Potent sirtuin inhibition with 1,2,5-trisubstituted benzimidazoles

Article abstract of DOI:10.1039/c6md00378h

Two series of compounds were synthesized based on the benzimidazole scaffold. The compounds were subsequently screened for their SIRT1, SIRT2 and SIRT3 activities. Three of the compounds showed good inhibitory activity against SIRT2 in this study with the most potent compound (5i) having an IC₅₀ value of 2.9 μM. Molecular docking analysis demonstrated that 5i was able to inhibit SIRT2 by displacing the co-factor NAD⁺ in the active site. This was further confirmed experimentally by ligand-NAD⁺ competitive assay.

Full text of DOI:10.1039/c6md00378h

View Article Online
View Journal
MedChemComm
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: K. Y. Yeong, H.
Osman and S. C. Tan, Med. Chem. Commun., 2016, DOI: 10.1039/C6MD00378H.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/medchemcomm

Please do not adjust margins
MedChemComm
Page 1 of 8
View Article Online
DOI: 10.1039/C6MD00378H
Journal Name
COMMUNICATION
†
Potent sirtuin inhibition with 1,2,5-trisubstituted benzimidazoles
Received 00th January 20xx,
Accepted 00th January 20xx
a*
b
a
DOI: 10.1039/x0xx00000x
Y. K. Yoon , H. Osman , T. S. Choon
www.rsc.org/
12,13
14
15
Two series of compounds were synthesized based on the disorders such as cancer
,
Parkinson’s , Alzheimer
and
benzimidazole scaffold. The compounds were subsequently cardiovascular diseases. Owing to their broad involvement in
screened for their SIRT1, SIRT2 and SIRT3 activities. Three of various diseases, sirtuins were actively researched.To date, several
the compounds showed good inhibitory activity against SIRT2 classes of sirtuin inhibitors have been identified such as the
1
6
in this study with the most potent compound (5i) having an nicotinamide,
hydroxynaphthyldehyde derivatives such as
1
7,18
19
IC50 value of 2.9 µM. Molecular docking analysis performed salermide and cambinol,
2
demonstrated that 5i was able to inhibit SIRT2 by displacing splitomicin analogs and tenovins.
pyrimidine-based inhibitors,
21
0
+
the co-factor NAD in the active site. This was further
In our previous studies, we have shown that the reported scaffold
Figure 1) gave varying degree of inhibitory activity with changes in
+
confirmed experimentally by ligand-NAD competitive assay.
(
1
2
the R and R positions. In most cases, strong electron donating
2
basic terminal at the R position would enhance the sirtuin
2
2,23
inhibitory activity
. It was also revealed that sirtuin inhibitors
based on the benzimidazole scaffold showed that they mostly
occupied the adenosine diphosphate (A pocket) and ribose (B
Introduction
Benzimidazole is a heterocycle which contained a phenyl ring fused pocket) binding sites.
to an imidazole ring. The benzimidazole core structure is an
In view of this, longer chains (> 6 atoms length) were applied at the
1
interesting platform for drug discovery as they were shown to
1
R position in the effort to increase the inhibitory potential of the
possess a wide spectrum of pharmacological activities such as
compounds as this may allow the side chain to occupy the
nicotinamide pocket (C pocket) of the active binding site. In the
present study, the synthesis and sirtuin inhibitory activities of
2
3,4
5
antiviral , anti-inflammatory and anticancer properties. More
applications for benzimidazoles or benzimidazole-containing
compounds have recently been unraveled. Among the new
discoveries was the potential use of benzimidazole analogues in
inhibiting sirtuin enzymatic activity as recently reported by our
1
compounds with long chains at the R position were reported.
Molecular docking experiments were also carried out to provide
insights into the binding mode of this series of benzimidazoles.
6
group .
