4-Indolyl-N-hydroxyphenylacrylamides as potent HDAC class I and IIB inhibitors in?vitro and in?vivo

Article abstract of DOI:10.1016/j.ejmech.2017.03.079

A series of 4,5-indolyl-N-hydroxyphenylacrylamides, as HDAC inhibitors, has been synthesized and evaluated in?vitro and in?vivo. 4-Indolyl compounds 13 and 17 functions as potent inhibitors of HDAC1 (IC₅₀ 1.28?nM and 1.34?nM) and HDAC 2 (IC

Full text of DOI:10.1016/j.ejmech.2017.03.079

Accepted Manuscript
4-Indolyl-N-hydroxyphenylacrylamides as potent HDAC class I and IIB inhibitors in
vitro and in vivo
Samir Mehndiratta, Ruei-Shian Wang, Han-Li Huang, Chih-Jou Su, Chia-Ming Hsu,
Yi-Wen Wu, Shiow-Lin Pan, Jing-Ping Liou
PII:
S0223-5234(17)30247-7
10.1016/j.ejmech.2017.03.079
EJMECH 9339
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 December 2016
Revised Date: 28 March 2017
Accepted Date: 30 March 2017
Please cite this article as: S. Mehndiratta, R.-S. Wang, H.-L. Huang, C.-J. Su, C.-M. Hsu, Y.-W. Wu, S.-
L. Pan, J.-P. Liou, 4-Indolyl-N-hydroxyphenylacrylamides as potent HDAC class I and IIB inhibitors in
vitro and in vivo, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.03.079.
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Graphical Abstract
O
4
OCH3
5
N
H
OCH3
OCH3
O
4 or 5-(3 ', 4 ', 5 ' -Trimethoxybenzoyl)indole
Novel class of antitubulin agents (7, 8)
NHOH
H
N
HN
S
O2
o
180
O
S
N
13: PC3= 0.14 µM
A549= 0.25 µM
MDA-MB-231= 0.32 µM
AsPC-1= 0.24 µM
O
NHOH
O
Linker
Zn Binding Group
Cap
9: 3-(1-Benzenesulfonyl-1H-indol-5-yl)-N-hydroxyacrylamide
10: 3-(1-Benzenesulfonyl-1H-indoline-5-yl)-N-hydroxyacryl amide

