BACE1 inhibition and neuroprotection, along with memory
retention.
Correspondence
Archana S. Gurjar, Department of
Pharmaceutical Chemistry, Principal
K. M. Kundnani College of Pharmacy,
23 Jote Joy, R. S. Marg, Cuffe Parade,
Mumbai 400 005, India.
Email: gurjaras@gmail.com
Funding information
University of Mumbai, Grant/Award
Numbers: Minor Research Project Grant
509, APD/ICD/2018-19
K E Y W O R D S
BACE1, docking, in silico ADME studies, in vitro LPO assay, memory model studies, toxicity
study
1
| INTRODUCTION
neurotransmitter imbalance, oxidative stress, and others
including calcium dysregulation and inflammation.
[3]
Alzheimer's disease (AD) is an advancing multifaceted
neurodegenerative disorder resulting in the loss of the
neurons and intellectual abilities including memory and
reasoning, which imposes tremendous emotional, social,
and economic burden on the patient, his or her family,
Amongst the etiological factors, beta amyloid cleavage
enzyme-1 (BACE1, beta-secretase) is the key enzyme
responsible for the formation of Aβ peptides. BACE1 is a
type I transmembrane enzyme with high concentration
in neurons and belongs to the same retroviral and pepsin
aspartic protease family. It has an optimum acidic pH
with the correct sequence specificity for the processing of
amyloid precursor protein (APP), leading to an increased
[1,2]
and the community.
Several hypotheses have been
proposed to be involved in the etiopathogenesis of AD
such as beta amyloid (Aβ) formation and its accumula-
tion, Tau (τ) hyperphosphorylation and aggregation,
[
4,5]
Aβ generation.
It regulates a range of neuronal
©
2019 The Chemical Society Located in Taipei & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
J Chin Chem Soc. 2019;1–10.
http://www.jccs.wiley-vch.de
1

2
GURJAR ET AL.
functions and the formation of a myelin sheath in periph-
eral nerve cells. BACE1 knockout mice do not produce
Aβ from APP in the brain but are viable, suggesting that
targeted inhibition of the BACE1 enzyme would consid-
O
H N
2
N
F
N
N
[6]
N
erably reduce the on-target toxicities. The BACE1 crys-
tal structures project the active site as a long cleft with
two catalytic aspartate residues (Asp32 and Asp228) for
N
N
N
N
[7,8]
substrate recognition.
Beta-secretase inhibitors
1
H N
2
OCF3
broadly belong to two categories: peptidomimetic and
nonpeptidic. The nonpeptide BACE1 inhibitors can cross
the blood–brain barrier and reach the target site more
easily compared to the peptidomimetic ones. Some of the
nonpeptide inhibitors reported in literature are:
LY2811376 [(S)-4-(2,4-difluoro-5-pyrimidin-5-yl-phenyl)-
2
N
R
N
H
[
9]
3
4
-methyl-5,6-dihydro-4H-[1,3]thiazin-2-ylamine],
Lan-
abecestat (AZD3293), biflavonoids (2,3-dihydroamentoflavone
[
10]
FIGURE 1 Aminoimidazole and benzimidazole scaffolds
and 2,3- dihydro-6-methylginkgetin),
and various
nonpeptidic scaffolds such as amidino- or guanidino-
heterocycles, which were found to be apt BACE1
inhibitors due to hydrogen bond formation with the
are carcinogenic, such as dioxane; costly (lanthanum
triflates); or unstable. A search of suitable reagents led to
ceric ammonium nitrate (CAN), an inexpensive, easily avail-
able oxidizing agent for many functional groups (alcohols,
phenols, and ethers), as well as C─H bonds, especially the
benzylic ones. Aqueous CAN in catalytic amounts can be
[
11]
catalytic aspartyl dyad.
2+
Research depicts that the Cu ion may induce the
conversion of normal Aβ42 to abnormal conformation, in
turn facilitating its aggregation into fibril. There exists an
involvement of metal ions in the formation of amyloid
fibrils, and this metallo-Aβ complex leads to oxidative
[29]
used for the efficient synthesis of heterocycles.
On the basis of the above literature review, 1H-benzo[d]
imidazole scaffold (3) has been explored, and the designed
series of benzimidazole analogues has been targeted for the
dual role of BACE1 inhibition and antioxidant activity
(Table 1). Based on molecular docking and in silico absorp-
tion, distribution, metabolism, and excretion (ADME) stud-
ies, analogues that exhibited better interactions and drug-
like properties were selected for the synthesis and pharma-
cological evaluation, which comprises in vitro LPO assay,
acute toxicity studies, and memory model studies.
[12,13]
stress in the brains of AD patients.
Aβ-mediated
neurotoxicity is a result of the intracellular accumulation
of reactive oxygen species, leading to lipid oxidation. Oxi-
dative stress and Aβ accumulation leads to the formation
of lipid peroxidation (LPO) product; hence, antioxidant
activity estimation would be beneficial to assessing the
[14,15]
neuroprotective role.
Nitrogen-containing heteroaromatics are crucial core
scaffolds used to design drugs and represent one of the
most biologically active class of compounds. Investigation
of the reported benzimidazole analogues showed a wide
range of pharmacological activities, such as antimicro-
bial, analgesic, anticonvulsant, antihypertensive, antican-
2 | RESULTS AND DISCUSSION
2.1 | Molecular docking
[16]
cer, antitumor, and antiviral.
Malamas (1) and
Gravenfors (2) et al have independently reported amino
Molecular docking is a valuable tool to anticipate ligand–
[
17,18]
imidazoles as potent BACE1 inhibitors (Figure 1).
