Design and synthesis of azaisoflavone analogs as phytoestrogen mimetics

Article abstract of DOI:10.1016/j.ejmech.2014.07.030

A series of azaisoflavone analogs were designed and synthesized and their transactivation activities and binding affinities for ERα and ERβ were investigated. Among these compounds, 2b and 3a were the most potent with 6.5 and 1.1 μM of EC₅₀, respectively. Molecular modeling study showed putative binding modes of the compound 3a in the active site of ERα and ERβ, which were similar with that of genistein and provided insight of the effect of N-alkyl substitution of azaisoflavones on ERβ activity. Also, a biphasic effect of azaisoflavone analogs on MCF-7 cell growth depending on their concentrations was investigated.

Full text of DOI:10.1016/j.ejmech.2014.07.030

European Journal of Medicinal Chemistry 85 (2014) 107e118
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design and synthesis of azaisoflavone analogs as phytoestrogen
mimetics
,
,
Hyo Jin Gim a 1, Hua Li a 1, So Ra Jung a, Yong Joo Park b, Jae-Ha Ryu a, Kyu Hyuck Chung b,
,
*
Raok Jeon a
a Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women's University, 52 Hyochangwon-Gil, Yongsan-Ku,
Seoul 140-742, Republic of Korea
b School of Pharmacy, Sungkyunkwan University, 2066, Seobu-ro, Jangan-gu, Suwon-si, Gyeonggi-do, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of azaisoflavone analogs were designed and synthesized and their transactivation activities and
Received 28 September 2013
Received in revised form
30 June 2014
Accepted 2 July 2014
Available online 15 July 2014
binding affinities for ER
potent with 6.5 and 1.1
modes of the compound 3a in the active site of ER
and provided insight of the effect of N-alkyl substitution of azaisoflavones on ER
a
and ER
M of EC50, respectively. Molecular modeling study showed putative binding
and ER , which were similar with that of genistein
activity. Also, a biphasic
b were investigated. Among these compounds, 2b and 3a were the most
m
a
b
b
effect of azaisoflavone analogs on MCF-7 cell growth depending on their concentrations was
investigated.
Keywords:
© 2014 Elsevier Masson SAS. All rights reserved.
Estrogen receptor
ER agonist
Azaisoflavones
Proliferation
1. Introduction
[4]. However, Women's Health Initiative (WHI) study showed
beneficial effects of estrogen could increase risk of certain cancers
Estrogen receptors (ERs), a member of the steroid nuclear hor-
mone receptor family, are ligand-activated transcription factor
which play a crucial role in the development, maintenance, and
function of the female reproductive system as well as other tissues
such as colon, prostate, bone, cardiovascular system and central
such as breast [5]. Selective ER modulators (SERMs) are therapeutic
agents for treating osteoporosis and breast cancer by modulating
both ERs [6]. Tamoxifen, the first clinically relevant SERM, antag-
onizes the ER in breast tissue via active metabolite, hydrox-
ytamoxifen and inhibits estrogenic effects such as proliferation of
cancer cell [7].
nervous systems [1]. There are two isoforms, ER
expressed in nearly all tissues whereas ER is mainly expressed in
the ovaries, uterus and oviduct of the female reproductive tract but
not in breast tissue [2]. ER and ER have different biological
a and ERb. ERa is
b
Recently, a number of researches have been demonstrated ER
is more active in driving breast cancer cell proliferation than ER
a
b
a
b
[8]. Meanwhile, it has been demonstrated that ERb has anti-
functions including a tissue-specific regulation as well as their
different gene expression patterns [3].
proliferative effect in breast cancer cells [9] and preventive effects
for prostate adenocarcinoma [10] and also modulates the immune
The hormone 17
b-estradiol (E2) binds to ligand binding domain
response [11]. Therefore, discovery of selective ERb agonists may
(LBD) of ERs, enters the nucleus of the cell, and regulates the target
gene expression related to proliferation and differentiation of the
cell by recruiting a various co-regulators. A deficiency of estrogen is
associated with hot flashes, sweating, heart attacks and bone loss
and hormone replacement therapy (HRT) is a medical treatment for
older postmenopausal women in order to reduce these symptoms
provide high potential to develop therapeutic agents for the various
diseases such as breast cancer, prostate cancer, endometriosis, and
inflammation. Recently, non-steroidal selective ER
b agonists
SERBA-1, ERB-041 and WAY-202196 have been reported their anti-
inflammatory activities and estrogenic activities (Fig. 1).
Phytoesterogens, naturally occurring estrogenic plant com-
pounds, have been considered as an alternative method of meno-
pause treatment. Among them, isoflavones are the well-known ER
agonistic phytoestrogens and exhibit a number of biological prop-
erties including the prevention of cancers [12], coronary heart
diseases [13], and osteoporosis [14]. Genistein is one of the potent
* Corresponding author.
E-mail address: rjeon@sookmyung.ac.kr (R. Jeon).
These authors contributed equally to this work.
1
http://dx.doi.org/10.1016/j.ejmech.2014.07.030
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

108
H.J. Gim et al. / European Journal of Medicinal Chemistry 85 (2014) 107e118
Fig. 1. Structures of ER modulators.
phytoestrogenic isoflavone, reveals 10e40 fold greater affinity for
ER than for ER [15], and inhibits cell proliferation in various
aminochalcones 5ae5c which were followed by N-acetylation to
afford 2’-acetamidochalcones 6ae6c. Rearrangement of 2’-acet-
amidochalcone 6ae6c in the presence of thallium nitrate and tri-
b
a
breast cancer cell lines including MCF-7, T47D, BT20, MD-MBA-231,
and SKBR3 [16]. The biphasic effect of genistein on cell growth has
been reported in several studies in which genistein increased the
MCF-7 cell growth at low concentrations but inhibited at higher
concentrations [16a,17].
methyl orthoformate gave
b-ketoacetal 7ae7c which were
subsequently cyclized under acidic condition to yield quinolones
8ae10a. The resulting quinolones 8ae10a were alkylated with
various alkyl halides in the presence of base to afford N-alkylated
quinolones 8be10d. The final hydroxyquinolones 1ae3d were
obtained by deprotection of methoxyquinolones 8ae10d under HBr
and acetic acid conditions, respectively.
ER
ligand biding domains (LBDs) are nearly conserved differing by only
two amino acids; Leu384 and Met421 in ER are replaced by
Met336 and Ile373 in ER , respectively (Fig. 2) [18]. The hydroxy
groups in both ends of E2 and genistein essentially interact with
key residues such as Glu353, Arg394 and His524 of ER by
hydrogen bonding within 2e3 Å (Glu305, Arg346 and His475 of
ER ). Their different residues Leu384/Met336 are positioned above
a and ERb are similar in sequence with 58% identity and both
a
b
3. Results and discussion
a
3.1. ER transactivation activity and binding affinities
b
the C-ring of genistein in the distance of 4e5 Å, and Met421/Ile373
are located below the A-ring near C-5 hydroxy group of genistein.
These two pairs of amino acids within the LBD of ERa and ERb make
slight differences and mainly contribute to ligand selectivity [9c,19]
(Fig. 3).
Regarding the previous findings, we became interested in
modifying C-ring of isoflavone to identify a novel selective ER
agonist. In this point of view, a previously reported compound
azaisoflavone became the object of our attention. In fact, azaiso-
flavones were originally derived from isoflavone scaffold and
designed as an iNOS inhibitor since inhibitory activity of several
naturally occurring isoflavone for NO production was reported [20].
We supposed that the azaisoflavone analogs could function as
phytoestrogens. Furthermore, N-substitution of azaisoflavone ring
ER agonist activities of the prepared compounds were evaluated
by in vitro transient transactivation assay and described in Table 1.
The efficacy of the tested compounds was compared to the refer-
ence compounds E2 and genistein.
At first, an importance of phenolic OH on A and B-ring of azai-
soflavones was investigated. Mono-hydroxy analogs 1aed, di-
hydroxy of daidzein analogs 2aec and tri-hydroxy of genistein
analogs 3aec were compared with the corresponding methoxy
compounds 8a, 9aec and 10aec, respectively. All methoxy com-
pounds showed lower activities than the respective hydroxy com-
pounds, and mono-hydroxy analog 1a was less active than daidzein
analog 2a and genistein analog 3a as expected.
When the oxygen of genistein was simply replaced with nitro-
gen as depicted in compound 3a, both ER activities were dropped,
may give selectivity for ligand binding domains of ER
a and ERb.
whereas the selectivity for ER
analog 3a showed the highest ER
over ER
b
b
was increased. Genistein type
activity with 2.9 fold selectivity
Herein we report synthesis of azaisoflavone analogs and their ERa/b
transactivation activities. Structure activity relationship study was
focused on the influence of phenolic OH of A and B-ring and N-
substitution of C-ring of azaisoflavones. The effect of azaisoflevones
on MCF-7 cell proliferation was also evaluated.
a.
Effect of N-alkyl substituent of azaisoflavone on ER activity was
evaluated. In the case of mono-hydroxy compounds 1aed, only N-
propylated compound 1d showed increased ERa and b activity than
the parent compound 1a while N-methyl or ethyl substitution
vanished activity. In the daidzein analogs 2aef, introduction of
2. Chemistry
alkyl substituent at nitrogen of C-ring enhanced ER
selectivity. Especially, N-methyl substituted compound 2b showed
promising ER activity with the highest ER selectivity at the ratio
of 6.4:1. When the size of substituent was increased, the ER ac-
tivity was decreased. In the genistein analogs 3aec, N-alkyl sub-
stitution lowered ER activity and selectivity. In contrast, N-alkyl
b activity and
Synthetic routes for the preparation of azaisoflavones 1a-3d are
outlined in Scheme 1. Aniline or methoxyaniline was acylated with
acetonitrile in the presence of boron trichloride and aluminum
trichloride by FriedeleCraft’s reaction. Condensation of the result-
ing ketones 4ae4c with p-anisaldehyde resulted in 2’-
b
b
b
b

H.J. Gim et al. / European Journal of Medicinal Chemistry 85 (2014) 107e118
109
Fig. 2. Differences between the crystal structures of ER
a
and ER
b complexed with genistein. Two different colors represented complexed structures of genistein with LBDs of ERa
(megenta) and ER
article.)
b (green) (PDB ID: ERa-1X7R and ERb-1X7J). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this
substitutions have relatively weak influence in the ER
a
activity of
3.2. Docking analysis
both daidzein and genistein analogs. Among these compounds, N-
methylated daidzein analog 2b and non-alkylated genistein analog
To compare binding modes between known isoflavone and
3a were the most potent ER
EC50 value, respectively.
