Design, synthesis, enzyme inhibition, and tumor cell growth inhibition of 2-anilinobenzamide derivatives as SIRT1 inhibitors

Article abstract of DOI:10.1016/j.bmc.2009.07.001

A series of 2-anilinobenzamide derivatives were designed, synthesized and evaluated for their SIRT1-inhibitory activity. Among these, compounds 3 and 5 inhibited SIRT1 activity in enzyme assays and suppressed the growth of Daudi and HCT116 cells.

Full text of DOI:10.1016/j.bmc.2009.07.001

Bioorganic & Medicinal Chemistry 17 (2009) 5900–5905
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, enzyme inhibition, and tumor cell growth inhibition
of 2-anilinobenzamide derivatives as SIRT1 inhibitors
a
a
b
b
a
Takayoshi Suzuki a, , Keiko Imai , Erika Imai , Shinsuke Iida , Ryuzo Ueda , Hiroki Tsumoto ,
*
Hidehiko Nakagawa a, Naoki Miyata a,
*
a Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467-8603, Japan
b Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya, Aichi 467-8601, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 2-anilinobenzamide derivatives were designed, synthesized and evaluated for their SIRT1-
inhibitory activity. Among these, compounds 3 and 5 inhibited SIRT1 activity in enzyme assays and sup-
pressed the growth of Daudi and HCT116 cells.
Received 18 June 2009
Revised 1 July 2009
Accepted 2 July 2009
Available online 7 July 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
SIRT1 inhibitor
Enzyme assay
Tumor cell growth inhibition
2-Anilinobenzamide derivative
Daudi cells
HCT116 cells
1. Introduction
2. Chemistry
Sirtuins are NAD+-dependent protein deacetylases that are in-
volved in a diversity of cellular functions, such as genome main-
tenance, longevity, and metabolism.1 SIRT1, the most well studied
human sirtuin, was reported to deacetylate a variety of sub-
The compounds prepared for this study (compounds 2–12) are
enumerated in Table 1. The routes used for the synthesis of the
compounds are shown in Schemes 1–3. Compounds 2, 3, and 8–
11 were prepared as shown in Scheme 1. The Buchwald–Hartwig
reaction between 2-aminobenzamide 13 and 3- or 4-substituted
bromobenzene 14a–f using 2-dicyclohexylphosphino-20,40,60-tri-
isopropyl biphenyl20 as a ligand gave compounds 2, 3, and 8–11.
The preparation of the other 2-anilinobenzamide derivatives is
outlined in Scheme 2. Coupling of 3-bromoiodobenzene and thio-
phenol in the presence of copper iodide21 afforded phenyl sulfide
16a, which was converted into compound 4 by the Buchwald–Har-
twig reaction. Phenyl ether 5 was obtained from 1,3-dibromoben-
zene by the combination of Ullman condensation and the
Buchwald–Hartwig reaction. 3-Bromocinnamic acid 18 was con-
verted into methyl ester 16c under an acidic conditions, and the
Buchwald–Hartwig reaction of 16c with 13 yielded compound 6.
Methyl ester 6 was hydrolyzed under an alkaline conditions to give
cinnamic acid derivative 7.
strates, including histone H4,2 p53,3 BCL6,4 HIV Tat,5 PGC-1a 6
,
AceCS1,7 and Rb.8 The protein deacetylation by SIRT1 has been
suggested to be associated with certain disease states, such as
cancer and HIV infection.4,5,9 Therefore, SIRT1 inhibitors are of
interest not only as tools for elucidating in detail the biological
functions of the enzyme, but also as potential therapeutic
agents.10
Several classes of small-molecule SIRT1 inhibitors have been
identified so far.11 These include nicotinamide,12 EX-527,13 sura-
min analogues,14 HR73,5 Ro 31-8220,15 cambinol,4 oxadiazole-car-
bonylaminothioureas,16 salermide17 and NCS-12k18 (Fig. 1). We
previously reported that NCS-7 1 (Fig. 1) is a SIRT1-selective
inhibitor.19 Here we report the design, synthesis, SIRT1 inhibition,
and tumor cell growth inhibition of 2-anilinobenzamide
derivatives.
