Synthesis and evaluation of novel benzimidazole derivatives as sirtuin inhibitors with antitumor activities

Article abstract of DOI:10.1016/j.bmc.2013.12.029

A total of 15 novel benzimidazole derivatives were designed, synthesized and evaluated for their SIRT1 and SIRT2 inhibitory activity. All compounds showed better inhibition on SIRT2 as compared to SIRT1. Among these, compound 5j displayed the best inhibitory activity for SIRT1 (IC₅₀ = 58.43 μM) as well as for SIRT2 (IC₅₀ = 45.12 μM). Cell cytotoxicity assays also showed that compound 5j possesses good antitumor activity against two different cancer cell lines derived from breast cancer (MCF-7 and MDA-MB-468). A simple structure-activity-relationship (SAR) study of the newly synthesized benzimidazole derivatives was also discussed.

Full text of DOI:10.1016/j.bmc.2013.12.029

Bioorganic & Medicinal Chemistry 22 (2014) 703–710
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of novel benzimidazole derivatives
as sirtuin inhibitors with antitumor activities
Yeong Keng Yoon a, , Mohamed Ashraf Ali a,b,c, Ang Chee Wei a, Tan Soo Choon a, Hasnah Osman d,
⇑
Keykavous Parang e, Amir Nasrolahi Shirazi e
a Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Minden 11800, Penang, Malaysia
b New Drug Discovery Research, Department of Medicinal Chemistry, Alwar Pharmacy College, Alwar 301030, Rajasthan, India
c New Drug Discovery Research, Department of Medicinal Chemistry, Sunrise University, Alwar 301030, Rajasthan, India
d School of Chemical Sciences, Universiti Sains Malaysia, Minden 11800, Penang, Malaysia
e Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, Kingston, RI 02881, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A total of 15 novel benzimidazole derivatives were designed, synthesized and evaluated for their SIRT1 and
SIRT2 inhibitory activity. All compounds showed better inhibition on SIRT2 as compared to SIRT1. Among
Received 12 October 2013
Revised 2 December 2013
Accepted 9 December 2013
Available online 19 December 2013
these, compound 5j displayed the best inhibitory activity for SIRT1 (IC50 = 58.43
lM) as well as for SIRT2
(IC50 = 45.12 M). Cell cytotoxicity assays also showed that compound 5j possesses good antitumor activity
l
against two different cancer cell lines derived from breast cancer (MCF-7 and MDA-MB-468). A simple struc-
ture–activity-relationship (SAR)study of thenewly synthesized benzimidazole derivativeswas alsodiscussed.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Sirtuin
Benzimidazole
Electron donating
Anti-proliferative
Breast cancer
1. Introduction
derivatives,17,18 splitomycin analogs19 and tenovins.20 A large
high-throughput screening effort led to the discovery of a series
Reversible protein acetylation is an important process that regu-
lates the function of histones as well as many non-histone proteins.
This modification is controlled by histone acetyltransferases and his-
tone deacetylases (HDACs).1 Sirtuins are class III HDACs which is
NAD+-dependant that catalyze the deacetylation of proteins.2 They
are involved in various cellular functions, such as longevity and
metabolism.3–5 Seven sirtuin members are found in human (SIRT1-
7)6 and among these, SIRT1 and SIRT2 are the most studied.7,8 SIRT1
and SIRT2 are reportedly associated with diseases such as cancer9
and neurodegenerative disorders.10,11 Since sirtuins have been found
upregulated in many tumor types, are able to inactivate some tumor
suppressor proteins such as p53 at transcriptional and post-transla-
tional level and maintain chromosomal stability, the inhibitors of sir-
tuins have been proposed as potential anti-cancer agents.12–14
Recently, a SIRT2 specific inhibitor (AGK-2) has also been proposed
as a useful agent for protection against alpha-synuclein-induced tox-
icity in different models of Parkinson’s disease.15 Therefore, potent
SIRT1 andSIRT2 modulators couldbeused asvaluabletoolsto gainin-
sight into the specific cellular functions of their effector proteins.
To date, several classes of sirtuin inhibitors have been identified
such as the physiological inhibitor nicotinamide,16 sirtinol and
of indole compounds as interesting inhibitors of SIRT1, including
one of the most potent compounds known so far, EX-527.21 Since
indole and benzimidazole share some structure similarities, we
embark to synthesize and evaluate the potential of utilizing benz-
imidazoles as sirtuin inhibitors. The pharmacokinetics of benzimi-
dazoles were also well studied, therefore they are a good starting
point in developing new drugs. Herein we would like to report a
new class of sirtuin inhibitors based on the benzimidazole scaffold.
To the best of our knowledge, there has been no report on utilizing
benzimidazole or their analogs as sirtuin inhibitors apart from
those reported by Sirtris (now GSK) as sirtuin modulators.22 In
addition, anti-proliferative activity of the novel benzimidazole
derivatives against two breast tumor cell lines (MCF-7 and
MDA-MB-468) were also reported. Additionally, systematic struc-
ture–activity relationship (SAR) study was also performed and
investigated with the 15 novel compounds.
2. Results and discussion
2.1. Chemistry
The procedure to synthesize benzimidazole derivatives was
adopted and modified from previously published literature
(Scheme 1).23,24 Our synthetic study into novel benzimidazoles
⇑
Corresponding author. Tel.: +60 4 6593140; fax: +60 4 6569796.
E-mail address: kyyeong@gmail.com (Y.K. Yoon).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.12.029

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Y. K. Yoon et al. / Bioorg. Med. Chem. 22 (2014) 703–710
started with 4-fluoro-3-nitro benzoic acid which was esterified in
the presence of catalytic sulfuric acid in ethanol by refluxing for
8 h to afford the ethyl ester 1 in 75% yield. Our research work takes
into account the basic functional groups that are amendable to
pro-drug design and strategy, including the ability to increase lipo-
philicity. This first step reaction to convert carboxylic acid group to
ester group was also an attempt to mask potential undesirable
drug properties such as low solubility in lipid membranes and
chemical instability.
The ethylbenzoate 1 was then treated with ethanolamine and
DIPEA in dry dichloromethane at room temperature yielded ethyl
4-(2-hydroxyethylamino)-3-nitrobenzoate 2, which was then
reduced to the amine 3 using ammonium formate and 10% Pd/C
by refluxing for 3 h. The structure of ethyl 4-(2-hydroxyethyl-
amino)-3-aminobenzoate
analysis.
3 was confirmed by spectroscopic
Figure 1. Hydrogen bonds between 5j and amino acid within the NAD+ cavity of
SIRT2.