The binding affinities of azaisoflavones for ER
determined by a radiometric competitive binding assay and
b
agonists showing 6.5
m
M and 1.1
m
M of
azaisoflavone and to explain the effect of N-alkyl group on the ER
selectivity, A docking analysis of azaisoflavones 2aef and 3aed
were carried out using Surflex Dock interfaced with SYBYL-X
version 2.0. Crystal structures of hER and hER in complex with
genistein (PDB code: ER
a/
b
a
and ERb
were
a
b
b
depicted in Table 1. Most of the azaisoflavone analogs revealed rare
a
ꢀ 1X7R, ER ꢀ 1X7J) were employed for
ER
istein analogs 3aec showed remarkable ER
with relatively lower ER binding affinity. Introduction and
lengthening of N-alkyl substituents of genistein analogs increased
both ER and binding affinities although selectivity was reversely
lowered. Thus, non-substituted genistein analog 3a showed the
most selective binding affinity for ER . IC50 values of genistein
analogs were determined; 3a (74.5 M for ER , 4.2 M for ER ), 3b
(35.6 M for ER , 0.72 M for ER ), 3c (18.4 M for ER , 0.10 M for
ER ), and 3d (16.6 M for ER , 0.15 M for ER ). Di-hydroxy of
daidzein analogs showed moderate binding affinity for ER with no
affinity for ER , and N-alkyl substitution slightly enhanced ER
a
binding affinity except tri-hydroxy of genistein analogs. Gen-
the docking study and internal default parameters of Surflex Dock
were used for all the variables.
b
binding affinity along
a
We investigated closely binding modes of azaisoflavones at the
active site of ER
took note of two points. First, the distance between centroid C-ring
of genistein and Met336 in ER is closer than that of genistein and
Leu384 in ER . It is known to causes relatively stronger Met-
aromatic interaction in ER
so it is consisted with the observed selectivity of genistein for ER
over ER
repulsive to the Met421 sulfur atom in ER
[22]. A number of ER selective agonists were reported [19]. Among
them, benzoxazole analog ERB-041 provides good example for the
role of Met421 in ER and Ile373 in ER to distinguish ER from
ER . ERB-041 has 200 folds selectivity for ER and was proposed
that the vinyl group of benzoxazole ring made steric clash with
Met421 in ER , which is relatively longer than Ile373 in ER . In
contrast, this vinyl substituent appropriately positioned near Ile373
a/b in comparison with that of genistein. Herein we
a
b
b
b
a
m
a
m
b
b
than Leu-aromatic interaction in ER
a
,
m
a
m
b
m
a
m
b
b
m
a
m
b
a
. Also, it has been reported genistein C-5 hydroxy group is
, but not to Ile373 in ERb
b
a
a
b
b
binding affinity.
Interestingly, these results of ER
b
binding affinity were not
a
b
a
correlated with transactivation activity mentioned above. There is a
quite discrepancy between transactivation activity and binding
affinity and it may be ascribed to the causes that the recep-
toreligand complex is only present in the binding assays, whereas
various interactions with the many coregulators are present in the
cellular transcription assays [21]. These interactions with cor-
egulators can modulate the transcriptional activities of ligand.
b
b
a
b
in ER
[23].
b, and even forming an additional hydrophobic interaction
Azaisoflavone 3a showed a similar binding mode with genistein
whose hydroxyl groups of A and B-ring interact with the critical
residues including His, Glu, Arg, and water molecule within
appropriate distance in the active site of both ERs as shown in Fig. 4.
Regarding the previous report, we also assume favorable Met336-
aromatic interaction of 3a in ER
a closer distance of 3.744Å from Sd of Met336 in ER
hand, 3.886 Å from Cd of Leu384 in ER . Additional H-bonding
interaction was observed between C-7 hydroxy group of compound
3a and Gly472 in ER
Fig. 5 exhibited binding modes of N-alkylated daidzein analogs
at the active site of ER and . N-alkylated genistein analogs
showed relatively low docking scores compared with N-alkyalted
b
. In fact, C4 atom of 3a located at
b
, on the other
a
2
b.
a
b
Fig. 3. Representative structure of azaisoflavone analogs.

110
H.J. Gim et al. / European Journal of Medicinal Chemistry 85 (2014) 107e118
Scheme 1. Synthesis of azaisoflavone analogs. Reaction conditions: a) CH3CN, BCl3, AlCl3, dichloroethane, 80 ꢁC, 20 h; b) p-anisaldehyde, NaOH, EtOH, rt, 10 h; c) Ac2O, pyridine, rt,
3 h; d) thallium(III)nitrate, HC(OCH3)3, rt, 4 h; e) HCl, EtOH, reflux, 2 h; f) R3-X, NaH, DMF, rt, 3e12 h; g) HBr, AcOH, 120 ꢁC, 3 days.
daidzein analogs in accordance with transactivation activities. In
the case of ER , when alkyl substituents were introduced at the
nitrogen of daidzein analogs, they occupied an empty space near
Leu384 which might be more acceptable than longer Met336 for
these alkyl groups.
MCF-7 (ATCC HTB-22) and CV-1 (ATCC CCL-7) cells were main-
tained at 37 ꢁC in a humidified atmosphere of 5% CO2 in DMEM
supplemented with 10% fetal bovine serum (FBS; GE Healthcare).
The cells were cultured in phenol red-free DMEM containing 5%
charcoal-dextran stripped FBS (CD-FBS) for 3 days to eliminate any
estrogenic source before treatment of samples.
a
On the other hand, binding modes in ER
b LBD were quite
different with those in ER . The daidzein analogs 2bed were flip-
a
Effect of azaisoflavones on cell growth was not correspondingly
related to the ER activity. Among tested compounds, azaiso-
flavones 1d, 2c, 2d, 3a, 3b and 3c showed biphasic effects with
stimulation of cell growth at low concentrations and inhibition at
high concentrations as genistein did (Fig. 6). Meanwhile, com-
pounds 2e, 2f and 3d were only observed inhibitory activity at the
given doses.
ped over retaining their key H-bonding interactions with Glu305,
Arg346, His475, and water molecule. Methyl, ethyl and propyl
groups were positioned near Ile373 instead of Met336 as shown in
Fig. 5. The docking scores of 2bed decreased with lengthened alkyl
substituents showing Me (6.8615), Et (6.8290), and Pr (6.6300).
Methyl group of azaisoflavone 2b seemed to be the most suitable
substituent for the cavity near Ile373. Taken together, docking
result gave an account of the transactivation activity and selectivity
of the discussed compounds.
4. Conclusion
In summary, we have designed and synthesized a series of
azaisoflavones based on phytoestrogenic isoflavone scaffold and
evaluated their estrogenic activity and binding affinities in the LBD
3.3. Effects of MCF-7 cell growth
Some phytoestrogens are known to modulate survival and death
of breast cancer cell in an ER-dependent or -independent manner
[24]. Interestingly, genistein and daidzein have been found to
stimulate breast cancer cell growth in vitro and in vivo studies at
of ERa and b. We found that several azaisoflavone analogs displayed
promising estrogenic activity and subtype selectivity. Also, most of
the azaisoflavone analogs were found to modulate the proliferation
of MCF-7 breast cancer cells in a dose dependent manner. SAR
study indicated that presence of hydroxy group is critical for the ER
activity while the potency and selectivity of the compounds were
highly influenced within a very narrow structural change. Inter-
estingly, introduction of optimal length of N-alkyl substituent in the
daidzein anlalogs increased ER activity, meanwhile genistein ana-
low concentrations (0.1e10
mM) while they inhibit the cell prolif-
eration at high concentrations (20e100
mM) [17,25]. However, the
mechanism of the biphasic action of genistein on the growth of
breast cancer cells is not fully understood yet.
The proliferative and/or anti-proliferative activity of mostly ER
active azaisoflavone analogs on MCF-7 cell was also evaluated.
logs lowered ERb activity with N-substitution. Molecular modeling

H.J. Gim et al. / European Journal of Medicinal Chemistry 85 (2014) 107e118
111
Table 1
In vitro functional transactivation activities and competitive binding affinities of azaisoflavones on human ER
a
/b
.
Compds
R1
R2
R3
R4
ER transactivation activity
ER binding affinity
% max (20
M)b
ER ERb
% max (20
ER
m
M)a
b
/a
ratio
m
b/a ratio
a
ER
b
a
Genistein
1a
1b
1c
1d
2a
2b
2c
2d
2e
2f
3a
3b
3c
3d
8a
9a
OH
H
H
H
H
H
H
H
H
OH
H
H
H
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OMe
OMe
OMe
OMe
OMe
OMe
OMe
e
172.8
NAc
NA
232.8
19.6
NA
1.3
93.4
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
6.9
35.6
57.0
78.5
NA
NA
NA
NA
NA
NA
NA
100
95.1
10.7
18.4
18.3
23.0
24.1
40.6
40.4
37.2
38.5
39.2
82.9
93.0
92.2
106.6
21.3
42.5
NA
1.0
H
Me
Et
Pr
H
Me
Et
Pr
i-pentyl
Bn
H
Me
Et
Pr
H
H
Me
Et
H
Me
Et
NA
NA
79.0
24.5
16.1
33.1
42.3
13.0
31.7
46.2
50.4
60.5
56.9
NA
NA
NA
NA
10.1
NA
42.4
31.9
102.1
95.4
67.0
44.8
33.1
132.1
63.2
47.2
16.7
10.1
16.2
10.3
NA
0.5
1.3
6.4
2.9
1.6
3.4
1.0
2.9
1.3
0.8
0.3
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
OMe
OMe
OMe
OMe
OMe
OMe
H
H
OH
OH
OH
OH
H
H
H
H
12.0
2.6
1.6
1.4
9b
9c
10a
10b
10c
E2d
12.8
47.2
NA
13.6
100
OMe
OMe
OMe
10.9
NA
13.2
100
NA
100
1.0
1.0
a
Fold activation relative to maximum activation obtained with E2 (20 nM) for ER
a
and b. The compounds were tested in at least three separate experiments.
b
Binding affinities were determined by a competitive binding assay using [3H]-E2. Fold affinities relative to maximum activation obtained with E2 (1 nM) for ER
a
and
b
. The
compounds were tested in at least three separate experiments.
c
NA means not active, which is for compounds producing transactivation activity or binding to ER lower lower than 10% at 20 mM.
