Discovery of orally available runt-related transcription factor 3 (RUNX3) modulators for anticancer chemotherapy by epigenetic activation and protein stabilization

Article abstract of DOI:10.1021/acs.jmedchem.5b00062

Recently, we identified a novel strategy for anticancer chemotherapy by restoring runt-related transcription factor 3 (RUNX3) levels via lactam-based histone deacetylase (HDAC) inhibitors that stabilize RUNX3. Described here are the synthesis, biological evaluation, and pharmacokinetic evaluation of new synthetic small molecules based on pyridone-based HDAC inhibitors that specifically stabilize RUNX3 by acetylation and regulate its function. Many of the newly synthesized compounds showed favorable RUNX activities, HDAC inhibitory activities, and inhibitory activities on the growth of human cancer cell lines. Notably, one of these new derivatives, (E)-N-hydroxy-3-(2-oxo-1-(quinolin-2-ylmethyl)-1,2-dihydropyridin-3-yl)acrylamide (4l), significantly restored RUNX3 in a dose-dependent manner and showed high metabolic stability, a good pharmacokinetic profile with high oral bioavailability and long half-life, and strong antitumor activity. This study suggests that pyridone-based analogues modulate RUNX3 activity through epigenetic regulation as well as strong transcriptional and post-translational regulation of RUNX3 and could be potential clinical candidates as orally available RUNX3 modulators for the treatment of cancer.

Full text of DOI:10.1021/acs.jmedchem.5b00062

Subscriber access provided by University of Guelph Library
Article
Discovery of orally available runt-related transcription
factor 3 (RUNX3) modulators for anticancer chemotherapy
by epigenetic activation and protein stabilization
Jee Sun Yang, Chulho Lee, Misun Cho, Hyuntae Kim, Jae Hyun Kim, Seonghwi Choi,
Soo Jin Oh, Jong Soon Kang, Jin-Hyun Jeong, Hyun-Jung Kim, and Gyoonhee Han
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00062 • Publication Date (Web): 26 Mar 2015
Downloaded from http://pubs.acs.org on April 6, 2015
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 33
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Discovery of orally available runtꢀrelated
transcription factor 3 (RUNX3) modulators for
anticancer chemotherapy by epigenetic activation
and protein stabilization
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Jee Sun Yang§, Chulho Lee§, Misun Cho§, Hyuntae Kim‡, Jae Hyun Kim‡, Seonghwi Choi‡, Soo
Jin Oh†, Jong Soon Kang†, JinꢀHyun Jeong#,*, HyunꢀJung Kimfl, Gyoonhee Han§, ‡,*
§ Translational Research Center for Protein Function Control, Department of Biotechnology,
Yonsei University, Seodaemunꢀgu, Seoul 120ꢀ749, Republic of Korea
† Bioevaluation Center, Korea Research Institute of Bioscience and Biotechnology, Ochang,
Cheongwon, Chungbuk 363ꢀ883, Republic of Korea
#
College of Pharmacy and Yonsei Institute of Pharmaceutical Sciences, Yonsei University,
Yeonsuꢀgu, Incheon 406ꢀ840, Republic of Korea
fl College of Pharmacy, Gachon University of Medicine and Science, Incheon 406ꢀ799, Republic
of Korea
‡ Department of Biomedical Sciences (WCU Program), Yonsei University, Seodaemunꢀgu,
Seoul 120ꢀ749, Republic of Korea
ACS Paragon Plus Environment
1

Journal of Medicinal Chemistry
Page 2 of 33
1
2
3
4
5
6
KEYWORD: runtꢀrelated transcription factor 3 (RUNX3), histone deacetylase inhibitors,
hydroxamic acids, pyridones, property, metabolic stability
7
8
9
ABSTRACT. Recently, we identified a novel strategy for anticancer chemotherapy by restoring
runtꢀrelated transcription factor 3 (RUNX3) levels via lactamꢀbased histone deacetylase (HDAC)
inhibitors that stabilize RUNX3. Described here are the synthesis, biological evaluation, and
pharmacokinetic evaluation of new synthetic small molecules based on pyridoneꢀbased HDAC
inhibitors that specifically stabilize RUNX3 by acetylation and regulate its function. Many of the
newly synthesized compounds showed favorable RUNX activities, HDAC inhibitory activities,
and inhibitory activities on the growth of human cancer cell lines. Notably, one of these new
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
derivatives,
(E)ꢀNꢀhydroxyꢀ3ꢀ(2ꢀoxoꢀ1ꢀ(quinolinꢀ2ꢀylmethyl)ꢀ1,2ꢀdihydropyridinꢀ3ꢀ
yl)acrylamide (4l), significantly restored RUNX3 in a doseꢀdependent manner and showed high
metabolic stability, a good pharmacokinetic profile with high oral bioavailability and long halfꢀ
life, and strong antitumor activity. This study suggests that pyridoneꢀbased analogues modulate
RUNX3 activity through epigenetic regulation as well as strong transcriptional and postꢀ
translational regulation of RUNX3, and could be potential clinical candidates as orally available
RUNX3 modulators for the treatment of cancer.
INTRODUCTION
The dynamic and reversible process of epigenetic regulation can contribute to multiple human
diseases such as cancer, inflammation, and metabolic diseases.1ꢀ5 Histone deacetylase (HDAC)
proteins function as epigenetic erasers that catalyze the removal of acetyl groups from lysine
residues of target histones to regulate many cellular processes. In cancer cells, in particular,
histone deacetylase (HDAC) isoenzymes and the corresponding hypoacetylation of histones are
ACS Paragon Plus Environment
2

Page 3 of 33
Journal of Medicinal Chemistry
1
2
typically seen at high levels.6ꢀ8 Significant efforts have gone into developing HDAC inhibitors in
the field of oncology. Various HDAC inhibitors have shown potent anticancer efficacy for solid
and hematological cancers; most of these are hydroxamic acid derivatives, such as
suberoylanilide hydroxamic acid (SAHA; Vorinostat, Zolinza®) and trichostatin A (TSA) (Figure
1).9 However, most potent epigenetic drugs, which are mainly targeted at histone proteins, show
substantial limitations, such as epigenetic nonꢀspecificity (pleiotropic effects) and drug
resistance.1, 10, 11 To overcome these problems of epigenetic therapy, many researchers have
focused on modifying nonꢀhistone proteins – transcription factors, chaperones, structural
proteins, and tumor suppressors – for the treatment of various cancers.12ꢀ16
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In recent studies, runtꢀrelated transcription factor 3 (RUNX3), which is associated with the
transforming growth factor (TGF)ꢀβ signaling pathway, was reported as a tumor suppressor in
multiple solid tumors, particularly gastric cancer.17ꢀ19 RUNX3 activity is induced by the
transcriptional activation and postꢀtranslational stabilization of RUNX3, and these processes are
in turn controlled by the opposing actions of histone deacetylase (HDAC) and histone
acetyltransferase (HAT).20 Hypermethylation of the CpG islands in the RUNX3 promoter region
and methylation of histones H3K9 and H3K27 lead to the recruitment of HDACs, and
deacetylation of histone H3 by HDACs induces repression of the transcriptional expression of
RUNX3 genes.17, 21 On the other hand, RUNX3 acetylation by p300 (HAT) results in RUNX3
stabilization via protection against ubiquitinꢀmediated degradation.17, 22ꢀ24 Therefore, epigenetic
regulation and postꢀtranslational modification by HDACs are highly related to the tumor
suppressor activity of RUNX3. We have previously reported that the restoration and stabilization
of RUNX3 levels by HDAC inhibition using lactamꢀbased HDAC inhibitors have the potential
become the new approach for antiꢀcancer chemotherapy.25
ACS Paragon Plus Environment
3

