Synthesis, pharmacophores, and mechanism study of pyridin-2(1H)-one derivatives as regulators of translation initiation factor 3A

Article abstract of DOI:10.1002/ardp.201300138

Twenty-seven 1,5-disubstituted-pyridin-2(1H)-one derivatives were synthesized and evaluated for their anti-cancer and anti-fibrosis activity by A549 and NIH3T3 cell viability assays, respectively. To study the selectivity between the cancer and fibrosis cell lines, pharmacophore models (F ₁-F₄) were built in advance for compounds with pyridin-2(1H)-one scaffold, which revealed the relationship between the occupation of the aromatic sub-site F₄ and potent anti-cancer activity. The relationship between structure and anti-cancer activity for all target compounds is also reported herein: 1-Phenyl-5-((m-tolylamino)methyl) pyridine-2(1H)-one (22) displayed both potency and selectivity (IC₅₀ = 0.13 mM) toward the A549 cell line through the inhibition of translation initiation, especially by eIF3a suppression, and can be treated as a lead for the design of novel eIF3a regulators and anti-lung cancer agents. A series of pyridin-2(1H)-one derivatives were synthesized. Elucidation of their pharmacophores and mechanism of action suggested that structures with F ₄ occupation displayed more selective anti-cancer than anti-fibrosis activity and that they interrupt the initiation phase of translation by acting on the eukaryotic translation initiation factor 3, subunit A.

Full text of DOI:10.1002/ardp.201300138

654
Arch. Pharm. Chem. Life Sci. 2013, 346, 654–666
Full Paper
Synthesis, Pharmacophores, and Mechanism Study of
Pyridin-2(1H)-one Derivatives as Regulators of Translation
Initiation Factor 3A
Weixing Zhu1, Jie Shen2, Qianbin Li1, Qi Pei2, Jun Chen1, Zhuo Chen1, Zhaoqian Liu2, and
Gaoyun Hu1
1
Chemistry Section, Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Central South
University, Changsha, Hunan, China
Pharmacology Section, Institute of Clinical Pharmacology, Central South University, Changsha, Hunan, China
2
Twenty-seven 1,5-disubstituted-pyridin-2(1H)-one derivatives were synthesized and evaluated for their
anti-cancer and anti-fibrosis activity by A549 and NIH3T3 cell viability assays, respectively. To study the
selectivity between the cancer and fibrosis cell lines, pharmacophore models (F1–F4) were built in
advance for compounds with pyridin-2(1H)-one scaffold, which revealed the relationship between the
occupation of the aromatic sub-site F4 and potent anti-cancer activity. The relationship between
structure and anti-cancer activity for all target compounds is also reported herein: 1-Phenyl-5-((m-
tolylamino)methyl)pyridine-2(1H)-one (22) displayed both potency and selectivity (IC50 ¼ 0.13 mM)
toward the A549 cell line through the inhibition of translation initiation, especially by eIF3a
suppression, and can be treated as a lead for the design of novel eIF3a regulators and anti-lung cancer
agents.
Keywords: Eukaryotic translation initiation factor 3, subunit A (eIF3a) / Fibrosis / Lung cancer / Pyridin-2(1H)-one /
Regulators
Received: April 21, 2013; Revised: June 19, 2013; Accepted: June 28, 2013
DOI 10.1002/ardp.201300138
Introduction
(eIFs). In eukaryotes, at least 13 factors have been identified to
participate in the translation initiation step [3, 4]. Among
these factors, eIF3 is the largest and the most complex one
with molecular weight of about 550–700 kDa [3, 4]. eIF3a is
the largest subunit of eIF3 and has been suggested to play
critical roles in regulating synthesis of proteins including
a-tubulin, ribonucleotide reductase M2, and p27 as well as in
regulating cell cycle progression and cell proliferation and
differentiation [5–8]. Nevertheless, eIF3a is essential for cancer
cells to maintain malignant phenotype [6] and ectopic over-
expression of eIF3a transformed NIH3T3 cells in vitro [9].
Although over-expression of eIF3a has been found in many
cancers such as cancers of breast [10], cervix [11], stomach [12],
and esophagus [13], eIF3a appeared to be specifically over-
expressed in human lung cancer and played vital roles in
pathogenesis and prognosis of this disease [14, 15]. Further-
more, it is discovered that lung cancer cell is more sensitive
to cisplatin treatment due to higher levels of eIF3a, which
further reveal the close relationship between eIF3a and lung
cancer. eIF3a also regulates the expression and activity of
Lung cancer is the leading cause of cancer-related mortality
with an estimated yearly incidence of 1.2 million new cases
worldwide and its 5-year overall survival rate is approximately
16% [1]. The low efficiency of conventional therapies in
achieving long-term survival of patients with lung cancer calls
for the development of novel treatment/prevention options.
In this regard, chemoprevention using naturally occurring or
synthetic compounds to arrest or reverse the carcinogenic
process maybe extremely important as a lung cancer
prevention strategy [2].
Protein synthesis in eukaryotes is a complicated process
that involves many eukaryotic translation initiation factors
Correspondence: Prof. Gaoyun Hu, Department of Medicinal Chemistry,
School of Pharmaceutical Sciences, Central South University, No. 172,
Tongzipo Road, Changsha 410013, Hunan, China.
E-mail: hugaoyun@csu.edu.cn
Fax: þ86 731 82650388
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2013, 346, 654–666
1,5-Disubstituted Pyridine-2(1H)-ones as eIF3a Regulators
655
nucleotide excision repair (NER) proteins, which mediate the
effect of eIF3a on cellular response to DNA-damaging anti-
cancer drugs [15]. Therefore, eIF3a is recognized as an
attractive bio-marker in the treatment of proliferative
diseases, especially lung cancer.
However, little is known about small molecule eIF3a
regulators up to date. The only compound reported is
L-mimosine, a plant amino acid, which was developed as cell
cycle blocker (Fig. 1). L-Mimosine can specifically and
reversibly block mammalian cells at late G1 phase [16].
Recently, it has been suggested that eIF3a acts as a mediator of
L-mimosine effect on the protein synthesis [5]. The decreased
expression of eIF3a by L-mimosine is due to its iron-chelating
function of 3-hydroxy-4-carbonyl group, which results in up-
regulated expression of CDK inhibitor (p27), down-regulated
expression of ribonucleotide reductase M2 [6] and de novo
synthesis of tyrosinated a-tubulin [5].
Figure 2. Designing strategy for pyridin-2(1H)-one derivative.
anti-fibrosis activity and selectivity using MTT methods.
Pharmacophore models were also built using Molecular
Operating Environment (MOE, Chemical Computing Group,
Inc., Canada) to elucidate differences on selectivity. Further-
more, to further evaluate whether eIF3a protein was involved
in the effect of potent compounds against cell proliferation,
the levels of eIF3a mRNA and protein upon drug treatment
were determined by real-time PCR and immunoblot analysis
assay in cancer cell line.
Additionally, as an analog, pyridin-2(1H)-one scaffold is also
widely applied in the design of novel drugs because of its
potency of anti-inflammatory [17] and anti-fibrosis activity [18,
19]. In recent years, it is suggested that pulmonary fibrosis
share similar pathological ground to lung cancer, which is Chemistry
mediated by growth factors [20] and is associated with an
increased risk of lung cancer [21, 22]. Another pyridin-2(1H)-
one derivative, 5-methyl-1-phenylpyridin-2(1H)-one (pirfeni-
done, PFD) is the only small molecular drug coming into
market for anti-fibrosis therapy (Fig. 1). In our previous efforts
to study the mechanism and modification of PFD, we
discovered 1-(3-fluorophenyl)-5-methylpyridin-2(1H)-one (fluo-
rofenidone, AKF-PD), which shows better anti-fibrosis activity
than that of PFD (Fig. 1) [23, 24]. In recent studies, PFD was
reported to display antitumor effects on malignant gliomas
by inhibition of the expression of TGF-b [25] and EGFR
phosphorylation [26].
To further conduct structure modification and anti-
proliferation activity study based on the pyridin-2(1H)-one
scaffold, we designed and synthesized a novel series of 1,5-
disubstituted-pyridone derivatives using PFD as a lead (Fig. 2).
In vitro cell viability assays were conducted on human A549
and mice NIH3T3 cell lines to evaluate their anti-cancer and
Amides 5a–5c were synthesized from malic acid following the
methods reported previously as shown in Scheme 1 [27]. In
brief, compound 1 was prepared in high yield and treated
with aniline in ethanol to obtain 1-substituded pyridin-2(1H)-
one 2 in the form of ester [28], which was further hydrolyzed
and acidified to obtain acid 3. The final compound 5 was
obtained following classical methods for the synthesis of
amides under basic condition from acyl chloride 4.
The synthetic procedures for amines (compounds 9–29) and
ethers (compounds 30–32) consisted of two main reactions:
Ullmann reaction [29, 30] and bromination reaction [31, 32] as
illustrated in Scheme 2 to yield intermediates 7a–7c, which
were finally SN2 substituted with different aniline derivatives
or sodium salt of substituted phenol in acetonitrile.
Additionally, other than to obtain 7a, the bromination
reaction for 6a will also yield products 7e and 7f because C-3 is
also active under the same radical conditions, and 7d as well,
which is consistent with [31]. This can be further confirmed by
the purification of 8a from the mixture of bromination
reaction for 6a with the treatment of CH3ONa. Furthermore,
during the synthesis of compound 9, C-5 aldehyde derivative
8b was also obtained from the hydrolysis of imine formed
between C-5 and benzyl group because of the di-brominated
product 7d (Scheme 3) [33].
Results and discussion
To investigate the anti-proliferation activities of pyridin-2(1H)-
one derivatives, the in vitro cell viability assay was conducted
Figure 1. The chemical structures of L-mimosine, pifenidone (PFD),
and fluorofenidone (AKF-PD).
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

656
W. Zhu et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 654–666
H
COOC
3
H
O
COOMe
a
c
N
O
b
H
COO
H
OOC
O
O
1
2
O
H
COO
l
COC
R
N
H
e
d
N
O
N
O
N
O
a
5
:
=
R
R
R
Scheme 1. Reagents and conditions: (a) (i) H2SO4,
SO3, 98–104°C, 1.5 h; (ii) CH3OH, 95–100°C, 2 h,
65%; (b) (i) aniline, C2H5OH; (ii) 15% Na2CO3, 73%;
(c) (i) 10% NaOH, 3 h; (ii) 5% HCl, 87%; (d) SOCl2,
reflux, 4 h; (e) RNH2, CH2Cl2, reflux, 4 h.