Benzimidazole derivatives fused with the oxadiazole ring
enzyme interactions. A literature survey indicates the role of
the two catalytic aspartyl BACE1 active site residues (Asp32
[19]
[30]
system exhibited moderate antioxidant activity.
Con-
and Asp228) in enzymatic catalysis.
The X-ray crystal
ventional methods for the synthesis of benzimidazoles
make use of harsh strong acids or mineral acids for the
reaction of o-aryldiamines with carboxylic acids and their
structure of BACE1 complexed with octapeptide inhibitor
OM00-3 (PDB: 1m4h) projected eight subsites that can be
[31]
associated during ligand–enzyme interactions.
[20]
derivatives.
Other methods have reported the direct
The PDB 1m4h structure showcased a good activity
condensation of o-aryldiamines and aldehydes in the
presence of various reagents or catalysts such as
profile with K 0.3 nM and was thus considered for the
i
[
31]
docking studies.
The docking protocol was validated
[21]
[22]
[23]
dioxane,
MgCl .6H O,
copper
hydroxide,
NH Cl,
4
with an root mean square deviation of 1.51 (Figure 2). The
[
24]
[25]
[26]
0
0
octapeptide inhibitor OM00-3 occupied S , S , S , and S
2
H O and HCl,
boric acid,
lantha-
2
2
2
2
1
2
1
[
27]
[20,28]
num chloride,
and triflates.
Some of the reagents
sub sites and interacted primarily with the catalytic

GURJAR ET AL.
3
TABLE 1 In silico ADME results of 2-substituted-1H-benzo[d]imidazole analogues
Mol.
wt.
Donor
HB
QPlogP
AccptHB o/w
QP
logBB
Rule
% human oral
absorption
a
No.
4
R
QPPMDCK of five
H
118.14
132.17
146.19
160.22
166.61
194.24
1
1
1
1
1
1
2
2
2
2
2
2
1.32
1.52
2.55
2.62
2.46
3.26
0.10
0.18
0.17
0.21
0.38
0.28
1,402.18
1868.13
2,247.21
2,588.39
5,166.26
2,698.87
0
0
0
0
0
0
95.86
100
5
CH
3
2
6
CH
CH
3
100
7
CH(CH
3
)
2
100
8
CH
2
Cl
100
9
Ph
100
10
11
12
13
14
15
16
17
18
19
20
21
22
23
184.20
239.23
226.23
209.25
270.29
239.23
224.26
210.24
210.24
224.26
254.29
284.31
228.68
228.68
1
1
3
3
2
1
1
2
2
1
1
1
1
1
2
3
3
3
4
3
2
2
2
2
3
4
2
2
2.57
2.62
1.72
2.21
2.81
2.53
3.34
2.52
2.42
3.34
3.5
0.23
−0.54
−0.74
−0.33
−0.27
−0.66
0.21
2,233.4
365.19
0
0
0
0
0
0
0
0
0
0
0
0
0
0
100
93.81
85.70
95.32
100
247.49
628.52
1,130.27
272.58
91.14
100
2,702.77
1,052
−0.13
−0.27
0.21
100
742.27
100
2,696.25
2,695.2
2,695.96
5,347.96
6,664.27
100
0.14
100
3.58
3.62
3.68
0.08
100
0.39
100
0.45
100
a
g/mol.

4
GURJAR ET AL.
FIGURE 2 Docking pose of OM00-3 and analogue 11 in BACE1 active site
aspartate dyad, Asp32 and/or Asp228, which plays a cru-
cial role in enzymatic catalysis and inhibitor binding. The
amino acid residues of various subsites are as follows: S1:
118 to 284 with an outlier analogue 4. The number of
donor hydrogen bonds (DonorHB) were found to be
1–3, while acceptor hydrogen bonds (accptHB) were 2–4.
The QPlogPo/w parameter is an important factor for par-
titioning of drugs between lipophilic and hydrophilic
phases. The QPlogPo/w values were favorable as observed,
1.32–3.68. Similarly, the QPlogBB values for all the ana-
logues were obtained in a small range of −0.74 to 0.39.
The QPPMDCK model modestly mimics the blood–brain
barrier. The QPPMDCK values were gauged as optimum
to great. None of these analogues violated Lipinski's rule
of five for drug-like properties. The percentage of human
oral absorption was found to be high, that is, more than
85%, for all the analogues. Thus, these analogues can be
presumed to be potential drug candidates with desirable
pharmacokinetic properties.
Leu30, Asp32, Tyr71, Gln73, Phe108, Asp228, Gly230; S :
2
0
Tyr71, Thr72, Gln73, Gly230, Thr231, Arg235; S : Gly34,
1
0
Tyr71, Thr72, Asp228; and S : Gly34, Ser35, Val69, Pro70,
2
[31]
Tyr71, Tyr198.
In the present study, a series was designed using 1H-
benzo[d]imidazole scaffold with various substituents at
2
-position in the imidazolyl ring. (Table 1) The substitu-
ents have been varied from simple alkyl groups such as
methyl and ethyl to phenyl and heteroaryl. Furthermore,
mono-, di-, and trisubstitutions with different functional
groups have been tried at various positions in the phenyl
ring. In the designed series, most of the analogues have
interacted with BACE1 active site residues. The analogues
0
mainly occupied S and S subsites, analogues 5, 8, 9, 18,
1
1
and 19 have occupied the S subsite probably due to inter-
2
action with Arg235 residue of the BACE1 active site. Some
analogues, that is, 11, 12, 13, and 14, interacted well with
the catalytic aspartate dyad. Amongst them, substituents
such as amino, hydroxy, and methoxy groups formed H-
bonds with the active site residues, while nitro-substituted
analogue interacted through the NH of benzimidazole
ring. Thus, these benzimidazole analogues might be prom-
ising candidates for AD.