Journal of Medicinal Chemistry
Page 4 of 33
1
2
3
4
5
6
7
8
9
In this paper, we tried to identify an orally available RUNX3 modulator using a series of
pyridoneꢀbased HDAC inhibitors which showed remarkable metabolic stability than lactamꢀ
based HDAC inhibitors.26 Pyridoneꢀbased HDAC inhibitors were screened by a cellꢀbased
RUNX3 transcriptional activation assay system to investigate the relationship between HDAC
inhibitory activity and RUNX3 activity. Cellꢀbased mechanistic studies, including metabolic
stability measurements, as well as an in vivo xenograft assay, were performed using the screened
compounds to identify candidates. Our results demonstrate that our pyridoneꢀbased derivatives
contribute to RUNX3 stabilization through epigenetic regulation and strong postꢀtranslational
acetylation of RUNX3. Furthermore, the selected compounds caused attenuation of lung cancer
pathogenesis. The high potency, stability, pharmacokinetic profiles, and in vivo efficacy of these
compounds indicate the possibility of orally available RUNX3 modulators for treating cancer.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
RESULTS AND DISCUSSION
Chemistry. The general procedure for the synthesis of pyridoneꢀbased RUNX3 modulators is
outlined in Scheme 1. Commercially available pyridone carbaldehyde and Wittig reagent were
refluxed in dichloromethane by the Wittig reaction to give (E)ꢀmethyl 3ꢀ(2ꢀoxoꢀ1,2ꢀ
dihydropyridinꢀ3ꢀyl)acrylate (1). Compound 3 was produced from an Nꢀalkylation reaction
between commercial or synthetic aromatic alkyl halide 2 and compound 1. The hydroxamic acidꢀ
based RUNX3 modulator series represented by 4 was obtained by reacting the esters of the
pyridone with KONH2 (1.7 M in MeOH) in MeOH at low temperature.
Thirtyꢀone pyridoneꢀbased analogues were newly designed and synthesized as RUNX3
modulators based on our previously synthesized HDAC inhibitors25ꢀ27. The pyridone core and a
conjugated system with an olefin connecting the hydroxamic acid moiety were retained for
ACS Paragon Plus Environment
4

Page 5 of 33
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
metabolic stability against hydrolysis. For structureꢀactivity relationship studies, the carbon chain
linker between the cap group and the pyridone core was varied in length from one to three.
Furthermore, diverse functional groups, such as bromophenyl, ethylphenyl, methoxyphenyl, or a
solubilizing group (amineꢀcontaining heterocyclic group, such as pyridinyl or quinolinyl) were
introduced as the cap group in these HDAC inhibitors to improve their RUNX activity and
pharmacokinetic profiles.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Biological evaluation. RUNX transcriptional activity in the presence of synthesized pyridoneꢀ
based analogues was evaluated using the luciferase assay system in 6xOSE2ꢀC2C12 stable cells
which are very efficient and useful system for screening of the transcriptional activity of
RUNX2.25, 28 Fibroblast growth factor 2 (FGFꢀ2), which stimulates RUNX2 expression and
function, was used as a positive control. RUNX activities are tabulated as percentage of
activation compared to that seen with FGFꢀ2. Inhibition of HDAC activity and cancer cell
growth by pyridoneꢀbased analogues were monitored by the HDAC Fluorescent Activity Assay
kit and the sulforhodamine B (SRB) assay, respectively. Six human cancer cell lines, PCꢀ3
(prostate), MDAꢀMBꢀ231 (breast), ACHN (renal), HCTꢀ15 (colon), NCIꢀH23 (nonꢀsmall cell
lung), and NUGCꢀ3 (gastric), were used in the SRB assay. The results are tabulated as 50%
inhibitory concentration (IC50) or 50% growth inhibition (GI50) values in the micromolar range
(Table 1). For these activity tests, SAHA was used as a reference for the comparison of in vitro
activities.
As shown in Table 1, most of the evaluated compounds, with the exception of compounds
containing a pyridinyl moiety on the cap group (4i, 4j, 4k, 4u, 4v, and 4e’), showed favorable
RUNX activities, HDAC inhibitory activities, and growth inhibitory activities against human
cancer cells. Among these compounds, 9 of the pyridoneꢀbased analogues (4g, 4l, 4o, 4p, 4t, 4w,
ACS Paragon Plus Environment
5

Journal of Medicinal Chemistry
Page 6 of 33
1
2
3
4
5
6
7
8
9
4x, 4b’, and 4d’) induced RUNX activation by over 100% while SAHA showed moderate
RUNX activity at 80.2%. Moreover, three analogues, 4g, 4l, and 4w, induced RUNX activity to
greater than 150% (200.0%, 687.5%, and 162.8%, respectively). All of these RUNXꢀactivating
analogues also showed potent HDAC inhibition with an average IC50 value of 0.12 ꢁM. In
particular, compound 4l activated RUNX and inhibited the HDAC enzyme as well as the growth
of six cancer cell types even more thoroughly than did SAHA.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Structure-activity relationship (SAR) study of pyridone-based analogues. Initially, in order
to assess the effects of the length of carbon chain between the pyridone core and the cap group,
the compounds were divided into three groups based on the length of the spacer: the oneꢀcarbon
linker group (4a-4h and 4l), twoꢀcarbon linker group (4m-4t), and threeꢀcarbon linker group
(4w-4d’) (Table 1). The activity data of pyridinylꢀsubstituted compounds were not grouped with
those of other compounds because of their extraordinarily poor activities on RUNX and on
cancer cell growth inhibition. In terms of RUNX activity, compounds with oneꢀcarbon linkers
were almost twoꢀfold more active than compounds with twoꢀ or threeꢀcarbon linkers. On the
other hand, the HDAC inhibition and cell growth inhibition activities of these compounds were
almost the same regardless of their carbon chain length. If we fix the substituents of compounds
to 3ꢀmethoxyphenyl, it is certain that compound with oneꢀcarbon linker (4g) possesses more
potent RUNX activity than other compounds with higherꢀcarbon linkers (4q and 4b’). Also, their
HDAC inhibitory activities are varied from 0.03 to 0.15 ꢁM (4g = 0.15, 4q = 0.07, and 4b’ =
0.03 ꢁM). Especially, compound 4g exhibited the lowest HDAC inhibitory activity while it
induced RUNX activation by 200%. These results show that HDAC inhibitory activity of the
synthesized compounds are not correlated to the RUNX activities although our compounds were
designed on the model of HDAC inhibitors,
ACS Paragon Plus Environment
6