-
-
:
:
=
=
5b
c
5
H
H
H
H
H
C
C
2C 2C
3
H
2C 2OC
3
_
a
c
5
5
3
4
NH
Br
I
a
N
c
O
N
O
b
O
N
R2
+
R1
O
N
H
R1
R1
R1
7a−7c
6a−6c
9: R1=m-F,R2=H
9−29
6a,7a: R1= m-F
6b,7b: R1=H
20: R1=H,R2=H
10: R1=m-F,R2=p-COMe
11: R1=m-F,R2=p-Me
12: R1=m-F,R2=p-OMe
13: R1=m-F,R2=m-Me
14: R1=m-F,R2=o-Me
15: R1=m-F,R2=p-Cl
16: R1=H,R2=o-Me
21: R1=H,R2=p-Cl
22: R1=H,R2=m-Me
23: R1=p-OMe,R2=p-Cl
6c,7c: R1=p-OMe
24: R1=p-OMe,R2=p-COMe
25: R1=p-OMe,R2=p-OMe
26: R1=p-OMe,R2=H
Scheme 2. Reagents and conditions: (a) Cu,
K2CO3, DMF, 160–170°C, reflux, 20 h, 61–75%;
(b) N-bromosuccinimide (NBS), azobisisobutyronitrile
(AIBN), CCl4, medium-pressure mercury (450-W),
reflux, 4 h; (c) substituted aniline, CH3CN, r.t.,
2.5 h.
27: R1=p-OMe,R2=p-Me
28: R1=p-OMe,R2=o-Me
29: R1=p-OMe,R2=m-Me
17: R1=H,R2=p-Me
18: R1=H,R2=p-OMe
19: R1=H,R2=p-COMe
Br
Br
Br
Br
Br
Br
+
N
N
O
N
O
O
N
O
N
O
a
+
+
F
F
F
F
F
a
7
7
d
7e
7
f
6a
O
c
b
r
B
H
O
N
N
O
N
O
c
N
O
Scheme 3. Reagents and conditions: (a)
N-bromosuccinimide (NBS), azobisisobutyro-
nitrile (AIBN), CCl4, medium-pressure mercury
(450-W), reflux, 4 h; (b) m-fluoroaniline, CH3CN,
r.t., 2.5 h; c. CH3OH, NaOH.
F
F
F
8b
8a
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2013, 346, 654–666
1,5-Disubstituted Pyridine-2(1H)-ones as eIF3a Regulators
657
on two cell lines with different states of eIF3a expression:
the 4-pharmacophore query (F1–F4) was built using flexible
alignment and pharmacophore query within MOE as shown
in Fig. 3a. Compound 5a and 9 shared similar F4 occupation
(Fig. 3b). However, F4 aromatic/hydrophobic feature cannot be
occupied by PFD due to its vacancy on the C-5 position of
pyridin-2(1H)-one, which suggest the critical role in the
selectivity profiles for F4 occupation (Fig. 3c).
A549,
a human lung adenocarcinoma cell with high
expression of eIF3a, and NIH3T3, a mouse embryonic
fibroblast cell line with low expression of eIF3a. Cisplatin
and PFD were used as control. The results for compounds 5
and 9 are shown in Table 1.
Structure–selectivity relationship (SSR) for pyridin-2
(1H)-one derivatives with different substituents at R2
Cell viability assays for compound 5 and 9
Western blot assay for compounds 5, 9, and AKF-PD
As mentioned before, eIF3a is a specific bio-marker of lung
cancer. The possible reason for the selectivity change for
pyridin-2(1H)-one derivatives may partially be due to the
over-expression of eIF3a in lung cancer, which can extremely
increase the sensitivity of anti-cancer agents toward A549
cell line as described previously [24]. To further evaluate
whether eIF3a is involved in the effect of novel pyridin-2(1H)-
one derivatives, we next examined the expression levels of
eIF3a protein upon treatment with 5a (C-5 amide deriva-
tives), 9 (C-5 amine derivatives), and AKF-PD in A549 cells by
Western blot methods as described previously [15]. As shown
in Fig. 4, AKF-PD (with no substituent at R1 on C-5 position)
exhibited no inhibitory effects on eIF3a expression, whereas
5a and 9 significantly and consistently suppressed the eIF3a
level at 600 mM, which means that F4 occupation is a
necessary requirement for the inhibition of eIF3a expression.
Secondly, the existence of C-5 aromatic substituent occupy-
ing F4 sub-site is more favorable for eIF3a inhibition for
amine analogs (9, with inhibitory rate of 80%) than for amide
group (5a, with inhibitory rate of 30%), which make amine
derivatives with aromatic ring attractive to develop more
potent and selective anti-cancer agents as eIF3a regulators.
PFD was developed as an anti-fibrosis agent and, in recent
years, it was further reported to display antitumor effects on
malignant glioma cells. In order to get insight into the
influencing factors on the selectivity of pyridin-2(1H)-one
derivatives toward different cells, aromatic ring or aliphatic
chain were introduced into substituents at R2 (Scheme 1). As
shown in Table 1, PFD shows good selectivity against NIH3T3
cell line which is consistent with its anti-fibrosis applica-
tion, whereas compound 5a reversed the selectivity from
fibroblast cell to cancer cell, which show 63 times selectivity
value toward A549 cell line compared to that of PFD. When
compared with compounds with aliphatic substituents at R2
(5b, 5c), the experimental data for those with aromatic
substituents at R2 (5a, 9) demonstrated that the existence of
aromatic ring at R2 is more favorable and selective for the
inhibition of cancer cell proliferation.
Selectivity study based on pharmacophore building for
PFD, 5a and 9
Herein, to get further insight into the changes in selectivity
based on structure modification, 5a and 9 were aligned and
Table 1. In vitro cell viability assay results for compound 5 and 9
R2
N
O
R1
IC50 (mM)a)
Substituents
R2
Ratio of values
NCE
R1
NIH3T3
A549
NIA
NCP
5a
5b
5c
9
PFD
Cisplatin
H
H
H
F
–CONHPh
–CONHC3H7
–CONH(CH2)2OCH3
–CH2NHPh
564.32 ꢀ 25.32
3.47 ꢀ 1.25
6.47 ꢀ 1.46
0.39 ꢀ 0.12
2.27 ꢀ 0.58
0.015 ꢀ 0.007
31.5 ꢀ 12.34
8.08 ꢀ 1.97
14.08 ꢀ 2.79
0.46 ꢀ 0.19
8.32 ꢀ 2.53
0.06 ꢀ 0.01
17.33
0.43
0.46
0.85
0.27
0.25
64.15
1.59
1.70
3.14
1.00
0.93
H
–CH3
a)
The data shown are representative of three individual experiments. NIA ¼ IC50(NIH3T3)/IC50(A549); NCP ¼ ratio IC50(NCE)/ratio IC50(PFD)
.
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

658
W. Zhu et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 654–666
Figure 3. Selectivity study based on pharmacophore modeling: (a) 4-point pharmacophore features built from 5a and 9 (F1: hydrogen bond
acceptor; F2–F4: hydrophobic or aromatic sub-site); (b) alignment for compounds 5a and 9 to pharmacophore features; (c) alignment for PFD
with pharmacophore features. All the models were built using MOE software.
Structure–activity relationship (SAR) for pyridin-2(1H)-
one derivatives 10–32
shows 79.3 times for IC50 ratio value and makes it the most
selective and potent inhibitor with pyridin-2(1H)-one scaffold
for lung cancer cell line up to now.
To further obtain novel chemical entries with more structure
varieties occupying F4 sub-site and with higher selectivity and
potency, the R2 substituent on C-5 position of pyridin-2(1H)-
one was modified to substituted aniline (10–29, Scheme 2). As
shown in Table 2, most compounds showed strong inhibitory
activity against both cell lines with IC50 values of 0.13–
14.50 mM. However, the selectivity toward A549 was retained,
which confirmed that the higher level expression of eIF3a in
lung cancer results in more sensitive effect for pyridin-2(1H)-
one derivatives with F4 occupation.
Structure–activity relationship (SAR) study indicated that
both anti-fibrosis and anti-cancer activity for pyridin-2(1H)-one
derivatives are not related with types of substituents at R1 or
R2. Both 21 and 22 displayed better selectivity and potency
toward A549 cell line. The most potent compound 22
displayed similar activity (IC50 ¼ 0.13 mM) to that of cisplatin
(IC50 ¼ 0.06 mM). Additionally, what is most notable for
compound 22 is that this compound possesses highest
selectivity toward A549 cell line compared with PFD, which
To further study whether the hydrogen bond donor (NH)
on C-5 position is important for the activity profile, the NH
group was isosterically replaced by oxygen to obtain a series
of 1-substituted phenyl-5-substituted phenyloxymethyl-2(1H)-
pyridinone. What was interesting is that compound 30 and 31
displayed most potency against NIH3T3 cell proliferation with
IC50 value of 0.016 and 0.014 mM, respectively (Table 3). This
suggested that the hydrogen bond acceptor (O) on C-5 position
may be more favorable for the anti-fibrosis activity of pyridin-2
(1H)-one. Regardless of the loss of selectivity for compound 30,
however, it still arouse our interests in its anti-fibrosis activity
and further structure modification work about compound 30
in our group is under the way.
Suppression of eIF3a expression for L-mimosine and
pyridin-2(1H)-one derivatives
Dysregulation of protein synthesis, especially hyperactivity in
translation or post-translational modification procession, is
deeply involved in tumorigenesis and therapeutic effects of
cancer and restoration of translation regulation provide a
promising pattern for cancer therapy [34]. Up to date, there
are quite a few of antitumor agents suppressing translation
including retinoic acid, rapamycin, wortmannin, homohar-
ringtonine, bruceantin, and didemnin B, which down-
regulate translation through different mechanisms [35–37].
Additionally, Dong and co-workers reported L-mimosine
(Fig. 1) inhibited protein synthesis during initiation of
translation, which is associated with the inhibition of protein
synthesis of eIF3a [5].
To further evaluate the mechanism of action on the
inhibition of eIF3a expression (whether transcription or post-
translational level) for pyridin-2(1H)-one derivatives, Western
blot analysis and real-time PCR analysis in A549 cells
were accessed for novel active compounds in different
Figure 4. Western blot analysis results for pyridin-2(1H)-one
derivatives (5a, 9, and AKF-PD) in A549 cells. Each bar represents
the mean ꢀ SD of at least three different experiments. The bars for
eIF3a protein are plotted with the percentage about ratios value of
eIF3a/b-actin of DMSO control.
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2013, 346, 654–666
1,5-Disubstituted Pyridine-2(1H)-ones as eIF3a Regulators
659
Table 2. In vitro cell viability assay results for compound 10–29.