2.3 | Synthesis and characterization
On the basis of computational results, designed ana-
logues were chosen for the synthesis using a CAN
reagent and further characterized with infrared (IR),
nuclear magnetic resonance (NMR), and mass spectros-
copy. The synthesized analogues IR spectra portrayed
absorption bands nearly identical to the literature for the
1
13
functional group present in them. HNMR and CNMR
spectra exhibited predictable delta values for the protons
[
34]
2.2 | In silico ADME studies
and carbon atoms, respectively.
Mass analysis of the
analogues showed the expected molecular mass values.
Thus, all the results of identification and characterization
optimistically confirms the formation of synthesized ana-
logues and the correctness of their anticipated structure.
The spectra have been provided in the Supplementary
Information.
The drug-like properties of the analogues were assessed
by performing in silico ADME studies. The in silico
ADME study results were found to be very much within
[
32,33]
the QikProp recommended ranges (Table 1).
The
molecular weight (Mol. wt.) in the series varied from

GURJAR ET AL.
5
2
.4 | Pharmacological evaluation
.4.1 | In vitro LPO assay
2.5 | Memory model studies
2
2.5.1 | Elevated plus maze model
As discussed earlier, oxidative stress and Aβ accumulation
leads to the formation of LPO product, so antioxidant activity
was determined by means of in vitro LPO assay to portray
the neuroprotective role exhibited by the analogues. The IC50
value of the reference compound ascorbic acid was observed
to be 47.77 μM and in the range of 25–265 μM for the benz-
imidazole analogues (Table 2). Amongst them, two ana-
logues (11 and 14) exhibited IC50 values of 25.68 and
This model was used to judge the effect of the synthe-
sized compounds on the memory and learning of Swiss
albino mice. Individual groups consisting of six mice
(three male and three female) were treated with analogue
11 at a dose of 50 and 100 mg/kg and analogue 14 at a
dose of 50 mg/kg (Table 3). There was substantial
decrease in the transfer latency (TL) compared to the tox-
icant group. The results demonstrate successful retention
of memory in the mice by both these analogues. How-
ever, TL recorded for analogue 11 at 100 mg/kg dose level
was more effective.
53.82 μM, better than others in the series. The in vitro LPO
assay results for analogue 16 were incoherent and were
therefore not considered. Thus, the in vitro LPO assay results
for these analogues showcased their neuroprotective role.
2.5.2 | Passive avoidance test
2.4.2 | Correlation of docking and LPO
assay results
The test was conducted after 24 hr (on ninth day) without
shock, and the time required to step down on the grid
floor from the wooden box was recorded as step-down
latency (SDL), a measure of learning. On comparison with
the toxicant, analogues 11 and 14 retained memory as the
respective dosed animals took prolonged time to step
down from the block. Furthermore, relating the two test
compounds, analogue 11 showcased better results, analo-
gous to the elevated plus maze (EPM) results (Figure 3).
Data for the above two tests were analyzed by one-way
analysis of variance. A significant statistical difference was
found between control and test groups (p value > .01).
Hence, post-test was applied to the data using Dunnett
multiple comparison tests. According to Dunnett test, if q
is greater than 2.730, then p value is less than .05.
The docking results of benzimidazole analogues 11, 13,
and 14 correlated with the in vitro antioxidant activity
results. These analogues exhibited interactions with the
catalytic dyad, which plays a vital role in inhibitor binding
and enzymatic catalysis. Analogously, these compounds
exhibited better IC50 values in the in vitro LPO assay.
2.4.3 | Acute toxicity studies
Based on the above correlation from the three analogues,
1 and 14 with good LPO assay IC50 values were selected
1
for the in vivo studies. Following the guidelines, an acute
toxicity study of representative benzimidazole analogue
1
1 led to identification of a safe and nonlethal dose of
1
,000 mg/kg in Swiss albino mice.
3 | EXPERIMENTAL
3
.1 | Structure-based drug design and
TABLE 2 In vitro LPO assay results
molecular docking
a
Analogue no.
IC50 (μM)
127.12
84.18
For ligand and protein preparation, Sybyl version 8.1.1
and Biopolymer module (Tripos International, St Louis,
MO) running on Red Hat Enterprise Linux (RHEL)
workstation were used, respectively.
5.2.2 (CCDC Ltd., England, UK) was used for docking
4
9
1
1
1
1
1
1
1
2
3
4
5
7
25.68
[35]
GOLD version
127.37
69.50
[
36]
studies.
53.82
265.08
81.41
3.2 | Ligand and protein preparation
Ascorbic acid
47.77
All the ligands and protein were prepared as indicated in
our earlier published article.
a
[37]
Values are the average of three experiments.

6
GURJAR ET AL.
250
TABLE 3 Effect of test compounds on transfer latency in
elevated plus-maze model
200
1
1
50
00
Mean transfer
S. No. Groups
latency ± SD in s
31.600 ± 3.209
21.600 ± 2.302
50
1
2
Control
0
Scopolamin
e HBr (0.4
mg/kg)
Vehicle
control
Piracetam
(200mg/kg)
Analogue 11 Analogue 11 Analogue 14
Standard (piracetam
(50mg/kg)
(100mg/kg)
(50mg/kg)
2
00 mg/kg)
mean
135.6
188.6
95.8
147.4
165.6
116.6
3
Toxicant (scopolamine
HBr: 0.4 mg/kg)
80.200 ± 5.070
FIGURE 3 Passive avoidance test results
4
5
6
Analogue 11 (50 mg/kg)
Analogue 11 (100 mg/kg)
Analogue 14 (50 mg/kg)
59.400 ± 2.302
46.600 ± 2.966
66.000 ± 3.873
13
CNMR in DMSO. The spectrometer used for recording the
mass spectra was 410 Prostar Binary LC with 500 MS IT
PDA detectors (Varian Inc).
