Synthesis and biological evaluation of 1-arylsulfonyl-5-(N -hydroxyacrylamide)indoles as potent histone deacetylase inhibitors with antitumor activity in vivo

Article abstract of DOI:10.1021/jm300197a

A series of 1-arylsulfonyl-5-(N-hydroxyacrylamide)indoles has been identified as a new class of histone deacetylase inhibitors. Compounds 8, 11, 12, 13, and 14 demonstrated stronger antiproliferative activities than 1 (SAHA) with GI₅₀ values ranging from 0.36 to 1.21 μM against Hep3B, MDA-MB-231, PC-3, and A549 human cancer cell lines. Lead compound 8 showed remarkable HDAC 1, 2, and 6 isoenzymes inhibitory activities with IC ₅₀ values of 12.3, 4.0, 1.0 nM, respectively, which are comparable to 1. In in vivo efficacy evaluation against lung A549 xenograft model, 8 displayed better antitumor activity than compound 1.

Full text of DOI:10.1021/jm300197a

Article
pubs.acs.org/jmc
Synthesis and Biological Evaluation of 1-Arylsulfonyl-5-(N-
hydroxyacrylamide)indoles as Potent Histone Deacetylase Inhibitors
with Antitumor Activity in Vivo
Mei-Jung Lai,†,● Han-Li Huang,§,● Shiow-Lin Pan,#,∞,● Yi-Min Liu,† Chieh-Yu Peng,‡,●
Hsueh-Yun Lee,† Teng-Kuang Yeh,# Po-Hsien Huang,∥,⊥ Che-Ming Teng,§ Ching-Shih Chen,∥
Hsun-Yueh Chuang,† and Jing-Ping Liou*,†
†School of Pharmacy, College of Pharmacy, Taipei Medical University, 250 Wuxing Street, Taipei 11031, Taiwan
‡School of Chinese Medicine, College of Chinese Medicine, China Medical University, Taichung 404, Taiwan
§Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan
∥Division of Medicinal Chemistry, College of Pharmacy, The Ohio State University, 500 West 12th Avenue, Columbus, Ohio 43210,
United States
⊥Epigenomics and Cancer Risk Factors (C010), German Cancer Research Center (DKFZ), Heidelberg, Germany
#Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Zhunan Town, Miaoli County, Taiwan
∞Graduate Institute of Pharmacology, Taipei Medical University, Taipei, Taiwan
S
* Supporting Information
ABSTRACT: A series of 1-arylsulfonyl-5-(N-hydroxyacrylamide)indoles has been identified as a
new class of histone deacetylase inhibitors. Compounds 8, 11, 12, 13, and 14 demonstrated
stronger antiproliferative activities than 1 (SAHA) with GI50 values ranging from 0.36 to 1.21 μM
against Hep3B, MDA-MB-231, PC-3, and A549 human cancer cell lines. Lead compound 8
showed remarkable HDAC 1, 2, and 6 isoenzymes inhibitory activities with IC50 values of 12.3, 4.0,
1.0 nM, respectively, which are comparable to 1. In in vivo efficacy evaluation against lung A549
xenograft model, 8 displayed better antitumor activity than compound 1.
indicated the hydroxamic acid or the N-hydroxyacrylamide
group plays an important role for activity.13,14 Our previous
studies of antitubulin agents, for example, 3-aroylindoles,15a 5-
aroylindoles,15b 1-aroylindoles,15c 3-arylthioindoles,15d and 1-
aryl(1-benzyl)indoles,15e−g showed substantial anticancer activ-
ity. The literature16 reported that the 2-aroyl-5-(N-
hydroxyacrylamide)indoles exhibited potent histone deacetyla-
tion inhibition. On the basis of these observations, we
attempted to explore the structure−activity relationships
between the indole moiety and the N-hydroxyacrylamide
group. Using the 1-arylsulfonylindole as a base coupled with
the N-hydroxyacrylamide group at the C-3, -4, -5, -6, or -7
position of indole ring provided the regioisomers of indolyl-N-
hydroxyacrylamides and allowed evaluation of bioactivity
(Figure 2). Herein we describe the discovery of 1-
arylsulfonyl-5-(N-hydroxyacrylamide)indoles as a new class of
histone deacetylase inhibitors showing antitumor activity in
vivo.
INTRODUCTION
■
Histone acetylation and deacetylation play a crucial role for
regulating protein function of eukaryotic cells, which is
correlated with two classes of enzymes: histone deacetylases
(HDACs) and histone acetyltransferases (HATs). They
affected the removal (HDAC) and addition (HAT) of the
acetyl group at specific lysine residues. The acetylation status of
histone is important in modulating gene expression and cell life,
and its dysregulation is involved in the development of several
cancers. Histone hyperacetylation leads to transcriptional
activation of suppressed genes, some of them being associated
with cell cycle arrest, differentiation, or apotosis in tumor cells.
Recent studies have shown that acetylation of non-histone
proteins is also relevant for tumorigenesis, cancer cell
proliferation, and immune functions. Hence, inhibition of
HDAC has arisen as an efficient therapeutic strategy to adjust
aberrant epigenetic changes associated with cancer.1−5
In October 2006, small molecule 1 (SAHA, vorinostat) was
approved by FDA for the treatment of refractory cutaneous T-
cell lymphorma.6 In November 2009, prodrug 2 (FK-228,
romidepsin) also gained approval from FDA for the treatment
of cutaneous T-cell lymphoma.7 Many small molecular agents,
for example, 3 (LBH-589),8 4 (PXD101),9 and 5 (SB939),10
are undergoing human clinical trials11,12 (Figure 1). A close
view of these small molecule histone deacetylase inhibitors
RESULTS AND DISCUSSION
Chemistry. The synthesis of 3-(N-hydroxyacrylamide)-
indole (6) is depicted in Scheme 1. Compound 22 was reacted
■
Received: December 7, 2011
Published: March 22, 2012
© 2012 American Chemical Society
3777
dx.doi.org/10.1021/jm300197a | J. Med. Chem. 2012, 55, 3777−3791

Journal of Medicinal Chemistry
Article
indoles 7−10. The 1-substituted phenylsulfonyl compounds
11−14 were synthesized in 41−61% yields from compound 25
by reacting with the corresponding arylsulfonyl chloride in the
presence of KOH and tetrabutylammonium hydrogen sulfate
(TBAHS) at room temperature. Compound 15, with a
hydroxyamide group at the C-5 position of the indole ring,
was synthesized in 40% yield by treatment of indole 5-
methylcarboxylate (44) with 1-phenylsulfonylation, LiOH-
mediated hydroxylation, NH2OTHP/PYBOP coupling reac-
tion, and TFA deprotection (Scheme 3). Similar to the
preparation of compound 8, compounds 16, 17, and 18, with a
benzoyl, benzyl, and phenyl group at the N1-position, were
obtained in 5−63% yield through a five-step synthesis as shown
in the Scheme 4. The 1-benzoylation and benzylation of indole-
5-carboxyaldehyde (25) was achieved by using KO-t-Bu as base
in the presence of KI and DMF at room temperature. The 1-
arylation of the indole ring was achieved in 28% yield by the
treatment of 25 and 4-iodobenzene with CuO/K2CO3 in DMF.
As shown in the Scheme 5, the treatment of compound 8 with
KOH and Pd/C afforded the desired compounds 19 and 20,
respectively. The synthesis of 3-(benzenesulfonyl)-5-(N-
hydroxyacrylamide)indole (21) is described in Scheme 6.
Starting from the indole-5-carboxyaldehyde (25), the 3-
arylsulfonyl-5-(methylacrylate)indole 54 was obtained by
methyl(triphenylphosphoranylidene)acetate-mediated Wittig
reaction, diphenyl disulfide mediated electrophilic substitution,
and mCPBA oxidation. The conversion of methyl acrylate (54)
to N-hydroxyacrylamide (21) was accomplished by a reaction
sequence, including the LiOH hydrolysis, NH2OTHP/PYBOP
coupling reaction, and TFA deprotection.
Biological Evaluation. (A) HeLa Nuclear HDAC Enzyme
Inhibition. First, we evaluated the position effect of the N-
hydroxyacrylamide group in the 1-benzenesulfonylindole
system as shown in Table 1. The regioisomers 3-, 4-, 5-, 6-,
and 7-(N-hydroxyacrylamide)-1-benzenesulfonylindoles (6, 7,
8, 9, and 10, respectively) were evaluated for the inhibition of
HeLa nuclear HDAC activity (which consists of pan-HDAC
isoenzymes). The N-hydroxyacrylamide moiety located at the
C-5 position on the indole ring resulted in the best activity
among the five regioisomers with 1-benzenesulfonyl-5-(N-
hydroxyacrylamide)indole (8) showing IC50 values of 29.5 nM
on HeLa HDAC enzyme inhibition, which was more potent
than 1 (IC50 = 96.4 nM). Shifting of the N-hydroxyacrylamide
Figure 1. Examples of histone deacetylase inhibitor.
with the O-(tetrahydro-2H-pyran-2-yl)hydroxylamine
(NH2OTHP) in the presence of benzotriazol-1-yloxytripyrro-
lidinophosphonium hexafluorophosphate (PYBOP) and Et3N
to prepare the tetrahydropyran-protected compound 23, which
was further subjected to the N1-benzenesulfonylation of indole
in the presence of KOH and tetrabutylammonium hydrogen
sulfate (TBAHS) at room temperature and then trifluoroacetic
acid (TFA) mediated deprotection to afford the desired 6. The
general method for the syntheses of 4-, 5-, 6-, and 7-(N-
hydroxyacrylamide)indoles 7−10 and 1-arylsulfonyl-5-(N-
hydroxyacrylamide)indoles 11−14 are shown in the Scheme
2. The preparation of compounds 7−10 involved a five-step
reaction sequence, with an overall yield of 34−52%. The
various commercially available indolecarboxyaldehydes (24−
27) with benzenesulfonyl chloride yielded the related 1-
benzenesulfonylindoles (28−31). These indoles-1-sulfona-
mides were subject to the Wittig reaction with methyl
(triphenylphosphorylidene)acetate followed by LiOH hydrol-
ysis and PYBOP-mediated amide formation, and the reaction
sequence was completed by TFA-mediated deprotection to
afford the desired 1-benzenesulfonyl(N-hydroxyacrylamide)-
Figure 2. Synthetic indolyl-N-hydroxyacrylamides and indolylhydroxyamide.
3778
dx.doi.org/10.1021/jm300197a | J. Med. Chem. 2012, 55, 3777−3791

Journal of Medicinal Chemistry
Article
a
Scheme 1
a
Reagents and conditions: (a) NH2OTHP, PYBOP, Et3N, DMF, room temp, 68%; (b) (i) benzenesulfonyl chloride, KOH, TBAHS, CH2Cl2, room
temp; (ii) TFA, CH3OH, room temp, two steps, 32%.
a
Scheme 2
a
Reagents and conditions: (a) benzenesulfonyl chloride, KOH, TBAHS, CH2Cl2, room temp, 66−91%; (b) (i) methyl
(triphenylphosphoranylidene)acetate, CH2Cl2, room temp; (ii) 1 M LiOH(aq), dioxane, 40 °C, two steps, 79−90%; (c) (i) NH2OTHP,
PYBOP, Et3N, DMF, room temp; (ii) TFA, CH3OH, room temp, two steps, 53−78%; (d) substituted arylsulfonyl chloride, KOH, TBAHS, CH2Cl2,
room temp, 76−82%.
a
Scheme 3
a
Reagents and conditions: (a) (i) benzenesulfonyl chloride, KOH, TBAHS, CH2Cl2, room temp, two steps, 76%; (ii) 1 M LiOH(aq), dioxane, 40
°C; (b) (i) NH2OTHP, PYBOP, Et3N, DMF, room temp; (ii) TFA, CH3OH, room temp, two steps, 53%.
group to the C-4 (7) and C-6 (9) positions resulted in a
decrease in enzyme activity to the 100 nM level, while moving
to the C-3 and C-7 positions, as in compounds 6 and 10,
respectively, resulted in the loss of HDAC enzyme activity.
Second, we expanded the panel construction using indole 8 as
motif. Further investigation of substitution effects on the N1-
benzenesulfonyl group of compound 8 revealed that 11 with an
additional methoxy group at the C-4′ position, 12 with the
dimethoxy substitution at the C-3′ and C-4′ positions, 13 with a
fluoro group at the C-4′ position, and 14 with a nitro group at
C-4′ position all exhibited substantial activities with IC50 values
of 6.8, 9.7, 16.8, and 12.6 nM, respectively. In an effort to
further understand the role of N-hydroxyacrylamide substituent
at the C-5 position of indole ring, compound 15 (with two
carbons shorter than 8) and compound 20 (with a saturated
double from 8) were prepared. They exhibited a decrease in
potency, thus emphasizing the importance of N-hydroxyacry-
lamide at the C-5 position of indole in inhibiting HDAC
enzyme activity. In order to understand the role of 1-sulfonyl
functionality in 8, 1-benzoyl, 1-benzyl, and 1-phenyl substituted
compounds 16, 17, and 18, respectively, were prepared.
Compound 17, with a benzyl group, retained HDAC enzyme
activity comparable to that of 8, but benzoyl (16) or phenyl
(18) substitution resulted in a decrease in activity, thus
revealing that the 1-benzenesulfonyl and 1-benzyl group in the
indol-5-yl-N-hydroxyacrylamide series were preferable. The
removal of 1-benzenesulfonyl group in 8, for example,
compound 19, resulted in a dramatic loss of activity, indicating
the 1-arylsulfonyl group played a vital role for activity in this
series. Changing the position of arylsulfonyl group from N-1 to
C-3 on the indole ring, for example, compound 21, caused a
reduced potency.
(B) In Vitro Cell Growth Inhibitory Activity. The synthesized
indolyl-N-hydroxyacrylamides 6−14, 16−19, 21 and indolylhy-
droxamic acids 15, 20 were evaluated for antiproliferative
activities against human liver carcinoma Hep3B cells, breast
3779
dx.doi.org/10.1021/jm300197a | J. Med. Chem. 2012, 55, 3777−3791

