Benzimidazoles as new scaffold of sirtuin inhibitors: Green synthesis, in vitro studies, molecular docking analysis and evaluation of their anti-cancer properties

Article abstract of DOI:10.1016/j.ejmech.2014.06.060

Two series of novel benzimidazole derivatives were designed, synthesized and evaluated for their SIRT1 and SIRT2 inhibitory activity. Among the newly synthesized compounds, compound 4j displayed the best inhibitory activity for SIRT1 (IC₅₀ = 54

Full text of DOI:10.1016/j.ejmech.2014.06.060

Accepted Manuscript
Benzimidazoles as new scaffold of sirtuin inhibitors: green synthesis, in vitro studies,
molecular docking analysis and evaluation of their anti-cancer properties
Yeong Keng Yoon, Mohamed Ashraf Ali, Ang Chee Wei, Amir Nasrolahi Shirazi,
Keykavous Parang, Tan Soo Choon
PII:
S0223-5234(14)00589-3
10.1016/j.ejmech.2014.06.060
EJMECH 7103
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 March 2014
Revised Date: 23 June 2014
Accepted Date: 26 June 2014
Please cite this article as: Y.K. Yoon, M.A. Ali, A.C. Wei, A.N. Shirazi, K. Parang, T.S. Choon,
Benzimidazoles as new scaffold of sirtuin inhibitors: green synthesis, in vitro studies, molecular docking
analysis and evaluation of their anti-cancer properties, European Journal of Medicinal Chemistry (2014),
doi: 10.1016/j.ejmech.2014.06.060.
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ACCEPTED MANUSCRIPT
GRAPHICAL ABSTRACT
The most active compound (4j) showed potent SIRT1 and SIRT2 inhibition with IC50 of
54.21 µM and 26.85 µM respectively. It possessed good anti-proliferative activity against
three cancer cell lines tested.