24
Compounds 5a-g were previously synthesized and evaluated for
Sirtuins are NAD-positive-dependent class III HDACs that share their antimycobacterium activity following established protocol
7
extensive homologies with the yeast HDAC Sir2 . Seven human (Figure 2). However, since the compounds have a long and flexible
8
1
sirtuins have been identified (SIRT1-7) to date . Collectively, they chain substitution at R which may be suitable for inhibiting sirtuins,
9
10
controlled many downstream targets such as NF-ĸB , p53 and the compounds were included in this study.
FOXO proteins . Sirtuins have recently been linked to age-related
1
1
O
N
N
R1
O
R2
Figure 1. Benzimidazole-based core structure for sirtuin inhibition
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 2 of 8
View Article Online
DOI: 10.1039/C6MD00378H
COMMUNICATION
Journal Name
overnight.The precipitate formed was filtered and dried to afford
sodium bisulfite adducts (55%-90%).
Experimental
Chemistry
General procedure for the preparation of 2-substituted
benzimidazole derivatives (5a-j and 8a-j)
All chemicals were supplied by Sigma-Aldrich (U.S.A.), Merck
Chemicals (Germany) and Acros Organics (U.S.A.). Elemental
analyses were measured on Perkin Elmer 2400 Series II CHN
Elemental Analyzer and were within ± 0.4% of the calculated values.
Ethyl-3-amino-4-(2-substituted amino)benzoate (1 mmol) and
various sodium bisulfite adducts (1.5 mmol) were dissolved in DMF
o
1
13
1
(5 mL). The reaction mixture was stirred at 90 C under N
2
H and C NMR were performed on Bruker Avance 500 ( H: 500
13
atmosphere for 24-48 hours. After completion of reaction, the
reaction mixture was diluted in ethyl acetate (25 mL) and washed
with water (10 mL x 3). The organic layer was collected, dried over
Na SO and evaporated under reduced pressure to afford the
3
MHz, C: 125 MHz) spectrometer in CDCl . Mass spectra were
recorded on Varian 320-MS TQ LC/MS in positive ESI mode.
2
4
desired final compounds.
Procedure for the preparation of Ethyl-4-fluoro-3-nitrobenzoate
4-Fluoro-3-nitrobenzoic acid (5 g, 27 mmol) was refluxed in ethanol Sirtuin in vitro enzymatic fluorescence assay
(
50 mL) and concentrated H SO (2 mL) for 8 hours. After
2
4
completion of reaction, the solvent was evaporated under reduced Sirtuin substrates were derived from human p53 sequences (SIRT1:
pressure. The aqueous layer was extracted with ethyl acetate (25 amino acids 379-382 conjugated to 7-dimethylamino-4-
mL x 3). The organic layer was dried over Na
under reduced pressure to yield 1 as cream-coloured powder (75%). dimethylamino-4-methylcoumarin, AMC; SIRT3: amino acids 317-
20 conjugated to 7-dimethylamino-4-methylcoumarin, AMC).
2 4
SO and concentrated methylcoumarin, AMC; SIRT2: amino acids 317-320 conjugated to 7-
3
Sirtuin substrate (125 µM), NAD+ (3 mM), test compounds (50 µM)
General procedure for the preparation of 4-(2-substituted amino)-
-nitro-ethylbenzoate
and sirtuin human recombinant were incubated for 45 minutes at
o
3
3
7 C. 50 µL of stop solution consisting nicotinamide and sirtuin
developer was then added and the mixture was incubated for a
o
further 30 minutes at 37 C. Fluorescence was measured at 355 nm
Ethyl-4-fluoro-3-nitrobenzoate, 1 (0.5 g, 2.34 mmol), primary
amines [Series 5: N-(2-aminoethyl)piperazine, 0.15 mL, 1.16 mmol;
(
excitation) and 460 nm (emission) and the inhibition was
calculated as the ratio of absorbance under each experimental
condition to that of the control.