Page 7 of 33
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
To evaluate the effects of different substituents at the cap group of the title compounds, diverse
substituents, such as bromophenyl, ethylphenyl, pyridinyl, quinolinyl, tꢀbutyl phenyl,
ethoxyphenyl and methoxyphenyl, were introduced. On the whole, compounds with methoxy or
quinolinyl substituents at the cap group showed favorable RUNX activity and HDAC inhibitory
activities. In contrast, compounds with pyridinyl groups showed poor RUNX activities (<70 %)
and HDAC inhibitory activities as mentioned above. Also, to assess the effects of the position of
the substituents, bromophenyl substituted compounds (4a – 4c) were prepared. The carbon chain
length of the selected compounds was fixed to one carbon, which was previously stated to have
superior RUNX activity than the compounds with twoꢀ or threeꢀcarbon linkers. They activated
RUNX similarly (around 90 %) regardless of the position of the substituents (orthoꢀ, metaꢀ, or
paraꢀposition) and exhibited HDAC inhibitory activities with IC50 value of 0.19, 0.38, and 0.10
ꢁM, respectively. These results show that the position of the substituents does not seriously
affect the RUNX activities and it was affirmed again that the RUNX activation has no
correlation with the HDAC inhibitory activity. .
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Among the synthesized compounds, 4l (n=1, R=2ꢀquinolinyl), 4o (n=2, R=4ꢀethyl), 4p (n=2,
R=4ꢀtꢀbutyl), 4b’ (n=3, R=3ꢀmethoxyphenyl) and 4d’(n=3, R=2ꢀethoxy) showed comparatively
high inhibition not only of RUNX activity but also of human cancer cell growth. Compound 4l
exhibited the most potent cancer cell growth inhibitory activities (GI50 = 0.10–0.71 ꢁM) and
HDAC inhibitory activity (IC50 = 30 nM) as well as RUNX activity (relative RUNX
transcriptional activation to FGFꢀ2 = 687.5%).
Regulation of RUNX3 acetylation. RUNX3 acetylation is reported to be essential for the
maintenance of RUNX3 stability.22 To assess RUNX3 stability, we evaluated RUNX3
acetylation and protein levels after treating cells with our synthesized compounds. Nine
ACS Paragon Plus Environment
7

Journal of Medicinal Chemistry
Page 8 of 33
1
2
3
4
5
6
7
8
9
compounds with over 100% RUNX activity were selected for evaluation from among 31
synthesized compounds. The wellꢀcharacterized antiꢀcancer drug TSA, a HDAC inhibitor, was
used as a positive control.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Among the tested compounds, 4g, 4l, 4w, and 4d’ significantly increased the levels of acetylated
RUNX3 compared to the positive control (Figure 2). These compounds also achieved strong
stabilization of the RUNX3 protein. The RUNX3 acetylation levels of these compounds were
even higher than that of SAHA, which showed a 14.1ꢀfold greater RUNX3 acetylation level over
that of the reference compound, TSA. In particular, compound 4l dramatically increased the
acetylation of RUNX3 to 77.5ꢀfold higher than that seen with TSA (Figure 2B). However, the
HDAC inhibitory activities of these four compounds displayed a wide spectrum (from 0.03 ꢁM
to 0.48 ꢁM), indicating that HDAC inhibitory activities and RUNX3 acetylation do not have a
quantitative relationship.
RUNX3 mRNA expression. According to the previous experiments, compound 4l was chosen
for further evaluation of RUNX3 mRNA expression and RUNX3 stability to determine whether
it regulates RUNX3 at the transcriptional level. The expression of RUNX3 mRNA was
determined in a lung cancer cell line, NCIꢀH460, by RTꢀPCR. In this cell line, due to
hypermethylation of the CpG island in the promoter region, RUNX3 is not expressed in the
normal condition. However, when treated with an HDAC inhibitor RUNX3 expression is
restored.21 SNU16 gastric cancer cells, which do not have a deletion in the RUNX3 locus and
normally express RUNX3, were used as a positive control.
As shown in Figure 3, Compound 4l induced RUNX3 mRNA expression in a doseꢀdependent
manner. The increased levels of RUNX3 mRNA induced by 0.1 and 1 ꢁM 4l were comparable to
ACS Paragon Plus Environment
8

Page 9 of 33
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
that induced by SAHA. Furthermore, 10 ꢁM 4l showed strong effects on RUNX3 mRNA
expression in comparison with SAHA. This result shows that compound 4l successfully
reactivated the tumor suppressor gene RUNX3.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Metabolic stability in mouse, rat, and human liver microsomes. In parallel with the RUNX3
acetylation experiments, metabolic stability profiles were evaluated in mouse liver microsomes
for the nine selected compounds that exhibited the greatest RUNX transcriptional activation over
100%. In addition, we calculated the values of lipophilicity (AlogP) and polar surface area (PSA)
of the selected compounds using Discovery Studio 4.0™ (Table S2).
We previously confirmed that the enzymatic hydrolysis properties of these compounds can be
improved by replacing the gammaꢀ or deltaꢀlactam core with a pyridone core.26 The double bond
conjugation system of hydroxamic acid with the pyridone structure improved metabolic stability
against hydrolysis of the carboxyl acid. As a consequence, the newly synthesized pyridoneꢀbased
analogues (4g, 4l, 4o, 4p, 4t, 4w, 4x, 4b’, and 4d’) were resistant to hydrolysis (Table 2). In the
presence of NADPH in mouse liver microsomes, which are involved in the NADPHꢀdependent
cytochrome P450 (CYP450)ꢀmediated oxidative reaction, 4g, 4l, 4t and 4b’ (61.5%, 90.3%,
59.6%, and 88.4% remaining, respectively) were more stable than other compounds.
Furthermore, the compounds that showed metabolic instability had higher lipophilicity (AlogP
values >2.0) than comparably stable compounds. The instability of these compounds can be
explained based on the previous observation that metabolic instability results from the high
lipophilicity of compounds in mouse liver microsomes29.
In vitro phase I and II metabolic stability in rat liver microsomes was assessed for compounds 4l
and 4b’, which showed the greatest stability in mouse liver microsomes, and the data are
ACS Paragon Plus Environment
9

Journal of Medicinal Chemistry
Page 10 of 33
1
2
3
4
5
6
7
8
9
summarized in Table 3. In phase I metabolism, both compounds exhibited good stability against
metabolic hydrolysis and oxidative reactions, as seen in mouse liver microsomes. However,
uridine 5'ꢀdiphosphoꢀglucuronic acid (UDPGA)ꢀdependent phase II metabolism was observed
for compound 4b’ (45.7% remaining). Based on several studies of the metabolism of hydroxamic
acid derivatives30ꢀ32, the hydroxamic acid moiety of 4b’ seems to be metabolized by Oꢀ
glucuronidation. Compound 4l, however, was stable against UDPGAꢀdependent metabolism in
rat liver microsomes, with 72.4% remaining. Similar stability results with respect to the phase I
and phase II metabolism of 4l, and even better values with respect to glucuronidation, were seen
in human liver microsomes. Among these, 4l, which contains a quinolinyl moiety, emerged as
the most stable compound against NADPHꢀ and UDPGAꢀdependent metabolism. As a result,
introducing a quinolinyl moiety at the cap group in pyridoneꢀbased analogues improved
microsomal stability.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Pharmacokinetic profiles. Compound 4l, which caused high RUNX3 acetylation level and high
stability against enzymatic hydrolysis and oxidation, was selected for in vivo pharmacokinetic
evaluation. The pharmacokinetics of 4l administered intravenously and orally at 2 mg/kg and 10
mg/kg, respectively, was evaluated in male SpragueꢀDawley (SD) rats. The systemic exposure,
clearance, and steady state volume of distribution of 4l were 354.5 ngꢂhr/ml, 5.6 l/hr/kg, and 4.4
l/kg, respectively (Table 4). After oral administration, 4l exhibited high exposure, as 863.3
ngꢂhr/ml. This high oral exposure level seems to be correlated with the stability in liver
microsomes. The terminal halfꢀlives of 4l were approximately 4.4 and 10.3 h after intravenous
and oral dosing, respectively. Moreover, the oral bioavailability for 4l was found to be 48.7% at
10 mg/kg oral dose. Compound 4l showed significant improvement in t1/2 and oral bioavailability,
and these values were competitive with those of SAHA.33
ACS Paragon Plus Environment
10