ACCEPTED MANUSCRIPT
Benzimidazoles as new scaffold of sirtuin inhibitors: green
synthesis, in vitro studies, molecular docking analysis and
evaluation of their anti-cancer properties
Yeong Keng Yoona*, Mohamed Ashraf Alia,b,c, Ang Chee Weia, Amir Nasrolahi Shirazid,
Keykavous Parangd, Tan Soo Choona
aInstitute for Research in Molecular Medicine, Universiti Sains Malaysia, Minden 11800,
Penang, Malaysia.
bNew Drug Discovery Research, Department of Medicinal Chemistry, Alwar Pharmacy
College, Alwar, Rajasthan-301030, India.
cNew Drug Discovery Research, Department of Medicinal Chemistry, Sunrise University,
Alwar, Rajasthan-301030, India.
dDepartment of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University
of Rhode Island, Kingston, RI, 02881, United States.
ABSTRACT
Two series of novel benzimidazole derivatives were designed, synthesized and evaluated for
their SIRT1 and SIRT2 inhibitory activity. Among the newly synthesized compounds,
compound 4j displayed the best inhibitory activity for SIRT1 (IC50 = 54.21 µM) as well as
for SIRT2 (IC50 = 26.85 µM). Cell proliferation assay showed that compound 4j possessed
good antitumor activity against three different types of cancer cells derived from colon
(HCT-116), breast (MDA-MB-468) and blood-leukemia (CCRF-CEM) with cell viability of
40.0%, 53.2% and 27.2% respectively at 50 µM. Docking analysis of representative
compound 4j into SIRT2 indicated that the interaction with receptor was primarily due to
hydrogen bonding and π-π stacking interactions.
Keywords: Sirtuin, Benzimidazole, Anti-proliferative, Green chemistry synthesis
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* Corresponding author:
Yeong Keng Yoon
Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Minden, 11800,
Penang, Malaysia.
Phone: +604 6593140
Fax: +604 6569796
Email: kyyeong@gmail.com
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1.Introduction
Sirtuins are a family of NAD+ -dependent deacetylases and/or ADP-ribosyl transferases that
modify a broad range of protein substrates [1-3]. The mammalian sirtuin family consists of
seven family members (SIRT1-7), characterized by a conserved 275 amino acid catalytic core
and unique additional N-terminal and/or C-terminal sequences of variable length [4]. Of the
seven human sirtuin isoforms that have been discovered, SIRT1 [5] and SIRT2 [6] are the
most studied. Between them, SIRT1 & SIRT2 have been shown to deacetylate more than 35
different substrates such as H4K16, p53, forkhead box class O (FOXO), nuclear factor-ĸb
(NK-ĸB), and many others [7-9]. Since SIRT1 & SIRT2 are involved in so many downstream
enzyme activities, it is not surprising that both SIRT1 and SIRT2 have been implicated with
numerous disorders such as cancer [10,11] and neurological diseases [12,13]. Therefore,
potent SIRT1 and SIRT2 modulators could be used as valuable tools to gain insight into the
specific cellular functions of their effector proteins.
In view of the importance of these enzymes, sirtuin biology has been advancing at a
tremendous pace in recent times. Many new discoveries were made at the genetic level for
studying these important enzymes [14-16]. Several classes of small molecule sirtuin
inhibitors have been identified so far including sirtinol [17], splitomycin analogs [18] and
tenovins [19]. Recently, a large high-throughput screening effort led to the discovery of a
potent indole-based SIRT1 inhibitor (EX-527) [20]. Since many benzimidazole-containing
compounds exhibit important biological properties [21,22], and the fact that indole and
benzimidazole share some structure similarities the potential of benzimidazole and its
derivatives exhibiting sirtuin inhibitory activities is an interesting proposition.
As benzimidazole is a very important class of heterocyclic compounds in the area of drug
design [23], the synthesis of benzimidazoles has received considerable attention in the past
few decades [24]. However, the diversified synthesis of regiodefined 1,2-disubstituted
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benzimidazoles remains complicated. The commonly used approach of condensation between
4-substituted-phenylenediamine and aldehyde often leads to mixture of 1,2-disubstituted, 2-
substituted benzimidazoles and bis dihydrobenzimidazoles [25].
Herein, we would like to report a new green method where the synthetic regiodefined ethyl-
(1,2-disubstituted)-5-carboxylate reaction of benzimidazole is totally carried out at ambient
temperature utilizing water/ethanol as solvent. Sirtuin inhibitory activities of the newly
synthesized benzimidazoles were reported. Docking studies for 4b and 4j (the most potent
compound for 3-aminopropyl-2-pyrrolidinone and phenyl substituted series respectively)
with SIRT2 were also performed. In addition, anti proliferation activity of the benzimidazole
derivatives against three different tumor cell lines belonging to colon (HCT-116), breast
(MDA-MB-468) and leukemia (CCRF-CEM) were also reported.
2. Results and Discussion
2.1 Chemistry
The synthetic scheme is a four-step pathway leading to the formation of a variety of
benzimidazole derivatives (Scheme 1). This methodology was applied to synthesize both
series of ethyl-(1,2-disubstituted)-5-carboxylate benzimidazole derivatives.
The electronic effects of the different substituted aldehydes have also been investigated. It
has been found that both electron withdrawing groups (-nitro, -trifluoromethyl) as well as
electron donating groups (-methyl, -hydroxy, -dimethylamino) do not affect the efficiency of
the transformation. Halogen substituents are also well tolerated. This showed the versatility
of the protocol over a broad range of substituents from weak to strong electron
withdrawing/donating groups. Excellent yields for the synthesized ethyl-(1,2-disubstituted)-
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5-carboxylate benzimidazoles were obtained and products were easily isolated as they tend to
precipitate out in water and require minimum purification.
2.2 Enzymatic Assays
The in vitro enzymatic screening assay for SIRT1 and SIRT2 inhibitory activity were
performed using Sensolyte® fluorimetric drug discovery kits (AnaSpec, Fremont, CA)
according to the manufacturer’s protocol. Cambinol and Tenovin-6, two of the few sirtuin
inhibitors which showed antitumor activities in mouse xenograft models, were used as
standard control for both the SIRT1 and SIRT2 assays while DMSO was used as a vehicle
control. IC50 values were determined for all compounds which showed over 50% inhibition
for either SIRT1 or SIRT2 at 50 µM. Preliminary structure-activity relationship (SAR) was
established when in vitro screening on the compounds showed that compounds with strong
electron donating group (dimethylamino) at R2 possessed the best sirtuin inhibitory activities.
The importance of strong electron donating group is highlighted as it is instrumental in
providing good sirtuin inhibition. Removal of the dimethylamino group led to a drastic drop
in sirtuin inhibitory activities. Generally, the novel benzimidazole derivatives showed better
inhibition on SIRT2 as compared to SIRT1 (Table 1). Experiments were performed in
triplicates. Standard deviation obtained for all experiments are less than 20%.
The most potent inhibitor for SIRT1 as well as SIRT2 was found to be 4j (SIRT1 IC50
=
54.21 µM; SIRT2 IC50 = 26.85 µM). Compound 4j showed better SIRT2 inhibitory activity
compared to the standard controls used (cambinol and Tenovin-6). It is also equipotent
compared with cambinol and Tenovin-6 in terms of SIRT1 inhibitory activities.
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2.3 Molecular Docking Analysis
In an attempt to predict the binding mode of this novel chemical series, docking study of
representative compound 4b (the most potent for 3-aminopropyl-2-pyrrolidinone substituted
series) and 4j (the most potent for phenyl substituted series) into the active site of human
SIRT2 (PDB entry code: 3ZGV, x-ray resolution = 2.30 Å) was performed [26]. The receptor
and the drug candidate were optimized before actual docking in Autodock 4.2 using standard
procedure of the software.
Analysis of the top-ranked pose of compound 4j docked within the SIRT2-ADPr cofactor
binding site demonstrated several plausible molecular interactions between 4j and the
receptor. The docking analysis reveals that the compound 4j interact with receptor primarily
due to hydrogen bonding as well as π-π stacking interactions. The N-H group of SER263 is
hydrogen bonded strongly to the oxygen from the ester chain of compound 4j .Other
hydrogen bonds which could be observed include interactions with Thr262, Leu264, Gln265,
Arg97, and Gln167 (Figure 1). This is relatively consistent with the hydrogen bonds
observed between SIRT2 and ADPr complex [26]. Other SIRT2-inhibitor predictions
including salermide and NF-675 also reported similar bonding results [27].
The benzene ring on the position-2 of the benzimidazole core was stabilized through π-π
stacking interactions between the imidazole group of His187 and benzene ring of Phe235.
The importance of the diaminobenzene substituent was highlighted through its interactions
with Val233 and Phe119. Lone pair oxygen-π interactions could also be observed through
Val233 and the phenyl ring of dimethylaminobenzene substituent as well as the N from the
dimethylamino group with benzene ring from Phe119.
As for compound 4b, the mode of interaction with receptor was deemed to be different as
compared to compound 4j. The ketone oxygen from the pyrrolidinone moeity of compound
4b was hydrogen bonded strongly to Lys287. Another strong hydrogen bond observed was
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between the oxygen from the ethyl ester group of compound 4b with Ala85. Other hydrogen
bond interactions which could be observe included interaction of 4b with Asn286, Ser98,
Arg97, Ser263 and Gln167. However, as shown in Figure 2, the benzene ring at position-2
was situated out from the receptor cavity. Therefore, substituents on the benzene ring would
deem not have a significant effect on the sirtuin inhibitory activity on SIRT2 enzyme as
shown by compounds 4a-h in Table 1.
2.4 Cellular Assays
The antitumor activity of various known sirtuin inhibitors has been previously demonstrated
in the literature [28,29]. We measure the entire library of newly synthesized compounds’
cytotoxicity in a panel of human cancer cell lines to probe the relationship between
SIRT1/SIRT2 inhibition and cancer cell anti-proliferative activity. The three representative
compounds which possessed good sirtuin inhibitory activities (4b, 4j and 4l) were then
screened for their anti-proliferative effect against three different cancer cell lines derived
from colon (HCT-116), breast (MDA-MB-468) and blood-leukemia (CCRF-CEM) at 50 µM
concentration. Interestingly as shown in Figure 3, the most potent compound (4j) gave the
best cytotoxic activity against all three cancer cells while less pronounced effect was
observed for the other two compounds with inferior SIRT2 inhibition. Compound 4j
possessed anti-proliferative activity against all three different types of cancer cells tested (cell
viability 40.0% for HCT-116, 53.2% for MDA-MB-468, 27.2% for CCRF-CEM, after 72 hrs
treatment), which showed its broad spectrum ability in inhibiting cancer cell growth.
Standard deviation obtained for all experiments are less than 20%.
From the results obtained, although it seems SIRT2 could potentially play a more important
role in inhibiting cancer cells, we cannot rule out the possibility that combinational inhibitory
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effects of SIRT1 and SIRT2 activity may contribute to the observed cytotoxicity as has been