Ethyl 4-(2-hydroxyethylamino)-3-aminobenzoate 3 was then
refluxed with various substituted bisulfite adducts of aromatic
aldehydes 4a–l in DMF overnight to afford benzimidazole deriva-
tives 5a–l in good yields (75–95%).25 The structure of the novel
benzimidazoles were confirmed by spectroscopic analysis.
SIRT1/SIRT2 inhibitory activities as direct comparison to their ester
derivatives. The screening results revealed that the carboxylic acid
derivatives showed poorer inhibitory activity against both SIRT1
and SIRT2.
2.2. Enzymatic assays
The in vitro screening of the total 15 novel benzimidazole deriv-
atives led to the identification of three potent SIRT1/SIRT2 inhibi-
tors (5j, 5k and 5l) and the results are shown in Table 1.
Experiments were performed in triplicates. Standard deviation ob-
tained for all experiments are less than 15%.
Basically, all the novel benzimidazole derivatives showed better
inhibition on SIRT2 as compared to SIRT1. The most potent inhib-
itor for SIRT2 as well as SIRT1 was found to be 5j (SIRT1
The in vitro enzymatic screening assay for SIRT1 and SIRT2
inhibitory activity were performed using SensolyteÒ fluorimetric
drug discovery kits (AnaSpec, Fremont, CA) according to the man-
ufacturer’s protocol. Cambinol was used as standard control 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 lM. Initial in vitro screening on 11 of the com-
pounds (5a–k) showed that compounds with strong electron
donating group such as dimethylamino at R1 possessed the best
sirtuin inhibitory activities. The inhibition activity was however,
greatly affected when the substitution was replaced by weaker
electron donating groups such as methyl or even hydroxyl and
methoxyl groups. To explore the importance of the strong electron
donating effect on the sirtuin inhibitory activity, we then proceed
to synthesize a compound with two electron donating substituents
on the phenyl ring (1,3-dioxole, 5l). The inhibitory effect was en-
hanced when another electron donating group was added to the
phenyl ring. Although knowing well the potential downside of
the carboxylic acid derivatives of the benzimidazoles in terms of
bioavailability, we find it is worth investigating the carboxylic acid
derivatives of compounds 5j, 5k and 5l (6j, 6k and 6l, respectively).
The compounds were then synthesized and screened for their
IC50 = 58.43 lM; SIRT2 IC50 = 45.12 lM). As shown in Table 1, com-
pound 5l also showed good SIRT2 inhibitory activity but its po-
tency was slightly less that that of compound 5j. Overall,
compound 5j showed better SIRT2 inhibitory activity compared
to the standard control used (cambinol). However, none of the
compounds screened in the present derivatives are found to be
more potent than cambinol in inhibiting SIRT1.
2.3. Molecular docking
In an attempt to predict the binding mode of this novel chemi-
cal series, the most active compound (5j) was docked into the
active site of human SIRT2. Since the X-ray crystal structure of
SIRT2-substrate complex structure has recently been reported,26
Table 1
SIRT1 and SIRT2 inhibitory activities of novel benzimidazole derivatives
Compound
SIRT1 inhibition (%) at 50
lM
IC50 SIRT1 inhibition (
l
M)
SIRT2 inhibition (%) at 50
lM
IC50 SIRT2 inhibition (lM)
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
12.73
14.72
5.32
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
58.43
74.52
80.11
N.D.
N.D.
N.D.
47.90
22.73
22.49
11.60
19.23
23.46
36.29
29.03
15.32
18.29
68.50
64.19
61.47
26.78
25.70
21.99
68.32
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
45.12
60.34
56.05
N.D.
N.D.
N.D.
52.89
11.74
23.22
33.77
24.94
12.65
17.79
48.70
43.33
39.63
19.65
24.00
13.97
70.77
6j
6k
6l
Cambinol
N.D. = Not determined.

Y. K. Yoon et al. / Bioorg. Med. Chem. 22 (2014) 703–710
705
Figure 2. Molecular interactions between 5j and SIRT2.
we used that X-ray crystal structure in this docking study (PDB en-
try code: 3ZGV, X-ray resolution = 2.30 Å) instead of the apo-struc-
ture. 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 5j within the
SIRT2-ADPr cofactor binding site demonstrated several plausible
molecular interactions. The docking analysis reveals that the com-
pound 5j interact with receptor primarily due to hydrogen bonding
as well as hydrophobic and mild polar interactions. The O–H group
of compound 5j is hydrogen bonded strongly to Glu288. Other
hydrogen bonds which could be observe within 3.5 Å include inter-
actions with ASN286, THR262, ARG97, SER263, GLN167 and
CYS324 (Fig. 1). This is relatively consistent with the hydrogen
bonds observed between SIRT2 and ADPr complex26 as well as
other SIRT2-inhibitor predictions including salermide and
NF-675.27 Hydrogen bonds between H from CYS324 and to a lesser
extent H from LYS287 with N from dimethylamino group from the
benzimidazole also helped to stabilized the binding complex.
Interactions between lone pair oxygen-p could also be observed
through ASP95 and the imidazole ring as well as GLY86 and ben-
zene ring. Apart from these, some hydrophobic and mild polar
interactions could be observed between compound 5j and
THR89, ALA85, PHE96, GLU323 and VAL266 (Fig. 2).
As for compound 5k, which also showed relatively good SIRT2
inhibition, the docking pose showed a different orientation as
Figure 3. Molecular interactions between 5k and SIRT2.

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Y. K. Yoon et al. / Bioorg. Med. Chem. 22 (2014) 703–710
COOH
COOCH2CH3
COOCH2CH3
OH
COOCH2CH3
H2N
C2H5OH
H2SO4
HCOONH4
Pd/C
DIPEA, DCM
overnight, RT
NO2
NO2
NO2
NH
NH2
NH
F
F
1
HO
HO
2
3
sodium
HO
SO3Na
DMF
metabisulfite
R1
CHO
90 o
C
ethanol
R1
4
O
N
N
O
R1
OH
5
R1:
a = H
b = Br
c = Cl
d = OCF3
e = CF3
f = NO2
g = CH3
h = OH
i = OCH3
j = dimethylamino
k = piperidine
l = 1,3-dioxole
Scheme 1. Protocol for synthesis of titled compounds.
compared to 5j (Fig. 3). Docking simulation indicated that the
dimethylamino group from compound 5j was able to form a hydro-
gen bond to CYS324. However, 5k has a different binding mode as
the bulkier piperidine moiety was unable to fit into the cavity to
form hydrogen to CYS324, which was located at the end of the
pocket. It was able to form strong hydrogen bonds with ASP95,
ARG97 and LYS287.
revealed that compound 5j is competitive with respect to NAD+
which imply that the inhibitor competes with NAD+ to occupy
the same binding site in the receptor (see Supporting information).
