Synthesis and Biological Evaluation of 3-(4-Substituted-phenyl)-N-hydroxy-2-propenamides, a New Class of Histone Deacetylase Inhibitors

Article abstract of DOI:10.1021/jm030377q

Inhibitors of histone deacetylases (HDACs) have been shown to induce differentiation and/or apoptosis of human tumor cells. Novel 3-(4-substituted-phenyl)-N-hydroxy-2-propenamides have been prepared as a new class of HDAC inhibitors and evaluated for their antiproliferative activity and HDAC inhibitory activity. Incorporation of a 1,4-phenylene carboxamide linker, shown by 5, and a 4-(dimethylamino)phenyl or 4-(pyrrolidin-1-yl)phenyl group as a cap substructure generated highly potent hydroxamic acid-based HDAC inhibitors 5a and 5b.

Full text of DOI:10.1021/jm030377q

J . Med. Chem. 2003, 46, 5745-5751
5745
Syn th esis a n d Biologica l Eva lu a tion of
3-(4-Su bstitu ted -p h en yl)-N-h yd r oxy-2-p r op en a m id es, a New Cla ss of Histon e
Dea cetyla se In h ibitor s
Dae-Kee Kim,*,† J u Young Lee,‡ J ae-Sun Kim,‡ J e-Ho Ryu,‡ J in-Young Choi,‡ J un Won Lee,‡ Guang-J in Im,‡
Tae-Kon Kim,‡ J ung Woo Seo,‡ Hyun-J u Park,§ J akyung Yoo,§ J ung-Hyun Park,| Tae-You Kim,| and
Yung-J ue Bang|
In2Gen Co., Ltd., 28 Yongon-dong, Chongno-gu, Seoul 110-799, Korea, Life Science Research Center, SK Chemicals,
600 J ungja-dong, Changan-gu, Suwon, Kyungki-do 440-745, Korea, College of Pharmacy, Sungkyunkwan University,
Suwon, Kyungki-do 440-746, Korea, and National Research Laboratory for Cancer Epigenetics, Cancer Research Institute,
Seoul National University College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-799, Korea
Received August 7, 2003
Inhibitors of histone deacetylases (HDACs) have been shown to induce differentiation and/or
apoptosis of human tumor cells. Novel 3-(4-substituted-phenyl)-N-hydroxy-2-propenamides have
been prepared as a new class of HDAC inhibitors and evaluated for their antiproliferative
activity and HDAC inhibitory activity. Incorporation of a 1,4-phenylene carboxamide linker,
shown by 5, and a 4-(dimethylamino)phenyl or 4-(pyrrolidin-1-yl)phenyl group as a cap
substructure generated highly potent hydroxamic acid-based HDAC inhibitors 5a and 5b.
In tr od u ction
the hydroxamic acid functionality penetrates a narrow,
hydrophobic channel and chelates a buried zinc ion.26
HDAC inhibitors typically possess a metal-binding
functionality, a cap substructure that interacts with
amino acids at the entrance rim of the N-acetyllysine
binding channel, and a linker connecting the cap and
the metal-binding functionality.26 A careful analysis of
the structure-activity relationships (SAR) on the hy-
droxamic acid- and the anilide-based HDAC inhibitors
suggests that the 1,4-phenylene carboxamide linker
shown as the target compounds 5 would be the most
optimized one.13,16-22,24,25 In addition to this assumption,
we further refined the cap substructure with 4-substi-
tuted phenyl or pyridyl groups. Since TSA has a
dimethylamino group at the 4-position of the phenyl
ring, we were particularly interested in introducing an
acyclic or a cyclic tertiary amine at the same position.
Thus, dimethylamino, pyrrolidinyl, and 4-substituted-
piperazinyl groups were chosen for this purpose. In
general, hydroxamates have poor bioavailability;27,28
therefore, we adopted a pyridyl ring as a cap substruc-
ture, which would allow better physicochemical proper-
ties due to its both lipophilic and hydrophilic character,
as shown in MS-275.
Acetylation and deacetylation of histones of the core
proteins of nucleosomes in chromatin play an important
role in the regulation of gene expression.1-3 The level
of acetylation of histones is established and maintained
by two classes of enzymes, histone acetyltransferases
(HATs) and histone deacetylases (HDACs), which have
been identified as transcriptional coactivators and
transcriptional corepressors, respectively.4,5 There is
increasing evidence that aberrant histone acetylation
has been linked to various malignant diseases.6-8
HDACs are zinc hydrolases that modulate deacetyl-
ation of the ꢀ-N-acetyl groups of lysine residues in the
N-terminal tails of core histones. Natural products such
as trichostatin A (TSA) (1, Chart 1),9 trapoxin B,10 and
depsipeptide11 strongly inhibit HDACs, induce dif-
ferentiation of cancer cells, and suppress cell prolifera-
tion. Depsipeptide is now under clinical evaluation;
however, TSA and trapoxin B have not been developed
clinically owing to their instability in vivo and toxicity.12
A number of the hydroxamic acid-based synthetic
inhibitors, such as suberoylanilide hydroxamic acid
(SAHA) (2),13-15 oxamflatin (3)16 and others,17-22 and
the anilide-based synthetic inhibitors, such as MS-275
(4)23,24 and its analogues,25 have also been reported to
induce differentiation of cancer cells and inhibit cell
proliferation. Among them, SAHA14,15 and MS-27523
show potent in vivo antitumor effects in tumor-bearing
animals and are now under clinical evaluation. Crystal
structures of a bacterial HDAC homologue (HDAC-like
protein, HDLP) bound to TSA and SAHA reveal that
Resu lts a n d Discu ssion
Ch em ica l Syn th esis. The hydroxamic acids 5a -d
were prepared as shown in Scheme 1. Coupling of the
carboxylic acids 6a -c with an amino alcohol 729 by
using either bis(2-oxo-3-oxazolidinyl)phosphinic chloride
(BOP-Cl) and Et3N in CH2Cl2 (for 8a ) or 1-(3-dimethyl-
aminopropyl)-3-ethylcarbodiimide hydrochloride (EDC)
and 1-hydroxybenzotriazole (HOBT) in the presence of
DMAP in pyridine (for 8b and 8c) gave the amides 8a -
c. Swern oxidation of the hydroxy group of 8a -c with
(COCl)2 and DMSO in THF followed by Wittig reaction
of the resulting aldehydes with (carbethoxymethylene)-
triphenylphosphorane in CH3CN afforded the 2-prope-
noic acid esters 9a -c. Coupling of picolinic acid (6d )
* To whom correspondence should be addressed. Present address:
College of Pharmacy, Ewha Womans University, 11-1 Daehyun-dong,
Seodaemun-gu, Seoul 120-750, Korea, Tel: 82 2 3277 3025. Fax: 82 2
3277 2467. E-mail: dkkim@ewha.ac.kr.