NH
N
O
R2
R1
Substituents
IC50 (mM)a)
Ratio of values
NCP
NCE
R1
R2
NIH3T3
A549
NIA
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
m-F
m-F
m-F
m-F
m-F
m-F
H
H
H
H
H
H
H
p-OMe
p-OMe
p-OMe
p-OMe
p-OMe
p-OMe
p-OMe
p-COMe
p-Me
p-OMe
m-Me
o-Me
p-Cl
o-Me
p-Me
p-OMe
p-COMe
H
p-Cl
m-Me
p-Cl
p-COMe
p-OMe
H
p-Me
o-Me
7.20 ꢀ 1.89
28.40 ꢀ 3.49
147.10 ꢀ 5.74
0.36 ꢀ 0.15
0.34 ꢀ 0.08
5.10 ꢀ 1.09
0.41 ꢀ 0.21
0.23 ꢀ 0.04
1.57 ꢀ 0.59
1.16 ꢀ 0.97
3.50 ꢀ 1.59
4.09 ꢀ 1.67
1.52 ꢀ 0.37
3.75 ꢀ 0.88
0.57 ꢀ 0.27
3.18 ꢀ 0.89
2.54 ꢀ 1.23
3.52 ꢀ 2.64
1.63 ꢀ 0.59
21.34 ꢀ 3.49
7.06 ꢀ 2.34
–
1.02
–
3.78
–
5.39 ꢀ 0.97
0.38 ꢀ 0.24
0.29 ꢀ 0.19
14.50 ꢀ 2.56
0.32 ꢀ 0.07
0.28 ꢀ 0.03
1.01 ꢀ 0.21
5.92 ꢀ 1.11
0.80 ꢀ 0.32
0.32 ꢀ 0.13
0.13 ꢀ 0.04
2.17 ꢀ 0.34
0.95 ꢀ 0.06
1.51 ꢀ 0.12
1.70 ꢀ 0.38
1.53 ꢀ 0.64
1.91 ꢀ 0.31
1.92 ꢀ 0.22
27.29
0.95
1.17
0.35
1.28
0.82
1.55
0.20
4.38
12.78
11.69
1.72
0.6
101.07
3.52
4.33
1.29
4.74
3.04
5.74
0.74
16.22
47.33
43.29
6.37
2.22
7.81
5.52
8.52
3.15
41.14
2.11
1.49
2.30
0.85
11.11
m-Me
a)
The data shown are representative of three individual experiments. NIA ¼ IC50(NIH3T3)/IC50(A549); NCP ¼ ratio IC50(NCE)/ratio IC50(PFD)
.
concentrations: PFD in high concentration (300 mM), 22 and
L-mimosine (200 mM), and 30 in low concentration (100 mM)
using conventional methods as described previously [15].
As shown in Fig. 5, after incubation of A549 cell with
different compounds for 8 h, L-mimosine and PFD in high
concentration caused a decrease in mRNA levels of eIF3a to
about a half of control whereas compound 22 and 30 in low
concentration displayed less influence to the levels of eIF3a
mRNA. However, what interested us was that the suppression
of protein did not proportionate with the level of mRNA,
which showed opposite results: the eIF3a protein level of A549
cells were down-regulated by 22 and 30 substantively in low
Table 3. In vitro cell viability assay results for compound 30–32.
O
N
O
R
Substituents
R
IC50 (mM)a)
Ratio of values
NCE
NIH3T3
A549
NIA
NCP
30
31
32
m-Me
p-Cl
H
0.016 ꢀ 0.002
0.014 ꢀ 0.003
0.043 ꢀ 0.005
0.28 ꢀ 0.13
0.22 ꢀ 0.10
0.38 ꢀ 0.12
0.06
0.06
0.11
0.21
0.24
0.41
a)
The data shown are representative of three individual experiments. NIA ¼ IC50(NIH3T3)/IC50(A549); NCP ¼ ratio IC50(NCE)/ratio IC50(PFD)
.
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

660
W. Zhu et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 654–666
sensitivity of lung cancer cell line, which is superior to
fibroblast cell line; (ii) pyridin-2(1H)-one derivative, especially
compounds with F4 occupation, demonstrates better anti-
proliferation activity. Of all the three series derivatives,
compound 22 most selectively and effectually inhibits the
cancer cell proliferation; (iii) the functional activities of active
compounds with F4 occupation are associated, at least
partially, with their inhibition of translation initiation by
suppressing eIF3a as demonstrated by RT-PCR and Western
blot analysis.
In summary, given the important roles of eIF3a protein in
cell growth, development and tumorigenesis, for the first time
our studies suggest that eIF3a is an attractive therapeutic
target in the treatment of lung cancer and also demonstrate
the relationship between the F4 occupation and eIF3a protein
level, which are important for selectivity toward cancer cells.
The structure with F4 occupation especially 1-substitued
phenyl-5-((substituted phenylamino)methyl)pyridine-2(1H)-
one derivatives can be considered as a promising scaffold
to develop small molecule eIF3a regulators as potential anti-
lung cancer agents.
Figure 5. Western blot analysis and mRNA RT-PCR results for
pyridin-2(1H)-one derivatives (PFD, 22, 30) and L-mimosine in A549
cells. Each bar represents the mean ꢀ SD of at least three different
experiments. The bars for eIF3a protein are plotted with the
percentage about ratios value of eIF3a/b-actin of DMSO control.
The bars for mRNA levels are plotted with the percentage of DMSO
control.
concentration, whereas L-mimosine and PFD in high concen-
tration displayed less effect on the protein level. The
interesting results indicated a post-translational level about
the suppression of eIF3a expression for pyridin-2(1H)-one
derivative (22 and 30), which is a quite different manner
from that of L-mimosine demonstrated previously to be a
transcription inhibitor of eIF3a expression.
These results indicated that the mechanism of inhibition on
protein synthesis by pyridin-2(1H)-one derivative is also much
distinct from the known inhibitors of protein synthesis,
which is likely to be operating at the initiation phase of
translation through eIF3a. Additionally, pyridin-2(1H)-one
may not be fully functional of pyridin-4(1H)-one as exempli-
fied by L-mimosine. Unraveling this mechanism may open a
window for new therapeutic opportunities for the treatment
of tumors, especially lung cancer. Pyridin-2(1H)-one deriva-
tives may also be used in combination with therapeutic
agents that show different mechanisms of action and multi-
targeted therapeutic strategy, as well as overcoming the drug
resistance characteristics.
Experimental
Chemistry
All chemicals and solvents were used as received from commer-
cial sources without further treatment unless otherwise stated.
All reactions were monitored by TLC with 0.25 mm silica gel
plates (60GF-254). UV light was used to visualize the spots. 1H NMR
and 13C NMR spectra were recorded on Bruker DRX 400 NMR
instruments with TMS as an internal standard, d in parts per
million and J in Hertz. Mass spectra were recorded on Qstar LC/MS
instrument. Melting points were determined uncorrected on a
BUCHI electrothermal melting point apparatus. Column chro-
matography was performed with commercial silica gel (300–
400 mesh) for purification. All tested compounds are >95% pure
by HPLC analysis performed on an Agilent 1100 HPLC instrument
using an Agela Promosil C18 column (250 mm ꢁ 4.6 mm, 5 mm)
according to one of the following methods over 20 min, with
detection at 254 nm and a flow rate of 1.0 mL/min. Method A:
Compounds 5a–5c, 9–12, 18–20, and 23–25 were eluted with 60%
methanol/40% water. Method B: Compounds 13–17, 21–22, and 27
were eluted with 65% methanol/35% water. Method C: Compounds
26 and 28–32 were eluted with 70% methanol/30% water.
Conclusion
General procedure for the preparation of C-5 amides
derivatives (5a–5c)
Synthesis of methylcoumalate (1)
To 134 g of malic acid was added 155 mL of concentrated sulfuric
acid followed by 70 mL of 50% oleum. The resulting slurry was
heated at 98–104°C for 1.5 h. The solution was cooled to room
temperature and 170 mL of dry methanol was added slowly. The
resulting solution was heated at 95–100°C for 2 h, allowed to cool
to room temperature and then poured into 120 mL of cold water.
After standing for 0.5 h, the solution was extracted with
chloroform and upon evaporation of the solvent, recrystallization
A novel series of PFD analogs with different substitutions
on the N-1 position and C-5 position of pyridin-2(1H)-one
scaffold were designed and synthesized to conduct structure–
selectivity relationship (SSR) and SAR as well as mechanism
studies.
Cell-based in vitro viability assays demonstrated that F4
occupation can improve the sensitivity of most compounds to
lung cancer cell line and change the selectivity of PFD analogs.
The reasons lie in: (i) eIF3a is a specific bio-marker for the
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2013, 346, 654–666
1,5-Disubstituted Pyridine-2(1H)-ones as eIF3a Regulators
661
from EtOAc to give compound 1. Slight yellow solid, 65% yield.
mp: 68.9–70.7°C ([38]; 65–66°C).
8.10 (d, 1H, J ¼ 2.8 Hz). 13C NMR (100 MHz, CDCl3): d 39.6, 58.7,
70.9, 113.4, 120.5, 126.4, 129.0, 129.4 (2C), 137.1, 140.2, 140.8,
162.0, 163.9. MS m/z: 272 [M]þ.
Synthesis of methyl 6-oxo-1-phenyl-1,6-dihydropyridine-3-
carboxylate (2)
General procedure for the synthesis of C-5 amines
derivatives (9–29)
Synthesis of 1-(3-fluorophenyl)-5-methylpyridin-2(1H)-one
11.2 g of aniline was added slowly to a solution of 15.4 g of
methylcoumalate in 150 mL of alcohol, after stirring for a few
minutes, crystallization of a yellow solid occurred, which was
filtered-off and dried for 4 h under vacuum at 45°C. The crude
solid was dissolved in 140 mL of 15% Na2CO3, heated to 40–45°C,
cooled, and the solid formed was filtered-off and recrystallized
from isopropyl alcohol to obtain 2. White solid, 73% yield. mp:
198.7–200.4°C ([28]; 100–105°C).
(6a)
To 9.6 g of anhydrous DMF was added 45.0 g (0.2 mol) of 1-fluoro-
3-iodophenyl, 21.8 g (0.2 mol) of 5-methyl-2-pyridone, 28 g of
K2CO3 and 1.0 g of copper. The mixture was stirred and heated to
160–170°C and kept refluxing for 20 h. When the suspension was
cooled naturally to 110–120°C, about 330 mL of hot water (80–
90°C) was slowly poured in. Reaction mixture was cooled to 20°C
and filtered; residue was dissolved in 300 mL ethyl acetate
ultrasonically. After the filtration, the solid cake was washed with
ethyl acetate (20 mL ꢁ 2) and the combined ethyl acetate phase
was washed with water (50 mL ꢁ 3). The organic layer was
evaporated and to the residues was added 85 mL of hot petroleum
ether and then cooled to 15–20°C. The temperature was kept for
1.5 h and the mixture was filtered to obtain crude product of 6a,
which was recrystallized from 20% ethanol. White solid, 75%
yield. mp: 132.1–133.7°C.
Synthesis of 6-oxo-1-phenyl-1,6-dihydropyridine-3-
carboxylic acid (3)
One gram of methyl 6-oxo-1-phenyl-1,6-dihydropyridine-3-carbox-
ylate (2) was heated at 50°C and dissolved in 32 mL of 10% NaOH,
cooled, and stirred for 3 h. The solution’s pH was modulated by
diluted HCl to 2–3 to form white solid, which was filtered-off and
recrystallized from methanol. White solid, 87% yield. mp: 218.5–
220.0°C.