Series 8: octylamine, 0.38 mL, 2.30 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, the
reaction mixture was washed with water (10 mL x 2) followed by
Molecular docking
1
0% Na
2
CO
4
and concentrated under reduced pressure to afford 2 as The crystal structure of human SIRT1 (PDB code: 4IGI), SIRT2 (PDB
3
solution (10 mL). The organic layer was dried over
Na SO
2
brown oil (91%) or 6 as brown solid (86%).
code: 3ZGV; 4RMG; 5FYQ) and SIRT3 (PDB code: 4JSR) were taken
from the Protein Data Bank. The enzymes and ligands were
structurally optimized prior to the actual docking simulation. After
removing the co-crystallized water molecules, hydrogen atoms
were added to the protein structure. Ligands were energy
minimized with Chem 3D Pro 13.0 using the MM2 forcefield.
Docking was carried out using Autodock 4.2. For each molecule, 10
docking runs were performed. The top-ranked pose for each ligand
were retained and further analyzed with VMD 1.9.1 molecular
graphics software.
General procedure for the preparation of Ethyl-3-amino-4-(2-
substituted amino)benzoate
4-(2-substituted
amino)-3-nitro-ethylbenzoate
(1
mmol),
ammonium formate (0.378 g, 6 mmol) and Pd/C (50 mg) were
mixed in ethanol (10 mL). The reaction mixture was refluxed 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
2 4
with water, dried over Na SO and evaporated to dryness to yield
ethyl-3-amino-4-(2-substituted amino)benzoate3 (70%) or 7 (79%).
Results and discussion
Synthesis of compounds
General procedure for the preparation of sodium bisulfite addcuts
of 4-substituted benzaldehyde
The benzimidazole derivatives were synthesized according to the
synthetic scheme shown in Figure 2. Briefly, the synthetic scheme
Appropriate benzaldehyde (10 mmol) was dissolved in ethanol (20
mL). Sodium metabisulfite (15 mmol) in 5 mL water was added in
follows
a four-step pathway. The first step involves the
esterification of the starting material 4-fluoro-3-nitro benzoic acid.
The second step involves amination while reduction of the nitro
portion over 5 minutes. The reaction mixture was stirred at room
o
temperature for
1
hour and subsequently stirred at 4 C
2
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 3 of 8
Pleas Me de od Cn oh te amd Cj u os tm mma rgins
View Article Online
DOI: 10.1039/C6MD00378H
Journal NameCOMMUNICATION
group to the amino follows in the third step. Finally, the last step Experiments were performed in triplicates. Generally, the
involves cyclization and formation of the benzimidazole core. The synthesized benzimidazole derivatives showed better inhibition
sodium bisulfite adducts were attached with different substitution against SIRT2 compared to SIRT1 or SIRT3 as shown in Table 1. It
1
at the R position to yield the desired final compounds.
was also revealed that compounds from Series 5 showed better
sirtuin inhibitory activity than compounds from Series 8.
As compounds from Series 5 were shown to have better sirtuin
COOCH
2
CH
3
inhibitory activities, three novel derivatives with different
2
COOH
COOCH
2
CH
3
substituent at
R
position (5h: 2-methoxyphenol, 5i: N,N-
(i)
(ii)
NO
2
dimethylbenzeneamine, 5j: phenylpiperidine) were synthesized
following similar procedure and were subsequently evaluated for
their in vitro SIRT1, 2, and 3 inhibition.Structure of the novel
compounds were determined by NMR, elemental as well as MS
analysis. The dimethylamino and piperidine functional groups were
chosen mainly based on our previous results which showed potent
NH
NO
2
NO
2
F
F
N
N
1
(
iii)
H
3 2
CH COOC
NO
2
COOCH
2 3
CH
2
(iv)
2
NO
2
sirtuin inhibitory activity when the R position was substituted with
NH
7
2
2,23
amino functionalized groups
. The fact that 5c and 5f (which
CH
3
2 3
COOCH CH
6
were doubly substituted on the phenyl ring) were found to be the
two most potent inhibitors from this series also prompted us to
synthesize and evaluate compound 5h. Apart from that, analogous
compounds 8h-j were also synthesized and evaluated for
comparison purposes. The sirtuin inhibitory results of the six novel
compounds were tabulated in Table 2.
