Indole-3-ethylsulfamoylphenylacrylamides: Potent histone deacetylase inhibitors with anti-inflammatory activity

Article abstract of DOI:10.1016/j.ejmech.2014.08.020

A series of 2-methyl-1H-indol-3-ethylsulfamoylphenylacrylamides based on LBH589-PXD101 core have been synthesized and evaluated for their histone deacetylase (HDAC) inhibitory and anti-inflammatory activity. In vitro, compounds 9-12 show 2.6-fold better H

Full text of DOI:10.1016/j.ejmech.2014.08.020

European Journal of Medicinal Chemistry 85 (2014) 468e479
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Indole-3-ethylsulfamoylphenylacrylamides: Potent histone
deacetylase inhibitors with anti-inflammatory activity
Samir Mehndiratta a, Yi-Ling Hsieh b, Yi-Min Liu a, Amber Weiching Wang b,
,
*
Hsueh-Yun Lee a, Lung-Yu Liang b, Sunil Kumar a, Che-Ming Teng c, Chia-Ron Yang b
,
,
*
Jing-Ping Liou a
a School of Pharmacy, College of Pharmacy, Taipei Medical University, Taipei 11031, Taiwan
b School of Pharmacy, College of Medicine, National Taiwan University, Taipei, Taiwan
c Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 2-methyl-1H-indol-3-ethylsulfamoylphenylacrylamides based on LBH589ePXD101 core have
been synthesized and evaluated for their histone deacetylase (HDAC) inhibitory and anti-inflammatory
activity. In vitro, compounds 9e12 show 2.6-fold better HDAC inhibition and 3-fold better IL-6 sup-
pression compared to LBH589$HCl (1$HCl). Furthermore, these compounds did not show apparent cell
viability suppression on macrophages while in contrast, treatment with 1$HCl resulted in significant
reduction in cell viability as demonstrated by an MTT assay. Repressed expression of iNOS, COX-2 and
reduced phosphorylation of p65 revealed the inhibitory effect of these analogues on inflammatory
mediator release which is related to inhibited NF-kB signals. (N-Hydroxy-3-{3-[2-(2-methyl-1H-indol-3-
yl)-ethylsulfamoyl]-phenyl}-acrylamide) (9), exhibited ability superior to that of 1$HCl, was able to
reduce carrageenan-induced acute inflammation in an animal model. Compounds 9e12 have potential
anti-inflammatory activity and compound 9 can serve as lead compound for further development.
© 2014 Published by Elsevier Masson SAS.
Received 22 April 2014
Received in revised form
5 August 2014
Accepted 6 August 2014
Available online 7 August 2014
Keywords:
Anti-inflammatory activity
Histone deacetylase inhibitors
Indole
1. Introduction
expression of granulocyteemacrophage colony-stimulating factor
(GM-CSF), an inflammatory gene. HDACs counteract the action of
Histone acetylation finds its roots in the 1960s when it was
recognized that acetylation of histones and remodeling of the
compact chromatin structure is associated with gene induction.
This initiated a quest for development of inhibitors of histone
deacetylase (HDAC) as novel drug candidates. Intrinsic histone
acetyltransferase (HAT) activity of several transcription factors,
such as camp response element binding protein (CREB/p300 fam-
ilies), regulates the acetylation levels of lysine residues on the N-
terminal tails of nuclear histones H3 and H4. Dysregulation of
acetylation status of histones has been shown to be highly corre-
lated with development of cancer, and this has attracted much
scientific attention [1e3]. In the last decade; however, we have
gained a better understanding of the molecular mechanisms
associated with activation of inflammatory genes. These mecha-
nisms involve activation of a cascade of steps ultimately leading to
gene transcription by acetylating histone, thus increasing
HAT by reversing the hyperacetylation and suppressing the genes
or gene silencing [2,4].
Although HDAC inhibitors (Fig. 1) inhibit the action of HDACs
and thus increase the hyperacetylation, reports have suggested that
almost equal numbers of genes are induced as are inhibited. The
effects of HDAC inhibitors vary according to cell type and stimulus
[2,5] and this supports the idea that they have other potential
therapeutic applications such as anti-inflammation, immune
modulation [6], anti-rheumatic [7], and anti-HIV activity [8e9].
Numerous studies involving mainly SAHA (2) [10e11], TSA (3)
[12e13], MS-275 (4) [14e15], and ITF2357 (5) [16e17] have shown
promising evidence of their anti-inflammatory properties. Song
et al. have reported that Panobinostat (LBH589, 1) suppresses
various pro-inflammatory cytokines, indicating its immunomodu-
latory and anti-inflammatory potential [18] but a large amount of
work and rigorous discussion are still necessary to establish this
compound as an anti-inflammatory agent.
The sulfonamide moiety has been recognized as contributing
diverse types of pharmacological activity to various drugs, such as
diuretics, antibacterials and anticancer agents [19e20] and is a key
* Corresponding authors.
E-mail addresses: cryang@ntu.edu.tw (C.-R. Yang), jpl@tmu.edu.tw (J.-P. Liou).
http://dx.doi.org/10.1016/j.ejmech.2014.08.020
0223-5234/© 2014 Published by Elsevier Masson SAS.

S. Mehndiratta et al. / European Journal of Medicinal Chemistry 85 (2014) 468e479
469
Fig. 1. Examples of histone deacetylase inhibitors and sulfonamide containing compounds.
structural feature of various drug skeletons like Celecoxib (6) and
ABT751 (7) are to name but a few. This group has been utilized in
the development of potent HDAC inhibitors such as compound 8
and
1-arylsulfonyl-5-(N-hydroxyacrylamide)indoles/indolines
[21e22]. In our experience, the eSO2e group serves as a useful
linker which maintains the activity of the compounds [23].
Furthermore, clinical phase-II results obtained with compound 8
are very encouraging for treatment of T-cell lymphoma [24]. The
main structural feature of compound 8 is the sulfonamide linkage
and the precise structure of the linkage is crucial to its activity, as a
dramatic (>6 times) decrease in activity was observed on reversal
of the linkage. The reverse sulfonamide, eNHSO2e is favored over
the forward sulfonamide eSO2NHe (Fig. 2) [25]. The meta-
relationship of the sulfonamide and the N-hydroxyacrylamide is
also a pre-requisite for the highest activity.
Fig. 2. Reversal of sulfonamide linkage.
Compound 1, another potent HDAC inhibitor, is currently in
phase II/III trials for treatment of various malignancies [23,26].
Compound 1 has a 2-methyltryptamine unit attached to the para-
position of N-hydroxyphenylacrylamide through a eCH2e link-
age. The methylene linkage thus could potentially be a site for
modification and this prompted an effort to convert the eCH2e in 1
to eSO2e as in 8 while maintaining the order of the linkage, thus
generating a series of LBH589ePXD101 based synthetics (Fig. 3). A
recently published report on the metabolism of compound 1
revealed that the aliphatic secondary amino group actively partic-
ipates in the formation of cyclized metabolites, could be a “soft
spot” in the structure [27] and such results encouraged our ongoing
efforts.
Fig. 3. LBH589ePXD101 core based model.
meta- or the para-position, chain length effect, ring restriction,
substituent effect of both the sulfonamide- and the indole-NH, as
well as the significance of methyl group at C2 in the indole ring.
This led to a potent series of compounds with marked anti-
inflammatory activity (Fig. 4). To the best of our knowledge, only
one report has been published to date [18] exclusively considering
the inflammatory mediator suppression effect of compound 1. No
results for PXD101 have been published. This led us to synthesize a
Compounds 9e20 were designed and synthesized on the basis
of the regio effect of the N-hydroxyacrylamide chain at either the

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Fig. 4. 2-Methyl-1H-indol-3-ethylsulfamoylphenylacrylamide derivatives.
series of compounds based on LBH589ePXD101 core model and in
this report, we disclose results obtained from compounds based on
this model and sharing structural features of both parent com-
pounds. This work proves the validity of this model and rationally
designed molecules, and helps to establish the anti-inflammatory
activity of this class of HDAC inhibitors.
affording the corresponding protected N-hydroxyamide. Depro-
tection of OTHP group was achieved with TFA to yield compounds 9
and 10 in 49 and 54% yield, respectively.
The synthesis of compounds 11e13 is summarized in Scheme 2.
Selective protection of compound 24 was achieved by reacting with
di-t-butyl dicarbonate in the presence of DMAP as a catalyst to
obtain compound 27 in quantitative yield. Compound 27 under-
went a Heck reaction with t-butyl acrylate, and this was followed by
alkylation at N1 with various alkyl halides, affording compounds
29e31. The resulting products underwent TFA hydrolysis, amida-
tion, and OTHP deprotection as described in Scheme 1 to afford
compounds 11e13.
2. Results and discussion
2.1. Chemistry
The synthesis of compounds 9 and 10 is shown in Scheme 1. 2-
Methyltryptamine (22) was allowed to react with 3-
bromobenzenesulfonyl chloride or 4-bromobenzenesulfonyl chlo-
ride in the presence of pyridine and acetonitrile to respectively
yield compounds 23 or 24 which were subjected to Heck coupling
with t-butyl acrylate in the presence of tris(dibenzylideneacetone)
dipalladium and tri-t-butylphosphonium tetrafluoroborate to
afford the cinnamates 25 and 26. Hydrolysis of the t-butyl group in
25 and 26 was achieved by treatment with trifluoroacetic acid
(TFA), yielding the corresponding acids which were subject to the
amidation with NH2OTHP in the presence of EDC$HCl and HOBt,
The sulfonamide in 24 was alkylated directly using appropriate
alkyl halides as alkylating agents and Cs2CO3 in DMF, yielding
compounds 32e34. Heck coupling using methyl acrylate was per-
formed with the remainder of the reaction conditions the same as
were used in the case of compound 10 to obtain compounds 35e37.
The methyl ester hydrolysis was achieved with aqueous lithium
hydroxide solution. The same reactions and reaction conditions
were used for subsequent steps to obtain the final compounds 14,
15, and 16 in 44e52% yield, as outlined in Scheme 2.
Scheme 1. Synthetic approaches to compounds 9 and 10a.