ACCEPTED MANUSCRIPT
European Journal of Medicinal Chemistry
4-Indolyl-N-hydroxyphenylacrylamides as potent HDAC Class I and IIB
inhibitors in Vitro and in Vivo.
Samir Mehndirattaa,1, Ruei-Shian Wangb,c,1, Han-Li Huangb, Chih-Jou Sub, Chia-Ming Hsub, Yi-Wen
Wub, Shiow-Lin Panb,d, Jing-Ping Liou*,a
* Corresponding author. School of Pharmacy, College of Pharmacy, Taipei Medical University,
Taiwan. Email address: jpl@tmu.edu.tw
1 Contributed equally to this work.
a School of Pharmacy, College of Pharmacy, Taipei Medical University (TMU).
b PhD Program for Cancer Biology and Drug Discovery, College of Medical Science and
Technology, TMU
c Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University.
d Department of Pharmacology, College of Medicine, Taipei Medical University.
Abstract. A series of 4,5-indolyl-N-hydroxyphenylacrylamides, as HDAC inhibitors, has been
synthesized and evaluated in vitro and in vivo. 4-Indolyl compounds 13 and 17 functions as potent
inhibitors of HDAC1 (IC50 1.28 nM and 1.34 nM) and HDAC 2 (IC50 0.90 and 0.53 nM).
N-Hydroxy-3-{4-[2-(1H-indol-4-yl)-ethylsulfamoyl]-phenyl}-acrylamide (13) inhibited the human
cancer cell growth of PC3, A549, MDA-MB-231 and AsPC-1 with a GI50 of 0.14, 0.25, 0.32, and
0.24 µM, respectively. In in vivo evaluations bearing prostate PC3 xenografts nude mice model,
compound 13 suppressed tumor growth with a tumor growth inhibition (TGI) of 62.2%.
Immunohistochemistry of protein expressions, in PC-3 xenograft model indicated elevated
acetyl-histone 3 and prominently inhibited HDAC2 protein expressions. Therefore, compound 13
could be a suitable lead for further investigation and the development of selective HDAC 2 inhibitors
as potent anti-cancer compounds.
KEYWORDS. Indole, HDAC, Prostate cancer, Hydroxamic acid, Cancer, Acrylamide
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1. Introduction
Prostate cancer, a solid tumor, is the most death causing disease in men second only to lung
cancer. Histone deacetylases (HDACs) are overexpressed in many cancer types including prostate
cancer and are associated with increased cancer cell proliferation and survival by repressing growth
suppressive genes.[1] A study by Weichart et al comprising 192 samples of prostate cancer reported
overexpressed class 1 HDAC isoforms in most of them (HDAC1: 69.8%, HDAC2: 74%, HDAC3:
94.8%); concluding the important role of HDAC1 and 2 in controlling prostate tumor
dedifferentiation and indicating the highly significant prognostic value of HDAC2.[1f] Recent
studies have indicated the role of over expressed HDAC 2 not only in development of prostate cancer
but also in neurodegeneration leading to cognitive impairment. Studies explore the potential and
thereof the need of HDAC2 inhibitors for the treatment of neurodegenerative diseases such as
Alzheimer’s disease.[1-3] Upregulated HDAC1 levels [4-6] has been observed in pre malignant and
malignant lesions of prostate cancers.
Many studies have concluded that inhibition of class I and II HDACs by histone deacetylase
inhibitors (HDIs), such as suberoylanilide hydroxamic acid (SAHA, 1) and valproic acid (5), led to
growth arrest, differentiation and/or apoptosis of prostate tumor cells (Figure 1).[5,7-9] PXD101 (3),
a sulfonamide based HDI has shown promising results for the treatment of prostatic adenocarcinoma
and has completed a phase 1 clinical trial for advanced solid tumors including prostate cancer.[10-12]
including various other HDIs such as LBH589 (4), valproic acid (5), MS275 (6). Except FK-228
(Romidepsin, 2), all other FDA approved HDIs possess a similar Zn2+ binding Group (ZBG) e.g.
hydroxamic acid; however, they differ significantly in their surface recognition cap and linker.
Indole moiety has the privilege to be the most explored heterocyclic compound for the development
of various anticancer agents e.g. LBH589 (4). However, a lot of focus has been laid on exploring the
indole C2 and C3 position unlike indole -4, -5, -6, and -7 which remain comparatively unexplored.
Some published reports have supported the idea that the position -4, -5, -6 and -7 of indole could be
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potential sites for attaching a connector unit with surface recognition cap for developing potent
HDAC inhibitors.[13-17]
We previously have explored the regioisomeric effects of various positions of indole on tubulin
polymerization inhibition and have developed a novel class of anti-tubulin agents (7, 8).[18] In
addition, we have also reported the syntheses of potent HDIs with indole/indoline (9, 10) as a linker
and a phenyl group as a surface recognition cap.[19-20] All of these studies have revealed that the
compounds with substitution at -4 and -5 position of indole were more potent than other analogues of
respective series. Close observation of these HDIs indicated that type of connector unit e.g. rigid (9
or 10) or flexible (13) could affect the potency of compounds (Figure 2). Prompted from the results
we decided to explore the regioisomeric effect of indole -4 and -5 positions on HDAC inhibitory
activity by interchanging the cap and linker of our previously reported HDAC inhibitors. This has led
to generation of a potent series of 4,5-indolyl-N-hydroxyphenylacrylamides, with indole as surface
recognition cap and phenyl as linker, possessing potent activity against prostate cancer (Figure 3).
Apart from regioisomeric effect of meta and para substitutions of acrylamide side chain with respect
to connector unit (CU); length of CU and substitution at indole N1 has also been studied to complete
the SAR of these hydoxamides.
2. Results and discussion
2.1. Chemistry
To synthesize amine 20, 4-nitroindole was reduced using iron (Fe) and ammonium chloride
(NH4Cl) and corresponding amine was extracted using ethyl acetate (EtOAc) and water. Organic
layer was then collected, dried and directly used for next step. To synthesize amine 21, 4-cynoindole
was reduced using 10% Pd/C in MeOH over a hydrogenator at 40-42 psi and stirred overnight,
removed carefully from hydrogenator, filtered through celite and purified by column
chromatography. Reduction using borane-tetrahydrofuran complex or Pd/C at room temperature did
not yield satisfactory results (Scheme 1).
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To synthesize the amines from 22 to 25; corresponding aldehydes underwent henry reaction to
give corresponding nitro alkenes Attempts to reduce these nitro alkenes using lithium aluminum
hydride (LAH) or Pd/C in methanol (MeOH) produced only poor yields of desired products. Thus,
nitroalkenes were first reduced to nitroalkanes using NaBH4 in methanol followed by nitro group
reduction using Fe-NH4Cl in a mixture of water and isopropyl alcohol (IPA) (1:4) to give 22 to 25 in
quantitative yield (Scheme 1).
The synthesis of compounds 11 to18 is shown in Scheme 2. Amines 20 to 25 were allowed to
react with 3-or 4-bromobenzenesulfonyl chloride or 4- in the presence of pyridine and acetonitrile
(ACN) to respectively yield compounds 35 to 42. Heck coupling with t-butyl acrylate in the presence
of tris(dibenzylideneacetone)dipalladium [Pd2(dba)3] and tri-t-butylphosphonium tetrafluoroborate
{[(t-Bu)3PH]BF4]} afforded cinnamates 43 to 50. Hydrolysis of the methyl ester group in 43 to 50
was achieved by treatment with 1N LiOH solution yielding the corresponding acids which were
subject to amidation with NH2OTHP in the presence of EDC•HCl and HOBt affording the
corresponding protected N-hydroxyamides. De-protection of the OTHP group was achieved with
TFA in dichloromethane (DCM) to yield compounds 11 to 18 in 38 to 56% yield.
2.2. Biological evaluation
2.2.1. HeLa nuclear HDAC enzyme inhibition.
Using
HeLa
nuclear
extract
as
HDAC
source,
the
ability
of
4,5-indolyl-N-hydroxyphenylacrylamides were evaluated to inhibit HDAC activity (Table 1).
Compound 11, where sulfonamide is directly linked to indole C-4, failed to show any potent activity
against HeLa nuclear extract (HDAC IC50 >1000 nM). Increasing the connecter unit (CU) length
from n=0 to n=1, by addition of one methylene group between sulfonamide and indole C-4 (12,
HDAC IC50= 7.13 nM), showed a marked increase in HDAC inhibitory activity compared to its
precursor 11 with ~18 and ~11 fold more potency compared to SAHA (1, HDAC IC50= 131.26 nM)
and PXD101 (3, HDAC IC50= 80 nM) respectively. 12, was found to be more potent than reference
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compound 9 (HDAC IC50= 29.5 nM) and MS275 (6, HDAC IC50 >1000 nM) and comparable with
LBH589.HCl (4, HDAC IC50 =4.90 nM). These results indicated that a flexible CU was well
tolerated in this series of molecules and addition of one carbon distance was a favorable modification.
Compound 13 (n=2, HDAC IC50= 8.56 nM) showed comparable HDAC inhibitory activity with its
precursor 12 and 4.HCl but was found to be more potent against various cancer cell lines compared
to 12. To compare the regioisomeric effect of N-hydroxyacrylamide side chain, compound 14 was
synthesized. Replacement of N-hydroxyacrylamide side chain from para (13) to meta position (14)
led to decrease in HDAC inhibitory activity indicating that a para substitution is favored unlike
PXD101. However 14 (HDAC IC50= 30.45 nM) was still more potent than 1 (HDAC IC50= 131.26
nM), 3 (HDAC IC50= 80 nM), 6 (HDAC IC50 >1000 nM), and comparable with 9 (HDAC IC50= 29.5
nM). Regioisomers at indole C-5 (15 and 16) were less potent compared to 13, however comparable
with reference compounds. Substituting the Indole-N1 with a methyl group (17) maintained HDAC
inhibitory activity as 13. Substituting N-1 with 4-methoxybenzenesulfonyl moiety (18) led to
decrease in HDAC inhibitory activity indicating bulky group substitution at N-1 is not a suitable
modification. In nutshell, all of these compounds, except 11, showed overall better HDAC HeLa
nuclear extract inhibitory activity compared to standard compounds like SAHA, PXD101 and
MS275.
2.2.2. In Vitro Cell Growth Inhibitory Activity.
The synthesized 4,5-indolyl-N-hydroxyphenylacrylamides, 11 to 18, were evaluated for
anti-proliferative activities against prostate carcinoma PC-3 cells, lung carcinoma A549 cells, breast
carcinoma MDA-MB-231 cells and pancreatic carcinoma AsPC-1 cells (Table 1) The cancer cell
growth inhibitory results were consistent with the HDAC extract enzyme inhibitory activity.
Compounds such as 13, 14, 16 and 17 showed better to comparable activities with standard
compounds like SAHA, PXD101 and MS275. Compound 12 (n=1) and 13 (n=2) showed comparable
HDAC inhibitory activities, however the anti-proliferative activities of compound 13 (PC-3; GI50 =
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0.14 µM) was better than 12 (PC-3; GI50 = 0.86 µM). Compound 13 was overall more potent than its
parent compound 9. Being consistent with HDAC inhibitory activity, 14 showed diminished
anti-cancer activity compared to 13; indicating that a para substitution is preferred for optimal
activity. Substitution of N-1 with methyl group (17) showed comparable anti-proliferative activity to
that of 13, except against breast carcinoma MDA-MB-231 cells where 17 (IC50 = 0.08 µM) showed
slight better activity as that of 13 (IC50 = 0.32 µM). Compound 18 was overall less potent than its
precursor compound 17.
2.2.3. HDAC Isoform Inhibition.
Compounds 13 and 17 were tested for HDAC isoform enzyme-inhibitory activity including
HDAC isoform 1 and 2, which are generally overexpressed in prostate cancer cells, using compound
1, 3 and 6 as reference compounds (Table 2). 13 and 17 showed potent HDAC inhibitory activity
against HDAC 1 and 2. These results are consistent with their high efficacy against PC-3 cell lines.
13 (HDAC 1 = 1.28 nM; HDAC 2 = 0.90 nM) and 17 (HDAC 1 = 1.34 nM; HDAC 2 = 0.53 nM)
were 15 to 85 fold more potent than standard compounds 1 (HDAC 1 = 110 nM; HDAC 2 = 120 nM)
and 3 (HDAC 1 = 20 nM; HDAC 2 = 50 nM) against HDAC 1 and 55 to 226 fold more potent for
HDAC 2. Furthermore, results have also indicated that a methyl substitution (17) is favored for the
HDAC inhibitory activity.
2.2.4. Western blot analysis.
In an effort to further validate the target in the 4,5-indolyl-N-hydroxyphenylacrylamide series,
compound 13 was evaluated for the expression of histone 3 and α-tubulin acetylation PC-3 cell line;
which were important biomarkers associated with intracellular HDAC inhibition. Western blot
analysis indicated that compound 13 increased acetylation levels of histone 3 and α-tubulin and was
found equipotent as standard compound 1 in PC-3 cells (Fig. 4A-C).
2.2.5. In vivo Efficacy in Human Prostate PC3 Xenograft.
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We evaluated the in vivo efficacy on compounds 13.Using human prostate PC3 xenograft nude
mouse model (Fig. 5).Once a tumor was approximately 40 mm3 in size and palpable, the mice were
randomized into vehicle control and treatment groups (7-8 mice per group) and control mice received
the vehicle (1.0% carboxymethyl cellulose/0.5% Tween80). Results have indicated (Fig. 5A) that
administration of 13 (20 mg/kg and 40 mg/kg, ip, q2d) significantly reduced tumor volume dose
dependently in nude mice model. The percent of tumor growth inhibition (TGI) of 13 (20 mg/kg and
40 mg/kg) was 51.6% (*p < 0.05) and 62.2% (**p < 0.01) respectively. In addition, no significant
differences in weight loss were observed during 13 treatment periods (Figure 5B).
2.2.6. Effect on Protein Expression in Xenograft Model
After in vivo prostate PC-3 xenograft study, we further conducted the immunohistochemistry of
protein expressions as shown Figure 6. Results indicated the effect of 13 on HDAC inhibition
marker- acetyl-histone 3(Ac-H3) and expression of HDAC isoforms 1 and 2 in xenograft tumors.
Data revealed the information that compound 13 increased the acetyl-histone 3 (Ac-H3) expression
dose-dependently and decreased the HDAC 2 expression in 40 mg/kg dose group. No significant
change in the expression of HDAC 1 was observed.
3. Conclusion
We have designed and evaluated a novel series of 4,5-indolyl-N-hydroxyphenylacrylamide as
potent histone deacetylase inhibitors. Lead compound 13 showed anti-proliferative activity, with
GI50 values ranging from 0.14 to 0.32 µM in a variety of human cancer cell lines from different
organs. 13, has demonstrated better activity than reference compounds 1, 3 and 6. Compound 13
exhibited low nanomolar IC50 values against HDAC 1 and 2 isoforms with 1.28 and 0.90
respectively. Higher potency against HDAC 1 and 2 corresponds to better activity of 13 against
prostate cancer PC-3 cell line. Structure-activity relationship information revealed that the
N-hydroxyacrylamide group placed at the C-4 position of indole displayed the best activity compared
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to C-5 position and a para substitution is a prerequisite for the potent activity. Flexible connector
unit (CU) with n=2 was found to be more potent than its parent compound 9 (rigid CU) and other
molecules of the series including reference compounds. In in vivo efficacy evaluation against human
xenograft model in nude mice bearing PC-3 cancer cell line, compound 13 (40 mg/kg, ip, q2d) not
only demonstrated substantial antitumor activity with a TGI of 62.2% but also found to upregulate
acetyl-histone 3 and α-tubulin expression. Compound 13, is found to be a potent inhibitor of HDAC
Class I and IIB and can decrease level of HDAC2 more selectively than HDAC1 as indicated by
immunohistochemistry
analysis.
In
summary,
N-Hydroxy-3-{4-[2-(1H-indol-4-yl)-ethylsulfamoyl]-phenyl}-acrylamide (13) could be a potential
lead for further investigation to develop selective HDAC2 inhibitors as potent anticancer agents.
4. Experimental section
4.1. Chemistry
Nuclear magnetic resonance spectra were obtained with Bruker DRX-500 spectrometer
(operating at 300 MHz and 500 MHz), with chemical shift in parts per million (ppm, δ) downfield
from TMS as an internal standard. High-resolution mass spectra (HRMS) were measured with a
JEOL (JMS-700) electron impact (EI) mass spectrometer. Purity of the final compounds were
determined using an Hitachi 2000 series HPLC system using C-18 column (Agilent ZORBAX
Eclipse XDB-C18 5 µm. 4.6 mm × 150 mm) and were found to be ≥ 95%. Flash column
chromatography was done using silica gel (Merck Kieselgel 60, No. 9385, 230-400 mesh ASTM).
All reactions were carried out under an atmosphere of dry nitrogen.
General procedures for reduction of nitro group - Method A. Nitro compound (1 mmol) was
dissolve in a 0.1 M mixture of IPA: H2O (4:1) and iron powder (Fe, 3 mmol), ammonium chloride
(NH4Cl, 2 mmol) were added and refluxed for two hours. After cooling down to room temperature
reaction mixture was filtered through celite, extracted with EtOAc (25 mL × 3). The combined
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organic layer was dried over anhydrous MgSO4 and concentrated under reduced pressure and
purified by column chromatography. Method B. 4-cynoindole (1.0 mmol) was dissolved in MeOH in
a hydrogenator reaction flask, 10% Pd/C (10 mg) was added and hydrogenator was shook at 40-42
psi overnight. Reaction mixture was carefully taken out from hydrogenator, filtered through celite
and purified by column chromatography (EtOAc: MeOH; 9.5 : 0.5) with few drops of aqueous
ammonia .
General procedure for the synthesis of amines 22 to 25. Henry reaction was performed as
reported in the literature.[21] Thus obtained nitroalkene (1 mmol) was dissolved in methanol (15 mL)
and this solution was added sodium borohydride (NaBH4, 4.4 mmol) portion wise. Reaction was
stirred for 45 mins and was quenched using 1mL acetic acid. Compound was purified from a short
column to give an off-white residue. Thus obtained nitro products were reduced using method – A.
General procedure for sulfonamide synthesis. To a mixture of amine (1 mmol), pyridine (1 mL)
and acetonitrile (0.25 mL) was added 4-bromobenzenesulfonyl chloride (1.1 mmol) portion wise,
and then was allowed to stir at room temperature for 2 h. The reaction was quenched with water and
was acidified with dropwise addition of 3N HCl. The reaction mixture was then extracted with
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 = 3: 7)
General procedure for Heck reaction. A mixture of bromo compound (1 mmol), Pd2(dba)3 (0.1
mmol, [(t-Bu)3PH]BF4 (0. 2 mmol), K2CO3 (1mmol), TEA (3 mmol), methyl acrylate (1 mmol), and
o
DMF (5 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 gel
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General Procedure for hydroxamic acid synthesis. A mixture of 1N LiOH (3 mL) and Heck ester
(1 mmol) was stirred at 40 oC for 2 hours. The reaction was concentrated under reduced pressure and
then was added water. The mixture was acidified with 3N HCl to give off white precipitates. The
off-white solid (1mmol) was dissolved in DMF (10 mL) and was added EDC•HCl (1.5 mmol), HOBt
hydrate (1.5 mmol) and TEA (3 mmol). After being stirred at room temperature for 30 min,
NH2OTHP (1.2 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 chromatography (EtOAc: n-hexane = 1: 1) to give
colorless liquid. To the resulting product dissolved in CH3OH (5mL) was added of 10% TFA (5 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
compounds.
4.1.1. Syntheses of amines 20-25.
4.1.1.1. 4-Aminoindole (20). Compound 20 was synthesized using 4-nitroindole (19, 0.5 g, 3.08
mmol), iron powder (Fe, 0.5 g, 9.24 mmol) and ammonium chloride (NH4Cl, 0.3 g, 6.16 mmol)
followed by method-A in 89 % yield; 1H NMR (300 MHz, CD3OD): δ 6.38 (dd, J = 6.9 Hz, J = 0.9
Hz, 1H), 6.50 (dd, J = 6.0 Hz, J = 3.3 Hz, 1H), 6.81-6.92 (m, 1H), 7.10 (d, J = 3.3 Hz, 1H).
4.1.1.2. 1H-Indol-4-yl-methylamine (21). Compound 20 was synthesized using 4-cynoindole (26, 0.2
g, 1.40 mmol) and 10 % pd/c followed by method-B in 82% yield; 1H NMR (300 MHz, CD3OD): δ
4.07 (s, 1H), 6.56 (dd, J = 3.0 Hz, J = 3.3 Hz, 1H), 7.99 (d, J = 7.2 Hz, 1H), 7.09 (d, J = 7.2 Hz, 1H),
7.26 (d, J = 3.3 Hz, 1H), 7.32 (d, J = 8.1 Hz, 1H).
4.1.1.3. 4-(2-amino-ethyl)-1H-indole (22). Compound 22 was synthesized using nitro alkene (29,
1.32 g, 6.53 mmol) obtained by reported procedure[3] and further followed by general procedure for
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1
the synthesis of amines 22 to 25 in 65% over all yield (from 27); H NMR (500 MHz, MeOD): δ
2.99 - 3.03 (m, 2H), 3.10 (t, J = 8.0 Hz, 2H), 6.51 (d, J = 3.0 Hz, 1H), 6.83 (d, J = 7.0 Hz, 1H), 7.03
(d, J = 8.0 Hz, 1H), 7.21 (d, J = 3.5 Hz, 1H), ), 7.25 (d, J = 8.0 Hz, 1H).
4.1.1.4. 5-(2-amino-ethyl)-1H-indole (23) Compound 23 was synthesized using nitro alkene (30, 1.32
g, 6.53 mmol) obtained by reported procedure and further followed by general procedure for the
synthesis of amines 22 to 25 in 61% over all yield (from 28); 1H NMR (500 MHz, MeOD): δ 2.84 (t,
J = 7.0 Hz, 2H), 2.94 (t, J = 7.0 Hz, 2H), 6.36 (d, J = 3.0 Hz, 1H), 6.96 (dd, J = 8.0 Hz, J = 8.5 Hz,
1H), 7.18 (d, J = 3.0 Hz, 1H), 7.31 (d, J = 8.0 Hz, 1H), ), 7.38 (s, 1H).
4.1.1.5. N-Methyl-4-formylindole (31) To the solution of 28 in dimethylformamide (DMF)
maintained at 0 oC was added sodium hydride (NaH, 0.36 g, 10.32 mmol) and was stirred for 5 mins
before adding methylidodide (MeI, 0.44 mL, 8.25 mmol) and the reaction was stirred for 30 mins.
o
Reaction was quenched by slow addition of water at 0 C and extracted using EtOAc (25 mL × 3).
The combined organic layer was collected, dried over anhydrous MgSO4 and concentrated under
reduced pressure to give a pale yellow residue, which was purified by silica gel chromatography
(EtOAc: Hexane, 4:1) to give 31 as off white residue in 89% yield; 1H NMR (300 MHz, CDCl3): δ
3.89 (s, 3H), 7.26-7.28 (m, 2H), 7.38 (t, J = 8.1 Hz, 1H), 7.46 (t, J = 8.1 Hz, 2H), 10.27 (s, 1H).
4.1.1.6. 1-(4-Methoxy-benzenesulfonyl)-1H-indole-4-carbaldehyde (32) To the solution of 28 (1 g,
6.89 mmol) in dichloromethane (DCM) was added tetrabutylammonium hydrogen sulfate (TBAHS,
0.35 g, 1.03 mmol), potassium hydroxide (KOH, 0.77 g, 13.78 mmol) and stirred for 20 mins before
adding 4-Methoxybenzenesulfonyl chloride (2.14 g, 10.33 mmol) and the reaction was stirred for 12
hours at room temperature. Reaction was quenched by addition of water and neutralized with 3N
HCl and extracted using EtOAc (25 mL × 3). The combined organic layer was collected, dried over
anhydrous MgSO4 and concentrated under reduced pressure to give a pale yellow residue, which was
purified by silica gel chromatography (EtOAc: Hexane, 4:1) to give 32 as off-white residue in 45%
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yield; 1H NMR (300 MHz, CDCl3): δ 3.81 (s, 3H), 6.90 (d, J = 9.0 Hz, 2H), 7.46 - 7.51 (m, 2H), 7.72
- 7.77 (m, 2H), 7.82 – 7.85 (m, 2H), 8.28 (t, J = 8.1 Hz, 1H), 10.91 (s, 1H).
4.1.1.7. 2-(1-Methyl-1H-indol-4-yl)-ethylamine (24) Compound 24 was synthesized using nitro
alkene (33, 1.32 g, 6.53 mmol) obtained by reported procedure and further followed by general
procedure for the synthesis of amines 22 to 25 in 62% over all yield (from 31); 1H NMR (300 MHz,
MeOD): δ 2.72. (t, J = 7.5 Hz, 2H), 2.92 (t, J = 7.5 Hz, 2H), 3.82 (s, 3H), 6.51 (dd, J = 3.3 Hz, J =
3.3 Hz, 1H), 6.96 (d, J = 7.2 Hz, 1H), 7.12 (d, J = 3.0 Hz, 1H), 7.16-722 (m, 1H), 7.27-7.30 (m, 1H).
4.1.1.8. 2-[1-(4-Methoxy-benzenesulfonyl)-1H-indol-4-yl]-ethylamine (25) Compound 25 was
synthesized using nitro alkene (34, 0.51 g, 1.42 mmol) obtained by reported procedure and further
followed by general procedure for the synthesis of amines 22 to 25 in 58% over all yield (from 32);
1H NMR (300 MHz, MeOD): δ 2.74. (t, J = 7.0 Hz, 2H), 2.85 (t, J = 7.5 Hz, 2H), 3.81 (s, 3H), 6.61
(dd, J = 3.3 Hz, J = 3.0 Hz, 1H), 6.73 (d, J = 6.9 Hz, 2H), 6.89-7.13 (m, 2H), 7.38 (d, J = 7.5 Hz,
2H), 7.79 (m, 2H).
4.1.2. Syntheses of sulfonamides 35-42.
4.1.2.1. 4-Bromo-N-(1H-indol-4-yl)-benzenesulfonamide (35) Compound 35 was synthesized using a
mixture of 4-aminoindole (20, 1.0 g, 7.56 mmol), pyridine (4 mL), acetonitrile (1 mL) and
4-bromobenzenesulfonyl chloride (2.12 g, 8.32 mmol) followed by general procedure for
sulfonamide synthesis in 61 % yield; 1H NMR (300 MHz, CDCl3): δ 6.34 (t, J = 2.1 Hz, 1H), 6.70 (s,
1H), 7.05 (d, J = 6.9 Hz, 1H), 7.10 (d, J = 7.8 Hz, 1H), 7.15-7.17 (m, 1H), 7.25 (d, J = 6.0 Hz, 1H), 7.
51 (d, J = 8.7 Hz, 1H), 7.63 (d, J = 8.7 Hz, 1H), 8.25 (bs, 1H).
4.1.2.2. 4-Bromo-N-(1H-indol-4-ylmethyl)-benzenesulfonamide (36) Compound 36 was synthesized
using a mixture of 1H-Indol-4-yl-methylamine (21, 1.0 g, 6.84 mmol), pyridine (4 mL), acetonitrile
(1 mL) and 4-bromobenzenesulfonyl chloride (2.24 g, 7.52 mmol) followed by general procedure for
1
sulfonamide synthesis in 71 % yield; H NMR (300 MHz, CDCl3): δ 4.45 (d, J = 5.7 Hz, 2H), 4.74
12