† In2Gen Co., Ltd.
‡ Life Science Research Center, SK Chemicals.
§ Sungkyunkwan University.
| Seoul National University College of Medicine.
10.1021/jm030377q CCC: $25.00 © 2003 American Chemical Society
Published on Web 11/22/2003

5746 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26
Kim et al.
Ch a r t 1
Sch em e 1a
Sch em e 2a
a
(a) BOP-Cl, Et3N, CH2Cl2, rt, overnight (for 8a ), or EDC,
HOBT, DMAP, pyridine, rt, overnight (for 8b and 8c); (b) (i)
(COCl)2, DMSO, THF, -78 °C, 1 h, then warmed to -35 °C, 10
min, then cooled to -78 °C, add Et3N, 0 °C, 1 h; (ii) Ph3Pd
CHCO2Et, CH3CN, 70 °C, 2 h; (c) EDC, DMAP, DMF, rt, 4 h; (d)
NH2OH, MeOH, rt, overnight.
a
(a) anhyd K2CO3, DMF, 80 °C, overnight; (b) conc HCl, 80 °C,
overnight; (c) EDC, HOBT, DMAP, pyridine, rt, overnight; (d)
NH2OH, MeOH, rt, overnight.
with an amino ester 1030 under similar reaction condi-
tions as for 8b and 8c produced the ester 9d . Treatment
of 9a -d with NH2OH in MeOH yielded the hydroxamic
acids 5a -d . The hydroxamic acids 5e-g were synthe-
sized in four steps, as shown in Scheme 2. Reaction of
1-(pyridylmethyl)piperazines 11a -c31 with 4-fluoroben-
zonitrile (12) in the presence of anhydrous K2CO3 in
DMF provided the benzonitriles 13a -c. Hydrolysis of
13a -c with concentrated HCl followed by coupling
with 10 under the same reaction conditions as for
8b and 8c afforded the esters 14a -c, which were
treated with NH2OH in MeOH to give the hydroxamic
acids 5e-g.
Biologica l Eva lu a tion . Antiproliferative activities
of the new HDAC inhibitors 5a -g against human lung
cancer (A549), breast cancer (SK-BR-3), and stomach
cancer (MKN45) cell lines were evaluated using the
MTT assay32,33 (spectrophotometric quantification of
viable cells as measured by mitochondrion-catalyzed
formation of a formazan from a tetrazolium salt) in
comparison to TSA, oxamflatin, and MS-275. The re-
sults are given as IC50 values and are shown in Table
1. The 4-(dimethylamino)phenyl compound 5a and the
4-(pyrrolidin-1-yl)phenyl compound 5b showed better
antiproliferative activity against all the three cancer cell

3-(4-Substituted-phenyl)-N-hydroxy-2-propenamides
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26 5747
Ta ble 1. Antiproliferative and HDAC Inhibitory Activities of Compounds 5a-g, Oxamflatin, TSA, and MS-275
IC50 (µM)a,b
HDAC
compd
A549
SK-BR-3
MKN45
inhibnc (nM)b
5a
5b
5c
5d
5e
5f
0.48 ( 0.13
0.35 ( 0.22
2.89 ( 0.31
2.23 ( 0.99
11.98 ( 3.97
1.65 ( 0.32
3.02 ( 0.95
0.08 ( 0.02
0.84 ( 0.19
3.58 ( 0.50
0.16 ( 0.06
0.11 ( 0.01
1.28 ( 0.23
0.87 ( 0.04
3.19 ( 0.95
0.27 ( 0.08
1.72 ( 0.33
0.02 ( 0.01
0.59 ( 0.04
0.87 ( 0.10
0.83 ( 0.28
0.80 ( 0.27
4.98 ( 0.44
3.64 ( 1.74
38.59 ( 15.85
2.94 ( 0.91
56.05 ( 16.51
0.10 ( 0.03
1.07 ( 0.20
4.16 ( 1.74
172.02 ( 8.22
205.27 ( 8.46
370.97 ( 4.54
941.61 ( 7.98
569.97 ( 4.13
5g
TSA (1)
oxamflatin (3)
MS-275 (4)
53.29 ( 3.68
233.15 ( 4.86
2194.59 ( 6.54
a
Antiproliferative activity against human cancer cell lines was evaluated using the MTT assay, and the concentration of compound
causing 50% cell growth inhibition (IC50) was determined. Data represent the mean ( SD (n ) 3). c HDAC activity was analyzed at
various concentrations of compound by measuring a fluorescent HDAC substrate. Test compounds were diluted serially, and the SNU-16
cell nuclear extract was added. Substrate was added and the mixture was incubated for 10 min at 25 °C prior to being assayed for HDAC
activity.
b
lines tested than the pyridyl compounds 5c and 5d and
the 4-(4-pyridylmethylpiperazin-1-yl)phenyl compounds
5e-g. Compound 5b showed the most significant anti-
proliferative activity with IC50 values of 0.35 ( 0.22,
0.11 ( 0.01, and 0.80 ( 0.27 µM against A549, SK-BR-
3, and MKN45 cancer cell lines, respectively. These
values were 5-10-fold lower than those seen with MS-
275 and even lower than those seen with oxamflatin,
one of the most potent synthetic hydroxamic acid-based
HDAC inhibitors known. Furthermore, compound 5b
was only 4-8-fold less potent than TSA. The inhibitory
potency of compound 5a [IC50 ) 0.48 ( 0.13 (A549), 0.16
( 0.06 (SK-BR-3), 0.83 ( 0.28 (MKN45) µM] was
F igu r e 1. Compound 5a (green) superimposed onto TSA
comparable to that of compound 5b. Among the pyridyl-
(magenta) within the HDLP binding site. Oxygen atoms of the
ligands are red and nitrogen atoms are blue.
methyl-substituted piperazine derivatives 5e-g, the
3-pyridyl derivative 5f was more potent as an antipro-
liferative agent than the 2-pyridyl (5e) and the 4-pyridyl
(5g) derivatives.
