Ring-opened tetrahydro-γ-carbolines display cytotoxicity and selectivity with histone deacetylase isoforms

Article abstract of DOI:10.1016/j.ejmech.2016.12.039

This study is focused on modification of the indole moiety and the N1-zinc binding domain of tubastatin A, and the effects of such changes on biological activity. Fourteen N-substituted indoles (5–18) were synthesized and structure-activity relationship studies indicated that the change of the tetrahydro-γ-carboline in tubastatin A led to substituted indoles (compounds 7, 11, and 15) which showed significant improvements of selective inhibition for HDAC6 over HDAC1 and HDAC2 in comparison to ACY1215, a compound undergoing clinical trials. In addition, attachment of different hydroxamic acid groups, the zinc binding motif at the N1 position, contributes to the antiproliferative activity in cancer cells. Several synthetic compounds exhibited potent growth inhibition in a broad spectrum of tumor cell lines, induced irreversible growth arrest capacities by suppressing colony formation ability and activated the apoptosis pathway. The data provide compelling evidence that our newly synthesized compounds with type B to D hydroxamic acid groups as the zinc binding motif at the N1 position are potent selective inhibitors of HDAC6 and could be investigated preclinically as potential anticancer drugs.

Full text of DOI:10.1016/j.ejmech.2016.12.039

European Journal of Medicinal Chemistry 127 (2017) 115e127
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Ring-opened tetrahydro-g-carbolines display cytotoxicity and
selectivity with histone deacetylase isoforms
,
,
Kunal Nepali a 1, Hsueh-Yun Lee a 1, Mei-Jung Lai b, Ritu Ojha a, Tung-Yun Wu c,
Gu-Xian Wu a, Mei-Chuan Chen c *, Jing-Ping Liou a
,
, d, **
a School of Pharmacy, College of Pharmacy, Taipei Medical University, Taiwan
b Center for Translational Medicine, Taipei Medical University, Taiwan
c Ph.D. Program for the Clinical Drug Discovery from Botanical Herbs, College of Pharmacy, Taipei Medical University, Taiwan
d School of Pharmacy, National Defense Medical Center, Taipei, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
This study is focused on modification of the indole moiety and the N1-zinc binding domain of tubastatin
A, and the effects of such changes on biological activity. Fourteen N-substituted indoles (5e18) were
synthesized and structure-activity relationship studies indicated that the change of the tetrahydro-g-
Received 4 October 2016
Received in revised form
17 December 2016
carboline in tubastatin A led to substituted indoles (compounds 7, 11, and 15) which showed significant
improvements of selective inhibition for HDAC6 over HDAC1 and HDAC2 in comparison to ACY1215, a
compound undergoing clinical trials. In addition, attachment of different hydroxamic acid groups, the
zinc binding motif at the N1 position, contributes to the antiproliferative activity in cancer cells. Several
synthetic compounds exhibited potent growth inhibition in a broad spectrum of tumor cell lines, induced
irreversible growth arrest capacities by suppressing colony formation ability and activated the apoptosis
pathway. The data provide compelling evidence that our newly synthesized compounds with type B to D
hydroxamic acid groups as the zinc binding motif at the N1 position are potent selective inhibitors of
HDAC6 and could be investigated preclinically as potential anticancer drugs.
Accepted 19 December 2016
Available online 21 December 2016
Keywords:
Anticancer agents
Histone deacetylase inhibitors
© 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction
trials [8]. ACY1215 (1) was developed as an HDAC6 inhibitor and, in
combination with bortezomib, has shown potent inhibitory activity
Histone deacetylase (HDAC) is considered as a potential target
for regulation of epigenetic aberrance [1]. HDAC inhibitors have
been used mainly in treatment of cancer and since 2007, SAHA,
PXD101, FK228, and LBH589 have been approved by US FDA for
cancer treatment [2e5]. Eighteen iso-types of HDAC and their
functions have been identified which are grouped into four classes
(I, II, III, and IV) based on distribution and homology with respect to
the original yeast enzymes [6,7]. Histone deacetylase 6 (HDAC6) has
been recognized as a promising target for several disorders such as
tumorigenesis, inflammation, and neurodegeneration. Currently,
two HDAC6 inhibitors, ACY1215 (1) and ACY241 (2), are in clinical
against multiple myeloma [9]. ACY241 (2) has been used alone and
in combination with pomalidomide and dexamethasone to treat
multiple myeloma in a clinical study. Significant attention is now
being paid to the development of selective HDAC6 inhibitors
(Fig. 1).
Tubastatin A (3) was identified as a potent HDAC6 inhibitor with
neuroprotective functions (Fig. 1) [10]. HDAC6 belongs to class II
HDAC which requires the assistance of zinc ion in its catalytic
process. The structure of zinc-dependent HDAC inhibitors com-
prises three domains: cap, linker group, and zinc binding domain
[11]. The 3-methyl-tetrahydro-g-carboline and hydroxamic acid of
tubastatin A function as cap group and zinc binding domain,
respectively. Second-generation analogs of tubastatin A with
diverse substituents at the indole nitrogen (N3) of the tetrahydro-
* Corresponding author.
** Corresponding author. School of Pharmacy, College of Pharmacy, Taipei Medical
University, Taiwan.
g
-carboline display improved selective HDAC6 activity and show an
immunosuppressive effect with Foxp3þ regulatory T cells [12].
Shen et al. replaced the 3-methyl-tetrahydro- -carboline of com-
pound 3 with bicyclic heterocyclic units without changing the
g
E-mail addresses: mcchen1250@tmu.edu.tw (M.-C. Chen), jpl@tmu.edu.tw
(J.-P. Liou).
1
Contributed equally to this work.
http://dx.doi.org/10.1016/j.ejmech.2016.12.039
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

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K. Nepali et al. / European Journal of Medicinal Chemistry 127 (2017) 115e127
Fig. 1. Structures of selective HDAC6 inhibitors.
linker and the zinc binding domain which resulted in compounds
with improved HDAC6 selectivity [13]. Our previous study on
azaindolylsulfonamides identified compound 4 as a potent anti-
cancer agent with selective HDAC6 activity (Fig. 1) [14]. Literature
surveys [12e14] led us to modify the cap of compound 3 using a
ring-opening strategy to provide compounds with N,N-dimethyl-1-
2. Results and discussion
2.1. Chemistry
Scheme 1 describes the synthetic approach to compounds 5 and
6. Substitution of 3-methyl-tetrahydro-g-carboline (19) obtained
(2-methyl-1H-indol-3-yl)methanamine,
dimethylmethanamine, or
1-(1H-indol-3-yl)-N,N-
N,N-dimethyl-1H-indole-3-
based on Butler's methodology [10] was carried out using NaH and
3-bromobenzenesulfonyl chloride or 1-bromo-4-(bromomethyl)
benzene to give 20a or 20b. The Heck reaction between 20a or 20b
and methyl acrylate gave the corresponding cinnamates (21a, 21b).
The resulting products were hydrolyzed in the presence of LiOH to
yield the carboxylic acid (22a, 22b) which was subjected to ami-
dation with NH2OTHP and subsequent deprotection with TFA to
carboxamide as cap regions in this study. In addition, four types
of zinc binding motifs (A-D in Fig. 2) were attached to previously
studied caps. As a result, compounds 5e18 were developed and
their structure-activity relationships has been established in this
paper.
Fig. 2. Designed compounds 5e18.

K. Nepali et al. / European Journal of Medicinal Chemistry 127 (2017) 115e127
117
Scheme 1. Reagents and conditions: (a) 3-bromobenzenesulfonyl chloride or 1-bromo-4-(bromomethyl)benzene, NaH, DMF, rt; (b) Pd2(dba)3, [(t-Bu)3P]BF4, K2CO3, TEA, DMF,
100 ꢀC; (c) 1M LiOH(aq), p-dioxane, 40 ꢀC; (d) i. NH2OTHP, EDC.HCl, HOBt, DIPEA, DMF, rt; ii. 10% TFA(aq), MeOH, rt.
give the anticipated hydroxamic acids (5, 6). Scheme 2 shows the
preparation of compounds 8, 9, 12, 13, 16, and 17. Substitution of
commercially available 3-formylindoles (23, 24) followed by
reductive amination with dimethylamine in the presence of NaB-
H(OAc)3 gave compounds 26a-26d. Reaction of 25 which is ob-
tained based on Acheson's methodology [15] with the appropriate
Scheme 2. Reagents and conditions: (a) For 26a-d: i. bromobenzenesulfonyl chlorides, NaH, DMF, rt; ii. dimethylamine, NaBH(OAc)3, CH2Cl2, rt. For 26e-f: bromobenzenesulfonyl
chlorides, NaH, DMF, rt; (b) Pd2(dba)3, [(t-Bu)3P]BF4, K2CO3, TEA, DMF, 100 ꢀC; (c) 1M LiOH(aq), p-dioxane, 40 ꢀC; (d) i. NH2OTHP, EDC.HCl, HOBt, DIPEA, DMF, rt; ii. 10% TFA(aq),
MeOH, rt.