Page 11 of 33
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Reduced tumor growth in xenograft models. We evaluated the most active and stable
compound 4l in in vivo xenograft experiments to assess antitumor activity and toxicity (body
weight loss). NCIꢀH460 cells were subcutaneously implanted into nude mice. Compound 11-8,
for which we have previously reported antitumor activity in gastric cancer cells following
intravenous injection, was used as a positive control.25 Compounds 4l and 11-8 were
administered orally at 20 mg/kg daily for 2 weeks. Tumor volumes were measured every 2 or 3
days for 14 days.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
As shown in Figures 4A and 4B, treatment with 4l caused a dramatic reduction in tumor growth
without significant body weight loss. Notably, 4l showed increased antitumor activity compared
to 11-8 (45.71% and 38.08%, respectively), indicating that its improved metabolic stability and
activity may contribute to increased clinical availability. As shown in Figure 4C, 4l significantly
decreased the tumor weight by 45.70%, while 11-8 resulted in a 37.93% reduction. These results
imply that this new class of RUNX3 modulators could be applied in new cancer therapeutics. In
pharmacokineticꢀpharmacodynamic (PK–PD) studies, twoꢀfold higher levels of 4l were also
observed in tumors as compared to 11-8 (Supplementary Figure S1), which correlates with the
antiꢀtumor effect observed in the xenograft model.
CONCLUSION
Thirtyꢀone pyridoneꢀbased analogues were designed and synthesized as RUNX3 modulators to
improve potency and pharmacokinetic profiles. Most of the pyridoneꢀbased compounds
excluding those with pyridinyl substituents at the cap group exhibited favorable RUNX activity
and cancer cell growth inhibitory activities. RUNX3 stability and metabolic stability were
evaluated for nine compounds which exhibited outstanding RUNX activities. Among them, 4l
ACS Paragon Plus Environment
11

Journal of Medicinal Chemistry
Page 12 of 33
1
2
3
4
5
6
7
8
9
resulted in remarkably increased RUNX3 acetylation level and RUNX3 mRNA expression,
showing that it can successfully stabilize RUNX3 through epigenetic regulation and strong postꢀ
translation regulation of RUNX3. Furthermore, 4l emerged as the most stable compound against
NADPHꢀ and UDPGAꢀdependent metabolism. Introducing a quinolinyl moiety is expected to
result in metabolic blocking of CYPꢀmediated oxidation and glucuronidation. The superior
metabolic stability of 4l resulted in a good pharmacokinetic profile with high oral bioavailability
and long halfꢀlife and understandably reduced tumor growth. Taken together, the data presented
here suggest that compound 4l represents a promising clinical candidate as an orally available
RUNX3 modulator for the treatment of cancer.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
EXPERIMENTAL SECTION
Chemistry. All chemicals were obtained from commercial suppliers and used without further
purification. All reactions were monitored by thinꢀlayer chromatography (TLC) on preꢀcoated
silica gel 60 F254 (mesh) (Merck, Mumbai, India), and spots were visualized under UV light (254
nm). Flash column chromatography was performed with silica (Merck EM9385, 230ꢀ400 mesh).
1H and 13C nuclear magnetic resonance (NMR, Varian 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; m,
multiplet; br, broad. Liquid chromatographyꢀmass spectrometry (LCꢀMS) spectra were recorded
by electrospray ionization (ESI) probe using a Shimadzu LC–MS2010 instrument with an
Agilent C18 column, 50×4.6 mm, 5 ꢁm particle size; mobile phase: 0.1% formic acid in H2O/0.1%
formic acid in CH3CN (1:9) over 10 min; flow rate: 0.2 ml minꢀ1; scan mode (0–500 amuzꢀ1).
The detected ion peaks were (M+z)/z in positive where M represents the molecular weight of the
ACS Paragon Plus Environment
12

Page 13 of 33
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
compound and z represents the charge (number of protons). Highꢀresolution ESIꢀMS
measurements were performed on a Micromass QuadrupoleꢀTime of Flight (QꢀTOF) Acquity
UPLCꢀMass System at Yonsei University; positive mode. To determine the purity of the final
compounds (4a−4e’), high performance liquid chromatography (HPLC) experiments were
conducted using the Agilent analytical column EclipseꢀXDBꢀC18 (150×4.6 mm, 5 ꢁm) on
Shimadzu HPLC 2010 instruments. All compounds tested in biological assays were ≥95% pure.
Conditions and retention times are described in Supporting Information (Table S1). Cell lines
were obtained from R&D Systems (Minneapolis, MN, USA).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
General procedure for the preparation of title compounds 4a-4e’. To a solution of acrylate
3aꢀ3f’ in anhydrous methanol (10.0 ml/mmol), under N2 atmosphere, 1.7 M KONH2 (5.0 equiv)
was added at 0°C. The mixture was stirred for 12 h. The mixture was concentrated in vacuo and
the crude product was purified by column chromatography (silica gel, 5 % methanol in
dichloromethane) to afford the final pure product.
(E)-N-hydroxy-3-(2-oxo-1-(quinolin-2-ylmethyl)-1,2-dihydropyridin-3-yl)acrylamide (4l).
Yellow solid (26 mg, 22%). 1H NMR (500 MHz, DMSOꢀd6) δ 8.34 (d, 1H, J =8.0 Hz), 7.97ꢀ
7.94 (m, 2H), 7.88 (d, 1H, J =7.5 Hz), 7.78ꢀ7.71 (m, 2H), 7.57 (t, 1H, J =5.0 Hz), 7.41 (d, 1H, J
=9.0 Hz), 7.33 (d, 1H, J =15.0 Hz), 7.06 (d, 1H, J =15.5 Hz), 6.43 (t, 1H, J =7.0 Hz), 5.44 (s,
2H); ESI (m/z) 322 (MH+), 344 (MNa+); HRMS (ESI) calculated for C18H15N3O3 [MH+]
322.1191, found 322.1185; HPLC (tR : purity = 9.606 min, 99.7 %).
(E)-N-hydroxy-3-(1-(3-(3-methoxyphenyl)propyl)-2-oxo-1,2-dihydropyridin-3-
yl)acrylamide (4b’). Ivory solid (171 mg, 45%). 1H NMR (400 MHz, DMSOꢀd6) δ 10.71 (s,
1H), 8.91 (s, 1H), 7.73 (d, 1H, J = 8.0 Hz), 7.66 (d, 1H, J = 6.8 Hz), 7.31 (d, 1H, J = 15.6 Hz),
ACS Paragon Plus Environment
13