3.3 | Molecular docking
The same steps were followed to prepare and validate the
docking protocol as mentioned in our previous article.
3.7 | Method
[
37]
3.7.1 | Synthesis of 1H-benzo[d]
imidazole (1)
3.4 | In silico ADME studies
The synthesis was carried out as per the standard refer-
ence procedure, and the reactions were monitored using
an n-hexane: ethyl acetate (1:1) TLC system.
In silico ADME studies were carried out on the RHEL
[
39]
workstation with QikProp software version 3.3
[38]
(Schrodinger LLC, New York, NY).
3
.7.2 | Synthesis of substituted-2-phenyl-
1H-benzo[d]imidazole analogues (9, 11, 12,
4–17)
3.5 | Method
1
LigPrep was used to preprocess the structures for input to
QikProp for in silico ADME prediction. The structures
were optimized by means of the OPLS-2005 force field.
The results can be viewed in the generated-OUT file.
A total of 0.5 g. o-phenylenediamine (4.629 mmol) and
substituted aromatic aldehydes (4.629 mmol) were stirred
in 15 ml ethanol in the presence of cerric ammonium
nitrate (0.1 mmol) for 48 hr. The reaction was monitored
by TLC technique using ethyl acetate: n-hexane (1:1). The
reaction mixture was then poured in brine solution for
precipitation. The precipitate was then filtered, washed
with cold water, dried using vacuum, and recrystallized
with ethanol: water (1:1) mixture to obtain the title com-
pounds. The synthesized compounds purity was confirmed
3.6 | Synthesis and characterization
All materials used were acquired from commercial sources.
The purity of starting materials was assessed by thin-layer
chromatography (TLC), as well as by determination of their
physical constants. Reactions were routinely monitored by
thin-layer chromatography on Merck silica gel F254 plates,
and spots were visualized under an ultraviolet lamp at
1
13
by TLC, IR, HNMR, CNMR, and mass spectroscopy.
2
54 nm. Analab melting point apparatus μThermoCal10
3.7.3 | Synthesis of 2-(4-aminophenyl)-
1H benzo[d]imidazole (13)
was used to determine the physical constants, and a
Shimadzu IR Affinity-1 fourier transform infra red (FTIR)
spectrophotometer was used to record IR spectra. HNMR
spectra were registered in parts per million (ppm) with tetra-
methyl silane as an internal standard on Mercury Plus
1
A total of 1.9 g. of o-phenylenediamine (17.592 mmol)
and 2.25 g. of p-aminobenzoic acid (16.423 mmol) were
refluxed for 2 hr in the presence of o-phosphoric acid.
The cooled reaction mixture was then poured on crushed
ice with stirring. The pH of the solution was adjusted to
300 MHz (Varian) and Bruker 300 Ultra shield NMR Spec-
trometer by using acetone/ dimethyl sulfoxide (DMSO) sol-
vent. Bruker BioSpin GmbH at 100 MHz was utilized for
[
40]
7 using 5 M sodium hydroxide solution.
The brown

GURJAR ET AL.
7
1
color precipitate was filtered, dried, and recrystallized
from methanol. The purity of the compound was checked
by TLC and confirmed by IR, HNMR, CNMR and
mass. TLC system used was n-hexane: ethyl acetate (1:1).
1,450.53 (C C). HNMR: (ppm, 300 MHz, DMSO-d ), δ
6
12.576(s, 1H), 6.832–6.859 (d, J = 8.1 Hz), 6.637–6.663
(d, J = 7.8 Hz), 7.635–7.657 (d, J = 6.6 Hz), 7.469–7.444
(d, J = 7.5 Hz), 6.984–7.011 (d, J = 8.1 Hz), 8.893 (s,
1
13
1
3
OH). CNMR (ppm, 100 MHz, DMSO-d ): δ 147.13,
6
1
45.43, 145.36, 144.57, 142.66, 135.89, 127.73, (121.96,
121.76-d), 120.33, 118.72, (117.08, 116.62-d), (115.69,
15.55-d), 113.44. MS: (M+2): 228.3 (Figures S10, S11,
3.8 | Spectral and analytical data
1
3.8.1 | 1H-benzo[d]imidazole (4)
and S12).
ꢀ
Buff white (s), Melting point—172 C, Yield—78%, R —
f
−
1
0
.157, 0.162, FTIR (cm ): 3,064.06 (NH), 2,794.97
3.8.5 | 2-(4-aminophenyl)-1H-benzo[d]
imidazole (13)
(
C─H), 1,246.07 (C─N), 1,587.48 (C N), 1,409.06 (C C),
13
CNMR (ppm, 100 MHz, DMSO-d ): δ 141.86, 121.51,
6
ꢀ
1
18.91, 111.57. MS: (M+1): 119.2 (Figures S1 and S2).
