Benzimidazole derivatives protect against cytokine-induced apoptosis in pancreatic β-Cells

Article abstract of DOI:10.1016/j.bmcl.2015.08.022

Apoptotic cell death is the cause of the loss of insulin-producing β-cells in all forms of diabetes mellitus. The identification of small molecules capable of protecting cytokine-induced apoptosis could form the basis of useful therapeutic interventions.

Full text of DOI:10.1016/j.bmcl.2015.08.022

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Benzimidazole derivatives protect against cytokine-induced
apoptosis in pancreatic b-Cells
a,b,
Nik Khairunissa Nik Abdullah Zawawi a,b,y, Sajid Ali Rajput c,y, Muhammad Taha a,b, , Norizan Ahmat
⇑
⇑
,
Nor Hadiani Ismail a,b, Noraishah Abdullah b, Khalid Mohammed Khan d, M. Iqbal Choudhary a,c,d,
⇑
a Atta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor D.E., Malaysia
b Faculty of Applied Science, UiTM Shah Alam, 40450 Shah Alam, Selangor D.E., Malaysia
c Dr. Panjwani Center for Molecular Medicine and Drug Research, International Centre for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan
d H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, 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:
Apoptotic cell death is the cause of the loss of insulin-producing b-cells in all forms of diabetes mellitus.
The identification of small molecules capable of protecting cytokine-induced apoptosis could form the
basis of useful therapeutic interventions. Here in, we present the discovery and synthesis of new benz-
imidazole derivatives, capable of rescuing pancreatic b-cells from cytokine-induced apoptosis. Three
hydrazone derivatives of benzimidazole significantly increased the cellular ATP levels, reduced
caspase-3 activity, reduced nitrite production and increased glucose-stimulated insulin secretion in the
presence of proinflammatory cytokines. These findings suggest that these compounds may protect b-cells
from the harmful effects of cytokines and may serve as candidates for therapeutic intervention for
diabetes.
Received 29 May 2015
Revised 6 August 2015
Accepted 8 August 2015
Available online xxxx
Keywords:
Pancreatic b-cells
Benzimidazole
Cytokines
Apoptosis
Ó 2015 Elsevier Ltd. All rights reserved.
Glucose-stimulated insulin secretion
Diabetes mellitus is a chronic metabolic disorder, characterized
by the presence of persistent hyperglycaemia resulting from
defects in insulin secretion, insulin action or both.1,2 The decline
in b-cell mass due to apoptosis and insulin producing function
underlie much of the pathology of both type 1 and type 2 diabetes
mellitus. Apoptotic death of pancreatic b-cells is a hallmark of all
forms of diabetes. The b-cells are highly sensitive to apoptotic
damages induced by multiple stress factors, such as inflammatory
and oxidative assault.3–5 The inflammatory cytokines, such as
duction, increase ROS production and decrease in insulin biosyn-
thesis and secretion. Caspase-3 also mediates b-cell apoptosis
and cause
a
loss of glucose-stimulated insulin secretion
(GSIS).10,11,5,12
Current treatments for diabetes fail to halt the decline in
functional b-cell mass; therefore, strategies to prevent b-cell dys-
function and apoptosis are urgently needed. Strategies to discover
small molecules, capable of inhibiting b cell apoptosis, may have a
great therapeutic potential, and can be used in combination with
traditional diabetic therapies. Benzimidazole nucleus is an
important pharmacophore with unique chemical and biological
properties.13–17 Benzimidazoles have been found to possess
anti-inflammatory, antispasmodic, antihistaminic, analgesic,
antimicrobial, antiproliferative, antitumor, anti-HIV-RT, antiulcer,
anticancer, anti-tubercular, and cycloxygenase inhibitor activi-
ties.18–22 In our continuous efforts to discover bioactive com-
pounds.23–29 We screened broad range of compounds and found
benzimidazoles as prominent class of compounds on the bases of
that we synthesized a series of benzimidazole derivatives and
investigated their ability to protect the rat insulinoma cell line
INS-1E from cytokine-induced apoptosis.30 However, the mecha-
nism of protection by which the compound protected b-cells
against cytokine-induced cell death is not reported in this
communication.