S. Mehndiratta et al. / European Journal of Medicinal Chemistry 85 (2014) 468e479
471
Scheme 2. Synthetic approaches to compounds 11e16a.
Using tryptamine or N-methyltryptamine as the primary amine,
the same procedure as was used for the synthesis of compound 10
was followed to produce compounds 17 and 18 (Scheme 3).
compared to 1$HCl. Based on this observation, additional com-
pounds with N-hydroxyacrylamide para- to the sulfonamide were
synthesized. Comparison of compounds 11e13 revealed the sub-
stitution effect of the indole-NH. Compounds 11 (3.3 nM) and 12
(3.4 nM) have up to a 2.2 fold better HDAC inhibitory activity
compared to that of 1$HCl (7.5 nM) and the results for 1$HCl and 13
(7.3 nM) were very similar to one another. This result suggests that
bulky substitution at the N1 position is not well tolerated. All of
these compounds were found to exhibit lower (IL-6) or similar
2,3,4,9-Tetrahydro-1H-pyrido[3,4-b]indole (tetrahydro-b-car-
boline, 44) was used to rigidify the structure and prevent rotation
around the CeC single bond. Compound 44 was allowed to react
with 4-bromobenzenesulfonyl chloride to obtain 45, which un-
derwent Heck coupling and subsequent reactions described in
Scheme 3, to produce compound 19.
Compound
22
was
allowed
to
react
with
4-
(nitric oxide, TNF-a and PGE2) IC50 values when compared to 1$HCl.
methoxycarbonylbenzenesulfonyl chloride in the presence of pyr-
idine and acetonitrile to generate compound 47. This was then
hydrolyzed followed by amide coupling with NH2OTHP. Depro-
tection was achieved under acidic conditions with TFA to yield
compound 20 (Scheme 4).
The effect of substitution on the sulfonamide was evaluated from
the comparison of compounds 14e16. All of these compounds
caused diminished inhibitory activity of both HDAC and inflam-
matory agents. We expanded the panel by synthesizing 17 and 18
using tryptamine and 2-methyltryptamine respectively to evaluate
the effect of C2 methyl on the activity of these series of compounds.
Comparison of 17 and 18 with 1$HCl showed a slight increase in
HDAC inhibitory activity and comparable results for NO and IL-6
suppression. Constraint of the core, as in 19 and a shorter side
chain (20) are detrimental to the inhibitory activity of HDAC and
inflammatory cytokines.
2.2. Biological evaluation
2.2.1. HeLa nuclear HDAC enzyme inhibition
Using HeLa nuclear extract as HDAC source, we evaluated the
ability
ethylsulfamoylphenylacrylamides to inhibit HDAC activity
(Table 1). In addition, the inflammatory factors (NO, TNF- , IL-6 and
of
the
2-methyl-1H-indol-3-
In summary seven compounds (9e13, 18, and 19) of this series
were found to exhibit more potent or similar HDAC activity inhi-
bition when compared with compound 1$HCl. Although 1$HCl
a
PGE2) produced in LPS-activated (25 ng/mL) macrophages were
measured after treatment with the same compounds. First, we
evaluated the regio effect of the N-hydroxyacrylamide moiety at
the meta- or para-position with respect to the sulfonamide. Two
regioisomers 9 and 10 were evaluated for inhibition of HeLa nuclear
HDAC inhibitory activity (Table 1). Compound 9 gave results com-
parable to those obtained with 1$HCl while 10, with an IC50 value of
2.8 nM showed a 2.6 fold increase in activity, indicating that the
para-N-hydroxyacrylamide is favored. Compound 9 therefore dif-
fers from compound 8 in which the meta-N-hydroxyacrylamide is
favored. Compared to 1$HCl, both compounds showed 1.5e3 fold
better IL-6 suppression and comparable results with NO and PGE2
exhibited slightly more active in the reduction of NO, TNF-a, and
PGE2; Compounds 9e13 showed lower or comparable results for IL-
6 suppression (Table 1) signifying that these compounds have
similar anti-inflammatory effects.
2.2.2. Cell viability assay (MTT assay)
In an MTT assay, macrophages were treated for 24 h with 2-
methyl-1H-indol-3-ethylsulfamoylphenylacrylamides,
cytotoxicity was then evaluated. Four compounds, 9e12, fail to
show suppression of cell viability at 0.01e1 M (Fig. 5) while
treatment with compound 1$HCl shows significant reduction in
whose
m

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S. Mehndiratta et al. / European Journal of Medicinal Chemistry 85 (2014) 468e479
Scheme 3. Synthetic approaches to compounds 17e19a.
Scheme 4. Synthetic approaches to compounds 20a.
cell viability at 1
have diminished cytotoxicity.
m
M. This result suggested that compounds 9e12
observed to markedly increase acetylation of histone 3 above
0.1 M (Fig. 6B), confirming that the inhibition by analogues is due
m
to suppression of HDAC activity.
2.2.3. Western blot assay
A Western blot assay was performed to determine whether the
inhibitory effects of analogues on inflammatory factor (NO, PGE2)
release are related to suppression of iNOS and COX-2 expression
and whether they inhibit NF-kB signals. It was observed that the 2-
2.2.4. Carrageenan-induced rat hind paw edema model
To investigate the effects on inflammation models, rats were
initially treated orally with 20 mg/kg of 9e12, and 1$HCl for 1 h
then subjected to carrageenan-induced acute inflammatory hind
paw edema. Our results showed 9e12 and 1$HCl significantly
prevented carrageenan-induced paw thickness (Fig. 7), and com-
pound 9 exhibited more inhibition than other analogues or com-
pound 1$HCl. Indomethacin, used as positive control, also
methyl-1H-indol-3-ethylsulfamoylphenylacrylamide
analogues
9e12 not only significantly repress the expression of iNOS and COX-
2, but also dose-dependently reduce the phosphorylation of p65
(0.01e1 mM; Fig. 6A). Furthermore, analogues of 9e12 were also