Buff white (s), Melting point—245 C. Yield—51%,
−
1
R —0.264, 0.230, FTIR (cm ): 3,350 (NH), 3,439.23
f
(
NH ), 2,977.26 (C─H), 1,273.07 (C─N), 1,608.70
2
1
3
.8.2 | 2-phenyl-1H-benzo[d]
(C N), 1,444.75 (C C). HNMR: (ppm, 300 MHz,
DMSO-d6),
imidazole (9)
δ
11.676 (s, 1H), 7.494–7.523 (dd,
J = 3.0 Hz), 7.117–7.159 (dd, J = 3.0 Hz), 7.948–8.008
ꢀ
Pale yellow (s), Melting point—288 C, Yield—79%, R —0.157,
0
(dd, J = 4.65 Hz), 6.773–6.841 (dd, J = 2.04 Hz), 5.123
f
−1
13
.162, FTIR (cm ): 3,220 (NH), 3,118.90 (C─H), 1,315.45
(s, NH2).
CNMR (ppm, 100 MHz, DMSO-d ): δ
6
1
(C─N), 1,540 (C N), 1,436.97 (C C), HNMR (ppm,
152.52, 150.50, 127.68, (121.28, 120.99-d), 117.88,
117.28, 113.48, 110.51. MS: (M+1): 210.2 (Figures S13,
S14, and S15).
3
00 MHz, DMSO-d ): δ 11.914 (s, 1H), 7.235–7.252 (t, J = 2.55
6
Hz), 7.710–7.725 (t, J = 2.25 Hz), 8.252–8.268 (d, J = 4.8 Hz),
7
DMSO-d ): δ 150.37, 130.86, 129.08, 128.81, 126.96, 126.12,
13
.523–7.588 (m, J = 9.45 Hz). CNMR (ppm, 100 MHz,
6
123.22, 114.72. MS: (M+1): 195.2 (Figures S3, S4, S5, and S6).
3.8.6 | 2-(4-hydroxy-
3
,5-dimethoxyphenyl)-1H-benzo[d]
3.8.3 | 2-(2-nitrophenyl)-1H-benzo[d]
imidazole (14)
imidazole (11)
ꢀ
Brown (s), Melting point—177 C, Yield—78%, R —0.230,
f
ꢀ
−1
Brown (s), Melting point—270 C, Yield—91.6%, R —
0.264, FTIR (cm ): 3,345.67 (OH), 3,201.97 (NH), 2,970
(C─H), 1,275.90 (C─N), 1,218.10, 1,112.01 (C─O─C),
1,602.91 (C N), 1,465.96 (C C). HNMR: (ppm,
f
−
1
0
.512, 0.3928, FTIR (cm ): 3,153.61(NH), 3,072.60
1
(
C─H), 1,246.02 (C─N), 1,541.12, 1,307.74 (NO ),
2
1
1
3
,651.07 (C N), 1,436.97 (C C), HNMR (ppm,
00 MHz, DMSO-d ): δ 13.051(s, 1H), 7.560–7.585 (d,
300 MHz, DMSO-d ), δ 12.691 (s, 1H), 7.485–7.515 (t,
6
J = 4.5 Hz), 7.158–7.188 (dd, J = 2.85 Hz), 7.599–7.627 (s,
6
J = 7.5 Hz), 7.216–7.280 (t, J = 9.6 Hz), 7.839–7.893
1H), 6.989 (s, 1H), 6.344 (s, 1H), 8.902 (s, 1H), 3.884 (s,
13
(
td, J = 9.6 Hz), 7.649–7.673 (d, J = 7.2 Hz),
.649–7.673 (dd, J = 1.35 Hz), 8.015–8.045 (dd,
J = 1.2 Hz), 7.963–7.999 (dd, J = 1.35 Hz), 7.728–7.733
1H). CNMR (ppm, 100 MHz, DMSO-d ): δ 148.14,
6
7
143.83, 134.91, (122.27, 121.93-d), 121.34, (118.86,
118.30-d), 110.82, 106.69, 104.11. MS: (M+2): 272.2
(Figures S16, S17, and S18).
1
3
(
d, J = 1.5 Hz). CNMR (ppm, 100 MHz, DMSO-d ): δ
6
1
48.92, 147.26, 132.58, 130.88, 130.83, 124.24, 123.02,
1
21.83, 119.20, 111.61. MS: (M+2): 241.2 (Figures S7,
S8, and S9).
3.8.7 | 2-(3-nitrophenyl)-1H-benzo[d]
imidazole (15)
ꢀ
3
.8.4 | 2-(3, 4-dihydroxyphenyl)-1H-
Light brown (s), Melting point—205 C, Yield—73.2%,
f
−
1
benzo[d]imidazole (12)
R —0.710, 0.6, FTIR (cm ): 3,286.70 (NH), 3,086.11
C─H), 1,292.31 (C─N), 1,550.71, 1,550.71 (NO ),
1,643.35 (C N), 1,643.35(C C). HNMR: (ppm,
300 MHz, DMSO-d ), δ 13.295 (s, 1H), 7.589–7.709 (d,
J = 4.8 Hz), 7.250–7.266 (d, J = 4.8 Hz), 9.012–9.024
(
2
ꢀ
1
Pale yellow (s), Melting point—236 C, Yield-66.66%,
−
1
R —0.081, 0.085, FTIR (cm ): 3,379.43 (OH), 3,100
f
6
(
NH), 2,924.21 (C─H), 1,280.79 (C─N), 1,605.81 (C N),

8
GURJAR ET AL.
(
t, J = 1.8 Hz), 9.012–9.024 (dt, J = 3.2 Hz), 7.83–7.88
the above mixture, and its final volume was adjusted to
4 ml with distilled water. This solution was heated in a
water bath for 60 min at 95 C. Then, the solution was
(
3s, 1H), 8.312–8.350 (2 dd, J = 1.05 Hz, 0.75 Hz).