An HDAC inhibition assay34,35 was performed using
a nuclear extract from SNU-16 (human gastric adeno-
carcinoma) cells (Table 1). Expectedly, compounds with
better antiproliferative profiles (5a and 5b) inhibited
HDACs more than the less active antiproliferative
agents (5c, 5d , and 5f). Compound 5a was the most
active derivative (IC50 ) 172.02 ( 8.22 nM), showing
13- and 1.4-fold more HDAC inhibition than MS-275
(IC50 ) 2194.59 ( 6.54 nM) and oxamflatin (IC50
)
233.15 ( 4.86 nM), respectively. Compound 5a exhibited
only a 3.2-fold lower potency than TSA (IC50 ) 53.29 (
3.68 nM). Compound 5b (IC50 ) 205.27 ( 8.46 nM) was
slightly weaker as an HDAC inhibitor than compound
5a .
Dock in g Stu d y. To examine a possible binding mode
of 5a at the active site of HDAC, flexible docking was
conducted using the FlexX program implanted in Sybyl
6.9 (Tripos Inc.).36 The reference protein coordinate used
for docking was taken from the X-ray structure (PDB
entry 1c3r) of bacterial HDLP complexed with TSA.26
Three-dimensional structures of 5a and reference mol-
ecule (TSA) were prepared and then docked into the
protein active site. A superposition of FlexX-docked TSA
onto the crystallographic geometry yielded a root-mean-
square (rms) deviation of 0.75 Å and revealed that FlexX
performed well in reproducing the binding conformation
of TSA (result not shown). To compare the binding
conformation of 5a identified by FlexX with the X-ray
pose of TSA, the top-ranked conformer of 5a was
retrieved and overlayed over TSA in the active site of
F igu r e 2. Binding pose of 5a at the active site of HDLP,
generated by FlexX. The amino acid residues within the HDLP
binding site are represented in line form, and the zinc ion is
shown as a yellow ball. Yellow dotted lines are coordination
with Zn2+ (<2.7 Å), and arrows are hydrogen-bonding interac-
tions (<2.5 Å).
HDLP. As shown in Figure 1, their binding poses are
very similar.
The FlexX-docked pose of 5a in the active site of
HDLP is demonstrated in Figure 2. Compound 5a
snugly fits into the catalytic pocket of HDLP occupied

5748 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26
Kim et al.
8a (1.04 g, 66%): 1H NMR (DMSO-d6) δ 2.97 (s, 6 H, 2 NCH3),
4.41-4.47 (m, 4 H, NCH2 and OCH2), 5.10 (t, 1 H, J ) 5.7 Hz,
OH), 6.70 (m, 2 H, 2 Ar-H), 7.25 (apparent s, 4 H, 4 Ar-H),
7.76 (m, 2 H, 2 Ar-H), 8.63 (br t, 1 H, J ) 6.0 Hz, NH); IR
(neat) 3318 (NH and OH), 1622 (CO) cm-1; MS (FAB) m/z 285
(MH+). Anal. (C17H20N2O2) C, H, N.
by TSA in the X-ray crystal structure, and the hydrox-
amic acid group anchors into the bottom of the pocket.
In the X-ray structure of the TSA-HDLP complex, the
hydroxamic acid group is coordinated to the zinc ion,
which is critical for the HDAC catalysis, and forms
hydrogen bonds with Asp168, His170, and Tyr297.25
Similarly, the hydroxamic acid of 5a is coordinated to
zinc ion through its carbonyl (2.4 Å) and hydroxyl (2.7
Å) oxygen atoms. In addition, the hydroxyl and amino
groups of hydroxamic acid form hydrogen bonds with
Asp168 and Tyr297, respectively (Figure 2). The phen-
ylene linker moiety of 5a , which is equivalent to the
methyl-substituted vinyl group of TSA, intercalates
between Phe141 and Phe198 and possibly plays an
important role in enhancing the affinity in the tube-
like pocket of HDLP through the π-π interaction.
The binding mode of 5a generated by FlexX revealed
that the structure of the molecule is compatible with
the TSA-binding cavity of HDLP, and it would be related
to the experimental result showing that 5a has HDAC
inhibitory activity comparable to that of TSA.
N -(4-H yd r oxym e t h ylb e n zyl)-4-p yr r olid in -1-ylb e n z-
a m id e (8b). To a mixture of 4-pyrrolidin-1-ylbenzoic acid (6b)
(2.00 g, 10.5 mmol), 7 (4.30 g, 31.4 mmol), 1-hydroxybenzo-
triazole (1.70 g, 12.6 mmol), and DMAP (256 mg, 2.1 mmol)
in pyridine (50 mL) was added EDC (3.00 g, 15.7 mmol), and
the mixture was stirred at room temperature overnight. The
reaction mixture was concentrated under reduced pressure,
and an aqueous NaHCO3 solution (50 mL) was added. The
mixture was extracted with 5% MeOH in CHCl3 (150 mL ×
2), and the organic layer was dried (MgSO4) and evaporated
under reduced pressure. The crude product was purified by
MPLC on silica gel (3% MeOH in CHCl3) to afford 8b (2.56 g,
79%): 1H NMR (DMSO-d6) δ 1.96 (m, 4 H, 2 CH2), 3.28 (m, 4
H, 2 NCH2), 4.41-4.47 (m, 4 H, NCH2 and OCH2), 5.10 (t, 1H,
J ) 5.7 Hz, OH), 6.53 (m, 2 H, 2 Ar-H), 7.25 (apparent s, 4 H,
4 Ar-H), 7.75 (m, 2 H, 2 Ar-H), 8.59 (br t, 1H, J ) 6.0 Hz,
NH); IR (neat) 3258 (NH and OH), 1615 (CO) cm-1; MS (FAB)
m/ z 311 (MH+). Anal. (C19H22N2O2) C, H, N.
N-(4-Hydr oxym eth ylben zyl)n icotin am ide (8c) was simi-
larly obtained from 6c as for 8b: yield 86%; 1H NMR (DMSO-
d6) δ 4.46-4.49 (m, 4 H, NCH2 and OCH2), 5.13 (br t, 1H, J )
5.6 Hz, OH), 7.28 (apparent s, 4 H, 4 Ar-H), 7.51 (dd, 1 H, J )
7.8 Hz, 4.8 Hz, Pyr-H), 8.22 (m, 1 H, Pyr-H), 8.71 (dd, 1 H, J
) 4.8 Hz, 1.5 Hz, Pyr-H), 9.04 (d, 1 H, J ) 2.1 Hz, Pyr-H),
9.21 (br t, 1 H, J ) 5.7 Hz, NH); IR (neat) 3310 (NH and OH),
1631 (CO) cm-1; MS (FAB) m/z 243 (MH+). Anal. (C14H14N2O2)
C, H, N.
