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J=1.5 Hz, 1H), 8.26 (d, J=8.0 Hz, 1H), 8.01 (dt, J=1.5, 8.0 Hz, 1H),
7.69 (t, J=8.0 Hz, 1H), 7.44 (d, J=7.0 Hz, 2H), 7.30 (t, J=7.5 Hz,
2H), 7.23 (t, J=7.5 Hz, 1H), 4.26 ppm (s, 2H); 13C NMR (125 MHz,
CD3OD): d=176.6, 170.3, 166.6, 135.8, 135.0, 132.5, 131.9, 131.8,
130.9, 130.2, 128.3, 127.7, 125.7, 29.8 ppm; EIMS: m/z 327 [M+];
Anal: calcd for C16H13N3O3S: C 58.70, H 4.00, N 12.84, found: C
58.31, H 4.14, N 12.53.
tration in vacuo gave 811 mg (quant.) of 35 as a brown oil:
1H NMR (500 MHz, CDCl3): d=8.24 (s, 1H), 7.96 (dt, J=1.4, 7.7 Hz,
1H), 7.74 (ddd, J=1.2, 2.0, 6.1 Hz, 1H), 7.21 (d, J=4.0 Hz, 1H), 7.10
(d, J=3.5 Hz, 1H), 4.75 (s, 2H), 3.95 ppm (s, 3H); EIMS: m/z 310,
312 [M+].
3-(5-Phenylthiomethylthiophen-2-yl)-N-hydroxybenzamide (12):
Compound 12 (36% yield) was prepared from compound 35 ac-
cording to the procedure for the preparation of compound 8: mp:
3-Azido benzoic acid ethyl ester (31): An aqueous solution
(30 mL) of NaNO2 (3.69 g, 53.4 mmol) was added to a solution of 3-
amino benzoic acid ethyl ester 30 (2.02 g, 12.2 mmol) in TFA
(17 mL) at 08C. The mixture was stirred at 08C for 1.5 h, then an
aqueous solution (14 mL) of NaN3 (4.05 g, 62.3 mmol) was added.
The solution was stirred at room temperature for 2.5 h. The mix-
ture was poured into 2 N aqueous HCl and extracted with EtOAc.
The organic layer was washed with brine and dried over Na2SO4.
Filtration, concentration in vacuo, and purification by silica gel
flash column chromatography (eluting with a 2–20% EtOAc/n-
1
154–1558C: H NMR (500 MHz, [D6]DMSO): d=11.32 (s, 1H), 7.92 (s,
1H), 7.72 (d, J=7.5 Hz, 1H), 7.65 (d, J=8.5 Hz, 1H), 7.47 (t, J=
7.5 Hz, 1H), 7.40–7.36 (m, 3H), 7.32 (t, J=7.7 Hz, 2H), 7.21 (t, J=
7.2 Hz, 1H), 7.01 (d, J=3.5 Hz, 1H), 4.51 ppm (s, 2H); 13C NMR
(125 MHz, CDCl3): d=163.6, 141.5, 141.5, 135.2, 133.7, 133.4, 129.2,
128.9, 128.6, 127.9, 127.5, 126.2, 125.8, 123.7, 123.2, 31.6 ppm;
EIMS: m/z 341 [M+]; Anal: calcd for C18H15NO2S2·0.5H2O: C 61.19, H
4.60, N 4.00, found: C 61.77, H 4.76, N 4.10.
1
hexane gradient) gave 2.20 g (94%) of 31 as a yellow oil: H NMR
(500 MHz, CDCl3): d=7.82 (dt, J=1.0, 8.5 Hz, 1H), 7.70 (t, J=1.7 Hz,
1H), 7.68 (d, J=8.0 Hz, 1H), 7.42 (t, J=8.0 Hz, 1H), 7.19 (ddd, J=
0.9, 2.4, 6.4 Hz, 1H), 4.39 (q, J=7.3 Hz, 2H), 1.40 ppm (t, J=7.0 Hz,
3H); FTIR (neat): n˜ =2102 cmÀ1
.
Biology
Enzyme assays: HDAC activity assays were performed using an
HDACs/HDAC8 deacetylase fluorimetric assay kit (CY-1150/CY-1158,
Cyclex Company Limited), an HDAC1/HDAC6 fluorescent activity
drug discovery kit (AK-511/AK-516, BIOMOL Research Laboratories),
a Fluorescent SIRT1 Activity Assay/Drug Discovery Kit (AK-555,
3-[4-(Phenylthiomethyl)-1H-1,2,3-triazol-1-yl]benzoic acid ethyl
ester (32): An aqueous solution (10 mL) of CuSO4 (83.7 mg,
523 mmol) and sodium ascorbate (0.585 g, 2.95 mmol) was added
to a solution of phenyl propargyl sulfide (0.775 g, 5.23 mmol) and
azide 31 (0.980 g, 5.12 mmol) in MeOH (10 mL). The reaction mix-
ture was stirred for 21 h at room temperature, then filtered
through Celite. After removal of the solvent, the residue was puri-
fied by silica gel flash column chromatography (eluting with a 5–
40% EtOAc/n-hexane gradient) to give 1.17 g (67%) of 32 as
BIOMOL Research Laboratories) or and
a Fluorogenic HDAC
Class2a Assay Kit (BPS Bioscience Incorporated), with total HDACs
(CY-1150, Cyclex Company Limited), HDAC1 (SE-456, BIOMOL Re-
search Laboratories), HDAC2 (SE-500, BIOMOL Research Laborato-
ries), HDAC3/NCOR1 complex (SE-515, BIOMOL Research Laborato-
ries), HDAC4 (BPS Bioscience Incorporated), HDAC6 (SE-508,
BIOMOL Research Laboratories), and HDAC8 (CY-1158, Cyclex Com-
pany Limited), according to the supplier’s instructions. Fluores-
cence of the wells was measured on a fluorimetric reader with ex-
citation set at 360 nm and emission detection set at 460 nm, and
the percent inhibition was calculated from the fluorescence read-
ings of inhibited wells relative to those of control wells. The con-
centration of a compound that results in 50% inhibition (IC50) was
determined by plotting log[inhibitor] versus the log function of
percent inhibition.