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K. Nepali et al. / European Journal of Medicinal Chemistry 127 (2017) 115e127
bromobenzenesulfonyl chlorides yielded compounds 26e and 26f.
The subsequent Heck reaction of 26a-f with methyl acrylate in the
presence of Pd2(dba)3 and [(t-Bu)3P]BF4 yielded the cinnamates
27a-f which were hydrolyzed to obtain the corresponding car-
boxylic acids 28a-f. The resulting products subsequently under-
went amidation with NH2OTHP and TFA-mediated deprotection to
afford the anticipated hydroxamic acids (8, 9, 12, 13, 16, and 17). The
synthesis of 10, 14, and 18 is shown in Scheme 3. Compounds 23
and 24 underwent substitution with 4-bromobenzyl bromide fol-
lowed by reductive amination with dimethylamine to afford 29a-b.
Reaction of 25 with 4-bromobenzyl bromide yielded compound
29c. The resulting products were developed via a synthetic route
similar to that shown in Scheme 2, i.e. Heck reaction, ester hydro-
lysis, amidation with NH2OTHP, and deprotection with TFA, to yield
the designed hydroxamic acids 10, 14, and 18. Compounds 7, 11, and
15 lacking a C¼C bond between the hydroxamic acid group and the
phenyl ring was synthesized based on the approach shown in
Scheme 4. Substitution of 23e25 with methyl 4-(bromomethyl)
benzoate was carried out in the presence of NaH to generate the
benzoates 32a-c, which were subjected to hydrolysis by LiOH to
afford the corresponding carboxylic acids 33a-c. Compounds 33a
and 33b underwent amidation with NH2OBn, reductive amination
with dimethylamine, and debenzylation, which yielded the
designed compounds 7 and 11. Amidation of compound 33c with
NH2OBn followed by debenzylation furnished the hydroxamic acid
15.
negligible antiproliferative activity against tested cancer cell lines.
HCT116 cells were more sensitive than A549 and PC3 cells to
compounds 5e18 (Figs. 3e5). The data also demonstrated that
compounds with a B-, C-and D-type hydroxamic acid group have
antiproliferative activity more potent than the compounds with an
A-type hydroxamic acid group, indicating the replacement of the
type A moiety of compound 3 with B- and D-type hydroxamic acid
(5 and 6) unit led to improvement of the in vitro antitumor activity.
Compounds 5 and 6 inhibited the growth of HCT116 cells with GI50
values of 0.64 and 0.73 mM, respectively. The attachment of type B,
C or D hydroxamic acid groups to N,N-dimethyl-1-(2-methyl-1H-
indol-3-yl)methanamine (8e10), 1-(1H-indol-3-yl)-N,N-dimethyl-
methanamine (12e14), or N,N-dimethyl-1H-indole-3-carboxamide
(16e18) also led to enhancement of the antiproliferative activity as
compared to compounds bearing A-type hydroxamic acid group (7,
11, and 15). This result indicated that an N1 hydroxamic acid moiety
significantly affects the cellular activity of this series of compounds.
The change from rigid tetrahydro-g-carboline to flexible
substituted indoles on the other hand marginally influenced the
cellular activity. The antiproliferative activities in three different
cell lines (Figs. 3e5) of compounds 5, 8, 12, and 16 revealed that the
N,N-dimethyl-1H-indole-3-carboxamide structure probably results
in decrease of cellular activity. It could also be seen that the N,N-
dimethyl-1-(2-methyl-1H-indol-3-yl)methanamine and 1-(1H-
indol-3-yl)-N,N-dimethylmethan-amine moieties favor the B- and
C-type hydroxamic acids, while N,N-dimethyl-1H-indole-3-
carboxamide favors the D-type hydroxamic acid. Collectively, the
data suggest that the hydroxamic acid groups at N1 position of
substituted indoles play crucial roles in determination of the
expression of cytotoxicity.
2.2. Biological evaluation
2.2.1. In vitro cell growth inhibitory activity
The ability of synthetic compounds (5e18) to inhibit the growth
of human colorectal HCT116 cells (Fig. 3), human prostate cancer
PC3 cells (Fig. 4), and lung adenocarcinoma A549 cells (Fig. 5), was
evaluated using compound 3 as a reference compound. The results
indicated that compounds with hydroxyaminocarbonyl-p-benzyl
group (type A hydroxamic acid group; 3, 7, 11, and 15) exhibited
Based on these results, we chose several compounds from B-
type hydroxamic acid moiety (8 and 12), C-type hydroxamic acid
moiety (9 and 13), and D-type hydroxamic acid moiety (18) to
investigate their effect on the colony forming ability of
HCT116 cells. As shown in Fig. 6, the selected compounds show a
concentration-dependent decrease in colony forming numbers,
Scheme 3. Reagents and conditions: (a) For 29a-b: i. 4-bromobenzyl bromide, NaH, DMF, rt; ii. dimethylamine, NaBH(OAc)3, CH2Cl2, rt. For 29c: methyl 4-(bromomethyl)benzoate,
NaH, DMF, rt; (b) Pd2(dba)3, [(t-Bu)3P]BF4, K2CO3, TEA, DMF, 100 ꢀC; (c) 1M LiOH(aq), p-dioxane, 40 ꢀC; (d) i. NH2OTHP, EDC.HCl, HOBt, DIPEA, DMF, rt; ii. 10% TFA(aq), MeOH, rt.

K. Nepali et al. / European Journal of Medicinal Chemistry 127 (2017) 115e127
119
Scheme 4. Reagents and conditions: (a) methyl 4-(bromomethyl)benzoate, NaH, DMF, rt; (b) 1 M LiOH(aq), p-dioxane, 40 ꢀC; (c) For 7and 11: i. NH2OBn.HCl, EDC.HCl, HOBt, DIPEA,
DMF, rt; ii. dimethylamine, NaBH(OAc)3, CH2Cl2, rt; 10% Pd/C, H2, MeOH, rt. For 15: i. NH2OBn.HCl, EDC.HCl, HOBt, DIPEA, DMF, rt; ii. 10% Pd/C, H2, MeOH, rt.
Fig. 3. Antiproliferative activity (GI50) of tested compounds against human colorectalHCT116 cells.
indicating the potential of these compounds to induce irreversible
growth arrest capacity in HCT116 cells.
selectivity than our previously synthesized compound 4. This result
indicates that the A-type hydroxamic acid plays a vital role,
contributing to the HDAC6 selectivity. In addition, the strategy of
ring-opening of the tetrahydro-g-carboline of compound 3 pro-
2.2.2. HDAC isoform inhibition
vides potent selective HDAC6 inhibitors. Although replacement of
A-type hydroxamic acid moieties with B-, C-, and D-types resulted
in a decrease in HDAC6 selectivity, most of this series of compounds
showed better HDAC6 selectivity than ACY1215. Taken together the
results of cellular and enzymatic activity, it is assumed that A-type
hydroxamic acid is significant for HDAC6 selectivity while it causes
loss of antiproliferative activity. In addition, B-, C-, and D-types of
In order to examine the HDAC6 selective inhibitory activity of
our compounds, all the synthetic compounds (5e18), compound 4,
and tubastatin A (3) were assayed for their activities on HDAC1,
HDAC2, and HDAC6 (Table 1). Compounds 7 and 15 showed slightly
more selective HDAC6 activity than compound 3, and compound 11
displayed selective HDAC6 activity comparable to that of compound
3. Compounds 7, 11, and 15 showed remarkably more HDAC6

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K. Nepali et al. / European Journal of Medicinal Chemistry 127 (2017) 115e127
Fig. 4. Antiproliferative activity (GI50) of tested compounds against human prostate cancer PC3 cells.
Fig. 5. Antiproliferative activity (GI50) of tested compounds against lung carcinoma A549 cells.
hydroxamic acid are beneficial for cellular activity and have slight
repression by these compounds, the effects of compounds on cell
effect on HDAC6 selectivity.
cycle progression were assessed by flow cytometry. As shown in
Fig. 7B and C, treatment with 1.25 mM or 2.5 mM of the selected
compounds for 48 h caused accumulation of HCT116 cells subG1
phase in a dose-dependent manner and exposure to higher con-
centrations (5 mM) of compound 3 for 48 h had a similar effect
2.2.3. Western blotting and flow cytometry
The biomarkers of HDAC inhibition are acetylation of histone
and non-histone proteins. Class I HDAC enzymatic inhibition results
in hyperacetylation of histone H3 and HDAC6 inhibition could lead
to an increase in acetylated tubulin. To confirm the HDAC isozyme
selectivity, we performed western blot analysis to evaluate the
protein levels of HDAC inhibition biomarkers, acetyl-histone H3
and acetyl-tubulin, in HCT116 cells treated with different com-
pounds. The synthetic compounds (8, 9, 12, 13, and 18), which have
been evaluated in previous colony formation assay, exhibited
stronger inhibition of the HDAC6 enzyme, resulting in upregulation
of acetyl-tubulin without significant induction of acetyl-histone H3
(Fig. 7A).
(Fig. 7B), indicating that synthetic compounds (8, 9, 12, 13, and 18)
exerted a more potent cell cycle deregulation effect than tubastatin
A (3). In addition, the selected compounds (8, 9, 12, 13, and 18) also
activate apoptotic markers PARP and caspase 3 whereas tubastatin
A failed to cause apparent cytotoxicity to HCT116 cells (Fig. 7D).
These results indicated that the novel synthetic compounds (8, 9,
12,13, and 18) exhibit a broad spectrum of antitumor activities with
selectivity for HDAC6 over HDAC1 and HDAC2.
To investigate the underlying mechanism of cell growth