interleukin-1b (IL-1b), interferon-c (INF-c) and tumour necrosis
factor- (TNF- ) are important mediators in impaired function,
a
a
apoptosis and progressive loss of b-cells. These cytokines activate
intracellular signalling pathways that drive b-cell apoptosis.6–9
IL-1b, TNF-
a
and INF-c induce the expression of transcription
factor NF-
j
B and STAT1. Whereas, the downstream signalling
was reported to occur through nitric oxide (NO), which disrupts
mitochondrial physiology by inhibiting the electron-transport
chain, resulting in a decrease in glucose oxidation rates, ATP pro-
⇑
Corresponding authors. Tel.: +60 182901765 (M.T.); tel.: +60 03 55435590; fax:
+60 03 55444562 (N.A.); tel.: +92 (21) 4824924/4824925; fax: +92 (21) 4819018/
4819019 (M.I.C.).
E-mail addresses: taha_hej@yahoo.com, muhamm9000@puncakalam.uitm.edu.
my (M. Taha), noriz118@salam.uitm.edu.my (N. Ahmat), hej@cyber.net.pk, iqbal.
choudhary@iccs.edu (M.I. Choudhary).
y
Authors equally contributed.
http://dx.doi.org/10.1016/j.bmcl.2015.08.022
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Zawawi, N. K. N. A.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.08.022

2
N.K.N.A. Zawawi et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
cellular ATP levels as a surrogate for cell viability.32 After 48 h incu-
bation with a cytokine cocktail of IL-1b, INF- and TNF- resulted
NH2
NH2
O
O
O
H
O
O
OH
c
a
Na2S2O5, MeOH
H2O 80%, rt
+
in more than 2-fold decreased in ATP levels (Fig. 1A). However,
only seven of the compounds tested protect against cytokine
effects in b-cells (Fig. 1A). In dose-dependency study, compound
SO3Na
DMF, reflux
6 hours
19 completely restored the b-cell ATP levels at 2
lM concentration
(Fig. 2A) with an EC50 value of 0.968 M (Table 2). Similarly, com-
l
O
Hydrazine
MeOH
O
O
N
N
pounds 4 and 5 also increased b-cell ATP levels by more than 90%
of untreated controls (Fig. 2B), whereas compounds 6 and 7 were
slightly less potent in increasing the cellular ATP levels with their
N
H
N
H
HN NH2
RCHO
Acetic acid, n-butanol
reflux 3 hours
EC50 values of 2.57, 3.17, 7.28 and 16.71 lM, respectively (Table 2).
3
Structure–activity relationship (SAR) of active benzimidazole
analogues was determined from the primary screening. It was
observed that increases in the cellular ATP levels in b-cells mainly
depend upon three parameters: the functional groups on substi-
tuted phenyl moiety, and the number and the position of the
functional groups on the phenyl ring. As shown in Table 2, hydro-
xyl (–OH) and methoxy (–OCH3) groups and their respective posi-
tions on the phenyl ring were the preferred substitution required
for the activity. The benzimidazole derivatives with hydroxyl
group on the phenyl ring were found to have an activity pattern,
such that the more hydroxyl group, the greater is the activity. Thus,
the tri-hydroxyl containing compounds were found to be more
potent than di- and mono-hydroxyl group containing analogues.
Compounds 4, 5, 6, and 7, all having dihydroxyl group on the phe-
nyl moiety showed excellent to moderate activity in increasing the
cellular ATP levels, depending upon the position of the hydroxyl
groups (Table 2). These compounds had one hydroxyl group at
meta position, while the other hydroxyl group was moved to car-
bon at 3rd, 4th, and 5th positions on the phenyl moiety. Compound
19, which has the tri-hydroxyl group on phenyl ring, was found to
be the most potent suppressor of b-cell apoptosis among all
4
7
2'
3'
O
N
N
HN
N
6'
H
5'
R
1
(1-20)
Scheme 1. Synthesis of benzimidazole benzohydrazide (1–20).
The synthesis of the target compounds began with the synthesis
of sodium metasulfite adduct according to literature protocol.31
The resulting sulfite adduct was refluxed with 4,5-dimethyl-O-
phenylenediamine in DMF for 6 h to give the arylester substituted
benzimidazole. The benzohydrazide of benzimidazole was formed
by refluxing arylester of benzimidazole with methanolic hydrazine
hydrate (Scheme 1). The synthesis of benzimidazole benzohy-
drazide Schiff bases (1–20) was accomplished by reacting different
aldehydes (Table 1) with benzimidazole benzohydrazide in
n-butanol in the presence of catalytic amount of acetic acid as
shown in Scheme 1.