ACCEPTED MANUSCRIPT
(bs, 1H), 6.47 (t, J = 1.2 Hz, 1H), 6.90 (d, J = 6.9 Hz, 1H), 7.09 (t, J = 7.8 Hz, 1H), 7.23 (t, J = 2.7
Hz, 1H), 7.34 (d, J = 8.1 Hz, 1H), 7.59-7.61 (m, 2H), 7.68-7.72 (m, 2H), 8.28 (bs, 1H).
4.1.2.3. 4-Bromo-N-[2-(1H-indol-4-yl)-ethyl]-benzenesulfonamide (37) Compound 37 was
synthesized using a mixture of 2-(1H-Indol-4-yl)-ethylamine (22, 1.0 g, 6.24 mmol), pyridine (4 mL),
acetonitrile (1 mL) and 4-bromobenzenesulfonyl chloride (1.78 g, 6.86 mmol) followed by general
1
procedure for sulfonamide synthesis in 78 % yield; H NMR (500 MHz, CDCl3): δ 3.04 (t, J = 7.0
Hz, 2H), 3.33-3.37 (m, 2H), 6.40 (t, J = 1.0 Hz, 1H), 6.79 (d, J = 7.5 Hz, 1H), 7.08 (t, J = 7.5 Hz,
1H), 7.17 (t, J = 3.0 Hz, 1H), 7.25-7.29 (m, 1H), 7.46-7.47 (m, 4H), 8.27 (bs, 1H).
4.1.2.4. 3-Bromo-N-[2-(1H-indol-4-yl)-ethyl]-benzenesulfonamide (38) Compound 38 was
synthesized using a mixture of 2-(1H-Indol-4-yl)-ethylamine (22, 1.0 g, 6.24 mmol), pyridine (4 mL),
acetonitrile (1 mL) and 3-bromobenzenesulfonyl chloride (1.78 g, 6.86 mmol) followed by general
1
procedure for sulfonamide synthesis in 75 % yield; H NMR (500 MHz, CDCl3): δ 3.06 (t, J = 7.0
Hz, 2H), 3.36-3.40 (m, 2H), 6.40 (s, 1H), 6.80 (d, J = 7.0 Hz, 1H), 7.09 (t, J = 7.5 Hz, 1H), 7.19 (t, J
= 2.5 Hz, 1H), 7.25-7.29 (m, 3H), 7.62-6.64 (m,; 2H), 7.85 (s, 1H), 8.25 (bs, 1H).
4.1.2.5. 4-Bromo-N-[2-(1H-indol-5-yl)-ethyl]-benzenesulfonamide (39). Compound 37 was
synthesized using a mixture of 2-(1H-Indol-5-yl)-ethylamine (23, 1.0 g, 6.24 mmol), pyridine (4 mL),
acetonitrile (1 mL) and 4-bromobenzenesulfonyl chloride (1.78 g, 6.86 mmol) followed by general
1
procedure for sulfonamide synthesis in 66 % yield; H NMR (300 MHz, CDCl3): δ 2.86 (t, J = 6.3
Hz, 2H), 3.25-3.31 (m, 2H), 4.40 (t, J = 5.7 Hz, 1H), 6.48-6.90 (m, 1H), 6.87-6.89 (m, 1H), 7.24 (d, J
= 2.7 Hz, 1H), 7.28-7.31 (m, 2H), 7.52-7.60 (m, 4H), 8.27 (bs, 1H).
4.1.2.6. 3-Bromo-N-[2-(1H-indol-5-yl)-ethyl]-benzenesulfonamide (40). Compound 38 was
synthesized using a mixture of 2-(1H-Indol-5-yl)-ethylamine (23, 1.0 g, 6.24 mmol), pyridine (4 mL),
acetonitrile (1 mL) and 3-bromobenzenesulfonyl chloride (1.78 g, 6.86 mmol) followed by general
13