K. Nepali et al. / European Journal of Medicinal Chemistry 127 (2017) 115e127
121
Fig. 6. Colony formation ability of compounds in HCT116 cells. (A) The compounds selected suppressed colony formation and the numbers of colonies were determined and are
shown in (B). Clonogenic survival was assessed as described in the Experimental section.
Table 1
HDAC inhibition Activity and isoform selectivity of compounds 4e18.
compd
IC50(M SD)a
Selectivity ratio
HDAC1/HDAC6
HDAC1
HDAC2
HDAC6
HDAC2/HDAC6
4b
5
6
7
8
9
10
11
12
13
14
15
16
17
18
3.10 ꢁ 10ꢃ7
2.70 ꢁ 10ꢃ6
1.60 ꢁ 10ꢃ6
9.73 ꢁ 10ꢃ6
1.85 ꢁ 10ꢃ6
4.01 ꢁ 10ꢃ6
2.13 ꢁ 10ꢃ6
7.13 ꢁ 10ꢃ6
1.51 ꢁ 10ꢃ6
1.93 ꢁ 10ꢃ6
2.25 ꢁ 10ꢃ6
5.72 ꢁ 10ꢃ6
6.91 ꢁ 10ꢃ6
2.01 ꢁ 10ꢃ6
6.27 ꢁ 10ꢃ7
>1.0 ꢁ 10ꢃ5
0.058
1.16 ꢁ 10ꢃ6
3.13 ꢁ 10ꢃ6
1.65 ꢁ 10ꢃ6
>1.0 ꢁ 10ꢃ5
1.34 ꢁ 10ꢃ6
1.27 ꢁ 10ꢃ6
2.38 ꢁ 10ꢃ6
1.22 ꢁ 10ꢃ5
1.64 ꢁ 10ꢃ6
9.70 ꢁ 10ꢃ7
1.12 ꢁ 10ꢃ6
>1.0 ꢁ 10ꢃ5
>1.0 ꢁ 10ꢃ5
1.46 ꢁ 10ꢃ6
1.04 ꢁ 10ꢃ6
>1.0 ꢁ 10ꢃ5
0.048
5.20 ꢁ 10ꢃ9
1.89 ꢁ 10ꢃ7
4.18 ꢁ 10ꢃ8
6.17 ꢁ 10ꢃ9
8.49 ꢁ 10ꢃ8
6.30 ꢁ 10ꢃ8
9.64 ꢁ 10ꢃ8
2.17 ꢁ 10ꢃ8
8.93 ꢁ 10ꢃ8
1.67 ꢁ 10ꢃ8
4.31 ꢁ 10ꢃ8
8.01 ꢁ 10ꢃ9
2.01 ꢁ 10ꢃ7
1.08 ꢁ 10ꢃ8
8.50 ꢁ 10-9
1.37 ꢁ 10ꢃ8
0.0047
60
14
38
1576
21
63
22
328
16
115
52
714
34
186
73
>729
12
223
16
39
>1620
15
20
24
562
18
58
25
>1248
>49
135
122
>729
10
Tubastatin A
ACY1215c
a
These assays were conducted by the Reaction Biology Corporation, Malvern, PA. All compounds were dissolved in DMSO and tested in 10-dose IC50mode with 3-fold serial
dilution starting at 10
m
M.
b
Data from ref [14].
c
Datafrom ref [9].
3. Conclusion
4. Experimental section
This study modified the cap of tubastatin A using a ring-opening
strategy, which gave three substituted indoles (N,N-dimethyl-1-(2-
methyl-1H-indol-3-yl)methanamine, 1-(1H-indol-3-yl)-N,N-dime-
thylmethanamine, and N,N-dimethyl-1H-indole-3-carboxamide).
Meanwhile, four types of zinc binding moieties (A-D) were bound
to the preceding compounds, and their biological effects were
examined. Structure-activity relationship studies revealed that
compounds 7, 11, and 15 with substituted indoles as the cap motif
also showed selective HDAC6 activity, comparable to that of
tubastatin A (3). The replacement of A-type hydroxamic acid group
with B-, C-, or D-type groups led to enhancement of anti-
proliferative activity; for example, in compounds 8, 9,12, 13, and 18,
and activation of apoptosis. All compounds in this study displayed
better HDAC6 selectivity than the compound in clinical trials
(ACY1215). This study led to novel HDAC inhibitors exhibiting not
only potent antiproliferative activity but also HDAC6 selectivity.
4.1. Chemistry
Nuclear magnetic resonance (1H and 13CNMR) spectra were
obtained with Bruker Fourier 300 NMR spectrometers, and are
reported as chemical shifts in parts per million (ppm, d) downfield
from TMS as an internal standard. High-resolution mass spectra
(HRMS) were recorded with a Finnigan MAT 95S mass spectrom-
eter. The purities of the final compounds were determined using a
Hitachi 2000 series HPLC system using an Agilent Zorbax Eclipse
XDB-C18 column (5
m
m, 4.6 mm ꢁ 150 mm) with the solvent system
consisting of acetonitrile (mobile phase A) and water containing
0.1% formic acid and 10 mmol NH4OAc (mobile phase B), and were
found to be ꢂ 95%. Flash column chromatography was performed
using silica gel (Merck Kieselgel 60, no. 9385, 230e400 mesh
ASTM). All reactions were carried out under an atmosphere of dry
nitrogen.

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K. Nepali et al. / European Journal of Medicinal Chemistry 127 (2017) 115e127
Fig. 7. Effect of selected compounds on HDAC inhibition biomarkers, cell cycle distribution, and activation of apoptosis. (A)Accumulation of acetyltubulin after 6 h treatment with
selected compounds. (B) Sub-G1 phase induction and (C) the mean percentage of subG1 fraction populations with 1.25 M and 2.5 M of compounds for 48 h in HCT116 cells. (D)
m
m
Apoptosis induction with different concentrations of compounds for 48 h in HCT116 cells. Cells were treated with indicated concentrations for indicated times, then assessed by flow
cytometry or western blot analysis as described in Materials and Methods.
4.1.1. N-Hydroxy-3-[3-(2-methyl-1,2,3,4-tetrahydro-pyrido[4,3-b]
indole-5-sulfonyl)-phenyl]-acrylamide (5)
410.1175; found, 410.1171.
A mixture of compound 22a (0.55 g, 1.4 mmol), EDC.HCl (0.32 g,
2.1 mmol), HOBt (0.22 g, 1.7 mmol) and DIPEA (0.48 mL, 2.8 mmol)
in DMF (5.0 mL) was stirred at room temperature for 30 min before
adding NH2OTHP (0.19 g, 1.7 mmol). After being stirred for a further
5 h, the reaction mixture was quenched with water and extracted
with EtOAc (50 mL x 3). The combined organic layer was dried over
anhydrous MgSO4 and was purified by silica gel chromatography
(EtOAc) to give a semisolid residue. The resulting product was
dissolved in CH3OH (10 mL) and to this was added 10% TFA(aq)
(5 mL). The reaction was stirred at room temperature for 5 h and
the reaction mixture was concentrated under reduced pressure to
give a white precipitate which was recrystallized from CH3OH to
4.1.2. (E)-3-(4-((1,2,3,4-Tetrahydro-2-methylpyrido[4,3-b]indol-5-
yl)methyl)phenyl)-N-hydroxyacrylamide (6)
The title compound was obtained in 51% yield from compound
22b in a manner similar to that described for the synthesis of
compound 5; HPLC purity: 97.56% (tR
¼
16.25 min). mp:
2.79 (s, 3H), 2.98 (t,
213e214 ꢀC. 1HNMR (300 MHz, CD3OD)
d
J ¼ 6.0 Hz, 2H), 3.23 (t, J ¼ 6.0 Hz, 2H), 4.09 (s, 2H), 5.37 (s, 2H), 6. 42
(d, J ¼ 15.9 Hz, 1H), 7.04 (d, J ¼ 8.1 Hz, 2H), 7.08e7.17 (m, 2H), 7.32
(d, J ¼ 7.5 Hz, 1H), 7.44e7.54 (m, 4H). 13CNMR (75 MHz, DMSO-d6)
21.27, 40.91, 46.41, 47.17, 48.72, 111.73, 116.48, 120.09, 121.79, 122.76,
124.67, 125.18, 128.48, 129.94, 130.67, 132.98, 133.73, 134.48, 135.76,
159.75. HRMS (ESI) for C22H22N3O2 (M-Hþ): calcd, 360.1712; found,
360.1707.
afford compound
5
in 52% yield; HPLC Purity: 97.98%
(tR ¼ 19.26 min). mp: 224e225 ꢀC. 1HNMR (300 MHz, DMSO-d6)
d
2.52 (s, 3H), 2.73 (bs, 2H), 3.18 (bs, 2H), 4.02 (s, 2H), 6.63 (d,
4.1.3. 4-((3-((Dimethylamino)methyl)-2-methyl-1H-indol-1-yl)
methyl)-N-hydroxybenzamide (7)
J ¼ 15.9 Hz, 1H), 7.29 (t, J ¼ 7.5 Hz 1H), 7.38 (t, J ¼ 7.5 Hz, 1H),
7.45e7.54 (m, 2H), 7.60 (t, J ¼ 8.1 Hz, 1H), 7.88 (d, J ¼ 8.1 Hz, 2H),
8.04 (d, J ¼ 8.4 Hz, 2H), 9.18 (s, 1H, NH, D2O exchangeable proton),
10.92 (s, 1H, NH, D2O exchangeable proton). 13CNMR (75 MHz,
To the stirred solution of compound 33a (0.5 g, 1.2 mmol) in
CH3OH (10 mL) was added a catalytic amount of 10% palladium on
carbon and the reaction mixture was stirred at room temperature
for 6 h under hydrogen. The reaction mixture was filtered over
celite and the filtrate was dried in vacuum to afford compound 7 in
75% yield; HPLC purity: 95.05% (tR ¼ 19.04 min). mp: 130e131 ꢀC.
DMSO-d6) d 20.93, 43.15, 49.26, 50.81, 113.72, 118.44, 122.17, 123.74,
124.67, 125.17, 126.63, 127.29, 130.47, 132.12, 134.67, 134.98, 135.59,
136.46, 137.78, 161.73. HRMS (ESI) for C21H20N3O4S (M-Hþ): calcd,