The ability of these compounds in protecting the rat insulinoma
cell line INS-1E from cytokine-induced apoptosis were tested using
Table 1
Benzimidazole benzohydrazide derivatives 1–20
Compd no.
R
Yield (%)
Compd no.
R
Yield (%)
89
Compd no.
R
Yield (%)
90
OH
2''
3''
1
91
8
15
Cl
4''
OH
2''
3''
2
90
9
90
16
92
HO
OH
4''
3''
4''
OCH3
NO2
2''
OH
2''
3
4
5
92
91
92
10
11
12
91
90
90
17
18
19
92
89
93
3''
H3CO
4''
4''
4''
OCH3
OCH3
OH
2''
3''
5''
HO
F
N
4''
OH
2'' OH
2''
3''
OH
HO5''
OH
H3CO5''
3''
4''
OH
OH
2''
2''
3''
6
7
92
89
13
14
93
93
20
90
4''
Br
4''
OH
OH
3''
3''
OH
4''
OH
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N.K.N.A. Zawawi et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
25000
20000
15000
10000
5000
0
60
50
40
30
20
10
0
A
B
C
1000000
900000
800000
700000
600000
500000
400000
300000
200000
100000
0
§
§
§
§
§
§
§
§
§
‡
§
§
§
*
§
§
+
§
+
§
§
Cytokines
Cmpd
-
+
+
+
+
+
+
+
Cytokines
Cmpd
-
+
+
+
+
+
+
+
Cytokines
Cmpd
-
+
+
+
+
+
+
+
+
8000
7000
6000
5000
4000
3000
2000
1000
0
D
E
1.20
1.00
0.80
0.60
0.40
0.20
0.00
F
1.20
1.00
0.80
0.60
0.40
0.20
0.00
§
§
§
§
*
§
§
§
§
‡
§
‡
‡
§
§
*
+
§
*
§
§
§
+
Cytokines
Cmpd
-
+
+
+
+
+
+
+
Cytokines
Cmpd
-
+
+
+
+
+
+
+
Cytokines
Cmpd
-
+
+
+
+
+
+
+
+
Figure 1. Effects of benzimidazole derivatives on cellular ATP levels, caspase-3 activity, cellular nitrite production, and mitochondrial physiological parameters in b-cells and
after exposure to a cytokine cocktail. (A–F) INS-1E cells were treated for 48 h with a cocktail of proinflammatory cytokines (IL-1b 20 ng/mL, INF- 50 ng/mL, and TNF- 50 ng/
mL) in the presence or absence of benzimidazole derivatives, and were assessed for cellular ATP levels (A), caspase-3 activity (B), and nitrite production (C), mitochondrial
membrane potential ( m) (D), reactive oxygen species (ROS) production (E), and MTT activity (F). Concentrations used were based on molecular weight and as follows:
M for compounds 4, 5, and 19; 10 M for compounds 6 and 7; and 20 M for compounds 10 and 12. Data represent the mean standard deviation of 24 independent
c
a
D
W
5
l
l
l
wells. ⁄ indicates p<0.05, à <0.01 and § <0.001, respectively, as compared to the cytokine treatment alone.
Figure 2. Dose-dependency study for cellular ATP levels and caspase-3 activity. INS-1E cells were treated with cytokine cocktail (see Supporting data) in the presence of
increasing concentrations of compounds 4, 5, 6 and 19 (A–D). Cellular ATP levels (A–B) and caspase-3 activities (C–D) were measured and normalized to untreated controls.
Data are presented as the mean standard deviation of 12 independent wells. ⁄ indicates p<0.05, à <0.01 and § <0.001, respectively, relative to cytokine-treated cells.
benzimidazole derivatives (Table 2). Whereas, compounds 10 and
12, having a single hydroxyl group on phenyl ring, only showed
partial activity (Table 2).