Journal of Medicinal Chemistry
Article
a
extract enzyme activity except for compound 17. Only the
series of 1-(arylsulfonyl)-5-(N-hydroxyacrylamide)indoles 8,
11, 12, 13, and 14 showed more potent antiproliferative
activities than SAHA (1). For example, compound 8
demonstrated GI50 of 0.41, 0.48, 0.62, and 1 μM against the
Hep3B, MDA-MB-231, PC-3, and A549 cell lines, respectively.
Compound 12 with dimethoxybenzenesulfonyl group displayed
antiproliferative activity in four cancer cell lines with GI50 values
of 0.36, 0.37, 0.93, and 0.56 μM, respectively. The 1-
(arylbenzyl)-5-(N-hydroxyacrylamide)indole 17 displayed
about 2-fold decrease in activities compared to 8 and slight
decrease in activities compared to 1. Other compounds with
the N-hydroxyacrylamide group at the C-3, C-4, C-6, C-7
position of 1-(arylsulfonyl)indoles (6, 7, 9, 10), benzoyl,
phenyl, and no substitution at the N1 of 5-(N-
hydroxyacrylamide)indoles (16, 18, 19), and modifications to
the C-5 position from 8 (15, 20) had significantly less
antiproliferative activities.
(C) HDAC Isoform Inhibition. The 1-arylsulfonyl-5-(N-
hydroxyacrylamide)indoles 8, 11, 12, 13, 14, and 17 were
tested for HDAC isoform enzyme-inhibitory activity (HDACs
1, 2, and 6) using compound 1 as a reference (Table 2).
Compound 8 with 1-benzenesulfonyl group had low nanomolar
IC50 values against HDACs 2 and 6 with IC50 values of 1 and 4
nM, respectively. Compound 11 with 4′-methoxyphenylsulfonyl
group displayed high activity on HDAC 6 (IC50 = 3.3 nM) and
40- to 100-fold selectivity for HDACs 1 and 2. However,
compound 12 with a dimethoxy group at both C-3′ and C-4′
positions exhibited selective and potent inhibitory activity for
HDAC 2, with IC50 of 2.3 nM, among HDACs 1, 2, and 6. The
results indicated that the position of introduced methoxy group
in the 1-arylsulfonyl group of 5-(N-hydroxyacrylamide)indoles
seems to affect selectivity between HDAC isoforms. Com-
pounds 13 and 14, with a fluoro and nitro substitution at the 4′-
position of the arylsulfonyl moiety, respectively, exhibited
diminished activities against HDACs 1, 2, and 6 compared with
parent compound 8.
Scheme 4
a
Reagents and conditions: (a) benzoyl bromide or benzyl chloride,
KO-t-Bu, KI, DMF, room temp, 85−87%; (b) 4-iodobenzene, K2CO3,
C u O , D M F , r e fl u x , 2 8 % ; ( c ) ( i ) m e t h y l
(triphenylphosphoranylidene)acetate, CH2Cl2, room temp; (ii) 1 M
LiOH(aq), dioxane, 40 °C; (d) (i) NH2OTHP, PYBOP, Et3N, DMF,
room temp; (ii) TFA, CH3OH, room temp, two steps, 44−81%.
a
Scheme 5
a
Reagents and conditions: (a) 1 M KOH(aq), CH3OH, reflux, 51%; (b)
H2, 10% Pd/C, CH2Cl2, room temp, 30%.
carcinoma MDA-MB-231 cells, prostate carcinoma PC-3 cells,
and lung carcinoma A549 cells (Table 1). The cancer cell
growth inhibitory results were proportional to the HDAC
(D) Up-Regulation Effect of Histone and α-Tubulin. In an
effort to further validate the target in the 1-(arylsulfonyl)-5-(N-
a
Scheme 6
a
Reagents and conditions: (a) methyl (triphenylphosphoranylidene)acetate, CH2Cl2, room temp, 99%; (b) NaH, Ph-S-S-Ph, DMF, 0 °C to room
temp, 42%; (c) mCPBA, CH2Cl2, 0 °C to room temp, 46%; (d) 1 M LiOH(aq), MeOH, 40 °C, 52%; (e) (i) NH2OTHP, EDC−HCl, HOBt, N-
methylmorphine, DMF, room temp; (ii) 10% TFA(aq), MeOH, room temp, two steps, 26%.
3780
dx.doi.org/10.1021/jm300197a | J. Med. Chem. 2012, 55, 3777−3791

Journal of Medicinal Chemistry
Article
Table 1. Inhibition of HeLa Nuclear Extract HDAC Activity and Antiproliferative Activity against Human Cancer Cell Lines by
Compounds 6−21 and SAHA (1)
a
GI50 SD (μM)
a
IC50 SD (nM)
HeLa nuclear HDAC
compd
Hep3B
MDA-MB-231
PC-3
A549
6
>1000
8.87 0.2
4.32 0.1
0.41 0.1
2.12 0.1
3.82 0.4
0.55 0.1
0.36 0.1
0.64 0.1
0.56 0.1
>10
8.14 0.7
4.16 0.3
0.48 0.1
2.41 0.3
7.71 0.6
0.75 0.1
0.37 0.1
0.66 0.2
0.75 0.2
5.68 0.3
3.18 0.4
1.18 0.2
1.73 0.1
4.37 0.5
2.21 0.4
1.25 0.1
0.97 0.2
5.77 0.5
3.67 0.3
0.62 0.2
2.40 0.3
4.75 0.4
0.80 0.2
0.93 0.3
0.39 0.1
0.85 0.2
7.26 0.6
2.32 0.2
1.23 0.2
1.54 0.2
4.80 0.1
2.07 0.1
1.26 0.1
1.21 0.3
7.06 0.1
6.70 0.2
1.02 0.2
5.30 0.7
5.50 0.4
1.18 0.2
0.56 0.1
0.75 0.7
1.21 0.2
>10
7
170.9 42.7
29.5 4.5
254.0 84.6
>1000
8
9
10
11
12
13
14
15
16
17
18
19
20
21
1
6.8 1.7
9.7 2.0
16.8 3.8
12.6 2.6
>1000
757.6 92.8
32.1 9.3
186.5 20.9
>1000
2.40 0.3
0.86 0.1
1.32 0.2
3.65 0.4
2.56 0.3
1.27 0.1
0.69 0.2
5.80 0.8
2.37 0.1
2.85 0.8
9.07 0.9
4.04 0.2
3.33 0.2
1.85 0.3
407.5 170.8
275.2 31.7
96.4 10.3
a
SD: standard deviation. All experiments were independently performed at least three times.
Table 2. Activities of Compounds 8, 11−14, 17 and
Table 4. Pharmacokinetics Parameters of 8 in Rat
Reference Compound 1 against HDAC Isoforms 1, 2, and 6
parameter
iv
po
a
a
IC50 SD (nM)
dose (mg/kg)
2
20
CL (mL min−1 kg−1
)
35.5 3.7
2.1 0.1
2.6 0.2
927 80
compd
HDAC1
HDAC2
HDAC6
Vss (l/kg)
8
12.3 4.2
124.8 9.8
18.1 2.5
33.2 6.6
69.9 12.0
71.2 14.2
8.9 3.3
4.0 0.7
1.0 0.2
3.3 0.7
45.4 6.4
59.2 1.5
18.3 3.6
40.4 12.4
5.0 0.4
T1/2 (h)
5.5 1.9
916 183
1.7 0.6
217 116
10
11
12
13
14
17
1
361.8 22.2
2.3 0.3
AUC(0−inf) (ng mL−1 h)
Tmax (h)
199.4 24.7
189.3 70.6
381.1 36.8
1.7 0.2
Cmax (ng/mL)
F (%)
a
Compound was dissolved in PEG400/DMSO (80:20,v/v) for
a
intravenous administration and in 1% carboxylmethyl cellulose and
0.5% Tween 80 for oral dosing.
SD: standard deviation. All experiments were independently
performed at least three times.
and 51% at 10 μM, respectively. Compound 8 has a solubility
of <1 μg/mL in water and 939 μg/mL in EtOH.
(E.2) In Vivo Rat PK Profiling of 8. The PK parameters of 8
in male Sprague−Dawley rats, following single dose intravenous
(iv) and oral dosing, are shown in Table 4. Following iv
administration, 8 showed a mean T1/2 of 2.6 h. After a single
hydroxyacrylamide)indole series, compound 8 was evaluated
for the expression of histone and α-tubulin acetylation in PC-3
prostate cancer cell lines and A549 non-small-cell lung cancer
cells, which were important biomarkers associated with
intracellular HDAC inhibition. Western blot analysis of the
cell lysates revealed that compound 8, like compound 1, was a
pan-HDAC inhibitor on the basis of its ability to up-regulate
H3 and tubulin acetylation in dose-dependent manner (Figure
3).
(E) CYP Inhibition and Pharmacokinetic Study. (E.1)
Effect of 8 on Cytochrome P450 Isozymes. Compound 8 was
tested for its inhibition of CYP1A2, 2C19, 2C8, 2C9, 2D6, 2E1,
and 3A4 at 10 μM (Table 3). The CYP inhibition of 8 was
greatest for CYP2C19 and CYP2C8 with 78% at 10 μM. Both
CYP3A4 and CYP1A2 were also affected to a lesser extent, 65%
oral dose, compound 8 showed rapid absorption with Tmax
1.7 h, T1/2 = 5.5 h, and bioavailability F = 10%.
=
(F) In Vivo Efficacy in Human Xenograft. Compound 8 and
reference compound 1 were evaluated for in vivo efficacy of
tumor xenograft in nude mice bearing human A549 cancer cell
line expressed by tumor growth delay (TGD; Figure 4A, Table
5) and tumor growth inhibition (TGI; Figure 4B). Once tumor
was palpable with a size of ∼65 mm3, mice were randomized
into vehicle control and treatment groups of seven animals
each. Control mice received vehicle (0.5% carboxymethyl
cellulose). Compounds 8 and 1 were suspended in the 0.5%
carboxymethyl cellulose. All tumors in mice grew to the 1000
mm3 end point volume. The median TTE (time to end point)
for control mice was 27.0 days. Compound 1, at 200 mg/kg po
daily to end, produced a median TTE of 30.0 days,
corresponding to a 3.0 day T − C and a TGD (tumor growth
delay) of 11%. On the basis of the log rank analysis, compound
Table 3. CYP Inhibition and Solubility of Compound 8
a
% CYP inhibition at 10 μM
sol (μg/mL)
compd 1A2 2C19 2C8 2C9 2D6 2E1 3A4
H2O
<1
EtOH
939
8
51
78
78
22
31
22
65
a
The studies were performed by Ricerca Biosciences.
3781
dx.doi.org/10.1021/jm300197a | J. Med. Chem. 2012, 55, 3777−3791

Journal of Medicinal Chemistry
Article
Figure 3. Effect of α-tubulin acetylation and histone H3 acetylation in cultured (A) human prostate cells (PC-3) and (B) non-small-cell lung cancer
cells (A549) by compounds 8 and 1 using Western blot analysis. Quantitative analysis of Western blot was done with ImageQuant (Molecular
Dynamics, U.S.). Acetyl-histone H3 (C, D) and acetyl α-tubulin (E, F) were analyzed in PC-3 and A549 cells, respectively.
Figure 4. Inhibition of human A549 lung cancer xenograft growth in nude mice (n = 7). Compounds 8 and 1 were suspended in the 0.5%
carboxymethyl cellulose. All tumors in mice grew to the 1000 mm3 end point volume. (A) Tumor growth delay (TGD): 100 mg/kg compound 8 po
◆
●
▼
▲
daily ( ); 200 mg/kg compound 8 po daily ( ); 200 mg/kg compound 1 po daily ( ). (B) % of tumor growth inhibition (%TGI): control ( );
▽
▼
○
100 mg/kg compound 8 po daily ( ); 200 mg/kg compound 8 po daily ( ); 200 mg/kg compound 1 po daily ( ). (∗) P < 0.05, (∗∗) P < 0.01
compared with control group at the indicated times.
3782
dx.doi.org/10.1021/jm300197a | J. Med. Chem. 2012, 55, 3777−3791