1
3
ꢀ
CNMR (ppm, 100 MHz, DMSO-d ): δ 149.02, 148.35,
6
1
(
1
35.25, 132.45, 131.71, 130.66, (124.46, 124.17-d),
122.80, 122.54-d), (121.56, 121.54-d), (121.25,
20.79-d), 117.32. MS: (M+2): 241.2 (Figures S19, S20,
and S21).
cooled to room temperature, and the final volume was
again increased to 5 ml with distilled water. For extrac-
tion purposes, 5 ml mixture of n-butanol: pyridine (15:1)
was utilized. After centrifugation at 4,000 rpm for
1
0 min, the upper organic solution's optical density was
[
41]
noted at 532 nm against an appropriate blank.
The ref-
3
.8.8 | 2-(3-methoxyphenyl)-1H-benzo[d]
erence standard and all analogues were assayed by the
same method. The following formula was used for calcu-
lating the percentage inhibition, that is, decrease in TBA
reacting substance formation against an appropriate
control.
imidazole (16)
ꢀ
Dark brown (s), Melting point—204 C, Yield—67.17%,
−1
R —0.756, 0.8, FTIR (cm ): 3,097.68 (NH), 2,985.81
f
(C─H), 1,271.09 (C─N), 1,238.30, 1,010 (C─O─C),
1
1
,620.21 (C N), 1,462.04 (C C). HNMR: (ppm,
Percentage inhibition
À
Á
3
00 MHz, DMSO-d ), δ 12.93 (s, 1H), 7.462–7.489 (d,
6
= Abs
−Abssample × 100=Abscontrol
:
control
J = 8.1 Hz), 7.043–7.055 (t, J = 4.05 Hz), 7.749–7.760 (t,
J = 1.65 Hz), 7.238–7.434 (d, J = 1.65 Hz), 7.772–7.776 (d,
J = 1.2 Hz), 7.577–7.618 (dd, J = 3.3 Hz), 7.194–7.225 (dd,
J = 3.0 Hz), 7.070–7.082 (dd, J = 0.9 Hz), 3.865 (s, 3H)
IC50 values were calculated from an average of tripli-
cate analysis.
(Figures S22 and S23).
3.11 | Acute toxicity studies
3.8.9 | 2-(2-hydroxyphenyl)-1H-benzo[d]
imidazole (17)
3.11.1 | Animals
ꢀ
Pale yellow (s), Melting point—237 C, Yield—74%, R —
Swiss albino mice weighing 20–25 g of male and female
genders were procured from the registered breeder Bom-
bay Veterinary College (BVC), Mumbai. The animals
were maintained in hygienic condition in the animal
house in group of six in clean protected plastic mice cages
containing husk bedding. The animals were fed standard
pellet food and aqua distil water. Animal house environ-
f
−
1
0
2
1
1
1
.842, 0.88, FTIR (cm ): 3,326.39 (OH), 3,057.30 (NH),
,924.21 (C─H), 1,261.50 (C─N), 1,591.34 (C N),
,454.39 (C C). CNMR (ppm, 100 MHz, DMSO-d ): δ
57.97, 131.68, 131.33, 130.17, 126.66, 126.16, (122.58,
13
6
22.50-d), 119.06, 117.13, 115.01, 112.54, 110.78. MS: (M
+
1): 211.2 (Figures S24 and S25).
ꢀ
mental conditions were maintained between 22 ± 2 C
with 60 ± 10% relative humidity in a 12-hr light and
12-hr dark cycle. In order to acclimatize, prior to the
experimental studies, the animals were housed for
15 days (protocol number: KMKCI/IAEC/08/2017).
3
.9 | Pharmacological evaluation
.9.1 | In vitro LPO assay
3
Commercial-grade chemicals and solvents were pur-
chased. Remi centrifuge (R-4C DX) was used for centrifu-
gation of the solutions. Absorbance was recorded on
Thermo scientific ultraviolet visible spectrophotometer
3.12 | Method
An acute toxicity study was performed on two male and
two female albino mice at an initial dose of 1,000 mg/kg
p.o to study morbidity and mortality, if any, as per
(Thermosci. Evolution 300).
[
42,43]
OECD-423 guidelines.
The aqueous suspension of
3
.10 | Method
test compound in 1% wt/vol sodium carboxymethylcellu-
lose was formulated and administered with a dose vol-
ume not exceeding 1 ml/100 g of mice body weight.
Initially, mice were critically observed at different time
intervals, such as 15, 30 min, 1, 2, 4, 24, 48, and 72 hr,
which was continued for 14 days. In the acute toxicity
A 0.1 ml test sample, 0.2 ml brain tissue homogenate,
and 0.2 ml of 8.1% sodium dodecyl sulfate were mixed
together. Furthermore, 1.5 ml of 20% acetic acid and
1
.5 ml of 8% 2-thiobarbituric acid (TBA) were added to

GURJAR ET AL.
9
study, they were observed for any clinical or behavioral
changes and mortality to help determine the nonlethal
dose on completion of this study.
they climbed back. On the eighth day after administration
of toxicant, the training session was implemented, and after
24 hr, that is, on the ninth day, SDL was recorded to assess
[45]
memory retention of the learned activity.
3.12.1 | Memory model studies
4
| CONCLUSIONS
Gift samples for the standard drugs piracetam and toxi-
cant (scopolamine HBr) were procured from Abbott
Healthcare Pvt. Ltd. and Cipla Pvt. Ltd respectively.
The docking results of benzimidazole analogues depict inter-
actions with BACE1 active site residues. As pointed out ear-
lier, BACE1 enzyme has a pivotal role in AD pathogenesis.
All the analogues in the series exhibited drug-like properties.