Journal of Medicinal Chemistry
Page 14 of 33
1
2
3
4
5
6
7
8
9
7.15 (t, 1H, J = 8.0 Hz), 7.08 (d, 1H, J = 15.2 Hz ), 6.755ꢀ6.705 (m, 3H), 6.29 (t, 1H, J = 6.8
Hz), 3.94 (t, 2H, J = 7.2 Hz), 3.69 (s, 3H), 2.55 (t, 2H, 7.8 Hz), 1.966ꢀ1.891 (m, 2H); ESI (m/z)
329 (MH+), 351 (MNa+); HRMS (ESI) calculated for C18H20N2O4 [MH+] 329.1501, found
329.1493; HPLC (tR : purity = 11.335 min, 99.0 %).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(E)-3-(1-(3-(2-ethoxyphenyl)propyl)-2-oxo-1,2-dihydropyridin-3-yl)-N-hydroxyacrylamide
(4d’). Brown solid (145 mg, 44%). 1H NMR (400 MHz, DMSOꢀd6) δ 10,71 (s, 1H), 8.88 (s, 1H),
7.74 (d, 1H, J = 6.4 Hz), 7.66 (d, 1H, J = 7.2 Hz), 7.31 (d, 1H, J = 15.6Hz), 7.11ꢀ7.07 (m, 3H),
6.87 (d, 1H, J = 8.4 Hz), 6.81 (t, 1H, J = 7.4 Hz), 6.29 (t, 1H, J = 6.6 Hz), 3.97ꢀ3.91 (m, 4H),
2.52 (t, 2H, J = 7.4 Hz), 1.88 (t, 2H, J = 7.4 Hz), 1.23 (t, 3H, J = 7.0 Hz); ESI (m/z) 343 (MH+),
365 (MNa+); HRMS (ESI) calculated for C19H22N2O4 [MH+] 343.1658, found 343.1649; HPLC
(tR : purity = 12.367 min, 98.4 %).
Cell Cultures. All tissue culture media and antibiotics were purchased from Hyclone (Logan,
UT, USA). HEK293 and C2C12ꢀ6xOSE cells were maintained in Dulbecco’s modified Eagle
medium (DMEM) with 10% fetal bovine serum (FBS) and antibiotics [penicillin (100 IU/ml),
streptomycin (100 mg/ml)] (Invitrogen, Grand Island, NY, USA) at 37°C in a 5% CO2
atmosphere. NCIꢀH460 and SNU16 cells were maintained in RPMI1640 media with 10% FBS
and antibiotics. Treatment with 2 mM isopropylꢀβꢀDꢀthiogalactopyranoside (IPTG, Sigmaꢀ
Aldrich, St. Louis, MO, USA) was performed for 24 h to induce RUNX3 expression. Drugs were
then added to cell cultures for an additional 24 h to assess effects on RUNX3 stability.
Screening for RUNX activity. The percentage of RUNX transcriptional activation relative to
FGFꢀ2 was determined using the C2C12ꢀ6xOSE cell line with the luciferase assay system. The
C2C12ꢀ6xOSE cell line was kindly provided by Prof. HyunꢀMo Ryoo (Seoul National
ACS Paragon Plus Environment
14

Page 15 of 33
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
University, Seoul, South Korea). Cells were plated at 1 × 104 cells per well in 96ꢀwell plates.
Fibroblast growth factor (FGF)ꢀ2 and drugs (1 ꢁM each) were added to cells the next day. After
24 h, cells were harvested and analyzed by luciferase assay with the BrightꢀGlo Luciferase Assay
System according to the manufacturer’s protocol (Promega, Madison, WI, USA). Lysates were
analyzed with the GloMaxꢀmulti Detection System (Promega). FGFꢀ2 was used as a positive
control for RUNX activation.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
HDAC inhibitory assays. The IC50 values of HDAC inhibitors were determined using the
HDAC fluorometric assay/drug discovery kit (BioMol, now ENZO Life Science, 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 deacetylases (HDAC)
activity. In this assay, we used nuclear extracts from the human cervical cancer cell line HeLa
(from 6 to 9 mg protein/ml 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).34 The HeLa cell nuclear extract contained all
class I HDACs (HDAC1, HDAC2, HDAC3 and HDAC8), class II HDACs (HDAC4–7, 9 and
10), and human homolog of yeast Sir2 , SIRT1. SAHA was used as the positive control. A
nuclear fraction of HeLa cells (15 ꢁl of 30×) was diluted to 10 ꢁl with the assay buffer
containing the HDAC inhibitors and the Fluor de lysⓇ substrate (lysine with an acetylated amino
group on the side chain) at a final concentration of 100 ꢁM. 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 microtiterꢀplateꢀreading fluorimeter capable of excitation at a
wavelength at 360 nm and detection of emitted light at 460 nm.
ACS Paragon Plus Environment
15

Journal of Medicinal Chemistry
Page 16 of 33
1
2
3
4
5
6
7
8
9
Cancer cell growth inhibitory assays. The final concentrations of reagent used to treat cells
were 0.1, 0.3, 1, 3, and 10 ꢁg/ml. Cells were harvested from exponential phase cultures by
trypsinization, counted, and plated into 96ꢀwell plates. Optimal seeding densities for various
human tumor cell lines (HCTꢀ15, PCꢀ3, NUGCꢀ3, ACHN, MDAꢀMBꢀ231, and NCIꢀH23) were
determined to ensure exponential growth during a 5ꢀday assay. The sulforhodamine B assay was
performed with minor modifications.35 The culture medium was aspirated prior to fixation of the
cells by the addition of 50 ꢁl 50% cold trichloroacetic acid. After 1 h incubation at 4°C, cells
were washed five times with deionized water. After 24 h, the cells were stained with 100 ꢁl 1×
Sulforhodamine B (SRB, SigmaꢀAldrich, 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; subsequently, bound protein stain was
solubilized with 100 ꢁl of 10 mM unbuffered Tris and the optical density (OD) was read at 540
nm.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Liver microsomal stability assay. Microsomal incubations were conducted in triplicate in 0.1
M potassium phosphate buffer (pH 7.4) in 8ꢀwell tube strips placed in a 8×12 rack (1.2 ml; VWR,
Emeryville, CA). To study NADPHꢀdependent oxidative metabolism, the test compound (1 µM)
was incubated with pooled mouse, rat, or human liver microsomes (final concentration: 0.5
mg/ml) in the presence of NADPH (final concentration: 1 mM) in a final volume of 160 µl. The
reaction was started by adding 1 mM βꢀNADPH and was terminated at 0 and 30 min by the
addition of 160 ꢁl iceꢀcold acetonitrile containing an internal standard (IS). Buspirone was used
as a positive control. The incubation mixtures were centrifuged at 910×g for 10 min. The
supernatant was analyzed by liquid chromatography electrospray ionization tandem mass
spectrometry (LCꢀESI/MS/MS). The percent compound remaining was calculated from the peak
ACS Paragon Plus Environment
16

Page 17 of 33
Journal of Medicinal Chemistry
1
2
3
4
5
6
area ratios of the analyte to the IS. These experiments were performed at the KRIBB Bioꢀ
Evaluation Center (Chungbuk, Korea).
7
8
9
In silico physiological profiles. The AlogP, number of single rotatable bonds, and fast PSA
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
were calculated by Discovery Studio 4.0 (Accelrys, San Diego, CA).
Western blot analysis. After cells were lysed with radioꢀimmunoprecipitation assay (RIPA)
buffer (25 mM TrisꢀHCl, pH 7.6, 150 mM NaCl, 1% NPꢀ40, 1% sodium deoxycholate, 0.1%
sodium dodecyl sulfate, SDS), protein concentrations were determined with a bicinchoninic iacid
assay (BCA) protein assay kit (Pierce, Rockford, IL, USA). Equal amounts of protein were
fractionated by SDSꢀpolyacrylamide gel electrophoresis (PAGE) and transferred onto a
nitrocellulose membrane (BioRad, Richmond, CA, USA). After blocking with 5% skim milk in
phosphateꢀbuffered saline (PBS) containing 0.1% Tween 20, membranes were incubated with
the appropriate primary antibodies at 4°C overnight. Proteins were detected with horseradish
peroxidase (HRP)ꢀconjugated secondary antibodies and an enhanced chemiluminescence (ECL)
kit (Amersham Pharmacia Biotech, Buckinghamshire, UK). Antibodies against myc (9E10) and
tubulin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antiꢀacetylated lysine
antibody was purchased from Cell Signaling (Danvers, MA, USA).
Immunoprecipitation analysis. The mycꢀRUNX3 expression vector was kindly provided by
Prof. SukꢀChul Bae (Chungbuk National University, Cheongju, South Korea). The mycꢀRUNX3
expression vector was transfected into HEK293 cells with Lipofectamine 2000 (Invitrogen).
Transfected cells were treated with HDAC inhibitors at 1 ꢁM the next day for an additional 24 h.
Cells were lysed in iceꢀcold lysis buffer (25 mM HEPES, pH 7.5, 150 mM NaCl, 1% Nonidet Pꢀ
40, 0.25% sodium deoxycholate, 10% glycerol, 25 mM NaF, 1 mM EDTA, 1 mM Na3VO4) and
ACS Paragon Plus Environment
17

