Ring size changes in the development of class I HDAC inhibitors

Article abstract of DOI:10.1080/14756366.2021.1941920

Five pathways involving different ring structures led to generation of fourteen thienylbenzamides (7–20) which display the structure-activity relationships of class I HDAC inhibitors. All the synthesised compounds inhibit HDAC1 and HDAC2 selectively over other isoforms and many inhibit DLD1 and HCT116 cells more effectively than a parent compound. Compounds 8 and 16 inhibit HCT116 cells by activation of the apoptosis pathway.

Full text of DOI:10.1080/14756366.2021.1941920

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Ring size changes in the development of class I
HDAC inhibitors
Er-Chieh Cho, Chi-Yuan Liu, Di-Wei Tang & Hsueh-Yun Lee
To cite this article: Er-Chieh Cho, Chi-Yuan Liu, Di-Wei Tang & Hsueh-Yun Lee (2021) Ring size
changes in the development of class I HDAC inhibitors, Journal of Enzyme Inhibition and Medicinal
Chemistry, 36:1, 1387-1401, DOI: 10.1080/14756366.2021.1941920
To link to this article: https://doi.org/10.1080/14756366.2021.1941920
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2021, VOL. 36, NO. 1, 1387–1401
https://doi.org/10.1080/14756366.2021.1941920
RESEARCH PAPER
Ring size changes in the development of class I HDAC inhibitors
Er-Chieh Choa,b,c#, Chi-Yuan Liua#, Di-Wei Tanga and Hsueh-Yun Leea,d
b
aSchool of Pharmacy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan; Master Program in Clinical Genomics and Proteomics,
c
College of Pharmacy, Taipei Medical University, Taipei, Taiwan; Cancer Center, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan;
dPh.D. Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, Taipei, Taiwan
ABSTRACT
ARTICLE HISTORY
Received 24 March 2021
Revised 31 May 2021
Accepted 8 June 2021
Five pathways involving different ring structures led to generation of fourteen thienylbenzamides (7–20)
which display the structure-activity relationships of class I HDAC inhibitors. All the synthesised compounds
inhibit HDAC1 and HDAC2 selectively over other isoforms and many inhibit DLD1 and HCT116 cells more
effectively than a parent compound. Compounds 8 and 16 inhibit HCT116 cells by activation of the apop-
tosis pathway.
KEYWORDS
Thienylbenzamides; ring
transformation; HDAC;
colon cancer
GRAPHICAL ABSTRACT
DLD1
2.98 µM
3.17 µM
HCT116
1.01 µM
0.77 µM
HDAC1
31.4 nM
34.6 nM
HDAC2
177 nM
201 nM
8
16
1. Introduction
focus on the development of HDACis with class or isoform select-
ivity in order to reduce their adverse effects and raise their thera-
peutic index. The relationship between each HDAC isoform and
human cancer is quite complicated and not yet fully understood;
nevertheless, it is certain that HDAC1 and HDAC2 are significantly
involved in several types of malignancies and serve as important
targets for cancer treatment6. HDAC1 is highly expressed in liver
cancer cell lines for example, and its inactivation impairs cell cycle
transition and causes autophagic cell death7. HDAC2 is found in
pancreatic ductal adenocarcinoma (PDAC) and confers resistance
towards etoposide in PDAC cells8. A program in our institute has
tried to identify selective inhibitors that target HDAC1/2 preferen-
tially. In general, the structure of an HDACi is divided into a cap
region, a linker, and a zinc binding group (ZBG) and modification
of each of these three parts is likely to cause a considerable
impact on the selectivity of an HDACi9. After transforming the
hydroxamic acid ZBG on vorinostat for example, into an o-aminoa-
Epigenetic regulation involves a variety of DNA based processes
such as DNA replication, transcription and DNA repair and is crit-
ical to cellular development1–2. Targeting epigenetic regulators
has emerged in recent decades as an attractive strategy for treat-
ment of human diseases. The dysregulation of histone deacetylase
(HDAC) activity is linked to neurological syndromes, inflammation
and carcinogenesis. HDAC, an epigenetic enzyme, plays an import-
ant role in gene expression by removing the acetyl group of ace-
tylated lysine residues on histones and some other proteins3. To
date, 18 members of HDAC groups have been identified in
humans, and can be categorised into four classes (I-IV) depending
on their cellular localisation and homologies with yeast. So far,
most HDAC inhibitors (HDACi) such as the FDA-approved drugs
vorinostat, belinostat and panobinostat are pan-HDACis which
non-selectively inhibit class I and II members. Use of pan-HDACis
can lead to some side effects, including gastrointestinal syndrome,
fatigue, and thrombocytopenia4–5. Researchers have begun to nilide, the molecule became inactive towards HDAC610. Further
CONTACT Hsueh-Yun Lee
hyl@tmu.edu.tw
School of Pharmacy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan;
Ph.D. Program in Drug
Discovery and Development Industry, College of Pharmacy, Taipei Medical University, Taipei, Taiwan
#Contributed equally to this work.
Supplemental data for this article can be accessed here.
ß 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

1388
E.-C. CHO ET AL.
studies suggest that structures containing an o-aminoanilide as but not in HDAC3 due to the difference in the amino acid
the ZBG generally inhibit class I HDACs. Such inhibitors are exem- sequence of the latter (Figure 1).
Ring constriction is a comprehensive strategy in the process of
structural optimisation. In our previous study, we utilised this
strategy to modify the antitubulin agent ABT751, obtaining fura-
nylazaindoles such as 7 as potent antitubulin agents17. In an
enzyme complex with compound 4, the amide group of 4 is
located at an open area of the enzyme, which is an area that can
accommodate various scaffolds. In order to understand the details
of the SAR study and seek structural possibilities, this study
plified by benzamide compounds 1 (MS-275), 2 (MGCD0103), and
3 (CI-994)11,12. In a survey of the literature, we noted that a 14 Å
internal cavity, which is located near the catalytic active site, is
crucial for the selectivity of HDAC proteins. This internal cavity
was first studied by Wang et al.13 It was hypothesised that this
cavity accommodates the acetate by-product of deacetylation and
provides it with an exit pathway. Researchers discovered that
attached substituents on the o-aminoanilide can access the 14 Å
internal cavity, and this led to the development of biaryl type applied the strategy of ring transformation to the modification of
benzamides including 4 (Merck60), 5 (MRLB-223) and compound the benzamide moiety (Figure 2). There are four ring closing path-
614–16. These molecules specifically attained selectivity towards ways envisaged in the study, which respectively give derivatives
HDAC1 and HDAC2 by adding a small hydrophobic aromatic unit of indole (7, pathway a), indoline-/tetrahydroquinoline (8–13,
such as a phenyl or thienyl group to the para position of the anil- pathway b), pyridine (14, pathway c), and lactam-containing moi-
ine. According to the homology models of HDAC1 and HDAC3 eties (15–16, pathway d). In addition, the amide moiety was modi-
based on the crystal structure of an HDAC8 mutant, the additional fied by reversing the motif followed by ring restriction, which
aromatic unit is able to occupy the 14 Å internal cavity in HDAC1 provides compounds 17–20. In addition to the synthetic routes to
O
O
O
N
N
N
N
H
O
N
H
H
H
H
N
N
NH2
O
N
NH2
NH2
N
H
N
O
3
2
1
S
S
O
O
N
O
H
N
NH2
H
H
N
N
O
N
NH2
O
N
O
H
O
NH2
O
N
H
HN
6
5
4
Figure 1. Class I HDAC inhibitors (1–6).
Figure 2. Structural modification and structures of compounds 7–20.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1389
these designed compounds (7–20), an investigation of their bio- presence of acetic acid to generate the indole-5-carboxylate (29)
which was subjected to hydrolysis to obtain the corresponding
indole-5-carboxylic acid (30). The amidation of 30 with 25, with
the assistance of HATU gave compound 31 which was subjected
to TFA-medicated Boc-deprotection to afford compound 7.
Scheme 3 shows the synthesis of indoline-containing com-
pounds (8–12). The N-substitution of methyl indoline-5-carboxyl-
ate (32) was carried out by the reaction with acetic anhydride,
butyryl chloride, isobutyryl chloride, methanesulfonyl chloride or
benzenesulfonyl chloride affording 33a–33e. The subsequent
hydrolysis of the ester group in 33a–33e using LiOH yielded the
corresponding carboxylic acids (34a–34e). The subsequent amida-
tion of 34a–34e with 25 gave compounds 35a–35e which were
subjected to TFA-mediated deprotection to obtain com-
pounds 8–12.
logical activity will be examined in this study.
2. Results and discussion
2.1. Chemistry
In the synthesis of compounds 7–20, the Boc-masked benzene-
1,2-diamine (25) is a crucial building block which is obtained from
the synthetic route shown in Scheme 1. 2-Nitroaniline (21) was
brominated with NBS, and the resulting product (22) was pro-
tected by a Boc group to provide compound 23. The subsequent
Suzuki arylation of 23 with 2-thienylboronic acid gave 24, which
was subjected to reduction of its nitro group to afford com-
pound 25.
Scheme 2 shows the synthesis of compound 7. The condensa-
tion between 4-methyl-3-nitrobenzoate (26) and DMF-DMA
yielded the enamine 27, which was then treated with acetyl chlor-
ide and subsequent hydrolysis generated compound 28. The
Production of a tetrahydroquinoline was also achieved, and is
shown in Scheme 4. With a catalytic amount of 10% Pd/C, reduc-
tion of quinoline-6-carboxylic acid (36) by ammonium formate
was carried out in refluxing MeOH to obtain the acid (37). The
resulting product was then reacted with iron powder in the resulting product was treated with acetyl chloride to yield 38
Scheme 1. Synthetic approach to compound 25. Reagents and conditions: (a) NBS, acetic acid, 0 ꢀC; (b) i. Boc2O, DMAP, THF, rt; ii. 5N NaOH(aq), THF, 70 ꢀC; (c) 2-thie-
nylboronic acid, Pd(PPh3)4, P(o-tol)3, K2CO3, H2O, DME, reflux; (d) H2, 10% Pd/C, MeOH, rt.
Scheme 2. Synthetic approach to compound 7. Reagents and conditions: (a) DMF-DMA, reflux; (b) i. acetyl chloride, pyridine, DCM, rt; ii. p-dioxane, H2O, reflux; (c) Fe
powder, CH3COOH, 90 ꢀC; (d) NaOH, H2O, MeOH, reflux; (e) 25, HATU, DIPEA, DMF, 70 ꢀC; (f) TFA, DCM, rt.

1390
E.-C. CHO ET AL.
Scheme 3. Synthetic approach to compounds 8–12. Reagents and conditions: (a) for 33a: acetic anhydride, TEA, DCM, 130 ꢀC; for 33b: butyryl chloride, TEA, DCM,
0 ꢀC; for 33c: isobutyryl chloride, TEA, DCM, 0 ꢀC; for 33d: methanesulfonyl chloride; pyridine, 0 ꢀC; for 33e: benzenesulfonyl chloride; pyridine, rt; (b) 1N LiOH(aq), THF,
rt; (c) 25, HATU, DIPEA, DMF, 60 ꢀC; (d) TFA, DCM, rt.
reacted with toluenesulfonylmethyl isocyanide (TosMIC) under the
conditions of the van Leusen reaction to afford compound 41f,
which is a regioisomer of 41e. Alternatively, 40d was reacted with
o-phenylenediamine in the presence of Na2S2O4 to produce the
benzimidazole ester (41g). All synthesised substituted benzoates
(41a–41g) underwent hydrolysis by LiOH or NaOH, which gener-
ated the corresponding carboxylic acids 42a–42g. The resulting
products were converted into the final products (14–20) through
routes similar to those in Scheme 1.