ACCEPTED MANUSCRIPT
1
procedure for sulfonamide synthesis in 61 % yield; H NMR (500 MHz, CDCl3): δ 2.83 (t, J = 7.0
Hz, 2H), 3.25-3.29 (m, 2H), 4.59 (t, J = 6.0 Hz, 1H), 6.45 (d, J = 2.5 Hz, 1H), 6.86-6.88 (m; 1H),
7.19 (t, J = 2.5 Hz, 1H), 7.26-7.29 (m, 3H), 7.62-7.66 (m, 2H), 7.90 (s, 1H), 8.29 (bs, 1H).
4.1.2.7. 4-Bromo-N-[2-(1-methyl-1H-indol-4-yl)-ethyl]-benzenesulfonamide (41). Compound 41 was
synthesized using a mixture of 2-(1-Methyl-1H-indol-4-yl)-ethylamine (24, 1.0 g, 5.74 mmol) in
pyridine (4 mL), acetonitrile (1 mL) and 4-bromobenzenesulfonyl chloride (1.88 g, 6.32 mmol)
followed by general procedure for sulfonamide synthesis in 68 % yield; 1H NMR (300 MHz, CDCl3):
δ 3.06 (t, J = 6.3 Hz, 2H), 3.33-3.40 (m, 2H), 3.80 (s, 1H), 4.35 (t, J = 6.0 Hz, 1H), 6.34 (dd, J = 3.0
Hz, J = 3.3 Hz, 1H), 6.81 (d, J = 7.2 Hz, 1H), 7.04 (d, J = 3.3 Hz, 1H), 7.11-7.16 (m, 1H), 7.22- 7.27
(m, 1H), 7.49 (bs, 4H).
4.1.2.8. 4-Bromo-N-[2-(1-methyl-1H-indol-4-yl)-ethyl]-benzenesulfonamide (42). Compound 42 was
synthesized using a mixture of 2-[1-(4-Methoxy-benzenesulfonyl)-1H-indol-4-yl]-ethylamine (25,
0.36 g, 1.08 mmol), pyridine (1 mL), acetonitrile (0.5 mL) and 4-bromobenzenesulfonyl chloride
1
(0.31 g, 1.18 mmol) followed by general procedure for sulfonamide synthesis in 49 % yield; H
NMR (300 MHz, CDCl3): δ 3.01 (t, J = 7.2 Hz, 2H), 3.32-3.41 (m, 2H), 3.78 (s, 3H), 6.64-6.67 (m,
3H), 6.92 (dd, J = 6.9 Hz, J = 3.0 Hz, 1H), 7.12 (dd, J = 3.0 Hz, J = 3.3 Hz 1H), 7.22 (m, 1H),
7.61-7.64 (m, 2H), 7.68-7.72 (m, 2H), 7.78 (d, J = 7.5 Hz, 1H), 7.84-7.89 (m, 2H).
4.1.3. Syntheses of Heck products (methyl esters) 43-50.
4.1.3.1 3-[4-(1H-Indol-4-ylsulfamoyl)-phenyl]-acrylic acid methyl ester (43). Compound 43 was
synthesized using a mixture of 35 (1.0 g, 2.85 mmol), Pd2(dba)3, (0.26 g, 0.28 mmol),
[(t-Bu)3PH]BF4 (0.16 g, 0.57 mmol), K2CO3 (0.39 g, 2.85 mmol), TEA (1.17 mL, 8.55 mmol),
methyl acrylate (0.30 mL, 3.42 mmol), and DMF (10 mL) followed by general procedure for Heck
1
reaction in 89 % yield; H NMR (300 MHz, CDCl3): δ 3.82 (s, 3H), 6.33-6.35 (m, 1H), 6.45(d, J =
16.2 Hz, 1H), 6.75 (s, 1H), 7.04-7.14 (m, 3H), 7.22 -7.25 (m, 1H), 7.49 (d, J = 8.7 Hz, 1H), 7. 61 (d,
14