Synthesis of 6-oxo-N,1-diphenyl-1,6-dihydropyridine-3-
carboxamide (5a)
Synthesis of 5-methyl-1-phenylpyridin-2(1H)-one (6b)
Following the same procedure as reported for the synthesis of 6a
and recrystallized from water. White solid, 73% yield. mp: 108.9–
110.6°C.
4.3 g (0.02 mol) of 3 was added and dissolved to 50 mL SOCl2,
refluxed for 4 h. To the slight yellow oil obtained after complete
removal of SOCl2 was added 30 mL of CH2Cl2, 3.7 g (0.04 mol) of
aniline. After heating at reflux for 4 h, the solvent was removed
and the crude product of 5a was purified upon column
chromatography (EtOAc/petroleum ether). White solid, 69% yield.
HPLC: Rt ¼ 7.48 min, 99.4%. mp: 196.1–197.8°C. IR (KBr, cmꢂ1):
3340, 3051, 1663, 1608, 1597, 1523, 1493, 1483, 1327, 1276, 1128.
1H NMR (400 MHz, CDCl3): d 6.64 (d, 1H, J ¼ 9.6 Hz), 7.14 (s, 1H),
7.30 (m, 3H), 7.48 (m, 4H), 7.55 (d, 2H, J ¼ 8.0 Hz), 7.85 (d, 1H,
J ¼ 9.6 Hz), 8.02 (d, 1H, J ¼ 9.6 Hz), 8.17 (d, 1H, J ¼ 2.4 Hz);
13C NMR (100 MHz, CDCl3): d 114.2, 120.4, 120.5 (2C), 124.7,
126.4, 129.0 (2C), 129.2, 129.5, 137.7, 140.1, 141.3, 162.1, 162.2.
MS m/z: 290 [M]þ.
Synthesis of 1-(4-methoxyphenyl)-5-methylpyridin-2(1H)-
one (6c)
Following the same procedure as reported for the synthesis of 6a
and purified upon column chromatography (EtOAc/petroleum
ether). White solid, 61% yield. HPLC: Rt ¼ 18.42 min, 99.3%. mp:
1
101.8–103.8°C. H NMR (400 MHz, DMSO-d6): d 2.03 (s, 3H), 3.79
(s, 3H), 6.39 (d, 1H, J ¼ 9.2 Hz), 7.02 (d, 2H, J ¼ 8.8 Hz), 7.2 8
(d, 2H, J ¼ 8.8 Hz), 7.35 (dd, 1H, J ¼ 9.2 Hz, 2.4 Hz), 7.38 (s, 1H).
13C NMR (100 MHz, DMSO-d6): d 16.3, 54.4, 113.8, 114.1 (2C), 120.1,
127.9, 133.9 (2C), 136.5, 142.9, 158.6, 160.6. MS m/z: 216 [MþH]þ.
Synthesis of 6-oxo-1-phenyl-N-propyl-1,6-dihydropyridine-
3-carboxamide (5b)
Synthesis of 1-(3-fluorophenyl)-5-((phenylamino)methyl)-
pyridin-2(1H)-one (9)
Following the same procedure as reported for the synthesis of 5a.
White solid, 65% yield. HPLC: Rt ¼ 4.44 min, 98.9%. mp: 121.7–
123.5°C. IR (KBr, cmꢂ1): 3348, 3073, 2970, 2926, 2874, 1682, 1652,
1608, 1589, 1519, 1486, 1360, 1316, 1294, 1153, 1116. 1H NMR
(400 MHz, CDCl3): d 0.92 (m, 3H), 1.52 (m, 2H), 3.25 (m, 2H), 6.51
(m, 2H), 7.33 (m, 2H), 7.47 (m, 3H), 7.72 (m, 1H), 8.09 (s, 1H).
13C NMR (100 MHz, CDCl3): d 11.4, 22.8, 41.7, 113.8, 120.3,
126.4 (2C), 129.0, 129.4 (2C), 137.3, 140.2, 140.7, 162.0, 163.8.
MS m/z: 256 [M]þ.
In a dried flask was placed 0.01 mol of 6a and 150 mL of
anhydrous CCl4. To the solution was added 0.01 mol N-
bromosuccinimide, and 0.5 g of azobisisobutyronitrile. The
mixture was heated at reflux under the irradiation of 450-W
lamp for 4 h. When cooled to room temperature, the mixture was
filtered and the filtrate was evaporated to obtain 7a in yellow oil.
To the oil of 7a was added 60 mL CH3CN followed by 0.02 mol of
aniline. The mixture was stirred air tightly at room temperature
for 2.5 h, and then filtered. The filtrate was evaporated to a
colorful solid mixture and upon column chromatography (EtOAc/
petroleum ether) to obtain 9.
Synthesis of N-(2-methoxyethyl)-6-oxo-1-phenyl-1,6-
dihydropyridine-3-carboxamide (5c)
Slight yellow solid, 25% yield. HPLC: Rt ¼ 9.13 min, 97.3%. mp:
White solid, 60% yield. HPLC: Rt ¼ 6.68 min, 99.6%. mp: 116.8–
124.9–126.5°C; IR (KBr, cmꢂ1): 3059, 1523, 1490, 1449, 1312,
118.4°C. IR (KBr, cmꢂ1): 3325, 3066, 2948, 2836, 1671, 1637, 1527,
1286, 1264, 1235, 1153, 1113. H NMR (400 MHz, CDCl3): d 4.00
1
1
1316, 1260, 1194, 1153, 1120. H NMR (400 MHz, CDCl3): d 3.35
(s, 1H), 4.12 (s, 2H), 6.66 (dd, 3H, J ¼ 9.6 Hz, 12.4 Hz), 6.77 (t, 1H,
J ¼ 7.2 Hz), 7.14 (m, 3H), 7.20 (t, 2H, J ¼ 8.0 Hz), 7.30 (d, 1H,
J ¼ 1.6 Hz), 7.43 (m, 2H). 13C NMR (100 MHz, CDCl3): d 45.0, 113.1
(s, 3H), 3.53 (t, 2H), 3.56 (m, 2H), 6.60 (d, 2H, J ¼ 7.2 Hz), 7.35
(d, 2H, J ¼ 8.8 Hz), 7.47 (m, 3H), 7.72 (dd, 1H, J ¼ 9.6 Hz, 2.4 Hz),
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

662
W. Zhu et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 654–666
(2C), 114.4–114.6 (F-Ar), 115.6–115.8 (F-Ar), 117.2, 118.4, 122.2,
122.3 (F-Ar), 129.4 (2C), 130.5–130.6 (F-Ar), 135.4, 140.4, 142.0–
142.1 (F-Ar), 147.4, 161, 161.8–163.9 (F-Ar). MS m/z: 294 [MþH]þ.
1-(3-Fluorophenyl)-5-((4-methoxyphenylamino)methyl)-
pyridin-2(1H)-one (12)
Hoary solid, 27% yield. HPLC: Rt ¼ 6.79 min, 99.4%. mp: 126.7–
127.9°C. IR (KBr, cmꢂ1): 3311, 3073, 3051, 3007, 2940, 2918, 2829,
1667, 1600, 1587, 1538, 1512, 1486, 1445, 1294, 1242, 1253, 1175,
1150, 1131. 1H NMR (400 MHz, DMSO-d6): d 3.62 (s, 3H), 3.98
(d, 2H, J ¼ 6.0 Hz), 5.65 (t, 1H, J ¼ 6.0 Hz), 6.50 (d, 1H, J ¼ 6.0 Hz),
6.58 (d, 2H, J ¼ 8.8 Hz), 6.70 (d, 2H, J ¼ 8.8 Hz), 7.24 (d, 1H,
J ¼ 8.0 Hz), 7.30 (d, 1H, J ¼ 8.4 Hz), 7.35 (d, 2H, J ¼ 10 Hz), 7.55
(d, 1H, J ¼ 8.4 Hz), 7.58 (d, 2H, J ¼ 10 Hz), 7.65 (s, 1H). 13C NMR
(100 MHz, DMSO-d6): d 43.6, 55.2, 113.6 (2C), 114.2–114.5 (F-Ar),
114.5 (2C), 115.0–115.2 (F-Ar), 117.4, 120.4, 122.9 (F-Ar), 130.7–
130.8 (F-Ar), 136.1, 141.4, 142.1–142.2 (F-Ar), 142.4, 150.9, 160.6,
160.6–163.0 (F-Ar). MS m/z: 324 [M]þ.
1-(3-Fluorophenyl)-6-oxo-1,6-dihydropyridine-3-
carbaldehyde (8a)
Light yellow solid, 7% yield. HPLC: Rt ¼ 5.11 min, 98.3%. mp:
166.6–170.2°C. IR (KBr, cmꢂ1): 3311, 3118, 3022, 1667, 1604, 1586,
1523, 1497, 1482, 1449, 1320, 1286, 1253, 1224, 1150, 1127.
1H NMR (400 MHz, CDCl3): d 6.70–6.73 (d, 1H, J ¼ 9.6 Hz), 7.17–
7.27 (m, 3H), 7.50–7.56 (m, 1H), 7.88–7.91 (dd, 1H, J ¼ 9.6, 2.4 Hz),
7.96–7.97 (d, 1H, J ¼ 2.4 Hz), 9.69 (s, 1H, CHO). 13C NMR
(400 MHz, CDCl3): d 114.4–114.6 (F-Ar), 116.6, 116.8 (F-Ar),
118.5, 122.0, 122.2 (F-Ar), 122.2, 131.0, 131.1 (F-Ar), 136.1,
140.8, 140.9 (F-Ar), 146.7, 161.5, 161.9, 164.0 (F-Ar), 185.9 (CHO).
MS m/z: 217 [M]þ.
1-(3-Fluorophenyl)-5-((m-tolylamino)methyl)pyridin-2(1H)-
one (13)
White solid, 30% yield. HPLC: Rt ¼ 9.64 min, 98.5%. mp: 113.6–
115.2°C. IR (KBr, cmꢂ1): 3348, 3073, 3066, 2918, 2822, 1674, 1600,
1578, 1515, 1482, 1453, 1427, 1320, 1297, 1283, 1257, 1227, 1164,
1150, 1131, 1116. 1H NMR (400 MHz, DMSO-d6): d 2.17 (s, 3H), 4.02
(d, 2H, J ¼ 6.0 Hz), 5.96 (t, 1H, J ¼ 6.0 Hz), 6.37 (d, 1H, J ¼ 7.2 Hz),
6.44 (m, 2H), 6.50 (d, 1H, J ¼ 9.6 Hz), 6.95 (t, 1H, J ¼ 8.0 Hz), 7.24
(d, 1H, J ¼ 8.8 Hz), 7.33 (m, 2H), 7.56 (m, 2H), 7.66 (d, 1H,
J ¼ 2.0 Hz). 13C NMR (100 MHz, DMSO-d6): d 21.4, 42.8, 109.8,
113.3, 114.3–114.5 (F-Ar), 115.1–115.3 (F-Ar), 117.2, 117.4, 120.5,
122.9–123.0 (F-Ar), 128.8, 130.8–130.9 (F-Ar), 136.2, 137.9, 141.5,
142.1–142.2 (F-Ar), 148.3, 160.7, 160.7–163.1 (F-Ar). MS m/z: 308
[M]þ.