2.2. Biological evaluation
2.2.1. In vitro cell growth inhibitory activity
All synthetic compounds (7–20) were examined for their cellular
Scheme 4. Synthetic approach to compound 13. Reagents and conditions: (a)
activity against two colorectal cancer cells, DLD1 and HCT116. The
IC50 values shown in Table 1 were determined after DLD1 and
HCT116 cells were exposed to each tested compound for 48 h or
72 h. IC50 curves of these compounds in DLD1 and HCT116 cells
were analysed and shown in the Supplementary Figure 1. The
results in the first column reveal that most of the compounds
tested, with the exception of 10, 17, and 18, showed better DLD1
inhibitory activity than the thiophenylaniline (4). In the third col-
umn it can be seen that most of tested compounds exhibited
comparable or weaker activity against HCT116 cells, when com-
pared with 4, but compounds 8, 16, 17, and 18 inhibit the
growth of HCT116 cells with IC50 values of 4.13, 0.34, 6.12, and
1.84 lM, respectively therefore displaying improved HCT116 inhibi-
tory activity. In order to understand the influence of exposure
time on antiproliferative activity, compounds that showed activity
against both DLD1 and HCT116 cells were selected and tested for
ammonium formate, 10% Pd/C, MeOH, reflux; (b) acetyl chloride, TEA, DCM, rt;
(c) 25, HATU, DIPEA, DMF, rt, 6 h, 50 ꢀC; (d) TFA, DCM, rt.
which was subjected to the synthetic routes to the anticipated 4-
thienyl-2-aminobenzamide shown in Scheme 1 to obtain com-
pound 13.
Scheme 5 shows the synthetic route to compounds 14–20. The
reaction of methyl 4-aminobenzoate (40a) with 2-bromopyridine
gave the corresponding benzoate (41a). Methyl 4-bromobenzoate
(40b) underwent Buchwald-Hartwig amination with 2-pyrrolidone
and 2-piperidone yielding compounds 41b and 41c, respectively.
The reverse amide moiety was obtained from the reaction of 40c
with methylamine hydrochloride with assistance of EDCꢁHCl, gen-
erating compound 41d. Compound 40c, upon amidation with
aminoacetaldehyde dimethyl acetal followed by treatment with
MeSO3H and phosphorus pentoxide (P4O10), yielded the oxazole- their cellular activity in a 72 h exposure. The result shows that
substituted compound (41e). Methyl 4-formylbenzoate (40d) was most of the selected compounds displayed slightly better potency

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1391
Scheme 5. Synthetic approach to compounds 14–20. Reagents and conditions: (a) for compound 41a: 40a, 2-bromopyridine, 140ꢀC; for compound 41b: 40b, 2-pyr-
rolidone, Pd2(dba)3, xantphos, Cs2CO3, p-dioxane, reflux; for compound 41c: 40b, 2-piperidone, Pd2(dba)3, xantphos, Cs2CO3, p-dioxane, reflux; for compound 41d: 40c,
methylamine hydrochloride, EDCꢁHCl, DMAP, DMF, rt; for compound 41e: i. 40c, aminoacetaldehyde dimethyl acetal, EDCꢁHCl, NMM, DCM, rt; ii. MeSO3H, P4O10, N2,
140 ꢀC; for compound 41f: 40d, TosMIC, K2CO3, MeOH, reflux; for compound 41g: 40d, o-phenylenediamine, Na2S2O4, DMF, 70 ꢀC; (b) for compounds 41a, 41d, 41g:
1N LiOH(aq), p-dioxane, rt; for compounds 41b, 41c, 41e, 41f: 1N NaOH(aq), MeOH, rt; (c) 25, HATU, DIPEA, DMF, rt; (d) TFA, DCM, rt.
Table 1. Inhibitory activity (IC50, lMa) against colorectal cancer cells after the
treatment of tested compounds for 48 h or 72 h.
and IMR90 cells, to address this issue. Cells were treated with ten
of the compounds with great cancer cell inhibition activity for
48 h, analysed by MTT assay, and then calculated for IC50. Our
results showed that the IC50 of these compounds in non-cancer-
ous cells were much higher than that in cancer cells, suggested
that our compounds exhibit high cancer targeting specificity. In
summary, the synthesised compounds derived from ring trans-
formation of the amide moiety act more rapidly than the parent
compound, and the selected compounds (8–16) display a slightly
better DLD1 inhibitory activity than 4.
Due to the distinct differences in the results from the 48 h
experiment, the following discussion is focussed on these results.
The acetyl-substituted indoline molecule (8, from path b in Figure
2) showed approximately a 10-fold and 3-fold rate increase in
cytotoxicity against DLD1 and HCT116 cells, respectively when
compared to compound 4. After 48 h treatment of 8, the com-
pound inhibited the growth of DLD1 and HCT116 cells with IC50
values of 2.87 and 4.13 lM, respectively. Comparison of 8 with 9
and 10 suggests that the decrease of activity can probably be
DLD1
HCT116
Compd
48 h
72 h
NDb
48 h
72 h
ND
7
8
9
18.68 0.73
2.87 0.20
18.47 2.45
29.58 23.6
10.03 0.64
3.82 0.15
3.29 0.12
3.98 0.14
7.66 1.09
3.76 0.94
26.28 1.99
31.32 1.58
14.84 2.41
3.55 0.99
29.28 3.41
16.31 2.04
4.13 0.14
25.63 1.41
15.56 1.92
13.67 1.36
36.54 0.54
14.55 0.81
13.04 0.40
11.13 1.71
6.12 0.86
0.34 0.14
1.84 0.76
12.02 0.73
24.88 0.09
13.6 0.68
2.98 0.20
2.45 0.49
5.17 3.13
1.64 0.07
1.39 0.58
4.83 0.50
3.16 0.27
3.33 0.30
3.17 0.11
ND
1.01 0.42
2.46 0.39
2.61 0.42
2.30 0.37
2.72 0.43
1.21 0.02
1.31 0.07
1.44 0.93
0.77 0.01
ND
10
11
12
13
14
15
16
17
18
19
20
4
ND
ND
ND
6.20 0.26
ND
ND
ND
1.05 0.10
aThe IC50 was estimated using GraphPad Prism 7 software and shown as
mean SD from at last three independent experiments. bND: not determined.
than 4 towards DLD1 cells, while only compound 16 showed bet- attributed to the bulky nature of the N-substituents in the indo-
ter HCT116 inhibitory activity than compound 4. Moreover, in line scaffold. The expansion of ring size from indoline (8) to tetra-
order to evaluate the cancer targeting specificity of these com- hydroquinoline (13) indeed led to
a slight reduction of
pounds, we applied two non-cancerous human cell lines, 293 T HCT116 inhibition.

1392
E.-C. CHO ET AL.
Table 2. Inhibitory activity (IC50, M) of compounds against HDAC1, HDAC2,
HDAC6 and HDAC8.
apoptotic cells. The results showed that there was significant
(ꢂ20%) induction of the sub-G1 population in both groups, one
treated at 1 lM for 48 h, the other at 0.2 lM for 72 h (Figure 3(A)),
suggesting that both compounds can inhibit cancer cells by acti-
vating the apoptosis pathway. A dose dependent effect was
observed with both compounds (Figure 3(A)). The morphology of
cells in each group was monitored and recorded as a control in
the flow cytometry (Figure 3(B)).
Compd
HDAC1
HDAC2
HDAC6
HDAC8
7
8
9
10
11
12
13
14
15
16
17
18
19
20
4
7.80 ꢃ 10ꢄ8
3.14 ꢃ 10ꢄ8
9.27 ꢃ 10ꢄ8
4.62 ꢃ 10ꢄ8
3.07 ꢃ 10ꢄ8
1.11 ꢃ 10ꢄ7
3.88 ꢃ 10ꢄ8
5.84 ꢃ 10ꢄ8
6.71 ꢃ 10ꢄ8
3.46 ꢃ 10ꢄ8
3.25 ꢃ 10ꢄ8
5.51 ꢃ 10ꢄ8
3.68 ꢃ 10ꢄ8
8.94 ꢃ 10ꢄ8
3.11 ꢃ 10ꢄ8
1.51 ꢃ 10ꢄ8
2.60 ꢃ 10ꢄ7
1.77 ꢃ 10ꢄ7
4.58 ꢃ 10ꢄ7
2.18 ꢃ 10ꢄ7
1.52 ꢃ 10ꢄ7
5.35 ꢃ 10ꢄ7
2.29 ꢃ 10ꢄ7
1.88 ꢃ 10ꢄ7
3.15 ꢃ 10ꢄ7
2.01 ꢃ 10ꢄ7
8.53 ꢃ 10ꢄ8
2.83 ꢃ 10ꢄ7
1.63 ꢃ 10ꢄ7
3.61 ꢃ 10ꢄ7
1.53 ꢃ 10ꢄ7
3.20 ꢃ 10ꢄ8
>1.0 ꢃ 10ꢄ5
>1.0 ꢃ 10ꢄ5
>1.0 ꢃ 10ꢄ5
>1.0 ꢃ 10ꢄ5
>1.0 ꢃ 10ꢄ5
>1.0 ꢃ 10ꢄ5
>1.0 ꢃ 10ꢄ5
>1.0 ꢃ 10ꢄ5
>1.0 ꢃ 10ꢄ5
>1.0 ꢃ 10ꢄ5
>1.0 ꢃ 10ꢄ5
>1.0 ꢃ 10ꢄ5
>1.0 ꢃ 10ꢄ5
>1.0 ꢃ 10ꢄ5
>1.0 ꢃ 10ꢄ5
1.45 ꢃ 10ꢄ9
>1.0 ꢃ 10ꢄ5
>1.0 ꢃ 10ꢄ5
>1.0 ꢃ 10ꢄ5
>1.0 ꢃ 10ꢄ5
>1.0 ꢃ 10ꢄ5
>1.0 ꢃ 10ꢄ5
>1.0 ꢃ 10ꢄ5
>1.0 ꢃ 10ꢄ5
>1.0 ꢃ 10ꢄ5
>1.0 ꢃ 10ꢄ5
>1.0 ꢃ 10ꢄ5
2.2.4. Western blot analysis
A Western blot assay was also performed. HCT116 cells were
>1.0 ꢃ 10ꢄ5 treated with compounds 8 and 16 at different concentrations and
>1.0 ꢃ 10ꢄ5
harvested at the 48h time point for analysis. The signal of acety-
lated histone H3 induced in cells being treated (Figure 4(A)), sug-
>1.0 ꢃ 10ꢄ5
>1.0 ꢃ 10ꢄ5
8.71 ꢃ 10ꢄ7
gested that these two compounds exhibited class
I HDACs
Trichostatin A
inhibition capacity. There was also slight induction of the acetylated
a-tubulin signal, which suggested that these two compounds might
exhibit a certain level of HDAC6 inhibitory capacity (Figure 4(A)).
The data showed that under the treatment of compounds 8 and
16, the signal of apoptotic markers, including the PARP cleaved
form and PUMA, and cell cycle arrest marker p21 were all induced
(Figure 4(A)). This suggested that compounds 8 and 16 are able to
activate an apoptosis pathway and affect the cell cycle regulation
in colon cancer cells. The signals from Western blot assay were
quantified and analysed by image J software compared to GAPDH,
the loading control, and are shown in Figure 4(B).
Compounds 15 and 16, designed from path d in Figure 2 dis-
played better activity than 4, and also showed that the six-mem-
bered lactam (16) is favoured for cellular activity. Compounds
17–20, obtained through a reverse amide route have inconsistent
inhibitory activities on DLD1 and HCT116 cells. Compounds 17
and 18 for instance, show remarkable antiproliferative activity
against HCT116 cells but are inactive towards DLD1 cells. Notably,
among compounds 7–20, compound 17, with an IC50 value of
0.34 lM is the most active inhibitor of HCT116 cells.
