Synthesis and bioactivity evaluation of 2-arylbenzimidazole analogues

Article abstract of DOI:10.14233/ajchem.2014.15551

Benzimidazole is an important drug intermediate. Benzimidazole analogues have shown antibacterial, antiviral, resisting parasites medicinal activity. In this paper, we used phenylenediamine as a starting material through cyclization, acylation reaction to

Full text of DOI:10.14233/ajchem.2014.15551

Asian Journal of Chemistry; Vol. 26, No. 7 (2014), 1891-1894
ASIAN JOURNAL OF CHEMISTRY
http://dx.doi.org/10.14233/ajchem.2014.15551
Synthesis and Bioactivity Evaluation of 2-ArylbenzimidazoleAnalogues
*
XU ZHANG, JIE HUANG, FEI HU, PENGYU and ERBING HUA
Key Laboratory of Industrial Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, 13th
Avenue, Tianjin Economic and Technological DevelopmentArea (TEDA), Tianjin 300457, P.R. China
*Corresponding author: E-mail: huarb@tust.edu.cn
Received: 29 March 2013;
Accepted: 1 September 2013;
Published online: 22 March 2014;
AJC-14923
Benzimidazole is an important drug intermediate. Benzimidazole analogues have shown antibacterial, antiviral, resisting parasites medicinal
activity. In this paper, we used phenylenediamine as a starting material through cyclization, acylation reaction to synthetize 2-arybenzimidazole
analogues. Twenty-two analogues were synthesized and characterized on the basis of 1H NMR spectra. All this compounds were tested
through novel yeast-based screening method to evaluate their bioactivities.
Keywords: Synthesis, 2-Arybenzimidazole, Yeast-based screening.
flash chromatography. 1H spectra were recorded on Bruker
AM-400 NMR spectrometer in deuterated chloroform and
deuterated DMSO.
INTRODUCTION
Benzimidazole derivatives with their unique biological
activity1, such as sterilization, antiinflammatory, antioxidant
activity are widely used as many important pharmaceutical
intermediates2-5. In this paper, we mainly study the bioactivity
of 2-arybenzimidazole analogues as SIRT1 activators. SIRT1,
an NAD+-dependent sirtuin deacetylase, has emerged as
potential therapeutic targets for treatment of human illnesses
such as type II diabetes6,7, cardiovascular8 and neurodege-
nerative diseases9. SRT1720 (Fig. 1), an imidazothiazole
derivative, recently made as the most potent SIRT1 activator
is structurally unrelated to resveratrol10. In yeast, sir2 is the
closet homolog of SIRT111. A mechanistic explanation has
been proposed whereby calorie restriction slows aging by
activating Sir212. Over-expression of Sir2 increases life span
by 30-40 %13,14. According to report that using sir2 activators
can increase the yeast replicative lifespan and reduce chrono-
logical lifespan, the characterization is their gradient of growth
curves increased15-18. On this basis, we report a novel yeast-
based screening method to detect the Sirt1 activators. The
aim of this work is to quickly screen lower biology toxic and
new scaffold structure for novel potent SIRT1 activators.
N
N
N
OH
O
HO
HN
S
N
OH
N
HN
resveratrol
SRT1720
Fig. 1. Structures of the reported SIRT1 activators
Synthsis route of the target compounds (Scheme-I)
First step (a) : To a flask 1,2-diaminobenzene (3.24 g, 30
mmol), 3-aminobenzoic acid (4.93 g, 36 mmol) and PPA (33 g)
were added, then the reaction mixture was allowed to be heated
to 200 °C by oil bath and kept reflux for 5 h.After the reaction
was completely finished, the reaction mixture was slowly
poured into ice water and the resulting mixture was basified
with solid NaOH and NaHCO3.At pH 8-10, the precipitate was
filtered, washed with water and dried to obtain the crude product,
which purified by flash column chromatography (silica gel,
pure CH2Cl2) to afford intermediate.
EXPERIMENTAL
All reagents and solvents used were of reagent grade.