K. Nepali et al. / European Journal of Medicinal Chemistry 127 (2017) 115e127
123
1HNMR (300 MHz, DMSO-d6)
d
2.18 (s, 6H), 2.33 (s, 3H), 3.57 (s, 2H),
1HNMR (300 MHz, CD3OD)
d
2.52 (s, 6H), 4.00 (s, 2H), 6.55 (d,
5.48 (s, 2H), 6.99e7.01 (m, 4H), 7.34 (m, 1H), 7.57 (dd, J ¼ 3.0, 6.9 Hz,
J ¼ 15.9 Hz, 1H), 7.33 (t, J ¼ 7.5 Hz, 1H), 7.42 (t, J ¼ 8.1 Hz, 1H),
7.52e7.58 (m, 2H), 7.71 (d, J ¼ 7.8 Hz,1H), 7.80e7.85 (m, 2H), 7.94 (d,
J ¼ 7.2 Hz,1H), 8.06 (d, J ¼ 8.4 Hz, 1H), 8.11 (s, 1H). 13CNMR (75 MHz,
1H), 7.66 (d, J ¼ 8.1 Hz, 2H). 13CNMR (75 MHz, DMSO-d6)
d 7.98,
42.15, 43.31, 50.60, 105.70, 107.14, 116.21, 116.81, 118.41, 123.44,
123.78, 126.03, 127.33, 129.52, 133.18, 133.79, 139.49, 161.67. HRMS
(ESI) for C20H22N3O2 (M-Hþ): calcd, 336.1712; found, 336.1709.
CD3OD)
d 40.27, 49.19, 110.79, 113.57, 116.96, 117.66, 121.01, 122.44,
122.83, 124.55, 124.63, 127.25, 127.69, 129.82, 132.44, 133.87, 134.79,
135.74, 161.19. HRMS (ESI) for
398.1175; found, 398.1167.
C
20H20N3O4S (M-Hþ): calcd,
4.1.4. 3-[3-(3-Dimethylaminomethyl-2-methyl-indole-1-sulfonyl)-
phenyl]-N-hydroxy-acrylamide (8)
The title compound was obtained in 58% yield from compound
28a in a manner similar to that described for the synthesis of
compound 5. HPLC purity: 97.18% (tR ¼ 14.51 min). mp: 180e181 ꢀC.
4.1.9. 3-[4-(3-Dimethylaminomethyl-indole-1-sulfonyl)-phenyl]-N-
hydroxy-acrylamide (13)
The title compound was obtained in 58% yield from compound
28d in a manner similar to that described for the synthesis of
1HNMR (300 MHz, CD3OD)
d 2.76 (s, 3H), 2.81 (s, 6H), 4.41 (s, 2H),
6.53 (d, J ¼ 15.6 Hz, 1H), 7.36e7.47 (m, 2H), 7.51e7.68 (m, 2H), 7.69
compound 5; HPLC purity: 98.66% (tR
¼
18.40 min). mp:
(d, J ¼ 6.9 Hz, 1H), 7.84e7.85 (m, 2H), 8.02 (bs, 1H), 8.27 (d,
179e180 ꢀC. 1HNMR (300 MHz, CD3OD)
d
2.58 (s, 6H), 4.08 (s, 2H),
J ¼ 8.1 Hz, 1H). 13CNMR (75 MHz, CD3OD)
d
9.65, 39.34, 47.59,
6.56 (d, J ¼ 15.6 Hz,1H), 7.31 (t, J ¼ 7.5 Hz,1H), 7.39 (t, J ¼ 7.2 Hz,1H),
7.47 (d, J ¼ 15.6 Hz, 1H), 7.62 (d, J ¼ 8.1 Hz, 2H), 7.71 (d, J ¼ 7.8 Hz,
1H), 7.88 (s, 1H), 7.93 (d, J ¼ 8.1 Hz, 2H), 8.01 (d, J ¼ 8.4 Hz, 1H).
108.30, 111.74, 115.74, 117.72, 121.45, 122.24, 122.42, 124.11, 126.46,
127.50, 129.88, 133.39, 133.97, 134.73, 136.45, 136.55, 161.09. HRMS
(ESI) for C21H22N3O4S (M-Hþ): calcd, 412.1331; found, 412.1329.
13CNMR (75 MHz, DMSO-d6)
d 41.20, 49.51, 111.03, 118.43, 121.23,
121.47, 123.07, 125.05, 125.90, 126.36, 127.06, 128.02, 132.13, 133.65,
134.34, 138.72, 164.86. HRMS (ESI) for C20H20N3O4S (M-Hþ): calcd,
398.1175; found, 398.1168.
4.1.5. 3-[4-(3-Dimethylaminomethyl-2-methyl-indole-1-sulfonyl)-
phenyl]-N-hydroxy-acrylamide (9)
The title compound was obtained in 58% yield from compound
28b in a manner similar to that described for the synthesis of
compound 5. HPLC purity: 98.07% (tR ¼ 17.04 min). mp: 191e192 ꢀC.
4.1.10. (E)-3-(4-((3-((Dimethylamino)methyl)-1H-indol-1-yl)
methyl)phenyl)-N-hydroxyacrylamide (14)
The title compound was obtained in 55% yield from compound
31b in a manner similar to that described for the synthesis of
1HNMR (300 MHz, DMSO-d6)
d
2.71 (s, 9H), 4.39 (s, 2H), 6.59 (d,
J ¼ 15.9 Hz, 1H), 7.36e7.41 (m, 2H), 7.46 (d, J ¼ 15.9 Hz, 1H), 7.75 (d,
J ¼ 8.4 Hz, 2H), 7.80 (d, J ¼ 7.2 Hz,1H) 7.93 (d, J ¼ 8.4 Hz, 2H), 8.11 (d,
J ¼ 7.5 Hz, 1H), 9.17 (s, 1H, D2O exchangeable proton), 10.95 (s, 1H,
compound 5; HPLC purity: 99.14% (tR
¼
19.14 min). mp:
168e169 ꢀC. 1HNMR (300 MHz, CD3OD)
d
2.86 (s, 6H), 4.50 (s, 2H),
D2O exchangeable proton). 13CNMR (75 MHz, CD3OD)
d
9.84, 39.25,
5.46 (s, 2H), 6.48 (d, J ¼ 15.9 Hz, 1H), 7.18e7.24 (m, 4H), 7.41 (d,
J ¼ 6.9 Hz, 1H), 7.46e7.54 (m, 3H), 7.62 (s, 1H), 7.77 (dd, J ¼ 2.7,
47.43, 107.57, 111.69, 112.27, 115.84, 116.16, 118.96, 121.51, 122.26,
124.13, 125.74 126.41, 133.20, 134.79, 135.60, 136.95, 138.05, 158.88.
HRMS (ESI) for C21H22N3O4S (M-Hþ): calcd, 412.1331; found,
412.1330.
6.3 Hz, 1H). 13CNMR (75 MHz, CD3OD)
d 38.49, 46.67, 49.30, 100.58,
107.68, 114.68, 115.51, 117.83, 119.83, 124.12, 124.39, 125.14, 125.20,
128.77, 131.73, 134.00, 136.85, 161.62. HRMS (ESI) for C21H22N3O2
(M-Hþ): calcd, 348.1712; found, 348.1714.
4.1.6. (E)-3-(4-((3-((Dimethylamino)methyl)-2-methyl-1H-indol-
1-yl)methyl)phenyl)-N-hydroxyacrylamide (10)
4.1.11. 1-(4-(Hydroxycarbamoyl)benzyl)-N,N-dimethyl-1H-indole-
3-carboxamide (15)
The title compound was obtained in 52% yield from compound
31a in a manner similar to that described for the synthesis of
compound 5. HPLC purity: 97.05% (tR ¼ 16.99 min). mp: 174e175 ꢀC.
The title compounds was obtained in 70% yield from compound
33c in a manner similar to that described for the synthesis of
compound 7. HPLC purity: 98.12% (tR ¼ 17.06 min). mp: 197e198 ꢀC.
1HNMR (300 MHz, CD3OD)
d 2.44 (s, 3H) 2.90 (s, 6H), 4.53 (s, 2H),
5.41 (s, 2H), 6.46 (d, J ¼ 15.6 Hz, 1H), 6.97e7.05 (m, 2H) 7.16e7.18
1HNMR (300 MHz, DMSO-d6)
d 3.12 (s, 6H), 5.54 (s, 2H), 7.10e7.20
(m, 2H), 7.32 (d, J ¼ 6.0 Hz, 1H), 7.39e7.49 (m, 3H), 7.71 (d,
(m, 2H), 7.31 (d, J ¼ 8.1 Hz, 2H), 7.48 (dd, J ¼ 1.2, 6.9 Hz, 1H), 7.70 (d,
J ¼ 6.3 Hz,1H). 13CNMR (75 MHz, CD3OD)
d 6.86, 38.59, 43.30, 48.77,
97.88, 106.97, 114.91, 117.85, 119.26, 123.78, 124.98, 125.26, 125.48,
131.36, 131.70, 134.09, 136.53, 136.69, 162.21. HRMS (ESI) for
J ¼ 8.1 Hz, 2H). 7.82 (dd, J ¼ 1.8, 8.1 Hz, 1H), 8.00 (s, 1H). 13CNMR
(75 MHz, DMSO-d6) d 37.06, 46.80, 107.47, 108.39, 118.33, 118.98,
119.95, 124.85, 125.10, 129.23, 129.90, 133.20, 138.55, 161.75, 163.54.
C
22H24N3O2 (M-Hþ): calcd, 362.1869; found, 362.1866.
HRMS (ESI) for
336.1345.
C
19H18N3O3 (M-Hþ): calcd, 336.1348; found,
4.1.7. 4-((3-((Dimethylamino)methyl)-1H-indol-1-yl)methyl)-N-
hydroxybenzamide (11)
4.1.12. 1-[3-(2-Hydroxycarbamoyl-vinyl)-benzenesulfonyl]-1H-
indole-3-carboxylic acid dimethylamide (16)
The title compound was obtained in 72% yield from compound
33b in a manner similar to that described for the synthesis of
compound 7. HPLC purity: 98.70% (tR ¼ 18.69 min). mp: 171e172 ꢀC.
The title compound was obtained in 53% yield from compound
28e in a manner similar to that described for the synthesis of
compound 5. HPLC purity: 97.41% (tR ¼ 17.09 min). mp: 129e130 ꢀC.
1HNMR (300 MHz, CD3OD)
d 2.37 (s, 6H), 3.81 (s, 2H), 5.48 (s, 2H),
7.09e7.18 (m, 2H), 7.22 (d, J ¼ 8.1 Hz, 2H), 7.30e7.34 (m, 2H),
1HNMR (300 MHz, CD3OD)
d
3.15 (s, 6H), 6.56 (d, J ¼ 15.9 Hz, 1H),
7.67e7.70 (m, 3H). 13CNMR (75 MHz, CD3OD)
d
40.57, 46.29, 50.14,
7.34 (t, J ¼ 7.2 Hz,1H), 7.43 (t, J ¼ 7.2 Hz,1H), 7.53 (d, J ¼ 15.9 Hz,1H),
106.73, 106.96, 112.55, 115.99, 116.71, 118.68, 123.96, 124.41, 125.24,
125.91, 126.43, 133.85, 139.28, 163.62. HRMS (ESI) for C19H20N3O2
(M-Hþ): calcd, 322.1556; found, 322.1553.
7.63e7.71 (m, 3H), 8.01e8.04 (m, 4H). 13CNMR (75 MHz, CD3OD)
d
46.75, 107.00, 107.57, 114.66, 117.93, 118.13, 119.75, 124.33, 124.52,
124.91, 125.12, 128.30, 131.69, 133.37, 136.43, 136.91, 162.14, 165.71.
HRMS (ESI) for C20H18N3O5S: (M-Hþ): calcd, 412.0967; found,
412.0963.
4.1.8. 3-[3-(3-Dimethylaminomethyl-indole-1-sulfonyl)-phenyl]-N-
hydroxy-acrylamide (12)
The title compound was obtained in 53% yield from compound
28c in a manner similar to that described for the synthesis of
compound 5. HPLC purity: 97.54% (tR ¼ 17.01 min). mp: 160e161 ꢀC.
4.1.13. 1-[4-(2-Hydroxycarbamoyl-vinyl)-benzenesulfonyl]-1H-
indole-3-carboxylic acid dimethylamide (17)
The title compound was obtained in 59% yield from compound