Con clu sion
Incorporation of the 1,4-phenylene carboxamide linker
shown in compounds 5a -g and a 4-(dimethylamino)-
phenyl or 4-(pyrrolidin-1-yl)phenyl group as a cap
substructure generated the highly potent hydroxamic
acid-based HDAC inhibitors 5a and 5b.
Compounds 5a and 5b have been selected as preclini-
cal candidates, and work is underway to evaluate their
effects on various cancer cells. Cancer cells sensitive to
compound 5b showed relatively higher levels of HDAC
1, suggesting that 5b might selectively target the HDAC
1 isotype.37 Compound 5b has been found to induce cell
cycle arrest at G1 and G2/M phases and to induce
mitochondrial- and caspase-dependent apoptotic cell
death.37 These data and ongoing animal experiments
will be published elsewhere in due course.
3-{4-[(4-Dim eth ylam in oben zoylam in o)m eth yl]ph en yl}-
a cr ylic Acid Eth yl Ester (9a ). To a solution of oxalyl chloride
(0.95 mL, 10.8 mmol) in THF (10 mL) at -78 °C was added a
solution of DMSO (1.68 mL, 23.6 mmol) in THF (10 mL), and
the reaction mixture was stirred for 30 min. A solution of 8a
(1.40 g, 4.8 mmol) in THF (80 mL) was added to it, and then
the mixture was stirred for 1 h and warmed to -35 °C. After
o
20 min, the mixture was cooled to -78 C, and triethylamine
(3.43 mL, 24.6 mmol) was added. After stirring at 0 °C for 30
min, the mixture was diluted with water (100 mL), and then
THF was evaporated under reduced pressure. The resulting
residue was extracted with 5% MeOH in CHCl3 (200 mL × 2),
and the organic layer was dried (MgSO4) and evaporated to
dryness under reduced pressure to give a crude product which
was crystallized from MeOH/CHCl3/Et2O to afford the corre-
sponding aldehyde (1.10 g, 80%) as a white solid.
Exp er im en ta l Section
Ch em istr y. 1H NMR spectra were recorded on a Varian
Unity 300 spectrometer. The chemical shifts are reported in
parts per million (ppm) relative to internal tetramethylsilane
in CDCl3 or DMSO-d6. Infrared spectra were recorded on a
Magna 750 FTIR spectrophotometer. Fast-atom bombardment
mass spectra (FAB-MS) were obtained on a VG Quattro mass
spectrometer. Analytical thin-layer chromatography (TLC) was
performed on Merck silica gel 60F-254 glass plates. Medium-
pressure chromatography (MPLC) was performed using Merck
silica gel 60 (230-400 mesh) with a VSP-2200 ceramic pump
(Eyela). Elemental analyses were performed on a Carlo Erba
1106 elemental analyzer. Where indicated by the symbols of
the elements, analyses were within (0.4% of theoretical
values.
4-(Dim eth yla m in o)-N-(4-h yd r oxym eth ylben zyl)ben z-
a m id e (8a ). To a mixture of 4-(dimethylamino)benzoic acid
(6a ) (915 mg, 5.54 mmol) and triethylamine (772 µL, 5.54
mmol) in CH2Cl2 (25 mL) at 0 °C was added BOP-Cl (1.50 g,
6.09 mmol), and the mixture was stirred at room temperature
for 20 min. To the reaction mixture were added (4-amino-
methylphenyl)methanol (7) (760 mg, 5.54 mmol) and triethyl-
amine (1.54 mL, 11.08 mmol), and the mixture was stirred at
room temperature overnight. To the reaction mixture was
added a 50% aqueous NaHCO3 solution (50 mL), and the
mixture was extracted with 5% MeOH in CHCl3 (100 mL × 1,
40 mL × 2). The organic layer was dried (MgSO4) and
evaporated under reduced pressure. The crude product was
purified by MPLC on silica gel (2% MeOH in CHCl3) to afford
A solution of the aldehyde (800 mg, 2.83 mmol) above and
(Ph)3PdCHCO2Et (1.48 g, 4.25 mmol) in CH3CN (40 mL) was
stirred at 60 °C for 2 h. The reaction mixture was concentrated
under reduced pressure, and the residue was purified by
MPLC on silica gel (2% MeOH in CHCl3) to afford 9a (916
mg, 92%): 1H NMR (DMSO-d6) δ 1.25 (t, 3 H, J ) 7.2 Hz,
CH2CH3), 2.97 (s, 6 H, 2 NCH3), 4.18 (q, 2 H, J ) 7.2 Hz, CH2-
CH3), 4.46 (d, 2 H, J ) 6.0 Hz, NCH2), 6.59 (d, 1 H, J ) 15.9
Hz, CH), 6.71 (m, 2 H, 2 Ar-H), 7.33 (d, 2 H, J ) 8.1 Hz, 2
Ar-H), 7.63 (d, 1 H, J ) 15.9 Hz, CH), 7.67 (d, 2 H, J ) 8.1
Hz, 2 Ar-H), 7.77 (m, 2 H, 2 Ar-H), 8.70 (br t, 1 H, J ) 6.0 Hz,
NH); IR (neat) 3247 (NH), 1704 (CO), 1634 (CO) cm-1; MS
(FAB) m/z 353 (MH+). Anal. (C21H24N2O3) C, H, N.
3-{4-[(4-P yr r olid in -1-ylben zoyla m in o)m eth yl]p h en yl}-
a cr ylic a cid eth yl ester (9b) was similarly obtained from
1
8b as for 9a : yield 47%; H NMR (DMSO-d6) δ 1.25 (t, 3 H, J
) 7.2 Hz, CH2CH3), 1.96 (m, 4 H, 2 CH2), 3.28 (m, 4 H, 2
NCH2), 4.18 (q, 2 H, J ) 7.2 Hz, CH2CH3), 4.46 (d, 2 H, J )
5.7 Hz, NHCH2), 6.54 (m, 2 H, 2 Ar-H), 6.58 (d, 1 H, J ) 16.5
Hz, CH), 7.33 (d, 2 H, J ) 8.1 Hz, 2 Ar-H), 7.62 (d, 1 H, J )
16.5 Hz, CH), 7.67 (d, 2 H, J ) 8.1 Hz, 2 Ar-H), 7.76 (m, 2 H,
2 Ar-H), 8.66 (br t, 1 H, J ) 5.7 Hz, NH); IR (neat) 3256 (NH),
1707 (CO), 1629 (CO) cm-1; MS (FAB) m/z 379 (MH+). Anal.