compounds 4 and 5 reduced the expression of cleaved caspase-3
activity (Fig. 2C) with IC50 values of 8.81 and 9.48 M, respectively
(Table 2). Whereas compounds 6 and 19 were only partially effec-
l
In the follow-up study with the initial hit compounds, caspase-
3 activity was measured as a direct readout of apoptosis. Caspase-3
activity was found to have increased by more than 5-fold after 48 h
exposure of INS-1E cells to cytokine cocktail (Fig. 1B). This increase
in caspase-3 activity was largely suppressed by compounds 4, 5, 6,
and 19, which significantly decreased the caspase-3 activity
(Table 2). However, compounds 7 and 10 were found to be not
effective, whereas compound 12 even caused increased caspase-3
activity (Fig. 1B). In dose-dependency study, we demonstrated that
tive in reducing caspase-3 activity (Fig. 1D) with IC50 values of
16.38 and 18.47 lM, respectively (Table 2). These results indicated
that compounds 4, 5, 6 and 19 were able to halt the apoptotic pro-
cess in the presence of inflammatory cytokines.
Pro-inflammatory cytokines were reported to induce the pro-
duction of nitric oxide, which drives b-cell death by both apoptosis
and necrosis.33 Cellular production of nitrite was used as a
surrogate for NO levels.24 In the cellular nitrite production assay,
cellular nitrite levels were found to be increased by more than
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4
N.K.N.A. Zawawi et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Table 2
Effect of benzimidazole derivatives on induced cytokines in INS-1E cells
Compd no.
R
INS-1E cells viability target potency (
Caspase-3 Nitrite
IC50
lM)
ATP
GSIS
Max. act.
93
EC50
(lM)
Max. act.
IC50
(lM)
Max. act.
(l
M)
Max. act.
71
EC50 (lM)
OH
2''
4
5
2.57
84
82
8.81
71
17.28
11.42
13.28
8.32
5''
3''
HO
OH
2''
89
86
3.17
7.28
9.48
79
72
84
64
OH
OH
2''
6
7
75
22
16.38
>30
17.48
17.59
ND
ND
4''
OH
3''OH
73
16.71
68
53
4''
OH
2''
OH
10
12
19
54
61
98
29.28
25.28
15
12
68
>30
18
20
85
>30
>30
9.47
IA
IA
91
ND
4''
OCH3
2''
OH
>30
ND
H3CO5''
3''
5''
0.968
18.47
5.41
HO
OH
4''
OH
Max. act. = maximum activity, IA = inactive, ND = not determine.
Figure 3. Correlation between inhibition of cellular nitrite production and restoration of glucose-stimulated insulin secretion (GSIS). Cellular nitrite production was
measured after treatment with cytokine cocktail (see Supporting data) with increasing concentrations of (A) compounds 5 and 19, (B) compounds 4, 6, and 7. Data represent
the mean standard deviation of 12 independent wells. (C) Glucose-stimulated insulin secretion was measured in the presence of 2 lM glucose and 16 lM glucose in
the absence or presence of cytokines and benzimidazole derivatives. (D) Compounds 4, 5, and 19 were also assessed for their dose-dependent increase in insulin secretory
function of b-cells. Data represent the mean standard deviation of 2 independent experiments. ⁄ indicates p<0.05, à <0.01 and § <0.001, respectively, relative to
cytokine-treated cells.
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N.K.N.A. Zawawi et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
5
6-fold following 48 h cytokine exposure, which was significantly
References and notes
decreased by many of the test compounds in varying levels
(Fig. 1C). Compounds 19 and 5 induced a dose-dependent decrease
in the production of nitrite (Fig. 3A) and were an excellent suppres-
sors of the series with IC50 values of 9.47 and 11.42 lM, respec-
tively (Table 2). Compounds 4, 6 and 7 moderately inhibited the
cellular nitrite production (Fig. 3B) with IC50 values of 17.28,
17.48 and 17.59 lM, respectively (Table 2). In contrast to cas-
pase-3 activity, compound 7 was found to be active in nitrite assay.
These results suggest that only certain position of hydroxyl group
on benzimidazole derivatives are considered important in assess-
ing this activity and as expected, compounds 10 and 12 exhibited
less effect on cellular nitrite production (Fig. 1C).
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INS-1E cells were generously provided by Paolo Meda,
Department of Morphology, University of Geneva Medical School,
Switzerland. Authors would like to acknowledge the Ministry of
Education Malaysia and Universiti Teknologi MARA for the finan-
cial support under RAGS grant 600-RMI/RAGS/5/3/ (2/2012).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.08.
022.
Please cite this article in press as: Zawawi, N. K. N. A.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.08.022