124
K. Nepali et al. / European Journal of Medicinal Chemistry 127 (2017) 115e127
28f in a manner similar to that described for the synthesis of
compound 5. HPLC purity: 99.12% (tR ¼ 18.71 min). mp: 141e142 ꢀC.
compound 21a. 1HNMR (300 MHz, CDCl3)
d 2.60 (s, 3H), 2.86e2.87
(m, 4H), 3.79 (s, 2H), 3.82 (s, 3H), 5.27 (s, 2H), 6.40 (d, J ¼ 16.2 Hz,
1H), 7.01 (d, J ¼ 8.1 Hz, 2H), 7.11e7.20 (m, 3H), 7.41 (d, J ¼ 8.1 Hz,
2H), 7.49 (m, 1H), 7.65 (d, J ¼ 16.2 Hz, 1H).
1HNMR (300 MHz, DMSO-d6)
d
3.06 (s, 6H), 6.58 (d, J ¼ 15.9 Hz, 1H),
7.33 (t, J ¼ 7.2 Hz,1H), 7.40e7.47 (m, 2H), 7.68 (d, J ¼ 7.5 Hz,1H), 7.78
(d, J ¼ 8.4 Hz, 2H), 7.99 (d, J ¼ 8.1 Hz, 1H), 8.04 (d, J ¼ 8.7 Hz, 2H),
8.13 (s, 1H). 13CNMR (75 MHz, DMSO-d6)
d
38.16, 110.95, 115.07,
4.1.19. 3-[3-(2-Methyl-1,2,3,4-tetrahydro-pyrido[4,3-b]indole-5-
sulfonyl)-phenyl]-acrylic acid (22a)
119.38, 121.90, 123.29, 124.48, 125.40, 126.43, 126.60, 131.42, 133.51,
134.20, 134.24, 139.00, 159. 42, 161.45. HRMS (ESI) for C20H18N3O5S:
(M-Hþ): calcd, 412.0967; found, 412.0966.
A mixture of 21a (0.6 g, 1.5 mmol), 1 M LiOH(aq) (6.0 mL), and
dioxane (15 mL) was stirred at 40 ꢀC for 2 h. The reaction was
concentrated under reduced pressure and water was added. The
mixture was acidified with 3 N HCl and extracted with EtOAc
(50 mL x 3). The combined organic layer was dried over anhydrous
MgSO4 and concentrated under reduced pressure to yield com-
4.1.14. 1-(4-((E)-2-(Hydroxycarbamoyl)vinyl)benzyl)-N,N-
dimethyl-1H-indole-3-carboxamide (18)
The title compound was obtained in 54% yield from compound
31c in a manner similar to that described for the synthesis of
pound 22a in 94% yield; 1HNMR (300 MHz, CD3OD)
d 2.51 (s, 3H),
compound 5; HPLC purity: 95.19% (tR
180e181 ꢀC. 1HNMR (300 MHz, CD3OD)
3.22 (s, 6H), 5.50 (s, 2H),
6.45 (d, J ¼ 15.9 Hz, 1H), 7.19e7.25 (m, 4H), 7.40 (m, 1H), 7.50e7.58
(m, 3H), 7.77e7.81 (m, 2H). 13CNMR (75 MHz, CD3OD)
44.12,
¼
17.42 min). mp:
2.83 (t, J ¼ 6.0 Hz, 2H), 3.21 (t, J ¼ 6.0 Hz, 2H), 3.57 (bs, 2H), 6.42 (d,
J ¼ 15.9 Hz,1H), 7.19e7.31 (m, 3H), 7.35e7.57 (m, 3H), 7.87e7.99 (m,
2H), 8.11 (d, J ¼ 8.1 Hz, 1H).
d
d
46.75, 107.00, 107.57, 114.66, 117.93, 118.13, 119.75, 124.33, 124.52,
125.12, 128.30, 131.69, 133.37, 136.43, 136.91, 162.14, 165.71. HRMS
(ESI) for C21H20N3O3: (M-Hþ): calcd, 362.1505; found, 362.1502.
4.1.20. (E)-3-(4-((1,2,3,4-Tetrahydro-2-methylpyrido[4,3-b]indol-5-
yl)methyl)phenyl)acrylic acid (22b)
The title compound was obtained in 96% yield from compound
21b in a manner similar to that described for the synthesis of
4.1.15. 5-(3-Bromo-benzenesulfonyl)-2-methyl-2,3,4,5-tetrahydro-
1H-pyrido[4,3-b]indole (20a)
compound 22a. 1HNMR (300 MHz, CD3OD)
d 2.75 (s, 3H), 2.92 (t,
J ¼ 6.0 Hz, 2H), 3.19 (t, J ¼ 6.0 Hz, 2H), 4.07 (s, 2H), 5.31 (s, 2H), 6.41
(d, J ¼ 15.9 Hz, 1H), 7.01e7.23 (m, 4H), 7.29 (d, J ¼ 7.5 Hz, 2H),
7.37e7.43 (m, 3H).
To a mixture of NaH (0.12 g, 3.0 mmol), 19 (0.5 g, 2.6 mmol), and
DMF (3 mL), 3-bromobenzenesulfonyl chloride (0.75 g, 3.0 mmol)
was added dropwise and stirred at room temperature for 2.5 h. The
reaction was quenched with water and then extracted with EtOAc
(50 mL x 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 4: 1) to give compound 20a in 80% yield; 1HNMR
4.1.21. [1-(3-Bromo-benzenesulfonyl)-2-methyl-1H-indol-3-
ylmethyl]-dimethyl-amine (26a)
A mixture of 23 (0.5 g, 3.1 mmol), NaH (0.15 g, 3.7 mmol), DMF
(3 mL), and 3-bromobenzenesulfonyl chloride (0.8 g, 3.2 mmol)
was added dropwise and stirred at room temperature for 2.5 h. The
reaction was quenched with water and then extracted with EtOAc
(50 mL x 3). The combined organic layer was dried over anhydrous
MgSO4 and concentrated under reduced pressure. The resulting
residue was dissolved in DCM (25 mL), and then sodium tri-
acetoxyborohydride (1.0 g, 4.7 mmol) and dimethyl amine (5 M
solution in EtOH, 1.0 mL, 5.0 mmol) were added and stirred at room
temperature for 12 h. The reaction mixture was quenched with
water and was extracted with DCM (50 mL x 3). The combined
organic layer was dried over anhydrous MgSO4 and purified by
silica gel chromatography to give compound 26a in 68% yield;
(300 MHz, CDCl3)
J ¼ 5.7 Hz, 2H), 3. 59 (s, 2H), 7.32e7.35 (m, 2H), 7.64e7.70 (m, 3H),
8.00 (t, J ¼ 3.0 Hz, 1H), 8.12e8.15 (m, 2H).
d
2.57 (s, 3H), 2.85 (t, J ¼ 5.7 Hz, 2H), 3.19 (t,
4.1.16. 5-(4-Bromobenzyl)-2,3,4,5-tetrahydro-2-methyl-1H-pyrido
[4,3-b]indole (20b)
The title compound was obtained in 74% yield from compound
19 by using 4-bromobenzyl bromide in a manner similar to that
described for the synthesis of compound 20a. 1HNMR (300 MHz,
CDCl3)
d 2.62 (s, 3H), 2.81e2.85 (m, 4H), 3.81 (s, 2H), 5.29 (s, 2H),
7.07 (d, J ¼ 8.1 Hz, 2H), 7.09e7.23 (m, 3H), 7.43 (d, J ¼ 8.1 Hz, 2H),
1HNMR (300 MHz, CDCl3)
d 2.18 (s, 6H), 2.54 (s, 3H) 3.39 (s, 2H),
7.47 (m, 1H).
7.19e7.23 (m, 2H), 7.36e7.53 (m, 4H), 7.69 (d, J ¼ 8.4 Hz,1H), 8.14 (d,
J ¼ 7.8 Hz, 1H).
4.1.17. 3-[3-(2-Methyl-1,2,3,4-tetrahydro-pyrido [4,3-b]indole-5-
sulfonyl)-phenyl]-acrylic acid methyl ester (21a)
4.1.22. [1-(4-Bromo-benzenesulfonyl)-2-methyl-1H-indol-3-
ylmethyl]-dimethyl-amine (26b)
A mixture of 20a (0.8 g, 2.0 mmol), tris(dibenzylideneacetone)
dipalladium (0.36 g, 0.39 mmol), tri-t-butylphosphonium tetra-
fluoroborate (0.23 g, 0.79 mmol), K2CO3 (0.55 g, 4.0 mmol), TEA
(0.68 mL, 4.9 mmol), methyl acrylate (0.26 mL, 3.0 mmol), and DMF
(15 mL) was stirred at 100 ꢀC for 12 h. The reaction was quenched
with water and extracted with EtOAc (50 mL x 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) to give compound 21a in 83% yield;
The title compounds was obtained in 71% yield from compound
23 in a manner similar to that described for the synthesis of com-
pound 26a. 1HNMR (300 MHz, CDCl3)
d 2.29 (s, 6H), 2.41 (s, 3H),
3.59 (s, 2H), 7.24e7.41 (m, 2H), 7.56 (d, J ¼ 8.7 Hz, 2H), 7.67 (m, 1H),
7.78 (d, J ¼ 8.7 Hz, 2H), 8.01 (m, 1H).
4.1.23. [1-(3-Bromo-benzenesulfonyl)-1H-indol-3-ylmethyl]-
dimethyl-amine (26c)
1HNMR (300 MHz, CDCl3)
d
2.47 (s, 3H), 2.75 (t, J ¼ 6.0 Hz, 2H), 3.13
The title compound was obtained in 65% yield from compound
24 in a manner similar to that described for the synthesis of com-
(t, J ¼ 6.0 Hz, 2H), 3.48 (bs, 2H), 3.76 (s, 3H), 6.37 (d, J ¼ 15.9 Hz, 1H),
7.18e7.35 (m, 3H), 7.38e7.59 (m, 3H), 7.89e7.97 (m, 2H), 8.09 (d,
J ¼ 8.1 Hz, 1H).
pound 26a. 1HNMR (300 MHz, CDCl3)
d 2.29 (s, 6H), 3.59 (s, 2H),
7.35e7.37 (m, 2H), 7.38e7.47 (m, 2H), 7.65e7.67 (m, 2H), 7.89 (d,
J ¼ 1.2 Hz, 1H), 8.00e8.03 (m, 2H).
4.1.18. (E)-Methyl 3-(4-((1,2,3,4-tetrahydro-2-methylpyrido[4,3-b]
indol-5-yl)methyl)phenyl)acrylate (21b)
The title compound was obtained in 74% yield from compound
20b in a manner similar to that described for the synthesis of
4.1.24. [1-(4-Bromo-benzenesulfonyl)-1H-indol-3-ylmethyl]-
dimethyl-amine (26d)
The title compound was obtained in 68% yield from compound