Reaction temperatures were controlled by oil bath temperature
modulator. Thin layer chromatography (TLC) was performed
using E. Merck silica gel 60 GF254 precoated plates (0.25
mm). Silica gel (particle size 200-400 mesh) was used for
Second step (b): In a round-bottomed flask, aromatic
carboxylic acid (1.2 mmol) was dissolved in suitable amount
of CH2Cl2 (25 mL), followed by adding ethylenediamine

1892 Zhang et al.
Asian J. Chem.
R2
O
COOH
NH2
NH
H
N
H
N
a
NH2
NH2
b
N
N
R1
R1
NH2
R1
Scheme-I: Reagents and conditions: (a) PPA, 200 °C, 6 h (yield 85-93 %); (b) RCOOH,CH2Cl2, EDCI, DMAP, Et3N, 60 °C, 6 h (yield 32-86 %)
chloride (EDCl) (1.5 mmol), Et3N (2 mmol), DMAP (0.5 mmol)
and intermediate 1 (1 mmol). Then the reaction mixture was
allowed to heat at 80 °C and kept reflux for 6 h. After cooling
the reaction mixture was poured into a 100 mL separating
funnel, and water was added, then extracted by CH2Cl2, the
organic layer was washed by water, saturated salt water and
dried by sodium sulfate. The organic phase was evaporated
under vacuum to remove the CH2Cl2, providing the crude
product. Then it was purified by flash column chromatography
to afford the target compounds 1-22.
TABLE-1
STRUCTURES AND YIELDS OF TARGET COMPOUNDS
Compound R1 R2
Yield
Compound R1
R2
Yield
*
*
H
H
1
3
Me
Me
62.2%
2
4
48.2%
*
*
59.4%
53.4%
O
*
O
*
Synthesis of the target compounds 1-22 is outlined in
H
H
5
7
Me
Me
Table-1
45.8%
72.6%
6
8
35.9%
42.9%
O
O
O
O
O
O
O
O
1H NMR data of compounds 1-22
*
*
Compound 1: 1H NMR (400 MHz CDCl3): δ (ppm) 8.41
(t, 1H, J = 4.2 Hz), 8.08 (s, 1H), 7.88-7.86 (m, 3H), 7.65-7.63
(m, 3H), 7.59-7.56 (m, 1H), 7.52-7.48 (m, 2H), 7.43 (t, 1H, J =
7.8 Hz), 7.29-7.25 (m, 3H).
Compound 2: 1H NMR (400 MHz CDCl3): δ (ppm) 10.45
(s, 1H), 8.48 (s, 1H), 8.01 (s, 1H), 7.92 (d, 1H, J = 6.4 Hz), 7.88
(d, 2H, J = 8.8 Hz), 7.82 (s, 1H), 7.56 (s, 1H), 7.51 (d, 2H, J =
12.8 Hz), 7.26 (s, 2H), 7.01 (d, 2H, J = 9.2 Hz), 3.90 (s, 3H).
Compound 3: 1H NMR (400 MHz DMSO): δ (ppm) 12.76
(d, 1H, J = 15.2 Hz), 10.43 (s, 1H), 8.68 (s, 1H), 8.03 (d, 2H,
J = 7.2 Hz), 7.86-7.84 (m, 2H), 7.64-7.31 (m, 6H), 7.06-7.01
(m, 1H), 2.44-2.43 (m, 3H).
Compound 4: 1H NMR (400 MHz CDCl3): δ (ppm) 8.39
(s, 1H), 7.99 (s, 1H), 7.87-7.84 (m, 3H), 7.62 (d, 1H, J = 8 Hz),
7.54 (d, 1H, J = 8 Hz), 7.44 (t, 1H, J = 8 Hz), 7.40 (s, 1H), 7.10 (d,
1H, J = 8.4 Hz), 6.99 (d, 2H, J = 8.8 Hz), 3.89 (s, 3H), 2.49 (s, 3H).
Compound 5: 1H NMR (400 MHz CDCl3): δ (ppm) 8.34
(s, 1H), 8.11 (s, 1H), 7.81 (d, 1H, J = 8 Hz), 7.71-7.67 (m, 2H),
7.38 (t, 1H, J = 8 Hz), 7.25-7.24 (m, 1H), 6.96 (d, 2H, J = 2 Hz),
6.60 (t, 1H, J = 2.2 Hz), 3.80 (s, 6H).