ACCEPTED MANUSCRIPT
J = 16.2 Hz, 1H), 7.78 (d, J = 8.4 Hz, 1H), 8.24 (bs, 1H).
4.1.3.2. 3-{4-[(1H-Indol-4-ylmethyl)-sulfamoyl]-phenyl}-acrylic acid methyl ester (44). Compound
44 was synthesized using a mixture of 36 (1.2 g, 3.28 mmol), Pd2(dba)3 (0.30 g, 0.33 mmol),
[(t-Bu)3PH]BF4 (0.19 g, 0.66 mmol), K2CO3 (0.36 g, 2.63 mmol), TEA (1.56 mL, 9.84 mmol),
methyl acrylate (0.31 mL, 3.93 mmol) and DMF (10 mL) followed by general procedure for Heck
reaction in 61 % yield; 1H NMR (300 MHz, CDCl3): δ 3.85 (s, 3H), 4.67 (d, J = 6.0 Hz, 2H), 4.72 (t,
J = 6.0 Hz, 1H), 6.47-6.59 (m, 1H), 6.56 (t, J = 16.2 Hz, 1H), 6.90 (d, J = 7.2 Hz, 1H), 7.08 (t, J =
8.1 Hz, 1H), 7.22 (t, J = 3.0 Hz, 1H), 7.38 (d, J = 8.4 Hz, 1H), 7.60 (d, J = 8.4 Hz, 2H), 7.70 (d, J =
16.2 Hz, 1H), 7.88 (d, J = 8.4 Hz, 2H).
4.1.3.3. 3-{4-[2-(1H-Indol-4-yl)-ethylsulfamoyl]-phenyl}-acrylic acid methyl ester (45). Compound
45 was synthesized using a mixture of 37 (1.0 g, 2.63 mmol), Pd2(dba)3 (0.24 g, 0.26 mmol,
[(t-Bu)3PH]BF4 (0.15 g, 0.52 mmol), K2CO3 (0.36 g, 2.63 mmol), TEA (0.74 mL, 5.26 mmol),
methyl acrylate (0.28 mL, 2.63 mmol), and DMF (10 mL) followed by general procedure for Heck
1
reaction in 92 % yield; H NMR (300 MHz, MeOD): δ 2.99 (t, J = 7.2 Hz, 2H), 3.20-3.25 (m, 2H),
3.79 (s, 3H), 6.36-6.37 (m, 1H), 6.61 (d, J = 15.6 Hz, 1H), 6.74 (d, J = 6.9 Hz, 1H), 6.75-7.00 (m,
1H), 7.17 (d, J = 3.3 Hz, 1H), 7.23 (d, J = 8.1 Hz, 1H), 7.61-7.66 (m, 3H), 7.75 (d, J = 8.7 Hz, 2H).
4.1.3.4. 3-{3-[2-(1H-Indol-4-yl)-ethylsulfamoyl]-phenyl}-acrylic acid methyl ester (46). Compound
46 was synthesized using a mixture of 38 (1.0 g, 2.63 mmol), Pd2(dba)3 (0.24 g, 0.26 mmol,
[(t-Bu)3PH]BF4 (0.15 g, 0.52 mmol), K2CO3 (0.36 g, 2.63 mmol), TEA (0.74 mL, 5.26 mmol),
methyl acrylate (0.28 mL, 2.63 mmol), and DMF (10 mL) followed by general procedure for Heck
reaction in 88 % yield; 1H NMR (500 MHz, DMSO): δ 2.89 (t, J = 7.5 Hz, 2H), 3.03 (t, J = 7.5 Hz,
2H),3.72 (s, 3H), 6.30 (s, 1H), 6.68-6.73 (m, 2H), 6.93 (t, J = 7.5 Hz, 1H), 7.20 (d, J = 8.0 Hz, 1H),
7.24 (t, J = 2.0 Hz, 1H), 7.60 (t, J = 7.5 Hz, 1H), 7.70 (d, J = 16.0 Hz, 1H) 7.78-7.79 (m, 2H),
7.92-7.96 (m,3H), 8.04 (s, 1H).
15

ACCEPTED MANUSCRIPT
4.1.3.5. 3-{4-[2-(1H-Indol-5-yl)-ethylsulfamoyl]-phenyl}-acrylic acid methyl ester (47). Compound
47 was synthesized using a mixture of 39 (1.0 g, 2.63 mmol), Pd2(dba)3 (0.24 g, 0.26 mmol,
[(t-Bu)3PH]BF4 (0.15 g, 0.52 mmol), K2CO3 (0.36 g, 2.63 mmol), TEA (0.74 mL, 5.26 mmol),
methyl acrylate (0.28 mL, 2.63 mmol), and DMF (10 mL) followed by general procedure for Heck
1
reaction in 89 % yield; H NMR (500 MHz, CDCl3): δ 2.84 (t, J = 6.5 Hz, 2H), 3.25-3.29 (m, 2H),
3.82 (s, 3H), 6.44 (t, J = 2.0 Hz, 1H), 6.48 (d, J = 16.0 Hz, 1H), 6.85-6.87 (m, 1H), 7.19 (d, J = 2.5
Hz, 1H), 7.25-7.28 (m, 2H), 7.51 (d, J = 8.5 Hz, 1H), 7.65 (d, J = 16.0 Hz, 1H), 7.71 (d, J = 8.0 Hz,
1H), 8.27 (bs, 1H).
4.1.3.6. 3-{3-[2-(1H-Indol-5-yl)-ethylsulfamoyl]-phenyl}-acrylic acid methyl ester (48). Compound
48 was synthesized using a mixture of 40 (1.0 g, 2.63 mmol), Pd2(dba)3 (0.24 g, 0.26 mmol,
[(t-Bu)3PH]BF4 (0.15 g, 0.52 mmol), K2CO3 (0.36 g, 2.63 mmol), TEA (0.74 mL, 5.26 mmol),
methyl acrylate (0.28 mL, 2.63 mmol), and DMF (10 mL) followed by general procedure for Heck
1
reaction in 81 % yield; H NMR (500 MHz, CDCl3): δ 2.84 (t, J = 6.5 Hz, 2H), 3.25-3.29 (m, 2H),
3.83 (s, 3H), 6.42-6.45 (m, 2H), 6.83-6.85 (m, 1H), 7.20 (t, J = 2.5 Hz, 1H), 7.26-7.29 (m, 2H), 7.45
(t, J = 7.5 Hz, 1H), 7.57 (d, J = 16.5 Hz, 1H), 7.63 (d, J = 7.5 Hz, 1H), 7.74-7.75 (m, 2H).
4.1.3.7. 3-{4-[2-(1-Methyl-1H-indol-4-yl)-ethylsulfamoyl]-phenyl}-acrylic acid methyl ester (49).
Compound 49 was synthesized using a mixture of 41 (1.0 g, 2.85 mmol), Pd2(dba)3 (0.26 g, 0.28
mmol), [(t-Bu)3PH]BF4 ] (0.16 g, 0.56 mmol), K2CO3 (0.39 g, 2.85 mmol), TEA (0.93 mL, 8.55
mmol), methyl acrylate (0.42 mL, 3.42 mmol), and DMF (10 mL) followed by general procedure for
Heck reaction in 56 % yield; 1H NMR (300 MHz, CDCl3): δ 2.67 (t, J = 6.9 Hz, 2H), 3.17 (t, J = 7.2
Hz, 2H), 3.69 (s, 3H), 3.84 (s, 3H), 6.50 (d, J = 15.9 Hz, 1H), 6.54-6.56 (m, 1H), 6.89 (d, J = 6.3 Hz,
1H), 6.75 (t, J = 3.0 Hz, 1H),7.14-7.23 (m, 2H), 7.58 (d, J = 8.4 Hz, 2H), 7.66 (d, J = 15.9 Hz, 1H),
7.79 (d, J = 8.4 Hz, 2H).
4.1.3.8. 3-(4-{2-[1-(4-Methoxy-benzenesulfonyl)-1H-indol-4-yl]-ethylsulfamoyl}-phenyl)-acrylic acid
16

ACCEPTED MANUSCRIPT
methyl ester (50). Compound 50 was synthesized using a mixture of 42 (0.3 g, 0.62 mmol), Pd2(dba)3
(0.06 g, 0.06 mmol), [(t-Bu)3PH]BF4] (0.03 g, 0.12 mmol), K2CO3 (0.08 g, 0.62 mmol), TEA (0.23
mL, 1.67 mmol), methyl acrylate (0.11 mL, 0.93 mmol), and DMF (5 mL) followed by general
procedure for Heck reaction in 61 % yield; 1H NMR (300 MHz, CDCl3): δ 2.95 - 2.97 (m, 2H), 3.13
- 3.16 (m, 2H), 3.80 (s, 3H), 3.82 (s, 3H), 6.63 - 6.72 (m, 2H), 6.97-6.99 (m, 3H), 7.18 (t, J = 7.8 Hz,
1H), 7.62-7.72 (m, 6H), 7.81-7.99 (m, 4H).
4.1.4. Syntheses of hydroxamic acids 11-18.
4.1.4.1. N-Hydroxy-3-[4-(1H-indol-4-ylsulfamoyl)-phenyl]-acrylamide (11). Compound 11 was
synthesized using free acid (0.82 g, 2.39 mmol), obtained after hydrolysis of compound 43 (1 g, 1.81
mmol), and a mixture of EDC•HCl (0.55 g, 3.58 mmol), HOBt (0.48 g, 3.58 mmol), NMM (0.92 mL,
8.36 mmol) and NH2OTHP (0.31 g, 2.63 mmol) followed by general procedure for hydroxamic acid
synthesis in 49% yield (from 43); mp: 158-159 oC; purity: 95.13 %. 1H NMR (300 MHz, CD3OD): δ
6.40 (t, J = 3.0 Hz, 1H), 6.53 (d, J = 15.9 Hz, 1H), 6.84 (d, J = 6.9 Hz, 1H), 6.93 (t, J = 7.8 Hz, 1H),
7.05 (d, J = 3.3 Hz, 1H), 7.11 (d, J = 7.1 Hz, 1H), 7.22 (d, J = 15.9 Hz, 1H), 7.47 (d, J = 8.4 Hz, 2H),
7.68 (d, J = 8.4 Hz, 2H). MS (ESI) m/z: 356.1 (M-H+). HRMS (ESI) for C17H15N3O4S (M-H+): calcd,
356.0895; found, 356.1869.
4.1.4.2. N-Hydroxy-3-{4-[(1H-indol-4-ylmethyl)-sulfamoyl]-phenyl}-acrylamide (12). Compound 12
was synthesized using free acid (1.1 g, 2.08 mmol), obtained after hydrolysis of compound 44 (1.2 g,
3.02 mmol), and a mixture of EDC•HCl (0.72 g, 7.28 mmol), HOBt hydrate (0.62 g, 3.12 mmol),
NMM (1.2 mL, 7.28 mmol) and NH2OTHP (0.40 g, 3.08 mmol) followed by general procedure for
o
1
hydroxamic acid synthesis 53% yield (overall from 44); mp: 161-163 C; purity: 98.45 %. H NMR
(300 MHz, CD3OD): δ 4.35 (s, 2H), 6.46 (d, J = 3.0 Hz, 1H), 6.55 (d, J = 15.6 Hz, 1H), 6.84 (d, J =
7.5 Hz, 1H), 6.96 (t, J = 8.1 Hz, 1H), 7.19 (d, J = 3.0 Hz, 1H), 7.24 (d, J = 8.1 Hz, 1H), 7.55-7.61 (m,
3H), 7.76 (d, J = 8.1 Hz, 2H). MS (ESI) m/z: 370.1 (M-H+). HRMS (ESI) for C18H17N3O4S (M-H+):
17