S. Mehndiratta et al. / European Journal of Medicinal Chemistry 85 (2014) 468e479
473
Table 1
Inhibition of HeLa nuclear extract HDAC activity and LPS-induced inflammatory factors expression in macrophages by compounds 9e20 and reference 1·HCl, 8.
Compd
HeLa nuclear HDACs
IC50 (nM SEMa)
NO
TNF-
a
IL-6
PGE2
IC50
(
mM
SEMa)
9
7.9 0.6
2.8 0.2
3.3 0.2
3.4 0.3
7.3 1.1
47.4 3.8
29.6 0.9
9.2 0.9
6.6 0.3
5.7 0.7
43.9 3.3
771.8 19.5
7.5 0.6
26.4 1.3
0.36 0.03
0.74 0.06
0.30 0.03
0.26 0.03
3.1 0.24
1.0 0.08
1.2 0.09
1.6 0.42
0.74 0.06
0.29 0.03
3.7 0.25
>10
1.5 0.21
0.48 0.03
0.8 0.06
0.73 0.05
1.5 0.23
>10
>10
>10
1.2 0.09
0.62 0.07
>10
0.013 6.2 ꢀ 10ꢂ4
0.020 8.6 ꢀ 10ꢂ4
0.014 7.5 ꢀ 10ꢂ4
0.021 5.9 ꢀ 10ꢂ4
0.14 9.6 ꢀ 10ꢂ3
0.55 3.2 ꢀ 10ꢂ2
0.59 4.3 ꢀ 10ꢂ2
0.45 2.8 ꢀ 10ꢂ2
0.14 9.6 ꢀ 10ꢂ4
0.032 6.6 ꢀ 10ꢂ4
0.031 6.6 ꢀ 10ꢂ4
2.39 2.1 ꢀ 10ꢂ2
0.034 5.7 ꢀ 10ꢂ4
0.059 1.7 ꢀ 10ꢂ3
0.32 0.21
10
11
12
13
14
15
16
17
18
19
20
1$HCl
8
0.33 0.18
0.15 0.02
0.27 0.02
3.1 0.26
2.9 0.19
2.9 0.21
7.4 0.48
2.2 0.22
1.5 0.16
7.2 0.43
>10
0.11 0.02
4.7 0.25
>10
0.054 0.01
8.28 0.24
0.24 0.14
2.2 0.32
a
SEM: Mean of standard deviation. All experiments were independently performed at least three times.
300 or 500 MHz. Chemical shifts are reported in parts per million
(ppm, ) downfield from TMS as an internal standard. High-
d
resolution mass spectra (HRMS) were measured with a JEOL
(JMS-700) electron impact (EI) mass spectrometer. Purity of the
final compounds was determined using an Hitachi 2000 series
HPLC system using C-18 column (Agilent ZORBAX Eclipse XDB-C18
5
m
m. 4.6 mm ꢀ 150 mm). Flash column chromatography was
accomplished on silica gel (Merck Kieselgel 60, No. 9385, 230e400
mesh ASTM). All reactions were carried out under an atmosphere of
dry nitrogen.
4.1.1. Synthesis of 2-methyl-1H-indol-3-
ethylsulfamoylphenylacrylamide derivatives
4.1.1.1. Method A: typical procedure for indole N-alkylation (29e31).
To a solution of compound 28 (0.92 mmol) in DMF (2 mL) was
added NaH (1.10 mmol) at 0 ꢁC, and then was allowed to stir at the
same temperature for 10 min before the addition of an alkyl iodide
(1.38 mmol). The ice bath was removed after 5 min and was stirred
for additional 1 h. The reaction was quenched with slow addition of
water and then was neutralized with 3 N HCl. The reaction mixture
was then extracted with DCM (20 mL ꢀ 3) and the combined
organic layer was dried over anhydrous MgSO4 and concentrated
under reduced pressure. The resulting residue was purified by silica
gel chromatography (EtOAc: n-hexane ¼ 1: 4).
Fig. 5. Cell viability of macrophages were determined after 24 h treatment with
0.01e1 M of 9e12 and 1$HCl, 8 using the MTT assay. The data are the mean SEM,
with n ¼ 3. *p < 0.05 compared to the basal group.
m
suppressed hind paw edema. These results suggest that the 2-
methyl-1H-indol-3-ethylsulfamoyl-phenylacrylamide analogues
9e12 exhibit potent anti-inflammatory activity.
3. Conclusion
We have synthesized
a series of 2-methyl-1H-indol-3-
ethylsulfamoylphenylacrylamide derivatives as potent HDAC in-
hibitors with potential anti-inflammatory activity. When compared
to 1$HCl, compounds 9e12 and 16e18 show comparable or better
HDAC inhibitory activity, in the single digit nM range. Compound 10
showed 2.6-fold better HDAC inhibition and 1.7-fold improved IL-6
suppression compared to 1$HCl. In addition, compounds 9e12 did
not show apparent cell viability suppression in an MTT assay,
denoting diminished cellular toxicitycompared to 1$HCl. Compound
9 has the best in vivo activity when compared to other analogues of
the series and to 1$HCl. We have also observed markedly increased
acetylation of histone 3, confirming that the inhibition by analogues
is dueto suppression of HDAC activity. Our results are consistentwith
previous published reports on LBH589 and indicate that PXD101 and
LBH589 may also have non-oncological use. Compound 9 has po-
tential in further investigations as an anti-inflammatory agent.
4.1.1.2. Method B: typical procedure for N-alkylation of the sulfon-
amide (32e34). A mixture of compound 24 (1.15 mmol), Cs2CO3
(1.72 mmol), and DMF (1 mL) was stirred at ambient temperature
for 30 min. To the mixture was added an alkyl iodide (2.3 mmol)
and then was allowed to stir at room temperature until the reaction
complete as indicated by TLC monitoring (in the case of isopropyl
iodide reaction was carried out at 60 ꢁC). The reaction was
quenched with water, neutralized with 3 N HCl and then extracted
with DCM (20 mL ꢀ 3). The combined organic layer was dried over
anhydrous MgSO4 and concentrated under reduced pressure. The
resulting residue was purified by silica gel chromatography
(EtOAc:n-hexane ¼ 1:4).
4.1.2. 3-Bromo-N-[2-(2-methyl-1H-indol-3-yl)-ethyl]-
benzenesulfonamide (23)
4. Experimental section
To a mixture of 2-methyltryptamine (22, 1.0 g, 5.74 mmol),
pyridine (4 mL), and acetonitrile (1 mL) was added 3-
bromobenzenesulfonyl chloride (1.88 g, 6.32 mmol) dropwise,
and then was allowed to stir at room temperature for 2 h. The re-
action was quenched with water and was acidified with dropwise
addition of 3 N HCl. The reaction mixture was then extracted with
4.1. Chemistry
Nuclear magnetic resonance (1H NMR and 13C NMR) spectra
were obtained with a Bruker DRX-500 spectrometer operating at

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S. Mehndiratta et al. / European Journal of Medicinal Chemistry 85 (2014) 468e479
Fig. 6. The suppressive effects of 9e12 and 1$HCl, 8 on the LPS-induced protein levels of nitric oxide synthase (iNOS), cyclooxygenase (COX-2) and phosphorylation of p65 NF-kB.
(A) RAW 264.7 cells (1 ꢀ 106) in 6-well plates were incubated with 0.01e1
mM of the indicated compounds for 30 min, followed by stimulation with LPS (25 ng/mL) for 24 h. Then
the cells were harvested and whole cell extracts were prepared for Western blot analysis for iNOS, COX-2 and the phosphorylation of p65. (B) RAW 264.7 cells (1 ꢀ 106) in 6-well
plates were incubated for the indicated compounds (0.1e1 mM) for 24 h, then were harvested and cell lysates prepared for Western blot analysis for the indicated proteins. The
relative protein expressions are presented as mean SEM for three replicates. *p < 0.05 compared to the LPS-treated group (A) or non-treated group (B).
EtOAc (20 mL ꢀ 3). The combined organic layer was dried over
anhydrous MgSO4 and concentrated under reduced pressure. The
resulting residue was purified by silica gel chromatography (EtOAc:
n-hexane ¼ 3: 7) to give the compound 23, in 76% yield; 1H NMR
(s, 3H), 2.90 (t, J ¼ 6.5 Hz, 2H), 3.22e3.26 (m, 2H), 4.33 (t, J ¼ 5.5 Hz,
1H), 7.02 (t, J ¼ 7.5 Hz, 1H), 7.12 (t, J ¼ 7.0 Hz, 1H), 7.25e7.28 (m, 3H),
7.49e7.54 (m, 4H), 7.78 (s, 1H).
(500 MHz, CDCl3):
d
2.27 (s, 3H), 2.85 (t, J ¼ 6.5 Hz, 2H), 3.18e3.22
4.1.4. 3-{3-[2-(2-Methyl-1H-indol-3-yl)-ethylsulfamoyl]-phenyl}-
acrylic acid tert-butyl ester (25)
(m, 2H), 4.63 (t, J ¼ 6.5 Hz, 1H), 7.01 (t, J ¼ 7.5 Hz, 1H), 7.08 (t,
J ¼ 7.5 Hz, 1H), 7.19e7.22 (m, 2H), 7.28 (d, J ¼ 8 Hz, 1H), 7.56e7.59
(m, 2H), 7.86 (t, J ¼ 7.0 Hz, 1H), 7.90 (s, 1H).
A mixture of 23 (1.70 g, 4.33 mmol), tris(dibenzylideneacetone)
dipalladium (0.40 g, 0.43 mmol), tri-t-butylphosphonium tetra-
fluoroborate (0.25 g, 0.86 mmol), K2CO3 (1.19 g, 8.66 mmol), TEA
(1.64 mL, 11.69 mmol), t-butyl acrylate (1 mL, 7.34 mmol), and DMF
(10 mL) was stirred at 100 ꢁC for 12 h. The reaction was quenched
with water, extracted with CH2Cl2 (20 mL ꢀ 3) and the combined
organic layer was dried over anhydrous MgSO4 and concentrated
under reduced pressure. The resulting residue was purified by silica
4.1.3. 4-Bromo-N-[2-(2-methyl-1H-indol-3-yl)-ethyl]-
benzenesulfonamide (24)
The title compound was obtained in 50% yield by using 4-
bromobenzenesulfonyl chloride in
a
manner similar to that
2.34
described for the synthesis of 23. 1H NMR (500 MHz, CDCl3):
d