Compound 6: 1H NMR (400 MHz CDCl3): δ (ppm) 8.40
(t, 1H, J = 1.8 Hz), 8.02 (s, 1H), 7.87 (d, 1H, J = 8 Hz), 7.66-7.63
(m, 1H), 7.55 (s, 1H), 7.47 (t, 2H, J = 8 Hz), 7.41 (s, 1H), 7.12-
7.09 (m, 1H), 6.99 (d, 2H, J = 2.4 Hz), 6.65 (t, 1H, J = 2.2 Hz),
3.86 (s, 6H), 2.49 (s, 3H).
Compound 7: 1H NMR (400 MHz CDCl3): δ (ppm) 8.77 (s,
1H), 8.27 (s, 1H), 7.71 (t, 2H, J = 6.8 Hz), 7.55-7.53 (m, 2H), 7.28
(s, 1H), 7.25 (t, 1H, J = 7.8 Hz), 7.22-7.20 (m, 2H), 3.87 (s, 3H),
3.75 (s, 6H).
Compound 8: 1H NMR (400 MHz CDCl3): δ (ppm) 8.34
(t, 1H, J = 1.8 Hz), 8.07 (s, 1H), 8.82-7.77 (m, 2H), 7.55 (s, 1H),
7.48 (t, 2H, J = 8 Hz), 7.40 (s, 1H), 7.11 (s, 3H), 3.94 (s, 6H),
3.93 (s, 3H), 2.49 (s, 3H).
Compound 9: 1H NMR (400 MHz CDCl3): δ (ppm) 9.88
(s, 1H), 8.46 (s, 1H), 7.90 (d, 1H, J = 7.6 Hz), 7.81 (s, 1H), 7.65
(d, 2H, J = 2.4 Hz), 7.51-7.49 (m, 3H), 7.39 (s, 1H), 7.30-7.27
(m, 4H), 2.53 (s, 3H).
O
O
*
*
H
H
9
47.9%
86.1%
10
12
Me
Me
63.9%
43.1%
*
*
11
Br
*
Br
*
13
H
35.1%
Me
14
56.1%
NO2
NO2
*
*
*
*
O2N
O2N
Me
H
H
15
17
55.3%
40.6%
16
18
60.7%
39.6%
Me
O
O
O
O
O
*
O
*
O
O
Me
Me
H
H
20
22
19
21
53.3%
48.3%
40.3%
32.3%
H3C
*
H3C
*
Compound 10: 1H NMR (400 MHz CDCl3): δ (ppm) 8.75-
8.74 (m, 2H), 8.47-8.45 (m, 2H), 8.41-8.39 (m, 2H), 7.64-7.60
(m, 2H), 7.47 (t, 1H, J = 4.2 Hz), 7.45 (s, 1H), 7.43 (s, 1H), 7.41
(d, 1H, J = 0,4 Hz), 7.39 (s, 1H), 2.68 (s, 6H).
Compound 11: 1H NMR (400 MHz CDCl3): δ (ppm) 8.41
(t, 1H, J = 2 Hz), 7.92 (s, 1H), 7.86 (d, 1H, J = 7.6 Hz), 7.78-7.75
(m, 3H), 7.68-7.66 (m, 2H), 7.52 (t, 1H, J = 8 Hz), 7.32-7.26 (m,
3H).
Compound 12: 1H NMR (400 MHz CDCl3): δ (ppm) 8.35
(s, 1H), 8.12 (s, 1H), 7.86-7.81 (m, 3H), 7.61-7.60 (m, 1H),
7.59-7.51 (m, 2H), 7.48 (t, 2H, J = 7.4 Hz), 7.38 (t, 2H, J = 7.8
Hz), 7.09-7.07 (m, 1H), 2.46 (s, 3H).