3-Bromo-1-(3-fluorophenyl)-5-(methoxymethyl)pyridin-2-
(1H)-one (8b)
In a dried flask was placed 0.1 mol of 6b and 1500 mL of dry CCl4.
To the solution was added 0.1 mol of NBS and 0.5 g of AIBN. The
mixture was heated at reflux with a 450-W lamp for 4 h. Then
0.05 mol NBS was added again and refluxed for one more hour.
When cooled to room temperature, the mixture was filtered, the
filtrate was evaporated to a yellow oil, which was dissolved in
60 mL CH3OH, refluxed for 4 h, cooled below 10°C, standing
overnight. Crystal occurred, 8b was obtained by filtering [33].
White solid, 3% yield. HPLC: Rt ¼ 4.27 min, 98.9%. mp: 82.3–
84.2°C. 1H NMR (400 MHz, CDCl3): d 3.40 (s, 3H), 4.21 (s, 2H), 7.16
(m, 3H), 7.32 (t, 1H), 7.46 (m, 1H), 7.84 (d, 1H, J ¼ 2.4 Hz). 13C NMR
(100 MHz, CDCl3): d 58.3, 70.6, 114.3–114.5 (F-Ar), 115.9–116.1
(F-Ar), 116.5, 117.7, 122.2 (F-Ar), 130.6–130.7 (F-Ar), 135.4, 141.8–
141.9 (F-Ar), 142.4, 158.1, 161.4–163.8 (F-Ar). MS m/z: 312 [M]þ.
1-(3-Fluorophenyl)-5-((o-tolylamino)methyl)pyridin-2(1H)-
one (14)
White solid, 29% yield. HPLC: Rt ¼ 9.74 min, 99.4%. mp: 94.7–
96.1°C. IR (KBr, cmꢂ1): 3370, 3073, 2999, 2962, 2866, 2362, 1682,
1612, 1597, 1575, 1538, 1515, 1482, 1449, 1309, 1283, 1249, 1227,
1142, 1127. 1H NMR (400 MHz, DMSO-d6): d 2.11 (s, 3H), 4.11
(d, 2H, J ¼ 6.0 Hz), 5.43 (t, 1H, J ¼ 6.0 Hz), 6.50 (m, 2H), 6.58
(d, 1H, J ¼ 8.0 Hz), 6.96 (d, 1H, J ¼ 7.2 Hz), 6.98 (d, 1H,
J ¼ 8.0 Hz), 7.24 (d, 1H, J ¼ 8.8 Hz), 7.30 (dd, 1H, J ¼ 2.4 Hz,
8.4 Hz), 7.36 (dt, 1H, J ¼ 2.0 Hz, 10.0 Hz), 7.54 (d, 1H, J ¼ 8.0 Hz),
7.63 (dt, 1H, J ¼ 2.4 Hz, 8.4 Hz), 7.68 (d, 1H, J ¼ 1.6 Hz). 13C NMR
(100 MHz, DMSO-d6): d 17.8, 42.7, 109.8, 114.3–114.5 (F-Ar), 115.0–
115.2 (F-Ar), 116.1, 117.4, 120.4, 122.0, 122.9 (F-Ar), 126.7, 129.5,
130.7–130.8 (F-Ar), 136.2, 141.2, 142.1–142.2 (F-Ar), 145.8, 160.6,
160.6–163.0 (F-Ar). MS m/z: 308 [M]þ.
5-((4-Acetylphenylamino)methyl)-1-(3-fluorophenyl)-
pyridin-2(1H)-one (10)
Tawny solid, 29% yield. HPLC: Rt ¼ 4.47 min, 98.2%. mp: 155.9–
157.4°C. IR (KBr, cmꢂ1): 3318, 1582, 1523, 1490, 1453, 1423, 1316,
1
1294, 1253, 1224, 1146, 1120. H NMR (400 MHz, CDCl3): d 2.51
(s, 3H), 4.20 (s, 2H), 6.62 (d, 2H, J ¼ 8.8 Hz), 6.69 (d, 1H, J ¼ 9.6 Hz),
7.14 (t, 3H, J ¼ 6.0 Hz), 7.30 (s, 1H), 7.43 (dd, 1H, J ¼ 2.4, 9.6 Hz),
7.46 (m, 1H). 13C NMR (100 MHz, CDCl3): d 26.0, 44.1, 111.7 (2C),
114.3–114.5 (F-Ar), 115.7–115.9 (F-Ar), 116.3, 122.2–122.3 (F-Ar),
128.8, 130.6 (2C), 130.7–130.8 (F-Ar), 135.5, 140.1–140.2 (F-Ar),
142.0, 151.4, 161.3, 161.6–163.8 (F-Ar), 196.2. MS m/z: 336 [M]þ.
5-((4-Chlorophenylamino)methyl)-1-(3-fluorophenyl)-
pyridin-2(1H)-one (15)
1-(3-Fluorophenyl)-5-((p-tolylamino)methyl)pyridin-2(1H)-
one (11)
Slight sage green solid, 42% yield. HPLC: Rt ¼ 11.39 min, 99.2%.
mp: 130.9–132.1°C. IR (KBr, cmꢂ1): 3303, 3165, 3083, 3032, 2837,
1669, 1598, 1582, 1536, 1526, 1490, 1460, 1429, 1398, 1316, 1291,
1244, 1224, 1173, 1127. 1H NMR (400 MHz, DMSO-d6): d 4.02
(d, 2H, J ¼ 6.0 Hz), 6.28 (t, 1H, J ¼ 6.0 Hz), 6.51 (d, 1H, J ¼ 9.2 Hz),
6.64 (d, 2H, J ¼ 9.2 Hz), 7.09 (d, 2H, J ¼ 8.8 Hz), 7.25 (dd, 1H,
J ¼ 0.8 Hz, 8.0 Hz), 7.31 (dd, 1H, J ¼ 2.4 Hz, 8.4 Hz), 7.36 (dt, 1H,
J ¼ 2.4 Hz, 10.0 Hz), 7.59 (d, 1H, J ¼ 8.4 Hz), 7.69 (d, 1H,
J ¼ 2.0 Hz). 13C NMR (100 MHz, DMSO-d6): d 42.8, 113.8 (2C),
114.3–114.5 (F-Ar), 115.0–115.2 (F-Ar), 116.7, 119.4, 120.6, 122.9
(F-Ar), 128.6 (2C), 130.7–130.8 (F-Ar), 136.3, 141.3, 142.0–142.1
(F-Ar), 147.2, 160.6, 160.6–163.0 (F-Ar). MS m/z: 328 [M]þ.
White solid, 28% yield. HPLC: Rt ¼ 14.21 min, 99.4%. mp: 135.1–
136.5°C. IR (KBr, cmꢂ1): 3339, 1654, 1603, 1572, 1541, 1424, 1357,
1285, 1244, 1183, 1163. 1H NMR (400 MHz, DMSO): d 2.13 (s, 3H),
4.00 (d, 2H, J ¼ 6.0 Hz), 5.85 (t, 1H, J ¼ 6.0 Hz), 6.49 (d, 1H,
J ¼ 9.6 Hz), 6.54 (d, 2H, J ¼ 6.0 Hz), 6.88 (d, 2H, J ¼ 6.0 Hz), 7.24
(dd, 1H, J ¼ 0.8 Hz, 8.0 Hz), 7.31 (d, 1H, J ¼ 8.8 Hz), 7.35 (d, 1H,
J ¼ 9.6 Hz), 7.54 (dd, 1H, J ¼ 2.4 Hz, 8.8 Hz), 7.58 (d, 1H,
J ¼ 6.0 Hz), 7.65 (s, 1H). 13C NMR (100 MHz, DMSO-d6): d 20.4,
43.4, 113.0 (2C), 114.6–114.8 (F-Ar), 115.3–115.5 (F-Ar), 120.8, 123.2
(F-Ar), 124.8, 129.6 (2C), 131.0–131.1 (F-Ar), 136.5, 141.7, 142.4–
142.5 (F-Ar), 146.3, 160.9, 161.0–163.3 (F-Ar). MS m/z: 308 [M]þ.
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2013, 346, 654–666
1,5-Disubstituted Pyridine-2(1H)-ones as eIF3a Regulators
663
1-Phenyl-5-((o-tolylamino)methyl)pyridin-2(1H)-one (16)
White solid, 38% yield. HPLC: Rt ¼ 8.49 min, 96.8%. mp: 121.9–
122.6°C. IR (KBr, cmꢂ1): 3362, 3036, 2896, 1741, 1663, 1604,
1582, 1527, 1490, 1453, 1382, 1323, 1279, 1260, 1201, 1127.
1H NMR (400 MHz, DMSO-d6): d 2.10 (s, 3H), 4.12 (d, 2H,
J ¼ 6.0 Hz), 5.38 (s, 1H), 6.48 (m, 2H), 6.57 (d, 1H, J ¼ 7.6 Hz),
6.97 (br, 2H), 7.49 (m, 7H). 13C NMR (100 MHz, DMSO-d6): d 17.7,
42.8, 109.8, 116.0, 117.1, 120.4, 122.0, 126.6 (3C), 128.0,
129.1, 129.8 (2C), 136.4, 140.9, 141.0, 145.8, 160.8. MS m/z:
290 [M]þ.
5-((4-Chlorophenylamino)methyl)-1-phenylpyridin-2(1H)-
one (21)
Slight sage green solid, 38% yield. HPLC: Rt ¼ 9.91 min, 99.0%.
mp: 135.6–137.4°C. IR (KBr, cmꢂ1): 3296, 1660, 1597, 1575, 1530,
1515, 1486, 1449, 1397, 1382, 1312, 1279, 1260, 1172, 1124.
1H NMR (400 MHz, DMSO-d6): d 4.02 (d, 2H, J ¼ 6.0 Hz), 6.22
(d, 1H, J ¼ 6.0 Hz), 6.48 (d, 1H, J ¼ 9.6 Hz), 6.63 (m, 2H), 7.08
(m, 2H), 7.37 (m, 2H), 7.44 (m, 1H), 7.52 (dt, 3H, J ¼ 6.4 Hz, 2.0 Hz),
7.63 (d, 1H, J ¼ 2.0 Hz). 13C NMR (100 MHz, DMSO-d6): d 42.8,
113.8 (2C), 116.5, 119.3, 120.5, 126.6 (2C), 128.1, 128.6 (2C), 129.1
(2C), 136.6, 140.9, 141.1, 147.2, 160.8. MS m/z: 310 [M]þ.