2.2.2. HDAC isoform inhibitory activity
2.2.5. Docking study of compounds 4, 8, and 16
Table 2 indicates the inhibitory activity against HDAC isoforms.
Compounds with cellular activity were selected for this enzymatic
activity, and their results are compared with those from compound
4. With exception of 12, all other tested compounds display HDAC1
and HDAC2 inhibitory activity comparable to that of 4, which
reveals that this series of compounds retain class I HDAC isoform
activity over other HDAC isoforms. This result also reveals that with
the presence of a thienylbenzamide moiety the tested compounds
retain class I HDAC inhibitory activity, and the modification of sur-
face recognition area provokes only a very slight change in the
HDAC isoform inhibition. In the first column it can be seen that
HDAC1 is more sensitive to compounds having bulkier substitution.
For example, 9, 12, and 20 exhibit weaker HDAC1 inhibitory activity
than others, with IC50 values of 92.7, 111.0, and 89.4 nM, respect-
ively. Despite 9, 12, and 20 also result in weaker HDAC2 inhibitory
activity, the extent of influence is less than that shown in the first
column. In addition to the influence of bulkiness of substituents on
the inhibition of HDAC subtypes, the orientation of amide moiety is
also observed. For instance, 17 possessing a reverse amide motif
shows comparable HDAC1 activity to that of 4 and results in 2-fold
increase of HDAC2 potency than 4. Compound 17 exhibits the
highest inhibitory activity against HDAC1 and HDAC2, with IC50 val-
ues of 32.5 and 85.3nM, respectively.
Figure 5 demonstrates the binding modes of compounds 4 (yel-
low), 8 (purple), and 16 (green) in the binding site of HDAC2 (PDB
ID: 5IX0), which was defined using the Glide module of
€
Schrodinger Maestro. The result indicates that compounds (8 and
16) designed with ring closure, have similar spatial alignments to
that of 4 in the binding site. The 2-aminobenzamide moiety of 4,
8, and 16 forms H-bonds with His145 and His146 of the target
enzyme. The amide motif of 4, 8, and 16 interacts with Gly154
residue via H-bonding. The similar interactions of 4, 8, and 16
explain their HDAC isoform inhibitory activity shown in Table 2.
3. Conclusion
This study utilised a ring restriction strategy to modify the amide
motif of reported HDAC inhibitors, and led to various heterocycle-
containing replacements, including indole (7), indolines (8–12)/tet-
rahydroquinoline (13), pyridine (4), lactams (15–16), and oxazoles
(18–19). Many of synthesised compounds show a faster effect in
antiproliferative assays than that of 4, and displayed distinct anti-
proliferative activity after a 48 h treatment. The structure-activity
relationship study indicated that N-acetylindoline derivative (8)
and the piperidinone-containing compound (16) exhibit marked
antiproliferative activity against DLD1 and HCT116 cells. After 48 h
treatment, 8 has IC50 values of 2.87 and 4.13 lM towards DLD1
and HCT116 cells and 16 has IC50 values of 3.76 and 6.12 lM
towards DLD1 and HCT116 cells. The anticancer activity of these
compounds stems from their activation of the apoptosis pathway.
Compounds 8 and 16 show HDAC1 and HDAC2 inhibitory activity
comparable to that of 4, and have the similar spatial alignment
and interaction in the active site of HDAC enzyme. The results
cited above indicate that the strategy of ring closure contributes
2.2.3. Flow cytometry analysis
Biological assays were carried out to investigate the inhibitory and
regulatory mechanisms of compounds 8 and 16 towards cancer
cells, because 8 and 16 show potent activity against both DLD1
and HCT116 in a 48 h exposure (Table 1). Firstly, flow cytometry
analysis was performed. HCT116 colon cancer cells were treated
with compounds 8 or 16 at different concentrations and har-
vested at various time points for analysis (Figure 3(A)). The cell to the decrease in the response time, and is probably due to the
cycle profile was analysed, and the sub-G1 population represents change of their physicochemical properties. Our work reports a

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1393
Figure 3. Bright-field microscopy of untreated/treated HCT116 cells (A) and flow cytometric analysis (B). Compounds 8 and 16 induced subG1 cell accumulation of
HCT116 in a dose- and time-dependent manner. HCT116 cells were treated with 8 (0.2 mM, 1.0 mM) and 16 (0.2 mM, 1.0 mM) for indicated times, then the cell cycle
distribution and DNA content were analysed by flow cytometry. Quantitative data were evaluated on the flow cytometric histograms and are shown as mean SD.
detailed study of the structure-activity relationships at the cap 700) electron impact (EI) mass spectrometer. Purity of the final com-
pounds was determined using a Hitachi 2000 series HPLC system
using C-18 column (Agilent ZORBAX Eclipse XDB-C18 5 lm. 4.6 mm
ꢃ 150 mm) and was found to be ꢅ 95% in all cases. Flash column
chromatography was carried out using silica gel (Merck Kieselgel 60,
No. 9385, 230–400 mesh ASTM). All reactions were done under an
atmosphere of dry nitrogen.
region and could benefit the development of class I HDAC inhibi-
tors with concerning pharmcokinetic features.
4. Experimental section
4.1. Chemistry
Nuclear magnetic resonance spectra were obtained with Bruker DRX-
300 spectrometer operating at 300 MHz and Agilent DD2 600 MHz 4.1.1. N-(2-Amino-5-(thiophen-2-yl)phenyl)-2-methyl-1H-indole-5-
NMR operating at 600 MHz, with chemical shifts reported in parts
carboxamide (7)
per million (ppm, d) downfield from TMS, an internal standard. High-
Trifluoroactic acid (TFA, 0.6 ml) was added to a solution of com-
resolution mass spectra (HRMS) were measured with a JEOL (JMS- pound 31 (0.34 g, 0.76 mmol) in dichloromethane (DCM, 8 ml) and

1394
E.-C. CHO ET AL.
Figure 4. After pre-treatment, compounds 8 and 16 suppressed the cell growth effect of HCT116 cells. (A) Western blot analysis of acetylated a-tubulin, a-tubulin, ace-
tylated histone H3, histone H3, PARP, PUMA, and p21 after treatment for 48h with increasing doses of 8 and 16 in HCT116 cells. GAPDH was used as a loading con-
trol. (B) Graphical representation of the protein expression intensity normalised with corresponding loading control.
137.2, 128.2, 128.0, 125.0, 124.4, 123.7, 123.5, 123.2, 122.4, 121.0,
120.2, 119.5, 116.5, 110.0, 100.2, 13.4. HRMS (ESI) for C20H18N3OS
(M þ Hþ) calcd 348.1165, found 338.1172.
4.1.2. 1-Acetyl-N-(2-amino-5-(thiophen-2-yl)phenyl)indoline-5-car-
boxamide (8)
The title compound was obtained in 51% overall yield from com-
pound 35a in a manner similar to that described for the prepar-
1
ation of 7: mp 219 ꢀC. H NMR (300 MHz, DMSO-d6) d 9.63 (s, 1H),
8.10 (d, J ¼ 8.4 Hz, 1H), 7.88–7.84 (m, 2H), 7.46 (d, J ¼ 1.8 Hz, 1H),
7.38–7.34 (m, 1H), 7.29 (dd, J ¼ 8.7, 2.1 Hz, 1H), 7.26–7.22 (m, 1H),
7.05 (dd, J ¼ 5.1, 3.6 Hz, 1H), 6.81 (d, J ¼ 8.4 Hz, 1H), 5.12 (s, 2H),
4.17 (t, J ¼ 8.4 Hz, 2H), 3.21 (t, J ¼ 8.4 Hz, 2H), 2.20 (s, 3H). 13C NMR
(150 MHz, DMSO-d6) d 169.1, 165.0, 145.6, 144.2, 142.9, 131.9,
129.0, 128.2, 127.6, 124.5, 123.8, 123.8, 123.7, 123.2, 122.3, 121.0,
116.4, 114.8, 48.6, 27.1, 24.0. HRMS (ESI) for C21H20N3O2S (M þ Hþ)
calcd 378.1271, found 378.1276.
Figure 5. Overlay of compounds 4 (yellow), 8 (purple), and 16 (green) in the
binding site of HDAC2 (PDB ID: 5IX0).
4.1.3. N-(2-Amino-5-(thiophen-2-yl)phenyl)-1-butyrylindoline-5-car-
boxamide (9)
The title compound was obtained in 99% overall yield from com-
pound 35b in a manner similar to that described for the prepar-
ation of 7: 1H NMR (300 MHz, DMSO-d6) d 9.62 (s, 1H), 8.14 (d,
J ¼ 8.4 Hz, 1H), 7.88–7.80 (m, 2H), 7.46 (d, J ¼ 2.1 Hz, 1H), 7.35 (dd,
J ¼ 5.1, 1.2 Hz, 1H), 7.28 (dd, J ¼ 8.4, 2.4 Hz, 1H), 7.24 (dd, J ¼ 3.6,
0.9 Hz, 1H), 7.04 (dd, J ¼ 5.1, 3.6 Hz, 1H), 6.81 (d, J ¼ 8.4 Hz, 1H),
5.11 (s, 2H), 4.16 (t, J ¼ 8.4 Hz, 2H), 3.20 (t, J ¼ 8.4 Hz, 2H),
2.50–2.40 (m, 2H), 1.69–1.58 (m, 2H), 0.95 (t, J ¼ 7.5 Hz, 3H). 13C
NMR (150 MHz, DMSO-d6) d 171.4, 165.00, 145.7, 144.2, 142.9,
stirred at room temperature (rt) for 6 h. After completion of the
reaction, the mixture was neutralised with sat. NaHCO3(aq) and
extracted with EtOAc. The organic layer was dried over anhydrous
MgSO4, and the solvent was removed. The residue was purified by
flash column chromatography (DCM/MeOH ¼ 250/1) to afford the
title compound (0.14 g, 53%); mp 106 ꢀC (dec). 1H NMR (300 MHz,
DMSO-d6) d 11.21 (s, 1H), 9.61 (s, 1H), 8.17 (s, 1H), 7.70 (dd, J ¼ 8.4,
1.8 Hz, 1H), 7.53 (d, J ¼ 2.1 Hz, 1H), 7.38–7.33 (m, 2H), 7.29 (dd,
J ¼ 8.4, 2.1 Hz, 1H), 7.25 (dd, J ¼ 3.6, 1.2 Hz, 1H), 7.05 (dd, J ¼ 5.1, 131.8, 128.8, 128.2, 127.6, 124.5, 123.8, 123.7, 123.6, 123.2, 122.3,
3.6 Hz, 1H), 6.83 (d, J ¼ 8.1 Hz, 1H), 6.27 (s, 1H), 5.12 (s, 2H), 2.42 (s, 121.0, 116.4, 114.8, 47.8, 39.1, 27.1, 17.2, 13.7. HRMS (ESI) for
3H). 13C NMR (75 MHz, DMSO-d6) d 166.6, 144.4, 142.8, 137.9, C23H24N3O2S (M þ Hþ) calcd 406.1589, found 406.1592.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1395
4.1.4. N-(2-Amino-5-(thiophen-2-yl)phenyl)-1-isobutyrylindoline-5- 4.1.8. N-(2-Amino-5-(thiophen-2-yl)phenyl)-4-(pyridin-2-ylamino)-
carboxamide (10) benzamide (14)
The title compound was obtained in 92% overall yield from com- The title compound was obtained in 41% overall yield from com-
pound 35c in a manner similar to that described for the prepar- pound 43a in a manner similar to that described for the prepar-
1
ation of 7: 1H NMR (300 MHz, DMSO-d6) d 9.63 (s, 1H), 8.16 (d, ation of 7: mp 202 ꢀC. H NMR (300 MHz, DMSO-d6) d 9.56 (s, 1H),
J ¼ 7.5 Hz, 1H), 7.89–7.84 (m, 2H), 7.46 (d, J ¼ 7.5 Hz, 1H), 7.35 (dd, 9.41 (s, 1H), 8.25–8.22 (m, 1H), 7.96 (d, J ¼ 8.7 Hz, 2H), 7.84 (d,
J ¼ 5.4, 1.2 Hz, 1H), 7.29 (dd, J ¼ 8.1, 2.1 Hz, 1H), 7.24 (dd, J ¼ 3.6, J ¼ 8.7 Hz, 2H), 7.66–7.59 (m, 1H), 7.50 (d, J ¼ 2.4 Hz, 1H), 7.35 (dd,
1.2 Hz, 1H), 7.07–7.03 (m, 1H), 6.81 (d, J ¼ 8.1 Hz, 1H), 5.11 (s, 2H), J ¼ 5.1, 1.2 Hz, 1H), 7.30 (dd, J ¼ 8.4, 2.1 Hz, 1H), 7.25 (dd, J ¼ 3.6,
4.24 (t, J ¼ 8.4 Hz, 2H), 3.23–3.19 (m, 2H), 2.90–2.85 (m, 1H), 1.12 1.2 Hz, 1H), 7.05 (dd, J ¼ 5.1, 3.6 Hz, 1H), 6.92 (d, J ¼ 8.4 Hz, 1H),
(d, J ¼ 6.6 Hz, 6H). 13C NMR (150 MHz, DMSO-d6) d 175.5, 164.9, 6.86–6.80 (m, 2H), 5.13 (s, 2H). 13C NMR (75 MHz, DMSO-d6) d
145.8, 144.2, 142.9, 132.0, 128.9, 128.1, 127.5, 124.4, 123.8, 123.7, 165.1, 155.3, 147.3, 144.8, 144.3, 142.9, 137.5, 128.8, 128.2, 125.5,
123.6, 123.1, 122.3, 120.9, 116.4, 115.2, 47.8, 32.5, 30.6, 27.1, 18.9. 123.9, 123.8, 123.7, 123.2, 122.4, 121.0, 116.5, 116.4, 115.2, 111.5.