The concentration of E2 for transactivation activity is 20 nM and that of E2 for the competitive binding affinity assay is 1 nM.
d
also demonstrated that, despite the high homology, ligands can be
made to discriminate between the two ER subtypes. These findings
suggest that further elaboration of the azaisoflavone scaffold as
well as more sophisticate tuning with the appropriate substitution
will be the next strategy to explore those applications. We antici-
pate that these azaisoflavones might work as a useful pharmaco-
logical tool for development of therapeutic agents to treat ER
mediated disease.
Fig. 4. A comparison of putative binding modes of azaisoflavone 3a with those of genistein in the active sites of hERa and hERb.

112
H.J. Gim et al. / European Journal of Medicinal Chemistry 85 (2014) 107e118
Fig. 5. Putative binding modes of N-alkylated daidzein analogs 2bed when docked into the X-ray crystal structure of ERa and ERb.
5. Experimental
and/or multiple resonances), number of protons and coupling
constant in hertz (Hz). Mass spectra were recorded on a JEOL
GCmate II spectrometer with glycerol as a matrix for the FAB-MS.
5.1. General procedures
Most of the reagents and solvents were purchased from Aldrich
chemicals and used without purification. Unless indicated, all
anhydrous solvents were distilled under nitrogen condition.
Acetonitrile, dichloroethane, pyridine and dimethylformamide
were distilled from calcium hydride under nitrogen. Column
chromatography was performed using silica gel 60 (230e400 mesh,
Merck) with the indicated solvents. Thin-layer chromatography
(TLC) was performed using Kieselgel 60 F254 plates (Merck). IR
spectra were recorded on a JASCO FT/IR 430 spectrophotometer.
NMR spectra were recorded on a Varian YH 400 spectrometer (1H
400 MHz and 13C 100 MHz). Chemical shifts are expressed in parts
5.2. Synthesis of compounds
5.2.1. 3-(4-hydroxyphenyl)-1H-quinolin-4-one (1a)
A solution of quinolone 8a (100 mg, 0.39 mmol), acetic acid and
HBr (0.1 ml, 1.984 mmol) was stirred at 120 ꢁC for 3 days. The re-
action mixture was cooled to rt to form precipitates. The resulted
precipitates were filtered and washed with water and EtOAc thor-
oughly. The residue was purified by column chromatography
(EtOAc/toluene/EtOH ¼ 1:1:1) to give quinolone 1a (79 mg, 84%): IR
(neat, cmꢀ1) 3414, 1614, 1577, 1373, 1262, 1181; 1H NMR (400 MHz,
DMSO-d6)
d
12.23 (1H, s), 8.20 (1H, dd, J ¼ 8.4, 0.8 Hz), 8.16 (1H, s),
per million (ppm, d) relative to an internal standard, tetrame-
7.59e7.68 (2H, m), 7.49e7.46 (2H, m), 7.36 (1H, dt, J ¼ 7.2, 1.6 Hz),
thylsilane. 1H NMR data are reported in the order of chemical shift,
multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet
6.76 (2H, dt, J ¼ 8.8, 3.0 Hz); 13C NMR (100 MHz, DMSO-d6)
d 175.0,
156.8, 139.8, 138.1, 132.1, 130.2, 127.2, 126.2, 126.0, 123.9, 120.6,
Fig. 6. Cytotoxic effects of genistein, daidzein, and azaisoflavone analogs on MCF-7 breast cancer cells. Each experiment was performed three times independently and results were
expressed as means SD. One-way ANOVA with Dunnett's posttest was performed for each compound. Treatment means that are significantly different from control are indicated
as *p < 0.05, **p < 0.01 and ***p < 0.001.

H.J. Gim et al. / European Journal of Medicinal Chemistry 85 (2014) 107e118
113
118.9, 115.4; LRMS (FAB) m/z 238.1 (M þ Hþ); HRMS (FAB) calcd for
LRMS (FAB) m/z 268.1 (M þ Hþ); HRMS (FAB) calcd for C16H14N O3,
268.0974; found: 268.0975 (M þ H)þ.
C
15H12N O2, 238.0868; found: 238.0865 (M þ H)þ.
5.2.7. 7-hydroxy-3-(4-hydroxyphenyl)-1-ethyl-1H-quinolin-4-one
(2c)
5.2.2. 1-Methyl-3-(4-hydroxyphenyl)-1H-quinolin-4-one (1b)
This compound was prepared from 8b using the same procedure
as described for the preparation of 1a: Yield 75%; IR (neat, cmꢀ1
)
This compound was prepared from 9c using the same procedure
as described for the preparation of 1a: Yield 83%; IR (neat, cmꢀ1
)
3408, 1644, 1518, 1451, 1350, 1159; 1H NMR (400 MHz, DMSO-d6)
3337, 2855, 1734, 1457, 1257, 1031; 1H NMR (400 MHz, CD3OD)
d
10.39 (1H, s), 9.40 (1H, s), 8.10 (1H, s), 7.42 (2H, d, J ¼ 8.4 Hz), 6.75
(2H, d, J ¼ 8.4 Hz), 6.22 (1H, s), 6.09 (1H, s), 3.72 (3H, s); 13C NMR
d
8.26 (1H, d, J ¼ 8.8 Hz), 8.01 (1H, s), 7.44 (2H, d, J ¼ 8.8 Hz), 6.95
(1H, d, J ¼ 2.4 Hz), 6.93 (1H, dd, J ¼ 2.4,8.8 Hz), 6.82 (2H, d,
(100 MHz, DMSO-d6) d 179.0, 164.8, 163.2, 157.0, 144.2, 142.8, 130.3,
J ¼ 8.8 Hz), 4.31 (2H, q, J ¼ 14.4 Hz), 1.48 (3H, t, J ¼ 7.2 Hz); 13C NMR
129.6, 125.7, 118.6, 115.5, 108.2, 98.4, 91.1, 41.4; LRMS (FAB) m/z
252.1 (M þ Hþ); HRMS (FAB) calcd for C16H14N O2, 252.1025; found:
252.1027 (M þ H)þ.
(100 MHz, CD3OD)
d 183.2, 171.4, 161.3, 156.1, 143.8, 135.1, 131.1,
119.4,116.0,107.5,100.5, 97.3, 88.4, 71.5, 59.8, 30.7, 22.8; LRMS (FAB)
m/z 282.1 (M þ Hþ); HRMS (FAB) calcd for C17H16N O3, 282.1130;
found: 282.1129 (M þ H)þ.
5.2.3. 1-Ethyl-3-(4-hydroxyphenyl)-1H-quinolin-4-one (1c)
This compound was prepared from 8c using the same procedure
5.2.8. 7-hydroxy-3-(4-hydroxyphenyl)-1-propyl-1H-quinolin-4-
one (2d)
as described for the preparation of 1a: Yield 81%; IR (neat, cmꢀ1
)
3380, 1612, 1515, 1269, 838, 763; 1H NMR (400 MHz, DMSO-d6)
This compound was prepared from 9d sing the same procedure
d
8.27 (1H, d, J ¼ 8.0 Hz), 8.22 (1H, s), 7.75e7.69 (2H, m), 7.51 (2H, d,
as described for the preparation of 1a: Yield 82%; IR (neat, cmꢀ1
)
J ¼ 8.4 Hz), 7.36 (1H, t, J ¼ 7.2 Hz), 6.75 (2H, d, J ¼ 8.4 Hz), 4.35 (2H,
3393, 2955, 2922, 2850, 1739, 1462, 1244, 1021; 1H NMR (400 MHz,
CD3OD)
q, J ¼ 6.8 Hz), 3.72 (1H, s),1.34 (1H, t, J ¼ 6.8 Hz); 13C NMR (100 MHz,
d
8.27 (1H, d, J ¼ 8.8 Hz), 8.01 (1H, s), 7.44 (2H, d, J ¼ 8.8 Hz),
DMSO-d6)
d 174.5, 156.9, 142.9, 139.1, 132.5, 130.3, 129.0, 127.0,
6.96e6.94 (2H, m), 6.84 (2H, d, J ¼ 8.8 Hz), 4.23 (2H, t, J ¼ 7.2 Hz),
126.9, 123.9, 120.9, 117.0, 115.3, 47.9, 15.1; LRMS (FAB) m/z 266.1
(M þ Hþ); HRMS (FAB) calcd for C17H16N O2, 266.1181; found:
266.1180 (M þ H)þ.
1.92 (2H, q, J ¼ 7.2 Hz), 1.02 (3H, t, J ¼ 7.2 Hz); 13C NMR (100 MHz,
CD3OD)
d 177.2, 162.8, 157.7, 144.3, 142.5, 131.1, 129.8, 128.0, 122.5,
121.4, 116.0, 115.6, 100.7, 55.7, 23.1, 11.2; LRMS (FAB) m/z 296.1
(M þ Hþ); HRMS (FAB) calcd for C18H18N O3, 296.1287; found:
296.1280 (M þ H)þ.
