Identification of benzofuran-3-yl(phenyl)methanones as novel SIRT1 inhibitors: Binding mode, inhibitory mechanism and biological action

Article abstract of DOI:10.1016/j.ejmech.2012.12.026

SIRT1 is a NAD⁺-dependent deacetylase. Here we described new SIRT1 inhibitors with the scaffold of benzofuran-3-yl(phenyl)methanone. The inhibitors were predicted to bind in C-pocket of SIRT1, forming hydrophobic interactions with Phe273, Phe312 and Ile347. Introducing hydroxyl to meta position of phenyl may form H-bond with Asn346. Indeed, (2,5-dihydroxyphenyl)(5- hydroxy-1-benzofuran-3-yl)methanone (16), an analogue with hydroxyls at ortho and meta positions, showed greater inhibition. The binding mode was validated by structural modifications and kinetic studies. Since C-pocket is the site where the nicotinamide moiety of NAD⁺ binds and the hydrolysis takes place, binding of 16 in C-pocket would block the transformation of NAD⁺ to productive conformation and hence inhibit the deacetylase activity. Consistently, 16 inhibited SIRT1 through up-regulating p53 acetylation on cellular level.

Full text of DOI:10.1016/j.ejmech.2012.12.026

European Journal of Medicinal Chemistry 60 (2013) 441e450
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Identification of benzofuran-3-yl(phenyl)methanones as novel SIRT1
inhibitors: Binding mode, inhibitory mechanism and biological action
,
a,
Jiahui Wu a, Yi Li b, Kaixian Chen c, Hualiang Jiang c, Ming-Hua Xu b , Dongxiang Liu
*
*
a Department of Pharmacology III, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China
b Department of Medicinal Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China
c Drug Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China
a r t i c l e i n f o
a b s t r a c t
SIRT1 is a NADþ-dependent deacetylase. Here we described new SIRT1 inhibitors with the scaffold of
benzofuran-3-yl(phenyl)methanone. The inhibitors were predicted to bind in C-pocket of SIRT1, forming
hydrophobic interactions with Phe273, Phe312 and Ile347. Introducing hydroxyl to meta position of
phenyl may form H-bond with Asn346. Indeed, (2,5-dihydroxyphenyl)(5-hydroxy-1-benzofuran-3-yl)
methanone (16), an analogue with hydroxyls at ortho and meta positions, showed greater inhibition. The
binding mode was validated by structural modifications and kinetic studies. Since C-pocket is the site
where the nicotinamide moiety of NADþ binds and the hydrolysis takes place, binding of 16 in C-pocket
would block the transformation of NADþ to productive conformation and hence inhibit the deacetylase
activity. Consistently, 16 inhibited SIRT1 through up-regulating p53 acetylation on cellular level.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Article history:
Received 10 September 2012
Received in revised form
8 December 2012
Accepted 12 December 2012
Available online 20 December 2012
Keywords:
Benzofuran-3-yl(phenyl)methanones
Binding mode
Deacetylation
Inhibitor
SIRT1
1. Introduction
benzofuran-3-yl) methanone (16), was designed with an IC50 of
2.8
mM. The interactions between the inhibitor and SIRT1 were
SIRT1 is a nuclear nicotinamide adenine dinucleotide (NADþ)-
dependent deacetylase that deacetylates histones, tumour
suppressor protein p53 and several other transcription factors [1,2].
It plays important roles in many biological functions such as fat
mobilization [3], life span regulation [4,5], cellular stress responses
[5,6], inflammation [7] and apoptosis [8,9], which renders it
a potential target for the design of drugs against cancer, diabetes
and age-related diseases. Up to present, numbers of SIRT1 inhibi-
tors, such as nicotinamide [10], Ex527 [11], sirtinol [12] and spli-
tomicin derivatives [13] have been reported. These inhibitors had
been used to explore the biological functions of SIRT1 [14e16], and
been demonstrated to increase p53 acetylation and thus induce
apoptosis of cancer cells [17e19]. Here we describe a series of new
SIRT1 inhibitors with the scaffold of benzofuran-3-yl(phenyl)
methanone, which were discovered from our in-house compound
library. Through the structureeactivity relationship (SAR) analysis
and docking simulation, we predicted that the inhibitors bind in the
C-pocket of SIRT1. Based on the interaction model, another more
potent SIRT1 inhibitor, (2, 5-dihydroxyphenyl) (5-hydroxy-1-
further supported by structural modifications of 16 and enzyme
kinetics studies. Moreover, we also demonstrated that compound
16 inhibits SIRT1 deacetylase activity and up-regulates the acety-
lation of p53 on cellular level, suggesting that benzofuran-3-
yl(phenyl)methanone may serve as a novel structural scaffold for
developing potent SIRT1 inhibitors that have potential application
in cancer treatment.
2. Results and discussion
2.1. Identification, SAR analysis and binding mode prediction of new
SIRT1 inhibitor
We employed a 4-amino-7-methylcoumarin (AMC)-based fluo-
rescent assay to identify the compounds that inhibit SIRT1 enzyme
activity [20]. The acetyl peptide substrate, ac-RHKKac-AMC, is
derived from the p53 sequence and its C-terminus was conjugated
with AMC. The AMC fluorophore can be cleaved from the peptide by
trypsin only when the -lysine of the peptide is deacetylated. The
3
fluorescence from the free AMC can be quantified at 490 nm with the
excitation wavelength of 340 nm. A known SIRT1 inhibitor, nico-
tinamide, was used as the positive control. 15 benzofuran-3-
yl(phenyl)methanone derivatives with SIRT1 inhibitory activities
* Corresponding authors. Fax: þ86 21 50807088.
E-mail addresses: xumh@mail.shcnc.ac.cn (M.-H. Xu), dxl@mail.shcnc.ac.cn,
dongxiangliu@hotmail.com (D. Liu).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.12.026

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J. Wu et al. / European Journal of Medicinal Chemistry 60 (2013) 441e450
were identified from our in-house compound library. These
analogues share the same structural scaffold but differ by several
substitutions. Their inhibitory activities at 100 mM and IC50 values for
the inhibition of SIRT1 were determined (Table 1). Compound 11
exhibited similar inhibitory activity as nicotinamide.