Journal of Medicinal Chemistry
Article
Table 5. Summary Responses of Compounds 1 and 8 in the Human A549 Lung Xenograft Model
a
b
c
d
e
f
agent (n )
dose (mg/kg)
route
schedule
median TTE (day)
T − C (day)
% TGD
log rank significance
P
control (7)
1 (7)
po
po
po
po
q.d. to end point
q.d. to end point
q.d. to end point
q.d. to end point
27.0
30.0
39.8
g
200
200
3.0
11
47
ns
0.3925
0.0274
0.1688
h
8 (7)
12.8
9.2
∗
ns
8 (7)
100
36.2
34
a
b
c
d
n = number of animals. Study end point = 1000 mm3. Days in progress = 48. TTE = time to end point. T − C = difference between median TTE
e
f
g
(days) of treated versus control group. % TGD (tumor growth delay) = [(T − C)/C] × 100. Statistical significance = log rank test. ns = not
significant. ∗ indicates P < 0.05 compared with control.
h
water containing 0.1% formic acid +10 mmol NH4OAc) and was
found to be ≥95%. Flash column chromatography was done using
silica gel (Merck Kieselgel 60, no. 9385, 230−400 mesh ASTM). All
reactions were carried out under an atmosphere of dry nitrogen.
Syntheses of 1-Phenylsulfonyl-(N-hydroxyacrylamide)-
indoles 6−10. 3-(1H-Indol-3-yl)-N-(tetrahydropyran-2-yloxy)-
acrylamide (23). To a solution of trans-3-indoleacrylic acid
(22, 0.50 g, 2.67 mmol), PYBOP (1.47 g, 2.83 mmol), and
triethylamine (0.74 mL, 6.41 mmol) in THF (25 mL) was
added NH2OTHP (0.38 g, 3.21 mmol), and the mixture was
stirred at room temperature. After being stirred for 2 h, the
reaction mixture was concentrated under reduced pressure. The
residue was dissolved in EtOAc and quenched with water,
followed by extraction with EtOAc (20 mL × 3). The
combined organic layer was dried over anhydrous MgSO4
and concentrated under reduced pressure. The residue was
purified by silica gel chromatography ((EtOAc/n-hexane =
1.5:1)/1% NH3(aq)) to give compound 23. Yield 68%. 1H NMR
(500 MHz, CD3OD): δ 7.84−7.87 (m, 2H), 7.59 (s, 1H), 7.41
(d, J = 7.8 Hz, 1H), 7.15−7.21 (m, 2H), 6.47 (d, J = 14.9 Hz,
1H), 4.97−4.98 (m, 1H), 4.03−4.08 (m, 1H), 3.63−3.65 (m,
1H), 1.79−1.90 (m, 3H), 1.58−1.70 (m, 3H).
3-(1-Benzenesulfonyl-1H-indol-3-yl)-N-hydroxyacrylamide (6).
After a suspension of 23 (0.52 g, 1.82 mmol), TBAHS (0.09 g, 0.27
mmol), and KOH (0.20 g, 3.63 mmol) in CH2Cl2 (15 mL) was stirred
for 20 min, benzenesulfonyl chloride (0.35 mL, 2.72 mmol) was
added. The mixture was stirred at room temperature for 16 h. The
reaction was quenched with water, and extraction was with CH2Cl2
(20 mL × 3). The combined organic layer was dried over anhydrous
MgSO4, concentrated under reduced pressure, and dried with vacuum
to give a yellow residue without purification, which was dissolved in
methanol (50 mL) and treated with TFA (2.2 mL, 29.8 mmol). The
mixture was stirred at room temperature for 12 h. The reaction
mixture was concentrated under reduced pressure to give a yellow
residue, which was recrystallized by CH3OH to afford the desired
compound 6. Yield 32%; mp 195−197 °C. 1H NMR (500 MHz,
DMSO): δ 10.62 (s, 1H), 9.03 (s, 1H), 8.27 (s, 1H), 7.97−8.01 (m,
3H), 7.85 (d, J = 8.0 Hz, 1H), 7.69 (t, J = 7.5 Hz, 1H), 7.56−7.61 (m,
3H), 7.42 (t, J = 8.0 Hz, 1H), 7.37 (t, J = 8.0 Hz, 1H), 6.62 (d, J = 16.0
Hz, 1H). MS (EI) m/z: 342 (M+, 22%), 77 (100%). HRMS (EI) for
C17H14N2O4S (M+): calcd, 342.0674; found, 342.0673.
1 did not produce significant antitumor activity (P = 0.3925).
However, compound 8, at 200 and 100 mg/kg po daily to end,
produced a median TTE of 39.8 and 36.2 days, corresponding
to a 12.8 and 9.2 day T − C and a TGD of 47% and 34%,
respectively. On the basis of the log rank analysis, compound 8
exhibits significant antitumor activity at 200 mg/kg (P =
0.0274) but not at 100 mg/kg (P = 0.1668). Results indicated
that there was a dose-dependent delay of tumor growth in 8.
Compound 8 with TGI of 32.1% at 200 mg/kg, po (P < 0.01)
apparently demonstrated more potent antitumor activity than
reference compound 1 with TGI of 4.7% at 200 mg/kg po on
day 20. In summary, compound 8 displayed better efficacy in in
vivo lung A549 xenografts model than 1. No significant body
weight difference as indicated in the Supporting Information
and no adverse effects were observed.
CONCLUSION
■
We have identified a new class of 1-arylsulfonyl-5-(N-
hydroxyacrylamide)indoles as potent histone deacetylase
inhibitors. Lead compound 8 (MPT0E014) showed anti-
proliferative activity, with GI50 values ranging from 0.41 to 1
μM in a variety of human cancer cell lines from different
organs. It has better activity than compound 1. Compound 8
also exhibited low nanomolar IC50 values against HDACs 1, 2,
and 6 enzymes with 1.0, 4.0, and 12.3 nM, respectively,
comparable to compound 1. Structure−activity relationship
information revealed that the N-hydroxyacrylamide group
located at the C-5 position of indole ring displayed the best
activity compared to other positions such as the C-3, -4, -6, and
-7 positions. The 1-arylsulfonyl substitution pattern contributed
to a significant extent for maximal activity, for example,
compounds 8, 11, 12, 13, and 14 showed better activity
compared to benzoyl, benzyl, or phenyl group substituted
compounds. In an in vivo efficacy evaluation against human
xenograft model in nude mice bearing lung A549 cancer cell
line, compound 8 demonstrated greater antitumor activity than
compound 1. There was a 47% tumor growth delay (32%
tumor growth inhibition) on treatment with 8 daily at 200 mg/
kg po. In summary, compound 8 has potential in further
investigations as anticancer agents.
1-Benzenesulfonyl-1H-indole-4-carbaldehyde (28). After a sus-
pension of 1H-indole-4-carbaldehyde (24, 0.5 g, 3.44 mmol), TBAHS
(0.18 g, 0.52 mmol), and KOH (0.39 g, 6.89 mmol) in CH2Cl2 (15
mL) was stirred for 20 min, benzenesulfonyl chloride (0.66 mL, 5.17
mmol) was added, and the mixture was stirred at room temperature
overnight. The reaction was quenched with water, and extraction was
with CH2Cl2 (20 mL × 3). The combined organic layer was dried over
anhydrous MgSO4 and concentrated under reduced pressure to give a
yellow residue, which was purified by silica gel chromatography
(EtOAc/n-hexane = 1:3) to afford compound 28. Yield 85%. 1H NMR
(500 MHz, CDCl3): δ 10.17 (s, 1H), 8.28 (d, J = 8.4 Hz, 2H), 7.87−
7.89 (m, 2H), 7.76 (d, J = 3.6 Hz, 1H), 7.72 (d, J = 7.5 Hz, 1H), 7.44−
7.57 (m, 5H).
EXPERIMENTAL SECTION
■
Chemistry. Nuclear magnetic resonance (1H NMR) spectra were
obtained with a Bruker DRX-500 spectrometer (operating at 500
MHz), with chemical shift in parts per million (ppm, δ) downfield
from TMS as an internal standard. Mass spectrometry (MS) data were
measured on a JEOL JMS-700 mass spectrometer (HRMS-EI and MS-
EI), Finnigan TSQ 700 mass spectrometer (MS-EI), and micrOTOF
orthogonal ESI-TOF mass spectrometer (HRMS-ESI). Purity of the
final compounds was determined using a Hitachi 2000 series HPLC
system using C-18 column (Agilent ZORBAX Eclipse XDB-C18 5 μm,
4.6 mm × 150 mm) with the solvent system (elution conditions:
mobile phase A consisting of acetonitrile; mobile phase B consisting of
3-(1-Benzenesulfonyl-1H-indol-4-yl)acrylic Acid (32). To a
solution of 28 (0.2 g, 0.7 mmol) in CH2Cl2 (10 mL) was added
methyl (triphenylphosphoranylidene)acetate (0.28 g, 0.84 mmol), and
the mixture was stirred at room temperature. After being stirred for 16
3783
dx.doi.org/10.1021/jm300197a | J. Med. Chem. 2012, 55, 3777−3791

Journal of Medicinal Chemistry
Article
h, the reaction mixture was quenched with water and extracted with
CH2Cl2 (25 mL × 3). The combined organic layer was dried over
anhydrous MgSO4 and concentrated under reduced pressure to give a
yellow residue, which was purified by silica gel chromatography
(EtOAc/n-hexane = 1:2) to give the acrylic acid methyl ester
7.72 (m, 2H), 7.61 (d, J = 3.7 Hz, 1H), 7.55−7.58 (m, 1H), 7.52 (dd, J
= 8.7, 1.4 Hz, 1H), 7.45−7.48 (m, 2H), 6.71 (d, J = 3.7 Hz, 1H), 6.39
(d, J = 16.1 Hz, 1H).
3-(1-Benzenesulfonyl-1H-indol-5-yl)-N-hydroxyacrylamide (8).
To a solution of 33 (1.00 g, 3.05 mmol), PYBOP (1.69 g, 3.24
mmol), and triethylamine (1.02 mL, 7.33 mmol) in DMF (1.5 mL)
was added NH2OTHP (0.43 g, 3.67 mmol), and the mixture was
stirred at room temperature. After the mixture was stirred for 3 h, the
reaction was quenched with water, followed by extraction with EtOAc
(20 mL × 3). The combined organic layer was dried over anhydrous
MgSO4 and concentrated under reduced pressure. The residue was
purified by silica gel chromatography ((CH2Cl2/CH3OH = 30:1)/1%
NH3(aq)) to give a white solid, which was treated with TFA (6.90 mL,
92.90 mmol) in the presence of CH3OH (140 mL) and stirred at
room temperature for 12 h. The reaction mixture was concentrated
under reduced pressure to give a white residue, which was
recrystallized by EtOAc/EtOH to afford the desired 8. Yield 71%;
1
compound. Yield 88%. H NMR (500 MHz, CDCl3): δ 8.03 (d, J =
8.3 Hz, 1H), 7.96 (d, J = 16.0 Hz, 1H), 7.88−7.89 (m, 2H), 7.68 (d, J
= 3.7 Hz, 1H), 7.54−7.57 (m, 1H), 7.44−7.49 (m, 3H), 7.32−7.35 (m,
1H), 6.94 (d, J = 3.7 Hz, 1H), 6.51 (d, J = 16.0 Hz, 1H), 3.82 (s, 3H).
To a solution of ester compound (0.2 g, 0.58 mmol) in dioxane (10
mL), 1 M LiOH(aq) (1.2 mL) was added. The mixture was stirred at 40
°C. After being stirred for 12 h, the mixture was concentrated under
reduced pressure. The residue was dissolved in water. Concentrated
HCl was added to obtain acidic pH to produce a precipitate, which was
recrystallized by CH3OH and dried by vacuum to afford compound
1
32. Yield 90%. H NMR (500 MHz, CD3OD): δ 8.03 (d, J = 8.0 Hz,
1H), 7.96 (d, J = 15.9 Hz, 1H), 7.93 (d, J = 8.0 Hz, 2H), 7.87 (d, J =
3.8 Hz, 1H), 7.59−7.62 (m, 1H), 7.55 (d, J = 7.6 Hz, 1H), 7.49−7.52
(m, 2H), 7.33−7.34 (m, 1H), 7.03 (d, J = 3.8 Hz, 1H), 6.54 (d, J =
15.9 Hz, 1H).
1
mp 165−167 °C. H NMR (500 MHz, CD3OD): δ 7.98 (d, J = 8.5
Hz, 1H), 7.93 (d, J = 7.5 Hz, 2H), 7.68−7.72 (m, 2H) 7.59−7.63 (m,
2H), 7.49−7.54 (m, 3H), 6.75 (d, J = 3.5 Hz, 1H), 6.43 (d, J = 16.0
Hz, 1H). MS (EI) m/z: 342 (M+, 3%), 327 (100%). HRMS (EI) for
C17H14N2O4S (M+): calcd, 342.0674; found, 342.0673.
3-(1-Benzenesulfonyl-1H-indol-4-yl)-N-hydroxyacrylamide (7).
To a solution of 32 (0.2 g, 0.61 mmol), PYBOP (0.34 g, 0.65
mmol), and triethylamine (0.2 mL, 1.47 mmol) in DMF (1.5 mL) was
added NH2OTHP (0.09 g, 0.73 mmol), and the mixture was stirred at
room temperature. After the mixture was stirred for 2 h, the reaction
was quenched with water, followed by extraction with EtOAc (20 mL
× 3). The combined organic layer was dried over anhydrous MgSO4
and concentrated under reduced pressure. The residue was purified by
silica gel chromatography ((CH2Cl2/CH3OH = 30:1)/1% NH3(aq)) to
give a white solid, which was treated with TFA (1.9 mL, 25.58 mmol)
in the presence of CH3OH (35 mL). The mixture was stirred at room
temperature for 12 h. The reaction mixture was concentrated under
reduced pressure to give a white residue, which was recrystallized by
CH3OH to afford the desired 7. Yield 72%; mp 100−102 °C. 1H NMR
(500 MHz, CD3OD): δ 7.96 (d, J = 8.5 Hz, 1H), 7.88 (d, J = 8.0 Hz,
2H), 7.84 (d, J = 16.0 Hz, 1H), 7.73 (d, J = 3.5 Hz, 1H), 7.56 (t, J =
7.5 Hz, 1H), 7.44−7.48 (m, 3H), 7.29 (t, J = 7.5 Hz, 1H), 6.98 (d, J =
3.5 Hz, 1H), 6.50 (d, J = 16.0 Hz, 1H). MS (EI) m/z: 342 (M+, 17%),
77 (100%). HRMS (EI) for C17H14N2O4S (M+): calcd, 342.0674;
found, 342.0674.
1-Benzenesulfonyl-1H-indole-5-carbaldehyde (29). After a sus-
pension of 1H-indole-5-carbaldehyde (25, 1.00 g, 6.89 mmol), TBAHS
(0.35 g, 1.03 mmol), and KOH (0.77 g, 13.78 mmol) in CH2Cl2 (30
mL) was stirred for 20 min, benzenesulfonyl chloride (1.32 mL, 10.33
mmol) was added. The mixture was stirred at room temperature for 16
h. The reaction was quenched with water, and extraction was with
CH2Cl2 (20 mL × 3). The combined organic layer was dried over
anhydrous MgSO4 and concentrated under reduced pressure to give a
yellow residue, which was purified by silica gel chromatography
(EtOAc/n-hexane = 1:2) to afford compound 29. Yield 91%. 1H NMR
(500 MHz, CDCl3): δ 10.03 (s, 1H), 8.11 (d, J = 8.6 Hz, 1H), 8.06 (s,
1H), 7.89 (d, J = 7.6 Hz, 2H), 7.85−7.87 (m, 1H), 7.67 (d, J = 3.7 Hz,
1H), 7.55−7.58 (m, 1H), 7.45−7.48 (m, 2H), 6.78 (d, J = 3.7 Hz,
1H).
1-Benzenesulfonyl-1H-indole-6-carbaldehyde (30). After a sus-
pension of 1H-indole-6-carbaldehyde (26, 0.5 g, 3.44 mmol), TBAHS
(0.18 g, 0.52 mmol), and KOH (0.39 g, 6.89 mmol) in CH2Cl2 (15
mL) was stirred for 20 min, benzenesulfonyl chloride (0.66 mL, 5.17
mmol) was added, and the mixture was stirred at room temperature
for 16 h. The reaction was quenched with water, and extraction was
with CH2Cl2 (20 mL × 3). The combined organic layer was dried over
anhydrous MgSO4 and concentrated under reduced pressure to give a
yellow residue, which was purified by silica gel chromatography
(EtOAc/n-hexane = 1:3) to afford compound 30. Yield 87%. 1H NMR
(500 MHz, CDCl3): δ 10.08 (s, 1H), 8.49 (s, 1H), 7.91 (dd, J = 8.5,
1.0 Hz, 2H), 7.77−7.79 (m, 2H), 7.65 (d, J = 8.5 Hz, 1H), 7.56−7.59
(m, 1H), 7.46−7.49 (m, 2H), 6.74 (d, J = 3.5 Hz, 1H).
3-(1-(Phenylsulfonyl)-1H-indol-6-yl)acrylic Acid (34). To a sol-
ution of 30 (1.0 g, 3.5 mmol) in CH2Cl2 (20 mL) was added methyl
(triphenylphosphoranylidene)acetate (1.4 g, 4.2 mmol), and the
mixture was stirred at room temperature. After being stirred for 16 h,
the reaction mixture was quenched with water and extracted with
CH2Cl2 (25 mL × 3). The combined organic layer was dried over
anhydrous MgSO4 and concentrated under reduced pressure to give
the methyl ester compound as a yellow residue for the next reaction.
To a solution of crude methyl ester in dioxane (20 mL) was added 1
M LiOH(aq) (7 mL), and the mixture was stirred at 40 °C. After being
stirred for 12 h, the mixture was concentrated under reduced pressure.
The residue was dissolved in water, and concentrated HCl was added
to give acidic pH to produce the precipitate, which was recrystallized
by CH3OH and dried by vacuum to afford compound 34. Yield 79%
1
(two steps). H NMR (500 MHz, CD3OD, DMSO-d6): δ 8.14 (s,
1H), 7.96 (d, J = 7.6 Hz, 2H), 7.77 (d, J = 15.9 Hz, 1H), 7.75 (d, J =
3.6 Hz, 1H), 7.51−7.63 (m, 5H), 6.76 (d, J = 3.6 Hz, 1H), 6.49 (d, J =
15.9 Hz, 1H).
3-(1-Benzenesulfonyl-1H-indol-6-yl)-N-hydroxyacrylamide (9).
To a solution of 34 (0.3 g, 0.92 mmol), PYBOP (0.51 g, 0.97
mmol), and triethylamine (0.31 mL, 2.2 mmol) in DMF (2 mL) was
added NH2OTHP (0.13 g, 1.10 mmol), and the mixture was stirred at
room temperature. After the mixture was stirred for 3 h, the reaction
was quenched with water, followed by extraction with EtOAc (20 mL
× 3). The combined organic layer was dried over anhydrous MgSO4
and concentrated under reduced pressure. The residue was purified by
silica gel chromatography ((CH2Cl2/CH3OH = 30:1)/1% NH3(aq)) to
give a white solid, which was treated with TFA (2.1 mL, 28.27 mmol)
in the presence of CH3OH (42 mL). The mixture was stirred at room
temperature for 16 h. The reaction mixture was concentrated under
reduced pressure to give a red residue, which was recrystallized by
CH3OH to afford the desired 9. Yield 64%; mp 166−168 °C. 1H NMR
(500 MHz, CD3OD): δ 8.13 (s, 1H), 7.94 (d, J = 8.0 Hz, 2H), 7.72 (d,
J = 3.5 Hz, 1H), 7.67 (d, J = 15.5 Hz, 1H), 7.60 (t, J = 7.5 Hz, 1H),
7.50−7.56 (m, 3H), 7.44 (d, J = 8.0 Hz, 1H), 6.74 (d, J = 3.5 Hz, 1H),
3-(1-Benzenesulfonyl-1H-indol-5-yl)acrylic Acid (33). To a
solution of 29 (1.79 g, 6.27 mmol) in CH2Cl2 (25 mL), methyl
(triphenylphosphoranylidene)acetate (2.52 g, 7.53 mmol) was added,
and the mixture was stirred at room temperature. After being stirred
overnight, the reaction mixture was quenched with water and extracted
with CH2Cl2 (25 mL × 3). The combined organic layer was dried over
anhydrous MgSO4 and concentrated under reduced pressure to give
the methyl ester compound as a yellow residue for the next reaction.
To a solution of methyl ester residue in dioxane (20 mL), 1 M
LiOH aqueous solution (11.7 mL) was added. The mixture was stirred
at 40 °C. After being stirred for 16 h, the mixture was concentrated
under reduced pressure. The residue was dissolved in water, and
concentrated HCl was added to obtain acidic pH to produce the
precipitate, which was recrystallized by CH3OH and dried by vacuum
1
to afford compound 33. Yield 80% (two steps). H NMR (500 MHz,
CDCl3): δ 7.96 (d, J = 8.7 Hz, 1H), 7.89 (d, J = 8.9 Hz, 2H), 7.67−
3784
dx.doi.org/10.1021/jm300197a | J. Med. Chem. 2012, 55, 3777−3791