Journal of Medicinal Chemistry
Page 18 of 33
1
2
3
4
5
6
7
8
9
cleared by centrifugation. For immunoprecipitation experiments, 500 mg of protein was
incubated with antiꢀmyc 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.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Reverse transcription polymerase chain reaction (RT-PCR). NCIꢀH460 cells were plated and
treated with drugs the next day. After 24 h, cells were harvested. Total RNA was extracted with
an RNeasy mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol.
Firstꢀstrand cDNA synthesis was performed on 1 ꢀg of total RNA with Superscript III reverse
transcriptase according to the manufacturer’s instructions (Invitrogen). The resulting firstꢀstrand
cDNA was amplified to measure mRNA levels of RUNX3 with specific primers. Equal
quantities of each sample were analyzed by 1.5% agarose gel electrophoresis and visualized with
ethidium bromide staining and UV illumination. The mRNA level of glyceraldehyde phosphate
dehydrogenase (GAPDH) served as an internal control for RTꢀPCR analysis. The following
primers were used: 5′ꢀGCA GGC AAT GAC GAG AAC TAꢀ3′ (RUNX3 forward primer), 5′ꢀ
GTC TGG TCC TCC AGC TTC TGꢀ3′ (RUNX3 reverse primer), 5′ꢀCAA AGT TCT CAT GGA
TGA CCꢀ3′ (GAPDH forward primer), 5′ꢀCCA TGG AGA AGG CTG GGGꢀ3′ (GAPDH
reverse primer).
In vivo pharmacokinetic studies. Animal studies were approved by the Institutional Animal
Care and Use Committee at KRIBB and conducted as described previously36 with minor
modifications. Briefly, specific pathogenꢀfree male Sprague–Dawley (SD) rats (10 weeks old,
body weight 298–315 g), purchased from Koatech Co. (Kyeonggi, Republic of Korea), were
given a single dose of 4l intravenously (i.v.; 2 mg/kg, n = 3) or orally (p.o.; 10 mg/kg, n = 3).
ACS Paragon Plus Environment
18

Page 19 of 33
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Dosing solutions were prepared in dimethylacetamide (DMAC)/20% 2ꢀhydroxypropylꢀβꢀ
cyclodextrin (10/90, v/v%) for i.v. administration, and DMAC/Vitamin E TPGS 80/25% solutol
HS 15 (10/10/80, v/v%) for p.o. administration, and administered at dosing volumes of 5 and 10
ml/kg for i.v. and p.o., respectively. A 100ꢀꢁl aliquot of each plasma sample was prepared and
three volumes of iceꢀcold acetonitrile containing carbamazepine (internal standard) were added.
The mixture was centrifuged at 910 ×g for 10 min, and the supernatant was subjected to LCꢀ
MS/MS analysis. Pharmacokinetic parameters were calculated by standard noncompartmental
analysis of plasma concentration–time profiles using KineticaTM4.4.1 (Thermo Fisher Scientific,
Inc., Woburn, MA, USA). The areas under the plasma concentration–time curves (AUC) were
calculated by the linear–trapezoidal method. Systemic plasma clearance (CLp) was calculated as
follows: CLp = Dose/AUCinf. Terminal elimination halfꢀlife (t1/2) was calculated by the following
equation: t1/2 = 0.693/λz where λz is the terminal disposition rate constant. Volume of distribution
at steady state (Vss) was calculated as follows: Vss = Dose × AUMCinf/(AUCinf)2,
where AUMCinf is the area under the first moment of the plasma concentration–time curve
extrapolated to infinity. Oral bioavailability (F) was calculated as follows: F (%) =
(AUCp.o./AUCi.v.) × (Dosei.v./Dosep.o.) × 100.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In vivo xenograft experiment.
6ꢀweekꢀold female specifiedꢀpathogenꢀfree (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 Bioscience and
Biotechnology (KRIBB). NCIꢀH460 cells (3 × 107/animal) were injected subcutaneously into the
flank area. When tumors reached a volume of 50–80 mm3, as measured with a digital caliper,
ACS Paragon Plus Environment
19

Journal of Medicinal Chemistry
Page 20 of 33
1
2
3
4
5
6
7
8
9
mice were randomized into groups of four animals each to receive either vehicle control or 4l.
The drugs were orally administered at 20 mg/kg in 10% DMAC and 10% Tween 80 (Sigma) in
saline daily for two 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 = 0.523 × (long dimension) × (short
dimension)2.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Statistics software. The level of protein expression was quantified with Scion image
densitometry software (Scion Corporation, Frederick, MD). Data were analyzed with a
commercial statistics software package (SigmaStat, SPSS Science, Chicago, IL, USA). Student’s
tꢀtest was performed for individual comparisons. Multiple comparisons were assessed by oneꢀ
way ANOVA or regression analysis.
ACS Paragon Plus Environment
20

Page 21 of 33
Journal of Medicinal Chemistry
1
2
3
4
FIGURES
5
6
7
8
9
O
O
O
H
N
OH
OH
N
N
H
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
N
SAHA
TSA
O
O
n
OH
R
N
N
H
cap
group
lactam
core
zinc binding
moiety
pyridone-based HDAC inhibitors
Figure 1. Chemical structures of hydroxamic acidꢀbased HDAC inhibitors.
Figure 2. RUNX3 acetylation of selected pyridoneꢀbased HDAC inhibitors. A) Mycꢀtagged
RUNX3 (MycꢀRUNX3) was expressed in HEK293 cells. Acetylated RUNX3 levels were
analyzed by immunoprecipitation in HEK293 cells following treatment with 1 ꢁM of the
indicated test compound; B) Relative expression of RUNX3 acetylation based on the results of
ACS Paragon Plus Environment
21

Journal of Medicinal Chemistry
Page 22 of 33
1
2
3
4
5
the western blot shown in Figure 2A (with 1 ꢁM of the indicated test compound); C) Selected
compounds based on the RUNX3 acetylation assay.
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3. Doseꢀdependent effects of 4l on RUNX3 mRNA expression in NCIꢀH460 cells.
SNU16 gastric cancer cells were used as a positive control.
Figure 4. Effect of 4l, 20 mg/kg, q1d × 14 on the growth of a NCIꢀH460 human lung tumor
xenograft in nude mice: A) average body weight changes, B) average tumor volume, and C)
average tumor weight in mice treated with vehicle control (V.C.), 11-8, and 4l. **p<0.01,
***p<0.001, Student tꢀtest. The standard deviations of each value are provided in table S6 and
S7. ꢃVt (tumor volume) = Vt (measurement of the tumor volume) – V0 (initial tumor volume).
ACS Paragon Plus Environment
22