(C23H26N2O3) C, H, N.

3-(4-Substituted-phenyl)-N-hydroxy-2-propenamides
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26 5749
3-{4-[(Nicotin oylam in o)m eth yl]ph en yl}acr ylic acid eth -
yl ester (9c) was similarly obtained from 8c as for 9a : yield
39%; 1H NMR (DMSO-d6) δ 1.26 (t, 3 H, J ) 7.2 Hz, CH2CH3),
4.19 (q, 2 H, J ) 7.2 Hz, CH2CH3), 4.53 (d, 2 H, J ) 6.0 Hz,
NCH2), 6.60 (d, 1 H, J ) 15.9 Hz, CH), 7.38 (d, 2 H, J ) 8.1
Hz, 2 Ar-H), 7.52 (m, 1 H, Pyr-H), 7.63 (d, 1 H, J ) 15.9 Hz,
CH), 7.69 (d, 2 H, J ) 8.1 Hz, 2 Ar-H), 8.23 (m, 1 H, Pyr-H),
8.72 (m, 1 H, Pyr-H), 9.06 (m, 1 H, Pyr-H), 9.26 (br t, 1 H, J
) 6.0 Hz, NH); IR (neat) 3279 (NH), 1703 (CO), 1632 (CO)
cm-1; MS (FAB) m/z 311 (MH+). Anal. (C18H18N2O3) C, H, N.
4-(4-P yr id in -2-ylm e t h ylp ip e r a zin -1-yl)b e n zon it r ile
(13a ). A suspension of 1-(pyridin-2-ylmethyl)piperazine (11a )
(638 mg, 3.60 mmol), 4-fluorobenzonitrile (436 mg, 3.60 mmol),
and K2CO3 (746 mg, 5.40 mmol) in DMF (50 mL) was stirred
at 80 °C overnight. The reaction mixture was cooled to room
temperature and filtered, and the filtrate was washed with
EtOAc (30 mL). The combined filtrate and washings were
evaporated to dryness under reduced pressure. The crude
residue was purified by MPLC on silica gel (3% MeOH in CH2-
Cl2) to afford 13a (317 mg, 32%): 1H NMR (CDCl3) δ 2.66
(apparent t, 4 H, J ) 5.1 Hz, 2 NCH2), 3.36 (apparent t, 4 H,
J ) 5.1 Hz, 2 NCH2), 3.72 (s, 2 H, NCH2Pyr), 6.84 (m, 2 H, 2
Ar-H), 7.19 (m, 1 H, Pyr-H), 7.42 (m, 1 H, Pyr-H), 7.48 (m, 2
H, 2 Ar-H), 7.68 (m, 1 H, Pyr-H), 8.59 (m, 1 H, Pyr-H); IR (neat)
2216 (CN) cm-1; MS (FAB) m/z 279 (MH+). Anal. (C17H18N4)
C, H, N.
4-(4-P yr idin -3-ylm eth ylpiper azin -1-yl)ben zon itr ile (13b)
was similarly obtained from 11b as for 13a : yield 38%; 1H
NMR (CDCl3) δ 2.59 (apparent t, 4 H, J ) 5.1 Hz, 2 NCH2),
3.33 (apparent t, 4 H, J ) 5.1 Hz, 2 NCH2), 3.57 (s, 2 H, NCH2-
Pyr), 6.85 (m, 2 H, 2 Ar-H), 7.27 (m, 1 H, Pyr-H), 7.48 (m, 2
H, 2 Ar-H), 7.69 (m, 1 H, Pyr-H), 8.53 (m, 1 H, Pyr-H), 8.57
(m, 1 H, Pyr-H); IR (neat) 2206 (CN) cm-1; MS (FAB) m/z 279
(MH+). Anal. (C17H18N4) C, H, N.
3-{4-[(P icolin oylam in o)m eth yl]ph en yl}acr ylic Acid Eth -
yl Ester (9d ). To a mixture of picolinic acid (6d ) (170 mg, 1.4
mmol), 10 (284 mg, 1.4 mmol), and DMAP (34 mg, 2.1 mmol)
in DMF (10 mL) was added EDC (291 mg, 15.7 mmol), and
the mixture was stirred at room temperature for 4 h. The
reaction mixture was concentrated under reduced pressure,
and an aqueous NaHCO3 solution (20 mL) was added. The
mixture was extracted with 5% MeOH in CHCl3 (30 mL × 3),
and the organic layer was dried (Na2SO4) and evaporated
under reduced pressure. The crude product was purified by
MPLC on silica gel (1% MeOH in CHCl3) to afford 9d (247
mg, 58%): 1H NMR (CDCl3) δ 1.34 (t, 3 H, J ) 7.2 Hz,
CH2CH3), 4.26 (q, 2 H, J ) 7.2 Hz, CH2CH3), 4.69 (d, 2 H, J )
6.3 Hz, NCH2), 6.42 (d, 1 H, J ) 15.9 Hz, CH), 7.38 (d, 2 H, J
) 8.1 Hz, 2 Ar-H), 7.44 (m, 1 H, Pyr-H), 7.50 (d, 2 H, J ) 8.1
Hz, 2 Ar-H), 7.67 (d, 1 H, J ) 15.9 Hz, CH), 7.87 (m, 1 H,
Pyr-H), 8.24 (m, 1 H, Pyr-H), 8.42 (m, 1 H, NH), 8.54 (m, 1 H,
Pyr-H); IR (neat) 3384 (NH), 1717 (CO), 1636 (CO) cm-1; MS
(FAB) m/z 311 (MH+). Anal. (C18H18N2O3) C, H, N.