ACCEPTED MANUSCRIPT
calcd, 370.0940; found, 370.0862.
4.1.4.3. N-Hydroxy-3-{4-[2-(1H-indol-4-yl)-ethylsulfamoyl]-phenyl}-acrylamide (13). Compound 13
was synthesized using free acid (1.0 g, 2.70 mmol), obtained after hydrolysis of compound 45 (1 g,
2.60 mmol), and a mixture of EDC•HCl (0.77 g, 4.05 mmol), HOBt hydrate (0.62 g, 4.05 mmol),
NMM (1.13 mL, 8.1 mmol) and NH2OTHP (0.38 g, 3.24 mmol) followed by general procedure for
o
1
hydroxamic acid synthesis 56% yield (overall from 45); mp: 164-165 C; purity: 97.52 %. H NMR
(300 MHz, CD3OD): δ 2.96-3.01 (m, 2H), 3.23 (t, J = 8.1 Hz, 2H), 6.37 (d, J = 3.0 Hz, 1H), 6.57 (d,
J = 15.6 Hz, 1H), 6.74 (d, J = 6.9 Hz, 1H), 6.97 (d, J = 8.1 Hz, 1H), 7.17 (d, J = 3.0 Hz, 1H), 7.21 (d,
J = 7.2 Hz, 1H), 7.56-7.64 (m, 3H), 6.74 (d, J = 8.1 Hz, 2H). MS (ESI) m/z: 386.1 (M+H+). HRMS
(ESI) for C19H19N3O4S (M+H+): calcd, 386.1095; found, 386.1169.
4.1.4.4. N-Hydroxy-3-{3-[2-(1H-indol-4-yl)-ethylsulfamoyl]-phenyl}-acrylamide (14). Compound 14
was synthesized using free acid (1.0 g, 2.70 mmol), obtained after hydrolysis of compound 46 (1 g,
2.60 mmol), and a mixture of EDC•HCl (0.77 g, 4.05 mmol), HOBt hydrate (0.62 g, 4.05 mmol),
NMM (1.13 mL, 8.1 mmol) and NH2OTHP (0.38 g, 3.24 mmol) followed by general procedure for
hydroxamic acid synthesis 51% yield (overall from 46); mp: 161-163 oC; purity: 100.00 %. 1H NMR
(500 MHz, DMSO): δ 2.70 (t, J = 7.0 Hz, 2H), 2.93-2.98 (m, 2H), 6.29 (s, 1H), 6.62 (d, J = 16.0 Hz,
1H), 6.83 (t, J = 8.5 Hz, 1H), 7.23-7.25 (m, 3H), 7.59 (d, J = 16.0 Hz, 1H), 7.75 (d, J = 8.5 Hz, 3H),
7.84 (d, J = 8.0 Hz, 2H). MS (ESI) m/z: 386.1 (M+H+). HRMS (ESI) for C19H19N3O4S (M+H+):
calcd, 386.1095; found, 386.1169.
4.1.4.5. N-Hydroxy-3-{4-[2-(1H-indol-5-yl)-ethylsulfamoyl]-phenyl}-acrylamide (15). Compound 15
was synthesized using free acid (1.0 g, 2.70 mmol), obtained after hydrolysis of compound 47 (1 g,
2.60 mmol), and a mixture of EDC•HCl (0.77 g, 4.05 mmol), HOBt hydrate (0.62 g, 4.05 mmol),
NMM (1.13 mL, 8.1 mmol) and NH2OTHP (0.38 g, 3.24 mmol) followed by general procedure for
hydroxamic acid synthesis 48 % yield (overall from 47); mp: 159-160 oC; purity: 96.10 %. 1H NMR
18

ACCEPTED MANUSCRIPT
(300 MHz, MeOD): δ 2.78 (t, J = 7.2 Hz, 2H), 3.15 (t, J = 7.5 Hz, 2H), 7.73 (d, J = 3.0 Hz, 1H), 6.57
(d, J = 15.6 Hz, 1H), 6.81-6.85 (m, 1H), 7.18-7.24 (m, 1H), 7.44 (d, J = 15.6 Hz, 1H), 7.56 (d, J =
8.4 Hz, 1H), 7.71 (d, J = 8.1 Hz, 2H). MS (ESI) m/z: 386.1 (M+H+). HRMS (ESI) for C19H19N3O4S
(M+H+): calcd, 386.1095; found, 386.1169.
4.1.4.6. N-Hydroxy-3-{3-[2-(1H-indol-5-yl)-ethylsulfamoyl]-phenyl}-acrylamide (16). Compound 16
was synthesized using free acid (1.0 g, 2.70 mmol), obtained after hydrolysis of compound 48 (1 g,
2.60 mmol), and a mixture of EDC•HCl (0.77 g, 4.05 mmol), HOBt hydrate (0.62 g, 4.05 mmol),
NMM (1.13 mL, 8.1 mmol) and NH2OTHP (0.38 g, 3.24 mmol) followed by general procedure for
hydroxamic acid synthesis 38 % yield (overall from 48); mp: 162-163 oC; purity: 97.86 %. 1H NMR
(500 MHz, MeOD): δ 2.73 (t, J = 7.5 Hz, 2H), 3.15 (t, J = 7.5 Hz, 2H), 6.30 (d, J = 3.0 Hz, 1H), 6.50
(d, J = 16.0 Hz, 1H), 6.81 (d, J = 8.5 Hz, 1H), 7.15 (d, J = 3.0 Hz, 1H), 7.20-7.21 (m, 2H), 7.46 (t, J
= 7.5 Hz, 1H), 7.54 (d, J = 15.5 Hz, 1H), 7.67-7.71 (m, 2H), 7.88 (s, 1H). MS (ESI) m/z: 386.1
(M+H+). HRMS (ESI) for C19H19N3O4S (M+H+): calcd, 386.1095; found, 386.1169.
4.1.4.7. N-Hydroxy-3-{4-[2-(1-methyl-1H-indol-4-yl)-ethylsulfamoyl]-phenyl}-acrylamide (17).
Compound 17 was synthesized using free acid (0.15 g, 0.39 mmol), obtained after hydrolysis of
compound 49 (1 g, 2.50 mmol), and a mixture of EDC•HCl (0.09 g, 0.58 mmol), HOBt hydrate (0.08
g, 0.58 mmol), NMM (0.11 mL, 1.05 mmol) and NH2OTHP (0.05 g, 0.43 mmol) followed by
o
general procedure for hydroxamic acid synthesis 55 % yield (overall from 49); mp: 147-148 C;
1
purity: 98.80 %. H NMR (300 MHz, MeOD): δ 2.98 (t, J = 6.9 Hz, 2H), 3.22 (t, J = 7.1 Hz, 2H),
3.75 (s, 3H), 6.34 (d, J = 3.3 Hz, 1H), 6.57(d, J = 15.6 Hz, 1H), 6.78 (d, J = 7.2 Hz, 1H), 7.03 (d, J =
7.8 Hz, 1H), 7.07-7.09 (m, 2H), 7.19 (d, J = 8.1 Hz, 1H), 7.56-7.62 (m, 3H), 7.72 (d, J = 8.1Hz, 2H).
MS (ESI) m/z: 400.1 (M+H+). HRMS (ESI) for C20H22N3O4S (M+H+): calcd, 400.1331; found,
400.1321.
19