reported previously by Peck et al [30].
As literature highlights that benzimidazole and its derivatives might also be toxic to normal
cells [31,32], the cytotoxocity of the newly synthesized compounds on human fibroblast cells
GM00637 were also evaluated. The tolerable toxicity of the compounds 4a-p was confirmed
by the cytotoxicity test at concentrations up to 50 µM. After 72 hours of exposure, viability
was assessed on the basis of cellular conversion of MTS into a formazan product using the
Promega Cell Titer 96 Non-radioactive Cell proliferation method according to manufacturer’s
protocol. All the compounds were found to be non-toxic up to 50 ꢀM.
3.Conclusion
In conclusion, we have discovered three novel benzimidazole derivatives which showed good
SIRT1/SIRT2 inhibition activity with micromolar IC50 values. Moreover, they possessed
good antitumor activity against three cancer cell lines evaluated in this study. The most
potent compound discovered in this study, 4j, showed better SIRT2 inhibitory activity
compared to the standard controls used. More importantly, the correlation between in vitro
SIRT2 (and to a lesser extent SIRT1) inhibition and cancer cell cytotoxicity using small
molecule sirtuin inhibitors was established. Further studies to explore the mechanism of
action of these potent small molecule sirtuin inhibitors on cancer cells are currently underway
in our laboratory. Compounds with potent SIRT2 inhibition and which demonstrates
cyototoxic effect such as 4j is prime candidates for modifications to further improve their
activities.
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4. Experimental
4.1. Chemistry
All general chemicals were supplied by Sigma-Aldrich (U.S.A) and Merck Chemicals
(Germany). Standard control cambinol and Tenovin-6 were obtained from Cayman
Chemicals (U.S.A). Purity of the compounds was checked on thin layer chromatography
(TLC) plates (silica gel G) in the solvent system chloroform-methanol (9:1). The spots were
located under short (254nm)/long (365nm) UV light. Elemental analyses were performed on
Perkin Elmer 2400 Series II CHN Elemental Analyzer and were within ± 0.4% of the
1
13
calculated values. H and C NMR were performed on Bruker Avance 500 spectrometer in
CDCl3 using TMS as internal standard. Mass spectra were recorded on Varian 320-MS TQ
LC/MS using ESI mode.
4.1.1 General procedure for synthesis of ethyl-(1,2-disubstituted)-5-carboxylate: The
starting material, 4-fluoro-3-nitro benzoic acid was esterified in the presence of catalytic
sulfuric acid in ethanol by refluxing for 6 hours to afford 1 in 70% yield. Ethyl-4-fluoro-3-
nitrobenzoate, 1 (0.5 g, 2.34 mmol) and amine (2.58 mmol) [a-h: 1-(3-aminopropyl)-2-
pyrrolidinone; i-p: aniline] were mixed in ethanol (5 mL) and stirred at room temperature for
1 hour. The solvent was evaporated under reduced pressure and resuspended in ethyl acetate.
The organic layer was then washed with water (10 mL x 3), dried over Na2SO4 and
evaporated to dryness to yield 2 (90%). The nitroethyl ester, 2 (1 mmol), ammonium formate
(3 mmol) and 10% Pd/C (100 mg) were mixed in ethanol (10 mL). The reaction mixture was
stirred at room temperature until completion (solution turned colourless). The reaction
mixture was then filtered through Celite 545. The filtrate was evaporated under reduced
pressure. It was resuspended in ethyl acetate and washed with water, dried over Na2SO4 and
evaporated to dryness to yield 3 (71%). The aminoethyl ester, 3 (1 mmol) and various
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benzaldehydes (1 mmol) and sodium metabisulfite (1 mmol) were mixed in water (5 mL).
The reaction mixture was stirred at room temperature for 2 hours. Precipitate formed were
then filtered, washed with cold water and dried under reduced pressure to yield final
compounds 4a-p (76-90%).
4.1.2
Ethyl
1-(3-(2-oxopyrrolidin-1-yl)propyl)-2-phenyl-1H-benzo[d]imidazole-5-
1
carboxylate (4a) Yield: 89%; H NMR (500 MHz, CDCl3): δ 1.45 (3H, t, J = 6.9 Hz), 1.90-
2.10 (4H, m), 2.35 (2H, t, J = 6.0 Hz), 3.34 (2H, t, J = 5.0 Hz), 3.50 (2H, t, J = 6.0 Hz), 4.31
(2H, t, J = 6.5 Hz), 4.48 (2H, q, J = 6.9 Hz), 6.90-7.10 (5H, m), 7.48 (1H, d, J = 9.0 Hz), 8.10
(1H, dd, J = 1.5 Hz, J = 9.0 Hz), 8.55 (1H, s). 13C NMR (125 MHz, CDCl3): δ 14.78, 18.24,
28.07, 31.15, 40.22, 42.93, 47.28, 61.28, 109.80, 122.78, 124.86, 125.53, 129.29, 129.63,
130.43, 130.59, 138.97, 143.13, 155.51, 167.44, 175.71. ESI-MS: m/z 392.3. [M+H]+. Anal.
Calc for C23H25N3O3: C, 70.56%; H, 6.46%; N, 10.71%. Found: C, 70.53%; H, 6.42%; N,
10.80%.
4.1.3
Ethyl
2-(4-(dimethylamino)phenyl)-1-(3-(2-oxopyrrolidin-1-yl)propyl)-1H-
benzo[d]imidazole-5-carboxylate (4b) Yield: 85%; 1H NMR (500 MHz, CDCl3): δ 1.43 (3H,
t, J = 6.9 Hz), 1.90-2.10 (4H, m), 2.35 (2H, t, J = 6.0 Hz), 3.05 (6H, s), 3.22 (2H, t, J = 5.0
Hz), 3.32 (2H, t, J = 6.0 Hz), 4.30 (2H, t, J = 6.5 Hz), 4.42 (2H, q, J = 6.9 Hz), 6.80 (2H, d, J
= 9.0 Hz), 7.37 (1H, d, J = 9.0 Hz), 7.61 (2H, d, J = 9.0 Hz), 8.01 (1H, dd, J = 1.5 Hz, J = 9.0
13
Hz), 8.51 (1H, s). C NMR (125 MHz, CDCl3): δ 14.39, 17.85, 27.56, 30.82, 39.92, 40.17,
42.64, 46.96, 60.81, 109.14, 111.83, 116.40, 121.57, 123.98, 124.87, 130.22, 138.70, 142.43,
151.46, 155.92, 167.15, 175.29. ESI-MS: m/z 435.3. [M+H]+. Anal. Calc for C25H30N4O3: C,
69.12%; H, 7.01%; N, 12.90%. Found: C, 69.12%; H, 7.03%; N, 12.87%.
4.1.4 Ethyl 2-(4-hydroxyphenyl)-1-(3-(2-oxopyrrolidin-1-yl)propyl)-1H-benzo[d]imidazole-
5-carboxylate (4c) Yield: 80%; 1H NMR (500 MHz, CDCl3): δ 1.43 (3H, t, J = 6.9 Hz), 1.90-
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2.10 (4H, m), 2.46 (2H, t, J = 6.0 Hz), 3.30 (2H, t, J = 5.0 Hz), 3.45 (2H, t, J = 6.0 Hz), 4.30
(2H, t, J = 6.5 Hz), 4.47 (2H, q, J = 6.9 Hz), 6.83 (2H, d, J = 9.0 Hz), 7.47 (1H, d, J = 9.0
13
Hz), 7.60 (2H, d, J = 9.0 Hz), 8.01 (1H, dd, J = 1.5 Hz, J = 9.0 Hz), 8.52 (1H, s). C NMR
(125 MHz, CDCl3): δ 14.62, 18.29, 28.75, 32.01, 41.23, 43.43, 48.28, 62.02, 107.83, 109.87,
110.10, 111.04, 123.05, 126.59, 153.40, 157.70, 167.99, 175.16. ESI-MS: m/z 408.2.
[M+H]+. Anal. Calc for C25H30N4O3: C, 67.81%; H, 6.21%; N, 10.34%. Found: C, 67.92%;
H, 6.14%; N, 10.41%.
4.1.5
Ethyl
1-(3-(2-oxopyrrolidin-1-yl)propyl)-2-p-tolyl-1H-benzo[d]imidazole-5-
1
carboxylate (4d) Yield: 82%; H NMR (500 MHz, CDCl3): δ 1.44 (3H, t, J = 6.9 Hz), 2.10-
2.20 (4H, m), 2.44 (2H, t, J = 6.0 Hz), 2.62 (3H, s), 3.30 (2H, t, J = 5.0 Hz), 3.43 (2H, t, J =
6.0 Hz), 4.30 (2H, t, J = 6.5 Hz), 4.45 (2H, q, J = 6.9 Hz), 7.01 (2H, d, J = 9.0 Hz), 7.40 (1H,
d, J = 9.0 Hz), 7.65 (2H, d, J = 9.0 Hz), 7.97 (1H, dd, J = 1.5 Hz, J = 9.0 Hz), 8.52 (1H, s).
13C NMR (125 MHz, CDCl3): δ 14.60, 18.17, 24.50, 28.80, 32.01, 41.15, 43.25, 48.33, 62.51,
107.83, 109.87, 110.10, 111.04, 123.05, 126.59, 129.88, 153.42, 167.67, 175.10. ESI-MS:
m/z 406.2. [M+H]+. Anal. Calc for C24H27N3O3: C, 71.09%; H, 6.71%; N, 10.36%. Found:
C, 71.22%; H, 6.64%; N, 10.40%.
4.1.6
Ethyl
2-(4-(trifluoromethyl)phenyl)-1-(3-(2-oxopyrrolidin-1-yl)propyl)-1H-
benzo[d]imidazole-5-carboxylate (4e) Yield: 90%; 1H NMR (500 MHz, CDCl3): δ 1.45 (3H,
t, J = 6.9 Hz), 2.20-2.30 (4H, m), 2.64 (2H, t, J = 6.0 Hz), 3.45 (2H, t, J = 5.0 Hz), 3.60 (2H,
t, J = 6.0 Hz), 4.33 (2H, t, J = 6.5 Hz), 4.44 (2H, q, J = 6.9 Hz), 7.45 (1H, d, J = 9.0 Hz), 7.85
(2H, d, J = 9.0 Hz), 7.90 (2H, d, J = 9.0 Hz), 8.12 (1H, dd, J = 1.5 Hz, J = 9.0 Hz), 8.70 (1H,
13
s). C NMR (125 MHz, CDCl3): δ 14.78, 18.01, 28.12, 31.00, 40.01, 42.76, 46.15, 61.27,
110.17, 121.54, 122.80, 125.00, 125.73, 127.89, 129.33, 131.64, 139.05, 143.02, 150.81,
167.36, 175.10. ESI-MS: m/z 460.2. [M+H]+. Anal. Calc for C24H24N3O3F3: C, 62.72%; H,
5.33%; N, 9.24%. Found: C, 62.70%; H, 5.33%; N, 9.19%.
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4.1.7 Ethyl 2-(4-nitrophenyl)-1-(3-(2-oxopyrrolidin-1-yl)propyl)-1H-benzo[d]imidazole-5-
1
carboxylate (4f) Yield: 80%; H NMR (500 MHz, CDCl3): δ 1.45 (3H, t, J = 6.9 Hz), 2.00-
2.20 (4H, m), 2.39 (2H, t, J = 6.0 Hz), 3.35 (2H, t, J = 5.0 Hz), 3.46 (2H, t, J = 6.0 Hz), 4.40
(2H, t, J = 6.5 Hz), 4.52 (2H, q, J = 6.9 Hz), 7.43 (1H, d, J = 9.0 Hz), 7.68 (2H, d, J = 9.0
Hz), 7.88 (1H, d, J = 9.0 Hz), 8.20 (1H, dd, J = 1.5 Hz, J = 9.0 Hz), 8.59 (1H, s). 13C NMR
(125 MHz, CDCl3): δ 14.58, 18.23, 28.56, 28.68, 31.01, 38.55, 41.37, 46.79, 61.50, 109.96,
122.50, 125.56, 125.88, 126.18, 129.72, 129.99, 138.68, 142.51, 154.25, 167.03, 175.01. ESI-
MS: m/z 437.2. [M+H]+. Anal. Calc for C23H24N4O5: C, 63.32%; H, 5.54%; N, 12.80%.
Found: C, 63.45%; H, 5.59%; N, 12.72%.
4.1.8 Ethyl 2-(4-chlorophenyl)-1-(3-(2-oxopyrrolidin-1-yl)propyl)-1H-benzo[d]imidazole-5-
1
carboxylate (4g) Yield: 88%; H NMR (500 MHz, CDCl3): δ 1.45 (3H, t, J = 6.9 Hz), 1.90-
2.10 (4H, m), 2.48 (2H, t, J = 6.0 Hz), 3.43 (2H, t, J = 5.0 Hz), 3.55 (2H, t, J = 6.0 Hz), 4.30
(2H, t, J = 6.5 Hz), 4.48 (2H, q, J = 6.9 Hz), 7.46 (3H, m), 7.80 (2H, d, J = 9.0 Hz), 8.11 (1H,
dd, J = 1.5 Hz, J = 9.0 Hz), 8.49 (1H, s). 13C NMR (125 MHz, CDCl3): δ 14.40, 18.13, 27.97,
30.75, 39.96, 42.03, 46.88, 61.01, 109.93, 122.44, 124.67, 125.92, 126.13, 127.45, 137.97,
142.72, 154.53, 167.50, 174.99. ESI-MS: m/z 426.2. [M+H]+. Anal. Calc for C23H24N3O3Cl:
C, 64.89%; H, 5.72%; N, 9.91%. Found: C, 65.05%; H, 5.83%; N, 9.82%.
4.1.9 Ethyl 2-(4-bromophenyl)-1-(3-(2-oxopyrrolidin-1-yl)propyl)-1H-benzo[d]imidazole-5-
1
carboxylate (4h) Yield: 84%; H NMR (500 MHz, CDCl3): δ 1.45 (3H, t, J = 6.9 Hz), 2.00-
2.10 (4H, m), 2.46 (2H, t, J = 6.0 Hz), 3.37 (2H, t, J = 5.0 Hz), 3.47 (2H, t, J = 6.0 Hz), 4.32
(2H, t, J = 6.5 Hz), 4.45 (2H, q, J = 6.9 Hz), 7.46 (2H, d, J = 9.0 Hz), 7.60 (1H, d, J = 9.0
Hz), 8.14 (1H, dd, J = 1.5 Hz, J = 9.0 Hz), 8.60 (1H, s). 13C NMR (125 MHz, CDCl3): δ
14.58, 18.17, 28.04, 30.16, 40.23, 41.79, 46.4, 61.03, 108.10, 122.65, 124.78, 125.40, 126.19,
128.77, 129.21, 129.82, 130.56, 139.11, 143.13, 156.25, 167.57, 174.99. ESI-MS: m/z 470.1
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[M+H]+. Anal. Calc for C23H24N3O3Br: C, 58.67%; H, 5.13%; N, 8.91%. Found: C, 58.62%;
H, 5.12%; N, 8.81%.
1
4.1.10 Ethyl 1,2-diphenyl-1H-benzo[d]imidazole-5-carboxylate (4i) Yield: 76%; H NMR
(500 MHz, CDCl3): δ 1.45 (3H, t, J = 7.1 Hz), 4.43 (2H, q, J = 7.1 Hz), 7.10 (1H, d, J = 9
Hz), 7.30-7.70 (10H, m), 7.87 (1H, dd, J = 1.5 Hz, 9 Hz), 8.55 (1H, s). 13C NMR (125 MHz,
CDCl3): 14.51, 61.42, 110.34, 111.80, 111.99, 112.56, 113.14, 113.91 115.67, 118.82,
119.05, 130.24, 131.00, 136.56, 140.28, 142.44, 152.16, 167.88. ESI-MS: m/z 343.1 [M+H]+.
Anal. Calc for C22H18N2O2: C, 77.17%; H, 5.30%; N, 8.18%. Found: C, 77.06%; H, 5.35%;
N, 8.32%.
4.1.11 Ethyl 2-(4-(dimethylamino)phenyl)-1-phenyl-1H-benzo[d]imidazole-5-carboxylate
1
(4j) Yield: 87%; H NMR (500 MHz, CDCl3): δ 1.43 (3H, t, J = 7.1 Hz), 2.97 (6H, s), 4.41
(2H, q, J = 7.1 Hz), 6.58 (2H, d, J = 9 Hz), 7.18 (1H, d, J = 9 Hz), 7.35-7.60 (5H, m), 7.46
(2H, d, J = 9 Hz), 7.92 (1H, dd, J = 1.5 Hz, 9 Hz), 8.56 (1H, s). 13C NMR (125 MHz, CDCl3):
14.79, 40.43, 61.22, 110.04, 111.74, 116.39, 121.56, 124.65, 125.67, 127.91, 129.21, 130.42,
130.99, 137.49, 140.76, 142.71, 151.55, 155.13, 167.57. ESI-MS: m/z 386.1 [M+H]+. Anal.
Calc for C24H23N3O2: C, 74.78%; H, 6.01%; N, 10.90%. Found: C, 74.76%; H, 6.02%; N,
10.90%.
4.1.12 Ethyl 2-(4-hydroxyphenyl)-1-phenyl-1H-benzo[d]imidazole-5-carboxylate (4k)
Yield: 77%; 1H NMR (500 MHz, CDCl3): δ 1.43 (3H, t, J = 7.1 Hz), 4.42 (2H, q, J = 7.1 Hz),
7.19 (2H, d, J = 9 Hz), 7.28 (1H, d, J = 9 Hz), 7.35-7.60 (5H, m), 7.47 (2H, d, J = 9 Hz), 7.99
(1H, dd, J = 1.5 Hz, 9 Hz), 8.56 (1H, s). 13C NMR (125 MHz, CDCl3): 14.26, 61.05, 118.95,
120.87, 122.54, 124.79, 126.95, 127.26, 128.08, 129.17, 130.34, 132.96, 136.15, 140.28,
143.03, 158.69, 167.00. ESI-MS: m/z 359.1 [M+H]+. Anal. Calc for C22H18N2O3: C, 73.73%;
H, 5.06%; N, 7.82%. Found: C, 73.56%; H, 5.16%; N, 7.88%.
13