4-(4-P yr idin -4-ylm eth ylpiper azin -1-yl)ben zon itr ile (13c)
was similarly obtained from 11c as for 13a : yield 59%; 1H NMR
(CDCl3) δ 2.59 (apparent t, 4 H, J ) 5.1 Hz, 2 NCH2), 3.35
(apparent t, 4 H, J ) 5.1 Hz, 2 NCH2), 3.56 (s, 2 H, NCH2-
Pyr), 6.85 (m, 2 H, 2 Ar-H), 7.30 (m, 2 H, 2 Pyr-H), 7.49 (m, 2
H, 2 Ar-H), 8.56 (m, 2 H, 2 Pyr-H); IR (neat) 2213 (CN) cm-1
;
4-Dim eth ylam in o-N-[4-(2-h ydr oxycar bam oylvin yl)ben z-
yl]ben za m id e (5a ). To a solution of 1.76 M NH2OH in MeOH
(0.97 mL) was added 9a (100 mg, 0.28 mmol), and the mixture
was stirred at room temperature overnight. The reaction
mixture was neutralized by adding 1 N HCl aqueous solution
(pH 7) at 0 °C. The solid precipitated was collected by filtration,
washed with H2O and Et2O, dried under vacuum, and crystal-
lized from MeOH/CH2Cl2 to afford 5a (55 mg, 58%): 1H NMR
(DMSO-d6) δ 2.97 (s, 6 H, 2 NCH3), 4.45 (d, 2 H, J ) 5.7 Hz,
NCH2), 6.42 (d, 1 H, J ) 15.9 Hz, CH), 6.71 (m, 2 H, 2 Ar-H),
7.33 (d, 2 H, J ) 8.1 Hz, 2 Ar-H), 7.43 (d, 1 H, J ) 15.9 Hz,
CH), 7.51 (d, 2 H, J ) 8.1 Hz, 2 Ar-H), 7.77 (m, 2 H, 2 Ar-H),
8.68 (br t, 1 H, J ) 5.7 Hz, CONH), 9.00 (br s, 1 H, NHOH),
10.71 (br s, 1 H, NHOH); IR (neat) 3178 (NH), 1633 (CO), 1608
(CO) cm-1; MS (FAB) m/z 340 (MH+). Anal. (C19H21N3O3) C,
H, N.
MS (FAB) m/z 279 (MH+). Anal. (C17H18N4) C, H, N.
3-(4-{[4-(4-P yr id in -2-ylm et h ylp ip er a zin -1-yl)b en zoyl-
a m in o]m eth yl}p h en yl)a cr ylic Acid Eth yl Ester (14a ). A
solution of 13a (330 mg, 1.19 mmol) in concentrated HCl (20
mL) was stirred at 80 °C overnight. The reaction mixture was
cooled to room temperature and then evaporated to dryness
under reduced pressure. The residue was dissolved in satu-
rated LiOH solution (pH 9) and then evaporated to dryness
under reduced pressure. The residue was dissolved in 10%
aqueous HCl solution (pH 2), evaporated, and dried under
vacuum to give the corresponding acid, which was used in the
next step without further purification.
To a mixture of the acid above, 3-(4-aminomethylphenyl)-
acrylic acid ethyl ester hydrochloride (10) (305 mg, 1.26 mmol),
1-hydroxybenzotriazole (195 mg, 1.44 mmol), and DMAP (22
mg, 0.18 mmol) in pyridine (12 mL) was added EDC (276 mg,
1.44 mmol), and the mixture was stirred at room temperature
for 20 h. The reaction mixture was concentrated under reduced
pressure, and to it an aqueous NaHCO3 solution (20 mL) was
added. The mixture was extracted with 10% MeOH in CHCl3
(30 mL × 3), and the organic layer was dried (Na2SO4) and
evaporated under reduced pressure. The crude product was
purified by MPLC on silica gel (5% MeOH in CHCl3) to afford
14a (220 mg, 38%): 1H NMR (DMSO-d6) δ 1.25 (t, 3 H, J )
7.2 Hz, CH2CH3), 2.57 (m, 4 H, 2 NCH2), 3.28 (m, 4 H, 2 NCH2),
3.66 (s, 2 H, NCH2Pyr), 4.18 (q, 2 H, J ) 7.2 Hz, CH2CH3),
4.46 (d, 2 H, J ) 5.7 Hz, NHCH2), 6.59 (d, 1 H, J ) 16.2 Hz,
CH), 6.96 (m, 2 H, 2 Ar-H), 7.26-7.34 (m, 3 H, Pyr-H and 2
Ar-H), 7.48 (m, 1 H, Pyr-H), 7.60-7.68 (m, 3 H, CH and 2 Ar-
H), 7.75-7.81 (m, 3 H, 2 Ar-H and Pyr-H), 8.51 (m, 1 H, Pyr-
H), 8.80 (br t, 1 H, J ) 5.7 Hz, NH); IR (neat) 3350 (NH), 1702
(CO), 1637 (CO) cm-1; MS (FAB) m/z 485 (MH+). Anal.
(C29H32N4O3) C, H, N.
N-[4-(2-Hyd r oxyca r ba m oylvin yl)ben zyl]-4-p yr r olid in -
1-ylben za m id e (5b) was similarly obtained from 9b as for
5a : yield 35%; 1H NMR (DMSO-d6) δ 1.96 (m, 4 H, 2 CH2),
3.28 (m, 4 H, 2 NCH2), 4.45 (d, 2 H, J ) 5.7 Hz, NHCH2), 6.42
(d, 1 H, J ) 15.9 Hz, CH), 6.54 (m, 2 H, 2 Ar-H), 7.32 (d, 2 H,
J ) 8.1 Hz, 2 Ar-H), 7.43 (d, 1 H, J ) 15.9 Hz, CH), 7.50 (d, 2
H, J ) 8.1 Hz, 2 Ar-H), 7.76 (m, 2 H, 2 Ar-H), 8.64 (br t, 1 H,
J ) 5.7 Hz, CONH), 9.00 (br s, 1 H, NHOH), 10.70 (br s, 1 H,
NHOH); IR (neat) 3302 (NH), 1630 (CO), 1608 (CO) cm-1; MS
(FAB) m/z 366 (MH+). Anal. (C21H23N3O3) C, H, N.
N -[4-(2-H y d r o x y c a r b a m o y lv in y l)b e n z y l]n ic o t in -
a m id e (5c) was similarly obtained from 9c as for 5a : yield
74%; 1H NMR (DMSO-d6) δ 4.51 (m, 2 H, NCH2), 6.43 (d, 1 H,
J ) 15.9 Hz, CH), 7.37 (d, 2 H, J ) 8.1 Hz, 2 Ar-H), 7.44 (d, 1
H, J ) 15.9 Hz, CH), 7.52 (m, 1 H, Pyr-H), 7.53 (d, 2 H, J )
8.1 Hz, 2 Ar-H), 8.23 (m, 1 H, Pyr-H), 8.72 (m, 1 H, Pyr-H),
9.05 (m, 1 H, Pyr-H), 9.25 (m, 1 H, CONH); IR (neat) 3249
(NH), 1653 (CO), 1626 (CO) cm-1; MS (FAB) m/z 298 (MH+).
Anal. (C16H15N3O3) C, H, N.