Journal of Medicinal Chemistry
Article
6.52 (d, J = 16.0 Hz, 1H). MS (EI) m/z: 342 (M+, 11%), 77 (100%).
HRMS (EI) for C17H14N2O4S (M+): calcd, 342.0674; found,
342.0674.
3-[1-(4-Methoxybenzenesulfonyl)-1H-indol-5-yl]acrylic Acid (40).
To a solution of 36 (1.91 g, 6.06 mmol) in CH2Cl2 (20 mL) was
added methyl (triphenylphosphoranylidene)acetate (2.43 g, 7.27
mmol), and the mixture was stirred at room temperature. After
being stirred overnight, the reaction mixture was quenched with water
and extracted with CH2Cl2 (25 mL × 3). The combined organic layer
was dried over anhydrous MgSO4 and concentrated under reduced
pressure to give the methyl ester as a yellow residue for the next
reaction.
1-Benzenesulfonyl-1H-indole-7-carbaldehyde (31). After a sus-
pension of 1H-indole-7-carbaldehyde (27, 0.8 g, 5.51 mmol), TBAHS
(0.28 g, 0.83 mmol), and KOH (0.62 g, 11.0 mmol) in CH2Cl2 (20
mL) was stirred for 20 min, benzenesulfonyl chloride (1.05 mL, 8.27
mmol) was added, and the mixture was stirred at room temperature
for 16 h. The reaction was quenched with water, and extraction was
with CH2Cl2 (20 mL × 3). The combined organic layer was dried over
anhydrous MgSO4 and concentrated under reduced pressure to give a
yellow residue, which was purified by silica gel chromatography
(EtOAc/n-hexane = 1:4) to afford compound 31. Yield 66%. 1H NMR
(500 MHz, CDCl3): δ 10.69 (s, 1H), 7.80 (d, J = 7.5 Hz, 1H), 7.69 (d,
J = 7.6 Hz, 1H), 7.67 (d, J = 3.7 Hz, 1H), 7.57 (d, J = 7.7 Hz, 2H),
7.49−7.52 (m, 1H), 7.35−7.38 (m, 3H), 6.78 (d, J = 3.7 Hz, 1H).
3-(1-Benzenesulfonyl-1H-indol-7-yl)acrylic Acid (35). To a
solution of 31 (1.04 g, 3.65 mmol) in CH2Cl2 (20 mL) was added
methyl (triphenylphosphoranylidene)acetate (1.46 g, 4.38 mmol), and
the mixture was stirred at room temperature. After being stirred
overnight, the reaction mixture was quenched with water and extracted
with CH2Cl2 (25 mL × 3). The combined organic layer was dried over
anhydrous MgSO4 and concentrated under reduced pressure to give
the methyl ester as a yellow residue for the next reaction.
To a solution of crude methyl ester in dioxane (40 mL) was added 1
M LiOH(aq) (12.2 mL), and the mixture was stirred at 40 °C. After
being stirred for 12 h, the mixture was concentrated under reduced
pressure. The residue was dissolved in water, and concentrated HCl
was added to obtain acidic pH to produce the precipitate, which was
recrystallized and dried by vacuum to afford compound 40. Yield 89%
1
(two steps). H NMR (500 MHz, CD3OD): δ 7.98 (d, J = 8.7 Hz,
1H), 7.85−7.88 (m, 2H), 7.76 (s, 1H), 7.71 (d, J = 16.0 Hz, 1H), 7.66
(d, J = 3.6 Hz, 1H), 7.56 (d, J = 8.5 Hz, 1H), 6.98−7.01 (m, 2H), 6.74
(d, J = 3.6 Hz, 1H), 6.44 (d, J = 16.0 Hz, 1H), 3.79 (s, 3H).
N-Hydroxy-3-[1-(4-methoxybenzenesulfonyl)-1H-indol-5-yl]-
acrylamide (11). To a solution of 40 (0.2 g, 0.56 mmol), PYBOP
(0.32 g, 0.62 mmol), and triethylamine (0.19 mL, 1.34 mmol) in DMF
(2 mL) was added NH2OTHP (0.08 g, 0.67 mmol), and the mixture
was stirred at room temperature. After the mixture was stirred for 3 h,
the reaction was quenched with water, followed by extraction with
EtOAc (20 mL × 3). The combined organic layer was dried over
anhydrous MgSO4 and concentrated under reduced pressure. The
residue was purified by silica gel chromatography ((CH2Cl2/CH3OH
= 30:1)/1% NH3(aq)) to give a white solid, which was treated with
TFA (1.3 mL, 17.34 mmol) in the presence of CH3OH (27 mL). The
mixture was stirred at room temperature for 16 h. The reaction
mixture was concentrated under reduced pressure to give a white
residue, which was recrystallized by CH3OH to afford compound 11.
Yield 78%; mp 90−92 °C. 1H NMR (500 MHz, CD3OD): δ 7.96 (d, J
= 8.5 Hz, 1H), 7.84−7.87 (m, 2H), 7.71 (s, 1H), 7.64 (d, J = 3.5 Hz,
1H), 7.61 (d, J = 15.5 Hz, 1H), 7.52 (d, J = 8.5 Hz, 1H), 6.97−6.99
(m, 2H), 6.72 (d, J = 3.5 Hz, 1H), 6.43 (d, J = 15.5 Hz, 1H), 3.78 (s,
3H). MS (EI) m/z: 372 (M+, 5%), 171 (100%). HRMS (EI) for
C18H16N2O5 (M+): calcd, 372.0780; found, 372.0779.
3-[1-(3,4-Dimethoxybenzenesulfonyl)-1H-indol-5-yl]acrylic Acid
(41). After a suspension of 1H-indole-5-carbaldehyde (25, 0.4 g,
2.76 mmol), TBAHS (0.14 g, 0.41 mmol), and KOH (0.31 g, 5.51
mmol) in CH2Cl2 (15 mL) was stirred for 20 min, 3,4-
dimethoxybenzenesulfonyl chloride (0.98 g, 4.13 mmol) was added,
and the mixture was stirred at room temperature for 16 h. The
reaction was quenched with water, and extraction was with CH2Cl2
(20 mL × 3). The combined organic layer was dried over anhydrous
MgSO4 and concentrated under reduced pressure to give a yellow
residue 37, which was dissolved in CH2Cl2 (15 mL) and then treated
with methyl (triphenylphosphoranylidene)acetate (1.11 g, 3.31 mmol)
at room temperature. After being stirred overnight, the reaction
mixture was quenched with water and extracted with CH2Cl2 (25 mL
× 3). The combined organic layer was dried over anhydrous MgSO4
and concentrated under reduced pressure to give the methyl ester
compound as a yellow residue for the next reaction.
To a solution of crude methyl ester in dioxane (25 mL) was added 1
M LiOH(aq) (7.3 mL), and the mixture was stirred at 40 °C. After
being stirred for 12 h, the mixture was concentrated under reduced
pressure. The residue was dissolved in water, and concentrated HCl
was added to obtain acidic pH to produce the precipitate, which was
recrystallized and dried by vacuum to afford compound 35. Yield 85%
1
(two steps). H NMR (500 MHz, CDCl3): δ 8.72 (d, J = 15.5 Hz,
1H), 7.85 (d, J = 3.8 Hz, 1H), 7.74 (d, J = 7.5 Hz, 2H), 7.53−7.61 (m,
2H), 7.38−7.43 (m, 3H), 7.25−7.27 (m, 1H), 6.75 (d, J = 3.8 Hz,
1H), 6.14 (d, J = 15.5 Hz, 1H).
3-(1-Benzenesulfonyl-1H-indol-7-yl)-N-hydroxyacrylamide (10).
To a solution of 35 (0.2 g, 0.61 mmol), PYBOP (0.34 g, 0.65
mmol), and triethylamine (0.2 mL, 1.47 mmol) in DMF (1.5 mL) was
added NH2OTHP (0.09 g, 0.73 mmol), and the mixture was stirred at
room temperature. After the mixture was stirred for 3 h, the reaction
was quenched with water, followed by extraction with EtOAc (20 mL
× 3). The combined organic layer was dried over anhydrous MgSO4
and concentrated under reduced pressure. The residue was purified by
silica gel chromatography ((CH2Cl2/CH3OH = 30:1)/1% NH3(aq)) to
give a white solid, which was treated with TFA (1.4 mL, 18.85 mmol)
in the presence of CH3OH (28 mL). The mixture was stirred at room
temperature for 16 h. The reaction mixture was concentrated under
reduced pressure to give a white residue, which was recrystallized by
CH3OH to afford the desired compound 10. Yield 62%; mp 177−178
°C. 1H NMR (500 MHz, CD3OD): δ 8.55 (d, J = 15.5 Hz, 1H), 7.88
(d, J = 3.5 Hz, 1H), 7.73 (m, 2H), 7.55−7.59 (m, 2H), 7.43 (t, J = 7.5
Hz, 2H), 7.30 (d, J = 7.5 Hz, 1H), 7.21 (t, J = 7.5 Hz, 1H), 6.81 (d, J =
3.5 Hz, 1H), 6.00 (d, J = 15.5 Hz, 1H). MS (EI) m/z: 342 (M+, 16%),
77 (100%). HRMS (EI) for C17H14N2O4S (M+): calcd, 342.0674;
found, 342.0672.
Syntheses of 1-Arylsulfonyl-5-(N-hydroxyacrylamide)-
indoles 11−14. 1-(4-Methoxybenzenesulfonyl)-1H-indole-5-car-
baldehyde (36). After a suspension of 1H-indole-5-carbaldehyde
(25, 1.0 g, 6.89 mmol), TBAHS (0.35 g, 1.03 mmol), and KOH
(0.77 g, 13.78 mmol) in CH2Cl2 (20 mL) was stirred for 20
min, 4-methoxybenzenesulfonyl chloride (2.14 g, 10.33 mmol)
was added, and the mixture was stirred at room temperature
overnight. The reaction was quenched with water, and
extraction was with CH2Cl2 (20 mL × 3). The combined
organic layer was dried over anhydrous MgSO4 and
concentrated under reduced pressure to give a yellow residue,
which was purified by silica gel chromatography (EtOAc/n-
To a solution of crude methyl ester in dioxane (25 mL) was added 1
M LiOH(aq) (5.5 mL), and the mixture was stirred at 40 °C. After the
mixture was stirred for 12 h, the reaction was concentrated under
reduced pressure. The residue was dissolved in water, and
concentrated HCl was added to obtain acidic pH to produce the
precipitate, which was recrystallized and dried by vacuum to afford
compound 41. Yield 78% (three steps). 1H NMR (500 MHz,
CD3OD): δ 8.00 (d, J = 8.7 Hz, 1H), 7.77 (s, 1H), 7.71 (d, J = 16.0
Hz, 1H), 7.68 (d, J = 3.6 Hz, 1H), 7.55−7.59 (m, 2H), 7.34 (d, J = 2.3
Hz, 1H), 6.99−7.02 (m, 1H), 6.74 (d, J = 3.6 Hz, 1H), 6.44 (d, J =
16.0 Hz, 1H), 3.81 (s, 3H), 3.78 (s, 3H).
1
hexane = 1:2) to afford compound 36. Yield 88%. H NMR
3-[1-(3,4-Dimethoxybenzenesulfonyl)-1H-indol-5-yl]-N-hydrox-
yacrylamide (12). To a solution of 41 (0.2 g, 0.52 mmol), PYBOP
(0.3 g, 0.57 mmol), and triethylamine (0.17 mL, 1.25 mmol) in DMF
(2 mL) was added NH2OTHP (0.07 g, 0.62 mmol), and the mixture
(500 MHz, CDCl3): δ 10.03 (s, 1H), 8.10 (d, J = 8.6 Hz, 1H),
8.06 (s, 1H), 7.83−7.86 (m, 3H), 7.66 (d, J = 3.6 Hz, 1H),
6.89−6.92 (m, 2H), 6.77 (d, J = 3.6 Hz, 1H), 3.80 (s, 3H).
3785
dx.doi.org/10.1021/jm300197a | J. Med. Chem. 2012, 55, 3777−3791

