Improving potency and metabolic stability by introducing an alkenyl linker to pyridine-based histone deacetylase inhibitors for orally available RUNX3 modulators

Article abstract of DOI:10.1016/j.ejmech.2016.11.055

RUNX3, a tumor suppressor, is suppressed in various cancers by abnormal epigenetic changes. Histone deacetylases (HDACs) can deacetylate the lysine residues of RUNX3, followed by degradation via a ubiquitin-mediated pathway. Inhibition of HDAC leads to functional restoration of the RUNX3 protein by epigenetic expression and RUNX3 protein stabilization. We previously reported a series of HDAC inhibitors that restored RUNX3 function. In the present study, we introduced an alkenyl linker group to pyridine-based HDAC inhibitors to improve their potencies and chemical properties. This alkenyl linker made the compounds more rigid, facilitating a better fit than alkyl moieties to the active site of HDAC proteins. Most compounds in this series exhibited potent RUNX activities, HDAC inhibitory activities, and inhibitory activities towards the growth of human cancer cell lines. Notably, one of these derivatives, (E)-3-(1-cinnamyl-2-oxo-1,2-dihydropyridin-3-yl)-N-hydroxyacrylamide (7k), showed excellent properties in a microsomal stability study, in a xenograft study, and in an in vivo pharmacokinetic evaluation. Modulation of RUNX3 therefore results in highly potent and orally available anticancer chemotherapeutic agents.

Full text of DOI:10.1016/j.ejmech.2016.11.055

European Journal of Medicinal Chemistry 126 (2017) 997e1010
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Improving potency and metabolic stability by introducing an alkenyl
linker to pyridine-based histone deacetylase inhibitors for orally
available RUNX3 modulators
,
Doona Song a, Chulho Lee a, Yoon Jeong Kook a c, Soo Jin Oh b, Jong Soon Kang b,
, c, *
Hyun-Jung Kim a, Gyoonhee Han a
a Translational Research Center for Protein Function Control, Department of Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea
b Bioevaluation Center, Korea Research Institute of Bioscience and Biotechnology, Ochang, Cheongwon, Chungbuk 363-883, Republic of Korea
c Department of Integrated OMICS for Biomedical Sciences (WCU Program), Yonsei University, Seoul 120-749, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
RUNX3, a tumor suppressor, is suppressed in various cancers by abnormal epigenetic changes. Histone
deacetylases (HDACs) can deacetylate the lysine residues of RUNX3, followed by degradation via a
ubiquitin-mediated pathway. Inhibition of HDAC leads to functional restoration of the RUNX3 protein by
epigenetic expression and RUNX3 protein stabilization. We previously reported a series of HDAC in-
hibitors that restored RUNX3 function. In the present study, we introduced an alkenyl linker group to
pyridine-based HDAC inhibitors to improve their potencies and chemical properties. This alkenyl linker
made the compounds more rigid, facilitating a better fit than alkyl moieties to the active site of HDAC
proteins. Most compounds in this series exhibited potent RUNX activities, HDAC inhibitory activities, and
inhibitory activities towards the growth of human cancer cell lines. Notably, one of these derivatives, (E)-
3-(1-cinnamyl-2-oxo-1,2-dihydropyridin-3-yl)-N-hydroxyacrylamide (7k), showed excellent properties
in a microsomal stability study, in a xenograft study, and in an in vivo pharmacokinetic evaluation.
Modulation of RUNX3 therefore results in highly potent and orally available anticancer chemothera-
peutic agents.
Received 24 June 2016
Received in revised form
10 November 2016
Accepted 29 November 2016
Available online 1 December 2016
Keywords:
RUNX3
RUNX3 acetylation
HDAC inhibition
Epigenetic control
Protein stabilization
© 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction
many ways involving protein-protein interactions, post-
translational modifications, subcellular localization, and the avail-
Epigenetics is currently defined as an inheritable change
without DNA sequence modifications [1]. Disorders in such changes
affect a wide variety of pathologies including cancers [2,3]. There
are two major mechanisms of epigenetic regulations involving DNA
methylation and covalent histone modification. Histones can un-
dergo multiple posttranslational modifications by diverse enzymes
such as histone acetyltransferases (HATs), histone deacetylases
(HDACs), histone methyltransferases (HMTs), and histone deme-
thylases (HDMs) [4]. HATs and HDACs exert their functions within
complexes that include multiple HATs, HDACs, transcription coac-
tivators, and corepressors [5]. HDAC activities are modulated in
ability of metabolic cofactors [6]. Although the relationships
between aberrant expression of various HDACs and cancer remain
largely correlative, the altered expression of HDACs can play an
important role in tumor onset and progression, so HDACs are
attractive targets for the development of therapeutic agents [7].
Regarding acetylation of multiple HDAC substrates,
a single
pathway or multiple pathways may be involved in HDAC inhibitor
(HDACi)-induced cell death. HDACi can be classified into several
structural classes including hydroxamates, cyclic peptides, aliphatic
acids, and benzamides [6]. Trichostatin A (TSA, Fig. 1) was the first
natural product isolated and shown to inhibit HDACs [8]. SAHA, a
structurally close analogue to TSA, was the first HDACi to be
approved for clinical use by the Food and Drug Administration
(Fig. 1) [9]. A series of hydroxamic acids has been shown to block
HDACs and affect cell proliferation at nanomolar concentrations
[10]. Despite the outstanding efficacy of HDACs, they have major
disadvantages such as nonspecificity and resistance, which are
* Corresponding author. Translational Research Center for Protein Function
Control, Department of Biotechnology, Yonsei University, Seoul 120-749, Republic of
Korea.
E-mail address: gyoonhee@yonsei.ac.kr (G. Han).
http://dx.doi.org/10.1016/j.ejmech.2016.11.055
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

998
D. Song et al. / European Journal of Medicinal Chemistry 126 (2017) 997e1010
Fig. 1. Chemical structures of hydroxamic acid-based deacetylase inhibitors. A248 was reported in previous study [29].
common disadvantages in epigenetic therapies [11e13]. A new
approach is therefore needed to eliminate these disadvantages.
Epigenetic alteration can lead to a variety of transcriptional ef-
fects involving the imbalance between HDACs and HATs. Tumor
suppressors, such as RUNX3, are suppressed in various cancers by
abnormal epigenetic changes [14,15]. All RUNX family members
share the central Runt domain, which is a well-conserved domain
that recognizes specific DNA sequences. RUNX1 and RUNX2 are
involved in hematopoiesis and osteogenesis, respectively, and are
genetically altered in leukemia and bone disease [16]. In contrast,
RUNX3 is required for the control of cell proliferation of the gastric
epithelium, neurogenesis of the dorsal root ganglia, and differen-
tiation of T-cells [17]. RUNX3 interacts with p300 HAT and is
acetylated at lysine residues, which is necessary for its transcrip-
tional activity. This acetylation is reversed by HDACs that are in
competition with Smurf1-mediated ubiquitination [18]. HDACs
cause the deacetylation of RUNX3, followed by degradation by the
ubiquitin-mediated pathway. HDAC inhibitors therefore increase
the acetylation of RUNX3, and improve the stability and tran-
scriptional activity of RUNX3 [19]. RUNX3 inactivation is usually
caused by epigenetic changes, rather than rare mutations [15].
RUNX3 may therefore be an excellent target for anti-cancer therapy
because it functions as a tumor suppressor and can be recovered by
RUNX3-targeted agents [20].
In a previous study, we reported that the restoration and sta-
bilization of RUNX3 levels by HDAC inhibition could be a new
approach in cancer treatment [21]. Furthermore, a series of HDAC
inhibitors introduced a pyridone core and an olefin moiety to
improve chemical and metabolic stabilities [22]. It is probable that
the olefin moiety of HDAC inhibitors is important for activity. In the
present study, we designed a new series of pyridone-based HDAC
inhibitors that contained an alkenyl group as a linker group to
improve the potencies and efficacies as an anticancer agent. A total
of 22 compounds were synthesized and tested using various bio-
logical assays. We conducted in vitro biological evaluations
including evaluations of RUNX transcriptional activation levels and
HDAC inhibitory activities, plus cancer cell growth inhibitory ac-
tivities. The synthesized compounds were tested for their effects on
the posttranslational acetylation of RUNX3 involving protein sta-
bilization through epigenetic regulation, and were tested for
microsomal stability to monitor their physicochemical properties.
Furthermore, in vivo xenograft studies and pharmacokinetic pro-
files were performed using the screened compounds to identify
orally available candidates. The high potency, stability, and in vivo
efficacy of synthesized compounds suggested that the RUNX3
modulators can be used for cancer therapy.
2. Results, methods, and discussion
2.1. Strategies and chemistry
In a previous study, the pyridone core HDAC inhibitors with a
hydroxamate conjugated system were reported to improve the
chemical and metabolic stabilities of hydroxamic acid [22]. A three-
dimensional quantitative structure-activity relationship (3D QSAR)
and docking simulation study indicated that hydroxamic acid
chelated to zinc ion in the HDAC active site passes through a narrow
hydrophobic tunnel, and that the bulky aromatic cap groups are
important for inhibitory activity [23]. Based on these studies, we
synthesized potent and orally available RUNX3 modulators based
on HDAC inhibitors (Fig. 1). We designed and synthesized a new
scaffold composed of an alkenyl group on the three carbon linker
group between the cap group and the pyridone core. This olefin
moiety made the compounds more rigid and more stable than the
alkyl moiety. Moreover, this rigidity facilitated a better fit to the
HDAC active site, and the increased chemical stability protected the
compounds from enzymatic hydrolysis to improve their activities
and physicochemical properties as RUNX3 modulators. These
compounds also contained diverse functional phenyl, bromo-
phenyl,
chlorophenyl,
methoxyphenyl,
naphthyl,
tri-
fluoromethylphenyl, and trifluoromethoxyphenyl groups as the cap
groups that were evaluated in structure-activity relationship
studies (SARs).
The general procedures for the synthesis of pyridone-based
RUNX3 modulators are outlined in Scheme 1. Based on the previ-
ous SAR study, more alkyl linker and new alkenyl linker derivatives
were designed and synthesized [22]. Scheme 1 shows the proced-
ure for the olefin linker (between the cap group and the pyridone
core) in the pyridone-based derivatives that were used to produce a
new series of RUNX3 modulators. Commercially available carbal-
dehyde and Wittig reagent were refluxed in dichloromethane to
produce the Wittig products, 1. Compounds 3 and 6 were produced
by an N-alkylation reaction of compound 1 [(E)-methyl 3-(2-oxo-
1,2-dihydropyridin-3-yl) acrylate] with synthetic or commercial
aromatic alkyl halides 2 and 5, respectively. The hydroxamic acid
analogues, 4 and 7, were obtained by reacting the esters of the
pyridones with KONH2 (1.7
temperature.
M in MeOH) in MeOH at low

D. Song et al. / European Journal of Medicinal Chemistry 126 (2017) 997e1010
999
Scheme 1. General procedure for synthesis of pyridone core-based deacetylase inhibitors with an alkyl linker moiety (4) or conjugated linker moiety (7). Reagents: (a) CH2Cl2,
reflux; (b) K2CO3, acetone, reflux; (c) KONH2 (1.7 M in MeOH), MeOH, 0 ꢀC.
2.2. In vitro biological evaluation
known upregulator of RUNX2 expression. This assay system was
useful for estimating the transcriptional activity of RUNX3, because
all three RUNX proteins shared the same consensus sequences in
the promoter regions of the target genes [24]. A total of 29 com-
pounds were evaluated, showing 40e470% of the relative RUNX3
transcriptional activity compared to FGF-2 (Table 1). They were
classified into three groups based on these results, except for
compounds with RUNX activation <100%. Remarkably, most olefin
linker analogues were excellent activators of RUNX3 transcription
except 7d (R ¼ 4-bromophenyl). Among the alkyl linker derivatives,
All synthesized compounds (4ae4k and 7ae7k) were tested for
in vitro HDAC inhibitory activities involving RUNX transcriptional
and cancer cell growth inhibitory activities. The results are sum-
marized in Table 1, using SAHA as a positive control.
Inhibition of HDAC activity by all of the synthesized compounds
was monitored by the HDAC fluorescent activity assay kit using
whole HDACs from HeLa cell extracts. While HDAC inhibitory ac-
tivities of alkyl linker compounds varied from 0.07 to 0.48
those of alkenyl-linker analogues varied from 0.001 to 0.1 M. All
alkenyl linker analogues exhibited better HDAC inhibitory activities
than SAHA, with an IC50 of 0.11 M. RUNX transcriptional activities
mM,
m
compound 4c (R
¼
4-trifluoromethlyphenyl), 4f (R
¼
4-
chlorophenyl), and 4k (R ¼ 4-trifluoromethoxyphenyl) showed
much lower activities than FGF-2. All alkenyl linker analogues
showed better RUNX transcription activities than SAHA, which
showed a moderate RUNX activity of 80.2%. Compared to alkyl
linker compounds, alkenyl linker analogues exhibited high average
m
of the synthesized compounds were monitored using the luciferase
assay system and expressed as the percentage of activation
compared to a positive control, fibroblast growth factor 2 (FGF-2), a