As shown in Table 1, the phenylmethanone was linked to
benzofuran or naphtho[1,2-b]furan fragment at C-3 position. For
the substitutions at the benzofuran ring, the hydroxyl at C-5 posi-
tion seemed to be important in maintaining the inhibitory activity,
since acetoxylation of the hydroxyl caused negligible activities (5,
6). Introducing di-bromo to C-4 and C-6 positions of the benzofuran
increased the hydrophobicity of the derivatives, which was
preferred over the introduction of a 4-methylmorpholine ring at C-
hydrophobicity of the benzofuran is beneficial, and big group
substitution is not favourable at C-4 position. In contrast, intro-
ducing methyl to C-2 position of the benzofuran ring did not affect
the activity (1 and 2). For the naphtho[1,2-b]furan derivatives, the
hydroxyl at C-5 is important as aforementioned, for compound 15
exhibited a reduced activity as compared to 13. Overall, the naph-
tho[1,2-b]furan substitutions displayed better activities than the
benzofuran when comparing 13 with 1, indicating that higher
hydrophobicity is preferred. In term of the substitutions at the
phenylmethanone, introducing halogen atoms to R3 position was
favourable, for 11 and 13 showed improved activities than 12. The
long chained phenyl group was not well tolerant at R3 (8 and 7), and
introducing dimethoxy to R2 and R4 were not favourable as well (7
and 9).
4
position (7 and 3). This indicated that enhancing the
Table 1
The inhibitory activities of compounds 1e15 and nicotinamide at concentration of 100
mM and their IC50 data.
Compound
R1
R2
H
R3
Br
R4
H
SIRT1 activity %a
IC50 ꢀ SDb
(mM)
NAM
1
0.55 ꢀ 0.04
0.28 ꢀ 0.17
90 ꢀ 7
240 ꢀ 16
2
3
H
H
Br
H
H
H
0.26 ꢀ 0.04
0.19 ꢀ 0.03
243 ꢀ 25
>300
4
OMe
H
OMe
0.14 ꢀ 0.06
>300
5
6
7
OMe
H
H
H
H
OMe
H
0.16 ꢀ 0.09
0.17 ꢀ 0.12
0.30 ꢀ 0.02
>300
>300
H
H
198 ꢀ 13
8
H
H
0.23 ꢀ 0.06
>300

J. Wu et al. / European Journal of Medicinal Chemistry 60 (2013) 441e450
443
Table 1 (continued )
Compound
R1
R2
R3
R4
SIRT1 activity %a
IC50 ꢀ SDb
(mM)
9
OMe
H
H
OMe
0.17 ꢀ 0.13
0.25 ꢀ 0.07
>300
>300
10
H
H
11
12
H
H
Cl
H
H
0.54 ꢀ 0.06
0.16 ꢀ 0.04
88 ꢀ 21
>300
Me
13
14
H
H
H
Br
H
H
H
0.36 ꢀ 0.09
0.29 ꢀ 0.13
0.17 ꢀ 0.03
146 ꢀ 18
256 ꢀ 27
>300
OMe
15
Br
a
Inhibition of SIRT1 activity ꢀ SD at 100
m
M. SD represents standard deviation of three independent measurements.
b
SD, standard deviation of at least two independent measurements, each measurement was carried out in triplicate.
To further improve the activity, we determined to predict the
473) was in the disallowed region (Fig. S2). This residue is on a loop
of the Rossmann fold domain and far away from the catalytic core.
Thus, the quality of the SIRT1 model was acceptable for further
study.
binding mode of the benzofuran-3-yl(phenyl)methanone deriva-
tives with SIRT1 by molecular modelling. Currently, the three
dimensional structure of SIRT1 is unsolved. However, the structures
of mammalian sirtuins are highly conserved in the Rossmann fold
domain. Among them, SIRT1 shares close homology with SIRT3,
particularly at the Zinc-finger motif. Until now, only the complex
structure of SIRT5 and its inhibitor suramin was solved [21].
Therefore, the structure model of SIRT1 was created by protein
homology modelling with the crystal structures of SIRT3 (PDB ID:
3GLS) [22] and SIRT5-suramin complex (PDB ID: 2NYR) as
templates. First, the structures of SIRT3 and SIRT5 were super-
imposed. Then, SIRT1 residues in the Zinc-binding domain and
Rossmann fold domain were aligned to the corresponding amino
acids of SIRT3 and SIRT5, respectively (see Supporting information,
Fig. S1). The model structure of SIRT1 was constructed and refined
using the “Build Homology Models” module in Discovery Studio 2.5
(Fig. 1A). The model was subjected to evaluation by Profiles-3D
[23]. The overall compatibility score of the model was 103.3,
which was much better than the expected low score of 51.1. The
Ramachandran plot analysis revealed that only one residue (i.e. His
Compound 1 was docked into the apo-SIRT1 model using the
autodock software [24]. Combining with the above SAR analysis,
we determined the most rational binding pose of the compound.
As shown in Fig. 1A, compound 1 binds in the cleft between the
Rossmann fold domain and the Zinc-binding domain of SIRT1.
The benzofuran ring of compound 1 was surrounded by hydro-
phobic residues in the C-pocket (Fig. 1B). Specifically, the benzyls
of Phe 273, Phe 297 and Phe 312 hang as a ceiling over 1, while Ile
270 and Ile 316 function as the hydrophobic wall which interacts
with the benzofuran ring. His 363 points to the side of the
benzene ring while Ile 347 lies under it. The hydroxyl of
compound 1 may form hydrogen bond (H-bond) with residue
Asp 348.
The binding mode of the compound is consistent with the SAR
analysis of the analogues. As shown in Table 1, the IC50 values of
compounds 1 and 13 were 240
mM and 146 mM, respectively,
suggesting that the naphtho[1,2-b]furan substitution at R1 position

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J. Wu et al. / European Journal of Medicinal Chemistry 60 (2013) 441e450
improved the inhibitory activity. According to the binding mode
(Fig. 1B), the benzofuran fragment at R1 position is surrounded by
hydrophobic residues such as Phe 312, Ile 270, Ile 316 and Ile 347,
indicating that increase of hydrophobicity at R1 position should
improve the inhibitory activity. In addition, substitution with
methyl at C-2 position of the benzofuran did not affect the
inhibitory activity (240
mM for 1 and 243 mM for 2, Table 1).
Consistently, the space formed by the neighbouring residues of
Phe 273 and Phe 297 could accommodate the methyl at C-2
position. Moreover, substitution of a hydroxyl at the C-5 position
of benzofuran ring gave stronger inhibition than that of
a methoxyl (146 mM for 13 and IC50 >300 mM for 15), indicating
that the hydroxyl may form H-bond with SIRT1 residue(s).
According to the binding mode, residue Asp 348 was near the
benzofuran ring. The side chain of the residue should have chance
to interact with the hydroxyl at C-5 position of benzofuran frag-
ment through H-bond (Fig. 1B), also, the backbone amino of Asp
348 may H-bond with the hydroxyl. Notably, the derivatives with
methoxyl substitutions at R2 and R4 exhibit significant loss of the
activities, regardless of the substitutions at R1 (IC50>300
mM for 4,
5 and 9). We predicted that the substituents at R2/R4 may be
crucial for the inhibitory activity. In the binding pocket, residues
Gln 345 and Asn 346 acted as the floor of the compound.