HRMS (ESI) for C23H24N3O2S (M þ Hþ) calcd 406.1589, HRMS (ESI) for C22H19N4OS (M þ Hþ) calcd 387.1274,
found 406.1588.
found 387.1276.
4.1.5. N-(2-Amino-5-(thiophen-2-yl)phenyl)-1-(methylsulfonyl)indo-
line-5-carboxamide (11)
4.1.9.
N-(2-Amino-5-(thiophen-2-yl)phenyl)-4-(2-oxopyrrolidin-1-
yl)benzamide (15)
The title compound was obtained in 70% overall yield from com-
pound 35d in a manner similar to that described for the prepar-
ation of 7: 1H NMR (300 MHz, DMSO-d6) d 9.64 (s, 1H), 7.93–7.88
(m, 2H), 7.45 (d, J ¼ 2.1 Hz, 1H), 7.37–7.32 (m, 2H), 7.29 (dd, J ¼ 8.4,
2.1 Hz, 1H), 7.23 (dd, J ¼ 3.6, 0.9 Hz, 1H), 7.07–7.03 (m, 1H), 6.81 (d,
J ¼ 8.4 Hz, 1H), 5.12 (s, 2H), 4.02 (t, J ¼ 8.4 Hz, 2H), 3.19 (t,
J ¼ 8.4 Hz, 2H), 3.07 (s, 3H). 13C NMR (150 MHz, DMSO-d6) d 164.8,
144.7, 144.2, 142.9, 131.8, 129.4, 128.2, 128.1, 125.2, 123.9, 123.8,
123.5, 123.1, 122.2, 120.9, 116.3, 112.1, 50.2, 34.6, 27.0. HRMS (ESI)
for C20H20N3O3S2 (M þ Hþ) calcd 414.0946, found 414.0948.
The title compound was obtained in 55% overall yield from com-
pound 43b in a manner similar to that described for the prepar-
1
ation of 7: mp 223 ꢀC. H NMR (300 MHz, DMSO-d6) d 9.77 (s, 1H),
8.05 (d, J ¼ 8.7 Hz, 2H), 7.80 (d, J ¼ 9.0 Hz, 2H), 7.48 (d, J ¼ 2.1 Hz,
1H), 7.35 (dd, J ¼ 5.1, 1.2 Hz, 1H), 7.29 (dd, J ¼ 8.4, 2.1 Hz, 1H), 7.25
(dd, J ¼ 3.6, 1.2 Hz, 1H), 7.05 (dd, J ¼ 5.1, 3.6 Hz, 1H), 6.82 (d,
J ¼ 8.4 Hz, 1H), 5.16 (s, 2H), 3.90 (t, J ¼ 7.2 Hz, 2H), 2.55 (t,
J ¼ 7.8 Hz, 2H), 2.15–2.04 (m, 2H). 13C NMR (150 MHz, DMSO-d6) d
174.4, 164.8, 144.3, 143.0, 142.2, 129.2, 128.6, 128.2, 123.9, 123.8,
123.6, 123.2, 122.3, 121.0, 118.2, 116.4, 48.0, 32.4, 17.3. HRMS (ESI)
for C21H20N3O2S (M þ Hþ) calcd 378.1271, found 378.1275.
4.1.6. N-(2-Amino-5-(thiophen-2-yl)phenyl)-1-(phenylsulfonyl)indo-
line-5-carboxamide (12)
4.1.10.
N-(2-Amino-5-(thiophen-2-yl)phenyl)-4-(2-oxopiperidin-1-
The title compound was obtained in 93% overall yield from com-
pound 35e in a manner similar to that described for the prepar-
ation of 7: 1H NMR (300 MHz, DMSO-d6) d 9.71 (s, 1H), 7.91–7.86
(m, 3H), 7.80 (s, 1H), 7.75–7.68 (m, 1H), 7.64–7.55 (m, 3H), 7.45 (d,
J ¼ 2.1 Hz, 1H), 7.38 (dd, J ¼ 5.1, 1.2 Hz, 1H), 7.32 (dd, J ¼ 5.1,
2.4 Hz, 1H), 7.26 (dd, J ¼ 3.6, 1.2 Hz, 1H), 7.05 (dd, J ¼ 5.1, 3.6 Hz,
1H), 6.87 (d, J ¼ 8.4 Hz, 1H), 4.01 (d, J ¼ 8.4 Hz, 2H), 3.03 (d,
J ¼ 8.4 Hz, 2H). 13C NMR (150 MHz, DMSO-d6) d 164.8, 144.0, 143.8,
140.5, 135.9, 133.9, 132.0, 129.5, 128.2, 128.1, 127.1, 125.2, 124.6,
123.9, 123.8, 123.7, 123.6, 121.4, 117.5, 112.9, 50.3, 26.8. HRMS
(ESI) for C25H22N3O3S2 (M þ Hþ) calcd 476.1103, found 476.1104.
yl)benzamide (16)
The title compound was obtained in 42% overall yield from com-
pound 43c in a manner similar to that described for the prepar-
ation of 7: mp 97 ꢀC (dec). H NMR (300 MHz, CDCl3) d 8.29 (s, 1H),
7.90 (d, J ¼ 8.4 Hz, 2H), 7.53 (d, J ¼ 2.1 Hz, 1H), 7.37–7.32 (m, 3H),
7.20–7.17 (m, 2H), 7.03 (dd, J ¼ 5.1, 3.6 Hz, 1H), 6.83 (d, J ¼ 8.7 Hz,
1H), 3.66 (t, J ¼ 6.0 Hz, 2H), 2.55 (t, J ¼ 6.0 Hz, 2H), 2.00–1.86 (m,
4H). 13C NMR (150 MHz, CDCl3) d 170.5, 165.3, 146.5, 144.4, 140.7,
132.1, 128.5, 128.0, 126.4, 126.2, 125.1, 124.8, 123.7, 123.2, 122.1,
118.7, 51.4, 33.1, 23.6, 21.4. HRMS (ESI) for C22H22N3O2S (M þ Hþ)
calcd 392.1427, found 392.1432.
1
4.1.7. 1-Acetyl-N-(2-amino-5-(thiophen-2-yl)phenyl)-1,2,3,4-tetrahy-
droquinoline-6-carboxamide (13)
The title compound was obtained in 50% overall yield from com-
4.1.11. N1-(2-Amino-5-(thiophen-2-yl)phenyl)-N4-methylterephtha-
lamide (17)
pound 39 in a manner similar to that described for the prepar- The title compound was obtained in 88% overall yield from com-
1
ation of 7: mp 107–109 ꢀC. H NMR (300 MHz, DMSO-d6) d 9.67 (s, pound 43d in a manner similar to that described for the prepar-
1
1H), 7.84 (s, 1H), 7.81 (d, J ¼ 8.4 Hz, 1H), 7.73–7.60 (m, 1H), 7.46 (d, ation of 7: mp 252 ꢀC. H NMR (300 MHz, DMSO-d6) d 9.83 (s, 1H),
J ¼ 2.1 Hz, 1H), 7.35 (dd, J ¼ 5.1, 1.2 Hz, 1H), 7.30 (dd, J ¼ 8.4, 8.59 (d, J ¼ 4.5 Hz, 1H), 8.07 (d, J ¼ 8.1 Hz, 2H), 7.95 (d, J ¼ 8.4 Hz,
2.1 Hz, 1H), 7.24 (dd, J ¼ 3.6, 1.2 Hz, 1H), 7.05 (dd, J ¼ 5.4, 3.9 Hz, 2H), 7.48 (d, J ¼ 1.8 Hz, 1H), 7.35 (dd, J ¼ 5.1, 1.2 Hz, 1H), 7.31 (dd,
1H), 6.81 (d, J ¼ 8.4 Hz, 1H), 5.13 (s, 2H), 3.72 (t, J ¼ 6.3 Hz, 2H), J ¼ 8.4, 2.4 Hz, 1H), 7.25 (dd, J ¼ 3.6, 0.9 Hz, 1H), 7.05 (dd, J ¼ 5.1,
2.80 (t, J ¼ 6.3 Hz, 2H), 2.23 (s, 3H), 1.91 (p, J ¼ 6.6 Hz, 2H). 13C NMR 3.6 Hz, 1H), 6.82 (d, J ¼ 8.4 Hz, 1H), 5.18 (s, 2H), 2.82 (d, J ¼ 4.5 Hz,
(150 MHz, DMSO-d6) d 169.6, 165.0, 144.2, 143.0, 141.4, 131.3, 3H). 13C NMR (75 MHz, DMSO-d6) d 165.9, 165.0, 144.2, 143.2,
129.9, 128.2, 125.4, 123.9, 123.9, 123.5, 123.2, 122.3, 121.0, 116.4, 136.9, 136.7, 128.2, 127.9, 127.0, 124.1, 124.1, 123.2, 123.1, 122.2,
44.1, 26.6, 23.4, 23.3. HRMS (ESI) for C22H22N3O2S (M þ Hþ) calcd 121.0, 116.3, 26.3. HRMS (ESI) for C19H18N3O2S (M þ Hþ) calcd
392.1427, found 392.1432.
352.1114, found 352.1120.

1396
E.-C. CHO ET AL.
4.1.12. N-(2-Amino-5-(thiophen-2-yl)phenyl)-4-(oxazol-2-yl)benza- purification. 1H NMR (300 MHz, DMSO-d6) d 9.67 (s, 1H), 8.12 (d,
J ¼ 2.4 Hz, 1H), 7.86 (dd, J ¼ 9.0, 2.4 Hz, 1H), 7.59 (d, J ¼ 8.7 Hz, 1H),
mide (18)
1.44 (s, 9H).