1000
D. Song et al. / European Journal of Medicinal Chemistry 126 (2017) 997e1010
Table 1
Percentage of RUNX transcriptional activation relative to fibroblast growth factor (FGF)-2, inhibition of deacetylase enzyme activity and cancer cell growth by pyridone-based
analogues.
Compd
linker
R
Activation (%)
RUNX
Group
IC50
(
m
M)
GI50
(m
M)
MDA-MB-231
HDAC
PC-3
ACHN
HCT-15
NCI-H23
NUGC-3
4a
4b
4c
4d
4e
4f
4g
4h
4i
alkyl
alkyl
alkyl
alkyl
alkyl
alkyl
alkyl
alkyl
alkyl
alkyl
alkyl
alkyl
alkyl
alkyl
alkyl
alkyl
alkyl
alkyl
olefin
olefin
olefin
olefin
olefin
olefin
olefin
olefin
olefin
olefin
olefin
2-Trifluoromethylphenyl
3-Trifluoromethylphenyl
4-Trifluoromethylphenyl
2-Chlrophenyl
3-Chlrophenyl
4-Chlrophenyl
2-Methoxyphenyl
1-Naphthyl
2-Trifluoromethoxylphenyl
3-Trifluoromethoxylphenyl
4-Trifluoromethoxylphenyl
2-Bromophenyl
3-Bromophenyl
4-Bromophenyl
3-Methoxyphenyl
4-Methoxyphenyl
2-Naphthyl
Phenyl
3-Trifluoromethylphenyl
2-Bromophenyl
3-Bromophenyl
4-Bromophenyl
105.6
118.5
39.4
127.4
129.0
53.1
152.4
225.2
88.7
III
III
e
III
III
e
II
0.11
0.13
0.37
0.08
0.07
0.14
0.08
0.03
0.09
0.12
0.28
0.48
0.10
0.21
0.03
0.14
0.07
0.34
0.03
0.07
0.07
0.08
0.02
0.03
0.04
0.001
0.02
0.04
0.09
1.29
1.14
1.98
0.76
0.83
1.41
0.86
0.06
0.22
0.41
0.56
1.86
1.26
3.97
0.70
1.78
0.35
3.93
0.28
0.05
0.36
0.57
0.13
0.39
0.57
0.03
0.28
0.23
0.47
1.35
1.69
3.94
0.55
0.41
1.10
0.64
0.08
0.30
0.58
1.37
2.89
1.85
4.38
0.72
1.36
0.31
3.53
0.38
0.19
0.52
0.81
0.14
0.57
0.61
0.07
0.20
0.16
0.73
0.95
0.85
2.32
0.58
0.63
1.48
0.48
0.02
0.27
0.52
1.49
1.92
0.74
4.19
0.76
0.98
0.19
3.84
0.28
0.05
0.11
0.40
0.17
0.38
0.56
0.03
0.14
0.23
0.69
0.88
0.72
1.01
1.35
1.19
1.96
0.47
0.11
0.53
0.89
1.10
1.58
1.16
7.45
1.59
0.90
0.38
2.82
0.72
0.31
0.36
0.63
0.68
0.49
0.63
0.03
0.25
0.89
1.16
1.14
0.98
3.22
0.50
0.48
1.33
0.68
0.07
0.33
0.64
1.63
1.88
1.22
4.88
0.87
1.31
0.29
3.80
0.31
0.10
0.49
0.76
0.28
0.15
0.24
0.03
0.03
0.23
1.11
0.99
0.83
2.36
0.31
0.71
1.57
0.29
0.03
0.14
0.41
0.96
1.50
1.20
4.02
1.05
0.97
0.14
2.59
0.29
0.19
0.26
0.56
0.21
0.32
0.56
0.07
0.16
0.11
0.35
I
e
e
e
II
III
e
III
e
III
e
II
4j
72.6
31.2
4k
4la
4ma
4na
4oa
4pa
4qb
4rb
7a
7b
7c
7d
7e
7f
7g
7h
7i
162.8
100.8
53.8
142.1
97.6
131.9
81.8
183.0
180.2
164.8
89.2
247.6
331.1
148.1
461.8
230.7
324.9
111.0
II
II
e
3-Chlrophenyl
I
I
III
I
I
3-Methoxyphenyl
3-Trifluoromethoxylphenyl
1-Naphthyl
2-Naphthyl
2-Quinolinyl
7j
7k
I
III
Phenyl
SAHA
A248
80.2
0.11
0.11
2.69
2.94
2.00
0.60
4.22
0.41
2.49
1.65
2.34
3.46
2.94
NDc
102.6
Average value of olefin linker analogues
Average value of alkyl linker analogues
Average value on HDAC inhibition of group I
Average value on HDAC inhibition of group II
Average value on HDAC inhibition of group III
224.76
110.63
0.02
0.15
0.08
0.04
0.16
0.31
1.24
0.40
1.45
0.27
1.20
0.56
1.44
0.34
1.36
0.28
1.08
a
Compounds were reported in our previous work [30].
Compounds reported in our previous work [22].
No Data.
b
c
values of RUNX3 transcriptional activation (Table 1). Compounds
4h, 7h, 7i, and 7j, which had a simple polycyclic group in common
on the cap moiety, strongly activated the RUNX3 transcription at
225.2%, 461.8%, 230.7%, and 324.9%, respectively. All synthesized
compounds were classified into three groups based on their HDAC
inhibitory activities and RUNX activation levels [21]. For RUNX
activation, seven compounds in group I showed strong RUNX
transcriptional activation > 200%. For group II, the compounds
showed RUNX transcriptional activation levels between 150 and
200%. For group III, the compounds showed RUNX transcriptional
activation levels between 100 and 150%. The HDAC inhibitory ac-
tivities showed a similar tendency, based upon the group involved
in RUNX activation. The averages for each group, were Group I,
antiproliferative activities than the alkyl linker analogues.
Taken together, the results from in vitro assays (RUNX tran-
scriptional activation, HDAC inhibitory activity, and cancer cell
growth inhibitory activity) showed that the synthesized com-
pounds with alkenyl-linker moieties were more potent than those
with alkyl linker moieties.
2.3. SAR
To identify even more effective compounds, a SAR study was
conducted based on in vitro activities of the functional groups, the
cap groups, and the various linker group modifications between the
pyridone-based core and the cap group. The various cap group
substituents involved halogenphenyl, trifluoromethylphenyl, tri-
fluoromethoxyphenyl, methoxyphenyl, naphthyl, and quinolinyl
groups. Overall, compounds with naphthyl, quinolinyl, methox-
yphenyl, and chlorophenyl substituents as the cap group showed
the best HDAC inhibitory activities, RUNX transcriptional activities,
and cell growth inhibitory activities. In addition, all compounds
with a simple polycyclic group on the cap moiety (4h, 7h, 7i, and 7j)
strongly induced RUNX transcription > 200% (4h ¼ 225.2%,
7h ¼ 461.8%, 7i ¼ 230.7%, and 7j ¼ 324.9%) and inhibited the HDAC
0.02 mM; Group II, 0.15 mM; and Group III, 0.08 mM (Table 1).
It was possible that the potency of HDAC inhibition was corre-
lated with the potency of RUNX activation. Inhibition of cancer cell
growth of the prepared analogues was therefore evaluated using
the sulforhodamine B (SRB) assay that showed GI50 values using
PC-3 (prostate), MDA-MB-231 (breast), ACHN (renal), HCT-15 (co-
lon), NCI-H23 (non-small cell lung), and NUGC-3 (gastric) cancer
cell lines. As shown in Table 1, most compounds were more active
than SAHA in the six cancer cell lines, and were the most potent
against the NUGC-3 cell line. Based upon the average of GI50 values,
almost all alkenyl linker compounds showed better
activity with an IC50 < 0.04
m
M (4h ¼ 0.03
m
M, 7h ¼ 0.001
mM,
7i ¼ 0.02 M, and 7j ¼ 0.04
m
m
M). Compared to alkyl linker

D. Song et al. / European Journal of Medicinal Chemistry 126 (2017) 997e1010
1001
compounds, the alkenyl linker analogues exhibited a higher
average value of RUNX transcriptional activation.
To evaluate the effect on the position of the substituent phenyl
group, the substituents were introduced to the ortho, meta, or para
positions. For the CF3 group in the cap group, the relative RUNX
transcriptional and HDAC inhibitory activities showed activities of
compound 7g (148.1%, also a strong RUNX activity). Based on these
results, the compounds with high HDAC inhibitory activity tended
to show nonspecific activity because of strong epigenetic and
transcriptional activations. Among the alkenyl compounds, 7i and
7k showed the best ratio of Ac-RUNX3/para position-Ac-histone of
4.3 and 3.8, respectively.
105.6%/0.11
mM (4a, ortho position), 118.5%/0.13
mM (4b, meta po-
sition), and 39.4%/0.37
mM (4c, para position). In a similar manner,
2.5. Microsomal stability
the RUNX activation and HDAC inhibitory activities showed similar
tendencies in analogues with chlorophenyl (4de4f), tri-
fluoromethoxyphenyl (4ie4k), and methoxyphenyl (4g, 4o, and
4p) substituents. Most compounds which had ortho- and meta-
substituted analogues therefore showed better activities of HDAC
inhibition, RUNX activation, and cell growth inhibitory activities
than para-substituted analogues.
In a previous study, the introduction of an olefin system be-
tween the pyridone core and hydroxamate group was crucial for
optimizing biological potency and improving pharmacokinetic
properties. In the present study, the effects of the alkenyl system
between the cap and core structures were examined. Alkenyl
groups made the compounds more rigid and more stable than alkyl
moieties (Fig. S1, Table S1). This rigidity could lead to a better fit to
the HDAC active site, and the chemical stability could protect the
compounds from enzymatic hydrolysis. Furthermore, the intro-
duction of a double bond on the linker moiety enhanced the po-
tency as an RUNX3 modulator (Table 1).
Based on results of in vitro assays, the alkenyl analogues were
potential RUNX modulators. The metabolic stability profiles were
then determined to identify the effects of the olefin group between
the cap group and the core on ADME (Absorption, Distribution,
Metabolism, and Excretion) properties. Metabolic stability profiles
of five olefin-linker analogues (7b, 7c, 7i, 7j, and 7k) that exhibited
Ac-RUNX3/Ac-H3 ratios > 1 were evaluated in mouse liver micro-
somes (Table 2). During oxidative conditions using mouse liver
microsomes involved in NADPH-dependent cytochrome P450
(CYP450)-mediated oxidative reactions, most olefin linker ana-
logues showed increases of metabolic stability > 10e20% compared
with those of their corresponding alkyl linker compounds, except 7i
(Table 2). For example, compounds 4o, 7c, 7j, and 7k showed high
microsomal stability (65.1%, 40.9%, 65.8%, and 88.2%, respectively,
remaining after 30 min), and compounds 4n, 7b and 7i also showed
moderate metabolic stability (35.5%, 33.6%, and 27.9%, respectively,
remaining after 30 min). Taken together, the alkenyl-linker ana-
logues showed higher stability than the alkyl-linker analogues
against enzymatic hydrolysis.
All compounds showed excellent HDAC inhibitory activities
with IC50 values < 0.1
mM and RUNX activation values > 100%,
except 7d (4-bromophenyl). Most of the synthesized compounds
with an alkenyl linker had improved HDAC inhibition, RUNX3
activation, and cancer cell inhibition. Most notably, compound 7k
2.6. In vivo antitumor activity
increased the IC50 (0.09
vation (111.0%) compared to compound 4r (0.34
and 81.8%, respectively). The average RUNX activation and HDAC
inhibition changed from 103.92%/0.14 M (alkyl linker) to 229.33%/
0.04 M (alkenyl linker). The growth inhibition of cancer cells was
also improved. The GI50 values were decreased more than half for
most of the cancer cell lines. In addition, the alkenyl and alkyl linker
analogues with the same R groups could be directly compared.
Based on various in vitro assays, the introduction of an alkenyl
group on the linker moiety therefore resulted in the best activity.
m
M), GI50 (0.34e1.10
m
M), and RUNX acti-
To determine the pharmacological efficacy of the selected
RUNX3 modulators, we tested these compounds in an in vivo
xenograft model. Based on in vitro biological evaluations and
evaluations of microsomal stability, the alkenyl linker analogues
were more potent than the alkyl linker analogues, so three com-
pounds (7c, 7j, and 7k) were selected for further evaluation. These
compounds had relatively better cancer cell growth inhibition ac-
tivities in gastric cancer cell lines, so the MKN28 cell line was
selected as a xenograft model, which is the same type of gastric
cancer cell without expression of RUNX3 because of epigenetic
inactivation by DNA methylation [25]. Reactivation of RUNX3 by
AZA and TSA in MKN28 cells has been previously reported [15], so
this in vivo MKN28 xenograft regression model monitored the ef-
fects of the compounds involving reactivation and stabilization of
RUNX3. These compounds represented the three different groups
involved in RUNX transcriptional activation (7j, group I with strong
HDAC inhibition; 7c, group II with mild HDAC inhibition; and 7k,
group III with mild HDAC inhibition).
Female S.P.F BALB/c nude mice were injected subcutaneously
(s.c.) with 4 ꢁ 107 cells/mL (MKN28 cell line) before treatment with
oral feeding once a day for 2 weeks with 10 mL/kg (30 mg/kg) for
each compound. Three compounds (7c, 7j, and 7k) were considered
to be relatively nontoxic because of good animal survival and no
loss of weight (Fig. 3A). The 7k compound showed significantly
decreased the tumor weight by 43.7% compared to 7c and 7j.
(20.2%, and 12.4%, respectively; Table 3). Compound 7k showed
111.0% RUNX transcriptional activation (group III) and mild HDAC
inhibition, while 7c and 7j showed 164.8% and 324.9% RUNX
transcriptional activity with mild and strong HDAC inhibition,
respectively. It seems that strong HDAC inhibitors with lower ac-
tivity to restore the RUNX3 protein showed poor in vivo efficacy.
This selective stabilization of RUNX3 stabilization over nonspecific
gene expression was an important factor in tumor suppression.
Furthermore, 7k showed greater metabolic stability compared with
m
M, 2.58e3.92 M,
m
m
m
2.4. RUNX3 stabilization
Acetylation of RUNX3 was recently reported to be essential for
the retention of RUNX3 stability [19]. To examine the stabilization
of RUNX3, we evaluated the acetylation level of RUNX3 after
treating cells with 13 compounds (4d-4h, 7a, 7c, and 7e-7k) by
comparing them with SAHA as a positive control that had HDAC
inhibition and RUNX3 transcriptional activation activities of
<0.1 mM and >100%, respectively. In parallel, we characterized the
expression of RNX3 and the acetylation of histone (H3), to deter-
mine if these compounds were nonspecific epigenetic activators.
Acetylation of H3 resulted in HDAC inhibition and hyperacetylation
of H3 that led to upregulation of nonspecific gene expression. The
ratio of Ac-RUNX3 to Ac-histone (H3) was also estimated to
determine the specificity of the compounds for RUNX3 stabilization
over nonspecific gene expression. All selected analogues showed
higher ratios of acetylated RUNX3/acetylated H3 than SAHA.
(Fig. 2C). Most notably, alkyl derivatives 4d, 4g, and 4h showed 4e6
fold more acetylated RUNX protein than SAHA. Most alkenyl de-
rivatives showed 2e4 fold greater RUNX3 acetylation levels
compared with that of SAHA. With a low ratio of Ac-RUNX3/Ac-
histone < 1, all compounds (7a, 7e, 7f, 7h, and 7g) showed strong
inhibition of HDAC (IC50 < 0.04 mM) and RUNX3 (over 150%) except