Substitution at R4 with methoxyl group is not preferred for the
interactions with these polar residues. In terms of R3 substitution
of the benzofuran, big hydrophobic group such as benzyloxy was
not favourable in the binding pocket (198
mM for 7 and
IC50>300
m
M for 8). As shown in Fig. 1B, big substituent at R3 may
have steric bump with residues Gln 345 and Ala 262. However, the
pocket seems to be fairly tolerant of substituents with halogen
atoms. According to the IC50 data, substitution with halogen atom
contributed to the inhibitory potency (88
mM for 11, 146 mM for 13
and IC50>300 M for 10), chlorine and bromine atoms may act as
m
the H-bond acceptors to interact with the amino group at the side
chain of Gln 345 [25].
From the above analysis, we can see that the main driving forces
for the binding of the inhibitor are hydrophobic interactions and H-
bonds. As shown in Fig. 1B, the side chains of some polar or charged
residues such as Gln 345 and Asn 346, are not far away from
compound 1 (Fig. 1B). We speculated that substituents at R2 and R4
positions may have the chance to form H-bonds with the side-
chains of the residues. Particularly, the carboxamide group at the
side chain of Asn 346 is close to the R4 position of compound 1. In
search of more potent SIRT1 inhibitors, we defined the hydroxyl
group at the R4 position and searched the SPECS commercial
compound database (http//www.specs.net). (2,5-dihydroxyphenyl)
(5-hydroxy-1-benzofuran-3-yl) methanone (compound 16, MDL
No. AG-690/03072013) was retrieved and purchased from the
company. The fluorescent assay showed that 16 was a more potent
SIRT1 inhibitor than nicotinamide. The IC50 values of 16 and nico-
tinamide were 2.8 mM and 90 mM, respectively (Table 2).
2.2. Compound 16 is a noncompetitive SIRT1 inhibitor for acetyl
peptide and a mixed-type competitive inhibitor for NADþ
In previous publications, several SIRT1 activators were found to
bind to the fluorophore covalently attached to the acetyl peptide
colored in yellow. C-pocket of SIRT1 is circled by a red oval. B) Details of the interaction
ꢀ
of 1 with SIRT1. Amino acids within 5 A of the ligand were shown as sticks. The black
dashed lines represent H-bonds formed between the hydroxyl or carbonyl oxygen
atoms of the ligand and SIRT1 residues. C) Details of the binding mode of 16 with
SIRT1. (For interpretation of the references to color in this figure legend, the reader is
referred to the web version of this article.)
Fig. 1. A) Binding site of compound 1 in SIRT1. The Zinc-binding domain of SIRT1 is
colored in blue, the large Rossmann fold domain is colored in cyan, the purple lines
represent the loops connecting the two domains, Zinc ion is highlighted in red and 1 is

J. Wu et al. / European Journal of Medicinal Chemistry 60 (2013) 441e450
445
and lower the Km of the peptide, but they did not enhance the
deacetylation of the native peptide by SIRT1 [26,27]. To avoid any
false positive results caused by the AMC fluorophore labelled on the
peptide, compound 16 was assayed by quantifying NADþ, using
a method developed by our laboratory [28]. Similar results were
obtained with ac-RHKKac-NH2 as the substrate (Fig. S3). Compound
16 was confirmed to be a potent SIRT1 inhibitor with almost 30-
fold greater potency than nicotinamide.
To study the inhibitory mechanism of benzofuran-3-
yl(phenyl)methanones, we determined the reversibility of 16
binding to SIRT1. DMSO or 10-fold the IC50 concentration of 16
was incubated with 100
mM SIRT1 at room temperature for
30 min. The mixtures were then rapidly diluted 100-fold into
the reaction buffer containing NADþ and ac-RHKKac-AMC. The
amount of deacetyl peptide was monitored in time, and two
straight lines were drawn through the data points (Fig. 2). The
reaction wells with DMSO were served as control. If the binding
of 16 with SIRT1 was rapidly reversible, the slope of the experi-
mental curve should be 91% of the slope of the control curve,
since the final concentration of the compound was 0.1 ꢁ the IC50
and enzyme activity should only be inhibited by 9%. If the binding
of 16 was slowly reversible or irreversible over the timescale of
the experiment, 91% enzyme activity should be inhibited by 10-
fold the IC50 of the inhibitor before dilution and only 9% of the
starting deacetylase activity should be detected. In fact, the slope
of the compound curve was 89% of the control slope, indicating
that 16 binding is rapidly reversible and does not involve covalent
bonds.
To further examine the mode of inhibition of 16, we deter-
mined its inhibition pattern by means of LineweavereBurk plot.
Since NADþ-dependent deactylation is a double substrate reac-
tion, the concentration of one substrate was fixed; the velocities
of the reaction were then measured at different concentrations
of 16, and reciprocal velocities were plotted against reciprocal
concentrations of another substrate. As shown in Fig. 3A,
increasing concentrations of 16 reduced Vmax but not Km, indi-
cating that 16 is a noncompetitive inhibitor of SIRT1 with respect
to the ac-RHKKac-AMC peptide. However for another substrate
NADþ, both Vmax and Km changed (Fig. 3B), indicating that 16 is
a mixed-type inhibitor of SIRT1 with respect to NADþ. It displayed
different affinities for the free enzyme and the enzymeesubstrate
complex.
Fig. 3. Inhibition of SIRT1 by different concentrations of 16 as a function of the
concentrations of ac-RHKKac-AMC and NADþ. A) Double-reciprocal lines of 16 for
peptide substrate. NADþ was fixed with gradient concentrations of ac-RHKKac-AMC at
indicated concentrations of 16. B) Double-reciprocal lines of 16 for NADþ. Ac-RHKKac
-
AMC was fixed with gradient concentrations of NADþ at indicated concentrations of 16.
16 is a noncompetitive inhibitor with respect to peptide substrate, and a mixed-type
inhibitor with respect to NADþ
.
2.3. Validation of the binding mode
To verify the binding modes of the inhibitors, compound 16
was docked into the apo-SIRT1 model (Fig. 1C). Comparing the
binding modes of compound 16 and 1, both compounds bind in
the same site of SIRT1, forming hydrophobic interactions with Phe
273, Phe 293, Phe 312, Ile 316 and Ile 347. The hydroxyl at meta-
position of the phenyl of compound 16 may form H-bond with
the side chain of Asn 346 or the backbone amino of Ala 262. The
hydroxyl at ortho-position may H-bond with the imidazole ring of
His 363. To confirm the binding mode, we intended to modify
the two hydroxyls on the phenyl ring of compound 16, as well as
to mutate the residues of SIRT1 that may interact with the
compound.