2-(Phenylthio)benzamide 12 was synthesized as depicted in
Scheme 3. Coupling of 2-bromobenzoic acid 19 and diphenyldisul-
fide 20 in the presence of copper and copper iodide yielded 2-
(phenylthio)benzoic acid 21. Benzamide 12 was obtained by the
coupling of carboxylic acid 21 and ammonia in the presence of
EDCI and HOBt.
* Corresponding authors. Tel./fax: +81 52 836 3407 (N.M.).
E-mail addresses: suzuki@phar.nagoya-cu.ac.jp (T. Suzuki), miyata-n@phar.
nagoya-cu.ac.jp (N. Miyata).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.07.001

T. Suzuki et al. / Bioorg. Med. Chem. 17 (2009) 5900–5905
5901
O
O
H
N
NH2
O
NH2
Cl
O
N
nicotinamide
EX-527
Br
HR73
CH3
H
N
H
N
H
N
S
NH
O
O
O
N
O
N
N
CH3
NH
OH
OH
H2N
S
Ro 31-8220
salermide
cambinol
O
O
NH2
O
HN
O
O
NH
H
N
N
H
OEt
O
NCS-7 (1)
NCS-12k
Figure 1. Examples of SIRT1 inhibitors.
O
Table 1
SIRT1 inhibition data for compounds 1–12a
NH2
NH
Br
O
a
O
NH2
NH2
+
NH2
R
X
R
14a
: R = 3-Me
13
2
14b: R = 3-Ph
: R = 3-Me
14c
14d
3: R = 3-Ph
: R = 3-CH2OH
8
9
: R = 3-CH2CH2OH
: R = 3-CH2OH
R
14e: R = 4-Ph
: R = 3-CH2CH2OH
14f
10: R = 4-Ph
: R = 4-OPh
Compound
R
X
IC50 (lM)
11
: R = 4-OPh
1
2
3
4
5
6
7
8
H
–NH–
–NH–
–NH–
–NH–
–NH–
–NH–
–NH–
–NH–
–NH–
–NH–
–NH–
–S–
56
68
52
89
58
65
58
60
72
Scheme 1. Reagents: (a) Pd2dba3, 2-dicyclohexylphosphino-20,40,60-triisopropyl
biphenyl, K2CO3, tert-BuOH, reflux.
3-Me
3-Ph
3-SPh
3-OPh
3-(E)-CH@CH–COOMe
3-(E)-CH@CH–COOH
3-CH2OH
3-CH2CH2OH
4-Ph
4-OPh
H
Br
Br
Br
9
10
11
12
>300b
>300c
78
I
Br
COOH
15
17
18
a
Values are means of at least three separate experiments.
b
a
c
b
c
36% Inhibition at 300
0% Inhibition at 300
l
M.
M.
l
O
Br
NH2
NH
d
3. Results and discussion
3.1. Enzyme assays
R
16a: R = SPh
16b: R = OPh
R
16c: R = (E)-CH=CHCOOMe
4: R = SPh
The compounds synthesized in this study were subjected to an
in vitro assay using recombinant SIRT1 and the results are summa-
rized in Table 1. Compound 1 was used as positive control, which
5: R = OPh
6: R = (E)-CH=CHCOOMe
7: R = (E)-CH=CHCOOH
e
inhibited SIRT1 with an IC50 of 56 lM.
Scheme 2. Reagents and conditions: (a) thiophenol, CuI, K2CO3, ethylene glycol, i-
PrOH, 80 °C; (b) phenol, Cu, K2CO3, 165 °C; (c) concd HCl, MeOH, 80 °C; (d) 2-
aminobenzamide (13), Pd2dba3, 2-dicyclohexylphosphino-20,40,60-triisopropyl
biphenyl, K2CO3, tert-BuOH, reflux; (e) LiOHꢀH2O, MeOH, THF, H2O, rt.