1002
D. Song et al. / European Journal of Medicinal Chemistry 126 (2017) 997e1010
Fig. 2. RUNX3 acetylation of selected pyridone-based deacetylase inhibitors.
the other compounds (7c, 7j, and 7k showed 40.9%, 65.8%, and
88.2%, respectively, remaining after 30 min). As a result, 7k in the
xenograft model showed antitumor activity that was greater than
two times that of 7c and 7j.

D. Song et al. / European Journal of Medicinal Chemistry 126 (2017) 997e1010
1003
Table 2
clearance and steady state volume of 7k during intravenous
administration were 4.1 0.5 l/h/kg and 11.4 2.6 l/kg, respec-
tively. The terminal half-lives of 7k were approximately 3.4 h and
5.2 h after intravenous and oral dosing, respectively. The oral
bioavailability for 7k was 54.4%. These results were consistent with
the pharmacological effects of 7k in the xenograft regression
model, and strongly suggested that compound 7k could be an
effective orally administered anti-cancer agent.
Microsomal stability of pyridone-based analogues.
Cpd
linker
R
% remaining after 30 min
(ꢂ) NADPH
(þ) NADPH
4la
alkyl
alkyl
alkyl
alkyl
olefin
olefin
olefin
olefin
olefin
2-Bromophenyl
3-Bromophenyl
2-Naphthyl
Phenyl
2-Bromophenyl
3-Bromophenyl
2-Naphthyl
2-Quinolinyl
Phenyl
93.3 0.0
24.2 0.0
4ma
96.7 0.0
20.7 0.0
4qb
90.3 4.5
35.5 2.7
4r
7b
7c
7i
7j
7k
95.35 1.64
93.28 2.47
89.93 4.29
89.57 1.69
82.80 1.46
99.12 3.09
99.07 1.39
65.10 0.74
33.61 1.31
40.91 1.27
27.93 0.35
65.84 4.10
88.21 1.05
0.29 0.11
3. Conclusions
We report a new series of pyridone-based RUNX3 modulators
with an alkenyl group connecting to a cap group that was shown to
improve the biological potency and pharmacokinetic profiles.
Twenty-two pyridone-based analogues were synthesized and
evaluated using various biological assays. All compounds with the
alkenyl linker between the cap group and core structures showed
improved activities for HDAC inhibition, RUNX transcriptional
activation, and cancer cell growth inhibition compared to those
with one alkyl linker. The ratio of Ac-RUNX3/Ac-histone (H3) was
also estimated to determine the selectivity of RUNX3 stabilization
compared with nonspecific gene expression. This could be an
important factor for antitumor activity through RUNX3 modula-
tion. Among the alkenyl compounds, 7i and 7k showed the best
ratio of Ac-RUNX3/Ac-histone at 4.3 and 3.8, respectively. The
metabolic stability profiles showed that the alkenyl linker ana-
logues had better stability than the alkyl linker analogues. Based on
the results of the in vitro assays, three compounds were selected to
buspirone
a
Compounds were reported in our previous work [30].
Compounds from reported in our previous work [22].
b
2.7. Pharmacokinetics profiling
Based on the results of metabolic stability profiles and in vivo
xenograft studies, compound 7k was selected for in vivo pharma-
cokinetic evaluation (Table 4). Compound 7k was administered
intravenously and orally at 2 and 10 mg/kg, respectively, in male ICR
mice. The plasma concentration profile of 7k showed a peak plasma
concentration of Cmax (627.0
360.9 ng/mL) and Tmax (18 min)
using oral administration. The AUCinf (Area Under the Curve) values
of 7k from the intravenous and oral administrations were
496.3 53.2 ng/h/mL and 1350.8 291.7 ng/h/mL, respectively. The
Fig. 3. In vivo study of the human stomach tumor (MKN28) xenograft regression model.

1004
D. Song et al. / European Journal of Medicinal Chemistry 126 (2017) 997e1010
Table 3
Effect of 7k, 7c, and 7j, 30 mg/kg, 4 ꢁ 107 cells/ml on the growth of a human stomach tumor (MKN28) xenograft regression model.
Group
Dose
Tumor volume(Vt-Vo)y
Tumor weight (mg)
(n ¼ 4)
(mg/kg)
day 0
2
4
7
11
14
14
ꢂ124
ꢂ126
ꢂ128
ꢂ131
ꢂ204
ꢂ207
ꢂ207
Vehicle Control
(D10 þ T10 þ D80%)
7k
0
0
0.0
0
25.9
5.3
18.3
2.8
65.9
10.6
45.2
5.8
118.1
9.9
80.1
7.1
415.5
54.3
260.1
24.4
659.1
61.1
369.2
46.2
***
1716
223.7
965.6
97.2
30
0.0
*
*
***
**
***
29.30%
22.3
7.1
31.40%
57.3
9.9
32.20%
101.3
14.9
37.40%
359.3
27.9
44.00%
533.4
75.0
*
19.10%
570.9
25.9
*
13.40%
298
21.8
***
43.70%
1368.8
85.4
7c
30
30
30
0
0.0
*
13.80%
25.2
5.4
12.90%
61.4
11.7
14.30%
108.7
18.4
13.50%
375.7
12.0
20.20%
1503.2
179.1
7j
0
0.0
2.80%
16.5
3.0
6.80%
40.8
4.6
8.00%
73.8
7.1
9.60%
212.2
21.9
12.40%
794.6
81.7
A248
0
0.0
*
**
***
***
***
36.40%
38.00%
37.60%
48.90%
54.80%
53.70%
Significant figures (t-TEST) : *p < 0.05, **p < 0.01, ***p < 0.001 vs Vehicle Control.
y : △t ¼ VtꢂVo, Vt (Measurement of the tumor volume), Vo (Initial tumor volume).
treat mice to monitor the antitumor effects of a human gastric
tumor (MKN28) xenograft regression model. When the mice were
treated with 7k (R, phenyl; olefin linker; moderate HDAC inhibitory
activity, and group II in RUNX transcriptional activity), a significant
in vivo antitumor activity was observed. In experiments, the results
consistently provide that the in vivo efficacy is quite related to
RUNX3 stability with weak or moderate activation of RUNX3 when
the xenograft experiments using MKN28 which are RUNX3-off
cells. Based on our results, strong HDAC inhibitors with lower ac-
tivity to restore the RUNX3 protein showed poor in vivo efficacy.
The 7k compound also showed a long half-life (3.4 h and 5.2 h after
intravenous and oral dosing, respectively) and potent oral
bioavailability (54.4%) using an in vivo pharmacokinetic study. By
modulation of RUNX3 activity, the results strongly suggested that
7k could be a highly potent and orally available anticancer
chemotherapeutic agent.
4. Experimental
4.1. Chemistry
4.1.1. Experimental section
All chemicals were obtained from commercial suppliers and
used without further purification. All reactions were monitored by
thin-layer chromatography (TLC) on pre-coated silica gel 60 F254
(mesh) (E. Merck, Mumbai, India), and spots were visualized under
UV light (254 nm). Flash column chromatography was performed
with silica (Merck EM9385, 230e400 mesh). 1H and 13C nuclear
magnetic resonance (NMR, Varan Unity Inova) spectra were
recorded at 400 and 100 MHz, or at 500 and 125 MHz. Proton and
carbon chemical shifts are expressed in ppm relative to internal
tetramethylsilane, and coupling constants (J) are expressed in
Hertz. Splitting patterns were presented as s, singlet; d, doublet; t,
triplet; q,quartet; dd, double of doublets; dt, double of triplet; m,
multiplet; br, broad. LCeMS (Liquid chromatography-mass spec-
trometry) spectra were recorded by electrospray ionization (ESI)
probe using a Shimadzu LCeMS2010 instrument with an Agilent
Table 4
C18 column, 50*4.6mm, 5
mm particle size; mobile phase: 0.1%
Oral (PO) and intravenous (IV) pharmacokinetic parameters of 7k in male ICR mice.
formic acid in H2O/0.1% formic acid in CH3CN (1:9) over 10 min;
flow rate: 0.2 mLmin-1; scan mode (0e500 amuzꢂ1). The detected
ion peaks were (M þ z)/z in positive where M represents the mo-
lecular weight of the compound and z represents the charge
(number of protons). High-resolution ESI-MS measurements were
performed on a Micromass Quadrupole-Time of Flight (Q-TOF)
Acquity UPLC-Mass System at Yonsei University; positive mode.
PK parametera
IV
PO
mean
SD
n
3
mean
SD
n
Dose
tmax
Cmax
AUCinf
CL
(mg/kg)
(hr)
(ng/ml)
(ng.hr/ml)
(l/hr/kg)
(l/kg)
2
10
0.3
627.0
1350.8
NA
NA
5.2
54.4
3
3
3
3
NA
NA
496.3
4.1
11.4
3.4
NA
0.0
360.9
291.7
b
53.2
0.5
2.6
3
3
3
3
Vss
t1/2
(hr)
0.2
1.6
11.8
3
3
F
(%)
4.1.2. General procedure for 3 and 6
a
PK parameters were based on mean plasma concentrationꢂtime profiles of
three animals per time points. PK parameters were calculated by non-
compartmental analysis using PK Solutions 2.0 (Summit Research Services, Mon-
trose, CO, USA).
A
solution of (E)-methyl 3-(2-oxo-1,2-dihydropyridin-3-yl)
acrylate 1 (0.2 g, 1.12 mmol) in acetone (0.2 m) was added to alkyl
halide 2 or alkenyl halide 5 (1.67 mmol) in the presence of K2CO3
(0.46 g, 3.35 mmol). The reaction mixture was stirred at reflux for
12 h, then was diluted with acetone, filtered over a Celite pad to
remove K2CO3, and concentrated. The crude product was purified
by flash column chromatography (EtOAc/CH2Cl2, 1:5e1:1).
b
AUC after intravenous (iv) administration was calculated from 0 to infinity,
whereas the oral AUC was calculated from 0 to 5 h. Since the purpose of this study is
to find orally available anticancer reagent, the bioavailability of the compound is
considered to be important because it shows potential of this compound to be orally
administed. Therefore, it was bolded to give an emphasis.