2.3.1. Synthesis of the derivatives of compound 16
Five compounds were synthesized based on the scaffold of
compound 16. As shown in Table 2, the hydroxyls at the phenyl-
methanone were either removed or methylated. The 3-benzoyl-5-
hydroxybenzofuran derivatives (20e22) were synthesized accord-
ing to Scheme 1 [29]. Briefly, the condensation of substituted
Fig. 2. Determination of the reversibility of 16 binding. The activity of SIRT1 in the
presence of DMSO (squares) and 16 (circles) is plotted versus time.

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J. Wu et al. / European Journal of Medicinal Chemistry 60 (2013) 441e450
Table 2
The inhibitory activities of the derivatives of compound 16.
Compound
R1
R2
IC50 ꢀ SDa(
mM)
NAM
16
20
21
22
90.4 ꢀ 7.1
2.8 ꢀ 0.4
71.4 ꢀ 7.4
31.2 ꢀ 5.6
>300
OH
OH
H
OMe
OH
OMe
OH
H
OH
OMe
OMe
OH
23
24
45.1 ꢀ 6.3
41.7 ꢀ 1.3
a
SD, standard deviation of at least two independent measurements, each
measurement was carried out in triplicate.
Fig. 4. A) Sequence alignment of SIRT1, SIRT3 and Sir2Tm. Amino acids in dark grey are
conserved among the three homologues. Amino acids in light grey are conserved in
two of the homologues. The amino acids labelled with asterisks were chosen to mutate
in SIRT1. B) Inhibition of SIRT3 and mutant SIRT3L195F by compound 16 at concentra-
acetophenones (17aec) with dimethylformamide dimethyl acetal
18 at 120 ꢂC in DMF for 8 h afforded the intermediate enaminones
19 with no further purification. Subsequently, the enaminones 19
were reacted with benzoquinone in acetic acid at room tempera-
ture for 3 h to form the corresponding products (20e22) in 58e75%
yield. Further, compounds (16, 23 and 24) were obtained in
moderate yields via demethylation of the corresponding ether 22
using boron tribromide solution in dichloromethane at low
temperature. All synthesized compounds (16, 20e24) were char-
acterized by 1H, 13C NMR, ESI-MS and ESI-HRMS.
The inhibitory activities of the derivatives were tested against
SIRT1 as shown in Table 2. The removal of a hydroxyl resulted in
a significant loss of the activity (20e24 and 16), which indicated the
important roles of the hydroxyls in the interaction. As discussed
above, the two hydroxyls are highly likely to form H-bonds with
SIRT1 residues (i.e. Asn 346, His 363). However, single hydroxyl
substitutition at R1 or R2 exhibited different activities. The ortho
substitution of the phenyl seems to be inferior to the meta
substitution (20 and 21). The substitution of dimethoxyls to R1 and
R2 caused significantly reduced activity (22); however, introducing
a single methoxyl to the phenol fragment did not affect the
inhibitory activities too much (20 and 23, 21 and 24), indicating
that maintaining the dihydroxyls on the phenyl ring of the scaffold
is crucial for the interaction.
tions of 0 mM, 50 mM and 100 mM.
2.3.2. Binding of 16 in the C-pocket was supported by mutagenesis
of SIRT1
The C-pocket of SIRT1 consists of several conserved amino
acids, such as residues Phe 273, Phe 312, Asn 346, Ile 347 and
His 363 (Fig. 4A). These residues were selected for mutagenesis
based on the above binding mode and also for the following
reasons: Phe 312 of SIRT1 corresponds to Leu 195 of SIRT3
(Fig. 4A); since the SIRT3L195F substitution mutant is more sensi-
tive to compound 16 than SIRT3 wild type (Fig. 4B), we changed
Phe 312 to leucine in SIRT1. Also, Phe 273 of SIRT1 corresponds to
Phe 33 of Sir2Tm (Fig. 4A), and the latter residue is thought to act
as a gate-keeper in preventing the O-alkylamidate intermediate
from base exchange with nicotinamide and converting the inter-
mediate to the 10,20-bicyclic intermediate so ensuring the
completion of deacetylation [30]. Therefore, we changed the
phenylalanine in SIRT1 to leucine to see if the side chain of Phe
273 would affect the inhibition. Since the mutant SIRT1H363A is
deacetylase-inactive, the mutation was abandoned for further
experiment [31].
HO
R1
R2
O
R1
R2
O
O
R1
R2
OMe
OMe
Me
a
b
N
O
N
17a R1 = OTBS, R2 = H
17b R1 = H, R2 = OTBS
17c R1 = R2 = OMe
20 R1 = OH, R2 = H
21 R1 = H, R2 = OH
22 R1 = R2 = OMe
18
19
23 R1 = OH, R2 = OMe
24 R1 = OMe, R2 = OH
16 R1 = OH, R2 = OH
c
d
Scheme 1. The synthetic procedures of compounds 20e24 and 16. Reagents and conditions: a) DMF, 120ꢂC, 8h; b) for 20e22: benzoquinone, HOAc, rt, 3h; c) for 23e24: 2M BBr3,
CH2Cl2, -78ꢂC, 2h; d) for 16: 2M BBr3, CH2Cl2, -78ꢂC, 30min, then 0ꢂC, 2h.

J. Wu et al. / European Journal of Medicinal Chemistry 60 (2013) 441e450
447
The SIRT1 mutants had reduced level of deacetylase activities. For
IC50 values determination, appropriate amounts of SIRT1 and the
mutants were used to achieve the same catalytic activity (Fig. 5A). The
Ki values of 16 for SIRT1 and the mutants were determined from
secondary plots of the kinetics data as following. First, the concen-
tration of the acetyl peptide was fixed; the velocities of the reaction
for wild type SIRT1 and the mutants were measured at different
concentrations of 16, and reciprocal velocities were plotted against
reciprocal concentrations of NADþ (Fig. S4). Then the slopes of the
double-reciprocal lines were plotted versus the concentration of 16.
The values of ꢃKi were calculated from the x intercepts of secondary
plots for wild type SIRT1 and the mutants (Fig. 5B). As shown in Fig. 5,
the IC50 and Ki values were much higher in themutants, indicatingthe
hydrophobic side chains of Phe 273, Phe 312 and Ile 347 play
important roles in the inhibition of 16, which interact directly with 16
through hydrophobic interactions. Also, the data indicated that the
H-bond between the amino group at side chain of Asn 346 and
the hydroxyl of 16 is important for the inhibition. The structural
modifications of the compounds in combination of the mutagenesis
studies of SIRT1 have validated the rationality of the binding
mode and proved that the inhibitors bind in the C-pocket of SIRT1.