Previously, we reported that compound 1 acts as an inhibitor by
competing for the acetylated lysine substrate. The molecular mod-
el of compound 1 docked into the yeast Hst2 (homologue of SIRTs)

5902
T. Suzuki et al. / Bioorg. Med. Chem. 17 (2009) 5900–5905
O
S
amine of Tyr 229 (Hst2 numbering) (Fig. 3, left). To explore the novel
structure of the SIRT1 inhibitor, we designed compound 12 in which
the NH group of compound 1 is replaced with a sulfur atom. The con-
formation of compound 12 could be fixed by an intramolecular Sꢀ ꢀ ꢀO
interaction,22 and the CO group and the NH group of 12 could form
hydrogen bonds with Ala 227 and Tyr 229 (Fig. 3, right). As shown
in Table 1, compound 12 showed SIRT1-inhibitory activity, but its
potency of 12 was slightly less than that of 1.
O
OH
S
a
OH
2
Br
20
19
21
O
S
NH2
3.2. Cellular assays
b
Although several reports have suggested that SIRT1 is associ-
ated with cancer,3,4,9 only cambinol and salermide (Fig. 1) have
been reported to show anticancer activity.4,17 To investigate the
effectiveness of our SIRT1 inhibitors as anticancer agents, com-
pounds 1, 3, 5, and 7–9 were evaluated for their ability to inhibit
growth of tumor cells. Human colon cancer HCT116 cells and Bur-
kitt’s lymphoma Daudi cells were used as tumor cells, because the
silencing of SIRT1 in HCT116 cells has been reported to induce
apoptosis and BCL6, one of the substrates of SIRT1,9 has been re-
ported to be expressed in Daudi cells.4 As shown in Table 2, com-
pounds 1, 3, and 5 exerted tumor cell growth inhibitory activity.
Compared to compounds 1 and 7–9 that were weakly active to-
12
Scheme 3. Reagents and conditions: (a) Cu, CuI, N-methyl pyrrolidone, 200 °C; (b)
NH4Cl, Et3N, EDCI, HOBt, THF, rt.
catalytic core suggested that the NH2 group and the CO group of 1
form hydrogen bonds with the backbone carbonyl of Ala 227 and
with the backbone amine of Tyr 229, and the phenyl group of 1
blocks the entrance to the acetylated lysine substrate binding
pocket by interacting with hydrophobic amino acid residues19
(Fig. 2). Inspection of the simulated Hst2/1 complex also suggested
that the presence of a large space around the 3-position of the phe-
nyl ring of compound 1, which is formed by hydrophobic amino
acid residues (Val 182 and Phe 184) and hydrophilic amino acid
residues (His 135 and Glu 186) (Fig. 2). Therefore, we designed
compounds 2–9 in which hydrophobic (compounds 2–5) and
hydrophilic (compounds 6–9) substituents are introduced to the
3-position of the phenyl ring of 1. We anticipated that these sub-
stituents would interact with the amino acids forming the large
space, which might lead to a potent inhibition of SIRT1. As shown
in Table 1, compounds 2–9 showed similar SIRT1-inhibitory activ-
ity to compound 1. Among these, 3-phenyl analogue 3 displayed
wards HCT116 cells (4–21% inhibition at 100
lM), compounds 3
and 5 showed potent activity with GI50 of 43
l
M and 50 M,
l
respectively. Similarly, compounds 3 and 5 were more potent cell
growth inhibitors than 1 and 7–9 in the case of Daudi cells. These
results suggested that SIRT1 inhibitors bearing the 2-anilinobenza-
mide moiety are potential antitumor agents. Although the reason
for the more potent activity of compounds 3 and 5 is unclear, it
is reasonable to assume that it was due to the more efficient mem-
brane permeability resulting from the highly lipophilic character of
these compounds.
4. Conclusion
the highest activity with an IC50 of 52 lM. We also synthesized
We have designed and prepared a series of 2-anilinobenzamide
compounds 10 and 11, the para-isomers of compounds 3 and 5,
respectively, but they were much weaker inhibitors than com-
pounds 3 and 5, highlighting the importance of the substitution
at the 3-position of the phenyl ring.