This is in agreement with our molecular docking prediction.
2.5. Cellular assays
Weaker hydrogen bonds were also observed between H from
GLN167 with N from piperidine moiety as well as THR89 and
CYS324 with O the from ester chain. Apart from these, p–p stack-
ing interaction was also indicated between the piperidine ring of
5k and PHE96.
The antitumor activity of various known sirtuin inhibitors has
been previously demonstrated in the literature.20,29 To investi-
gate the effectiveness of our compounds as antitumor agents,
they were evaluated for their ability to inhibit growth of tumor
cells. Human breast cancer MCF-7 cells and MDA-MB-468 cells
were used as tumor cells, because the inhibition of SIRT2 has been
shown to completely inhibit MCF-7 cell proliferation and could
also inhibit tumor cell growth in a mouse xenograft model of tri-
ple negative breast cancer.30 Interestingly as shown in Figure 4,
compounds 5j, 5k and 5l exerted potent anti-proliferative activity
against MCF-7 tumor cells compared to the other compounds
tested. Similar growth inhibitory trend was observed against
MDA-MB-468 tumor cells. This showed that not only the newly
2.4. Competition analysis
Besides molecular modeling, the binding mode of compound 5j
towards SIRT2 was also analyzed using competition analysis fol-
lowing method adopted from Lai et al.28 The inhibition of 5j was
tested with increasing concentration of NAD+ while the other
parameters of the assay were kept constant. Competition analysis

Y. K. Yoon et al. / Bioorg. Med. Chem. 22 (2014) 703–710
707
120
100
80
60
40
20
0
prime candidates for modifications to further improve their activ-
ities. An extension of the study in future may contribute to the
development of useful anticancer agents in this series.
4. Experimental
4.1. Chemistry
MCF7
MDA-MB-468
All general chemicals were supplied by Sigma–Aldrich (U.S.A)
and Merck Chemicals (Germany). Cambinol was obtained from
Cayman Chemicals (U.S.A). Thin layer chromatography (silica gel
G) was ran in the solvent system chloroform–methanol (9:1). The
spots were located under short (254 nm)/long (365 nm) UV light.
Elemental analyses were performed on Perkin Elmer 2400 Series
II CHN Elemental Analyzer and were within 0.4% of the calculated
values. 1H and 13C NMR were performed on Bruker Avance 300
spectrometer in CDCl3 using TMS as internal standard. Mass spec-
tra were recorded on Varian 320-MS TQ LC/MS using ESI mode.
Column chromatography purification was done in solvent system
chloroform–methanol (9:1) using Silica Gel 60 (0.063–0.200 mm).
Figure 4. Inhibitory activity of compounds 5a–l against MCF-7 and MDA-MB-468
cell line.
Table 2
Anti-proliferative activities of selected benzimidazole derivatives against MCF-7 and
MDA-MB-468 cancer cell lines
4.1.1. Procedure for the preparation of ethyl-4-fluoro-3-nitro
benzoate (1)
Compound
Cell inhibition (%) at 50 lM
4-Fluoro-3-nitrobenzoic acid (5 g, 27 mmol) was refluxed in
ethanol (50 mL) and concentrated H2SO4 (2 mL) for 8 hours. After
completion of reaction (as evident from TLC), the solvent was evap-
orated under reduced pressure. The aqueous layer was extracted
with ethyl acetate (25 mL Â 3). The organic layer was dried over
Na2SO4 and concentrated under reduced pressure to yield 1 as
cream-coloured powder (75%).
MCF-7
MDA-MB-468
5j
5k
5l
49.63
42.37
62.43
38.26
46.33
45.51
42.30
22.09
Cambinol
Data for 1. Yield: 75%; 1H NMR (300 MHz, CDCl3): d 1.44 (3H, t,
J = 7.2 Hz, CH3CH2O–), 4.45 (2H, q, J = 7.2 Hz, CH3CH2O–), 7.41 (1H,
d, J = 8.4 Hz, H arom.), 8.70 (1H, dd, J = 1.5 Hz, 8.4 Hz, H arom.), 8.89
(1H, s, H arom.). ESI-MS: m/z 214.1 [M+H]+. Anal. Calcd for C9H8-
NO4F: C, 50.71; H, 3.78; N, 6.57. Found: C, 50.65%; H, 3.83%; N, 6.60.
synthesized compounds are able to inhibit the luminal subtype
breast cancer cells (MCF-7) but they are also able to inhibit the
triple-negative breast cancer cells (MDA-MB-468) which target
therapies currently do not exist. Taken together, these results
suggested that sirtuin inhibitors especially SIRT1 and SIRT2 inhib-
itors bearing the 2-phenylbenzimidazole moiety with strong
electron donating group on the para-position are potential antitu-
mor agents. Results for the selected compounds which showed
good anti-proliferative activity (5j, 5k, 5l) against both cancer cell
lines are tabulated in Table 2.
SIRT2 could potentially play a more important role in inhibiting
breast cancer MCF-7 and MDA-MB-468 cells as compared to SIRT1.
This is due to the observation that compounds with good cytotoxic
effect have potent SIRT2 inhibition. However, it should be noted
that these compounds also exhibited inhibitory effects to SIRT1, al-
beit at higher concentrations. We cannot rule out the possibility
that combinational inhibitory effects of SIRT1 and SIRT2 activity
may contribute to the observed cytotoxicity as has been reported
previously by Peck et al.31
4.1.2. Procedure for the preparation of ethyl 4-(2-hydroxyethy
lamino)-3-nitrobenzoate (2)
Ethyl-4-fluoro-3-nitrobenzoate, 1 (0.5 g, 2.34 mmol), ethanol-
amine (2.58 mmol) and N,N-Diisopropylethylamine, DIPEA
(0.49 mL, 2.78 mmol) were mixed in dichloromethane (10 mL).
The reaction mixture was stirred overnight at room temperature.
After completion of reaction (as evident from TLC), the reaction
mixture was washed with water (10 mL Â 2) followed by 10% Na2-
CO3 solution (10 mL). The organic layer was dried over Na2SO4 and
concentrated under reduced pressure to afford 2 as yellow solid
(89%).
Data for 2. Yield: 89%; 1H NMR (300 MHz, CDCl3): d 1.44 (3H, t,
J = 7.2 Hz, CH3CH2O–), 3.48 (2H, t, J = 5.7 Hz, –NCH2–), 3.88 (2H, t,
J = 5.7 Hz, –CH2OH), 4.45 (2H, q, J = 7.2 Hz, CH3CH2O–), 6.82 (1H,
d, J = 8.4 Hz, H arom.), 8.25 (1H, dd, J = 1.5 Hz, 8.4 Hz, H arom.),
8.71 (1H, s, H arom.). ESI-MS: m/z 255.2 [M+H]+. Anal. Calcd for
3. Conclusion
C11H14N2O5: C, 51.97; H, 5.55; N, 11.02%. Found: C, 51.95; H,
5.56; N, 11.05.