Some analogues exhibited better IC50 values in the in vitro
LPO assay. Thus, it can be envisaged that analogues 11 and
14, which interact with the catalytic aspartate dyad and
exhibit better antioxidant activity, help to retain memory in
mice and can be considered potential leads for further opti-
mization and development of more promising analogues
with dual role of BACE1 inhibition and neuroprotection.
3.12.2 | Groups
The Swiss albino mice considered for both the model
studies were divided into six groups, each with six ani-
mals (three male and three females per group):
Group 1: Control group (untreated)
Group 2: Toxicant group (scopolamine 0.4 mg/kg, i.p.)
Group 3: Standard treatment group (piracetam
2
00 mg/kg, p.o.)
Group 4: Analogue 11 at dose level one (50 mg/kg, p.o.)
Group 5: Analogue 11 at dose level two (100 mg/kg, p.o.)
Group 6: Analogue 14 at dose level one (50 mg/kg, p.o.)
ACKNOWLEDGMENTS
We acknowledge the University of Mumbai for sanction
of Minor Research Project Grant (Research Project No.:
509, Sanction letter No.: APD/ICD/2018-19/593 dated
March 16, 2019).
3.12.3 | Elevated plus maze model
ORCID
Archana S. Gurjar https://orcid.org/0000-0002-3352-
Animals in all groups were subjected to five trials at intervals
of 10 min to establish TL on the EPM. Then, all groups,
except toxicant, were administered vehicle/standard/test drug
for eight consecutive days. On the eighth day, an hour after
administration of the last dose, toxicant (scopolamine HBr)
was injected to all groups except the vehicle control. After
3567
REFERENCES
[1] B. M. Gupta, A. Bala, Ann. Neurosci. 2013, 20(2), 71.
[2] R. Anand, K. D. Gill, A. A. Mahdi, Neuropharmacology 2014,
76, 27.
45 min, the animals were subjected to a training session of
[
3] C. P. Ferri, M. Prince, C. Brayne, H. Brodaty, L. Fratiglioni,
M. Ganguli, K. Hall, K. Hasegawa, H. Hendrie, Y. Huang,
A. Jorm, C. Mathers, P. R. Menezes, E. Rimmer, M. Scazufca,
Alzheimer's Disease International, Lancet (London, England)
3
min, in which they were individually placed at the end of
one open arm facing away from the central platform, and
TL in s was calculated from the time the mice moved from
an open arm to any enclosed arms. The central platform
and the time animals took to move from an open arm to
either of the enclosed arms is the TL. On the ninth day,
that is, after 24 hr, retention of this learning task was
2005, 366, 2112.
[
[
[
[
4] A. Kumar, A. Singh, Ekavali, Pharmacol. Rep. 2015, 67(2), 195.
5] R. Macleod, R. T. Cameron, Future Sci. OA 2015, 1(3), 1.
6] B. De Strooper, R. Vassar, T. Golde, Nat. Rev. Neurol. 2010, 6, 99.
7] T. A. Dineen, M. M. Weiss, T. Williamson, P. Acton, S. Babu-
Khan, M. D. Bartberger, J. Brown, K. Chen, Y. Cheng,
M. Citron, M. D. Croghan, R. T. Dunn II., J. Esmay,
R. F. Graceffa, S. S. Harried, D. Hickman, S. A. Hitchcock,
D. B. Horne, H. Huang, R. Imbeah-Ampiah, T. Judd,
M. R. Kaller, C. R. Kreiman, D. S. la, V. Li, P. Lopez, S. Louie,
H. Monenschein, T. T. Nguyen, L. D. Pennington, T. San
Miguel, E. A. Sickmier, H. M. Vargas, R. C. Wahl, P. H. Wen,
D. A. Whittington, S. Wood, Q. Xue, B. H. Yang, V. F. Patel,
W. Zhong, J. Med. Chem. 2012, 55, 9025.
[44]
noted in terms of TL.
3.12.4 | Passive avoidance test
Similar to the EPM study, SDL in seconds was estimated
using Cook's pole apparatus by the passive avoidance test.
In the 3-min training session, animal's latency to step down
from the platform on the grid was observed. On stepping
down on the grid with all the four paws, for 15 s, they were
instantly given electric shocks (1 Hz, 0.5 s, 45 V DC) until
[
8] A. Cavalli, M. L. Bolognesi, A. Minarini, M. Rosini,
V. Tumiatti, M. Recanatini, C. Melchiorre, J. Med. Chem. 2008,
51(3), 347.

10
GURJAR ET AL.
[
9] D. Kumar, A. Ganeshpurkar, D. Kumar, G. Modi, S. K. Gupta,
[31] L. Hong, R. T. Turner, G. Koelsch, D. Shin, A. K. Ghosh,
J. Tang, J. Biochem. 2002, 41, 10963.
S. K. Singh, Eur. J. Med. Chem. 2018, 148, 436.
[
[
[
10] S. H. Omara, C. J. Scotta, A. S. Hamlinc, H. K. Obieda,
[32] M. A. Abdelsalam, O. M. AboulWafa, E.-S. A. M. Badawey,
M. S. El-Shoukrofy, M. M. El-Miligy, N. Gouda,
M. M. Elaasser, Future Med. Chem. 2018, 10, 1159.
[33] A. Boobis, U. Gundert-Remy, P. Kremers, P. Macheras,
O. Pelkonen, Eur. J. Pharm. Sci. 2002, 17(4), 183.
[34] P. S. Kalsi, Spectroscopy of Organic Compounds, 6th ed., New
Delhi, India: New Age International Publications, p. 286, 2004.
[35] Sybyl Version 8.1.1, Tripos International, St. Louis, MO, 2010.
[36] GOLD Version 5.2.2, CCDC Ltd., England: UK, 2014.