5.2.4. 1-propyl-3-(4-hydroxyphenyl)-1H-quinolin-4-one (1d)
This compound was prepared from 8d using the same procedure
as described for the preparation of 1a: Yield 72%; IR (neat, cmꢀ1
)
5.2.9. 7-hydroxy-3-(4-hydroxyphenyl)-1-(3-methylbutyl)-1H-
quinolin-4-one (2e)
3195, 1610, 1566, 1541, 1512, 1492, 1330, 1231, 840, 750; 1H NMR
(400 MHz, CDCl3 þ CD3OD) 8.52 (1H, d, J ¼ 8.4 Hz), 7.67e7.71 (1H,
d
This compound was prepared from 9e using the same procedure
m), 7.51 (1H, d, J ¼ 8.4 Hz), 7.34e7.46 (2H, m), 7.45 (2H, d,
J ¼ 8.8 Hz), 6.89 (2H, d, J ¼ 8.8 Hz), 4.19 (2H, dd, J ¼ 7.6, 7.2 Hz), 1.94
(2H, m), 1.03 (3H, dd, J ¼ 7.6, 7.2 Hz); 13C NMR (100 MHz,
as described for the preparation of 1a: Yield 52%; IR (neat, cmꢀ1
)
3120, 2956,1617,1515,1443,1267,1025; 1H NMR (400 MHz, CD3OD)
d
8.27 (1H, d, J ¼ 8.8 Hz), 8.06 (1H, s), 7.79 (1H, d, J ¼ 2.4 Hz), 7.43
CDCl3 þ CD3OD)
d 176.3, 156.2, 142.3, 138.9, 130.0, 127.3, 126.9,
(1H, dd, J ¼ 2.4,8.8 Hz), 7.00e6.93 (4H, m), 4.29 (2H, t, J ¼ 7.2 Hz),
7.00e6.93 (4H, m), 3.97 (3H, s), 1.78 (3H, m), 1.06 (3H, s), 1.04 (3H,
126.3, 123.8, 122.0, 115.3, 115.2, 55.0, 22.2, 11.0; LRMS (FAB) m/z
280.1 (M þ Hþ); HRMS (FAB) calcd for C18H18N O2, 280.1338; found:
280.1336 (M þ H)þ.
s); 13C NMR (100 MHz, CD3OD)
d 177.1, 163.7, 157.7, 144.0, 142.4,
131.2, 129.7, 128.0, 122.6, 121.1, 116.1, 116.0, 100.7, 52.8, 38.6, 27.2,
22.7; LRMS (FAB) m/z 324.2 (M þ Hþ); HRMS (FAB) calcd for
20H22N O3, 324.1600; found: 324.1602 (M þ H)þ.
5.2.5. 7-Hydroxy-3-(4-hydroxyphenyl)-1H-quinolin-4-one (2a)
A solution of quinolone 9a (9.5 mg, 0.034 mmol), pyridine
(0.5 mL) and HBr (1 mL) was stirred at 80 ꢁC overnight. The reaction
mixture was cooled to rt, diluted and extracted with EtOAc. The
organic layer was washed with water and brine, then dried over
anhydrous MgSO4 and concentrated in vacuo. The residue was
purified by column chromatography (CHCl3/EtOAc/MtOH ¼ 20:1:2)
to give 2a (2.3 mg, 27%): IR (neat, cmꢀ1) 3263, 2924, 1591, 1460,
C
5.2.10. 7-hydroxy-3-(4-hydroxyphenyl)-1-benzyl-1H-quinolin-4-
one (2f)
This compound was prepared from 9f using the same procedure
as described for the preparation of 1a: Yield 37%; IR (neat, cmꢀ1
)
3423, 3214, 1616, 1516, 1327, 1270, 1222, 837; 1H NMR (400 MHz,
CD3OD)
d
8.24 (1H, d, J ¼ 8.8 Hz), 8.15 (1H, s), 7.46 (2H, d, J ¼ 8.4 Hz),
1235, 840; 1H NMR (400 MHz, DMSO-d6)
d
11.57 (1H, d, J ¼ 5.6 Hz),
7.32e7.35 (2H, m), 7.24e7.28 (1H, m), 7.19e7.21 (2H, m), 6.87 (1H,
dd, J ¼ 8.8, 2.4 Hz), 6.80e6.83 (3H, m), 5.48 (2H, s)); 13C NMR
10.20 (1H, br s), 9.29 (1H, s), 8.00 (1H, d, J ¼ 8.8 Hz), 7.87 (1H, d,
J ¼ 5.6 Hz), 7.48 (2H, d, J ¼ 8.8 Hz), 6.82 (1H, d, J ¼ 2.4 Hz), 6.73e6.78
(100 MHz, CDCl3 þ CD3OD)
d 176.2, 161.0, 156.0, 142.4, 141.3, 134.9,
(3H, m); 13C NMR (100 MHz, DMSO-d6)
d 175.0, 160.6, 156.3, 141.5,
129.9, 128.9, 128.7, 127.9, 126.3, 125.9, 121.7, 114.9, 114.4, 100.1, 56.4;
LRMS (FAB) m/z 344.1 (M þ Hþ); HRMS (FAB) calcd for C22H18N O3,
344.1287; found: 344.1285 (M þ H)þ.
137.0, 129.9, 128.0, 127.4, 119.8, 119.6, 115.0, 114.3, 101.2; LRMS (FAB)
m/z 254.1 (M þ Hþ); HRMS (FAB) calcd for C15H12N O3, 254.0817;
found: 254.0814 (M þ H)þ.
5.2.11. 5,7-dihydroxy-3-(4-hydroxyphenyl)-1H-quinolin-4-one
(3a)
5.2.6. 7-hydroxy-3-(4-hydroxyphenyl)-1-methyl-1H-quinolin-4-
one (2b)
This compound was prepared from 10a using the same proce-
dure as described for the preparation of 1a: Yield 87%; IR (neat,
cmꢀ1) 3294, 1654, 616, 1566, 1514, 1440,1382, 1275, 1199, 884, 829,
This compound was prepared from 9b using the same procedure
as described for the preparation of 1a: Yield 58%; IR (neat, cmꢀ1
)
3350, 2920, 1621, 1535, 1517, 1331, 1254, 1193; 1H NMR (400 MHz,
CD3OD)
8.27 (1H, d, J ¼ 8.8 Hz), 8.00 (1H, s), 7.45 (2H, d, J ¼ 8.8 Hz),
695, 655; 1H NMR (400 MHz, DMSO-d6)
d
12.02 (1H, d, J ¼ 6 Hz),
d
10.11 (1H, s), 7.43-7.41 (1H, m), 7.42 (2H, dd, J ¼ 6.8,2.0 Hz), 6.74
6.97 (1H, dd, J ¼ 2.4, 8.8 Hz), 6.94 (1H, d, J ¼ 2.4 Hz), 6.84 (1H, d,
(2H, dd, J ¼ 6.8, 2.0 Hz), 6.26 (1H, d, J ¼ 2.0 Hz), 5.97 (1H, d,
J ¼ 8.8 Hz), 3.89 (3H, s); 13C NMR (100 MHz, CD3OD)
d
195.7, 180.0,
J ¼ 2.0 Hz); 13C NMR (100 MHz, DMSO-d6)
d 179.7, 163.7, 156.9,
164.8, 151.7, 144.9, 131.2, 120.9, 116.0, 101.3, 100.7, 65.7, 54.0, 41.1;
142.2, 138.5, 130.3, 126.1, 118.6, 115.4, 108.1, 97.9, 91.9; LRMS (FAB)

114
H.J. Gim et al. / European Journal of Medicinal Chemistry 85 (2014) 107e118
m/z 270.1 (M þ Hþ); HRMS (FAB) calcd for C15H12N O4, 270.0766;
found: 270.0762 (M þ H)þ.
5.2.17. 1-(2-amino-4,6-dimethoxyphenyl)ethanone (4c)
This compound was prepared from 3,5-dimethoxyaniline using
the same procedure as described for the preparation of 4a: Yield
81%; IR (neat, cmꢀ1) 3435, 3282, 2973, 1615, 1578, 1456, 1426, 1363,
1322, 1247, 1208,1168, 1139, 953, 813; 1H NMR (400 MHz, CDCl3)
5.2.12. 5,7-dihydroxy-3-(4-hydroxyphenyl)-1-methyl-1H-quinolin-
4-one (3b)
This compound was prepared from 10b using the same proce-
dure as described for the preparation of 1a: Yield 88%; IR (neat,
cmꢀ1) 3407, 1644, 1561, 1517, 1451, 1349, 1159; 1H NMR (400 MHz,
d
6.34 (2H, s), 5.73 (1H, d, J ¼ 2.4 Hz), 5.68 (1H, d, J ¼ 2.4 Hz), 3.79
(3H, s), 3.75 (3H, s), 2.51 (3H, s); 13C NMR (100 MHz, CDCl3)
164.2, 163.7, 153.8, 105.9, 92.2, 88.5, 55.5, 55.4, 34.1.
d 200.4,
DMSO-d6)
J ¼ 8.4 Hz), 6.75 (2H, d, J ¼ 8.4 Hz), 6.22 (1H, s), 6.09 (1H, s), 3.72
(3H, s); 13C NMR (100 MHz, DMSO-d6)
179.0, 164.8, 163.2, 157.0,
144.2, 142.8, 130.3, 129.6, 125.7, 118.6, 115.5, 108.2, 98.4, 91.1; LRMS
(FAB) m/z 284.1 (M þ Hþ); HRMS (FAB) calcd for C16H14N O4,
284.0923; found: 284.0918 (M þ H)þ.
d 10.39 (1H, s), 9.40 (1H, s), 8.10 (1H, s), 7.43 (2H, d,
5.2.18. 1-(2-aminophenyl)-3-(4-methoxyphenyl)propenone (5a)
A mixture of aminophenyl ketone 4a (2.50 g, 18.51 mmol), p-
anisaldehyde (2.52 g, 18.51 mmol), a small amount of sodium hy-
droxide (1-2 pallets), and ethanol (3 mL) was stirred at rt for 10 h.