3-(4-{[4-(4-P yr id in -3-ylm et h ylp ip er a zin -1-yl)b en zoyl-
a m in o]m eth yl}p h en yl)a cr ylic a cid eth yl ester (14b) was
1
N -[4-(2-H y d r o x y c a r b a m o y lv i n y l)b e n z y l]p i c o li n -
a m id e (5d ) was similarly obtained from 9d as for 5a : yield
29%; 1H NMR (DMSO-d6) δ 4.51 (m, 2 H, NCH2), 6.45 (d, 1 H,
J ) 15.9 Hz, CH), 7.35 (d, 2 H, J ) 8.1 Hz, 2 Ar-H), 7.42 (d, 1
H, J ) 15.9 Hz, CH), 7.50 (d, 2 H, J ) 8.1 Hz, 2 Ar-H), 7.59-
7.64 (m, 1 H, Pyr-H), 7.97-8.06 (m, 2 H, 2 Pyr-H), 8.66 (m, 1
H, Pyr-H), 9.02 (br s, 1 H, NHOH), 9.36 (m, 1 H, CONH), 10.76
(br s, 1 H, NHOH); IR (neat) 3254 (NH), 1653 (CO), 1636 (CO)
cm-1; MS (FAB) m/z 298 (MH+). Anal. (C16H15N3O3) C, H, N.
similarly obtained from 13b as for 14a : yield 84%; H NMR
(CDCl3) δ 1.34 (t, 3 H, J ) 7.2 Hz, CH2CH3), 2.60 (apparent t,
4 H, J ) 5.1 Hz, 2 NCH2), 3.30 (apparent t, 4 H, J ) 5.1 Hz,
2 NCH2), 3.57 (s, 2 H, NCH2Pyr), 4.26 (q, 2 H, J ) 7.2 Hz,
CH2CH3), 4.64 (d, 2 H, J ) 5.7 Hz, NHCH2), 6.32 (br t, 1 H, J
) 5.7 Hz, NH), 6.41 (d, 1 H, J ) 15.9 Hz, CH), 6.88 (m, 2 H,
2 Ar-H), 7.27 (m, 1 H, Pyr-H), 7.36 (d, 2 H, J ) 8.1 Hz, 2 Ar-
H), 7.49 (d, 2 H, J ) 8.1 Hz, 2 Ar-H), 7.64-7.72 (m, 4 H, CH,
Pyr-H, and 2 Ar-H), 8.53 (m, 1 H, Pyr-H), 8.57 (m, 1 H, Pyr-

5750 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26
Kim et al.
H); IR (neat) 3349 (NH), 1707 (CO), 1638 (CO) cm-1; MS (FAB)
formazan crystals. The plates were read immediately at 550
nm on an Elisa reader (Dynatech, MR 5000). The IC50 was
defined as the concentration of compounds that produced a
50% reduction in surviving cells and was calculated from the
expansion of control cultures over 4 days (number of cells at
day 4 minus the number of cells at day 0) by quantal probit
analysis of pharmacologic calculations with a computer pro-
gram.
m/z 485 (MH+). Anal. (C29H32N4O3) C, H, N.
3-(4-{[4-(4-P yr id in -4-ylm et h ylp ip er a zin -1-yl)ben zoyl-
a m in o]m eth yl}p h en yl)a cr ylic a cid eth yl ester (14c) was
1
similarly obtained from 13c as for 14a : yield 94%; H NMR
(CDCl3) δ 1.33 (t, 3 H, J ) 7.2 Hz, CH2CH3), 2.60 (apparent t,
4 H, J ) 5.1 Hz, 2 NCH2), 3.31 (apparent t, 4 H, J ) 5.1 Hz,
2 NCH2), 3.56 (s, 2 H, NCH2Pyr), 4.26 (q, 2 H, J ) 7.2 Hz,
CH2CH3), 4.65 (d, 2 H, J ) 5.7 Hz, NHCH2), 6.36 (br t, 1 H, J
) 5.7 Hz, NH), 6.41 (d, 1 H, J ) 15.9 Hz, CH), 6.88 (m, 2 H,
2 Ar-H), 7.30 (d, 2 H, J ) 6.0 Hz, 2 Pyr-H), 7.36 (d, 2 H, J )
8.1 Hz, 2 Ar-H), 7.49 (d, 2 H, J ) 8.1 Hz, 2 Ar-H), 7.66 (d, 1 H,
J ) 15.9 Hz, CH), 7.72 (m, 2 H, 2 Ar-H), 8.56 (m, 2 H, 2 Pyr-
H); IR (neat) 3356 (NH), 1701 (CO), 1634 (CO) cm-1; MS (FAB)
m/z 485 (MH+). Anal. (C29H32N4O3) C, H, N.
HDAC In h ibition . SNU-16 (human gastric adenocarci-
noma) cells were quickly cooled by placing the plates on ice
and were then washed twice with ice-cold PBS. Collected cells
were centrifuged and resuspended in five packed cell volumes
of buffer A (20 mM Tris pH 7.6, 10 mM KCl, 0.2 mM EDTA,
20% glycerol, 1.5 mM MgCl2, 2 mM dithiothreitol (DTT), 0.4
mM phenylmethylsulfonyl fluoride (PMSF), 1 mM Na3VO4,
and 2 µg/mL each of leupeptin, pepstatin, and aprotinin).
Nuclei were pelleted (2500g, 10 min) and resuspended in two
packed cell volumes of buffer B (identical to buffer A except
that the KCl level was increased to 0.42 M). Nuclear debris
was removed by centrifugation (15 000g, 20 min). HDAC
inhibition assay was performed using an HDAC Fluorescent
Activity Assay Kit (BIOMOL Research Laboratories, Inc.,
Plymouth Meeting, PA) according to the manufacturer’s
recommendation. In brief, 5 µg of SNU-16 nuclear extract was
added to the diluted HDAC inhibitor and then substrate was
added. Samples were incubated for 10 min at 25 °C, then the
reaction was stopped by addition of developer. Fluorescence
was analyzed using LS 55 Luminescence spectrometer (Perkin-
Elmer, Wellesley, MA).
P r ep a r a tion of Th r ee-Dim en sion a l Da ta ba se a n d Ta r -
get P r otein Str u ctu r e. All docking experiments have been
performed with Sybyl (Tripos Inc., version 6.9) on a SGI-
Octane 2 with a single 475-MHz processor and 128MB main
memory. The structures of TSA and 5a were prepared in mol2
format using the sketcher module of Sybyl 6.9, and Gasteiger-
Huckel charges were assigned to the ligand atoms. The
minimization was run until it converged to a maximum
derivative of 0.001 kcal mol-1 Å-1, and the final coordinate
was stored in database. The X-ray coordinate of HDLP-TSA
complex (1c3r) was retrieved from the PDB,26 and all crystal-
lographic water molecules were removed. The active site was
defined as all the amino acid residues and zinc ion enclosed
within a 6.5 Å radius sphere centered by the bound ligand,
TSA.