(
iv)
NH
2
NH
COOCH
2
CH
3
N
N
NH
2
NH
7
H
3
CH
2
COOC
NH
2
3
CH
3
7
Compound 5i was found to be the most potent inhibitor of the
sodium
CHO
R2
sodium
HO
SO
3
Na
HO
3
SO Na
metabisulfite
metabisulfite
series (SIRT1 IC50 = 10.22 µM; SIRT2 IC50 = 2.92 µM; SIRT3 IC50
=
(v)
(v)
ethanol
ethanol
1
0.02 µM). It is gratifying to note that 5i is more inhibitory than
AGK2, one of the most potent SIRT2 inhibitors commercially
available. In fact, 5i is the most potent sirtuin inhibitor derived from
the benzimidazole scaffold reported thus far. It was found to be a
pan-SIRT1/2/3 inhibitor, albeit with a preference to inhibit SIRT2. Its
selectivity for SIRT2 is more than 3-fold over both SIRT1 and SIRT3.
R2
R2
4a-j
4
a-j
2 3
COOCH CH
2 3
COOCH CH
N
N
N
N
7
CH
3
N
N
NH
R2
R2
H
3 2
CH COOC
2
8a-j
5a-j
Figure 2. Synthesis protocol of compounds 5a-j and 8a-j. (i) ethanol/H
2
4
SO ,
reflux, 8 h; (ii) N-(2-aminoethyl)piperazine, DIPEA, dichloromethane, room
temperature, 18 h; (iii) octylamine, DIPEA, dichloromethane, room
o
temperature, 18 h (iv) ammonium formate, Pd/C, ethanol, 60 C, 1h; (v)
o
DMF, 90 C, 24-48 h.
Enzymatic Assays
The synthesized compounds were evaluated for their in vitro SIRT1,
2
and 3 inhibitory activities using commercially available fluorescent
assay kits (Cayman Chemicals, Ann Arbor, MI). EX-527 (SIRT1
selective inhibitor), AGK-2 (SIRT2 selective inhibitor) and Tenovin-6
(pan-SIRT1,2, and 3 inhibitor), were used as standard control while
DMSO was used as a vehicle control. IC50 values were determined
for all compounds which showed over 50% inhibition at 50 µM.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 4 of 8
View Article Online
DOI: 10.1039/C6MD00378H
COMMUNICATION
Journal Name
Table 1. SIRT1, SIRT2 and SIRT3 inhibitory activities of synthesized
compounds.
2
R
SIRT1 IC50 (µM) or % of
inhibition at 50 µM
31.09 ± 4.23 %
23.89 ± 4.16 %
28.19 ± 4.64 %
24.34 ± 5.61 %
19.96 µM
SIRT2 IC50 (µM) or % of
inhibition at 50 µM
26.77 ± 5.18 %
30.02 ± 2.22 %
43.30 ± 5.52 %
38.34 ± 4.50 %
7.87 µM
SIRT3 IC50 (µM) or % of
inhibition at 50 µM
25.15 ± 3.03 %
20.39 ± 2.87 %
37.96 ± 7.38 %
24.88 ± 2.29 %
23.26 µM
5
8
5
8
5
8
a
a
b
b
c
Cl
HO
c
29.03 ± 3.63 %
45.94 ± 1.26 %
31.54 ± 1.82 %
OH
5
8
5
8
5
8
d
d
e
e
f
24.56 ± 3.09 %
25.02 ± 1.94 %
28.38 ± 5.40 %
15.30 ± 2.77 %
29.80 µM
37.16 ± 2.86 %
35.59 ± 2.04 %
42.98 ± 6.03 %
30.33 ± 2.10 %
16.12 µM
20.59 ± 3.28 %
16.85 ± 1.03 %
30.71 ± 2.46 %
19.44 ± 3.29 %
27.03 µM
OCF
3
CH
O
3
O
f
>50 µM
41.40 µM
>50 µM
F
5
8
g
g
20.60 ± 6.99 %
22.01 ± 2.30 %
20.92 ± 7.89 %
13.96 ± 2.94 %
18.44 ± 4.65 %
24.40 ± 3.05 %
N
EX-527
AGK2
Tenovin-6
-
-
-
0.30 µM
N.D.