1-Phenyl-5-((p-tolylamino)methyl)pyridin-2(1H)-one (17)
White solid, 41% yield. HPLC: Rt ¼ 8.42 min, 98.5%. mp: 132.4–
133.9°C. IR (KBr, cmꢂ1): 3318, 3044, 2999, 2918, 2844, 1671, 1608,
1582, 1519, 1493, 1453, 1430, 1379, 1316, 1275, 1249, 1205, 1124.
1H NMR (400 MHz, DMSO-d6): d 2.13 (s, 3H), 4.00 (d, 2H,
J ¼ 6.4 Hz), 5.86 (t, 1H, J ¼ 6.0 Hz), 6.48 (d, 1H, J ¼ 9.2 Hz),
6.54 (d, 2H, J ¼ 8.4 Hz), 6.88 (d, 2H, J ¼ 8.0 Hz), 7.36 (d, 2H,
J ¼ 7.2 Hz), 7.45 (t, 1H, J ¼ 7.2 Hz), 7.52 (m, 3H), 7.62 (d, 1H,
J ¼ 1.6 Hz). 13C NMR (100 MHz, DMSO-d6): d 20.4, 43.4, 113.0 (2C),
117.5, 120.7 (2C), 124.8, 127.0, 128.4, 129.4 (2C), 129.7 (2C), 136.8,
141.2, 141.6, 146.3, 161.1. MS m/z: 290 [M]þ.
1-Phenyl-5-((m-tolylamino)methyl)pyridin-2(1H)-one (22)
White solid, 29% yield. HPLC: Rt ¼ 8.44 min, 98.9%. mp: 118.5–
119.0°C. IR (KBr, cmꢂ1): 3303, 1680, 1598, 1577, 1536, 1490, 1449,
1403, 1280, 1260, 1163, 1132. 1H NMR (400 MHz, DMSO-d6): d 2.17
(s, 3H), 4.01 (d, 2H, J ¼ 6.0 Hz), 5.96 (t, 1H, J ¼ 6.0 Hz), 6.94 (t, 1H,
J ¼ 8.0 Hz), 7.36 (d, 2H, J ¼ 8.0 Hz), 7.44 (t, 1H, J ¼ 7, 6 Hz), 7.51
(d, 2H, J ¼ 7.6 Hz), 7.54 (d, 1H, J ¼ 2.8 Hz), 7.63 (s, 1H). 13C NMR
(100 MHz, DMSO-d6): d 21.3, 42.8, 109.7, 113.2, 117.0, 117.0, 120.4,
126.6 (2C), 128.0, 128.7, 129.1 (2C), 136.5, 137.8, 140.9, 141.2,
148.3, 160.8. MS m/z: 290 [M]þ.
5-((4-Methoxyphenylamino)methyl)-1-phenylpyridin-2-
5-((4-Chlorophenylamino)methyl)-1-(4-methoxyphenyl)-
(1H)-one (18)
pyridin-2(1H)-one (23)
Hoary solid, 27% yield. HPLC: Rt ¼ 5.90 min, 98.7%. mp: 138.4–
139.7°C. IR (KBr, cmꢂ1): 3296, 2992, 2836, 1667, 1600, 1582, 1530,
1515, 1453, 1401, 1297, 1275, 1249, 1235, 1179. 1H NMR
(400 MHz, DMSO-d6): d 3.62 (s, 3H), 3.98 (s, 2H), 5.65 (br, 1H),
6.48 (d, 1H, J ¼ 9.6 Hz), 6.58 (d, 2H, J ¼ 8.8 Hz), 6.71 (d, 2H,
J ¼ 8.8 Hz), 7.37 (d, 2H, J ¼ 7.6 Hz), 7.44 (t, 1H, J ¼ 7.6 Hz), 7.52
(m, 3H), 7.61 (s, 1H). 13C NMR (100 MHz, DMSO-d6): d 43.6, 55.2,
113.6 (2C), 114.5 (2C), 117.2, 126.6 (2C), 128.0, 129.0 (2C), 136.4,
140.9, 141.2, 142.4, 150.9, 160.8. MS m/z: 306 [M]þ.
White solid, 35% yield. HPLC: Rt ¼ 21.03 min, 99.2%. mp: 151.0–
151.8°C. IR (KBr, cmꢂ1): 3288, 3016, 2955, 2837, 1669, 1598, 1582,
1506, 1485, 1460, 1398, 1311, 1244, 1173, 1111. 1H NMR
(400 MHz, DMSO-d6): d 3.80 (s, 3H), 4.01 (d, 2H, J ¼ 5.6 Hz),
6.27 (t, 1H, J ¼ 5.6 Hz), 6.47 (d, 1H, J ¼ 9.2 Hz), 6.63 (d, 2H,
J ¼ 8.8 Hz), 7.04 (d, 1H, J ¼ 8.8 Hz), 7.09 (d, 1H, J ¼ 8.8 Hz), 29
(d, 1H, J ¼ 8.8 Hz), 7.50 (dd, 1H, J ¼ 2.4 Hz, 9.2 Hz), 7.60 (s, 1H).
13C NMR (100 MHz, DMSO-d6): d 42.8, 55.4, 113.8 (2C), 114.2 (2C),
116.3, 119.3, 120.4, 127.7 (2C), 128.6 (2C), 133.7, 137.0, 141.0,
147.2, 158.7, 161.0. MS m/z: 340 [M]þ.
5-((4-Acetylphenylamino)methyl)-1-phenylpyridin-2(1H)-
one (19)
5-((4-Acetylphenylamino)methyl)-1-(4-methoxyphenyl)-
pyridin-2(1H)-one (24)
Yellow solid, 24% yield. HPLC: Rt ¼ 4.45 min, 98.2%. mp: 166.5–
168.8°C. IR (KBr, cmꢂ1): 3318, 1664, 1588, 1536, 1495, 1454,
1357, 1321, 1270, 1173, 1116. 1H NMR (400 MHz, DMSO-d6):
d 2.39 (s, 3H), 4.14 (d, 2H, J ¼ 5.6 Hz), 6.50 (d, 1H, J ¼ 9.6 Hz),
6.67 (d, 2H, J ¼ 8.8 Hz), 6.96 (t, 1H, J ¼ 6.0 Hz), 7.38 (d, 2H,
J ¼ 8.8 Hz), 7.44 (t, 1H, J ¼ 7.2 Hz), 7.52 (m, 3H), 7.68 (d, 1H,
J ¼ 2.4 Hz), 7.72 (d, 2H, J ¼ 8.8 Hz). 13C NMR (100 MHz,
DMSO-d6): d 26.1, 42.4, 111.4 (2C), 116.6, 120.7 (2C), 125.5,
126.8 (2C), 128.4, 129.3 (2C), 130.6 (2C), 136.9, 140.9, 141.3,
152.6, 161.1, 195.6. MS m/z: 318 [M]þ.
Slight yellow solid, 33% yield. HPLC: Rt ¼ 4.99 min, 98.9%. mp:
119.4–120.7°C. IR (KBr, cmꢂ1): 3272, 2965, 2832, 1664, 1593, 1577,
1531, 1511, 1454, 1357, 1270, 1250, 1173. 1H NMR (400 MHz,
DMSO-d6): d 2.39 (s, 3H), 3.80 (s, 3H), 4.12 (d, 2H, J ¼ 5.6 Hz), 6.48
(d, 1H, J ¼ 9.6 Hz), 6.66 (d, 2H, J ¼ 8.8 Hz), 6.20 (t, 1H, J ¼ 5.6 Hz),
7.05 (d, 2H, J ¼ 8.8 Hz), 7.29 (d, 2H, J ¼ 8.8 Hz), 7.50
(dd, 1H, J ¼ 9.6 Hz, 2.8 Hz), 7.66 (d, 1H, J ¼ 2.0 Hz), 7.72
(d, 2H, J ¼ 8.8 Hz). 13C NMR (100 MHz, DMSO-d6): d 25.8, 42.2,
55.4, 111.2 (2C), 114.1 (2C), 115.9, 120.4, 125.3, 127.7 (2C), 130.3
(2C), 133.6, 137.0, 140.8, 152.3, 158.7, 160.9, 195.0. MS m/z: 349
[MþH]þ.
1-Phenyl-5-((phenylamino)methyl)pyridin-2(1H)-one (20)
White solid, 45% yield. HPLC: Rt ¼ 8.22 min, 98.7%. mp: 138.2–
139.2°C. IR (KBr, cmꢂ1): 3303, 3059, 3022, 2925, 1667, 1600,
1578, 1534, 1519, 1497, 1453, 1279, 1257, 1179, 1157, 1131.
1H NMR (400 MHz, DMSO-d6): d 4.02 (d, 2H, J ¼ 6.0), 6.06 (t, 1H,
J ¼ 6.0), 6.49 (d, 1H, J ¼ 9.2 Hz), 6.54 (t, 1H, J ¼ 7.6 Hz), 6.62
(d, 2H, J ¼ 7.6 Hz), 7.06 (t, 2H, J ¼ 8.0 Hz), 7.37 (d, 2H,
J ¼ 8.0 Hz), 7.44 (m, 2H), 7.53 (m, 3H), 7.64 (d, 1H, J ¼ 2.0 Hz).
13C NMR (100 MHz, DMSO-d6): d 42.8, 112.5 (2C), 116.1, 117.0,
120.4, 126.6 (2C), 128.1, 128.9 (2C), 129.1 (2C), 136.5, 140.9,
141.2, 148.3, 160.8. MS m/z: 276 [M]þ.
1-(4-Methoxyphenyl)-5-((4-methoxyphenylamino)methyl)-
pyridin-2(1H)-one (25)
White solid, 31% yield. HPLC: Rt ¼ 6.76 min, 95.8%. mp: 169.0–
170.3°C. IR (KBr, cmꢂ1): 3288, 3098, 3016, 2945, 2899, 2832, 1669,
1598, 1577, 1511, 1454, 1449, 1393, 1306, 1244, 1178, 1106.
1H NMR (400 MHz, DMSO-d6): d 3.62 (s, 3H), 3.80 (s, 3H), 3.96 (d, 2H,
J ¼ 5.6), 5.64 (t, 1H, J ¼ 5.6 Hz), 6.46 (d, 1H, J ¼ 9.2 Hz), 6.58
(dd, 2H, J ¼ 7.2 Hz, 2.4 Hz), 6.71 (d, 2H, J ¼ 9.2 Hz), 7.04 (dd, 2H,
J ¼ 6.8 Hz, 2.0 Hz), 7.28 (dd, 2H, J ¼ 6.8 Hz, 2.0 Hz), 7.52 (dd, 1H,
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

664
W. Zhu et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 654–666
J ¼ 9.6 Hz, 2.4 Hz), 7.57 (d, 1H, J ¼ 2.0 Hz).13C NMR (100 MHz,
DMSO-d6): d 43.7, 55.2, 55.4, 113.6 (2C), 114.2 (2C), 114.6 (2C),
117.0, 120.2, 127.7 (2C), 133.8, 136.8, 141.1, 142.5, 150.9, 158.7,
161.0. MS m/z: 336 [M]þ.
General procedure for the synthesis of C-5 ethers
derivatives (30–32)
Synthesis of 1-phenyl-5-(m-tolyloxymethyl)pyridin-2(1H)-
one (30)
0.1 mol m-cresol was heated at reflux in 40 mL 10.5% NaOH
for 1 h, then the water was removed to obtain white salt. To
the salt in 30 mL CH3CN was added 7b in 30 mL CH3CN, stirred
and refluxed 2.5 h. The mixture was filtered, the filtrate was
evaporated to dryness and 30 was obtained upon column
chromatography (EtOAc/petroleum ether).