After the reaction was completed, the solvent was removed in
vacuo. The residue was diluted and extracted with EtOAc. The
organic layer was washed with water and brine, then dried over
anhydrous MgSO4 and concentrated in vacuo to give 2'-amino-
chalcone 5a (4.27 g, 91%) as a yellow solid : IR (neat, cmꢀ1) 3463,
2925, 1568, 1259, 1028, 800, 748, 657; 1H NMR (400 MHz, CDCl3)
d
5.2.13. 5,7-dihydroxy-3-(4-hydroxyphenyl)-1-ethyl-1H-quinolin-4-
one (3c)
This compound was prepared from 10c using the same proce-
dure as described for the preparation of 1a: Yield 85%; IR (neat,
cmꢀ1) 2971, 1612, 1509, 1459, 1354, 1288, 1245, 1161, 1121, 1073,
d
7.79 (1H, d, J ¼ 8.4 Hz), 7.65 (1H, d, J ¼ 15.6 Hz), 7.52 (2H, d,
J ¼ 7.6 Hz), 7.43 (1H, d, J ¼ 15.6 Hz), 7.20 (1H, m), 6.86 (2H, d,
1031, 951; 1H NMR (400 MHz, DMSO-d6)
d 9.42 (1H, s), 8.11 (1H, s),
J ¼ 8.8 Hz), 6.63 (2H, d, J ¼ 8.0 Hz), 6.23 (2H, s), 3.79 (3H, s); 13C
7.44 (2H, d, J ¼ 8.8 Hz), 6.74 (2H, d, J ¼ 8.8 Hz), 6.29 (1H, d,
NMR (100 MHz, CDCl3) d 192.0,151.1, 143.0,134.3, 131.1, 130.2, 128.2,
J ¼ 2.0 Hz), 6.07 (1H, d, J ¼ 2.0 Hz), 4.19-4.17 (2H, m),1.31 (3H, t,
120.9, 117.5, 116.0, 114.6, 55.6, 29.9.
J ¼ 7.2 Hz); 13C NMR (100 MHz, DMSO-d6)
d 179.0, 165.0, 163.2,
157.0, 143.2, 141.6, 130.4, 129.6, 128.9, 125.7, 119.0, 115.4, 108.5, 98.2,
90.8, 48.5, 14.5; LRMS (FAB) m/z 298.1 (M þ Hþ); HRMS (FAB) calcd
for C17H16N O4, 298.1079; found: 298.1074 (M þ H)þ.
5.2.19. 1-(2-amino-4-methoxyphenyl)-3-(4-methoxyphenyl)
propenone (5b)
This compound was prepared from 4b using the same procedure
as described for the preparation of 5a: Yield 81%; IR (neat, cmꢀ1
)
5.2.14. 5,7-dihydroxy-3-(4-hydroxyphenyl)-1-propyl-1H-quinolin-
4-one (3d)
This compound was prepared from 10d using the same proce-
dure as described for the preparation of 1a: Yield 32%; IR (neat,
cmꢀ1) 3258, 1642, 1560, 1515, 1446, 1362, 1157, 836; 1H NMR
3406, 3290, 2964, 2836, 1635, 1568, 1509, 1421, 1380, 1348, 1288,
1250, 1211, 1176, 1144, 990, 957; 1H NMR (400 MHz, CDCl3)
d
7.81
(1H, d, J ¼ 8.8 Hz), 7.69 (1H, d, J ¼ 15.2 Hz), 7.57 (2H, d, J ¼ 8.8 Hz),
7.47 (2H, d, J ¼ 15.2 Hz), 6.92 (2H, d, J ¼ 8.8 Hz), 6.56 (2H,brs), 6.27
(1H, dd, J ¼ 8.8, 2.4 Hz), 6.11 (1H, d, J ¼ 3.0 Hz), 3.84 (3H, s), 3.81 (3H,
(400 MHz, CD3OD)
d
7.84 (1H, s), 7.36 (2H, d, J ¼ 8.4 Hz), 6.79 (2H, d,
s); 13C NMR (100 MHz, CDCl3)
d 181.9, 169.9, 164.8, 161.9, 144.5,
J ¼ 8.4 Hz), 6.27 (1H, s), 6.14 (1H, s), 4.06 (2H, t, J ¼ 7.2 Hz), 1.83 (2H,
144.4, 132.6, 130.5, 127.8, 120.4, 116.5, 114.7, 109.7, 104.5, 55.8, 55.6,
31.1, 25.9.
m), 0.96 (3H, t, J ¼ 7.2 Hz); 13C NMR (100 MHz, CD3OD)
d 178.9,
164.0, 162.9, 156.5, 142.9, 141.6, 129.8, 125.5, 119.7, 114.7, 108.3, 97.7,
90.2, 54.8, 21.3, 9.7; LRMS (FAB) m/z 312.1 (M þ Hþ); HRMS (FAB)
calcd for C18H18N O4, 312.1236; found: 312.1243 (M þ H)þ.
5.2.20. 1-(2-amino-4,6-dimethoxyphenyl)-3-(4-methoxyphenyl)
propenone (5c)
This compound was prepared from 4c using the same procedure
as described for the preparation of 5a: Yield 56%; IR (neat, cmꢀ1
)
5.2.15. 1-(2-aminophenyl)ethanone (4a)
To a solution of boron trichloride (2.77 g, 23.63 mmol) in
3432, 3333, 2938, 2360, 1571, 1509, 1457, 1419, 1339, 1282, 1245,
1211, 1163, 1026, 970; 1H NMR (400 MHz, CDCl3)
d
7.56 (1H, d,
dichloromethane was added
a solution of aniline (2.00 g,
21.48 mmol) in 1,2-dichloroethane (15 mL) at 0 ꢁC. To the mixture
was added aluminum chloride (3.15 g, 23.63 mmol) and acetonitrile
(0.88 g, 21.48 mmol) and the mixture was refluxed at 80 ꢁC for 20 h.
After the reaction was completed, the reaction mixture was cooled
to 0 ꢁC, then 2 N HCl was added. The mixture was stirred at 80 ꢁC for
30 min. The reaction mixture was extracted with dichloromethane.
The organic layer was washed with 1 N NaOH and brine, dried over
anhydrous MgSO4, and concentrated in vacuo to give amino phe-
nylketone 4a (2.67 g, 92%) as a yellow solid: IR (neat); 3460, 3341,
J ¼ 15.6 Hz), 7.50 (2H, d, J ¼ 8.8 Hz), 7.36 (1H, d, J ¼ 15.6 Hz), 6.87
(2H, d, J ¼ 8.8 Hz), 5.99 (2H, s), 5.80 (1H, d, J ¼ 2.4 Hz), 5.75 (1H, d,
J ¼ 2.4 Hz), 3.82 (3H, s), 3.81 (3H, s), 3.77 (3H, s); 13C NMR
(100 MHz, CDCl3)
d 191.6, 164.1, 162.8, 161.0, 153.1, 139.5, 129.9,
128.9, 127.6, 114.4, 107.2, 92.5, 89.0.
5.2.21. N-[2-[3-(4-methoxyphenyl)-1-oxo-2-propenyl]phenyl]
acetamide (6a)
To a solution of 2'-aminochalcone 5a (4.20 g, 16.58 mmol) in
pyridine (40 mL) was added acetic anhydride (40 mL). The mixture
was stirred at rt for 3 h. After the reaction was completed, the re-
action mixture was extracted with EtOAc. The organic layer was
washed with water and brine, dried over anhydrous MgSO4, and
concentrated in vacuo to give a yellow solid. The residue was
recrystallized with EtOAc and n-hexane to give 2'-acet-
amidochalcone 6a (3.27 g, 76%) as a pale yellow solid: IR (neat,
cmꢀ1) 3158, 1687, 1641, 1576, 1510, 1455, 1308, 1255, 1204, 1012,
1644, 1615, 1242, 1162; 1H NMR (400 MHz, CDCl3)
d 7.71 (1H, dd,
J ¼ 8.4, 1.6 Hz), 7.26 (1H, ddd, J ¼ 8.4, 1.6 Hz), 6.65 (2H, m), 6.27 (2H,
br s), 2.58 (3H, s); 13C NMR (100 MHz, CDCl3)
132.0, 118.2, 117.2, 115.7, 27.9.
d 200.8, 150.2, 134.4,
5.2.16. 1-(2-amino-4-methoxyphenyl)ethanone (4b)
This compound was prepared from m-anisidine using the same
procedure as described for the preparation of 4a: Yield 56%; IR
(neat, cmꢀ1) 3450, 3332, 1619, 1223; 1H NMR (400 MHz, CDCl3)
838, 812; 1H NMR (400 MHz, CDCl3)
d 11.53 (1H, s), 8.66 (1H, d,
d
7.63 (1H, d,, J ¼ 8.8 Hz), 6.41 (2H, brs), 6.23 (1H, dd, J ¼ 8.8, 2.4 Hz),
J ¼ 8.0 Hz), 7.93 (1H, dd, J ¼ 8.0,1.4 Hz), 7.75 (1H, d, J ¼ 15.6 Hz), 7.59
(2H, d, J ¼ 3.2 Hz), 7.52 (1H, dt, J ¼ 7.8, 1.6 Hz), 7.41 (1H, d,
J ¼ 15.6 Hz), 7.13 (1H, dt, J ¼ 8.2,1.0 Hz), 6.92 (2H, dd, J ¼ 8.8, 4.8 Hz),
6.06 (1H, d, J ¼ 2.4 Hz), 3.79 (3H, s), 2.51 (3H, s); 13C NMR (100 MHz,
CDCl3)
d 198.8, 164.2, 152.7, 134.0, 112.7, 104.3, 99.0, 55.0, 27.5.