S. Mehndiratta et al. / European Journal of Medicinal Chemistry 85 (2014) 468e479
475
resulting product dissolved in CH3OH (10 mL) was added of 10% TFA
(10 mL) and was allowed to stir at room temperature for 5 h. The
reaction mixture was concentrated under reduced pressure to give
a white residue, which was recrystallized from CH3OH to afford the
desired compound 9, in 54% yield (from 25); mp: 165e166 ꢁC. 1H
NMR (300 MHz, CD3OD):
d
2.27 (s, 3H), 2.80 (t, J ¼ 7.2 Hz, 2H), 3.10
(t, J ¼ 7.8 Hz, 2H), 6.52 (d, J ¼ 15.9 Hz, 1H), 6.86e6.99 (m, 2H), 7.17
(d, J ¼ 7.8 Hz, 1H), 7.25 (d, J ¼ 7.8 Hz, 1H), 7.48e7.60 (m, 2H),
7.72e7.77 (m, 2H), 7.91 (s, 1H). 13C NMR (300 MHz, CD3OD): 11.02,
25.67, 44.45, 107.66, 111.08, 117.79, 119.23, 120.37, 121.07, 126.30,
128.39,129.32,130.48,132.09,133.09,136.74,136.98,139.56,142.75,
165.38. MS (ESI) m/z: 400.1 (MþHþ). HRMS (ESI) for C20H22N3O4S
(MþHþ): calcd, 400.1331; found, 400.1321.
4.1.7. N-hydroxy-3-{4-[2-(2-methyl-1H-indol-3-yl)-
ethylsulfamoyl]-phenyl}-acrylamide (10)
The title compound was obtained in 49% yield from compound
26 in a manner similar to that described for the synthesis of 9; mp:
Fig. 7. 2-Methyl-1H-indol-3-ethylsulfamoylphenylacrylamide derivatives suppress
carrageenan-induced hind paw edema in rats. A 0.5% (w/v) suspension of carrageenan
in normal saline was administered to male Wistar rats (7 weeks) by intradermal in-
jection into the base of the right hind paw. One hour prior to carrageenan injection,
rats were orally administered vehicle (1% carboxymethyl cellulose and 0.5% Tween 80)
or a fine suspension of 9e12 and 1$HCl (20 mg/kg) in vehicle. A positive control group
was included in which rats were pretreated with 1 mg/kg indomethacin. Three hours
after carrageenan administration, changes in the volume of the right hind paw were
measured by digital caliper and digital plethysmometer (Diagnostic & Research In-
struments CO., Ltd, Taipei, Taiwan), respectively. The results are expressed as the
mean SEM, with n ¼ 3 (except for vehicle group, in which n ¼ 4). *p < 0.05 compared
to the basal group; #p < 0.05 for the comparison of the indicated groups.
168e169 ꢁC. 1H NMR (500 MHz, CD3OD):
d 2.26 (s, 3H), 2.80 (t,
J ¼ 7.5 Hz, 2H), 3.08 (t, J ¼ 7.0 Hz, 2H), 6.54 (d, J ¼ 15.5 Hz, 1H), 6.87
(t, J ¼ 7.0 Hz, 1H), 6.95 (t, J ¼ 7.5 Hz, 1H), 7.15 (d, J ¼ 8.5 Hz, 1H), 7.24
(d, J ¼ 8.0 Hz, 1H), 7.55e7.60 (m, 3H), 7.71 (d, J ¼ 8.5 Hz, 2H). 13
C
NMR (300 MHz, CD3OD): 9.88, 24.57, 43.30, 106.57, 109.95, 116.70,
118.06, 119.91, 120.04, 126.95, 127.73, 128.21, 131.96, 135.63, 138.34,
138.57, 141.24, 164.17. MS (ESI) m/z: 400.1 (MþHþ). HRMS (ESI) for
C
20H22N3O4S (MþHþ): calcd, 400.1331; found, 400.1319.
4.1.8. 4-Bromo-N-[2-(2-methyl-1H-indol-3-yl)-ethyl]-benzene-N-
tert-butoxycarbonylsulfonamide (27)
gel chromatography (EtOAc: n-hexane ¼ 3: 7) to give compound 25,
in 83% yield; 1H NMR (500 MHz, CDCl3):
d 1.54 (s, 9H), 2.32 (s, 3H),
To a solution of compound 24 (4.8 g,11.11 mmol) in DCM (50 mL)
was added di-t-butyl dicarbonate (2.42 g, 11.11 mmol) and DMAP
(0.27 g, 2.22 mmol) and the mixture was stirred at room temper-
ature for 4 h. The reaction was quenched with water and was
extracted with EtOAc (20 mL ꢀ 3). The combined organic layer was
dried over anhydrous MgSO4 and concentrated under reduced
pressure to give a pale yellow liquid in quantitative yield. 1H NMR
2.90 (t, J ¼ 6.5 Hz, 2H), 3.22e3.26 (m, 2H), 4.40 (t, J ¼ 6.5 Hz, 1H),
6.37 (d, J ¼ 16.0 Hz,1H), 7.00 (t, J ¼ 7.0 Hz,1H), 7.09 (t, J ¼ 7.5 Hz,1H),
7.23e7.29 (m, 3H), 7.41 (t, J ¼ 8.0 Hz, 1H), 7.50 (d, J ¼ 16.0 Hz, 1H),
7.60 (d, J ¼ 7.5 Hz, 1H), 7.68 (d, J ¼ 8.0 Hz, 1H), 7.79 (s, 1H), 7.86 (s,
1H).
(500 MHz, CDCl3):
d 1.37 (s, 9H), 2.40 (s, 3H), 3.12e3.15 (m, 2H),
4.1.5. 3-{4-[2-(2-Methyl-1H-indol-3-yl)-ethylsulfamoyl]-phenyl}-
acrylic acid tert-butyl ester (26)
3.96e3.99 (m, 2H), 7.07e7.13 (m, 2H), 7.27 (d, J ¼ 7.5 Hz, 2H),
7.56e7.61 (m, 3H), 7.67 (d, J ¼ 9.0 Hz, 2H), 7.78 (bs, 1H).
The title compound was obtained in 76% yield from compound
24 in a manner similar to that described for the synthesis of 25. 1H
4.1.9. 3-{4-[2-(2-Methyl-1H-indol-3-yl)-ethyl-N-tert-
NMR (500 MHz, CDCl3):
d
1.55 (s, 9H), 2.29 (s, 3H), 2.88 (t, J ¼ 6.5 Hz,
butoxycarbonylsulfamoyl]-phenyl}-acrylic acid tert-butyl ester (28)
The title compound was obtained in 84% yield from compound
27 in a manner similar to that described for the synthesis of 25. 1H
2H), 3.20e3.24 (m, 2H), 4.61 (t, J ¼ 5.5 Hz, 1H), 6.41 (d, J ¼ 16.0 Hz,
1H), 6.99 (t, J ¼ 7.5 Hz, 1H), 7.08 (t, J ¼ 7.5 Hz, 1H), 7.21 (d, J ¼ 8.0,
1H), 7.28 (d, J ¼ 8.0 Hz, 1H), 7.46 (d, J ¼ 8.0 Hz, 2H), 7.54 (d,
J ¼ 16.0 Hz, 1H), 7.66 (d, J ¼ 8.0 Hz, 2H), 7.97 (s, 1H).
NMR (500 MHz, CDCl3): d 1.29 (s, 9H), 1.52 (s, 9H), 2.32 (s, 3H), 3.09
(t, J ¼ 7.5 Hz, 2H), 4.00 (t, J ¼ 8.0 Hz, 2H), 6.56 (d, J ¼ 16.5 Hz, 1H),
6.94 (t, J ¼ 7.5 Hz, 1H), 7.00 (t, J ¼ 8.0 Hz, 1H), 7.24 (d, J ¼ 8.0, 1H),
7.48 (d, J ¼ 8.0 Hz, 1H), 7.58 (d, J ¼ 16.0 Hz, 1H), 7.66 (d, J ¼ 9.0 Hz,
2H), 7.72 (d, J ¼ 8.0 Hz, 2H).
4.1.6. N-hydroxy-3-{3-[2-(2-methyl-1H-indol-3-yl)-
ethylsulfamoyl]-phenyl}-acrylamide (9)
A mixture of TFA (15 mL) and 25 (2 g, 4.5 mmol) was stirred at
0
ꢁC for 20 min. The reaction was quenched with water and was
4.1.10. 3-{4-[2-(1,2-Dimethyl-1H-indol-3-yl)-ethyl-N-tert-
neutralized with saturated sodium bicarbonate solution. The
resulting white precipitate was collected by filtration and was used
directly in the next step. The off-white solid (1.75 g, 4.55 mmol) was
dissolved DMF (10 mL) and was added 1-(3-dimethylaminopropyl)-
3-ethylcarbodiimide hydrochloride (EDC$HCl, 1.30 g, 6.83 mmol),
hydroxybenzotriazole (HOBt, 0.92 g, 6.83 mmol), and N-methyl-
morpholine (NMM, 1.35 mL, 11.74 mmol). After being stirred at
room temperature for 30 min, NH2OTHP (0.59 g, 5.00 mmol) was
added and allowed to stir for additional 5 h. The reaction mixture
was quenched with water and was extracted with EtOAc
(25 mL ꢀ 3). The combined organic layer was collected, dried over
anhydrous MgSO4 and concentrated under reduced pressure to give
a light yellow residue, which was purified by silica gel chroma-
tography (EtOAc:n-hexane ¼ 3:1) to give colorless liquid. To the
butoxycarbonylsulfamoyl]-phenyl}-acrylic acid tert-butyl ester (29)
The title compound was obtained in 72% yield by using 28 (0.5 g,
0.92 mmol), NaH (0.68 g,1.10 mmol) and MeI (0.086 mL,1.38 mmol)
in DMF (2 mL) in a manner similar to that as described in method A.
1H NMR (500 MHz, CDCl3):
d 1.35 (s, 9H), 1.52 (s, 12H), 2.39 (s, 3H),
3.18 (t, J ¼ 8.0 Hz, 2H), 3.64 (s, 3H), 3.96 (t, J ¼ 8.0 Hz, 2H), 6.44 (d,
J ¼ 16 Hz, 1H), 7.08 (t, J ¼ 7.0 Hz, 1H), 7.16 (t, J ¼ 7.5 Hz, 1H),
7.24e7.28 (m, 1H), 7.56 (m, 3H), 7.64 (d, J ¼ 7.5 Hz, 1H), 7.81 (d,
J ¼ 8.5 Hz, 2H).
4.1.11. 3-{4-[2-(1-Ethyl-2-methyl-1H-indol-3-yl)-ethyl-N-tert-
butoxycarbonylsulfamoyl]-phenyl}-acrylic acid tert-butyl ester (30)
The title compound was obtained in 62% yield by using 28 (0.5 g,
0.92 mmol), NaH (0.68 g, 1.10 mmol) and EtI (0.11 mL, 1.38 mmol) in