N-[4-(2-Hyd r oxyca r ba m oylvin yl)ben zyl]-4-(4-p yr id in -
2-ylm eth ylp ip er a zin -1-yl)ben za m id e (5e) was similarly
1
obtained from 14a as for 5a : yield 59%; H NMR (DMSO-d6)
δ 2.59 (m, 4 H, 2 NCH2), 3.28 (m, 4 H, 2 NCH2), 3.68 (br s, 2
H, NCH2Pyr), 4.45 (d, 2 H, J ) 5.7 Hz, NHCH2), 6.42 (d, 1 H,
J ) 15.9 Hz, CH), 6.96 (m, 2 H, 2 Ar-H), 7.26-7.34 (m, 3 H, 2
Ar-H and Pyr-H), 7.43 (d, 1 H, J ) 15.9 Hz, CH), 7.47-7.52
(m, 3 H, 2 Ar-H and Pyr-H), 7.77-7.81 (m, 3 H, 2 Ar-H and
Pyr-H), 8.51 (m, 1 H, Pyr-H), 8.78 (br t, 1 H, J ) 5.7 Hz,
CONH), 9.02 (br s, 1 H, NHOH), 10.73 (br s, 1 H, NHOH); IR
(neat) 3270 (NH), 1631 (CO), 1608 (CO) cm-1; MS (FAB) m/z
472 (MH+). Anal. (C27H29N5O3) C, H, N.
N-[4-(2-Hyd r oxyca r ba m oylvin yl)ben zyl]-4-(4-p yr id in -
3-ylm eth ylp ip er a zin -1-yl)ben za m id e (5f) was similarly
1
obtained from 14b as for 5a : yield 46%; H NMR (DMSO-d6)
δ 2.56 (m, 4 H, 2 NCH2), 3.28 (m, 4 H, 2 NCH2), 3.62 (br s, 2
H, NCH2Pyr), 4.45 (d, 2 H, J ) 5.7 Hz, NHCH2), 6.42 (d, 1 H,
J ) 15.9 Hz, CH), 6.96 (m, 2 H, 2 Ar-H), 7.31 (d, 2 H, J ) 8.1
Hz, 2 Ar-H), 7.37-7.45 (m, 2 H, CH and Pyr-H), 7.50 (d, 2 H,
J ) 8.1 Hz, 2 Ar-H), 7.76-7.79 (m, 3 H, 2 Ar-H and Pyr-H),
8.50 (m, 1 H, Pyr-H), 8.54 (m, 1 H, Pyr-H), 8.78 (br t, 1 H, J
) 5.7 Hz, CONH), 9.01 (br s, 1 H, NHOH), 10.73 (br s, 1 H,
NHOH); IR(neat) 3278 (NH), 1631 (CO), 1607 (CO) cm-1; MS
(FAB) m/z 472 (MH+). Anal. (C27H29N5O3) C, H, N.
N-[4-(2-Hyd r oxyca r ba m oylvin yl)ben zyl]-4-(4-p yr id in -
4-ylm eth ylp ip er a zin -1-yl)ben za m id e (5g) was similarly
obtained from 14c as for 5a : yield 54%; 1H NMR (DMSO-d6) δ
2.52 (m, 4 H, 2 NCH2), 3.28 (m, 4 H, 2 NCH2), 3.57 (s, 2 H,
NCH2Pyr), 4.45 (d, 2 H, J ) 5.7 Hz, NHCH2), 6.42 (d, 1 H, J
) 15.9 Hz, CH), 6.96 (m, 2 H, 2 Ar-H), 7.32 (d, 2 H, J ) 8.1
Hz, 2 Ar-H), 7.36 (d, 2 H, J ) 6.0 Hz, 2 Pyr-H), 7.42 (d, 1 H,
J ) 15.9 Hz, CH), 7.51 (d, 2 H, J ) 8.1 Hz, 2 Ar-H), 7.78 (m,
2 H, 2 Ar-H), 8.53 (d, 2 H, J ) 6.0 Hz, 2 Pyr-H), 8.78 (br t, 1
H, J ) 5.7 Hz, CONH), 9.02 (br s, 1 H, NHOH), 10.73 (br s, 1
F lexible Dock in g. The docking and subsequent scoring
were performed using the default parameters of the FlexX
programs implanted in Sybyl 6.9.36 For the docking of ligand
into the target active site, the main settings are 1000 solutions
per iteration during the incremental construction algorithm
and a maximum protein-ligand atom-atom overlap of 2.5 Å3.
Final scores for all FlexX solutions (up to 1000) per compound
were calculated by a standard scoring function and used for
database ranking.
H, NHOH); IR (neat) 3335 (NH), 1635 (CO), 1609 (CO) cm-1
MS (FAB) m/z 472 (MH+). Anal. (C27H29N5O3) C, H, N.
;
An tip r olifer a tive Activity. Three human cancer cell lines
(lung cancer, A549; breast cancer, SK-BR-3; and stomach
cancer, MKN45) were tested in the MTT assay. A549 and SK-
BR-3 were obtained from the American Type Culture Collec-
tion (Bethesda, MD), and MKN45 was kindly provided by Dr.
N. Saijo, National Cancer Center Hospital and Research
Institute, J apan. These cell lines were grown in RPMI 1640
medium containing penicillin-streptomycin (100 units/mL)
and 10% heat-inactivated fetal bovine serum at 37 °C in an
atmosphere of 5% CO2. Single-cell suspensions were prepared
by trypsinization and pipet disaggregation. The number of cells
for each cell line plated in 96-well microtiter plates was
determined from the growth curve obtained in the MTT assay.
Test compounds were diluted from stock solution in DMSO
into fresh medium to a 10-fold concentration. Cells were
inoculated into each well in 180 µL of medium, and eight
different concentrations of 20 µL of test compounds were added
to each well. The plates were then incubated for 4 days at 37
°C in an atmosphere of 5% CO2. After 4 days of culture, 0.1
mg (20 µL of 5 mg/mL) of MTT was added to each well. The
plates were then incubated at 37 °C for 4 h. After the plates
were centrifuged at 1000 rpm for 10 min, the supernatant was
aspirated. DMSO (150 µL) was added to each well to solubilize
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