In conclusion, we have discovered some novel benzimidazole
derivatives which showed good SIRT1/SIRT2 inhibition activity
with micromolar IC50 values. Moreover, they possess good antitu-
mor activity against both MCF-7 (luminal) as well as MDA-MB-
468 (basal-A subtype) breast cancer cell lines evaluated in this
study. More importantly, we are able to correlate between
in vitro SIRT2 (and to a lesser extent SIRT1) inhibition and cancer
cell cytotoxicity using small molecule sirtuin inhibitors. 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 cyototoxicity activities such as 5j and 5l are
4.1.3. Procedure for the preparation of ethyl 4-(2-hydroxyethy
lamino)-3-aminobenzoate (3)
Ethyl 4-(2-hydroxyethylamino)-3-nitrobenzoate,
2
(1 mmol),
ammonium formate (3 mmol) and Pd/C (50 mg) were mixed in
ethanol (10 mL). The reaction mixture was refluxed until comple-
tion (solution turned colourless). The reaction mixture was then
filtered through Celite 545. The filtrate was evaporated under re-
duced pressure. It was resuspended in ethyl acetate and washed
with water, dried over Na2SO4 and evaporated to dryness to yield
3 (75%).

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Y. K. Yoon et al. / Bioorg. Med. Chem. 22 (2014) 703–710
Data for 3. Yield: 75%; 1H NMR (300 MHz, CDCl3): d 1.43 (3H, t,
J = 5.7 Hz, –CH2OH), 4.45 (2H, q, J = 7.2 Hz, CH3CH2O–), 6.86 (1H,
d, J = 8.4 Hz, H aromatic), 6.95 (1H, d, J = 8.4 Hz, H aromatic), 7.36
(1H, d, J = 8.4 Hz, H aromatic), 7.70 (1H, d, J = 8.4 Hz, H aromatic),
7.75 (1H, dd, J = 1.5 Hz, 8.4 Hz, H arom.), 7.78 (1H, d, J = 8.4 Hz, H
aromatic), 8.00 (1H, s, H arom.). 13C NMR (75 MHz, CDCl3): 14.54,
47.58, 60.34, 60.72, 109.45, 111.92, 112.07, 122.66, 122.80,
126.19, 127.87, 129.15, 130.82, 133.02, 139.11, 142.03, 156.25,
159.60, 166.79. ESI-MS: m/z 395.3 [M+H]+. Anal. Calcd for C19H17-
N2O4F3: C, 57.87; H, 4.35; N, 7.10. Found: C, 57.69; H, 4.19; N, 7.14.
Data for 5e. Yield: 91%; 1H NMR (300 MHz, CDCl3): d 1.48 (3H, t,
J = 7.2 Hz, CH3CH2O–), 4.18 (2H, t, J = 5.7 Hz, –NCH2–), 4.34 (2H, t,
J = 5.7 Hz, –CH2OH), 4.45 (2H, q, J = 7.2 Hz, CH3CH2O–), 6.88 (1H,
d, J = 8.4 Hz, H aromatic), 6.95 (1H, d, J = 8.4 Hz, H aromatic), 7.36
(1H, d, J = 8.4 Hz, H aromatic), 7.70 (1H, d, J = 8.4 Hz, H aromatic),
7.75 (1H, dd, J = 1.5 Hz, 8.4 Hz, H arom.), 7.79 (1H, d, J = 8.4 Hz, H
aromatic), 8.05 (1H, s, H arom.). 13C NMR (75 MHz, CDCl3): 14.55,
47.60, 60.31, 60.72, 110.15, 111.93, 112.71, 123.10, 123.80,
126.19, 127.87, 129.33, 130.82, 131.88, 133.02, 139.11, 142.09,
157.43, 166.81. ESI-MS: m/z 378.3 [M+H]+. Anal. Calcd for C19H17-
N2O3F3: C, 60.32; H, 4.53; N, 7.40. Found: C, 60.35; H, 4.55; N, 7.38.
Data for 5f. Yield: 80%; 1H NMR (300 MHz, CDCl3): d 1.49 (3H, t,
J = 7.2 Hz, CH3CH2O–), 4.20 (2H, t, J = 5.7 Hz, –NCH2–), 4.34 (2H, t,
J = 5.7 Hz, –CH2OH), 4.48 (2H, q, J = 7.2 Hz, CH3CH2O–), 6.88 (1H, d,
J = 8.4 Hz, H aromatic), 6.95 (1H, d, J = 8.4 Hz, H aromatic), 7.36
(1H, d, J = 8.4 Hz, H aromatic), 7.73 (1H, d, J = 8.4 Hz, H aromatic),
7.75 (1H, dd, J = 1.5 Hz, 8.4 Hz, H arom.), 7.80 (1H, d, J = 8.4 Hz, H aro-
matic), 8.06 (1H, s, H arom.). 13C NMR (75 MHz, CDCl3): 14.53, 47.56,
60.31, 60.72, 110.10, 111.93, 112.71, 123.10, 123.69, 126.15, 126.97,
128.94, 131.88, 133.02, 139.11, 142.08, 149.86, 157.34, 166.80. ESI-
MS: m/z 356.2 [M+H]+. Anal. Calcd for C18H17N3O5: C, 60.84; H, 4.82;
N, 11.83. Found: C, 60.91; H, 4.78; N, 11.83.
J = 7.2 Hz, CH3CH2O–), 3.48 (2H, t, J = 5.7 Hz, –NCH2–), 3.88 (2H, t,
J = 5.7 Hz, –CH2OH), 4.44 (2H, q, J = 7.2 Hz, CH3CH2O–), 6.56 (1H,
d, J = 8.4 Hz, H arom.), 7.13 (1H, s, H arom.), 7.25 (1H, dd,
J = 1.5 Hz, 8.4 Hz, H arom.). ESI-MS: m/z 225.2 [M+H]+. Anal. Calcd
for C11H14N2O5: C, 58.91; H, 7.19; N, 12.49. Found: C, 58.78; H,
7.29; N, 12.42.
4.1.4. General procedure for the preparation of sodium bisulfite
addicts of 4-substituted benzaldehyde (4)
Appropriate benzaldehyde (10 mmol) was dissolved in ethanol
(20 mL). Sodium metabisulfite (15 mmol) in 5 mL water was added
in portion over 5 min. The reaction mixture was stirred at room
temperature for 1 h and subsequently stirred at 4 °C overnight.