476
S. Mehndiratta et al. / European Journal of Medicinal Chemistry 85 (2014) 468e479
DMF (2 mL) in a manner similar to that described in method A. 1H
NMR (300 MHz, CDCl3):
9H), 2.34 (s, 2H), 2.95 (t, J ¼ 6.6 Hz, 2H), 3.23e3.24 (m, 2H),
4.07e4.15 (m, 2H), 6.43 (d, J ¼ 16.2 Hz, 1H), 7.01 (t, J ¼ 6.9 Hz, 1H),
7.15 (t, J ¼ 6.9 Hz, 1H), 7.26e7.33 (m, 4H), 7.51e7.56 (m, 3H), 7.71 (d,
J ¼ 8.4 Hz, 2H).
4.1.16. 4-Bromo-N-methyl-N-[2-(2-methyl-1H-indol-3-yl)-ethyl]-
benzenesulfonamide (32)
The title compound was obtain din 65% yield by using 24 (0.5 g,
1.15 mmol), Cs2CO3 (0.56 g, 1.72 mmol) and MeI (0.10 mL, 2.3 mmol)
in DMF (2 mL) in a manner similar to that described in method B. 1H
d
1.33 (t, J ¼ 7.2 Hz, 3H), 1.38 (s, 9H), 1.55 (s,
NMR (300 MHz, CDCl3):
d 2.40 (s, 3H), 2.85 (s, 3H), 2.97e3.02 (m,
2H), 3.25e3.30 (m, 2H), 7.06e7.15 (m, 2H), 7.26e7.31 (m, 3H),
7.65e7.69 (m, 4H), 7.35 (bs, 1H).
4.1.12. 3-(4-{2-[1-(2-Diethylamino-ethyl)-2-methyl-1H-indol-3-
yl]-ethyl-N-tert-butoxycarbonylsulfamoyl}-phenyl)-acrylic acid
tert-butyl ester (31)
4.1.17. 4-Bromo-N-ethyl-N-[2-(2-methyl-1H-indol-3-yl)-ethyl]-
benzenesulfonamide (33)
The title compound was obtained in 83% yield by using 28 (0.2 g,
0.37 mmol), NaH (0.01 g, 0.44 mmol) and 2-bromo-N,N-dieth-
ylethylamine hydrobromide (0.14 mg, 0.55 mmol) in DMF (2 mL), in
a manner similar to that described in method A. 1H NMR (500 MHz,
The title compound was obtained in quantitative yield by using
24 (0.5 g, 1.15 mmol), Cs2CO3 (0.56 g, 1.72 mmol) and EtI (0.18 mL,
2.3 mmol) in DMF (2 mL) in a manner similar to that described in
method B. 1H NMR (300 MHz, CDCl3):
d
1.17 (t, J ¼ 7.2 Hz, 3H), 2.39
CD3OD):
d
1.05 (t, J ¼ 7.0 Hz, 6H), 1.28 (s, 9H), 1.52 (s, 9H), 2.38 (s,
(s, 3H), 2.98e3.03 (m, 2H), 3.29e3.35 (m, 4H), 7.07e7.16 (m, 2H),
7.26e7.29 (m, 1H), 7.45e7.48 (m, 1H), 7.56e7.59 (m, 2H), 7.63e7.65
(m, 2H).
3H), 2.62e2.67 (m, 6H), 3.12 (t, J ¼ 7.0 Hz, 2H), 3.99 (t, J ¼ 7.5 Hz,
2H), 4.18 (t, J ¼ 8.0 Hz, 2H), 6.56 (d, J ¼ 16 Hz, 1H), 7.00 (t, J ¼ 7.5 Hz,
1H), 7.10 (t, J ¼ 7.5 Hz, 1H), 7.30 (d, J ¼ 8.0 Hz, 1H), 7.53 (d, J ¼ 7.5 Hz,
1H), 7.58 (d, J ¼ 16 Hz,1H), 7.66 (d, J ¼ 8.5 Hz, 2H), 7.73 (d, J ¼ 6.5 Hz,
2H).
4.1.18. 4-Bromo-N-isopropyl-N-[2-(2-methyl-1H-indol-3-yl)-
ethyl]-benzenesulfonamide (34)
The title compound was obtained in 95% yield by using 24 (0.5 g,
1.15 mmol), Cs2CO3 (0.56 g, 1.72 mmol) and MeI (0.23 mL,
2.3 mmol) in DMF (2 mL) in a manner similar to that described in
4.1.13. 3-{4-[2-(1,2-Dimethyl-1H-indol-3-yl)-ethyl-N-tert-
butoxycarbonylsulfamoyl]-phenyl}-N-hydroxy-acrylamide (11)
The title compound was obtained in 61% yield from compound
29 in a manner similar to that described for the synthesis of 9; mp:
method B. 1H NMR (300 MHz, CDCl3):
d
1.09 (d, J ¼ 7.2 Hz, 6H), 2.47
(s, 3H), 3.09e3.14 (m, 2H), 3.19e3.26 (m, 2H), 4.11e4.16 (m, 1H),
7.09e7.16 (m, 2H), 7.28e7.31 (m, 1H), 7.54e7.57 (m, 1H), 7.59e7.63
(m, 2H), 7.70e7.74 (2H), 7.86 (s, 1H).
168e169 ꢁC. 1H NMR (500 MHz, CD3OD):
d 2.25 (s, 3H), 2.82 (t,
J ¼ 7.0 Hz, 2H), 3.09 (t, J ¼ 7.5 Hz, 2H), 3.56 (s, 3H), 6.53 (d, J ¼ 16 Hz,
1H), 6.92 (t, J ¼ 7.0 Hz, 1H), 7.03 (t, J ¼ 7.5 Hz, 1H), 7.18 (d, J ¼ 8.0 Hz,
1H), 7.29 (d, J ¼ 7.5 Hz, 1H), 7.53e7.57 (m, 3H), 7.65 (d, J ¼ 8.0 Hz,
2H). 13C NMR (300 MHz, CD3OD): 8.69, 24.76, 28.21, 43.42, 106.65,
108.19, 116.88, 118.26, 120.05, 126.83, 127.44, 127.62, 133.54, 136.66,
138.41, 141.29, 164.15. MS (ESI) m/z: 414.1 (MþHþ). HRMS (ESI) for
4.1.19. 3-(4-{Methyl-[2-(2-methyl-1H-indol-3-yl)-ethyl]-
sulfamoyl}-phenyl)-acrylic acid methyl ester (35)
The title compound was obtained in 80% yield from compound