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1
4.1.13 Ethyl 1-phenyl-2-p-tolyl-1H-benzo[d]imidazole-5-carboxylate (4l) Yield: 84%; H
NMR (500 MHz, CDCl3): δ 1.41 (3H, t, J = 7.1 Hz), 2.34 (3H, s), 4.40 (2H, q, J = 7.1 Hz),
7.26 (2H, d, J = 9 Hz), 7.31 (1H, d, J = 9 Hz), 7.35-7.60 (5H, m), 7.53 (2H, d, J = 9 Hz), 7.99
13
(1H, dd, J = 1.5 Hz, 9 Hz), 8.55 (1H, s). C NMR (125 MHz, CDCl3): 14.24, 24.36, 60.96,
118.92, 120.75, 122.53, 124.82, 126.95, 127.23, 128.00, 129.17, 130.33, 132.96, 136.15,
140.28, 143.03, 151.95, 166.97. ESI-MS: m/z 357.1 [M+H]+. Anal. Calc for C23H20N2O2: C,
77.51%; H, 5.66%; N, 7.86%. Found: C, 77.62%; H, 5.62%; N, 8.78%.
4.1.14 Ethyl 2-(4-(trifluoromethyl)phenyl)-1-phenyl-1H-benzo[d]imidazole-5-carboxylate
1
(4m) Yield: 89%; H NMR (500 MHz, CDCl3): δ 1.44 (3H, t, J = 7.1 Hz), 4.44 (2H, q, J =
7.1 Hz), 7.27 (1H, d, J = 9 Hz), 7.33-7.60 (5H, m), 7.58 (2H, d, J = 9 Hz), 7.70 (2H, d, J = 9
Hz), 8.03 (1H, dd, J = 1.5 Hz, 9 Hz), 8.63 (1H, s). 13C NMR (125 MHz, CDCl3): 14.38,
61.03, 110.32, 122.45, 122.67, 124.84, 125.33, 125.36, 125.41, 125.97, 127.29, 130.30,
132.89, 136.15, 140.28, 142.43, 152.26, 166.89. ESI-MS: m/z 410.1 [M+H]+. Anal. Calc for
C24H23N3O2: C, 67.31%; H, 4.18%; N, 6.83%. Found: C, 67.30%; H, 4.12%; N, 6.88%.
4.1.15 Ethyl 2-(4-nitrophenyl)-1-phenyl-1H-benzo[d]imidazole-5-carboxylate (4n) Yield:
90%; 1H NMR (500 MHz, CDCl3): δ 1.45 (3H, t, J = 7.1 Hz), 4.47 (2H, q, J = 7.1 Hz), 7.15
(1H, d, J = 9 Hz), 7.35-7.60 (5H, m), 7.50 (2H, d, J = 9 Hz), 7.63 (2H, d, J = 9 Hz), 8.01 (1H,
dd, J = 1.5 Hz, 9 Hz), 8.62 (1H, s). 13C NMR (125 MHz, CDCl3): 14.38, 61.11, 111.06,
122.59, 124.84, 125.33, 125.36, 125.41, 125.97, 127.37, 130.33, 132.96, 136.15, 140.27,
142.42, 150.49, 151.99, 167.10. ESI-MS: m/z 388.1 [M+H]+. Anal. Calc for C22H17N3O4: C,
68.21%; H, 4.42%; N, 10.85%. Found: C, 68.20%; H, 4.42%; N, 10.87%.
4.1.16 Ethyl 2-(4-chlorophenyl)-1-phenyl-1H-benzo[d]imidazole-5-carboxylate (4o) Yield:
81%; 1H NMR (500 MHz, CDCl3): δ 1.43 (3H, t, J = 7.1 Hz), 4.42 (2H, q, J = 7.1 Hz), 7.25
(1H, d, J = 9 Hz), 7.28 (2H, d, J = 9 Hz), 7.40-7.60 (5H, m), 7.54 (2H, d, J = 9 Hz), 8.00 (1H,
dd, J = 1.5 Hz, 9 Hz), 8.52 (1H, s). 13C NMR (125 MHz, CDCl3): 14.40, 61.64, 110.28,
14