Fig. 6. Western blot analysis of acetyl-p53 and total p53 in MCF-7 cells treated with
gradient concentrations of 16. The total p53 were loaded the same but acetyl-p53
increased as a function of 16.
apoptosis [32]. It has been shown that SIRT1 inhibitors up-
regulate p53 acetylation by inhibiting SIRT1 deacetylase
activity [17,18,33]. To investigate the inhibitory activity of
compound 16 on cellular level, we measured the acetylation of
p53 in MCF-7 cancer cells treated with various concentrations of
16. As a positive control, we showed that EX527, a known SIRT1
inhibitor, increased p53 acetylation at low concentration [17].
Apoptosis was induced by doxorubicin (DOX). Trichostatin (TSA)
was employed to selectively inhibit class I and II mammalian
histone deacetylases (HDACs). Acetyl-p53 and total p53 were
detected by western blotting. Total p53 remained constant while
acetyl-p53 increased as a function of 16 concentration (Fig. 6),
indicating that 16 inhibits SIRT1 deacetylase activity on cellular
level.
2.4. Compound 16 inhibits p53 deacetylation on cellular level
SIRT1 negatively regulates tumour suppressor protein p53,
which rescues cells from DNA damage and stress-induced
3. Conclusions
In this paper we identified a new class of SIRT1 inhibitors with
the benzofuran-3-yl(phenyl)methanone scaffold by high through-
put screening. The binding mode of the inhibitors was predicted
through the SAR analysis and docking simulations, based on which
another more potent SIRT1 inhibitor, (2, 5-dihydroxyphenyl) (5-
hydroxy-1-benzofuran-3-yl) methanone (16), was found and
confirmed to be a noncompetitive inhibitor for acetyl peptide and
a mixed-type competitive inhibitor for NADþ. The SAR results,
docking simulations, mutagenesis experiments and enzyme
kinetics studies, indicated that 16 bound in the C-pocket of SIRT1.
Structural modifications of the two hydroxyls on the phenyl
fragment of 16 had proved their crucial roles in the inhibitory
activities, which may form H-bonds with SIRT1 residues. Single-
site mutations of Phe 273, Phe 312, Asn 346 and Ile 347 reduced
the susceptibility of SIRT1 to the inhibition of 16. Compound 16
was proved to have hydrophobic interactions with Phe 273, Phe
312 and Ile 347. Meanwhile, the hydroxyl substitution at meta-
position of the phenyl of the compound is vital for the inhibi-
tory activity, which may interact with Asn 346 through H-bond.
The imidazole ring of His 363 may also form H-bond with the
hydroxyl at ortho-position of the phenyl ring. According to the
catalytic mechanism of Sir2 enzymes, C-pocket is the site where
the nicotinamide moiety of NADþ binds and the hydrolysis takes
place [14,21,34]. SIRT1 residues Phe 273, Asn 346, Ile 347 and His
363 are the constituent of the C-pocket and play important roles
in the catalysis of the deacetylation [21]. If compound 16 occupies
the C-pocket and interacts with above residues, transformation of
NADþ from non-productive conformation to productive confor-
mation will be blocked, thus the subsequent hydrolysis and
nicotinamide release are prevented. As the consequence, the
deacetylase activity will be inhibited. Consistent with the in vitro
results, 16 inhibits SIRT1 deacetylase activity and up-regulates
acetylation of p53 protein on cellular level. Compound 16 could
serve as a lead compound for the development of more potent
SIRT1 inhibitors, which may have potential applications in the
treatment of cancer.
Fig. 5. A) Inhibition of SIRT1 wild type and mutants by 16. B) Secondary plots for
determining Ki values of 16 for mixed-type inhibition of SIRT1 and its mutants. The
first plots (double-reciprocal plots) for SIRT1 wild type are shown in Fig. 3B, the first
plots for SIRT1 mutants are shown in the Supporting information (Fig. S4).

448
J. Wu et al. / European Journal of Medicinal Chemistry 60 (2013) 441e450
4. Experimental section
were assayed the same as for the wild type with indicated
concentrations of 16.
4.1. Materials and measurements
4.2. Homology modelling and docking simulation
4.1.1. Protein expression and purification
The coding region of SIRT1(156-664) was inserted between the
NdeI and XhoI restriction sites in pET28a vector together with an N-
terminal hexa-histidine tag. The plasmid was transformed into
Escherichia coli BL21 (DE3) competent cells. A single colony was
Apo-SIRT1 structure was modelled by using the software
Accelrys Discovery Studio Client 2.5 (DS 2.5). SIRT1 sequence
(SIRT1-244-498) was retrieved from SWISS-prot database (Uni-
ProtKB code: Q96EB6, http://www.uniprot.org/). The crystal
structures of SIRT5 and suramin complex (PDB ID: 2NYR) and SIRT3
(PDB ID: 3GLS) were used as the templates. Automatic sequence
alignment was performed with ClustalW2 [35]. Adjustment was
made manually in the loop regions according to the hydrophobicity
and polarity of the residues (Fig. S1). The distances between the
four cysteines of the Zinc-finger motif were restrained with devi-
inoculated in LB medium containing 50 m
g/mL kanamysin at 37 ꢂC.
When OD600 reached 0.8e1.0, the culture was induced with 0.5 mM
IPTG at 16 ꢂC overnight. The protein was purified on a Ni-NTA
column and stored at ꢃ80 ꢂC. SIRT1 mutations SIRT1F273L
,
SIRT1F312L, SIRT1N346A and SIRT1I347A were generated by site-
directed mutagenesis of SIRT1(156-664) DNA fragment and the
mutant proteins were expressed and purified as for wild type
SIRT1(156-664).
ꢀ
ation of 0.6 A. The Zinc ion was copied to the model. Then SIRT1
model was built and refined by using Homology models module.
The model was validated by Profiles-3D and Ramachandran plot
analysis. Compounds were built by using the Simulate Structures
module of DS 2.5. Hydrogen atoms were added by applying
CHARMm forcefield to the compounds. Afterwards, the structures
of compounds were optimized by energy minimization. Autodock
4.2 was used to predict the binding modes of compounds in SIRT1.
The pdb file of protein model applied with CHARm forcefield and
the mol2 files of compounds were pre-treated with AutoDock Tools
1.5.4. Genetic algorithm (GA) was used in the docking procedure.
After autogrid and autodock, possible binding poses were obtained.
4.1.2. Screening for SIRT1 inhibitors
Compounds were dissolved in DMSO to prepare 10 mM stock
solutions; a fluorescent-based assay was used to screen compounds
that inhibit the deacetylase activity of SIRT1. The C-terminus of the
acetyl substrate acetyl-ArgeHiseLyseLys ( -acetyl) was labelled
3
with 4-amino-7-methylcoumarin (AMC). When the substrate is
deacetylated by SIRT1, AMC can be cleaved from the peptide by
trypsin and detected by its fluorescence. The reaction conditions
were as follows: 750 m mM ac-RHKKac-AMC and 1 mM
M NADþ, 31.25
enzyme in buffer containing 25 mM Tris (pH 8.0), 137 mM NaCl,
2.7 mM KCl and 1 mM MgCl2. The reactions were carried out for
45 min at room temperature, stopped with 10 mM nicotinamide
and developed with 0.5 mg/mL trypsin. After incubation for 30 min,
fluorescence intensities were measured with a microplate reader
(POLARstar OPTIMA, BMG Labtech) at an excitation wavelength of
340 nm and an emission wavelength of 490 nm, with appropriate
positive and negative controls.