42.10 µM
N.D.
8.34 µM
25.60 µM
N.D.
N.D.
82.65 µM
Table 2. SIRT1, SIRT2 and SIRT3 inhibitory activities of 5h-j and 8h-j.
Molecular docking
SIRT1 IC50
µM)
SIRT2 IC50
SIRT3 IC50
2
R
(
(µM)
(µM)
Based on the previous results, the reported scaffold with shorter
OCH
3
35.07 ± 2.42 µM
50 µM
8.61 ± 1.29 µM
46.95 ± 3.05 µM
27.91 ± 2.21 µM
>50 µM
5
8
h
h
1
substitution at the R position were not able to effectively occupy
>
OH
N
the nicotinamide binding site (C pocket). Instead, the shorter
substituents actually distorted the confirmation of the
benzimidazole base structure when docked into the active site
which resulted in weaker inhibitory activity (Supplementary data).
5
8
5
8
i
i
j
j
10.22 ± 0.98 µM
50 µM
36.97 ± 1.82 µM
50 µM
2.92 ± 0.33 µM
38.10 ± 3.10 µM
10.02 ± 1.11 µM
>50 µM
>
13.48 ± 1.75 µM
36.76 ± 2.90 µM
29.24 ± 2.48 µM
>50 µM
N
>
Therefore, in an attempt to rationalize the observed in vitro
enzymatic activities, docking study of compound the most potent
compound of the series (5i) into the active site of human SIRT2 was
2
5
performed (PDB entry code: 3ZGV) . The receptor and the drug
candidates were optimized before actual docking in Autodock 4.2.
Analysis of the top-ranked pose of compound 5i demonstrated
several plausible molecular interactions between the ligand and the
enzyme.
Compound 5i was found to occupy the location of the co-factor
+
NAD in the active site. The docking analysis reveals that 5i interacts
with residue Arg97 and Pro94 through hydrogen bonding. Hydrogen
bond was also observed between the ester group from the
benzimidazole structure with Ser263 and Ser98. In general, the
planar benzimidazole scaffold occupied the adenosine binding site
(
A pocket) and ribose binding site (B pocket) of the active site while
the N,N-dimethylbenzeneamine substituent was located at the
gauge of the acetyl lysine channel. The importance of the N,N-
dimethylbenzeneamine substituent was further highlighted through
hydrogen bonding with Phe235. Lone pair oxygen-π interactions
4
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 5 of 8
Pleas Me de od Cn oh te amd Cj u os tm mma rgins
View Article Online
DOI: 10.1039/C6MD00378H
Journal NameCOMMUNICATION
could also be observed between the nitrogen atom from the N,N
-
scaffold was located at the opening of the acetyl lysine channel
dimethylbenzeneamine group with benzene ring from Phe235. formed by Phe414, His363 and Phe297. Hydrogen bonds were
Moreover, the side chain of ethylpiperizinyl benzoate was located formed between the amino group of 5i with Ile347 and Asp348 in
deep inside the nicotinamide binding site (C pocket) of the active the C pocket.
site which was deemed important for controlling the activity of the
enzyme (Figure 3). This was not seen In comparison, compound 8i
1
which has a linear octyl chain at the R position was found to have a
different conformation when docked inside the active site of SIRT2
compared to compound 5i (See supplementary data). In the
process, it lost important interactions with some residues such as
Arg97, making it a less potent inhibitor compared with compound
5
i.