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111.80, 116.26, 122.70, 122.97, 124.65, 125.67, 127.91, 129.20, 130.35, 131.18, 137.50,
140.76, 142.71, 152.68, 167.48. ESI-MS: m/z 377.1 [M+H]+. Anal. Calc for C22H17N2O2Cl:
C, 70.12%; H, 4.55%; N, 7.43%. Found: C, 70.10%; H, 4.52%; N, 7.54%.
4.1.17 Ethyl 2-(4-bromophenyl)-1-phenyl-1H-benzo[d]imidazole-5-carboxylate (4p) Yield:
79%; 1H NMR (500 MHz, CDCl3): δ 1.44 (3H, t, J = 7.1 Hz), 4.44 (2H, q, J = 7.1 Hz), 7.24
(1H, d, J = 9 Hz), 7.32 (2H, d, J = 9 Hz), 7.40-7.60 (5H, m), 7.58 (2H, d, J = 9 Hz), 8.00 (1H,
dd, J = 1.5 Hz, 9 Hz), 8.60 (1H, s). 13C NMR (125 MHz, CDCl3): 14.39, 61.65, 110.30,
111.82, 116.37, 122.75, 123.89, 124.65, 125.67, 127.91, 129.20, 130.32, 131.08, 137.50,
140.76, 142.71, 152.69, 167.50. ESI-MS: m/z 421.1 [M+H]+. Anal. Calc for C22H17N2O2Br:
C, 62.72%; H, 4.07%; N, 6.65%. Found: C, 62.76%; H, 4.02%; N, 6.73%.
4.2 Biology
Fluorescent optical density for in vitro assay was measured on Tecan Infinite M200. Optical
density for cell proliferative assay was measured with Thermo Scientific MultiSkan FC
microplate reader.
4.2.1. SIRT1 in vitro Assay
3.3 µM of SIRT1 substrate derived from human p53 sequences, 66.7 µM NAD+, 50 µM of
interested compounds (all final concentration) and 0.5 µg of SIRT1 human recombinant
(GenBank Accession #: NM_012238) with 193- 741 amino acids and GST tag at its N-
terminal, were incubated for 45 minutes at 37oC. 50 µL of stop solution consisting
nicotinamide and SIRT1 developer was then added and the mixture was incubated for a
further 10 minutes at 37oC. Fluorescence was measured at 490 nm (excitation) and 520 nm
(emission) and the inhibition was calculated as the ratio of absorbance under each
experimental condition to that of the control.
15