Vol. 26, No. 7 (2014)
Synthesis and Bioactivity Evaluation of 2-ArylbenzimidazoleAnalogues 1893
Compound 13: 1H NMR (400 MHz DMSO): δ (ppm) 12.95
(s, 1H), 10.79 (s, 1H), 8.88 (t, 1H, J = 1.8 Hz), 8.71 (t, 1H, J =
1.6 Hz), 8.49-8.46 (m, 2H), 7.94-7.86 (m, 3H), 7.67 (d, 1H, J =
7.2 Hz), 7.60-7.54 (m, 2H), 7.24-7.20 (m, 2H).
Compound 14: 1H NMR (400 MHz DMSO): δ (ppm) 12.79
(d, 1H, J = 16 Hz), 10.78 (s, 1H), 8.88 (d, 1H, J = 1.2 Hz), 8.68
(s, 1H), 8.48-8.46 (m, 2H), 7.94-7.86 (m, 3H), 7.58-7.32 (m,
3H), 7.07-7.01 (m, 1H), 2.44 (d, 3H, J = 7.6 Hz).
by subtracting the initial OD value at t = 0 from each
subsequent OD reading. The concentration of DMSO in the
growth media was kept below 0.1 %, which had no detectable
effect on yeast growth. Growth curves were displayed with
Excel/Growth Curves software.
RESULTS AND DISCUSSION
Compound 15: 1H NMR (400 MHz DMSO): δ (ppm) 12.94
(s, 1H), 10.85 (s, 1H), 8.64 (s, 1H), 8.19-8.17 (m, 1H), 7.91-
7.88 (m, 2H), 7.84-7.76 (m, 2H), 7.68 (t, 2H, J = 8.8 Hz), 7.54
(t, 2H, J = 8 Hz), 7.25-7.17 (m, 2H).
Compound 16: 1H NMR (400 MHz DMSO): δ (ppm) 11.06
(s, 1H), 8.69 (s, 1H), 8.20 (d, 1H, J = 8 Hz), 7.96-7.91 (m, 2H),
7.83(t, 2H, J = 6.4 Hz), 7.79-7.71 (m, 2H), 7.68 (d, 2H, J = 10.8
Hz), 7.58 (s, 1H), 7.33 (d, 1H, J = 7.6 Hz), 3.17 (s, 3H).
Compound 17: 1H NMR (400 MHz CDCl3): δ (ppm) 8.42
(s, 1H), 7.99 (s, 1H), 7.86 (d, 1H, J = 8 Hz), 7.73 (d, 1H, J = 8
Hz), 7.52-7.48 (m, 2H), 7.44-7.41 (m, 1H), 7.30-7.26 (m, 5H),
6.94 (d, 1H, J = 8 Hz), 3.97 (s, 6H).
Compound 18: 1H NMR (400 MHz DMSO): δ (ppm) 10.76
(s, 1H), 8.02 (s, 1H), 7.99 (s, 1H), 7.73 (t, 3H, J = 9.8 Hz), 7.61
(s, 1H), 7.55 (t, 1H, J = 8 Hz), 7.36 (t, 4H, J = 8.6 Hz), 2.51 (s,
6H), 2.33 (s, 3H).
Compound 19: 1H NMR (400 MHz CDCl3): δ (ppm) 10.56
(s, 1H), 8.44 (s, 1H), 8.02 (s, 1H), 7.87 (d, 1H, J = 8 Hz), 7.58 (d,
3H, J = 8 Hz), 7.36 (t, 1H, J = 8 Hz), 7.28-7.22 (m, 3H), 6.51 (d,
2H, J = 8.4 Hz), 3.75 (s, 6H).
Compound 20: 1H NMR (400 MHz CDCl3): δ (ppm) 8.55
(s, 1H), 8.31 (s, 1H), 7.77-7.67 (m, 2H), 7.44 (s, 1H), 7.26-7.16
(m, 3H), 7.00 (d, 1H, J = 8 Hz), 6.38 (s, 2H), 3.62-3.49 (m, 6H),
2.42 (s, 3H).
Compound 21: 1H NMR (400 MHz CDCl3): δ (ppm) 8.33
(s, 1H), 7.88 (s, 1H), 7.64 (s, 2H), 7.44 (d, 3H, J = 4.4 Hz), 7.29-
7.26 (m, 3H), 2.22 (s, 3H).
Compound 22: 1H NMR (400 MHz CDCl3): δ (ppm) 8.27
(s, 1H), 7.83 (d, 1H, J = 6.8 Hz), 7.53 (d, 2H, J = 9.6 Hz), 7.45-
7.411 (m, 4H), 7.09 (d, 1H, J = 8 Hz), 2.48 (s, 3H), 2.20 (s, 3H).