D. Song et al. / European Journal of Medicinal Chemistry 126 (2017) 997e1010
1005
4.1.2.1. (E)-methyl
3-(2-oxo-1-(3-(2-(trifluoromethyl)phenyl)pro-
(MHþ), 350 (MNaþ).
pyl)-1,2-dihydropyridin-3-yl)acrylate (3a). Brown oil (212 mg,
57.0%). 1H NMR (500 MHz, DMSO-d6)
d
8.13 (d, 1H, J ¼ 5.0 Hz),
4.1.2.8. (E)-methyl 3-(1-(3-(naphthalen-1-yl)propyl)-2-oxo-1,2-
7.64e7.28 (m, 3H), 7.16 (d, 1H, J ¼ 12.5 Hz), 7.11 (d, 1H, J ¼ 3.5 Hz),
6.75 (td,1H, JA ¼ 55.0 Hz, JB ¼ 5.5 Hz), 6.23 (t,1H, J ¼ 6.5 Hz), 6.00 (d,
1H, J ¼ 13.0 Hz), 4.14e4.09 (m, 2H), 3.77 (s, 3H), 2.84 (t, 2H,
J ¼ 7.5 Hz), 2.11e2.09 (m, 2H); 13C NMR (125 MHz, DMSO-d6)
dihydropyridin-3-yl)acrylate (3h). Yellow oil (34.2 mg, 90.9%). 1H
NMR (500 MHz, DMSO-d6)
d
8.01 (d, 1H, J ¼ 8.0 Hz), 7.93e7.88 (m,
2H), 7.76 (d, 1H, J ¼ 8.0 Hz), 7.60 (d, 1H, J ¼ 16.0 Hz), 7.54e7.48 (m,
2H), 7.43e7.37 (m, 2H), 7.07 (d, 1H, J ¼ 16.0 Hz), 6.37 (t, 1H,
J ¼ 6.8 Hz), 5.99 (d, 1H, J ¼ 13.0 Hz), 4.11 (t, 2H, J ¼ 7.2 Hz), 3.69 (s,
3H), 3.09e3.05 (m, 2H), 2.09e2.03 (m, 2H); 13C NMR (125 MHz,
d
168.2, 160.8, 140.8, 140.3, 138.7, 138.5, 138.2, 131.9, 130.8, 130.0,
126.5, 126.3, 125.7, 120.4, 105.8, 51.5, 51.3, 30.7, 21.0; ESI (m/z) 366
(MHþ), 388 (MNaþ).
CDCl3) d 168.0, 166.8, 161.1, 160.8, 142.8, 141.8, 141.6, 137.9, 134.2,
132.0, 129.3, 127.3, 126.7, 126.3, 124.2, 119.2, 106.3, 52.1, 51.8, 50.0,
4.1.2.2. (E)-methyl
pyl)-1,2-dihydropyridin-3-yl)acrylate (3b). Yellow oil (154 mg,
50.3%). 1H NMR (500 MHz, DMSO-d6)
7.99e7.81 (m, 2H),
3-(2-oxo-1-(3-(3-(trifluoromethyl)phenyl)pro-
30.4, 29.9; ESI (m/z) 348 (MHþ), 370 (MNaþ).
d
4.1.2.9. (E)-methyl 3-(2-oxo-1-(3-(2-(trifluoromethoxy)phenyl)pro-
pyl)-1,2-dihydropyridin-3-yl)acrylate (3i). Yellow oil (34.2 mg,
7.60e7.32 (m, 5H), 7.13 (d, 1H, J ¼ 16.0 Hz), 6.28 (t, 1H, J ¼ 6.5 Hz),
3.98 (t, 2H, J ¼ 8.0 Hz), 3.71 (s, 3H), 2.71 (t, 2H, J ¼ 7.5 Hz), 2.02e1.94
90.9%). 1H NMR (500 MHz, CDCl3)
d 8.04e8.02 (m, 1H), 7.53e7.41
(m, 2H); 13C NMR (125 MHz, DMSO-d6)
d
168.0, 161.1, 140.4, 140.0,
(m, 1H), 7.40e7.23 (m, 2H), 7.21 (dd, 1H, JA ¼ 9.0 Hz, JB ¼ 4.5 Hz),
7.19e7.16 (m, 2H), 7.06e7.02 (m, 3H), 6.13e6.08 (m, 1H), 3.92e3.85
(m, 2H), 3.64 (s, 3H), 2.63 (t, 2H, J ¼ 8.0 Hz), 1.97 (d, 2H, J ¼ 5.5 Hz);
ESI (m/z) 382 (MHþ), 404 (MNaþ).
138.2, 138.0, 137.6, 131.5, 130.2, 129.6, 126.7, 126.4, 125.8, 120.6,
105.2, 51.1, 50.4, 30.5, 20.7; ESI (m/z) 366 (MHþ), 388 (MNaþ).
4.1.2.3. (E)-methyl
pyl)-1,2-dihydropyridin-3-yl)acrylate (3c). Brown oil (224 mg,
71.8%). 1H NMR (500 MHz, DMSO-d6)
3-(2-oxo-1-(3-(4-(trifluoromethyl)phenyl)pro-
4.1.2.10. (E)-methyl 3-(2-oxo-1-(3-(3-(trifluoromethoxy)phenyl)pro-
pyl)-1,2-dihydropyridin-3-yl)acrylate (3j). Yellow oil (96.8 mg,
d
8.13 (d, 1H, J ¼ 7.0 Hz), 7.62
(d, 1H, J ¼ 16.0 Hz), 7.54e7.52 (m, 2H), 7.31e7.24 (m, 2H), 7.12 (dd,
1H, JA ¼ 13.0 Hz, JB ¼ 3.5 Hz), 6.28 (t, 1H, J ¼ 8.5 Hz), 6.01 (d, 1H,
J ¼ 13.0 Hz), 3.99 (t, 2H, J ¼ 8.0 Hz), 3.77 (s, 3H), 2.75 (t, 2H,
J ¼ 7.0 Hz), 2.14e2.11 (m, 2H); 13C NMR (125 MHz, DMSO-d6)
12.76%). 1H NMR (400 MHz, DMSO-d6)
d
7.87 (t, 2H, J ¼ 5.2 Hz), 7.59
(d, 1H, J ¼ 15.6 Hz), 7.31 (d, 4H, J ¼ 8.4 Hz), 7.07 (d, 1H, J ¼ 16.0 Hz),
6.36 (t, 1H, J ¼ 6.8 Hz), 4.2 (t, 2H, J ¼ 7.2 Hz), 3.71 (s, 3H), 2.66 (t, 2H,
J ¼ 7.6 Hz), 2.03e1.95 (m, 2H); 13C NMR (100 MHz, DMSO-d6)
d
166.6, 161.4, 140.8, 140.2, 138.7, 138.4, 138.2, 128.6(3), 125.4(2),
d 167.7, 160.5, 147.0, 142.5, 141.4, 141.2, 141.0, 130.4(3), 123.6,
119.9, 105.7, 51.6, 49.9, 32.6, 30.1; ESI (m/z) 366 (MHþ), 388 (MNaþ).
121.3(2), 118.9, 106.0, 51.8, 49.2, 31.9, 30.3; ESI (m/z) 382 (MHþ), 404
(MNaþ).
4.1.2.4. (E)-methyl 3-(1-(3-(2-chlorophenyl)propyl)-2-oxo-1,2-
dihydropyridin-3-yl)acrylate (3d). Yellow oil (130 mg, 83.2%). 1H
4.1.2.11. (E)-methyl 3-(2-oxo-1-(3-(4-(trifluoromethoxy)phenyl)pro-
pyl)-1,2-dihydropyridin-3-yl)acrylate (3k). Yellow oil (100 mg,
NMR (500 MHz, CDCl3)
d
7.63 (d, 1H, J ¼ 16.0 Hz), 7.52 (dd, 1H,
JA ¼ 7.25 Hz, JB ¼ 2.5 Hz), 7.34e7.32 (m, 1H), 7.30e7.28 (m, 1H), 7.24
(dt, 1H, JA ¼ 7.5 Hz, JB ¼ 4.0 Hz), 7.19 (dd, 1H, JA ¼ 7.25 Hz,
JB ¼ 1.0 Hz), 7.16e7.15 (m, 1H), 7.11 (d, 1H, J ¼ 15.5 Hz), 6.23 (q, 1H,
JA ¼ 7.0 Hz, JB ¼ 4.0 Hz), 4.06e3.99 (m, 2H), 3.74 (d, 3H, J ¼ 36.0 Hz),
2.82e2.79 (m, 2H), 2.14e2.08 (m, 2H); ESI (m/z) 332 (MHþ), 354
(MNaþ).
50.1%). 1H NMR (500 MHz, CDCl3)
d
7.54 (d, 1H, J ¼ 16.0 Hz), 7.43
(dd, 1H, JA ¼ 7.0 Hz, JB ¼ 1.5 Hz), 7.22e7.18 (m, 2H), 7.18e7.12 (m,
2H), 7.04 (q, 2H, JA ¼ 9.0 Hz, JB ¼ 17.5 Hz), 6.13 (q, 1H, JA ¼ 7.0 Hz,
JB ¼ 14.0 Hz), 3.90 (dt, 2H, JA ¼ 19.0 Hz, JB ¼ 7.5 Hz), 3.69 (s, 3H),
2.63e2.60 (m, 2H), 2.04e2.00 (m, 2H); ESI (m/z) 382 (MHþ), 404
(MNaþ).
4.1.2.5. (E)-methyl 3-(1-(3-(3-chlorophenyl)propyl)-2-oxo-1,2-
4.1.2.12. (E)-methyl 3-(2-oxo-1-((E)-3-(3-(trifluoromethyl)phenyl)
allyl)-1,2-dihydropyridin-3-yl)acrylate (6a). Yellow oil (270 mg,
dihydropyridin-3-yl)acrylate (3e). Yellow oil (907.5 mg, 47.8%). 1H
NMR (400 MHz, DMSO-d6)
d
7.89e7.85 (m, 2H), 7.60 (d, 1H,
21.7%). 1H NMR (500 MHz, CDCl3)
d
7.65 (d, 1H, J ¼ 16.0 Hz), 7.61 (s,
J ¼ 15.6 Hz), 7.33e7.19 (m, 4H), 7.07 (d, 1H, J ¼ 16.0), 6.36 (t, 1H,
J ¼ 6.8 Hz), 4.00 (t, 2H, J ¼ 7.4 Hz), 3.71 (s, 3H), 2.64 (t, 2H,
J ¼ 7.6 Hz), 2.02e1.95 (m, 2H); 13C NMR (100 MHz, DMSO-d6)
1H), 7.58e7.54 (m, 2H), 7.51 (d, 1H, J ¼ 8.0 Hz), 7.45e7.41 (m, 2H),
7.14 (d, 1H, J ¼ 15.5 Hz), 6.63 (d, 1H, J ¼ 16.0 Hz), 6.45e6.39 (m, 1H),
6.30 (t, 1H, J ¼ 7.0 Hz), 4.80 (dd, 2H, JA ¼ 6.0 Hz, JB ¼ 1.0 Hz), 3.79 (s,
d
167.7, 160.4, 144.0, 142.4, 141.3, 141.2, 133.3, 130.5, 128.4, 27.3,
3H); 13C NMR (125 MHz, CDCl3)
d 168.4, 160.9, 141.3, 140.4, 138.3,
126.3, 123.5, 118.8, 105.9, 51.7, 49.1, 32.1, 30.2; ESI (m/z) 355 (MNaþ).
136.9, 133.0, 130.0, 129.4, 125.9, 125.5, 125.0, 123.6, 120.9, 106.4,
51.8, 51.5; ESI (m/z) 364 (MHþ), 386 (MNaþ).
4.1.2.6. (E)-methyl 3-(1-(3-(4-chlorophenyl)propyl)-2-oxo-1,2-
dihydropyridin-3-yl)acrylate (3f). Yellow oil (1.4 mg, 5.6%). 1H
4.1.2.13. (E)-methyl 3-(1-((E)-3-(2-bromophenyl)allyl)-2-oxo-1,2-
NMR (400 MHz, DMSO-d6)
d
7.89e7.85 (m, 2H), 7.59 (d, 1H,
dihydropyridin-3-yl)acrylate (6b). Yellow oil (260 mg, 36.5%). 1H
J ¼ 15.6 Hz), 7.33 (d, 2H, J ¼ 8.4 Hz), 7.25 (d, 2H, J ¼ 8.4 Hz), 7.06 (d,
1H, J ¼ 15.6 Hz), 6.36 (t,1H, J ¼ 15.6), 3.98 (t, 2H, J ¼ 15.6 Hz), 3.70 (s,
3H), 2.61 (t, 2H, J ¼ 7.8 Hz), 2.00e1.92 (m, 2H); 13C NMR (100 MHz,
NMR (500 MHz, CDCl3) d 7.64e7.61 (m, 1H), 7.53e7.43 (m, 4H),
7.24e7.21 (m, 1H), 7.14e7.07 (m, 2H), 6.93 (d, 1H, J ¼ 16.0 Hz),
6.30e6.23 (m, 2H), 4.79 (dd, 2H, JA ¼ 5.5 Hz, JB ¼ 1.0 Hz), 3.76e3.75
DMSO-d6)
d
167.7, 160.4, 142.4, 141.4, 141.2, 140.4, 130.9, 130.5 (2),
(m, 3H); 13C NMR (125 MHz, CDCl3)
d 167.7, 160.3, 142.7, 141.1(2),
128.6 (2), 123.5, 118.8, 105.9, 51.7, 49.2, 31.8, 30.3; ESI (m/z) 332
135.7, 133.2, 131.0, 130.1, 128.4, 128.4, 127.7, 123.2, 119.1, 106.3, 51.8,
(MHþ), 354 (MNaþ).
50.7; ESI (m/z) 374 (MHþ), 396 (MNaþ).
4.1.2.7. (E)-methyl 3-(1-(3-(2-methoxyphenyl)propyl)-2-oxo-1,2-
4.1.2.14. (E)-methyl 3-(1-((E)-3-(3-bromophenyl)allyl)-2-oxo-1,2-
dihydropyridin-3-yl)acrylate (3g). Brown oil (154 mg, 94.4%). 1H
dihydropyridin-3-yl)acrylate (6c). Yellow oil (200 mg, 38.2%). 1H
NMR (500 MHz, DMSO-d6)
d
7.47 (d, 1H, J ¼ 12.5 Hz), 7.45e7.39 (m,
NMR (500 MHz, CDCl3) d 7.66e7.62 (m, 1H), 7.55 (dd, 1H,
2H), 7.25 (d, 1H, J ¼ 15.0 Hz), 7.10e7.07 (m, 2H), 7.01e6.97 (m, 2H),
6.65 (d, 1H, J ¼ 7.5 Hz), 3.95 (t, 2H, J ¼ 7.5 Hz), 3.73 (d, 6H,
J ¼ 11.5 Hz), 2.55 (t, 2H, J ¼ 7.5 Hz), 2.10e2.05 (m, 2H); ESI (m/z) 328
JA ¼ 5.0 Hz, JB ¼ 2.0 Hz), 7.50 (br t, 1H, J ¼ 2.0 Hz), 7.42e7.39 (m, 1H),
7.38e7.36 (m, 1H), 7.28e7.27 (m, 1H), 7.19e7.11 (m, 2H), 6.52 (d, 1H,
J ¼ 16.0 Hz), 6.35e6.26 (m, 2H), 4.77e4.76 (m, 2H), 3.78e3.77 (m,