White solid, 47% yield. HPLC: Rt ¼ 24.43 min, 95.5%. mp: 93.4–
95.0°C. IR (KBr, cmꢂ1): 3032, 2909, 2888, 1680, 1623, 1577, 1541,
1485, 1449, 1393, 1367, 1296, 1260, 1152, 1127. 1H NMR
(400 MHz, DMSO-d6): d 2.27 (s, 3H), 4.83 (s, 2H), 6.54 (d, 2H,
J ¼ 9.2 Hz), 6.80 (m, 3H), 7.17 (t, 1H), 7.40 (d, 2H, J ¼ 7.6 Hz), 7.45
(d, 2H, J ¼ 6.8 Hz), 7.52 (t, 2H, J ¼ 7.2 Hz), 7.59 (d, 1H, J ¼ 9.2 Hz),
7.82 (s, 1H). 13C NMR (100 MHz, DMSO-d6): d 21.1, 66.0, 111.8,
114.5, 115.5, 120.6, 121.6, 126.8 (2C), 128.3, 129.1 (2C), 129.3,
138.4, 139.0, 140.7, 141.4, 158.2, 160.9. MS m/z: 291 [M]þ.
1-(4-Methoxyphenyl)-5-((phenylamino)methyl)pyridin-2-
(1H)-one (26)
White solid, 32% yield. HPLC: Rt ¼ 6.91 min, 99.2%. mp: 139.7–
140.5°C. IR (KBr, cmꢂ1): 3375, 3298, 3108, 3011, 2934, 2837, 2356,
2340, 1664, 1603, 1582, 1541, 1506, 1454, 1306, 1244, 1173, 1106.
1H NMR (400 MHz, DMSO-d6): d 3.80 (s, 3H), 4.01 (d, 2H,
J ¼ 6.0 Hz), 6.05 (t, 1H, J ¼ 6.0 Hz), 6.46 (d, 1H, J ¼ 9.2 Hz),
6.54 (t, 1H, J ¼ 7.2 Hz), 6.61 (d, 2H, J ¼ 8.0 Hz), 7.04 (d, 2H,
J ¼ 8.8 Hz), 7.07 (d, 2H, J ¼ 8.0 Hz), 7.28 (d, 2H, J ¼ 8.8 Hz), 7.52
(dd, 1H, J ¼ 2.0 Hz, 9.2 Hz), 7.59 (s, 1H). 13C NMR (100 MHz,
DMSO-d6): d 42.8, 55.4, 112.5 (2C), 114.2 (2C), 116.1, 116.8, 120.3,
127.7 (2C), 128.9 (2C), 133.8, 136.7, 141.0, 148.3, 150.9, 158.7,
161.0. MS m/z: 306 [M]þ.
1-(4-Methoxyphenyl)-5-((p-tolylamino)methyl)pyridin-2-
5-((4-Chlorophenoxy)methyl)-1-phenylpyridin-2(1H)-one
(31)
(1H)-one (27)
White solid, 41% yield. HPLC: Rt ¼ 10.93 min, 99.0%. mp: 155.0–
156.1°C. IR (KBr, cmꢂ1): 3288, 3016, 2945, 2904, 2832, 1675, 1598,
1588, 1516, 1506, 1460, 1403, 1296, 1250, 1178, 1101. 1H NMR
(400 MHz, DMSO-d6): d 2.12 (s, 3H), 3.79 (s, 3H), 3.98 (d, 2H,
J ¼ 6.0 Hz), 5.86 (t, 1H, J ¼ 6.0 Hz), 6.43 (d, 1H, J ¼ 9.2 Hz), 6.52
(d, 2H, J ¼ 8.4 Hz), 6.87 (d, 2H, J ¼ 8.0 Hz), 7.03 (d, 2H, J ¼
8.8 Hz), 7.26 (d, 2H, J ¼ 8.8 Hz), 7.50 (dd, 1H, J ¼ 9.6 Hz, 2.4 Hz),
7.54 (s, 1H). 13C NMR (100 MHz, DMSO-d6): d 20.0, 43.1, 55.4, 112.6
(2C), 114.1 (2C), 116.8, 120.2, 124.4, 127.6 (2C), 129.2 (2C), 133.7,
136.6, 140.9, 146.0, 150.9, 158.6, 160.9. MS m/z: 320 [M]þ.
White solid, 44% yield. HPLC: Rt ¼ 9.44 min, 99.1%. mp: 112.9–
114.7°C. IR (KBr, cmꢂ1): 3129, 1675, 1613, 1588, 1526, 1485, 1388,
1291, 1234, 1173, 1132. 1H NMR (400 MHz, DMSO-d6): d 4.86
(s, 2H), 6.53 (d, 1H, J ¼ 9.6 Hz), 7.04 (d, 2H, J ¼ 8.8 Hz), 7.34 (d, 2H,
J ¼ 8.4 Hz), 7.42 (d, 2H, J ¼ 7.6 Hz), 7.46 (t, 1H), 7.51–7.55 (t, 2H),
7.62 (dd, 1H, J ¼ 9.6 Hz, 2.4 Hz), 7.86 (d, 1H, J ¼ 2.0 Hz). 13C NMR
(100 MHz, DMSO-d6): d 66.5, 114.0, 116.6 (2C), 120.6, 124.5, 126.8
(2C), 128.2, 129.1 (2C), 129.2 (2C), 138.8, 140.6, 141.4, 157.0, 160.8.
MS m/z: 311 [M]þ.
1-(4-Methoxyphenyl)-5-((o-tolylamino)methyl)pyridin-2-
(1H)-one (28)
5-(Phenoxymethyl)-1-phenylpyridin-2(1H)-one (32)
White solid, 42% yield. HPLC: Rt ¼ 6.15 min, 99.6%. mp: 109.6–
110.4°C. IR (KBr, cmꢂ1): 3021, 1685, 1613, 1598, 1526, 1501, 1454,
1398, 1357, 1229, 1168, 1137. 1H NMR (400 MHz, DMSO-d6): d 4.86
(s, 2H), 6.54 (d, 1H, J ¼ 9.6 Hz), 6.95 (t, 1H), 7.01 (d, 2H, J ¼ 7.6 Hz),
7.30 (t, 2H), 7.42 (d, 2H, J ¼ 8.4 Hz), 7.46 (m, 1H), 7.52 (t, 2H), 7.62
(dd, 1H, J ¼ 8.8 Hz, 2.4 Hz), 7.86 (d, 1H, J ¼ 2.0 Hz). 13C NMR
(100 MHz, DMSO-d6): d 66.0, 114.4, 114.8 (2C), 120.6, 120.8,
126.7 (2C), 128.2, 129.1, 129.5, 138.5, 140.6, 141.4, 158.1, 160.8.
MS m/z: 277 [M]þ.
White solid, 36% yield. HPLC: Rt ¼ 6.94 min, 94.8%. mp: 130.6–
132.3°C. IR (KBr, cmꢂ1): 3318, 3032, 3006, 2965, 2929, 2837, 1664,
1603, 1582, 1506, 1454, 1398, 1311, 1244, 1168, 1132. 1H NMR
(400 MHz, DMSO-d6): d 2.10 (s, 3H), 3.79 (s, 3H), 4.10 (d, 2H,
J ¼ 6.0 Hz), 5.43 (t, 1H, J ¼ 6.0 Hz), 6.45 (d, 1H, J ¼ 8.8 Hz), 6.50
(t, 1H, J ¼ 7.6 Hz), 6.57 (d, 1H, J ¼ 7.6 Hz), 6.96 (dd, 2H,
J ¼ 7.8 Hz, 2.8 Hz), 7.03 (dd, 2H, J ¼ 6.8 Hz, 2.4 Hz), 7.27
(dd, 2H, J ¼ 6.8 Hz, 2.4 Hz), 7.55 (dd, 1H, J ¼ 9.2 Hz, 2.4 Hz),
7.59 (s, 1H). 13C NMR (100 MHz, DMSO-d6): d 17.8, 42.8, 55.4, 109.8,
114.1 (2C), 116.0, 116.9, 120.3, 122.0, 126.6, 127.7 (2C), 129.8,
133.8, 136.7, 140.8, 145.8, 158.7, 161.0. MS m/z: 320 [M]þ.
Pharmacophore modeling
The structures of compound 5a and 9 were built by MOE. The
hydrogens and partial charges of the compounds were prelim-
inarily adjusted under forcefield MMFF94x. The compounds were
flexibly aligned and the pharmacophore features were created by
“Pharmacophore Query Editors” modules for the best alignment
with default parameters.
1-(4-Methoxyphenyl)-5-((m-tolylamino)methyl)pyridin-2-
(1H)-one (29)
White solid, 38% yield. HPLC: Rt ¼ 6.71 min, 98.0%. mp: 104.1–
105.3°C. IR (KBr, cmꢂ1): 3318, 3006, 2919, 2832, 1664, 1597, 1582,
1536, 1506, 1460, 1429, 1393, 1250, 1188, 1135, 1116. 1H NMR
(400 MHz, DMSO-d6): d 2.17 (s, 3H), 3.80 (s, 3H), 4.00 (d, 2H,
J ¼ 5.6 Hz), 5.95 (t, 1H, J ¼ 6.0 Hz), 6.36 (d, 1H, J ¼ 7.6 Hz), 6.41
(d, 1H, J ¼ 8.0 Hz), 6.46 (d, 2H, J ¼ 9.6 Hz), 6.94 (t, 1H, J ¼ 8.0 Hz),
7.04 (dd, 2H, J ¼ 9.6 Hz, 2.0 Hz), 7.28 (dd, 2H, J ¼ 6.8 Hz, 2.4 Hz),
7.46 (dd, 2H, J ¼ 9.2 Hz, 2.8 Hz), 7.58 (d, 1H, J ¼ 1.6 Hz). 13C NMR
(400 MHz, DMSO-d6): d 21.3, 42.8, 55.4, 109.7, 113.2, 114.2 (2C),
116.8, 117.0, 120.3, 127.7 (2C), 128.7, 133.8, 136.8, 137.8, 141.0,
148.3, 158.7, 161.0. MS m/z: 320 [M]þ.