The title compound was obtained in 88% overall yield from com-
pound 43e in a manner similar to that described for the prepar-
1
ation of 7: mp 209–211 ꢀC. H NMR (300 MHz, DMSO-d6) d 9.88 (s,
4.1.17. Tert-Butyl (2-nitro-4-(thiophen-2-yl)phenyl)carbamate (24)
A mixture of 23 (1.23 g, 3.88 mmol), 2-thienylboronic acid (0.66 g,
5.16 mmol), Pd(PPh3)4 (0.27 g, 0.23 mmol), P(o-tol)3 (0.36 g,
1.17 mmol), K2CO3 (1.64 g, 11.87 mmol), H2O (7 ml) and DME
(20 ml) was stirred at reflux for 4.5 h. After completion of the reac-
tion, the mixture was filtered through celite and washed with
EtOAc. Water was added to the filtrate, and the two layers were
separated. The organic layer was dried over anhydrous MgSO4,
and the solvent was removed. The crude material was purified by
flash column chromatography (Hex/DCM ¼ 5/1) to afford the title
compound as a yellow solid (1.02 g, 82%). 1H NMR (300 MHz,
DMSO-d6) d 9.64 (s, 1 H), 8.14 (d, J ¼ 2.4 Hz, 1H), 7.94 (dd, J ¼ 8.7,
2.1 Hz, 1H), 7.69 (d, J ¼ 8.7 Hz, 1H), 7.64–7.60 (m, 2H), 7.17 (dd,
J ¼ 5.1, 3.6 Hz, 1 H), 1.45 (s, 9H).
1H), 8.30 (d, J ¼ 0.6 Hz, 1H), 8.17 (d, J ¼ 8.4 Hz, 2H), 8.12 (d,
J ¼ 8.4 Hz, 2H), 7.49 (d, J ¼ 2.1 Hz, 1H), 7.46 (d, J ¼ 0.6 Hz, 1H), 7.36
(dd, J ¼ 5.1, 1.2 Hz, 1H), 7.31 (dd, J ¼ 8.4, 2.1 Hz, 1H), 7.25 (dd,
J ¼ 3.6, 1.2 Hz, 1H), 7.05 (dd, J ¼ 5.1, 3.6 Hz, 1H), 6.82 (d, J ¼ 8.4 Hz,
1H), 5.20 (s, 2H). 13C NMR (75 MHz, DMSO-d6) d 164.8, 160.2, 144.2,
143.2, 140.7, 136.0, 129.3, 128.9, 128.8, 128.2, 125.7, 124.2, 124.1,
123.2, 123.1, 122.2, 121.0, 116.4. HRMS (ESI) for C20H16N3O2S
(M þ Hþ) calcd 362.0958, found 362.0964.
4.1.13. N-(2-Amino-5-(thiophen-2-yl)phenyl)-4-(oxazol-5-yl)benza-
mide (19)
The title compound was obtained in 69% overall yield from com-
pound 43f in a manner similar to that described for the prepar-
1
ation of 7: mp 209 ꢀC. H NMR (300 MHz, DMSO-d6) d 9.82 (s, 1H),
8.53 (s, 1H), 8.12 (d, J ¼ 8.4 Hz, 2H), 7.90–7.86 (m, 3H), 7.48 (d,
J ¼ 2.1 Hz, 1H), 7.35 (dd, 5.1, 1.2 Hz, 1H), 7.31 (dd, J ¼ 8.4, 2.1 Hz,
1H), 7.25 (dd, J ¼ 3.6, 0.9 Hz, 1H), 7.05 (dd, J ¼ 5.1, 3.6 Hz, 1H), 6.82
(d, J ¼ 8.4 Hz, 1H), 5.18 (s, 2H). 13C NMR (150 MHz, DMSO-d6) d
164.8, 152.4, 149.9, 144.2, 143.1, 134.2, 130.0, 128.7, 128.2, 124.1,
124.0, 123.8, 123.5, 123.2, 123.2, 122.3, 121.0, 116.4. HRMS (ESI) for
C20H16N3O2S (M þ Hþ) calcd 362.0958, found 362.0962.
4.1.18. Tert-Butyl (2-amino-4-(thiophen-2-yl)phenyl)carbamate (25)
A catalytic amount of 10% Pd/C was added to a suspension of 24
(1.02 g, 3.18 mmol) in MeOH (10 ml), and the mixture was stirred
at rt for 4 h under a hydrogen balloon. After completion of the
reaction, the mixture was filtered through celite, and the solvent
was removed in vacuo to afford the title compound as a light yel-
1
low solid (0.92 g, 99%). H NMR (300 MHz, DMSO-d6) d 8.33 (s, 1H),
7.44 (dd, J ¼ 5.1, 1.2 Hz, 1H), 7.29 (dd, J ¼ 3.6, 1.2 Hz, 1H), 7.26 (d,
J ¼ 8.4 Hz, 1H), 7.08 (dd, J ¼ 5.1, 3.6 Hz, 1H), 6.97 (d, J ¼ 2.1 Hz, 1H),
6.84 (d, J ¼ 8.4, 2.1 Hz, 1H), 5.00 (s, 2H), 1.46 (s, 9H).
4.1.14. N-(2-Amino-5-(thiophen-2-yl)phenyl)-4-(1H-benzo[d]imida-
zol-2-yl)benzamide(20)
The title compound was obtained in 60% overall yield from com-
pound 43g in a manner similar to that described for the prepar-
1
ation of 7: mp 356 ꢀC (dec). H NMR (300 MHz, DMSO-d6) d 9.97 (s,
4.1.19. Methyl (E)-3-(2-(dimethylamino)vinyl)-4-nitrobenzoate (27)
A
mixture of 26 (5 g, 25.63 mmol) and DMF-DMA (8 ml,
1H), 8.34 (d, 2H), 8.24 (d, 2H), 7.75–7.70 (m, 2H), 7.53 (d, J ¼ 2.1 Hz,
1H), 7.41–7.33 (m, 4H), 7.28 (dd, J ¼ 3.6, 0.9 Hz, 1H), 7.07 (dd,
J ¼ 5.1, 3.6 Hz, 1H), 6.89 (d, J ¼ 8.4 Hz, 1H). 13C NMR (150 MHz,
DMSO-d6) d 164.8, 149.5, 144.0, 141.6, 136.5, 136.5, 129.9, 128.8,
128.3, 126.9, 124.2, 124.0, 123.9, 123.6, 123.3, 121.4, 117.2, 114.9.
HRMS (ESI) for C24H19N4OS (M þ Hþ) calcd 411.1274,
found 411.1277.
60.09 mmol) was stirred at reflux for 31 h under an inert atmos-
phere. After completion of the reaction, the solvent was removed
in vacuo, and the residue was recrystallized from MeOH to yield
the title compound as a dark purple solid (3.9 g, 61%). 1H NMR
(300 MHz, DMSO-d6) d 8.16 (d, J ¼ 1.8 Hz, 1H), 7.84 (d, J ¼ 8.4 Hz,
1H), 7.5 (d, J ¼ 13.2 Hz, 1H), 7.43 (dd, J ¼ 8.4, 1.8 Hz, 1H), 5.51 (d,
J ¼ 13.5 Hz, 1H), 3.88 (s, 3H), 2.91 (s, 6H).
4.1.15. 4-Bromo-2-nitroaniline (22)
4.1.20. Methyl 4-nitro-3-(2-oxopropyl)benzoate (28)
NBS (4.83 g, 27.14 mmol) was added portionwise to a solution of
2-nitroaniline (21, 3.75 g, 27.17 mmol) in AcOH (24 ml) in an ice
bath, and stirred at 0 ꢀC for 9 min. The mixture was then poured
into ice-cold water, and the suspension was filtered to obtain the
title compound as an orange solid (5.52 g, 94%). 1H NMR
(300 MHz, DMSO-d6) d 8.06 (d, J ¼ 2.4 Hz, 1H), 7.57 (s, 2H), 7.53 (dd,
J ¼ 9.0, 2.4 Hz, 1H), 6.99 (d, J ¼ 9.0 Hz, 1H).
Acetyl chloride (1.44 ml, 20.36 mmol) was added dropwise to a
solution of 27 (3.6 g, 14.39 mmol) and pyridine (1.85 ml,
22.96 mmol) in DCM (60 ml). After stirring at rt for 24 h, water was
added to the mixture and the organic layer was collected and
concentrated. Water (9.6 ml) and dioxane (18 ml) were added to
the residue, which was then heated to reflux for 15 h. After cool-
ing down, the dioxane was then evaporated under reduced pres-
sure, and the residue was extracted with EtOAc. The organic layer
was collected, dried over anhydrous MgSO4, concentrated, and
purified by flash column chromatography (Hex/EtOAc ¼ 5/1) to
4.1.16. Tert-Butyl (4-bromo-2-nitrophenyl)carbamate (23)
A mixture of 22 (5.52 g, 25.44 mmol), Boc2O (16.8 g, 76.98 mmol),
and DMAP (0.31 g, 2.54 mmol) in THF (50 ml) was stirred at rt for
2 days, and then the solvent was removed in vacuo to obtain a
dark purple oil which was redissolved in THF (50 ml), treated with
5 N NaOH(aq) (16 ml, 80 mmol), and heated to 70 ꢀC for 15 h. After
completion of the reaction, the solvent was removed under
reduced pressure. Water was added water to the residue, which
was then extracted with DCM, and dried over anhydrous MgSO4.
1
afford the title compound (2.2 g, 64%). H NMR (300 MHz, CDCl3) d
8.14 (d, J ¼ 8.4 Hz, 1H), 8.09 (dd, J ¼ 8.4, 1.8 Hz, 1H), 7.95 (d,
J ¼ 1.8 Hz, 1H), 4.18 (s, 2H), 3.96 (s, 1H), 2.33 (s, 1H).
4.1.21. Methyl 2-methyl-1H-indole-5-carboxylate (29)
A mixture of 28 (2.8 g, 11.80 mmol) and Fe powder (6.53 g,
The solvent was then concentrated to afford the title compound 116.61 mmol) in AcOH (30 ml) was stirred at 90 ꢀC for 12.5 h. After
(8.00 g, 99%) which was used in next step without further cooling down, the reaction mixture was filtered through celite,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1397
neutralised with sat. NaHCO3(aq), and extracted with EtOAc. The 4.1.27. 1-Acetylindoline-5-carboxylic acid (34a)
1N LiOH(aq) (2.65 ml) was added to a solution of compound 33a
(0.2 g, 0.91 mmol) in THF (5 ml), and the mixture was stirred at rt
for 5 h. After completion of the reaction, water was added to the
organic layer was dried over anhydrous MgSO4, and the solvent
was removed. The crude material was purified by flash column
chromatography (Hex/EtOAc ¼ 6/1) to afford the title compound
(1.83 g, 82%). 1H NMR (300 MHz, DMSO-d6) d 11.30 (s, 1H), mixture which was then acidified with 3 N HCl(aq) to pH ꢆ 4. The
resulting suspension was filtered to yield the title compound
8.11–8.09 (m, 1 H), 7.64 (dd, J ¼ 8.4, 1.8 Hz, 1H), 7.33 (dt, J ¼ 8.4,
(0.14 g, 75%). 1H NMR (300 MHz, DMSO-d6) d 8.07 (d, 8.7 Hz, 1H),
0.6 Hz, 1H), 6.27–6.26 (m, 1H), 3.81 (s, 3H), 2.39 (d, J ¼ 0.6 Hz, 3H).
7.79–7.76 (m, 2H), 4.14 (t, J ¼ 8.7 Hz, 2H), 3.17 (t, J ¼ 8.7 Hz, 2H),
2.18 (s, 3H).