Journal of Medicinal Chemistry
Article
was stirred at room temperature. After the mixture was stirred for 2 h,
the reaction was quenched with water, followed by extraction with
EtOAc (20 mL × 3). The combined organic layer was dried over
anhydrous MgSO4 and concentrated under reduced pressure. The
residue was purified by silica gel chromatography ((CH2Cl2/CH3OH
= 30:1)/1% NH3(aq)) to give a white solid, which was treated with
TFA (1.2 mL, 16.1 mmol) in the presence of CH3OH (25 mL). The
mixture was stirred at room temperature for 16 h. The reaction
mixture was concentrated under reduced pressure to give a yellow
residue, which was recrystallized by CH3OH to afford the desired
TBAHS (0.53 g, 1.55 mmol), and KOH (1.16 g, 20.67 mmol) in
CH2Cl2 (25 mL) was stirred for 20 min, 4-nitrobenzenesulfonyl
chloride (3.44 g, 15.5 mmol) was added. The mixture was stirred at
room temperature for 16 h. The reaction was quenched with water,
and extraction was with CH2Cl2 (20 mL × 3). The combined organic
layer was dried over anhydrous MgSO4 and concentrated under
reduced pressure to give a yellow residue, which was purified by silica
gel chromatography (EtOAc/n-hexane = 1:3) to afford compound 39.
1
Yield 82%. H NMR (500 MHz, CDCl3): δ 10.04 (s, 1H), 8.29−8.32
(m, 2H), 8.07−8.13 (m, 4H), 7.89 (dd, J = 9.0, 2.0 Hz, 1H), 7.65 (d, J
1
compound 12. Yield 53%; mp 146−148 °C. H NMR (500 MHz,
= 3.5 Hz, 1H), 6.85 (d, J = 3.5 Hz, 1H).
CD3OD): δ 7.99 (d, J = 8.5 Hz, 1H), 7.72 (s, 1H), 7.67 (d, J = 3.5 Hz,
1H) 7.62 (d, J = 16.0 Hz, 1H), 7.55 (dd, J = 2.0, 8.5 Hz, 1H), 7.53 (d,
J = 8.5 Hz, 1H), 7.33 (d, J = 2.0 Hz, 1H), 7.01 (d, J = 8.5 Hz, 1H),
6.73 (d, J = 3.5 Hz, 1H), 6.43 (d, J = 16.0 Hz, 1H), 3.81 (s, 3H), 3.78
(s, 3H). MS (EI) m/z: 387 (M+ − 15, 59%), 137 (100%). HRMS (EI)
for C19H18N2O6S (M+): calcd, 402.0886; found, 402.0884.
3-[1-(4-Nitrobenzenesulfonyl)-1H-indol-5-yl]acrylic Acid (43). To
a solution of 39 (0.50 g, 1.51 mmol) in CH2Cl2 (15 mL) was added
methyl (triphenylphosphoranylidene)acetate (0.61 g, 1.82 mmol), and
the mixture was stirred at room temperature. After being stirred
overnight, the reaction mixture was quenched with water and extracted
with CH2Cl2 (25 mL × 3). The combined organic layer was dried over
anhydrous MgSO4 and concentrated under reduced pressure to give a
methyl ester yellow residue for the next reaction.
1-(4-Fluorobenzenesulfonyl)-1H-indole-5-carbaldehyde (38).
After a suspension of 1H-indole-5-carbaldehyde (25, 1.0 g, 6.89
mmol), TBAHS (0.35 g, 1.03 mmol), and KOH (0.77 g, 13.78 mmol)
in CH2Cl2 (20 mL) was stirred for 20 min, 4-fluorobenzenesulfonyl
chloride (2.01 g, 10.33 mmol) was added. The mixture was stirred at
room temperature for 16 h. The reaction was quenched with water,
and extraction was with CH2Cl2 (20 mL × 3). The combined organic
layer was dried over anhydrous MgSO4 and concentrated under
reduced pressure to give a yellow residue, which was purified by silica
gel chromatography (EtOAc/n-hexane = 1:5) to afford compound 38.
To a solution of crude methyl ester in dioxane (15 mL) was added 1
M LiOH(aq) (3.1 mL), and the mixture was stirred at 40 °C. After
being stirred for 12 h, the mixture was concentrated under reduced
pressure. The residue was dissolved in water, and concentrated HCl
was added to obtain acidic pH to produce the precipitate, which was
recrystallized and dried by vacuum to afford compound 43. Yield 82%
1
(two steps). H NMR (500 MHz, DMSO-d6): δ 8.34−8.35 (m, 2H),
8.24−8.27 (m, 2H), 7.94 (d, J = 8.5 Hz, 1H), 7.92 (s, 1H), 7.89 (d, J =
3.5 Hz, 1H), 7.70 (dd, J = 9.0, 1.5 Hz, 1H), 7.61 (d, J = 16.0 Hz, 1H),
6.91 (d, J = 3.5 Hz, 1H), 6.48 (d, J = 16.0 Hz, 1H).
1
Yield 76%. H NMR (500 MHz, CDCl3): δ 10.04 (s, 1H), 8.08−8.11
(m, 2H), 7.92−7.94 (m, 2H), 7.87 (d, J = 8.7 Hz, 1H), 7.65 (d, J = 3.6
Hz, 1H), 7.08−7.16 (m, 2H), 6.80 (d, J = 3.6 Hz, 1H).
N-Hydroxy-3-[1-(4-nitrobenzenesulfonyl)-1H-indol-5-yl]-
acrylamide (14). To a solution of 43 (0.2 g, 0.54 mmol), PYBOP
(0.31 g, 0.59 mmol), and triethylamine (0.18 mL, 1.3 mmol) in DMF
(1.5 mL) was added NH2OTHP (0.08 g, 0.65 mmol), and the mixture
was stirred at room temperature. After the mixture was stirred for 3 h,
the reaction was quenched with water, followed by extraction with
EtOAc (20 mL × 3). The combined organic layer was dried over
anhydrous MgSO4 and concentrated under reduced pressure. The
residue was purified by silica gel chromatography ((CH2Cl2/CH3OH
= 30:1)/1% NH3(aq)) to give a white solid, which was treated with
TFA (1.2 mL, 16.7 mmol) in the presence of CH3OH (27 mL). The
mixture was stirred at room temperature for 16 h. The reaction
mixture was concentrated under reduced pressure to give a white
residue, which was recrystallized by CH3OH to afford the desired
3-[1-(4-Fluorobenzenesulfonyl)-1H-indol-5-yl]acrylic Acid (42).
To a solution of 38 (1.0 g, 3.30 mmol) in CH2Cl2 (25 mL) was
added methyl (triphenylphosphoranylidene)acetate (1.32 g, 3.96
mmol), and the mixture was stirred at room temperature for 16 h.
The reaction mixture was quenched with water and extracted with
CH2Cl2 (25 mL × 3). The combined organic layer was dried over
anhydrous MgSO4 and concentrated under reduced pressure to give
the methyl ester yellow residue for the next reaction.
To a solution of crude methyl ester in dioxane (30 mL) was added 1
M LiOH(aq) (6.6 mL) was added, and the mixture was stirred at 40 °C.
After being stirred for 12 h, the mixture was concentrated under
reduced pressure. The residue was dissolved in water, and
concentrated HCl was added to obtain acidic pH to produce the
precipitate, which was recrystallized and dried by vacuum to afford
compound 42. Yield 90% (two steps). 1H NMR (500 MHz, CD3OD):
δ 8.00−8.03 (m, 3H), 7.79 (s, 1H), 7.71 (d, J = 15.9 Hz, 1H), 7.69 (d,
J = 3.7 Hz, 1H), 7.59 (d, J = 8.8 Hz, 1H), 7.24−7.28 (m, 2H), 6.78 (d,
J = 3.7 Hz, 1H), 6.45 (d, J = 15.9 Hz, 1H).
1
compound 14. Yield 65%; mp 154−156 °C. H NMR (500 MHz,
CD3OD): δ 8.33 (d, J = 8.5 Hz, 2H), 8.19 (d, J = 8.5 Hz, 2H), 8.01 (d,
J = 8.5 Hz, 1H), 7.72−7.74 (m, 2H), 7.61 (d, J = 16.0 Hz, 1H), 7.57
(d, J = 8.5 Hz, 1H), 6.82 (d, J = 3.5 Hz, 1H), 6.45 (d, J = 16.0 Hz,
1H). MS (EI) m/z: 387 (M+, 0.21%), 372 (100%). HRMS (EI) for
C17H13N3O6S (M+): calcd, 387.0525; found, 387.0523.
3-[1-(4-Fluorobenzenesulfonyl)-1H-indol-5-yl]-N-hydroxyacryla-
mide (13). To a solution of 42 (0.2 g, 0.58 mmol), PYBOP (0.33 g,
0.64 mmol), and triethylamine (0.19 mL, 1.39 mmol) in DMF (1.5
mL) was added NH2OTHP (0.08 g, 0.70 mmol), and the mixture was
stirred at room temperature. After the mixture was stirred for 3 h, the
reaction was quenched with water, followed by extraction with EtOAc
(20 mL × 3). The combined organic layer was dried over anhydrous
MgSO4 and concentrated under reduced pressure. The residue was
purified by silica gel chromatography ((CH2Cl2/CH3OH = 30:1)/1%
NH3(aq)) to give a white solid, which was treated with TFA (1.3 mL,
18 mmol) in the presence of CH3OH (28 mL). The mixture was
stirred at room temperature for 16 h. The reaction mixture was
concentrated under reduced pressure to give a white residue, which
was recrystallized by CH3OH to afford the desired compound 13.
Yield 76%; mp 138−140 °C. 1H NMR (500 MHz, CD3OD): δ 7.97−
8.01 (m, 3H), 7.72 (s, 1H), 7.67 (d, J = 3.5 Hz, 1H), 7.62 (d, J = 15.5
Hz, 1H), 7.54 (t, J = 8.5 Hz, 1H), 7.24 (d, J = 8.5 Hz, 2H), 6.76 (d, J =
3.5 Hz, 1H), 6.44 (d, J = 15.5 Hz, 1H). MS (EI) m/z: 360 (M+, 0.6%),
345 (100%). HRMS (EI) for C17H13FN2O4S (M+): calcd, 360.0580;
found, 360.0580.
Synthesis of 1-Phenylsulfonyl-5-hydroxyamideindole
(15). 1-Benzenesulfonyl-1H-indole-5-carboxylic Acid (45). After
a suspension of methyl indole-5-carboxylate (44, 0.30 g, 1.71
mmol), TBAHS (0.19 g, 0.26 mmol), and KOH (0.19 g, 3.42
mmol) in CH2Cl2 (15 mL) was stirred for 20 min,
benzenesulfonyl chloride (0.32 mL, 2.57 mmol) was added,
and the mixture was stirred at room temperature for 16 h. The
reaction was quenched with water, and extraction was with
CH2Cl2 (20 mL × 3). The combined organic layer was dried
over anhydrous MgSO4 and concentrated under reduced
pressure to give a yellow residue, which was purified by silica
gel chromatography (EtOAc/n-hexane = 1:4) to afford the 1-
phenylsulfonyl indole compound as a white solid. Yield 89%.
1H NMR (500 MHz, CDCl3): δ 8.26 (s, 1H), 7.99−8.04 (m,
2H), 7.87−7.89 (m, 2H), 7.62 (d, J = 3.5 Hz, 1H), 7.54−7.57
(m, 1H), 7.43−7.47 (m, 2H), 6.73 (d, J = 3.5 Hz, 1H), 3.91 (s,
3H).
To a solution of 1-phenylsulfonylindole (0.52 g, 1.65 mmol) in
dioxane (15 mL) was added 1 M LiOH aqueous solution (3.8 mL),
and the mixture was stirred at 40 °C. After being stirred overnight, the
mixture was concentrated under reduced pressure. The residue was
1-(4-Nitrobenzenesulfonyl)-1H-indole-5-carbaldehyde (39). After
a suspension of 1H-indole-5-carbaldehyde (25, 1.5 g, 10.33 mmol),
3786
dx.doi.org/10.1021/jm300197a | J. Med. Chem. 2012, 55, 3777−3791