In the calculated Hst2/1 complex, an intramolecular hydrogen
bond was observed between the CO group and the NH group of 119
(Fig. 2). The fixation of the conformation by the intramolecular
hydrogen bond is thought to enable the inhibitor to form a hydrogen
bond with the backbone carbonyl of Ala 227 and with the backbone
derivatives on the basis of the simulated Hst2/1 complex. Among
Ala 227
Ala 227
O
O
H
H
H
H
O
N
N
Tyr 229
N
S
Tyr 229
N
N
H
H
O
H
1
12
Figure 3. Stable conformations of 1 and 12 and their interactions with Ala 227 and
Tyr 229.
Table 2
Growth inhibition of HCT116 cells and Daudi cells by compounds 1, 3, 5, and 7–9a
Compound
HCT116
% Inhibition
Daudi
% Inhibition
GI50
(
lM)
GI50 (lM)
at 100
lM
at 100 lM
1
3
5
7
8
9
21
126
126
0
4
12
>100
43
50
>100
>100
>100
74
108
103
0
0
6
77
26
31
>100
>100
>100
Figure 2. View of the conformation of 1 (ball and stick) docked into the yeast Hst2
(homologue of SIRTs) catalytic core.
a
Values are means of at least three separate experiments.

T. Suzuki et al. / Bioorg. Med. Chem. 17 (2009) 5900–5905
5903
them, compounds 3 and 5 inhibited SIRT1 in enzyme assays and
suppressed the growth of Daudi and HCT116 cells.
In conclusion, we have identified novel lead structures from
which more potent SIRT1 inhibitors can be developed. The findings
of this study should pave the way for the development of new anti-
cancer drugs. Detailed studies of compounds 3 and 5 are under
way.
5.1.4. 2-[3-(2-Hydroxylethyl)phenylamino]benzamide (9)
Yield 15%; colorless oil: 1H NMR (CDCl3, 500 MHz, d; ppm) 9.52
(1H, broad s), 7.47 (1H, dd, J = 7.8 Hz, 1.4 Hz), 7.35–7.24 (3H, m),
7.11 (1H, d, J = 7.9 Hz), 7.08 (1H, s), 6.91 (1H, d, J = 7.6 Hz), 6.76
(1H, td, J = 7.4 Hz, 1.4 Hz), 3.87 (2H, t, J = 9.4 Hz), 2.85 (2H, t,
J = 6.4 Hz); MS (EI) m/z: 256 (M+); HRMS Calcd for C15H16N2O2:
256.121, Found: 256.121.
5.1.5. 2-(Biphenyl-4-ylamino)benzamide (10)
Yield 75%; mp 167–168 °C; 1H NMR (CDCl3, 500 MHz, d; ppm)
9.61 (1H, s), 7.59 (2H, d, J = 7.4 Hz), 7.56 (2H, d, J = 8.6 Hz), 7.49
(1H, dd, J = 8.0 Hz, 1.2 Hz), 7.46–7.38 (3H, m), 7.36–7.26 (4H, m),
6.78 (1H, td, J = 7.5 Hz, 1.2 Hz); MS (GC) m/z: 228 (M+); Anal. Calcd
for C19H16N2O: C, 79.14; H, 5.59; N, 9.72. Found: C, 78.88; H, 5.79;
N, 9.95.
5. Experimental
5.1. Chemistry
Melting points were determined using a Yanagimoto micro
melting point apparatus or a Büchi 545 melting point apparatus
and were left uncorrected. Proton nuclear magnetic resonance
spectra (1H NMR) were recorded on a JEOL JNM-LA500 spectrome-
ter in solvent as indicated. Chemical shifts (d) were reported in
parts per million relative to the internal standard tetramethylsil-
ane. Elemental analysis was performed with a Yanaco CHN COR-
DER NT-5 analyzer, and all values were within 0.4% of the
calculated values. Electron Ionization mass spectra (EI-MS) and
High-resolution mass spectra (HRMS) were recorded on a JEOL
JMS-SX102A mass spectrometer. GC–MS analyses were performed
on a Shimadzu GCMS-QP2010. Reagents and solvents were pur-
chased from Aldrich, Tokyo Kasei Kogyo, Wako Pure Chemical
Industries, and Kanto Kagaku and used without purification. Flash
column chromatography was performed using Silica Gel 60 (parti-
cle size 0.046–0.063 mm) supplied by Merck.