1006
D. Song et al. / European Journal of Medicinal Chemistry 126 (2017) 997e1010
3H); 13C NMR (125 MHz, CDCl3)
d
167.7, 160.3, 142.7, 141.2, 141.1,
4.1.2.21. (E)-methyl 3-(2-oxo-1-((E)-3-(quinolin-2-yl)allyl)-1,2-
dihydropyridin-3-yl)acrylate (6j). Red solid (2.12 g, 97.1%). 1H NMR
(500 MHz, CDCl3)
7.81 (d, 1H, J ¼ 8.0 Hz), 7.73 (d, 2H, J ¼ 7.0 Hz), 7.64 (d, 1H,
J ¼ 16.0 Hz), 7.56e7.54 (m, 3H), 7.11 (d, 1H, J ¼ 16.0 Hz), 6.29 (t, 1H,
J ¼ 7.0 Hz), 5.49 (s, 2H), 3.77 (s, 3H); ESI (m/z) 347 (MHþ), 349
(MNaþ).
138.9, 131.6, 131.1, 130.9, 129.4, 126.8, 125.9, 123.8, 122.6, 119.0,
106.3, 51.8, 50.6; ESI (m/z) 374 (MHþ), 396 (MNaþ).
d
8.16 (d, 1H, J ¼ 8.5 Hz), 8.06 (d, 1H, J ¼ 8.5 Hz),
4.1.2.15. (E)-methyl 3-(1-((E)-3-(4-bromophenyl)allyl)-2-oxo-1,2-
dihydropyridin-3-yl)acrylate (6d). Yellow oil (100 mg, 22.1%). 1H
NMR (500 MHz, CDCl3)
d
7.64 (dd, 1H, JA ¼ 16.0 Hz, JB ¼ 0.5 Hz), 7.55
(dd, 1H, JA ¼ 5.5 Hz, JB ¼ 2.0 Hz), 7.44e7.42 (m, 2H), 7.41 (dd, 1H,
JA ¼ 4.5 Hz, JB ¼ 2.0 Hz), 7.24e7.22 (m, 2H), 7.13 (d, 1H, J ¼ 16.0 Hz),
6.54 (d,1H, J ¼ 15.5 Hz), 6.35e6.27 (m, 2H), 4.76 (dd, 2H, JA ¼ 5.0 Hz,
4.1.2.22. (E)-methyl 3-(1-cinnamyl-2-oxo-1,2-dihydropyridin-3-yl)
acrylate (6k). Yellow oil (420 mg, 93.5%). 1H NMR (500 MHz, CDCl3)
JB ¼ 1.5 Hz), 3.79 (s, 3H); 13C NMR (125 MHz, CDCl3)
d
167.7, 160.34,
d
8.09 (dd, 1H, JA ¼ 7.0 Hz, JB ¼ 1.5 Hz), 7.46 (dd, 1H, JA ¼ 7.0 Hz,
142.7, 141.2, 141.1, 135.7, 132.0, 131.9(2), 128.9(2), 125.9, 123.8, 121.3,
JB ¼ 1.5 Hz), 7.34 (dd, 1H, JA ¼ 6.5 Hz, JB ¼ 2.0 Hz), 7.31 (d, 1H,
J ¼ 2.0 Hz), 7.30e7.24 (m, 2H), 7.21 (t, 2H, J ¼ 7.5 Hz), 7.15e7.11 (m,
1H), 6.53 (dd, 1H, JA ¼ 16.0 Hz, JB ¼ 5.0 Hz), 6.27e6.15 (m, 2H),
119.0, 106.3, 51.8, 50.71; ESI (m/z) 374 (MHþ), 396 (MNaþ).
4.1.2.16. (E)-methyl 3-(1-((E)-3-(3-chlorophenyl)allyl)-2-oxo-1,2-
4.70e4.65 (m, 2H), 3.70 (s, 3H); 13C NMR (125 MHz, CDCl3)
d 168.4,
dihydropyridin-3-yl)acrylate (6e). Yellow oil (470 mg, 33.3%). 1H
160.9, 141.1, 140.4, 138.3, 136.0, 134.8, 128.8, 128.4, 126.8, 125.6,
NMR (500 MHz, CDCl3)
d
7.63 (d, 1H, J ¼ 15.5 Hz), 7.54 (d, 1H,
123.3, 120.6, 106.202, 51.8, 51.4; ESI (m/z) 296 (MHþ), 318 (MNaþ).
J ¼ 7.0 Hz), 7.40 (d, 1H, J ¼ 7.0 Hz), 7.33 (s, 1H), 7.21 (s, 3H), 7.12 (d,
1H, J ¼ 15.5 Hz), 6.52 (d, 1H, J ¼ 15.5 Hz), 6.35e6.26 (m, 2H), 4.76 (d,
4.1.3. General procedure for 4 and 7
2H, J ¼ 6.5 Hz), 3.77 (s, 3H); 13C NMR (125 MHz, CDCl3)
d
168.4,
NH2OK (1.7 m suspension in MeOH, 1.6 mL) was added to a
solution of 3 or 6 (0.14 mmol) in anhydrous MeOH (0.2 m), and the
mixture was stirred for 12 h at 0 ꢀC. The mixture was concentrated,
and the crude product was purified by flash column chromatog-
raphy (5e10% MeOH in CH2Cl2).
160.9, 141.3, 140.4, 138.4, 137.9, 134.8, 133.1, 130.1, 128.3, 126.7, 125.7,
125.0, 120.747, 106.4, 51.8, 51.4; ESI (m/z) 330 (MHþ), 352 (MNaþ).
4.1.2.17. (E)-methyl 3-(1-((E)-3-(3-methoxyphenyl)allyl)-2-oxo-1,2-
dihydropyridin-3-yl)acrylate (6f). Orange oil (380 mg, 24.6%). 1H
NMR (500 MHz, CDCl3)
d
7.64 (d, 1H, J ¼ 15.5 Hz), 7.54 (dd, 1H,
4.1.3.1. (E)-N-hydroxy-3-(2-oxo-1-(3-(2-(trifluoromethyl)phenyl)
propyl)-1,2-dihydropyridin-3-yl)acrylamide (4a). Light brown solid
JA ¼ 6.0 Hz, JB ¼ 1.0 Hz), 7.42 (dd, 1H, JA ¼ 5.5 Hz, JB ¼ 1.5 Hz), 7.22 (t,
1H, J ¼ 7.5 Hz), 7.15e7.10 (m,1H), 6.96 (d,1H, J ¼ 8.0 Hz), 6.90 (s,1H),
6.83e6.80 (m, 1H), 6.59 (d, 1H, J ¼ 16.0 Hz), 6.34e6.25 (m, 2H), 4.76
(d, 2H, J ¼ 6.5 Hz), 3.79 (s, 3H), 3.78 (s, 3H); 13C NMR (125 MHz,
(80 mg, 40%). MP: 45e48 ꢀC;1H NMR (500 MHz, DMSO-d6)
d 7.82 (d,
1H, J ¼ 6.5 Hz), 7.83e7.35 (m, 6H), 7.13 (d, 1H, J ¼ 15.0 Hz), 6.30 (dt,
1H, JA ¼ 34.0 Hz, JB ¼ 7.0 Hz), 4.04 (dt, 2H, J ¼ 27.0 Hz, 7.5 Hz), 2.77
(t, 2H, J ¼ 7.5 Hz), 1.97 (q, 2H, J ¼ 7.5 Hz); ESI (m/z) 367 (MHþ), 389
(MNaþ); HRMS (ESI) calculated for C18H17F3N2O3 (MHþ), found.
CDCl3)
d 168.2, 160.7, 159.8, 141.0, 140.3, 138.1, 137.3, 134.5, 129.6,
125.5, 123.4, 120.5, 119.3, 114.057, 111.7, 106.0, 55.3, 51.6, 51.1; ESI
(m/z) 326 (MHþ), 348 (MNaþ).
4.1.3.2. (E)-N-hydroxy-3-(2-oxo-1-(3-(3-(trifluoromethyl)phenyl)
4.1.2.18. (E)-methyl 3-(2-oxo-1-((E)-3-(3-(trifluoromethoxy)phenyl)
allyl)-1,2-dihydropyridin-3-yl)acrylate (6g). Yellow solid (500 mg,
propyl)-1,2-dihydropyridin-3-yl)acrylamide
(4b). Brown
solid
(46 mg, 47.4%). MP: 135e137 ꢀC; 1H NMR (500 MHz, DMSO-d6)
48.1%). 1H NMR (500 MHz, CDCl3)
d
7.63 (d, 1H, J ¼ 15.5 Hz), 7.55
d
7.78 (d, 1H, J ¼ 4.0 Hz), 7.72e7.60 (m, 3H), 7.47 (d, 2H, J ¼ 7.0 Hz),
(dd, 1H, JA ¼ 5.0 Hz, JB ¼ 2.0 Hz), 7.41 (dd, 1H, JA ¼ 4.5 Hz,
JB ¼ 2.0 Hz), 7.34e7.28 (m, 2H), 7.20 (s, 1H), 7.13 (d, 1H, J ¼ 15.5 Hz),
7.11e7.08 (m, 1H), 6.57 (d, 1H, J ¼ 16.0 Hz), 6.35 (dt, 1H, JA ¼ 15.5 Hz,
JB ¼ 6.5 Hz), 6.28 (t, 1H, J ¼ 6.5 Hz), 4.77 (dd, 2H, JA ¼ 5.0 Hz,
7.37 (t, 1H, J ¼ 9.0 Hz), 7.12e6.77 (m, 1H), 6.29 (dt, 1H, JA ¼ 42.0 Hz,
JB ¼ 6.0 Hz), 3.98 (dt, 2H, JA ¼ 26.0 Hz, JB ¼ 7.0 Hz), 2.73 (t, 2H,
J ¼ 8.5 Hz), 2.04e1.97 (m, 2H); ESI (m/z) 367 (MHþ), 389 (MNaþ);
HRMS (ESI) calculated for C18H17F3N2O3 (MNaþ) 389.1083, found
389.1082.
JB ¼ 1.5 Hz), 3.77 (s, 3H); 13C NMR (125 MHz, CDCl3)
d 168.1, 160.6,
149.6,141.0,140.1,138.1,138.0,132.8,130.0,125.6,125.1,125.0,120.6,
120.4, 119.4, 118.9, 106.2, 51.6, 51.1; ESI (m/z) 380 (MHþ), 402
(MNaþ).
4.1.3.3. (E)-N-hydroxy-3-(2-oxo-1-(3-(4-(trifluoromethyl)phenyl)
propyl)-1,2-dihydropyridin-3-yl)acrylamide
(4c). Yellow
solid
(74 mg, 33.2%). MP: 47e49 ꢀC; 1H NMR (500 MHz, DMSO-d6)
4.1.2.19. (E)-methyl 3-(1-((E)-3-(naphthalen-1-yl)allyl)-2-oxo-1,2-
d
7.79e7.67 (m, 2H), 7.56 (dd, 4H, JA ¼ 22.0 Hz, 5.5 Hz), 7.45e7.32
dihydropyridin-3-yl)acrylate (6h). Yellow oil (90 mg, 5.8%). 1H
(m, 1H), 7.11 (dd, 1H, JA ¼ 15.0 Hz, JB ¼ 7.0 Hz), 6.29 (dt, 1H,
JA ¼ 39.0 Hz, JB ¼ 7.0 Hz), 3.98 (dt, 2H, JA ¼ 26.0 Hz, JB ¼ 7.0 Hz), 2.73
(t, 2H, J ¼ 7.5 Hz), 2.09e1.98 (m, 2H); ESI (m/z) 367 (MHþ), 389
(MNa); HRMS (ESI) calcd for C18H17F3N2O3 (MHþ) 367.1264, found
367.1266.
NMR (500 MHz, CDCl3)
d
8.06 (d, 1H, J ¼ 8.0 Hz), 7.85 (d, 1H,
J ¼ 8.0 Hz), 7.79 (d, 1H, J ¼ 8.5 Hz), 7.67 (d, 1H, J ¼ 16.0 Hz), 7.59 (d,
1H, J ¼ 7.0 Hz), 7.54 (t, 2H, J ¼ 6.5 Hz), 7.51e7.48 (m, 2H), 7.43 (t, 1H,
J ¼ 8.0 Hz), 7.39 (d, 1H, J ¼ 15.5 Hz), 7.16 (dd, 1H, JA ¼ 15.5 Hz,
JB ¼ 0.5 Hz), 6.38e6.32 (m, 1H), 6.30e6.27 (m, 1H), 4.89 (d, 2H,
J ¼ 6.5 Hz), 3.79 (s, 3H); 13C NMR (125 MHz, CDCl3)
d
168.2, 160.7,
4.1.3.4. (E)-3-(1-(3-(2-chlorophenyl)propyl)-2-oxo-1,2-
141.0, 140.3, 138.1, 133.6, 133.5, 131.9, 131.0, 128.6, 128.565, 126.3,
125.9, 125.5, 124.2, 123.5, 120.5, 106.1, 51.6, 51.3; ESI (m/z) 346
(MHþ), 368 (MNaþ).
dihydropyridin-3-yl)-N-hydroxyacrylamide (4d). Orange oil (44 mg,
44.7%). 1H NMR (500 MHz, DMSO-d6)
d 10.76 (s, 1H), 8.92 (d, 1H,
J ¼ 14.0 Hz), 7.80e7.68 (m, 2H), 7.39e7.34 (m, 3H), 7.26e7.19 (m,
2H), 7.13 (d, 1H, J ¼ 15.5 Hz), 6.32 (d, 1H, J ¼ 6.0 Hz), 4.00 (dt, 2H,
JA ¼ 25.0 Hz, JB ¼ 6.5 Hz), 2.70 (t, 2H, J ¼ 7.5 Hz), 1.94 (d, 2H,
J ¼ 6.5 Hz); ESI (m/z) 333 (MHþ), 355 (MNaþ); HRMS (ESI) calcu-
lated for C17H17ClN2O3 (MHþ) 333.1000, found 333.0997.
4.1.2.20. (E)-methyl 3-(1-((E)-3-(naphthalen-2-yl)allyl)-2-oxo-1,2-
dihydropyridin-3-yl)acrylate (6i). Yellow solid (70 mg, 7.7%). 1H
NMR (500 MHz, CDCl3)
d
7.80e7.79 (m, 2H), 7.75 (d, 2H, J ¼ 15.5 Hz),
7.70e7.66 (m, 1H), 7.59e7.54 (m, 2H), 7.48e7.43 (m, 3H), 7.19e7.15
(m, 1H), 6.77 (d, 1H, J ¼ 16.0 Hz), 6.48e6.42 (m, 1H), 6.28e6.25 (m,
1H), 4.82 (d, 2H, J ¼ 6.5 Hz), 3.81e3.80 (m, 3H); ESI (m/z) 346
(MHþ), 368 (MNaþ).
4.1.3.5. (E)-3-(1-(3-(3-chlorophenyl)propyl)-2-oxo-1,2-
dihydropyridin-3-yl)-N-hydroxyacrylamide (4e). Light brown solid
(238.0 mg, 27.81%). MP: 137e139 ꢀC; 1H NMR (400 MHz, DMSO-d6)