Figure 4. Compound 5i docked in human SIRT1. (PDB entry code: 4I5I)
As for SIRT3, 5i has strong interactions with residues primarily in the
acetyl lysine channel and the C pocket of the active site (Figure 5).
The conformation was supported by the strong hydrogen bonds
formed with Asp231 and Gly295. Weaker interactions were also
observed with Phe157, Leu298 and Glu296. Residue His248 which is
3
0,31
critical for deacetylation activity of the enzyme
bonded to the NH group of the ligand.
was also weakly
Figure 3. Compound 5i docked in human SIRT2. (PDB entry code: 3ZGV)
2
When compared against other available SIRT2 structures [SIRT2-
SirReal2 (extended C-pocket) PDB: 4RMG; SIRT2-Ran TFAcK37–
1
3-mer peptide (lysine channel) PDB: 5FYQ], it was found that 5i
adopt different conformations. In the extended C-pocket (4RMG), 5i
was highly distorted and did not have strong contact with Tyr139
26
and Phe190 as reported by Jung et al . Therefore, the exceptional
SIRT2 selectivity shown by the SirReal2 ligand was not observed
2
7
here. On the other hand, using the crystal structure of 5FYQ , it
was found that only part of 5i was docked in the lysine channel,
with the benzimidazole moiety located out from the active site. This
is plausible as 5i is not a peptidomimetic-based inhibitor. Weaker
binding of 5i in both sites were indicated from their higher overall
free-binding energy, further supporting the docking of 5i in the
+
NAD binding site (Supplementary data).
Figure 5. Compound 5i docked in human SIRT3. (PDB entry code: 4JSR)
To test the hypothesis that the binding mode of 5i towards SIRT2
Besides SIRT2, 5i was also docked into the active site of human
2
8
29
SIRT1 (PDB entry code: 4I5I) and SIRT3 (PDB entry code: 4JSR) to
determine its interaction with both receptors. The mode of
interaction with SIRT1 was unlike those observed for SIRT2 as 5i
was oriented differently in the active site of SIRT1. Docking
simulation indicated that the N,N-dimethylbenzeneamine group
was inverted “top-down” and now occupy part of the A pocket
+
was by displacing the co-factor, a ligand-NAD competitive assay
3
2
was carried out following previously reported method . The
+
inhibition of 5i was tested with increasing concentration of NAD
(
Figure 4) while the ester group attached to the benzimidazole
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 6 of 8
View Article Online
DOI: 10.1039/C6MD00378H
COMMUNICATION
Journal Name
while the other parameters of the assay were kept constant (Figure was funded through RUC Research Grant (1001/PSK/8620012)
and FRGS Research Grant (203/PKIMIA/6711462).
6
).
Notes and references
1
.
Taniuos F.A., Hamelberg D., Bailly C., Czarny A., Boykin
D.W., Wilson W.D. J. Am. Chem. Soc., 2004, 126, 143-153 .
Tonelli M., Simone M., Tasso B., Novelli F., Boido V.,
Sparatore F., Paglietti G., Pricl S., Giliberti G., Blois S., Ibba
C., Sanna G., Loddo R., La Colla P. Bioorg. Med. Chem.,
2
.
2
010, 18, 2937–2953.
Lazer E.S., Matteo M.R., Possanza G.J. J. Med. Chem
987, 30, 726-729.
Chen G., Liu Z., Zhang Y., Shan X., Jiang L., Zhao Y., He W.,
3
4
.
.
,
1
Feng Z., Yang S., Liang G. ACS Med. Chem. Lett.,2013, 4,
Figure 5. Decreasing SIRT2 inhibition by 5i with increasing concentrations of
69-74.
+
NAD
5. Easmon J., Puerstinger G., Roth T., Fiebig H.H., Jenny M.,
Jaeger W., Heinisch G., Hofmann J. Int. J. Cancer, 2001, 94
9–96.