H.J. Gim et al. / European Journal of Medicinal Chemistry 85 (2014) 107e118
115
3.84 (3H, s), 2.22 (3H, s); 13C NMR (100 MHz, CDCl3)
162.2, 145.8, 141.1, 134.6, 130.7, 130.6, 127.5, 123.9, 122.6, 121.4,
120.6, 114.7, 55.7, 25.7.
d
193.6, 169.6,
cmꢀ1) 3395, 1700, 1654, 1614, 1578, 1511, 1451, 1430, 1335,1294,
1242, 1161, 1109, 1069, 807; 1H NMR (400 MHz, CDCl3)
10.64 (1H,
s), 7.81 (1H, s), 7.31 (2H, d, J ¼ 8.8 Hz), 6.83 (2H, d, J ¼ 8.8 Hz), 6.11
(1H, d, J ¼ 2.4 Hz), 4.93 (1H, d, J ¼ 8.8 Hz), 4.81 (1H, d, J ¼ 8.4 Hz),
3.80 (3H, s), 3.75 (3H, s), 3.75 (3H, s), 3.13 (3H, s), 2.16 (3H, s); 13C
d
5.2.22. N-{5-methoxy-2-[3-(4-methoxyphenyl)acryloyl]phenyl}
acetamide (6b)
NMR (100 MHz, CDCl3)
d 203.6, 169.4, 164.3, 161.1, 158.9, 141.9,
This compound was prepared from 5b using the same procedure
130.2, 128.0, 113.9, 107.8, 97.5, 94.5, 60.5, 55.8, 55.6, 55.4, 55.1, 54.5,
25.7.
as described for the preparation of 6a: Yield 61%; IR (neat, cmꢀ1
)
1700, 1605, 1512, 1452, 1419, 1291, 1235, 1202, 1176, 1158, 1235,
1202, 978, 835; 1H NMR (400 MHz, CDCl3)
d
12.20 (1H, br s), 8.35
5.2.27. 3-(4-methoxyphenyl)-4(1H)-quinolinone (8a)
(1H, d, J ¼ 2.4 Hz), 7.82 (1H, d, J ¼ 8.8 Hz), 7.64 (1H, d, J ¼ 15.2 Hz),
7.48 (2H, d, J ¼ 8.8 Hz), 7.34 (1H, d, J ¼ 15.2 Hz), 6.83 (2H, d,
J ¼ 8.8 Hz), 6.54 (1H, dd, J ¼ 8.8, 2.4 Hz), 3.79 (3H, s), 3.75 (3H, s),
To a solution of acetal 7a (2.37 g, 6.63 mmol) in ethanol was
added 5% HCl and the mixture was stirred at rt for 10 min, then
warmed to 50 ꢁC and stirred for 2 h. The reaction mixture was
cooled to rt to form light brown precipitates. The precipitates were
filtered and recrystallized with ethanol to give quinolone 8a (1.52 g,
91%): IR (neat, cmꢀ1) 3062, 2360, 1562, 1515, 1340, 1296,1244,1022,
2.17 (3H, s); 13C NMR (100 MHz, CDCl3)
d 191.8, 169.9, 164.7, 161.9,
144.5, 144.4, 132.6, 130.5, 127.8, 120.4, 116.5, 114.7, 109.7, 104.5, 55.8,
55.6, 25.9.
819, 754, 615; 1H NMR (400 MHz, DMSO-d6)
d
8.16 (1H, dd, J ¼ 8.0,
5.2.23. N-{3,5-dimethoxy-2-[3-(4-methoxyphenyl)acryloyl]phenyl}
acetamide (6c)
1.2 Hz), 8.06 (1H, d, J ¼ 4.0 Hz), 7.62 (3H, m), 7.53 (1H, dd, J ¼ 7.6,
0.6 Hz), 7.29 (1H, dt, J ¼ 7.4, 1.2 Hz), 6.91 (2H, td, J ¼ 8.0, 3.4 Hz), 3.74
(3H, s); 13C NMR (100 MHz, DMSO-d6)
d 175.4, 158.6, 139.9, 138.1,
This compound was prepared from 5c using the same procedure
as described for the preparation of 6a: Yield 89%; IR (neat, cmꢀ1
)
132.1, 120.1, 129.1, 126.2, 123.8, 120.2, 118.8, 118.7, 113.9, 55.7; LRMS
(FAB) m/z 252.1 (M þ Hþ); HRMS (FAB) calcd for C16H14N O2,
252.1025; found: 252.1017 (M þ H)þ.
2941, 1699, 1605, 1578, 1513, 1471, 1365, 1336, 1294, 1262, 1234,
1203, 1176, 1158, 1108, 1031, 975; 1H NMR (400 MHz, CDCl3)
d
10.99
(1H, s), 7.88 (1H, d, J ¼ 2.4 Hz), 7.62 (1H, d, J ¼ 15.6 Hz), 7.49e7.52
(2H, m), 7.22e7.27 (1H, m), 6.87e6.91 (2H, m), 6.22 (1H, d,
J ¼ 2.0 Hz), 3.86 (3H, s), 3.85 (3H, s), 3.82 (3H, s), 2.17 (3H, s); 13C
5.2.28. 3-(4-methoxyphenyl)-1-methyl-1H-quinolin-4-one (8b)
To a solution of quinolone 8a (100 mg, 0.321 mmol) and NaH
(60% dispersion in mineral oil, 19.3 mg, 0.482 mmol) in DMF
(1.5 mL) was added iodoethane (91.2 mg, 0.642 mmol). The mixture
was stirred at rt for 3 h. The reaction was quenched with water and
extracted with EtOAc. The organic layer was washed with water,
dried over MgSO4, and concentrated in vacuo. The residue was
NMR (100 MHz, CDCl3) d 200.1, 170.3, 164.5, 161.8, 142.3, 141.8, 131.1,
128.2, 126.0, 114.6, 110.2, 97.8, 94.1, 58.1, 57.6, 57.0, 24.1, 38.9.
5.2.24. N-[2-[3,3-dimethoxy-2-(4-methoxyphenyl)-1-oxopropyl]
phenyl]acetamide (7a)
A solution of thallium(III) nitrate trihydrate (3.31 g, 7.45 mmol)
in trimethyl orthoformate (7 mL) was added to a solution of 2'-
acetamidochalcone 6a (2.00 g, 6.77 mmol) in trimethyl ortho-
formate (15 mL) over a period of 15 min and the mixture was stirred
at rt for 4 h. After the reaction was completed, thallium(I) was
removed through the filtration. The filtrate was neutralized by
addition of 5% aqueous solution of NaOH and extracted with
dichloromethane. The combined extracts were washed with water
and brine, dried over anhydrous MgSO4, and concentrated in vacuo
purified
by
column
chromatography
(CHCl3/EtOAc/
MeOH ¼ 2:1:0.2) to give 8b (102.4 mg, 98%): IR (neat, cmꢀ1) 3512,
2925, 1613, 1574, 1510, 1460, 1242, 1177, 1070, 1028; 1H NMR
(400 MHz, DMSO-d6)
d, J ¼ 8.8 Hz), 6.45 (1H, s), 6.39 (1H, s), 3.87 (3H, s), 3.75 (3H, s), 3.72
(6H, s); 13C NMR (100 MHz, DMSO-d6)
174.6, 162.7, 162.6, 158.5,
144.6, 141.7, 130.3, 129.1, 121.9, 113.7, 112.4, 95.1, 91.2, 56.4, 56.2,
55.7, 41.5; LRMS (FAB) m/z 266.1 (M þ Hþ); HRMS (FAB) calcd for
C17H16N O2, 266.1181; found: 266.1173 (M þ H)þ.
d
7.89 (1H, s), 7.49 (2H, d, J ¼ 8.8 Hz), 6.87 (2H,
d
to give acetal 7a (2.37 g, 98%) as a brown semisolid: IR (neat, cmꢀ1
)
3445, 1515, 1382, 1243; 1H NMR (400 MHz, CDCl3)
d
11.53 (1H, s),
5.2.29. 3-(4-methoxyphenyl)-1-ethyl-1H-quinolin-4-one (8c)
This compound was prepared from 8a using the same procedure
8.66 (1H, dd, J ¼ 8.8, 0.8 Hz), 7.94 (1H, dd, J ¼ 8.4, 1.6 Hz), 7.46 (1H,
dt, J ¼ 7.6, 1.6 Hz), 7.28 (2H, dd, J ¼ 6.4, 2.0 Hz), 7.03 (1H, dd, J ¼ 7.8,
1.4 Hz), 6.83 (2H, dd, J ¼ 6.4, 2.0 Hz), 5.02 (1H, d, J ¼ 8.4 Hz), 4.85
(1H, d, J ¼ 8.4 Hz), 4.09 (1H, q, J ¼ 7.2 Hz), 3.74 (3H, s), 3.39 (3H, s),
as described for the preparation of 8b: Yield 81%: IR (neat, cmꢀ1
)
2990, 2833, 1571, 1512, 1329, 1241, 1176, 1031, 835, 758; 1H NMR
(400 MHz, DMSO-d6)
3.18 (3H, s), 2.21 (3H, s); 13C NMR (100 MHz, CDCl3)
d 135.2, 131.2,
d
8.27 (1H, d, J ¼ 8 Hz), 8.23 (1H, s), 7.49e7.65
(4H, m), 7.36 (1H, t, J ¼ 7.4 Hz), 6.93 (2H, d, J ¼ 7.2 Hz), 4.35 (2H, q,
130.0, 122.6, 121.0, 114.6, 106.7, 56.8, 55.8, 55.4, 54.7, 25.8.
J ¼ 6.8 Hz), 3.75 (3H, s), 1.35 (3H, t, J ¼ 6.8 Hz); 13C NMR (100 MHz,
DMSO-d6)
d 174.9, 158.7, 142.9, 139.2, 132.8, 130.2, 128.8, 127.5,
5.2.25. N-{2-[3,3-dimethoxy-2-(4-methoxyphenyl)propionyl]-5-
methoxyphenyl}acetamide (7b)
127.2, 123.8, 120.4, 117.0, 113.9, 55.9, 47.8, 15.1; LRMS (FAB) m/z
280.1 (M þ Hþ); HRMS (FAB) calcd for C18H18N O2, 280.1338; found:
280.1347 (M þ H)þ.