D. Song et al. / European Journal of Medicinal Chemistry 126 (2017) 997e1010
1007
d
10.74 (s, 1H), 8.94 (s, 1H), 7.77 (d, 1H, J ¼ 6.0 Hz), 7.69 (d, 1H,
4.1.3.12. (E)-N-hydroxy-3-(2-oxo-1-((E)-3-(3-(trifluoromethyl)
phenyl)allyl)-1,2-dihydropyridin-3-yl)acrylamide (7a). Pale gray
solid (150 mg, 62.0%). MP: 169e172 ꢀC; 1H NMR (500 MHz, DMSO-
J ¼ 6.8 Hz), 7.37e7.29 (m, 3H), 7.25 (d, 1H, J ¼ 8.4 Hz), 7.19 (d, 1H,
J ¼ 7.6 Hz), 7.12 (d, 1H, J ¼ 15.6 Hz), 6.33 (t, 1H, J ¼ 6.8 Hz), 3.98 (t,
2H, J ¼ 7.2 Hz), 2.63 (t, 2H, J ¼ 7.6 Hz), 2.01e1.94 (m, 2H); ESI (m/z)
333 (MHþ), 355 (MNaþ); HRMS (ESI) calculated for C17H17ClN2O3
(MHþ) 333.1000, found 333.0994.
d6)
d
10.75 (s, 1H), 8.93 (s, 1H), 7.80 (d, 2H, J ¼ 11.0 Hz), 7.75 (t, 2H,
J ¼ 8.5 Hz), 7.60 (d,1H, J ¼ 7.5 Hz), 7.56 (d,1H, J ¼ 8.5 Hz), 7.37 (d,1H,
J ¼ 15.5 Hz), 7.13 (d, 1H, J ¼ 15.5 Hz), 6.67e6.57 (m, 2H), 6.38 (t, 1H,
J ¼ 7.0 Hz), 4.78 (d, 2H, J ¼ 5.5 Hz); ESI (m/z) 365 (MHþ), 387
(MNaþ); HRMS (ESI) calculated for C18H15F3N2O3 (MHþ) 365.1108,
found 365.1103.
4.1.3.6. (E)-3-(1-(3-(4-chlorophenyl)propyl)-2-oxo-1,2-
dihydropyridin-3-yl)-N-hydroxyacrylamide (4f). Light brown solid
(242.0 mg, 45.25%). MP: 140e143 ꢀC;1H NMR (400 MHz, DMSO-d6)
4.1.3.13. (E)-3-(1-((E)-3-(2-bromophenyl)allyl)-2-oxo-1,2-
d
10.75 (s, 1H), 8.95 (s, 1H), 7.78 (d, 1H, J ¼ 6.4 Hz), 7.69 (d, 1H,
dihydropyridin-3-yl)-N-hydroxyacrylamide
(7b). Orange
solid
J ¼ 6.8 Hz), 7.37e7.24 (m, 5H), 7.12 (d, 1H, J ¼ 15.6 Hz), 6.33 (t, 1H,
J ¼ 6.6 Hz), 3.97 (t, 2H, J ¼ 7.2 Hz), 2.61 (t, 2H, J ¼ 7.6 Hz), 1.99e1.92
(100 mg, 52.5%). 1H NMR (500 MHz, DMSO-d6)
d
10.76 (s, 1H), 7.84
(m, 2H); 13C NMR (100 MHz, DMSO-d6)
d 167.7, 160.4, 144.0, 142.4,
(d,1H, J ¼ 6.5 Hz), 7.75 (d,1H, J ¼ 7.0 Hz), 7.68 (d,1H, J ¼ 8.0 Hz), 7.61
(dt, 1H, JA ¼ 8.0 Hz, JB ¼ 1.0 Hz), 7.38e7.33 (m, 2H), 7.22e7.19 (m,
1H), 7.14 (d, 1H, J ¼ 15.5 Hz), 6.78 (d, 1H, J ¼ 15.5 Hz), 6.48e6.41 (m,
1H), 6.38 (t, 1H, J ¼ 6.0 Hz), 4.81 (d, 2H, J ¼ 6.0 Hz); ESI (m/z) 375
(MHþ), 399 (MNaþ); HRMS (ESI) calculated for C17H15BrN2O3
(MHþ) 375.0339, found 375.0329.
141.3, 133.3, 130.5, 128.5, 127.3, 126.3, 123.5, 118.8, 105.9, 51.7, 49.2,
32.1, 30.2; ESI (m/z) 334 (MHþ), 356 (MNaþ), 332 (MHꢂ).
4.1.3.7. (E)-N-hydroxy-3-(1-(3-(2-methoxyphenyl)propyl)-2-oxo-
1,2-dihydropyridin-3-yl)acrylamide (4g). Orange solid (31.9 mg,
21.2%). MP: 150e152 ꢀC; 1H NMR (500 MHz, DMSO-d6)
d 10.75 (s,
4.1.3.14. (E)-3-(1-((E)-3-(3-bromophenyl)allyl)-2-oxo-1,2-
1H), 8.94 (s, 1H), 7.79e7.69 (m, 2H), 7.34 (d, 1H, J ¼ 16.0 Hz),
7.19e7.14 (m, 3H), 6.94 (d, 1H, J ¼ 7.5 Hz), 6.86 (t, 2H, J ¼ 7.2 Hz),
6.33 (t, 1H, J ¼ 6.8 Hz), 3.98 (t, 2H, J ¼ 7.5 Hz), 3.76 (s, 3H), 2.56 (t,
2H, J ¼ 7.8 Hz), 1.91 (t, 2H, J ¼ 7.5 Hz); ESI (m/z) 328 (MHþ), 351
(MNaþ); HRMS (ESI) calculated for C18H20N2O4 (MHþ) 329.1496,
found 329.1494.
dihydropyridin-3-yl)-N-hydroxyacrylamide
(7c). Orange
solid
(100 mg, 51.5%). MP: 159e162 ꢀC; 1H NMR (500 MHz, DMSO-d6)
d
10.76 (s,1H), 8.94 (s,1H), 7.80 (dd,1H, JA ¼ 5.0 Hz, JB ¼ 2.0 Hz), 7.73
(dd, 1H, JA ¼ 5.5 Hz, JB ¼ 1.5 Hz), 7.67 (s, 1H), 7.45e7.43 (m, 2H), 7.37
(d, 1H, J ¼ 15.5 Hz), 7.28 (t, 1H, J ¼ 8.0 Hz), 7.13 (d, 1H, J ¼ 15.5 Hz),
6.52e6.50 (m, 2H), 6.38 (t, 1H, J ¼ 7.0 Hz), 4.75 (d, 2H, J ¼ 5.0 Hz);
ESI (m/z) 375 (MHþ), 399 (MNaþ); HRMS (ESI) calculated for
4.1.3.8. (E)-N-hydroxy-3-(1-(3-(naphthalen-1-yl)propyl)-2-oxo-1,2-
C
17H15BrN2O3 (MHþ) 375.0339, found 375.0334.
dihydropyridin-3-yl)acrylamide (4h). Yellow solid (8.1 mg, 8.7%).
MP: 182e184 ꢀC; 1H NMR (500 MHz, DMSO-d6)
d
10.74 (s, 1H), 8.94
4.1.3.15. (E)-3-(1-((E)-3-(4-bromophenyl)allyl)-2-oxo-1,2-
dihydropyridin-3-yl)-N-hydroxyacrylamide (7d). Orange solid
(70 mg, 29.7%). MP: 108e111 ꢀC; 1H NMR (500 MHz, DMSO-d6)
(s, 1H), 8.01 (d, 1H), 7.93e7.91 (m, 1H), 7.87e7.85 (m, 1H), 7.78 (d,
1H, J ¼ 7.5 Hz), 7.71 (d, 1H, J ¼ 7.5 Hz), 7.55e7.50 (m, 2H), 7.44e7.38
(m, 2H), 7.35 (d, 1H, J ¼ 15.5 Hz), 7.13 (d, 1H, J ¼ 15.5 Hz), 6.35 (t, 1H,
J ¼ 6.8 Hz), 4.11 (t, 2H, J ¼ 7.2 Hz), 3.08 (t, 2H, J ¼ 7.8 Hz), 2.07 (t, 2H,
J ¼ 7.2 Hz); ESI (m/z) 349 (MHþ), 371 (MNaþ); HRMS (ESI) calcu-
lated for C21H20N2O3 (MHþ) 349.1547, found 349.1542.
d
10.76 (s,1H), 8.94 (s,1H), 7.81 (dd,1H, JA ¼ 5.0 Hz, JB ¼ 2.0 Hz), 7.73
(d,1H, J ¼ 7.0 Hz), 7.51 (d, 2H, J ¼ 8.5 Hz), 7.40 (d, 2H, J ¼ 8.5 Hz), 7.19
(dd, 1H, JA ¼ 6.0 Hz, JB ¼ 2.5 Hz), 6.55 (d, 1H, J ¼ 16.0 Hz), 6.38 (t, 1H,
J ¼ 6.5 Hz), 4.73 (d, 2H, J ¼ 6.0 Hz); ESI (m/z) 375 (MHþ), 399
(MNaþ); HRMS (ESI) calculated for C17H15BrN2O3 (MHþ) 375.0339,
found 375.0335.
4.1.3.9. (E)-N-hydroxy-3-(2-oxo-1-(3-(2-(trifluoromethoxy)phenyl)
propyl)-1,2-dihydropyridin-3-yl)acrylamide (4i). Light brown solid
(220 mg, 73.8%). MP: 91e93 ꢀC; 1H NMR (400 MHz, DMSO-d6)
4.1.3.16. (E)-3-(1-((E)-3-(3-chlorophenyl)allyl)-2-oxo-1,2-
dihydropyridin-3-yl)-N-hydroxyacrylamide (7e). Pale gray solid
(190 mg, 47.4%). MP: 150e155 ꢀC; 1H NMR (500 MHz, DMSO-d6)
d
10.75 (s, 1H), 8.94 (s, 1H), 7.78 (d, 1H, J ¼ 6.0 Hz), 7.69 (d, 1H,
J ¼ 6.4 Hz), 7.36e7.25 (m, 5H), 7.12 (d, 1H, J ¼ 15.6 Hz), 6.33 (t, 1H,
J ¼ 6.6 Hz), 3.99 (t, 2H, J ¼ 7.0 Hz), 2.65 (t, 2H, J ¼ 7.6 Hz), 2.02e1.94
(m, 2H); ESI (m/z) 383 (MHþ), 405 (MNaþ), 381 (MHꢂ); HRMS (ESI)
calculated for C18H17F3N2O4 (MNaþ) 405.1033, found 405.1030.
d
10.75 (s,1H), 8.94 (s,1H), 7.81e7.79 (m,1H), 7.73 (d,1H, J ¼ 7.0 Hz),
7.53 (s, 1H), 7.40e7.29 (m, 4H), 7.13 (d, 1H, J ¼ 15.5 Hz), 6.57e6.48
(m, 2H), 6.38 (t, 1H, J ¼ 6.5 Hz), 4.75 (d, 2H, J ¼ 5.0 Hz); ESI (m/z) 331
(MHþ), 353 (MNaþ); HRMS (ESI) calculated for C17H15ClN2O3 (MHþ)
331.0844, found 331.0839.
4.1.3.10. (E)-N-hydroxy-3-(2-oxo-1-(3-(3-(trifluoromethoxy)phenyl)
propyl)-1,2-dihydropyridin-3-yl)acrylamide (4j). Light brown solid
4.1.3.17. (E)-N-hydroxy-3-(1-((E)-3-(3-methoxyphenyl)allyl)-2-oxo-
(17.0 mg, 5.7%). 1H NMR (400 MHz, DMSO-d6)
d 10.75 (s,1H), 8.94 (s,
1,2-dihydropyridin-3-yl)acrylamide (7f). Pale yellow solid (140 mg,
37.6%). 1H NMR (500 MHz, DMSO-d6)
d 10.74e10.73 (m, 1H), 8.91 (s,
1H), 7.78 (d, 1H, J ¼ 6.0 Hz), 7.69 (d, 1H, J ¼ 6.4 Hz), 7.36e7.25 (m,
5H), 7.12 (d, 1H, J ¼ 15.6 Hz), 6.33 (t, 1H, J ¼ 6.6 Hz), 3.99 (t, 2H,
J ¼ 7.0 Hz), 2.65 (t, 2H, J ¼ 7.6 Hz), 2.02e1.94 (m, 2H); ESI (m/z) 383
(MHþ), 405 (MNaþ), 381 (MHꢂ); HRMS (ESI) calculated for
1H), 7.80 (dd, 1H, JA ¼ 4.5 Hz, JB ¼ 2.0 Hz), 7.72 (dd, 1H, JA ¼ 5.0 Hz,
JB ¼ 2.0 Hz), 7.36 (d, 1H, J ¼ 15.5 Hz), 7.25e7.21 (m, 1H), 7.15e7.12
(m, 1H), 6.99e6.98 (m, 2H), 6.86e6.80 (m, 1H), 6.54 (d, 1H,
J ¼ 16.0 Hz), 6.44e6.36 (m, 2H), 4.74 (d, 2H, J ¼ 5.5 Hz), 3.74 (s, 3H);
ESI (m/z) 327 (MHþ), 349 (MNaþ); HRMS (ESI) calculated for
C
18H17F3N2O4 (MNaþ) 405.1033, found 405.1035.
4.1.3.11. (E)-N-hydroxy-3-(2-oxo-1-(3-(4-(trifluoromethoxy)phenyl)
C
18H18N2O4 (MHþ) 327.1339, found 327.1336.
propyl)-1,2-dihydropyridin-3-yl)acrylamide
(4k). Orange
oil
(220 mg, 73.8%). 1H NMR (500 MHz, DMSO-d6)
d
10.75 (s, 1H), 8.92
4.1.3.18. (E)-N-hydroxy-3-(2-oxo-1-((E)-3-(3-(trifluoromethoxy)
phenyl)allyl)-1,2-dihydropyridin-3-yl)acrylamide (7g). Pale yellow
solid (240 mg, 53.1%). MP: 159e162 ꢀC; 1H NMR (500 MHz, DMSO-
(d, 1H, J ¼ 15.5 Hz), 7.78 (d, 1H, J ¼ 6.5 Hz), 7.71 (t, 1H, J ¼ 7.0 Hz),
7.34 (dd, 3H, JA ¼ 5.5 Hz, JB ¼ 9.0 Hz), 7.27 (d, 2H, J ¼ 8.0 Hz), 6.33 (t,
1H, J ¼ 7.0 Hz), 3.98 (dt, 2H, JA ¼ 25.0 Hz, JB ¼ 7.5 Hz), 2.68e2.64 (m,
2H), 2.10 (s, 1H), 2.02e1.94 (m, 2H); ESI (m/z) 383 (MHþ), 405
(MNaþ).
d6)
d
10.74 (s, 1H), 8.92 (s, 1H), 7.80 (dd, 1H, JA ¼ 4.5 Hz, JB ¼ 1.5 Hz),
7.73 (d, 1H, J ¼ 5.5 Hz), 7.48e7.43 (m, 3H), 7.36 (d, 1H, J ¼ 15.5 Hz),
7.24 (d, 1H, J ¼ 7.5 Hz), 7.12 (d, 1H, J ¼ 15.5 Hz), 6.61e6.51 (m, 2H),