Journal of Medicinal Chemistry
Article
dissolved in water, and concentrated HCl was added to obtain acidic
pH to produce the precipitate, which was dried by vacuum to afford
compound 45. Yield 85%. 1H NMR (500 MHz, CD3OD): δ 8.25 (d, J
= 0.8 Hz, 1H), 8.03 (d, J = 8.9 Hz, 1H), 7.97 (dd, J = 8.9, 1.5 Hz, 1H),
7.95 (d, J = 7.6 Hz, 2H), 7.75 (d, J = 3.7 Hz, 1H), 7.60−7.63 (m, 1H),
7.51−7.54 (m, 2H), 6.83 (d, J = 3.7 Hz, 1H).
in the presence of CH3OH (16 mL). The mixture was stirred
overnight at room temperature. The reaction mixture was concen-
trated under reduced pressure to give a white residue, which was
recrystallized by CH3OH to afford the desired 16. Yield 49%; mp
146−148 °C. 1H NMR (500 MHz, DMSO): δ 9.00 (s, 1H), 8.25 (d, J
= 8.5 Hz, 1H), 7.85 (s, 1H), 7.76 (d, J = 7.5 Hz, 2H), 7.69 (t, J = 7.5
Hz, 1H), 7.54−7.61 (m, 4H), 7.42 (d, J = 3.5 Hz, 1H), 6.78 (d, J = 3.5
Hz, 1H), 6.48 (d, J = 15.5 Hz, 1H). MS (EI) m/z: 306 (M+, 17%), 105
(100%). HRMS (EI) for C18H14N2O3 (M+): calcd, 306.1004; found,
306.1006.
1-Benzyl-1H-indole-5-carbaldehyde (47). After a suspension of
1H-indole-5-carbaldehyde (25, 0.5 g, 3.44 mmol), KI (0.29 g, 1.72
mmol), and potassium tert-butoxide (0.77 g, 6.89 mmol) in DMF (3
mL) was stirred for 15 min, benzyl chloride (0.8 mL, 6.89 mmol) was
added. The mixture was stirred at room temperature for 16 h. The
reaction was quenched with water, and extraction was with CH2Cl2
(20 mL × 3). The combined organic layer was dried over anhydrous
MgSO4, concentrated under reduced pressure, and dried with vacuum
to give a yellow residue, which was purified by silica gel
chromatography (EtOAc/n-hexane = 1:3) to afford compound 47.
Yield 85%. 1H NMR (500 MHz, CDCl3): δ 10.02 (s, 1H), 8.17 (d, J =
1.1 Hz, 1H), 7.73 (dd, J = 8.6, 1.1 Hz, 1H), 7.36 (d, J = 8.6 Hz, 1H),
7.26−7.33 (m, 3H), 7.22 (d, J = 3.0 Hz, 1H), 7.10−7.12 (m, 2H), 6.71
(d, J = 3.0 Hz, 1H), 5.36 (s, 2H).
3-(1-Benzyl-1H-indol-5-yl)acrylic Acid (50). To a solution of 47
(0.3 g, 1.28 mmol) in CH2Cl2 (10 mL) was added methyl
(triphenylphosphoranylidene)acetate (0.51 g, 1.53 mmol), and the
mixture was stirred at room temperature. After being stirred for 16 h,
the reaction mixture was quenched with water and extracted with
CH2Cl2 (25 mL × 3). The combined organic layer was dried over
anhydrous MgSO4, concentrated under reduced pressure, and dried
with vacuum to give a brown residue, which was purified by silica gel
chromatography (EtOAc/n-hexane = 1:3) to afford the methyl ester
compound as an orange solid. Yield 85%. 1H NMR (500 MHz,
CDCl3): δ 7.82 (d, J = 16.0 Hz, 1H), 7.80 (s, 1H), 7.39 (dd, J = 8.5,
1.5 Hz, 1H), 7.27−7.32 (m, 4H), 7.14 (d, J = 3.0 Hz, 1H), 7.10 (d, J =
7.0 Hz, 2H), 6.58 (d, J = 3.0 Hz, 1H), 6.39 (d, J = 16.0 Hz, 1H), 5.32
(s, 2H), 3.81 (s, 3H).
1-Benzenesulfonyl-1H-indole-5-carboxylic Acid Hydroxyamide
(15). To a solution of 45 (0.18 g, 0.60 mmol), PYBOP (0.33 g,
0.63 mmol), and triethylamine (0.20 mL, 1.43 mmol) in DMF (1.5
mL) was added NH2OTHP (0.08 g, 0.72 mmol), and the mixture was
stirred at room temperature. After the mixture was stirred for 2 h, the
reaction was quenched with water, followed by extraction with EtOAc
(15 mL × 3). The combined organic layer was dried over anhydrous
MgSO4 and concentrated under reduced pressure. The residue was
purified by silica gel chromatography ((CH2Cl2/CH3OH = 30:1)/1%
NH3(aq)) to give a white solid, which was treated with TFA (1.70 mL,
22.89 mmol) in the presence of CH3OH (31 mL). The mixture was
stirred at room temperature for 12 h. The reaction mixture was
concentrated under reduced pressure to give a white residue, which
was purified by silica gel chromatography (CH2Cl2/CH3OH = 30:1)
1
to afford the desired 15 as a colorless oil. Yield 53%. H NMR (500
MHz, CD3OD): δ 8.05 (d, J = 8.0 Hz, 1H), 7.93 (d, J = 7.5 Hz, 3H),
7.74 (d, J = 3.5 Hz, 1H), 7.68 (d, J = 7.5 Hz, 1H), 7.61 (t, J = 7.5 Hz,
1H), 7.51 (t, J = 8.0 Hz, 2H), 6.81 (d, J = 3.5 Hz, 1H). MS (EI) m/z:
316 (M+, 6%), 315 (24%), 77 (100%). HRMS (EI) for C15H12N2O4S
(M+): calcd, 316.0518; found, 316.0518.
Syntheses of 1-Benzoyl, Benzyl, and Phenyl-5-(N-
hydroxyacrylamide)indoles 16−18. 1-Benzoyl-1H-indole-5-car-
baldehyde (46). After a suspension of 1H-indole-5-carbaldehyde
(25, 0.50 g, 2.85 mmol), KI (0.64 g, 1.43 mmol), and potassium
tert-butoxide (0.64 g, 5.71 mmol) in DMF (2 mL) was stirred
for 15 min, benzoyl bromide (0.50 mL, 4.28 mmol) was added,
and the mixture was stirred at room temperature for 16 h. The
reaction was quenched with water, and extraction was with
CH2Cl2 (20 mL × 3). The combined organic layer was dried
over anhydrous MgSO4, concentrated under reduced pressure,
and dried with vacuum to give a yellow residue, which was
purified by silica gel chromatography (EtOAc/n-hexane = 1:2)
1
to afford compound 46. Yield 87%. H NMR (500 MHz,
To a solution of methyl ester (0.32 g, 1.10 mmol) in dioxane (10
mL) was added 1 M LiOH(aq) (2.2 mL), and the mixture was stirred at
40 °C. After being stirred overnight, the mixture was concentrated
under reduced pressure. The residue was dissolved in water, and
concentrated HCl was added to obtain acidic pH to produce the
precipitate, which was dried by vacuum to afford compound 50. Yield
91%. 1H NMR (500 MHz, CD3OD): δ 7.76−7.79 (m, 2H), 7.39 (dd, J
= 8.5, 1.5 Hz, 1H), 7.22−7.34 (m, 5H), 7.12 (d, J = 7.0 Hz, 2H), 6.55
(d, J = 3.0 Hz, 1H), 6.35 (d, J = 16.0 Hz, 1H), 5.39 (s, 2H).
3-(1-Benzyl-1H-indol-5-yl)-N-hydroxyacrylamide (17). To a sol-
ution of 50 (0.1 g, 0.4 mmol), PYBOP (0.23 g, 0.44 mmol), and
triethylamine (0.13 mL, 0.96 mmol) in DMF (1 mL) was added
NH2OTHP (0.06 g, 0.48 mmol), and the mixture was stirred at room
temperature. After the mixture was stirred for 1 h, the reaction was
quenched with water, followed by extraction with EtOAc (20 mL × 3).
The combined organic layer was dried over anhydrous MgSO4 and
concentrated under reduced pressure. The residue was purified by
silica gel chromatography ((CH2Cl2/CH3OH = 30:1)/1% NH3(aq)) to
give a white solid, which was treated with TFA (0.92 mL, 12.4 mmol)
in the presence of CH3OH (19 mL). The mixture was stirred at room
temperature for 12 h. The reaction mixture was concentrated under
reduced pressure to give a white residue, which was recrystallized by
CDCl3): δ 10.10 (s, 1H), 8.50 (d, J = 8.5 Hz, 1H), 8.15 (d, J =
2.0 Hz, 1H), 7.92 (dd, J = 9.0, 2.0 Hz, 1H), 7.75−7.77 (m, 2H),
7.63−7.66 (m, 1H), 7.54−7.57 (m, 2H), 7.42 (d, J = 3.5 Hz,
1H), 6.74 (d, J = 3.5 Hz, 1H).
3-(1-Benzoyl-1H-indol-5-yl)acrylic Acid (49). To a solution of 46
(0.62 g, 2.03 mmol) in CH2Cl2 (15 mL) was added methyl
(triphenylphosphoranylidene)acetate (0.81 g, 2.44 mmol), and the
mixture was stirred at room temperature. After being stirred for 16 h,
the reaction mixture was quenched with water and extracted with
CH2Cl2 (25 mL × 3). The combined organic layer was dried over
anhydrous MgSO4 and concentrated under reduced pressure to give a
methyl ester yellow residue for the next reaction.
To a solution of crude methyl ester in dioxane (10 mL) was added 1
M LiOH(aq) (4.1 mL), and the mixture was stirred at 40 °C. After
being stirred for 12 h, the mixture was concentrated under reduced
pressure. The residue was dissolved in water, and concentrated HCl
was added to obtain acidic pH to produce the precipitate, which was
recrystallized and dried by vacuum to afford compound 49. Yield 82%
(two steps). 1H NMR (500 MHz, CDCl3): δ 8.28 (d, J = 8.6 Hz, 1H),
7.66−7.76 (m, 4H), 7.47−7.58 (m, 4H), 7.27 (d, J = 3.7 Hz, 1H), 6.58
(d, J = 3.7 Hz, 1H), 6.39 (d, J = 15.9 Hz, 1H).
3-(1-Benzoyl-1H-indol-5-yl)-N-hydroxyacrylamide (16). To a
solution of 49 (0.1 g, 0.34 mmol), PYBOP (0.05 g, 0.41 mmol),
and triethylamine (0.12 mL, 0.82 mmol) in DMF (1.5 mL) was added
NH2OTHP (0.05 g, 0.41 mmol), and the mixture was stirred at room
temperature. After the mixture was stirred for 3 h, the reaction was
quenched with water, followed by extraction with EtOAc (20 mL × 3).
The combined organic layer was dried over anhydrous MgSO4 and
concentrated under reduced pressure. The residue was purified by
silica gel chromatography ((CH2Cl2/CH3OH = 30:1)/1% NH3(aq)) to
give a white solid, which was treated with TFA (0.78 mL, 10.5 mmol)
1
CH3OH to afford the desired 17. Yield 81%; mp 133−135 °C. H
NMR (500 MHz, CDCl3): δ 7.75 (s, 1H), 7.66 (d, J = 15.0 Hz, 1H),
7.20−7.36 (m, 6H), 7.12 (d, J = 7.0 Hz, 2H), 6.53 (d, J = 3.0 Hz, 1H),
6.37 (d, J = 15.0 Hz, 1H), 5.38 (s, 2H). MS (EI) m/z: 292 (M+, 19%),
91 (100%). HRMS (EI) for C18H16N2O2 (M+): calcd, 292.1212;
found, 292.1213.
1-Phenyl-1H-indole-5-carbaldehyde (48). A suspension of 1H-
indole-5-carbaldehyde (25, 0.70 g, 4.82 mmol), 4-iodobenzene (0.65
mL, 5.79 mmol), and K2CO3 (0.93 g, 6.75 mmol), CuO (0.04 g, 0.48
mmol) in DMF (2 mL) was refluxed for 2 days. The reaction mixture
3787
dx.doi.org/10.1021/jm300197a | J. Med. Chem. 2012, 55, 3777−3791