This compound was prepared from 6b using the same procedure
as described for the preparation of 7a: Yield 32%; IR (neat, cmꢀ1
)
2935, 1612, 1512, 1457, 1245, 1110, 818; 1H NMR (400 MHz, CDCl3)
d
12.07 (1H, br s), 8.38 (1H, d, J ¼ 2.4 Hz), 7.92 (1H, d, J ¼ 8.8 Hz), 7.31
5.2.30. 3-(4-methoxyphenyl)-1-propyl-1H-quinolin-4-one (8d)
This compound was prepared from 8a using the same procedure
(2H, d, J ¼ 8.8 Hz), 6.83 (2H, d, J ¼ 8.8 Hz), 6.55 (1H, dd, J ¼ 8.8,
2.4 Hz), 5.05 (1H, d, J ¼ 8.8 Hz), 4.81 (1H, d, J ¼ 8.8 Hz), 3.81 (3H, s),
3.74 (3H, s), 3.41 (3H, s), 3.20 (3H, s), 2.24 (3H, s); 13C NMR
(100 MHz, CDCl3)
114.8, 114.5, 109.8, 106.7, 104.3, 56.3, 55.8, 55.7, 55.4, 54.8, 51.5, 26.0.
as described for the preparation of 8b: Yield 98%: IR (neat, cmꢀ1
)
2963, 2933, 1623, 1583, 1512, 1490, 1246, 1178, 1035, 835, 760; 1H
NMR (400 MHz, CDCl3)
d
170.0, 165.0, 159.2, 144.8, 133.4, 129.9, 127.5,
d
8.49 (1H, d, J ¼ 8.0 Hz), 7.60 (1H, d,
J ¼ 2.0 Hz), 7.54 (2H, d, J ¼ 8.8 Hz), 7.51e7.56 (1H, m), 7.25e7.33 (2H,
m), 6.85 (2H, d, J ¼ 8.8 Hz), 3.99 (2H, t, J ¼ 6.8 Hz), 3.73 (3H, s), 1.79
5.2.26. N-{2-[3,3-dimethoxy-2-(4-methoxyphenyl)propionyl]-3,5-
dimethoxyphenyl}acetamide (7c)
This compound was prepared from 6c using the same proce-
dure as described for the preparation of 7a: Yield 98%; IR (neat,
(2H, m), 0.90 (3H, t, J ¼ 7.6 Hz),; 13C NMR (100 MHz, CDCl)
d 175.7,
158.6, 141.7, 138.9, 131.7, 129.7, 128.0, 127.5, 127.3, 123.3, 121.1, 115.2,
113.6, 55.2, 54.7, 22.2, 11.1; LRMS (FAB) m/z 294.1 (M þ Hþ); HRMS
(FAB) calcd for C19H20N O2, 294.1494; found: 294.1495 (M þ H)þ.

116
H.J. Gim et al. / European Journal of Medicinal Chemistry 85 (2014) 107e118
5.2.31. 7-methoxy-3-(4-methoxyphenyl)-2,3-dihydro-1H-quinolin-
4-one (9a)
7.18 (1H, dd, J ¼ 2.4,8.8 Hz), 7.08 (1H, d, J ¼ 2.4 Hz), 6.76 (2H, d,
J ¼ 8.8 Hz), 5.39, (1H, t, J ¼ 7.2 Hz), 5.14 (2H, d, J ¼ 6.4 Hz), 4.03 (3H,
s), 3.64 (3H, s), 1.95 (3H, s), 1.83 (3H, s). And then to a solution of N-
isoprenylated compound (30 mg, 0.08 mmol) in ethanol was added
Pd(OH)2 (24 mg, 80 wt%) and ammoniumformate (64.2 mg,
0.80 mmol) in H2O (0.5 mL) and stirred at rt for 18 h. The mixture
was filtered through Celite pad and washed with ethanol. The li-
quor was concentrated in vacuo and purified by column chroma-
tography (n-hexane/EtOAc ¼ 3:2) to give 9e (28 mg, 92%): 1H NMR
This compound was prepared using from 7b the same procedure
as described for the preparation of 8a: Yield 30%; IR (neat) 2965,
1632, 1560, 1515, 1350, 1289, 1250, 1180, 1029 cm-1; IR (neat, cmꢀ1
)
3398, 3073, 2967, 1634, 1517, 1252, 1181, 1030, 829; 1H NMR
(400 MHz, DMSO-d6)
d
11.78 (1H, d, J ¼ 3.6 Hz), 8.05 (1H, d,
J ¼ 9.2 Hz), 7.99 (1H, t, J ¼ 2.4 Hz), 7.60 (2H, d, J ¼ 8.4 Hz), 6.91 (4H,
d, J ¼ 4.8 Hz), 3.83 (3H, s), 3.74 (3H, s); 13C NMR (100 MHz, DMSO-
d6)
d
174.9, 162.3, 158.6, 141.6, 141.5, 137.8, 137.6, 130.1, 129.1, 128.1,
(400 MHz, CDCl3)
d
8.34 (1H, d, J ¼ 8.8 Hz), 8.13 (1H, s), 7.47 (2H, d,
120.7, 120.0, 114.1, 113.9, 99.5, 55.1, 55.7; LRMS (FAB) m/z 282.1
(M þ Hþ); HRMS (FAB) calcd for C17H16N O3, 282.1130; found:
282.1134 (M þ H)þ.
J ¼ 8.8 Hz), 7.18 (1H, dd, J ¼ 2.4,8.8 Hz), 7.08 (1H, d, J ¼ 2.4 Hz), 6.76
(2H, d, J ¼ 8.8 Hz), 4.35 (2H, t, J ¼ 7.2 Hz), 4.03 (3H, s), 3.64 (3H, s),
1.80e1.76 (3H, m), 1.95 (3H, s), 1.83 (3H, s); LRMS (FAB) m/z 352.2
(M þ Hþ); HRMS (FAB) calcd for C22H26NO3, 352.1913; found:
352.1913 (M þ H)þ.
5.2.32. 7-methoxy-3-(4-methoxyphenyl)-1-methyl-1H-quinolin-4-
one (9b)
This compound was prepared from 9a using the same procedure
5.2.36. 1-methoxy-7-benzyloxy-3-(4-benzyloxyphenyl)-1H-
quinolin-4-one (9f)
as described for the preparation of 8b: Yield 95%: IR (neat, cmꢀ1
)
3584, 2924, 1618, 1575, 1509, 1482, 1331, 1252, 1213, 1178, 1123,
To a solution of quinolone 9a (65.0 mg, 0.231 mmol) and NaH
60% dispersion in mineral oil (18.5 mg, 0.462 mmol) in DMF (2 mL)
was added benzyl bromide (33.6 mL, 0.277 mmol). The mixture was
1065,1033, 831; 1H NMR (400 MHz, CDCl3)
d
8.48 (1H, d, J ¼ 8.8 Hz),
7.59 (3H, d, J ¼ 8.8 Hz), 7.00 (1H, dd, J ¼ 2.4,8.8 Hz), 6.94 (2H, d,
J ¼ 8.8 Hz), 6.71 (1H, d, J ¼ 2.4 Hz), 3.94 (3H, s), 3.83 (3H, s), 3.78
stirred at rt for 5 h. The reaction was quenched with water and
extracted with EtOAc. The organic layer was washed with water,
dried over MgSO4, and concentrated in vacuo. The residue was
purified by column chromatography (n-hexane/EtOAc ¼ 2:3) to
(3H, s); 13C NMR (100 MHz, CDCl3)
d 175.3, 162.4, 162.3, 158.4, 141.7,
129.5, 129.0, 127.8, 121.1, 113.4, 112.2, 97.4, 55.4, 55.1, 40.5; LRMS
(FAB) m/z 296.1 (M þ Hþ); HRMS (FAB) calcd for C18H18N O3,
296.1287; found: 296.1284 (M þ H)þ.
give 9f (53.5 mg, 62%): 1H NMR (400 MHz, CDCl3)
d 8.45 (1H, d,
J ¼ 9.2 Hz), 7.71 (1H, s), 7.61 (2H, d, J ¼ 8.8 Hz), 7.29e7.35 (3H, m),
7.17 (2H, d, J ¼ 6.8 Hz), 6.92 (2H, d, J ¼ 8.8 Hz), 6.90e6.93 (1H, m),
6.61 (1H, d, J ¼ 2.0 Hz), 5.26 (2H, s), 3.80 (3H, s), 3.72 (2H, s); 13C
5.2.33. 7-methoxy-3-(4-methoxy-phenyl)- 1-ethyl-1H-quinolin-4-
one (9c)
This compound was prepared from 9a using the same procedure
NMR (100 MHz, CDCl3) d 175.6,162.4,158.7,141.7,141.0,135.3,129.8,
as described for the preparation of 8b: Yield 85% 1;H NMR
129.4, 129.2, 128.2, 127.8, 126.1, 121.7, 121.6, 113.7, 112.4, 98.6, 56.7,
(400 MHz, CDCl3)
d
8.49 (1H, d, J ¼ 9.2 Hz), 7.62 (1H, s), 7.60 (2H, d,
55.4, 55.3; LRMS (FAB) m/z 372.2 (M þ Hþ); HRMS (FAB) calcd for
J ¼ 8.8 Hz), 6.98 (1H, dd, J ¼ 2.4,8.8 Hz), 6.94 (2H, d, J ¼ 8.8 Hz), 6.76
C
24H22N O3, 372.1600; found: 372.1600 (M þ H)þ.
(1H, d, J ¼ 2.4 Hz), 4.15 (2H, q, J ¼ 14.4 Hz), 3.93 (3H, s), 3.83 (3H, s),
1.50 (3H, t, J ¼ 7.2 Hz); 13C NMR (100 MHz, CDCl3)
d
175.3, 162.4,
5.2.37. 5,7-dimethoxy-3-(4-methoxyphenyl)-1H-quinolin-4-one
(10a)
162.3, 158.5, 140.5, 140.3, 129.7, 129.6, 127.9, 121.7, 121.6, 113.6, 111.8,
104.9, 97.7, 55.5, 55.2, 47.9, 14.2; LRMS (FAB) m/z 310.1 (M þ Hþ);
HRMS (FAB) calcd for C19H20N O3, 310.1443; found: 310.1445
(M þ H)þ.