1008
D. Song et al. / European Journal of Medicinal Chemistry 126 (2017) 997e1010
6.37 (t, 1H, J ¼ 7.0 Hz), 4.76 (d, 2H, J ¼ 5.0 Hz); ESI (m/z) 381 (MHþ),
403 (MNaþ); HRMS (ESI) calculated for C18H15F3N2O4 (MHþ)
381.1057, found 381.1052.
amino group on the side chain) at a final concentration of 100
The samples were incubated for 30 min at 37 ꢀC and then added to
the Fluor de Lys developer (25 L) for 10 min at rt). The samples
were measured using microtiter-plate-reading fluorimeter
mM.
m
a
4.1.3.19. (E)-N-hydroxy-3-(1-((E)-3-(naphthalen-1-yl)allyl)-2-oxo-
capable of excitation at a wavelength at 360 nm and detection of
emitted light at 460 nm.
1,2-dihydropyridin-3-yl)acrylamide (7h). Pale yellow solid (10 mg,
12.2%). MP: 146e149 ꢀC; 1H NMR (500 MHz, DMSO-d6)
d 10.77 (s,
1H), 8.93 (s, 1H), 8.14 (d, 1H, J ¼ 8.5 Hz), 7.94e7.92 (m, 2H), 7.87 (d,
1H, J ¼ 8.5 Hz), 7.76e7.74 (m, 1H), 7.67 (d, 1H, J ¼ 7.0 Hz), 7.58e7.52
(m, 2H), 7.47 (dd, 2H, JA ¼ 20.5 Hz, JB ¼ 7.5 Hz), 7.38 (d, 1H,
J ¼ 15.5 Hz), 7.16 (d, 1H, J ¼ 15.5 Hz), 6.43 (dt, 2H, JA ¼ 17.0 Hz,
JB ¼ 6.0 Hz), 4.88 (d, 2H, J ¼ 6.0 Hz); ESI (m/z) 347 (MHþ), 369
(MNaþ); HRMS (ESI) calcd for C21H18N2O3 (MHþ) 347.1390, found
347.1390.
4.2.2. RUNX3 transcriptional activation
The percentage of RUNX transcriptional activation relative to
FGF-2 was determined by using the C2C12-6xOSE cell line with the
luciferase assay system. The C2C12-6xOSE cell line was kindly
provided by Prof. Hyun-Mo Ryoo (Seoul National University, Seoul,
South Korea). Cells were plated at 1 ꢁ 104 cells per well in 96-well
plates. Fibroblast growth factor (FGF)-2 and drugs (1 mM each) were
added to cells the next day. After 24 h, cells were harvested and
analyzed by luciferase assay with the Bright-Glo luciferase assay
system according to the manufacturer's protocol (Promega, Madi-
son, WI, USA). Lysates were analyzed with the GloMax-Multi
Detection System (Promega). FGF-2 was used as a positive control
for RUNX activation.
4.1.3.20. (E)-N-hydroxy-3-(1-((E)-3-(naphthalen-2-yl)allyl)-2-oxo-
1,2-dihydropyridin-3-yl)acrylamide (7i). Pale orange solid (30 mg,
33.3%). MP: 199e202 ꢀC; 1H NMR (500 MHz, DMSO-d6)
d 10.74 (s,
1H), 3.91 (s, 1H), 7.88e7.85 (m, 5H), 7.75e7.73 (m, 1H), 7.68 (dd, 1H,
JA ¼ 7.5 Hz, JB ¼ 1.5 Hz), 7.50e7.45 (m, 2H), 7.37 (d, 1H, J ¼ 15.5 Hz),
7.15 (d, 1H, J ¼ 15.5 Hz), 6.72 (d, 1H, J ¼ 16.0 Hz), 6.59e6.53 (m, 1H),
6.39 (t, 1H, J ¼ 7.0 Hz), 4.81 (d, 2H, J ¼ 5.5 Hz). 13C NMR (125 MHz,
4.2.3. Cancer cell growth inhibitory assays
DMSO-d6)
d
164.0, 160.4, 141.66, 139.8, 135.4, 133.9, 133.5, 133.1,
The final concentrations of reagent used to treat cells were 0.1,
133.0, 128.6, 128.3, 128.0, 126.8, 126.7, 126.53, 125.6, 124.7123.9,
121.1, 106.2, 50.8; ESI (m/z) 346 (MHþ), 368 (MNaþ); HRMS (ESI)
calcd for C21H18N2O3 (MNaþ) 369.1210, found 369.1207.
0.3,1, 3, and 10 mg/mL. Cells were harvested from exponential phase
cultures by trypsinization, counted, and plated into 96-well plates.
Optimal seeding densities for various human tumor cell lines (HCT-
15, PC-3, NUGC-3, ACHN, MDA-MB-231, and NCI-H23) were
determined to ensure exponential growth during a 5-day assay. The
sulforhodamine B assay was performed with minor modifications
[27]. The culture medium was aspirated prior to fixation of the cells
4.1.3.21. (E)-N-hydroxy-3-(2-oxo-1-((E)-3-(quinolin-2-yl)allyl)-1,2-
dihydropyridin-3-yl)acrylamide (7j). Brown solid (72 mg, 48.1%). 1H
NMR (400 MHz, DMSO-d6)
d 10.73 (s, 1H), 8.91 (s, 1H), 8.28 (d, 1H,
J ¼ 8.4 Hz), 7.89 (d, 2H, J ¼ 8.0 Hz), 7.84 (d, 1H, J ¼ 6.0 Hz), 7.75e7.67
(m, 3H), 7.51 (t, 1H, J ¼ 7.4 Hz), 7.35 (d, 1H, J ¼ 16.0 Hz), 7.12 (d, 1H,
J ¼ 15.6 Hz), 7.02e6.95 (m, 1H), 6.64 (d, 1H, J ¼ 16.0 Hz), 6.38 (t, 1H,
J ¼ 6.8 Hz), 4.84 (d, 2H, J ¼ 5.2 Hz); ESI (m/z) 347 (MHþ), 349
(MNaþ); HRMS (ESI) calcd for C20H17N3O3 (MNaþ) 370.1162, found
370.1161.
by the addition of 50
incubation at 4 ꢀC, cells were washed five times with deionized
water. After 24 h, the cells were stained with 100 of
1 ꢁ sulforhodamine B (SRB, Sigma-Aldrich, St. Louis, MO, USA)
dissolved in 1% acetic acid for at least 30 min and subsequently
washed four times with 1% acetic acid to remove unbound stain.
The plates were left to dry at room temperature for 24 h; subse-
mL of 50% cold trichloroacetic acid. After 1 h
mL
4.1.3.22. (E)-3-(1-cinnamyl-2-oxo-1,2-dihydropyridin-3-yl)-N-
quently, bound protein stain was solubilized with 100 mL of 10 mM
hydroxyacrylamide (7k). Orange solid (200 mg, 44.0%). MP:
unbuffered Tris and the optical density (OD) was read at 540 nm.
144e147 ꢀC; 1H 1H NMR (500 MHz, DMSO-d6)
d 7.73 (t, 1H,
J ¼ 6.5 Hz), 7.42e7.13 (m, 6H), 6.57 (dd,1H, JA ¼ 12.5 Hz, JB ¼ 3.5 Hz),
4.2.4. Liver microsomal stability assay
6.42e6.35 (m, 2H), 4.72 (dd, 2H, JA ¼ 17.5 Hz, JB ¼ 6.0 Hz); 13C NMR
Microsomal incubations were conducted in triplicate in 0.1 M
potassium phosphate buffer (pH 7.4) in eight-well tube strips. To
study NADPH-dependent metabolic stability, test compounds
(1 mm) were incubated with pooled mouse liver microsomes at a
final concentration of 0.5 mgmLꢂ1 (BD Genetest, USA), in the
presence of NADPH (SigmaeAldrich, St. Louis, MO, USA) in a final
volume of 160 mL. Buspirone was used as a positive control. The
test compounds and microsomes were pre-incubated at 37 ꢀC for
5 min, and the reaction was initiated by adding NADPH. The reac-
tion was terminated at 0 min and 30 min by the addition of 160 mL
of ice-cold CH3CN. The values for % remaining were analyzed by a
liquid chromatography electrospray ionization tandem mass
spectrometry (LC-ESI-MS/MS) for quantitation of compounds.
These experiments were performed at the KRIBB Bio-Evaluation
Center (Chungbuk, Korea).
(125 MHz, DMSO-d6)
d 164.0, 160.4, 141.6, 139.7, 136.3, 135.4, 133.3,
129.08(2),126.32,126.81(2),124.9,124.7,121.1,106.2, 50.8; ESI (m/z)
297 (MHþ), 319 (MNaþ); HRMS (ESI) calcd for C17H16N2O3 (MHþ)
297.1234, found 297.1232.
4.2. Biological assays
4.2.1. HDAC inhibition
4.2.1.1. HDAC inhibitory assays. The IC50 values of HDAC inhibitors
were determined using the HDAC fluorometric assay/drug discov-
ery kit (BioMol, now ENZO Life Sciences, Plymouth Meeting, PA,
USA) which is based on the unique Fluor de Lys system (fluorogenic
histone deacetylase lysyl substrate/developer). It is designed to
measure histone deacetylase (HDAC) activity. In this assay, we used
nuclear extracts from the human cervical cancer cell line HeLa
(from 6 to 9 mg protein/mLꢂ1 in 0.1 M KCl, 20 mM HEPES/NaOH, pH
7.9, 20% (v/v) glycerol, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF)
[26]. The HeLa cell nuclear extract contained all class I HDACs
(HDAC1, HDAC2, HDAC3, and HDAC8), class II HDACs (HDAC4ꢂ7, 9,
and 10), and human homolog of yeast Sir2, SIRT1. SAHA was used as
4.2.5. Western blot analysis
4.2.5.1. Western blot analysis. After cells were lysed with
radioimmuno-precipitation assay (RIPA) buffer (25 mM Tris-HCl,
pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% so-
dium dodecyl sulfate, SDS), protein concentrations were deter-
mined with a bicinchoninic acid assay (BCA) protein assay kit
(Pierce, Rockford, IL, USA). Equal amounts of protein were frac-
tionated by SDSꢂpolyacrylamide gel electrophoresis (PAGE) and
the positive control. A nuclear fraction of HeLa cells (15
was diluted to 10 L with the assay buffer containing the HDAC
inhibitors and the Fluor de Lys substrate (lysine with an acetylated
mL of 30 ꢁ )
m