5.1.6. 2-(4-Phenoxyphenylamino)benzamide (11)
Yield 22%; mp 146–147 °C; 1H NMR (DMSO-d6, 500 MHz, d;
ppm) 10.0 (1H, s), 8.10 (1H, broad s), 7.70 (1H, dd, J = 7.9 Hz,
1.5 Hz), 7.45 (1H, broad s), 7.38 (2H, t, J = 7.3 Hz), 7.30 (1H, t,
J = 7.0 Hz), 7.20 (2H, d, J = 8.9 Hz), 7.17 (1H, d, J = 8.6 Hz), 7.10
(1H, t, J = 7.6 Hz), 7.00 (2H, d, J = 8.9 Hz), 6.99 (2H, d, J = 8.6 Hz),
6.76 (1H, t, J = 7.0 Hz); MS (EI) m/z: 304 (M+); Anal. Calcd for
C19H16N2O2ꢀ1/3H2O: C, 73.53; H, 5.41; N, 9.03. Found: C, 73.70;
H, 5.39; N, 9.06.
5.1.7. 3-Bromophenyl phenyl sulfide (16a)
A mixture of 3-bromoiodobenzene (15) (900
thiophenol (728 L, 7.07 mmol), CuI (69.0 mg, 0.36 mmol), K2CO3
(1.95 g, 14.1 mmol) and anhydrous ethylene glycol (789 L,
lL, 7.07 mmol),
l
l
5.1.1. 2-(3-Tolyamino)benzamide (2)
14.1 mmol) in i-PrOH (5 mL) was stirred at 80 °C for 18 h under
Ar. The reaction mixture was poured into water, and the whole
was extracted with AcOEt. The AcOEt layer was separated, washed
with brine, and dried over Na2SO4. Filtration, concentration in va-
cuo and purification by silica gel flash column chromatography
(n-hexane only) gave 1.19 g (64%) of 16a as a colorless oil: 1H
NMR (CDCl3, 500 MHz, d; ppm) 7.40–7.28 (7H, m), 7.25–7.11 (2H,
m); MS (GC) m/z: 263 (M+), 265 (M++2).
A mixture of 3-bromotoluene (14a) (357 lL, 2.94 mmol), 2-ami-
nobenzamide (13) (999 mg, 7.34 mmol), Pd2dba3 (80 mg,
0.088 mmol), 2-dicyclohexylphosphino-20,40,60-triisopropylbiphe-
nyl (211 mg, 0.44 mmol), K2CO3 (1.0 g, 7.36 mmol) in tert-BuOH
(6 mL) was refluxed at 80 °C for 18 h. The reaction mixture was
poured into 2 N aqueous HCl and the whole was extracted with
CHCl3. The CHCl3 layer was separated, washed with water and
brine, and dried over Na2SO4. Filtration, concentration in vacuo
and purification by silica gel flash column chromatography
(AcOEt/n-hexane = 2/3) gave 116 mg (17%) of 2 as an off-white so-
lid. The solid was recrystallized from AcOEt/n-hexane to give
58 mg of 2 as colorless crystals: mp 93–95 °C; 1H NMR (CDCl3,
500 MHz, d; ppm) 9.46 (1H, s), 7.46 (1H, dd, J = 7.9 Hz, 1.5 Hz),
7.33–7.28 (2H, m), 7.20 (1H, t, J = 8.1 Hz), 7.05–6.98 (2H, m), 6.86
(1H, d, J = 7.6 Hz), 6.74 (1H, td, J = 7.3 Hz, 1.5 Hz), 2.33 (3H, s);
MS (EI) m/z: 226 (M+); HRMS Calcd for C14H14N2O 226.111, Found
226.111.