This compound was prepared from 7c using the same procedure
as described for the preparation of 8a: Yield 93%; IR (neat, cmꢀ1
)
3368, 3232, 2592, 1644, 1601, 1514, 1472, 1411, 1310, 1243, 1203,
1183, 1164, 1108, 1073, 1037, 977, 945, 867, 832; 1H NMR (400 MHz,
5.2.34. 7-methoxy-3-(4-methoxyphenyl)-1-propyl-1H-quinolin-4-
one (9d)
DMSO-d6)
(2H, d, J ¼ 7.2 Hz), 6.52 (1H, s), 6.35 (1H, s), 3.80 (3H, s), 3.78 (3H, s),
3.73 (3H, s); 13C NMR (100 MHz, DMSO-d6)
173.7, 162.8, 161.5,
158.7, 143.9, 137.0, 130.5, 128.7, 121.6, 113.9, 111.2, 95.8, 91.9, 56.6,
56.1, 55.7, 31.4; LRMS (FAB) m/z 312.1 (M þ Hþ); HRMS (FAB) calcd
for C18H18N O4, 312.1236; found: 312.1241 (M þ H)þ.
d
11.89 (1H, s), 7.89 (1H, s), 7.49 (2H, d, J ¼ 7.2 Hz), 6.89
This compound was prepared from 9a using the same procedure
d
as described for the preparation of 8b: Yield 67% 1;H NMR
(400 MHz, CD3OD)
d
8.32 (1H, d, J ¼ 8.8 Hz), 8.02 (1H, s), 7.53 (2H, d,
J ¼ 8.8 Hz), 7.05 (1H, dd, J ¼ 2.4,8.8 Hz), 6.99 (1H, d, J ¼ 2.4 Hz), 6.94
(2H, d, J ¼ 8.8 Hz), 4.26 (2H, t, J ¼ 7.2 Hz), 3.94 (3H, s), 3.80 (3H, s),
1.90 (2H, q, J ¼ 14.4 Hz), 1.48 (3H, t, J ¼ 7.2 Hz); 13C NMR (100 MHz,
5.2.38. 5,7-dimethoxy-3-(4-methoxyphenyl)-1-methyl-1H-
quinolin-4-one (10b)
CD3OD)
d 175.3, 162.3, 158.5, 141.3, 140.5, 129.6, 129.5, 127.9, 121.6,
121.0, 113.5, 111.6, 98.0, 55.4, 55.2, 54.7, 21.9, 11.1; LRMS (FAB) m/z
324.2 (M þ Hþ); HRMS (FAB) calcd for C20H22N O3, 324.1600; found:
324.1600 (M þ H)þ.
This compound was prepared from 10a using the same proce-
dure as described for the preparation of 8b: Yield 83%; IR (neat,
cmꢀ1) 3512, 2925, 1613, 1574, 1510, 1459, 1344, 1289, 1241, 1177,
1070, 1027; 1H NMR (400 MHz, DMSO-d6)
d 7.89 (1H, s), 7.49 (2H, d,
5.2.35. 7-methoxy-3-(4-methoxyphenyl)-1-(3-methylbutyl)-1H-
quinolin-4-one (9e)
To a solution of quinolone 9a (50 mg, 0.17 mmol) and NaH 60%
dispersion in mineral oil (12 mg, 0.53 mmol) in DMF (1 mL) was
J ¼ 8.4 Hz), 6.87 (2H, d, J ¼ 8.4 Hz), 6.45 (1H, d, J ¼ 2.0 Hz), 6.39 (1H,
d, J ¼ 2.0 Hz), 3.87 (3H, s), 3.75 (3H, s), 3.73 (3H, s), 3.72 (3H, s); 13
C
NMR (100 MHz, DMSO-d6) d 174.6, 162.7, 162.6, 158.5, 144.6, 141.7,
130.3, 129.1, 121.9, 113.8, 112.4, 95.1, 91.2, 56.4, 56.1, 55.7, 41.5; LRMS
(FAB) m/z 326.1 (M þ Hþ); HRMS (FAB) calcd for C19H20N O4,
326.1392; found: 326.1391 (M þ H)þ.
added dimethylallyl bromide (41 mL, 0.35 mmol). The mixture was
stirred at rt for 2 h. The reaction was quenched with water and
extracted with EtOAc. The organic layer was washed with water,
dried over MgSO4, and concentrated in vacuo. The residue was
purified by column chromatography (n-hexane/EtOAc ¼ 5:2) to
give N-isoprenylated compound (53 mg, 85%): IR (neat, cmꢀ1) 3584,
1617, 1576, 1510, 1472, 1258, 1177, 1034, 831; 1H NMR (400 MHz,
5.2.39. 5,7-dimethoxy-3-(4-methoxyphenyl)-1-ethyl-1H-quinolin-
4-one (10c)
This compound was prepared from 10a using the same proce-
dure as described for the preparation of 8b: Yield 88%; IR (neat,
cmꢀ1) 3284, 1641, 1562, 1515, 1449, 1362, 1159, 836; 1H NMR
CDCl3)
d
8.33 (1H, d, J ¼ 8.8 Hz), 8.13 (1H, s), 7.47 (2H, d, J ¼ 8.8 Hz),

H.J. Gim et al. / European Journal of Medicinal Chemistry 85 (2014) 107e118
117
(400 MHz, DMSO-d6)
d
7.82 (1H, s), 7.49 (2H, d, J ¼ 8.4 Hz), 6.89 (2H,
normalize the transfection efficiency,
co-transfected. The luciferase activities were measured using
luciferase assay system (Promega Corp., Madison, WI) and the
galactosidase activities were measured as the absorbance at 410 nm
by using an ELISA plate reader. Data are reported as relative lucif-
erase activity divided by the b-galactosidase activity.
b
-galactosidase plasmid was
d, J ¼ 8.4 Hz), 6.50 (1H, d, J ¼ 2.0 Hz), 6.45 (1H, d, J ¼ 2.0 Hz), 4.21
(2H, q, J ¼ 7.2 Hz), 3.91 (3H, s), 3.88 (3H, s), 3.78 (3H, s), 1.42 (3H, t);
b
-
13C NMR (100 MHz, DMSO-d6)
d
174.6, 162.8, 162.7, 158.5, 143.3,
140.7, 130.4, 129.1, 122.3, 113.7, 112.8, 94.9, 90.9, 56.4, 56.2, 48.3,
14.6; LRMS (FAB) m/z 340.1 (M þ Hþ); HRMS (FAB) calcd for
C
20H22N O4, 340.1549; found: 340.1550 (M þ H)þ.
5.5. ER competitive binding affinity assays
5.2.40. 5,7-dimethoxy-3-(4-methoxyphenyl)-1-propyl-1H-
quinolin-4-one (10d)
The competitive ER binding assay was performed as previously
This compound was prepared from 10a using the same proce-
dure as described for the preparation of 8b: Yield 84%; IR (neat,
cmꢀ1) 2963, 2934, 1632, 1612, 1591, 1463, 1289, 1245, 1163, 1038,
described (Arcaro et al., 1999). Briefly, 6 nM of the recombinant
human ER (rhER a, b Invitrogen Life Technology, Inc., CA, USA) was
incubated with test compounds for 9 h at room temperature in the
834; 1H NMR (400 MHz, CDCl3)
d
7.57 (2H, t, J ¼ 8.4 Hz), 7.44 (1H, s),
presence of 2.5 nM of [2,4,6,7-3H]-E2 (81.0 ci/mmol). Subsequently,
6.90 (2H, d, J ¼ 8.4 Hz), 6.34 (1H, d, J ¼ 2.0 Hz), 6.29 (1H, d,
J ¼ 2.0 Hz), 3.97 (2H, q, J ¼ 7.2 Hz), 3.94 (3H, s), 3.90 (3H, s), 3.81 (3H,
s), 1.89 (2H, tq, J ¼ 7.2 Hz), 1.00 (3H, t); 13C NMR (100 MHz, CDCl3)
100 mL of 50% (v/v) hydroxyapatite slurry was added to the reaction
mixture for 20 min at 4 ꢁC. Bound and free radioligand were
separated by centrifugation at 12,000 ꢂ g for 2 min, and the
radioactivity of the supernatant was determined using a liquid
scintillation counter (LS-6500, Beckman counter, CA, USA). The
amount of the receptor-bound [3H]-E2 in the presence of the test
compounds was presented relative to maximum receptor bound
ligand (10-7 M E2). The data are expressed as the ratio of the
receptor-bound [3H]-E2 in the presence of the compounds to the
0.1% DMSO used as a control. The IC50 values were determined from
doseeresponse curves obtained from three separate experiments.
d
175.7, 163.2, 162.4, 158.5, 143.1, 139.7, 130.1, 128.2, 123.1, 113.4,
112.9, 93.9, 90.0, 56.2, 55.7, 55.3, 21.7, 11.2; LRMS (FAB) m/z 354.2
(M þ Hþ); HRMS (FAB) calcd for C21H24N O4, 354.1705; found:
354.1740 (M þ H)þ.
5.3. Molecular modeling
5.3.1. Minimization of molecules
All the molecules for docking were sketched in the SYBYL-X and
energy minimizations were performed using Tripos Force field and
Gasteiger-Huckel charge with 100,000 iterations of conjugate
gradient method with convergence criterion of 0.05 kcal/mol. After
minimization is completed, all molecules are included in a database
for docking analysis.
5.6. MTT assay
MCF-7 cells were seeded in 96-well plate at a density of
1.5 ꢂ 103 cell/well for 24 h and followed by the treatment of 0.1 nM
E2, 100 nM ICI and the indicated concentrations of samples. After 4
days, MTT solution (5 mg/mL in phosphate buffered saline) was
added to each well and further incubated for 2 h. The absorbance of
5.3.2. Protein preparation
Crystal structures of genistein bound to ERa and ERb (PDB code
the formazan crystals dissolved in 100 mL of DMSO was measured at
1X7R and 1X7J) were refined using the following protocol. All
hydrogen was added to selected protein as random orientation and
the charge was loaded to protein and ligands with MMFF94 and
Gasteiger-Huckel, respectively. Sidechain amide was fixed and then
staged minimization of biopolymer hydrogens and ligands was
performed with 100 iterations of conjugate gradients method to a
gradient of 0.5 kcal using MMFF94s Force field and charges. For
next docking study, originally bound ligand was extracted from
prepared protein.
540 nm using the microplate reader (VERSAmax Molecular Devices,
CA, USA).
Acknowledgments
This work was supported by the National Research Foundation
of Korea grant funded by the Korea government (Project No. 2011-
0030074) and the SRC Research Center for Women's Diseases of
Sookmyung Women's University (2011).
5.3.3. Docking studies
Appendix A. Supplementary data
All molecular modeling calculations were carried out using the
Surflex Dock interfaced with SYBYL-X version 2.0. In this automated
docking program, the flexibility of the ligands is considered while
the protein or biomolecule is considered as a rigid structure. The
ligand is built in an incremental fashion, where each new fragment
is added in all possible positions and conformations to a pre-placed
base fragment inside the active site.
The protomol was generated using the crystallized ligand with a
threshold of 0.50 as default settings. All other parameters accepted
the default settings. The 3D coordinates of the active site were
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.07.030.
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