D. Song et al. / European Journal of Medicinal Chemistry 126 (2017) 997e1010
1009
transferred onto a nitrocellulose membrane (BioRad, Richmond,
CA, USA). After blocking with 5% skim milk in phosphate-buffered
saline (PBS) containing 0.1% Tween 20, membranes were incu-
bated with the appropriate primary antibodies at 4 ꢀC overnight.
Proteins were detected with horseradish peroxidase (HRP) conju-
gated secondary antibodies and an enhanced chemiluminescence
(ECL) kit (Amer-sham Pharmacia Biotech, Buckinghamshire, U.K.).
Antibodies against myc (9E10) and tubulin were from Santa Cruz
Biotechnology (Santa Cruz, CA, USA). Antiacetylated lysine antibody
was purchased from Cell Signaling (Danvers, MA, USA).
was calculated as follows: CLp ¼ dose/AUCinf. Terminal elimination
half-life (t1/2 was calculated by the following equation: t1/
)
¼ 0.693/lz where lz is the terminal disposition rate constant.
2
Volume of distribution at steady state (Vss) was calculated as fol-
lows: Vss ¼ dose ꢁ AUMCinf/(AUCinf)2, where AUMCinf is the area
under the first moment of the plasma concentrationꢂtime curve
extrapolated to infinity. Oral bioavailability (F) was calculated as
follows: F (%) ¼ (AUCpo/AUCiv) (doseiv/dosepo) ꢁ 100.
4.3. In silico studies
4.2.5.2. Immunoprecipitation analysis. The myc-RUNX3 expression
vector was kindly provided by Prof. Suk-Chul Bae (Chungbuk Na-
tional University, Cheongju, South Korea). The myc-RUNX3
expression vector was transfected into HEK293 cells with
Lipofect-amine 2000 (Invitrogen). Transfected cells were treated
Molecular docking studies were performed using the docking
software LigandFit and Calculate Binding Energies within the
software package Discovery Studio 2017 (Accelrys, San Diego, CA,
USA). The crystal structure of human HDAC2 (PDB code: 3MAX)
obtained from the Protein Data Bank was refined to remove water
molecules and add hydrogen atoms to the entire enzyme at pH 7.4.
A binding pocket of the native ligand (SAHA) was selected as the
binding site for the study. The consistent force field (CFF) function
was selected as the energy grid, and the Monte Carlo trial method
was used to roughly search the conformations when pyridone-
based HDAC inhibitors were docked into the HDAC enzyme. The
ligands were then optimized using the CHARMm force field
function.
with HDAC inhibitors at 1 mM the next day for an additional 24 h.
Cells were lysed in ice-cold lysis buffer (25 mM HEPES, pH 7.5,
150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 10%
glycerol, 25 mM NaF, 1 mM EDTA, 1 mM Na3VO4) and cleared by
centrifugation. For immunoprecipitation experiments, 500 mg of
protein was incubated with anti-myc antibody and precipitated
with protein G beads (Pierce) at 4 ꢀC. The beads were washed three
times with cold lysis buffer, and the immunoprecipitates were
analyzed by Western blot with an antibody against acetylated
lysine.
Acknowledgement
4.2.6. In vivo xenograft studies
This research was supported by a grant from the Translational
Research Center for Protein Function Control (TRCP
2016R1A5A1004694), the Ministry of Science, ICT and Future
Planning (Grant NRF-2013M3A6A4072536), the Basic Science
Research Program through the National Research Foundation of
Korea funded by the Ministry of Education (Grant NRF-
2015R1A6A3A01020077), the Korea Health Technology R&D Proj-
ect through the Korea Health Industry Development Institute
(KHIDI), funded by the Ministry of Health & Welfare, Republic of
Korea (HI14C1324), the Yonsei University Research Fund (Post Doc.
Researcher Supporting Program 2015-12-0005), and the KRIBB
Research Initiative Program.
Six-week-old female specified-pathogen-free (SPF) BALB/c nude
mice were purchased from Charles River Laboratories (Yokohama,
Japan) and maintained in accordance with the Animal Research
Committee's Guidelines at the Korea Research Institute of Biosci-
ence and Biotechnology (KRIBB). MKN28 cells (4 ꢁ 107/animal)
were injected subcutaneously into the flank area. When tumors
reached a volume of 50e80 mm3, as measured with a digital
caliper, mice were randomized into groups of four animals each to
receive either vehicle control, 7c, 7j, or 7k. The drugs were orally
administered at 30 mg/kg in 10% DMAC and 10% Tween80 (Sigma)
in saline daily for 2 weeks. Tumor growth was measured three
times a week until the end of the study. The length and width of the
tumor were measured with calipers. Tumor volume was calculated
according to the formula for an ellipse, volume ¼ 0.523 ꢁ (long
dimension) ꢁ (short dimension)2.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.11.055.
Myc-tagged RUNX3 (Myc-RUNX3) was expressed in
HEK293 cells. Acetylated RUNX3 levels were analyzed by immu-
4.2.7. In vivo pharmacokinetic studies
Animal studies were approved by the Institutional Animal Care
and Use Committee at KRIBB and conducted as described previ-
ously with minor modifications [28]. Briefly, male ICR mice (5e6
weeks old, body weight 25e30 g), purchased from Koatech Co.
(Kyeonggi, Republic of Korea), were given a single dose of 7k
intravenously (iv, 2 mg/kg, n ¼ 3) or orally (po, 10 mg/kg, n ¼ 3).
Dosing solutions were prepared in dimethylacetamide (DMAC)/20%
2-hydroxypropyl-b-cyclodextrin (10/90, v/v %) for iv administra-
tion, and DMAC/vitamin E TPGS 80/25% Solutol HS 15 (10/10/80, v/v
%) for po administration, and administered at dosing volumes of 5
noprecipitation in HEK293 cells following treatment with 1
mM
indicated test compound. (A) the Western blot of alkyl linker de-
rivatives; (B) the Western blot of olefin linker derivatives; (C)
relative expression of RUNX3 acetylation based on the results of the
Western blot.
30 mg/kg, q1d ꢁ 14 on the growth of a MKN28 human stomach
tumor xenograft in nude mice; (A) Average body weight changes;
(B) average tumor volume; (C) average tumor weight in mice
treated with vehicle control (V.C.).
and 10 mL/kg for iv and po, respectively. A 100 mL aliquot of each
plasma sample was prepared, and three volumes of ice-cold
acetonitrile containing carbamazepine (internal standard) were
added. The mixture was centrifuged at 910g for 10 min, and the
supernatant was subjected to LCꢂMS/MS analysis. Pharmacoki-
netic parameters were calculated by standard noncompartmental
analysis of plasma concentrationꢂtime profiles using Kinetica 4.4.1
(Thermo Fisher Scientific, Inc., Woburn, MA, USA). The areas under
the plasma concentrationꢂtime curves (AUC) were calculated by
the linearꢂtrapezoidal method. Systemic plasma clearance (CLp)
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