ACCEPTED MANUSCRIPT
4.1.4.8.
N-Hydroxy-3-(4-{2-[1-(4-methoxy-benzenesulfonyl)-1H-indol-4-yl]-ethylsulfamoyl}-phenyl)-acrylam
ide (18). Compound 18 was synthesized using free acid (0.2 g, 0.38 mmol), obtained after hydrolysis
of compound 50 (0.21 g, 0.34 mmol), and a mixture of EDC•HCl (0.09 g, 0.58 mmol), HOBt hydrate
(0.08 g, 0.58 mmol), NMM (0.11 mL, 1.05 mmol) and NH2OTHP (0.05 g, 0.43 mmol) followed by
o
general procedure for hydroxamic acid synthesis 49 % yield (overall from 50); mp: 142-144 C;
purity: 97.13 %. 1H NMR (300 MHz, MeOD): δ 2.95 - 2.97 (m, 2H), 3.12- 3.15 (m, 2H), 3.80 (s, 3H),
6.57 (d, J = 15.3 Hz, 1H), 6.72 (s, 1H), 6.97- 7.00 (m, 3H), 7.18(t, J = 7.2 Hz, 1H), 7.56-7.63 (m,
4H), 7.70 - 7.73 (m, 2H), 7.79 -7.86 (m, 3H). MS (ESI) m/z: 554.2 (M-H+). HRMS (ESI) for
C26H25N3O7S2 (M-H+): calcd, 554.1134; found, 555.1053.
4.2. Biology
4.2.1. Tumor Cell Culture.
All human cancer cells were maintained in RPMI 1640 medium supplemented with 10% FBS
and penicillin (100 units/ml)/streptomycin (100 µg/ml)/amphotericin B (0.25 µg/ml). All cells were
maintained in humidified air containing 5% CO2 at 37°C and cultured every 2-3 days. All cells were
cultured in tissue culture flasks in a humidified air containing 5% CO2 and 95% air at 37 ° C and
cultured every 2-3 days.
4.2.2. The Sulforhodamine B Assays
Cells were seeded at the density of 5000 cells/well into 96-plate overnight. Basal cells were
fixed with 10% trichloroacetic acid (TCA) to represent cell population at the time of compound
addition (T0). After additional incubation of DMSO (C) or different doses of test compounds (Tx) for
48 h, cells were fixed with 10 % TCA and stained with SRB at 0.4 % (w/v) in 1 % acetic acid.
Unbound SRB was washed out using 1 % acetic acid and SRB bound cells were solubilized with 10
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mM Trizma base. The absorbance was read at a wavelength of 515 nm. The 50% growth inhibition
(GI50) was calculated by 100 − [(Tx – T0) / (C – T0)] × 100.
4.2.3. HeLa Nuclear Extract HDAC Activity Assay
HDAC Fluorescent Activity Assay Kit (BioVision, CA) was used to detect HeLa nuclear
extract HDAC activity according to manufacturer’s instructions. Briefly, the HDAC fluorometric
substrate and assay buffer were added to HeLa nuclear extracts in a 96-well format and incubated at
37°C for 30 min. The reaction was stopped by adding lysine developer, and the mixture was
incubated for another 30 min at 37°C. Additional negative controls included incubation without the
nuclear extract, without the substrate, or without both. A fluorescence plate reader with excitation at
355 nm and emission at 460 nm was used to quantify HDAC activity.
4.2.4. HDAC Biochemical Assays.
The HDACs in vitro activities of human recombinant HDAC 1, 2, 4, 6, and 8 were conducted
by Eurofin Panlabs (Taipei, Taiwan). In brief, indicated compounds were incubated with specific
HDAC enzyme and Fluor-de-Lys deacetylase substrate. Fluor-de-Lys deacetylsubstrate were
spectrofluorimetric quantitated compared to control.
4.2.5. Western Blot Analysis
Cells were incubated with indicated compounds for 24 h and lysed with ice-cold lysis buffer (20
mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% Sodium
Deoxycholate, 0.1% SDS, 2.5 mM β-glycerolphosphate, 1 mM Na4P2O7, 5 mM NaF, 1 mM Na3VO4
and protease inhibitor cocktail from Millipore) on ice for 30 min followed by centrifugation at 13000
rpm for 30 min. Protein concentrations were determined and equal amounts of protein were
separated by 8-15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and
transferred to poly(vinylidene difluoride) (PVDF) membranes. Membranes were immunoblotted with
specific antibodies overnight at 4°C and then applied to appropriate horseradish
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peroxidase-conjugated anti-mouse or anti-rabbit IgG secondary antibodies for 1 h at room
temperature. Signals were detected using an enhanced chemiluminescence (Amersham,
Buckinghamshire, UK).
4.2.6. Immunohistochemistry (IHC).
Formalin-fixed paraffin-embeded tumor sections were submitted to Rapid Science Co., Ltd. to
conduct the immunohistochemistry staining. In brief, tumor sections were deparaffinized,
rehydration and immersed in 3% H2O2 to inactivate endogenous peroxidase. For antigen retrival,
slides were incubated in buffer with pH 9 at 80°C overnight. After PBS washing, the slides were
blocked with fatty-free milk and incubated with primary antibody at 4°C overnight. The secondary
antibody was added and then Mayer's Hematoxylin solution was used for counterstaining. For
hematoxylin and eosin stain (H&E stain), the tumor sections were incubated in hematoxylin solution
and then counterstained with eosin.
4.2.7. Antitumor Activity in vivo.
Male nude mice were injected subcutaneously with the same volume of BD Matrigel Matrix HC
(catalog 354248, BD bioscience), and PC3 cells (1×107cell/mouse) into the flank of each animal.
When the tumors had grown to around 100 mm3, animals were divided into three groups (n=6) and
receive the following treatment by intraperitoneal injection for 18 days during the study: (a) vehicle
alone, Compound 13 at (b) 20 mg/kg or (c) 40 mg/kg every other day (q2d). Compound 13 was
dissolved in vehicle (5% Cremophor EL + 5% DMSO + 90% dextrose). Tumor size was measured
twice weekly and calculated from V = l*w2/2, where w = width (w) and l = length (l). The mice were
housed on Taipei Medical University Laboratory Animal Center, TMU, on a 12-hour light cycle at
21–23 °C and 60–85% humidity. Nude mice were maintained in accordance with the Institutional
Animal Care and Use Committee procedures and guidelines (IACUC no. LAC-2013-0139).
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4.2.8. Statistical and Graphical Analyses.
Each experiment was performed independently at least three times and the data are presented as
mean ± SEM for the indicated number of separate experiments. Student’s t-test was used to compare
the mean of each group with that of the control group in experiments and one-way ANOVA was used
in animal study. P-values less than 0.05 were considered significant (*P < 0.05, **P < 0.01, ***P <
0.001).
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).
Abbreviations
HDAC, histone deacetylase; NH2OTHP, O-(tetrahydro-2H-pyran-2-yl)hydroxylamine; EDC,
N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide;
HOBt,
1-hydroxybenzotriazole;
TFA,
trifluoroacetic acid; NMM, N-methylmorpholine; DMF, N,N-dimethylformamide; TGI, tumor
growth inhibition; IP, intraperitoneal.
References
[1] (a) K. Narita, K. Matsuhara, J. Itoh, Y. Akiyama, S. Dan, T. Yamori, A. Ito, M. Yoshida, T. Katoh,
Synthesis and biological evaluation of novel FK228 analogues as potential isoform selective
HDAC inhibitors, Eur. J. Med. Chem.,121 (2016) 592-609. (b) F. Thaler, L. Moretti, R. Amici, A.
Abate, A. Colombo, G. Carenzi, M.C. Fulco, R. Boggio, G. Dondio, S. Gagliardi, Synthesis,
biological characterization and molecular modeling insights of spirochromanes as potent HDAC
inhibitors, Eur. J. Med. Chem., 108 (2016) 53-67 (c) F.-W. Peng, J. Xuan, T.-T. Wu, J.-Y. Xue,
Z.-W. Ren, D.-K. Liu, X.-Q. Wang, X.-H. Chen, J.-W. Zhang, Y.-G. Xu, Design, synthesis and
biological evaluation of N-phenylquinazolin-4-amine hybrids as dual inhibitors of VEGFR-2
and HDAC, Eur. J. Med. Chem.,109 (2016) 1-12. (d) R. Cincinelli, V. Zwick, L. Musso, V. Zuco,
M. De Cesare, F. Zunino, C. Simoes-Pires, A. Nurisso, G. Giannini, M. Cuendet,
Biphenyl-4-yl-acrylohydroxamic acids: Identification of a novel indolyl-substituted HDAC
inhibitor with antitumor activity, Eur. J. Med. Chem.,112 (2016) 99-105. (e) J. Roche, P.
Bertrand, Inside HDACs with more selective HDAC inhibitors, Eur. J. Med. Chem., 121 (2016)
451-483. (f) W. Weichert, A. Röske, V. Gekeler, T. Beckers, C. Stephan, K. Jung, F. Fritzsche, S.
Niesporek, C. Denkert, M. Dietel, Histone deacetylases 1, 2 and 3 are highly expressed in
prostate cancer and HDAC2 expression is associated with shorter PSA relapse time after radical
prostatectomy, Br. J. Cancer, 98 (2008) 604-610.
[2] J. Gräff, D. Rei, J.-S. Guan, W.-Y. Wang, J. Seo, K.M. Hennig, T.J. Nieland, D.M. Fass, P.F.
Kao, M. Kahn, An epigenetic blockade of cognitive functions in the neurodegenerating brain,
Nature, 483 (2012) 222-226.
23