The precipitate formed was filtered and dried to afford sodium
bisulfite adducts (85–98%).
Data for 4j. Yield: 75%; 1H NMR (300 MHz, CDCl3): d 3.00 (3.00
(6H, s, –N(CH3)2), 6.87 (2H, d, J = 8.4 Hz, H arom.), 7.45 (2H, d,
J = 8.4 Hz, H arom.). ESI-MS: m/z 253.2 [M]+.
4.1.5. General procedure for the preparation of 2-substituted
benzimidazole derivatives (5)
Ethyl 4-(2-hydroxyethylamino)-3-aminobenzoate, 3 (1 mmol) and
various sodium bisulfite adducts, 4 (1.5 mmol) were dissolved in
DMF (5 mL). The reaction mixture was stirred at 90 °C under N2
atmosphere for 24–48 h. After completion of reaction (evident by
TLC), the reaction mixture was diluted in ethyl acetate (25 mL)
and washed with water (10 mL Â 3). The organic layer was col-
lected, dried over Na2SO4 and evaporated under reduced pressure
to afford crude products. Final compounds 5–6 were obtained in
75–95% yields after recrystallisation from ethanol or column puri-
fication in solvent system chloroform–methanol (9:1) using Silica
Gel 60 (0.063–0.200 mm).
Data for 5g. Yield: 75%; 1H NMR (300 MHz, CDCl3): d 1.46 (3H, t,
J = 7.2 Hz, CH3CH2O–), 2.35 (3H, s, –CH3 arom.), 4.16 (2H, t,
J = 5.7 Hz, –NCH2–), 4.29 (2H, t, J = 5.7 Hz, –CH2OH), 4.40 (2H, q,
J = 7.2 Hz, CH3CH2O–), 6.75 (1H, d, J = 8.4 Hz, H aromatic), 6.80
(1H, d, J = 8.4 Hz, H aromatic), 7.26 (1H, d, J = 8.4 Hz, H aromatic),
7.60 (1H, d, J = 8.4 Hz, H aromatic), 7.69 (1H, dd, J = 1.5 Hz,
8.4 Hz, H arom.), 7.77 (1H, d, J = 8.4 Hz, H aromatic), 7.89 (1H, s,
H arom.). 13C NMR (75 MHz, CDCl3): 14.48, 25.34, 47.59, 60.59,
60.70, 108.14, 109.77, 111.48, 120.91, 123.22, 125.05, 129.69,
131.50, 138.03, 138.24, 141.86, 157.39, 166.81. ESI-MS: m/z
325.2 [M+H]+. Anal. Calcd for C19H20N2O3: C, 70.35; H, 6.21; N,
8.64. Found: C, 70.13; H, 6.45; N, 8.46.
Data for 5a. Yield: 95%; 1H NMR (300 MHz, CDCl3): d 1.46 (3H, t,
J = 7.2 Hz, CH3CH2O–), 4.14 (2H, t, J = 5.7 Hz, –NCH2–), 4.29 (2H, t,
J = 5.7 Hz, –CH2OH), 4.42 (2H, q, J = 7.2 Hz, CH3CH2O–), 6.70–7.60
(6H, m, H arom.), 7.81 (1H, dd, J = 1.5 Hz, 8.4 Hz, H arom.), 7.93
(1H, s, H arom.). 13C NMR (75 MHz, CDCl3): 14.57, 47.31, 60.39,
60.50, 115.21, 116.79, 119.72, 120.45, 121.25, 121.75, 121.84,
124.30, 131.75, 137.13, 139.38, 152.79, 166.00. ESI-MS: m/z
311.2 [M+H]+. Anal. Calcd for C18H18N2O3: C, 69.66; H, 5.85; N,
9.03. Found: C, 69.56; H, 5.90; N, 9.02.
Data for 5b. Yield: 82%; 1H NMR (300 MHz, CDCl3): d 1.48 (3H, t,
J = 7.2 Hz, CH3CH2O–), 4.16 (2H, t, J = 5.7 Hz, –NCH2–), 4.30 (2H, t,
J = 5.7 Hz, –CH2OH), 4.46 (2H, q, J = 7.2 Hz, CH3CH2O–), 6.82 (1H,
d, J = 8.4 Hz, H aromatic), 6.94 (1H, d, J = 8.4 Hz, H aromatic), 7.35
(1H, d, J = 8.4 Hz, H aromatic), 7.60 (1H, d, J = 8.4 Hz, H aromatic),
7.73 (1H, dd, J = 1.5 Hz, 8.4 Hz, H arom.), 7.76 (1H, d, J = 8.4 Hz, H
aromatic), 7.97 (1H, s, H arom.). 13C NMR (75 MHz, CDCl3): 14.50,
47.57, 60.31, 60.67, 108.94, 111.85, 111.95, 122.65, 123.66,
126.19, 127.80, 128.10, 129.82, 130.56, 139.11, 142.03, 156.25,
166.54. ESI-MS: m/z 389.4 [M+H]+. Anal. Calcd for C18H17N2O3Br:
C, 55.54; H, 4.40; N, 7.20. Found: C, 55.59; H, 5.26; N, 7.27.
Data for 5c. Yield: 86%; 1H NMR (300 MHz, CDCl3): d 1.48 (3H, t,
J = 7.2 Hz, CH3CH2O–), 4.18 (2H, t, J = 5.7 Hz, –NCH2–), 4.30 (2H, t,
J = 5.7 Hz, –CH2OH), 4.47 (2H, q, J = 7.2 Hz, CH3CH2O–), 6.85 (1H,
d, J = 8.4 Hz, H aromatic), 6.95 (1H, d, J = 8.4 Hz, H aromatic), 7.36
(1H, d, J = 8.4 Hz, H aromatic), 7.69 (1H, d, J = 8.4 Hz, H aromatic),
7.75 (1H, dd, J = 1.5 Hz, 8.4 Hz, H arom.), 7.80 (1H, d, J = 8.4 Hz, H
aromatic), 8.01 (1H, s, H arom.). 13C NMR (75 MHz, CDCl3): 14.54,
47.60, 60.30, 60.72, 109.46, 111.90, 112.07, 122.65, 123.68,
126.19, 127.87, 128.10, 130.82, 133.02, 139.11, 142.03, 156.25,
166.57. ESI-MS: m/z 345.2 [M+H]+. Anal. Calcd for C18H17N2O3Cl:
C, 62.70; H, 4.97; N, 8.12. Found: C, 62.87; H, 5.14; N, 8.19.