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4.2.2. SIRT2 in vitro Assay
6.7 µM of SIRT2 substrate derived from human p53 sequences, 333 µM NAD+, 50 µM of
interested compounds (all final concentration) and 0.5 µg of SIRT2 human recombinant
(GenBank Accession #: NM_030593) with 13- 319 amino acids and His tag at its C-terminal,
were incubated for 45 minutes at 37oC. 50 µL of stop solution consisting nicotinamide and
SIRT1 developer was then added and the mixture was incubated for a further 10 minutes at
37oC. Fluorescence was measured at 490 nm (excitation) and 520 nm (emission) and the
inhibition was calculated as the ratio of absorbance under each experimental condition to that
of the control.
4.2.3. Cell Proliferation Assay
All cell lines were obtained from the American Type Culture Collection (Rockville, MD).
Cells were seeded in 96-well plates at a density of 5 × 103 per well (for MDA-MB-468 and
HCT-116) or 4 x 104 per well (for CCRF-CEM). The cells were treated with 50 µM of
interested compounds and allowed to adhere for 72 hours. Then, the proliferative activity was
determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
2H-tetrazolium, inner salt assay (CellTiter 96 Non-Radioactive Cell Proliferation Assay;
Promega, Madison, WI) to monitor the number of viable cells according to the manufacturer's
instructions.
Briefly,
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
sulfophenyl)-2H-tetrazolium, inner salt solution was added at 20 ꢀL/well, and after 1 hour of
incubation at 37°C in a humidified 5% CO2 atmosphere, the conversion of 3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner
salt to formazan was measured in a plate reader at 490 nm. All experiments were done in
triplicate, and the proliferation rate was calculated as the ratio of absorbance under each
experimental condition to that of the control nontransfectant.
16