32 in a manner similar to that described for the synthesis of 25. 1H
NMR (300 MHz, CDCl3):
d 2.40 (s, 3H), 2.84 (s, 3H), 2.97e3.02 (m,
C
21H24N3O4S (MþHþ): calcd, 414.1488; found, 414.1479.
2H), 3.23e3.29 (m, 2H), 3.83 (s, 3H), 6.50 (d, J ¼ 16.2 Hz, 1H),
7.05e7.15 (m, 2H), 7.26e7.28 (m, 3H), 7.45 (d, J ¼ 7.5 Hz,1H), 7.58 (d,
J ¼ 8.1 Hz, 2H), 7.67 (d, J ¼ 15.9 Hz, 1H), 7.75 (d, J ¼ 8.4 Hz, 2H), 7.80
(s, 1H).
4.1.14. 3-{4-[2-(1-Ethyl-2-methyl-1H-indol-3-yl)-ethyl-N-tert-
butoxycarbonylsulfamoyl]-phenyl}-N-hydroxy-acrylamide (12)
The title compound was obtained in 59% yield from compound
30 in a manner similar to that described for the synthesis of 9; mp:
4.1.20. 3-(4-{Ethyl-[2-(2-methyl-1H-indol-3-yl)-ethyl]-sulfamoyl}-
phenyl)-acrylic acid methyl ester (36)
171e172 ꢁC. 1H NMR (500 MHz, CD3OD):
d
1.23 (t, J ¼ 7.0 Hz, 3H),
The title compound was obtained in 88% yield from compound
2.28 (s, 3H), 2.83 (t, J ¼ 7.5 Hz, 2H), 3.07 (t, J ¼ 7.5 Hz, 2H), 4.06e4.10
(m, 2H), 6.56 (d, J ¼ 15.5 Hz, 1H), 6.92 (t, J ¼ 7.5 Hz, 1H), 7.03 (t,
J ¼ 7.5 Hz, 1H), 7.22 (d, J ¼ 8.0 Hz, 1H), 7.30 (d, J ¼ 8.0 Hz, 1H),
7.56e7.62 (m, 3H), 7.72 (d, J ¼ 8.5 Hz, 2H). 13C NMR (300 MHz,
CD3OD): 9.56, 15.21, 25.67, 37.97, 44.27, 107.79, 109.33, 118.02,
119.20, 121.00, 121.36, 127.84, 128.46, 128.68, 133.68, 136.24, 138.82,
139.34, 142.09, 164.48. MS (ESI) m/z: 428.2 (MþHþ). HRMS (ESI) for
33 in a manner similar to that described for the synthesis of 25. 1H
NMR (300 MHz, CDCl3):
d
1.17 (t, J ¼ 7.2 Hz, 3H), 2.40 (s, 3H),
2.96e3.04 (m, 2H), 3.30e3.37 (m, 4H), 3.83 (s, 3H), 6.50 (d,
J ¼ 15.9 Hz,1H), 7.08e7.13 (m, 2H), 7.25e7.28 (m, 2H), 7.45e7.48 (m,
1H), 7.57 (d, J ¼ 8.4 Hz, 2H), 7.67 (d, J ¼ 16.2 Hz, 1H), 7.78e7.81 (m,
3H).
C
22H26N3O4S (MþHþ): calcd, 428.1644; found, 428.1638.
4.1.21. 3-(4-{Isopropyl-[2-(2-methyl-1H-indol-3-yl)-ethyl]-
sulfamoyl}-phenyl)-acrylic acid methyl ester (37)
4.1.15. 3-(4-{2-[1-(2-Diethylamino-ethyl)-2-methyl-1H-indol-3-
yl]-ethyl-N-tert-butoxycarbonylsulfamoyl}-phenyl)-N-hydroxy-
acrylamide (13)
The title compound was obtained in 89% yield from compound
34 in a manner similar to that described for the synthesis of 25. 1H
NMR (300 MHz, CDCl3): d 1.07e1.10 (m, 3H), 2.45 (s, 3H), 3.09e3.18
The title compound was obtained in 41% yield from compound
31 in a manner similar to that described for the synthesis of 9; mp:
(m, 2H), 3.21e3.27 (m, 2H), 3.83 (s, 3H), 4.12e4.21 (m, 1H), 6.51 (d,
J ¼ 15.9 Hz, 1H), 7.11e7.16 (m, 2H), 7.28e7.31 (m, 1H), 7.55e7.62 (m,
3H), 7.68 (d, J ¼ 16.2 Hz, 1H), 7.84e7.89 (m, 3H).
173e174 ꢁC. 1H NMR (500 MHz, CD3OD):
d
1.02 (t, J ¼ 7.0 Hz, 6H),
2.30 (s, 3H), 2.57e2.65 (m, 6H), 2.82 (t, J ¼ 7.5 Hz, 2H), 3.05 (t,
J ¼ 7.5 Hz, 2H), 4.09e4.14 (m, 2H), 6.60 (d, J ¼ 16.0 Hz, 1H), 6.94 (t,
J ¼ 7.5 Hz, 1H), 7.05 (t, J ¼ 8.0 Hz, 1H), 7.22e7.31 (m, 3H), 7.54 (d,
J ¼ 8.0 Hz, 2H), 7.66 (d, J ¼ 8.5 Hz, 2H). 13C NMR (300 MHz, CD3OD):
8.99, 10.61, 24.92, 40.83, 43.50, 51.66, 107.36, 108.48, 117.35, 118.63,
120.39, 127.05, 127.79, 127.85, 133.26, 135.93, 138.03, 138.61, 141.43,
163.81. MS (ESI) m/z: 499.2 (MþHþ). HRMS (ESI) for C26H35N4O4S
(MþHþ): calcd, 499.2379; found, 499.2357.
4.1.22. N-hydroxy-3-(4-{methyl-[2-(2-methyl-1H-indol-3-yl)-
ethyl]-sulfamoyl}-phenyl)-acrylamide (14)
The title compound was obtained in 51% yield from compound
35 in a manner similar to that described for the synthesis of 9; mp:
132e133 ꢁC. 1H NMR (300 MHz, CD3OD):
d 2.34 (s, 3H), 2.81 (s, 3H)
2.95 (t, J ¼ 6.6 Hz, 2H), 3.25 (t, J ¼ 7.8 Hz, 2H), 6.56 (d, J ¼ 15.6 Hz,
1H), 6.91e7.02 (m, 2H), 7.20e7.22 (m, 1H), 7.37e7.39 (m, 1H), 7.58

