Structure modification and synthesis of

benzimidazole derivatives as potential

antidiabetic drugs

Nik Khairunissa’ Nik Abdullah Zawawi, Norizan Ahmat, Muhammad Taha,

Hamizah Muhamad Nazeri, Isna Athirah Othman
Atta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA (UiTM),
Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor D. E. Malaysia.
 INTRODUCTION

• Type-1 diabetes is caused by the autoimmune destruction of insulin

producing β-cells in the pancreas.
 INTRODUCTION

• β-cell apoptosis involves a set of signaling cascades initiated by interleukin-

1β (IL-1β), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α).
• IL-1β and TNF-α induce nuclear factor kappa-light-chain-enhancer of
activated B cells (NFκB) expression, and downstream activation of gene
expression is thought to occur through nitric oxide (NO) signaling, which both
increases endoplasmic reticulum stress-response pathways and decreases
cell-specific functions.
• NO is a highly reactive molecule that inhibits the electron-transport chain,
leading to decreases in glucose oxidation rates, ATP generation, and insulin
production; cellular nitrite is more stable and serves as a surrogate marker
for NO.
• NFκB activation and IFN-γ-induced STAT-1 signaling work together to effect
β-cell apoptosis, mainly involving the intrinsic apoptotic pathway in both
rodents and humans.
• The downstream effector of this cascade, caspase-3, results in apoptosis and
the loss of the ability to secrete insulin in response to glucose stimulation.
 INTRODUCTION

Figure 1. Assay development and screening for cellular

ATP levels in the presence of inflammatory cytokines.
 OBJECTIVES

• To synthesize a new series of benzimidazole

derivatives
• To investigate their ability to protect the rat
insulinoma cell line INS-1E from cytokine-induced
apoptosis.
 MATERIALS AND METHODS
NH2
O O Na2S2O5, MeOH O OH NH2
+
O H H2O 80%, rt O SO3Na

DMF, reflux
6 hours

N O Hydrazine N O

N HN NH2 MeOH N O
H H

RSCN,
THF, rt
4 3
2' 3'
N O

N HN NH
7 H 6' 5'
S
1
(1-18) HN
R
Scheme 1: Synthesis of benzimidazole thiourea derivatives 1-
18
Formation of benzimidazole benzoyl ester
Proposed mechanism

O
O OH NH2 - SO3Na NH2
+ O
O SO3Na NH2 NH

OH
- H2O

cyclization O
H
N O NH2
Thermal decomposition
O
of NaSO3 to SO2
N O N
H

H H
N O - H2SO2 N O

N O N O
SO2H
Mechanism: Formation of Benzimidazole benzoylhydrazide

N O  N O
NH2NH 2 95%, 195 C
NH 2
N O MeOH, reflux 12.0 h N N
H H H
40 mmol
(5)
( 4)

Proposed mechanism
 Nucleophilic acyl substitution (basic)
O O- O O
R1 O R1 O R1 +
NHNH 2 R1 NHNH 2
+NH
2NH2
H
NH2 NH2
NH2 NH2
H
R 1= N

N
Mechanism: Formation of Benzimidazole thiourea derivatives

N O
N O
S C N
N HN NH2 R
H N HN NH2
H S
R N
Proton transfer

N O Rearrangement
N O
N HN NH
H S N HN NH H
HN H S
R N
R

• Nucleophilic attack at the electrophilic carbon of the

thiocyanate ion by the amine.
 Assay Principal
• Under specified conditions (pH = 6.8; T = 37 °C),
α-glucosidase will catalyse the conversion of the substrate
4-nitrophenyl-α-D-glucopyranoside to α-D-glucopyranoside
and p-nitrophenol, as shown below (Equation 1).
• [PNPG + α-glucosidase --> α-D-glucopyranoside + PNP
(yellow)]
• The yellow colour of the latter product is measured
spectrophotometrically at 405 nm.