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4.2.4. Autofluorescence
Compounds in DMSO at 50 µM concentration (100 µL) were pipetted into Nunc Microwell
96-wells plate. DMSO was used as control well. Excitation wavelength was set at 490 nm
and emission wavelength at 520 nm. The criteria for a compound being considered
autofluorescent was defined as having >50% fluorescence of the control wells. None of the
analyzed compounds were found to be autofluorescent.
Acknowledgements
The authors wish to express their gratitude and appreciation to Pharmacogenetics and Novel
Therapeutics Research Cluster, Institute for Research in Molecular Medicine, Universiti
Sains Malaysia, Penang for supporting this work. This work was funded through FRGS Grant
No (203/C1PPM/6711335) and HiCoE research Grant No (311.CIPPM.4401005).
The authors hereby declare there is no conflict of interests.
17

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Tables, Figures and Scheme:
1. Table 1. SIRT1 and SIRT2 inhibitory activities of novel benzimidazole derivatives.
2. Figure 1. Molecular interactions between 4j and SIRT2.
3. Figure 2. Molecular interactions between 4b and SIRT2.
4. Figure 3. Inhibitory activity of representative compounds 4b, 4j and 4l against HCT-
116, MDA-MB-468 and CCRF-CEM cell line.
5. Scheme 1. Synthesis protocol of titled compounds 4a-p
22