S. Mehndiratta et al. / European Journal of Medicinal Chemistry 85 (2014) 468e479
477
(d, J ¼ 15.9 Hz, 1H), 7.65e7.72 (m, 4H). 13C NMR (300 MHz, CD3OD):
4.1.28. 3-{4-[2-(1-Methyl-1H-indol-3-yl)-ethylsulfamoyl]-phenyl}-
acrylic acid tert-butyl ester (43)
9.97, 23.19, 34.19, 50.46, 106.57, 110.02, 116.82, 118.21, 120.04,
120.26, 127.36, 127.87, 128.27, 131.98, 135.67, 138.20, 138.53, 138.87,
164.08. MS (ESI) m/z: 414.1 (MþHþ). HRMS (ESI) for C21H24N3O4S
(MþHþ): calcd, 414.1488; found, 414.1484.
The title compound was obtained in 21% yield from compound
41 in a manner similar to that described for the synthesis of 25. 1H
NMR (300 MHz, CDCl3):
d
1.55 (s, 9H), 2.95 (t, J ¼ 6.3 Hz, 2H),
3.27e3.34 (m, 2H), 3.74 (s, 3H), 6.44 (d, J ¼ 16.2 Hz, 1H), 6.83 (s, 1H),
7.04e7.07 (m, 1H), 7.21e7.31 (m, 5H), 7.39 (d, J ¼ 7.8 Hz, 1H),
7.51e7.59 (m, 2H), 7.72 (d, J ¼ 8.4 Hz, 2H).
4.1.23. 3-(4-{Ethyl-[2-(2-methyl-1H-indol-3-yl)-ethyl]-sulfamoyl}-
phenyl)-N-hydroxy-acrylamide (15)
The title compound was obtained in 52% over all yield from
compound 36 in a manner similar to that described for the syn-
4.1.29. N-hydroxy-3-{4-[2-(1H-indol-3-yl)-ethylsulfamoyl]-
phenyl}-acrylamide (17)
thesis of 9; mp: 131e132 ꢁC. 1H NMR (300 MHz, d6-acetone):
d 1.16
The title compound was obtained in 19% over all yield from
compound 42 in a manner similar to that described for the syn-
(t, J ¼ 7.2 Hz, 3H), 2.37 (s, 3H), 2.96e3.01 (m, 2H), 3.31e3.37 (m, 4H),
6.69 (d, J ¼ 15.3 Hz, 1H), 6.94e7.04 (m, 2H), 7.25e7.28 (m, 1H),
7.45e7.48 (m, 1H), 7.60 (d, J ¼ 15.6 Hz, 1H), 7.75 (d, J ¼ 8.4 Hz, 2H),
7.83 (d, J ¼ 8.4 Hz, 2H). 13C NMR (300 MHz, d-Acetone): 10.62,13.76,
24.58, 43.10, 47.98, 107.24, 110.40, 110.46, 117.29, 118.63, 120.37,
120.75, 127.46, 128.21, 128.54, 132.24, 135.77, 137.90, 138.85, 140.94,
163.04. MS (ESI) m/z: 428.2 (MþHþ). HRMS (ESI) for C22H26N3O4S
(MþHþ): calcd, 428.1644; found, 428.1650.
thesis of 9; mp: 157e158 ꢁC. 1H NMR (300 MHz, CD3OD):
d 2.88 (t,
J ¼ 7.2 Hz, 2H), 3.19 (t, J ¼ 7.2 Hz, 2H), 6.55 (d, J ¼ 15.6 Hz, 1H),
6.91e6.98 (m, 2H), 7.05 (t, J ¼ 8.1 Hz, 1H), 7.28 (d, J ¼ 8.1 Hz, 1H),
7.37 (d, J ¼ 7.8 Hz, 1H), 7.56e7.62 (m, 3H), 7.75 (d, J ¼ 8.1 Hz, 2H). 13
C
NMR (300 MHz, CD3OD): 25.41, 43.42, 110.92, 111.03, 117.65, 118.24,
120.02, 120.91, 122.30, 126.98, 127.02, 127.73, 136.66, 138.39, 138.55,
141.10, 164.20. MS (ESI) m/z: 386.1 (MþHþ). HRMS (ESI) for
C
19H20N3O4S (MþHþ): calcd, 386.1175; found, 386.1165.
4.1.24. N-hydroxy-3-(4-{isopropyl-[2-(2-methyl-1H-indol-3-yl)-
ethyl]-sulfamoyl}-phenyl)-acrylamide (16)
4.1.30. N-hydroxy-3-{4-[2-(1-Methyl-1H-indol-3-yl)-
ethylsulfamoyl]-phenyl}-acrylamide (18)
The title compound was obtained in 44% over all yield from
compound 37 in a manner similar to that described for the syn-
The title compound was obtained in 17% over all yield from
compound 43 in a manner similar to that described for the syn-
thesis of 9; mp: 129e130 ꢁC. 1H NMR (300 MHz, d6-acetone):
d 1.09
thesis of 9; mp: 153e154 ꢁC. 1H NMR (300 MHz, CDCl3):
d 2.86 (t,
(t, J ¼ 6.6 Hz, 6H), 2.43 (s, 3H), 3.06e3.11 (m, 2H), 3.23e3.28 (m,
2H), 4.12e4.21 (m, 1H), 6.69 (d, J ¼ 17.4 Hz, 1H), 6.99e7.05 (m, 2H),
7.26e7.29 (m, 1H), 7.53e7.55 (m, 1H), 7.61 (d, J ¼ 15.6 Hz, 1H), 7.79
(d, J ¼ 8.1 Hz, 2H), 7.92 (d, J ¼ 8.4 Hz, 2H), 9.90 (s, 1H). 13C NMR
(300 MHz, CD3OD): 10.05, 19.96, 27.09, 43.22, 49.69, 107.24, 110.03,
116.91, 118.24, 120.03, 120.25, 127.28, 128.00, 128.37, 131.90, 138.15,
138.83, 141.50, 164.06. MS (ESI) m/z: 442.2 (MþHþ). HRMS (ESI) for
J ¼ 7.5 Hz, 2H), 3.20 (t, J ¼ 6.9 Hz, 2H), 3.68e3.69 (d, J ¼ 1.5 Hz, 3H),
6.55 (d, J ¼ 16.2 Hz, 1H), 6.87 (s, 1H), 6.97 (t, J ¼ 6.9 Hz, 1H), 7.12 (t,
J ¼ 8.1 Hz, 1H), 7.25 (d, J ¼ 8.1 Hz, 1H), 7.38 (d, J ¼ 8.1 Hz, 1H),
7.55e7.59 (m, 3H), 7.71 (d, J ¼ 8.4 Hz, 2H). 13C NMR (300 MHz,
CD3OD): 25.22, 35.18, 43.46, 108.78, 110.46, 117.90, 118.22, 120.03,
120.98, 126.79, 126.92, 127.56, 127.63, 134.15, 138.35, 138.45, 141.25
MS (ESI) m/z: 400.1 (MþHþ). HRMS (EI) for C20H22N3O4S (MþHþ):
calcd, 400.1331; found, 400.1321.
C
23H28N3O4S (MþHþ): calcd, 442.1801; found, 442.1787.
4.1.31. 2-(4-Bromo-benzenesulfonyl)-2,3,4,9-tetrahydro-1H-b-
carboline (45)
4.1.25. 4-Bromo-N-[2-(1H-indol-3-yl)-ethyl]-benzenesulfonamide
(40)
The title compound was obtained in 49% yield by using trypt-
amine and 4-bromobenzenesulfonyl chloride in a manner similar
to that described for the synthesis of 23. 1H NMR (500 MHz, CDCl3):
The title compound was obtained in 95% yield by using 2,3,4,9-
tetrahydro-1H-pyrido[3,4-b]indole and 4-bromobenzenesulfonyl
chloride in a manner similar to that described for the synthesis of
23. 1H NMR (300 MHz, CDCl3):
J ¼ 5.7 Hz, 2H), 4.42 (s, 2H), 7.08e7.21 (m, 2H), 7.30e7.33 (m, 1H),
7.44 (d, J ¼ 7.2 Hz, 1H), 7.64e7.68 (m, 2H), 7.70e7.74 (m, 2H), 7.77 (s,
1H).
d
2.85 (t, J ¼ 5.7 Hz, 2H), 3.54 (t,
d
1.55 (s, 3H), 2.95 (t, J ¼ 6.5 Hz, 2H), 3.27e3.31 (m, 2H), 6.97 (s, 1H),
7.07 (t, J ¼ 7.5 Hz, 1H), 7.20 (t, J ¼ 7.5 Hz, 1H), 7.35 (d, J ¼ 8.5 Hz, 1H),
7.39 (d, J ¼ 7.0 Hz, 1H), 7.51 (d, J ¼ 8.5 Hz, 2H), 7.55 (d, J ¼ 8.5 Hz,
2H), 8.02 (s, 1H).
4.1.32. 3-[4-(1,3,4,9-Tetrahydro-b-carboline-2-sulfonyl)-phenyl]-
acrylic acid methyl ester (46)
4.1.26. 4-Bromo-N-[2-(1-methyl-1H-indol-3-yl)-ethyl]-
benzenesulfonamide (41)
The title compound was obtained in 53% yield by using N-
methyltryptamine and 4-bromobenzenesulfonyl chloride, in a
manner similar to that described for the synthesis of 23. 1H NMR
The title compound was obtained in 76% yield from compound
45 in a manner similar to that described for the synthesis of 25. 1H
NMR (300 MHz, CD3OD):
d
2.21 (t, J ¼ 5.1 Hz, 2H), 2.93 (t, J ¼ 5.4 Hz,
2H), 3.21 (s, 3H), 3.81 (s, 2H), 5.89 (d, J ¼ 15.9 Hz, 1H), 6.42 (t,
J ¼ 7.5 Hz, 1H), 6.50 (t, J ¼ 7.8 Hz, 1H), 6.69 (d, J ¼ 7.8 Hz, 1H),
7.02e7.08 (m, 3H), 7.23 (d, J ¼ 8.4 Hz, 2H).
(300 MHz, CDCl3):
(s, 3H), 6.83 (s, 1H), 7.05e7.10 (m, 1H), 7.22e7.32 (m, 3H), 7.37e7.40
(m, 1H), 7.51e7.55 (m, 4H).
d
2.95 (t, J ¼ 6.3 Hz, 2H), 3.26e3.32 (m, 2H), 3.75
4.1.33. N-hydroxy-3-[4-(1,3,4,9-tetrahydro-b-carboline-2-
sulfonyl)-phenyl]-acrylamide (19)
4.1.27. 3-{4-[2-(1H-indol-3-yl)-ethylsulfamoyl]-phenyl}-acrylic
acid tert-butyl ester (42)
The title compound was obtained in 26% over all yield from
compound 46 in a manner similar to that described for the syn-
thesis of 9; mp: 193e194 ꢁC (decompose). 1H NMR (300 MHz,
The title compound was obtained in 23% yield from compound
40 in a manner similar to that described for the synthesis of 25. 1H
DMSO-d6):
d
2.73 (bs, 2H), 3.45 (t, J ¼ 5.4 Hz, 2H), 4.32 (s, 2H), 6.57
NMR (300 MHz, CD3OD):
d
1.55 (s, 9H), 2.85e2.90 (m, 2H),
(d, J ¼ 15.9 Hz, 1H), 6.94 (t, J ¼ 7.2 Hz, 1H), 7.03 (t, J ¼ 8.1 Hz, 1H),
7.28e7.36 (m, 2H), 7.50 (d, J ¼ 15.6 Hz, 1H), 7.78 (d, J ¼ 8.4 Hz, 2H),
7.85 (d, J ¼ 8.1 Hz, 2H), 9.13 (bs, 1H), 10.79e10.85 (m, 2H). 13C NMR
(300 MHz, CD3OD): 26.02, 35.81, 46.18, 111.21, 117.86, 119.18, 121.63,
3.17e3.22 (m, 2H), 6.54 (d, J ¼ 16.2 Hz, 1H), 6.91e6.98 (m, 2H),
7.02e7.07 (m, 1H), 7.26e7.29 (m, 1H), 7.36e7.39 (m, 1H), 7.55e7.65
(m, 3H), 7.74 (d, J ¼ 8.7 Hz, 2H).