Biological assays
Materials
The cisplatin and L-mimosine were purchased from Sigma.
Antibodies against b-actin were from Abcam (Cambridge, MA,
USA). Cell culture media and reagents were obtained from
Hyclone. All other reagents were of molecular biology grade from
Sigma (St Louis, MO, USA) or Invitrogen (Carlsbad, CA, USA).
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2013, 346, 654–666
1,5-Disubstituted Pyridine-2(1H)-ones as eIF3a Regulators
665
Table 4. Primers and annealing temperature used for real-time
PCR.
in a Stratagene Mx3000p Multiplex Quantitative PCR System
(Stratagene, La Jolla, CA, USA) using SYBR GreenER Qpcr
Supermix Universal Kit (Invitrogen) according to the manu-
script’s instructions. The complete sequences of the oligonucleo-
tide primers, annealing temperature are given in Table 4. The Ct
value was defined as the PCR cycle number at which the reporter
fluorescence crosses the threshold. The Ct value of each product
was determined and normalized against that of internal control,
b-actin.
Annealing
Gene
Primer sequence
temperature
Human F: 50-TGGCACCCAGCACAATGAA-30
60°C
b-actin R: 50-CTAAGTCATAGTCCGCCTAGAAGCA-30
eIF3a
F: 50-TCAAGTCGCCGGGACGATA-30
R: 50-CCTGTCATCAGCACGTCTCCA-30
60°C
This work was supported by National Natural Science Foundation of
China (Grant No. 20972194). We also thank Dr. Tao Huang
(Department of Molecular Modeling, Drug Design & Cheminformatics,
CloudScientific Technology Co. Ltd.) for flexible assignment and
pharmacophore building with MOE system.
Cell viability assay
Cell proliferation assay was determined using CellTiter 961
AQueous One Solution Cell Proliferation Assay Kit, according to
the manufacturer’s instructions (Promega, Madison, WI, USA).
Briefly, A549 or NIH3T3 cells were seeded in 96-well plates and
allowed to grow for 24 h followed by incubation with compounds
for 24 h. Culture medium was then removed. Pipet 20 mL of MTS
solution into each well of the 96-well assay plate containing the
samples in 100 mL of culture medium. Incubate the plate in a
humidified, 5% CO2 atmosphere for 2 h at 37°C. Record the
absorbance at 490 nm using a Multiskan Ascent 354 microplate
reader (Thermo Labsystems, Helsinki, Finland). The absorption
value was determined by Ascent SoftwareTM (Thermo Labsystems,
Helsinki, Finland) and IC50 values were obtained from the dose–
response curves using GraphPad PrismTM 5.0 software (GraphPad
Software, Inc., La Jolla, CA, USA).
The authors have declared no conflict of interest.
References
[1] R. Siegel, E. Ward, O. Brawley, A. Jemal, CA Cancer J. Clin. 2011,
61, 212–236.
[2] M. B. Sporn, K. T. Liby, Nat. Clin. Pract. Oncol. 2005, 2, 518–
525.
[3] Z. Dong, J. T. Zhang, Crit. Rev. Oncol. Hematol. 2006, 59, 169–
180.
[4] J. Y. Yin, Z. Dong, Z. Q. Liu, J. T. Zhang, Biosci. Rep. 2011, 31,
1–15.
Immunoblot analysis assay
A549 cells (1 ꢁ 105/well) were treated with compound 5a, 9,
22, 30, L-mimosine, PFD or AKF-PD for 8 h. After protein sample
preparation, expression of eIF3a and b-actin were examined by
Western blot analysis as previously described. Briefly, cells were
lysed in radio immuno precipitation assay (RIPA) lysis buffer
[50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium
deoxycholate, 0.1% SDS, 1% NP-40, 1 mM sodium orthovanadate,
1 mM EDTA, 1 mM sodium fluoride, 100 mg/mL phenylmethyl-
sulfonylfluoride (PMSF), 100 mg/mL dithiothreitol (DTT)] at 4°C
for 30 min. The lysates were clarified by centrifugation at
10,000 g for 10 min at 4°C; protein concentration of the lysate
samples was determined using Bio-Rad Protein Assay kit. For
Western blot analysis, protein samples were separated by SDS–
PAGE and transferred to polyvinylidene difluoride membranes
(Millipore). The membranes were then blocked with 5% skimmed
milk and incubated with primary antibodies (anti-eIF3a and anti-
b-actin) overnight followed by washing. The reaction was detected
by IRDye1 800CW, conjugated goat (polyclonal) anti-mouse or
rabbit antibody (LI-COR Biosciences, Lincoln, NE, USA) and
visualized by Odyssey Scanner (LI-COR Biosciences).
[5] Z. Dong, J. T. Zhang, Mol. Biol. Cell 2003, 14, 3942–3951.
[6] Z. Dong, L. H. Liu, B. Han, R. Pincheira, J. T. Zhang, Oncogene
2004, 23, 3790–3801.
[7] Z. Dong, Z. Liu, P. Cui, R. Pincheira, Y. Yang, J. Liu, J. T. Zhang,
Exp. Cell Res. 2009, 315, 1889–1894.
[8] Z. Liu, Z. Dong, Z. Yang, Q. Chen, Y. Pan, Y. Yang, P. Cui,
X. Zhang, J. T. Zhang, Differentiation 2007, 75, 652–661.
[9] L. Zhang, X. Pan, J. W. Hershey, J. Biol. Chem. 2007, 282, 5790–
5800.
[10] F. Bachmann, R. Banziger, M. M. Burger, Cancer Res. 1997, 57,
988–994.
[11] A. Dellas, J. Torhorst, F. Bachmann, R. Banziger,
E. Schultheiss, M. M. Burger, Cancer 1998, 83, 1376–1383.
[12] G. Chen, M. M. Burger, Int. J. Cancer 2004, 112, 393–398.
[13] G. Chen, M. M. Burger, Int. J. Cancer 1999, 84, 95–100.
[14] R. Pincheira, Q. Chen, J. T. Zhang, Br. J. Cancer 2001, 84, 1520–
1527.
[15] J. Y. Yin, J. Shen, Z. Z. Dong, Q. Huang, M. Z. Zhong, D. Y. Feng,
H. H. Zhou, J. T. Zhang, Z. Q. Liu, Clin. Cancer Res. 2011, 17,
4600–4609.
Real-time reverse transcriptase PCR
A549 cells (1 ꢁ 105/well) were treated with 22, 30, L-mimosine,
PFD for 8 h. mRNA levels of eIF3a were analyzed by real-time
RT x2010;PCR as described previously using specific primers and
normalized to that of b-actin. Briefly, the cells were harvested
and total RNA was isolated using Trizol reagent (Invitrogen).
One microgram of total RNAs were reverse transcribed using
Reverse Transcription System Kit (Promega) according to the
manuscript’s instructions. The real-time PCR were carried out
[16] M. Lalande, Exp. Cell Res. 1990, 186, 332–339.
[17] J. Hynes, Jr., K. Leftheri, Curr. Top. Med. Chem. 2005, 5, 967–985.
[18] Y. A. Ammar, M. M. Ismail, H. M. El-Sehrawi, E. Noaman, A. H.
Bayomi, T. Z. Shawer, Arch. Pharm. (Weinheim) 2006, 339, 429–
436.
[19] L. Richeldi, U. Yasothan, P. Kirkpatrick, Nat. Rev. Drug Discov.
2011, 10, 489–490.
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

666
W. Zhu et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 654–666
[20] N. Ueki, M. Nakazato, T. Ohkawa, T. Ikeda, Y. Amuro, T. Hada,
[28] Y. K. Ko, S. C. Lee, D. W. Koo, M. Jung, D.-W. Kim, Bull. Korean
Chem. Soc. 2001, 22, 234–236.
K. Higashino, Biochim. Biophys. Acta 1992, 1137, 189–196.
[21] J. Park, D. S. Kim, T. S. Shim, C. M. Lim, Y. Koh, S. D. Lee,
W. S. Kim, W. D. Kim, J. S. Lee, K. S. Song, Eur. Respir. J. 2001, 17,
1216–1219.
[29] A. Klapars, X. Huang, S. L. Buchwald, J. Am. Chem. Soc. 2002,
124, 7421–7428.
[30] M. E. Jung, L. J. Street, Y. Usui, J. Am. Chem. Soc. 1986, 108,
6810–6811.
[22] M. C. Aubry, J. L. Myers, W. W. Douglas, H. D. Tazelaar,
T. L. Washington Stephens, T. E. Hartman, C. Deschamps,
V. S. Pankratz, Mayo Clin. Proc. 2002, 77, 763–770.
[31] D. L. Comins, R. E. Lyle, J. Org. Chem. 1976, 41, 2065–2066.
[32] C. J. Morrow, H. Rapoport, J. Org. Chem. 1974, 39, 2116–2118.
[33] D. Bankston, Synthesis 2004, 2004, 283–289.
[23] Z. Z. Peng, G. Y. Hu, H. Shen, L. Wang, W. B. Ning, Y. Y. Xie,
N. S. Wang, B. X. Li, Y. T. Tang, L. J. Tao, Nephrology (Carlton)
2009, 14, 565–572.
[34] S. P. Blagden, A. E. Willis, Nat. Rev. Clin. Oncol. 2011, 8, 280–291.
[35] Y. Ma, Q. Feng, D. Sekula, J. A. Diehl, S. J. Freemantle,
[24] Y. Tang, B. Li, N. Wang, Y. Xie, L. Wang, Q. Yuan, F. Zhang, J. Qin,
Z. Peng, W. Ning, G. Hu, J. Li, L. Tao, Int. Immunopharmacol. 2010,
10, 580–583.
E. Dmitrovsky, Cancer Res. 2005, 65, 6476–6483.
[36] R. M. Tujebajeva, D. M. Graifer, N. B. Matasova, O. S.
Fedorova, V. B. Odintsov, N. A. Ajtkhozhina, G. G. Karpova,
Biochim. Biophys. Acta 1992, 1129, 177–182.
[25] I. Burghardt, F. Tritschler, C. A. Opitz, B. Frank, M. Weller,
W. Wick, Biochem. Biophys. Res. Commun. 2007, 354, 542–547.
[37] C. M. Crews, J. L. Collins, W. S. Lane, M. L. Snapper, S. L.
[26] S. Krishnan, J. M. Goble, L. A. Frederick, C. D. James, J.
H. Uhm, S. H. Kaufmann, D. Babovic-Vuksanovic, J. Appl. Res.
2007, 7, 58–68.
Schreiber, J. Biol. Chem. 1994, 269, 15411–15414.
[38] V. Plemenkov, R. Ashirov, O. Lodochnikova, I. Litvinov,
R. Zagidullin, Russ. J. Org. Chem. 2006, 42, 969–972.
[27] T. Kaminski, G. Kirsch, J. Heterocycl. Chem. 2008, 45, 229–234.
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com