5.1.8. 1-Bromo-3-phenoxybenzene (16b)
A mixture of 1,3-dibromobenzene (17) (25 g, 106 mmol), phe-
nol (998 lL, 0.21 mmol), K2CO3 (1.46 g, 106 mmol) and Cu (2.0 g,
31.8 mmol) was stirred at 165 °C. After 1.5 h, the mixture was di-
luted with THF. The mixture was poured into water, and the whole
was extracted with AcOEt. The AcOEt layer was separated, washed
with brine, and dried over Na2SO4. Filtration, concentration in va-
cuo and purification by silica gel flash column chromatography
(n-hexane only to AcOEt/n-hexane = 1/1) gave 475 mg (18%) of
16b as a yellow oil: 1H NMR (CDCl3, 500 MHz, d; ppm) 7.36 (2H,
t, J = 8.0 Hz), 7.26–7.13 (4H, m), 7.02 (2H, dd, J = 8.7 Hz, 1.1 Hz),
6.93 (1H, m); MS (GC) m/z: 248 (M+), 250 (M++2).
Compounds 3 and 8–11 were prepared from 2-aminobenzam-
ide (13) and an appropriate bromobenzene (13b–f) using the pro-
cedure described for 2.
5.1.2. 2-(Biphenyl-3-ylamino)benzamide (3)
5.1.9. 3-(3-Bromophenyl)acrylic acid methyl ester (16c)
To a solution of trans-3-bromocinnamic acid (18) (201 mg,
0.89 mmol) in MeOH (5 mL) was added concentrated HCl
(0.1 mL). The solution was heated at 80 °C for 17 h. After cooling
the solution, the acid was neutralized with saturated aqueous
Na2CO3. The aqueous solution was extracted with ether. The organ-
ic phase was washed with brine, dried over Na2SO4, filtered and
concentrated in vacuo to give 184 mg (86%) of 16c as a white solid:
1H NMR (CDCl3, 500 MHz, d; ppm) 7.67 (1H, s), 7.61 (1H, d,
J = 16.2 Hz), 7.51 (1H, d, J = 7.9 Hz), 7.44 (1H, d, J = 7.9 Hz), 7.26
(1H, t, J = 7.9 Hz), 6.43 (1H, d, J = 16.1 Hz), 3.81 (3H, s); MS (GC)
m/z: 240 (M+), 242 (M++2).
Yield 29%; mp 143–144 °C; 1H NMR (CDCl3, 500 MHz, d; ppm)
9.61 (1H, s), 7.57 (2H, dd, J = 8.1 Hz, 1.1 Hz), 7.49–7.28 (10H, m),
7.21 (1H, d, J = 6.4 Hz), 6.77 (1H, td, J = 7.5 Hz, 1.2 Hz); MS (GC)
m/z: 228 (M+); Anal. Calcd for C19H16N2Oꢀ1/7H2O: C, 78.44; H,
5.64; N, 9.63. Found: C, 78.44; H, 5.69; N, 9.59.
5.1.3. 2-(3-Hydroxymethylphenylamino)benzamide (8)
Yield 14%; mp 117–119 °C; 1H NMR (CDCl3, 500 MHz, d; ppm)
9.54 (1H, broad s), 7.47 (1H, dd, J = 7.9 Hz, 1.5 Hz), 7.35–7.28 (3H,
m), 7.23 (1H, s), 7.15 (1H, d, J = 8.2 Hz), 7.03 (1H, d, J = 7.3 Hz),
6.77 (1 H, t, J = 8.1 Hz, 1.2 Hz), 6.01 (2H, broad s), 4.68 (2H, s);
MS (GC) m/z: 242 (M+); Anal. Calcd for C14H14N2O2: C, 69.41; H,
5.82; N, 11.56. Found: C, 69.16; H, 6.06; N, 11.38.
Compounds 4–6 were prepared from 2-aminobenzamide (13)
and 16a–c using the procedure described for 2.