K. Nepali et al. / European Journal of Medicinal Chemistry 127 (2017) 115e127
125
24 in a manner similar to that described for the synthesis of com-
pound 26a. 1HNMR (300 MHz, CDCl3)
2.28 (s, 6H), 3.56 (s, 2H),
compound 21a. 1HNMR (300 MHz, CDCl3)
d
3.16 (s, 6H), 3.82 (s, 3H),
d
6.47 (d, J ¼ 16.2 Hz, 1H), 7.35e7.42 (m, 2H), 7.60 (d, J ¼ 8.1 Hz, 2H),
7.26e7.39 (m, 2H), 7.47 (s, 1H), 7.58 (d, J ¼ 8.7 Hz, 2H), 7.65 (m, 1H),
7.62e7.77 (m, 2H), 7.95 (d, J ¼ 8.1 Hz, 2H), 7.99e8.01 (m, 2H).
7.74 (d, J ¼ 8.7 Hz, 2H), 7.97 (m, 1H).
4.1.33. 3-[3-(3-Dimethylaminomethyl-2-methyl-indole-1-
sulfonyl)-phenyl]-acrylic acid (28a)
4.1.25. 1-(3-Bromo-benzenesulfonyl)-1H-indole-3-carboxylic acid
dimethylamide (26e)
The title compound was obtained in 95% yield from compound
27a in a manner similar to that described for the synthesis of
The title compound was obtained in 80% yield from compound
24 in a manner similar to that described for the synthesis of com-
compound 22a. 1HNMR (300 MHz, CD3OD)
d 2.79 (s, 3H), 2.89 (s,
pound 20a. 1HNMR (300 MHz, CDCl3)
d
3.16 (s, 6H), 7.17e7.39 (m,
6H), 4.51 (s, 2H), 6.53 (d, J ¼ 16.2 Hz, 1H), 7.40e7.46 (m, 2H),
3H), 7.42e7.48 (m, 3H), 7.79 (m, 1H), 7.82e7.89 (m, 2H).
7.60e7.71 (m, 3H), 7.89e7.91 (m, 2H), 8.06 (m, 1H), 8.28 (m, 1H).
4.1.26. 1-(4-Bromo-benzenesulfonyl)-1H-indole-3-carboxylic acid
dimethylamide (26f)
4.1.34. 3-[4-(3-Dimethylaminomethyl-2-methyl-indole-1-
sulfonyl)-phenyl]-acrylic acid (28b)
The title compound was obtained in 82% yield from compound
25 in a manner similar to that described for the synthesis of com-
The title compound was obtained in 95% yield from compound
27b in a manner similar to that described for the synthesis of
pound 20a. 1HNMR (300 MHz, CDCl3)
d
3.18 (s, 6H), 7.25e7.32 (m,
compound 22a. 1HNMR (300 MHz, CD3OD)
d 2.56 (s, 3H), 2.89 (s,
2H), 7.49 (d, J ¼ 8.1 Hz, 2H), 7.58 (m, 1H), 7.75 (d, J ¼ 8.1 Hz, 2H),
6H), 4.59 (s, 2H), 6.67 (d, J ¼ 16.2 Hz,1H), 7.37e7.82 (m, 4H), 8.06 (d,
7.79e7.85 (m, 2H).
J ¼ 8.4 Hz, 2H), 8.10e8.11 (m, 2H), 8.29 (s, 1H).
4.1.27. 3-[3-(3-Dimethylaminomethyl-2-methyl-indole-1-
sulfonyl)-phenyl]-acrylic acid methyl ester (27a)
4.1.35. 3-[3-(3-Dimethylaminomethyl-indole-1-sulfonyl)-phenyl]-
acrylic acid (28c)
The title compound was obtained in 65% yield from compound
26a in a manner similar to that described for the synthesis of
The title compound was obtained in 92% yield from compound
27c in a manner similar to that described for the synthesis of
compound 21a. 1HNMR (300 MHz, CDCl3)
d
2.11 (s, 6H), 2.52 (s, 3H),
compound 22a. 1HNMR (300 MHz, CD3OD)
d 2.91 (s, 6H), 4.54 (s,
3.37 (s, 2H), 3.71 (s, 3H), 6.35 (d, J ¼ 16.2 Hz, 1H), 7.17e7.21 (m, 2H),
2H), 6.55 (d, J ¼ 16.2 Hz, 1H), 7.42e7.7.49 (m, 2H), 7.60e7.71 (m,
7.39e7.52 (m, 5H), 7.66 (d, J ¼ 8.4 Hz, 1H), 8.12 (d, J ¼ 7.8 Hz, 1H).
4H), 7.90e7.93 (m, 2H), 8.09 (m, 1H), 8.31 (m, 1H).
4.1.28. 3-[4-(3-Dimethylaminomethyl-2-methyl-indole-1-
sulfonyl)-phenyl]-acrylic acid methyl ester (27b)
4.1.36. 3-[4-(3-Dimethylaminomethyl-indole-1-sulfonyl)-phenyl]-
acrylic acid (28d)
The title compound was obtained in 67% yield from compound
26b in a manner similar to that described for the synthesis of
The title compound was obtained in 90% yield from compound
27d in a manner similar to that described for the synthesis of
compound 21a. 1HNMR (300 MHz, CDCl3)
d
2.22 (s, 6H), 2.54 (s, 3H),
compound 22a. 1HNMR (300 MHz, CD3OD)
d 2.88 (s, 6H), 4.54 (s,
3.37 (s, 2H), 3.71 (s, 3H), 6.35 (d, J ¼ 16.2 Hz, 1H), 7.17e7.21 (m, 2H),
2H), 6.65 (d, J ¼ 16.2 Hz, 1H), 7.39e7.47 (m, 2H), 7.56 (d, J ¼ 16.2 Hz,
1H), 7.74e7.80 (m, 2H), 8.02 (d, J ¼ 8.4 Hz, 2H), 8.08e8.10 (m, 2H),
8.27 (s, 1H).
7.39e7.52 (m, 4H), 7.66 (d, J ¼ 8.4 Hz, 2H), 8.12 (d, J ¼ 7.8 Hz, 1H).
4.1.29. 3-[3-(3-Dimethylaminomethyl-indole-1-sulfonyl)-phenyl]-
acrylic acid methyl ester (26c)
4.1.37. 3-[3-(3-Dimethylcarbamoyl-indole-1-sulfonyl)-phenyl]-
acrylic acid (28e)
The title compound was obtained in 62% yield from compound
26c in a manner similar to that described for the synthesis of
The title compound was obtained in 94% yield from compound
27e in a manner similar to that described for the synthesis of
compound 21a. 1HNMR (300 MHz, CDCl3)
d 2.29 (s, 6H), 3.59 (s, 2H),
3.84 (s, 3H), 6.44 (d, J ¼ 15.9 Hz, 1H), 7.35e7.37 (m, 2H), 7.38e7.47
(m, 2H), 7.65e7.67 (m, 3H), 7.89 (d, J ¼ 1.2 Hz, 1H), 8.00e8.03 (m,
2H).
compound 22a. 1HNMR (300 MHz, CD3OD)
d 3.21 (s, 6H), 6.58 (d,
J ¼ 16.2 Hz, 1H), 7.39e7.59 (m, 3H), 7.68e7.79 (m, 4H), 7.98 (m, 1H),
8.00e8.11 (m, 2H).
4.1.30. 3-[4-(3-Dimethylaminomethyl-indole-1-sulfonyl)-phenyl]-
acrylic acid methyl ester (27d)
4.1.38. 3-[4-(3-Dimethylcarbamoyl-indole-1-sulfonyl)-phenyl]-
acrylic acid (28f)
The title compound was obtained in 65% yield from compound
26d in a manner similar to that described for the synthesis of
The title compound was obtained in 90% yield from compound
27f in a manner similar to that described for the synthesis of
compound 21a. 1HNMR (300 MHz, CDCl3)
d
2.28 (s, 6H), 3.56 (s, 2H),
compound 22a. 1HNMR (300 MHz, CD3OD)
d 3.26 (s, 6H), 6.59 (d,
3.82 (s, 3H), 6.45 (d, J ¼ 15.9 Hz, 1H), 7.24e7.38 (m, 2H), 7.50e7.58
J ¼ 16.2 Hz, 1H), 7.39e7.48 (m, 2H), 7.66 (d, J ¼ 8.1 Hz, 2H),
(m, 5H), 7.64 (d, J ¼ 8.4 Hz, 2H), 7.76 (d, J ¼ 8.1 Hz, 1H).
7.69e7.81 (m, 2H), 7.99 (d, J ¼ 8.1 Hz, 2H), 8.01e8.18 (m, 2H).
4.1.31. 3-[3-(3-Dimethylcarbamoyl-indole-1-sulfonyl)-phenyl]-
acrylic acid methyl ester (27e)
4.1.39. (1-(4-Bromobenzyl)-2-methyl-1H-indol-3-yl)-N,N-
dimethylmethanamine (29a)
The title compound was obtained in 64% yield from compound
26e in a manner similar to that described for the synthesis of
The title compound was obtained in 68% yield from compound
23 in a manner similar to that described for the synthesis of com-
compound 21a. 1HNMR (300 MHz, CDCl3)
d
3.16 (s, 6H), 3.85 (s, 3H),
pound 26a. 1HNMR (300 MHz, CDCl3)
d 2.39 (s, 6H), 2.41 (s, 3H),
6.48 (d, J ¼ 16.2 Hz, 1H), 7.29e7.54 (m, 3H), 7.62e7.68 (m, 4H), 7.91
3.75 (s, 2H), 5.34 (s, 2H), 6.98 (d, J ¼ 8.1 Hz, 2H), 7.15 (m, 3H), 7.43 (d,
(m, 1H), 7.92e8.00 (m, 2H).
J ¼ 8.1 Hz, 2H), 7.70 (m, 1H).
4.1.32. 3-[4-(3-Dimethylcarbamoyl-indole-1-sulfonyl)-phenyl]-
acrylic acid methyl ester (27f)
4.1.40. (1-(4-bromobenzyl)-1H-indol-3-yl)-N,N-
dimethylmethanamine (29b)
The title compound was obtained in 67% yield from compound
26f in a manner similar to that described for the synthesis of
The title compounds was obtained in 70% yield from compound
24 in a manner similar to that described for the synthesis of