Journal of Medicinal Chemistry
Article
1
was quenched with water, followed by extraction with EtOAc (20 mL
× 3). The combined organic layer was dried over anhydrous MgSO4
and concentrated under reduced pressure to give a yellow residue,
which was purified by silica gel chromatography (EtOAc/n-hexane =
1:4) to afford compound 48. Yield 28%. 1H NMR (500 MHz,
CD3OD): δ 10.06 (s, 1H), 8.21 (d, J = 1.5 Hz, 1H), 7.78 (dd, J = 9.0,
1.5 Hz, 1H), 7.54−7.60 (m, 3H), 7.49−7.51 (m, 2H) 7.42−7.44 (m,
2H), 6.83 (d, J = 3.2 Hz, 1H).
°C. H NMR (500 MHz, CD3OD): δ 7.86−7.89 (m, 3H),
7.57−7.60 (m, 2H), 7.48 (t, J = 7.0 Hz, 2H), 7.37 (s, 1H), 7.17
(dd, J = 1.5 Hz, 8.5 Hz, 1H), 6.66 (d, J = 3.5 Hz, 1H), 2.96 (t, J
= 7.5 Hz, 2H), 2.36 (t, J = 7.5 Hz, 2H). HRMS (ESI) for
C17H15N2O4S (M − H)−: calcd, 343.0758; found, 343.0737.
S y n t h e s i s o f 3 - B e n z e n e s u l f o n y l - 5 - ( N -
hydroxypropanamide)indole 21. Methyl 3-(1H-Indol-5-yl)-
acrylate (52). To a mixture of compound 25 (2.00 g, 13.78
mmol) and CH2Cl2 (20 mL) was added methyl
(triphenylphosphoranylidene)acetate (6.91 g, 20.67 mmol).
The mixture was stirred at room temperature for overnight.
The reaction was quenched by water, and extraction was with
CH2Cl2 (30 mL × 3). The organic layer was collected and dried
over anhydrous MgSO4 and concentrated in vacuum to yield a
yellow product. The residue was purified by flash column over
3-(1-Phenyl-1H-indol-5-yl)acrylic Acid (51). To a solution of 48
(0.29 g, 1.31 mmol) in CH2Cl2 (10 mL) was added methyl
(triphenylphosphoranylidene)acetate (0.52 g, 1.57 mmol), and the
mixture was stirred at room temperature. After being stirred for 16 h,
the reaction mixture was quenched with water and extracted with
CH2Cl2 (25 mL × 3). The combined organic layer was dried over
anhydrous MgSO4, concentrated under reduced pressure, and dried
with vacuum to give a brown residue, which was purified by silica gel
chromatography (EtOAc/n-hexane = 1:5) to afford the methyl ester
compound as a yellow liquid. Yield 92%. 1H NMR (500 MHz,
CDCl3): δ 7.83−7.86 (m, 2H), 7.35−7.55 (m, 8H), 6.71 (d, J = 3.1
Hz, 1H), 6.42 (d, J = 15.9 Hz, 1H), 3.81 (s, 3H).
1
silica gel (EtOAc/n-hexane = 2:1) to afford 52. Yield 99%; H
NMR (500 MHz, CDCl3): δ 8.32 (br, 1H), 7.84 (d, J = 15.5
Hz, 1H), 7.81 (s, 1H), 7.43 (dd, J = 2.0 Hz, 10.5 Hz, 1H), 7.38
(d, J = 8.5 Hz, 1H), 7.24 (t, d, J = 3.0 Hz, 1H), 6.59 (s, 1H),
6.42 (d, J = 15.5 Hz, 1H), 3.80 (s, 3H).
Methyl 3-(3-(Phenylthio)-1H-indol-5-yl)acrylate (53). To a
mixture of NaH (0.30 g, 2.24 mmol) and DMF (2 mL) was added
52 (0.30 g, 1.49 mmol) at 0 °C, and the mixture was stirred at room
temperature for 90 min. To the mixture was added phenyl disulfide
(0.36 g, 1.64 mmol), and the mixture was stirred at room temperature
for overnight. The reaction was quenched by water at 0 °C, and
extraction was with ethyl acetate (30 mL × 3). The organic layer was
collected and dried over anhydrous MgSO4 and concentrated in
vacuum to yield an orange product. The residue was purified by flash
column over silica gel (EtOAc/n-hexane = 1:4) to afford 53. Yield
To a solution of the methyl ester compound (0.31 g, 1.12 mmol) in
dioxane (10 mL) was added 1 M LiOH(aq) (2.3 mL), and the mixture
was stirred at 40 °C. After being stirred for 12 h, the mixture was
concentrated under reduced pressure. The residue was dissolved in
water, and concentrated HCl was added to obtain acidic pH to
produce the precipitate, which was dried by vacuum to afford
compound 51. Yield 48%. H NMR (500 MHz, CDCl3): δ 7.85 (s,
1H), 7.78 (d, J = 15.9 Hz, 1H), 7.47−7.57 (m, 7H), 7.38−7.41 (m,
1H), 6.71 (d, J = 3.0 Hz, 1H), 6.41 (d, J = 15.9 Hz, 1H).
1
N-Hydroxy-3-(1-phenyl-1H-indol-5-yl)acrylamide (18). To a sol-
ution of 51 (0.2 g, 0.76 mmol), PYBOP (0.43 g, 0.84 mmol), and
triethylamine (0.25 mL, 1.82 mmol) in DMF (1.5 mL) was added
NH2OTHP (0.11 g, 0.91 mmol), and the mixture was stirred at room
temperature. After the mixture was stirred for 2 h, the reaction was
quenched with water, followed by extraction with EtOAc (20 mL × 3).
The combined organic layer was dried over anhydrous MgSO4 and
concentrated under reduced pressure. The residue was purified by
silica gel chromatography ((CH2Cl2/CH3OH = 30:1)/1% NH3(aq)) to
give a white solid, which was treated with TFA (1.8 mL, 24.2 mmol) in
the presence of CH3OH (36 mL). The mixture was stirred at room
temperature for 16 h. The reaction mixture was concentrated under
reduced pressure to give a white residue, which was recrystallized by
1
42%. H NMR (500 MHz, CDCl3): δ 8.58 (br, 1H), 7.76−7.80 (m,
2H), 7.52 (d, J = 2.5 Hz, 1H), 7.48 (dd, J = 1.0 Hz, 8.5 Hz, 1H), 7.43
(d, J = 8.0 Hz, 1H), 7.17 (t, J = 8.0 Hz, 2H), 7.11 (d, J = 7.0 Hz, 2H),
7.07 (t, J = 7.0 Hz, 1H), 6.39 (d, J = 16.0 Hz, 1H), 3.78 (s, 3H).
Methyl 3-(3-(Phenylsulfonyl)-1H-indol-5-yl)acrylate (54). To a
mixture of 53 (0.30 g, 097 mmol) and CH2Cl2 (10 mL) was added
mCPBA (0.50 g, 2.91 mmol) at 0 °C, and the mixture was stirred at
room temperature overnight. The reaction was quenched by saturated
NaHCO3 (aq), and extraction was with CH2Cl2 (30 mL × 3). The
organic layer was collected and dried over anhydrous MgSO4 and
concentrated in vacuum to yield an orange product. The residue was
purified by flash column over silica gel (EtOAc/n-hexane = 1:1) to
afford 54. Yield 46%. 1H NMR (500 MHz, DMSO-d6): δ 8.22 (s, 1H),
8.03−8.07 (m, 3H), 7.81 (d, J = 16.0 Hz, 1H), 7.67 (dd, J = 1.5 Hz, 8.0
Hz, 1H), 7.54−7.60 (m, 3H), 7.51 (d, J = 8.5 Hz, 1H), 6.57 (d, J =
16.0 Hz, 1H), 3.73 (s, 3H).
1
CH3OH to afford the desired 18. Yield 44%; mp 97−100 °C. H
NMR (500 MHz, CD3OD): δ 7.82 (s, 1H), 7.70 (d, J = 15.5 Hz, 1H),
7.51−7.58 (m, 5H), 7.48 (d, J = 3.0 Hz, 1H), 7.44 (d, J = 8.5 Hz, 1H),
7.39−7.42 (m, 1H), 6.71 (d, J = 3.0 Hz, 1H), 6.42 (d, J = 15.5 Hz,
1H). HRMS (EI) for C17H14N2O2 (M+): calcd, 278.1055; found,
278.1055.
3-(3-(Phenylsulfonyl)-1H-indol-5-yl)acrylic Acid (55). To a mixture
of 54 (0.20 g, 0.59 mmol) and dioxane (1 mL) was added 1 M LiOH
(aq) (1.18 mL, 1.18 mmol), and the mixture was stirred overnight at
40 °C. Solvent was removed, and the remainder was dissolved in
water. To the water layer was added 3 N HCl (aq), and gravity
filtration yielded a white product. The product, without more
Synthesis of 5-(N-Hydroxyacrylamide)indole 19. N-Hy-
droxy-3-(1H-indol-5-yl)acrylamide (19). 8 (0.20 g, 0.58 mmol)
was dissolved in MeOH (8 mL), and to the mixture was added
1 M KOH (aq) (1.16 mL, 1.16 mmol). The mixture was stirred
and refluxed for 1 h. The reaction was quenched by water, and
extraction was with ethyl acetate (30 mL × 3). The organic
layer was collected and dried over anhydrous MgSO4 and
concentrated in vacuo to yield a colorless product. The residue
was purified by flash column chromatography over silica gel
(EtOAc/n-hexane = 2:1) to afford 19. Yield 51%; mp 162−164
°C. 1H NMR (500 MHz, CD3OD): δ 8.17 (d, J = 9.0 Hz, 1H),
7.75 (s, 1H), 7.65−7.68 (m, 2H), 7.52 (d, J = 3.0 Hz, 1H), 6.65
(d, J = 4.0 Hz, 1H), 6.46 (d, J = 15.5 Hz, 1H). HRMS (ESI) for
C11H9N2O2 (M − H)−: calcd, 201.0670; found, 201.0681.
S y n t h e s i s o f 1 - B e n z e n e s u l f o n y l - 5 - ( N -
hydroxypropanamide)indole 20. N-Hydroxy-3-(1-(phenylsul-
fonyl)-1H-indol-5-yl)propanamide (20). 8 (0.20 g, 0.58 mmol)
was dissolved in CH2Cl2 (10 mL), and to the mixture was
added 10% Pd/C (0.01 g, 0.06 mmol). The mixture was stirred
at room temperature for 1 h and filtered via Celite by CH2Cl2
wash. The residue was purified by flash column over silica gel
(EtOAc/n-hexane = 2:1) to afford 20. Yield 30%; mp 116−118
1
purification, was 55. Yield 52%. H NMR (500 MHz, DMSO-d6): δ
8.21 (s, 1H), 8.03−8.05 (m, 2H), 7.97 (s, 1H), 7.80 (d, J = 16.0 Hz,
1H), 7.54−7.62 (m, 4H), 7.51 (d, J = 8.5 Hz, 1H), 6.46 (d, J = 16.0
Hz, 1H).
3-(3-Benzenesulfonyl-1H-indol-5-yl)-N-hydroxyacrylamide (21).
A mixture of 55 (0.60 g, 1.83 mmol), EDC (0.53 g, 2.75 mmol),
HOBt (0.30 g, 2.20 mmol), N-methylmorphine (0.48 mL, 4.39
mmol), and DMF (1.5 mL) was stirred for a while. Then to the
mixture was added o-(tetrahydro-2H-pyran-2-yl)hydroxylamine (0.26
g, 2.20 mmol), and the mixture was stirred at room temperature
overnight. The residue was purified by flash column over silica gel
(EtOAc/n-hexane = 4: 1) to yield the colorless oil product. The
product was dissolved in MeOH (2 mL), and to the mixture was
added 10% TFA (aq) (2 mL). The mixture was stirred at room
temperature overnight. To the mixture was added water, and gravity
1
filtration afforded 21. Yield 26%; mp 180−182 °C. H NMR (500
MHz, DMSO-d6): δ 8.21 (s, 1H), 8.01 (d, J = 7.0 Hz, 2H), 7.95 (s,
1H), 7.54−7.60 (m, 4H), 7.51 (d, J = 9.0 Hz, 1H), 7.45 (d, J = 8.5 Hz,
3788
dx.doi.org/10.1021/jm300197a | J. Med. Chem. 2012, 55, 3777−3791

Journal of Medicinal Chemistry
Article
1H), 6.45 (d, J = 15.5 Hz, 1H). MS (EI) m/z: 342 (M+, 3%), 327
(100%). HRMS (EI) for C17H14N2O4S (M+): calcd, 342.0674; found,
342.0673.
compound was added into water or ethanol (1 mL). The mixture was
sonicated for 10 min and then shaken for 24 h at room temperature.
After centrifugation, an aliquot of the clear supernatant was transferred
into vials and the target compound present in the sample was
determined by reversed phase HPLC and UV detection at 328 nm.
Pharmacokinetic Evaluation. The in vivo pharmacokinetic study
was approved by Institutional Animal Care and Use Committee of
National Health Research Institutes. A 1 and 5- mg/mL doing solution
was preparing by dissolving appropriate amount compound in a
mixture of PEG400/DMSO (80:20,v/v) for intravenous adminis-
tration and 1% carboxylmethyl cellulose, 0.5% Tween 80 for oral
dosing, respectively. Male Sprague−Dawley rats, weighing 250−350 g
each (8−10 weeks old), were obtained from BioLASCO, Ilan, Taiwan.
Tested compound was separately administered to group of three male
rats intravenously (2 mg/kg dose) by a bolus injection to the jugular
vein or periorally (20 mg/kg dose). The volume of dosing solution
administered was adjusted according to the body weight recorded
before dose administration. At 0 (prior to dosing), 2, 5 (IV only), 15,
and 30 min and at 1, 2, 4, 6, 8, and 24 h after dosing, a blood sample
(∼150 μL) was collected from each animal via the jugular-vein cannula
and stored in ice (0−4 °C). Plasma was separated from the blood by
centrifugation (14,000 × g for 15 min at 4 °C) and stored in a freezer
(−80 °C). All samples were analyzed for the test compound by LC-
MS/MS (ABI4000). Data were acquired via multiple reactions
monitoring. Plasma concentration data were analyzed with standard
noncompartmental method.
Biology. HeLa Nuclear Extract HDAC Activity Assay.17 The
HeLa nuclear extract HDAC activity was measured by using the
HDAC fluorescent activity assay kit (BioVision, CA) according to the
manufacturer’s instructions. Briefly, the HDAC fluorometric substrate
and assay buffer were added to HeLa nuclear extracts in a 96-well
format and incubated at 37 °C for 30 min. The reaction was stopped
by adding lysine developer, and the mixture was incubated for another
30 min at 37 °C. Additional negative controls included incubation
without the nuclear extract, without the substrate, or without both.
TSA at 1 μM served as the positive control. A fluorescence plate reader
with excitation at 355 nm and emission at 460 nm was used to quantify
HDAC activity.
HDAC Biochemical Assays.18 The HDAC in vitro activities of
recombinant human HDACs 1, 2, 6, and 8 (BPS Biosciences) were
detected by fluorigenic release of 7-amino-4-methylcoumarin from
substrate upon deacetylase enzymatic activity.
Tumor Cell Culture. All human cancer cells were maintained in
RPMI 1640 medium containing 100 units/mL penicillin G sodium,
100 μg/mL streptomycin sulfate, 0.25 μg/mL amphotericin B, and 25
μg/mL gentamicin. The medium was supplemented with 10% fetal
bovine serum and 2 mM glutamine. The cells were cultured in tissue
culture flasks in a humidified incubator at 37 °C, in an atmosphere of
5% CO2 and 95% air.
Sulforhodamine B Assays.19 Human cancer A549 (non-small-
cell lung cancer), MDA-MB-231 (estrogen-independent breast
cacner), Hep 3B (hepatoma), and PC-3 (prostate) cells were seeded
in 96-well plates in medium with 5% FBS. After 24 h, cells were fixed
with 10% trichloroacetic acid (TCA) to represent cell population at
the time of compound addition (T0). After additional incubation of
DMSO or test compound for 48 h, cells were fixed with 10% TCA,
and SRB at 0.4% (w/v) in 1% acetic acid was added to stain cells.
Unbound SRB was washed out by 1% acetic acid, and SRB bound cells
were solubilized with 10 mM Trizma base. The absorbance was read at
a wavelength of 515 nm. Using the absorbance parameters time zero
(T0), control growth (C), and cell growth in the presence of the
compound (Tx), the percentage growth was calculated at each of the
compound concentrations levels. Growth inhibition of 50% (GI50) was
calculated from [(Ti − Tz)/(C − Tz)] × 100 = 50, which was the
compound concentration resulting in a 50% reduction in the net
protein increase (as measured by SRB staining) in control cells during
the compound incubation.
Antitumor Activity in Vivo.21 Female nude mice (NTUH Animal
Facility) were 8 weeks old. The animals were fed ad libitum water
(reverse osmosis, 1 ppm Cl) and PicoLab rodent diet 20 modified and
irradiated LabDiet consisting of 20.0% crude protein, 9.9% crude fat,
and 4.7% crude fiber. The mice were housed at National Taiwan
University Laboratory Animal Center, NTUMC, on a 12 h light cycle
at 21−23 °C and 60−85% humidity. Nude mice were maintained in
accordance with the Institutional Animal Care and Use Committee
procedures and guidelines. Mice were sorted into four groups of seven
mice. All doses were administered at a volume of 10 mL/kg (0.2 mL/
20 g mouse), scaled to the body weight of each animal. Control group
1 mice received vehicle daily po to the end point. Group 2 received
reference 1 daily at 200 mg/kg po to the end point. Groups 3 and 4
received compound 8 daily at 200 and 100 mg/kg po, respectively, to
the end schedule.
The human A549 lung adenocarcinoma cells used for implantation
were harvested during log phase growth and resuspended in
phosphate-buffered saline at 5 × 107 cells/mL. Each mouse was
injected sc in the right flank with 1 × 107 cells (0.2 mL cell
suspension). Tumor size, in mm3, was calculated from the following:
tumor volume = w2l /2, where w is the width and l is the length in mm
of the tumor. Tumor weight can be estimated with the assumption that
1 mg is equivalent to 1 mm3 of tumor volume. Mice were sorted into
four groups of seven mice. Each animal was euthanized when the
tumors reached the predetermined end point size of 1000 mm3. The
time to end point (TTE) for each mouse was calculated by the
following equation: TTE = (log10(end point volume) − b)/m, where
TTE is expressed in days, end point volume is in mm3, b is the
intercept, and m is the slope of the line obtained by linear regression of
a log-transformed tumor growth data set. The data set is comprised of
the first observation that exceeded the study end point volume and the
three consecutive observations that immediately preceded the
attainment of the end point volume. The calculated TTE is usually
less than the day on which an animal is euthanized for tumor size.
Treatment efficacy was determined from tumor growth delay (TGD),
which is defined as the increase in the median TTE for a treatment
group compared to the control group, TGD = T − C, expressed in
days, or as a percentage of the median TTE of the control group, %
TGD = (T − C)/C × 100, where T is the median TTE for a treatment
group, C is the median TTE for control group 1. Animals were
weighed daily for the first 5 days, then twice weekly until the
completion of the study. In addition, we also determined tumor
growth inhibition (TGI), whose antitumor effects are expressed as %
T/C (treated versus control), dividing the tumor volumes from
Western Blot Analysis.20 PC-3 and A549 cells were treated with
vehicle (0.1% DMSO) and a test compound at 1, 2.5, 5, 10, and 20 μM
in RPMI 1640 supplemented with 10% FBS for 48 h. The cells were
collected and sonicated. Protein concentrations in the resultant lysates
were determined by a Bradford protein assay kit (Bio-Rad, Hercules,
CA). The protein lysates, containing the same amount of proteins,
were subjected to 10% SDS−polyacrylamide gel (10%) electro-
phoresis. The proteins on the gel were then transferred onto an
Immobilon nitrocellulose membrane (Millipore, Bellerica, MA) in a
semidry transfer cell. The transblotted membrane was washed twice
with Tris-buffered saline containing 0.1% Tween 20 (TBST). After
being blocked with TBST containing 5% nonfat milk for 40 min, the
membrane was incubated with a primary antibody specific to acetyl-H3
(antibody obtained from Upstate Biotechnology, Inc., Lake Placid,
NY), H3 (Upstate Biotechnology), acetyl α-tubulin (Sigma-Aldrich, St.
Louis, MO), or α-tubulin (Sigma-Aldrich, St. Louis, MO) (1:3000
dilution) in TBST/1% nonfat milk at 4 °C overnight. The membrane
was washed three times with TBST for a total of 15 min and then
incubated with a goat anti-rabbit or anti-mouse IgG antibody
conjugated with horseradish (diluted 1:3000) for 1 h at room
temperature. After the samples were washed at least three times with
TBST, the signals were developed using the enhanced chemilumi-
nescence system (Pierce). Quantitative analysis of Western blot was
done with ImageQuant (Molecular Dynamics, U.S.)
Solubility Determination in Water and Ethanol. Solubility was
determinations by the shake-flask method. Briefly, 1 mg of a test
3789
dx.doi.org/10.1021/jm300197a | J. Med. Chem. 2012, 55, 3777−3791