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T. Suzuki et al. / Bioorg. Med. Chem. 17 (2009) 5900–5905
5.1.10. 2-(3-Phenylsulfanylphenylamino)benzamide (4)
Yield 6%; colorless oil: 1H NMR (CDCl3, 500 MHz, d; ppm) 9.52
(1H, broad s), 7.45 (1H, d, J = 7.6 Hz), 7.39 (2H, d, J = 7.3 Hz), 7.32
(2H, t, J = 7.3 Hz), 7.23 (3H, t, J = 7.9 Hz), 7.18 (1H, s), 7.06 (1H, d,
J = 7.3 Hz), 6.97 (2H, d, J = 8.7 Hz), 6.76 (1H, td, J = 6.8 Hz, 2.2 Hz);
MS (EI) m/z: 320 (M+); HRMS Calcd for C19H16N2OS: 320.098,
Found: 320.099.
AcOEt to give 209 mg of 12 as colorless crystals: mp 170–174 °C;
1H NMR (DMSO-d6, 500 MHz, d; ppm) 7.90 (1H, broad s), 7.53
(1H, dd, J = 7.6 Hz, 1.5 Hz), 7.48 (1H, broad s), 7.43–7.39 (5H, m),
7.30 (1 H, td, J = 7.6 Hz, 1.5 Hz), 7.23 (1H, td, J = 7.4 Hz, 1.1 Hz),
6.91 (1H, dd, J = 7.9 Hz, 0.7 Hz); MS (GC) m/z: 229 (M+); Anal. Calcd
for C13H11NOSꢀ1/6H2O: C, 67.21; H, 4.92; N, 6.03. Found: C, 67.13;
H, 4.94; N, 6.10.
5.1.11. 2-(3-Phenoxyphenylamino)benzamide (5)
5.2. Biology
Yield 42%; mp 104–105 °C; 1H NMR (CDCl3, 500 MHz, d; ppm)
9.53 (1H, broad s), 7.46 (1H, dd, J = 7.9 Hz, 1.5 Hz), 7.38–7.23 (5H,
m), 7.10 (1H, t, J = 7.3 Hz), 7.04 (2H, dd, J = 8.7 Hz, 1.1 Hz), 6.94
(1H, dd, J = 8.2 Hz, 1.2 Hz), 6.88 (1H, t, J = 2.3 Hz), 6.77 (1H, td,
J = 7.5 Hz, 1.1 Hz), 6.66 (1H, dd, J = 8.2 Hz, 1.5 Hz); MS (GC) m/z:
304 (M+); Anal. Calcd for C19H16N2O2ꢀ1/10H2O: C, 74.54; H, 5.33;
N, 9.15. Found: C, 74.54; H, 5.63; N, 9.01.
5.2.1. SIRT1-inhibitory activity assay
The SIRT1 activity assay was performed using SIRT fluorimetric
drug discovery kit (AK-555, BIOMOL Research Laboratories),
according to the supplier’s protocol. SIRT1 (human, recombinant)
(0.067 U/
de Lys-SIRT1 substrate, 25
l
L, 5
l
l/well) was incubated at 37 °C with 25
lM of Fluor
l
M of NAD+ and various concentrations
of samples. Reactions were stopped after 60 min by adding Fluor
de LysTM Developer II with nicotinamide which stops further
deacetylation. Then, 45 min after addition of this developer, the
fluorescence of the wells was measured on a fluorometric reader
with excitation set at 360 nm and emission detection set at
460 nm, and the % inhibition was calculated from the fluorescence
readings of inhibited wells relative to those of control wells. The
compound concentration resulting in 50% inhibition was deter-
mined by plotting the log [Inh] versus the logit function of the%
inhibition. IC50 values were determined using a regression analysis
of the concentration/inhibition data.
5.1.12. 3-[3-(2-Carbamoylphenylamino)phenyl]acrylic acid
methyl ester (6)
Yield 46%; mp 119–120 °C; 1H NMR (CDCl3, 500 MHz, d; ppm)
9.60 (1H, broad s), 7.65 (1H, d, J = 15.8 Hz), 7.49 (1H, d,
J = 8.5 Hz), 7.38 (1H, s), 7.34–7.31 (3H, m), 7.22 (1H, d, J = 8.2 Hz),
7.19 (1H, d, J = 7.6 Hz), 6.81 (1H, td, J = 7.0 Hz, 2.3 Hz), 6.41 (1H,
d, J = 16.1 Hz), 3.81 (3H, s); MS (GC) m/z: 296 (M+); Anal. Calcd
for C17H16N2O3: C, 68.91; H, 5.44; N, 9.45. Found: C, 68.62; H,
5.56; N, 9.34.