Yoon, Y. K.; Ali, M. A.; Wei, A. C.; Choon, T. S.; Osman,
H.; Parang, K.; Shirazi, A. N. Bioorg. Med. Chem.,2014
2, 703-710.
7. Blander, G.; Guarente, L. Annu. Rev. Biochem.2004, 73,
17-435
Fyre R.A. Biochem. Biophys. Res. Commun.,2000, 273,
93-798.
,
8
6
.
Competition analysis revealed that compound 5i is competitive with
,
+
respect to NAD which implies that the inhibitor competes with
2
+
NAD to occupy the same binding site in the receptor. This is in
4
agreement with our molecular docking prediction. The competition
analysis of 5i with the peptide substrate revealed that the
percentage of inhibition plateau at approximately 65% even with
increments of peptide substrate concentration, thus indicating the
inability of the peptide substrate to further dislodge 5i from binding
to the enzyme’s active site (Supplementary data). Altough this is not
a full kinetic study, the preliminary data shown here supported the
molecular docking prediction.
8
9
.
.
7
Yeung, F.; Hoberg, J.E.; Ramsey, C.S.; Keller, M.D.;
Jones, D.R.; Frye, R.A.; Mayo. M.W. EMBO J., 2004, 23,
2
369-2380
0. Luo, J.; Su, F.; Chen, D.; Shiloh, A.; Gu, W. Nature, 2000
08, 377-281
1. Berdichevsky, A; Guarente, L. Cell Cycle, 2006, 5, 2588-
2591.
2. Saunders, L.R.; Verdin, E. Oncogene, 2007, 26, 5489-
1
1
1
1
1
,
4
5
504
3. Bosch-Presegue, L.; Vaquero, A. Genes Cancer, 2011, 2,
48-662
4. Chen, J.; Zhou, Y.; Mueller-Steiner, S.; Chen, L. F.;
Kwon, H.; Yi, S.; Mucke, L.; Gan, L. J. Biol. Chem., 2005
80, 40364-40374
Conclusions
6
,
In conclusion, we have discovered several novel benzimidazole
derivatives which showed potent sirtuin inhibition activity,
with preference towards SIRT2. The most potent compound
found in this study, 5i, showed better SIRT2 inhibitory activity
compared to the standard controls used. It is also the most
potent sirtuin inhibitor from the 1,2,5-trisubstituted
benzimidazole scaffold reported to date. Molecular docking
analysis performed demonstrated that 5i was able to inhibit
2
15. Garske, A. L.; Smith, B. C.; Denu, J. M. ACS Chem. Biol.,
2007, 2, 529-532
16. Bitterman, K.J.; Anderson, R.M.; Cohen, H.Y.; Latorre-
Esteves, M.; Sinclair, D.A. J. Biol. Chem., 2002, 277,
4
5099-45107.
1
7. Lara, E.; Mai, A.; Calvanese, V.; Altucci, L.; Lopez-Nieva,
P.; Martinez-Chantar, M.L.; Varela-Rey, M.; Rotili, D.;
Nebbioso, A.; Ropero, S.; Montoya, G.; Oyarzabal, J.;
Velasco, S.; Serrano, M.; Witt, M.; Villar-Garea, A.;
Inhof, A.; Mato, J.M.; Esteller, M.; Fraga, M.F.
Oncogene, 2009, 28, 781-791.
+
SIRT2 by occupy the position of co-factor NAD in the active
site. This was further confirmed experimentally by ligand-NAD
+
competitive assay. The importance of a long and flexible chain
at R position was highlighted with the increment of inhibitory
1
1
1
8. 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-
potency as the compounds were able to occupy part of the C
pocket in the active site.
4
377.