4.3. Synthesis of compounds
Unless otherwise specified, all reactions were carried out in
flame-dried glassware with magnetic stirring under an atmosphere
of nitrogen. Solvents were dried and distilled by standard proce-
dures. NMR spectra were recorded on a Varian spectrometer
(300 MHz for 1H, and 100 MHz for 13C). Chemical shifts are reported
in
d
ppm referenced to methanol-d (
d
3.31) for 1H NMR and
4.1.3. Determination of the IC50 values
methanol-d (
d
49.0) for 13C NMR.
Compounds were dissolved and diluted with DMSO. To deter-
mine the IC50 of the benzofuran derivatives and NAM for wild type
4.3.1. General procedure for the synthesis of compounds 20e22
Under nitrogen atmosphere, a solution of the appropriate ace-
tophenone 17 (2.0 mmol) and N,N-dimethylformamide dimethyl
acetal 18 (2.0 mmol) in DMF (5.0 mL) was heated to 120 ꢂC and the
colorless solution turned yellow during the reaction. After being
stirred for 8 h at this temperature, the solvent was removed under
reduced pressure to give the crude product 19 with no further
purification. Subsequently, the mixture of compound 19 and ben-
zoquinone in acetic acid (3.0 mL) was stirred at room temperature
under N2 atmosphere for 3 h and monitored by TLC. When the
reaction was over, a saturated aq. NaCl was added and the mixture
was extracted with EtOAc (10 mL ꢁ 3). The combined organic phase
was dried over Na2SO4, filtered, and concentrated. The residue was
purified by silica gel flash chromatography to afford the corre-
sponding products 20e22.
SIRT1, 10
reaction system contained 750
AMC, 1 M SIRT1 and different concentrations of compounds. To
determine the IC50 of 16 for SIRT1 mutants, 5 L compound in
m
L compound was added into 40
m
L reaction buffer. The
m
M NADþ, 31.25
mM ac-RHKKac-
m
m
DMSO was added into 45 mL reaction buffer. The final concentra-
tions of NADþ and ac-RHKKac-AMC in the reaction system were the
same as the above. To achieve the same deacetylase activity, the
concentrations of wild type SIRT1, SIRT1F273L
,
SIRT1F312L
M, 18.2
M, respectively. IC50 were evaluated by fitting the exper-
,
SIRT1N346A, and SIRT1I347A was set 1
and 2.5
mM, 11.1
mM, 13.8
m
mM
m
imental data using software Origin 7.0.
4.1.4. Determination of the type of 16 inhibition
To determine the inhibition type of ac-RHKKac-AMC by 16, the
concentrations of SIRT1 and NADþ were fixed at 1
mM and 1 mM,
respectively, and the concentrations of ac-RHKKac-AMC were set at
4.3.1.1. (5-hydroxybenzofuran-3-yl)(2-hydroxyphenyl)methanone
(20). This compound was prepared from 1-(2-(tert-butyldime-
thylsilyloxy)phenyl)ethanone 17a. Yield: 58%, brown solid. 1H NMR
66.7
33.3
m
M, 100
m
M, 150 M and 300
m
mM, and those of 16 at 100 mM,
mM, 11.1
mM and 0 mM. A double reciprocal plot of 1/V versus 1/
[ac-RHKKac-AMC] (where V is the initial reaction velocity and [ac-
RHKKac-AMC] is the concentration of ac-RHKKac-AMC peptide) was
analysed. Inhibition of NADþ by 16 was examined in a similar
manner. The concentrations of SIRT1 and ac-RHKKac-AMC were
(300 MHz, CD3OD)
(d, J ¼ 8.1 Hz,1H), 7.79e7.84 (m,1H), 8.22e8.25 (m,1H), 8.29 (s,1H).
13C NMR (100 MHz, CD3OD):
117.6, 118.2, 118.7, 119.4, 121.0, 124.3,
125.2, 126.7, 126.8, 135.4, 149.6, 151.4, 157.6, 157.8, 178.8. ESI-MS (m/
z, %) 255 [M þ H]þ.
d
: 6.94e6.80 (m, 3H), 7.51 (t, J ¼ 8.1 Hz, 1H), 7.64
d
fixed at 1
were set to 143
concentrations of 16 were set to 60
M. The double-reciprocal plots of 16 for the four SIRT1 mutants
mM and 400
m
M, respectively. The concentrations of NADþ
M, 333 M and 1000 M, and the
M, 26.67 M, 17.78 M and
mM, 200
m
m
m
m
m
m
4.3.1.2. (5-Hydroxybenzofuran-3-yl)(3-hydroxyphenyl)methanone
0
m
(21). This
compound
was
prepared
from
1-(3-(tert-

J. Wu et al. / European Journal of Medicinal Chemistry 60 (2013) 441e450
449
butyldimethylsilyloxy)phenyl)ethanone 17b. Yield: 65%, brown
solid. 1H NMR (300 MHz, CD3OD)
: 6.89 (d, J ¼ 8.4 Hz, 1H), 7.06 (d,
J ¼ 7.8 Hz, 1H), 7.26 (s, 1H), 7.31e7.42 (m, 3H), 7.55 (s, 1H), 8.27 (s,
1H). 13C NMR (100 MHz, CD3OD):
108.1, 112.8, 115.7, 116.2, 120.7,
2H), 7.80 (d, J ¼ 8.4 Hz, 2H), 8.323 (s, 1H). 13C NMR (100 MHz,
d
CD3OD): d 108.1, 112.8, 115.8, 121.8, 127.1, 128.2, 131.6, 133.1, 139.4,
151.5, 155.6, 156.1, 190.9. ESI-MS (m/z, %) 317 [M þ H]þ.
d
121.0, 122.1, 127.2, 130.9, 141.9, 151.5, 155.5, 156.0, 159.1, 192.3. ESI-
4.4.2. (4-Bromophenyl)(5-hydroxy-2-methylbenzofuran-3-yl)
MS (m/z, %) 255 [M þ H]þ.
methanone (2)
1H NMR (300 MHz, CD3OD)
d: 2.47 (s, 3H), 6.74(s, 1H), 6.75e6.78
4.3.1.3. (2,5-Dimethoxyphenyl)(5-hydroxybenzofuran-3-yl)meth-
anone (22). This compound was prepared from 1-(2,5-
dimethoxyphenyl)ethanone 17c. Yield: 75%, pale white solid. 1H
(m, 1H), 7.31 (d, J ¼ 9.3 Hz, 1H), 7.68 (d, J ¼ 8.7 Hz, 2H), 7.73 (d,
J ¼ 8.7 Hz, 2H). 13C NMR (100 MHz, CD3OD):
d 14.9, 106.9, 112.3,
114.3, 117.7, 128.5, 128.6, 131.8, 133.1, 139.7, 149.5, 155.3, 164.6, 192.7.