126
K. Nepali et al. / European Journal of Medicinal Chemistry 127 (2017) 115e127
compound 26a. 1HNMR (300 MHz, CDCl3)
5.37 (s, 2H), 7.15e7.26 (m, 6H), 7.48 (d, J ¼ 8.1 Hz, 2H), 7.71 (m, 1H).
d
2.39 (s, 6H), 3.65 (s, 2H),
4.1.48. Methyl 4-((3-formyl-2-methyl-1H-indol-1-yl)methyl)
benzoate (32a)
The title compound was obtained in 82% yield from compound
23 in a manner similar to that described for the synthesis of com-
4.1.41. 1-(4-Bromobenzyl)-N,N-dimethyl-1H-indole-3-carboxamide
(29c)
pound 20a. 1HNMR (300 MHz, CDCl3)
d 2.78 (s, 3H), 3.91 (s, 3H),
5.41 (s, 2H), 7.23 (d, J ¼ 8.1 Hz, 2H), 7.28e7.39 (m, 3H), 7.68 (d,
The title compound was obtained in 84% yield from compound
25 in a manner similar to that described for the synthesis of 26e.
J ¼ 8.1 Hz, 2H), 8.28 (m, 1H), 10.22 (s, 1H).
1HNMR (300 MHz, CDCl3)
d 3.21 (s, 6H), 5.43 (s, 2H), 7.28 (d,
4.1.49. Methyl 4-((3-formyl-1H-indol-1-yl)methyl)benzoate (32b)
The title compound was obtained in 80% yield from compound
24 in a manner similar to that described for the synthesis of com-
J ¼ 8.1 Hz, 2H), 7.23e7.41 (m, 3H), 7.69 (d, J ¼ 8.1 Hz, 2H), 7.79 (s,
1H), 8.32 (m, 1H).
pound 20a. 1HNMR (300 MHz, CDCl3)
d 3.88 (s, 3H), 5.44 (s, 2H),
4.1.42. (E)-Methyl 3-(4-((3-((dimethylamino)methyl)-2-methyl-
1H-indol-1-yl)methyl)phenyl)acrylate (30a)
7.21 (d, J ¼ 8.1 Hz, 2H), 7.32e7.46 (m, 3H), 7.66 (d, J ¼ 8.1 Hz, 2H),
7.75 (s, 1H), 8.35 (m, 1H), 10.05 (s, 1H).
The title compounds was obtained in 68% yield from compound
29a in a manner similar to that described for the synthesis of
4.1.50. Methyl 4-((3-(dimethylcarbamoyl)-1H-indol-1-yl)methyl)
benzoate (32c)
compound 21a. 1HNMR (300 MHz, CDCl3)
d 2.37 (s, 6H), 2.39 (s, 3H),
3.75 (s, 2H), 3.82 (s, 3H), 5.36 (s, 2H), 6.43 (d, J ¼ 15.9 Hz, 1H), 6.99
(d, J ¼ 8.1 Hz, 2H), 7.15e7.21 (m, 3H), 7.41 (d, J ¼ 8.1 Hz, 2H), 7.69 (d,
J ¼ 15.9 Hz, 1H), 7.70 (m, 1H).
The title compound was obtained in 68% yield from compound
25 in a manner similar to that described for the synthesis of com-
pound 20a. 1HNMR (300 MHz, CDCl3)
d 3.20 (s, 6H), 3.93 (s, 3H),
5.42 (s, 2H), 7.20e7.29 (m, 5H), 7.51 (s, 1H), 7.81 (dd, J ¼ 2.4, 9.0 Hz,
4.1.43. (E)-Methyl 3-(4-((3-((dimethylamino)methyl)-1H-indol-1-
yl)methyl)phenyl)acrylate (30b)
1H), 8.01 (d, J ¼ 8.4 Hz, 2H).
The title compound was obtained in 65% yield from compound
29b in a manner similar to that described for the synthesis of
4.1.51. 4-((3-Formyl-2-methyl-1H-indol-1-yl)methyl)benzoic acid
(33a)
compound 21a. 1HNMR (300 MHz, CDCl3)
d 2.34 (s, 6H), 3.69 (s, 2H),
The title compound was obtained in 88% yield from compounds
32a in a manner similar to that described for the synthesis of
3.83 (s, 3H), 5.33 (s, 2H), 6.44 (d, J ¼ 16.2 Hz, 1H), 7.11e7.26 (m, 6H),
7.46 (d, J ¼ 8.1 Hz, 2H), 7.74 (d, J ¼ 16.2 Hz, 1H), 7.78 (dd, J ¼ 1.2,
7.2 Hz, 1H).
compound 22a. 1HNMR (300 MHz, CD3OD)
d 2.81 (s, 3H), 5.47 (s,
2H), 7.28 (d, J ¼ 8.1 Hz, 2H), 7.31e7.36 (m, 3H), 7.79 (d, J ¼ 8.1 Hz,
2H), 8.32 (m, 1H), 10.27 (s, 1H).
4.1.44. (E)-Methyl 3-(4-((3-(dimethylcarbamoyl)-1H-indol-1yl)
methyl)phenyl)acrylate (30c)
4.1.52. 4-((3-Formyl-1H-indol-1-yl)methyl)benzoic acid (33b)
The title compound was obtained in 90% yield from compounds
32b in a manner similar to that described for the synthesis of
The title compound was obtained in 64% yield from compound
29c in a manner similar to that described for the synthesis of
compound 22a. 1HNMR (300 MHz, CD3OD)
d 5.49 (s, 2H), 7.27 (d,
compound 21a. 1HNMR (300 MHz, CDCl3)
d 3.19 (s, 6H), 3.83 (s, 3H),
J ¼ 8.1 Hz, 2H), 7.29e7.49 (m, 3H), 7.69 (d, J ¼ 8.1 Hz, 2H), 7.72 (s,
5.38 (s, 2H), 6.43 (d, J ¼ 16.2 Hz, 1H), 7.16 (d, J ¼ 8.1 Hz, 2H),
7.24e7.27 (m, 3H), 7.48e7.50 (m, 3H), 7.68 (d, J ¼ 16.4 Hz, 1H), 7.82
(m, 1H).
1H), 8.39 (m, 1H), 10.15 (s, 1H).
4.1.53. 4-((3-(Dimethylcarbamoyl)-1H-indol-1-yl)methyl)benzoic
acid (33c)
4.1.45. (E)-3-(4-((3-((Dimethylamino)methyl)-2-methyl-1H-indol-
1-yl)methyl)phenyl)acrylic acid (31a)
The title compound was obtained in 90% yield from compound
32c in a manner similar to that described for the synthesis of
The title compound was obtained in 93% yield from compounds
30a in a manner similar to that described for the synthesis of
compound 22a. 1HNMR (300 MHz, CD3OD)
d 3.39 (s, 6H), 5.56 (s,
2H), 7.25e7.39 (m, 5H), 7.59 (s, 1H), 7.87 (dd, J ¼ 2.4, 9.0 Hz, 1H),
compound 22a. 1HNMR (300 MHz, CD3OD)
d 2.36 (s, 6H), 2.41 (s,
8.21 (d, J ¼ 8.4 Hz, 2H).
3H), 3.79 (s, 2H), 5.32 (s, 2H), 6.48 (d, J ¼ 15.9 Hz, 1H), 7.01 (d,
J ¼ 8.1 Hz, 2H), 7.19e7.25 (m, 3H), 7.46 (d, J ¼ 8.1 Hz, 2H), 7.78 (d,
J ¼ 15.9 Hz, 1H), 7.81 (m, 1H).
4.2. Biology
4.2.1. Materials
4.1.46. (E)-3-(4-((3-((Dimethylamino)methyl)-1H-indol-1-yl)
methyl)phenyl)acrylic acid (31b)
Sulforhodamine B (SRB), crystal violet, and propidium iodide
(PI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). An-
tibodies to acetyl-tubulin was purchased from Cell Signaling
(Danvers, MA, USA), PARP was from Santa Cruz (Dallas, Texas, USA),
caspase 3 was from Novus (Littleton, CO, USA), acetyl-Histone H3,
The title compound was obtained in 95% yield from compound
30b in a manner similar to that described for the synthesis of
compound 22a. 1HNMR (300 MHz, CD3OD)
d 2.39 (s, 6H), 3.78 (s,
2H), 5.29 (s, 2H), 6.49 (d, J ¼ 16.2 Hz, 1H), 7.14e7.29 (m, 6H), 7.49 (d,
and b-actin were purchased from Millipore (Billerica, MA, USA).
J ¼ 8.1 Hz, 2H), 7.72 (d, J ¼ 16.2 Hz, 1H), 7.76 (dd, J ¼ 1.2, 7.2 Hz, 1H).
4.2.2. Cell culture
4.1.47. (E)-3-(4-((3-(Dimethylcarbamoyl)-1H-indol-1-yl)methyl)
phenyl)acrylic acid (31c)
Human NSCLC cell line A549, colorectal carcinoma HCT116 cells,
and human prostate cancer cell line PC-3 cells were obtained from
American Type Culture Collection (ATCC) (Manassas, VA). Cells
were maintained in RPMI 1640 medium with 10% fetal bovine
The title compound was obtained in 94% yield from compound
30c in a manner similar to that described for the synthesis of
compound 22a. 1HNMR (300 MHz, CD3OD)
d
3.31 (s, 6H), 5.47 (s,
serum (FBS) and penicillin (100 U/ml)/streptomycin (100
mg/mL)/
2H), 6.51 (d, J ¼ 16.2 Hz, 1H), 7.23 (d, J ¼ 8.1 Hz, 2H), 7.29e7.41 (m,
amphotericin (0.25
5% CO2.
m
g/mL) at 37 ꢀC in a humidified incubator with
3H), 7.53e7.59 (m, 3H), 7.78 (d, J ¼ 16.4 Hz, 1H), 7.92 (m, 1H).