9. Disch, J.S.; Evindar, G.; Chiu, C.H.; Blum, C.A.; Dai, H.;
Jin, L.;Schuman, E.; Lind, K.E.; Belyanaskaya, S.L.; Deng,
J.; Coppo, F.; Aquilani, L.; Grabill, T.L.; Cuozzo, J.W.;
Lavu, S.; Mao, C.; Vlasuk, G.P.; Perni, R.B. J. Med.
Chem., 2013, 56, 3666-3679.
Acknowledgements
The authors also wish to thank Institute for Research in
Molecular Medicine and School of Chemical Sciences,
Universiti Sains Malaysia, for supporting this work. This work
2
0. Posakony, J.; Hirao, M.; Stevens, S.; Simon, J.A.;
Bedalov, A. J. Med. Chem., 2004, 47, 2635-2644.
6
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 7 of 8
Pleas Me de od Cn oh te amd Cj u os tm mma rgins
View Article Online
DOI: 10.1039/C6MD00378H
Journal NameCOMMUNICATION
2
1. 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, 454-463.
2
2
2
2. Yoon Y.K.,Ali M.A., Wei A.C., Choon T.S., Shirazi A.N.,
Parang K. Med.Chem.Commun., 2015,6, 1857-1863.
3. Yoon, Y. K.; Ali, M. A.; Wei, A. C.; Shirazi, A. N., Parang,
K.; Choon, T.S. Eur. J. Med. Chem., 2014, 83, 448-454.
4. Yoon Y.K., Ali M.A., Choon T.S., Ismail R., Wei A.C., Kumar
R.S., Osman H., Beevi F. BioMed Res. Int., 2013, 926309.
5. Moniot, S.; Schutkowski, M.; Steegborn, C. J. Struct.
Biol., 2013, 182, 136-143
6. Rumpf T., Schiedel M., Karaman B., Roessler C., North
B., Lehotzky A., Oláh J., Ladwein K., Schmidtkunz K.,
Gajer M., Pannek M., Steegborn C., Sinclair D.,
Gerhardt S., Ovádi J., Schutkowski M., Sippl W., Einsle
O.Jung M. Nat. Commun., 2015, 6, 6263.
2
2
2
7. Knyphausen P., de Boor S., Kuhlmann N., Scislowski L.,
Extra A., Baldus L., Schacherl M., Baumann U.,
Neundorf I., Lammers M. J. Biol. Chem., 2016, 291,
1
4677-14694.
2
2
8. Zhao X., Allison D., Condon B., Zhang F., Gheyi T., Zhang
A., Ashok S., Russell M., MacEwan I., Qian Y., Jamison
J.A., Luz J.G. J. Med. Chem., 2013, 56, 963-969.
9. Disch J.S., Evindar G., Chiu C.H., Blum C.A., Dai H., Jin
L., Schuman E., Lind K.E., Belyanskaya S.L., Deng
J., Coppo F., Aquilani L., Graybill T.L., Cuozzo J.W., Lavu
S., Mao C., Vlasuk G.P., Perni R.B. J. Med. Chem.,2013
,
5
0. Chang J.H., Kim H.C., Hwang K.Y., Lee J.W., Jackson S.P.,
6, 3666-3679.
3
3
3
Bell S.D., Cho Y., J. Biol. Chem., 2002, 277, 34489-
3
4498.
1. Jin L., Wei W., Jiang Y., Peng H., Cai J., Mao C., Dai H.,
Choy W., Bemis J.E., Jirousek M.R., Milne J.C., Westphal
C.H., Perni R.B., J. Biol. Chem., 2009, 284, 24394-24405.
2. Lai, C. J.; Wu, J. C. Assay Drug Dev. Technol.,2003, 1,
5
27-535.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
Please do not adjust margins

MedChemComm
Page 8 of 8
View Article Online
DOI: 10.1039/C6MD00378H
GRAPHICAL ABSTRACT
Benzimidazole analogs were previously shown to inhibit sirtuin activity. Novel compound 5i
was found to be a potent SIRT2 inhibitor with an IC50 value of 2.92 μM.