1
a white solid: H NMR (500 MHz, CDCl3): d=8.27 (t, J=1.7 Hz, 1H),
8.10 (dt, J=1.2, 8.0 Hz, 1H), 7.95 (ddd, J=1.0, 2.2, 6.2 Hz, 1H), 7.84
(s, 1H), 7.60 (t, J=8.0 Hz, 1H), 7.38 (d, J=7.5 Hz, 2H),7.30 (t, J=
7.7 Hz, 2H), 7.22 (t, J=7.5 Hz, 1H), 4.43 (q, J=7.3 Hz, 2H), 4.33 (s,
2H), 1.43 ppm (t, J=7.2 Hz, 3H); EIMS: m/z 339 [M+].
3-[4-(Phenylthiomethyl)-1H-1,2,3-triazol-1-yl]-N-hydroxybenza-
mide (11): Compound 11 (48% yield) was prepared from com-
pound 32 according to the procedure for the preparation of com-
1
pound 8: mp: 179–1808C: H NMR (500 MHz, [D6]DMSO): d=11.40
(s, 1H), 9.22 (s, 1H), 8.74 (s, 1H), 8.22 (s, 1H), 8.01 (d, J=8.0 Hz,
1H), 7.84 (d, J=7.5 Hz, 1H), 7.67 (d, J=8.0 Hz, 1H), 7.41 (d, J=
8.0 Hz, 2H), 7.33 (t, J=7.7 Hz, 2H), 7.20 (t, J=7.2 Hz, 1H), 4.39 ppm
(s, 2H); 13C NMR (125 MHz, [D6]DMSO): d=162.8, 144.9, 136.4,
135.5, 134.3, 130.1, 129.0, 128.2, 126.8, 126.0, 122.4, 121.5, 118.3,
27.1 ppm; EIMS: m/z 326 [M+]; Anal: calcd for C16H14N4O2S: C
58.88, H 4.32, N 17.17, found: C 58.69, H 4.54, N 17.00.
Western blot analysis: The cohesin, a-tubulin, or H3K9 acetylating
activities of the test compounds were assayed according to pub-
lished methods.[3,19]
.
Cell growth inhibition assay: Cells were plated at an initial density
of 2ꢃ105 cells per well (50 mL per well) in 96-well plates in RPMI
1640 medium with 10% fetal bovine serum, and were exposed to
inhibitors for 72 h at 378C in a 5% CO2 incubator. A solution of 3-
(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-
phenyl)-2H-tetrazolium inner salt (MTS) was then added (20 mL per
well), and incubation was continued for 2 h. The solubilized dye
was quantified by colorimetric reading at 490 nm using a reference
wavelength of 650 nm. The absorbance values of control wells (C)
and test wells (T) were measured. The absorbance of the test wells
was also measured at time 0 (T0, addition of compounds). Using
these measurements, cell growth inhibition (percentage of growth)
by a test inhibitor at each concentration used was calculated as:
%growth=100ꢃ[(TÀT0)/(CÀT0)], when T>T0 and %growth=100ꢃ
[(TÀT0)/T], when T<T0. Computer analysis of the percent growth
values afforded a 50% growth inhibition parameter (GI50). The GI50
was calculated as 100ꢃ[(TÀT0)/(CÀT0)]=50.
3-(5-Methylthiophen-2-yl)benzoic acid methyl ester (34): Com-
pound 34 (69% yield) was prepared from 3-(methoxycarbonyl)phe-
nylboronic acid (14) and 2-iodo-3-methylthiophene (33) according
to the procedure for the preparation of compound 15: 1H NMR
(500 MHz, CDCl3): d=8.22 (s, 1H), 7.90 (d, J=8.0 Hz, 1H), 7.72 (d,
J=7.5 Hz, 1H), 7.42 (t, J=7.7 Hz, 1H), 7.18 (d, J=3.5 Hz, 1H), 6.75–
6.74 (m, 1H), 3.94 (s, 3H), 2.52 ppm (s, 3H); EIMS: m/z 232 [M+].
3-(5-Bromomethylthiophen-2-yl)benzoic acid methyl ester (35):
NBS (0.554 g, 3.11 mmol) and AIBN (19.2 mg, 0.116 mmol) were
added to a solution of compound 34 (0.601 g, 2.61 mmol) in CCl4
(20 mL). The mixture was stirred at reflux for 1 h, then diluted with
CCl4 and filtered. The filtrate was evaporated, poured into H2O, and
extracted with EtOAc. The organic layer was separated, washed
with H2O and brine, and dried over Na2SO4. Filtration and concen-
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ChemMedChem 2014, 9, 657 – 664 663