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Table 1. SIRT1 and SIRT2 inhibitory activities of synthesized benzimidazole derivatives
SIRT1 inhibition IC50 SIRT1 SIRT2 inhibition IC50 SIRT2
Entry Compound at 50 µM ± S.D.
(%)
inhibition
(µM)
at 50 µM ± S.D.
(%)
inhibition
(µM)
1
2
30.03 ± 9.08
49.15 ±10.12
30.93 ± 5.13
33.56 ± 11.24
32.84 ± 7.09
37.36 ± 3.45
27.16 ± 8.73
27.30 ± 4.94
21.03 ± 3.69
60.59 ± 8.26
11.01 ± 10.29
43.93 ± 5.96
33.76 ± 2.43
34.20 ± 10.87
19.82 ± 3.49
26.70 ± 7.33
67.59 ± 4.85
44.00 ± 7.84
N.D.
43.91 ± 4.45
57.90 ± 6.80
43.81 ± 6.77
45.40 ± 13.31
33.17 ± 2.21
42.98 ± 3.18
34.50 ± 5.68
40.06 ± 8.20
34.31 ± 4.91
71.69 ± 6.74
22.94 ± 8.40
50.79 ± 11.94
45.52 ± 8.47
48.03 ± 9.93
32.52 ± 3.54
41.31 ± 15.83
62.04 ± 9.80
66.20 ± 5.98
N.D.
4a
60.31 ± 4.22
N.D.
52.78 ± 5.07
N.D.
4b
3
4c
4
N.D.
N.D.
4d
5
N.D.
N.D.
4e
6
N.D.
N.D.
4f
7
N.D.
N.D.
4g
8
N.D.
N.D.
4h
9
N.D.
N.D.
4i
10
11
12
13
14
15
16
17
18
49.12 ± 3.33
N.D.
26.85 ± 1.92
N.D.
4j
4k
79.87 ± 8.10
N.D.
60.60 ± 2.42
N.D.
4l
4m
N.D.
N.D.
4n
N.D.
N.D.
4o
N.D.
N.D.
4p
47.90 ± 4.55
58.10 ± 3.67
45.86 ± 3.31
29.73 ± 1.84
Cambinol
Tenovin-6
N.D. = Not determined

ACCEPTED MANUSCRIPT
Figure 1. Molecular interactions between 4j and SIRT2.

ACCEPTED MANUSCRIPT
Figure 2. Molecular interactions between 4b and SIRT2

ACCEPTED MANUSCRIPT
Figure 3. Inhibitory activity of representative compounds 4b, 4j and 4l against HCT-116,
MDA-MB-468 and CCRF-CEM cell line.

ACCEPTED MANUSCRIPT
Scheme 1. Synthesis protocol of titled compounds 4a-p
O
NH2
R1
EtOH /
conc. H2SO4
O
NO2
HOOC
NO2
F
O
NO2
F
O
reflux
6 hrs
EtOH / R.T.
1 hr
NH
R1
1
4-fluoro-3-nitrobenzoic
acid
2
HCOONH4
10% Pd/C
EtOH / R.T.
1-2 hrs
O
O
NH2
NH
R1
3
R2
NaHS03
water / R.T.
1 hr
CHO
O
(3)
(4)
N
(2)
O
(5)
R2
(6)
(1)
N
R1
(7)
4
4a
4b
4c
4d
4e
4f
R1 = 3-(2-oxopyrrolidin-1-yl)propyl
R1 = 3-(2-oxopyrrolidin-1-yl)propyl
R1 = 3-(2-oxopyrrolidin-1-yl)propyl
R1 = 3-(2-oxopyrrolidin-1-yl)propyl
R1 = 3-(2-oxopyrrolidin-1-yl)propyl
R1 = 3-(2-oxopyrrolidin-1-yl)propyl
R1 = 3-(2-oxopyrrolidin-1-yl)propyl
R1 = 3-(2-oxopyrrolidin-1-yl)propyl
R1 = phenyl
R2 = -H
R2 = -N(CH3)2
R2 = -OH
R2 = -CH3
R2 = -CF3
R2 = -NO2
R2 = -Cl
4g
4h
4i
R2 = -Br
R2 = -H
4j
R1 = phenyl
R1 = phenyl
R1 = phenyl
R1 = phenyl
R1 = phenyl
R1 = phenyl
R1 = phenyl
R2 = -N(CH3)2
R2 = -OH
R2 = -CH3
R2 = -CF3
R2 = -NO2
R2 = -Cl
4k
4l
4m
4n
4o
4p
R2 = -Br

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HIGHLIGHTS:
•
•
•
•
•
Compounds with better in vitro SIRT2 inhibition than cambinol and Tenovin-6
Good anti proliferative effect against three different cancer cell lines
Binding mode of potent compounds with SIRT2 was established
Novel green chemistry method in benzimidazole synthesis
Benzimidazoles as potential anti-cancer agent