Journal of Medicinal Chemistry
Article
treatment groups by those of the control groups and multiplied by
100. The mice were examined frequently for overt signs of any adverse,
drug-related side effects.
Statistical and Graphical Analyses. The log rank test was used
to determine the statistical significance of the difference between the
TTE values of two groups. Statistical and graphical analyses were
performed with Prism 3.03 (GraphPad) for Windows. The two-tailed
statistical analyses were conducted at P = 0.05. Kaplan−Meier plots
show the percentage of animals remaining in the study versus time.
The Kaplan−Meier plots use the same data set as the log rank test.
(6) Marks, P. A. Discovery and development of SAHA as an
anticancer agent. Oncogene 2007, 26, 1351−1356.
(7) Grant, C.; Rahman, F.; Piekarz, R.; Peer, C.; Frye, R.; Robey, R.
W.; Gardner, E. R.; Figg, W. D.; Bates, S. E. Expert Rev. Anticancer
Ther. 2010, 10, 997−1008.
(8) Atadja, P. Development of the pan-HDAC inhibitor panobinostat
(LBH589): success and challenges. Cancer Lett. 2009, 280, 233−241.
(9) Plumb, J. A.; Finn, P. W.; Williams, R. J.; Bandara, M. J.; Romero,
M. R.; Watkins, C. J.; Thangue, N. B. L.; Brown, R. Pharmacodynamic
response and inhibition of growth of human tumor xenografts by the
novel histone deacetylase inhibitor PXD101. Mol. Cancer Ther. 2003,
2, 721−728.
(10) (a) Wang, H.; Yu, N.; Chen, D.; Lee, K. C. L.; Lye, P. L.; Chang,
J. W. W.; Deng, W.; Ng, M. C. Y.; Lu, T.; Khoo, M. L.; Poulsen, A.;
Sangthongpitag, K.; Wu, X.; Hu, C.; Goh, K. C.; Wang, X.; Fang, L.;
Goh, K. L.; Khng, H. H.; Goh, S. K.; Yeo, P.; Liu, X.; Bonday, Z.;
Wood, J. M.; Dymock, B. W.; Ethirajulu, K.; Sun, E. T. J. Med. Chem.
2011, 54, 4694−4720. (b) Novotny-Diermayr, V.; Sangthongpitag, K.;
Hu, C. Y.; Wu, X.; Sausgruber, N.; Yeo, P.; Greicius, G.; Pettersson, S.;
Liang, A. L.; Loh, Y. K.; Bonday, Z.; Goh, K. C.; Hentze, H.; Hart, S.;
Wang, H.; Ethirajulu, K.; Wood, J. M. Mol. Cancer Ther. 2010, 9, 642−
652.
ASSOCIATED CONTENT
* Supporting Information
■
S
NMR spectra of compound 8, HPLC purity data for target
compounds, animal body weight change of compounds 8 and 1
treatment in vivo, in vivo efficacy evaluation of compounds 8,
11, 13 (oral route), and 1 (by ip and oral routes) against A549
xenografts, activities of compounds 8, 11−14, 17, and 1 against
HDAC 8, the CYP inhibition and solubility of compounds 11−
14, and in vitro metabolic stability in rat microsomes. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(11) Ma, X.; Ezzeldin, H. H.; Diasio, R. B. Histone deacetylase
inhibitors current status and overview of recent clinical trials. Drugs
2009, 69, 1911−1934.
AUTHOR INFORMATION
Corresponding Author
*Phone: 886-2-2736-1661, extension 6130. Fax: 866-2-
27369558. E-mail: jpl@tmu.edu.tw.
■
(12) Wagner, J. M.; Hackanson, B.; Lubbert, M.; Jung, M. Histone
̈
deacetylase (HDAC) inhibitors in recent clinical trials for cancer
therapy. Clin. Epigenet. 2010, 1, 117−136.
(13) Paris, M.; Porcelloni, M.; Binaschi, M.; Fattori, D. Histone
deacetylase inhibitors: from bench to clinic. J. Med. Chem. 2008, 51,
1505−1529.
Author Contributions
●Contributed equally to this work.
(14) Venugopal, B.; Evans, T. R. J. Developing histone deacetylase
inhibitors as anti-cancer therapeutics. Curr. Med. Chem. 2011, 18,
1658−1671.
Notes
The authors declare no competing financial interest.
(15) (a) Liou, J. P.; Chang, Y. L.; Kuo, F. M.; Chang, C. W.; Tseng,
H. Y.; Wang, C. C.; Yang, Y. N.; Chang, J. Y.; Lee, S. J.; Hsieh, H. P.
Concise synthesis and structure−activity relationships of combretas-
tatin A-4 analogues, 1-aroylindoles and 3-aroylindoles, as novel classes
of potent antitubulin agents. J. Med. Chem. 2004, 47, 4247−4257.
(b) Liou, J. P.; Wu, C. Y.; Hsieh, H. P.; Chang, C. Y.; Chen, C. M.;
Kuo, C. C.; Chang, J. Y. 4- and 5-Aroylindoles as Novel Classes of
Potent Antitubulin Agents. J. Med. Chem. 2007, 50, 4548−4552.
(c) Liou, J. P.; Wu, Z. Y.; Kuo, C. C.; Chang, C. Y.; Lu, P. Y.; Chen, C.
M.; Hsieh, H. P.; Chang, J. Y. Discovery of 4-amino and 4-hydroxy-1-
aroylindoles as potent tubulin polymerization inhibitors. J. Med. Chem.
2008, 51, 4351−4355. (d) Liou, J. P.; Mahindroo, N.; Chang, C. W.;
Guo, F. M.; Lee, S. W. H.; Yeh, T. K.; Tan, U. K.; Kuo, C. C.; Chang,
Y. W.; Lu, P. H.; Tung, Y. S.; Chang, J. Y.; Hsieh, H. P. Structure−
activity relationship studies of 3-aroylindoles as potent antimitotic
agents. ChemMedChem 2006, 1, 1106−1118. (e) Lee, H. Y.; Chang, J.
Y.; Chang, L. Y.; Lai, W. Y.; Lai, M. J.; Shih, K. H.; Kuo, C. C.; Chang,
C. Y.; Liou, J. P. Concise syntheses of N-aryl-5,6,7-trimethoxyindoles
as antimitoic and vascular disrupting agents: application of the copper-
mediated Ullmann-type arylation. Org. Biomol. Chem. 2011, 9, 3154−
3157. (f) Lai, M. J.; Kuo, C. C.; Yeh, T. K.; Hsieh, H. P.; Chen, L. T.;
Pan, W. Y.; Hsu, K. Y.; Chang, J. Y.; Liou, J. P. Synthesis and
structure−activity relationships of 1-benzyl-4,5,6-trimethoxyindoles as
a novel class of potent antimitotic agents. ChemMedChem 2009, 4,
588−593. (g) Lai, M. J.; Chang, J. Y.; Lee, H. Y.; Kuo, C. C.; Lin, M.
H.; Hsieh, H. P.; Chang, C. Y.; Wu, J. S.; Wu, S. Y.; Shey, K. S.; Liou, J.
P. Synthesis and biological evaluation of 1-(4′-indolyl and 6′-
quinolinyl) indoles as a new class of potent anticancer agents. Eur. J.
Med. Chem. 2011, 46, 3623−3629.
ACKNOWLEDGMENTS
■
This research were supported by the National Science Council,
Taiwan (Grants NSC 100-2325-B-038-002, NSC 100-2628-M-
038-001-MY3, NSC 100-2325-B-038-005-CC2, and NSC 99-
2323-B-038-005-CC2).
ABBREVIATIONS USED
■
HDAC, histone deacetylase; HAT, histone acetyltransferase;
NH2OTHP, O-(tetrahydro-2H-pyran-2-yl)hydroxylamine;
PYBOP, benzotriazol-1-yl-oxytripyrrolidinophosphonium hexa-
fluorophosphate; TBAHS, tetrabutylammonium hydrogen
sulfate; EDC, N-(3-dimethylaminopropyl)-N′-ethylcarbodii-
mide; HOBt, 1-hydroxybenzotriazole; TFA, trifluoroacetic
acid; TTE, time to end point; TGD, tumor growth delay;
TGI, tumor growth inhibition
REFERENCES
■
(1) (a) Paris, M.; Porcelloni, M.; Binaschi, M.; Fattori, D. Histone
deacetylase inhibitors: from bench to clinic. J. Med. Chem. 2008, 51,
1505−1529. (b) Marks, P. A. The clinical development of histone
deacetylase inhibitors as targeted anticancer drugs. Expert Opin. Invest.
Drugs 2010, 19, 1049−1066.
(2) Minucci, S.; Pelicci, P. G. Histone deacetylase inhibitors and the
promise of epigenetic (and more) treatments for cancer. Nat. Rev.
Cancer 2006, 6, 38−51.
(3) Gallinari, P.; Marco, S. D.; Jones, P.; Pallaoro, M.; Steinkuhler, C.
̈
HDACs, histone deacetylation and gene transcription: from molecular
biology to cancer therapeutics. Cell Res. 2007, 17, 195−211.
(4) Lane, A. A.; Chabner, B. A. Histone deacetylase inhibitors in
cancer therapy. J. Clin. Oncol. 2009, 27, 5459−5468.
(5) Glaser, K. B. HDAC inhibitors: clinical update and mechanism-
based potential. Biochem. Pharmacol. 2007, 74, 659−671.
(16) Mahboobi, S.; Sellmer, A.; Hocher, H.; Garhammer, C.;
̈
Pongratz, H.; Maier, T.; Ciossek, T.; Beckers, T. 2-Aroylindoles and 2-
arolbenzofurans with N-hydroxyacrylamide substructures as a novel
series of rationally designed histone deacetylase inhibitors. J. Med.
Chem. 2007, 50, 4405−4418.
3790
dx.doi.org/10.1021/jm300197a | J. Med. Chem. 2012, 55, 3777−3791

Journal of Medicinal Chemistry
Article
(17) Hassig, C. A.; Symons, K. T.; Guo, X.; Nguyen, P. M.; Annable,
T.; Wash, P. L.; Payne, J. E.; Jenkins, D. A.; Bonnefous, C.; Trotter, C.;
Wang, Y.; Anzola, J. V.; Milkova, E. L.; Hoffman, T. Z.; Dozier, S. J.;
Wiley, B. M.; Saven, A.; Malecha, J. W.; Davis, R. L.; Muhammad, J.;
Shiau, A. K.; Noble, S. A.; Rao, T. S.; Smith, N. D.; Hager, J. H.
KD5170, a novel mercaptoketone-based histone deacetylase inhibitor
that exhibits broad spectrum antitumor activity in vitro and in vivo.
Mol. Cancer Ther. 2008, 7, 1054−1065.
(18) Olsen, C. A.; Ghadiri, M. R. Discovery of potent and selective
histone deacetylase inhibitors via focused combinatorial libraries of
cyclic alpha3beta-tetrapeptides. J. Med. Chem. 2009, 52, 7836−7846.
(19) Wu, S. Y.; Pan, S. L.; Chen, T. H.; Liao, C. H.; Huang, D. Y.;
Guh, J. H.; Chang, Y. L.; Kuo, S. C.; Lee, F. Y.; Teng, C. M. YC-1
induces apoptosis of human renal carcinoma A498 cells in vitro and in
vivo through activation of the JNK pathway. Br. J. Pharmacol. 2008,
155, 505−513.
(20) Tsai, A. C.; Pan, S. L.; Sun, H. L.; Wang, C. Y.; Peng, C. Y.;
Wang, S. W.; Chang, Y. L.; Kuo, S. C.; Lee, K. H.; Teng, C. M. CHM-
1, a new vascular targeting agent, induces apoptosis of human
umbilical vein endothelial cells via p53-mediated death receptor 5 up-
regulation. J. Biol. Chem. 2010, 285, 5497−5506.
(21) Tsai, A. C.; Pan, S. L.; Liao, C. H.; Guh, J. H.; Wang, S. W.; Sun,
H. L.; Liu, Y. N.; Chen, C. C.; Shen, C. C.; Chang, Y. L.; Teng, C. M.
Moscatilin, a bibenzyl derivative from the India orchid Dendrobrium
loddigesii, suppresses tumor angiogenesis and growth in vitro and in
vivo. Cancer Lett. 2010, 292, 163−170.
3791
dx.doi.org/10.1021/jm300197a | J. Med. Chem. 2012, 55, 3777−3791