4.1.22. 2-Methyl-1H-indole-5-carboxylic acid (30)
NaOH (0.5 g, 12.50 mmol) and H2O (6 ml) were added to a solution
4.1.28. 1-Butyrylindoline-5-carboxylic acid (34b)
of 29 (0.6 g, 3.17 mmol) in MeOH (15 ml). The mixture was stirred
at rt for 1.5 h, then acidified with 3 N HCl(aq). The resulting suspen-
sion was filtered to afford the title compound (0.55 g, 98%). 1H
NMR (300 MHz, DMSO-d6) d 12.29 (s, 1H), 11.25 (s, 1H), 8.07 (d,
J ¼ 1.8 Hz, 1H), 7.62 (dd, J ¼ 8.4, 1.8 Hz, 1H), 7.30 (d, J ¼ 8.4 Hz, 1H),
6.25–6.23 (m, 1H), 2.39 (d, J ¼ 0.9 Hz, 3H).
The title compound was obtained from compound 33b in a man-
ner similar to that described for the preparation of 34a: 1H NMR
(300 MHz, DMSO-d6) d 12.61 (s, 1H), 8.11 (d, J ¼ 8.7 Hz, 1H),
7.79–7.75 (m, 2H), 4.13 (t, J ¼ 8.4 Hz, 2H), 3.16 (t, J ¼ 8.4 Hz, 2H),
2.45 (t, J ¼ 7.5 Hz, 2H), 1.67–1.54 (m, 2H), 0.94 (t, J ¼ 7.5 Hz, 3H).
4.1.29. 1-Isobutyrylindoline-5-carboxylic acid (34c)
4.1.23. Tert-Butyl (2-(2-methyl-1H-indole-5-carboxamido)-4-(thio-
phen-2-yl)phenyl) carbamate (31)
The title compound was obtained from compound 33c in a man-
ner similar to that described for the preparation of 34a: 1H NMR
(300 MHz, DMSO-d6) d 12.63 (s, 1H), 8.13 (d, J ¼ 7.8 Hz, 1H),
7.79–7.75 (m, 2H), 4.21 (t, J ¼ 8.4 Hz, 2H), 3.18 (t, J ¼ 8.4 Hz, 2H),
2.90–2.80 (m, 1H), 1.10 (d, J ¼ 6.6 Hz, 6H).
DIPEA (2.48 ml, 14.24 mmol) was added to a solution of 30 (0.5 g,
2.85 mmol), 25 (1.13 g, 3.89 mmol), and HATU (2.17 g, 5.71 mmol)
in DMF (14 ml), and the mixture was stirred at 70 ꢀC for 20 h. The
reaction was quenched with water, followed by extraction with
EtOAc. The organic layer was dried over anhydrous MgSO4, con-
centrated, and purified by flash column chromatography to afford
the title compound (0.48 g, 38%). 1H NMR (300 MHz, DMSO-d6) d
11.27 (s, 1H), 9.82 (s, 1H), 8.78 (s, 1H), 8.11 (d, J ¼ 1.5 Hz, 1H), 7.89
(d, J ¼ 2.1 Hz, 1H), 7.66 (dd, J ¼ 8.7, 1.5 Hz, 1H), 7.56 (d, J ¼ 8.7 Hz,
1H), 7.53 (dd, J ¼ 5.1, 1.2 Hz, 1H), 7.49 (dd, J ¼ 8.4, 2.1 Hz, 1H), 7.46
(dd, 3.6, 1.2 Hz, 1H), 7.37 (d, J ¼ 8.7 Hz, 1H), 7.13 (dd, J ¼ 5.1, 3.6 Hz,
1H), 6.27 (s, 1H), 2.42 (d, J ¼ 0.6 Hz, 3H), 1.47 (s, 9H).
4.1.30. 1-(Methylsulfonyl)indoline-5-carboxylic acid (34d)
The title compound was obtained from compound 33d in a man-
ner similar to that described for the preparation of 34a: 1H NMR
(300 MHz, DMSO-d6) d 12.71 (s, 1H), 7.84–7.80 (m, 2H), 7.33–7.29
(m, 1H), 4.00 (t, J ¼ 8.4 Hz, 2H), 3.15 (t, J ¼ 8.4 Hz, 2H), 3.06 (s, 3H).
4.1.31. 1-(Phenylsulfonyl)indoline-5-carboxylic acid (34e)
The title compound was obtained from compound 33e in a man-
ner similar to that described for the preparation of 34a: 1H NMR
(300 MHz, DMSO-d6) d 12.72 (s, 1H), 7.89–7.85 (m, 2H), 7.80 (dd,
J ¼ 8.4, 1.8 Hz, 1H), 7.74–7.67 (m, 2H), 7.63–7.56 (m, 2H), 7.53 (d,
J ¼ 8.4 Hz, 1H), 3.97 (t, J ¼ 8.4 Hz, 2H), 3.00 (t, J ¼ 8.4 Hz, 2H).
4.1.24. Methyl 1-acetylindoline-5-carboxylate (33a)
A mixture of methyl indoline-5-carboxylate (32, 1.0 g, 5.64 mmol)
and acetic anhydride (Ac2O, 3.5 ml) was stirred at 130 ꢀC for 2 h.
After completion of the reaction, the mixture was poured into ice-
cold water, and the resulting suspension was filtered. The solid
was recrystallized from MeOH to afford the title compound
1
(1.08 g, 87%). H NMR (300 MHz, DMSO-d6) d 8.10 (d, J ¼ 9 Hz, 1H),
4.1.32. Tert-Butyl (2-(1-acetylindoline-5-carboxamido)-4-(thiophen-
2-yl)phenyl) carbamate (35a)
7.82–7.78 (m, 2H), 4.15 (t, J ¼ 8.7 Hz, 2H), 3.81 (s, 3H), 3.18 (t,
J ¼ 8.4 Hz, 2H), 2.19 (s, 3H).
The title compound was obtained in 90% overall yield from com-
pound 34a in a manner similar to that described for the prepar-
ation of 31: 1H NMR (300 MHz, DMSO-d6) d 9.84 (s, 1H), 8.74 (s,
1H), 8.13 (d, J ¼ 9.0 Hz, 1H), 7.86–7.80 (m, 3H), 7.58 (d, J ¼ 8.4 Hz,
1H), 7.54–7.48 (m, 2H), 7.45 (dd, J ¼ 3.6, 1.2 Hz, 1H), 7.13 (dd,
J ¼ 5.1, 3.6 Hz, 1H), 4.18 (t, J ¼ 8.7 Hz, 2H), 3.22 (t, J ¼ 8.7 Hz, 2H),
2.20 (s, 3H), 1.46 (s, 9H).
4.1.25. Methyl 1-(methylsulfonyl)indoline-5-carboxylate (33d)
The title compound was obtained from compound 32 in a man-
ner similar to that described for the preparation of 33a: 1H NMR
(300 MHz, CDCl3) d 7.93–7.86 (m, 2H), 7.40 (d, J ¼ 8.4 Hz, 1H), 4.04
(t, J ¼ 8.7 Hz, 2H), 3.88 (s, 3H), 3.18 (t, J ¼ 8.7 Hz, 2H), 2.92 (s, 3H).
4.1.33. Tert-Butyl (2-(1-butyrylindoline-5-carboxamido)-4-(thio-
phen-2-yl)phenyl)carbamate (35b)
4.1.26. Methyl 1-(methyl-(k1-oxidanyl)-(phenyl)sulfinyl)indoline-5-
The title compound was obtained from compound 34b in a man-
carboxylate (33e)
The title compound was obtained from compound 32 in a man- ner similar to that described for the preparation of 31: 1H NMR
ner similar to that described for the preparation of 33a: 1H NMR (300 MHz, DMSO-d6) d 9.83 (s, 1H), 8.74 (s, 1H), 8.16 (d, J ¼ 8.4 Hz,
(300 MHz, DMSO-d6) d 7.90–7.81 (m, 3H), 7.74–7.67 (m, 2H), 1H), 7.85–7.82 (m, 3H), 7.60–7.44 (m,4H), 7.15–7.11 (m, 1H), 4.17 (t,
7.63–7.54 (m, 3H), 3.98 (t, J ¼ 8.4 Hz, 2H), 3.79 (s, 3H), 3.02 (t, J ¼ 8.4 Hz, 2H), 3.21 (t, J ¼ 8.4 Hz, 2H), 2.48–2.45 (m, 1H), 1.67–1.59
J ¼ 8.4 Hz, 2H).
(m, 2H), 1.56 (s, 9H), 0.95 (t, J ¼ 7.5 Hz, 3H).

1398
E.-C. CHO ET AL.
1
4.1.34. Tert-Butyl (2-(1-isobutyrylindoline-5-carboxamido)-4-(thio- preparation of 31: H NMR (300 MHz, DMSO-d6) d 9.88 (s, 1H), 8.71
(s, 1H), 7.83–7.76 (m, 3H), 7.74–7.68 (m, 1H), 7.60 (d, J ¼ 8.4 Hz,
phen-2-yl)phenyl)carbamate (35c)
The title compound was obtained in 90% overall yield from com-
1H), 7.54–7.48 (m, 2H), 7.45 (dd, J ¼ 3.6, 0.9 Hz, 1H), 7.13 (dd,
pound 34c in a manner similar to that described for the prepar-
J ¼ 5.1, 3.6 Hz, 1H), 3.73 (t, J ¼ 6.3 Hz, 2H), 2.78 (t, J ¼ 6.3 Hz, 2H),
ation of 31: 1H NMR (300 MHz, DMSO-d6) d 9.83 (s, 1H), 8.75 (s,
2.23 (s, 3H), 1.91 (p, J ¼ 6.3 Hz, 2H), 1.46 (s, 9H).
1H), 8.19 (d, J ¼ 6.9 Hz, 1H), 7.85–7.81 (m, 3H), 7.59–7.44 (m, 4H),
7.15–7.11 (m, 1H), 4.25 (t, J ¼ 8.7 Hz, 2H), 3.23 (t, J ¼ 8.7 Hz, 2H),
2.89–2.85 (m, 1H), 1.46 (s, 9H), 1.12 (d, J ¼ 6.9 Hz, 6H).
4.1.40. Methyl 4-(pyridin-2-ylamino)benzoate (41a)
A mixture of methyl-4-aminobenzoate (40a, 2.1 g, 13.3 mmol) and
2-bromopyridine (1 g, 6.33 mmol) was heated to 140 ꢀC and
reacted for 2 h. After cooling down, water and 1 N HCl(aq) (13 ml)
was added to the mixture, and the solution was washed with
EtOAc. The organic layer was removed, and the aqueous layer was
basified with 5 N NaOH(aq), followed by extraction with EtOAc. The
organic layer was collected, dried over anhydrous MgSO4, concen-
4.1.35. Tert-Butyl (2-(1-(methylsulfonyl)indoline-5-carboxamido)-4-
(thiophen-2-yl)phenyl)-carbamate (35d)
The title compound was obtained in 90% overall yield from com-
pound 34d in a manner similar to that described for the prepar-
ation of 31: 1H NMR (300 MHz, DMSO-d6) d 9.85 (s, 1H), 8.70 (s,
1H), 7.89–7.86 (m, 2H), 7.80 (d, J ¼ 2.1 Hz, 1H), 7.60 (d, J ¼ 8.4 Hz,
trated, and purified by flash column chromatography (DCM then
1H), 7.54–7.48 (m, 2H), 7.44 (dd, J ¼ 3.6, 0.9 Hz, 1H), 7.36 (d,
1
DCM/MeOH ¼ 20/1) to yield the title compound (0.87 g, 60%). H
NMR (300 MHz, DMSO-d6) d 9.52 (s, 1H), 8.25–8.21 (m, 1H),
7.89–7.80 (m, 4H), 7.67–7.60 (m, 1H), 6.94–6.90 (m, 1H), 6.88–6.82
(m, 1H), 3.80 (s, 3H).