ACCEPTED MANUSCRIPT
[3] J. Gräff, L.-H. Tsai, The potential of HDAC inhibitors as cognitive enhancers, Annu. Rev.
Pharmacol. Toxicol., 53 (2013) 311-330.
[4] K. Halkidou, L. Gaughan, S. Cook, H. Y. Leung, D. E. Neal, C. N. Robson, Upregulation and
nuclear recruitment of HDAC1 in hormone refractory prostate cancer, Prostate, 59 (2004)
177-189.
[5] A. Abbas, S. Gupta, The role of histone deacetylases in prostate cancer, Epigenetics, 3 (2008)
300-309.
[6] E. Noonan, R. Place, D. Pookot, S. Basak, J. Whitson, H. Hirata, C. Giardina, R. Dahiya,
miR-449a targets HDAC-1 and induces growth arrest in prostate cancer, Oncogene, 28 (2009)
1714-1724.
[7] M. Kirschbaum, P. Frankel, L. Popplewell, J. Zain, M. Delioukina, V. Pullarkat, D. Matsuoka, B.
Pulone, A. J. Rotter, I. Espinoza-Delgado, Phase II study of vorinostat for treatment of relapsed
or refractory indolent non-Hodgkin's lymphoma and mantle cell lymphoma, J. Clin. Oncol., 29
(2011) 1198-1203.
[8] L. M. Butler, D. B. Agus, H. I. Scher, B. Higgins, A. Rose, C. Cordon-Cardo, H. T. Thaler, R. A.
Rifkind, P. A. Marks, V. M. Richon, Suberoylanilide hydroxamic acid, an inhibitor of histone
deacetylase, suppresses the growth of prostate cancer cells in vitro and in vivo, Cancer Res., 60
(2000) 5165-5170.
[9] V. Lakshmikanthan, I. Kaddour, Djebbar, R. W. Lewis, M. V. Kumar, SAHA-sensitized prostate
cancer cells to TNFα-related apoptosis inducing ligand (TRAIL): Mechanisms leading to
synergistic apoptosis, Int. J. Can., 119 (2006) 221-228.
[10] https://clinicaltrials.gov/ct2/show/results/NCT00413075, in.
[11] G. L. Gravina, F. Marampon, I. Giusti, E. Carosa, S. D. Sante, E. Ricevuto, V. Dolo, V.
Tombolini, E. A. Jannini, C. Festuccia, Differential effects of PXD101 (belinostat) on
androgen-dependent and androgen-independent prostate cancer models, Int. J. Oncol., 40 (2012)
711.
[12] G. L. Gravina, F. Marampon, P. Muzi, A. Mancini, M. Piccolella, P. Negri-Cesi, M. Motta, A.
Lenzi, E. Di Cesare, V. Tombolini, PXD101 potentiates hormonal therapy and prevents the onset
of castration-resistant phenotype modulating androgen receptor, HSP90, and CRM1 in preclinical
models of prostate cancer, Endocr. Relat. Cancer, 20 (2013) 321-337.
[13] (a) Y. Zhang, P. Yang, C. J. Chou, C. Liu, X. Wang, W. Xu, Development of
N-hydroxycinnamamide-based histone deacetylase inhibitors with an indole-containing cap
group, ACS Med. Chem.Lett., 4 (2013) 235-238. (b) S. Mehndiratta, Y. L. Hsieh, Y. M. Liu, A.
W. Wang, H. Y. Lee, L. Y. Liang, S. Kumar, C. M. Teng, C. R. Yang, J. P. Liou,
Indole-3-ethylsulfamoylphenylacrylamides: Potent histone deacetylase inhibitors with
anti-inflammatory activity, Eur. J. Med. Chem., 85 (2014) 468-479. (c) Y. C. Huang, F. I. Huang,
S. Mehndiratta, S. C. Lai, J. P. Liou, C. R. Yang, Anticancer activity of MPT0G157, a derivative
of indolylbenzenesulfonamide, inhibits tumor growth and angiogenesis, Oncotarget, 6 (2015)
18590-18601. (d) S. Mehndiratta, S. L. Pan, S. Kumar, J. P. Liou,
Indole-3-ethylsulfamoylphenylacrylamides with Potent Anti-proliferative and Anti-angiogenic
Activities” Anticancer Agents Med. Chem., 16 (2016), 907-913.
[14] Y. S. Cho, L. Whitehead, J. Li, C. H. -T. Chen, L. Jiang, M. Vögtle, E. Francotte, P. Richert, T.
Wagner, M. Traebert, Conformational refinement of hydroxamate-based histone deacetylase
inhibitors and exploration of 3-piperidin-3-ylindole analogues of dacinostat (LAQ824), J. Med.
Chem., 53 (2010) 2952-2963.
[15] T. A. Miller, D. J. Witter, S. Belvedere, Histone deacetylase inhibitors, J. Med. Chem., 46
(2003) 5097-5116.
[16] Y. Guo, J. Xiao, Z. Guo, F. Chu, Y. Cheng, S. Wu, Exploration of a binding mode of indole
amide analogues as potent histone deacetylase inhibitors and 3D-QSAR analyses, Bioorg.
Med.Chem., 13 (2005) 5424-5434.
24

ACCEPTED MANUSCRIPT
[17] S. Mahboobi, A. Sellmer, H. Höcher, C. Garhammer, H. Pongratz, T. Maier, T. Ciossek, T.
Beckers, 2-Aroylindoles and 2-aroylbenzofurans with N-hydroxyacrylamide substructures as a
novel series of rationally designed histone deacetylase inhibitors, J. Med. Chem., 50 (2007)
4405-4418.
[18] J. -P. Liou, C. -Y. Wu, H. -P. Hsieh, C. -Y. Chang, C. -M. Chen, C. -C. Kuo, J. -Y. Chang,
4-and 5-aroylindoles as novel classes of potent antitubulin agents, J. Med. Chem., 50 (2007)
4548-4552.
[19] M. -J. Lai, H. -L. Huang, S. -L. Pan, Y. -M. Liu, C. -Y. Peng, H. -Y. Lee, T. -K. Yeh, P.-H.
Huang, C. -M. Teng, C. -S. Chen, J. -P. Liou, Synthesis and biological evaluation of
1-arylsulfonyl-5-(N-hydroxyacrylamide) indoles as potent histone deacetylase inhibitors with
antitumor activity in vivo, J. Med. Chem., 55 (2012) 3777-3791.
[20] H. Y. Lee, C. R. Yang, M. J. Lai, H. L. Huang, Y. L. Hsieh, Y. M. Liu, T. K. Yeh, Y. H. Li, S.
Mehndiratta, C.M. Teng, J. P. Liou,1-Arylsulfonyl-5-(N-hydroxyacrylamide) indolines Histone
Deacetylase Inhibitors Are Potent Cytokine Release Suppressors, ChemBioChem, 14 (2013)
1248-1254.
[21] X. Zhao, X. Liu, H. Mei, J. Guo, L. Lin, X. Feng, Asymmetric Dearomatization of Indoles
through a Michael/Friedel–Crafts-Type Cascade To Construct Polycyclic Spiroindolines, Angew.
Chem. Int. Ed. Engl, 54 (2015) 4032-4035.
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Figure Caption
Figure 1. Various examples of HDIs
Figure 2. Design and rationale of the study
Figure 3. Series of 4,5-indolyl-N-hydroxyphenylacrylamides
Figure 4. (A) Effect of α-tubulin acetylation and histone H3 acetylation in cultured
human prostate cells (PC-3) by compound 13 and reference 1. Cells were treated with
1 μM of compound 13 and reference 1 for 24 h and subjected to western blot analysis.
Quantification analysis of western blot using ImageJ software; (B) acetyl-α-tubulin
and (C) acetyl-histone 3 were analyzed in both cells
Figure 5. Inhibition of prostate cancer (PC-3) cells xenograft growth in nude mice (n
= 7-8). Compounds 12 was suspended in 1% carboxymethyl cellulose and 0.5%
Tween 80. Left panel, tumor growth of PC3 xenograft nude mice treated with or
without compound 13 by ip (20 and 40 mg/kg) with a frequency of q2d. Tumor
growth is tracked by the mean tumor volume (mm3) ± S.E and calculated as % tumor
growth inhibition (%TGI). Tumor volume was determined using caliper
measurements and was calculated as the product of 1/2 x length x width2. Right panel,
body weight (g) of the mice. *, p < 0.05, and ** p < 0.01 as compared with the control
group.
Figure 6. Immunohistochemistry of protein expressions in PC-3 xenograft model.
Formalin-fixed paraffin-embedded sections from PC3 xenograft tumors were stained
with H&E stain, acetyl-hisotne 3, HDAC 1 and 2 antibodies.

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Table 1 Inhibition of HeLa Nuclear Extract HDAC Activity and Antiproliferative
Activity against Human Cancer Cell Lines by 11 to 18 and Reference Compounds
Cell type (GI50 ± SD, μMa)
IC50 ± SD
(nMa)
Breast
MDA-MB-2
31
Prostate
PC-3
Lung
A549
Pancreatic
AsPC-1
HeLa nuclear
HDAC
Cpds.
11
> 1000
3.49 ± 0.17
0.86 ± 0.07
0.14 ± 0.02
0.31 ± 0.08
0.31 ±0.05
0.33 ± 0.01
0.15 ± 0.03
2.88 ± 0.39
0.62 ± 0.2
>10
>10
>10
12
7.17 ± 3.13
8.56 ± 3.67
30.45 ± 7.93
76.13 ± 33.3
52.17 ± 10.2
14.3 ± 3.91
60 ± 12
1.65 ± 0.27 3.55 ± 0.29
0.25 ± 0.01 0.32 ± 0.07
0.87 ± 0.03 2.06 ± 0.66
0.60 ± 0.16 9.91 ± 2.04
0.82 ± 0.02 2.47 ± 0.38
0.25 ± 0.01 0.08 ± 0.01
4.56 ± 1.91
0.24 ± 0.03
3.85 ± 1.05
3.97 ± 1.22
2.78 ± 1.36
0.16 ± 0.02
13
14
15
16
17
18
919
4.35 ± 0.63 5.85 ± 0.09 12.46 ± 2.24
29.5 ± 4.5
1.02 ± 0.2
0.48 ± 0.1
ND
SAHA (1)
PXD101 (3)
LBH589.HCl (4)
131.26 ± 30.0 0.60 ± 0.06
1.73 ± 0.02 2.67 ± 0.50
0.23 ± 0.01 0.28 ± 0.04
5.44 ± 0.49
0.65 ± 0.12
0.03±0.002
81 ± 21
0.31 ± 0.02
0.07±0.01
4.90±1.74
0.15±0.01
0.06±0.03
MS-275 (6)
> 1000
0.43 ± 0.06
5.76 ± 0.57 1.41 ± 0.13
6.90 ± 1.30
aSD: standard deviation. All experiments were independently performed at least three times.