Yeast strains and culture condition: Yeast strains used
throughout this study were Yeast Parental strain-BY4743
(Sir2) ordered from Thermoscientific, USA. All yeast cells
were grown at 30 °C. The yeast strains were grown on YPD
(yeast peptone dextrose) medium (1 % yeast extract, 2 %
peptone, 2 % glucose). All components were of analytical
quality. All compounds tested were dissolved in DMSO. Drug
screening assay was determined in yeast peptone dextrose
medium.
In step (a) we used PPA to catalyze carboxylic acid
compounds and o-phenylendiamine reaction. We optimized
and confirmed 200 °C, 6h and n(benzoic acid): n(diamino-
benzene) = 1.2:1 are the best reaction conditions, the yield
increased about more 15-20 % than other methods19-21. This
method has many advantages, such as lower cost, few side
effect and easier purification.
In step (b), a majority of target compounds can be obtained
through EDCI, DMAP catalyzing acylation reaction. However,
compound 15, 16, 19, 20 had larger steric hindrance and did
not react. We changed the method and chose TsCl and Py
catalyze reaction for making 15, 16, 19, 20, the yield was 40-
60 %.
Yeast-based drug screening assay: Yeast as model of
drug screening for SIRT1 activators is based on the homologue
between yeast Sir2 and human SIRT111. Here we used the Bio-
screen CMBR machine for measuring yeast growth curves by
monitoring outgrowth of yeast cells22. The results showed
that this method provides growth curves with decreased
variability comparable to traditional one. If compounds can
activate Sir2 leading to increase replicative life span and reduce
chronological lifespan, which could have higher gradient of
growth curves than blank yeast15-18
.
Primary SAR analysis outlined based on Fig. 2 as follows:
Based on growth curves, many compounds have distinct effect
on the growth curve of yeast. Compared with resveratrol and
blank, the curve show that resveratrol could promote the
proliferation of yeast, but has no effect on the final biomass
and implied no cytotoxicity on yeast. Most of the compounds
had inhibition on the growth of yeast, showing that there
were some cytotoxicities. Among them,compounds 6, 11, 14
had obvious inhibitory effects on yeast growth and may have
stronger cytotoxicities. Fortunately, the data showed that
compounds 2 have higher activity than resveratrol and non-
cytotoxicity.
The result of the gradient of growth curves22 is outlined
on Fig. 3. Gradient = (OD12 h-OD4 h) / (t12-t4), OD12 h and
OD4 h indicate the absorption of yeast growth after 12 h and
4 h, respectively..
Base on the gradient of growth curves, para-methoxyl
group at benzene has the best activity, and the more the
number of methoxyl group the less the activity is. In addition,
wherever two methoxyl groups are on beneze ring, the activity
didn't change obviously. Ortho-nitro group at benzene ring
also had better effect than meta-position. According to effects
of 1-22 on Yeast strain date, methyl group at benzene may be
an active group, because compounds 2 and 4 have the most
active property for activating Sir2 yeast, but compound 2 has
a little cytotoxicity on yeast. In all, we obtained one lead
compound 4 based on above analysis.
Growth curve of the yeast: A Bioscreen C MBR machine
(Growth Curves USA, Piscataway, NJ) was used for all out-
growth assays. For outgrowth of aged cells, 5 µL of the aging
culture was inoculated into 145 µL of rich yeast peptone
dextrose medium in a Bioscreen Honeycomb 100-well plate
(cat no. 9502550). Compounds were added to corresponding
number of tubes to a final concentration of 100 µM/L. Incu-
bation of the plate was kept constant at 30 °C, with the shaking
module set to high continuous shaking. Absorbance readings
at 600 nm (wideband range) were taken every 0.5 h for 24 h.
OD data were normalized for background prior to presentation

1894 Zhang et al.
Asian J. Chem.
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0
Res. Blank
1
2
3
4
5
6
7
8
9
10 11
12
13 14
15
16 17 18 19 20
21 22
Fig. 3. Gradient of growth curves of yeast with compounds (100 µM L–1)
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
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ACKNOWLEDGEMENTS
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Funding for this study was provided by National Natural
Science Foundation of China (No: 81072521) and Tianjin
University of Science and Technology (No: 20100411).