478
S. Mehndiratta et al. / European Journal of Medicinal Chemistry 85 (2014) 468e479
121.10, 128.34, 128.57, 130.02, 136.95, 138.19, 138.69, 144.49. MS
(ESI) m/z: 398.1 (MþHþ). HRMS (ESI) for C20H20N3O4S (MþHþ):
calcd, 398.1175; found, 398.1167.
mL of penicillin, and 100 mg/mL of streptomycin (Biological In-
dustries, Kibbutz Beit Haemek, Israel) at 37 ꢁC in a humidified at-
mosphere of 5% CO2 in air.
4.1.34. 4-[2-(2-Methyl-1H-indol-3-yl)-ethylsulfamoyl]-benzoic acid
methyl ester (47)
The title compound was obtained in 39% yield by using 2-
methyltryptamine and 4-methoxycarbonylbenzenesulfonyl chlo-
ride in a manner similar to that described for the synthesis of 23. 1H
4.2.1. HeLa nuclear extract HDAC activity assay
The HeLa nuclear extract HDAC activity was measured with the
HDAC Fluorescent Activity Assay Kit (BioVision, CA, USA) according
to manufacturer's instructions. 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 a
further 30 min at 37 ꢁC. Additional negative controls included in-
cubation without the nuclear extract, without the substrate, or
NMR (300 MHz, CDCl3):
d
2.35 (s, 3H), 2.92 (t, J ¼ 6.6 Hz, 2H),
3.25e3.32 (m, 2H), 3.97 (s, 3H), 4.41 (t, J ¼ 6.3 Hz, 1H), 6.99e7.04
(m, 1H), 7.09e7.14 (m, 1H), 7.24e7.30 (m, 3H), 7.75e7.79 (m, 2H),
7.82 (bs, 1H), 8.06 (t, J ¼ 8.4 Hz, 2H).
without both. TSA at 1 mM served as the positive control. A fluo-
4.1.35. 4-[2-(2-Methyl-1H-indol-3-yl)-ethylsulfamoyl]-benzoic acid
(48)
rescence plate reader with excitation at 355 nm and emission at
460 nm was used to quantify HDAC activity.
A mixture of compound 47 (0.40 g, 1.07 mmol), 1 M LiOH(aq)
(4 mL), and dioxane (10 mL) was stirred at 40 ꢁC for 2 h. The re-
action was concentrated under reduced pressure and then was
added water. The mixture was acidified with 3 N HCl to give a white
precipitate. The resulting solid was collected by filtration and was
dried to yield compound 48 as an off-white solid, in 96% yield; 1H
4.2.2. Western blot analysis
RAW 264.7 cells (1 ꢀ106) were treated with test compounds for
24 h, then were incubated for 10 min at 4 ꢁC in lysis buffer (20 mM
HEPES, pH 7.4, 2 mM EGTA, 50 mM
X-100, 10% glycerol, 1 mM DTT, 1
b
-glycerophosphate, 0.1% Triton
m
g/mL of leupeptin, 5 g/mL of
m
NMR (300 MHz, CD3OD):
(m, 2H), 6.87e6.92 (m, 1H), 6.94e6.99 (m, 1H), 7.17 (d, J ¼ 7.2 Hz,
1H), 7.26 (d, J ¼ 8.7 Hz, 1H), 7.77e7.80 (m, 2H), 8.04e8.07 (m, 2H).
d
2.29 (s, 3H), 2.82 (t, J ¼ 7.2 Hz, 2H), 3.12
aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 1 mM sodium
orthovanadate), and then were scraped off, incubated on ice for a
further 10 min, and centrifuged at 17,000 g for 30 min at 4 ꢁC. The
protein concentration was determined using a Bradford protein
4.1.36. N-hydroxy-4-[2-(2-methyl-1H-indol-3-yl)-ethylsulfamoyl]-
benzamide (20)
assay kit (Bio-Rad, Hercules, CA, USA) and protein samples (80 mg)
subjected to 10% sodium dodecyl sulfate-polyacrylamide gel elec-
trophoresis and finally transferred onto a nitrocellulose membrane
(Millipore, Bellerica, MA, USA). The 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 primary antibodies 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 goat
anti-rabbit or anti-mouse IgG antibodies conjugated with horse-
radish (diluted 1:1000) for 1 h at room temperature. Signals were
developed with ECL reagents (Advansta Corp., Menlo Park, CA, USA)
and exposure to photographic film.
A mixture of compound 48 (0.37 g, 1.03 mmol), EDC$HCl (0.24 g,
1.54 mmol), HOBt (0.21 g, 1.54 mmol), NMM (0.30 mL, 2.78 mmol),
and DMF (5 mL) was stirred at room temperature for 30 min before
adding NH2OTHP (0.13 g, 1.13 mmol). After being stirred for a
further 5 h, the reaction mixture was quenched with water and
extracted with EtOAc (25 mL ꢀ 3). The combined organic layer was
dried over anhydrous MgSO4 and was purified by silica gel chro-
matography (EtOAc:n-hexane ¼ 1:1) to give a colorless liquid. The
resulting product was dissolved in CH3OH (10 mL) and was added
10% TFA (5 mL). The reaction was stirred at room temperature for
2 h and was concentrated under reduced pressure to give a white
residue, which was recrystallized from CH3OH to afford the desired
compound 20, in 52% yield (from 48); mp: 167e168 ꢁC. 1H NMR
4.2.3. Nitric oxide (NO) assay
(300 MHz, CD3OD):
d
2.28 (s, 3H), 2.82 (t, J ¼ 7.2 Hz, 2H), 3.10 (t,
RAW 264.7 cells (1 ꢀ 106) were plated and pretreated with the
J ¼ 7.8 Hz, 2H), 6.88e6.93 (m, 1H), 6.94e7.00 (m, 1H), 7.18 (d,
J ¼ 7.5 Hz, 1H), 7.27 (d, J ¼ 8.1 Hz, 1H), 7.79 (s, 4H). 13C NMR
(300 MHz, CD3OD): 10.05, 24.63, 43.38, 106.62, 110.09, 110.14,
116.82, 118.24, 120.07, 126.63, 127.46, 128.23, 128.27, 132.14, 135.53,
135.57, 143.37, 165.29. MS (ESI) m/z: 374.1 (MþHþ). HRMS (ESI) for
indicated concentrations of compound 9 for 1 h, and subjected to
stimulation with LPS (25 ng/mL) for 24 h, and then 100
reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% naph-
thylethylenediamine dihydrochloride) was mixed with 100 L of
mL of Griess
m
the cell supernatant and the optical density at 550 nm was
measured. Nitrite concentration was determined using a dilution of
sodium nitrite as a standard.
C
18H20N3O4S (MþHþ): calcd, 374.1175; found, 374.1153.
4.2. Biology
4.2.4. PGE2 determination
ELISA used in cytokine measurements was acquired from R&D
Systems (Minneapolis, MN, USA). Anti-iNOS was purchased from
Santa Cruz Biotechnology (Santa Cruz, CA, USA), COX-2 and -actin
To investigate the effect of compounds 9e20 and 1$HCl on PGE2
levels in LPS stimulated cells, RAW 264.7 cells (1 ꢀ 106) were
treated in the presence or absence of test compounds for 1 h, and
then stimulated with LPS (25 ng/mL) for 24 h at 37 ꢁC. The con-
centrations of PGE2 in the supernatants of RAW 264.7 cell cultures
were determined using an EIA kit (R&D Systems, Minneapolis, MN,
USA).
b
antibodies were purchased from Epitomics Inc. (Burlingame, CA,
USA); histone H3 and acetyl-histone H3 antibodies were purchased
from Cell Signaling Technology (Danvers, MA, USA) and Millipore
(Temecula, CA, USA), respectively. Horseradish peroxidase-
conjugated goat anti-mouse or anti-rabbit IgG antibodies were
obtained from Jackson ImmunoResearch Inc. (Cambridgeshire, UK).
Murine macrophage cell line RAW 264.7 was obtained from Bio-
resource Collection and Research Center (Hsinchu, Taiwan) and
maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco
Laboratories Inc.) supplemented with 10% (v/v) fetal bovine serum
(FBS; Invitrogen™ Life Technologies, Carlsbad, CA, USA) with 100 U/
4.2.5. IL-6 and TNF-
a determination
To determine the effect of test compounds on the production of
cytokines IL-6 and TNF-
a from LPS-stimulated cells, RAW 264.7
cells (1 ꢀ 106) were plated and pretreated in the presence or
absence of compound 9 and SAHA for 1 h, and then stimulated with
LPS (25 ng/mL) for 24 h at 37 ꢁC. Supernatants were collected and

S. Mehndiratta et al. / European Journal of Medicinal Chemistry 85 (2014) 468e479
479
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4.2.7. Statistical analyses
Results are expressed as the mean
SEM for the indicated
number of separate experiments. Means were checked for statis-
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significant.
Acknowledgments
This research were supported by the Ministry of Science and
Technology, Taiwan (grant no. MOST 103-2113-M-038-001-MY3
and MOST 103-2320-B-002-008-MY3).
Appendix A. Supplementary data
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