Data for 5d. Yield: 89%; 1H NMR (300 MHz, CDCl3): d 1.47 (3H, t,
J = 7.2 Hz, CH3CH2O–), 4.17 (2H, t, J = 5.7 Hz, –NCH2–), 4.32 (2H, t,
Data for 5h. Yield: 77%; 1H NMR (300 MHz, CDCl3): d 1.46 (3H, t,
J = 7.2 Hz, CH3CH2O–), 4.15 (2H, t, J = 5.7 Hz, –NCH2–), 4.29 (2H, t,
J = 5.7 Hz, –CH2OH), 4.40 (2H, q, J = 7.2 Hz, CH3CH2O–), 6.73 (1H,
d, J = 8.4 Hz, H aromatic), 6.78 (1H, d, J = 8.4 Hz, H aromatic), 7.26
(1H, d, J = 8.4 Hz, H aromatic), 7.61 (1H, d, J = 8.4 Hz, H aromatic),
7.70 (1H, dd, J = 1.5 Hz, 8.4 Hz, H arom.), 7.74 (1H, d, J = 8.4 Hz, H
aromatic), 7.90 (1H, s, H arom.). 13C NMR (75 MHz, CDCl3): 14.47,
47.60, 60.61, 60.69, 109.25, 110.06, 111.48, 120.35, 123.78,
124.05, 125.69, 131.50, 132.03, 138.24, 141.86, 157.39, 159.85,
166.79. ESI-MS: m/z 327.2 [M+H]+. Anal. Calcd for C18H18N2O4: C,
66.25; H, 5.56; N, 8.58. Found: C, 66.19; H, 5.77; N, 8.50.
Data for 5i. Yield: 84%; 1H NMR (300 MHz, CDCl3): d 1.46 (3H, t,
J = 7.2 Hz, CH3CH2O–), 3.70 (3H, s, –OCH3), 4.14 (2H, t, J = 5.7 Hz, –
NCH2–), 4.28 (2H, t, J = 5.7 Hz, –CH2OH), 4.40 (2H, q, J = 7.2 Hz, CH3-
CH2O–), 6.74 (1H, d, J = 8.4 Hz, H aromatic), 6.78 (1H, d, J = 8.4 Hz, H
aromatic), 7.25 (1H, d, J = 8.4 Hz,
H aromatic), 7.61 (1H, d,
J = 8.4 Hz, H aromatic), 7.70 (1H, dd, J = 1.5 Hz, 8.4 Hz, H arom.),
7.76 (1H, d, J = 8.4 Hz, H aromatic), 7.92 (1H, s, H arom.). 13C
NMR (75 MHz, CDCl3): 14.47, 47.60, 56.90, 60.62, 60.71, 109.25,
110.19, 111.48, 120.46, 123.78, 124.05, 125.69, 131.50, 132.17,
138.24, 141.86, 157.39, 160.75, 166.80. ESI-MS: m/z 341.2
[M+H]+. Anal. Calcd for C19H20N2O4: C, 67.05; H, 5.92; N, 8.23.
Found: C, 67.18; H, 5.76; N, 8.33.

Y. K. Yoon et al. / Bioorg. Med. Chem. 22 (2014) 703–710
709
Data for 5j. Yield: 92%; 1H NMR (300 MHz, CDCl3): d 1.47 (3H, t,
J = 7.2 Hz, CH3CH2O–), 3.00 (6H, s, –N(CH3)2), 4.23 (2H, t, J = 5.7 Hz,
–NCH2–), 4.30 (2H, t, J = 5.7 Hz, –CH2OH), 4.40 (2H, q, J = 7.2 Hz,
CH3CH2O–), 6.56 (1H, d, J = 8.4 Hz, H aromatic), 6.70 (1H, d,
J = 8.4 Hz, H aromatic), 7.22 (1H, d, J = 8.4 Hz, H aromatic), 7.67
(1H, d, J = 8.4 Hz, H aromatic), 7.72 (1H, dd, J = 1.5 Hz, 8.4 Hz, H
arom.), 7.73 (1H, d, J = 8.4 Hz, H aromatic), 7.87 (1H, s, H arom.).
13C NMR (75 MHz, CDCl3): 14.46, 40.02, 47.19, 56.90, 60.62,
60.69, 109.22, 111.01, 111.54, 120.55, 123.42, 124.44, 125.19,
131.10, 131.98, 138.31, 141.39, 151.07, 156.45, 166.80. ESI-MS:
m/z 354.1 [M+H]+. Anal. Calcd for C20H23N3O3: C, 67.97; H, 6.56;
N, 11.89%. Found: C, 68.06; H, 6.55; N, 11.90.
J = 8.4 Hz, H aromatic), 7.32 (1H, d, J = 8.4 Hz, H aromatic), 7.40
(1H, dd, J = 1.5 Hz, 8.4 Hz, H arom.), 7.55 (1H, d, J = 8.4 Hz, H aro-
matic), 7.79 (1H, dd, J = 1.5 Hz, 8.4 Hz, H aromatic), 7.89 (1H, s, H
arom.). 13C NMR (75 MHz, CDCl3): 47.07, 60.77, 101.54, 108.14,
109.27, 110.44, 120.78, 122.37, 123.78, 124.85, 125.15, 138.75,
143.03, 147.32, 150.28, 155.39, 167.84. ESI-MS: m/z 327.2
[M+H]+. Anal. Calcd for C17H14N2O5: C, 62.57; H, 4.32; N, 8.59.
Found: C, 62.55; H, 4.28; N, 8.60.
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.
Data for 5k. Yield: 87%; 1H NMR (300 MHz, CDCl3): d 1.44 (3H, t,
J = 7.2 Hz, CH3CH2O–), 1.65 (2H, t, J = 5.7 Hz, H piperidine), 1.73
(4H, m, H piperidine), 3.32 (4H, t, J = 5.7 Hz, H piperidine), 4.04
(2H, t, J = 5.7 Hz, –NCH2–), 4.26 (2H, t, J = 5.7 Hz, –CH2OH), 4.40
(2H, q, J = 7.2 Hz, CH3CH2O–), 7.00 (1H, d, J = 8.4 Hz, H aromatic),
7.28 (1H, d, J = 8.4 Hz, H aromatic), 7.31 (1H, d, J = 8.4 Hz, H aro-
matic), 7.65 (1H, d, J = 8.4 Hz, H aromatic), 7.79 (1H, d, J = 8.4 Hz,
H aromatic), 7.85 (1H, dd, J = 1.5 Hz, 8.4 Hz, H arom.), 7.90 (1H, s,
H arom.). 13C NMR (75 MHz, CDCl3): 14.42, 24.31, 25.55, 47.55,
48.28, 49.04, 60.32, 60.86, 109.10, 114.81, 119.90, 121.59, 123.95,
124.31, 124.65, 131.41, 136.20, 142.00, 144.88, 152.89, 166.55.