% Inhibition = (At30 – At0)control – (At30 – At0)sample X 100

(At30 – At0)control
A = Absorbance measure at λ 405nm
 Baker’s Yeast α-glucosidase
inhibition assay [Shibano et al., 1997]
10 μL of (sample)
25μL of α-glucosidase enzyme (0.0625 U/ml)
95μL of buffer (pH 6.8)

Pre-incubated at 37°C for 10 minutes

[Light sensitive] Add 25μL of 5mM

P-Nitrophenyl-α-glucopyranose (substrate)

Measure absorbance at λ405 nm (0 min)

Measure absorbance of reaction mixture at 37°C for 30

min at λ405 nm
 RESULTS & DISCUSSION
• 18 benzimidazole thiourea derivatives have been
synthesized in good yield and characterized by IR,
1
H NMR, MS and melting point.

• The synthesized compounds were evaluated for their

α-glucosidase inhibitory potential.

• Four compounds (5, 8, 10 & 14) showed significant

inhibitory effects with IC50 less than 100 μM;
61.93±0.22, 71.82±0.28, 50.57±0.81 and 35.83±0.66
μM, respectively.
 No Substituent IC50±SEM No Substituent IC50±SEM No Substituent IC50±SEM
(μM) (μM) (μM)
7 13
1 2'' Br 129.45±0.46 246.83±98 305.25±1.19
3''
F 4''
OCH3
8 14
2 2'' Cl 284.47±1.10 71.82±0.28 35.83±0.66
3''
OCH3 4''
NO2
9 15
3 2'' F 230.68±0.92 117.26±0.44 163.39±0.59
3''
NO2 4''

F F
F
10 16
4 2'' OCH3 345.65±1.35 50.57±0.81 218.84±0.88
3''

4''
Br
11 17
5 61.93±0.22 186.68±0.71 2'' *NA
3''
4'' Cl 4''
Cl Cl
12 18
6 177.99±0.63 276.82±0.64 297.99±1.20
3''
Br 4'' 4''
F
*NA= not active 19 Acarbose 774.5±1.94

Table 1: α-Glucosidase inhibitory activity of benzimidazole thiourea

derivatives 1-18
 10.75 1
H NMR spectrum of compound 14
10.17

8.29
8.27
8.23
8.21
8.13
8.11
7.96
7.94

7.41

2.34
2.34 7.41
N O
10.17
N HN NH
2.34 7.41 H S
5, 6-Me
10.75 HN
8.29
8.27
8.23
8.21
8.13
8.11

7.96
7.94

7.41
N+ O-
O

aromatic H

1
H NMR (500 MHz, DMSO) δ 10.75
2.06
2.07

2.03

1.95

2.00

8.5 8.4 8.3 8.2 8.1 8.0 7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2
(s, 1H), 10.17 (s, 1H), 8.27 (d, J = 9.2
f1 (ppm)
H-4, 7
Hz, 2H), 8.22 (d, J = 9.1 Hz, 2H), 8.11
(t, J = 8.6 Hz, 2H), 7.95 (d, J = 8.5
Hz, 2H), 7.41 (s, 2H), 2.34 (s, 6H).
NH NH
0.93

1.26

2.06
2.07
2.03
1.95

2.00

6.19
11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5
f1 (ppm)
 • Yield 91%.
• m.p. 196.5°C
• IR(KBr) (νmax, cm-1):
3167, 1592, 1508,
1465, 1323, 1248,
1176, 1112, 1001.
IR spectrum of comp. 14
Intens.
• EI-MS: 461.2 (M+1).
+MS, 0.3min #16
x107
461.2
2.5

2.0

1.5

1.0

0.5 281.1 324.9

619.0
158.9 357.3 386.9 430.9
0.0
100 200 300 400 500 600 700 800 900 1000 m/z

Mass spectrum of comp. 14

 CONCLUSION

• 18 benzimidazole thiourea derivatives have been

synthesized and evaluated for their AGI potential.

• Compound 5, 8, 10 & 14 showed significant inhibitory

effects with IC50 less than 100 μM.

• On the basis of in-vitro testing, we demonstrated

that benzimidazole thiourea derivatives are
potential α-glucosidase inhibitors that exert
stronger inhibitory effects than does acarbose
(IC50 =774.5.±1.94 μM)
 ACKNOWLEDGEMENT

• Authors would like to acknowledge the Ministry of

Education Malaysia and Universiti Teknologi MARA
for the financial support under RAGS grant 600-
RMI/RAGS/5/3/ (2/2012).
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