[37] A. S. Gurjar, V. Andrisano, A. D. Simone, V. S. Velingkar, Bio-
org. Chem. 2014, 57, 90.
[38] Suite, Small-Molecule Drug Discovery, QikProp, Version 3.3,
User Manual, Schrödinger, LLC, New York, NY 2013, p. 2013.
[39] S. Furniss Brian, J. Hannaford Antony, W. G. Smith peter,
T. A. R. Vogel's Textbook of Practical Organic Chemistry, 5th
ed., London, UK: Longman Group UK Limited, 1989.
[40] N. Kumar, P. Sharma, A. Pareek, A. Prasad, Int. J. Chem.
Pharm. Sci. 2013, 4(1), 12.
Fitoterapia 2018, 128, 118.
11] S. Azimi, A. Zonouzi, O. Firuzi, A. Iraji, M. Saeedi,
M. Mahdavi, N. Edraki, Eur. J. Med. Chem. 2017, 138, 729.
12] R. J. Lin, T. R. Jinn, S. Jang, F. D. Mai, F. Y. Li, J. Chin. Chem.
Soc. 2013, 60(7), 891.
13] L. J. Ming, J. Chin. Chem. Soc. 2010, 57(3A), 285.
14] C. Shen, Y. Chen, H. Liu, K. Zhang, T. Zhang, A. Lin, N. Jing,
J. Biol. Chem. 2008, 283, 17721.
[
[
[
[
[
15] D. Allan Butterfield, A. Castegna, C. M. Lauderback, J. Drake,
Neurobiol. Aging 2002, 23(5), 655.
16] A. Kamil, S. Akhtar, S. Jahan, A. Karim, K. Rafiq, S. Hassan,
Int. J. Sci. Eng. Res. 2013, 4, 1674.
17] M. S. Malamas, J. Erdei, I. Gunawan, K. Barnes,
M. Johnson, Y. Hui, J. Turner, Y. Hu, E. Wagner, K. Fan,
A. Olland, J. Bard, A. J. Robichaud, J. Med. Chem. 2009, 52,
6
314.
[
18] Y. Gravenfors, J. Viklund, J. Blid, T. Ginman, S. Karlström,
J. Kihlström, K. Kolmodin, J. Lindström, S. von Berg, F. von
Kieseritzky, K. Bogar, C. Slivo, B. M. Swahn, L. L. Olsson,
P. Johansson, S. Eketjäll, J. Fälting, F. Jeppsson, K. Strömberg,
J. Janson, F. Rahm, J. Med. Chem. 2012, 55, 9297.
[41] M. N. Alam, N. J. Bristi, M. Rafiquzzaman, Saudi Pharm. J.
2013, 21(2), 143.
[42] A. V. Smirnov, A. F. Kucheryavenko, A. A. Spasov, Bull. Exp.
Biol. Med. 2014, 157(5), 580.
[
19] S. Rajasekaran, G. Rao, A. Chatterjee, Int. J. Drug Dev. Res.
[43] B. Mathew, J. Suresh, S. Anbazhagan, J. Saudi Chem. Soc.
2016, 20, S132.
[44] J. Itoh, T. Nabeshima, T. Kameyama, Psychopharmacology
(Berl) 1990, 101, 27.
2
012, 4(3), 324.
20] R. Trivedi, S. K. De, R. A. Gibbs, J. Mol. Catal. A Chem. 2006,
45(1–2), 8.
[
2
[
21] S. Lin, L. Yang, Tetrahedron Lett. 2005, 46, 4315.
[45] M. R. Zarrindast, M. Bananej, A. Khalilzadeh, S. Fazli-Tabaei,
A. Haeri-Rohani, A. Rezayof, Neurobiol. Learn. Mem. 2006, 86
(3),286.
[
22] M. A. Chari, D. Shobha, S. Malayalama, Int. J. Org. Chem.
2
013, 3(December), 243.
[
23] D. Kathirvelan, P. Yuvaraj, K. Babu, A. S. Nagarajan,
R. Reddy, Indian J. Chem. 2013, 52(August), 1152.
[
[
24] P. Ghosh, R. Subba, Tetrahedron Lett. 2015, 56, 1.
25] K. Bahrami, M. M. Khodaei, I. Kavianinia, Synthesis (Stuttg)
SUPPORTING INFORMATION
Additional supporting information may be found online
in the Supporting Information section at the end of this
article.
2
007, 4, 547.
[
26] Z. Karimi-Jaberi, M. Amiri, E-J. Chem. 2012, 9(1), 167.
27] Y. Venkateswarlu, S. R. Kumar, P. Leelavathi, Org. Med. Chem.
Lett. 2013, 3(1), 7.
[
[
[
[
28] R. Srinivasulu, K. Ravi Kumar, P. Veera Venkata
Satyanarayana, Green Sustain. Chem. 2014, 4, 33.
29] D. Kesselring, K. Maurer, K. D. Moeller, J. Am. Chem. Soc.
How to cite this article: Gurjar AS, Solanki VS,
Meshram AR, Vishwakarma SS. Exploring beta
amyloid cleavage enzyme-1 inhibition and
neuroprotective role of benzimidazole analogues as
anti-alzheimer agents. J Chin Chem Soc. 2019;1–10.
https://doi.org/10.1002/jccs.201900200
2
008, 130, 11290.
30] L. M. Munter, H. Sieg, T. Bethge, F. Liebsch, F. S. Bierkandt,
M. Schleeger, H. J. Bittner, J. Heberle, N. Jakubowski,
P. W. Hildebrand, G. Multhaup, J. Am. Chem. Soc. 2013, 135,
1
9354.