ESI-MS: m/z 394.2 [M+H]+. Anal. Calcd for C23H27N3O3: C, 70.21;
H, 6.92; N, 10.68. Found: C, 70.12; H, 6.89; N, 10.78.
4.2.1. SIRT1 in vitro assay
3.3
66.7
tration) and 0.5
sion #: NM_012238) with 193–741 amino acids and GST tag at its
N-terminal, were incubated for 45 min at 37 °C. 50 L of stop solu-
l
M of SIRT1 substrate derived from human p53 sequences,
M NAD+, 50
M of interested compounds (all final concen-
g of SIRT1 human recombinant (GenBank Acces-
l
l
l
l
tion consisting nicotinamide and SIRT1 developer was then added
and the mixture was incubated for a further 10 min at 37 °C. Fluo-
rescence was measured at 490 nm (excitation) and 520 nm (emis-
sion) and the inhibition was calculated as the ratio of absorbance
under each experimental condition to that of the control.
Data for 5l. Yield: 90%; 1H NMR (300 MHz, CDCl3): d 1.47 (3H, t,
J = 7.2 Hz, CH3CH2O–), 4.27-4.33 (4H, m, –OCH2CH2OH), 4.42 (2H, q,
J = 7.2 Hz, CH3CH2O–), 6.02 (2H, s, –OCH2O–), 6.72 (1H, d, J = 8.4 Hz,
H aromatic), 7.19 (1H, d, J = 8.4 Hz, H aromatic), 7.32 (1H, dd,
J = 1.5 Hz, 8.4 Hz, H arom.), 7.40 (1H, d, J = 8.4 Hz, H aromatic),
7.70 (1H, dd, J = 1.5 Hz, 8.4 Hz, H aromatic), 7.74 (1H, s, H arom.).
13C NMR (75 MHz, CDCl3): 14.46, 47.07, 60.25, 60.77, 101.54,
108.14, 109.27, 110.44, 120.78, 122.37, 123.78, 124.85, 125.15,
137.62, 141.03, 147.48, 149.13, 155.37, 166.49. ESI-MS: m/z
355.2 [M+H]+. Anal. Calcd for C19H18N2O5: C, 64.40; H, 5.12; N,
7.91. Found: C, 64.43; H, 5.20; N, 7.89.
4.2.2. SIRT2 in vitro assay
6.7
333
M NAD+, 50
tion) and 0.5 g of SIRT2 human recombinant (GenBank Accession
#: NM_030593) with 13–319 amino acids and His tag at its C-ter-
minal, were incubated for 45 min at 37 °C. 50 L of stop solution
lM of SIRT2 substrate derived from human p53 sequences,
l
lM of interested compounds (all final concentra-
l
l
consisting nicotinamide and SIRT1 developer was then added and
the mixture was incubated for a further 10 min at 37 °C. Fluores-
cence 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.
4.1.6. General procedure for the preparation of 5-carboxylic
acid-2-substituted benzimidazole derivatives (6)
Compounds 6j–6l were synthesized as according to procedure
4.1.2 to 4.1.5 above. The esterification step of 4.1.1 was omitted.
Data for 6j. Yield: 86%; 1H NMR (300 MHz, CDCl3): d 3.00 (6H, s,
–N(CH3)2), 4.23 (2H, t, J = 5.7 Hz, –NCH2–), 4.39 (2H, t, J = 5.7 Hz, –
CH2OH), 6.56 (1H, d, J = 8.4 Hz, H aromatic), 6.92 (1H, d, J = 8.4 Hz,
H aromatic), 7.42 (1H, d, J = 8.4 Hz, H aromatic), 7.70 (1H, d,
J = 8.4 Hz, H aromatic), 7.81 (1H, dd, J = 1.5 Hz, 8.4 Hz, H arom.),
7.85 (1H, d, J = 8.4 Hz, H aromatic), 8.00 (1H, s, H arom.). 13C
NMR (75 MHz, CDCl3): 40.04, 47.19, 60.62, 60.69, 109.22, 111.01,
111.54, 120.55, 123.42, 124.44, 125.19, 132.10, 132.98, 138.87,
142.19, 151.32, 156.94, 167.47. ESI-MS: m/z 325.1 [M+H]+. Anal.
Calcd for C18H19N3O3: C, 66.45; H, 5.89; N, 12.91. Found: C,
66.30; H, 5.97; N, 12.99.
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 Â 103 per well. The cells were treated with
50 lM of interested compounds and allowed to adhere for 72 h.
Then, the proliferative activity was determined by 3-(4,5-dimeth-
ylthiazol-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 solu tion was added at
Data for 5k. Yield: 90%; 1H NMR (300 MHz, CDCl3): d 1.65 (2H, t,
J = 5.7 Hz, H piperidine), 1.73 (4H, m, H piperidine), 3.32 (4H, t,
J = 5.7 Hz, H piperidine), 4.04 (2H, t, J = 5.7 Hz, –NCH2–), 4.26 (2H,
t, J = 5.7 Hz, –CH2OH), 7.11 (1H, d, J = 8.4 Hz, H aromatic), 7.35
(1H, d, J = 8.4 Hz, H aromatic), 7.41 (1H, d, J = 8.4 Hz, H aromatic),
7.77 (1H, d, J = 8.4 Hz, H aromatic), 7.86 (1H, d, J = 8.4 Hz, H aro-
matic), 7.94 (1H, dd, J = 1.5 Hz, 8.4 Hz, H arom.), 8.03 (1H, s, H
arom.). 13C NMR (75 MHz, CDCl3): 24.33, 25.55, 47.55, 48.28,
49.04, 60.86, 109.10, 114.81, 119.90, 121.59, 123.95, 124.31,
124.65, 131.41, 136.54, 142.39, 145.98, 153.02, 167.98. ESI-MS:
m/z 366.2 [M+H]+. Anal. Calcd for C21H23N3O3: C, 69.02; H, 6.34;
N, 11.50. Found: C, 69.12; H, 6.55; N, 11.38.
20 lL/well, and after 1 h 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-tetrazo-
lium, inner salt to formazan was measured in a plate reader at
490 nm. All experiments were done in triplicate, and the prolifera-
tion rate was calculated as the ratio of absorbance under each
experimental condition to that of the control nontransfectant.