J ¼ 9.0 Hz, 1H), 7.15–7.11 (m, 1H), 4.03 (t, J ¼ 8.4 Hz, 2H), 3.20 (t,
J ¼ 8.4 Hz, 2H), 3.08 (s, 3H), 1.46 (s, 9H).
4.1.36. Tert-Butyl (2-(1-(phenylsulfonyl)indoline-5-carboxamido)-4-
(thiophen-3-yl)phenyl)-carbamate (35e)
The title compound was obtained in 90% overall yield from com-
pound 34e in a manner similar to that described for the prepar-
ation of 31: 1H NMR (300 MHz, DMSO-d6) d 9.81 (s, 1H), 8.69 (s,
1H), 7.91–7.84 (m, 3H), 7.77–7.69 (m, 3H), 7.64–7.57 (m, 4H),
7.53–7.47 (m, 2H), 7.43 (dd, J ¼ 3.6, 1.2 Hz, 1H), 7.12 (dd, J ¼ 5.1,
3.6 Hz, 1H), 4.01 (t, J ¼ 8.4 Hz, 2H), 3.04 (t, J ¼ 8.4 Hz, 2H), 1.44
(s, 9H).
4.1.41. Methyl 4-(2-oxopyrrolidin-1-yl)benzoate (41b)
A mixture of methyl 4-bromobenzoate (40b, 2.4 g, 11.2 mmol), 2-
pyrrolidone (0.8 g, 9.4 mmol), Pd2(dba)3 (0.22 g, 0.24 mmol), xant-
phos (0.4 g, 0.71 mmol), Cs2CO3 (4.3 g,13.16 mmol) and p-dioxane
(30 ml) was stirred at 90 ꢀC for 2 h. After completion of the reac-
tion, the mixture was filtered through celite, and the solvent was
removed in vacuo. The residue was purified by flash column chro-
matography (Hex/EtOAc ¼ 3/2) to afford the title compound
(2.02 g, 98%). 1H NMR (300 MHz, CDCl3) d 8.03 (d, J ¼ 9.0 Hz, 2H),
7.72 (d, J ¼ 9.0 Hz, 2H), 3.92–3.87 (m, 5H), 2.64 (t, J ¼ 8.1 Hz, 2H),
2.24–2.13 (m, 2H).
4.1.37. 1,2,3,4-Tetrahydroquinoline-6-carboxylic acid (37)
A mixture of quinoline-6-carboxylic acid (36, 1 g, 5.8 mmol),
ammonium formate (1.82 g, 28.9 mmol) and 10% Pd/C (0.61 g,
0.58 mmol) in MeOH (30 ml) was refluxed for 2 h. After completion
of the reaction, the mixture was filtered through celite, and the
solvent was removed in vacuo. Water was added to the residue
which was extracted several times with DCM and EtOAc. The
organic layers were combined, dried over anhydrous MgSO4, and
evaporated under reduced pressure to yield the title compound
4.1.42. Methyl 4-(2-oxopiperidin-1-yl)benzoate (41c)
A mixture of 2-piperdone (0.5 g, 5 mmol), methyl 4-bromoben-
zoate (40b, 1.26 g, 5.85 mmol), Pd2(dba)3 (0.12 g, 0.13 mmol), xant-
phos (0.22 g, 0.38 mmol), Cs2CO3 (2.3 g, 7 mmol) and p-dioxane
(16 ml) was stirred at reflux for 3 h. After completion of the reac-
tion, the mixture was filtered through celite, and the solvent was
removed in vacuo. The residue was purified by flash column chro-
matography (Hex/EtOAc ¼ 1/1) to afford the title compound
(1.11 g, 99%). 1H NMR (300 MHz, DMSO-d6) d 8.05 (d, J ¼ 8.7 Hz,
2H), 7.36 (d, J ¼ 9.0 Hz, 2H), 3.91 (s, 3H), 3.71–3.66 (m, 2H),
2.61–2.56 (m, 2H), 1.98–1.92 (m, 4H).
1
(0.5 g, 49%). H NMR (300 MHz, DMSO-d6) d 11.83 (s, 1H), 7.46–7.41
(m, 2H), 6.49 (s, 1H), 6.42–6.38 (m, 1H), 3.25–3.19 (m, 2H), 2.66 (t,
J ¼ 6.0 Hz, 2H), 7.82–7.73 (m, 2H).
4.1.38. 1-Acetyl-1,2,3,4-tetrahydroquinoline-6-carboxylic acid (38)
Acetyl chloride (CH3COCl, 0.08 ml, 1.13 mmol) was added to a solu-
tion of compound 37 (0.2 g, 1.13 mmol) and TEA (0.16 ml,
1.13 mmol) in DCM (6 ml), and the mixture was stirred at rt for
20 min. After the reaction, 5ꢂ6 equivalents of 1 N NaOH(aq) were
added, and the mixture water was added to the mixture which
was then washed with EtOAc. The aqueous layer was then acidi-
fied with 1 N HCl(aq) and extracted with EtOAc. The organic layers
were collected, dried over anhydrous MgSO4 and concentrated to
4.1.43. Methyl 4-(methylcarbamoyl)benzoate (41d)
A
mixture of 4-(methoxycarbonyl)benzoic acid (40c, 0.5 g,
2.8 mmol), methylamine hydrochloride (0.2 g, 3 mmol), EDCꢁHCl (1-
ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride, 1 g,
5.6 mmol) and DMAP (4-dimethylaminopyridine, 1.5 g, 12.6 mmol)
in DMF (5 ml) was stirred at rt for 5 h. The reaction was quenched
with water, added 3 N HCl(aq), and extracted with EtOAc. The
organic layer was washed with sat. NaHCO3(aq), dried over anhyd-
rous MgSO4 and concentrated to afford the title compound
(0.28 g, 52%). 1H NMR (300 MHz, CDCl3) d 8.08 (d, J ¼ 8.4 Hz, 2H),
7.81 (d, J ¼ 8.4 Hz, 2H), 6.27 (s, 1H), 3.93 (s, 3H), 3.03 (d,
1
afford the title compound (0.15 g, 61%). H NMR (300 MHz, DMSO-
d6) d 7.80–7.60 (m, 3H), 3.70 (t, J ¼ 6.3 Hz, 2H), 2.76 (t, J ¼ 6.6 Hz,
2H), 2.21 (s, 3 H), 1.92–1.83 (m, 2H).
4.1.39. Tert-Butyl (2-(1-acetyl-1,2,3,4-tetrahydroquinoline-6-carbox-
amido)-4-(thiophen-2- l)phenyl) carbamate (39)
The title compound was obtained in 80% overall yield from com-
pound 38 in
a
manner similar to that described for the J ¼ 4.8 Hz, 3H).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1399
4.1.44. Methyl 4-(oxazol-2-yl)benzoate (41e)
4.1.50. 4-(Methylcarbamoyl)benzoic acid (42d)
A
mixture of 4-(methoxycarbonyl)benzoic acid (40c, 6 g, The title compound was obtained in 99% overall yield from com-
33.3 mmol),
aminoacetaldehyde
dimethyl
acetal
(3.52 g, pound 41d in a manner similar to that described for the prepar-
33.3 mmol), EDCꢁHCl (12.8 g, 66.6 mmol), and NMM (N-methylmor- ation of 34a: 1H NMR (300 MHz, DMSO-d6) d 8.59 (d, J ¼ 4.5 Hz,
pholine, 9 ml, 81.8 mmol) in DCM (67 ml) was stirred at rt for 22 h. 1H), 8.00 (d, J ¼ 8.4 Hz, 2H), 7.91 (d, J ¼ 8.4 Hz, 2H), 2.79 (d,
The reaction was quenched with water, acidified with 3 N HCl(aq) J ¼ 4.5 Hz, 3H).
to pH ꢆ 2, and extracted with DCM. The organic layer was dried
over anhydrous MgSO4, and the solvent was removed in vacuo to
4.1.51. 4-(Oxazol-2-yl)benzoic acid (42e)
afford the crude product. Phosphorus pentoxide (P4O10, 15 g,
52.8 mmol) was added to a solution of the crude product (3 g,
11.2 mmol) in MeSO3H (22 ml) in an ice bath, and the mixture was
heated to 140 ꢀC for 9 h under an inert atmosphere. After cooling
down, water was added to the mixture, which was then extracted
with DCM, and dried over anhydrous MgSO4. After removal of the
solvent, the crude material was purified by flash column chroma-
tography (Hex/EtOAc ¼ 3/2) to afford the title compound (0.23 g,
7.8%). 1H NMR (300 MHz, CDCl3) d 8.13 (s, 4H), 7.76 (d, J ¼ 0.9 Hz,
1H), 7.29 (d, J ¼ 0.6 Hz, 1H), 3.95 (s, 3H).
The title compound was obtained in 89% overall yield from com-
pound 41e in a manner similar to that described for the prepar-
1
ation of 30: H NMR (300 MHz, DMSO-d6) d 8.29 (d, J ¼ 0.6 Hz, 1H),
8.12–8.05 (m, 4H), 7.45 (d, J ¼ 0.6 Hz, 1H).
4.1.52. 4-(Oxazol-5-yl)benzoic acid (42f)
The title compound was obtained in 100% overall yield from com-
pound 41f in a manner similar to that described for the prepar-
ation of 30: 1H NMR (300 MHz, DMSO-d6) d 8.53 (s, 1H), 8.03 (d,
J ¼ 8.4 Hz, 2H), 7.87–7.83 (m, 3H).
4.1.45. Methyl 4-(oxazol-5-yl)benzoate (41f)
A mixture of methyl 4-formylbenzoate (40d, 0.6 g, 3.66 mmol),
TosMIC (0.86 g, 4.39 mmol), and K2CO3 (0.66 g, 4.75 mmol) in
MeOH (10 ml) was stirred at reflux for 3 h. After cooling down, the
resulting suspension was filtered to afford the title compound
(0.48 g, 65%). 1H NMR (300 MHz, CDCl3) d 8.10 (d, J ¼ 9.0 Hz, 2H),
7.97 (s, 1H), 7.73 (d, J ¼ 8.7 Hz, 2H), 7.48 (s, 1H), 3.94 (s, 3H).
4.1.53. 4-(1h-Benzo[d]imidazol-2-yl)benzoic acid (42g)
The title compound was obtained in 77% overall yield from com-
pound 41g in a manner similar to that described for the prepar-
ation of 34a: 1H NMR (300 MHz, DMSO-d6) d 8.29 (d, J ¼ 8.4 Hz,
2H), 8.11 (d, J ¼ 8.4 Hz, 2H), 7.66–7.61 (m, 2H), 7.29–7.23 (m, 2H).
4.1.54. Tert-Butyl (2-(4-(pyridin-2-ylamino)benzamido)-4-(thiophen-
2-yl)phenyl) carbamate (43a)
4.1.46. Methyl 4-(1H-benzo[d]imidazol-2-yl)benzoate (41g)
A mixture of methyl 4-formylbenzoate (40d, 1.5 g, 9.14 mmol), o-
phenylenediamine (1 g, 9.25 mmol) and Na2S2O4 (1.6 g, 9.2 mmol)
in DMF (6 ml) was stirred at 70 ꢀC for 18 h. After completion of the
reaction, ice-cold water was added, and the resulting suspension
was filtered to afford the title compound (2.21 g, 95%). 1H NMR
(300 MHz, DMSO-d6) d 8.31 (d, J ¼ 8.7 Hz, 2H), 8.12 (d, J ¼ 8.7 Hz,
2H), 7.65–7.61 (m, 2H), 7.28–7.21 (m, 2H), 3.89 (s, 3H).