K. Nepali et al. / European Journal of Medicinal Chemistry 127 (2017) 115e127
127
4.2.3. Sulforhodamine B (SRB) assay
4.2.8. Statistical analysis
All cells were seeded in 96-well culture plates at a density of
3e5 x 103 cells/well. After attachment, cells were fixed with 10%
trichloroacetic acid (TCA) to provide a measurement of the cell
population at the time of drug addition (T0). After additional in-
cubation of DMSO or compounds for 48 h, cells were fixed with 10%
TCA and stained with 0.4% (w/v) SRB dissolved in 1% AcOH. The
protein-bound dye was subsequently extracted with 10 mM trizma
base to determine the absorbance at a wavelength of 515 nm. The
percentage of cell growth was calculated with O.D. value from
different treatments, such as time zero (T0), control growth (C), and
cell growth in the presence of the drug (Tx). Percentage growth was
Results are expressed as the mean
SD for the indicated
number of separate experiments. Means were assessed for signifi-
cant differences using t-test and P-values < 0.05 were considered
significant.
Acknowledgments
This research were supported by the Ministry of Science and
Technology of the Republic of China (grant no. MOST 103-2113-M-
038-001-MY3
and
MOST-103-2320-B-038-030-MY3),
and
TMU102-AE1-B43 by Taipei Medical University.
calculated as [(Tx-T0)/(C-T0)]*100. Growth inhibition of 50% (GI50
)
is determined at the drug concentration that results in 50%
reduction of total protein increase in control cells during the
compound incubation.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.12.039.
4.2.4. HDAC enzymes inhibition assays
Enzyme inhibition assays were performed by the Reaction
Biology Corporation, Malvern, PA (http://www.reactionbiology.
com). The substrate for HDAC-1, -2, and -6 is a fluorogenic pep-
tide derived from p53 residues 379e382 [RHKK(Ac)]. Compounds
were dissolved in DMSO and tested in 10-dose IC50 mode with 3-
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