4.2.4. Autofluorescence
Compounds in DMSO at 50 lM concentration (100 lL) 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% fluores-
Data for 5l. Yield: 91%; 1H NMR (300 MHz, CDCl3): d 4.27–4.33
(4H, m, –OCH2CH2OH), 6.02 (2H, s, –OCH2O–), 6.88 (1H, d,

710
Y. K. Yoon et al. / Bioorg. Med. Chem. 22 (2014) 703–710
12. Fraga, M. F.; Agrelo, R.; Esteller, M. Ann. N.Y. Acad. Sci. 2007, 1100, 60.
13. Saunders, L. R.; Verdin, E. Oncogene 2007, 26, 5489.
14. Zhao, W.; Kruse, J. P.; Tang, Y.; Jung, S. Y.; Qin, J.; Gu, W. Nature 2008, 451,
587.
cence of the control wells. None of the analyzed compounds were
found to be autofluorescent.
15. Outeiro, T. F.; Kontopoulos, E.; Altmann, S. M.; Kufareva, I.; Strathearn, K. E.;
Amore, A. M.; Volk, C. B.; Maxwell, M. M.; Rochet, J. C.; McLean, P. J.; Young, A.
B.; Abagyan, R.; Feany, M. B.; Hyman, B. T.; Kazantsev, A. G. Science 2007, 317,
516.
16. Bitterman, K. J.; Anderson, R. M.; Cohen, H. Y.; Latorre-Esteves, M.; Sinclair, D.
A. J. Biol. Chem. 2002, 277, 45099.
5. Conflict of interest
The authors hereby declare there is no conflict of interests.
Acknowledgments
17. Grozinger, C. M.; Chao, E. D.; Blackwell, H. E.; Moazed, D.; Schreiber, S. L. J. Biol.
Chem. 2001, 276, 38837.
18. Mai, A.; Massa, S.; Lavu, S.; Pezzi, R.; Simeoni, S.; Ragno, R.; Mariotti, F. R.;
Chiani, F.; Camilloni, G.; Sinclair, D. A. J. Med. Chem. 2005, 48, 7789.
19. Posakony, J.; Hirao, M.; Stevens, S.; Simon, J. A.; Bedalov, A. J. Med. Chem. 2004,
47, 2635.
20. Lain, S.; Hollick, J. J.; Campbell, J.; Staples, O. D.; Higgins, M.; Aoubala, M.;
McCarthy, A.; Appleyard, V.; Murray, K. E.; Baker, L.; Thompson, A.; Mathers, J.;
Holland, S. J.; Stark, M. J. R.; Pass, G.; Woods, J.; Lane, D. P.; Westwood, N. J.
Cancer Cell 2008, 13, 454.
The authors wish to express their gratitude and appreciation to
Pharmacogenetics and Novel Therapeutics Research Cluster, Insti-
tute for Research in Molecular Medicine, Universiti Sains Malaysia,
Penang for supporting this work. This work was funded through
Research Grant No. RUC (1001/PSK/8620012) and HiCoE research
Grant No. (311.CIPPM.4401005).
21. Napper, A. D.; Hixon, J.; McDonagh, T.; Keavey, K.; Pons, J. F.; Barker, J.; Yau, W.
T.; Amouzegh, P.; Flegg, A.; Hamelin, E.; Thomas, R. J.; Kates, M.; Jones, S.;
Navia, M. A.; Saunders, J. O.; DiStefano, P. S.; Curtis, R. J. Med. Chem. 2005, 48,
8045.
Supplementary data
22. Nunes, J. J.; Milne, J.; Bemis, J.; Xie, R.; Vu, C. B.; Ng, P. Y.; Disch, J. S. WIPO
Patent 2007, WO/2207/019416.
23. Özden, S.; Atabey, D.; Yildiz, S.; Göker, H. Bioorg. Med. Chem. 2005, 13, 1587.
24. Yeong, K. Y.; Ali, M. A.; Ang, C. W.; Tan, S. C.; Khaw, K. Y.; Murugaiyah, V.;
Osman, H.; Masand, V. H. Bioorg. Chem. 2013, 49, 33.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.12.029.
References and notes
25. Gomez, H. T.; Nunez, E. H.; Rivera, I. L.; Alvarez, J. G.; Cedillo-Rivera, R.; Puc, R.
M.; Ramos, R. A.; Rodriguez-Gutierrez, M. C.; Chan-Bacab, M. J.; Navarrete-
Vazquez, G. Bioorg. Med. Chem. Lett. 2008, 18, 3147.
1. Blander, G.; Guarente, L. Annu. Rev. Biochem. 2004, 73, 417.
2. Michan, S.; Sinclair, D. Biochem. J. 2007, 404, 1.
26. Moniot, S.; Schutkowski, M.; Steegborn, C. J. Struct. Biol. 2013, 182, 136.
27. Sakkiah, S.; Arooj, M.; Kumar, M. R.; Eom, S. H.; Lee, K. W. PLoS ONE 2013, 8,
e51429.
28. Lai, C. J.; Wu, J. C. Assay Drug Dev. Technol. 2003, 1, 527.
29. Heltweg, B.; Gatbonton, T.; Schuler, A. D.; Posakony, J.; Li, H.; Goehle, S.;
Kollipara, R.; Depinho, R. A.; Gu, Y.; Simon, J. A.; Bedalov, A. Cancer Res. 2006,
66, 4368.
30. Lin, H. Sirtuin Inhibitors as Anticancer Agents, 7th Drug Design and Medicinal
Chemistry, Boston, MA, 2013.
31. Peck, B.; Chen, C. Y.; Ho, K. K.; Di Fruscia, P.; Myatt, S. S.; Coombes, R. C.;
Fuchter, M. J.; Hsiao, C. D.; Eric Lam, W. F. Mol. Cancer Ther. 2010, 9, 844.
3. Denu, J. M. Curr. Opin. Chem. Biol. 2005, 9, 431.
4. Haigis, M. C.; Guarente, L. P. Genes Dev. 2011, 20, 2913.
5. Sebastian, C.; Satterstrom, F. K.; Haigis, M. C.; Mostoslavsky, R. J. Biol. Chem.
2012, 287, 42444.
6. Fyre, R. A. Biochem. Biophys. Res. Commun. 2000, 273, 793.
7. Milner, J. Curr. Pharm. Des. 2009, 15, 39.
8. Inoue, T.; Hiratsuka, M.; Osaki, M.; Oshimura, M. Cell Cycle 2007, 6, 1011.
9. Bosch-Presegue, L.; Vaquero, A. Genes Cancer 2011, 2, 648.
10. Garske, A. L.; Smith, B. C.; Denu, J. M. ACS Chem. Biol. 2007, 2, 529.
11. Chen, J.; Zhou, Y.; Mueller-Steiner, S.; Chen, L. F.; Kwon, H.; Yi, S.; Mucke, L.;
Gan, L. J. Biol. Chem. 2005, 280, 40364.