Page 23 of 33
Journal of Medicinal Chemistry
1
2
3
4
SCHEMES
5
6
7
Scheme 1. General procedure for synthesis of pyridoneꢀbased HDAC inhibitorsa
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
aReagents: (a) CH2Cl2, reflux; (b) K2CO3, acetone, reflux; (c) KONH2 (1.7 M in MeOH), MeOH,
0°C
ACS Paragon Plus Environment
23

Journal of Medicinal Chemistry
Page 24 of 33
1
2
3
4
TABLES.
5
6
7
8
9
Table 1. Percentage of RUNX transcriptional activation relative to FGFꢀ2, inhibition of HDAC
enzyme activity, and cancer cell growth by pyridoneꢀbased analogues. The standard deviations of
each value are provided in table S3–S5.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
HDAC
IC50 (µM)a
RUNX
(%)b
GI50 (µM)a
ACHN HCT-15
Compound
PC-3
MDA-MB-231
NCI-H23
NUGC-3
0.19
0.38
0.10
0.23
0.10
86.0
91.6
99.2
52.3
74.3
2.88
4.84
1.88
5.02
2.07
1.59
3.07
2.49
6.95
3.68
1.53
3.03
1.48
3.24
1.54
2.40
2.73
4.31
3.93
1.86
2.30
3.04
3.00
4.96
2.35
1.66
2.78
2.11
5.41
1.67
4a
4b
4c
4d
4e
0.09
46.4
3.04
2.59
2.80
2.40
2.81
1.82
4f
0.15
0.38
0.46
0.30
0.42
200.0
84.6
35.6
32.3
69.1
1.82
4.70
10.22
5.33
9.17
3.18
6.26
>10
1.26
4.73
6.44
4.16
9.49
2.60
4.52
>10
7.51
>10
2.69
5.30
>10
6.70
3.01
1.69
3.88
>10
5.94
9.19
4g
4h
4i
10.00
4.92
4j
4k
0.03
687.5
0.49
0.10
0.25
0.38
0.13
0.71
4l
0.16
0.36
0.07
0.11
0.07
74.0
53.5
3.58
4.84
0.53
0.50
5.87
4.90
6.39
0.72
0.84
6.94
2.00
2.14
0.70
0.70
2.93
7.29
8.60
0.69
0.71
>10
3.30
3.04
0.69
0.91
5.06
1.99
2.83
0.74
0.47
2.91
4m
4n
4o
106.0
104.7
53.8
4p
4q
0.32
30.9
4.92
3.89
3.59
6.06
4.92
3.92
4r
0.12
0.07
3.94
>10
0.48
76.9
119.5
26.7
1.34
0.14
>10
>10
1.86
1.74
2.05
>10
>10
2.89
0.94
2.34
>10
>10
1.92
0.92
1.00
>10
>10
1.58
1.81
1.05
>10
>10
1.88
1.42
1.28
>10
>10
1.50
4s
4t
4u
4v
4w
19.6
162.8
0.10
100.8
1.26
1.85
0.74
1.16
1.22
1.20
4x
0.21
0.31
0.38
0.03
0.14
53.8
14.3
34.4
142.1
97.6
3.97
1.85
1.48
0.70
1.78
4.38
2.88
2.23
0.72
1.36
4.19
1.71
1.83
0.76
0.98
7.45
1.06
1.42
1.59
0.90
4.88
2.15
1.78
0.87
1.31
4.02
1.74
1.31
1.05
0.97
4y
4z
4a'
4b'
4c'
0.05
0.57
0.11
105.5
30.0
80.2
0.18
>10
2.69
0.56
>10
2.00
0.32
>10
4.22
0.66
>10
2.49
0.46
5.21
2.34
0.47
>10
2.94
4d'
4e'
SAHA
Average
1ꢀcarbon linkerc
2ꢀcarbon linkerd
0.18
0.16
0.21
158.0
77.4
88.9
2.97
2.71
1.64
3.32
3.43
2.11
2.21
1.92
1.56
2.79
3.61
1.98
2.95
2.60
1.82
2.42
1.95
1.53
3ꢀcarbon linkere
a Values are the means of a minimum of three independent experiments; Values are the means
b
of a minimum of two independent experiments; Average activity of compounds 4aꢀ4h and 4l; d
c
Average activity of compounds 4mꢀ4t; e Average activity of compounds 4wꢀ4d’.
ACS Paragon Plus Environment
24

Page 25 of 33
Journal of Medicinal Chemistry
1
2
3
4
5
6
Table 2. Mouse liver microsomal stability of pyridoneꢀbased compounds
7
8
9
% Remaining, 30 minsa
Compound
NADPH(-)
98.6 ± 0.0
100.0 ± 8.7
96.5 ± 0.0
103.1 ± 0.0
104.6 ± 0.0
93.3 ± 0.0
96.7 ± 0.0
109.9 ± 4.0
103.6 ± 1.3
100.8 ± 2.8
NADPH(+)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61.5 ± 0.0
90.3 ± 4.4
11.1 ± 0.0
28.3 ± 0.0
59.6 ± 0.0
24.2 ± 0.0
20.7 ± 0.0
88.4 ± 2.0
31.5 ± 1.0
0.4 ± 0.0
4g
4l
4o
4p
4t
4w
4x
4b'
4d'
Buspironeb
a
A 1ꢀꢁM aliquot of the test compound was incubated with 0.5 mg/ml pooled mouse liver
microsomes for 30 min at 37°C; b positive control.
Table 3. The rat and human liver microsomal stability of pyridoneꢀbased compounds
% Remaining, 60 minsa
Compound
Species
NADPH(-)
92.0 ± 1.7
74.7 ± 0.0
116.0 ± 4.0
NADPH(+)
83.5 ± 1.7
75.5 ± 2.2
72.7 ± 5.2
UDPGA(-) UDPGA(+)
88.9 ± 3.0 72.4 ± 2.3
95.2 ± 1.8 99.7 ± 4.5
81.3 ± 9.5 45.7 ± 5.7
Rat
Human
Rat
4l
4b’
a Microsomal stability was determined in liver microsomes (0.5 mg/ml) at 37°C using 1 ꢀM test
compounds (n = 2–3)
Table 4. Oral (PO) and intravenous (IV) pharmacokinetic (PK) parameters of 4l in SD rats
ACS Paragon Plus Environment
25

Journal of Medicinal Chemistry
Page 26 of 33
1
2
3
4
5
6
4l
PK parametera
IV
PO
10
7
8
9
(mg/kg)
(hr)
2
Dose
tmax
Cmax
AUCinf
CL
NA
0.67 ± 0.14
142.1 ± 81.0
863.3 ± 73.3
NA
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(ng/ml)
(ng.hr/ml)
(l/hr/kg)
(l/kg)
NA
b
354.5 ± 5.6
5.6 ± 0.1
14.0 ± 2.2
4.4 ± 0.8
NA
NA
Vss
(hr)
10.3 ± 0.8
t1/2
(%)
F
48.7 ± 4.1
a PK parameters were based on mean plasma concentrationꢀtime profiles of three animals per
time points. PK parameters were calculated by noncompartmental analysis using PK Solutions
AUC after intravenous (ꢀIV)
administration was calculated from 0 to infinity, whereas the oral AUC was calculated from 0 to
5 h.
b
2.0 (Summit Research Services, Montrose, CO, USA);
ASSOCIATED CONTENT
Supporting Information
Structural data of intermediates 3aꢀ3e’, final compounds 4aꢀ4e’, HPLC conditions and retention
times of final compounds 4aꢀ4e’, in silico properties of 4aꢀ4e’, in vitro relative RUNX
transcriptional activity in response to FGFꢀ2, in vitro inhibitory activities against HDAC enzyme
and human cancer cell lines of 4aꢀ4e’, and HDAC isoform selectivity of 4l. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
ACS Paragon Plus Environment
26