NMR (300 MHz, CD3OD)
J ¼ 8.7 Hz,1H), 6.97 (s,1H), 7.08-7.09 (m, 2H), 7.38 (d, J ¼ 8.7 Hz,1H),
7.52 (s, 1H), 8.06 (s, 1H). 13C NMR (100 MHz, CD3OD):
56.4, 56.8,
d: 3.74 (s, 3H), 3.79 (s, 3H), 6.87 (d,
ESI-MS (m/z, %) 331 [M þ H]þ.
d
4.4.3. (4,6-dibromo-5-hydroxybenzofuran-3-yl)(phenyl)
107.9, 112.9, 114.5, 115.3, 115.5, 118.3, 123.6, 126.4, 131.3, 151.6, 152.3,
methanone (7)
154.9, 155.9, 157.0, 192.4. ESI-MS (m/z, %) 299 [M þ H]þ.
1H NMR (300 MHz, CD3OD)
d
: 7.51e7.56 (m, 2H), 7.64e7.70 (m,
1H), 7.89 (s, 1H), 7.90e7.93 (m, 2H), 8.14 (s, 1H). 13C NMR (100 MHz,
4.3.2. General procedure for the synthesis of compound 23e24
Compound 22 (1.0 mmol) was dissolved in 5.0 mL of dry
dichloromethane at ꢃ78 ꢂC and 2 M boron tribromide solution in
dichloromethane (3.0 mmol) was added under nitrogen. The
mixture was stirred at ꢃ78 ꢂC for 2.0 h. After completion, the
reaction was quenched with 20% aqueous NaOH, then the mixture
was adjusted to pH 1e2 by slow addition of 1N HCl and extracted
with CH2Cl2 three times. The combined organic phase was dried
over Na2SO4, filtered, and evaporated under reduced pressure. The
residue was purified by flash chromatography to give the corre-
sponding products 23 and 24, respectively.
CD3OD): d 102.9, 111.0, 116.3, 123.4, 127.6, 129.9, 131.0, 134.9, 139.9,
149.6, 150.7, 151.2, 191.0. ESI-MS (m/z, %) 397 [M þ H]þ.
4.4.4. (4-Chlorophenyl)(5-hydroxynaphtho[1,2-b]furan-3-yl)
methanone (11)
1H NMR (300 MHz, (CD3)2SO)
d: 7.56e7.61 (m, 2H), 7.65e7.73
(m, 1H), 7.67 (d, J ¼ 8.4 Hz, 2H), 7.96 (d, J ¼ 8.4 Hz, 2H), 8.21e
8.28 (m, 2H), 8.75 (s, 1H), 10.33 (s, 1H). 13C NMR (100 MHz,
(CD3)2SO):
d 100.0, 119.4, 120.8, 120.9, 121.1, 123.4, 123.5, 125.2,
125.9, 127.6, 128.9, 130.7, 138.8, 144.6, 151.0, 153.2, 188.8. ESI-MS (m/
z, %) 323 [M þ H]þ.
4.3.2.1. (2-Hydroxy-5-methoxyphenyl)(5-hydroxybenzofuran-3-yl)
4.4.5. (4-bromophenyl)(5-hydroxynaphtho[1,2-b]furan-3-yl)
methanone (23). Yield: 53%, pale white solid. 1H NMR (300 MHz,
methanone (13)
CD3OD)
d
: 3.91 (s, 3H), 6.70e6.80 (m, 3H), 7.39e7.44 (m, 1H), 7.57e
1H NMR (300 MHz, (CD3)2SO)
d: 7.56e7.61 (m, 2H), 7.68e7.73
7.64 (m, 2H), 8.28 (s, 1H). 13C NMR (100 MHz, CD3OD):
d
56.4, 106.1,
(m, 1H), 7.81 (d, J ¼ 8.4 Hz, 2H), 7.88 (d, J ¼ 7.8 Hz, 2H), 8.20e
117.5, 118.3, 118.7, 120.9, 121.2, 123.5, 125.2, 125.8, 149.6, 151.4, 152.7,
8.28 (m, 2H), 8.76 (s, 1H), 10.33 (s, 1H). 13C NMR (100 MHz,
157.4, 158.8, 178.6. ESI-MS (m/z, %) 285 [M þ H]þ.
(CD3)2SO): d 100.0, 119.4, 120.8, 120.9, 121.1, 123.4, 123.5, 125.2,
126.6, 127.6, 130.9, 131.9, 137.7, 144.6, 151.0, 153.3, 188.9. ESI-MS (m/
4.3.2.2. (5-Hydroxy-2-methoxyphenyl)(5-hydroxybenzofuran-3-yl)
z, %) 367 [M þ H]þ.
methanone (24). Yield: 35%, yellow solid.1H NMR (300 MHz, CD3OD)
d
: 3.77 (s, 3H), 6.89 (dd, J ¼ 2.1 Hz, 9.3 Hz,1H), 6.95 (d, J ¼ 8.7 Hz,1H),
4.4.6. (5-Hydroxynaphtho[1,2-b]furan-3-yl)(4-methoxyphenyl)
7.15 (dd, J ¼ 2.7 Hz, 9.3 Hz, 1H), 7.29 (d, J ¼ 2.4 Hz, 1H), 7.41 (d,
methanone (14)
J ¼ 8.7 Hz,1H), 7.44 (d, J ¼ 1.8 Hz,1H), 8.37 (s,1H).13C NMR (100 MHz,
1H NMR (300 MHz, (CD3)2SO)
d
: 3.86 (s, 3H), 7.11 (d, J ¼ 8.7 Hz,
CD3OD):
d
56.3, 107.9, 112.9, 115.2, 115.7, 119.8, 121.9, 122.9, 123.7,
2H), 7.53e7.59 (m, 2H), 7.65-7.70 (m, 1H), 7.96 (d, J ¼ 9.0 Hz, 2H),
127.1, 151.3, 153.7, 154.2, 155.9, 194.2. ESI-MS (m/z, %) 285 [M þ H]þ.