The title compound was obtained from compound 42a in a man-
ner similar to that described for the preparation of 31: 1H NMR
(600 MHz, DMSO-d6) d 9.76 (s, 1H), 9.45 (s, 1H), 8.71 (s, 1H), 8.24
(dd, J ¼ 5.4, 1.8 Hz, 1H), 7.92 (d, J ¼ 7.2 Hz, 2H), 7.85 (d, J ¼ 9.0 Hz,
2H), 7.84 (d, J ¼ 2.4 Hz, 1H), 7.66–7.62 (m, 1H), 7.59 (d, J ¼ 8.4 Hz,
1H), 7.53 (dd, J ¼ 4.8, 1.2 Hz, 1H), 7.50 (dd, J ¼ 8.4, 1.4 Hz, 1H), 7.45
(dd, J ¼ 4.2, 1.2 Hz, 1H), 7.13 (dd, J ¼ 5.4, 3.6 Hz, 1H), 6.92 (d,
J ¼ 8.4 Hz, 1H), 6.86–6.83 (m, 1H), 1.47 (s, 9H).
4.1.47. 4-(Pyridin-2-ylamino)benzoic acid (42a)
A mixture of compound 41a (0.55 g, 2.40 mmol) and 1 N LiOH(aq)
(7.2 ml) was stirred in p-dioxane (10 ml) at rt for 3 h. The solution
4.1.55. Tert-Butyl (2-(4-(2-oxopyrrolidin-1-yl)benzamido)-4-(thio-
was quenched with water and neutralised with 3 N HCl(aq). The phen-2-yl)phenyl) carbamate (43b)
The title compound was obtained in 85% overall yield from com-
solvent was removed under reduced pressure, and the crude
product (0.93 g) was used in next step without purification.
pound 42b in a manner similar to that described for the prepar-
ation of 31: 1H NMR (300 MHz, DMSO-d6) d 9.89 (s, 1H), 8.72 (s,
1H), 8.00 (d, J ¼ 8.7 Hz, 2H), 7.84 (d, J ¼ 8.7 Hz, 2H), 7.82 (d,
J ¼ 2.1 Hz, 1H), 7.61 (d, J ¼ 8.4 Hz, 1H), 7.54–7.49 (m, 2H), 7.46 (dd,
J ¼ 3.6, 1.2 Hz, 1H), 7.13 (dd, J ¼ 5.1, 3.6 Hz, 1H), 3.90 (t, J ¼ 7.2 Hz,
2H), 2.55 (t, J ¼ 7.8 Hz, 2H), 2.15–2.04 (m, 2H), 1.45 (s, 9H).
4.1.48. 4-(2-Oxopyrrolidin-1-yl)benzoic acid (42b)
The title compound was obtained in 76% overall yield from com-
pound 41b in a manner similar to that described for the prepar-
ation of 30: 1H NMR (300 MHz, MeOH-d4 þ CDCl3) d 8.02 (d,
J ¼ 9.0 Hz, 2H), 7.75 (d, J ¼ 9.0 Hz, 2H), 3.96 (t, J ¼ 7.2 Hz, 2H), 2.63
(t, J ¼ 7.8 Hz, 2H), 2.25–2.14 (m, 2H).
4.1.56. Tert-Butyl (2-(4-(2-oxopiperidin-1-yl)benzamido)-4-(thio-
phen-2-yl)phenyl) carbamate (43c)
The title compound was obtained in 82% overall yield from com-
pound 42c in a manner similar to that described for the prepar-
4.1.49. 4-(2-Oxopiperidin-1-yl)benzoic acid (42c)
The title compound was obtained in 68% overall yield from com- ation of 31: 1H NMR (300 MHz, DMSO-d6) d 9.91 (s, 1H), 8.71 (s,
pound 41c in a manner similar to that described for the prepar- 1H), 7.98 (d, J ¼ 8.7 Hz, 2H), 7.82 (d, J ¼ 2.1 Hz, 1H), 7.63 (d,
1
ation of 30: H NMR (300 MHz, DMSO-d6) d 7.93 (d, J ¼ 8.4 Hz, 2H), J ¼ 8.7 Hz, 1H), 7.53–7.50 (m, 2H), 7.48 (d, J ¼ 8.7 Hz, 2H), 7.45 (dd,
7.42 (d, J ¼ 8.4 Hz, 2H), 3.65 (t, J ¼ 5.7 Hz, 2H), 2.42 (t, J ¼ 6.0 Hz, J ¼ 3.6, 1.2 Hz, 1H), 7.13 (dd, J ¼ 5.1, 3.6 Hz, 1H), 3.68 (t, J ¼ 6.0 Hz,
2H), 1.91–1.82 (m, 4H).
2H), 2.43 (t, J ¼ 6.3 Hz, 2H), 1.93–1.80 (m, 4H), 1.46 (s, 9H).

1400
E.-C. CHO ET AL.
4.1.57. Tert-Butyl (2-(4-(methylcarbamoyl)benzamido)-4-(thiophen- 4.2.2. In vitro cell cycle assay
HCT116 cells (2.5 ꢃ 105) were seeded into 6-well plates and
2-yl)phenyl) carbamate (43d)
exposed with increasing doses (0.2 mM, 1.0 mM) of 8 and 16. After
48 h of treatment, cells were harvested and fixed in pre-chilled
70% (v/v) EtOH at ꢄ20 ꢀC. Then, the samples were washed with
PBS twice, and stained by propidium iodide (50 mg/mL) containing
RNase A (100 mg/mL). DNA content was detected by flow cytomet-
ric analysis with an AttuneTM NxT Acoustic Focussing Cytometer
(Thermo Fisher Scientific). Sub G1 peaks were analysed as indica-
tive of apoptotic population.
The title compound was obtained in 70% overall yield from com-
pound 42d in a manner similar to that described for the prepar-
ation of 31: 1H NMR (300 MHz, DMSO-d6) d 9.99 (s, 1H), 8.77 (s,
1H), 8.61 (d, J ¼ 4.5 Hz, 1H), 8.05 (d, J ¼ 8.4 Hz, 2H), 7.98 (d,
J ¼ 8.7 Hz, 2H), 7.82 (d, J ¼ 2.1 Hz, 1H), 7.64 (d, J ¼ 8.7 Hz, 1H),
7.54–7.50 (m, 2H), 7.46 (dd, J ¼ 3.6, 1.2 Hz, 1H), 7.13 (dd, 5.1,
3.6 Hz, 1H), 2.82 (d, J ¼ 4.5 Hz, 3H), 1.46 (s, 9H).
4.1.58. Tert-Butyl (2-(4-(oxazol-2-yl)benzamido)-4-(thiophen-2-
yl)phenyl)carbamate (43e)
4.2.3. Western blot analysis
The title compound was obtained in 74% overall yield from com-
pound 42e in a manner similar to that described for the prepar-
ation of 31: 1H NMR (300 MHz, DMSO-d6) d 9.84 (s, 1H), 8.35 (s,
1H), 8.20 (s, 4H), 8.11 (d, J ¼ 0.6 Hz, 1H), 8.07 (d, J ¼ 2.1 Hz, 1H),
7.67 (d, J ¼ 8.4 Hz, 1H), 7.55 (dd, J ¼ 8.4, 2.1 Hz, 1H), 7.46–7.44 (m,
1H), 7.44 (s, 1H), 7.38 (d, J ¼ 0.6 Hz, 1H), 7.15–7.11 (m, 1H), 1.51
(s, 9H).
HCT116 cells were treated with as described previously before har-
vesting and lysis. Protein concentrations were quantified using a
Bio-Rad protein assay. For Western blotting, protein extracts were
separated by SDS polyacrylamide gel electrophoresis and trans-
ferred to PVDF membranes. The membranes were blocked with
5% non-fat dried milk in 1 ꢃ TBS containing 0.1% Tween 20 for
1 h at rt then probed with the indicated primary antibodies. The
employed antibodies were obtained from the following sources:
Acetyl-Histone H3 (Lys9), acetyl-a-tubulin (Lys40) were obtained
from Cell Signalling Technology. Histone H3, a-tubulin, PARP
(poly-ADP-ribose polymerase), PUMA (the p53 upregulated modu-
lator of apoptosis), GAPDH were purchased from GeneTex.
p21Cip1/WAF1 was purchased from Santa Cruz. Signal intensity of
protein expression was quantified using the ImageJ software.
4.1.59. Tert-Butyl (2-(4-(oxazol-5-yl)benzamido)-4-(thiophen-2-
yl)phenyl)carbamate (43f)
The title compound was obtained in 80% overall yield from com-
pound 42f in a manner similar to that described for the prepar-
1
ation of 31: H NMR (300 MHz, CDCl3) d 9.37 (s, 1H), 8.08–8.02 (m,
3H), 7.98 (s, 1H), 7.73 (d, J ¼ 8.7 Hz, 2H), 7.47 (s, 1H), 7.38 (dd,
J ¼ 8.4, 2.4 Hz, 1H), 7.28–7.26 (m, 1H), 7.24 (dd, J ¼ 5.1, 1.2 Hz, 1H),
7.23 (d, J ¼ 2.4 Hz, 1H), 7.03 (dd, J ¼ 5.1, 3.6 Hz, 1H), 6.87 (s, 1H),
1.53 (s, 9H).
Disclosure statement
The authors report no declarations of interest.
4.1.60. Tert-Butyl (2-(4-(1H-benzo[d]imidazol-2-yl)benzamido)-4-
(thiophen-2-yl)phenyl)-carbamate (43g)
Funding
The title compound was obtained in 80% overall yield from com-
pound 42g in a manner similar to that described for the prepar-
This research was supported by the Ministry of Science and
Technology, Taiwan [grant no. MOST108-2320-B-038-042-MY3 and
1
ation of 31: H NMR (300 MHz, DMSO-d6) d 13.07 (s, 1H), 10.02 (s,
1H), 8.79 (s, 1H), 8.35 (d, J ¼ 8.4 Hz, 2H), 8.16 (d, J ¼ 8.4 Hz, 2H), MOST 109-2320-B-038-038], Taiwan, and WanFang Hospital, Chi-
7.85 (d, J ¼ 2.1 Hz, 1H), 7.77–7.57 (m, 3H), 7.56–7.51 (m, 2H), 7.47
Mei Medical Center, and Hualien Tzu-Chi Hospital Joint Cancer
Center Grant-Focus on Colon Cancer Research [MOHW109-TDU-B-
212-134020, supported by Health and welfare surcharge of
tobacco products], Taiwan.
(dd, J ¼ 3.6, 1.2 Hz, 1H), 7.30–7.20 (m, 2H), 7.14 (dd, J ¼ 5.1, 3.6 Hz,
1H), 1.47 (s, 9H).
4.2. Biology
4.2.1. In vitro cell viability assay
References
Human colorectal tumour cell lines (HCT116 and DLD1), human
non-cancerous cell lines, kidney cells (293 T) and lung cells
(IMR90), were obtained from the America Type Culture Collection
(ATCC). All cell lines were cultivated in DMEM or RPMI 1640
medium (supplemented with 10% foetal bovine serum and 1%
antibiotic-antimycotic) and maintained at 37 ꢀC in a 5% CO2
humidified incubator. 4,500 cells/well were seeded into 96-well
plates. After an incubation time of 24 h, cells were treated with
varying concentrations of synthetic compounds in quadruple
wells. For indicated 48 h or 72 h treatments, the cell viability was
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bated for 4–6 h, and the insoluble crystals were dissolved after
adding dimethyl sulfoxide (DMSO). The absorbance value was
detected by a spectrophotometer (BioTek Instruments) at 570 nm.
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