Page 27 of 33
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
* Tel.: +82 2 2123 2882; fax: +82 2 362 7265; eꢀmail: gyoonhee@yonsei.ac.kr (G. Han)
* Tel.: +82 32 749 4509; fax: +82 32 749 4105; eꢀmail: organicjeong@yonsei.ac.kr (J. H. Jeong)
Author Contributions
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The manuscript includes contributions from all authors. All authors have approved the final
version of the manuscript.
ACKNOWLEDGMENT
We thank Prof. So Hee Kwon for helpful discussions. This research was supported by a grant
from the Translational Research Center for Protein Function Control (NRFꢀ2009ꢀ0083522), the
Basic Science Research Program through the National Research Foundation of Korea (NRF)
funded by the Ministry of Education (NRFꢀ2013R1A1A2008165), Ministry of Health & Welfare
(A120478), the Ministry of Science, ICT & Future Planning (NRFꢀ2013M3A6A4072536), and
the KRIBB Research Initiative Program.
ABBREVIATIONS
RUNX, runt related transcription factor; HDAC, histone deacetylase; HAT, histone
acetyltransferase; TGF, transforming growth factor; TSA, trichostatin A; SAHA, suberoylanilide
hydroxamic acid; PK, pharmacokinetics; EDC, Nꢀ(3ꢀdimethylaminopropyl)ꢀN’ꢀ
ethylcarbodiimide hydrochloride; DMAP, 4ꢀ(dimethylamino)pyridine; PSA, polar surface area;
UDPGA, uridine 5'ꢀdiphosphoꢀglucuronic acid.
REFERENCES
(1) Arrowsmith, C. H.; Bountra, C.; Fish, P. V.; Lee, K.; Schapira, M. Epigenetic protein
families: a new frontier for drug discovery. Nat. Rev. Drug Discov. 2012, 11, 384ꢀ400.
ACS Paragon Plus Environment
27

Journal of Medicinal Chemistry
Page 28 of 33
1
2
3
4
5
6
7
8
9
(2) Fleming, C. L.; Ashton, T. D.; Gaur, V.; McGee, S. L.; Pfeffer, F. M. Improved synthesis and
structural reassignment of MC1568: a class IIa selective HDAC inhibitor. J. Med. Chem.
2014, 57, 1132ꢀ1135.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(3) McKinsey, T. A. Isoformꢀselective HDAC inhibitors: closing in on translational medicine for
the heart. J. Mol. Cell. Cardiol. 2011, 51, 491ꢀ496.
(4) Miller, T. A.; Witter, D. J.; Belvedere, S. Histone deacetylase inhibitors. J. Med. Chem. 2003,
46, 5097ꢀ5116.
(5) Paris, M.; Porcelloni, M.; Binaschi, M.; Fattori, D. Histone deacetylase inhibitors: from
bench to clinic. J. Med. Chem. 2008, 51, 1505ꢀ1529.
(6) Lane, A. A.; Chabner, B. A. Histone deacetylase inhibitors in cancer therapy. J. Clin. Oncol.
2009, 27, 5459ꢀ5468.
(7) Nakagawa, M.; Oda, Y.; Eguchi, T.; Aishima, S.; Yao, T.; Hosoi, F.; Basaki, Y.; Ono, M.;
Kuwano, M.; Tanaka, M.; Tsuneyoshi, M. Expression profile of class I histone deacetylases in
human cancer tissues. Oncol. Rep. 2007, 18, 769ꢀ774.
(8) Yoo, C. B.; Jones, P. A. Epigenetic therapy of cancer: past, present and future. Nat. Rev.
Drug Discov. 2006, 5, 37ꢀ50.
(9) Vigushin, D. M.; Coombes, R. C. Targeted histone deacetylase inhibition for cancer therapy.
Curr. Cancer Drug Targets 2004, 4, 205ꢀ218.
(10) Popovic, R.; Licht, J. D. Emerging epigenetic targets and therapies in cancer medicine.
Cancer Discov. 2012, 2, 405ꢀ413.
(11) Wilting, R. H.; Dannenberg, J. H. Epigenetic mechanisms in tumorigenesis, tumor cell
heterogeneity and drug resistance. Drug Resist. Updat. 2012, 15, 21ꢀ38.
ACS Paragon Plus Environment
28

Page 29 of 33
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(12) Ivanov, M.; Kacevska, M.; IngelmanꢀSundberg, M. Epigenomics and interindividual
differences in drug response. Clin. Pharmacol. Ther. 2012, 92, 727ꢀ736.
(13) New, M.; Olzscha, H.; La Thangue, N. B. HDAC inhibitorꢀbased therapies: can we interpret
the code? Mol. Oncol. 2012, 6, 637ꢀ656.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(14) Rius, M.; Lyko, F. Epigenetic cancer therapy: rationales, targets and drugs. Oncogene 2012,
31, 4257ꢀ4265.
(15) Singh, B. N.; Zhang, G.; Hwa, Y. L.; Li, J.; Dowdy, S. C.; Jiang, S. W. Nonhistone protein
acetylation as cancer therapy targets. Expert Rev. Anticancer Ther. 2010, 10, 935ꢀ954.
(16) Tang, J.; Yan, H.; Zhuang, S. Histone deacetylases as targets for treatment of multiple
diseases. Clin. Sci. (Lond) 2013, 124, 651ꢀ662.
(17) Chuang, L. S.; Ito, Y. RUNX3 is multifunctional in carcinogenesis of multiple solid tumors.
Oncogene 2010, 29, 2605ꢀ2615.
(18) Li, Q. L.; Ito, K.; Sakakura, C.; Fukamachi, H.; Inoue, K.; Chi, X. Z.; Lee, K. Y.; Nomura,
S.; Lee, C. W.; Han, S. B.; Kim, H. M.; Kim, W. J.; Yamamoto, H.; Yamashita, N.; Yano, T.;
Ikeda, T.; Itohara, S.; Inazawa, J.; Abe, T.; Hagiwara, A.; Yamagishi, H.; Ooe, A.; Kaneda,
A.; Sugimura, T.; Ushijima, T.; Bae, S. C.; Ito, Y. Causal relationship between the loss of
RUNX3 expression and gastric cancer. Cell 2002, 109, 113ꢀ124.
(19) Tsang, Y. H.; Lamb, A.; Chen, L. F. New insights into the inactivation of gastric tumor
suppressor RUNX3: the role of H. pylori infection. J. Cell Biochem. 2011, 112, 381ꢀ386.
(20) Bae, S. C.; Lee, Y. H. Phosphorylation, acetylation and ubiquitination: the molecular basis
of RUNX regulation. Gene 2006, 366, 58ꢀ66.
(21) Li, Q. L.; Kim, H. R.; Kim, W. J.; Choi, J. K.; Lee, Y. H.; Kim, H. M.; Li, L. S.; Kim, H.;
Chang, J.; Ito, Y.; Youl Lee, K.; Bae, S. C. Transcriptional silencing of the RUNX3 gene by
ACS Paragon Plus Environment
29