8.12e8.26 (m, 2H), 8.67 (s, 1H), 10.25 (s, 1H). 13C NMR (100 MHz,
(CD3)2SO): d 55.6, 100.2, 114.1, 119.3, 120.8, 121.0, 121.5, 123.3, 123.5,
4.3.3. General procedure for the synthesis of compound 16
125.0, 127.4, 131.2, 144.5, 150.7, 151.8, 162.9, 188.3. ESI-MS (m/z, %)
2 Mboron tribromidesolutionindichloromethane(6.0 mmol)was
added dropwise to a solution of the compound 22 (1.0 mmol) in
CH2Cl2 (5.0 mL) at ꢃ78 ꢂC, then the mixture was allowed to warm to
0 ꢂC and stirred for 2 h. After completion, the reaction was quenched
with 20% aqueous NaOH, then the mixture was adjusted to pH 1e2 by
slow addition of 1N HCl. The precipitated gray solid was collected by
filtration and purified by flash chromatography to give the product 16.
319 [M þ H]þ.
4.5. Western blotting
Cells of the human cancer cell line MCF-7 were cultured in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with
10% fetal calf serum. Cells were seeded at concentration of 1 ꢁ 105
in six-well plates and incubated in a humidified atmosphere con-
taining 5% CO2 at 37 ꢂC for 24 h. Gradient concentrations of 16 in
4.3.3.1. (2,5-Dihydroxyphenyl)(5-hydroxybenzofuran-3-yl)meth-
anone (16). Yield: 51%, brown solid. 1H NMR (300 MHz, CD3OD)
d:
combination with 0.1 mM trichostatin (TSA) and 5 mM doxorubicin
6.70e6.79 (m, 3H), 7.27e7.31 (m, 1H), 7.51e7.54 (m, 2H), 8.25 (s,
(DOX) were added to MCF-7 cells. After 6 h of incubation at 37 ꢂC,
total cell protein was extracted using a kit from Active Motif
(Carlsbad,CA). Total protein was determined with a BCA kit (catalog
no. P0012, Beyotime Institute of Biotechnology, Shanghai, China).
1H). 13C NMR (100 MHz, CD3OD):
d
109.1, 117.5, 118.4, 118.7, 120.7,
121.4, 123.3, 124.8, 126.0, 150.0, 151.5, 151.8, 156.7, 157.5, 178.8. ESI-
MS (m/z, %) 271 [M þ H]þ.
Samples containing 50 mg protein were loaded onto a 9% gel. After
4.4. Characterization of the active methanone derivatives (1, 2, 7,
11, 13 and 14) purchased from SPECS company
electrophoresis, the proteins were transferred to a nitrocellulose
membrane (Whatman, UK). Non-specific reactivity was blocked
with 5% non-fat milk in TBST (10 mM Tris, 150 mM NaCl, 0.05%
Tween-20, pH 7.5) at room temperature for 1 h. The membrane was
then incubated with polyclonal antibody against acetyl-p53 (Cell
Signaling Technology, catalog No. #2525) at 4 ꢂC overnight and
4.4.1. (4-bromophenyl)(5-hydroxybenzofuran-3-yl)methanone (1)
1H NMR (300 MHz, CD3OD)
7.42 (d, J ¼ 8.7 Hz, 1H), 7.54 (d, J ¼ 2.7 Hz, 1H), 7.73 (d, J ¼ 8.7 Hz,
d
: 6.90 (dd, J ¼ 2.7 Hz, 9.0 Hz, 1H),

450
J. Wu et al. / European Journal of Medicinal Chemistry 60 (2013) 441e450
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(2002) 45099e45107.
[15] B.D. Sanders, K. Zhao, J.T. Slama, R. Marmorstein, Structural basis for nico-
tinamide inhibition and base exchange in Sir2 enzymes, Mol. Cell 25 (2007)
463e472.
[16] H. Ota, E. Tokunaga, K. Chang, M. Hikasa, K. Iijima, M. Eto, K. Kozaki,
M. Akishita, Y. Ouchi, M. Kaneki, Sirt1 inhibitor, Sirtinol, induces senescence-
like growth arrest with attenuated Ras-MAPK signaling in human cancer cells,
Oncogene 25 (2006) 176e185.
immune complexes were detected with horseradish peroxidase-
conjugated goat anti-rabbit IgG antibody and visualized with
a SuperSignal West Dura Kit (Pierce). The same blot was re-probed
and p53 antibody (Cell Signaling Technology, catalog No. #2527)
was used to detect the total p53 protein.
Acknowledgement
[17] J.M. Solomon, R. Pasupuleti, L. Xu, T. McDonagh, R. Curtis, P.S. DiStefano,
L.J. Huber, Inhibition of SIRT1 catalytic activity increases p53 acetylation but
does not alter cell survival following DNA damage, Mol. Cell. Biol. 26 (2006)
28e38.
[18] B. Peck, C.Y. Chen, K.K. Ho, P. Di Fruscia, S.S. Myatt, R.C. Coombes,
M.J. Fuchter, C.D. Hsiao, E.W. Lam, SIRT inhibitors induce cell death and p53
acetylation through targeting both SIRT1 and SIRT2, Mol. Cancer Ther. 9
(2010) 844e855.
This work was supported by the CAS “Introducing Outstanding
Oversea Scientists Project”, the National Science Fund for Creative
Research Groups (Grant 21021063), and the Science and Tech-
nology Commission of Shanghai Municipality (Grant 08JC1422100).
[19] E. Lara, A. Mai, V. Calvanese, L. Altucci, P. Lopez-Nieva, M.L. Martinez-Chantar,
M. Varela-Rey, D. Rotili, A. Nebbioso, S. Ropero, G. Montoya, J. Oyarzabal,
S. Velasco, M. Serrano, M. Witt, A. Villar-Garea, A. Inhof, J.M. Mato, M. Esteller,
M.F. Fraga, Salermide, a Sirtuin inhibitor with a strong cancer-specific pro-
apoptotic effect, Oncogene 28 (2008) 781e791.
Appendix A. Supplementary material
Supplementary material related to this article can be found at
http://dx.doi.org/10.1016/j.ejmech.2012.12.026.
[20] SIRT1 fluorimetric drug discovery kit, in: B.R. Laboratories (Ed.), A fluor de lys
fluorescent assay system, 2009.
[21] J.L. Avalos, K.M. Bever, C. Wolberger, Mechanism of sirtuin inhibition by
nicotinamide: altering the NADþ Cosubstrate Specificity of a Sir2 enzyme,
Mol. Cell 17 (2005) 855e868.
[22] L. Jin, W. Wei, Y. Jiang, H. Peng, J. Cai, C. Mao, H. Dai, W. Choy, J.E. Bemis,
M.R. Jirousek, J.C. Milne, C.H. Westphal, R.B. Perni, Crystal structures of human
SIRT3 displaying substrate-induced conformational changes, J. Biol. Chem. 284
(2009) 24394e24405.
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