5.1.13. 3-[3-(2-Carbamoylphenylamino)phenyl]acrylic acid (7)
To a solution of 6 (411 mg, 1.39 mmol) in MeOH (3 mL), H2O
(3 mL) and THF (2.0 mL) was added LiOHꢀH2O (598 mg,
14.25 mmol). The reaction mixture was stirred at room tempera-
ture for 51 h. The mixture was diluted with AcOEt and the whole
was extracted with water. The aqueous layer was neutralized with
2 N aqueous HCl, and extracted with AcOEt. The AcOEt layer was
separated, washed with water and brine, and dried over Na2SO4.
Filtration, concentration in vacuo and purification by silica gel flash
column chromatography (CHCl3/MeOH = 19/1) gave 7 as a pale yel-
low solid: mp 246–249 °C; 1H NMR (DMSO-d6, 500 MHz, d; ppm)
10.03 (1H, s), 8.07 (1H, broad s), 7.71 (1H, d, J = 7.9 Hz), 7.55 (1H,
d, J = 15.8 Hz), 7.49 (1H, broad s), 7.44 (1H, s), 7.37–7.28 (4H, m),
7.21 (1H, d, J = 8.8 Hz), 6.83 (1H, t, J = 7.3 Hz), 6.50 (1H, d,
J = 16.1 Hz); MS (GC) m/z: 282 (M+); Anal. Calcd for C16H14N2O3:
C, 68.07; H, 5.00; N, 9.92. Found: C, 67.83; H, 5.38; N, 9.52.
5.2.2. Cell growth inhibition assay
HCT116 human colon cancer cells and Daudi human lymph
cancer cells were purchased from American Type Culture Collec-
tion (ATCC, Manassas, VA) and cultured in McCoy’s 5A culture
medium (Sigma) and RPMI culture medium (GIBCO) containing
penicillin and streptomycin supplemented with fetal bovine ser-
um as described in the ATCC instructions, respectively. HCT116
cells and Daudi cells were plated in 96-well plates at initial den-
sities of 5000 cells/ well (50
After 24 h, cells were exposed to a solution of test compounds
in McCoy’s medium or RPMI medium (50 L) at various concen-
trations at 37 °C in 5% CO2 for 72 h. Then, 10
L of alamarBlueTM
lL/ well) and incubated at 37 °C.
l
l
was added, and cells were incubated at 37 °C for 3 h. The fluores-
cence of the wells was measured by a florometric platereader
with excitation set at 530 nm and emission detection set at
590 nm, and the percentage of cell growth was calculated for
the fluorescence reading.
5.1.14. 2-Phenylsulfanylbenzoic acid (21)
A mixture of 2-bromobenzoic acid (19) (483 mg, 2.40 mmol),
Ph2S2 (20) (220 mg, 1.01 mmol), Cu (65 mg, 1.02 mmol) and CuI
(190 mg, 1.00 mmol) in NMP (10 mL) was heated at 200 °C for
1 h. The resultant solution was acidified to pH 2 with 2 N aqueous
HCl, and extracted with AcOEt. The AcOEt layer was separated,
washed with brine and dried over Na2SO4. Filtration and concen-
tration in vacuo gave 522 mg (95%) of 21 as a crude solid: 1H
NMR (DMSO-d6, 500 MHz, d; ppm) 7.91 (1H, dd, J = 7.8 Hz,
1.4 Hz), 7.55–7.50 (5H, m), 7.36 (1H, t, J = 7.6 Hz), 7.21 (1H, t,
J = 7.7 Hz), 6.72 (1H, dd, J = 8.1 Hz, 0.8 Hz).
Acknowledgements
This work was supported in part by a Grant-in-Aid for Scientific
Research from Japan Society for the Promotion of Science, a Grant-
in Aid for Research in Nagoya City University, and a grant from
Ichihara International Scholarship Foundation.
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